Compositions and methods for treating pancreatic cancer

Abstract

The present disclosure provides methods comprising a combination therapeutic strategy for treating cancer. In some embodiments, the methods comprise administering a composition comprising a glutathione biosynthesis inhibitor and a composition comprising a mitochondrial inhibitor to a subject to treat pancreatic cancer.

Description

Docket No.27013 / 70192 COMPOSITIONS AND METHODS FOR TREATING PANCREATIC CANCER BACKGROUND

[0001] Pancreatic Ductal Adenocarcinoma (PDAC), the most common type of pancreatic cancer, is a highly lethal cancer with an overall five-year survival rate of 13% (1). Long- established chemotherapeutic regimes remain the standard-of-care, which generally target genome integrity and have only a modest survival advantage. Other therapeutic approaches, such as immunotherapies and targeted therapies have not been clinically effective. Emerging evidence suggests that the tumor microenvironment (TME) in pancreatic cancer is a driving force for observed chemotherapy resistance and poor clinical outcomes (2-4). Cancer cells’ access to the nutrient supply is substantially decreased (5-8), due to a dense and tenacious stroma (9), increased interstitial fluid pressure (10, 11), and disorganized vascularization (5, 12). When PDAC cells are exposed to nutrient-limited microenvironment, a condition that mimics PDAC tumor microenvironment, survival mechanisms are prioritized (2, 3, 7, 13-16), as opposed to proliferative pathways, and these pro-survival mechanisms promote resistance to conventional therapies, such as chemotherapies (2, 3). A better understanding of how PDAC cells adapt to steep glucose and glutamine gradients in the TME may uncover important therapeutic opportunities associated with a therapeutic window since these survival mechanisms are likely less important to well-perfused and unstressed normal tissues. There thus remains a need in the art for new and effective treatments for pancreatic cancer. SUMMARY

[0002] The present disclosure provides materials and methods comprising a combination therapy for the treatment of cancer. In one embodiment, the disclosure provides a method of treating cancer comprising the steps of administering to a subject in need thereof (a) a composition comprising a therapeutically effective amount of a glutathione biosynthesis inhibitor, and (b) a composition comprising a therapeutically effective amount of a mitochondrial inhibitor.

[0003] In one aspect, the cancer is a metastatic cancer. In another aspect, the cancer comprises one or more cancerous tumors. In yet another aspect, the cancer comprises a cancerous tumor, and wherein the cancerous tumor is a lymphoma or a cancerous tumor of a tissue or organ selected from the group consisting of skin, head and neck, esophagus, stomach, liver, colon, rectum, pancreas, lung, breast, cervix, ovary, kidney, bladder, prostate, thyroid, brain, muscle, and bone. In still another aspect, the cancer is pancreatic cancer, lung cancer, or liver cancer. In another aspect, the cancer is pancreatic cancer. In yet another aspect, the pancreatic cancer is Pancreatic Ductal Adenocarcinoma (PDAC).Docket No.27013 / 70192

[0004] In one embodiment, the glutathione biosynthesis inhibitor comprises one or more of an inhibitor of glutamate-cystine ligase, an inhibitor of the catalytic subunit of glutamate- cysteine ligase (GCL), and an inhibitor of the modifier subunit of glutamate-cysteine ligase (GCL). In another embodiment, the glutathione biosynthesis inhibitors are an inhibitor of GCLC, and an inhibitor of GCLM. In yet another embodiment, the glutathione biosynthesis inhibitor is selected from the group consisting of L-buthionine sulfoximine (BSO), DL-BSO, and EN25.

[0005] In one embodiment, the mitochondrial inhibitor is one or more of a mitochondrial oxidative phosphorylation (OXPHOS) inhibitor, a tricarboxylic acid (TCA) cycle inhibitor, and a glutaminase inhibitor. In another embodiment, the mitochondrial inhibitor is an OXPHOS inhibitor. In an aspect, the OXPHOS inhibitor is an inhibitor of mitochondrial electron transport complex I, II, III, and / or V. In another aspect, the OXPHOS inhibitor is an inhibitor of mitochondrial electron transport complex I. In yet another aspect, the inhibitor of mitochondrial electron transport complex I is selected from the group consisting of metformin, phenformin, rotenone, and IACS-010759. In still another aspect, the mitochondrial inhibitor is an inhibitor of gene expression or gene product activity and wherein the gene is one or more of the genes provided in Table 1.

[0006] In one embodiment, the method of treating cancer further comprises administering to a subject in need thereof one or more additional therapies comprising chemotherapy, surgery, immunotherapy, targeted therapy, and radiotherapy. In another embodiment, the one or more additional therapies comprises a therapeutically effective amount of a checkpoint inhibitor. In yet another embodiment, the checkpoint inhibitor is selected from the group consisting of an inhibitor of CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L), PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, BTLA, SIGLEC9, and 2B4. In still another embodiment, the checkpoint inhibitor is selected from the group consisting of pembrolizumab, ipilimumab, avelumab, atezolizumab, cetrelimab, dostarlimab, cemiplimab, spartalizumab, camrelizumab, durvalumab, and nivolumab.

[0007] In one embodiment, the compositions for treating cancer are administered to the subject by a route selected from the group consisting of parenteral, enteral, oral, intramuscular, intradermal, subcutaneous, intratumoral, intranodal, intranasal, transdermal, inhalation, mucosal, and topical. In another embodiment, the compositions are administered approximately at the same time.

[0008] The present disclosure additionally provides a method of treating Pancreatic Ductal Adenocarcinoma (PDAC), said method comprising the steps of administering to the subjectDocket No.27013 / 70192 in need thereof (a) a composition comprising a therapeutically effective amount of L- buthionine sulfoximine (BSO) and (b) a composition comprising a therapeutically effective amount of metformin.

[0009] In one embodiment, the subject is a mammal. In another embodiment, the mammal is a human.

[0010] Various kits are contemplated and provided by the present disclosure. In one embodiment, the present disclosure provides a kit comprising (a) a therapeutically effective amount of a composition comprising a glutathione biosynthesis inhibitor, and (b) a composition comprising a therapeutically effective amount of a mitochondrial inhibitor, and instructions for using the same for the treatment of pancreatic cancer. In another embodiment, the present disclosure further provides a kit comprising (a) a composition comprising a therapeutically effective amount of L-buthionine sulfoximine (BSO), and (b) a composition comprising a therapeutically effective amount of metformin, and instructions for using the same for the treatment of pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 shows that glutathione reduction, mediated by GCLC inhibitor BSO, displayed no therapeutic impact in mice with PDAC. (A) BSO (L-buthionine sulfoximine) blocks the catalytic component of GCL (GCLC), the rate-limiting enzyme of glutathione biosynthesis pathway. (B) Total glutathione pool (GSH and GSSG) in KPC orthotopic tumors after 10 days of receiving indicated treatments. (C) Overall survival of KPC orthotopic- bearing C57BL / 6 mice (CTRL (n=10), BSO (n=8).

[0012] Figure 2 depicts PDAC cell resistance to BSO under low glucose conditions. Survival of MiaPaCa-2 PDAC cells under indicated treatments and conditions were detected via clonogenic assay (crystal violet staining), BSO (250μM), GSH (1 mM). Results are averages of 3 independent biologic replicates ± S.D. *** representing p <0.001.

[0013] Figure 3 demonstrates that pharmacologic inhibitors of mitochondria significantly sensitized PDAC cells to BSO under nutrient limitation. A drug screening study identified cellular processes causing resistance to BSO in human and murine PDAC (PANC-1 and KPC) cells under low glucose conditions. Top hits were mitochondrial inhibitors.

[0014] Figure 4 shows an upregulation of genes associated with OXPHOS in PDAC cells under BSO treatment. (A) Gene set enrichment analysis of genes associated with OXPHOS derived from RNA sequencing (NES: 1.87, FDR q Value: 0.0013). (B-D) Relative mRNA normalized with 18S, and protein levels of TOMM20 via qPCR and Western Blotting, and mitochondrial mass detected by Mitotracker Green in MiaPaCa-2 PDAC cells underDocket No.27013 / 70192 indicated treatment and low glucose conditions. Results in B-D are averages of 3 independent biologic replicates ± S.D.

[0015] Figure 5 shows that PDAC cells, but not normal cells, upregulate mitochondrial function under glutathione limitation. Relative basal oxygen consumption rate (A, D), ATP level (B, E), and mitochondrial membrane potential (C, F) under indicated treatment for 48 hours. BSO (250 μM). Results are averages of 3 independent biologic replicates ± S.D. ** representing p <0.01 and *** representing p <0.001.

[0016] Figure 6 demonstrates that elevated expression of genes associated with ETC complex I in PDAC cells treated with BSO under low glucose state. Red bars indicate genes encoding components of ETC complex I.

[0017] Figure 7 depicts that complex I inhibitor metformin significantly improved BSO efficacy in PDAC cells under low glucose conditions. Cells were cultured under low glucose conditions for 30 hours, followed by indicated treatments for an additional 96 hours. BSO (250 μM), Metformin (0.5 mM). Cell viability was detected via clonogenic assay. Results are averages of 3 independent biologic replicates ± S.D. ** representing p <0.01 and *** representing p <0.001.

[0018] Figure 8 shows that complex I inhibitor phenformin sensitized PDAC cells to GCLC inhibitor under low glucose conditions. Cells were cultured under low glucose conditions for 30 hours, followed by indicated treatments for an additional 96 hours. BSO (250 μM), phenformin (1 μM). Cell viability was detected via clonogenic assay. Results are averages of 3 independent biologic replicates ± S.D. *** representing p <0.001.

[0019] Figure 9 shows that BSO and metformin combination therapy increased mitochondrial ROS production. MiaPaCa-2 cells were treated with BSO (250 μM) under low glucose conditions for 24 hours followed by other indicated treatments for an additional 48 hours. Metformin (0.5 mM), Mito-tempo (1 μM). Cell viability was detected via clonogenic assay. Results are averages of 3 independent biologic replicates ± S.D. *** representing p <0.001.

[0020] Figure 10 demonstrates that NADK2 silencing sensitized PDAC cells to BSO. RNA seq analysis identified mRNA expression of ME2, NADK2, and MTHFD2 was significantly altered in cells treated with BSO compared to Ctrl. Relative mRNA levels of genes, normalized to 18S, in cells transiently transfected with indicated siRNAs. (C, D) Cell viability was detected via MTT assay following 72 hours of BSO treatment (250 μM), and / or metformin (0.5 mM). Results in B-D are averages of 3 independent biologic replicates ± S.D. *** representing p <0.001.Docket No.27013 / 70192

[0021] Figure 11 shows that mitochondrial respiratory complex I is a metabolic vulnerability in Gclc-knockout murine KPC cells. Gclc- / - KPC PDAC cells were generated via CRISPR-Cas9 approach. (B) Total glutathione pool (GSH and GSSG), and (C) relative viability was assessed via clonogenic assay after 4 days of receiving indicated treatments (Met 0.5 mM). (D) KPC allograft growth in C57BL / 6 mice treated with vehicle or metformin (1.25 mg / mL in drinking water) n=4 mice per group. Results in B and C are averages of 3 independent biologic replicates ± S.D. *** representing p <0.001.

[0022] Figure 12 demonstrates that BSO and metformin combination therapy inhibited PDAC tumor growth. (A) Body weights of non-tumor bearing C57BL / 6 mice treated with indicated treatments for the indicated time. BSO (4.4 mg / mL in drinking water), metformin (1.25 mg / mL in drinking water). (B) Growth of subcutaneous KPC allografts in C57 / BL6 mice treated for the indicated time. Results are average ± S.E.M. *** representing p < 0.001.

[0023] Figure 13 shows that upregulation of Gclc in orthotopic primary tumors and liver metastases generated from PDAC. (A) Bioluminescence imaging for a mouse with primary orthotopic KPC tumor and liver metastases indicated with red and yellow arrows, respectively. (B) Relative mRNA, normalized with 18S. Results in B are averages of five corresponding tissues ± S.E.M. * representing p <0.05 and ** representing p <0.01. DETAILED DESCRIPTION

[0024] Disclosed herein are compositions and methods comprising a combination therapeutic strategy for the treatment of cancer that concomitantly targets glutathione biosynthesis and mitochondrial function. In some cases, the methods provided herein comprise the steps of administering to a subject in need thereof (a) a composition comprising a therapeutically effective amount of a glutathione biosynthesis inhibitor, and (b) a composition comprising a therapeutically effective amount of a mitochondrial inhibitor.

[0025] As will be appreciated by those of skill in the art, the present disclosure also contemplates methods of improving survival, e.g., of a subject afflicted with pancreatic cancer. Similarly, the present disclosure contemplates compositions and methods for reducing tumor burden and / or tumor volume in a subject.

[0026] The five-year survival rate for patients presenting with metastatic pancreatic cancer is only 3%, the worst survival rate for all cancers (1), and the lack of effective available therapeutics dramatically impacts pancreatic cancer patients’ lives. The relative ineffectiveness of standard of care chemotherapy in pre-clinical murine PDAC models also mirrors the poor patient outcomes (5). There is significant room in this field for improvement. Disclosed herein is the discovery that mitochondrial electron transport complex (ETC) I comprises a metabolic vulnerability in PDAC cells under glutathione limitation. PancreaticDocket No.27013 / 70192 tumors exhibit reduced levels of elemental nutrients compared to normal adjacent tissue, potentially rendering them more susceptible to genetic ablation or pharmacologic inhibition of mitochondrial metabolism and glutathione de novo biosynthesis; however, preliminary data showed neither administration of a GCLC inhibitor nor mitochondrial complex I inhibitor metformin alone led to a significant inhibition in xenograft tumor growth, in agreement with prior studies (17, 18). Disclosed herein is the discovery that co-targeting these metabolic nodes is well-tolerated in animals, and co-targeting effectively inhibited the growth of allograft tumors in syngeneic models, demonstrating significant efficacy in a model of pancreatic cancer.

[0027] In this disclosure, “comprises”, “comprising”, “containing”, “having”, and the like have the meaning ascribed to them in U.S. patent law and mean “includes”, “including”, and the like; the terms “consisting essentially of” or “consists essentially” likewise have the meaning ascribed in U.S. patent law and these terms are open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited are not changed by the presence of more than that which is recited, but excluding prior art embodiments.

[0028] Unless specifically otherwise stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.

[0029] As used herein, the term “tumor” refers to an accumulation or mass of abnormal cells. Tumors may be benign (non-cancerous), premalignant (pre-cancerous, including hyperplasia, atypia, metaplasia, dysplasia and carcinoma in situ), or malignant (cancerous).

[0030] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive.

[0031] As used herein, the terms “patient”, “subject”, “recipient”, and the like are used interchangeably herein to refer to a vertebrate, a mammal, a primate, or a human. Mammals include, without limitation, humans, non-human primates, domestic and farm animals, and other animals, including, but not limited to dogs, horses, cats, cattle, sheep, pigs, mice, rats, and goats. Primates include, for example, chimpanzees, cynomologous monkeys, spider monkeys, and macaques. Exemplary subjects are humans, including adults, children, and the elderly. A subject can be one who is currently being treated for, or seeking treatment, monitoring, adjustment or modification of an existing therapeutic treatment.,

[0032] The phrases “therapeutically effective amount”, “effective amount”, “immunologically effective amount”, “anti-tumor effective amount”, and the like, as used herein, indicate an amount necessary to administer to a subject, or to a cell, tissue, or organ of a subject, to achieve a therapeutic effect, such as an ameliorating or a curative effect.Docket No.27013 / 70192 The therapeutically effective amount is sufficient to elicit the biological or medical response of a cell, tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor, clinician, or healthcare provider.

[0033] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50(the dose lethal to 50% of the population) and the ED50(the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50- Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the antibody or antigen binding fragment thereof), which achieves a half-maximal inhibition of symptoms as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[0034] The terms “administering”, “administer”, “administration”, and the like, as used herein, refer to any mode of transferring, delivering, introducing, or transporting a therapeutic agent to a subject in need of treatment with such an agent. Such modes include, but are not limited to, oral, topical, intravenous, intraarterial, intraperitoneal, intramuscular, intratumoral, intradermal, intranasal, and subcutaneous administration.

[0035] Embodiments of the methods and compositions provided herein are useful for treating a tumor or cancer, meaning that tumor growth is significantly inhibited as demonstrated by various techniques well-known in the art such as, for example, by a reduction in tumor volume. Tumor volume may be determined by various known procedures, (e.g., obtaining two dimensional measurements with a dial caliper). Preventing and / or treating a tumor can result in the prolonged survival of the subject being treated.

[0036] Maintaining redox homeostasis is crucial for cancer growth, especially when cancer cells experience nutrient limitation (7, 19) and rely on mitochondrial function for survival (7, 20). Furthermore, metabolic stress within the tumor microenvironment (TME) in PDAC is a direct stimulant of oxidative stress. Since glucose is the principal precursor for NADPH production in cancer cells (7, 21-23), it follows that glucose deprivation leads to theDocket No.27013 / 70192 induction of ROS (reactive oxygen species) levels (7). Like NADPH, glutathione is a key ingredient for antioxidant defense (2, 7, 24, 25).

[0037] Employing glutathione biosynthesis inhibitors with inhibitors that block mitochondrial function may represent an important therapeutic opportunity to address a broad range of cancers, for example those that exhibit a nutrient-limited tumor microenvironment (TME) (8). In some cases, the cancer targeted by the methods disclosed herein is a metastatic cancer, and / or wherein the cancer comprises one or more cancerous tumors. In some embodiments, the cancer targeted by the methods disclosed herein comprises a cancerous tumor, wherein the cancerous tumor is a lymphoma or a cancerous tumor of a tissue or organ selected from the group consisting of skin, head and neck, esophagus, stomach, liver, colon, rectum, pancreas, lung, breast, cervix, ovary, kidney, bladder, prostate, thyroid, brain, muscle, and bone. In various embodiments, the cancer is pancreatic cancer, lung cancer, or liver cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the pancreatic cancer is Pancreatic Ductal Adenocarcinoma (PDAC).

[0038] The terms “cell”, “cell line”, “cancer cell line”, “tumor cell line”, and the like as used interchangeably herein refers to a cell line that originated from a cancerous tumor as described herein, and / or originates from a parental cell line of a tumor originating from a specific source / organ / tissue. Glutathione Biosynthesis Inhibitors

[0039] Under harsh metabolic stress, including a nutrient-limited tumor microenvironment, glutathione production is increased (7) due to glutamate-cysteine ligase (GCL) upregulation (2), which maintains the redox state required for continued survival and growth in PDAC cells. Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme for the de novo glutathione biosynthesis pathway, which is a crucial upstream component of antioxidant defense. GCL is a heterodimeric protein composed of a catalytic (GCLC) and modifier (GCLM) subunits. The catalytic subunit of GCL (GCLC) catalyzes the union of glutamate and cysteine. The modifier subunit of GCL (GCLM) increases the efficiency of GCLC (26). Increased glutathione synthesis has also been documented in human patient samples (2, 17), suggesting an alternative survival pathway upregulated under low glucose conditions. BSO has been reported to successfully inhibit GCLC and impair glutathione synthesis, which is especially important for nutrient-deprived PDAC (27). The safety of BSO in combination with chemotherapy has been established in multiple myeloma and neuroblastoma clinical trials (28, 29), although the efficacy of such a combination has not been fully characterized. While GCL inhibition using a pharmacologic approach is shown to be effective inDocket No.27013 / 70192 downregulating glutathione synthesis pathway in preclinical models, it does not display antitumor activity as a single-agent modality (17, Fig.11D, 12B). However, as disclosed herein, glutathione biosynthesis can be targeted concomitantly with other pathways to effectively target cancers that, for example, exhibit nutrient limitation.

[0040] In some embodiments, the glutathione biosynthesis inhibitor comprises one or more of an inhibitor of glutamate-cysteine ligase (GCL), an inhibitor of the catalytic subunit of glutamate-cysteine ligase (GCLC), and an inhibitor of modifier subunit of glutamate-cysteine ligase (GCLM). In some embodiments, the glutathione biosynthesis inhibitor is an inhibitor of GCLC and GCLM. In some embodiments, the inhibitor is selected from a group consisting of L-buthionine sulfoximine (BSO), DL-BSO, and EN25. In still other embodiments, an inhibitor of glutathione itself, such as an antibody or small molecule that binds to and prevents / inhibits glutathione activity, is contemplated by the present disclosure.

[0041] In PDAC models, cancer cells upregulate several metabolic activities associated with NADPH and glutathione production in order to maintain redox when the cells experience glucose limitation. Glutathione synthesis possesses an important role in cancer cell growth (30, 31), prompting assessment of the therapeutic impact of GCLC suppression via pharmacologic tools in preclinical models of PDAC. GCLC blockage alone was not sufficient to suppress the growth of PDAC tumors, and it was discovered that pancreatic cancer cells exhibit resistance to the anti-growth inhibitory effects of selective inhibitor of GCLC, BSO (L- buthionine sulfoximine), under low glucose conditions, underscoring the contribution of PDAC cells to observed metabolic adaptation. Mitochondrial Inhibitors

[0042] Pro-oxidative effects of classical chemotherapeutic agents are well known and believed to be a key mechanism of anticancer activity by cytotoxic agents (32). The mechanisms underlying reactive oxygen species (ROS) induction vary for different chemotherapeutics. In some instances, ROS generation is even attributable to pharmacologic effects on non-cancer cells, like immune cells, but the generally accepted mechanisms of ROS induction for most anti-neoplastic agents relate to the direct effects on mitochondria and impaired antioxidant machinery (33). Chemotherapeutics induce apoptosis, which leads to the release of cytochrome c from mitochondria, and which in turn diverts electrons from the electron transport chain to generate free radicals (34). Cancer cells rely on robust antioxidant machinery to overcome chemotherapy-associated oxidative stress, highlighting such machinery as a therapeutically exploitable target that has, until now, been overlooked.Docket No.27013 / 70192

[0043] Although mitochondrial respiration is a major source of ROS production (35-37), preliminary data disclosed herein showed mitochondrial ETC I playing a crucial role in maintaining mitochondrial redox under glutathione limitation. In line with these observations, prior studies have shown that glutathione is an important regulator of mitochondrial redox (38, 39). Such results suggest a novel role of ETC complex I for enabling the mitochondria to maintain a redox state when glutathione is limited. These results further suggest that blocking mitochondrial complex I in cells under glutathione limitation is a synthetic lethal approach. Indeed, silencing ETC complex I through small molecule inhibitors or a genetic approach rendered PDAC cells vulnerable to oxidative stress resulting from glutathione limitation, as demonstrated herein. This points to an ETC complex I-mediated metabolic circuit as a critical metabolic dependency related to the entire cellular redox balance.

[0044] The combination therapeutic strategy disclosed herein comprises a glutathione biosynthesis inhibitor and a mitochondrial inhibitor. In some embodiments, the mitochondrial inhibitor is one or more of a mitochondrial oxidative phosphorylation (OXPHOS) inhibitor, a TCA cycle inhibitor, and a glutaminase inhibitor. In various embodiments, the mitochondrial inhibitor is an OXPHOS inhibitor. In some embodiments, the OXPHOS inhibitor is an inhibitor of mitochondrial electron transport complex I, II, III, and / or V. In various embodiments, the OXPHOS inhibitor is an inhibitor of mitochondrial electron transport complex I. In some embodiments, the inhibitor of mitochondrial electron transport complex I is selected from the group consisting of metformin, phenformin, rotenone, and IACS-01759. In various embodiments, the mitochondrial inhibitor is an inhibitor of gene expression or gene product activity, and wherein the gene is one or more of the genes provided in Table 1. Additional Therapies

[0045] In various embodiment, the present disclosure provides methods of treating a cancer in a subject comprising administering to the subject a therapeutically effective amount of two compositions, wherein at least one composition comprises a glutathione biosynthesis inhibitor, and wherein at least one composition comprises a mitochondrial inhibitor. In some embodiments, the method comprises administering one or more additional therapeutic agents. In various embodiments, an additional therapeutic agent can be an anti- cancer agent including, for example, chemotherapy, surgery, immunotherapy, targeted therapy, and radiotherapy.

[0046] In one embodiment, the present disclosure provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a composition described herein, and further comprising administering to the subject a therapeutically effective amount of a checkpoint inhibitor. In another embodiment, theDocket No.27013 / 70192 checkpoint inhibitor is selected from the group consisting of an inhibitor of CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L), PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, BTLA, SIGLEC9, and 2B4. In some embodiments, the checkpoint inhibitor is selected from the group consisting of pembrolizumab, ipilimumab, avelumab, atezolizumab, cetrelimab, dostarlimab, cemiplimab, spartalizumab, camrelizumab, durvalumab, and nivolumab. Administration

[0047] In various embodiments, the present disclosure provides an aforementioned method wherein the composition or compositions are administered to the subject by a route selected from the group consisting of parenteral, enteral, oral, intramuscular, intradermal, subcutaneous, intratumoral, intranodal, intranasal, transdermal, inhalation, mucosal, and topical. In one embodiment, the route is intradermal. In some embodiments, the composition or compositions are administered to an administration site on the subject selected from the group consisting of arm or arms, thigh or thighs, and back. In some embodiments, the composition or compositions are administered orally or intravenously. In some embodiments, oral administration comprises administering the composition or compositions as a pill. In an embodiment, the composition or compositions are administered intraperitoneally. In another embodiment, the compositions are intradermally administered at different administration sites on the subject. In another embodiment, the composition is intradermally administered by injection with a syringe positioned at an angle between 5 and 15 degrees from the surface of the administration site. In some embodiments, a method of treating cancer in a subject is provided comprising administering to the subject a therapeutically effective amount of a first dose and therapeutically effective amounts of subsequent doses of one or more compositions provided herein, wherein the one or more compositions are administered 1-24 times in year one, 1-16 times in year two, and 1-14 times in year three. In another embodiment, the present disclosure provides a method of stimulating an immune response in a subject comprising administering to the subject a first dose of a therapeutically effective amount of two compositions provided herein, wherein the first four doses are administered every 21 days up to day 63, and then every 42 days for three additional doses up to day 189. In one embodiment, the method further comprises administering five additional doses at 42-day intervals up to day 399, and then at least at two 84-day intervals thereafter.

[0048] Embodiments of the compositions of the disclosure are formulated to be compatible with their intended route of administration (i.e., parenteral, intravenous, intra- arterial, intradermal, subcutaneous, oral, inhalation, transdermal, topical, intratumoral, transmucosal, intraperitoneal or intra-pleural, and / or rectal administration). Solutions orDocket No.27013 / 70192 suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; dimethyl sulfoxide (DMSO); antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes, or one or more vials comprising glass or polymer (e.g., polypropylene). The term “vial” as used herein means any kind of vessel, container, tube, bottle, or the like that is adapted to store embodiments of the vaccine composition as described herein.

[0049] In various embodiments, the methods of treatment with combination therapeutics disclosed herein comprise administration of two compositions. In some embodiments, the methods of treatment with combination therapeutics disclosed herein comprise administration of two or more compositions. Embodiments of the combination therapeutics described herein may comprise administration of one therapeutic simultaneously with, prior to, or after administration of another therapeutic. When in separate compositions, the two or more compositions of the “dose” are meant to be administered “concurrently”. In some embodiments, the two or more compositions are administered at different sites on the subject (e.g., arm, thigh, or back). As used herein, “concurrent” administration of two compositions or therapeutic agents indicates that within about 30 minutes of administration of a first composition or therapeutic agent, the second composition or therapeutic agent is administered. In cases where more than two compositions and / or therapeutic agents are administered concurrently, each composition or agent is administered within 30 minutes, wherein timing of such administration begins with the administration of the first composition or agent and ends with the beginning of administration of the last composition or agent. In some cases, concurrent administration can be completed (i.e., administration of the last composition or agent begins) within about 30 minutes, or within 15 minutes, or within 10 minutes, or within 5 minutes of start of administration of first composition or agent. Administration of a second (or multiple) therapeutic agents or compositions “prior to” or “subsequent to” administration of a first composition means that the administration of the first composition and another therapeutic agent is separated by at least 30 minutes, e.g., at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 18 hours, at least 24 hours, or at least 48 hours.Docket No.27013 / 70192

[0050] In some embodiments, the disclosure provides a method of treating Pancreatic Ductal Adenocarcinoma (PDAC), said method comprising the steps of administering to a subject in need thereof (a) a composition comprising a therapeutically effective amount of L- buthionine sulfoximine (BSO), and (b) a composition comprising a therapeutically effective amount of metformin. In some embodiments, BSO is administered in a fixed dose as an intravenous (IV) bolus of 3 g / m2, over a period of 30 minutes. In other embodiments, metformin is administered as a 72 hour infusion of 1 g / m2. In still other embodiments, BSO is administered as an IV bolus of 3 g / m2over a period of 30 minutes, followed by a 72 hour infusion of 1 g / m2(28). In some embodiments, metformin is administered at a dose of 500 mg per day, wherein metformin is administered on a daily schedule.

[0051] The terms “treat”, “treating”, “treatment”, and the like, as used herein, unless otherwise indicated, refers to reversing, alleviating, inhibiting the process of disease, disorder or condition to which such term applies, or one or more symptoms of such disease, disorder or condition and includes the administration of any of the compositions, pharmaceutical compositions, or dosage forms described herein, to prevent the onset of the symptoms or the complications, alleviate the symptoms or the complications, or eliminate the disease, condition, or disorder in a subject in need thereof. As used herein, treatment can be curative or ameliorating. In some embodiments, the subject in need thereof is a mammal. In some embodiments, the subject in need thereof is a human. Kits

[0052] The present disclosure provides kits for carrying out the methods disclosed herein. Such kits may be used for treating or prolonging the survival of a subject. In some embodiments, the present disclosure provides a kit comprising two or more compositions provided herein. In some embodiments, the kit further comprises instructions for use. In some embodiments, the kit is used for the treatment of cancer. In various embodiments, the present disclosure provides a kit comprising a therapeutically effective amount of a composition comprising a glutathione biosynthesis inhibitor, and a composition comprising a therapeutically effective amount of a mitochondrial inhibitor, and instructions for using the same for the treatment of pancreatic cancer. In one embodiment, the present disclosure provides a kit comprising a composition comprising a therapeutically effective amount of L- buthionine sulfoximine (BSO), and a composition comprising a therapeutically effective amount of metformin, and instructions for using the same for the treatment of pancreatic cancer.Docket No.27013 / 70192 EXAMPLES Example 1: Reduced BSO-mediated cell death induction in PDAC cells under nutrient limitation

[0053] Consistent with prior reports showing GCLC suppression via its potent and selective inhibitor BSO (L-buthionine sulfoximine) (2, 40-43) effectively reduces glutathione levels in PDAC cells (17, 18), it was demonstrated herein that the total glutathione levels are indeed lower in pancreatic tumors receiving BSO relative to the control group (Fig.1A, B); however, BSO administration did not exhibit any therapeutic impact (Fig.1C). These data support the contribution of a compensatory mechanism that enables tumor growth under glutathione limitation. Glucose is likely more limiting than glutamine, as cancer cells can extract glutamine from ambient protein content via cellular processes like autophagy, micropinocytosis, or stromal cells (6, 44-46). Decreased levels of glucose, to a greater extent than glutamine, have been detected in pancreatic tumor interstitial fluid compared to levels in the plasma (8). Extending these findings requires understanding whether cell culture condition is a determinant of BSO efficacy in PDAC cells.

[0054] BSO treatment dramatically inhibited the viability of PDAC cells under nutrient abundance, and active form of glutathione (GSH) rescued cells from the harsh oxidative effects of BSO, indicating a selective, on-target effect of BSO (Fig.2A). However, pretreatment of cells with low glucose conditions (2.5 mM glucose), to mimic TME of PDAC, plays a critical role in BSO effectiveness in cultured PDAC cells. Under this condition, PDAC cells were quite resistant to the anti-cancer effects of BSO (Fig.2B), although a significant reduction in glutathione levels in cells cultured in the same low glucose conditions was observed (Fig.2C), suggesting the involvement of a compensatory mechanism protecting cancer cells against glutathione limitation. Example 2: PDAC cells adapt to glutathione-limited conditions by compensatory induction of mitochondrial respiratory complex I

[0055] To elucidate the underlying mechanism of this observed resistance under nutrient limitation, a cell-based drug screening was used to identify the compounds that enhance the sensitivity of PDAC cells to BSO. Employing this unbiased approach using two different PDAC cells (human PANC-1 and murine KPC PDAC cells) identified mitochondrial inhibitors, such as mitochondrial OXPHOS (oxidative phosphorylation) inhibitors, TCA cycle inhibitors, and glutaminase inhibitors, as the top positive hits (Fig.3). Subsequent RNA sequencing was performed in PDAC cells treated with BSO and control. Gene Set Enrichment Analysis (GSEA) identified significant upregulation of genes associated with mitochondrial OXPHOS in PDAC cells treated with BSO compared to control (Fig.4A). However, no alteration in totalDocket No.27013 / 70192 mitochondrial mass was observed. Measuring mitochondrial marker TOMM20 mRNA and protein levels (Fig.4B, C) and mitochondria staining via MitoTracker Green (Fig.4D) showed that there was no change in mitochondrial mass in PDAC cells under glutathione depletion. Mitochondrial OXPHOS upregulation was corroborated by mitochondrial activity assays, where it was shown that ATP production, mitochondrial membrane potential, and oxygen consumption rate (OCR), were significantly increased in PDAC cells receiving BSO compared to control (Fig.5A-C); however, mitochondrial hyperactivity was not observed in a non-cancer cell line (HPNE immortalized normal pancreatic cells) cultured in the same condition (Fig.5D-F). It has been previously documented that mitochondrial function is imperative for the survival of glucose-deprived pancreatic cancer cells to maximize energy production when nutrients are limited (7). These findings indicate that PDAC cells rely more on mitochondrial function to survive harsh metabolic stress. Further investigation revealed the upregulation of genes associated with electron transport complex (ETC) I as the most significantly altered genes in cells treated with BSO compared to control (Fig.6, Table 1), which may reflect compensatory changes. To further interrogate whether the upregulation of complex I plays an important role in PDAC cell adaptation to glutathione limitation under low glucose conditions, PDAC cells were treated with pharmacologic inhibitors of GCLC and complex I. While treatment of PDAC cells with either BSO or sublethal doses of complex I inhibitor metformin (47-52) had negligible effects on PDAC cell survival, the combination of these two compounds dramatically reduced the viability of cultured human PDAC cells under low glucose conditions (Fig.7, 8), indicating targeting metabolic adaptation provides important therapeutic opportunities. Given the role of ETC complex I as NADH dehydrogenase that converts NADH to NAD+, its function can regenerate adequate levels of NAD+ required for sustaining the TCA cycle, as observed herein (Fig.5C), in addition to its activity being involved in mitochondrial redox homeostasis (53-55). It was further examined whether mitochondrial redox relies on the function of this complex when glutathione is limited. Under glutathione-limited conditions, inhibition of complex I leads to detrimental production of mitochondrial ROS that reduced PDAC cell viability (Fig.9). Upon treatment with BSO and metformin, PDAC cells experienced an elevation of mitochondrial ROS, which led to a significant reduction in cell viability (Fig.9A, B), as treatment of cells with mitochondrial-specific antioxidant, Mito-Tempo, restored cell viability (Fig.9C). These results showed the critical role of complex I in maintaining redox state in PDAC cells, especially under glutathione limitation.Docket No.27013 / 70192 Table 1: Elevated Expression of Genes Associated with ETC complex I in PDAC cells treated with BSO under low glucose state. PDAC cell elevated gene expression post- BSO ETC Complex I Genes Other Genes MT-ND4L CYB5B NDUFA7 FH MT-ND6 COX7A2 NDUFS5 MT-ND2 NDUFA8 MT-ND1 NDUFC2 MT-ND5 NDUFA1 NDUFA11 Example 3: Functional interaction between ETC complex I and NADK2 sustains redox balance under glutathione limitation

[0056] To further investigate if upregulation of mitochondrial complex I is an alternative mechanism to maintain redox state when glutathione is limiting, it was investigated how NAD+ production via complex I sustains redox balance in the mitochondria. Maintaining redox homeostasis in mitochondria relies on sufficient production of NADPH in the mitochondria as cytosolic NADPH cannot enter the mitochondria (21, 56, 57). To elucidate how complex I upregulation plays a crucial role in maintaining redox in mitochondria, the expression of genes encoding NADPH-generating enzymes in BSO-treated and control cells were compared. Expression of ME2 (malic enzyme 2), NADK2 (mitochondrial isoform of NAD kinase), and MTHFD2 (mitochondrial isoform of methylenetetrahydrofolate dehydrogenase) were significantly elevated in cells treated with BSO compared to control (Fig.10A). Using a selected siRNA screening, a differential role was identified for NADK2, as silencing of this enzyme sensitized PDAC cells to BSO under low glucose condition (Fig.Docket No.27013 / 70192 10B). NADK2, the mitochondrial isoform of NAD kinase, converts NAD(H) to NADP(H) (58, 59). Silencing of other genes did not show any substantial changes in the viability of BSO- treated cells compared to the control. Notably, siRNA efficacy was >80% in all cases (Fig. 10C). To further determine whether NADK2 and complex I are functionally linked, the efficacy of complex I inhibitor metformin in PDAC cells with reduced NADK2 expression was evaluated. Silencing NADK2 did not offer any additional impact on cell viability then treatment of single-agent metformin, indicating NADK2 function in maintaining mitochondrial redox is dependent on ETC complex I (Fig.10D). Collectively, these data suggest PDAC cells can overcome the oxidative stress results from glutathione limitation by a compensatory increase in complex I expression and activity that leads to the production of NAD+, which, in turn, can be used as a substrate for NADK2 to generate a robust antioxidant potential required for mitochondrial redox. Example 4: Co-targeting glutathione biosynthesis and mitochondrial complex I reduced tumor burden in a syngeneic mouse model of PDAC

[0057] To further investigate whether mitochondrial complex I is also metabolic dependency in PDAC cells lacking GCLC expression, Gclc knockout KPC cells were developed using CRISPR Cas-9-base approach (Fig.11A). As expected, total glutathione levels were markedly reduced in Gclc-deficient KPC cells (in pool of Gclc- / - knockout cells including KO1 and KO2 cells) compared to isogenic control cells (Gclc+ / +) (Fig.11B). Similar to parental PDAC cells under BSO, the survival of Gclc-deficient cells depend on complex I activity. Inhibition of complex I using metformin dramatically reduced viability of Gclc-knockout cells under glucose limitation (Fig.11C). The therapeutic impact of complex I inhibitor metformin was evaluated (60, 61) in mice bearing KPC allografts derived from Gclc+ / + and Gclc- / - KPC cells in mice with immunocompetent background. In this study, animals were provided with metformin at 1.25 mg / ml in their drinking water, which is equivalent to 250 mg / kg metformin in mice (61). In vivo (Fig.1C), no significant difference in the growth rate of Gclc-proficient vs deficient tumors was observed. Interestingly, metformin administration significantly blocked Gclc-deficient allograft tumor growth but had no impact on the growth rate of Gclc-proficient tumors (Fig.11D). These observations provide additional evidence that complex I hyperactivity under glutathione limitation is a PDAC cell- driven mechanism.

[0058] Next, to evaluate the synergistic role of combination treatments of GCLC inhibitor and complex I inhibitor compared to single-agent BSO or metformin alone, an in vivo study was performed to determine whether animals tolerate such a combination treatment strategy. For toxicity testing study, non-tumor bearing mice provided with selective GCLC inhibitor BSO (4.4 mg / ml in drinking water), complex I inhibitor metformin (1.25 mg / ml in theirDocket No.27013 / 70192 drinking water), combination of these agents, and control mice remained untreated, as shown in Fig.12A. The results indicate that the single and combination therapy were well tolerated in non-tumor bearing mice. No behavioral, morphological, or body weight loss was observed in indicated experimental arms, and no blood glucose changes were observed in animals. In an independent in vivo study in mice with KPC allograft tumors, combination of both treatments exerted a profound therapeutic response in a syngeneic mouse model. Collectively, these preliminary data demonstrate the important role of mitochondrial complex I as a compensatory mechanism when PDAC cells experience glutathione limitation.

[0059] PDAC Metastatic Models: The long-term survival rate for pancreatic cancer patients presenting with metastatic disease is only 3%. PDAC is typically a systemic disease at presentation, as over 70% of all PDAC cases metastasize to the liver as the main site of pancreatic cancer metastasis. It is critically important to assess the impact of therapeutics that interact with the tumor microenvironment; however, the metabolic and cellular profiles of the PDAC microenvironment in primary versus metastases have not been well characterized. It has been demonstrated that GCLC expression was highly upregulated in metastatic lesions generated by splenic injection of murine pancreatic cancer KPC cells. (Fig.13). This observation comports with a prior report that GCLC expression is elevated in cultured PDAC cells under matrix-detached condition (62), which resembles an early step of cancer metastasis (63, 64), suggesting GCLC is an important component of antioxidant defense machinery that is required for cancer metastasis (65-69).Docket No.27013 / 70192 REFERENCES 1. Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin.2024 Jan- Feb;74(1):12-49. doi: 10.3322 / caac.21820. Epub 2024 Jan 17. PMID: 38230766. 2. Vaziri-Gohar A, Hue JJ, Abbas A, Graor HJ, Hajihassani O, Zarei M, Titomihelakis G, Feczko J, Rathore M, Chelstowska S, Loftus AW, Wang R, Zarei M, Goudarzi M, Zhang R, Willard B, Zhang L, Kresak A, Willis JE, Wang GM, Tatsuoka C, Salvino JM, Bederman I, Brunengraber H, Lyssiotis CA, Brody JR, Winter JM. Increased glucose availability sensitizes pancreatic cancer to chemotherapy. Nat Commun.2023 Jun 28;14(1):3823. doi: 10.1038 / s41467-023-38921-8. PMID: 37380658; PMCID: PMC10307839. 3. Zarei M, Lal S, Parker SJ, Nevler A, Vaziri-Gohar A, Dukleska K, Mambelli-Lisboa NC, Moffat C, Blanco FF, Chand SN, Jimbo M, Cozzitorto JA, Jiang W, Yeo CJ, Londin ER, Seifert EL, Metallo CM, Brody JR, Winter JM. Posttranscriptional Upregulation of IDH1 by HuR Establishes a Powerful Survival Phenotype in Pancreatic Cancer Cells. Cancer Res. 2017 Aug 15;77(16):4460-4471. doi: 10.1158 / 0008-5472.CAN-17-0015. Epub 2017 Jun 26. PMID: 28652247; PMCID: PMC5922269. 4. Jain A, Bhardwaj V. Therapeutic resistance in pancreatic ductal adenocarcinoma: Current challenges and future opportunities. World J Gastroenterol.2021 Oct 21;27(39):6527-6550. doi: 10.3748 / wjg.v27.i39.6527. PMID: 34754151; PMCID: PMC8554400. 5. Olive KP, Jacobetz MA, Davidson CJ, Gopinathan A, McIntyre D, Honess D, Madhu B, Goldgraben MA, Caldwell ME, Allard D, Frese KK, Denicola G, Feig C, Combs C, Winter SP, Ireland-Zecchini H, Reichelt S, Howat WJ, Chang A, Dhara M, Wang L, Rückert F, Grützmann R, Pilarsky C, Izeradjene K, Hingorani SR, Huang P, Davies SE, Plunkett W, Egorin M, Hruban RH, Whitebread N, McGovern K, Adams J, Iacobuzio-Donahue C, Griffiths J, Tuveson DA. Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science.2009 Jun 12;324(5933):1457-61. doi: 10.1126 / science.1171362. Epub 2009 May 21. PMID: 19460966; PMCID: PMC2998180. 6. Kamphorst JJ, Nofal M, Commisso C, Hackett SR, Lu W, Grabocka E, Vander Heiden MG, Miller G, Drebin JA, Bar-Sagi D, Thompson CB, Rabinowitz JD. Human pancreatic cancer tumors are nutrient poor and tumor cells actively scavenge extracellular protein. Cancer Res.2015 Feb 1;75(3):544-53. doi: 10.1158 / 0008- 5472.CAN-14-2211. PMID: 25644265; PMCID: PMC4316379.Docket No.27013 / 70192 7. Vaziri-Gohar A, Cassel J, Mohammed FS, Zarei M, Hue JJ, Hajihassani O, Graor HJ, Srikanth YVV, Karim SA, Abbas A, Prendergast E, Chen V, Katayama ES, Dukleska K, Khokhar I, Andren A, Zhang L, Wu C, Erokwu B, Flask CA, Zarei M, Wang R, Rothermel LD, Romani AMP, Bowers J, Getts R, Tatsuoka C, Morton JP, Bederman I, Brunengraber H, Lyssiotis CA, Salvino JM, Brody JR, Winter JM. Limited nutrient availability in the tumor microenvironment renders pancreatic tumors sensitive to allosteric IDH1 inhibitors. Nat Cancer.2022 Jul;3(7):852-865. doi: 10.1038 / s43018-022-00393-y. Epub 2022 Jun 9. PMID: 35681100; PMCID: PMC9325670. 8. Sullivan MR, Danai LV, Lewis CA, Chan SH, Gui DY, Kunchok T, Dennstedt EA, Vander Heiden MG, Muir A. Quantification of microenvironmental metabolites in murine cancers reveals determinants of tumor nutrient availability. Elife.2019 Apr 16;8:e44235. doi: 10.7554 / eLife.44235. PMID: 30990168; PMCID: PMC6510537. 9. Koong AC, Mehta VK, Le QT, Fisher GA, Terris DJ, Brown JM, Bastidas AJ, Vierra M. Pancreatic tumors show high levels of hypoxia. Int J Radiat Oncol Biol Phys.2000 Nov 1;48(4):919-22. doi: 10.1016 / s0360- 3016(00)00803-8. PMID: 11072146. 10. Zhang Z, Ji S, Zhang B, Liu J, Qin Y, Xu J, Yu X. Role of angiogenesis in pancreatic cancer biology and therapy. Biomed Pharmacother.2018 Dec;108:1135-1140. doi: 10.1016 / j.biopha.2018.09.136. Epub 2018 Oct 1. PMID: 30372814. 11. Rhim AD, Oberstein PE, Thomas DH, Mirek ET, Palermo CF, Sastra SA, Dekleva EN, Saunders T, Becerra CP, Tattersall IW, Westphalen CB, Kitajewski J, Fernandez-Barrena MG, Fernandez-Zapico ME, Iacobuzio-Donahue C, Olive KP, Stanger BZ. Stromal elements act to restrain, rather than support, pancreatic ductal adenocarcinoma. Cancer Cell.2014 Jun 16;25(6):735-47. doi: 10.1016 / j.ccr.2014.04.021. Epub 2014 May 22. PMID: 24856585; PMCID: PMC4096698. 12. Provenzano PP, Cuevas C, Chang AE, Goel VK, Von Hoff DD, Hingorani SR. Enzymatic targeting of the stroma ablates physical barriers to treatment of pancreatic ductal adenocarcinoma. Cancer Cell.2012 Mar 20;21(3):418-29. doi: 10.1016 / j.ccr.2012.01.007. PMID: 22439937; PMCID: PMC3371414.Docket No.27013 / 70192 13. Vaziri-Gohar A, Zarei M, Brody JR, Winter JM. Metabolic Dependencies in Pancreatic Cancer. Front Oncol.2018 Dec 12;8:617. doi: 10.3389 / fonc.2018.00617. Erratum in: Front Oncol.2019 Jan 31;8:672. PMID: 30631752; PMCID: PMC6315177. 14. Subramani E, Dominic A, Bhattacharya PK, Frigo DE, Bederman I, Vaziri-Gohar A. Editorial: Metabolic regulation under oxidative stress in cancer. Front Oncol.2023 Sep 12;13:1286086. doi: 10.3389 / fonc.2023.1286086. PMID: 37771445; PMCID: PMC10523380. 15. Nwosu ZC, Ward MH, Sajjakulnukit P, Poudel P, Ragulan C, Kasperek S, Radyk M, Sutton D, Menjivar RE, Andren A, Apiz-Saab JJ, Tolstyka Z, Brown K, Lee HJ, Dzierozynski LN, He X, Ps H, Ugras J, Nyamundanda G, Zhang L, Halbrook CJ, Carpenter ES, Shi J, Shriver LP, Patti GJ, Muir A, Pasca di Magliano M, Sadanandam A, Lyssiotis CA. Uridine- derived ribose fuels glucose-restricted pancreatic cancer. Nature.2023 Jun;618(7963):151- 158. doi: 10.1038 / s41586-023-06073-w. Epub 2023 May 17. PMID: 37198494; PMCID: PMC10232363. 16. Birsoy K, Possemato R, Lorbeer FK, Bayraktar EC, Thiru P, Yucel B, Wang T, Chen WW, Clish CB, Sabatini DM. Metabolic determinants of cancer cell sensitivity to glucose limitation and biguanides. Nature.2014 Apr 3;508(7494):108-12. doi: 10.1038 / nature13110. Epub 2014 Mar 16. PMID: 24670634; PMCID: PMC4012432. 17. Kremer DM, Nelson BS, Lin L, Yarosz EL, Halbrook CJ, Kerk SA, Sajjakulnukit P, Myers A, Thurston G, Hou SW, Carpenter ES, Andren AC, Nwosu ZC, Cusmano N, Wisner S, Mbah NE, Shan M, Das NK, Magnuson B, Little AC, Savani MR, Ramos J, Gao T, Sastra SA, Palermo CF, Badgley MA, Zhang L, Asara JM, McBrayer SK, di Magliano MP, Crawford HC, Shah YM, Olive KP, Lyssiotis CA. GOT1 inhibition promotes pancreatic cancer cell death by ferroptosis. Nat Commun.2021 Aug 11;12(1):4860. doi: 10.1038 / s41467-021- 24859-2. PMID: 34381026; PMCID: PMC8357841. 18. Hopkins BD, Pauli C, Du X, Wang DG, Li X, Wu D, Amadiume SC, Goncalves MD, Hodakoski C, Lundquist MR, Bareja R, Ma Y, Harris EM, Sboner A, Beltran H, Rubin MA, Mukherjee S, Cantley LC. Suppression of insulin feedback enhances the efficacy of PI3K inhibitors. Nature.2018 Aug;560(7719):499-503. doi: 10.1038 / s41586-018-0343-4. Epub 2018 Jul 4. Erratum in: Nature.2018 Aug 29;: PMID: 30051890; PMCID: PMC6197057. 19. Ahmad IM, Aykin-Burns N, Sim JE, Walsh SA, Higashikubo R, Buettner GR, Venkataraman S, Mackey MA, Flanagan SW, Oberley LW, Spitz DR. Mitochondrial O2*- andDocket No.27013 / 70192 H2O2 mediate glucose deprivation-induced stress in human cancer cells. J Biol Chem.2005 Feb 11;280(6):4254-63. doi: 10.1074 / jbc.M411662200. Epub 2004 Nov 23. PMID: 15561720. 20. Rossignol R, Gilkerson R, Aggeler R, Yamagata K, Remington SJ, Capaldi RA. Energy substrate modulates mitochondrial structure and oxidative capacity in cancer cells. Cancer Res.2004 Feb 1;64(3):985- 93. doi: 10.1158 / 0008-5472.can-03-1101. PMID: 14871829. 21. Lewis CA, Parker SJ, Fiske BP, McCloskey D, Gui DY, Green CR, Vokes NI, Feist AM, Vander Heiden MG, Metallo CM. Tracing compartmentalized NADPH metabolism in the cytosol and mitochondria of mammalian cells. Mol Cell.2014 Jul 17;55(2):253-63. doi: 10.1016 / j.molcel.2014.05.008. Epub 2014 May 29. PMID: 24882210; PMCID: PMC4106038. 22. Chandel NS. NADPH-The Forgotten Reducing Equivalent. Cold Spring Harb Perspect Biol.2021 Jun 1;13(6):a040550. doi: 10.1101 / cshperspect.a040550. PMID: 34074675; PMCID: PMC8168431. 23. TeSlaa T, Ralser M, Fan J, Rabinowitz JD. The pentose phosphate pathway in health and disease. Nat Metab.2023 Aug;5(8):1275-1289. doi: 10.1038 / s42255-023-00863-2. Epub 2023 Aug 23. PMID: 37612403. 24. Amores-Sánchez MI, Medina MA. Glutamine, as a precursor of glutathione, and oxidative stress. Mol Genet Metab.1999 Jun;67(2):100-5. doi: 10.1006 / mgme.1999.2857. PMID: 10356308. 25. Lu SC. Glutathione synthesis. Biochim Biophys Acta.2013;1830(5):3143-53. Epub 20120917. doi: 10.1016 / j.bbagen.2012.09.008. PubMed PMID: 22995213; PubMed Central PMCID: PMC3549305. 26. Franklin CC, Backos DS, Mohar I, White CC, Forman HJ, Kavanagh TJ. Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase. Mol Aspects Med.2009 Feb-Apr;30(1-2):86-98. doi: 10.1016 / j.mam.2008.08.009. Epub 2008 Sep 6. PMID: 18812186; PMCID: PMC2714364. 27. Bansal A, Simon MC. Glutathione metabolism in cancer progression and treatment resistance. J Cell Biol.2018 Jul 2;217(7):2291-2298. doi: 10.1083 / jcb.201804161. Epub 2018 Jun 18. PMID: 29915025; PMCID: PMC6028537.Docket No.27013 / 70192 28. Tagde A, Singh H, Kang MH, Reynolds CP. The glutathione synthesis inhibitor buthionine sulfoximine synergistically enhanced melphalan activity against preclinical models of multiple myeloma. Blood Cancer J.2014 Jul 18;4(7):e229. doi: 10.1038 / bcj.2014.45. PMID: 25036800; PMCID: PMC4219442. 29. Villablanca JG, Volchenboum SL, Cho H, Kang MH, Cohn SL, Anderson CP, Marachelian A, Groshen S, Tsao-Wei D, Matthay KK, Maris JM, Hasenauer CE, Czarnecki S, Lai H, Goodarzian F, Shimada H, Reynolds CP. A Phase I New Approaches to Neuroblastoma Therapy Study of Buthionine Sulfoximine and Melphalan With Autologous Stem Cells for Recurrent / Refractory High-Risk Neuroblastoma. Pediatr Blood Cancer.2016 Aug;63(8):1349-56. doi: 10.1002 / pbc.25994. Epub 2016 Apr 19. PMID: 27092812; PMCID: PMC8992729. 30. Kennedy L, Sandhu JK, Harper ME, Cuperlovic-Culf M. Role of Glutathione in Cancer: From Mechanisms to Therapies. Biomolecules.2020 Oct 9;10(10):1429. doi: 10.3390 / biom10101429. PMID: 33050144; PMCID: PMC7600400. 31. Traverso N, Ricciarelli R, Nitti M, Marengo B, Furfaro AL, Pronzato MA, Marinari UM, Domenicotti C. Role of glutathione in cancer progression and chemoresistance. Oxid Med Cell Longev.2013;2013:972913. doi: 10.1155 / 2013 / 972913. Epub 2013 May 20. PMID: 23766865; PMCID: PMC3673338. 32. Yang H, Villani RM, Wang H, Simpson MJ, Roberts MS, Tang M, Liang X. The role of cellular reactive oxygen species in cancer chemotherapy. J Exp Clin Cancer Res.2018 Nov 1;37(1):266. doi: 10.1186 / s13046-018-0909-x. PMID: 30382874; PMCID: PMC6211502. 33. Sangeetha P, Das UN, Koratkar R, Suryaprabha P. Increase in free radical generation and lipid peroxidation following chemotherapy in patients with cancer. Free Radic Biol Med. 1990;8(1):15-9. doi: 10.1016 / 0891-5849(90)90139-a. PMID: 2157633. 34. Conklin KA. Chemotherapy-associated oxidative stress: impact on chemotherapeutic effectiveness. Integr Cancer Ther.2004 Dec;3(4):294-300. doi: 10.1177 / 1534735404270335. PMID: 15523100. 35. Tirichen H, Yaigoub H, Xu W, Wu C, Li R, Li Y. Mitochondrial Reactive Oxygen Species and Their Contribution in Chronic Kidney Disease Progression Through Oxidative Stress.Docket No.27013 / 70192 Front Physiol.2021 Apr 23;12:627837. doi: 10.3389 / fphys.2021.627837. PMID: 33967820; PMCID: PMC8103168. 36. Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release. Physiol Rev.2014 Jul;94(3):909-50. doi: 10.1152 / physrev.00026.2013. PMID: 24987008; PMCID: PMC4101632. 37. Sullivan LB, Chandel NS. Mitochondrial reactive oxygen species and cancer. Cancer Metab.2014 Nov 28;2:17. doi: 10.1186 / 2049-3002-2-17. PMID: 25671107; PMCID: PMC4323058. 38. Wang Y, Yen FS, Zhu XG, Timson RC, Weber R, Xing C, Liu Y, Allwein B, Luo H, Yeh HW, Heissel S, Unlu G, Gamazon ER, Kharas MG, Hite R, Birsoy K. SLC25A39 is necessary for mitochondrial glutathione import in mammalian cells. Nature.2021 Nov;599(7883):136-140. doi: 10.1038 / s41586-021-04025-w. Epub 2021 Oct 27. PMID: 34707288. 39. Liu Y, Liu S, Tomar A, Yen FS, Unlu G, Ropek N, Weber RA, Wang Y, Khan A, Gad M, Peng J, Terzi E, Alwaseem H, Pagano AE, Heissel S, Molina H, Allwein B, Kenny TC, Possemato RL, Zhao L, Hite RK, Vinogradova EV, Mansy SS, Birsoy K. Autoregulatory control of mitochondrial glutathione homeostasis. Science.2023 Nov 17;382(6672):820-828. doi: 10.1126 / science.adf4154. Epub 2023 Nov 2. PMID: 37917749. 40. Harris IS, Endress JE, Coloff JL, Selfors LM, McBrayer SK, Rosenbluth JM, Takahashi N, Dhakal S, Koduri V, Oser MG, Schauer NJ, Doherty LM, Hong AL, Kang YP, Younger ST, Doench JG, Hahn WC, Buhrlage SJ, DeNicola GM, Kaelin WG Jr, Brugge JS. Deubiquitinases Maintain Protein Homeostasis and Survival of Cancer Cells upon Glutathione Depletion. Cell Metab.2019 May 7;29(5):1166-1181.e6. doi: 10.1016 / j.cmet.2019.01.020. Epub 2019 Feb 21. PMID: 30799286; PMCID: PMC6506399. 41. Miess H, Dankworth B, Gouw AM, Rosenfeldt M, Schmitz W, Jiang M, Saunders B, Howell M, Downward J, Felsher DW, Peck B, Schulze A. The glutathione redox system is essential to prevent ferroptosis caused by impaired lipid metabolism in clear cell renal cell carcinoma. Oncogene.2018 Oct;37(40):5435-5450. doi: 10.1038 / s41388-018-0315-z. Epub 2018 Jun 5. PMID: 29872221; PMCID: PMC6173300.Docket No.27013 / 70192 42. Griffith OW, Meister A. Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (Sn-butyl homocysteine sulfoximine). J Biol Chem.1979 Aug 25;254(16):7558-60. PMID: 38242. 43. Friedman HS, Colvin OM, Griffith OW, Lippitz B, Elion GB, Schold SC Jr, Hilton J, Bigner DD. Increased melphalan activity in intracranial human medulloblastoma and glioma xenografts following buthionine sulfoximine-mediated glutathione depletion. J Natl Cancer Inst.1989 Apr 5;81(7):524-7. doi: 10.1093 / jnci / 81.7.524. PMID: 2921776. 44. Yang S, Wang X, Contino G, Liesa M, Sahin E, Ying H, Bause A, Li Y, Stommel JM, Dell'antonio G, Mautner J, Tonon G, Haigis M, Shirihai OS, Doglioni C, Bardeesy N, Kimmelman AC. Pancreatic cancers require autophagy for tumor growth. Genes Dev.2011 Apr 1;25(7):717-29. doi: 10.1101 / gad.2016111. Epub 2011 Mar 15. PMID: 21406549; PMCID: PMC3070934. 45. Commisso C, Davidson SM, Soydaner-Azeloglu RG, Parker SJ, Kamphorst JJ, Hackett S, Grabocka E, Nofal M, Drebin JA, Thompson CB, Rabinowitz JD, Metallo CM, Vander Heiden MG, Bar-Sagi D. Macropinocytosis of protein is an amino acid supply route in Ras- transformed cells. Nature.2013 May 30;497(7451):633-7. doi: 10.1038 / nature12138. Epub 2013 May 12. PMID: 23665962; PMCID: PMC3810415. 46. Sousa CM, Biancur DE, Wang X, Halbrook CJ, Sherman MH, Zhang L, Kremer D, Hwang RF, Witkiewicz AK, Ying H, Asara JM, Evans RM, Cantley LC, Lyssiotis CA, Kimmelman AC. Pancreatic stellate cells support tumour metabolism through autophagic alanine secretion. Nature.2016 Aug 25;536(7617):479-83. doi: 10.1038 / nature19084. Epub 2016 Aug 10. Erratum in: Nature.2016 Dec 1;540(7631):150. PMID: 27509858; PMCID: PMC5228623. 47. Ota S, Horigome K, Ishii T, Nakai M, Hayashi K, Kawamura T, Kishino A, Taiji M, Kimura T. Metformin suppresses glucose-6-phosphatase expression by a complex I inhibition and AMPK activation-independent mechanism. Biochem Biophys Res Commun.2009 Oct 16;388(2):311-6. doi: 10.1016 / j.bbrc.2009.07.164. Epub 2009 Aug 5. PMID: 19664596. 48. El-Mir MY, Nogueira V, Fontaine E, Avéret N, Rigoulet M, Leverve X. Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I. J Biol Chem.2000 Jan 7;275(1):223-8. doi: 10.1074 / jbc.275.1.223. PMID: 10617608.Docket No.27013 / 70192 49. Owen MR, Doran E, Halestrap AP. Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain. Biochem J. 2000 Jun 15;348 Pt 3(Pt 3):607-14. PMID: 10839993; PMCID: PMC1221104. 50. Fontaine E. Metformin-Induced Mitochondrial Complex I Inhibition: Facts, Uncertainties, and Consequences. Front Endocrinol (Lausanne).2018 Dec 17;9:753. doi: 10.3389 / fendo.2018.00753. PMID: 30619086; PMCID: PMC6304344. 51. Vial G, Detaille D, Guigas B. Role of Mitochondria in the Mechanism(s) of Action of Metformin. Front Endocrinol (Lausanne).2019 May 7;10:294. doi: 10.3389 / fendo.2019.00294. PMID: 31133988; PMCID: PMC6514102. 52. Luengo A, Sullivan LB, Heiden MG. Understanding the complex-I-ty of metformin action: limiting mitochondrial respiration to improve cancer therapy. BMC Biol.2014 Oct 24;12:82. doi: 10.1186 / s12915-014- 0082-4. PMID: 25347702; PMCID: PMC4207883. 53. Fato R, Bergamini C, Bortolus M, Maniero AL, Leoni S, Ohnishi T, Lenaz G. Differential effects of mitochondrial Complex I inhibitors on production of reactive oxygen species. Biochim Biophys Acta.2009 May;1787(5):384-92. doi: 10.1016 / j.bbabio.2008.11.003. Epub 2008 Nov 14. PMID: 19059197; PMCID: PMC2724837. 54. Li N, Ragheb K, Lawler G, Sturgis J, Rajwa B, Melendez JA, Robinson JP. Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. J Biol Chem.2003 Mar 7;278(10):8516-25. doi: 10.1074 / jbc.M210432200. Epub 2002 Dec 20. PMID: 12496265. 55. Balsa E, Perry EA, Bennett CF, Jedrychowski M, Gygi SP, Doench JG, Puigserver P. Defective NADPH production in mitochondrial disease complex I causes inflammation and cell death. Nat Commun.2020 Jun 1;11(1):2714. doi: 10.1038 / s41467-020-16423-1. PMID: 32483148; PMCID: PMC7264245. 56. Pollak M. Potential applications for biguanides in oncology. J Clin Invest.2013 Sep;123(9):3693-700. doi: 10.1172 / JCI67232. Epub 2013 Sep 3. PMID: 23999444; PMCID: PMC3754250. 57. Nikiforov A, Dölle C, Niere M, Ziegler M. Pathways and subcellular compartmentation of NAD biosynthesis in human cells: from entry of extracellular precursors to mitochondrialDocket No.27013 / 70192 NAD generation. J Biol Chem.2011 Jun 17;286(24):21767-78. doi: 10.1074 / jbc.M110.213298. Epub 2011 Apr 19. PMID: 21504897; PMCID: PMC3122232. 58. Zhu J, Schwörer S, Berisa M, Kyung YJ, Ryu KW, Yi J, Jiang X, Cross JR, Thompson CB. Mitochondrial NADP(H) generation is essential for proline biosynthesis. Science.2021 May 28;372(6545):968-972. doi: 10.1126 / science.abd5491. Epub 2021 Apr 22. PMID: 33888598; PMCID: PMC8241437. 59. Du J, Estrella M, Solorio-Kirpichyan K, Jeffrey PD, Korennykh A. Structure of human NADK2 reveals atypical assembly and regulation of NAD kinases from animal mitochondria. Proc Natl Acad Sci U S A.2022 Jun 28;119(26):e2200923119. doi: 10.1073 / pnas.2200923119. Epub 2022 Jun 21. PMID: 35733246; PMCID: PMC9245612. 60. Wheaton WW, Weinberg SE, Hamanaka RB, Soberanes S, Sullivan LB, Anso E, Glasauer A, Dufour E, Mutlu GM, Budigner GS, Chandel NS. Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis. Elife.2014 May 13;3:e02242. doi: 10.7554 / eLife.02242. PMID: 24843020; PMCID: PMC4017650. 61. Chandel NS, Avizonis D, Reczek CR, Weinberg SE, Menz S, Neuhaus R, Christian S, Haegebarth A, Algire C, Pollak M. Are Metformin Doses Used in Murine Cancer Models Clinically Relevant? Cell Metab.2016 Apr 12;23(4):569-70. doi: 10.1016 / j.cmet.2016.03.010. PMID: 27076070. 62. Zhang Y, Xu Y, Lu W, Li J, Yu S, Brown EJ, Stanger BZ, Rabinowitz JD, Yang X. G6PD- mediated increase in de novo NADP+ biosynthesis promotes antioxidant defense and tumor metastasis. Sci Adv.2022 Jul 22;8(29):eabo0404. doi: 10.1126 / sciadv.abo0404. Epub 2022 Jul 20. PMID: 35857842; PMCID: PMC9299539. 63. Schafer ZT, Grassian AR, Song L, Jiang Z, Gerhart-Hines Z, Irie HY, Gao S, Puigserver P, Brugge JS. Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment. Nature.2009 Sep 3;461(7260):109-13. doi: 10.1038 / nature08268. Epub 2009 Aug 19. PMID: 19693011; PMCID: PMC2931797. 64. Vanharanta S, Massagué J. Origins of metastatic traits. Cancer Cell.2013 Oct 14;24(4):410-21. doi: 10.1016 / j.ccr.2013.09.007. PMID: 24135279; PMCID: PMC3998120.Docket No.27013 / 70192 65. Sayin VI, Ibrahim MX, Larsson E, Nilsson JA, Lindahl P, Bergo MO. Antioxidants accelerate lung cancer progression in mice. Sci Transl Med.2014 Jan 29;6(221):221ra15. doi: 10.1126 / scitranslmed.3007653. PMID: 24477002. 66. Piskounova E, Agathocleous M, Murphy MM, Hu Z, Huddlestun SE, Zhao Z, Leitch AM, Johnson TM, DeBerardinis RJ, Morrison SJ. Oxidative stress inhibits distant metastasis by human melanoma cells. Nature.2015 Nov 12;527(7577):186-91. doi: 10.1038 / nature15726. Epub 2015 Oct 14. PMID: 26466563; PMCID: PMC4644103. 67. Le Gal K, Ibrahim MX, Wiel C, Sayin VI, Akula MK, Karlsson C, Dalin MG, Akyürek LM, Lindahl P, Nilsson J, Bergo MO. Antioxidants can increase melanoma metastasis in mice. Sci Transl Med.2015 Oct 7;7(308):308re8. doi: 10.1126 / scitranslmed.aad3740. PMID: 26446958. 68. Wiel C, Le Gal K, Ibrahim MX, Jahangir CA, Kashif M, Yao H, Ziegler DV, Xu X, Ghosh T, Mondal T, Kanduri C, Lindahl P, Sayin VI, Bergo MO. BACH1 Stabilization by Antioxidants Stimulates Lung Cancer Metastasis. Cell.2019 Jul 11;178(2):330-345.e22. doi: 10.1016 / j.cell.2019.06.005. Epub 2019 Jun 27. PMID: 31257027. 69. Breau M, Houssaini A, Lipskaia L, Abid S, Born E, Marcos E, Czibik G, Attwe A, Beaulieu D, Palazzo A, Flaman JM, Bourachot B, Collin G, Tran Van Nhieu J, Bernard D, Mechta-Grigoriou F, Adnot S. The antioxidant N-acetylcysteine protects from lung emphysema but induces lung adenocarcinoma in mice. JCI Insight.2019 Oct 3;4(19):e127647. doi: 10.1172 / jci.insight.127647. PMID: 31578304; PMCID: PMC6795405.

Claims

Docket No.27013 / 70192 What is claimed is:

1. A method of treating cancer, said method comprising the steps of: administering to a subject in need thereof (a) a composition comprising a therapeutically effective amount of a glutathione biosynthesis inhibitor, and (b) a composition comprising a therapeutically effective amount of a mitochondrial inhibitor.

2. The method of claim 1, wherein the cancer is a metastatic cancer and / or wherein the cancer comprises one or more cancerous tumors.

3. The method of claim 2, wherein the cancer comprises a cancerous tumor, and wherein the cancerous tumor is a lymphoma or a cancerous tumor of a tissue or organ selected from the group consisting of skin, head and neck, esophagus, stomach, liver, colon, rectum, pancreas, lung, breast, cervix, ovary, kidney, bladder, prostate, thyroid, brain, muscle, and bone.

4. The method of any one of the preceding claims, wherein the cancer is pancreatic cancer, lung cancer, or liver cancer.

5. The method of any one of the preceding claims, wherein the cancer is pancreatic cancer.

6. The method of claim 5, wherein the pancreatic cancer is Pancreatic Ductal Adenocarcinoma (PDAC).

7. The method of any one of the preceding claims, wherein the glutathione biosynthesis inhibitor comprises one or more of an inhibitor of glutamate-cysteine ligase (GCL), an inhibitor of the catalytic subunit of glutamate-cysteine ligase (GCLC), and an inhibitor of modifier subunit of glutamate-cysteine ligase (GCLM).

8. The method of claim 7, wherein the glutathione biosynthesis inhibitor is an inhibitor of GCLC and / or GCLM.

9. The method of claim 7, wherein the inhibitor is selected from the group consisting of L-buthionine sulfoximine (BSO), DL-BSO, and EN25.

10. The method of any one of the preceding claims, wherein the mitochondrial inhibitor is one or more of a mitochondrial oxidative phosphorylation (OXPHOS) inhibitor, a tricarboxylic acid (TCA) cycle inhibitor, and a glutaminase inhibitor.

11. The method of claim 10, wherein the mitochondrial inhibitor is an OXPHOS inhibitor.

12. The method of claim 11 wherein the OXPHOS inhibitor is an inhibitor of mitochondrial electron transport complex I , II, III, and / or V.Docket No.27013 / 70192 13. The method of claim 12 wherein the OXPHOS inhibitor is an inhibitor of mitochondrial electron transport complex I.

14. The method of claim 13, wherein the inhibitor of mitochondrial electron transport complex I is selected from the group consisting of metformin, phenformin, rotenone, and IACS-010759.

15. The method of claim 10, wherein the mitochondrial inhibitor is an inhibitor of gene expression or gene product activity and wherein the gene is one or more of the genes provided in Table 1.

16. The method of any one of the preceding claims, further comprising administering to the subject in need thereof one or more additional therapies comprising chemotherapy, surgery, immunotherapy, targeted therapy, and radiotherapy.

17. The method of claim 16, wherein the one or more additional therapies comprises a therapeutically effective amount of a checkpoint inhibitor.

18. The method of claim 17, wherein the checkpoint inhibitor is selected from the group consisting of an inhibitor of CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L), PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, BTLA, SIGLEC9, and 2B4.

19. The method of claim 17, wherein the checkpoint inhibitor is selected from the group consisting of pembrolizumab, ipilimumab, avelumab, atezolizumab, cetrelimab, dostarlimab, cemiplimab, spartalizumab, camrelizumab, durvalumab, and nivolumab.

20. The method of any one of the preceding claims, wherein the compositions are administered to the subject by a route selected from the group consisting of parenteral, enteral, oral, intramuscular, intradermal, subcutaneous, intratumoral, intranodal, intranasal, transdermal, inhalation, mucosal, and topical.

21. The method of claim 20, wherein the compositions are administered approximately at the same time.

22. A method of treating Pancreatic Ductal Adenocarcinoma (PDAC), said method comprising the steps of: administering to a subject in need thereof (a) a composition comprising a therapeutically effective amount of L-buthionine sulfoximine (BSO), and (b) a composition comprising a therapeutically effective amount of metformin.

23. The method of any one of the preceding claims wherein the subject is a mammal.

24. The method of claim 23, wherein the mammal is a human.Docket No.27013 / 70192 25. A kit comprising (a) a therapeutically effective amount of a composition comprising a glutathione biosynthesis inhibitor, and (b) a composition comprising a therapeutically effective amount of a mitochondrial inhibitor, and instructions for using the same for the treatment of pancreatic cancer.

26. A kit comprising (a) a composition comprising a therapeutically effective amount of L- buthionine sulfoximine (BSO), and (b) a composition comprising a therapeutically effective amount of metformin, and instructions for using the same for the treatment of pancreatic cancer.

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