Methods for treating cancer having an EGFR activating mutation

Abstract

The present disclosure provides methods for treating cancer in patients determined to have an EGFR activating mutation by administering a CDK inhibitor and / or a SAC factor inhibitor.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 642,472, filed Mar. 13, 2018, which is hereby incorporated by reference in its entirety.

[0002] This invention was made with government support under grant number CA190628 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] 1. Field The present invention relates generally to the fields of cancer biology and medicine. More specifically, the present invention relates to methods for treating cancers having activating EGFR mutations.

Background Art

[0004] 2. Description of Related Art EGFR-mutant NSCLC patients initially respond to EGFR-targeted therapies, but resistant disease invariably emerges. In nearly half of the resistant cases, the tumors lack EGFR secondary mutations such as T790M and are resistant to second- and third-generation EGFR tyrosine kinase inhibitors (TKIs). Identifying treatment regimens that are effective against cancers that are resistant to TKIs, such as T790M-negative resistance, remains a major clinical challenge.

Summary of the Invention

[0005] Summary In some embodiments, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of a cyclin-dependent kinase (CDK) inhibitor and / or a spindle assembly checkpoint (SAC) factor inhibitor, wherein the subject is determined to have one or more activating EGFR mutations. In some aspects, the subject is determined to have two, three, or four activating EGFR mutations. In certain aspects, the subject is human.

[0006] In some embodiments, one or more EGFR activating mutations are selected from the group consisting of L858R, exon 19 deletions, and exon 20 insertions. In certain embodiments, the exon 19 deletion is an in-frame deletion between L747 and L749, such as, for example, an E746 - A750 deletion, an L747 - E749 deletion, or an A750P. In certain embodiments, the exon 20 insertion is N771Del Ins FH. In some embodiments, the EGFR mutation is G719S, G719A, S768I, E709A, R776H, or L861Q.

[0007] In certain embodiments, a subject is determined to have an EGFR activating mutation by analyzing a patient - derived genomic sample. In some embodiments, the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. In some embodiments, the presence of an EGFR activating mutation is determined by nucleic acid sequencing or PCR analysis.

[0008] In some embodiments, the CDK inhibitor is further defined as a CDK2 inhibitor, a CDK5 inhibitor, a CDK1 inhibitor, or a CDK9 inhibitor. In certain embodiments, the CDK inhibitor is dinaciclib (SCH727965), alvocidib (flavopiridol), roscovitine (seliciclib, CYC202), SNS - 032 (BMS - 387032), LY2857785, ADZ5438, BMS - 265246, NU6027, LDC000067, wogonin, or RO - 3306. In certain embodiments, the CDK inhibitor is MSC2530818, senexin A, LDC4297 (LDC044297), PHA - 793887, BS - 181 HCl, PHA - 767491, THX1 2HCl, or XL413. In some embodiments, the CDK inhibitor is dinaciclib or alvocidib. In certain embodiments, the CDK inhibitor is not a CDK4 inhibitor and / or not a CDK6 inhibitor. In certain embodiments, the CDK inhibitor is not palbociclib (PD0332991), abemaciclib (LY2835219), or ribociclib.

[0009] In certain embodiments, the SAC factor inhibitor is further defined as a polo-like kinase 1 (PLK1) inhibitor, an Aurora kinase inhibitor, a survivin and / or a KSP inhibitor. In some embodiments, the PLK1 inhibitor is BI 2536, volasertib, GSK461364, ON-01910, GW 843682X, or HMN-214. In some embodiments, the PLK1 inhibitor is volasertib or ON-01910. In other embodiments, the SAC inhibitor is not a PLK1 inhibitor. In some embodiments, the Aurora kinase inhibitor is a pan-Aurora inhibitor, an Aurora A / B inhibitor, or an Aurora A inhibitor. In certain embodiments, the Aurora kinase inhibitor is AMG 900, alisertib, PF-03814735, tozasertib, MLN8054, or SNS-314 mesylate. In some embodiments, the Aurora kinase inhibitor is AMG-900 or alisertib. In some embodiments, the KSP inhibitor is ispinesib or SB743921. In some embodiments, the survivin inhibitor is YM155.

[0010] In some embodiments, the treatment results in the accumulation of cells in G2 / M phase, an increase in nuclear size, and / or polyploidy.

[0011] In certain embodiments, the cancer is resistant to one or more tyrosine kinase inhibitors (TKIs). In some embodiments, the one or more TKIs are selected from the group consisting of osimertinib, erlotinib, gefitinib, afatinib, poziotinib, dacomitinib, and CO-1686. In some embodiments, the cancer has acquired broad drug resistance. In some embodiments, the cancer is resistant to pemetrexed, irinotecan, vinblastine, and / or gemcitabine. In certain embodiments, the cancer has acquired a mutation to poziotinib and / or other TKIs. In some embodiments, the acquired mutation to poziotinib includes an EGFR exon 20 insertion. In some embodiments, the cancer is undergoing epithelial-mesenchymal transition (EMT). In some embodiments, EMT is evidenced by a decrease in E-cadherin expression, an increase in vimentin and / or Axl expression, and / or an increase in an invasive phenotype.

[0012] In further embodiments, the subject is further determined to include secondary mutations. In some embodiments, the secondary mutation is a T790M resistance mutation. In other embodiments, the subject is determined to not have secondary mutations. In some embodiments, the subject is determined to not have a T790M resistance mutation.

[0013] In further embodiments, the method further includes administering at least one additional anti-cancer therapy. In some embodiments, the at least one additional anti-cancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy, or immunotherapy. In some embodiments, the at least one additional anti-cancer therapy is a TKI and / or chemotherapy. In certain embodiments, the TKI is osimertinib, erlotinib, gefitinib, afatinib, dacomitinib, or CO-1686. In certain embodiments, the chemotherapy is pemetrexed, irinotecan, vinblastine, or gemcitabine.

[0014] In some embodiments, the CDK inhibitor, SAC factor inhibitor, and / or anticancer therapy is administered intravenously, subcutaneously, intraosseously, orally, transdermally, by sustained release, by controlled release, by delayed release, as a suppository, or sublingually. In some embodiments, administration of the CDK inhibitor, SAC factor inhibitor, and / or anticancer therapy includes local administration, topical administration, or systemic administration. In certain embodiments, the CDK inhibitor, SAC factor inhibitor, and / or anticancer therapy is administered two or more times.

[0015] In some embodiments, the cancer is oral cancer, hypopharyngeal cancer, nasopharyngeal cancer, respiratory cancer, genitourinary cancer, gastrointestinal cancer, cancer of the central or peripheral nervous system tissues, endocrine or neuroendocrine cancer or hematopoietic cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, hypopharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, pancreatic islet cell cancer, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, multiple endocrine neoplasia types I and II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. In certain embodiments, the cancer is non-small cell lung cancer.

[0016] In another embodiment, there is provided a pharmaceutical composition comprising a CDK inhibitor and / or an SAC factor inhibitor for use in a subject determined to have one or more EGFR activating mutations.

[0017] In some embodiments, the one or more EGFR activating mutations are selected from the group consisting of L858R, exon 19 deletion, and exon 20 insertion. In certain embodiments, the exon 19 deletion is an in-frame deletion between L747 and L749. In certain embodiments, the exon 20 insertion is N771Del Ins FH.

[0018] In certain embodiments, the subject was determined to have an EGFR activating mutation by analyzing a genomic sample from the patient. In some embodiments, the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. In some embodiments, the presence of the EGFR activating mutation is determined by nucleic acid sequencing or PCR analysis.

[0019] In some embodiments, the CDK inhibitor is further defined as a CDK2 inhibitor, a CDK5 inhibitor, a CDK1 inhibitor, or a CDK9 inhibitor. In certain embodiments, the CDK inhibitor is dinaciclib (SCH727965), alvocidib (flavopiridol), roscovitine (seliciclib, CYC202), SNS-032 (BMS-387032), LY2857785, ADZ5438, BMS-265246, NU6027, LDC000067, wogonin, or RO-3306. In certain embodiments, the CDK inhibitor is MSC2530818, senexin A, LDC4297 (LDC044297), PHA-793887, BS-181 HCl, PHA-767491, THX1 2HCl, or XL413. In some embodiments, the CDK inhibitor is dinaciclib or alvocidib. In certain embodiments, the CDK inhibitor is not a CDK4 inhibitor and / or not a CDK6 inhibitor. In certain embodiments, the CDK inhibitor is not palbociclib (PD0332991), abemaciclib (LY2835219), or ribociclib.

[0020] In certain embodiments, the SAC factor inhibitor is further defined as a polo-like kinase 1 (PLK1) inhibitor, an Aurora kinase inhibitor, a survivin and / or a KSP inhibitor. In some embodiments, the PLK1 inhibitor is BI 2536, volasertib, GSK461364, ON-01910, GW 843682X, or HMN-214. In some embodiments, the PLK1 inhibitor is volasertib or ON-01910. In other embodiments, the SAC inhibitor is not a PLK1 inhibitor. In some embodiments, the Aurora kinase inhibitor is a pan-Aurora inhibitor, an Aurora A / B inhibitor, or an Aurora A inhibitor. In certain embodiments, the Aurora kinase inhibitor is AMG 900, alisertib, PF-03814735, tozasertib, MLN8054, or SNS-314 mesylate. In some embodiments, the Aurora kinase inhibitor is AMG-900 or alisertib. In some embodiments, the KSP inhibitor is ispinesib or SB743921. In some embodiments, the survivin inhibitor is YM155.

[0021] In certain embodiments, the cancer is resistant to one or more tyrosine kinase inhibitors (TKIs). In some embodiments, the one or more TKIs are selected from the group consisting of osimertinib, erlotinib, gefitinib, afatinib, poziotinib, dacomitinib, and CO-1686. In some embodiments, the cancer has acquired broad drug resistance. In some embodiments, the cancer is resistant to pemetrexed, irinotecan, vinblastine, and / or gemcitabine. In certain embodiments, the cancer has acquired mutations to poziotinib and other TKIs. In some embodiments, the acquired mutations to poziotinib include EGFR exon 20 insertions. In some embodiments, the cancer is undergoing epithelial-mesenchymal transition (EMT). In some embodiments, EMT is demonstrated by a decrease in E-cadherin expression, an increase in vimentin and / or Axl expression, and / or an increase in an invasive phenotype.

[0022] In a further aspect, the subject is further determined to include a secondary mutation. In some aspects, the secondary mutation is a T790M resistance mutation, a C797S resistance mutation, or an L792H resistance mutation. In other aspects, the subject is determined not to have a secondary mutation. In some aspects, the subject is determined not to have a T790M resistance mutation.

[0023] In a further aspect, the composition further includes at least one additional anti-cancer therapy. In some aspects, the at least one additional anti-cancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy, or immunotherapy. In some aspects, the at least one additional anti-cancer therapy is a TKI and / or chemotherapy. In certain aspects, the TKI is osimertinib, erlotinib, gefitinib, afatinib, dacomitinib, or CO-1686. In a particular aspect, the chemotherapy is pemetrexed, irinotecan, vinblastine, or gemcitabine.

[0024] In some aspects, the cancer is oral cancer, oropharyngeal cancer, hypopharyngeal cancer, respiratory cancer, urogenital cancer, gastrointestinal cancer, cancer of the central or peripheral nervous system tissues, endocrine or neuroendocrine cancer or hematopoietic cancer, glioma, sarcoma, cancer tumor, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, hypopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, pancreatic islet cell cancer, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, multiple endocrine neoplasia types I and II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. In a particular aspect, the cancer is non-small cell lung cancer.

[0025] In another embodiment, a method of predicting responsiveness to a CDK inhibitor and / or an SAC factor inhibitor alone or in combination with a second anti-cancer therapy in a subject having cancer, the method comprising detecting an EGFR activating mutation in a genomic sample obtained from the patient, wherein if the sample is positive for the presence of the EGFR activating mutation, the patient is predicted to have a favorable responsiveness to the CDK inhibitor and / or the SAC factor inhibitor alone or in combination with an anti-cancer therapy, is provided.

[0026] In some embodiments, favorable responsiveness to a CDK inhibitor and / or an SAC factor inhibitor alone or in combination with these and an anti-cancer therapy includes a decrease in tumor size or tumor burden, inhibition of tumor growth, reduction of tumor-related pain, reduction of cancer-related conditions, reduction of cancer-related symptoms, non-progression of cancer, prolongation of disease-free period, prolongation of the period to progression, induction of remission, reduction of metastasis, or improvement of patient survival.

[0027] In some embodiments, one or more EGFR activating mutations are selected from the group consisting of L858R, exon 19 deletion, and exon 20 insertion. In certain embodiments, the exon 19 deletion is an in-frame deletion between L747 and L749. In certain embodiments, the exon 20 insertion is N771Del Ins FH.

[0028] In certain embodiments, the subject is determined to have an EGFR activating mutation by analyzing a genomic sample derived from the patient. In some embodiments, the genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. In some embodiments, the presence of the EGFR activating mutation is determined by nucleic acid sequencing or PCR analysis.

[0029] In some embodiments, the CDK inhibitor is further defined as a CDK2 inhibitor, a CDK5 inhibitor, a CDK1 inhibitor, or a CDK9 inhibitor. In certain embodiments, the CDK inhibitor is dinaciclib (SCH727965), alvocidib (flavopiridol), roscovitine (seliciclib, CYC202), SNS-032 (BMS-387032), LY2857785, ADZ5438, BMS-265246, NU6027, LDC000067, wogonin, or RO-3306. In certain embodiments, the CDK inhibitor is MSC2530818, senexin A, LDC4297 (LDC044297), PHA-793887, BS-181 HCl, PHA-767491, THX1 2HCl, or XL413. In some embodiments, the CDK inhibitor is dinaciclib or alvocidib. In certain embodiments, the CDK inhibitor is not a CDK4 inhibitor and / or not a CDK6 inhibitor. In certain embodiments, the CDK inhibitor is not palbociclib (PD0332991), abemaciclib (LY2835219), or ribociclib.

[0030] In certain embodiments, the SAC factor inhibitor is further defined as a polo-like kinase 1 (PLK1) inhibitor, an Aurora kinase inhibitor, a survivin and / or a KSP inhibitor. In some embodiments, the PLK1 inhibitor is BI 2536, volasertib, GSK461364, ON-01910, GW 843682X, or HMN-214. In some embodiments, the PLK1 inhibitor is volasertib or ON-01910. In other embodiments, the SAC inhibitor is not a PLK1 inhibitor. In some embodiments, the Aurora kinase inhibitor is a pan-Aurora inhibitor, an Aurora A / B inhibitor, or an Aurora A inhibitor. In certain embodiments, the Aurora kinase inhibitor is AMG 900, alisertib, PF-03814735, tozasertib, MLN8054, or mesylate SNS-314. In some embodiments, the Aurora kinase inhibitor is AMG-900 or alisertib. In some embodiments, the KSP inhibitor is ispinesib or SB743921. In some embodiments, the survivin inhibitor is YM155.

[0031] In certain embodiments, the cancer is resistant to one or more tyrosine kinase inhibitors (TKIs). In some embodiments, the one or more TKIs are selected from the group consisting of osimertinib, erlotinib, gefitinib, afatinib, poziotinib, dacomitinib, and CO-1686. In some embodiments, the cancer has acquired broad drug resistance. In some embodiments, the cancer is resistant to pemetrexed, irinotecan, vinblastine, and / or gemcitabine. In certain embodiments, the cancer has acquired a mutation to poziotinib and other TKIs. In some embodiments, the acquired mutation to poziotinib includes an EGFR exon 20 insertion. In some embodiments, the cancer is undergoing epithelial-mesenchymal transition (EMT). In some embodiments, EMT is demonstrated by a decrease in E-cadherin expression, an increase in vimentin and / or Axl expression, and / or an increase in an invasive phenotype.

[0032] In a further aspect, the subject is further determined to include a secondary mutation. In some aspects, the secondary mutation is a T790M resistance mutation. In other aspects, the subject is determined not to have a secondary mutation. In some aspects, the subject is determined not to have a T790M resistance mutation.

[0033] In a further aspect, the method further includes administering a CDK inhibitor and / or a SAC factor inhibitor alone or in combination with a second anti-cancer therapy to a patient predicted to have a favorable response. In some aspects, at least one additional anti-cancer therapy is chemotherapy, radiation therapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy, or immunotherapy. In some aspects, at least one additional anti-cancer therapy is a TKI and / or chemotherapy. In certain aspects, the TKI is osimertinib, erlotinib, gefitinib, afatinib, dacomitinib, or CO-1686. In certain aspects, the chemotherapy is pemetrexed, irinotecan, vinblastine, or gemcitabine.

[0034] In some aspects, the CDK inhibitor, SAC factor inhibitor, and / or anti-cancer therapy is administered intravenously, subcutaneously, intraosseously, orally, transdermally, by sustained release, by controlled release, by delayed release, as a suppository, or sublingually. In some aspects, the administration of the CDK inhibitor, SAC factor inhibitor, and / or anti-cancer therapy includes topical administration, regional administration, or systemic administration. In certain aspects, the CDK inhibitor, SAC factor inhibitor, and / or anti-cancer therapy is administered more than once.

[0035] In some embodiments, the cancer is oral cancer, oropharyngeal cancer, hypopharyngeal cancer, respiratory cancer, urogenital cancer, gastrointestinal cancer, cancer of the central or peripheral nervous system tissue, endocrine or neuroendocrine cancer or hematopoietic cancer, glioma, sarcoma, carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, oropharyngeal cancer, hypopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, pancreatic islet cell cancer, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, multiple endocrine neoplasia types I and II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. In certain embodiments, the cancer is non-small cell lung cancer.

[0036] [The present invention 1001] A method for treating cancer in a subject, comprising administering to the subject an effective amount of a cyclin-dependent kinase (CDK) inhibitor and / or a spindle assembly checkpoint (SAC) factor inhibitor, wherein the subject is determined to have one or more EGFR activating mutations, said method. [The present invention 1002] The method of the present invention 1001, wherein said one or more EGFR activating mutations are selected from the group consisting of L858R, exon 19 deletion, and exon 20 insertion. [The present invention 1003] The method of the present invention 1002, wherein said exon 19 deletion is E746 - A750 deletion, L747 - E749 deletion, or A750P. [The present invention 1004] The method of the present invention 1002, wherein said exon 20 insertion is N771Del Ins FH. [The present invention 1005] The method according to any one of the present inventions 1001 - 1004, wherein the subject is determined to have two, three, or four EGFR activating mutations. [The present invention 1006] The method of the present invention 1001, wherein the subject is determined to have an EGFR activating mutation by analyzing a genomic sample derived from the subject. [The present invention 1007] The method of the present invention 1006, wherein said genomic sample is isolated from saliva, blood, urine, normal tissue, or tumor tissue. [The present invention 1008] The method of the present invention 1006, wherein the presence of an EGFR activating mutation is determined by nucleic acid sequencing or PCR analysis. [The present invention 1009] The method of the present invention 1001, wherein said CDK inhibitor is further defined as a CDK2 inhibitor, a CDK5 inhibitor, a CDK1 inhibitor, or a CDK9 inhibitor. [The present invention 1010] The method of the present invention 1001, wherein said CDK inhibitor is dinaciclib (SCH727965), alvocidib (flavopiridol), roscovitine (seliciclib, CYC202), SNS - 032 (BMS - 387032), LY2857785, ADZ5438, BMS - 265246, NU6027, LDC000067, wogonin, or RO - 3306. [The present invention 1011] The method of the present invention 1001, wherein said CDK inhibitor is MSC2530818, senexin A, LDC4297 (LDC044297), PHA - 793887, BS - 181 HCl, PHA - 767491, THX1 2HCl, or XL413. [The present invention 1012] The method of the present invention 1001, wherein the CDK inhibitor is dinaciclib or albosidib. [The present invention 1013] The method of the present invention 1001, wherein the CDK inhibitor is not a CDK4 inhibitor and / or not a CDK6 inhibitor. [The present invention 1014] The method of the present invention 1001, wherein the CDK inhibitor is not palbociclib (PD0332991), abemaciclib (LY2835219), or ribociclib. [The present invention 1015] The method of the present invention 1001, wherein the SAC factor inhibitor is further defined as a polo-like kinase 1 (PLK1) inhibitor, an Aurora kinase inhibitor, survivin, and / or a KSP inhibitor. [The present invention 1016] The method of the present invention 1015, wherein the PLK1 inhibitor is BI 2536, volasertib, GSK461364, ON-01910, GW 843682X, or HMN-214. [The present invention 1017] The method of the present invention 1015, wherein the PLK1 inhibitor is volasertib or ON-01910. [The present invention 1018] The method of the present invention 1001, wherein the SAC inhibitor is not a PLK1 inhibitor. [The present invention 1019] The method of the present invention 1015, wherein the Aurora kinase inhibitor is a pan-Aurora inhibitor, an Aurora A / B inhibitor, or an Aurora A inhibitor. [The present invention 1020] The method of the present invention 1015, wherein the Aurora kinase inhibitor is AMG 900, alisertib, PF-03814735, tozasertib, MLN8054, or SNS-314 mesylate. [The present invention 1021] The method of the present invention 1001, wherein the Aurora kinase inhibitor is AMG-900 or alisertib. [The present invention 1022] The method of the present invention 1015, wherein the KSP inhibitor is ispinesib or SB743921. [The present invention 1023] The method of the present invention 1015, wherein the survivin inhibitor is YM155. [The present invention 1024] The method according to any one of the present inventions 1001 to 1023, wherein the treatment results in accumulation of cells in the G2 / M phase, enlargement of nuclear size, and / or polyploidy. [The present invention 1025] The method of the present invention 1001, wherein the cancer is resistant to one or more tyrosine kinase inhibitors (TKIs). [The present invention 1026] The method of the present invention 1025, wherein the one or more TKIs are selected from the group consisting of osimertinib, erlotinib, gefitinib, afatinib, poziotinib, dacomitinib, and CO-1686. [The present invention 1027] The method of the present invention 1001, wherein the cancer has acquired broad-spectrum drug resistance. [The present invention 1028] The method of the present invention 1001, wherein the cancer is resistant to pemetrexed, irinotecan, vinblastine, and / or gemcitabine. [The present invention 1029] The method of the present invention 1001, wherein the cancer has acquired a mutation to poziotinib, erlotinib, or osimertinib. [The present invention 1030] The method of the present invention 1029, wherein the acquired mutation to poziotinib, erlotinib, or osimertinib includes an EGFR exon 20 insertion. [The present invention 1031] The method of the present invention 1001, wherein the cancer has undergone epithelial-mesenchymal transition (EMT). [The present invention 1032] The method of the present invention 1031, wherein EMT is indicated by a decrease in E-cadherin expression, an increase in vimentin and / or Axl expression, and / or an increase in an invasive phenotype. [The present invention 1033] The method of the present invention 1001, wherein the subject is further determined to include secondary mutations. [The present invention 1034] The method of the present invention 1033, wherein the secondary mutation is a T790M resistance mutation, a C797S resistance mutation, or an L792H resistance mutation. [The present invention 1035] The method of the present invention 1001, wherein the cancer does not include secondary mutations. [The present invention 1036] The method of the present invention 1001, wherein the cancer does not include a T790M resistance mutation. [The present invention 1037] The method according to any one of the present inventions 1001 to 1036, further comprising administering at least one additional anti-cancer therapy. [The present invention 1038] The method of the present invention 1037, wherein the at least one additional anti-cancer therapy is chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy, or immunotherapy. [The present invention 1039] The method of the present invention 1037, wherein the at least one additional anti-cancer therapy is a TKI and / or chemotherapy. [The present invention 1040] The method of the present invention 1039, wherein the TKI is osimertinib, erlotinib, gefitinib, afatinib, dacomitinib, or CO-1686. [The present invention 1041] The method of the present invention 1039, wherein the chemotherapy is pemetrexed, irinotecan, vinblastine, or gemcitabine. [The present invention 1042] The method of the present invention 1039, wherein the CDK inhibitor, SAC factor inhibitor, and / or anticancer therapy is administered intravenously, subcutaneously, intraosseously, orally, transdermally, by sustained release, by controlled release, by delayed release, as a suppository, or sublingually. [The present invention 1043] The method of the present invention 1039, wherein the administration of the CDK inhibitor, SAC factor inhibitor, and / or anticancer therapy includes local administration, topical administration, or systemic administration. [The present invention 1044] The method of the present invention 1039, wherein the CDK inhibitor, SAC factor inhibitor, and / or anticancer therapy is administered two or more times. [The present invention 1045] The method of the present invention 1001, wherein the subject is human. [The present invention 1046] The method of the present invention 1045, wherein the subject has cancer. [The present invention 1047] The method of the present invention 1046, wherein the cancer is oral cancer, hypopharyngeal cancer, nasopharyngeal cancer, respiratory cancer, urogenital cancer, gastrointestinal cancer, cancer of the central or peripheral nervous system tissue, endocrine or neuroendocrine cancer or hematopoietic cancer, glioma, sarcoma, cancer tumor, lymphoma, melanoma, fibroma, meningioma, brain cancer, hypopharyngeal cancer, nasopharyngeal cancer, kidney cancer, biliary tract cancer, pheochromocytoma, pancreatic islet cell cancer, Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumor, adrenal tumor, osteosarcoma, multiple endocrine neoplasia type I and type II, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, gastric cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. [The present invention 1048] The method of the present invention 1046, wherein the cancer is non-small cell lung cancer. [The present invention 1049] A pharmaceutical composition comprising a CDK inhibitor and / or an SAC factor inhibitor for use in a subject determined to have one or more EGFR activating mutations. [The present invention 1050] The composition of the present invention 1049, wherein the one or more EGFR activating mutations are selected from the group consisting of L858R, exon 19 deletion, and exon 20 insertion. [The present invention 1051] The composition of the present invention 1050, wherein the exon 19 deletion is E746~A750 deletion, L747~E749 deletion, or A750P. [The present invention 1052] The composition of the present invention 1050, wherein the exon 20 insertion is N771Del Ins FH. [The present invention 1053] The composition of the present invention 1049, wherein the subject is determined to have two, three, or four EGFR activating mutations. [The present invention 1054] The composition of the present invention 1049, wherein the CDK inhibitor is further defined as a CDK2 inhibitor, a CDK5 inhibitor, a CDK1 inhibitor, or a CDK9 inhibitor. [The present invention 1055] The composition of the present invention 1049, wherein the CDK inhibitor is dinaciclib (SCH727965), alvocidib (flavopiridol), roscovitine (seliciclib, CYC202), SNS-032 (BMS-387032), LY2857785, ADZ5438, BMS-265246, NU6027, LDC000067, wogonin, or RO-3306. [The present invention 1056] The composition of the present invention 1049, wherein the CDK inhibitor is MSC2530818, senexin A, LDC4297 (LDC044297), PHA-793887, BS-181 HCl, PHA-767491, THX1 2HCl, or XL413. [The present invention 1057] The composition of the present invention 1049, wherein the CDK inhibitor is dinaciclib or alvocidib. [The present invention 1058] The composition of the present invention 1049, wherein the CDK inhibitor is not a CDK4 inhibitor and / or not a CDK6 inhibitor. [The present invention 1059] The composition of the present invention 1049, wherein the CDK inhibitor is not palbociclib (PD0332991), not abemaciclib (LY2835219), and not ribociclib. [The present invention 1060] The composition of the present invention 1049, wherein the SAC factor inhibitor is further defined as a polo-like kinase 1 (PLK1) inhibitor, an Aurora kinase inhibitor, a survivin and / or a KSP inhibitor. [The present invention 1061] The composition of the present invention 1060, wherein the PLK1 inhibitor is BI 2536, volasertib, GSK461364, ON-01910, GW 843682X, or HMN-214. [The present invention 1062] The composition of the present invention 1060, wherein the PLK1 inhibitor is volasertib or ON-01910. [The present invention 1063] The composition of the present invention 1049, wherein the SAC inhibitor is not a PLK1 inhibitor. [The present invention 1064] The composition of the present invention 1060, wherein the Aurora kinase inhibitor is a pan-Aurora inhibitor, an Aurora A / B inhibitor, or an Aurora A inhibitor. [The present invention 1065] The composition of the present invention 1060, wherein the Aurora kinase inhibitor is AMG 900, alisertib, PF-03814735, tozasertib, MLN8054, or mesylate SNS-314. [The present invention 1066] The composition of the present invention 1049, wherein the Aurora kinase inhibitor is AMG-900 or alisertib. [The present invention 1067] The composition of the present invention 1060, wherein the KSP inhibitor is ispinesib or SB743921. [The present invention 1068] The composition of the present invention 1060, wherein the survivin inhibitor is YM155. [The present invention 1069] The composition of the present invention 1049, wherein the cancer does not contain the T790M resistance mutation. [The present invention 1070] The composition of the present invention 1049, further comprising administering at least one additional anti-cancer therapy. [The present invention 1071] The composition of the present invention 1070, wherein the at least one additional anti-cancer therapy is chemotherapy, radiotherapy, gene therapy, surgery, hormone therapy, anti-angiogenesis therapy, or immunotherapy. [The present invention 1072] The composition of the present invention 1070, wherein the at least one additional anti-cancer therapy is TKI and / or chemotherapy. [The present invention 1073] The composition of the present invention 1072, wherein the TKI is osimertinib, erlotinib, gefitinib, afatinib, dacomitinib, or CO-1686. [The present invention 1074] The composition of the present invention 1072, wherein the chemotherapy is pemetrexed, irinotecan, vinblastine, or gemcitabine. [The present invention 1075] The composition of the present invention 1049, wherein the cancer is non-small cell lung cancer. [The present invention 1076] The composition of the present invention 1049, wherein the subject is human. [The present invention 1077] A method for predicting the responsiveness of a CDK inhibitor and / or a SAC factor inhibitor alone or in combination with a second anti-cancer therapy in a subject having cancer, the method comprising detecting an EGFR activating mutation in a genomic sample obtained from the subject, and predicting that the subject has a favorable responsiveness to the CDK inhibitor and / or the SAC factor inhibitor alone or in combination with an anti-cancer therapy when the sample is positive for the presence of the EGFR activating mutation. [The present invention 1078] The method of the present invention 1077, wherein the anti-cancer therapy is TKI and / or chemotherapy. [The present invention 1079] The method of the present invention 1077, wherein a CDK inhibitor and / or an SAC factor inhibitor alone or in combination with an anticancer therapy has a favorable responsiveness, including a reduction in tumor size or tumor burden, inhibition of tumor growth, alleviation of tumor-related pain, alleviation of cancer-related conditions, alleviation of cancer-related symptoms, non-progression of cancer, extension of the disease-free period, extension of the period until progression, induction of remission, reduction of metastasis, or improvement of the viability of the subject. [The present invention 1080] The method of the present invention 1077, further comprising the step of administering to the subject predicted to have a favorable responsiveness a CDK inhibitor and / or an SAC factor inhibitor alone or in combination with a second anticancer therapy. [The present invention 1081] The method of the present invention 1080, wherein the second anticancer therapy is a TKI or chemotherapy. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, since various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art from the detailed description, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are for purposes of illustration only.

Brief Description of the Drawings

[0037] The following drawings form a part of this specification and are included to further demonstrate certain specific aspects of the present invention. The present invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein.

[0038] The patent or application file includes at least one drawing created in color. Copies of this patent or patent application publication with color drawings are provided by the Patent Office upon payment of the claims and the required fees.

[0039]

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Mode for Carrying Out the Invention

[0040] Description of Exemplary Embodiments To address the unmet needs for treatment regimens for TKI-resistant cancers, a panel of NSCLC cell lines that had acquired resistance to the EGFR TKI erlotinib was obtained in this study. A subset of EGFR TKI-resistant mutants was negative for EGFR secondary mutations, resistant to second- and third-generation EGFR TKIs including osimertinib, afatinib, and dacomitinib, and underwent epithelial-mesenchymal transition (EMT) demonstrated by decreased E-cadherin, increased expression of N-cadherin and Axl, and increased invasive phenotype measured by the Boyden chamber assay. Proteomic profiling revealed that EGFR TKI-resistant cells showed similar mesenchymal and invasive phenotypes, but there were significant heterogeneities in protein expression and pathway activation among resistant mutants derived from the same parental cell line.

[0041] To identify therapeutic agents active against EMT-related EGFR TKI resistance, high-throughput drug screening was performed and the efficacy of 1,321 compounds was tested. EMT-related EGFR TKI resistance was associated with the acquisition of broad-spectrum drug resistance. Compared with parental cells, mesenchymal EGFR TKI-resistant cells had significantly stronger resistance to chemotherapeutic agents used to treat NSCLC, including pemetrexed, irinotecan, vinblastine, and gemcitabine. EGFR TKI-resistant cells showed acquisition of resistance to inhibitors of 147 other tyrosine kinases and serine / threonine kinases. In contrast, both parental cells and mesenchymal EGFR TKI-resistant mutants had high sensitivity to CDK inhibitors and agents targeting spindle assembly checkpoint (SAC) factors including PLK1, Aurora, KSP, and survivin. These findings were confirmed by MTS and clonogenic assays. Treatment with SAC inhibitors induced accumulation of cells in G2 / M phase, enlargement of nuclear size, and polyploidy.

[0042] To clinically confirm these findings, a cell line (MDA-011) was established from the pleural effusion of an EGFR-mutant NSCLC patient who was negative for T790M and resistant to erlotinib. In vitro, MDA-011 cells were resistant to erlotinib and osimertinib. MDA-011 cells had high sensitivity to CDK inhibitors and SAC inhibitors as determined by MTS and clonogenic assays. These data indicated that EMT-related resistance to EGFR TKI is associated with broad-spectrum drug resistance, but vulnerability to inhibition of CDK and SAC may be exploitable to overcome resistant disease in NSCLC patients.

[0043] Accordingly, in certain embodiments, the present disclosure provides a method for treating cancers having an activated EGFR mutation, such as NSCLC, with an inhibitor of CDK and / or an inhibitor of SAC factors. EGFR mutant NSCLC cells may or may not have acquired resistance to EGFR TKIs. Exemplary CDK inhibitors include dinaciclib and albosidib, and exemplary agents targeting spindle assembly checkpoint (SAC) factors include those targeting PLK1 (volasertib), those targeting Aurora A (AMG-900 and alisertib), and those targeting KSP (ispinesib). In some embodiments, cancer cells such as NSCLC express EGFR activating mutations such as L858R, exon 19 deletion, and exon 20 insertion. Cancer cells may have acquired resistance and / or broad-spectrum drug resistance to EGFR TKIs, for example, by T790M mutation and / or epithelial-mesenchymal transition.

[0044] I. Definitions "EGFR activating mutation" as used herein refers to a somatic mutation that may lead to the development of cancers such as lung cancer and is found in exons 18-21 of EGFR. Exemplary EGFR activating mutations include single nucleotide substitutions, such as the change of L858R, V765A, T783, or G719 to serine, alanine, or cysteine, exon 19 deletion (e.g., in-frame deletion of exon 19 between L747 and L749), and exon 20 insertion and / or duplication (e.g., D770_N771(ins NPG), D770_(ins SVQ), and D770_(ins G) N771T). Other "resistance mutations" such as T790M may be acquired.

[0045] As used herein, "a" or "an" can include one or more. When used in conjunction with the word "comprising" in the claims of this specification, the words "a" or "an" can mean one or more.

[0046] The present disclosure supports alternatives as well as definitions that refer to "and / or", but the use of the term "or" in the claims is used to mean "and / or" unless it is clearly indicated that only the alternatives are meant or that the alternatives are mutually exclusive. As used herein, "another" can mean at least a second, or more. The terms "about", "substantially", and "approximately" generally mean ±5% of the indicated value.

[0047] "Treatment" or "treating" includes (1) inhibiting a disease in a subject or patient who is experiencing or exhibiting a disease state or overall symptoms of the disease (e.g., preventing further progression of the disease state and / or overall symptoms), (2) improving a disease in a subject or patient who is experiencing or exhibiting a disease state or overall symptoms of the disease (e.g., reversing the disease state and / or overall symptoms), and / or (3) acting on any measurable reduction of the disease in a subject or patient who is experiencing or exhibiting a disease state or overall symptoms of the disease.

[0048] "Prevention" or "preventing" includes (1) inhibiting the onset of a disease in a subject or patient who has a risk of the disease and / or is likely to contract the disease, but has not yet experienced or shown any or all of the disease state or overall symptoms of the disease, and / or (2) delaying the onset of the disease state or overall symptoms of the disease in a subject or patient who has a risk of the disease and / or is likely to contract the disease, but has not yet experienced or shown any or all of the disease state or overall symptoms of the disease.

[0049] As used herein, the terms "patient" or "subject" refer to a living mammalian organism, e.g., a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a genetically engineered species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, minors, infants, and fetuses.

[0050] When the term "effective" is used in this specification and / or claims, it means sufficient to achieve a desired, expected, or intended result. When used in the context of treating a patient or subject with a compound, an "effective amount", "therapeutically effective amount", or "pharmaceutically effective amount" means an amount of the compound that is sufficient to act on the treatment or prevention of the disease when administered to the subject or patient for treating or preventing the disease.

[0051] As used herein, the term "IC 50 " refers to the inhibitory dose that is 50% of the maximum response obtained. This quantitative measurement indicates how much of a particular drug, or other substance (inhibitor), is required to inhibit by half a particular biological, biochemical, or chemical process (or component of a process, i.e., an enzyme, cell, cell receptor, or microorganism).

[0052] An "anticancer" agent can, for example, promote the killing of cancer cells, induce apoptosis of cancer cells, reduce the growth rate of cancer cells, reduce the incidence or number of metastases, shrink the size of tumors, inhibit tumor growth, reduce the blood supply to tumors or cancer cells, promote the immune response against cancer cells or tumors, prevent or inhibit the progression of cancer, or extend the survival period of a subject having cancer, thereby having a negative impact on cancer cells / tumors in a subject.

[0053] The term "insertion" or "insertion mutation" refers to the addition of one or more nucleotide base pairs into a DNA sequence. For example, an insertion mutation in exon 20 of EGFR can be present between amino acids 767-774 and can be about 2-21 base pairs in length.

[0054] "Detect," "detectable," and their grammatical equivalents refer to methods for determining the presence, and / or amount, and / or homology of a target nucleic acid sequence. In some embodiments, detection results in amplification of the target nucleic acid sequence. In other embodiments, determination of the sequence of the target nucleic acid can be characterized as "detecting" the target nucleic acid. The label bound to the probe can include any of a variety of labels well known in the art that are detectable, for example, by chemical or physical means. Labels that can be attached to the probe include, for example, fluorescent and luminescent materials.

[0055] "Amplify," "amplification," and their grammatical equivalents refer to any method of replicating at least a portion of a target nucleic acid sequence in a template-dependent manner, including, but not limited to, a wide range of techniques for amplifying nucleic acid sequences either linearly or exponentially. Exemplary means for performing the amplification step include ligase chain reaction (LCR), ligase detection reaction (LDR), ligation followed by Q-replicase amplification, PCR, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid sequence-based amplification (NASBA), two-step multiplex amplification, rolling circle amplification (RCA), recombinase-polymerase amplification (RPA) (TwistDx, Cambridg, UK), and self-sustained sequence replication (3SR), as well as their multiplex versions or combinations, such as, but not limited to, OLA / PCR, PCR / OLA, LDR / PCR, PCR / PCR / LDR, PCR / LDR, LCR / PCR, PCR / LCR (also known as composite chain reaction - CCR), etc. Descriptions of such techniques can be found elsewhere, in Sambrook et al., Molecular Cloning, 3 rd Edition).

[0056] As used generally in this specification, "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, with a reasonable benefit / risk ratio, are suitable for use in contact with human and animal tissues, organs, and / or bodily fluids without undue toxicity, irritation, allergic response, or other problems or complications.

[0057] "Pharmaceutically acceptable salts" means salts of the compounds of the present invention that are pharmaceutically acceptable as described above and have the desired pharmacological activity. Non-limiting examples of such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid; or 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphor-sulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiary butylacetic acid, and trimethylacetic acid. Pharmaceutically acceptable salts also include base addition salts that can be formed when the acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Non-limiting examples of acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, and N-methylglucamine. It should be recognized that specific anions or cations forming part of any salt of the present invention are not dangerous as long as the salt as a whole is pharmaceutically acceptable.Further examples of pharmaceutically acceptable salts and their preparation and use are provided in Handbook of Pharmaceutical Salts: Properties, and Use (P.H. Stahl & C.G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0058] II. EGFR Activating Mutations Certain embodiments of the present disclosure relate to determining whether a subject has one or more EGFR activating mutations, such as L858R, exon 19 deletions, or exon 20 mutations, such as insertion mutations, particularly one or more insertion mutations. The subject may have two, three, four, or more activating EGFR mutations. Methods for detecting mutations are known in the art and include PCR analysis and nucleic acid sequencing, as well as FISH and CGH. In certain embodiments, exon 20 mutations are detected, for example, by DNA sequencing from, e.g., tumor or plasma-derived circulating free DNA.

[0059] EGFR exon 19 mutations can include one or more point mutations, insertions, and / or deletions of 3 to 18 nucleotides, in-frame deletions of exon 19, at amino acids 747 - 749.

[0060] EGFR exon 20 mutations can include one or more point mutations, insertions, and / or deletions of 3 to 18 nucleotides at amino acids 763 - 778. One or more EGFR exon 20 mutations can be located at one or more residues selected from the group consisting of A763, A767, S768, V769, D770, N771, P772, and H773.

[0061] EGFR exon 20 insertions can include H773_V774insH, A767_v769ASV, N771_P772insH, D770_N771insG, H779_V774insH, N771delinsHH, S768_D770dupDVD, A767_V769dupASV, A767_V769dupASV, P772_H773dup, N771_H773dupNPH, S768_D770dupSVD, N771delinsGY, S768_D770delinsSVD, D770_D770delinsGY, A767_V769dupASV, and / or H773dup. In certain embodiments, the exon 20 mutation is A763insFQEA, A767insASV, S768dupSVD, V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH, and N771dupNPH.

[0062] A patient sample can be any bodily tissue or fluid that contains nucleic acids derived from lung cancer in a subject. In certain embodiments, the sample is a blood sample that contains circulating tumor cells or cell-free DNA. In other embodiments, the sample can be a tissue, e.g., lung tissue. The lung tissue can be derived from tumor tissue and can be fresh frozen or formalin-fixed and paraffin-embedded (FFPE). In certain embodiments, a lung tumor FFPE sample is obtained.

[0063] Samples suitable for use in the methods described herein include genetic material, e.g., genomic DNA (gDNA). Genomic DNA is typically extracted from biological samples such as blood or buccal mucosal scrapings, but can also be extracted from other biological samples such as urine, tumors, or sputum-containing expectorants. The sample itself typically comprises nucleated cells (e.g., blood or buccal cells) or tissue removed from a subject, including normal or tumor tissue. Methods and reagents for obtaining, processing, and analyzing samples are well known in the art. In some embodiments, the sample is obtained with the cooperation of a medical institution, e.g., by collecting blood. In some embodiments, the sample is obtained without the cooperation of a medical institution, e.g., the sample is obtained non-invasively, such as a sample containing buccal cells obtained using a buccal swab or brush, or an oral rinse sample.

[0064] In some cases, biological samples can be processed for DNA isolation. For example, DNA in a cell or tissue sample can be separated from other components of the sample. Cells can be recovered from biological samples using techniques well known in the art. For example, cells can be recovered by centrifuging a cell sample and resuspending the pelleted cells. Cells can be resuspended in a buffer such as phosphate buffered saline (PBS). After centrifuging the cell suspension to obtain a cell pellet, the cells can be lysed to extract DNA, e.g., gDNA. See, e.g., Ausubel et al. (2003). Samples can be concentrated and / or purified to isolate DNA. All samples obtained from a subject, including those subjected to any kind of further processing, are considered to be those obtained from the subject. For example, genomic DNA can be extracted from biological samples using conventional methods, including phenol extraction. Alternatively, genomic DNA can be extracted using kits such as the QIAamp® Tissue Kit (Qiagen, Chatsworth, Calif.) and the Wizard® Genomic DNA Purification Kit (Promega). Non-limiting examples of sample sources include urine, blood, and tissue.

[0065] The presence or absence of an EGFR activating mutation as described herein can be determined using methods well known in the art. For example, the presence or absence of an insertion mutation can be detected using gel electrophoresis, capillary electrophoresis, size exclusion chromatography, sequencing, and / or arrays. If desired, amplification of the nucleic acid can be accomplished using methods well known in the art, such as PCR. In one example, a sample (e.g., a sample containing genomic DNA) is obtained from a subject. The DNA in the sample is then tested to determine the characteristics of the insertion mutation as described herein. The insertion mutation can be detected by any method described herein, e.g., by sequencing or by hybridization to a nucleic acid probe of a gene in genomic DNA, RNA, or cDNA, e.g., a DNA probe (including cDNA and oligonucleotide probes) or an RNA probe. The nucleic acid probe can be designed to hybridize specifically or preferentially to a particular variant.

[0066] A set of probes typically refers to a set of primers (usually primer pairs) and / or detectably labeled probes used to detect target gene mutations (e.g., EGFR activating mutations) used in the recommended treatments enabled by the present disclosure. The primer pairs are used in an amplification reaction to define amplicons spanning regions for the target gene mutations for each of the genes. The set of amplicons is detected by a set of matching probes. In an exemplary embodiment, the method can use a TaqMan™ (Roche Molecular Systems, Pleasanton, Calif.) assay to detect a set of target gene mutations (e.g., EGFR activating mutations). In one embodiment, the set of probes is a set of primers used to produce amplicons detected by a nucleic acid sequencing reaction such as a next-generation sequencing reaction. In these embodiments, for example, AmpliSEQ™ (Life Technologies / Ion Torrent, Carlsbad, Calif.) or TruSEQ™ (Illumina, San Diego, Calif.) technology can be utilized.

[0067] Analysis of nucleic acid markers can be carried out using techniques well known in the art, including but not limited to sequence analysis and electrophoretic analysis. Non-limiting examples of sequence analysis include sequencing with mass spectrometry such as Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al., 1992), solid-phase sequencing (Zimmerman et al., 1992), matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS; Fu et al., 1998), as well as sequencing by hybridization (Chee et al., 1996; Drmanac et al., 1993; Drmanac et al., 1998). Non-limiting examples of electrophoretic analysis include slab gel electrophoresis, such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis. Furthermore, next-generation sequencing methods can be carried out using kits and devices commercially available from companies such as the Life Technologies / Ion Torrent PGM or Proton, the Illumina HiSEQ or MiSEQ, and the Roche / 454 next-generation sequencing system.

[0068] Other methods of nucleic acid analysis include direct manual sequencing (Church and Gilbert, 1988; Sanger et al., 1977; U.S. Patent No. 5,288,644); automated fluorescent sequencing; single-strand conformation polymorphism assay (SSCP) (Schafer et al., 1995); clamped denaturing gel electrophoresis (CDGE); two-dimensional gel electrophoresis (2DGE or TDGE); conformation-sensitive gel electrophoresis (CSGE); denaturing gradient gel electrophoresis (DGGE) (Sheffield et al., 1989); denaturing high-performance liquid chromatography (DHPLC, Underhill et al., 1997) infrared matrix-assisted laser desorption / ionization (IR-MALDI) mass spectrometry (WO99 / 57318); mobility shift analysis (Orita et al., 1989); restriction enzyme analysis (Flavell et al., 1978; Geever et al., 1981); quantitative real-time PCR (Raca et al., 2004); heteroduplex analysis; chemical mismatch cleavage (CMC) (Cotton et al., 1985); RNase protection assay (Myers et al., 1985); use of polypeptides that recognize nucleotide mismatches, such as the E. coli mutS protein; allele-specific PCR, and combinations of such methods may be included. See, for example, U.S. Patent Publication No. 2004 / 0014095. This patent is hereby incorporated by reference in its entirety.

[0069] In one example, a method for identifying an EGFR activating mutation in a sample includes contacting a nucleic acid derived from such a sample with a nucleic acid probe that can specifically hybridize to a nucleic acid encoding a mutated EGFR protein or a fragment thereof containing the mutation, and detecting the hybridization. In a particular embodiment, such a probe is, for example, a radioisotope ( 3 H, 32 P, or 33P), a fluorescent agent (rhodamine or fluorescein), or a chromogenic agent, and is detectably labeled. In certain embodiments, the probe is an antisense oligomer such as a PNA, morpholino-phosphoramidate, LNA, or 2'-alkoxyalkoxy. The probe can be from about 8 nucleotides to about 100 nucleotides, or from about 10 to about 75, or from about 15 to about 50, or from about 20 to about 30. In another aspect, such a probe of the present disclosure is provided in a kit for identifying EGFR activating mutations in a sample, and such a kit contains oligonucleotides that specifically hybridize to or are adjacent to the mutation sites in the EGFR gene. The kit further contains instructions for treating a patient having a tumor containing an EGFR activating mutation with a CDK inhibitor and / or an SAC inhibitor based on the results of a hybridization assay using the kit.

[0070] In another aspect, a method for detecting an exon 20 mutation in a sample includes amplifying such a nucleic acid sample corresponding to exon 20 of the EGFR gene or a fragment thereof that is considered to contain the mutation, and comparing the electrophoretic mobility of the amplified nucleic acid with the electrophoretic mobility of the corresponding wild-type EGFR or a fragment thereof. A difference in mobility indicates the presence of a mutation in the amplified nucleic acid sequence. The electrophoretic mobility can be measured on a polyacrylamide gel.

[0071] Alternatively, nucleic acids can be analyzed for mutations using Enzymatic Mutation Detection (EMD) (Del Tito et al., 1998). EMD uses bacteriophage resolvase T4 endonuclease VII, which scans along double-stranded DNA until it detects and resolves the structural distortions caused by base pair mismatches due to point mutations, insertions, and deletions. Detection of two short fragments formed, for example, by gel electrophoresis upon resolvase cleavage indicates the presence of a mutation. The advantage of the EMD method is that it is assayed directly from the PCR reaction, eliminating the need for sample purification, shortening hybridization times, increasing the signal-to-noise ratio, and is a single protocol for identifying point mutations, deletions, and insertions. Mixed samples containing up to 20-fold the expression of normal DNA and fragments up to 4 kb in size can be assayed. However, EMD scanning does not identify the specific base changes that occur in mutation-positive samples and, if necessary, further sequencing procedures are required to identify the mutations. As demonstrated in U.S. Patent No. 5,869,245, the CEL I enzyme can be used in a similar manner to bacteriophage resolvase T4 endonuclease VII.

[0072] III. Treatment Methods Also provided herein is a method for treating or delaying the progression of cancer in an individual, the method comprising administering to the individual, a subject determined to have an EGFR activating mutation (e.g., L858R mutation, exon 20 deletion, and / or exon 20 insertion), an effective amount of a CDK inhibitor, a SAC factor inhibitor, or a structurally similar inhibitor. The subject can have one or more EGFR activating mutations.

[0073] Examples of cancers contemplated for treatment include lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, lymphoma, pre-neoplastic lesions of the lung, colon cancer, melanoma, and bladder cancer. In certain embodiments, the cancer is non-small cell lung cancer.

[0074] In some embodiments, the subject is a mammal, such as a primate, preferably a higher primate, such as a human (e.g., a patient having or at risk of having the disorders described herein). In one embodiment, the subject is in need of enhancing an immune response. In certain embodiments, the subject is susceptible or at risk of being susceptible. For example, the subject has received or is receiving chemotherapy and / or radiation therapy. Alternatively, or in combination, the subject is susceptible or at risk of being susceptible as a result of an infection.

[0075] The CDK inhibitor can be a CDK2 inhibitor, a CDK5 inhibitor, a CDK1 inhibitor, or a CDK9 inhibitor. The CDK inhibitor can be a CDK inhibitor such as dinaciclib (SCH727965), alvocidib (flavopiridol), roscovitine (seliciclib, CYC202), SNS-032 (BMS-387032), LY2857785, ADZ5438, BMS-265246, NU6027, LDC000067, wogonin, or RO-3306. Further exemplary CDK inhibitors include MSC2530818, senexin A, LDC4297 (LDC044297), PHA-793887, BS-181 HCl, PHA-767491, THX1 2HCl, or XL413. In certain aspects, the CDK inhibitor can be dinaciclib or alvocidib.

[0076] The SAC factor inhibitor can be a polo-like kinase 1 (PLK1) inhibitor, an Aurora kinase inhibitor, a survivin and / or a KSP inhibitor. The PLK1 inhibitor can be BI 2536, volasertib, GSK461364, ON-01910, GW 843682X, or HMN-214. The Aurora kinase inhibitor can be a pan-Aurora inhibitor, an Aurora A / B inhibitor, or an Aurora A inhibitor. The Aurora kinase inhibitor can be AMG 900, alisertib, PF-03814735, tozasertib, MLN8054, or SNS-314 mesylate. The KSP inhibitor can be ispinesib or SB743921. The survivin inhibitor can be YM155. In some embodiments, the SAC factor inhibitor is not a MAD2 inhibitor.

[0077] Certain embodiments relate to the administration of a TKI in combination with a CDK inhibitor and / or a SAC factor inhibitor. The TKI can be osimertinib, erlotinib, gefitinib, afatinib, poziotinib, dacomitinib, and CO-1686, or other TKIs known in the art. In some embodiments, the TKI is poziotinib (also known as HM781-36B, HM781-36, and 1-[4-[4-(3,4-dichloro-2-fluoroanilino)-7-methoxyquinazolin-6-yl]oxypiperidin-1-yl]prop-2-en-1-one) is administered. Poziotinib is a quinazoline-based pan-HER inhibitor that irreversibly inhibits signaling through the HER family of tyrosine kinase receptors, including HER1, HER2, and HER4. Poziotinib or structurally similar compounds (e.g., U.S. Patent No. 8,188,102 and U.S. Patent Publication No. 2013 / 0071452; incorporated herein by reference) may be used in the methods of the present invention.

[0078] A. Pharmaceutical Composition Also provided herein are pharmaceutical compositions and formulations for a subject determined to have an EGFR activating mutation, such as an L858 mutation, an exon 19 deletion, or an exon 20 insertion, comprising a CDK inhibitor and / or a SAC factor inhibitor and a pharmaceutically acceptable carrier.

[0079] The pharmaceutical compositions and formulations described herein comprise an active ingredient (e.g., an antibody or polypeptide) having a desired degree of purity, in the form of a lyophilized formulation or an aqueous solution, in one or more optional pharmaceutically acceptable carriers (Remington’s Pharmaceutical Sciences 22 ndIt can be prepared by mixing with (edition, 2012). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations used, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (such as zinc-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG), but are not limited thereto. In the present specification, an exemplary pharmaceutically acceptable carrier further includes an interstitial drug dispersant such as a soluble neutral-active hyaluronidase glycoprotein (sHASEGP), such as a human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use including rHuPH20 are described in U.S. Patent Publications Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, the sHASEGP is combined with one or more additional glucosaminoglycanases such as chondroitinase.

[0080] B. Combination Therapy In certain embodiments, the compositions and methods of the present embodiment include a CDK inhibitor and / or an SAC factor inhibitor in combination with at least one additional therapy. The additional therapy can be radiotherapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination thereof. The additional therapy can be in the form of adjuvant or neoadjuvant therapy.

[0081] In some embodiments, the additional therapy is the administration of a small molecule enzyme inhibitor or a metastasis inhibitor. In some embodiments, the additional therapy is the administration of an agent that reduces side effects (e.g., an agent intended to reduce the occurrence and / or severity of side effects of a treatment, such as an antiemetic). In some embodiments, the additional therapy is radiotherapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiotherapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional therapy can be one or more chemotherapeutic agents well known in the art.

[0082] The inhibitor can be administered before, during, after, or in various combinations with further cancer therapies such as immune checkpoint therapy. Administration can range from co - administration to intervals of minutes, days, or weeks. In embodiments where the inhibitor is provided to the patient separately from a further therapeutic agent, generally, a significant period does not elapse between each delivery time to ensure that the two compounds can provide an advantageous combined effect to the patient. In such cases, it is contemplated that antibody therapy and anti - cancer therapy may be provided to the patient within about 12 - 24 or 72 hours of each other, more specifically within about 6 - 12 hours of each other. In some situations, it may be desirable to space the intervals between each administration by several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) to significantly extend the treatment period.

[0083] Various combinations can be utilized. In the following examples, the CDK inhibitor and / or the SAC factor inhibitor is "A" and the anti - cancer therapy is "B". TIFF0007698418000001.tif17128

[0084] The administration of any compound or the implementation of a therapy of this embodiment to a patient follows the general protocol for the administration of such a compound, taking into account the toxicity of the agent if present. Thus, in some embodiments, there is a step of monitoring the toxicity resulting from the combination therapy.

[0085] 1. Chemotherapy According to this embodiment, a variety of chemotherapeutic agents can be used. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to denote a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within the cell, for example, whether they affect the cell cycle and at which stage of the cell cycle they act. Alternatively, the agents may be characterized based on their ability to directly crosslink to DNA, to intervene in DNA, or to induce chromosomal and mitotic abnormalities by acting on nucleic acid synthesis.

[0086] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially, bratasin and bratasinone); camptothecin (including synthetic analog topotecan); bryostatin; calicheamicin; CC-1065 (including its azaglycosine, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chloronaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially, calicheamicin gammaII and calicheamicin omegaI1); dynemicin including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein-engineered antibiotic chromophores, actinomycin, actinomycin, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, calminomycin, cardinophilin, chromomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, for example, mitomycin C, mycophenolic acid, nogalarnycin, olivomycin, peplomycin, potfiromycin, puromycin, querramycin, rhodomycin, streptonigrin, streptozocin, tubercidin, ubenimex, dinostatin, and zorubicin; antimetabolites, for example, methotrexate and 5-fluorouracil (5-FU); folic acid analogs, for example, denopterin, pteropterin, and trimetrexate; purine analogs, for example, fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, for example, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxuridine, doxifluridine, enocitabine, and floxuridine; androgens, for example, calusterone, drostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal, for example, mitotane and trilostane; folic acid supplements, for example, folic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; dexamethasone; diaziquone; elfomithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; maytansinoids, for example, maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;Roxanthrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; lysoxine; schizophyllan; spirigermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichloroethylamine; trichothecene (especially, T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoid, for example, paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complex, for example, cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (for example, CPT-11); topoisomerase inhibitor RFS2000; difluorodifluoromethylornithine (DMFO); retinoid, for example, retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein transferase inhibitor, transplatin, and any pharmaceutically acceptable salt, acid, or derivative of the foregoing are included.;

[0087] 2. Radiation therapy Other factors that cause DNA damage and are widely used include those known as gamma rays, X-rays, and / or the targeted delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents are contemplated, such as electron ranges, proton beam irradiation (U.S. Pat. Nos. 5,760,395 and 4,870,287), and UV irradiation. All of these agents are likely to affect a wide range of damage to DNA, to precursors of DNA, to DNA replication and repair, and to the assembly and maintenance of chromosomes. The dose range for X-rays spans from a daily dose of 50-200 roentgens for a long period (3-4 weeks) to a single dose of 2000-6000 roentgens. The dose range for radioisotopes varies widely and depends on the half-life of the isotope, the intensity and type of the emitted radiation, and the uptake by neoplastic cells.

[0088] 3. Immunotherapy One of ordinary skill in the art will understand that additional immunotherapies can be used in combination with or in conjunction with the methods of the present embodiment. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is an example. The immune effector can be, for example, an antibody specific for some marker on the surface of the tumor cell. The antibody alone can be used as an effector of the therapy, or the antibody may recruit other cells that actually act in cell killing. The antibody can also be conjugated to a drug or toxin (such as a chemotherapeutic agent, a radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and function as a molecular targeting agent. Alternatively, the effector can be a lymphocyte having surface molecules that interact directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0089] Antibody-drug conjugates have emerged as an epoch-making approach in the development of cancer therapy. Cancer is one of the leading causes of death worldwide. Antibody-drug conjugates (ADCs) contain monoclonal antibodies (MAbs) covalently linked to cell-killing drugs. This approach combines the high specificity of the Mabs for their antigen targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver the payload (drug) to tumor cells along with abundant levels of antigen. Targeted delivery of the drug also minimizes drug exposure in normal tissues, leading to reduced toxicity and improved therapeutic indices. The approval of two ADC drugs, ADCETRIS® (brentuximab vedotin), approved by the FDA in 2011, and KADCYLA® (trastuzumab emtansine or T-DM1), approved in 2013, has validated this approach. Currently, more than 30 ADC drug candidates are at various stages of clinical trials for cancer therapy (Leal et al., 2014). As antibody engineering and linker-payload optimization become increasingly mature, the drug discovery and development of new ADCs rely heavily on the identification and validation of new targets suitable for this approach and the generation of targeted MAbs. Two criteria for ADC targets are upregulated / high-level expression in tumor cells and robust internalization.

[0090] In one aspect of immunotherapy, tumor cells need to have several markers that are suitable for targeting (i.e., not present in most other cells). There are many tumor markers, and any of these could be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative aspect of immunotherapy is to combine an anti-cancer effect and an immune-stimulating effect. There are also immune-stimulating molecules, including cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

[0091] Examples of immunotherapy include immunoadjuvants such as Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy such as interferons α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy such as TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and 5,846,945); and monoclonal antibodies such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Pat. No. 5,824,311). It is contemplated that one or more anti-cancer therapies can be used in combination with the antibody therapy described herein.

[0092] In some embodiments, the immunotherapy can be an immune checkpoint inhibitor. Immune checkpoints either enhance or weaken a signal (e.g., a costimulatory molecule). Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig suppressor of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 system and / or CTLA-4.

[0093] The immune checkpoint inhibitor can be a drug, e.g., a small molecule, a recombinant form of a ligand or receptor, or in particular, an antibody, e.g., a human antibody (e.g., International Patent Publication No. WO2015 / 016718; Pardoll, Nat Rev Cancer, 12(4):252-64, 2012; both are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or their analogs may be used, in particular, chimeric, humanized, or human forms of antibodies may be used. Those skilled in the art will appreciate that alternative and / or equivalent names may be used for the specific antibodies referred to in this disclosure. Such alternative and / or equivalent names are interchangeable in the context of this specification. For example, pembrolizumab is known to also be known as MK-3475 and lambrolizumab as alternative and equivalent names.

[0094] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a particular aspect, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In a particular aspect, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In a particular aspect, the PDL2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 system antagonists for use in the methods provided herein are well known in the art and are described, for example, in U.S. Patent Publications Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all of which are incorporated herein by reference.

[0095] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO2006 / 121168. Pembrolizumab, also known as MK-3475, Merck3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0096] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to the T cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86, also known as B7-1 and B7-2, respectively, on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. T cell activation via the T cell receptor and CD28 leads to increased expression of the inhibitory receptor for CTLA-4, the B7 molecules.

[0097] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0098] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present method can be prepared using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can be used. For example, U.S. Patent No. 8,119,129; International Patent Publications WO01 / 14424, WO98 / 42752, and WO00 / 37504 (CP675,206, tremelimumab; also known as previous ticilimumab); U.S. Patent No. 6,207,156; Hurwitz et al., 1998; Camacho et al., 2004; and anti-CTLA-4 antibodies disclosed in Mokyr et al., 1998 can be used in the methods disclosed herein. The teachings of each of the foregoing publications are incorporated herein by reference. Antibodies that compete with these antibodies recognized in the art for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Applications WO2001 / 014424, and WO2000 / 037504, and U.S. Patent No. 8,017,114, all of which are incorporated herein by reference.

[0099] Exemplary anti-CTLA-4 antibodies are ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and / or binds to the same epitope on CTLA-4 as the above-described antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence homology with the above-described antibodies (e.g., at least about 90%, 95%, or 99% variable region homology with ipilimumab).

[0100] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors, such as those described in U.S. Patent Nos. 5,844,905, 5,885,796, and International Patent Applications Nos. WO1995 / 001994 and WO1998 / 042752; all incorporated herein by reference, as well as immunoadhesins, such as those described in U.S. Patent No. 8,329,867, incorporated herein by reference.

[0101] 4. Surgery Approximately 60% of people with cancer undergo some type of surgery, including prophylactic, diagnostic or staging, therapeutic, and palliative surgery. Therapeutic surgery includes resection in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and can also be combined with other treatment methods, such as the treatment of the present embodiment, chemotherapy, radiotherapy, hormone therapy, gene therapy, immunotherapy, and / or alternative treatment methods. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatment includes laser surgery, cryosurgery, electro-surgery, and microscope-controlled surgery (Mohs surgery).

[0102] In the resection of some or all of the cancer cells, tissue, or tumor, a cavity may be formed in the body. Treatment can be achieved by perfusion, direct injection, or topical application in the area using additional anti-cancer therapies. Such treatments can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be varied in dosage.

[0103] 5. Other Agents It is contemplated that other agents may be used in combination with specific aspects of the present embodiment to improve the therapeutic effect of the treatment. These additional agents include agents that act on the upregulation of cell surface receptors and GAP junctions, cell growth inhibitors and differentiating agents, inhibitors of cell adhesion, agents that increase the sensitivity of hyperproliferative cells to apoptosis-inducing agents, or other biological agents. An increase in intracellular signaling by increasing the number of GAP junctions may increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, a cell growth inhibitor or differentiating agent can be used in combination with specific aspects of the present embodiment to improve the anti-hyperproliferative effect of the treatment. Inhibitors of cell adhesion are contemplated to improve the effects of the present embodiment. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, can be used in combination with specific aspects of the present embodiment to improve the therapeutic effect.

[0104] IV. Kit Kits for detecting EGFR activation mutations, such as those disclosed herein, are also within the scope of the present disclosure. An example of such a kit may include a set of primers specific for L858R, exon 19 deletions, and / or exon 20 mutations. The kit may further include instructions for using the primers to detect the presence or absence of specific EFGR activation mutations described herein. The kit may further include instructions for diagnostic purposes indicating that identification of a positive for the EGFR activation mutations described herein in a sample from a cancer patient is an indicator of sensitivity to a CDK inhibitor and / or an SAC factor inhibitor. The kit may further include instructions indicating that identification of a positive for the EGFR activation mutations described herein in a sample from a cancer patient indicates that the patient should be treated with a CDK inhibitor and / or an SAC factor inhibitor.

Examples

[0105] V. Examples The following examples are included to demonstrate preferred embodiments of the present invention. Those skilled in the art should understand that the techniques disclosed in the following examples are those found by the inventors to function well in the practice of the present invention and can, therefore, be considered to constitute preferred modes for its practice. However, those skilled in the art should understand that, in light of the present disclosure, numerous changes can be made to the specific embodiments disclosed herein and that similar or analogous results can still be obtained without departing from the spirit and scope of the present invention.

[0106] Example 1 - Identification of a Method for Treating Cancer with EGFR Activation Mutations EGFR - mutated NSCLC patients initially respond to EGFR - targeted therapy, but resistant disease invariably emerges. In almost half of the resistant cases, the tumors lack secondary EGFR mutations such as T790M and are resistant to second - and third - generation EGFR tyrosine kinase inhibitors (TKIs). Identification of a treatment regimen effective against T790M - negative resistance remains a major clinical challenge. To address this unmet need, a panel of NSCLC cell lines that had acquired resistance to the EGFR TKIs erlotinib, gefitinib, osimertinib, or poziotinib was obtained.

[0107] Proteomic profiling of the resistant cells was performed by reverse - phase protein array (RPPA) and Western blotting for markers of epithelial - mesenchymal transition (EMT). The Cell Titer glo assay in a high - throughput 384 - well plate system was used for drug screening. The long - term effects of inhibitors of SAC and CDK were evaluated by a 14 - day clonogenic assay.

[0108] Using HCC827 and HCC4006 (EGFR-mutant NSCLC cells), EGFR TKI-resistant variants were obtained in vitro by continuously culturing the cells with increasing concentrations of TKI (e.g., erlotinib). Erlotinib-resistant cells (ER) were negative for EGFR secondary mutations, resistant to second- and third-generation EGFR TKIs including osimertinib, afatinib, and dacomitinib (Figure 1), and had undergone epithelial-mesenchymal transition (EMT) as demonstrated by decreased E-cadherin, enhanced expression of N-cadherin and Axl, and increased invasive phenotype measured by the Boyden chamber assay. Proteomic profiling revealed that EGFR TKI-resistant cells showed similar mesenchymal and invasive phenotypes, but there was significant heterogeneity in protein expression and pathway activation among resistant variants derived from the same parental cell line.

[0109] To identify therapeutic agents active against EMT-related EGFR TKI resistance, high-throughput drug screening was performed to test the efficacy of 1,321 compounds. EMT-related EGFR TKI resistance was associated with the acquisition of broad-spectrum drug resistance. Compared with parental cells, mesenchymal EGFR TKI-resistant cells had significantly stronger resistance to chemotherapeutic agents used to treat NSCLC, including pemetrexed, irinotecan, vinblastine, and gemcitabine. EGFR TKI-resistant cells showed acquisition of resistance to inhibitors of 147 other tyrosine kinases and serine / threonine kinases. In contrast, both parental cells and mesenchymal EGFR TKI-resistant variants had high sensitivity to CDK inhibitors and agents targeting spindle assembly checkpoint (SAC) factors including PLK1, Aurora, KSP, and survivin. These findings were confirmed by MTS and clonogenic assays. Treatment with SAC inhibitors induced accumulation of cells in G2 / M phase, enlargement of nuclear size, and polyploidy.

[0110] To clinically confirm these findings, a cell line (MDA-011) was established from the pleural effusion of an EGFR-mutated NSCLC patient with T790M-negative erlotinib resistance. In vitro, MDA-011 cells were resistant to erlotinib and osimertinib. MDA-011 cells had high sensitivity to CDK inhibitors and SAC inhibitors as determined by MTS and clonogenic assays. These data indicated that EMT-related resistance to EGFR TKIs is associated with broad drug resistance but is vulnerable to the inhibition of CDK and SAC, which can be utilized to overcome resistant diseases in NSCLC patients.

[0111] (Table 1) IC50 of SAC inhibitor in 5-day Cell Titer Glo assay TIFF0007698418000002.tif159128

[0112] (Table 2) IC50 of SAC inhibitor in 5-day Cell Titer Glo assay TIFF0007698418000003.tif168129

[0113] (Table 3) IC50 of CDK inhibitor in 5-day Cell Titer Glo assay TIFF0007698418000004.tif119128

[0114] Next, H1975 cells (EGFR-mutated positive with T790M) were cultured in the presence of osimertinib until resistant mutants emerged. Osimertinib-resistant cells (H1975 OR5 and H1975 OR16) had undergone EMT and were sensitive to CDK inhibitors and drugs targeting SAC factors including PLK1 (volasertib), Aurora (AMG-900 and alisertib), and KSP (ispinesib) (Figure 7).

[0115] In addition, CDK and SAC inhibitors were investigated in the context of having acquired resistance to inhibitors having activity against EGFR exon 20 insertion mutations. YUL-0019 cells harbor the EGFR exon 20 insertion mutation and have high sensitivity to the EGFR inhibitor poziotinib. YUL-0019 cells were continuously cultured in the presence of poziotinib until resistant cells emerged. YUL-0019 cells and YUL-0019 PR8 (poziotinib-resistant) cells had high sensitivity to SAC inhibitors including CDK inhibitors (including dinaciclib and albosidib), PLK1 (volasertib), Aurora (AMG-900 and alisertib), and KSP (ispinesib) (Figure 8). These data indicate that EMT-related resistance to EGFR TKI is associated with broad drug resistance but is vulnerable to inhibition of CDK and SAC that can be utilized to overcome resistant diseases in NSCLC patients.

[0116] All of the methods disclosed and claimed herein can be constructed and implemented without undue experimentation in light of the present disclosure. Although the compositions and methods of the invention have been described in connection with preferred embodiments, it will be apparent to those skilled in the art that changes may be applied to the methods and processes described herein, or to the order of the steps of such methods, without departing from the concept, spirit, and scope of the invention. More specifically, certain chemically and physiologically related agents may be used in place of the agents described herein, and it will be apparent that they may achieve the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the scope of the spirit, scope, and concept of the invention as defined by the appended claims.

[0117] References The following references provide exemplary procedural or other details that supplement the references set forth herein and are specifically incorporated herein by reference. TIFF0007698418000005.tif189149TIFF0007698418000006.tif230106TIFF0007698418000007.tif45128

Claims

1. A pharmaceutical composition for treating cancer in a subject, comprising an effective amount of a cyclin-dependent kinase (CDK) inhibitor and / or a spindle assembly checkpoint (SAC) factor inhibitor, wherein the cancer is a cancer having one or more EGFR activating mutations, does not include the T790M resistance mutation, and undergoes epithelial-mesenchymal transition (EMT), the one or more EGFR activating mutations are selected from the group consisting of L858R, E746-A750 deletion, L747-E749 deletion, A750P, and N771Del Ins FH, the CDK inhibitor is selected from the group consisting of dinaciclib (SCH727965) and alvocidib (flavopiridol), and the SAC factor inhibitor is selected from the group consisting of volasertib, ON-01910, AMG-900, alisertib, ispinesib, and SB743921, the pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the EGFR activating mutation is an E746-A750 deletion, an L747-E749 deletion, or A750P.

3. The pharmaceutical composition according to claim 1, wherein the EGFR activating mutation is N771Del Ins FH.

4. The pharmaceutical composition according to claim 1, wherein the SAC factor inhibitor is volasertib or ON-01910.

5. The pharmaceutical composition according to claim 1, wherein the SAC factor inhibitor is AMG-900 or alisertib.

6. The pharmaceutical composition according to claim 1, wherein the SAC factor inhibitor is ispinesib or SB743921.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the treatment results in accumulation of cells in the G2 / M phase, enlargement of nuclear size, and / or polyploidy.

8. The pharmaceutical composition according to claim 1, wherein the cancer is resistant to one or more tyrosine kinase inhibitors (TKIs).

9. The pharmaceutical composition according to claim 8, wherein the one or more TKIs are selected from the group consisting of osimertinib, erlotinib, gefitinib, afatinib, poziotinib, dacomitinib, and CO-1686.

10. The pharmaceutical composition according to claim 1, wherein the cancer is resistant to pemetrexed, irinotecan, vinblastine, and / or gemcitabine.

11. The pharmaceutical composition according to claim 1, wherein EMT is indicated by a decrease in E-cadherin expression, an increase in vimentin and / or Axl expression, and / or an increase in an invasive phenotype.

12. The pharmaceutical composition according to claim 1, wherein the subject is determined to have a secondary mutation including a C797S resistance mutation or an L792H resistance mutation.

13. The pharmaceutical composition according to claim 1, wherein the cancer does not contain a secondary mutation.

14. The pharmaceutical composition according to claim 1, which is used in combination with a TKI and / or chemotherapy.

15. The pharmaceutical composition according to claim 14, wherein the TKI is osimertinib, erlotinib, gefitinib, afatinib, dacomitinib, or CO-1686.

16. The pharmaceutical composition according to claim 14, wherein the chemotherapy is pemetrexed, irinotecan, vinblastine, or gemcitabine.

17. The pharmaceutical composition according to claim 1, wherein the cancer is non-small cell lung cancer.

18. A method for assisting in predicting the response of a subject having cancer to a CDK inhibitor and / or a SAC factor inhibitor alone or in combination with a second anti-cancer therapy, the method comprising detecting an EGFR activation mutation selected from the group consisting of L858R, E746-A750 deletion, L747-E749 deletion, A750P, and N771Del Ins FH in a genomic sample obtained from the subject, wherein the cancer undergoes epithelial-mesenchymal transition (EMT), the sample is positive for the presence of the EGFR activation mutation, and negative for the presence of the T790M resistance mutation, which indicates that the subject has a favorable response to the CDK inhibitor and / or the SAC factor inhibitor alone or in combination with an anti-cancer therapy, the CDK inhibitor is selected from the group consisting of dinaciclib (SCH727965) and albosidib (flavopiridol), and the SAC factor inhibitor is selected from the group consisting of volasertib, ON-01910, AMG-900, alisertib, ispinesib, and SB743921. The method.

19. The method according to claim 18, wherein the anti-cancer therapy is a TKI and / or chemotherapy.

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