NZ620634B2 - Uses of labeled hsp90 inhibitors - Google Patents

Abstract

method for determining whether a patient with a blood cancer will likely respond to therapy with an HSP90 inhibitor comprises (a) contacting a sample containing blood cancer cells and non-cancerous blood cells from the patient with a cell permeable fluorescently labeled HSP90 inhibitor that binds directly and preferentially to a tumor-specific form of HSP90 present in the cancer cells of the patient; (b) measuring the amount of fluorescently labeled HSP90 inhibitor bound to HSP90 protein in the cancer cells and non-cancerous cells in the sample; and (c) comparing the amount of the fluorescently labeled HSP90 inhibitor bound to the cancer cells with the amount of the fluorescently labeled HSP90 inhibitor bound to the non-cancerous cells. A greater amount of fluorescently labeled HSP90 inhibitor bound to the cancer cells than the non-cancerous cells indicates presence of the tumor specific form of HSP90 in the cancer cells; thereby determining that the blood cancer will likely respond to the HSP90 inhibitor. directly and preferentially to a tumor-specific form of HSP90 present in the cancer cells of the patient; (b) measuring the amount of fluorescently labeled HSP90 inhibitor bound to HSP90 protein in the cancer cells and non-cancerous cells in the sample; and (c) comparing the amount of the fluorescently labeled HSP90 inhibitor bound to the cancer cells with the amount of the fluorescently labeled HSP90 inhibitor bound to the non-cancerous cells. A greater amount of fluorescently labeled HSP90 inhibitor bound to the cancer cells than the non-cancerous cells indicates presence of the tumor specific form of HSP90 in the cancer cells; thereby determining that the blood cancer will likely respond to the HSP90 inhibitor.

Description

WO 09657 USE§ OF LABELED HSP90 INHIBITORS This application claims the benefit under 35 U.S.C. § 119(e) ofUS. ional Application no 61/506,010, filed July 8, 2011, the contents of all of which are orated herein in their entireties by reference thereto. 2. BACKGROUND To maintain homeostasis, cells employ ate molecular machineries sed of thousands of proteins programmed to execute wellndefined functions. Dysreguiation of these pathways, through protein mis-expression or mutation, can lead to biological advantages that confer a malignant phenotype. Although at the cellular level such dysregulation may be beneficial (122., favoring increased survival), at the molecular level this es cells to invest energy in maintaining the ity and function of these proteins. It is believed that to maintain these proteins in a pseudo‘ stable state, cancer cells co-opt molecular chaperones, including P15139032”.

In support of this hypothesis, HSP90 is recognized to play important roles in maintaining the transformed phenotypen”. HSP90 and its associated co—chaperones assist in the correct conformational folding of cellular proteins, collectively referred to as “client proteins", many of which are effectors of signal n'ansduction pathways controlling cell growth, differentiation, the DNA damage response, and cell survival. Tumor cell addiction to deregulated proteins (Le, through mutations, aberrant expression, er cellular translocation etc) can thus become critically dependent on HSP90”.

The rationale for HSP90 therapy in various forms of cancers is now well-supported by preclinical and clinical studies including in disease resistant to standard therapyglm. For instance, studies have demonstrated a notable sensitivity of certain HER2+ tumors to HSP90 inhibitors95'”. In these tumors, l7-AAG (also called Tanespimycin) and l7-DMAG (Alvespirnycin) elicited responses even, and in ular, in patients with progressive e after trastuzumab therapy”. Other HSP90 inhibitors, such as PU.H7l , when tested pre-clinically in a number of -negan've breast cancer mouse models, delivered the most potent targeted singleaagent anti~turnor effect yet reported in this difficult-to—treat breast cancer subtypem.

While these data strongly support the use of HSP90 inhibitors in cancer, there is at the moment no clear consensus on how to identify those patients most-likely to benefit from HSP90 therapy'm'm. This is especially problematic knowing that for a successfirl pment of targeted agents it is essential to define the patient ulation that should receive the drug (122., tumors with EGFR mutations for tareeva). Such selection may reduce the number of patients receiving ineffective treatment and decrease the staggering number of targeted oncology agents that fail in late-stage clinical trials.

Further, there is no clinical assay that can non-invasively ascertain HSP90-target inhibition.

While pharmacodynamic monitoring of peripheral blood lymphocytes has provided a readily accessible and reproducible index of in viva biologic activity of HSP90 inhibitors in clinical , drug effects in normal tissue do not predict tumor-specific activity97"""’"2. ous use of biopsies to measure pharmacodynamic changes has remained an important way to assay for target modulation, but this method remains limited e of the logistical and ethical issues associated with invasive assays. As an alternative, changes in the levels of tumor HERZ and VEGF levels is now being investigated using zirconium 89 labeled antibodiesm‘m and of soluble HERZ extracellular domain levels in patient sera by ELISA“, but these studies are restricted to the subset of breast tumors that s these biomarkers.

Accordingly, there exists a strong need for kers in HSP90 targeted therapy: The majority of cancer patients are treated with novel, experimental therapies, in many cases with little insight into the mechanism of action of the specific agent, the suitability of a particular treatment for different disease subsets, and little knowledge into optimal dose and scheduling of therapeutics in different malignant gs. The end result is c clinical investigation, in which patients with refractory malignancies are treated with a spectrum of novel agents without knowledge of which therapeutic ches are best for different clinical ts.

HSP90 is a highly sought target in cancer because of its critical role in stabilizing and folding proteins involved in oncogenic transformation. Given their potential to e a number of ent oncoproteins and affect multiple signaling pathways, HSP90 inhibitors (HSP90i) have been esized to be active in a wide variety of cancers. Early clinical trials have continued the therapeutic potential of this approach in a subset of tumors, but finding kers to predict which cancers and patient populations will be most sensitive to such treatment has proven nging. Such poor understanding and selection of the adequate patient population has led to a large number of emerging HSP90 cancer therapeutics progressing slowly or g to continue development.

Immediate efforts to fy the sive population and to develop a companion diagnostic assay for HSP90 therapy are therefore urgently needed.

The design of a proper dose and schedule needed to achieve anti—tumor efficacy is also poorly understood in HSP90 therapy. Plasma pharrnacokinetics generally provide data informative for the design of therapeutic dosing, with the plasma area under the curve (AUC) oflen as a metric of systemic drug exposure. However, for HSP90 inhibitors, the tration and duration of retention of drugs in tumor tissues, and not in blood, determines their umor efi'ect'mm. Specifically, most HSP90 inhibitors are characterized by an atypical pharmacokinetic profile of rapid clearance from plasma and normal tissues but relatively prolonged dnig retention in tumors (i.e. for over 12-48 h post—administration). As such, clinical tanding of tumor response to HSP90 therapy remains severely limited if response is correlated with the injected dose, rather than the tumor dose. The d value of plasma pharmacokinetics and the importance oftumor dose for tumor response t the need for the al development of an assay of tumor pharmacokinetics for HSP90 tors. A validated, clinically practical vasive assay of tumor HSP90 would enable eutic dosing to focus upon achieving a steady-state tumor drug concentration, rather than a surrogate steady-state plasma concentration. Such assay could indicate if at or below a m permitted dosage, therapeutically effective tumor concentrations could be achieved. In case of contrary, ts could pursue an alternative treatment sparing them needless exposure to potential drug toxicity without a al benefit. [0010} To overcome these limitations associated with HSP90 therapy, we here design and develop a nonvinvasive assay that we propose will facilitate the optimal clinical implementation, development and use of HSP90 inhibitors in cancers. 3. SQMMARY 0F DIS§L0§URE This invention provides methods ofusing labeled HSP90 tors to improve treatment of cancer patients with HSP90 inhibitors.

The disclosure provides evidence that the abundance of this particular “oncogenic HSP90” species, which is not dictated by HSP90 expression alone, predicts for sensitivity to HSP90 inhibition therapy, and thus is a biomarker for HSP90 therapy. The disclosure also provides evidence that fying and measuring the abundance of this oncogenic HSP90 species in tumors predicts of the HSP90 fraction that response to HSP90 therapy. “Oncogenic HSP90” is defined herein as represents a cell stress specific form of chaperone complex, that is expanded and constitutively maintained in the tumor cell context, and that may execute ons necessary to maintain the malignant phenotype. Such roles are not only to regulate the folding of overexpresscd (i. e. HERZ), mutated (Le. vaRaf) or chimeric proteins (Le. Bcr—Abl), but also to tate scaffolding and complex formation of molecules involved in aberrantly ted signaling complexes (i.e. STATS, BCL6). While the tumor s addicted to survival on a network of HSP90—oncoprotcins, these proteins become dependent on enic HSP90" for functioning and stability. This symbiotic interdependence suggests that addiction of tumors to HSP90 oncoproteins equals addiction to “oncogenic HSP90”. Measuring the abundance of the latter is a read-out of the first, and therefore, in accordance with the present disclosure, is a biomarker for HSP90 therapy enrichment.

Furthermore, we show that HSP90 forms biochemically distinct complexes in malignant cells.

A major fraction of cancer cell HSP90 retains “housekeeping” chaperone functions similar to normal cells, whereas a functionally ct HSP90 pool enriched or expanded in cancer cells (112., “oncogenic HSPQO”) specifically interacts with oncogenic proteins required to maintain tumor cell survival, nt proliferative features and invasive and metastatic behavior.

To measure in a by~tumor manner the abundance of the “oncogenic I-ISPQO”, the invention also provides chemical tools. Such tools include fluorescently labeled and ANCAvlabelcd HSP90 inhibitors, biotinylated HSP90 inhibitors and radiolabelcd tors that specifically identify and interact with this tumor “oncogenic HSPQO” species, making it feasible to measure the abundance of the “oncogenic HSP90” species in different types of tumors, tumor cells, tumor-supporting cells and tumor-associated biological ions, such as in hematologic malignancies, solid tumors and liquid tumors, and thus, measure and predict ivity to HSP90 inhibition therapy. These may be in the form of but not limited to cancer cells in a solid or liquid tumor, cancer stem cells, circulating tumor cells, tumor supporting immune cells, es and supporting progenitor cells.

In one aspect, the disclosure provides a method for determining r a tumor will likely respond to therapy with an HSP90 tor which ses the following steps: (a) ting the tumor or a sample containing cells from the tumor with a detectably labeled HSP90 inhibitor which binds preferentially to a tumor-specific form of HSP90 present in a tumor or tumor cells; (b) measuring the amount of labeled HSP90 inhibitor bound to the tumor or the tumor cells in the sample; and (c) comparing the amount of labeled HSP90 inhibitor bound to the tumor or the tumor cells in the sample measured in step (b) to the amount of labeled I-ISPQO inhibitor bound to a reference ; wherein a greater amount of labeled HSP90 tor bound to the tumor or the tumor cells measured in step (b) as compared with the reference amount indicates the tumor will likely respond to the HSPQO inhibitor.

In one embodiment the reference is from cells of the same patient with the tumor. The reference can be normal cells from the cancer patient For instance, the normal cells can be lymphocytes from a patient with a blood tumor, leukocytes from a patient with circulating tumor cells or normal tissue surrounding a solid tumor. In another embodiment the reference is a tumor cell or another cell from the cancer patient with little to no expression of the nic HSP90. In another embodiment, the reference is from cells of a different patient than the patient with the tumor. For instance, the reference can be from cells of a healthy individual or cells with little to no expression of the oncogenic HSP90 from a cancer patient other than the patient with the tumor to be measured.

In one aspect, the disclosure provides a method for determining whether a tumor will likely respond to therapy with an HSP90 tor which comprises the following steps: (a) contacting the tumor or a sample containing cells from the tumor with a first detectably labeled HSP90 inhibitor which binds preferentially to a tumor-specific form of HSP90 present in a tumor or tumor cells and a second detectably labeled inhibitor which has minimal or no binding to the tumor-specific form ofHSP90; (b) measuring the amount of first labeled inhibitor and second labeled tor bound to the tumor or the tumor cells in the sample; and (c) comparing the amount of first labeled inhibitor bound to the tumor or the humor cells with the amount of second labeled tor bound to the tumor or tumor cells, wherein a greater amount of first labeled inhibitor bound to the tumor or tumor cells as compared with the second labeled inhibitor bound to the tumor or tumor cells indicates the tumor will likely d to the HSP9O inhibitor.

In one ment, the d HSP90 inhibitor is fluorescently labeled or abeled inhibitor that is cell permeable and that selectively binds to “oncogenic HSP90”. For e, different fluorescently labeled and ANCA—labeled versions of the HSP90 inhibitor PU—H7l are provided that have been optimized for use in flow cytometry and for the analysis of cancer cells found in or isolated from a solid or liquid tumor, cancer stem cells, circulating tumor cells, tumor supporting immune cells, exosomes and supporting progenitor cells, and for use in tissue staining for samples obtained by several interventional methods such as biopsies, surgeries and fine needle aspirates.

In one such embodiment, we show that fluorescently labeled inhibitors such as PU-H7l- FITCZ [Section 5.2, l .1.) can be used to measure the abundance of “oncogenic HSP9D” in tissues obtained from such sources as biopsies and surgery specimens. In another embodiment, we show that fluorescently labeled inhibitors such as PU—H7l-FITC2 can be used to measure the abundance of “oncogenic HSP90” in established cancer cell lines or in primary cancer cells. In still another embodiment we show that fluorescently labeled inhibitors can be used to measure the abundance of “oncogenic HSP90” in cells isolated fiom cancer specimens such as from tumors, in cancer stem cells, in circulating tumor cells and in cancer cells ed from fine needle tes. In still other embodiments, we show that the other cently labeled, ANCA—labeled and biotinylated HSP90 inhibitors that are also useful to perform the above mentioned measurements. [0020! In mother ment, the labeled HSP90 inhibitor is a radiolabeled inhibitor that ively binds to “oncogenic HSP90”. For example, different versions of radiolabeled PU—H7l have been optimized for PET imaging. In a particular embodiment, iodine 124 radiolabeled versions of PU—H7l are for PET imaging of solid and liquid tumors. The abeled inhibitors can be used to image numerous types of primary and metastatic cancers including but not limited to colorectal cancer, pancreatic cancer, d cancer, basal cell carcinoma, melanoma, renal cell carcinoma, bladder and non-small cell lung cancer, cancer, prostate cancer, a lung cancer including small cell lung cancer breast cancer, neuroblastoma, gastrointestinal cancers including gastrointestinal stromal tumors, geal cancer, stomach cancer, liver cancer, gallbladder cancer, anal cancer, brain tumors including s. lymphomas including follicular lymphoma and diffuse large B—cell lymphoma, leukemias, myelomas, myeloproliferative neoplasms and gynecologic cancers including n, al, and endometrial cancers.

The disclosure further provides means to measure in a nunor-by—tumor manner the abundance of the “oncogenic HSP90" in solid tumo1s such as, but not limited to, those tumors listed above and in liquid tumors, such as, but not limited to, those associated with lymphomas, leukemias, myelomas and roliferative neoplasms. In one embodiment, the invention shows that by the use of Iodine 124 labeled HSP90 inhibitors that specifically interact with the “oncogenic HSP90" it is possible to use non-invasively PET imaging and quantify the “oncogenic HSP90” in patients, in solid tumors and liquid tumors.

In one aspect , the disclosure provides a method for determining whether a patient with hematologic malignancies such as blood cancer (e.g., leukemias) will likely respond to y with an HSP90 tor which comprises contacting a sample containing cancer cells from the patient and reference ncer cells with a cell permeable fluorescently labeled HSP90 inhibitor that binds preferentially to a tumor-specific form of HSP90 present in the cancer cells of the patient, measuring the amount of fluorescently labeled HSP90 inhibitor bound to the cancer cells and non-cancer in the sample, and comparing the amount of the fluorescently labeled HSP90 inhibitor bound to the cancer cells with the amount of the fluorescently labeled HSP90 inhibitor bound to the ncer cells, wherein a greater amount of fluorescently labeled HSP90 inhibitor bound to the cancer cells than the non-cancer cells tes the tumor will likely respond to the HSP90 inhibitor.

In one such embodiment the reference normal cells are normal cells (e.g., lymphocytes) which are from the same patient as the cancer cells. In another embodiment, the reference non-cancer cells are ed from a different patient than the cancer patient. [0024) In another aspect. the sure provides a method for determining whether a t with a solid tumor will likely respond to therapy with an HSP90 inhibitor which comprises contacting a sample, such as obtained from biopsy, surgery, fine needle tes or other interventional procedure, containing cancer cells and ncer cells from the patient (e.g. surrounding stroma, benign cells or other types of normal cells in the specimen) with a cell permeable fluorescently labeled HSP90 inhibitor that binds preferentially to a tumoraspecific form ofHSP90 present in the cancer cells of the patient, measuring the amount of cently labeled HSP90 inhibitor bound to the cancer cells and labeled HSP90 non’cancer cells in the sample, and comparing the amount of the fluorescently inhibitor bound to the cancer cells with the amount ofthe fluorescently labeled HSP90 inhibitor bound to the non—cancer cells. wherein a greater amount of fluorescently d HSP90 inhibitor bound to the cancer cells than the non-cancer cells indicate that the tumor will likely respond to the HSP90 inhibitor.

The disclosure also provides a method for determining whether a patient with a solid tumor will likely d to therapy with an HSP90 inhibitor which comprises contacting a sample containing circulating cancer cells and non-cancer cells (e.g., leukocytes) from the patient with a cell permeable fluorescently labeled HSP90 inhibitor that binds preferentially to a tumor—specific form of HSP90 t in the cancer cells of the patient, measuring the amount of fluorescently labeled HSP90 inhibitor bound to the cancer cells and non-cancer in the sample, and comparing the amount of the cently labeled HSP9O inhibitor bound to the cancer cells with the amount of the fluorescently labeled HSP90 inhibitor bound to the non-cancer cells, wherein a greater amount of fluorescently labeled HSP90 inhibitor bound to the cancer cells than the non-cancer cells indicates the tumor will likely d to the HSP90 inhibitor.

In an alternative embodiment, the reference non—cancer cells are obtained from a patient other than the patient with the tumor In another aspect, the disclosure provides methods for using abeled HSP90 inhibitors to ine patients who will be susceptible to HSP90 inhibition therapy.

In one such embodiment, the disclosure provides s for determining r a cancer patient with an imageable tumor will likely respond to therapy with an tor of HSP90 which comprises the following steps: (a) administering to the patient a radiolabeled HSP9O inhibitor which binds preferentially to a tumor—specific form of HSP90 present in the tumor or in tumor cells of the tumor; (11) measuring uptake of the radiolabeled HSP9O inhibitor by the patient’s tumor at one or more time points afler the administration in step (a); (c) measuring uptake of the radiolabeled HSP90 inhibitor by a predetermined y tissue or blood of the patient at said one or more time points after the administration in step (a); (d) computing a ratio of the uptake ed at one or multiple time points in step (b) with the uptake measured at the same time points in step (c); and (e) determining the likelihood the cancer patient will respond to therapy with the inhibitor of HSP90, n a ratio greater than 2 computed in step (d) at one or multiple time points indicates that the patient will likely respond.

In another embodiment, the disclosure provides a method for determining whether a cancer patient with an imageable tumor will likely respond to therapy with an inhibitor of HSP90 which comprises the following steps: (a) administering to the patient a radiolabeled HSP90 inhibitor which binds preferentially to a tumor-specific form of HSP90 present in the tumor or in tumor cells of the tumor; (b) measuring uptake of the radiolabeled HSP90 inhibitor by the patient’s tumor at one or more time points more than 4 hours afier the administration in step (a), wherein an uptake of the inhibitor at said one or more time points relative to the uptake in healthy tissue surrounding the tumor indicates that the t will likely respond to therapy with an inhibitor of HSP90.

In yet another embodiment, the disclosure provides a method for ining whether an ble tumor will likely respond to therapy with an inhibitor of HSP90 which ses the following steps: (a) administering to the patient a radiolabeled HSP90 inhibitor which binds preferentially to a tumor~specific form of HSP90 t in the tumor or in tumor cells of the tumor, (b) visually inspecting by PET the uptake of the radiolabeled inhibitor in the tumor or in tumor cells of the tumor at one or more time points 2 hours or more following administration of the radiolabeled HSP90 inhibitor in step (a), (c) comparing the PET image obtained in step (b) with the PET image obtained in healthy tissue surrounding the tumor at said one or more time ; wherein the presence of an illuminated region in the PET image in the tumor or in tumor cells of the tumor at said one or more time points indicates that the patient will likely respond to HSP9O inhibition therapy.

In still r embodiment, the disclosure provides a method for determining whether a specific cancer t with a tumor sing the oncogenic HSP90 will likely respond to therapy with a defined dose of an inhibitor of HSP90 which comprises the following steps: (a) administering to the patient a abeled form of the HSP90 inhibitor which binds preferentially to a tumor—specific form of HSP90 present in the tumor or in tumor cells of the tumor; (b) measuring uptake of the radiolabeled form of the HSP90 inhibitor by the patient’s tumor at one or more time points afier the administration in step (a); (c) ating for the defined dose of the HSP90 tor, the concentrations of the HSP90 inhibitor which would be t in the patient’s tumor at each of said one or more time points, based on the uptake measured at said one or more time points in step (b); and (d) comparing the concentrations of the HSP90 inhibitor calculated in step (c) with reference concentrations of the HSP90 inhibitor which would need to be present in the tumor at said one or more time points for the HSP90 inhibitor to be effective in treating the tumor, wherein the patient will likely respond to therapy with the defined dose of the HSP90 tor if the concentrations of the HSP90 inhibitor calculated in step (c) would equal or exceed the concentrations of the HSP90 inhibitor needed to effectively treat the tumor.

In r aspect, we show that radiolabeled HSP90 inhibitors can be used to determine efi'ective doses and dosing schedules of HSP90 inhibitors.

In one such embodiment, the disclosure provides a method for determining whether a specific cancer patient with a tumor expressing the oncogenic HSP90 will likely respond to therapy with a defined dose of an inhibitor of HSP90 which comprises the following steps: (a) administering to the patient a radiolabeled form of the HSP90 inhibitor which binds entially to a tumor-specific form of HSP90 present in the tumor or in tumor cells of the tumor; (b) measuring uptake of the radiolabeled form of the HSP90 inhibitor by the patient’s tumor at one or more time points afier the administration in step (a); (c) calculating for the defined dose of the HSP90 inhibitor, the concentrations of the HSP90 inhibitor which would be t in the patient’s tumor at each of said one or more time points, based on the uptake measured at said one or more time points in step (b); and (d) comparing the re of the tumor to the HSP90 inhibitor calculated in step (c) with a reference re to the HSP90 inhibitor which would need to be present in the tumor at said one or more time points for the HSP90 inhibitor to be effective in treating the tumor, wherein the patient will likely respond to therapy with the defined dose of the HSP90 inhibitor if the tumor exposure to the HSP90 inhibitor calculated in step (c) would equal or exceed the tumor exposure to the HSP90 inhibitor needed to efiectively treat the tumor.

Additional embodiments include a method for determining, for a specific cancer patient with an imageable tumor, an ive dose and frequency of administration for therapy with an inhibitor of HSP90; a method for determining the concentration of a HSP90 inhibitor present in an imageable tumor in a cancer patient; and a method for determining or monitoring the responsiveness to therapy with an inhibitor of HSP90 of a tumor in a cancer patient.

In yet another embodiment, this sure provides a method for determining, for a specific cancer patient with a tumor that expresses the oncogenic HSP90, an ive dose and frequency of administration for y with an inhibitor of HSP90 which comprises the following steps: (a) administering to the patient a radiolabeled form of the HSP90 inhibitor which binds preferentially to a specific form of HSP90 present in a tumor or tumor cells; (b) measuring uptake of the radiolabeled form of the HSP90 tor by the patient’s tumor at one or more time points after the administration in step (a); and (c) calculating the dose and frequency of administration needed to maintain in the tumor at each of said one or more time points a concentration of the HSP90 inhibitor effective to treat the tumor, based on the uptake ed at said one or more time points in step (b), thereby mining, for the cancer patient, the effective dose and frequency of administration for therapy with the inhibitor of HSP90.

In still another embodiment, this disclosure provides a method for determining, for a c cancer patient with a tumor expressing the oncogenic HSP90, an effective dose and frequency of administration for therapy with an inhibitor of HSP90 which comprises the following steps: (a) administering to the patient a radiolabeled form of the HSP90 inhibitor which binds preferentially to a turner-specific form of HSP90 present in a tumor or tumor cells; (b) measuring uptake of the radiolabeled form of the HSP90 inhibitor by the patient’s tumor at one or more time points afier the administration in step (a); and (c) calculating the dose and frequency of administration needed to maintain in the tumor over the period of ent an average tumor concentration of the HSP90 inhibitor effective to treat the tumor, based on the uptake measured at said one or more time points in step (b), thereby determining, for the cancer patient, the effective dose and frequency of administration for therapy with the tor of HSP90.

In a still further aspect, the disclosure provides a method for determining the concentration of an HSP90 inhibitor t in a tumor expressing the oncogenic HSP90 in a cancer patient which comprises the following steps: (a) inistering to the patient a predetermined amount of the HSP90 inhibitor and an amount of a radiolabeled form of the HSP90 inhibitor which binds preferentially to a tumor-specific form of HSP90 present in a tumor or tumor cells; (b) periodically measuring the uptake of the radiolabeled HSP90 inhibitor by the patient’s tumor at one or more time point(s) afier the co-administration in step (a); (c) ining the tration of the HSP90 inhibitor present in the tumor at any such time point based on the ements of the uptake of the radiolabeled HSP90 inhibitor in step (b).

In yet another aspect, the disclosure es a method for determining the responsiveness to therapy with an inhibitor ofHSP90 of a tumor expressing the oncogenic HSP90 in a cancer patient which comprises the following steps: (a) administering a radiolabeled form of the HSP90 inhibitor which binds preferentially to a tumor-specific form of HSP90 present in a tumor or tumor cells, to the patient at one or more time points within the period during which the t is receiving the inhibitor of HSP90 as therapy; (b) measuring the concentration of the radiolabeled HSP90 inhibitor in the patient’s tumor at said one or more time points afier the administration in step (a); and (c) comparing the trations of the radiolabeled HSP90 inhibitor measured in step (b) with the minimum concentration of the HSP90 inhibitor needed to effectively treat the tumor, n measured concentrations greater than the minimum needed to treat the tumor indicate that the patient is likely to respond to therapy with the HSP90 inhibitor.

In another aspect, the disclosure provides method for determining whether a patient suffering from a neurodegenerative disease will likely respond to therapy with an HSP90 tor which comprises the following steps: (a) contacting the brain with a radiolabeled HSP90 inhibitor which binds preferentially to a enic form of HSP90 present in a brain cells of the patient; (b) measuring the amount of labeled HSP90 inhibitor bound to the brain cells in the sample; and (c) comparing the amount of d HSP90 inhibitor bound to the brain cells in the sample measured in step (b) to a reference amount; wherein a greater amount of labeled HSP90 inhibitor bound to the brain cells measured in step (b) as compared with the reference amount indicates the patient will likely respond to the HSP90 inhibitor. (0040] In another aspect, the disclosure provides methods of treating HSP90 dependent cancers with the HSP90 inhibitor PU—l-I7l. In particular embodiments, methods of ng HSP90 dependent cancers to e specific tumor exposures of PU—H7l are provided. In other embodiments, novel dosing regimens of PU-H7l are provided.

In another aspect. the disclosure provides a method for determining whether a human cancer which comprises: present in a patient will likely respond to therapy with an HSP90 inhibitor (a) obtaining a sample containing cells from the patient’s cancer, which cells express HSP90 protein alone or in addition to HSP70 protein; (b) assessing for the cells present in the sample obtained in step (a) the presence of at least one of the following parameters: an activated AKT pathway, a defect in P’TEN tumor suppressor fiinction or expression, an activated STATS y, or Bcl-xL protein expression; and (c) comparing the assessment obtained in step (b) with a predetermined reference ment of the same parameter or parameters assessed in step (b) for human cancer cells from one or more cancer patient(s) who responded to therapy with the HSP90 inhibitor so as to thereby determine r the patient’s cancer will likely respond to y with the HSP9O tor.

In one embodiment. human cancers currently of erable interest for use of this particular method are breast cancer, pancreatic cancer and acute myeloid leukemia.

Methods for assessing each of the ters are well known in the art and readily available.

However a correlation of one or more of these particular parameters with predicting the efiicacy of a HSP90 tor has not been shown previously. Although in theory a single parameter may be ent to enable a skilled practitioner to predict the efficacy of any given HSP90 inhibitor, it is these parameters will need to be more likely that at least 2, perhaps at least 3 or more or even all of taken into account to make a sound prediction of y. 4. BRIEF DESCRIPTION OF FIGURES Figure l. PUrH7l preferentially interacts with a restricted fi‘action of HSP90 that is more abundant in cancer cells. (a) Sequential immune-purification steps with H9010, an anti-HSP90 antibody, e HSP90 in the MDA-MB—468 cell extract. Lysate = control cell extract. (b) HSP9O from MDA-MB—468 extracts was isolated through sequential chemical- and immuno-purification and values were steps. The amount of HSP90 in each pool was fied by densitometry normalized to an internal standard. (c) Saturation studies were performed with ”‘I-PU-H7l in the ted cells. All the isolated cell samples were counted and the specific uptake of ”'I-PU-H71 determined. These data were plotted against the tration of l3'I-PU171 to give a saturation binding curve. Representative data of four separate repeats is presented (lower). Expression of HSP90 in the indicated cells was analyzed by Western blot (upper). ((1) Primary AML and CML, CD34+ cord blood cells (CB), or K562 cells were pre-treated with the indicated doses of PU-H7l for 24 h. Post—treatment cells were treated with 1 uM PU—FITC. Binding of PU-FITC to the cells was evaluated by flow try and is represented as mean fluorescence ity (MFI). TEG-FITC is shown as a non—specific binding control. CD45 vs. SSC gating was used to distinguish binding to blast or lymphocytes from the primary specimens. (e) Percent viability relative to untreated control for primary AML and CML, CD34+ CB or K562 cells afier ent at the indicated doses of PU- 1-171. Cell viability was ted by annexin V/7-AAD staining 96 h post-treatment. Data are presented as means t SE (n = 3).

Figure 2. PU-H71 is selective for and isolates HSP90 in complex with oncoproteins and co- chaperones. (a) HSP90 xes in K562 extracts were isolated by precipitation with H9010, a non- specific IgG, or by PU—H7 1— or Control—beads. Control beads contain ethanolamine, an HSP90-inert molecule. Proteins in pull-downs were ed by Western blot. (b,c) Single or sequential - and chemical—precipitations, as indicated, were conducted in K562 extracts with H9010 and PU—beads at the indicated frequency and in the showu sequence. Proteins in the pull~downs and in the remaining supernatant were analyzed by WB. NS = ecific. (d) K562 cell were treated for 24h with vehicle (-) or PU-H7l (+), and proteins ed by Western blot. (e) sion of proteins in Hsp70—knocked-down cells was ed by n blot (left) and changes in protein levels presented in relative luminescence units (RLU) (right). Control = scramble siRNA. (f) Sequential cal—precipitations, as indicated, were conducted in K562 extracts with GM-, SNX- and NV?- beads at the indicated fi-equency and in the shown sequence. Proteins in the pull-downs and in the remaining supernatant were ed by Western blot. (g) HSP90 in K562 cells exists in complex with both aberrant, Bcr-Abl, and normal1 c-Abl, proteins. PU-H7l, but not H9010, selects for the HSP90 population that is Bcr-Abl onco—protein bound.

Figure 3. (a,b) HSP90 from breast cancer and CML cell extracts (120 pg) was isolated througt serial chemical- and immuno-purification steps, as indicated. The supernatant was isolated to analyze the lefl—over HSP90. HSP90 in each fi-action was analyzed by Western blot. Lysate = endogenous protein content; PU-, GM— and Control-beads indicate proteins isolated on the particular beads. H9010 and IgG indicate protein isolated by the particular Ab. Control beads contain an HSP90 inert molecule. The data are consistent with those ed from multiple repeat experiments (11 2 2). (c) HSP90 binding ofPE conjugated antibody vs PU-H7 l-FITC. The percent of total cellular HSP90 isolated by PU-H7l is indicated for each cell line above the data bar. (d) Sequential chemical- and immuno—purification steps were performed in eral blood leukocyte (PBL) extracts (250 pg) n blot. to isolate PU-H7 l and H9010-specific HSP90 species. All samples were analyzed by (upper). Binding to HSP90 in PBL was evaluated by flow cytometry using an HSP90—PE antibody and PU-H71—FITC. FlTC-TEG = control for nonspecific binding (lower). (e) Correlation for binding of PU-H7l-FITC (1 M to HSP90 versus percent viability afier treatment with PU-l-I71 in a panel of 14 leukemia cell lines: Kasumi-l, Kasumi—4, KCL—22, REH, TF-l , KG-l , HL—60, OCI- AMLS, K562, MOLM-l 3, TUR, THP-l, U937 and MV4—l 1. Total HSP90 levels in these cells are similar, as demonstrated by Western blot (not shown).

Figure 4. (2) Flow cytometric dot plots demonstrate the gating strategy used for primary chronic myeloid leukemia (CML) samples to distinguish blast (CD45dim, red circles) and non- malignant cytes (blue circles). (b) Ratio for PU-H71-FITC binding to HSP90 in CML blasts to normal lymphocytes from the primary CML patient samples shown in (a). (c) Percent viability of CML blasts (red) or normal lymphocytes (blue) relative to untreated control for the primary CML samples shown in (I) after treatment at the indicated time points and doses ofPU-H7l. (d) Flow cytometric dot plots demonstrate the gating strategy used for primary chronic phase CML (cpCML) s to distinguish blast (CD45dim, red circle) and lignant lymphocytes (blue circle) and to analyze binding of CD34+ cells (red square) within the blast gate (CD45dim, red circle). CD45 vs.

SSC dot plots were ted on viable cells based on 7—AAD discrimination. (e) Ratio for PU-H71- FITC binding to HSP90 in chronic phase CML (cpCML) CD34+ cells and to normal lymphocytes. (1) Percent Viability of cpCML CD34+ cells (red) and normal lymphocytes (blue) relative to ted control afler treatment for 48h with luM PU—H71-FITC or TEG-FITC. (g) Ratio for PU—H7l —FlTC binding to HSP90 in CD34+ cells and lymphocytes from normal cord blood, chronic phase CML ) and blast phase (prML) cells (n=5). (h) Percent viability afler 48h treatment with PU—H71 (1 uM) of blast and chronic CML CD34+ cells, and normal CD34+ cells (from cord blood; CB) ve to untreated control. Cell Viability in panels c, f, and h was evaluated by annexin V/7-AAD staining. Data are presented as means t SE (n = 3).

Figure 5. (3) Within normal cells, tutive expression ofHSP90 is required for its evolutionarily conserved housekeeping fimction of folding and ocating cellular proteins to their cellular proper cellular compamnent (“housekeeping complex”). Upon malignant transformation, ns are perturbed through mutations, hyperactivity, ion in incorrect cellular compartments and maintain or other means. The presence of these functionally altered proteins is required to initiate the malignant phenotype, and it is these oncogenic proteins that are specifically maintained by a subset of stress d HSP90 (“oncogenic complex"). PU—H7l specifically binds to the fraction of HSP90 that chaperones oncogenic proteins (“oncogenic complex"). (I!) HSP90 and its interacting co- chaperones were isolated in K562 cell extracts using PU~ and Control-beads, and H9010 and IgG— immobilized Abs. Control beads contain an HSP90 inert le. (c) HSP90 from K562 cell ts was isolated through three serial immune-purification steps with the H9010 HSP90 specific antibody. The remaining supernatant was isolated to analyze the left-over proteins. Proteins in each on were analyzed by Western blot. Lysate a endogenous protein content. The data are consistent with those obtained from multiple repeat experiments (n 2 2). [0049} Figure 6. GM and PU’H71 are selective for aberrant protein/HSP90 species. (a) Bcr-Abl and Ab] bound HSP90 species were monitored in experiments where a constant volume of PU-H7l beads (80 pL) was probed with indicated amounts ofK562 cell lysate (left), or where a constant amount of lysate (1 mg) was probed with the indicated s ofPU—H7l beads (right). (b) (left) PU— and GM~beads (80 pL) recognize the HSP90-mutant B~Raf complex in the SKMelZS melanoma cell extract (300 pg), but fail to interact with the HSP90—WT B—Raf complex found in the normal colon fibroblast CCDl 8C0 extracts (300 pg). H9010 HSP90 Ab izes both HSP90 species. (c) In MDA-MB-468 cell extracts (300 pg), PU— and (EM—beads (80 p1) interact with HERB and Raf—l kinase but not with the non-oncogenic tyrosine-protein kinase CSK, a c-Src related tyrosine kinase, and p38. ((1) (right) PU-beads (80 pl.) interact with v-Src/l-ISP9O but not c-Src/HSP90 species. To facilitate c-Src detection, a n in lower abundance than v—Src, higher s of c—Src expressing 3T3 cell lysate (1,000 pg) were used when ed to the v-Src transformed 3T3 cell (250 pg), providing explanation for the higher HSP90 levels detected in the 3T3 cells (Lysate, 3T3 fibroblasts vs v~Src 3T3 fibroblasts). Lysate = endogenous protein t; PU—, GM- and Control-beads indicate proteins isolated on the particular beads. HSP90 Ab and IgG indicate protein isolated by the particular Ab. Control beads contain an HSP90 inert molecule. The data are tent with those obtained from multiple repeat experiments (n 2 2).

Figure 7. Single chemicaluprecipitations were conducted in Bcr—Abl—expressing CML cell lines (a) and in primary CML cell extracts (b) with PU- and l-beads. ns in the pull- downs were analyzed by Western blot. Several Bcr-Abl cleavage products are noted in the primary CML samples as reported”. N/A : not available.

Figure 8. Structures of several HSP90 inhibitors.

Figure 9. Fluorescent ligands for the heat shock protein 90 (HSP90) synthesized ning either fluorescein isothiocyanate (FITC), 4—nitrobenzo[1,2,5]oxadiazole (NBD) or the red shifted dye sulforhodamine 101 (Texas Red) conjugated to PU-H7l .

Figure 10. Reagents and conditions for the reaction scheme shown: (a) FITC, EtJN, DMF, rt, 12 h, 40%; (b) Texas Red sulfonyl chloride, DMF, 0—10°C, 12 h, 61%; (c) DMF, rt, 20 h, 47%.

Figure 11. Reagents and conditions for the reaction scheme shown: (a) FITC, Et3N, DMF, rt, h, 72%; (b) NED-C1, Et3N, DMF, rt, 12 h, 40%.

Figure 12. Reagents and ions for the reaction scheme shown: (a) N-(3—bromopropyl)- phthalimide, CszCO], DMF, rt, 34%; (b) hydrazine hydrate, MeOH, CHZCIz, rt, 64%; (c) FITC, EtJN, DMF, rt, 12 h, 74%; (d) NED-C1, Eth, DMF, rt, 12 h, 42%. [0056) Figure 13. (A) MOLM-l3 cells were treated with the indicated PU—H7l-fluorescent derivatives (1 M) at 37°C for 4b and g to live cells (DAPI negative) measured by flow cytometry. The extent of binding is shown as mean fluorescence intensity (Ml—‘1). (B) MOLM-l 3 cells were treated with the indicated PU-H7l-fluorescent derivatives (1 M) at 37°C for 24h. Their viability was determined by DAPI exclusion (C) MOLM-l3 cells were d with the indicated PU-H71-fluorescent derivatives (1 pM) for 2411. The steady-state level of the HSP90 client proteins n1FLT3 and Raf-l was analyzed by Western blot. B-Actin was used to normalize for equal protein loading.

Figure 14. (A) Confocal fluorescence microscopy of leukemia cells stained with PU’H71— FITC2 shows ent intracellular localimtion. (B) A primary acute myeloid ia sample was pre-treated with the indicated dose of PU—H7l or vehicle (Untreated) for 24 h. Post-treatment cells were treated with 1 uM PU-H71—FITC2 or TEG»FITC. Binding ofPUvH7l-FITC2 and TEG-FITC to the cells was ted by flow cytometry and is ented as mean cence intensity (MFI).

TEG-FITC is shown as a non-specific binding control. CD45 vs. SSC gating was used to guish binding to blast (malignant cells) or lymphocytes (normal cells) from the primary specimens. [0001} Figure 15. Fluorescence emission spectrum of PU—ANCA in a normal cell (left) and a breast cancer cell (right). The spectral emission profile of breast cancer cells resulted in a fluorescent emission peak at approximately 530nm wavelength, the representative fluorescence on of the bound PU-H7 l-ANCA.

Figure 16. (:1) Ratio for PU-H7l—FITC binding to HSP90 in cord blood and CML blasts to nomial lymphocytes from healthy donors (cord blood) and chronic and blast phase CML patients (cpCML and prML, respectively). Note no significant g to cord blood from healthy patients vs increased binding in CML that correlates with disease progression. (h) Percent viability after 48h treatment with PU-H7l (1 M) ofblast and chronic CML CD34+ cells, and normal CD34+ cells (from cord blood; CB) relative to untreated control. Cell viability was evaluated by annexin V/7— AAD staining. Data are presented as means iSE (n = 3). (c) Correlation for binding of PU-H7 l- FITC (1 uM) to HSP90 versus percent viability after treatment with SOOnM PU-H7l for 48h in a set of 19 primary AML patient samples. Each dot represents a primary AML . Each experiment was performed at least in ate. These cells express similar total HSP90 .

Figure 17. Xenotransplant assays suggest in vivo sensitivity to PU-H7l treatment ofAML samples with high-PU—FITC binding. (:1) Percent viability at 48h for in vitro PU-H7l treatment of two y AML samples that show low and high-PU—FITC uptake (11). Viability was determined by Annexin/7AAD assays. ([1) Bone marrow cells from xenotransplanted animals (for AML s shown in panel a) were stained with human specific antibodies to determine PU'FITC binding. PU- FITC binding is represented as a ratio of human mia)/murine (normal) cells. (it) Percent CD34+ tumor cells in animals d with 75mg/kg PU—H7l 3xweek for 3 weeks.

Figure 181 Use of labeled~PU~H7l to detect and quantify the “oncogenic HSP90” and predict sensitivity of tumor cells to HSP90 inhibitors. (A) Correlation for binding of PU-H71-FITC (1 M) to HSP90 versus percent viability after treatment with 1 uM PU—H7l, SNX-2112 or NVP—AUY922 for 48h in a panel of pancreatic and breast cancer cell lines. Binding is measured as a ratio of PU- FITC uptake in the respective cancer cell and the uptake in the reference cells, the HSP90 resistant leukemia cells HL60 (see panel D). (B) sion of total tumor HSP90 was measured by Western blot and plotted against PU~FITC binding. (C) Percent ity afler treatment with 1 uM PU—H7l, SNX«2112 or NVP-AUY922 for 48h in a panel of pancreatic and breast cancer cell lines was plotted against expression of total tumor HSP90. (D) HSP90 binding of PE conjugated antibody vs - FITC in a low-binding and sensitivity (HL»60) and high-binding and sensitivity (MV4—1 1) AML cell line. Data are presented as means iSE (n = 3).

] Figure 19. Ratio of PU»H7l—F1TC2 binding to tumor cells and to reference HL-60 ia cells. A responsive (>50% reduced viability) from sponsive (<50% reduced viability) cells could be differentiated by a ratio of binding to PU~H71vFITC2 from about 2.7 to about 5.87 or above for responsive cells compared to about 1.23 to about 2.07 or below for ponsive cells.

Figure 20. PU-H7l—F1TC2 accumulation in EpCAM+ Circulating Tumor Cells: PBMC‘s isolated from whole blood were pre—treated with PU-FITC or controls (PU—FITC9, DMSO, 1M 2 x106 cells/m1, 4hrs). Cells were then stained with CD45, CD14 and EpCAM antibodies. (A) Cells are gated to exclude dead cells. Viable cells are then gated to determine EpCAM+ vs CD45+ cells.

Monocytes are excluded from the analysis by gating the CD45+ cells further as FSC vs. CD14. (B) Histogram plots showing the C Median floreseence intensity (MFI) ofCD45+CD14~ cells (blue) and EpCAM+ cells (Red). The dmg accumulation in EpCAM+ cells is calculated as the ratio of MFI EpCAM+/MP] CD45+CD14- lating tumor cellslleukocytes) afler subtracting the values of the DMSO and C9 controls (used to control for non-specific and ound binding).

Figure 21. Shows the uptake of PU-H71-FITC2 by different Lyl clones and is expressed as median fluorescence intensity. The sensitivity of these cells to HSP90 inhibitors correlates with their uptake of labeled-PU-H7l. Expression of total tumor HSP90 in these tumor cells was measured by Western blot (inset).

Figure 22 (a-c). Correlation in PU-H71 Binding and Toxicity in Pancreatic Cancer Cells.

(A) Binding in Live Cells; Pancreatic Cancer cells (1x106 cells) were treated for 6hrs with PUT'ITCZ (luM) or controls [TEG-FITC (luM) or DMSO] .

Cells were washed twice with FACS buffer (PBS, 0.05% FBS), and stained with lug/ml of DAPI (Invitrogen) in FACS buffer at room temperature, prior to analysis. The fluorescence ities from live cells (DAPI negative) representing PU-H71- fluorescent derivative g were captured by flow cytometry (LSR—II, BD Biosciences), and analyzed by FlowJo software (Tree Star, d, OR). Value represent mean florescent intensities subtracted from the DMSO and TC controls. (B) Toxicity; Pancreatic Cancer cells (1x105 cells) were treated for 43m with PU~FITC2 (lpM). Cells were washed twice with FACS buffer (PBS, 0.05% PBS), and stained with lug/m] ofDAPI (Invitrogen) in FACS buffer at room temperature and captured by flow cytometry (LSR—II, BD ences). Values represent the %live cells (DAPI negative) normalized to the values from the DMSO control. (C) Cor-relative analysis; MFI and toxicity obtained from A and B were plotted on the x and y axis tively and a correlative linear regression analysis performed.

Figure 23. (A) Binding ofPUH7l—FITC2 to leukemia stem cells (LSCs, CD34+CD38- m). Primary AML s were incubated with 1 pM PU-H7l-FITC2 at 37°C for 4 h. Cells were stained with CD34, CD38, CD45 and 7-AAD followed by flow cytometry analysis. Binding PU—H7l to LSCs is shown as the mean fluorescence intensity (MFD in live cells (7—AAD negative).

(B) Percent viability ofLSCs ve to the untreated control from three primary AML samples alter 48 hour treatment with laM PUl-I7l. Cells were stained with CD45, CD34 and CD38 prior to Annexin V and 7—AAD staining. Viability LSCs was ed by flow cytometry and determined as the percentage of AnnexinV«/7AAD- of the CD45dim CD34+CD38- gate.

Figure 24. Tumors have distinct [ml]»PU-H7l uptake indicating differences in their “oncogenic HSP90” t and thus in their potential to respond to HSP90 therapy. [ml]-PU—H7l PET images at 24h ‘2‘I]—PU-H7l injection were measured as Maximal Standardized Uptake Values (SUVM) in several patients with breast cancer. BC = breast cancer. TNBCvtriple~negative {0067] Figure 25. Tumor:muscle SUV ratio for a select number of patients that are responsive to HSP90 inhibition therapy as determined by PET following administration of ['Z‘H-PU-H7l. In these patients, the tumonmuscle SUV increases over time. Values averaged for several positive and negative tumors are presented Figure 26. FDG/CT and [‘"Il-PU—H7l PET/CT of patient with mantle cell lymphoma. The patient shows clear visualization of the lesion a: 30min afier ['“I]-PU-H71-injection. No [mm-PU— H71 uptake was seen in this tumor at later times (3.5—24h and beyond).

Figure 27. —H7l PET/CT of patient with ent breast cancer in the two indicated lymph nodes (LN). PET images at several times post—['2‘11-PU-H71 ion (0.1, 0.4, 0.6, 3.5 and 21 .4h) were quantified and SUVrnax data obtained for [mm—PU-H71 were converted to HSP90i concentrations for an administered dose of PU-H7l of lOmg/mz. The exposure of the two tumors to PU-H7l over the time of 0 to 24h was also calculated and represented as the area~under—the—curve (AUC). CT (lefi), PU-PET/CT (middle), and FDG-PET/CT fusion (right) transaxial images trate [ml]—PU-H7l-avidity in one of the diseased lymph nodes but not the other suggesting that the lesion in the left tracheobronchial anterior angle lymph node (TAALN) is less likely than the led tracheobronchial angle LN (TALN) to respond to HSP90 therapy.

Figure 28. A triple-negative breast cancer patient was imaged with ‘z‘I-PU-H7l PET. At 20min post-injection, uptake is noted in a lung mass (left arrow), and a bone lesion (right arrow) (A), but at 2411 uptake is seen only in the lung lesion (B). Both the lung and bone tumors are confirmed by CT and FDG-scans (C). The patient started treatment with the HSP90 inhibitor STA—9090. Twenty days post HSP90 inhibitor treatment, the lung but not the bone lesion is remarkably reduced in size, as evidenced by both CT and FDGePET (D).

Figure 29. [”‘n—PU-Hn PET/CT ofa patient with metastatic HERZ breast cancer in the paratrachenl node. PET images at the indicated times post-[‘z‘fl-PU-H'Il or post ection of [ml]- PU-H7l with lOmg/m2 PUeH7l were measured as l Standardized Uptake Values (SUVM).

SUV data obtained for [mu-Putin were converted to HSP90i concentrations for an administered dose of PUeH7l of lOmg/mz. The exposure of the tumor to PU-H7l over the time of0 to 48h was also calculated and represented as the area-under-the-curve (AUC). Tumor trations of PU-H71 ( in micromolar values) as estimated from ['24I]-PU-H7l PET or as determined from [IZ4I]—PU—H7l PET afler ection of [ml]-PU-H7l/PU-H 71 are comparable. (0072] Figure 30. [m1]-PU-H71 PET is a vasive assay for HSP90 inhibitors. (in) The chemical structure ofPU—H7l and ['24I]—PU-H7l. (b) Representative PET scan of ['“I]-PU-H7l in lvfl)A—MB-468 tumor-bearing mice. location of the tumor is indicated by a red arrow. (c) The [ml]— PU-H7l tumor—to-organ activity concentration ratios for the indicated times post-administration. (d) Biodistribution of ['3'I]ePU-H7l in MDA—MZB-468 tumors and plasma (n=5). Tumor - S and Tumor - L, small and large tumors, respectively. (inset) The 24 to 14011 slow terminal clearance phase of PU—H7l from tumors was analyzed using a linear regression curve fit as implemented in GraphPad Prism.

Figure 31. U—H7l PET accurately ts the ry of therapeutically effective PU«H7 1 concentrations in tumor. (n) Predicted PU-H7l tumor distribution based on the mean %ID/g generated by ['Z‘H-PU-H7l PET (lower). The predicted tumor concentration at ted times p.a. of indicated PU-H71 doses (upper). (h) Tumor PU-H7l concentrations (n=5) following administration of 75 mg/kg agent as predicted by [mI]-PU-H7l PET, and determined by LC-MS/MS and by ['“11-PU—H71 PET following co—injection of ['"I]-PU-H71 and PU—H7l. (c,d) Representative Western blot analyses ofMDA—MB—468 tumors administered PU-H7l at the indicated doses and analyzed at 24 h p.a. (c) and ofMDA-MB-468 cells d for 24h with the indicated concentrations of PU-H7l (d). Blots (n=3) were quantified by densitometry and the change in protein levels d versus the concentration of PU-H7l (right panels). (e,f) Target ncy at 24h p.a. as predicted by [ml]-PUeH71 PET following co-administration of tracer amounts of [ml]—PU~H71 mixed with the indicated doses of PU—H7l. [0074} Figure 32. [ml]-PU—H7l PET ts the design of an efficacious dose regimen for HSP90 therapy. (:1) Predicted Ple-l7 1 tumor distribution when administered for 2 weeks at the indicated doses on a 3xweelc (Mon-Wed-Fri with Sat/Sun oft) le based on the mean tumor ty concentration ) derived by [”‘11-PU-H71 PET. (inset) The AUCs for PU—H71 in tumors were calculated using GraphPad Prism. ([1) The viability ofMDA-MB-468 cells treated for 48h with the indicated doses of PU-H7 l was analyzed by Ethydium BromideJAcridine Orange staining (upper).

Estimated tumor apoptosis induced by the [ml]-PU-H7l PET ted indicated average and minimtun PU-H7l tumor concentrations. (c) MDAeMB-468 tumor—bearing mice (n = 5) were administered i.p. the indicated doses of 7 l on a 3xweek schedule. Tumor volume and mouse weight were monitored over the indicated treatment . (d) MDA—MB-468 tumor-bearing mice (n r- 5) were administered i.p. the ted doses of PU-H7l on a 3xweek schedule. Tumor volume and mouse weight were monitored over the indicated treatment period. (c) Predicted PU-H71 tumor distribution based on the mean tumor ty concentration ('anD/g) derived by [mI]—PU-H7 1 PET when administered at the indicated dose on a 3xweek (Mon-Wed-Fri with Sat]Sun oft) schedule. (f) Western blot analysis of the Vehicle (Control) and PU—H7 l (5 mg/kg)etreated tumors sacrificed on Thu, at 24 h p.a. of the last dose. PU-H7l tumor concentrations, as determined by LC—MS/MS, are indicated for each tumor. (1') is of data (rt-‘5) from panel (d).

Figure 33. [ml]—PUoH71 PET predicts the design of an efficacious le regimen for HSP90 therapy. (3) Predicted PU-H7l tumor distribution based on the mean tumor activity concentration (%lD/g) derived by PU-H71 PET when administered at 75 mg/ltg on the indicated schedules. ([1) ted tumor sis induced by the indicated average and minimum PU-H7l tumor concentrations as ted from in vitra analyses. (c) MDA-MB-468 tumor—bearing mice (n = ) were administered i.p. PU-H7l (75 mg/kg) on the indicated schedules. Tumor volume and mouse weight were monitored over the indicated treatment period. ((1) Western blot and (e) LC-MS/MS analysis of the PU—l—l71 (75 mg/kg)-treated tumors on the lxweek schedule sacrificed on Thu, at 24h and Thu, at 96h p.a. of the last dose. Control; vehicle only treated mice.

Figure 34. Tumor exposure to PU—H7l as predicted by PU-PET for a pancreatic patient with metastatic disease to lung and indicated lymph nodes. Tumor concentrations are calculated based on an administered dose of 20mg/m2. Plasma exposure is also shown in red. Calculated AUCs (uM-h) for the period 0 to l92h are tabulated on the right.

Figure 35. [mI]-PU-H71 PET/CT ofa patient with pancreatic cancer with recurrent disease in the lung. PET images at the indicated times 'ZAIJ-PU-H71 injection (48 and l96h, left panels) were quantified and SVU data obtained for [ml]-PU‘H71 were converted to PU-H71 concentrations in the tumor for the ted administered doses of . The exposure of two tumors, one in the lefi lung and another in the right hilum LN, to PU-H7l over the time of 0 to 33611 for a two-week ent on a twice-week (Tue and Fri) schedule and an administered dose of 20, 60 and 80 mg/m2 (upper right and bottom panels) was also calculated and represented as the area-under~the»curve (AUC) and as an average tumor concentration {0073] Figure 36. Panel A shows the biodistrihution of '"I-PU~H71 over 0 to 72h in the tumor ofa breast cancer patient as obtained from . The data was used to te the tumor exposure to weekend off, three an administered dose of 10mg/m2 when given twice a week for two weeks with times a week for two weeks with weekend off, once a week for two weeks with weekend off and five times a week for two weeks with weekend off. {0079] Figure 37. [”‘fl—PU-l-Ul PET predicts the magnitude of response to HSP90 therapy. (a) Predicted average occupancy of tumor HSP90 sites by PU-H7I when administered at the indicated doses and on the ted schedules. (b,c) Correlation of tumor HSP90 sites occupancy with the observed anti-tumor effect was ed in GraphPad Prism.

Figure 33. The use of ”‘I—PU—H7l PET assay in the clinical development ofHSP90 tors: (a) in ining the dose of HSP90 inhibitor needed to achieve effective tumor concentrations, selection of patient eligible for HSP90 therapy and in designing an efficacious dose and schedule regimen; (b) in assaying the actual concentration of the drug delivered to the tumor and predicting al outcome on HSP90 y and (c) in determining the “maximum tumor dose".

CR 3 complete response, PR = partial response, NR = no response. [00311 Figure 39. In vivo PET imaging of [‘“I]-PU-D213 and ['2‘1]—PU—H7l in MDA—MB-468 xenografi TNBC mice. PET imaging was conducted on either an R4 or a Focus 120 dedicated small animal PET scanner (Concord Microsystems, Inc., Knoxville, TN); separate anatomical imaging was conducted on a dedicated small animal CT scanner (ImTek, Inc., Oak Ridge, TN), using a custom built stereotactic restraint device. Maximum intensity projection (MlP) of CT and PET image datasets were ically registered, and overlay images were generated using an alpha transparency blend of PET and CT data.

Figure 40. Cases of BC show a dose-dependent response to PU<H71. H&E stained slides display significant areas of apoptosis containing both pyknotic cells (indicative of early stage sis) and katyorrhexic cells (representative of late phase apoptosis) when the tumor is highly sensitive to PU-H71. (A) Apoptosis/cell death in TNBC specimens treated for 48h with the indicated concentrations of PU—H7l was quantified and d against the concentration of PU-H7 1. Both apoptotic and necrotic/late apoptotic cells Were d and added to the %apoptosis as depicted on the y-axis. Note a clustering of cases in three sensitivity groups (steepest, top curves, most sensitive, LN15 and LNlG; middle curves, sensitive, PTlZ, PTl7, PT25, PTZS and LNIO and lower curves, less sensitive, PTlO, PTlS, PT16 and PT30). Interestingly, the lymph node metastases showed a higher sensitivity than the primary tumor at the equivalent dose. mary tumor, LN=lymph node.

Tumors most sensitive to PU'H71 also stain high for p-Akt. (B) Same as for (A) with specimens from HER2+, TNEC and ER+ BC patients treated for 24 or 48h with PU~H71.

Figure 41. Apoptotic sensitivity to HSP90 inhibition correlates with addiction of cells for al on the AKT- and STAT« but not MEK-pathways. (A),(B) Representative AML cells were incubated for the indicated times with the indicated concentrations of the HSP90, AKT, JAK and MEK inhibitors and apoptosis was assessed using the Acridine OrangeJEthydium Bromide method.

The data are consistent with those obtained from multiple repeat experiments (n 2 3). Points, mean; bars, s.d (C) % Apoptosis values from cell treated for 72h with the AKTi, MEKi and JAKi alone were plotted against those obtained upon HSP90 inhibitor treatment and a linear regression analysis, as implemented in Prism 4.0 was performed. {0084] Figure 42. AML primary cells with higiest levels of p-STAT5 are also most sensitive to PU- H71. (A) Phospho—STATS levels in blast cells (CD45dim gated) represented as mean fluorescence intensity (MFI) for three different primary AML samples. Phosphorylation level of Stat5 was assessed by flow try. (B) Percent ity ofAML blast relative to the unheated l from three y AML s afier 48 hour treatment with luM PUH71. Cells were stained with CD45 prior to Annexin V and 7-AAD staining. Viability ofAML blast cells was measured by flow cytometry and determined as the percentage of AnnexinV—/7AAD- of the CD45dim vs SSC gate for AML blast.

Figure 43. 8-10 mo 35ng mice were administered (A) 75mg/kg or (B) the indicated dose of the HSP90 inhibitor PU—HZl5l and the PD marker, HSP70, was measured in hippocarnpus, an afflicted brain region in this model ofAD, at 24h post-administration. HSP70 is induced when HSP90 is inhibited and its ion is an indicator that therapeutic levels of the HSP90 inhibitor were red to the brain region of interest. (0 Levels ofthe HSP90 inhibitor in the indicated brain regions and plasma were determined by LC—MS/MS after the administration of 50mg/kg PU-HZlSl.

Hsp90 inhibitor levels at different times after single ip injection are shown in micromolar units. Brain exposure was also measured as the area under the curve (AUC).

Figure 44. PU—H7l is selective for HSP90. (A) Coomassie stained gel of l HSP90 inhibitor bead—pulldowns. K562 lysates (60 pg) were ted with 25 pl. of the ted beads.

Following washing with the indicated buffer, proteins in the pull—downs were applied to an SDS- PAGE gel. (B) PU~H71 (10 M) was tested in the X screen (Ambit) against 359 kinases.

The TREEsgmt'rM Interaction Map for PU-H71 is presented. Only SNARK (NUAK family SNFl—like kinase 2) (red dot on the kinase tree) appears as a potential low affinity kinase hit of the small molecule.

. DETAILED DESCRIPTlg!N .1. Oncogenlc HSP90 as a tumor specific blornarker The disclosure provides evidence that the abundance of this particular “oncogenic HSP90" species, which is not dictated by HSP90 expression alone, predicts for sensitivity to HSP90 inhibition therapy, and thus is a ker for HSP90 therapy. The invention also provides evidence that identifying and measuring the abundance of this oncogenic HSP90 species in tumors predicts of response to HSP90 therapy.

In the following sections, we show that the HSP90 inhibitor PU-H7l s tumor-enriched HSP90 complexes and affinity-captures HSP90—dependent oncogenic client ns. The compound PU-H7l was disclosed in US. Patent No. 7,834,181, which is hereby incorporated by reference. PU- H71 has the following chemical structure: NH; . °7 titres ° [01001 A PU-H71 PU.H71 can be administered as a free base or as a pharmaceutically able salt.

In addition, we show that the abundance of the PU-H7l-enriched HSP90 species, which is not dictated by HSP90 expression alone, is predictive of the cell’s sensitivity to HSP90 inhibition by PU— H71 and other HSP90 inhibitors. .1.1. Heterogeneous HSP90 tation in cancer cells To investigate the interaction of small molecule HSP90 inhibitors with tumor HSP90 complexes, we made use of agarose beads covalently ed to either amycin (GM) or PU- H7l (GM- and PU-beads, respectively) (Figures 1, 2). Both GM and , chemically distinct agents, interact with and inhibit HSP90 by binding to its N-terminal domain regulatory ”. For ison, we also generated G protein agarose-beads coupled to an anti-HSP90 antibody (H9010).

First we evaluated the binding of these agents to HSP90 in breast cancer and in chronic myeloid leukemia (CML) cell lysates. Four consecutive immunoprecipitation (IP) steps with H9010, but not with a non—specific IgG, efficiently depleted l-ISP90 from these extracts (Figure la, 4xH9010 and not shown). in contrast, sequential pull-downs with PU— or ds removed only a fraction of the total cellular HSP90 (Flgures lb, 3:, 3b). Specifically, in MDAeMB-468 breast cancer cells, the combined PUehead fractions represented imately 20-30% of the total cellular HSP90 pool, and fiirther on of fresh d aliquots failed to precipitate the remaining HSP90 in the lysate (Figure lb, PU-beads). This PU-depleted, remaining HSP90 fraction, while inaccessible to the small molecule, maintained affinity for H9010 (Figure lb, . From this we conclude that a significant fraction of HSP90 in the MDA—MB-468 cell extracts was still in a native conformation but not reactive with . [0092) To exclude the possibility that changes in HSP90 configuration in cell lysates make it unavailable for binding to immobilized PU—H7l but not to the antibody, we analyzed g of radiolabeled ”II-PUel-l7l to HSP90 in intact cancer cells (Figure 1:, lower). The chemical structures of ”‘I—PU-H7l and PUvH71 are identical: PU—H7l contains a stable iodine atom (”71) and ”'I-PU- H71 contains radioactive iodine; thus, isotopically labeled '“I-PU-H7l has identical al and biological properties to the unlabeled PU—H7l. Binding of '“I-PU-ml to HSP90 in several cancer cell lines became saturated at a well-defined, gh distinct, number of sites per cell (Figure 1e, lower). We quantified the fraction of ar HSP90 that was bound by PU-H7l in -468 cells. First, we determined that HSP90 represented 2.66-3.33% of the total cellular protein in these cells, a value in close agreement with the reported abundance of HSP90 in other tumor cells”.

Approximately 41.65x106 MDA-MB-468 cells were lysed to yield 3875 pg of protein, ofwhich 10107-12904 pg was HSP90. One cell, therefore, contained (2.47-3.09)xlo*“ ug, (2.74-3.43)x10‘” umols or (l.t54—2.06)xlo7 molecules of HSP90. In MDA-MB-468 cells, ”'I—PU-H7l bound at most to .5)(106 of the available cellular binding sites (Figure to, , which amounts to 26.6-33.5% of the total cellular HSP90 (calculated as 5.5x106/(l.64-2.06)x107*100). This value is remarkably similar to the one obtained with PU~bead pull-downs in cell extracts (Figure lb), ming that PUeH71 binds to a fraction ofHSP90 in -468 cells that represents approximately 30% of the total HSP90 pool and validating the use of PU~heads to efficiently e this pool. 1n K562 and other established t(9;22)+ CML cell lines, PU—H7l bound 10.3—23% of the total cellular HSP90 es 1:, 3b, 3c).

Next, we extended our studies to several primary leukemia cells and to normal blood cells.

Among these were primary chronic and blast phase CML and acute myeloid leukemia (AML) samples that contained both blasts (malignant cell population) and lymphocytes (normal cell population), CD34+ cells isolated from the cord blood of healthy donors, total mononuclear cells from peripheral blood and also peripheral blood leukocytes (PBLS) (Figures le—e, 3, 4). We used a fluorescein labeled PU-H7l (PU—FITC). This chemical tool allows for the flow tric analysis, in heterogeneous cell populations, of PU-H7l binding to distinct cell populations using cell surface WO 09657 markers, as well as the investigation of cells’ sensitivity to PU-H71. A tetraethylene glycol derivatized FlTC (FITC-TEG) was used to l for non-specific binding (Figure 1d).

PUmH71 efficiently bound to HSP90 in K562 cells and in CML and AM]. blasts with a half relative binding affinity (ECm) of 116, 201 and 425 nM, respectively (Figure Id). In contrast, its affinity for normal blood cells was weaker, with EC“), higher than 2,000nM (Figures 1d, 3d). HSP90 remains highly expressed in these normal blood cells as indicated by substantial binding to the HSP90 antibody e 3d).

Cells with t avidity for PU—H7 1 were also most sensitive to killing by the agent (Figures 1e, 3e, 4). When evaluated in a panel ofCML and AML cell lines and primary samples, a significant correlation between the ability of PU—H7l to bind HSP90 and the cell killing ial of PUeH‘ll t these cells was noted (Figures 3e, 4).

Collectively, these data show that certain HSP90 inhibitors, such as PU—H71, preferentially bind to a subset of HSP90 species that is more abundant in cancer cells than in normal cells (Figure !). The abundance of this HSP90 species, which is not dictated by HSP90 expression level alone, is predictive of the cell’s ivity to HSP90 inhibition, thus the abundance of this tumor HSP90 species can be used as a biomarker predictive of response to HSP90 therapy. .1.2. Onco- and WT-protein bound HSP90 species eo-exist in cancer cells, but PU-H7l selects for the oneo-protein/HSP90 species To explore the biochemical functions associated with these HSP90 species, we performed immunoprecipitations (11’s) and chemical precipitations (CPs) with antibody— and HSP90-inhibitor beads, respectively, and we analysed the ability of HSP90 bound in these contexts to co-precipitate with a chosen subset of known clients. K562 CML cells were first investigated because this cell line co-expresses the aberrant Bcr-Abl protein. a constitutively active kinase, and its normal counterpart c- Abl. These two Abl species are clearly separable by lar weight and thus easily distinguishable by Western blot (Figure 2a, Lysate), facilitating the is of HSP90 onco- and wild type (WT)~ clients in the same cellular context. We observed that H9010, but not a non-specific IgG, isolated HSP90 in complex with both Bcr-Abl and Abl (Figures 2a, Sc, H9010). Comparison of immunoprecipitated Bcr—Abl and Abl (Figures 2a, 2b, 121?, H9010) with the fraction of each protein remaining in the atant (Figure 2b, leg/l, Remaining supematant), indicated that the antibody did not preferentially enrich for HSP90 bound to either mutant or WT forms ofAb] in K562 cells. {0098] In contrast, PU~bound HSP90 preferentially isolated the Bcr~Abl protein (Figures 2a, 2b, right, ds). Following PU—bead depletion of the HSP90/Ber—Abl species (Figure 211, right, PU- beads), H9010 precipitated the remaining Abl species (Figure 2b, right, H9010). PU-beads retained ivity for Ecr-Abl species at substantially ting conditions (i.e. excess of lysate, Figure 63, left, and beads, Figure 6:, right). As r confirmation of the biochemical selectivity of PU—H7l for the Bcr—AbVHSP90 species, Ber-Abl was much more susceptible to degradation by PU-H71 than was Abl (Figure 2d). The selectivity of PU—H71 for the aberrant Abl species extended to other established t(9;22)+ CML cell lines (Figure 78), as well as to y s (Figure 71)). .1.3. The onco- but not WT-protein bound HSP90 species are most ent on co-chnperone recruitment for client protein regulation by HSP90 To further differentiate between the PU-H7l- and antibody-associated HSP90 fractious, we performed tial depletion experiments and evaluated the co—chaperone tuency of the two s”. The fraction ofHSP90 containing the HSP90/Ber—Abl complexes bound several co— chapcrones, including Hsp70, Hsp40, HOP and HIP (Figure 2c, PU-beads). PU-bead pull-downs These findings strongly were also enriched for several additional HSP90 co—chaperone species. suggest that PU—H71 izes co—chaperone-bound HSP90. The PU—beads—depleted, remaining HSP90 pool, shown to include HSP90/Abl species, was not associated with w-chaperones (Figure 2c, H9010), although their nt expression was detected in the lysate (Figure 2c, Remaining supernatant) Co—chaperones are however isolated by H9010 in the total cellular extract (Figures 5b, 5c).

These s suggest the existence of distinct pools of HSP90 preferentially bound to either Bcr—Abl or Abl in CML cells (Figure 2). H9010 binds to both the l and the Abl containing HSP90 species, whereas PU—H71 is selective for the Bcr—Abl/HSP90 species. Our data also t that HSP90 may utilize and require more acutely the classical co—chaperones Hsp70, Hsp40 and HOP when it modulates the activity of aberrant (i.e. Bcr-Abl) but not normal (i.e. Abl) proteins (Figure 5a). In accord with this hypothesis, we find that Ber-Abl is more ive than Abl to down of Hsp70, an HSP90 co-chaperone, in K562 cells (Figure 2e). .1.4. The onco—proteianSP90 species selectivity and the complex trapping ability of PU—H7l are not shared by all HSP90 inhibitors We next evaluated whether other inhibitors that interact with the N—terminal regulatory pocket of HSP90 in a manner similar to PU-H7l, including the tic inhibitors SNX-21 12 and WP- AUY922, and the natural product GM”, could selectively isolate similar HSP90 species (Figure 20‘ SNX-beads trated selectivity for Bcr»Abl/HSP90, whereas NVP-beads d similarly to H9010 and did not discriminate between Bcr—Abl/HSP90 and Abl/HSP90 species (see SNX- versus NVP-beads, respectively; Figure 21'). While GM—beads also recognized a subpopulation ofHSP90 in cell lysates (Figure 321), they were much less efficient than were PU-beads in cipitating Bcr- Abl (Figure 2f, GM—beads). Similar ineffectiveness for GM in trapping HSP90/client protein complexes was previously reported”. .1.5. The rotein/HSP90 species selectivity and the complex trapping ability of PU~H71 is not cted to Bcr-Ahl/HSP90 species To determine whether selectivity towards coco-proteins was not restricted to Bcr—Abl, we tested several additional well-defined HSP90 client proteins in other tumor cell lines (Figures 6h- d)“. In ent with our results in K562 cells, H9010 precipitated HSP90 complexed with both mutant B’Raf expressed in SKMe128 melanoma cells and WT B—Raf expressed in CCD18Co normal colon fibroblasts (Figure 6!), H9010). PU- and GM—bmds r, selectively recognized HSP90/mutant B~Raf, showing little recognition of HSP90/WT B-Raf (Figure 6b, PU-beads and ds). However, as was the casein K562 cells, GM—beads were significantly less efficient than PU—beads in «so-precipitating the mutant client protein. Similar s were obtained for other HSP90 clients (Figures 6c, 6d). ]0l03] In summary, PU-H7l enriches a broad cross-section of proteins that participate in signaling ys vital to the maligth ype in CML. The interaction of PU—bound HSP90 with the aberrant CML signalosome was retained in primary CML samples. .1.6. PU-H7l identified proteins and networks are those important for the malignant phenotype We hypothesize that the presence of these proteins in the PU—bead pull-downs is functionally significant and suggests a role for HSP90 in broadly supporting the malignant signalosome in CML cells.

To demonstrate that the networks identified by PU-beads are important for transformation in K562, we next showed that inhibitors of key nodal proteins fi'om dual ks Bcr—Abl, NFKB, mTOR, MEK and CAMIIK) sh the growth and proliferation potential of K562 cells.

Next we demonstrated that PU—beads identified HSP90 interactors with yet no assigned role in CML, also contribute to the transformed phenotype. The histone—arginine methyltransferase CARMI, a transcriptional c0eactivator of many genes”, was validated in the PU-bead pull-downs from CML cell lines and primary CML cells. This is the first reported link between HSP90 and CARM], although other arginine methyltransferases, such as , have been shown to be HSP90 clients in ovarian cancer cells”. While elevated CARM] levels are implicated in the development of prostate and breast cancers, little is known on the importance of CARMl in CML leukomogenesis”.

We found CARM] essentially entirely ed by the HSP90 species ized by PU’beads and also sensitive to degradation by PU-H7 1. CARM] therefore, may be a novel HSP90 onco'protein in CML. Indeed, down experiments with CARM] but not control shRNAs, demonstrate reduced viability and induction of apoptosis in K562 but not in normal CD34+ cells (not shown), supporting this hypothesis. qPCR data ed that the CARMl mRNA levels were markedly reduced by the two different shRNAs (data not shown). {0107] To demonstrate that the presence of proteins in the PU-pulldowns is due to their participation in aberrantly ted signaling and not merely their abundant expression, we compared d pulldowns from K562 and Mia-PaCa-2, a pancreatic cancer cell line. While both cells express higt levels of STATS n, activation of the STATS pathway, as demonstrated by STATS phosphorylation and DNA-binding”, was noted only in the K562 cells. In accordance, this protein was identified only in the K562 PU‘bead pulldowns. In st, activated STAT3 was identified in PU—HSP90 xes from both K562 and Mia-PaCa~2 cells extracts.

The mTOR y was identified by the PU’beads in both K562 and Mia—PaCa—2 cells, and indeed, its pharmacologic tion by PP242, a selective inhibitor that targets the ATP domain of mTORG", is toxic to both cells. On the other hand, the Abl inhibitor Gleevec“ was toxic only to K562 cells. Both cells express Abl but only K562 has the oncogenic Bcr—Abl and PU~beads identify Abl, as Ber—Abl, in K562 but not in Mia-PaCa-2 cells. .1.7. PU-H7l flel a novel mechanism of oncogenic STAT-actlvntion {0109] PU«bead pull—downs contain several proteins, including Bcr—Abl‘“, CAMKIIy”, FAK”, vav— l62 and PRKD2“ that are constitutively activated in CML leukemogenesis. These are cal HSP90-regulated clients that depend on HSP9O for their stability because their steady-state levels decrease upon HSP90 inhibitionn’“. Constitutive tion of STAT3 and STAT5 is also reported in CML“‘“. These proteins, however, do not fit the criteria of classical HSP90 client proteins because STATS and STAT3 levels remain essentially modified upon HSP90 inhibition. The PU—pull—downs also contain proteins isolated potentially as part of an active signaling mega-complex, such as rn'DOR, VSP32, VSPlS and RAPTOR“. mTOR activity, as measured by cellular levels OR, also appears to be more sensitive to HSP90 inhibition than are the complex ents (i.e. compare the relative decrease in p-mTOR and RAPTOR in PU—H7l treated cells. Further, PU-HSP90 complexes contain adapter ns such as GRBZ, DOCK, CRKL and EPSIS, which link Bcr-Abl to key effectors of multiple ntly activated signaling pathways in K56230'“. Their expression also remains ged upon HSP90 inhibition. We therefore wondered whether the contribution of HSP90 to certain oncogenic pathways extends beyond its classical folding actions. Specifically, we show that HSP90 also acts as a scaffolding molecule that maintains signaling complexes in their active configuration, as has been previously postulated’w‘“. .1.8. HSP90 binds to and influences the conformation of STAT5.

To investigate this hypothesis further we d on STATS, which is constitutively phosphorylated in CML“. The overall level of p—STATS is determined by the balance of phosphorylation and dephosphorylation events. Thus, the high levels of p- STATS in K562 cells may reflect either an increase in upstream kinase activity or a decrease in protein tyrosine phosphatase ase) activity. A direct interaction between HSP90 and p—STATS could also modulate the cellular levels of p—STATS.

To dissect the relative contribution of these potential mechanisms, we first investigated the effect of PUflH7l on the main kinases and PTPases that regulate STATS phosphorylation in K562 cells. Bcr’Abl directly tes STATS without the need for JAK phosphorylation“. Concordantly, STATS-phosphorylation y decreased in the presence of the Bcr-Abl inhibitor c. While HSP90 regulates Bcr-Abl stability, the reduction in steady~state Ber-Abl levels following HSP90 inhibition requires more than 3 h“. Indeed no change in Bcr—Abl expression or function, as evidenced by no decrease in CRKL orylation, was ed with PU-H7 l in the time interval it reduced S levels. Also, no change in the activity and expression of HCK, a kinase activator of STATS in 32Dcl3 cells transfected with BcraAbl“, was noted.

Thus reduction of p-STATS phoSphorylation by PU-H7l in the 0 to 90 min interval is unlikely to be explained by destabilization of Bcr—Abl or other kinasei. [0113! We ore examined whether the rapid decrease in p—STATS levels in the presence of PU- H7l may be accounted for by an increase in PTPase activity. The expression and activity of SHIPZ, the major cytosolic STATS phosphatase”, were also not altered within this time al. Similarly, the levels of SOCS1 and SOCS3, which form a negative feedback loop that switches off STAT~ signaling“ were unaffected by PU-H71. [01 14] Thus no effect on STATS in the interval 0-90rnin can likely be attributed to a change in kinase or phosphatase activity towards STATS upon HSP90 inhibition. As an alternative mechanism, and because the majority of S but not STATS is HSP90 bound in CML cells, we hypothesized that the ar levels of activated STATS are fine-tuned by direct binding to HSP90.

The activation/inactivation cycle of STATS entails their transition between different dimer conformations. Phosphorylation of STATs occurs in an anti-parallel dimer conformation that upon phosphorylation triggers a parallel dimer conformation. Dephosphorylation of STATS on the other hand require extensive spatial reorientation, in that the ne phosphorylated STAT dimers must shifi from parallel to anti-parallel configuration to expose the phospho—tyrosine as a better target for phosphatases“. We find that STATS is more susceptible to trypsin cleavage when bound to HSP90, indicating that binding ofHSP90 directly modulates the conformational state of STATS, potentially to keep STATS in a conformation unfavorable for dephosphorylation and/or favorable for phosphorylation.

To investigate this possibility we used a pulse-chase strategy in which orthovanadate (Na3V04), a non-specific PTPase inhibitor, was added to cells to block the dephosphorylation of STATS. The residual level of p-STATS was then determined at several later time points. In the absence 71, p—STATS accumulated rapidly, s in its presence, cellular p-STATS levels were diminished. The kinetics of this process were similar to the rate of p—STATS steady~state reduction. .1.9. HSP90 maintains STATS in an active conformation directly within containing transcriptional xes. [01171 In addition to STATS phosphorylation and dimerization, the biological activity of STATS requires its nuclear translocation and direct binding to its various target genesém. We wondered therefore, whether HSP90 might also facilitate the transcriptional activation of STATS genes, and thus participate in er-associated STATS transcription complexes. Using an ELISA-based assay, we found that STATS is constitutively active in K562 cells and binds to a STATS binding consensus sequence (S’-TTCCCGGAA-3’). STATS activation and DNA binding is partially abrogated, in a dose-dependent manner, upon HSP90 inhibition with PU—H7l. Furthermore, quantitative ChIP assays in K562 cells revealed the presence of both HSP90 and STATS at the critical STATS targets MYC and CCNDZ, Neither protein was present at intergenic control s (not shown). ingly, PU- H71 (1 uM) decreased the mRNA abundance of the STATS target genes CCNDZ, MYC, CCNDI, BCL-fl and MCLI“, but not of the control genes HPRT and GAPDH. tively, these data show that STATS activity is positively regulated by HSP90 in CML cells. Our findings are consistent with a io whereby HSP90 binding to STATS modulates the conformation of the protein and by this mechanism it alters STATS phosphorylation/ dephosphorylation kinetics, shifiing the balance towards increased levels ofp-STATS. In addition, HSP90 ins STATS in an active conformation ly within STATS-containing transcriptional complexes. Considering the complexity of the STAT-pathway, other potential mechanisms however, cannot be excluded. Therefore, in addition to its role in promoting protein stability, HSPQO promotes oncogenesis by maintaining client proteins in an active configuration.

More broadly, the data reveal that it is the PU—H7l—HSP90 fraction of cellular HSP90 that is most closely involved in supporting oncogenic protein functions in tumor cells, and a labeled PUaH71 can be used to identify this tumor HSP90 species that is bound to a broad cross-section ofthe protein ys required to maintain the ant phenotype in specific tumor cells. .1.10. HSP90 is present in two distinct forms in tumor cell: The methods presented above take advantage of several properties of PU~H71 which i) binds preferentially to the fraction ofHSP90 that is ated with oncogenic client ns, and ii) locks HSP90 in an once-client bound configuration. fication of HSP90 clients required for tumor cell survival may also serve as tumor- specific biomarkers for selection of patients likely to benefit from HSP90 therapy and for phan'nacodynamic monitoring ofHSP90 inhibitor efficacy during al trials (122., clients whose expression or phosphorylation changes upon HSP90 inhibition). Tumor specific HSP90 client profiling provide one approach for personalized therapeutic targeting of tumors.

This work substantiates and significantly extends the work of Kamal er al, providing a more sophisticated understanding of the original model in which HSP90 in tumors is described as present entirely in multi-chaperone complexes, whereas HSP90 from normal tissues exists in a , uncomplexed state“. We show that HSP90 forms mically distinct complexes in cancer cells (Figure 51). In this View, a major fraction of cancer cell HSP90 retains “housekeeping" one functions similar to normal cells, s a functionally distinct HSP90 pool enriched or expanded in cancer cells specifically interacts with oncogeuic proteins required to maintain tumor cell survival.

Perhaps this HSP90 fraction represents a cell stress specific form of chaperone complex that is expanded and constitutively ined in the tumor cell context. Our data t that it may execute fimctions necessary to in the malignant phenotype. One such role is to regulate the g ofmutated (112., mB«Raf) or chimeric proteins (122., Ber-A131)“. We now present experimental evidence for an additional role; that is, to facilitate scaffolding and complex formation of molecules involved in aberrantly activated signaling xes. Herein we describe such a role for HSP90 in ining constitutive STATS signaling in CML. These data are consistent with previous work in which we showed that HSP90 was required to maintain functional transcriptional repression complexes by the BCLIS oncogenic transcriptional repressor in B cell lymphoma cells").

What distinguishes the ding fraction ofHSP90 from the non-PU-binding fraction? This is a very complex question that remains under active investigation. gh both HSPQOu. and HSP9OB isoforms are recognized by PU-H7l, our data provide evidence for at least one difference between Bcr-Abl/HSP90 (PU—preferring) and Abl/HSP90 (PU-non-preferring) chaperone xes.

That is, Bcr—Abl/HSP90 chaperone complexes contain a number of co—chaperones (suggesting that an active chaperoning process is underway, further supported by the sensitivity of Ecr‘Abl to the silencing of , while Abl/HSP90 complexes lack associated co—chaperones (likely representing sequestered but not ly chapemned Ab], supported by the insensitivity ofAbl to Hsp'lO knockdown) (see Figure 2e). rmore, we have observed that HSP90 that is mutated to more avidly bind to its client proteins also binds more avidly than does wild type HSP90 to PU-beads (manuscript in preparation). Finally, we have observed a differential impact of HSP90 phosphorylation on PU-H7l and geldanamycin binding. These findings, which are being pursued further, suggest that various HSP90 inhibitors may be ly affected by specific post~translational modifications to the chaperone. Taken together, these preliminary observations show that PU-H7l recognizes an HSP90 fraction that is participating in an active one cycle, and that this characteristic is not necessarily shared by other HSP90 inhibitors. .2. Labeled HSP90 inhibitors for diagnostic and stic applications To measure in a tumor-by-tumor manner the abundance of the “oncogenic HSP90”, the disclosure provides several chemical tools (see Sections 5.2.1. and 5.2.2.) that can be used for diagnostic and prognostic purposes. Additionally, the chemical tools provide new insight into the heterogeneity of tumor ated HSP90 and harnesses the biochemical features of a particular HSP90 inhibitor to identify tumor-specific HSP90 that regulates the tumor-promoting biological pathways and proteins. Such tools e labeled HSP90 inhibitors that specifically identify and interact with this tumor “oncogenic HSP90” species, making it feasible to measure the abundance of the “oncogenic HSP90” species in difi'erent subpopulations in tumors and thus, measure and predict sensitivity to HSP90 inhibition therapy. Moreover, measuring the abundance of “oncogenic HSP90” provides a means of determining whether a tumor is ent on HSP90.

In one aspect, the disclosure provides a method for ining whether a tumor will likely d to therapy with an HSP90 inhibitor which comprises the following steps: (a) contacting the tumor or a sample containing cells from the tumor with a detectably labeled HSP90 inhibitor which binds preferentially to a tumor—specific form of HSP90 present in a tumor or tumor cells; (b) measuring the amount of labeled HSP90 inhibitor bound to the tumor or the tumor cells in the sample; and (c) comparing the amount of labeled HSP90 inhibitor bound to the tumor or the tumor cells in the sample measured in step (b) to a nce amount of the labeled HSP90 inhibitor bound to normal cells; wherein a greater amount of labeled HSP90 inhibitor bound to the tumor or the minor cells measured in step (b) as compared with the reference amount indicates the tumor will likely respond to the HSP90 inhibitor.

The method es measuring in a tumor the nce of an HSP90 s, the “oncogenic HSPQO”, as a biomarker for HSP90 therapy. The abundance of this HSP90 species does not necessarily correspond with the total HSP90 sion in the tumor. The disclosure provides several solutions to measuring the abundance of “oncogenic HSP90”. In one such embodiment, labeled derivatives of certain HSP90 inhibitors can be used as tools to measure its presence and its abundance.

Further, in this particular method the greater the ratio of the amount of labeled HSP90 inhibitor bound to the tumor or tumor cells measured in step (b) as compared to the reference amount, the greater the ude of the likely response to the HSP90 tor therapy. [0128) Still, further in this ular method the greater the amount of d HSP90 inhibitor bound to the tumor or the tumor cells measured in step (a), the greater the magnitude of the likely response to the HSP90 inhibitor y.

In one embodiment of this particular , the reference amount of the labeled HSP90 inhibitor bound to normal cells is the amount of the labeled HSP90 inhibitor bound to normal cells in the sample containing cells from the tumor.

In another embodiment, the reference amount of the labeled HSP90 inhibitor bound to normal cells is a predetermined amount of the labeled HSP90 inhibitor bound to normal cells in a reference sample.

In another aspect, the disclosure provides a method for determining whether a tumor will likely respond to therapy with an HSP90 inhibitor which comprises the ing steps: (a) ting the tumor or a sample containing cells from the tumor with a detectably labeled HSP90 inhibitor which binds preferentially to a specific form of HSP90 present in a tumor or tumor cells; (b) measuring the amount of labeled HSP90 inhibitor bound to the tumor or the tumor cells in the sample; and (c) comparing the amount of labeled HSP90 inhibitor bound to the tumor or the tumor cells in the sample measured in step (b) to a reference; wherein a greater amount of labeled HSP90 inhibitor bound to the tumor or the tumor cells measured in step (b) as compared with the reference amount tes the tumor will likely respond to the HSP90 inhibitor. [0132} In one embodiment of this particular method, the reference sample are cancer cells with no to little “oncogenic HSP90" expression In another embodiment the reference is a correspondingly labeled nd with little to no binding to the “oncogenic HSP90”, The detectably labeled HSP90 inhibitor may be labeled with any detectable label, and many such labels are well known in the art. For example, the detectably labeled HSP90 tor may be fluorescently labeled, biotin labeled, ANCA—labeled or radioactively labeled.

In the practice of this particular method, the tumor may be any tumor or tumor-derived biological formation that contain the enic HSP90", such as exosomes. For example, the tumor and the other cells or tumor-derived biological formations that contain the enic HSP90” may be associated with, indicative of, or derived from any cancer selected from the group consisting of ctal cancer, pancreatic cancer, thyroid cancer, a leukemia including acute myeloid leukemia, acute lymphoblastic leukemia and c myeloid leukemia, lymphoid leukemia, multiple myeloma, basal cell carcinoma, melanoma, renal cell carcinoma, bladder cancer, prostate cancer, a lung cancer including small cell lung cancer and non-small cell lung cancer, breast cancer, neuroblastoma, myeloproliferative disorders, intestinal cancers including gastrointestinal stromal tumors, esophageal cancer, h cancer, liver cancer, gallbladder cancer, anal cancer, brain tumors including gliomas, lymphomas including follicular lymphoma and e large B—cell lymphoma, and gynecologic cancers including ovarian, cervical, and endometrial cancers.

In the ce of this particular method, the tumor, the tumor cell or the associated cell or biological formation may be present in a subject or may be isolated from a t. Thus, the tumor, tumor cell or tumor«associated cell to be contacted may be in the form of a solid tumor per se in vivo or in the form of an attached cell such as in a tissue sample or as within a liquid tumor or biological fluid; a sample ed during a blood draw, bone marrow aspirate, biopsy, a fine needle aspiration or a surgical procedure; a biological fluid; blood or bone marrow. The cells to be contacted with the labeled HSP90 inhibitor may be present in any form including as ted cells, live cells, frozen cells, fixed and permeabilized cells, or formalin—fixed paraffin-embedded cells. |0136l The detectably labeled HSP90 inhibitor may be a d form of the HSP90 inhibitor which is to be administered as therapy, or may be a labeled form of a different HSP90 inhibitor including a chemically unrelated HSP90 inhibitor or a d form of an analog, homolog or derivative of the HSP90 inhibitor to be administered. Subject only to the requirement that the detectably labeled HSP90 inhibitor and most likely the unlabeled HSP90 tor to which is corresponds binds preferentially to a tumorspecific form ofHSP90 present in many tumor and tumor cells. In this regard, “preferentially” means the HSP90 inhibitor binds with ntially greater affinity to the tumor—specific form of HSP90 as compared to the affinity, if any, with which it binds to HSP90 characteristic of normal or non-tumor cells. tly. one HSP90 inhibitor considered likely to be administered as therapy is PU-H71 or an analog, homolog or derivative of PU—H7l. See for example, US. Patents 7,820,658 B2; 7,834,181 B2; and 7,906,657 82, which are all hereby incorporated by reference in their entireties, for descriptions of illustrative HSP90 inhibitors.

In one embodiment the HSP90 inhibitor is PU-H71 and the detectably labeled HSP90 inhibitor is a form of PU—H71 or of an analog, homolog, or derivative 71. Examples of forms of PU-H7l which may be the detectably labeled HSP90 inhibitor include, but are not limited to, ['2‘ - PU-H71, PU-H7l-FITC2 or PU—H71-NBD1, or a biotinylated analog of PU-H7l such as PU-H71- biotin-5, PU—H71-biotin—6, PU—H71-biotin-8 or PU-H71—biotin-9, which are described below.

A labeled derivative of PU—H71, such as abeled ['“I1-PU-H71, [‘3‘1]_PU—H71, [”31]— PU-H71, fluorescently-labeled , biotinvlabeled-PU-H7 1, or ANCA-labeled inhibitor can therefore be employed as a tool to identify and quantify the tumor-specific HSP90 species. The nce of this tumor HSP90 species can be used as a biomarker predictive of response to HSP90 therapy. .2.1. Fluorescent, Biotlnylated and ANCA-labeled Probes for ing Oncogenic HSP90 The disclosure provides fluorescently labeled, biotjnylated probes and ANCA—labeled probe: that are e of detecting oncogenic HSP90 in cancer cells. Section 5.2.1.1. describes the production of various types of probes to be used in ance with the present sure. Section .2.1.2. describes the use of such probes in prognostic and diagnostic assays. .2.1.1. Production of Probes The sure provides fluorescently labeled. biotinylated and ANCA-labeled inhibitors that are cell permeable and that selectively bind to “oncogenic HSP90”. Cell permeable inhibitors are capable of penetrating the cell membrane of a cell and binding HSP90 within the cytoplasm of the cell. To be useful in die methods of the invention, the labeled inhibitor has to penetrate the cells in an amount that is memurable by the methods of ion known to the person skilled in the art. Section 52.111. describes the pment of different fluorescently labeled probes that are cell permeable and are capable of selectively binding to “oncogenic HSP90”. Section 5.2.1.1.2. bes the development of different biotinylated pmbes that are cell permeable and are capable of selectively binding to “oncogenic HSP90”. Section 5.2.1.1.}. describes the pment of different ANCA- labeled probes that are cell permeable and are capable of selectively binding to “oncogenic HSP90”. .2.1.1.]. Fluorescently Labeled Probes Fluorescently labeled inhibitors of HSP90 have already been reported, with analogs of geldanamycin (GMJilTC,l3 ipy,” GM—cy3b”) as well as pyrazole 1 (fluorescein analog, VER-00045864)” used as ligands in fluorescence polarization assays (Figure 8). A cell—impermeable GM—FITC derivative was used to identify cell surface HSP90 by fluorescence microscopy. ‘6 HSP90 r, is mainly a cytoplasmic protein with cell e expression detected only in certain cells. ['2 Fluorescent probes are thus needed to analyze both intracellular and cell surface HSP90.

An HSP90 cell’permeable probe that specifically and tightly interacts with “oncogenic HSP90” is favored for flow cytometry measurements of this potential biomarker e fixation/penneabilization methods used for die ion of intracellular ns by flow cytometry may result in the ction of the “oncogenic HSP90 complexes” and of the cellular morphology and surface imrnunoreactivity, properties useful in flow cytomen-y for the characterization of cells in heterogeneous populations. To solve this issue the disclosure provides methods for the synthesis, characterization and evaluation of fluorescently labeled HSP90 tors that permeate live cells and bind to the target.

WO 09657 This present sure provides various new fluorescently labeled derivatives of PU—H7 1 (2), a purine-scaffold inhibitor of HSP90 (Figure 8) and bes their biological application as probes for studying HSP90 by fluorescence-activated flow cytometry and fluorescence microscopy. Several HSP90 inhibitors based on the purine-scaffold, including BIIBOZl, MPC-3100, PU-H7l and Debio 0932 (formerly CUDC-305) are currently in clinical development for caucers."“9 {0145] Fluorescent s for the heat shock protein 90 (HSP90) were synthesized containing either fluorescein isothiocyanate (FITC), 4-niuobenzo[l,2,5]oxadiazole (NED) or the red shined dye sulforhodamine 101 (Texas Red) conjugated to PU~H7l (Figure 9). Two of the compounds, PU- H7l-FITC2 (9) and PU-H7l-NBD1 (8), were shown to be le for fluorescenceeactivated flow cytometry and fluorescence microscopy. Thus these molecules serve as useful probes for studying HSP90 in heterogeneous live cell populations. |0146] For the development of small dye—labeled ligands, the selection of an optimal fluorophore and its site of attachment are relevant. Particularly in small molecules the uced dye can significantly affect the biochemical and pharrnacologic characteristics of the ligand. According to the X—ray crystal ure of PU—H7l (2) bound to HSP90,z° the N9-alkylamino chain of the ligand is oriented towards solvent. As a result of this, as well as previous SAR, several of the nds synthesized in the present disclosure contain the fluorescent label attached to the N9 position. In ular embodiments, as described below, derivatives ofPU-H7l with different linkers were labeled with either FlTC, NED or Texas Red (TR) (see Figure l).

In one embodiment of the present disclosure, 3 six carbon spacer was appended to the constituent amine of an inhibitor based on the purine scaffold, thereby providing Compound 3 (Scheme 1 and Figure 10). We had usly used this linker to attach PU~H71 to solid support and showed that Compound 3 retains good affinity for Hsp9o.“ As depicted in Scheme 1, nd 3 was reacted with FlTC in DMF/‘Eth to give Compound 4 (PU—H7l-FITCl) in 40% yield following purification by HPLC.

NH; l 0 N \ o> & / F“! b E a s PU—H71-TR N0, K / NH2 | 0 K :0 51>-sN 13;; N c M fl § H~ NH 6 7 z"? 3 FU-H71-NBD1 Scheme 1: Synthesis of PU-H71-FITC and PU-H7l-NBD1 In another ment, PU-H7l—Texas Red und 5; PU-H7l-TR) was synthesized by the reaction of 3 with sulforhodamine 101 sulfonyl chloride in DMF to give Compound 5 in 61% yield following purification by HPLC (Scheme 1). In the case of the N'BD analog, bromide 6 was reacted with Compound 7 in DMF to give Compound 8 (PU-H71~NBD1) in 47% yield (Scheme 1). nd 6“ and NBD derivative 722 were prepared as previously described.

In another embodiment, we took advantage of the secondary amine present in PU—H7l23 and reacted it directly with FITC or NED-C1 to give Compound 9 (PU-H7l-FITC2) (72%) or nd (PU-H7l-NBD2) (40%), respectively (Scheme 2 and Figure 11). It was hypothesized that attachment of the dye directly to the amine would result in more cell permeable analogs, owing to the presence of the ionizable amine functionality. onally, derivatives containing an isopropyl group (e.g., nd 9 and Compound 10) in place of a hydrogen render the compounds more ilic and enhance their cell permeability.

NH; l S finsg?N NH, l 0 \ O > Ni” ., 1:1?“5 § “L «- é —- “A ,N N SANH is j: 0 C00” 02” ZPUAHH in PU-HTi—NBDZ O \O HO 0 o 9 PU-H71-FITC2 Scheme 2: Synthesis of PU-H7l-FITC2 and PU—H71-NBD2 {0150] In yet another embodiment, as depicted in Scheme 3 and Figure 12, desisopropyl—PU«H7] (Compound 13) was reacted with FITC or NED-C1 to give Compound 14 (PU—H7l-FITC3) (74%) or Compound 15 (PU-H7l-NBD3) (42%), respectively. Compound 13 was synthesized by N9- alkylation of Compound 11 with r0mopropyl)—phthalimide and subsequent removal of phthalimide with hydrazine (Scheme 3).

PU-H71-NBI33 14 PU-H7H=ITI23 Scheme 3: Synthesis of PU—H'll-FITCS and -NBD3. [0151! Additional compounds analogous to PU-H7l-FITC3 (shown in Scheme 3) but with different linker lengths were prepared, as depicted in Scheme 4.

NH, I NH, l o N N N \ o N \ D> N)\j:N SH!) N N 0 a N N N a 0 n n o N NH: 11 O 17. n—Z 17!: n=4 in re 16- 11:2 1!“: n=4 16¢ "=6 H:5 by} \LN’ N #9 h“ 5i”NH COOH O ‘O H0 0 0 1a. PU~H71-FITCA n:2 15b PU-H71-FITCS n=4 15c PU—H71-FITC6 n=6 Scheme 4: Synthesis of PU-H71-FITC4, PU-H‘Tl-FITCS, and PU-H7l-FITC6 The compounds ed in Schemes 14 were assessed for their ability to permeate cells and bind to HSP9O within the cells. Cell permeable probes are favored because fixation/permeabilization s used for the detection of intracellular antigens by flow cytometry often result in the destruction of cellular morphology and surface immunoreactivity, properties usefill in flow cytometry for the terization of cells in heterogeneous populations. Thus, it is of particular interest to find cell-permeable ligands that interact with the target in live cells without the requirement of fixation and penneabilization steps. [0153} To investigate which of the above synthesized fluorescently labeled PU-H7l derivatives retained the cells permeability profile of the parent compound , we examined the cellular permeability of these HSP90 probes in human acute myelogenous leukemia (AML) cell lines, W4- 11 and MOLM-l3. Of the ten fluorescent tives of PU~H7l prepared in Schemes 1-4, we find that PU-H71-FITC2 (9) and —NBD1 (8) have the highest y to permeate cells and bind to HSP90 (Figure 13). Specifically, we show efficient staining of live cells by these two derivatives (Figures 13A) as well as biological activity in these cells indicative of target (HSP90) inhibition (Figures 13]}, 13C). in particular, we show that both PU-H7l-FITC2 (9) and PU—H7 l-NBD] (8) decrease the viability of MOLM-13 cells (Figure 133), effect associated with degradation of HSP90— client proteins such as mutant FLT3 and Raf-l (Figure 13C) indicating intracellular HSPQO tion in these cancer cells.H rmore, al fluorescence copy of leukemia cells stained with PU-H7l» FITC2 (9) showed prominent intracellular localization (Figure 14A). In these experiments, DAPI was used as a viability dye to minate between viable and non~viable cells. This dye is impermeable in live cells at the tested concentration, but permeates non-viable cells and binds specific regions of DNA. DAPI is excited in most instruments with a UV laser. Similar data were generated with PU-H7 l-NBDI (8) (not shown).

Flow cytometry is ly used to separate and distinguish different cell populations in normal and malignant hematopoiesis by the use of specific markers. As an example, blast cells are often quantified and characterized by dim CD45 staining (CD45dim), in contrast to the circulating ast cell populations, which are bright for CD45 staining (CD45hi).“ These cells, gated and separated by the presence of their identifying markers, we show here can also be stained for the target, HSPQO, with —FITC2 (Figure 143). In accord with previous reports indicating the ive binding ofPU-H7l to tumor cell HSP90,”3 PU—H7l-FITC2 preferentially stained the malignant cell (blasts) and not the normal cell (lymphocytes) population in a primary acute myeloid leukemia sample (Figure 143).

Accordingly, we show that PU-H71—FITC2 (9) and PU-H7l-N'BD1 (8) permeate live cells and bind to the target. Specifically, we show that PU-H7l-F1TC2 and PU—H7laNBDl stain live cells (Figure 13A), reduce the viability of leukemia cells e 133), inhibit the intracellular HSP90 as indicated by degradation ofHSP90 client ns (Figure 13C), are localized intracellularly as indicated by confocal copy (Figure 14A) and bind specifically to tumor versus normal cell HSP90 as indicated by flow cytometry (Figure 143), to e ample evidence that these probes permeate the cell and bind specifically to the tumor HSP90 target, similarly to PU—H7l. Examples such as provided in Figure 4, Figure 15, Figurelfi and Figure 18 also trate that these fluorescent derivatives of PU—H7l interact with the “oncogenic HSP90” species and moreover provide a means to quantify this species in a large spectnrm of cancer cells. As discussed in Section .2.1.2., these fluorescent derivatives of PU~H71 can be d as probes for fluorescence—activated flow try or as tools for monitoring real—time interaction ofHSP90 with the target by fluorescence microscopy.

Based on the results discussed above, we designed various other cell permeable probes that can interact with HSP90 and thus, can be used as diagnostic and/or prognostic tools. In one embodiment, compounds r to PU—H7l—FITC2 but with a ent substituent on the benzo[d][l,3]dioxole ring were synthesized in a manner similar to PU-H7l-FITC2, as shown in Scheme 5.

N": X ~ XI0) "s“ )‘—s 0 N Frro, 5m Scheme 5: Synthesis of Compounds Analogous to PU-H71-FITC2 In r embodiment, compounds similar to compounds depicted in Scheme 5 but where the pyrimidine ring on the purine-scaffold is replaced with a pyridine ring, wen: synthesized in [runner similar to PU-H7l-FITC2, as shown in Scheme 6.

COO" HO O O ,N, hg”:b::::"N\ I a, “15:41:1Q? tays'tlji "Bili’qg H‘bfi::/k 03 203 37:5: ‘3 "bi; :NNA 3&3?“ Scheme 6: Synthesis of Compounds Analogous to PU-H7l-FITC2 In another embodiment, Compound PU-FITC7 is prepared, as depicted in Scheme 7.

CF: HEN N F35 0 NC", T 003 ”2"“ N s amen" "LN, N 0.3 [MFA N/k ‘“ Q “O H0 0 O N PU-FITC‘I Scheme 7: Synthesis of PU-FITC'I In another ment, Compound PU—FITC8 is prepared, as depicted in Scheme 8.

N 5 0 NW LN’ " N Q3 i ,k 8 man," N N ———- LN] 54km ”KL mam NH COOH 21 O \Q H0 0 o uPu-Frrca Scheme 8: Synthesis of PU-FITCB In another embodiment, Compound C9 is prepared, as depicted in Scheme 9.

WO 09657 N YOV‘ocus LN“ N \ ”Yaw N OCH, ‘L / N NJ\ N FITC,Et3N M NH DMF,rt A coon " O \O HO 0 o 14 PU-FITCS Scheme 9: Synthesis of PU—FITC9 In still another embodiment, Compound DZl3-FITC1 (PU‘DZIJ-FITC) is prepared, as depicted in Scheme 10.

NH; I ) ”“2 ' g FJ‘N/"*1“N )L / FITC.Et3N S F N s N N\u g wan ct,NIH o —{ O O0H 0 DZ13-FITC1 Scheme 10: Synthesis of Compound DZl3-FITC1 [0163) In still another embodiment, Compound SNX—FITC is prepared, as depicted in Scheme 11. 0 NH; 0 NH; ‘0” FITC. EtaN s n, 0 NH: ———~—~> M DMF, rt N"W N'N H n o \ OOH F30 F3 0 o O0 26 H0 SNXuFlTC Scheme 11: Synthesis of Compound SNX-FITC .2.1.1.2. Synthesis of ylated probes for detecting oneogenic HSP90 A series of biotinylated s of PU-H71 (2) and desisopropyl-PU—H7l (13) were prepared with the e of obtaining compounds that are capable of permeating cell membranes and bind to intracellular HSP90 in live cells. The HSP90 inhibitors 13 and 2 were conjugated to biotin through a linker. The type of linker, as well as its length, were systematically altered so as to identify compounds capable ofpermeating into live cells and binding to HSP90.

The biotin tag enables for pull down experiments through subsequent binding to streptavidin The linker should be of sufficient length to enable the concomitant binding to HSP90 and streptavidin.

The biotin tag also s for detection using a labeled avidin or avidin antibody, and thus the biotinylated HSP90 inhibitors can be useful in staining tissues to detect the “oncogenic HSP90”. nd 13 and Compound 2 contain an amine functionality which enables for the direct attachment of biotin and biotin containing linkers through me formation of an amide bond. In one ment, biotinylated molecules were prepared with no linker (i. e., direct ment to biotin).

The synthesis oftwo such compounds, referred to as PU-H71-biotln2 and PU—H71-biotin3, is depicted in Scheme 12. The compounds may be prepared from Compound 13 or Compound 2, respectively, by DCC coupling with D~biotjn under sonication.

WO 09657 NH; 1 o “fit >4N > N N NH, I 7 N O N \ a ‘L / ys —" N N O NHR ”RHHN 13 R=H 2 R=isopropyl 0 PU-H71-biofin3 R= H Pu-Hn-biounz R= isopropyl Scheme 12: Synthesis of PUrH7l-biotin2 and -biotin3 In r embodiment, biotinylated molecules were prepared by covalently attaching PU- 1-171 (2) or desisopropyl—PU»H71 (13) to biotin through a 6—carbon chain spacer group to produce PU- H7l-biutin4 or PU—H71-biotin7, as depicted in Scheme 13. PU-H7l-biotln4 and PU-H7l~blofln7 may be prepared by reacting Compound 13 or Compound 2, respectively, with the commercially available N—hydroxysuccinimide active ester containing biotin molecule referred to as EZ—Link® NHS-LGBiotin, in the presence of a base. lfiijysfl"NH, 1 ) 13 R= H 2 R= isopmpyl PU-H11-biotin4 R=H PU-H71-blatln7 R=isopropyl Scheme 13: sis of PU-H71—biofln4 and PU—H71-biot'ln7 In still another embodiment, biotinylated molecules were prepared by covalently ing PU—H7l (2) or desisopropyl—PU—H7 1 (13) to biotin through an extended carbon chain spacer group to produce PU-H7l-hiot'm5 or PU-H71-blotin8, as depicted in Scheme 14. PU-H71-biatin5 and PU- H7l-biotin8 may be prepared by reacting Compound 13 or Compound 2, respectively, with the commercially available N—hydroxysuccinimide active ester containing biotin le referred to as k® NHS—LC-LC-Biotin, in the presence of a base.

N“: l 0‘7 O N O t“, f5 a -‘—. 13 R=H 2 R=isopropyl PU-H71-hiofln5 R=H PU-H71-blotlna R=isopropyl Scheme 14: Synthesis of PU-H71-biotin5 and PU-H71-biotin8 In yet another embodiment, biotinylated molecules were ed by covalently attaching PU—H71 (2) or desisopropyl—PU»H71 (13) to biotin through a hylene glycol chain to e PU-H7l—biotin6 or PU—H7l-blotin9, as depicted in Scheme 15. PU—H7l-biotln6 and PU-H7l- biotin9 may be prepared by reacting Compound 13 or Compound 2, respectively, with the commercially available N-hydroxysuccinimide active ester ning biotin molecule referred to as EZ—Link® NHSvPEGA-Biotin, in the presence of a base.

WJZMQ"NH; . °\, _ 0 Z DVD [:2 Y(0 m 13 R=H 2 R=isopropyl z/\/O\/\o/\/O\/\I “39NH -blofln6 R=H PU-H71-biotin9 ropyl Scheme 15: Synthesis of PU-H7l-hiotin6 and PU-H7l-hiotin9 In yet another embodiment depicted in Scheme 16, an amine linked biotin analog, referred to as PU—H71-biotin, was synthesized by the reaction of bromide compound 6 with EZ-Link® Amine- PE03-Biotin SIysQ) SIfag) —a* ENS Br 2 PU-H71 -bioti rI Scheme 16: Synthesis of PU—H7l-hiotin To ensure the biotinylated nds still retain afi'mity for HSP90, they were each evaluated in a fluorescence polarization assay using SKBr3 cancer cell lysate. As can be seen each of the compounds retain good affinity for HSP90 with ICso’s in the range 31—154 nM (Table l; PU—H7l, 1C5" = 25 nM).

Table 1. Properties of Biotinylated nds Compound EC“ (nM); SKBr3 HSP90—streptavidin TPSA ClogP HSP90 binding binding with K562 assa l sate cells as - I“ 153.5 No .12738.66 146.24 - No -696.58 155.03 - N0 -80974 18413 - 31.4 Yes Yes 922.90 n.d hiotin9 n.a.= not applicable n.d.= not determined; TPSA and Clog P values were determined with Chemdraw and n.d. Indicates that it was not possible to determine a value for the given ure.

Two general trends can be observed. First, compared to PU-H71 analogs the desisopropyl analogs bind on average with approximately 2—fold greater affinity (i.e. PU-H7l—hiotin3 vs -2, -4 vs - 7, -5 vs —8, -6 vs -9). Second, in terms of the linkers the carbon series is more potent than the ne glycol series (i.e. PU-H71~hintin4 and -5 vs -6, -7 and -8 vs -9)‘ In sum, all of the compounds prepared retain good afiinity with HSP90 and were suitable for further analysis.

Having shown that each of the ed biotinylated molecules retain good afiinity to HSP90 we next wanted to determine whether the chain length was sufficient to maintain concomitant binding to HSP90 and streptavidin. K562 lysate (500 ug protein) was treated overnight with a mixture of streptavidin beads and 100 uM of each of the compounds. Following sufficient washing to remove any unbound material, the remaining bead pellet was analyzed by SDS~ PAGE. The gel was washed and stained with coomasie blue for l h. PU—H71-hiofin—5, —6, -B, -9 as well as PU—H71-hiotin show a band at approximately 90 kDa, indicating concomitant binding to HSP90 and streptavidin. Analogs without a linker l-hiofin2 and -3) and with a 6—carbon spacer group (PU-H7l-hiotin4 and -7) did not show a band at 90 kDa, indicating that the linker was too short. In contrast, nds ning an extended carbon chain spacer group l-hiotin5 and .8 and a polyethylene chain (PU-H7l-biotin6 and —9) were of sufficient length to enable itant binding.

Having shown that some of the les bind concomitantly to HSP90 and streptavidin, we next investigated whether this can similarly be accomplished in live cells. In this case, binding in K562 cells was first determined by treatment with 100 pM of PU-H7l-biotin-5, -6, -8, -9 as well as PU—H7l-blotin for 4 h then analyzed by SDS-PAGE. Of the compounds evaluated only PU—H7 1- biotin failed to maintain binding in live cells. Interestingly, PU—H7l-hiofin contains an ionizable amine which limits its permeability and may be a y factor for its failure to bind. In contrast, PU-H7l-biotin-5, -6, -8, -9 do not contain an ionizable amine and are able to permeate the cell ne. The active compounds were evaluated at 50, 25, and 10 1.1M and show that PU-H7l- WO 09657 -6 and 9 maintain good binding even at 10 uM. These two compounds were further evaluated at 5, 2.5 and 1 uM and even at the lowest concentration a faint band is still present at imawa 90 kDa. PU~H71~biotln—6 still shows a faint hand at 0.5 uM, ting concomitant binding is still maintained at this low tration.

It appears that compounds ning extended carbon chain spacer groups (PU-H7l-blotin- S, -8) or polyethylene glycol chain linkers (PU—H71—blofln—6, -9), ective of whether 13 or 2 is attached, are able to te the membrane ofK562 cells, bind to HSP90 and subsequently bind to streptavidin beads. Furthermore, it appears as if compounds containing hylene glycol chain linkers (PU-H71—bintin—6, -9) may be preferred. .2.1.1.3. Synthesis of ANCA-Labeled Probes The present disclosure further provides probes for detecting oncogenic HSP9O by labeling inhibitors with amino naphthalenyl~2~cyano-acrylate (ANCA). ANCA is a fluorescent probe that can bind to and stain amyioid plaques in human tissue. ANCA is oflen referred to as a molecular rotor.

Molecular rotors are probes where the fluorescence quantum yield is dependent on the surrounding environment. The structural motif of the molecular rotor is such that when brought in to close proximity of a olecule the internal molecular rotation is hindered (increase in rigidity) resulting in a change in fluorescent emission i. e. bound and unbound molecular rotors have different fluorescence emission peaks (see Figure 15). This physical aspect can be exploited when conjugated to PU-H71, which has specificity to the “oncogenic H5p90”. The molecular rotor conjugated to PU- H7l allows one to n in a heterogeneous population of cancer cells, the cells with “oncogenic Hsp90” and allows the quantitation of such species in the cells present in specimens obtained from interventions such as biopsy, surgery or fine needle aspirates.

In one embodiment, desisopropyl—PU‘H71 (13), PU-H7l (2) or nds analogs of 13 or 2 may be labeled with ANCA, as depicted in Scheme 17. In Scheme 17, desisopropyl-PU-H71 (13) is reacted with cyanoacetic acid to produce Compound 26 In the next step, Compound 26 is reacted with Compound 27 at elevated temperature to afford Compound 28 (PU-ANCA).

Scheme 17: sis of PU—ANCA In yet another embodiment, ANCA labeled HSP90 inhibitors useful in the invention, such as those based on purine are shown in Scheme 18. 2012/045864 'L , >4 N " o b n N N _-_, / 2.", E 03 g O 0 0 0 CN CN CN CN / / / / 0 O O O Q Q Q Q «"3 Q Q «"3 2 05%" LEE?5m"12%;? NH, "C 1:15;“"2%03 IRE? 0 Scheme 18: Synthesis of ANCA-labeled HSP90 inhibitors based on purine In yet another embodiment, ANCA labeled HSP90 inhibitors useful in the invention, such as those based on imidazopyridine ate shown in Scheme 19. g o g o 0 i g NH NH NH NH NH 0 O O O 0 CH CN cN CH / c” / / / 0 O O 0 Scheme 19: Synthesis of ANCA-lnbeled HSP90 inhlbitor: based on imldazopyridine .2.1.2. Utilization of probes in cancer prognosis and treatment .2.1.2.1. Hematologic ancies [0181) s discussed in Section 5.1, confirm that certain HSP90 inhibitors bind preferentially to a subset ofHSP90 species, the “oncogenic HSP90” that is more abundant in cancer cells than in normal cells. Abundance of this species is not dictated solely by the amount ofHSP90 expression and is tive of cellular sensitivity to HSP90 inhibition. Thus, determining the proportion of the HSP90 population in a t’s cancer cells that is available for binding to a tagged inhibitor that s for this “oncogenic HSP90”, such as PU—H7l, predicts sensitivity to HSP90 inhibitors in the clinic and s the level to which the cancer cells are dependent on HSP90.

Specifically, the disclosure shows that cell permeable fluorescently labeled HSP90 inhibitors such as PU-H7l-FITC derivatives (e.g., PU-FITC; PU-H7 l—FIT02) label live cells as early as one hour after exposure, s the viability of ia cells at 24-48h, inhibits the intracellular rumor HSP90 as indicated by degradation of HSP90 client oncoproteins, are zed intracellularly as indicated by confocal microscopy and bind specifically to tumor versus normal cell HSP90 as indicated by flow try. Furthermore, the fluorescently labeled compounds of the present disclosure bind to the “oncogenic HSP90” species, which provide ample evidence that this probe permeates the cell and binds cally to the tumor “oncogenic HSP90" target, similarly to PUe H71.

The methods of the present disclosure may be used to determine if a patient with a hematologic malignancy (e.g., leukemia) or a myeloproliferative disorder will be responsive to HSP90 inhibition therapy. The method may be applied to different hematologic malignancies including, but not limited to, leukemia including acute myeloid leukemia, acute lymphoblastic leukemia and chronic d leukemia, to lymphoid leukemias, to multiple myeloma and myeloproliferative sms and disorders.

The disclosure provides a method for determining whether a patient with a blood cancer will likely respond to therapy with an HSP90 inhibitor which comprises contacting a sample containing cancer cells and non—cancer cells (e.g., lymphocytes) from the patient with a cell permeable fluorescently labeled HSP90 tor which binds preferentially to a tumor—specific form ofHSP90 present in the cancer cells of the patient, measuring the amount of fluorescently d HSP90 inhibitor bound to the cancer cells and non-cancer cells in the sample, and comparing the amount of the fluorescently labeled HSP90 inhibitor bound to the cancer cells with the amount of the fluorescently labeled HSP90 tor bound to the non-cancer cells, wherein a greater amount of fluorescently labeled HSP90 inhibitor bound to the cancer cells than the non-cancer cells indicates the tumor will likely respond to the HSP90 inhibitor. In certain embodiments, the amount of binding to the cell permeable cently labeled HSP90 inhibitor is determined using flow cytometry.

In some embodiments, a ratio of binding blood cancer cells to normal lymphocytes of about 1.5 or greater indicates that a cancer patient will be susceptible to HSP90 tion therapy. In other embodiments, a ratio of binding blood cancer cells to normal lymphocytes of about 2 or greater indicates that a cancer patient will be susceptible to HSP90 inhibition therapy. In still other embodiments, a ratio of g blood cancer cells to normal lymphocytes of about 2.5 or greater indicates that a cancer patient will be susceptible to HSP90 inhibition therapy. In still other embodiments, a ratio of binding blood cancer cells to normal lymphocytes of about 3 or greater indicates that a cancer patient will be susceptible to HSP90 inhibition y. In still other embodiments, a ratio of binding blood cancer cells to normal lymphocytes of about 4 or greater indicates that a cancer patient will be susceptible to HSP90 tion therapy. In still other ments, a ratio of binding blood cancer cells to normal lymphocytes of about 5 or greater indicates that a cancer patient will be susceptible to HSP90 tion therapy.

A large number of established cell lines and primary tumor samples were igated by conducting a correlative analysis n binding of a cell permeable fluorescently labeled HSP90 inhibitor (e.g., PUH7l—FITC2) and cell viability in vitro upon expowre to HSP90 inhibitors. To determine PUH71-FITC2 binding to a panel of cell lines and primary leukemia samples, we used multipatameter flow cytometry analysis. We also tested the sensitivity of these cells to HSP90 inhibitors by performing viability assays 48h afler drug exposure.

Fluorescence-activated flow try, remains a method of choice for enumerating, ing and analyzing cells.9"° In fact, a multitude of measurements can be performed now by flow cytometry, and recent technical advances allow these emean to be made simultaneously on dual cells within heterogeneous populations.‘l Such arameter analysis is quite powerful as it provides more data fiom less sample, a key consideration when patient samples are limited.

Multiparameter analysis also allows more accurate identification of populations, by excluding unwanted cells that bind some reagents?“ The method is thus optimal for analyzing the g of HSP90 ligands, when fluorescently labeled, to distinct cell populations. scently labeled ligands have historically had a wide variety of uses in biology and pharmacology,” and offer the advantage of retaining the pharmacological properties of the unlabeled ligand. In addition to in vitro investigations of ligand—receptor binding, small molecule cent probes allow for real time and non-invasive monitoring of the interaction between the target and the ligand in living cell populations, such as by means of flow cytometry.

Fluorescent dyes absorb light at certain wavelengths and in turn emit their cence energy at a higher wavelength. Each dye has a distinct on spectrum, which can be exploited for multicolor analysis by flow cytometry. Among the most used are fluorescein isothiocyanate (FITC), 4-nitrobenzo[l ,2,5]oxadiazole (NED) or the red shifled dye sulforhodamine 101 (Texas Red).

FlTC and NED are detected in the FL] channel on most instruments and are also a good choice for fluorescence microscopy (excitation 495 and 466 nM and emission 519 and 539 nM, respectively), whereas Texas Red is detected in FL3 on single laser instruments (excitation 589 nM and emission 615 nM).

In Section , we discussed studies with several primary leukemia cells and normal blood cells. In particular, we analyzed primary chronic and blast phase CML and acute myeloid leukemia (AML) samples that contained both blasts (malignant cell population) and cytes (normal cell population), CD34+ cells isolated from the cord blood of healthy donors, total mononuclear cells from peripheral blood and also peripheral blood leukocytes (PBLs) (Figures lc-e, 3, 4). We used a fluorescein d PU-H7l (PUH7 l-FITCZ) as a tool to perform multiparameter flow cytometric analysis, in heterogeneous cell populations. As shown in Figure 4a, a gating gy is used to distinguish between the normal cell population (lymphocytes) and the malignant cell population (blasts). The flow cytometric dot blots are shown for three different patients. In Figure 4b, the ratio ofPU—H7l-FITC2 binding to HSP90 in CML blasts to normal lymphocytes from the primary patient s is shown.

In Figure 4d, flow cytomertry is again used to distinguish between blasts and normal lymphocytes and to e g ofCD34+ cells within the blast gate. In Figure 4e and 4g, the ratio ofPU—H7l-FITC2 binding to HSP90 CD34+ blasts to normal lymphocytes in six leukemia patients and in three y patients was determined. The nine patients were treated with either PU- H71¢FITC2 or a control (TEG-FITC) (Figure 4t and 4h). As shown in Figure 4b, ts who had the highest ratio (referred to as CML03106, 0614 and 0124; ratios averaged in Figure 4g as “bcCML”) were more sensitive than those with a lower ratio (referred to as CMLOl 18, 0128 and 0222; ratios averaged in Figure 4g as “cpCML”). It is noted that healthy patients had a ratio nearing one (Figure 4g) and their cells in cord blood were not significantly sensitive to PU-H71 (Figure 4h, referred to as CBl,2,3). The s displayed in Figure 4[ indicate that the viability of the CD34+ blasts was significantly reduced in the patients while the normal lymphocytes were not afl‘ected.

Similarly, the control compound (TEG—FITC) did not reduce the viability of either the CD34+ blasts or the normal lymphocytes.

In primary samples, we analyzed both the blast populations and normal lymphocytes within the same patient. We found that in a panel comprised of primary leukemia cells (primary chronic~ and blast—phase chronic enous leukemia (CML) and acute myelogeuous leukemia (AML) samples), and healthy blood cells (including CD34+ cord blood cells, and total peripheral blood mononuclear cells isolated from healthy donors), cells with the highest y for PUH7 l-FITCZ were also the most sensitive to g by this agent (Figure 16). Itnportantly, normal lymphocytes present within the leukemia blood samples, show low binding to PU—FITC and were not affected by PU-H71. Thus, we rationalized that the use of the relative binding ofPU-FITC in leukemia cells compared to normal cytes within the same patient can be used as a normalized value to compare PUH71—FITC2 binding across samples. Specifically, when ted in CML samples, blast crisis CML (bcCML) cells ted the highest binding to PU-FITC (over 4 fold relative to normal lymphocytes) and demonstrated the highest sensitivity to PU-H7l treatment when compared to chronic phase (cpCML) (Figure 16). In contrast, PU—H7 l bound weakly to HSP90 in normal blood cells (ICw values higher than 2,000nM vs ~lOOnM in thML) and was non-toxic in these cells at concentrations that were toxic to the cancer cells (Figures ld ,e and 163, C). Figure 16C shows the graph correlating the ratios ed by analyzing the binding of PU-H7l-FITC2 to blasts and to normal lymphocytes in 19 primary AML samples (reported as Fold PU binding on the X-axis) and the measured viability of the blasts when d with PU—H7l. Responsive (>50% reduced viability) from sponsive (<50% reduced viability) tumors cells could be differentiated by a ratio of about 2.31 to about 7.43 or above compared to about 0.65 to about 2.22 or below, respectively.

Furthermore, in a panel of 14 leukemia cell lines we also noted a significant ation between PU—H71~FITC2 binding (as presented in mean fluorescence intensity) and the sensitivity of these cells to HSP9O inhibition by PU—H7l (Figure 3e).

Based on the data collected for the 19 primary AML specimens, we have calculated the sensitivity and accuracy curves to determine the probability of the assay to tly identify the sensitive and resistant AML specimens. We performed a classification performance analysis using an arbitrary cut—off value of 2 or higher for PU—FITC binding (blast!lymphocyte) and less than 50% viability as a predicted outcome, and observed the following values: Accuracy: 83.3% (53.2 - 93.8%; 95% CI); Sensitivity: 91.7% (72.8 - 99.5%; 95% CI); Specificity: 66.7% (28.9% — 82.4%; 95% CI); Positive predictive value: 84.6% (67.2 - 91.9%; 95% CI); Negative predictive value: 80% (34.7% - 98.9%; 95% CI); Fisher exact test, p = 0.022. These ations suggest that PU—FITC has a good classification performance; this tion will be repeated with a larger cohort of samples to obtain more accurate and precise performance estimates. To minimize assay differences due to experimental or instrument ion, we will use the following: (1) BD Cytometer Setup & Tracking (CST) beads to allow for automated performance adjustments and e day‘to—day cytometer performance and consistency. CST beads will be run prior every new experimental set. (2) Positive control MV4ll (sensitive cell line~high binding) and a negative control HL60 (low sensitivity cell line-low g) will be included in the assays.

To determine whether the in vitro observations in leukemia cells can be confirmed in animal preclinical models, we set up xenotransplants using primary AML samples with different sensitivities (high and low) to PU—H7l evaluated in vitro and or predicted by PU-FITC binding. Primary AML cells were injected into sub—lethally irradiated NOD/SCID mice (n=8). Three to four weeks afier ion, when the human leukemia cells bave engrafled in the bone marrow (BM) of the mouse, treatment with PU—H7l or vebicle control was d (75mg/kg 3xweek) and ued for four weeks. Mice were sacrificed and leukemia engraftment evaluated using anti-human CD45 and CD34.

To ine the ability of the surviving cells to give rise to e, we transplanted equal numbers of buman cells into sub-lethally irradiated NOD/SCID mice. This experiment determines whether PU~H7l treatment for the high binding— high in vitro sensitivity cells ts further tumor initiation.

If that is the case, it will suggest that treatment will decrease the likelihood of relapse. Because the xenografls may alter the biology of the ia , FITC2 binding to the primary cells was ted prior to injection of the engrafted cells (4 weeks afler transplant).

Results from the xenotransplant experiments are depicted in Flgure 17. In experiments using two primary AMI. samples (high sensitivity and low sensitivity, Figures 17a), we found that the high sensitivity sample has higher PU'H71-FITC2 binding than the low sensitivity sample in the xenografted AML sample (Figure 171)) and responds significantly better to treatment with an HSP9O inhibitor (Figure 17c). In addition we found that cells from the PU—high sensitivity AMI. showed significantly decreased engraflment in secondary transplants (p=0.016). The results show that HSP90 involvement in the survival and eration of leukemia cells of patients at similar stages of the disease may be substantially different. Additionally, the effect ofHSP90 inhibition therapy may be predicted from using fluorescently labeled probes of the present disclosure. .2.1.2.2. Solid and Liquid Tumors Fluorescently labeled, ANCA-labelcd and biotinylated probes of the present sure also have prognostic and stic applications for solid tumors and lymphomas and other liquid tumor associated cancers. Examples of such tumors are those associated with a cancer selected from the group consisting of colorectal cancer, pancreatic cancer, thyroid cancer, basal cell carcinoma, melanoma, renal cell carcinoma, bladder cancer, prostate , a lung cancer including small cell lung cancer and non-small cell lung cancer, breast cancer, neuroblastoma, intestinal s including gastrointestinal stromal tumors, esophageal cancer, stomach cancer, liver cancer, gallbladder , anal cancer, brain tumors including s, lymphomas including follicular lymphoma and diffuse large B-cell lymphoma, and gynecologic cancers including ovarian, cervical, and endomen-ial s, particularly breast cancer, gastric cancer, or pancreatic cancer. A person skilled in the art will recognize that labeling can be performed on tumor cells that are part of a tissue slice such as obtained from a biopsy or surgical resection of a tumor. In this case, tumor cells will be surrounded by cells of the stroma, benign tissue, vessels and other cells such as lymphocytes, macrophages. Labeling can also be performed in iated tumor cells such as those obtained from tissues that contain such tumor cells. Labeling can also be performed in tumor cells such as those obtained fi'orn established cancer cell lines. Not last, labeling can also be performed in tumor cells such as those ed from biological fluids that contain such tumor cells ing plasma and pleura. In one embodiment, labeling can be performed in tumor cell and tumor-associated cells and biologic bodies such as those found in the circulation of cancer patients, cells ed by fine needle aspirates or other interventional procedures that result in a biospecimen containing cancer cells or other types of cells or biological formations that contain the “oncogenic HSP90”. In yet another embodiment, labeling can be performed in other cells associated with malignant transformation or biologic bodies that incorporate the oncogenic HSP90, such as the tumor exosomes. For instance, Section 6.3.8. describes isolating tissue for staining from a patient with c cancer and breast cancer after surgical resection and Section 5.2.1.2.4. describes isolating circulating tumor cells from a cancer t.

Experiments in pancreatic and breast cancer cell lines indicate that analyses conducted in blood tumors are also valid in solid tumors and lymphomas. Thus labeled cell permeable HSP90 inhibitors, can detect and quantify the “oncogenic HSP90” present in the solid tumor cells or lymphoma cells. Moreover, the inhibitors can be used to predict the sensitivity of solid or liquid tumor cells to HSP90 inhibition therapy. A person skilled in the art will recognize that liquid tumors are associated but not limited to leukemias, lymphomas, myelomas and myeloproliferative neoplasms.

Such person will also recognize that certain liquid tumors can also form solid tumors, and that in addition to the blood, cancer cells associated with these diseases can spread to the lymph nodes, spleen, liver, bone marrow and other sites.

In one example, a panel of pancreatic and breast cancer cells were tested for (l) sensitivity to several distinct HSP90 inhibitors such as PU-H7l, SNX-2l 12 and NVP-AUY922 (see Figure 2) ; (2) binding to PU-H7l —FITC2; and (3) expression of total HSP90 in these tumor cells. Figure 18 shows a significant correlation between PU~H7 2 binding and sensitivity of these cells to PU—H7l, SNX—2 l 12 and NVP~AUY922 (r1 = 0.59, 0.62 and 0.61, respectively Figure 18A). In contrast no significant ation was determined between the sensitivity to HSP90 inhibitors and the expression of total tumor HSP90 in these cells (Figure 18C). Similarly, no significant correlation could be ished n the expression of enic HSP90" as determined by PU-FI'IC and the expression of total tumor HSP90 in these cells e 183). The I'LL-60 leukemia cells are resistant to PUAH71 and other HSP90 inhibitors and show low to no binding to PU—FITC. Thus, we rationalized that the use of the relative binding of a labeled HSP90 inhibitor (e.g., PU—H7l-FITC2) in cancer cells compared to HL~60 can also be used as a normalized value to compare C binding across samples and experiments (Figure 18D). Figure 18 shows such analysis using the ratio of labeled-PUH'I] (e.g., -FITC2) binding to the respective cancer cell and to HL60 in several pancreatic and breast cancer cells. Collectively, these data indicate that: (l) PU-FITC is an appropriate tool to measure the abundance of the “oncogenic ; (2) measuring the abundance of the “oncogenic HSP90" predicts for sensitivity to HSP90i; and (3) the abundance of total tumor HSP90 is not predictive of response to HSP90 inhibitors nor it ates with the abundance of the “oncogenic HSP90” as measured by labeled PU-H7l. [0200) The labeled HSP90 inhibitors of the t disclosure can be used to determine if a patient will benefit from HSP90 inhibition therapy. In one ment, binding of the labeled HSP90 inhibitor to the patient’s tumor cells can be compared with binding to control cells. Increased binding relative to the control indicates that the t will be amenable to HSP90 inhibition therapy. As shown in Figure 19, responsive (>50% reduced viability) fi'orn non-responsive (<50% reduced WO 09657 viability) cells could be differentiated by a ratio of PUmH71-FITC2 binding to tumor cells and reference HL60 cells from about 2.7 to about 5.87 or above for responsive cells compared to about 1.23 or about 2.07 or below for nonresponsive cells. It will be understood that these ratios for determining responsiveness to the HSP90 inhibitor will depend on the nature of the labeled HSP90 inhibitor and the reference specimen (i.e. HL60 cells, normal leukocytes,CD45+CDl4- cells, or normal lymphocytes in the blood) and/or control derivative (i.e. PUFITC9 or FITC-TEG used to account for non-specific/background binding) used in the assay.

A more detailed description of the invention in labeling the oncogenic HSP90 in ating tumor cells is given in section 5.2.1 .2.4. Figure 20 shows the use of PUFITC9 as a PU derivative designed to have low to no binding to oncogenic HSP90, and thus to account for non- specific/background binding. It also shows the use of the patient’s leukocytes (CD45+CD14- cells) as a reference cell (cells with low to no oncogenic HSP90).

Experiments in diffuse large B—cell lymphoma (DLBCL) cells also indicate that sensitivity of these cells to HSP90 inhibitors correlates with their uptake of labeled-PU-H71 but not with the expression of total tumor HSP90 in the cell (Figure 21). Specifically, OCI-Ly7 and OCI-Lyl are two DLBCL cells highly sensitive to HSP90 tion (Cerchietti et al Nature ne 2009). They are both avid PU—H7l binders. We treated these cells for an extended period of time with sub-therapeutic concentrations 0 tors and were able to select clones that exhibited 5 to 10~times lower sensitivity than the parental cells to several tested HSP90 inhibitors, sucb as PU-H7l , PU—DZl 3 and 17DMAG (Figure 8). Figure 21 shows that, while these clones express total tumor HSP90 levels similar to the parental Lyl cells, they have lower “oncogenic HSP90” levels as measured by labeled PU«H7l-uptake. The binding experiment was carried out in the presence and absence of PSC333 (2.5 uM), a P-gP inhibitor, to demonstrate that ential uptake was a result of distinct “oncogenic HSP90” levels and not an indirect measure of drug pump—mediated efflux. 221. Pancreatic ducts] adenoeareinomn Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer-related mortality in the United States. The ar survival rate is the lowest among all cancers, with estimates g from 0.4 to 4 percent. In 2009, an estimated 42,470 new cases of PDAC were diagnosed, and an estimated 35,240 patients died as a result of their disease. Because of the aggressiveness of this cancer, the inability to diagnose it early, and the t lack of outcome altering therapies, ity rates from PDAC closely mirror incidence rates. The only potentially curative treatment for PDAC is surgical resection. Because the disease is generally ed at presentation, only 10 to 20% of patients are eligible for curative ion. In these ts who undergo pancreaticoduodenectomy, five—year survival remains dismal, approximately 20%.

Development of effective chemotherapeutic agents to treat PDAC has been enormously challenging.

Traditional cytotoxic agents are largely ineffective at controlling tumor growth, improving quality life and prolonging patient survival. [02041 To tolerate the complex load of aberrant pathways and molecules, PDACs become dependent assists for survival on molecular chaperones. The major chaperone, heat shock n 90 (HSP90), and abets once-proteins g malignant processes in PDAC, such as proliferation, al asis, and allow for the pment of a cancer phenotype. In addition, HSP90 helps cancer cells build resistance to other therapies by increasing the apoptotic threshold. These hensive biological functions pmpose an important role for anti.HSP90-targeted therapy in PDAC. of the Consequently, these tumors are appropriate candidates for treatment with inhibitors of one major cancer chaperones, HSP90.

Identification of the abundance of tumor HSP90 species required for pancreatic cancer HSP90 survival by means of HSP90 inhibitors, such as PU-H7l that preferentially bind the oncogenic from species, will serve as a tumor—specific biomarker for selection of patients likely to benefit HSP90—therapy and to personalize eutic targeting of tumors Indeed, the sensitivity of pancreatic cell lines to HSP90 inhibitors correlates with tumor HSP90 s abundance, as measured by cellular uptake of fluorescein labeled PU—H7l 1- FITCZ) (Figure 22). Cells that take up the highest amount of PU-H71~FITC2 are also those most sensitive to the HSP90 inhibitors.

Similar to studies with blood cancers (Section 5 2.1.2.1.), the higher the relative binding of reference the labeled HSP90 inhibitor (e.g. PU—H7l-FITC2) in pancreatic cancer cells compared to derivative or reference cells (e.g., HL60 or normal cells), the more tible the atic tumor or tumor cells will be to HSP90 inhibitor y (Figure 19). In some embodiments, a ratio of binding will be tumor cells to reference cells of about 2 or greater indicates that a pancreatic cancer patient susceptible to HSP90 inhibition therapy. In other embodiments, a ratio of binding pancreatic cancer will be tumor or tumor cells to reference cells of 2.5 or greater indicates that a cancer patient susceptible to HSP90 inhibition therapy. In other embodiments, a ratio of binding atic cancer will be susceptible tumor or tumor cells to reference cells of 3 or greater indicates that a cancer patient to HSP90 inhibition therapy. .2.1.2.3. Cancer Stem Cells The present disclosure provides methods of detemtining the amount of “oncogenic HSP90” and thereby determining if CSCs cancer stem cells (CSCs) relative to normal cells (e.g., lymphocytes) that cancer stem cells (CSCs) are responsive to HSP90 inhibitor therapy. Recent ce suggests it has been shown are able to originate and maintain disease for a diverse type of cancers. Moreover, that these cells are resistant to common chemotherapeutic agents and thus more likely to result disease e or metastasis. ore, it is critical to identify therapies that can abIate CSCs in order to obtain better therapeutic es. Heat shock proteins (HSPs) play an important llance role in protein synthesis, maintenance and degradation. In Figure 23, we provide data in acute myeloid leukemia (AML) stem cells that shows that CSC populations are sensitive to HSP90 inhibition and that ivity correlates with the abundance of the oncogenic tumor HSP90 species, as recognized by a labeled PU~H71.

Figure 23A displays the ratio of g of PU-FITC to leukemia stem cells (LSCs, CD34+CD38- CD45dim) and to lymphocytes. Primary AML samples were incubated with luM PU- H7l-FITC2 at 37°C for 4 h. Cells were stained with CD34, CD38, CD45 and 7-AAD followed by flow cytometry analysis. Figure 23]] displays the percent viability of LSCs relative to the untreated control from three primary AML s after 43 hour treatment with luM PU-H71. Cells were d with CD45, CD34 and CD33 prior to Annexin V and 7-AAD ng. Viability in LSCs was measured by flow cytometry and determined as the percentage of AnnexinV—/7AAD— of the CD45din1 CD34+CD33— gate. Notably, the cells with the higher binding to PU-H7l—FITC2 were most susceptible to treatment with the HSP90 inhibitor.

Similar to studies with blood cancers (Section 5.2.1.2.1.), the higher the relative binding of the fluorescently labeled HSP90 inhibitor (eg. PU—H7l-FITC2) in CSCs compared to normal cells (e.g., lymphocytes) within the same patient, the more susceptible the CSCs will be to HSP90 inhibitor therapy. In some embodiments, a ratio of g CSCs to normal lymphocytes of 1.5 or greater indicates that a cancer t will be susceptible to HSP90 inhibition y. In other embodiments, a ratio of binding CSCs to normal lymphocytes of 2 or r indicates that a cancer patient will be susceptible to HSP90 inhibition therapy. .2.1.2.4. Circulating Tumor Cells Circulating tumor cells (CTCs) are cells that have detached from a primary tumor and circulate in the bloodstream. CTCs may constitute seeds for subsequent growth of onal tumors tasis) in different tissues. Figure 20 shows labeling of CTCs isolated from a patient with HER2+ metastatic breast cancer. The tumor cells isolated from her plasma bind around 84-fold more ‘C than the leukocytes (CD45+CD14— cells) also isolated from her , ting that these tumor cells have high levels of the oncogenic HSP90 and that therapy with an HSP90 inhibitor would be effective at killing them. Indeed, twenty—four hours after this patient received a dose of 20rng/m2 PU-H7l, a 6-fold drop in the number of CTCs in the blood was measured. .2.2. Radiolabeled Probes for Detecting Oncogenlc HSP90 The disclosure provides for using radiolabeled probes that are capable of detecting oncogenic HSP90 in cancer cells. Section 5.2.2.1 describes the various types of probes to be used in accordance with the present disclosure. Section 52.2.2 describes the use of such probes in prognostic and diagnostic assays. .2.2.1. nheled Probes HSP90 inhibitors that can be labeled without changing the affinity, selectivity or biodistribution profile of the inhibitor are ideal probes for stic and/or diagnostic purposes. In one embodiment, the probe is an iodine 124 radiolabeled versions of the HSP90 inhibitor. In another embodiment, the probe is an iodine 131 radiolabeled n of the HSP90 inhibitor. In another embodiment, the probe is an iodine 123 radiolabeled version of the HSP90 inhibitor. In another embodiment. the probe is an iodine 125 radiolabeled version of the HSP90 inhibitor.

In one embodiment. the radiolabeled probe is a compound of the following a: flZ \ Z;__ 21\ 5’ Y Xa A/ NFY X9 x4 22 N .‘ (IA) (1B) or a pharmaceutically able salt thereof, wherein: (a) each of 2., Z; and Z; is independently CH or N; (13) Y is CH;, O, or S; (c) Xa, Xb, Xe and Xd are independently selected from CH, CH2, 0, N, NH, S, carbonyl, fluoromethylene, and difluorornethylene selected so as to satisfy valence, wherein each bond to an X group is either a single bond or a double bond; (d) X; is mL 1241, 1251 01,1311; (e) X. is hydrogen or halogen; and (t) R is straight-chain— or branched— substituted or unsubstituted alkyl, straight—cham- or branched— substituted or unsubstituted alkenyl, straight—Chaim or branched- substituted or unsubstituted alkynyl, or tuted or tituted cycloalkyl, n the R group is optionally interrupted by -S(O)N(RA)-, -NRAS(O)-, -SO;N(RA)-, —NRASOI-, -C(O)N(RA)-, or -NRAC(O)-, and/or the R group is optionally terminated by —S(O)NRAR5, -NRAS(O)RE, -SOZNRARB, -NRASOZRB, — C(O)NRARB, or -NRAC(O)RB, wherein each RA and RE is independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C5 alkynyl, cycloalkyl, heteroalkyl, heterocycloalkyl, aryl, heteroaryl, ryl, kyl, alkylheteroaryl, heteroarylalkyl, and alkylheteroarylalkyl.

In another embodiment, the radiolabeled probe is a compound of the following formula: "*2 MHZ X2 X2 iii»: \ 23 Z, \ Z1 \ ‘ Y )53 N)_Y Xic 2 NAZ/ 2 . k Xch l? Xb'“Xd (1A) ([3) or a pharmaceutically acceptable salt thereof, wherein: (a) each of 21, Z; and Z; is independently CH or N; (b) Y is CH2, 0, or S; (c) X3, Xb, Xe and Xd are independently selected from CH, CH1, 0, N, NE, S, carbonyl, fluoromethylene, and difluoromethylene selected so as to satisfy valence, wherein each bond to an X group is either a single bond or a double bond; ((1) x2 is IZII’ I241, l251 orllll; (e) X.. is hydrogen or halogen; and (l) R is {CH2}..,—N-R,uR“R.2 or ...-N-RmR”, where m is 2 or 3 and where RWRH are independently selected from hydrogen, methyl, ethyl, ethenyl, ethynyl, propyl, yalkyl, isopropyl, t~butyl, isobutyl, entyl, a 3—membered ring including the nitrogen or a 6- membered ring including the N and optionally an additional heteroatom with substituents to satisfy valence, with the proviso that when all of RIO-Ru are present the nd further comprises a pharmaceutically acceptable counter ion.

In another embodiment, the radiolabeled probe is a compound of the following formula: N \1% W x?4:}03 or a pharmaceutically acceptable salt thereof, wherein: Y is CH; or S; X. is H or halogen X; is ”31,1241,- ' 1’I orml; and R is «(CH2)m’N~RmR, ‘Ru or -(CHg)m'N-R]0R] ,, where in is 2 or 3 and where Rm’Ru are independently selected from hydrogen, methyl, ethyl, ethenyl, ethynyl, propyl, hydroxyalkyl, isopropyl, l, isobutyl, cyclopentyl, a 3-membered ring including the nitrogen or a 6- mernbered ring including the N and optionally an additional heteroatom with tuents to satisfy valence, with the proviso that when all of Rm—Ru are t the compound further comprises a pharmaceutically acceptable counter ion.

In one embodiment, the radiolabeled probe is a nd ofthe following formula: N \IJI\ KAN/ N\R>—~Qv (0002] or a phannaceutically acceptable salt thereof, wherein: Y is CH; or S; X. is H or halogen; X1 is 1231 1241 1151 Grill]. and R is 2-ethanesulfonic acid isopropylamide, 2«ethanesulfonic acid ethylamide, 2-ethanesulfonic acid methylarnide, 2-ethanesulfonic acid amide, 2-ethanesulfonic acid t-butylamide, 2- ethanesulfonic acid isobutylamide, 2—ethanesulfonic acid cyclopropylamide, isopropanesulfonic acid 2-ethylamide, ethanesulfonic acid 2-ethylarnide, N-2 ethyl esulfonamide, 2-inethyl- e-chulfonic acid 2—ethylamide, Z‘methyl-pmpane-Zesulfinic acid 2-ethylamide, 2- methyl-propane-l-sulfonic acid 2—ethylamide, cyclopropanesufonic acid 2-ethylamide, 3- propane-l-snlfonic acid isopropylamide, 3vpropane-l-sulfonic acid ethylarnide, 3-propane»l- sulfonic acid methylamide, 3-propane—1—sulfonjc acid amide, ane»1-sulfonic acid t~ butylamide, 3’propane—l-sulfonic acid isobutylarnide, ane»I—sulfonic acid cyclopropylamide, propane—Z-sulfonic acid 3-propylarnide, ethanesulfonic acid ylamide, N—3apropyl methanesulfonamide, 2-methyl-propane—2-sulfonic acid 3~propylamide, 2-methylpropane-Z-sulftnic acid 3-propylamide, 2-methyl-propane-l-sulfonic acid 3-propylatnide, cyclopropanesulfonic acid ylamidc, 3—N~isopropyl propionamide, 3-N-ethy1 namide, 3-Nvmethyl propionamide, 3-propionamide, 3-N—t-butyl propionamide, 3—Nv isobutyl propionamide, 3-N-cyclopropyl propionamide, N—2-ethyl isobutyramide, N—2—ethyl propionamide, Nethyl acetamide, Nethyl fonnami de, hyl 2,2-dirnethyl— propionamjde, hyl 3-methylbutyramide, or cyclopropane carboxylic acid 2-ethyl-amide.

In another embodiment, the radiolabcled probe is a compound of the following formula: NH2 X2 N \(:VY x{ N N J . x5 or a pharmaceutically able salt thereof, wherein: one of Xa and Xb is O and the other is CH2; Y is CH2 or S; X.. is en or halogen; and x2 is I”I, 12“I, ”51 ormI ; and R is 2-ethanesulfonic acid isopropyiamide, Z—ethanesulfonic acid efllylamide, 2—ethanesulfonic acid methylamide, 2-ethanesulfonic acid amide, 2—ethanesulfonic acid t-butylamide, 2-ethanesulfonic acid isobmylamide, 2-ethanesulfonic acid cyclopropylamide, isopropanesulfonic acid 2—ethylamide, ethanesulfonic acid 2~ethylamide, N-2 ethyl methanesulfonamide, 2—methyI-propane-2—sulfonic acid 2—ethylamide, 2—methyl-propanesulfmic acid 2-ethylamide, 2-methyl-propane-l-sulfonic acid 2- ethylamide, cyclopropanesufonic acid 2-ethylamide, aneol-sulfonic acid isopropylamide, 3- propane—l-sulfonic acid ethylarnide, 3—propane—l—sulfonic acid methylamide, ane-l-sulfonic acid amide, 3-propane-l-sulfonic acid lamide, 3—propane-l—sulfonic acid isobutylamide, 3- propane-l—sulfonic acid cyclopropylamide, propane-Z—sulfonic acid 3-propylamide, ethanesulfonic acid 3—propylamide, N—3-propyl esulfonamide, 2—methyl-propane—2-sulfonic acid 3- propylamide, yl-propane‘2—sulfinic acid ylamide, 2-methyl-propane—l-sulfonjc acid 3- propylamide, cyclopropanesulfonic acid 3-propylamide, 3-N-isopropyl propionamide, 3—N—ethyl propionamide, 3-N—methyl propionamide, 3-propionamide, 3—N-t-butyl propionamide, 3-N-isobutyl propionamide, 3-N-cyclopropyl propionamide, Nethyl isobutyramide, N-Z—ethyl propionamide, N- 2-ethyl acetamide, Nethyl formamide, N~2-ethyl 2,2-djmethyl-propionamide, hyl 3- methylbutyramide, or cyclopropane carboxylic acid 2—ethyl-amide.

In another embodiment, the radiolabeled probe is a compound of the following formula: ft\w x2 N X4 N/ N\ or a pharmaceutically able salt thereof, wherein: Xa-Xc-Xb is CHz-CHz—CHZ, CH=CH-CH2, or =CH; Y is CH2 or S; X: is ”31,1241, 1251 OIIJII; and R is Z—ethanesulfonic acid isopropylamide, nesulfonic acid ethylamide, 2‘ ethanesulfonic acid methylamide, 2~ethanesulfonic acid amide, Z-ethanesulfonic acid t-butylamide, 2- ethanesulfonic acid isobutylamide, Z—ethanesulfonic acid cyclopropylamide, isopropanesulfonic acid Z—ethylamide, ethanesulfonic acid Z—ethylamide, N—Z ethyl methanesulfonamide, 2—methyl—propaue sulfonic acid lamide, 2-methyl-propanesulfinic acid 2—ethylamide, 2-methyl-propaue—l- sulfonic acid Z—ethylamide, cyclopropanesufonic acid 2-ethylamide, 3~propane—l-sulfonic acid isopropylamide, 3-propane-l—sulfom'c acid ethylamide, 3-propaneal-sulfonic acid methylarnide, 3- propaue-l-sulfonic acid amide, 3—propane—l—sulfonic acid lamide, anc-l-sulfonic acid isobutyiamide, ane-l‘sulfonic acid cyclopropylamide, propane-Z-sulfonic acid 3-propylamide, ethanesulfonic acid 3-propylamide, N—3-propyl methanesulfonamide, 2-methyl-propanesulfonic acid 3-propylarnide, 2-methyl-propane—2-sulfinic acid Sopropylamide, 2-methyl-propane-l-sulfonic acid 3-propylamide, cyclopropanesulfonic acid ylarnide, 3~N~isopropyl propionamide, 3-N— ethyl propionamide, 3-N-methyl namide, 3-propionamide, 3-N-t-butyl propionamide, 3-N- isobutyl propionamidc, 3-N-cyclopropyl propionamide, N—Z-ethyl isobutyramide, N~2-ethyl propionamide, N~2-cthyl acetamide, N—Z-ethyl formamide, N—Z-ethyl 2,2»dimethyl-propionamide, N— Z—ethyl 3-methylbutyramide, or cyclopropane carboxylic acid Z-ethyl-amide.

In another embodiment, the radiolabeled probe is a compound ofthe following formula: fiyaX; N x“ N N\ \ or a phmnaceutically acceptable salt thereof, n: X3 is CH2, CF}, S, SO, 50;, O, NH, or NR1, wherein R2 is alkyl; x2 is IE] ‘24] 1251 Drill].

X4 is hydrogen or halogen; X5 is O or CH2; R is 3-isopropylaminopropyl, 3-(isopropyl(methyl)amjno)propyl, 3—(isopropyl(ethyl)amino)propyl, 3- ((2«hydroxyethleisopropyl)amino)propyl, 3-(methyl(prop—2~ynyl)amino)propyl, 3- (allyl(methyl)amino)propyl, 3-(ethyl(methyl)amino)propyl, 3-(cyclopropyl(propyl)amjno)propyl, 3- (cyelohexyl(2—hydroxyethyl)amino)propyl, 3-(2—methylaziridin—l-yl)propyl, 3-(piperidin—l-yl)propyl, 3~(4-(2-hydroxyefl1yl)piperazin—l‘yl)propyl, 3-morpholinopropyl, 3-(trimethylammonio)propyl, 2- (isopropylamino)ethyl, butylamino)ethyl, 2-(neopentylamino)ethyl, 2< (cyclopropylmethylamino)ethyl, 2-(ethyl(methyl)amino)ethyl, 2«(isobutyl(methyl)amino)ethyl, or 2- (methyl(propynyl)amino)ethyl; and nislor2.

In another embodiment, the radiolabeled probe is selected from a nd having the following formulas: In another embodiment, the radiolabeled probe is selected from a compound having the ing formulas: NH; ‘2‘! NH, 131, NH: ml NH; ’3‘! N\N\ |,>~g:Q'O N\N Q0N\N F N N 2 o>n,\>—cH; ).,\>—c:Q'ON\ Q0) N o F N H2 0)a,fiF N 2 0 NH, ‘2‘: NH, 131.

N \ 0) N > N \ F’l ’ > 3 N “2 FJLN/ N ”2 H06 H6 {0224] In still another ment, the radiolaheled probe is selected from a compound having the following formulas: H5 H6 In still another embodiment, the abeled probe is selected from a compound having the following fon-nulas: NH; ml "kl—cl?) Whit} fix—9:1?) @343chNH; 131; NH; iul NH; 151, FN/NgH’ 0FN”>”2 OFN/NeH’ OFN’N?”2 0 NH NH NH NH In still another embodiment, the radiolabeled probe is selected from a nd having the following formulas: "'1" 33:12: 9:121 &:;;m:n $4112: €§§Zlao~3izz~>lm fibfli} ~» $113] "" s": «3 <3 In still another embodiment, the radiolabeled probe is ed from a compound having the following formulas: W0 2013/009657 H2N o HZN o "‘2N 0 H H H 1mgN ion/ON '13” H300 N N N IN \ N N erg CF3 \Q—KCF: 0 o O HZN o H2N o H,N o Methods of synthesizing the radiotracers in the above embodiments can be found for instance in US. Patent No. 7,834,181, , and PCT ation PCT/U52012/032371, the contents of all of which are hereby incorporated by referene in their entirety. Specific examples of radiolabeled probes are described in Sections 5.2.2.2.1. and 2.2. 2. Utilization of Radiolabeled Probes in Cancer Treatment To non-invasively measure the sion of the HSP90 tumor species (“oncogenic HSP90”), determine the dependence of the tumor on HSP90 and to ain target inhibition, a positron emission aphy (PET) assay, that is based on HSP90 specific inhibitors that selectively bind to “oncogenic HSP90" in cancer cells is used. For a number of compelling reasons, positron emission tomography (PET) is well-suited for measuring the cokinetics and retention of drug in tumor in individual patientsm'm. PET is a quantitative method with higher resolution and ivity compared with other forms of nuclear imaging. It allows non—invasive three-dimensional imaging, yielding reliable estimates of tissue concentrations (e.g. uCi or percent of the injected dose per gram (%ID)) of an administered radiolabeled compound in tumors and normal organs, regardless of their depth in the bodyl "H ’3. PET can therefore provide spatially and temporally resolved tumor uptake, concentration and clearance, as well as whole—body distribution of the tracer. Since PET does not necessarily provide detailed anatomical information. the PET assay is ofien combined with a CAT scan. The CAT scan provides a hensive view of the structural anatomy of the body. The PET scan imagery can be overlaid on top of the CAT scan to determine exactly where in the body the radiolabeled inhibitor goes. The combined use of a PET scan and CAT scan will be referred to herein as PET/CT.

Detection and quantification methods other than PET may also be used. In one ment, SPECT imaging (Single Photon Emission Computed Tomography) s, such as iodine 131, iodine 123, and iodine 125 can be used. In particular embodiments, '“I—PU—H7 1, ”31-PU-H7 1 or '"I—PU—H7 can be used as radiolabeled inhibitors for SPECT imaging. [0231) Methods of the present sure are applicable to any tumor which may be imaged with the clearest applicability being for solid and liquid tumors or lymphomas. Examples of such tumors are those associated with a cancer selected from the group ting of colorectal cancer, pancreatic cancer, thyroid cancer, basal cell carcinoma, melanoma, renal cell carcinoma, bladder cancer, te cancer, a lung cancer including small cell lung cancer and non—small cell lung cancer, breast cancer, neuroblastoma, gastrointestinal cancers ing gastrointestinal stromal tumors, esophageal cancer, stomach cancer, liver cancer, gallbladder cancer, antfl cancer, brain tumors including s, lymphomas including follicular ma and diffuse large B-cell lymphoma, leukemias, mas, multiple myeloma, myeloproliferative neoplasms and gynecologic cancers including ovarian, cervical, and endornen'ial cancers, particularly breast cancer, gastric cancer, or atic cancer. As discussed below, we show that the uptake and exposure of tumors to the radiolabeled HSP90 inhibitor (e.g., '“I—PU-H7l) varies in a manner that is predictive of response to 1151390 therapy and will distinguish patients likely to have either a favorable or unfavorable therapeutic response to PU—H71 or other HSP90 therapies. Specifically, tumors that demonstrate minimal uptake and/or rapid clearance of the radiolabeled HSP90 inhibitor (e.g., mI-PU-H71) may be inaccessible or resistant to PU-H71 or other HSP90 inhibitors. Alternatively, such tumors may not depend on HSP90 for survival (i.e., “low abundance of “oncogenic HSP90”), making HSP90 therapy inappropriate.

Conversely, tumors with high uptakes and long retention of the abeled HSP90 inhibitor (e.g., corresponding to high tumor-to-blood ratios at later time points or high tumor AUC for the al of 0 to 24 or 48h or beyond) would be predicted to be more sensitive to targeting by HSP90 inhibitors.

Patient selection can be further guided if the eutic doses and schedules required to e effective tumor trations, as predicted by PET, would result in prohibitive toxicities (e.g., the effective dose is higher than the maximum tolerated dose (MTD) or if 15 to 100% of the “oncogenic HSP90” is occupied only by doses higher than the MTD.

The abundance of the HSP90 nic complex (i.e., “oncogenic HSP90”) as measured by uptake of the radiolabeled inhibitor, is reflective of the sensitivity of the tumor to HSP90 inhibition.

Thus, in ance with one aspect of the present disclosure, the abundance of “oncogenic HSP90” in tumors is used as a biomarker of response to HSP90 inhibition. As discussed above, PET allows for non-invasive, reliable estimates of tissue concentrations of radiolabeled nd in tumors and normal organs. [‘“11—PU-H71 and other HSP90 inhibitors that preferentially bind to the HSP90 oncogenic s can thus be used to measure non-invasively their tumor uptake, a feature that allows, similarly to the above described use of fluorescently labeled , the quantification of “oncogenic x HSP90“. Thus, high tumor uptake of the radiolabeled inhibitor will identify patients with tumors that are most likely to d to HSP90 inhibitors. PU—H7l tracer accumulation in tumors is quantified from PET imagery using techniques known to persons skilled in the art.

Tumor accumulation ofPU—H71 tracer can be quantified fi'om analysis of tumor tracer concentrations at a single time-point or multiple time points. Tracer concentration refers to the amount of tracer t in a particular volume of tissue. There are various mathematical forms for expression of tracer concentration widely-known in the state of the art as known to persons skilled therein. Tracer- amount and/or tissue-volume each may be expressed as a on of a reference value. For example, the commonly used standardized uptake value, SUV, expresses the n'acer-amount as a fraction of the total tracer-dose administered to the patient; and expresses the tissue-volume as a fraction of a body reference value (e.g., body mass or body surface area).

In the present disclosure, we show that cancer patients demonstrate variable ‘avidity’ (uptake and retention) for radiolabeled inhibitors that bind selectively to “oncogenic HSPQO”. Cancer patients with similar types and stages of cancer can have substantially different uptakes of the radiolabeled inhibitor, which indicates different levels of involvement of HSP90 in the survival and proliferation of cancer cells. As an example, twelve breast cancer patients were evaluated for their uptake of [”41]- PU—H71 afler 24 hours. The results of these studies are depicted in Figure 24. Each bar on the graph indicates the maximal standardized uptake value (SUVm) of ['Z‘U-PU—H7l, as ined h PET. The SUV“, varies significantly from patient to patient, which indicates differences in the amount of “oncogenic HSP90" in the patients’ . ts with higher SUVmall values are more likely to respond to HSP90 inhibition therapy. For instance, patients with an SUV“ of [‘Z4I]-PU-H7l of about 0.25 or greater when ed 24 hours following administration of the radiotracer are potential candidates for HSP90 inhibition therapy. Patients with an SUVM of ['l‘I]-PU-H71 of about 0.75 or greater when measured 24 hours afler administration of the compound are strong candidates for HSP90 inhibition therapy. Patients with an SUV,“ of ['"11-PU-H71 of about 1.5 or greater when measured 24 hours afler administration of the compound are very strong candidates for HSP90 inhibition therapy.

Based on our finding that cancer patients demonstrate variable avidity for particular HSP90 inhibitors (122., those that bind preferentially to “oucogenic HSP90”), radiolabeled HSP90 inhibitors can be used to distinguish patients likely to respond to HSP90 inhibition y from patients who are unlikely to respond. Accordingly, the present disclosure provides a method for determining whether a tumor will likely respond to y with an HSP90 tor which ses contacting the tumor or a sample ning cells from the tumor with a detectably labeled HSP90 inhibitor which binds preferentially to a tumor-specific form of HSP90 present in a tumor or tumor cells, measuring the amount of labeled HSP90 inhibitor bound to the tumor or the tumor cells in the sample, and comparing the amount of labeled HSP90 inhibitor bound to the tumor or the tumor cells in the sample to a reference . A greater amount of labeled HSP90 inhibitor bound to the tumor or the tumor cells as compared with the reference amount indicates the tumor will likely d to the HSP90 inhibitor.

Measuring the amount of labeled HSP90 inhibitor bound to the tumor or tumor cells may be conducted in a number of different ways. For instance, in one embodiment, as discussed above, the SUV...“ (or SUV“) of the radiolabeled nd is calculated at a particular time point. For instance, the SUV may be calculated at a time 4 hours or more following administration of the radiolabeled tor. In some embodiments, the SUV may be calculated at a time 8 hours or more following administration of the radiolabeled inhibitor. In particular embodiments, the SUV may be calculated at a time 16 hours or more following administration ofthe radiolabeled inhibitor (e.g., 16 hours, 20 hours, 24 hours,48 hours, 72 hours, 192 hours). The SUV may be calculated in a range bounded by any of the two foregoing values, e.g,, at a time ranging from 8 hours to 16 hours, from 16 hours to 24 hours, from 16 hours to 48 hours, etc.

The SUV may be compared to a reference amount of the labeled HSP9O tor bound to normal cells. In one embodiment, the reference SUV may be an average level taken from healthy individuals or from measurements on the normal cells and tissues of cancer patients in a control population at a particular time point. As discussed in Section 5.1., normal cells have l or no “oncogenic HSP90”. Hence, the uptake of the radiolabeled inhibitor specific for “oncogenic HSP90” in the cells of healthy individuals or the healthy tissues or organs of a cancer patient is minimal. It will be appreciated by a person skilled in the art that a measurement at the time the labeled inhibitor has cleared the blood circulation is preferred. It is also appreciated by a person skilled in the art that the ement can be performed in any normal tissues but that those that are not ed in the labeled inhibitor metabolism and clearance are preferred. In one embodiment, such preferred measurement is from one or more areas as selected from al muscle, bone or heart blood pool.

In another embodiment, the maximum uptake (122., SUVmJ of the radiolabeled inhibitor in a patient’s tumor (referred to herein as “tumor SUV’) may be compared with the uptake of the radiolabeled inhibitor in the patient’s healthy cells. For example, in one embodiment, the SUV data from the tumor of a patient taken at a particular time point may be compared to the SUV from the blood or select areas from the bone or from the muscle of the patient. The term “blood SUV" refers to the e SUV of the contents of the heart derived from the PET assay. The term “muscle SUV" refers to the e SUV of the skeletal ature of the patient d from the PET assay. The heart and the skeletal musculature were chosen because they are representative of the ‘background’ activity surrounding tumor sites.

Use ofPETAssay Far Patient Selection and Treatment We have found that in patients with tumors dependent on HSP90, the tumor:musele and the tumortblood SUV ratios derived from PET se in a time ent manner following injection or In these a radiolabeled inhibitor that cally binds “oncogenic HSP90” (e.g., [‘1‘11—PUvH71). patients, the tumorzmuscle and the tumorrblood SUV ratios are generally close to 1:1 following injection of the radiolabeled inhibitor and the ratio increases over time. Data derived from PET on a select number of patients with various types of solid tumors and liquid tumors who are sive to HSP90 inhibition therapy are shown in Figure 25. For each patient, the maximum tumor SUV (SUVM) and average muscle SUV at multiple times following administration of ['”I]—PU-H71 were obtained from the PET assay. Figure 25 shows the mean tumorzmuscle SUV ratio and standard deviation values for the cancer patients. The tumor:muscle SUV ratio increases from 0 to 48 hours.

In addition to solid , the method allowed the imaging of liquid tumors such as is the case for a patient diagnosed with marginal zone lymphoma and chronic lymphocytic leukemia stage IV who presented massive splenomegaly imageable by PUcPET.

Based on this accumulation of data, we have determined that cancer patients with a tumor:muscle SUV and/or tumorzblood SUV ratios greater than 2 following administration of an HSP90 tor that cally binds “oncogenic HSP90”are likely to respond to HSP90 inhibition therapy. The ratio is ably ated at one or more times at more than 4 hours following administration of the radiolabeled inhibitor. For instance, the ratio may be ated at a time 8 hours, 16 hours, 24 hours or 48 hours following administration of the radiolabeled inhibitor. In ular ments, a tumorzmuscle or tumorzblood SUV ratio of 2.5 or greater at a time of 24 hours ing administration of the radiolabeled inhibitor indicates that the patient is likely to respond to HSP90 inhibition therapy. In other embodiments, a tumormuscle or tumor2blood SUV ratio of 4 or greater at a time of 24 hours following administration of the radiolabeled inhibitor indicates that the patient is likely to d to HSP90 inhibition therapy. In still other embodiments, a turnonmuscle or tumor:blood SUV ratio of 5 or greater at a time of24 hours following stration of the radiolabeled inhibitor indicates that the patient is likely to respond to HSP90 inhibition therapy. In these embodiments, the SUV in the tumor is the “ and the SUV in the muscle or blood is the average SUV (122., SUVWE).

In another embodiment, the PET image obtained in the tumor is compared to healthy (La, non-cancerous) tissue of the patient. Preferably, in this embodiment, the reference PET scan is taken in the same organ as the tumor. For instance, if the patient has a tumor in the spine, the spinal tumor is compared to normal spinal bones. If the tumor is dependent upon HSP90, a greater concentration of the radiolabeled inhibitor will he found in the tumor than in the healthy tissue. The amounts of the radiolabeled inhibitor can be determined quantitatively using the PET scan. The SUV values for the tumor can be compared to the SUV values for the healthy surrounding tissue at a particular time point or at a plurality of time points following ion of the radiolabeled inhibitor. Alternatively, the PET image from the tumor and the PET image from the healthy tissue can be compared by visual tion. If the tumor retains the radiolabeled inhibitor, then, visually, on the PET imagery, the tumor will ‘light up’ and look like a ‘hotspot’ (see, for e. Figure 26 and Figure 27).

We have determined that the presence of a ‘hotspot’ or ‘hotspots‘ at particular times following administration of the radiolabeled inhibitor ates HSP9O involvement in the patient’s cancer and provides an indication that the patient will be amenable to HSP90 inhibition therapy. time at least 1.5 hours presence of hotspots in the PET imagery is preferably determined at a ing administration of the mdiolabeled tor. For instance, the hot spot may be detected at 2 hours, 4 hours, 6 hours, 8 hours, 16 hours, 24 hours, 48 hours, 72 hours, 165 hours or 192 hours following administration of the radiolabeled inhibitor. The presence of a hot spot may be detected between a range bounded by any of the two foregoing values, e,g., at a time ranging from 2 hours to 4 hours, from 4 hours to 8 hours, from 16 hours to 24 hours, etc. The ce of a hotspot in a patient’s tumor at time points less than 2 hours does not necessarily indicate that the patient will be a good candidate for HSP90 inhibition therapy. For instance, Figure 26 (right panel) depicts a [m1]- PU—H7l PET/CT of a patient with mantle cell lymphoma taken 30 minutes after ['Z‘I]—PU—H7l ion. The PET scan shows clear visualization after 30 minutes. However, no uptake of [”AIJ-PU- H7l was ed at later times (3.5 — 24 hours). Accordingly, the patient is not a likely candidate for HSP90 therapy.

The PET assay of the present disclosure may also be used to ine which metastatic or primary solid tumors and liquid tumors are more susceptible to HSP9O inhibition y. For example, Figure 27 shows the [‘I‘H-PU—H7l PET/Cf of patient with recurrent breast cancer in the two indicated lyrrrph nodes (LN). PET images at the indicated times post—[‘Z‘H—PU-H7l injection Were quantified and SUV data obtained for [mlj-PU-H7l were converted to HSP90 inhibitor concentrations for a hypothetical administered dose of PU—H7l of lOmg/ml. The exposure of the two tumors to PU-H71 over the time of 0 to 24h was also calculated and represented as the nder-the— curve (AUC). in the lower panel of Figure 27, CT (lefl), PU<PET/CT (middle), and T/CT fusion (right) transaxial images demonstrate U-H71—avidity in one of the lymph nodes but not the other. PU—PET imaging is at 24h post-[ml]-PU—H7l injection. Interestingly, PU—avidity does not overlap with FDG-avidity in this case. This is not a unique case, and in several of the analyzed patient’s FDG— and PU—avidity correlated for some tumors but not all. Location of the tumors is indicated by arrows. PET images at the indicated times post-[ml]-PU-H7l injection were measured as Maximal Standardized Uptake Values (SUVM). The results fiom the PET assay indicate that the left tracheobronchial angle lymph node is expected to be more susceptible to HSP9O inhibition y than the laion of the lefi tracheobronchial anterior angle lymph node.

In mother aspect of the present invention, patients who are identified as being ates for HSP90 therapy are treated with a pharmaceutically effective amount of an HSP9O tor. We have determined that cancer patients that are determined to be candidates to HSP90 inhibition therapy d highly favorably to HSP90 inhibition therapy. If a t has multiple tumors, then only those tumors with a sufficient avidity for the radiolabeled HSP90 inhibitor are expected to respond to HSPQO inhibition therapy. For example, Figure 28 shows the image obtained for a 48 year old breast cancer patient with lung and bone metastases who was imaged with ['“I]-PU—H7l and then treated with an HSP90 tor, Specifically, when the patient was imaged with ['2‘1].PU—H71 PET, the scan showed targeting in dominant right lung metastasis but not in the spine metastasis.

When the patient went on HSP90 therapy with STA9090 (ganetespib), an HSP9O inhibitor chemically distinct from PU-H7l, early partial se was demonstrated by FDG PET-CT studies in the lung mass but not in the spinal lesion (Figure 28), in accord with the prediction by [”411-PU-H71 PET.

Similar results were obtained in patients with lymphoma, pancreatic cancer and neuroblastoma patients.

Use ofPETAssayfor Dosage Determination The present disclosure provides s of determining an effective dose and frequency of stration for therapy with an inhibitor of HSP90 which comprises administering to the t a radiolabeled form of the HSP90 tor which binds preferentially to a tumor—specific form of HSP90 present in a tumor or tumor cells, measuring uptake of the radiolabeled form of the HSP90 tor by the patient‘s tumor at one or more lime points, and calculating the dose and frequency of administration needed to maintain in the tumor at each time point a concentration of the HSP90 inhibitor effective to treat the tumor. The uptake of the radiolabeled form of the HSP90 inhibitor can be detemtined using a PET assay, as sed above. The methodology can be applied to numerous types of solid and liquid tutnors including but not limited to colorectal cancer, pancreatic cancer, thyroid cancer, basal cell carcinoma, melanoma, renal cell carcinoma, bladder cancer, prostate cancer, a lung cancer including small cell lung cancer and all cell lung cancer, breast cancer, neuroblastoma, gastrointestinal cancers including intestinal stromal tumors, esophageal cancer, stomach cancer, liver cancer, gallbladder cancer, anal cancer, brain tumors including gliomas, lymphomas including follicular lymphoma and diffuse large B-cell lymphoma, leukemias, myelomas and myeloproliferative sms and gynecologic cancers including ovarian, cervical, and endometrial cancers.

In one embodiment of the disclosure, the SUV of the radiolabeled tor derived fiom PET can be ted to molar concentrations of the drug in the tumor according to the following equation: 3 1 1 iHSWDtnhtbttormJt =HSP9CI 1mm («1050) x -T-——~x”X“ w MW In the above equation, [HSP90 inhibitor]t is the molar concentration of the inhibitor in the tumor at a time t following ion of the radiolabeled inhibitor. The term HSP 90 tor (dose) is the injected therapeutic dose. The term W is the tumor water space. The term MW is the lar weight of the injected drug. The term [AmUr]t is the %-injected radiolabeled dose in the tumor at time t, a value obtained from the SUV obtained from the PET image. Specifically, the term [Am] can be derived from the SUV in the tumor (SUme) by the following equation; Maud/100% = SUV,,,..,,/[body weighr(g)] In the above equation, [body weight] refers to the body weight of the patient.

In one aspect, the t disclosure provides a method for determining the concentration of an HSP90 inhibitor present in an imageable tumor in a cancer patient. A solution of the radiolabeled inhibitor (also referred to herein as “hot” drug) can be injected into the patient without concomitant injection of the drug (i.e., non-radiolabeled form of the drug, also referred to herein as “cold" drug).

In such cases, the concentration of the drug [HSP90 inhibitornmJt can be determined using the equation above. In one embodiment, the radiolabeled inhibitor (“hot drug”) is the d form of the injected drug (“cold drug”). For instance, the radiolabeled inhibitor can be ['“I]~PU~H71 and the administered drug can be PU—H7l. In another embodiment, the radiolabeled inhibitor can be different than the injected drug. The determination of the concentration of the drug in the tumor [HSP90 inhibitormmh can be ine at a single time point or a plurality of time points ing ion of the abeled tor and the therapeutic drug. By comparing the concentration of the drug in the tumor [HSP90 inhibitormuk with known efficacious doses obtained from preclinical studies (e.g, half—inhibitory concentrations (ICsn)), one can determined if the administered dose will be efficacious.

A doctor can then adjust the dose accordingly to ensure that the desired amount of the drug is in the tumor.

In the embodiment where the radiolabeled inhibitor is the radiolabeled form of the drug to be administered to the patient, the concentration of the drug in the tumor [HSP90 inhibitormJt can be determined without actually administering the cold drug. In such cases, following determination of [AWN]: from the PET assay, different hypothetical injected dose values (D) can be imputed into the equation above to determine the concentration of the drug in the tumor [HSP90 inhibitormmh. An effective dose can thereby be determined by comparing the concentration of the drug in the tumor [HSP90 inhibitormmrfl with known efficacious doses obtained from preclinical studies, as discussed above. Moreover. as discussed in detail in Section 5.2.1.2.1., the methodology can be utilized to design an efficacious dosing regimen for HSP90 therapy.

We have determined that calculations of tumor concentration or drug exposure (e.g., AUC) following administration ofjust the hot drug and inputting hypothetical amount of the HSP90 inhibitor provide similar results to experiments in which the cold and the hot drug are co- administered. As an example, Figure 29 shows the concentration of PU—H71 in a diseased paratracheal node over the time of imaging (0-72h) as obtained by the two s. Remarkably similar tumor concentrations and thus tumor exposure to PU~H71 is measured by the two methods (AUCMh 24.9 vs. 22.3uM—h).

The present disclosure also provides methods of determining the dose of an HSP90 inhibitor that is needed to saturate the oncogenic HSP90 receptors in the tumor. As described above, the PET hot drug) and a specific assay can be conducted by co—injecting a abeled HSP90 inhibitor (i.e., amount of the eutic drug (i.e., cold drug). If the dose of the injected drug is sufficiently high to the uptake of the radiolabeled occupy most or all of the enic HSP90“ in the tumor, then inhibitor is suppressed. The point at which uptake of the radiolabeled inhibitor is suppressed can be used to determine the target-saturating dose of the inhibitor, which would also be the ‘maximum tumor dose‘ that a single dose of the drug can deliver or the lly effective single dose of the drug. As shown in Equation (4) of Section 52.2.2. 1 ., below, the number of tumor sites occupied by the HSP90 inhibitor can be calculated and converted to a percent occupancy. If the HSP90 inhibitor is delivered in an amount that approaches full occupancy of the HSP90 sites, additional drug would not be expected to provide increased levels of efficacy. Hence, the methodology es a means of determining a dose of the inhibitor that can occupy most or all of the oncogenic HSP90 in the tumor.

As discussed in more detail in Section 5.2.2.2. 1., the above described methodology provides a more rational and effective dosing gy that is based on PET-derived lly effective tumor tration rather than conventional maximum tolerated dose (MTD). The approach avoids dose escalation and limits the toxicological problems ated with the drug. .2.2.2.1. [‘“Il-PUvH71 In one aspect of the present disclosure, the abeled HSP90 inhibitor to be employed for PET imaging is a radiolabeled form of PU-H71, such as ['1411—PU-H71. As discussed below, PET imaging with ['z‘H-PU-H7l can be used to inform doctors if cancer patients will be susceptible to HSP90 therapy. Moreover, the s obtained from PET imaging with ['Z‘I]’PU—H7l can be used to determine the dose of an HSP90 inhibitor that should be administered to a ular cancer patient.

Additionally, the results obtained from PET imaging with [‘2‘I]-PU-H7l can be used to determine a dosing schedule of an HSP90 inhibitor. The HSP90 inhibitor to be administered as therapy is PU-H7l or an analog, homolog, or derivative of PU-H7l.

[”‘Il-PU-Hfl PETassay as a potential non-invasive tumor HSP90 assay Introduction of PET for non-invasive assay of HSP90 inhibitors is feasible because one of the HSP90 inhibitors, , contains an endogenous iodine atom, the naturally occurring stable e iodine-127 (”71) (Figure 30a)"‘. This can be isompically substituted for PET with the long- lived on emitter iodine-124 (“1), an isotope with a four-day al half~life e 308).

Such isotopic labeling does not change the affinity, selectivity or biodistribution profile of this compound. In fact, the PET radiopharmaceutical, ['“I]-PU—H71, is the same molecule as the therapeutic compound, PUcH7l, and should therefore predict its pharmacokinetics. A single administration of trace (microgram) amounts of radiolabeled drug is also completely non-perturbing ically, and allows serial PET imaging for monitoring tissue tracer—concentrations over multiple days.

The relatively long half—life of ”‘1 is ideal for monitoring the reported prolonged tumor pharmacoldnetics of HSP90 inhibitors because it allows for satisfactory quantitative PET g for up to one week following administration. In addition, l2"I is now cially available in the United States, and its half-life ensures that [IZ‘I]-PU-H7l can be made available in medical s worldwide.

Hence, [”‘I]»PU-H7l is wellcsuited for use as a true r’ of PU-H7I and as a target biomarker for other HSP90 inhibitors.

The HSP90 inhibitors are a promising class of targeted cancer y, but their optiml clinical development requires elopment of pharmacometric assays specific to the HSP90 target.

Because of its endogenous iodine, we use the HSP90 inhibitor PU-H71 to develop a first—ofeits-kind non—invasive imaging assay for tumor HSP90 based on positron emission tomography. We show in mouse models of breast cancer that [mI]-PU—H7l is a true tracer for the intratumoral pharmacoldnetics and pharmacodynamics of the parent drug. We demonstrate its use in determining the dose of HSP90 inhibitor needed to achieve effective tumor concentrations, in assaying the actual concentration of the dmg red to the tumor and in designing an efficacious dose and schedule regimen. The assay also informs on a tumor target saturating dose, promising to lead HSP90 therapy beyond the conventional maximum ted dose. Based on this work we e that the assay will provide clinicians an improved ability for trial enrichment and to tailor drug dosage and schedule to 2012/045864 the individual patient, promising to fill an unmet clinical need and to positively-impact clinical decision-making with HSPQOetargeted agents.

{”‘Il-pU—Hn biadisrriburton and clearance s that ofPU-H71 To evaluate the use of [”‘fl-PU-HH in predicting and monitoring the in viva kinetic profile of PU—H71 and other HSP90 tors, we have produced [”‘lj-PU-H7l and performed serial small animal PET imaging studies of tumor-bearing mice (human breast cancer MDA-MB—46B xenografts), deriving time-activity ([2 %ID/g versus time post-administration) curves for tumors and various normal tissues. Corroborative biodistribution s of PU-H7i or [um—Point were performed by cing s of animals at ed times post-injection and harvesting and intravenous (i.v.) gamma counting oftumors and selected normal tissues (Figures 30b-c). Following with or intraperitoneal (i.p.) administration, the agent y distributed to tissues (Figure 30b; 1-2h), subsequent fast clearance from blood and other normal tissues (Figure 30b; 4 to 100h). At 4h post administration Qua), it was present at 40-, 9-, 18—, 6- and 10-fold higher concentrations in tumor than in brain, bone, muscle, spleen and heart, respectively (Figure 30:). By 24h, the tumor—to—norrnal tissue ratios increased to 50 to 100 for brain, muscle, spleen, heart and blood. Uptakes (%lD/g) in both smaller (~100 mm3 in volume) and larger (~200 rum1 in volume) tumors were similar (Figure 3011). [”41]—PU~H71 cleared fi'om tumors in a onential fashion (Figure 30d). An initial rapid phase, attributable to clearance of blood-borne ty (Figures 30b,d), was followed by a slow terminal clearance phase (half time ~ 60 h) attributable to c tumor retention e 30d, inset), consistent with the previously reported tandem liqtu'd chromatography mass spectrum (LC-MS/MS)— based tumor cokinetic data for PU—H7l 1004:5413. These data support the concept that within the tumor, the therapeutic and the radiotracer forms ofthe drug behave identically.

In contrast to tumors, ['“I]-PUH71 cleared rapidly from the total body in a monoexponential fashion, with no evidence of plasma protein-binding (Figures Job-d). ['Z‘H-PU—Hfl excretion occurred via the hepatobiliary and urinary routes (Figures 30b). Hepatic activity was cleared more slowly than total-body activity, presumably due to hepatic drug metabolism and hepatobiliary excretion. Activity in the c region likely represented both intact tracer and metabolites in the biliary tree and not [mI]-PU-H71 in hepatocytes, consistent with previously reported findings that intact PU-H7l disappears y from the mouse liver'oo" '7. Activity in the gastrointestinal tract accounted for over 50% of the administered activity (Figure 3%) but was almost entirely excreted activity (not shown).

Activity in non-gastrointestinal and non-genitourinary organs (i.e., the kidneys, ureters and urinary bladder) accounted for ~l% of the administered activity. The remaining 49% (50% in gastrointestinal organs minus 1% in urinary organs) were likely ed via the urinary tract.

This observation is supported by the 4h in viva [’Z‘I]~PU-H7l PET data e 30h) derived using a region-of-interest (ROI) scribing the entire mouse and accounting for only 40-50% of the administered activity. Because intestinal transit time in mice is usually longer than 411' w, it is likely that the balance of the administered activity (1. e, the 40«50% not in the 4b whole-body ROI) was excreted via the urinary tract, Radioactivity was visualized in the thyroid region which was ssed if mice received a saturating dose of potassium iodide prior to administration of the radionacer (Figure 30h), indicating production of free radioiodine (is. radio~iodide) in viva. In mice that did not receive potassium , the maximum thyroid activity between 4 and 28h postinjection was <0.4% of the administered activity. The normal mouse thyroid lates up to ~4—7% of administered radioiodide’zo'm.

Thus, a thyroid uptake of <0.4% suggests that the amount of free radioiodine released in viva fiom [‘2°1]-PU-H7l is small % of the administered activity). The release of a small amount of fi‘ee radioiodine is not on for radioiodinated tracers, and in al practice, thyroid uptake is routinely and effectively blocked using oral administration of a saturated on of potassium iodide prior to a radion'acer administration.

[‘"Ij-PU—Hn allawsfar clear tumor HSPM visualization by PET In viva PET imaging of [mI]-PU-H7l in bearing mice provided clear tumor visualization, and thus potential tumor HSP90 targeting and tumor retention from 2h post injection (Figure 30b, arrows). The mean (+ standard deviation) uptake values (%lD/g) ed from ROI analysis were 0.35 :t 0.07, 0.083 a: 0.02, 0.058 :t 0.02, 0.031 :t 0.01 and 0.024 d: 0.008 at 4, 24, 48, 72 and 100 h respectively, consistent with the values obtained in biodistribution studies (Figure 30d), continuing that non-invasive monitoring of PU—H7l in vivo and le quantification of its time— dependent tumor concentration is possible by ['Z4I]-PU-H7l PET.

Use of (“'II-PU-Hfl PET imaging to detennine the dose of PU-H71 needed ta achieve a phannacnlogically efl‘ective PU-H71 tumor concentration As noted above, the phannacokinetics of vPU-H71, and therefore unlabeled PU-H7l, in tumor and in blood are notably different: tumor drug concentrations stabilized by ~24h post-injection afier an initial rapid exponential clearance attributable largely to drug clearance from the tumor blood pool. Blood drug levels, in contrast, exhibited rapid and continuous exponential clearance es 30b, 30d). The tumor~to—blood activity concentration ratios of —PU—H71 reached values of ~1 0 and greater by ~12h postinjection (Figure 301:). Thus the integrated blood levels ofPU—H71 (i.e. the area under the blood time-activity concentration curve, AUCPU’m'bW) were not reliable surrogates of the tumor levels of PU—H7 1. Indeed, the area under the tumor time-activity concentration curve, AUCPU‘H7IMD, is 15-fold larger than the AUCPW‘M (Figure 30a, 11.4 versus 0.78 van/gm for tumor and blood, respectively). Blood phannacokinetics ore, are not reliable for designing a dosing regimen for achieving a patient-specific therapeutically effective tumor concentration.

We therefore investigated the ability of['2‘I]-PU—H71 PET to predict the administered PU- H71 dose required to achieve and maintain a eutically ive tumor concentration over a selected period of time. For an administered dose of PU—H71 (PUvH7lm, in mg), the drug concentration (mg/mL or g/L) in the tumor water space (using an average water space, W = 0.8 mIJg) at a time t post-administration, [PUH71..,.,.,,],, is calculated from the PET-derived [‘2‘l]-PU-H71 activity concentration in tumor (in %ID/g), [Amm],, at equilibrium and achieved at time t post- administration: [Au-writ [PU-H71w]. = PU—H71m - - V17 (1) 100% (0265] The drug concentration (in M) in the tumor water space, [PU~H71..m,] at the time t postadministration, is therefore: [An-tall 1 1x10‘ [PU—H71m], = PU-H71m. - _ 100% 0.8 512 "M (13) where the factor 512 is the molecular weight of PU—H71 and the factor 1x106 converts the concentration to uMl Conversely, to achieve a ed therapeutically—efi'cctive tumor PU-H7I concentration, the required dose of PU-H71 to ster (in mg) for an individual patient can be calculated based upon his or her PET—derived activity concentration in tumor at time tpost—adrninistration: PU-H71w = [Pu-m1WL.0.a-[i~—-f’°°‘il%e (2) Using the tumor-activity data in Figure 30d, equation (la) was employed to ate the time—dependent PU—H7l concentrations in tumor from 0 to 160h post—administration (p.a.) for administered doses of l, 5, 25, 50 and 75 mg/kg PU—H7l (0.02, 0.1, 0.5, l and 1.5mg for a 20g mouse) (Figure 31a, anel). From preclinical studies with PU—H71 in established cancer cells it is known that a 72h-exposure of several established breast cancer cells to the HSP90 inhibitor leads to cell growth inhibition with recorded half-inhibitory concentrations (leo) of 0.05 to 0.25uM, depending on the cell type'm. Thus, a single dose, at each of the foregoing dose , was predicted by [mI]-PU-H7l PET to achieve eutically effective trations in tumor through 72h pa.

(Figure 31:, upperpanel). For administered doses of PU-H7l of 5, 25, 50 and 75 mg/kg and a [AmmJPm value of ~0. l4i0.05 ”MD/g (Figure 31d), equation (la) yields the tumor concentrations of PU-H7l of 0.37, 1.83, 3.67 and 5.51pM, respectively, consistent with trations measured in these tumors by LC-MS/MS (Figure 3“: and not shown). A dose of lmykg yields tumor concentrations of less than 0.05M at 48h and beyond (Figure 31a) and perhaps thus, represents the lower limit for a therapeutically effective dose in this tumor model.

[’"Ij-PU—H7I PET accurately predicts the delivery of therapeutically efl'ective PUH7I concentrations in tumor To validate that PET accurately predicted tumor concentrations achieved afier injection of 5 to Bing/kg PU-H71, and that these concentrations were therapeutically effective in viva, igated the codynamic effects associated with these doses (Figures 31c, 31d). In accord with the above findings suggesting delivery of pharmacologically effective tumor concentration of PU-H7l , stration of PU—H71 doses of 5 to 75 mg/kg to mice bearing MDA»MB-468 tumors led to downregulation and/or tion of Akt and Raf-l and induction of apoptosis, as evidenced by PARP cleavage (Figure 31:, In viva). These pharmacodynamic changes are similar to those observed in tissue culture, where exposure ofMDA—MB-468 cells for 24h to concentrations of PU~H71 above 0.1pM resulted in HSP90 inhibition, as demonstrated by a dose-dependent depletion of HSP90— ent onco-proteins (tie. Raf-l, Akt) (Reference 100 and Figure 31d, In vitro). The half inhibitory concentrations determined in vitro to result in onco—client protein degradation (ECSQP‘MN'I = 0. z and E050“ = 0.15i0.02pM; Figure 31d) are similar to those determined by run—Pu- H71 PET to be in tumors and result in the measured pharmacodynamic effects (EC5OMH = 0.24:0.03pM and ECmA“ = 0.09tODSpM; Figure 31c).

Collectively, the consistent PET-predicted tumor concentrations validated by MS measurements and by Western blot pharmacodynarnic analyses, trate the ability and the accuracy of the [ml]—PU-H71 PET assay to inform the selection of the administered dose of this HSP90 inhibitor required to achieve a therapeutically effective tumor tration. -dose [l“II-PU-H71 accuratelypredicts PU-H71 tumor concentrations over a range s, up to target saturation in tumors The pharmacokinetics of tracer, microdose amounts of ['“I]-PU-H71 might not correlate with those of the copic therapeutic doses. At high PU—H7l doses, factors such as HSP90 target saturation in tumors, distinct plasma protein binding profile or changes in drug metabolism due to ial inhibition of liver metabolizing enzymes, may alter the phannacokinetics of the agent.

We therefore examined whether at a potentially saturating dose, PET-based predictions of PUvH71 tration correlate with those experimentally determined by LC-MS/MS (Figure 31b).

Administration of 75mg/kg PU—H7l to the MDA—MB—468 tumors results in tumor regression and curesm, and thus at this curative dose, saturation of the tumor HSP90 target or at least occupancy of a therapeutically significant number of target molecules is presumably achieved Based on tumor concentrations derived from [”‘ij—PU—Hn PET studies or PU-H7l biodistribution studies, administration to tumorebearing mice of 75 mg/kg inhibitor yielded tumor concentrations of 5r5lil.78, 3.50i0i27, 2.18£1.78, 1.29:0.29 and O.69:I:0.25pM at 24, 48, 72, 96 and 120k ministration, respectively (Figure 31b). These values are in good agreement with the actual PU-H7l tumor concentrations measured by LC-MS/MS (Figure 31b, LC~MSIMS), demonstrating the reliability of the PET assay predictions over a range of doses up to those ing in maximally effective target tion (Figure 31c).

Use offull—FUJI71 in patient selectionfor ESPN therapy In addition to providing a clinically practical PET-based approach for monitoring the biodistribution of PU-H7l and informing on the tumor pharmacoln'netics of PU—H7l, the foregoing analyses suggests an approach to patient screening, distinguishing patients likely to have either a favorable or unfavorable therapeutic response to PUeH71 or other HSP90 therapies.

Specifically, tumors that demonstrate minimal uptake and/or rapid nce of ['“I]—PU—H71 may be ssible to PU—H7l or other HSP90 inhibitors. atively, such finding may also indicate that the tumor does not depend on HSP90 for survival and thus HSP90 therapy is not apropiateu'w. Conversely, tumors with high uptakes and long retention of [ml]—PU—H71 (e.g., corresponding to high mmor-to—blood ratios at later time points, Figure 30) would be predicted to be more sensitive to targeting by HSP90 inhibitors. Patient selection can be further guided if the therapeutic doses required to achieve effective tumor concentrations, as predicted by ['"I]-PU—H71 PET, would result in prohibitive toxicities (eg., the efiective dose is higher than the maximum ted dose).

In conclusion the ability of the tumor to retain the HSP90 inhibitor at an effective tration over a prolonged period of time and to achieve such concentrations at non—toxic inhibitor doses, are two key criteria for HSP90 therapy entry that can be reliably measured by [”‘I]- PU-H7l PET.

Us: ofmicrodase ['"II-PU-H71 err-injected with therapeutic-dose PU-H71 to assay PU-H71 tumor concentrations by PET While [”41]oPU-H7l PET estimates well the dose of HSP90 inhibitor needed to result in cious tumor concentrations, we investigated whether [1241]-PU-H7l in tracer amounts (~6.5ng/g) inistered with therapeutic amounts of PU—H71 g to 75mg/kg or 5,000ng/ g to 75,000ng/g), could reliably assay the amount ofPU—H71 essentially delivered to the tumor e 31b, PET following co-injection of PU-H71 and PU~H71). The ability to e drug exposure in tissues of drug activity could provide critical information for predicting potential response (ex. what concentration has been delivered to the tumor and whether it is sufficient for marked 2012/045864 pharmacodynamic response). Sequential measurements over the time of ent could also be used as an indicator of whether tumor y, and thus responsiveness, has been altered (ex. 3 decrease in the concentration delivered to the tumor could indicate potential pment of resistance to the HSP90 inhibitor).

Because the radiotracer ['I‘I]-PU-H7l and the non-radioactive PU-H7l are injected in a ratio of ~l:l0,000, we can reasonably assume that the latter is essentially the only significant form of the drug in the tumor. Thus, for a co—administered dose of PU—H7l (PU—H714“, in mg) and a tracer amount of [mI]—PU»H71, the drug concentration in the tumor water space (again using an average water space, W 3 0.8 ml/g and a MW of512), is: [Annult [UP-H71m] = 1m100%- - 2.4r4x103 uM (3) Solving equation (3) for doses of PU-H7l of 5, 25, 50 and 75 rug/kg (0.1, 0.5, l and 1.5mg for a 20g mouse) yields the actual tumor concentrations of the HSP90 inhibitor (Flgure 31b, shown only for 75mg/kg). These values correlate well with the PU-H71 tumor concentrations estimated by —PU-H7l PET and by [‘“I]-PU-H7l tracer biodistributions and ted by LC—MS/MS (Figure 31b and not shown).

Collectively, these data show that the microdose ['uH-PUcH71 PET assay can yield both the dose of PU—H7l needed to result in a specific tumor concentration and the actual concentration of therapeutic PU—H7l delivered to a tumor.

Use of ["‘Il-PU—HTI to assay the maximum tumor dose, the dose that delivers tumor target saturation by an HSP90 drug [”‘I]-PU-H7l PET of cavinjected [mI]~PU-H7l and PU-H71 could potentially evaluate the occupancy of tumor HSP90 targets by an HSP90 inhibitor. For example, demonstration by PET that a given therapeutic dose of HSP90 inhibitor completely or significantly sses tumor uptake of [ml]-PU-H7l, may te that the therapeutic dose has saturated the tumor HSP90 targets, and that the administered dose delivers the ‘maximum tumor dose’ that a single dose of drug can deliver. This target—saturating dose may also be ed to as the maximally effective single dose of drug.

To investigate this possibility, we calculated the number of tumor HSP90 sites occupied by PU-H7 l per gram of tumor (HSP90 sites/g tumor), for a tracer dose (~6.5nyg) of [ml]—PU-H7l co- administered with therapeutic doses (5 to 75mg/kg or 5,000 to 75,000nyg), PU-H7ldm, of .

This was obtained using the formula: . A..." 1 (Hsp90 srteslg tumor): [755%]: - PU-H‘Hm 'W nmovg (4) WO 09657 Solving equation (4) for cmadministered doses of non—radioactive doses of PUH71, PU- H71mu, of 5, 25, 50 and 75 mg/kg (5,000, 25,000, 50,000. and 75,000ng/g, tively) yields the number ofPU-H7 1 molecules (in nmol) bound per gram of tumor. Because one molecule of ligand occupies the pocket of and binds to one HSP90 molecule97.l09 also yields the number of , equation (4) tumor HSP90 sites (in nmol) occupied by PU—H7l/g tumor (Figure 31c).

Analysis of the binding curve at 24h post-injection suggests that occupancy of the ble HSP90 sites is nearing saturation at a PU-H7ldme of 75mg/kg, with one gram of tumor containing a maximum of 160.7x10‘3 nmols ofHSP90 (Figure 31e, BMAX), corresponding to 960x10” HSP90 molecules. Using this value, we next calculated the percentage oftumor HSP9O sites occupied by different administered doses ofPU-H7l and determined that administration of 5, 25, 50 and 75mg/kg PU—H7 1 resulted in 12.1, 57.7, 88.7 and 92.7% of the available HSP9D tumor sites, respectively, being ed by the inhibitor (Figure 310. With near—complete saturation of tumor uptake achieved by a single therapeutic dose of PU—H7l of 75mg/kg, that dose has occupies most of the available tumor HSP9O sites and therefore increasing the dose fiirther would not be expected to result in increased amounts of drug localizing in tumor but would increase systemic drug exposure and potential patient toxicity.

In summary, analysis of the ‘maximum tumor dose’, as demonstrated here by [‘z‘H-PU-H7l PET, provides ation more le in trial design than the um tolerated dose’.

Specifically it would indicate a dose that results in maximal tumor (not whole body) exposure, for a single dose stered, leading to the best possible antiotumor effect while minimizing toxicity ated with a single dose administered. Furthermore, it suggests a new approach to selection of therapeutic dosing frequency, wherein once the maximal tumor dose has been fied, therapeutic dosing frequency could be increased to an endpoint ofmaximum tolerated frequency (rather than the conventional maximum ted dose, MTD), thereby maximizing tumor exposure temporally Use I-PU-H71 PET to dmign an efficacious dose regimenfor HSP90 therapy The preceding results demonstrated that [‘Z‘I]—PU-H7l PET can be used to predict the dose needed to achieve and maintain a specific tumor concentration over a selected time-period afier a single PU-H7l administration. Because tumors demonstrate prolonged retention of PU—H7l, ed administration of PU—H71 at sufficient frequency would ially result, with each successive dose, in higher cumulative tumor concentrations of PU-H7l. A steady-state tumor PU-H7l concentration, specific to the dosage and schedule used, would eventually be achieved. Hence, the data suggests a ial role for [mI]-PU—H7l PET g oftumor pharmacokinetics in guiding PUH7l dosing design, analogous to the use of plasma phannacokinetics in guiding dosing towards achieving steady— state plasma concentrations.

To explore this, we estimated the tumor concentrations of PU-H7l that would result upon administration of 5, 25, 50 and 75 mg/kg PU-H7l on a 3 administrations-per—week schedule (3xweek; MondayMednesdayfFriday) with weekends off (Figure 323). Simulations were performed to determine the tumor concentrations of PU~H7I when administered on this schedule and at the indicated doses (Figure 32a). The tumor AUC, and the average and the minimum tumor concentrations of PU—H7l ([PU-H7l]m and [PUH7l]mm, respectively) which resulted on this schedule and at these doses were also determined (Figure 32a, inset). Predicted PU—H‘7l trations in tumors ranged from [PU-H7l]mt.. = 0.17 to 2‘54uM and [PU«H7 l L“, = 0.49 to 7.45M, for administered doses of 5 to g, respectively (see inset Flgure 32a). [0286) As mentioned, in vitro exposure of IvaA—MB—468 breast cancer cells to lower PU—H7l concentrations (0.05 to lpM) results in potent cell growth inhibition with a reported 1C5.) of 60 to lOOan. At higher PU-H7l concentrations (>2uM), and when these cancer cells are exposed to the drug for 48h, massive cancer cell killing by apoptosis was noted (i.e. >70% cells oing apoptosis) (Figure 32b). In light of these in wire analyses, it is predicted that on the 3xweek schedule, administration of doses of 5 to 75mg/kg PU-H7l will result in therapeutically effective tumor drug trations that span values predicting mainly tumor tory effects (at 5 rug/kg) to potent tumor apoptosis (75mg/kg) (Figure 32b). Indeed, when mice bearing MDA—MB-468 xenografied tumors were treated with PU—H7l as described above, a dose-dependent response was observed in the PU—H71-treated tumors (Figure 32c). Afier a 7—week treatment period, a significant tumor response was noted with 63, 82 and 99% tumor growth inhibition (TGI) observed on the 5, 25 and 50 rug/kg doses, tively, and a 100% sion at the 75mg/kg dose e 32c).

We continued treatment until tumors in the control arm reached the maximum size permitted by our utional Animal Care and Use Committee (IUCAC) (Figure 32d) and sacrificed the mice on a ay (24h afler the last day administered dose). Only animals on the 5 mg/kg arm harbored tumors sufficiently large for analysis by n blot and LC—MS/MS (tumor volume: 139i66mrn3), gh cantly smaller than those treated with vehicle only (tumor volume: 1 l26i396mm3) (Figure 32d).

Solving equation (la) for administered doses of PU—H7l of 5 tug/kg and using the measured time-activity data for [”‘Il-PU—H7l in tumor (Figure 30d), yields the PU-H7l tumor concentrations over the treatment period (Figure 32c). Our simulations t that the tumor concentration of PU- H7l at the time of sacrifice should be 0.43uM (Figure 32c), which is remarkably similar to the actual concentration of 0.52i0.13uM determined in these tumors by LC-MS/MS (Figures 321', 32g).

Further, the observed pharrnacodynamic , namely HSP90 inhibition as demonstrated by significant Alct degradation (a 57% decrease; P=0.0017; Figure 321' and Figure 32g, leftpane!) and PARP cleavage (Figure 320 in tumors was consistent with a therapeutically effective PU—H7l concentration in tumors at this time (Flgure 32g, rightpanel). These findings show that the tumor uptake of PU-H‘l I remained unchanged over the 12 weeks of treatment, consistent with the persisting responsiveness of these tumors to PU-H7l (Figure 32d and Reference 100), indicating that [m1].

PUH71 PET may be used to monitor response persistence or conversely, the potential of acquired resistance to HSP90 therapy.

Use of1'”II-PU-H71 PETia design an efficacious schedule regimen for HSP90 therapy Considering the prolonged retention of PU-H7l by tumors, we asked whether less frequent administration of the agent would maintain its effectiveness. In the al setting, this may be useful as a rationale for continuing y at a lower dosage in patients encing ty or rationally balancing dosage and dose frequency in designing dosing schedules.

Simulations were performed for PUaH7l administered at 75 mg/kg on a le of 3- (Mon- i), 2~ (Mon and Fri) or 1- (Mon) administration(s) per week (Figure 33a). These calculations suggest that tumors will be exposed to a [PU—H7lmm,]m of 7.45, 5.41 and 2.88uM, on the 3xweek, 2xweek and lxweel: schedules, respectively (Figure 331:), indicating that the less frequent administration schedule should still r therapeutically effective concentrations to the tumor (Figure 33b). Indeed, significant tumor growth inhibition was obtained on the lxweek schedule, while the 2xweek administration led to tumor regressions over the 5 weeks of treatment (Figure 331:).

In the evaluable tumors (i.e. Control and 5 rug/kg treatment arms), changes in phamracodynamic markers Were ed when mice were sacrificed at 24 and 96h following the last administered dose.

Significant and near total depletion of coco—proteins (95% level decrease, P=0.0021 and P=0.0025 at 24b and 96h, respectively) was observed at both time points (Flgure 33d), suggesting that target saturating PU—H7l concentrations (>2pM) should be present in tumors at these time .

In these tumors, the PET-predicted tumor concentration of PU-H7l is 5.51 and 2.18uM at 24b and 96h, tively (Figure 33a, lxweek panel), which agrees closely with the actual tumor concentrations of 5.53i0.26 and 309$] .40, respectively, measured by MS (Figure 33c).

The methodology described above is readily applicable to human patients. One example is depicted in Figure 34 and Flgure 35. Figure 34 shows U-H7l PET/CT of a patient with recurrent pancreatic cancer with disease metastatic to lungs and adjacent lymph nodes. PET images at several times post«[mI]-PU-H7l injection (4, 24, 4s and ms) were quantified and SUV data obtained for ['Z‘I]-PU-H7l Were converted to trations for an administered dose of PU-H71 of 20mg/ml. These tumors show very good uptake of [[2‘I]-PU-H7l with retention and visualization even at l92h (8-days after administration). The ['2‘l]—PU—H7l PET/CT predicts that this t is likely to respond to HSP90 therapy. The ion of PU—H7l in the tumor for times beyond l92h (8 days) was not predicted by the mouse experiments, where as noted in Figure 30, ml—PU-H7l was cleared from the MDA—MB-468 tumors by 150h post-administration. Calculation 7l concentrations at various time points fiom 0 to 192h also allows for the calculation of a tumor area under the curve (AUC). In Figure 34, the area under the curve for the various lung nodules and lymph nodes (LN) is calculated from 0 to 192 hours.

The exposure of these ttunors to PU—H7l as detennjned by PU-PET allows for determining and optimal dose and schedule for treating this patient. Specifically, the re to PU~H71 of two characteristic tumors, one in the left lung and another in the right hilum LN over the time of the two- week treatment regimen when administered a dose of 20mg/m2 on a twice-week (Tue and Fri) schedule were calculated and represented as the nder—the—curve (AUC) and as an average tumor concentration e 35, top panel). The tumors show good drug exposure with AUCs values of 190 and @9th, respectively and average tumor concentrations of the Hsp90 inhibitor of 0.71 and 1.57 uM, respectively. In preclinical models of pancreatic cancer such concentrations were effective at inhibiting the growth, ng their invasive and metastatic potential and inducing apoptosis in pancreatic cancer cells, suggesting that the dose of 20mymz given on a 2xweek schedule (Tue-Fri) is likely to benefit the patient.

As also shown in Figure 35 (bottom panels, similar simulations were performed at a dose of 40 mg/m2 and BOmg/mz on a week (Tue and Fri) schedule. The most optimal as per the PU- PET prediction is a dose of 80mg/m2given at a 2xweek schedule (Tuesday and ), where the tumor re and the average tumor concentration over the 0336 days would reach above 760 and 1998uM-h and 2.8 and 6.3uM (Figure 35, bottom panel), respectively. These values are predicted by the preclinical studies (Figures 32 and 33) to result in significant regressions and cures.

Figure 36 shows similar calculations for a HER2+ breast cancer patient with disease metastasized to the lung. The top panels show results from the PET assay with [”‘l]—PU-H71. The middle and bottom panels sbow tumor concentrations of PU-H71 at various times based on an administered PU-H7l dosage of 10 mg/m2. The pharmacokinetic data is reported in terms of the AUC of the PU-H7l in the tumor between 0 or 336 hours or the average concentration of the drug in the tumor over that time period. The PU-PET data predict that when given twice a week for two weeks, a dose of lOmg/m2 would deliver a tumor AUCMM of 103 uM-h and maintain an average tumor concentration of 0.59uM and thus on this schedule, a dose of mg/m2 and beyond would be required for favorable response. When given three times a week for two weeks, a dose of 10mym2 would deliver a tumor AUCMM of 140.8nM—h and maintain an average tumor tration of 0.71ttM and thus on this schedule, a dose of 60mg/‘m2 and beyond would be required for favorable response. When given once a week for two weeks, a dose of lOmg/mZ would deliver a tumor AUC; thus on this am. of only 54uM‘h and maintain an average tumor concentration of 0.41 uM and scbedule, a dose of Zt‘lflmg/rn2 and beyond would be required for favorable se. When given 5 times a week (daily with weekend off) for two weeks, a dose of lOmg/m2 would deliver a tumor AUCMM of M-h and maintain an average tumor concentration of 0.81uM and thus on this schedule, a dose of 50mg/mZ and beyond would be ed for a favorable response.

WO 09657 Collectively, these data demonstrate the utility ]-PU—H7l PET in informing on the design of an efficacious dose and dose-schedule regimen for the personalized clinical use ofHSP90 therapies.

Tumor masure m PU-H71, as determined by [’“II-PU-Hfl per, reliably predicts anti-tumor response to HSP90 therapy Understanding the number of target sites that require inhibition over the time of treatment to result in cure remains a major challenge in targeted therapy. We therefore simulated, for the several dose and schedule regimens investigated above, the occupancy ofHSP90 sites by inhibitor over the period of treatment (Figure 3711). We then attempted to conduct a correlative analysis of tumor HSP90 occupancy with the observed anti-tumor response (Figure 371)).

The number of tumor HSP90 sites occupied by PU—H7l per gram of tumor (HSP90 sites/g) when PU-H7l is administered in therapeutic doses (5 to 75mgfkg or 5,000 to ng/g in mice) can be obtained using Equation (4). Because one gram of the MDA—MB-468 tumor contains a maximum of 10‘3 nmols ofHSP90 (Figure 31d, BMAX) and given that occupancy of more than 100% of the sites is not possible, we could simulate the occupancy ofHSP90 sites over the time of treatment for each dose and schedule regimen (Figure 37a).

Target occupancy, measured as the average %HSP90 sites occupied and recognized by PU- H71 ((% Occupied HSP90 sites)m) over the time of ent (Figure 37b). as the average tumor concentration ofPU-H7l recorded over the time of treatment ([PU-H7 1]""mavg; Figure 37c) and as the tumor exposure over the time of treatment as calculated by tumor AUC ated significantly well with the magnitude of the observed anti-tumor effect (r2=0.7559,0.8162 and 0.8188, respectively). Our analyses t that occupancy of over 80% of the tumor HSP90 sites averaged over the time of treatment (Figure 37b), or maintenance of an average tumor concentration of 5M of the HSP90 inhibitor e 37c), is needed for MDA-MB-468 tumor sion and cure. Lower occupancy however, as obtained by an average of 15% and above occupied sites or achieved by maintaining an average tumor concentration over the time of treatment of0.5M and above, may still lead to l response (Figures 37b, 37c).

Discussion Based on the foregoing analysis, we have ed and developed the first non-invasive sed assay with ial use in the clinical development of HSP90 inhibitors. We demonstrate its use in ining the intratumoral phannacokinetics and pharmacodynamics of the parent drug, in determining the dose of HSP90 inhibitor needed to achieve ive tumor concentrations, in assaying the actual concentration of the drug delivered to the tumor and in designing an efficacious dose and schedule regimen based on target modulation efficiency and not maximum tolerated dose (MTD). We also demonstrate its use in selecting patients most likely to have tumor response to HSP90-targeting.

Others have shown in preclinical or clinical studies that carbon 11-, fluorine 18— and nitrogen 13—labeled drugs, such as -methyl imatinib‘”, 3-N-methyl and a carbonyl-[NC]- temozolomidem, N»[2-(dimethylnmino)ethyl]acridine—4—carboxamide (["C]DACA)'“, [”‘F]5-FU”‘, [mF]fluorine derivative of dasatinib'” and [”N]cisplatin‘26 were useful to estimate by PET pharmacokinetic parameters for agents whose half-lives are cantly less than the total sampling time during the scan. For HSP90 inhibitors, whose tumor halfulives (i.e. >24h) are longer than the sampling duration permitted by these isotopes (i.e. 10min, 20min and 110min for ”C, ”N and ”F, respectively), PET pharmacokinetic tions using these radioisotopes are opriate. Because of the relatively long half-life of I2"I, the PU-H7l PET assay is thus the first reported assay that is able to non-invasively and quantitatively monitor the tumor pharmacoltjnetics of HSP90 inhibitors (Figure 38).

In addition, the U-H7l PET assay is a true materialization of the concept of targeted imaging for targeted therapy using radiolabeled biologically-inactive trace amounts of the targeted therapeutic agents for medical imaging'”"“. While the concept is well recognized and highly advocated for the future of drug development, providing a path towards personalized medicine, there is no precedent for the use of an oncology small-molecule therapeutic agent as an imaging agent to select patients most responsive to its drug action and to advise on the schedule of its administered dose Because of the presence of iodine in the native structure of PU—H71 and therefore the absence of any perturbation of its structure and biological behavior by incorporation of a radioiodine label, the PU-H7l PET assay is to our knowledge one of the first such assays. cally, the observed biodistribution profile of ['24I]ePU-H7l, namely tumor ion with rapid clearance from non—tumor , mirrors that of the therapeutic agent PU-H71. In addition, while formation of drug metabolites limits the application of labeled drugs for PET“, our data demonstrate that the ['“I]—PU-H7l PET both microdose and therapeutic—dose assay accurately measures tumor PU-H7l concentrations at levels, indicating that PU-H7l metabolites do not contribute significantly to the PET-measured tumor activities. Altogether, these findings suggest that [mI]-PU~H71 is well-suited for use as a true in viva ‘tracer‘ ofPU—H7 l.

In the development of targeted cancer therapy it is well understood that clinical trials require knowledge on whether effective tumor concentrations are achieved and whether the target is appropriately modulated. Our data demonstrate that the [mI]—PU'H71 PET assay quantitatively measures tumor HSP90 inhibitor phannacokinetics ng for tumor dose—tumor response correlations that are more informative than conventional tumor response ations with injected dose or plasma pharmacokinetics. Our data also demonstrate that for , tumor cokinetics mirror tumor phamacodynamics, identifying U-H7l PET as a non—invasive WO 09657 ement of both parameters. Thus, tumor pharmacokinetics, also indicative of target occupancy, have tive power in understanding both immediate (i.e. target modulation following a one dose inhibitor) and long-term (lie. target modulation over the designed schedule) response to an HSP90 inhibitor treatment regimen. In addition, by evaluating [Warn—n71 tumor pharmacokinetics in individual patients being ered for HSP90 therapy, one may also identify patients most likely to benefit from HSP90 treatment and consequently adjust the therapeutic dose and schedule, based upon tumor uptake and clearance of ['"I]-PU-H7l (Figure 38a).

We also show that the assay can guide the development of individualized dosing regimens based on PET-derived tumor HSP90 pharmacokinetics, as an imaging biomarker of the extent of tumor HSP90 targeting and its saturation by an HSP90 inhibitor therapeutic dose, with potential for predicting the anti-tumor effect and outcome over the course of HSP90 y e 38h). Due to these characteristics, the [morn—H71 PET assay is optimal for use in HSP90 inhibitor clinical studies as a pharmacometric tool for understanding (Figure 38a) and, potentially, predicting tumor se to HSP90 treatment (Figure 38b) The U—H7 1 data also es a more rational and effective dosing gy in HSP90- ed therapy based on PETederived maximally effective tumor tration rather the conventional maximum tolerated dose (MTD). This approach aims towards a new dosing goal of achieving a maximum tumor dose, an optimal dose level at which tumor targets are nearly saturated by drug, as visualized by PET (Figure 38c). This approach would avoid fiirther dose escalation that may only increase patient toxicity not anti-tumor efficacy. If tumor saturation is observed at a therapeutic dose less than a maximum tolerated dose (Ml'D) (as determined by a dose-escalation trial, for example), then a dosing strategy might pursue finding the maximum toleratedfrequency of dosing at the saturating maximally effective tumor concentration as determined by PET. [ml]—PU—H7l PET might explore the duration of tumor tion afler a maximally efl'ective tumor dose to further inform selection of dosing frequency. [mI]-PU-H7l PET might be similarly used to guide dose and frequency selection of other HSP9O inhibitors. The ability of a therapeutic dose of an HSP90-targeted agent to itively inhibit tumor—targeting by [ml]—PU—H71 tracer might provide an index of tumor drug saturation. For clinical development of HSP90 inhibitors, [ml]—PU-H7l PET might be used to gather tumor pharmacokinetic data from a Phase 1/2 trial population to derive a generally applicable dosing strategy or it might be used on an individual-patient basis for truly ‘personalized’ dose-schedule selection. PU-H7l PET imaging, we believe, is a clinical tool well suited to g these hypotheses. With a Phase 0 microdose trial of [mI]-PU-H71 currently undenNay, and the [ml]— PU-H71 PET assay being incorporated in an upcoming Phase 1 clinical study ofPU-H7l at Memorial Sloan-Kettering Cancer Center, this concept will soon be evaluated in a al setting.

In conclusion, [mI]-PU-H71 PET, as a targeted assay of tumor HSP90, may dramatically and rationally advance patient selection, dose selection, tumor sis and tion of tumor response WO 09657 at the level of the molecular target in HSP90-targeted therapy. As such, the novel ['ul]ePU-H71 clinical development and use of HSP90 assay should facilitate the rational, cost-effective, and optimal inhibitors in cancer. The use of ['“I]-PU—H71 PET represents a major advance in the design of clinical trials 0 inhibitors and promote the paradigm of targeted imaging phhnnacometrics for development of other targeted therapeutics. 52.2.2.2. [”‘n-PU—Dzn and [‘“Il-PU-Hzm {0307] Other HSP90 inhibitors with endogenous iodine where evaluated for their ability to perform in the HSP90 PET assay. Two such compounds e [‘2‘1].PU—Dz13 and ['Z‘I]-PU»HZISI, were synthesized. as shown in Schemes 16 and 17.

Jii'jflg h3g3 itiNST g n3 ” Wé fi ‘6 ¥ n) 0213 Sn-PU-0213 NH; 12‘! 7 NH; 124' 07 1,1ugiulSn-DZ-13inMe0H JL / JL / F N N F N N 2.[‘ MM24 4 TFA, 70°C 1 hr ‘ ‘ 3. Chloremine-T (on, 10 min g g 12“I-F'u-Iazu Scheme 16: Synthesis of [‘“Ii-PU—Dzn NH: 1 NH, g NH: )3 31 sflN N SQ N N \ o N \ N \ O ‘> . o) 1‘ /. >\~s N’ My [\N/ Ny N o) a b $ ' S _’ i HN Boo—N Boom PU—HZ151 NH2 12" NH: 124' “ii—SQkN’l 0) N/ 0) c SN d SN Boo-N HN 12"l-Pu—Hz15i1 SCHEME 17:3ynthesis of [”‘Il—PU-HZISI. Reagents and conditions: a. sun, , cnzch, n; b.

Pd(PPh3)a, hexamethylditin, dioxane, 90°C; 0. [‘“n—Nal,chlo1-amine—T, n, 10 min; d. TFA, 70°C, 60 min. [0308) The hemical yields ofthese nds were 36.96 a 12.97% ([‘”I]—PU-DZl3), 36.45 a 15.75-1/n ([mH-PU-HZISI) and 45.33 a 15.76% ([‘1‘11—PU—H7l); radioehernical purity (>98%) was confirmed by HPLC. The specific activities were 633 rnCi/pmol ([‘1‘11-0213), 576 mCi/urnol ([mn- H2151) and 1000 mCi/umol ]-PU—H7l). [03091 In order to assess the in vitro stability of [‘2‘11—PU.H71 and ['“I]-PU-DZI3, the compounds were incubated in human sera at 37°C over five days, and analyzed by l'I‘LC to determine if dehalogenation occurred. It was determined that both [ml]-PU-H7l and [mm-PU-DZB were stable (> 98%) over five days (120 h).

The tumor s ['“I]—PU—DZI3 when it is administered by both the IV and [P routes, however, tumor retention is higher by IV administration, with tical significance (P < 0.05) (24 h post administration, intraperitoneal versus intravenous routes). In the C5 713116] non-tumor bearing mouse. ['“11-PU-D213 quickly clears cardiac blood (“/oID/g was 3.33 a 0.13 at 2 min and 0.013 0.00 by 24 h post administration), and has the greatest uptake by the stomach and intestines (3-8% ID/g). Liver and spleen uptakes were not significantly different (P < 0.05), suggesting reticuloendothelial system (RES) involvement. The kidney uptake of [lJ‘I]-PU—DZI3, however, implied urinary clearance (%[D/g dropped from 5.53 i 0.34 at 2 min (data not shown) to 0.06 i 0.01 by 24 h post administration). In the -468 mac mouse model, [”‘I1—Pu—Dzn was retained by the tumor longer when administered by the intravenous route as compared with the inunperitoneal route. By IV administration, the tumor uptake of ['3‘11—PU-D213 (1.47 e 0.22 %ID/g at l h), slowly decreased (%ID/g 3 056 i 0.14, 4 h; 0.40 t 0.03, 12 h,- 0.09 i 0.03, 24 h) over 72 h (0.05 t 0.00 “/uID/g). As with nonemmor bearing mice, intestinal and RES uptakes of ['3‘I]-PU- oz 13, and renal clearance was seen in MBA-MD- 468. The in viva biodistn'bution of [mu—PU- DZlB, when inistered with PU-DZl3 at 25 lug/kg (24 h post administration, intraperitoneal versus intravenous routes), shows that PU—PET predicted tumor concentrations compare favorably with the values determined by LCMS-MS. [0311) In vivo PET g ed MDA-MB—468 tumors with [‘"I]-PU«H71 and [”‘I1-PU-Dz13 HSP90 inhibitors. Figure 39 shows the PET imaging results at 48 h post injection. of mice systemically injected with either inhibitor ([‘Z‘HrU—Hn or [”‘l1-PU-D213). Both radioiodinated HSP90 inhibitors detected the tumors with PET at each time point. The uptake %ID/g of the two inhibitors were not significantly different in the tumor masses, although PU-DZIB qualitatively appeared to have less cific abdominal uptake. [0312} Unlike [mu—Punzu and ['“I]-PU—H7l, [”‘HrU—Hzm could not detect the tumors in mice possibly because of its rapid metabolism by liver. .3. Treating cancer patients with PU-H'Il The methods described in Section 5.2.1. indicate that radiolabeled HSP90 tors such as [‘Z‘H-PUJ-WI can be used to identify patients that are likely to respond to HSP90 inhibition therapy and to design optimized dosing regimens for individual ts. The dosing regimens are based on such factors as tumor exposure of the inhibitor and occupancy of HSP90 by the inhibitor. These pharmacokinetic parameters are readily assessed using the abeled inhibitors of the t disclosure. Owing to the fact that pharmacokinetic data can be easily obtained from a large pool of individual cancer patients, We have the ability to detemiine a range of pharmacokinetic parameters that will be suitable for achieving a desired level of efficacy of a particular HSP90 inhibitor without concomitant toxicological problems caused by overdosing with the HSP90 inhibitor.

Accordingly, the sure further provides methods of treating patients with solid tumors, hematologic malignancies, and lymphomas with HSP90 inhibitors, particularly PU—H7l, to achieve a particular pharmacokinetic . The disclosure also provides methods of treating solid tumors, hematologic malignancies, and lymphomas with HSP90 tors, particularly PU-H'Il, by administering the inhibitor at particular dosage levels arid/or particular dosing schedules. In particular ments, the tumor to be treated with the HSP90 inhibitor is an “HSP90 ent tumor”. As discussed above, an HSP90 dependent tumor is a tumor whose physiology utilizes HSP90. An HSP90 dependent tumor contains a signifi cant amount of “oncogenic HSP90" relative to the normal housekeeping HSPQO. The methodology described in this section can be applied to numerous types of basal cell s including but not limited to colorectal cancer, pancreatic cancer, thyroid cancer, carcinoma, melanoma, renal cell carcinoma, bladder cancer, prostate cancer, lung cancer including small cell lung cancer, all cell lung cancer and arcinoma, breast cancer of all subtypes, neuroblastoma, gastrointestinal s including intestinal stromal tumors, esophageal cancer, stomach cancer, liver cancer, gallbladder , anal cancer, brain tumors including gliomas, lymphomas including follicular lymphoma and diffuse large B—cell lymphoma, multiple myeloma, including other plasma cell ers, leukemias, myeloproliferative neoplasms and gynecologic cancers including ovarian, cervical, and endometrial cancers.

In one embodiment, the sure provides methods of treating a human patient having a solid tumor, lymphoma or hematologic malignancy comprising administering a sufficient amount of PU—H7l to the patient to provide an ncy of 15% or greater of the oncogenic HSP90 in the patient’s tumor, an occupancy of 30% or greater of the oncogenic HSP90 in the patient’s tumor, an HSP90 in the patient’s tumor, or an occupancy of 60% occupancy of 50% or greater of the oncogenic in time between about or greater of the oncogenic HSP90 in the patient’s tumor at at least one point 16 hours to about 24 hours following administration of the drug. For instance, the stration of PU—H7l can provide an occupancy of at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or 100% of the oncogenic HSP90 in the t at at least one point in time between 16 and 24 hours ing administration of the drug. in one particular embodiment, the administration 7l can provide at least 60%, at an ncy of at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, least 70%, at least 80%, at least 85%, at least 90%, at least 95% or 100% of the oncogenic HSP90 in the patient at about 24 hours following administration of the drug. In another embodiment, the administration of PU-H71 can provide an occupancy of at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or 100% of the oncogenic HSP90 in the patient in the entire range between 16 hours and 24 hours following administration of the drug. PU-H71 can be stered to provide an occupancy of oncogenic HSP90 bounded by any of the two foregoing values at at least one point in time or in the entire range between about 16 hours to about 24 hours following administration of the drug, e.g., an of from about 30% to about 80%, an occupancy of from about 20% to about 80%, an occupancy ofbetween 40% and 90%, an occupancy of from occupancy of between 40% and 80%, an ncy about 50% to about 80%, an occupancy of from about 50% to about 90%, an occupancy of from about 60% to about 80%, an occupancy of from about 60% to about 99%, an occupancy of from about 50% 70% to to about 99%, an occupancy of from about 50% to about 99.9%, an occupancy of from about about 99.9%, etc. In another embodiment, PU-H71 is administered at the minimum dosage to achieve 100% occupancy of the oncogenic HSP90. As discussed in Section 5.2.1.1., administering PU—H71 to provide the above oncogenic HSP90 occupancies results in efficacious doses of . In one ular embodiment, the patient with the tumor is a human patient with an HSP90 dependent tumor.

In another embodiment, the disclosure provides methods of treating a patient having a solid tumor, lymphoma or hematologic malignancy comprising administering a sufficient amount of PU- H7l to the patient to provide a tumor concentration at 24 hours post administration in the range from about 0.3 M to about 7.5 M. For instance, the PU—H7l concentration in the tumor about 24 hours following stration of PU—H7l can be 0.3 M, 1 pM, 3 Md, 5 uM or 7 pM. PU—H7l can be administered to e a tumor concentration of the drug after about 24 hours between any of the two foregoing values, e.g., a tumor concentration fi'om about 1 pM to about 3 pM, a tumor concentration from about 1 pM to about 5 uM, a tumor concentration from about 3 M to about 5 pM, a tumor concentration from about 3 pM to about 7 pM, etc. In one particular embodiment, the patient with the tumor is a human patient with an HSP90 dependent tumor.

In another embodiment, the sure provides methods of treating a patient having a solid tumor, lymphoma or hematologic malignancy sing administering a sufficient amount ofPU- H7l to the t to provide a tumor concentration at about 48 hours p0st administration in the range of from about 0.05 MA to about 3.5 M. For instance, the PU-H7l concentration in the tumor at about 48 hours following administration of PU—H7l can be about 0.5 pM, about 1 pM, about 1.5 pM, about 2 M or about 3 pM. PU-H7l can be administered to provide a tumor concentration of the drug afier about 43 hours n any of the two foregoing values, 2.3., a tumor concentration from about 1 pM to about 2 M, a tumor concentration from about 1 pM to about 3 pM, a tumor tration fi'om about 0.5 pM to about 2 pM, a tumor concentration from about 0.25 pM to about 2 pM, etc. In one particular embodiment, the patient with the tumor is a human patient with an HSP90 dependent tumor. {0318] In another embodiment, the disclosure provides methods of treating a patient having a solid tumor, lymphoma or logic malignancy comprising administering a sufficient amount of PU- H7l to the patient to provide a tumor concentration of at about 24 hours post administration in the range of from about 0.3 pM to about 7.5 pM and at about 48 hours post administration in the range from about 0.05 MA to about 3.5 M. In one ular embodiment. the patient with the tumor is a human patient with an HSP90 dependent tumor.

As discussed in Section 5.2.1.1., the radiolabeled assay provides a convenient means of determining the tumor exposure of PU'H71. The tumor exposure can be measured using various cokinetic parameters such as AUC in the tumor and average tumor concentration of the drug over a particular time period. Based on a wealth of pharmacokinetic data gathered on patients with us solid tumors, we have determined that measuring the AUC and average tumor concentration over a particular treatment period (cg, two weeks) provides important information regarding the efficacy and toxicology of the drug. As discussed above, the term “tumor AUC” refers to the cumulative intracellular concentration of the drug over the time period from administration the drug to another point in time. For instance, the AUC in the water space of the tumor over a time period of 0 hours to 336 hours is referred to herein as tumor AUCMM... The “0” time point can refer to the time when the drug is first administered at the onset of a new treatment cycle. Alternatively, the “0” time point can refer to the time point the drug is administered in the middle of a treatment cycle. It will be understood that multiple doses of the drug can be administered at various times between the 0 time point and the 336 hour time point. As discussed below, tumor AUC values and efiicacious doses. average tumor trations that fall within particular ranges provide In one embodiment, the disclosure provides methods of treating a patient having a solid of PU— tumor, lymphoma or hematologic malignancy comprising administering a sufficient amount H71 to the patient that provides an AUCMM from about 150 to about 4,000 pM—h. For ce the tumor AUCMM of PU-H7l can be about 300 pM—h, about 500 pM-b, about 800 pM-h, about 1200 pM-h, about 1500 pM-h, about 2000 pM-h, about 3000 uM-h or about 4000 pM—h. PU—H7l can be administered to provide a tumor AUCMM between any of the two foregoing values, e.g., a tumor AUCMM ranging from about 300 uM—h to about 800 pM-h, a tumor AUCa.336h ranging from about 500 uM-h to about 800 vah, a tumor AUCmm ranging from about 500 pM—h to about 1000 uM—h, a tumor AUCMm. ranging from about 1000 uM-h to about 1500 pM-h, a tumor AUCMm ranging from about 1000 pM-h to about 2000 pM-h, a tumor AUCMM ranging from about 1500 pM—h to about 2000 uM-h, a tumor AUCMM g from about 2000 pM-h to about 3000 pM-h, a tumor AUCMM. ranging from about 2000 uM-h to about 4000 pM-h, a tumor AUCMM g from about 3000 Man to about 4000 uM-h, etc. In one embodiment, the “0” time point is the start of a new treatment cycle. In one particular embodiment, the patient with the tumor is a human patient with an HSP90 dependent tumor.

In another embodiment, the disclosure provides methods of treating a patient having a solid ofPU- tumor, ma or hematologic malignancy comprising administering a sufficient amount H71 to the patient that provides an AUCMW. from about 75 uM-h to about 2,000 pM-h. For instance the tumor AUCMm of PU«H71 can be about 75 pM-h, about 250 pM-h, about 400 uM-h, about 600 uM-h, about 750 pM-h, about 1000 uM—h, about 1500 uM-h or about 2000 pM-h. PU-H7l can be administered to provide a tumor AUCmm between any of the two foregoing values, e.g., a tumor AUCmm. g from about 150 uM-h to about 400 uM—h, a tumor AUCMW. ranging from about 250 uM-h to about 400 pM-h, a tumor AUCmm ranging from about 200 pM-h to about 500 pM—h, a tumor AUCHm. g from about 500 uM~h to about 750 pM-h, a tumor AUCmm ranging from about 500 pM-h to about 1000 pM—h, a tumor AUCMm ranging from about 750 uM—h to about 1000 uM‘h, a tumor AUCmm| ranging from about 1000 uM-h to about 1500 pM-h, a tumor AUCMsah ranging from about 1000 uM-h to about 2000 uM-h, a tumor AUCmm ranging from about 1500 M. 2012/045864 h to about 2000 pM-h, etc. In one embodiment, the “0” time point is the start of a new ent cycle. In one particular embodiment, the patient with the tumor is a human patient with an HSP90 dependent tumor.

In another embodiment, the disclosure provides methods of treating a patient having a solid sufficient amount of PU- tumor, lymphoma or hematologic malignancy comprising administering a H7l to the patient that es an AUCM“. from about 10 to about 300 pM-h. For instance the tumor AUCmn of PU—H71 can be about 15 pM-h, about 20 pM—h, about 25 pM-h about 30 pM—h, about 40 pM—h, about 50 pM-h, about 80 pM—h, about 100 pM-h, about 150 pM-h or about 200 pM—h.

PU-H71 can be administered to provide a tumor h between any of the two foregoing values, about 100 uM-h, a tumor AUCMH. ranging e.g., a tumor AUCME‘ ranging from about 10 pM-h to from about 10 uM-h to about 80 uM—h, a tumor AUCM“, ranging from about 15 pM—h to about 80 pM—h, a tumor AUCMgh ranging from about 15 uM-h to about 50 uM-h, a tumor AUCMgnranging from about 20 pM-h to about 50 pM«h, a tumor AUCM“ ranging from about 20 pM-h to about 40 pM—h, a tumor AUCMU“ ranging from about 20 pM-h to about 30 pM-h, etc. In one embodiment, the “0” time point is the start of a new ent cycle. In one particular embodiment, the patient with the tumor is a human patient with an HSP90 dependent tumor.

In another embodiment, the disclosure provides methods of treating a patient having a a solid of PU- tumor, lymphoma or hematologic malignancy comprising administering a sufiicient amount H71 to the patient that provides an average tumor tration of PU-H71 (referred to herein as [PU- H71]m) between 0 and 336 hours from about 0.5 M to about 7.5 M. For instance, the [PU—H71]_vg between 0 and 336 hours can be about 1 pM, about 3 pM, about 5 nM, or about 7 pM. PU—H71 can be administered to provide a 1]...E between any of two foregoing values, e.g.. a [PU—H71]...,E (measured between 0 hours and 336 hours) g from about 1 M to about 5 M, from about 3 M to 7 pM, born about 3 M to about 5 pM, etc. In one embodiment, the “0” time point is the start of a new treatment cycle. In one particular embodiment, the patient with the tumor is a human patient with an HSP90 dependent tumor.

In another embodiment, the disclosure provides s of ng a patient having a solid of PU- tumor, lymphoma or hematologic ancy comprising administering a sufficient amount H71 to the patient that provides an average tumor concentration of PUvH71 ([PU‘H71],VS) between 0 and 168 hours from abOut 0.25 M to about 3.75 M. For instance, the [PU-I~I71].,,g between 0 and 168 hours can be about 0.5 pM, about 1.5 uM, about 2.5 M, or about 3.5 pM. PU-H71 can be administered to provide a [I’U-H71].vll between any of two ing values, e.g., a [PU—H71].,,s (measured between 0 hours and 168 hours) ranging from about 0.5 M to about 2.5 M, from about 1.5 M to 3.5 M, from about 1.5 M to about 2.5 uM, etc. In one embodiment, the “0" time point is the start of a new treatment cycle. In one particular embodiment, the patient with the tumor is a human patient with an HSP90 dependent tumor.

WO 09657 2012/045864 {0325] As will be understood by a person skilled in the art, the total amount of PU-H7l that needs to be administered to achieve the desired enic HSP90” occupancy, tumor AUC or [PU-H71].m is dependent by both the route of administration and the dosing schedule. PU-H7l may be administered by s injectable routes including intravenously, subcutaneously, intramuscularly and intraperitoneally. Alternatively, PU—H7l can be administered orally.

In one embodiment of the present disclosure, PU-H71 is administered intravenously to a human patient having a solid tumor, lymphoma or hematologic malignancy at a dosage ranging from about 5 mg/ml to about 250 rug/m2 according to a dosing schedule selected from once weekly, twice weekly, three times weekly, four times weekly or five times weekly. In particular ments, PU« H7] is administered intravenously to a human patient at a dosage from about 20 mg/m2 to about 60 mg/ml according to a dosing le selected from once weekly, twice weekly, three times weekly, four times weekly or five times weekly. In particular ments, PU-H71 is administered intravenously to a human patient at a dosage from about 50 mg/ml to about 250 mg/m2 according to a dosing schedule selected from once weekly, twice weekly, three times weekly, four times weekly or five times weekly, In other embodiments, PU-H7l is stered intravenously to a human patient at a dosage from about 50 mg/m2 to about 100 mg/m2 according to a dosing schedule selected from once weekly, twice weekly, three times weekly, four times weekly or five times weekly. In other embodiments, PU~H71 is administered intravenously to a human patient at a dosage from about 75 mg/ml to about 200 mg/ml according to a dosing schedule selected from once weekly, twice weekly, three times weekly, four times weekly or five times weekly. In still other embodiments, PU-H71 is administered intravenously to a human patient at a dosage from about 75 mg/m2 to about 150 mg/m2 ing to a dosing schedule selected from once weekly, twice weekly, three times weekly, four times weekly or five times weekly. In one ular embodiment, the t with the tumor is a human patient with an HSP90 dependent tumor.

In preferred ments, PU-H7l is administered intravenously to a human patient having a solid tumor, lymphoma or hematologic ancy according to a dosing schedule of once weekly, twice weekly or three times weekly. In a particular embodiment, PU—H7l is administered intravenously in an amount ranging from about 50 mg/m2 to about 150 mg/m2 or from about 70 mg/m2 to about 125 mg/mZ according to a dosing schedule of twice weekly. In another particular embodiment, PU-H7 l is administered intravenously in an amount ranging fi'om about 20 mg/m2 to about 100 rag/m2 or from about 40 mg/m2 to about 80 rug/n12 according to a dosing schedule of three times weekly. In another embodiment, PU—H7l is administered intravenously in an amount ranging from about 90 mg/m2 to about 190 mg/mZ or fi'om about 100 mg/m2 to about 250 mg/ml according to a dosing le of once weekly. In one particular embodiment, the patient with the tumor is a human patient with an HSP90 dependent tumor. [0328) In one embodiment, PU—H71 is administered intravenously to a human t having a solid tumor, lymphoma or logic ancy according to a dosing schedule of once weekly or twice weekly for Mo weeks followed by one week off. In another particular embodiment, PU-H7l is stered at a dosing schedule of once weekly or twice weekly for one week followed by one week off. Alternatively, PU-l-I7l can be administered once weekly or twice weekly without any weeks off in between. .4. Assessing pathways and oteins dependent on HSP90 for prognostic and diagnostic applications [0329} As discussed in Section 5.1., information on the ratio between “oncogenic HSP90” and normal HSP90 in cancer cells can be used to determine the contribution of HSP90 in the survival and proliferation of the cancer cells. Additionally, we have identified particular proteins and pathways that often depend on HSP90 for survival. Identification of the expression levels of these proteins and or these pathways in the cancer cells of a patient can provide important information on the role of HSP90 protein in the patient’s cancer, particularly when assessed against patients who have responded to HSP90 therapy. Accordingly, the present sure provides methods for determining whether a human cancer present in a patient will likely respond to therapy with an HSP90 inhibitor which comprises (a) obtaining a sample containing cells expressing HSP90 protein from the patient’s cancer; (b) ing for the cells present in the sample the presence of at least one of the following parameters: an activated AKT pathway, a defect in P’I‘EN tumor ssor function or expression, an ted STATS pathway, or a 3ch family member, such as Bel-XL, protein expression; and (c) comparing the assessment ed in step (b) with a predetermined reference assessment of the same parameter or parameters assessed in step (b) for human cancer cells from one or more cancer t(s) who responded to therapy with the HSP90 inhibitor so as to thereby determine whether the patient’s cancer will likely d to therapy with the HSP90 inhibitor. In particular embodiments, the cells are breast cancer cells or acute myeloid ia (AML) cells.

Despite of the large number of potential new agents entering clinical evaluation every year, only 5% to 8% ever reach registration. Of particular concern is the high rate of failures in Phase 3, where an estimated 50% ofoncology agents are stopped in development. Such failures are especially expensive and e many patients of potentially more effective treatments. These dire statistics clearly speak for the need to discover and implement tive kers for patient selection and trial enrichment.

What have we learned from the results obtained in the last years with targeted agents? When a new drug has been administered, either as a single agent or in addition to herapy in a study population not selected by any biomarker, most trials have produced negative results, while in a small minority of Cases a statistically significant benefit has been demonstrated. This benefit, however, consisted, at best, of a small or moderate absolute prolongation of overall survival. On the other hand, examples of a greater absolute t obtained with the use of ed agents based on a biomarker— driven patient selection are constantly increasing. Biomarkers provide the possibility to use tumor and patient characteristics to integrate an accurate predictor of efficacy with a specific ism based therapy, guiding the selection of treatment for each individual patient. In particular, a validated predictive marker can prospectively identify individuals who are likely to have a positive clinical e from a specific treatment.

The t disclosure recognizes these issues and proposes to develop and validate biomarkers for a biomarker—driven patient selection and trial enrichment in the implementation of HSP90 inhibitors into the treatment of cancers. .4.1. Markers predictive of apoptotic ivity to HSP90 in breast cancer Depending on the genetic making of the tumor, either a cytostatic or a cytotoxic effect may result from HSP90 inhibition in BC. In clinic however, a highly apoptotic not a atic response to ent is most desired. Thus, to identify the breast cancer tumors more likely to undergo apoptosis when challenged with PU—H7 l and other HSP90 inhibitors, we have conducted inary studies in cell lines to fy molecular lesions that are associated with highest apoptotic response upon HSP90 inhibition.

These studies propose an directed tion of activated Akt and elevated Bcl—xL and/or 3ch and/or Mcl-las major elements conferring apoptotic sensitivity of tumors to HSP90 inhibition (i.e. biomarkers predictive of se). A person skilled in the art will appreciate that measurement ofan activated Akt pathway may require measuring expression and/or phosphorylation status of one of more proteins associated with this pathway, such as but not limited to Akt, 56, PRAS40, Bcll, mTOR, IKK1 NFkB. Detailed information on the Akt pathway and its activation may be found online in the KEGG PATHWAY database; and the al Cancer Institute’s Nature Pathway Interaction Database. See also the websites of Cell Signaling Technology, Beverly, Mass; BioCarta, San Diego, Calif; and Invitrogen/Life Technologies Corporation, Clarsbad, Calif. This pathway is composed of, but not restricted to l-phosphatidyl-D—myo-inositol 4,5-bisphosphate, 14—3- 3, l43-Cdknlb, Akt, BAD, BCLZ, BCLZLl, CCNDl, CDC37, CDKNlA, CDKNlB, citrulline, CTNN'Bl, EIF4E, EIF4EBP1, ERKl/Z, FKHR, GABl/Z, GDFlS, Glycogen synthase, GRBZ, Gsk3, Ikb, IkB-kaB, IKK (complex), ILK, in, JAK, L-arginine, LIMSl, MAPZKl/Z, MAP3K5, MAPSKB, MAPKBIPl, MCLl, MDM2, MTOR, NANOG, NFkB (complex), nitric oxide, N083, P110, p70 86k, PDPKl, phosphatidylinositol-3,4,5-tn'phosphate, PI3K p85, PPZA, PTEN, PTGSZ, RAFl, Ras, RHEB, SFN, SHCl (includes EG:20416), , Sos, THEM4, '1'P53 (includes EG:22059), TSCl, Tscl-Tsc2, TSC2, YWHAE WO 09657 A person skilled in the art will appreciate that measuring the expression of one or more Bel-2 family anti-apoptotic molecules, such as Bel-2, Bel-XL, Mclml may be ary to appreciate the contribution of this anti~apoptotic family.

While studies in established BC cells provide valuable information on the BCs more likely to respond to HSP90 therapy, cultured cells cannot entirely recapitulate the real clinical e.

Experimental models of breast cancer encompass a very small number of cell lines, which were developed several decades ago. Although some cell lines retain most of the original features, we found a discrepancy in levels of HSP90 and other features, between patient samples and cell lines, which in part can be a consequence of culture stress. In addition1 cultured cell lines do not recapitulate the effect the environment has on tumor cells. All together, we believe that primary explants can resemble the tumor features and response to treatment more faithfully than cell lines.

Thus, to address the on: “What is the spectrum of BC tumors most ive to HSP90 therapy7”, our studies include evaluation 71 in clinical breast cancer tumor specimens obtained from die-identified ogy discards.

In these samples we established a correlative relationship between sensitivity of BC tumors to HSP90 inhibitors and expression of select biomarkers. The following phase would be to move forward and e to use this for patient selection in the next trial. Once the scoring system is defined and ted, patient ion could ultimately be done on FFPE or Cl‘Cs to correlate the marker of interest with predicted response. Such diagnostic measures can then be introduced as common practice in selection of BC tumors more likely to respond to HSP90 therapy, in the same fashion HERZ—scoring is used to guide patient selection for Trastuzumab therapy.

Test a vivo the sensitivity of BC samples to PU—H7l. Fresh tissue sections of BC patient tumors are exposed ex viva to PU-H71 to assess the overall sensitivity of cancer cells and effect on normal cells (:12. vessels, benign ducts if present in section). Concentrations of PU»H71 and with this agent, where it was exposure times are based on both prior in vilra and in viva PK analyses determined that up to micromolar concentrations of PU—H7l are delivered to and ed into tumors at 24h and 48h post administration. Response are determined by one or two measures: 1. quantification in H&E stained s of cells exhibiting morphologic changes indicative of apoptosis and 2. quantification of positive cells. t tissue procurement: pathology discards from de-identified samples and needle core es during HSP90 inhibitor trials are obtained in accordance with the guidelines and approval of the utional Review Board. The freshly procured tissue will be used immediately.

Ex vivo sensitivity examination: Immediately following surgical l of the mastectomy specimen the tissue is transported to the Tissue Procurement Services (TPS) area of the Pathology suite. Once the lesion is located, tissue is harvested under sterile conditions. The specimen size removed for evaluation is lly S-IOmm x 5—10 mm. Every effort is made to sample the most viable area. Distant from the lesion, a specimen of equivalent size is removed representative of normal breast epithelial tissue. Both specimens are placed in minimal essential media (MEM) with 1% penicillin/streptomycin. A small portion of the lesion and the entire piece of normal breast epithelial tissue o a “snap” freeze for future molecular evaluation by WB. The remaining portion of the lesion (mastectomy) is processed for pathological evaluation. For every lesion pathology provides IHC for receptor status, proliferation s, epithelial markers and one hematoxylin/eOsin (H&E) stained slide accompanied by 10 unstained to be further assessed for non- standard biomarkers (e.g. pAKT, Bcle, HSP90 and Hsp70).

From preliminary analyses we have learned that fresh tissue slicing provides a quick and ion. In more efficient ex vivo method for HSP90 inhibition evaluation than primary cell ' addition, it preserves the cancer cells in the endogenous environment of the surrounding . This is important since the interaction between stromal cells and tumor cells is known to play a major role in cancer growth and ssion. In this method, the tissue (i.e. lesion) is placed in a plastic mold and embedded in 6% agarose tissue is then mounted on the stage of the . The agnrose-embedded Vibratome that is submersed in a chilled reservoir (for tissue preservation) containing MEM with 1% penicillin/ streptomycin. The tissue is then sliced using metal blades producing serial sections of the lesion that are 200 pm thick. Each section (minus the surrounding agarose-embedding media) is immediately placed in a 24-well tissue culture plate containing MEM with 1% penicillin/streptomycin. From a 5mm x 5mm piece of tissue approximately 25 sections are produced.

This allows for replicate analyses of tissue sections treated with a minimum of 4 doses of the HSP90 inhibitor and one with vehicle only Replicates can be assayed by both IHC as well as viability assays atic plate reader or cytospin preparation) once tissue section undergoes enzymatic dissociation by brief exposure to dispase.

To date, forty—two specimens encompassing all BC subtypes have been acquired. Out of these, nine were of receptor status negative. Both primary tumors (PT) and lymph node metastases (LN) (if t) have been ed where “fresh tissue sections” 200 urn thick were exposed to sing doses of PU-H7l. Treatment of triple negative infiltrating ductal oma (lDC) with PU—H71 attained, in a dose-dependent , apoptosis of both the primary tumors and lymph node metastases. Interestingly, LN mets appear more ive to PU—H7l than the corresponding PT.

Most significantly, normal (e.g. vessels, lymphocytes) and benign (e.g. ducts, lobules) tissue remained unaltered following a 48 hour exposure to PU-H7l. Data show a ring ofthe TN'BC PU~H71 cases in 4 distinct sensitivity groups: very sensitive, with 100% apoptosis noted at 0.5uM (top curve, Figure 40 A), sensitive with 100% apoptosis at luM PU-H7l (middle curve, Figure 40 A), partly resistant with ~50% apoptosis noted at M (bottom curve, Figure 40 A) and resistant (PT#14, no apoptosis noted at any of the tested concentrations, not shown).

As noted in Figure 40]], several tumors are more sensitive to HSP90 inhibition than predicted from the preliminary data generated on cell lines. Specifically, while -46B is one of the most sensitive breast cancer cell line (Caldas et al PNAS 2009), the studies presented in this invention show that tumor cells ofmuch higher sensitivity to PU—H7l can be fotmd in primary specimens obtained from human breast cancer patients. Specifically, while a 43h treatment of 0.5uM PU—H7l is ed in the MDA—MB-468 cells to observe about 50% ofthem oing apoptosis, we find that for several HER2+, triple—negative and ER+ breast cancers, trations as low as 0.05 pM induce a similar effect. In addition, while 3 43h treatment of about 5uM PU-H7l is ed in the MDA— MB468 cells to observe about 100% of them undergoing sis (Figure 32b), we find that for several 12+, -negative and ER+ breast s, concentrations as low as 0.5 uM induce a similar effect. Such s provide information on the required tumor concenn‘ations of PU~H71 that are expected to provide a eutic effect.

Investigations in GI and pancreatic cancer resulted in similar gs. .4.1.1. Investigate the expression of proposed biomarkers by [HC and WB, and score samples by biomarker expression.

H-IC scores s based on low to high expression of HSP90, Hsp70, p—Akt/Akt and BclxL.

Adequate negative controls are obtained by replacing the primary antibody with antibody dilution buffer. HSP90, p—Akt/Akt, Bcl-xL and Hsp70 staining intensity will be scored (2 times) for each specimen on a scale oft) to 3, in which 0 represents negative, 1 weakly positive, 2 moderately positive, and 3 ly positivesstaining While IHC alone could be somewhat problematic since scoring is oflen quite subjective, its use in parallel with a second method like Western blot will "min" and validate the IHC to make correlations with the ex vivo and in patient response. If the amount of protein obtained is not enough for the classic membrane-WE, the ultra-sensitive capillary—WE logy is used. A typical core needle biopsy specimen yields between 20 and 40 mg of tissue, which is sufficient for the proposed IHC, and potentially for capillary WB analyses. This information will be analyzed in the context of clinical response, guiding on the validity of the proposed scoring method. Namely, we will have the ability to correlate clinical response with response predicted by biomarker evaluation. Once the scoring system is defined and validated, patient selection could be ultimately done on FFPE to correlate the marker of interest with predicted response. Such diagnostic measure can then be introduced as common practice in selection of TNBC tumors more likely to respond to HSP90 therapy, in the same fashion as HERZ-scoring is used to guide patient selection for Trastuzumab therapy.

For some patients, es may not be possible, either due to an inaccessible tumor (deep internal metastasis) or no granted consent. For these cases, we will aim to probe whether circulating tumor cells (CTCs) harvested from the blood may be of informative value. Depending on the stage of the disease, for advanced~cancer patients, we expect to recover between 1,000 and 10,000 BC cells with this technique. This number of cells is enough for capillary—WE analysis of proteins (or real time qPCR analysis, if needed) and for scoring of HSP90, Hsp70, p—Akt and Bcl—xL expression by immunomagnetic enrichment then flow try.

Figure 40 shows that breast cancer tumors with activated Akt, as evidenced by high staining with phospho-Akt, Ser473, are also those very sensitive to HSP90 inhibition. .4.2. Determinants of apoptotic sensitivity to HSP90 inhibition in Acute Myeloid Leukemia (AML) Targeted therapies that are designed to induce apoptosis in leukemic cells are the most promising anti-leukemia strategies. We explored biomarkers predictive of apoptotic sensitivity to heat shock protein 90 (HSPQO) y in AML. We found that addition of HSP90 inhibitors to a panel of genetically distinct AML cell lines potently inhibited cell growth and induced the degradation of several AML cell—specific onco-proteins such as mutant FLT3, TEL-TRKC, AMLl - ETO, mutant c~KIT and mutant JAKZ. Notably, the vity for these cells to undergo apoptosis upon HSP90 inhibition varied considerably. The most sensitive cell lines were , MV—4~ll and M0-9l cells, and for each of these cell lines we observed near 100% killing of the initial cell population afier 48-72 h ofHSP90 inhibitor treatment. In st, only 20% death was seen in HEL and HL-60 cells under these conditions. We next made use of specific inhibitors of known oncogenic signaling pathways known to be ulated in AML to demonstrate that apoptotic sensitivity of AML cells to HSP90 inhibition ated with PISK—Akt and STATS activation, but not with activation of the RafuMAPK pathway. Irnportantly, similar results were observed in cells lines, xenograf’t models and isogenic cell line systems. We also found that dual activation of these two pathways, even in the context of Bcl-xL overexpression1 lowers the apoptotic old ofAML when HSP90 is inhibited. Taken together, our findings suggest that AML patients with tion of Akt and STATS signaling are most likely to benefit from HSP90 inhibitor therapy, and clinical trials should aim to enroll patients with specific activation of these important signaling pathways.

Irnportantly, 50- 70% of patients with AML display phosphorylation of both Thr3 08 and Ser473 Akt. This molecule contributes to proliferation, survival and drug resistance in AML, and is associated with adverse e. Taken together, our findings suggest that AML patients with activation of AKT and STATS signaling are most likely to benefit from HSPQO tor therapy, and clinical trials should aim to enroll patients with c activation ofthese important ing pathways. .4.2.1. HSP90 inhibition induces cell-type specific killing in AML cell lines A number of chemically distinct small molecule HSP90 tors have been reported, and several are in al or late-stage preclinical igation (Chiosis et a1., 2008). Among these are the ansamycin natural product derivatives l7—AAG and l7-DMAG, and the synthetic compounds GNP-2024 (BIIBOZI) and PU'H71, all in clinical evaluation, and PU-DZIB, a close derivative of PU- H71 in pic-clinical pment.

To evaluate the sensitivity um ofAML cell lines to HSP90 tors, and to investigate a posxible relationship between their genetic background and induction of apoptosis by HSP90 therapy, we made use of a varied cell panel. Specifically, we chose Kasutni—l and SKNO—l, cell lines that contain the AMLl-ETO fiision and the mutated c-Kl'l' (N822K) ns; MOLM-13, a human cell line established from the peripheral blood of a patient at relapse of AML which had evolved from MDS, that contains both the MLL-AF9 fusion protein and the FLT3 11'D mutation; M0-9l that contains the TEL-TRKC fiision protein and also harbors a constitutively activated STATS; and finally, HEL that contains the JAKZ V61 7F mutation. The HSP90 inhibitors potently inhibited, in a dose- and cell-dependent manner the growth of each tested AML cell line and also induced cell killing, with notable differences observed among the cell lines. Most sensitive were the MOLM-13 and M0-9l cells where 100% killing of the initial cell population was noted after 72 h, followed by Kasumi—l and SKNO-l , with 50-80% and HEL with 20%. Nominal peripheral blood leukocytes were unaffected at similar concentrations.

The ability of the distinct HSP90 inhibitors to kill the AML cell lines was r, suggesting cytotoxicity of the compounds occurs through a common mechanism of action, namely HSP90 inhibition. .4.2.2 HSP90 inhibition induces apoptosis in AML cells PU-H71 was thus chosen to further investigate the mechanisms accountable for AML cell- g by HSP90 inhibitors. As evidenced by dual acridine orange/ethidium bromide ng, PARP cleavage and activation of caspase 3,7, cytotoxicty of PU—H'Il in AML occurred mainly through induction of apoptosis. The number of cells undergoing apoptosis after 72 h of treatment with PU— H'l’l, neared 100% for 3 and M0-9l, 50-60% for SKNO—l and —l, and 30% for HEL, values in good ent with the observed cell killing. A ten-fold, in MOLM-l3 and M0-9l, and two-fold1 in Kasumi-l and SKNO—l, increase in caspase-3,7 activation was observed as early as 24 h. ially no live cells were ed for the M0-9l cell line afier 48 h of HSP90 tor treatment.

In the most sensitive cells, MOLM—13 and M0—91, apoptosis was associated with downregulation of the anti-apoptotic molecule Bcl-xL. .4.2.3. Inhibition of HSP90 depletes key AML onto-proteins but this effect fails to correlate with apoptotic sensitivity The distinct apoptotic sensitivity of AML cell lines towards HSP90 inhibitors could be due to effective HSP90 tion in certain cell lines but ineffective in others. To test this hypothesis we evaluated the effect of PU—H7l on two proteins demonstrated to be HSP90-dependent in a majority of and markedly reduced cancers, the RAF—l and AKT kinases. The HSP90 inhibitor dose-dependently the steady—state levels of these proteins in all the tested cells. This is in accord with the established mechanism y HSP90 is required for the stability and function of these kinases in cancer cells.

In on to these “pan-cancer” HSP90 client proteins, PUeH71 also led to the degradation of specific leukomogenesis drivers, such as mutant FLT3 in MOLM-l3, TEL-TRKC in M0-9l, AML1~ETO and mutant cKIT in Kasumi-l and SKNO—l, and mutant JAK2 in HEL (AML-cell AMLl-ETO specific HSP90 once-clients). Mutant FLT3, cKIT and JAK2, and the fiision protein AML or other transformed cells. The were previously reported to be sensitive to HSP90 inhibition in fiision protein TEL»TRKC however, is a novel client of HSP90, as ted by our s showing line potent degradation ofTELJI‘RKC by PU-H7l in the M0—9l cell Collectively, our findings indicate that the HSP90 inhibitors deplete AML cells of key malignancy driving ns, including the 'two hits’ postulated to be necessary events for leukemogenesis, but no correlation is evident between this effect and the ability of HSP90 inhibitors to induce apoptosis in AML cells. .4.2.4. Apoptotic sensitivity to tion of HSP90, PI3K/AKT and JAR/STAT pathways overlaps in AML cells Because a relationship between the genetic p and the apoptotic sensitivity to HSP90 anti- inhibition is not t, a potential answer could lie in the functional differences that lead to an apoptotic phenotype or in a differential expression of certain anti—apoptotic molecules among these cells. Three main pathways have been linked to regulation of apoptosis in AML: the PISK/AKT/N'FkB, the JAR/STAT and the ras/MAPK ys. More importantly, HSP90 regulates several key molecules along these pathways, and tion of HSP90 can lend to combinatorial inhibition of these molecules, such as p~AKT, p-STAT and p—ERK.

To probe the significance of individual pathways to apoptosis in AML cell lines, we used specific small les, such as the Akt inhibitor VIII, a quinoxaline compound that potently and selectively inhibits Aktl/AktZ activity , the MAP kinase MEK inhibitor PD98059 (MEKi) the pan-Jak inhibitor 2-(1,l-Dimethylethyly9-fluoro-3 ,6-dihydIo-7H-benz[h]-in1idaz[4,5- flisoquinolin—7-one (JAKi)). We also ed the AML cell pool by the addition of thee additional lines: HL60, a widely studied promyelocytic cell line positive for myc ne expression, THl’J, 21 cell line that came from the peripheral blood of a one-year old infant male with monocytic AML, and MV4-l l, a cell line that contains a 4;11 translocation and a FLT3 lTD mutation.

The number of apoptotic cells upon ent with the AKT, JAK and MEK inhibitors (AKTi, JAKi and MEKi) and the HSP90 inhibitor PU-H7l, was quantified at 24, 48 and 72 h following the addition of c inhibitors. Modest or little induction of apoptosis ensued upon the on of the MEK inhibitor. AKT and JAK inhibitors on the other hand, had variable but potent effects on sis. Analysis of apoptosis indicated that cells sensitive to AKTi were also the ones most likely to apoptose when HSP90 was inhibited (slope = 0.9023 1 2), suggesting that apoptotic sensitivity to HSP90 inhibition potentially correlates with sensitivity to PI3K/AKT pathway inhibition in AML. The correlation between JAK/STAT pathway and HSP90 inhibition was also good (slope = 0.8245 d: 0.1490), gh two cell lines, MV4—11 and THP-l were clear outliners.

These findings suggest that AML cell addiction for survival on either of or both the PISK/AKT and JAK/STAT pathway correlates with and potentially dictates apoptotic sensitivity to HSP90 inhibition. .4.2.5. cs and potency of in viva inhibition of AKT and STAT5 SP90 inhibition correlate with tumor apoptosis We next analyzed the phannacodynamic effects of HSP90 inhibition in both HEL and M0-91 tumors afled in mice. Unlike the cultured cells, the use of the in vivo model allows real-time monitoring of dependent pathways inhibition. Because pathways most dependent on HSP90 are also most sensitive to its pharmacologic inhibition, they remain inhibited by PU-H7l for the longest period of time in tumors. Accordingly, in the M0-91 tumors that harbor elevated p-AKT and , and appear addicted to the activation ofboth pathways, PU—H7l d marked apoptosis.

Apoptosis in M0—9l lasted for 96 h post—administration of one dose of PU-H7l, mirroring the potent inhibition of both AKT and STAT. Highest level of cleaved PARP was observed in the al of 12-72 h post-PU—H7l administration, when both p-AKT and p—STATS levels were reduced by 70 to 100% of the initial levels. The effect ofPU—H7l on PARP declined by 96 h, when p-AKT, but not p- STATS, recovered to baseline levels.

HEL afied tumors were less sensitive that M0—9l tumors to apoptosis induction by PU» H71. Under culture ions, HEL cells express elevated p-STAT5, and inhibition of the JAK/STAT pathways by the JAKi or by PU—H71 commits 20-30% of cells to undergo apoptosis. The AKTi on the other hand, has little to no effect in these cells. Accordingly, limited PARP cleavage and caspase-3 activation is noted upon HSP90 inhibition in these cells.

Nonetheless, and in contrast to tissue culture, when xenografied in nude mice, HEL cells demonstrate low to te expression level of p-AKT. This is not surprising, as it was reported that AKT activity can be stimulated in AML cells by the environment, such as by cytokines, and in vivo tumors may be more addicted on AKT activity for survival because of stressors unique to tumor tissue, such as hypoxia, acidity and al vascularization. Elevation of AKT ty in xenografted HEL cells appears to be necessary for tumor survival because PU—I-l7l induces markedly higher apoptosis in HEL tumors than in cultured HEL cells. As with M0—91 tumors, highest level of cleaved PAR? was observed when both p—AKT and p—STATS levels were reduced by PU—H7l by 70 to 100% of the l levels (in the interval of 12-48 h post-PU-H7l administration). Cleavage of PARP diminished significantly when p—AKT but not p—STATS recovered to baseline levels (72 h).

Collectively, our data suggest that the apoptotic activity of HSP90 inhibitors in AML correlates with and is a measure of downregulation of the activated p-AKT and p—STATS species.

Besides p-Akt [i.e. Ser 473] the activation state of the Ala—pathway can be determined as a measure of the orylation status of 56, 56k or mTOR. also downregulated by PU-H7l treatment. observations also imply that additive ion of AML cells to AKT and STAT-pathway activation also renders them more sensitive to HSP90 tion. 6. HSP90 inhibition induces apoptosis in cells addicted for survival on the PISK/AKT and JAK/STAT pathways\ To demonstrate this hypothesis we made use of FLS. 12 isogenic cell lines. FL5.12 was derived as an interleukin-3 (IL-3 )«dependent cell line with a functional JAK/STAT pathway and it has characteristic features of an early lymphocytic progenitor. Both the parental cells and the transfected cells express moderate-levels of active AKT and STAT5, as evidenced by AKT orylation on Ser473 and STAT5 phosphorylation on Tyr694, respectively. The level of p—STATS but not p~AKT is dependent on the ce of IL-3. Introduction of a constitutively ted, oylatcd form of AKT (mAKT) under the control of a doxycycline (DOX)—inducible promoter further allows for the regulation of p-AKT levels in these cells. All together, these cells are a good isogenic model to evaluate the dependence of HSP90 inhibitor apoptotic sensitivity on activated AKT and STATS- pathways.

When the ruAKT-transfected cells were treated with the AKTi, an increase in apoptotic cells from 5-7% to 15—20% was noted. This value reflects the contribution of the endogenous vaKT to the survival of these cells. When AKT ty was increased by addition of Box, cells became more addicted to AKT for al and the AKTi led to 30% apoptotic cells (P = 0.015).

When the mAKT-transfected cells were treated with the HSP90 inhibitor, approximately 35- 40% tic cells were detected. This value reflects the combined contribution of the nous p-AKT and p—STATS to the survival of these cells. Further increase in p—AKT levels by Dox, led to an increase in apoptotic cells from 35-40 to 50% upon PU-H71 addition.

Together these findings demonstrate that apoptotic sensitivity of AML cells to HSP90 inhibitors is a reflection of the cell’s addiction for survival on the AKT and STAT-pathways. .4.2.7. Bel-XL overexpression fails to inhibit the apoptotie effect of HSP90 inhibition in AML Constitutively high levels of BchL have been associated with resistance of leukemia cells to various categories of herapeutic agents. We therefore investigated whether introduction of Bcl—xL would overcome dependence of the FLSJZ transfected cells for sturdval on AKT and STAT and would render them resistant to inhibition of these pathways by PU—H71. To investigate this hypothesis, we made use of FL5.12.mAKT cells stably transfected with an expression vector containing the apoptotic inhibitor Bcl-xL. These cells remain dependent on IL-3 for proliferation in vitro. In these cells, similar to M0—91 cells, a itant activation of STATS and AKT—pathways and overexpression of Bcl-xL is ed. As the case in M09], HSP90 inhibition by PU-H7l led to a reduction in the activity and steady~state levels of these proteins and retained its apoptotic effect. .4.2.8. Discussion e of the large number of ial new agents entering clinical evaluation every year, only 5% to 8% ever reach ration. Of particular concern is the high rate of failures in Phase 3, where an estimated 50% ofoncology agents are stopped in pment. Such failures are especially expensive and deprive many patients of potentially more effective ents. These dire statistics y speak for the need to discover and implement tive biomarkers for patient selection and trial enrichment. Our study addresses this problem in AML and indicates that apoptotic sensitivity to HSP90 inhibition correlates with accumulative addiction of cells for survival on signaling pathways with anti-apoptotic roles. We fy activated Akt and STAT as major ys in this regard.

AKT signaling is frequently activated in acute AML patient blasts and strongly contributes to proliferation, survival and drug resistance of these cells. From 50 to 70% of patients with AML display phosphorylation of both Thr308 and Ser473 AKT. Both the disease-free and the overall survival time for patients demonstrating AKT activation was significantly shorter when ed to patients with no AKT activation, collectively suggesting that AKT-inactivation may be a powerful gy in AML. HSP90 tes this pathway and several of its key elements, likely in a transfonnation—dependent manner. Accordingly, a significant correlation was observed between the expression ofHSP90 and that of pAKT in primary acute myeloid leukemia (AML) cells, suggesting that HSP90 overexpression is necessary to the AML cell to buffer the increased activity and dependence of the cell on the AKT-pathway.

Constitutive STAT activation also occurs in approximately 70% ofAML samples. STAT activation in AML cells has been ated with, but not restricted to, FLT3 lTDs and an autocrine stimulation of IL-6. However, other upstream modulators of STAT pathways may also be playing a role in the activation of STAT. Indeed, KIT mutations have also been found to te JAK]STAT pathways. AML cases with high STATS and FLT3 phosphorylation demonstrated, in general, a lower percentage of spontaneous apoptosis, compared to AML blasts with no spontaneous STAT5 phosphorylation. Translocations involving JAKJ‘STAT genes provide another link n STAT activation and leukemogenesis. The t(9;12) ocation, which combines the erization domain of the TEL gene with the catalytic domain of JAKZ, has been found in both lyrnphocytic and myeloid leukemia. This translocation constitutively activates downstream effectors such as STATS and induces cytokine-independent growth in transfection models. As usly reported and also shown here, several of these STAT-activating proteins require HSP90 to facilitate their aberrant activity, Taken together, addiction for al of aggressive AML clones on several activating pathways and molecules, such as AKT and STAT5, renders them also most addicted to HSP90.

HSP90 inhibition thus, becomes most efi'ective in killing these cells. Our findings also suggest that concomitant overexpression of the anti-apoptofic Bcl-xL in the context of activated AKT and STAT5 does not cantly alter the sensitivity of these cells towards HSP90. Bcl-xL overexpression is a major contributor to drug ance in AML. Overexpression ofantiapoptotic proteins of the BeloZ family (Bcl«2, Bcl-x(L)) causes drug resistance to 122 "standard" chemotherapy agents and is associated with a worse clinical outcome in AML patients.

In conclusion, our findings suggest that AML patients with activation of AKT and STAT5 signaling are most likely to benefit from HSP90 inhibitor therapy (see s 41 and 42) and clinical trials should aim to enroll patients with c activation of these important signaling pathways. Our findings also suggest that introduction of HSP90 inhibitors is ted in combination with other treatments in Bcl-xL overexpressing AMLs, as a means to lower their apoptotic threshold. .5. Use of radiolabeled HSP90 inhlbltors to select neurodegenerafive patients who will be susceptible to HSP90 inhibition therapy The use of radiolabeled HSP90 inhibitors to select patients who will be susceptible to HSP90 inhibition therapy was bes in Section 5.2.1. r methodology can be used to identify patients ing from neurodegenerative diseases that are likely to respond to HSP90 therapy.

Accordingly, the disclosure provides method for detemtining whether a patient suffering from a neurodegenerative disease will likely respond to therapy with an HSP90 inhibitor which comprises the following steps: (a) contacting the brain with a radiolabeled HSP90 inhibitor which binds preferentially to a pathogenic form of HSP90 present in a brain cells of the patient; (b) measuring the amount of labeled HSP90 inhibitor bound to the brain cells in the sample; and WO 09657 (c) comparing the amount of labeled HSP90 inhibitor bound to the brain cells in the sample measured in step (b) to a reference ; wherein a greater amount of labeled HSP90 inhibitor bound to the brain cells measured in will likely respond to the step (b) as compared with the reference amount indicates the patient HSP90 inhibitor.

In one embodiment the reference is from cells of the same patient with the neurodegenerative diseases. For instance, we have determined that normal neurons have little or no “pathogenic HSP90: Accordingly, the nce amount can be determined using normal neurons as the patient in a non- ed brain region. In another embodiment, the reference can be from cells of a healthy individual.

In another embodiment. the nce amount can be measured from a study tion of healthy individuals.

Both malignant transformation and neurodegeneration, as it occurs in Alzheimer‘s e, Parkinson’s, frontotemporal dementia and other dementi as, spinal and bulbar muscular atrophy are complex and lengthy multistep processes characterized by abnormal expression, post-translational modification, and processing of certain ns. To maintain and allow the accumulation of these dysregulated processes, and to facilitate the step-wise evolution of the disease phenotype, cells must this role has been attributed to heat shock co—opt a compensatory regulatory mechanism. In cancer, protein 90 (HSP90). In this sense, at the phenotypic level, HSP90 s to serve as a mical buffer for the numerous -specific lesions that are characteristic of diverse tumors. A similar role exists for HSP90 in neurodegeneration and thus the PET assay described in Section 5.2.1. can be used to identify the “pathogenic HSP90” in the diseased brain. The “pathogenic HSP90” in egenerative disease plays a role similar to the “oncogenic HSP90" in cancer. The use of HSP90 inhibitors the ent of neurodegenerative diseases is describe in US. Published Application No. 2009/0298857, which is hereby incorporated by reference.

As the HSP90 inhibitor PU~HZ151 shows high binding affinity to neurodegenerative brain HSP90, is capable of strongly inducing HSP70 levels, and is estimated to be brain permeable, we selected it for further in viva evaluation. PU~HZISl was described in and it has the following chemical structure: iii»— .

PU—H2151 [0100! Indeed, when administered intraperitoneally to 3ng AD mice, Sl resulted in significant target modulation as demonstrated by HST-V70 induction in the hippocampus (Figure 43A). detected at as low The effect was dose dependent (Figure 43!!) with a significant induction of HSP70 as the lOmg/kg administered dose. levels We next determined in the brain and plasma of3ng AD mice, the HSP90 inhibitor associated with these codynamic effects (Figure 43C). When administered intraperitoneally at 4h, at 50 mg/kg to 3ng mice, PUAHZISI levels in the cortex reached 3.33:0.9 pg/g (~5,000 nM) 0.051008 ug/g (~l70 nM) at 12h, 0030.03 pg/g (~60 nM) at 24b and 0.02:!:0.02 ug/g (~53 nM) at 48h post-administration. In comparison, PU-DZB, a less effective HSP90 tor, administered at a 0.2 pg/g similar dose (75 mg/kg) reached a brain concentration of only 0.35 ug/g (~700 nM) at 4h and (~390 nM) at 12h, and was cted in the cortex by 24 hours post-administration.

In the plasma, PU—HZlSl reached 2. 110.1 ugJg (4,000 nM) at 4h, but was undetectable beyond 8h. The exposure of the cortex to PU-HZlSl over the al of 0 to 48h, as measured by the plasma (17.5 versus 7‘1 uM-h). area under the curve (AUC), was 2.5-times higher than that of ofplasma The levels in the cerebellum (disease cted brain region in this model) paralleled those in this model). This observation more closely than those recorded in the cortex (diseased brain region is also supported by the extended retention of inhibitor PU—HZlSl to over 48h post~administration this brain region, findings similar to those ed with inhibitors ofthis class, such as PU—H7l, tquOl‘S. l2"I—PU—HZISI and other radiolabeled HSP90 inhibitors can therefore be used to select patients afflicted by an HSP90-dependent neurodegenerative disease and identify those more likely benefit from such y. It can also be used in a fashion similar to PU-H7l in , to determine the pathogenic brain exposure to the HSP90 inhibitor and determine an optimal dose and schedule administration.

A person skilled in the art can appreciate that the uses described by this invention for PU-H7l in cancer can be achieved in neurodegenerative diseases as well with a radiolabeled, brain permeable HSP90 inhibitor. 6. Materials and Methods 6.1. Synthetic Methods 6.1.1. Synthesis of Fluorescently Labeled Probes [0383) ‘H NMR spectra were ed on a Bruker 500 or 600 MHz instrument. Chemical shifts are reported in 5 values in ppm downfield from TMS as the internal standard. ‘H data are ed as follows: chemical shift, multiplicity (s :- t, d = doublet, t == triplet, q = quartet, br = broad, in = multiplet), ng constant (Hz), integration. High resolution mass spectra were recorded on a Waters LCT Premier system. Low resolution mass spectra were ed on a Waters Acquity Ultra Performance LC with ospray ionization and SQ detector. High-performance liquid chromatography analyses were performed on a Waters Autopuriflcation system with FDA, MicroMass 20, and ELSD detector, and a reversed phase column (Waters X-Bridge C18, 4.6 x 150 mm, 5 pm) using a gradient of; method A (a) H20 + 0.1% TFA and (b) CH3CN + 0.1% TFA, 5 to 95% b over 10 s at 1.2 mUmin; method B (a) H20 + 0.1% TFA and (b) CH3CN + 0.1% TFA, to 95% b over 13 minutes at 1.2 mUrnin. Column chromatography was performed using 230-400 mesh silica gel (EMD). All reactions were performed under argon protection. Fluorescein isothiocyanate (FITC), sulforhodamine 101 sulfonyl de (Texas Red—Cl) and 4—chloro-7—nitro- 1,2,3-benzoxadiazole (NED-Cl) were purchased from Aldrich.

PU—H71-F1TC1 [4] (Scheme 1). Compound 32' (15 mg, 0.0263 mmol), FITC (11.3 mg, 0.0289 mmol) and Et3N (0.1 mL) in DMF (0.2 mL) was stirred for 12 h at It. The reaction e was concentrated under reduced pressure and the residue was purified by HPLC to give 10.1 mg (40%) of4. ‘H NMR (500 MHz, MeOH-d‘) 5 8.17 (s, 1H), 8.05 (s, 1H), 7.93 (s, 1H), 7.65~7.74 (m, 1H), 7.40 (s, 1H), 7.08-7.16 (m, 2H), 6.76—6.89 (m, 2H), 6.66 (s, 2H), 6.50-6.59 (m, 2H), 6.02 (s, 2H), 4.35 (t,J= 6.9 Hz, 2H), 3.96 (t, J= 6.4 Hz, 2H), 3.78 (br s, 2H), 3.62 (br s, 2H), 2.31 (m, 2H), 1.77 (m, 2H), 1.69 (m, 2H), 1.45 (m, 4H); HRMS (ESI) m/z [M+H]+ calcd. for Cal-LOINEO782, 959.1506; found 959.1530; HPLC (method A) R. = 4.52 (96%).

PU—H7l-Texas Red [5]. Compound 3“ (4.6 mg, 0.008 mmol) in DMF (0.25 mL) was cooled to 0°C by ioelwater bath. Then sulforhodamine 101 sulfonyl chloride (3 mg, 0.005 mmol) was added and the solution was stirred for 12 11, allowing the ature to slowly rise from 0 to 10°C. The on mixture was directly purified by HPLC to give 3.4 mg (61%) of 5 as a dark purple solid. ‘H NMR (500 MHz, MeOH~d;) 5 8.56 (d, J= 1.4 Hz, 1H), 8.31 (s, 1H), 8.16 (dd, J= 1.6, 7.9 Hz, 1H), 7.48 (5, 11-1), 7.46 (d, J: 7.9 Hz, 1H), 7.28 (s, 1H), 6.58 (3, 21-1), 6.08 (s, 2H), 4.47 (t, J = 6.8 Hz, 2H), 3.56 (t, J: 5.4 Hz, 4H), 3.52 (t, J: 5.6 Hz, 4H), 3.15 (t, J= 7.6 Hz, 21-1), 3.08 (m, 4H), 3.01 (t, J: 7.7 Hz, 2H), 2.93 (t, J= 6.7 Hz, 2H), 2.68 (m, 4H), 2.35 (m, 2H), 2.11 (m, 41!), 1.90200 (m, 4H), 1.66 (m, 2H), 1.27-1.45 (m, 6H); HRMS (ESI) m/z [Md-H]+ calcd. for C51H571N903S3, 537; found 11582534; HPLC (method B) R. = 9.40 (99%).

PU-H'Il-NBDI [3] (Scheme 1). Compound 62‘ (12.2 mg, 0.0229 mmol) and 722 (32 mg, 0.1145 mmol) were dissolved in DMF (0.4 mL) and stirred at 11 for 20 h. Solvent was removed under reduced pressure and the resulting residue was purified by preparntory TLC (CH2C122MeOH—NH3 (7N), 10:1) to give 7.9 mg (47%) of 8. ‘H NMR (500 MHz, CDClg/MeOH-dl) 6 8.32 (d, J: 8.8 Hz, 1H), 8.00 (s, 1H), 7.21 (s,1H),6.89(s, 1H), 6.04 (d, J= 8.8 Hz. 1H), 5.89 (s, 2H), 4.13 (t, J= 6.9 Hz 2H), 3.32 (m, 2H), 2.51 (t,J 2 6.9 Hz, 2H), 2.47 (t. J = 7.4 Hz, 2H), 1.94 (m, 2H), 1.63 (m, 2H), 1.36- 1.45 (m, 2H), 1.21-1.35 (m, 4H); HRMS (ESI) m/z {M+H]* calcd. for C27H301NmOsS, 733.1166; found 733.1171; HPLC (method B) R. = 3.80 (98%).

PUnH71~FITC2 [9] (Scheme 2). Compound 223 (16.7 mg, 0.0326 mmol), FITC (14.0 mg, 0.0359 mmol) and Eth (0.1 mL) in DMF (02 mL) was stirred for 5 h at rt. The reaction mixture was trated under reduced pressure and the residue was purified by HPLC to give 21.2 mg (72%) of 9. ‘H NMR (500 MHz, CDC13)5 8.15 (s, 1H), 7.86 (s, 1H), 7.77 (d, J: 7.9 Hz, 1H), 7.34 (s, 1H), 7.09 (d, J= 7.9 Hz, 1H), 7.01 (s, 1H), 6.63«6.71 (m, 4H), 6.51 (d, J= 7.3 Hz, 2H), 6.02 (s, 2H), 5.53 (br s, 2H), 4.30 (br s, 2H), 3.64 (br s, 2H), 2.85 (br 5, 1H), 2.27 (m, 2H), 1.23 (d, J = 6.2 Hz, 6H); HRMS (ESI) m/z [M+H]+ calcd. for C39HDIN70751, 902.0928; found 902.0942; I-[PLC (method B) R. = 9.90 (99%).

PU-H'll-NBD2 (10}. Compound 223 (25.4 mg, 0.050 mmol), l (10.0 mg, 0.05 mrnol) and Eth (7.6 11L, 0.055 mrnol) in DMF (0.35 mL) was stirred for 12 h at rt. The reaction mixture was trated under reduced pressure and the residue was purified by preparatory TLC (CHZC12:MeOH-NH3 (7N), 25:1) to give 13.4 mg (40%) of 10. 'H NMR (600 MHz,CDC1,/MeOH-d,) 8.25 (d, J= 8.9 Hz, 1H), 8.06 (s, 1H), 7.18 (s, 1H), 6.85 (s, 1H), 6.07 (d, J: 8.9 Hz, 1H), 5.87 (s, 2H), 4.24 (t, J= 6.9 Hz, 2H), 3.74 (11.1, 21-1), 3.13 (m, 1H), 2.12 (m, 210, 1.22 (d, J= 6.5 Hz, 6H); HRMS (ESI) m/z [M+H]* calcd. for C24H231N905S, 676.0588; found 676.0593; HPLC (method 13) R. = 10.37 (99%). 2-(3—(6—amino—8—(6—iodobenzold][1,3]dlox01ylth10)-9H—purinyl)propyl)lsoindo|1ne- 1,3-dione [12] (Scheme 3). 50 mg (0.121 mmol) of Compound 1123 was dissolved in BM? (2 mL). 43.4 mg (0.1331 mmol) of C51CO3 and 162 mg (0.605 mmol) of N—(3 -bromopropyl)-phthalimide were added and the mixture was stirred at rt for 30 s. Then onal C52C03 (8 mg, 0.0242 mmol) was added and the mixture was stirred for 30 minutes. Then additional CsZC03 (3 mg, 0.0242 mmol) was added and the mixture was stirred for 30 minutes. Solvent was removed under reduced re and the resulting residue was purified by preparatory TLC (CHzclzzMeoflzAcOH, 15: 1 :0.5) to give 25 mg (34%) of 12. 'H NMR (500 MHz, CDC13) 6 8.25 (s, 1H), 7.85 (dd. J: 3.0, 5.5 Hz, 2H), 7.74 (dd, J2 3.0, 5.4 Hz, 2H), 7.11 (s, 1H), 6.80 (s, 1H), 6.10 (br s, 2H), 6.00 (s, 2H), 4.27 (t, J: 7.6 calcd. for CnHIEIN604S, Hz, 2H), 3.77 (t, J = 6.7 Hz, 2H), 2.15 (m, 2H); HRMS (ESI) m/z [M+H]+ 601.0155; found 601.0169; HPLC (method A) R. x 7.74. minopropyl)—8—(6-lodobenzo[d][1,3]d10xolylthio)-9H—purin-é-amine [13] e 3). To a suspension of Compound 12 (34 mg, 0.0566 mmol) in MeOH/CHZCII (0.7:0.1 mL) was added hydrazine hydrate (41 11L, 42.5 mg, 0.849 mmol) and the mixture was stirred at It for overnight. Solvent was removed under reduced pressure and the ing residue was purified by of 13. !H NW (500 MHz, preparatory TLC (CH3C121MeOH~NH; (7N), 10:1) to give 17 mg (64%) CDClg/MeOH-d.) 6 8.22 (s, 1H), 7.38 (s, 1H), 7.06 (s, 1H), 6.05 (s, 2H), 4.31 (t, J = 6.9 Hz, 2H), 2.76 471.0100; found (t, J = 6.6 Hz, 2H), 2.05 (m, 2H); HRMS (ESI) m/z [1171+I-1]+ calcd. for C.sl-1..[N.015, 471.0086; HPLC (method A) R. = 5.78.

PU-H71«F1TC3 [14] (Scheme 3). Compound 13 (8.4 mg, 0.0179 mmol), FITC (7.7 mg, mixture 0.0196 mmol) and Eth (0.1 mL) in DMF (02 mL) was stirred for 12 h at 11. The reaction HPLC to give 11.4 mg was concentrated under d pressure and the residue was purified by (74%) of 14. ‘11 NMR (600 MHz, Maori-d.) s 8.23 (s, 1H), 8.11 (s, 1H), 7.68 (d, J= 8.0 Hz, 11-1), 7.35 (s, 1H), 7.20 (s, 1H), 7.09 (d, J= 8.0 Hz, 11-1), 6.63-6.70 (m, 4H), 6.50 (d, J= 8.3 Hz, 2H), 5.97 (s, 2H), 4.34 (t, J= 6.5 Hz, 2H), 3.61 (m, 2H), 2.21 (t, J= 6.5 Hz, 2H); M5 (E81) m/z 860.1 [Mi-HT; HPLC (method B) R.

HRMS (ESI) m/z [Me-H]+ calcd. for C351-Iz71N707SZ, 58; found 860.0451; = 9.48 (96%).

NED-C1 (3.1 mg, PU—H71—NBD3 [15] (Scheme 3). Compound 13 (7.2 mg, 0.0153 mmol), 0.0213 nunol) and Eth (2.3 11L, 0.0168 mmol) in DMF (0.2 mL) was stirred for 12 h at rt. reaction mixture was concentrated under reduced pressure and the residue was purified by preparatory TLC (CI-12C111MeOI-1~NH3 (7N), 20:1) to give 4.] mg (42%) of 15. lH NMR (600 MHz, DMF~d7) 6.76 (5, 9.54 (br 5, 1H), 8.53 (d, J= 8.8 Hz, 1H), 8.22 (s, 1H), 7.51 (br 5, 21-1), 7.28 (s, 1H), 11-1), 6.42 HRMS (ESI) (d, J= 7.9 Hz, 1H), 6.10 (s, 2H), 4.47 (t, J3 7.0 Hz, 2H), 3.67 (m, 2H), 2.35 (m, 2H); 9.57 (99%). m/z [Md-HT calcd. for Cleljl-Ngoss, 634.0118; found 30; HPLC (method B) R. = Synthesis of tetrnethylene glycol-FITC (TEG-FITC). FITC (20 mg, 0.051 mrnol), for 12 h tetraethyiene glycol (49.9 mg, 0.257 mmol) and Eth (0.1 mL) in DMF (0.4 mL) was stirred and the residue was purified by at 11. The reaction mixture was concentrated under reduced pressure HPLC to give 17.3 mg (58%) of TEG-FITC. ‘H NMR (600 MHz, “) 5 7.53—8.25 (m, 2H), 3.77 (m, 2H), 7.14 (d, J: 8.2 Hz, 1H), 6.72—6.91 (m, 4H), 6.65 (d, J= 6.8 Hz, 2H), 4.60 (br s, 2H), 3.31-3.63 (m, 12H); HRMS (ESI) m/z [M+I-1]+ calcd. for ngHmNOmS, 584.1590; found 584.1570; HPLC (method 13) R. = 8.97 (99%). 6Anflno-B—((6-iodobenzo[d]Il,3]dloxol—5-yl)thio)-9H-purinyl)butyl)isoindollne- in DMF (8 mL). 1,3-dlone (16a) (Scheme 4). 200 mg (0.484 mmol) of Compound 11 was dissolved 466 mg (1.43 mmol) of C52C03 and 683 mg (2.42 mmol) of N-(4—br0mobutyl)phthalimide were added and the mixture was ted for 30 min. 31.5 mg (0.097 mmol) of C52C03 was added and the mixture was again sonicated for 30 min. This was repeated two more times for a total reaction time of 2 h. DMF was removed and the ing residue was purified by preparatory TLC (CH2C1,:MeOH:AcOH, 15:1:0.5) to give 134 mg (45%) ofCompound 16:. 'H NMR (500 MHz, CDCl,) 6 8.18 (s, 1H), 7.84 (dd, J= 5.5, 3.1 Hz, 2H), 7.72 (dd, J= 5.5, 3.1 Hz, 2H), 7.22 (s, 1H), 6.89 (s, 1H), 6.76 (br s, 2H), 5.99 (s, 2H), 4.23 (t,J= 7.1 Hz, 2H), 3.69 (t, J= 7.0 Hz, 21-1), 1.67—1.83 (m, 4H); MS (ESI) m/z 615.2 [Mu-11+. 9-(4-Aminohutyl)((6-iodobenzo[d]I1,3]dioxol-S-yl)thlo)-9H-purln-G-nmine (17a) (Scheme 4). To a suspension of nd 16a (38.9 mg, 0.063 nunol) in 2 mL MeOH/CHZCIZ (7:1 mL) was added ine hydrate (46 uL, 0.950 rnmol) and the mixture was stirred at rt for 12 h.

Solvent was d under d pressure and the resulting residue was purified by preparatory TLC (CH2C1;:MeOH-NH3 (7N), 10:1) to give 18 mg (59%) of Compound 173. 'H NMR (500 MHz, CDClglMeOH-ab) 8 8.22 (s, 1H), 7.38 (s, 1H), 7.04 (s, 1H), 6.05 (s, 2H), 4.23 (t, J = 7.4 Hz, 2H), 2.78 (t, J: 7.1 Hz, 2H), 1.82-1.91 (m, 2H), 1.55-1.63 (m, 2H); MS (ESI) m/z 485.0 [M+H]“.

PU-H7l-FITC4 (18:) e 4): Compound 17:! (9.7 mg, 0.020 rnmol), FITC (8.57 mg (0.022 mmol) and Eth (0.1 mL) in DMF (0.2 ml.) was stirred for 3 h at It. The reaction mixture was directly purified by HPLC to give 5.2 mg (30%) of Compound 188. 1H NNIR (600 MHz, MeOH-dl) 5 8.22 (s, 1H), 8.00 (s, 1H), 7.61 (d, J2 7.6 Hz, 1H), 7.37 (s, 1H), 7.19 (s, 1H), 7.06 (d, J= 8.2 Hz, 1H), 6.58-6.67 (m, 4H), 6.48 (dd, J= 8.7, 2.0 Hz, 2H), 5.97 (s, 2H), 4.30 (t, J= 7.0 Hz, 2H), 3.58 (br s, 2H), 1.90-2.00 (m, 2H), .70 (m, 2H); HRMS (ESI) m/z [M+H]+ calcd. for C37H191N707Sz, 874.0615; found 874.0610; HPLC R, = 9.57 (98%). 2-(6—(6-Amino—8~((6—iodobenzo[d][ l,3]dioxol-5—yl)thio)-9H‘purin-9—yl)hexyl)isoindoline— 1,3-dione (16b) e 4). 200 mg (0.484 mmol) of Compound 11 was dissolved in DMF (8 mL). 466 mg (1.43 mmol) of C52C03 and 751 mg (2.42 mmol) N-(6—bromohexyl)phthalimide were added and the mixture was sonicated for 2 h. Solvent was d under reduced pressure and the resulting residue was purified by preparatory TLC (CH2C12:MeOH:AcOH, 15:1:0.5) to give 100 mg (32%) of Compound 16b. 1H NMR (500 MHz, CDCl;) 5 8.26 (s, 1H), 7.83 (dd,J = 5.4, 3.1 Hz, 2H), 7.70 (dd, J : 5.4, 3.0 Hz, 2H), 7.26 (s, 1H), 6.87 (s, 1H), 6.36 (br s, 2H), 5.96 (s, 2H), 4.18 (t, J= 7.5 Hz, 2H), 3.66 (t,J: 7.2 Hz, 2H), 1.70-1.79 (m, 2H), 1.60-1.68 (m, 2H), 1.32-1.43 (m, 4H); MS (ESI) m/z 643.2 [M+H]*. 9-(6-Aminohexyl)-H(6-iodobenzo[d][1,3]dioxol-S-yl)thlo)-9H-purin-G-alnine (17b) (Scheme 4). To a suspension of Compound 16b (97 mg, 0.1511 mrnol) in 4 mL MeOH/CH1C12 (7:1 mL) was added hydrazine hydrate (110 uL, 2.27 mmol) and the mixture was stirred at rt for 12 h.

Solvent was removed under reduced pressure and the resulting residue was purified by preparatory TLC 1:MeOH-NH3 (TN), 10:1) to give 47 mg (61%) of 171). 1H NMR (500 MHz, CDCl;) 7.5 Hz, 2H), 2.67 (t, J 8.32 (s, 1H), 7.31 (s, 1H), 6.90 (s, 1H), 5.99 (s, 2H), 5.34 (br s, 2H), 4.20 (t, J= = 6.5 Hz, 2H), 1.72-1.84 (in, 2H), 1.31—1.45 (m, 6H); MS (ESI) m/z 513.0 .

PU‘H7l-FITC5 (Compound 18h) (Scheme 4). Compound 17b (9.7 mg, 0.01894 mmol), for 3h at rt. The FITC (8.11 mg, 0.0208 mmol) and Eth (0.1 mL) in DMF (02 mL) Was stirred 181). 'H NMR reaction mixture was directly purified by HPLC to give 8.0 mg (47%) of Compound 7.16 (s, 1H), (600 MHz, MeOH-do 5 8.23 (s, 1H), 8.09 (s, 1H), 7.65 (d, J= 7.9 Hz, 1H), 7.35 (s, 1H), J: 8.8, 2.2 Hz, 7.08 (d, J= 8.3 Hz, 1H), 6.71 (d, J= 8.8 Hz, 2H), 6.67 (d, J= 2.2 Hz, 2H), 6.53 (dd, 2H), 5.96 (s, 2H), 4.24 (t, J2 7.1 Hz, 2H), 3.50 (br- s, 2H), 1.79-1.88 (m, 2H), 1.52-1.61 (m, 2H), 1.31-1.42 (m, 4H); HRMS (ESI) m/z [M+H]+ calcd for C19H331N7O7sz, 902.0928; found 902.0939; HPLC R, = 10.02 (99%). 2-(8-(6—Amiuo—8—«6—iodobenzoId][13]dinxnl-S-yl)thin)—9H-purin-9—yl)octyl)isoiudoline- in DMF (8 mL). 1,3-dinne (16:) (Scheme 4). 200 mg (0.484 mmol) of Compound 11 was dissolved added 466 mg (1.43 mmol) of C52C03 and 819 mg (2.42 mmol) N-(S—bromooctylmhthalimidn were and the and the mixture was sonicated for 1.5 h. Solvent was removed under reduced pressure 120 mg resulting residue was purified by preparatory TLC (CHZClzzMeOHerOH, 15: 1 20.5) to give J: 5.5, 3.1 Hz, 2H), (34%) of nd 16c. ‘H NMR (500 MHz, CDC13)6 8.29 (s, 1H), 7.84 (dd, 4.18 (t, J= 7.5 7.70 (dd, J: 5.5, 3.1 Hz, 2H), 7.28 (s, 1H), 6.87 (s, 1H), 6.29 (br s, 2H), 5.96 (s, 2H), m/z 671.3 [M+H]+.

Hz, 2H), 3.67 (t, J= 7.3 Hz, 2H), 1.62-1.77 (m, 4H), 1.25-1.36 (m, 811); MS (ESI) nduooctyl)-8—((6—indobeuzold][l,3]dioxol-S-ylfihio}9H-purin—B—amine (17¢). To a added suspension of Compound 16c (90.1 mg, 0.1345 mmol) in 4 mL MeOH/CH2C11 (7:1 mL) was hydrazine hydrate (98 11L, 2.017 mmol) and the mixture was stirred at It for 12 h. t was removed under reduced pressure and the resulting e was purified by preparatory 8.33 (s, (CH2C12:MeOH-NH3 (7N), 10:1) to give 25 mg (34%) of 17c. ‘H NMR (500 MHz, CDCl;) J= 7.1 1H), 7.31 (s, 1H), 6.90 (s, 1H), 5.99 (s, 2H), 5.72 (br s, 2H), 4.20 (t, J= 7.5 Hz, 2H), 2.66 (t, Hz, 2H), 1.70—1.80 (m, 2H), 1.36-1.45 (m, 21-1), l.21-1.35(1r1, 8H); MS (ESI) n1/z541.1 [M+H]*. 0.028 Synthesis of PU-H7l-FITC6 (Compound 18c) (Scheme 4): Compound 17c (15.0 mg, for 4 h at rt. mmol), FITC (11.9 mg, 0.031 mmol) and 13th (0.1 mL) in DMF (0.2 mL) was stirred 18c. 1H The reaction mixture was directly purified by I-IPLC to give 16.9 mg (66%) of Compound NMR (600 MHz, MeOH-d4) 5 3.22 (s, 1H), 8.11 (s, 1H), 7.68 (d, J= 7.3 Hz, 1H), 7.34 (s, 1H), 7.12 6.53 (dd, J: 8.7, (s, 1H), 7.09 (d, J: 8.2 Hz, 1H), 6.72 (d, J: 8.7 Hz, 2H), 6.67 (d, J: 2.0 Hz, 2H), 1.52~1.59 (m, 2.0 Hz, 2H), 5.96 (s, 2H), 4.20 (t, J= 7.1 Hz, 2H), 3.50 (br s, 2H), .81 (m, 2H), found 2H), 1.23-1.35 (m, 8H); HRMS (ESI) m/z [M+H]+ calcd. for CHI-13711470752, 930.1241; 930.1231; HPLC R. = 10.60 (96%).

Synthesis of PU-FITC7 und 20) (Scheme 7). Compound 19 (15.0 mg, 0.025 mmol), for 8 h at rt. The FITC (10.7 mg, 0.0275 mmol) and Et3N (0.1 mL) in DMF (0.3 mL) was d IH NMR (600 reaction e was ly purified by HPLC to give 23.5 mg (95%) of PU-FITC7. 7.19—7.23 MHz, MeOH-d4, 2 rotamers) 6 8.18—8.22 (m. 1H), 7.75-7.87 (m, 4H), 7.53«7.58 (m. 1H), .55 (m, (m, 1H), 7.05 (d, J= 8.2 Hz, 1H), 6.98 (8, 0.1511), 6.95 (s, 0.85H), 6.57-6.75 (m, 4H), 2.03- 2H), 6.05 (s, 0.311), 6.00 (s, 1.7H), 3.95-4.05 (m, 2H), 3.55—3.64 (m, 1.711), 2.86-2.92 (m, , HRMS 2.12(m,1.7H), 1.93—2.00 (m, 0.311), 1.18 (d, .1a 6.5 Hz, 0.911), 1.13 (d, J= 6.5 Hz, 5.111); (ESI) m/z [M-t-Hr calcd. for C47H35F6N70752, 988.2022; found 988.2005; HPLC R. = 11.00 (99%).

Synthesis of PUaFlTCS (Compound 22) (Scheme 8): Compound 21 (19.4 mg, 0.050 for 14 11 at rt. mmol), FITC (21.4 mg, 0.055 mmol) and Eth (0.1 mL) in DMF (04 mL) was stirred 'H NMR The reaction mixture was ly purified by I-IPLC to give 34.3 mg (88%) of CB. 7.20 (dd, J= 8.1, (600 MHz, MeOH—d4) 5 8.35 (s, 1H), 7.97 (s, 1H), 7.69 (dd, J: 8.2, 1.9 Hz, 1H), 6.01 1.9 Hz, 1H), 7.14-7.18 (m, 2H), 6.91 (d, J= 8.0 Hz, 1H), 6.76-6.85 (m, 4H), 6.59-6.65 (m, 2H), (s, 2H), 4.40 (t, J= 6.7 Hz, 2H), 3.82 (t, J= 7.4 Hz, 2H), 2.35-2.43 (m, 2H), 1.31 (d, J= 6.7 Hz 6H); = 10.13 HRMS (E31) 111/: [M+H]+ calcd. for Gal-13418170752, 776.1961; found 78; HPLC R. (98%).

Synthesis of PU—FITC9 (Compound 24) (Scheme 9): Compound 23 (10.0 mg, 0.032 mmol), FITC (13.9 mg, 0.036 mmol) and Eth (0.1 mL) in DMF (03 mL) was stirred at rt PU-FITC9. overnight. The reaction e was directly purified by HPLC to give 18.3 mg (82%) of IH NMR (600 MHz, 4) 5 8.33 (s, 1H), 7.92 (s, 1H), 7.66 (dd, J: 8.1, 1.8 Hz, 1H), 7.15 (d, J 6.5 Hz, 2H), = 8.2 Hz, 1H), 6.73—6.83 (m, 4H), 6.58-6.65 (m, 2H), 4.73-4.76 (m, 2H), 4.23 (t, J= 3.81—3.85 (in, 2H), 3.74-3.81 (m, 2H), 3.41 (s, 3H), 2.28—2.37 (m, 2H), 1.30 (d, J= 6.6 Hz, 6H); 9.20 (99%).

HRMS (ESI) m/z [MM—I]+ calcd. for C35H35N7O7S, 698.2397; found 698.2399; HPLC R. = Synthesis ofDZl3-FITC1 (Scheme 10). PU-DZl3 (20.8 mg, 0.0406 mmol), FITC (17.4 in DMF (0.3 mL) was stirred for 12 h at rt. The reaction mg, 0.0447 mmol) and Eth (0.1 mL) mixture was directly purified by HPLC to give 33.7 mg (92%) of DZl3—FITC1. |H NMR (500 MHz, DMF—d7) 8 9.46 (s, 1H), 8.05 (dd, J= 7.0, 1.8 Hz, 1H), 7.76-7.82 (m, 1H), 7.44 (s, 1H), 7.26 (d, J: 6.0 Hz, 2H), 4.42 7.3 Hz, 1H), 6.90 (s, 1H), 6.78 (m, 2H), 6.67-6.72 (m, 4H), 6.11 (s, 2H), 4.59 (t, 0.92 (d, J: 6.7 Hz, 6H); (s, 2H), 4.39 (t, J: 6.0 Hz, 2H), 3.53 (d, J= 6.9 Hz, 2H), 2.17-2.28 (m, 1H), = 11.77 HRMS (ESI) m/z [M+H]+ calcd. for CwH341-‘1N707S, 902.1269; found 902.1293; HPLC R. (98%).

Synthesis of SNX—FITC (Scheme 11). Compound 25 (9.5 mg, 0.0205 mmol), FITC (8.8 mg, reaction mixture was 0.0225 mmol) and Eth (0.1 mL) in DMF (0.2 mL) was stirred for 4 h at rt. The concentrated under reduced pressure and the residue was purified by HPLC to give 13.5 mg (77%) found 853.2630. an orange solid. RMS (ESI) m/z [M+I-1}I]+ calcd. for CunF3N507S, 853.2631; 2012/045864 6.1.2. Synthesis of biotinylated compounds [0408) PU-H71-biotin3. 13 (9.1 mg, 0.0193 mmol), D-biotin (7.1 mg, 0.0290 mmol), DCC (3 mg, sonicated for 5 h. The reaction 0.0386 mmol) and a catalytic amount ofDMAP in CHIC]; (1 mL) was residue was purified by mixture was concentrated under reduced pressure and the ing 7.5 mg (56%) of PU-H71-biotln3. lH preparatory TLC (CHzclzzMeOH-NHJ (7N), 10:1) to give NMR (600 MHz. CDClJMeOH-do 15 7.97 (s, 1H), 7.17 (s, 1H), 6.86 (s, 1H), 5.34 (s, 2H), 4.23-4.27 6.4 Hz, 2H), 2.90—2.97 (m, 1H), 2.67 (m, 1H), 4.05—4.09 (m, 1H), 4.03 (t, J= 7.2 Hz, 2H), 3.02 (t, J= 2H), (dd, J= 4.9, 12.8 112,111), 2.49 (d, J: 12.8 Hz, 1H), 2.01 (t,J= 7.5 Hz, 2H),1.75-1.83(m, 1.34-1.54 (m, 4H), 1.18-1.27 (m, 2H); MS : m/z 697.1 [M+H]*. 0.117 PU-H71~biotin2. 2 (30 mg, 0.059 mmol), D-biotin (19 mg, 0.078 mmol), DCC (24 mg, h. The reaction mmol) and a catalytic amount ofDMAP in CH2C12 (1 mL) was sonicated for 9 mixture was concentrated under reduced pressure and the resulting residue was purified by 43.2 mg (99%) of PU—H7l-biotin2. ‘H preparatory TLC (CH2C12:MeOH-NH3 (7N), 10:1) to give NMR (600 MHz, CDClg, 2 rotamers) 15 8.22 (s, 1H), 7.22 (s, 0.6H), 7.21 (s, 0.4H), 6.87 (s, 0.6H), 6.76 .85 (br s, 0.6H), 5.73 (br 5, 0.411), (5, 0.411), 6.25 (br s, 0.6H), 6.16 (br s, 0.4H), 5.88-5.96 (m, 21-1), 4.00-4.07 (111, 4.54-4.63 (m, 0.6H), 4.32-4.45 (m, 1.6H), .25 (m, 0.4H), 4.11-4.19 (m, 1.4H), 2.62-2.68 , 3.88—3.95 (111, 041-1), 2.97322 (m, 2.411), 2.78-2.84 (m, 1H), 2.69-2.77 (m, 0.6H), 1,434.72 (m, 311), 1.16—1.40 (m, 111), 2.22-2.27 (m, 0.6H), 1.94—2.05 (m, 1.4H), 1.74-139 (m, 1.4H), m/z 739.2 [M+H]+. (m, 3.611), 1.00-1.06 (m, 4H), 0.97 (d, J= 6.7 Hz, 2H); MS (ESI): 0.0394 -biotiu4. 13 (16.9 mg, 0.0359 mmol), EZ-Link® NHS—LC-Biotin (17.9 mg, stirred at rt for 1 h. The mmol) and DIEA (9.3 mg, 12.5 uL, 0.0713 mmol) in DMF (0.5 mL) was residue was purified by reaction mixture was concentrated under reduced pressure and the resulting .8 mg (72%) of PU—H71-biotin4. 'H preparatory TLC (CHzClzzMeOH-Nflg (7N), 10:1) to give 7.03 (s, 1H), 6.66 (t, J NMR (500 MHz, CDCl;) 8 8.22 (s, 1H), 7.52 (t, J= 5.6 Hz, 1H), 7.36 (s, 1H), 4.25 (t, J: 6.8 Hz, = 5.5 Hz, 1H), 6.25 (br s, 2H), 6.03 (s, 2H), 4.47-4.52 (m, 1H), 4.28-4.33 (m, 1H), 2.63-2.79 (m, 1H), 2.24 (t, 2H), 3.17325 (m, 4H), 3.11-3.17 (m, 1H), 2.90 (dd, J= 5.0, 12.9 Hz, 1H), 1.48-1.56 (m, 2H), 1.31- J= 7.4 Hz, 2H), 2.13-2.19 (m, 2H), 1.94—2.02 (m, 2H), 1.58-1.74 (m, 6H), 1.46 (m, 4H); MS (ESI): m/z 810.3 .

PU—H7l-biotin7. 2 (15 mg, 0.0292 mmol), Isl—Link® NHS‘LC-Biotin (14.6 mg, 0.0321 heated at 35 “C for 6 h. The mmol) and DIEA (7.5 mg, 10.2 11L, 0.0584 mmol) in DMF (0.5 mL) was e was purified by reaction mixture was concentrated under reduced pressure and the ing .3 mg (41%) of -biotin7. In atory TLC (CH2ClzzMeOH—NH3 (7N), 10:1) to give IH NMR (500 MHz, addition, 6.9 mg of unreacted 2 was recovered to give an actual yield of 77%. 6.85 (s, 0.6H), CDCl;, 2 rotamers) 8 8.26-8.29 (m, 1H), 7.29 (s, 0.4H), 7.28 (s, 0.6H), 6.87 (s, 0.4H), .68 (br s, 0.411), 5.58 (hr 6.76 (br 5, 0.411), 6.74 (br s, 0.6H), 6.51—6.63 (bx s, 2H), 5.96—6.00 (m, 2H), , 0.611), 4.56-4.64 (m, 0.4H), 4.45-4.52 (m, 1H), 4.28-4.36 (m, 1H), 4.20-4.27 (m, 2H), 4.01-4.09 (111, 061-1), 3.08-3.32 (m, 5H), 2.86-2.94 (m, 1H), 2.69-2.76 (m, 1H), 2.31-2.37 (m, 1H), 1.96-2.22 (m, 4H), 1.89-1.96 (m, 1H), 1.30-1.80 (m, 12H), 1.10-1.16 (m, 4H), 1.04-1.09 (m, 2H); MS (E81): 111/: 852.3 [M+H]*. -bioflns. 13 (16.6 mg, 0.0352 mmol), EZ-Link" -LC-Biotin (22.0 mg, 0.0387 mmol) and DIEA (9.1 mg, 12.3 1.1L, 0.0704 mmol) in DMF (0.5 mL) was stirred at It for 1 h.

The reaction mixture was concentrated under reduced re and the resulting residue was purified by preparatory TLC z:MeOH-NH3 (7N), 10: 1) to give 27.8 mg (86%) of PU-HTl-biotinS. ‘H NMR (500 MHz, CDClg/MeOH-zb) 5 8.12 (s, 1H), 7.60 (m, 1H), 7.30 (s, 1H), 7.09 (m, 1H), 6.98 (s, 1H), 5.97 (s, 2H), .44 (m, 1H), 4.20-4.24 (m, 1H), 4.17 (t, J= 7.1 Hz, 2H), 3.04-3.18 (m, 7H), 2.83 (dd, J= 5.0, 12.9 Hz, 1H), 2.64 (cL J: 12.8 Hz, 1H), 2.16 (t, J= 7.5 Hz, 2H), 2.03-2.12 (m, 4H), 1.88-1.96 (m, 2H), .66 (m, 18H); M3 (E81): 111/: 923.4 [M+H]*.

PU-HTl—biotins. 2 (15 mg, 0.0292 mmol), EZ-Liiik°D NHS-LC-LC-Biotin (18.2 mg, 0.0321 mmol) and D1EA(7.5 mg, 10.2 uL, 0.0584 mmol) in DMF (05 mL) was heated at 35 °C for 6 h. The reaction mixture was concentrated under d pressure and the ing residue was purified by preparatory TLC (CHZClzzMeOH-NHg (7N), 10:1) to give 8.2 mg (29%) of PU—H7l-biotln8. In addition, 9.6 mg of unreacted 2 was recovered to give an actual yield of 81%. ‘H NMR (500 MHz, CDC13/MeOH—d4, 2 rotamers) 6 8.18 (5, 0.411), 8.16 (s, 0.6H), 7.31 (s, 1H), 6.98 (s, 0.6H), 6.95 (s, 0.4H), 6.80—6.90 (m, 2H), 5.98 (s, 2H), 4.47-4.55 (m, 0.4H), .47 (m, 1H), 4.23-4.27 (m, 1H), 4.16-4.22 (m, 2H), 3.95—4.03 (m, 0.6H), 3.31-3.34 (m, 0.6H), 3.19-3.24 (m, 1.4H), 3.07-3.17 (m, 5H), 2.82-2.89 (m, 1H), 2.64-2.70 (m, 1H), 2.25-2.32 (m, 1H), 1.94-2.16 (m, 7H), .70 (m, 18H), 1.09 (cL J= 6.7 Hz, 4H), 1.03 (d, J = 6.8 Hz, 2H); MS (ESI): m/z 965.5 . {0414] PU-H71—biotin6. 13 (17.6 mg, 0.0374 mmol), EZ-Linlrtm NHS-PEGr—Biotin (24.2 mg, 0.0411 mmol) and DIEA (9.7 mg, 13 pL, 0.0704 mmol) in DMF (05 mL) was stirred at rt for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue was purified by preparatory TLC (CH2C12:MeOH-NH3 (7N), 10:1) to give 31.0 mg (88%) of PU-H7l-biotin6. 'H NMR (500 MHz, CDC13) 6 8.29 (s, 1H), 7.51 (t, J= 5.8 Hz, 1H), 7.32 (s, 1H), 7.03 (t, J= 5.3 Hz, 1H), 6.90 (s, 1H), 6.79 (s, 1H), 6.57 (br s, 2H), 6.01 (s, 2H), 5.97 (s, 1H), 4.48-4.53 (m, 1H), 4.25- 4.35 (m, 3H), 3.79 (t, J= 6.1 Hz, 2H), 3.59-3.68 (m, 12H), 3.57 (t, J= 5.1 Hz, 2H), 3.40-3.46 (m, 2H), 3.18-3.24 (m, 2H), 3.12-3.18 (m, 1H), 2.90 (dd, J= 5.0, 12.8 Hz, 1H), 275 (CL J: 12.7 Hz, 1H), 2.54 (t,J= 6.0 Hz, 2H), 2.20 (t, J: 7.4 Hz, 2H), 1.40-2.01 (m, 2H), 1.59-1.79 (m, 4H), 1.38-1.48 (m, 2H); MS (ESI): m/z 944.4 [M+H]*.

PU-H71—biot1n9. 2 (15 mg, 0.0292 mmol), EZ-Link" NHS-PEGr-Biotin (18.9 mg, 0.0321 mmol) and DIEA (7.5 mg, 10.2 1.1L, 0.0584 mmol) in DMF (0.5 mL) was heated at 35 °C for 6 h. The reaction mixture was concentrated under reduced pressure and the resulting residue was purified by of PU—H7l-biotin9. In preparatory TLC (CH1C11:MeOH—NH3 (7N), 10:1) to give 9.3 mg (32%) 'H NMR (500 MHz, addition, 9.0 mg of unreacted 2 was recovered to give an actual yield of 81%.

CDC13/MeOH—d4, 2 rotamers) 5 8.18 (5, 0.411), 8.16 (5, 061-1), 7.30-7.32 (m, 1H), 6.98 (s, 0.6H), 6.96 4.15-4.21 (m, 2H), (s, 0.4H), 5.98 (s, 2H), 4.49-4.56 (m, 0411), 4.39-4.46 (m, 1H), 4.22-4.27 (m, 1H), 3.99-4.07 (m, 0.6H), .71 (m, 2H), 3.51-3.61 (m, 1211), 3.45—3.50 (m, 2H), 3.29-3.38 (m, 2H), .25 (m, 2H), .12 (m, 1H), 2.81—2.88 (m, 1H), 2.63-2.68 (m, 1H), 2.57-2.63 (111, 121-1), 6.7 Hz, 2.41-2.47 (m, 0.8H), .18 (m, 4H), 1.52—1.70 (m, 4H), 1.32-1.41 (m, 2H), 1.08 (d, J: 4H), 1.02 (d, J = 6.8 Hz, 2H); MS (1551): m/z 986.5 [M+H]*.

PU~H7l-biotin. 6 (4.2 mg, 0.0086 mmol) and EZ-I.inkm Amine~PE03-Biotin (5.4 mg, 0.0129 concentrated and the mmol) in DMF (0.2 mL) was stirred at rt for 24 h. The reaction mixture was residue tographed (CHC13:MeOH-NH3 (7N), 5:1) to give 1.1 mg (16%) of PU-H7l-hiofln.

NMR (CDC13) 5 8.30 (s, 1H), 8.10 (s, 1H), 7.31 (s, 1H), 6.87 (s, 1H), 6.73 (br , 6.36 (br s, 1H), 3.43 6.16 (br s, 2H), 6.00 (s, 2H), 4.52 (m, 1H), .37 (m, 3H), 3.58-3.77 (m, 10H), 3.55 (m, 2H), 2.17 (t, J= 7.0 Hz, (m, 2H), 3.16 (m, 1H), 2.92 (m, 1H), 2.80 (m, 2H), 2.72 (m, 1H), 2.66 (m, 2H), 2H), 2.04 (m, 2H), 1.35~1.80 (m, 6H); MS (ESI): m/z 872.2 [M+H]+/ 6.1.3. Synthesis of ANCA—lnbclcd compounds Synthesis of 6-Amino-8—«6—iodobcnzold][1,3ldloxol-S—yl)thio)-9H—purin-9— yl)prnpy1)-2—cyunoacetamide (Compound 26) (Scheme 17). Compound 13‘ (120.3 mg, 0.256 0.307 mmol) in CHZCIZ (4 mL) was added cyanoacetic acid (26 mg, 0.307 mmol) and DCC (63 mg, mmol) and stirred at rt for 5 h. The reaction e was concentrated and purified by chromatography (CH2C12:MeOH—NH3 (7N), 100:1 to 50: 1) to give 131 mg (95%) of Compound 1H NMR (600 MHz, CDC13/MeOH-d4): 5 8.25 (s, 1H), 7.40 (s, 1H), 7.08 (s, 1H), 6.07 (s, 2H), 4.27 (t, MS (m/z): [M+H]+ 538.0.

J= 5.9 Hz, 2H), 3.57 (s, 2H), 3.27 (t, J= 5.1 Hz, 2H), 1.98—2.06 (m, 2H); Synthesis of PUoANCA (Compound 28 (Scheme 17). Compound 326 (44 mg, 0.0825 mmoi) in DMF (1 mL) was added 27 (19 mg, 0.075 mmol) and piperidine (10 11L) and heated at EC for 24 h. The reaction mixture was concentrated and purified by preparatory TLC (CH1C11:MeOH- ‘H NMR (600 MHz, NH; (7N), 12.5:1) to give 24.3 mg (42%) of Compound 28 as an orange solid.

DMF—d7): 5 8.73 (t, J= 5.8 Hz, 1H), 8.38 (d, J= 1.1 Hz, 1H), 8.36 (s, 1H), 8.29(s,1H), 8.18 (dd, J2 8.8, 1.7 Hz, 1H), 7.95 (d, J= 9.2 Hz, 1H), 7.92 (d, J= 8.8 Hz, 1H), 7.56 (dd, J= 9.2, 2.5 Hz, 1H), 7.52 (br s, 2H), 7.49 (s, 1H), 7.34 (d, J= 2.2 Hz, 1H), 6.95 (s, 1H), 6.16 (s, 2H), 4.40 (t,J= 6.9 Hz, 2H), 3,444.47 (m, 4H), 3.33.143 (m, 2H), 2.53-2.58 (m, 4H), 2.30 (s, 3H), 2.09—2.15 (m, 2H); NMR(150 MHz, DMF'd7): 5 161.5, 156.0, 153.5, 151.7, 151.5,151.2,149.5, 148.8, 144.4, 137.2, 133.6,130.3, 129.2, 127.4,127.0,126.5, 125.2,120.1,119.3, 118.9, 117.4, 111.0, 108.5,103.0, 102.9, 774.1472; 89.4, 54.9, 47.9, 45.6, 41.3, 40.5, 37.2; HRMS (ESI) m/z [M+H]+ calcd. for C34H331N9035, found 774.1473. 6.1.4. Synthesis of Radlolabeled Compounds le for The parent compounds 7l, PU-HZlS 1 and 3 were synthesized as shown in radioiodination (i.e. Sn—precursors). For radioiodination1 the synthesis follows the ion and Sl; Scheme 19. Briefly, the PU—compounds were solvated in methanol (25 pg PU—H7l for biodjstribution), pg PU-DZIB), and added to Nal (5-10 1.1L) ([“411 isotope for imaging, ['“I] in acetic followed by ion with Chloramine T (CT, 10 pL, 10 min) in acidic media (2 lug/ml. acid). The hot (radiolabeled) compounds were synthesized with the amine protecting group (tert—Butyloxycarbonyl), which was removed under acidic conditions (e.g. trifluroacetic acid (TFA), hloric acid HCl)) for each compound and purified using high re liquid chromatography methanol (MeOH) (HPLC). The PU-DZ13 and PU-HZISI precursors were abeled using 15 pL afier the addition of the radiolabel, to solvate, 10 min incubation at room temperature (RT) in CT “C. The PU—H7l which was followed by the addition of 50 pL of TFA, and one h incubation at 70 MeOH and 15 uL CT directly following the addition of the precursor was radiolabeled with 20 uL allowed to cool for 2 radiolabel, afier which time the solution was heated at 50 °C for 5 min, and then in H10) and 10 pL min. Aflerwards, 10 pL of methionine methyl ester (formulated from 0.5 g/mL concentrated HCl were added prior to tion at 50 "C (l h). The radiolabeled products were ted and the solvents were removed under reduced pressure, using a rotary evaporator. specific activity of [‘z‘fluPUJUl was ~1000 mCi/umol, which was in line with our previous experiences with this class of [ml] compound. For in viva administration, the ['2‘1]-PU-compounds were formulated in sterile 0.9% saline solution. 6.2. Evaluating the role of HSP90 in Cancer Cells The methods described in this section relate to the disclosure in Section 5.1. and KU182, Cell Lines and Primary Cells: The CML cell lines K562, Kasumi—4, MEG—01 triple-negative breast cancer cell line MDA-MB—468, HER2+ breast cancer cell line SKBI'3, cell melanoma cell line SK—Mel—2 8, prostate cancer cell lines LNCaP and DU145, pancreatic cancer American Type line Mia-PaCa-Z, colon fibroblast, CCCD18Co cell lines were obtained from the Collection of Culture Collection. The CML cell line KCL—22 was obtained from the Japanese Cells Research Bioresources. The NIH-3T3 fibroblast cells were transfected as previously described“.

RPMI (K562, SK—Mel—ZS, were cultured in IZ (MDA-MB—468, SKBr3 and Mia—PaCa-Z), 1% L—glutamine, LNCaP, DU145 and NIH-3T3) or MEM (CCDlSCo) supplemented with 10% PBS, with 20% 1% penicillin and streptomycin. Kasumi—4 cells were maintained in IMDM supplemented leen/Strep. PBL PBS, 10 ng/ml Granulocyte macrophage colony-stimulating factor (GM—CSF) and blood (human peripheral blood leukocytes) (n=3) and cord blood (n=5) were ed from patient layered over purchased from the New York Blood Center. Thirty five ml of the cell suspension was at 2,000 rpm for 40 min at 4 ml of Ficoll-Paque plus (GE Healthcare). Samples were fuged in RPMI medium with 10% FBS and °C, and the leukocyte interface was collected. Cells were plated CML and AML cells were obtained with used as ted. Primary human chronic and blast crisis the University of informed consent. The manipulation and analysis of specimens was approved by Institutional Review ter, Weill Cornell Medical College and University of Pennsylvania Piscataway, NY) Boards. Mononuclear cells were isolated using Ficoll-Plaque (Pharmacia Biotech, of Iscove’s density nt separation. Cells were cryopreserved in freezing medium ting and 10% dimethylsulfoxide modified Dulbecco medium (IMDM), 40% fetal bovine serum (FBS), in a fied (DMSO) or in CryoStorTM CS«10 (Biolife). When cultured, cells were kept atmosphere of 5% C02 at 37°C. them in Cell lysis for chemical and lmmuno—precipitation: Cells were lysed by collecting NazMoO4 Felts Buffer (HEPES ZOmM, KCl SOmM, MgClz 5111M, NP40 0.01%, y ed and aprotinin), followed by ZOmM, pH 7.2—7.3) with added lug/1.1L of protease inhibitors (leupeptin concentration was determined using three successive freeze (in dry ice) and thaw steps. Total protein the BCA kit (Pierce) ing to the manufacturer’s instructions.

Cruz [mmunoprecipitntionz The Hsp90 antibody (H9010) or normal IgG (Santa of cell lysate together with Biotechnology) was added at a volume of 10 uL to the indicated amount incubated at 4°C overnight, The beads 40 uL ofprotein G agarose beads (Upstate), and the mixture and separated by SDS-PAGE, followed by a standard were washed five times with Felts lysis buffer western blotting procedure.

HSP90 Chemical precipitation: HSP90 inhibitors beads or l beads, containing an washed three times in lysis inactive chemical (ethanolamine) conjugated to agarose beads, were then incubated at 4“C with the buffer. Unless otherwise ted, the bead conjugates (80uL) were 200 uL with lysis indicated amounts of cell lysates (120-500 pg), and the volume was adjusted to with the lysis buffer and proteins buffer. Following incubation, bead conjugates were washed 5 times 2-4 successive chemical in the pull—down analyzed by Western blot. For depletion studies, indicated. precipitations were performed, ed by immunoprecipitation steps, where and the fluorescein—labeled Reagents: The HSP90 tors, the solid-support immobilized Gleevec from LC derivatives were synthesized as previously reportedfl‘fl. We purchased EMS-345541 and sodium Laboratories, AS703026 from Selleck, KN-93 from Tocris, and PP242, vanadate from Sigma. All compounds were used as DMSO stocks.

DMSO (vehicle) for 24 h and Western ng: Cells were either treated with PU-H71 or buffer supplemented with leupeptin lysed in 50 mM Tris, pH 7.4, 150 mM NaCl and 1% NP40 lysis determined using BCA (Sigma Aldrich) and aprotinin (Sigma h). Protein concentrations were kit (Pierce) according to the manufacturer’s instructions, Protein lysates (15-200 pg) were with electrophoretically resolved by SDS/PAGE, transferred to nitrocellulose membrane and probed Bcr—Abl (1:75, the following primary antibodies against: HSP90 (1:2000, SMC—107A/B; StressMarq), Santa 011:), p- 554148; BD Pharmingen), P13K(1:1000, 06-195; e), mTOR (1:200, Sc~1549; mTOR (1:1000, 2971; Cell Signaling), STAT3 (1:1000, 9132; Cell Signaling), p-STAT3 (12000, Cell Signaling), 9145; Cell Signaling), STATS (1:500, Sc-835; Santa sz), p—STAT5 (1:1000, 9351; Cell Signaling), RICTOR (122000, NBlOO-ol 1; Novus Biologicals), RAPTOR (1:1000, 2280; P90RSK (1:1000, 9347; Cell Signaling), Raf-l ( 1:300, Sc-l33; Santa Cruz), CARMl (121000, 09- Cell Signaling), FAK 818; Millipore), CRKL (1:200, Sc-3l9; Santa Cnlz), GRB2 (111000, 3972; Cell (121000, Sc-1688; Santa Cruz), BTK (1:1000, 3533; Cell Signaling), A-Raf(1:1000, 4432; Signaling), PRKDZ (1:200, 415, Santa Cruz), HCK (1:500, 06-833; Milipore), p~HCK (1:500, incubated with a ab52203; Abcam) and n (112000, A1978; Sigma). The membranes were then Detection 1:3000 dilution of a corresponding horseradish peroxidase conjugated secondary antibody. ion System (Amersham was performed using the ECL'Enhanced Chemiluminescence Bioscieriees) according to cturer’s instructions. densitometric Densitometry: Gels were scanned in Adobe Photoshop 7.0.1 and quantitative is was performed using Un—Scanclt 5.1 software (Silk ific). studies were Radioisotope binding studies and HSP90 quantification s: Saturation MRC-S and performed with —mi and cells (K562, MDA—MB-468, SKBr3, LNCaP, DU—l45, of mI-PU—H7l either PBL). Briefly, triplicate samples of cells wae mixed with increasing amount afier 1 hr with or without 1 pM led PU’H71. The solutions were shaken in an orbital shaker and Brandel cell harvester. the cells were isolated and washed with ice cold Tris—buffered saline using a ml—l’U—I-I71 determined. These All the ed cell samples Were counted and the specific uptake of For the data were plotted against the concentration of I3‘l-I’U-I-I7l to give a saturation binding curve, 2.55xio7 KU182 quantification of PU-bound HSP90, 7 K562 cells, 07 KCL—22 cells, of total protein, cells and 07 MEG-01 cells were lysed to result in 6382, 3225, 1349 and 3414 pg respectively. To calculate the percentage of HSP90, cellular HSP90 expression was quantified by using rd curves created of recombinant HSP90 purified from HeLa cells (StressgeniiADLSPP— 770).

Pulse-Chase. K562 cells were treated with Na3VO4 (1 mM) with or without PU-H71 (5 M), in 50 mM Tris pH 7.4, 150 mM NaCl as indicated. Cells were collected at indicated times and lysed and 1% NP-40 lysis , and Were then subjected to western blotting procedure.

Cells Tryptic digestion: K562 cells were treated for 30 min with vehicle or PU-H7l (50 pM). 1% NP-40 lysis buffer. STATS were collected and lysed in 50 mM Tris pH 7.4, 150 mM NaCl, protein was immunoprecipitated from 500 pg of total protein lysate with an anti—STATS antibody washed with trypsin (Santa Cruz, sc-835). Protein precipitates bound to protein G agarose beads were each sample. The buffer (50 mM Tris pH 8.0, 20 mM CaClz) and 33 ng of trypsin has been added to Protein samples were incubated at 37°C and aliquots were collected at the indicated time points. aliquots were ted to SDS-PAGE and blotted for STATS.

STATSb ] Activated STATS DNA binding assay: The DNA—binding capacity of STAT5a and was assayed by an ELISA-based assay (TransAM, Active Motif, ad, CA) following manufacturer instructions. Briefly, 5x10‘s K562 cells were treated with PU-H7l l and 10 M or STAT control for 24 h. Ten micrograms of cell lysates were added to wells containing pre-adsorbed treated cells the assay was performed consensus oligonucleotides (5’-TTCCCGGAA-3’). For control either a wild-type in the absence or presence of 20 pmol of competitor oligonucleotides that contains HeLa cells (5 pg per well) were used as or mutated STAT consensus binding site. Interferon-treated anti- ve controls for the assay. Afier incubation and washing, rabbit polyclonal anti-STATSa or STAT5b antibodies 0, Active Motif) was added to each well, followed by l-[PR-anti-rabbit secondary antibody (1:1000, Active Motif). Afier HRP substrate addition, absorbance was read at In this assay the nm with a reference wavelength of 655 run (Synergy4, , Winooski, VT). factor present in the absorbance is directly proportional to the quantity of DNA-bound ription units sample. Experiments were carried out in four replicates. Results were expressed as arbitrary (AU) from the mean absorbance values with SEM.

Quantitative Chromatin lmrrrunoprecipitatlon P) : Q-ChIP was made as previously described with modifications“. Briefly, 10“ K562 cells were fixed with 1% dehyde, lysed and sonicated on sonicator, Branson). STAT5 N20 (Santa Cruz) and HSP90 (Zymed) antibodies were added to the pre-cleared sample and incubated overnight at 4 °C. Then, protein—A or G beads were added, and the sample was eluted from the beads followed by detcrosslinidng. the ChIP products was DNA was purified using PCR purification columns (Qiagen). Quantification of performed by tative PCR (Applied Biosystems ) using Fast SYBR Green (Applied Biosystems). Target genes containing STAT g site were detected with the following primers: CCND2 (5-G'1'1'GTTCTGGTCCCTTI'AATCG and 5-ACCTCGCATACCCAGAGA), MYC (5- ATGCGTTGCTGGGTTA'ITIT and 5-CAGAGCGTGGGATGTTAGTG) and for the intergenic control region (5-CCACCTGAGTCTGCAATGAG and 5-CAGTCTCCAGCC'ITI‘G'1'1'CC).

Real time QPCR: RNA was extracted from PU—H71—treated and control K562 cells using RNeasy Plus kit (Qiagcn) following the cturer instructions. cDNA was synthesized using High Capacity RNA-to'cDNA kit (Applied Biosystems). We amplified c genes with the following s: MYC (5-AGAAGAGCATC'1'1'CCGCATC and 5-CC'1'1'TAAACAGTGCCCAAGC), BCL—XL (5- CCND2 (S-TGAGCTGCTGGCTAAGATCA and 5-ACGGTACTGCTGCAGGCTAT), CTT'ITGTGGAACTCTATGGGAACA and 5-CAGCGGTTGAAGCGTTCCT), MCLl (5- AGACCTTACGACGGGTTGG and S-ACATTCCTGATGCCACCTTC), CCNDl (5- CCTGTCCTACTACCGCCTCA and 5*GGCTTCGATCTGCTCCTG), HPRT (5- CGTC’I’I'GCTCGAGATGTGATG and S-GCACACAGAGGGCTACAATGTG), GAPDH (5- CGACCAC'ITTGTCAAGCTCA and 5-CCCTGTTGCTGTAGCCAAAT), (5- TGAGTGAAAGGGAGCCAGAAG and 5—CAGATGCCCCACTCACAAGA). Transcript abundance of 20 sec at 95 °C followed by 40 was detected using the Fast SYBR Green conditions (initial step cycles of 1 sec at 95 °C and 20 sec at 60 °C). The CT value of the housekeeping gene (RPLISA) was of the difference subtracted from the correspondent genes of st (ACT). The standard deviation the ACT values of the was calculated from the standard deviation of the CT values (replicates). Then, PU—H7l-treated cells were expressed relative to their respective control—treated cells using the AACT treated method. The fold expression for each gene in cells treated with the drug relative to l with the cells is determined by the sion: 2M”. Results were represented as fold sion standard error of the mean for replicates.

HSP70 knock-down. Transfections were carried out by electroporation (Amaxa) and the knockdown Nucleofector on V (Amaxa), ing to manufacturer’s instructions. HSP70 studies were performed using siRNAs designed as previously reported“ against the open reading u'ansfected frame of HSP70 (HSPAIA; accession number NM~005345). Negative control cells were active with inverted control siRNA sequence (HSP70C; con RNA technologies). The HSP70A (5’-GGACGAGUUUGAGCACAAG-3’) sequences against HSWO used for the study are is HSP70C (5'- and HSP7OB (5’- CCAAGCAGACGCAGAUCUUJ’). Sequence for the control GGACGAGUUGUAGCACAAG-3’). Three n cells in 2 mL media (RPMI supplemented with 1% L-glutamine, 1% penicillin and streptomycin) were transfected with 0.5 M siRNA according the manufacturer’s instructions. Transfected cells were maintained in 6—well plates and at 84h, lysed ed by standard Western blot procedures.

Kinnse screen85 (Figure 44). For most assays, kinase-tagged T7 phage s were grown parallel in 24-well blocks in an E. coli host derived from the BL21 strain. E. coli were grown to log— and incubated phase and infected with T7 phage from a frozen stock (multiplicity of infection = 0.4) and filtered with g at 32°C until lysis 0 min). The lysates were centrifuged (6,000 x g) cells and (0.2 pm) to remove cell debris. The remaining kjnases were produced in HEK-293 uently tagged with DNA for qPCR detection. Streptavidin’coated magnetic beads were treated with biotinylated small molecule ligands for 30 minutes at room ature to generate affinity resins for kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05 % Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non—specific phage binding. Binding reactions were assembled by combining 0.17x kinases, liganded afl'mity beads, and test compounds in 1x binding buffer (20 % SeaBlock, PBS, 0.05 % Tween 20, 6 mM DTT). Test compounds were prepared as 40x stocks in 100% DMSO and directly diluted into the assay. All reactions were performed in polypropylene 384-well plates in a final volume of 0.04 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the y beads were washed with wash buffer (lit PBS, 0.05 % Tween 20). The beads were then re~suspended in elution buffer (1x PBS, 0.05 % Tween 20, 0.5 tun non—biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR. KINOMErcan's ivity score (S) is a quantitative measure of compound selectivity. It is calculated by dividing the number of kinases that bind to the compound by the total number of distinct s tested, excluding mutant ts. TREErpoF" is a proprietary data ization sofiware tool developed by KINOMEscan“. Kinases found to bind are marked with circles in Figure 44, where larger circles indicate higher—affinity binding. The kinase dendrograrn was adapted and is reproduced with permission from Science and Cell ing Technology, Inc.

Lentivirul vectors, lentiviral production and K562 cells transduction. iral constructs of shRNA knock-down of CARMI were sed from the TRC lentiviral shRNA ies of Openbiosystem: pLKOJ-shCARMl-KDI (catalog No: RHSS979-9576107) and pLKO.l- shCARMl—KDZ (catalog No: RHSS979—9576108). The control shRNA (shRNA scramble) was Addgene plasmid 1864. GFP was cloned in to replace puromycin as the selection marker. Lentiviruses were produced by transient transfection of 293T as in the previously described protocol“. Viral supernatant was collected, filtered through a OAS-urn filter and concentrated. K562 cells were infected with high—titer lentiviral trated suspensions, in the presence of 8 ug/ml ene (Aldrich). Transduced K562 cells were sorted for green fluorescence (GFP) afier 72 hours transfection.

RNA extraction and quantitative Real-Time PCR (qRT-PCR). For R, total RNA was isolated from 106 cells using the RNeasy mini kit (QIAGEN, Germany), and then subjected to reverse-transcription with random hexamers (SuperScript Ill kit, Invitrogen). ime PCR reactions were performed using an ABI 7500 sequence detection . The PCR products were detected using either Sybr green I chemistry or TaqMan methodology (PE d tems, Norwallr1 CT).

Details for real—time PCR assays were described elsewhere“. The primer sequences for CARMl qPCR are TGATGGCCAAGTCTGTCAAG(forwa.rd) and TGAAAGCAACGTCAAACCAG(reverse).

Cell viablllty, Apoptosis, and Proliferation assay. Viability assessment in K562 cells untransfected or transfected with CARMl shRNA or scramble was performed using Trypan Blue.

This chromophore is negatively charged and does not interact with the cell unless the membrane is damaged. Therefore, all the cells that exclude the dye are Viable. Apoptosis analysis was assessed using fluorescence microscopy by mixing 2 pL of acridine orange (100 ug/mL), 2 11L of ethidium bromide (100 ug/mL), and 20 uL of the cell suspension. A minimum of 200 cells was counted in at least five random fields. Live apoptotic cells were entiated from dead apoptotic, ic, and normal cells by examining the changes in cellular morphology on the basis of ctive r and cytoplasmic cence. Viable cells display intact plasma membrane (green color), whereas dead cells display damaged plasma membrane (orange color). An appearance of ultrastructural changes, including shrinkage, heterochromatin condensation, and r degranulation, are more consistent with apoptosis and ted cytoplasmic membrane with necrosis. The percentage of apoptotic cells (apoptotic index) was calculated as: % Apoptotic cells = (total number of cells with apoptotic nuclei/total number of cells counted) x 100. For the proliferation assay, 5 x 103 K562 cells were plated on a 96-well solid black plate (Coming). The assay was performed according to the cturer’s indications (CellTiter—Glo scent Cell Viability Assay, Promega). All experiments were repeated three times. Where indicated, growth inhibition studies were performed using the Alamar blue assay. This reagent offers a rapid ive measure of cell viability in cell culture, and it uses the indicator dye resazurin to measure the metabolic capacity of cells, an indicator of cell viability.

Briefly, exponentially growing cells were plated in microtiter plates (Corning # 3603) and incubated for the indicated times at 37 “C. Drugs were added in triplicates at the ted concentrations, and the plate was incubated for 72 h. Resazurin (55 pM) was added, and the plate read 6 h later using the Analyst GT (Fluorescence intensity mode, excitation 530nm, emission 580nm, with 560nm dichroic mirror). Results were analyzed using the ax Pro and the GraphPad Prism sofiwares. The percentage cell growth inhibition was ated by comparing fluorescence readings obtained from treated versus control cells. The [CM was calculated as the drug concentration that inhibits cell growth by 50%.

Quantitative analysis of synergy between mTOR and HSP90 tors: To determine the drug interaction between pp242 (mTOR inhibitor) and PU—H71 (HSP90 inhibitor), the combination index (CI) isobologram method of Chou—Talalay was used as previously described"? This method, based on the median-effect principle of the law of mass action, quantifies synergism or antagonism for two or more drug combinations, regardless of the mechanisms of each drug, by computerized simulation. Based on algorithms, the computer software ys median-effect plots, ation index plots and normalized isobolograms (where non constant ratio combinations of 2 drugs are used).

PU—H71 (0.5, 0.25, 0.125, 0.0625, 0.03125, 0.0125 pM) and pp242 (0.5, 0.125, 0.03125, 0.0008, 0.002, 0.001 pM) were used as single agents in the concentrations mentioned or combined in a non constant ratio (PU~H71: pp242; 1:1, 1:2, 1:4, 1:7.8, 1215.6, 1112.5). The Fa (fraction killed cells) was calculated using the formulae Fa=1-Fu; Fu is the fraction of unaffected cells and was used for a dose effect is using the computer software (CompuSyn, Pararnus,New Jersey, USA). 6.3. Fluorescently labeled probes in cellular assays 6.3.1. Flow cytometry analysis of fluorescent-PU-l-Ul binding The human acute myelogenous leukemia (AML) cell lines MOLM~13 and MV4-11 cells were a gift from Dr. Stephen D. Nimer, MSKCC, and were maintained in RPM11640 medium supplemented with 10% fetal bovine serum (FBS) and leen/Strep in a humidified atmosphere of 5% CO; at 37°C. Cells were plated in 6-well plates at the density of 5 X 105 cells/mL, and treated with the ted derivatives (1 pM) at 37 °C for 4 h, For detection ofHSP90 binding in live cells, cells were washed twice with FACS buffer (PBS, 0.05% FBS), and prior to analysis, stained with lug/ml of DAPI (Invitrogen) in FACS buffer at room temperature. The cence intensities fiom live cells (DAPI ve) representing PU—H71-fluorescent derivative binding were captured by flow cytometry (LSR-II, BD Bioseiences), and analyzed by FlowJo software (Tree Star, Ashland, OR). For evaluation of HSP90 binding in fixed cells, cells were , fixed for 30 min with ED Cytofix buffer (BD, Biosciences, San Jose, CA), and then pen-neabilized for 30 min on ice using BD Perm buffer HI (BD Biosciences, San Jose, CA). Complete cell permeabilization was determined with DAPI. Cells were analyzed by flow cytometry as mentioned above. For competition tests, primary AML s at the y of 2><106 cells/ml were treated with 1 HM unconjugated PU-H7l for 4 h followed by treatment of 1 pM PU-H7l-F1TC2 for l h. Cells were collected, washed twice, stained for CD45 to distinguish blasts from normal lymphocytes incubated for 30 min at 4°C, washed and stained with 7-AAD in FACS butter to be analyzed by flow cytometry. 6.3.2. Flow cytometry. CD34 isolation CD34+ cell isolation was performed using CD34 MicroBead Kit and the automated magnetic cell sorter autoMACS according to the manufacturer's instructions (Miltenyi Biotech, Auburn, CA).

Viability assay~ CML cells lines were plated in 48-well plates at the density of 5X105 cells/ml, and d with indicated doses ofPU-H7l. Cells were ted every 24 h, stained with Annexin V- V450 (BD Biosciences) and 7-AAD (Invitrogen) in Annexin V buffer (10 mM HEPES/NaOH, 0.14 M NaCl, 2.5 mM CaClz). Cell viability was analyzed by flow n'y (BD Biosciences). For patient samples, primary blast crisis CML cells were plated in 48—well plates at 2><106 ml, and treated with indicated doses of PU-H7l for up to 96 h. Cells were stained with CD34—APC, CD3 8-PE- CY7 and CD45-APC-H7 antibodies (BD Biosciences) in FACS buffer (PBS, 0.05% FBS) at 4 °C for min prior to Annexin V/7-AAD staining. PU»H7I binding assay — CML cells lines were plated in 48-well plates at the density of 5X105 cells/ml, and treated with 1 uM PU-H71-FITC. At 4 h post treatment, cells were washed twice with FACS . To measure PU-H71-FITC binding in live cells, cells were stained with 7-AAD in FACS buffer at room temperature for 10 min, and analyzed by flow cytometry (BD Biosciences). At 48h, and 96 h post PU~H7l-FITC ent, cells were stained with n V—V450 (BD Biosciences) and 7-AAD in Annexin V buffer, and subjected to flow cytometry to measure viability deter-mined by AnnexinV/7AAD double negative gates. To evaluate the binding of —FITC to leukemia patient samples, y bp or cpCML cells were plated in 48-well plates at 2><106 ml, and treated with 1 uM PU~H71-FITC. At 24 h post treatment, cells were washed twice, and stained with CD34-APC (or CD34—PECy7), E-CY7 (or CD38-PE) and CD45-APC-H7 antibodies in FACS buffer at 4°C for 30 min prior to 7—AAD staining. At 48h, and 96 h post treatment, cells were stained with CD34—APC (or CD34-PECy7), CD38~PE—CY7 (or CD38-PE) and CD45-APC-H7 antibodies followed by Annexin V—V450 and 7-AAD staining to measure cell viability in blast, lymphocytes and CD34+ cell tions. For competition test, CML cell lines at the density of 5X 105 cells/ml or y CML samples at the density of 2X10fi cells/ml were treated with 1 HM unconjugated PU—H71 for 4 h followed by treatment of 1 HM PU-H71-FITC for l h. Cells were collected, washed twice, stained for 7-AAD in FACS buffer, and analyzed by flow cytometry. HSP90 ng — Cells were fixed with fixation buffer (BD Biosciences) at 4°C for 30 min, and perrneabilized in Penn Buffer III (BD Biosciences) on ice for 30 min. Cells were stained with anti—HSP90 phycoerythrin conjugate (PE) (F-S clone, Santa Cruz Biotechnologies; CA) for 60 minutes Cells were washed and then analyzed by flow cytometry. Normal mouse IgGZa—PE was used as isotype l. 6.3.3. Fluorescent microscopy analysis of PU—H7l-FITC2 (9) binding MV4—ll cells were plated in 48—well plates at the density of 5 X 105 cells/ml, and treated with 1 HM PU—H71~FITC2 or PU-H71-NBD1. At 24 h post treatment, cells were blocked with 3% BSA/FACS buffer at room temperature for 30 min and incubated with NaVKtATPase u. 1 antibody (Novus Biologicals) in 3% BSA/FACS buffer at room temperature for 30 min. Cells were washed three times with FACS buffer and ted with goat anti-rabbit Alexa Fluor 568 (Invitrogen) in 3% BSA/FACS buffer at room temperature for 20 min. Cells were then washed three times with FACS bufl‘er, incubated with 1 [lyml DAPI in FACS buffer for 10 min, mounted on slides, and observed under al microscope (Zeiss).

Western Blotting: Cells were either treated with the fluorescent PU-H7l derivatives, TEG- FITC or DMSO (vehicle) for 24 h and lysed in 50 mM Tris, pH 7.4, 150 mM NaCl and 1% NP40 lysis buffer supplemented with leupeptin (Sigma Aldrich) and aprotinin (Sigma Aldrich). Protein concentiations were deten'nined using BCA kit (Pierce) aCcording to the manufacturer’s instructions.

Protein lysates (50 ug) were ophoretically resolved by SDS/PAGE, transferred to nitrocellulose membrane and probed with the following y antibodies against: Raf-1 (1:300, Sc-l33; Santa Cruz), FLT3 (l : 1000, 50-430; Santa Cruz) and Bvactin (122000, A1978; Sigma). The membranes were then incubated with a 1:3000 dilution of a corresponding horseradish peroxidase conjugated secondary antibody. ion was performed using the ECL—Enhanced Chemiluminescence Detection System ham Biosciences) according to manufacturer’s instructions.

WO 09657 6.3.4. Tumor stem cell assays PU-H7I binding assay -— Primary samples were plated in 48-well plates at 2H0“ cells/ml, and treated with 1 uM PU-H71-F1TC2 or TEG-FITC. At 4 h post treatment, cells were washed once with FACS buffer (PBS, 0.05% FBS), and stained with CD34-APC, CD38-PE-CY7 and CD45-APC- H7 antibodies (BD Biosciences) in FACS buffer (PBS + 0.5% FBS) at 4°C for 30 min prior to 7—AAD (Invitrogen) staining. The MFI of bound PU-H7l-FITC2 was evaluated in the BD-LSR II flow cytometer) and normalized to TEG-FITC. Values were represented as ratio of binding of PUH7 1- FITC in LSCs (CD45dim. CD34+, CD38- gate) relative to lymphocytes (CD45hi vs SSC gate).

Stem cell viability assay - Primary cells were plated in l plates at 2X10‘S cells/ml, and treated with 1 uM PU-H71 for 48 h. Cells were stained with PC, CD38-PE—CY7 and CD45- APC—H7 antibodies (BD Biosciencas) in FACS buffer (PBS, 0.05% FBS) at 4 EC for 30 min prior to Annexin V-V450 (BD Biosciences) and 7-AAD staining in Annexin V buffer (10 mM I-[EPES/NaOH, 0.14 M NaCl, 2.5 mM CaClz). Cell ity was determined as the percentage of annexin V-/7AAD- cells normalized to untreated cells.

Statistical Analysis. Unless otherwise indicated, data were analyzed by unpaired ed t tests as implemented in GraphPad Prism (version 4; GraphPad Sofiware). A P value of less than 0.05 was considered significant. Unless ise noted, data are presented as the meaniSD or meantSEM ofduplicate or triplicate ates. Error bars ent the SD or SEM of the mean. If a single panel is presented, data are representative of 2 or 3 individual experiments. 6.3.5. tion of PU—FlTC binding in primary leukemia samples and in leukemia and solid tumor cell lines Procedure: Peripheral blood (PE) or bone marrow (BM) mononuclear cells from leukemia ts are either isolated from fresh samples using Ficoll density gradients or from viably frozen aliquotes. Cells are treated with 1 uM PU-FTTC and at 4h post-treatment cells will be washed and d with antibodies to distinguish different subpopulations (CD45~APC-H7 to identify blasts (CD45 dim vs SSC gate) and lymphocytes (CD45hi vs. SSC gate» and 7—AAD staining to discriminate dead cells. PU—FITC binding will be ted using a BD LSR~II instrument. The instrument is set up using CST beads prior the experiment. A calibration curve using commercially available beads labeled with difi‘erent fluorescent intensities (Quantum Alexa Fluor 488 kit) is used for the quantitation of AF488/F[TC fluorescence intensities for PU-FITC binding. Binding to lymphocytes is used to ine background binding of PU~FTTC for each patient sample. PU-FITC binding is evaluated as the fold difference in mean fluorescence intensity (MFI) of blasts relative to lymphocytes. A non—specific l ("IEG-FITC) is used to determine non—specific binding. We propose to evaluate at least 100 primary leukemia samples. For each assay we use a minimum of 900,000 total mononuclear cells. We collect at least 50,000 events for the analysis. Analysis is performed using FlowJo software. Binding of PU-FITC on a larger panel of commercially ble cell lines (lymphomas, multiple myeloma, breast cancer, prostate, pancreatic and lung cancer cell lines) can be evaluated. Because cell lines do not have their own al l, we use the calculated delta MFI for TEG—FITC substracted from PU—FITC (quantified using the calibration curves performed with the fluorescent beads) or the binding to HL—60 cells as described above.

Emigration gt gym-m”' Ill :9 mgrum§: To determine the sensitivity of all samples evaluated for PU-FITC binding, cells are plated in 96-well plates and treated with sing doses of PU—H71. In cell lines and a select subset of primary s, where sufficient material is available, their sensitivity is also tested to other chemically distinct Hsp90i (l7-AAG, NVP-AUY922 and 0 or other Hsp90i currently in clinical evaluation) (5). Cells are collected 48h later and stained with CD45eAPGH7 to distinguish blast and normal lymphocytes (for primary cells). Cells are then washed and stained with n V—V450 and 7-AAD in Annexin V buffer (10 mM HEPES/NaOH, 0.14 M NaCl. 2.5 mM CaC12). Cell viability will be analyzed by flow cytometry.

Stgtim‘I gansiderations: The primary goal is to evaluate our assay for fold PU binding so that it best distinguishes Hsp90i responders versus nonresponders. These experiments are done in vitro, using 150 samples, including primary samples and cell lines. Response is defined if greater than 50% of cells are alive. The area under the receiver operating characteristic (ROC) curve is calculated to assess the ability of fold PU binding in distinguishing se versus no response. For our purposes, we define an AUC of .37 or higher (compared to a null AUC of .75) to indicate that fold PU binding can distinguish Hsp90i responders from the ponders. For a sample of 150, and assuming a 30% response rate, we will have 80% power to detect a difference in AUC from .75 to .87 with a type I error of 5%. As the response rate decreases, the power also ses. As an example, if we assume a 20% se rate, we will have 80% power to detect a difference in AUC fi'om .75 to .89 with a type I error of5%. 6.3.6. Measuring PU-FITC binding in Live Tumor cell lines in the presence or absence of pgp inhibitors Adherent cancer cell lines are plated and allowed to adhere ght at 37°C. 5% C02. Cells are either pre-treated (SuM) with the pgp inhibitors (PSC 833, Tocris Biosciences or Reversan, Tocris ences) or DMSO vehicle for 2hrs prior to the addition of media containing luM of DMSO, PU- FITC9 (a negative PU—FITC control) and C2 (PU-H71 drug labeled with FITC) . Cells are incubated with the FITC drug or control conjugates for an additional 4hrs, 37“C, 5% C02. Cells are then trypsinized, washed twice with lXFACS buffer (lXPBS+0.5% FBS) and pellet resuspended in soon 1X FACS buffer containing a cell viability dye (lug/ml DAPI). Samples are run on the BD LSRII flow ter and 10-20,000 events collected. Prior to each experimental run, lasers are normalized on the LSRJI with CST beads (BD Bioscicnces). The binding ofPU~FITC in tumor cells WO 09657 is determined in DAPl~ve live cells by measuring the FTTC median fluorescence intensity (MFI). The non~specific FITC signal from the FITC9 and DMSO controls is subtracted from the PU»FITC signal. 6.3.7 Dissociated Tumor Cells and Circulating Tumor Cells [0451) Dissociation of Tumor Cells and measurement of PU-FITC binding- EGFR+ tumors ed from mice were dissociated according to manufacturer’s protocol (tissue dissociation kit, Millipore). Briefly, 1 g of fresh tissue is cut into small pieces (6.3., ~10-20 pieces per g tissue) using a l. The minced tissue is washed twice in PBS and transferred in iation solution for 50mins at 37°C with gentle agitation. Following dissociation, the cells are washed, strained using a sieve and un-dissociuted tissue fi'agments discarded. The dissociated tissue is transferred to a fresh tube containing 1X Dissociation Buffer with se Inhibitors. Cells are washed twice in the dissociation buffer containing protease inhibitors. Cells are re-suspended to l x 106 cells/ml in media. Samples are equally divided into 3 tubes cells and d with luM (per 1 x 106 cells/ml) ofDMSO, PU—FITC9 (negative FITC control) and PU—FITCZ (PU-H7] dnlg labelled to FlTC) for 4hrs at 37°C, 5% C01. l EGFR+ cell lines [Aspcl (Low binding); BxPc3 (high binding)] spiked with thawed PBMC’s obtained from the blood bank are stained concurrently with the dissociated tumor cells as mentioned above. Cells are washed twice with lXFACS buffer (lXPBS+O.5% FBS) and stained for 30mins on ice with anti-human EGFR—PE (BD Biosciences), anti-human CDl4-APC-Cy7 (ebiosciences) or CD14— as Red (invitrogen) and anti-human CD45-APC (ebiosciences). Cells are then washed twice with IXFACS buffer (lXPBS+O.5“/o FBS) and pellet re—suspended in SOOul 1X FACS buffer containing a cell viability dye (1‘1ng DAPI). Samples are run on the BD LSRH flow cytometer and 100~200,000 events collected. Lasers are normalized on the LSRII with CST beads (BD ences) prior to each experimental run. The drug accumulation ofPU—FITC in tumor cells is determined by measuring the FITC Median Fluorescence Intensity (MFD in EGFR+ cells (EGFR+CD45-) and EGFR— cells (EGFR-CD45+CDl4—). The non-specific FTTC signal from the PU-FITC9 and DMSO ls is subtracted. Values are calculated as a ratio of the MFI ofTumor (EGFR+CD45—CDl4-): MFI of EGFR’CD45+CD14- cells. To normalize MFI values across patient Quantum FITC standardizations beads (Bangs laboratories) will be run with each sample and a standard curve ted. This allows quantitation of the FITC fluorescence intensity in molecules of equivalent soluble hrome (MESF units). PU-FlTCZ accumulation in each sample can be quantitated across samples by extrapolating values generated from the standard curve [0452) Measure PU—FITC binding to EpCAM+ circulating tumor cells in PBMC’s: All experimental blood ng procedures were performed under the Institutional Review Board— approved protocols at Memorial Sloan Kettering Cancer Center. 8mls of peripheral blood is withdrawn into anticoagulant EDTA tubes from cancer patients enrolled on PU-H7l clinical trials.

PBMC’s are ed on ficoll gradients, cells are counted and viability ined by trypan blue dye exclusion assay. PBMC’s are spun down and pended to 2 x 106 cells/ml. Samples are equally 2012/045864 divided into 3 tubes and treated with IuM (per 2 x 106 cells/ml) of DMSO. PU—FITC9 (negative FITC l) and PU-FITC2 (PU-H7] drug labelled to FITC) for 4hrs at 37°C, 5% C01. Controls EpCAM+ cell lines [AspcI (Low binding); BxPc3 (high binding)] are spiked with freshly thawed PBMC's obtained from the blood bank and stained concurrently with the patient PBMC’s mentioned above. Cells are washed twice with lXFACS buffer (lXPBS+0.5% FBS) and stained for 30mins on ice with anti-human EpCAM-PE (miltenyi biotech), anti-human PC—Cy7 (ebiosciences) or CD14- PE-Texas Red (invitrogen) and anti~hurnan CD45-APC (ebiosciences). Cells are then washed twice with lXFACS buffer (lXPBS+O5% FBS) and pellet ended in 50011] 1X FACS buffer containing a cell viability dye (lug/ml DAPI). Samples are run on the BD LSRII flow cytometer and IOU-200,000 events collected. Lasers are normalized on the LSRH with CST beads (BD Biosciences) prior to each experimental run. The binding ofPUvFITC in tumor cells is determined by measuring the FITC Median Fluorescence Intensity (MFI) in EpCAM+ cells (EpCAM+CD45-) and EpCAMv cells (EpCAM-CD45+CDI4»). The non-specific FITC signal from the FITC9 and DMSO controls is subtracted. Values are calculated as a ratio of the MFI of Tumor (EpCAM+CD45-CDl4—): MFI of EpCAM-CD45+CD14— cells. To normalize MFI values across patient Quantum FITC standardizations beads (Bangs laboratories) will be run with each patient sample and a standard curve generated. This allows quantitation of the FITC fluorescence intensity in molecules of equivalent soluble fluorochrome (MESF . PU—FITC2 binding in each sample be quantitated across samples by extrapolating values generated from the rd curve.

Modifications to protocol: PBMC’s obtained from patients will be split into 2 tubes. Both will be stained with PU—FITC as mentioned above, and d with EpCAM-PE or the isotypic control and CDl4-PE-Texas red and CD45-APC. Samples will be processed as mentioned above.

Threshold ratio values will be determined using PU-FITC low and high accumulating cell lines spiked with PBMC’s 6.3.8. Analysis of PU-H'Il binding in tissues Examination of the sensitivity of gastric patient tumor specimens to HSP90 inhibitors using a viva tumor tissue resources and correlate with l-FITC staining: Immediately following surgical removal of the gastrectorny specimen the tissue is orted to the Tissue ement Services (TPS) area of the Pathology suite. Once the lesion is located, tissue is harvested under e conditions. The en size d for evaluation is typically 5-]0mm x 5-10 mm.

Every effort is made to sample the most viable area. Distant from the lesion, a specimen of equivalent size is d representative of normal gastric epithelial tissue. Both specimens are placed in minimal essential media (MEM) with 1% penicillin/streptomycin. A small portion of the lesion the entire piece of normal c epithelial tissue undergo a “snap" freeze for future molecular evaluation by WB. The remaining portion of the lesion (gastrectomy) is processed for pathological evaluation For every lesion pathology provides IHC for proliferation markers, epithelial markers and one hemawxylin/eosin (H&E) stained slide accompanied by 10 unstained to be further ed for staining with fluorescein labeled PU—H7l (PU-FITC). Formalin-fixed paraffin-embedded sections or frozen sections are analyzed for PUH7l-FITC2 staining. In parallel, a portion of the tissue is prepared for ex vivo analysis of the sensitivity of the tumor to PU—l-{7 l . From preliminary analyses we have learned that fresh tissue slicing preserves the cancer cells in the endogenous environment of the surrounding tissue. In this method, the tissue (i.e. lesion) is placed in a plastic mold and embedded in 6% agarose. The e~embedded tissue is then mounted on the stage of the Vibratome that is submersed in a chilled reservoir (for tissue preservation) containing MEM with 1% penicillin/ streptomycin. The tissue is then sliced using metal blades produc'mg serial sections of the lesion that are 200 pm thick. Each section (minus the surrounding agaroseembedding media) is immediately placed in a l tissue culture plate containing MEM with 1% penicillin/streptomycin. From a 5mm x 5mm piece of tissue approximately 25 sections are produced. This allows for replicate analyses of tissue sections d with a minimum of 4 doses of the Hsp90 tor and one with vehicle only. Replicates can be assayed by both ll-IC as well as viability assays (automatic plate reader or cytospin preparation) once tissue section undergoes enzymatic dissociation by brief exposure to dispase. The degree of apoptosis induced by PUvH7l in these gastric tumor slices will then be correlated with PU—H7l—FITC staining. PU-H7l uptake (as measured by lHC scoring of PU—H7l- FITC staining) correlates with the sensitivity of these tumors to Hsp90 inhibition. Similar protocols have been developed for breast and pancreatic cancer. 6.4. abeled probes in cellular assays scent probe treatment: Adherent cells were treated when 70% confluent with SuM PUH7l—ANCA for 1 hour under standard tissue culture ions. Following treatment the media was aspirated, the slides washed 3 times with PBS and then ished with complete media.

Fluorescence on Spectrum: Fluorescence emission was determined using an inverted fluorescent confocal cope (Leica SP5) and scanning cancer and ncerous cells on chamber slides at Snm increments from 400mm to 600nm Confocal Microscopy: Following treatment cence of cells was observed using an inverted fluorescent confocal microscope (Leica SP5). Confocal images were ed at the appropriate fluorescence emission peak of the bound high affinity species (~530nm). Images were uploaded into NIH Image] sofiware. The fluorescence ity of duals cells from several random fields were measured and corrected for background.

Response Modeling: A standard curve of [CM vs. fluorescence density integrity was established for 12 breast cancer cell lines and 2 normal breast cell lines. Cell lines grown on militi- well plates where five wells were analyzed for response (vehicle only, 0.25pM, 0.50 pM, 1,0 pM and 2.5 uM PUH7 l—treated) and one well was analyzed for fluorescence intensity of bound (PUH7l— ANCA—treated). The lCm of all cell lines was plotted on the y-axis and the density integrity (fluorescence intensity) was plotted on the x-axis, This standard curve is used to model and predict response of breast cancer specimens, such as those obtained from biopsy, surgery or fine needle aspirates, to HSP90 therapy.

Response Measurement in Cancer Biopsies: Patient biopsies, once procured are placed in sterile saline and delivered immediately to the tissue ement area of the ogy Department A portion of the lesion is taken for fresh tissue ning med on a ome (Leica VT1000).

Sections 200nm thick are cut and immediately placed in multi-wcll plates in minimal essential media with growth factors and otics and placed in 37°C in an air*5% C02 atmosphere at constant humidity. The sections are then treated with the PUH7l-ANCA for 45 minutes to 1 hour. Following treatment the 'section is washed 2X with PBS and then OCTembedded (fresh frozen). The OCT— embedded en is then cut into several 4 pm thick sections and transferred to charged . The nuclear counterstain, DAPI is then applied to the slide. The slides are then observed on a confocal microscope (Leica SP5) and ed at 530nm fluorescent emission peak to determine the percentage ofprobe—bound to the oncogenic HSP90 species. 6.5. Studies with radiolabeled HSP90 inhibitors Reagents. I”‘I]—PU—H7l and {'Z‘H—PUHH were synthesized and purified as previously reported'”.

Cell Lines. The MDA-MB-468 human breast cancer cell line was obtained from the American Type Culture Collection. Cells were cultured routinely in DME-HG supplemented with % FBS, 1% L-glutamine, 1% penicillin, and streptomycin.

In V‘rvo Studies. All animal studies were conducted in compliance with MSKCC’s Institutional Animal Care and Use Conunittee (IACUC) guidelines. Female athymic nu/nu mice -M, 20 — 25 g, 6 weeks old) were ed from Harlan Laboratories and were allowed to atize at the MSKCC Vivarium for 1 week prior to implanting tumors. Mice were provided with food and water ad libitum. MDA~MB-468 tumor xenogmfis were established on the forelimbs ofmice by sub-cutaneous (s.c.) injection of l X107 cells in a 200 itL cell suspension of a 1:1 v/v e of PBS with reconstituted basement membrane (BD MauigelTM, Collaborative Biomedical Products Inc., Bedford, MA). Before administration, a solution of PU—H7l was formulated in PBS (pH 7.4).

In viva Blodistribution Studies. For acute in viva biodistribution studies, mice (n=5) with MDA-MB-468 breast tumor xenografis on forelimbs were ed intravenously in the tail vein with 0.93—1.1 MBq (25 - 50 “C0 of [‘Z‘u-PU—Hfl or [‘3'I]-PU»H71 in 200 uL of saline. For dose estimation experiments groups of mice (n=5) were injected with [mI]-PU-H71 or [mI]~PU-H7l diluted in a sterile solution containing PU.H7l corresponding to 5, 25, 50 or 75 mg/kg ofbody weight of mice. Activity in the syringe before and alter administration was assayed in a dose calibrator (CRCoISR; Capintec) to determine the activity stered to each animal. Animals (n4 per group) of the tracer and were euthanized by CO; asphyxiation at different time points poshadministration Part of tumor tissue was frozen immediately organs including tumor(s) were harvested and weighed. post—harvesting for further mical and histological analyses. ”"1 was measured in a scintillation 400-600 keV y—counter (Perkin Elmer 1480 Wizard 3 Auto Gamma counter, m, MA) using and decay-corrected to the time of injection, and the energy window. Count data were background measured percent injected dose per gram (%ID/g) for each tissue sample was calculated by using a calibration factor to convert count rate to activity and the ty was ized to the activity injected to yield the activity concentration in %lD/g.

Small-Animal PET Imaging. For animal imaging studies mice with MDA-MB-468 breast cancer xenogrnfts on forelimbs were used. g was performed with a dedicated small- animal PET scanner (Focus 120 microPET; Concorde Microsystems, Knoxville, TN). Mice were maintained under 2% isoflurane (Baxter Healthcare, Deerfield, IL) anesthesia in oxygen at 2 Umin during the entire scanning period. In suitable cases, to reduce the thyroid uptake of free iodide arising from metabolism of tracer mice received 0.01% potassium iodide solution in their drinking water starting 48 h prior to tracer administration. For PET g each mouse was administered 9.25 MBq (250 uCi) of [ml]~PU-H7l via the tail vein. Sequential list—mode acquisitions (10-30 min) were obtained for each animal at various time points post tracer administration. An energy window of420— 580 keV and a coincidence timing window of 6 us were used. The resulting list~mode data were sorted into 2-dirnensional histograms by Fourier rebinning; transverse images were reconstructed by filtered back projection (FBP). The image data were corrected for non-uniformity of scanner response, dead—time count losses, and al decay to the time of ion. There was no correction applied for attenuation, scatter or partial-volume averaging. The measured reconstructed spatial resolution of the Focus 120 is l.6-mm FWHM at the center of the field of view. ROI analysis of the reconstructed images was performed using ASIPro sofiware (Concorde Microsystems, Knoxville, TN), and the maximum pixel value was recorded for each tissue/organ ROI. A system calibration factor (i.e., uCi/mIJcps/voxel) that was derived from reconstructed images of a mousesize filled cylinder containing ”F and was used to convert the l2"I voxel count rates to activity concentrations (after adjustment for the ”‘1 positron ing ratio). The resulting image data were then normalized to the administered activity to pararneterize the microPET images in terms of %ID/g (corrected for decay to the time of injection).

LC—MS/MS analyses. Frozen tissue was dried and weigled prior to homogenization in acetonitrile/HZO (3:7). PU-H7l was extracted in methylene chloride, and the organic layer was separated and dried under vacuum. Samples were reconstituted in mobile phase. Concentrations of PU—H7l in tissue or plasma were determined by erformance MS. PU-H7 l-da was added as the internal standardm. Compound is was med on the 6410 LC-MSfMS system (Agilent Technologies) in multiple reaction monitoring (MRM) mode using positive-ion electrospray ionization. For tissue samples, a Zorbax Eclipse XDB-C 1 8 column (2.1 x 50 mm, 3.5 um) was used for the LC separation, and the analyte was eluted under an isocratic condition (80% HzO+O.l% HCOOH: 20% CH3CN) for 3 minutes at a flow rate of 0.4 mI/min. For plasma s, a Zorbax Eclipse XDB—Cls column (4.6 x 50 mm, 5 tun) was used for the LC separation, and the analyte was eluted under a gradient condition (H;O+0. 1% HCOOH2CH3CN, 95:5 to 70:30) at a flow rate of 0.35 ml/tnin.

Pharmacndynamic analyses. For protein analysis, tumors were homogenized in SDS lysis buffer (50 mM Tris, pH 7.4, 2% SDS) and subjected to Western blot analysis. Protein concentrations were determined using the BCA kit e) according to the manufacturer’s instructions. Protein lysates (20—100 ug) were ophoreticallyr ed by SDS/PAGE, transferred to nitrocellulose membrane, and probed with the indicated primary antibodies: Anti-Hsp90 from mouse , SPA— 830; Stressgen), anti~Akt from rabbit (1:500, 9272; Cell Signaling), anti—phospho~A1a (Ser 473) fi'om rabbit (1:500, 92718; Cell Signaling), anti-PARP (p85 fragment) from rabbit (1:250, G734l; Promega). Membranes were then incubated with a 1:5,000 dilution of a peroxidase-conjugated corresponding secondary antibody. Equal loading of the protein samples was confirmed by parallel Western blots for B-actin (115,000, ab8227—50; Abcam). Detection was performed using the ECL Enhanced Chemiluminescence Detection System (Amersham Biosciences) according to the manufacturer’s instructions.

Densitometry. Gels were scanned in Adobe Photoshop and quantitative densitometric analysis was performed using n-It 5. l. y Studies. Mice (1115) hearing -468 tumors reaching a volume of 100—150 mm3 were treated i.p. (i.p.) using different doses and schedules, as indicated. Tumor volume was determined by measurement with Vernier calipers, and tumor volume was calculated as the product of its length x width2 x 0.4. Tumor volume was expressed on indicated days as the median tumor volume :1: SD indicated for groups ofmice. t (%) tumor growth inhibition values were ed on the final day of study for drug~treated compared with vehicle treated mice and are calculated as 100 x { l atedpim 4., _ Treatedmy .)/(Controlpi,,.1 m - Controlmy .)]} . Tumor regression values were determined by calculating the ratio of median tumor volumes at the time when ent was ted to median tumor volume on the final day of study for a given treatment group. Percent (%) tumor regression is 100 x [1 —— edpim. mflrmtedm, .)].

Acr'ldlne Orange/Ethidium Bromide Cell Viability Assay. The Easycount ViaSure kit (Immunocon) was used in conjunction with the Easycount system to count dead and live cells automatically. The ViaSure Staining Reagent uses a mixture of ready‘to-use nucleic acid dyes, acridine orange and ethidium bromide, to identify live and dead cells, respectively, in a single test. ne orange is taken up by both viable and non-viable cells and emits green fluorescence if intercalated into double~stranded nucleic acid (DNA) or red fluorescence ifbound to -stranded nucleic acid (RNA). Ethidium bromide is taken up only by non-viable cells and emits red fluorescence by intercalation into DNA. Viable cells have uniform green nuclei with organized structure. Early apoptotic cells (which still have intact membranes but have d to undergo DNA cleavage) have green nuclei, but perinuclear chromatin condensation is visible as bright green patches or fragments. Late apoptotic cells have orange to red nuclei with condensed or fragmented chromatin.

Necrotic cells have a uniformly orange to red nuclei with organized structure. A total of at least 200 cells per condition were counted.

Simulations. A double exponential fimction x(t) = a. exp(-fllt) + a; exp(—flzt) was fitted to the measurements In general, sum of exponentials have been used to analyze pharmacokinetics data'”.

The lized Nonlinear Model package (http://cran.rproject.org/web/packages/gnm/index.htrnl) in R statistical ge (wwwrprojectorg) has been used to fit the model to the data. Several fits were initially sought, and the best one was ed for further simulations of different drug administration scenarios, A script in R has been developed.

Statistical Analysis. Unless ise ted, data were analyzed by unpaired 2-tailed t tests as implemented in GraphPad Prism (version 4; GraphPad Sofiware). A P value of less than 0.05 was considered significant. Unless otherwise noted, data are presented as the meanistandard deviation (SD) or mearrtstandard error of the mean (SEM) of duplicate or triplicate replicates. Error bars represent the SD or SEM of the mean. If a single panel is presented, data are entative of 2 or 3 individual experiments.

Human Studies. A once-daily dose of a potassium iodide solution (SSKI) is administered for 14 days beginning day before ion. A single tracer-injection 0 mCi; < 100 pg) is administered by slow peripheral IV bolus. Patients undergo non-invasive PET-based assays at 0h (dynamic scan), 3—4h, 24b and 40-80 h and/or 160200 b (static . In our PU-PET pilot trial, a more demanding schedule has been well-tolerated by patients without difficulties in t recruitment or ‘drop-out’. Timevpoints are selected based upon our human ml—PUH71 PK data: (1) I2"I-PUH71 clears rapidly from the blood circulation, in a bi-exponential manner — a rapid clearance (blood tm320 min), ed by slow clearance at negligibly-low blood- levels; (2) tumor PUH7l-tracer concentrations by PET imaging, have been variable, being either quantitatively greater than or equivalent to background tissue tracervlevels. with differential uptake and/or retention of tracer being evident from 4-to-24 or 48 and beyond 72h afier tracer-injection; (3) PUH7l-avid tumor concentrations have been either sustained or have shown a monoexponential-rype clearance, over a period of days.

We have optimized our mI~PUH7l PET image acquisition protocol to ensure robust count— statistics. A dedicated research PET/CT scanner (GE Discovery DSTE) obtains quantitative biodisn-ibution images with attenuation-, decay- and scatter-corrections and adjustment for system ivity. CT scans for attenuation correction and anatomic co-registration are performed prior to tracer—injection, scaled to body weight (up to 85 mA for 281 kg). The CT protocol is designed to suffice for anatomic localization of tracer-signal and for attenuation correction, while minimizing radiation exposure. No intravenous or oral radiographic contrast is administered. PET data are reconstructed using a standard ordered subset expected maximization ive algorithm.

Emission data is ted for scatter, attenuation and decay.

PU-tumor avidity is a binary outcome defined on visual inspection of PET imagery, as tumor tracer-signal judged to be distinctly-higher than reference blood pool or organ background, at any time-point. Tumor PU-H7l concentrations are assessed quantitatively from PET data by (1) t tumor Standardized Uptake Value (SUV) at any time-point; and (2) the integral oftumor SUV as a on of time post PU—H71—inj ection between the first and last PET time-points (i. e. PU-PET SUVmax readings or molar concentrations calculated from such SUV readings are graphed as a function of time post-PU stration and AUC values and average tumor concentrations ofHsp90 inhibitor ated as shown in Figure 27).

These parameters (or other as d) are then correlated with response on the therapeutic trials with HSP90 inhibitors. On these studies, tumor response is evaluated per-tumor and per-patient.

The timing oftumor response assessments are made as per therapy trial protocol. Tumor response assessment is from al imaging performed closest to the end of 1“ cycle (one cycle typically being 3-5 weeks). Tumor response is defined by RECIST 1.1, for CT or MRI, and/or PERCIST 1.0 for FDG PET—CT (36,37). If discordant, the more favorable response will be used. Clinical response will be judged according to —specific therapy trial response criteria.

Tumor PUH71-avidity data on PET imagery is dichotomized in two ways: (1) by using a binary e of ative/visual judgment of tumors as being ‘avid’ or ‘non-avid’ (discussed ; (2) by using ROC curves calculated for clustered data, a cutpoint for tumor-avidity that has the best operating characteristics in discriminating between tumor~response versus no response will be calculated (40).

For both dichotomizations, the sensitivity, specificity and other measures of classification are calculated using patient response as the truth. If each patient has a single tumor, a sample size of 40 patients will produce a two-sided confidence interval with a maximum width of .324. As the number of tumors per patient increases, the confidence interval width will generally decrease overall. An exploratory analysis designed to find a patient-level summary statistic for tumor-avidity that best correlates with overall RECIST patient—level response is performed. Patient-level summary statistics for avidity that the investigated include 1) the highest tumor SUV and/or AUC and 2) the e of all tumor SUV and/or AUCs. This patient-level data is used to construct an ROC curve and a cutoff for t summarized tumor-avidity that is based on the Youden index and the point closest to (0,1) will be calculated.

In preclinical studies, uptake and retention at a n time point or over a period of time is best correlated with the observed response. Preliminary data suggest that both prolonged tumor retention (for over 24-4811) as well as tumorcexposure as measured by AUC are pertinent parameters to t tumor response to Hsp90 inhibition therapy. Specifically, in preliminary investigations in MDA-MB-468 tumors, several parameters were ated such as the tumor area—under-the-curve (AUC), the average and minimum tumor concentrations ofPU-H71, target occupancy measured as the average %Hsp90 sites occupied and recognized by PU-H71 ((% Occupied Hsp90 sites),.,§) over the time oftreatment. We found that the average tumor concentration of PU-H71 recorded over the time of treatment, the tumor AUC and the %”oncogenic Hsp90” occupancy correlated cantly well with the magnitude of the ed anti—tumor efl'ect (r2= 0.8162, 0.8188 and 0.7559, tively) (Figure 37). These suggest that most appropriate parameters to predict response to Hsp90i are those that measure ependent exposure, i. e. uptake and retention. 6.6. s to identify markers tive of npoptotic sensitivity to HSP90 in breast cancer and AML Cells: Kasurni-l, SKNO-l, MOLM-l3, M0—91, HEL, HL-60, THP-l, MV4-11 were grown in RPMI media supplemented with 10inM HEPES, 4.5g/L glucose, 1.5 g/L sodium bicarbonate, 1mM sodium pyruvate, 10% FBS, and 1% perncillin/sneptomycin. The stable transfectants of FL5.12 were previously described (18-Karnauskas 2003). In brief, for the generation ofFL5.mAKT the myristylated AKT was cloned in the doxycycline ble vector pRevTRE (Clontech) (this clone is designated mAKT). As a l, cell were cted with vector alone (this clone is designated Vector). For tion of FL5.mAlct.Bcl-xL, the human Bcl-xL cDNA was cloned into the EcoRI site of the pBabePuro vector, and transfected into FL5.mAkt3 as described (18) (this clone is designated mAKTBcl—xL). These lines were cultured as described previously using DME-HG media supplemented with 10mM HEPES, 10% FBS media containing 1 ngml Geneticin (G418) (Sigma #69516) and 1 or 2 nymL of IL—3 (RD Systems#403 ML).

PBL (human eral blood ytes) were isolated from patient blood sed fi-om the New York Blood Center. Thirty five ml of the cell sion was layered over 15 ml of Ficoll— Paque plus (GE Healthcare). Samples were centrifuged at 2,000 rpm for 40 min at 4 “C, and the leukocyte interface was collected. Cells were plated in RPMI medium with 10% FBS and treated next day with appropriate concentrations of PU-H7l for the indicated times.

Reagents: The Hsp90 inhibitors were synthesized as previously reported. We purchased from Calbiochem the Akt Inhibitor VIII, e-Selective, Akti-l/2 (#124018), the PD98059 MEK inhibitor (#513000) and the pan-JAK Inhibitor I (#420099). These compounds were used at concentrations as indicated by the vendor to result in inhibition of their target pathways. All compounds, except for in vivo studies, were used as DMSO stocks.

Growth Inhibition: Growth tion studies were performed using the Alamar blue assay.

This reagent offers a rapid objective measure of cell ity in cell culture, and it uses the indicator dye resazurin to e the metabolic capacity of cells, an indicator of cell viability. Briefly, exponentially growing AML cell lines were plated at 2x104 cells/well in microtiter plates (Corning # 3650) and incubated for the indicated times at 37 °C. Drugs were added in triplicates at the indicated concentrations, and the plate was incubated for 72 h. Resazurin (55 pM) was added, and the plate read 6 h later using the Analyst GT (Fluorescence intensity mode, excitation 530nm, emission 580nm1 with 560nm dichroic mirror). Results were analyzed using the Soflmax Pro re. The percentage cell growth inhibition was calculated by comparing fluorescence readings obtained fi'om treated versus control cells, accounting for initial cell population (time zero). The ICso was ated as the drug concentration that inhibits cell growth by 50%.

Apoptosis Assay: Cells were treated for 24, 48 or 72 h with vehicle (DMSO) or inhibitors as indicated. Following staining with Acrldine Orange and Ethidium Bromide (1:1 mix of 100 CI g/ml), cells were visualized with a fluorescent microscope (Zeiss Axiovert 40 CFL) and counted. Percentage of apoptotic cells was determined from 200-300 cells counted in each group. The tage of tic cells was calculated as: % Apoptotic cells = (total number of cells with apoptotic nuclei / total number of cells counted) x 100. Acxidine orange is taken up by both viable and nonviable cells and emits green fluorescence if intercalated into double stranded nucleic acid (DNA) or red fluorescence if bound to single stranded c acid (RNA). Ethidium bromide is taken up only by nonviable cells and emits red fluorescence by intercalation into DNA. Viable cells have uniform green nuclei with organized structure. Early apoptotic cells (which still have intact nes but have started to o DNA cleavage) have green nuclei, but pen'nuclear chromatin condensation is visible as bright green patches or fi'agrnents. Late apoptotic cells have orange to red nuclei with condensed or fragmented chromatin. ic cells have a uniformly orange to red nuclei with organized structure. 2012/045864 Caspase 3,7 Activation Assay: Cells were plated and d as described in the growth inhibition assay section. Following a 24h or 48h exposure of cells to Hsp90 inhibitors, 100 uL buffer containing 10 mM Hepes, pH 7.5, 2 mM EDTA, 0.1% CHAPS, 0.1 mg’mL PMSF, Complete Protease Inhibitor mix (#1697498; Roche), and the caspase substrate Z-DEVD-RllO (#R22120; Molecular Probes) at 25 M was added to each well. Plates were placed on an orbital shaker to promote cell lysis and reaction. The fluorescence signal of each well was measured in an Analyst GT (Molecular Dev ices) microplate reader (excitation 485 nm; emission at 530 nm). The percentage increase in caspase— 3,7 activity was calculated by comparison of the fluorescence reading obtained from treated versus control cells. All experimental data were analyzed using SOFTmax Pro 4.3.1 and plotted using Prism 4.0 pad Soflware Inc., San Diego, CA).

Western blot: Cells were grown to 60—70% nce and treated with inhibitor or vehicle for the indicated times. n lysates were prepared in 50 mM Tris pH 7.4, 150 mM NaCl and 1% NP—40 lysis buffer. Protein concentrations were measured using the BCA kit (Pierce) according to the manufacnirer's instructions. Protein lysates (10—100 ug) were ed by SDS—PAGE, transferred onto nitrocellulose membrane and incubated with the ted primary dies. To activate AKT, FL5.12-derived cell lines Were pretreated with l pig/m1 Box for 18h prior to treatment with inhibitors.

Antibodies: Anti-AKT from rabbit (1:500, 9272; Cell Signaling), anti-phospho—AKT (Ser 473) from rabbit (1:500, 92718; Cell Signaling), anti-RAF-l from rabbit (1:300, sc»133; Santa Cruz Biotechnology), anti-PAR? (p85 fragment) from rabbit (1:250, G734]; Promega), anti-Ecl-xL from rabbit (1: 1,000, 2762; Cell Signaling), anti-JAK2 from rabbit (1:500, 3773; Cell Signaling), anti-c— KIT from mouse (1: 1,000, 3308; Cell Signaling), MLl from rabbit (1:500, 4334; Cell Signaling), anti—FLT3 from rabbit (1 :1,000, 3462; Cell Signaling), RKC fi'om rabbit (1:1,000, ab43078; Abcam), STATS, p—STATS, p—ERK and anti-B—Actin from mouse (1:3,000, ab8227——50; Abcam). The membranes were then incubated with 1:3,000 dilution of a perioxidase-conjugated corresponding secondary antibody and proteins were detected via ECL—Enhanced Chemiluminescence Detection System (Amersham ences).

Pharmacodynamle Study: Four— to 6-week—old nu/nu athymic female mice were obtained from Taconic Farms. Experiments were d out under an Institutional Animal Care and Use tee-approved ol, and institutional guidelines for the proper and humane use of animals in research were followed. HEL and M0—91 cells were subcutaneously implanted in the right flank of mice using a 20-gauge needle and allowed to grow. Before administration, a solution ofPU-H71'HC1 was formulated in sterile PBSl For this assay, tumors were allowed to reach 6-8 mm in er before treatment. Mice bearing M0-91 and HEL tumors were administered eritoneally (i.p.) g PU-H7l. Animals were sacrificed by C02 euthanasia at 12, 24, 48, 72, and 96h post administration of PU-H7l. Tumors were homogenized and proteins analyzed by westem blot as described above. [0488! Densitometry. Gels were scanned in Adobe Photoshop 7.0.] and quantitative densitometric analysis was performed using n-It 5.1.

Statistics. We performed tical analysis and graph plotting with Prism 4.0 (GraphPad e).We presented all data as mean :t s.d. A P value of less than 0.05 was considered significant.

StatSpImsphoflaw -— Primary cells were stained with CD34~APC, CD38—PECY7 and CD45- APC-H? antibodies (BD Biosciences) in FACS buffer at 4°C for 30 min Cells were washed once, fixed with 4% paraformaldehyde at room temperature for 30 min, and permeabilized with PBS/0. 1% Triton X—100 on ice for 10 min‘ Cells were d with p—StatS-PE or isotype control (BD Biosciences) at 4°C overnight. Cells were then washed once with PBS, and subjected to flow try analysis using BDnLSR [1 flow cytometer (BD Biosciences). The MFI of p—Stat5 staining was normalized to isotype control.

Stem cell viability assay - Primary cells were plated in 48-well plates at 2><10‘s cells/ml, and treated with 1 uM PU«H7 l for 48 h. Cells were stained with CD34-APC, CD38-PE—CY7 and CD45- APC-H7 antibodies (BD Biosciences) in FACS buffer (PBS, 0.05% FBS) at 4 °C for 30 min prior to Annexin V-V450 (BD Biosciences) and 7«AAD staining in Annexin V buffer (10 mM HEPES/NaOH, 0.14 M NaCl, 2.5 111M C302). Cell viability was normalized to untreated cells. 6.7. Studies to identify neurodegeuerative patients susceptible to HSP90 inhibition therapy The transgenic model ofAD (3ng—AD), which expresses the human APPswe, PSlMl46V and 1 L, progressively develops both Abeta and tau pathology in an age-dependent manner in e-relevant brain regions ngs et 31., 2005; Oddo et al., 2005; Oddo et a1., 2003). This mouse model is established by co—injection of APP and tau eDNA constructs into PSlMl46 knockin mouse embryos. These mice develop intracellular AB preceding amyloid plaque deposition, consistent with the observations in patients with mild cognitive impairment (MCI) and patients with Down’s me. They also p extracellular Abeta deposit prior to tangle formation, allowing us to study the events involved in these two AD development stages. The tau pathology is first apparent in pyramidal cells of hippocampus CA1 region and sses into cortex, mimicking distribution pattern in human AD brain (Mesulam, 2000). Therefore, the AD 3x-tg mouse model shows many rities to human AD and provides the opportunity to study the effect and retention of Hsp90 inhibitors in the enically affected and in the normal brain regions.

Determination of brain and plasma drug concentrations. The aqueous solution of compound PUJ-IZlSl as HCl salt was administered i.p. to 3XTg AD mice (35 g e body weight) 2012/045864 at the indicated doses Mice were killed by C01 euthanasia at different time points afler the ent according to ols approved by MSKCC Institutional Animal Care and Use Committee.

Hemibrains were separated into corticolimbic and subcortical regions, quickly frozen in liquid nitrogen and stored at ~80 l’C. Plasma was obtained from blood that was collected into a 1.5 ml.

Eppendorftube cooled in ice and subject to centrifuge. Frozen brain tissue was dried and weighed prior to homogenization in acetonitrile/l-IZO (3:7) The mixture was extracted with methylene chloride, the organic layer separated and dried under . Plasma (50 uL) was mixed with acetonitrile (0.25 mL) and fuged. The resulting supematants were dried under vacuum. Samples were reconstituted in mobile phase. Concentrations of compound in brain and plasma were determined by highuperformance LC-MS/MS. ridol was added as the internal standard. Compound analysis was performed on the 6410 LC-MS/MS system (Agilent Technologies) in multiple reaction monitoring (MRM) mode using positive-ion electrospray ionization. A Zorbax Eclipse XDB-CIB column (2.1 x 50 mm. 3.5 pm) was used for the DC separation, and the analyte was eluted under an isocratic condition (65% H20+O.l% HCOOH: 35% CH3CN) for 5 minutes at a flow rate of 0.35 ml/min.

Pharmacodynamic analyses. For protein analysis, ed brain region (hippocampus) was homogenized in SDS lysis buffer (50 mM Tris pH 7.4, 2% SDS) and subjected to Western blot analysis. Levels of ns were be analyzed by immunoblotting with anti—Hsp70 and Hsp90 antibodies. 7. EMBODIMENTS The invention can be illustrated by the following embodiments ated in the numbered paragraphs below: 1. A method for determining whether a tumor will likely respond to therapy with an HSP90 inhibitor which comprises the following steps: (a) contacting the tumor or a sample containing cells from the tumor with a detectably labeled HSP90 inhibitor which binds preferentially to a tumor-specific form of HSP90 present in a tumor or tumor cells; (b) measuring the amount of labeled HSP90 inhibitor bound to the tumor or the tumor cells in the ; and (c) ing the amount of labeled HSP90 inhibitor bound to the tumor or the tumor cells in the sample measured in step (b) to a reference amount of the labeled HSP90 inhibitor bound to normal cells; wherein a greater amount of labeled HSP9O inhibitor bound to the tumor or the tumor cells measured in step (b) as compared with the reference amount indicates the tumor will likely respond to the HSP90 inhibitor. 2. A method of embodiment I, wherein the r the ratio of the amount of labeled HSP90 inhibitor bound to the tumor or the tumor cells measured in step (b) as compared with the reference amount, the greater the magnitude of the likely response to the HSP9O inhibitor 3. A method ofembodiment 1, wherein the greater the amount of labeled HSP90 inhibitor bound to the tumor or the tumor cells measured in step (a), the greater the magnitude of the likely response to the HSP90 inhibitor therapy. 4. A method of embodiment 1, wherein the reference amount of the labeled HSP9O inhibitor bound to normal cells is the amount of the labeled HSP90 inhibitor bound to normal cells in the sample containing cells from the tumor.

. A method of embodiment 1, wherein the reference amount of the labeled HSP9O tor bound to normal cells is a predetermined amount of the labeled HSP9O inhibitor bound to normal cells in a reference sample. 6. A method of embodiment 1, wherein the detectably labeled HSP90 inhibitor is fluorescently labeled. 7. A method diment 1, wherein the detectably labeled HSP9O inhibitor is biotin»labeled. 8. A method of embodiment 1, wherein the ably labeled HSP90 inhibitor is radioactively 9. A method of embodiment 1. wherein the tumor is associated with a cancer selected from the group ting of colorectal cancer, pancreatic cancer, thyroid cancer, a leukemia including acute myeloid ia, acute lymphoblastic leukemia and chronic myeloid leukemia, multiple myeloma, basal cell carcinoma, melanoma, renal cell carcinoma, bladder cancer, te cancer, a lung cancer including small cell lung cancer and non-small cell lung cancer, breast , neuroblastoma, myeloproliferative disorders, intestinal cancers including gastrointestinal stromal , esophageal cancer, stomach cancer, liver cancer, gallbladder cancer, anal cancer, brain tumors including gliomas, lymphomas ing follicular lymphoma and diffiise large B~cell lymphoma, and gynecologic s including ovarian, cervical, and endometrial cancers.

. A method of embodiment 9, wherein the cancer is breast . 11, A method of embodiment 9, wherein the cancer is a leukemia. 12. A method of embodiment 11, wherein the leukemia is chronic myeloid leukemia. 13. A method of embodiment 9, wherein the cancer is gastric cancer. 14. A method ofembodiment 9, wherein the cancer is atic cancer.

. A method of embodiment 1, wherein the tumor cell is a tumor stem cell. 16. A method of embodiment 1, wherein in step (a) the tumor is contacted, and is present, in a subject. 17. A method of embodiment 1, wherein in step (a) the sample containing tumor cells is contacted and is a tissue sample. 18. A method of embodiment l, where the tissue sample is a sample obtained during a biopsy, a fine needle aspiration or a surgical procedure. 19. A method of embodiment 1, wherein in step (a) the sample is contacted and comprises a biological fluid.

. A method of embodiment 19, wherein the biological fluid is blood or bone marrow. 21. A method of embodiment 1, n in step (a) the sample is contacted and comprises disrupted tumor cells. 22. A method of embodiment 1, wherein in step (a) the sample is contacted and comprises live cells. 23. A method of embodiment 1, wherein in step (a) the sample is contacted and comprises frozen cells. 24. A method of embodiment 1, wherein in step (a) the sample is contacted and comprises fixed and penneabilized cells.

. A method of embodiment 1, wherein in step (a) the sample is ted and comprises fonnnlinwfixed, pamffirkembedded cells. 26. A method of ment 1, wherein the detectably labeled HSP90 inhibitor is a labeled form of the HSP90 inhibitor to be administered as therapy. 27. A method of embodiment 1, wherein the HSP90 inhibitor to be administered as therapy is PU‘H71 or an analog, homolog or derivative ofPU-H7l. 28. A method of embodiment 27, wherein the HSP90 inhibitor is PU—H7l. 291 A method of embodimmt 1, wherein the detectably labeled HSP90 inhibitor is a form of PU- H7l or of an analog, homolog, or derivative ofPU—H7l.

. A method of embodiment 1, wherein the detectably labeled HSP90 inhibitor is a form of PU- H71. 311 A method of ment 30, wherein the detectably labeled HSP90 inhibitor is [unpu- H71. 32. A method of embodiment 30, wherein the detectably d HSP90 inhibitor is PU-H7l- FITCZ or PU—H7l-NBD1. 33. A method of embodiment 30, n the detectably labeled HSP90 inhibitor is a biotinylated analog ofPU-H7 l. 34. A method of embodiment 33, wherein the biotinylated analog ofPU—H7l is PU-H7l-biotin-5, PU~H7 l-biotin-6, -biotin-8 or PU—H7l-biotin-9.

. A method for determining whether a cancer patient with an imageable tumor will likely d to therapy with an tor of HSP90 which comprises the following steps: 2012/045864 (a) administering to the patient a radiolabeled HSP90 inhibitor which binds preferentially to a tumor-specific form of HSP90 present in the tumor or in tumor cells of the tumor; (1)) ing uptake of the radiolabeled HSP90 inhibitor by the t’s tumor at a plurality of time points after the stration in step (a); (c) measuring uptake of the radiolabeled HSP90 inhibitor by a predetermined y tissue of the patient at the same plurality of time points afier the administration in step (a); (d) computing a ratio of the uptake measured at multiple time points in step (b) with the uptake measured at the same time points in step (c); and (e) determining the likelihood the cancer patient will respond to therapy with the inhibitor of HSP90, wherein a ratio greater than 2 ed in step (d) at multiple time points indicates that the patient will likely d. 36. A method of embodiment 35, wherein the tumor is associated with a cancer selected from the group consisting of colorectal cancer, atic cancer, thyroid cancer, basal cell carcinoma, melanoma, renal cell carcinoma, bladder cancer, prostate cancer, a lung cancer including small cell lung cancer and non—small cell lung cancer, breast cancer, neuroblastoma, gastrointestinal cancers including gastrointestinal stromal tumors, esophageal cancer, stomach cancer, liver cancer, gallbladder cancer, anal cancer, brain tumors including gliomas, lymphomas including follicular lymphoma and diffuse large B—cell lymphoma, and gynecologic cancers ing ovarian, cervical, and endometrial cancers. 37. A method of ment 36, wherein the cancer is breast cancer, lymphoma, neuroblastoma, gastric cancer, or pancreatic cancer. 38. A method of embodiment 35, wherein the radiolabeled HSP90 tor is a radiolabeled form of the HSP90 inhibitor to be administered as therapy. 39. A method of embodiment 36, wherein the HSP90 inhibitor to be administered as therapy is PU-H7l or an analog, homolog, or derivative of PU-H7l. 40. A method of embodiment 35, wherein the abeled HSP90 inhibitor is a radiolabeled form of PU-H7l. 41. A method of embodiment 40, wherein the radiolabeled form ofPU-H71 is ['"Ij-PU-H7l. 42. A method for determining whether a cancer patient with an imageable tumor will likely respond to therapy with a predetermined dose of an inhibitor of HSP90 which ses the following steps: (a) administering to the patient a radiolabeled form of the HSP90 inhibitor which binds preferentially to a tumor-specific form of HSP90 t in the tumor or in tumor cells of the tumor; (b) measuring uptake of the radiolabeled form of the HSP90 inhibitor by the patient’s tumor at a plurality of time points afler the administration in step (a); (c) calculating for the predetermined dose of the HSP90 inhibitor, the concentrations of the HSP90 tor which would be present in the patient’s tumor at each of such plurality of time points, based on the uptake measured at such plurality of time points in step (b); and (d) comparing the concentrations of the HSP90 inhibitor calculated in step (c) with reference concentrations of the HSP90 inhibitor which would need to be present in the tumor at such plurality of time points for the HSP90 inhibitor to be effective in treating the tumor wherein the patient will likely respond to therapy with the predetermined dose of the HSP90 inhibitor if the trations of the HSP90 inhibitor calculated in step (c) would equal or exceed the concentrations of the HSP90 inhibitor needed to effectively treat the tumor and would not be toxic to the patient. 43. A method of ment 40, wherein the tumor is associated with a cancer selected from the group consisting of colorectal cancer, pancreatic cancer, thyroid cancer, basal cell carcinoma, melanoma, renal cell carcinoma, bladder cancer, prostate cancer, a lung cancer ing small cell lung cancer and non-small cell lung , breast cancer, neuroblastoma, gastrointestinal cancers including gastrointestinal stromal tumors, esophageal cancer, stomach cancer, liver cancer, gallbladder cancer, anal cancer, brain tumors ing gliomas, lymphoma ing follicular lymphoma and diffiise large B-cell lymphoma, and gynecologic cancers including ovarian, cervical, and endometrial cancers. 44. A method of ment 43, wherein the cancer is breast cancer, lymphoma, neuroblastorna, gastric cancer, or pancreatic cancer. 45. A method of ment 42, wherein the HSP90 tor to be administered as therapy is PU—H7l or an analog, homolog, or derivative of PU—H7l. 46. A method of ment 42, wherein the radiolabeled HSP90 inhibitor is a radiolabeled form of PU—H7 l . 47. A method ofembodiment 46, wherein the rndiolabeled form of PU-l-I‘ll is [‘“nru—Hn. 48. A method for determining, for a specific cancer patient with an imageable tumor, an ive dose and frequency of administration for therapy with an tor of HSP90 which comprises the following steps: (a) administering to the patient a radiolabeled form of the HSP90 tor which binds preferentially to a tumor-specific form of HSP90 present in a tumor or tumor cells; (b) measuring uptake of the radiolabeled form of the HSP90 inhibitor by the patient’s tumor at a plurality of time points afier the administration in step (a); and (c) calculating the dose and frequency of administration needed to maintain in the tumor at each of such plurality of time points a concenu'ation of the HSP inhibitor efi‘ective to treat the tumor, based on the uptake measured at such time points in step (b), thereby ining, for the cancer patient, the effective dose and ncy of administration for therapy with the inhibitor of HSP90. 49. A method of embodiment 48, n the tumor is ated with a cancer selected from the group consisting of colorectal cancer, pancreatic cancer, thyroid cancer, basal cell carcinoma, melanoma, renal cell carcinoma, bladder cancer, prostate cancer, a lung cancer including small cell lung cancer and non-sn1all cell lung cancer, breast , neuroblastoma, gastrointestinal cancers including gastrointestinal stromal tumors, esophageal cancer, h cancer, liver cancer, gallbladder cancer, anal cancer, brain tumors including gliomas, lymphomas including follicular lymphoma and diffiise large Bucell lymphoma, and gynecologic cancers including ovarian, cervical, and endometrial cancers. 50. A method of embodiment 49, n the cancer is breast cancer, lymphoma, neuroblastoma, gastric cancer, or pancreatic cancer. 51. A method of embodiment 48, wherein the radiolabeled HSP90 tor is a radiolabeled form of the HSP90 inhibitor to be administered as therapy. 52. A method of embodiment 48, wherein the HSP90 inhibitor to be administered as therapy is PU—H71 or an analog, homolog, or tive of PU-H71. form 53. A method of embodiment 48, wherein the radiolabeled HSP90 inhibitor is a radiolabeled of PU-H71. 54. A method of embodiment 53, wherein the radiolabeled form of PU—H7l is ['z‘fl—PU-H7l. 55. A method for ining the concentration of a HSP90 inhibitor present in an imageable tumor in a cancer patient which ses the ing steps: (a) co-administering to the patient a predetermined amount of the HSP90 inhibitor and a predetermined amount of a radiolabeled form of the HSP90 inhibitor which binds preferentially to a tuniorvspecific form ofHSP90 present in a tumor or tumor cells; 03) periodically measuring the uptake of the radiolabeled HSP90 inhibitor by the patient’s tumor at one or more predefined time point(s) afler the co-administmtion in step (a); and (c) determining the concentration of the HSP90 inhibitor present in the tumor at any such time point based on the measurements of the uptake of the radiolabeled HSP90 tor in step (b). 56. A method of ment 54, wherein the tumor is associated with a cancer selected from basal cell oma, group consisting of colorectal cancer, pancreatic , thyroid cancer, melanoma, renal cell carcinoma, bladder cancer, prostate cancer, a lung cancer including small cell lung cancer and non-small cell lung cancer, breast cancer, neuroblastoma, gastrointestinal cancers ing gastrointestinal l , esophageal cancer, stomach brain tumors , liver cancer, gallbladder cancer, anal cancer. including gliomas, lymphomas including follicular lymphoma and diffuse large B-cell lymphoma, and gynecologic cancers including ovarian, cervical, and endometrial cancers. 57. A method of embodiment 56, wherein the cancer is breast cancer, lymphoma, neumblastoma, gastric cancer, or pancreatic cancer. 58. A method of embodiment 55, wherein the radiolabeled HSP90 inhibitor is a radiolabeled form of the HSP90 inhibitor to be administered as therapy. 59. A method of embodiment 55, wherein the HSP90 inhibitor to be stered as therapy PU—H7 l or an analog, homolog, or derivative ofPU—H7 1. 60. A method of embodiment 55, wherein the radiolabeled HSP90 inhibitor is a radiolabeled form of PU—H7 l . 61. A method ofembodiment 60, wherein the radiolabeled form ofPU-H71 is [ml]—PU-H7 1. 62. A method for determining the responsiveness to therapy with mt inhibitor of HSP90 of an imageable tumor in a cancer patient which comprises the following steps: (a) administering a radiolabeled form of the HSP90 inhibitor which binds preferentially to a specific form of HSP90 present in a tumor or tumor cells, to the t at multiple time points within the period during which the patient is ing the inhibitor ofHSP90 as therapy; and (b) measuring the concentration of the radiolabeled HSP90 inhibitor in the patient’s tumor at such multiple time points after the administration in step (a); and (c) comparing the concentrations of the radiolabeled HSP90 inhibitor measured in step (b) with the minimum concentrations of the HSP90 inhibitor needed to effectively treat the tumor, wherein measured concentrations greater than the minimum needed to treat the tumor indicate that the t is likely to respond to therapy with the HSP90 tor. 63. A method of embodiment 62, wherein the tumor is associated with a cancer selected from the group consisting of colorectal cancer, atic cancer, thyroid cancer, basal cell carcinoma, melanoma, renal cell carcinoma, bladder cancer, prostate cancer, a lung cancer including small cell lung cancer and non-small cell lung cancer, breast , neuroblastorna, gastrointestinal cancers including gastrointestinal stromal tumors, esophageal cancer, stomach cancer, liver cancer, gallbladder , anal cancer, brain tumors including gliomas, lymphomas including follicular lymphoma and diffuse large Becell lymphoma, and gynecologic cancers including ovarian, cervical, and endometrial cancers. 64. A method ofembodiment 63, wherein the cancer is breast cancer, lymphoma, lastoma, gastric , or pancreatic cancer. 65. A method of embodiment 62, wherein the radiolabeled HSP90 tor is a radiolabeled form of the HSP90 inhibitor to be administered as y. 66. A method of embodiment 62, wherein the HSP90 inhibitor to be administered as therapy is PU-H7l or an analog, g, or derivative of PU-H7 l. 67. A method ofembodiment 62, wherein the radiolabeled HSP90 inhibitor is a radiolabeled form of PU-H7 l . 68. A method of embodiment 67, wherein the radiolabeled form ofPU—H7l is {‘241]-PU-H71. 69. A method for determining whether a human cancer present in a t will likely respond to therapy with an HSP90 inhibitor which comprises: (a) obtaining a sample containing cells from the patient’s , which cells express HSP90 protein alone or in addition to HSP70 protein; (b) assessing for the cells present in the sample obtained in step (a) the presence of at least one of the following parameters: an activated AKT pathway, a defect in PTEN tumor suppressor function or expression, an activated STATS pathway, or Bcl-xL protein sion; and (c) comparing the assessment obtained in step (b) with a predetermined reference assessment of the same parameter or ters assessed in step (b) for human cancer cells from one or more cancer patient(s) who responded to therapy with the HSP90 tor so as to thereby determine whether the patient’s cancer will likely respond to therapy with the HSP90 inhibitor. 70. A method of embodiment 69, wherein the human cancer is breast cancer. 71. 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Claims (21)

1. A method for determining whether a patient with a blood cancer will likely respond to therapy with an HSP90 inhibitor which comprises: (a) contacting a sample containing blood cancer cells and non-cancerous blood cells from the patient with a cell permeable fluorescently labeled HSP90 tor that binds directly and preferentially to a tumor-specific form of HSP90 present in the cancer cells of the patient; (b) measuring the amount of fluorescently labeled HSP90 inhibitor bound to HSP90 n in the cancer cells and non-cancerous cells in the sample; and (c) ing the amount of the scently labeled HSP90 inhibitor bound to the cancer cells with the amount of the fluorescently d HSP90 inhibitor bound to the non-cancerous cells, wherein a greater amount of fluorescently labeled HSP90 inhibitor bound to the cancer cells than the non-cancerous cells indicates presence of the tumor specific form of HSP90 in the cancer cells; thereby determining that the blood cancer will likely respond to the HSP90 inhibitor.

2. The method of claim 1, wherein the non-cancer cells are lymphocytes.

3. The method of claim 1 or claim 2, wherein said measuring is conducted by flow cytometry.

4. The method of any one of claims 1-3 , wherein the r the ratio of the amount of fluorescently labeled HSP90 inhibitor bound to the cancer cells measured in step (b) as compared with the fluorescently labeled HSP90 tor bound to the non-cancerous cells, the greater the magnitude of the likely response to the HSP90 inhibitor therapy.

5. The method of any one of claims 1-4, n the blood cancer is a leukemia.

6. The method of claim 5, wherein the leukemia is chronic myeloid leukemia.

7. The method of any one of claims 1-6, wherein the sample comprises live cells.

8. The method of any one of claims 1-7, wherein the sample comprises a biological fluid.

9. The method of claim 8, wherein the biological fluid is blood or bone marrow.

10. The method of claim 8, wherein the biological fluid is blood.

11. The method of claim 1, n the sample comprises disrupted tumor cells.

12. The method of claim 1, wherein the sample comprises frozen cells.

13. The method of claim 1, wherein the sample comprises fixed and permeabilized cells.

14. The method of any one of claims 1-13, wherein the fluorescently labeled HSP90 inhibitor is a labeled form of the HSP90 inhibitor to be administered as y.

15. The method of claim 14, wherein the HSP90 inhibitor to be administered as therapy is PUH71 or an analog, homolog or derivative of PU-H71.

16. The method of claim 15, wherein the HSP90 inhibitor is PU-H71.

17. The method of any one of claims 1-16, wherein the fluorescently d HSP90 inhibitor is a form of PU-H71 or of an analog, homolog, or derivative of PU-H71.

18. The method of any one of claims 1-17, wherein the fluorescently labeled HSP90 inhibitor is a form of PU-H71.

19. The method of any one of claims 1-18, wherein the fluorescently labeled HSP90 inhibitor is PU-H71-FITC2.

20. The method of any one of claims 1-18, wherein the fluorescently labeled HSP90 inhibitor is PU-H71-NBD1.

21. The method of claim 1, substantially as herein bed with reference to any one of the Examples and/or