WO2015095834A2 - Cancer treatments using erk1/2 and bcl-2 family inhibitors - Google Patents

Cancer treatments using erk1/2 and bcl-2 family inhibitors Download PDF

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WO2015095834A2
WO2015095834A2 PCT/US2014/071736 US2014071736W WO2015095834A2 WO 2015095834 A2 WO2015095834 A2 WO 2015095834A2 US 2014071736 W US2014071736 W US 2014071736W WO 2015095834 A2 WO2015095834 A2 WO 2015095834A2
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cas
inhibitors
cancer
delta
therapeutics
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PCT/US2014/071736
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French (fr)
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WO2015095834A3 (en
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Saurabh Saha
Dean WELSCH
Gary Decrescenzo
Jeffrey James ROIX
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Biomed Valley Discoveries, Inc.
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Publication of WO2015095834A3 publication Critical patent/WO2015095834A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • the present invention provides, inter alia, methods, and kits and compositions for treating or ameliorating the effects of a cancer in a subject in need thereof by administering to the subject an effective amount of (i) an ERK1/2 inhibitor or a pharmaceutically acceptable salt thereof and (ii) a BCL- 2 family inhibitor or a pharmaceutically acceptable salt thereof. Methods for selecting a subject with cancer that may benefit from such a combination drug therapy are also provided.
  • sequence listing text file "0375606.txt”, file size of 221 KB, created on December 18, 2014.
  • sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. ⁇ 1 .52(e)(5).
  • BCL-2 B-cell CLL/lymphoma 2 proteins
  • MOMP mitochondrial outer membrane permeabilization
  • Extracellular-signal-regulated kinases are protein kinases that are involved in cell cycle regulation, including the regulation of meiosis, mitosis, and postmitotic functions in differentiated cells. Disruption of the ERK pathway is common in cancers. However, to date, little progress has been made developing effective ERK inhibitors for the treatment of cancer.
  • One embodiment of the present invention is a method of treating or ameliorating the effects of a cancer in a subject in need thereof.
  • This method comprises administering to the subject an effective amount of (i) a first anti-cancer agent, which is an ERK1/2 inhibitor or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof, to treat or ameliorate the effects of the cancer.
  • Another embodiment of the present invention is a method of treating or ameliorating the effects of a cancer in a subject in need thereof.
  • This method comprises administering to the subject an effective amount of (i) BVD-523 or a pharmaceutically acceptable salt thereof and (ii) navitoclax or a pharmaceutically acceptable salt thereof, to treat or ameliorate the effects of the cancer.
  • An additional embodiment of the present invention is a method of effecting cancer cell death.
  • This method comprises contacting the cancer cell with an effective amount of (i) a first anti-cancer agent, which is BVD-523 or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof.
  • a further embodiment of the present invention is a kit for treating or ameliorating the effects of a cancer in a subject in need thereof.
  • This kit comprises an effective amount of (i) an ERK1/2 inhibitor or a pharmaceutically acceptable salt thereof and (ii) a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof, packaged together with instructions for their use.
  • Another embodiment of the present invention is a pharmaceutical composition for treating or ameliorating the effects of a cancer in a subject in need thereof.
  • This pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier and an effective amount of (i) a first anti-cancer agent, which is an ERK 1/2 inhibitor or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof, wherein administration of the first and second anti-cancer agents provides a synergistic effect compared to administration of either anti-cancer agent alone.
  • a further embodiment of the present invention is a method for selecting a subject with cancer that may benefit from a combination drug therapy. This method comprises:
  • Figure 1A is a dose matrix showing % inhibition of the ABT263 (navitoclax, a BCL-2 inhibitor)/BVD-523 (an ERK1/2 inhibitor) combination in a human malignant melanoma cell line (A375 cells) using the Alamar Blue cell viability assay.
  • Figure 1 B is a dose matrix showing excess over Bliss for the ABT263/BVD-523 combination.
  • Figure 1 C shows % viability relative to DMSO only treated controls for ABT263 and BVD-523 single agent treatments in A375 cells using the Alamar Blue cell viability assay.
  • Figure 1 D shows % viability relative to DMSO only treated controls for ABT263/BVD-523 combination treatments in A375 cells using the Alamar Blue cell viability assay.
  • Figure 2A is a dose matrix showing % inhibition of the ABT263/BVD-523 combination in a human colorectal carcinoma cell line (HCT1 16 cells) using the Alamar Blue cell viability assay.
  • Figure 2B is a dose matrix showing excess over Bliss for the ABT263/BVD-523 combination.
  • Figure 2C shows % viability relative to DMSO only treated controls for ABT263 and BVD-523 single agent treatments in HCT1 16 cells using the Alamar Blue cell viability assay.
  • Figure 2D shows % viability relative to DMSO only treated controls for ABT263/BVD-523 combination treatments in HCT1 16 cells using the Alamar Blue cell viability assay.
  • Figure 3A is a dose matrix showing % inhibition of the ABT263 (BCL-2 inhibitor)/dabrafenib (BRAF inhibitor) combination in A375 cells using the Alamar Blue cell viability assay.
  • Figure 3B is a dose matrix showing excess over Bliss for the ABT263/dabrafenib combination.
  • Figure 3C shows % viability relative to DMSO only treated controls for ABT263 and dabrafenib single agent treatments in A375 cells using the Alamar Blue cell viability assay.
  • Figure 3D shows % viability relative to DMSO only treated controls for ABT263/dabrafenib combination treatments in A375 cells using the Alamar Blue cell viability assay.
  • Figure 4A is a dose matrix showing % inhibition of the ABT263/dabrafenib combination in HCT1 16 cells using the Alamar Blue cell viability assay.
  • Figure 4B is a dose matrix showing excess over Bliss for the ABT263/dabrafenib combination.
  • Figure 4C shows % viability relative to DMSO only treated controls for ABT263 and dabrafenib single agent treatments in HCT1 16 cells using the Alamar Blue cell viability assay.
  • Figure 4D shows % viability relative to DMSO only treated controls for ABT263/dabrafenib combination treatments in HCT1 16 cells using the Alamar Blue cell viability assay.
  • Figure 5A is a dose matrix showing % inhibition of the ABT263 (BCL-2 inhibitor)/trametinib (type 2 MEK inhibitor) combination in A375 cells using the Alamar Blue cell viability assay.
  • Figure 5B is a dose matrix showing excess over Bliss for the ABT263/trametinib combination.
  • Figure 5C shows % viability relative to DMSO only treated controls for ABT263 and trametinib single agent treatments in A375 cells using the Alamar Blue cell viability assay.
  • Figure 5D shows % viability relative to DMSO only treated controls for ABT263/trametinib combination treatments in A375 cells using the Alamar Blue cell viability assay.
  • Figure 6A is a dose matrix showing % inhibition of the ABT263/trametinib combination in HCT1 16 cells using the Alamar Blue cell viability assay.
  • Figure 6B is a dose matrix showing excess over Bliss for the ABT263/trametinib combination.
  • Figure 6C shows % viability relative to DMSO only treated controls for ABT263 and trametinib single agent treatments in HCT1 16 cells using the Alamar Blue cell viability assay.
  • Figure 6D shows % viability relative to DMSO only treated controls for ABT263/trametinib combination treatments in HCT1 16 cells using the Alamar Blue cell viability assay.
  • Figure 7 is a histogram showing viability of HCT1 16 cells after 96 hours of incubation with various amounts of BVD-523 or BVD-523 in combination with 3 ⁇ ABT-263. The Bliss Scores are shown in the yellow boxes.
  • Figure 8 is a histogram showing caspase activity in HCT1 16 cells after 24 hours of incubation with various amounts of BVD-523 or BVD- 523 in combination with 3 ⁇ ABT-263.
  • Figure 9 is a histogram showing caspase activity in HCT1 16 cells after 48 hours of incubation with various amounts of BVD-523 or BVD- 523 in combination with 3 ⁇ ABT-263.
  • Figure 10A is a dose matrix showing % inhibition of the ABT199 (another BCL-2 inhibitor)/BVD-523 combination in A375 cells using the Alamar Blue cell viability assay.
  • Figure 10B is a dose matrix showing excess over Bliss for the ABT199/BVD-523 combination.
  • Figure 10C shows % viability relative to DMSO only treated controls for ABT199 and BVD-523 single agent treatments in A375 cells using the Alamar Blue cell viability assay.
  • Figure 10D shows % viability relative to DMSO only treated controls for ABT199/BVD-523 combination treatments in A375 cells using the Alamar Blue cell viability assay.
  • Figure 1 1A is a dose matrix showing % inhibition of the ABT199/BVD-523 combination in A375 cells using the CellTiter-Glo cell viability assay.
  • Figure 1 1 B is a dose matrix showing excess over Bliss for the ABT199/BVD-523 combination.
  • Figure 1 1 C shows % viability relative to DMSO only treated controls for ABT199 and BVD-523 single agent treatments in A375 cells using the CellTiter-Glo cell viability assay.
  • Figure 1 1 D shows % viability relative to DMSO only treated controls for ABT199/BVD-523 combination treatments in A375 cells using the CellTiter-Glo cell viability assay.
  • Figure 12A is a dose matrix showing % inhibition of the ABT199/dabrafenib combination in A375 cells using the Alamar Blue cell viability assay.
  • Figure 12B is a dose matrix showing excess over Bliss for the ABT199/dabrafenib combination.
  • Figure 12C shows % viability relative to DMSO only treated controls for ABT199 and dabrafenib single agent treatments in A375 cells using the Alamar Blue cell viability assay.
  • Figure 12D shows % viability relative to DMSO only treated controls for ABT199/dabrafenib combination treatments in A375 cells using the Alamar Blue cell viability assay.
  • Figure 13A is a dose matrix showing % inhibition of the ABT199/dabrafenib combination in A375 cells using the CellTiter-Glo cell viability assay.
  • Figure 13B is a dose matrix showing excess over Bliss for the ABT199/dabrafenib combination.
  • Figure 13C shows % viability relative to DMSO only treated controls for ABT199 and dabrafenib single agent treatments in A375 cells using the CellTiter-Glo cell viability assay.
  • Figure 13D shows % viability relative to DMSO only treated controls for ABT199/dabrafenib combination treatments in A375 cells using the CellTiter- Glo cell viability assay.
  • Figure 14A is a dose matrix showing % inhibition of the ABT199/trametinib combination in A375 cells using the Alamar Blue cell viability assay.
  • Figure 14B is a dose matrix showing excess over Bliss for the ABT199/trametinib combination.
  • Figure 14C shows % viability relative to DMSO only treated controls for ABT199 and trametinib single agent treatments in A375 cells using the Alamar Blue cell viability assay.
  • Figure 14D shows % viability relative to DMSO only treated controls for ABT199/trametinib combination treatments in A375 cells using the Alamar Blue cell viability assay.
  • Figure 15A is a dose matrix showing % inhibition of the ABT199/trametinib combination in A375 cells using the CellTiter-Glo cell viability assay.
  • Figure 15B is a dose matrix showing excess over Bliss for the ABT199/trametinib combination.
  • Figure 15C shows % viability relative to DMSO only treated controls for ABT199 and trametinib single agent treatments in A375 cells using the CellTiter-Glo cell viability assay.
  • Figure 15D shows % viability relative to DMSO only treated controls for ABT199/trametinib combination treatments in A375 cells using the CellTiter- Glo cell viability assay.
  • Figure 16 shows that both direct ERK substrate phosphorylation and known effector pathways are modulated following acute and prolonged treatment with BVD-523 in vitro.
  • Western blots were performed using a variety of antibodies to detect changes in whole-cell lysates of cancer lines exposed to BVD-523.
  • A375 BRAF mutant cell line a human melanoma cell line
  • HCT1 16 KRAS mutant cell line a human colorectal carcinoma cell line
  • Figure 17 shows the results of the combination of BVD-523 and ABT-263.
  • Figure 17A shows a dose matrix showing inhibition (%) for the combination in A549 cells.
  • Figure 17B - Figure 17C show the results of single agent proliferation assays for the combination in 17A.
  • Figure 17D shows Loewe excess for the combination in 17A and
  • Figure 17E shows Bliss excess for the combination in 17A.
  • Figure 17F shows a dose matrix showing inhibition (%) for the combination in H1650 cells.
  • Figure 17G - Figure 17H show the results of single agent proliferation assays for the combination in 17F.
  • Figure 171 shows Loewe excess for the combination in 17F and
  • Figure 17J shows Bliss excess for the combination in 17F.
  • Figure 17K shows a dose matrix showing inhibition (%) for the combination in H226 cells.
  • Figure 17L - Figure 17M show the results of single agent proliferation assays for the combination in 17K.
  • Figure 17N shows Loewe excess for the combination in 17K and Figure 170 shows Bliss excess for the combination in 17K.
  • Figure 17P shows a dose matrix showing inhibition (%) for the combination in H460 cells.
  • Figure 17Q - Figure 17R show the results of single agent proliferation assays for the combination in 17P.
  • FIG. 17S shows Loewe excess for the combination in 17P and Figure 17T shows Bliss excess for the combination in 17P.
  • Figure 18 shows the results of the combination of SCH772984 and ABT-263.
  • Figure 18A shows a dose matrix showing inhibition (%) for the combination in A549 cells.
  • Figure 18B - Figure 18C show the results of single agent proliferation assays for the combination in 18A.
  • Figure 18D shows Loewe excess for the combination in 18A and
  • Figure 18E shows Bliss excess for the combination in 18A.
  • Figure 18F shows a dose matrix showing inhibition (%) for the combination in H1650 cells.
  • Figure 18G - Figure 18H show the results of single agent proliferation assays for the combination in 18F.
  • Figure 181 shows Loewe excess for the combination in 18F and Figure 18J shows Bliss excess for the combination in 18F.
  • Figure 18K shows a dose matrix showing inhibition (%) for the combination in H226 cells.
  • Figure 18L - Figure 18M show the results of single agent proliferation assays for the combination in 18K.
  • Figure 18N shows Loewe excess for the combination in 18K and Figure 180 shows Bliss excess for the combination in 18K.
  • Figure 18P shows a dose matrix showing inhibition (%) for the combination in H460 cells.
  • Figure 18Q - Figure 18R show the results of single agent proliferation assays for the combination in 18P.
  • FIG. 18S shows Loewe excess for the combination in 18P and Figure 18T shows Bliss excess for the combination in 18P.
  • Figure 19 shows the results of the combination of BVD-523 and ABT-263.
  • Figure 19A shows a dose matrix showing inhibition (%) for the combination in A549 cells.
  • Figure 19B - Figure 19C show the results of single agent proliferation assays for the combination in 19A.
  • Figure 19D shows Loewe excess for the combination in 19A and
  • Figure 19E shows Bliss excess for the combination in 19A.
  • Figure 19F shows a dose matrix showing inhibition (%) for the combination in H1650 cells.
  • Figure 19G - Figure 19H show the results of single agent proliferation assays for the combination in 19F.
  • Figure 191 shows Loewe excess for the combination in 19F and
  • Figure 19J shows Bliss excess for the combination in 19F.
  • Figure 19K shows a dose matrix showing inhibition (%) for the combination in H226 cells.
  • Figure 19L - Figure 19M show the results of single agent proliferation assays for the combination in 19K.
  • Figure 19N shows Loewe excess for the combination in 19K and Figure 190 shows Bliss excess for the combination in 19K.
  • Figure 19P shows a dose matrix showing inhibition (%) for the combination in H460 cells.
  • Figure 19Q - Figure 19R show the results of single agent proliferation assays for the combination in 19P.
  • FIG. 19S shows Loewe excess for the combination in 19P and Figure 19T shows Bliss excess for the combination in 19P.
  • Figure 20A shows Lowe Volumes for the combinations of BVD- 523, SCH772984 and Trametinib with ABT-263.
  • Figure 20B shows Bliss Volumes for the combinations of BVD-523, SCH772984 and Trametinib with ABT-263.
  • Figure 20C shows Synergy Scores for the combinations of BVD- 523, SCH772984 and Trametinib with ABT-263.
  • Figure 21 shows the results of the combination of BVD-523 and SCH772984.
  • Figure 21 A shows a dose matrix showing inhibition (%) for the combination in A375 cells.
  • Figure 21 B - Figure 21 C show the results of single agent proliferation assays for the combination in 21 A.
  • Figure 21 D shows Loewe excess for the combination in 21 A and
  • Figure 21 E shows Bliss excess for the combination in 21A.
  • One embodiment of the present invention is a method of treating or ameliorating the effects of a cancer in a subject in need thereof.
  • This method comprises administering to the subject an effective amount of (i) a first anti-cancer agent, which is an ERK1/2 inhibitor or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof, to treat or ameliorate the effects of the cancer.
  • the terms "treat,” “treating,” “treatment” and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient.
  • the methods and compositions of the present invention may be used to slow the development of disease symptoms or delay the onset of the disease or condition, or halt the progression of disease development.
  • every treated subject may not respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population, e.g., patient population. Accordingly, a given subject or subject population, e.g., patient population, may fail to respond or respond inadequately to treatment.
  • ameliorate means to decrease the severity of the symptoms of a disease in a subject.
  • a "subject" is a mammal, preferably, a human.
  • categories of mammals within the scope of the present invention include, for example, farm animals, domestic animals, laboratory animals, etc.
  • farm animals include cows, pigs, horses, goats, etc.
  • domestic animals include dogs, cats, etc.
  • laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc.
  • cancers include both solid and hemotologic cancers.
  • solid cancers include adrenocortical carcinoma, anal cancer, bladder cancer, bone cancer (such as osteosarcoma), brain cancer, breast cancer, carcinoid cancer, carcinoma, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewing family of cancers, extracranial germ cell cancer, eye cancer, gallbladder cancer, gastric cancer, germ cell tumor, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma, kidney cancer, large intestine cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, malignant mesothelioma, Merkel cell carcinoma, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma
  • the subject's cancer is preferably selected from the group consisting of colorectal cancer, breast cancer, pancreatic cancer, lymphoma, lung cancer, testicular cancer, thyroid cancer, hematologic malignancies, and endometrial cancers. More preferably, the cancer is colorectal cancer.
  • Non-limiting examples of hematologic malignancies include RAS mutant myelodysplastic syndromes (MDS), lymphoid cancers, and myeloid cancers.
  • hematologic malignancies also include, e.g., Adult Acute Megakaryoblastic Leukemia (M7), Adult Acute Minimally Differentiated Myeloid Leukemia (M0), Adult Acute Monoblastic Leukemia (M5a), Adult Acute Monocytic Leukemia (M5b), Adult Acute Myeloblasts Leukemia With Maturation (M2), Adult Acute Myeloblasts Leukemia Without Maturation (M1 ), Adult Acute Myeloid Leukemia With 1 1 q23 (MLL) Abnormalities, Adult Acute Myeloid Leukemia With Del(5q), Adult Acute Myeloid Leukemia With Inv(16)(p13;q22), Adult Acute Myeloid Leukemia With t(16;16)(p13
  • an "ERK1/2 inhibitor” means those substances that (i) directly interact with ERK1 and/or ERK2, e.g., by binding to ERK1/2 and (ii) decrease the expression or the activity of ERK1 and/or ERK2 protein kinases. Therefore, inhibitors that act upstream of ERK1/2, such as MEK inhibitors and RAF inhibitors, are not ERK1/2 inhibitors according to the present invention. Preferred ERK1/2 inhibitors of the present invention do not decrease the amount of phosphorylated ERK1 and/or ERK2 but decrease the activity of phosphorylated ERK1 and/or ERK2.
  • Non-limiting examples of ERK1/2 inhibitors include AEZS-131 (Aeterna Zentaris), AEZS-136 (Aeterna Zentaris), BVD-523 (BioMed Valley Discoveries, Inc.), SCH-722984 (Merck & Co.), SCH-772984 (Merck & Co.), SCH-900353 (MK-8353) (Merck & Co.), pharmaceutically acceptable salts thereof, and combinations thereof.
  • the ERK1/2 inhibitor is BVD-523 or a pharmaceutically acceptable salt thereof.
  • BVD-523 a preferred ERK1/2 inhibitor, corresponds to a compound according to formula (I):
  • BVD-523 may be synthesized according to the methods disclosed, e.g., in U.S. Patent No. 7,354,939. Enantiomers and racemic mixtures of both enantiomers of BVD-523 are also contemplated within the scope of the present invention.
  • BVD-523 is a preferred ERK1/2 inhibitor of the present invention because its mechanism of action is believed to be, inter alia, unique and distinct from certain other ERK1/2 inhibitors, such as SCH772984.
  • SCH772984 inhibits autophosphorylation of ERK (Morris et al., 2013), whereas BVD-523 allows for the autophosphorylation of ERK while still inhibiting ERK. (See, e.g., Figure 16).
  • a "BLC2 family inhibitor” means those substances that (i) directly interact with BLC2 family members, e.g., by binding to BLC2 family members and (ii) decrease the expression or the activity of BLC2 family members.
  • BCL-2 family inhibitors of the present inventions include (-)-epigallocatechin gallate (Sigma Aldrich, St.
  • the ERK 1/2 inhibitor is BVD- 523 or a pharmaceutically acceptable salt thereof and the BCL-2 family inhibitor is navitoclax or a pharmaceutically acceptable salt thereof.
  • the ERK1/2 inhibitors and/or the BCL-2 family inhibitors may be administered according to the methods of the present invention as a part of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent.
  • the subject with cancer has a somatic RAS mutation.
  • "somatic mutation” means a change occurring in any cell that is not destined to become a germ cell.
  • the mutation may be, e.g., a substitution, deletion, insertion, or a fusion.
  • the RAS mutation is a mutation in H-RAS, N-RAS, or K-RAS. Table 1 below shows the distribution and frequency of these RAS mutations in human tumors. These data were obtained from the Sanger Catalogue of Somatic Mutations in Cancer.
  • CML Crohn's disease
  • Tables 2, 3, and 4 below show the SEQ ID Nos. of representative nucleic acid and amino acid sequences of wild type H-RAS, K- RAS, and N-RAS from various animals, respectively. These sequences may be used in methods for identifying subjects with a mutant RAS genotype (such as in the methods set forth below).
  • Bos taurus variant X1 33 nucleic acid cow, Bos taurus variant X1
  • Nucleic acids may be obtained from biological samples.
  • biological samples include, but are not limited to, blood, plasma, urine, skin, saliva, and biopsies.
  • Biological samples are obtained from a subject by routine procedures and methods which are known in the art.
  • Non-limiting examples of methods for identifying mutations include PCR, sequencing, hybrid capture, in-solution capture, molecular inversion probes, fluorescent in situ hybridization (FISH) assay, and combinations thereof.
  • Various sequencing methods are known in the art. These include, but are not limited to, Sanger sequencing (also referred to as dideoxy sequencing) and various sequencing-by-synthesis (SBS) methods as disclosed in, e.g., Metzker, 2005, sequencing by hybridization, by ligation (for example, WO 2005021786), by degradation (for example, U.S. Patent Nos. 5,622,824 and 6,140,053) and nanopore sequencing (which is commercially available from Oxford Nanopore Technologies, UK).
  • SBS sequencing-by-synthesis
  • a given nucleotide in the sequence is read more than once during the sequencing process. Deep sequencing techniques are disclosed in e.g., U.S. Patent Publication No. 20120264632 and International Patent Publication No. WO2012125848.
  • PCR-based methods for detecting mutations are known in the art and employ PCR amplification, where each target sequence in the sample has a corresponding pair of unique, sequence-specific primers.
  • PCR-RFLP polymerase chain reaction-restriction fragment length polymorphism
  • the mutation is discriminated by digestion with specific restriction endonucleases and is identified by electrophoresis. See, e.g., Ota et al., 2007. Mutations may also be detected using real time PCR. See, e.g., International Application publication No. WO2012046981 .
  • Hybrid capture methods are known in the art and are disclosed in e.g., U.S. Patent Publication No. 20130203632 and U.S. Patent Nos. 8,389,219 and 8,288,520. These methods are based on the selective hybridization of the target genomic regions to user-designed oligonucleotides.
  • the hybridization can be to oligonucleotides immobilized on high or low density microarrays (on-array capture), or solution-phase hybridization to oligonucleotides modified with a ligand (e.g. biotin) which can subsequently be immobilized to a solid surface, such as a bead (in-solution capture).
  • a ligand e.g. biotin
  • MIP Molecular Inversion Probe
  • genomic homology regions are ligated by undergoing an inversion in configuration (as suggested by the name of the technique) and creating a circular molecule. After the first restriction, all molecules are amplified with universal primers. Amplicons are restricted again to ensure short fragments for hybridization on a microarray. Generated short fragments are labeled and, through a Tag sequence, hybridized to a cTag (complementary strand for index) on an array. After the formation of Tag-cTag duplex, a signal is detected.
  • cTag complementary strand for index
  • the method further comprises administering to the subject at least one additional therapeutic agent effective for treating or ameliorating the effects of the cancer.
  • the additional therapeutic agent may be selected from the group consisting of an antibody or fragment thereof, a cytotoxic agent, a toxin, a radionuclide, an immunomodulator, a photoactive therapeutic agent, a radiosensitizing agent, a hormone, an anti-angiogenesis agent, and combinations thereof.
  • an "antibody” encompasses naturally occurring immunoglobulins as well as non-naturally occurring immunoglobulins, including, for example, single chain antibodies, chimeric antibodies ⁇ e.g., humanized murine antibodies), and heteroconjugate antibodies ⁇ e.g., bispecific antibodies). Fragments of antibodies include those that bind antigen, ⁇ e.g., Fab', F(ab') 2 , Fab, Fv, and rlgG). See also, e.g., Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, III.); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York (1998). The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. The term "antibody” further includes both polyclonal and monoclonal antibodies.
  • therapeutic antibodies examples include rituximab (Rituxan), Cetuximab (Erbitux), bevacizumab (Avastin), and Ibritumomab (Zevalin).
  • Cytotoxic agents according to the present invention include DNA damaging agents, antimetabolites, anti-microtubule agents, antibiotic agents, etc.
  • DNA damaging agents include alkylating agents, platinum-based agents, intercalating agents, and inhibitors of DNA replication.
  • Non-limiting examples of DNA alkylating agents include cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, ifosfamide, carmustine, lomustine, streptozocin, busulfan, temozolomide, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof.
  • Non-limiting examples of platinum-based agents include cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, triplatin tetranitrate, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof.
  • Non-limiting examples of intercalating agents include doxorubicin, daunorubicin, idarubicin, mitoxantrone, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof.
  • Non-limiting examples of inhibitors of DNA replication include irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof.
  • Antimetabolites include folate antagonists such as methotrexate and premetrexed, purine antagonists such as 6-mercaptopurine, dacarbazine, and fludarabine, and pyrimidine antagonists such as 5-fluorouracil, arabinosylcytosine, capecitabine, gemcitabine, decitabine, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof.
  • Anti- microtubule agents include without limitation vinca alkaloids, paclitaxel (Taxol®), docetaxel (Taxotere®), and ixabepilone (Ixempra®).
  • Antibiotic agents include without limitation actinomycin, anthracyclines, valrubicin, epirubicin, bleomycin, plicamycin, mitomycin, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof.
  • a preferred additional therapeutics agent is an inhibitor of the PI3K/Akt pathway.
  • Non-limiting examples of an inhibitor of the PI3K/Akt pathway include A-674563 (CAS # 552325-73-2), AGL 2263, AMG-319 (Amgen, Thousand Oaks, CA), AS-041 164 (5- benzo[1 ,3]dioxol-5-ylmethylene-thiazolidine-2,4-dione), AS-604850 (5-(2,2- Difluoro-benzo[1 ,3]dioxol-5-ylmethylene)-thiazolidine-2,4-dione), AS-605240 (5-quinoxilin-6-methylene-1 ,3-thiazolidine-2,4-dione), AT7867 (CAS # 857531 -00-1 ), benzimidazole series, Genentech (Roche Holdings Inc., South San Francisco, CA), BML-257 (CAS # 32387-96-5), CAL-120 (Gilead Sciences, Foster City, CA), CAL-129 (Gilead Sciences), CAL-130 (
  • PI3 kinase delta inhibitors-2 Incozen (Incozen Therapeutics), PI3 kinase inhibitor, Roche-4 (Roche Holdings Inc.), PI3 kinase inhibitors, Roche (Roche Holdings Inc.), PI3 kinase inhibitors, Roche-5 (Roche Holdings Inc.), PI3-alpha/delta inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd., South San Francisco, CA), PI3-delta inhibitors, Cellzome (Cellzome AG, Heidelberg, Germany), PI3-delta inhibitors, Intellikine (Intellikine Inc., La Jolla, CA), PI3-delta inhibitors, Pathway Therapeutics-1 (Pathway Therapeutics Ltd.), PI3-delta inhibitors, Pathway Therapeutics-2 (Pathway Therapeutics Ltd.), PI3-delta/gamma inhibitors, Cellzome (Cellzome AG), PI3-delta/gamma inhibitors, Cellz
  • the term "toxin” means an antigenic poison or venom of plant or animal origin.
  • An example is diphtheria toxin or portions thereof.
  • the term “radionuclide” means a radioactive substance administered to the patient, e.g., intravenously or orally, after which it penetrates via the patient's normal metabolism into the target organ or tissue, where it delivers local radiation for a short time.
  • radionuclides include, but are not limited to, 1-125, At-21 1 , Lu-177, Cu-67, I- 131 , Sm-153, Re-186, P-32, Re-188, ln-1 14m, and Y-90.
  • the term "immunomodulator” means a substance that alters the immune response by augmenting or reducing the ability of the immune system to produce antibodies or sensitized cells that recognize and react with the antigen that initiated their production.
  • Immunomodulators may be recombinant, synthetic, or natural preparations and include cytokines, corticosteroids, cytotoxic agents, thymosin, and immunoglobulins. Some immunomodulators are naturally present in the body, and certain of these are available in pharmacologic preparations.
  • immunomodulators include, but are not limited to, granulocyte colony- stimulating factor (G-CSF), interferons, imiquimod and cellular membrane fractions from bacteria, IL-2, IL-7, IL-12, CCL3, CCL26, CXCL7, and synthetic cytosine phosphate-guanosine (CpG).
  • G-CSF granulocyte colony- stimulating factor
  • interferons imiquimod and cellular membrane fractions from bacteria
  • IL-2, IL-7, IL-12, CCL3, CCL26, CXCL7 and synthetic cytosine phosphate-guanosine (CpG).
  • the term "photoactive therapeutic agent” means compounds and compositions that become active upon exposure to light. Certain examples of photoactive therapeutic agents are disclosed in, e.g., U.S. Patent Application Serial No. 201 1/0152230 A1 , "Photoactive Metal Nitrosyls For Blood Pressure Regulation And Cancer Therapy.”
  • the term “radiosensitizing agent” means a compound that makes tumor cells more sensitive to radiation therapy. Examples of radiosensitizing agents include misonidazole, metronidazole, tirapazamine, and trans sodium crocetinate.
  • hormone means a substance released by cells in one part of a body that affects cells in another part of the body.
  • hormones include, but are not limited to, prostaglandins, leukotrienes, prostacyclin, thromboxane, amylin, antimullerian hormone, adiponectin, adrenocorticotropic hormone, angiotensinogen, angiotensin, vasopressin, atriopeptin, brain natriuretic peptide, calcitonin, cholecystokinin, corticotropin-releasing hormone, encephalin, endothelin, erythropoietin, follicle-stimulating hormone, galanin, gastrin, ghrelin, glucagon, gonadotropin- releasing hormone, growth hormone-releasing hormone, human chorionic gonadotropin, human placental lactogen, growth hormone, in
  • Some compounds interfere with the activity of certain hormones or stop the production of certain hormones.
  • These hormone-interfering compounds include, but are not limited to, tamoxifen (Nolvadex®), anastrozole (Arimidex®), letrozole (Femara®), and fulvestrant (Faslodex®). Such compounds are also within the meaning of hormone in the present invention.
  • an "anti-angiogenesis” agent means a substance that reduces or inhibits the growth of new blood vessels, such as, e.g., an inhibitor of vascular endothelial growth factor (VEGF) and an inhibitor of endothelial cell migration.
  • VEGF vascular endothelial growth factor
  • Anti-angiogenesis agents include without limitation 2-methoxyestradiol, angiostatin, bevacizumab, cartilage-derived angiogenesis inhibitory factor, endostatin, IFN-a, IL-12, itraconazole, linomide, platelet factor-4, prolactin, SU5416, suramin, tasquinimod, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, thrombospondin, TNP-470, ziv-aflibercept, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof.
  • administration of the first and second anti-cancer agents provides a synergistic effect compared to administration of either anti-cancer agent alone.
  • synergistic means more than additive. Synergistic effects may be measured by various assays known in the art, including but not limited to those disclosed herein, , such as the excess over bliss assay.
  • Another embodiment of the present invention is a method of treating or ameliorating the effects of a cancer in a subject in need thereof.
  • This method comprises administering to the subject an effective amount of (i) BVD-523 or a pharmaceutically acceptable salt thereof and (ii) navitoclax or a pharmaceutically acceptable salt thereof, to treat or ameliorate the effects of the cancer.
  • the BVD-523 and navitoclax, or their respective pharmaceutically acceptable salts may be administered in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or diluent.
  • Suitable and preferred subjects and various types of cancer are as disclosed herein.
  • the methods may be used to treat the cancers disclosed above, including those cancers with the mutational backgrounds identified above. Methods of identifying such mutations are also as set forth above.
  • the method further comprises administering at least one additional therapeutic agent, preferably an inhibitor of the PI3K/Akt pathway, as disclosed herein.
  • administration of the BVD-523 and navitoclax provides a synergistic effect compared to administration of either agent alone.
  • An additional embodiment of the present invention is a method of effecting cancer cell death.
  • This method comprises contacting the cancer cell with an effective amount of (i) a first anti-cancer agent, which is BVD-523 or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof.
  • "contacting" means bringing the ERK1/2 inhibitors, BCL-2 family inhibitors, and optionally one or more additional therapeutic agents into close proximity to the cancer cells.
  • ERK1/2 inhibitors e.g., providing the ERK1/2 inhibitors, BCL-2 family inhibitors, and optionally other therapeutic agents to a culture media in which the cancer cells are located.
  • Suitable and preferred ERK1/2 inhibitors, BCL-2 family inhibitors, and combinations of the foregoing are as disclosed herein.
  • the methods of this embodiment may be carried out in vitro or in vivo, and may be used to effect cancer cell death in cells of the types of cancer disclosed herein.
  • effecting cancer cell death may be accomplished in cancer cells having various mutational backgrounds and/or that are characterized as disclosed above. Methods of identifying such mutations are also as set forth above.
  • the cancer cell is a mammalian cancer cell.
  • the mammalian cancer cell is obtained from a mammal selected from the group consisting of humans, primates, farm animals, and domestic animals. More preferably, the mammalian cancer cell is a human cancer cell.
  • the method may be used to effect cell death in any of the cancers disclosed herein, preferably colorectal cancer cells.
  • the method further comprises contacting the cancer cell with at least one additional therapeutic agent, preferably an inhibitor of the PI3K/Akt pathway, as disclosed herein.
  • contacting the cancer cell with the first and second ant-cancer agents provides a synergistic effect compared to contacting the cancer cell with either anti-cancer agent alone.
  • a further embodiment of the present invention is a kit for treating or ameliorating the effects of a cancer in a subject in need thereof.
  • This kit comprises an effective amount of (i) an ERK1/2 inhibitor or a pharmaceutically acceptable salt thereof and (ii) a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof, packaged together with instructions for their use.
  • kits may also include suitable storage containers, e.g., ampules, vials, tubes, etc., for each anti-cancer agent of the present invention (which may be, e.g., in the form of pharmaceutical compositions) and other reagents, e.g., buffers, balanced salt solutions, etc., for use in administering the anti-cancer agents to subjects.
  • suitable storage containers e.g., ampules, vials, tubes, etc.
  • other reagents e.g., buffers, balanced salt solutions, etc.
  • the kits may further include a packaging container, optionally having one or more partitions for housing the pharmaceutical composition(s) and other optional reagents.
  • kits of the invention may be used to treat any of the cancers disclosed herein, including those cancers having mutational backgrounds and/or that are characterized as disclosed above. Methods of identifying such mutations are as set forth above.
  • the kit further comprises at least one additional therapeutic agent, preferably an inhibitor of the PI3K/Akt pathway, as disclosed herein.
  • administration of the first and second anti-cancer agents provides a synergistic effect compared to administration of either anti-cancer agent alone.
  • Another embodiment of the present invention is a pharmaceutical composition for treating or ameliorating the effects of a cancer in a subject in need thereof.
  • This pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier and an effective amount of (i) a first anti-cancer agent, which is an ERK 1/2 inhibitor or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof, wherein administration of the first and second anti-cancer agents provides a synergistic effect compared to administration of either anti-cancer agent alone.
  • This pharmaceutical composition may further comprise a pharmaceutically acceptable diluent or carrier.
  • ERK1/2 inhibitors ERK1/2 inhibitors
  • BCL-2 family inhibitors ERK1/2 inhibitors
  • combinations of ERK1/2 inhibitors and BCL-2 family inhibitors are as disclosed herein.
  • the pharmaceutical compositions of the invention may be used to treat any of the cancers disclosed herein, including those cancers having various mutational backgrounds and/or that are characterized as disclosed above.
  • the pharmaceutical composition further comprises at least one additional therapeutic agent, preferably an inhibitor of the PI3K/Akt pathway, as disclosed herein.
  • compositions according to the present invention may be in a unit dosage form comprising both anti-cancer agents.
  • first anti-cancer agent is in a first unit dosage form and the second anti-cancer agent is in a second unit dosage form, separate from the first.
  • the first and second anti-cancer agents may be co-administered to the subject, either simultaneously or at different times, as deemed most appropriate by a physician. If the first and second anti-cancer agents are administered at different times, for example, by serial administration, the first anti-cancer agent may be administered to the subject before the second anticancer agent. Alternatively, the second anti-cancer agent may be administered to the subject before the first anti-cancer agent.
  • a further embodiment of the present invention is a method for selecting a subject with cancer that may benefit from a combination drug therapy. This method comprises:
  • EMT epithelial-to-mesenchymal transition
  • TGF transforming growth fact
  • PDGF platelet-derived growth factor
  • modulations of downstream transcription factors including the Goosecoid (Gsc), Snail (Snail), Snai2 (Slug), Twist 1 (Twist), FOXC1 , FOXC2, Zeb1 , Sip1 (Zeb2), as well as the members of the miR-200 family of microRNAs, may lead to EMT.
  • Determining an EMT gene signature is within the skill of the art and may be accomplished according to the method set forth in Taube et al., 2010 Determining the presence of a K-RAS mutation may be accomplished by using one of the methods disclosed herein.
  • ERK1/2 inhibitors ERK1/2 inhibitors, BCL-2 family inhibitors, combinations of ERK1/2 inhibitors and BCL-2 family inhibitors are as disclosed herein.
  • the methods may be used to treat the cancers disclosed above, including those cancers with the mutational backgrounds identified above. Methods of identifying such mutations are also as set forth above
  • the method further comprises administering at least one additional therapeutic agent, preferably an inhibitor of the PI3K/Akt pathway, as disclosed herein.
  • an "effective amount” or a "therapeutically effective amount” of an anti-cancer agent of the invention is an amount of such agent or composition that is sufficient to effect beneficial or desired results as described herein when administered to a subject.
  • Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the route of administration, the rate of excretion, the duration of the treatment, the identity of any other drugs being administered, the age, size, and species of mammal, e.g., human patient, and like factors well known in the arts of medicine and veterinary medicine.
  • a suitable dose of an agent or composition according to the invention will be that amount of the agent or composition, which is the lowest dose effective to produce the desired effect.
  • the effective dose of an agent or composition of the present invention may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.
  • a suitable, non-limiting example of a dosage of an ERK1/2 inhibitor, a BCL-2 family inhibitor, or another anti-cancer agent disclosed herein is from about 1 mg/kg to about 2400 mg/kg per day, such as from about 1 mg/kg to about 1200 mg/kg per day, 75 mg/kg per day to about 300 mg/kg per day, including from about 1 mg/kg to about 100 mg/kg per day.
  • Other representative dosages of such agents include about 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg, 1 100 mg/kg, 1200 mg/kg, 1300 mg/kg, 1400 mg/kg, 1500 mg/kg, 1600 mg/kg, 1700 mg/kg, 1800 mg/kg, 1900 mg/kg, 2000 mg/kg, 2100 mg/kg, 2200 mg/kg, and 2300 mg/kg per day.
  • the effective dose of an ERK1/2 inhibitor, a BCL-2 family inhibitor, or another anti-cancer agent disclosed herein, e.g., BVD-523 and navitoclax, may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.
  • the ERK1/2 inhibitors, BCL-2 family inhibitors, or other anticancer agents or pharmaceutical compositions containing same of the present invention may be administered in any desired and effective manner: for oral ingestion, or as an ointment or drop for local administration to the eyes, or for parenteral or other administration in any appropriate manner such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic.
  • the ERK1/2 inhibitors, BCL-2 family inhibitors, or other anti-cancer agents or pharmaceutical compositions containing same of the present invention may be administered in conjunction with other treatments.
  • the ERK1/2 inhibitors, BCL-2 family inhibitors, or other anticancer agents or the pharmaceutical compositions of the present invention may be encapsulated or otherwise protected against gastric or other secretions, if desired.
  • compositions of the invention comprise one or more active ingredients, e.g. anti-cancer agents, in admixture with one or more pharmaceutically-acceptable diluents or carriers and, optionally, one or more other compounds, drugs, ingredients and/or materials.
  • active ingredients e.g. anti-cancer agents
  • pharmaceutically-acceptable diluents or carriers optionally, one or more other compounds, drugs, ingredients and/or materials.
  • the agents/compounds of the present invention are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g., Remington, The Science and Practice of Pharmacy (21 st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.).
  • Pharmaceutically acceptable diluents or carriers are well known in the art (see, e.g., Remington, The Science and Practice of Pharmacy (21 st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.) and The National Formulary (American Pharmaceutical Association, Washington, D.C.)) and include sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starches, cellulose preparations, calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogen phosphate), sodium citrate, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and
  • Each pharmaceutically acceptable diluent or carrier used in a pharmaceutical composition of the invention must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.
  • Diluents or carriers suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable diluents or carriers for a chosen dosage form and method of administration can be determined using ordinary skill in the art.
  • compositions of the invention may, optionally, contain additional ingredients and/or materials commonly used in pharmaceutical compositions.
  • ingredients and materials are well known in the art and include (1 ) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8)
  • compositions of the present invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules, a solution or a suspension in an aqueous or nonaqueous liquid, an oil-in-water or water-in-oil liquid emulsion, an elixir or syrup, a pastille, a bolus, an electuary or a paste.
  • These formulations may be prepared by methods known in the art, e.g., by means of conventional pan- coating, mixing, granulation or lyophilization processes.
  • Solid dosage forms for oral administration may be prepared, e.g., by mixing the active ingredient(s) with one or more pharmaceutically-acceptable diluents or carriers and, optionally, one or more fillers, extenders, binders, humectants, disintegrating agents, solution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants, and/or coloring agents.
  • Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using a suitable excipient.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using a suitable binder, lubricant, inert diluent, preservative, disintegrant, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine.
  • the tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical- formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein. They may be sterilized by, for example, filtration through a bacteria-retaining filter.
  • compositions may also optionally contain opacifying agents and may be of a composition such that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • the active ingredient(s) may also be in microencapsulated form.
  • Liquid dosage forms for oral administration include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain suitable inert diluents commonly used in the art.
  • the oral compositions may also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions may contain suspending agents.
  • compositions of the present invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more active ingredient(s) with one or more suitable nonirritating diluents or carriers which are solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • suitable nonirritating diluents or carriers which are solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • the pharmaceutical compositions of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such pharmaceutically-acceptable diluents or carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants.
  • the active agent(s)/compound(s) may be mixed under sterile conditions with a suitable pharmaceutically-acceptable diluent or carrier.
  • the ointments, pastes, creams and gels may contain excipients.
  • Powders and sprays may contain excipients and propellants.
  • compositions of the present invention suitable for parenteral administration may comprise one or more agent(s)/compound(s) in combination with one or more pharmaceutically- acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.
  • suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.
  • Proper fluidity can be maintained, for example, by the use of coating materials, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain suitable adjuvants, such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption.
  • a drug e.g., pharmaceutical formulation
  • the rate of absorption of the active agent/drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form.
  • delayed absorption of a parenterally- administered agent/drug may be accomplished by dissolving or suspending the active agent/drug in an oil vehicle.
  • injectable depot forms may be made by forming microencapsule matrices of the active ingredient in biodegradable polymers. Depending on the ratio of the active ingredient to polymer, and the nature of the particular polymer employed, the rate of active ingredient release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid diluent or carrier, for example water for injection, immediately prior to use.
  • sterile liquid diluent or carrier for example water for injection
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.
  • the present invention provides combinations shown to enhance the effects of ERK inhibitors.
  • applicants have also shown that the combination of different ERK inhibitors is likewise synergistic. Therefore, it is contemplated that the effects of the combinations described herein can be further improved by the use of one or more additional ERK inhibitors. Accordingly, some embodiments of the present invention include one or more additional ERK inhibitors.
  • HCT1 16 cells K-RAS mutation human colorectal carcinoma cells
  • A375 cells BRAF V600 E human malignant melanoma
  • HCT1 16 studies cells were seeded into triplicate 96-well plates at a cell density of 1500 cells/well in McCoy's 5A Medium plus 10% fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • A375 studies cells were seeded at a density of 3000 cells/well in Dulbecco's Modified Eagle Medium (DMEM) plus 10% FBS. In each case, cells were allowed to adhere overnight prior to addition of test compound or vehicle control.
  • DMEM Dulbecco's Modified Eagle Medium
  • ABT263 (ranging from 0-3 ⁇ ) with BVD-0523 (ranging from 0 to 10 ⁇ ), ABT263 (ranging from 0-3 ⁇ ) with dabrafenib (ranging from 0 to 1 ⁇ ), and ABT263 (ranging from 0-3 ⁇ ) with trametinib ((ranging from 0 to 0.010 ⁇ ).
  • the final concentration of DMSO was 0.2%. The compounds were incubated with the cells for 96 hours.
  • A375 cells were seeded into triplicate 96-well plates at a cell density of 3000 cells/well in McCoy's 5A plus 10% FBS. Cells were allowed to adhere overnight prior to addition of test compound or control.
  • ABT199 (ranging from 0-1 ⁇ ) with BVD-0523 (0 to 10 ⁇ )
  • ABT199 (ranging from 0-1 ⁇ ) with dabrafenib (ranging from 0 to 1 ⁇ )
  • ABT199 (ranging from 0-1 ⁇ ) with trametinib (ranging from 0 to 0.1 ⁇ ).
  • the final concentration of DMSO was 0.2%.
  • the compounds were incubated with the cells for 96 hours.
  • HCT1 16 cells were seeded in triplicate in white 96-well plates at a cell density of 5000 cells/well in McCoy's 5A plus 10% FBS.
  • A375 cells were seeded at a density of 5000 cells/well in DMEM plus 10% FBS. Cells were allowed to adhere overnight prior to addition of test compound or vehicle control. The final concentration of DMSO was 0.2%, and 800 nM staurosporine was included as a positive control. 24 and 48 hour assay incubation periods were used.
  • Cbiiss is the fractional inhibition that would be expected if the combination of the two drugs were exactly additive.
  • Cbiiss values are subtracted from the experimentally observed fractional inhibition values to give an 'excess over Bliss' value. Excess over Bliss values greater than 0 indicate synergy, whereas values less than 0 indicate antagonism. Excess over Bliss values are plotted as heat maps ⁇ SD.
  • ERK kinase inhibition using BVD-523 in combination with the BCL-2 family inhibitor ABT-263/navitoclax is effective to inhibit cancer cell growth
  • ERK kinase inhibition exemplified using BVD-523, in combination with the BCL-2 family inhibitor ABT-263/navitoclax, is effective to inhibit cancer cell growth.
  • the closely related BCL- 2 family inhibitor ABT-199 did not appear effective in combination in the same background.
  • the finding may be highly specific, and that preferential combination effects may require disruption of particular client BCL family proteins, such as BCL-XL.
  • none of these combinations appear effective in BRAF mutant A375 melanoma cells, suggesting the specificity observed here may be linked to cancer types, genetic markers or the status of biomarkers relevant the BCL-2 family.
  • BCL- 2 specific inhibition ⁇ e.g., ABT-263
  • ERK exhibition may prove effective in treating lymphomas; other cancers may also be sensitive due to modulation of various BCL-2 family members ⁇ e.g., prosurvival MCL1 , proapopotic BIM), when combined with ERK inhibition.
  • BCL-2 family members e.g., prosurvival MCL1 , proapopotic BIM
  • Tumor types identified using the combination of MEK and BCL-2 family inhibition include lung breast and hematologic malignancies.
  • BVD-523 altered markers of MAPK kinase activity and effector function
  • HCT1 16 cells (5 x 10 6 ) were seeded into 10 cm dishes in McCoy's 5A plus 10% FBS.
  • A375 cells (2.5 x 10 6 ) were seeded into 10 cm dishes in DMEM plus 10% FBS. Cells were allowed to adhere overnight prior to addition of the indicated amount of test compound (BVD-523) or vehicle control. Cells were treated for either 4 or 24 hours before isolation of whole-cell protein lysates, as specified below. Cells were harvested by trypsinisation, pelleted and snap frozen.
  • Lysates were prepared with RIPA (Radio-lmmunoprecipitation Assay) buffer, clarified by centrifugation and quantitated by bicinchoninic acid assay (BCA) assay. 20- 50 g of protein was resolved by SDS-PAGE electrophoresis, blotted onto PVDF membrane and probed using the antibodies detailed in Table 5 (for the 4-hour treatment) and Table 6 (for the 24-hour treatment) below.
  • RIPA Radio-lmmunoprecipitation Assay
  • BCA bicinchoninic acid assay
  • Figure 16 shows Western blot analyses of cells treated with BVD-523 at various concentrations for the following: 1 ) MAPK signaling components in A375 cells after 4 hours; 2) cell cycle and apoptosis signaling in A375 24 hours treatment with various amounts of BVD-523; and 3) MAPK signaling in HCT-1 16 cells treated for 4 hours.
  • the results show that acute and prolonged treatment with BVD-523 in RAF and RAS mutant cancer cells in-vitro affects both substrate phosphorylation and effector targets of ERK kinases.
  • the concentrations of BVD-523 required to induce these changes is typically in the low micromolar range.
  • BVD-523 treatment induces complex changes in the MAPK feedback phosphatase, DUSP6: slowly migrating protein isoforms are reduced following acute treatment, while total protein levels are greatly reduced following prolonged BVD-523 treatment. Both of these findings are consistent with reduced activity of ERK kinases, which control DUSP6 function through both post-translational and transcriptional mechanisms. Overall, despite increases in cellular forms of ERK that are typically thought to be active, it appears likely that cellular ERK enzyme activity is fully inhibited following either acute or prolonged treatment with BVD-523.
  • effector genes that require MAPK pathway signaling are altered following treatment with BVD-523.
  • the G1/S cell-cycle apparatus is regulated at both post-translational and transcriptional levels by MAPK signaling, and cyclin-D1 protein levels are greatly reduced following prolonged BVD-523 treatment.
  • gene expression and protein abundance of apoptosis effectors often require intact MAPK signaling, and total levels of Bim-EL increase following prolonged BVD- 523 treatment.
  • Figure 16 shows that BVD-523 inhibits the MAPK signaling pathway and may be more favorable compared to RAF or MEK inhibition in this setting.
  • BVD-523 properties of BVD-523 may make this a preferred agent for use as an ERK inhibitor, compared to other agents with a similar activity.
  • kinase inhibitor drugs display unique and specific interactions with their enzyme targets, and that drug efficacy is strongly influenced by both the mode of direct inhibition, as well as susceptibility to adaptive changes that occur following treatment.
  • inhibitors of ABL, KIT, EGFR and ALK kinases are effective only when their cognate target is found in active or inactive configurations.
  • certain of these inhibitors are uniquely sensitive to either secondary genetic mutation, or post-translational adaptive changes, of the protein target.
  • RAF inhibitors show differential potency to RAF kinases present in certain protein complexes and/or subcellular localizations.
  • ERK kinases are similarly known to exist in diverse, variable, and complex biochemical states, it appears likely that BVD-523 may interact with and inhibit these targets in a fashion that is distinct and highly preferable to other agents.
  • Test compounds were incubated with the cells for 72h at 37°C, 5% CO2 in a humidified atmosphere.
  • CellTiter-Glo® reagent Promega, Madison, Wl
  • BMG FLUOstar plate reader BMG Labtech, Ortenberg, Germany
  • This volume score shows whether the overall response to a combination is synergistic (positive values), antagonistic (negative values) or additive (values ⁇ 0).
  • A549 cells were most sensitive to BVD-523 as a single agent.
  • the single agent ABT-263 responses were consistent with the GDSC/Sanger data.
  • Volume scores for the combinations tested are shown in Figure 20 and Tables 9-1 1 and are consistent with the conclusions drawn from the heat maps.
  • RAF mutant melanoma cell line A375 cells were cultured in DMEM with 10% FBS and seeded into triplicate 96-well plates at an initial density of 2000 cells per well. Combination interactions between ERK inhibitors BVD-523 and SCH772984 were analized after 72 hours as described above in Example 4. Viability was determined using CellTiter-Glo® reagent (Promega, Madison, Wl) according to manufacturer's instructions and luminescence was detected using the BMG FLUOstar plate reader (BMG Labtech, Ortenberg, Germany).
  • ABSALAN Farnaz; Mostafa Ronaghi (2008). Molecular Inversion Probe Assay. Methods in Molecular Biology 396. Humana Press, pp. 315- 330.

Abstract

The present invention provides, inter alia, methods for treating or ameliorating the effects of a cancer in a subject in need thereof. These methods include administering to the subject an effective amount of (i) an ERK1/2 inhibitor (such as BVD-523) or a pharmaceutically acceptable salt thereof and (ii) a BCL-2 family inhibitor (such as navitoclax) or a pharmaceutically acceptable salt thereof, to treat or ameliorate the effects of the cancer. Kits and pharmaceutical compositions for treating or ameliorating the effects of a cancer in a subject and methods for selecting a subject with cancer that may benefit from a combination drug therapy are also provided.

Description

CANCER TREATMENTS USING ERK1/2 AND BCL-2 FAMILY INHIBITORS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Patent Application Serial No. 61/919,148, filed on December 20, 2013 which application is incorporated by reference herein in its entirety.
FIELD OF INVENTION
[0002] The present invention provides, inter alia, methods, and kits and compositions for treating or ameliorating the effects of a cancer in a subject in need thereof by administering to the subject an effective amount of (i) an ERK1/2 inhibitor or a pharmaceutically acceptable salt thereof and (ii) a BCL- 2 family inhibitor or a pharmaceutically acceptable salt thereof. Methods for selecting a subject with cancer that may benefit from such a combination drug therapy are also provided.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0003] This application contains references to amino acids and/or nucleic acid sequences that have been filed concurrently herewith as sequence listing text file "0375606.txt", file size of 221 KB, created on December 18, 2014. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1 .52(e)(5).
BACKGROUND OF THE INVENTION
[0004] Members of the family of B-cell CLL/lymphoma 2 proteins (BCL- 2) are apoptosis regulators. These proteins control mitochondrial outer membrane permeabilization (MOMP). Expression of BCL-2 protein blocks cell death in response to various cellular injuries. A number of cancers, including melanoma, breast, prostate, chronic lymphocytic leukemia, and lung cancer, may be caused by damage to the BCL-2 gene. Mutations in BCL-2 may also be a cause of resistance to cancer treatments. Unfortunately, resistance can quickly develop using conventional BCL-2 inhibitor therapies to treat cancer.
[0005] Extracellular-signal-regulated kinases (ERKs) are protein kinases that are involved in cell cycle regulation, including the regulation of meiosis, mitosis, and postmitotic functions in differentiated cells. Disruption of the ERK pathway is common in cancers. However, to date, little progress has been made developing effective ERK inhibitors for the treatment of cancer.
[0006] As the understanding of the molecular basis of cancer grows, there is an increased emphasis on developing drugs that specifically target particular nodes in pathways that lead to cancer. In view of the deficiencies noted above, there is, inter alia, a need for effective molecularly targeted cancer treatments, including combination therapies. The present invention is directed to meeting these and other needs.
SUMMARY OF THE INVENTION
[0007] One embodiment of the present invention is a method of treating or ameliorating the effects of a cancer in a subject in need thereof. This method comprises administering to the subject an effective amount of (i) a first anti-cancer agent, which is an ERK1/2 inhibitor or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof, to treat or ameliorate the effects of the cancer.
[0008] Another embodiment of the present invention is a method of treating or ameliorating the effects of a cancer in a subject in need thereof. This method comprises administering to the subject an effective amount of (i) BVD-523 or a pharmaceutically acceptable salt thereof and (ii) navitoclax or a pharmaceutically acceptable salt thereof, to treat or ameliorate the effects of the cancer.
[0009] An additional embodiment of the present invention is a method of effecting cancer cell death. This method comprises contacting the cancer cell with an effective amount of (i) a first anti-cancer agent, which is BVD-523 or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof.
[0010] A further embodiment of the present invention is a kit for treating or ameliorating the effects of a cancer in a subject in need thereof. This kit comprises an effective amount of (i) an ERK1/2 inhibitor or a pharmaceutically acceptable salt thereof and (ii) a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof, packaged together with instructions for their use.
[0011] Another embodiment of the present invention is a pharmaceutical composition for treating or ameliorating the effects of a cancer in a subject in need thereof. This pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier and an effective amount of (i) a first anti-cancer agent, which is an ERK 1/2 inhibitor or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof, wherein administration of the first and second anti-cancer agents provides a synergistic effect compared to administration of either anti-cancer agent alone.
[0012] A further embodiment of the present invention is a method for selecting a subject with cancer that may benefit from a combination drug therapy. This method comprises:
(a) selecting a subject with a somatic K-RAS mutation;
(b) determining whether the subject with a K-RAS mutation has an epithelial-to-mesenchymal transition gene signature, wherein subjects having a K-RAS mutation and an epithelial-to-mesenchymal transition gene signature are likely to benefit from the combination drug therapy; and
(c) administering to the subject having a K-RAS mutation and an epithelial-to-mesenchymal transition gene signature the combination drug therapy which comprises an effective amount of (i) an ERK1/2 inhibitor or a pharmaceutically acceptable salt thereof and (ii) a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0014] Figure 1A is a dose matrix showing % inhibition of the ABT263 (navitoclax, a BCL-2 inhibitor)/BVD-523 (an ERK1/2 inhibitor) combination in a human malignant melanoma cell line (A375 cells) using the Alamar Blue cell viability assay. Figure 1 B is a dose matrix showing excess over Bliss for the ABT263/BVD-523 combination. Figure 1 C shows % viability relative to DMSO only treated controls for ABT263 and BVD-523 single agent treatments in A375 cells using the Alamar Blue cell viability assay. Figure 1 D shows % viability relative to DMSO only treated controls for ABT263/BVD-523 combination treatments in A375 cells using the Alamar Blue cell viability assay.
[0015] Figure 2A is a dose matrix showing % inhibition of the ABT263/BVD-523 combination in a human colorectal carcinoma cell line (HCT1 16 cells) using the Alamar Blue cell viability assay. Figure 2B is a dose matrix showing excess over Bliss for the ABT263/BVD-523 combination. Figure 2C shows % viability relative to DMSO only treated controls for ABT263 and BVD-523 single agent treatments in HCT1 16 cells using the Alamar Blue cell viability assay. Figure 2D shows % viability relative to DMSO only treated controls for ABT263/BVD-523 combination treatments in HCT1 16 cells using the Alamar Blue cell viability assay.
[0016] Figure 3A is a dose matrix showing % inhibition of the ABT263 (BCL-2 inhibitor)/dabrafenib (BRAF inhibitor) combination in A375 cells using the Alamar Blue cell viability assay. Figure 3B is a dose matrix showing excess over Bliss for the ABT263/dabrafenib combination. Figure 3C shows % viability relative to DMSO only treated controls for ABT263 and dabrafenib single agent treatments in A375 cells using the Alamar Blue cell viability assay. Figure 3D shows % viability relative to DMSO only treated controls for ABT263/dabrafenib combination treatments in A375 cells using the Alamar Blue cell viability assay.
[0017] Figure 4A is a dose matrix showing % inhibition of the ABT263/dabrafenib combination in HCT1 16 cells using the Alamar Blue cell viability assay. Figure 4B is a dose matrix showing excess over Bliss for the ABT263/dabrafenib combination. Figure 4C shows % viability relative to DMSO only treated controls for ABT263 and dabrafenib single agent treatments in HCT1 16 cells using the Alamar Blue cell viability assay. Figure 4D shows % viability relative to DMSO only treated controls for ABT263/dabrafenib combination treatments in HCT1 16 cells using the Alamar Blue cell viability assay.
[0018] Figure 5A is a dose matrix showing % inhibition of the ABT263 (BCL-2 inhibitor)/trametinib (type 2 MEK inhibitor) combination in A375 cells using the Alamar Blue cell viability assay. Figure 5B is a dose matrix showing excess over Bliss for the ABT263/trametinib combination. Figure 5C shows % viability relative to DMSO only treated controls for ABT263 and trametinib single agent treatments in A375 cells using the Alamar Blue cell viability assay. Figure 5D shows % viability relative to DMSO only treated controls for ABT263/trametinib combination treatments in A375 cells using the Alamar Blue cell viability assay.
[0019] Figure 6A is a dose matrix showing % inhibition of the ABT263/trametinib combination in HCT1 16 cells using the Alamar Blue cell viability assay. Figure 6B is a dose matrix showing excess over Bliss for the ABT263/trametinib combination. Figure 6C shows % viability relative to DMSO only treated controls for ABT263 and trametinib single agent treatments in HCT1 16 cells using the Alamar Blue cell viability assay. Figure 6D shows % viability relative to DMSO only treated controls for ABT263/trametinib combination treatments in HCT1 16 cells using the Alamar Blue cell viability assay.
[0020] Figure 7 is a histogram showing viability of HCT1 16 cells after 96 hours of incubation with various amounts of BVD-523 or BVD-523 in combination with 3 μΜ ABT-263. The Bliss Scores are shown in the yellow boxes.
[0021] Figure 8 is a histogram showing caspase activity in HCT1 16 cells after 24 hours of incubation with various amounts of BVD-523 or BVD- 523 in combination with 3 μΜ ABT-263.
[0022] Figure 9 is a histogram showing caspase activity in HCT1 16 cells after 48 hours of incubation with various amounts of BVD-523 or BVD- 523 in combination with 3 μΜ ABT-263.
[0023] Figure 10A is a dose matrix showing % inhibition of the ABT199 (another BCL-2 inhibitor)/BVD-523 combination in A375 cells using the Alamar Blue cell viability assay. Figure 10B is a dose matrix showing excess over Bliss for the ABT199/BVD-523 combination. Figure 10C shows % viability relative to DMSO only treated controls for ABT199 and BVD-523 single agent treatments in A375 cells using the Alamar Blue cell viability assay. Figure 10D shows % viability relative to DMSO only treated controls for ABT199/BVD-523 combination treatments in A375 cells using the Alamar Blue cell viability assay.
[0024] Figure 1 1A is a dose matrix showing % inhibition of the ABT199/BVD-523 combination in A375 cells using the CellTiter-Glo cell viability assay. Figure 1 1 B is a dose matrix showing excess over Bliss for the ABT199/BVD-523 combination. Figure 1 1 C shows % viability relative to DMSO only treated controls for ABT199 and BVD-523 single agent treatments in A375 cells using the CellTiter-Glo cell viability assay. Figure 1 1 D shows % viability relative to DMSO only treated controls for ABT199/BVD-523 combination treatments in A375 cells using the CellTiter-Glo cell viability assay.
[0025] Figure 12A is a dose matrix showing % inhibition of the ABT199/dabrafenib combination in A375 cells using the Alamar Blue cell viability assay. Figure 12B is a dose matrix showing excess over Bliss for the ABT199/dabrafenib combination. Figure 12C shows % viability relative to DMSO only treated controls for ABT199 and dabrafenib single agent treatments in A375 cells using the Alamar Blue cell viability assay. Figure 12D shows % viability relative to DMSO only treated controls for ABT199/dabrafenib combination treatments in A375 cells using the Alamar Blue cell viability assay.
[0026] Figure 13A is a dose matrix showing % inhibition of the ABT199/dabrafenib combination in A375 cells using the CellTiter-Glo cell viability assay. Figure 13B is a dose matrix showing excess over Bliss for the ABT199/dabrafenib combination. Figure 13C shows % viability relative to DMSO only treated controls for ABT199 and dabrafenib single agent treatments in A375 cells using the CellTiter-Glo cell viability assay. Figure 13D shows % viability relative to DMSO only treated controls for ABT199/dabrafenib combination treatments in A375 cells using the CellTiter- Glo cell viability assay. [0027] Figure 14A is a dose matrix showing % inhibition of the ABT199/trametinib combination in A375 cells using the Alamar Blue cell viability assay. Figure 14B is a dose matrix showing excess over Bliss for the ABT199/trametinib combination. Figure 14C shows % viability relative to DMSO only treated controls for ABT199 and trametinib single agent treatments in A375 cells using the Alamar Blue cell viability assay. Figure 14D shows % viability relative to DMSO only treated controls for ABT199/trametinib combination treatments in A375 cells using the Alamar Blue cell viability assay.
[0028] Figure 15A is a dose matrix showing % inhibition of the ABT199/trametinib combination in A375 cells using the CellTiter-Glo cell viability assay. Figure 15B is a dose matrix showing excess over Bliss for the ABT199/trametinib combination. Figure 15C shows % viability relative to DMSO only treated controls for ABT199 and trametinib single agent treatments in A375 cells using the CellTiter-Glo cell viability assay. Figure 15D shows % viability relative to DMSO only treated controls for ABT199/trametinib combination treatments in A375 cells using the CellTiter- Glo cell viability assay.
[0029] Figure 16 shows that both direct ERK substrate phosphorylation and known effector pathways are modulated following acute and prolonged treatment with BVD-523 in vitro. Western blots were performed using a variety of antibodies to detect changes in whole-cell lysates of cancer lines exposed to BVD-523. In the A375 BRAF mutant cell line (a human melanoma cell line) and in the HCT1 16 KRAS mutant cell line (a human colorectal carcinoma cell line), phosphorylation of ERK-dependent residues (T359/S363) in RSK 1 and 2 proteins was reduced after 4 hours of treatment with BVD-523 at micromolar concentrations. Following 24 hours of treatment, direct substrate inhibition was maintained in BRAF mutant cell lines, and the MAPK feedback phosphatase DUSP6 was greatly reduced, suggesting durable and nearly complete MAPK pathway inhibition. Lastly, consistent with cytostatic effects of BVD-523 across multiple cell line backgrounds, the MAPK effector and G1/S-cell-cycle determinant gene cyclin-D1 was greatly reduced after 24 hours of treatment. In the A375 cell line, while the apoptosis effector and ERK substrate Bim-EL was increased following prolonged treatment, increased apoptosis was not observed, consistent with a lack of PARP cleavage, as well as other observations (not shown) that additional factors influence the capacity for BVD-523 to induce cell death.
[0030] Figure 17 shows the results of the combination of BVD-523 and ABT-263. Figure 17A shows a dose matrix showing inhibition (%) for the combination in A549 cells. Figure 17B - Figure 17C show the results of single agent proliferation assays for the combination in 17A. Figure 17D shows Loewe excess for the combination in 17A and Figure 17E shows Bliss excess for the combination in 17A. Figure 17F shows a dose matrix showing inhibition (%) for the combination in H1650 cells. Figure 17G - Figure 17H show the results of single agent proliferation assays for the combination in 17F. Figure 171 shows Loewe excess for the combination in 17F and Figure 17J shows Bliss excess for the combination in 17F. Figure 17K shows a dose matrix showing inhibition (%) for the combination in H226 cells. Figure 17L - Figure 17M show the results of single agent proliferation assays for the combination in 17K. Figure 17N shows Loewe excess for the combination in 17K and Figure 170 shows Bliss excess for the combination in 17K. Figure 17P shows a dose matrix showing inhibition (%) for the combination in H460 cells. Figure 17Q - Figure 17R show the results of single agent proliferation assays for the combination in 17P. FIG. 17S shows Loewe excess for the combination in 17P and Figure 17T shows Bliss excess for the combination in 17P.
[0031] Figure 18 shows the results of the combination of SCH772984 and ABT-263. Figure 18A shows a dose matrix showing inhibition (%) for the combination in A549 cells. Figure 18B - Figure 18C show the results of single agent proliferation assays for the combination in 18A. Figure 18D shows Loewe excess for the combination in 18A and Figure 18E shows Bliss excess for the combination in 18A. Figure 18F shows a dose matrix showing inhibition (%) for the combination in H1650 cells. Figure 18G - Figure 18H show the results of single agent proliferation assays for the combination in 18F. Figure 181 shows Loewe excess for the combination in 18F and Figure 18J shows Bliss excess for the combination in 18F. Figure 18K shows a dose matrix showing inhibition (%) for the combination in H226 cells. Figure 18L - Figure 18M show the results of single agent proliferation assays for the combination in 18K. Figure 18N shows Loewe excess for the combination in 18K and Figure 180 shows Bliss excess for the combination in 18K. Figure 18P shows a dose matrix showing inhibition (%) for the combination in H460 cells. Figure 18Q - Figure 18R show the results of single agent proliferation assays for the combination in 18P. FIG. 18S shows Loewe excess for the combination in 18P and Figure 18T shows Bliss excess for the combination in 18P. [0032] Figure 19 shows the results of the combination of BVD-523 and ABT-263. Figure 19A shows a dose matrix showing inhibition (%) for the combination in A549 cells. Figure 19B - Figure 19C show the results of single agent proliferation assays for the combination in 19A. Figure 19D shows Loewe excess for the combination in 19A and Figure 19E shows Bliss excess for the combination in 19A. Figure 19F shows a dose matrix showing inhibition (%) for the combination in H1650 cells. Figure 19G - Figure 19H show the results of single agent proliferation assays for the combination in 19F. Figure 191 shows Loewe excess for the combination in 19F and Figure 19J shows Bliss excess for the combination in 19F. Figure 19K shows a dose matrix showing inhibition (%) for the combination in H226 cells. Figure 19L - Figure 19M show the results of single agent proliferation assays for the combination in 19K. Figure 19N shows Loewe excess for the combination in 19K and Figure 190 shows Bliss excess for the combination in 19K. Figure 19P shows a dose matrix showing inhibition (%) for the combination in H460 cells. Figure 19Q - Figure 19R show the results of single agent proliferation assays for the combination in 19P. FIG. 19S shows Loewe excess for the combination in 19P and Figure 19T shows Bliss excess for the combination in 19P.
[0033] Figure 20A shows Lowe Volumes for the combinations of BVD- 523, SCH772984 and Trametinib with ABT-263. Figure 20B shows Bliss Volumes for the combinations of BVD-523, SCH772984 and Trametinib with ABT-263. Figure 20C shows Synergy Scores for the combinations of BVD- 523, SCH772984 and Trametinib with ABT-263. [0034] Figure 21 shows the results of the combination of BVD-523 and SCH772984. Figure 21 A shows a dose matrix showing inhibition (%) for the combination in A375 cells. Figure 21 B - Figure 21 C show the results of single agent proliferation assays for the combination in 21 A. Figure 21 D shows Loewe excess for the combination in 21 A and Figure 21 E shows Bliss excess for the combination in 21A.
DETAILED DESCRIPTION OF THE INVENTION
[0035] One embodiment of the present invention is a method of treating or ameliorating the effects of a cancer in a subject in need thereof. This method comprises administering to the subject an effective amount of (i) a first anti-cancer agent, which is an ERK1/2 inhibitor or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof, to treat or ameliorate the effects of the cancer.
[0036] As used herein, the terms "treat," "treating," "treatment" and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient. In particular, the methods and compositions of the present invention may be used to slow the development of disease symptoms or delay the onset of the disease or condition, or halt the progression of disease development. However, because every treated subject may not respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population, e.g., patient population. Accordingly, a given subject or subject population, e.g., patient population, may fail to respond or respond inadequately to treatment.
[0037] As used herein, the terms "ameliorate", "ameliorating" and grammatical variations thereof mean to decrease the severity of the symptoms of a disease in a subject.
[0038] As used herein, a "subject" is a mammal, preferably, a human. In addition to humans, categories of mammals within the scope of the present invention include, for example, farm animals, domestic animals, laboratory animals, etc. Some examples of farm animals include cows, pigs, horses, goats, etc. Some examples of domestic animals include dogs, cats, etc. Some examples of laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc.
[0039] In the present invention, cancers include both solid and hemotologic cancers. Non-limiting examples of solid cancers include adrenocortical carcinoma, anal cancer, bladder cancer, bone cancer (such as osteosarcoma), brain cancer, breast cancer, carcinoid cancer, carcinoma, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewing family of cancers, extracranial germ cell cancer, eye cancer, gallbladder cancer, gastric cancer, germ cell tumor, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma, kidney cancer, large intestine cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, malignant mesothelioma, Merkel cell carcinoma, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian epithelial cancer, ovarian germ cell cancer, pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pituitary cancer, plasma cell neoplasm, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell cancer, transitional cell cancer of the renal pelvis and ureter, salivary gland cancer, Sezary syndrome, skin cancers (such as cutaneous t-cell lymphoma, Kaposi's sarcoma, mast cell tumor,and melanoma), small intestine cancer, soft tissue sarcoma, stomach cancer, testicular cancer, thymoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.
[0040] In the present invention, the subject's cancer is preferably selected from the group consisting of colorectal cancer, breast cancer, pancreatic cancer, lymphoma, lung cancer, testicular cancer, thyroid cancer, hematologic malignancies, and endometrial cancers. More preferably, the cancer is colorectal cancer.
[0041] Non-limiting examples of hematologic malignancies according to the present invention include RAS mutant myelodysplastic syndromes (MDS), lymphoid cancers, and myeloid cancers. In the present invention, hematologic malignancies also include, e.g., Adult Acute Megakaryoblastic Leukemia (M7), Adult Acute Minimally Differentiated Myeloid Leukemia (M0), Adult Acute Monoblastic Leukemia (M5a), Adult Acute Monocytic Leukemia (M5b), Adult Acute Myeloblasts Leukemia With Maturation (M2), Adult Acute Myeloblasts Leukemia Without Maturation (M1 ), Adult Acute Myeloid Leukemia With 1 1 q23 (MLL) Abnormalities, Adult Acute Myeloid Leukemia With Del(5q), Adult Acute Myeloid Leukemia With Inv(16)(p13;q22), Adult Acute Myeloid Leukemia With t(16;16)(p13;q22), Adult Acute Myeloid Leukemia With t(8;21 )(q22;q22), Adult Acute Myelomonocytic Leukemia (M4), Adult Erythroleukemia (M6a), Adult Pure Erythroid Leukemia (M6b), Recurrent Adult Acute Myeloid Leukemia, and Untreated Adult Acute Myeloid Leukemia.
[0042] As used herein, an "ERK1/2 inhibitor" means those substances that (i) directly interact with ERK1 and/or ERK2, e.g., by binding to ERK1/2 and (ii) decrease the expression or the activity of ERK1 and/or ERK2 protein kinases. Therefore, inhibitors that act upstream of ERK1/2, such as MEK inhibitors and RAF inhibitors, are not ERK1/2 inhibitors according to the present invention. Preferred ERK1/2 inhibitors of the present invention do not decrease the amount of phosphorylated ERK1 and/or ERK2 but decrease the activity of phosphorylated ERK1 and/or ERK2. Non-limiting examples of ERK1/2 inhibitors include AEZS-131 (Aeterna Zentaris), AEZS-136 (Aeterna Zentaris), BVD-523 (BioMed Valley Discoveries, Inc.), SCH-722984 (Merck & Co.), SCH-772984 (Merck & Co.), SCH-900353 (MK-8353) (Merck & Co.), pharmaceutically acceptable salts thereof, and combinations thereof. Preferably, the ERK1/2 inhibitor is BVD-523 or a pharmaceutically acceptable salt thereof. In the present invention, BVD-523, a preferred ERK1/2 inhibitor, corresponds to a compound according to formula (I):
Figure imgf000017_0001
and pharmaceutically acceptable salts thereof. BVD-523 may be synthesized according to the methods disclosed, e.g., in U.S. Patent No. 7,354,939. Enantiomers and racemic mixtures of both enantiomers of BVD-523 are also contemplated within the scope of the present invention. BVD-523 is a preferred ERK1/2 inhibitor of the present invention because its mechanism of action is believed to be, inter alia, unique and distinct from certain other ERK1/2 inhibitors, such as SCH772984. For example, SCH772984 inhibits autophosphorylation of ERK (Morris et al., 2013), whereas BVD-523 allows for the autophosphorylation of ERK while still inhibiting ERK. (See, e.g., Figure 16).
[0043] As used herein, a "BLC2 family inhibitor" means those substances that (i) directly interact with BLC2 family members, e.g., by binding to BLC2 family members and (ii) decrease the expression or the activity of BLC2 family members. Non-limiting examples of BCL-2 family inhibitors of the present inventions include (-)-epigallocatechin gallate (Sigma Aldrich, St. Louis, MO), ABT-737 (Abbott), antimycin A (Sigma Aldrich), apogossypolone (Selleck Chemicals, Boston, MA), CAS # 383860-03-5 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), CAS # 810659-53-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), CAS #141266-44-6 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), BP-100-1 .02 (Bio-Path Holdings), chelerythrine chloride (Tocris Bioscience, Bristol, UK), HA14-1 (CAS # 65673- 63-4), licochalcone-A (CAS #58749-22-7), navitoclax (Abbott), obatoclax (GX15-080), R-(-)-gossypol (Ascenta Therapeutics), S-44563 (Servier), sabutoclax (Oncothyreon), TW-37, VAL-101 (ValiRx), WEHI-539 (Hoffmann- La Roche, Inc.), pharmaceutically acceptable salts thereof, and combinations thereof. Preferably, the BCL-2 family inhibitor is navitoclax (ABT263) or a pharmaceutically acceptable salt thereof.
[0044] In one aspect of this embodiment, the ERK 1/2 inhibitor is BVD- 523 or a pharmaceutically acceptable salt thereof and the BCL-2 family inhibitor is navitoclax or a pharmaceutically acceptable salt thereof.
[0045] The ERK1/2 inhibitors and/or the BCL-2 family inhibitors may be administered according to the methods of the present invention as a part of a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent.
[0046] In another aspect of this embodiment, the subject with cancer has a somatic RAS mutation. As used herein, "somatic mutation" means a change occurring in any cell that is not destined to become a germ cell. The mutation may be, e.g., a substitution, deletion, insertion, or a fusion. Preferably, the RAS mutation is a mutation in H-RAS, N-RAS, or K-RAS. Table 1 below shows the distribution and frequency of these RAS mutations in human tumors. These data were obtained from the Sanger Catalogue of Somatic Mutations in Cancer.
Table 1
Figure imgf000019_0001
Organ/Tissue Tumor Type H-RAS N-RAS K-RAS
Other N/A N/A 27 (298)
Leukemias acute myelogenous 0 (1216) 12 (3404) 4 (1778) leukemia
(chronic myeloid 0 (265) 3 (532) 2 (313) leukemia) CML
chronic 1 (1 18) 15 (157) 1 1 (84) myelomonocytic
leukemia
juvenile 0 (41 ) 19 (165) 7 (143) myelomonocytic
myeloid leukemia
Lymphomas ALL 0 (284) 10 (703) 7 (549)
Burkitt's lymphoma 0 (30) 10 (30) 3 (30)
Hodgkin's 2 (44) 16 (45) 0 (44) lymphoma
Plasma cell 2 (185) 20 (484) 6 (403) myeloma
Liver Hepatocellular 0 (163) 4 (202) 4 (307) carcinoma
Lung Large cell carcinoma 4 (50) 4 (49) 21 (189)
Non small cell 0 (683) 1 (695) 16 (3575) carcinoma
Squamous cell 1 (261 ) 0 (360) 6 (1407) carcinoma
Other (neoplasia) N/A N/A 22 (563)
Pancreas Ductal 0 (1 10) 1 (138) 69 (3483) adenocarcinoma
Endocrine tumor 0 (2) 75 (4) 1 (68)
Prostate Adenocarcinoma 6 (489) 2 (509) 8 (1002)
Skin Basal cell carcinoma 7 (180) 1 (147) 4 (147)
Squamous cell 9 (236) 7 (107) 5 (107) carcinoma
Malignant 1 (904) 20 (3466) 2 (924) melanoma
Soft tissue Angiosarcoma 0 (6) 0 (6) 49 (53)
Leiomyosarcoma 3 (30) 0 (13) 8 (173)
Liposarcoma 6 (72) 0 (21 ) 4 (45) Organ/Tissue Tumor Type H-RAS N-RAS K-RAS
Rhabdomyosarcoma 4 (158) 1 1 (151 ) 4 (162)
Myxoma 0 (19) 0 (19) 1 1 (19)
Malignant fibrous 15 (1 17) 2 (57) 16 (131 ) histiocytoma- pleomorphic
sarcoma
Stomach Adenocarcinoma 4 (218) 2 (205) 6 (2054)
Other 1 1 (9) 0 (1 ) 6 (241 )
Testis Germinoma 0 (56) 7 (1 15) 7 (190)
Seminoma 17 (30) 0 (30) 0 (23)
Thyroid Anaplastic 4 (440) 17 (436) 9 (433) carcinoma
Follicular carcinoma 5 (381 ) 17 (392) 4 (372)
Papillary carcinoma 2 (1525) 4 (1941 ) 2 (1654)
Hurthle cell 16 (44) 4 (26) 0 (41 ) carcinoma
N/A = not available.
[0047] The following Tables 2, 3, and 4 below show the SEQ ID Nos. of representative nucleic acid and amino acid sequences of wild type H-RAS, K- RAS, and N-RAS from various animals, respectively. These sequences may be used in methods for identifying subjects with a mutant RAS genotype (such as in the methods set forth below).
Table 2 H-RAS sequences
SEQ ID polypeptide or nucleic Organism Other
No. acid sequence Information
1 nucleic acid Human isoform 1
2 Polypeptide Human isoform 1
3 nucleic acid Human isoform 2
4 Polypeptide Human isoform 2
5 nucleic acid Human isoform 3
6 Polypeptide Human isoform 3
7 nucleic acid rat (Rattus variant 1
norvegicus)
8 polypeptide rat (Rattus variant 1 SEQ ID polypeptide or nucleic Organism Other No. acid sequence Information
norvegicus)
9 nucleic acid rat (Rattus variant 2 norvegicus)
10 polypeptide rat (Rattus variant 2 norvegicus)
1 1 nucleic acid mouse, Mus
musculus
12 polypeptide mouse, Mus
musculus
13 nucleic acid guinea pig, Cavia variant 1 porcellus
14 polypeptide guinea pig, Cavia variant 1 porcellus
15 nucleic acid guinea pig, Cavia variant 2 porcellus
16 polypeptide guinea pig, Cavia variant 2 porcellus
17 nucleic acid guinea pig, Cavia variant 3 porcellus
18 polypeptide guinea pig, Cavia variant 3 porcellus
19 nucleic acid guinea pig, Cavia variant 4 porcellus
20 polypeptide guinea pig, Cavia variant 4 porcellus
21 nucleic acid dog, Canis lupus variant 1 familiaris
22 polypeptide dog, Canis lupus variant 1 familiaris
23 nucleic acid dog, Canis lupus variant 2 familiaris
24 polypeptide dog, Canis lupus variant 2 familiaris
25 nucleic acid cat, Felis catus variant 1
26 polypeptide cat, Felis catus variant 1
27 nucleic acid cat, Felis catus variant 2
28 polypeptide cat, Felis catus variant 2
29 nucleic acid cow, Bos taurus variant 1
30 polypeptide cow, Bos taurus variant 1
31 nucleic acid cow, Bos taurus variant 2
32 polypeptide cow, Bos taurus variant 2
33 nucleic acid cow, Bos taurus variant X1
34 polypeptide cow, Bos taurus variant X1
35 nucleic acid chicken, Gallus
gallus
36 polypeptide chicken, Gallus
gallus Table 3 K-RAS sequences
SEQ ID polypeptide or nucleic Organism Other No. acid sequence Information
37 nucleic acid Human isoform a
38 polypeptide Human isoform a
39 nucleic acid Human isoform b
40 polypeptide Human isoform b
41 nucleic acid rat (Rattus
norvegicus)
42 polypeptide rat (Rattus
norvegicus)
43 nucleic acid mouse, Mus
musculus
44 polypeptide mouse, Mus
musculus
45 nucleic acid rabbit, Oryctolagus
cuniculus
46 polypeptide rabbit, Oryctolagus
cuniculus
47 nucleic acid guinea pig, Cavia variant 1 porcellus
48 polypeptide guinea pig, Cavia variant 1 porcellus
49 nucleic acid guinea pig, Cavia variant 2 porcellus
50 polypeptide guinea pig, Cavia variant 2 porcellus
51 nucleic acid dog, Canis lupus variant 1 familiaris
52 polypeptide dog, Canis lupus variant 1 familiaris
53 nucleic acid dog, Canis lupus variant 2 familiaris
54 polypeptide dog, Canis lupus variant 2 familiaris
55 nucleic acid cat, Felis catus variant 1
56 polypeptide cat, Felis catus variant 1
57 nucleic acid cat, Felis catus variant 2
58 polypeptide cat, Felis catus variant 2
59 nucleic acid cow, Bos taurus
60 polypeptide cow, Bos taurus
61 nucleic acid cow, Bos taurus variant X2
62 polypeptide cow, Bos taurus variant X2
63 nucleic acid cow, Bos taurus variant X3
64 polypeptide cow, Bos taurus variant X3
65 nucleic acid chicken, Gallus
gallus SEQ ID polypeptide or nucleic Organism Other
No. acid sequence Information
66 polypeptide chicken, Gallus
gallus
Table 4 N-RAS sequences
SEQ ID polypeptide or nucleic Organism Other
No. acid sequence Information
67 nucleic acid Human
68 polypeptide Human
69 nucleic acid rat (Rattus
norvegicus)
70 polypeptide rat (Rattus
norvegicus)
71 nucleic acid mouse, Mus
musculus
72 polypeptide mouse, Mus
musculus
73 nucleic acid guinea pig, Cavia
porcellus
74 polypeptide guinea pig, Cavia
porcellus
75 nucleic acid guinea pig, Cavia variant X1
porcellus
76 polypeptide guinea pig, Cavia variant X1
porcellus
77 nucleic acid dog, Canis lupus
familiaris
78 polypeptide dog, Canis lupus
familiaris
79 nucleic acid cat, Felis catus
80 polypeptide cat, Felis catus
81 nucleic acid cow, Bos taurus
82 polypeptide cow, Bos taurus
83 nucleic acid chicken, Gallus
gallus
84 polypeptide chicken, Gallus gallus
[0048] Methods for identifying mutations in nucleic acids, such as the above identified RAS genes, are known in the art. Nucleic acids may be obtained from biological samples. In the present invention, biological samples include, but are not limited to, blood, plasma, urine, skin, saliva, and biopsies. Biological samples are obtained from a subject by routine procedures and methods which are known in the art.
[0049] Non-limiting examples of methods for identifying mutations include PCR, sequencing, hybrid capture, in-solution capture, molecular inversion probes, fluorescent in situ hybridization (FISH) assay, and combinations thereof.
[0050] Various sequencing methods are known in the art. These include, but are not limited to, Sanger sequencing (also referred to as dideoxy sequencing) and various sequencing-by-synthesis (SBS) methods as disclosed in, e.g., Metzker, 2005, sequencing by hybridization, by ligation (for example, WO 2005021786), by degradation (for example, U.S. Patent Nos. 5,622,824 and 6,140,053) and nanopore sequencing (which is commercially available from Oxford Nanopore Technologies, UK). In deep sequencing techniques, a given nucleotide in the sequence is read more than once during the sequencing process. Deep sequencing techniques are disclosed in e.g., U.S. Patent Publication No. 20120264632 and International Patent Publication No. WO2012125848.
[0051] PCR-based methods for detecting mutations are known in the art and employ PCR amplification, where each target sequence in the sample has a corresponding pair of unique, sequence-specific primers. For example, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method allows for rapid detection of mutations after the genomic sequences are amplified by PCR. The mutation is discriminated by digestion with specific restriction endonucleases and is identified by electrophoresis. See, e.g., Ota et al., 2007. Mutations may also be detected using real time PCR. See, e.g., International Application publication No. WO2012046981 .
[0052] Hybrid capture methods are known in the art and are disclosed in e.g., U.S. Patent Publication No. 20130203632 and U.S. Patent Nos. 8,389,219 and 8,288,520. These methods are based on the selective hybridization of the target genomic regions to user-designed oligonucleotides. The hybridization can be to oligonucleotides immobilized on high or low density microarrays (on-array capture), or solution-phase hybridization to oligonucleotides modified with a ligand (e.g. biotin) which can subsequently be immobilized to a solid surface, such as a bead (in-solution capture).
[0053] Molecular Inversion Probe (MIP) techniques are known in the art and are disclosed in e.g., Absalan et al., 2008. This method uses MIP molecules, which are special "padlock" probes (Nilsson et al., 1994) for genotyping. A MIP molecule is a linear oligonucleotide that contains specific regions, universal sequences, restriction sites and a Tag (index) sequence (16-22 bp). A MIP hybridizes directly around the genetic marker/SNP of interest. The MIP method may also use a number of "padlock" probe sets that hybridize to genomic DNA in parallel (Hardenbol et al., 2003). In case of a perfect match, genomic homology regions are ligated by undergoing an inversion in configuration (as suggested by the name of the technique) and creating a circular molecule. After the first restriction, all molecules are amplified with universal primers. Amplicons are restricted again to ensure short fragments for hybridization on a microarray. Generated short fragments are labeled and, through a Tag sequence, hybridized to a cTag (complementary strand for index) on an array. After the formation of Tag-cTag duplex, a signal is detected.
[0054] In another aspect of this embodiment, the method further comprises administering to the subject at least one additional therapeutic agent effective for treating or ameliorating the effects of the cancer. The additional therapeutic agent may be selected from the group consisting of an antibody or fragment thereof, a cytotoxic agent, a toxin, a radionuclide, an immunomodulator, a photoactive therapeutic agent, a radiosensitizing agent, a hormone, an anti-angiogenesis agent, and combinations thereof.
[0055] As used herein, an "antibody" encompasses naturally occurring immunoglobulins as well as non-naturally occurring immunoglobulins, including, for example, single chain antibodies, chimeric antibodies {e.g., humanized murine antibodies), and heteroconjugate antibodies {e.g., bispecific antibodies). Fragments of antibodies include those that bind antigen, {e.g., Fab', F(ab')2, Fab, Fv, and rlgG). See also, e.g., Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, III.); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York (1998). The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. The term "antibody" further includes both polyclonal and monoclonal antibodies.
[0056] Examples of therapeutic antibodies that may be used in the present invention include rituximab (Rituxan), Cetuximab (Erbitux), bevacizumab (Avastin), and Ibritumomab (Zevalin).
[0057] Cytotoxic agents according to the present invention include DNA damaging agents, antimetabolites, anti-microtubule agents, antibiotic agents, etc. DNA damaging agents include alkylating agents, platinum-based agents, intercalating agents, and inhibitors of DNA replication. Non-limiting examples of DNA alkylating agents include cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, ifosfamide, carmustine, lomustine, streptozocin, busulfan, temozolomide, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof. Non-limiting examples of platinum-based agents include cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, triplatin tetranitrate, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof. Non-limiting examples of intercalating agents include doxorubicin, daunorubicin, idarubicin, mitoxantrone, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof. Non-limiting examples of inhibitors of DNA replication include irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof. Antimetabolites include folate antagonists such as methotrexate and premetrexed, purine antagonists such as 6-mercaptopurine, dacarbazine, and fludarabine, and pyrimidine antagonists such as 5-fluorouracil, arabinosylcytosine, capecitabine, gemcitabine, decitabine, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof. Anti- microtubule agents include without limitation vinca alkaloids, paclitaxel (Taxol®), docetaxel (Taxotere®), and ixabepilone (Ixempra®). Antibiotic agents include without limitation actinomycin, anthracyclines, valrubicin, epirubicin, bleomycin, plicamycin, mitomycin, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof. [0058] In the present invention, a preferred additional therapeutics agent is an inhibitor of the PI3K/Akt pathway. Non-limiting examples of an inhibitor of the PI3K/Akt pathway include A-674563 (CAS # 552325-73-2), AGL 2263, AMG-319 (Amgen, Thousand Oaks, CA), AS-041 164 (5- benzo[1 ,3]dioxol-5-ylmethylene-thiazolidine-2,4-dione), AS-604850 (5-(2,2- Difluoro-benzo[1 ,3]dioxol-5-ylmethylene)-thiazolidine-2,4-dione), AS-605240 (5-quinoxilin-6-methylene-1 ,3-thiazolidine-2,4-dione), AT7867 (CAS # 857531 -00-1 ), benzimidazole series, Genentech (Roche Holdings Inc., South San Francisco, CA), BML-257 (CAS # 32387-96-5), CAL-120 (Gilead Sciences, Foster City, CA), CAL-129 (Gilead Sciences), CAL-130 (Gilead Sciences), CAL-253 (Gilead Sciences), CAL-263 (Gilead Sciences), CAS # 612847-09-3, CAS # 681281 -88-9, CAS # 75747-14-7, CAS # 925681 -41 -0, CAS # 98510-80-6, CCT128930 (CAS # 885499-61 -6), CH5132799 (CAS # 1007207-67-1 ), CHR-4432 (Chroma Therapeutics, Ltd., Abingdon, UK), FPA 124 (CAS # 902779-59-3), GS-1 101 (CAL-101 ) (Gilead Sciences), GSK 690693 (CAS # 937174-76-0), H-89 (CAS # 127243-85-0), Honokiol, IC871 14 (Gilead Science), IPI-145 (Intellikine Inc.), KAR-4139 (Karus Therapeutics, Chilworth, UK), KAR-4141 (Karus Therapeutics), KIN-1 (Karus Therapeutics), KT 5720 (CAS # 108068-98-0), Miltefosine, MK-2206 dihydrochloride (CAS # 1032350-13-2), ML-9 (CAS # 105637-50-1 ), Naltrindole Hydrochloride, OXY- 1 1 1 A (NormOxys Inc., Brighton, MA), perifosine, PHT-427 (CAS # 1 191951 - 57-1 ), PI3 kinase delta inhibitor, Merck KGaA (Merck & Co., Whitehouse Station, NJ), PI3 kinase delta inhibitors, Genentech (Roche Holdings Inc.), PI3 kinase delta inhibitors, Incozen (Incozen Therapeutics, Pvt. Ltd., Hyderabad, India), PI3 kinase delta inhibitors-2, Incozen (Incozen Therapeutics), PI3 kinase inhibitor, Roche-4 (Roche Holdings Inc.), PI3 kinase inhibitors, Roche (Roche Holdings Inc.), PI3 kinase inhibitors, Roche-5 (Roche Holdings Inc.), PI3-alpha/delta inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd., South San Francisco, CA), PI3-delta inhibitors, Cellzome (Cellzome AG, Heidelberg, Germany), PI3-delta inhibitors, Intellikine (Intellikine Inc., La Jolla, CA), PI3-delta inhibitors, Pathway Therapeutics-1 (Pathway Therapeutics Ltd.), PI3-delta inhibitors, Pathway Therapeutics-2 (Pathway Therapeutics Ltd.), PI3-delta/gamma inhibitors, Cellzome (Cellzome AG), PI3-delta/gamma inhibitors, Cellzome (Cellzome AG), PI3-delta/gamma inhibitors, Intellikine (Intellikine Inc.), PI3-delta/gamma inhibitors, Intellikine (Intellikine Inc.), PI3- delta/gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3-delta/gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3-gamma inhibitor Evotec (Evotec), PI3-gamma inhibitor, Cellzome (Cellzome AG), PI3-gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3K delta/gamma inhibitors, lntellikine-1 (Intellikine Inc.), PI3K delta/gamma inhibitors, lntellikine-1 (Intellikine Inc.), pictilisib (Roche Holdings Inc.), PIK-90 (CAS # 677338-12-4), SC-103980 (Pfizer, New York, NY), SF-1 126 (Semafore Pharmaceuticals, Indianapolis, IN), SH-5, SH-6, Tetrahydro Curcumin, TG100-1 15 (Targegen Inc., San Diego, CA), Triciribine, X-339 (Xcovery, West Palm Beach, FL), XL-499 (Evotech, Hamburg, Germany), pharmaceutically acceptable salts thereof, and combinations thereof.
[0059] In the present invention, the term "toxin" means an antigenic poison or venom of plant or animal origin. An example is diphtheria toxin or portions thereof. [0060] In the present invention, the term "radionuclide" means a radioactive substance administered to the patient, e.g., intravenously or orally, after which it penetrates via the patient's normal metabolism into the target organ or tissue, where it delivers local radiation for a short time. Examples of radionuclides include, but are not limited to, 1-125, At-21 1 , Lu-177, Cu-67, I- 131 , Sm-153, Re-186, P-32, Re-188, ln-1 14m, and Y-90.
[0061] In the present invention, the term "immunomodulator" means a substance that alters the immune response by augmenting or reducing the ability of the immune system to produce antibodies or sensitized cells that recognize and react with the antigen that initiated their production. Immunomodulators may be recombinant, synthetic, or natural preparations and include cytokines, corticosteroids, cytotoxic agents, thymosin, and immunoglobulins. Some immunomodulators are naturally present in the body, and certain of these are available in pharmacologic preparations. Examples of immunomodulators include, but are not limited to, granulocyte colony- stimulating factor (G-CSF), interferons, imiquimod and cellular membrane fractions from bacteria, IL-2, IL-7, IL-12, CCL3, CCL26, CXCL7, and synthetic cytosine phosphate-guanosine (CpG).
[0062] In the present invention, the term "photoactive therapeutic agent" means compounds and compositions that become active upon exposure to light. Certain examples of photoactive therapeutic agents are disclosed in, e.g., U.S. Patent Application Serial No. 201 1/0152230 A1 , "Photoactive Metal Nitrosyls For Blood Pressure Regulation And Cancer Therapy." [0063] In the present invention, the term "radiosensitizing agent" means a compound that makes tumor cells more sensitive to radiation therapy. Examples of radiosensitizing agents include misonidazole, metronidazole, tirapazamine, and trans sodium crocetinate.
[0064] In the present invention, the term "hormone" means a substance released by cells in one part of a body that affects cells in another part of the body. Examples of hormones include, but are not limited to, prostaglandins, leukotrienes, prostacyclin, thromboxane, amylin, antimullerian hormone, adiponectin, adrenocorticotropic hormone, angiotensinogen, angiotensin, vasopressin, atriopeptin, brain natriuretic peptide, calcitonin, cholecystokinin, corticotropin-releasing hormone, encephalin, endothelin, erythropoietin, follicle-stimulating hormone, galanin, gastrin, ghrelin, glucagon, gonadotropin- releasing hormone, growth hormone-releasing hormone, human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin, insulin, somatomedin, leptin, liptropin, luteinizing hormone, melanocyte stimulating hormone, motilin, orexin, oxytocin, pancreatic polypeptide, parathyroid hormone, prolactin, prolactin releasing hormone, relaxin, renin, secretin, somatostain, thrombopoietin, thyroid-stimulating hormone, testosterone, dehydroepiandrosterone, androstenedione, dihydrotestosterone, aldosterone, estradiol, estrone, estriol, Cortisol, progesterone, calcitriol, and calcidiol.
[0065] Some compounds interfere with the activity of certain hormones or stop the production of certain hormones. These hormone-interfering compounds include, but are not limited to, tamoxifen (Nolvadex®), anastrozole (Arimidex®), letrozole (Femara®), and fulvestrant (Faslodex®). Such compounds are also within the meaning of hormone in the present invention.
[0066] As used herein, an "anti-angiogenesis" agent means a substance that reduces or inhibits the growth of new blood vessels, such as, e.g., an inhibitor of vascular endothelial growth factor (VEGF) and an inhibitor of endothelial cell migration. Anti-angiogenesis agents include without limitation 2-methoxyestradiol, angiostatin, bevacizumab, cartilage-derived angiogenesis inhibitory factor, endostatin, IFN-a, IL-12, itraconazole, linomide, platelet factor-4, prolactin, SU5416, suramin, tasquinimod, tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, thrombospondin, TNP-470, ziv-aflibercept, pharmaceutically acceptable salts thereof, prodrugs, and combinations thereof.
[0067] In an additional aspect of this embodiment, administration of the first and second anti-cancer agents provides a synergistic effect compared to administration of either anti-cancer agent alone. As used herein, "synergistic" means more than additive. Synergistic effects may be measured by various assays known in the art, including but not limited to those disclosed herein, , such as the excess over bliss assay.
[0068] Another embodiment of the present invention is a method of treating or ameliorating the effects of a cancer in a subject in need thereof. This method comprises administering to the subject an effective amount of (i) BVD-523 or a pharmaceutically acceptable salt thereof and (ii) navitoclax or a pharmaceutically acceptable salt thereof, to treat or ameliorate the effects of the cancer. In this embodiment, the BVD-523 and navitoclax, or their respective pharmaceutically acceptable salts, may be administered in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or diluent.
[0069] Suitable and preferred subjects and various types of cancer are as disclosed herein. In this embodiment, the methods may be used to treat the cancers disclosed above, including those cancers with the mutational backgrounds identified above. Methods of identifying such mutations are also as set forth above.
[0070] In one aspect of this embodiment, the method further comprises administering at least one additional therapeutic agent, preferably an inhibitor of the PI3K/Akt pathway, as disclosed herein.
[0071] In a further aspect of this embodiment, administration of the BVD-523 and navitoclax provides a synergistic effect compared to administration of either agent alone.
[0072] An additional embodiment of the present invention is a method of effecting cancer cell death. This method comprises contacting the cancer cell with an effective amount of (i) a first anti-cancer agent, which is BVD-523 or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof. In this embodiment, "contacting" means bringing the ERK1/2 inhibitors, BCL-2 family inhibitors, and optionally one or more additional therapeutic agents into close proximity to the cancer cells. This may be accomplished using conventional techniques of drug delivery to mammals or in the in vitro situation by, e.g., providing the ERK1/2 inhibitors, BCL-2 family inhibitors, and optionally other therapeutic agents to a culture media in which the cancer cells are located. [0073] Suitable and preferred ERK1/2 inhibitors, BCL-2 family inhibitors, and combinations of the foregoing are as disclosed herein. In addition, the methods of this embodiment may be carried out in vitro or in vivo, and may be used to effect cancer cell death in cells of the types of cancer disclosed herein.
[0074] In this embodiment, effecting cancer cell death may be accomplished in cancer cells having various mutational backgrounds and/or that are characterized as disclosed above. Methods of identifying such mutations are also as set forth above.
[0075] In one aspect of this embodiment, the cancer cell is a mammalian cancer cell. Preferably, the mammalian cancer cell is obtained from a mammal selected from the group consisting of humans, primates, farm animals, and domestic animals. More preferably, the mammalian cancer cell is a human cancer cell. In this aspect of the invention, the method may be used to effect cell death in any of the cancers disclosed herein, preferably colorectal cancer cells.
[0076] In another aspect of this embodiment, the method further comprises contacting the cancer cell with at least one additional therapeutic agent, preferably an inhibitor of the PI3K/Akt pathway, as disclosed herein.
[0077] In a further aspect of this embodiment, contacting the cancer cell with the first and second ant-cancer agents provides a synergistic effect compared to contacting the cancer cell with either anti-cancer agent alone.
[0078] A further embodiment of the present invention is a kit for treating or ameliorating the effects of a cancer in a subject in need thereof. This kit comprises an effective amount of (i) an ERK1/2 inhibitor or a pharmaceutically acceptable salt thereof and (ii) a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof, packaged together with instructions for their use.
[0079] The kits may also include suitable storage containers, e.g., ampules, vials, tubes, etc., for each anti-cancer agent of the present invention (which may be, e.g., in the form of pharmaceutical compositions) and other reagents, e.g., buffers, balanced salt solutions, etc., for use in administering the anti-cancer agents to subjects. The anti-cancer agents of the invention and other reagents may be present in the kits in any convenient form, such as, e.g., in a solution or in a powder form. The kits may further include a packaging container, optionally having one or more partitions for housing the pharmaceutical composition(s) and other optional reagents.
[0080] Suitable and preferred subjects, ERK1/2 inhibitors, BCL-2 family inhibitors, and combinations of the foregoing are as disclosed herein. The kits of the invention may be used to treat any of the cancers disclosed herein, including those cancers having mutational backgrounds and/or that are characterized as disclosed above. Methods of identifying such mutations are as set forth above.
[0081] In one aspect of this embodiment, the kit further comprises at least one additional therapeutic agent, preferably an inhibitor of the PI3K/Akt pathway, as disclosed herein.
[0082] In a further aspect of this embodiment, administration of the first and second anti-cancer agents provides a synergistic effect compared to administration of either anti-cancer agent alone.
[0083] Another embodiment of the present invention is a pharmaceutical composition for treating or ameliorating the effects of a cancer in a subject in need thereof. This pharmaceutical composition comprises a pharmaceutically acceptable diluent or carrier and an effective amount of (i) a first anti-cancer agent, which is an ERK 1/2 inhibitor or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof, wherein administration of the first and second anti-cancer agents provides a synergistic effect compared to administration of either anti-cancer agent alone. This pharmaceutical composition may further comprise a pharmaceutically acceptable diluent or carrier.
[0084] Suitable and preferred subjects, ERK1/2 inhibitors, BCL-2 family inhibitors, combinations of ERK1/2 inhibitors and BCL-2 family inhibitors are as disclosed herein. The pharmaceutical compositions of the invention may be used to treat any of the cancers disclosed herein, including those cancers having various mutational backgrounds and/or that are characterized as disclosed above.
[0085] In one aspect of this embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent, preferably an inhibitor of the PI3K/Akt pathway, as disclosed herein.
[0086] The pharmaceutical compositions according to the present invention may be in a unit dosage form comprising both anti-cancer agents. In another aspect of this embodiment, the first anti-cancer agent is in a first unit dosage form and the second anti-cancer agent is in a second unit dosage form, separate from the first.
[0087] The first and second anti-cancer agents may be co-administered to the subject, either simultaneously or at different times, as deemed most appropriate by a physician. If the first and second anti-cancer agents are administered at different times, for example, by serial administration, the first anti-cancer agent may be administered to the subject before the second anticancer agent. Alternatively, the second anti-cancer agent may be administered to the subject before the first anti-cancer agent.
[0088] A further embodiment of the present invention is a method for selecting a subject with cancer that may benefit from a combination drug therapy. This method comprises:
(a) selecting a subject with a somatic K-RAS mutation;
(b) determining whether the subject with a K-RAS mutation has an epithelial-to-mesenchymal transition gene signature, wherein subjects having a K-RAS mutation and an epithelial-to-mesenchymal transition gene signature are likely to benefit from the combination drug therapy; and
(c) administering to the subject having a K-RAS mutation and an epithelial-to-mesenchymal transition gene signature the combination drug therapy which comprises an effective amount of (i) an ERK1/2 inhibitor or a pharmaceutically acceptable salt thereof and (ii) a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof.
[0089] As used herein, the "epithelial-to-mesenchymal transition (EMT)" refers to a process in which adherent epithelial cells shed their epithelial characteristics and acquire mesenchymal cell properties. EMT has been linked to metastatic progression and acquisition of stem-cell characteristics. Loss of E-cadherin is considered to be a fundamental event in EMT. Exposure to growth factors, such as transforming growth fact (TGF) β1 , hepatocyte growth factor, and platelet-derived growth factor (PDGF), or modulations of downstream transcription factors, including the Goosecoid (Gsc), Snail (Snail), Snai2 (Slug), Twist 1 (Twist), FOXC1 , FOXC2, Zeb1 , Sip1 (Zeb2), as well as the members of the miR-200 family of microRNAs, may lead to EMT. Determining an EMT gene signature is within the skill of the art and may be accomplished according to the method set forth in Taube et al., 2010 Determining the presence of a K-RAS mutation may be accomplished by using one of the methods disclosed herein.
[0090] Suitable and preferred subjects, ERK1/2 inhibitors, BCL-2 family inhibitors, combinations of ERK1/2 inhibitors and BCL-2 family inhibitors are as disclosed herein. In this embodiment, the methods may be used to treat the cancers disclosed above, including those cancers with the mutational backgrounds identified above. Methods of identifying such mutations are also as set forth above
[0091] In one aspect of this embodiment, the method further comprises administering at least one additional therapeutic agent, preferably an inhibitor of the PI3K/Akt pathway, as disclosed herein.
[0092] In the present invention, an "effective amount" or a "therapeutically effective amount" of an anti-cancer agent of the invention, including pharmaceutical compositions containing same that are disclosed herein, is an amount of such agent or composition that is sufficient to effect beneficial or desired results as described herein when administered to a subject. Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the route of administration, the rate of excretion, the duration of the treatment, the identity of any other drugs being administered, the age, size, and species of mammal, e.g., human patient, and like factors well known in the arts of medicine and veterinary medicine. In general, a suitable dose of an agent or composition according to the invention will be that amount of the agent or composition, which is the lowest dose effective to produce the desired effect. The effective dose of an agent or composition of the present invention may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.
[0093] A suitable, non-limiting example of a dosage of an ERK1/2 inhibitor, a BCL-2 family inhibitor, or another anti-cancer agent disclosed herein is from about 1 mg/kg to about 2400 mg/kg per day, such as from about 1 mg/kg to about 1200 mg/kg per day, 75 mg/kg per day to about 300 mg/kg per day, including from about 1 mg/kg to about 100 mg/kg per day. Other representative dosages of such agents include about 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg, 1 100 mg/kg, 1200 mg/kg, 1300 mg/kg, 1400 mg/kg, 1500 mg/kg, 1600 mg/kg, 1700 mg/kg, 1800 mg/kg, 1900 mg/kg, 2000 mg/kg, 2100 mg/kg, 2200 mg/kg, and 2300 mg/kg per day. The effective dose of an ERK1/2 inhibitor, a BCL-2 family inhibitor, or another anti-cancer agent disclosed herein, e.g., BVD-523 and navitoclax, may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.
[0094] The ERK1/2 inhibitors, BCL-2 family inhibitors, or other anticancer agents or pharmaceutical compositions containing same of the present invention may be administered in any desired and effective manner: for oral ingestion, or as an ointment or drop for local administration to the eyes, or for parenteral or other administration in any appropriate manner such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. Further, the ERK1/2 inhibitors, BCL-2 family inhibitors, or other anti-cancer agents or pharmaceutical compositions containing same of the present invention may be administered in conjunction with other treatments. The ERK1/2 inhibitors, BCL-2 family inhibitors, or other anticancer agents or the pharmaceutical compositions of the present invention may be encapsulated or otherwise protected against gastric or other secretions, if desired.
[0095] The pharmaceutical compositions of the invention comprise one or more active ingredients, e.g. anti-cancer agents, in admixture with one or more pharmaceutically-acceptable diluents or carriers and, optionally, one or more other compounds, drugs, ingredients and/or materials. Regardless of the route of administration selected, the agents/compounds of the present invention are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g., Remington, The Science and Practice of Pharmacy (21 st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.). [0096] Pharmaceutically acceptable diluents or carriers are well known in the art (see, e.g., Remington, The Science and Practice of Pharmacy (21 st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.) and The National Formulary (American Pharmaceutical Association, Washington, D.C.)) and include sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starches, cellulose preparations, calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogen phosphate), sodium citrate, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), organic esters (e.g., ethyl oleate and tryglycerides), biodegradable polymers (e.g., polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils (e.g., corn, germ, olive, castor, sesame, cottonseed, and groundnut), cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones, talc, silicylate, etc. Each pharmaceutically acceptable diluent or carrier used in a pharmaceutical composition of the invention must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Diluents or carriers suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable diluents or carriers for a chosen dosage form and method of administration can be determined using ordinary skill in the art.
[0097] The pharmaceutical compositions of the invention may, optionally, contain additional ingredients and/or materials commonly used in pharmaceutical compositions. These ingredients and materials are well known in the art and include (1 ) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate; (10) suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth; (1 1 ) buffering agents; (12) excipients, such as lactose, milk sugars, polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins, cocoa butter, starches, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonites, silicic acid, talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, and polyamide powder; (13) inert diluents, such as water or other solvents; (14) preservatives; (15) surface-active agents; (16) dispersing agents; (17) control-release or absorption-delaying agents, such as hydroxypropylmethyl cellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monostearate, gelatin, and waxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21 ) emulsifying and suspending agents; (22), solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan; (23) propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane; (24) antioxidants; (25) agents which render the formulation isotonic with the blood of the intended recipient, such as sugars and sodium chloride; (26) thickening agents; (27) coating materials, such as lecithin; and (28) sweetening, flavoring, coloring, perfuming and preservative agents. Each such ingredient or material must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Ingredients and materials suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable ingredients and materials for a chosen dosage form and method of administration may be determined using ordinary skill in the art.
[0098] The pharmaceutical compositions of the present invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules, a solution or a suspension in an aqueous or nonaqueous liquid, an oil-in-water or water-in-oil liquid emulsion, an elixir or syrup, a pastille, a bolus, an electuary or a paste. These formulations may be prepared by methods known in the art, e.g., by means of conventional pan- coating, mixing, granulation or lyophilization processes. [0099] Solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like) may be prepared, e.g., by mixing the active ingredient(s) with one or more pharmaceutically-acceptable diluents or carriers and, optionally, one or more fillers, extenders, binders, humectants, disintegrating agents, solution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants, and/or coloring agents. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using a suitable excipient. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using a suitable binder, lubricant, inert diluent, preservative, disintegrant, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine. The tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical- formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein. They may be sterilized by, for example, filtration through a bacteria-retaining filter. These compositions may also optionally contain opacifying agents and may be of a composition such that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. The active ingredient(s) may also be in microencapsulated form.
[0100] Liquid dosage forms for oral administration include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. The liquid dosage forms may contain suitable inert diluents commonly used in the art. Besides inert diluents, the oral compositions may also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions may contain suspending agents.
[0101] The pharmaceutical compositions of the present invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more active ingredient(s) with one or more suitable nonirritating diluents or carriers which are solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. The pharmaceutical compositions of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such pharmaceutically-acceptable diluents or carriers as are known in the art to be appropriate.
[0102] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants. The active agent(s)/compound(s) may be mixed under sterile conditions with a suitable pharmaceutically-acceptable diluent or carrier. The ointments, pastes, creams and gels may contain excipients. Powders and sprays may contain excipients and propellants.
[0103] The pharmaceutical compositions of the present invention suitable for parenteral administration may comprise one or more agent(s)/compound(s) in combination with one or more pharmaceutically- acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Proper fluidity can be maintained, for example, by the use of coating materials, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These pharmaceutical compositions may also contain suitable adjuvants, such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption.
[0104] In some cases, in order to prolong the effect of a drug (e.g., pharmaceutical formulation), it is desirable to slow its absorption from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility.
[0105] The rate of absorption of the active agent/drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally- administered agent/drug may be accomplished by dissolving or suspending the active agent/drug in an oil vehicle. Injectable depot forms may be made by forming microencapsule matrices of the active ingredient in biodegradable polymers. Depending on the ratio of the active ingredient to polymer, and the nature of the particular polymer employed, the rate of active ingredient release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.
[0106] The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid diluent or carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.
[0107] The present invention provides combinations shown to enhance the effects of ERK inhibitors. Herein, applicants have also shown that the combination of different ERK inhibitors is likewise synergistic. Therefore, it is contemplated that the effects of the combinations described herein can be further improved by the use of one or more additional ERK inhibitors. Accordingly, some embodiments of the present invention include one or more additional ERK inhibitors.
[0108] The following examples are provided to further illustrate the methods of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any way.
EXAMPLES
Example 1
Materials and Methods
Combination Assays
[0109] For combination studies with ABT263, HCT1 16 cells (K-RAS mutation human colorectal carcinoma cells) and A375 cells (BRAF V600 E human malignant melanoma) were used. For the HCT1 16 studies, cells were seeded into triplicate 96-well plates at a cell density of 1500 cells/well in McCoy's 5A Medium plus 10% fetal bovine serum (FBS). For the A375 studies, cells were seeded at a density of 3000 cells/well in Dulbecco's Modified Eagle Medium (DMEM) plus 10% FBS. In each case, cells were allowed to adhere overnight prior to addition of test compound or vehicle control.
[0110] The following combinations were tested in both HCT1 16 and A375 cells using a 10 x 8 dose matrix: ABT263 (ranging from 0-3 μΜ) with BVD-0523 (ranging from 0 to 10 μΜ), ABT263 (ranging from 0-3 μΜ) with dabrafenib (ranging from 0 to 1 μΜ), and ABT263 (ranging from 0-3 μΜ) with trametinib ((ranging from 0 to 0.010 μΜ). The final concentration of DMSO was 0.2%. The compounds were incubated with the cells for 96 hours.
[0111] For combination studies with ABT199, A375 cells were seeded into triplicate 96-well plates at a cell density of 3000 cells/well in McCoy's 5A plus 10% FBS. Cells were allowed to adhere overnight prior to addition of test compound or control.
[0112] The following combinations were tested using a 10 x 8 dose matrix: ABT199 (ranging from 0-1 μΜ) with BVD-0523 (0 to 10 μΜ), ABT199 (ranging from 0-1 μΜ) with dabrafenib (ranging from 0 to 1 μΜ), and ABT199 (ranging from 0-1 μΜ) with trametinib (ranging from 0 to 0.1 μΜ). The final concentration of DMSO was 0.2%. The compounds were incubated with the cells for 96 hours.
[0113] Next, Alamar Blue 10% (v/v) was added and incubated with the cells for 4 hours prior to reading on a fluorescent plate reader. [0114] After reading Alamar Blue, the medium/Alannar Blue mix was flicked off, 100 μΙ of CellTiter-Glo/PBS (1 :1 ) was added, and the plates were processed as per the manufacturers instructions (Promega, Madison, Wl). Media only background values were subtracted before the data was analyzed. Caspase-Glo 3/7 assays
[0115] Caspase-Glo 3/7 assays were performed in both HCT1 16 and A375 cells. In brief, HCT1 16 cells were seeded in triplicate in white 96-well plates at a cell density of 5000 cells/well in McCoy's 5A plus 10% FBS. A375 cells were seeded at a density of 5000 cells/well in DMEM plus 10% FBS. Cells were allowed to adhere overnight prior to addition of test compound or vehicle control. The final concentration of DMSO was 0.2%, and 800 nM staurosporine was included as a positive control. 24 and 48 hour assay incubation periods were used. Then, Caspase-Glo® 3/7 50% (v/v) was added, plates were mixed for 5 minutes on an orbital shaker and incubated for 1 hour at room temperature prior to reading on a luminescent plate reader. Media only background values were subtracted before the data was analysed. Data Analysis
[0116] The single and combination data are presented as dose- response curves generated in GraphPad Prism (plotted using % viability relative to DMSO only treated controls).
[0117] Predicted fractional inhibition values for combined inhibition were calculated using the equation Cbiiss =A + B - (A x B) where A and B are the fractional inhibitions obtained by drug A alone or drug B alone at specific concentrations. Cbiiss is the fractional inhibition that would be expected if the combination of the two drugs were exactly additive. Cbiiss values are subtracted from the experimentally observed fractional inhibition values to give an 'excess over Bliss' value. Excess over Bliss values greater than 0 indicate synergy, whereas values less than 0 indicate antagonism. Excess over Bliss values are plotted as heat maps ± SD.
Example 2
ERK kinase inhibition using BVD-523 in combination with the BCL-2 family inhibitor ABT-263/navitoclax is effective to inhibit cancer cell growth
[0118] The results of the combination assays are shown in Figures 1 -6. As shown in Figures 2 and 6, in HCT1 16 cells, there was synergy between ABT263 and trametinib (Figures 6C and 6D) and synergy between ABT263 and BVD-523 (Figures 2C-2D). There was also synergy between ABT263 and trametinib in A375 cells, as shown in Figures 5C and 5D. [0119] The results of the Caspase-Glo 3/7 assays performed in HCT1 16 cell lines are shown in Figure 7-9. As shown in Figure 7, there was synergy between ABT263 and BVD-523.
[0120] The results of combination assays with BVD-523 and the BCL-2 family inhibitor ABT-199 are shown in Figures 10-15.
[0121] As the figures show, ERK kinase inhibition, exemplified using BVD-523, in combination with the BCL-2 family inhibitor ABT-263/navitoclax, is effective to inhibit cancer cell growth. Additionally, the closely related BCL- 2 family inhibitor ABT-199 did not appear effective in combination in the same background. Thus, the finding may be highly specific, and that preferential combination effects may require disruption of particular client BCL family proteins, such as BCL-XL. Lastly, none of these combinations appear effective in BRAF mutant A375 melanoma cells, suggesting the specificity observed here may be linked to cancer types, genetic markers or the status of biomarkers relevant the BCL-2 family.
[0122] The results above may be explained by unique synthetic lethality in RAS mutant malignancies, which may display a high dependency on BCL- XL for prevention of apoptosis following MAPK inhibition. Studies have suggested that the status BH3-fold effectors may influence apoptosis more broadly during various types of cell stress like MAPK inhibition. While our current findings disclosed herein are focused on colorectal and RAS mutant backgrounds, we expect this combination may have broader application: BCL- 2 specific inhibition {e.g., ABT-263) combined with ERK exhibition may prove effective in treating lymphomas; other cancers may also be sensitive due to modulation of various BCL-2 family members {e.g., prosurvival MCL1 , proapopotic BIM), when combined with ERK inhibition. Tumor types identified using the combination of MEK and BCL-2 family inhibition include lung breast and hematologic malignancies.
Example 3
BVD-523 altered markers of MAPK kinase activity and effector function
[0123] For Western blot studies, HCT1 16 cells (5 x 106) were seeded into 10 cm dishes in McCoy's 5A plus 10% FBS. A375 cells (2.5 x 106) were seeded into 10 cm dishes in DMEM plus 10% FBS. Cells were allowed to adhere overnight prior to addition of the indicated amount of test compound (BVD-523) or vehicle control. Cells were treated for either 4 or 24 hours before isolation of whole-cell protein lysates, as specified below. Cells were harvested by trypsinisation, pelleted and snap frozen. Lysates were prepared with RIPA (Radio-lmmunoprecipitation Assay) buffer, clarified by centrifugation and quantitated by bicinchoninic acid assay (BCA) assay. 20- 50 g of protein was resolved by SDS-PAGE electrophoresis, blotted onto PVDF membrane and probed using the antibodies detailed in Table 5 (for the 4-hour treatment) and Table 6 (for the 24-hour treatment) below.
Table 5 - Antibody Details
Figure imgf000053_0001
Figure imgf000054_0001
0 milk
Table 6 - Antibody details
Figure imgf000054_0002
Figure imgf000055_0001
0 milk mouse
[0124] Figure 16 shows Western blot analyses of cells treated with BVD-523 at various concentrations for the following: 1 ) MAPK signaling components in A375 cells after 4 hours; 2) cell cycle and apoptosis signaling in A375 24 hours treatment with various amounts of BVD-523; and 3) MAPK signaling in HCT-1 16 cells treated for 4 hours. The results show that acute and prolonged treatment with BVD-523 in RAF and RAS mutant cancer cells in-vitro affects both substrate phosphorylation and effector targets of ERK kinases. The concentrations of BVD-523 required to induce these changes is typically in the low micromolar range.
[0125] Changes in several specific activity markers are noteworthy. First, the abundance of slowly migrating isoforms of ERK kinase increase following BVD-523 treatment; modest changes can be observed acutely, and increase following prolonged treatment. While this could indicate an increase in enzymatically active, phosphorylated forms of ERK, it remains noteworthy that multiple proteins subject to both direct and indirect regulation by ERK remain "off' following BVD-523 treatment. First, RSK1/2 proteins exhibit reduced phosphorylation at residues that are strictly dependent on ERK for protein modification (T359/S363). Second, BVD-523 treatment induces complex changes in the MAPK feedback phosphatase, DUSP6: slowly migrating protein isoforms are reduced following acute treatment, while total protein levels are greatly reduced following prolonged BVD-523 treatment. Both of these findings are consistent with reduced activity of ERK kinases, which control DUSP6 function through both post-translational and transcriptional mechanisms. Overall, despite increases in cellular forms of ERK that are typically thought to be active, it appears likely that cellular ERK enzyme activity is fully inhibited following either acute or prolonged treatment with BVD-523.
[0126] Consistent with these observations, effector genes that require MAPK pathway signaling are altered following treatment with BVD-523. The G1/S cell-cycle apparatus is regulated at both post-translational and transcriptional levels by MAPK signaling, and cyclin-D1 protein levels are greatly reduced following prolonged BVD-523 treatment. Similarly, gene expression and protein abundance of apoptosis effectors often require intact MAPK signaling, and total levels of Bim-EL increase following prolonged BVD- 523 treatment. As noted above, however, PARP protein cleavage and increased apoptosis were not noted in the A375 cell background; this suggests that additional factors may influence whether changes in BVD- 523/ERK-dependent effector signaling are translated into definitive events such as cell death and cell cycle arrest. [0127] Consistent with the cellular activity of BVD-523, marker analysis suggests that ERK inhibition alters a variety of molecular signaling events in cancer cells, making them susceptible to both decreased cell proliferation and survival.
[0128] In sum, Figure 16 shows that BVD-523 inhibits the MAPK signaling pathway and may be more favorable compared to RAF or MEK inhibition in this setting.
[0129] Finally, properties of BVD-523 may make this a preferred agent for use as an ERK inhibitor, compared to other agents with a similar activity. It is known that kinase inhibitor drugs display unique and specific interactions with their enzyme targets, and that drug efficacy is strongly influenced by both the mode of direct inhibition, as well as susceptibility to adaptive changes that occur following treatment. For example, inhibitors of ABL, KIT, EGFR and ALK kinases are effective only when their cognate target is found in active or inactive configurations. Likewise, certain of these inhibitors are uniquely sensitive to either secondary genetic mutation, or post-translational adaptive changes, of the protein target. Finally, RAF inhibitors show differential potency to RAF kinases present in certain protein complexes and/or subcellular localizations. In summary, as ERK kinases are similarly known to exist in diverse, variable, and complex biochemical states, it appears likely that BVD-523 may interact with and inhibit these targets in a fashion that is distinct and highly preferable to other agents. Example 4
Cell culture studies of BCL-2 and ERK inhibitors
Combination Proliferation Assay
[0130] Cells were seeded into triplicate 96-well plates at the densities indicated in Table 7 in RPMI media containing 10% FBS and allowed to adhere overnight prior to addition of test compound or vehicle control. Combinations were tested using a 10x8 dose matrix. The final DMSO concentration was constant at 0.2%.
[0131] Test compounds were incubated with the cells for 72h at 37°C, 5% CO2 in a humidified atmosphere. CellTiter-Glo® reagent (Promega, Madison, Wl) was added according to manufacturer's instructions and luminescence detected using the BMG FLUOstar plate reader (BMG Labtech, Ortenberg, Germany). The average media only background value was deducted and the data analyzed.
[0132] Combination interactions across the dose matrix were determined by the Loewe Additivity and Bliss independence models using Chalice™ Combination Analysis Software (Horizon Discovery Group, Cambridge, MA) as outlined in the user manual (available at chalice.horizondiscovery.com/chalice- portal/documentation/analyzer/home.jsp). Synergy is determined by comparing the experimentally observed level of inhibition at each combination point with the value expected for additivity, which is derived from the single- agent responses along the edges of the matrix. Potential synergistic interactions were identified by displaying the calculated excess inhibition over that predicted as being additive across the dose matrix as a heat map, and by reporting a quantitative 'Synergy Score' based on the Loewe model. The single agent data derived from the combination assay plates were presented as dose-response curves generated in Chalice™.
[0133] Synergy was visualized by plotting the calculated excess inhibition over expected, at each test point in the matrix, as a heat map, where brighter colors are indicative of higher activity levels. Excess activity over that predicted by the Loewe model if a combination was additive was also quantitated using a simple volume score, which calculates the volume between the measured and the predicted response surface.
[0134] This volume score shows whether the overall response to a combination is synergistic (positive values), antagonistic (negative values) or additive (values ~ 0).
[0135] The single agent dose-response curves from the combination assay plates and calculated relative IC50 values were generated using a 4- parameter logistic equation in GraphPad Prism.
Table 7 - Cell Line Seeding Density
Figure imgf000059_0001
[0136] The aim of this study was to assess the effects of combining the ERK inhibitor BVD-523 with the Bcl-2-family inhibitor ABT-263 across a panel of four lung cancer cell lines, with differing KRAS status and varying sensitivities to ABT-263 or MEK inhibition (Table 8).
Table 8 - Details of Cell Lines Tested
Figure imgf000060_0001
"Reported sensitivit in GDSC/Sanger data sets.
[0137] The effects of the combinations on cell viability were assessed after 72h by quantitating cellular ATP levels using CellTiter-Glo® (Promega, Madison, Wl).
[0138] Combination interactions across a matrix of concentrations were determined by the Loewe Additivity and Bliss Independence models using Horizon's Chalice™ Bioinformatics Software. Synergy was visualized by displaying the calculated excess inhibition over that predicted as being additive at each test point across the matrix as a heat map.
[0139] Activity over Loewe additivity was also quantified in Chalice™ using a simple volume score, which effectively calculates a volume between the measured and Loewe additive response surfaces, and emphasizes the overall synergistic (positive values) or antagonistic (negative values) effect of the combination (Figure 17 - Figure 19).
[0140] Visualization of the Loewe 'excess inhibition' heat maps and the Loewe volume scores suggested that the interaction between BVD-523 with ABT263 across the dose matrix varied between cell lines. There was evidence for a window of synergy at higher BVD-523 concentrations in H1650 cells. Whereas interactions in A549 and H460 were mostly additive, and in H226 cells net antagonistic. A similar pattern of results was obtained with another ERK inhibitor SCH772984 and MEK inhibitor trametinib.
[0141] A549 cells were most sensitive to BVD-523 as a single agent. The single agent ABT-263 responses were consistent with the GDSC/Sanger data. Volume scores for the combinations tested are shown in Figure 20 and Tables 9-1 1 and are consistent with the conclusions drawn from the heat maps.
Table 9 - Loewe Volumes
Figure imgf000061_0001
Table 10 - Bliss Volumes
Figure imgf000061_0002
Table 1 1 - Synergy Scores
Figure imgf000062_0001
In summary, these results suggest that in a subset of lung cancer cell lines interactions between BVD-523 and ABT-263 can be at least additive and, in some cases, synergistic.
Example 5
Combination Interactions Between ERK inhibitors
[0142] RAF mutant melanoma cell line A375 cells were cultured in DMEM with 10% FBS and seeded into triplicate 96-well plates at an initial density of 2000 cells per well. Combination interactions between ERK inhibitors BVD-523 and SCH772984 were analized after 72 hours as described above in Example 4. Viability was determined using CellTiter-Glo® reagent (Promega, Madison, Wl) according to manufacturer's instructions and luminescence was detected using the BMG FLUOstar plate reader (BMG Labtech, Ortenberg, Germany).
[0143] Visualization of the Loewe and Bliss 'excess inhibition' heat maps suggested that the combination of BVD-523 and SCH772984 was mainly additive with windows of potential synergy in mid-range doses (Figure 21 ).
[0144] In summary, these results suggest that interactions between BVD-523 and SCH772984 are at least additive, and in some cases synergistic.
Documents
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HAMPTON M, Melvin RG, Kendall AH, Kirkpatrick BR, Peterson N, Andrews MT (201 1 ). "Deep sequencing the transcriptome reveals seasonal adaptive mechanisms in a hibernating mammal". PLoS One. 6 (10).
HARDENBOL, P., et al. Multiplexed genotyping with sequence-tagged molecular inversion probes. Nat. Biotechnol., 2003, no.21 , pp. 673-678.
METZKER, Emerging technologies in DNA sequencing Genome Res. 2005.
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MEYERSON, M.; Gabriel, S.; Getz, G. (2010). "Advances in understanding cancer genomes through second-generation sequencing". Nature
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[0145] All documents cited in this application are hereby incorporated by reference as if recited in full herein.
[0146] Although illustrative embodiments of the present invention have been described herein, it should be understood that the invention is not limited to those described, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the invention.

Claims

What is claimed is:
1 . A method of treating or ameliorating the effects of a cancer in a subject in need thereof comprising administering to the subject an effective amount of (i) a first anti-cancer agent, which is an ERK 1/2 inhibitor or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof, to treat or ameliorate the effects of the cancer.
2. The method according to clam 1 , wherein the subject is a mammal.
3. The method according to claim 2, wherein the mammal is selected from the group consisting of humans, primates, farm animals, and domestic animals.
4. The method according to claim 2, wherein the mammal is a human.
5. The method according to claim 1 , wherein the ERK1/2 inhibitor is selected from the group consisting of AEZS-131 (Aeterna Zentaris), AEZS- 136 (Aeterna Zentaris), BVD-523 (BioMed Valley Discoveries, Inc.), SCH- 722984 (Merck & Co.), SCH-772984 (Merck & Co.), SCH-900353 (MK-8353) (Merck & Co.), pharmaceutically acceptable salts thereof, and combinations thereof.
6. The method according to claim 1 , wherein the ERK1/2 inhibitor is BVD- 523 or a pharmaceutically acceptable salt thereof.
7. The method according to claim 1 , wherein the BCL-2 family inhibitor is selected from the group consisting of (-)-epigallocatechin gallate, ABT-737 (Abbott), antimycin A, apogossypolone, CAS # 383860-03-5, CAS # 810659- 53-1 , CAS #141266-44-6, BP-100-1 .02 (Bio-Path Holdings), chelerythrine chloride, HA14-1 (CAS # 65673-63-4), licochalcone-A (CAS #58749-22-7), navitoclax (Abbott), obatoclax (GX15-080), R-(-)-gossypol (Ascenta Therapeutics), S-44563 (Servier), sabutoclax (Oncothyreon), TW-37, VAL-101 (ValiRx), WEHI-539 (Hoffmann-La Roche, Inc.), pharmaceutically acceptable salts thereof, and combinations thereof.
8. The method according to claim 1 , wherein the BCL-2 family inhibitor is navitoclax or a pharmaceutically acceptable salt thereof.
9. The method according to claim 1 , wherein the ERK 1/2 inhibitor is BVD-523 or a pharmaceutically acceptable salt thereof, and the BCL-2 family inhibitor is navitoclax or a pharmaceutically acceptable salt thereof.
10. The method according to claim 1 , wherein the subject with cancer has a somatic RAS mutation.
1 1 . The method according to claim 1 , wherein the cancer is selected from the group consisting of colorectal cancer, breast cancer, pancreatic cancer, lymphoma, lung cancer, testicular cancer, thyroid cancer, hematologic malignancies, and endometrial cancers.
12. The method according to claim 1 1 , wherein the hematologic malignancy is selected from the group consisting of RAS mutant myelodysplastic syndromes (MDS), lymphoid cancers, and myeloid cancers.
13. The method according to claim 1 1 , wherein the hematologic malignancy is selected from the group consisting of Adult Acute Megakaryoblastic Leukemia (M7), Adult Acute Minimally Differentiated Myeloid Leukemia (MO), Adult Acute Monoblastic Leukemia (M5a), Adult Acute Monocytic Leukemia (M5b), Adult Acute Myeloblasts Leukemia With Maturation (M2), Adult Acute Myeloblasts Leukemia Without Maturation (M1 ), Adult Acute Myeloid Leukemia With 1 1 q23 (MLL) Abnormalities, Adult Acute Myeloid Leukemia With Del(5q), Adult Acute Myeloid Leukemia With lnv(16)(p13;q22), Adult Acute Myeloid Leukemia With t(16;16)(p13;q22), Adult Acute Myeloid Leukemia With t(8;21 )(q22;q22), Adult Acute Myelomonocytic Leukemia (M4), Adult Erythroleukemia (M6a), Adult Pure Erythroid Leukemia (M6b), Recurrent Adult Acute Myeloid Leukemia, and Untreated Adult Acute Myeloid Leukemia.
14. The method according to claim 1 , wherein the cancer is colorectal cancer.
15. The method according to claim 1 further comprising administering at least one additional therapeutic agent selected from the group consisting of an antibody or fragment thereof, a cytotoxic agent, a toxin, a radionuclide, an immunomodulator, a photoactive therapeutic agent, a radiosensitizing agent, a hormone, an anti-angiogenesis agent, and combinations thereof.
16. The method according to claim 15, wherein the additional therapeutic agent is an inhibitor of the PI3K/Akt pathway.
17. The method according to claim 16, wherein the inhibitor of the PI3K/Akt pathway is selected from the group consisting of A-674563 (CAS # 552325- 73-2), AGL 2263, AMG-319 (Amgen, Thousand Oaks, CA), AS-041 164 (5- benzo[1 ,3]dioxol-5-ylmethylene-thiazolidine-2,4-dione), AS-604850 (5-(2,2- Difluoro-benzo[1 ,3]dioxol-5-ylmethylene)-thiazolidine-2,4-dione), AS-605240 (5-quinoxilin-6-methylene-1 ,3-thiazolidine-2,4-dione), AT7867 (CAS # 857531 -00-1 ), benzimidazole series, Genentech (Roche Holdings Inc., South San Francisco, CA), BML-257 (CAS # 32387-96-5), CAL-120 (Gilead Sciences, Foster City, CA), CAL-129 (Gilead Sciences), CAL-130 (Gilead Sciences), CAL-253 (Gilead Sciences), CAL-263 (Gilead Sciences), CAS # 612847-09-3, CAS # 681281 -88-9, CAS # 75747-14-7, CAS # 925681 -41 -0, CAS # 98510-80-6, CCT128930 (CAS # 885499-61 -6), CH5132799 (CAS # 1007207-67-1 ), CHR-4432 (Chroma Therapeutics, Ltd., Abingdon, UK), FPA 124 (CAS # 902779-59-3), GS-1 101 (CAL-101 ) (Gilead Sciences), GSK 690693 (CAS # 937174-76-0), H-89 (CAS # 127243-85-0), Honokiol, IC871 14 (Gilead Science), IPI-145 (Intellikine Inc.), KAR-4139 (Karus Therapeutics, Chilworth, UK), KAR-4141 (Karus Therapeutics), KIN-1 (Karus Therapeutics), KT 5720 (CAS # 108068-98-0), Miltefosine, MK-2206 dihydrochloride (CAS # 1032350-13-2), ML-9 (CAS # 105637-50-1 ), Naltrindole Hydrochloride, OXY- 1 1 1 A (NormOxys Inc., Brighton, MA), perifosine, PHT-427 (CAS # 1 191951 - 57-1 ), PI3 kinase delta inhibitor, Merck KGaA (Merck & Co., Whitehouse Station, NJ), PI3 kinase delta inhibitors, Genentech (Roche Holdings Inc.), PI3 kinase delta inhibitors, Incozen (Incozen Therapeutics, Pvt. Ltd., Hyderabad, India), PI3 kinase delta inhibitors-2, Incozen (Incozen Therapeutics), PI3 kinase inhibitor, Roche-4 (Roche Holdings Inc.), PI3 kinase inhibitors, Roche (Roche Holdings Inc.), PI3 kinase inhibitors, Roche-5 (Roche Holdings Inc.), PI3-alpha/delta inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd., South San Francisco, CA), PI3-delta inhibitors, Cellzome (Cellzome AG, Heidelberg, Germany), PI3-delta inhibitors, Intellikine (Intellikine Inc., La Jolla, CA), PI3-delta inhibitors, Pathway Therapeutics-1 (Pathway Therapeutics Ltd.), PI3-delta inhibitors, Pathway Therapeutics-2 (Pathway Therapeutics Ltd.), PI3-delta/gamnna inhibitors, Cellzome (Cellzome AG), PI3-delta/gamnna inhibitors, Cellzome (Cellzome AG), PI3-delta/gamma inhibitors, Intellikine (Intellikine Inc.), PI3-delta/gamma inhibitors, Intellikine (Intellikine Inc.), PI3- delta/gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3-delta/gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3-gamma inhibitor Evotec (Evotec), PI3-gamma inhibitor, Cellzome (Cellzome AG), PI3-gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3K delta/gamma inhibitors, lntellikine-1 (Intellikine Inc.), PI3K delta/gamma inhibitors, lntellikine-1 (Intellikine Inc.), pictilisib (Roche Holdings Inc.), PIK-90 (CAS # 677338-12-4), SC-103980 (Pfizer, New York, NY), SF-1 126 (Semafore Pharmaceuticals, Indianapolis, IN), SH-5, SH-6, Tetrahydro Curcumin, TG100-1 15 (Targegen Inc., San Diego, CA), Triciribine, X-339 (Xcovery, West Palm Beach, FL), XL-499 (Evotech, Hamburg, Germany), pharmaceutically acceptable salts thereof, and combinations thereof.
18. The method according to claim 1 , wherein administration of the first and second anti-cancer agents provides a synergistic effect compared to administration of either anti-cancer agent alone.
19. A method of treating or ameliorating the effects of a cancer in a subject in need thereof comprising administering to the subject an effective amount of (i) BVD-523 or a pharmaceutically acceptable salt thereof and (ii) navitoclax or a pharnnaceutically acceptable salt thereof, to treat or ameliorate the effects of the cancer.
20. The method according to clam 19, wherein the subject is a mammal.
21 . The method according to claim 20, wherein the mammal is selected from the group consisting of humans, primates, farm animals, and domestic animals.
22. The method according to claim 20, wherein the mammal is a human.
23. The method according to claim 19, wherein the BVD-523 or a pharmaceutically acceptable salt thereof is administered in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or diluent.
24. The method according to claim 19, wherein the navitoclax or a pharmaceutically acceptable salt thereof is administered in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or diluent.
25. The method according to claim 19, wherein the subject with cancer has a somatic RAS mutation.
26. The method according to claim 19, wherein the cancer is selected from the group consisting of colorectal cancer, breast cancer, pancreatic cancer, lymphoma, lung cancer, testicular cancer, thyroid cancer, hematologic malignancies, and endometrial cancers.
27. The method according to claim 26, wherein the hematologic malignancy is selected from the group consisting of RAS mutant myelodysplastic syndromes (MDS), lymphoid cancers, and myeloid cancers.
28. The method according to claim 26, wherein the hematologic malignancy is selected from the group consisting of Adult Acute Megakaryoblastic Leukemia (M7), Adult Acute Minimally Differentiated Myeloid Leukemia (MO), Adult Acute Monoblastic Leukemia (M5a), Adult Acute Monocytic Leukemia (M5b), Adult Acute Myeloblasts Leukemia With Maturation (M2), Adult Acute Myeloblasts Leukemia Without Maturation (M1 ), Adult Acute Myeloid Leukemia With 1 1 q23 (MLL) Abnormalities, Adult Acute Myeloid Leukemia With Del(5q), Adult Acute Myeloid Leukemia With lnv(16)(p13;q22), Adult Acute Myeloid Leukemia With t(16;16)(p13;q22), Adult Acute Myeloid Leukemia With t(8;21 )(q22;q22), Adult Acute Myelomonocytic Leukemia (M4), Adult Erythroleukemia (M6a), Adult Pure Erythroid Leukemia (M6b), Recurrent Adult Acute Myeloid Leukemia, and Untreated Adult Acute Myeloid Leukemia.
29. The method according to claim 19, wherein the cancer is colorectal cancer.
30. The method according to claim 19 further comprising administering at least one additional therapeutic agent selected from the group consisting of an antibody or fragment thereof, a cytotoxic agent, a toxin, a radionuclide, an immunomodulator, a photoactive therapeutic agent, a radiosensitizing agent, a hormone, and combinations thereof.
31 . The method according to claim 30, wherein the additional therapeutic agent is an inhibitor of the PI3K/Akt pathway.
32. The method according to claim 31 , wherein the inhibitor of the PI3K/Akt pathway is selected from the group consisting of A-674563 (CAS # 552325- 73-2), AGL 2263, AMG-319 (Amgen, Thousand Oaks, CA), AS-041 164 (5- benzo[1 ,3]dioxol-5-ylmethylene-thiazolidine-2,4-dione), AS-604850 (5-(2,2- Difluoro-benzo[1 ,3]dioxol-5-ylmethylene)-thiazolidine-2,4-dione), AS-605240 (5-quinoxilin-6-methylene-1 ,3-thiazolidine-2,4-dione), AT7867 (CAS # 857531 -00-1 ), benzimidazole series, Genentech (Roche Holdings Inc., South San Francisco, CA), BML-257 (CAS # 32387-96-5), CAL-120 (Gilead Sciences, Foster City, CA), CAL-129 (Gilead Sciences), CAL-130 (Gilead Sciences), CAL-253 (Gilead Sciences), CAL-263 (Gilead Sciences), CAS # 612847-09-3, CAS # 681281 -88-9, CAS # 75747-14-7, CAS # 925681 -41 -0, CAS # 98510-80-6, CCT128930 (CAS # 885499-61 -6), CH5132799 (CAS # 1007207-67-1 ), CHR-4432 (Chroma Therapeutics, Ltd., Abingdon, UK), FPA 124 (CAS # 902779-59-3), GS-1 101 (CAL-101 ) (Gilead Sciences), GSK 690693 (CAS # 937174-76-0), H-89 (CAS # 127243-85-0), Honokiol, IC871 14 (Gilead Science), IPI-145 (Intellikine Inc.), KAR-4139 (Karus Therapeutics, Chilworth, UK), KAR-4141 (Karus Therapeutics), KIN-1 (Karus Therapeutics), KT 5720 (CAS # 108068-98-0), Miltefosine, MK-2206 dihydrochloride (CAS # 1032350-13-2), ML-9 (CAS # 105637-50-1 ), Naltrindole Hydrochloride, OXY- 1 1 1 A (NormOxys Inc., Brighton, MA), perifosine, PHT-427 (CAS # 1 191951 - 57-1 ), PI3 kinase delta inhibitor, Merck KGaA (Merck & Co., Whitehouse Station, NJ), PI3 kinase delta inhibitors, Genentech (Roche Holdings Inc.), PI3 kinase delta inhibitors, Incozen (Incozen Therapeutics, Pvt. Ltd., Hyderabad, India), PI3 kinase delta inhibitors-2, Incozen (Incozen Therapeutics), PI3 kinase inhibitor, Roche-4 (Roche Holdings Inc.), PI3 kinase inhibitors, Roche (Roche Holdings Inc.), PI3 kinase inhibitors, Roche-5 (Roche Holdings Inc.), PI3-alpha/delta inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd., South San Francisco, CA), PI3-delta inhibitors, Cellzome (Cellzome AG, Heidelberg, Germany), PI3-delta inhibitors, Intellikine (Intellikine Inc., La Jolla, CA), PI3-delta inhibitors, Pathway Therapeutics-1 (Pathway Therapeutics Ltd.), PI3-delta inhibitors, Pathway Therapeutics-2 (Pathway Therapeutics Ltd.), PI3-delta/gamnna inhibitors, Cellzome (Cellzome AG), PI3-delta/gamma inhibitors, Cellzome (Cellzome AG), PI3-delta/gamma inhibitors, Intellikine (Intellikine Inc.), PI3-delta/gamma inhibitors, Intellikine (Intellikine Inc.), PI3- delta/gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3-delta/gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3-gamma inhibitor Evotec (Evotec), PI3-gamma inhibitor, Cellzome (Cellzome AG), PI3-gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3K delta/gamma inhibitors, lntellikine-1 (Intellikine Inc.), PI3K delta/gamma inhibitors, lntellikine-1 (Intellikine Inc.), pictilisib (Roche Holdings Inc.), PIK-90 (CAS # 677338-12-4), SC-103980 (Pfizer, New York, NY), SF-1 126 (Semafore Pharmaceuticals, Indianapolis, IN), SH-5, SH-6, Tetrahydro Curcumin, TG100-1 15 (Targegen Inc., San Diego, CA), Triciribine, X-339 (Xcovery, West Palm Beach, FL), XL-499 (Evotech, Hamburg, Germany), pharmaceutically acceptable salts thereof, and combinations thereof.
33. The method according to claim 19, wherein administration of the first and second anti-cancer agents provides a synergistic effect compared to administration of either anti-cancer agent alone.
34. A method of effecting cancer cell death comprising contacting the cancer cell with an effective amount of (i) a first anti-cancer agent, which is BVD-523 or a pharmaceutically acceptable salt thereof and (ii) a second anticancer agent, which is a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof.
35. The method according to clam 34, wherein the cancer cell is a mammalian cancer cell.
36. The method according to claim 35, wherein the mammalian cancer cell is obtained a mammal selected from the group consisting of humans, primates, farm animals, and domestic animals.
37. The method according to claim 35, wherein the mammalian cancer cell is a human cancer cell.
38. The method according to claim 34, wherein the ERK1/2 inhibitor is selected from the group consisting of AEZS-131 (Aeterna Zentaris), AEZS- 136, BVD-523 (BioMed Valley Discoveries, Inc.), SCH-722984 (Merck & Co.), SCH-772984 (Merck & Co.), SCH-900353 (MK-8353) (Merck & Co.), pharmaceutically acceptable salts thereof, and combinations thereof.
39. The method according to claim 34, wherein the ERK1/2 inhibitor is BVD-523 or a pharmaceutically acceptable salt thereof.
40. The method according to claim 34, wherein the BCL-2 family inhibitor is selected from the group consisting of (-)-epigallocatechin gallate, ABT-737 (Abbott), antimycin A, apogossypolone, CAS # 383860-03-5, CAS # 810659- 53-1 , CAS #141266-44-6, BP-100-1 .02 (Bio-Path Holdings), chelerythrine chloride, HA14-1 (CAS # 65673-63-4), licochalcone-A (CAS #58749-22-7), navitoclax (Abbott), obatoclax (GX15-080), R-(-)-gossypol (Ascenta Therapeutics), S-44563 (Servier), sabutoclax (Oncothyreon), TW-37, VAL-101 (ValiRx), WEHI-539 (Hoffmann-La Roche, Inc.), pharmaceutically acceptable salts thereof, and combinations thereof.
41 . The method according to claim 34, wherein the BCL-2 family inhibitor is navitoclax or a pharmaceutically acceptable salt thereof.
42. The method according to claim 34, wherein the ERK 1/2 inhibitor is BVD-523 or a pharmaceutically acceptable salt thereof, and the BCL-2 family inhibitor is navitoclax or a pharmaceutically acceptable salt thereof.
43. The method according to claim 34, wherein the subject with cancer has a somatic RAS mutation.
44. The method according to claim 34, wherein the cancer is selected from the group consisting of colorectal cancer, breast cancer, pancreatic cancer, lymphoma, lung cancer, testicular cancer, thyroid cancer, hematologic malignancies, and endometrial cancers.
45. The method according to claim 44, wherein the hematologic malignancy is selected from the group consisting of RAS mutant myelodysplastic syndromes (MDS), lymphoid cancers, and myeloid cancers.
46. The method according to claim 44, wherein the hematologic malignancy is selected from the group consisting of Adult Acute Megakaryoblastic Leukemia (M7), Adult Acute Minimally Differentiated Myeloid Leukemia (MO), Adult Acute Monoblastic Leukemia (M5a), Adult Acute Monocytic Leukemia (M5b), Adult Acute Myeloblasts Leukemia With Maturation (M2), Adult Acute Myeloblasts Leukemia Without Maturation (M1 ), Adult Acute Myeloid Leukemia With 1 1 q23 (MLL) Abnormalities, Adult Acute Myeloid Leukemia With Del(5q), Adult Acute Myeloid Leukemia With lnv(16)(p13;q22), Adult Acute Myeloid Leukemia With t(16;16)(p13;q22), Adult Acute Myeloid Leukemia With t(8;21 )(q22;q22), Adult Acute Myelomonocytic Leukemia (M4), Adult Erythroleukemia (M6a), Adult Pure Erythroid Leukemia (M6b), Recurrent Adult Acute Myeloid Leukemia, and Untreated Adult Acute Myeloid Leukemia.
47. The method according to claim 34, wherein the cancer is colorectal cancer.
48. The method according to claim 34 further comprising contacting the cancer cell with at least one additional therapeutic agent selected from the group consisting of an antibody or fragment thereof, a cytotoxic agent, a toxin, a radionuclide, an immunomodulator, a photoactive therapeutic agent, a radiosensitizing agent, a hormone, an anti-angiogenesis agent, and combinations thereof.
49. The method according to claim 48, wherein the additional therapeutic agent is an inhibitor of the PI3K/Akt pathway.
50. The method according to claim 49, wherein the inhibitor of the PI3K/Akt pathway is selected from the group consisting of A-674563 (CAS # 552325- 73-2), AGL 2263, AMG-319 (Amgen, Thousand Oaks, CA), AS-041 164 (5- benzo[1 ,3]dioxol-5-ylmethylene-thiazolidine-2,4-dione), AS-604850 (5-(2,2- Difluoro-benzo[1 ,3]dioxol-5-ylmethylene)-thiazolidine-2,4-dione), AS-605240 (5-quinoxilin-6-methylene-1 ,3-thiazolidine-2,4-dione), AT7867 (CAS # 857531 -00-1 ), benzimidazole series, Genentech (Roche Holdings Inc., South San Francisco, CA), BML-257 (CAS # 32387-96-5), CAL-120 (Gilead Sciences, Foster City, CA), CAL-129 (Gilead Sciences), CAL-130 (Gilead Sciences), CAL-253 (Gilead Sciences), CAL-263 (Gilead Sciences), CAS # 612847-09-3, CAS # 681281 -88-9, CAS # 75747-14-7, CAS # 925681 -41 -0, CAS # 98510-80-6, CCT128930 (CAS # 885499-61 -6), CH5132799 (CAS # 1007207-67-1 ), CHR-4432 (Chroma Therapeutics, Ltd., Abingdon, UK), FPA 124 (CAS # 902779-59-3), GS-1 101 (CAL-101 ) (Gilead Sciences), GSK 690693 (CAS # 937174-76-0), H-89 (CAS # 127243-85-0), Honokiol, IC871 14 (Gilead Science), IPI-145 (Intellikine Inc.), KAR-4139 (Karus Therapeutics, Chilworth, UK), KAR-4141 (Karus Therapeutics), KIN-1 (Karus Therapeutics), KT 5720 (CAS # 108068-98-0), Miltefosine, MK-2206 dihydrochloride (CAS # 1032350-13-2), ML-9 (CAS # 105637-50-1 ), Naltrindole Hydrochloride, OXY- 1 1 1 A (NormOxys Inc., Brighton, MA), perifosine, PHT-427 (CAS # 1 191951 - 57-1 ), PI3 kinase delta inhibitor, Merck KGaA (Merck & Co., Whitehouse Station, NJ), PI3 kinase delta inhibitors, Genentech (Roche Holdings Inc.), PI3 kinase delta inhibitors, Incozen (Incozen Therapeutics, Pvt. Ltd., Hyderabad, India), PI3 kinase delta inhibitors-2, Incozen (Incozen Therapeutics), PI3 kinase inhibitor, Roche-4 (Roche Holdings Inc.), PI3 kinase inhibitors, Roche (Roche Holdings Inc.), PI3 kinase inhibitors, Roche-5 (Roche Holdings Inc.), PI3-alpha/delta inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd., South San Francisco, CA), PI3-delta inhibitors, Cellzome (Cellzome AG, Heidelberg, Germany), PI3-delta inhibitors, Intellikine (Intellikine Inc., La Jolla, CA), PI3-delta inhibitors, Pathway Therapeutics-1 (Pathway Therapeutics Ltd.), PI3-delta inhibitors, Pathway Therapeutics-2 (Pathway Therapeutics Ltd.), PI3-delta/gamnna inhibitors, Cellzome (Cellzome AG), PI3-delta/gamma inhibitors, Cellzome (Cellzome AG), PI3-delta/gamma inhibitors, Intellikine (Intellikine Inc.), PI3-delta/gamma inhibitors, Intellikine (Intellikine Inc.), PI3- delta/gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3-delta/gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3-gamma inhibitor Evotec (Evotec), PI3-gamma inhibitor, Cellzome (Cellzome AG), PI3-gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3K delta/gamma inhibitors, lntellikine-1 (Intellikine Inc.), PI3K delta/gamma inhibitors, lntellikine-1 (Intellikine Inc.), pictilisib (Roche Holdings Inc.), PIK-90 (CAS # 677338-12-4), SC-103980 (Pfizer, New York, NY), SF-1 126 (Semafore Pharmaceuticals, Indianapolis, IN), SH-5, SH-6, Tetrahydro Curcumin, TG100-1 15 (Targegen Inc., San Diego, CA), Triciribine, X-339 (Xcovery, West Palm Beach, FL), XL-499 (Evotech, Hamburg, Germany), pharmaceutically acceptable salts thereof, and combinations thereof.
51 . The method according to claim 34, wherein contacting the cancer cell with the first and second ant-cancer agents provides a synergistic effect compared to contacting the cancer cell with either anti-cancer agent alone.
52. A kit for treating or ameliorating the effects of a cancer in a subject in need thereof comprising an effective amount of (i) a first anti-cancer agent, which is an ERK 1/2 inhibitor or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof, packaged together with instructions for their use.
53. The kit according to clam 52, wherein the subject is a mammal.
54. The kit according to claim 53, wherein the mammal is selected from the group consisting of humans, primates, farm animals, and domestic animals.
55. The kit according to claim 53, wherein the mammal is a human.
56. The kit according to claim 52, wherein the ERK1/2 inhibitor is selected from the group consisting of AEZS-131 (Aeterna Zentaris), AEZS-136, BVD- 523 (BioMed Valley Discoveries, Inc.), SCH-722984 (Merck & Co.), SCH- 772984 (Merck & Co.), SCH-900353 (MK-8353) (Merck & Co.), pharmaceutically acceptable salts thereof, and combinations thereof.
57. The kit according to claim 52, wherein the ERK1/2 inhibitor is BVD-523 or a pharmaceutically acceptable salt thereof.
58. The kit according to claim 52, wherein the BCL-2 family inhibitor is selected from the group consisting of (-)-epigallocatechin gallate, ABT-737 (Abbott), antimycin A, apogossypolone, CAS # 383860-03-5, CAS # 810659- 53-1 , CAS #141266-44-6, BP-100-1 .02 (Bio-Path Holdings), chelerythrine chloride, HA14-1 (CAS # 65673-63-4), licochalcone-A (CAS #58749-22-7), navitoclax (Abbott), obatoclax (GX15-080), R-(-)-gossypol (Ascenta Therapeutics), S-44563 (Servier), sabutoclax (Oncothyreon), TW-37, VAL-101 (ValiRx), WEHI-539 (Hoffmann-La Roche, Inc.), pharmaceutically acceptable salts thereof, and combinations thereof.
59. The kit according to claim 52, wherein the BCL-2 family inhibitor is navitoclax or a pharmaceutically acceptable salt thereof.
60. The kit according to claim 52, wherein the ERK 1/2 inhibitor is BVD-523 or a pharmaceutically acceptable salt thereof, and the BCL-2 family inhibitor is navitoclax or a pharmaceutically acceptable salt thereof.
61 . The kit according to claim 52, wherein the subject with cancer has a somatic RAS mutation.
62. The method according to claim 52, wherein the cancer is selected from the group consisting of colorectal cancer, breast cancer, pancreatic cancer, lymphoma, lung cancer, testicular cancer, thyroid cancer, hematologic malignancies, and endometrial cancers.
63. The kit according to claim 62, wherein the hematologic malignancy is selected from the group consisting of RAS mutant myelodysplastic syndromes (MDS), lymphoid cancers, and myeloid cancers.
64. The kit according to claim 62, wherein the hematologic malignancy is selected from the group consisting of Adult Acute Megakaryoblastic Leukemia (M7), Adult Acute Minimally Differentiated Myeloid Leukemia (MO), Adult Acute Monoblastic Leukemia (M5a), Adult Acute Monocytic Leukemia (M5b), Adult Acute Myeloblastic Leukemia With Maturation (M2), Adult Acute Myeloblastic Leukemia Without Maturation (M1 ), Adult Acute Myeloid Leukemia With 1 1 q23 (MLL) Abnormalities, Adult Acute Myeloid Leukemia With Del(5q), Adult Acute Myeloid Leukemia With Inv(16)(p13;q22), Adult Acute Myeloid Leukemia With t(16;16)(p13;q22), Adult Acute Myeloid Leukemia With t(8;21 )(q22;q22), Adult Acute Myelomonocytic Leukemia (M4), Adult Erythroleukemia (M6a), Adult Pure Erythroid Leukemia (M6b), Recurrent Adult Acute Myeloid Leukemia, and Untreated Adult Acute Myeloid Leukemia.
65. The kit according to claim 52, wherein the cancer is colorectal cancer.
66. The kit according to claim 52 further comprising at least one additional therapeutic agent selected from the group consisting of an antibody or fragment thereof, a cytotoxic agent, a toxin, a radionuclide, an immunomodulator, a photoactive therapeutic agent, a radiosensitizing agent, a hormone, an anti-angiogenesis agent, and combinations thereof.
67. The kit according to claim 66, wherein the additional therapeutic agent is an inhibitor of the PI3K/Akt pathway.
68. The kit according to claim 67, wherein the inhibitor of the PI3K/Akt pathway is selected from the group consisting of A-674563 (CAS # 552325- 73-2), AGL 2263, AMG-319 (Amgen, Thousand Oaks, CA), AS-041 164 (5- benzo[1 ,3]dioxol-5-ylmethylene-thiazolidine-2,4-dione), AS-604850 (5-(2,2- Difluoro-benzo[1 ,3]dioxol-5-ylmethylene)-thiazolidine-2,4-dione), AS-605240 (5-quinoxilin-6-methylene-1 ,3-thiazolidine-2,4-dione), AT7867 (CAS # 857531 -00-1 ), benzimidazole series, Genentech (Roche Holdings Inc., South San Francisco, CA), BML-257 (CAS # 32387-96-5), CAL-120 (Gilead Sciences, Foster City, CA), CAL-129 (Gilead Sciences), CAL-130 (Gilead Sciences), CAL-253 (Gilead Sciences), CAL-263 (Gilead Sciences), CAS # 612847-09-3, CAS # 681281 -88-9, CAS # 75747-14-7, CAS # 925681 -41 -0, CAS # 98510-80-6, CCT128930 (CAS # 885499-61 -6), CH5132799 (CAS # 1007207-67-1 ), CHR-4432 (Chroma Therapeutics, Ltd., Abingdon, UK), FPA 124 (CAS # 902779-59-3), GS-1 101 (CAL-101 ) (Gilead Sciences), GSK 690693 (CAS # 937174-76-0), H-89 (CAS # 127243-85-0), Honokiol, IC871 14 (Gilead Science), IPI-145 (Intellikine Inc.), KAR-4139 (Karus Therapeutics, Chilworth, UK), KAR-4141 (Karus Therapeutics), KIN-1 (Karus Therapeutics), KT 5720 (CAS # 108068-98-0), Miltefosine, MK-2206 dihydrochloride (CAS # 1032350-13-2), ML-9 (CAS # 105637-50-1 ), Naltrindole Hydrochloride, OXY- 1 1 1 A (NormOxys Inc., Brighton, MA), perifosine, PHT-427 (CAS # 1 191951 - 57-1 ), PI3 kinase delta inhibitor, Merck KGaA (Merck & Co., Whitehouse Station, NJ), PI3 kinase delta inhibitors, Genentech (Roche Holdings Inc.), PI3 kinase delta inhibitors, Incozen (Incozen Therapeutics, Pvt. Ltd., Hyderabad, India), PI3 kinase delta inhibitors-2, Incozen (Incozen Therapeutics), PI3 kinase inhibitor, Roche-4 (Roche Holdings Inc.), PI3 kinase inhibitors, Roche (Roche Holdings Inc.), PI3 kinase inhibitors, Roche-5 (Roche Holdings Inc.), PI3-alpha/delta inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd., South San Francisco, CA), PI3-delta inhibitors, Cellzome (Cellzome AG, Heidelberg, Germany), PI3-delta inhibitors, Intellikine (Intellikine Inc., La Jolla, CA), PI3-delta inhibitors, Pathway Therapeutics-1 (Pathway Therapeutics Ltd.), PI3-delta inhibitors, Pathway Therapeutics-2 (Pathway Therapeutics Ltd.), PI3-delta/gamma inhibitors, Cellzome (Cellzome AG), PI3-delta/gamma inhibitors, Cellzome (Cellzome AG), PI3-delta/gamma inhibitors, Intellikine (Intellikine Inc.), PI3-delta/gamma inhibitors, Intellikine (Intellikine Inc.), PI3- delta/gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3-delta/gamnna inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3-gamnna inhibitor Evotec (Evotec), PI3-gamnna inhibitor, Cellzome (Cellzome AG), PI3-gamnna inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3K delta/gamma inhibitors, lntellikine-1 (Intellikine Inc.), PI3K delta/gamma inhibitors, lntellikine-1 (Intellikine Inc.), pictilisib (Roche Holdings Inc.), PIK-90 (CAS # 677338-12-4), SC-103980 (Pfizer, New York, NY), SF-1 126 (Semafore Pharmaceuticals, Indianapolis, IN), SH-5, SH-6, Tetrahydro Curcumin, TG100-1 15 (Targegen Inc., San Diego, CA), Triciribine, X-339 (Xcovery, West Palm Beach, FL), XL-499 (Evotech, Hamburg, Germany), pharmaceutically acceptable salts thereof, and combinations thereof.
69. The kit according to claim 52, wherein administration of the first and second anti-cancer agents provides a synergistic effect compared to administration of either anti-cancer agent alone.
70. A pharmaceutical composition for treating or ameliorating the effects of a cancer in a subject in need thereof, the pharmaceutical composition comprising a pharmaceutically acceptable diluent or carrier and an effective amount of (i) a first anti-cancer agent, which is an ERK 1/2 inhibitor or a pharmaceutically acceptable salt thereof and (ii) a second anti-cancer agent, which is a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof, wherein administration of the first and second anti-cancer agents provides a synergistic effect compared to administration of either anti-cancer agent alone.
71 . The pharmaceutical composition according to clam 70, wherein the subject is a mammal.
72. The pharmaceutical composition according to claim 71 , wherein the mammal is selected from the group consisting of humans, primates, farm animals, and domestic animals.
73. The pharmaceutical composition according to claim 71 , wherein the mammal is a human.
74. The pharmaceutical composition according to claim 70, wherein the ERK1/2 inhibitor is selected from the group consisting of AEZS-131 (Aeterna Zentaris), AEZS-136, BVD-523 (BioMed Valley Discoveries, Inc.), SCH- 722984 (Merck & Co.), SCH-772984 (Merck & Co.), SCH-900353 (MK-8353) (Merck & Co.), pharmaceutically acceptable salts thereof, and combinations thereof.
75. The pharmaceutical composition according to claim 70, wherein the ERK1/2 inhibitor is BVD-523 or a pharmaceutically acceptable salt thereof.
76. The pharmaceutical composition according to claim 70, wherein the BCL-2 family inhibitor is selected from the group consisting of (-)- epigallocatechin gallate, ABT-737 (Abbott), antimycin A, apogossypolone, CAS # 383860-03-5, CAS # 810659-53-1 , CAS #141266-44-6, BP-100-1 .02 (Bio-Path Holdings), chelerythrine chloride, HA14-1 (CAS # 65673-63-4), licochalcone-A (CAS #58749-22-7), navitoclax (Abbott), obatoclax (GX15- 080), R-(-)-gossypol (Ascenta Therapeutics), S-44563 (Servier), sabutoclax (Oncothyreon), TW-37, VAL-101 (ValiRx), WEHI-539 (Hoffmann-La Roche, Inc.), pharmaceutically acceptable salts thereof, and combinations thereof.
77. The pharmaceutical composition according to claim 70, wherein the BCL-2 family inhibitor is navitoclax or a pharmaceutically acceptable salt thereof.
78. The pharmaceutical composition according to claim 70, wherein the ERK 1/2 inhibitor is BVD-523 or a pharmaceutically acceptable salt thereof, and the BCL-2 family inhibitor is navitoclax or a pharmaceutically acceptable salt thereof.
79. The pharmaceutical composition according to claim 70, wherein the subject with cancer has a somatic RAS mutation.
80. The pharmaceutical composition according to claim 70, wherein the cancer is selected from the group consisting of colorectal cancer, breast cancer, pancreatic cancer, lymphoma, lung cancer, testicular cancer, thyroid cancer, hematologic malignancies, and endometrial cancers.
81 . The pharmaceutical composition according to claim 80, wherein the hematologic malignancy is selected from the group consisting of RAS mutant myelodysplastic syndromes (MDS), lymphoid cancers, and myeloid cancers.
82. The pharmaceutical composition according to claim 80, wherein the hematologic malignancy is selected from the group consisting of Adult Acute Megakaryoblastic Leukemia (M7), Adult Acute Minimally Differentiated Myeloid Leukemia (M0), Adult Acute Monoblastic Leukemia (M5a), Adult Acute Monocytic Leukemia (M5b), Adult Acute Myeloblasts Leukemia With Maturation (M2), Adult Acute Myeloblasts Leukemia Without Maturation (M1 ), Adult Acute Myeloid Leukemia With 1 1 q23 (MLL) Abnormalities, Adult Acute Myeloid Leukemia With Del(5q), Adult Acute Myeloid Leukemia With lnv(16)(p13;q22), Adult Acute Myeloid Leukemia With t(16;16)(p13;q22), Adult Acute Myeloid Leukemia With t(8;21 )(q22;q22), Adult Acute Myelomonocytic Leukemia (M4), Adult Erythroleukemia (M6a), Adult Pure Erythroid Leukemia (M6b), Recurrent Adult Acute Myeloid Leukemia, and Untreated Adult Acute Myeloid Leukemia.
83. The pharmaceutical composition according to claim 70, wherein the cancer is colorectal cancer.
84. The pharmaceutical composition according to claim 70 further comprising at least one additional therapeutic agent selected from the group consisting of an antibody or fragment thereof, a cytotoxic agent, a toxin, a radionuclide, an immunomodulator, a photoactive therapeutic agent, a radiosensitizing agent, a hormone, an anti-angiogenesis agent, and combinations thereof.
85. The pharmaceutical composition according to claim 84, wherein the additional therapeutic agent is an inhibitor of the PI3K/Akt pathway.
86. The pharmaceutical composition according to claim 85, wherein the inhibitor of the PI3K/Akt pathway is selected from the group consisting of A- 674563 (CAS # 552325-73-2), AGL 2263, AMG-319 (Amgen, Thousand Oaks, CA), AS-041 164 (5-benzo[1 ,3]dioxol-5-ylmethylene-thiazolidine-2,4- dione), AS-604850 (5-(2,2-Difluoro-benzo[1 ,3]dioxol-5-ylmethylene)- thiazolidine-2,4-dione), AS-605240 (5-quinoxilin-6-methylene-1 ,3-thiazolidine- 2,4-dione), AT7867 (CAS # 857531 -00-1 ), benzimidazole series, Genentech (Roche Holdings Inc., South San Francisco, CA), BML-257 (CAS # 32387-96- 5), CAL-120 (Gilead Sciences, Foster City, CA), CAL-129 (Gilead Sciences), CAL-130 (Gilead Sciences), CAL-253 (Gilead Sciences), CAL-263 (Gilead Sciences), CAS # 612847-09-3, CAS # 681281 -88-9, CAS # 75747-14-7, CAS # 925681 -41 -0, CAS # 98510-80-6, CCT128930 (CAS # 885499-61 -6), CH5132799 (CAS # 1007207-67-1 ), CHR-4432 (Chroma Therapeutics, Ltd., Abingdon, UK), FPA 124 (CAS # 902779-59-3), GS-1 101 (CAL-101 ) (Gilead Sciences), GSK 690693 (CAS # 937174-76-0), H-89 (CAS # 127243-85-0), Honokiol, IC871 14 (Gilead Science), IPI-145 (Intellikine Inc.), KAR-4139 (Karus Therapeutics, Chilworth, UK), KAR-4141 (Karus Therapeutics), KIN-1 (Karus Therapeutics), KT 5720 (CAS # 108068-98-0), Miltefosine, MK-2206 dihydrochloride (CAS # 1032350-13-2), ML-9 (CAS # 105637-50-1 ), Naltrindole Hydrochloride, OXY-1 1 1A (NormOxys Inc., Brighton, MA), perifosine, PHT-427 (CAS # 1 191951 -57-1 ), PI3 kinase delta inhibitor, Merck KGaA (Merck & Co., Whitehouse Station, NJ), PI3 kinase delta inhibitors, Genentech (Roche Holdings Inc.), PI3 kinase delta inhibitors, Incozen (Incozen Therapeutics, Pvt. Ltd., Hyderabad, India), PI3 kinase delta inhibitors-2, Incozen (Incozen Therapeutics), PI3 kinase inhibitor, Roche-4 (Roche Holdings Inc.), PI3 kinase inhibitors, Roche (Roche Holdings Inc.), PI3 kinase inhibitors, Roche-5 (Roche Holdings Inc.), PI3-alpha/delta inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd., South San Francisco, CA), PI3-delta inhibitors, Cellzome (Cellzome AG, Heidelberg, Germany), PI3- delta inhibitors, Intellikine (Intellikine Inc., La Jolla, CA), PI3-delta inhibitors, Pathway Therapeutics-1 (Pathway Therapeutics Ltd.), PI3-delta inhibitors, Pathway Therapeutics-2 (Pathway Therapeutics Ltd.), PI3-delta/gamnna inhibitors, Cellzome (Cellzome AG), PI3-delta/gamnna inhibitors, Cellzome (Cellzome AG), PI3-delta/gamnna inhibitors, Intellikine (Intellikine Inc.), PI3- delta/gamma inhibitors, Intellikine (Intellikine Inc.), PI3-delta/gamnna inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3-delta/gamnna inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3-gamnna inhibitor Evotec (Evotec), PI3-gamnna inhibitor, Cellzome (Cellzome AG), PI3- gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3K delta/gamma inhibitors, lntellikine-1 (Intellikine Inc.), PI3K delta/gamma inhibitors, lntellikine-1 (Intellikine Inc.), pictilisib (Roche Holdings Inc.), PIK-90 (CAS # 677338-12-4), SC-103980 (Pfizer, New York, NY), SF-1 126 (Semafore Pharmaceuticals, Indianapolis, IN), SH-5, SH-6, Tetrahydro Curcumin, TG100-1 15 (Targegen Inc., San Diego, CA), Triciribine, X-339 (Xcovery, West Palm Beach, FL), XL-499 (Evotech, Hamburg, Germany), pharmaceutically acceptable salts thereof, and combinations thereof.
87. The pharmaceutical composition according to claim 70, which is in a unit dosage form comprising both anti-cancer agents.
88. The pharmaceutical composition according to claim 70 in which the first anti-cancer agent is in a first unit dosage form and the second anti-cancer agent is in a second unit dosage form, separate from the first.
89. The pharmaceutical composition according to claim 70, wherein the first and second anti-cancer agents are co-administered to the subject.
90. The pharmaceutical composition according to claim 70, wherein the first and second anti-cancer agents are administered to the subject serially.
91 . The pharmaceutical composition according to claim 90, wherein the first anti-cancer agent is administered to the subject before the second anticancer agent.
92. The pharmaceutical composition according to claim 90, wherein the second anti-cancer agent is administered to the subject before the first anticancer agent.
93. A method for selecting a subject with cancer that may benefit from a combination drug therapy comprising:
(a) selecting a subject with a somatic K-RAS mutation;
(b) determining whether the subject with a K-RAS mutation has an epithelial-to-mesenchymal transition gene signature, wherein subjects having a K-RAS mutation and an epithelial-to-mesenchymal transition gene signature are likely to benefit from the combination drug therapy; and
(c) administering to the subject having a K-RAS mutation and an epithelial-to-mesenchymal transition gene signature the combination drug therapy which comprises an effective amount of (i) an ERK1/2 inhibitor or a pharmaceutically acceptable salt thereof and (ii) a BCL-2 family inhibitor or a pharmaceutically acceptable salt thereof.
94. The method according to clam 93, wherein the subject is a mammal.
95. The method according to claim 94, wherein the mammal is selected from the group consisting of humans, primates, farm animals, and domestic animals.
96. The method according to claim 94, wherein the mammal is a human.
97. The method according to claim 93, wherein the ERK1/2 inhibitor is selected from the group consisting of AEZS-131 (Aeterna Zentaris), AEZS- 136, BVD-523 (BioMed Valley Discoveries, Inc.), SCH-722984 (Merck & Co.), SCH-772984 (Merck & Co.), SCH-900353 (MK-8353) (Merck & Co.), pharmaceutically acceptable salts thereof, and combinations thereof.
98. The method according to claim 93, wherein the ERK1/2 inhibitor is BVD-523 or a pharmaceutically acceptable salt thereof.
99. The method according to claim 93, wherein the BCL-2 family inhibitor is selected from the group consisting of (-)-epigallocatechin gallate, ABT-737 (Abbott), antimycin A, apogossypolone, CAS # 383860-03-5, CAS # 810659- 53-1 , CAS #141266-44-6, BP-100-1 .02 (Bio-Path Holdings), chelerythrine chloride, HA14-1 (CAS # 65673-63-4), licochalcone-A (CAS #58749-22-7), navitoclax (Abbott), obatoclax (GX15-080), R-(-)-gossypol (Ascenta Therapeutics), S-44563 (Servier), sabutoclax (Oncothyreon), TW-37, VAL-101 (ValiRx), WEHI-539 (Hoffmann-La Roche, Inc.), pharmaceutically acceptable salts thereof, and combinations thereof.
100. The method according to claim 93, wherein the BCL-2 family inhibitor is navitoclax or a pharmaceutically acceptable salt thereof.
101 . The method according to claim 93, wherein the ERK 1/2 inhibitor is BVD-523 or a pharmaceutically acceptable salt thereof and the BCL-2 family inhibitor is navitoclax or a pharmaceutically acceptable salt thereof.
102. The method according to claim 93, wherein the cancer is selected from the group consisting of colorectal cancer, breast cancer, pancreatic cancer, lymphoma, lung cancer, testicular cancer, thyroid cancer, hematologic malignancies, and endometrial cancers.
103. The method according to claim 102, wherein the hematologic malignancy is selected from the group consisting of RAS mutant myelodysplastic syndromes (MDS), lymphoid cancers, and myeloid cancers.
104. The method according to claim 102, wherein the hematologic malignancy is selected from the group consisting of Adult Acute Megakaryoblastic Leukemia (M7), Adult Acute Minimally Differentiated Myeloid Leukemia (M0), Adult Acute Monoblastic Leukemia (M5a), Adult Acute Monocytic Leukemia (M5b), Adult Acute Myeloblasts Leukemia With Maturation (M2), Adult Acute Myeloblasts Leukemia Without Maturation (M1 ), Adult Acute Myeloid Leukemia With 1 1 q23 (MLL) Abnormalities, Adult Acute Myeloid Leukemia With Del(5q), Adult Acute Myeloid Leukemia With lnv(16)(p13;q22), Adult Acute Myeloid Leukemia With t(16;16)(p13;q22), Adult Acute Myeloid Leukemia With t(8;21 )(q22;q22), Adult Acute Myelomonocytic Leukemia (M4), Adult Erythroleukemia (M6a), Adult Pure Erythroid Leukemia (M6b), Recurrent Adult Acute Myeloid Leukemia, and Untreated Adult Acute Myeloid Leukemia.
105. The method according to claim 93, wherein the cancer is colorectal cancer.
106. The method according to claim 93 further comprising administering at least one additional therapeutic agent selected from the group consisting of an antibody or fragment thereof, a cytotoxic agent, a toxin, a radionuclide, an immunomodulator, a photoactive therapeutic agent, a radiosensitizing agent, a hormone, and combinations thereof.
107. The method according to claim 106, wherein the additional therapeutic agent is an inhibitor of the PI3K/Akt pathway.
108. The method according to claim 107, wherein the inhibitor of the PI3K/Akt pathway is selected from the group consisting of - A-674563 (CAS # 552325-73-2), AGL 2263, AMG-319 (Amgen, Thousand Oaks, CA), AS- 041 164 (5-benzo[1 ,3]dioxol-5-ylmethylene-thiazolidine-2,4-dione), AS-604850 (5-(2,2-Difluoro-benzo[1 ,3]dioxol-5-ylmethylene)-thiazolidine-2,4-dione), AS- 605240 (5-quinoxilin-6-methylene-1 ,3-thiazolidine-2,4-dione), AT7867 (CAS # 857531 -00-1 ), benzimidazole series, Genentech (Roche Holdings Inc., South San Francisco, CA), BML-257 (CAS # 32387-96-5), CAL-120 (Gilead Sciences, Foster City, CA), CAL-129 (Gilead Sciences), CAL-130 (Gilead Sciences), CAL-253 (Gilead Sciences), CAL-263 (Gilead Sciences), CAS # 612847-09-3, CAS # 681281 -88-9, CAS # 75747-14-7, CAS # 925681 -41 -0, CAS # 98510-80-6, CCT128930 (CAS # 885499-61 -6), CH5132799 (CAS # 1007207-67-1 ), CHR-4432 (Chroma Therapeutics, Ltd., Abingdon, UK), FPA 124 (CAS # 902779-59-3), GS-1 101 (CAL-101 ) (Gilead Sciences), GSK 690693 (CAS # 937174-76-0), H-89 (CAS # 127243-85-0), Honokiol, IC871 14 (Gilead Science), IPI-145 (Intellikine Inc.), KAR-4139 (Karus Therapeutics, Chilworth, UK), KAR-4141 (Karus Therapeutics), KIN-1 (Karus Therapeutics), KT 5720 (CAS # 108068-98-0), Miltefosine, MK-2206 dihydrochloride (CAS # 1032350-13-2), ML-9 (CAS # 105637-50-1 ), Naltrindole Hydrochloride, OXY- 1 1 1 A (NormOxys Inc., Brighton, MA), perifosine, PHT-427 (CAS # 1 191951 - 57-1 ), PI3 kinase delta inhibitor, Merck KGaA (Merck & Co., Whitehouse Station, NJ), PI3 kinase delta inhibitors, Genentech (Roche Holdings Inc.), PI3 kinase delta inhibitors, Incozen (Incozen Therapeutics, Pvt. Ltd., Hyderabad, India), PI3 kinase delta inhibitors-2, Incozen (Incozen Therapeutics), PI3 kinase inhibitor, Roche-4 (Roche Holdings Inc.), PI3 kinase inhibitors, Roche (Roche Holdings Inc.), PI3 kinase inhibitors, Roche-5 (Roche Holdings Inc.), PI3-alpha/delta inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd., South San Francisco, CA), PI3-delta inhibitors, Cellzome (Cellzome AG, Heidelberg, Germany), PI3-delta inhibitors, Intellikine (Intellikine Inc., La Jolla, CA), PI3-delta inhibitors, Pathway Therapeutics-1 (Pathway Therapeutics Ltd.), PI3-delta inhibitors, Pathway Therapeutics-2 (Pathway Therapeutics Ltd.), PI3-delta/gamnna inhibitors, Cellzome (Cellzome AG), PI3-delta/gamma inhibitors, Cellzome (Cellzome AG), PI3-delta/gamma inhibitors, Intellikine (Intellikine Inc.), PI3-delta/gamma inhibitors, Intellikine (Intellikine Inc.), PI3- delta/gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3-delta/gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3-gamma inhibitor Evotec (Evotec), PI3-gamma inhibitor, Cellzome (Cellzome AG), PI3-gamma inhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3K delta/gamma inhibitors, lntellikine-1 (Intellikine Inc.), PI3K delta/gamma inhibitors, lntellikine-1 (Intellikine Inc.), pictilisib (Roche Holdings Inc.), PIK-90 (CAS # 677338-12-4), SC-103980 (Pfizer, New York, NY), SF-1 126 (Semafore Pharmaceuticals, Indianapolis, IN), SH-5, SH-6, Tetrahydro Curcumin, TG100-1 15 (Targegen Inc., San Diego, CA), Triciribine, X-339 (Xcovery, West Palm Beach, FL), XL-499 (Evotech, Hamburg, Germany), pharmaceutically acceptable thereof, and combinations thereof.
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