WO2015183956A1 - Use of abl1 inhibitors with ampk activators for treating fumarate hydratase-deficient cancer - Google Patents

Abstract

The disclosure provides a method of treating cancer in a subject in need thereof or a subject having a hereditary cancer syndrome, wherein the subject has a germline fumarate hydratase gene mutation, comprising administering to the subject a therapeutically effective amount of an inhibitor of ABL1, e.g., vandetanib, and an activator of AMPK, e.g., metformin. The disclosure also provides a pharmaceutical composition and a kit comprising an inhibitor of ABL1 and an activator of AMPK.

Description

USE OF ABL1 INHIBITORS WITH AMPK ACTIVATORS FOR TREATING

FUMARATE HYDRATASE-DEFICIENT CANCER

CROSS-REFERENCE TO A RELATED APPLICATION

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 62/003,319, filed May 27, 2014, which is incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] Cancers depend on altered metabolic programs and on up-regulated stress response pathways necessary to cope with the consequences of deregulated metabolism. Utilizing genetically defined cancers with well-characterized metabolic adaptations provides a unique opportunity to identify novel therapeutic interventions. Hereditary kidney cancers provide models that are particularly well-suited for this purpose, as two types of kidney cancer are characterized by mutation of the TCA cycle genes, fumarate hydratase (FH) and succinate dehydrogenase (SDH), respectively. Inactivation of either enzyme disrupts mitochondrial respiration and promotes dependence on aerobic glycolysis, a characteristic of aggressive kidney cancer (Linehan et al., 2013; The Cancer Genome Atlas Research

Network, 2013). Mutations in FH are found in the germline of patients with hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a hereditary cancer syndrome in which affected individuals are at risk for developing cutaneous and uterine leiomyomas and a highly aggressive form of type 2 papillary kidney cancer (Grubb, III et al., 2007). No effective therapy is currently available for patients with advanced FH-deficient kidney cancer.

[0003] FH-deficient kidney cancers and their cell line models are highly glycolytic and display increased glucose dependence, lactate production, elevated levels of the

hypoxiastimulated transcription factor HIF l a and decreased activity of AMP-activated kinase (AMPK) (Yang et al., 2010; Tong et al., 201 1 ; Yang et al., 2012). This metabolic adaptation is, at least in part, a direct consequence of the intracellular accumulation of the

oncometabolite, fumarate (Isaacs et al., 2005; Frezza et al., 201 1 ) (Tong et al., 201 1 ). By inhibiting HIF prolyl hydroxylase, fumarate stabilizes HIF l a which leads to the transcription of multiple genes, including those that encode for glucose transporters 1 and 4 (Glutl , Glut4), and vascular endothelial growth factor VEGF.

[0004] Although increased HIF l a transcriptional activity supports rapid anabolic growth of HLRCC tumors (Koivunen et al., 2007; Isaacs et al., 2005), their high energetic demands and increased glycolytic activity cause redox homeostasis to become unbalanced due to elevated production of reactive oxygen species (ROS) (Sudarshan et al., 2009) (Sullivan et al., 2013). To survive this proteotoxic stress, FH-deficient kidney cancer cells utilize the oxidative branch of the pentose phosphate pathway, driving NADPH production and glutathione synthesis (Yang et al., 2013). Excess fumarate also stabilizes the master regulator of the antioxidant response, the transcription factor nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or NRF2) (Ooi et al., 201 1 ; Adam et al., 201 1). NRF2 activation in cancer has been reported to be either beneficial or detrimental depending on the context and/or the tumor type (Sporn and Liby, 2012). In HLRCC tumors, NRF2 activation appears to be critical for tumor growth and survival (Frezza et al., 201 1) (Ooi et al., 2011) (Adam et al, 2011). In addition, increased fumarate levels result in succinated glutathione, resulting in decreased NADPH levels and further accumulation of ROS (Sullivan et al., 2013).

[0005] Although FH mutation has been reported almost solely in HLRCC cancers, reduced FH activity has been found in other cancers, including clear cell kidney cancer (Sudarshan et al., 201 1), and induction of pseudo-hypoxia and deregulated redox homeostasis are common features of most aggressive epithelial cancers (Denko, 2008; Cairns et al., 2011). Although several studies have identified components of these pathways as potential molecular targets in FH-deficient tumors (Xie et al., 2009; Sourbier et al., 2010; Frezza et al., 201 1), the identification of an effective form of therapy for HLRCC cancer patients still remains elusive.

[0006] The foregoing shows that there is an unmet need for the treatment of cancers in patients with germline fumarate hydratase mutation.

BRIEF SUMMARY OF THE INVENTION

[0007] The invention provides a method of treating cancer in a subject in need thereof or a subject having a hereditary cancer syndrome, wherein the subject has a germline fumarate hydratase gene mutation, comprising administering to the subject a therapeutically effective amount of an inhibitor of ABL1 and an activator of AMPK.

[0008] The invention also provides a kit for treating cancer in a subject in need thereof or a subject having a hereditary cancer syndrome, wherein the subject has a germline fumarate hydratase gene mutation, comprising therapeutically effective amounts of an inhibitor of ABL1 and an activator of AMPK.

[0009] The invention further provides a pharmaceutical composition comprising an inhibitor of ABL1 , an activator of AMPK, and a pharmeutically acceptable carrier. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0010] Figure 1A illustrates cell viability following vandetanib treatment of FH-deficient cells (UO 262 and UOK268) and their paired molecularly restored counterparts

(UOK262WT and UOK268WT).

[0011] Figure IB illustrates the effect of vandetanib (5 nM) on UO 262 invasiveness monitored in real time using the xCELLingence platform.

[0012] Figure 1C illustrates HIFla expression in UOK262 and UOK268 cells as assessed by ELISA after vandetanib treatment or siRNA-mediated silencing of ABLl for 16 h in each case.

[0013] Figure ID shows that mTOR-mediated phosphorylation of S6K is decreased following vandetanib treatment and siRNA-mediated silencing of ABLl as visualized by immunoblotting. Phosphorylation of CRKII is used as a marker of ABLl activity.

Vandetanib (VAN) was used at 20 nM; imatinib (IMA) was used at 200 nM.

[0014] Figure IE shows ABLl kinase activity as determined by in vitro assay using ABLl immunoprecipitated from UOK262 or UO 268 cells. ABLl kinase activity was measured by quantifying substrate phosphorylation.

[0015] Figure 1 f shows ABLl kinase activity as determined by in vitro assay using purified ABLl protein. ABLl kinase activity was measured by quantifying substrate phosphorylation.

[0016] Figure 1G illustrates steady-sTate phosphorylation of ABLl in UOK262 and UOK268 cells following vandetanib treatment as visualized by immunoblotting.

[0017] Figure 2A illustrates the activation status in HLRCC tumor specimens as determined by immunoblotting for phosphorylated ABLl following immunoprecipitation of ABLl protein from tissue lysates. The intensity ratio of phosphorylated ABLl to total ABLl was assessed by densitometric analysis.

[0018] Figure 2B illustrates the ABLl activation status in a panel of cell lines as described for Figure 2A.

[0019] Figure 2C shows that overexpression of ABLl in UOK262 cells reversed vandetanib cytotoxicity when treated for 24 h with 50 nM vandatanib. VAN: vandetanib.

[0020] Figures 2D and 2E show that silencing of ABLl is cytotoxic for FH-deficient cells (Figure 2D) but not for UOK262WT cells (Figure 2E). [0021] Figure 2F shows the importance of ABLl for soft agar colony growth of FH- deficient cells as assessed by measuring the number of colonies visible 4 weeks after seeding with cells treated as shown. VAN, vandetanib; IMA, Imatinib.

1 0221 Figure 3A shows expression of the glucose transporters Glutl and Glut4 following vandetanib treatment (24 h) in UOK262 cells

[0023] Figure 3B shows glucose uptake as measured in UOK262 cells using the non- degradable fluorescent glucose analog 2-NBDG (20 μΜ) after vandetanib (VAN; 20 nM, 16 h) or imatinib (IMA; 200 nM, 16 h) treatment, or after silencing either ABLl or HIFla.

[0024] Figure 3C shows decreased lactate secretion after vandetanib treatment (20 nM, 16 h).

[0025] Figure 3D shows the effect of ABLl modulation on aerobic glycolysis as measured by recording the extracellular acidification rate (ECAR, a surrogate for lactate secretion) after silencing or over-expressing ABLl in UO 262 and HEK293, respectively.

[0026] Figure 4A shows the lactate/pyruvate ratio in mice harboring UOK262 xenografts before and 2 days after treatment with vandetanib as determined by C-hyperpolarized imaging..

[0027] Figure 4B shows growth of UOK262 xenografts treated once weekly with vehicle (DMSO.PBS) or vandetanib (100 mg/kg).

[0028] Figure 4C shows ABLl kinase activity (measured by quantifying ABLl phosphorylation status) in tumor tissue excised from vehicle- or vandetanib-treated mice.

[0029] Figure 5 A depicts basal in vitro ABLl kinase activity in UOK262WT and UOK262 cells.

[0030] Figure 5B shows the effect of intracellular fumarate accumulation on ABLl activity in HEK293 cells (DMF, dimethyl-fumarate; Fum, fumarate; 0.25 mM, 4 h).

[0031] Figure 5C shows the effect of ABLl activity on NRF2 nuclear/cytoplasmic distribution as assessed by immunoblotting of both nuclear and cytoplasmic extracts.

100321 Figure 5D shows NRF2 transcriptional activity as assessed in HEK293 cells following treatment with vandetanib (VAN, 50 nM, 4 h) and/or DMF (*: significance compared to CTL; #: significance compared to DMF-induced NRF2 transcriptional activity).

[0033] Figure 5E depicts the role of ABLl in regulating NRF2 transcriptional activity as assessed as in Figure 5D following transient silencing of ABLl using siRNA.

[0034] Figure 5F depicts the effect of NAC pre-treatment on the viability of vandetanib- treated UOK262 cells (50 nM, 16 h). [0035] Figure 5G illustrates that NAC (5 mM) and vandetanib (20 niM) equivalently reduce HIFla protein expression within 4 h.

[0036] Figure 5H illustrates NQOl expression in UOK262 cells after vandetanib or ABL1 silencing.

[0037] Figure 6A shows NRF2 transcriptional activity as assessed in HEK293 cells following treatment with the AMPK activators metformin (METF; 1 mM, 6 h), and AICAR (50 μΜ, 6 h) in the presence of DMF (0.25 mM).

[0038] Figure 6B illustrates that metformin and SIRT1 knockdown exert opposing effects on NRF2 acetylation status as visualized by immunoblotting following immunoprecipitation of NRF2 using an antibody recognizing the acetylation of lysine residues ("AcK").

[0039] Figure 6C shows the effect of vandetanib (5 nM) and metformin (5 μΜ) treatment, singly or together, on NRF2 transcriptional activity.

[0040] Figure 6D shows the effect of combining metformin (METF, 5 μΜ) and vandetanib (VAN, 5 nM) on UOK262 viability after 24 h.

[0041] Figure 6E shows the effect of metformin and vandetanib alone or in combination on UOK262 xenograft growth in mice. This study used 1/10^th of the vandetanib dose used in the experiment shown in Figures 4A-C.

[0042] Figure 6F depicts averaged data from two independent experiments (8 animals per group).

[0043] Figure 6G shows the percent overall survival of mice receiving treatments described in Figures 6E and 6F.

[0044] Figure 7A illustrates the HIFla mRNA level in UOK262 cells after vandetanib treatment.

[0045] Figure 7B shows that the decrease in HIFl a protein expression is not rescued by proteasome inhibition with Velcade.

[0046] Figure 7C illustrates the efficiency of silencing ABL1 with siRNA as visualized by immunoblotting.

[0047] Figure 7D shows regulation of the mTOR/ HIFl a by ABL1 in HEK293 cells transiently overexpressing ABL1 as visualized by immunoblotting.

[0048] Figure 8 A illustrates the activation status of ABL1 by immunoblotting in normal (N) and leiomyoma (L) skin samples obtained from HLRCC patients.

[0049] Figure 8B illustrates the activation status of ABL1 in normal uterine tissues compared to uterine leiomyomas. [0050] Figure 8C shows the silencing efficiency of ABLl in UOK262WT cells as visualized by immunoblotting.

[0051] Figure 8D shows UOK262 invasiveness after ABLl silencing or vandetanib treatment.

[0052] Figure 9A illustrates the silencing efficiency of / HIFla siRNA in UOK262 cells as visualized by immunoblotting.

[0053] Figure 9B illustrates the effect of vandetanib on the phosphorylation status of ABLl in the VHL -/- clear cell kidney cancer line UOL150 as visualized by immunoblotting.

[0054] Figure 9C shows the effect of vandetanib (1 μΜ, 16 h) on glucose uptake in UOK150 cells.

[0055] Figure 10A illustrates expression of HIFl in excised xenografts.

[0056] Figure 10B illustrates expression of Glutl in excised xenografts.

[0057] Figure IOC shows the phosphorylation status of S6K in excised xenografts.

[0058] Figure 10D shows VEGF expression in protein extracts from tumor xenografts as assessed by immunoblotting.

[0059] Figure 10E illustrates apoptosis as measured by terminal deoxynucleotidyl transferase (Tdt) immunohistochemistry in tumor xenografts excised from mice treated with vehicle or vandetanib.

[0060] Figure 10F shows the phosphorylation status of EGFR. HER2. HER3, and VDGFR2 in tumor tissue extracts following treatment with vandetanib or vehicle.

[0061] Figure 1 1 A shows the effect of NAC (5 mM, 24h) on pAbl as visualized by immunoblotting.

[0062] Figure 1 I B shows the effect of ABLl overexpression on DMF toxicity in

HEK293 cells.

[0063] Figure 1 1C illustrates representative immunohistochemistry of NQOl expression in clear cell renal cell carcinoma (VHL-/-), papillary type I kidney tumor (Met mutation), and HLRCC kidney tumor specimens (FH-/-).

[0064] Figure 1 ID is a magnification of the HLRCC image shown in Figure 1 1C.

[0065] Figure 1 IE shows NQOl expression in tissue lysates from HLRCC xemografts excised from mice treated with vehicle or vandetanib.

[0066] Figure 12A shows NRF2 trascriptional activity as assessed in HEK293 cells following treatment with nicotinamide (NAM, ImM, 6h), silencing of SIRT1 , or treatment with vandetanib (VAN, 20 nM), in presence of DMF (0.25 mM). [0067] Figure 12B shows the effect of SIRT1 knockdown and metformin treatment (METF, ImM) on NQOl expression in UOK262 cells as visualized by immunoblotting.

[0068] Figure 12C shows the effect of combining metformin (METF, 5 μΜ) with vandetanib (VAN, 5 nM) on NQOl expression in UOK262 cells.

[0069] Figure 12D shows the effect of metformin (METF, 5 μΜ) on the viability of UO 262 cells treated with ABL1 siRNA for 24 h.

[0070] Figure 12E shows the effect of AICAR (5 μΜ) on the viability of UOK262 cells treated with ABL1 siRNA for 24 h.

[0071] Figure 12F shows the effect of metformin (5 μΜ) on the sensitivity of UOK262 cells to imatinib.

[0072] Figures 13A and 13B illustrate data from the TCGA database showing the percentage of tumors bearing a mutation within the ABL1-NRF2 axis, including ABL1,

NFE2L2, KEAP1, CUL3, SIRT1, or FH mutations, per tumor type.

[0073] Figure 13C shows the activation of the ABL1-NRF2 axis as assessed by immunoblotting in a panel of non-small cell lung cancers-derived cell lines.

[0074] Figure 13D shows a viability assay following siRNA-mediated silencing of ABL1 or vandetanib treatment (36 h, vandetanib 100 nM). Only the cell line lacking NQOl expression (i.e., NCI-H292), which is an indicator of NRF2 activity, is insensitive to either treatment.

[0075] Figure 14A shows cell viability of UOK262 infected with murine ABL constructs (wild type (WT), kinase-dead ( 290M), kinase inhibitor-resistant (T315I), and constitutively active ABL (P 1311)) in the presence and absence of vandetanib. Scrambled miRNA

(miSCR) was used as an miRNA control and empty pBABE vector (BABE) served as the control for infection with the various murine ABL constructs.

[0076] Figure 14B depicts an immunoblot showing reduction in ABL1 protein expression 72 hr post miABL infection and subsequent expression level of re-introduced murine ABL proteins.

100771 Figures 15A-15D depict dose-response curves for cell viability in medium lacking pyruvate after 48 hr treatment with various concentrations of several different ABL1 inhibitors in UOK262 and UOK262WT cells. VAN = vandetanib, PON = ponatinib, DASA = dasatinib, NILO = nilitinib.

[0078] Figures 16A-16D depict dose-response curves for cell viability of UOK262 cells in medium with pymvate ("PYR") and without pyruvate "NO PYR") after 48 hr treatment with several ABLl inhibitors. VAN = vandetanib, PON = ponatinib, DASA = dasatinib, NILO = nilitinib.

[0079] Figure 17 shows ABLl kinase activity in xenograft tissues on day post-treatment with vehicle and vandetanib.

[0080] Figure 18 shows the NRF2 nuclear staining for UO 262 cells treated with vandatenib as compared with control in graphical form.

DETAILED DESCRIPTION OF THE INVENTION

[0081] The invention provides a method of treating cancer in a subject in need thereof or a subject having a hereditary cancer syndrome, wherein the subject has a germline fumarate hydratase gene mutation, comprising administering to the subject a therapeutically effective amount of an inhibitor of ABLl and an activator of AMPK.

[0082] The inhibitor of ABLl can be any suitable inhibitor of ABLl . In certain embodiments, the inhibitor of ABLl is a tyrosine kinase inhibitor. Non-limiting examples of suitable tyrosine kinase inhibitors include vandetanib, imatinib, nilotinib, ponatinib and dasatinib. In a preferred embodiment, the inhibitor of ABLl is vandetanib.

[0083] The activator of AMPK can be any suitable activator. Non-limiting examples of suitable activators of AMPK include metformin, buformin, phenformin, and AICAR (5- amino-l-P-D-ribofuranosyl-imidazole-4-carboxamide). In a preferred embodiment, the activator of AMPK is metformin.

[0084] The term "ABLl " refers to a tyrosine kinase encoded by the Abelson (ABLl) proto-oncogene.

[0085] The term "AMPK" refers to 5' AMP-activated protein kinase or AMPK or 5' adenosine monophosphate-activated protein kinase.

[0086] In certain preferred embodiments, the inhibitor of ABLl and the activator of AMPK are administered in amounts that are lower than therapeutically effective amounts of the inhibitor of ABLl and the activator of AMPK when administered separately.

[0087] In an embodiment, the cancer is lung squamous cell carcinoma, lung

adenocarcinoma, bladder urothelial carcinoma, head and neck squamous cell carcinoma, papillary renal carcinoma, or clear cell clear renal carcinoma.

[0088] In a preferred embodiment, the subject has a hereditary cancer syndrome.

[0089] In a more preferred embodiment, the hereditary cancer syndrome is hereditary leiomyomatosis and renal cell carcinoma (HLRCC). [0090] In certain embodiments, the subject has a least one kidney tumor.

[0091] In certain embodiments, the treating results in regression of the at least one kidney tumor.

[0092] In certain embodiments, the subject has at least one tumor bearing a mutation within the aB l-NRL2 axis. In certain embodiments, the mutation is an ABL1, NFE2L2, KEAP1, COLS, SIRT1, or an FH mutation. In certain embodiments, the tumor is associated with lung squamous cell carcinoma, lung adenocarcinoma, bladder urothelial carcinoma, head and neck squamous cell carcinoma, papillary renal carcinoma, or clear cell clear renal carcinoma.

[0093] "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. As used herein, the term "ameliorating," with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease. Treatment of cancer can be evidenced, for example, by a reduction in tumor size, a reduction in tumor burden, a reduction in clinical symptoms resulting from the cancer, increase in longevity, increase in tumor free survival time, and the like. Treating in embodiments, can include inhibiting the development or progression of a cancer, in particular kidney cancer.

[0094] The terms "administer," "administering", "administration," and the like, as used herein, refer to the methods that may be used to enable delivery of agents or compositions to the desired site of biological action. These methods include, but are not limited to oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular or infusion), topical and rectal administration. Administration techniques that are optionally employed with the agents and methods described herein, include e.g., as discussed in Goodman and Gilman, The Pharmacological Basis of Therapeutics; Pergamon; and Remington's Pharmaceutical Sciences , Mack

Publishing Co., Easton, Pa. In certain embodiments, the agents and compositions described herein are administered orally.

[0095] By the term "coadminister" is meant that each of the inhibitor of ABL1 and the activator of AMPK are administered during a time frame wherein the respective periods of biological activity overlap. Thus, the term includes sequential as well as coextensive administration of the inhibitor of ABL1 and the activator of AMP . The inhibitor of ABL1 and the activator of AMPK can be administered simultaneously, separately (chronologically staggered), cyclically, or sequentially and in any order, e.g., before or after.

[0096] One skilled in the art will appreciate that any suitable methods of administering the inhibitor of ABL1 and the activator of AMPK to a human for the treatment or prevention of disease states, in particular, kidney cancer, can be used. Although more than one route can be used to administer the inhibitor of ABL1 and the activator of AMPK, a particular route can provide a more immediate and more effective reaction than another route. Accordingly, the described methods are merely exemplary and are in no way limiting.

[0097] The doses of the inhibitor of ABL1 and the activator of AMPK administered to a mammal, particularly, a human, in accordance with the present invention should be sufficient to effect a desired response. Such a response includes reversal or prevention of the adverse effects of kidney cancer. One skilled in the art will recognize that dosage will depend upon a variety of factors, including the age, condition, and body weight of the human, as well as the source, particular type of the disease, and extent of the disease in the human. The size of the doses will also be determined by the route, timing and frequency of administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular compound and the desired physiological effect. It will be appreciated by one of skill in the art that various conditions or disease states may require prolonged treatment involving multiple administrations.

[0098] Suitable doses and dosage regimens can be determined by conventional range- finding techniques known to those of ordinary skill in the art. Generally, treatment is initiated with smaller dosages that are less than the optimum dose of the compound.

Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. The present inventive method typically will involve the administration of about 0.1 to about 300 rag of each of the compounds described above per kg body weight of the animal or mammal.

[0099] The therapeutically effective amount of the inhibitor of ABL1 and the activator of AMPK administered can vary depending upon the desired effects and the factors noted above. Typically, dosages of each agent will be between 0.01 mg/kg and 250 mg/kg of the subject's body weight, and more typically between about 0.05 mg/kg and 100 mg/kg, such as from about 0.2 to about 80 mg/kg, from about 5 to about 40 mg/kg or from about 10 to about 30 mg/kg of the subject's body weight. Thus, unit dosage forms can be formulated based upon the suitable ranges recited above and the subject's body weight. The term "unit dosage form" as used herein refers to a physically discrete unit of therapeutic agent appropriate for the subject to be treated.

[0100] Alternatively, dosages are calculated based on body surface area and from about 1 mg/m² to about 200 mg/m², such as from about 5 mg/m² to about 100 mg/m² will be administered to the subject per day. In particular embodiments, administration of the therapeutically effective amount of the inhibitor of ABL1 and the activator of AMPK involves administering to the subject from about 5 mg/m² to about 50 mg/m², such as from about 10 mg/m² to about 40 mg/m² per day. Thus, unit dosage forms also can be calculated using a subject's body surface area based on the suitable ranges recited above and the desired dosing schedule.

[0101] In some embodiments, the inhibitor of ABL1 and the activator of AMPK are administered to inhibit the growth of a target cell. In some embodiments, the growth of a target cell is about 1%, e.g., about 2%, about 3%, about 4%, about 5%, about 10%, about 20%), about 30%, about 40%, about 50%, about 60%o, about 70%, about 80%o, about 90%, or about 100%), inhibited relative to the growth rate preceding administration of a compound and/or composition disclosed herein.

[0102] In some embodiments, the inhibitor of ABL1 and the activator of AMPK are is administered to degrade a target cell. In some embodiments, a compound and/or composition disclosed herein is administered to degrade a plurality of target cells. In some embodiments, 1 %, e.g., about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, of the target cells are degraded. In some embodiments, essentially all of the target cells are degraded.

[0103] In some embodiments, the inhibitor of ABL1 and the activator of AMPK are is administered to kill a target cell. In some embodiments, a compound and/or composition disclosed herein is administered to kill a plurality of target cells. In some embodiments, 1 %, e.g., about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, of the target cells are killed. [0104] In some embodiments, the inhibitor of ABLl and the activator of AMPK are is administered to reduce the size of a tumor, inhibit tumor growth, reduce metastasis or prevent metastasis in an individual in need thereof.

[0105] In some embodiments, the size of a tumor is reduced. In some embodiments, the size of a tumor is reduced by at least 1%, e.g., about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%.

[0106] In some embodiments, tumor growth is inhibited. In some embodiments, tumor growth is inhibited by at least 1 %, e.g., about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%, relative to the growth rate preceding administration of a compound and/or composition disclosed herein.

[0107] In some embodiments, metastasis is inhibited. In some embodiments, metastasis is inhibited by at least 1 %, e.g., about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%, relative to the growth rate preceding administration of a compound and/or composition disclosed herein. In some embodiments, metastasis is inhibited by at least 2% relative to the growth rate preceding administration of a compound and/or composition disclosed herein.

[0108] In certain embodiments, the combination of the inhibitor of ABLl and the activator of AMPK advantageously allows for the use of a lower dose of the inhibitor of ABLl than a clinically effective dose of the inhibitor of ABLl when used alone.

[0109] In an embodiment, the invention provides a pharmaceutical composition comprising a combination of an inhibitor of ABLl or a salt thereof, an activator of AMPK or a salt thereof, and a pharmaceutically acceptable carrier.

[01 10] It is preferred that the pharmaceutically acceptable carrier be one that is chemically inert to the active compounds and one that has no detrimental side effects or toxicity under the conditions of use.

[0111] The choice of carrier will be determined in part by the particular inhibitor of ABLl and particular activator of AMPK chosen, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention. The following formulations for oral, aerosol, nasal, pulmonary, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intratumoral, topical, rectal, and vaginal administration are merely exemplary and are in no way limiting.

[0112] The pharmaceutical composition can be administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration that comprise a solution or suspension of the inhibitor of ABL1 and activator or salts thereof dissolved or suspended in an acceptable carrier suitable for parenteral administration, including aqueous and non-aqueous isotonic sterile injection solutions.

[0113] Overall, the requirements for effective pharmaceutical carriers for parenteral compositions are well known to those of ordinary skill in the art. See, e.g., Banker and Chalmers, eds., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company,

Philadelphia, pp. 238-250 (1982), and Toissel, ASHP Handbook on Injectable Drugs, 4th ed., pp. 622-630 (1986). Such solutions can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The inhibitor of ABL1 and activator of AMPK or salts thereof may be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol,

dimethylsulfoxide, glycerol ketals, such as 2,2-dimethyl-l ,3-dioxolane-4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a phamiaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

|01 14] Oils useful in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils useful in such formulations include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0115] Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-beta-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

[0116] The parenteral formulations can contain preservatives and buffers. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0117] Topical formulations, including those that are useful for transdermal drug release, are well-known to those of skill in the art and are suitable in the context of the invention for application to skin. Topically applied compositions are generally in the form of liquids, creams, pastes, lotions and gels. Topical administration includes application to the oral mucosa, which includes the oral cavity, oral epithelium, palate, gingival, and the nasal mucosa. In some embodiments, the composition contains at least one active component and a suitable vehicle or carrier. It may also contain other components, such as an anti-irritant. The carrier can be a liquid, solid or semi-solid. In embodiments, the composition is an aqueous solution. Alternatively, the composition can be a dispersion, emulsion, gel, lotion or cream vehicle for the various components. In one embodiment, the primary vehicle is water or a biocompatible solvent that is substantially neutral or that has been rendered substantially neutral. The liquid vehicle can include other materials, such as buffers, alcohols, glycerin, and mineral oils with various emulsifiers or dispersing agents as known in the art to obtain the desired pH, consistency and viscosity. It is possible that the compositions can be produced as solids, such as powders or granules. The solids can be applied directly or dissolved in water or a biocompatible solvent prior to use to form a solution that is substantially neutral or that has been rendered substantially neutral and that can then be applied to the target site. In embodiments of the invention, the vehicle for topical application to the skin can include water, buffered solutions, various alcohols, glycols such as glycerin, lipid materials such as fatty acids, mineral oils, phosphoglycerides, collagen, gelatin and silicone based materials.

[0118] Formulations suitable for oral administration can consist of (a) liquid solutions, such as a therapeutically effective amount of the inventive compound dissolved in diluents, such as water, saline, or orange juice, (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredients, as solids or granules, (c) powders, (d) suspensions in an appropriate liquid, and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a

pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.

[0119] The inhibitor of ABL1 and activator of AMP or salts thereof, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. The compounds are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of active compound are 0.01 %- 20% by weight, preferably 1 %-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such surfactants are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1 %-20% by weight of the composition, preferably 0.25%-5%. The balance of the composition is ordinarily propellant. A carrier can also be included as desired, e.g., lecithin for intranasal delivery. These aerosol formulations can be placed into acceptable pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non- pressured preparations, such as in a nebulizer or an atomizer. Such spray formulations may be used to spray mucosa.

[0120] Additionally, the inhibitor of ABLl and activator of AMPK or salts thereof may be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.

[0121] It will be appreciated by one of ordinary skill in the art that, in addition to the aforedescribed pharmaceutical compositions, the inhibitor of ABLl and activator of AMPK or salts thereof may be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes. Liposomes serve to target the compounds to a particular tissue, such as lymphoid tissue or cancerous hepatic cells. Liposomes can also be used to increase the half-life of the inhibitor of ABLl and activator of AMPK or salts thereof. Liposomes useful in the present invention include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the active agent to be delivered is incorporated as part of a liposome, alone or in conjunction with a suitable chemotherapeutic agent. Thus, liposomes filled with the inhibitor of ABLl and activator of AMPK or salts thereof, can be directed to the site of a specific tissue type, hepatic cells, for example, where the liposomes then deliver the selected compositions. Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, for example, liposome size and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9, 467 (1980), and U.S. Patents 4,235,871 , 4,501 ,728, 4,837,028, and 5,019,369. For targeting to the cells of a particular tissue type, a ligand to be incorporated into the liposome can include, for example, antibodies or fragments thereof specific for cell surface determinants of the targeted tissue type. A liposome suspension containing a compound or salt of the present invention may be administered intravenously, locally, topically, etc. in a dose that varies according to the mode of administration, the agent being delivered, and the stage of disease being treated.

[0122] In an embodiment, the invention provides a kit for treating kidney cancer in a subject in need thereof, wherein the subject has a germline fumarate hydratase gene mutation, comprising therapeutically effective amounts of an inhibitor of ABL1 and an activator of AMP . In certain embodiments, the kit further includes instructions for use.

[0123] Here, it has been shown that ABL1 is indirectly activated by intracellular fumarate (not a consequence of succination, data not shown) and that the kinase is an important, SIRT1 -independent determinant of NRF2 nuclear translocation and optimal transcriptional activity both in vitro and in vivo (Figure 7).

[0124] The sensitivity of NRF2 to AMPK activation is likely a consequence of AMPK- dependent SIRT1 activation, since metformin decreased nicotinamide-sensitive NRF2 acetylation. Notably, in an HLRCC xenograft model, inclusion of metformin (at a clinically achievable dose) reduced the effective vandetanib concentration by 90% (compared to single agent vandetanib administration). Weekly administration of this drug combination for 8 weeks caused tumor regression in 100% of treated mice and extended tumor- free survival for more than one year after cessation of treatment.

[0125] The data set forth herein show that ABL1 is a critical, pharmacologically tractable modulator of the cellular response to excess fumarate accumulation. Inhibiting this kinase provides a clinically viable strategy to simultaneously interfere with aerobic glycolysis and the anti-oxidant response pathway, upon which highly glycolytic HLRCC tumors depend for survival.

[0126] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0127] Methods

[0128] Cell lines and cell culture. UOK262, UOK268, UOK262WT, UOK268WT, and UOK150 cell lines were established in the Urologic Oncology Branch from surgically resected tumor specimens (National Cancer Institute, Bethesda, MD) (Yang et al., 2010; Anglard et al., 1992; Yang et al., 2012). All other cell lines were purchased from ATCC (Manassas, VA). Cells were cultured in high glucose DMEM without pyruvate supplemented with 10% FBS. The cells were harvested or treated when they reached 70-80% confluence. [0129] Chemical agents. Vandetanib was generously provided by Astra Zeneca. PTHrP- neutralizing antibody was from Bachem (Torrance, CA). All other compounds used were from Sigma-Aldrich (St. Louis, MO) or Selleck Chemicals (Houston, TX).

[0130] Cell viability. Cell viability was measured using a cytotoxicity assay kit purchased from Promega Biosciences, Inc. (San Luis Obispo, CA), following the

manufacturer's protocol and as previously described (Sourbier et al., 2010).

[0131] Invasion assay. Invasiveness of UOK262 cells was evaluated as previously described (Tong et al., 201 1 ; Yang et al., 2010) using the RT-CIM™ system (Acea

Biosciences, San Diego, CA). Cells were cultured overnight without serum prior to assay.

Invasion was monitored in real time for several days in a humidified incubator at 37°C and 5

% C02.

[0132] Immunoblotting. Ten to twenty micrograms of protein were loaded in 4-20% polyacrylamide gels (Biorad, Hercules, CA). After electrophoresis, proteins were transferred to PVDF membranes, blocked with 5% fat-free milk for at least lh, and incubated with primary antibodies overnight at 4°C. After several washes with TBS-Tween, blots were incubated with horseradish peroxidase-linked secondary antibodies (Sigma-Aldrich) for l -2h before development with the ECL protein detection system (Thermo Fisher Scientific, Rockford, IL). Rabbit antibodies against CREB (#9197), β-actin (#4970), ABL1 (#2862), p- Abl (Y412) (#2865), pCRKII and CRKII (#3491 and 3492), pAMP and total AMPK (#2535 and 2603), phospho and total S6K (#9208, 9202), NRF2 (#12721 ), and mouse antibodies against acetylated-lysine (#9681 ), NQOl (#3187) and a-tubulin (#3873) were from Cell Signaling Technology, Inc (Danvers, MA). Mouse HIFl a antibody was from BD Transduction Labs (#610958; San Jose, CA), Glutl (#GTX15309) and p53 (#GTX70216) antibodies were from GeneTex, Inc. (Irvine, CA). All antibodies were used at 1 : 1000 dilution.

[0133] ABL1 kinase activity assay. Five nanograms of purified ABL1 protein and the peptide substrate paxillin were incubated for lh at 30°C with 10 μΜ ATP in kinase assay buffer. The reaction was stopped by adding denaturing sample buffer and phosphorylation of the recognition motif I/V/L-Y-X-X-P/F was assessed by immunoblot analysis.

[0134] Colony formation assay. Anchorage-independent colony formation was assessed using soft-agar as previously described (Sourbier et al., 2012) with the following

modifications. Two layered agarose gels of 0.5% (bottom) and 0.4% (top) low melting agarose were poured into 60 mm dishes. Three hundred thousand UOK262 cells were added to the top layer and cultured until they formed colonies. Colonies with a diameter of > 0.1 mm were counted in 5 random high-power fields using a phase contrast microscope.

[0135] Glucose uptake assay. Glucose uptake was measured using the fluorescent D- glucose analog 2-[N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2- NBDG) as previously described (O'Neil et al., 2005). Briefly, 5,000 cells were seeded in black-well 96-well plates. After treatment as indicated in results or figure legends, cells were incubated in KREB buffer containing lg/L glucose in presence or absence of 2-NBDG (20 μΜ). After 20 minutes incubation and several washes, uptake of 2-NBDG was measured by fluorescence spectrometry.

[0136] Lactate secretion assay. Spent media from cells were collected after treatment as indicated. After concentration of the media (Amicon ultracentrifugal units, Millipore, Temecula, CA), 20 iL of the samples were loaded in a 96-well plate and the amount of lactate contained was measured using the lactate secretion assay kit from BioAssay Systems (Hayward, CA), following the manufacturer's protocol.

[0137] Measurements of ExtraCellular Acidification Rate (ECAR). Extracellular acidification rates (ECAR) were measured using the XF96 Extracellular Flux Analyzer from Seahorse Bioscience (Chicopee, MA) using XF96 microplates as previously described (Yang et al., 2010). Briefly, cells were seeded at 30,000 per well and treated as described in the figure legends. Twenty-four hours later, culture media were removed from the XF culture plates, wells were washed three times and cells were incubated with bicarbonate-free DMEM supplemented with glucose (4.5 g/L) and sodium pyruvate (2 mM) (pH 7.4 at 37°C).

[0138] Animal study. Animal experiments were performed in accordance with the guidelines of the Animal Care and Use Committee of the National Institutes of Health. For the first experiment, 16 female athymic nude mice (Taconic, Germantown, NY) were injected in the right flank with 4 million UOK262 cells diluted in an equal mix of PBS/matrigel (v/v; BD Bioscience, San Jose, CA). Six weeks after injection, tumor volume reached 100 mm³. Mice were randomized into two groups of eight mice each. One group was treated with vandetanib (l OOmg/kg once weekly by intraperitoneal (i.p.) injection) and the second group was treated vehicle (PBS/DMSO) following an identical schedule. When tumors from the vehicle-treated mice reached 1.5 cm , the experiment was stopped and all tumors were excised and frozen or paraffin-embedded for further studies. Blood was also collected at this time for measurement of secreted VEGF-A. For the second experiment, 40 female athymic nude mice were injected with 4 million UOK262 cells as described above. When tumor volumes reached 100 mm , mice were randomized into 4 groups of 8 mice. One group was treated with vandetanib alone (lOmg/kg once weekly, i.p.), a second group was treated vehicle (PBS/DMSO) following an identical schedule, a third group was treated with metformin alone (5mg/kg once weekly, i.p.) and the fourth group was treated with both metformin and vandetanib simultaneously (once weekly, i.p.). When tumors in vehicle- treated mice reached 1.5 cm , all treatments were stopped; mice in the first 3 groups were sacrificed and tumors were excised for further study. Mice that had been treated with both metformin and vandetanib were monitored for long-term survival without further treatment.

[0139] RTK protein array kit. Proteins were extracted from tumor xenografts using the lysis buffer provided by the RTK array kit (Cell Signaling Technology Inc, Danvers, MA). EGFR family and VEGF receptor phosphorylation status was assessed following the manufacturer's protocol and quantified using a densitometer (GS-800, Bio-Rad, Hercules, CA).

[0140] Measurement of secreted human VEGF-A. The amount of Human VEGFA secreted in spent media or in mouse plasma was measured using adapted MesoScale

Discovery plates (MesoScale Discovery, Gaithersburg, MD), according to the manufacturer's protocol. Briefly, samples (20 μί) were incubated for 2h in a 96-well plate pre-coated with the capture VEGF antibody, and in the presence of a sulfo-tag secondary antibody. After five washes, the plates were read using an MS2400 machine (MesoScale Discovery).

[0141] Immunohistochemistry. Five-micron tissue sections cut from formalin-fixed, paraffin embedded (FFPE) samples were utilized for immunohistochemical analysis performed by the NCI Pathology/Histotechnology Laboratory (National Cancer Institute, Frederick, MD) as described on their web-site (http://web.ncifcrf.gov/rtp/lasp/phl/immuno/). Apoptotic cells were visualized using ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Millipore, Billerica, MA) following the manufacturer's protocol and as previously described (Sourbier et al., 2010). The percentage of positive nuclei over the total number of tumor cells was obtained from five to ten high-power fields. Subcellular localization of immunostaining was also evaluated.

[0142] 13 C MR1 of hyperpolarized 13 C-labeled pyruvate metabolism. Samples of [1- 1 3 C] pyruvic acid (30 \L) containing 15 mM triarylmethyl radical (TAM) and 2.5 mM gadolium chelate ProHance (Bracco Diagnostics, Milano, Italy) were polarized at 3.35 T and 1.4 K in a Hypersense DNP Polarizer (Oxford Instruments), according to the manufacturer's instructions. After 40-60 min, the hyperpolarized sample was rapidly dissolved in 4.5 mL of a superheated alkaline buffer comprising 40 mM 4-(2-hydroxyethyl)-l - piperazineethanesulfonic acid (HEPES), 30 mM NaCl and 100 mg/L

ethylendiaminetetraacetic acid (EDTA). NaOH was added to the dissolution buffer to adjust pH to 7.4 after mixture with [1- 13 C] pyruvic acid. Hyperpolarized [1- 13 C] pyruvate solution (12 body weight) was intravenously injected through a catheter placed in the tail vein of the mouse. Hyperpolarized ¹³C MRI studies were performed on a 7 T scanner (Bruker Bio- Spin MRI GmbH) using a 17 mm home-built ¹³C solenoid coil placed inside of a saddle coil for ^lH. The ¹³C two-dimensional spectroscopic images were acquired 30 seconds after the start of pyruvate injection from a 28 x 28 mm field of view in a 8 mm coronal slice through the tumor, with matrix size of 16 x 16, spectral width of 8 kHz, repetition time (TR) 78 ms, 0.2 ms Gaussian excitation pulse with a flip angle of 10°. The total time required to acquire an image was 20 seconds.

[0143] ATP assay. ATP levels were determined using the ATP Lite assay (PerkinElmer, Shelton, Connecticut), following the manufacturer's protocol.

[0144] NRF2 reporter assay. NRF2 transcriptional activity was measured using the pGL4.37[luc2/ARE/Hygro] vector (Promega, Madison, WI), following the manufacturer protocol. Briefly, 5000 HEK293 cells were plated in white-clear view plates (Perkin-Elmer) and were transfected the following day with 100 ng of DNA per well using XTreme Gene HP transfection reagent (Roche Applied Science, Indianapolis, IN). After 24 h, cells were treated as indicated in Figure Legends. Luminescence was measured as an indicator of NRF2 transcriptional activity using the One-Glo Luciferase Assay System (Promega).

[0145] Statistics. All values are expressed as mean ± standard error. All experiments were performed three times, with exception of the animal study which was performed two times. Values were compared using the Student-Newman-Keul's test. P < 0.05 was considered significant.

EXAMPLE 1

[0146] This example demonstrates that vandetanib is highly cytotoxic for H-deficient HLRCC kidney cancer cells.

[0147] In order to uncover novel therapeutic strategies for patients with highly aggressive HLRCC-associated kidney cancer, the HLRCC-derived UOK262 cell line was utilized to screen a panel of 17 agents targeting diverse signaling pathways (Table 1). The tyrosine kinase inhibitor vandetanib (IC₅₀ = 16 nM) proved by far to be the most potent compound tested. Vandetanib displayed synthetic lethality for FH-deficient cells, since stable reintroduction of wild-type FH into two independently derived HLRCC cell lines (UOK262 and UO 268) abrogated its cytotoxicity (Figure 1A).

Table 1

*neither IC50 nor GI50 was reached

[0148] It was previously reported that HIFl a expression is necessary to maintain the invasive phenotype of HLRCC cells (Tong et al., 201 1). Vandetanib fully inhibited the invasiveness of UOK262 cells (Figure IB) and markedly decreased their HIFl a protein expression to levels seen in wild-type FH-restored cells (Figure 1C), but its mechanism of action involved neither protein degradation nor modulation of HIFl a mRNA levels (Figures 7A and 7B). These data suggested that vandetanib may affect HIFl a translation, a process regulated by the protein kinase mTOR (Powis and Kirkpatrick, 2004). Indeed, it was found that steady-state phosphorylation of S6 kinase, a downstream target of mTOR, was markedly decreased following vandetanib treatment (Figure ID).

[0149] Since ABLl is reported to regulate mTOR (Cilloni and Saglio, 2012; Kim et al., 2005; Markova et al., 2010) and vandetanib potently inhibits ABLl in vitro (Davis et al., 2011 ; Karaman et al., 2008), it was a question if vandetanib-mediated inhibition of the mTOR/HIFla pathway might be a consequence of ABLl inhibition. Using UOK262 cells in which ABLl was silenced with siRNA and HEK293 cells in which ABLl was transiently over-expressed, it was confirmed that reduced expression of ABLl , like vandetanib treatment, inhibited HIFl expression and mTOR activity, while transient ABLl over- expression upregulated both mTOR activity and HIF-dependent gene expression (Figures 1 C and ID and Figures 7C and 7D). Next, it was found that vandetanib and the ABLl inhibitor imatinib both inhibited the in vitro kinase activity of endogenous ABLl that was

immunopurified from both FH-deficient renal cell carcinoma cell lines (UOK262 and UOK268), as well as the activity of purified ABLl protein (Figure IE and IF, respectively). Notably, and in agreement with other reports (Davis et al., 2011 ; Karaman et al., 2008), the data demonstrate vandetanib to be a significantly more potent ABLl inhibitor than is imatinib (IC₅₀ = 16 nM and 200 nM, respectively). These data were confirmed by assessing the phosphorylation status of CRKII, a substrate of ABLl (Figure ID), and by assessing the phosphorylation status of ABLl immunopurified from UOK262 and UOK268 cell lines previously treated for 4 hours with vandetanib (Figure 1G). Finally, ABLl -specific siRNA reduced HIFl to normal levels in HLRCC-derived cell lines, similarly to vandetanib and restoration of wild-type FH (Figure 1 C).

EXAMPLE 2

[0150] This example demonstrates that ALB1 is a novel therapeutic target in

FH-deficient tumor cells.

[0151] The Abelson (ABL) family of nonreceptor tyrosine kinases, comprised of ABLl and ABL2, affects diverse signaling pathways involved in cell growth, invasion and migration. Activation of ABLl and ABL2 have been detected in a number of different types of cancer (Greuber et al., 2013). Although the role of oncogenic Bcr-Abl fusin protein has been extensively studied in cancer (Cilloni and Saglio, 2012), much less is known about the physiological and/or pathological role of wild-type ABLl . Based on the preceding data, ABLl was immunoprecipitated from protein lysates of 5 HLRCC tumors surgically removed from 3 patients, as well as from lysates of normal tissues, and ABLl phosphorylation normalized to total immunoprecipitated ABLl protein as an indicator of its activation state were compared (Figure 2A). The ABLl activation state in 5 of 5 tumor specimens was significantly greater than that in the normal tissues. A similar comparison in 2 HLRCC- derived cell lines and their wild-type FH-restored counterparts was performed (Figure 2B). Constitutive ABLl phosphorylation, while clearly evident in both HLRCC-derived cell lines, was markedly reduced upon restoration of wild-type FH (non-transformed HE 293 kidney cells are included for comparison), although this did not affect the expression of total ABLl protein. The phosphorylation status of ABLl in skin and uterine leiomyomas, two classical manifestations of HLRCC disease, compared with their normal counterparts was also assessed (Figures 8A and 8B). ABLl was constitutively phosphorylated in skin leiomyomas but not consistently in uterine leiomyomas, suggesting that treatment of these manifestations might require alternative targeting approaches.

[0152] Given these findings, it was investigated whether the cytotoxicity of vandetanib in FH-deficient tumor cells was ABLl -dependent. While over-expression of ABLl in UOK262 cells significantly reversed vandetanib cytotoxicity in vitro (Figure 2C), the silencing of ABLl dramatically decreased cell viability in the absence of vandetanib (Figure 2D) and thus was consistent with the hypothesis that vandetanib toxicity is mediated, at least in part, by ABLl inhibition. Finally, like vandetanib cytotoxicity, the cytotoxicity incurred by ABLl knockdown was abrogated by restoration of wild-type FHm ^' UOK262 cells (Figure 2E and Figure 8C). To assess the importance of ABLl in maintaining the tumorigenic phenotype of FH-deficient cells, their anchorage-independent growth and invasiveness after ABLl inhibition or silencing was measured. These data show that ABLl expression and activity are necessary to sustain the clonogenicity and invasive potential of FH-deficient tumor cells (Figure 2F and Figure 8D).

EXAMPLE 3

[0153] This example demonstrates that ABLl promotes aerobic glycolysis.

[0154] Based on the data linking ABLl with HIFl a expression (Figure 1 C), it was investigated whether ABLl inhibition affected the aerobic glycolysis on which FH-deficient cells depend for generation of ATP and cellular building blocks (Yang et al., 2010; Tong et al., 201 1). First, it was found that vandetanib significantly decreased expression of the glucose transporters Glutl and Glut4 in UO 262 cells (Figure 3A), concomitant with decreased glucose uptake and lactate secretion (Figure 3B and 3C, respectively). Transient silencing of ABLl or HIFla decreased glucose uptake to a similar degree (Figure 3B and Figure 9A). To confirm that ABLl promotes aerobic glycolysis, the impact of ABLl modulation on the extracellular acidification rate (ECAR, a surrogate for lactate secretion) was assessed. In agreement with the earlier results, silencing or pharmacologic inhibition of ABLl significantly decreased ECAR in UOK262 cells (Figure 3D). In contrast, transient over-expression of ABLl in HEK293 cells caused a significant increase in ECAR, consistent with increased glycolysis (Figure 3D). Supporting a relationship between ABLl activity and glycolysis, vandetanib concomitantly inhibited both ABLl activity and glucose uptake in UOK150, a glycolytic clear cell line derived from a non-HLRCC kidney cancer harboring an inactivating VHL mutation (Sourbier et al., 2012) (Figures 9B and 9C).

EXAMPLE 4

[0155] This example demonstrates that vandetanib inhibits ABLl , glycolysis and FH -/- RCC tumor growth in vivo.

[0156] In order to visualize the dynamic impact of vandetanib on HLRCC metabolism in vivo, magnetic resonance spectroscopic imaging (MRSI) to monitor the metabolism of intravenously injected hyperpolarized [1-¹³C] pyruvate was used, as previously described (Golman et al., 2006; Chen et al., 2007). MRSI detected increased conversion of pyruvate to lactate in tumors, most notably in those characterized by a high rate of glycolysis and impaired oxidative phosphorylation.

[0157] Next, the antitumor efficacy of vandetanib was assessed in an HLRCC murine xenograft model. As shown in Figure 4A, vandetanib (100 mg/kg, once weekly, i.p.) caused marked tumor regression in 80% (12 of 15, two independent experiments) of treated mice. Using tumor tissue remaining after 10 weeks of treatment, the molecular effects of vandetanib were interrogated in vivo. The activation status of ABLl was significantly reduced in tumors excised from vandetanib-treated mice compared with vehicle-treated control animals (Figure 4B). Also, similar to earlier in vitro observations, activity of the mTOR/HIFla pathway was decreased in tumors excised from vandetanib-treated mice. Tumors from vandetanib-treated mice were also more apoptotic and displayed less activation of EGFR family and VEGF receptor kinases compared with tumors excised from vehicle- treated animals. Also, similar to earlier in vitro observations, activity of the mTOR/HIFl a pathway was decreased in tumors excised from vandetanib-treated mice (Figures 10A-10D). Tumors from vandetanib-treated mice were also more apoptotic and displayed less activation of EGFR family and VEGF receptor kinases compared with tumors excised from vehicle- treated animals (Figures 10E and 10F).

EXAMPLE 5

[0158] This example demonstrates that Fumarate-mediated ABLl activation upregulates NRF2 transcriptional activity in vitro and in vivo.

[0159] The constitutively elevated level of phosphorylated ABLl in HLRCC cell lines is reversed by restoration of wild-type FH expression (see Figure 2B), and the in vitro kinase activity of ABLl immunopurified from HLRCC cells is greater than that of the kinase immunopurified from FH-restored HLRCC cells (Figure 5A). Therefore, it was investigated whether intracellular accumulation of fumarate influenced ABLl kinase activity, either directly or indirectly. It was observed that treatment of cells expressing functional FH protein (HEK293 or UOK262WT) with a cell-permeable form of fumarate (dimethylfumarate, DMF) resulted in increased ABLl phosphorylation (Figure 5B). However, neither fumarate nor DMF were able to directly stimulate ABLl in vitro, suggesting that fumarate-mediated ABLl activation in cells is indirect (data not shown). Since ABLl is activated by oxidative stress and excess intracellular fumarate increases ROS (Sullivan et al., 2013), fumarate-induced ABLl activation may be ROS-dependent. Consistent with this model, treatment of UO 262 cells with the ROS scavenger N-acetylcysteine (NAC) reduced steady-state ABLl phosphorylation to a level approximating that in HEK293 and UOK262WT cells (Figure 1 1 A). However, it cannot be excluded that the possibility that additional factors may contribute to the increased activation state of ABLl in these tumor cells.

[0160] Accumulation of intracellular fumarate has been shown to stabilize expression of the antioxidant response transcription factor NRF2 via inactivation of its endogenous inhibitor KEAPl (Adam et al., 201 1 ; Ooi et al., 201 1). Since the Src kinase family modulates NRF2 nuclear translocation (Jain and Jaiswal, 2006; Shelton and Jaiswal, 2013), it was examined whether ABLl might play a similar role in FH-deficient tumor cells. By evaluating nuclear and cytoplasmic fractions prepared from cells treated (or not) with vandetanib, it was observed that vandetanib caused a time-dependent redistribution of NRF2 from nucleus to cytosol (Figure 5C). Next, using a luciferase-based NRF2 reporter assay, it was found that DMF stimulated, while vandetanib inhibited NRF2 transcriptional activity (baseline and DMF-stimulated, Figure 5D). Importantly, using the same assay it was confirmed that silencing of ABLl abrogated DMF-induced NRF2 transcriptional activity (Figure 5E).

Taken together, these data support a role for ABLl in activating NRF2-mediated

transcription in response to elevated intracellular fumarate levels. Consistent with a role for ABLl in protecting cells from the toxicity of excess fumarate, the significant impact of DMF on HEK293 cells viability was nearly completely reversed by over-expression of ABLl (Figure 1 IB).

[0161] HLRCC tumor cells constitutively express high levels of ROS, and ROS-inducing agents are particularly cytotoxic to these cells (Sourbier et al., 2010). Because NRF2 plays a critical role in cellular defense against ROS (Sporn and Liby, 2012), it was tested whether the vandetanib sensitivity of these cells might be affected by cellular ROS level. UOK262 cells were pretreated with NAC for 2 h prior to treatment with vandetanib for an additional 16 h. NAC pre-treatment, did not itself affect cell viability, but it significantly protected the cells from vandetanib toxicity (Figure 5F). Since ABLl also promotes HIF l a-mediated glycolysis (vide supra), it was probed whether vandetanib-mediated inhibition of HIF la contributed to cell death. However, NAC reduced HIF la expression as effectively as did vandetanib, indicating that vandetanib toxicity is most likely a consequence of inhibiting NRF2 activity (Figure 5F, G). In support of this hypothesis, both ABLl inhibition and silencing decreased expression of the endogenous NRF2 transcriptional target NQOl (Figure 5H).

[0162] To assess whether these findings are relevant in vivo, the effect of vandetanib on NQOl expression in HLRCC xenografts was examined. NQOl is highly expressed in HLRCC tumor tissue (Figure 1 1 C) (Adam et al., 201 1). Its expression was significantly decreased in UOK262 xenografts excised from mice treated with vandetanib compared with tumor tissue excised from vehicle- treated mice (Figure 1 ID).

EXAMPLE 6

[0163] This example demonstrates that AMPK activation inhibits NRF2 activity synergistically with vandetanib to inhibit FH -/- RCC tumor growth.

[0164] SIRT1 -mediated deacetylation of NRF2 is reported to inhibit its transcriptional activity (Kawai et al., 201 1). Since AMPK positively regulates SIRT1 and is constitutively hypoactivated in FH-deficient tumors (Tong et al., 201 1) (Fulco and Sartorelli, 2008), it was of interest whether pharmacologic activation of AMPK in these cells might enhance SIRT1 deacetylase activity to inhibit NRF2 independently from vandetanib. First, the effect of AMPK modulation on NRF2 transcriptional activity was assessed using a luciferase-reporter assay. The AMPK activators metformin and AICAR decreased DMF-induced NRF2 activity, while knockdown/inhibition of SIRT1 with siRNA or nicotinamide, respectively, had the opposite effect (although the increased NRF2 activity remained sensitive to vandetanib) (Figures 6 A, 12A, and 12B). Consistent with these data, metformin treatment decreased NRF2 acetylation while silencing SIRT1 had the opposite effect (Figure 6B).

[0165] Next, it was of interest whether metformin and vandetanib synergistically inhibited NRF2. Data obtained using a luciferase-reporter in HEK293 cells and by monitoring endogenous NQOl expression in UOK262 cells suggested that this is the case (Figures 6C and 12C). Supporting these observations, it was found that metformin, at a concentration lacking single agent toxicity, significantly decreased the viability of UOK262 cells when combined with a low concentration of vandetanib (Figure 6D). Since both vandetanib and metformin have multiple effects in cells, it was confirmed that their synergistic impact on NRF2 was due to ABLl inhibition combined with AMPK activation. Thus, both metformin and AICAR cooperated with knockdown of ABLl to markedly reduce cell viability, while, in the absence of ABLl silencing, neither drug was effective. Further, imatinib displayed greater cytotoxicity at lower concentrations in combination with metformin compared with its single agent activity (Figures 12D-12F). Finally, in vivo analysis confirmed that low dose vandetanib (10 mg/kg, once weekly; 1/10th of the dose used in the experiment shown in Figure 4) combined with metformin (5 mg/kg, once weekly) induced complete tumor regression in 100% of treated mice (Figures 6E-F, 12 of 12 mice from 2 independent experiments). Nine of ten mice treated with the vandetanib/metformin combination for 8 weeks remained tumor- free for more than 13 months after treatment cessation (while vehicle-treated mice all had to be sacrificed by 8 weeks due to tumor size, Figure 6G).

EXAMPLE 7

[0166] This example demonstrates that targeting ABLl may be a useful therapeutic strategy for tumors exhibiting aberrant activation of NRF2 signaling.

[0167] Aberrant activation of NRF2 signaling occurs in multiple tumor types as shown in Figures 13A-B. Activation of the ABL1 -NRF2 axis was assessed by immunoblotting in a panel of non-small cell lung cancer-derived cell lines (A549, NC1-H23, NCI-H292, and NCI-H838), and the results shown in Figure 13C. A viability assay following siRNA-mediated silencing of ABL1 or vandetanib treatment for 36 h at a vandetanib concentration of 100 nM was conducted, and the results illustrated in Figure 13D.

[0168] As is apparent from the results shown in Figures 13C and 13D, cell lines expressing NQOl (i.e., UOK150, A549, NCI-H23, and NCI-H838) were affected by vandetanib and siAbl.

EXAMPLE 8

[0169] This example demonstrates the rescue with various infected murine ABL proteins performed 72 hr after lentiviral infection of UOK262 cells with ABL-targeted miRNA (miABL). Scrambled miRNA (miSCR) was used as an miRNA control and empty pBABE vector (BABE) served as the control for infection with the various murine ABL constructs. Murine ABL constructs included wild type (WT), kinase-dead ( 290M), kinase inhibitor- resistant (T3151), and constitutively active ABL (P 1311). Cell viability in presence and absence of vandetanib was assessed (24 hr, 50 nM; VAN: vandetanib). The results are depicted in Figure 14A.

[0170] An immunoblot showing reduction in ABL1 protein expression 72 hr post miABL infection, and subsequent expression level of re-introduced murine ABL proteins is depicted in Figure 14B. The mTOR phosphorylation status is correlated with expression of competent ABL1 protein.

EXAMPLE 9

[0171] This example demonstrates cell viability (in medium lacking pyruvate, the standard culture condition) after 48 hr treatment with several ABL1 inhibitors in UOK262 and UOK262WT cells. The results are depicted in Figures 15A-15D.

EXAMPLE 10

101721 This example demonstrates a comparison of the effect of pyruvate in culture medium on UOK262 cell viability after 48 hr treatment with several ABL1 inhibitors. VAN = vandetanib, PON = ponatinib, DASA = dasatinib, NILO = nilitinib. The results are depicted in Figures 16A-16D. Calculated IC50 values in UOK262 cells are shown. EXAMPLE 1 1

101731 This example demonstrates the effect of vantinib on ABLl kinase activity. ABLl kinase activity was measured by kinase assay after immunopurification of ABLl protein from tumor tissues excised from vehicle-treated and vandetanib-treated mice 2 days

post-treatment. * p<0.05; VAN, vandetanib. The results are depicted graphically in Figure 17. As is apparent from the results shown in Figure 17, ABLl kinase activity in

vandetanib-treated mice was approximately 40% of the activity observed for the

vehicle-treated mice.

EXAMPLE 12

[0174] This example demonstrates the impact of vandetanib on NRF2

nuclear/cytoplasmic distribution as assessed by confocal immunofluorescence microscopy. The microscopy results showed that NRF2 nuclear staining was reduced for UOK262 cells treated with vandatenib as compared with control. NRF2 nuclear staining for vantanib treatment as compared to control is shown in Figure 18, confirming the microscopy results.

[0175] Aspects of the Invention include the following:

1. A method of treating kidney cancer in a subject in need thereof or a subject having a hereditary cancer syndrome, wherein the subject has a germline fumarate hydratase gene mutation, comprising administering to the subject a therapeutically effective amount of an inhibitor of ABLl and an activator of AMPK.

2. The method of aspect 1 , wherein the inhibitor of ABLl is vandetanib.

3. The method of aspect 1 or 2, wherein the activator of AMPK is metformin.

4. The method of any one of aspects 1-3, wherein the inhibitor of ABLl and the activator of AMPK are administered in amounts that are lower than therapeutically effective amounts of the inhibitor of ABLl and the activator of AMPK when administered separately.

5. The method of any one of aspects 1 -4, wherein the cancer is lung squamous cell carcinoma, lung adenocarcinoma, bladder urothelial carcinoma, head and neck squamous cell carcinoma, papillary renal carcinoma, or clear cell clear renal carcinoma.

6. The method of any one of aspects 1 -4, wherein the subject has a hereditary cancer syndrome.

7. The method of aspect 6, wherein the hereditary cancer syndrome is hereditary leiomyomatosis and renal cell carcinoma. 8. The method of any one of aspects 1-7, wherein the subject has at least one kidney tumor.

9. The method of aspect 8, wherein the treating results in regression of the at least one kidney tumor.

10. A kit for treating cancer in a subject in need thereof or a subject having a hereditary cancer syndrome, wherein the subject has a germline fumarate hydratase gene mutation, comprising therapeutically effective amounts of an inhibitor of ABL1 and an activator of AMPK.

1 1. The kit of aspect 10, wherein the inhibitor of ABL1 is vandetanib.

12. The kit of aspect 10, wherein the activator of AMPK is metformin.

13. The kit of any one of aspects 10-12, further comprising instructions for use.

14. The kit of any one of aspects 10-13, wherein the cancer is lung squamous cell carcinoma, lung adenocarcinoma, bladder urothelial carcinoma, head and neck squamous cell carcinoma, papillary renal carcinoma, or clear cell clear renal carcinoma.

15. The kit of any one of aspects 10-13, wherein the subject has a hereditary cancer syndrome.

16. The kit of aspect 15, wherein the hereditary cancer syndrome is hereditary leiomyomatosis and renal cell carcinoma

17. A pharmaceutical composition comprising a combination of an inhibitor of ABL1 or a salt thereof, an activator of AMPK or a salt thereof, and a pharmaceutically acceptable carrier.

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[0213] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0214] The use of the terms "a" and "an" and "the" and "at least one" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (for example, "at least one of A and B") is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly

contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0215] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):

1. A combination of an inhibitor of ABLl and an activator of AMPK for use in treating cancer in a subject in need thereof or a subject having a hereditary cancer syndrome, wherein the subject has a germline fumarate hydratase gene mutation.

2. The combination for use according to claim 1 , wherein the inhibitor of ABLl is vandetanib.

3. The combination for use according to claim 1 or 2, wherein the activator of AMPK is metformin.

4. The combination for use according to any one of claims 1-3, wherein the inhibitor of ABLl and the activator of AMPK are used in amounts that are lower than therapeutically effective amounts of the inhibitor of ABLl and the activator of AMPK when used separately.

5. The combination for use according to any one of claims 1-4, wherein the cancer is lung squamous cell carcinoma, lung adenocarcinoma, bladder urothelial carcinoma, head and neck squamous cell carcinoma, papillary renal carcinoma, or clear cell clear renal carcinoma.

6. The combination for use according to any one of claims 1-4, wherein the subject has a hereditary cancer syndrome.

7. The combination for use according to claim 6, wherein the hereditary cancer syndrome is hereditary leiomyomatosis and renal cell carcinoma.

8. The combination for use according to any one of claims 1 -4, wherein the subject has at least one kidney tumor.

9. The combination for use according to claim 8, wherein the treating results in regression of the at least one kidney tumor.

10. A kit for treating cancer in a subject in need thereof or a subject having a hereditary cancer syndrome, wherein the subject has a germline fumarate hydratase gene mutation, comprising therapeutically effective amounts of an inhibitor of ABLl and an activator of AMPK.

1 1. The kit of claim 10, wherein the inhibitor of ABLl is vandetanib.

12. The kit of claim 10, wherein the activator of AMPK is metformin.

13. The kit of any one of claims 10-12, further comprising instructions for use.

14. The kit of any one of claims 10-13, wherein the cancer is lung squamous cell carcinoma, lung adenocarcinoma, bladder urothelial carcinoma, head and neck squamous cell carcinoma, papillary renal carcinoma, or clear cell clear renal carcinoma.

15. The kit of any one of claims 10-13, wherein the subject has a hereditary cancer syndrome.

16. The kit of claim 15, wherein the hereditary cancer syndrome is hereditary leiomyomatosis and renal cell carcinoma

17. A pharmaceutical composition comprising a combination of an inhibitor of ABLl or a salt thereof, an activator of AMPK or a salt thereof, and a pharmaceutically acceptable carrier.