Combination therapy for the treatment of gastrointestinal stromal tumors

Abstract

To provide a method of treating a tumor having one or more c-KIT mutations.SOLUTION: A method includes the use of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea or a pharmaceutically acceptable salt thereof, in combination with a MAPKAP kinase inhibitor. Preferably, the MAPKAP kinase inhibitor is selected from the groups consisting of a mitogen-activated protein kinase inhibitor (MEK inhibitor) and an effective amount of an extracellular signal regulated kinase inhibitor (ERK inhibitor).SELECTED DRAWING: None

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 62 / 624,448, filed January 31, 2018, which is incorporated by reference in its entirety.

[0002] c-KIT (also known as KIT, CD117, and stem cell factor receptor) is a 145 kDa transmembrane tyrosine kinase protein that acts as a type III receptor. The c-KIT proto-oncogene, located on chromosome 4q11-21, encodes the c-KIT receptor, whose ligand is stem cell factor (SCF, hematopoietic stem cell factor, kit ligand, mast cell growth factor). The receptor has tyrosine protein kinase activity, and binding of the ligand SCF leads to the autophosphorylation of c-KIT and its association with substrates such as phosphatidylinositol 3-kinase (PI3K). Tyrosine phosphorylation by protein tyrosine kinases is particularly important in cell signaling and can mediate signals for key intracellular processes such as proliferation, survival, differentiation, apoptosis, adhesion, invasion, and migration.

[0003] The role of c-KIT expression and activity has been studied in hematological and solid tumors, such as acute leukemia and gastrointestinal stromal tumors (GISTs). Most GISTs are accompanied by primary activating mutations in the genes encoding the closely related RTKs, c-KIT (75–80% of GISTs) or PDGFRα (8% of non-c-KIT mutant GISTs), and gain of function mutations in the c-KIT gene and expression of constitutively phosphorylated c-KIT are found in many GISTs. The majority of c-KIT primary mutations causing GISTs affect the juxtamembrane (JM) region of the protein encoded by exon 11 and consist of in-frame deletions or insertions, or missense mutations (i.e., V560D). Exon 11 mutations in c-KIT have been identified as primary mutations in approximately 65% ​​of GISTs. Such JM domain mutations disrupt the autoinhibitory mechanism of c-KIT kinase, leading to constitutive kinase activity and the causation of cell transformation events in GISTs. Other c-KIT primary mutations causing GIST are located in exon 9 (AY501-502 duplication / insertion, 8%), exon 13 (mutation, 1%), and exon 17 (mutation, 1%).

[0004] The clinical importance of c-KIT expression in malignant tumors was demonstrated in studies with a specific tyrosine kinase receptor inhibitor, Gleevec® (imatinib mesylate, STI571 (signal transduction inhibitor number 571), Novartis Pharma AG, Basel, Switzerland). A further clinically relevant breakthrough was the finding of an antitumor effect of this compound in GIST, a group of tumors that are generally considered resistant to conventional chemotherapy. However, while major responses have been seen after first-line treatment of GIST with the c-KIT inhibitor Gleevec®, and a significant number of patients with metastatic and / or inoperable GIST benefit from treatment with Gleevec®, complete tumor remissions are rare, with approximately 50% of patients experiencing disease recurrence within 2 years of treatment. It has also been reported that the combination of the c-KIT inhibitor imatinib and the MEK kinase inhibitor MEK162 resulted in increased in vitro growth inhibition and in vivo tumor regression in various GIST cancer cell lines compared with treatment with either agent alone.

[0005] GISTs become resistant to Gleevec® in most cases and Thus, targeted small molecule therapy targeting c-KIT secondary mutations remains elusive. GIST patients who relapse after treatment with Gleevec® or Sutent® still have disease caused by c-KIT mutations. These secondary mutations occur on the same allele as the primary mutation in the JM region and therefore represent an activated form of c-KIT that is even more aggressive than the original primary mutation. These secondary mutations of c-KIT identified in GIST lead to acquired drug resistance. Secondary mutations are found in the ATP binding pocket (exon 13, i.e., K642E, V654A; exon 14, i.e., T670I), and the activation loop (exon 17, i.e., N822K, D816H, D816V, D820A; exon 18 A829P). These various c-KIT secondary mutations have been reported as follows: Sunitinib malate (Sutent™, Pfizer Inc.) is an inhibitor of multiple RTKs, in this context especially c-KIT and PDGFRα, and has been shown to be effective against certain imatinib-resistant c-KIT mutants, such as the ATP-binding pocket mutants V654A and T670I. Certain Gleevec®-resistant mutants are also resistant to sunitinib, such as D816H and D816V, located in the activation loop of the c-KIT catalytic domain encoded by exon 17. The median survival time after progression due to Gleevec® resistance remains relatively short.

[0006] Complex, multiple c-KIT secondary mutations have been demonstrated to arise and change within individual patients, and these changes in c-KIT mutation status have been demonstrated by biopsy samples obtained from various progressing metastases within each patient. The complex heterogeneity of c-KIT mutability within individual patients highlights the unmet medical need to identify c-KIT kinase inhibitors that are effective across a broad range of c-KIT primary and secondary mutations. Furthermore, there is a need to identify therapies that are cytotoxic or cytodestructive against c-KIT-mediated GISTs, as opposed to simply cytotoxic, and that result in disease remission and / or reduced disease recurrence. Summary of the Invention

[0007] The present disclosure is directed to the combination of c-KIT inhibitor compound A or c-KIT inhibitor compound B with inhibitors of MAPKAP kinase signaling pathway. In this specification, MAPKAP pathway is defined as signaling through kinase RAF→MEK→ERK. It has been unexpectedly demonstrated that the combination of c-KIT inhibitor compound A or compound B with MEK inhibitors including trametinib, ERK inhibitors including ulixertinib, or RAF inhibitors including LY3009120 leads to cell death, apoptosis or prolonged cell stasis of GIST cells, enhanced GIST tumor regression in vivo, or eradication of GIST cells in colony formation assays, compared to the combination of imatinib and MEK inhibitors or compared to monotherapy with compound A, imatinib or MEK inhibitors. Furthermore, it has been demonstrated that the combination of c-KIT inhibitor compound A with MEK inhibitors leads to enhanced cell death or apoptosis and eradication of GIST cancer cell lines that are resistant to imatinib in combination with MEK inhibitors. In colony growth studies in GIST cells, Compound A exhibited superior synergy in combination with a MEK inhibitor compared to imatinib in combination with a MEK inhibitor.The present disclosure relates, in part, to a method of treating a tumor in a patient using Compound A, or a pharma- ceutically acceptable salt thereof, as described herein.

[0008] For example, a method of treating a tumor with one or more c-KIT mutations in a patient in need thereof may include administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, or a pharma- ceutical acceptable salt thereof, and an effective amount of a mitogen-activated protein kinase inhibitor (MEK inhibitor) and / or or administering an effective amount of an extracellular signal-regulated kinase inhibitor (ERK inhibitor).

[0009] The present disclosure also provides a method of treating a solid tumor in an imatinib-resistant patient, comprising administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea or a pharma- ceutically acceptable salt thereof; and administering to the patient an effective amount of a MEK inhibitor or an ERK inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib, and ulixertinib, wherein the solid tumor is selected from the group consisting of lung adenocarcinoma, lung squamous cell carcinoma, glioblastoma, pediatric glioma, astrocytoma, sarcoma, gastrointestinal stromal tumor (GIST), and melanoma.

[0010] Also contemplated herein is a method of treating imatinib-resistant gastrointestinal stromal tumor or imatinib-resistant melanoma in a patient in need thereof, comprising administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, or a pharma- ceutically acceptable salt thereof, and administering to the patient an effective amount of a MEK or ERK inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib, and ulixertinib.

[0011] The present disclosure also provides a method of treating a solid tumor in a patient in need thereof, comprising administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea or a pharma- ceutically acceptable salt thereof; and administering to the patient an effective amount of a MEK inhibitor or an ERK inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib, and ulixertinib, wherein the solid tumor is selected from the group consisting of lung adenocarcinoma, lung squamous cell carcinoma, glioblastoma, pediatric glioma, astrocytoma, sarcoma, gastrointestinal stromal tumor (GIST), and melanoma.

[0012] Also contemplated herein is a method of treating gastrointestinal stromal tumor or melanoma in a patient in need thereof, comprising administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, or a pharma- ceutically acceptable salt thereof, and administering to the patient an effective amount of a MEK or ERK inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib, and ulixertinib.

[0013] Further contemplated herein is a method of treating a solid tumor in a patient in need thereof, comprising administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, or a pharma- ceutically acceptable salt thereof, and administering to the patient an effective amount of a RAF inhibitor. [Brief description of the drawings]

[0014] [Figure 1-1]FIG. 1A shows a graphical representation of caspase activity after various treatments with Compound A and trametinib over a 24 hour period in GIST-T1 cells.

[0015] [Figure 1-2] FIG. 1B is a matrix diagram of synergy based on the combination index method for various treatments with Compound A and trametinib over 24 hours in GIST-T1 cells. The combination index plot shows synergy graphed as combination index (CI) on the y-axis and fraction affected (Fa) on the x-axis.

[0016] [Figure 1-3] FIG. 1C shows a graphical representation of caspase activity after various treatments with Compound A and trametinib in GIST-T1 cells over a 48 hour period.

[0017] FIG. 1D is a matrix diagram of synergy based on the combination index method for various treatments with Compound A and trametinib over 48 hours in GIST-T1 cells. The combination index plot shows synergy graphed as combination index (CI) on the y-axis and percent action (Fa) on the x-axis.

[0018] [Figure 1-4] FIG. 1E shows a graphical representation of caspase activity after various treatments with Compound A and trametinib in GIST-T1 / D816E imatinib-resistant cells over a 24-hour period.

[0019] FIG. 1F is a matrix diagram of synergy based on the combination index method for various treatments with Compound A and trametinib in GIST-T1 / D816E imatinib-resistant cells over 24 hours. The combination index plot shows synergy graphed as combination index (CI) on the y-axis and percent action (Fa) on the x-axis.

[0020] [Figure 1-5]FIG. 1G shows a graphical representation of caspase activity after various treatments with Compound A and trametinib in GIST-T1 / T670I imatinib-resistant cells over a 24-hour period.

[0021] FIG. 1H is a matrix diagram of synergy based on the combination index method for various treatments with Compound A and Trametinib in GIST-T1 / T670I imatinib-resistant cells over 24 hours. The combination index plot shows synergy graphed as combination index (CI) on the y-axis and percent action (Fa) on the x-axis.

[0022] [Figure 2-1] FIG. 2A shows a graphical representation of caspase activity after various treatments with Compound B and trametinib over a 24 hour period in GIST-T1 cells.

[0023] FIG. 2B is a matrix diagram of synergy based on the combination index method for various treatments with Compound B and trametinib over 24 hours in GIST-T1 cells. The combination index plot shows synergy graphed as combination index (CI) on the y-axis and percent action (Fa) on the x-axis.

[0024] [Figure 2-2] FIG. 2C shows a graphical representation of caspase activity after various treatments with Compound B and trametinib in GIST-T1 / D816E imatinib-resistant cells over a 24 hour period.

[0025] FIG. 2D is a matrix diagram of synergy based on the combination index method for various treatments with Compound B and Trametinib over 24 hours in GIST-T1 / D816E imatinib-resistant cells. The combination index plot shows synergy graphed as combination index (CI) on the y-axis and percent action (Fa) on the x-axis.

[0026] [Figure 2-3]FIG. 2E shows a graphical representation of caspase activity after various treatments with Compound B and trametinib in GIST-T1 / T670I imatinib-resistant cells over a 24 hour period.

[0027] FIG. 2F is a matrix diagram of synergy based on the combination index method for various treatments with compound B and trametinib in GIST-T1 / T670I imatinib-resistant cells over 24 hours. The combination index plot shows synergy graphed as combination index (CI) on the y-axis and percent action (Fa) on the x-axis.

[0028] [Figure 3-1] FIG. 3A shows a graphical representation of caspase activity after various treatments with Compound A and binimetinib over a 24 hour period in GIST-T1 cells.

[0029] FIG. 3B is a matrix diagram of synergy based on the combination index method for various treatments of GIST-T1 cells with Compound A and binimetinib over a 24-hour period. The combination index plot shows synergy graphed as combination index (CI) on the y-axis and percent effect (Fa) on the x-axis.

[0030] [Figure 3-2] FIG. 3C shows a graphical representation of caspase activity after various treatments with Compound A and binimetinib in GIST-T1 / D816E imatinib-resistant cells over a 24-hour period.

[0031] FIG. 3D is a matrix representation of synergy based on the combination index method for various treatments with Compound A and binimetinib over 24 hours in GIST-T1 / D816E imatinib-resistant cells. The combination index plot shows synergy graphed as combination index (CI) on the y-axis and percent action (Fa) on the x-axis.

[0032] [Figure 3-3]FIG. 3E shows a graphical representation of caspase activity after various treatments with Compound A and binimetinib in GIST-T1 / T670I imatinib-resistant cells over a 24-hour period.

[0033] FIG. 3F is a matrix diagram of synergy based on the combination index method for various treatments with Compound A and binimetinib over 24 hours in GIST-T1 / T670I imatinib-resistant cells. The combination index plot shows synergy graphed as combination index (CI) on the y-axis and percent action (Fa) on the x-axis.

[0034] [Figure 4-1] FIG. 4A shows a graphical representation of caspase activity after various treatments with Compound B and binimetinib over a 24 hour period in GIST-T1 cells.

[0035] FIG. 4B is a matrix diagram of synergy based on the combination index method for various treatments with Compound B and binimetinib over 24 hours in GIST-T1 cells. The combination index plot shows synergy graphed as combination index (CI) on the y-axis and percent action (Fa) on the x-axis.

[0036] [Figure 4-2] FIG. 4C shows a graphical representation of caspase activity after various treatments with Compound B and binimetinib in GIST-T1 / D816E imatinib-resistant cells over a 24 hour period.

[0037] FIG. 4D is a matrix diagram of synergy based on the combination index method for various treatments with Compound B and binimetinib over 24 hours in GIST-T1 / D816E imatinib-resistant cells. The combination index plot shows synergy graphed as combination index (CI) on the y-axis and percent action (Fa) on the x-axis.

[0038] [Figure 4-3]FIG. 4E shows a graphical representation of caspase activity after various treatments with Compound B and binimetinib in GIST-T1 / T670I imatinib-resistant cells over a 24 hour period.

[0039] FIG. 4F is a matrix diagram of synergy based on the combination index method for various treatments with Compound B and binimetinib over 24 hours in GIST-T1 / T670I imatinib-resistant cells. The combination index plot shows synergy graphed as combination index (CI) on the y-axis and percent action (Fa) on the x-axis.

[0040] [Figure 5-1] FIG. 5A shows a graphical representation of caspase activity after various treatments with Compound A and cobimetinib over a 24 hour period in GIST-T1 cells.

[0041] FIG. 5B is a matrix diagram of synergy based on the combination index method for various treatments with Compound A and cobimetinib over 24 hours in GIST-T1 cells. The combination index plot shows synergy graphed as combination index (CI) on the y-axis and percent action (Fa) on the x-axis.

[0042] [Figure 5-2] FIG. 5C shows a graphical representation of caspase activity after various treatments with Compound A and cobimetinib in GIST-T1 / D816E imatinib-resistant cells over a 24-hour period.

[0043] FIG. 5D is a matrix representation of synergy based on the combination index method for various treatments with Compound A and cobimetinib in GIST-T1 / D816E imatinib-resistant cells over 24 hours. The combination index plot shows synergy graphed as combination index (CI) on the y-axis and percent action (Fa) on the x-axis.

[0044] [Figure 5-3]FIG. 5E shows a graphical representation of caspase activity after various treatments with Compound A and cobimetinib in GIST-T1 / T670I imatinib-resistant cells over a 24-hour period.

[0045] FIG. 5F is a matrix diagram of synergy based on the combination index method for various treatments with Compound A and cobimetinib in GIST-T1 / T670I imatinib-resistant cells over 24 hours. The combination index plot shows synergy graphed as combination index (CI) on the y-axis and percent action (Fa) on the x-axis.

[0046] [Figure 6-1] FIG. 6A shows a graphical representation of caspase activity after various treatments with Compound B and cobimetinib over a 24 hour period in GIST-T1 cells.

[0047] FIG. 6B is a matrix diagram of synergy based on the combination index method for various treatments of GIST-T1 cells with Compound B and cobimetinib over a 24-hour period. The combination index plot shows synergy graphed as combination index (CI) on the y-axis and percent action (Fa) on the x-axis.

[0048] [Figure 6-2] FIG. 6C shows a graphical representation of caspase activity after various treatments with Compound B and cobimetinib in GIST-T1 / D816E imatinib-resistant cells over a 24-hour period.

[0049] FIG. 6D is a matrix diagram of synergy based on the combination index method for various treatments with Compound B and cobimetinib in GIST-T1 / D816E imatinib-resistant cells over 24 hours. The combination index plot is plotted with the combination index (CI) on the y-axis and the activity rate ( Shown is the synergy graphed as Fa.

[0050] [Figure 6-3]FIG. 6E shows a graphical representation of caspase activity after various treatments with Compound B and cobimetinib in GIST-T1 / T670I imatinib-resistant cells over a 24-hour period.

[0051] FIG. 6F is a matrix diagram of synergy based on the combination index method for various treatments with Compound B and cobimetinib in GIST-T1 / T670I imatinib-resistant cells over 24 hours. The combination index plot shows synergy graphed as combination index (CI) on the y-axis and percent action (Fa) on the x-axis.

[0052] [Figure 7-1] FIG. 7A shows a graphical representation of caspase activity after various treatments with Compound A and the ERK inhibitor ulixertinib in GIST-T1 cells over a 24 hour period.

[0053] FIG. 7B is a matrix diagram of synergy based on the combination index method for various treatments with Compound A and ulixertinib over 24 hours in GIST-T1 cells. The combination index plot shows synergy graphed as combination index (CI) on the y-axis and percent effect (Fa) on the x-axis.

[0054] [Figure 7-2] FIG. 7C shows a graphical representation of caspase activity after various treatments with Compound A and ulixertinib in GIST-T1 / T670I imatinib-resistant cells over a 24-hour period.

[0055] FIG. 7D is a synergy matrix diagram based on the combination index method for various treatments with Compound A and ulixertinib in GIST-T1 / T670I imatinib-resistant cells over 24 hours.

[0056] [Figure 8-1]FIG. 8A shows images of representative culture plates and a graphical representation of the number of GIST-T1 colonies counted after various treatments with Compound A, imatinib, and trametinib over a 2-week period followed by a 9-day recovery period.

[0057] [Figure 8-2] Figure 8B shows a representative culture plate image and a graphical representation of the number of GIST-T1 / D816E colonies counted after various treatments with Compound A, imatinib, and trametinib for 2 weeks followed by a 10-day recovery period. The top right panel shows a representative culture plate after an additional 10 days of recovery.

[0058] [Figure 8-3] FIG. 8C shows images of representative culture plates and a graphical representation of the number of GIST-T1 / D816E colonies counted after various treatments with Compound A, imatinib (IM), and trametinib over a 2-week period followed by a 10-day recovery period.

[0059] FIG. 8D shows images of representative culture plates and a graphical representation of the number of GIST-T1 / T670I colonies counted after various treatments with Compound A, imatinib, and trametinib over a 2-week period followed by a 10-day recovery period.

[0060] [Figure 9-1] FIG. 9A shows images of representative culture plates and a graphical representation of the number of GIST-T1 colonies counted after various treatments with Compound B and trametinib over a 2-week period followed by a 10-day recovery period.

[0061] [Figure 9-2] Figure 9B shows images of representative culture plates and a graphical representation of the number of GIST-T1 / D816E colonies counted after various treatments with Compound B and trametinib over a 2-week period followed by a 10-day recovery period. The top right panel shows a representative culture plate after an additional 10 days of recovery.

[0062] [Figure 9-3] FIG. 9C shows images of representative culture plates showing the number of GIST-T1 / T670I colonies counted after various treatments with Compound B and trametinib over a 2-week period followed by a 10-day recovery period.

[0063] [Figure 10-1] Figure 10A shows a representative culture plate image and a graphical representation of the number of GIST-T1 colonies counted after various treatments with Compound A, imatinib, and binimetinib for 2 weeks followed by a 10-day recovery period. The top right panel shows a representative culture plate after an additional 10 days of recovery.

[0064] [Figure 10-2] Figure 10B shows a representative culture plate image and a graphical representation of the number of GIST-T1 / D816E colonies counted after various treatments with Compound A, imatinib, and binimetinib for 2 weeks followed by a 10-day recovery period. The top right panel shows a representative culture plate after an additional 10 days of recovery.

[0065] [Figure 10-3] Figure 10C shows a representative culture plate image and a graphical representation of the colony counts of GIST-T1 / T670I after various treatments with Compound A, imatinib, and binimetinib for 2 weeks followed by a 10-day recovery period. The top right panel shows a representative culture plate after an additional 10 days of recovery.

[0066] [Figure 11-1] Figure 11A shows a representative culture plate image and a graphical representation of the number of GIST-T1 colonies counted after various treatments with Compound B and binimetinib for 2 weeks followed by a 10-day recovery period. The top right panel shows a representative culture plate after an additional 10 days of recovery.

[0067] [Figure 11-2] Figure 11B shows a representative culture plate image and a graphical representation of the number of GIST-T1 / D816E colonies counted after various treatments with Compound B and binimetinib for 2 weeks followed by a 10-day recovery period. The top right panel shows a representative culture plate after an additional 10 days of recovery.

[0068] [Figure 11-3] Figure 11C shows a representative culture plate image and a graphical representation of the number of GIST-T1 / D816E colonies counted after various treatments with Compound B and binimetinib for 2 weeks followed by a 10-day recovery period. The top right panel shows a representative culture plate after an additional 10 days of recovery.

[0069] [Figure 12-1] Figure 12A shows a representative culture plate image and a graphical representation of the number of GIST-T1 colonies counted after various treatments with Compound A, imatinib, and cobimetinib for 2 weeks followed by a 10-day recovery period. The top right panel shows a representative culture plate after an additional 10 days of recovery.

[0070] [Figure 12-2] Figure 12B shows images of representative culture plates and a graphical representation of the number of GIST-T1 / D816E colonies counted after various treatments with Compound A, imatinib, and cobimetinib for 2 weeks followed by a 10-day recovery period. The top right panel shows a representative culture plate after an additional 10 days of recovery.

[0071] [Figure 12-3] Figure 12C shows an image of a representative culture plate and a graphical representation of the number of GIST-T1 / T670I colonies counted after various treatments with Compound A, imatinib, and cobimetinib for 2 weeks followed by a 10-day recovery period. The top right panel shows a representative culture plate after an additional 10 days of recovery.

[0072] [Figure 13-1] Figure 13A shows images of representative culture plates and a graphical representation of the number of GIST-T1 colonies counted after various treatments with Compound B and cobimetinib over a 2-week period followed by a 10-day recovery period. The top right panel shows a representative culture plate after an additional 10 days of recovery.

[0073] [Figure 13-2] Figure 13B shows images of representative culture plates and a graphical representation of the number of GIST-T1 / D816E colonies counted after various treatments with Compound B and cobimetinib over a 2-week period followed by a 10-day recovery period. The top right panel shows a representative culture plate after an additional 10 days of recovery.

[0074] [Figure 13-3] Figure 13C shows images of representative culture plates showing GIST-T1 / T670I colony counts after various treatments with Compound B and cobimetinib over a 2-week period followed by a 10-day recovery period. The top right panel shows a representative culture plate after an additional 10 days of recovery.

[0075] [Figure 14-1] FIG. 14A shows images of representative culture plates and a graphical representation of the number of GIST-T1 colonies counted after various treatments with Compound A and the ERK inhibitor ulixertinib over a 2-week period followed by a 10-day recovery period.

[0076] [Figure 14-2] FIG. 14B shows images of representative culture plates and a graphical representation of the number of GIST-T1 / D816E colonies counted after various treatments with Compound A and ulixertinib over a 2-week period followed by a 10-day recovery period.

[0077] [Figure 14-3]Figure 14C shows a representative culture plate image and a graphical representation of the number of GIST-T1 / T670I colonies counted after various treatments with Compound A and ulixertinib for 2 weeks followed by a 10-day recovery period. The top right panel shows a representative culture plate after an additional 10 days of recovery.

[0078] [Figure 15] FIG. 15 shows images of representative culture plates and a graphical representation of the number of GIST-T1 / D816E colonies counted after various treatments with Compound B and ulixertinib over a 2-week period.

[0079] [Figure 16-1] FIG. 16A shows a graphical representation of caspase activity from various treatments with Compound A and trametinib over 48 hours in GIST-T1 cells transfected with vector control or N-ras G12D.

[0080] FIG. 16B shows images of representative culture plates of GIST-T1 colonies transfected with vector control (FIG. 16B.1) or N-ras G12D (FIG. 16B.2) after various treatments with Compound A, imatinib, and trametinib followed by a 10-day recovery period.

[0081] [Figure 16-2] FIG. 16C shows a graphical representation of the colony numbers of GIST-T1 transfected with vector control (FIG. 16C.1) or N-ras G12D (FIG. 16C.2) counted after various treatments with Compound A, imatinib, and trametinib followed by a 10-day recovery period.

[0082] FIG. 16D shows various treatments with Compound A and trametinib followed by extended 21 Shown is an image of a representative culture plate of N-ras G12D transfected GIST-T1 colonies after a day recovery period.

[0083] [Figure 17] FIG. 17A is a graphical representation of Ba / F3 V560D KIT cell growth with c-KIT secondary mutations at T670I, K807E, or D816V, or cell growth harboring only the original c-KIT V560D mutation combined with additional non-c-KIT resistance mechanisms, upon saturation mutagenesis followed by single-agent treatment with either imatinib (left panel) or Compound A (right panel).

[0084] FIG. 17B is a graphical representation of Ba / F3 V560D KIT cell growth with c-KIT secondary mutations at T670I, K807E, or D816V, or cell growth harboring only the original c-KIT V560D mutation combined with additional non-c-KIT resistance mechanisms, upon saturation mutagenesis followed by combination treatment with either imatinib and trametinib (left panel) or Compound A and trametinib (right panel).

[0085] [Figure 18] FIG. 18A provides a graphical representation of GIST T1 xenograft tumor growth following treatment with single agent Compound A, single agent trametinib, or the combination of Compound A and trametinib.

[0086] FIG. 18B is an expanded graphical representation from FIG. 18A, showing the recovery of efficacy on tumor regression following treatment with single agent Compound A, single agent trametinib, or the combination of Compound A and trametinib. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0087]

[00087] 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo It has been found that the combination of -1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea (Compound A) with a MAPKAP kinase pathway inhibitor, such as trametinib, unexpectedly synergizes to induce cell death, apoptosis, or long-term cell stasis of GIST cells, induce tumor cell eradication, induce tumor regression, reduce tumor volume, inhibit tumor regrowth, and / or enhance cell death, apoptosis, or cell stasis or eradication of GIST cancer cell lines that are resistant to imatinib in combination with MEK inhibitors in the accompanying examples.In addition, the combination therapy disclosed herein appears to be cytocidal as opposed to merely cytostatic.

[0088] Without wishing to be bound by any particular theory, it is believed that many c-KIT inhibitors inhibit only certain mutant forms of c-KIT, such as the prominent exon 11 mutation observed in GIST. Other mutant forms of c-KIT are resistant to many c-KIT inhibitors, and often arise as secondary mutations in exons 13, 14, 17, and 18, rendering the tumor resistant to treatment with c-KIT inhibitors. The present disclosure provides a method of treating tumors, e.g., c-KIT-mediated tumors, such as GIST, by inhibiting both c-KIT and MAPKAP pathway kinases using a c-KIT inhibitor disclosed herein as Compound A or a pharma- ceutically acceptable salt thereof, or Compound B or a pharma-ceutically acceptable salt thereof. Surprisingly, Compound A and Compound B (and pharma- ceutically acceptable salts thereof) synergize with MEK inhibitors, ERK inhibitors, or RAF inhibitors to induce cell death, apoptosis, or prolonged cell stasis in GIST cells, induce tumor cell eradication to the limit of detection, reduce tumor volume, inhibit tumor regrowth, and / or, in combination with MAPKAP kinase inhibitors, induce enhanced cell death, apoptosis, cell stasis, or eradication to the limit of detection in GIST cancer cell lines resistant to imatinib. Compound A inhibits c-KIT resistance. In GIST cells containing mutations, it shows superior potency and synergy in combination with MEK inhibition compared to imatinib in combination with MEK inhibition. Moreover, the level of potency and synergy, and the extent of long-term cell stasis or eradication of GIST tumor cells in combination with MEK inhibition, are superior to those of imatinib in combination with MEK inhibition, even against cell lines known to be sensitive to imatinib. Again, without wishing to be bound by any particular theory, it is believed that compound A can inhibit a broader range of mutant forms of c-KIT in GIST cells than previous c-KIT inhibitors, including imatinib, in tumor cells, e.g., GIST cells, which may be through a mechanism involving the inhibition of drug efflux pumps, including BCRP efflux pumps. Imatinib is a substrate of BCRP efflux pump, leading to a decrease in intracellular concentration in tumor cells where this efflux pump is present (Eechoute, K, et al, Clin Cancer Res. 2015, 17, 406-15). GIST tumors have been demonstrated to overexpress the BCRP efflux pump in 93% (42 / 45) of GIST patient tumors evaluated (Feldman, R, et al. J Clin Oncol. 2015, 33, 58). Compound A is a potent inhibitor of the BCRP efflux transporter and has an IC 50 The value represents 40 nM.

[0089] Thus, in certain embodiments, the present disclosure provides a method of inducing long-term cell stasis, inducing cell death, inducing apoptosis of tumor cells, inducing eradication of tumor cells, inducing tumor regression, reducing tumor volume, inhibiting tumor regrowth, or inhibiting the growth of resistant tumor cells, comprising administering to a patient in need thereof an effective amount of (i) 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl ester (FPA) or (ii) 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl ester (FPA) or and (ii) a MAPKAP kinase inhibitor, e.g., the MEK inhibitors trametinib, binimetinib, or cobimetinib, the ERK inhibitor ulixertinib, or a RAF inhibitor. In certain embodiments of any of the methods disclosed herein, the tumor is a c-KIT-mediated solid tumor, e.g., a c-KIT-mediated GIST or melanoma. Definitions

[0090]

[00090] As used herein, Compound A and Compound B refer to 1-[4 -bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, and 1-(5-(7-amino-1-ethyl-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl)-4-bromo-2-fluorophenyl)-3-phenylurea, respectively. Pharmaceutically acceptable salts, tautomers, hydrates, and solvates of Compound A and Compound B are also contemplated in the present disclosure. The structures of Compound A and Compound B are shown below: [ka] 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea (Compound A) [ka] 1-(5-(7-amino-1-ethyl-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl)-4-bromo-2-fluorophenyl)-3-phenylurea (Compound B)

[0091] Methods for preparing Compound A and Compound B are disclosed in US Pat. No. 8,461,179 B1, the contents of which are incorporated herein by reference.

[0092] Exemplary methods and materials are described herein. In the specification and the appended claims, unless otherwise indicated by the context, the singular form includes the plural form. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0093] Throughout this disclosure, various patents, patent applications, and publications are referenced. The disclosures of these patents, patent applications, and publications are incorporated by reference in their entireties into this disclosure to more fully describe the state of the art as known to those skilled in the art as of the date of this disclosure. In the event of any inconsistency between these patents, patent applications, and publications and this disclosure, the present disclosure shall control.

[0094] For convenience, certain terms employed in the present specification, examples and claims are summarized here.Unless otherwise specified, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.The initial definition provided for a group or word provided in this disclosure applies to that group or word individually or as part of another group throughout this disclosure, unless otherwise indicated.

[0095]

[00095] "Pharmaceutically acceptable carriers, diluents or excipients" include those suitable for use in humans or animals. The term "adjuvant" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the United States Food and Drug Administration as acceptable for use in livestock.

[0096] "Pharmaceutically acceptable salts" include acid addition salts.

[0097] "Pharmaceutically acceptable acid addition salts" refers to salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which include, for example, but not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and for example, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, It is formed from such organic acids as formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid.

[0098] "Pharmaceutical composition" refers to a formulation of a compound described herein, such as Compound A or a pharma- ceutically acceptable salt thereof, with a vehicle generally acceptable in the art for the delivery of biologically active compounds to a mammal, such as a human. Such vehicles include any pharma- ceutically acceptable carrier, diluent, or excipient therefor.

[0099] Subjects or patients "in need of treatment" with the combination therapy of the present disclosure, e.g., compound A in combination with a MEK inhibitor, include patients with diseases and / or conditions that can be treated with the combination disclosed herein to achieve beneficial therapeutic results, e.g., GIST patients. Beneficial outcomes include objective responses, increased progression-free survival, increased survival, prolonged stable disease, and / or reduced severity of symptoms or delayed onset of symptoms. In certain embodiments, the patient in need of treatment is afflicted with tumor growth or tumor progression, and the patient is afflicted with, but is not limited to, lung adenocarcinoma, lung squamous cell carcinoma, glioblastoma, pediatric glioma, astrocytoma, sarcoma, melanoma, or gastrointestinal stromal tumor.

[0100]

[00100] When used in connection with the compounds or other therapeutic agents disclosed herein, " The term "effective amount" refers to an amount of a therapeutic agent, either alone or in combination, that is useful for treating or preventing a disease or disorder, e.g., Compound A or a MEK inhibitor. An effective amount of a therapeutic agent used in combination therapy is an amount of each therapeutic agent that is useful for treating or preventing a disease or disorder when used in combination therapy, even if the amount of one or both of the therapeutic agents in the absence of the other agent is not effective for treating or preventing the disease or disorder. In certain embodiments, an effective amount is an amount that results in long-term cell stasis of GIST cells, cytolethal GIST cell death, apoptosis of GIST cells, eradication of GIST cells, regression of GIST, reduction of GIST tumor volume, inhibition of GIST regrowth, and / or leads to long-term cell stasis, cell death, apoptosis, or eradication to the limit of detection of GIST cancer cell lines that are resistant to imatinib in combination with a MEK inhibitor, and / or leads to a beneficial clinical outcome for the condition treated with the compound compared to no treatment. An "effective amount" can vary depending on the form of administration, the specific location of the disease or disorder, and the age, weight, and general health of the subject. The amount of compound to be administered will depend on the extent, severity and type of disease or condition, the amount of treatment desired, and the release characteristics of the pharmaceutical preparation.It will also depend on the subject's health condition, size, weight, age, sex and tolerance to drugs.Typically, the compound is administered for a period of time sufficient to achieve the desired therapeutic effect.

[0101] The terms "treatment," "treat," and "treating" refer to the therapeutic effects of, as used herein, inducing long-term cytostasis of GIST cells, inducing cytolethal GIST cell death, inducing apoptosis of GIST cells, inducing eradication of GIST tumor cells to the limit of visual detection as determined by stereomicroscopy at 5x magnification, causing regression of GIST tumors in a patient, reducing GIST tumor volume, inhibiting regrowth of GIST, and / or alleviating, delaying, or reversing, for example, one or more symptoms. It is meant to include the full range of interventions in patients with "cancer" with the intent of inhibiting the growth of resistant GIST cells in a given treatment, such as administering a combination therapy as disclosed herein, as well as inducing regression of GIST, even if it does not actually eliminate GIST. In some embodiments, treating includes completely eliminating the disease or disorder, e.g., GIST. Treating may be curing, improving, or at least partially alleviating the disorder.

[0102] "Cancer," as defined herein, refers to a neoplasm that has the ability to invade surrounding tissues, metastasize (spread to other organs), and ultimately, if untreated, may cause death to the patient. In certain embodiments, the cancer may be a solid tumor.

[0103] As used herein, "tumor" refers to a mass. It is a term that can refer to a benign (generally harmless) or malignant (cancerous) growth. Malignant growths can originate in solid organs or bone marrow.

[0104] "Tumor growth", as defined herein, refers to the growth of a tumor mass caused by genomic mutations in the c-KIT gene, which may alter the expression and / or activity of the c-KIT protein.

[0105]

[00105] As defined herein, "tumor progression" refers to the progression of a pre-existing c- It refers to KIT-dependent tumor growth, where such growth of an existing tumor mass can result from further genomic alterations of c-KIT that are resistant to treatment.

[0106] As defined herein, "tumor regression," "complete response," and "partial response" refer to a reduction in tumor size as determined by weight or volume as determined by RECIST 1.1 or Choi criteria.

[0107] Eradication of existing c-KIT-mediated tumors, such as c-KIT-mediated GIST, is defined as "complete cell lethality cell killing" of tumors to the limit of detection as determined by stereomicroscopy at 5x magnification for in vitro evaluation, or as complete remission as determined by RECIST 1.1 or Choi criteria for in vivo preclinical or clinical evaluation without the possibility of tumor regrowth under preclinical or clinical conditions. Thus, "eradication of c-KIT-mediated tumors" indicates that all cells of c-KIT-mediated tumors are killed or removed to the limit of detection without the possibility of c-KIT-mediated tumor regrowth.

[0108] As used herein, "tumor regrowth" refers to the growth of a tumor that previously stopped growing or regressed following treatment, e.g., Gleevec® or Sutent®. In certain embodiments, tumor regrowth occurs due to the introduction of a secondary c-Kit mutation in tumor cells. In other embodiments, tumor regrowth occurs due to the activation or mutation of a different signaling pathway, including, but not limited to, activation of the MAPKAP signaling pathway, including signaling through MEK kinase.

[0109] As used herein, "cell stasis" refers to cells that have stopped dividing and remain in a dormant, non-replicating state.

[0110]

[00110] As used herein, "apoptosis" refers to programmed cell death. Hallmarks of apoptosis detectable by histological and histochemical methods include cell shrinkage, increased membrane permeability, nuclear and cytoplasmic condensation, endolytic cleavage of nuclear DNA into oligonucleosomal fragments, and endocytic cleavage by macrophages. The process involves the eventual formation of apoptotic bodies that are absorbed and removed by the cellular nucleus. Apoptosis is primarily mediated by caspases, which are aspartate-specific serine proteases. Apoptosis can be induced through intrinsic genetic programming in response to various conditions, e.g., DNA damage or removal of growth factors, or apoptosis can be induced by exogenous factors, such as damage to cellular DNA by irradiation and some cytotoxic drugs used in cancer treatment. It can be inhibited by naturally occurring factors (e.g., cytokines) and by some drugs (e.g., protease inhibitors). Apoptosis typically does not occur or is impaired in malignant cells. In certain embodiments, apoptosis refers to programmed cell death determined by an increase in cleaved and activated caspase 3 and caspase 7.

[0111] "Combination therapy" refers to a treatment that includes administration of two or more therapeutic agents to a patient, e.g., Compound A and a MEK inhibitor. Two or more therapeutic agents may be delivered simultaneously, e.g., in separate pharmaceutical compositions or in the same pharmaceutical composition, or two or more therapeutic agents may be delivered at different times. For example, two or more therapeutic agents may be delivered simultaneously or during overlapping times, and / or one therapeutic agent may be delivered before or after the other therapeutic agent. Treatment involving the combination of a KIT inhibitor, such as Compound A, and a MEK inhibitor may include treatment with either single agent, preceded by or followed by a period of simultaneous treatment with both agents. However, it is intended that during some period of time, an effective amount of two or more therapeutic agents will be present in the patient's body.

[0112] "MAPKAP pathway inhibitors" are inhibitors of the MAP kinase signaling pathway. Inhibitors of this pathway include RAS inhibitors, RAF inhibitors (e.g., dabrafenib, vemurafenib, LY3009120), MEK inhibitors (e.g., trametinib, binimetinib, cobimetinib), and ERK inhibitors (e.g., ulixertinib). The terms "MAPKAP pathway inhibitors" and "MAPKAP kinase inhibitors" are used interchangeably herein. Treatment method

[0113] The compounds and compositions described herein can be used to treat tumors in patients in need thereof. For example, a method for treating tumors with one or more c-KIT mutations in a patient in need thereof is provided, comprising administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea or its pharma- ceutically acceptable salt and an effective amount of one or more MAPKAP kinase inhibitors. In one embodiment, the MAPKAP kinase inhibitor is selected from the group consisting of a mitogen-activated protein kinase inhibitor (MEK inhibitor) and an effective amount of an extracellular signal-regulated kinase inhibitor (ERK inhibitor).

[0114] The c-KIT mutation can be a primary mutation in exon 9, exon 11, exon 13, or exon 17 of the c-KIT gene. In another embodiment, the c-KIT mutation is a deletion mutation.

[0115]

[00115] Additionally, the tumor may have one or more secondary resistance mutations in the c-KIT gene. In some embodiments, the resistance secondary mutation is in exon 13, exon 14, exon 17, or exon 18 in the c-KIT gene. In some embodiments, the resistance secondary mutation is in exon 17 in the c-KIT gene. In some embodiments, the resistance secondary mutation is a substitution of aspartic acid at codon 816, or a substitution of asparagine at codon 822. In some embodiments, the resistance secondary mutation is D816V, D816E, D816H, ... 20A, T670I, or N822V. In some embodiments, the resistance secondary mutation was acquired after prior administration of imatinib, sunitib, or regorafenib, or a pharma- ceutically acceptable salt thereof, to the patient.

[0116] The disclosed method further comprises determining whether the tumor is associated with a c-KIT secondary mutation. In some embodiments, determining whether the tumor is associated with a c-KIT secondary mutation comprises identifying a mutation in DNA extracted from a tumor sample. In some embodiments, determining whether the tumor is associated with a c-KIT secondary mutation comprises identifying a mutation in circulating tumor DNA. In another embodiment, the tumor is resistant to treatment with imatinib mesylate, sunitinib malate, or regorafenib.

[0117] Furthermore, the tumor may be selected from the group consisting of lung adenocarcinoma, lung squamous cell carcinoma, glioblastoma, pediatric glioma, astrocytoma, sarcoma, gastrointestinal stromal tumor (GIST), and melanoma. In some embodiments, the tumor is melanoma. In some embodiments, the tumor is GIST.

[0118] The method may further include administering to the patient a cancer targeted therapeutic, a cancer targeted biologic, an immune checkpoint inhibitor, and / or a chemotherapeutic agent. The method may also further include administering to the patient a RAF inhibitor.

[0119] In another embodiment, 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl-3-phenylurea, or a pharma- ceutically acceptable salt thereof, and the MAPKAP kinase inhibitor are administered substantially simultaneously or sequentially.

[0120]

[00120] The MEK inhibitor in the disclosed method is trametinib, selumetinib, co- In some embodiments, the MEK inhibitor is binimetinib. In some embodiments, the MEK inhibitor is trametinib. In some embodiments, the ERK inhibitor is selected from the group consisting of ulixertinib, SCH772984, and LY3214996.

[0121] Administration for 2 weeks or more according to these disclosed methods may result in a partial reduction in tumor volume of at least 30% in a patient, hi some embodiments, treatment results in a complete reduction in tumor volume.

[0122] The disclosed method may further comprise determining whether the tumor or tumor cell comprises a primary mutation of the c-KIT gene. In some embodiments, the primary mutation is in exon 11 of the c-KIT gene. In some embodiments, the primary mutation is in exon 9 of the c-KIT gene. In some embodiments, the primary mutation is a deletion mutation. In some embodiments, the primary mutation is V560D. In other embodiments, there are one or more additional secondary mutations of the c-KIT mutation.

[0123] The present disclosure provides a method of treating a solid tumor in a patient resistant to imatinib, comprising administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, or a pharma- ceutically acceptable salt thereof, and ... selected from the group consisting of trametinib, binimetinib, cobimetinib, and ulixertinib. and administering an effective amount of a MAPKAP kinase inhibitor to the solid tumor, wherein the solid tumor is selected from the group consisting of lung adenocarcinoma, lung squamous cell carcinoma, glioblastoma, pediatric glioma, astrocytoma, sarcoma, gastrointestinal stromal tumor (GIST), and melanoma. In some embodiments, the method further comprises administering a RAF inhibitor. In some embodiments, the RAF inhibitor is a pan-RAF inhibitor.

[0124] Also provided herein is a method of treating imatinib-resistant gastrointestinal stromal tumor or imatinib-resistant melanoma in a patient in need thereof, comprising administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, or a pharma- ceutically acceptable salt thereof, and administering to the patient an effective amount of a MAPKAP kinase inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib, and ulixertinib.

[0125]

[00125] In some embodiments, the method comprises determining whether the tumor is associated with a mutation in the c-KIT gene. In some embodiments, the mutation is in exon 17 of the c-KIT gene. In some embodiments, the c-KIT mutation is a substitution of aspartic acid at codon 816 or an asparagine at codon 822. In some embodiments, the mutation is one of D816V, D816E, D816H, D820A, T670I, or N822V.

[0126] Further provided is a method of treating a solid tumor in a patient in need thereof, comprising administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, or a pharma- ceutically acceptable salt thereof, and administering to the patient an effective amount of a RAF inhibitor.

[0127] In such disclosed methods, the solid tumor may be selected from the group consisting of lung adenocarcinoma, lung squamous cell carcinoma, GIST, and melanoma. In some embodiments, the solid tumor is associated with one or more mutations of the c-KIT gene.

[0128] Furthermore, the RAF inhibitor can be a pan-RAF inhibitor. In another embodiment, the RAF inhibitor is dabrafenib, vemurafenib, or LY3009120.

[0129] The present disclosure also provides a method of treating a solid tumor in a patient in need thereof, comprising administering to the patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea or a pharma- ceutically acceptable salt thereof, and administering to the patient an effective amount of a MAPKAP kinase inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib and ulixertinib, wherein the solid tumor is selected from the group consisting of lung adenocarcinoma, lung squamous cell carcinoma, glioblastoma, pediatric glioma, astrocytoma, sarcoma, gastrointestinal stromal tumor (GIST), and melanoma. In some embodiments, the method further comprises administering a RAF inhibitor. In some embodiments, the RAF inhibitor is a pan-RAF inhibitor.

[0130]

[00130] Provided herein is a method for treating gastrointestinal stromal tumors or A method of treating melanoma comprising administering to a patient an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, or a pharma- ceutical acceptable salt thereof, and administering to the patient trametinib, binimetinib, cobimetinib, and administering to the patient an effective amount of a MAPKAP kinase inhibitor selected from the group consisting of: ribavirinib, ribavirinib, and ulixertinib.

[0131] In some embodiments, the method further comprises determining whether the tumor is associated with a mutation of the c-KIT gene. In some embodiments, the mutation is in exon 17 of the c-KIT gene. In some embodiments, the c-KIT mutation is a substitution of aspartic acid at codon 816 or a substitution of asparagine at codon 822. In some embodiments, the mutation is one of D816V, D816E, D816H, D820A, T670I, or N822V.

[0132] In one embodiment, the disclosure provides a method of treating or preventing a tumor, optionally a c-KIT mediated tumor, in a patient, e.g., GIST, comprising administering to a patient in need thereof an effective amount of Compound A or a pharmaceutically acceptable salt thereof in combination with an effective amount of a MEK inhibitor, e.g., trametinib. In a related embodiment, the disclosure provides a method of treating or preventing a tumor, optionally a c-KIT mediated tumor, in a patient, e.g., GIST, comprising administering to a patient in need thereof an effective amount of Compound B or a pharmaceutically acceptable salt thereof in combination with an effective amount of a MEK inhibitor, e.g., trametinib.

[0133] In certain embodiments, the methods include methods for inducing long-term stasis of tumor cells, e.g. GIST cells, killing tumor cells, e.g. GIST cells, inducing apoptosis of tumor cells, e.g. GIST cells, inducing eradication of tumor cells, e.g. GIST cells, to the limit of detection, inducing tumor cell regression, e.g. GIST regression, reducing tumor volume, e.g. GIST tumor volume, inhibiting tumor regrowth, e.g. GIST regrowth. In another particular embodiment, the methods include methods for inducing long-term stasis of tumor cells, e.g. GIST cells. In another particular embodiment, the methods include methods for killing tumor cells, e.g. GIST cells. In another particular embodiment, the methods include methods for inducing apoptosis of tumor cells, e.g. GIST cells. In another particular embodiment, the methods include methods for inducing eradication of tumor cells, e.g. GIST cells, to the limit of detection. In another particular embodiment, the methods include methods for inducing tumor regression, e.g. GIST regression. In another particular embodiment, the methods include methods for reducing tumor volume, e.g. GIST tumor volume. In another particular embodiment, the methods include methods for inhibiting tumor regrowth, e.g. GIST regrowth. In another specific embodiment, these methods include a method of inhibiting the growth of drug-resistant tumor cells, e.g., drug-resistant GIST cells. In a specific embodiment, the method includes a method of eradicating tumors, e.g., GIST, to the limit of detection in a subject. In a specific embodiment of any of the methods disclosed herein, tumor growth or tumor progression in a patient is caused by c-KIT overexpression, constitutive phosphorylation of c-KIT, increased c-KIT activity, oncogenic c-KIT missense mutations, oncogenic deletion c-KIT mutations, oncogenic nucleotide duplications / insertions, oncogenic c-KIT gene rearrangements leading to c-KIT fusion proteins, c-KIT intragenic in-frame deletions, and / or oncogenic c-KIT gene amplification. In one embodiment, tumor growth or tumor progression is caused by constitutive phosphorylation of c-KIT.In certain embodiments, the tumor comprises one or more of the c-KIT primary activating mutations and / or c-KIT secondary mutations disclosed herein. In another specific embodiment, the tumor comprises one or more mutations in genes other than c-KIT that cause tumor growth by signaling through the MAPKAP pathway, which involves RAF, MEK or ERK kinase activation.

[0134] The methods described herein include, but are not limited to, administering to a subject with Compound A or a pharma- ceutically acceptable salt thereof, When referring to treatment with compound A or a pharma- ceutically acceptable salt thereof, it is meant that only one of compound A or a pharma- ceutically acceptable salt thereof, or compound B or a pharma- ceutically acceptable salt thereof, is required. However, it is understood that these methods encompass administering to a patient both compound A or a pharma- ceutically acceptable salt thereof, and compound B or a pharma- ceutically acceptable salt thereof, in combination with a MEK inhibitor, an ERK inhibitor, or a RAF inhibitor. It is further understood that when compound A is administered to a subject in combination with a MEK inhibitor, an ERK inhibitor, or a RAF inhibitor, a portion of compound A is metabolized in vivo to compound B, and that an in vivo mixture of compound A and compound B can also be used to effectively treat a subject in combination with a MEK inhibitor, an ERK inhibitor, or a RAF inhibitor.

[0135]

[00135] Exemplary MEK inhibitors that can be used in accordance with the methods and compositions of the present disclosure include Examples include, but are not limited to, trametinib, selumetinib, cobimetinib, and binimetinib.

[0136] Exemplary ERK inhibitors that may be used in accordance with the methods and compositions of the present disclosure include, but are not limited to, ulixertinib, SCH772984, LY3214996, ravoxertinib, and VX-11e.

[0137] Exemplary RAF inhibitors that may be used in accordance with the methods and compositions of the disclosure include, but are not limited to, LY3009120, dabrafenib, and vemurafenib.

[0138] In one embodiment, compound A or a pharma- ceutically acceptable salt thereof and a MEK inhibitor, e.g., trametinib, are administered to a patient suffering from a c-KIT-mediated tumor, e.g., GIST. In another embodiment, compound B or a pharma- ceutically acceptable salt thereof and a MEK inhibitor, e.g., trametinib, are administered to a patient suffering from a c-KIT-mediated tumor, e.g., GIST.

[0139] In a related embodiment, compound A or a pharma- ceutically acceptable salt thereof and a MEK inhibitor, e.g., trametinib, are administered to a patient suffering from a tumor, e.g., a patient suffering from GIST, wherein the tumor growth or tumor progression is caused by a c-KIT primary activating mutation and / or a c-KIT secondary mutation. In another embodiment, compound B or a pharma- ceutically acceptable salt thereof and a MEK inhibitor, e.g., trametinib, are administered to a patient suffering from a tumor, e.g., a patient suffering from GIST, wherein the tumor growth or tumor progression is caused by a c-KIT primary activating mutation and / or a c-KIT secondary mutation. In certain embodiments, the c-KIT primary activating mutation is an exon 11 mutation (e.g., a 57 base pair exon 11 deletion). In certain embodiments, the c-KIT primary activating mutation is a duplication of A-Y502-503 in exon 9. In certain embodiments, the c-KIT primary activating mutation is an exon 13 mutation. In certain embodiments, the c-KIT primary activating mutation is an exon 17 mutation. In certain embodiments, the c-KIT secondary mutation is any resistance mutation disclosed herein, such as a T670I mutation or a D816E mutation. In certain embodiments, multiple c-KIT secondary resistance mutations are present simultaneously in a subject.

[0140]

[00140] In certain embodiments, compound A or a pharma- ceutically acceptable salt thereof and In certain embodiments, compound B or a pharmaceutically acceptable salt thereof and a MEK inhibitor, e.g., trametinib, are administered to a cancer patient. In certain embodiments, compound B or a pharmaceutically acceptable salt thereof and a MEK inhibitor, e.g., trametinib, are administered to a cancer patient. In embodiments, the tumor or cancer is a lung adenocarcinoma, a lung squamous cell carcinoma, a glioblastoma, a pediatric glioma, an astrocytoma, a sarcoma, a melanoma, or a gastrointestinal stromal tumor (GIST). In one embodiment, the cancer is a melanoma. In another embodiment, the tumor or cancer is a gastrointestinal stromal tumor (GIST). In certain embodiments of any of these methods, the tumor or cancer is a c-KIT-mediated cancer, for example a c-KIT-mediated GIST or melanoma.

[0141] Treatment with compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof in combination with a MEK inhibitor, for example trametinib, includes administering compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof, before, after, simultaneously with, or during the overlapping period with administering the MEK inhibitor. It is understood that the effective amount of any of compound A or a pharmaceutically acceptable salt thereof, compound B or a pharmaceutically acceptable salt thereof, or a MEK inhibitor, for example trametinib, may vary when used in combination as disclosed herein, compared to when any of these agents are used by themselves for the same purpose, for example to treat or prevent tumors. In certain embodiments, the effective amount of compound A or a pharmaceutically acceptable salt thereof, or compound B or a pharmaceutically acceptable salt thereof, when administered as a combination therapy with a MEK inhibitor, for example trametinib, is less than when administered as a monotherapy, for example to treat or prevent GIST. In certain embodiments, the effective amount of a MEK inhibitor, e.g., trametinib, when administered in combination therapy with Compound A, or a pharma- ceutically acceptable salt thereof, or when administered in combination therapy with Compound B, or a pharma-ceutically acceptable salt thereof, for treating or preventing GIST, is a lower amount.

[0142] Any of the methods disclosed herein may further comprise determining whether the tumor to be treated is associated with one or more c-KIT gene mutations. Such determination may be made by conventional methods for determining the presence of gene mutations in a biological sample obtained from a patient, e.g., a tumor sample, a blood sample, or a plasma sample. Furthermore, such determination may be made by reviewing the results of tests performed to determine the presence of one or more c-KIT gene mutations in a biological sample obtained from a patient, e.g., a tumor sample, a blood sample, or a plasma sample. In certain embodiments of any of the methods disclosed herein, the methods are performed on a patient whose tumor has been identified as associated with one or more c-KIT gene mutations. The c-KIT gene mutations include, but are not limited to, any of those specifically described herein.

[0143] In various embodiments of any of the methods disclosed herein, treatment with either compound A or a pharma- ceutically acceptable salt thereof, or compound B or a pharma- ceutically acceptable salt thereof, in combination with a MEK inhibitor, e.g., trametinib, induces long-term cell stasis of tumor cells, e.g., GIST cells, induces death of tumor cells, e.g., GIST cells, induces apoptosis of tumor cells, e.g., GIST cells, induces eradication of tumor cells, e.g., GIST cells, to the limit of detection, induces regression of tumor cells, e.g., GIST cells, reduces tumor weight or volume, e.g., GIST tumors, inhibits regrowth of tumors, e.g., GIST tumors. In another embodiment of any of the methods disclosed herein, treatment with either compound A or a pharma- ceutically acceptable salt thereof, or compound B or a pharma- ceutically acceptable salt thereof, in combination with a MEK inhibitor, e.g., trametinib, induces long-term cell stasis in tumor cells, e.g., GIST cells, induces death of tumor cells, e.g., GIST cells, induces apoptosis of tumor cells, e.g., GIST cells, induces eradication of tumor cells, e.g., GIST cells, to the limit of detection, induces regression of tumor cells, e.g., GIST cells, reduces tumor weight or volume, e.g., GIST tumors, inhibits tumor regrowth, e.g., GIST tumors, in drug-resistant tumors, e.g., drug-resistant GIST. In another embodiment of any of the methods disclosed herein In this case, treatment with either compound A or a pharma- ceutically acceptable salt thereof, or compound B or a pharma- ceutically acceptable salt thereof, in combination with a MEK inhibitor, e.g., trametinib, eradicates tumors to the limit of detection in treated patients, e.g., GIST patients. Methods for measuring or determining the amount of tumor cell stasis, tumor cell death, tumor cell apoptosis, tumor regression, tumor weight or volume, tumor regrowth, growth of resistant tumor cells, and tumor eradication are known in the art and include any of the methods described herein.

[0144] In certain embodiments, the combination treatment of compound A or a pharma- ceutically acceptable salt thereof, or compound B or a pharma-ceutically acceptable salt thereof; and a MEK inhibitor, for example trametinib, results in an increase in the amount of tumor cell stasis, tumor cell death, or tumor cell apoptosis, for example GIST cells, compared to the amount of tumor cell stasis, cell death, or apoptosis of the same type or tumor type that is either treated or not treated with a MEK inhibitor, for example trametinib, alone, or with a c-KIT inhibitor, for example imatinib, alone, or with a MEK inhibitor, for example trametinib, in combination with a c-KIT inhibitor, imatinib. For example, cell stasis, cell death, or apoptosis can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20-fold. In certain embodiments, the amount of apoptosis is determined by measuring caspase activity in the tumor cells.

[0145]

[00145] In certain embodiments, the treatment is with a combination of: Compound A or A pharmaceutically acceptable salt thereof, or compound B or its pharmaceutically acceptable salt; and a MEK inhibitor, for example trametinib, cause increased tumor regression or a decrease in tumor size or volume (for example GIST) compared to the size, for example weight or mass, of the same type of tumor or the same tumor that is either treated or not treated with only a MEK inhibitor, for example trametinib, or only a c-KIT inhibitor, for example imatinib.For example, tumor weight or volume can be decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0146] In certain embodiments, the combination treatment of: Compound A or its pharmaceutically acceptable salt, or Compound B or its pharmaceutically acceptable salt; and MEK inhibitor, for example trametinib, inhibits the amount of tumor growth or regrowth, for example GIST growth or regrowth, to a greater extent than the amount of tumor growth or regrowth of the same type or the same tumor that is treated or not treated with MEK inhibitor alone, for example trametinib, or with c-KIT inhibitor alone, for example imatinib.For example, tumor growth or regrowth can be inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0147] In a particular embodiment, the treatment with the combination of: Compound A or a pharma- ceutically acceptable salt thereof, or Compound B or a pharma- ceutically acceptable salt thereof; and a MEK inhibitor, e.g., trametinib, increases the proliferation of resistant tumor cells, e.g., resistant GIST cells, to a greater extent compared to the amount of proliferation of resistant tumor cells of the same type or tumor that are either treated or not treated with a MEK inhibitor alone, e.g., trametinib, or with a c-KIT inhibitor alone, e.g., imatinib, or with a combination of a MEK inhibitor, e.g., trametinib, and a c-KIT inhibitor, e.g., imatinib. The resistant tumor cells inhibit proliferation. For example, the amount or number of proliferation of resistant tumor cells can be inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In certain embodiments, the resistant tumor cells are resistant to treatment with a c-KIT inhibitor, e.g., imatinib, and / or a MEK inhibitor, e.g., trametinib. In certain embodiments, the resistant tumor cells comprise a c-KIT secondary mutation. In certain embodiments, the c-KIT secondary mutation is a mutation in any of the following amino acid residues of c-KIT: V654, N655, T670, L783, D816, D820, N822, Y823, A829, and / or T847, including but not limited to any of the amino acid substitutions shown in the accompanying figures. In certain embodiments, the resistant tumor cells contain an activated MAPKAP kinase pathway, which in certain embodiments, includes a mutation in a RAS gene, e.g., the N-RAS or K-RAS gene, a fibroblast growth factor receptor (FGFR) gene, and / or a mutation in the neurofibromin-1 (NF1) gene. In certain embodiments, the mutation is an N-RAS G12D mutation.

[0148] In certain embodiments, the combination treatment of compound A or its pharma- ceutically acceptable salt, or compound B or its pharma-ceutically acceptable salt, in combination with MEK inhibitor, for example trametinib, results in the eradication of tumor, for example GIST, to the limit of detection. In certain embodiments, the eradication of tumor means that there is no more tumor detectable in the patient to the limit of detection. In certain embodiments, after the eradication of tumor, for example GIST, by the combination therapy disclosed herein, there is no more tumor detectable in the patient for at least 6 months, at least 1 year, at least 2 years, at least 5 years, or at least 10 years. The eradication of tumor can be determined by optical emission tomography (PET), CT scan, the absence of cell-free DNA (cfDNA) in blood containing c-KIT mutation, the absence of blood tumor cells (CTC) in the vasculature of the subject, or the absence of cancer cell biomarkers in the vasculature of the circulating blood of the subject.

[0149] In various embodiments of any of the methods disclosed herein, treatment with either compound A or a pharma- ceutically acceptable salt thereof, or compound B or a pharma- ceutically acceptable salt thereof, in combination with an ERK inhibitor, e.g., ulixertinib, induces long-term cell stasis of tumor cells, e.g., GIST cells, induces death of tumor cells, e.g., GIST cells, induces apoptosis of tumor cells, e.g., GIST cells, induces eradication of tumor cells, e.g., GIST cells, to the limit of detection, induces regression of tumors, e.g., GIST tumors, reduces the weight or volume of tumors, e.g., GIST tumors, inhibits regrowth of tumors, e.g., GIST tumors. In another embodiment of any of the methods disclosed herein, treatment with either compound A or a pharma- ceutically acceptable salt thereof, or compound B or a pharma-ceutically acceptable salt thereof, in combination with an ERK inhibitor, e.g., ulixertinib, induces long-term cell stasis in tumor cells, e.g., GIST cells, induces death of tumor cells, e.g., GIST cells, induces apoptosis of tumor cells, e.g., GIST cells, induces eradication of tumor cells, e.g., GIST cells, to the limit of detection, induces regression of tumors, e.g., GIST tumors, reduces tumor weight or volume, e.g., GIST tumors, inhibits tumor regrowth, e.g., GIST tumors, in drug-resistant tumors, e.g., drug-resistant GIST. In another embodiment of any of the methods disclosed herein, treatment with either compound A or a pharma-ceutically acceptable salt thereof, or compound B or a pharma-ceutically acceptable salt thereof, in combination with an ERK inhibitor, e.g., ulixertinib, eradicates tumors to the limit of detection in treated patients, e.g., GIST patients. Methods for measuring or determining the amount of tumor cell stasis, tumor cell death, tumor cell apoptosis, tumor regression, tumor weight or volume, tumor regrowth, growth of resistant tumor cells, and tumor eradication are known in the art and include any of the methods described herein.

[0150]

[00150] In certain embodiments, the treatment is with a combination of: Compound A or A pharmaceutically acceptable salt, or compound B or its pharmaceutically acceptable salt; and an ERK inhibitor, for example ulixertinib, cause an increase in the amount of tumor cell stasis, tumor cell death or tumor cell apoptosis, for example GIST cells, compared to the amount of stasis, cell death or apoptosis of tumor cells of the same type or of the same tumor type that are either treated or not treated with an ERK inhibitor alone, for example ulixertinib, or with a c-KIT inhibitor alone, for example imatinib, or with an ERK inhibitor, for example ulixertinib, in combination with a c-KIT inhibitor imatinib. For example, cell stasis, cell death or apoptosis can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20-fold. In certain embodiments, the amount of apoptosis is determined by measuring caspase activity in the tumor cells.

[0151] In certain embodiments, the combination treatment of compound A or its pharmaceutically acceptable salt, or compound B or its pharmaceutically acceptable salt; and an ERK inhibitor, for example ulixertinib, results in increased tumor regression or a decrease in tumor size or volume (for example GIST) compared to the tumor size, for example weight or volume, of the same type or same tumor that is either treated or not treated with only an ERK inhibitor, for example imatinib, or only a c-KIT inhibitor, for example imatinib.For example, tumor weight or volume can be decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0152] In certain embodiments, the combination treatment of: Compound A or its pharmaceutically acceptable salt, or Compound B or its pharmaceutically acceptable salt; and an ERK inhibitor, for example ulixertinib, inhibits the amount of tumor growth or regrowth, for example GIST growth or regrowth, to a greater extent than the amount of tumor growth or regrowth of the same type or the same tumor that is treated or not treated with an ERK inhibitor alone, for example ulixertinib, or with a c-KIT inhibitor alone, for example imatinib. For example, tumor growth or regrowth can be inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0153] In certain embodiments, the combination treatment of: compound A or its pharmaceutically acceptable salt, or compound B or its pharmaceutically acceptable salt; and an ERK inhibitor, for example ulixertinib, inhibits the proliferation of resistant tumor cells, for example resistant GIST cells, to a greater extent than the amount of proliferation of resistant tumor cells of the same type or the same tumor that are either treated or not treated with an ERK inhibitor alone, for example ulixertinib, or with a c-KIT inhibitor alone, for example imatinib, or with a combination of an ERK inhibitor, for example ulixertinib, and a c-KIT inhibitor, for example imatinib.For example, the amount or number of proliferation of resistant tumor cells can be inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In certain embodiments, the resistant tumor cells are resistant to treatment with a c-KIT inhibitor, e.g., imatinib, and / or an ERK inhibitor, e.g., ulixertinib. In certain embodiments, the resistant tumor cells comprise a c-KIT secondary mutation. In certain embodiments, the c-KIT secondary mutation is any of the following amino acid residues of c-KIT: The mutation is one of the following: V654, N655, T670, L783, D816, D820, N822, Y823, A829, and / or T847, including but not limited to any of the amino acid substitutions shown in the attached figures. In certain embodiments, the resistant tumor cells contain an activated MAPKAP kinase pathway, which in certain embodiments include a mutation in a RAS gene, e.g., the N-RAS or K-RAS gene, a fibroblast growth factor receptor (FGFR) gene, and / or a mutation in the neurofibromin-1 (NF1) gene. In certain embodiments, the mutation is an N-RAS G12D mutation.

[0154] In certain embodiments, the combination treatment of compound A or its pharma- ceutically acceptable salt, or compound B or its pharma-ceutically acceptable salt, in combination with an ERK inhibitor, for example ulixertinib, results in the eradication of tumors, for example GIST, to the limit of detection. In certain embodiments, the eradication of tumors means that there is no more tumor detectable in the patient to the limit of detection. In certain embodiments, after the eradication of tumors, for example GIST, by the combination therapy disclosed herein, there is no more tumor detectable in the patient for at least 6 months, at least 1 year, at least 2 years, at least 5 years, or at least 10 years. The eradication of tumors can be determined by optical emission tomography (PET), CT scan, the absence of cell-free DNA (cfDNA) in blood containing c-KIT mutation, the absence of tumor cells (CTC) in blood in the vasculature of the subject, or the absence of cancer cell biomarkers in the vasculature of the circulating blood of the subject.

[0155]

[00155] In various embodiments of any of the methods disclosed herein, a double layer Treatment with compound A or a pharma- ceutically acceptable salt thereof, or compound B or a pharma-ceutically acceptable salt thereof, in combination with a RAF inhibitor, which is a nib, induces long-term cell stasis of tumor cells, e.g., GIST cells, induces death of tumor cells, e.g., GIST cells, induces apoptosis of tumor cells, e.g., GIST cells, induces eradication of tumor cells, e.g., GIST cells, to the limit of detection, induces regression of tumors, e.g., GIST tumors, reduces tumor weight or volume, e.g., GIST tumors, inhibits regrowth of tumors, e.g., GIST tumors. In another embodiment of any of the methods disclosed herein, treatment with compound A or its pharmaceutically acceptable salt, or compound B or its pharmaceutically acceptable salt, in combination with a RAF inhibitor, e.g., dabrafenib, induces long-term cell stasis of tumor cells, e.g., GIST cells, induces death of tumor cells, e.g., GIST cells, induces apoptosis of tumor cells, e.g., GIST cells, induces eradication of tumor cells, e.g., GIST cells, to the limit of detection, induces regression of tumors, e.g., GIST tumors, reduces tumor weight or volume, e.g., GIST tumors, inhibits tumor regrowth, e.g., GIST tumors, in drug-resistant tumors, e.g., drug-resistant GIST. In another embodiment of any of the methods disclosed herein, treatment with compound A or its pharmaceutically acceptable salt, or compound B or its pharmaceutically acceptable salt, in combination with a RAF inhibitor, e.g., dabrafenib, eradicates tumors to the limit of detection in treated patients, e.g., GIST patients. Methods for measuring or determining the amount of tumor cell stasis, tumor cell death, tumor cell apoptosis, tumor regression, tumor weight or volume, tumor regrowth, growth of resistant tumor cells, and tumor eradication are known in the art and include any of the methods described herein.

[0156] In a particular embodiment, the treatment is with the following combination: Compound A or a pharma- ceutically acceptable salt thereof, or Compound B or a pharma- ceutically acceptable salt thereof; and a RAF inhibitor, e.g., dabrafenib, is treated or is being treated with a RAF inhibitor, e.g., dabrafenib, alone, or with a c-KIT inhibitor, e.g., imatinib, alone, or with a RAF inhibitor, e.g., dabrafenib, in combination with the c-KIT inhibitor imatinib. The amount of tumor cell retention, tumor cell death or tumor cell apoptosis, for example, GIST cells, is increased compared to the amount of tumor cell retention, cell death or apoptosis of the same type or the same tumor type that is either absent or not present. For example, cell retention, cell death or apoptosis can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20-fold. In certain embodiments, the amount of apoptosis is determined by measuring the caspase activity of tumor cells.

[0157] In certain embodiments, the combination treatment of compound A or its pharmaceutically acceptable salt, or compound B or its pharmaceutically acceptable salt; and a RAF inhibitor, for example dabrafenib, results in increased tumor regression or a decrease in tumor size or volume (for example GIST) compared to the tumor size, for example weight or volume, of the same type or same tumor that is either treated or not treated with only a RAF inhibitor, for example dabrafenib, or only a c-KIT inhibitor, for example imatinib.For example, tumor weight or volume can be decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0158] In certain embodiments, the combination treatment of compound A or its pharmaceutically acceptable salt, or compound B or its pharmaceutically acceptable salt; and a RAF inhibitor, for example dabrafenib, inhibits the amount of tumor growth or re-growth, for example GIST growth or re-growth, to a greater extent than the amount of tumor growth or re-growth of the same type or the same tumor that is treated or not treated with only a RAF inhibitor, for example dabrafenib, or only a c-KIT inhibitor, for example imatinib.For example, tumor growth or re-growth can be inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0159] In certain embodiments, the treatment with the combination of: compound A or its pharma- ceutically acceptable salt, or compound B or its pharma-ceutically acceptable salt; and a RAF inhibitor, for example dabrafenib, inhibits the proliferation of resistant tumor cells, for example resistant GIST cells, to a greater extent than the proliferation of resistant tumor cells of the same type or tumor that are either treated or not treated with a RAF inhibitor alone, for example dabrafenib, or with a c-KIT inhibitor alone, for example imatinib, or with a combination of a RAF inhibitor, for example dabrafenib, and a c-KIT inhibitor, for example imatinib.For example, the proliferation amount or number of resistant tumor cells can be inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.In certain embodiments, resistant tumor cells are resistant to treatment with a c-KIT inhibitor, for example imatinib, and / or a RAF inhibitor, for example dabrafenib. In certain embodiments, the resistant tumor cells comprise a c-KIT secondary mutation. In certain embodiments, the c-KIT secondary mutation is a mutation in any of the following amino acid residues of c-KIT: V654, N655, T670, L783, D816, D820, N822, Y823, A829, and / or T847, including but not limited to any of the amino acid substitutions shown in the accompanying figures. In certain embodiments, the resistant tumor cells comprise an activated MAPKAP kinase pathway, which in certain embodiments are a mutation in the RAS gene, e.g., the N-RAS or K-RAS gene, the fibroblast growth factor receptor (FGFR) gene, and / or the neurofibromin-1 (NF1) gene. In certain embodiments, the mutation is an N-RAS G12D mutation.

[0160]

[00160] In certain embodiments, the compound is combined with a RAF inhibitor, e.g., dabrafenib. Treatment with the combination of either compound A or its pharma- ceutically acceptable salt, or compound B or its pharma-ceutically acceptable salt, results in the eradication of tumors, for example GIST, to the limit of detection. In certain embodiments, eradication of tumor means that there is no more tumor detectable in the patient to the limit of detection. In certain embodiments, after the eradication of tumors, for example GIST, by the combination therapy disclosed herein, there is no more tumor detectable in the patient for at least 6 months, at least 1 year, at least 2 years, at least 5 years, or at least 10 years. Eradication of tumor can be determined by optical emission tomography (PET), CT scan, absence of cell-free DNA (cfDNA) in blood containing c-KIT mutation, absence of blood tumor cells (CTC) in the vasculature of the subject, or absence of cancer cell biomarkers in the vasculature of the circulating blood of the subject.

[0161] The present disclosure describes combination therapy involving administration of compound A or a pharma- ceutically acceptable salt thereof, or compound B or a pharma- ceutically acceptable salt thereof, with one or more MAPKAP kinase inhibitors, e.g., MEK inhibitors, ERK inhibitors, or RAF inhibitors. The combination therapy described herein can be used by itself or in further combination with one or more additional therapeutic agents (e.g., one or more additional therapeutic agents described below). For example, either compound A or a pharma- ceutically acceptable salt thereof, or compound B or a pharma- ceutically acceptable salt thereof, and a MEK inhibitor can be administered with a cancer targeting therapeutic agent, a cancer targeting biological agent, an immune checkpoint inhibitor, or a chemotherapeutic agent. In another embodiment, compound A or compound B and a MEK inhibitor are administered without other therapeutic agents. The therapeutic agent can be administered together with another therapeutic agent described herein in the combination therapy, or sequentially with the therapeutic agent.

[0162] Combination therapy can be achieved by administering two or more therapeutic agents, each of which is formulated and administered separately, or by administering two or more therapeutic agents in a single formulation. Other combinations are also encompassed in combination therapy. Two or more therapeutic agents in combination therapy can be administered simultaneously, but this is not necessary. For example, administration of a first agent (or combination of agents) can precede administration of a second agent (or combination of agents) by minutes, hours, days, or weeks. Thus, two or more agents can be administered within minutes of each other, or within 1, 2, 3, 6, 9, 12, 15, 18, or 24 hours of each other, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days of each other, or within 2, 3, 4, 5, 6, 7, 8, 9 weeks or more of each other. In some cases, even longer intervals are possible. In many cases, it is desirable, but not necessary, that the two or more agents used in combination therapy be present in the patient's body at the same time.

[0163] Combination therapy can also include more than one administration of one or more of the agents used in the combination, with the component agents in a different order, e.g., when agent X and agent Y are used in combination, they can be administered one or more times sequentially in any combination, e.g., in the order XYX, XXY, YXY, YYX, XXYY, etc.

[0164] The one or more additional therapeutic agents that may be administered in accordance with the present disclosure include cytotoxic agents, cisplatin, doxorubicin, etoposide, irinotecan, topotecan, paclitaxel, docetaxel, epothilones, tamoxifen, 5-fluorouracil, methotrexate, temozolomide, cyclophosphamide, lonafarib, tipifarnib, 4-((5 -((4-(3-chlorophenyl)-3-oxopiperazin-1-yl)methyl)-1H-imidazol-1-yl)methyl)benzonitrile hydrochloride, (R)-1-((1H-imidazol-5-yl)methyl)-3-benzyl-4-(thiophen-2-ylsulfonyl)-2,3,4,5-Tetrahydro-1H-benzodiazepine-7-carbonitrile, cetuximab, imatinib, interferon alpha-2b, pegylated interferon alpha-2b, aromatase combination, gemcitabine, uracil mustard, chlormethine, ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, carmustine, lomustine, streptozocin, decarba dinamin, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, leucovorin, oxaliplatin, pentostatin, vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, epirubicin, idarubicin, mithramycin, deoxycoformycin, mitomycin-C, L-asparaginase, teniposide 17α-ethynyl estradiol, diethylstilbestrol, testosterone, prednisone, fluoxymethasone Terone, dromostanolone propionate, testolactone, megestrol acetate, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, 17α-hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide acetate, flutamide, toremifene citrate, goserelin acetate, carboplatin, hydroxyurea, amsacrine, procarbazine, mitotane, mitoxantrone, levamisole, vinorelbine These include, but are not limited to, rubin, anastrazole, letrozole, capecitabine, raloxifene, droloxafine, hexamethylmelamine, bevacizumab, trastuzumab, tositumomab, bortezomib, ibritumomab tiuxetan, arsenic trioxide, porfimer sodium, cetuximab, thioTEPA, altretamine, fulvestrant, exemestane, rituximab, alemtuzumab, dexamethasone, bicalutamide, chlorambucil, and valrubicin.

[0165]

[00165] The one or more additional therapeutic agents that may be administered include, but are not limited to, Without any side effects, AKT inhibitors, alkylating agents, all-trans retinoic acid, antiandrogens, azacytidine, BCL2 inhibitors, BCL-XL inhibitors, BCR-ABL inhibitors, BTK inhibitors, BTK / LCK / LYN inhibitors, CDK1 / 2 / 4 / 6 / 7 / 9 inhibitors, CDK4 / 6 inhibitors, CDK9 inhibitors, CBP / p300 inhibitors, EGFR inhibitors, endothelin receptor antagonists, RAF inhibitors, MEK inhibitors, ERK inhibitors, farnesyltransferase inhibitors, FLT3 inhibitors, glucocorticoid receptor agonists, HDM2 inhibitors, histone deacetylase inhibitors, IKKβ inhibitors, immunomodulatory drugs (IMiDs), ingenol, ITK inhibitors, JAK1 / JAK2 / JAK3 / TYK2 inhibitors, MTOR inhibitors, PI3 kinase inhibitors, dual PI3 kinase / MTOR inhibitors, proteasome inhibitors, protein kinase C agonists, SUV39H1 inhibitors, TRAIL, VEGFR2 inhibitors, Wnt / β-catenin signaling inhibitors, decitabine, and anti-CD20 monoclonal antibodies.

[0166] In certain embodiments, the additional therapeutic agent is a CTLA4 inhibitor, e.g., but not limited to, ipilimumab and tremelimumab; a PD1 inhibitor, e.g., but not limited to, pembrolizumab and nivolumab, e.g., atezolizumab (formerly MPDL3280A), durvalumab (formerly MEDI4736), avelumab, a PDL1 inhibitor, e.g., but not limited to, PDR001, e.g., urelumab and PF-05082566, a 4 1BB or 4 1BB ligand inhibitor, e.g., but not limited to, MEDI6469, an OX40 ligand agonist, e.g., but not limited to, TRX518, a GITR agent, e.g., but not limited to, varlilumab CD27 inhibitors not limited to, TNFRSF25 or TL1A inhibitors, such as but not limited to CP-870893, CD40 agonists, HVEM or LIGHT or LTA or BTLA or CD160 inhibitors, such as but not limited to BMS-986016, LAG3 inhibitors, TIM3 inhibitors, Siglecs inhibitors, ICOS or ICOS ligand agonists, such as but not limited to MGA271, B7 H3 inhibitors, B7 H4 inhibitors, VISTA inhibitors, HHLA2 or TMIGD2 inhibitors, inhibitors of butyrophilins including BTNL2 inhibitors, CD244 or CD48 inhibitors, KIR inhibitors such as but not limited to lirilumab, inhibitors of ILT and LIR such as but not limited to IPH2201, NKG2D and NKG2A inhibitors, MICA and MICB inhibitors, CD244 inhibitors such as but not limited to emacutuzumab, cabirarizumab, pexidartinib, ARRY382, CSF1R inhibitors such as but not limited to BLZ945, IDO inhibitors such as but not limited to INCB024360, thalidomide, lenalidomide, TGFβ inhibitors such as but not limited to galunisertib, adenosine, or CD39 or CD73 inhibitors such as urocuprumab and (3S,6 S,9S,12R,17R,20S,23S,26S,29S,34aS)-N-((S)-1-amino-5-guanidino-1-oxopentan-2-yl)-26,29-bis(4-aminobutyl)-17-((S)-2-((S)-2-((S)-2-(4-fluorobenzamido)-5-guanidinopentanamido)-5-guanidinopentanamido)-3-(naphthalen-2-yl)propanamido)-6-(3-guanidin dinopropyl)-3,20-bis(4-hydroxybenzyl)-1,4,7,10,18,21,24,27,30-nonaoxo-9,23-bis(3-ureidopropyl)triacontahydro-1H,16H-pyrrole[2,1-p][1,2]dithia[5,8,11,14,17,20,23,26,29]nonaazacyclodotriacontine-12-carboxamide The immunomodulatory agent is selected from the group consisting of a CXCR4 or CXCL12 inhibitor, such as but not limited to BKT140, a phosphatidylserine inhibitor, such as but not limited to bavituximab, a SIRPA or CD47 inhibitor, such as but not limited to CC-90002, a VEGF inhibitor, such as but not limited to bevacizumab, and a neuropilin inhibitor, such as but not limited to MNRP1685A.

[0167] Pharmaceutical Compositions Aspects of the present disclosure are directed to therapeutic methods that include the administration of combinations of compounds disclosed herein, or one or more pharmaceutical compositions that include such compounds and pharmaceutically acceptable diluents, excipients or carriers.In certain embodiments, the methods disclosed herein involve administering a first pharmaceutical composition that includes compound A or any of its pharmaceutically acceptable salts and a pharmaceutically acceptable diluent, excipients or carriers, and a second pharmaceutical composition that includes a MEK inhibitor, for example trametinib, an ERK inhibitor, for example ulixertinib, a RAF inhibitor, for example LY3009120, or its pharmaceutically acceptable salts, and a pharmaceutically acceptable diluent, excipients or carriers. In certain embodiments, the methods disclosed herein involve administering a first pharmaceutical composition comprising compound B or a pharma- ceutically acceptable salt thereof and a pharma- ceutically acceptable diluent, excipient or carrier, and a second pharmaceutical composition comprising an MEK inhibitor, e.g., trametinib, an ERK inhibitor, e.g., ulixertinib, an RAF inhibitor, e.g., LY3009120, or a pharma- ceutically acceptable salt thereof and a pharma- ceutically acceptable diluent, excipient or carrier. In certain embodiments, the methods disclosed herein involve administering a pharmaceutical composition comprising compound A or a pharma- ceutically acceptable salt thereof, an MEK inhibitor, e.g., trametinib, and a pharma- ceutically acceptable diluent, excipient or carrier. In certain embodiments, the methods disclosed herein involve administering a pharmaceutical composition comprising compound B or a pharma- ceutically acceptable salt thereof, an MEK inhibitor, e.g., trametinib, and a pharma- ceutically acceptable diluent, excipient or carrier. involves administering a pharmaceutical composition.

[0168] In certain embodiments, the methods disclosed herein involve administering a pharmaceutical composition comprising Compound A, or a pharma- ceutical acceptable salt thereof, an ERK inhibitor, e.g., ulixertinib, and a pharma- ceutical acceptable diluent, excipient, or carrier. In certain embodiments, the methods disclosed herein involve administering a pharmaceutical composition comprising Compound B, or a pharma- ceutical acceptable salt thereof, an ERK inhibitor, e.g., ulixertinib, and a pharma- ceutical acceptable diluent, excipient, or carrier.

[0169] In certain embodiments, the methods disclosed herein involve administering a pharmaceutical composition comprising Compound A, or a pharma- ceutically acceptable salt thereof, a RAF inhibitor, e.g., LY3009120, and a pharma- ceutically acceptable diluent, excipient, or carrier. In certain embodiments, the methods disclosed herein involve administering a pharmaceutical composition comprising Compound B, or a pharma- ceutically acceptable salt thereof, a RAF inhibitor, e.g., LY3001290, and a pharma- ceutically acceptable diluent, excipient, or carrier.

[0170]

[00170] In using the pharmaceutical compositions of the compounds described herein, pharma- ceutically acceptable Possible carriers can be either solid or liquid. Solid forms include powders, tablets, dispersible granules, capsules, cachets, and suppositories. Powders and tablets may be comprised of about 5 to about 95 percent active ingredient. Suitable solid carriers are known in the art, and include, for example, magnesium carbonate, magnesium stearate, talc, sugar, or lactose. Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable for oral administration. Examples of pharma-ceutically acceptable carriers and methods of manufacturing various compositions can be found in A. Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th Edition, (1990), Mack Publishing Co., Easton, Pa., which is incorporated herein by reference in its entirety.

[0171] Liquid form preparations include solutions, suspensions and emulsions.For example, water or water-propylene glycol solutions for parenteral injection, or the addition of sweeteners and opacifiers for oral solutions, suspensions and emulsions.Liquid form preparations also include solutions for intranasal administration.

[0172] In particular, injectable, liquid compositions can be prepared, for example, by dissolving, dispersing, etc. For example, the disclosed compounds are dissolved or mixed in a pharma- ceutically acceptable solvent, such as, for example, water, saline, aqueous dextrose, glycerin, ethanol, etc., to form an injectable isotonic solution or suspension. Proteins, such as albumin, chylomicron particles, or serum proteins, can be used to solubilize the disclosed compounds.

[0173] Parenteral injectable administration is generally used for subcutaneous, intramuscular, or intravenous injections and infusions. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, or solid forms suitable for dissolving in liquid prior to injection.

[0174] Aerosol preparations suitable for inhalation may also be used. These preparations may include solutions and solids in powder form, which may be in combination with a pharma- ceutically acceptable carrier, such as an inert compressed gas, for example nitrogen.

[0175]

[00175] Also, with regard to use, liquid forms for either oral or parenteral administration are Also contemplated are solid form preparations which are intended to be converted immediately prior to use into liquid preparations. Forms include solutions, suspensions and emulsions.

[0176] dose In some embodiments, when Compound A or Compound B (or a pharma- ceutically acceptable salt thereof) is used in combination with, for example, a MEK inhibitor (e.g., trametinib) for a treatment protocol, the two therapeutic agents may be administered together, or in a "dual regimen," the two therapeutic agents may be administered in a dual regimen, where the two therapeutic agents are dosed and administered separately. When Compound A or B (or a pharma- ceutically acceptable salt thereof) and a MEK inhibitor are dosed separately, a typical dose of Compound A or Compound B (or a pharma- ceutically acceptable salt thereof) administered to a subject in need of treatment is typically about 5 mg / day to about 5000 mg / day, and in other embodiments, about 50 mg / day to about 1000 mg / day. Other doses may be from about 10 mmol up to about 250 mmol / day, about 20 mmol to about 70 mmol / day, or about 30 mmol to about 60 mmol / day. The effective dosage of the disclosed compounds, when used with respect to the indicated effects, ranges from about 0.5 mg to about 5000 mg of the disclosed compounds required to treat the condition. Compositions for in vivo or in vitro use can contain about 0.5, 5, 20, 50, 75, 100, 150, 250, 500, 750, 1000, 1250, 2500, 3500, or 5000 mg of the disclosed compounds, or can contain a range of amounts from one to another listed in the dosage list. For oral administration, a typical recommended daily dosage regimen can range from about 1 mg / day to about 500 mg / day, or 1 mg / day to 200 mg / day, in a single dose or in 2 to 4 divided doses. In one embodiment, a typical daily oral dosage regimen is 150 mg.

[0177] In certain embodiments, the dose of the MEK inhibitor is consistent with the doses disclosed above and / or approved for use by the Food and Drug Administration. In other embodiments, the dose of the MEK inhibitor is less than the previously approved dose, e.g., about 20%, about 50%, or about 80% of the approved dose. In certain embodiments, the dose of trametinib is about .5 mg to 20 mg orally per day, e.g., about 1 mg per day, or about 2 mg per day. In certain embodiments, the dose of cobimetinib is about 10 mg to 200 mg per day, e.g., about 30 mg per day, or about 60 mg per day. In certain embodiments, the dose of binimetinib is about 10 mg to 200 mg twice per day, e.g., about 25 mg or about 45 mg twice per day. In certain embodiments, the dose of selumetinib is about 10 mg to 200 mg per day, for example about 30 mg or about 75 mg twice per day.

[0178] The amount and frequency of administration of the compounds described herein and / or pharma- ceutically acceptable salts thereof and other therapeutic agents will be adjusted according to the judgment of the attending physician, taking into account factors such as the age, condition and size of the patient, and the severity of the condition being treated.

[0179] The compounds of the present disclosure (e.g., Compound A or Compound B (and pharma- ceutically acceptable salts thereof), MEK inhibitors, and other therapeutic agents) may be administered by any suitable route. The compounds can be administered orally (e.g., with food) in capsules, suspensions, tablets, pills, dragees, liquids, gels, syrups, slurries, and the like. Methods for encapsulating compositions (such as in hard gelatin or cyclodextran coatings) are known in the art (see Baker, et al., "Controlled Release of Biological The compounds can be administered to a subject in conjunction with an acceptable pharmaceutical carrier as part of a pharmaceutical composition. The dosage form of the pharmaceutical composition will vary according to the route of administration selected. A suitable pharmaceutical carrier may contain inactive ingredients that do not interact with the compounds. The carrier should be biocompatible and i.e., it is non-toxic, non-inflammatory, non-immunogenic and does not produce other undesirable reactions at the site of administration.

[0180]

[00180] Exemplary pharmaceutical compositions include a compound described herein and a pharma- ceutically acceptable carrier. a) diluents such as purified water, triglyceride oils, e.g. hydrogenated or partially hydrogenated vegetable oils or mixtures thereof, corn oil, olive oil, sunflower oil, safflower oil, fish oils or their esters, e.g. EPA or DHA, or triglycerides or mixtures thereof, omega-3 fatty acids or derivatives thereof, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, sodium, saccharin, glucose and / or glycine; b) diluents such as silica, talc, stearic acid, magnesium or calcium salts thereof, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and / or poly lubricants, such as ethylene glycol, also for tablets; c) binders, if desired, such as magnesium aluminium silicate, starch paste, gelatine, tragacanth, methylcellulose, sodium carboxymethylcellulose, magnesium carbonate, natural sugars, such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, waxes and / or polyvinylpyrrolidone; d) disintegrants, such as starch, agar, methylcellulose, bentonite, xanthan gum, alginic acid or its sodium salt, or effervescent mixtures; e) absorbents, colouring agents, flavouring agents and sweetening agents; f) binders, such as Tween 80, Labrasol, HPMC, DOSS, Caproyl and / or g) an emulsifier or dispersing agent, such as, for example, cyclodextrin, hydroxypropyl cyclodextrin, PEG 400, PEG 200, or the like, which is an agent that enhances absorption of the compound.

[0181] If prepared as a fixed dose, such combinations will employ the compounds described herein within the dosage ranges described herein or known to those of skill in the art.

[0182] Since the compounds described herein (e.g., Compound A and Compound B, and MAPKAP kinase inhibitors, including MEK inhibitors) are intended for use in pharmaceutical compositions, those skilled in the art will appreciate that they can be provided in substantially pure form, e.g., at least 60% pure, at least 75% pure, at least 85% pure, and at least 98% pure (w / w). Pharmaceutical preparations may be in unit dosage form. In such preparations, they are prepared by dividing them into suitably sized unit doses containing an appropriate amount of Compound A or Compound B, e.g., an effective amount to achieve the desired purpose as described herein. EXAMPLES

[0183] Treatment of c-KIT-mediated tumor cells with either Compound A or a pharma- ceutically acceptable salt thereof in combination with a MEK inhibitor or an ERK inhibitor or a RAF inhibitor was unexpectedly found to synergistically induce apoptosis of tumor cells. Moreover, this combination therapy inhibits the proliferation of tumor cells, including tumor cells with secondary mutations that confer resistance to other c-KIT inhibitors and / or MEK, ERK or RAF inhibitors. Moreover, the combination therapy disclosed herein appeared to have a long-term effect on tumor cell quiescence, as opposed to rapid tumor regrowth without drug combination treatment. Moreover, the combination therapy disclosed herein appeared to have a cytotoxic effect on tumor cells, as opposed to a purely cytoplasmic effect. Moreover, the combination therapy disclosed herein appeared to have a long-term effect on tumor cells, as opposed to a purely cytoplasmic effect. Moreover, the combination therapy disclosed herein appeared to have a long-term effect on tumor cells, as opposed to rapid tumor regrowth, as opposed to rapid tumor regrowth without drug combination treatment. The combination therapy appeared to eradicate GIST tumor cells to the limit of detection, with no tumor cell colony growth following removal of the combination therapy, including a drug-free recovery period of up to 21 days. Identification of this unexpected finding was performed in biochemical and cellular assays, including those described herein.

[0184] The present disclosure is further illustrated by the following examples, which should not be considered as limiting the present disclosure in scope or spirit to the specific procedures described herein. It should be understood that the examples are presented to illustrate specific embodiments and are not intended to limit the scope of the present disclosure. It should also be understood that various other embodiments, modifications and equivalents that may be suggested to those skilled in the art may be made without departing from the spirit of the present disclosure and / or the scope of the appended claims.

[0185] Example 1. Combination treatment of Compound A with trametinib induces apoptosis in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E and GIST-T1 / T670I.

[00185] Combination treatment with compound A and trametinib improved GIST-T1 (57bp Tests were performed to demonstrate that Compound A induces apoptosis in GIST-T1 (exon 11 deletion) imatinib-sensitive cells, GIST-T1 / D816E imatinib-resistant cells, and GIST-T1 / T670I (imatinib-resistant cells). Assays were performed in 96-well plates seeded with 10,000 GIST-T1, GIST-T1 / D816E, or GIST-T1 / T670I cells per well. Cells were treated with vehicle control, Compound A, trametinib, or combinations thereof at various concentrations, and cells were grown for 24 and 48 hours in the presence of drug treatment. Apoptosis was assessed by measuring caspase 3 / 7 activity.

[0186] Figures 1A and 1C are graphical representations showing the relative percentage of caspase activity (compared to vehicle control set at 100%) determined for various treatments of GIST-T1 cells. Figures 1B and 1D are matrix diagrams of synergy and combination index plots based on the combination index (CI) method described by Chou and Talalay (1984) and computer software of Chou and Martin (2005). CI<1 indicates synergy, CI=1 indicates additive effect, and CI>1 indicates antagonism. Combined treatment with compound A and trametinib for 24 hours (Figures 1A, B) and 48 hours (Figures 1C, D) showed strong synergy in inducing apoptosis in GIST-T1 cells.

[0187] Figure 1E is a graph showing caspase activity from various treatments of GIST-T1 / D816E imatinib-resistant cells. Figure 1F is a matrix diagram of synergy and combination index plot based on the combination index (CI) method described by Chou and Talalay (1984) and Chou and Martin computer software (2005). CI<1 indicates synergy, CI=1 indicates additive effect, and CI>1 indicates antagonism. 24-hour combination treatment with Compound A and Trametinib shows strong synergy to induce apoptosis of GIST-T1 / D816E imatinib-resistant cells.

[0188] Figure 1G is a graphical representation showing caspase activity from various treatments of GIST-T1 / T670I imatinib-resistant cells. Figure 1H is a matrix diagram of synergy and a combination index plot based on the combination index (CI) method described by Chou and Talalay (1984) and the computer software of Chou and Martin (2005). CI<1 indicates synergy, CI=1 indicates additivity, and CI>1 indicates antagonism. Combination treatment with Compound A and trametinib for 24 hours significantly increased the caspase activity of GIST-T1 / T670I imatinib-resistant cells. A strong synergistic effect was demonstrated in inducing apoptosis in ST-T1 / T670I imatinib-resistant cells.

[0189] Example 2. Combination treatment of Compound B with trametinib induces apoptosis in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E and GIST-T1 / T670I. Figure 2A is a graph showing the relative percentage of caspase activity (compared to vehicle control set at 100%) determined for various treatments of GIST-T1 cells. Figure 2B is a matrix diagram of synergy and combination index plot as described in Example 1, in which the combination treatment of compound B and trametinib for 24 hours (Figure 2A, B) shows strong synergy in inducing apoptosis in GIST-T1 cells.

[0190]

[00190] Figure 2C shows the results of various treatments of imatinib-resistant GIST-T1 / D816E cells. Figure 2D is a graphical representation of the caspase activity of the imatinib-resistant GIST-T1 / D816E cells. Figure 2C is a matrix diagram of synergy and combination index plot. Combination treatment with Compound B and Trametinib for 24 hours showed strong synergy in inducing apoptosis in GIST-T1 / D816E imatinib-resistant cells.

[0191] Figure 2E is a graph showing caspase activity from various treatments of GIST-T1 / T670I imatinib-resistant cells. Figure 2F is a matrix diagram of synergy and combination index plot. Compound B and trametinib combined treatment for 24 hours showed strong synergy in inducing apoptosis of GIST-T1 / T670I imatinib-resistant cells.

[0192] Example 3. Combination treatment of Compound A with binimetinib induces apoptosis in imatinib-resistant cells of GIST-T1 and GIST-T1 / D816E and in imatinib-resistant cells of GIST-T1 / T670I Figure 3A is a graph showing the relative percentage of caspase activity (compared to vehicle control set at 100%) determined for various treatments of GIST-T1 cells. Figure 3B shows the matrix diagram of synergy and combination index plot based on the combination index (CI) method described in Example 1. Combination treatment with compound A and binimetinib for 24 hours (Figures 3A, 3B) showed strong synergy to induce apoptosis in GIST-T1 cells.

[0193] Figure 3C is a graph showing caspase activity from various treatments of GIST-T1 / D816E imatinib-resistant cells. Figure 3D is a matrix diagram of synergy and combination index plot based on the combination index (CI) method described in Example 1. Compound A and binimetinib combination treatment for 24 hours showed strong synergy to induce apoptosis of GIST-T1 / D816E imatinib-resistant cells.

[0194] Figure 3E is a graph showing caspase activity from various treatments of GIST-T1 / T670I imatinib-resistant cells. Figure 3F is a matrix diagram of synergy and combination index plot. Compound A and binimetinib combination treatment for 24 hours showed strong synergy to induce apoptosis of GIST-T1 / T670I imatinib-resistant cells.

[0195] Example 4. Combination treatment of Compound B with binimetinib induces apoptosis in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E and GIST-T1 / T670I.

[00195] Figure 4A shows the caspase activity determined upon various treatments of GIST-T1 cells. The graph shows the relative percentage of activity (compared to the vehicle control set at 100%). Figure 4B is a matrix diagram of synergy and combination index plot based on the combination index (CI) method described by Chou and Talalay (1984). Combination treatment with compound B and binimetinib for 24 hours (Figure 4A, B) showed strong synergistic effect in inducing apoptosis in GIST-T1 cells.

[0196] Figure 4C is a graph showing caspase activity from various treatments of GIST-T1 / D816E imatinib-resistant cells. Figure 4D is a matrix diagram of synergy and combination index plot. Compound B and binimetinib combined treatment for 24 hours showed strong synergy to induce apoptosis of GIST-T1 / D816E imatinib-resistant cells.

[0197] Figure 4E is a graph showing caspase activity from various treatments of GIST-T1 / T670I imatinib-resistant cells. Figure 4F is a matrix diagram of synergy and combination index plot. Compound B and binimetinib combined treatment for 24 hours showed strong synergy to induce apoptosis of GIST-T1 / T670I imatinib-resistant cells.

[0198] Example 5. Combination treatment of Compound A with cobimetinib induces apoptosis in GIST-T1 imatinib-sensitive cells, GIST-T1 / D816E imatinib-resistant cells, and GIST-T1 / T670I imatinib-resistant cells Figure 5A is a graphical representation showing the relative percentage of caspase activity (compared to vehicle control set at 100%) determined for various treatments of GIST-T1 cells. Figure 5B shows a matrix diagram of synergy and a combination index plot based on the combination index (CI) method described in Example 1. Combination treatment with compound A and cobimetinib for 24 hours (Figures 5A, 5B) showed strong synergy in inducing apoptosis in GIST-T1 cells.

[0199] Figure 5C is a graph showing caspase activity from various treatments of GIST-T1 / D816E imatinib-resistant cells. Figure 5D is a matrix diagram of synergy and combination index plot based on the combination index (CI) method described in Example 1. 24-hour combination treatment with Compound A and Cobimetinib showed strong synergy to induce apoptosis of GIST-T1 / D816E imatinib-resistant cells.

[0200]

[00200] Figure 5E shows the results of various treatments of imatinib-resistant GIST-T1 / T670I cells. Figure 5F is a graph showing the caspase activity of the CI-based synergy matrix and combination index plots based on the combination index (CI) method described in Example 1. Compound A and cobimetinib combination treatment for 24 hours showed strong synergistic effect in inducing apoptosis of GIST-T1 / T670I imatinib-resistant cells.

[0201] Example 6. Combination treatment of Compound B with cobimetinib induces apoptosis in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E and GIST-T1 / T670I. Figure 6A is a graphical representation showing the relative percentage of caspase activity (compared to vehicle control set at 100%) determined for various treatments of GIST-T1 cells. Figure 6B is a matrix diagram of synergy and combination index plot. Combination treatment with Compound B and Cobimetinib for 24 hours (Figures 6A, 6B) showed strong synergy in inducing apoptosis in GIST-T1 cells.

[0202] FIG. 6C shows caspases from various treatments of GIST-T1 / D816E imatinib-resistant cells. Figure 6D is a graphical representation showing the enzyme activity. Figure 6D is a matrix diagram of synergy and combination index plot. Combination treatment with Compound B and Cobimetinib for 24 hours showed strong synergy in inducing apoptosis in GIST-T1 / D816E imatinib-resistant cells.

[0203] Figure 6E is a graph showing caspase activity from various treatments of GIST-T1 / T670I imatinib-resistant cells. Figure 6F is a matrix diagram of synergy and combination index plot. Compound B and cobimetinib combination treatment for 24 hours showed strong synergy in inducing apoptosis of GIST-T1 / T670I imatinib-resistant cells.

[0204] Example 7. Combination treatment of Compound A with ulixertinib (BVD-523) induces apoptosis in GIST-T1 and GIST-T1 / T670I imatinib-resistant cells Figure 7A is a graphical representation showing the relative percentage of caspase activity (compared to vehicle control set at 100%) determined for various treatments of GIST-T1 cells. Figure 7B shows a matrix diagram of synergy and combination index plot based on the combination index (CI) method described in Example 1. Combination treatment with Compound A and ulixertinib for 24 hours (Figures 7A, 7B) showed strong synergy at higher concentrations to induce apoptosis in GIST-T1 cells.

[0205]

[00205] Figure 7C shows the results of various treatments of imatinib-resistant GIST-T1 / T670I cells. Figure 7D is a graph showing the caspase activity of the IL-1000-binding domains of Compound A and ulixertinib. Figure 7D is a matrix diagram of synergy and combination index plot based on the combination index (CI) method described in Example 1. 24-hour combination treatment with Compound A and ulixertinib showed strong synergistic effect in inducing apoptosis of GIST-T1 / T670I imatinib-resistant cells.

[0206] Example 8. Combination treatment of Compound B with ulixertinib (Bvd-523) induces apoptosis in GIST-T1 and GIST-T1 / T670I imatinib-resistant cells The synergy diagram of caspase activity and combination index plots can be used to show the synergy of the combination of Compound B and ulixertinib in inducing apoptosis in GIST-T1, GIST-T1 / D816E imatinib-resistant cells and GIST-T1 / T670I imatinib-resistant cells.

[0207] Example 9. Combination treatment of Compound A with SCH772984 induces apoptosis in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E and GIST-T1 / T670I. The synergy diagram of caspase activity and combination index plots can be used to show the synergy of the combination of Compound A and SCH772984 in inducing apoptosis in GIST-T1, GIST-T1 / D816E imatinib-resistant cells and GIST-T1 / T670I imatinib-resistant cells.

[0208] Example 10. Combination treatment of Compound B with SCH772984 induces apoptosis in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E and GIST-T1 / T670I. Using the synergy diagram of caspase activity and combination index plot, compound B and SCH77298 in inducing apoptosis in GIST-T1, GIST-T1 / D816E imatinib-resistant cells and GIST-T1 / T670I imatinib-resistant cells were compared. There may be a synergistic effect for the combination of 4.

[0209] Example 11. Combination treatment of Compound A with LY3009120 induces apoptosis in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E and GIST-T1 / T670I The caspase activity synergy diagram and combination index plot can be used to show the synergy of the combination of Compound A and LY3009120 in inducing apoptosis in GIST-T1, GIST-T1 / D816E imatinib-resistant cells and GIST-T1 / T670I imatinib-resistant cells.

[0210] Example 12. Combination treatment of Compound B with LY3009120 induces apoptosis in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E and GIST-T1 / T670I

[00210] Using the synergy diagram and combination index plot of caspase activity, GIS There may be a synergistic effect of the combination of Compound B and LY3009120 in inducing apoptosis in T-T1, GIST-T1 / D816E imatinib-resistant cells and GIST-T1 / T670I imatinib-resistant cells.

[0211] Example 13. Combined treatment of Compound A with Dabrafenib induces apoptosis in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E and GIST-T1 / T670I. The caspase activity synergy diagram and combination index plot can be used to show the synergy of the combination of Compound A and Dabrafenib in inducing apoptosis in GIST-T1, GIST-T1 / D816E imatinib-resistant cells and GIST-T1 / T670I imatinib-resistant cells. .

[0212] Example 14. Combination treatment of Compound B with Dabrafenib induces apoptosis in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E and GIST-T1 / T670I. The caspase activity synergy diagram and combination index plot can be used to show the synergy of the combination of Compound B and Dabrafenib in inducing apoptosis in GIST-T1, GIST-T1 / D816E imatinib-resistant cells and GIST-T1 / T670I imatinib-resistant cells.

[0213] Example 15. Combination treatment prevents colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E, and GIST-T1 / T670I Studies were conducted to demonstrate that combined treatment with Compound A and Trametinib prevents cell proliferation in GIST-T1 (57bp exon 11 deletion) imatinib-sensitive cells, GIST-T1 / D816E imatinib-resistant cells, and GIST-T1 / T670I imatinib-resistant cells. Assays were performed in 6-well plates seeded with 100 cells per well. Cells were treated with vehicle control, Compound A, Trametinib, Imatinib (IM) or combinations at various concentrations, and cells were cultured for 2 weeks. After treatment, drugs were washed out and cells were cultured in normal medium for 1-3 weeks. Proliferated cell colonies were stained with crystal violet and counted.

[0214] FIG. 8A shows a representative image of a culture plate and a graphical representation of the number of GIST-T1 colonies counted from various treatments. GIST T1 cells are sensitive to imatinib and Compound A as single agents. Imatinib and Compound A each as single agents show a similar reduction in GIST T1 colony growth to 23-30% of vehicle control. It should be noted that 2 weeks of combined treatment with 50nM compound A and either 50nM or 100nM trametinib unexpectedly led to complete cell stasis or eradication of GIST T1 colony growth to the detection limit as visualized by stereomicroscopy at 5x magnification, and no colonies were detected 9 days after combined treatment was removed (shown by arrows in Figure 8A).In contrast, 2 weeks of combined treatment with 100nM imatinib and either 50nM or 100nM trametinib did not lead to complete tumor cell stasis or eradication 9 days after combined treatment was removed.

[0215]

[00215] Figure 8B shows images of representative culture plates and GI counts from various treatments. Figure 8B shows a graphical representation of colony counts of GIST T1 / D816E. Note that imatinib (500nM) and Compound A (100nM or 250nM) as single agents each induce colony growth to approximately 61-72% of vehicle control, indicating a similar lack of cytocidal efficacy in GIST T1 / D816E (Figure 8B). Two weeks of combined treatment with compound A (100 nM or 250 nM) and trametinib (100 nM) led to almost complete cytostasis with compound A (100 nM), and complete eradication of colony growth in GIST-T1 / D816E cells with trametinib (100 nM) and compound A (250 nM) combinations to the limit of detection as visualized by stereomicroscopy at 5x after 10 days of recovery (see arrows in Figure 8B), whereas two weeks of combined treatment with imatinib (500 nM) and trametinib (50 nM or 100 nM) did not lead to complete cytostasis or eradication of tumor cells (see graph in Figure 8B). This was evident when cells were cultured for an additional 10 days without drugs, resulting in the growth of approximately 20-25 colonies. Figure 8C shows representative images of culture plates where compound A was further reduced to 25 nM, 50 nM, or 100 nM and evaluated in combination with 50 nM trametinib. Complete tumor cell stasis or eradication to the limit of detection as visualized by stereomicroscopy at 5-fold tumor colony growth was achieved with 100 nM compound A in combination with trametinib after 10 days of recovery (see arrow in Figure 8C), near complete tumor cell stasis or near eradication (1% of vehicle control) was achieved with 50 nM compound A in combination with trametinib (see arrow in Figure 8C), and significant tumor cell stasis or death was achieved with 25 nM compound A (11% of vehicle control). In contrast, the combination of 100 nM imatinib with 50 nM trametinib did not eradicate tumor colony growth and achieved mild tumor cell stasis or death after 10 days of recovery (60% of vehicle control).

[0216] Figure 8D shows representative culture plate images and a graphical representation of the number of GIST-T1 / T670I colonies counted from various treatments. Note that imatinib (500 nM) and compound A (250 nM or 500 nM) as single agents each show a similar reduction in GIST T1 / T670I colony growth to about 44-49% of vehicle control. Two weeks of treatment with 250 nM or 500 nM compound A in combination with either 50 nM or 100 nM trametinib led to complete cell stasis or eradication of GIST T1 / T670I colony growth to the limit of detection as visualized by stereomicroscopy at 5x magnification, with no colonies detected 10 days after removal of the combined treatment (indicated by arrows in Figure 8D). In contrast, 2 weeks of treatment with 500 nM imatinib in combination with either 50 nM or 100 nM trametinib did not lead to complete tumor cell stasis or eradication 9 days after removal of the combined treatment.

[0217] Example 16. Combination treatment of Compound B with trametinib prevents colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E and GIST-T1 / T670I The experiments described in Example 8 were also performed with combined treatment with Compound B and trametinib in three GIST cell lines.

[0218] Figure 9A shows images of representative culture plates and a graphical representation of the number of GIST-T1 colonies counted from the various treatments. GIST T1 cells were sensitive to compound B as a single agent, showing a 42-54% reduction in GIST T1 colony growth compared to vehicle controls. Two weeks of combination treatment with 50 nM or 100 nM compound B and either 50 nM or 100 nM trametinib led to significant cytostasis with almost no colony growth, whereas combination treatment with 250 nM compound A and either 50 nM or 100 nM trametinib led to complete cytostasis or eradication of GIST T1 colony growth to the limit of detection as visualized by stereomicroscopy at 5x, with no colonies detected 10 days after removal of combination treatment (indicated by arrows in Figure 9A). Colony growth was prevented even after extended, prolonged recovery for a total of 20 days.

[0219] Figure 9B shows the representative culture plate images and a graphical representation of the colony numbers of GIST-T1 / D816E counted from various treatments. Note that compound B (50nM, 100nM or 250nM) as a single agent shows cytocidal efficacy against GIST T1 / D816E, with colony growth of about 59-84% of vehicle control (Figure 9B). Two weeks of combined treatment with compound B (250 nM) and trametinib (50 nM) or compound B (100 nM or 250 nM) and trametinib (100 nM) led to >90% cell stasis or eradication of colony growth in GIST-T1 / D816E cells as visualized by stereomicroscopy at 5x magnification after 10 days of recovery (see arrows in Figure 9B), and even after an extended period of 20 days, treatment with compound B (250 nM) maintained cell stasis or cell death in combination with trametinib (100 nM).

[0220]

[00220] Figure 9C shows images of representative culture plates and GI counts from various treatments. A graphical representation of the number of ST-T1 / T670I colonies is shown. Note that compound B (50 nM, 100 nM or 250 nM) as a single agent shows colony growth of GIST T1 / T670I to approximately 75-78% of vehicle control. Two weeks of treatment with either 100 nM or 250 nM compound B in combination with either 50 nM or 100 nM trametinib led to complete cell stasis or eradication of GIST T1 / T670I colony growth to the limit of detection as visualized by stereomicroscopy at 5x, with no colonies detected 10 days after removal of the combined treatment (indicated by arrows in Figure 9C). Growth inhibition was maintained even after an extended 20 days without drug.

[0221] Example 17. Combination treatment of Compound A with binimetinib prevents colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E and GIST-T1 / T670I A study was conducted to demonstrate that combination treatment with Compound A and binimetinib prevents colony growth in three GIST cell lines as described in Example 15.

[0222] Figure 10A shows images of representative culture plates and a graphical representation of the number of GIST-T1 colonies counted from the various treatments. Note that imatinib and compound A as single agents each show a similar reduction in GIST T1 colony growth to 36-41% of vehicle control. Two-week combination treatments with 100 nM or 250 nM compound A and either 500 nM, 1 uM, 2 uM or 3 uM binimetinib were evaluated. Combination of compound A (100 nM or 250 nM) with binimetinib (2 uM or 3 uM) led to complete cell stasis or eradication of GIST T1 colony growth to the limit of detection as visualized by stereomicroscopy at 5x, with no colonies detected 10 days after removal of the combination treatment (indicated by arrows in Figure 10A). In contrast, imatinib at 500 nM led to complete cell stasis or eradication of GIST T1 colony growth to the limit of detection as visualized by stereomicroscopy at 5x, with no colonies detected 10 days after removal of the combination treatment (indicated by arrows in Figure 10A). Two weeks of combination treatment with imatinib and either 500nM, 1uM, 2um or 3uM binimetinib did not result in complete tumor cell stasis or eradication 10 days after removal of the combination treatment. The effect was more evident after extended periods of incubation without drug, with approximately 10-15 colonies visible with imatinib and no colony growth observed with compound A.

[0223] Figure 10B shows a representative image of the culture plate and a graphical representation of the number of colonies of GIST-T1 / D816E counted from the various treatments. Note that imatinib (500 nM) and Compound A (100 nM or 250 nM) as single agents each had colony growth that was approximately 60-95% of vehicle control, indicating a lack of cytocidal efficacy for GIST T1 / D816E (Figure 10B). Two-week combination treatments with Compound A (100 nM or 250 nM) and binimetinib (500 nM, 1 uM, 2 uM or 3 uM) were evaluated. The combination of compound A (100nM or 250nM) with binimetinib (3uM) led to complete cell stasis or eradication of colony growth in GIST-T1 / D816E cells to the limit of detection as visualized by stereomicroscopy at 5x magnification after 10 days of recovery (see arrow in Figure 10B), whereas combination treatment with imatinib (500nM) and binimetinib (500nM, 1uM, 2uM or 3uM) for 2 weeks did not lead to complete cell stasis or eradication of tumor cells. The effect was more evident after extended periods of incubation, where treatment with imatinib did not lead to complete inhibition, whereas compound A maintained cell stasis or cell death even after 20 days.

[0224] FIG. 10C shows a representative image of the culture plate and a graphical representation of the number of GIST-T1 / T670I colonies counted from the various treatments. Note that imatinib (500 nM) as a single agent did not show any reduction in colonies, while compound A (100 nM or 250 nM) as a single agent showed a dose-dependent reduction in colony growth to approximately 78-89% of vehicle control. Two weeks of treatment with 250 nM compound A in combination with 1 uM, 2 uM or 3 uM binimetinib led to complete cell stasis or eradication of GIST T1 / T670I colony growth to the limit of detection as visualized by stereomicroscopy at 5x magnification, with no colonies detected 10 days after removal of the combined treatment (indicated by the arrow in FIG. 10C). Two weeks of treatment with 100 nM compound A in combination with 3 uM binimetinib led to complete cell stasis or eradication of GIST T1 / T670I colony growth to the limit of detection as visualized by stereomicroscopy at 5x magnification, with no colonies detected 10 days after removal of the combined treatment (indicated by arrows in Figure 10C). Cell stasis was maintained even after an extended 20-day period after removal of the drug. In contrast, two weeks of treatment with 500 nM imatinib in combination with either 500 nM, 1 uM, 2 uM or 3 uM binimetinib did not lead to complete tumor cell stasis or eradication 10 days after removal of the combined treatment.

[0225] Example 18. Combination treatment of Compound B with binimetinib prevents colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E and GIST-T1 / T670I

[00225] Combination treatment of Compound B and binimetinib is described in Example 15. Studies were performed to demonstrate that IFN-γ-blockade prevents colony growth in three GIST cell lines.

[0226] Figure 11A shows a representative image of the culture plate and a graphical representation of the number of GIST-T1 colonies counted from the various treatments. Note that each concentration of Compound B as a single agent shows a similar reduction in GIST T1 colony growth, ranging from 27 to 31% of the vehicle control. 250 nM Compound B and 2 uM or 3 uM binimetinib reduced GIST T1 colony growth by 27 to 31% of the vehicle control. A week of combined treatment led to complete cell stasis or eradication of GIST T1 colony growth to the limit of detection as visualized by stereomicroscopy at 5x magnification, with no colonies detected 10 days after combined treatment was removed (indicated by arrows in Figure 11A), and significant cell stasis or cell death of GIST-T1 cells was maintained even after an extended, prolonged recovery period of 20 days (upper right panel of Figure 11A). A two-week combined treatment with 100 nM compound B and 3 uM binimetinib led to complete cell stasis or eradication of GIST T1 colony growth to the limit of detection as visualized by stereomicroscopy at 5x magnification, with no colonies detected 10 days after combined treatment was removed (indicated by arrows in Figure 11A).

[0227] Figure 11B shows a representative image of the culture plate and a graphical representation of the number of GIST-T1 / D816E colonies counted from the various treatments. Note that compound B (100 nM or 250 nM) as a single agent showed a similar lack of cytocidal efficacy in GIST T1 / D816E, with colony growth of about 74-83% of vehicle control (Figure 11B). Two weeks of combined treatment with 100 nM or 250 nM compound B and either 2 uM or 3 uM binimetinib led to complete cytostasis or eradication of colony growth in GIST-T1 / D816E cells to the limit of detection as visualized by stereomicroscopy at 5x after 10 days of recovery (see arrow in Figure 11B). Cytostasis was maintained even after an extended 20-day period with higher concentrations of compound B.

[0228] Figure 11C shows a representative image of the culture plate and a graphical representation of the number of GIST-T1 / T670I colonies counted from the various treatments. Note that compound B (100 nM or 250 nM) as a single agent showed a reduction in GIST T1 / T670I colony growth to approximately 72-78% of vehicle control. Unexpectedly, 2 weeks of treatment with either 100 nM or 250 nM compound B and 3 uM binimetinib led to complete cell stasis or eradication of GIST T1 / T670I colony growth to the limit of detection as visualized by stereomicroscopy at 5x magnification, with no colonies detected 10 days after removal of the combined treatment (indicated by the arrow in Figure 11C). Unexpectedly, 2 weeks of treatment with 250 nM compound B and 2 uM binimetinib led to complete cell stasis or eradication of GIST T1 / T670I colony growth to the limit of detection as visualized by stereomicroscopy at 5x magnification, with no colonies detected 10 days after removal of the combined treatment (indicated by arrows in Figure 11C). Cell stasis was maintained even after an extended 20-day period after drug removal.

[0229] Example 19. Combination treatment of Compound A with cobimetinib prevents colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E, and GIST-T1 / T670I A study was conducted to demonstrate that combination treatment with Compound A and cobimetinib prevents colony growth in three GIST cell lines as described in Example 15.

[0230]

[00230] Figure 12A shows images of representative culture plates and G counts from various treatments. A graphical representation of IST-T1 colony counts is shown. Note that imatinib and compound A as single agents each show a similar reduction in GIST T1 colony growth to 18-23% of vehicle control. Two weeks of combination treatment with 250 nM compound A and either 100 nM, 200 nM or 500 nM cobimetinib led to complete cell stasis or eradication of GIST T1 colony growth to the limit of detection as visualized by stereomicroscopy at 5x, with no colonies detected 10 days after removal of combination treatment (indicated by arrows in FIG. 12A). Two weeks of combination treatment with 100 nM compound A and 500 nM cobimetinib led to complete cell stasis or eradication of GIST T1 colony growth to the limit of detection as visualized by stereomicroscopy at 5x, with no colonies detected 10 days after removal of combination treatment (indicated by arrows in FIG. 12A). The combination treatment led to complete cell stasis or eradication of GIST T1 colony growth to the limit of detection, and no colonies were detected 10 days after removal of the combination treatment (indicated by arrows in FIG. 12A). In contrast, combination treatment with 500 nM imatinib and 100 nM, 200 nM, or 500 nM cobimetinib for 2 weeks did not lead to complete tumor cell stasis or eradication 10 days after removal of the combination treatment.

[0231] The effect was more evident after extended periods of incubation, with approximately 10-15 colonies growing with 500 nM imatinib, and no colony growth observed with 500 nM compound A and 500 nM cobimetinib.

[0232] Figure 12B shows the representative culture plate images and a graphical representation of the colony numbers of GIST-T1 / D816E counted from various treatments. Note that imatinib (500nM) and compound A (100nM or 250nM) as single agents each show approximately 65-74% colony growth of vehicle control, indicating a similar lack of cytocidal efficacy of GIST T1 / D816E (Figure 12B). Two weeks of combined treatment with compound A (250 nM) and cobimetinib (100 nM, 200 nM or 500 nM) led to complete cell stasis or eradication of colony growth in GIST-T1 / D816E cells to the limit of detection as visualized by stereomicroscopy at 5x magnification after 10 days of recovery (see arrow in Figure 12B), whereas two weeks of combined treatment with imatinib (500 nM) and cobimetinib (100 nM, 200 nM or 500 nM) did not lead to complete cell stasis or eradication of tumor cells. The effect was more evident after extended periods of incubation, where treatment with imatinib did not lead to complete inhibition, whereas compound A showed significant maintenance of cell stasis or cell death even 20 days after removal of the drug.

[0233] Figure 12C shows representative images of culture plates and a graphical representation of the number of GIST-T1 / T670I colonies counted from various treatments. Unexpectedly, 2 weeks of treatment with either 50nM or 100nM compound A in combination with cobimetinib (200nM or 500nM) led to >99% inhibition of GIST T1 / T670I colony growth as visualized by stereomicroscopy at 5x (indicated by arrows in Figure 12C). Cell stasis was maintained even after an extended 20-day period after drug removal. In contrast, 2 weeks of treatment with 500nM imatinib in combination with up to 500nM cobimetinib did not lead to robust cell stasis or cell eradication 10 days after removal of combined treatment.

[0234] Example 20. Combination treatment of Compound B with cobimetinib prevents colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E, and GIST-T1 / T670I A study was conducted to demonstrate that combination treatment with Compound B and cobimetinib prevents colony growth in three GIST cell lines as described in Example 15.

[0235]

[00235] Figure 13A shows images of representative culture plates and G counts from various treatments. A graphical representation of GIST-T1 colony counts is shown. Note that compound B as a single agent shows a similar reduction in GIST T1 colony growth to 42-54% of vehicle control. Two weeks of combination treatment with 50 nM, 100 nM or 250 nM compound B and either 200 nM or 500 nM cobimetinib led to complete or near complete cell stasis or eradication of GIST T1 colony growth to the limit of detection as visualized by stereomicroscopy at 5x, with no colonies detected 10 days after removal of combination treatment (indicated by arrows in Figure 13A). Two weeks of combination treatment with 100 nM or 250 nM compound B and either 200 nM or 500 nM cobimetinib led to complete or near complete cell stasis or eradication of GIST T1 colony growth to the limit of detection as visualized by stereomicroscopy at 5x, with no colonies detected 10 days after removal of combination treatment (indicated by arrows in Figure 13A). Thus, significant cell stasis or cell death of GIST-T1 cells was maintained even after an extended, prolonged recovery period of 20 days.

[0236] Figure 13B shows images of representative culture plates and a graphical representation of the number of GIST-T1 / D816E colonies counted from the various treatments. Note that compound B (100 nM or 250 nM) as a single agent was associated with colony growth of approximately 58-84% of vehicle control, indicating a lack of cytocidal efficacy in GIST T1 / D816E (Figure 13B). Two weeks of combined treatment with compound B at 50 nM, 100 nM or 250 nM and cobimetinib at either 200 nM or 500 nM led to >90% inhibition of colony growth in GIST-T1 / D816E cells as visualized by stereomicroscopy at 5x after 10 days of recovery (see arrows in Figure 13B). Cytostasis was significantly maintained with compound B at higher concentrations, even after an extended period of 20 days.

[0237] Figure 13C shows images of representative culture plates and a graphical representation of the number of GIST-T1 / T670I colonies counted from the various treatments. Note that compound B (100 nM or 250 nM) as a single agent showed a reduction in GIST T1 / T670I colony growth to approximately 75-78% of vehicle control. Two weeks of treatment with either 50 nM, 100 nM or 250 nM compound B and 200 nM or 500 nM cobimetinib led to complete cell stasis or eradication of GIST T1 / T670I colony growth to the limit of detection as visualized by stereomicroscopy at 5x, with no colonies detected 10 days after removal of the combined treatment (indicated by arrows in Figure 13C). Cell stasis was maintained even after an extended 20-day period after drug removal.

[0238] Example 21. Combination treatment of Compound A with ulixertinib prevents colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / D816E and GIST-T1 / T670I A study was conducted to demonstrate that combination treatment with Compound A and ulixertinib prevents colony growth in three GIST cell lines as described in Example 15.

[0239] Figure 14A shows a representative image of the culture plate and a graphical representation of the number of GIST-T1 colonies counted from various treatments. Note that compound A as a single agent shows a similar reduction in GIST T1 colony growth to 37-41% of vehicle control. Two weeks of combined treatment with 50nM, 100nM or 250nM compound A and either 1uM, 2uM or 3uM ulixertinib led to a significant reduction in GIST T1 colony growth as visualized by stereomicroscopy at 5x (shown by arrow in Figure 14A).

[0240]

[00240] Figure 14B shows images of representative culture plates and G counts from various treatments. A graphical representation of colony counts of GIST-T1 / D816E is shown. Note that compound A (50nM, 100nM or 250nM) as a single agent shows a similar lack of cytocidal efficacy in GIST T1 / D816E, with colony growth of about 81-93% of vehicle control (Figure 14B). Two weeks of combined treatment with compound A (250nM) and ulixertinib (2uM or 3uM) led to complete cytostasis or eradication of colony growth in GIST-T1 / D816E cells to the limit of detection as visualized by stereomicroscopy at 5x magnification after 10 days of recovery (see arrow in Figure 14B).

[0241] FIG. 14C shows an image of a representative culture plate and a graphical representation of the number of GIST-T1 / T670I colonies counted from the various treatments. Unexpectedly, two weeks of treatment with Compound A and ulixertinib (2uM or 3uM) led to complete cell stasis or eradication of GIST T1 / T670I colony growth to the limit of detection as visualized by stereomicroscopy at 5x magnification, with no colonies detected 10 days after removal of the combined treatment (indicated by arrows in Figure 14C). Cell stasis was maintained even after an extended 20-day period after drug removal.

[0242] Example 22. Combination treatment of Compound B with ulixertinib prevents colony growth in imatinib-resistant cells of GIST-T1 / D816E Figure 15 shows representative culture plate images and a graphical representation of the number of GIST-T1 / D816E colonies counted from various treatments. Note that compound B (50 nM, 100 nM or 250 nM) as a single agent shows a similar lack of cytocidal efficacy of GIST T1 / D816E, with colony growth of about 52-95% of vehicle control (Figure 15). Two weeks of combination treatment with compound B (250 nM) and ulixertinib (3 uM) led to complete cytostasis or eradication of GIST T1 / D816E colony growth to the limit of detection as visualized by stereomicroscopy at 5x magnification, with no colonies detected 10 days after removal of combination treatment (indicated by arrow in Figure 15).

[0243] Example 23. Combined treatment with Compound A and the ERK inhibitor SCH772984 prevents colony growth in imatinib-resistant cells of GIST-T1 / D816E Using the protocol outlined in Example 15, a synergistic effect can be demonstrated for the combination of Compound A and SCH772984 in preventing colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / T670I and GIST-T1 / D816E.

[0244] Example 24. Combined treatment with Compound B and the ERK inhibitor SCH772984 prevents colony growth in imatinib-resistant cells of GIST-T1 / D816E Using the protocol outlined in Example 15, synergy can be demonstrated for the combination of Compound B and SCH772984 in preventing colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / T670I and GIST-T1 / D816E.

[0245] Example 25. Combined treatment with Compound A and the RAF inhibitor LY3009120 prevents colony growth in imatinib-resistant cells of GIST-T1 / D816E

[00245] Using the protocol outlined in Example 15, GIST-T1, GIST-T The combination of Compound A and LY3009120 can show synergy in preventing colony growth in imatinib-resistant cells of GIST-T1 / T670I and GIST-T1 / D816E.

[0246] Example 26. Combined treatment with compound B and the RAF inhibitor LY3009120 prevents colony growth in imatinib-resistant cells of GIST-T1 / D816E Using the protocol outlined in Example 15, synergy can be demonstrated for the combination of Compound B and LY3009120 in preventing colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / T670I and GIST-T1 / D816E.

[0247] Example 27. Combined treatment with Compound A and the RAF inhibitor dabrafenib prevents colony growth in imatinib-resistant cells of GIST-T1 / D816E Using the protocol outlined in Example 15, we were able to demonstrate synergy between the combination of Compound A and dabrafenib in preventing colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / T670I, and GIST-T1 / D816E. do.

[0248] Example 28. Combined treatment with compound B and the RAF inhibitor dabrafenib prevents colony growth in imatinib-resistant cells of GIST-T1 / D816E Using the protocol outlined in Example 15, synergy can be demonstrated for the combination of Compound B and dabrafenib in preventing colony growth in imatinib-resistant cells of GIST-T1, GIST-T1 / T670I and GIST-T1 / D816E.

[0249] Example 29. Combination treatment induces apoptosis in N-ras G12D-transfected GIST-T1 cells A study was conducted to demonstrate that the combined treatment of Compound A and Trametinib induces apoptosis in empty vector control (EV) and mutant N-ras G12D transfected GIST-T1 cells. The assay was performed in 96-well plates seeded with 10,000 cells per well for GIST-T1 cells transfected with vector control or N-ras G12D. Cells were treated with vehicle control, Compound A, Trametinib, or the combination at various concentrations and cells were allowed to grow for 48 hours. Apoptosis was assessed by measuring caspase 3 / 7 activity.

[0250]

[00250] Figure 16A provides a graphical representation of caspase activity measurements after various treatments. Combined treatment with 50 nM compound A and trametinib (50 nM or 100 nM) for 48 h induced increased apoptosis in mutant N-ras G12D-transfected GIST-T1 cells compared to cells treated with either single-agent compound A or trametinib.

[0251] Example 30. Combination treatment prevents colony growth in N-ras G12D-transfected GIST-T1 cells A study was performed to demonstrate that combined treatment with compound A and trametinib prevents resistant colony growth in empty vector control and mutant N-ras G12D transfected GIST-T1 cells. The assay was performed in 6-well plates seeded with 100 cells per well. Cells were treated with vehicle control, 50 nM compound A, 50 nM or 100 nM trametinib, or a combination thereof, and the cells were cultured for 2 weeks. In the same experiment, cells were treated with vehicle control, 100 nM imatinib, 50 nM or 100 nM trametinib, or a combination thereof. After 2 weeks, the drugs were washed out and the cells were cultured in normal medium for 1 to 3 weeks. Colonies were stained with crystal violet and counted.

[0252] Figure 16B shows a representative image of the culture plate, and Figure 16C shows a graphical representation of the number of GIST-T1 colonies transfected with vector control (Figure 16B.1) and mutant N-ras G12D (Figure 16B.2) counted after various treatments. Quantification of colony growth in GIST T-1 cells transfected with vector control and N-ras G12D is shown in Figure 16C.1 and Figure 16C.2, respectively. Combined treatment with 100nM imatinib and 50nM trametinib resulted in colony growth (35% of vehicle control), and combination of 100nM imatinib with 100nM trametinib also resulted in colony growth (19% of vehicle). In contrast, combination of Compound A with trametinib unexpectedly resulted in superior cell stasis or cell death compared to combination with imatinib. Two weeks of combined treatment with 50 nM Compound A and 50 nM trametinib led to nearly complete cell stasis or cell death (2% of vehicle control), and combination of 50 nM Compound A with 100 nM trametinib led to complete (0% of colony growth of vehicle control) cell stasis or cell death to the limit of detection as visualized by stereomicroscopy at 5x after 10 days of drug washout and recovery (see arrow in Figure 16C.2). see).

[0253] FIG. 16D shows mutant N-ras counted after an extended drug-free recovery period. The representative culture plate image of the number of GIST-T1 colonies transfected with N-ras G12D is shown.The combination treatment of 100nM compound A and 50nM or 100nM trametinib for 2 weeks leads to almost complete inhibition of colony growth in GIST-T1 cells transfected with N-ras G12D after 21 days of extended recovery period.

[0254] Example 32. Combination treatment prevents colony growth of drug-resistant GIST cells Saturation mutagenesis studies were performed in Ba / F3 cells transfected with oncogenic KIT V560D mutant. DNA nicking was induced with N-ethyl-N-nitrosourea (ENU) for 18 hours to randomly generate additional mutations in the KIT gene or other genes. Assays were performed in 6-well plates seeded with 100 cells per well. After ENU washing, wells were incubated with 100 nM or 250 nM or 500 nM imatinib, 100 nM or 250 nM or 500 nM imatinib in combination with 10 nM trametinib, 25 nM or 100 nM or 250 nM compound A, or 25 nM, 100 nM or 250 nM compound A in combination with 10 nM trametinib. Wells showing resistance to drug treatment indicated proliferation of Ba / F3 cells. These cells were subjected to PCR and sequencing of the KIT gene to determine the presence of secondary resistance mutations induced by ENU treatment.

[0255]

[00255] Figure 17A shows the growth of Ba / F3 colonies resistant to imatinib. Figure 17A (right panel) is a graphical representation of a saturation mutagenesis study of Ba / F3 cells with compound A. Single-agent treatment with 25 nM, 100 nM, or 250 nM compound A did not lead to the proliferation of any new resistance secondary mutations as determined by PCR and DNA sequencing. Only Ba / F3 cells containing the original V560D (parent) KIT mutant were shown to proliferate after exposure to compound A, likely reflecting mutations in genes other than KIT (Figure 17A, right panel). Figure 17B is a graphical representation showing Ba / F3 cell colony growth with imatinib in the presence of trametinib or compound A in the presence of trametinib. Combination of imatinib at 250 nM or 500 nM with trametinib at 10 nM did not lead to the growth of any new resistance secondary mutations, but did lead to the growth of the original KIT V560D (parent) cells, likely reflecting mutations in genes other than KIT (Figure 17B, left panel). Significantly, in contrast to the combination study of imatinib with trametinib, combination of 25, 100, or 250 nM compound A with trametinib at 10 nM led to complete cell stasis or cell death without any cell growth to the limit of detection as determined by visual inspection of any well (Figure 17B, right panel).

[0256] Example 33. In vivo xenograft study of Compound A in combination with trametinib The GIST T1 xenograft model was performed in accordance with all National Institutes of Health (NIH) laws, regulations, and guidelines, approval of the Animal Care AND Use Committee at MI Bioresearch (Ann Arbor, MI, USA), and approval of an AAALAC-accredited facility. All mice were provided with food and water ad libitum. All mice were observed for clinical signs at least once daily. Envigo female nude mice (HsdCrl: athymic nude-NU-Foxn1nu, 6-7 weeks old) were inoculated subcutaneously with 5 million cells in Dulbecco's phosphate-buffered saline mixed with an equal volume of Matrigel using a 27-gauge needle and syringe just under the right axilla. Tumor burden was 117 cm in diameter at 10 days on average. mm 3 Upon reaching 10 mg / kg / day, mice were randomly assigned to groups such that the mean tumor burden for all groups was within 10% of the overall mean tumor burden for the study population. Groups received the following treatments on days 10-27: vehicle control diet (n=10); Compound A was formulated in the mouse diet to achieve approximately 100 mg / kg / day of Compound A (n=10); or Compound A was formulated in the mouse diet to achieve approximately 25 mg / kg / day of Compound A (n=10); trametinib was administered orally at 0.5 mg / kg BID and fed a vehicle control diet (n=10); trametinib was administered orally at 0.5 mg / kg BID and fed a Compound A formulated diet (to achieve approximately 100 mg / kg / day of Compound A treatment) (n=10); or trametinib was administered orally at 0.5 mg / kg BID and fed a Compound A formulated diet (to achieve approximately 100 mg / kg / day of Compound A treatment) (n=10). The animals were orally dosed BID and fed a diet formulated with Compound A (to achieve treatment with Compound A at approximately 25 mg / kg / day (n=10). On day 27, all animals were placed on a control diet and observed for tumor regrowth. Tumor volume and body weight were measured three times a week. Tumor burden (mg) was calculated using the formula: tumor burden (mg=mm 3 )=(length x width 2 ) / 2.

[0257] Figures 18A and 18B are graphical representations showing tumor growth inhibition compared to vehicle control. Figure 18B shows the same data as Figure 18A, but expanded to show the difference between cohorts treated with Compound A or Compound A / Trametinib. Treatment with Trametinib led to slight tumor growth inhibition compared to vehicle control. At high doses of Compound A (approximately 100 mg / kg / day), 6 / 10 mice had complete tumor regression, and the remaining 4 / 10 mice had partial tumor regression during the treatment period. At low doses of Compound A (approximately 25 mg / kg / day), 2 / 10 mice had complete tumor regression, and 6 / 10 mice had partial tumor regression. At high doses of Compound A (approximately 100 mg / kg / day) in combination with Trametinib, 10 / 10 mice had complete tumor regression during the treatment period. At low doses of Compound A (approximately 25 mg / kg / day) in combination with trametinib, 5 / 10 mice had complete tumor regression and 5 / 10 mice had partial tumor regression. Furthermore, after the dosing period, all cohorts treated with Compound A had slower tumor regrowth than the initial tumor growth of the vehicle control, indicating a long-term effect on tumor cell growth until the end of the study on day 66. At high doses of Compound A (approximately 100 mg / kg / day), 1 / 10 mice remained in partial tumor regression at the end of the study. At low doses of Compound A (approximately 25 mg / kg / day), 2 / 10 mice remained in partial tumor regression at the end of the study. At high doses of Compound A (approximately 100 mg / kg / day) in combination with trametinib, 1 / 10 mice remained in complete tumor regression and 4 / 10 mice remained in partial tumor regression at the end of the study. At low doses of Compound A (approximately 25 mg / kg / day) in combination with trametinib, 2 / 10 mice remained in partial tumor regression at the end of the study. These data indicate that the combination of Compound A and trametinib induces cell death and / or achieves prolonged cell stasis for at least 40 days after administration.

[0258] Example 34. Compound A is a potent inhibitor of the BCRP efflux transporter To examine the inhibition of the BCRP drug efflux transporter with compound A, a vesicular trafficking inhibition assay was performed using inside-out membrane vesicles prepared in BCRP-expressing cells in the presence of a low-permeability probe substrate and ATP. The potential of compound A to alter the uptake of the probe substrate into transporter-containing vesicles was measured.

[0259] The in vitro interaction potential of Compound A with the human efflux transporter BCRP was investigated at seven concentrations in a vesicular transport inhibition assay. Compound A effectively inhibited the transport of BCRP probe substrates, with 44% inhibition observed at the lowest concentration tested here (0.04 μM). The IC50 value was estimated to be approximately 0.04 μM.

[0260] Equivalent

[00260] Those of skill in the art will appreciate that numerous modifications to the specific embodiments specifically described in this disclosure may be possible. Equivalents to the above may be recognized or ascertained with no more than routine experimentation, and such equivalents are intended to be encompassed by the scope of the following claims.

Claims

1. For use in treating a tumor with one or more c-KIT mutations in a patient in need thereof. an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, or a pharma- ceutically acceptable salt thereof; A pharmaceutical comprising an effective amount of one or more MAPKAP kinase inhibitors selected from the group consisting of trametinib, cobimetinib, binimetinib and ulixertinib, The tumor is a gastrointestinal stromal tumor (GIST).

2. The method of claim 1, wherein the c-KIT mutation is a primary mutation in exon 9, exon 11, exon 13, or exon 17 of the c-KIT gene.

3. The method of claim 1 or 2, wherein the tumor is associated with one or more secondary resistance mutations in the c-KIT gene.

4. The pharmaceutical described in claim 3, wherein the secondary resistance mutation is in exon 13, exon 14, exon 17 or exon 18 of the c-KIT gene.

5. The pharmaceutical described in claim 4, wherein the secondary resistance mutation is in exon 17 of the c-KIT gene.

6. The pharmaceutical agent according to any one of claims 1 to 5, wherein the c-KIT mutation is a deletion mutation.

7. The pharmaceutical described in any one of claims 3 to 5, wherein the resistance secondary mutation is a substitution of aspartic acid at codon 816 or a substitution of asparagine at codon 822.

8. The pharmaceutical agent described in any one of claims 3 to 5, wherein the secondary resistance mutation is one of D816V, D816E, D816H, D820A, T670I, or N822V.

9. The pharmaceutical described in claim 3 or 4, wherein the secondary resistance mutation is one of V654A or T670I.

10. The pharmaceutical agent according to any one of claims 3 to 5 and 7 to 9, wherein the secondary resistance mutation was acquired after previous administration of imatinib, sunitinib or regorafenib, or a pharma- ceutically acceptable salt thereof, to the patient.

11. The method of any one of claims 3 to 5 and 7 to 10, wherein the treatment further comprises determining whether the tumor is associated with a secondary resistance mutation in c-KIT.

12. The pharmaceutical described in claim 11, wherein determining whether the tumor is associated with a resistance secondary mutation in c-KIT includes identifying the mutation in DNA extracted from the tumor sample.

13. The method of claim 11, wherein determining whether the tumor is associated with a secondary resistance mutation in c-KIT comprises identifying the mutation in circulating tumor DNA.

14. The pharmaceutical composition according to any one of claims 3 to 13, wherein the tumor has shown resistance to imatinib mesylate, sunitinib malate, or regorafenib.

15. The pharmaceutical of any one of claims 1 to 14, further comprising a cancer targeted therapeutic agent, a cancer targeted biologic, an immune checkpoint inhibitor, an immunomodulatory agent, and / or a chemotherapeutic agent.

16. The pharmaceutical composition according to any one of claims 1 to 15, further comprising a RAF inhibitor.

17. The pharmaceutical agent according to any one of claims 1 to 16, wherein the 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea or a pharma- ceutically acceptable salt thereof and the MAPKAP kinase inhibitor are administered substantially simultaneously or successively.

18. A pharmaceutical described in any one of claims 1 to 17, wherein the MAPKAP kinase inhibitor is binimetinib.

19. A pharmaceutical described in any one of claims 1 to 17, wherein the MAPKAP kinase inhibitor is trametinib.

20. A pharmaceutical agent described in any one of claims 1 to 19, wherein the patient experiences a partial reduction in tumor volume of at least 30% after use of the pharmaceutical agent for more than two weeks.

21. A pharmaceutical described in any one of claims 1 to 20, which brings about a complete reduction in tumor volume.

22. The method of any one of claims 1 to 21, wherein the treatment further comprises determining whether the tumor or tumor cells contain a primary mutation in the c-KIT gene.

23. The pharmaceutical described in claim 22, wherein the primary mutation is in exon 11 of the c-KIT gene.

24. The pharmaceutical described in claim 22, wherein the primary mutation is in exon 9 of the c-KIT gene.

25. The method according to any one of claims 22 to 24, wherein the primary mutation is a deletion mutation.

26. A pharmaceutical described in any one of claims 22 to 24, wherein the primary mutation is V560D.

27. The method of any one of claims 1 to 26, wherein one or more additional secondary mutations in c-KIT are present.

28. for use in the treatment of solid tumors in patients resistant to imatinib; an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea or a pharma- ceutically acceptable salt thereof; A pharmaceutical composition comprising an effective amount of a MAPKAP kinase inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib and ulixertinib, The solid tumor is a gastrointestinal stromal tumor (GIST).

29. The pharmaceutical agent of claim 28, further comprising a RAF inhibitor.

30. The pharmaceutical composition of claim 29, wherein the RAF inhibitor is a pan-RAF inhibitor.

31. For use in the treatment of imatinib-resistant gastrointestinal stromal tumors in a patient in need thereof. an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea, or a pharma- ceutically acceptable salt thereof; A medicine comprising in combination an effective amount of a MAPKAP kinase inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib, and ulixertinib.

32. The method of claim 31, wherein the treatment further comprises determining whether the tumor is associated with a mutation in the c-KIT gene.

33. The method of claim 32, wherein the mutation is in exon 17 of the c-KIT gene.

34. The pharmaceutical described in claim 32 or 33, wherein the c-KIT mutation is a substitution of aspartic acid at codon 816 or a substitution of asparagine at codon 822.

35. The pharmaceutical described in claim 32 or 33, wherein the mutation is one of D816V, D816E, D816H, D820A, T670I, or N822V.

36. The pharmaceutical composition of claim 32, wherein the mutation is a V654A mutation in exon 13 or a T670I mutation in exon 14.

37. For use in the treatment of a solid tumor in a patient in need thereof. an effective amount of 1-[4-bromo-5-[1-ethyl-7-(methylamino)-2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl]-2-fluorophenyl]-3-phenylurea or a pharma- ceutically acceptable salt thereof; A pharmaceutical composition comprising an effective amount of a MAPKAP kinase inhibitor selected from the group consisting of trametinib, binimetinib, cobimetinib, and ulixertinib, The solid tumor is a gastrointestinal stromal tumor (GIST).

38. The pharmaceutical agent of claim 37, further comprising a RAF inhibitor.

39. The pharmaceutical composition of claim 38, wherein the RAF inhibitor is a pan-RAF inhibitor.

40. The method of claim 37, wherein the treatment further comprises determining whether the tumor is associated with a mutation in the c-KIT gene.

41. The pharmaceutical composition described in claim 40, wherein the mutation is in exon 17 of the c-KIT gene.

42. The pharmaceutical described in claim 40 or 41, wherein the c-KIT mutation is a substitution of aspartic acid at codon 816 or a substitution of asparagine at codon 822.

43. The pharmaceutical described in claim 40 or 41, wherein the mutation is one of D816V, D816E, D816H, D820A, T670I, or N822V.

44. The pharmaceutical described in claim 40, wherein the mutation is a V654A mutation in exon 13 or a T670I mutation in exon 14.

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