WO2019246163A1

WO2019246163A1 - Methods of inducing regulated cell death by administering mlkl modulators

Info

Publication number: WO2019246163A1
Application number: PCT/US2019/037817
Authority: WO
Inventors: Amir SCHAJNOVITZ; David T. Scadden
Original assignee: President And Fellows Of Harvard College; The General Hospital Corporation
Priority date: 2018-06-18
Filing date: 2019-06-18
Publication date: 2019-12-26

Abstract

Disclosed are methods of treating cancer with an MLKL modulator in vitro and in vivo. In some aspects, methods of treating small cell lung carcinoma, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer are provided. In some aspects, methods of treating cancer with elevated MLKL, differentially phosphorylated MLKL, or an isoform of MLKL are provided.

Description

METHODS OF INDUCING REGULATED CELL DEATH BY

ADMINISTERING MLKL MODULATORS

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 62/686,652, filed on June 18, 2018 and U.S. Provisional Application No. 62/687,209, filed on June 19, 2018, the entire teachings of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The pseudokinase mixed lineage kinase domain-like (MLKL) gene belongs to the protein kinase superfamily. The encoded protein contains a protein kinase-like domain that is believed to be inactive because it lacks several residues required for activity; MLKL is not known to have protein kinase activity. Activation of MLKL upon its phosphorylation by the protein kinase RIPK3 (a key signaling molecule in the necroptosis pathway) triggers necroptosis, a programmed cell death process. High levels of MLKL and RIP3 are associated with inflammatory bowel disease in children.

SUMMARY OF THE INVENTION

[0003] Work described herein has shown that carbenoxolone (CBX), a small molecule glycyrrhetinic acid derivative, differentially activates Mixed Lineage Kinase Domain -like pseudokinase (MLKL) in cancer cells. This non-canonical MLKL activation displays different kinetics and utilizes different mediators than previously reported for MLKL activation. Further, this activation induces a previously unknown form of regulated cell death (e.g., RCD, rPCD), revealing an opportunity for therapeutic intervention and modalities useful for treating disorders including cancers and other disorders characterized by cells having aberrant redox metabolism. The previously unknown form of rapid programmed cell death (rPCD) has distinct kinetics compared to known programmed cell death pathways and has distinct downstream executers signatures. CBX- induced rPCD is executed by MLKL-regulated proteolysis, possibly via MLKL regulation of calcium sensing cysteine protease activity. MLKL appears to be differentially phosphorylated in certain cancer cells as compared with normal cells, and non-canonical activation induces cell death in the affected cell(s).

[0004] MLKL phosphorylation appears to correlate with its activation. MLKL must self-oligomerize into trimeric or tetrameric form, which is the active form. MLKL dimerizes by making disulphide bonds at residue C86 in human MLKL. Thus, MLKL dimerization, and hence activation, is mediated by oxidation. CBX may therefore induce MLKL dimerization and multimerization via acute release of reactive oxygen species.

[0005] Results disclosed herein strongly suggest that succinate dehydrogenase (SDH) is the molecular target for CBX (a succinate- sterol). Specifically, it is shown herein that, using SDH shRNA to knockdown the expression of SDH (subunit D), conferred a complete resistance to CBX in a human AML cell line. Further, in primary mouse AML cells (eASl2), SDH knockdown resulted in 35% live cells after 24 hours exposure to 200mM CBX (compared to only 1% live cells in the shRNA control cells).

[0006] Further results shown herein (e.g., FIG. 50) show that inhibition of MLKL activity (e.g., inhibition of dimerization) selectively kills cancer cells.

Without being bound by theory, the inventors postulate that cancer cells are at least partially dependent upon MLKL activity for survival. Thus, inhibition of MLKL activity (as well as an increase in MLKL activity as shown above) leads to cell death.

[0007] Some aspects of the disclosure are directed to a method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an MLKL modulator. In some embodiments, the cancer cells are not leukemic cells. In some embodiments, the MLKL modulator is not carbenoxolone.

[0008] In some embodiments, the MLKL modulator modulates MLKL phosphorylation, dimerization, or multimerization. In some embodiments, the MLKL modulator increases dimerization or multimerization of MLKL. In some embodiments, the MLKL modulator increases MLKL activity. In some embodiments, the MLKL modulator selectively increases MLKL activity in cancer cells. In some embodiments, the MLKL modulator selectively causes metabolic arrest in cancer cells. In some embodiments, the MLKL modulator causes rapid programmed cell death in cancer cells.

[0009] In some embodiments, the MLKL modulator decreases MLKL activity. In some embodiments, the MLKL modulator selectively decreases MLKL activity in cancer cells. In some embodiments, the MLKL modulator inhibits MLKL dimerization. In some embodiments, the MLKL modulator selectively inhibits MLKL dimerization in cancer cells. In some embodiments, the MLKL modulator does not selectively inhibit MLKL dimerization in cancer cells.

[0010] In some embodiments, the MLKL modulator causes acute release of reactive oxygen species. In some embodiments, the anti-cancer activity of the MLKL modulator is decreased by calcium chelation. In some embodiments, the MLKL modulator causes (e.g., selectively causes) intracellular release of calcium ions. In some embodiments, the anti-cancer activity of the MLKL modulator is decreased by iron chelation.

[0011] In some embodiments, the anti-cancer activity of the MLKL modulator is decreased by an MLKL inhibitor.

[0012] In some embodiments, the MLKL modulator selectively causes reactive oxygen species generation in cancer cell mitochondria. In some

embodiments, the MLKL modulator selectively causes lipid peroxidation in cancer cells. In some embodiments, the MLKL modulator selectively causes double stranded DNA breaks in the genome of cancer cells. In some embodiments, the MLKL modulator selectively causes phosphatidylserine exposure in cancer cells.

[0013] In some embodiments, the anti-cancer activity of the MLKL modulator is cysteine protease dependent. In some embodiments, the MLKL modulator causes (e.g., specifically causes) intracellular PARP-l cleavage prior to CASP-3 cleavage in cancer cells. In some embodiments, the MLKL modulator causes (e.g., specifically causes) intracellular phosphorylation of g-H2AC.

[0014] In some embodiments, the MLKL modulator selectively causes rPCD in cancer cells. In some embodiments, the MLKL modulator selectively causes cancer cell death within 1 hour of contact between the cancer cell and the MLKL modulator.

[0015] In some embodiments, the cancer cells are selected from the group consisting of leukemia, small cell lung carcinoma cells, colon cancer cells, CNS cancer cells, melanoma cells, ovarian cancer cells, renal cancer cells, prostate cancer cells, and breast cancer cells. In some embodiments, the cancer cells are not leukemia cells. In some embodiments, the cancer cells are not small cell lung carcinoma cells. In some embodiments, the cancer cells are not colon cancer cells. In some

embodiments, the cancer cells are not CNS cancer cells. In some embodiments, the cancer cells are not melanoma cells. In some embodiments, the cancer cells are not ovarian cancer cells. In some embodiments, the cancer cells are not renal cancer cells. In some embodiments, the cancer cells are not prostate cancer cells. In some embodiments, the cancer cells are not breast cancer cells. In some embodiments, the cancer cells are not cervical cancer cells.

[0016] In some embodiments, the cancer cells have elevated levels of phosphorylated MLKL as compared to non-cancerous cells. In some embodiments, the phosphorylated MLKL is phosphorylated at residue serine 345 (murine) or the corresponding residue in human MLKL. In some embodiments, the MLKL modulator selectively modulates phosphorylated MLKL. In some embodiments, the cancer cells have elevated levels of MLKL isoform. In some embodiments, the MLKL isoform has a mass of about 30 kda. In some embodiments, the cancer cells have elevated levels of multimeric MLKL. In some embodiments, the multimeric MLKL has a mass of about 100 kda. In some embodiments, the multimeric MLKL has a mass of about 400-600 kda. In some embodiments, the multimeric MLKL is REF PMID: 28827318. In some embodiments, the cancer cells have elevated expression of SDH (e.g., as compared to non-cancerous cells). In some embodiments, the MLKL modulator binds to SDH (e.g., subunit D of SDH). In some embodiments, the MLKL modulator modulates SDH activity (e.g., inhibits SDH activity). In some embodiments, the anti-cancer activity of the MLKL modulator is dependent upon SDH expression.

[0017] In some embodiments, the MLKL modulator is contacted with the cancer cells in vivo (e.g., in a human or mouse, in a cancer patient). [0018] Some aspects of the disclosure are related to a method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an agent, wherein the agent selectively increases calcium flux in cancer cells. In some embodiments, the cancer cells are not leukemic cells. In some embodiments, the agent is not carbenoxolone.

[0019] Some aspects of the disclosure are related to a method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an agent, wherein the agent selectively causes transient metabolic arrest in cancer cells. In some embodiments, the cancer cells are not leukemic cells. In some embodiments, the agent is not carbenoxolone.

[0020] Some aspects of the disclosure are related to a method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an agent, wherein the agent selectively causes mitochondrial reactive oxygen species generation in cancer cells. In some embodiments, the cancer cells are not leukemic cells. In some embodiments, the agent is not carbenoxolone.

[0021] Some aspects of the disclosure are related to a method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an agent, wherein the agent selectively causes lipid peroxidation in cancer cells. In some embodiments, the cancer cells are not leukemic cells. In some embodiments, the agent is not carbenoxolone.

[0022] Some aspects of the disclosure are related to a method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an agent, wherein the agent selectively causes double stranded DNA breaks in cancer cells. In some embodiments, the cancer cells are not leukemic cells. In some embodiments, the agent is not carbenoxolone.

[0023] Some aspects of the disclosure are related to a method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an agent, wherein the agent inhibits SDH activity. In some embodiments, the cancer cells are not leukemic cells. In some embodiments, the agent is not carbenoxolone.

[0024] Some aspects of the disclosure are related to a method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an agent, wherein the agent selectively causes

phosphatidylserine exposure in cancer cells. In some embodiments, the cancer cells are not leukemic cells. In some embodiments, the agent is not carbenoxolone.

[0025] In some embodiments of methods disclosed herein, the agent modulates MLKL phosphorylation, dimerization, or multimerization. In some embodiments, the agent increases MLKL dimerization. In some embodiments, the agent increases MLKL multimerization. In some embodiments of methods disclosed herein, the agent increases MLKL activity. In some embodiments of the methods disclosed herein, the MLKL modulator selectively modulates phosphorylated MLKL. In some embodiments of methods disclosed herein, the agent selectively increases MLKL activity in cancer cells. In some embodiments of methods disclosed herein, the agent selectively causes metabolic arrest in cancer cells. In some embodiments of methods disclosed herein, the anti-cancer activity of the agent is decreased by calcium chelation. In some embodiments of methods disclosed herein, the anti-cancer activity of the agent is decreased by iron chelation. In some embodiments of methods disclosed herein, the anti-cancer activity of the agent is decreased by an MLKL inhibitor. In some embodiments of methods disclosed herein, the agent selectively causes reactive oxygen species generation in cancer cell mitochondria. In some embodiments of methods disclosed herein, the agent selectively causes lipid peroxidation in cancer cells. In some embodiments of methods disclosed herein, the agent selectively causes double stranded DNA breaks in the genome of cancer cells.

In some embodiments of methods disclosed herein, the agent selectively causes phosphatidylserine exposure in cancer cells. In some embodiments, the agent decreases MLKL dimerization. In some embodiments, the anti-cancer agent is an MLKL dimerization inhibitor.

[0026] In some embodiments of methods disclosed herein, the cancer cells are selected from the group consisting of leukemia, small cell lung carcinoma cells, colon cancer cells, CNS cancer cells, melanoma cells, ovarian cancer cells, renal cancer cells, prostate cancer cells, and breast cancer cells. In some embodiments, the cancer cells are not leukemia cells. In some embodiments, the cancer cells are not small cell lung carcinoma cells. In some embodiments, the cancer cells are not colon cancer cells. In some embodiments, the cancer cells are not CNS cancer cells. In some embodiments, the cancer cells are not melanoma cells. In some embodiments, the cancer cells are not ovarian cancer cells. In some embodiments, the cancer cells are not renal cancer cells. In some embodiments, the cancer cells are not prostate cancer cells. In some embodiments, the cancer cells are not breast cancer cells.

[0027] In some embodiments of methods disclosed herein, the agent is contacted with the cancer cells in vivo. In some embodiments, the agent is administered to a subject (e.g., a human or mouse). In some embodiments, the subject has cancer. In some embodiments, the agent causes (e.g., selectively causes) rPCD in cancer cells. In some embodiments, rPCD is defined as a novel cell death mechanism characterize by 4 distinct features: (1) Kinetics = l-hour exposure to an rPCD agonist is sufficient to induce cell death, irreversibly. (2) Down Stream Signature = cleaved- PARP-l POSITIVE / cleaved-Caspase-3 NEGATIVE cell population (detectable by FACS). The rPCD- signature population is detectable as early as after 15 minutes of exposure to an rPCD agonist, peaking after 1 hour of exposure. (3) Selectivity = rPCD is cancer specific, and is not triggered in normal cells. rPCD is not triggered by standard induction chemotherapy (iCT), apoptosis agonist (staurosporine,; pan-protein kinase inhibitor), Ferroptosis agonist (RSL3) or Necroptosis agonists (TZS). (4) Dependency = rPCD is MLKL-dependent.

[0028] In some embodiments, the anti-cancer activity of the agent is cysteine protease dependent. In some embodiments, the agent causes (e.g., specifically causes) intracellular PARP-l cleavage prior to CASP-3 cleavage in cancer cells. In some embodiments, the agent causes (e.g., specifically causes) intracellular phosphorylation of g-H2AC. In some embodiments, the agent inhibits MLKL dimerization.

[0029] Some aspects of the disclosure are related to a method of screening one or more test agents for a candidate anti-cancer agent, comprising contacting the test agent with a cancer cell; assessing whether the cancer cell undergoes regulated cell death (ie., rPCD); and determining that the test agent is a candidate anti-cancer agent if the cancer cell undergoes regulated cell death. In some embodiments, the step of "assessing whether the cancer cell undergoes regulated cell death" comprises measuring gene expression levels in the contacted cancer cell. In some embodiments, the step of "assessing whether the cancer cell undergoes regulated cell death" comprises measuring MLKL activation in the contacted cancer cell. In some embodiments, the step of "assessing whether the cancer cell undergoes regulated cell death" comprises measuring MLKL dimerization and/or multimerization in the contacted cancer cell.

[0030] In some embodiments, the cancer cell comprises phosphorylated MLKL. In some embodiments, the cancer cell is a leukemia cell, a small cell lung carcinoma cell, a colon cancer cell, a CNS cancer cell, a melanoma cell, an ovarian cancer cell, a renal cancer cell, a prostate cancer cell, or a breast cancer cell.

[0031] Some aspects of the disclosure are related to a method of screening for a cancer cell susceptible to treatment with an anti-cancer agent, comprising detecting phosphorylated MLKL in the cancer cell, wherein if phosphorylated MLKL is detected in the cancer cell then the cancer cell is determined to be susceptible to treatment with the anti-cancer agent.

[0032] In some embodiments, phosphorylated MLKL is detected with an antibody. In some embodiments, phosphorylated MLKL is detected by

immunoprecipitation .

[0033] In some embodiments, the anti-cancer agent is an MLKL modulator. In some embodiments, the anti-cancer agent is an MLKL activator. In some

embodiments, the anti-cancer agent is an MLKL dimerization inhibitor.

[0034] In some embodiments, the method further comprises measuring MLKL dimerization and/or multimerization in the cancer cell.

[0035] In some embodiments, the cancer cell is a leukemia cell, a small cell lung carcinoma cell, a colon cancer cell, a CNS cancer cell, a melanoma cell, an ovarian cancer cell, a renal cancer cell, a prostate cancer cell, or a breast cancer cell.

In some embodiments, the cancer cell is obtained from a subject having cancer. In some embodiments, the cancer cell is obtained during a biopsy from a subject. In some embodiments, the method further comprises treating the subject with the anti cancer agent.

[0036] Some aspects of the disclosure are related to a method of screening for a cancer cell susceptible to treatment with an anti-cancer agent, comprising detecting an elevated level of MLKL in the cancer cell as compared to a control, wherein if an elevated MLKL is detected in the cancer cell then the cancer cell is determined to be susceptible to treatment with the anti-cancer agent. In some embodiments, the control is the level of MLKL in a non-cancerous cell (or population of non-cancerous cells) from which the cancerous cell is derived from. In some embodiments, the control is the level of MLKL in a CBX resistance cancer cell (or population of cells).

[0037] In some embodiments, MLKL is detected with an antibody. In some embodiments, MLKL is detected by immunoprecipitation.

[0038] In some embodiments, the anti-cancer agent is an MLKL modulator. In some embodiments, the anti-cancer agent is an MLKL activator. In some

embodiments, the anti-cancer agent is an MLKL dimerization inhibitor.

[0039] In some embodiments, the cancer cell is a leukemia cell, a small cell lung carcinoma cell, a colon cancer cell, a CNS cancer cell, a melanoma cell, an ovarian cancer cell, a renal cancer cell, a prostate cancer cell, a cervical cancer cell, or a breast cancer cell. In some embodiments, the cancer cell is obtained from a subject having cancer. In some embodiments, the cancer cell is obtained during a biopsy. In some embodiments, the method further comprises treating the subject with the anti cancer agent.

[0040] The practice of the present invention will typically employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant nucleic acid (e.g., DNA) technology, immunology, and RNA interference (RNAi) which are within the skill of the art. Non-limiting descriptions of certain of these techniques are found in the following publications: Ausubel, F., et ah, (eds.), Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, all John Wiley & Sons, N.Y., edition as of December 2008; Sambrook, Russell, and Sambrook, Molecular Cloning: A

Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and Lane, D., Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988; Freshney, R.I.,“Culture of Animal Cells, A Manual of Basic Technique”, 5th ed., John Wiley & Sons, Hoboken, NJ, 2005. Non-limiting information regarding therapeutic agents and human diseases is found in Goodman and Gilman’s The Pharmacological Basis of Therapeutics, l lth Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill/ Appleton & Lange; lOth ed. (2006) or l lth edition (July 2009). Non-limiting information regarding genes and genetic disorders is found in McKusick, V.A.: Mendelian Inheritance in Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (l2th edition) or the more recent online database: Online Mendelian Inheritance in Man, OMIM™. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University

(Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD), as of May 1, 2010, available on the World Wide Web at ncbi.nlm.nih.gov/omim/ and in Online Mendelian Inheritance in Animals (OMIA), a database of genes, inherited disorders and traits in animal species (other than human and mouse), at omia.angis.org.au/contact.shtml.

[0041] All patents, patent applications, and other publications (e.g., scientific articles, books, websites, and databases) mentioned herein are incorporated by reference in their entirety. In case of a conflict between the specification and any of the incorporated references, the specification (including any amendments thereof, which may be based on an incorporated reference), shall control. Standard art- accepted meanings of terms are used herein unless indicated otherwise. Standard abbreviations for various terms are used herein.

[0042] The above discussed, and many other features and attendant advantages of the present inventions will become better understood by reference to the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0044] FIG. 1 shows the results of varying doses of CBX on 4 leukemia cell lines, 9 Non-Small Cell Lung Cancer cell lines; 6 colon cancer cell lines, 6 Central Nervous System cancer cell lines, 8 Melanoma cell lines, 7 Ovarian cancer cell lines,

8 renal cancer cell lines, 2 prostate cancer cell lines, and 5 breast cancer cell lines. X- axis- log¹⁰ molar concentration of CBX; Y-axis- percent growth of cell line (negative values correspond to percent of cells killed). [0045] FIG. 2 shows the mean optical densities, percent growth, GI50 (50% Growth Inhibition), TGI (Total Growth Inhibition), and LC50 (Concentration killing 50% of cells) for the cancer cell lines of FIG. 1. Concentrations shown are log¹⁰ molar concentration of CBX.

[0046] FIG. 3 shows the GI50, TGI and LC50 log¹⁰ molar concentrations of CBX for the cell lines of FIG. 1. The accompanying bar graph illustrates the deviation in CBX concentrations from the average value for the cell lines of a specific cancer tested. For example, the CNS cell lines have very close GI50 values, and more variable LC50 values.

[0047] FIG. 4 shows the percent growth of all the cancer cell lines described in FIG. 1 at various CBX concentrations (left panel) and the percent growth of 6 normal cell lines at various CBX concentrations (right panel). X-axis- Log¹⁰ molar concentrations of CBX. Y-axis- percent growth (negative values indicate cell killing) with IC50, TGI and LC50 shown.

[0048] FIG. 5 shows the LC50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) for the cancer cell lines of FIG. 1 indicating a net loss of cells following treatment. LC50 is calculated from [(Ti-Tz)/Tz] x 100 = -50. Values are calculated for each of these three parameters if the level of activity is reached;

however, if the effect is not reached or is exceeded, the value for that parameter is expressed as greater or less than the maximum or minimum concentration tested.

[0049] FIG. 6 shows the CBX concentration required to block 100% of cell growth/proliferation (TGI) for the cancer cell lines of FIG. 1. The y-axis shows mM concentration of CBX. The drug concentration resulting in total growth inhibition (TGI) is calculated from Ti = Tz ()

[0050] FIG. 7 shows the CBX concentration required to block 50% of cell growth/proliferation (GI50) for the cancer cell lines of FIG. 1. The y-axis shows pM concentration of CBX. Growth inhibition of 50 % (GI50) is calculated from [(Ti- Tz)/(C-Tz)] x 100 = 50, which is the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation. [0051] FIG. 8 shows the CBX concentration (mM) required to achieve a 50% overall effect for the cancer cell lines of FIG. 1.

[0052] FIGS. 9A-9C show that MLKL protein is over-expresses in Acute Myeloid Leukemia (AML) cells and that CBX differentially activates MLKL. FIG. 9A shows that AML cells overexpress MLKL protein versus normal bone marrow. FIG. 9B shows that CBX differentially activates MLKL in leukemia versus normal bone marrow. FIG.9C shows that an MLKL inhibitor blocks CBX activity.

[0053] FIG. 10 shows that CBX (200 mM) induces caspase dependent cell death in mouse MLL-AF9 cells.

[0054] FIG. 11 shows that CBX induces Ca²⁺ influx in mouse and human leukemia cells, but not in normal human PB-MNC (Peripheral Blood Mononuclear Cells).

[0055] FIGS. 12A-12D show that CBX blocks mitochondrial respiration in mouse and human leukemia cells. Calculation of oxygen consumption rates at steady state in human (FIG. 12A) and mouse (FIG. 12B) cells. Average of >4 samples. FIG. 12C (human cells) and FIG. 12D (mouse cells): oxygen consumption rates before and after PBS or CBX injections. Arrows indicate time of compound injection (as indicated). Similar results were documented in all human and mouse cell lines tested.

[0056] FIGS. 13A-13D show a comparison of mitochondrial stress and CBX effects on oxygen consumption in normal human PB-MNC (FIG. 13A), Human leukemia (FIG. 13B), normal mouse PB-MNC (FIG. 13C), and mouse primary leukemia (FIG. 13D).

[0057] FIG. 14 shows that anti-mycin-mediated mitochondrial stress is not toxic to leukemia cells.

[0058] FIG. 15 shows that anti-mycin-mediated mitochondrial arrest does not induce Ca²⁺ influx in leukemia cells.

[0059] FIG. 16 shows that CBX activity is Ca²⁺ dependent. Human MLL- AF9 cell line Nomo-l was treated with DMSO (control) or DMSO+BAPTA-AM (Ca²⁺ chelator) followed by treatment with PBS (blue-control) or CBX (200 mM- red) fifteen minutes later. Measurements taken 3 hours after CBX treatment. The results show that Ca²⁺ chelation blocks CBX activity as measured by the prevalence of apoptotic cells (Annexin-V+/7ADDD-). [0060] FIG. 17 shows that CBX-mediated mitochondrial arrest is transient.

[0061] FIG. 18 shows that CBX induces oxidative stress in liver mitochondria, which is responsible for pore opening. Adopted from Salvi et al, Endocrinology 145(5):2305-2312 (2005).

[0062] FIGS. 19A-19C show that CBX induces mitochondrial reactive oxygen species generation. FIG. 19A shows that CBX causes over a 5-fold increase in mitochondrial ROS in leukemia cells versus normal PB-MNC. FIG. 19B shows that CBX causes the generation of peroxyl radicals (lipid peroxidation) in leukemia cells but not in normal PB-MNC. FIG. 19C shows CBX causes double-stranded DNA breaks in leukemia cells but not in normal PB-MNC.

[0063] FIG. 20 shows that CBX-induced lipid peroxidation may be mediated by lipoxygenase activity. Human MLL-AF9 cell line Nomo-l was treated with DMSO (control) or DMSO+Baicalein (lipoxygenase inhibitor) followed by treatment with PBS (control- blue) or CBX (200 mM- red) fifteen minutes later. Measurements taken 3 hours after CBX treatment. The results show that lipoxygenase inhibition reduces CBX activity as measured by the prevalence of apoptotic cells (Annexin- V+/7ADDD-).

[0064] FIG. 21 shows that CBX activity is iron-dependent but not ferroptosis- dependent. Control (blue) and CBX-treated (red) leukemia cells were pretreated with DFO (deferoxamine- iron chelator), LXN1 (liproxstatin-l- inhibitor of GPX4- mediated ferroptosis, or aToc (a- tocopherol/vitamin E- lipophilic antioxidant) for 1 hour. Then 200 pM CBX was added and cell viability (7AAD) was measured 24 hours later. X-axis shows concentration (pM) of inhibitor added. The results show that CBX activity is iron dependent but not dependent upon ferroptosis.

[0065] FIG. 22 shows that MLKL inhibition but not RIPK1 inhibition blocks CBX activity. Leukemia cells were pretreated with DMSO (control), Neel (inhibitor of RIPK1 (Serine-threonine kinase which transduces inflammatory and cell-death signals)), or NSA (MLKL inhibitor) and then treated with PBS(control- blue) or 200 pM CBX.

[0066] Fig. 23 shows necrosulfonamide (a MKLK inhibitor) blocks CBX cell death induction in Human AML (MLL-AF9) cells. [0067] FIG. 24 shows that MLKL is phosphorylated in leukemia cells but not in normal cells.

[0068] FIG. 25 shows that CBX induces cell death through a number of different pathways.

[0069] FIG. 26 shows that different cells have a different response to CBX. Different types of cancer cells undergo rPCD, previously known programmed cell death, or proliferation arrest. Non-cancerous cells did not have adverse effects caused by CBX. Modified from Galluzzi at al, 2018, Figure 1 (Cell Death Differ. 2018 Mar; 25(3): 486-541).

[0070] FIG. 27 shows that melanoma (A375), which undergoes rPCD in response to CBX, exhibits an about 100 kda MLKL band (total MLKL) while breast cancer cells (MDA-MB-231), which do not undergo rPCD in response to MLKL, exhibits an about 55 kda MLKL band (total MLKL), suggesting a simple western blot of a patient's cancer cells may predict susceptibility to an MLKL modulator.

[0071] FIG. 28 shows that CBX-induced cell death is regulated by cysteine protease activity.

[0072] FIG. 29 shows that in the Nomol human AML cell line, PARP-l cleavage proceeds CASP-3 cleavage after treatment with 200 mM CBX. Times shown are post CBX addition. FACS plot.

[0073] FIG. 30 shows that in the Nomol human AML cell line, PARP-l cleavage leads to rapid DNA damage signal (cleaved CASP-3) after treatment with 200 pM CBX. Times shown are post CBX addition. FACS plot.

[0074] FIG. 31 shows that in the Nomol human AML cell line, knockdown of MLKL expression inhibits PARP-l and CASP-3 cleavage caused by treatment with 200 pM CBX. Times shown are post CBX addition. FACS plot.

[0075] FIG. 32 shows that in the Nomol human AML cell line, knockdown of MLKL expression inhibits PARP-l cleavage and CASP-3 phosphorylation caused by treatment with 200 pM CBX. Times shown are post CBX addition. FACS plot.

[0076] FIG. 33 is flowcharts showing that CBX-induced rPCD is executed by MLKL-regulated proteolysis. rPCD has an execution time of 30-60 minutes. Known Programmed Cell Death (PCD) leading to DNA breakdown has an execution time of 4-8 hours. As illustrated, MLKL is an upstream activator of a currently unidentified calcium sensing cysteine protease. Further, in rPCD, PARP-l is cleaved prior to CASP-3, unlike PCD.

[0077] FIG. 34 shows the structure of MLKL. Adapted from Dondelinger et al., Cell Reports, Vol. 7, pp. 971-981 (2014).

[0078] FIG. 35 shows MLKL activation and membrane rupture. Adapted from Quarato et al., Mol. Cell, Vol. 61, No. 14, pp. 589-601 (2016).

[0079] FIG. 36 illustrates Alternative splicing forms of human and mouse MLKL. Included is a putative murine MLKL isoform having a mass of about 20-30 kda .

[0080] FIG. 37 provides the molecular weights (kda) for mouse and human MLKL isoforms, including the estimated kda for MLKL heterogeneous and homogeneous multimers. FL=Full Length isoform; S= Short isoform.

[0081] FIG. 38 provides the sequence of the amino acid region deleted from the human MLKL short isoform. This sequence is a protein kinase domain that is catalytically inactive but contains an unusual pseudoactive site with an interaction between Lys-230 and Gln-356 residues. Upon phosphorylation by RIPK3, the protein kinase domain undergoes an active conformation. The sequence of the human short isoform differs from the canonical sequence as follows: 179-205:

YLPPKCMQEIPQEQIKEIKKEQLS GSPS LES S S GKSPLEISRFKVKNVKTGS AS

(SEQ ID NO: 1).

[0082] FIG. 39 illustrates the anti-MLKL antibodies used for detection of MLKL in mouse and human cells.

[0083] FIG. 40 shows total MLKL in murine bone marrow cells and primary MLL-AF9 leukemia after CBX treatment (200 mM). Red arrow signifies putative short MLKL isoform at around 30 kda. Automatic exposure.

[0084] FIG. 41 shows total MLKL in murine bone marrow cells and primary MLL-AF9 leukemia after CBX treatment (200 pM). Red arrow signifies putative short MLKL isoform at around 30 kda. Maximum exposure for increased sensitivity.

[0085] FIG. 42 shows phosphorylated MLKL in murine bone marrow cells and primary MLL-AF9 leukemia after CBX treatment (200 pM). Red arrow signifies putative short MLKL isoform at around 30 kda. [0086] FIGS. 43A-43D. FIG. 43A shows SDH expression (e.g.,

hyperactivity) is associated with poor prognosis. FIG. 43B shows SDH protein is differentially expressed in mouse MLL-AF9 AML compared to normal myeloid progenitor cells. FIG. 43C shows SDH is hyperactive in blood cancer cells compared to normal cells. FIG. 43D shows CBX inhibits SDH activity in human leukemia lysates in vitro. Y-axis= activity mU/ml.

[0087] FIG. 44 shows knockdown of SDH via shRNA suppresses CBX- induced cell death in mouse primary AML and Nomo-l (human AML cell line).

[0088] FIG. 45 illustrates that MLKL knockdown in mouse primary leukemia cells (eASl2) suppresses loss of cell viability due to CBX treatment.

[0089] FIG. 46 illustrates that MLKL knockdown in mouse primary leukemia cells (eASl2) and human NOMOl AML cells suppresses loss of cell viability due to CBX treatment.

[0090] FIG. 47 illustrates a schematic for identifying the calcium sensing cysteine protease downstream of MLKL leading to rPCD.

[0091] FIG. 48 illustrates the time course of downstream rPCD executers in mouse leukemia cells contacted with CBX and various inhibitors.

[0092] FIG. 49 illustrates the time course of downstream rPCD executers in human leukemia cell line NOMOl contacted with CBX and various inhibitors.

[0093] FIG. 50 shows that interference with MLKL dimerization selectively kills tumor cells. NSA, which blocks MLKL dimerization in human cells (see, Reynoso et ah, J Biol Chem. 20l7;292(42): 17514-17524), selectively kills human tumor cells (AML) but not cord blood cells, or human peripheral blood cells. NSA is specific for human MLKL but, at higher concentrations, has non-specific effects on mouse AML cells.

[0094] FIGS. 51A-51D show Oxygen Consumption Rate of Human and Mouse Leukemia and Normal Cells. ATP = adenosine triphosphate; BM-MNC =bone marrow mononuclear cells; CBX = carbenoxolone disodium salt; PB-MNC = peripheral blood mononuclear cells; PBS = phosphate buffered saline.

[0095] FIGS. 52A-52B show a Comparison of Mitochondrial Stress and CBX Effects on Oxygen Consumption in Normal (A) or Leukemia (B) Human Cells. CBX = carbenoxolone disodium salt; PBS = phosphate buffered saline; Mito-Stress = Mitochondrial stress was induced by ATP synthase inhibitor, FCCP H⁺ ionophor, or antimycin complex III inhibitor. The green and red arrows indicate introduction of treatment.

[0096] FIG. 53 shows a comparison of CBX effects on glycolytic function in normal mouse BM-MNCs and primary leukemia cells (eASl2).

[0097] FIG. 54 shows glycolytic consumption rate of human normal BM- MNC and leukemia cells.

[0098] FIG. 55 shows that CBX-senstitive primary leukemia and CBX- resistant leukemia have similar glycolytic function at base line but different responses to mitochondrial stress.

[0099] FIG. 56 shows that normal and leukemia cells display different metabolic response to CBX.

[0100] FIGS. 57A-57C show Superoxide Radicals, Peroxyl Radicals, and Double-strand DNA Breaks in Normal and Leukemic Cells Following Incubation with CBX. CBX = carbenoxolone disodium salt; DNA = deoxyribonucleic acid; ROS = reactive oxygen species.

[0101] FIG. 58 shows calcium (Ca²⁺) influx in human healthy peripheral blood mononuclear cCells versus leukemia cells following incubation with CBX.

CBX = carbenoxolone disodium salt; min. = minute; Mito-Stress = Mitochondrial stress was induced by ATP synthase inhibitor; FCCP H⁺ ionophor, or antimycin complex III inhibitor; PB-MNC = peripheral blood mononuclear cells; PBS = phosphate buffered saline. The green and red arrows indicate introduction of treatment.

[0102] FIG. 59 shows that MLKL knockdown causes a shift from oxygen consumption to extracellular acidification.

[0103] FIG. 60 shows that rPCD is a new form of caspase independent cell death (CICD). Mouse AML cells harvested from bone marrow were exposed to the listed agents for one hour, followed by washing and plating the exposed cells. As shown, exposure for 1 hour to CBX, but not iCT or RSL3, caused significant AML cell death. Rapid cell death is a hallmark of rPCD.

[0104] FIG. 61 shows a downstream signature of rPCD via exposure to CBX: cleaved PARP1 followed by cleaved CASP3. [0105] FIG. 62 shows a downstream signature of rPCD in AML cells exposed to CBX.

[0106] FIG. 63 shows that CBX selectivity for AML cells versus selectivity of other anti-cancer agents.

[0107] FIG. 64 shows that CBX sensitivy melanoma cell line A375 has MLKL polymers.

[0108] FIG. 65A-65D shows 4 features of rPCD. : (FIG. 65 A) Kinetics = 1- hour exposure to an rPCD agonist is sufficient to induce cell death, irreversibly. (FIG. 65B) Down Stream Signature = cleaved-PARP- 1 POSITIVE / cleaved-Caspase-3 NEGATIVE cell population (detectable by FACS). The rPCD- signature population is detectable as early as after 15 minutes of exposure to an rPCD agonist, peaking after 1 hour of exposure. (FIG. 65C) Selectivity = rPCD is cancer specific, and is not triggered in normal cells. rPCD is not triggered by standard induction chemotherapy (iCT), apoptosis agonist (staurosporine,; pan-protein kinase inhibitor),

Ferroptosis agonist (RSL3) or Necroptosis agonists (TZS). (FIG. 65D) Dependency = rPCD is MLKL-dependent.

DETAILED DESCRIPTION OF THE INVENTION

[0109] MLKL Modulator Methods

[0110] Some aspects of the disclosure are directed to a method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an MLKL modulator. In some embodiments, the cancer cells are not leukemic cells. In some embodiments, the MLKL modulator is not carbenoxolone or a derivative or analog thereof. In some embodiments, the MLKL modulator is not 1 S-P-glycyrrhctinic acid or a derivative thereof. In some

embodiments, the derivative of 1 S-P-glycyrrhctinic acid is selected from the group consisting of glycyrrhizine, glycyrrhizinic acid, carbenoxolone or 2-hydroxyethyl- 1 SP-glycyrrhctinic acid amide. In some embodiments, the MLKL modulator is not a gap junction blocker as described in US Publication No. 2016/0367578, published December 22, 2016 (incorporated herein by reference in its entirety).

[0111] In some embodiments, the cancer cells have elevated levels of phosphorylated MLKL as compared to non-cancerous cells. In some embodiments, the phosphorylated MLKL is phosphorylated at residue serine 345 (murine) or the corresponding residue in human MLKL. In some embodiments, the MLKL modulator selectively modulates MLKL in cancer cells.

[0112] Example of MLKL modulators, include, e.g., small molecules, polypeptides, nucleic acids (e.g., RNAi agents, antisense oligonucleotide, aptamers), lipids, polysaccharides, etc. In general, MLKL modulators may be obtained using any suitable method known in the art. The ordinary skilled artisan will select an appropriate method based, e.g., on the nature of the MLKL modulators. A MLKL modulator may be at least partly purified. In some embodiments a MLKL modulator may be provided as part of a composition, which may contain, e.g., a counter-ion, aqueous or non-aqueous diluent or carrier, buffer, preservative, or other ingredient, in addition to the agent, in various embodiments. In some embodiments a MLKL modulator may be provided as a salt, ester, hydrate, or solvate. In some embodiments a MLKL modulator is cell-permeable, e.g., within the range of typical agents that are taken up by cells and act intracellularly, e.g., within mammalian cells, to produce a biological effect. Certain compounds may exist in particular geometric or

stereoisomeric forms. Such compounds, including cis- and trans-isomers, E- and Z- isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, (-)- and (+)- isomers, racemic mixtures thereof, and other mixtures thereof are encompassed by this disclosure in various embodiments unless otherwise indicated. Certain compounds may exist in a variety or protonation states, may have a variety of configurations, may exist as solvates (e.g., with water (i.e. hydrates) or common solvents) and/or may have different crystalline forms (e.g., polymorphs) or different tautomeric forms. Embodiments exhibiting such alternative protonation states, configurations, solvates, and forms are encompassed by the present disclosure where applicable. In some embodiments, "selectively" is used herein to mean at least about a 1.1 -fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or lO-fold preference for the identified subgenus (e.g., cancer cell) versus the genus (e.g., cell). In some embodiments, "selectively" is used herein to mean at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or as much as 100% for the identified subgenus (e.g., cancer cell) versus the genus (e.g., cell).

[0113] In some instances, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or as much as 100% of the cancer cells in the population of cells are eradicated, reduced, or inhibited (e.g., growth/proliferation inhibited) by exposure to or contact with the MLKL modulator. In some embodiments, at least 20% of the cancer cells in the population of cells are eradicated, reduced, or inhibited. In some embodiments, at least 50% of the cancer cells in the population of cells are eradicated, reduced, or inhibited. In some embodiments, at least 70% of the cancer cells in the population of cells are eradicated, reduced, or inhibited. In some embodiments, all of the cancer cells in the population of cells are eradicated, reduced, or inhibited.

[0114] The term“cancer” as used herein is defined as a hyperproliferation of cells whose unique trait— loss of normal controls— results in unregulated growth, lack of differentiation, local tissue invasion, and metastasis. With respect to the inventive methods, the cancer can be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma,

gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, leukemia, liquid tumors, liver cancer, lung cancer, lymphoma, malignant mesothelioma, mastocytoma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, and urinary bladder cancer. As used herein, the term“tumor” refers to an abnormal growth of cells or tissues of the malignant type, unless otherwise specifically indicated and does not include a benign type tissue.

[0115] In some embodiments, the cancer cells are solid tumor cancer cells. In some embodiments, the solid tumor is a liver, breast, gastrointestinal tract (e.g., colon cancer, esophageal cancer), cervical, ovarian, pancreatic, renal, prostate, esophageal, lung, or brain cancer (e.g., glioblastoma). In some embodiments, the cancer cells are selected from the group consisting of small cell lung carcinoma cells, colon cancer cells, CNS cancer cells, melanoma cells, ovarian cancer cells, renal cancer cells, prostate cancer cells, and breast cancer cells. In some embodiments, the cancer cells are selected from the group consisting of small cell lung carcinoma cells, colon cancer cells, CNS cancer cells, ovarian cancer cells, and renal cancer cells. In some embodiments, the cancer cells are not breast cancer cells. In some embodiments, the cancer cells are not melanoma cells. In some embodiments, the cancer cells are not prostate cancer cells. In some embodiments, the cancer cells are not cervix cancer cells. In some embodiments, the cancer cells are not cervical cancer cells. In some embodiments, the cancer cells are not lung cancer cells. In some embodiments, the cancer cells are not leukemia.

[0116] As used herein“modulating” (and verb forms thereof, such as “modulates”) means causing or facilitating a qualitative or quantitative change, alteration, or modification in a molecule, a process, pathway, or phenomenon of interest. Without limitation, such change may be an increase, decrease, a change in binding characteristics, or change in relative strength or activity of different components or branches of the process, pathway, or phenomenon.

[0117] In some embodiments, the MLKL modulator modulates MLKL phosphorylation, dimerization, or multimerization. In some embodiments, MLKL phosphorylation, dimerization, or multimerization is selectively modulated in cancer cells. [0118] In some embodiments, the MLKL modulator increases MLKL phosphorylation, dimerization, or multimerization. In some embodiments, MLKL phosphorylation, dimerization, or multimerization is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 500%, 600% or more. In some embodiments, MLKL phosphorylation, dimerization, or multimerization is decreased by about l-fold, 2-fold, 3 -fold, 4-fold, 5-fold, 6-fold or more.

[0119] In some embodiments, the MLKL modulator decreases MLKL phosphorylation, dimerization, or multimerization. MLKL phosphorylation, dimerization, or multimerization is decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,

93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some embodiments, MLKL phosphorylation, dimerization, or multimerization is decreased by about l-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold or more.

[0120] In some embodiments, the MKLK modulator increases MLKL activity. In some embodiments, MLKL activity is selectively modulated in cancer cells. In some embodiments, MLKL activity is increased by about 10%, 20%, 30%, 40%,

50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 500%, 600% or more. In some embodiments, MLKL activity is decreased by about l-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold or more. In some embodiments, the MLKL modulator selectively increases MLKL activity in cancer cells. In some

embodiments, the MLKL modulator causes minimal or no increases in MLKL activity in non-cancerous cells.

[0121] In some embodiments, the MLKL modulator selectively causes metabolic arrest in cancer cells. In some embodiments, the MLKL modulator causes minimal or no metabolic arrest in non-cancerous cells. In some embodiments, metabolic arrest comprises a reduction or elimination of mitochondrial respiration (e.g., oxygen consumption rate). In some embodiments, metabolic arrest comprises a reduction or stoppage of the tricarboxylic acid cycle (TCA cycle). In some embodiments, a reduction or stoppage of the TCA cycle comprises a reduction, relative to untreated cells, in the metabolite level of one or more of glutamine, glutamate, alpha-KG, succinate, fumarate, and malate. In some embodiments, metabolic arrest comprises a reduction or stoppage of glycolysis. In some

embodiments, a reduction or stoppage of glycolysis comprises a reduction, relative to untreated cells, in the metabolite level of one or more of glucose, lactate, and pyruvate. In some embodiments, the MLKL modulator causes transient metabolic arrest (e.g., transient mitochondrial metabolic arrest). In some embodiments, the transient arrest occurs for less than about 2 hours, less than about 1.5 hours, less than about 1 hour, less than about 40 minutes, less than about 30 minutes, less than about 25 minutes, less than about 20 minutes, or less than about 10 minutes after contact with the MLKL modulator.

[0122] In some embodiments, the anti-cancer activity (i.e., the ability of the MLKL modulator to eradicate, reduce, or inhibit cancer cells) of the MLKL modulator is decreased or eliminated by calcium chelation. In some embodiments, the calcium chelator is l,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester)( BAPTA-AM). The calcium chelator is not limited and may be any calcium chelator in the art. In some embodiments, the anti-cancer activity of the MLKL modulator is decreased or eliminated by iron chelation. In some embodiments, the iron chelator is Desferoxamine (DFO). The iron chelator is not limited and may be any iron chelator in the art. In some embodiments, the anti-cancer activity of the MLKL modulator is not decreased (e.g., significantly decreased) or eliminated by a ferroptosis inhibitor (e.g, a non-iron chelating ferroptosis inhibitor).

In some embodiments, the ferroptosis inhibitor is liprox statin- 1 (LXN1) or a- tocopherol (aToc). The ferroptosis inhibitor is not limited and may be any ferroptosis inhibitor in the art.

[0123] In some embodiments, the anti-cancer activity of the MLKL modulator is decreased or eliminated by an MLKL inhibitor. In some embodiments, the MLKL inhibitor is Necrosulfonamide (NSA). In some embodiments, the MLKL inhibitor is TC 13172, or NTB451. The MLKL inhibitor is not limited and may be any ferroptosis inhibitor in the art. In some embodiments, the anti-cancer activity of the MLKL modulator is not decreased or eliminated by Necrostatin-l.

[0124] In some embodiments, the MLKL modulator selectively causes reactive oxygen species (ROS) generation in cancer cell mitochondria. In some embodiments, the MLKL modulator cause minimal or no ROS generation in non- cancerous cell mitochondria. In some embodiments, the MLKL modulator preferentially causes about a 1.5-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or lO-fold increase in ROS generation in the mitochondria of the cancer cells over non-cancer cells.

[0125] In some embodiments, the MLKL modulator selectively causes lipid peroxidation in cancer cells. In some embodiments, the MLKL modulator causes minimal or no lipid peroxidation in non-cancer cells. In some embodiments, the MLKL modulator preferentially causes about a 1.5-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or lO-fold increase in lipid peroxidation in the cancer cells over non-cancer cells.

[0126] In some embodiments, the MLKL modulator selectively causes double stranded DNA breaks in the genome of cancer cells. In some embodiments, the MLKL modulator causes minimal or no double stranded DNA breaks in non-cancer cells. In some embodiments, the MLKL modulator preferentially causes about a 1.5- fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,

9-fold, or lO-fold increase in double stranded DNA breaks in the genomes of the cancer cells over non-cancer cells.

[0127] In some embodiments, the MLKL modulator selectively causes phosphatidylserine exposure in cancer cells. In some embodiments, the MLKL modulator causes minimal or no phosphatidylserine exposure in non-cancer cells. In some embodiments, the MLKL modulator preferentially causes about a 1.5-fold, 1.7- fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or

10-fold increase phosphatidylserine exposure in the cancer cells over non-cancer cells.

[0128] In some embodiments, the MLKL modulator is contacted with the cancer cells in vivo. In some embodiments, the MLKL modulator is administered to a subject (e.g., a human or mouse). In some embodiments, the subject is a mouse. In some embodiments, the subject is a human. As used herein, a“subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents.

[0129] In some embodiments, the subject suffers from cancer. In some embodiments, the subject suffers from leukemia, small cell lung carcinoma, colon cancer, CNS (e.g., brain) cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, or breast cancer. In some embodiments, the subject suffers from small cell lung carcinoma, colon cancer, CNS cancer, ovarian cancer, or renal cancer. In some embodiments, the subject does not suffer from leukemia, breast cancer, melanoma, prostate cancer, or cervix cancer. The cancer is not limited and may be any cancer described herein.

[0130] Anti-Cancer Agent Methods

[0131] Some aspects of the disclosure are related to a method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an agent, wherein the agent selectively increases calcium flux in cancer cells. In some embodiments, the cancer cells are not leukemic cells. In some embodiments, the agent is not carbenoxolone or a derivative or analog thereof. In some embodiments, the agent is not 1 S-P-glycyrrhctinic acid or a derivative thereof.

In some embodiments, the derivative of 1 S-P-glycyrrhctinic acid is selected from the group consisting of glycyrrhizine, glycyrrhizinic acid, carbenoxolone or 2- hydroxycthyl- 18P-glycyrrhctinic acid amide. In some embodiments, the agent is not a gap junction blocker as described in US Publication No. 2016/0367578, published December 22, 2016. In some embodiments, the agent is an MLKL modulator as described herein. Other example of agents, include, e.g., small molecules, polypeptides, nucleic acids (e.g., RNAi agents, antisense oligonucleotide, aptamers), lipids, polysaccharides, etc.

[0132] Some aspects of the disclosure are related to a method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an agent, wherein the agent selectively causes transient metabolic arrest in cancer cells. In some embodiments, the cancer cells are not leukemic cells. In some embodiments, the agent is not carbenoxolone or a derivative or analog thereof. In some embodiments, the agent is not 1 S-P-glycyrrhctinic acid or a derivative thereof. In some embodiments, the derivative of 1 S-P-glycyrrhctinic acid is selected from the group consisting of glycyrrhizine, glycyrrhizinic acid,

carbenoxolone or 2-hydroxycthyl-l 8P-glycyn hctinic acid amide. In some

embodiments, the agent is not a gap junction blocker as described in US Publication No. 2016/0367578, published December 22, 2016. In some embodiments, the agent is an MLKL modulator as described herein.

[0133] Some aspects of the disclosure are related to a method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an agent, wherein the agent selectively causes mitochondrial reactive oxygen species generation in cancer cells. In some embodiments, the cancer cells are not leukemic cells. In some embodiments, the agent is not carbenoxolone or a derivative or analog thereof. In some embodiments, the agent is not 18-b- glycyrrhetinic acid or a derivative thereof. In some embodiments, the derivative of l8-P-glycyrrhetinic acid is selected from the group consisting of glycyrrhizine, glycyrrhizinic acid, carbenoxolone or 2-hydroxycthyl-l 8P-glycyn hctinic acid amide. In some embodiments, the agent is not a gap junction blocker as described in US Publication No. 2016/0367578, published December 22, 2016. In some

embodiments, the agent is an MLKL modulator as described herein.

[0134] Some aspects of the disclosure are related to a method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an agent, wherein the agent selectively causes lipid peroxidation in cancer cells. In some embodiments, the cancer cells are not leukemic cells. In some embodiments, the agent is not carbenoxolone or a derivative or analog thereof. In some embodiments, the agent is not 1 S-P-glycyrrhctinic acid or a derivative thereof. In some embodiments, the derivative of 1 S-P-glycyrrhctinic acid is selected from the group consisting of glycyrrhizine, glycyrrhizinic acid,

carbenoxolone or 2-hydroxycthyl-l SP-glycyrrhctinic acid amide. In some

[0135] Some aspects of the disclosure are related to a method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an agent, wherein the agent selectively causes double stranded DNA breaks in cancer cells. In some embodiments, the cancer cells are not leukemic cells. In some embodiments, the agent is not carbenoxolone or a derivative or analog thereof. In some embodiments, the agent is not 1 S-P-glycyrrhctinic acid or a derivative thereof. In some embodiments, the derivative of 1 S-P-glycyrrhctinic acid is selected from the group consisting of glycyrrhizine, glycyrrhizinic acid,

carbenoxolone or 2-hydroxycthyl-l SP-glycyrrhctinic acid amide. In some

[0136] Some aspects of the disclosure are related to a method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an agent, wherein the agent selectively causes

phosphatidylserine exposure in cancer cells. In some embodiments, the cancer cells are not leukemic cells. In some embodiments, the agent is not carbenoxolone or a derivative or analog thereof. In some embodiments, the agent is not 18-b- glycyrrhetinic acid or a derivative thereof. In some embodiments, the derivative of l8-P-glycyrrhetinic acid is selected from the group consisting of glycyrrhizine, glycyrrhizinic acid, carbenoxolone or 2-hydroxycthyl-l SP-glycyrrhctinic acid amide. In some embodiments, the agent is not a gap junction blocker as described in US Publication No. 2016/0367578, published December 22, 2016. In some embodiments, the agent is an MLKL modulator as described herein. [0137] In some embodiments, the agent modulates MLKL phosphorylation, dimerization, or multimerization. In some embodiments, the agent selectively modulates MLKL phosphorylation, dimerization, or multimerization in cancer cells.

[0138] In some embodiments, the agent increases MLKL phosphorylation, dimerization, or multimerization. In some embodiments, MLKL phosphorylation, dimerization, or multimerization is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 500%, 600% or more. In some embodiments, MLKL phosphorylation, dimerization, or

multimerization is decreased by about l-fold, 2-fold, 3 -fold, 4-fold, 5-fold, 6-fold or more.

[0139] In some embodiments, the agent decreases MLKL phosphorylation, dimerization, or multimerization. In some embodiments, MLKL phosphorylation, dimerization, or multimerization is decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,

93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some embodiments, MLKL phosphorylation, dimerization, or multimerization is decreased by about l-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold or more. In some embodiments, MLKL

dimerization is decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,

50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,

96%, 97%, 98%, 99% or more. In some embodiments, MLKL dimerization is decreased by about l-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold or more.

[0140] In some embodiments, the agent increases MLKL activity. In some embodiments, MLKL activity is increased by about 10%, 20%, 30%, 40%, 50%,

60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 500%, 600% or more. In some embodiments, MLKL activity is decreased by about l-fold, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold or more. In some embodiments, the agent selectively increases MLKL activity in cancer cells. In some embodiments, the agent causes minimal or no increases in MLKL activity in non-cancerous cells. [0141] In some embodiments, the agent selectively causes metabolic arrest in cancer cells. In some embodiments, the agent causes minimal or no metabolic arrest in non-cancerous cells. In some embodiments, metabolic arrest comprises a reduction or elimination of mitochondrial respiration (e.g., oxygen consumption rate). In some embodiments, metabolic arrest comprises a reduction or stoppage of the tricarboxylic acid cycle (TCA cycle). In some embodiments, a reduction or stoppage of the TCA cycle comprises a reduction, relative to untreated cells, in the metabolite level of one or more of glutamine, glutamate, alpha- KG, succinate, fumarate, and malate. In some embodiments, metabolic arrest comprises a reduction or stoppage of glycolysis. In some embodiments, a reduction or stoppage of glycolysis comprises a reduction, relative to untreated cells, in the metabolite level of one or more of glucose, lactate, and pyruvate. In some embodiments, the agent causes transient metabolic arrest (e.g., transient mitochondrial metabolic arrest). In some embodiments, the transient arrest occurs for less than about 2 hours, less than about 1.5 hours, less than about 1 hour, less than about 40 minutes, less than about 30 minutes, less than about 25 minutes, less than about 20 minutes, or less than about 10 minutes after contact with the agent.

[0142] In some embodiments, the anti-cancer activity (i.e., the ability of the agent to eradicate, reduce, or inhibit cancer cells) of the agent is decreased or eliminated by calcium chelation. In some embodiments, the calcium chelator is 1,2- Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester)( BAPTA-AM). The calcium chelator is not limited and may be any calcium chelator in the art. In some embodiments, the anti-cancer activity of the agent is decreased or eliminated by iron chelation. In some embodiments, the iron chelator is

Desferoxamine (DFO). The iron chelator is not limited and may be any iron chelator in the art. In some embodiments, the anti-cancer activity of the agent is not decreased (e.g., significantly decreased) or eliminated by a ferroptosis inhibitor (e.g., a non-iron chelating ferroptosis inhibitor). In some embodiments, the ferroptosis inhibitor is liprox statin- 1 (LXN1) or a-tocopherol (aToc). The ferroptosis inhibitor is not limited and may be any ferroptosis inhibitor in the art.

[0143] In some embodiments, the anti-cancer activity of the agent is decreased or eliminated by an MLKL inhibitor. In some embodiments, the MLKL inhibitor is Necrosulfonamide (NSA). The MLKL inhibitor is not limited and may be any ferroptosis inhibitor in the art. In some embodiments, the anti-cancer activity of the agent is not decreased or eliminated by Necrostatin-l.

[0144] In some embodiments, the agent selectively causes reactive oxygen species (ROS) generation in cancer cell mitochondria. In some embodiments, the agent cause minimal or no ROS generation in non-cancerous cell mitochondria. In some embodiments, the agent preferentially causes about a 1.5-fold, 1.7-fold, 1.8- fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or lO-fold increase in ROS generation in the mitochondria of the cancer cells over non-cancer cells.

[0145] In some embodiments, the agent selectively causes lipid peroxidation in cancer cells. In some embodiments, the agent causes minimal or no lipid peroxidation in non-cancer cells. In some embodiments, the agent preferentially causes about a 1.5-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, or lO-fold increase in lipid peroxidation in the cancer cells over non-cancer cells.

[0146] In some embodiments, the agent selectively causes double stranded DNA breaks in the genome of cancer cells. In some embodiments, the agent causes minimal or no double stranded DNA breaks in non-cancer cells. In some

embodiments, the agent preferentially causes about a 1.5-fold, 1.7-fold, 1.8-fold, 1.9- fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or lO-fold increase in double stranded DNA breaks in the genomes of the cancer cells over non-cancer cells.

[0147] In some embodiments, the agent selectively causes phosphatidylserine exposure in cancer cells. In some embodiments, the agent causes minimal or no phosphatidylserine exposure in non-cancer cells. In some embodiments, the agent preferentially causes about a 1.5-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or lO-fold increase phosphatidylserine exposure in the cancer cells over non-cancer cells.

[0148] In some embodiments, the agent is contacted with the cancer cells in vivo. In some embodiments, the agent is administered to a subject (e.g., a human or mouse). In some embodiments, the subject is a mouse. In some embodiments, the subject is a human. [0149] In some embodiments of methods disclosed herein, the cancer cells are selected from the group consisting of leukemia, small cell lung carcinoma cells, colon cancer cells, CNS cancer cells, melanoma cells, ovarian cancer cells, renal cancer cells, prostate cancer cells, and breast cancer cells. In some embodiments of methods disclosed herein, the cancer cells are not selected from the group consisting of leukemia, small cell lung carcinoma cells, colon cancer cells, CNS cancer cells, melanoma cells, ovarian cancer cells, renal cancer cells, prostate cancer cells, and breast cancer cells. In some embodiments of methods disclosed herein, the agent is contacted with the cancer cells in vivo. In some embodiments, the agent is administered to a subject (e.g., a human or mouse). In some embodiments, the subject has cancer.

[0150] Combination Methods

[0151] In some aspects of the disclosure, the methods disclosed herein further comprise contacting the population of cancer cells with a second anti-cancer agent.

An MLKL modulator or agent as described herein can be administered concurrently with, prior to, or subsequent to, one or more other additional agents. In some embodiments, the one or more additional agent is venetoclax or a hypomethylating agent.

[0152] In general, each therapeutic agent (e.g., MLKL modulator, agent) will be administered at a dose and/or on a time schedule determined for that particular agent. The particular combination to employ in a regimen will take into account compatibility of the MLKL modulator or agent with the additional agent and/or the desired therapeutic effect to be achieved.

[0153] Additional agents of the disclosure may include, but are not limited to chemotherapy agents, antibody-based agents, kinase inhibitors (e.g., tyrosine kinase inhibitors, serine/threonine kinase inhibitors, etc.), immunomodulatory agents and biologic agents or combinations thereof. Chemotherapy agents include, but are not limited to actinomycin D, aldesleukin, alitretinoin, all-trans retinoic acid/ATRA, altretamine, amascrine, asparaginase, azacitidine, azathioprine, bacillus calmette- guerin/BCG, bendamustine hydrochloride, bexarotene, bicalutamide, bleomycin, bortezomib, busulfan, capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil, cisplatin/cisplatinum, cladribine, cyclophosphamide/cytophosphane, cytabarine, dacarbazine, daunorubicin/daunomycin, denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil (5-FU), gemcitabine, goserelin, hydrocortisone, hydroxyurea, idarubicin, ifosfamide, interferon alfa, irinotecan CPT-l l, lapatinib, lenalidomide, leuprolide,

mechlorethamine/chlormethine/mustine/HN2, mercaptopurine, methotrexate, methylprednisolone, mitomycin, mitotane, mitoxantrone, octreotide, oprelvekin, oxaliplatin, paclitaxel, pamidronate, pazopanib, pegaspargase, pegfilgrastim, PEG interferon, pemetrexed, pentostatin, phenylalanine mustard, plicamycin/mithramycin, prednisone, prednisolone, procarbazine, raloxifene, romiplostim, sargramostim, streptozocin, tamoxifen, temozolomide, temsirolimus, teniposide, thalidomide, thioguanine, thiophosphoamide/thiotepa, thiotepa, topotecan hydrochloride, toremifene, tretinoin, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, vorinostat, zoledronic acid, or combinations thereof. Antibody-based agents include, but are not limited to alemtuzumab, bevacizumab, cetuximab, fresolimumab, gemtuzumab ozogamicin, ibritumomab tiuxetan, ofatumumab, panitumumab, rituximab, tositumomab, trastuzumab, trastuzumab DM1, and combinations thereof. Immunomodulatory compounds include, but are not limited to small organic molecules that inhibit TNFa , LPS induced monocyte IL1 b , IL12, and IL6 production. In some embodiments, immunomodulatory compounds include but are not limited to methotrexate, leflunomide, cyclophosphamide, cyclosporine A, minocycline, azathioprine, an antibiotic (e.g., tacrolimus), methylprednisolone, a corticosteroid, a steroid, mycophenolate mofetil, rapamycin, mizoribine,

deoxyspergualin, brequinar, a T cell receptor modulator, or a cytokine receptor modulator, and a Toll-like receptor (TLR) agonist. In some embodiments, immunomodulatory compounds include, but are not limited to 5,6- dimethylxanthenone-4-acetic acid (DMXAA), thalidomide, lenalidomide, pomalidomide, lactoferrin, poly adenosine-polyuridy lie acid (poly AU), rintatolimod (polyI:polyCl2U; Hemispherx Biopharma), polyinosinic-polycytidylic acid stabilized with poly-L-lysine and carboxymethylcellulose (Poly-ICLC, Hiltonol®), imiquimod (3M)and resiquimod (R848; 3M), unmethylated CpG dinucleotide (CpG-ODN), and ipilumumab. Biologic agents include monoclonal antibodies (MABs), CSFs, interferons and interleukins. In some embodiments, the biologic agent is IL-2, IL-3, erythropoietin, G-CSF, filgrastim, interferon alfa, alemtuzumab, bevacizumab, cetuximab, gemtuzumab ozogamicin, ibritumomab tiuxetan, ofatumumab, panitumumab, rituximab, tositumomab or trastuzumab.

[0154] Kinase inhibitors (e.g., tyrosine kinase inhibitors, serine/threonine kinase inhibitors, etc.) include, but are not limited to axitinib, bafetinib, bosutinib, cediranib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, neratinib, nilotinib, ponatinib, quizartinib, regorafenib, sorafenib, sunitinib, vandetanib, vatalanib, vemurafinib, and combinations thereof.

[0155] In some embodiments, the additional agent is a JAK kinase inhibitor such as, but not limited to AC-430, AZD1480, baricitinib, BMS-911453, CEP-33779, CYT387, GLPG-0634, lestaurtinib, LY2784544, NS-018, pacritinib, R-348, R723, ruxolitinib, TG101348 (SAR302503), tofacitinib, and VX-509.

[0156] In certain embodiments, the additional agent includes but is not limited to anti-metabolites (e.g., 5-fluoro-uracil, cytarabine, methotrexate, fludarabine and others), antimicrotubule agents (e.g., vinca alkaloids such as vincristine, vinblastine; taxanes such as paclitaxel and docetaxel), alkylating agents (e.g., cyclophosphamide, melphalan, carmustine, nitrosoureas such as bischloroethylnitrosurea and

hydroxyurea), platinum agents (e.g. cisplatin, carboplatin, oxaliplatin, satraplatin and CI-973), anthracyclines (e.g., doxrubicin and daunorubicin), antitumor antibiotics (e.g., mitomycin, idarubicin, adriamycin and daunomycin), topoisomerase inhibitors (e.g., etoposide and camptothecins), anti-angiogenesis agents (e.g., sunitinib, sorafenib and bevacizumab) or any other cytotoxic agents, (e.g. estramustine phosphate, prednimu stine), hormones or hormone agonists, antagonists, partial agonists or partial antagonists, kinase inhibitors (such as imatinib), and radiation treatment.

[0157] Methods of Screening

[0158] Some aspects of the disclosure are related to a method of screening one or more test agents for a candidate anti-cancer agent, comprising contacting the test agent with a cancer cell; assessing whether the cancer cell undergoes regulated cell death; and determining that the test agent is a candidate anti-cancer agent if the cancer cell undergoes regulated cell death. In some embodiments, the step of "assessing whether the cancer cell undergoes regulated cell death" comprises measuring gene expression levels in the contacted cancer cell. In some embodiments, the step of "assessing whether the cancer cell undergoes regulated cell death" comprises measuring MLKL activation in the contacted cancer cell. In some embodiments, the step of "assessing whether the cancer cell undergoes regulated cell death" comprises measuring MLKL dimerization and/or multimerization in the contacted cancer cell.

[0159] Example of test agents, include, e.g., small molecules, polypeptides, nucleic acids (e.g., RNAi agents, antisense oligonucleotide, aptamers), lipids, polysaccharides, etc.

[0160] In some embodiments, the cancer cell comprises phosphorylated MLKL. In some embodiments, the phosphorylated MLKL is phosphorylated at residue serine 345 (murine) or the corresponding residue in human MLKL. In some embodiments, the cancer cell is a leukemia cell, a small cell lung carcinoma cell, a colon cancer cell, a CNS cancer cell, a melanoma cell, an ovarian cancer cell, a renal cancer cell, a prostate cancer cell, or a breast cancer cell. In some embodiments, the cancer cell is a cancer cell line cell. In some embodiments, the cancer cell line is UACC-62, RXF-393, A498, OVCAR-3, UO-31, SF-295, HCC-2998, SK-MEF-2, HF-60(TB) or SNB-75.

[0161] In some embodiments, the method of screening further comprises contacting the test agent with a second cancer cell line cell resistant to carbenoxolone treatment. The ability of the test agent to inhibit, suppress or eradicate the second cancer cell line cell resistant to carbenoxolone treatment may indicate that the test agent does not induce regulated cell death. In some embodiments, the second cancer cell line is RPMI-8226, K-562, SW-620, MDA-MB-435, OVCAR-8, NCFADR-RES, MDA-MB-23 l/ATCC, HS-578T, MDA-MB-468 or NCI-H226.

[0162] Some aspects of the disclosure are related to a method of screening for a cancer cell susceptible to treatment with an anti-cancer agent, comprising detecting phosphorylated MFKF in the cancer cell, wherein if phosphorylated MFKF is detected in the cancer cell then the cancer cell is determined to be susceptible to treatment with the anti-cancer agent. In some embodiments, the phosphorylated MFKF is phosphorylated at residue serine 345 (murine) or the corresponding residue in human MLKL. In some embodiments, the method further comprises treating the subject with the anti-cancer agent. In some embodiments, phosphorylated MLKL is detected with an antibody. In some embodiments, phosphorylated MLKL is detected by immunoprecipitation.

[0163] In some embodiments, the anti-cancer agent is an MLKL modulator. In some embodiments, the anti-cancer agent is an MLKL activator. In some

embodiments, the method further comprises measuring MLKL dimerization and/or multimerization in the cancer cell. Any suitable method available in the art can be used to measure MLKL dimerization and/or multimerization and is not limited.

[0164] Some aspects of the disclosure are related to a method of screening for a cancer cell susceptible to treatment with an anti-cancer agent, comprising detecting an elevated level of MLKL in the cancer cell as compared to a control, wherein if an elevated MLKL is detected in the cancer cell then the cancer cell is determined to be susceptible to treatment with the anti-cancer agent. In some embodiments, the control is the level of MLKL in a non-cancerous cell (or population of non-cancerous cells) from which the cancerous cell is derived from. In some embodiments, the control is the level of MLKL in a CBX resistance cancer cell (or population of cells).

[0165] In some embodiments, MLKL is detected with an antibody. In some embodiments, MLKL is detected by immunoprecipitation.

[0166] In some embodiments, the anti-cancer agent is an MLKL modulator. In some embodiments, the anti-cancer agent is an MLKL activator. In some

embodiments, the anti-cancer agent is an MLKL dimerization inhibitor.

[0167] In some embodiments, the cancer cell is a leukemia cell, a small cell lung carcinoma cell, a colon cancer cell, a CNS cancer cell, a melanoma cell, an ovarian cancer cell, a renal cancer cell, a prostate cancer cell, a cervical cancer cell, or a breast cancer cell. In some embodiments, the cancer cell is obtained from a subject having cancer. In some embodiments, the cancer cell is obtained during a biopsy. In some embodiments, the method further comprises treating the subject with the anti cancer agent. [0168] Compositions

[0169] Agents, compositions and MLKL modulators disclosed herein and/or identified or validated using a method described herein may be administered by any suitable means such as orally, intranasally, subcutaneously, intramuscularly, intravenously, intra-arterially, parenterally, intraperitoneally, intrathecally, intratracheally, ocularly, sublingually, vaginally, rectally, dermally, or by inhalation, e.g., as an aerosol. Depending upon the type of condition (e.g., type of cancer) to be treated, agents may, for example, be inhaled, ingested or administered by systemic routes. Thus, a variety of administration modes, or routes, are available. The particular mode selected will depend, of course, upon the particular agent or MLKL modulator selected, the particular condition being treated and the dosage required for therapeutic efficacy. The methods, generally speaking, may be practiced using any mode of administration that is medically or veterinarily acceptable, meaning any mode that produces acceptable levels of efficacy without causing clinically unacceptable (e.g., medically or veterinarily unacceptable) adverse effects. The term “parenteral” includes intravenous, intramuscular, intraperitoneal, subcutaneous, intraosseus, and intrasternal administration, e.g., by injection or infusion techniques. In some embodiments, a route of administration is parenteral or oral. Optionally, a route or location of administration is selected based at least in part on the location of the cancer cells. For example, an agent or MLKL modulator such as a small molecule, RNAi agent, or gene therapy vector may be administered locally to a target tissue or organ, e.g., a tissue or organ that exhibits evidence or symptoms of mitochondrial dysfunction or that typically exhibits evidence of dysfunction in individuals who have a particular mitochondrial disorder. “Local administration” encompasses (1) administration directly into or near a target tissue or organ, (2) into or near a blood vessel that directly supplies a target tissue or organ, or (3) into a fluid- filled extravascular compartment in fluid communication with the target tissue or organ (including inhalational administration where the target tissue or organ is a component of respiratory system such as the lung, intrathecal or intraventricular administration where the target organ or tissue is a component of the central nervous system such as the brain). “Near” in this context refers to locations up to 1 cm, 5 cm, or 10 cm from an edge or border of the target tissue, organ, or blood vessel. In some embodiments an agent or MLKL modulator, e.g., a small molecule, RNAi agent, or gene therapy vector, is locally administered to the liver, e.g., by injection or infusion injection into the portal vein or hepatic artery or directly into the liver parenchyma, e.g., to treat a subject with a cancer that affects the liver. In some embodiments, inhaled medications are of use. Such administration allows direct delivery to the lung, although it could also be used to achieve systemic delivery, e.g., to treat a disease affecting the liver, nervous system, muscles, etc. In some embodiments, intrathecal or intraventricular administration may be of use, e.g., in a subject with a cancer affecting the central nervous system. In some embodiments local

administration to the brain is performed by stereotactic injection into the parenchyma of the brain or by intrathecal or intraventricular injection, infusion, or implantation.

In some embodiments convection-enhanced delivery or step cannulae may be used to enhance delivery to the brain. In some embodiments nasal administration is used to deliver an agent or MLKL modulator to the brain. Other appropriate routes and devices for administering therapeutic agents will be apparent to one of ordinary skill in the art.

[0170] Suitable preparations, e.g., substantially pure preparations, of an active agent or MLKL modulator may be combined with one or more pharmaceutically acceptable carriers or excipients, etc., to produce an appropriate pharmaceutical composition. The term“pharmaceutically acceptable carrier or excipient” refers to a carrier (which term encompasses carriers, media, diluents, solvents, vehicles, etc.) or excipient which does not significantly interfere with the biological activity or effectiveness of the active ingredient(s) of a composition and which is not excessively toxic to the host at the concentrations at which it is used or administered. Other pharmaceutically acceptable ingredients can be present in the composition as well. Suitable substances and their use for the formulation of pharmaceutically active compounds is well-known in the art (see, for example, "Remington 's Pharmaceutical Sciences", E. W. Martin, l9th Ed., 1995, Mack Publishing Co.: Easton, PA, and more recent editions or versions thereof, such as Remington: The Science and Practice of Pharmacy. 21 st Edition. Philadelphia, PA. Lippincott Williams & Wilkins, 2005, for additional discussion of pharmaceutically acceptable substances and methods of preparing pharmaceutical compositions of various types). [0171] A pharmaceutical composition is typically formulated to be compatible with its intended route of administration. For example, preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media, e.g., sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's. Examples of non- aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; preservatives, e.g., antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Such parenteral preparations can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutical compositions and agents for use in such compositions may be manufactured under conditions that meet standards or criteria prescribed by a regulatory agency such as the US FDA (or similar agency in another jurisdiction) having authority over the manufacturing, sale, and/or use of therapeutic agents. For example, such compositions and agents may be manufactured according to Good Manufacturing Practices (GMP) and/or subjected to quality control procedures appropriate for pharmaceutical agents to be administered to humans.

[0172] For oral administration, agents or MLKL modulators can be formulated by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Suitable excipients for oral dosage forms are, e.g., fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.

Dyestuffs or pigments may be added to the tablets or dragee coatings for

identification or to characterize different combinations of active compound doses.

[0173] Pharmaceutical preparations which can be used orally include push fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art.

[0174] Formulations for oral delivery may incorporate agents to improve stability in the gastrointestinal tract and/or to enhance absorption.

[0175] For administration by inhalation, pharmaceutical compositions may be delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, a

fluorocarbon, or a nebulizer. Liquid or dry aerosol (e.g., dry powders, large porous particles, etc.) can be used. The disclosure contemplates delivery of compositions using a nasal spray or other forms of nasal administration. Several types of metered dose inhalers are regularly used for administration by inhalation. These types of devices include metered dose inhalers (MDI), breath-actuated MDI, dry powder inhaler (DPI), spacer/holding chambers in combination with MDI, and nebulizers.

[0176] For topical applications, pharmaceutical compositions may be formulated in a suitable ointment, lotion, gel, or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers suitable for use in such composition.

[0177] For local administration to the eye, pharmaceutical compositions may be formulated as solutions or micronized suspensions in isotonic, pH adjusted sterile saline, e.g., for use in eye drops, or in an ointment. In some embodiments intraocular administration is used. Routes of intraocular administration include, e.g., intravitreal injection, retrobulbar injection, peribulbar injection, subretinal, sub-Tenon injection, and subconjunctival injection. In some embodiments an intraocular implant

(sometimes termed an“insert”) is used to deliver an agent to the eye. In some embodiments a gene therapy vector is administered by subretinal injection.

[0178] Pharmaceutical compositions may be formulated for transmucosal or transdermal delivery. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated may be used in the formulation. Such penetrants are generally known in the art. Pharmaceutical compositions may be formulated as suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or as retention enemas for rectal delivery.

[0179] In some embodiments, a pharmaceutical composition includes one or more agents intended to protect the active agent(s) or MLKL modulators against rapid elimination from the body, such as a controlled release formulation, implant, microencapsulated delivery system, etc. Compounds may be encapsulated or incorporated into particles, e.g., microparticles or nanoparticles. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, PLGA, collagen, polyorthoesters, polyethers, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. For example, and without limitation, a number of particle -based delivery systems are known in the art for delivery of siRNA. Use of such compositions is

contemplated. In some embodiments lipidoid particles are used. In some

embodiments non-lipid particles are used. Liposomes or other lipid-based particles can also be used as pharmaceutically acceptable carriers. In some embodiments a macroscopic implant is used to deliver an agent systemically or locally. In some embodiments the implant is implanted in the CNS, e.g., in the brain. [0180] In some embodiments, a pharmaceutically acceptable derivative of an MLKL modulator or agent described herein or identified or validated as described herein, is provided. As used herein, a pharmaceutically acceptable derivative of a particular agent or MLKL modulator includes, but is not limited to, pharmaceutically acceptable salts, esters, salts of such esters, or any other adduct or derivative which upon administration to a subject in need thereof is capable of providing the compound, directly or indirectly. Thus, pharmaceutically acceptable derivatives can include salts, prodrugs, and/or active metabolites. The term“pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and/or lower animals without undue toxicity, irritation, allergic response and the like, and which are commensurate with a reasonable benefit/risk ratio. A wide variety of appropriate pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts include, but are not limited to, those derived from suitable inorganic and organic acids and bases.

[0181] Pharmaceutical compositions, when administered to a subject in need of treatment for a disorder are, in at least some embodiments, administered for a time and in an amount sufficient to treat the disease or condition (e.g., cancer) for which they are administered. Therapeutic efficacy and toxicity of active agents can be assessed by standard pharmaceutical procedures in cell cultures or experimental animals. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans or other subjects. Different doses for human administration can be further tested in clinical trials in humans as known in the art. The dose used may be the maximum tolerated dose or a lower dose. A therapeutically effective dose of an active agent or MLKL modulator in a pharmaceutical composition may be within a range of about 0.001 to about 100 mg/kg body weight, about 0.01 to about 25 mg/kg body weight, about 0.1 to about 20 mg/kg body weight, about 1 to about 10 mg/kg. Other doses include, for example, about 1 pg/kg to about 500 mg/kg, and about 100 pg/kg to about 5 mg/kg. In some embodiments a single dose is administered while in other embodiments multiple doses are administered. Those of ordinary skill in the art will appreciate that appropriate doses in any particular circumstance depend upon the potency of the agent(s) utilized, and may optionally be tailored to the particular recipient. The specific dose level for a subject may depend upon a variety of factors including the activity of the specific agent(s) employed, severity of the disease or disorder, the age, body weight, general health of the subject, etc.

[0182] It may be desirable to formulate pharmaceutical compositions, particularly those for oral or parenteral compositions, in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form, as that term is used herein, refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active agent(s) calculated to produce the desired therapeutic effect in association with an appropriate pharmaceutically acceptable carrier.

[0183] It will be understood that a therapeutic regimen may include administration of multiple unit dosage forms over a period of time. In some embodiments, a subject is treated for between 1-7 days. In some embodiments a subject is treated for between 7-14 days. In some embodiments a subject is treated for betweenl4-28 days. In other embodiments, a longer course of therapy is

administered, e.g., over between about 4 and about 10 weeks, 10-26 weeks, 26-52 weeks, or longer. In some embodiments, treatment is continued for 1-5 years, 1-10 years, 1-20 years, or more. In some embodiments, treatment may be continued indefinitely. For example, a subject at risk of a cancer may be treated for any period during which such risk exists or the subject desires to avoid developing or to control the severity of cancer. A subject may receive one or more doses a day, or may receive doses every other day or less frequently, within a treatment period. Treatment courses may be intermittent. For example, a subject may be treated when symptoms recur or may be monitored and treated when an indicator of impending symptoms or worsening of a disorder is detected.

[0184] In some embodiments, two or more different agents or MLKL modulators are administered. In some embodiments, an agent or MLKL modulator is administered in combination with a second compound useful for treating cancer. In some embodiments“in combination” refers to administration of two or more agents with the knowledge that the two agents are useful for treating a particular disorder or each agent is administered for the purpose of treating or contributing to treatment of the disorder. In some embodiments of combined administration (i) a dose of the second compound is administered before more than 90% of the most recently administered dose of the first agent has been metabolized to an inactive form or excreted from the body; or (ii) doses of the first and second compound are

administered at least once within 24 hours to 2 weeks of each other, or (iii) the agents are administered during overlapping time periods (e.g., by continuous or intermittent infusion); or (iv) any combination of the foregoing. The agent may be, but need not be, administered together as components of a single composition. In some

embodiments, they may be administered individually at substantially the same time (by which is meant within less than 10 minutes of one another). In some

embodiments they may be administered individually within a short time of one another (by which is meant less than 3 hours, sometimes less than 1 hour, apart). The agents may be, but need not, be administered by the same route of administration. When administered in combination with a second agent, the effective amount of a first agent needed to elicit a particular biological response may be less or more than the effective amount of the first agent when administered in the absence of the second compound (or vice versa), thereby allowing an adjustment of the amount dose of the either or both agent(s) relative to the amount that would be needed if one agent were administered in the absence of the other. For example, when agents are administered in combination (e.g., agents or MLKL modulator and a second agent), a sub- therapeutic dosage of either of the agents, or a sub-therapeutic dosage of both, may be used in certain embodiments. A“sub-therapeutic amount” as used herein refers to an amount which is less than that amount which would be expected to produce a therapeutic result in the subject if administered in the absence of the other agent, e.g., less than a recommended amount. The effects of multiple agents may, but need not be, additive or synergistic. One or more of the compounds may be administered multiple times.

[0185] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed

substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description.

[0186] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0187] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or prior publication, or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0188] One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The details of the description and the examples herein are representative of certain embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention. It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0189] The articles“a” and“an” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

Furthermore, it is to be understood that the invention provides all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. It is contemplated that all embodiments described herein are applicable to all different aspects of the invention where appropriate. It is also contemplated that any of the embodiments or aspects can be freely combined with one or more other such embodiments or aspects whenever appropriate. Where elements are presented as lists, e.g., in Markush group or similar format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been

specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. For example, any one or more active agents, additives, ingredients, optional agents, types of organism, disorders, subjects, or combinations thereof, can be excluded.

[0190] Where the claims or description relate to a composition of matter, it is to be understood that methods of making or using the composition of matter according to any of the methods disclosed herein, and methods of using the composition of matter for any of the purposes disclosed herein are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where the claims or description relate to a method, e.g., it is to be understood that methods of making compositions useful for performing the method, and products produced according to the method, are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0191] Where ranges are given herein, the invention includes embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other is excluded. It should be assumed that both endpoints are included unless indicated otherwise.

Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also understood that where a series of numerical values is stated herein, the invention includes embodiments that relate analogously to any intervening value or range defined by any two values in the series, and that the lowest value may be taken as a minimum and the greatest value may be taken as a maximum. Numerical values, as used herein, include values expressed as percentages. For any embodiment of the invention in which a numerical value is prefaced by“about” or“approximately”, the invention includes an embodiment in which the exact value is recited. For any embodiment of the invention in which a numerical value is not prefaced by“about” or “approximately”, the invention includes an embodiment in which the value is prefaced by“about” or“approximately”. [0192]“Approximately” or“about” generally includes numbers that fall within a range of 1% or in some embodiments within a range of 5% of a number or in some embodiments within a range of 10% of a number in either direction (greater than or less than the number) unless otherwise stated or otherwise evident from the context (except where such number would impermissibly exceed 100% of a possible value). It should be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited, but the invention includes embodiments in which the order is so limited. It should also be understood that unless otherwise indicated or evident from the context, any product or composition described herein may be considered“isolated”.

[0193] Examples

[0194] Example 1- Mitochondrial Respiration

[0195] The objective of these studies was to investigate the effects of carbenoxolone disodium salt (CBX) on mitochondrial respiration in mouse and human leukemia cell lines. Comparative analyses of oxygen consumption were conducted under various conditions using the live-cell metabolic assay platform Agilent Seahorse XF (Agilent Technologies, Inc.). Additionally, the glycolytic function and mitochondrial respiration of the various cell lines were assessed using timed injections of specific compounds according to manufacturer- specified protocols.

[0196] Materials and Methods

[0197] Test Article and Control Article

[0198] All materials were stored per manufacturer recommendations and standard handling precautions, lab coat, gloves, and eye protection were employed.

[0199] Preparation of Test and Control Articles

[0200] CBX was dissolved in the vehicle (phosphate buffered saline [PBS]) to achieve nominal stock concentration of 200 mM. The control article was used as received from the manufacturer.

[0201] Cell Lines and Culture Conditions

[0202] Generation of Primary GFP-Luciferase-MLL-AF9 Cell Line (eAS 12)

[0203] The primary GFP-Luciferase-MLL-AF9 cell line (eAS 12) were generated from a C57B1/6 luciferase-GFP double reporter transgenic mouse line by backcrossing mice expressing a modified firefly luciferase enzyme (Actb-luciferase, Caliper Life Sciences) which were then crossed onto the Ubiquitin-GFP mouse strain (Jackson Laboratories). This method was followed to avoid the limitations and variability of expression associated with viral transduced indicators. The resulting luciferase-GFP reporter mouse was crossed with the MLL-AF9 knock-in mouse, which at 6-7 months of age developed AML that faithfully recapitulates the phenotype associated with the t(9;l l)(p22;q23) translocation in humans. The MLL- AF9 cells were harvested from terminally ill trigenic mice and were used for inducing the disease in secondary recipients in the experiment described below.

[0204] The primary GFP-Luciferase-MLL-AF9 cell line was maintained in Roswell Park Memorial Institute medium (RPMI) supplemented with 20% FBS (Sigma).

[0205] CBX-Resistant Cell Line (eAS 12R)

[0206] In order to identify the molecular mechanism of CBX-mediated leukemia cell death, CBX-resistant leukemia cells were generated by long-term exposure of the sensitive primary MLL-AF9 leukemia cells (eAS 12) in culture to a low, sub-optimal, CBX concentration that was then gradually increased to more toxic concentrations over 25 weeks, for resistance induction and selection.

[0207] The CBX-resistant cell line (eAS 12R) was maintained in RPMI medium supplemented with 20% FBS (Sigma).

[0208] Bone Marrow Mononuclear Cell [0209] Human cell lines (bone marrow mononuclear cells, NB4, K562, MOLM-14, NOMO-l) were obtained from ATCC (Manassas, VA) and maintained in RPMI medium supplemented with 10% FBS (Sigma)

[0210] Experimental Study Design and Treatment

[0211] Mouse and human leukemic cells were plated in a 96-well plate containing seahorse medium. Baseline oxygen consumption rates were measured using the live-cell metabolic assay platform Agilent Seahorse XF. The instrument automatically“injected” the depicted drugs (as indicated by the arrow heads in Error! Reference source not found, and Error! Reference source not found.) while acquiring oxygen consumption and extracellular acidification rate. Additionally, the glycolytic function and mitochondrial respiration of the various cell lines were assessed using commercially available kits, which involve automated timed injections of specific compounds according to manufacturer- specified protocols (see below). A brief overview of the study design is presented in Table 1.

[0212] Table 1. CR130, CR133, and CR137: Study Design Summary

AML = acute myeloid leukemia; BM-MNC =bone marrow mononuclear cells; CBX = carbenoxolone disodium salt; PBS = phosphate buffered saline; mM = micromolar

a Glycolytic stress - timed injections of glucose, oligomycin, and 2-Deoxy-D-glucose (2-DG) to measure glycolytic function in cells

b Mitochondrial stress - timed injections of oligomycin, carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and antimycin A and rotenone (AA&R) to measure mitochondrial respiration in cells

[0213] Glycolytic and Mitochondrial Stress Tests

[0214] Agilent Seahorse XF Mito Stress Test Kit (Kit 103015-100) was used to assess mitochondrial function in cells, and experiments were conducted according to manufacturer- specified procedures in the User Guide. Briefly, cells were exposed to modulators of respiration that target components of the electron transport chain (ETC) in the mitochondria to evaluate key parameters of metabolic function. The XF instrument measures and reports the oxygen consumption rate (OCR) of cells. The injected compounds, their target in the ETC, and their measured endpoint are summarized below (Table 2).

Table 2. Description of Mitochondrial Function Assessment - Methods

ATP = adenosine triphosphate; FCCP = carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone

[0215] Agilent Seahorse XF Glycolysis Stress Test Kit (Kit 103020-100) was used to assess glycolytic function in cells, and experiments were conducted according to manufacturer- specified procedures in the User Guide. Briefly, cells were exposed to modulators of glycolysis to evaluate key parameters of glycolytic flux. The XF instrument measures the acidification rate and reports results as the extracellular acidification rate (ECAR). The injected compounds, their target in glycolytic function, and their measured endpoint are summarized below (Table 3).

[0216] Table 3. Description of Glycolytic Function Assessment - Methods

ATP = adenosine triphosphate; 2-DG = 2-Deoxy-D-glucose

[0217] Results

[0218] Oxygen Consumption and Mitochondrial Respiration

[0219] Comparative analysis revealed that human and mouse leukemia cells consume more oxygen than their normal healthy cell counterparts, which was mostly utilized for adenosine triphosphate (ATP) generation (FIGS. 51A-51B). Importantly, rapidly dividing CBX-resistant leukemia cells (eASl2R) consumed significantly less oxygen than CBX-sensitive primary leukemia cells (eASl2, Error! Reference source not found.FIG. 51B). This suggests that leukemia cells produce excess amounts of ATP to“fuel” additional cellular processes, other than rapid cell division. When human and mouse leukemia cells were treated with CBX, an immediate mitochondrial respiration arrest occurred, but normal cells were unaffected (FIGS. 51C-51D).

[0220] The effect of CBX in leukemia cells was comparable to the effect of Antimycin, a potent inhibitor of the mitochondrial respiration complex III (FIG. 52B, brown circle).

[0221] Glycolytic Function

[0222] In mouse cell lines, CBX treatment induced a transient increase in glycolysis in mouse primary leukemia cell lines (eASl2), but did not impact glycolysis in normal mouse BM-MNCs (FIG. 53).

[0223] Similarly, in human cell lines, CBX treatment induced a transient increase in glycolysis in human leukemic cells, while in healthy human bone marrow mononuclear cells, no CBX-related changes in glycolysis were observed (Error! Reference source not found.)·

[0224] Conclusion

[0225] In conclusion, when human and mouse leukemia cells were treated with CBX an immediate mitochondrial respiration arrest occurred, but normal cells were unaffected. The effect of CBX in leukemia cells was similar to the effect of Antimycin, a potent inhibitor of the mitochondrial respiration complex III. Similarly, CBX induced a transient increase in glycolysis in both human and mouse leukemia cells, but did not impact glycolysis in normal cells.

[0226] Example 2- Reactive Oxygen Species

[0227] Introduction

[0228] The objective of this study was to evaluate the formation of reactive oxygen species (ROS) and deoxyribonucleic acid (DNA) damage in response to CBX-treatment in mouse leukemia (infrared fluorescent protein [iRFP] -expressing primary MLL-AF9 cells, referred to as FM9) versus normal bone marrow

mononuclear cells (BM-MNC). ROS are required for the regulation of various cellular processes, such as differentiation and migration; however, acute increases in ROS levels can trigger apoptosis or regulated necrosis of cells.

[0229] Materials and Methods

[0230] Test Article and Control Article

[0231] All materials were stored per manufacturer recommendations and standard handling precautions, lab coat, gloves, and eye protection were employed.

[0232] Preparation of Test and Control Articles

[0233] CBX was dissolved in the vehicle (PBS) to achieve nominal stock concentration of 200 mM. The control article was used as received from the manufacturer.

[0234] Cell Lines and Culture Conditions

[0235] MLL-AF9 cells were obtained from a collaborator at MGH (Francois Mercier, MD) and BM-MNC cells were harvested from a C57B16 mice. Both were maintained in Roswell Park Memorial Institute medium (RPMI) and supplemented with 20% phosphate buffered saline (PBS, Sigma), 10 ng/ml IL3 (interleukin 3), 10 ng/ml IL-6 (interleukin 6) and 20 ng/ml stem cell factor at 37°C in 5% C0₂. Both types of cells were cultured with cytokines IL-3 (10 ng/mL), IL-6 (10 ng/mL), and stem cell factor (SCF; 20 ng/mL) to induce proliferation.

[0236] Experimental Study Design and Treatment

[0237] FM9 cells and normal primary mouse BM-MNC were incubated with 200 mM CBX for 1, 2, 4, and 8 hours and then analyzed by fluorescence-activated cell sorting (FACS) to quantify ROS formation and DNA damage. A 0 hour time point was included as control (PBS control; no CBX included). A brief overview of the study design is presented in Table Table 1.

[0238] Table 4. CR112: Study Design Summary

[0239] CBX = carbenoxolone disodium salt; BM-MNCs = bone marrow mononuclear cells; peripheral blood mononuclear cells; FM9 = infrared fluorescent protein [iRFP] -expressing primary MLL-AF9 cells; mM = micromolar

[0240] Stains and Analysis

[0241] Following incubation for 0 (control), 1, 2, 4, and 8 hours with CBX, cells were measured for levels of superoxide, peroxyl radicals, and phosphorylation of gH2AC (double- strand DNA breaks indicator).

[0242] Superoxide Measurement

[0243] Superoxide levels were measured using MitoSOX™ Red

mitochondrial superoxide indicator. Cells were incubated with MitoSOX Red reagent (diluted 1:1000 in DMSO) for a maximum of 15 minutes at 37°C. Cells were then analyzed by flow cytometry for staining with MitoSOX Red using FACS.

[0244] Peroxyl Radicals Measurement

[0245] Peroxyl radicals as a measure of lipid peroxidation were measured using BODIPY^® 581/591 Cl 1 Lipid Probes. Cells were incubated with BODIPY Lipid Probes (diluted 1:1000, 10 mM final concentration) for 30 minutes at 37°C.

Cells were then sorted for staining with BODIPY Lipid Probes using FACS.

[0246] DNA Damage

[0247] Phosphorylated gH2AC as a measure of double- strand DNA breaks was measured using an Anti-phospho-Histone H2A.X (Serl39) Antibody (Millipore Sigma Catalogue #16-202A). Cells were incubated with antibody per manufacturer- specified procedures and sorted using FACS.

[0248] Results

[0249] As shown in FIGS. 57A-57C, incubation of mouse leukemia cells (FM9 cells) with CBX induced a rapid increase in superoxide levels, increased levels of peroxyl radicals and gH2AC phosphorylation. These effects generally increased with increasing duration of CBX exposure. CBX did not induce ROS formation or DNA damage in normal BM-MNCs following incubation for up to 8 hours.

[0250] Conclusion

[0251] Incubation of mouse leukemia cells with CBX for 1 to 8 hours led to an increase in measures of oxidative stress, including mitochondrial ROS formation, lipid peroxidation, and double-strand DNA breaks. However, CBX treatment did not induce oxidative-stress in normal mouse BM-MNC.

[0252] Example 3

[0253] The objective of this study was to investigate the potential of carbenoxolone disodium salt (CBX) to induce intracellular calcium (Ca²⁺) influx in human leukemia cells versus healthy peripheral blood mononuclear cells (PBMCs). Leukemic cells (NB4, THP1, U937, K562, MOLM-14, NOMO-l, MONOMAC-6) and normal PBMCs were incubated in 96-well plates with vehicle control (phosphate buffered saline [PBS]), mitochondrial stress agents, or CBX (200 mM) and intracellular Ca²⁺ was measured with a Fluo-4 No Wash (NW) Calcium Assay Kit.

[0254] Materials and Methods

[0255] Test Article and Control Article

[0256] All materials were stored per manufacturer recommendations and standard handling precautions, lab coat, gloves, and eye protection were employed.

[0257] Preparation of Test and Control Articles

[0258] CBX was dissolved in the vehicle (PBS) to achieve nominal stock concentration of 200 pM. The control article was used as received from the manufacturer.

[0259] Cell Lines and Culture Conditions

[0260] Human PBMCs and leukemic cell lines (NB4, THP1, U937, K562, MOLM-14, NOMO-l, MONOMAC-6) were obtained from the American Type Culture Collection (ATCC) and maintained in Roswell Park Memorial Institute medium (RPMI) supplemented with 10% FBS (Sigma).

[0261] Experimental Study Design and Treatment [0262] Normal human PBMCs or leukemic cells (NB4, THP1, U937, K562, MOLM-14, NOMO-l, MONOMAC-6) were incubated in 96-well plates with vehicle control (PBS), mitochondrial stress agents (oligomycin; carbonyl cyanide-4

(trifluoromethoxy) phenylhydrazone [FCCP]; and rotenone/antimycin A), or CBX (200 mM) and intracellular Ca²⁺ was measured with a Fluo-4 NW Calcium Assay Kit (CYTATION 3 imaging reader, BioTek).

[0263] The drugs were added to the cells while acquiring calcium levels (depicted by arrow heads in the figure)

[0264] A brief overview of the study design is presented in Table .

[0265] Table 5. CR131: Study Design Summary

[0266] CBX = carbenoxolone disodium salt; PBMCs = peripheral blood mononuclear cells; mM = micromolar. ^a Mitochondrial stress - timed injections of oligomycin, carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP), and antimycin A and rotenone (AA&R).

[0267] Mitochondrial Stress Agents

[0268] Cells were exposed to modulators of respiration that target components of the electron transport chain (ETC) in the mitochondria to evaluate key parameters of metabolic function. The various agents, and their target in the ETC are

summarized in Table .

[0269] Table 6. Study Design: Evaluation of Metabolic Function

[0270] ATP = adenosine triphosphate; FCCP = carbonyl cyanide-4

(trifluoromethoxy) phenylhydrazone .

[0271] Fluo-4 Calcium Assay Kit [0272] Fluo-4 acetoxymethyl ester (AM) is a fluorescent Ca2+ indicator that is widely used for in-cell measurement of calcium signaling in high-throughput screening. Experiments were conducted in accordance with manufacturer-specified procedures in the Fluo-4 AM Calcium Assay Kit Manual. Fluorescence was measured once every minute using CYTATION 3 imaging reader (BioTek).

[0273] Results

[0274] As shown in Error! Reference source not found., mitochondrial stress agents (oligomycin, FCCP, and rotenone/antimycin A) did not induce Ca²⁺ influx in healthy human PBMCs or leukemia cells during incubation. Conversely, CBX treatment induced an acute Ca²⁺ influx in human leukemia cells and did not impact healthy PBMCs.

[0275] Conclusions

[0276] In conclusion, CBX treatment generated an acute calcium (Ca²⁺) influx in human leukemia cells and did not impact healthy PBMCs.

Claims

CLAIMS What is claimed is:

1. A method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an MLKL modulator, wherein the cancer cells are not leukemic cells, or wherein the MLKL modulator is not carbenoxolone.

2. The method of claim 1, wherein the MLKL modulator modulates MLKL

phosphorylation, dimerization, or multimerization.

3. The method of claims 1-2, wherein the MKLK modulator increases MLKL

activity.

4. The method of claim 3, wherein the MLKL modulator selectively increases MLKL activity in cancer cells.

5. The method of claims 1-4, wherein the MLKL modulator selectively causes metabolic arrest in cancer cells.

6. The method of claims 1-5, wherein the anti-cancer activity of the MLKL

modulator is decreased by calcium chelation.

7. The method of claims 1-6, wherein the anti-cancer activity of the MLKL

modulator is decreased by iron chelation.

8. The method of claims 1-7, wherein the anti-cancer activity of the MLKL

modulator is decreased by an MLKL inhibitor.

9. The method of claims 1-8, wherein the MLKL modulator selectively causes reactive oxygen species generation in cancer cell mitochondria.

10. The method of claims 1-9, wherein the MLKL modulator selectively causes lipid peroxidation in cancer cells.

11. The method of claims 1-10, wherein the MLKL modulator selectively causes double stranded DNA breaks in the genome of cancer cells.

12. The method of claims 1-11, wherein the MLKL modulator selectively causes phosphatidylserine exposure in cancer cells.

13. The method of claims 1-12, wherein the anti-cancer activity of the MLKL

modulator is decreased by a cysteine protease inhibitor.

14. The method of claims 1-13, wherein the MLKL modulator inhibits succinate dehydrogenase activity.

15. The method of claims 1-14, wherein the MKLK modulator selectively causes rapid programmed cell death (rPCD) in the cancer cells.

16. The method of claims 1-2, wherein the MKLK modulator inhibits MLKL

dimerization.

17. The method of claims 1-16, wherein the cancer cells are selected from the group consisting of leukemia, small cell lung carcinoma cells, colon cancer cells, CNS cancer cells, melanoma cells, ovarian cancer cells, renal cancer cells, prostate cancer cells, cervical cancer cells, and breast cancer cells.

18. The method of claims 1-17, wherein the cancer cells express an about 30 kda MLKL protein.

19. The method of claims 1-18, wherein the cancer cells have high levels of MLKL multimers, wherein the multimers preferably have a molecular weight of about 100 kda.

20. The method of claims 1-19, wherein the cancer cells have high levels of MLKL multimers or polymers, wherein the multimers preferably have a molecular weight of about 400-600 kDa.

21. The method of claims 1-20, wherein the MLKL modulator is contacted with the cancer cells in vivo.

22. A method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an agent, wherein the agent selectively increases calcium flux in cancer cells, and wherein the cancer cells are not leukemic cells or wherein the agent is not carbenoxolone.

23. A method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an agent, wherein the agent selectively causes transient metabolic arrest in cancer cells, and wherein the cancer cells are not leukemic cells or wherein the agent is not carbenoxolone.

24. A method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an agent, wherein the agent selectively causes mitochondrial reactive oxygen species generation in cancer cells, and wherein the cancer cells are not leukemic cells or wherein the agent is not carbenoxolone.

25. A method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an agent, wherein the agent selectively causes lipid peroxidation in cancer cells, and wherein the cancer cells are not leukemic cells or wherein the agent is not carbenoxolone.

26. A method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an agent, wherein the agent selectively causes double stranded DNA breaks in cancer cells, and wherein the cancer cells are not leukemic cells or wherein the agent is not carbenoxolone.

27. A method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an agent, wherein the agent selectively causes phosphatidylserine exposure in cancer cells, and wherein the cancer cells are not leukemic cells or wherein the agent is not carbenoxolone.

28. A method of eradicating or arresting the growth of cancer cells in a population of cells, comprising contacting the population of cells with an agent, wherein the agent selectively inhibits succinate dehydrogenase activity in cancer cells, and wherein the cancer cells are not leukemic cells or wherein the agent is not carbenoxolone.

29. The method of claims 22-28, wherein the agent modulates MLKL

phosphorylation, dimerization, or multimerization.

30. The method of claims 22-29, wherein the agent increases MLKL activity.

31. The method of claim 22-30, wherein the agent selectively increases MLKL

activity in cancer cells.

32. The method of claims 22-31, wherein the agent selectively causes metabolic arrest in cancer cells.

33. The method of claims 22-32, wherein the anti-cancer activity of the agent is

decreased by calcium chelation.

34. The method of claims 22-33, wherein the anti-cancer activity of the agent is

decreased by iron chelation.

35. The method of claims 22-34, wherein the anti-cancer activity of the agent is decreased by an MLKL inhibitor.

36. The method of claims 22-35, wherein the agent selectively causes reactive oxygen species generation in cancer cell mitochondria.

37. The method of claims 22-36, wherein the agent selectively causes lipid

peroxidation in cancer cells.

38. The method of claims 22-37, wherein the agent selectively causes double stranded DNA breaks in the genome of cancer cells.

39. The method of claims 22-38, wherein the agent selectively causes

phosphatidylserine exposure in cancer cells.

40. The method of claims 22-29, wherein the agent inhibits MLKL dimerization.

41. The method of claims 22-40, wherein the cancer cells are selected from the group consisting of leukemia, small cell lung carcinoma cells, colon cancer cells, CNS cancer cells, melanoma cells, ovarian cancer cells, renal cancer cells, prostate cancer cells, cervical cancer cells, and breast cancer cells.

42. The method of claims 22-41, wherein the cancer cells express an about 30 kda isoform of MLKL.

43. The method of claims 22-42, wherein the cancer cells have increased SDH

activity.

44. The method of claims 22-43, wherein the agent is contacted with the cancer cells in vivo.

45. A method of screening one or more test agents for a candidate anti-cancer agent, comprising

a) contacting the test agent with a cancer cell;

b) assessing whether the cancer cell undergoes regulated programmed cell death; and

c) determining that the test agent is a candidate anti-cancer agent if the cancer cell undergoes regulated cell death.

46. The method of claim 45, wherein step b) comprises measuring gene expression levels in the contacted cancer cell.

47. The method of claims 45-46, wherein step (b) comprises assessing whether the cancer cells undergo rapid programmed cell death.

48. The method of claims 45-47, wherein step b) comprises measuring MLKL

activation in the contacted cancer cell.

49. The method of claims 45-48, wherein step b) comprises measuring MLKL

dimerization and/or multimerization in the contacted cancer cell.

50. The method of claims 45-49, wherein the cancer cell comprises phosphorylated MLKL.

51. The method of claims 45-50, wherein the cancer cell comprises an MLKL isoform of about 30 kda.

52. The method of claims 45-51, wherein the cancer cell is a leukemia cell, a small cell lung carcinoma cell, a colon cancer cell, a CNS cancer cell, a melanoma cell, an ovarian cancer cell, a renal cancer cell, a prostate cancer cell, a cervical cancer cell, or a breast cancer cell.

53. A method of screening for a cancer cell susceptible to treatment with an anti cancer agent, comprising detecting phosphorylated MLKL in the cancer cell, wherein if phosphorylated MLKL is detected in the cancer cell then the cancer cell is determined to be susceptible to treatment with the anti-cancer agent.

54. The method of claim 53, wherein phosphorylated MLKL is detected with an antibody.

55. The method of claim 53, wherein phosphorylated MLKL is detected by

immunoprecipitation .

56. The method of claims 53-55, wherein the anti-cancer agent is an MLKL

modulator.

57. The method of claims 56, wherein the anti-cancer agent is an MLKL activator.

58. The method of claims 53-57, further comprising measuring MLKL dimerization and/or multimerization in the cancer cell.

59. The method of claims 53-58, wherein the cancer cell is a leukemia cell, a small cell lung carcinoma cell, a colon cancer cell, a CNS cancer cell, a melanoma cell, an ovarian cancer cell, a renal cancer cell, a prostate cancer cell, a cervical cancer cell, or a breast cancer cell.

60. The method of claims 53-59, wherein the cancer cell is obtained from a subject having cancer.

61. The method of claim 60, wherein the cancer cell is obtained during a biopsy.

62. The method of claims 53-61, further comprising treating the subject with the anti cancer agent.

63. A method of screening for a cancer cell susceptible to treatment with an anti cancer agent, comprising detecting an about 30 kda MLKL isoform in the cancer cell, wherein if MLKL is detected in the cancer cell then the cancer cell is determined to be susceptible to treatment with the anti-cancer agent.

64. The method of claim 63, wherein the MLKL isoform is detected with an antibody.

65. The method of claim 63, wherein the MLKL isoform is detected by

immunoprecipitation .

66. The method of claims 63-65, wherein the anti-cancer agent is an MLKL

modulator.

67. The method of claims 66, wherein the anti-cancer agent is an MLKL activator.

68. The method of claims 63-67, further comprising measuring MLKL dimerization and/or multimerization in the cancer cell.

69. The method of claims 63-68, wherein the cancer cell is a leukemia cell, a small cell lung carcinoma cell, a colon cancer cell, a CNS cancer cell, a melanoma cell, an ovarian cancer cell, a renal cancer cell, a prostate cancer cell, a cervical cancer cell, or a breast cancer cell.

70. The method of claims 63-69, wherein the cancer cell is obtained from a subject having cancer.

71. The method of claim 70, wherein the cancer cell is obtained during a biopsy.

72. The method of claims 63-71, further comprising treating the subject with the anti cancer agent.

73. A method of screening for a cancer cell susceptible to treatment with an anti cancer agent, comprising detecting an elevated level of MLKL in the cancer cell as compared to a control, wherein if an elevated MLKL is detected in the cancer cell then the cancer cell is determined to be susceptible to treatment with the anti cancer agent.

74. The method of claim 73, wherein the MLKL is detected with an antibody.

75. The method of claim 73, wherein the MLKL is detected by immunoprecipitation.

76. The method of claims 73-75, wherein the anti-cancer agent is an MLKL

modulator.

77. The method of claims 76, wherein the anti-cancer agent is an MLKL activator.

78. The method of claim 76, wherein the anti-cancer agent is an MLKL dimerization inhibitor.

79. The method of claims 73-78, further comprising measuring MLKL dimerization and/or multimerization in the cancer cell.

80. The method of claims 73-79, wherein the cancer cell is a leukemia cell, a small cell lung carcinoma cell, a colon cancer cell, a CNS cancer cell, a melanoma cell, an ovarian cancer cell, a renal cancer cell, a prostate cancer cell, a cervical cancer cell, or a breast cancer cell.

81. The method of claims 73-80, wherein the cancer cell is obtained from a subject having cancer.

82. The method of claim 81, wherein the cancer cell is obtained during a biopsy.

83. The method of claims 73-82, further comprising treating the subject with the anti cancer agent.