Novel medicine for treating osteosarcoma

Abstract

The present invention addresses the problem of developing a novel medicine for treating osteosarcoma. The present invention also addresses the problem of developing a therapy marker for examining the therapeutic effect of medicine in the treatment of osteosarcoma.　According to the present invention, in order to solve the problems, a compound capable of inhibiting the proliferation of osteosarcoma cells has been discovered and it has been demonstrated that a novel pharmaceutical composition for treating osteosarcoma can be provided. Furthermore, it has been found that the osteosarcoma to be treated with a novel pharmaceutical composition is osteosarcoma in which the number of gene copies of MCL1 gene is increased or the production of Mcl-1 protein is increased in cells or osteosarcoma in which the number of gene copies of BCL2L1 gene is increased or the production of Bcl-xL protein is increased in cells, and it has been demonstrated that a therapy marker for examining the therapeutic effect of the novel pharmaceutical composition in the treatment of osteosarcoma can be provided by detecting each of the above-mentioned increased events.

Description

Novel drugs for treating osteosarcoma

[0001] The present invention relates to a novel drug for treating osteosarcoma. The present invention also relates to the development of a therapeutic marker for examining the therapeutic effect of a drug in treating osteosarcoma.

[0002] Osteosarcoma (OS) is the most common primary malignant bone tumor, primarily affecting children, adolescents, and young adults. Treatment of osteosarcoma, consisting of a four-drug combination of adriamycin (DOX), cisplatin (CDDP), high-dose methotrexate (MTX), and ifosfamide, combined with surgery and chemotherapy, was established in the 1970s and remains the standard of care (Non-Patent Documents 1 and 2).

[0003] With the establishment of this multidisciplinary approach, the 5-year overall survival rate for patients without distant metastasis at initial diagnosis has reached approximately 70%. However, few effective treatments exist for patients with metastatic or recurrent osteosarcoma, and the 5-year survival rate is only 20% to 30%, resulting in a poor prognosis. Furthermore, established treatments consisting of combination chemotherapy and surgical resection are associated with significant toxicity and morbidity. A phase III trial of intensive postoperative chemotherapy in high-risk patients did not improve event-free survival but increased toxicity (Non-Patent Document 3).

[0004] Furthermore, recent clinical trials have shown that immune checkpoint inhibitors, including pembrolizumab, nivolumab, and / or ipilimumab, have no activity against osteosarcoma (Non-Patent Documents 4 and 5), highlighting the continued critical need for new therapeutic strategies.

[0005] Link, MP et al. N Engl J Med 314, 1600-1606, (1986)Meyers, PA et al. J Clin Oncol 10, 5-15, (1992).Marina, NM et al. Lancet Oncol 17, 1396-1408, (2016).D'Angelo, SP et al. Lancet Oncol 19, 416-426, (2018).Tawbi, HA et al. Lancet Oncol 18, 1493-1501, (2017).

[0006] An objective of the present invention is to develop a novel drug for treating osteosarcoma. Another objective of the present invention is to develop a therapeutic marker for examining the therapeutic effect of a drug in treating osteosarcoma.

[0007] In order to solve the above-mentioned problems, the present invention has discovered a compound that can inhibit osteosarcoma cell proliferation, and has demonstrated that it is possible to provide a new pharmaceutical composition for treating osteosarcoma. Furthermore, it has been found that osteosarcoma, which is the target of treatment with the new pharmaceutical composition, has an amplified gene copy number of the MCL1 gene or increased Mcl-1 protein production, or an amplified gene copy number of the BCL2L1 gene or increased Bcl-xL protein production in cells. It has been demonstrated that detecting these can be used as a therapeutic marker for examining the therapeutic effect of the new pharmaceutical composition when treating osteosarcoma.

[0008] More specifically, to solve the above-mentioned problems, the present application provides the following aspects: [1]: A pharmaceutical composition for treating osteosarcoma, comprising an Mcl-1 inhibitor; [2]: The pharmaceutical composition according to [1], wherein the Mcl-1 inhibitor is selected from the group consisting of obatoclax, AZD5991, MIK665, siRNA against the MCL1 gene, or a combination thereof; [3]: The pharmaceutical composition according to [1] or [2], wherein the osteosarcoma has an amplified gene copy number of the MCL1 gene or increased production of Mcl-1 protein in the cells; [4]: ​​The pharmaceutical composition according to [3], wherein the gene copy number of the MCL1 gene in the cells is measured by PCR or FISH; [5]: The pharmaceutical composition according to [3], wherein the production of Mcl-1 protein in the cells is measured by immunoblotting or immunohistochemical staining; [6]: A pharmaceutical composition for treating osteosarcoma for which an Mcl-1 inhibitor is ineffective, comprising a Bcl-xL inhibitor. [7]: The pharmaceutical composition of [6], wherein the Bcl-xL inhibitor is selected from the group consisting of A-1331852, siRNA against the BCL2L1 gene, or a combination thereof. [8]: The pharmaceutical composition of [6] or [7], wherein the Mcl-1 inhibitor is selected from the group consisting of obatoclax, AZD5991, MIK665, siRNA against the MCL1 gene, or a combination thereof. [9]: The pharmaceutical composition of [6] or [7], wherein the osteosarcoma has an amplified gene copy number of the BCL2L1 gene or increased Bcl-xL protein production in the cells.

[10] : The pharmaceutical composition of [9], wherein the gene copy number of the BCL2L1 gene in the cells is measured by PCR or FISH.

[11] : The pharmaceutical composition of [9], wherein the Bcl-xL protein production in the cells is measured by immunoblotting or immunohistochemical staining.

[12] : A pharmaceutical composition for treating osteosarcoma, comprising an ingredient selected from the group consisting of obatoclax, tipifarnib, bortezomib, carfilzomib, SN-38, A-1331852, and derivatives thereof, or combinations thereof;

[13] : The pharmaceutical composition according to

[12] , wherein the osteosarcoma is one in which the gene copy number of the MCL1 gene or the gene copy number of the BCL2L1 gene is amplified in the cells or the production of Mcl-1 protein is increased, or one in which the gene copy number of the BCL2L1 gene is amplified in the cells or the production of Bcl-xL protein is increased;

[14] : The pharmaceutical composition according to

[13] , wherein the gene copy number of the MCL1 gene or the gene copy number of the BCL2L1 gene is measured by PCR or FISH;

[15] : The pharmaceutical composition according to

[13] , wherein the production of Mcl-1 protein or Bcl-xL protein is measured in the cells by immunoblotting or immunohistochemical staining;

[16] : A pharmaceutical composition for treating osteosarcoma, comprising an Mcl-1 inhibitor in combination with a Bcl-xL inhibitor or an IGF-1R inhibitor;

[17] : The pharmaceutical composition of

[16] , wherein the Mcl-1 inhibitor is selected from the group consisting of obatoclax, AZD5991, MIK665, an siRNA against the MCL1 gene, or a combination thereof;

[18] : The pharmaceutical composition of

[16] , wherein the Bcl-xL inhibitor is selected from the group consisting of A-1331852 and an siRNA against the Bcl2L1 gene;

[19] : The pharmaceutical composition of

[16] , wherein the IGF-1R inhibitor is selected from the group consisting of OSI906, AEW541, AZD3463, and an siRNA against the IGF-1R gene.

[20] : The pharmaceutical composition according to any one of

[16] to

[19] , wherein the osteosarcoma has an amplified gene copy number of the MCL1 gene or an increased production of Mcl-1 protein, or an amplified gene copy number of the BCL2L1 gene or an increased production of Bcl-xL protein in the cells;

[21] : The pharmaceutical composition according to

[20] , wherein the gene copy number of the MCL1 gene or the gene copy number of the BCL2L1 gene is measured by PCR or FISH;

[22] : The pharmaceutical composition according to

[20] , wherein the production of Mcl-1 protein or Bcl-xL protein in the cells is measured by immunoblotting or immunohistochemical staining;

[23] : A method for detecting an amplification of the gene copy number of the MCL1 gene or an increase in the production of Mcl-1 protein, an amplification of the gene copy number of the BCL2L1 gene, or an increase in the production of Bcl-xL protein in osteosarcoma tissue or blood collected from a patient suffering from osteosarcoma, which serves as an indicator of whether or not the patient is a target for administration of a pharmaceutical composition comprising an ingredient selected from the group consisting of an Mcl-1 inhibitor, a Bcl-xL inhibitor, obatoclax, tipifarnib, bortezomib, carfilzomib, SN-38, A-1331852, and derivatives thereof, or a combination thereof, or comprising an Mcl-1 inhibitor in combination with a Bcl-xL inhibitor or an IGF-1R inhibitor;

[24] : The method according to

[23] , in which the gene copy number of the MCL1 gene or the gene copy number of the BCL2L1 gene in cells is measured by PCR or FISH;

[25] The method according to

[23] , in which intracellular production of Mcl-1 protein or Bcl-xL protein is measured by immunoblotting or immunohistochemical staining.

[0009] The present invention has demonstrated that a compound capable of inhibiting osteosarcoma cell proliferation can be found, thereby providing a new pharmaceutical composition for treating osteosarcoma. Furthermore, it has been found that osteosarcoma, which is the target of treatment with the new pharmaceutical composition, exhibits an amplified copy number of the MCL1 gene or increased production of Mcl-1 protein, or an amplified copy number of the BCL2L1 gene or increased production of Bcl-xL protein in cells. It has been demonstrated that detecting these compounds can be used as therapeutic markers for examining the therapeutic effect of the new pharmaceutical composition when treating osteosarcoma.

[0010] Figure 1A shows the drug sensitivity profiles of osteosarcoma cell lines and PDCs using an inhibitor library. Figure 1B shows a bar graph of cell viability values ​​for each cell line after treatment with obatoclax or navitoclax. Figure 1C shows the protein expression levels of Bcl-2 family proteins in osteosarcoma cells. Figure 1D shows copy number changes of MCL1 and BCL2L1 in osteosarcoma cells. Figure 1E shows MCL1 amplification in osteosarcoma cells. Figure 2 shows the effect of MIK665, an Mcl-1 inhibitor, on osteosarcoma cells. Figure 3-1 shows the effect of RNAi-mediated gene knockdown in NOS-10, Sa-xeno147, and G-292 cells confirmed by immunoblotting for various proteins. Figure 3-2 shows the efficiency of RNAi-mediated gene knockdown in Sa-xeno147 and G-292 cells confirmed by qPCR for Bcl-2. Figure 3-3 shows the effect of siRNA on caspase-3 / 7 activity in osteosarcoma cells. Figure 3-4 shows the effect of siRNA on the viability of osteosarcoma cells. Figure 3-5 shows the concentration-dependent cell viability of osteosarcoma cells when AZD5991 is used as an Mcl-1 inhibitor and A-1331852 is used as a Bcl-xL inhibitor. Figure 3-6 shows the concentration-dependent cell viability of osteosarcoma cells when MIK665 is used as an Mcl-1 inhibitor and A-1331852 is used as a Bcl-xL inhibitor. Figure 4-1 shows the results of evaluating the drug sensitivity of NOS-10 cells, Sa-xeno-147 cells, G-292 cells, and Sa-xeno-184 cells to obatoclax, navitoclax, venetoclax, A-1331852, AZD5991, or MIK665. Figure 4-2 shows the combined effect of AZD5991 and 3 μM navitoclax on cell viability of NOS-10 cells. Figure 4-3 shows the IC50 values ​​of obatoclax in osteosarcoma and non-small cell lung cancer cell lines.Figure 4-4 shows that the combination of obatoclax with DOX, CDDP, or MTX does not induce synergistic cytotoxic effects in NOS-10 cells. Figures 4-5 show that the combination of obatoclax with DOX, CDDP, or MTX does not induce synergistic cytotoxic effects in Sa-xeno-147 cells. Figure 5-1 shows that IGF-1R inhibitors, such as OSI906, AEW541, and AZD3463, enhance growth inhibitory effects when combined with Mcl-1 inhibitors, such as obatoclax, AZD5991, and MIK665. Figure 5-2 shows the expression levels of IGF-1Rβ or insulin receptor (IR)β proteins in osteosarcoma cell lines. The expression of each protein was detected using immunoblotting with the indicated antibodies. Figure 5-3 shows the combined effects of obatoclax and OSI906 on the expression of IGF1R downstream pathways and apoptosis-related proteins in NOS-10 and Sa-xeno-147 cells. Figure 5-4 shows results demonstrating that activation of IGF signaling with IGF-1 significantly inhibits obatoclax-induced apoptosis and conferred drug resistance to NOS-10 cells. Figure 6-1 shows the experimental scheme for this example using obatoclax as an Mcl-1 inhibitor. Figure 6-2 shows the changes in tumor volume in NOS-10 cells (B) and Sa-xeno-147 cells (C) following drug treatment with obatoclax as an Mcl-1 inhibitor. Figure 6-3 shows the experimental scheme for this example and the changes in tumor volume in NOS-10 cells following drug treatment with AZD5991 as an Mcl-1 inhibitor. Figure 7-1 shows the classification of the MCL1 to 1p13.1 ratio in 41 osteosarcoma patients, with high-level amplification defined as an MCL1 / 1p13.1 ratio of ≥ 3.0 and low-level amplification defined as an MCL1 / 1p13.1 ratio of 2.0 ≤ < 3.0. Figure 7-2 shows the results of FISH analysis of MCL1 genomic copy number in osteosarcoma patients. Figure 7-3 shows the results of immunohistochemical staining of MCL1 protein expression in osteosarcoma patients.

[0011] Pharmaceutical Compositions for Single-Agent Treatment of Osteosarcoma In this study, we screened a library of known compounds against 11 osteosarcoma cell lines and selected compounds capable of inhibiting cell proliferation in almost all osteosarcoma cells. As a result, six molecular targeted drugs, including the BH3 mimetic obatoclax, the farnesyltransferase inhibitor tipifarnib, the proteasome inhibitors bortezomib and carfilzomib, the topoisomerase inhibitor SN-38, and the Bcl-xL inhibitor A-1331852, as well as Mcl-1 inhibitors, were identified as compounds capable of inhibiting osteosarcoma cell proliferation.

[0012] Thus, in a first aspect, the present invention provides a pharmaceutical composition for treating osteosarcoma, comprising an Mcl-1 inhibitor.

[0013] In a second aspect, the present invention also provides a pharmaceutical composition comprising a Bcl-xL inhibitor for treating osteosarcoma for which an Mcl-1 inhibitor is ineffective.

[0014] In a third aspect, the present invention also provides a pharmaceutical composition for treating osteosarcoma, comprising an ingredient selected from the group consisting of obatoclax, tipifarnib, bortezomib, carfilzomib, SN-38, A-1331852, and derivatives thereof, or combinations thereof.

[0015] An Mcl-1 inhibitor refers to a substance that can inhibit the expression of the MCL1 gene or the production or function of the Mcl-1 protein. In the examples of the present application, obatoclax was obtained as a result of screening a compound library using the reduction in cell viability of osteosarcoma cells as an indicator, and it was revealed that the MCL1 gene is widely upregulated in osteosarcoma cells. Since obatoclax is an Mcl-1 inhibitor, we investigated whether Mcl-1 inhibitors other than obatoclax can also reduce the cell viability of osteosarcoma cells. Two Mcl-1 inhibitors tested (AZD5991 and MIK665) also showed similar effects of reducing the cell viability of osteosarcoma cells.

[0016] In the pharmaceutical composition of the first aspect of the present invention, the term "Mcl-1 inhibitor" may be any known Mcl-1 inhibitor, such as obatoclax, AZD5991, MIK665, marinopyrrole A (maritoclax), VU661013, S63845, A-1210477, UMI-77, motixafortide (BL-8040), (R)-(-)-gossypol acetic acid, sabutoclax, TW-37, gambogic acid, or siRNA against the MCL1 gene.

[0017] Obatoclax is a substance known as a pan-Bcl-2 inhibitor and BH3 mimetic, and is a compound known to be used as an anti-cancer drug or BCL-2 inhibitor to induce apoptosis in cancer cells such as hepatocellular carcinoma.

[0018] AZD5991 is a molecule known to be a highly potent macrocyclic Mcl-1 inhibitor with affinity for Mcl-1 at subnanomolar concentrations.

[0019] MIK665 is an inhibitor of Mcl-1, a molecule known to have proapoptotic and antitumor effects.

[0020] Marinopyrrole A (malitoclax) is a selective Mcl-1 antagonist that binds to Mcl-1 but not Bcl-XL and is known to target Mcl-1 for proteasomal degradation.

[0021] VU661013 is a potent and selective inhibitor of human Mcl-1, with a Ki of 97±30 pM in a TR-FRET assay. However, VU661013 does not significantly inhibit BCL-xL or BCL-2. VU661013 is known to destabilize the BIM / Mcl-1 association and induce apoptosis in AML.

[0022] S63845 is a potent and selective inhibitor of Mcl-1, but is known not to bind to other BCL-2 components, BCL-2 or BCL-XL.

[0023] A-1210477 is a potent and selective Mcl-1 inhibitor known to be over 100-fold more selective than other Bcl-2 family members.

[0024] UMI-77 is a selective Mcl-1 inhibitor known to exhibit selectivity over other members of the Bcl-2 family.

[0025] Motixafortide (BL-8040, BKT140, TF 14016, 4-fluorobenzoyl, 4F-benzoyl-TN14003, T140) is a CXCR4 antagonist known to induce apoptosis in AML blasts by downregulating ERK, BCL-2, Mcl-1, and cyclin D1 via altering miR-15a / 16-1 expression.

[0026] (R)-(-)-gossypol (AT-101) acetate, the R-(-) enantiomer of gossypol acetate, is a molecule known to bind to Bcl-2, Bcl-xL, and Mcl-1, and simultaneously induce apoptosis and cytoprotective autophagy.

[0027] Sabutoclax (BI-97C1) is a molecule known to be a pan-Bcl-2 inhibitor, including Bcl-xL, Bcl-2, Mcl-1, and Bfl-1.

[0028] TW-37 is a molecule known as a non-peptide inhibitor of Bcl-2, Bcl-xL, and Mcl-1.

[0029] Gamboformic acid (guttatic acid, gutinic acid, beta-gutiferin) is a molecule known to activate caspases and competitively inhibit Bcl-XL, Bcl-2, Bcl-W, Bcl-B, Bfl-1, and Mcl-1.

[0030] Examples of siRNAs that can be used against the MCL1 gene include siRNAs that target the signal sequence of the MCL1 gene and siRNAs that target the open reading frame sequence of the MCL1 gene. For example, a commercially available siRNA reagent (Dharmacon ON-TARGETplus siRNA SMARTPool) that contains a mixture of four types of siRNAs that target different sites in the MCL1 gene can be used.

[0031] The present invention also found that some osteosarcoma cells exhibit only slight amplification of the MCL1 gene, but instead exhibit significant amplification of the BCL2L1 gene or increased production of Bcl-xL protein. It was revealed that, for such osteosarcoma cells, application of an Mcl-1 inhibitor did not reduce cell viability, whereas application of a Bcl-xL inhibitor reduced cell viability. Based on this finding, the present invention provides the second aspect described above.

[0032] Examples of Bcl-xL inhibitors that can be used in the pharmaceutical composition of the second aspect of the present invention include A-1331852, siRNA against the BCL2L1 gene, and the like, but are not limited to these.

[0033] A-1331852 is a potent and selective BCL-XL inhibitor, a compound believed to have potential for the treatment of cancer, immune disorders, and autoimmune diseases.

[0034] Examples of siRNAs that can be used against the BCL2L1 gene include siRNAs that target the signal sequence of the BCL2L1 gene and siRNAs that target the open reading frame sequence of the BCL2L1 gene. For example, a commercially available siRNA reagent (Dharmacon ON-TARGETplus siRNA SMARTPool) that contains a mixture of four types of siRNAs that target different sites in the BCL2L1 gene can be used.

[0035] In a third aspect, the present invention relates to obatoclax, tipifarnib, bortezomib, carfilzomib, SN-38, and A-1331852, which, although their activities differ and their mechanisms of action are only partially clear, all have the ability to reduce the cell viability of osteosarcoma cells.

[0036] Tipifarnib is a highly selective inhibitor of farnesyltransferase, an enzyme required for activation of the proto-oncogene HRAS, and research into its clinical application in head and neck squamous cell carcinoma and other cancers is currently underway.

[0037] Bortezomib is a proteasome inhibitor that inhibits myeloma and mantle cell lymphoma cells and is commercially available as a drug for multiple myeloma, primary macroglobulinemia, and lymphoplasmacytic lymphoma. Carfilzomib is a selective proteasome inhibitor that is commercially available as a drug for multiple myeloma. These proteasome inhibitors inhibit tumor growth by activating the accumulation of polyubiquitinated proteins, which are proteasome substrates, thereby arresting the cell cycle and inducing apoptosis in tumor cells. However, proteasome inhibitors are highly toxic to other cancer cell lines, such as colorectal cancer and lung cancer, and are not selective for osteosarcoma.

[0038] SN-38 is a topoisomerase inhibitor and a compound with antitumor effects. It is an active metabolite of irinotecan, and is known to be 1,000 times more active than irinotecan itself.

[0039] In the pharmaceutical composition of the first aspect of the present invention, any one of the Mcl-1 inhibitors contained as active ingredients may be the active ingredient, or two or more Mcl-1 inhibitor compounds may be combined as the active ingredient.

[0040] In the pharmaceutical composition of the second aspect of the present invention, any one of the Mcl-1 inhibitors contained as an active ingredient may be used as the active ingredient, or multiple Bcl-xL inhibitor compounds may be used in combination as the active ingredient.

[0041] In the pharmaceutical composition of the third aspect of the present invention, any one of these six compounds contained as an active ingredient may be the active ingredient, or two or more compounds selected from the six compounds may be combined to form the active ingredient.

[0042] The pharmaceutical composition of the first aspect, the pharmaceutical composition of the second aspect, and the pharmaceutical composition of the third aspect of the present invention (hereinafter simply referred to as the "pharmaceutical composition of the present invention") may be administered by any administration route, and among these, oral administration and intravenous administration are commonly used.

[0043] As shown in the Examples herein, the difference in drug sensitivity between obatoclax and navitoclax indicates that Mcl-1 is important for osteosarcoma cell survival and that simultaneous inhibition of Bcl-xL by Mcl-1 leads to efficient induction of apoptosis in osteosarcoma cells.In this study, MCL1 expression was upregulated in 10 of 11 osteosarcoma cell lines, and increased MCL1 copy number was observed in 5 of the cell lines.

[0044] Mutations in p53 pathway proteins have been identified in over 75% of osteosarcomas. Furthermore, the PI3K / AKT / mTOR pathway is known to promote MCL1 expression at multiple levels, including transcription via activation of the CREB transcription factor, activation of mTORC1 and subsequent phosphorylation of 4E-BP1, and proteasomal degradation via inhibition of GSK3-induced phosphorylation. These characteristic genomic abnormalities and genetic mutations indicate widespread enhancement of Mcl-1-dependent evasion of programmed cell death in osteosarcoma. Meanwhile, as shown in the Examples herein, G-292 cells with BCL2L1 amplification were highly sensitive to obatoclax and Bcl-xL-selective BH3 mimetics, suggesting that Mcl-1 and Bcl-xL complement each other in their functions in osteosarcoma.

[0045] Based on these findings, it has been revealed that osteosarcomas to be treated with the pharmaceutical composition of the present invention are preferably those in which the gene copy number of the MCL1 gene is amplified or the production of Mcl-1 protein is increased in cells, or those in which the gene copy number of the BCL2L1 gene is amplified or the production of Bcl-xL protein is increased in cells. Osteosarcoma with such properties exhibits strong inhibition of cell proliferation by the five compounds that are the active ingredients of the present invention, and administration of the pharmaceutical composition of the present invention provides a high therapeutic effect.

[0046] The intracellular copy number of the MCL1 gene or the BCL2L1 gene can be measured using common techniques for detecting nucleic acids in the art, such as PCR or FISH. When measured by PCR, the copy number can be measured by amplifying a portion of the nucleotide sequence of the MCL1 gene or the BCL2L1 gene using a primer. When measured by FISH, the copy number can be measured using a portion of the nucleotide sequence of the MCL1 gene or the BCL2L1 gene as a probe.

[0047] Intracellular Mcl-1 or Bcl-xL production can be measured using common protein detection techniques in the art, such as immunoblotting or immunohistochemical staining. Specifically, immunoblotting involves electrophoresing a cell lysate sample on an SDS-polyacrylamide gel, transferring the sample to a PVDF membrane, and then reacting the membrane with an antibody against Mcl-1 protein as a primary antibody, followed by detection of the primary antibody.

[0048] Pharmaceutical Composition for Treating Osteosarcoma Using Combination Agents In the examples of the present invention, other drugs that are effective in combination with Mcl-1 inhibitors such as obatoclax were screened, and Bcl-xL inhibitors or insulin-like growth factor 1 receptor (IGF-1R) inhibitors were identified as compounds that exhibit synergistic effects with Mcl-1 inhibitors such as obatoclax AZD5991 and MIK665.

[0049] The studies in the Examples of the present application showed that production of Mcl-1 protein and Bcl-xL protein was increased in almost all osteosarcoma cell lines examined, suggesting that inhibiting the function of both molecules may be an effective combination therapy for osteosarcoma.

[0050] Furthermore, IGF-1R activates the survival signaling pathway, PI3K / Akt / mTOR, which confers resistance to apoptosis. The chromosome 15q26.3 region where IGF-1R resides is often amplified in osteosarcoma. In the present example, IGF treatment significantly increased the phosphorylation level of Akt, which is downstream of IGF-1R, and abolished obatoclax-induced apoptosis, demonstrating that activation of the IGF-1R / PI3K / Akt / mTOR pathway plays an important role in osteosarcoma cell survival. Furthermore, in the present example, it was found that either IGF1R or insulin receptor (IR) is expressed in osteosarcoma cells, suggesting that combination therapy of obatoclax with a dual inhibitor of IGF-1R and IR, such as OSI906, may be an effective combination therapy for osteosarcoma.

[0051] Prior to the filing date of this application, it was not known that an IGF-1R inhibitor alone had an antitumor effect against osteosarcoma. However, in the examples of this application, it was shown that by using an active ingredient of the pharmaceutical composition in the first aspect of the present invention, such as obatoclax, in combination with an IGF-1R inhibitor, the IGF-1R inhibitor has the effect of enhancing the action of the active ingredient of the pharmaceutical composition in the first aspect of the present invention, such as obatoclax, against osteosarcoma.

[0052] Based on these possibilities, in the examples of the present invention, we treated osteosarcoma cell lines with a combination of an Mcl-1 inhibitor and a Bcl-xL inhibitor or an IGF-1R inhibitor, and found that cell viability was significantly reduced.

[0053] Based on this, in a fourth aspect, the present invention can also provide a pharmaceutical composition for treating osteosarcoma, comprising an Mcl-1 inhibitor in combination with a Bcl-xL inhibitor or an IGF-1R inhibitor.

[0054] The Mcl-1 inhibitors and Bcl-xL inhibitors that can be used in the pharmaceutical composition of the present invention are as described above.

[0055] Examples of IGF-1R inhibitors that can be used in the pharmaceutical composition of the present invention include, but are not limited to, OSI906, AEW541, AZD3463, and siRNA against the IGF-1R gene.

[0056] OSI906 is a potent, selective, orally bioavailable dual inhibitor of IGF-1R and insulin receptor (IR) kinases. It inhibits IGF-1R autophosphorylation and downstream activation of Akt, ERK1 / 2, and S6 kinase, and is known to inhibit the proliferation of tumor cell lines.

[0057] AEW541 is an orally available IGF-1R and insulin receptor (IR) inhibitor with known antitumor activity.

[0058] AZD3463 is an orally available ALK / IGF1R inhibitor that suppresses cell viability by inducing both cell apoptosis and autophagy.

[0059] Examples of siRNAs that can be used for the IGF-1R gene include siRNAs that target the signal sequence of the IGF-1R gene and siRNAs that target the open reading frame sequence of the IGF-1R gene. For example, a commercially available siRNA reagent (Dharmacon ON-TARGETplus siRNA SMARTPool) that contains a mixture of four types of siRNAs that target different sites on the IGF-1R gene can be used.

[0060] In the pharmaceutical composition of the fourth aspect of the present invention in combination with an IGF-1R inhibitor, the osteosarcoma to be treated has the same characteristics as the osteosarcoma to be treated in the pharmaceutical composition of the first aspect and the pharmaceutical composition of the second aspect.

[0061] In the present invention, osteosarcoma to be treated is characterized by an intracellular amplification of the gene copy number of the MCL1 gene or an increase in Mcl-1 protein production, or an intracellular amplification of the gene copy number of the BCL2L1 gene or an increase in Bcl-xL protein production, as described above. By examining osteosarcoma tissues or blood collected from patients with osteosarcoma for these characteristics, it can be confirmed whether the osteosarcoma is a target for treatment with the pharmaceutical composition of the present invention. In other words, this characteristic serves as an indicator of whether osteosarcoma is a target for administration of the pharmaceutical composition of the present invention.

[0062] Therefore, in a fifth aspect, the present invention provides a method for detecting an amplification of the gene copy number of the MCL1 gene or an increase in Mcl-1 protein production, or an amplification of the gene copy number of the BCL2L1 gene or an increase in Bcl-xL protein production in osteosarcoma tissue or blood collected from a patient suffering from osteosarcoma, which serves as an indicator of whether or not the patient is a target for administration of a pharmaceutical composition comprising an ingredient selected from the group consisting of an Mcl-1 inhibitor, obatoclax, tipifarnib, bortezomib, carfilzomib, SN-38, A-1331852, and derivatives thereof, or a combination thereof, or comprising an Mcl-1 inhibitor in combination with a Bcl-xL inhibitor or an IGF-1R inhibitor.

[0063] If this characteristic is present in osteosarcoma tissue or blood collected from a patient suffering from osteosarcoma, the patient will be a candidate for administration of the pharmaceutical composition of the present invention. However, if this characteristic is not present, the pharmaceutical composition of the present invention may not be sufficiently effective, and therefore, a decision can be made to select another treatment.

[0064] In this embodiment, the intracellular copy number of the MCL1 gene or the BCL2L1 gene can be measured by PCR or FISH, and the intracellular production of Mcl-1 protein or Bcl-xL protein can be measured by immunoblotting or immunohistochemical staining.

[0065] The present invention will be specifically illustrated by the following examples, which are not intended to limit the present invention in any way.

[0066] Example 1: Identification of molecular targeted drugs that inhibit the proliferation of osteosarcoma cells In this example, compounds that can inhibit the proliferation of osteosarcoma cells were identified from among known molecular targeted drugs.

[0067] To identify molecular targeted drugs that broadly suppress osteosarcoma cell proliferation, we performed drug screening using an inhibitor library containing 92 molecular targeted drugs, evaluating their effects on osteosarcoma cell proliferation, using nine osteosarcoma cell lines available from public cell banks and two osteosarcoma patient-derived cell lines (PDCs) established in our laboratory.

[0068] Nine human osteosarcoma cell lines (MG-63, NY, HOS, HuO9, HuO-3N1, SJSA-1, NOS-1, NOS-10, and G-292 clone A141B1 (G-292) cells) were purchased from the American Type Culture Collection, the RIKEN BioResource Research Center, or the Japanese Collection of Research Bioresources Cell Bank (JCRC) and used at lower passage numbers.

[0069] Specifically, MG-63 cells, NY cells, and HOS cells were cultured in 96-well plates using MEM (Wako) containing 10% FBS (Corning), 1% non-essential amino acid solution (Wako), and 100 μg / mL kanamycin (Meiji). HuO9 cells, HuO-3N1 cells, SJSA-1 cells, NOS-1 cells, and NOS-10 cells were cultured in 96-well plates using RPMI-1640 medium (Wako) containing 10% FBS and 100 μg / mL kanamycin. G-292 clone A141B1 (G-292) cells were cultured in 96-well plates using McCoy's 5a medium containing 10% FBS. Sa-xeno-147 and Sa-xeno-184 cell lines, developed from patient-derived xenograft tumors, were cultured in collagen-coated dishes in RPMI 1640 / Ham's F12 (1:1) medium containing 15% FBS, 20 mM HEPES, and antibiotic-antimycotic solution (Sigma-Aldrich). Cells were cultured at 37°C in 5% CO2.

[0070] Cells were seeded in triplicate at a density of 2000 cells / well in 96-well plates (Greiner) or type I collagen-coated plates (IWAKI) and cultured for at least 24 hours. After 72 hours of incubation with one of the 96 compounds from the inhibitor library, the cells were incubated with CellTiter-Glo reagent (Promega) on a Titramax 100 plate shaker (Heidolph) for 2 minutes. After 10 minutes of equilibration, cell viability assays were performed on each osteosarcoma cell by measuring luminescence using a Tristar LB941 multimode microplate reader (Berthold Technologies). Cell viability was calculated as a ratio to the DMSO control, and their effects on osteosarcoma cell proliferation were evaluated (n = 2).

[0071] For cell viability, data were analyzed using GraphPad Prism, and the average values ​​from two independent experiments were displayed graphically as heatmaps using heatmap.2 with the gplots package in ComplexHeatmap version 2.4.3 (R version 4.0.2).

[0072] The effects of the compounds on osteosarcoma cell proliferation are shown in Figure 1A. Heatmap comparison of cell viability confirmed that five drugs (obatoclax, tipifarnib, bortezomib, carfilzomib, and SN-38) showed broad-spectrum growth inhibition of osteosarcoma cells.

[0073] Of these five compounds, we further investigated the pan-BH3 mimetic obatoclax. Specifically, osteosarcoma cells (11 types) were seeded in triplicate at a density of 2000 cells / well in 96-well plates (Greiner) or type I collagen-coated plates (IWAKI) and cultured for at least 24 hours. After treatment with 1 μM obatoclax for 72 hours, cell viability was measured using the CellTiter-Glo assay reagent. Obatoclax exhibited potent growth inhibitory effects on all osteosarcoma cell lines. In contrast, treatment with 1 μM navitoclax, a dual BH3 mimetic, had little effect on osteosarcoma cell proliferation (Figure 1B), indicating significant differences in the drug sensitivity of osteosarcoma cells to BH3 mimetics.

[0074] Next, the expression of apoptosis-related proteins in obatoclax-treated osteosarcoma cells was confirmed by immunoblotting.

[0075] Specifically, cells were harvested using a cell scraper, lysed in SDS lysis buffer [100 mM Tris-HCl (pH 7.5), 10% glycerol, 1% SDS], and boiled for 10 minutes. Protein concentrations were measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) after centrifugation at 20,000 × g for 10 minutes. Equal amounts of cell lysates were electrophoresed on an SDS-polyacrylamide gel. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and immunoblotted with the following antibodies (primary antibodies): antibody against Mcl-1 (CST #5453); antibody against Bcl-xL (CST #2764); antibody against Bcl-2 (CST #4223); antibody against Bid (CST #2002); antibody against Bim (CST #2933); and antibody against NOXA (CST #2933). #14766); antibody against Bax (CST #5023); antibody against Bak (CST #12105); mAb against β-actin (Sigma #A1978).

[0076] Next, the respective antibodies were detected using HRP-conjugated anti-mouse IgG or anti-rabbit IgG and enhanced chemiluminescence reagent (ECL prime, GE Healthcare). The chemiluminescent signal was detected using an AI600 image analyzer (GE Healthcare). In some experiments, cells were resuspended in NP-40 lysis buffer [20 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM EDTA, 1 mM EGTA, and protease and phosphatase inhibitors], incubated on ice for 10 minutes, and centrifuged at 20,000 × g for 5 minutes.

[0077] The results are shown in Figure 1C. Obatoclax targets three antiapoptotic Bcl-2 family proteins (Bcl-2, Bcl-xL, and Mcl-1), whereas navitoclax is more selective for Bcl-2 and Bcl-xL, suggesting that Mcl-1 is important for osteosarcoma cell survival. Immunoblotting showed that Mcl-1 was highly expressed and Bcl-xL was commonly expressed in all osteosarcoma cell lines except G-292 cells. Bcl-2 expression levels were below the detection limit in most osteosarcoma cell lines.

[0078] In osteosarcoma, the chromosome 1q21.2-3 region, where MCL1 is located, is often amplified, and it has been reported that increased genomic copy number of MCL1 contributes to cancer progression. Therefore, we investigated copy number alterations of various IGF1R / PI3K / Akt / mTOR pathway-related genes located in the chromosome 1q21.2-3 region, including MCL1, TARS2, ENSA, PIP5K1A, and OTUD7B, in each cell line. Furthermore, since the above results confirmed that not only Mcl-1 but also Bcl-xL are widely and highly expressed in osteosarcoma cell lines, we investigated copy number alterations of MCL1 and BCL2L1.

[0079] Genomic DNA from each cell line was prepared using ReverTra Ace qPCR RT Master Mix (TOYOBO) and DNeasy Blood & Tissue Kits (QIAGEN), respectively, according to the manufacturer's protocol. Quantitative real-time polymerase chain reaction (qPCR) was performed using FastStart Essential DNA Green Master (Roche) and LightCycle 96 (Roche) using the ΔΔCt method. Each copy number was quantified relative to the level of the repeat element LINE1, and DNA copy numbers were calculated and expressed as log2 ratios. Copy number changes of human IGF1R, located on chromosome 15q26.3, were examined as a control. Data are shown as mean ± SD (n = 3).

[0080] The primer pairs for detecting DNA copy number were as follows: human MCL1 forward, 5'-cttccaaggtaagggggttc-3' (SEQ ID NO: 1); human MCL1 reverse, 5'-actgactcgtttccggtttcc-3' (SEQ ID NO: 2); human TARS2 forward, 5'-atgggacgtgtgttacctgc-3' (SEQ ID NO: 3); human TARS2 reverse, 5'-tagtccggggttccttctgt-3' (SEQ ID NO: 4); human ENSA forward, 5'-ttcttctgtgttcgtccgca-3' (SEQ ID NO: 5); human ENSA reverse, 5'-gacaccaacaggaaagggct-3' (SEQ ID NO: 6); Human PIP5K1A forward, 5'-agggaggatgggtggtaact-3' (SEQ ID NO: 7); human PIP5K1A reverse, 5'-cgaatgttcttgccacctgc-3' (SEQ ID NO: 8); human OTUD7B forward, 5'-acccctattgccctttggtg-3' (SEQ ID NO: 9); human OTUD7B reverse, 5'-ctgccatgcaagaaagcgtt-3' (SEQ ID NO: 10); human BCL2L1 forward, 5'-cctctcccgacctgtgatac-3' (SEQ ID NO: 11); human BCL2L1 reverse, 5'-cttcctcggaaagtcactcc-3' (SEQ ID NO: 12); LINE1 element forward, 5'-aaagccgctcaactacatgg-3' (SEQ ID NO: 13); LINE1 element reverse, 5'-tgctttgaatgcgtcccagag-3' (SEQ ID NO: 14); human IGF1R forward, 5'-atccgccattctcatgcctt-3' (SEQ ID NO: 15); human IGF1R reverse, 5'-tttaacaaggcagggagggc-3' (SEQ ID NO: 16). Human genomic DNA (PROMEGA) was used as a PCR positive control template.

[0081] The results are shown in the upper and lower panels of Figure 1D. Using genomic DNA-targeted qPCR analysis, we observed an increase in MCL1 copy number at a high frequency of 45% in 5 of 11 osteosarcoma cell lines, and an increase in BCL2L1 copy number was observed in 1 of 11 osteosarcoma cell lines, G-292 cells (Figure 1D).

[0082] Furthermore, among the five cell types shown to have increased MCL-1 copy number, FISH analysis was performed on NOS-10 cells and Sa-xeno-184 cells, which showed MCL-1 amplification, as well as Sa-xeno-147 and G-292 cells, which did not show MCL-1 amplification.

[0083] The MCL1 gene copy number status was determined in tissue microarrays or cell blocks from osteosarcoma cell lines using in-house probes generated from BAC clones containing ChromaTide TexasRed-12-dUTP (Thermo Fisher Science) (red) targeting the human MCL1 gene located on chromosome 1q21.2 and Fluorescein-12-dUTP (Roche) (green) targeting a sequence in the control chromosomal region 1p13.1. Images were captured using an Olympus BX51 fluorescence microscope equipped with a charge-coupled device camera (DP71; Olympus, Tokyo, Japan). MCL1 gene gain was defined as an MCL1 / 1p13.1 ratio of ≥ 2.0, a high-level gain as an MCL1 / 1p13.1 ratio of ≥ 3.0, and a low-level gain as an MCL1 / 1p13.1 ratio of 2.0 or less and < 3.0.

[0084] The results are shown in Figure 1E. FISH analysis confirmed MCL1 amplification in NOS-10 and Sa-xeno-184 cells, but not in Sa-xeno-147 or G-292 cells (Figure 1E). Scale bar: 20 μm.

[0085] Example 2: Search for other candidate osteosarcoma therapeutic agents Since the present invention revealed that inhibition of Mcl-1 can suppress the viability of MCL1-amplified osteosarcoma cells, in this example, other compounds capable of inhibiting Mcl-1 were investigated.

[0086] In this example, we evaluated the drug sensitivity of NOS-10 cells to the Mcl-1 inhibitor MIK665. Various concentrations of MIK665 (TargetMol) (0, 1, 3, 10, 30, 100, 300, 1000, 3000, 10,000, and 30,000 nM) were administered. Cell viability was measured 72 hours after drug treatment using the CellTiter-Glo assay reagent. MG-63 cells, osteosarcoma cells lacking MCL1 copy number amplification, were used as a control. Data are presented as mean ± SD (n = 3) for MG-63 cells.

[0087] The results are shown in Figure 2. It was found that treatment with low concentrations of MIK665 reduced cell viability in NOS-10 cells with MCL1 amplification, compared with MG-63 cells without MCL1 amplification.

[0088] Example 3: Effect of Mcl-1 on osteosarcoma cell survival In this example, to evaluate the effect of Mcl-1 on osteosarcoma cell survival, we investigated whether inhibiting MCL1 expression induces cell apoptosis.

[0089] In this example, three types of cells were selected: NOS-10 cells, in which MCL1 amplification was observed in Example 1; Sa-xeno-147 cells, in which both MCL1 and BCL2L1 were normal in Example 1; and G-292 cells, in which BCL2L1 amplification was observed in Example 1. siRNA specific to BCL-2, BCL-xL, or MCL1 was introduced into these cells.

[0090] The following siRNAs were purchased from Dharmacon (Lafayette): SMARTpool siRNA for human BCL2 (L-003307-00-0005), SMARTpool siRNA for human BCL2L1 (L-003458-00-0005), and SMARTpool siRNA for human MCL1 (L-004501-00-0005). Silencer Negative Control No. 1 siRNA (Thermo) was used as a control.

[0091] Specifically, Opti-MEM I reduced serum medium (Thermo Fisher Scientific) containing 2.4 nM of each siRNA and 1.0 μL of Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) was incubated for 10 minutes at 24°C. This was then mixed into each cell suspension diluted to 2000 cells / 100 μL and incubated at room temperature for 3 minutes. After incubation, each cell was seeded into a 96-well microplate with a type I collagen-coated lid (IWAKI) at 2000 cells / 100 μL / well for cell viability assays or 4 × 10 cells / 100 μL for immunoblotting and qPCR. 5 Cells were seeded at 2 mL / well onto a type I collagen-coated 6-well microplate (IWAKI). After 48 hours of incubation, cell lysates were collected.

[0092] The efficiency of RNAi-mediated gene knockdown of MCL1, BCL-xL, or BCL-2 was confirmed 48 hours after siRNA treatment by immunoblotting in NOS-10, Sa-xeno-147, and G-292 cells (Figure 3-1(A)–(C)) and qPCR in Sa-xeno-147 and G-292 cells (Figure 3-2). Immunoblotting was performed with the following antibodies: Mcl-1 (CST #5453), Bcl-xL (CST #2764), Bcl-2 (CST #4223), PARP (CST #9542), and cleaved PARP (Asp214, CST #9541). mAb against β-actin (Sigma #A1978).

[0093] The knockdown effect of Bcl-2 in Sa-xeno-147 and G-292 cells was confirmed at the mRNA level by RT-qPCR. The mRNA level was quantified relative to that of GAPDH. Data are shown as mean ± SD (n = 3). The primer pairs used to detect BCL2 gene expression were as follows: human BCL2 forward, 5'-ccttctttgagttcggtggg-3' (SEQ ID NO: 17); human BCL2 reverse, 5'-tcttcagagacagccaggag-3' (SEQ ID NO: 18); human GAPDH forward, 5'-atggggaaggtgaaggtcg-3' (SEQ ID NO: 19); human GAPDH reverse, 5'-ggggtcattgatggcaacaata-3' (SEQ ID NO: 20).

[0094] The results are shown in Figures 3-1(A) to (C) and 3-2. In Figure 3-2, data are presented as mean ± SD (n = 3). The data were determined by Student's t-test (**p < 0.01). In these figures, simultaneous inhibition of Mcl-1 and Bcl-xL induced apoptosis in the cells examined, suggesting that increased copy number of either MCL1 or BCL2L1 may be a potential vulnerability of osteosarcoma cells.

[0095] To further confirm this possibility, we examined how knockdown of MCL1 by siRNA altered caspase-3 / 7 activity in osteosarcoma cells (NOS-10, Sa-xeno-147, and G-292 cells).

[0096] Caspase-3 / 7 activity was measured as follows: cells were incubated with Caspase-Glo 3 / 7 Assay Reagent (Promega) on a Titramax 100 plate shaker (Heidolph) for 2 minutes, and after 30 minutes of equilibration, luminescence was measured using a Tristar LB941 multimode microplate reader (Berthold Technologies). Relative cell viability was calculated as a ratio to the DMSO control.

[0097] The results are shown in Figure 3-3. Data are presented as mean ± SD (n = 6). The significance was determined by Student's t-test (**p < 0.01). The levels of apoptosis markers, cleaved PARP and caspase-3 / 7 activity, were significantly increased in NOS-10 cells (Figure 3-1(A) and Figure 3-3(D)), but were barely increased in Sa-xeno-147 cells (Figure 3-1(B) and Figure 3-3(E)) and G-292 cells (Figure 3-1(C) and Figure 3-3(F)). Furthermore, BCL-xL knockdown increased cleaved PARP levels and caspase-3 / 7 activity only in G-292 cells. These results suggest that increased copy number of either MCL1 or BCL2L1 may contribute to osteosarcoma cell vulnerability.

[0098] These results indicated that simultaneous inhibition of Mcl-1 and Bcl-xL induced apoptosis, and we therefore confirmed the effect of double knockdown of BCL-xL and MCL1 on the cell viability of osteosarcoma cells.

[0099] Specifically, three types of osteosarcoma cells (NOS-10, Sa-xeno-147, and G-292) were treated with siRNAs for BCL-xL and MCL1, and cell viability was measured 48 hours after treatment using the CellTiter-Glo Assay reagent.

[0100] The data are shown in Figures 2-3. Data for siRNA treatment are presented as mean ± SD (n = 6). The results were determined by Student's t-test (**p<0.01). The viability of all three types of osteosarcoma cells was significantly reduced (Figures 3-4(G) to 3-4(I)). These results indicate that Bcl-xL and Mcl-1 play important roles in osteosarcoma cell survival and that increasing the copy number of either gene may be a potential molecular target in osteosarcoma cells.

[0101] Because the above results indicated that simultaneous inhibition of Mcl-1 and Bcl-xL induced apoptosis, we evaluated the effect of combined use of an Mcl-1 inhibitor and a Bcl-xL inhibitor on the viability of osteosarcoma cells.

[0102] In this example, AZD5991 or MIK665 was used as an Mcl-1 inhibitor, and A-1331852 was used as a Bcl-xL inhibitor. The results for AZD5991 are shown in Figure 3-5, and the results for MIK665 are shown in Figure 3-6.

[0103] These results indicate that A-1331852 alone had little effect on cell viability, but when used in combination with AZD5991 or MIK665, it significantly reduced cell viability, suggesting that the combined use of an Mcl-1 inhibitor and a Bcl-xL inhibitor is effective in treating osteosarcoma.

[0104] Example 4: Inhibition of Mcl-1 and Bcl-xL suppresses osteosarcoma cell viability In this example, the effect of pharmacological inhibition of anti-apoptotic Bcl-2 family proteins on osteosarcoma cell proliferation was investigated.

[0105] In addition to obatoclax and navitoclax, BH3 mimetics with high selectivity for Bcl-2, Bcl-xL, or Mcl-1 have been developed, such as venetoclax, A-1331852, AZD5991, and MIK665, respectively. Osteosarcoma cells were treated with these BH3 mimetics to determine whether they altered cell viability.

[0106] Specifically, four types of osteosarcoma cells (NOS-10, Sa-xeno-147, G-292, and Sa-xeno-184) were treated with six BH3 mimetics (obatoclax, navitoclax, venetoclax, A-1331852, AZD5991, and MIK665), and cell viability of the osteosarcoma cells was measured.

[0107] The results are shown in Figure 4-1. In NOS-10 and Sa-xeno-184 cells with MCL1 amplification, AZD5991 and MIK665 reduced cell viability at relatively low concentrations, similar to obatoclax treatment (Figure 4-1(A)). A-1331852 was most effective in reducing cell viability in G-292 cells with BCL2L1 amplification, while AZD5991 and MIK665 showed moderate inhibitory effects in Sa-xeno-147 cells (Figure 4-1(B) and Figure 4-1(C)).

[0108] The combined effect of AZD5991 and 3 μM Navitoclax on cell viability of NOS-10 cells was also investigated. Cell viability was measured 72 hours after drug treatment using the CellTiter-Glo assay reagent. Data are shown as mean ± SD (n = 3). The combination of AZD5991 and Navitoclax enhanced the inhibitory effect on cell viability in NOS-10 cells (Figure 4-2(D)). This suggests that simultaneous inhibition of Bcl-xL and Mcl-1 is expected to induce greater growth inhibition than inhibition of Mcl-1 alone in MCL1-amplified osteosarcoma cells.

[0109] Furthermore, since it has been found that MCL1 copy number gain frequently occurs in lung adenocarcinoma and can be a therapeutic target, the inventors of the present invention decided to compare the IC50 values ​​of obatoclax in lung adenocarcinoma cell lines and osteosarcoma cell lines.

[0110] In this example, G-292 cells, NY cells, NOS-1 cells, NOS-10 cells, HuO9 cells, SJSA-1 cells, MG-63 cells, HuO-3N1 cells, HOS cells, Sa-xeno-147 cells, and Sa-xeno-184 cells were used as osteosarcoma cell lines, and LC-2 / ad cells, H2228 cells, H226 cells, H3122 cells, and A549 cells were used as lung adenocarcinoma cell lines.

[0111] These cells were treated with obatoclax for 72 hours, and cell viability was measured using the CellTiter-Glo Assay reagent. IC50 values ​​were calculated using Prism software. The maximum serum concentration (Cmax) of obatoclax (176 nM) is indicated by the dotted line.

[0112] The results are shown in Figure 4-3. The mean IC50 values ​​of obatoclax in osteosarcoma cell lines were lower than those in lung adenocarcinoma cell lines and were lower than the maximum serum concentration (Cmax) of obatoclax, 176 nM, indicating that the therapeutic effect of obatoclax against osteosarcoma can be achieved in vivo.

[0113] In contrast, cell viability after treatment with obatoclax in lung adenocarcinoma cell lines varied widely, with no clear trend observed. These results suggest that the presence of increased copy numbers of anti-apoptotic Bcl-2 family proteins can be used to predict the specific inhibitory effects of BH3 mimetics on osteosarcoma cell survival.

[0114] Various BH3 mimetics (e.g., navitoclax, obatoclax, venetoclax, A-1331852, and AZD5991) are currently being investigated in combination with standard therapies, and venetoclax has shown great clinical success in acute myeloid leukemia in combination with cytarabine or hypomethylating agents. Doxorubicin (DOX), cisplatin (CDDP), methotrexate (MTX), and ifosfamide (IFO) are used as standard treatments for osteosarcoma; IFO is a prodrug alkylating agent that is metabolized and activated by hepatic cytochrome P450 in the body.

[0115] Based on these findings, we investigated whether DOX, CDDP, or MTX, when combined with obatoclax, exhibited synergistic growth inhibition of osteosarcoma cells in vitro. Specifically, we investigated the effects of 40 nM obatoclax combined with DOX (Figure 4-4(E), Figure 4-5(A)), CDDP (Figure 4-4(F), Figure 4-5(B)), and MTX (Figure 4-4(G), Figure 4-5(C)) on cell viability of NOS-10 and Sa-xeno-147 cells. Cell viability was measured 72 hours after each drug treatment using the CellTiter-Glo assay reagent. All data are presented as mean ± SD (n = 3). IC50 values ​​were calculated and the data were graphed using GraphPad Prism.

[0116] The IC50 values ​​of DOX, CDDP, or MTX were not reduced in the presence of obatoclax (Figures 4-4(E) to 4-4(G) and 4-5), suggesting that standard chemotherapeutic agents for osteosarcoma may not induce synergistic cytotoxic effects when combined with obatoclax.

[0117] Example 5: Search for suitable combination partners of obatoclax for the treatment of osteosarcoma In this example, suitable combination partners of Mcl-1 inhibitors for the treatment of osteosarcoma were identified.

[0118] To identify suitable combination partners for Mcl-1 inhibitors such as obatoclax for osteosarcoma treatment, further drug screening was performed using an in-house inhibitor library in the presence of obatoclax in osteosarcoma cells. Specifically, the effect of 35 nM obatoclax combined with an inhibitor library containing 94 molecular targeted drugs on cell viability of NOS-10 cells was examined. Cell viability was measured 72 hours after treatment with each drug using the CellTiter-Glo assay reagent. All data are presented as mean ± SD (n = 3). GraphPad Prism was used to calculate IC50 values ​​and display the data graphically.

[0119] The results showed that IGF-1R inhibitors such as OSI906, AEW541, and AZD3463 enhanced the growth inhibitory effect and significantly reduced the IC50 value when used in combination with Mcl-1 inhibitors such as obatoclax, AZD5991, and MIK665 (Figure 5-1).

[0120] Immunoblotting analysis of various osteosarcoma cell lines using the following antibodies: antibody against IGF-1Rβ (CST #3027); antibody against insulin receptor β (CST #3025); mAb against β-actin (Sigma #A1978); confirmed that at least one of IGF-1R and insulin receptor (IR) was expressed in the osteosarcoma cell lines (Figure 4B).

[0121] Based on this, we next investigated the effects of combined treatment of obatoclax with OSI906, a dual inhibitor of IGF-1R and insulin receptors, on osteosarcoma cells in NOS-10 and Sa-xeno-147 cells.

[0122] NOS-10 and Sa-xeno-147 cells were pretreated with 40 nM obatoclax or 4 μM OSI906, followed by treatment with 40 ng / mL IGF. Cell samples were harvested 24 hours after drug treatment. Expression of each protein in these cells was confirmed using the following antibodies: IGF-1Rβ (CST #3027); p-Akt (S473, CST #4060); Akt (CST #4691); p-PRAS40 (CST #2997); PRAS40 (CST #2691); p-S6 (CST #5364); S6 (CST #2217); p-ERK (CST #9101); ERK (CST #9102); Mcl-1 (CST #5453); Bcl-xL (CST #2764); Bax (CST #5023); PARP (CST #9542); and cleaved PARP (Asp214, CST #9541). Evaluation was performed using immunoblot with mAb against β-actin (Sigma #A1978);

[0123] The results showed that the combination of obatoclax and OSI906 suppressed the phosphorylation levels of IGF-1R downstream molecules, such as Akt, PRAS40, and S6 protein, and increased the amount of cleaved PARP. Furthermore, the expression levels of phosphorylated ERK, Mcl-1, Bcl-xL, and Bax remained unchanged under these conditions (Figures 5-3(B) and 5-3(C)). Furthermore, the obatoclax-induced increase in cleaved PARP was counteracted by IGF ligand stimulation, which was overcome in the presence of OSI906 (Figures 5-2(C) and 5-3(C)).

[0124] These results suggest that the combination of OSI906 and obatoclax enhances apoptosis induced by obatoclax by overcoming drug resistance mediated by IGF signaling activation in osteosarcoma cells. To further confirm this possibility, NOS-10 cells were pretreated with 40 nM obatoclax and / or 4 μM OSI906 for 30 min, followed by treatment with 40 ng / mL IGF-1 to examine the effects of caspase-3 / 7 activity, which is an apoptotic signaling pathway.

[0125] Caspase activity was measured 24 hours after IGF-1 treatment using the Caspase-Glo 3 / 7 assay reagent. Data are presented as mean ± SD (n = 4). **p < 0.01 was determined by Student's t-test.

[0126] The results are shown in Figure 5-4. We found that activation of IGF signaling with IGF-1 significantly inhibited obatoclax-induced apoptosis and conferred drug resistance to NOS-10 cells. This drug resistance could be overcome by the combined use of obatoclax and OSI906, which enhanced obatoclax-induced apoptosis.

[0127] These results indicate that the combination of an IGF-1R inhibitor such as OSI906 with obatoclax enhanced obatoclax-induced apoptosis and synergistically reduced osteosarcoma cell viability by overcoming drug resistance mediated by activated IGF signaling in osteosarcoma cells.

[0128] Example 6: Examination of the combination of an Mcl-1 inhibitor and OSI906 in vivo In this example, we investigated whether the combination of an Mcl-1 inhibitor and OSI906 synergistically suppresses the growth of MCL1-amplified osteosarcoma tumors in a mouse xenograft model.

[0129] We used obatoclax and AZD5991 as Mcl-1 inhibitors to determine the efficacy of obatoclax or AZD5991 in combination with OSI906 in mouse xenograft models of osteosarcoma with and without MCL1 amplification (NOS-10 cells and Sa-xeno-147 cells, respectively). To prevent side effects such as weight loss, the drugs were administered at relatively low concentrations three times a week (experimental schemes are shown in Figures 5A and 5D).

[0130] The changes in tumor volume of NOS-10 and Sa-xeno-147 cells due to drug treatment were examined as follows: NOS-10 or Sa-xeno-147 cells were subcutaneously inoculated (5-6 × 10 cells) into immunodeficient SCID beige mice (CB17.Cg-PrkdcscidLystbg-J / CrlCrlj, Charles River Laboratories, Inc.). 6 cells / mouse or 1 x 10 6 Cells / mouse), vehicle (30% PEG400, 0.5% Tween 80, 5% propylene glycol in water), obatoclax (5 mg / kg), OSI906 (25 mg / kg), or obatoclax plus OSI906 (5 mg / kg and 25 mg / kg, respectively) were orally treated three times a week. AZD5991 and OSI906 were administered in combination with vehicle (30% HP-β-CD in water, pH 9.0) for AZD5991, vehicle (30% PEG400, 0.5% Tween 80, 5% propylene glycol in water) for OSI906, AZD5991 (30 mg / kg), OSI906 (25 mg / kg), or AZD5991 + OSI906 (30 mg / kg and 25 mg / kg, respectively). AZD5991 was administered intraperitoneally once weekly, and OSI906 was administered orally five times weekly. All data are presented as mean ± SD (n = 4, n = 5, or n = 6). *p < 0.05 and **p < 0.01, as determined using the Mann-Whitney U test.

[0131] The results for the use of obatoclax as an Mcl-1 inhibitor are shown in Figures 5B and 5C, and for AZD5991 in Figure 5D. In an NOS-10 cell xenograft model, obatoclax or OSI906 treatment alone had little effect on tumor growth. However, the combination of obatoclax and OSI906 significantly suppressed tumor growth (Figure 6-2(B)). However, neither treatment reduced tumor growth in Sa-xeno-147 cells (Figure 6-2(C)). These results suggest that the combination of obatoclax and OSI906 may be a potential therapeutic intervention for MCL1-amplified osteosarcoma.

[0132] Example 7 Genomic Copy Number and Protein Expression of MCL1 in Osteosarcoma Patients In this example, the genomic copy number and protein expression of MCL1 were evaluated in formalin-fixed, paraffin-embedded specimens from osteosarcoma patients by FISH analysis and immunohistochemical staining analysis using tissue microarrays.

[0133] Formalin-fixed, paraffin-embedded specimens from osteosarcoma patients were used as samples. Small tissue fragments were extracted from the formalin-fixed, paraffin-embedded tissue using a 2 mm stainless steel biopsy needle (manual tissue puncher; Beecher Instruments, Silver Spring, Maryland). These fragments were then placed into standard-sized recipient array blocks. After assembly of the array blocks, multiple 4 μm sections were cut and subjected to fluorescence in situ hybridization (FISH) and immunohistochemical staining analysis, as described below.

[0134] To analyze the genomic copy number of the human MCL1 gene and measure the MCL1 copy number / 1p13.1 ratio in samples from 41 osteosarcoma patients, FISH targeting the human MCL1 gene and sequences in chromosomal region 1p13.1 was performed. This FISH was performed according to the method described in Example 1. As in Example 1, an increase in the MCL1 gene was defined as an MCL1 / 1p13.1 ratio of ≥ 2.0, a high-level increase as ≥ 3.0, and a low-level increase as ≥ 2.0 and < 3.0.

[0135] The results showed that, of the 41 patients examined, 14.6% (n = 6) had high-level MCL1 amplification, and 31.7% (n = 13) had low-level MCL1 amplification (Figure 7-1). amp Three types of samples were defined as amplified (GTA479-19, GTA525-19, and GTA476-30) and without MCL1 amplification (without MCL1 amp The FISH results are shown for one sample (GTA525-2) defined as . ) as an example (Figure 7-2 (A)).

[0136] Furthermore, to confirm Mcl-1 protein expression, immunohistochemical staining was performed on specimens in which MCL1 was amplified by FISH analysis. Thin-section slides were subjected to antigen retrieval by heating at 95°C for 30 minutes in an antigen retrieval solution (Nichirei Biosciences, Inc., Tokyo, Japan) at pH 9.0. The sections were then reacted with a 1:200 dilution of the primary antibody, anti-Mcl-1 monoclonal antibody (CST, clone D5V5L), to form an antigen-antibody complex. This antigen-antibody complex was visualized using Histofine Simple Stain MAX PO detection reagent (Nichirei Biosciences, Inc.). The staining procedure was performed using a Histostainer 36A automated staining system (Nichirei Biosciences, Inc.).

[0137] The results are shown in Figure 7-3 (B). Scale bar: 20 μm. As a result, high-level MCL1 amplification (with MCL1 amp In three samples (GTA479-19, GTA525-19, and GTA476-30) defined as "without MCL1 amplification," Mcl-1 was also detectable at the protein level, whereas FISH revealed no MCL1 amplification (without MCL1 amplification). amp One sample (GTA525-2), defined as "non-specific," had no detectable Mcl-1 even at the protein level.

[0138] These results indicate that the amplification of the MCL1 gene copy number detected at the nucleic acid level in osteosarcoma patient samples is consistent with the detection pattern of Mcl-1 protein detected at the protein level in osteosarcoma patient samples, and that this could potentially be used as a biomarker to predict efficacy in patients receiving obatoclax treatment or a combination of obatoclax and OSI906 treatment.

[0139] The present invention has demonstrated that a compound capable of inhibiting osteosarcoma cell proliferation can be found, thereby providing a new pharmaceutical composition for treating osteosarcoma. Furthermore, it has been found that osteosarcoma, which is the target of treatment with the new pharmaceutical composition, exhibits an amplified copy number of the MCL1 gene or increased production of Mcl-1 protein, or an amplified copy number of the BCL2L1 gene or increased production of Bcl-xL protein in cells. It has been demonstrated that detecting these compounds can be used as therapeutic markers for examining the therapeutic effect of the new pharmaceutical composition when treating osteosarcoma.

Claims

1. A pharmaceutical composition for treating osteosarcoma, comprising an Mcl-1 inhibitor.

2. The pharmaceutical composition of claim 1, wherein the Mcl-1 inhibitor is selected from the group consisting of obatoclax, AZD5991, MIK665, siRNA against the MCL1 gene, or a combination thereof.

3. The pharmaceutical composition according to claim 1 or 2, wherein the osteosarcoma has an amplified gene copy number of the MCL1 gene or an increased production of Mcl-1 protein in cells.

4. The pharmaceutical composition according to claim 3, wherein the copy number of the MCL1 gene in cells is measured by PCR or FISH.

5. The pharmaceutical composition of claim 3, wherein intracellular production of Mcl-1 protein is measured by immunoblotting or immunohistochemical staining.

6. A pharmaceutical composition for treating osteosarcoma in which an Mcl-1 inhibitor is ineffective, comprising a Bcl-xL inhibitor.

7. The pharmaceutical composition of claim 6, wherein the Bcl-xL inhibitor is selected from the group consisting of A-1331852, siRNA against the BCL2L1 gene, or a combination thereof.

8. The pharmaceutical composition of claim 6 or 7, wherein the Mcl-1 inhibitor is selected from the group consisting of obatoclax, AZD5991, MIK665, siRNA against the MCL1 gene, or a combination thereof.

9. The pharmaceutical composition according to claim 6 or 7, wherein the osteosarcoma has an amplified gene copy number of the BCL2L1 gene or an increased production of Bcl-xL protein in cells.

10. The pharmaceutical composition according to claim 9, wherein the intracellular BCL2L1 gene copy number is measured by PCR or FISH.

11. The pharmaceutical composition according to claim 9, wherein intracellular Bcl-xL protein production is measured by immunoblotting or immunohistochemical staining.

12. A pharmaceutical composition for treating osteosarcoma, comprising an ingredient selected from the group consisting of obatoclax, tipifarnib, bortezomib, carfilzomib, SN-38, A-1331852 and derivatives thereof, or combinations thereof.

13. The pharmaceutical composition according to claim 12, wherein the osteosarcoma has an amplified gene copy number of the MCL1 gene or an increased production of Mcl-1 protein, or an amplified gene copy number of the BCL2L1 gene or an increased production of Bcl-xL protein in the cells.

14. The pharmaceutical composition according to claim 13, wherein the intracellular copy number of the MCL1 gene or the BCL2L1 gene is measured by PCR or FISH.

15. The pharmaceutical composition according to claim 13, wherein intracellular Mcl-1 protein production or Bcl-xL protein production is measured by immunoblotting or immunohistochemical staining.

16. A pharmaceutical composition for treating osteosarcoma comprising an Mcl-1 inhibitor in combination with a Bcl-xL inhibitor, or an IGF-1R inhibitor.

17. The pharmaceutical composition of claim 16, wherein the Mcl-1 inhibitor is selected from the group consisting of obatoclax, AZD5991, MIK665, siRNA against the MCL1 gene, or a combination thereof.

18. The pharmaceutical composition according to claim 16, wherein the Bcl-xL inhibitor is selected from the group consisting of A-1331852 and siRNA against the BCL2L1 gene.

19. The pharmaceutical composition of claim 16, wherein the IGF-1R inhibitor is selected from the group consisting of OSI906, AEW541, AZD3463, and siRNA against the IGF-1R gene.

20. The pharmaceutical composition according to any one of claims 16 to 19, wherein the osteosarcoma has an amplified gene copy number of the MCL1 gene or an increased production of Mcl-1 protein, or an amplified gene copy number of the BCL2L1 gene or an increased production of Bcl-xL protein in cells.

21. The pharmaceutical composition according to claim 20, wherein the intracellular copy number of the MCL1 gene or the BCL2L1 gene is measured by PCR or FISH.

22. The pharmaceutical composition according to claim 20, wherein intracellular Mcl-1 protein production or Bcl-xL protein production is measured by immunoblotting or immunohistochemical staining.

23. A method for detecting an amplification of the gene copy number of the MCL1 gene or an increase in the production of Mcl-1 protein, an amplification of the gene copy number of the BCL2L1 gene, or an increase in the production of Bcl-xL protein in osteosarcoma tissue or blood collected from a patient suffering from osteosarcoma, which is an indicator of whether or not the patient is a target for administration of a pharmaceutical composition comprising an ingredient selected from the group consisting of an Mcl-1 inhibitor, a Bcl-xL inhibitor, obatoclax, tipifarnib, bortezomib, carfilzomib, SN-38, A-1331852, and derivatives thereof, or a combination thereof, or comprising an Mcl-1 inhibitor in combination with a Bcl-xL inhibitor or an IGF-1R inhibitor.

24. The method according to claim 23, wherein the gene copy number of the MCL1 gene or the gene copy number of the BCL2L1 gene in the cells is measured by PCR or FISH.

25. The method of claim 23, wherein intracellular Mcl-1 protein production or Bcl-xL protein production is measured by immunoblotting or immunohistochemical staining.

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