WO2017117386A1

WO2017117386A1 - Methods of treating cancer using network brakes

Info

Publication number: WO2017117386A1
Application number: PCT/US2016/069198
Authority: WO
Inventors: Ross CAGAN; Tirtha Kamal DAS
Original assignee: Icahn School Of Medicine At Mount Sinai
Priority date: 2015-12-30
Filing date: 2016-12-29
Publication date: 2017-07-06
Also published as: WO2017117386A8

Abstract

The invention relates to improved methods of treating a cancer in a patient in need thereof using combination drug regimens that can improve therapeutic efficacy of kinase inhibitor(s). Such combination drug regimens may also reduce side effects of kinase inhibitor(s) and/or prevent or reduce cancer resistance to the kinase inhibitor(s).

Description

METHODS OF TREATING CANCER USING NETWORK BRAKES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of provisional application No. 62/273,089, filed on December 30, 2015, which is incorporated by reference herein in its entirety.

GOVERNMENT RIGHTS STATEMENT

[0002] This invention was made with government support under grant numbers R01- CA109730, R01-CA170495, and R01-CA084309 awarded by the National Institutes of Health. The Government has certain rights in the invention.

1. FIELD

[0003] The invention relates to improved methods of treating a cancer in a patient in need thereof using combination drug regimens that can improve therapeutic efficacy of kinase inhibitor(s). Such combination drug regimens may also reduce side effects of kinase inhibitor(s) and/or prevent or reduce cancer resistance to the kinase inhibitor(s).

2. BACKGROUND

[0004] Genetic analysis of cancer has led to the identification of certain mutations thought to be the drivers responsible for cancer development and/or progression, and many of such mutations are in genes encoding kinases or phosphatases, which lead to aberrant activation of certain signaling pathways.

[0005] Therefore, inactivating such signaling pathways by inhibiting the kinases involved is one of the best ways to treat cancer. Indeed, target-driven drug discovery has led to the development of a number of kinase inhibitors for the treatment of cancer.

[0006] However, many kinase inhibitors do not work efficiently, despite the fact that the patients being treated have aberrant activation of the corresponding kinases. Or, even when a kinase inhibitor works initially, the patient's cancer will often develop resistance to the kinase inhibitor. Therefore, there is a need for therapies that improve the therapeutic efficacy of kinase inhibitors and preferably also prevent or correct resistance to them. 3. SUMMARY OF THE INVENTION

[0007] The invention relates to improved methods of treating a cancer in a patient in need thereof using combination drug regimens that can improve therapeutic efficacy of kinase inhibitor(s).

[0008] The invention is based, in part, on the discovery that treatments with kinase inhibitors at clinical doses (i.e., doses commonly used in the standard-of-care therapy for the cancer to be treated) often surprisingly result in: (i) hyper-activation of kinases, which can be visualized by testing for the kinase activity of a set of specific kinases, which functionally counteracts the ability of the kinase inhibitor to inhibit cancer development and/or progression, thereby reducing the therapeutic efficacy of the kinase inhibitor; and (ii) activation of cancer stem cells, which promote cancer growth and metastasis. Thus, the use of many kinase inhibitors at clinical doses to treat a patient's cancer increases the resistance of the cancer cells to the kinase inhibitor and actually promotes cancer development and/or progression.

[0009] The invention is also based, in part, on the discovery that the administration of (1) a combination of a proteasome inhibitor at a subtherapeutic dose, an HDAC (histone deacetylase) inhibitor at a subtherapeutic dose and a kinase inhibitor, (2) a combination of a proteasome inhibitor at a subtherapeutic dose, an HDAC-PI3K dual inhibitor at a subtherapeutic dose and a kinase inhibitor, (3)a combination of an HDAC inhibitor at a subtherapeutic dose, an Hsp90 inhibitor at a subtherapeutic dose and a kinase inhibitor, and (4) a proteasome inhibitor at a subtherapeutic dose, an inhibitor of SPl -class transcriptions factors at a subtherapeutic dose, and a kinase inhibitor, can all restrain the signaling network activities of cancer cells (as assessed by, e.g., the kinase activity of a specific set of kinases), and prevent or reduce the development of resistance to treatment with the kinase inhibitor and/or prevent or reduce the activation of cancer stem cells. See Example 1 (Section 6), infra. Therefore, the administration of such a

combination with a kinase inhibitor: (1) results in therapeutic efficacy at lower doses of the kinase inhibitor than may otherwise be at the standard-of-care or clinically acceptable doses; and/or (2) permits higher doses of the kinase inhibitor than the standard-of-care or clinically acceptable doses to be used in the treatment of cancer without the toxicity and/or side effects {e.g., adverse side effects) associated with such higher doses.

[0010] In one aspect, provided herein is a method for treating a human patient diagnosed with cancer, comprising administering to a human patient in need thereof: (a) a Network Brake at a subtherapeutic dose; and (b) a kinase inhibitor at an effective dose, so that overall or progression-free survival of the patient is increased, wherein the Network Brake is a compound or a combination of compounds that reduces the hyper-activation of the cancer signaling network induced by the kinase inhibitor, and prevents the upregulation of one or more cancer stem cell markers induced by the kinase inhibitor. In a specific embodiment, the one or more cancer stem cell markers is Sox2, Oct4, Nanog, LIN28, c-Myc, or KLF4. In another specific embodiment, the one or more cancer stem cell markers is a combination of two or more of the following: Sox2, Oct4, Nanog, LIN28, c-Myc, or KLF4. In specific embodiments, overall or progression- free survival of the patient is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more relative to the average overall or progression-free survival of patients with the same type of cancer or patients with the same type of cancer in a similar position healthwise. In certain embodiments, the patients with the same type of cancer or patients with the same type of cancer in a similar position healthwise have received an accepted standard of care therapy for the cancer. In certain embodiments, the patients with the same type of cancer or patients with the same type of cancer in a similar position healthwise have not been treated with the kinase inhibitor or a combination of the kinase inhibitor and the Network Brake. In specific embodiments, overall or progression-free survival of the patient is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more relative to the average overall or progression-free survival of patients with the same type of cancer or patients with the same type of cancer in a similar position healthwise, who are administered with the kinase inhibitor at the effective dose but are not administered with the Network Brake.

[0011] In another aspect, provided herein is a method for treating a human patient diagnosed with cancer, comprising administering to a human patient in need thereof: (a) a Network Brake at a subtherapeutic dose; and (b) a kinase inhibitor at a dose equal to or greater than the kinase inhibitor's maximum tolerated dose as assessed in the absence of the Network Brake, so that overall or progression-free survival of the patient is increased, wherein the Network Brake is a compound or a combination of compounds that reduces the hyper-activation of the cancer signaling network induced by the kinase inhibitor, and prevents the upregulation of one or more cancer stem cell markers induced by the kinase inhibitor. In a specific embodiment, the one or more cancer stem cell markers is Sox2, Oct4, Nanog, LIN28, c-Myc, or KLF4. In another specific embodiment, the one or more cancer stem cell markers is a combination of two or more of the following: Sox2, Oct4, Nanog, LIN28, c-Myc, or KLF4. In specific embodiments, overall or progression-free survival of the patient is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more relative to the average overall or progression-free survival of patients with the same type of cancer or patients with the same type of cancer in a similar position healthwise. In certain embodiments, the patients with the same type of cancer or patients with the same type of cancer in a similar position healthwise have received an accepted standard of care therapy for the cancer. In certain embodiments, the patients with the same type of cancer or patients with the same type of cancer in a similar position healthwise have not been treated with the kinase inhibitor or a combination of the kinase inhibitor and the Network Brake. In specific embodiments, overall or progression- free survival of the patient is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more relative to the average overall or progression-free survival of patients with the same type of cancer or patients with the same type of cancer in a similar position healthwise, who are administered with the kinase inhibitor at the dose equal to or greater than the kinase inhibitor's maximum tolerated dose but not are administered with the Network Brake.

[0012] In another aspect, provided herein is a method for treating a human patient diagnosed with cancer, comprising administering to a human patient in need thereof: (a) a Network Brake at a subtherapeutic dose; and (b) a kinase inhibitor at a subclinical dose, so that overall or progression-free survival of the patient is increased, wherein the Network Brake is a compound or a combination of compounds that reduces the hyper-activation of the cancer signaling network induced by the kinase inhibitor, and prevents the upregulation of one or more cancer stem cell markers induced by the kinase inhibitor. In a specific embodiment, the one or more cancer stem cell markers is Sox2, Oct4, Nanog, LIN28, c-Myc, or KLF4. In another specific embodiment, the one or more cancer stem cell markers is a combination of two or more of the following: Sox2, Oct4, Nanog, LIN28, c-Myc, or KLF4. In specific embodiments, overall or progression- free survival of the patient is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more relative to the average overall or progression-free survival of patients with the same type of cancer or patients with the same type of cancer in a similar position healthwise. In certain embodiments, the patients with the same type of cancer or patients with the same type of cancer in a similar position healthwise have received an accepted standard of care therapy for the cancer. In certain embodiments, the patients with the same type of cancer or patients with the same type of cancer in a similar position healthwise have not been treated with the kinase inhibitor or a combination of the kinase inhibitor and the Network Brake. In specific embodiments, overall or progression-free survival of the patient is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more relative to the average overall or progression-free survival of patients with the same type of cancer or patients with the same type of cancer in a similar position healthwise, who are administered with the kinase inhibitor at the subclinical dose but are not administered with the Network Brake.

[0013] In a specific embodiment of any of the preceding aspects, a reduction in the hyper- activation of the cancer signaling network induced by the kinase inhibitor is assessed by an assay comprising: (1) culturing a first population of cancer cells in the presence of the kinase inhibitor at a specific concentration equivalent to the clinical dose for a period of time; (2) culturing a second population of cancer cells of the same type in the presence of the compound or the combination of compounds at a concentration(s) equivalent to the subtherapeutic dose(s), and the kinase inhibitor at the same specific concentration for said period of time; and (3) analyzing the level of activity of a certain set of kinases, which are members of the cancer signaling network, in the first and second populations of cancer cells at the end of said period of time, wherein a decrease in the overall, median, or mean activity level of the kinases in the second population of cancer cells relative to the overall, median or mean activity of the same kinases in the first population of cancer cells indicates that the compound or the combination of compounds reduces the hyper-activation of the cancer signaling network induced by the kinase inhibitor. In another specific embodiment of any of the preceding aspects, a reduction in the hyper-activation of the cancer signaling network induced by the kinase inhibitor is assessed by an assay comprising: (1) culturing a first population of cancer cells in the presence of the kinase inhibitor at a specific concentration equivalent to the clinical dose for a period of time; (2) culturing a second population of cancer cells of the same type in the presence of the compound or the combination of compounds at a concentration(s) equivalent to the subtherapeutic dose(s), and the kinase inhibitor at the same specific concentration for said period of time; and (3) analyzing the level of phosphorylation of a certain set of proteins, which are members of the cancer signaling network, in the first and second populations of cancer cells at the end of said period of time, wherein a decrease in the overall, median, or mean level of phosphorylation in the second population of cancer cells relative to the overall, median or mean level of phosphorylation in the first population of cancer cells indicates that the compound or the combination of compounds reduces the hyper-activation of the cancer signaling network induced by the kinase inhibitor.

[0014] In a specific embodiment in accordance with the methods described herein, the prevention of the upregulation of the one or more cancer stem cell markers induced by the kinase inhibitor is assessed by an assay comprising: (1) culturing a first population of cancer cells in the presence of the kinase inhibitor at a specific concentration equivalent to the clinical dosefor a period of time; (2) culturing a second population of cancer cells of the same type in the presence of the compound or the combination of compounds at a concentration(s) equivalent to the subtherapeutic dose(s), and the kinase inhibitor at the same specific concentration for said period of time; (3) culturing a third population of cancer cells of the same type without any treatment; and (4) analyzing the expression level of the one or more cancer stem cell markers in the first, second, and third populations of cancer cells at the end of said period of time, wherein the overall, median, or mean expression level of the one or more cancer stem cell markers in the first population of cancer cells is higher than the overall, median, or mean expression level of the same cancer stem cell marker(s) in the third population of cancer cells, and wherein a decrease in the overall, median, or mean expression level of the one or more cancer stem cell markers in the second population of cancer cells relative to the overall, median or mean expression level of the same cancer stem cell marker(s) in the first population of cancer cells indicates that the compound or the combination of compounds prevents the upregulation of the one or more cancer stem cell markers induced by the kinase inhibitor.

[0015] In a specific embodiment in accordance with the methods described herein, the cancer is a solid tumor cancer.

[0016] In a specific embodiment in accordance with the methods described herein, the Network Brake is a proteasome inhibitor. In a further specific embodiment, the proteasome inhibitor is bortezomib.

[0017] In another specific embodiment in accordance with the methods described herein, the Network Brake is a histone deacetylase inhibitor. In a further specific embodiment, the histone deacetylase inhibitor is vorinostat, belinostat, entinostat, panobinostat, or RG2833. [0018] In another specific embodiment in accordance with the methods described herein, the Network Brake is a combination of a proteasome inhibitor and a histone deacetylase inhibitor. In a further specific embodiment, the proteasome inhibitor is Bortezomib. In another further specific embodiment, the histone deacetylase inhibitor is vorinostat, belinostat, entinostat, panobinostat, or RG2833. In another further specific embodiment, the proteasome inhibitor is bortezomib and the histone deacetylase inhibitor is vorinostat. In another further specific embodiment, the proteasome inhibitor is bortezomib and the histone deacetylase inhibitor is belinostat. In another further specific embodiment, the proteasome inhibitor is bortezomib and the histone deacetylase inhibitor is entinostat. In another further specific embodiment, the proteasome inhibitor is bortezomib and the histone deacetylase inhibitor is panobinostat.

[0019] In another specific embodiment in accordance with the methods described herein, the Network Brake is a proteasome inhibitor and an inhibitor of SPl -class transcription factors. In a further specific embodiment, the proteasome inhibitor is bortezomib and the inhibitor of SP1- class transcription factors is mithramycin.

[0020] In another specific embodiment in accordance with the methods described herein, the Network Brake is a histone deacetylase inhibitor (HDAC) and an Hsp90 inhibitor. In a further specific embodiment, the histone deacetylase inhibitor is vorinostat and the Hsp90 inhibitor is AUY922.

[0021] In another specific embodiment in accordance with the methods described herein, the Network Brake is a proteasome inhibitor and an HDAC-PI3K inhibitor. In a further specific embodiment, the proteasome inhibitor is bortezomib and the HDAC-PI3K inhibitor is CUDC- 907.

[0022] In a specific embodiment in accordance with the methods described herein, the kinase inhibitor is afatinib, aflibercept, axitinib, bevacizumab, BEZ235, bosutinib, cabozantinib, cetuximab, crizotinib, dasatinib, erlotinib, everolimus, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, SU6656, sunitinib, tofacitinib, trametinib, trastuzumab, vandetanib, vemurafenib, or vismodegib. In another specific embodiment in accordance with the methods described herein, the kinase inhibitor is sorafenib. In another specific embodiment in accordance with the methods described herein, the kinase inhibitor is trametinib or erlotinib. [0023] In a specific embodiment in accordance with the methods described herein, the Network Brake is administered to the patient concurrently with the kinase inhibitor. In another specific embodiment in accordance with the methods described herein, the Network Brake is administered to the patient prior to the administration of the kinase inhibitor.

[0024] In another aspect, provided herein is a method for treating thyroid cancer to a human patient in need thereof, comprising: (a) administering to the patient bortezomib at a

subtherapeutic dose and vorinostat at a subtherapeutic dose; and (b) administering to the patient sorafenib at an effective dose. In a specific embodiment, the bortezomib and vorniostat are administered to the patient prior to the administration of the sorafenib to the patient. In another specific embodiment, the bortezomib and vorniostat are administered to the patient concurrently with the administration of the sorafenib to the patient. In certain embodiments, the thyroid cancer is associated with a RET mutation.

[0025] In another aspect, provided herein is a method for treating lung cancer to a patient in need thereof, comprising: (a) administering to the patient bortezomib at a subtherapeutic dose and vorinostat at a subtherapeutic dose; and (b) administering to the patient trametinib at an effective dose. In a specific embodiment, the bortezomib and vorniostat are administered to the patient prior to the administration of the trametinib to the patient. In another specific embodiment, the bortezomib and vorniostat are administered to the patient concurrently with the administration of the trametinib to the patient. In certain embodiments, the lung cancer is associated with a Ras mutation. In certain embodiments, the lung cancer is a non-small cell lung cancer.

[0026] In another aspect, provided herein is a method for treating liver cancer to a patient in need thereof, comprising: (a) administering to the patient bortezomib at a subtherapeutic dose and vorinostat at a subtherapeutic dose; and (b) administering to the patient trametinib at an effective dose. In a specific embodiment, the bortezomib and vorniostat are administered to the patient prior to the administration of the trametinib to the patient. In another specific embodiment, the bortezomib and vorniostat are administered to the patient concurrently with the administration of the trametinib to the patient. In certain embodiments, the liver cancer is a heptocellular carcinoma.

[0027] In another aspect, provided herein is a method for treating lung cancer to a patient in need thereof, comprising: (a) administering to the patient bortezomib at a subtherapeutic dose and vorinostat at a subtherapeutic dose; and (b) administering to the patient erlotinib at an effective dose. In a specific embodiment, the bortezomib and vorniostat are administered to the patient prior to the administration of the erlotinib to the patient. In another specific embodiment, the bortezomib and vorniostat are administered to the patient concurrently with the

administration of the erlotinib to the patient. In certain embodiments, the lung cancer is associated with a Ras mutation. In certain embodiments, the lung cancer is ErbB3 positive. In certain embodiments, the lung cancer is a non-small cell lung cancer.

[0028] In another aspect, provided herein is a method for treating breast cancer to a patient in need thereof, comprising: (a) administering to the patient bortezomib at a subtherapeutic dose and vorinostat at a subtherapeutic dose; and (b) administering to the patient BEZ235 at an effective dose. In a specific embodiment, the bortezomib and vorniostat are administered to the patient prior to the administration of the BEZ235 to the patient. In another specific embodiment, the bortezomib and vorniostat are administered to the patient concurrently with the

administration of the BEZ235 to the patient. In certain embodiments, the breast cancer is associated with a PI3K mutation. In certain embodiments, the breast cancer is estrogen receptor positive (ER⁺).

[0029] In another aspect, provided herein is a method for treating melanoma to a patient in need thereof, comprising: (a) administering to the patient bortezomib at a subtherapeutic dose and vorinostat at a subtherapeutic dose; and (b) administering to the patient vemurafenib at an effective dose. In a specific embodiment, the bortezomib and vorniostat are administered to the patient prior to the administration of the vemurafenib to the patient. In another specific embodiment, the bortezomib and vorniostat are administered to the patient concurrently with the administration of the vemeurafinib to the patient. In certain embodiments, the melanoma is associated with a Raf mutation.

[0030] In another aspect, provided herein is a method for screening compounds to select those having Network Brake activity, comprising: (a) culturing a first population of cancer cells in the presence of a kinase inhibitor at a specific concentration for a period of time; (b) culturing a second population of cancer cells of the same type in the presence of a test compound or a combination of test compounds at a test concentration(s), and the kinase inhibitor at the same specific concentration for said period of time, wherein the test concentration(s) is equivalent to a subtherapeutic dose; and (c) analyzing the level of activity of a certain set of kinases in the first and second populations of cancer cells at the end of said period of time, wherein a decrease in the overall, median, or mean activity of the kinases in the second population of cancer cells relative to the overall, median or mean activity of the same kinases in the first population of cancer cells indicates that the test compound or the combination of test compounds has Network Brake activity.

[0031] In another aspect, provided herein is a method for screening compounds to select those having Network Brake activity, comprising: (a) culturing a first population of cancer cells in the presence of a kinase inhibitor at a specific concentration for a period of time; (b) culturing a second population of cancer cells of the same type in the presence of a test compound or a combination of test compounds at a test concentration(s), and the kinase inhibitor at the same specific concentration for said period of time, wherein the test concentration (s) is equivalent to a subtherapeutic dose(s); and (c) analyzing the level of phosphorylation of a set of proteins, which are indicative of the kinase activity of a set of kinases, in the first and second populations of cancer cells at the end of said period of time, wherein a decrease in the overall, median, or mean phosphorylation level of the set of proteins in the second population of cancer cells relative to the overall, median or mean phosphorylation level of the same set of proteins in the first population of cancer cells indicates that the test compound or the combination of test compounds has Network Brake activity.

3.1. Definitions

[0032] As used herein, the term "cancer signaling network" refers to the signaling network in cancer cells, which can be assessed by the kinase activity of a specific set of kinases. In a specific embodiment, the cancer signaling network is assessed by evaluating the activity of all or a subset of the following kinases: EGFR, HER2, HER3, FGFR1, FGFR3, FGFR4, InsR, IGF-IR, TrkA, TrkB, Met, Ron, Ret, ALK, PDGFR, c-Kit, FLT3, M-CSFR, EphAl, EphA2, EphA3, EphB l, EphB3, EphB4, Tyro3, Axl, Tie2, VEGFR2, Akt (e.g., evaluating the phosphorylation at Thr308 and/or Ser473), ERKl, ERK2, S6, c-Abl, IRS-1 , Zap-70, Src, Lck, Statl, and Stat3. In some embodiments, the subset of kinases includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15 or more of the following kinases: EGFR, HER2, HER3, FGFR1, FGFR3, FGFR4, InsR, IGF-IR, TrkA, TrkB, Met, Ron, Ret, ALK, PDGFR, c-Kit, FLT3, M-CSFR, EphAl, EphA2, EphA3, EphB l, EphB3, EphB4, Tyro3, Axl, Tie2, VEGFR2, Akt (e.g., evaluating the phosphorylation at Thr308 and/or Ser473), ERKl, ERK2, S6, c-Abl, IRS-1, Zap-70, Src, Lck, Statl, and Stat3 [0033] As used herein, the term "Network Brake" refers to a compound or a combination of compounds, wherein (1) the compound or the combination of compounds at a subtherapeutic dose(s) or subtherapeutic dose range(s) is able to improve the efficacy of a kinase inhibitor when used in combination with the kinase inhibitor at a specific dose or dose range (see, e.g., Section 5.3.1 and Section 6 for methods for assessing the therapeutic efficacy of the test compound or the combination of test compounds at a subtherapeutic dose(s) or subtherapeutic dose range(s) and the kinase inhibitor at a specific dose or dose range for treating cancer); and, in specific embodiments, (2) the compound or the combination of compounds at a subtherapeutic dose or subtherapeutic range when used in combination with a kinase inhibitor at a specific dose or dose range prevents the emergence of cancer cells refractory/resistant to such treatment (see, e.g., Section 5.3.7 and Section 6 for methods for assessing the development of cancer cells refractory to treat with the test compound or combination of test compounds at the subtherapeutic dose(s) or subtherapeutic dose range(s) and the kinase inhibitor at the specific dose or dose range). In a specific embodiment, a compound or a combination of compounds at a subtherapeutic dose is identified as a Network Brake if it reduces the hyper-activation of the cancer signaling network induced by a kinase inhibitor. A Phosphoprofile Assay can be used to determine if a compound or a combination of compounds reduces the hyper-activation of the cancer signaling network induced by a kinase inhibitor. In another specific embodiment, a compound or a combination of compounds at a subtherapeutic dose is identified as a Network Brake if it reduces the expression level of a cancer stem cell marker(s) (for example, Sox2, Oct4, Nanog, LIN28, c-Myc , and/or KLF4). A Cancer Stem Cell Marker Assay (e.g., a western blot assay for measuring cancer stem cell marker levels) can be used to determine if a compound or a combination of compounds at a subtherapeutic dose reduces the expression level of a cancer stem cell marker(s). In another specific embodiment, a compound or a combination of compounds at a subtherapeutic dose is identified as a Network Brake if it reduces the level of an active histone mark(s) (for example, H3K9-Ac, H4K5-Ac, H3K4-Me3, H4K12-Ac, and/or H3K64-Ac), and/or increases the level of a repressive histone mark(s) (for example, H3K27-Me3). A Histone Modification Assay (e.g., a western blot assay for measuring histone mark levels) can be used to determine if a compound or a combination of compounds at a subtherapeutic dose reduces the level of an active histone mark(s), and/or increases the level of a repressive histone mark(s). A Network Brake can be a proteasome inhibitor, an HD AC inhibitor, an HSP (Heat Shock Protein) (e.g. , Hsp90) inhibitor, an HDAC-PI3K dual inhibitor, or a combination thereof. In a specific embodiment, a Network Brake is a combination of a proteasome inhibitor and an HDAC inhibitor. In another specific embodiment, a Network Brake is a combination of a proteasome inhibitor and an HDAC-PI3K dual inhibitor. In another specific embodiment, a Network Brake is a combination of an HDAC inhibitor and an HSP (e.g., Hsp90) inhibitor.

[0034] As used herein, the phrase "Network Brake activity" is used to describe the ability of a compound or a combination of compounds at a subtherapeutic dose(s) or subtherapeutic dose range(s) to improve the efficacy of a kinase inhibitor. In a specific embodiment, a compound or a combination of compounds at a subtherapeutic dose or subtherapeutic dose range(s) has Network Brake activity if it reduces the hyper-activation of the cancer signaling network induced by a kinase inhibitor. A Phosphoprofile Assay can be used to determine if a compound or a combination of compounds reduces the hyper-activation of the cancer signaling network induced by a kinase inhibitor. In another specific embodiment, a compound or a combination of compounds at a subtherapeutic dose is identified as a Network Brake if it reduces the expression level of a cancer stem cell marker(s) (for example, Sox2, Oct4, Nanog, LIN28, c-Myc , and/or KLF4) induced by a kinase inhibitor. A Cancer Stem Cell Marker Assay (e.g., a western blot assay for measuring cancer stem cell marker expression levels, or a dot blot protein array for measuring cancer stem cell marker expression levels) can be used to determine if a compound or a combination of compounds at a subtherapeutic dose is identified as a Network Brake if it reduces the expression level of a cancer stem cell marker(s) induced by a kinase inhibitor. The following kit can be used in the Cancer Stem Cell Marker Assay: Profiler Human Pluripotent Stem Cell Array Kit (R&D Systems® Cat# ARY010). In another specific embodiment, a compound or a combination of compounds at a subtherapeutic dose is identified as a Network Brake if it reduces the level of an active histone mark(s) (for example, H3K9-Ac, H4K5-Ac, H3K4-Me3, H4K12-Ac, and/or H3K64-Ac) upregulated by a kinase inhibitor, and/or increases the level of a repressive histone mark(s) (for example, H3K27-Me3) downregulated by a kinase inhibitor. A Histone Modification Assay (e.g., a western blot assay for measuring histone mark levels) can be used to determine if a compound or a combination of compounds at a

subtherapeutic dose reduces the level of an active histone mark(s) upregulated by a kinase inhibitor, and/or increases the level of a repressive histone mark(s) downregulated by a kinase inhibitor. [0035] As used herein, the term "hyper-activation" refers to activation that is 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, or more above normal or a baseline (e.g., the level of activation in non-cancerous cells or healthy cells or untreated cancer cells).

[0036] As used herein, the term "clinical dose" refers to a dose commonly used in the standard-of-care therapy for the cancer to be treated.

[0037] As used herein, the term "subclinical dose" refers to a dose of a compound at which no clinical effect is detected when the compound is administered alone. In a specific

embodiment, the subclinical dose is lower than the dose commonly used in the standard-of-care therapy for the cancer to be treated.

[0038] As used herein, the term "effective dose" refers to a dose of a compound at which a therapeutic effect is detected, either when the compound is administered alone or when the compound is administered in combination with one or more other compounds.

[0039] As used herein, the term "subtherapeutic dose(s)" refers to a dose of a compound or doses of compounds in a combination which demonstrates no therapeutic benefit for treating cancer (see, e.g. , Section 5.3.2 and Section 6 for methods for assessing the therapeutic efficacy of a compound or a combination of compounds for treating cancer) and is minimally toxic to non-cancerous cells (see, e.g. , Section 5.3.3 and Section 6 for methods for assessing the toxicity of a compound or a combination of compounds on non-cancerous cells). In a preferred embodiment, a compound at a subtherapeutic dose or a combination of compounds at subtherapeutic doses improves the therapeutic efficacy of a kinase inhibitor at a specific dose or dose range.

[0040] As used herein, the term "minimally toxic" and "minimal toxicity" are used to describe a toxicity level that results in 20% or less (e.g., 15%, 10%, 5%, 4%, 3%, 2%, or 1%, or 10% to 15%, 5% to 10%, 5% to 15%, 1% to 5% or 1% to 10%) of death of the cells or animals being tested.

[0041] As used herein, the term "subtherapeutic dose range" refers to a range of doses, wherein each dose in the range is a subtherapeutic dose.

4. BRIEF DESCRIPTION OF FIGURES

[0042] Figures 1A-1C. An approach to identifying drug cocktails for cancer treatment.

(A) Western analysis of developing wing discs demonstrating that the major pathways downstream of oncogenic Ret were activated in our Drosophila Ret^2B model. (B) A Drosophila whole animal viability assay to identify single drugs and cocktails. Expression of oncogenic Ret was driven in various tissues of the developing larvae using a patched promoter, ptc>Ret^2B. Drugs were mixed into food; a fixed number of embryos were placed in vials and larvae began consuming drug food 2-3 days later. Without drug no animals reached adult stages due to the Ret^2B transgene. Efficacy of drugs that promoted viability based on emergence of pupae or adults was quantified by calculating the proportion of pupae/adults as a percentage of original embryos. (C) Screening with single agents that target aspects of the Ret signaling pathway. Multiple drugs improved the number of animals that survived to pupal stage. Only sorafenib yielded adults.

[0043] Figures 2A-2C. Restraining hyperactivation of cellular protein networks correlate with efficacy. (A) Reducing gene dosage of erk improved sorafenib efficacy increasing the proportion of embryos that reached adulthood. In the viability assay, ptc>Ret^2B erk^+/~ flies consistently gave higher number of adults compared to ptc>Ret^2B flies across the range of sorafenib doses tested. Doses listed represent μΜ. (B) Expressing oncogenic Ret throughout the developing wing epithelium (765>Ret^2B) resulted in wing venation defects in untreated samples (see Fig. 9A). Defects were binned into four groups and were quantified. Lower doses of sorafenib increased wing venation defects (red asterisks), indicating increased MAPK signaling; higher doses (black asterisk) suppressed wing vein defects. Reducing gene dosage of erk (765>Ret^2B erk^'/+) rescued wing venation defects across most doses tested (see Fig. 9A). Total numbers of adults counted for each class are: 765>Ret^2B, 0μΜ=93, 50μΜ=41, 100μΜ=48, 200μΜ=41, 400μΜ=49; 765>Ret^2B, erk^'/+, 0μΜ=67, 50μΜ=58, 100μΜ=49, 200μΜ=62, 400μΜ=69. (C) Western analysis of whole wing discs of indicated genotype with increasing doses of sorafenib represented as column graph. Western data for the antibodies tested is in Fig. 9B. The column graph is represented as the ratio of the signal— analyzed in Image J— of treated tissue: control tissue of the same genotype. At lower sorafenib doses in 765>Ret^2B flies, pERK levels elevated indicating increase in Ras pathway activity (see panel); in addition, an overall increase was observed in the activity levels of most proteins tested (see Fig. 9C-9E).

[0044] Figures 3A-3C. Genetic and pharmacological approach identified drugs that act as 'network brakes'. (A) ptc>Ret^2B flies were screened against a panel of drug combinations (Fig. 8). A subset of combinations including sorafenib/bortezomib, sorafenib/dasatinib, and sorafenib/wortmannin improved adult viability compared to drugs as single agents. Reducing a genomic copy of erk, dsorl, or rpdS or coexpressing InR°^N further improved viability of flies fed with most of these drug combinations. The 3-drug cocktail sorafenib/bortezomib/vorinostat strongly improved adult viability of ptc>Ret^2B flies. (B) Western analysis of control (765-GAL4) and 765>Ret^2B larvae exposed to sorafenib/bortezomib or sorafenib/bortezomib/vorinostat drug combinations. As in Figure 2C, western data is represented as column graph normalized to animals of the same genotype treated with DMSO alone. Both tissues exposed to the 3-drug cocktail showed reduced activation of the markers sampled compared to sorafenib/bortezomib combination, including EGFR, pAkt, pErk, Racl specifically in control tissues. This indicated the 3-drug cocktail was most potent in restraining pathway hyperactivation in normal as well as Ret-expressing cells. (C) Bortezomib/vonnostat improved viability of ptc>Ret^2B and control flies fed with kinase inhibitors that target Ret pathway components. Flies were fed either kinase inhibitors alone (AD57, AD80, Trametinib, or Sorafenib) or as a 3-drug cocktail with

bortezomib/vonnostat (B+V). All kinase inhibitors showed improved viability in the presence of bortezomib/vorinostat. Total number of animals tested per treatment: control- AD57 (n=75), AD57+B+V (n=116); AD80 (n=114), AD80+B+V (n=112); trametinib (n=123),

trametinib+B+V (n=131); sorafenib (n=118), sorafenib (n=120). Ret^2B- AD57 (n=205),

AD57+B+V (n=186); AD80 (n=78), AD80+B+V (n=92); trametinib (n=115), trametinib+B+V (n=89); sorafenib (n=97), sorafenib (n=89). (D) Presence of vorinostat/AUY922 improved viability of Ret^2B (ptc>Ret^2B) and control (ptc-GAL4) flies fed with Ret-pathway kinase inhibitors. Ret^2B and control flies were fed either kinase inhibitors alone (AD57, AD80,

Trametinib, or Sorafenib) or as a 3-drug cocktail with vorinostat/AUY922 (V+922). Represented on the right hand side is the relative improve in viability of the 3-drug cocktail compared to kinase inhibitor treatment alone in both models tested. All the kinase inhibitors showed improved viability in the presence of vorinostat/AUY922 in both models.

[0045] Figures 4A-4E. Cocktails with network brake drugs restrain human thyroid cancer networks. (A) MTT viability assay curves indicate that bortezomib/vorinostat reduced viability of MZ-CRC-1 cells at moderate as well as high doses of sorafenib, including significant reduction of IC50. IC50 are in parentheses; doses of bortezomib (6 nM) and vorinostat (50 nM) are indicated. (B) AD80 efficacy also improved in the presence of bortezomib/vorinostat.

Dosing, IC50s indicated. (C) Subcutaneous mouse xenograft assays using human TT cells showing median relative tumor growth of: i) vehicle treated controls, ii) sorafenib (40 mg/kg) + bortezomib (0.05 mg/kg) treated, and iii) sorafenib (40 mg/kg) + bortezomib (0.05 mg/kg) + vorinostat (10 mg/kg) treated animals. Ten animals per treatment were used to analyze relative tumor growth, defined as the difference between median size of tumors at initiation of drug dosing to indicated timepoints. (D) Phospho-protein array results of MZ-CRC-1 cells were treated with indicated drugs in the presence or absence of bortezomib (B, 6 nM), vorinostat (V, 6 nM), and sorafenib (S, 1 μΜ). Sorafenib treatment alone led to hyperactivation of almost all phospho-protein markers on the panel; sorafenib/bortezomib/vorinostat was most effective suppressing overall hyperactivation. (E) Additional broad acting inhibitors act as network brakes at low subtherapeutic doses (Fig. 15E for dose ranges). AUY922/vorinostat and bortezomib/CUDC-907 increased efficacy of different targeted therapies. IC50s in parentheses.

[0046] Figures 5A-5D. (A) Western analysis of MZ-CRC-1 cells demonstrating that bortezomib/vorinostat restrained sorafenib induced hyperactivation of the cancer stem cell marker Sox2. Levels of other stem cell markers were reduced by sorafenib and by combinations. Drug combinations promoted strong upregulation of the cell death marker cPARP. Data is quantitated in panel D. (B) Western analysis of TT cells demonstrating that

bortezomib/vorinostat restrained activation of stem cell marker cMyc and reduced levels of Oct4 and Nanog. The combination strongly potentiated sorafenib induced upregulation of cPARP. Data is quantitated in panel D. (C) Immunofluorescence staining showing that sorafenib-treated TT cells upregulated Sox2. This upregulation was moderately restrained in cells treated with sorafenib (0.5 μΜ) plus bortezomib (2 nM) plus vorinostat (2 nM). (D) Data from panels A, B were quantified by Image J to generate comparative bar graphs.

[0047] Figures 6A-6E. Drug cocktails restrained multiple cancer cell networks. (A)

H358 cells were treated with indicated drugs in the presence or absence of bortezomib (B, 6nM), vorinostat (V, 50nM), and erlotinib (E, 1 μΜ). Erlotinib induced hyperactivation of various phospho-proteins, which was restrained in the presence of bortezomib/vorinostat. (B) An MTT viability assay demonstrated that bortezomib/vorinostat significantly lowered the IC50 (in parentheses) of erlotinib on H358 cells. (C) Scatter plot (PRISM) summary of phospho-protein array data on indicated cancer lines treated with sorafenib(S), erlotinib (E), trametinib (T) alone or in combination with bortezomib/vorinostat. Each phospho-protein signal was compared between treatments and median level of entire network is indicated in blue, inter-quartile range is indicated. Bortezomib/vorinostat treatment consistently reduced median phospho-protein level of each cancer network. Paired t-tests between single and triple treated samples for each cell line indicated p-values <0.05 (asterisks). (D) IC50s of various kinase inhibitor drugs on different cancer lines was lowered significantly in the presence of indicated dose of

bortezomib/vorinostat. From Figs. 4A, 6B, 14A. (E) Western analysis of H358 cells shows that the bortezomib/vorinostat combination restrained erlotinib-induced upregulation of Sox2.

Erlotinib also upregulated cMyc which was moderately reduced by bortezomib/vorinostat. Other stem cell markers were kept below untreated levels. The cell death marker cPARP was significantly upregulated in the presence of bortezomib/vorinostat.

[0048] Figures 7A-7D. Network brake-containing cocktails delay emergence of resistance. (A) H358 cells treated chronically with 1 μΜ erlotinib for 65 days developed resistance to erlotinib (IC50=4 μΜ). Cells treated with erlotinib + bortezomib (6nM) + vorinostat (50nM) retained erlotinib sensitivity similar to the parental line. IC50s in parentheses. (B) Phase contrast images provide examples from Panel A. Only erlotinib/bortezomib/vorinostat treatment strongly reduced cell number. (C) Parental, erlotinib resistant, and

erlotinib/bortezomib/vorinostat (E+B+V) treated cell lysates analyzed on phospho-protein arrays. Outlined in red are phospho-proteins whose levels show much higher signals in the resistant line compared to the other two cell treatment conditions. Identities are listed beneath boxes; for example c-Met is a protein known to promote resistance to erlotinib treatment in NSCLC

(Engelman et al., 2007). (D) Western analysis of lysates from erlotinib resistant H358 NSCLC cells. Resistant lines (res.) upregulated Sox2, RhoA, and activity of β-catenin;

erlotinib/bortezomib/vorinostat treatment kept these below parental H358 cells, while increasing activity of the tumor suppressor MOB. Active histone marks H3K9-Ac, H3K9-Me3, and H4K5- Ac were upregulated in erlotinib resistant cells. Erlotinib/bortezomib/vorinostat blocked this upregulation while elevating the repressive mark H3K27Me3.

[0049] Figures 8. Drugs tested for ability to rescue viability of ptc>Re^^B animals.

Primary targets are listed; note that most drugs have multiple targets.

[0050] Figures 9A-9E. Reducing MAPK dosage improved sorafenib treatment by preventing hyperactivation of cellular protein networks. (A) Whole mount adult wing images showing overall wing venation pattern following uniform expression of oncogenic Ret (765 > Ret²³) across the developing wing epithelium (upper panels). 100 μΜ sorafenib increased wing venation thickening/defects considerably (asterisks) while 200 μΜ also increased wing venation defects to a lesser extent. High sorafenib dose, 400 μΜ, strongly suppressed wing venation defects. Reducing erk gene dosage, 765>Ret^2B erk^'/+, resulted in sorafenib dependent improvement of overall venation pattern across all doses tested (lower panels; 100 μΜ, 200 μΜ, 400 μΜ). Reducing erk gene dosage in the absence of sorafenib did not significantly improve venation pattern (lower panel). (B) Western analysis with 14 protein markers to analyze the effect of drug treatment on the cellular protein networks of tissues. Three different tissues with indicated genotype were analyzed. (C) Western analysis of entire wing discs of indicated genotype with increasing doses of sorafenib. The raw western data for all the antibodies tested is in Fig. 9B. The column graph is represented as the ratio of the signal (analyzed in Image J) of treatment with respect to control tissues (Lane 1 for each signal in Fig. 9D). (D) Scatter plot (PRISM) of values for each protein in each experiment from (B). Blue line is median with interquartile range indicated. Median level of protein network rose in oncogenic Ret tissues, 765>Ret^2B, following sorafenib treatment (50 μΜ, 100 μΜ). Reducing erk gene dosage, 765>Ret^2B erk^'/+, in sorafenib treated tissues (50 μΜ, 100 μΜ, 200 μΜ) lowers median level of the network below zero. This lower median level of the network matched the improved wing venation pattern observed in (A). (E) Scatter plot (PRISM) of values for each protein in each experiment from western in (B). Value of each marker signal is normalized to dmso treated WT control tissue (Lane 1 in (B)). Blue line is median; interquartile range is indicated. Median level of the protein network rose in lower sorafenib doses, 765>Ret^2B . Reducing erk gene dosage, 765>Ret^2B,erk^'/+, reduced median level of the network in sorafenib treated animals (compare median of pairs indicated by each asterisk). This matched the improved efficacy observed with sorafenib after reducing erk gene dosage in (A and B). Red circles represent the pERK values which are very similar between the two experiments (also see (C)).

[0051] Figures 10A-10D. Toxic drug combination hyperactivated cellular protein networks. (A) Western data in (C) represented as column graph showing vandetanib/rapamycin combination treated wild type tissue upregulated a number of protein markers including pERK and Cyclin D. In oncogenic Ret expressing tissues the combination restrained hyperactivation of the same protein network. (B, D) Toxic drug combination vandetanib/rapamycin hyperactivated cellular networks in normal cells (red asterisk). In (B) signal for each marker was normalized to signal from untreated tissue of same genotype. In (D) signal for each marker was normalized to signal from untreated WT tissue (Lane 1 in (C)). Note oncogenic Ret expressing tissues did not show network hyperactivation and instead show reduced median network levels. Raw western data in (C). Scatter plot was generated and the relative level of each protein was analyzed in the same manner as in Figure 2. Blue line is median with interquartile range indicated. (C) Western analysis with 14 protein markers to analyze the effect of drug treatment on the cellular protein networks of tissues treated with toxic combination of vandetanib/rapamycin. Two different tissues with indicated genotype were analyzed.

[0052] Figures 11A-11D. (A) Comparison of median level of protein network between sorafenib/bortezomib and sorafenib/dasatinib combinations. Scatter plot signals are represented as the ratio of the signal (western data in (C) and (D) analyzed in Image J) of treatment with respect to DMSO treated tissues of the same genotype (Lane 1 and 5 in (C)). It was generated and the relative level of each protein was analyzed in the same manner as in other figures. Blue line is median with interquartile range indicated. Both combinations maintain median protein level of the network below baseline (red line) and slightly lower than sorafenib alone treatment in Ret-expressing tissues (asterisk, 165>Ret^2B). But sorafenib/dasatinib combination displays a higher median protein level (M= +14) in normal tissues compared sorafenib/bortezomib combination (M= + 5.5). (B) Genetic improvement of sorafenib/dasatinib combination also correlated with tighter control of network hyperactivation. Scatter plot signals are represented as the ratio of the signal (western data in (D) analyzed in Image J) of treatment with respect to dmso treated tissues of the control genotype (Lane 1 in (D)). It was generated and the relative level of each protein was analyzed in the same manner as in other figures. Viability of oncogenic Ret flies consuming sorafenib/dasatinib combination improved by reducing erk dosage (Figure 3 A). Comparison of median network protein levels between these two conditions, 765>Ret^2B and 765>Ret^2Berk^'/+ , shows sorafenib/dasatinib combination maintains a much lower median level of the network (M= -17 vs. M= -5.7; asterisks) when erk gene dosage was reduced. (C) Raw western data in used to generate scatter plots in Fig. 11 A. (D) Raw western data in used to generate scatter plots in Fig. 11 A-B.

[0053] Figure 12A-12B. (A) Viability studies shows that pan HDAC inhibitors can improve the viability of control flies (ptc-GAL4) treated with sorafenib(400μM)/bortezomib(l μM) (μΜ = μπιοΐ = micromolar). Control flies consuming sorafenib/bortezomib combination on average showed 76% of embryos surviving to adults. When pan HDAC inhibitors were added to this combination there was a consistent increase of viability - i)+vorinostat-82%, ii)+belinostat-90%, iii)+panobinostat-82%, iv)+entinostat-84%, v)CUDC-907-93%. Paired t-tests (PRISM) between sorafenib/bortezomib treated and triple drug treated flies showed p-values <0.05. Approximately 50 flies were analyzed for each treatment. (B) Viability studies showing that MTM, an inhibitor of the SP1 -class of transcription factors, improves viability of ptc-GAL4>Ret^2B flies treated with sorafenib(400μM)/bortezomib(lμM). Effect on adult viability is further improved by reducing erk gene dosage.

[0054] Figures 13A-13B. Cocktails with network brake drugs restrain human thyroid cancer networks. (A) TT cancer cells were treated with indicated drugs in the presence or absence of bortezomib (B; 2 nM), vorinostat (V; 2 nM), and sorafenib (S; 0.5 μΜ). Cell lysates were collected at indicated time points and incubated with phoshpo-protein arrays. Bar graph was generated as mentioned above. (B) The order and identity of phospho-protein array markers used in our studies. The Cell Signaling RTK phospho-protein array used in our studies and represented in the bar graphs in Figure 4-6. Columns on bar graphs from left to right are numbered from 1-39 in this panel.

[0055] Figures 14A-14E. Network Brake cocktails restrain various cancer cell networks. (A) MTT viability assays of indicated cell lines with indicated drugs and doses. Trametinib (T); BEZ235 (BEZ); bortezomib (B); vorinostat (V). Bortezomib and vorinostat doses are in nM. IC50s in parentheses. (B) Western analysis of H1299-NSCLC cells shows that the bortezomib/vorinostat combination restrained stem cell markers c-Myc, KLF4. Vimentin was kept below untreated levels. Cell death marker cPARP was significantly upregulated in the presence of targeted therapy with bortezomib/vorinostat. (C) Western analysis of HepG2-HCC cells shows that the bortezomib/vorinostat combination restrained trametinib induced

upregulation of oncogenic stem cell marker Sox2. Other stem cell markers were kept below untreated levels. Cell death marker cPARP was significantly upregulated in the presence of targeted therapy with bortezomib/vorinostat. (D) Quantitation of western analysis in (C) showing lowest level of stem cell markers occur with co-treatment of targeted therapy with bortezomib/vorinostat in HepG2 cells. (E) Quantitation of phospho-array analysis of H1299- NSCLC cells treated with targeted therapy, trametinib, or trametinib in combination with low dose network brake drugs bortezomib(0.25nM)/vorinostat(0.25nM) as in (A). Presence of both network drugs provided the most optimal restraint of the kinase network hyperactivation. A rare signal showed higher levels in the presence of 3 drugs (red asterisk). This data is represented as PRISM scatter plot in Figure 6C.

[0056] Figures 15A-15E. (A) MTT cell viability analysis as in Figure 7A: H358 parental cells and H358 erlotinib resistant cells were treated with the Met inhibitor crizotinib. Parental H358 cells (with little Met activity) showed little response to crizotinib (IC50 4μΜ). In contrast erlotinib-resistant H358 cells, which had upregulated pMet levels, showed increased

responsiveness to crizotinib (IC50 1.5 μΜ). (B) Western analysis of H358-NSCLC cells chronically treated with erlotinib alone or combined erlotinib/bortezomib/vorinostat. "Resistant" indicates erlotinib resistant cells generated using two different doses of Erlotinib (0.5μΜ and ΙμΜ). Resistant cells consistently upregulated Sox2, activated-Peat, RhoA, Racl, which were kept retrained in cells chronically treated with erlotinib/bortezomib/vorinostat. (C) Quantitation of western data in (B) using Image J software. Signal for each protein in erlotinib-resistant (E) and E+B+V lines were compared to parental H358 lines to give each individual column. (D) Phase contrast images of 239-Melanoma cells lines treated with the indicated conditions for 120 days. Cells treated with 0.5 μΜ vemurafenib developed resistance to the drug and grew confluently similar to the parental line. 239 cells treated continuously with combined

vemurafenib/bortezomib/vorinostat (0.5 μΜ, 6 nm, and 50 nM, respectively) displayed minimal growth over 120 days of culturing. The combination of bortezomib/vorinostat (6 nM, 50 nM) without targeted therapy vemurafenib grew similar to untreated parental lines. (E) Drug response curve for each network brake drug - vorinostat, bortezomib, CUDC-907, AUY922- on various different cancer cell lines tested in our study. Shaded grey area indicated low nanomolar doses used for each of these drugs in these studies. At these doses individual network brake drugs have very little effect on cancer cell line viability.

5. DETAILED DESCRIPTION

[0057] The invention relates to improved methods of treating a cancer in a patient in need thereof using combination drug regimens that can improve therapeutic efficacy of kinase inhibitor(s).

5.1. Treatment Of Cancer Patients

5.1.1. Network Brakes Restrain Cancer Signaling Networks, Resulting In Greater Efficacy [0058] Treatment with a kinase inhibitor at a clinical dose often surprisingly results in: (i) hyper-activation of a cancer signaling network (i.e., hyper-activation of a set of specific kinases, which can be visualized by testing for the kinase activity of the set of specific kinases), which functionally counteracts the ability of the kinase inhibitor to inhibit cancer development and/or progression, thereby reducing the therapeutic efficacy of the kinase inhibitor; and (ii) activation of cancer stem cells, which promote cancer growth and metastasis. Thus, many kinase inhibitors used at the clinical doses are actually partly promoting cancer development and/or progression. In addition, as the level of hyper-activation of the cancer signaling network and cancer stem cells increases over time, the patient's cancer may become resistant to the kinase inhibitor.

[0059] A "Network Brake" when used at a subtherapeutic dose(s) or subtherapeutic dose range(s) can restrain the cancer signaling network activities, and can keep cancer stem cell fates under control, while the Network Brake at such subtherapeutic dose(s) or subtherapeutic dose range(s) has no or low efficacy in killing cancer cells (i.e., kills less than 20% cancer cells) if used as a single agent. Therefore, when a Network Brake is used at a subtherapeutic dose(s) or subtherapeutic dose range(s) in combination with a kinase inhibitor, the kinase inhibitor can have improved therapeutic efficacy in inhibiting cancer development and/or progression. In specific embodiments, the combination with a Network Brake reduces the IC50 of a kinase inhibitor relative to the kinase inhibitor alone, for example, by about 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater, or by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 50-fold, 100-fold, or greater. The IC50 is a measure of the efficacy of a drug, indicating how much of a drug is needed to inhibit a given biological process (or a component of a process, such as an enzyme, cell, cell receptor or microorganism) by half.

[0060] As a result, in situations where a patient's cancer has developed resistance to a kinase inhibitor, the cancer patient can be co-treated with a Network Brake at a subtherapeutic dose(s) or subtherapeutic dose range(s) to reduce resistance to the kinase inhibitor. In other cases, a cancer patient can be treated with the combination of a kinase inhibitor and a subtherapeutic dose(s) or subtherapeutic dose range(s) of Network Brake from the outset to prevent or reduce resistance to the kinase inhibitor from the outset.

[0061] In addition, when a Network Brake is used, therapeutic efficacy of certain kinase inhibitors can be achieved at doses lower than the clinical doses, thereby reducing toxicity of the kinase inhibitors. Alternatively, a Network Brakes may allow a kinase inhibitor to be used safely at higher doses that would be otherwise toxic to the patient.

5.1.2. Combination Regimens

[0062] Provided herein are methods of treating a cancer in a patient in need thereof, comprising: (a) administering to the patient a Network Brake at a subtherapeutic dose; and (b) administering to the patient a kinase inhibitor.

[0063] In a first aspect, provided herein are methods of treating a cancer in a patient in need thereof, comprising: (a) administering to the patient a Network Brake at a subtherapeutic dose; and (b) administering to the patient a kinase inhibitor subsequent to and/or concurrently with the administration of the Network Brake; wherein the therapeutic efficacy of the kinase inhibitor is improved relative to the therapeutic efficacy of the kinase inhibitor without administering to the patient the Network Brake. In a specific embodiment, the kinase inhibitor is administered at a clinical dose. In another specific embodiment, the kinase inhibitor is administered to the patient at a dose lower than a clinical dose. In another specific embodiment, the kinase inhibitor is administered to the patient at a dose higher than a clinical dose.

[0064] In a second aspect, provided herein are methods of reducing cancer resistance to a kinase inhibitor in a patient whose cancer has been treated with the kinase inhibitor and has developed resistance to the kinase inhibitor, comprising: (a) administering to the patient a Network Brake at a subtherapeutic dose; and (b) administering to the patient the kinase inhibitor subsequent to and/or concurrently with the administration of the Network Brake; wherein the resistance to the kinase inhibitor is reduced (e.g., by about 10%, 20%, 30%, 40%, 50%, 60%, 70%), 80%), 90%), or 100%>) after administering to the patient the Network Brake. In a specific embodiment, the kinase inhibitor is administered at a clinical dose. In another specific embodiment, the kinase inhibitor is administered to the patient at a dose lower than a clinical dose. In another specific embodiment, the kinase inhibitor is administered to the patient at a dose higher than a clinical dose.

[0065] In a third aspect, provided herein are methods of preventing or reducing cancer resistance to a kinase inhibitor in a patient whose cancer has not been treated with the kinase inhibitor, comprising: (a) administering to the patient a Network Brake at a subtherapeutic dose; and (b) administering to the patient a kinase inhibitor subsequently to and/or concurrently with the administration of the Network Brake; wherein the resistance to the kinase inhibitor is prevented or reduced (e.g., by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%,).

[0066] In a fourth aspect, provided herein are methods of reducing toxicity of a kinase inhibitor to a patient having cancer, comprising: (a) administering to the patient a Network Brake at a subtherapeutic dose; and (b) administering to the patient a kinase inhibitor at a dose lower than a clinical dose subsequent to and/or concurrently with the administration of the Network Brake; wherein the toxicity of the kinase inhibitor is reduced (e.g., by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) relative to the toxicity after administering to the patient the kinase inhibitor at the clinical dose without administering to the patient the Network Brake. In a specific embodiment, the methods of reducing toxicity of a kinase inhibitor to a patient having cancer comprise: (a) administering to the patient a Network Brake at a subtherapeutic dose; and (b) administering to the patient a kinase inhibitor at a dose lower than a clinical dose subsequent to and/or concurrently with the administration of the Network Brake; wherein the toxicity of the kinase is maintained or reduced (e.g., by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), and wherein the therapeutic efficacy is maintained or improved relative to the therapeutic efficacy experienced after administering to the patient the kinase inhibitor at the clinical dose without administering to the patient the Network Brake. In a specific embodiment, the reduction in toxicity of the kinase inhibitor when administered to the patient with the Network Brake is relative to the toxicity when the kinase inhibitor is

administered to the patient at the clinical dose without administering to the patient the Network Brake. In another specific embodiment, the maintenance or reduction in toxicity of the kinase inhibitor when administered with the Network Brake relative to the toxicity when the kinase inhibitor is administered without the Network Brake is assessed in cell culture and/or an animal model. Toxicity of a kinase inhibitor can be measured by any method known in the art for testing the adverse effects that a drug may have, and can be performed using cultured cells or animal models (e.g., flies, worms, mice, rats, and primates).

[0067] In a fifth aspect, provided herein are methods of improving therapeutic efficacy of a kinase inhibitor in treating a cancer in a patient in need thereof, without increasing toxicity of the kinase inhibitor, comprising: (a) administering to the patient a Network Brake at a

subtherapeutic dose; and (b) administering to the patient a kinase inhibitor at a dose higher than a clinical dose subsequent to and/or concurrently with the administration of the Network Brake; wherein the therapeutic efficacy of the kinase inhibitor is improved relative to the therapeutic efficacy experienced after administering to the patient the kinase inhibitor at the clinical dose without administering to the patient the Network Brake, and wherein the toxicity of the kinase inhibitor is maintained or reduced. In a specific embodiment, the maintenance or reduction in the toxicity of the kinase inhibitor when administered with the Network Brake is relative to the toxicity of the kinase inhibitor administered to the patient at the clinical dose without administering to the patient the Network Brake. In another specific embodiment, the

maintenance or reduction in toxicity of the kinase inhibitor when administered with the Network Brake relative to the toxicity when the kinase inhibitor is administered without the Network Brake is assessed in cell culture and/or an animal model.

[0068] In a sixth aspect, provided herein are methods for treating a patient diagnosed with a solid tumor cancer, comprising administering to a patient in need thereof (a) a Network Brake at a subtherapeutic dose; and (b) a kinase inhibitor at an effective dose, wherein the Network Brake is a compound or a combination of compounds that reduces the hyper-activation of the cancer signaling network induced by the kinase inhibitor, and in certain embodiments, prevents the upregulation of one or more cancer stem cell inhibitors induced by the kinase inhibitor. In specific embodiments, such methods of treating the patient result in an increase in overall or progression-free survival of the patient. In specific embodiments, overall or progression-free survival of the patient is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more relative to the average overall or progression-free survival of patients with the same type of solid tumor cancer or patients with the same type of solid tumor cancer in a similar position healthwise. In certain embodiments, the patients with the same type of solid tumor cancer or patients with the same type of solid tumor cancer in a similar position healthwise have received an accepted standard of care therapy for the solid tumor cancer. In certain embodiments, the patients with the same type of solid tumor cancer or patients with the same type of solid tumor cancer in a similar position healthwise have not been treated with the kinase inhibitor or a combination of the kinase inhibitor and the Network Brake. In specific embodiments, overall or progression-free survival of the patient is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%), 75%), 80%), 85%), 90%, 95% or more relative to the average overall or progression-free survival of patients with the same type of solid tumor cancer or patients with the same type of solid tumor cancer in a similar position healthwise, who are administered with the kinase inhibitor at the effective dose but are not administered with the Network Brake. In a specific embodiment, a reduction in the hyper-activation of the cancer signaling network induced by the kinase inhibitor is assessed by an assay comprising: (1) culturing a first population of cancer cells in the presence of the kinase inhibitor at a specific concentration equivalent to the clinical dose for a period of time; (2) culturing a second population of cancer cells of the same type in the presence of the compound or the combination of compounds at a concentration(s) equivalent to the subtherapeutic dose(s), and the kinase inhibitor at the same specific concentration for said period of time; and (3) analyzing the level of activity of a certain set of kinases, which are members of the cancer signaling network, in the first and second populations of cancer cells at the end of said period of time, wherein a decrease in the overall, median, or mean activity level of the kinases in the second population of cancer cells relative to the overall, median or mean activity of the same kinases in the first population of cancer cells indicates that the compound or the combination of compounds reduces the hyper-activation of the cancer signaling network. In another specific embodiment, a reduction in the hyper-activation of the cancer signaling network induced by the kinase inhibitor is assessed by an assay comprising: (1) culturing a first population of cancer cells in the presence of the kinase inhibitor at a specific concentration equivalent to the clinical dose for a period of time; (2) culturing a second population of cancer cells of the same type in the presence of the compound or the combination of compounds at a concentration(s) equivalent to the subtherapeutic dose(s), and the kinase inhibitor at the same specific concentration for said period of time; and (3) analyzing the level of phosphorylation of a certain set of proteins, which are members of the cancer signaling network, in the first and second populations of cancer cells at the end of said period of time, wherein a decrease in the overall, median, or mean level of phosphorylation in the second population of cancer cells relative to the overall, median or mean level of phosphorylation in the first population of cancer cells indicates that the compound or the combination of compounds reduces the hyper-activation of the cancer signaling network. In another specific embodiment, the prevention of the upregulation of the one or more cancer stem cell markers induced by the kinase inhibitor is assessed by an assay comprising: (1) culturing a first population of cancer cells in the presence of the kinase inhibitor at a specific concentration equivalent to the clinical dose for a period of time; (2) culturing a second population of cancer cells of the same type in the presence of the compound or the combination of compounds at a concentration(s) equivalent to the subtherapeutic dose(s), and the kinase inhibitor at the same specific concentration for said period of time; (3) culturing a third population of cancer cells of the same type without any treatment; and (4) analyzing the expression level of the one or more cancer stem cell markers in the first, second, and third populations of cancer cells at the end of said period of time, wherein the overall, median, or mean expression level of the one or more cancer stem cell markers in the first population of cancer cells is higher than the overall, median, or mean expression level of the same cancer stem cell marker(s) in the third population of cancer cells, and wherein a decrease in the overall, median, or mean expression level of the one or more cancer stem cell markers in the second population of cancer cells relative to the overall, median or mean expression level of the same cancer stem cell marker(s) in the first population of cancer cells indicates that the compound or the combination of compounds prevents the upregulation of the one or more cancer stem cell markers induced by the kinase inhibitor. In certain embodiments, the effective dose of the kinase inhibitor is the clinical dose. In some embodiments, the Network Brake is

administered to the patient prior to the administration of the kinase inhibitor. In other embodiments, the Network Brake and the kinase inhibitor are concurrently administered to the patient.

[0069] In a seventh aspect, provided herein are methods for treating a patient diagnosed with a solid tumor cancer, comprising administering to a patient in need thereof: (a) a Network Brake at a subtherapeutic dose; and (b) a kinase inhibitor at a dose equal to or greater than the kinase inhibitor's maximum tolerated dose as assessed in the absence of the Network Brake, wherein the Network Brake is a compound or a combination of compounds that reduces the hyper- activation of the cancer signaling network induced by the kinase inhibitor, and in certain embodiments, prevents the upregulation of one or more cancer stem cell markers induced by the kinase inhibitor. In specific embodiments, such methods of treating the patient result in an increase in overall or progression-free survival of the patient. In specific embodiments, overall or progression-free survival of the patient is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more relative to the average overall or progression-free survival of patients with the same type of solid tumor cancer or patients with the same type of solid tumor cancer in a similar position healthwise. In certain embodiments, the patients with the same type of solid tumor cancer or patients with the same type of solid tumor cancer in a similar position healthwise have received an accepted standard of care therapy for the solid tumor cancer. In certain embodiments, the patients with the same type of solid tumor cancer or patients with the same type of solid tumor cancer in a similar position healthwise have not been treated with the kinase inhibitor or a combination of the kinase inhibitor and the Network Brake. In specific embodiments, overall or progression-free survival of the patient is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more relative to the average overall or progression-free survival of patients with the same type of solid tumor cancer or patients with the same type of solid tumor cancer in a similar position healthwise, who are administered with the kinase inhibitor at the dose equal to or greater than the kinase inhibitor's maximum tolerated dose but are not administered with the Network Brake. In specific embodiments, such methods of treating the patient do not result in an increase in the toxicity and/or side effects of the kinase inhibitor experienced by the patient. In a specific embodiment, a reduction in the hyper- activation of the cancer signaling network induced by the kinase inhibitor is assessed by an assay comprising: (1) culturing a first population of cancer cells in the presence of the kinase inhibitor at a specific concentration equivalent to the maximum tolerated dose for a period of time; (2) culturing a second population of cancer cells of the same type in the presence of the compound or the combination of compounds at a concentration(s) equivalent to the subtherapeutic dose(s), and the kinase inhibitor at the same specific concentration for said period of time; and (3) analyzing the level of activity of a certain set of kinases, which are members of the cancer signaling network, in the first and second populations of cancer cells at the end of said period of time, wherein a decrease in the overall, median, or mean activity level of the kinases in the second population of cancer cells relative to the overall, median or mean activity of the same kinases in the first population of cancer cells indicates that the compound or the combination of compounds reduces the hyper-activation of the cancer signaling network. In another specific embodiment, a reduction in the hyper-activation of the cancer signaling network induced by the kinase inhibitor is assessed by an assay comprising: (1) culturing a first population of cancer cells in the presence of the kinase inhibitor at a specific concentration equivalent to the maximum tolerated dose for a period of time; (2) culturing a second population of cancer cells of the same type in the presence of the compound or the combination of compounds at a

concentration(s) equivalent to the subtherapeutic dose(s), and the kinase inhibitor at the same specific concentration for said period of time; and (3) analyzing the level of phosphorylation of a certain set of proteins in the first and second populations of cancer cells at the end of said period of time, wherein a decrease in the overall, median, or mean level of phosphorylation in the second population of cancer cells relative to the overall, median or mean level of

phosphorylation in the first population of cancer cells indicates that the compound or the combination of compounds reduces the hyper-activation of the cancer signaling network. In another specific embodiment, the prevention of the upregulation of the one or more cancer stem cell markers induced by the kinase inhibitor is assessed by an assay comprising: (1) culturing a first population of cancer cells in the presence of the kinase inhibitor at a specific concentration equivalent to the maximum tolerated dose for a period of time; (2) culturing a second population of cancer cells of the same type in the presence of the compound or the combination of compounds at a concentration(s) equivalent to the subtherapeutic dose(s), and the kinase inhibitor at the same specific concentration for said period of time; (3) culturing a third population of cancer cells of the same type without any treatment; and (4) analyzing the expression level of the one or more cancer stem cell markers in the first, second, and third populations of cancer cells at the end of said period of time, wherein the overall, median, or mean expression level of the one or more cancer stem cell markers in the first population of cancer cells is higher than the overall, median, or mean expression level of the same cancer stem cell marker(s) in the third population of cancer cells, and wherein a decrease in the overall, median, or mean expression level of the one or more cancer stem cell markers in the second population of cancer cells relative to the overall, median or mean expression level of the same cancer stem cell marker(s) in the first population of cancer cells indicates that the compound or the combination of compounds prevents the upregulation of the one or more cancer stem cell markers induced by the kinase inhibitor. In certain embodiments, the dose equal to or greater than the kinase inhibitor's maximum tolerated dose is the kinase inhibitor's maximum tolerated dose. In certain embodiments, the dose equal to or greater than the kinase inhibitor's maximum tolerated dose is a dose greater than the kinase inhibitor's maximum tolerated dose. In some embodiments, the Network Brake is administered to the patient prior to the administration of the kinase inhibitor. In other embodiments, the Network Brake and the kinase inhibitor are concurrently administered to the patient. [0070] In an eighth aspect, provided herein are methods for treating a patient diagnosed with a solid tumor cancer, comprising administering to a patient in need thereof: (a) a Network Brake at a subtherapeutic dose; and (b) a kinase inhibitor at a subclinical dose, wherein the Network Brake is a compound or a combination of compounds that reduces the hyper-activation of the cancer signaling network induced by the kinase inhibitor, and in certain embodiments, prevents the upregulation of one or more cancer stem cell markers induced by the kinase inhibitor. In specific embodiments, such methods of treating the patient result in an increase in overall or progression-free survival of the patient. In specific embodiments, overall or progression-free survival of the patient is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more relative to the average overall or progression-free survival of patients with the same type of solid tumor cancer or patients with the same type of solid tumor cancer in a similar position healthwise. In certain embodiments, the patients with the same type of solid tumor cancer or patients with the same type of solid tumor cancer in a similar position healthwise have received an accepted standard of care therapy for the solid tumor cancer. In certain embodiments, the patients with the same type of solid tumor cancer or patients with the same type of solid tumor cancer in a similar position healthwise have not been treated with the kinase inhibitor or a combination of the kinase inhibitor and the Network Brake. In specific embodiments, overall or progression-free survival of the patient is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%), 75%), 80%), 85%), 90%, 95% or more relative to the average overall or progression-free survival of patients with the same type of solid tumor cancer or patients with the same type of solid tumor cancer in a similar position healthwise, who are administered with the kinase inhibitor at the subclinical dose but are not administered with the Network Brake. In specific embodiments, such methods of treating the patient result in a decrease in the toxicity and/or side effects of the kinase inhibitor experienced by the patient. In a specific embodiment, a reduction in the hyper-activation of the cancer signaling network induced by the kinase inhibitor is assessed by an assay comprising: (1) culturing a first population of cancer cells in the presence of the kinase inhibitor at a specific concentration equivalent to the subclinical dose for a period of time; (2) culturing a second population of cancer cells of the same type in the presence of the compound or the combination of compounds at a concentration(s) equivalent to the

subtherapeutic dose(s), and the kinase inhibitor at the same specific concentration for said period of time; and (3) analyzing the level of activity of a certain set of kinases, which are members of the cancer signaling network, in the first and second populations of cancer cells at the end of said period of time, wherein a decrease in the overall, median, or mean activity level of the kinases in the second population of cancer cells relative to the overall, median or mean activity of the same kinases in the first population of cancer cells indicates that the compound or the combination of compounds reduces the hyper-activation of the cancer signaling network. In another specific embodiment, a reduction in the hyper-activation of the cancer signaling network induced by the kinase inhibitor is assessed by an assay comprising: (1) culturing a first population of cancer cells in the presence of the kinase inhibitor at a specific concentration equivalent to the subclinical dose for a period of time; (2) culturing a second population of cancer cells of the same type in the presence of the compound or the combination of compounds at a

concentration(s) equivalent to the subtherapeutic dose(s), and the kinase inhibitor at the same concentration for said period of time; and (3) analyzing the level of phosphorylation of a certain set of proteins, which are members of the cancer signaling network, in the first and second populations of cancer cells at the end of said period of time, wherein a decrease in the overall, median, or mean level of phosphorylation in the second population of cancer cells relative to the overall, median or mean level of phosphorylation in the first population of cancer cells indicates that the compound or the combination of compounds reduces the hyper-activation of the cancer signaling network. In another specific embodiment, the prevention of the upregulation of the one or more cancer stem cell markers induced by the kinase inhibitor is assessed by an assay comprising: (1) culturing a first population of cancer cells in the presence of the kinase inhibitor at a specific concentration equivalent to the subclinical dose for a period of time; (2) culturing a second population of cancer cells of the same type in the presence of the compound or the combination of compounds at a concentration(s) equivalent to the subtherapeutic dose(s), and the kinase inhibitor at the same specific concentration for said period of time; (3) culturing a third population of cancer cells of the same type without any treatment; and (4) analyzing the expression level of the one or more cancer stem cell markers in the first and second populations of cancer cells at the end of said period of time, wherein the overall, median, or mean expression level of the one or more cancer stem cell markers in the first population of cancer cells is higher than the overall, median, or mean expression level of the same cancer stem cell marker(s) in the third population of cancer cells, and wherein a decrease in the overall, median, or mean expression level of the one or more cancer stem cell markers in the second population of cancer cells relative to the overall, median or mean expression level of the same cancer stem cell marker(s) in the first population of cancer cells indicates that the compound or the combination of compounds prevents the upregulation of the one or more cancer stem cell markers induced by the kinase inhibitor. In some embodiments, the Network Brake is administered to the patient prior to the administration of the kinase inhibitor. In other embodiments, the Network Brake and the kinase inhibitor are concurrently administered to the patient.

[0071] In specific embodiments, the Network Brake comprises or consists essentially of an HDAC inhibitor (e.g., vorinostat), a proteasome inhibitor (e.g., bortezomib), an HSP (e.g., Hsp90) inhibitor (e.g., AUY922), an inhibitor of SPl -class transcription factors, an HDAC-PI3K dual inhibitor (e.g., CUDC-907), or a combination thereof. In a specific embodiment, the Network Brake comprises or consists essentially of an HDAC inhibitor (e.g., vorinostat). In another specific embodiment, the Network Brake comprises or consists essentially of a proteasome inhibitor (e.g., bortezomib). In another specific embodiment, the Network Brake comprises or consists essentially of an HSP (e.g., Hsp90) inhibitor (e.g., AUY922). In another specific embodiment, the Network Brake comprises or consists essentially of an HDAC-PI3K dual inhibitor (e.g., CUDC-907). In another specific embodiment, the Network Brake comprises or consists essentially of an HDAC inhibitor and a proteasome inhibitor (e.g., vorinostat and bortezomib). In another specific embodiment, the Network Brake comprises or consists essentially of a proteasome inhibitor and an HDAC-PI3K dual inhibitor (e.g., bortezomib and CUDC-907). In another specific embodiment, the Network Brake comprises or consists essentially of an HDAC inhibitor and an HSP (e.g., Hsp90) inhibitor (e.g., vorinostat and AUY922). In another specific embodiment, the Network Brake comprises or consists essentially of a proteasome inhibitor (e.g., bortezomib) and an inhibitor of SPl -class transcription factors (e.g., mithramycin). The HDAC inhibitor that can be used according to the methods described herein can be any pharmaceutical agent that inhibits the activity of histone deacetylase(s). Non- limiting exemplary HDAC inhibitors include 4SC-202, abexinostat, ACY-1215, AR-42, belinostat, CG200745, chidamide, CHR-2845, CHR-3996, CUDC-101, entinostat, givinostat, kevetrin, ME-344, mocetinostat, panobinostat, pracinostat, quisinostat, resminostat, romidepsin, sulforaphane, trichostatin A, valproic acid, vorinostat, and other inhibitors of histone

deacetylase(s) that are approved by regulatory agencies for use in human patients. In a specific embodiment, the HDAC inhibitor that can be used according to the methods described herein is vorinostat, belinostat, or entinostat. In a further specific embodiment, the HDAC inhibitor that can be used according to the methods described herein is vorinostat. The proteasome inhibitor that can be used according to the methods described herein can be any pharmaceutical agent that inhibits the action of proteasomes. Non-limiting exemplary proteasome inhibitors include bortezomib, carfilzomib, delanzomib, disulfiram, epigallocatechin gallat, epoxomicin, lactacystin, lxazomib citrate, MG132, oprozomib, salinosporamide A, and other inhibitors of proteasomes that are approved by regulatory agencies for use in human patients. In a specific embodiment, the proteasome inhibitor is bortezomib. In a specific embodiment, the proteasome inhibitor that can be used according to the methods described herein is bortezomib. The heat shock protein (HSP) inhibitor that can be used according to the methods described herein can be any pharmaceutical agent that inhibits the action of HSPs. Non-limiting exemplary HSP inhibitors include alvespimycin, AT13387, AUY922, CNF2024/BIIB021, CUDC-305, DS- 2248, ganetespib, HSP990, IPI-493, KW-2478, MPC-3100, PU-H71, retaspimycin, SNX-5422, tanespimycin, XL888, and other inhibitors of HSPs that are approved by regulatory agencies for use in human patients. In a specific embodiment, the HSP inhibitor is an HSP70 or HSP90 inhibitor. In a specific embodiment, the HSP (e.g., Hsp90) inhibitor that can be used according to the methods described herein is AUY922 or BIIB021. The HDAC-PI3K dual inhibitor that can be used according to the methods described herein can be any pharmaceutical agent that inhibits the action of both HDAC and PI3K. Non-limiting exemplary HDAC-PI3K dual inhibitors include CUDC-907, FK-A5, romidepsin, and other inhibitors of both HDAC and PI3K that are approved by regulatory agencies for use in human patients. In a specific embodiment, the HDAC-PI3K dual inhibitor that can be used according to the methods described herein is CUDC-907.

[0072] In certain embodiments of the methods described herein, the Network Brake can be a drug targeting cell cycle, metabolism, cytoskeleton, proteases, topoisomerases, or mitochondria.

[0073] The kinase inhibitor that can be used according to the methods described herein can be any pharmaceutical agent that inhibits kinase activity, and can be, for example, a small molecule, or an antibody or antigen-binding fragment thereof that binds to a kinase. Non- limiting exemplary kinase inhibitors include AD57, AD80, afatinib, aflibercept, axitinib, AZD6244, bevacizumab, bosutinib, cabozantinib, cetuximab, crizotinib, dactolisib (BEZ235), dasatinib, erlotinib, everolimus, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, LY294002, mubritinib, nilotinib, panitumumab, pazopanib, PD0325901, pegaptanib, PI-103, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, SU6656, sunitinib, tofacitinib, trametinib, trastuzumab, vandetanib, vemurafenib, vismodegib, wortmannin, and other inhibitors of kinase(s) that are approved by regulatory agencies for use in human patients. In specific embodiments, the kinase inhibitor is selected from the group consisting of dactolisib, erlotinib, sorafenib, trametinib and vemurafenib.

[0074] In a specific embodiment, the kinase inhibitor is sorafenib, and can be used to target Ret, Raf, VEGFR2, and/or BRAF. In another specific embodiment, the kinase inhibitor is erlotinib, and can be used to target EGFR, ErbB2, ErbB3, and/or ErbB4. In another specific embodiment, the kinase inhibitor is trametinib, and can be used to target MEK, Ras, and/or MAPK. In another specific embodiment, the kinase inhibitor is dactolisib, and can be used to target mTOR and/or PI3K. In another specific embodiment, the kinase inhibitor is vemurafenib, and can be used to target Raf. In another specific embodiment, the kinase inhibitor is ABT-869, and can be used to target FLT3, CSF1R, and/or VEGFR2. In another specific embodiment, the kinase inhibitor is AMG-706, and can be used to target VEGFR2, FLT1, FLT4, and/or KIT. In another specific embodiment, the kinase inhibitor is AST-487, and can be used to target FLT3 and/or KIT. In another specific embodiment, the kinase inhibitor is AZD-1152HQPA, and can be used to target AURKB. In another specific embodiment, the kinase inhibitor is BIRB-796, and can be used to target p38-alpha. In another specific embodiment, the kinase inhibitor is BMS-387032/SNS-032, and can be used to target CDK2. In another specific embodiment, the kinase inhibitor is CHIR-258/TKI-258, and can be used to target FLT3and/or FGFR3. In another specific embodiment, the kinase inhibitor is CHIR-265/RAF-265, and can be used to target BRAF and/or VEGFR2. In another specific embodiment, the kinase inhibitor is CI-1033, and can be used to target EGFR and/or ERBB2. In another specific embodiment, the kinase inhibitor is CP-690550, and can be used to target JAK3. In another specific embodiment, the kinase inhibitor is CP-724714, and can be used to target ERBB2. In another specific embodiment, the kinase inhibitor is dasatinib, and can be used to target ABLl and/or SRC. In another specific embodiment, the kinase inhibitor is EKB-569, and can be used to target EGFR. In another specific embodiment, the kinase inhibitor is flavopiridol, and can be used to target CDK2, CDK9, and/or another CDK family kinase. In another specific embodiment, the kinase inhibitor is gefitinib, and can be used to target EGFR. In another specific embodiment, the kinase inhibitor is GW-2580, and can be used to target CSF1R. In another specific embodiment, the kinase inhibitor is GW-786034, and can be used to target VEGFR2, FLT1, and/or FLT4. In another specific embodiment, the kinase inhibitor is imatinib, and can be used to target ABL1, KIT, and/or PDGFRB. In another specific embodiment, the kinase inhibitor is TNJ-7706621, and can be used to target CDK2, CDKl, and/or AURKB. In another specific embodiment, the kinase inhibitor is lapatinib, and can be used to target EGFR and/or ERBB2. In another specific embodiment, the kinase inhibitor is LY-333531, and can be used to target PRKCB 1. In another specific embodiment, the kinase inhibitor is MLN-518, and can be used to target FLT3 and/or KIT. In another specific embodiment, the kinase inhibitor is MLN-8054, and can be used to target AURKA. In another specific embodiment, the kinase inhibitor is PI-103, and can be used to target PIK3CA. In another specific embodiment, the kinase inhibitor is PKC-412, and can be used to target FLT3 and/or KIT. In another specific embodiment, the kinase inhibitor is PTK- 787, and can be used to target VEGFR2. In another specific embodiment, the kinase inhibitor is roscovitine/CYC-202, and can be used to target CDK2, CDKl, and/or CDK5. In another specific embodiment, the kinase inhibitor is SB-202190, and can be used to target p38-alpha. In another specific embodiment, the kinase inhibitor is SB-203580, and can be used to target p38- alpha. In another specific embodiment, the kinase inhibitor is SB-431542, and can be used to target ALK5 and/or ALK4. In another specific embodiment, the kinase inhibitor is

staurosporine, and can be used to target PRKCH. In another specific embodiment, the kinase inhibitor is SU-14813, and can be used to target VEGFR2, FLT1, PDGFRB, KIT, and/or FLT3. In another specific embodiment, the kinase inhibitor is sunitinib, and can be used to target KIT, VEGFR2, and/or FLT3. In another specific embodiment, the kinase inhibitor is VX-680/MK- 0457, and can be used to target AURKA, AURKB, and/or AURKC. In another specific embodiment, the kinase inhibitor is VX-745, and can be used to target p38-alpha. In another specific embodiment, the kinase inhibitor is ZD-6474, and can be used to target VEGFR2, EGFR, and/or RET. In a specific embodiment, the kinase inhibitor is an inhibitor that is disclosed in Example 1 (Section 6, infra) and/or targets a kinase disclosed in Example 1.

[0075] In a specific embodiment, the kinase inhibitor is selected from the group consisting of AD57, AD80, dactolisib, erlotinib, sorafenib, vandetanib, trametinib and vemurafenib, and the Network Brake is a combination of bortezomib and vorinostat. [0076] In specific embodiments, the kinase inhibitor is a kinase inhibitor listed in the first column of Table 1. In specific embodiments, the Network Brake is a compound or combination of compounds listed in the second column of Table 1. In specific embodiments, the kinase inhibitor is a kinase inhibitor listed in the first column of Table 1, and the Network Brake is a compound or combination of compounds listed in the second column of Table 1.

[0077] Table 1. Kinase inhibitor and Network Brake combinations.

[0078] In a specific embodiment, when the kinase inhibitor administered is bevacizumab, the Network Brake administered is not leucovorin calcium. In another specific embodiment, when the kinase inhibitor administered is bevacizumab, the Network Brake administered is not fluorouracil. In another specific embodiment, when the kinase inhibitor administered is bevacizumab, the Network Brake administered is not irinotecan hydrochloride. In another specific embodiment, when the kinase inhibitor administered is bevacizumab, the Network Brake administered is not a combination of leucovorin calcium, fluorouracil and irinotecan

hydrochloride.

[0079] In a specific embodiment, when the kinase inhibitor administered is cetuximab, the Network Brake administered is not leucovorin calcium. In another specific embodiment, when the kinase inhibitor administered is cetuximab, the Network Brake administered is not fluorouracil. In another specific embodiment, when the kinase inhibitor administered is cetuximab, the Network Brake administered is not irinotecan hydrochloride. In another specific embodiment, when the kinase inhibitor administered is cetuximab, the Network Brake administered is not a combination of leucovorin calcium, fluorouracil and irinotecan

hydrochloride.

[0080] In certain embodiments, when the kinase inhibitor is a PI3K kinase inhibitor, the Network Brake is not bortezomib, bortezomib and dactolisib, bortezomib and PI103, or bortezomib and LY294002. In some embodiments, when the kinase inhibitor is a PI3K kinase inhibitor to treat colorectal cancer, the Network Brake is not bortezomib, bortezomib and dactolisib, bortezomib and PI103, or bortezomib and LY294002.

[0081] In certain embodiments of the methods described herein, the kinase inhibitor can be replaced by an anti-cancer drug that is not a kinase inhibitor. For example, in certain

embodiments of the methods described herein, the kinase inhibitor is replaced with a targeted therapy that is not a kinase inhibitor. Examples of such targeted therapy include, but ar not limited to, drugs targeting cell cycle, metabolism, cytoskeleton, topoisom erases, and

mitochondria.

[0082] In a specific embodiment, provided herein is a method for treating cancer to a patient in need thereof, comprising (a) administering to the patient bortezomib at a subtherapeutic dose and vorinostat at a subtherapeutic dose; and (b) administering to the patient a kinase inhibitor. In a specific embodiment, the bortezomib and vorinostat are concurrently administered to the patient. In another specific embodiment, the bortezomib and vorinostat are sequentially administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours or 6 hours of each other. In another specific embodiment, the bortezomib, vorinostat and kinase inhibitor are concurrently administered to the patient. In another specific embodiment, the bortezomib and vorinostat are concurrently administered to the patient and then the kinase inhibitor is administered to the patient (e.g., the kinase inhibitor may be administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, or 3 days of the administration of the bortezomib and vorinostat). In another specific embodiment, the bortezomib and vorinostat are sequentially administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours or 6 hours of each other, and then the kinase inhibitor is administered to the patient (e.g., the kinase inhibitor may be administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, or 3 days of the administration of the bortezomib and vorinostat). In another specific embodiment, the subtherapeutic dose of bortezomib is about 2-fold, 5-fold, 10- fold, 20-fold, 50-fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating a particular cancer. In one embodiment, the subtherapeutic dose of bortezomib is about 0.6 mg/m², 0.5 mg/m², 0.4 mg/m², 0.3 mg/m², 0.2 mg/m², 0.1 mg/m², 0.05 mg/m², 0.01 mg/m²,

0.005 mg/m 2 , 0.001 mg/m 2 or lower, or about 0.6 mg/m 2 to 0.5 mg/m 2 , 0.5 mg/m 2 to 0.4 mg/m 2 , 0.4 mg/m² to 0.3 mg/m², 0.3 mg/m² to 0.2 mg/m², 0.2 mg/m² to 0.1 mg/m², 0.1 mg/m² to 0.05 mg/m², 0.05 mg/m² to 0.01 mg/m², 0.01 mg/m² to 0.005 mg/m², 0.005 mg/m² to 0.001 mg/m² or lower. In another specific embodiment, the subtherapeutic dose of vorinostat is about 2-fold, 5- fold, 10-fold, 20-fold, 50-fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating a particular cancer. In one embodiment, the subtherapeutic dose of vorinostat is about 200 mg/day, 150 mg/day, 100 mg/day, 50 mg/day, 25 mg/day, 10 mg/day, 5 mg/day, 2 mg/day, 1 mg/day or lower, or about 200 mg/day to 150 mg/day, 150 mg/day to 100 mg/day, 100 mg/day to 50 mg/day, 50 mg/day to 25 mg/day, 25 mg/day to 10 mg/day, 10 mg/day to 5 mg/day, 5 mg/day to 2 mg/day, 2 mg/day to 1 mg/day or lower. In a specific embodiment, the kinase inhibitor is sorafenib, trametinib, erlotinib, dactolisib, vandetanib or vemurafinab. See, e.g., Section 5.1.4, infra, for dose and frequency of administration information. In another specific embodiment, the cancer is a solid tumor cancer, such as thyroid cancer, lung cancer, liver cancer, breast cancer, or melanoma.

[0083] In a specific embodiment, provided herein is a method for treating thyroid cancer to a patient in need thereof, comprising (a) administering to the patient bortezomib at a

subtherapeutic dose and vorinostat at a subtherapeutic dose; and (b) administering to the patient sorafenib. In a specific embodiment, the bortezomib and vorinostat are concurrently

administered to the patient. In another specific embodiment, the bortezomib and vorinostat are sequentially administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours or 6 hours of each other. In another specific embodiment, the bortezomib, vorinostat and sorafenib are concurrently administered to the patient. In another specific embodiment, the bortezomib and vorinostat are concurrently administered to the patient and then sorafenib is administered to the patient {e.g., sorafenib may be administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, or 3 days of the administration of the bortezomib and vorinostat). In another specific embodiment, the bortezomib and vorinostat are sequentially administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours or 6 hours of each other, and then sorafenib is administered to the patient (e.g., sorafenib may be administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, or 3 days of the administration of the bortezomib and vorinostat). In another specific embodiment, the subtherapeutic dose of bortezomib is about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating thyroid cancer. In one embodiment, the subtherapeutic dose of bortezomib is about 0.6 mg/m², 0.5 mg/m², 0.4 mg/m², 0.3 mg/m², 0.2 mg/m², 0.1 mg/m², 0.05 mg/m², 0.01 mg/m², 0.005 mg/m², 0.001 mg/m² or

2 2 2 2 2 2 lower, or about 0.6 mg/m to 0.5 mg/m , 0.5 mg/m to 0.4 mg/m , 0.4 mg/m to 0.3 mg/m , 0.3 mg/m² to 0.2 mg/m², 0.2 mg/m² to 0.1 mg/m², 0.1 mg/m² to 0.05 mg/m², 0.05 mg/m² to 0.01

2 2 2 2 2

mg/m , 0.01 mg/m to 0.005 mg/m , 0.005 mg/m to 0.001 mg/m or lower. In another specific embodiment, the subtherapeutic dose of vorinostat is about 2-fold, 5-fold, 10-fold, 20-fold, 50- fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating thyroid cancer. In one embodiment, the subtherapeutic dose of vorinostat is about 200 mg/day, 150 mg/day, 100 mg/day, 50 mg/day, 25 mg/day, 10 mg/day, 5 mg/day, 2 mg/day, 1 mg/day or lower, or about 200 mg/day to 150 mg/day, 150 mg/day to 100 mg/day, 100 mg/day to 50 mg/day, 50 mg/day to 25 mg/day, 25 mg/day to 10 mg/day, 10 mg/day to 5 mg/day, 5 mg/day to 2 mg/day, 2 mg/day to 1 mg/day or lower. In specific embodiments, sorafenib is administered at an effective dose. In specific embodiments, sorafenib is administered at a subclinical dose. In a specific embodiment, sorafenib is administered to the patient at a dose about 2-fold, 5-fold, 10- fold, 20-fold, 50-fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating thyroid cancer. In one embodiment, sorafenib is administered at about 400 mg/day or higher. In another embodiment, sorafenib is administered at about 200 mg/day, 150 mg/day, 100 mg/day, 50 mg/day, 25 mg/day, 10 mg/day, 5 mg/day, 2 mg/day, 1 mg/day or lower, or about 200 mg/day to 150 mg/day, 150 mg/day to 100 mg/day, 100 mg/day to 50 mg/day, 50 mg/day to 25 mg/day, 25 mg/day to 10 mg/day, 10 mg/day to 5 mg/day, 5 mg/day to 2 mg/day, 2 mg/day to 1 mg/day or lower. In another specific embodiment, the thyroid cancer is associated with a RET mutation (e.g., a RET2A and/or RET2B mutation). The RET2A mutation covers a spectrum of mutations including mutations in codons C609, C61 1, C618, C620, and C634. The RET- MEN2B mutation is primarily a RET-M918T mutation.

[0084] In a specific embodiment, provided herein is a method for treating lung cancer to a patient in need thereof, comprising (a) administering to the patient bortezomib at a

subtherapeutic dose and vorinostat at a subtherapeutic dose; and (b) administering to the patient trametinib. In a specific embodiment, the bortezomib and vorinostat are concurrently

administered to the patient. In another specific embodiment, the bortezomib and vorinostat are sequentially administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours or 6 hours of each other. In another specific embodiment, the bortezomib, vorinostat and trametinib are concurrently administered to the patient. In another specific embodiment, the bortezomib and vorinostat are concurrently administered to the patient and then trametinib is administered to the patient (e.g., trametinib may be administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, or 3 days of the administration of the bortezomib and vorinostat). In another specific embodiment, the bortezomib and vorinostat are sequentially administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours or 6 hours of each other, and then trametinib is administered to the patient (e.g., trametinib may be administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, or 3 days of the administration of the bortezomib and vorinostat). In another specific embodiment, the subtherapeutic dose of bortezomib is about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating lung cancer. In one embodiment, the subtherapeutic dose of bortezomib is about 0.6 mg/m², 0.5 mg/m², 0.4 mg/m², 0.3 mg/m², 0.2 mg/m², 0.1 mg/m², 0.05 mg/m², 0.01 mg/m², 0.005 mg/m², 0.001 mg/m² or

2 2 2 2 2

mg/m , 0.01 mg/m to 0.005 mg/m , 0.005 mg/m to 0.001 mg/m or lower. In another specific embodiment, the subtherapeutic dose of vorinostat is about 2-fold, 5-fold, 10-fold, 20-fold, 50- fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating lung cancer. In one embodiment, the subtherapeutic dose of vorinostat is about 200 mg/day, 150 mg/day, 100 mg/day, 50 mg/day, 25 mg/day, 10 mg/day, 5 mg/day, 2 mg/day, 1 mg/day or lower, or about 200 mg/day to 150 mg/day, 150 mg/day to 100 mg/day, 100 mg/day to 50 mg/day, 50 mg/day to 25 mg/day, 25 mg/day to 10 mg/day, 10 mg/day to 5 mg/day, 5 mg/day to 2 mg/day, 2 mg/day to 1 mg/day or lower. In specific embodiments, trametinib is administered at an effective dose. In specific embodiments, trametinib is administered at a subclinical dose. In a specific

embodiment, trametinib is administered to the patient at a dose about 2-fold, 5-fold, 10-fold, 20- fold, 50-fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating lung cancer. In one embodiment, trametinib is administered at about 2 mg/day or higher. In another embodiment, trametinib is administered at about 1 mg/day, 0.5 mg/day, 0.1 mg/day, 0.05 mg/day, 0.01 mg/day, 0.005 mg/day, 0.001 mg/day or lower, or about 1 mg/day to 0.5 mg/day, 0.5 mg/day to 0.1 mg/day, 0.1 mg/day to 0.05 mg/day, 0.05 mg/day to 0.01 mg/day, 0.01 mg/day to 0.005 mg/day, 0.005 mg/day to 0.001 mg/day or lower. In another specific embodiment, the lung cancer is associated with a Ras mutation (e.g., a RAS-Q61K mutation). In a specific embodiment, the lung cancer is non-small cell lung cancer.

[0085] In a specific embodiment, provided herein is a method for treating lung cancer to a patient in need thereof, comprising (a) administering to the patient bortezomib at a

subtherapeutic dose and vorinostat at a subtherapeutic dose; and (b) administering to the patient erlotinib. In a specific embodiment, the bortezomib and vorinostat are concurrently administered to the patient. In another specific embodiment, the bortezomib and vorinostat are sequentially administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours or 6 hours of each other. In another specific embodiment, the bortezomib, vorinostat and erlotinib are concurrently administered to the patient. In another specific embodiment, the bortezomib and vorinostat are concurrently administered to the patient and then erlotinib is administered to the patient (e.g., erlotinib may be administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, or 3 days of the administration of the bortezomib and vorinostat). In another specific embodiment, the bortezomib and vorinostat are sequentially administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours or 6 hours of each other, and then erlotinib is administered to the patient (e.g., erlotinib may be administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, or 3 days of the administration of the bortezomib and vorinostat). In another specific embodiment, the subtherapeutic dose of bortezomib is about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating lung cancer. In one embodiment, the subtherapeutic dose of bortezomib is about 0.6 mg/m², 0.5 mg/m², 0.4 mg/m², 0.3 mg/m², 0.2 mg/m², 0.1 mg/m², 0.05 mg/m², 0.01 mg/m², 0.005 mg/m², 0.001 mg/m² or lower, or about 0.6 mg/m 2 to 0.5 mg/m 2 , 0.5 mg/m 2 to 0.4 mg/m 2 , 0.4 mg/m 2 to 0.3 mg/m 2 , 0.3 mg/m² to 0.2 mg/m², 0.2 mg/m² to 0.1 mg/m², 0.1 mg/m² to 0.05 mg/m², 0.05 mg/m² to 0.01 mg/m 2 , 0.01 mg/m 2 to 0.005 mg/m 2 , 0.005 mg/m 2 to 0.001 mg/m 2 or lower. In another specific embodiment, the subtherapeutic dose of vorinostat is about 2-fold, 5-fold, 10-fold, 20-fold, 50- fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating lung cancer. In one embodiment, the subtherapeutic dose of vorinostat is about 200 mg/day, 150 mg/day, 100 mg/day, 50 mg/day, 25 mg/day, 10 mg/day, 5 mg/day, 2 mg/day, 1 mg/day or lower, or about 200 mg/day to 150 mg/day, 150 mg/day to 100 mg/day, 100 mg/day to 50 mg/day, 50 mg/day to 25 mg/day, 25 mg/day to 10 mg/day, 10 mg/day to 5 mg/day, 5 mg/day to 2 mg/day, 2 mg/day to 1 mg/day or lower. In specific embodiments, erlotinib is administered at an effective dose. In specific embodiments, erlotinib is administered at a subclincal dose. In a specific embodiment, erlotinib is administered to the patient at a dose about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating lung cancer. In one embodiment, erlotinib is administered at about 150 mg/day or higher. In another

embodiment, erlotinib is administered at about 75 mg/day, 50 mg/day, 25 mg/day, 10 mg/day, 5 mg/day, 2 mg/day, 1 mg/day or lower, or about 75 mg/day to 50 mg/day, 50 mg/day to 25 mg/day, 25 mg/day to 10 mg/day, 10 mg/day to 5 mg/day, 5 mg/day to 2 mg/day, 2 mg/day to 1 mg/day or lower. In another specific embodiment, the lung cancer is ErbB3 positive. In a specific embodiment, the lung cancer is non-small cell lung cancer.

[0086] In a specific embodiment, provided herein is a method for treating liver cancer to a patient in need thereof, comprising (a) administering to the patient bortezomib at a

administered to the patient. In another specific embodiment, the bortezomib and vorinostat are sequentially administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours or 6 hours of each other. In another specific embodiment, the bortezomib, vorinostat and trametinib are concurrently administered to the patient. In another specific embodiment, the bortezomib and vorinostat are concurrently administered to the patient and then trametinib is administered to the patient (e.g., trametinib may be administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, or 3 days of the administration of the bortezomib and vorinostat). In another specific embodiment, the bortezomib and vorinostat are sequentially administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours or 6 hours of each other, and then trametinib is administered to the patient (e.g., trametinib may be administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, or 3 days of the administration of the bortezomib and vorinostat). In another specific embodiment, the subtherapeutic dose of bortezomib is about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating liver cancer. In one embodiment, the subtherapeutic dose of bortezomib is about 0.6 mg/m², 0.5 mg/m², 0.4 mg/m², 0.3 mg/m², 0.2 mg/m², 0.1 mg/m², 0.05 mg/m², 0.01 mg/m², 0.005 mg/m², 0.001 mg/m² or

2 2 2 2 2

mg/m , 0.01 mg/m to 0.005 mg/m , 0.005 mg/m to 0.001 mg/m or lower. In another specific embodiment, the subtherapeutic dose of vorinostat is about 2-fold, 5-fold, 10-fold, 20-fold, 50- fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating liver cancer. In one embodiment, the subtherapeutic dose of vorinostat is about 200 mg/day, 150 mg/day, 100 mg/day, 50 mg/day, 25 mg/day, 10 mg/day, 5 mg/day, 2 mg/day, 1 mg/day or lower, or about 200 mg/day to 150 mg/day, 150 mg/day to 100 mg/day, 100 mg/day to 50 mg/day, 50 mg/day to 25 mg/day, 25 mg/day to 10 mg/day, 10 mg/day to 5 mg/day, 5 mg/day to 2 mg/day, 2 mg/day to 1 mg/day or lower. In specific embodiments, trametinib is administered at an effective dose. In specific embodiments, trametinib is administered at a subclinical dose. In a specific

embodiment, trametinib is administered to the patient at a dose about 2-fold, 5-fold, 10-fold, 20- fold, 50-fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating liver cancer. In one embodiment, trametinib is administered at about 2 mg/day or higher. In another embodiment, trametinib is administered at about 1 mg/day, 0.5 mg/day, 0.1 mg/day, 0.05 mg/day, 0.01 mg/day, 0.005 mg/day, 0.001 mg/day or lower, or about 1 mg/day to 0.5 mg/day, 0.5 mg/day to 0.1 mg/day, 0.1 mg/day to 0.05 mg/day, 0.05 mg/day to 0.01 mg/day, 0.01 mg/day to 0.005 mg/day, 0.005 mg/day to 0.001 mg/day or lower. In a specific embodiment, the liver cancer is a hepatocellular carcinoma.

[0087] In a specific embodiment, provided herein is a method for treating breast cancer to a patient in need thereof, comprising (a) administering to the patient bortezomib at a

subtherapeutic dose and vorinostat at a subtherapeutic dose; and (b) administering to the patient dactolisib. In a specific embodiment, the bortezomib and vorinostat are concurrently

administered to the patient. In another specific embodiment, the bortezomib and vorinostat are sequentially administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours or 6 hours of each other. In another specific embodiment, the bortezomib, vorinostat and dactolisib are concurrently administered to the patient. In another specific embodiment, the bortezomib and vorinostat are concurrently administered to the patient and then dactolisib is administered to the patient (e.g., dactolisib may be administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, or 3 days of the administration of the bortezomib and vorinostat). In another specific embodiment, the bortezomib and vorinostat are sequentially administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours or 6 hours of each other, and then dactolisib is administered to the patient (e.g., dactolisib may be administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, or 3 days of the administration of the bortezomib and vorinostat). In another specific embodiment, the subtherapeutic dose of bortezomib is about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating breast cancer. In one embodiment, the subtherapeutic dose of bortezomib is about 0.6 mg/m², 0.5 mg/m², 0.4 mg/m², 0.3 mg/m², 0.2 mg/m², 0.1 mg/m², 0.05 mg/m², 0.01 mg/m², 0.005 mg/m², 0.001 mg/m² or

2 2 2 2 2

mg/m , 0.01 mg/m to 0.005 mg/m , 0.005 mg/m to 0.001 mg/m or lower. In another specific embodiment, the subtherapeutic dose of vorinostat is about 2-fold, 5-fold, 10-fold, 20-fold, 50- fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating breast cancer. In one embodiment, the subtherapeutic dose of vorinostat is about 200 mg/day, 150 mg/day, 100 mg/day, 50 mg/day, 25 mg/day, 10 mg/day, 5 mg/day, 2 mg/day, 1 mg/day or lower, or about 200 mg/day to 150 mg/day, 150 mg/day to 100 mg/day, 100 mg/day to 50 mg/day, 50 mg/day to 25 mg/day, 25 mg/day to 10 mg/day, 10 mg/day to 5 mg/day, 5 mg/day to

2 mg/day, 2 mg/day to 1 mg/day or lower. In specific embodiments, dactolisib is administered at an effective dose. In another embodiment, dactolisib is administered at a subclinical dose. In a specific embodiment, dactolisib is administered to the patient at a dose about 2-fold, 5-fold, 10- fold, 20-fold, 50-fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating breast cancer. In one embodiment, dactolisib is administered at about 40 mg/kg or higher. In another embodiment, dactolisib is administered at about 20 mg/kg, 10 mg/kg, 5 mg/kg, 1 mg/kg, 0.5 mg/kg, 0.1 mg/kg, 0.05 mg/kg, 0.01 mg/kg or lower, or about 20 mg/kg to 10 mg/kg, 10 mg/kg to 5 mg/kg, 5 mg/kg to 1 mg/kg, 1 mg/kg to 0.5 mg/kg, 0.5 mg/kg to 0.1 mg/kg, 0.1 mg/kg to 0.05 mg/kg, 0.05 mg/kg to 0.01 mg/kg or lower. In another specific embodiment, the breast cancer is associated with a PI3K mutation (e.g., a PI3KCA mutation).

[0088] In a specific embodiment, provided herein is a method for treating breast cancer to a patient in need thereof, comprising (a) administering to the patient bortezomib at a

subtherapeutic dose and vorinostat at a subtherapeutic dose; and (b) administering to the patient BEZ235. In a specific embodiment, the bortezomib and vorinostat are concurrently administered to the patient. In another specific embodiment, the bortezomib and vorinostat are sequentially administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours or 6 hours of each other. In another specific embodiment, the bortezomib, vorinostat and BEZ235 are concurrently administered to the patient. In another specific embodiment, the bortezomib and vorinostat are concurrently administered to the patient and then BEZ235 is administered to the patient (e.g., dactolisib may be administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, or 3 days of the administration of the bortezomib and vorinostat). In another specific embodiment, the bortezomib and vorinostat are sequentially administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours or 6 hours of each other, and then BEZ235 is administered to the patient (e.g., BEZ235 may be administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours,

3 hours, 4 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, or 3 days of the administration of the bortezomib and vorinostat). In another specific embodiment, the subtherapeutic dose of bortezomib is about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating breast cancer. In one embodiment, the subtherapeutic dose of bortezomib is about 0.6 mg/m², 0.5 mg/m², 0.4 mg/m², 0.3 mg/m², 0.2 mg/m², 0.1 mg/m², 0.05 mg/m², 0.01 mg/m², 0.005 mg/m², 0.001 mg/m² or

2 2 2 2 2

mg/m , 0.01 mg/m to 0.005 mg/m , 0.005 mg/m to 0.001 mg/m or lower. In another specific embodiment, the subtherapeutic dose of vorinostat is about 2-fold, 5-fold, 10-fold, 20-fold, 50- fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating breast cancer. In one embodiment, the subtherapeutic dose of vorinostat is about 200 mg/day, 150 mg/day, 100 mg/day, 50 mg/day, 25 mg/day, 10 mg/day, 5 mg/day, 2 mg/day, 1 mg/day or lower, or about 200 mg/day to 150 mg/day, 150 mg/day to 100 mg/day, 100 mg/day to 50 mg/day, 50 mg/day to 25 mg/day, 25 mg/day to 10 mg/day, 10 mg/day to 5 mg/day, 5 mg/day to 2 mg/day, 2 mg/day to 1 mg/day or lower. In specific embodiments, BEZ235 is administered at an effective dose. In another embodiment, BEZ235 is administered at a subclinical dose. In a specific embodiment, dactolisib (BEZ235) is administered to the patient at a dose about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating breast cancer. In one embodiment, dactolisib is administered at about 40 mg/kg or higher. In another embodiment, dactolisib is administered at about 20 mg/kg, 10 mg/kg, 5 mg/kg, 1 mg/kg, 0.5 mg/kg, 0.1 mg/kg, 0.05 mg/kg, 0.01 mg/kg or lower, or about 20 mg/kg to 10 mg/kg, 10 mg/kg to 5 mg/kg, 5 mg/kg to 1 mg/kg, 1 mg/kg to 0.5 mg/kg, 0.5 mg/kg to 0.1 mg/kg, 0.1 mg/kg to 0.05 mg/kg, 0.05 mg/kg to 0.01 mg/kg or lower. In another specific embodiment, the breast cancer is associated with a PI3K mutation (e.g., a PI3KCA mutation). In another specific embodiment, the breast cancer is estrogen receptor positive.

[0089] In a specific embodiment, provided herein is a method for treating melanoma to a patient in need thereof, comprising (a) administering to the patient bortezomib at a

subtherapeutic dose and vorinostat at a subtherapeutic dose; and (b) administering to the patient vemeurafinib. In a specific embodiment, the bortezomib and vorinostat are concurrently administered to the patient. In another specific embodiment, the bortezomib and vorinostat are sequentially administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours or 6 hours of each other. In another specific embodiment, the bortezomib, vorinostat and vemeurafinib are concurrently administered to the patient. In another specific embodiment, the bortezomib and vorinostat are concurrently administered to the patient and then vemeurafinib is administered to the patient (e.g., vemeurafinib may be administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, or 3 days of the administration of the bortezomib and vorinostat). In another specific embodiment, the bortezomib and vorinostat are sequentially administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours or 6 hours of each other, and then vemeurafinib is administered to the patient (e.g., vemurafenib may be administered to the patient within 10 minutes, 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, or 3 days of the administration of the bortezomib and vorinostat). In another specific embodiment, the subtherapeutic dose of bortezomib is about 2-fold, 5-fold, 10-fold, 20- fold, 50-fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating melanoma. In one embodiment, the subtherapeutic dose of bortezomib is about 0.6 mg/m², 0.5 mg/m², 0.4 mg/m², 0.3 mg/m², 0.2 mg/m², 0.1 mg/m², 0.05 mg/m², 0.01 mg/m², 0.005 mg/m²,

2 2 2 2 2 2

0.001 mg/m or lower, or about 0.6 mg/m to 0.5 mg/m , 0.5 mg/m to 0.4 mg/m , 0.4 mg/m to 0.3 mg/m², 0.3 mg/m² to 0.2 mg/m², 0.2 mg/m² to 0.1 mg/m², 0.1 mg/m² to 0.05 mg/m², 0.05 mg/m² to 0.01 mg/m², 0.01 mg/m² to 0.005 mg/m², 0.005 mg/m² to 0.001 mg/m² or lower. In another specific embodiment, the subtherapeutic dose of vorinostat is about 2-fold, 5-fold, 10- fold, 20-fold, 50-fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating melanoma. In one embodiment, the subtherapeutic dose of vorinostat is about 200 mg/day, 150 mg/day, 100 mg/day, 50 mg/day, 25 mg/day, 10 mg/day, 5 mg/day, 2 mg/day, 1 mg/day or lower, or about 200 mg/day to 150 mg/day, 150 mg/day to 100 mg/day, 100 mg/day to 50 mg/day, 50 mg/day to 25 mg/day, 25 mg/day to 10 mg/day, 10 mg/day to 5 mg/day, 5 mg/day to 2 mg/day, 2 mg/day to 1 mg/day or lower. In specific embodiments, vemurafenib is administered at an effective dose. In another specific embodiment, vemurafenib is administered at a subclinical dose. In a specific embodiment, vemurafenib is administered to the patient at a dose about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 75-fold, or 100-fold lower than the dosage proved to be efficacious for treating melanoma. In one embodiment, vemurafenib is

administered at about 960 mg/12hr (microgram per 12 hour) or higher. In another embodiment, vemurafenib is administered at about 480 mg/12hr, 240 mg/12hr, 120 mg/12hr, 60 mg/12hr, 30 mg/12hr, 15 mg/12hr, 5 mg/12hr, 2 mg/12hr or lower, or about 480 mg/12hr to 240 mg/12hr, 240 mg/12hr to 120 mg/12hr, 120 mg/12hr to 60 mg/12hr, 60 mg/12hr to 30 mg/12hr, 30 mg/12hr to 15 mg/12hr, 5 mg/12hr to 2 mg/12hr or lower. In another specific embodiment, the melanoma is associated with a Raf mutation (e.g., a Raf-V600E mutation).

[0090] In certain embodiments, a method of treating cancer as described herein results in one, two, three or more of the following effects: complete response, partial response, increase in overall survival, increase in disease free survival, increase in objective response rate, increase in time to progression, increase in progression-free survival, increase in time-to-treatment failure, and improvement or elimination of one or more symptoms of cancer. In a specific embodiment, a method of treating cancer as described herein results in an increase in overall survival. In another specific embodiment, a method of treating cancer as described herein results in an increase in progression-free survival. In another specific embodiment, a method of treating cancer as described herein results in an increase in overall survival and an increase in progression-free survival. In another specific embodiment, a method of treating cancer as described herein results in the improvement or elimination of one or more symptoms (e.g., clinical symptoms) of cancer. In some embodiment, a method of treating cancer as described herein results in a reduction in the number symptoms (e.g., clinical symptoms) of cancer. In specific embodiments, the increase in overall survival, increase in disease free survival, increase in objective response rate, increase in time to progression, increase in progression-free survival, and/or increase in time-to-treatment failure is relative to patients with the same type of cancer or patients with the same type of cancer in a similar position healthwise. In certain embodiments, the patients with the same type of cancer or patients with the same type of cancer in a similar position healthwise have received an accepted standard of care therapy for the cancer. In certain embodiments, the patients with the same type of cancer or patients with the same type of cancer in a similar position healthwise have not been treated with the kinase inhibitor or a combination of the kinase inhibitor and the Network Brake.

[0091] "Complete response" refers to an absence of clinically detectable disease with normalization of any previously abnormal radiographic studies, bone marrow, and cerebrospinal fluid (CSF) or abnormal monoclonal protein measurements.

[0092] "Partial response" refers to at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%), or 90% decrease in all measurable tumor burden (i.e., the number of malignant cells present in the subject, or the measured bulk of tumor masses or the quantity of abnormal monoclonal protein) in the absence of new lesions.

[0093] "Overall survival" is defined as the time from randomization until death from any cause, and is measured in the intent-to-treat population. Overall survival should be evaluated in randomized controlled studies. Demonstration of a statistically significant improvement in overall survival can be considered to be clinically significant if the toxicity profile is acceptable, and has often supported new drug approval.

[0094] Several endpoints are based on tumor assessments. These endpoints include disease free survival (DFS), objective response rate (ORR), time to progression (TTP), progression-free survival (PFS), and time-to-treatment failure (TTF). The collection and analysis of data on these time-dependent endpoints are based on indirect assessments, calculations, and estimates (e.g., tumor measurements).

[0095] Generally, "disease free survival" (DFS) is defined as the time from randomization until recurrence of tumor or death from any cause. Although overall survival is a conventional endpoint for most adjuvant settings, DFS can be an important endpoint in situations where survival may be prolonged, making a survival endpoint impractical. DFS can be a surrogate for clinical benefit or it can provide direct evidence of clinical benefit. This determination is based on the magnitude of the effect, its risk-benefit relationship, and the disease setting. The definition of DFS can be complicated, particularly when deaths are noted without prior tumor progression documentation. These events can be scored either as disease recurrences or as censored events. Although all methods for statistical analysis of deaths have some limitations, considering all deaths (deaths from all causes) as recurrences can minimize bias. DFS can be overestimated using this definition, especially in patients who die after a long period without observation. Bias can be introduced if the frequency of long-term follow-up visits is dissimilar between the study arms or if dropouts are not random because of toxicity.

[0096] "Objective response rate" (ORR) is defined as the proportion of patients with tumor size reduction of a predefined amount and for a minimum time period. Response duration usually is measured from the time of initial response until documented tumor progression. Generally, the FDA has defined ORR as the sum of partial responses plus complete responses. When defined in this manner, ORR is a direct measure of drug antitumor activity, which can be evaluated in a single-arm study. If available, standardized criteria should be used to ascertain response. A variety of response criteria have been considered appropriate (e.g., RECIST criteria) (Therasse et al., (2000) J. Natl. Cancer Inst, 92: 205-16). The significance of ORR is assessed by its magnitude and duration, and the percentage of complete responses (no detectable evidence of tumor).

[0097] "Time to progression" (TTP) and "progression-free survival" (PFS) have served as primary endpoints for drug approval. TTP is defined as the time from randomization until objective tumor progression; TTP does not include deaths. PFS is defined as the time from randomization until objective tumor progression or death. Compared with TTP, PFS is the preferred regulatory endpoint. PFS includes deaths and thus can be a better correlate to overall survival. PFS assumes patient deaths are randomly related to tumor progression. However, in situations where the majority of deaths are unrelated to cancer, TTP can be an acceptable endpoint.

[0098] As an endpoint to support drug approval, PFS can reflect tumor growth and be assessed before the determination of a survival benefit. Its determination is not confounded by subsequent therapy. For a given sample size, the magnitude of effect on PFS can be larger than the effect on overall survival. However, the formal validation of PFS as a surrogate for survival for the many different malignancies that exist can be difficult. Data are sometimes insufficient to allow a robust evaluation of the correlation between effects on survival and PFS. Cancer trials are often small, and proven survival benefits of existing drugs are generally modest. The role of PFS as an endpoint to support licensing approval varies in different cancer settings. Whether an improvement in PFS represents a direct clinical benefit or a surrogate for clinical benefit depends on the magnitude of the effect and the risk-benefit of the new treatment compared to available therapies.

[0099] "Time-to-treatment failure" (TTF) is defined as a composite endpoint measuring time from randomization to discontinuation of treatment for any reason, including disease

progression, treatment toxicity, and death. TTF is not recommended as a regulatory endpoint for drug approval. TTF does not adequately distinguish efficacy from these additional variables. A regulatory endpoint should clearly distinguish the efficacy of the drug from toxicity, patient or physician withdrawal, or patient intolerance.

5.1.3. Patient Populations [00100] The patient referred to in this disclosure, can be, but is not limited to, a human or non- human vertebrate such as a wild, domestic or farm animal. In certain embodiments, the patient is a mammal, e.g., a human, a cow, a dog, a cat, a goat, a horse, a sheep, a pig, a rabbit, a rat, a mouse, a fly, or a worm. In a specific embodiment, the patient is a human.

[00101] In certain embodiments, the patient has a cancer. In specific embodiments, the patient has been diagnosed with a cancer. In specific embodiments, the cancer is a solid tumor cancer. In specific embodiments, the cancer is a blood cancer.

[00102] The solid tumor cancer can be a sarcoma, a carcinoma, a lymphoma, a germ cell tumor, a blastoma, or a combination thereof. Non-limiting exemplary solid tumor cancers that can be treated or prevented using methods described herein include: adrenal cancer, anal cancer, basal and squamous cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophagus cancer, ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor, gestational trophoblastic disease, hodgkin disease, kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer (such as small cell lung cancer and non-small cell lung cancer), lung carcinoid tumor, melanoma, merkel cell carcinoma, mesothelioma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parotid cancer, penile cancer, pituitary tumors, prostate cancer, rectum cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, skin lymphoma, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterus cancer, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor.

[00103] In specific embodiments, the solid tumor cancer is a primary cancer. In a specific embodiment, the solid tumor cancer is a primary cancer and has not metastasized. In another specific embodiment, the solid tumor cancer is a primary cancer and has metastasized. In specific embodiments, the solid tumor cancer is a secondary cancer.

[00104] Non-limiting exemplary blood cancers that can be treated or prevented using methods described herein include: acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, hairy cell leukemia, T-cell

prolymphocytic leukemia, Large granular lymphocytic leukemia, adult T-cell leukemia, plasma cell leukemia, Hodgkin lymphoma, Non-Hodgkin lymphoma, and multiple myeloma, [00105] In specific embodiments, the cancer has oncogenic activation of RET, VEGFR, PDGFR, Raf, KIT, and/or FLT-3. In a specific embodiment, the cancer has oncogenic activation of RET, VEGFR, PDGFR, Raf, KIT, and/or FLT-3, the kinase inhibitor administered to the patient is sorafenib, and the Network Brake administered to the patient is a combination of bortezomib and vorinostat, a combination of bortezomib and CUDC-907, or a combination of vorinostat and AUY922.

[00106] In specific embodiments, the cancer has oncogenic activation of MAPK/ERK signaling. In a specific embodiment, the cancer has oncogenic activation of MAPK/ERK signaling, the kinase inhibitor administered to the patient is trametinib, and the Network Brake administered to the patient is a combination of bortezomib and vorinostat, a combination of bortezomib and CUDC-907, or a combination of vorinostat and AUY922.

[00107] In specific embodiments, the cancer has oncogenic activation of ErbB3, EGFR or EGFR signaling. In a specific embodiment, the cancer has oncogenic activation of ErbB3, EGFR or EGFR signaling, the kinase inhibitor administered to the patient is erlotinib, and the Network Brake administered to the patient is a combination of bortezomib and vorinostat, a combination of bortezomib and CUDC-907, or a combination of vorinostat and AUY922.

[00108] In specific embodiments, the cancer has oncogenic activation of the MAPK/ERK signaling. In a specific embodiment, the cancer has oncogenic activation of the MAPK/ERK signaling, the kinase inhibitor administered to the patient is trametinib, and the Network Brake administered to the patient is a combination of bortezomib and vorinostat, a combination of bortezomib and CUDC-907, or a combination of vorinostat and AUY922.

[00109] In specific embodiments, the cancer has oncogenic activation of PI3K or PI3K signling (e.g., due to a mutation in PI3K). In a specific embodiment, the cancer has oncogenic activation of PI3K or PI3K signaling (e.g., due to a mutation in PI3K), the kinase inhibitor administered to the patient is dactolisib, and the Network Brake administered to the patient is a combination of bortezomib and vorinostat, a combination of bortezomib and CUDC-907, or a combination of vorinostat and AUY922.

[00110] In specific embodiments, the cancer has oncogenic activation of B-Raf or a signaling through B-Raf (e.g., due to a mutation in B-Raf). In a specific embodiment, the cancer has oncogenic activation of B-Raf or a signaling through B-Raf (e.g., due to a mutation in B-Raf), the kinase inhibitor administered to the patient is vemurafenib, and the Network Brake administered to the patient is a combination of bortezomib and vorinostat, a combination of bortezomib and CUDC-907, or a combination of vorinostat and AUY922.

[00111] In a specific embodiment, the cancer has an oncogenic activation like an oncogenic activitation disclosed in Example 1 (Section 6, infra).

[00112] In specific embodiments, the solid tumor cancer is a thyroid cancer. In some embodiments, the patient has one or more of the following symptoms associated with thyroid cancer: hoarse voice, neck pain, neck swelling, ear pain, enlarged lymph nodes, difficulty in swallowing, difficulty in breathing, constant wheezing, and/or continuous cough not due to cold . In certain embodiments, the patient has not developed symptoms associated with thyroid cancer. In specific embodiments, the thyroid cancer is selected from the group consisting of papillary thyroid cancer, follicular thyroid cancer, medullary thyroid cancer, anaplastic thyroid cancer, and combinations thereof. In a specific embodiment, the patient has MEN2A (multiple endocrine neoplasia type 2A) syndrome, such as medullary thyroid carcinoma, pheochromocytoma, and mucosal neuromas. In another specific embodiment, the patient has MEN2B (multiple endocrine neoplasia type 2B) syndrome. In specific embodiments, the thyroid cancer has oncogenic activation of RET {e.g., due to a mutation in RET). In a specific embodiment, the thyroid cancer has oncogenic activation of RET {e.g., due to a mutation in RET), the kinase inhibitor administered to the patient is sorafenib, and the Network Brake administered to the patient is a combination of bortezomib and vorinostat, a combination of bortezomib and CUDC-907, or a combination of vorinostat and AUY922. In specific embodiments, the thyroid cancer has oncogenic activation of VEGFR, PDGFR, Raf, KIT, and/or FLT-3. In a specific embodiment, the thyroid cancer has oncogenic activation of VEGFR, PDGFR, Raf, KIT, and/or FLT-3, the kinase inhibitor administered to the patient is sorafenib, and the Network Brake administered to the patient is a combination of bortezomib and vorinostat, a combination of bortezomib and CUDC-907, or a combination of vorinostat and AUY922.

[00113] In specific embodiments, the solid tumor cancer is a lung cancer. In some

embodiments, the patient has one or more of the following symptoms associated with lung cancer: chronic, hacking and raspy coughing with occasional blood-streked mucus, recurring respiratory infections {e.g., bronchitis and/or pneumonia), increasing shortness of breath, wheezing, persistent chest pain, hoarseness, neck swelling, face swelling, pain and weakness in the shoulder, arm or hand, fatigue, loss of weight and appetite, intermittent fever, severe headaches, body pain, and/or difficulty in swallowing. In certain embodiments, the patient has not developed symptoms associated with lung cancer. In specific embodiments, the lung cancer is selected from the group consisting of small cell lung cancer, non-small cell lung cancer, lung carcinoid tumor, and combinations thereof. In specific embodiments, the lung cancer has oncogenic activation of Ras (e.g., due to a mutation in Ras). The Ras gene can be H-Ras, K-Ras, or N-Ras. In a specific embodiment, the lung cancer has oncogenic activation of Ras (e.g., due to a mutation in Ras), the kinase inhibitor administered to the patient is trametinib, and the Network Brake administered to the patient is a combination of bortezomib and vorinostat, a combination of bortezomib and CUDC-907, or a combination of vorinostat and AUY922. In specific embodiments, the lung cancer has oncogenic activation of MAPK/ERK signaling. In a specific embodiment, the lung cancer has oncogenic activation of MAPK/ERK signaling, the kinase inhibitor administered to the patient is trametinib, and the Network Brake administered to the patient is a combination of bortezomib and vorinostat, a combination of bortezomib and CUDC-907, or a combination of vorinostat and AUY922. In specific embodiments, the lung cancer has oncogenic activation of ErbB3 (e.g., due to a mutation in ErbB3). In a specific embodiment, the lung cancer has oncogenic activation of ErbB3 (e.g., due to a mutation in ErbB3), the kinase inhibitor administered to the patient is erlotinib, and the Network Brake administered to the patient is a combination of bortezomib and vorinostat, a combination of bortezomib and CUDC-907, or a combination of vorinostat and AUY922. In specific embodiments, the lung cancer has oncogenic activation of EGFR or EGFR signaling. In a specific embodiment, the lung cancer has oncogenic activation of EGFR or EGFR signaling, the kinase inhibitor administered to the patient is erlotinib, and the Network Brake administered to the patient is a combination of bortezomib and vorinostat, a combination of bortezomib and CUDC-907, or a combination of vorinostat and AUY922.

[00114] In specific embodiments, the solid tumor cancer is a liver cancer. In some

embodiments, the patient has one or more of the following symptoms associated with liver cancer: loss of weight, loss of appetite, feeling very full after a small meal, nausea, vomiting, enlarged liver felt as a mass under the ribs, pain in the abdomen or near the right shoulder blade, swelling or fluid build-up in the abdomen, itching, yellowing of the skin and eyes, fever, enlarged veins on the belly that can be seen through the skin, abnormal bruising, and/or abnormal bleeding. In certain embodiments, the patient has not developed symptoms associated with liver cancer. In specific embodiments, the liver cancer is selected from the group consisting of hepatocellular carcinoma, fibrolamellar hepatocellular carcinoma, cholangiocarcinoma, angiosarcoma, hepatoblastoma, and combinations thereof. In specific embodiments, the liver cancer has oncogenic activation of the MAPK/ERK signaling. In a specific embodiment, the liver cancer has oncogenic activation of the MAPK/ERK signaling, the kinase inhibitor administered to the patient is trametinib, and the Network Brake administered to the patient is a combination of bortezomib and vorinostat, a combination of bortezomib and CUDC-907, or a combination of vorinostat and AUY922.

[00115] In specific embodiments, the solid tumor cancer is a breast cancer. In some embodiments, the patient has one or more of the following symptoms associated with breast cancer: swelling of all or part of a breast, lump in a breast, skin irritation or dimpling, breast or nipple pain, nipple retraction, redness of the nipple or breast skin, scaliness of the nipple or breast skin, thickening of the nipple or breast skin, and/or nipple discharge. In certain embodiments, the patient has not developed symptoms associated with breast cancer. In specific embodiments, the breast cancer is selected from the group consisting of ductal carcinoma in situ, invasive (or infiltrating) ductal carcinoma (including tubular carcinoma of the breast, medullary carcinoma of the brest, mucinous carcinoma of the breast, papillary carcinoma of the breast, and cribriform carcinoma of the breast), invasive (or infiltrating) lobular carcinoma, inflammatory breast cancer, lobular carcinoma in situ, paget disease of the nipple, phyllodes tumor,

angiosarcoma, and combinations thereof. In specific embodiments, the breast cancer has oncogenic activation of PI3K or PI3K signling (e.g., due to a mutation in PI3K). In a specific embodiment, the breast cancer has oncogenic activation of PI3K or PI3K signaling (e.g., due to a mutation in PI3K), the kinase inhibitor administered to the patient is dactolisib or BEZ-235, and the Network Brake administered to the patient is a combination of bortezomib and vorinostat, a combination of bortezomib and CUDC-907, or a combination of vorinostat and AUY922.

[00116] In specific embodiments, the solid tumor cancer is a melanoma. In some

embodiments, the patient has one or more of the following symptoms associated with melanoma: new spot on the skin, change in size of a spot, change in shape of a spot, change in color of a spot, change in the surface of a spot (e.g., scaliness, oozing, bleeding, or the appearance of a bump or nodule), appearance of a spot different from all other spots on skin, non-asymmetric spot, spot with irregular edge, spot with blurred edge, spot with uneven color, spot with shades of brown or black, spot with patches of pink, red, white or blue, spot larger than 6 mm across, and/or spread of pigment from the border of a spot into surrounding skin. In certain

embodiments, the patient has not developed symptoms associated with melanoma. In specific embodiments, the melanoma is selected from the group consisting of superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral melanoma, acral lentiginous melanoma, amelanotic melanoma, ocular melanoma, anorectal melanoma, and combinations thereof. In specific embodiments, the melanoma has oncogenic activation of B-Raf or a signaling through B-Raf (e.g., due to a mutation in B-Raf). In a specific embodiment, the melanoma has oncogenic activation of B-Raf or a signaling through B-Raf (e.g., due to a mutation in B-Raf), the kinase inhibitor administered to the patient is vemurafenib, and the Network Brake administered to the patient is a combination of bortezomib and vorinostat, a combination of bortezomib and CUDC-907, or a combination of vorinostat and AUY922.

[00117] In specific embodiments, the patient has failed a previous treatment with the kinase inhibitor. In some specific embodiments, the patient has failed the previous treatment due to intolerance of the toxicity of the previous treatment with the kinase inhibitor. In such specific embodiments, the Network Brake can be administered in combination with a lower dose of the kinase inhibitor to avoid intolerance, while achieving the same or even higher therapeutic efficacy. In some specific embodiments, the patient has failed the previous treatment due to resistance to the kinase inhibitor. In such specific embodiments, the Network Brake can be administered in combination with the kinase inhibitor to reduce the resistance to the kinase inhibitor.

[00118] In specific embodiments, the patient has not been previously treated with the kinase inhibitor, and the Network Brake is administered as part of a front line therapy to avoid intolerance of the kinase inhibitor due to toxicity, and/or to prevent or to reduce resistance to the kinase inhibitor, while achieving similar or even higher therapeutic efficacy.

[00119] In a specific embodiment, the patient is over the age of 10. In another specific embodiment, the patient is over the age of 20. In another specific embodiment, the patient is over the age of 30. In another specific embodiment, the patient is over the age of 40. In another specific embodiment, the patient is over the age of 50. In another specific embodiment, the patient is over the age of 60. In another specific embodiment, the patient is over the age of 70. In another specific embodiment, the patient is over the age of 80. In another specific embodiment, the patient is under the age of 10. In another specific embodiment, the patient is under the age of 20. In another specific embodiment, the patient is under the age of 30. In another specific embodiment, the patient is under the age of 40. In another specific embodiment, the patient is under the age of 50. In another specific embodiment, the patient is under the age of 60.

[00120] In a specific embodiment, the patient is 2 to 10 years old. In another specific embodiment, the patient is 10 to 20 years old. In another specific embodiment, the patient is 18 to 70 years old. In another specific embodiment, the patient is 18 to 50 years old.

5.1.4. Dosage and Administration

[00121] The Network Brake and/or the kinase inhibitor for use in treating a specific cancer in a patient can be selected by a physician and based on the nature of the specific cancer and condition of the patient, and/or can be selected by screening methods as described in Section 5.3 and/or Section 6, infra. Moreover, the subtherapeutic dose(s) at which the Network Brake should be administered and the dosing frequency, and the dose at which the kinase inhibitor should be administered and the dosing frequency can also be determined by a physician and based on the nature of the specific cancer, condition of the patient, and route of administration, and/or determined by screening methods as described in Section 5.3 and/or Section 6, infra.

[00122] For personalized therapy, cells {e.g., cancer cells) from the patient, an animal model mimicking the patient's cancer {e.g., a fly cancer model or mouse/rat cancer model), or any other cancer models can be used to identify the specific combination of Network Brake and kinase inhibitor, and the appropriate dosage regimen for the patient.

[00123] For patients having thyroid cancer, fly-based animal models harboring a mutation(s) critical for thyroid cancer development and/or progression {e.g., flies with oncogenic activation of RET to model MEN2) can be used, for example those described in Das and Cagan, 2013, Drug Discov Today Technol 10:e65-e71, and Rudrapatna et al., 2011, Developmental Dynamics 241 : 107-118. Various mouse models of thyroid cancer can also be used, for example those described in Charles, 2015, Curr Protoc Microbiol 69: 14.33.1-14.33.14; Russo et al., 2012, Front Endocrinol (Lausanne) 2: 119; and Zhu and Cheng, 2009, Horm Metab Res 41 :488-499.

[00124] For patients having lung cancer, fly-based animal models harboring a mutation(s) critical for lung cancer development and/or progression {e.g., flies with oncogenic activation of Ras) can be used to identify the specific combination of Network Brake and kinase inhibitor, for example those described in Miles et al., 201 1 4:753-761, and Rudrapatna et al., 201 1, Developmental Dynamics 241 : 107-1 18. Various mouse models of lung cancer can also be used, for example those described in Kwon and Berns, 2013, Molecular Oncology 7: 165-177, and Wang et al., 2012, Prog Mol Biol Transl Sci 105 :21 1-226.

[00125] For patients having liver cancer, fly-based animal models harboring a mutation(s) critical for liver cancer development and/or progression (e.g., flies with oncogenic activation of MAPK/ERK signaling) can be used to identify the specific combination of Network Brake and kinase inhibitor, for example those described in Miles et al., 201 1 4:753-761, and Rudrapatna et al., 201 1, Developmental Dynamics 241 : 107-1 18. Various mouse models of liver cancer can also be used, for example those described in He et al., 2015, Oncotarget 6:23306-23322, and Caviglia and Schwabe, 2015, Chapter 8: Mouse Models of Liver Cancer In Eferl & Casanova (Eds.), Methods in Molecular Biology: Mouse Models of Cancer 165-183, New York, NY: Springer New York.

[00126] For patients having breast cancer, fly-based animal models harboring a mutation(s) critical for breast cancer development and/or progression (e.g., flies with oncogenic activation of PI3K) can be used to identify the specific combination of Network Brake and kinase inhibitor, for example those described in Miles et al., 201 1 4:753-761, and Ntziachristos et al., 2014, Cancer Cell 25 :318-334. Various mouse models of breast cancer can also be used, for example those described in Hollern and Andrechek, 2014, Breast Cancer Research, 16:R59, and

Klarenbeek et al., 2013, Molecular Oncology 7: 146-164.

[00127] For patients having melanoma, fly-based animal models harboring a mutation(s) critical for melanoma development and/or progression (e.g., flies with oncogenic activation of B- Raf) can be used to identify the specific combination of Network Brake and kinase inhibitor, for example those described in Holderfield et al., 2014, Nature Reviews Cancer 14:455-467 and Kassel et al., 1963, Ann N Y Acad Sci, 100:791-816. Various mouse models of melanoma can also be used, for example those described in Walker et al., 201 1, Pigment Cell & Melanoma Research, 24: 1 158-1 176, and Fairchild and Carson, 2010, Chapter 1 1 : Animal Models of Melanoma In Teicher (Ed), Cancer Drug Discovery and Development: Tumor Models in Cancer Research 259-285, New York, NY: Humana Press. [00128] In a specific embodiment, for patients having amultiple endocrine neoplasia type 2B, a fly model such as described in Example 1 (Section 6, infra) may be used to identify the specific combination of Network Brake and kinase inhibitor.

[00129] The Network Brake and the kinase inhibitor can be administered to the patient by any medically-acceptable route known in the art suitable to the administration of the Network Brake and the kinase inhibitor, respectively, including the route(s) employed in standard-of-care therapies. Non-limiting exemplary routes of administration that can be employed include:

infusion, injection, intraarterial administration, intracerebral administration,

intracerebroventricular administration, intraci sternal administration, intradermal administration, intrafemoral administration, intramuscularly administration, intranasal administration, intraperitoneal administration, intraspinal administration, intratracheal administration, intrathecal administration, intratumor administration, intravenous administration, intraventricular administration, ophthalmical administration, oral administration, parenteral administration, perispinal administration, pulmonary administration, subcutaneous administration, transdermal administration, and topical administration.

[00130] The kinase inhibitor is preferably administered subsequent to or concurrently (for example, at about the same time, the same day, or same week, or same period during which the Network Brake is administered, or on similar dosing schedules, or on different but overlapping dosing schedules) with the administration of the Network Brake. The kinase inhibitor and the Network Brake can be in the same pharmaceutical formulation or in separate formulations.

[00131] In specific embodiments, the kinase inhibitor is administered at about 0.1, 0.5, 1, 2, 3, 4, 5, 7.5, or 10 mg per dose. In specific embodiments, the kinase inhibitor is administered at about 0.1 to 0.5, 0.5 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 7.5, or 7.5 to 10 mg per dose. In specific embodiments, the kinase inhibitor is administered at about 10, 15, 25, 50, 75, 100, 125, 150, 175, or 200 mg per dose. In specific embodiments, the kinase inhibitor is administered at about 10 to 15, 15 to 25, 25 to 50, 50 to 75, 75 to 100, 100 to 125, 125 to 150, 150 to 175, or 175 to 200 mg per dose. In specific embodiments, the kinase inhibitor is administered at about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mg per dose. In specific embodiments, the kinase inhibitor is administered at about 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, 900 to 1000, 1000 to 1100, 1100 to 1200, 1200 to 1300, 1300 to 1400, 1400 to 1500, 1500 to 1600, 1600 to 1700, 1700 to 1800, 1800 to 1900, or 1900 to 2000 mg per dose. In specific embodiments, the kinase inhibitor is administered at lower than 0.1 to 0.5, 0.5 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 7.5, or 7.5 to 10 mg per dose. In specific embodiments, the kinase inhibitor is administered at lower than 10, 15, 25, 50, 75, 100, 125, 150, 175, or 200 mg per dose. In specific embodiments, the kinase inhibitor is administered at lower than 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mg per dose. In specific embodiments, the kinase inhibitor is administered at greater than 0.1 to 0.5, 0.5 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 7.5, or 7.5 to 10 mg per dose. In specific embodiments, the kinase inhibitor is administered at greater than 10, 15, 25, 50, 75, 100, 125, 150, 175, or 200 mg per dose. In specific embodiments, the kinase inhibitor is administered at greater than 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mg per dose. In certain embodiments, the kinase inhibitor is administered twice a day. In certain embodiments, the kinase inhibitor is administered daily. In certain embodiments, the kinase inhibitor is administered once every two days. In certain embodiments, the kinase inhibitor is administered twice a week. In certain embodiments, the kinase inhibitor is administered once a week.

[00132] When the kinase inhibitor is erlotinib, in specific embodiments, erlotinib is administered at about 150 mg per dose (e.g., when the cancer is non-small cell lung cancer). In specific embodiments, erlotinib is administered at about 100 mg per dose (e.g., when the cancer is pancreatic cancer). In specific embodiments, erlotinib is administered at about 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, or 900 mg per dose. In specific embodiments, erlotinib is administered at about 25 to 50, 50 to 75, 75 to 100, 100 to 125, 125 to 150, 150 to 175, 175 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 450, 450 to 500, 500 to 550, 550 to 600, 600 to 650, 650 to 700, 700 to 750, 750 to 800, 800 to 850, or 850 to 900 mg per dose. In specific embodiments, erlotinib is administered at about 75, 100, 125, 150, 175, or 200 mg per dose. In specific embodiments, erlotinib is administered at about 75 to 100, 100 to 125, 125 to 150, 150 to 175, or 175 to 200 mg per dose. In a specific embodiment, erlotinib is administered at lower than 100 mg per dose. In another specific embodiment, erlotinib is administered at lower than 150 mg per dose. In another specific embodiment, erlotinib is administered at greater than 100 mg per dose. In another specific embodiment, erlotinib is administered at greater than 150 mg per dose. In another specific embodiment, erlotinib is administered at greater than 300 mg per dose. In another specific embodiment, erlotinib is administered at greater than 450 mg per dose. In certain embodiments, erlotinib is administered twice a day, daily, once every two days, twice a week, or once a week. In specific embodiments, erlotinib is administered daily. In a specific

embodiment, erlotinib is administered at about 150 mg per dose daily (e.g., when the cancer is non-small cell lung cancer). In another specific embodiment, erlotinib is administered at about 100 mg per dose daily (e.g., when the cancer is pancreatic cancer).

[00133] When the kinase inhibitor is dactolisib, in specific embodiments, dactolisib is administered at about 25, 50, 100, 125, 150, 175, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1 100, 1200, 1300, 1400, 1500, or 1600 mg per dose. In specific embodiments, dactolisib is administered at about 25 to 50, 50 to 100, 100 to 125, 125 to 150, 150 to 175, 175 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, 900 to 1000, 1000 to 1 100, 1 100 to 1200, 1200 to 1300, 1300 to 1400, 1400 to 1500, or 1500 to 1600 mg per dose. In specific embodiments, dactolisib is administered at about 200, 300, or 400 mg per dose. In specific embodiments, dactolisib is administered at about 200 to 300, or 300 to 400 mg per dose. In a specific embodiment, dactolisib is administered at lower than 25 mg per dose. In another specific embodiment, dactolisib is administered at lower than 200 mg per dose. In another specific embodiment, dactolisib is administered at greater than 400 mg per dose. In another specific embodiment, dactolisib is administered at greater than 800 mg per dose. In another specific embodiment, dactolisib is administered at greater than 1600 mg per dose. In certain embodiments, dactolisib is administered twice a day, daily, once every two days, twice a week, or once a week. In specific embodiments, dactolisib is administered twice a day.

[00134] When the kinase inhibitor is sorafenib, in specific embodiments, sorafenib is administered at about 400 mg per dose (e.g., when the cancer is differentiated thyroid carcinoma, hepatocellular carcinoma, or renal cell carcinoma). In specific embodiments, sorafenib is administered at about 25, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1 100, or 1200 mg per dose. In specific embodiments, sorafenib is administered at about 25 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, 900 to 1000, 1000 to 1 100, or 1 100 to 1200 mg per dose. In specific

embodiments, sorafenib is administered at about 300, 400, or 500 mg per dose. In specific embodiments, sorafenib is administered at about 300 to 400, or 400 to 500 mg per dose. In a specific embodiment, sorafenib is administered at lower than 400 mg per dose. In another specific embodiment, sorafenib is administered at greater than 400 mg per dose. In another specific embodiment, sorafenib is administered at greater than 600 mg per dose. In another specific embodiment, sorafenib is administered at greater than 800 mg per dose. In certain embodiments, sorafenib is administered twice a day, daily, once every two days, twice a week, or once a week. In specific embodiments, sorafenib is administered twice a day. In a specific embodiment, sorafenib is administered at about 400 mg per dose twice a day (e.g., when the cancer is differentiated thyroid carcinoma, hepatocellular carcinoma, or renal cell carcinoma).

[00135] When the kinase inhibitor is trametinib, in specific embodiments, trametinib is administered at about 2 mg per dose. In specific embodiments, trametinib is administered at about 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, or 4 mg per dose. In specific embodiments, trametinib is administered at about 0.5 to 0.75, 0.75 to 1, 1 to 1.5, 1.5 to 2, 2 to 2.5, 2.5 to 3, 3 to 3.5, or 3.5 to 4 mg per dose. In specific embodiments, trametinib is administered at about 1.5, 2, or 2.5 mg per dose. In specific embodiments, trametinib is administered at about 1.5 to 2, or 2 to 2.5 mg per dose. In a specific embodiment, trametinib is administered at lower than 2 mg per dose. In another specific embodiment, trametinib is administered at greater than 2 mg per dose. In another specific embodiment, trametinib is administered at greater than 3 mg per dose. In another specific embodiment, trametinib is administered at greater than 4 mg per dose. In certain embodiments, trametinib is administered twice a day, daily, once every two days, twice a week, or once a week. In specific embodiments, trametinib is administered daily. In a specific embodiment, trametinib is administered at about 2 mg per dose daily.

[00136] When the kinase inhibitor is vemurafenib, in specific embodiments, vemurafenib is administered at about 960 mg per dose (e.g., when the cancer is melanoma). In specific embodiments, vemurafenib is administered at about 120, 240, 360, 480, 720, 960, 1200, 1440, 1680, or 1920 mg per dose. In specific embodiments, vemurafenib is administered at about 120 to 240, 120 to 360, 360 to 480, 480 to 720, 720 to 960, 960 to 1200, 1200 to 1440, 1440 to 1680, or 1680 to 1920 mg per dose. In specific embodiments, vemurafenib is administered at about 720, 960, or 1200 mg per dose. In specific embodiments, vemurafenib is administered at about 720 to 960, or 960 to 1200 mg per dose. In a specific embodiment, vemurafenib is administered at lower than 960 mg per dose. In another specific embodiment, vemurafenib is administered at greater than 960 mg per dose. In another specific embodiment, vemurafenib is administered at greater than 1440 mg per dose. In another specific embodiment, vemurafenib is administered at greater than 1920 mg per dose. In certain embodiments, vemurafenib is administered twice a day, daily, once every two days, twice a week, or once a week. In specific embodiments, vemurafenib is administered twice a day. In a specific embodiment, vemurafenib is administered at about 960 mg per dose twice a day (e.g., when the cancer is melanoma).

[00137] In specific embodiments, the Network Brake compound or one of the Network Break compounds is administered at about 0.1, 0.5, 1, 2, 3, 4, 5, 7.5, or 10 mg per dose. In specific embodiments, the Network Brake compound or one of the Network Break compounds is administered at about 0.1 to 0.5, 0.5 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 7.5, or 7.5 to 10 mg per dose. In specific embodiments, the Network Brake compound or one of the Network Break compounds is administered at about 10, 15, 25, 50, 75, 100, 125, 150, 175, or 200 mg per dose. In specific embodiments, the Network Brake compound or one of the Network Break compounds is administered at about 10 to 15, 15 to 25, 25 to 50, 50 to 75, 75 to 100, 100 to 125, 125 to 150, 150 to 175, or 175 to 200 mg per dose. In specific embodiments, the Network Brake compound or one of the Network Break compounds is administered at about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mg per dose. In specific embodiments, the Network Brake compound or one of the Network Break compounds is administered at about 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, 900 to 1000, 1000 to 1 100, 1 100 to 1200, 1200 to 1300, 1300 to 1400, 1400 to 1500, 1500 to 1600, 1600 to 1700, 1700 to 1800, 1800 to 1900, or 1900 to 2000 mg per dose. In specific embodiments, the Network Brake compound or one of the Network Break compounds is administered at lower than 0.1 to 0.5, 0.5 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 7.5, or 7.5 to 10 mg per dose. In specific embodiments, the Network Brake compound or one of the Network Break compounds is administered at lower than 10, 15, 25, 50, 75, 100, 125, 150, 175, or 200 mg per dose. In specific embodiments, the Network Brake compound or one of the Network Break compounds is administered at lower than 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mg per dose. In specific

embodiments, the Network Brake compound or one of the Network Break compounds is administered at greater than 0.1 to 0.5, 0.5 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 7.5, or 7.5 to 10 mg per dose. In specific embodiments, the Network Brake compound or one of the Network Break compounds is administered at greater than 10, 15, 25, 50, 75, 100, 125, 150, 175, or 200 mg per dose. In specific embodiments, the Network Brake compound or one of the Network Break compounds is administered at greater than 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mg per dose. In certain embodiments, the Network Brake compound or one of the Network Break compounds is administered twice a day. In certain embodiments, the Network Brake compound or one of the Network Break compounds is administered daily. In certain embodiments, the Network Brake compound or one of the Network Break compounds is administered once every two days. In certain embodiments, the Network Brake compound or one of the Network Break compounds is administered twice a week. In certain embodiments, the Network Brake compound or one of the Network Break compounds is administered once a week.

[00138] When the Network Brake is bortezomib or comprises bortezomib, in some

embodiments, bortezomib is administered at about 1.3 mg/m² per dose (e.g., by intravenous or subcutaneous administration). In specific embodiments, bortezomib is administered at about 0.3, 0.5, 0.7, 0.9, 1.1, 1.3, 1.5, 1.7, 1.9, 2.1, 2.3, 2.5 mg/m² per dose. In specific embodiments, bortezomib is administered at about 0.3 to 0.5, 0.5 to 0.7, 0.7 to 0.9, 0.9 to 1.1, 1.1 to 1.3, 1.3 to 1.5, 1.5 to 1.7, 1.7 to 1.9, 1.9 to 2.1, 2.1 to 2.3, or 2.3 to 2.5 mg/m² per dose. In specific embodiments, bortezomib is administered at about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, or 1.6 mg/m² per dose. In specific embodiments, bortezomib is administered at about 1.0 to 1.1, 1.1 to 1.2, 1.2 to 1.3, 1.3 to 1.4, 1.4 to 1.5, or 1.5 to 1.6 mg/m² per dose. In specific embodiments, bortezomib is administered at lower than 1.3 mg/m² per dose. In specific embodiments, bortezomib is administered at about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, or 1.2 mg/m² per dose. In specific embodiments, bortezomib is administered at about 0.2 to 0.4, 0.4 to 0.6, 0.6 to 0.8, 0.8 to 1.0, or 1.0 to 1.2 mg/m² per dose. When the Network Brake is bortezomib or comprises bortezomib and the cancer is multiple myeloma or mantle cell lymphoma, bortezomib is administered at lower than 1.3 mg/m² per dose. When the Network Brake is bortezomib or comprises bortezomib and the cancer is multiple myeloma or mantle cell lymphoma, in specific embodiments, bortezomib is administered at about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, or 1.2 mg/m² per dose. When the Network Brake is bortezomib or comprises bortezomib and the cancer is multiple myeloma or mantle cell lymphoma, in specific embodiments, bortezomib is administered at about 0.2 to 0.4, 0.4 to 0.6, 0.6 to 0.8, 0.8 to 1.0, or 1.0 to 1.2 mg/m² per dose. In certain embodiments, bortezomib is administered twice a day, daily, once every two days, twice a week, or once a week. In specific embodiments, bortezomib is administered twice a week. In specific embodiments, bortezomib is administered once a week. In a specific embodiment, bortezomib is administered at lower than 1.3 mg/m² per dose twice a week. In another specific embodiment, bortezomib is administered at lower than 1.3 mg/m² per dose once a week.

[00139] When the Network Brake is vorinostat or comprises vorinostat, in some

embodiments, vorinostat is administered at about 400 mg per dose. In specific embodiments, vorinostat is administered at about 25, 50, 100, 150, 200, 300, 400, 500, 600, 700, or 800 mg per dose. In specific embodiments, vorinostat is administered at about 25 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, or 700 to 800 mg per dose. In specific embodiments, vorinostat is administered at about 300, 400, or 500 mg per dose. In specific embodiments, vorinostat is administered at about 300 to 400, or 400 to 500 mg per dose. In specific embodiments, vorinostat is administered at lower than 400 mg per dose. In specific embodiments, vorinostat is administered at about 25, 50, 100, 150, 200, 250, 300, or 350 mg per dose. In specific embodiments, vorinostat is administered at about 25 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, or 300 to 350 mg per dose. When the Network Brake is vorinostat or comprises vorinostat and the cancer is cutaneous T-cell lymphoma, vorinostat is administered at lower than 400 mg per dose. When the Network Brake is vorinostat or comprises vorinostat and the cancer is cutaneous T-cell lymphoma, in specific embodiments, vorinostat is administered at about 25, 50, 100, 150, 200, 250, 300, or 350 mg per dose. When the Network Brake is vorinostat or comprises vorinostat and the cancer is cutaneous T-cell lymphoma, in specific embodiments, vorinostat is administered at about 25 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, or 300 to 350 mg per dose. In certain embodiments, vorinostat is administered twice a day, daily, once every two days, twice a week, or once a week. In specific embodiments, vorinostat is administered daily. In a specific embodiment, vorinostat is administered at lower than 400 mg per dose daily.

5.2. Companion Diagnostics

[00140] In another aspect, provided herein are methods of treating a cancer in a patient in need thereof, comprising: (a) administering to the patient a Network Brake at a subtherapeutic dose; (b) administering to the patient a kinase inhibitor subsequent to and/or concurrently with the administration of the Network Brake; and (c) measuring the change in the level of a biomarker positively associated with the cancer; wherein a decrease in the level of the biomarker indicates amelioration of the cancer. In a specific embodiment, the kinase inhibitor is administered at a clinical dose. In another specific embodiment, the kinase inhibitor is administered to the patient at a dose lower than a clinical dose. In another specific embodiment, the kinase inhibitor is administered to the patient at a dose higher than a clinical dose.

[00141] In another aspect, provided herein are methods of treating a cancer in a patient in need thereof, comprising: (a) administering to the patient a Network Brake at a subtherapeutic dose; (b) administering to the patient a kinase inhibitor subsequent to and/or concurrently with the administration of the Network Brake; and (c) measuring the changes in the levels of a plurality of biomarkers positively associated with the cancer; wherein a decrease in the overall, medium, or mean level of the plurality of biomarkers indicates amelioration of the cancer. In a specific embodiment, the kinase inhibitor is administered at a clinical dose. In another specific embodiment, the kinase inhibitor is administered to the patient at a dose lower than a clinical dose. In another specific embodiment, the kinase inhibitor is administered to the patient at a dose higher than a clinical dose.

5.3. Screening For Network Brakes And Combination Regimens

[00142] In another aspect, provided herein are methods for identifying a Network Brake(s). In certain embodiments, one, two, three, or more, or all of the following assays are conducted to identify a compound or a combination of compounds of compounds as a Network Brake: (1) the ability of the compound or combination of compounds at a test dose(s)/concentration(s) to improve the efficacy of a kinase inhibitor at a certain dose/concentration to treat cancer

("Efficacy Assay"); (2) the ability of the compound or combination of compounds alone at a test dose(s)/concentration(s) to kill cancer cells ("Viability Assay"); (3) the toxicity of the compound or combination of compounds at the test dose(s)/concentration(s) on non-cancerous cells ("Toxicity Assay"); (4) the phosphorylation levels of certain kinases when cancer cells are treated with the compound or combination of compounds at the test dose(s)/concentration(s) and a certain dose/concentration of a kinase inhibitor relative to the phosphorylation levels of the same kinases when the same type of cancer cells are treated with the same concentration of the same kinase inhibitor alone ("Phosphoprotein Assay"); (5) the expression level of certain cancer stem cell marker(s) when cancer cells are treated with the compound or combination of compounds at the test dose(s)/concentration(s) and a certain dose/concentration of a kinase inhibitor relative to the expression level of the same cancer stem cell marker(s) when the same type of cancer cells are treated with the same concentration of the same kinase inhibitor alone ("Cancer Stem Cell Marker Assay"); (6) the level of one or more active histone marks and/or the level of one or more suppressive histone marks when cancer cells are treated with the compound or combination of compounds at the test dose(s)/concentration(s) and a certain

dose/concentration of a kinase inhibitor relative to the level(s) of the same active and/or suppressive mark(s) when the same type of cancer cells are treated with the same concentration of the same kinase inhibitor alone ("Histone Modification Assay"); and (7) the emergence of cancer cells resistant to the kinase inhibitor over time when the cancer cells have been treated with the compound or combination of compounds at the test dose(s)/concentration(s) and the kinase inhibitor ("Resistance Assay"). In a particular embodiment, one, two, three, or more or all of the assays described in Example 1 (Section 6, infra) are used to identify a compound or a combination of compounds as a Network Brake.

[00143] In a specific embodiment, a method for identifying a compound or a combination of compounds as a Network Brake involves evaluating the ability of the compound or combination of compounds at a test dose(s)/concentration(s) to improve the efficacy of a kinase inhibitor at a certain dose/concentration to treat cancer ("Efficacy Assay"), the ability of the compound or combination of compounds alone at a test dose(s)/concentration(s) to kill cancer cells ("Viability Assay"), and the toxicity of the compound or combination of compounds at the test

dose(s)/concentration(s) on non-cancerous cells ("Toxicity Assay"), as well as evaluating one, two, three or all of the following: (i) the phosphorylation levels of certain kinases when cancer cells are treated with the compound or combination of compounds at the test

dose(s)/concentration(s) and a certain dose/concentration of a kinase inhibitor relative to the phosphorylation levels of the same kinases when the same type of cancer cells are treated with the same concentration of the same kinase inhibitor alone ("Phosphoprotein Assay"); (ii) the expression level of certain cancer stem cell marker(s) when cancer cells are treated with the compound or combination of compounds at the test dose(s)/concentration(s) and a certain dose/concentration of a kinase inhibitor relative to the expression level of the same cancer stem cell marker(s) when the same type of cancer cells are treated with the same concentration of the same kinase inhibitor alone ("Cancer Stem Cell Marker Assay"); (iii) the level of one or more active histone marks and/or the level of one or more suppressive histone marks when cancer cells are treated with the compound or combination of compounds at the test dose(s)/concentration(s) and a certain dose/concentration of a kinase inhibitor relative to the level(s) of the same active and/or suppressive mark(s) when the same type of cancer cells are treated with the same concentration of the same kinase inhibitor alone ("Histone Modification Assay"); and (iv) the emergence of cancer cells resistant to the kinase inhibitor over time when the cancer cells have been treated with the compound or combination of compounds at the test

dose(s)/concentration(s) and the kinase inhibitor ("Resistance Assay").

[00144] In specific embodiments, the Efficacy Assay involves assessing the increase in cancer cell death and/or decrease in tumor size when the cancer cells and/or cancer subjects (e.g., animals, such as non-human animals) are treated with the compound or combination of compounds at the test dose(s)/concentration(s) and the kinase inhibitor at the certain

dose/concentration relative to when the cancer cells and/or cancer subjects (e.g., animals, such as non-human animals) are treated with the same kinase inhibitor at the same dose/concentration alone. In specific embodiments, the Efficacy Assay involves assessing the increase in the percentage of viable adults developed from animal juveniles (e.g. fly larvae) over-expressing or having a hyper-activated oncogene(s), which prevents the animal juveniles from developing into viable adults, when the animal juveniles are treated with the compound or combination of compounds at the test dose(s)/concentration(s) and the kinase inhibitor at the certain

dose/concentration relative to when the animal juveniles are treated with the same kinase inhibitor at the same dose/concentration alone. See Sections 5.3.1 and 6, infra, for methods for conducting the Efficacy Assay.

[00145] In specific embodiments, the Viability Assay involves assessing the amount of cancer cell death/tumor size reduction resulting from treating cancer cells/cancer subjects (e.g., animals, such as non-human animals) with a test dose(s)/concentration(s) of a compound or a combination of compounds utilizing techniques known to one of skill in the art. In specific embodiments, the Viability Assay involves assessing the percentage of viable adults developed from animal juveniles (e.g. fly larvae) having an oncogenic mutation(s), which prevent the animal juveniles from developing into viable adults, resulting from treating the animal juveniles with a test dose(s)/concentration(s) of a compound or a combination of compounds utilizing techniques known to one of skill in the art. See Section 5.3.2, infra, for methods for conducting the

Viability Assay. [00146] In specific embodiments, the Toxicity Assay involves assessing the toxicity of a test dose(s)/concentration(s) of a compound or a combination of compounds to non-cancerous cells or subjects without cancer (e.g., animals without cancer, such as non-human animals without cancer). See Section 5.3.3, infra, for methods for conducting the Viability Assay.

[00147] In specific embodiments, the Efficacy Assay is performed first.

[00148] When the Efficacy Assay is performed first, in a specific embodiment, if the Efficacy Assay demonstrates that (1) there is an increase in the percentage of cancer cells that undergo cell death when the cancer cells are treated with the compound or combination of compounds at the test dose(s)/concentration(s) and the kinase inhibitor at the certain dose/concentration relative to the percentage of cancer cells that undergo cell death when the same type of cancer cells are treated with the same dose/concentration of the kinase inhibitor alone, (2) there is a decrease in tumor size when a cancer subject(s) (e.g., an animal, such as a non-human animal) are treated with the compound or the combination of compounds at the test dose(s) and the kinase inhibitor at the certain dose relative the tumor size in a subject(s) of the same type with the same type of cancer treated with the same dose of the same kinase inhibitor alone, and/or (3) there is an increase in the percentage of viable adults developed from animal juveniles having an oncogenic mutation(s), which prevent the animal juveniles from developing into viable adults, when the animal juveniles are treated with the compound or the combination of compounds at the test dose(s) and the kinase inhibitor at the certain dose relative to when the animal juveniles are treated with the same dose of the same kinase inhibitor alone, then the compound or combination of compounds at the test dose(s) is tested in a Viability Assay. If the Viability Assay

demonstrates that the compound or the combination of compounds at the test

dose(s)/concentration(s) (1) does not result in cell death of the cancer cells as assessed by a technique known to one skilled in the art or results in about 20% or less of the cancer cells undergoing cell death as assessed by a technique known to one skilled in the art, (2) does not reduce tumor size or results in a 20% or less increase in tumor size, and/or (3) does not increase or results in a 15% or less increase in the percentage of viable adults developed from animal juveniles having an oncogenic mutation(s), which prevented the animal juveniles from developing into viable adults, then the compound or combination of compounds at the test dose(s)/concentration(s) is tested in a Toxicity Assay. If the Toxicity Assay demonstrates that the compound or the combination of compounds at the test dose(s)/concentration(s) is minimally toxic to non-cancerous cells or subjects without cancer (e.g., animals without cancer, such as non-human animals without cancer), then the compound or the combination of compounds at the test dose(s)/concentration(s) is identified as a candidate Network Brake and may be tested in the Phosphoprotein Assay, the Cancer Stem Cell Marker Assay, the Histone Modification Assay and/or the Resistance Assay for Network Brake activity. See Sections 5.3.4, 5.3.5, 5.3.6, and 5.3.7, infra, for methods for conducting the Phosphoprotein Assay, the Cancer Stem Cell Marker Assay, the Histone Modification Assay and Resistance Assay, respectively. In certain embodiments, the candidate Network Brake is tested in the Resistance Assay and one or more of the following assays for Network Brake activity: the Phosphoprofile Assay, the Cancer Stem Cell Marker Assay and the Histone Assay.

[00149] When the Efficacy Assay is performed first, in another specific embodiment, if the Efficacy Assay demonstrates that (1) there is an increase in the percentage of cancer cells that undergo cell death when the cancer cells are treated with the compound or combination of compounds at the test dose(s)/concentration(s) and the kinase inhibitor at the certain

dose/concentration relative to the percentage of cancer cells that undergo cell death when the same type of cancer cells are treated with the same dose/concentration of the kinase inhibitor alone, (2) there is a decrease in tumor size when a cancer subject(s) (e.g., an animal, such as a non-human animal) are treated with the compound or the combination of compounds at the test dose(s) and the kinase inhibitor at the certain dose relative the tumor size in a subject(s) of the same type with the same type of cancer treated with the same dose of the same kinase inhibitor alone, and/or (3) there is an increase in the percentage of viable adults developed from animal juveniles having an oncogenic mutation(s), which prevent the animal juveniles from developing into viable adults, when the animal juveniles are treated with the compound or the combination of compounds at the test dose(s) and the kinase inhibitor at the certain dose relative to when the animal juveniles are treated with the same dose of the same kinase inhibitor alone, then the compound or combination of compounds at the test dose(s) is tested in a Toxicity Assay. If the Toxicity Assay demonstrates that the compound or the combination of compounds at the test dose(s)/concentration(s) is minimally toxic to non-cancerous cells or subjects without cancer (e.g., animals without cancer, such as non-human animals without cancer), then the compound or combination of compounds at the test dose(s)/concentration(s) is tested in a Viability Assay. If the Viability Assay demonstrates that the compound or the combination of compounds at the test dose(s)/concentration(s) (1) does not result in cell death of the cancer cells as assessed by a technique known to one skilled in the art or results in about 20% or less of the cancer cells undergoing cell death as assessed by a technique known to one skilled in the art, (2) does not reduce tumor size or results in a 20% or less increase in tumor size, and/or (3) does not increase or results in a 15% or less increase in the percentage of viable adults developed from animal juveniles having an oncogenic mutation(s), which prevented the animal juveniles from developing into viable adults, then the compound or the combination of compounds at the test dose(s)/concentration(s) is identified as a candidate Network Brake and may be tested in the Phosphoprotein Assay, the Cancer Stem Cell Marker Assay, the Histone Modification Assay and/or the Resistance Assay for Network Brake activity. See Sections 5.3.4, 5.3.5, 5.3.6, and 5.3.7, infra, for methods for conducting the Phosphoprotein Assay, the Cancer Stem Cell Marker Assay, the Histone Modification Assay and Resistance Assay, respectively. In certain embodiments, the candidate Network Brake is tested in the Resistance Assay and one or more of the following assays for Network Brake activity: the Phosphoprofile Assay, the Cancer Stem Cell Marker Assay and the Histone Assay.

[00150] In specific embodiments, the Viability Assay is performed first.

[00151] When the Viability Assay is performed first, in a specific embodiment, if the

Viability Assay demonstrates that the compound or the combination of compounds at the test dose(s)/concentration(s) (1) does not result in cell death of the cancer cells as assessed by a technique known to one skilled in the art or results in about 20% or less of the cancer cells undergoing cell death as assessed by a technique known to one skilled in the art, (2) does not reduce tumor size or results in a 20% or less increase in tumor size, and/or (3) does not increase or results in a 15% or less increase in the percentage of viable adults developed from animal juveniles over-expressing or having a hyper-activated oncogene(s), which prevented the animal juveniles from developing into viable adults, then the compound or combination of compounds at the test dose(s) is tested in an Efficacy Assay. If the Efficacy Assay demonstrates that (1) there is an increase in the percentage of cancer cells that undergo cell death when the cancer cells are treated with the compound or combination of compounds at the test dose(s)/concentration(s) and the kinase inhibitor at the certain dose/concentration relative to the percentage of cancer cells that undergo cell death when the same type of cancer cells are treated with the same

dose/concentration of the kinase inhibitor alone, (2) there is a decrease in tumor size when a cancer subject(s) (e.g., an animal, such as a non-human animal) are treated with the compound or the combination of compounds at the test dose(s) and the kinase inhibitor at the certain dose relative the tumor size in a subject(s) of the same type with the same type of cancer treated with the same dose of the same kinase inhibitor alone, and/or (3) there is an increase in the percentage (e.g., an increase of greater than 15%) of viable adults developed from animal juveniles over- expressing or having a hyper-activated oncogene(s), which prevents the animal juveniles from developing into viable adults, when the animal juveniles are treated with the compound or the combination of compounds at the test dose(s) and the kinase inhibitor at the certain dose relative to when the animal juveniles are treated with the same dose of the same kinase inhibitor alone, then the compound or combination of compounds at the test dose(s)/concentration(s) is tested in a Toxicity Assay. If the Toxicity Assay demonstrates that the compound or the combination of compounds at the test dose(s)/concentration(s) is minimally toxic to non-cancerous cells or subjects without cancer (e.g., animals without cancer, such as non-human animals without cancer), then the compound or the combination of compounds at the test dose(s)/concentration(s) is identified as a candidate Network Brake and may be tested in the Phosphoprotein Assay, the Cancer Stem Cell Marker Assay, the Histone Modification Assay and/or the Resistance Assay for Network Brake activity. See Sections 5.3.4, 5.3.5, 5.3.6, and 5.3.7, infra, for methods for conducting the Phosphoprotein Assay, the Cancer Stem Cell Marker Assay, the Histone

Modification Assay and Resistance Assay, respectively. In certain embodiments, the candidate Network Brake is tested in the Resistance Assay and one or more of the following assays for Network Brake activity: the Phosphoprofile Assay, the Cancer Stem Cell Marker Assay and the Histone Assay.

[00152] When the Viability Assay is performed first, in another specific embodiment, if the Viability Assay demonstrates that the compound or the combination of compounds at the test dose(s)/concentration(s) (1) does not result in cell death of the cancer cells as assessed by a technique known to one skilled in the art or results in about 20% or less of the cancer cells undergoing cell death as assessed by a technique known to one skilled in the art, (2) does not reduce tumor size or results in a 20% or less increase in tumor size, and/or (3) does not increase or results in a 15% or less increase in the percentage of viable adults developed from animal juveniles over-expressing or having a hyper-activated oncogene(s), which prevented the animal juveniles from developing into viable adults, then the compound or combination of compounds at the test dose(s) is tested in a Toxicity Assay. If the Toxicity Assay demonstrates that the compound or the combination of compounds at the test dose(s)/concentration(s) is minimally toxic to non-cancerous cells or subjects without cancer (e.g., animals without cancer, such as non-human animals without cancer), then the compound or combination of compounds at the test dose(s)/concentration(s) is tested in an Efficacy Assay. If the Efficacy Assay demonstrates that (1) there is an increase in the percentage of cancer cells that undergo cell death when the cancer cells are treated with the compound or combination of compounds at the test

dose(s)/concentration(s) and the kinase inhibitor at the certain dose/concentration relative to the percentage of cancer cells that undergo cell death when the same type of cancer cells are treated with the same dose/concentration of the kinase inhibitor alone, (2) there is a decrease in tumor size when a cancer subject(s) (e.g., an animal, such as a non-human animal) are treated with the compound or the combination of compounds at the test dose(s) and the kinase inhibitor at the certain dose relative the tumor size in a subject(s) of the same type with the same type of cancer treated with the same dose of the same kinase inhibitor alone, and/or (3) there is an increase in the percentage (e.g., an increase of greater than 15%) of viable adults developed from animal juveniles over-expressing or having a hyper-activated oncogene(s), which prevents the animal juveniles from developing into viable adults, when the animal juveniles are treated with the compound or the combination of compounds at the test dose(s) and the kinase inhibitor at the certain dose relative to when the animal juveniles are treated with the same dose of the same kinase inhibitor alone, then the compound or the combination of compounds at the test dose(s)/concentration(s) is identified as a candidate Network Brake and may be tested in the Phosphoprotein Assay, the Cancer Stem Cell Marker Assay, the Histone Modification Assay and/or the Resistance Assay for Network Brake activity. See Sections 5.3.4, 5.3.5, 5.3.6, and 5.3.7, infra, for methods for conducting the Phosphoprotein Assay, the Cancer Stem Cell Marker Assay, the Histone Modification Assay and Resistance Assay, respectively. In certain embodiments, the candidate Network Brake is tested in the Resistance Assay and one or more of the following assays for Network Brake activity: the Phosphoprofile Assay, the Cancer Stem Cell Marker Assay and the Histone Assay.

[00153] In specific embodiments, the Toxicity Assay is performed first.

[00154] When the Toxicity Assay is performed first, in a specific embodiment, if the Toxicity

Assay demonstrates that the compound or the combination of compounds at the test dose(s)/concentration(s) is minimally toxic to non-cancerous cells or subjects without cancer (e.g., animals without cancer, such as non-human animals without cancer), then the compound or combination of compounds at the test dose(s) is tested in an Efficacy Assay. If the Efficacy Assay demonstrates that (1) there is an increase in the percentage of cancer cells that undergo cell death when the cancer cells are treated with the compound or combination of compounds at the test dose(s)/concentration(s) and the kinase inhibitor at the certain dose/concentration relative to the percentage of cancer cells that undergo cell death when the same type of cancer cells are treated with the same dose/concentration of the kinase inhibitor alone, (2) there is a decrease in tumor size when a cancer subject(s) (e.g., an animal, such as a non-human animal) are treated with the compound or the combination of compounds at the test dose(s) and the kinase inhibitor at the certain dose relative the tumor size in a subject(s) of the same type with the same type of cancer treated with the same dose of the same kinase inhibitor alone, and/or (3) there is an increase in the percentage (e.g., an increase of greater than 15%) of viable adults developed from animal juveniles over-expressing or having a hyper-activated oncogene(s), which prevent the animal juveniles from developing into viable adults, when the animal juveniles are treated with the compound or the combination of compounds at the test dose(s) and the kinase inhibitor at the certain dose relative to when the animal juveniles are treated with the same dose of the same kinase inhibitor alone, then the compound or combination of compounds at the test

dose(s)/concentration(s) is tested in a Viability Assay. If the Viability Assay demonstrates that the compound or the combination of compounds at the test dose(s)/concentration(s) (1) does not result in cell death of the cancer cells as assessed by a technique known to one skilled in the art or results in about 20% or less of the cancer cells undergoing cell death as assessed by a technique known to one skilled in the art, (2) does not reduce tumor size or results in a 20% or less increase in tumor size, and/or (3) does not increase or results in a 15% or less increase in the percentage of viable adults developed from animal juveniles over-expressing or having a hyper- activated oncogene(s), which prevented the animal juveniles from developing into viable adults, then the compound or the combination of compounds at the test dose(s)/concentration(s) is identified as a candidate Network Brake and may be tested in the Phosphoprotein Assay, the Cancer Stem Cell Marker Assay, the Histone Modification Assay and/or the Resistance Assay for Network Brake activity. See Sections 5.3.4, 5.3.5, 5.3.6, and 5.3.7, infra, for methods for conducting the Phosphoprotein Assay, the Cancer Stem Cell Marker Assay, the Histone Modification Assay and Resistance Assay, respectively. In certain embodiments, the candidate Network Brake is tested in the Resistance Assay and one or more of the following assays for Network Brake activity: the Phosphoprofile Assay, the Cancer Stem Cell Marker Assay and the Hi stone Assay.

[00155] When the Toxicity Assay is performed first, in another specific embodiment, if the Toxicity Assay demonstrates that the compound or the combination of compounds at the test dose(s)/concentration(s) is minimally toxic to non-cancerous cells or subjects without cancer (e.g., animals without cancer, such as non-human animals without cancer), then the compound or combination of compounds at the test dose(s) is tested in a Viability Assay. If the Viability Assay demonstrates that the compound or the combination of compounds at the test

dose(s)/concentration(s) (1) does not result in cell death of the cancer cells as assessed by a technique known to one skilled in the art or results in about 20% or less of the cancer cells undergoing cell death as assessed by a technique known to one skilled in the art, (2) does not reduce tumor size or results in a 20% or less increase in tumor size, and/or (3) does not increase or results in a 15% or less increase in the percentage of viable adults developed from animal juveniles overexpressing or having a hyper-activated oncogene(s), which prevented the animal juveniles from developing into viable adults, then the compound or combination of compounds at the test dose(s)/concentration(s) is tested in an Efficacy Assay. If the Efficacy Assay demonstrates that (1) there is an increase in the percentage of cancer cells that undergo cell death when the cancer cells are treated with the compound or combination of compounds at the test dose(s)/concentration(s) and the kinase inhibitor at the certain dose/concentration relative to the percentage of cancer cells that undergo cell death when the same type of cancer cells are treated with the same dose/concentration of the kinase inhibitor alone, (2) there is a decrease in tumor size when a cancer subject(s) (e.g., an animal, such as a non-human animal) are treated with the compound or the combination of compounds at the test dose(s) and the kinase inhibitor at the certain dose relative the tumor size in a subject(s) of the same type with the same type of cancer treated with the same dose of the same kinase inhibitor alone, and/or (3) there is an increase in the percentage (e.g., an increase of greater than 15%) of viable adults developed from animal juveniles over-expressing or having a hyper-activated oncogene(s), which prevent the animal juveniles from developing into viable adults, when the animal juveniles are treated with the compound or the combination of compounds at the test dose(s) and the kinase inhibitor at the certain dose relative to when the animal juveniles are treated with the same dose of the same kinase inhibitor alone, then the compound or the combination of compounds at the test dose(s)/concentration(s) is identified as a candidate Network Brake and may be tested in the Phosphoprotein Assay, the Cancer Stem Cell Marker Assay, the Histone Modification Assay and/or the Resistance Assay for Network Brake activity. See Sections 5.3.4, 5.3.5, 5.3.6, and 5.3.7, infra, for methods for conducting the Phosphoprotein Assay, the Cancer Stem Cell Marker Assay, the Histone Modification Assay and Resistance Assay, respectively. In certain embodiments, the candidate Network Brake is tested in the Resistance Assay and one or more of the following assays for Network Brake activity: the Phosphoprofile Assay, the Cancer Stem Cell Marker Assay and the Histone Assay.

[00156] In a specific embodiment, the candidate Network Brake is tested in the

Phosphoprofile Assay for Network Brake activity. When the Phosphoprotein Assay is performed, if there is a decrease in the overall, mean or median phosphorylation level of certain kinases when cancer cells are treated with the compound or combination of compounds at the test dose(s)/concentration(s) and a certain dose/concentration of a kinase inhibitor relative to the overall, mean or median phosphorylation level of the same kinases when the same type of cancer cells are treated with the same dose/concentration of the same kinase inhibitor alone, then the compound or combination of compounds at the test dose(s)/concentration(s) is identified as a Network Brake. Alternatively or in addition, when the Cancer Stem Cell Marker Assay is performed, if there is a decrease in the expression level of a cancer stem cell marker(s) when the cancer cells are treated with the compound or the combination of compounds at the test dose(s)/concentration(s) and the kinase inhibitor at the certain dose/concentration relative to the expression level of the same cancer stem cell marker(s) when the same type of cancer cells are treated with the same dose/concentration of the kinase inhibitor alone, then the compound or combination of compounds at the test dose(s) is identified as a Network Brake. Alternatively or in addition, when the Histone Modification Assay is performed, (1) if there is a decrease in the level of an active histone mark(s) when the cancer cells are treated with the compound or the combination of compounds at the test dose(s)/concentration(s) and the kinase inhibitor at the certain dose/concentration relative to the level of the same active histone mark(s) when the same type of cancer cells are treated with the same dose/concentration of the kinase inhibitor alone, and/or (2) if there is an increase in the level of a suppressive histone mark(s) when the cancer cells are treated with the compound or the combination of compounds at the test

dose(s)/concentration(s) and the kinase inhibitor at the certain dose/concentration relative to the level of the same suppressive histone mark(s) when the same type of cancer cells are treated with the same dose/concentration of the kinase inhibitor alone, then the compound or combination of compounds at the test dose(s) is identified as a Network Brake. Further, alternatively or in addition, when the Resistance Assay is performed, if there is resistance to the emergence of cancer cells refractory to the kinase inhibitor over time when the cancer cells are treated with the compound or combination of compounds at the test dose(s)/concentration(s) and the kinase inhibitor, then the compound or combination of compounds at the test dose(s) is identified as a Network Brake.

[00157] In contrast, if (1) (i) there is no increase in the percentage of cancer cells that undergo cell death when the cancer cells are treated with the compound or combination of compounds at the test dose(s)/concentration(s) and the kinase inhibitor at the certain dose/concentration relative to the percentage of cancer cells that undergo cell death when the same type of cancer cells are treated with the same dose/concentration of the kinase inhibitor alone, (ii) there is no decrease in tumor size when a cancer subject(s) (e.g., an animal, such as a non-human animal) are treated with the compound or the combination of compounds at the test dose(s) and the kinase inhibitor at the certain dose relative the tumor size in a subject(s) of the same type with the same type of cancer treated with the same dose of the same kinase inhibitor alone, and/or (iii) there is no increase in the percentage of viable adults developed from animal juveniles over-expressing or having a hyper-activated oncogene(s), which prevent the animal juveniles from developing into viable adults, when the animal juveniles are treated with the compound or the combination of compounds at the test dose(s) and the kinase inhibitor at the certain dose relative when the animal juveniles are treated with the same dose of the same kinase inhibitor alone, as

demonstrated in the Efficacy Assay; (2) if (i) greater than 20% of the cancer cells undergo cell death at the test dose(s)/concentration(s) of the compound or combination of compounds, (ii) there is a greater than 20% decrease in tumor size in a cancer subject(s) (e.g., an animal, such as a non-human animal) treated with the compound or combinations of compounds, and/or (iii) there is a greater than 15% increase in the percentage of viable adults developed from animal juveniles over-expressing or having a hyper-activated oncogene(s), which prevented the animal juveniles from developing into viable adults, treated with the compound or combination of compounds alone, as demonstrated in the Viability Assay; and/or (3) the compound or combination of compounds at the test dose(s)/concentration(s) is more than minimally toxic to non-cancerous cells and/or subjects without cancer (e.g., animals without cancer, such as non- human animals without cancer), as demonstrated in the Toxicity Assay, then the compound or combination of compounds at the test dose(s)/concentration(s) is not identified as a candidate Network Brake.

[00158] Moreover, when the Phosphoprotein Assay is performed, if a candidate compound or combination of compounds at the test dose(s)/concentration(s) results in no change or an increase in the overall, mean or median phosphorylation activity of certain kinases when cancer cells are treated with the compound or combination of compounds at the test dose(s)/concentration(s) and a certain concentration of the kinase inhibitor relative to the phosphorylation activity of the same kinases when the same type of cancer cells are treated with the same concentration of the same kinase inhibitor alone, then the compound or combination of compounds at the test dose(s) is not identified as a Network Brake. When the Cancer Stem cell Marker Assay is performed, if a candidate compound or combination of compounds at the test dose(s)/concentrations(s) in combination a kinase inhibitor at a certain dose/concentration results in no decrease in the expression level of a cancer stem cell marker (s) relative to the expression level of the same cancer stem cell marker(s) when the same type of cancer cells are treated with the same dose/concentration of the kinase inhibitor alone, then the compound or combination of compounds is not identified as a Network Brake. When the Histone Modification Assay is performed, if there is no decrease in the level of an active histone mark(s) when the cancer cells are treated with the compound or the combination of compounds at the test

dose(s)/concentration(s) and the kinase inhibitor at the certain dose/concentration relative to the level of the same active histone mark(s) when the same type of cancer cells are treated with the same dose/concentration of the kinase inhibitor alone, and/or there is no increase in the level of a suppressive histone mark(s) when the cancer cells are treated with the compound or the combination of compounds at the test dose(s)/concentration(s) and the kinase inhibitor at the certain dose/concentration relative to the level of the same suppressive histone mark(s) when the same type of cancer cells are treated with the same dose/concentration of the kinase inhibitor alone, then the compound or combination of compounds at the test dose(s) is not identified as a Network Brake. Further, when the Resistance Assay is performed, if a candidate compound or combination of compounds at the test dose(s)/concentration(s) results in the emergence of cancer cells refractory to the kinase inhibitor over time, then the compound or combination of compounds at the test dose(s)/concentration(s) is not identified as a Network Brake.

[00159] In certain embodiments, if the subtherapeutic dose(s) and/or subtherapeutic dose range(s) for a compound or a combination of compounds is known to one of skill in the art, then the compound or the combination of compounds at the subtherapeutic dose(s) and/or

subtherapeutic dose range(s) can be assessed in one, two, three or all of the following assays to determine whether the compound or the combination of compounds at the subtherapeutic dose(s) or the subtherapeutic dose range(s) is a Network Brake: Resistance Assay, Efficacy Assay, Phosphoprofile Assay, and Histone Modification Assay. In a specific embodiment, such a compound or combination of compounds at the subtherapeutic dose(s) or subtherapeutic dose range(s) is assessed in the Resistance Assay and one, two or all of the following assays to determine whether the compound or the combination of compounds at the subtherapeutic dose(s) or subtherapeutic dose range(s) is a Network Brake.

[00160] In specific embodiments, the compound or combination of compounds evaluated in the methods for identifying a Network Brake comprises, consists of or consists essentially of a histone deacetylase ("HDAC") inhibitor, a proteasome inhibitor, a heat shock protein ("HSP"; e.g., Hsp90) inhibitor, an HDAC-PI3K dual inhibitor, an inhibitor of SP1 -class transcription factors, or a combination thereof. See Sections 5.1.2 and 6 for examples of such compounds. In a specific embodiment, the compound evaluated in the methods for identifying a Network Brake comprises, consists of or consists essentially of an HDAC inhibitor. In another specific embodiment, the compound evaluated in the methods for identifying a Network Brake comprises, consists of or consists essentially of a proteasome inhibitor. In another specific embodiment, the compound evaluated in the methods for identifying a Network Brake comprises, consists of or consists essentially of an HSP (e.g., Hsp90) inhibitor. In another specific embodiment, the compound evaluated in the methods for identifying a Network Brake comprises, consists of or consists essentially of an HDAC-PI3K dual inhibitor. In another specific embodiment, the combination of compounds evaluated in the methods for identifying a Network Brake comprises, consists of or consists essentially of an inhibitor of SP1 -class transcription factors. In another specific embodiment, the combination of compounds evaluated in the methods for identifying a Network Brake comprises, consists of or consists essentially of an HDAC inhibitor and a proteasome inhibitor. In another specific embodiment, the combination of compounds evaluated in the methods for identifying a Network Brake comprises, consists of or consists essentially of a proteasome inhibitor and an HDAC-PI3K dual inhibitor. In another specific embodiment, the combination of compounds evaluated in the methods for identifying a Network Brake comprises, consists of or consists essentially of an HDAC inhibitor and an HSP (e.g., Hsp90) inhibitor. In another specific embodiment, the combination of compounds evaluated in the methods for identifying a Network Brake comprises, consists of or consists essentially of an inhibitor of SPl -class transcription factors and a proteasome inhibitor.

[00161] In certain embodiments, the compound or combination of compounds evaluated in the methods for identifying a Network Brake are from or derived from a chemical library, such as a chemical library containing the scaffolds for known drugs. A non-limiting exemplary chemical library that can be used in accordance with the methods described herein is the Selleckchem library of FDA-approved drugs (Selleckchem, USA).

[00162] In all of the methods for identifying a compound or combination of compounds as a Network Brake described herein, the compound or the combination of compounds are tested at a specific test dose/concentration (e.g., a specific concentration if tested in cell culture). When a kinase inhibitor is tested in the methods for identifying a compound or combination of compounds as a Network Brake described herein, it is also tested at a particular test

dose/concentration (e.g., a particular concentration if tested in cell culture). Therefore, the efficacy, the toxicity, the Network Brake activity, and the resistance as described herein are conditioned upon the specific test dose/concentration of the compound or the combination of compounds tested, and the particular test dose/concentration of the particular kinase inhibitor (when tested). The test concentration used in cell culture can be used to calculate the dose to be administered to a cancer patient.

5.3.1 Efficacy Assay

[00163] The Efficacy Assay referenced herein can be any assay known in the art to test the efficacy of a drug for treating cancer (e.g., by measuring the killing of cancer cells, tumor size, or development of viable adults from animal juveniles over-expressing or having a hyper-activated oncogene(s) that prevents them from maturing into viable adults), and can be performed using cancer cells or animal models (e.g., flies, worms, mice, rats, rabbits, or primates). Provided in this Section 5.3.1 and Section 6, infra, are examples of various ways to conduct the Efficacy Assay.

[00164] In a specific embodiment, the Efficacy Assay is conducted as described in Example 1 (Section 6, infra).

[00165] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds and a kinase inhibitor for treating cancer comprises comprises: (i) culturing cancer cells in the presence of a test compound or a combination of test compounds at a test concentration(s) and a kinase inhibitor at a specific concentration for a period of time; and (ii) measuring the percentage of dead cancer cells at the end of said period of time, wherein an increase of greater than about 20% (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 98% or 25% to 50%, 50% to 75%, or 75% to 99%) in the percentage of dead cancer cells measured when the cancer cells are cultured in the presence of the test compound or the combination of test compounds at the test concentration(s) and the kinase inhibitor at the specific concentration relative to the percentage of dead cancer cells when the same type of cancer cells are cultured in the presence of a negative control, such as vehicle alone (e.g., phosphate buffered saline (PBS) or another buffer), and the same kinase inhibitor at the same specific concentration for the same period of time indicates that the test compound or the combination of test compounds at the test concentration(s) and kinase inhibitor at the specific concentration is efficacious for treating cancer. In contrast, no detectable change or an increase of about 20% or less in the percentage of dead cancer cells measured when the cancer cells are cultured in the presence of the test compound or the combination of test compounds at the test concentration(s) and the kinase inhibitor at the specific concentration relative to the percentage of dead cancer cells when the same type of cancer cells are cultured in the presence of a negative control, such as vehicle alone (e.g., phosphate buffered saline (PBS) or another buffer), and the same kinase inhibitor at the same specific concentration for the same period of time indicates that the test compound or the combination of test compounds at the test concentration(s) and kinase inhibitor at the specific concentration is not efficacious or has minimal efficacy for treating cancer. In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, the cancer cells are derived or obtained from a cancer cell line. In other embodiments, the cancer cells are derived or obtained from a cancer patient. In a specific embodiment, the cancer cells are from the cancer cell line A549, H1299, HepG2, MD-MB-231, T47D, A375, 239, MZ-MTC, TT-MTC, or H358-NSCLC. In another specific embodiment the cancer cells are from a cancer cell line described in Example 1 (Section 6, infra).

[00166] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds and a kinase inhibitor for treating cancer comprises: (i) culturing a first population of cancer cells in the presence of a test compound or a combination of test compounds at a test concentration(s) and a kinase inhibitor at a specific concentration for a period of time; (ii) culturing a second population of the same type of cancer cells in the presence of a negative control, such as vehicle alone {e.g., phosphate buffered saline (PBS) or another buffer), and the same kinase inhibitor at the same specific concentration for the same period of time; and (iii) analyzing the percentage of dead cells of the cancer cells cultured under the conditions in (i) and (ii) at the end of said period of time, wherein an increase of greater than about 20% (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 98% or 25% to 50%, 50% to 75%, or 75% to 99%) in the percentage of dead cancer cells in the first population relative to the percentage of dead cancer cells in the second population indicates that the test compound or the combination of test compounds at the test

concentration(s) and kinase inhibitor at the specific concentration is efficacious for treating cancer. In contrast, no detectable change or an increase of about 20% or less (e.g., 15%, 10%, 5%, 4%, 3%, 2%, or 1%, or 10% to 15%, or 1% to 5%) in the percentage of dead cancer cells in the first population relative to the second population indicates that the test compound or the combination of test compounds at the test concentration(s) and kinase inhibitor at the specific concentration is not efficacious or has minimal efficacy for treating cancer. In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, the cancer cells are derived or obtained from a cancer cell line. In other embodiments, the cancer cells are derived or obtained from a cancer patient. In a specific embodiment, the cancer cells are from the cancer cell line A549, H1299, HepG2, MD-MB-231, T47D, A375, 239, MZ-MTC, TT-MTC, or H358-NSCLC. In another specific embodiment the cancer cells are from a cancer cell line described in Example 1 (Section 6, infra).

[00167] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds and a kinase inhibitor for treating cancer comprises: (i) culturing cancer cells in the presence of a test compound or a combination of test compounds at a test concentration(s) and a kinase inhibitor at a specific concentration for a period of time; and (ii) measuring the percentage of dead cancer cells at the end of said period of time, wherein an increase of greater than about 20% (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 98% or 25% to 50%, 50% to 75%, or 75% to 99%) in the percentage of dead cancer cells measured when the cancer cells are cultured in the presence of the test compound or the combination of test compounds at the test concentration(s) and the kinase inhibitor at the specific concentration relative to the percentage of dead cancer cells when the same type of cancer cells are cultured in the absence of the test compound or test compounds and the presence of the same kinase inhibitor at the same specific concentration for the same period of time indicates that the test compound or the combination of test compounds at the test concentration(s) and kinase inhibitor at the specific concentration is efficacious for treating cancer. In contrast, no detectable change or an increase of about 20% or less (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 25% to 50%, 50% to 7%%, 25% to 75%, 75% to 95% or 90% to 98%) in the percentage of dead cancer cells measured when the cancer cells are cultured in the presence of the test compound or the combination of test compounds at the test concentration(s) and the kinase inhibitor at the specific concentration relative to the percentage of dead cancer cells when the same type of cancer cells are cultured in the absence of the test compound or test compounds and the presence of the same kinase inhibitor at the same specific concentration for the same period of time indicates that the test compound or the combination of test compounds at the test concentration(s) and kinase inhibitor at the specific concentration is not efficacious or has minimal efficacy for treating cancer. In certain embodiments, the cancer cells are derived or obtained from a cancer cell line. In other embodiments, the cancer cells are derived or obtained from a cancer patient. In a specific embodiment, the cancer cells are from the cancer cell line A549, H1299, HepG2, MD- MB-231, T47D, A375, 239, MZ-MTC, TT-MTC, or H358-NSCLC. In another specific embodiment the cancer cells are from a cancer cell line described in Example 1 (Section 6, infra).

[00168] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds and a kinase inhibitor for treating cancer comprises: (i) culturing a first population of cancer cells in the presence of a test compound or a combination of test compounds at a test concentration(s) and a kinase inhibitor at a specific concentration for a period of time; (ii) culturing a second population of the same type of cancer cells in the absence of the test compound or the combination of test compounds and the presence of the same kinase inhibitor at the same specific concentration for the same period of time; and (iii) analyzing the percentage of dead cells of the cancer cells cultured under the conditions in (i) and (ii) at the end of said period of time, wherein an increase of greater than about 20% (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 25% to 50%, 50% to 7%%, 25% to 75%, 75% to 95% or 90% to 98%) in the percentage of dead cancer cells in the first population relative to the percentage of dead cancer cells in the second population indicates that the test compound or the combination of test compounds at the test concentration(s) and kinase inhibitor at the specific concentration is efficacious for treating cancer. In contrast, no detectable change or an increase of about 20% or less (e.g., 15%, 10%, 5%, 4%, 3%, 2%, or 1%, or 10%) to 15%), or 1% to 5%) in the percentage of dead cancer cells indicates that he test compound or the combination of test compounds at the test concentration(s) and kinase inhibitor at the specific concentration is not efficacious or has minimal efficacy for treating cancer. In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, the cancer cells are derived or obtained from a cancer cell line. In other embodiments, the cancer cells are derived or obtained from a cancer patient. In a specific embodiment, the cancer cells are from the cancer cell line A549, H1299, HepG2, MD-MB-231, T47D, A375, 239, MZ-MTC, TT-MTC, or H358-NSCLC. In another specific embodiment the cancer cells are from a cancer cell line described in Example 1 (Section 6, infra).

[00169] In certain embodiments, the test concentration of the test compound or each compound in the combination of test compounds used in cell culture is 0.1 nmol to 75 nmol, 1 nmol to 75 nmol, 10 nmol to 75 nmol, 20 nmol to 75 nmol, 30 nmol to 75 nmol, 50 nmol to 75 nmol, 0.1 to 50 nmol, 1.5 nmol to 50 nmol, 5 nmol to 50 nmol, 10 nmol to 50 nmol, 25 nmol to 50 nmol, 1 nmol to 10 nmol, 1 nmol to 6 nmol, 1 to 5 nml, 0.1 to 6 nmol, or 1 to 3 nmol. In some embodiments, the test concentration of the test compound or each compound in the combination of test compounds used in cell culture is 0.1 nmol, 0.25 nmol, 0.5 nmol, 0.75 nmol, 1 nmol, 5 nmol, 7 nmol, 10 nmol, 15 nmol, 20 nmol, 25 nmol, 30 nmol, 35 nmol, 40 nmol, 45 nmol, 50 nmol, 55 nmol, 60 nmol, 65 nmol, 70 nmol, or 75 nmol. In certain embodiments, the test concentration of each compound in the combination of test compounds is different. In other embodiments, the test concentration of each compound in the combination of test compounds is the same. In certain embodiments, the specific concentration of the kinase inhibitor used in cell culture is about 0.005 μπιοΐ (micromolar) to 20 μπιοΐ, 0.01 μπιοΐ to 20 μπιοΐ, 0.1 μπιοΐ to 20 μπιοΐ, 0.5 μπιοΐ to 20 μπιοΐ, 1 μπιοΐ to 20 μπιοΐ, 2 μπιοΐ to 20 μπιοΐ, 5 μπιοΐ to 20 μπιοΐ, 10 μπιοΐ to 20μπιο1, 0.005 μπιοΐ to 10 μπιοΐ, 0.01 μπιοΐ to 10 μπιοΐ, 0.1 μπιοΐ to 10 μmol, 0.5 μπιοΐ to 10 μmol, 1 μπιοΐ to 10 μmol, 2 μπιοΐ to 10 μmol, 5 μπιοΐ to 10 μmol, 0.005 μπιοΐ to 5 μmol, 0.01 μπιοΐ to 5 μmol, 0.1 μπιοΐ to 5 μmol, 0.5 μπιοΐ to 5 μmol, 1 μπιοΐ to 5 μmol, 2 μπιοΐ to 5 μmol, 0.005 μπιοΐ to 2 μmol, 0.01 μπιοΐ to 2 μmol, 0.1 μπιοΐ to 2 μmol, 0.5 μπιοΐ to 2 μmol, 1 μπιοΐ to 2 μmol, 0.005 μπιοΐ to 1 μmol, 0.01 μπιοΐ to 1 μmol, 0.1 μπιοΐ to Ιμτηοΐ, 0.005 μπιοΐ to 0.5 μmol, 0.01 μπιοΐ to 0.5 μmol, 0.1 μπιοΐ to 0.5 μmol, 0.005 μπιοΐ to 0.1 μmol, 0.01 μπιοΐ to 0.1 μmol, or 0.005 μπιοΐ to 0.01 μmol. In certain embodiments, the specific concentration of the kinase inhibitor used in cell culture is about 0.005 μπιοΐ, 0.01 μπιοΐ, 0.1 μπιοΐ, 0.5 μπιοΐ, 1 μπιοΐ, 2 μπιοΐ, 5 μmol, 10 μπιοΐ, or 20 μmol. In a specific embodiment, the test concentration of the test compound or each compound in the combination of compounds is equivalent to a subtherapeutic dose(s).

[00170] The period of time that the cancer cells are cultured in an Efficacy Assay can be, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or 3-5, 4-5, 4-7, 5-7, 7-10 or 7-14 days. In a specific embodiment, the period of time that the cancer cells are cultured in an Efficacy Assay is 4 days. In another specific embodiment, the period of time that the cancer cells are cultured in an Efficacy Assay is 5 days. In another specific embodiment, the period of time that the cancer cells are cultured in an Efficacy Assay is 6 days. In another specific embodiment, the period of time that the cancer cells are cultured in an Efficacy Assay is 7 days.

[00171] In certain embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is added to cell culture prior to the addition of the kinase inhibitor. For example, the test compound or the combination of test compounds is add to cell culture 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, or 21 days prior to the addition of the kinase inhibitor. In another example, the test compound or the combination of test compounds is added to cell culture 1 to 6 hours, 2 to 6 hours, 4 to 6 hours, 6 to 12 hours, 12 to 24 hours, 1 to 2 days, 2 to 5 days, 5 to 7 days, 7 to 14 days or 14 to 21 days prior to the addition of the kinase inhibitor. In other embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is added to cell culture concurrently with the addition of the kinase inhibitor. In yet other embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is added to cell culture subsequent to the addition of the kinase inhibitor. For example, the test compound or the combination of test compounds is added to cell culture 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, or 21 days subsequent to the addition of the kinase inhibitor. In another example, the test compound or the combination of test compounds is added to cell culture 1 to 6 hours, 2 to 6 hours, 4 to 6 hours, 6 to 12 hours, 12 to 24 hours, 1 to 2 days, 2 to 5 days, 5 to 7 days, 7 to 14 days or 14 to 21 days subsequent to the addition of the kinase inhibitor.

[00172] In certain embodiments, one or more additional controls, e.g., positive and/or negative controls are included in an Efficacy Assay described herein. For example, a Network Brake described in Section 6, infra, may be used a positive control. In a specific embodiment, an Efficacy Assay is conducted as described in Section 6, infra.

[00173] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds and a kinase inhibitor for treating cancer comprises: (i) administering to a first group of animals, which are alive and have cancer, a test compound or a combination of test compounds at a test dose(s) and a kinase inhibitor at a specific dose; and (ii) analyzing the percentage of dead cancer cells of the first group of animals after a period of time, wherein an increase of greater than about 20% (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 25% to 50%, 50% to 7%%, 25% to 75%, 75%) to 95% or 90% to 98%) in the percentage of dead cancer cells in the first group of animals relative to the percentage of dead cancer cells in a second group of the same type of animals, which are alive and have the same type of cancer, administered a negative control, such as control vehicle, and the same kinase inhibitor at the same specific dose after the same period of time indicates that the test compound or the combination of test compounds and kinase inhibitor at the specific concentration is efficacious for treating cancer. In contrast, no detectable change or an increase of about 20% or less (e.g., 15%, 10%, 5%, 4%, 3%, 2%, or 1%, or 10% to 15%, or 1%) to 5%) in the percentage of dead cancer cells in the first group relative to the second group indicates that the test compound or the combination of test compounds and kinase inhibitor at the specific concentration is not efficacious or has minimal efficacy for treating cancer. In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, a tumor biopsy or blood sample obtained from an animal(s) is analyzed to assess the percentage of dead cancer cells. In a specific embodiment, the animals are animal models for a particular type of cancer (e.g., lung, liver, brain, kidney, uterine, breast, pancreatic, thyroid, testicular, skin, or bone). In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are non-human animals. In another

embodiment, the animals are xenografts, such as described in Example 1 (Section 6, infra).

[00174] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds and a kinase inhibitor for treating cancer comprises: (i) administering to a first group of animals, which are alive and have cancer, a test compound or a combination of test compounds at a test dose(s) and a kinase inhibitor at a specific dose; and (ii) analyzing tumor size in the first group of animals after a period of time, wherein a decrease in tumor size by greater than 20% (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 25% to 50%, 50% to 7%%, 25% to 75%, 75% to 95% or 90%) to 98%)) in the first group of animals relative to the tumor size in a second group of the same type of animals, which are alive and have the same type of cancer, administered a negative control, such as control vehicle, and the same kinase inhibitor at the same specific dose after the same period of time indicates that the test compound or the combination of test compounds and kinase inhibitor at the specific concentration is efficacious for treating cancer. In contrast, no detectable change or an increase in tumor size of about 20% or less (e.g., 15%, 10%, 5%, 4%, 3%, 2%, or 1%), or 10% to 15%, or 1% to 5%) in the first group of animals relative to the tumor size in the second group of animals indicates that the test compound or the combination of test compounds and kinase inhibitor at the specific concentration is not efficacious or has minimal efficacy for treating cancer. In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, the tumor size is assessed by an imaging technique known to one of skill in the art, e.g., MRI, X-ray, CAT/CT scan or PET scan. In a specific embodiment, the animals are animal models for a particular type of cancer (e.g., lung, liver, brain, kidney, uterine, breast, pancreatic, thyroid, testicular, skin, or bone). In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are non-human animals. In another embodiment, the animals are xenografts, such as described in Example 1 (Section 6, infra).

[00175] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds and a kinase inhibitor for treating cancer comprises: (i) administering to a first group of animals, which are alive and have cancer, a test compound or a combination of test compounds at a test dose(s) and a kinase inhibitor at a specific dose; (ii) administering to a second group of the same type of animals, which are alive and have the same type of cancer, a negative control, such as control vehicle, and the same kinase inhibitor at the same specific dose; and (iii) analyzing the percentage of dead cancer cells of the first group of animals and the percentage of dead cancer cells of the second group of animals after a period of time, wherein an increase of greater than about 20% {e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 25% to 50%, 50% to 7%%, 25% to 75%), 75%) to 95%) or 90% to 98%) in the percentage of dead cancer cells in the first group of animals relative to the percentage of dead cancer cells in the second group of animals indicates that the test compound or the combination of test compounds at the test dose(s) and kinase inhibitor at the specific concentration is efficacious for treating cancer. In contrast, no detectable change or an increase of about 20% or less (e.g., 15%, 10%, 5%, 4%, 3%, 2%, or 1%, or 10% to 15%), or 1%) to 5%) in the percentage of dead cancer cells in the first group of animals relative to the percentage of dead cancer cells in the second group of animals indicates that the test compound or the combination of test compounds at the test dose(s) and kinase inhibitor at the specific concentration is not efficacious or has minimal efficacy for treating cancer. In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, a tumor biopsy or blood sample obtained from an animal(s) is analyzed to assess the percentage of dead cancer cells. In a specific embodiment, the animals are animal models for a particular type of cancer (e.g., lung, liver, brain, kidney, uterine, breast, pancreatic, thyroid, testicular, skin, or bone). In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are non-human animals. In another embodiment, the animals are xenografts, such as described in Example 1 (Section 6, infra).

[00176] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds and a kinase inhibitor for treating cancer comprises: (i) administering to a first group of animals, which are alive and have cancer, a test compound or a combination of test compounds at a test dose(s) and a kinase inhibitor at a specific dose; (ii) administering to a second group of the same type of animals, which are alive and have the same type of cancer, a negative control, such as control vehicle, and the same kinase inhibitor at the same specific dose; and (iii) analyzing tumor size in the first and second groups of animals after a period of time, wherein an decrease in tumor size by greater than 20% {e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 25% to 50%, 50% to 7%%, 25% to 75%, 75% to 95% or 90% to 98%) in the first group of animals relative to the tumor size in the second group of animals indicates that the test compound or the combination of test compounds at the test dose(s) and kinase inhibitor at the specific concentration is efficacious for treating cancer. In contrast, no detectable change or an increase in tumor size of about 20% or less [(e.g., 15%, 10%, 5%, 4%, 3%, 2%, or 1%, or 10% to 15%, or 1% to 5%) in the first group of animals relative to the tumor size in the second group of animals indicates that the test compound or the combination of test compounds at the test dose(s) and kinase inhibitor at the specific concentration is not efficacious or has minimal efficacy for treating cancer. In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, the tumor size is assessed by an imaging technique known to one of skill in the art, e.g., MRI, X-ray, CAT/CT scan or PET scan. In a specific embodiment, the animals are animal models for a particular type of cancer (e.g., lung, liver, brain, kidney, uterine, breast, pancreatic, thyroid, testicular, skin, or bone). In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are non-human animals. In another

[00177] In certain embodiments, the test dose of the test compound or each compound in the combination of test compounds administered to the animals is about 2-fold, 5-fold, 10-fold, 20- fold, 50-fold, 75-fold, or 100-fold lower than the dosage efficacious for treating a particular cancer in the animals. In one embodiment, the test dose of the test compound or each compound in the combination of test compounds administered to the animals is about 40 mg/kg, 20 mg/kg, 10 mg/kg, 5 mg/kg, lmg/kg, 0.5 mg/kg, 0.1 mg/kg, 0.05 mg/kg, 0.01 mg/kg, 0.005 mg/kg, 0.001 mg/kg or lower, or about 40 mg/kg to 20 mg/kg, 20 mg/kg to 10 mg/kg, 10 mg/kg to 5 mg/kg, 5 mg/kg to 1 mg/kg, 1 mg/kg to 0.5 mg/kg, 0.5 mg/kg to 0.1 mg/kg, 0.1 mg/kg to 0.05 mg/kg, 0.05 mg/kg to 0.01 mg/kg, 0.01 mg/kg to 0.005 mg/kg, 0.005 mg/kg to 0.001 mg/kg or lower. In a specific embodiment, the test dose of the test compound or each compound in the combination of test compounds is the subtherapeutic dose(s). In some embodiments, the test dose of each compound in the combination of test compounds administer to the animal is different. In other embodiments, the test dose of each compound in the combination of test compounds

administered to the animal is the same. In some embodiments, the specific dose of the kinase inhibitor administered to the animals is an effective dose. In some embodiments, the specific dose of the kinase inhibitor administered to the animals is a non-effective dose. In a specific embodiment, the specific dose of the kinase inhibitor administered to the animals is about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 75-fold, or 100-fold lower than the dosage efficacious for treating a particular cancer in the animals. In one embodiment, the specific dose of the kinase inhibitor administered to the animals is about 200 mg/kg, 100 mg/kg, 50 mg/kg, 25 mg/kg, 10 mg/kg, 5 mg/kg, 1 mg/kg, 0.5 mg/kg, 0.1 mg/kg or lower, or about 200 mg/kg to 100 mg/kg, 100 mg/kg to 50 mg/kg, 50 mg/kg to 25 mg/kg, 25 mg/kg to 10 mg/kg, 10 mg/kg to 5 mg/kg, 5 mg/kg to 1 mg/kg, 1 mg/kg to 0.5 mg/kg, 0.5 mg/kg to 0.1 mg/kg or lower.

[00178] The period of time after which the group of animals can be assessed in an Efficacy Assay can be, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or 3-5, 4-5, 4-7, 5-7, 7-10 or 7-14 days. In a specific embodiment, the period of time after which the group of animals can be assessed in an Efficacy Assay can be is 4 days. In another specific embodiment, the period of time after which the group of animals can be assessed in an Efficacy Assay is 5 days. In another specific embodiment, the period of time after which the group of animals can be assessed in an Efficacy Assay is 6 days. In another specific embodiment, the period of time after which are group of animals can be assessed in an Efficacy Assay is 7 days.

[00179] In certain embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is administered to an animal prior to the administration of the kinase inhibitor. For example, the test compound or the combination of test compounds is administered to an animal 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, or 21 days prior to the administration of the kinase inhibitor. In another example, the test compound or the combination of test compounds is administered to an animal 1 to 6 hours, 2 to 6 hours, 4 to 6 hours, 6 to 12 hours, 12 to 24 hours, 1 to 2 days, 2 to 5 days, 5 to 7 days, 7 to 14 days or 14 to 21 days prior to the administration of the kinase inhibitor. In other embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is administered to an animal concurrently with the administration of the kinase inhibitor. In yet other embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is administered to an animal subsequent to the administration of the kinase inhibitor. For example, the test compound or the combination of test compounds is administered to an animal 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, or 21 days subsequent to the administration of the kinase inhibitor. In another example, the test compound or the combination of test compounds is administered to an animal 1 to 6 hours, 2 to 6 hours, 4 to 6 hours, 6 to 12 hours, 12 to 24 hours, 1 to 2 days, 2 to 5 days, 5 to 7 days, 7 to 14 days or 14 to 21 days subsequent to the administration of the kinase inhibitor.

[00180] In specific embodiments, the Efficacy Assay is performed using animal juveniles engineered to controllably over-express or hyper-activate an oncogene(s), which prevents the animal juveniles from developing into viable adults, and the Efficacy Assay measures adult viability rate as a proxy for inhibition of the oncogenic mutation(s). The animal juveniles as used herein can be young animals before reaching adulthood or embryos. In specific

embodiments, the animal juveniles are non-human juveniles. In specific embodiments, the animal juveniles are non-primate juveniles. In specific embodiments, the animal juveniles are fly larvae. In a specific embodiment, the animal juveniles are the PTC>Ret2B files described in Section 6 (see also Dar et al., 2012, Nature 486: 80-84, which is incorporated herein in its entirety.). In a specific embodiment, the Efficacy Assay is conducted in the flies described in Example 1 (Section 6, infra) using the methods described in Example 1.

[00181] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds and a kinase inhibitor for treating cancer comprises: (i) administering to a first group of animal juveniles over-expressing or having a hyper-activated oncogene(s), which prevents the animal juveniles from developing into viable adults, a test compound or a combination of test compounds at a test dose(s) and a kinase inhibitor at a specific dose; and (ii) analyzing the percentage of viable adults developed from the first group of animal juveniles after a period of time, wherein an increase of greater than about 15% (e.g., 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 20% to 30%, 25% to 50%, 50% to 75%, 25% to 75%, 75% to 95% or 90% to 98%) in the percentage of viable adults developed from the first group of animal juveniles relative to the percentage of viable adults developed from a second group of the same type of animal juveniles over-expressing the same oncogene(s) or having the same hyper-activated oncogene(s), administered a negative control, such as control vehicle, and the same kinase inhibitor at the same specific dose after the same period of time indicates that the test compound or the combination of test compounds and kinase inhibitor at the specific concentration is efficacious for treating cancer. In contrast, no detectable change or an increase of about 15% or less (e.g., 10%, 5%, 4%, 3%,2%, or 1%, or 10% to 15%, 5% to 10%, 5% to 15%, or 1% to 5%) in the pecentage of viable adults developed from the first group relative to the second group indicates that the test compound or the combination of test compounds and kinase inhibitor at the specific concentration is not efficacious or has minimal efficacy for treating cancer. In a specific embodiment, the expression of the oncogene is inducible (e.g., by temperature) and, in specific embodiments, the over-expression or hyper-activation of the oncogene is induced concurrently with the administration of the animal juveniles with the test compound or combination of test compounds. See, e.g., Example 1 (Section 6, infra) for a description of the induction of an oncogene. In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, the group of animal juveniles comprises about 2 to 4 animal juveniles, 4 to 6 animal juveniles, 5 to 8 animal juveniles, 8 to 10 animal juveniles, 10 to 20 animal juveniles, 20- 30 animal juveniles, 30 to 60 animal juveniles, or 60 to 100 animal juveniles.

[00182] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds and a kinase inhibitor for treating cancer comprises: (i) administering to a first group of animal juveniles over-expressing or having a hyper-activated oncogene(s), which prevents the animal juveniles from developing into viable adults, a test compound or a combination of test compounds at a test dose(s) and a kinase inhibitor at a specific dose; (ii) administering to a second group of the same type of animal juveniles over- expressing the same oncogene(s) or having the same hyper-activated oncogene(s) a negative control, such as control vehicle, and the same kinase inhibitor at the same specific dose; and (iii) analyzing the percentage of viable adults developed from the first group of animal juveniles after a period of time and the percentage of viable adults developed from the second group of animals after the same period of time, wherein an increase of greater than about 15% (e.g., 20%, 25%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 20% to 30%, 25% to 50%, 50% to 75%, 25% to 75%, 75% to 95%, or 50% to 95%) in the percentage of viable adults developed from the first group of animal juveniles relative to the percentage of viable adults developed from the second group of animal juveniles indicates that the test compound or the combination of test compounds at the test dose(s) and kinase inhibitor at the specific

concentration is efficacious for treating cancer. In contrast, no detectable change or an increase of about 15% or less (e.g., 10%, 5%, 4%, 3%, 2%, or 1%, or 10% to 15%, or 1% to 5%) in the percentage of viable adults developed from the first group of animal juveniles relative to the percentage of viable adults developed from the second group of animal juveniles indicates that the test compound or the combination of test compounds at the test dose(s) and kinase inhibitor at the specific concentration is not efficacious or has minimal efficacy for treating cancer. In a specific embodiment, the expression of the oncogene is inducible (e.g., by temperature) and, in specific embodiments, the over-expression or hyper-activation of the oncogene is induced concurrently with the administration of the animal juveniles with the test compound or combination of test compounds. See, e.g., Example 1 (Section 6, infra) for a description of the induction of an oncogene. In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, the group of animal juveniles comprises 2 to 4 animal juveniles, 4 to 6 animal juveniles, 5 to 8 animal juveniles, 8 to 10 animal juveniles, 10 to 20 animal juveniles, 20- 30 animal juveniles, 30 to 60 animal juveniles, or 60 to 100 animal juveniles.

[00183] In certain embodiments, the test dose of the test compound or each compound in the combination of test compounds administered to the animal juveniles is about 0.01 μπιοΐ to 50 μπιοΐ, 0.1 μπιοΐ to 50 μπιοΐ, 1 μπιοΐ to 50 μπιοΐ, 5 μπιοΐ to 50 μπιοΐ, 10 μπιοΐ to 50 μπιοΐ, 20 μπιοΐ to 50 μπιοΐ, 0.01 μπιοΐ to 20 μπιοΐ, 0.1 μπιοΐ to 20 μπιοΐ, 1 μπιοΐ to 20 μπιοΐ, 5 μπιοΐ to 20 μπιοΐ, 0.01 μπιοΐ to 10 μπιοΐ, 0.1 μπιοΐ to 10 μπιοΐ, 1 μπιοΐ to 10 μπιοΐ, 5 μπιοΐ to 10 μπιοΐ, 0.01 μηιοΐ to 5 μηιοΐ, 0.1 μηιοΐ to 5 μηιοΐ, 1 μηιοΐ to 5 μηιοΐ, 0.01 μηιοΐ to 1 μηιοΐ, 0.1 μηιοΐ to 1 μηιοΐ, 0.01 μηιοΐ to 0.1 μηιοΐ. In certain embodiments, the test dose of the test compound or each compound in the combination of test compounds administered to the animal juveniles is about 0.01 μπιοΐ, 0.1 μιηοΐ, 1 μιηοΐ, 5 μιηοΐ, 10 μιηοΐ, 20 μιηοΐ, 50 μιηοΐ. In a specific embodiment, the test dose of the test compound or each compound in the combination of test compounds is the subtherapeutic dose(s). In some embodiments, the specific dose of the kinase inhibitor administered to the animal juveniles is about 0.1 μπιοΐ to 500 μπιοΐ, 1 μπιοΐ to 500 μπιοΐ, 10 μπιοΐ to 500 μπιοΐ, 50 μπιοΐ to 500 μπιοΐ, 200 μπιοΐ to 500 μπιοΐ, 0.1 μπιοΐ to 200 μπιοΐ, 1 μπιοΐ to 200 μπιοΐ, 10 μπιοΐ to 200 μπιοΐ, 50 μπιοΐ to 200 μπιοΐ, 0.1 μmol to 50 μπιοΐ, 1 μmol to 50 μπιοΐ, 10 μmol to 50 μπιοΐ, 0.1 μmol to 10 μπιοΐ, 1 μmol to 10 μπιοΐ, 0.1 μmol to 1 μπιοΐ. In some embodiments, the specific dose of the kinase inhibitor administered to the animal juveniles is about 0.1 μπιοΐ, 1 μπιοΐ, 10 μπιοΐ, 50 μπιοΐ, 200 μπιοΐ, 500 μmol.

[00184] The period of time after which the percentage of viable adults can be assessed in an Efficacy Assay can be, for example, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, or 28 days, or about 3-5, 4-5, 4-7, 5-7, 7-10, 7-14, 10-21, 14-21, 14-28, or 21-28 days. In a specific

embodiment, the period of time after which the percentage of viable adults can be assessed in an Efficacy Assay can be is 4 days. In another specific embodiment, the period of time after which the percentage of viable adults can be assessed in an Efficacy Assay is 5 days. In another specific embodiment, the period of time after which the percentage of viable adults can be assessed in an Efficacy Assay is 6 days. In another specific embodiment, the period of time after which the percentage of viable adults can be assessed in an Efficacy Assay is 7 days.

[00185] In certain embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is administered to an animal juvenile prior to the administration of the kinase inhibitor. For example, the test compound or the combination of test compounds is administered to an animal juvenile 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, or 21 days prior to the administration of the kinase inhibitor. In another example, the test compound or the combination of test compounds is administered to an animal juvenile 1 to 6 hours, 2 to 6 hours, 4 to 6 hours, 6 to 12 hours, 12 to 24 hours, 1 to 2 days, 2 to 5 days, 5 to 7 days, 7 to 14 days or 14 to 21 days prior to the administration of the kinase inhibitor. In other embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is administered to an animal juvenile concurrently with the administration of the kinase inhibitor. In yet other embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is administered to an animal juvenile subsequent to the administration of the kinase inhibitor. For example, the test compound or the combination of test compounds is administered to an animal juvenile 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, or 21 days subsequent to the administration of the kinase inhibitor. In another example, the test compound or the combination of test compounds is administered to an animal juvenile 1 to 6 hours, 2 to 6 hours, 4 to 6 hours, 6 to 12 hours, 12 to 24 hours, 1 to 2 days, 2 to 5 days, 5 to 7 days, 7 to 14 days or 14 to 21 days subsequent to the administration of the kinase inhibitor.

5.3.2. Viability Assay

[00186] The Viability Assay referenced herein can be any assay known in the art to test the efficacy of a drug for treating cancer (e.g., by measuring the killing of cancer cells, reduction in tumor size, or development of viable adults from animal juveniles having an oncogenic mutation(s) that prevent them from maturing into viable adults), and can be performed using cancer cells or animal models (e.g., flies, worms, mice, rats, rabbits, or primates). Provided in this Section 5.3.2 and Section 6, infra, are examples of various ways to conduct the Viability Assays.

[00187] In a specific embodiment, the Viability Assay is performed as described in Example 1 (Section 6, infra).

[00188] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds for treating cancer comprises: (i) culturing cancer cells in the presence of a test compound or a combination of test compounds at a test concentration(s) for a period of time; and (ii) measuring the percentage of dead cancer cells at the end of said period of time, wherein no measurable change or an increase of about 20% or less (e.g., 15%, 10%, 5%, 4%, 3%, 2% or 1%, or 15% to 19.5%, 10 to 15%, 5% to 15%, 5% to 10%, or 1% to 5%) in the percentage of dead cancer cells measured when the cancer cells are cultured in the presence of the test compound or the combination of test compounds at the test concentration(s) relative to the percentage of dead cancer cells when the same type of cancer cells are cultured in the presence of a negative control, such as vehicle alone (e.g., phosphate buffered saline (PBS) or another buffer), for the same period of time indicates that the test compound or the combination of test compounds at the test concentration(s) has no or minimal efficacy alone. In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, the cancer cells are derived or obtained from a cancer cell line. In other embodiments, the cancer cells are derived or obtained from a cancer patient. In a specific embodiment, the cancer cells are from the cancer cell line A549, H1299, HepG2, MD-MB-231, T47D, A375, 239, MZ-MTC, TT-MTC, or H358-NSCLC. In another specific embodiment the cancer cells are from a cancer cell line described in Example 1 (Section 6, infra).

[00189] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds for treating cancer comprises: (i) culturing a first population of cancer cells in the presence of a test compound or a combination of test compounds at a test concentration(s) for a period of time; (ii) culturing a second population of the same type of cancer cells in the presence of a negative control, such as vehicle alone {e.g., phosphate buffered saline (PBS) or another buffer) for the same period of time; and (iii) measuring the percentage of dead cells of the cancer cells cultured under the conditions in (i) and (ii) at the end of said period of time, wherein no measurable change or an increase of about 20% or less {e.g., 15%, 10%, 5%, 4%, 3%, 2% or 1%, or 15% to 19.5%, 10 to 15%, 5% to 15%, 5% to 10%, or 1% to 5%) in the percentage of dead cancer cells measured when the first population of cancer cells are cultured in the presence of the test compound or the combination of test compounds at the test

concentration(s) relative to the percentage of dead cancer cells when the second population of the same type of cancer cells are cultured in the presence of the negative control, such as control vehicle {e.g., phosphate buffered saline (PBS) or another buffer), for the same period of time indicates that the test compound or the combination of test compounds at the test

concentration(s) has no or minimal efficacy alone. In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the

combination of test compounds. In certain embodiments, the cancer cells are derived or obtained from a cancer cell line. In other embodiments, the cancer cells are derived or obtained from a cancer patient. In a specific embodiment, the cancer cells are from the cancer cell line A549, H1299, HepG2, MD-MB-231, T47D, A375, 239, MZ-MTC, TT-MTC, or H358-NSCLC. In another specific embodiment the cancer cells are from a cancer cell line described in Example 1 (Section 6, infra). [00190] In specific embodiments a method for assessing the efficacy of a test compound or a combination of test compounds for treating cancer comprises: (i) culturing cancer cells in the presence of a test compound or a combination of test compounds at a test concentration(s) for a period of time; and (ii) measuring the percentage of dead cancer cells at the end of said period of time, wherein no measurable change or an increase of about 20% or less (e.g., 15%, 10%, 5%, 4%, 3%, 2% or 1%, or 15% to 19.5%, 10 to 15%, 5% to 15%, 5% to 10%, or 1% to 5%) in the percentage of dead cancer cells measured when the cancer cells are cultured in the presence of the test compound or the combination of test compounds at the test concentration(s) relative to the percentage of dead cancer cells when the same type of cancer cells are cultured in the absence of the test compound or test compounds, for the same period of time indicates that the test compound or the combination of test compounds at the test concentration(s) has no or minimal efficacy alone. In certain embodiments, the cancer cells are derived or obtained from a cancer cell line. In other embodiments, the cancer cells are derived or obtained from a cancer patient. In a specific embodiment, the cancer cells are from the cancer cell line A549, H1299, HepG2, MD-MB-231, T47D, A375, 239, MZ-MTC, TT-MTC, or H358-NSCLC. In another specific embodiment the cancer cells are from a cancer cell line described in Example 1 (Section 6, infra).

[00191] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds for treating cancer comprises: (i) culturing a first population of cancer cells in the presence of a test compound or a combination of test compounds at a test concentration(s) for a period of time; (ii) culturing a second population of the same type of cancer cells in the absence of the test compound or the combination of test compounds for the same period of time; and (iii) measuring the percentage of dead cells of the cancer cells cultured under the conditions in (i) and (ii) at the end of said period of time, wherein no measurable change or an increase of about 20% or less (e.g., 15%, 10%, 5%, 4%, 3%, 2% or 1%, or 15% to 19.5%, 10 to 15%, 5% to 15%, 5% to 10%, or 1% to 5%) in the percentage of dead cancer cells measured when the first population of cancer cells are cultured in the presence of the test compound or the combination of test compounds at the test concentration(s) relative to the percentage of dead cancer cells when the second population of the same type of cancer cells are cultured in the absence of the test compound or the combination of test compounds for the same period of time indicates that the test compound or the combination of test compounds at the test concentration(s) has no or minimal efficacy alone. In certain embodiments, the cancer cells are derived or obtained from a cancer cell line. In other embodiments, the cancer cells are derived or obtained from a cancer patient. In a specific embodiment, the cancer cells are from the cancer cell line A549, H1299, HepG2, MD-MB-231, T47D, A375, 239, MZ-MTC, TT-MTC, or H358- NSCLC. In another specific embodiment the cancer cells are from a cancer cell line described in Example 1 (Section 6, infra).

[00192] In certain embodiments, the test concentration of the test compound or each compound in the combination of test compounds used in cell culture is 0.1 nmol to 75 nmol, 1 nmol to 75 nmol, 10 nmol to 75 nmol, 20 nmol to 75 nmol, 30 nmol to 75 nmol, 50 nmol to 75 nmol, 0.1 to 50 nmol, 1.5 nmol to 50 nmol, 5 nmol to 50 nmol, 10 nmol to 50 nmol, 25 nmol to 50 nmol, 1 nmol to 10 nmol, 1 nmol to 6 nmol, 1 to 5 nml, 0.1 to 6 nmol, or 1 to 3 nmol. In some embodiments, the test concentration of the test compound or each compound in the combination of test compounds used in cell culture is 0.1 nmol, 0.25 nmol, 0.5 nmol, 0.75 nmol, 1 nmol, 5 nmol, 7 nmol, 10 nmol, 15 nmol, 20 nmol, 25 nmol, 30 nmol, 35 nmol, 40 nmol, 45 nmol, 50 nmol, 55 nmol, 60 nmol, 65 nmol, 70 nmol, or 75 nmol In specific embodiments, the test concentration of each compound in the combination of test compounds is different. In other specific embodiments, the test concentration of each compound in the combination of test compounds is the same.

[00193] The period of time that the cancer cells are cultured in a Viability Assay can be, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or 3-5, 4-5, 4-7, 5-7, 7-10 or 7-14 days. In a specific embodiment, the period of time that the cancer cells are cultured in a Viability Assay is 4-12 days. In another specific embodiment, the period of time that the cancer cells are cultured in a Viability Assay is 5-12 days. In another specific embodiment, the period of time that the cancer cells are cultured in a Viability Assay is 6-12 days. In another specific embodiment, the period of time that the cancer cells are cultured in a Viability Assay is 7-12 days.

[00194] In certain embodiments, one or more additional controls, e.g., positive and/or negative controls are included in a Viability Assay described herein. For example, the Network Brake described in Section 6, infra, may be used a positive control. In a specific embodiment, a Viability Assay is conducted as described in Section 6, infra.

[00195] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds for treating cancer comprises: (i) administering to a group of animals, which are alive and have cancer, a test compound or a combination of test compounds at a test dose(s); and (ii) analyzing the percentage of dead cancer cells after a period of time, wherein no detectable change or an increase of about 20% or less (e.g., 15%, 10%, 5%, 4%, 3%, 2% or 1%, or 15% to 19.5%, 10 to 15%, 5% to 15%, 5% to 10%, or 1% to 5%) in the percentage of dead cancer cells indicates that the test compound or the combination of test compounds at the test dose(s) has no or minimal efficacy alone. In certain embodiments, a tumor biopsy or blood sample obtained from an animal(s) is analyzed to assess the percentage of dead cancer cells. In a specific embodiment, the animals are animal models for a particular type of cancer (e.g., lung, liver, brain, kidney, uterine, breast, pancreatic, thyroid, testicular, skin, or bone). In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are non-human animals.

[00196] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds for treating cancer comprises: (i) administering to a group of animals which are alive and have cancer a test compound or a combination of test compounds at a test dose(s); and (ii) analyzing tumor size after a period of time, wherein no detectable change or an increase (e.g., an increase of about 5%, 10%, 15%, 20%, 25%, 30% or more) in the tumor size indicates that the test compound or the combination of test compounds at the test dose(s) has no or minimal efficacy alone. In certain embodiments, the tumor size is assessed by an imaging technique known to one of skill in the art, e.g., MRI, X-ray, CAT/CT scan or PET scan. In a specific embodiment, the animals are animal models for a particular type of cancer (e.g., lung, liver, brain, kidney, uterine, breast, pancreatic, thyroid, testicular, skin, or bone). In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are non-human animals.

[00197] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds for treating cancer comprises: (i) administering to a first group of animals, which are alive and have cancer, a test compound or a combination of test compounds at a test dose(s); and (ii) analyzing the percentage of dead cancer cells of the first group of animals after a period of time, wherein no detectable change or an increase of about 20% or less (e.g., 15%, 10%, 5%, 4%, 3%, 2% or 1%, or 15% to 19.5%, 10 to 15%, 5% to 15%, 5% to 10%, or 1% to 5%) in the percentage of dead cancer cells in the first group of animals relative to the percentage of dead cancer cells in a second group of the same type of animals, which are alive and have the same type of cancer, administered a negative control, such as control vehicle, after the same period of time indicates that the test compound or the combination of test compounds has no or minimal efficacy at the test dose(s) alone. In a specific

embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, a tumor biopsy or blood sample obtained from an animal(s) is analyzed to assess the percentage of dead cancer cells. In a specific embodiment, the animals are animal models for a particular type of cancer (e.g., lung, liver, brain, kidney, uterine, breast, pancreatic, thyroid, testicular, skin, or bone). In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are non-human animals. In another specific embodiment, the animals are xenografts, such as described in Example 1 (Section 6, infra).

[00198] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds for treating cancer comprises: (i) administering to a first group of animals, which are alive and have cancer, a test compound or a combination of test

compounds at a test dose(s); and (ii) analyzing tumor size in the first group of animals after a period of time, wherein no detectable change or an increase {e.g., an increase of about 5%, 10%, 15%), 20%), 25%), 30%o or more) in the tumor size in the first group of animals relative to tumor size in a second group of the same type of animals, which are alive and have the same type of cancer, administered a negative control, such as control vehicle, after the same period of time indicates that the test compound or the combination of test compounds has no or minimal efficacy at the test dose(s) alone. In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, the tumor size is assessed by an imaging technique known to one of skill in the art, e.g., MRI, X-ray, CAT/CT scan or PET scan. In a specific embodiment, the animals are animal models for a particular type of cancer {e.g., lung, liver, brain, kidney, uterine, breast, pancreatic, thyroid, testicular, skin, or bone). In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are non-human animals. In another specific embodiment, the animals are xenografts, such as described in Example 1 (Section 6, infra).

[00199] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds for treating cancer comprises: (i) administering to a first group of animals, which are alive and have cancer, a test compound or a combination of test compounds at a test dose(s); (ii) administering to a second group of the same type of animals, which are alive and have the same type of cancer, a negative control, such as control vehicle; and (iii) analyzing the percentage of dead cancer cells of the first group of animals and the percentage of dead cancer cells of the second group of animals after a period of time, wherein no detectable change or an increase of about 20% or less (e.g., 15%, 10%, 5%, 4%, 3%, 2% or 1%, or 15% to 19.5%, 10 to 15%, 5% to 15%, 5% to 10%, or 1% to 5%) in the percentage of dead cancer cells in the first group of animals relative to the percentage of dead cancer cells in the second group of animals indicates that the test compound or the combination of test compounds has no or minimal efficacy at the test dose(s) alone. In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, a tumor biopsy or blood sample obtained from an animal(s) is analyzed to assess the percentage of dead cancer cells. In a specific embodiment, the animals are animal models for a particular type of cancer (e.g., lung, liver, brain, kidney, uterine, breast, pancreatic, thyroid, testicular, skin, or bone). In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are non-human animals. In another specific embodiment, the animals are xenografts, such as described in Example 1 (Section 6, infra).

[00200] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds for treating cancer comprises: (i) administering to a first group of animals, which are alive and have cancer, a test compound or a combination of test compounds at a test dose(s); (ii) administering to a second group of the same type of animals, which are alive and have the same type of cancer, a negative control, such as control vehicle; and (iii) analyzing tumor size in the first and second groups of animals after a period of time, wherein no detectable change or an increase (e.g., an increase of about 5%, 10%, 15%, 20%, 25%, 30% or more) in the tumor size in the first group of animals relative to the tumor size in the second group of animals indicates that the test compound or the combination of test compounds has no or minimal efficacy at the test dose(s) alone. In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, the tumor size is assessed by an imaging technique known to one of skill in the art, e.g., MRI, X-ray, CAT/CT scan or PET scan. In a specific embodiment, the animals are animal models for a particular type of cancer (e.g., lung, liver, brain, kidney, uterine, breast, pancreatic, thyroid, testicular, skin, or bone). In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are non-human animals. In another specific embodiment, the animals are xenografts, such as described in Example 1 (Section 6, infra).

[00201] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds for treating cancer comprises: (i) administering to a first group of animals, which are alive and have cancer, a test compound or a combination of test

compounds at a test dose(s); and (ii) analyzing the percentage of dead cancer cells of the first group of animals after a period of time, wherein a no detectable change or an increase of about 20% or less (e.g., 15%, 10%, 5%, 4%, 3%, 2% or 1%, or 15% to 19.5%, 10 to 15%, 5% to 15%, 5% to 10%), or 1%) to 5%) in the percentage of dead cancer cells in the first group of animals relative the percentage of dead cancer cells in a second group of the same type of animals, which are alive and have the same type of cancer, but to which the test compound or the combination of test compounds was not administered after the same period of time indicates that the test compound or the combination of test compounds at the test dose(s) has no or minimal efficacy alone. In certain embodiments, a tumor biopsy or blood sample obtained from an animal(s) is analyzed to assess the percentage of dead cancer cells. In a specific embodiment, the animals are animal models for a particular type of cancer (e.g., lung, liver, brain, kidney, uterine, breast, pancreatic, thyroid, testicular, skin, or bone). In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are non- human animals. In another specific embodiment, the animals are xenografts, such as described in Example 1 (Section 6, infra). [00202] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds for treating cancer comprises: (i) administering to a first group of animals, which are alive and have cancer, a test compound or a combination of test compounds at a test dose(s); and (ii) analyzing tumor size in the first group of animals after a period of time, wherein a no detectable change or an increase (e.g., an increase of about 5%, 10%, 15%), 20%), 25%), 30%o or more) in the tumor size in the first group of animals relative to the tumor size in a second group of the same type of animals which are alive and have the same type of cancer, but to which the test compound or the combination of test compounds was not administered after the same period of time indicates that the test compound or the combination of test compounds at the test dose(s) has no or minimal efficacy alone. In certain embodiments, the tumor size is assessed by an imaging technique known to one of skill in the art, e.g., MRI, X-ray, CAT/CT scan or PET scan. In a specific embodiment, the animals are animal models for a particular type of cancer (e.g., lung, liver, brain, kidney, uterine, breast, pancreatic, thyroid, testicular, skin, or bone). In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are non-human animals. In another specific embodiment, the animals are xenografts, such as described in Example 1 (Section 6, infra).

[00203] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds for treating cancer comprises: (i) administering to a first group of animals, which are alive and have cancer, a test compound or a combination of test compounds at a test dose(s); (ii) administering to a second group of the same type of animals, which are alive and have the same type of cancer, no test compound or the combination of test compounds; and (iii) analyzing the percentage of dead cancer cells of the first group of animals and the percentage of dead cancer cells of the second group of animals after a period of time, wherein no detectable change or an increase of about 20% or less (e.g., 15%, 10%>, 5%, 4%, 3%, 2% or 1%, or 15% to 19.5%, 10 to 15%, 5% to 15%, 5% to 10%, or 1% to 5%) in the percentage of dead cancer cells in the first group of animals relative to the percentage of dead cancer cells in the second group of animals indicates that the test compound or the combination of test compounds has no or minimal efficacy at the test dose(s) alone. In certain embodiments, a tumor biopsy or blood sample obtained from an animal(s) is analyzed to assess the percentage of dead cancer cells. In a specific embodiment, the animals are animal models for a particular type of cancer (e.g., lung, liver, brain, kidney, uterine, breast, pancreatic, thyroid, testicular, skin, or bone). In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are non-human animals. In another specific embodiment, the animals are xenografts, such as described in Example 1 (Section 6, infra).

[00204] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds for treating cancer comprises: (i) administering to a first group of animals, which are alive and have cancer, a test compound or a combination of test compounds at a test dose(s); (ii) administering to a second group of the same type of animals, which are alive and have the same type of cancer, no test compound or the combination of test compounds; and (iii) analyzing tumor size in the first and second groups of animals after a period of time, wherein no detectable change or an increase {e.g., an increase of about 5%, 10%, 15%, 20%), 25%), 30%) or more) in the tumor size in the first group of animals relative to the tumor size in the second group of animals indicates that the test compound or the combination of test compounds has no or minimal efficacy at the test dose(s) alone. In certain embodiments, the tumor size is assessed by an imaging technique known to one of skill in the art, e.g., MRI, X-ray, CAT/CT scan or PET scan. In a specific embodiment, the animals are animal models for a particular type of cancer {e.g., lung, liver, brain, kidney, uterine, breast, pancreatic, thyroid, testicular, skin, or bone). In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are non-human animals. In another specific embodiment, the animals are xenografts, such as described in Example 1 (Section 6, infra).

[00205] In certain embodiments, the test dose of the test compound or each compound in the combination of test compounds administered to the animals is about 2-fold, 5-fold, 10-fold, 20- fold, 50-fold, 75-fold, or 100-fold lower than the dosage efficacious for treating a particular cancer in the animals. In one embodiment, the test dose of the test compound or each compound in the combination of test compounds administered to the animals is about 40 mg/kg, 20 mg/kg, 10 mg/kg, 5 mg/kg, lmg/kg, 0.5 mg/kg, 0.1 mg/kg, 0.05 mg/kg, 0.01 mg/kg, 0.005 mg/kg, 0.001 mg/kg or lower, or about 40 mg/kg to 20 mg/kg, 20 mg/kg to 10 mg/kg, 10 mg/kg to 5 mg/kg, 5 mg/kg to 1 mg/kg, 1 mg/kg to 0.5 mg/kg, 0.5 mg/kg to 0.1 mg/kg, 0.1 mg/kg to 0.05 mg/kg, 0.05 mg/kg to 0.01 mg/kg, 0.01 mg/kg to 0.005 mg/kg, 0.005 mg/kg to 0.001 mg/kg or lower. In a specific embodiment, the test dose of the test compound or each compound in the combination of test compounds is the subtherapeutic dose(s). In some embodiments, the test dose of each compound in the combination of test compounds administer to the animal is different. In other embodiments, the test dose of each compound in the combination of test compounds

administered to the animal is the same.

[00206] The period of time after which the group of animals can be assessed in a Viability Assay can be, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or 3-5, 4-5, 4-7, 5-7, 7-10 or 7-14 days. In a specific embodiment, the period of time after which the group of animals can be assessed in a Viability Assay is 4-12 days. In another specific embodiment, the period of time after which the group of animals can be assessed in a Viability Assay is 5-12 days. In another specific embodiment, the period of time after which the group of animals can be assessed in a Viability Assay is 6-12 days. In another specific embodiment, the period of time after which the group of animals can be assessed in a Viability Assay is 7-12 days.

[00207] In specific embodiments, the Viability Assay is performed using animal juveniles engineered to controllably over-express or hyper-activate an oncogene(s), which prevents the animal juveniles from developing into viable adults, and the Viability Assay measures adult viability rate as a proxy for inhibition of the oncogenic mutation(s). The animal juveniles as used herein can be young animals before reaching adulthood or embryos. In specific

embodiments, the animal juveniles are non-human juveniles. In specific embodiments, the animal juveniles are non-primate juveniles. In specific embodiments, the animal juveniles are fly larvae. In a specific embodiment, the animal juveniles are the PTC>Ret2B flies described in Section 6 (see also Dar et al., 2012, Nature 486: 80-84, which is incorporated herein by reference in its entirety). In a particular embodiment, the Viability Assay is performed the flies described in Example 1 (Section 6, infra) in the manner described in Example 1.

[00208] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds for treating cancer comprises: (i) administering to a first group of animal juveniles over-expressing or having a hyper-activated oncogene, which prevents the animal juveniles from developing into viable adults, a test compound or a combination of test compounds at a test dose(s); and (ii) analyzing the percentage of viable adults developed from the first group of animal juveniles after a period of time, wherein no detectable change or an increase of about 15% or less (e.g., 10%, 5%, 4%, 3%, 2% or 1%, or 10 to 15%, 5% to 15%, 5% to 10%), or 1%) to 5%) in the percentage of viable adults developed from the first group of animal juveniles relative to the percentage of viable adults developed from a second group of the same type of animal juveniles over-expressing the same oncogene(s) or having the same hyper- activated oncogene(s), administered a negative control, such as control vehicle, after the same period of time indicates that the test compound or the combination of test compounds has no or minimal efficacy at the test dose(s) alone. In a specific embodiment, the expression of the oncogene is inducible (e.g., by temperature) and, in specific embodiments, the over-expression or hyper-activation of the oncogene is induced concurrently with the administration of the animal juveniles with the test compound or combination of test compounds. See, e.g., Example 1 (Section 6, infra) for a description of the induction of an oncogene. In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, the group of animal juveniles comprises 2 to 4 animal juveniles, 4 to 6 animal juveniles, 5 to 8 animal juveniles, 8 to 10 animal juveniles, 10 to 20 animal juveniles, 20-30 animal juveniles, 30 to 60 animal juveniles, or 60 to 100 animal juveniles.

[00209] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds for treating cancer comprises: (i) administering to a first group of animal juveniles over-expressing or having a hyper-activated oncogene(s), which prevents the animal juveniles from developing into viable adults, a test compound or a combination of test compounds at a test dose(s); (ii) administering to a second group of the same type of animal juveniles having the same over-expressed oncogene(s) or the same hyper-activated oncogene(s) a negative control, such as control vehicle; and (iii) analyzing the percentage of viable adults developed from the first group of animal juveniles and the percentage of viable adults developed from the second group of animal juveniles after a period of time, wherein no detectable change or an increase of about 15% or less (e.g., 10%, 5%, 4%, 3%, 2% or 1%, or 10 to 15%, 5% to 15%), 5%> to 10%), or 1%> to 5%) in the percentage of viable adults developed from the first group of animal juveniles relative to the percentage of viable adults developed from the second group of animal juveniles indicates that the test compound or the combination of test compounds has no or minimal efficacy at the test dose(s) alone. In a specific embodiment, the expression of the oncogene is inducible (e.g., by temperature) and, in specific embodiments, the over-expression or hyper-activation of the oncogene is induced concurrently with the administration of the animal juveniles with the test compound or combination of test compounds. See, e.g., Example 1 (Section 6, infra) for a description of the induction of an oncogene. In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, the group of animal juveniles comprises 2 to 4 animal juveniles, 4 to 6 animal juveniles, 5 to 8 animal juveniles, 8 to 10 animal juveniles, 10 to 20 animal juveniles, 20-30 animal juveniles, 30 to 60 animal juveniles, or 60 to 100 animal juveniles.

[00210] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds for treating cancer comprises: (i) administering to a first group of animal juveniles over-expressing or having a hyper-activated oncogene(s), which prevents animal juveniles from developing into viable adults, a test compound or a combination of test compounds at a test dose(s); and (ii) analyzing the percentage of viable adults developed from the first group of animal juveniles after a period of time, wherein a no detectable change or an increase of about 15% or less (e.g., 10%, 5%, 4%, 3%, 2% or 1%, or 10 to 15%, 5% to 15%, 5% to 10%), or 1%) to 5%) in the percentage of viable adults developed from the first group of animal juveniles relative the percentage of viable adults developed from a second group of the same type of animal juveniles over-expressing the same oncogene(s) or having the same hyper- activated oncogene(s), but to which the test compound or the combination of test compounds was not administered after the same period of time indicates that the test compound or the combination of test compounds at the test dose(s) has no or minimal efficacy alone. In a specific embodiment, the expression of the oncogene is inducible (e.g., by temperature) and, in specific embodiments, the over-expression or hyper-activation of the oncogene is induced concurrently with the administration of the animal juveniles with the test compound or combination of test compounds. See, e.g., Example 1 (Section 6, infra) for a description of the induction of an oncogene. In certain embodiments, the group of animal juveniles comprises 2 to 4 animal juveniles, 4 to 6 animal juveniles, 5 to 8 animal juveniles, 8 to 10 animal juveniles, 10 to 20 animal juveniles, 20-30 animal juveniles, 30 to 60 animal juveniles, or 60 to 100 animal juveniles. [00211] In specific embodiments, a method for assessing the efficacy of a test compound or a combination of test compounds for treating cancer comprises: (i) administering to a first group of animal juveniles over-expressing or having a hyper-activated oncogene(s), which prevents the animal juveniles from developing into viable animals, a test compound or a combination of test compounds at a test dose(s); (ii) administering to a second group of the same type of animal juveniles over-expressing the same oncogene(s) or having the same hyper-activated oncogene(s), no test compound or the combination of test compounds; and (iii) analyzing the percentage of viable adults developed from the first group of animal juveniles and the percentage of viable adults developed from the second group of animal juveniles after a period of time, wherein no detectable change or an increase of about 15% or less (e.g., 10%, 5%, 4%, 3%, 2% or 1%, or 10 to 15%), 5%> to 15%), 5%) to 10%), or 1% to 5%) in the percentage of viable adults developed from the first group of animal juveniles relative to the percentage of viable adults developed from the second group of animal juveniles indicates that the test compound or the combination of test compounds has no or minimal efficacy at the test dose(s) alone. In a specific embodiment, the expression of the oncogene is inducible (e.g., by temperature) and, in specific embodiments, the over-expression or hyper-activation of the oncogene is induced concurrently with the

administration of the animal juveniles with the test compound or combination of test compounds. See, e.g., Example 1 (Section 6, infra) for a description of the induction of an oncogene. In certain embodiments, the group of animal juveniles comprises 2 to 4 animal juveniles, 4 to 6 animal juveniles, 5 to 8 animal juveniles, 8 to 10 animal juveniles, 10 to 20 animal juveniles, 20- 30 animal juveniles, 30 to 60 animal juveniles, or 60 to 100 animal juveniles.

[00212] In certain embodiments, the test dose of the test compound or each compound in the combination of test compounds administered to the animal juveniles is about 0.01 μπιοΐ to 50 μτηοΐ, 0.1 μπιοΐ to 50 μτηοΐ, 1 μπιοΐ to 50 μτηοΐ, 5 μπιοΐ to 50 μτηοΐ, 10 μπιοΐ to 50 μτηοΐ, 20 μπιοΐ to 50 μτηοΐ, 0.01 μπιοΐ to 20 μτηοΐ, 0.1 μπιοΐ to 20 μτηοΐ, 1 μπιοΐ to 20 μτηοΐ, 5 μπιοΐ to 20 μτηοΐ, 0.01 μπιοΐ to 10 μτηοΐ, 0.1 μπιοΐ to 10 μτηοΐ, 1 μπιοΐ to 10 μτηοΐ, 5 μπιοΐ to 10 μτηοΐ, 0.01 μπιοΐ to 5 μτηοΐ, 0.1 μπιοΐ to 5 μτηοΐ, 1 μπιοΐ to 5 μτηοΐ, 0.01 μπιοΐ to 1 μτηοΐ, 0.1 μπιοΐ to 1 μτηοΐ, 0.01 μπιοΐ to 0.1 μτηοΐ. In certain embodiments, the test dose of the test compound or each compound in the combination of test compounds administered to the animal juveniles is about 0.01 μτηοΐ, 0.1 μτηοΐ, 1 μτηοΐ, 5 μτηοΐ, 10 μτηοΐ, 20 μτηοΐ, 50 μτηοΐ. In some embodiments, the test dose of each compound in the combination of test compounds administer to the animal juveniles is different. In other embodiments, the test dose of each compound in the combination of test compounds administered to the animal juveniles is the same.

[00213] The period of time after which the percentage of viable adults can be assessed in a Viability Assay can be, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, or 28 days, or 3-5, 4-5, 4-7, 5-7, 7-10, 7-14, 10-21, 14-21, 14-28, or 21-28 days. In a specific embodiment, the period of time after which the percentage of viable adults can be assessed in a Viability Assay is 4-12 days. In another specific embodiment, the period of time after which the percentage of viable adults can be assessed in a Viability Assay is 5-12 days. In another specific embodiment, the period of time after which the percentage of viable adults can be assessed in a Viability Assay is 6-12 days. In another specific embodiment, the period of time after which the percentage of viable adults can be assessed in a Viability Assay is 7-12 days.

5.3.3 Toxicity Assay

[00214] The Toxicity Assay can be any assay known in the art for testing the adverse effects of a drug, and can be performed using non-cancerous cells or animal models (e.g., flies, worms, mice, rats, rabbits, or primates). Provided in this Section 5.3.3 and Section 6, infra, are examples of various ways to conduct the Toxicity Assays.

[00215] In a specific embodiment, the Toxicity Assay is conducted as described in Example 1 (Section 6, infra).

[00216] In specific embodiments, a method for assessing the toxicity of a compound or a combination of compounds for toxicity on non-cancerous cells comprises: (i) culturing noncancerous cells in the presence of a test compound or a combination of test compounds at a test concentration(s) for a period of time; and (ii) measuring the percentage of dead non-cancerous cells at the end of said period of time, wherein no measurable change or an increase of about 20% or less (e.g., 15%, 10%, 5%, 4%, 3%, 2% or 1%, or 15% to 19.5%, 10 to 15%, 5% to 15%, 5% to 10%), or 1%) to 5%) in the percentage of dead non-cancerous cells when the non-cancerous cells are cultured in presence of the test compound or the combination of test compounds at the test concentration(s) relative to the percentage of dead non-cancerous cells when the noncancerous cells are cultured in the presence of a negative control, such as vehicle alone (e.g., phosphate buffered saline (PBS) or another buffer), for the same period of time indicates that the test compound or the combination of test compounds at the test concentration(s) has no or minimal toxicity. In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, the non-cancerous cells are derived or obtained from a cell line. In other embodiments, the non-cancerous cells are derived or obtained from a subject, such as noncancerous subject (e.g., a healthy subject). In a specific embodiment, the subject is a human. In other embodiments, the subject is a non-human animal.

[00217] In specific embodiments, a method for assessing the toxicity of a compound or a combination of compounds for toxicity on non-cancerous cells comprises: (i) culturing a first population of non-cancerous cells in the presence of a test compound or a combination of test compounds at a test concentration(s) for a period of time; (ii) culturing a second population of the same type of non-cancerous cells in the presence of a negative control, such as vehicle alone (e.g., PBS or another buffer), for the same period of time; and (iii) measuring the percentage of dead cells of the cancer cells cultured under conditions (i) and (ii) at the end of said period of time, wherein no measurable change or an increase of about 20% or less (e.g., 15%, 10%, 5%, 4%, 3%, 2% or 1%, or 15% to 19.5%, 10 to 15%, 5% to 15%, 5% to 10%, or 1% to 5%) in the percentage of dead non-cancerous cells in the first population of non-cancerous cells relative to the percentage of dead non-cancerous cells in the second population of non-cancerous cancer cells indicates that the test compound or the combination of test compounds at the test concentration(s) has no or minimal toxicity. In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, the non-cancerous cells are derived or obtained from a cell line. In other embodiments, the non-cancerous cells are derived or obtained from a subject, such as non-cancerous subject (e.g., a healthy subject). In a specific embodiment, the subject is a human. In other embodiments, the subject is a non-human animal.

[00218] In specific embodiments, a method for assessing the toxicity of a compound or a combination of compounds for toxicity on non-cancerous cells comprises: (i) culturing noncancerous cells in the presence of a test compound or a combination of test compounds at a test concentration(s) for a period of time; and (ii) measuring the percentage of dead non-cancerous cells at the end of said period of time, wherein no measurable change or an increase of about 20% or less (e.g., 15%, 10%, 5%, 4%, 3%, 2% or 1%, or 15% to 19.5%, 10 to 15%, 5% to 15%, 5%) to 10%), or 1%) to 5%)) in the percentage of dead non-cancerous cells when the non-cancerous cells are cultured with the test compound or the combination of test compounds at the test concentration relative to the percentage of dead non-cancerous cells when non-cancerous cells are cultured in the absence of the test compound or the combination of test compounds for the same period of time indicates that the test compound or the combination of test compounds at the test concentration(s) has no or minimal toxicity. In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, the non-cancerous cells are derived or obtained from a cell line. In other embodiments, the non-cancerous cells are derived or obtained from a subject, such as non-cancerous subject (e.g., a healthy subject). In specific embodiments, the subject is a human. In other embodiments, the subject is a non-human animal.

[00219] In specific embodiments, a method for assessing the toxicity of a compound or a combination of compounds for toxicity on non-cancerous cells comprises: (i) culturing a first population of non-cancerous cells in the presence of a test compound or a combination of test compounds at a test concentration(s) for a period of time; (ii) culturing a second population of the same type of non-cancerous cells in the absence of the test compound or the combination of test compounds for the same period of time; and (iii) measuring the percentage of dead cells of the non-cancerous cells cultured under conditions (i) and (ii) at the end of said period of time, wherein no measurable change or an increase of about 20% or less (e.g., 15%, 10%, 5%, 4%, 3%, 2% or 1%, or 15% to 19.5%, 10 to 15%, 5% to 15%, 5% to 10%, or 1% to 5%) in the percentage of dead non-cancerous cells in the first population of non-cancerous cells relative to the percentage of dead non-cancerous cells in the second population of non-cancerous cells indicates that the test compound or the combination of test compounds at the test

concentration(s) has no or minimal toxicity. In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, the non-cancerous cells are derived or obtained from a cell line. In other embodiments, the non-cancerous cells are derived or obtained from a subject, such as non-cancerous subject (e.g., a healthy subject). In specific embodiments, the subject is a human. In other embodiments, the subject is a non-human animal.

[00220] In certain embodiments, the test concentration of the test compound or each compound in the combination of test compounds used in cell culture is 0.1 nmol to 75 nmol, 1 nmol to 75 nmol, 10 nmol to 75 nmol, 20 nmol to 75 nmol, 30 nmol to 75 nmol, 50 nmol to 75 nmol, 0.1 to 50 nmol, 1.5 nmol to 50 nmol, 5 nmol to 50 nmol, 10 nmol to 50 nmol, 25 nmol to 50 nmol, 1 nmol to 10 nmol, 1 nmol to 6 nmol, 1 to 5 nml, 0.1 to 6 nmol, or 1 to 3 nmol. In some embodiments, the test concentration of the test compound or each compound in the combination of test compounds used in cell culture is 0.1 nmol, 0.25 nmol, 0.5 nmol, 0.75 nmol, 1 nmol, 5 nmol, 7 nmol, 10 nmol, 15 nmol, 20 nmol, 25 nmol, 30 nmol, 35 nmol, 40 nmol, 45 nmol, 50 nmol, 55 nmol, 60 nmol, 65 nmol, 70 nmol, or 75 nmol. In certain embodiments, the concentration of each compound in the combination of test compounds is different. In other embodiments, the concentration of each compound in the combination of test compounds is the same. In a specific embodiment, the concentrations of the test compound or each compound in the combination of test compounds tested in a Toxicity Assay is the same concentration or concentration range tested in the Efficacy Assay discussed in Section 5.3.1, supra. In a specific embodiment, the concentrations of the test compound or each compound in the combination of test compounds tested in a Toxicity Assay is the same concentration or concentration range tested in the Viability Assay discussed in Section 5.3.2, supra.

[00221] The period of time that the cancer cells are cultured in a Toxicity Assay can be, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or 3-5, 4-5, 4-7, 5-7, 7-10 or 7-14 days. In a specific embodiment, the period of time that the cancer cells are cultured in a Toxicity Assay is 4-12 days. In another specific embodiment, the period of time that the cancer cells are cultured in a Toxicity Assay is 5-12 days. In another specific embodiment, the period of time that the cancer cells are cultured in a Toxicity Assay is 6-12 days. In another specific embodiment, the period of time that the cancer cells are cultured in a Toxicity Assay is 7-12 days.

[00222] In certain embodiments, one or more additional controls, e.g., positive and/or negative controls are included in a Toxicity Assay described herein. For example, a Network Brake described in Section 6, infra, may be used a positive control. In a specific embodiment, a Viability Assay is conducted as described in Section 6, infra.

[00223] In specific embodiments, a method for assessing the toxicity of a compound or a combination of compounds for toxicity on non-cancerous cells comprises: (i) administering to a first group of animals, which are alive and do not have cancer, a test compound or a combination of test compounds at a test dose(s); and (ii) analyzing the percentage of dead non-cancerous cells of the first group of animals after a period of time, wherein no detectable change or an increase of about 20% or less (e.g., 15%, 10%, 5%, 4%, 3%, 2% or 1%, or 15% to 19.5%, 10 to 15%, 5% to 15%), 5%> to 10%), or 1%> to 5%>) in the percentage of dead non-cancerous cells in the first group of animals relative to the percentage of dead non-cancerous cells in a second group of the same type of animals, which are alive and do not have cancer, administered a negative control, such as control vehicle, after the same period of time indicates that the test compound or the combination of test compounds has no or minimal toxicity at the test dose(s). In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, a blood sample obtained from an animal(s) is analyzed to assess the percentage of dead non-cancerous cells. In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are healthy animals (e.g., flies, worms, mice, rats, rabbits, or primates). In another specific embodiment, the animals are non-human animals. In another specific embodiment, the animals are wild-type flies.

[00224] In specific embodiments, a method for assessing the toxicity of a compound or a combination of compounds for toxicity on non-cancerous cells comprises: (i) administering to a first group of animals, which are alive and do not have cancer, a test compound or a combination of test compounds at a test dose(s); (ii) administering to a second group of the same type of animals, which are alive and do not have cancer, a negative control, such as control vehicle; and (iii) analyzing the percentage of dead non-cancerous cells of the first group of animals and the percentage of dead non-cancerous cells of the second group of animals after a period of time, wherein no detectable change or an increase of about 15% or less (e.g., 10%, 5%, 4%, 3%, 2% or 1%, or 10 to 15%, 5% to 15%, 5% to 10%, or 1% to 5%) in the percentage of dead noncancerous cells in the first group of animals relative to the percentage of dead non-cancerous cells in the second group of animals indicates that the test compound or the combination of test compounds has no or minimal toxicity at the test dose(s). In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, a blood sample obtained from an animal(s) is analyzed to assess the percentage of dead non-cancerous cells. In certain

embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are healthy animals (e.g., flies, worms, mice, rats, rabbits, or primates). In another specific embodiment, the animals are non-human animals. In another specific embodiment, the animals are wild-type flies.

[00225] In specific embodiments, a method for assessing the toxicity of a compound or a combination of compounds for toxicity on non-cancerous cells comprises: (i) administering to a first group of animals, which are alive and do not have cancer, a test compound or a combination of test compounds at a test dose(s); and (ii) analyzing the percentage of dead non-cancerous cells of the first group of animals after a period of time, wherein a no detectable change or an increase of about 15% or less (e.g., 10%, 5%, 4%, 3%, 2% or 1%, or 10 to 15%, 5% to 15%, 5% to 10%), or 1%) to 5%) in the percentage of dead non-cancerous cells in the first group of animals relative to a second group of the same type of animals, which are alive and do not have the same type of cancer, but to which the test compound or the combination of test compounds was not administered after the same period of time indicates that the test compound or the combination of test compounds at the test dose(s) has no or minimal toxicity. In certain embodiments, a blood sample obtained from an animal(s) is analyzed to assess the percentage of dead non-cancerous cells. In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are healthy animals (e.g., flies, worms, mice, rats, rabbits, or primates). In another specific embodiment, the animals are non-human animals. In another specific embodiment, the animals are wild-type flies.

[00226] In specific embodiments, a method for assessing the toxicity of a compound or a combination of compounds for toxicity on non-cancerous cells comprises: (i) administering to a first group of animals, which are alive and do not have cancer, a test compound or a combination of test compounds at a test dose(s); (ii) administering to a second group of the same type of animals, which are alive and do not have cancer, no test compound or the combination of test compounds; and (iii) analyzing the percentage of dead non-cancerous cells of the first group of animals and the percentage of dead non-cancerous cells of the second group of animals after a period of time, wherein no detectable change or an increase of about 15%> or less (e.g., 10%, 5%, 4%, 3%, 2% or 1%, or 10 to 15%, 5% to 15%, 5% to 10%, or 1% to 5%) in the percentage of dead non-cancerous cells in the first group of animals relative to the percentage of dead noncancerous cells in the second group of animals indicates that the test compound or the combination of test compounds has no or minimal efficacy at the test dose(s). In certain embodiments, a blood sample obtained from an animal(s) is analyzed to assess the percentage of dead non-cancerous cells. In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are healthy animals (e.g., flies, worms, mice, rats, rabbits, or primates). In another specific embodiment, the animals are non-human animals. In another specific embodiment, the animals are wild-type flies.

[00227] In specific embodiments, a method for assessing the toxicity of a compound or a combination of compounds for toxicity on non-cancerous cells comprises: (i) administering to a first group of animals, which are alive and do not have cancer, a test compound or a combination of test compounds at a test dose(s); and (ii) analyzing the percentage of dead animals of the first group of animals after a period of time, wherein no detectable change or an increase of about 15% or less (e.g., 10%, 5%, 4%, 3%, 2% or 1%, or 10 to 15%, 5% to 15%, 5% to 10%, or 1% to 5%) in the percentage of dead animals in the first group of animals relative to the percentage of dead animals in a second group of the same type of animals, which are alive before

administration and do not have cancer, administered a negative control, such as control vehicle, after the same period of time indicates that the test compound or the combination of test compounds has no or minimal toxicity at the test dose(s). In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are healthy animals (e.g., flies, worms, mice, rats, rabbits, or primates). In another specific embodiment, the animals are non-human animals. In another specific embodiment, the animals are wild-type flies.

[00228] In specific embodiments, a method for assessing the toxicity of a compound or a combination of compounds for toxicity on non-cancerous cells comprises: (i) administering to a first group of animals, which are alive and do not have cancer, a test compound or a combination of test compounds at a test dose(s); (ii) administering to a second group of the same type of animals, which are alive and do not have cancer, a negative control, such as control vehicle; and (iii) analyzing the percentage of dead animals of the first group of animals and the percentage of dead animals of the second group of animals after a period of time, wherein no detectable change or an increase of about 15% or less (e.g., 10%, 5%, 4%, 3%, 2% or 1%, or 10 to 15%, 5% to 15%, 5% to 10%, or 1% to 5%) in the percentage of dead animals in the first group of animals relative to the percentage of dead animals in the second group of animals indicates that the test compound or the combination of test compounds has no or minimal toxicity at the test dose(s). In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are healthy animals (e.g., flies, worms, mice, rats, rabbits, or primates). In another specific embodiment, the animals are non-human animals. In another specific embodiment, the animals are wild-type flies. In certain embodiments, general health of the animals is analyzed instead of the percentage of dead animals to assess toxicity of the compound or the combination of compounds.

[00229] In specific embodiments, a method for assessing the toxicity of a compound or a combination of compounds for toxicity on non-cancerous cells comprises: (i) administering to a first group of animals, which are alive and do not have cancer, a test compound or a combination of test compounds at a test dose(s); and (ii) analyzing the percentage of dead animals of the first group of animals after a period of time, wherein a no detectable change or an increase of about 15% or less (e.g., 10%, 5%, 4%, 3%, 2% or 1%, or 10 to 15%, 5% to 15%, 5% to 10%, or 1% to 5%) in the percentage of dead animals in the first group of animals relative to a second group of the same type of animals, which are alive before administration and do not have the same type of cancer, but to which the test compound or the combination of test compounds was not administered after the same period of time indicates that the test compound or the combination of test compounds at the test dose(s) has no or minimal toxicity. In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are healthy animals (e.g., flies, worms, mice, rats, rabbits, or primates). In another specific embodiment, the animals are non-human animals. In another specific embodiment, the animals are wild-type flies. In certain embodiments, general health of the animals is analyzed instead of the percentage of dead animals to assess toxicity of the compound or the combination of compounds. [00230] In specific embodiments, a method for assessing the toxicity of a compound or a combination of compounds for toxicity on non-cancerous cells comprises: (i) administering to a first group of animals, which are alive and do not have cancer, a test compound or a combination of test compounds at a test dose(s); (ii) administering to a second group of the same type of animals, which are alive and do not have cancer, no test compound or the combination of test compounds; and (iii) analyzing the percentage of dead animals of the first group of animals and the percentage of dead animals of the second group of animals after a period of time, wherein no detectable change or an increase of about 15% or less (e.g., 10%, 5%, 4%, 3%, 2%, or 1%, or 5% to 15%, 10% to 15%, 5% tol0% or 1% to 5%) in the percentage of dead animals in the first group of animals relative to the percentage of dead animals in the second group of animals indicates that the test compound or the combination of test compounds has no or minimal efficacy at the test dose(s). In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are healthy animals (e.g., flies, worms, mice, rats, rabbits, or primates). In another specific embodiment, the animals are non-human animals. In another specific embodiment, the animals are wild-type flies. In another specific embodiment, the animals are healthy animals (e.g., non-human animals). In certain embodiments, general health of the animals is analyzed instead of the percentage of dead animals to assess toxicity of the compound or the combination of compounds.

[00231] In certain embodiments, the test dose of the test compound or each compound in the combination of test compounds administered to the animals is about 0.01 μπιοΐ to 50 μπιοΐ, 0.1 μπιοΐ to 50 μπιοΐ, 1 μπιοΐ to 50 μπιοΐ, 5 μπιοΐ to 50 μπιοΐ, 10 μπιοΐ to 50 μπιοΐ, 20 μπιοΐ to 50 μπιοΐ, 0.01 μπιοΐ to 20 μπιοΐ, 0.1 μπιοΐ to 20 μπιοΐ, 1 μπιοΐ to 20 μπιοΐ, 5 μπιοΐ to 20 μπιοΐ, 10 μπιοΐ to 20 μπιοΐ, 0.01 μπιοΐ to 10 μπιοΐ, 0.1 μπιοΐ to 10 μπιοΐ, 1 μπιοΐ to 10 μπιοΐ, 5 μπιοΐ to 10 μπιοΐ, 0.01 μπιοΐ to 5 μπιοΐ, 0.1 μπιοΐ to 5 μπιοΐ, 1 μπιοΐ to 5 μπιοΐ, 0.01 μπιοΐ to 1 μπιοΐ, 0.1 μπιοΐ to 1 μπιοΐ, 0.01 μπιοΐ to 0.1 μπιοΐ. In certain embodiments, the test dose of the test compound or each compound in the combination of test compounds administered to the animals is about 0.01 μπιοΐ, 0.1 μπιοΐ, 1 μπιοΐ, 5 μπιοΐ, 10 μπιοΐ 20 μπιοΐ, 50 μπιοΐ. In certain

embodiments, the test dose of the test compound or each compound in the combination of test compounds administered to the animals is about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 75-fold, or 100-fold lower than the dosage efficacious for treating a particular cancer in the animals. In one embodiment, the test dose of the test compound or each compound in the combination of test compounds administered to the animals is about 40 mg/kg, 20 mg/kg, 10 mg/kg, 5 mg/kg, lmg/kg, 0.5 mg/kg, 0.1 mg/kg, 0.05 mg/kg, 0.01 mg/kg, 0.005 mg/kg, 0.001 mg/kg or lower, or about 40 mg/kg to 20 mg/kg, 20 mg/kg to 10 mg/kg, 10 mg/kg to 5 mg/kg, 5 mg/kg to 1 mg/kg, 1 mg/kg to 0.5 mg/kg, 0.5 mg/kg to 0.1 mg/kg, 0.1 mg/kg to 0.05 mg/kg, 0.05 mg/kg to 0.01 mg/kg, 0.01 mg/kg to 0.005 mg/kg, 0.005 mg/kg to 0.001 mg/kg or lower. In a specific embodiment, the test dose of the test compound or each compound in the combination of test compounds is the subtherapeutic dose(s). In some embodiments, the test dose of each compound in the combination of test compounds administer to the animal is different. In other

embodiments, the test dose of each compound in the combination of test compounds

administered to the animal is same.

[00232] The period of time after which the group of animals can be assessed in a Toxicity Assay can be, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or 3-5, 4-5, 4-7, 5-7, 7-10 or 7-14 days. In a specific embodiment, the period of time after which the group of animals can be assessed in a Toxicity Assay can be is 4 days. In another specific embodiment, the period of time after which the group of animals can be assessed in a Toxicity Assay is 5 days. In another specific embodiment, the period of time after which the group of animals can be assessed in a Toxicity Assay is 6 days. In another specific embodiment, the period of time after which the group of animals can be assessed in a Toxicity Assay is 7 days. In another specific embodiment, the period of time after which the group of animals can be assessed in a Toxicity Assay is 10-14 days.

[00233] In certain embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is administered to an animal prior to the administration of the kinase inhibitor. For example, the test compound or the combination of test compounds is administered to an animal 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, or 21 days prior to the administration of the kinase inhibitor. In another example, the test compound or the combination of test compounds is administered to an animal 1 to 6 hours, 2 to 6 hours, 4 to 6 hours, 6 to 12 hours, 12 to 24 hours, 1 to 2 days, 2 to 5 days, 5 to 7 days, 7 to 14 days or 14 to 21 days prior to the administration of the kinase inhibitor. In other embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is administered to an animal concurrently with the administration of the kinase inhibitor. In yet other embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is administered to an animal subsequent to the administration of the kinase inhibitor. For example, the test compound or the combination of test compounds is administered to an animal 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, or 21 days subsequent to the administration of the kinase inhibitor. In another example, the test compound or the combination of test compounds is administered to an animal 1 to 6 hours, 2 to 6 hours, 4 to 6 hours, 6 to 12 hours, 12 to 24 hours, 1 to 2 days, 2 to 5 days, 5 to 7 days, 7 to 14 days or 14 to 21 days subsequent to the administration of the kinase inhibitor.

5.3.4 Phosphoprofile Assay

[00234] The Phosphoprofile Assay can be any assay known in the art for assessing the activity of certain kinases. The phosphorylation activity of the kinases is indicative of the efficacy of a test compound or a combination of test compounds at a subtherapeutic dose(s) or subtherapeutic dose range(s) and a kinase inhibitor at a specific dose or dose range for treating cancer. Provided in this Section 5.3.4 and Section 6, infra, are examples of how the Phosphoprofile Assay can be conducted. The results from the Phosphoprofile Assay indicate whether a test compound or combination of compounds at a subtherapeutic dose(s) or subtherapeutic dose range(s) has Network Brake activity. A test compound or a combination of test compounds has Network Brake activity if there is a decrease in the overall, median, or mean phosphorylation level of kinases in cancer cells following treatment of the cancer cells with the test compound or the combination of test compounds at a concentration equivalent to a subtherapeutic dose(s) and a kinase inhibitor at a specific concentration relative to the overall, median or mean

phosphorylation level of kinases in the same type of cancer cells following treatment of the cancer cells with the kinase inhibitor alone at the specific concentration.

[00235] In a specific embodiment, the Phosphoprofile Assay is conducted as described in Example 1 (Section 6, infra).

[00236] In specific embodiments, a method of determining whether a compound or a combination of compounds has Network Brake activity comprises: (a) culturing a first population of cancer cells in the presence of a kinase inhibitor at a specific concentration for a period of time; (b) culturing a second population of cancer cells of the same type in the presence of a test compound or a combination of test compounds at a test concentration(s), and the kinase inhibitor at the same specific concentration for said period of time; and (c) analyzing the level of activity of a certain set of kinases in the first and second populations of cancer cells at the end of said period of time, wherein a decrease in the overall, median, or mean level of activity of the kinases in the second population of cancer cells relative to the overall, median or mean level of activity of the same kinases in the first population of cancer cells indicates that the test compound or the combination of test compounds has Network Brake activity. In contrast, no detectable change or an increase in the overall, mean or median activity of the kinases in the second population of cancer cells relative to the overall, median or mean activity of the same kinases in the first population of cancer cells indicates that the test compound or combination of test compounds does not have Network Brake activity. In certain embodiments, the cancer cells are derived or obtained from a cancer cell line. In other embodiments, the cancer cells are derived or obtained from a cancer patient. In a specific embodiment, the cancer cells are from the cancer cell line A549, H1299, HepG2, MD-MB-231, T47D, A375, 239, MZ-MTC, TT-MTC, or H358-NSCLC. In another specific embodiment the cancer cells are from a cancer cell line described in Example 1 (Section 6, infra). In one embodiment, the set of kinases is a plurality of, e.g., 5-10, 10-15, 10-20, 15-20, 20-25, 20-30, 20-40, 30-40, 25-50, 40-50, 50-75, 50-100, or 75- 100 kinases. In a specific embodiment, the activity of one, all or a subset of the following kinases are assessed: EGFR, HER2, HER3, FGFR1, FGFR3, FGFR4, InsR, IGF-IR, TrkA, TrkB, Met, Ron, Ret, ALK, PDGFR, c-Kit, FLT3, M-CSFR, EphAl, EphA2, EphA3, EphB l, EphB3, EphB4, Tyro3, Axl, Tie2, VEGFR2, Akt (e.g., evaluating the phosphorylation at Thr308 and/or Ser473), ERKl, ERK2, S6, c-Abl, IRS-1, Zap-70, Src, Lck, Statl, and Stat3. In certain embodiments, the activity of kinases is assessed using a phosphorylation assay known to one of skill in the art or described herein (e.g., in Section 6, infra).

[00237] In specific embodiments, a method of assessing a test compound or a combination of test compounds has Network Brake activity comprises: (a) culturing a first population of cancer cells in the presence of a kinase inhibitor at a specific concentration for a period of time; (b) culturing a second population of cancer cells of the same type in the presence of a test compound or a combination of test compounds at a test concentration(s), and the kinase inhibitor at the same specific concentration for said period of time; and (c) analyzing the level of

phosphorylation of a set of proteins, which are indicative of the kinase activity of a set of kinases, in the first and second populations of cancer cells at the end of said period of time, wherein a decrease in the overall, median, or mean level of phosphorylation of the set of proteins in the second population of cancer cells relative to the overall, median or mean level of phosphorylation of the same set of proteins in the first population of cancer cells indicates that the test compound or the combination of test compounds has Network Brake activity. In contrast, no detectable change or an increase in the overall, mean or median activity of the phosphorylation of the set of proteins in the second population of cancer cells relative to the overall, median or mean phosphorylation of the same set of proteins in the first population of cancer cells indicates that the test compound or combination of test compounds does not have Network Brake activity. In certain embodiments, the cancer cells are derived or obtained from a cancer cell line. In other embodiments, the cancer cells are derived or obtained from a cancer patient. In a specific embodiment, the cancer cells are from the cancer cell line A549, H1299, HepG2, MD-MB-231, T47D, A375, 239, MZ-MTC, TT-MTC, or H358-NSCLC. In another specific embodiment the cancer cells are from a cancer cell line described in Example 1 (Section 6, infra). In one embodiment, the level of phosphorylation of the set of proteins is indicative of the kinase activity of a plurality of, e.g., 5-10, 10-15, 10-20, 15-20, 20-25, 20-30, 20-40, 30-40, 25-50, 40-50, 50-75, 50-100, or 75-100 kinases. In a specific embodiment, the level of phosphorylation of the set of proteins is indicative of the kinase activity of all or a subset of the following kinases: EGFR, HER2, HER3, FGFR1, FGFR3, FGFR4, InsR, IGF-IR, TrkA, TrkB, Met, Ron, Ret, ALK, PDGFR, c-Kit, FLT3, M-CSFR, EphAl, EphA2, EphA3, EphBl, EphB3, EphB4, Tyro3, Axl, Tie2, VEGFR2, Akt {e.g., evaluating the phosphorylation at Thr308 and/or Ser473), ERKl, ERK2, S6, c-Abl, IRS-1, Zap-70, Src, Lck, Statl, and Stat3. In another specific embodiment, a kit that comprises antibodies that can bind to a certain set of kinases in their phosphorylated forms is used in the Phosphoprofile Assay. In a particular embodiment, the following kit is used in the Phosphoprofile Assay: PathScan® RTK Signaling Antibody Array Kit (Cell Signaling Technology® Cat# 7982).

[00238] Phosphorylation of each protein in a panel of proteins can be quantified by any method known in the art, for example, by quantifying the amount of an antibody that specifically binds to a phosphorylated form of the respective kinase for each kinase in the panel {e.g., using an array of such antibodies). [00239] In a specific embodiment, the activities of a plurality of kinases known to be activated in cancer development and/or progression are assessed in accordance with the methods described herein. In another specific embodiments, the activities of a plurality of kinases known to be activated in cancer development and/or progression, and belonging to one or more signaling pathways known to be involved in cancer development and/or progression are assessed in accordance with the methods described herein. In another specific embodiment, the plurality of kinases whose activities are assessed in accordance with the methods described herein are receptor tyrosine kinases (RTKs).

[00240] In certain embodiments, the test concentration of the test compound or each compound in the combination of test compounds used in cell culture is 0.1 nmol to 75 nmol, 1 nmol to 75 nmol, 10 nmol to 75 nmol, 20 nmol to 75 nmol, 30 nmol to 75 nmol, 50 nmol to 75 nmol, 0.1 to 50 nmol, 1.5 nmol to 50 nmol, 5 nmol to 50 nmol, 10 nmol to 50 nmol, 25 nmol to 50 nmol, 1 nmol to 10 nmol, 1 nmol to 6 nmol, 1 to 5 nml, 0.1 to 6 nmol, or 1 to 3 nmol. In some embodiments, the test concentration of the test compound or each compound in the combination of test compounds used in cell culture is 0.1 nmol, 0.25 nmol, 0.5 nmol, 0.75 nmol, 1 nmol, 5 nmol, 7 nmol, 10 nmol, 15 nmol, 20 nmol, 25 nmol, 30 nmol, 35 nmol, 40 nmol, 45 nmol, 50 nmol, 55 nmol, 60 nmol, 65 nmol, 70 nmol, or 75 nmol. In certain embodiments, the test concentration of each compound in the combination of test compounds is different. In other embodiments, the test concentration of each compound in the combination of test compounds is the same. In certain embodiments, the specific concentration of the kinase inhibitor used in cell culture is about 0.0001 μπιοΐ to 20 μπιοΐ, 0.001 μπιοΐ to 20 μπιοΐ, 0.01 μπιοΐ to 20 μπιοΐ, 0.1 μπιοΐ to 20 μπιοΐ, 1 μπιοΐ to 20 μπιοΐ, 10 μπιοΐ to 20 μπιοΐ, 0.0001 μπιοΐ to 10 μπιοΐ, 0.001 μπιοΐ to 10 μπιοΐ, 0.01 μπιοΐ to 10 μπιοΐ, 0.1 μπιοΐ to 10 μπιοΐ, 1 μπιοΐ to 10 μπιοΐ, 0.0001 μπιοΐ to 1 μπιοΐ, 0.001 μπιοΐ to 1 μπιοΐ, 0.01 μπιοΐ to 1 μπιοΐ, 0.1 μπιοΐ to 1 μπιοΐ, 0.0001 μπιοΐ to 0.1 μπιοΐ, 0.001 μιηοΐ to 0.1 μιηοΐ, 0.01 μιηοΐ to 0.1 μιηοΐ, 0.0001 μιηοΐ to 0.01 μιηοΐ, 0.001 μιηοΐ to 0.01 μπιοΐ, or 0.0001 μπιοΐ to 0.001 μπιοΐ. In certain embodiments, the specific concentration of the kinase inhibitor used in cell culture is about 0.0001 μπιοΐ, 0.001 μπιοΐ, 0.01 μπιοΐ, 0.1 μπιοΐ, 1 μπιοΐ, 10 μπιοΐ, or 20 μπιοΐ. In a specific embodiment, the test concentration of the test compound or each compound in the combination of compounds is equivalent to a subtherapeutic dose. In certain embodiments, the test concentration of the test compound or each compound in the combination of compounds is a concentration used in Example 1 (Section 6, infra). [00241] The period of time that the cancer cells are cultured in a Phosphoprofile Assay can be, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or 3-5, 4-5, 4-7, 5-7, 7-10 or 7-14 days. In a specific embodiment, the period of time that the cancer cells are cultured in a

Phosphoprofile Assay is 4 days. In another specific embodiment, the period of time that the cancer cells are cultured in a Phosphoprofile Assay is 5 days. In another specific embodiment, the period of time that the cancer cells are cultured in a Phosphoprofile Assay is 6 days. In another specific embodiment, the period of time that the cancer cells are cultured in a

Phosphoprofile Assay is 7 days.

[00242] In certain embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is added to cell culture prior to the addition of the kinase inhibitor. For example, the test compound or the combination of test compounds is add to cell culture 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, or 21 days prior to the addition of the kinase inhibitor. In another example, the test compound or the combination of test compounds is added to cell culture 1 to 6 hours, 2 to 6 hours, 4 to 6 hours, 6 to 12 hours, 12 to 24 hours, 1 to 2 days, 2 to 5 days, 5 to 7 days, 7 to 14 days or 14 to 21 days prior to the addition of the kinase inhibitor. In other embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is added to cell culture concurrently with the addition of the kinase inhibitor. In yet other embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is added to cell culture subsequent to the addition of the kinase inhibitor. For example, the test compound or the combination of test compounds is added to cell culture 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, or 21 days subsequent to the addition of the kinase inhibitor. In another example, the test compound or the combination of test compounds is added to cell culture 1 to 6 hours, 2 to 6 hours, 4 to 6 hours, 6 to 12 hours, 12 to 24 hours, 1 to 2 days, 2 to 5 days, 5 to 7 days, 7 to 14 days or 14 to 21 days subsequent to the addition of the kinase inhibitor.

[00243] In certain embodiments, one or more additional controls, e.g., positive and/or negative controls are included in a Phosphoprofile Assay described herein. For example, a Network Brake described in Section 6, infra, may be used a positive control. In a specific embodiment, a Phosphoprofile Assay is conducted as described in Section 6, infra. [00244] In specific embodiments, the test compound(s) or the combination of test compounds assessed in a Phosphoprofile Assay are the test compound(s) or the combination of test compounds which demonstrate therapeutic efficacy in combination with a kinase inhibitor in an Efficacy Assay, no therapeutic efficacy alone in a Viability Assay and are minimally toxic as assessed by a Toxicity Assay.

5.3.5 Cancer Stem Cell Marker Assay

[00245] The Cancer Stem Cell Marker Assay can be any assay known in the art for assessing the expression level of certain cancer stem cell marker(s). The expression level of the cancer stem cell marker(s) is indicative of the efficacy of a test compound or a combination of test compounds at a subtherapeutic dose(s) or subtherapeutic dose range(s) and a kinase inhibitor at a specific dose or dose range for treating cancer. Provided in this Section 5.3.5 and Section 6, infra, are examples of how the Cancer Stem Cell Marker Assay can be conducted. The results from the Cancer Stem Cell Marker Assay indicate whether a test compound or combination of compounds at a subtherapeutic dose(s) or subtherapeutic dose range(s) has Network Brake activity. A test compound or a combination of test compounds has Network Brake activity if there is a decrease in the overall, median, or mean expression level of cancer stem cell marker(s) in cancer cells following treatment of the cancer cells with the test compound or the combination of test compounds at a concentration equivalent to a subtherapeutic dose(s) and a kinase inhibitor at a specific concentration relative to the overall, median or mean expression level of the cancer stem cell marker(s) in the same type of cancer cells following treatment of the cancer cells with the kinase inhibitor alone at the specific concentration.

[00246] In a specific embodiment, the Cancer Stem Cell Marker Assay is conducted as described in Example 1 (Section 6, infra).

[00247] In specific embodiments, a method of determining whether a compound or a combination of compounds has Network Brake activity comprises: (a) culturing a first population of cancer cells in the presence of a kinase inhibitor at a specific concentration for a period of time; (b) culturing a second population of cancer cells of the same type in the presence of a test compound or a combination of test compounds at a test concentration(s), and the kinase inhibitor at the same specific concentration for said period of time; (c) culturing a third population of cancer cells of the same type without any treatment; and (d) analyzing the expression level of a cancer stem cell marker or a certain set of cancer stem cell marker(s) in the first, second, and third populations of cancer cells at the end of said period of time, wherein the overall, median, or mean expression level of the cancer stem cell marker(s) in the first population of cancer cells is higher than the overall, median, or mean expression level of the same cancer stem cell marker(s) in the third population of cancer cells, and wherein a decrease in the overall, median, or mean expression level of the cancer stem cell marker(s) in the second population of cancer cells relative to the overall, median or mean expression level of the same cancer stem cell marker(s) in the first population of cancer cells indicates that the test compound or the combination of test compounds has Network Brake activity. In contrast, no detectable change or an increase in the overall, mean or median expression level of the cancer stem cell marker(s) in the second population of cancer cells relative to the overall, median or mean expression level of the same cancer stem cell marker(s) in the first population of cancer cells indicates that the test compound or combination of test compounds does not have Network Brake activity. In certain embodiments, the cancer cells are derived or obtained from a cancer cell line. In other embodiments, the cancer cells are derived or obtained from a cancer patient. In a specific embodiment, the cancer cells are from the cancer cell line A549, H1299, HepG2, MD-MB-231, T47D, A375, 239, MZ-MTC, TT-MTC, or H358-NSCLC. In another specific embodiment the cancer cells are from a cancer cell line described in Example 1 (Section 6, infra). In one embodiment, the set of cancer stem cell marker(s) is a plurality of, e.g., 2-5, 5-10, 10-15, 10-20, 15-20, 20-25, 20-30, 20-40, 30-40, 25-50, 40-50, 50-75, 50-100, or 75-100 cancer stem cell markers. In one embodiment, the set of cancer stem cell marker(s) is a single cancer stem cell marker {e.g., Sox2). In a specific embodiment, the expression level of one, all or a subset of the following cancer stem cell markers are assessed: Sox2, Oct4, Nanog, LIN28, c-Myc, and KLF4. In certain embodiments, the expression level of cancer stem cell marker(s) is assessed using an assay known to one of skill in the art or described herein {e.g., in Section 6, infra).

[00248] The expression level of each cancer stem cell marker can be quantified by any method known in the art, for example, by quantifying the amount of an antibody that specifically binds to a cancer stem cell marker {e.g., using western blot or dot blot). In a specific

embodiment, the expression level of a cancer stem cell marker is assessed at the protein level. In another embodiment, the expression level of a cancer stem cell marker is assessed at the RNA level. In another embodiment, the expression level of a cancer stem cell marker is assessed at the protein and RNA levels. [00249] In a specific embodiment, the expression level of cancer stem cell marker(s) known to be upregulated in cancer development and/or progression are assessed in accordance with the methods described herein.

[00250] In certain embodiments, the test concentration of the test compound or each compound in the combination of test compounds used in cell culture is 0.1 nmol to 75 nmol, 1 nmol to 75 nmol, 10 nmol to 75 nmol, 20 nmol to 75 nmol, 30 nmol to 75 nmol, 50 nmol to 75 nmol, 0.1 to 50 nmol, 1.5 nmol to 50 nmol, 5 nmol to 50 nmol, 10 nmol to 50 nmol, 25 nmol to 50 nmol, 1 nmol to 10 nmol, 1 nmol to 6 nmol, 1 to 5 nml, 0.1 to 6 nmol, or 1 to 3 nmol. In some embodiments, the test concentration of the test compound or each compound in the combination of test compounds used in cell culture is 0.1 nmol, 0.25 nmol, 0.5 nmol, 0.75 nmol, 1 nmol, 5 nmol, 7 nmol, 10 nmol, 15 nmol, 20 nmol, 25 nmol, 30 nmol, 35 nmol, 40 nmol, 45 nmol, 50 nmol, 55 nmol, 60 nmol, 65 nmol, 70 nmol, or 75 nmol. In certain embodiments, the test concentration of each compound in the combination of test compounds is different. In other embodiments, the test concentration of each compound in the combination of test compounds is the same. In certain embodiments, the specific concentration of the kinase inhibitor used in cell culture is about 0.0001 μπιοΐ to 20 μπιοΐ, 0.001 μπιοΐ to 20 μπιοΐ, 0.01 μπιοΐ to 20 μπιοΐ, 0.1 μπιοΐ to 20 μπιοΐ, 1 μπιοΐ to 20 μπιοΐ, 10 μπιοΐ to 20 μπιοΐ, 0.0001 μπιοΐ to 10 μπιοΐ, 0.001 μπιοΐ to 10 μπιοΐ, 0.01 μπιοΐ to 10 μπιοΐ, 0.1 μπιοΐ to 10 μπιοΐ, 1 μπιοΐ to 10 μπιοΐ, 0.0001 μπιοΐ to 1 μπιοΐ, 0.001 μπιοΐ to 1 μπιοΐ, 0.01 μπιοΐ to 1 μπιοΐ, 0.1 μπιοΐ to 1 μπιοΐ, 0.0001 μπιοΐ to 0.1 μπιοΐ, 0.001 μιηοΐ to 0.1 μιηοΐ, 0.01 μιηοΐ to 0.1 μιηοΐ, 0.0001 μιηοΐ to 0.01 μιηοΐ, 0.001 μιηοΐ to 0.01 μπιοΐ, or 0.0001 μπιοΐ to 0.001 μπιοΐ. In certain embodiments, the specific concentration of the kinase inhibitor used in cell culture is about 0.0001 μπιοΐ, 0.001 μπιοΐ, 0.01 μπιοΐ, 0.1 μπιοΐ, 1 μπιοΐ, 10 μπιοΐ, or 20 μπιοΐ. In a specific embodiment, the test concentration of the test compound or each compound in the combination of compounds is equivalent to a subtherapeutic dose. In certain embodiments, the test concentration or each compound in the combination of compounds is a concentration used in Example 1 (Section 6, infra).

[00251] The period of time that the cancer cells are cultured in a Cancer Stem Cell Marker Assay can be, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or 3-5, 4-5, 4-7, 5-7, 7-10 or 7-14 days. In a specific embodiment, the period of time that the cancer cells are cultured in a Cancer Stem Cell Marker Assay is 4 days. In another specific embodiment, the period of time that the cancer cells are cultured in a Cancer Stem Cell Marker Assay is 5 days. In another specific embodiment, the period of time that the cancer cells are cultured in a Cancer Stem Cell Marker Assay is 6 days. In another specific embodiment, the period of time that the cancer cells are cultured in a Cancer Stem Cell Marker Assay is 7 days.

[00252] In certain embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is added to cell culture prior to the addition of the kinase inhibitor. For example, the test compound or the combination of test compounds is add to cell culture 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, or 21 days prior to the addition of the kinase inhibitor. In another example, the test compound or the combination of test compounds is added to cell culture 1 to 6 hours, 2 to 6 hours, 4 to 6 hours, 6 to 12 hours, 12 to 24 hours, 1 to 2 days, 2 to 5 days, 5 to 7 days, 7 to 14 days or 14 to 21 days prior to the addition of the kinase inhibitor. In other embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is added to cell culture concurrently with the addition of the kinase inhibitor. In yet other embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is added to cell culture subsequent to the addition of the kinase inhibitor. For example, the test compound or the combination of test compounds is added to cell culture 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, or 21 days subsequent to the addition of the kinase inhibitor. In another example, the test compound or the combination of test compounds is added to cell culture 1 to 6 hours, 2 to 6 hours, 4 to 6 hours, 6 to 12 hours, 12 to 24 hours, 1 to 2 days, 2 to 5 days, 5 to 7 days, 7 to 14 days or 14 to 21 days subsequent to the addition of the kinase inhibitor.

[00253] In certain embodiments, one or more additional controls, e.g., positive and/or negative controls are included in a Cancer Stem Cell Marker Assay described herein. For example, a Network Brake described in Section 6, infra, may be used a positive control. In a specific embodiment, a Cancer Stem Cell Marker Assay is conducted as described in Section 6, infra.

[00254] In specific embodiments, the test compound(s) or the combination of test compounds assessed in a Cancer Stem Cell Marker Assay are the test compound(s) or the combination of test compounds which demonstrate therapeutic efficacy in combination with a kinase inhibitor in an Efficacy Assay, no therapeutic efficacy alone in a Viability Assay and/or are minimally toxic as assessed by a Toxicity Assay.

5.3.6 Histone Modification Assay

[00255] The Histone Modification Assay can be any assay known in the art for assessing the modification (e.g., methylation, acetylation) level of certain histone mark(s). The level of the histone mark(s) is indicative of the efficacy of a test compound or a combination of test compounds at a subtherapeutic dose(s) or subtherapeutic dose range(s) and a kinase inhibitor at a specific dose or dose range for treating cancer. Provided in this Section 5.3.6 and Section 6, infra, are examples of how the Histone Modification Assay can be conducted. The results from the Histone Modification Assay indicate whether a test compound or combination of compounds at a subtherapeutic dose(s) or subtherapeutic dose range(s) has Network Brake activity. A test compound or a combination of test compounds has Network Brake activity if there is a decrease in the overall, median, or mean level of active histone mark(s) in cancer cells following treatment of the cancer cells with the test compound or the combination of test compounds at a concentration equivalent to a subtherapeutic dose(s) and a kinase inhibitor at a specific concentration relative to the overall, median or mean level of the same active histone mark(s) in the same type of cancer cells following treatment of the cancer cells with the kinase inhibitor alone at the specific concentration; and/or if if there is an increase in the overall, median, or mean level of suppressive histone mark(s) in cancer cells following treatment of the cancer cells with the test compound or the combination of test compounds at a concentration equivalent to a subtherapeutic dose(s) and a kinase inhibitor at a specific concentration relative to the overall, median or mean level of the same suppressive histone mark(s) in the same type of cancer cells following treatment of the cancer cells with the kinase inhibitor alone at the specific

concentration.

[00256] In specific embodiments, a method of determining whether a compound or a combination of compounds has Network Brake activity comprises: (a) culturing a first population of cancer cells in the presence of a kinase inhibitor at a specific concentration for a period of time; (b) culturing a second population of cancer cells of the same type in the presence of a test compound or a combination of test compounds at a test concentration(s), and the kinase inhibitor at the same specific concentration for said period of time; and (c) analyzing the level of an active histone mark(s) or a certain set of active histone mark(s) and/or a suppressive histone mark(s) or a certain set of suppressive histone mark(s) in the first and second populations of cancer cells at the end of said period of time, wherein a decrease in the overall, median, or mean level of the active histone mark(s) in the second population of cancer cells relative to the overall, median or mean level of the same active histone mark(s) in the first population of cancer cells, and/or an increase in the overall, median, or mean level of the suppressive histone mark(s) in the second population of cancer cells relative to the overall, median or mean level of the same suppressive histone mark(s) in the first population of cancer cells indicates that the test compound or the combination of test compounds has Network Brake activity. In contrast, no detectable change or an increase in the overall, mean or median level of the active histone mark(s) in the second population of cancer cells relative to the overall, median or mean level of the same active histone mark(s) in the first population of cancer cells, and/or no detectable change or a decrease in the overall, mean or median level of the suppressive histone mark(s) in the second population of cancer cells relative to the overall, median or mean level of the same suppressive histone mark(s) in the first population of cancer cells indicates that the test compound or combination of test compounds does not have Network Brake activity. In certain embodiments, the cancer cells are derived or obtained from a cancer cell line. In other embodiments, the cancer cells are derived or obtained from a cancer patient. In a specific embodiment, the cancer cells are from the cancer cell line A549, H1299, HepG2, MD-MB-231, T47D, A375, 239, MZ-MTC, TT-MTC, or H358-NSCLC. In another specific embodiment the cancer cells are from a cancer cell line described in Example 1 (Section 6, infra). In one embodiment, the set of active histone mark(s) is a plurality of, e.g., 1-5, 5-10, 10-15, 10-20, 15- 20, 20-25, 20-30, 20-40, 30-40, 25-50, 40-50, 50-75, 50-100, or 75-100 cancer stem cell markers. In one embodiment, the set of active histone mark(s) is a single active histone mark. In one embodiment, the set of suppressive histone mark(s) is a plurality of, e.g., 5-10, 10-15, 10-20, 15- 20, 20-25, 20-30, 20-40, 30-40, 25-50, 40-50, 50-75, 50-100, or 75-100 cancer stem cell markers. In one embodiment, the set of suppressive histone mark(s) is a single active histone mark {e.g., H3K27-Me3). In a specific embodiment, the level of one, all or a subset of the following active histone marks are assessed: H3K9-Ac, H4K5-Ac, H3K4-Me3, H4K12-Ac, and H3K64-Ac. In a specific embodiment, the level of one, all or a subset of the following active and suppressive histone marks are assessed: H3K9-Ac, H4K5-Ac, H3K4-Me3, H4K12-Ac, H3K64-Ac, and H3K27-Me3. In certain embodiments, the level of histone mark(s) is assessed using an assay known to one of skill in the art or described herein (e.g., in Section 6, infra).

[00257] The level of each histone mark can be quantified by any method known in the art, for example, by quantifying the amount of an antibody that specifically binds to a histone mark {e.g., using western blot). In a specific embodiment, the level expression of a histone mark(s) is assessed at the protein level. In another embodiment, the level of expression of a histone mark(s) is assessed at the RNA level. In another embodiment, the level of expression of a histone mark(s) is assessed at the protein and RNA levels.

[00258] In a specific embodiment, the level of a histone mark(s) known to be activated or suppressed in cancer development and/or progression is/are assessed in accordance with the methods described herein.

[00259] In certain embodiments, the test concentration of the test compound or each compound in the combination of test compounds used in cell culture is 0.1 nmol to 75 nmol, 1 nmol to 75 nmol, 10 nmol to 75 nmol, 20 nmol to 75 nmol, 30 nmol to 75 nmol, 50 nmol to 75 nmol, 0.1 to 50 nmol, 1.5 nmol to 50 nmol, 5 nmol to 50 nmol, 10 nmol to 50 nmol, 25 nmol to 50 nmol, 1 nmol to 10 nmol, 1 nmol to 6 nmol, 1 to 5 nml, 0.1 to 6 nmol, or 1 to 3 nmol. In some embodiments, the test concentration of the test compound or each compound in the combination of test compounds used in cell culture is 0.1 nmol, 0.25 nmol, 0.5 nmol, 0.75 nmol, 1 nmol, 5 nmol, 7 nmol, 10 nmol, 15 nmol, 20 nmol, 25 nmol, 30 nmol, 35 nmol, 40 nmol, 45 nmol, 50 nmol, 55 nmol, 60 nmol, 65 nmol, 70 nmol, or 75 nmol. In certain embodiments, the test concentration of each compound in the combination of test compounds is different. In other embodiments, the test concentration of each compound in the combination of test compounds is the same. In certain embodiments, the specific concentration of the kinase inhibitor used in cell culture is about 0.0001 μπιοΐ to 20 μπιοΐ, 0.001 μπιοΐ to 20 μπιοΐ, 0.01 μπιοΐ to 20 μπιοΐ, 0.1 μπιοΐ to 20 μπιοΐ, 1 μπιοΐ to 20 μπιοΐ, 10 μπιοΐ to 20 μπιοΐ, 0.0001 μπιοΐ to 10 μπιοΐ, 0.001 μπιοΐ to 10 μπιοΐ, 0.01 μπιοΐ to 10 μπιοΐ, 0.1 μπιοΐ to 10 μπιοΐ, 1 μπιοΐ to 10 μπιοΐ, 0.0001 μπιοΐ to 1 μπιοΐ, 0.001 μπιοΐ to 1 μπιοΐ, 0.01 μπιοΐ to 1 μπιοΐ, 0.1 μπιοΐ to 1 μπιοΐ, 0.0001 μπιοΐ to 0.1 μπιοΐ, 0.001 μιηοΐ to 0.1 μιηοΐ, 0.01 μιηοΐ to 0.1 μιηοΐ, 0.0001 μιηοΐ to 0.01 μιηοΐ, 0.001 μιηοΐ to 0.01 μπιοΐ, or 0.0001 μπιοΐ to 0.001 μπιοΐ. In certain embodiments, the specific concentration of the kinase inhibitor used in cell culture is about 0.0001 μπιοΐ, 0.001 μπιοΐ, 0.01 μπιοΐ, 0.1 μπιοΐ, 1 μπιοΐ, 10 μπιοΐ, or 20 μπιοΐ. In a specific embodiment, the test concentration of the test compound or each compound in the combination of compounds is equivalent to a subtherapeutic dose. In certain embodiments, the test concentration of the test compound or each compound in the combination of test compounds used in cell culture a concentration described in Example 1 (Section 6, infra).

[00260] The period of time that the cancer cells are cultured in a Histone Modification Assay can be, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or 3-5, 4-5, 4-7, 5-7, 7-10 or 7- 14 days. In a specific embodiment, the period of time that the cancer cells are cultured in a Histone Modification Assay is 4 days. In another specific embodiment, the period of time that the cancer cells are cultured in a Histone Modification Assay is 5 days. In another specific embodiment, the period of time that the cancer cells are cultured in a Histone Modification Assay is 6 days. In another specific embodiment, the period of time that the cancer cells are cultured in a Histone Modification Assay is 7 days.

[00261] In certain embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is added to cell culture prior to the addition of the kinase inhibitor. For example, the test compound or the combination of test compounds is add to cell culture 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, or 21 days prior to the addition of the kinase inhibitor. In another example, the test compound or the combination of test compounds is added to cell culture 1 to 6 hours, 2 to 6 hours, 4 to 6 hours, 6 to 12 hours, 12 to 24 hours, 1 to 2 days, 2 to 5 days, 5 to 7 days, 7 to 14 days or 14 to 21 days prior to the addition of the kinase inhibitor. In other embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is added to cell culture concurrently with the addition of the kinase inhibitor. In yet other embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is added to cell culture subsequent to the addition of the kinase inhibitor. For example, the test compound or the combination of test compounds is added to cell culture 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, or 21 days subsequent to the addition of the kinase inhibitor. In another example, the test compound or the combination of test compounds is added to cell culture 1 to 6 hours, 2 to 6 hours, 4 to 6 hours, 6 to 12 hours, 12 to 24 hours, 1 to 2 days, 2 to 5 days, 5 to 7 days, 7 to 14 days or 14 to 21 days subsequent to the addition of the kinase inhibitor. [00262] In certain embodiments, one or more additional controls, e.g., positive and/or negative controls are included in a Histone Modification Assay described herein. For example, a Network Brake described in Section 6, infra, may be used a positive control. In a specific embodiment, a Histone Modification Assay is conducted as described in Section 6, infra.

[00263] In specific embodiments, the test compound(s) or the combination of test compounds assessed in a Histone Modification Assay are the test compound(s) or the combination of test compounds which demonstrate therapeutic efficacy in combination with a kinase inhibitor in an Efficacy Assay, no therapeutic efficacy alone in a Viability Assay and/or are minimally toxic as assessed by a Toxicity Assay.

5.3.7 Resistance Assay

[00264] The Resistance Assay can be any assay known in the art for testing cancer resistance to a drug. Provided in this Section 5.3.7 and Section 6, infra, are examples of various of ways to conduct the Resistance Assays. The results from the Resistance Assays indicate whether a test compound or a combination of test compounds at a subtherapeutic dose(s) has Network Brake activity. A test compound or a combination of test compounds has Network Brake activity if there is resistance to the emergence of cancer cells refractory to treatment with the test compound or the combination of test compounds at a subtherapeutic dose(s) (or equivalent concentration(s)) and a kinase inhibitor at a specific dose/concentration.

[00265] In a specific embodiment, the Resistance Assay is conducted as described in Example 1 (Section 6, infra).

[00266] In specific embodiments, a method for determining resistance to the emergence of cancer cells refractory to treatment with a test compound or a combination of test compounds and a kinase inhibitor comprises: (i) culturing cancer cells in the presence of a test compound or a combination of test compounds at a test concentration(s) and a kinase inhibitor at a specific concentration for a period of time; and (ii) measuring the percentage of dead cancer cells over said period of time, wherein no detectable change or an increase in the percentage of dead cancer cells over the period time indicates that that there is prevention of the emergence of cancer cells refractory/resistant to treatment with the test compound or the combination of test compounds at the test concentration(s) and the kinase inhibitor at the specific concentration. In contrast, a decrease in the percentage of dead cancer cells over the period of time when the cancer cells are cultured in the presence of the test compound or the combination of test compounds at the test concentration(s) and the kinase inhibitor at the specific concentration indicates that there is no prevention of the emergence of cancer cells refractory/resistant to treatment with the test compound or the combination of test compounds at the test concentration(s) and the kinase inhibitor at the specific concentration. In certain embodiments, the percentage of dead cancer cells are assessed: (i) prior to the addition of the test compound or the combination of test compounds and the kinase inhibitor; (ii) within 6-12 hours, 12-24 hours, and/or 1-2 days of culturing the cancer cells in the presence of the test compound or the combination of test compounds and the kinase inhibitor; and/or (iii) after 2-5 days, 5-7 days, 7-14 days, 7-21 days 14-21 days, 21 to 28 days, 25 to 30 days, 30 to 40 days, 40 to 60 days, and/or 60 to 120 days of culturing the cancer cells in the presence of the test compound or the combination of test compounds and the kinase inhibitor. In certain embodiments, the cancer cells are derived or obtained from a cancer cell line. In other embodiments, the cancer cells are derived or obtained from a cancer patient. In a specific embodiment, the cancer cells are from the cancer cell line A549, H1299, HepG2, MD-MB-231, T47D, A375, 239, MZ-MTC, TT-MTC, or H358-NSCLC. In another specific embodiment the cancer cells are from a cancer cell line described in Example 1 (Section 6, infra).

[00267] In specific embodiments, a method for determining resistance to the emergence of cancer cells refractory to treatment with a test compound or a combination of test compounds and a kinase inhibitor comprises: (i) culturing a first population of cancer cells in the presence of a test compound or a combination of test compounds at a test concentration(s) and a kinase inhibitor at a specific concentration for a period of time; (ii) culturing a second population of the same type of cancer cells in the presence of a negative control, such as vehicle alone {e.g., phosphate buffered saline (PBS) or another buffer), and the same kinase inhibitor at the same specific concentration for the same period of time; and (iii) analyzing the percentage of dead cells of the cancer cells cultured under the conditions in (i) and (ii) at the over said period of time, wherein an increase of greater than about 20% in the percentage of dead cancer cells is sustained over the period of time in the first population relative to the percentage of dead cancer cells in the second population indicates that there is prevention of the emergence of cancer cells refractory/resistant to treatment with the test compound or the combination of test compounds at the test concentration(s) and the kinase inhibitor at the specific concentration. In contrast, an increase of about 20% or less or a decrease in the percentage of dead cancer cells in the first population relative to the second population is not sustained over the period of time indicates that there is no prevention of the emergence of cancer cells refractory/resistant to treatment with the test compound or the combination of test compounds at the test concentration(s) and the kinase inhibitor at the specific concentration. In certain embodiments, the percentage of dead cancer cells are assessed: (i) prior to culturing the cancer cells in the presence of the test compound or the combination of test compounds and the kinase inhibitor, and the prior to culturing the cancer cells in the presence of the negative control and the kinase inhibitor; (ii) within 6 to 12 hours, 12 to 24 hours, and/or 1 to 2 days of culturing the cancer cells in the presence of the test compound or the combination of test compounds and the kinase inhibitor, and within 6 to 12 hours, 12 to 24 hours, and/or 1 to 2 days of culturing the cancer cells in the presence of the negative control and the kinase inhibitor; and/or (iii) after 2 to 5 days, 5 to 7 days, 7 to 14 days, 7-21 days 14 to 21 days, 21 to 28 days, 25 to 30 days, 30 to 40 days, 40 to 60 days, and/or 60 to 120 days of culturing the cancer cells in the presence of the test compound or the combination of test compounds and the kinase inhibitor, and after 2 to 5 days, 5 to 7 days, 7 to 14 days, 7 to 21 days 14 to 21 days, 21 to 28 days, 25 to 30 days, 30 to 40 days, 40 to 60 days, and/or 60 to 120 days of culturing the cancer cells in the presence of the negative control and the kinase inhibitor. In a specific embodiment, the vehicle control is the solvent or mixture of solvents that are used to dissolve the test compound or the combination of test compounds. In certain embodiments, the cancer cells are derived or obtained from a cancer cell line. In other embodiments, the cancer cells are derived or obtained from a cancer patient. In a specific embodiment, the cancer cells are from the cancer cell line A549, H1299, HepG2, MD-MB-231, T47D, A375, 239, MZ-MTC, TT-MTC, or H358-NSCLC. In another specific embodiment the cancer cells are from a cancer cell line described in Example 1 (Section 6, infra).

[00268] In specific embodiments, a method for determining resistance to the emergence of cancer cells refractory to treatment with a test compound or a combination of test compounds and a kinase inhibitor comprises: (i) culturing cancer cells in the presence of a test compound or a combination of test compounds at a test concentration(s) and a kinase inhibitor at a specific concentration for a period of time; and (ii) measuring the phosphorylation activity of a set of kinases over said period of time, wherein no detectable change or a decrease the in overall, mean or median phosphorylation activity over the period time indicates that that there is prevention of the emergence of cancer cells refractory/resistant to treatment with the test compound or the combination of test compounds at the test concentration(s) and the kinase inhibitor at the specific concentration. In contrast, an increase in the overall, mean or median phosphorylation activity over the period of time when the cancer cells are cultured in the presence of the test compound or the combination of test compounds at the test concentration(s) and the kinase inhibitor at the specific concentration indicates that there is no prevention of the emergence of cancer cells refractory/resistant to treatment with the test compound or the combination of test compounds at the test concentration(s) and the kinase inhibitor at the specific concentration. In certain embodiments, the phosphorylation activity is assessed: (i) prior to the addition of the test compound or the combination of test compounds and the kinase inhibitor; (ii) within 6-12 hours, 12-24 hours, and/or 1-2 days of culturing the cancer cells in the presence of the test compound or the combination of test compounds and the kinase inhibitor; and/or (iii) after 2-5 days, 5-7 days, 7-14 days, 7-21 days 14-21 days, 21 to 28 days, 25 to 30 days, 30 to 40 days, 40 to 60 days, and/or 60 to 120 days of culturing the cancer cells in the presence of the test compound or the combination of test compounds and the kinase inhibitor. In certain embodiments, the cancer cells are derived or obtained from a cancer cell line. In other embodiments, the cancer cells are derived or obtained from a cancer patient. In a specific embodiment, the cancer cells are from the cancer cell line A549, H1299, HepG2, MD-MB-231, T47D, A375, 239, MZ-MTC, TT-MTC, or H358-NSCLC. In another specific embodiment the cancer cells are from a cancer cell line described in Example 1 (Section 6, infra). In one embodiment, the set of kinases is a plurality of, e.g., 5-10, 10-15, 10-20, 15-20, 20-25, 20-30, 20-40, 30-40, 25-50, 40-50, 50-75, 50-100, or 75- 100 kinases. In a specific embodiment, the activity of one, all or a subset of the following kinases are assessed: EGFR, HER2, HER3, FGFR1, FGFR3, FGFR4, InsR, IGF-IR, TrkA, TrkB, Met, Ron, Ret, ALK, PDGFR, c-Kit, FLT3, M-CSFR, EphAl, EphA2, EphA3, EphB l, EphB3, EphB4, Tyro3, Axl, Tie2, VEGFR2, Akt (e.g., evaluating the phosphorylation at Thr308 and/or Ser473), ERKl, ERK2, S6, c-Abl, IRS-1, Zap-70, Src, Lck, Statl, and Stat3. In certain embodiments, the activity of kinases is assessed using a phosphorylation assay known to one of skill in the art or described herein (e.g., in Section 6, infra).

[00269] In specific embodiments, a method for determining resistance to the emergence of cancer cells refractory to treatment with a test compound or a combination of test compounds and a kinase inhibitor comprises: (i) culturing cancer cells in the presence of a test compound or a combination of test compounds at a test concentration(s) and a kinase inhibitor at a specific concentration for a period of time; and (ii) measuring the level of phosphorylation of a set of proteins, which are indicative of the kinase activity of a set of kinases, over said period of time, wherein no detectable change or a decrease the in overall, mean or median phosphorylation level over the period time indicates that there is prevention of the emergence of cancer cells refractory/resistant to treatment with the test compound or the combination of test compounds at the test concentration(s) and the kinase inhibitor at the specific concentration. In contrast, an increase in the overall, mean or median phosphorylation level over the period of time when the cancer cells are cultured in the presence of the test compound or the combination of test compounds at the test concentration(s) and the kinase inhibitor at the specific concentration indicates that there is no prevention of the emergence of cancer cells refractory/resistant to treatment with the test compound or the combination of test compounds at the test

concentration(s) and the kinase inhibitor at the specific concentration. In certain embodiments, the level of phosphorylation is assessed: (i) prior to the addition of the test compound or the combination of test compounds and the kinase inhibitor; (ii) within 6-12 hours, 12-24 hours, and/or 1-2 days of culturing the cancer cells in the presence of the test compound or the combination of test compounds and the kinase inhibitor; and/or (iii) after 2-5 days, 5-7 days, 7- 14 days, 7-21 days 14-21 days, 21 to 28 days, 25 to 30 days, 30 to 40 days, 40 to 60 days, and/or 60 to 120 days of culturing the cancer cells in the presence of the test compound or the combination of test compounds and the kinase inhibitor. In certain embodiments, the cancer cells are derived or obtained from a cancer cell line. In other embodiments, the cancer cells are derived or obtained from a cancer patient. In a specific embodiment, the cancer cells are from the cancer cell line A549, H1299, HepG2, MD-MB-231, T47D, A375, 239, MZ-MTC, TT-MTC, or H358-NSCLC. In another specific embodiment the cancer cells are from a cancer cell line described in Example 1 (Section 6, infra). In one embodiment, the level of phosphorylation of the set of proteins is indicative of the kinase activity of a plurality of, e.g., 5-10, 10-15, 10-20, 15-20, 20-25, 20-30, 20-40, 30-40, 25-50, 40-50, 50-75, 50-100, or 75-100 kinases. In a specific embodiment, the level of phosphorylation of the set of proteins is indicative of the kinase activity of one, all or a subset of the following kinases: EGFR, HER2, HER3, FGFR1, FGFR3, FGFR4, InsR, IGF-IR, TrkA, TrkB, Met, Ron, Ret, ALK, PDGFR, c-Kit, FLT3, M-CSFR, EphAl, EphA2, EphA3, EphBl, EphB3, EphB4, Tyro3, Axl, Tie2, VEGFR2, Akt (e.g., evaluating the phosphorylation at Thr308 and/or Ser473), ERKl, ERK2, S6, c-Abl, IRS-1, Zap-70, Src, Lck, Statl, and Stat3.

[00270] In specific embodiments, a method for determining resistance to the emergence of cancer cells refractory to treatment with a test compound or a combination of test compounds and a kinase inhibitor comprises: (a) culturing a first population of cancer cells in the presence of a kinase inhibitor at a specific concentration for a period of time; (b) culturing a second population of cancer cells of the same type in the presence of a test compound or a combination of test compounds at a test concentration(s), and the kinase inhibitor at the same specific concentration for said period of time; and (c) analyzing the level of activity of a certain set of kinases in the first and second populations of cancer cells over the period of time, wherein a sustained decrease in the overall, median, or mean activity level of the set of kinases in the second population of cancer cells over the period of time relative to the overall, median or mean activity level of the same kinases in the first population of cancer cells over the period of time indicates prevention of the emergence of cancer cells refractory/resistant to treatment with the test compound or the combination of test compounds at the test concentration(s) and the kinase inhibitor at the specific concentration. In certain embodiments, the cancer cells are derived or obtained from a cancer cell line. In other embodiments, the cancer cells are derived or obtained from a cancer patient. In a specific embodiment, the cancer cells are from the cancer cell line A549, H1299, HepG2, MD-MB-231, T47D, A375, 239, MZ-MTC, TT-MTC, or H358-NSCLC. In another specific embodiment the cancer cells are from a cancer cell line described in Example 1 (Section 6, infra). In one embodiment, the set of kinases is a plurality of, e.g., 5-10, 10-15, 10- 20, 15-20, 20-25, 20-30, 20-40, 30-40, 25-50, 40-50, 50-75, 50-100, or 75-100 kinases. In a specific embodiment, the activity of one, all or a subset of the following kinases are assessed: EGFR, HER2, HER3, FGFR1, FGFR3, FGFR4, InsR, IGF-IR, TrkA, TrkB, Met, Ron, Ret, ALK, PDGFR, c-Kit, FLT3, M-CSFR, EphAl, EphA2, EphA3, EphB l, EphB3, EphB4, Tyro3, Axl, Tie2, VEGFR2, Akt (e.g., evaluating the phosphorylation at Thr308 and/or Ser473), ERKl, ERK2, S6, c-Abl, IRS-1, Zap-70, Src, Lck, Statl, and Stat3. In certain embodiments, the activity of kinases is assessed using a phosphorylation assay known to one of skill in the art or described herein (e.g., in Section 6, infra).

[00271] In specific embodiments, a method for determining resistance to the emergence of cancer cells refractory to treatment with a test compound or a combination of test compounds and a kinase inhibitor comprises: (a) culturing a first population of cancer cells in the presence of a kinase inhibitor at a specific concentration for a period of time; (b) culturing a second population of cancer cells of the same type in the presence of a test compound or a combination of test compounds at a test concentration(s), and the kinase inhibitor at the same specific concentration for said period of time; and (c) analyzing the level of phosphoryation of a certain set of proteins, which are indicative of the phosphorylation activity of a set of kinases, in the first and second populations of cancer cells over the period of time, wherein a sustained decrease in the overall, median, or mean phosphorylation level of the set of proteins in the second population of cancer cells over the period of time relative to the overall, median or mean activity of the phosphorylation level of the same set of proteins in the first population of cancer cells over the period of time indicates that there is prevention of the emergence of cancer cells

refractory/resistant to treatment with the test compound or the combination of test compounds at the test concentration(s) and the kinase inhibitor at the specific concentration. In certain embodiments, the cancer cells are derived or obtained from a cancer cell line. In other embodiments, the cancer cells are derived or obtained from a cancer patient. In a specific embodiment, the cancer cells are from the cancer cell line A549, H1299, HepG2, MD-MB-231, T47D, A375, 239, MZ-MTC, TT-MTC, or H358-NSCLC. In another specific embodiment the cancer cells are from a cancer cell line described in Example 1 (Section 6, infra). In one embodiment, the set of kinases is a plurality of, e.g., 5-10, 10-15, 10-20, 15-20, 20-25, 20-30, 20- 40, 30-40, 25-50, 40-50, 50-75, 50-100, or 75-100 kinases. In a specific embodiment, the activity of all or a subset of the following kinases are assessed: EGFR, HER2, HER3, FGFR1, FGFR3, FGFR4, InsR, IGF-IR, TrkA, TrkB, Met, Ron, Ret, ALK, PDGFR, c-Kit, FLT3, M- CSFR, EphAl, EphA2, EphA3, EphBl, EphB3, EphB4, Tyro3, Axl, Tie2, VEGFR2, Akt (e.g., evaluating the phosphorylation at Thr308 and/or Ser473), ERK1, ERK2, S6, c-Abl, IRS-1, Zap- 70, Src, Lck, Statl, and Stat3. In one embodiment, the level of phosphorylation of the set of proteins is indicative of the kinase activity of a plurality of, e.g., 5-10, 10-15, 10-20, 15-20, 20- 25, 20-30, 20-40, 30-40, 25-50, 40-50, 50-75, 50-100, or 75-100 kinases. In a specific embodiment, the level of phosphorylation of the set of proteins is indicative of the kinase activity of all or a subset of he following kinases: EGFR, HER2, HER3, FGFR1, FGFR3, FGFR4, InsR, IGF-IR, TrkA, TrkB, Met, Ron, Ret, ALK, PDGFR, c-Kit, FLT3, M-CSFR, EphAl, EphA2, EphA3, EphB l, EphB3, EphB4, Tyro3, Axl, Tie2, VEGFR2, Akt (e.g., evaluating the phosphorylation at Thr308 and/or Ser473), ERK1, ERK2, S6, c-Abl, IRS-1, Zap-70, Src, Lck, Statl, and Stat3.

[00272] In certain embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is added to cell culture prior to the addition of the kinase inhibitor. For example, the test compound or the combination of test compounds is added to cell culture 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, or 21 days prior to the addition of the kinase inhibitor. In another example, the test compound or the combination of test compounds is added to cell culture 1 to 6 hours, 2 to 6 hours, 4 to 6 hours, 6 to 12 hours, 12 to 24 hours, 1 to 2 days, 2 to 5 days, 5 to 7 days, 7 to 14 days or 14 to 21 days prior to the addition of the kinase inhibitor. In other embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is added to cell culture concurrently with the addition of the kinase inhibitor. In yet other embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is added to cell culture subsequent to the addition of the kinase inhibitor. For example, the test compound or the combination of test compounds is added to cell culture 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, or 21 days subsequent to the addition of the kinase inhibitor. In another example, the test compound or the combination of test compounds is added to cell culture 1 to 6 hours, 2 to 6 hours, 4 to 6 hours, 6 to 12 hours, 12 to 24 hours, 1 to 2 days, 2 to 5 days, 5 to 7 days, 7 to 14 days or 14 to 21 days subsequent to the addition of the kinase inhibitor.

[00273] In certain embodiments, the test concentration of the test compound or each compound in the combination of test compounds used in cell culture is 0.1 nmol to 75 nmol, 1 nmol to 75 nmol, 10 nmol to 75 nmol, 20 nmol to 75 nmol, 30 nmol to 75 nmol, 50 nmol to 75 nmol, 0.1 to 50 nmol, 1.5 nmol to 50 nmol, 5 nmol to 50 nmol, 10 nmol to 50 nmol, 25 nmol to 50 nmol, 1 nmol to 10 nmol, 1 nmol to 6 nmol, 1 to 5 nml, 0.1 to 6 nmol, or 1 to 3 nmol. In some embodiments, the test concentration of the test compound or each compound in the combination of test compounds used in cell culture is 0.1 nmol, 0.25 nmol, 0.5 nmol, 0.75 nmol, 1 nmol, 5 nmol, 7 nmol, 10 nmol, 15 nmol, 20 nmol, 25 nmol, 30 nmol, 35 nmol, 40 nmol, 45 nmol, 50 nmol, 55 nmol, 60 nmol, 65 nmol, 70 nmol, or 75 nmol. In certain embodiments, the test concentration of each compound in the combination of test compounds is different. In other embodiments, the test concentration of each compound in the combination of test compounds is the same. In certain embodiments, the specific concentration of the kinase inhibitor used in cell culture is about 0.0001 μπιοΐ to 20 μπιοΐ, 0.001 μπιοΐ to 20 μπιοΐ, 0.01 μπιοΐ to 20 μπιοΐ, 0.1 μπιοΐ to 20 μπιοΐ, 1 μπιοΐ to 20 μπιοΐ, 10 μπιοΐ to 20 μπιοΐ, 0.0001 μπιοΐ to 10 μπιοΐ, 0.001 μπιοΐ to 10 μmol, 0.01 μπιοΐ to 10 μmol, 0.1 μπιοΐ to 10 μmol, 1 μπιοΐ to 10 μmol, 0.0001 μπιοΐ to 1 μmol, 0.001 μπιοΐ to 1 μmol, 0.01 μπιοΐ to 1 μmol, 0.1 μπιοΐ to 1 μmol, 0.0001 μπιοΐ to 0.1 μmol, 0.001 μιηοΐ to 0.1 μιηοΐ, 0.01 μιηοΐ to 0.1 μιηοΐ, 0.0001 μιηοΐ to 0.01 μιηοΐ, 0.001 μιηοΐ to 0.01 μmol, or 0.0001 μπιοΐ to 0.001 μπιοΐ. In certain embodiments, the specific concentration of the kinase inhibitor used in cell culture is about 0.0001 μπιοΐ, 0.001 μπιοΐ, 0.01 μπιοΐ, 0.1 μπιοΐ, 1 μπιοΐ, 10 μmol, or 20 μπιοΐ. In a specific embodiment, the test concentration of the test compound or each compound in the combination of compounds is equivalent to a subtherapeutic dose.

[00274] In specific embodiments, a method for determining resistance to the emergence of cancer cells refractory to treatment with a test compound or a combination of test compounds and a kinase inhibitor comprises: (i) administering to a first group of animals, which are alive and have cancer, a test compound or a combination of test compounds at a test dose(s) and a kinase inhibitor at a specific dose over a period of time; and (ii) analyzing the percentage of dead cancer cells of the first group of animals over said period of time, wherein no detectable change or an increase in the percentage of dead cancer cells in the first group of animals over said period of time indicates that that there is prevention of the emergence of cancer cells refractory/resistant to treatment with the test compound or the combination of test compounds at the test

concentration(s) and the kinase inhibitor at the specific concentration. In contrast, a decrease in the percentage of dead cancer cells in the first group of animals over said period of time indicates that there is no prevention of the emergence of cancer cells refractory/resistant to treatment with the test compound or the combination of test compounds at the test concentration(s) and the kinase inhibitor at the specific concentration. In certain embodiments, the percentage of dead cancer cells are assessed: (i) prior to the administration of the test compound or the combination of test compounds and the kinase inhibitor to the animals; (ii) within 6 to 12 hours, 12 to 24 hours, and/or 1 to 2 days of following administration of the test compound or the combination of test compounds and the kinase inhibitor to the animals; and/or (iii) after 2 to 5 days, 5 to 7 days, 7 to 14 days, 7 to 21 days 14 to 21 days, 21 to 28 days, 25 to 30 days, 30 to 40 days, 40 to 60 days, and/or 60 to 120 days of the repeated administration of the test compound or the combination of test compounds and the kinase inhibitor to the animals. In certain embodiments, a tumor biopsy or blood sample obtained from an animal(s) is analyzed to assess the percentage of dead cancer cells. In a specific embodiment, the animals are animal models for a particular type of cancer (e.g., lung, liver, brain, kidney, uterine, breast, pancreatic, thyroid, testicular, skin, or bone). In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are non-human animals.

[00275] In specific embodiments, a method for determining resistance to the emergence of cancer cells refractory to treatment with a test compound or a combination of test compounds and a kinase inhibitor comprises: (i) administering to a first group of animals, which are alive and have cancer, a test compound or a combination of test compounds at a test dose(s) and a kinase inhibitor at a specific dose over a period of time; and (ii) analyzing tumor size in the first group of animals over said period of time, wherein no detectable change or a decrease in the size of the tumor in the first group of animals over said period of time indicates that that there is prevention of the emergence of cancer cells refractory/resistant to treatment with the test compound or the combination of test compounds at the test concentration(s) and the kinase inhibitor at the specific concentration. In contrast, an increase size of the tumor in the first group of animals over said period of time indicates that there is no prevention of the emergence of cancer cells refractory/resistant to treatment with the test compound or the combination of test compounds at the test concentration(s) and the kinase inhibitor at the specific concentration. In certain embodiments, tumor size in the animals assessed: (i) prior to the administration of the test compound or the combination of test compounds and the kinase inhibitor to the animals; (ii) within 6 to 12 hours, 12 to 24 hours, and/or 1 to 2 days of following administration of the test compound or the combination of test compounds and the kinase inhibitor to the animals; and/or (iii) after 2 to 5 days, 5 to 7 days, 7 to 14 days, 7 to 21 days 14 to 21 days, 21 to 28 days, 25 to 30 days, 30 to 40 days, 40 to 60 days, and/or 60 to 120 days of the repeated administration of the test compound or the combination of test compounds and the kinase inhibitor to the animals. In certain embodiments, the tumor size is assessed by an imaging technique known to one of skill in the art, e.g., MRI, X-ray, CAT/CT scan or PET scan. In a specific embodiment, the animals are animal models for a particular type of cancer (e.g., lung, liver, brain, kidney, uterine, breast, pancreatic, thyroid, testicular, skin, or bone). In certain embodiments, the group of animals comprises 2 to 4 animals, 4 to 6 animals, 5 to 8 animals, 8 to 10 animals, 10 to 20 animals, 20-30 animals, 30 to 60 animals, or 60 to 100 animals. In a specific embodiment, the animals are non- human animals.

[00276] In certain embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is administered to an animal prior to the administration of the kinase inhibitor. For example, the test compound or the combination of test compounds is administered to an animal 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, or 21 days prior to the administration of the kinase inhibitor. In another example, the test compound or the combination of test compounds is administered to an animal 1 to 6 hours, 2 to 6 hours, 4 to 6 hours, 6 to 12 hours, 12 to 24 hours, 1 to 2 days, 2 to 5 days, 5 to 7 days, 7 to 14 days or 14 to 21 days prior to the administration of the kinase inhibitor. In other embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is administered to an animal concurrently with the administration of the kinase inhibitor. In yet other embodiments, in accordance with the methods described herein, the test compound or the combination of test compounds is administered to an animal subsequent to the administration of the kinase inhibitor. For example, the test compound or the combination of test compounds is administered to an animal 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, or 21 days subsequent to the administration of the kinase inhibitor. In another example, the test compound or the combination of test compounds is administered to an animal 1 to 6 hours, 2 to 6 hours, 4 to 6 hours, 6 to 12 hours, 12 to 24 hours, 1 to 2 days, 2 to 5 days, 5 to 7 days, 7 to 14 days or 14 to 21 days subsequent to the administration of the kinase inhibitor.

[00277] In certain embodiments, the test dose of the test compound or each compound in the combination of test compounds administered to the animals is about 2-fold, 5-fold, 10-fold, 20- fold, 50-fold, 75-fold, or 100-fold lower than the dosage efficacious for treating a particular cancer in the animals. In one embodiment, the test dose of the test compound or each compound in the combination of test compounds administered to the animals is about 40 mg/kg, 20 mg/kg, 10 mg/kg, 5 mg/kg, lmg/kg, 0.5 mg/kg, 0.1 mg/kg, 0.05 mg/kg, 0.01 mg/kg, 0.005 mg/kg, 0.001 mg/kg or lower, or about 40 mg/kg to 20 mg/kg, 20 mg/kg to 10 mg/kg, 10 mg/kg to 5 mg/kg, 5 mg/kg to 1 mg/kg, 1 mg/kg to 0.5 mg/kg, 0.5 mg/kg to 0.1 mg/kg, 0.1 mg/kg to 0.05 mg/kg, 0.05 mg/kg to 0.01 mg/kg, 0.01 mg/kg to 0.005 mg/kg, 0.005 mg/kg to 0.001 mg/kg or lower. In a specific embodiment, the test dose of the test compound or each compound in the combination of test compounds is the subtherapeutic dose(s). In some embodiments, the test dose of each compound in the combination of test compounds administer to the animal is different. In other embodiments, the test dose of each compound in the combination of test compounds

[00278] In specific embodiments, the test compound(s) or the combination of test compounds assessed in a Resistance Assay are the test compound(s) or the combination of test compounds which demonstrate therapeutic efficacy in combination with a kinase inhibitor in an Efficacy Assay, no therapeutic efficacy alone in a Viability Assay and/or are minimally toxic as assessed by a Toxicity Assay.

[00279] In a specific embodiment, the Resistance Assay is conducted as described in Example 1 (Section 6, infra).

6. EXAMPLE 1

[00280] This example describes compounds that are Network Brakes and methods for identifying compounds that have Network Brake activity. This example also provides combinations of a Network Brake and a kinase inhibitor for treating cancer.

6.1 Materials and Methods

[00281] Antibodies

[00282] Antibodies used for Drosophila and human cancer line western analysis were: anti- pRet, anti-pJnk, anti-pAkt, anti-SOX2, anti-KLF4, anti-LIN28, anti-Oct4, anti-Nanog, anti- cMyc, anti-pMOB, anti-cleaved PARP (Cell Signaling), anti-pSrc(Y⁴¹⁸)(Invitrogen), anti-pERK (SIGMA), plus anti-Actin, , anti-E-cadherin, anti-a-Catenin, anti-Rhol, anti-Syntaxin, anti- CycD, anti-Argos, anti-B-tubulin (Developmental Studies Hybridoma Bank), anti-Actin , anti- GAPDH, anti-RhoA antibodies were purchased from Santa Cruz Biotechnology, anti-Racl antibody (BD biosciences), anti-EGFr (Julia Cordero), anti-activated-B-Catenin (Millipore). All histone modification antibodies were obtained from ActiveMotif.

[00283] Chronic drug treatment assays on human cancer lines

[00284] Human NSCLC cancer line H358 or melanoma line 239 were grown in 75cm² culture flasks. H358 cells were grown in the following conditions: i) 0.01% DMSO, ii) 0.5 μΜ erlotinib, iii) Ι μΜ erlotinib, iv) bortezomib (6 nM) + vorinostat (50 nM), v) erlotinib (1 μΜ) bortezomib (6 nM) + vorinostat (50nM). 239-melanoma cells were grown in the following different conditions: i) 0.01%DMSO, ii) 0.5μΜ vemurafenib, iii) bortezomib (6 nM) + vorinostat (50 nM), iv) vemurafenib (0.5 μΜ) bortezomib (6 nM) + vorinostat (50 nM). Initially cells were seeded in culture flasks at 20% confluency and incubated in RPMI1640 media with the above mentioned conditions. When cells reached confluency (DMSO controls) they were split and transferred to new culture flask with identical fresh media conditions once a week. Otherwise identical fresh media was provided to each flask once a week. When single drug treated cells (erlotinib or vemurafenib) started growing similar to DMSO control cells they were amplified for 3 generations and assessed for development of drug resistance by indicated assays. Resistant cells and triple drug cocktail treated cells were allowed to grow without selection for 3 generations to get enough cells for various assays.

[00285] Fly stocks, genetics, and subcloning

[00286] Fly stocks were obtained from Bloomington and VDRC Drosophila stock centers, C. Pfleger, M. Mlodzik. UAS-Re^^EN2B flies were generated and published previously (Dar et al., 2012).

[00287] Inhibitor studies in flies

[00288] Drugs were obtained from LC laboratories or Selleck Chemicals and were dissolved in DMSO as stock solutions ranging from 1-200μΜ. Drugs were then diluted in molten (-50- 60°C) enriched fly food, vortexed, mixed by pipette multiple times, aliquoted into 5ml vials and left to solidify at room temperature to yield the indicated final drug concentrations. 30-60 embryos of each genotype were raised on drug-containing food (500-1000 μΐ) in 5 ml vials until they matured as third-instar larvae (wing disc western assay assay) or allowed to proceed to adulthood (viability assay and wing vein quantitation assay). 5 vials per experiment were analyzed and repeated at least 3 times.

[00289] MTT assays using cancer lines

[00290] All cancer cell lines were cultured in RPMI1640 media, supplemented with 10% BSA and pen/strep antibiotics mix. Cells were grown in 75 cm² sterile polystyrene culture flasks to 80%) confluency, trypsinized, and re-seeded in equal aliquots into 96-well plates. After 2 days and -50% confluency, media was removed and replaced with DMSO or drug containing media. Cells were allowed to grow another 6 days (MZ-CRC-1 and TT) or 4 days (all other fast growing cancer lines) after which MTT assay was performed. Cell media was removed and replaced with Thiazolyl Blue Tetrazolium Bromide (MTT) containing media (1 mg/ml final concentration) and cells were allowed to grow at 37°C for another 3.5 hours. MTT media was removed, and MTT precipitate dissolved in 4 mM HC1, 0.1%> P40 in Isopropanol, solvent by shaking for 1 hr. Spectrophotometric readings at 590 nm and 630 nm using a 96-well plate reader were used to establish growth and viability of cells. Each drug dose was tested in quadruplicates and experiments repeated twice.

[00291] Phospho-protein array analysis

[00292] For assessment of kinase activity of human cancer cell lines, the PathScan RTK Signaling Antibody Array Kit (Cat # 7982) was used. Briefly, 100 cm² tissue culture plates were plated with human cancer cells at 50-60%> confluency in RPMI1640 media with or without drugs and allowed to incubate for 4, 5 days. Cells were washed with cold lx PBS, scraped with cell scrapers in lx Lysis buffer from kit, sonicated until cell debris cleared, and cellular coagulates spun down by centrifugation. Lysate supernatant was carefully removed to avoid pellet, and stored at -80C in small aliquots. Small amount of lysate for each treatment was used to assess protein concentration using BIORAD protein assay. Each antibody array was incubated with ly sates at 0.5mg/ml total protein concentration, as recommended by manufacturer, and developed according to manufacturer protocols. Doublet of each signal was quantitated using densitometric analysis on Image J program, and normalized to time matched untreated cells to generate bar graph.

[00293] Western analysis of fly wing discs [00294] 30 third-instar discs of each genotype were dissolved in Lysis Buffer (50mM Tris, 150mM NaCl, 1% Triton-XlOO, ImM EDTA) supplemented with protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktail (Sigma). For human cell lines, lysis was performed with RIP A buffer. Total protein in each sample was quantitated using BIORAD protein assay. Samples were boiled, resolved on Invitrogen NU-PAGE gradient SDS-page and transferred by standard protocols. Each lane was loaded with approximately 5-disc equivalent of lysates to yield a total of 6 gel replicates for each genotype collected. Membranes were stripped with SIGMA Restore stripping buffer and reprobed with other antibodies to assess signal under exactly the same loading conditions.

[00295] Western blotting of cancer cell lines

[00296] Human cancer cell lines were grown in 100cm² well plates in RPMI1640 media each supplemented with 10% heat-inactivated FBS and pen/strep antibiotics. Cells were treated for 4, 5 days with inhibitors or vehicle (0.1% DMSO). After treatment, media was removed, cells were washed twice with cold PBS, and then lysed in RIPA buffer (25mM Tris pH 7.6; 150mM NaCl; 1% P-40; 0.1% SDS) containing protease and phosphatase inhibitors and sonicated (Roche). Lysate protein concentration was assessed using BIORAD protein assay. 5 or lOug of total cell lysate were separated by SDS-PAGE, transferred to PVDF membrane and blotted for the indicated proteins using commercial antibodies. Membranes were stripped and probed as described above.

[00297] Whole mount imaging of fly and wings

[00298] For adult wing vein analysis, wings were dissected and kept in 100% ethanol overnight, mounted on slides in 80% glycerol in phosphate buffered saline solution, and imaged by regular light microscopy using Leica DM5500 Q microscope.

[00299] Xenograft Analysis

[00300] 5-10 x 10⁶ TT cells were injected subcutaneously into one flank of male nu/nu mice. 10 mice each showing established growing tumors were separated into vehicle or drug treatment groups. A similar range of tumor sizes was selected for each experiment. Vehicle, sorafenib (40 mg/kg), sorafenib(40 mg/kg)+bortezomib (0.05 mg/kg), or sorafenib(40 mg/kg)+bortezomib (0.05 mg/kg)+vorinostat (lOmg/kg) were administered by oral gavage (PO) once daily, 3 times a week. Tumor and body weight measurements were performed 3 times a week. Mouse experiments were carried out by Antitumor Assesment Facility at Memorial Sloan Kettering Cancer Center following Public Health Services guidelines, set forth by the Office for Laboratory and Animal Welfare (OLAW) division of the National Institutes of Health (NIH).

6.2 Results

[00301] Multiple Endocrine Neoplasia Type 2B (MEN2B) is an often aggressive disease characterized by a series of morbidities including medullary thyroid carcinoma,

pheochromocytoma, and mucosal neuromas. Most cases are associated with activated, oncogenic Ret^M918T; previous studies have shown that multiple downstream signaling pathways are activated to promote transformation(Read et al., 2005; Salvatore et al., 1993; Wells and Santoro, 2009). Ret^M918T is modeled by Drosophila Ret^M1117T; this oncogenic isoform is referred to as Ret^2B. Genetic modifier studies (Read et al., 2005) as well as western analysis confirmed that this Drosophila model (Fig. 1 A) recapitulated important signaling cascades also observed in vertebrate systems (Read et al., 2005; Salvatore et al., 1993). Using this model & Drosophila whole animal viability assay was developed to identify potent multi -targeted inhibitors of the Ret signaling cascade (Fig. IB; Dar et al., 2012). Using the patched-GAL4 driver to express UAS- Ret^2B in multiple developing tissues (ptc>Ret^2B) led to approximately 50% of counted embryos reaching pupal stages but none developing to adults (Fig. 1C). This assay provided a

quantitative measure of Ret^2B activity including transformation activity (Dar et al., 2012).

[00302] Sorafenib altered cellular networks in a Drosophila Ret^2B model

[00303] Mixing drugs into the flies' media, a panel of clinically relevant anti-cancer drugs was screened for improved ptc>Ret^2B viability (Fig. 8). Sorafenib exhibited the strongest rescue: a small fraction of embryos was rescued to adult stages (Fig. 1C). Sorafenib is a kinase inhibitor with multiple targets including Ret and its downstream effector Raf, and is effective against Ret- dependent human thyroid cancer cells(Krajewska et al., 2015). Previous work validated sorafenib as effective in suppressing Ret^2B-dependent oncogenic signaling in Drosophila tissues (Dar et al., 2012; Das and Cagan, 2010).

[00304] Previous work demonstrated that, at low doses, Raf inhibitors can activate Ras pathway signaling by promoting formation of active complexes (Poulikakos et al., 2010).

Indeed, low dose sorafenib activated the Ras/MAPK pathway in vivo as assessed by increased wing venation, a phenotype linked to elevated Ras pathway activity (Figs. 2A-B, 9A; Sawamoto et al., 1996) (Gui chard et al., 1999); few animals survived to pupariation (Fig. 1C). Interestingly, reducing gene dosage of the Ras/Raf downstream pathway effector erk (ptc>Ret^2B ;erk^+/~) significantly improved sorafenib efficacy even at low doses, resulting in improved wing venation and reduced toxicity as assessed by pupanation rates (Figs. 2A, 2B, 9A). The ability of sorafenib to demonstrate efficacy at low doses in the proper genetic background suggests that sorafenib is provoking toxicity at least in part due to mechanisms beyond directly promoting active complexes.

[00305] To explore the nature of sorafenib 's whole animal toxicity, oncogenic Ret was expressed throughout the larval wing disc epithelium (765>Ret^2B). Western analysis indicated that feeding sorafenib to 765>Ret^2B larvae led to activation of multiple signal transduction proteins including activated, phosphorylated forms of Ret, Erk, Akt, and Src (Figs. 2B-C, 9A-E). This contributed to sorafenib' s toxicity: similar to its effects on viability, reducing erk gene dosage (765>Ret^2B,erk^+/~) improved wing phenotypes and significantly reduced overall phosphorylation levels of most assayed proteins (Figs. 2C, 9B-E). It was concluded that altering sorafenib or its targets has broad effects on cellular networks, leading to cellular toxicity and poor efficacy. Restraining these networks genetically can lead to significant improvement of sorafenib' s overall efficacy. Therefore, a search was conducted for clinically relevant therapeutics that led to a similar network restraint.

[00306] Drug combinations can restrain hyperactivation of cellular protein networks

[00307] In a limited screen of combinations of clinically-relevant drugs, three drugs combined with sorafenib to further improve ptc>Ret^2B viability (Figs. 3 A, 8). Sorafenib/bortezomib and sorafenib/dasatinib were focused on as the most clinically relevant combinations (Fig. 3 A;

Kumar et al., 2013; Rao and Lauer, 2015; Sullivan et al., 2015; Schott, 2009; Tuma, 2006;

Kumar et al., 2013; Rao and Lauer, 2015; Sullivan et al., 2015). Western analyses indicated that each combination kept the median network activity level in transformed cells below the level observed with sorafenib alone (Fig. 11 A-B brackets, asterisk) (see raw data in Fig. 11C-D); importantly sorafenib/bortezomib also kept median protein network in normal cells closer to baseline state compared to sorafenib/dasatinib (Fig. 11 A-B brackets, asterisk; median = +5.5 vs +14, respectively) (see raw data in Fig. 11C-D). Sorafenib/dasatinib rescued fewer ptc>Ret^2B to pupariation, though a larger proportion of these pupae hatched to adults (Fig. 3 A). Based on improved pupal rescue, lower network activation in normal cells, and restraint of the overall signaling network, the focus was bortezomib, a proteasome inhibitor approved for multiple myeloma and mantle cell lymphoma. [00308] One drug combination (vandetanib/rapamycin) from the screen showed the opposite effect: decreased viability for ptc>Ret^2B flies and control flies, indicating overall animal toxicity (Fig. 10 A-D). Western analysis profiling indicated higher median level of the protein network in normal cells treated with this combination (Fig. 10B; asterisk). Thus while improved efficacy (Fig. 2A-C) restrained the protein networks towards a normal state, increased whole animal toxicity resulted from hyperactivation of the same networks.

[00309] Next, a genetic approach was used to determine whether sorafenib/bortezomib- mediated rescue could be further improved. Using a dominant genetic modifier screen, a subset of genes targeted by clinically relevant anti-cancer drugs was focused on. Reducing dosage of genes encoding ERK (rollec ^'), MEK (dsorI^+/~), or FID AC 1 (rpd3^+/~) orthologs, or expressing a dominant negative insulin receptor (InR^DN) by transgene, improved the viability of ptc>Ret^2B flies fed sorafenib/bortezomib (Fig. 3 A). The combination sorafenib/dasatinib also benefited from reducing erk gene dosage (Fig. 3 A), again lowering the median protein network level from -5.5 to -17 in transformed cells (Fig. 11 A-D).

[00310] The requirement for the histone deacetylase Rpd3 for network hyperactivation was interesting given the broad control of cellular networks by epigenetic complexes. Vorinostat is approved for the treatment of T-cell lymphoma and for multiple myeloma and is one of several pan-FDD AC inhibitors in clinical trials for other cancer types {e.g., Duvic et al., 2007; Fenichel, 2015; Fushida et al., 2015; Kaushik et al., 2015; Okam et al., 2015; Pasini et al., 2015; Rose, 2011). Addition of low dose vorinostat enhanced the viability of both ptc>Ret^2B flies and, notably, control flies treated with sorafenib/bortezomib (Figs. 3A, 12A). Other pan-HDAC inhibitors also enhanced the viability of control flies when combined with sorafenib/bortezomib, suggesting that mild inhibition of the FID AC network synergized with sorafenib/bortezomib (Fig. 12A) to restrain toxicity. Based on western analysis, the three drug combination

sorafenib/bortezomib/vorinostat further reduced cells' overall network activation. For example, the triple combination further reduced activation of AKT, ERK, and Racl in control cells compared to sorafenib/bortezomib; similar reductions were observed in Ret^2B- expressing tissues to further improve the median protein network level (Fig. 3B). Vorinostat regulates transcription of a large number of target genes (Chun, 2015; Lee et al., 2015), suggesting it restrains the cellular response to drugs by controlling changes in transcription. Consistent with this view Mithramycin (MTM), a chemical inhibitor of SP1 -class transcription factors, similarly improved efficacy of the sorafenib/bortezomib combination (Fig. 12B).

[00311] Together, this data supports a model in which sorafenib promotes strong

hyperactivation of the overall cellular network in both transformed and normal tissues. The result is limited efficacy and significant toxicity. Drug efficacy is strongly improved by restraining broad network hyperactivation in both transformed and wild type tissue. Both can be accomplished by selective use of genetics or through drugs that broadly control the cellular network including bortezomib and vorinostat, providing a 'network brake' to the effects of sorafenib. Importantly, these drugs were active at doses well below those required for significant efficacy. This work raised the question as to whether other targeted therapies were directing a similar change in cellular networks and, if so, whether bortezomib/vorinostat cocktails would restrain these changes to render targeted therapies more effective.

[00312] Bortezomib/vorinostat improved multiple targeted therapies in Drosophila

[00313] Other Ret pathway kinase inhibitors including trametinib (MEK inhibitor; approved for use in melanoma patients) and the experimental polypharmacological drugs AD57 and AD80 (Dar et al., 2012; Flaherty et al., 2012; Gilmartin et al., 2011) were examined. Each was tested in combination with bortezomib/vorinostat. Viability of both control (ptc-GAL4) and ptc>Ret^2B flies improved in the presence of each targeted therapy when combined with bortezomib and vorinostat (Fig. 3C).

[00314] Next, it was tested whether other drugs inhibiting broad protein networks also cooperated at sub-therapeutic doses. Bortezomib was substituted with Hsp90 inhibitor AUY922 and vorinostat with the pan HDAC inhibitor CUDC-907 and tested them in conjunction with targeted therapies. In Drosophila, AUY922/vorinostat paired with sorafenib, trametinib, AD57, and AD80 to improve viability of both control and ptc>Ret^2B flies (Fig. 3D) in a manner similar to bortezomib/vorinostat. It is concluded that broadly acting drugs including bortezomib, vorinostat, AUY922, and CUDC-907 can cooperate in modular fashion to reduce kinase network hyperactivation and improve efficacy of targeted therapies.

[00315] These data indicate that a diverse group of cancer relevant kinase inhibitors are improved in overall efficacy when administered in the presence of a bortezomib/vorinostat combination. Together they act as a 'network brake' cocktail: when paired together at low doses, they strongly reduced drug-induced network hyperactivation. [00316] 'Network brake' cocktails restrained human thyroid cancer networks to oppose tumor progression

[00317] Next, it was assessed whether the findings in Drosophila translated to human medullary thyroid cancer (MTC) models in which Ret acts as the primary oncogenic driver (Fig. 13). The MTC cancer cell line MZ-CRC-1 (Cool ey et al., 1995) harbors an activating mutation in Ret analogous to the Drosophila Ret^2B mutation. Sorafenib has been previously demonstrated to inhibit MTC cell line growth(Koh et al., 2012); this efficacy required high dose treatment (Fig. 4A). Protein lysates isolated from MZ-CRC-1 cells were treated with sorafenib and incubated with phospho-protein arrays to monitor activity levels of 39 proteins (Fig. 13B). Cells treated for 4 hrs showed inhibition of several intracellular pathway effectors including ERK; overall, a low hyperactivation of the signaling network was observed (Fig. 4D). Strikingly, after 4 days of chronic sorafenib treatment a strong shift was observed: nearly all assayed phospho-proteins were strongly upregulated (Fig. 4D). Hyperactivation of RTKs such as EGFR, FIER2/3, and MET were especially intriguing as these are known to promote drug resistance in different cancers (Engelman et al., 2007; Gravdal et al., 2012; Kanda et al., 2013; Li et al., 2013).

[00318] Low dose bortezomib/vorinostat enhanced the growth inhibitory effects of sorafenib: sorafenib alone had an IC50 of approximately 4 μΜ; inclusion of low dose bortezomib plus vorinostat (6 nM each) shifted sorafenib IC50 to 0.2 μΜ, a 20-fold reduction to achieve similar effects (Fig. 4A). Similar to the findings in Drosophila, the optimized polypharmacological inhibitor AD80 was also strongly improved in the presence of bortezomib/vorinostat: the IC50 on MZ-CRC-1 cancer cells was reduced from 6 nM to 0.6 nM in the presence of

bortezomib/vorinostat, a 10-fold change (Fig. 4B). Again, similar effects were observed with other broadly acting drugs: AUY922/vorinostat paired with sorafenib (MZ-CRC-1, MTC cell line) and trametinib (H1299, NSCLC cell line) to significantly reduce IC50s compared to targeted therapy treatment alone (Fig. 4E).

[00319] Presence of cancer stem cells are associated with resistance in many cancers (Borah et al., 2015; Reya et al., 2001). MZ-CRC-1 cells treated chronically with sorafenib upregulated Sox2 protein levels (Fig. 5A), a stem cell fate marker previously demonstrated to promote a broad program of tumorigenesis (Basu-Roy et al., 2012; Boumahdi et al., 2014; Guo et al., 2011; Leis et al., 2012; Siegle et al., 2014). Importantly, expression of Sox2 was strongly suppressed by addition of bortezomib alone or a bortezomib/vorinostat drug combination (Fig. 5 A, D). Similar results regarding growth and stem cell markers were observed with TT cells, MTC- derived cells that harbor the oncogenic Ret isoform Ret^C634W. Sorafenib had a moderate inhibitory effect on TT cell growth and the signaling network, with the stem cell markers Sox2 and c-Myc emerging after chronic treatment (Figs. 5B,D); high nuclear Sox2 was observed in a subset of cells (Fig. 5C). Co-treatment with bortezomib/vorinostat led to a further decrease in levels of the signaling phospho-proteins (Figs. 5B,D). Sox2 levels were suppressed by bortezomib and, to a lesser extent, by bortezomib/vorinostat (Figs. 5B,D); the latter prevented expression of nuclear Sox2 (Fig. 5C). Finally, co-treatment led to strong progressive

upregulation of cleaved-PARP in both MZ-CRC-1 and TT cells (Figs. 5A,B), indicating a significant increase in apoptotic cell death.

[00320] Next, the effects of pairing low dose bortezomib/vorinostat with sorafenib in a mouse thyroid cancer xenograft model were assessed. Growth of established TT cell xenografts in mice were potently inhibited with sorafenib/bortezomib/vorinostat compared to vehicle, sorafenib alone, or sorafenib/bortezomib treatments (Fig. 4C). Vehicle-alone treated animals showed a median 300% increase in tumor size, while sorafenib/bortezomib/vorinostat treated animals exhibited 10% tumor growth during the course of treatment. In these studies sorafenib worked potently at 40 mg/kg in combination with bortezomib/vorinostat, a concentration lower than most previous xenograft studies(Carlomagno et al., 2006; Salvatore et al., 2006). Together these data indicate that— similar to the whole animal Drosophila data— broadly acting cocktails such as bortezomib/vorinostat can act as 'network brakes' in at least two thyroid cancer cell lines. These cocktails restrain cellular networks, prevent emergence of cells with stem cell-like properties, and allow strong efficacy with lower doses of targeted therapies over long treatment periods.

[00321] Bortezomib/vorinostat was effective against multiple cell types

[00322] To assess the broader applicability of these results, a panel of cancer cell lines with defined genetic mutations that respond to different targeted kinase inhibitors was assembled. The non-small cell lung cancer cell line H358 exhibits high EGFR activity and is sensitive to erlotinib, an EGFR inhibitor approved for NSCLC(Bezjak et al., 2006; Shepherd et al., 2005). Chronic erlotinib treatment led to progressive hyperactivation of the kinase network; this effect was restrained and the IC50 of erlotinib reduced significantly in the presence of

bortezomib/vorinostat (Fig. 6A-C). A similar pattern was observed with the NSCLC cell line H1299, which contains an oncogenic isoform of K-RAS: the relevant standard-of-care drug trametinib led to network hyperactivation that was restrained by bortezomib/vorinostat (Figs. 14A, B, E). In each case this restraint coincided with an increase in efficacy. Broadening the survey, synergy was observed with H1299 cells (NSCLC), HepG2 and PLC5 (hepatocellular carcinoma), T47D (ER⁺ breast cancer) and A375 (melanoma) (Fig. 6C,D). The IC50s of each standard-of-care targeted therapy was lowered significantly in the presence of

bortezomib/vorinostat, ranging from modest reduction (5-fold in BEZ235 -treated T47D cells) to more potent reduction (83-fold in trametinib-treated H1299 cells; Figs. 6D, 14A).

[00323] Finally, it was assessed whether the targeted therapies were affecting markers of stem cell fate across different cell types. Erlotinib treated H358 NSCLC cells showed strong upregulation of Sox2 as well as increased Myc levels. Co-treatment with bortezomib/vorinostat strongly reduced Sox2 and moderately reduced Myc levels (Fig. 6E). In addition, the stem cell marker LIN28 and epithelial-to-mesenchymal (EMT) marker Vimentin were both kept below untreated levels with bortezomib/vorinostat co-treatment (Fig. 6E). In H1299 cells, levels of Myc and KLF4 were reduced with trametinib/bortezomib/vorinostat treatment (Fig. 14B) while HCC cell line HepG2 showed little alteration in these markers (Figs. 14C, D). In each cell line, high levels of cleaved PARP were observed with bortezomib/vorinostat co-treatment indicating apoptotic death. Together these results demonstrate that, across a large number of human cancer cell types, bortezomib/vorinostat co-treatment enhanced established therapies by restraining network hyperactivation and by reducing levels of pro-tumorigenic markers.

[00324] Bortezomib/vorinostat restrained emergence of drug resistance

[00325] Cancer cells progressively evade sensitivity to targeted kinase inhibitor therapies by upregulating different combinations of cellular kinases and RTK's, leading to drug resistance. Bortezomib/vorinostat co-treatment restrained hyperactivation of kinase networks, and it was hypothesized that would also delay or prevent drug resistance from arising over longer treatment periods.

[00326] Cellular resistance to erlotinib in clinics as well as in NSCLC human cancer cells such as H358 have been well documented (Fong et al., 2013; Haber et al., 2005; Halmos et al., 2015; Kobayashi et al., 2005; Kritikou et al., 2013). H358 cells became insensitive to erlotinib by approximately 35 days of chronic treatment: parental cells had an IC50 of 0.5 μΜ while resistant lines displayed an 8-fold increase to 4 μΜ (Figs. 7A,B). Chronic co-treatment with erlotinib/bortezomib/vorinostat prevented resistance from emerging as H358 cells retained sensitivity to erlotinib similar to parental cells (Fig. 7A,B; IC50 of 0.7 μΜ). Similar effects were observed with Mel-239, a melanoma cell line harboring oncogenic BRAF^V600E: 90 days of treatment led to resistance to the standard-of-care drug vemurafenib; bortezomib/vorinostat prevented resistance from arising and cells retained sensitivity to the BRAF inhibitor (Fig. 15D). It is concluded that cotreatment with 'network brake' drugs can significantly delay the emergence of resistance to targeted therapies in different cancer types.

[00327] To understand the mechanism by which 'network brake' drugs prevented drug resistance from arising phospho-protein array analysis was performed. Erlotinib-resistant H358 NSCLC cells showed upregulation of a large palette of kinases when compared to the parental cell line (Fig. 7C). This included increased phosphorylation levels of pathway effectors associated with drug resistance including ERBB3, Met, c-Kit, Src, and Stat3 (Engelman et al., 2007; Gravdal et al., 2012; Kanda et al., 2013; Li et al., 2013), suggesting that broad network upregulation contributes to cellular resistance. Upregulation of Met in Erlotinib resistant cells had functional consequences as these cells were almost 3-fold more responsive to the Met inhibitor crizotinib (Fig. 15 A). Erlotinib resistant cells also showed higher levels of the stem cell marker Sox2 as well as pro-tumorigenic proteins such as activated forms of β-Catenin and Rhol . Cells treated chronically with erlotinib/bortezomib/vorinostat did not display increased activity of these kinases; overall, a lower median level of activated kinases compared to resistant cells along with suppression of Sox2 levels was observed (Fig. 7D). Conversely,

erlotinib/bortezomib/vorinostat elevated levels of phosphorylated Mob, a key effector of the Hippo signaling pathway that suppresses growth (Figs. 7C, 15B, 15C; Chen et al., 2015b;

Lignitto et al., 2013). In general, addition of low dose bortezomib plus vorinostat blocked emergent resistance by restraining activation of pro-tumorigenic proteins and upregulating tumor suppressor activity.

[00328] HDAC inhibitors such as vorinostat alter hi stone modifications to exert broad effects on transcription(Bhadury et al., 2014; Chen et al., 2015a; Haberland et al., 2009). It was examined whether alterations in histone modifications could contribute to the broad network changes provoked by targeted therapies. Erlotinib-resistant lines showed strong upregulation of the active histone mark H3K9-Ac and sustained activation of the active marks H4K5-Ac and H3K4-Me3 (Fig. 7D). Treatment with erlotinib/bortezomib/vorinostat exhibited strongly reduced levels of these active marks and elevated levels of the inactive histone mark H3K27- Me3 (Fig. 7D). In summary, addition of bortezomib plus vorinostat in the long-term

experiments directed the histone code towards lowered transcription, prevented upregulation of overall cellular network activity, and achieving a balance of low pro-tumorigenic and high tumor suppressor signals.

6.3 Discussion

[00329] Treating cancer cells in the context of the whole body poses major challenges. A key challenge in the efforts to develop cancer therapeutics has been the emergence of resistance to drug treatment. Preclinical and clinical studies show that resistance eventually emerges even with kinase inhibitors with high target selectivity (Wilson et al., 2012). Cancer cells respond to inhibition of oncogene-addicted pathways by finding alternative mechanisms to provide high signaling through these addicted pathways, or by shifting their dependence to other pathways (Glickman and Sawyers, 2012). For example, at least six different mechanisms by which cancer cells develop resistance to drugs targeting BRAF^V600E have been identified (Haarberg and Smalley, 2014; Johannessen et al., 2010; Nazarian et al., 2010; Poulikakos et al., 2011;

Villanueva et al., 2010; Wilson et al., 2012; Yadav et al., 2012). This highlights the adaptability and high degree of connectivity within the kinase signaling network: inhibiting signaling in one part of the network provokes responses in other parts.

[00330] The findings described herein provide one path towards addressing these major challenges in cancer therapeutics. Using 'network brake' drugs as part of a cocktail would allow standard-of-care kinase inhibitors to be used at lower doses over longer periods. This would reduce the toxicity that may result from both strong on-target inhibition as well as off-target effects. In the analysis described herein, the IC50s of at least five different kinase inhibitors were reduced considerably in the presence of low dose bortezomib plus vorinostat. Further, sorafenib/bortezomib/vorinostat inhibited tumor growth in xenografted mice at low doses, indicating that sorafenib can work potently in vertebrate models if paired with at least one 'network brake' cocktail.

[00331] Another benefit of the 'network brake' drug combinations is their ability to restrain drug-induced hyperactivation of cellular signaling networks, a potential source of progressive drug resistance in tumor cells and toxicity in normal tissues (Belum et al., 2013). For example, both H358 cells and 239-mel cells developed resistance to targeted monotherapy; the addition of low dose 'network brake' drugs prevented upregulation of a family of kinases known to promote resistance. Finally restraining network hyperactivation prevented upregulation of stem cell markers, most prominently Sox2, a factor that controls stem cell fate during development and in cancer progression including EMT (Boumahdi et al., 2014; Mani et al., 2008; Siegle et al., 2014). Supporting these observations another study found that therapy-induced upregulation of cellular components such as integrins contribute to 'sternness' and drug resistance, a condition that can be restrained by co-treatment with bortezomib (Seguin et al., 2014). By allowing targeted therapies to function at lower doses, by restraining hyperactivation of the signaling network, by preventing the upregulation of stem cell markers, and by promoting increased death of cancer cells, 'network brake' drugs hold the potential for improving the therapeutic index and longevity of a broad palate of targeted therapies.

[00332] Cancer cells rely on a subset of the available cellular signaling pathways, a phenomenon termed 'oncogene addiction' . The mechanism by which cancer cells undergo apoptotic death when addicted pathways are inhibited is unclear. One theory is that cancer cells are reliant on fewer signaling pathways and therefore inhibition of the addicted pathways sends cancer cells into crisis thereby hastening death (Gillies et al., 2012; Pagliarini et al., 2015;

Pellicano et al., 2014; Seguin et al., 2014). It has been shown that targeted therapies can hyperactivate the overall cellular network; this may allow cancer cells to shift dependence to other pathways, providing a route for resistance. By restraining hyperactivation of the network in response to targeted therapies, the 'network brake' drugs may block this alternative path.

[00333] Modern cancer therapeutics has evolved from solely using chemotherapeutics to the current emphasis on targeted therapies. The latter promises a 'surgical precision' approach. However, emergent resistance has limited usefulness of these drugs and recent studies have indicated that targeted therapies show significant toxicity similar to or exceeding

chemotherapies. Low dose 'network brake' drugs may provide a useful tool for sustaining the activity and precision of targeted therapies. Multiple 'network brake' drugs have been described herein that can improve efficacy of targeted therapies: bortezomib (proteasome), vorinostat and CUDC-907 (histone deacetylases), MTM (Spl transcriptome), and AUY922 (Hsp90 inhibitor). A whole animal approach permits us to address the effects of these powerful drugs on

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[00403] Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[00404] All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[00405] The present invention is not to be limited in scope by the specific

embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Claims

What is claimed is:

1. A method for treating a human patient diagnosed with cancer, comprising administering to a human patient in need thereof:

(a) a Network Brake at a subtherapeutic dose; and

(b) a kinase inhibitor at an effective dose, so that overall or progression-free survival of the patient is increased, wherein the Network Brake is a compound or a combination of compounds that reduces the hyper-activation of the cancer signaling network induced by the kinase inhibitor, and prevents the upregulation of one or more cancer stem cell markers induced by the kinase inhibitor.

2. A method for treating a human patient diagnosed with cancer, comprising administering to a human patient in need thereof:

(a) a Network Brake at a subtherapeutic dose; and

(b) a kinase inhibitor at a dose equal to or greater than the kinase inhibitor's maximum tolerated dose as assessed in the absence of the Network Brake, so that overall or progression-free survival of the patient is increased, wherein the Network Brake is a compound or a combination of compounds that reduces the hyper-activation of the cancer signaling network induced by the kinase inhibitor, and prevents the upregulation of one or more cancer stem cell markers induced by the kinase inhibitor.

3. A method for treating a human patient diagnosed with cancer, comprising administering to a human patient in need thereof:

(a) a Network Brake at a subtherapeutic dose; and (b) a kinase inhibitor at a subclinical dose, so that overall or progression-free survival of the patient is increased, wherein the Network Brake is a compound or a combination of compounds that reduces the hyper-activation of the cancer signaling network induced by the kinase inhibitor, and prevents the upregulation of one or more cancer stem cell markers induced by the kinase inhibitor.

4. The method of any one of claims 1 to 3, wherein a reduction in the hyper-activation of the cancer signaling network induced by the kinase inhibitor is assessed by an assay comprising: (1) culturing a first population of cancer cells in the presence of the kinase inhibitor at a specific concentration equivalent to the clinical dose for a period of time; (2) culturing a second population of cancer cells of the same type in the presence of the compound or the combination of compounds at a concentration(s) equivalent to the subtherapeutic dose(s), and the kinase inhibitor at the same specific concentration for said period of time; and (3) analyzing the level of activity of a certain set of kinases, which are members of the cancer signaling network, in the first and second populations of cancer cells at the end of said period of time, wherein a decrease in the overall, median, or mean activity level of the kinases in the second population of cancer cells relative to the overall, median or mean activity of the same kinases in the first population of cancer cells indicates that the compound or the combination of compounds reduces the hyper- activation of the cancer signaling network induced by the kinase inhibitor.

5. The method of any one of claims 1 to 3, wherein a reduction in the hyper-activation of the cancer signaling network induced by the kinase inhibitor is assessed by an assay comprising: (1) culturing a first population of cancer cells in the presence of the kinase inhibitor at a specific concentration equivalent to the clinical dose for a period of time; (2) culturing a second population of cancer cells of the same type in the presence of the compound or the combination of compounds at a concentration(s) equivalent to the subtherapeutic dose(s), and the kinase inhibitor at the same specific concentration for said period of time; and (3) analyzing the level of phosphorylation of a certain set of proteins, which are members of the cancer signaling network, in the first and second populations of cancer cells at the end of said period of time, wherein a decrease in the overall, median, or mean level of phosphorylation in the second population of cancer cells relative to the overall, median or mean level of phosphorylation in the first population of cancer cells indicates that the compound or the combination of compounds reduces the hyper-activation of the cancer signaling network induced by the kinase inhibitor.

6. The method of any one of claims 1 to 5, wherein the one or more cancer stem cell markers is Sox2, Oct4, Nanog, LIN28, c-Myc, or KLF4.

7. The method of any one of claims 1 to 6, wherein the prevention of the upregulation of the one or more cancer stem cell markers induced by the kinase inhibitor is assessed by an assay comprising:

(1) culturing a first population of cancer cells in the presence of the kinase inhibitor at a specific concentration equivalent to the clinical dose for a period of time;

(2) culturing a second population of cancer cells of the same type in the presence of the compound or the combination of compounds at a concentration(s) equivalent to the

subtherapeutic dose(s), and the kinase inhibitor at the same specific concentration for said period of time;

(3) culturing a third population of cancer cells of the same type without any treatment; and

(4) analyzing the expression level of the one or more cancer stem cell markers in the first, second, and third populations of cancer cells at the end of said period of time, wherein the overall, median, or mean expression level of the one or more cancer stem cell markers in the first population of cancer cells is higher than the overall, median, or mean expression level of the same cancer stem cell marker(s) in the third population of cancer cells, and wherein a decrease in the overall, median, or mean expression level of the one or more cancer stem cell markers in the second population of cancer cells relative to the overall, median or mean expression level of the same cancer stem cell marker(s) in the first population of cancer cells indicates that the compound or the combination of compounds prevents the upregulation of the one or more cancer stem cell markers induced by the kinase inhibitor.

8. The method of any one of claims 1 to 7, wherein the cancer is a solid tumor cancer.

9. The method of any one of claims 1 to 8, wherein the Network Brake is a proteasome inhibitor.

10. The method of any one of claims 1 to 8, wherein the Network Brake is a histone deacetylase inhibitor.

11. The method of any one of claims 1 to 8, wherein the Network Brake is a combination of a proteasome inhibitor and a histone deacetylase inhibitor.

12. The method of claim 9 or 11, wherein the proteasome inhibitor is bortezomib.

13. The method of claim 10 or 11, wherein the histone deacetylase inhibitor is vorinostat, belinostat, entinostat, panobinostat, or RG2833.

14. The method of claim 11, wherein the proteasome inhibitor is bortezomib and the histone deacetylase inhibitor is vorinostat.

15. The method of claim 11, wherein the proteasome inhibitor is bortezomib and the histone deacetylase inhibitor is belinostat.

16. The method of claim 11, wherein the proteasome inhibitor is bortezomib and the histone deacetylase inhibitor is entinostat.

17. The method of claim 11, wherein the proteasome inhibitor is bortezomib and the histone deacetylase inhibitor is panobinostat.

18. The method of any one of claims 1 to 8, wherein the Network Brake is a proteasome inhibitor and an inhibitor of SP1 -class transcription factors.

19. The method of claim 18, wherein the proteasome inhibitor is bortezomib and the inhibitor of SP1 -class transcription factors is mithramycin.

20. The method of any one of claims 1 to 8, wherein the Network Brake is a histone deacetylase inhibitor (HDAC) and an Hsp90 inhibitor.

21. The method of claim 20, wherein the histone deacetylase inhibitor is vorinostat and the Hsp90 inhibitor is AUY922.

22. The method of any one of claims 1 to 8, wherein the Network Brake is a proteasome inhibitor and an HDAC-PI3K inhibitor.

23. The method of claim 22, wherein the proteasome inhibitor is bortezomib and the HDAC- PI3K inhibitor is CUDC-907.

24. The method of any one of claims 1 to 23, wherein the kinase inhibitor is afatinib, aflibercept, axitinib, bevacizumab, BEZ235, bosutinib, cabozantinib, cetuximab, crizotinib, dasatinib, erlotinib, everolimus, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, SU6656, sunitinib, tofacitinib, trametinib, trastuzumab, vandetanib, vemurafenib, or vismodegib.

25. The method of any one of claims 1 to 23, wherein the kinase inhibitor is sorafenib.

26. The method of any one of claims 1 to 23, wherein the kinase inhibitor is trametinib or erlotinib.

27. The method of any one of claims 1 to 26, wherein the Network Brake is administered to the patient concurrently with the kinase inhibitor.

28. The methods of any one of claims 1 to 26, wherein the Network Brake is administered to the patient prior to the administration of the kinase inhibitor.

29. A method for treating thyroid cancer to a human patient in need thereof, comprising: (a) administering to the patient bortezomib at a subtherapeutic dose and vorinostat at a

subtherapeutic dose; and (b) administering to the patient sorafenib at an effective dose.

30. The method of claim 29, wherein the bortezomib and vorniostat are administered to the patient prior to the administration of the sorafenib to the patient.

31. The method of claim 29, wherein the bortezomib and vorniostat are administered to the patient concurrently with the administration of the sorafenib to the patient.

32. The method of any one of claims 29 to 31, wherein the thyroid cancer is associated with a RET mutation.

33. A method for treating lung cancer to a patient in need thereof, comprising: (a) administering to the patient bortezomib at a subtherapeutic dose and vorinostat at a

subtherapeutic dose; and (b) administering to the patient trametinib at an effective dose.

34. The method of claim 33, wherein the lung cancer is associated with a Ras mutation.

35. The method of claim 33, wherein the lung cancer is a non-small cell lung cancer.

36. A method for treating liver cancer to a patient in need thereof, comprising: (a) administering to the patient bortezomib at a subtherapeutic dose and vorinostat at a

37. The method of claim 36, wherein the liver cancer is a heptocellular carcinoma.

38. The method of any one of claims 33 to 37, wherein the bortezomib and vorniostat are administered to the patient prior to the administration of the trametinib to the patient.

39. The method of any one of claims 33 to 37, wherein the bortezomib and vorniostat are administered to the patient concurrently with the administration of the trametinib to the patient.

40. A method for treating lung cancer to a patient in need thereof, comprising: (a) administering to the patient bortezomib at a subtherapeutic dose and vorinostat at a

subtherapeutic dose; and (b) administering to the patient erlotinib at an effective dose.

41. The method of claim 40, wherein the lung cancer is associated with a Ras mutation.

42. The method of claim 40, wherein the lung cancer is ErbB3 positive.

43. The method of claim 40, wherein the lung cancer is a non-small cell lung cancer.

44. The method of any one of claims 40 to 43, wherein the bortezomib and vorniostat are administered to the patient prior to the administration of the erlotinib to the patient.

45. The method of any one of claims 40 to 43, wherein the bortezomib and vorniostat are administered to the patient concurrently with the administration of the erlotinib to the patient.

46. A method for treating breast cancer to a patient in need thereof, comprising: (a) administering to the patient bortezomib at a subtherapeutic dose and vorinostat at a

subtherapeutic dose; and (b) administering to the patient BEZ235 at an effective dose.

47. The method of claim 46, wherein the bortezomib and vorniostat are administered to the patient prior to the administration of the BEZ235 to the patient.

48. The method of claim 46, wherein the bortezomib and vorniostat are administered to the patient concurrently with the administration of the BEZ235 to the patient.

49. The method of any one of claims 46 to 48, wherein the breast cancer is associated with a PI3K mutation.

50. The method of any one of claims 46 or 48, wherein the breast cancer is estrogen receptor positive (ER⁺).

51. A method for treating melanoma to a patient in need thereof, comprising: (a)

administering to the patient bortezomib at a subtherapeutic dose and vorinostat at a

subtherapeutic dose; and (b) administering to the patient vemurafenib at an effective dose.

52. The method of claim 51 , wherein the bortezomib and vorniostat are administered to the patient prior to the administration of the vemurafenib to the patient.

53. The method of claim 51 , wherein the bortezomib and vorniostat are administered to the patient concurrently with the administration of the vemeurafinib to the patient.

54. The method of claim 51 , wherein the melanoma is associated with a Raf mutation.

55. A method for screening compounds to select those having Network Brake activity, comprising:

(a) culturing a first population of cancer cells in the presence of a kinase inhibitor at a specific concentration for a period of time;

(b) culturing a second population of cancer cells of the same type in the presence of a test compound or a combination of test compounds at a test concentration(s), and the kinase inhibitor at the same specific concentration for said period of time, wherein the test concentration(s) is equivalent to a subtherapeutic dose; and

(c) analyzing the level of activity of a certain set of kinases in the first and second populations of cancer cells at the end of said period of time,

wherein a decrease in the overall, median, or mean activity of the kinases in the second population of cancer cells relative to the overall, median or mean activity of the same kinases in the first population of cancer cells indicates that the test compound or the combination of test compounds has Network Brake activity.

56. A method for screening compounds to select those having Network Brake activity, comprising:

(b) culturing a second population of cancer cells of the same type in the presence of a test compound or a combination of test compounds at a test concentration(s), and the kinase inhibitor at the same specific concentration for said period of time, wherein the test concentration (s) is equivalent to a subtherapeutic dose(s); and

(c) analyzing the level of phosphorylation of a set of proteins, which are indicative of the kinase activity of a set of kinases, in the first and second populations of cancer cells at the end of said period of time, wherein a decrease in the overall, median, or mean phosphorylation level of the set of proteins in the second population of cancer cells relative to the overall, median or mean phosphorylation level of the same set of proteins in the first population of cancer cells indicates that the test compound or the combination of test compounds has Network Brake activity.