JP7579564B2 - Pharmaceutical composition and kit for treating RB1-positive cancer - Google Patents

Description

The present invention relates to a pharmaceutical composition and a kit for treating RB1-positive cancer. More specifically, the present invention relates to a pharmaceutical composition for treating RB1-positive cancer, a kit for treating RB1-positive cancer, a method for testing the effectiveness of administering a medicine to a cancer patient, and a kit for testing the effectiveness of administering a medicine to a cancer patient.

Currently, the efficacy of single-agent administration of cyclin-dependent kinase (CDK) 4 and 6 inhibitors against various types of cancer, including hepatocellular carcinoma, is being investigated in phase II clinical trials (Non-Patent Document 1).

Schettini F., et al., CDK 4/6 Inhibitors as Single Agent in Advanced Solid Tumors, Front Oncol., 8, 608, 2018.

However, the inventors have demonstrated through in vitro and in vivo studies that single administration of CDK4 and 6 inhibitors to cancer cell lines is effective but not optimal. The present invention aims to provide a technology for effectively treating RB1-positive cancer.

The present invention includes the following aspects.
[1] A pharmaceutical composition for treating RB Transcriptional Corepressor 1 (RB1)-positive cancer, comprising a combination of a cyclin-dependent kinase (CDK) 4 and 6 inhibitor, and an NF-κB signaling pathway inhibitor or an AKT inhibitor as active ingredients.
[2] The pharmaceutical composition according to [1], wherein the CDK4 and CDK6 inhibitor is palbociclib (CAS number: 571190-30-2), ribociclib (CAS number: 1211441-98-3), abemaciclib (CAS number: 1231929-97-7), trilaciclib (CAS number: 1374743-00-6) or lerociclib (CAS number: 1628256-23-4).
[3] The pharmaceutical composition according to [1] or [2], which contains a combination of a CDK4 and 6 inhibitor and an NF-κB signaling pathway inhibitor as active ingredients, wherein the NF-κB signaling pathway inhibitor is an IKKβ inhibitor or an NF-κB inhibitor.
[4] The NF-κB signaling pathway inhibitor is an IKKβ inhibitor, and the IKKβ inhibitor is selected from the group consisting of Bay11-7082 (CAS number: 19542-67-7), IMD-0354 (CAS number: 978-62-1), IMD-1041 (CAS number: 1073666-73-5), IMD-2560, TPCA1 (CAS number: 507475-1 7-4), BOT-64 (CAS No.: 113760-29-5), BMS345541 (CAS No.: 445430-58-0), SC-514 (CAS No.: 354812-17-2), IKK-16 (CAS No.: 1186195-62-9), eltiprotafib (CAS No.: 251303-04-5) and Bay65-1942 (HCl salt) (CAS No.: 600734-06-3). The pharmaceutical composition according to [3].
[5] The NF-κB signaling pathway inhibitor is an NF-κB inhibitor, and the NF-κB inhibitor is selected from the group consisting of QNZ (CAS number: 545380-34-5), JSH-23 (CAS number: 749886-87-1), GYY4137 (CAS number: 106740-09-4), p-XSC (CAS number: 85539-83-9), CV3988 (CAS number: 85703-73-7), prostaglandin E2 (CAS number: 363-24-6), LY294002 (CAS number: 154447-36-6), Wortmannin (CAS number: 19545-26-7), mesalamine (CAS number: 89-57-6), rolipram (CAS number: 85416-75-7), gallic acid (CAS number: 149-91-7), anacardic acid (CAS number: 16611-84-0), MG-115 (CAS number: 133407-86-0), MG-132 (CAS number: 133407-82-6), lactacystin (CAS number: 133343-34-7), epoxomicin (CAS number: 134381-21-8), parthenolide (CAS number: No.: 20554-84-1), carfilzomib (CAS No.: 868540-17-4), bortezomib (CAS No.: 179324-69-7), MLN-4924 (CAS No.: 905579-51-3), delanzomib (CAS No.: 847499-27-8), ixazomib (CAS No.: 1239908-20-3), marizomib (CAS No.: 437742-34-2) and oprozomib (CAS No.: 935888-69-0). [3] The pharmaceutical composition according to [3].
[6] The pharmaceutical composition according to [1] or [2], comprising a combination of a CDK4 and 6 inhibitor and an AKT inhibitor as active ingredients, wherein the AKT inhibitor is selected from the group consisting of Akt Inhibitor X (CAS No.: 925681-41-0), ipatasertib (CAS No.: 1001264-89-6), capivasertib (CAS No.: 1143532-39-1), mirasertib (CAS No.: 1313881-70-7), afuresertib (CAS No.: 1047644-62-1), uprosertib (CAS No.: 1047634-65-0), triciribine phosphate (CAS No.: 61966-08-3) and MK2206 (CAS No.: 1032350-13-2).
[7] The pharmaceutical composition according to any one of [1] to [6], wherein the RB1-positive cancer is hepatocellular carcinoma, breast cancer, lung cancer or colon cancer.
[8] A kit for treating RB1-positive cancer, comprising a cyclin-dependent kinase (CDK) 4 and 6 inhibitor, and an NF-κB signaling pathway inhibitor or an AKT inhibitor.
[9] A method for testing the effectiveness of administering a combination of a CDK4 and 6 inhibitor, and an NF-κB signaling pathway inhibitor or an AKT inhibitor to a cancer patient, the method comprising the step of examining whether cancer cells derived from a cancer patient are RB1 positive, wherein the RB1 positivity of the cancer cells indicates that administration of the combination of the CDK4 and 6 inhibitor, and an NF-κB signaling pathway inhibitor or an AKT inhibitor is effective in treating the cancer patient.
[10] A kit for testing the effectiveness of administration of a combination of a CDK4 and 6 inhibitor, and an NF-κB signaling pathway inhibitor or an AKT inhibitor to a cancer patient, the kit comprising: a primer set for amplifying genomic DNA of the RB1 gene, a probe for genomic DNA of the RB1 gene, a primer set for amplifying cDNA of the RB1 gene, a probe for mRNA of the RB1 gene, or a substance that specifically binds to RB1 protein, the kit being used to test whether cancer cells derived from a cancer patient are RB1 positive, the kit indicating that administration of the combination of the CDK4 and 6 inhibitor, and an NF-κB signaling pathway inhibitor or an AKT inhibitor to the cancer patient is effective in treating the cancer patient.

The present invention provides a technology for effectively treating RB1-positive cancer.

FIG. 1 is a schematic diagram illustrating the action of CDK4 and 6 inhibitors on RB1. (a) is a schematic diagram illustrating the procedure for producing a QKO mouse, and (b) is a representative photograph of a tumor formed in the liver of a QKO mouse. 1A is a graph showing the results of flow cytometry in Experimental Example 2. FIG. 1B is a graph prepared based on the results of FIG. 13 is a graph showing the results of screening in Experimental Example 3. 1 is a graph showing cell viability in a study using Bay11-7082 in Experimental Example 4. 1 is a graph prepared based on the results of flow cytometry in the study using Bay11-7082 in Experimental Example 4. 1 is a graph prepared based on the results of flow cytometry in a study using IMD-0354 in Experimental Example 4. 13 is a graph prepared based on the results of flow cytometry in a study using shRNA against IKKβ in Experimental Example 4. 1 is a graph prepared based on the results of flow cytometry in a study using primary hepatocellular carcinoma cells (QKO PHCC) in Experimental Example 4. 13 is a graph prepared based on the results of flow cytometry in a study using Huh7 cells in Experimental Example 5. 1 is a graph prepared based on the results of flow cytometry in a study using Li-7 cells in Experimental Example 5. 1 is a graph prepared based on the results of flow cytometry in a study using H1975 cells in Experimental Example 5. 1 is a graph prepared based on the results of flow cytometry in a study using HCC1143 cells in Experimental Example 5. 1 is a graph prepared based on the results of flow cytometry in a study using T47D cells in Experimental Example 5. 1 is a graph prepared based on the results of flow cytometry in a study using HCT-116 cells in Experimental Example 5. 1 is a graph prepared based on the results of flow cytometry in a study using DLD-1 cells in Experimental Example 5. This is a graph prepared based on the results of flow cytometry in the study of combined use of ribociclib and Bay11-7082 in Experimental Example 5. 1 is a graph prepared based on the results of flow cytometry in the study of combined use of abemaciclib and Bay11-7082 in Experimental Example 5. 1 is a graph prepared based on the results of flow cytometry in Experimental Example 6. 13 is a graph prepared based on the results of flow cytometry in a study using HepG2 cells in Experimental Example 7. 1 is a graph prepared based on the results of flow cytometry in a study using SW480 cells in Experimental Example 7. 1 is a graph prepared based on the results of flow cytometry in a study using HCT-116 cells in Experimental Example 7. 1 is a graph prepared based on the results of flow cytometry in the study using DLD-1 cells in Experimental Example 7. 1 is a graph prepared based on the results of flow cytometry in a study using A549 cells in Experimental Example 7. 13 is a graph prepared based on the results of flow cytometry in Experimental Example 8. 1 is a graph showing the change in tumor volume over time in a study using a cancer-bearing mouse model transplanted with HepG2 cells in Experimental Example 9. 1 is a graph showing the change in tumor volume over time in a study using a cancer-bearing mouse model transplanted with Li-7 cells in Experimental Example 9. 13 is a graph showing the change in tumor volume over time in a study of Experimental Example 9 using a cancer-bearing mouse model transplanted with QKO PHCC cells expressing mouse Rb1 in a tetracycline-inducible manner.

[Gene and protein name notation]
In this specification, human genes and human proteins are represented by capital letters. Mouse genes are represented by capital letters for the first letter and lowercase letters thereafter. Mouse proteins are represented by capital letters. However, in some cases, human genes and mouse genes may be referred to without a strict distinction. In some cases, human proteins and mouse proteins may be referred to without a strict distinction.

[Pharmaceutical composition or kit]
In one embodiment, the present invention provides a pharmaceutical composition for treating RB1-positive cancer, comprising a combination of a CDK4 and 6 inhibitor and an NF-κB signaling pathway inhibitor or an AKT inhibitor as active ingredients.

The pharmaceutical composition may be a pharmaceutical composition containing a mixture of a CDK4 and 6 inhibitor and an NF-κB signaling pathway inhibitor. Alternatively, the pharmaceutical composition may be a pharmaceutical composition containing a mixture of a CDK4 and 6 inhibitor and an AKT inhibitor.

In one embodiment, the CDK4 and 6 inhibitors and the NF-κB signaling pathway inhibitor or AKT inhibitor may be unmixed and housed in separate containers to form a treatment kit. In the case of a treatment kit, these drugs are not mixed but are administered in combination.

The therapeutic kit may include a combination of a CDK4 and 6 inhibitor and an NF-κB signaling pathway inhibitor. Alternatively, the therapeutic kit may include a combination of a CDK4 and 6 inhibitor and an AKT inhibitor.

As described later in the Examples, the inventors have demonstrated that RB1-positive cancer cells can be effectively killed in vitro and in vivo by combining CDK4 and 6 inhibitors with an NF-κB signaling pathway inhibitor or an AKT inhibitor.

Figure 1 is a schematic diagram illustrating the action of CDK4 and 6 inhibitors on RB1. RB1 is dephosphorylated immediately after mitosis. Activated CDK4 and 6 monophosphorylate RB1. Once RB1 is monophosphorylated, the CDK2 complex achieves complete phosphorylation of RB1. Inhibition of the activity of CDK4 and 6 inhibits the monophosphorylation of RB1. As a result, RB1 is maintained in an unphosphorylated state for a long period of time. This results in not only cell cycle arrest, but also induction of senescence, apoptosis, enhanced immunogenicity, etc.

RB1-positive cancer refers to cancer in which the cancer cells that compose the tumor express RB1. The RB1 expressed by RB1-positive cancer may have a mutation, but it is preferable that the function of the wild-type RB1 described above is maintained, and wild-type RB1 is preferable. Specific examples of RB1-positive cancer include hepatocellular carcinoma, breast cancer, lung cancer, and colon cancer.

The accession number for human RB1 cDNA is NM_000321.3. The accession number for human RB1 protein is NP_000312.2. The accession number for mouse Rb1 cDNA is NM_009029.2. The accession number for mouse RB1 protein is NP_033055.2.

In the pharmaceutical composition of this embodiment, the CDK4 and 6 inhibitor may be a drug that inhibits the activity of a complex consisting of CDK4 and 6 and cyclin D. The CDK4 and 6 inhibitor may be a low molecular weight compound or an inhibitory nucleic acid against CDK4, CDK6 or cyclin D. Examples of inhibitory nucleic acids include shRNA and siRNA.

More specific examples of CDK4 and 6 inhibitors include palbociclib (CAS number: 571190-30-2), ribociclib (CAS number: 1211441-98-3), abemaciclib (CAS number: 1231929-97-7), trilaciclib (CAS number: 1374743-00-6), and relociclib (CAS number: 1628256-23-4). These may be used alone or in combination of two or more.

In addition, as an NF-κB signaling pathway inhibitor, a drug that blocks the NF-κB signaling pathway can be used.

The NF-κB signaling pathway is a pathway in which IκB kinase (IKK), consisting of IKKα, IKKβ, and IKKγ (NEMO), binds to and inactivates NF-κB, phosphorylating IκB, leading to ubiquitination and degradation, and as a result, NF-κB translocates from the cytoplasm to the nucleus, activating gene transcription.

The NF-κB signaling pathway inhibitor may be, for example, an IKKβ inhibitor or an NF-κB inhibitor. These may be used alone or in combination of two or more.

The NF-κB signaling pathway inhibitor may be a low molecular weight compound or an inhibitory nucleic acid against IKKα, IKKβ, IKKγ (NEMO), IκB, NF-κB, etc. Examples of inhibitory nucleic acids include shRNA, siRNA, etc.

Specific examples of IKKβ inhibitors include Bay11-7082 (CAS number: 19542-67-7), IMD-0354 (CAS number: 978-62-1), IMD-1041 (CAS number: 1073666-73-5), IMD-2560, TPCA1 (CAS number: 507475-17-4), BOT-64 (CAS number: 507475-17-4), and S number: 113760-29-5), BMS345541 (CAS number: 445430-58-0), SC-514 (CAS number: 354812-17-2), IKK-16 (CAS number: 1186195-62-9), eltiprotafib (CAS number: 251303-04-5), Bay65-1942 (HCl salt) (CAS number: 600734-06-3), etc.

Specific examples of NF-κB inhibitors include QNZ (CAS number: 545380-34-5), JSH-23 (CAS number: 749886-87-1), GYY4137 (CAS number: 106740-09-4), p-XSC (CAS number: 85539-83-9), CV3988 (CAS number: 85703-73-7), prostaglandin E2 (CAS number: 363-24-6), LY294002 (CAS number: 154447-36-6), Wortmannin (CAS number: 19545-26-7), mesalamine (CAS number: 89-57-6), rolipram (CAS number: 85416-75-7), gallic acid (CAS number: 149-91-7), anacardic acid (CAS number: 16611-84-0), MG-115 (CAS number: 133407-86-0), MG-132 (CAS number: 133407-82-6), lactacystin (CAS number: 133343-34-7), epoxomicin (CAS number: 134381-21-8), paclitaxel (CAS number: 134381-21-8), Examples include ruthenolide (CAS number: 20554-84-1), carfilzomib (CAS number: 868540-17-4), bortezomib (CAS number: 179324-69-7), MLN-4924 (CAS number: 905579-51-3), delanzomib (CAS number: 847499-27-8), ixazomib (CAS number: 1239908-20-3), marizomib (CAS number: 437742-34-2), and oprozomib (CAS number: 935888-69-0).

The pharmaceutical composition of this embodiment may contain a combination of a CDK4 and 6 inhibitor and an AKT inhibitor as active ingredients. The CDK4 and 6 inhibitor is the same as described above. The AKT inhibitor may be a low molecular weight compound or an inhibitory nucleic acid against AKT. Examples of inhibitory nucleic acids include shRNA and siRNA.

AKT inhibitors include Akt Inhibitor X (CAS number: 925681-41-0), ipatasertib (CAS number: 1001264-89-6), capivasertib (CAS number: 1143532-39-1), mirasertib (CAS number: 1313881-70-7), afuresertib (CAS number: 1047644-62-1), uprosertib (CAS number: 1047634-65-0), triciribine phosphate (CAS number: 61966-08-3), and MK2206 (CAS number: 1032350-13-2).

The CDK4 and 6 inhibitors, and the NF-κB signaling pathway inhibitors or AKT inhibitors may be mixed with a pharma- ceutical acceptable carrier and formulated as a pharmaceutical composition.

Alternatively, the CDK4 and 6 inhibitors, the NF-κB signaling pathway inhibitor, and the AKT inhibitor may be mixed with a pharma- ceutically acceptable carrier and separately formulated as pharmaceutical compositions to constitute a treatment kit.

The pharmaceutical composition is preferably formulated in a dosage form for oral use or a dosage form for parenteral use. The pharmaceutical composition may be in a dosage form for oral use or a dosage form for parenteral use. Examples of dosage forms for oral use include tablets, capsules, elixirs, microcapsules, etc. Examples of dosage forms for parenteral use include injections, inhalants, suppositories, patches, etc.

Examples of pharma- ceutically acceptable carriers include solvents such as sterile water and saline; binders such as gelatin, corn starch, gum tragacanth, and gum arabic; excipients such as crystalline cellulose; and leavening agents such as alginic acid.

The pharmaceutical composition may further contain additives. Examples of additives include lubricants such as magnesium stearate; sweeteners such as sucrose, lactose, and saccharin; flavorings such as peppermint and saffron oil; stabilizers such as benzyl alcohol and phenol; buffers such as phosphates and sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; and preservatives.

Pharmaceutical compositions can be formulated by combining the above-mentioned carriers and additives appropriately and mixing them in a unit dosage form required for generally accepted pharmaceutical practice.

Administration to a patient can be by methods known to those skilled in the art, such as intra-arterial injection, intravenous injection, subcutaneous injection, or intranasal, transbronchial, intramuscular, transdermal, or oral administration. The dosage varies depending on the patient's weight, age, symptoms, method of administration, etc., but a person skilled in the art can select an appropriate dosage.

The dosage of CDK4 and 6 inhibitors varies depending on the patient's symptoms, etc., but in the case of oral administration, it is generally considered appropriate to administer about 75 to 400 mg, for example about 75 to 125 mg, for example about 100 to 400 mg, once a day or in several divided doses to an adult (body weight 60 kg). In the case of parenteral administration, the dosage varies depending on the patient's symptoms, the target organ, the administration method, etc., but in the case of systemic administration, it is generally considered appropriate to administer about 30 to 200 mg, for example about 30 to 60 mg, for example about 50 to 200 mg, once a day to an adult (body weight 60 kg), once every 1 to 10 days. In the case of local administration, it is generally considered appropriate to administer about 7.5 to 40 mg, for example about 7.5 to 12.5 mg, for example about 10 to 40 mg, once a day to an adult (body weight 60 kg), once every 1 to 10 days.

The dosage of the NF-κB signaling pathway inhibitor varies depending on the patient's symptoms, etc., but in the case of oral administration, it is generally considered appropriate to administer about 50 to 900 mg, for example about 300 to 900 mg, for example about 2 to 5 mg, once a day or in several divided doses to an adult (body weight 60 kg). In the case of parenteral administration, the dosage varies depending on the patient's symptoms, the target organ, the administration method, etc., but in the case of systemic administration, it is generally considered appropriate to administer about 2 to 200 mg, for example about 2 to 5 mg, for example about 50 to 200 mg, once a day to an adult (body weight 60 kg), once every 1 to 10 days. In the case of local administration, it is generally considered appropriate to administer about 2 to 100 mg, for example about 2 to 50 mg, for example about 10 to 100 mg, once a day to an adult (body weight 60 kg).

The dosage of the AKT inhibitor varies depending on the patient's symptoms, etc., but in the case of oral administration, it is generally considered appropriate to administer about 200 to 600 mg, for example about 200 to 400 mg, for example about 300 to 600 mg, once a day or in several divided doses to an adult (body weight 60 kg). In the case of parenteral administration, the dosage varies depending on the patient's symptoms, the target organ, the administration method, etc., but in the case of systemic administration, it is generally considered appropriate to administer about 100 to 800 mg, for example about 100 to 500 mg, for example about 400 to 800 mg, once a day, once every 1 to 10 days to an adult (body weight 60 kg). In the case of local administration, it is generally considered appropriate to administer about 10 to 100 mg, for example about 10 to 25 mg, for example about 30 to 100 mg, once a day, once every 1 to 10 days to an adult (body weight 60 kg).

[Method of testing efficacy of administration to cancer patients]
In one embodiment, the present invention provides a method for testing the effectiveness of administration of a combination of a CDK4 and 6 inhibitor, and an NF-κB signaling pathway inhibitor or an AKT inhibitor to a cancer patient, comprising the step of examining whether cancer cells derived from a cancer patient are RB1 positive, wherein RB1 positivity of the cancer cells indicates that administration of the combination is effective in treating the cancer patient.

As described later in the Examples, it was revealed that when cancer cells are RB1 positive, the cancer cells can be effectively killed by administering a combination of CDK4 and 6 inhibitors and an NF-κB signaling pathway inhibitor or an AKT inhibitor.

When cancer cells are RB1 positive, it means that the cancer cells express RB1. The RB1 expressed by cancer cells may be mutated, but it is preferable that the RB1 expressed by cancer cells maintains the function of the wild-type RB1 described above, and it is preferable that the RB1 expressed by cancer cells is wild-type.

Whether or not a cancer cell is RB1 positive can be determined, for example, by PCR amplification of the RB1 locus on the genomic DNA of the cancer cell, PCR amplification of the cDNA of the RB1 gene, detection of the mRNA of the RB1 gene by microarray, ELISA of the RB1 protein, Western blotting, immunochemical staining, etc. The PCR amplified product may be sequenced to analyze whether or not a mutation is present in the RB1 gene.

[Kit for testing the efficacy of administering the combination to a cancer patient]
In one embodiment, the present invention provides a kit for testing the effectiveness of administration of a combination of a CDK4 and 6 inhibitor, and an NF-κB signaling pathway inhibitor or an AKT inhibitor to a cancer patient, the kit comprising a primer set for amplifying genomic DNA of the RB1 gene, a probe for genomic DNA of the RB1 gene, a primer set for amplifying cDNA of the RB1 gene, a probe for mRNA of the RB1 gene, or a substance that specifically binds to RB1 protein, the kit being used to test whether cancer cells derived from a cancer patient are RB1 positive, wherein RB1 positivity of the cancer cells indicates that administration of the combination of the CDK4 and 6 inhibitor, and an NF-κB signaling pathway inhibitor or an AKT inhibitor is effective in treating the cancer patient.

The kit of this embodiment can be suitably used to carry out the above-mentioned method of testing the effectiveness of administering a combination of CDK4 and 6 inhibitors and an NF-κB signaling pathway inhibitor or an AKT inhibitor to a cancer patient.

(Primer set for amplifying genomic DNA)
The kit of this embodiment may include a primer set for amplifying genomic DNA of the RB1 gene. The base sequences of the primer set are not particularly limited as long as they can amplify the RB1 gene on the genome.

When the genomic DNA of cancer cells derived from a cancer patient is amplified with a primer set for amplifying the genomic DNA of the RB1 gene, amplification of the RB1 gene is observed if the cancer cells retain the RB1 gene on their genomic DNA.

If cancer cells derived from a cancer patient retain the RB1 gene, it can be determined that administering a combination of CDK4 and 6 inhibitors and an NF-κB signaling pathway inhibitor or an AKT inhibitor to the cancer patient would be effective.

(Probes for Genomic DNA)
The kit of this embodiment may include a probe for the genomic DNA of the RB1 gene. The base sequence of the probe is not particularly limited as long as it can detect the RB1 gene on the genome. The probe may constitute a nucleic acid array.

When a probe for the genomic DNA of the RB1 gene is hybridized to the genomic DNA of cancer cells derived from a cancer patient, if the cancer cells have the RB1 gene on their genomic DNA, the probe hybridizes and the presence of the RB1 gene can be detected.

If cancer cells derived from a cancer patient retain the RB1 gene, it can be determined that administering a combination of CDK4 and 6 inhibitors and an NF-κB signaling pathway inhibitor or an AKT inhibitor to the cancer patient would be effective.

(Primer set for cDNA amplification)
The kit of this embodiment may contain a primer set for amplifying cDNA of RB1 gene. The base sequences of the primer set are not particularly limited as long as they can amplify cDNA of RB1 gene.

When RNA is extracted from cancer cells derived from a cancer patient and amplified by RT-PCR using a primer set for amplifying cDNA of the RB1 gene, amplification of cDNA of the RB1 gene is observed if the cancer cells express mRNA of the RB1 gene.

If cancer cells derived from a cancer patient express RB1 gene mRNA, it can be determined that administering a combination of CDK4 and 6 inhibitors and an NF-κB signaling pathway inhibitor or an AKT inhibitor to the cancer patient would be effective.

(Probes for mRNA)
The kit of this embodiment may include a probe for the mRNA of the RB1 gene. The base sequence of the probe is not particularly limited as long as it can detect the mRNA of the RB1 gene. The probe may constitute a nucleic acid array.

When RNA is extracted from cancer cells derived from a cancer patient and a probe for the mRNA of the RB1 gene is hybridized, if the cancer cells express the mRNA of the RB1 gene, the probe will hybridize and the presence of the mRNA of the RB1 gene can be detected.

If cancer cells derived from a cancer patient express RB1 gene mRNA, it can be determined that administering a combination of CDK4 and 6 inhibitors and an NF-κB signaling pathway inhibitor or an AKT inhibitor to the cancer patient would be effective.

(Specific binding substance)
The kit of this embodiment may contain a substance that specifically binds to RB1 protein. Examples of the specific binding substance include an antibody, an antibody fragment, an aptamer, and the like. Examples of the antibody fragment include F(ab') ₂ , Fab', Fab, Fv, scFv, and the like. The specific binding substance is not particularly limited as long as it can detect RB1 protein. The specific binding substance may be bound to a solid phase to constitute, for example, a flow strip, an array, and the like.

Proteins can be extracted from cancer cells derived from a cancer patient, and the presence of RB1 protein can be detected by ELISA or Western blotting using a substance that specifically binds to RB1 protein. Alternatively, the presence of RB1 protein can be detected by subjecting a section of cancer tissue derived from a cancer patient to immunochemical staining using a substance that specifically binds to RB1 protein.

If cancer cells derived from a cancer patient express RB1 protein, it can be determined that administering a combination of CDK4 and 6 inhibitors and an NF-κB signaling pathway inhibitor or an AKT inhibitor to the cancer patient would be effective.

[Other embodiments]
In one embodiment, the present invention provides a method for treating cancer, comprising the steps of: examining whether cancer cells derived from a patient are RB1 positive; and, if the cancer cells are RB1 positive, administering to the patient a combination of a CDK4 and 6 inhibitor, and an NF-κB signaling pathway inhibitor or an AKT inhibitor.

In one embodiment, the present invention provides the use of a combination of a CDK4 and 6 inhibitor and an NF-κB signaling pathway inhibitor or an AKT inhibitor for the treatment of RB1-positive cancer.

In one embodiment, the present invention provides the use of a combination of a CDK4 and 6 inhibitor and an NF-κB signaling pathway inhibitor or an AKT inhibitor for the manufacture of a pharmaceutical composition or treatment kit for RB1-positive cancer.

The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples. In the following examples, experiments using mice were performed in accordance with a protocol (AP-153426) approved by the Animal Experiment Committee of Kanazawa University.

[Experimental Example 1]
Inactivation of the RB pathway induces the development of hepatocellular carcinoma.
The RB family includes RB1, RB Transcriptional Corepressor Like 1 (RBL1), and RB Transcriptional Corepressor Like 2 (RBL2).

In this experimental example, mice lacking the RBL1 gene and capable of conditionally knocking out the Rb1 and Rb12 genes (hereinafter sometimes referred to as "TKO mice") were used, which also lacked the Tumor Protein P53 (Trp53) gene (hereinafter sometimes referred to as "QKO mice").

As shown in Figure 2(a), Rb1 ^lox/lox ; Rbl1 ^-/- ; Rbl2 ^lox/lox ; Trp53 ^-/- (QKO) mice were injected via the tail vein with a PiggyBac transposon vector construct (pCMV-Puro-Cre-IRES2-GFP) carrying genes encoding Cre recombinase, green fluorescent protein (GFP), and puromycin resistance protein (Puro) in tandem, and an expression vector encoding the transposase (pCMV-hyPBase) by hydrodynamic injection.

As a result, GFP fluorescence was observed in the liver 3 days after injection. As shown in Figure 2(b), tumor formation was typically observed in the liver 2.5 months after injection. In Figure 2(b), the arrow indicates the tumor.

Primary hepatocellular carcinoma cells (hereinafter sometimes referred to as "QKO PHCC") collected from the tumor showed high expression of Ki67 and alpha-fetoprotein (AFP), which are characteristic of hepatocellular carcinoma. These results demonstrated that inactivation of the RB pathway induces the development of hepatocellular carcinoma.

[Experimental Example 2]
(CDK4/6 inhibitors are not optimal)
RB7LP is a mutant of RB1 and is not phosphorylated. RB7LP lacks the N-terminal domain of RB1, but retains all of pockets A, B, and C, and is therefore capable of binding to proteins having E2F and LxCxE motifs. However, seven serine residues known to be phosphorylated have been replaced with amino acids that are not phosphorylated. SEQ ID NO:1 shows the amino acid sequence of RB7LP.

Therefore, RB7LP can mimic the RB1-dependent action of CDK4/6 inhibitors. Therefore, we expressed RB7LP in hepatocellular carcinoma cells and examined the induction of apoptosis.

Specifically, a tetracycline-inducible RB7LP expression vector (pTRE3G-puro-RB7LP-GFP) was introduced into the human hepatoma cell line HepG2. These cells (hereinafter sometimes referred to as "HepG2-pTRE3G-puro-RB7LP-GFP cells") express RB7LP upon the addition of doxycycline to the culture medium.

Next, 1 μg/mL doxycycline was added to the medium of HepG2-pTRE3G-puro-RB7LP-GFP cells and incubated for 3 days. For comparison, cells incubated in the same manner without the addition of doxycycline were used.

Next, annexin V-positive cells of each type of cell line were stained using eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific). Dead cells were also stained with propidium iodide (PI).

Then, each cell was analyzed using FACS Canto II (BD Biosciences). Cells with high Annexin V fluorescence and low PI fluorescence are early apoptotic cells, and cells with high Annexin V and PI fluorescence are late apoptotic cells.

Figure 3(a) is a graph showing the results of flow cytometry. In Figure 3(a), "DOX -" indicates the results in the absence of doxycycline, and "DOX +" indicates the results with the addition of doxycycline. The horizontal axis indicates the fluorescence intensity of annexin V, and the vertical axis indicates the fluorescence intensity of PI.

Figure 3(b) is a graph created based on the results of Figure 3(a). In Figure 3(b), the percentage of apoptotic cells is the sum of the percentage of cells in the early stage of apoptosis (annexin V positive, PI negative) and the percentage of cells in the late stage of apoptosis (annexin V positive, PI positive). In Figure 3(b), "DOX -" indicates the results in the absence of doxycycline, and "DOX +" indicates the results with the addition of doxycycline. Additionally, "***" indicates that there is a significant difference at P<0.001 as a result of an unpaired two-tailed Student's t-test.

As a result, it was revealed that induction of apoptosis by induction of RB7LP expression was insufficient. This result indicates that administration of a CDK4/6 inhibitor to cancer cells does not sufficiently induce apoptosis.

[Experimental Example 3]
(Screening for compounds that enhance the effect of RB7LP)
Compounds that specifically kill HepG2-pTRE3G-puro-RB7LP-GFP cells in the presence of doxycycline were screened from a compound library.

A commercially available library containing 80 types of kinase inhibitors (product name: SCREEN-WELL Kinase Inhibitor Library, model number: BML-2832, Enzo Life Sciences) was used as the compound library.

HepG2-pTRE3G-puro-RB7LP-GFP cells were cultured for 3 days in the presence or absence of 1 μg/mL doxycycline. Then, each cell was seeded at 20,000 cells/well in a 96-well plate, exposed to each compound at a final concentration of 1 μM in the presence or absence of 1 μg/mL doxycycline, and incubated for 24 hours. The viability of each cell was then measured using a commercially available kit (product name: Cell count reagent SF, Nacalai Tesque).

4 is a graph showing the results of the screening. The vertical axis of the graph shows the ratio shown in the following formula (F1). In the following formula (F1), "DOX(+) cells" show cells exposed to a compound in the presence of doxycycline, and "DOX(-) cells" show cells exposed to a compound in the absence of doxycycline.
Ratio = viability of DOX(-) cells / viability of DOX(+) cells ... (F1)

The horizontal axis indicates the compound number. A higher value on the vertical axis indicates a compound with DOX(+) cell-specific cytotoxicity. As a result, it was revealed that the IKKβ inhibitor Bay11-7082 (CAS number: 19542-67-7) has DOX(+) cell-specific cytotoxicity. In other words, it was revealed that Bay11-7082 enhances the cytotoxicity of HepG2 cells in which RB7LP expression was induced.

[Experimental Example 4]
(Examination of RB7LP expression and the effect of IKKβ inhibitors)
In HepG2 cells and primary hepatocellular carcinoma cells (QKO PHCC), the expression of RB7LP was induced and the cytotoxicity of IKKβ inhibitors was examined. The IKKβ inhibitors used were Bay11-7082, IMD-0354 (CAS number: 978-62-1), and shRNA against IKKβ.

<Study using Bay11-7082>
HepG2-pTRE3G-puro-RB7LP-GFP cells were cultured for 3 days in the presence or absence of 1 μg/mL doxycycline. Then, each cell was seeded in a 6-well plate, exposed to Bay11-7082 at a final concentration of 0, 5, 10, 15, 20, or 25 μM in the presence or absence of 1 μg/mL doxycycline, and incubated for 24 hours. Then, the viability of each cell was measured using a commercially available kit (product name "Cell count reagent SF", Nacalai Tesque).

Figure 5 is a graph showing the survival rate of each type of cell. As a result, it was revealed that 10 to 20 μM of Bay11-7082 showed the most synergistic effect on the induction of RB7LP expression.

Next, HepG2-pTRE3G-puro-RB7LP-GFP cells were exposed to 15 μM Bay11-7082 for 24 hours, and annexin V-positive cells were stained using eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific) in the same manner as in Experimental Example 2. Dead cells were also stained with propidium iodide (PI).

Next, flow cytometry analysis was performed to measure the percentage of cells that had undergone apoptosis. Figure 6 is a graph created based on the results of flow cytometry. In Figure 6, the percentage of cells that had undergone apoptosis was calculated as the sum of the percentage of cells in the early stage of apoptosis (annexin V positive, PI negative) and the percentage of cells in the late stage of apoptosis (annexin V positive, PI positive).

In FIG. 6, "DOX -" indicates the results in the absence of doxycycline, and "DOX +" indicates the results with the addition of doxycycline. "BAY -" indicates the results in the absence of Bay11-7082, and "BAY +" indicates the results in the presence of Bay11-7082 at a final concentration of 15 μM. "Z-VAD -" indicates the results in the absence of the caspase inhibitor Z-VAD-FMK (catalog number "3188-v", Peptide Institute, Inc.), and "Z-VAD +" indicates the results in the presence of Z-VAD-FMK at a final concentration of 20 μM. "*" and "****" indicate significant differences at P<0.05 and P<0.0001, respectively, as a result of two-way ANOVA followed by Tukey's post-hoc test.

The results showed that more than 80% of HepG2-pTRE3G-puro-RB7LP-GFP cells exposed to 15 μM Bay11-7082 in the presence of doxycycline underwent apoptosis. Furthermore, this apoptosis was significantly suppressed by the addition of Z-VAD-FMK.

These results further support the idea that Bay11-7082 significantly enhances the induction of apoptosis in HepG2 cells in which RB7LP expression was induced.

<<Study using IMD-0354>>
Next, a similar study was carried out using IKKβ inhibitor IMD-0354 (CAS number: 978-62-1) instead of Bay11-7082.

HepG2-pTRE3G-puro-RB7LP-GFP cells were cultured for 3 days in the presence or absence of 1 μg/mL doxycycline. Then, each cell was seeded in a 6-well plate, exposed to IMD-0354 at a final concentration of 15 μM in the presence or absence of 1 μg/mL doxycycline, and incubated for 24 hours.

Next, as in Experimental Example 2, annexin V-positive cells were stained using eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific). Dead cells were stained with propidium iodide (PI). Then, flow cytometry analysis was performed to measure the proportion of cells that had undergone apoptosis.

Figure 7 is a graph created based on the results of flow cytometry. In Figure 7, the percentage of cells that underwent apoptosis was calculated as the sum of the percentage of cells in the early stage of apoptosis (annexin V positive, PI negative) and the percentage of cells in the late stage of apoptosis (annexin V positive, PI positive).

In Figure 7, "DOX -" indicates the results in the absence of doxycycline, and "DOX +" indicates the results when doxycycline was added. "IMD -" indicates the results in the absence of IMD-0354, and "IMD +" indicates the results in the presence of IMD-0354 at a final concentration of 15 μM. "**" and "****" indicate that there is a significant difference at P<0.01 and P<0.0001, respectively, as a result of two-way ANOVA followed by Tukey's post-hoc test.

As a result, it was revealed that IMD-0354 significantly enhanced the induction of apoptosis in HepG2-pTRE3G-puro-RB7LP-GFP cells in which RB7LP expression was induced in the presence of doxycycline. In other words, it was revealed that IMD-0354 also exhibited an effect similar to that of Bay11-7082.

<<Study using shRNA against IKKβ>>
Next, instead of adding an IKKβ inhibitor, IKKβ was knocked down to examine whether induction of apoptosis in HepG2-pTRE3G-puro-RB7LP-GFP cells was enhanced.

For the shRNA against IKKβ, we used commercially available shRNA expression vectors (catalog numbers "#SHCLNG-NM_001556", "TRCN0000018917", "TRCN0000018915", "TRCN0000018916", and "TRCN0000018918") in which the puromycin resistance gene had been replaced with a blastidine resistance gene.

Four types of shRNA were introduced into HepG2-pTRE3G-puro-RB7LP-GFP cells, and the expression of IKKβ was confirmed by Western blotting. It was confirmed that IKKβ could be knocked down using any of the shRNAs. Therefore, one of these shRNAs was used in the next experiment.

HepG2-pTRE3G-puro-RB7LP-GFP cells, with or without the introduction of an expression vector for shRNA against IKKβ, were cultured for 3 days in the presence or absence of 1 μg/mL doxycycline. Then, each cell was seeded in a 6-well plate and incubated for an additional 24 hours in the presence or absence of 1 μg/mL doxycycline.

Next, as in Experimental Example 2, annexin V-positive cells were stained using eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific). Dead cells were stained with propidium iodide (PI). Then, flow cytometry analysis was performed to measure the proportion of cells that had undergone apoptosis.

Figure 8 is a graph created based on the results of flow cytometry. In Figure 8, the percentage of cells that underwent apoptosis was calculated as the sum of the percentage of cells in the early stage of apoptosis (annexin V positive, PI negative) and the percentage of cells in the late stage of apoptosis (annexin V positive, PI positive).

In Figure 8, "DOX -" indicates the results in the absence of doxycycline, and "DOX +" indicates the results with the addition of doxycycline. "shRNA -" indicates the results for cells in which shRNA against IKKβ was not introduced, and "shRNA +" indicates the results for cells in which shRNA against IKKβ was introduced. "****" indicates that there was a significant difference at P<0.0001 as a result of two-way ANOVA followed by Tukey's post-hoc test.

As a result, it was revealed that knockdown of IKKβ significantly enhanced the induction of apoptosis in HepG2-pTRE3G-puro-RB7LP-GFP cells in which RB7LP expression was induced in the presence of doxycycline. This result further supports the idea that inhibition of IKKβ function significantly enhances the induction of apoptosis in HepG2 cells in which RB7LP expression was induced.

<Study using primary hepatocellular carcinoma cells (QKO PHCC)>
An investigation was carried out using QKO PHCCs obtained in Experimental Example 1 instead of HepG2-pTRE3G-puro-RB7LP-GFP cells.

Cells were produced by introducing an RB7LP expression vector (plenti6.3-RB7LP) into QKO PHCC (hereinafter sometimes referred to as "RB7LP(+) QKO PHCC cells") and cells were produced by introducing a LacZ expression vector (plenti6.3/V5-LacZ) into QKO PHCC (hereinafter sometimes referred to as "RB7LP(-) QKO PHCC cells").

RB7LP(+)QKO PHCC cells and RB7LP(-)QKO PHCC cells were then exposed to Bay11-7082 at a final concentration of 15 μM and incubated for 24 hours.

Next, as in Experimental Example 2, annexin V-positive cells were stained using eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific). Dead cells were stained with propidium iodide (PI). Then, flow cytometry analysis was performed to measure the proportion of cells that had undergone apoptosis.

Figure 9 is a graph created based on the results of flow cytometry. In Figure 9, the percentage of cells that underwent apoptosis was calculated as the sum of the percentage of cells in the early stage of apoptosis (annexin V positive, PI negative) and the percentage of cells in the late stage of apoptosis (annexin V positive, PI positive).

In FIG. 9, "RB7LP -" indicates the results for RB7LP(-) QKO PHCC cells, and "RB7LP +" indicates the results for RB7LP(+) QKO PHCC cells. "BAY -" indicates the results in the absence of Bay11-7082, and "BAY +" indicates the results in the presence of Bay11-7082 at a final concentration of 15 μM. "*" and "****" indicate significant differences at P<0.05 and P<0.0001, respectively, as a result of two-way ANOVA followed by Tukey's post-hoc test.

As a result, it was revealed that Bay11-7082 significantly enhanced the induction of apoptosis in QKO PHCC cells expressing RB7LP. In other words, the same results were observed in QKO PHCC cells as in HepG2 cells.

[Experimental Example 5]
(Study of the combined effect of CDK4 and 6 inhibitors and IKKβ inhibitors)
Various cancer cell lines were incubated in the presence of CDK4 and 6 inhibitors and IKKβ inhibitors, and then Annexin V-positive cells were stained using eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific) in the same manner as in Experimental Example 2. Dead cells were stained with propidium iodide (PI). Then, flow cytometry analysis was performed to measure the proportion of cells that had undergone apoptosis.

The cancer cell lines used were human liver cancer cell lines Huh7, Li-7, and HepG2, human lung cancer cell line H1975, human breast cancer cell lines HCC1143 and T47D, and human colon cancer cell lines HCT-116 and DLD-1. All of these cells are known to be RB1 positive.

Palbociclib (CAS number: 571190-30-2), ribociclib (CAS number: 1211441-98-3), or abemaciclib (CAS number: 1231929-97-7) were used as CDK4 and 6 inhibitors. Bay11-7082 was used as an IKKβ inhibitor.

Combination use of palbociclib and Bay11-7082
Huh7 cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 10 μM and Bay11-7082 at a final concentration of 10 μM. Li-7 cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 10 μM and Bay11-7082 at a final concentration of 15 μM. H1975 cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 10 μM and Bay11-7082 at a final concentration of 10 μM. HCC1143 cells were incubated for 4 hours in the presence or absence of palbociclib at a final concentration of 10 μM and Bay11-7082 at a final concentration of 10 μM. T47D cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 10 μM and Bay11-7082 at a final concentration of 10 μM. HCT-116 cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 8 μM and Bay11-7082 at a final concentration of 15 μM. DLD-1 cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 8 μM and Bay11-7082 at a final concentration of 15 μM.

Figures 10 to 16 are graphs created based on the results of flow cytometry. In Figures 10 to 16, the percentage of cells that underwent apoptosis was calculated as the sum of the percentage of cells in the early stage of apoptosis (annexin V positive, PI negative) and the percentage of cells in the late stage of apoptosis (annexin V positive, PI positive).

Figure 10 shows the results for Huh7 cells, Figure 11 shows the results for Li-7 cells, Figure 12 shows the results for H1975 cells, Figure 13 shows the results for HCC1143 cells, Figure 14 shows the results for T47D cells, Figure 15 shows the results for HCT-116 cells, and Figure 16 shows the results for DLD-1 cells.

In Figures 10 to 16, "Palbo. -" indicates the results in the absence of palbociclib, and "Palbo. +" indicates the results in the presence of palbociclib. "BAY -" indicates the results in the absence of Bay11-7082, and "BAY +" indicates the results in the presence of Bay11-7082. "n.s.", "*", "**" and "****" indicate no significant difference, significant difference at P<0.05, P<0.01 and P<0.0001, respectively, as a result of two-way ANOVA followed by Tukey's post-hoc test.

As a result, it was found that in both cancer cell lines, the combined use of CDK4 and 6 inhibitors and IKKβ inhibitors significantly enhanced the induction of apoptosis. These results indicate that the combined use of CDK4 and 6 inhibitors and IKKβ inhibitors is effective not only in hepatocellular carcinoma, but also in lung cancer, breast cancer, and colon cancer.

Combination of ribociclib and Bay11-7082
A study was carried out using ribociclib as an inhibitor of CDK4 and 6. HepG2 cells were incubated for 24 hours in the presence or absence of ribociclib at a final concentration of 8 μM and Bay11-7082 at a final concentration of 15 μM.

Next, as in Experimental Example 2, annexin V-positive cells were stained using eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific). Dead cells were stained with propidium iodide (PI). Then, flow cytometry analysis was performed to measure the proportion of cells that had undergone apoptosis.

Figure 17 is a graph created based on the results of flow cytometry. In Figure 17, the percentage of cells that underwent apoptosis was calculated as the sum of the percentage of cells in the early stage of apoptosis (annexin V positive, PI negative) and the percentage of cells in the late stage of apoptosis (annexin V positive, PI positive).

In FIG. 17, "Ribo. -" indicates the results in the absence of ribociclib, and "Ribo. +" indicates the results in the presence of ribociclib. "BAY -" indicates the results in the absence of Bay11-7082, and "BAY +" indicates the results in the presence of Bay11-7082. "ns" and "****" indicate no significant difference and a significant difference at P<0.0001, respectively, as a result of two-way ANOVA followed by Tukey's post-hoc test.

As a result, it was found that even when ribociclib was used as a CDK4 and 6 inhibitor, the induction of apoptosis was significantly enhanced by combining it with an IKKβ inhibitor.

Combination use of abemaciclib and Bay11-7082
A study was carried out using abemaciclib as an inhibitor of CDK4 and 6. HepG2 cells were incubated for 24 hours in the presence or absence of abemaciclib at a final concentration of 6 μM and Bay11-7082 at a final concentration of 10 μM.

Next, as in Experimental Example 2, annexin V-positive cells were stained using eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific). Dead cells were stained with propidium iodide (PI). Then, flow cytometry analysis was performed to measure the proportion of cells that had undergone apoptosis.

Figure 18 is a graph created based on the results of flow cytometry. In Figure 18, the percentage of cells that underwent apoptosis was calculated as the sum of the percentage of cells in the early stage of apoptosis (annexin V positive, PI negative) and the percentage of cells in the late stage of apoptosis (annexin V positive, PI positive).

In FIG. 18, "Abema. -" indicates the results in the absence of abemaciclib, and "Abema. +" indicates the results in the presence of abemaciclib. "BAY -" indicates the results in the absence of Bay11-7082, and "BAY +" indicates the results in the presence of Bay11-7082. "****" indicates that there is a significant difference at P<0.0001 as a result of two-way ANOVA followed by Tukey's post-hoc test.

As a result, it was found that even when abemaciclib was used as a CDK4 and 6 inhibitor, the induction of apoptosis was significantly enhanced by combining it with an IKKβ inhibitor.

[Experimental Example 6]
(Examination of RB7LP expression and the effect of NF-κB inhibitors)
The induction of RB7LP expression and the induction of apoptosis by the addition of an NF-κB inhibitor were examined using an NF-κB inhibitor instead of an IKKβ inhibitor. QNZ (CAS number: 545380-34-5) was used as the NF-κB inhibitor.

HepG2-pTRE3G-puro-RB7LP-GFP cells were cultured for 3 days in the presence or absence of 1 μg/mL doxycycline. Then, each cell was seeded in a 6-well plate, exposed to a final concentration of 50 nM QNZ in the presence or absence of 1 μg/mL doxycycline, and incubated for 24 hours.

Next, as in Experimental Example 2, annexin V-positive cells were stained using eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific). Dead cells were stained with propidium iodide (PI). Then, flow cytometry analysis was performed to measure the proportion of cells that had undergone apoptosis.

Figure 19 is a graph created based on the results of flow cytometry. In Figure 19, the percentage of cells that underwent apoptosis was calculated as the sum of the percentage of cells in the early stage of apoptosis (annexin V positive, PI negative) and the percentage of cells in the late stage of apoptosis (annexin V positive, PI positive).

In Figure 19, "DOX -" indicates the results in the absence of doxycycline, and "DOX +" indicates the results with doxycycline added. "QNZ -" indicates the results in the absence of QNZ, and "QNZ +" indicates the results in the presence of QNZ. "**" and "****" indicate significant differences at P<0.01 and P<0.0001, respectively, as a result of two-way ANOVA followed by Tukey's post-hoc test.

As a result, it was revealed that QNZ significantly enhanced the induction of apoptosis in HepG2-pTRE3G-puro-RB7LP-GFP cells in which RB7LP expression was induced in the presence of doxycycline. In other words, it was revealed that NF-κB inhibitors also showed the same effect as IKKβ inhibitors.

[Experimental Example 7]
(Investigation of the combined effect of CDK4 and 6 inhibitors and AKT inhibitors)
An investigation was carried out using an AKT inhibitor instead of an IKKβ inhibitor. After incubating various cancer cell lines in the presence of CDK4 and 6 inhibitors and an AKT inhibitor, annexin V-positive cells were stained using eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific) in the same manner as in Experimental Example 2. In addition, dead cells were stained with propidium iodide (PI). Subsequently, flow cytometry analysis was performed to measure the proportion of cells that underwent apoptosis.

The cancer cell lines used were the human liver cancer cell line HepG2, the human colon cancer cell lines SW480, HCT-116, and DLD-1, and the human lung cancer cell line A549.

Palbociclib (CAS number: 571190-30-2) was used as the CDK4 and 6 inhibitor. Akt Inhibitor X (CAS number: 925681-41-0) was used as the AKT inhibitor.

HepG2 cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 10 μM and Akt Inhibitor X at a final concentration of 25 μM. SW480 cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 10 μM and Akt Inhibitor X at a final concentration of 20 μM. HCT-116 cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 10 μM and Akt Inhibitor X at a final concentration of 20 μM. DLD-1 cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 10 μM and Akt Inhibitor X at a final concentration of 20 μM. A549 cells were incubated for 24 hours in the presence or absence of palbociclib at a final concentration of 10 μM and Akt Inhibitor X at a final concentration of 20 μM.

Figures 20 to 24 are graphs created based on the results of flow cytometry. In Figures 20 to 24, the percentage of cells that underwent apoptosis was calculated as the sum of the percentage of cells in the early stage of apoptosis (annexin V positive, PI negative) and the percentage of cells in the late stage of apoptosis (annexin V positive, PI positive).

Figure 20 shows the results for HepG2 cells, Figure 21 shows the results for SW480 cells, Figure 22 shows the results for HCT-116 cells, Figure 23 shows the results for DLD-1 cells, and Figure 24 shows the results for A549 cells.

In Figures 20 to 24, "Palbo. -" indicates the results in the absence of palbociclib, and "Palbo. +" indicates the results in the presence of palbociclib. "CAS -" indicates the results in the absence of Akt Inhibitor X, and "CAS +" indicates the results in the presence of Akt Inhibitor X. "*", "***", and "***" indicate significant differences at P<0.05, P<0.001, and P<0.0001, respectively, as a result of two-way ANOVA followed by Tukey's post-hoc test.

As a result, it was found that in both cancer cell lines, the induction of apoptosis was significantly enhanced by the combined use of CDK4 and 6 inhibitors and AKT inhibitors. This result indicates that the combined use of CDK4 and 6 inhibitors and AKT inhibitors is effective not only in hepatocellular carcinoma, but also in colon cancer and lung cancer.

[Experimental Example 8]
(Investigation of the effect of RB1 expression on the combined effect of CDK4 and 6 inhibitors and IKKβ inhibitors)
The effect of RB1 expression on the combined effect of CDK4 and 6 inhibitors and an IKKβ inhibitor was examined.

First, a tetracycline-inducible Rb1 expression vector was introduced into QKO PHCC cells. These cells (hereinafter sometimes referred to as "QKO-PHCC-pTRE3G-blast-mouse Rb1-mcherry cells") express Rb1 upon the addition of doxycycline to the medium.

Next, QKO-PHCC-pTRE3G-blast-mouse Rb1-mcherry cells were cultured for 3 days in the presence or absence of 1 μg/mL doxycycline. Then, each cell was seeded in a 6-well plate and incubated for 24 hours in the presence or absence of 1 μg/mL doxycycline, in the presence or absence of palbociclib at a final concentration of 10 μM, and in the presence or absence of Bay11-7082 at a final concentration of 15 μM.

Next, as in Experimental Example 2, annexin V-positive cells were stained using eBioscience Annexin V-FITC Apoptosis Detection Kit (Thermo Fisher Scientific). Dead cells were stained with propidium iodide (PI). Then, flow cytometry analysis was performed to measure the proportion of cells that had undergone apoptosis.

Figure 25 is a graph created based on the results of flow cytometry. In Figure 25, the percentage of cells that underwent apoptosis was calculated as the sum of the percentage of cells in the early stage of apoptosis (annexin V positive, PI negative) and the percentage of cells in the late stage of apoptosis (annexin V positive, PI positive).

In FIG. 25, "DOX -" indicates the results in the absence of doxycycline, and "DOX +" indicates the results with the addition of doxycycline. "Palbo. -" indicates the results in the absence of palbociclib, and "Palbo. +" indicates the results in the presence of palbociclib. "BAY -" indicates the results in the absence of Bay11-7082, and "BAY +" indicates the results in the presence of Bay11-7082. "**" and "****" indicate significant differences at P<0.01 and P<0.0001, respectively, as a result of two-way ANOVA followed by Tukey's post-hoc test.

As a result, in the absence of Rb1, only a slight apoptosis-inducing effect was observed with the combined use of palbociclib and Bay11-7082. In contrast, in the presence of Rb1, it was revealed that the induction of apoptosis by the combined use of palbociclib and Bay11-7082 was significantly enhanced. These results indicate that the presence or absence of RB1 in cancer cells is a marker that indicates the effectiveness of the combined use of CDK4 and 6 inhibitors, and IKKβ inhibitors.

[Experimental Example 9]
(In vivo study of the combined effect of CDK4/6 inhibitor and IKKβ inhibitor)
A CDK4/6 inhibitor and an IKKβ inhibitor were administered to a tumor-bearing mouse model, and the change in tumor volume over time was measured.

The tumor-bearing mouse models used were a tumor-bearing mouse model transplanted with HepG2 cells, a tumor-bearing mouse model transplanted with Li-7 cells, and a tumor-bearing mouse model transplanted with QKO PHCC cells (QKO-PHCC-pTRE3G-blast-mouse Rb1-mcherry cells) that express mouse Rb1 in a tetracycline-inducible manner.

Palbociclib (CAS number: 571190-30-2) was used as a CDK4 and 6 inhibitor. Bay11-7082 was used as an IKKβ inhibitor.

<Study using a tumor-bearing mouse model transplanted with HepG2 cells>
1×10 ⁶ HepG2 cells were transplanted subcutaneously into the back of KSN/slc mice (6 weeks old, male) together with Matrigel (catalog number "354234", Corning) to prepare a tumor-bearing mouse model. After about 4 weeks, when the tumor size reached about 200 mm ³ , administration of palbociclib and Bay11-7082 to the mice in the test group was started. Palbociclib was administered daily by oral gavage at a dose of 80 mg/Kg. Bay11-7082 was dissolved in corn oil and administered every 2 days by subcutaneous injection at a dose of 20 mg/Kg. Vehicle (corn oil or L-sodium lactate) was administered to the control group. Tumor volume and body weight were measured every 2 days for each group of mice (n=5).

Figure 26 is a graph showing the change in tumor volume over time in mice of each group. In Figure 26, the vertical axis shows the increase rate (%) of tumor volume, and the horizontal axis shows the number of days after the start of drug administration.

In FIG. 26, "Palbo. -" indicates the results for mice that were not administered palbociclib, and "Palbo. +" indicates the results for mice that were administered palbociclib. "BAY -" indicates the results for mice that were not administered Bay11-7082, and "BAY +" indicates the results for mice that were administered Bay11-7082. "*", "***", and "***" indicate that there is a significant difference at P<0.05, P<0.01, and P<0.0001, respectively, as a result of two-way ANOVA followed by Tukey's post-hoc test.

As a result, administration of palbociclib alone slightly suppressed the increase in tumor volume compared to the non-administered group. It was also revealed that combined administration of palbociclib and Bay11-7082 almost completely suppressed the increase in tumor volume. Furthermore, no significant differences were observed in the body weight of mice in each group. These results indicate that the combined use of CDK4 and 6 inhibitors and IKKβ inhibitors is also effective in vivo.

<<Study using a tumor-bearing mouse model transplanted with Li-7 cells>>
1×10 ⁶ Li-7 cells were transplanted subcutaneously into the back of KSN/slc mice (6 weeks old, male) together with Matrigel (catalog number "354234", Corning) to prepare a tumor-bearing mouse model. After about 5 weeks, when the tumor size reached about 200 mm ³ , administration of palbociclib and Bay11-7082 to the mice in the test group was started. Palbociclib was administered daily by oral gavage at a dose of 80 mg/Kg. Bay11-7082 was dissolved in corn oil and administered every 2 days by subcutaneous injection at a dose of 20 mg/Kg. Vehicle (corn oil or L-sodium lactate) was administered to the control group. Tumor volume and body weight were measured every 2 days for each group of mice (n=5).

Figure 27 is a graph showing the change in tumor volume over time in mice of each group. In Figure 27, the vertical axis shows the increase rate (%) of tumor volume, and the horizontal axis shows the number of days after the start of drug administration.

In FIG. 27, "Palbo. -" indicates the results for mice that were not administered palbociclib, and "Palbo. +" indicates the results for mice that were administered palbociclib. "BAY -" indicates the results for mice that were not administered Bay11-7082, and "BAY +" indicates the results for mice that were administered Bay11-7082. "****" indicates that there is a significant difference at P<0.0001 as a result of two-way ANOVA followed by Tukey's post-hoc test.

As a result, administration of palbociclib alone significantly suppressed the increase in tumor volume compared to the non-administration group. It was also revealed that combined administration of palbociclib and Bay11-7082 significantly reduced tumor volume. Furthermore, no significant differences were observed in the body weight of mice in each group. These results indicate that combined use of CDK4 and 6 inhibitors and IKKβ inhibitors is effective in vivo, even in tumors derived from Li-7 cells.

<<Study using a tumor-bearing mouse model transplanted with QKO PHCC cells expressing mouse Rb1 in a tetracycline-inducible manner>>
^5x105 QKO-PHCC-pTRE3G-blast-mouse Rb1-mcherry cells were transplanted subcutaneously into the back of KSN/slc mice (6 weeks old, male) together with Matrigel (catalog number "354234", Corning) to prepare a tumor-bearing mouse model. After the tumor size reached approximately 200 ^mm3 , doxycycline, palbociclib, and Bay11-7082 were administered to the test group of mice.

Doxycycline (50 mg/Kg) and palbociclib (80 mg/Kg) were administered daily by oral gavage. Bay11-7082 was dissolved in corn oil and administered subcutaneously at a dose of 20 mg/Kg every 2 days. Vehicle (0.9% NaCl, corn oil, or sodium L-lactate) was administered to the control group. Tumor volume and body weight were measured every 2 days for each group of mice (n=5).

Figure 28 is a graph showing the change in tumor volume over time in mice of each group. In Figure 28, the vertical axis shows the increase rate (%) of tumor volume, and the horizontal axis shows the number of days after the start of drug administration.

In FIG. 28, "DOX -" indicates the results for mice that were not administered doxycycline, and "DOX +" indicates the results for mice that were administered doxycycline. "Palbo. -" indicates the results for mice that were not administered palbociclib, and "Palbo. +" indicates the results for mice that were administered palbociclib. "BAY -" indicates the results for mice that were not administered Bay11-7082, and "BAY +" indicates the results for mice that were administered Bay11-7082. "****" indicates that a significant difference was found at P<0.0001 as a result of two-way ANOVA followed by Tukey's post-hoc test.

As a result, administration of palbociclib alone significantly suppressed the increase in tumor volume compared to the non-administration group. It was also revealed that combined administration of palbociclib and Bay11-7082 significantly reduced tumor volume. Furthermore, no significant differences were observed in the body weight of mice in each group. These results indicate that combined use of CDK4 and 6 inhibitors and IKKβ inhibitors is effective in vivo, even in tumors derived from Li-7 cells.

As a result, in the absence of Rb1, the combined administration of palbociclib and Bay11-7082 only slightly suppressed the increase in tumor volume. In contrast, in the presence of Rb1, the combined administration of palbociclib and Bay11-7082 significantly reduced tumor volume. This result further supports the idea that the presence or absence of RB1 in cancer cells is a marker indicating the effectiveness of the combined use of CDK4 and 6 inhibitors and IKKβ inhibitors.

The present invention provides a technology for effectively treating RB1-positive cancer.

Claims (4)

A pharmaceutical composition for treating RB Transcriptional Corepressor 1 (RB1)-positive cancer, comprising a combination of a cyclin-dependent kinase (CDK) 4 and 6 inhibitor and an NF-κB signaling pathway inhibitor or an AKT inhibitor as active ingredients ,
The CDK4 and 6 inhibitor is palbociclib (CAS number: 571190-30-2), ribociclib (CAS number: 1211441-98-3) or abemaciclib (CAS number: 1231929-97-7);
the NF-κB signaling pathway inhibitor is an IKKβ inhibitor or an NF-κB inhibitor;
The IKKβ inhibitor is Bay11-7082 (CAS number: 19542-67-7), IMD-0354 (CAS number: 978-62-1), or an inhibitory nucleic acid against IKKβ;
The NF-κB inhibitor is QNZ (CAS number: 545380-34-5),
The AKT inhibitor is Akt Inhibitor X (CAS number: 925681-41-0),
The pharmaceutical composition, wherein the RB1 positive cancer is hepatocellular carcinoma or lung cancer . A kit for treating RB1-positive cancer , comprising an inhibitor of cyclin-dependent kinases (CDK) 4 and 6, and an inhibitor of the NF-κB signaling pathway or an inhibitor of AKT,
the CDK4 and 6 inhibitor is palbociclib, ribociclib, or abemaciclib;
the NF-κB signaling pathway inhibitor is an IKKβ inhibitor or an NF-κB inhibitor;
the IKKβ inhibitor is Bay11-7082, IMD-0354, or an inhibitory nucleic acid against IKKβ;
The NF-κB inhibitor is QNZ;
the AKT inhibitor is Akt Inhibitor X ,
The therapeutic kit, wherein the RB1-positive cancer is hepatocellular carcinoma or lung cancer . A method comprising the step of testing whether a cancer cell derived from a cancer patient is RB1 positive;
13. A method for testing the effectiveness of administering a combination of a CDK4 and 6 inhibitor and an NF-κB signaling pathway inhibitor or an AKT inhibitor to a cancer patient, wherein the cancer cells being RB1 positive indicates that the administration of the combination is effective in treating the cancer patient , comprising:
the CDK4 and 6 inhibitor is palbociclib, ribociclib, or abemaciclib;
the NF-κB signaling pathway inhibitor is an IKKβ inhibitor or an NF-κB inhibitor;
the IKKβ inhibitor is Bay11-7082, IMD-0354, or an inhibitory nucleic acid against IKKβ;
The NF-κB inhibitor is QNZ;
the AKT inhibitor is Akt Inhibitor X ,
The method, wherein the RB1 positive cancer is hepatocellular carcinoma or lung cancer . A primer set for amplifying genomic DNA of the RB1 gene;
A probe for the genomic DNA of the RB1 gene,
A primer set for amplifying cDNA of the RB1 gene,
A probe for the mRNA of the RB1 gene, or a substance that specifically binds to the RB1 protein,
Cancer cells derived from a cancer patient are used to test whether they are RB1 positive,
A kit for testing the effectiveness of administering a combination of a CDK4 and 6 inhibitor, and an NF-κB signaling pathway inhibitor or an AKT inhibitor to a cancer patient, wherein the cancer cells are RB1 positive, indicating that the administration of the combination is effective in treating the cancer patient ,
the CDK4 and 6 inhibitor is palbociclib, ribociclib, or abemaciclib;
the NF-κB signaling pathway inhibitor is an IKKβ inhibitor or an NF-κB inhibitor;
the IKKβ inhibitor is Bay11-7082, IMD-0354, or an inhibitory nucleic acid against IKKβ;
The NF-κB inhibitor is QNZ;
the AKT inhibitor is Akt Inhibitor X ,
The kit, wherein the RB1-positive cancer is hepatocellular carcinoma or lung cancer .

Images