Molecular targeted drug combination therapy in cancer patients having driver mutation

Abstract

The present invention addresses the problem of providing: a novel treatment strategy using a molecular targeted drug in cancer patients having a driver mutation; a novel method capable of identifying a treatment target patient group suitable for the novel treatment strategy, and capable of predicting, monitoring, or diagnosing a response to the novel treatment strategy; and a kit for use in the method. The present invention provides, as a means for solving the problem, a pharmaceutical composition which contains, as an active ingredient, a FAK inhibitor and a RAF / MEK dual inhibitor for use in the treatment of, for example, patients suffering from cancer, and which is for administration to patients who have been confirmed that an epithelial biomarker expression level in a tumor sample isolated from a patient is higher than the mesenchymal biomarker expression level.

Description

Combination targeted drug therapy for cancer patients with driver mutations

[0001] The present invention relates to combination therapy using molecular targeted drugs in cancer patients with driver mutations.

[0002] In recent years, various molecular targeted drugs have been developed for cancer treatment. Although the prognosis of cancers with driver mutations treated with molecular targeted drugs has shown a clear trend toward improvement compared to conventional anticancer drugs such as chemotherapy, the efficacy remains low, and further improvement is needed.

[0003] KRAS mutations are known as driver mutations, and are mutations of the oncogene KRAS commonly found in several cancers, including non-small cell lung cancer (NSCLC), colorectal cancer, and pancreatic cancer. The frequency of KRAS mutations in NSCLC varies by race, with KRAS mutations found in 20-30% of lung adenocarcinomas in Caucasians and 8-10% of Asians (see Non-Patent Documents 1 to 3).

[0004] Although EGFR, ALK, and BRAF inhibitors have been successfully developed, targeted therapies against KRAS have only recently been approved.

[0005] Currently, two KRAS G12C inhibitors, sotorasib and adagrasib, have been approved by the Food and Drug Administration for the treatment of patients with NSCLC harboring a KRAS G12C mutation (see Non-Patent Documents 4 and 5). A phase III clinical trial demonstrated that the KRAS G12C inhibitor sotorasib demonstrated significantly better progression-free survival (PFS) than docetaxel in patients with previously treated KRAS G12C-mutated NSCLC (see Non-Patent Document 6). Therefore, sotorasib has been approved for the treatment of KRAS G12C-mutated NSCLC in the United States, Japan, and other countries.

[0006] However, the objective response rates of sotorasib and adagrasib in patients with KRAS G12C mutant NSCLC are 28.1% (see Non-Patent Document 6) and 42.9% (see Non-Patent Document 5), respectively. In addition to sotorasib and adagrasib, several other KRAS inhibitors are under development, and further clinical applications are expected. However, alternative strategies for KRAS mutant cancers are attracting attention.

[0007] KRAS is one of the first oncogenes in which activating mutations were detected in human cancer. However, unlike other oncogenes such as EGFR, ALK, and ROS-1, KRAS has structural features such as a smooth surface and an extremely high affinity for GTP / GDP binding. Because of these characteristics, the development of therapeutic agents directly targeting KRAS has been unsuccessful for many years, and no therapeutic agent has yet been clinically approved (see Non-Patent Document 4).

[0008] However, in recent years, several inhibitors of the RAS GTPase family have been proposed as therapeutic agents applicable to solid cancers with KRAS mutations (see Non-Patent Documents 5 and 6).

[0009] In particular, a phase II clinical trial showed that sotorasib, a KRAS G12C inhibitor, provided a longer progression-free survival (PFS) than docetaxel in previously treated patients with NSCLC harboring a KRAS G12C mutation (see Non-Patent Document 7), and its use has been approved in the United States, Japan, and other countries for NSCLC patients harboring a KRAS G12C mutation.

[0010] However, the objective response rate of sotorasib in NSCLC patients with KRAS G12C mutation is only 32.2% (see Non-Patent Document 7), and the therapeutic effect is still insufficient.

[0011] Therefore, in addition to sotorasib, several other KRAS inhibitors have recently been developed, and further clinical applications are expected. At the same time, alternative strategies for KRAS-mutated cancers are attracting attention.

[0012] Much effort has been devoted to alternative strategies for KRAS mutant cancers, such as inhibiting downstream signaling through the mitogen-activated protein kinase (MAPK; RAF / MEK / ERK) pathway (see Non-Patent Document 7). Among several therapeutic strategies for KRAS mutant cancers, MEK inhibition is a potential treatment.

[0013] However, multiple clinical trials have shown that MEK inhibitor monotherapy does not provide convincing therapeutic effects for patients with KRAS-mutated NSCLC (see Non-Patent Documents 8 to 10). The limited efficacy observed with MEK inhibitors may be related to an adaptive feedback mechanism due to reactivation of MAPK signaling caused by blockade of ERK signaling (see Non-Patent Documents 11 to 13).

[0014] The novel RAF / MEK inhibitor avutometinib (VS-6766 / CH5126766 / RO5126766 / CKI27(2)) potently suppresses the MAPK pathway by inhibiting both RAF and MEK, making it an attractive molecular targeted drug for patients with KRAS gene mutations (see Non-Patent Documents 14 and 15). In a phase I trial, 30% of patients with KRAS-mutant NSCLC treated with avutometinib achieved an objective response. All of these patients experienced a low incidence of adverse events lasting 6 months or longer (see Non-Patent Document 16). Furthermore, combination therapy of avutometinib with the FAK inhibitor defactinib has demonstrated clinical efficacy in KRAS-mutant NSCLC and low-grade serous ovarian cancer (LGSOC) (see Non-Patent Documents 17-19). However, although the efficacy of avutometinib monotherapy in patients with KRAS-mutated NSCLC is higher than that of other MEK inhibitor monotherapies, the therapeutic effect is not fully satisfactory. Therefore, more effective treatment strategies are needed.

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[0016] The problems to be solved by the present invention are to provide a new therapeutic strategy using molecular targeted drugs for cancer patients with driver mutations, to identify patient groups suitable for treatment with the new therapeutic strategy, and to provide a new method for predicting, monitoring, or diagnosing response to the new therapeutic strategy, and a kit for use in the method.

[0017] As described above, the therapeutic effect of molecular targeted drugs developed for cancers with driver mutations, such as KRAS-mutated cancers, is insufficient. Therefore, there is still a need for more effective therapeutic strategies that can replace the administration of molecular targeted drugs in the treatment of cancers with driver mutations.

[0018] Therefore, the inventors of the present application have been conducting intensive research to establish appropriate sensitivity markers for cancers with such driver mutations, and to provide opportunities for early diagnosis of cancers with driver mutations using these sensitivity markers, as well as more appropriate cancer treatment by applying molecular targeted drugs to cancer patients with driver mutations corresponding to these markers.

[0019] In the course of their research, the inventors have now newly elucidated the feedback mechanism of the RAF / MEK dual inhibitor avutometinib via focal adhesion kinase (FAK) in non-small cell lung cancer (NSCLC) cells harboring driver mutations such as KRAS mutations.

[0020] Furthermore, the present inventors have now newly confirmed that a combined therapy of a RAF / MEK dual inhibitor and a FAK inhibitor has a stronger effect on NSCLC cells with an epithelial phenotype and KRAS mutation, and that this effect is mediated by Bim-mediated apoptosis.

[0021] Furthermore, the present inventors have now newly revealed that in in vivo testing, combined therapy of a RAF / MEK dual inhibitor and a FAK inhibitor for mutant cancers with an epithelial phenotype has the extremely remarkable and surprising effect of significantly shrinking mutant cancers with an epithelial phenotype, compared to RAF / MEK dual inhibitor monotherapy, and further has the extremely remarkable and surprising effect of delaying tumor regrowth after treatment is discontinued.

[0022] Based on these results, the present inventors demonstrated that combined therapy of a RAF / MEK dual inhibitor and a FAK inhibitor could be a promising treatment for NSCLC patients with KRAS mutations that exhibit an epithelial phenotype, and completed the present invention.

[0023] A first means of the present invention for solving the above problems is a pharmaceutical composition comprising, as active ingredients, a FAK inhibitor and a RAF / MEK dual inhibitor for use in treating a patient suffering from cancer, wherein the pharmaceutical composition is for administration to the patient in whom it has been confirmed that the expression level of epithelial biomarkers is higher than the expression level of mesenchymal biomarkers in a tumor sample isolated from the patient.

[0024] A second means of the present invention for solving the above-mentioned problems is a pharmaceutical composition comprising as active ingredients a FAK inhibitor and a RAF / MEK dual inhibitor for use in treating a patient suffering from cancer, the pharmaceutical composition being for administration to the patient in whom it has been confirmed that the epithelial biomarker expression level is higher than the mesenchymal biomarker expression level in a tumor sample isolated from the patient, and in whom the RAF / MEK dual inhibitor as a single agent is ineffective.

[0025] A third means of the present invention for solving the above-mentioned problems is a pharmaceutical composition comprising as active ingredients a FAK inhibitor and a RAF / MEK dual inhibitor for use in treating a patient suffering from cancer, the pharmaceutical composition being for administration to the patient in whom it has been confirmed that the epithelial biomarker expression level is higher than the mesenchymal biomarker expression level in a tumor sample isolated from the patient, and that the growth inhibitory effect of the RAF / MEK dual inhibitor alone after discontinuation of the drug is low.

[0026] A fourth means of the present invention for solving the above-mentioned problems is a pharmaceutical composition comprising, as an active ingredient, a RAF / MEK dual inhibitor used in combination with a FAK inhibitor for use in treating a patient suffering from cancer, wherein the pharmaceutical composition is for use in combination with a FAK inhibitor and is intended to be administered to the patient in whom it has been confirmed that the expression level of epithelial biomarkers is higher than the expression level of mesenchymal biomarkers in a tumor sample isolated from the patient.

[0027] A fifth aspect of the present invention for solving the above-mentioned problems is a pharmaceutical composition comprising, as an active ingredient, a RAF / MEK dual inhibitor to be used in combination with a FAK inhibitor for use in treating a patient suffering from cancer, wherein the pharmaceutical composition is to be used in combination with a FAK inhibitor for administration to the patient in whom it has been confirmed that the epithelial biomarker expression level is higher than the mesenchymal biomarker expression level in a tumor sample isolated from the patient, and in whom the RAF / MEK dual inhibitor as a single agent is ineffective.

[0028] A sixth aspect of the present invention for solving the above-mentioned problems is a pharmaceutical composition for use in treating a patient suffering from cancer, the pharmaceutical composition comprising as an active ingredient a RAF / MEK dual inhibitor to be used in combination with a FAK inhibitor, wherein the pharmaceutical composition is for administration to the patient in whom it has been confirmed that the epithelial biomarker expression level is higher than the mesenchymal biomarker expression level in a tumor sample isolated from the patient, and that the growth inhibitory effect of the RAF / MEK dual inhibitor alone after discontinuation of the drug is low.

[0029] A seventh aspect of the present invention for solving the above-mentioned problems is a pharmaceutical composition comprising an FAK inhibitor as an active ingredient, for use in treating a patient suffering from cancer, to be used in combination with a RAF / MEK dual inhibitor, wherein the pharmaceutical composition is for use in combination with a RAF / MEK dual inhibitor, and is for administration to the patient in whom it has been confirmed that the expression level of epithelial biomarkers is higher than the expression level of mesenchymal biomarkers in a tumor sample isolated from the patient.

[0030] An eighth means of the present invention for solving the above-mentioned problems is a pharmaceutical composition comprising an FAK inhibitor as an active ingredient, for use in treating a patient suffering from cancer, to be used in combination with a RAF / MEK dual inhibitor, wherein the pharmaceutical composition is for administration to the patient in whom the expression level of an epithelial biomarker is higher than the expression level of a mesenchymal biomarker in a tumor sample isolated from the patient, and in whom it has been confirmed that the RAF / MEK dual inhibitor alone is ineffective.

[0031] A ninth aspect of the present invention for solving the above-mentioned problems is a pharmaceutical composition comprising an FAK inhibitor as an active ingredient, for use in treating a patient suffering from cancer, to be used in combination with a RAF / MEK dual inhibitor, wherein the pharmaceutical composition is for administration to the patient in whom it has been confirmed that the epithelial biomarker expression level is higher than the mesenchymal biomarker expression level in a tumor sample isolated from the patient, and that the growth inhibitory effect of the RAF / MEK dual inhibitor alone after discontinuation of the drug is low.

[0032] A tenth aspect of the present invention for solving the above-mentioned problems is the pharmaceutical composition according to any one of the first to ninth aspects of the present invention, wherein the epithelial biomarker is selected from E-cadherin, Brk, γ-catenin, α1-catenin, α2-catenin, α3-catenin, keratin 8, keratin 18, connexin 31, plakophilin 3, stratafin 1, laminin alpha-5, and ST14.

[0033] An eleventh aspect of the present invention for solving the above-mentioned problems is the pharmaceutical composition according to any one of the first to tenth aspects of the present invention, wherein the mesenchymal biomarker is selected from vimentin, fibronectin, fibrillin-1, fibrillin-2, collagen alpha-2(IV), collagen alpha-2(V), LOXL1, nidogen, C11orf9, tenascin, N-cadherin, and embryonic EDB+fibronectin, tubulin alpha-3, and epimorphin.

[0034] A twelfth aspect of the present invention for solving the above-mentioned problems is the pharmaceutical composition according to any one of the first to eleventh aspects of the present invention, wherein the RAF / MEK dual inhibitor is avutometinib or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt.

[0035] A thirteenth aspect of the present invention for solving the above-mentioned problems is the pharmaceutical composition according to any one of the first to twelfth aspects of the present invention, wherein the FAK inhibitor is defactinib or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt.

[0036] A fourteenth aspect of the present invention for solving the above-mentioned problems is the pharmaceutical composition according to any one of the first to thirteenth aspects of the present invention, wherein the cancer is a cancer with a RAS mutation.

[0037] A fifteenth aspect of the present invention for solving the above-mentioned problems is the pharmaceutical composition according to any one of the first to fourteenth aspects of the present invention, wherein the cancer is a cancer with a KRAS mutation.

[0038] A sixteenth aspect of the present invention for solving the above-mentioned problems is the pharmaceutical composition according to any one of the first to fifteenth aspects of the present invention, wherein the cancer is selected from the group consisting of breast cancer, liver cancer, lung cancer including small cell lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, prostate cancer, malignant lymphoma, and leukemia.

[0039] A seventeenth aspect of the present invention for solving the above-mentioned problems is the use of an effective amount of a FAK inhibitor and an effective amount of a RAF / MEK dual inhibitor in the manufacture of a pharmaceutical for use in treating a patient suffering from cancer, wherein the pharmaceutical is for administration to the patient in whom it has been confirmed that the epithelial biomarker expression level is higher than the mesenchymal biomarker expression level in a tumor sample isolated from the patient.

[0040] An eighteenth means of the present invention for solving the above-mentioned problems is the use of an effective amount of a FAK inhibitor and an effective amount of a RAF / MEK dual inhibitor in the manufacture of a pharmaceutical for use in treating a patient suffering from cancer, wherein the pharmaceutical is for administration to the patient in whom it has been confirmed that the epithelial biomarker expression level is higher than the mesenchymal biomarker expression level in a tumor sample isolated from the patient, and in whom it has been confirmed that the RAF / MEK dual inhibitor is not effective as a single agent.

[0041] A nineteenth aspect of the present invention for solving the above-mentioned problems is the use of an effective amount of a FAK inhibitor and an effective amount of a RAF / MEK dual inhibitor in the manufacture of a pharmaceutical for use in treating a patient suffering from cancer, wherein the pharmaceutical is for administration to the patient in whom it has been confirmed that the epithelial biomarker expression level is higher than the mesenchymal biomarker expression level in a tumor sample isolated from the patient, and that the growth inhibitory effect of the RAF / MEK dual inhibitor alone is low after discontinuation of the drug.

[0042] A twentieth means of the present invention for solving the above-mentioned problems is the use of an effective amount of a RAF / MEK dual inhibitor in combination with a FAK inhibitor in the manufacture of a pharmaceutical for use in treating a patient suffering from cancer, wherein the pharmaceutical is for administration to the patient in whom it has been confirmed that the epithelial biomarker expression level is higher than the mesenchymal biomarker expression level in a tumor sample isolated from the patient.

[0043] A twenty-first means of the present invention for solving the above-mentioned problems is the use of an effective amount of a RAF / MEK dual inhibitor in combination with a FAK inhibitor in the manufacture of a pharmaceutical for use in treating a patient suffering from cancer, wherein the pharmaceutical is for administration to the patient in whom it has been confirmed that the epithelial biomarker expression level is higher than the mesenchymal biomarker expression level in a tumor sample isolated from the patient, and in whom it has been confirmed that the RAF / MEK dual inhibitor as a single agent is ineffective.

[0044] A twenty-second means of the present invention for solving the above-mentioned problems is the use of an effective amount of a RAF / MEK dual inhibitor in combination with a FAK inhibitor in the manufacture of a pharmaceutical for use in treating a patient suffering from cancer, wherein the pharmaceutical is for administration to the patient in whom it has been confirmed that the epithelial biomarker expression level is higher than the mesenchymal biomarker expression level in a tumor sample isolated from the patient, and that the growth inhibitory effect of the RAF / MEK dual inhibitor alone is low after discontinuation of the drug.

[0045] A twenty-third means of the present invention for solving the above-mentioned problems is the use of an effective amount of a FAK inhibitor in combination with a RAF / MEK dual inhibitor in the manufacture of a pharmaceutical for use in treating a patient suffering from cancer, wherein the pharmaceutical is for administration to the patient in whom it has been confirmed that the epithelial biomarker expression level is higher than the mesenchymal biomarker expression level in a tumor sample isolated from the patient.

[0046] A twenty-fourth means of the present invention for solving the above-mentioned problems is the use of an effective amount of a FAK inhibitor in combination with a RAF / MEK dual inhibitor in the manufacture of a pharmaceutical for use in treating a patient suffering from cancer, wherein the pharmaceutical is for administration to the patient in whom the epithelial biomarker expression level is higher than the mesenchymal biomarker expression level in a tumor sample isolated from the patient, and in whom it has been confirmed that the RAF / MEK dual inhibitor alone is ineffective.

[0047] A twenty-fifth means of the present invention for solving the above-mentioned problems is the use of an effective amount of a FAK inhibitor in combination with a RAF / MEK dual inhibitor in the manufacture of a pharmaceutical for use in treating a patient suffering from cancer, wherein the pharmaceutical is for administration to the patient in whom it has been confirmed that the epithelial biomarker expression level is higher than the mesenchymal biomarker expression level in a tumor sample isolated from the patient, and that the growth inhibitory effect of the RAF / MEK dual inhibitor alone is low after discontinuation of the drug.

[0048] A 26th means of the present invention for solving the above-mentioned problems is the use according to any one of the 17th to 25th means of the present invention, wherein the RAF / MEK dual inhibitor is avutometinib or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt.

[0049] A 27th means of the present invention for solving the above-mentioned problems is the use according to any one of the 17th to 26th means of the present invention, wherein the FAK inhibitor is defactinib or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt.

[0050] A 28th aspect of the present invention for solving the above-mentioned problems is the use according to any one of the 17th to 27th aspects of the present invention, wherein the cancer is a cancer with a RAS mutation.

[0051] A twenty-ninth aspect of the present invention for solving the above-mentioned problems is the use according to any one of the seventeenth to twenty-eighth aspects of the present invention, wherein the cancer is a cancer with a KRAS mutation.

[0052] A thirtieth aspect of the present invention for solving the above-mentioned problems is the use according to any one of the seventeenth to twenty-ninth aspects of the present invention, wherein the cancer is selected from the group consisting of breast cancer, liver cancer, lung cancer including small cell lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, prostate cancer, malignant lymphoma, and leukemia.

[0053] A thirty-first aspect of the present invention for solving the above-mentioned problems is a method for predicting sensitivity of a patient suffering from cancer to tumor cell growth inhibition by a FAK inhibitor and a RAF / MEK dual inhibitor, the method comprising the steps of: evaluating the expression levels of epithelial biomarkers and mesenchymal biomarkers expressed in a tumor sample isolated from the patient suffering from cancer; and predicting and indicating that the tumor cell growth will be highly sensitive to tumor cell growth inhibition by a FAK inhibitor and a RAF / MEK dual inhibitor if the epithelial biomarkers are at a higher expression level than the mesenchymal biomarkers; and predicting and indicating that the tumor cell growth will be less sensitive to inhibition by a FAK inhibitor and a RAF / MEK dual inhibitor if the epithelial biomarkers are at a lower expression level than the mesenchymal biomarkers.

[0054] A thirty-second aspect of the present invention for solving the above-mentioned problems is a method for predicting the sensitivity of a patient suffering from cancer to tumor cell growth inhibition by a FAK inhibitor and a RAF / MEK dual inhibitor, and suggesting the suitability of combined therapy with a FAK inhibitor and a RAF / MEK dual inhibitor, the method comprising the steps of: evaluating the expression levels of epithelial biomarkers and mesenchymal biomarkers expressed in a tumor sample isolated from the patient suffering from cancer; predicting that the tumor cell growth will be highly sensitive to inhibition by a FAK inhibitor and a RAF / MEK dual inhibitor when the epithelial biomarkers are at higher expression levels than the mesenchymal biomarkers, and suggesting that the tumor cell is suitable for combined anti-cancer therapy with a FAK inhibitor and a RAF / MEK dual inhibitor; and / or predicting that the tumor cell growth will be less sensitive to inhibition by a FAK inhibitor and a RAF / MEK dual inhibitor when the epithelial biomarkers are at lower expression levels than the mesenchymal biomarkers, and suggesting that the tumor cell is not suitable for combined anti-cancer therapy with a FAK inhibitor and a RAF / MEK dual inhibitor when the epithelial biomarkers are at lower expression levels than the mesenchymal biomarkers.

[0055] A thirty-third aspect of the present invention for solving the above-mentioned problems is a method according to the thirty-first or thirty-second aspect of the present invention, wherein the epithelial biomarker is selected from E-cadherin, Brk, γ-catenin, α1-catenin, α2-catenin, α3-catenin, keratin 8, keratin 18, connexin 31, plakophilin 3, stratafin 1, laminin alpha-5, and ST14.

[0056] A thirty-fourth aspect of the present invention for solving the above-mentioned problems is a method according to any one of the thirty-first to thirty-third aspects of the present invention, wherein the mesenchymal biomarker is selected from vimentin, fibronectin, fibrillin-1, fibrillin-2, collagen alpha-2(IV), collagen alpha-2(V), LOXL1, nidogen, C11orf9, tenascin, N-cadherin, and embryonic EDB+fibronectin, tubulin alpha-3, and epimorphin.

[0057] A thirty-fifth aspect of the present invention for solving the above-mentioned problems is a method according to any one of the thirty-first to thirty-fourth aspects of the present invention, wherein the cancer is a cancer with a RAS mutation.

[0058] A thirty-sixth aspect of the present invention for solving the above-mentioned problems is a method according to any one of the thirty-first to thirty-fifth aspects of the present invention, wherein the cancer is a cancer with a KRAS mutation.

[0059] A thirty-seventh aspect of the present invention for solving the above-mentioned problems is the method according to any one of the thirty-first to thirty-sixth aspects of the present invention, wherein the cancer is selected from the group consisting of breast cancer, liver cancer, lung cancer including small cell lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, prostate cancer, malignant lymphoma, and leukemia.

[0060] A thirty-eighth aspect of the present invention for solving the above-mentioned problems is a kit for predicting or monitoring sensitivity of a patient suffering from cancer to tumor cell growth inhibition by a FAK inhibitor and a RAF / MEK dual inhibitor, and / or for indicating or monitoring the suitability of anti-cancer treatment using a combination of a FAK inhibitor and a RAF / MEK dual inhibitor in a patient suffering from cancer, by the method according to the thirty-first or thirty-second aspect of the present invention, comprising an anti-epithelial biomarker antibody and / or a fragment thereof, and an anti-mesenchymal biomarker antibody and / or a fragment thereof.

[0061] A thirty-ninth aspect of the present invention for solving the above-mentioned problems is the kit according to the thirty-eighth aspect of the present invention, wherein the epithelial biomarker is selected from E-cadherin, Brk, γ-catenin, α1-catenin, α2-catenin, α3-catenin, keratin 8, keratin 18, connexin 31, plakophilin 3, stratafin 1, laminin alpha-5, and ST14.

[0062] A fortieth aspect of the present invention for solving the above-mentioned problems provides a kit according to the thirty-eighth or thirty-ninth aspect of the present invention, wherein the mesenchymal biomarker is selected from vimentin, fibronectin, fibrillin-1, fibrillin-2, collagen alpha-2(IV), collagen alpha-2(V), LOXL1, nidogen, C11orf9, tenascin, N-cadherin, and embryonic EDB+fibronectin, tubulin alpha-3, and epimorphin.

[0063] A forty-first means of the present invention for solving the above-mentioned problems is the kit according to any one of the thirty-eighth to fortieth means of the present invention, wherein the RAF / MEK dual inhibitor is avutometinib or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt.

[0064] A forty-second means of the present invention for solving the above-mentioned problems is the kit according to any one of the thirty-eighth to forty-first means of the present invention, wherein the FAK inhibitor is defactinib or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt.

[0065] A forty-third aspect of the present invention for solving the above-mentioned problems is a kit according to any one of the thirty-eighth to forty-second aspects of the present invention, wherein the cancer is a cancer with a RAS mutation.

[0066] A forty-fourth aspect of the present invention for solving the above-mentioned problems is a kit according to any one of the thirty-eighth to forty-third aspects of the present invention, wherein the cancer is a cancer with a KRAS mutation.

[0067] A forty-fifth aspect of the present invention for solving the above-mentioned problems is the kit according to any one of the thirty-eighth to forty-fourth aspects of the present invention, wherein the cancer is selected from the group consisting of breast cancer, liver cancer, lung cancer including small cell lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, prostate cancer, malignant lymphoma, and leukemia.

[0068] A 46th aspect of the present invention for solving the above-mentioned problems is a method for treating a patient suffering from cancer, comprising administering an effective amount of a FAK inhibitor and an effective amount of a RAF / MEK dual inhibitor to the patient in whom it has been confirmed that the expression level of an epithelial biomarker is higher than the expression level of a mesenchymal biomarker in a tumor sample isolated from the patient.

[0069] A forty-seventh aspect of the present invention for solving the above-mentioned problems is a method for treating a patient suffering from cancer, comprising administering an effective amount of a FAK inhibitor and an effective amount of a RAF / MEK dual inhibitor to the patient in whom it has been confirmed that the epithelial biomarker expression level is higher than the mesenchymal biomarker expression level in a tumor sample isolated from the patient and that a RAF / MEK dual inhibitor alone is not effective.

[0070] A forty-eighth means of the present invention for solving the above-mentioned problems is a method for treating a patient suffering from cancer, comprising administering an effective amount of a FAK inhibitor and an effective amount of a RAF / MEK dual inhibitor to the patient in whom it has been confirmed that the expression level of an epithelial biomarker is higher than the expression level of a mesenchymal biomarker in a tumor sample isolated from the patient and that the growth inhibitory effect of a single RAF / MEK dual inhibitor after discontinuation of the drug is low.

[0071] A forty-ninth aspect of the present invention for solving the above-mentioned problems is a treatment method according to any one of the forty-sixth to forty-eighth aspects of the present invention, wherein the epithelial biomarker is selected from E-cadherin, Brk, γ-catenin, α1-catenin, α2-catenin, α3-catenin, keratin 8, keratin 18, connexin 31, plakophilin 3, stratafin 1, laminin alpha-5, and ST14.

[0072] A fiftieth aspect of the present invention for solving the above-mentioned problems is a treatment method according to any one of the forty-sixth to forty-ninth aspects of the present invention, wherein the mesenchymal biomarker is selected from vimentin, fibronectin, fibrillin-1, fibrillin-2, collagen alpha-2(IV), collagen alpha-2(V), LOXL1, nidogen, C11orf9, tenascin, N-cadherin, and embryonic EDB+fibronectin, tubulin alpha-3, and epimorphin.

[0073] A fifty-first aspect of the present invention for solving the above-mentioned problems is a method for treatment according to any one of the forty-sixth to fiftieth aspects of the present invention, wherein the RAF / MEK dual inhibitor is avutometinib or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt.

[0074] A fifty-second means of the present invention for solving the above-mentioned problems is a treatment method according to any one of the forty-sixth to fifty-first means of the present invention, wherein the FAK inhibitor is defactinib or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt.

[0075] A fifty-third aspect of the present invention for solving the above-mentioned problems is a therapeutic method according to any one of the forty-sixth to fifty-second aspects of the present invention, wherein the cancer is a cancer having a RAS mutation.

[0076] A fifty-fourth aspect of the present invention for solving the above-mentioned problems is a therapeutic method according to any one of the forty-sixth to fifty-third aspects of the present invention, wherein the cancer is a cancer having a KRAS mutation.

[0077] A fifty-fifth aspect of the present invention for solving the above-mentioned problems is the method of treatment according to any one of the forty-sixth to fifty-fourth aspects of the present invention, wherein the cancer is selected from the group consisting of breast cancer, liver cancer, lung cancer including small cell lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, prostate cancer, malignant lymphoma, and leukemia.

[0078] According to the means of the present invention, a pharmaceutical composition containing a FAK inhibitor and a RAF / MEK dual inhibitor as active ingredients is provided to a patient suffering from cancer in which it has been confirmed that the expression level of epithelial biomarkers is higher than the expression level of mesenchymal biomarkers in a tumor sample isolated from the patient. By administering this pharmaceutical composition, a high level of antitumor effect that cannot be achieved by administering the RAF / MEK dual inhibitor or the FAK inhibitor alone is exerted, and the growth-suppressing effect continues to a certain extent after drug discontinuation, thereby providing a new means for achieving a remarkable anticancer effect. Furthermore, by the means of the present invention, it has become possible to predict and present the sensitivity of tumor cell proliferation to inhibition by a FAK inhibitor and a RAF / MEK dual inhibitor in a patient suffering from cancer, such that when the epithelial biomarker expressed in a tumor sample isolated from the patient is at a higher expression level than the mesenchymal biomarker, the sensitivity of tumor cell proliferation to inhibition by a FAK inhibitor and a RAF / MEK dual inhibitor is high, and when the epithelial biomarker is at a lower expression level than the mesenchymal biomarker, the sensitivity of tumor cell proliferation to inhibition by a FAK inhibitor and a RAF / MEK dual inhibitor is low. Furthermore, when the expression level of the epithelial biomarker is higher than that of the mesenchymal biomarker, it is predicted that the tumor cell proliferation will be highly sensitive to inhibition by a FAK inhibitor and a RAF / MEK dual inhibitor, and it is suggested that the combination of a FAK inhibitor and a RAF / MEK dual inhibitor is suitable for anti-cancer treatment, and when the expression level of the epithelial biomarker is lower than that of the mesenchymal biomarker, it is predicted that the tumor cell proliferation will be less sensitive to inhibition by a FAK inhibitor and a RAF / MEK dual inhibitor, and it is suggested that the combination of a FAK inhibitor and a RAF / MEK dual inhibitor is not suitable for anti-cancer treatment, thereby enabling the formulation and implementation of more appropriate treatment strategies. Furthermore, for patients with cancers that are not suitable for anti-cancer treatment with a FAK inhibitor and a RAF / MEK dual inhibitor, it is possible to avoid the unnecessary prescription of molecular targeted drugs and the resulting unintended acquisition of treatment resistance, and to provide an opportunity to select an alternative treatment option earlier, thereby significantly improving the success rate of cancer treatment and cure and significantly improving the patient's quality of life.

[0079] Phosphorylation of FAK by avutometinib alone in NCI-H358 cells. Changes in phosphorylation of FAK, AKT, and ERK by avutometinib alone in NCI-H358 and NCI-H2122 cells. Cell viability by FAK knockdown and avutometinib. Changes in phosphorylation of FAK, AKT, and ERK by FAK knockdown and avutometinib. Changes in IGF1 mRNA expression by avutometinib alone. Changes in IGF-1R phosphorylation and cMyc expression by drugs Western blot analysis in KRAS mutant lung cancer cell lines Effect of combined use of avutometinib and defactinib in KRAS mutant lung cancer cell lines Effect of combined use of avutometinib and defactinib in KRAS mutant lung cancer cell lines with high and low FAK phosphorylation expression, and in KRAS G12C point mutation and non-G12C point mutation groups EMT signature genes in KRAS mutant lung cancer cell lines Gene expression analysis Epithelial and mesenchymal scores in KRAS-mutated lung cancer cell lines Correlation between the combined effect of avutometinib and defactinib and EMT score in KRAS-mutated lung cancer cell lines Activation of FAK and AKT by avutometinib alone and reduction of FAK and AKT activity when combined with defactinib Induction of sub-G1 population when combined Induction of Bim expression and dephosphorylation of FoxO3a when combined Decreased FAK and AKT activity and induction of apoptosis when combined Changes in morphology of EMT-induced cells Changes in the expression of ZEB1, E-cadherin, and vimentin Changes in cell growth rate of EMT-induced cells Changes in the cell cycle of EMT-induced cells Changes in the combined effect of avutometinib and defactinib in EMT-induced cells Changes in FAK, AKT, and ERK phosphorylation in EMT-induced cells Tumor growth suppression effect of combination therapy in mice Antitumor effect of combination therapy in mice (macroscopic images of each tumor) Antitumor effect of combination therapy in mice (effect on tumor weight) Intratumoral FAK, AKT, and ERK phosphorylation by combination therapy in miceEffect on Bim expression levels Effect of combination therapy on the proportion of Ki-67-positive cells and TUNEL-positive cells in tumors in mice Effect of combination therapy on the proportion of Ki-67-positive cells and TUNEL-positive cells in tumors in mice Cell viability of NCI-H358 cells with avutometinib alone Changes in IGF1 mRNA expression upon Myc knockdown in KRAS-mutated lung cancer cell lines Western blotting analysis upon Myc knockdown in KRAS-mutated lung cancer cell lines Cell viability of KRAS-mutated lung cancer cell lines with avutometinib alone Cell viability of KRAS-mutated lung cancer cell lines with epithelial and mesenchymal phenotypes with avutometinib alone Cell viability of KRAS-mutated lung cancer cell lines with G12C point mutation and non-G12C point mutation with avutometinib alone Cell viability: Cell viability with avutometinib alone in KRAS mutant lung cancer cell lines with and without LKB1 mutations; Cell viability with avutometinib and defactinib in KRAS mutant lung cancer cell lines; Crystal violet staining results for KRAS mutant lung cancer cell lines after long-term exposure to avutometinib and defactinib; Effect of avutometinib and defactinib on the cell cycle of KRAS mutant lung cancer cell lines; Effect of avutometinib and defactinib on cell migration of KRAS mutant lung cancer cell lines; Evaluation of the effect of combination therapy on tumor regression in mice; Evaluation of the effect of combination therapy on body weight in mice; Mechanism of apoptosis induction by combination of avutometinib and defactinib in KRAS mutant lung cancer cell lines;

[0080] The present invention provides a new therapeutic strategy using molecular targeted drugs for cancer patients with driver mutations, as well as a new method for identifying patient groups suitable for treatment with the new therapeutic strategy, and for predicting, monitoring, or diagnosing response to the new therapeutic strategy, and a kit for use in the method.

[0081] As used herein, the term "treatment" means that administration of the pharmaceutical composition of the present invention to a subject results in the death of tumors or a reduction in the number of tumor cells, the inhibition of tumor growth, and the amelioration of various symptoms caused by tumors. Furthermore, in the present invention, the term "prevention" means the prevention of a tumor that has been reduced from growing again and thus increasing in number, and the prevention of the regrowth of a tumor whose growth has been inhibited.

[0082] In this specification, a halogen atom means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

[0083] In this specification, C1-6 alkyl group means a straight-chain or branched-chain alkyl group having 1 to 6 carbon atoms. Examples include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, a tert-butyl group, a 1-methylpropyl group, an n-pentyl group, a 1-methylbutyl group, a 2-methylbutyl group, a 3-methylbutyl group, a 1,1-dimethylpropyl group, a 2,2-dimethylpropyl group, a 1,2-dimethylpropyl group, a 1-ethylpropyl group, an n-hexyl group, a 1-methylpentyl group, a 2-methylpentyl group, a 3-methylpentyl group, a 4-methylpentyl group, a 1,1-dimethylbutyl group, a 1,2-dimethylbutyl group, a 1,3-dimethylbutyl group, a 2,2-dimethylbutyl group, a 2,3-dimethylbutyl group, a 3,3-dimethylbutyl group, a 1-ethylbutyl group, and a 2-ethylbutyl group.

[0084] In this specification, a C2-7 alkenyl group refers to a linear or branched alkenyl group having 2 to 7 carbon atoms, such as a vinyl group, an allyl group, a 1-butenyl group, a 2-butenyl group, a 3-butenyl group, a pentenyl group, a pentadienyl group, a hexenyl group, a hexadienyl group, a heptenyl group, a heptadienyl group, and a heptatrienyl group.

[0085] In this specification, a C2-7 alkynyl group refers to a straight-chain or branched-chain alkynyl group having 2 to 7 carbon atoms. Examples include an ethynyl group, a 1-propynyl group, a 2-propynyl group, a 1-butynyl group, a 2-butynyl group, a 3-butynyl group, a pentynyl group, a pentadiynyl group, a hexynyl group, a hexadiynyl group, a heptynyl group, a heptadiynyl group, and a heptatriynyl group.

[0086] In this specification, a C1-6 alkoxy group refers to an alkyloxy group having a linear or branched alkyl group having 1 to 6 carbon atoms. Examples include a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, a sec-butoxy group, a tert-butoxy group, an n-pentoxy group, and an n-hexoxy group.

[0087] In this specification, a C1-6 alkylthio group refers to an alkylthio group having a linear or branched alkyl group having 1 to 6 carbon atoms. Examples include a methylthio group, an ethylthio group, an n-propylthio group, an isopropylthio group, an n-butylthio group, a sec-butylthio group, a tert-butylthio group, an n-pentylthio group, and an n-hexylthio group.

[0088] As used herein, a C3-6 cycloalkyl group refers to a cyclic alkyl group having 3 to 6 atoms constituting the ring. It may be monocyclic or bicyclic, but unless otherwise specified, it refers to a monocyclic group. Examples of monocyclic groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups. Examples of bicyclic groups include bicyclo[1.1.1]pentanyl and bicyclo[2.1.1]hexyl groups.

[0089] As used herein, a C3-6 heterocycloalkyl group refers to a C3-6 cycloalkyl group in which at least one of the carbon atoms constituting the ring is replaced with a nitrogen atom, oxygen atom, or sulfur atom. It may be monocyclic or bicyclic, but unless otherwise specified, it refers to a monocyclic group. Examples of monocyclic groups include tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, piperidinyl, piperazinyl, and morpholinyl groups. Examples of bicyclic groups include oxabicyclo[3.1.0]hexan-6-yl and azabicyclo[2.1.1]hexanyl groups.

[0090] As used herein, the term "unsaturated five-membered heterocyclic ring" refers to an unsaturated five-membered ring containing at least one heteroatom selected from a nitrogen atom, an oxygen atom, and a sulfur atom, and examples thereof include furan, thiophene, pyrrole, imidazole, and thiazole.

[0091] In this specification, examples of pharmaceutically acceptable salts include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, etc.; sulfonates such as methanesulfonate, benzenesulfonate, toluenesulfonate, etc.; carboxylates such as formate, acetate, oxalate, maleate, fumarate, citrate, malate, succinate, malonate, gluconate, mandelate, benzoate, salicylate, fluoroacetate, trifluoroacetate, tartrate, propionate, glutarate, etc.; alkali metal salts such as lithium salt, sodium salt, potassium salt, cesium salt, rubidium salt, etc.; alkaline earth metal salts such as magnesium salt, calcium salt, etc.; and ammonium salts such as ammonium salt, alkylammonium salt, dialkylammonium salt, trialkylammonium salt, tetraalkylammonium salt, etc. Among them, alkali metal salts such as lithium salt, sodium salt, potassium salt, cesium salt, rubidium salt, etc. are preferred, and sodium salt and potassium salt are more preferred.

[0092] As used herein, a pharmaceutically acceptable solvate refers to, for example, a solvate with water, an alcohol (e.g., methanol, ethanol, 1-propanol, or 2-propanol), acetone, dimethylformamide, or dimethylacetamide. It may be a solvate with a single solvent or a solvate with multiple solvents. Preferred examples of the solvate include hydrates.

[0093] The FAK inhibitor is selected from the group consisting of, but not limited to, defactinib, TAE226, BI-853520, GSK2256098, PF-03814735, BI-4464, VS-4718, and APG-2449, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt. Preferably, the FAK inhibitor is defactinib or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt.

[0094] It is believed that agents that inhibit the MAPK (mitogen-activated protein kinase) pathway, which is activated by BRAF, K / N / H-RAS mutations, etc., may show results similar to those shown in this study with the RAF / MEK dual inhibitor. These MAPK pathway inhibitors include, but are not limited to, avutometinib (CKI27(2) / CH5126766 / RO5126766 / VS-6766), trametinib, IK-595, IMM-1-104, trametiglue, cobimetinib, binimetinib, selumetinib, dabrafenib, vemurafenib, encorafenib, or pharmaceutically acceptable salts thereof, or pharmaceutically acceptable solvates of the compounds or salts. Further, the compounds, salts, or solvates described in the following (A1) and (A2), which are RAF / MEK complex stabilizers or MEK inhibitors, also include MAPK pathway inhibitors. Preferably, the RAF / MEK dual inhibitor and MEK inhibitor are avutometinib or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt. The compounds, salts, or solvates described in the following (A1) and (A2) are arylamide derivatives that have RAF / MEK complex stabilizing activity and / or MEK inhibitory activity and are useful for the treatment or prevention of cell proliferative diseases, particularly cancer. (A1) A compound represented by the following general formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt: [wherein ring A is a group represented by the following general formula (2) or (4) (wherein bonds marked with *, ** and *** are -NH-, -CONH- and -CH ２ - is bonded to. X １ , X ２ , X ３ and X ４ are each independently -CR ２ = or -N=, and R ２ is a hydrogen atom, a halogen atom or a C1-6 alkyl group, and R １ is -S(=O) ２ -NH-R ８ Or -S(=O) ２ -R８ and R ８ is a hydrogen atom, a C1-6 alkyl group (the C1-6 alkyl group may be substituted with a halogen atom, a hydroxy group or a C1-6 alkoxy group), or a monocyclic C3-6 cycloalkyl group (the C3-6 cycloalkyl group may be substituted with a C1-6 alkyl group), and R ３ is a hydrogen atom, a C1-6 alkyl group, a C3-6 cycloalkyl group, or a C1-6 alkoxy group (the C1-6 alkoxy group may be substituted with a hydroxy group), and R ５ is a halogen atom or a C1-6 alkyl group, and R ６ is a hydrogen atom, a halogen atom or a C1-6 alkyl group, and R ４ is a halogen atom or a cyclopropyl group, R ７ is a hydrogen atom or a methyl group, R ９is a hydrogen atom, a halogen atom or a C1-6 alkyl group.] (A2) N-cyclopropyl-3,4-difluoro-2-(2-fluoro-4-iodoanilino)-5-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]benzamide, 2-(2-fluoro-4-iodoanilino)-5-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]-1-methyl-6-oxopyridine-3-carboxamide, 2-(4-cyclopropyl-2-fluoroanilino)-3,4-difluoro-5-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]benzamide, N-cyclopropyl-5-[[2-(ethylsulfamoylamino)-3-fluoropyridin-4-yl]methyl]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide, N-cyclopropyl-3,4-difluoro-5-[[3-fluoro-2-(2-fluoroethylsulfamoylamino)pyridin-4-yl]methyl]-2-(2-fluoro-4-iodoanilino)benzamide, 3,4-difluoro-2-(2-fluoro-4-iodoanilino)-5-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]-N-[(2-methylpropan-2-yl)oxy]benzamide, 3,4-difluoro-2-(2-fluoro-4-iodoanilino)-5-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]-N-methoxybenzamide, 3,4-difluoro-2-(2-fluoro-4-iodoanilino)-5-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]benzamide, 5-[[2-(cyclopropylsulfamoylamino)-3-fluoropyridin-4-yl]methyl]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide, 3,4-difluoro-2-(2-fluoro-4-iodoanilino)-5-[[3-fluoro-2-(propan-2-ylsulfamoylamino)pyridin-4-yl]methyl]benzamide,4-difluoro-2-(2-fluoro-4-iodoanilino)-5-[[3-fluoro-2-(2-methoxyethylsulfamoylamino)pyridin-4-yl]methyl]benzamide, 3,4-difluoro-2-(2-fluoro-4-iodoanilino)-5-[[3-fluoro-2-(2-methylpropylsulfamoylamino)pyridin-4-yl]methyl]benzamide, 3,4-difluoro-2-(2-fluoro-4-iodoanilino)-5-[[3-fluoro-2-[(1-methylcyclobutyl)sulfamoylamino]pyridin-4-yl]methyl]benzamide, 3,4-difluoro-2-(2-fluoro-4-iodoanilino)-5-[[3-fluoro-2-(propylsulfamoylamino)pyridin-4-yl]methyl]benzamide, 3,4-difluoro-2-(2-fluoro-4-iodoanilino)-5-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]-N-(2-hydroxyethoxy)benzamide, 5-[[3-(ethylsulfonylamino)-2-fluorophenyl]methyl]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)benzamide, 5-[[3-(ethylsulfonylamino)-2-fluorophenyl]methyl]-3,4-difluoro-2-(2-fluoro-4-iodoanilino)-N-methoxybenzamide, 3,4-difluoro-2-(2-fluoro-4-iodoanilino)-5-[[2-fluoro-3-(methylsulfamoylamino)phenyl]methyl]benzamide, 3,4-difluoro-2-(2-fluoro-4-iodoanilino)-5-[[2-fluoro-3-(methanesulfonamido)phenyl]methyl]benzamide, 4-fluoro-2-(2-fluoro-4-iodoanilino)-5-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]benzamide, N-cyclopropyl-2-(2-fluoro-4-iodoanilino)-5-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]-1-methyl-6-oxopyridine-3-carboxamide, 3,4-difluoro-2-(2-fluoro-4-iodoanilino)-5-[[3-fluoro-2-[(1-methylcyclobutyl)sulfamoylamino]pyridin-4-yl]methyl]-N-methoxybenzamide, 3,4-difluoro-5-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]-2-(2-fluoro-4-methylsulfanylanilino)benzamide, 2-(4-ethynyl-2-fluoroanilino)-3,4-difluoro-5-[[3-fluoro-2-(propylsulfamoylamino)pyridin-4-yl]methyl]benzamide, 2-(4-bromo-2-fluoroanilino)-3,4-difluoro-5-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]benzamide, 2-(2-chloro-4-iodoanilino)-5-[[3-(ethylsulfonylamino)-2-fluorophenyl]methyl]-3,4-difluoro-N-methoxybenzamide, 3,4-difluoro-2-(2-fluoro-4-iodoanilino)-5-[[2-fluoro-3-(oxan-4-ylsulfonylamino)phenyl]methyl]benzamide, 2-[4-(difluoromethylsulfanyl)-2-fluoroanilino]-3,4-difluoro-5-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]benzamide, 3,4-difluoro-2-[(4-fluoro-1-benzothiophen-5-yl)amino]-5-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]benzamide,4-difluoro-2-(2-fluoro-4-iodoanilino)-5-[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]oxybenzamide, 2-(2-fluoro-4-iodoanilino)-5-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]-N-methoxy-1-methyl-6-oxopyridine-3-carboxamide, 2-(2-fluoro-4-iodoanilino)-5-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]-1-methyl-N-[(2-methylpropan-2-yl)oxy]-6-oxopyridine-3-carboxamide, 5-[[2-(ethylsulfamoylamino)-3-fluoropyridin-4-yl]methyl]-2-(2-fluoro-4-iodoanilino)-1-methyl-6-oxopyridine-3-carboxamide, 5-(2-fluoro-4-iodoanilino)-2-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]pyridine-4-carboxamide, 5-(2-fluoro-4-iodoanilino)-8-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]imidazo[1,5-a]pyridine-6-carboxamide, 5-fluoro-4-(2-fluoro-4-iodoanilino)-1-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]-6-oxopyridine-3-carboxamide, 5-fluoro-4-(2-fluoro-4-iodoanilino)-1-[[3-fluoro-2-(propylsulfamoylamino)pyridin-4-yl]methyl]-6-oxopyridine-3-carboxamide, 4-(2-fluoro-4-iodoanilino)-1-[[3-fluoro-2-(methylsulfamoylamino)pyridin-4-yl]methyl]-5-methyl-6-oxopyridine-3-carboxamide, and 1-[[2-(ethylsulfonylamino)-3-fluoropyridin-4-yl]methyl]-4-(2-fluoro-4-iodoanilino)-5-methyl-6-oxopyridine-3-carboxamide, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt.

[0095] More preferably, the FAK inhibitor is defactinib or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, and the RAF / MEK dual inhibitor and MEK inhibitor are avutometinib or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt.

[0096] [Regarding Identification of Expression Levels of Epithelial and Mesenchymal Biomarkers] In the present invention, the expression levels of epithelial and mesenchymal biomarkers expressed in tumor samples isolated from cancer patients are identified in order to identify cancer patients with driver mutations who are suitable for treatment with a combination of a FAK inhibitor and a RAF / MEK dual inhibitor. Various anti-epithelial biomarker antibodies and / or fragments thereof, and anti-mesenchymal biomarker antibodies and / or fragments thereof can be used to identify these biomarkers. Antibodies that can be used include monoclonal antibodies, polyclonal antibodies, antibody fragments with binding specificity, and phage antibodies. Antibodies obtained by modifying the genes encoding the antibodies to improve specificity can also be used.

[0097] These antibodies and antibody fragments can be produced by administering a protein or peptide having an appropriate antigen or epitope to a host animal selected from mice, rats, pigs, cattle, horses, rabbits, goats, sheep, chickens, and the like, according to known methods. Alternatively, they can be produced as monoclonal antibodies by hybridoma technology (see, for example, Nature, 1975, 256:495-497; Immunology Today, 1983, 4:72; PNAS, 1983, 80:2026-2030; Monoclonal Antibodies and Cancer Therapy, 1985, 77-96, and the like), or can be prepared and isolated using any technique for producing antibody molecules (see, for example, Antibodies: A Laboratory Manual, 1988, Cold Spring Harbor Laboratory, Monoclonal Antibodies: Principles and Practice, 1986, Academic Press, etc. can be referenced. Antibodies prepared by methods such as those described above can also be used. Antibodies fragmented according to known methods can also be used. Commercially available antibodies can also be used.

[0098] When these antibodies and fragments thereof are used in a technique selected from the group consisting of Western blotting, immunohistochemical staining, immunofluorescence, immunoassay, ELISA, and mass spectrometry, they can be prepared and used by being labeled with an appropriate compound, fluorescent substance, etc. Furthermore, to increase the detection sensitivity, labeled secondary antibodies such as HRP-labeled secondary antibodies can also be used.

[0099] Additionally, in the methods of the present invention, the expression levels of epithelial biomarkers or mesenchymal biomarkers expressed by tumor cells can be assessed using any of the standard bioassay procedures known in the art, including detection methods using antibodies against epithelial biomarkers and mesenchymal biomarkers, such as ELISA, RIA, immunoprecipitation, immunoblotting, immunofluorescence microscopy, and other methods for measuring gene expression levels, such as RT-PCR, in situ hybridization, and cDNA microarrays. Generally, the terms "level of expression" and "expression level" are used interchangeably herein and generally refer to the amount of a biomarker in a sample. "Expression" generally refers to the process by which information (e.g., genetically encoded information and / or epigenetic information) is converted into structures present and operational in a cell. Thus, as used herein, "expression" can refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide).

[0100] [Regarding the process of measuring and comparing the amounts of epithelial and mesenchymal biomarker proteins] After obtaining written informed consent from a cancer patient, a specimen is removed from the area containing cancer tissue by a qualified professional such as a physician and provided. The amounts of epithelial and mesenchymal biomarker proteins are measured using Western blotting, immunohistochemical staining, immunofluorescence, immunoassay, ELISA, mass spectrometry, or the like. When immunohistochemical staining is performed, the specimen is optionally fixed with formalin or other methods, embedded in paraffin, and sections are prepared. A staining process is then performed using known techniques. The staining procedure may be performed as described in the Materials and Methods section of the Examples. Furthermore, counterstaining with hematoxylin or the like is performed as appropriate to ensure identification of the stained area.

[0101] Furthermore, the method can be performed using any fully automatic staining device and automatic staining processing device, such as the Ventana XT System Discovery, Ventana Benchmark ULTRA (Roche Diagnostics K.K.), the fully automatic antibody / in situ staining device Lica BOND, the fully automatic staining device Autostainer XL / Multistainer ST5020 (Leica Microsystems), etc., as well as a microscope and an automatic image processing system.

[0102] The present invention will be specifically explained below by way of examples, but the present invention should not be construed as being limited to these examples.

[0103] <Materials and Methods> [1. Cell Procurement] Nine human NSCLC cell lines (NCI-H358, NCI-H2122, NCI-H441, A549, SW1573, Calu-1, Calu-6, NCI-H23, and HOP-62) harboring mutations in KRAS were used.

[0104] The human cell lines NCI-H358, NCI-H2122, NCI-H441, A549, SW1573, Calu-1, and Calu-6 were all purchased from the American Type Culture Collection (Manassas, VA, USA). The human cell lines NCI-H23 and HOP-62 were obtained from the NCI-60 cancer cell line panel of the National Cancer Institute Developmental Therapeutics Program.

[0105] Information on LKB1 mutation status and type of point mutation in the KRAS oncogene was obtained from the Catalogue of somatic mutations in cancer (COSMIC) databases (https: / / cancer.sanger.ac.uk / cosmic / ).

[0106] [2. Cell Culture] All cell lines listed in 1. above were cultured at 37°C in 5% CO using Rothwell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mmol / L glutamine, 50 U / mL penicillin, and 100 μg / mL streptomycin. ２ The cells were cultured in an incubator under saturated water vapor pressure of 1000 kJ / min. After acquisition, a portion of each cell line was cryopreserved while it was still at a low passage number using the cryopreservation method specified for that cell line. Furthermore, for all cells used in the test, the cryopreserved cells with a low passage number were successively thawed and cultured for the test, so that the subculture period after thawing would be less than three months.

[0107] [3. Reagents] Avutometinib, a RAF / MEK dual inhibitor, was provided by Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan). Defactinib, a FAK inhibitor, was purchased from Selleck Chemicals (Houston, Texas, USA). These inhibitors were dissolved in dimethyl sulfoxide (DMSO) and stored at -20°C until use. Recombinant human transforming growth factor (TGF)-β1 protein was purchased from R&D Systems (Abingdon, UK).

[0108] 4. RTK Phosphorylation Antibody Array Cell lysates (500 μg / mL) were prepared from NCI-H358 cells exposed to either DMSO or the RAF / MEK dual inhibitor (avutometinib) for 72 hours.

[0109] The phosphorylation status of receptor tyrosine kinases (RTKs) in relevant pathways was analyzed using the RayBio C-Series Human RTK Phosphorylation Antibody Array C1 (RayBiotech Inc., Norcross, GA, USA) according to the manufacturer's instructions. The RTK phosphorylation antibody array was imaged and quantified using a ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA).

[0110] 5. Antibody and Western Blot Analysis Cells treated with each drug were lysed in RIPA buffer (50 mmol / L Tris-HCl, pH 8.0; 150 mmol / L NaCl; 1% NP-40; 0.5% deoxycholic acid; 0.1% SDS; 1 mmol / L dithiothreitol; and 0.5 mmol / L phenylmethylsulfonyl fluoride) supplemented with a phosphatase inhibitor (#07574; Nacalai Tesque, Kyoto, Japan) at 4°C for 30 minutes, followed by centrifugation.

[0111] Total protein concentration was measured using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). 10 μg protein aliquots were separated by SDS-PAGE.

[0112] Thereafter, the proteins were transferred to an Immobilon-P membrane (Millipore; Merck KGaA) and blocked with Tris-buffered saline (TBS) containing 5% skim milk at room temperature for 1 hour.

[0113] The membrane was incubated with the primary antibody overnight at 4°C.

[0114] The following primary antibodies were used as the antibodies. pFAK (Tyr397, #8556), FAK (#3285), pAKT (Ser473, #4060), AKT (#4691), pERK (#4370), ERK (#4696), Bim (#2933), pFoxO3a (Ser253, #9466), FoxO3a (#2497), Cleaved PARP (#5625), Cleaved Caspase 3 (#9661), pIGF-1R (#3021), IGF-1R (#9750), E-Cadherin (#3195S), Vimentin (#5741S) and ZEB1 (#3396S) (1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), β-actin (#A5441) (1:1000 dilution; Sigma-Aldrich, Burlington, MA, USA), and cMyc (#C2012) (1:1000 dilution; Abcam, Cambridge MA, UK).

[0115] After washing again three times with TBS-T, the membrane was incubated with HRP-conjugated species-specific secondary antibody for 1 hour at room temperature.

[0116] Immunoreactive bands were visualized using ECL chemiluminescent detection reagents (Immobilon Western Chemiluminescent HRP Substrate; Merck-Millipore) and imaged and quantified with a ChemiDoc Imaging System.

[0117] All immunoblot experiments were performed independently at least three times.

[0118] 6. Cell Viability Assay Cell viability was assessed using Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan).

[0119] cells (2-3 x 10 ３Cells (cells / well) were seeded into a 96-well plate and incubated with various compounds at the indicated concentrations for 72 hours. CCK-8 solution was added to each well, and after an additional 4 hours of incubation, the absorbance of the medium (450 nm) was measured using a SpectraMax® iD3 (Molecular Devices, LLC., San Jose, CA, USA). Cell viability was determined relative to that of the untreated control.

[0120] All cell viability assays were repeated at least three times with triplicate samples.

[0121] [7. Colony formation assay] Cells were plated in a 6-well plate at 5 x 10 ３ The cells were seeded at a density of 1000 cells / well. After 24 hours of incubation, the cells were treated with each drug for 15 days, with drug supplementation every 72 hours. After drug treatment, the 6-well plates were fixed with formalin and stained with 0.1% crystal violet, and the state of colony formation was visually confirmed.

[0122] Three independent experiments were performed, and a representative plate from three independent experiments is shown in the figure.

[0123] 8. Transfection Test: Duplexed Silencer Select siRNAs for MYC (s9129) and FAK (#1: PTK2HSS108800, #2: PTK2HSS183870) were used. These Duplexed Silencer Select siRNAs were purchased from Invitrogen (Carlsbad, CA, USA).

[0124] cells (1-2 x 10 ５Cells (1000 cells / well) were seeded into a 6-well plate. After 24 hours of incubation, the cells were transfected with these siRNAs (20 nmol / L) or Stealth RNAi (Invitrogen) as a negative control using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. After 6 hours of incubation, the Lipofectamine-containing medium was replaced with RPMI.

[0125] Each sample was tested in triplicate in three independent experiments.

[0126] 9. Quantitative Real-Time RT-PCR: Total intracellular RNA was extracted from NCI-H358 and HCI-H2122 cells treated with DMSO or the RAF / MEK dual inhibitor avutometinib (150 nmol / L) for 24 hours using Sepasol-RNA I Super G (Nacalai Tesque, Japan) according to the manufacturer's instructions. Complementary DNA (cDNA) was then synthesized from the extracted intracellular total RNA using the ReverTra Ace qPCR RT Master Mix kit (Toyobo, Osaka, Japan) according to the manufacturer's instructions.

[0127] Quantitative RT-PCR was performed using IGF1 (Hs01547656_m1) and GAPDH (Hs02758991_g1) cDNA and TaqMan probes (Applied Biosystems) using a QuantStudio 3 Real-Time PCR System (Thermoscience).

[0128] All gene expression levels were determined from the standard curve and calculated to determine the relative IGF1 / GAPDH ratio using the 2-ΔΔCT method.

[0129] Each sample was tested in triplicate in three independent experiments.

[0130] 10. Gene Set Variation Analysis (GSVA) ​​of Cell Lines Using Epithelial and Mesenchymal Gene Signature Sets RNA-seq gene expression data for nine NSCLC cell lines with KRAS mutations was obtained from the Cell Model Passports database (https: / / cellmodelpassports.sanger.ac.uk / ).

[0131] These data were log2-transformed from TMM-normalized count data. GSVA (see BMC Bioinformatics 2013;14:7 doi 10.1186 / 1471-2105-14-7) was used to calculate epithelial and mesenchymal scores. This scoring was implemented using the R package GSVA.

[0132] For each cell line (one expression data set), two scores were calculated using the epithelial gene list (288 genes) and the mesenchymal gene list (188 genes) (see EMBO Mol Med 2014;6(10):1279-93 doi 10.15252 / emmm.201404208). GSVA estimates the cumulative density function of each gene using all samples, ranks the genes across samples, and calculates a score of -1 or 1 for each set of genes using the Kolmogorov-Smirnov random walk statistic. The difference between the two scores was then calculated and used as the EMT score. Cell lines with a positive (high) EMT score are more likely to be mesenchymal, while cell lines with a negative (low) EMT score are more likely to be epithelial. Hierarchical clustering analysis was performed on the gene expression of the signature genes across all cell lines. The distance between clusters was measured using the Ward method and visualized in a heat map using Python and Python libraries (Pandas, Matplotlib, Seaborn).

[0133] 11. Quantification of cell cycle and apoptosis Cells (1-2 x 10 ５The 6-well plate on which the cells were seeded was incubated for 24 hours, and then the cells treated with each drug for 48 or 72 hours were harvested by trypsinization.

[0134] After washing twice with phosphate-buffered saline (PBS), the cells were suspended in PBS containing 0.1% Triton X-100 and 50 μg / mL propidium iodide, and the nuclei were stained.

[0135] Cells were analyzed using a BD Accuri C6 Flow Cytometer (BD Biosciences, San Jose, CA, USA) to quantify the extent of cells in G0-G1, S, G2-M, and sub-G1 phases, respectively.

[0136] [12. Scratch Assay] NCI-H358 cells and NCI-H2122 cells were cultured at 1 × 10 ５ The cells were seeded into 6-well plates at a concentration of 1000 cells / mL and cultured for 24 hours at 37° C. After the cell monolayers became nearly confluent, linear scratch wounds were made on each plate using a plastic pipette tip.

[0137] Images of the scratch wounds were taken before and 24 hours after treatment and the relative amount of migrating cells was assessed.

[0138] Three independent experiments were performed and evaluated individually.

[0139] 12. Cell Line-Derived Xenograft Mouse Test Female BALB / c nu / nu mice were obtained from Jackson Laboratory Japan Co., Ltd. (formerly Charles River Japan Co., Ltd., Japan). A suspension of NCI-H358 cells (4 x 10 ６ The mice were subcutaneously injected with 1000 guinea pigs (1000 / mouse) and maintained under pathogen-free conditions.

[0140] Tumors at the subcutaneous injection site were measured twice a week using calipers, and tumor volume was calculated using the following formula: 0.5 x length x width. ２ Tumor volume is approximately 70-120 mm ３At the time of reaching 28 days, mice were randomly assigned to one of four groups (n=6): 1) vehicle, 2) defactinib (10.0 mg / kg), 3) avutometinib (0.3 mg / kg), or 4) avutometinib + defactinib. Avutometinib and defactinib were dissolved in 20% hydroxypropyl-β-cyclodextrin / saline. The drugs were orally administered once daily until day 28, and the mice's weight and general condition were monitored daily.

[0141] The study using the mice was approved by the Kyoto Prefectural University of Medicine Institutional Review Board (approval number: M2021-38). In accordance with the institution's guidelines, animals were treated with an overdose of sodium pentobarbital before surgery.

[0142] 13. Quantification of Immunohistochemical Staining The tumors removed from the mice were fixed in formalin. After formalin fixation, they were embedded in paraffin to prepare paraffin-embedded tissue blocks. The prepared paraffin-embedded tissue blocks were cut into serial sections with a thickness of 4 μm, deparaffinized, and immunostained with Ki-67 (Clone MIB-1; Dako, Agilent, Santa Clara, CA, USA).

[0143] Furthermore, the degree of apoptosis was quantified by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method using the Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's instructions.

[0144] In addition to IHC staining, hematoxylin-eosin staining was performed to identify tumor cells. Five areas (0.1 mm ) containing the most positively stained cells were selected from each section. ２ Sections (100 mm x 100 mm) were randomly selected under a light microscope at 400x magnification. The percentage of positive cells for each immunohistochemical stain was assessed using ImageJ / Fiji.

[0145] 14. Statistical Analysis: The statistical significance of differences was analyzed using one-way analysis of variance and Spearman's rank correlation. Combination index (CI) values ​​were calculated using CompuSyn software (version 2.0; Biosoft, Cambridge, UK), and a combination was considered synergistic if its CI was <0.8. All statistical analyses were performed using GraphPad Prism Ver. 9.0 (GraphPad Software, Inc., San Diego, CA, USA), and a two-sided p value of <0.05 was considered statistically significant.

[0146] <Results> [1. The RAF / MEK dual inhibitor (avutometinib) activates FAK and AKT and reactivates ERK.]

[0147] Avutometinib is a RAF / MEK inhibitor that is expected to have a strong antitumor effect. However, when NCI-H358 cells, a human NSCLC cell line with a KRAS mutation, were treated with the RAF / MEK dual inhibitor (avutometinib) at a concentration of 1.5 μmol / L or higher for 72 hours, unexpectedly, approximately 45% of the cells remained viable (see Figure 6).

[0148] Therefore, to elucidate the mechanism by which the inhibitory effect of the RAF / MEK dual inhibitor (avutometinib) is circumvented, we used an RTK phosphorylation array kit to evaluate the differences in the phosphorylation status of molecules when NCI-H358 cells were treated with or without avutometinib for 72 hours.

[0149] As a result, it was found that treatment of NCI-H358 cells with the RAF / MEK dual inhibitor (avutometinib) for 72 hours increased the phosphorylation of focal adhesion kinase (FAK) (FIG. 1A).

[0150] This result suggests that in NCI-H358 cells, the FAK pathway may function as a bypass signal pathway for the blockade of the RAF / MEK pathway by avutometinib.

[0151] Further kinetic analysis confirmed that ERK phosphorylation was significantly inhibited by 3-hour treatment with the RAF / MEK dual inhibitor (avutometinib) in NCI-H2122 cells, as well as in NCI-H358 cells, but was reactivated at 72 hours (see Figure 1B).On the other hand, FAK and AKT phosphorylation steadily increased in these two cell lines after 72 hours of treatment with the RAF / MEK dual inhibitor (avutometinib) (see Figure 1B).

[0152] Next, FAK expression was silenced using siRNA, and the effect of knocking down FAK expression on cell viability of NCI-H358 and NCI-H2122 cells was evaluated.

[0153] The results confirmed that knockdown of FAK expression alone did not affect cell viability, whereas knockdown of FAK expression by treatment with the RAF / MEK dual inhibitor (avutometinib) significantly reduced the viability of both cancer cells (see Figure 1C).

[0154] The above test results suggest that in some NSCLC cells with KRAS mutations, even when the MAPK pathway signaling of the cells is inhibited by a RAF / MEK dual inhibitor, FAK expressed in the cells contributes in some way to the survival of the cells.

[0155] Therefore, we further knocked down FAK expression in these cells and confirmed that treatment with the RAF / MEK dual inhibitor (avutometinib) reduced the level of AKT phosphorylation in these cells, but not the level of ERK phosphorylation (see Figure 1D).

[0156] Previously, it has been reported that the FERM domain of FAK directly binds to the β subunit of IGF-1R, which contains the intracellular tyrosine kinase domain, and that IGF-1R activates FAK in human cells (Biochem Biophys Res Commun 2009; 388(2):301-5 doi 10.1016 / j.bbrc.2009.07.156 and Biochem Biophys Res Commun 2009; 387(1):36-41 doi 10.1016 / j.bbrc.2009.06.088). Another study using pancreatic cancer cells with a KRAS mutation showed that treatment with a MEK inhibitor increased IGF1 through the cMYC pathway, and further showed that IGF1 binds to and activates IGF-1R present on the cell membrane (Cell Rep 2017;18(9):2243-55 doi 10.1016 / j.celrep.2017.02.013).

[0157] Therefore, we treated NCI-H358 and NCI-H2122 cells with the RAF / MEK dual inhibitor (avutometinib). The results confirmed that the RAF / MEK dual inhibitor significantly increased IGF-1 mRNA levels in these cells (Figure 1E). Furthermore, we found that treatment with the RAF / MEK dual inhibitor (avutometinib) reduced cMYC expression and promoted IGF-1R phosphorylation over time (Figure 1F). Furthermore, knockdown of MYC expression increased IGF-1 mRNA levels and phosphorylation of IGF-1R, FAK, and AKT in both NCI-H358 and NCI-H2122 cells (Figures 7A and 7B).

[0158] 2. EMT status correlates with the synergistic effect of combined use of a FAK inhibitor and a RAF / MEK dual inhibitor.

[0159] First, we performed Western blot analysis of nine lung cancer cell lines with KRAS mutations, and obtained information on the type of point mutation in the KRAS oncogene and the LKB1 mutation status from the COSMIC database (see Figure 2A).

[0160] Next, the effect of monotherapy with the RAF / MEK dual inhibitor (avutometinib) on KRAS mutant NSCLC cells was confirmed (see Figure 8A).

[0161] Furthermore, detailed analysis of the efficacy of avutometinib alone revealed no significant differences between epithelial and mesenchymal phenotype cell lines, p = 0.456, or between KRAS G12C point mutation cell lines and non-G12C point mutation cell lines (p = 0.926). However, the difference between KRAS mutant NSCLC cell lines with mutant LKB1 and wild-type LKB1 (p = 0.005) confirmed that the antitumor effect of avutometinib alone was greater in KRAS mutant NSCLC cell lines with mutant LKB1 than in wild-type LKB1 (see also Figures 2A, 8B, 8C, and 8D).

[0162] Furthermore, we used CompuSyn to calculate the CI values ​​for combination therapy of the FAK inhibitor defactinib and the RAF / MEK dual inhibitor avutometinib. Among the nine NSCLC cell lines harboring KRAS mutations, NCI-H358, NCI-H2122, and NCI-H441 cells had CI values ​​of 0.7 or less, whereas the CI values ​​for the other cell lines were 0.7 to 1.2 (see Figure 2B).

[0163] We then investigated the antitumor effects of combined treatment with the RAF / MEK dual inhibitor avutometinib and the FAK inhibitor defactinib. We found that, regardless of the presence or absence of phosphorylated FAK expression, KRAS G12C mutation, or LKB1 mutation, the combined effect of the RAF / MEK dual inhibitor avutometinib and the FAK inhibitor defactinib was strong in epithelial cancer cells. However, no such combined effect was observed in mesenchymal cancer cells (see Figures 2A, B, and C).

[0164] From this, it can be understood that in combination therapy with a RAF / MEK dual inhibitor such as avutometinib and a FAK inhibitor such as defactinib, the state of epithelial-mesenchymal transition (EMT) to an epithelial phenotype may be an extremely useful biomarker for selecting appropriate patient groups.

[0165] Therefore, next, the EMT status of nine NSCLC cell lines with KRAS mutations was evaluated in more detail based on the mRNA expression levels of 416 EMT signature genes.

[0166] RNA-seq data were obtained from the database "Cell Model Passports." Hierarchical clustering analysis showed that the transcriptomes of the cell lines could be roughly divided into two groups, corresponding to epithelial or mesenchymal cell lines, based on the Western blotting results (see Figure 2D).

[0167] To assess the extent of EMT in these cells, we calculated a pair of scores summarizing the global transcriptional activity of epithelial and mesenchymal genes for each cell line. These scores were based on GSVA using a list of 228 epithelial genes and a list of 188 mesenchymal genes as signature gene sets (see EMBO Mol Med 2014;6(10):1279-93 doi 10.15252 / emmm.201404208).

[0168] Positive / high scores indicate high epithelial or mesenchymal characteristics, respectively. The results of calculating the scores revealed that the transcriptional status of the cell lines was biased toward the epithelial or mesenchymal regions in the epithelial-mesenchymal score space (see Figure 2E).

[0169] Therefore, we verified the classification of cell lines into epithelial or mesenchymal types based on Western blot results by analyzing the transcriptomes of 416 genes associated with EMT. Furthermore, we investigated the relationship between the CI value and the degree of EMT in these cell lines. To this end, we calculated the EMT score as a single score representing the degree of EMT, as described in the Materials and Methods section, and evaluated its correlation with the CI value.

[0170] As a result, as shown in Figure 2F, a strong positive correlation was confirmed between the two (R ２ = 0.7937). Cell lines with a positive / high EMT score were found to be strongly mesenchymal, while cell lines with a negative / low EMT score were found to be strongly epithelial. Therefore, cell lines with a CI value of 0.7 or higher were confirmed to be in a stronger mesenchymal state.

[0171] These results demonstrate that the biomarker for predicting the efficacy of combination therapy of a RAF / MEK dual inhibitor such as avutometinib with a FAK inhibitor such as defactinib is the EMT status, rather than the FAK phosphorylation status or KRAS point mutation type.

[0172] 3. Cancer cells exhibiting an epithelial phenotype were sensitized to RAF / MEK dual inhibitors by FAK inhibitors.

[0173] The combined effect of avutometinib, a RAF / MEK dual inhibitor, and defactinib, a FAK inhibitor, was evaluated in KRAS-mutated NSCLC cells.

[0174] As a result, the RAF / MEK dual inhibitor (avutometinib) alone did not significantly reduce cell viability in either epithelial or mesenchymal phenotypes of NSCLC cells harboring KRAS mutations (see Figure 8A).

[0175] However, it was confirmed that the combined use of the RAF / MEK dual inhibitor avutometinib and the FAK inhibitor defactinib significantly reduced the viability of NSCLC cells with KRAS mutations that exhibit an epithelial phenotype (see Figures 2B and 9A).

[0176] Furthermore, continuous treatment with the RAF / MEK dual inhibitor avutometinib and the FAK inhibitor defactinib for 15 days reduced the viability of KRAS-mutated NSCLC cells that exhibited an epithelial phenotype, but not of cells that exhibited a mesenchymal phenotype (see Figure 9B).

[0177] Next, the effects of drugs on the expression of various proteins were evaluated by Western blotting.

[0178] As a result, while treatment with avutometinib alone induced activation of FAK and AKT, 6 hours of combined treatment with the RAF / MEK dual inhibitor avutometinib and the FAK inhibitor defactinib suppressed the activation of FAK and AKT induced by treatment with avutometinib alone in cells exhibiting an epithelial phenotype (see Figure 3A), whereas no such suppression was observed in cells exhibiting a mesenchymal phenotype (see Figure 3A).

[0179] Next, we examined changes in the cell cycle and sub-G1 population caused by the combined use of the RAF / MEK dual inhibitor avutometinib and the FAK inhibitor defactinib.

[0180] As a result, sub-G1 population was induced in cells exhibiting an epithelial phenotype, but not in cells exhibiting a mesenchymal phenotype (see Figure 3B), and cell cycle progression was unaffected (see Figure 10). Furthermore, the addition of defactinib to avutometinib had no effect on cell migration in cells exhibiting an epithelial phenotype (see Figure 11).

[0181] Since the sub-G1 population was increased by combined treatment with the RAF / MEK dual inhibitor avutometinib and the FAK inhibitor defactinib, we evaluated the expression of the apoptosis-related Bim protein, and found that 24-hour combined treatment increased Bim protein more than avutometinib treatment alone (see Figure 3C).

[0182] Next, we evaluated the effect of combined treatment on the Bim-related protein FoxO3a. We found that avutometinib alone reduced FoxO3a phosphorylation in epithelial cells (Figure 3C). Furthermore, we confirmed that combined treatment with the RAF / MEK dual inhibitor avutometinib and the FAK inhibitor defactinib for 72 hours suppressed FAK and AKT activation induced by avutometinib alone in epithelial cells (Figure 3D).

[0183] Consistent with the flow cytometry results, 72-hour combined treatment with avutometinib and defactinib increased the cleavage of caspase-3 and PARP, markers of apoptotic cells, in cells exhibiting an epithelial phenotype compared with treatment with avutometinib alone (see Figure 3D).

[0184] Therefore, it is understood that the use of a FAK inhibitor in combination with a RAF / MEK dual inhibitor may induce apoptosis in epithelial phenotype KRAS mutant NSCLC cells via the FoxO3a-Bim pathway, regardless of the type of KRAS point mutation.

[0185] 4. The antitumor effect of combined treatment with a RAF / MEK dual inhibitor and a FAK inhibitor was reduced by the induction of EMT.

[0186] EMT, which plays an important role in cancer progression, has been reported to be induced by TGF-β (Cell Res 2009;19(2):156-72 doi 10.1038 / cr.2009.5). Therefore, we decided to evaluate the effect of combined treatment with avutometinib, a RAF / MEK dual inhibitor, and defactinib, a FAK inhibitor, on inducing a mesenchymal phenotype from epithelial to mesenchymal phenotype using TGF-β.

[0187] First, NCI-H358 cells were incubated with TGF-β1 for 2 weeks to induce EMT.

[0188] The results showed that EMT-induced cells (H358-TGF-β cells) exhibited spindle-shaped cell morphology, loss of polarity, and decreased cell-cell adhesion compared to parental cells (see Figure 4A). Furthermore, H358-TGF-β cells exhibited decreased E-cadherin expression and increased ZEB1 and vimentin expression compared to parental cells (see Figure 4B). Furthermore, TGF-β-induced EMT slowed cell growth and increased the proportion of cells in the G0-G1 phase (see Figures 4C and 4D).

[0189] Next, the CI values ​​of avutometinib + defactinib were calculated for both NCI-H358 cells and H358-TGF-β cells. The CI value for H358-TGF-β cells was significantly higher than that of the parent NCI-H358 cells (p=0.012) (see Figure 4E).

[0190] Thus, EMT appears to reduce the efficacy of the combination of avutometinib and defactinib. Treatment with avutometinib alone activated FAK in NCI-H358 cells, but not in H358-TGF-β cells (Figure 4F). However, 6-hour combined treatment with avutometinib and defactinib inhibited AKT phosphorylation induced by avutometinib alone in NCI-H358 cells, but not in H358-TGF-β cells (Figure 4F). Furthermore, although the steady-state level of AKT phosphorylation was higher in H358-TGF-β cells than in NCI-H358 cells, there was no difference in the inhibitory effect of avutometinib on phosphorylated ERK between NCI-H358 and H358-TGF-β cells (see Figure 4F).

[0191] These results suggest that the additive effect of defactinib on epithelial KRAS mutant cell lines treated with avutometinib is due to the contribution of the FAK-AKT mediated feedback mechanism when these cells are treated with avutometinib.

[0192] 5. Combination therapy of a RAF / MEK dual inhibitor and a FAK inhibitor sustained tumor regression in vivo.

[0193] In vivo experiments were carried out using NCI-H358 cells to further evaluate the combined effect of the RAF / MEK dual inhibitor avutometinib and the FAK inhibitor defactinib.

[0194] Mice were gavaged with avutometinib alone, defactinib alone, or the combination of these drugs at regular intervals daily for up to 28 days.

[0195] As a result, it was confirmed that both treatment with the RAF / MEK dual inhibitor avutometinib alone and treatment with the RAF / MEK dual inhibitor avutometinib in combination with the FAK inhibitor defactinib caused tumor regression during treatment.

[0196] After treatment was discontinued, tumors in the avutometinib monotherapy group rapidly regrew. However, surprisingly, tumors in the avutometinib and defactinib combination therapy group remained regressed. The combination therapy even caused tumor regression in two of the mice (see Figure 12A).

[0197] On the other hand, it was confirmed that treatment with the FAK inhibitor defactinib alone had little effect on tumor regression (see FIG. 5A).

[0198] During these treatment periods, no obvious side effects, including weight loss, were observed (see Figure 12B).

[0199] Furthermore, the size and weight of the excised tumors were measured at the end of the experiment (day 49), and the effects of avutometinib and defactinib were evaluated.

[0200] As a result, tumor regression was observed to a certain extent with avutometinib alone, while significantly stronger tumor regression was observed with the combination therapy of avutometinib and defactinib (see Figures 5A and 5B). The mean tumor weight in the combination therapy group was significantly lower than that in the avutometinib monotherapy group (p = 0.029) (see Figure 5C).

[0201] On the other hand, no tumor regression was observed with the FAK inhibitor defactinib alone, and no reduction in mean tumor weight was observed (see FIG. 5C).

[0202] To further investigate the molecular changes induced by avutometinib and defactinib, tumors were excised from mice administered these drugs for 4 days, and Western blotting and immunohistochemical staining were performed.

[0203] Similar to the in vitro results, in vivo experiments also showed that the combined use of defactinib and avutometinib suppressed avutometinib-activated FAK and AKT phosphorylation (Figure 5D). Furthermore, the number of Ki-67-positive proliferating tumor cells was significantly reduced in the combination group compared to the avutometinib-only group (p<0.001) (Figures 5E and 5F).

[0204] On the other hand, the number of TUNEL-positive tumor cells, i.e., apoptotic cells, was significantly higher in the combination group than in the avutometinib alone group (p<0.001) (see Figures 5E and 5F).

[0205] These in vivo analyses revealed that combination therapy with the RAF / MEK dual inhibitor avutometinib and the FAK inhibitor defactinib inhibited the AKT and MAPK pathways, induced apoptosis, and led to regression of KRAS-mutated NSCLC tumors with an epithelial phenotype (see Figure 13).

[0206] <Discussion> Although cancer cells carrying driver genes are effectively treated with molecular targeted drugs targeting those genes, they survive through mechanisms such as bypass signaling and activation of EMT, and are believed to ultimately acquire resistance to molecular targeted therapy (Cancer Discov 2016; 6(7): 754-69 doi 10.1158 / 2159-8290.Cd-15-1377., J Clin Invest 2011; 121(11): 4311-21 doi 10.1172 / jci57909).

[0207] The results of the study presented by the present inventors herein demonstrate that the effect of avutometinib on NSCLC cells harboring KRAS mutations differs depending on the cell line (see Figure 8A). In fact, avutometinib was more effective in lung cancer cell lines harboring LKB1 mutations than in lung cancer cell lines lacking LKB1 mutations (see Figures 2A and 8D). Trametinib has also been shown to be more effective in lung cancer cell lines harboring LKB1 mutations than in cell lines lacking LKB1 mutations (Cancer Res 2017;77(1):153-63 doi 10.1158 / 0008-5472.Can-16-1639). However, while MEK inhibitors have been reported to be more effective in epithelial cells ( Mol Cancer Ther 2012;11(3):720-9 doi 10.1158 / 1535-7163.Mct-11-0505.), our results showed no significant difference in the effect of avutometinib alone between epithelial and mesenchymal cells (see Figures 8A and 8B).

[0208] In a phase I trial, 30% of patients with KRAS-mutant NSCLC treated with avutometinib experienced objective responses. All patients experienced a low incidence of adverse events lasting 6 months or longer (Lancet Oncol 2020;21(11):1478-88 doi 10.1016 / s1470-2045(20)30464-2.). Although the efficacy of avutometinib monotherapy in patients with KRAS-mutant NSCLC was higher than that of other MEK inhibitor monotherapy, developing effective combination strategies will be important. Indeed, combination therapy of avutometinib and the FAK inhibitor defactinib has demonstrated clinical efficacy in NSCLC and LGSOC with KRAS mutations (Cancer Res 2021;81(13):suppl (July 01, 2021) CT019 doi 10.1158 / 1538-7445.AM2021-CT019., Journal of Clinical Oncology 2023;41(16):suppl (June 01, 2023) #5515 doi 10.1200 / JCO.2023.41.16_suppl.5515., Journal of Clinical Oncology 2023;41(16):suppl (June 01, 2023) #9100 doi 10.1200 / JCO. 2023.41.16_suppl. 9100. ).

[0209] FAK is a cytosolic non-receptor protein kinase belonging to the FAK family and was first identified in 1992 from Balb / c 3T3 cells using a homology-based cDNA cloning approach (Proc Natl Acad Sci U S A 1992;89(18):8487-91 doi 10.1073 / pnas.89.18.8487, Proc Natl Acad Sci U S A 1992;89(11):5192-6 doi 10.1073 / pnas.89.11.5192). FAK is activated by various transmembrane receptors, including integrins, growth factor receptors, and cytokine receptors (Curr Opin Cell Biol 2006;18(5):516-23 doi 10.1016 / j.ceb.2006.08.011), and the functional role of FAK is subsequently involved in cell proliferation, migration, and survival (IUBMB Life 2002;53(2):115-9 doi 10.1080 / 15216540211470).

[0210] Although the FAK inhibitor defactinib was indeed effective against NSCLC cells with KRAS mutations compared with treatment of NSCLC cells with wild-type KRAS (Clin Cancer Res 2016;22(23):5851-63 doi 10.1158 / 1078-0432.Ccr-15-2603), defactinib monotherapy was not sufficiently effective in patients with NSCLC with KRAS mutations (Lung Cancer 2020;139:60-7 doi 10.1016 / j.lungcan.2019.10.033). Therefore, it was necessary to identify an appropriate agent to be used in combination with defactinib for patients with NSCLC with KRAS mutations.

[0211] FAK is overexpressed in various cancers, including lung cancer, and is known to promote their progression, metastasis, and drug resistance (Biomed Res Int 2015;2015:690690 doi 10.1155 / 2015 / 690690., J Histochem Cytochem 2015;63(2):114-28 doi 10.1369 / 0022155414561498.). Furthermore, activation of FAK signaling in EGFR-mutated NSCLC cells induces acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs), and combination therapy of EGFR-TKIs and FAK inhibitors is effective against these cells (J Hematol Oncol 2020; 13(1): 169 doi 10.1186 / s13045-020-01009-7).

[0212] However, no effective anticancer therapeutic strategy was known for NSCLC cells with KRAS mutations that activate FAK when administered with a RAF / MEK dual inhibitor, or for cancer patients with such cells.

[0213] Under these circumstances, the inventors of the present application have found, through various trial and error efforts, that combined therapy with a RAF / MEK dual inhibitor and a FAK inhibitor is effective against KRAS mutant NSCLC cells in which FAK is activated when a RAF / MEK dual inhibitor is administered.

[0214] The description herein by the present inventors is the first to report that the efficacy of combined therapy of a RAF / MEK dual inhibitor and a FAK inhibitor against cancer has been evaluated and that this remarkable effect has been found.

[0215] Several signaling pathways, such as the TGF-β signaling pathway, the Wnt / β-catenin signaling pathway, and the integrin signaling pathway, play important roles in inducing EMT (Sci Signal 2014; 7(344):re8 doi 10.1126 / scisignal.2005189). Upon induction of EMT, transcription factors such as snail family transcriptional repressor (Snail), zinc finger E-box binding homeobox (ZEB), and the twist family bHLH transcription (Twist) suppress epithelial gene expression and induce mesenchymal gene expression (Cell 2011;145(1):162.e1 doi 10.1016 / j.cell.2011.03.029).

[0216] Treatment of KRAS-mutated NSCLC cells with MEK inhibitors activates RTKs as a physiological negative feedback loop, but several reports have shown that this feedback loop is mediated by different bypass signals in epithelial and mesenchymal phenotypes: epithelial cells activate IGF-1R and ERBB3 via c-MYC, whereas mesenchymal phenotype cells activate FRS2 via SPRY4 (Cancer Discov 2016;6(7):754-69 doi 10.1158 / 2159-8290.Cd-15-1377., Cell Rep 2014;7(1):86-93 doi 10.1016 / j.celrep.2014.02.045.). In KRAS-mutated pancreatic ductal carcinoma cells, KRAS inhibition reduced c-MYC expression, increased the expression of a set of genes related to the IGF-1 pathway in a gene array-based transcriptome analysis, and increased IGF-1R phosphorylation (Cell Rep 2017;18(9):2243-55 doi 10.1016 / j.celrep.2017.02.013). The FERM domain of FAK directly binds to the β subunit of IGF-1R, which contains the intracellular tyrosine kinase domain, and IGF-1R activates FAK in human cells (Biochem Biophys Res Commun 2009; 388(2): 301-5 doi 10.1016 / j.bbrc.2009.07.156., Biochem Biophys Res Commun 2009; 387(1): 36-41 doi 10.1016 / j.bbrc.2009.06.088.).

[0217] Therefore, our results demonstrate for the first time that the combination of the RAF / MEK dual inhibitor avutometinib and the FAK inhibitor defactinib is active against KRAS mutant NSCLC cells with an epithelial phenotype via the cMYC-IGF-1R-FAK pathway (Fig. 13). Furthermore, AKT activation by long-term exposure to TGF-β (Fig. 4F) is presumably responsible for the induction of EMT.

[0218] Previous reports have shown that the MEK inhibitor trametinib increases total Bim, a pro-apoptotic BH-3-only protein, in KRAS-mutant cancer cells (Cell Rep 2014;7(1):86-93 doi 10.1016 / j.celrep.2014.02.045., Mol Cancer Ther 2018;17(1):17-25 doi 10.1158 / 1535-7163.Mct-17-0146.). Similar to previous reports on MEK inhibitors, we showed that trametinib induces Bim expression in KRAS-mutant NSCLC cells with an epithelial phenotype (Figure 3C). Furthermore, we demonstrated that the combination of avutometinib and defactinib further increased Bim expression (see Figures 3C and 5D). To our knowledge, there have been no reports of increased Bim expression due to the administration of a FAK inhibitor. In the combination group, tumor regrowth was not observed even 3 weeks after discontinuation of administration, and the tumor-suppressing effect was sustained (see Figure 5A). This delay in tumor regrowth is thought to be due to the more potent apoptosis-inducing ability of both drugs.

[0219] Suppression of the activated AKT pathway activates the downstream FoxO3a pathway, restoring the transcriptional induction of Bim by FoxO3a (Blood 2010;115(12):2430-40 doi 10.1182 / blood-2009-07-232801., J Biol Chem 2003;278(50):49795-805 doi 10.1074 / jbc.M309523200.). In this study, as shown in Figure 3C, the combined use of defactinib and avutometinib also reduced phosphorylated FoxO3a, suggesting that Bim expression may be further elevated via this pathway. Therefore, by further inducing Bim expression, the combination therapy of avutometinib and defactinib was found to have a strong antitumor effect by inducing apoptosis in NSCLC cells with KRAS mutations that exhibit an epithelial phenotype (see Figure 13).

[0220] Several clinical trials have been conducted on MEK inhibitors in combination with other inhibitors in patients with KRAS-mutated NSCLC. Combination therapy with the pan-HER family tyrosine kinase inhibitor afatinib, which suppresses ERBB3 activation, and the MEK inhibitor selumetinib was a promising strategy that focused on feedback mechanisms. However, the efficacy of these combination therapies was limited due to adverse effects (Oncologist 2021;26(4):290-e545 doi 10.1002 / onco.13631).

[0221] On the other hand, the combination therapy of avutometinib and defactinib was reported to be generally well tolerated in a phase I trial (NCT03875820) (Cancer Res 2021; 81 (13): suppl (July 01, 2021) CT019 doi 10.1158 / 1538-7445.AM2021-CT019. and Cancer Res 2022; 82 (12): suppl (June 15, 2022) #3476 doi 10.1158 / 1538-7445.AM2022-3476.). Therefore, this combination therapy is promising and is currently undergoing clinical trials in patients with several cancers, including KRAS-mutated NSCLC and low-grade serous ovarian cancer (LGSOC). In this phase I / II trial, the objective response rate to the combination therapy was 15.0% in patients with KRAS-mutated NSCLC and 44.8% in patients with LGSOC (Cancer Res 2021;81(13):suppl (July 01, 2021) CT019 doi 10.1158 / 1538-7445.AM2021-CT019., Journal of Clinical Oncology 2023;41(16):suppl (June 01, 2023) #5515 doi 10.1200 / JCO.2023.41.16_suppl.5515., Cancer Res 2022;82(12):suppl (June 15, 2022) #3476 doi 10.1158 / 1538-7445. AM2022-3476. , Annals of Oncology 2021;32(5):suppl (Sep 01, 2021) #799 doi 10.1016 / j. annonc. 2021.08.1168. ).Two additional phase II trials are underway to evaluate the efficacy of avutometinib and defactinib combination therapy in patients with KRAS-mutated NSCLC and LGSOC (Cancer Res 2021;81(13):suppl (July 01, 2021) CT019 doi 10.1158 / 1538-7445.AM2021-CT019., Journal of Clinical Oncology 2023;41(16):suppl (June 01, 2023) #9100 doi 10.1200 / JCO.2023.41.16_suppl.9100., Future Oncol 2022;18(16):1907-15 doi 10.2217 / fon-2021-1582. There is an urgent need for biomarkers useful for this combination therapy in patients with KRAS-mutated NSCLC. As shown in Figure 2E, EMT status may be a viable biomarker.

[0222] For anti-cancer treatments using molecular targeted drugs, it is desirable to set a patient group that does not acquire resistance to the molecular targeted drug as the appropriate treatment target and to apply the drug to patients included in this group. There is a strong demand for the discovery of useful biomarkers to identify appropriate patient groups. The present inventors have demonstrated through test results that the FAK inhibitor defactinib suppresses bypass signals activated by treatment with the RAF / MEK inhibitor avutometinib in KRAS-mutated NSCLC cells with an epithelial phenotype, that the combined therapy effectively induces the FoxO3a and Bim pathways mediated by intrinsic apoptosis, and that the EMT state in cancers with driver mutations could be an actual biomarker.

[0223] Summary: As described above, the FAK inhibitor defactinib suppressed activated bypass signaling in NSCLC cells with KRAS mutations that exhibit an epithelial phenotype when combined with the RAF / MEK dual inhibitor avutometinib. Furthermore, we demonstrated that combination therapy of a FAK inhibitor and a RAF / MEK dual inhibitor, such as the combination of defactinib and avutometinib, is effective by inducing intrinsic apoptosis via the FoxO3a and Bim pathways. These results demonstrate that the EMT state, i.e., a state in which the expression level of epithelial biomarkers is higher than that of mesenchymal biomarkers, can be an actual biomarker for combination therapy. Further clinical studies and an increased number of cases are needed to confirm the usefulness of EMT as a biomarker for combination therapy.

[0224] According to the present invention, effective amounts of a FAK inhibitor and a RAF / MEK dual inhibitor are provided and administered to a cancer patient in whom the expression level of an epithelial biomarker in an isolated tumor sample has been confirmed to be higher than that of a mesenchymal biomarker. This provides a novel means for achieving a significant anticancer effect, exhibiting a high level of antitumor effect that cannot be achieved by administering a RAF / MEK dual inhibitor or a FAK inhibitor alone, while also maintaining a certain level of growth-suppressing effect after drug withdrawal. Furthermore, according to the present invention, it is possible to predict and demonstrate that when an isolated patient-derived tumor sample expresses an epithelial biomarker at a higher level than a mesenchymal biomarker, the sensitivity of the FAK inhibitor and the RAF / MEK dual inhibitor to tumor cell growth inhibition in cancer patients is high, and to demonstrate that the combination of a FAK inhibitor and a RAF / MEK dual inhibitor is suitable for anticancer treatment. On the other hand, if the epithelial biomarker is expressed at a lower level than the mesenchymal biomarker, it is possible to predict and indicate low sensitivity to a FAK inhibitor and a RAF / MEK dual inhibitor, and to indicate that the cancer is not suitable for anti-cancer treatment with a combination of a FAK inhibitor and a RAF / MEK dual inhibitor. For patients with cancers that are not suitable for anti-cancer treatment with a combination of a FAK inhibitor and a RAF / MEK dual inhibitor, this avoids the unnecessary prescription of molecular targeted drugs and the unintended acquisition of treatment resistance, and provides an opportunity to select an alternative treatment option earlier, thereby significantly improving the success rate of cancer treatment and cure and significantly improving the patient's quality of life. In this way, it becomes possible to formulate and implement more appropriate treatment strategies.

Claims

1. A pharmaceutical composition comprising as active ingredients a FAK inhibitor and a RAF / MEK dual inhibitor for use in the treatment of a patient suffering from cancer, said pharmaceutical composition for administration to said patient in whom an epithelial biomarker expression level has been identified as being higher than a mesenchymal biomarker expression level in a tumor sample isolated from said patient.

2. A pharmaceutical composition comprising as active ingredients a FAK inhibitor and a RAF / MEK dual inhibitor for use in the treatment of a patient suffering from cancer, said pharmaceutical composition being for administration to said patient in whom it has been determined that in a tumor sample isolated from said patient, the epithelial biomarker expression level is higher than the mesenchymal biomarker expression level, and in whom the RAF / MEK dual inhibitor as a single agent is ineffective.

3. A pharmaceutical composition comprising as active ingredients a FAK inhibitor and a RAF / MEK dual inhibitor for use in the treatment of a patient suffering from cancer, said pharmaceutical composition being for administration to said patient in whom it has been confirmed that in a tumor sample isolated from said patient, the expression level of an epithelial biomarker is higher than the expression level of a mesenchymal biomarker, and that the proliferation inhibitory effect of a single RAF / MEK dual inhibitor after discontinuation of said treatment is low.

4. A pharmaceutical composition comprising as an active ingredient a RAF / MEK dual inhibitor in combination with a FAK inhibitor for use in the treatment of a patient suffering from cancer, said pharmaceutical composition being for administration to said patient in whom an epithelial biomarker expression level has been identified as being higher than a mesenchymal biomarker expression level in a tumor sample isolated from said patient, said pharmaceutical composition being for use in combination with a FAK inhibitor.

5. A pharmaceutical composition comprising as an active ingredient a RAF / MEK dual inhibitor in combination with a FAK inhibitor for use in the treatment of a patient suffering from cancer, said pharmaceutical composition being for administration to said patient in whom it has been determined that in a tumor sample isolated from said patient, the epithelial biomarker expression level is higher than the mesenchymal biomarker expression level, and in whom the RAF / MEK dual inhibitor as a single agent is ineffective.

6. A pharmaceutical composition comprising as an active ingredient a RAF / MEK dual inhibitor in combination with a FAK inhibitor for use in the treatment of a patient suffering from cancer, said pharmaceutical composition being for administration to said patient in whom it has been confirmed that in a tumor sample isolated from said patient, the epithelial biomarker expression level is higher than the mesenchymal biomarker expression level and that the proliferation inhibitory effect of the RAF / MEK dual inhibitor alone after discontinuation of the drug is low, in combination with the FAK inhibitor.

7. A pharmaceutical composition comprising as an active ingredient a FAK inhibitor in combination with a RAF / MEK dual inhibitor for use in the treatment of a patient suffering from cancer, said pharmaceutical composition being in combination with a RAF / MEK dual inhibitor for administration to said patient in whom an epithelial biomarker expression level in a tumor sample isolated from said patient has been identified as being in a state where the epithelial biomarker expression level is higher than the mesenchymal biomarker expression level.

8. A pharmaceutical composition comprising as an active ingredient a FAK inhibitor in combination with a RAF / MEK dual inhibitor for use in the treatment of a patient suffering from cancer, said pharmaceutical composition being for administration to said patient in whom epithelial biomarker expression levels are higher than mesenchymal biomarker expression levels in a tumor sample isolated from said patient, and in whom the RAF / MEK dual inhibitor as a single agent has been shown to be ineffective.

9. A pharmaceutical composition comprising as an active ingredient a FAK inhibitor for use in the treatment of a patient suffering from cancer, in combination with a RAF / MEK dual inhibitor, said pharmaceutical composition being for administration to said patient in whom it has been confirmed that in a tumor sample isolated from said patient, the epithelial biomarker expression level is higher than the mesenchymal biomarker expression level and that the proliferation inhibitory effect of the RAF / MEK dual inhibitor alone after discontinuation of the drug is low.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the epithelial biomarker is selected from E-cadherin, Brk, gamma-catenin, alpha1-catenin, alpha2-catenin, alpha3-catenin, keratin 8, keratin 18, connexin 31, plakophilin 3, stratafin 1, laminin alpha-5 and ST14.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the mesenchymal biomarker is selected from vimentin, fibronectin, fibrillin-1, fibrillin-2, collagen alpha-2(IV), collagen alpha-2(V), LOXL1, nidogen, C11orf9, tenascin, N-cadherin and embryonic EDB+fibronectin, tubulin alpha-3 and epimorphin.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the RAF / MEK dual inhibitor is avutometinib or a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate of said compound or salt.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the FAK inhibitor is defactinib or a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate of said compound or salt.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the cancer is a cancer with a RAS mutation.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the cancer is a cancer having a KRAS mutation.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the cancer is selected from the group consisting of breast cancer, liver cancer, lung cancer including small cell lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, prostate cancer, malignant lymphoma and leukemia.

17. Use of an effective amount of a FAK inhibitor and an effective amount of a RAF / MEK dual inhibitor in the manufacture of a medicament for use in treating a patient suffering from cancer, wherein the medicament is for administration to the patient identified as having a condition in which an epithelial biomarker expression level is higher than a mesenchymal biomarker expression level in a tumor sample isolated from the patient.

18. Use of an effective amount of a FAK inhibitor and an effective amount of a RAF / MEK dual inhibitor in the manufacture of a medicament for use in treating a patient suffering from cancer, wherein the medicament is for administration to the patient in whom an epithelial biomarker expression level is higher than a mesenchymal biomarker expression level in a tumor sample isolated from the patient, and in whom the RAF / MEK dual inhibitor as a single agent has been identified as being ineffective.

19. Use of an effective amount of a FAK inhibitor and an effective amount of a RAF / MEK dual inhibitor in the manufacture of a medicament for use in treating a patient suffering from cancer, wherein the medicament is for administration to the patient in whom it has been determined that a tumor sample isolated from the patient has a higher epithelial biomarker expression level than a mesenchymal biomarker expression level and that the RAF / MEK dual inhibitor alone has a low antiproliferative effect after withdrawal.

20. Use of an effective amount of a RAF / MEK dual inhibitor in combination with a FAK inhibitor in the manufacture of a medicament for use in treating a patient suffering from cancer, wherein the medicament is for administration to the patient identified as having a condition in which an epithelial biomarker expression level is higher than a mesenchymal biomarker expression level in a tumor sample isolated from the patient, in combination with a FAK inhibitor.

21. Use of an effective amount of a RAF / MEK dual inhibitor in combination with a FAK inhibitor in the manufacture of a medicament for use in the treatment of a patient suffering from cancer, wherein the medicament is for administration to the patient in whom an epithelial biomarker expression level is higher than a mesenchymal biomarker expression level in a tumor sample isolated from the patient, and in whom the RAF / MEK dual inhibitor as a single agent has been identified as being ineffective.

22. Use of an effective amount of a RAF / MEK dual inhibitor in combination with a FAK inhibitor in the manufacture of a medicament for use in the treatment of a patient suffering from cancer, wherein the medicament is for administration to the patient in whom it has been determined that a tumor sample isolated from the patient has a higher epithelial biomarker expression level than a mesenchymal biomarker expression level and that the RAF / MEK dual inhibitor alone has a low antiproliferative effect after withdrawal of the drug, in combination with a FAK inhibitor.

23. Use of an effective amount of a FAK inhibitor in combination with a RAF / MEK dual inhibitor in the manufacture of a medicament for use in treating a patient suffering from cancer, wherein the medicament is for administration to the patient identified as having a condition in which an epithelial biomarker expression level is higher than a mesenchymal biomarker expression level in a tumor sample isolated from the patient.

24. Use of an effective amount of a FAK inhibitor in combination with a RAF / MEK dual inhibitor in the manufacture of a medicament for use in the treatment of a patient suffering from cancer, wherein the medicament is for administration to the patient in whom an epithelial biomarker expression level is higher than a mesenchymal biomarker expression level in a tumor sample isolated from the patient, and in whom the RAF / MEK dual inhibitor as a single agent has been identified as being ineffective.

25. Use of an effective amount of a FAK inhibitor active ingredient, in combination with a RAF / MEK dual inhibitor, in the manufacture of a medicament for use in the treatment of a patient suffering from cancer, wherein the medicament is for administration to a patient in whom epithelial biomarker expression levels are higher than mesenchymal biomarker expression levels in a tumor sample isolated from the patient, and in whom a RAF / MEK dual inhibitor alone has been identified as having low antiproliferative effects following withdrawal of the drug.

26. The use according to any one of claims 17 to 25, wherein the RAF / MEK dual inhibitor is avutometinib or a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate of said compound or salt.

27. The use according to any one of claims 17 to 26, wherein the FAK inhibitor is defactinib or a pharma- ceutically acceptable salt thereof, or a pharma- ceutically acceptable solvate of said compound or salt.

28. The use according to any one of claims 17 to 27, wherein the cancer is a cancer with a RAS mutation.

29. The use according to any one of claims 17 to 28, wherein the cancer is a cancer with a KRAS mutation.

30. The use according to any one of claims 17 to 29, wherein the cancer is selected from the group consisting of breast cancer, liver cancer, lung cancer including small cell lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, prostate cancer, malignant lymphoma and leukemia.

31. A method for predicting the sensitivity of tumor cell proliferation inhibition by a FAK inhibitor and a RAF / MEK dual inhibitor in a patient suffering from cancer, comprising the steps of: evaluating the expression levels of an epithelial biomarker and a mesenchymal biomarker expressed in a tumor sample isolated from a patient suffering from cancer, respectively; and predicting and indicating a high sensitivity of tumor cell proliferation inhibition by a FAK inhibitor and a RAF / MEK dual inhibitor if the epithelial biomarker is at a higher expression level than the mesenchymal biomarker, and predicting and indicating a low sensitivity of tumor cell proliferation to inhibition by a FAK inhibitor and a RAF / MEK dual inhibitor if the epithelial biomarker is at a lower expression level than the mesenchymal biomarker.

32. A method for predicting the sensitivity of a patient suffering from cancer to tumor cell proliferation inhibition by a FAK inhibitor and a RAF / MEK dual inhibitor and suggesting the suitability of a combination therapy with a FAK inhibitor and a RAF / MEK dual inhibitor, the method comprising the steps of: evaluating the expression levels of epithelial biomarkers and mesenchymal biomarkers expressed in a tumor sample isolated from a patient suffering from cancer; predicting high sensitivity of tumor cell proliferation inhibition by a FAK inhibitor and a RAF / MEK dual inhibitor when the epithelial biomarkers are at a higher expression level than the mesenchymal biomarkers, and suggesting suitability for anti-cancer treatment with a combination of a FAK inhibitor and a RAF / MEK dual inhibitor; and / or predicting low sensitivity of tumor cell proliferation to inhibition by a FAK inhibitor and a RAF / MEK dual inhibitor when the epithelial biomarkers are at a lower expression level than the mesenchymal biomarkers, and suggesting unsuitability for anti-cancer treatment with a combination of a FAK inhibitor and a RAF / MEK dual inhibitor.

33. The method of claim 31 or 32, wherein the epithelial biomarker is selected from E-cadherin, Brk, gamma-catenin, alpha1-catenin, alpha2-catenin, alpha3-catenin, keratin 8, keratin 18, connexin 31, plakophilin 3, stratafin 1, laminin alpha-5, and ST14.

34. The method of any one of claims 31 to 33, wherein the mesenchymal biomarkers are selected from vimentin, fibronectin, fibrillin-1, fibrillin-2, collagen alpha-2(IV), collagen alpha-2(V), LOXL1, nidogen, C11orf9, tenascin, N-cadherin and embryonic EDB+fibronectin, tubulin alpha-3 and epimorphin.

35. The method according to any one of claims 31 to 34, wherein the cancer is a cancer with a RAS mutation.

36. The method according to any one of claims 31 to 35, wherein the cancer is a cancer with a KRAS mutation.

37. The method according to any one of claims 31 to 36, wherein the cancer is selected from the group consisting of breast cancer, liver cancer, lung cancer including small cell lung cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, prostate cancer, malignant lymphoma and leukemia.

38. A kit for predicting or monitoring sensitivity to tumor cell growth inhibition by a FAK inhibitor and a RAF / MEK dual inhibitor in a patient suffering from cancer, and / or for suggesting or monitoring the suitability of anti-cancer treatment in a patient suffering from cancer using a combination of a FAK inhibitor and a RAF / MEK dual inhibitor, by the method of claim 31 or claim 32, comprising an anti-epithelial biomarker antibody and / or a fragment thereof, and an anti-mesenchymal biomarker antibody and / or a fragment thereof.

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