WO2025059108A1

WO2025059108A1 - Therapeutic combination and use thereof for treating cancer by manipulating pyruvate kinase activity and inhibiting cysteine/cystine metabolism

Info

Publication number: WO2025059108A1
Application number: PCT/US2024/046092
Authority: WO
Inventors: Sophia Y. LUNT; Elliot ENSINK; Tessa JORDAN; Hyllana MEDEIROS
Original assignee: Michigan State University MSU
Current assignee: Michigan State University MSU
Priority date: 2023-09-12
Filing date: 2024-09-11
Publication date: 2025-03-20
Anticipated expiration: 2026-03-12

Abstract

A therapeutic combination is provided herein that includes one or more cysteine/cystine metabolism inhibiting agents and one or more pyruvate kinase muscle isoform 2 (PKM2) activating agents. A pharmaceutical composition that includes the therapeutic combination is also provided herein. The present disclosure also provides a method of treating, preventing, or reducing the occurrence of cancer in a subject including administering to the subject an effective amount of the therapeutic combination or pharmaceutical composition provided herein.

Description

THERAPEUTIC COMBINATION AND USE THEREOF FOR TREATING CANCER BY MANIPULATING PYRUVATE KINASE ACTIVITY AND INHIBITING CYSTEINE/CYSTINE METABOLISM

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 63/537,874 filed on 12 September 2023, the entire content of which is hereby incorporated by reference.

GOVERNMENT SUPPORT STATEMENT

[0002] This invention was made with government support under R01CA270136 awarded by the National Institute of Health. The government may have certain rights in the invention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] This application contains references to nucleic acid sequences and/or amino acid sequences which have been submitted concurrently herewith as the sequence listing .xml file entitled “ST26_SL_9_Sept_2024.xml”, file size 13,000 Bytes (B), created on 9 September 2024. The aforementioned sequence listing is hereby incorporated by reference in its entirety.

FIELD

[0004] This disclosure generally relates to a therapeutic combination and pharmaceutical composition including a cysteine/cystine metabolism inhibiting agent and a pyruvate kinase muscle isoform 2 (PKM2) activating agent, and methods of treating cancer by administering said therapeutic combination or pharmaceutical composition.

BACKGROUND

[0005] This section provides background information related to the present disclosure which is not necessarily prior art.

[0006] Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with high mortality and limited efficacious therapeutic options. PDAC cells are capable of silently progressing and producing metastatic cells before clinical symptoms present or predictive biomarkers can be detected.

[0007] PDAC cells undergo metabolic alterations to survive within a nutrient-depleted tumor microenvironment. One metabolic shift in PDAC cells occurs through altered isoform expression of the glycolytic enzyme, pyruvate kinase (PK), which converts phosphoenolpyruvate to pyruvate and produces ATP. Pancreatic cancer cells preferentially upregulate pyruvate kinase muscle isoform 2 isoform (PKM2). The shift in PK isoform expression produces a profound reprogramming of complex networks of metabolic pathways, many of which are not well understood, including cysteine/cystine metabolism.

[0008] Cysteine is important for cell survival given its dual role in protein synthesis and defense against reactive oxygen species (ROS) as a precursor for tripeptide glutathione and coenzyme A. Healthy cells utilize circulating cysteine or cysteine biosynthesis to fulfill these roles, but cancer cells instead depend on exogenous cystine, the oxidized dimer of cysteine. Cystine is acquired predominantly through the cystine/glutamate antiporter (system Xc') which is a heterodimer of SLC7A11 (also known as xCT) and SLC3A2 and is overexpressed in many cancer cells. Depletion of extracellular cystine leads to a loss of intracellular glutathione supply and the accumulation of oxidative damage to membrane lipids. Uncontrolled lipid peroxidation propagates by reacting with ferrous iron and producing hydroxyl radicals leading to a form of cell death known as ferroptosis. Ferroptosis is fundamentally a product of aberrant metabolic behavior including changes in central carbon metabolism such as increased mitochondrial glutaminolysis and increased dependence on glucose flux through the pentose phosphate pathway to generate NADPH (reduced form of nicotinamide adenine dinucleotide phosphate (NADP). The impact of PKM2, which also plays a role in metabolic reprogramming, and the management of oxidative stress on ferroptosis in pancreatic cancer remains poorly characterized.

[0009] Recent advances in combinatorial chemotherapy and targeted immunotherapy have failed to significantly improve pancreatic cancer patient outcomes, thus, there is a need in the art for new and improved methods for preventing and treating PKM2-expressing cancers, such as PDAC.

SUMMARY

[0010] This section provides a general summary of the disclosure and is not a comprehensive disclosure of its full scope or all of its features.

[0011] In certain aspects, the present disclosure provides a therapeutic combination including one or more cysteine/cystine metabolism inhibiting agent(s) and one or more pyruvate kinase muscle isoform 2 (PKM2) activating agent(s). In some embodiments, the cysteine/cystine metabolism inhibiting agent may be imidazole ketone erastin (IKE). In some embodiments, the PKM2 activating agent may be TEPP-46. In certain aspects, the present disclosure also provides a therapeutic combination including a means for inhibiting cysteine/cystine metabolism, a means for activating PKM2, and a pharmaceutically acceptable carrier. [0012] In some embodiments, the therapeutic combination may include an additional agent including one or more anti-cancer agent(s). In some embodiments, the therapeutic combination may be used for treating, preventing, and/or reducing the occurrence of cancer; inhibiting survival of cancer cells; inducing tumor cell death, inducing ferroptosis in cancer cells, or a combination thereof. Also provided herein is a pharmaceutic composition that includes the therapeutic combination provided herein.

[0013] In further aspects, the present disclosure provides a method of treating, preventing, or reducing the occurrence of cancer in a subject in need thereof. In some embodiments, the method may include administering to the subject an effective amount of the therapeutic combination provided herein. In some embodiments, the method may include administering a means for inhibiting cysteine/cystine metabolism, a means for activating PKM2, and a pharmaceutically acceptable carrier. In some embodiments, the cancer may be a PKM2-expressing cancer.

[0014] Further areas of applicability will become apparent from the description provided herein. The description and specific examples in this summary are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0016] The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations and are not intended to limit the scope of the present disclosure.

[0017] FIG. 1 is a schematic view of mutually exclusive alternative splicing of PKM to produce PKM1 or PKM2. Targeting of exon 10 by clustered regularly interspaced short palindromic repeats (CRISPR) deletes PKM2 expression.

[0018] FIG. 2 is a Western blot of PKM1 and PKM2 in AsPCl and Panel control cells and PKM2 knock-out (PKM2KO) clones.

[0019] FIG. 3A and FIG. 3B are bar graphs showing relative viabilities of AsPCl (FIG. 3A) and Panel (FIG. 3B) control and respective PKM2KO clones in Dulbecco’s modified eagle medium (DMEM) without each individual amino acid as shown. Significance was assessed by two-way ANOVA. **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test. [0020] FIG. 4A and FIG. 4B include brightfield microscopy images of AsPCl control and PKM2KO cells (FIG. 4A) and Panel Control and PKM2KO cells (FIG. 4B) under either 200 pM (+) or 0 pM (-) cystine conditions. Scale bar = 100 pm.

[0021] FIG. 5A and FIG. 5B are bar graphs showing relative viabilities of AsPCl (FIG. 5A) and Panel (FIG. 5B) control and PKM2KO clones under 200 pM or 0 pM cystine. Significance was assessed by two-way ANOVA and Tukey test. **p<0.01, ***p<0.001.

[0022] FIG. 6 is a bar graph showing relative viabilities of AsPCl and Panel control and PKM2KO cells under 50 pM and 0 pM cystine determined using trypan blue. Significance was assessed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0023] FIG. 7A and FIG. 7B are bar graphs showing relative viabilities of AsPCl (FIG. 7A) and Panel (FIG. 7B) WT, control, and PKM2KO cells under 200 and 0 pM cystine. Significance was assessed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0024] FIG. 8A and FIG. 8B are line graphs showing proliferation analysis using Incucyte cell counts of both AsPCl (FIG. 8A) and Panel (FIG. 8B) control and their respective PKM2KO clones under 200 pM (+) or 0 pM (-) cystine. Significance was assessed by two-way ANOVA and Tukey test. Comparison between Control and PKM2KO cells at endpoint: *p<0.05, ***p<0.001. Comparison between 200 and 0 pM cystine conditions for each cell line at endpoint: ###p<0.001.

[0025] FIG. 9A and FIG. 9B are line graphs showing relative viabilities of AsPCl control and PKM2KO clones (FIG. 9A) and Panel control and PKM2KO clones (FIG. 9B) under a range of cystine concentrations from 200 pM to 0 pM. Significance was assessed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Dunnet test.

[0026] FIG. 10A and FIG. 10B are bar graphs showing relative viabilities of AsPCl (FIG. 10A) and Panel (FIG. 10B) control and PKM2KO cells under 50 pM cystine (+) and 0 pM cystine (-) co-treated with 5 pM ferrostatin-1 (FER), 100 pM trolox (TRO), 100 pM deferoxamine (DFO), 50 pM Z-VAD-FMK (ZVAD), or 10 pM necrostatin-lS (NEC). Significance by two-way ANOVA. *p<0.05, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0027] FIG. 11A and FIG. 11B are representative brightfield and fluorescent images of AsPCl control and PKM2KO cell lipid peroxidation quantified in FIG. 12A (FIG. 11 A) and Panel control and PKM2KO cell lipid peroxidation quantified in FIG. 12B (FIG. 11B). Scale bar = 50 pm. [0028] FIG. 12A and FIG. 12B are bar graphs showing relative lipid peroxidation of AsPCl control and PKM2KO cells (FIG. 12A) and Panel control and PKM2KO cells (FIG. 12B) under 50 pM cystine, 0 pM cystine, and 0 pM cystine with 5 pM FERI (FIG. 12A only), visualized by Cl l-BODIPY. Significance was assessed by two-way ANOVA. *p<0.05, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0029] FIG. 13A and FIG. 13B are bar graphs showing relative general ROS production measured by H2DCFDA in AsPCl (FIG. 13A) and Panel (FIG. 13B) control and PKM2KO cells under 50 and 0 pM cystine. Significance was assessed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0030] FIG. 14 is a Western blot of PKM2 and PKM1 expression in AsPCl and PKM2 control

+ vector, control + PKM1, PKM2KO + vector, and PKM2KO + PKM2.

[0031] FIG. 15A-FIG. 15D are bar graphs showing relative viabilities of AsPCl control + vector and control + PKM1 cells (FIG. 15A), AsPCl PKM2KO + vector and PKM2KO + PKM2 cells (FIG. 15B), Panel control + vector and control + PKM1 cells (FIG. 15C), and Panel PKM2KO + vector and PKM2KO + PKM2 cells (FIG. 15D) under 50 pM and 0 pM cystine. Significance was assessed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0032] FIG. 16A-FIG.16D are bar graphs showing Relative pyruvate kinase activity of: AsPCl control + vector and control + PKM1 cells (FIG. 16A), Panel control + vector and control + PKM1 cells (FIG. 16B), AsPCl PKM2KO + vector and PKM2KO + PKM2 cells (FIG. 16C), and Panel PKM2KO + vector and PKM2KO + PKM2 cells (FIG. 16D) under 50 pM (+) and 0 pM (-) cystine. Significance was assessed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0033] FIG. 17A and FIG. 17B are bar graphs showing relative PK activity in: AsPCl control and PKM2KO cells (FIG. 17A) and Panel control and PKM2KO cells (FIG. 17B) under 50 pM (+) and 0 (-) pM cystine. Significance was assessed by two-way ANOVA. *p<0.05, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0034] FIG. 18 is a schematic showing TEPP-46 promoting the formation of the active tetrameric form of PKM2 and compound 3k inhibiting tetramer formation, producing the less active dimeric form of PKM2.

[0035] FIG. 19A-19D are bar graphs showing relative viabilities at 50 pM and 0 pM cystine with (+) or without (-) treatment of 10 pM compound 3k in WT cells: AsPCl (FIG. 19A), Panel (FIG. 19B), MiaPaCa2 (FIG. 19C), and BxPC3 (FIG. 19D). Significance was assessed by two- way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0036] FIG. 20A-20D are bar graphs showing relative viability of AsPCl WT cells (FIG. 20A), Panel WT cells (FIG. 20B), MiaPaCa2 WT cells (FIG. 20C), and BxPC3 WT cells (FIG. 20D) treated with (+) or without (-) 2.5 pM compound 3k and 5 pM IKE. Significance was assessed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0037] FIG. 21A-21D are line graphs showing the effect of IKE and TEPP-46 combination treatment in the range of the indicated concentrations in AsPCl WT cells (FIG. 21A), Panel WT cells (FIG. 21B), MiaPaCa2 WT cells (FIG. 21C), and BxPC3 WT cells (FIG. 21D).

[0038] FIG. 22A-22D are bar graphs showing relative viability of: AsPCl WT cells with (+) or without (-) treatment with 5 pM IKE and 12.5 pM TEPP-46 (FIG. 22A), Panel WT cells with (+) or without (-) treatment with 0.625 pM IKE and 12.5 pM TEPP-46 (FIG. 22B), MiaPaCa2 WT cells with (+) or without (-) treatment with 0.625 pM IKE and 12.5 pM TEPP-46 (FIG. 22C), and BxPC3 WT cells with (+) or without (-) treatment with 5 pM IKE and 12.5 pM TEPP-46 (FIG. 22D). Significance was assessed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0039] FIG.23 is a Western blot of PKM1 and PKM2 expression in AsPCl, Panel, BxPC3, and MiaPaCa2 WT cells.

[0040] FIG. 24A-D are bar graphs showing relative viabilities of AsPCl WT cells (FIG. 24A) and BxPC3 WT cells (FIG. 24D) treated with (+) or without (-) 10 pM IKE and 50 pM TEPP-46, and Panel WT cells (FIG. 24B) and MiaPaCa2 WT cells (FIG. 24C) treated with (+) or without (-) 10 pM IKE and 50 pM TEPP-46. Cells each were co-treated with 5 pM ferrostatin-1 (FER), 50 pM Z-VAD-FMK (ZVAD), 10 pM necrostatin-lS (NEC). Significance was assessed by two- way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0041] FIG. 25 is a schematic of the mechanism of ferroptosis, the defense proteins xCT and GPX4 (the targets of imidazole ketone erastin (IKE) and Ras selective lethal 3 (RSL3), respectively), and the ferroptosis, apoptosis, and necroptosis inhibitors used in the study.

[0042] FIG. 26A and FIG. 26B are Western blots of xCT and GPX4 expression in AsPCl control and PKM2K0 cells (FIG. 26A) and Panel control and PKM2K0 cells (FIG. 26B) under 50 pM (+) and 0 pM (-) cystine. Cofilin and P-actin were used as loading controls. [0043] FIG. 27A-27D are line graphs showing concentration dependent response in viability of AsPCl (FIG. 27A) and Panel (FIG. 27C) control and PKM2K0 clones to a range of IKE concentrations from 50-0 pM (for Panel cells) and 100-0 pM (for AsPCl cells), and concentration dependent viability responses of AsPCl (FIG. 27B) and Panel (FIG. 27D) control and PKM2K0 clones to a range of RSL3 concentrations from 10-0 pM. Significance is determined two-way ANOVA. *p<0.05, ***p<0.01, ***p<0.001. Multiple hypothesis correction by the Dunnet test.

[0044] FIG. 28A and FIG. 28B are relative viabilities of AsPC 1 (FIG. 28A) and Pane 1 (FIG. 28B) control and PKM2K0 cells under 50 pM cystine with 5 pM IKE (+) co-treated with 5 pM ferrostatin-1 (FER), 100 pM trolox (TRO), 100 pM deferoxamine (DFO), 50 pM Z-VAD-FMK (ZVAD), or 10 pM necro statin- IS (NEC). Significance was assessed by two-way ANOVA. *p<0.05, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0045] FIG. 29 is a bar graph showing relative lipid peroxidation visualized by C 11 -BODIPY of AsPCl control and PKM2KO #1 under 50 pM cystine, 5 pM IKE, and 5 pM RSL3 treatment. Significance was assessed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0046] FIG. 30 is a bar graph showing relative viabilities of Panel control and PKM2KO cells under 50 pM cystine and 5 pM RSL3 co-treated with 5 pM ferrostatin-1 (FER), 100 pM trolox (TRO), 100 pM deferoxamine (DFO), 50 pM Z-VAD-FMK (ZVAD), 10 pM necrostatin-lS (NEC). Significance was assessed by two-way ANOVA. *p<0.05, **p<0.01. Multiple hypothesis correction by Tukey test.

[0047] FIG. 31 is a line graph showing the growth of xenograft tumors produced from AsPCl control and PKM2KO cells treated with vehicle control or 50 mg/kg IKE. Significance was assessed by two-way ANOVA at end point. *p<0.05, ***p<0.001, ns = non- significant. Multiple hypothesis correction by Tukey test.

[0048] FIG. 32A-32L are bar graphs showing stable isotope tracing of ¹³Ci,2-glucose under 50 pM (+) and 0 pM (-) cystine for 4 hours in AsPCl control and PKM2KO clone #1 to produce M+2 labeled hexose-phosphate (FIG. 32A), lactate (FIG. 32B), citrate (FIG. 32C), a- ketoglutarate (FIG. 32D), malate (FIG. 32E), and aspartate (FIG. 32F) and stable isotope tracing of ¹³Cs-glutamine under 50 pM (+) and 0 pM (-) cystine for 24 hours in AsPCl control and PKM2KO clone #1 to produce M+5 labeled glutamate (FIG. 32G), secreted glutamate (FIG. 32H), a-ketoglutarate (FIG. 321), glutathione (FIG. 32L), M+4 labeled malate (FIG. 32J), and aspartate (FIG. 32K). Significance was assessed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test. [0049] FIG. 33A-33E are bar graphs showing table isotope tracing of ¹³C5-glutamine under 50 pM (+) and 0 pM (-) cystine for 24 hours in AsPCl control and all PKM2K0 clones to produce M+4 labeled aspartate (FIG. 33A) and asparagine (FIG. 33B), and M+5 proline (FIG. 33C) and stable isotope tracing of ¹³Ci,2-glucose under 50 pM (+) and 0 pM (-) cystine for 4 hours in AsPCl control and all PKM2K0 clones to produce M+2 labeled aspartate (FIG. 33D) and alanine (FIG.

33E).

[0050] FIG. 34A-34F are bar graphs showing: stable isotope tracing of ¹³Ci,2-glucose under 50 pM (+) and 0 pM (-) cystine for 4 hours in AsPCl control and all PKM2K0 clones to produce M+l labeled ribose-5phosphate (FIG. 34A) and ribulose-5phosphate (FIG. 34B), stable isotope tracing of ¹³Ci,2-glucose under 50 pM (+) and 0 pM (-) cystine for 4 hours in Panel control and all PKM2K0 clones to produce M+l labeled ribose-5phosphate (FIG. 34C) and ribulose- 5phosphate (FIG. 34D), and stable isotope tracing of ¹³Ci,2-glucose under 50 pM (+) and 0 pM (- ) cystine for 4 hours in AsPCl (FIG. 34E) and Panel (FIG. 34F) control and all PKM2K0 clones to produce M+l labeled hexose-phosphate. Significance was assessed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0051] FIG. 35A and FIG. 35B are bar graphs showing stable table isotope tracing of ¹³Cs- glutamine under 50 pM (+) and 0 pM (-) cystine for 24 hours in AsPCl control and all PKM2K0 clones to produce M+5 labeled glutathione (FIG. 35A) and stable isotope tracing of ¹³Cs- glutamine under 50 pM (+) and 0 pM (-) cystine for 24 hours in Panel control and all PKM2K0 clones to produce M+5 labeled glutathione (FIG. 35B). Significance was assessed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0052] FIG. 36A and FIG. 36B are bar graphs showing relative viability of AsPCl WT cells (FIG. 36A) and Panel WT cells (FIG. 36B) under 50 pM (+) or 0 pM (-) cystine co-treated with 300 pM buthionine-sulfoximine (BSO), 1 mM glutathione-ethyl ester (GSH-EE), and/or 5 pM ferrostatin-1 (FER). Significance was assessed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0053] FIG. 37 is a bar graph showing relative viability of AsPCl control and PKM2K0 cells under 50 pM (+) and 0 pM (-) cystine supplemented with either 6 pM aspartate, 90 pM asparagine, 90 pM glutamate, or 240 pM proline. Significance was assessed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0054] FIG. 38 is a bar graph showing relative viability of AsPCl control and PKM2K0 clone #1 under 50 pM (+) and 0 pM (-) cystine with (+) or without (-) 250 pM glutamine. Significance was assessed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0055] FIG. 39A and FIG. 39B are bar graphs showing relative viability of AspCl (FIG. 39A) and Panel (FIG. 39B) control and PKM2KO cells under 50 pM cystine with (+) or without (-) 1 mM glutamine treated with 5 pM IKE. Significance was assessed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0056] FIG. 40 is a bar graph showing relative viability of AsPCl control and PKM2KO cells under 50 pM (+) and 0 pM (-) cystine with (+) or without (-) 1 mM glutamine. Significance was assessed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0057] FIG. 41 is a bar graph showing relative viability of AsPCl control and PKM2KO clone #1 under 50 pM (+) or 0 pM (-) cystine treated with 5 pM CB-839 (glutaminase inhibitor), supplemented with either 8 mM dimethyl- a-ketoglutarate (a-Kg), 8 mM dimethyl- succinate (Sue), or 32 mM dimethyl-malate (Mai). Significance was assessed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0058] FIG. 42 is a bar graph showing relative lipid peroxidation in AsPCl Control and PKM2KO clone #1 under 50 pM (+) or 0 pM (-) cystine with (+) or without (-) 1 mM glutamine. Significance was assessed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0059] FIG. 43A-43B are Western blots of xCT and GPX4 expression in AsPCl (FIG. 43A) and Panel (FIG. 43B) cells under 50 pM (+) and 0 pM (-) cystine with (+) or without (-) 1 mM glutamine. Vinculin was used as a loading control.

[0060] FIG. 44A-44D are bar graphs showing relative viabilities of AsPCl control (FIG. 44A) and PKM2KO clones (FIG. 44B, FIG. 44C, and FIG. 44C) under 50 pM (+) or 0 pM (-) cystine with (+) or without (-) 1 mM glutamine supplemented with either 8 mM dimethyl- a- ketoglutarate (aKG), 8 mM dimethyl-succinate (Sue), or 32 mM dimethyl-malate (Mai). Significance was assessed by one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Sidak test.

[0061] FIG. 45A-45C are bar graphs showing relative viabilities of Panel (FIG. 45A) and PKM2KO clones (FIG. 45B and FIG. 45C) under 50 pM (+) or 0 pM (-) cystine with or without 1 mM glutamine supplemented with either 8 mM DM-a-ketoglutarate (DM-akg), 8 mM DM- Succinate (DM-Suc), or 32 mM DM-Malate (DM-Mal). Significance was assessed by one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Sidak test.

[0062] FIG. 46A and FIG. 46B are bar graphs showing relative viabilities of AsPCl (FIG. 46A) and Panel (FIG. 46B) control and PKM2KO clones under 50 pM (+) or 0 pM (-) cystine supplemented with 8 mM dimethyl-malate (Mai). Significance was assessed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test.

[0063] FIG. 47A-47G are bar graphs showing relative viabilities of AsPCl (FIG. 47A-47D) and Panel (FIG. 47E-47G) control and PKM2KO clones under 50 pM cystine supplemented with either 8 mM DM-akg, 8 mM DM-Suc, or 32 mM DM-Mal. Significance was assessed by oneway ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Sidak test.

[0064] FIG. 48A and FIG. 48B are Western blots of malic enzyme 1 (MEI) expression in AsPCl (FIG. 48A) and Panel (FIG. 48B) control cells and PKM2KO clones under 50 pM (+) or 0 pM (-) cystine conditions.

[0065] FIG. 49A-49G are bar graphs showing relative viabilities of Panel control cells (FIG. 49A), Panel PKM2KO clone #1 (FIG. 49B), Panel PKM2KO clone #2 (FIG. 49C), AsPCl control cells (FIG. 49D), AsPCl PKM2KO clone #1 (FIG. 49E), AsPCl PKM2KO clone #2 (FIG. 49F), and AsPCl PKM2KO clone #3 (FIG. 49G) under 0 pM cystine treated with (+) or without (-) 50 pM malic enzyme 1 inhibitor (MEli) and co-treated with either 5 pM ferrostatin-1 (FER) or 1 mM N- acetylcysteine (NAC). Significance was assessed by one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Sidak test.

[0066] FIG. 50A-50G are bar graphs showing relative viabilities of AsPCl control (FIG. 50A), PKM2KO clone #1 (FIG. 50B), AsPCl PKM2KO clone #2 (FIG. 50C), AsPCl PKM2KO clone #3 (FIG. 50D), Panel control (FIG. 50E), Panel PKM2KO clone #1 (FIG. 50F), and PKM2KO clone #2 (FIG. 50G) under 50 pM (+) or 0 pM (-) cystine with (+) or without (-) 50 pM MEli and 32 mM dimethyl-malate (Mai) supplement. Significance was assessed by one-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Sidak test.

[0067] FIG. 51A and FIG. 51B are bar graphs showing relative NADPH abundance in AsPC 1 (FIG. 51A) and Panel (FIG. 51B) control and PKM2KO clones under 50 or 0 pM cystine. For J- K, significance was assessed by two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001. Multiple hypothesis correction by Tukey test. [0068] FIG. 52A and FIG. 52B are proposed models on how PK reprograms metabolism to influence cystine starvation induced ferroptosis under low PK activity (FIG. 52A) and high PK activity (FIG. 52B).

[0069] FIG. 53 is a treatment schematic for xenograft tumors formed from Panel WT cells, treated daily for 2 weeks with vehicle control, IKE, TEPP-46, or IKE and TEPP-46 combined. N=6 for each treatment group, except IKE and TEPP-46 combination with N=4.

[0070] FIG. 54 is a line graph showing tumor volume of xenograft tumors for each treatment group (shown in FIG. 53) over time. Significance was assessed by one-way ANOVA at end point. *p<0.05, **p<0.01, ns = non-significant. Multiple hypothesis correction by Sidak test.

[0071] FIG. 55 is a bar graph showing tumor weight at end point for each treatment group (shown in FIG. 53). Significance was assessed by one-way ANOVA at end point. *p<0.05, **p<0.01, ns = non-significant. Multiple hypothesis correction by Sidak test.

[0072] FIG. 56 includes images of tumors for each treatment group (shown in FIG. 53) at end point.

[0073] FIG. 57 is a line graph showing body weight of treated mice throughout the treatment course for each treatment group (shown in FIG. 53).

[0074] FIG. 58 is a schematic indicating the following: The human prostate cancer cell line LNCaP is characterized by androgen sensitivity and limited metastatic potential. To produce a metastatic castrate resistant prostate cancer cell line, LNCaP cells were inoculated in castrated mice. Cells isolated from tumors in these mice were then sequentially inoculated into additional castrated mice and bone metastatic cells were isolated to form the C4-2B cells.

[0075] FIG. 59 is a schematic representing the following: The human PKM gene encodes for the PKM1 and PKM2 isoforms through mutually exclusive alternative splicing. Inclusion of exon 9 produces the PKM1 isoform whereas inclusion of exon 10 produces PKM2. Targeted sgRNA for exon 10 were delivered using a lentiviral CRISPR/Cas9 system to knockout PKM2 expression.

[0076] FIG. 60 is a Western blot analysis of PKM1 and PKM2 expression in LNCaP and C4- 2B scramble control and PKM2KO cells.

[0077] FIG. 61A-61D are line graphs showing proliferation of LNCaP control and PKM2KO cells grown under 50 pM cystine conditions for 48 hours (FIG. 61A), proliferation of C4-2B control and PKM2KO cells grown under 50 pM cystine conditions for 48 hours (FIG. 61B), proliferation of LNCaP control and PKM2KO cells grown under 0 pM cystine conditions for 48 hours (FIG. 61C), and proliferation of C4-2B control and PKM2KO cells grown under 0 pM cystine conditions for 48 hours (FIG. 61D). Cell counts shown are relative to the 0 hour cell count immediately after switching to experimental media conditions. All data points represent the mean of three replicates with error bars representing the standard deviation. * = p-val < 0.05 ns = nonsignificant.

[0078] FIG. 62A-62F are bar graphs showing viability of LNCaP control and PKM2K0 cells grown under 50 and 0 pM cystine for 24 hours (FIG. 62A) and 48 hours (FIG. 62B), viability of C4-2B control and PKM2K0 cells grown under 50 (+) and 0 (-) pM cystine for 24 hours (FIG. 62C) and 48 hours (FIG. 62D), and viability of C4-2B control cells (FIG. 62E) and PKM2K0 cells (FIG. 62F) under cystine starvation treated with 1 mM N-acetyl-cysteine (NAC), 100 mM trolox (TRO), or 100 pM deferoxamine (DFO). Cystine starved alone were treated with an equivalent 0.1% DMSO vehicle treatment. Viability assay results are shown relative to the respective control cell line under the cystine present condition. Bar graphs represent the mean of three replicates with individual data points shown and error bars representing the standard deviation. * = p-val < 0.05, ** = p-val < 0.01, *** = p-val < 0.001.

[0079] FIG. 63A-63D are bar graphs showing relative viabilities of four different cancer cells lines after indicated treatment determined by trypan blue staining and cell counting. HCT-116 (FIG. 63A), A375 (FIG. 63B), A549 (FIG. 63C), and MDA-MD-231 (FIG. 63D) cells were incubated with vehicle, 10 pM IKE and/or 100 pM TEPP-46 as indicated for 24 h. Data are displayed as means +SD., *Statistical comparison between the treatment (IKE+/ TEPP-46+) and control groups (N = 6). **p-value <0.01, ***p-value <0.001, ****p-value < 0.0001, ns = nonsignificant.

[0080] FIG. 64A-64D are line graphs showing proliferation of HCT-116 (FIG. 64A), A375 (FIG. 64B), A549 (FIG. 64C), and MDA-MD-231 (FIG. 64D) cells were determined using Incucyte®. Cell counts shown are relative to the 0 hour cell count immediately after switching to experimental media containing vehicle, 10 pM of IKE and/or 100 pM TEPP-46 as indicated. Data are displayed as means +SD. *Statistical comparison between the treatment (IKE+/ TEPP-46+) and control groups (N = 3). ***p-value < 0.001.

DETAILED DESCRIPTION

A. Introduction

[0081] To further elucidate reprogrammed PDAC metabolism, the mechanisms by which PK activity impact cysteine/cystine, glucose, and glutamine metabolism in pancreatic cancer cells under nutrient restricted conditions were investigated. While increased PK activity sensitizes cells to oxidative stress, its activity has not yet been connected to ferroptosis. [0082] The inventors discovered PKM2 knock-out (PKM2KO) cells demonstrate a remarkable resistance to cystine starvation mediated ferroptosis. Without being bound by theory, this resistance to ferroptosis is caused by decreased PK activity, rather than an isoform-specific effect. It was found that PKM2K0 cells depend on glutamine metabolism to support antioxidant defenses against lipid peroxidation, primarily by increased glutamine flux through the malate aspartate shuttle and utilization of malic enzyme 1 (MEI) to produce NADPH (reduced form of nicotinamide adenine dinucleotide phosphate (NADP). Thus, cell death can be induced by the combination of PKM2 activation and inhibition of cysteine/cystine metabolism, and this mechanism can be used as a novel treatment strategy in vitro and in vivo for PKM2-expressing cancer cells, such as PDAC.

[0083] Example embodiments are provided so that this disclosure will be thorough and will fully convey the scope to those who are skilled in the art. Numerous specific details are set forth such as examples of specific compositions, components, and methods, to provide a thorough understanding of embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be employed, that example embodiments may be embodied in many different forms and that neither should be construed to limit the scope of the disclosure. In some example embodiments, well-known processes, well-known device structures, and well- known technologies are not described in detail.

B. Definitions

[0084] The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” may be intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms “comprises,” “comprising,” “including,” and “having,” are inclusive and therefore specify the presence of stated features, elements, compositions, steps, integers, operations, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Although the open-ended term “comprising,” is to be understood as a non-restrictive term used to describe and claim various embodiments set forth herein, in certain aspects, the term may alternatively be understood to instead be a more limiting and restrictive term, such as “consisting of’ or “consisting essentially of.” Thus, for any given embodiment reciting compositions, materials, components, elements, features, integers, operations, and/or process steps, the present disclosure also specifically includes embodiments consisting of, or consisting essentially of, such recited compositions, materials, components, elements, features, integers, operations, and/or process steps. In the case of “consisting of,” the alternative embodiment excludes any additional compositions, materials, components, elements, features, integers, operations, and/or process steps, while in the case of “consisting essentially of,” any additional compositions, materials, components, elements, features, integers, operations, and/or process steps that materially affect the basic and novel characteristics are excluded from such an embodiment, but any compositions, materials, components, elements, features, integers, operations, and/or process steps that do not materially affect the basic and novel characteristics can be included in the embodiment.

[0085] Any method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed, unless otherwise indicated.

[0086] The use of the term "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." As such, the terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or greater numbers of compounds.

[0087] The use of the term "at least one" will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100/1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal number terminology (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of differentiating between two or more items and is not meant to imply any sequence or order or importance to one item over another or any order of addition, for example.

[0088] The use of the term "or" in the claims is used to mean an inclusive "and/or" unless explicitly indicated to refer to alternatives only or unless the alternatives are mutually exclusive. For example, a condition "A or B" is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0089] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearance of the phrase "in some embodiments" or "one example" in various places in the specification is not necessarily all referring to the same embodiment, for example. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims.

[0090] Throughout this disclosure, the term "about" is used to indicate that a value includes the inherent variation of error for a composition/apparatus/device, the method being employed to determine the value, or the variation that exists among the study subjects. For example, but not by way of limitation, when the term "about" is utilized, the designated value may vary by plus or minus twenty percent, or fifteen percent, or twelve percent, or eleven percent, or ten percent, or nine percent, or eight percent, or seven percent, or six percent, or five percent, or four percent, or three percent, or two percent, or one percent from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art. Particularly in reference to a given quantity, number or percentage, “about” is meant to encompass deviations of plus or minus ten percent (± 10). For example, about 5% encompasses any value between 4.5% to 5.5%, such as 4.5, 4.6, 4.7, 4.8, 4.9, 5, 4.1, 5.2, 5.3, 5.4, or 5.5. Accordingly, unless otherwise indicated, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0091] The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0092] As will be understood by one skilled in the art, for any and all purpose, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Furthermore, as will be understood by one skilled in the art, a range includes each individual member.

[0093] Cysteine (also known as 2-amino-3-sulfhydrylpropanoic acid, CAS Number 52-90-4) is an amino acid that aids in protein synthesis and defense against reactive oxygen species (ROS) as a precursor for tripeptide glutathione and coenzyme A. Healthy cells utilize circulating cysteine or cysteine biosynthesis to fulfill these roles, while cancer cells instead depend on exogenous cystine, the oxidized dimer of cysteine (i.e., two cysteine molecules connected by a disulfide bond). Cystine is acquired predominantly through the cystine/glutamate antiporter (system Xc') which is a heterodimer of SLC7A11 (also known as xCT) and SLC3A2 and is overexpressed in many cancer cells. The use of “Cysteine/cystine” and “cystine/cysteine” as used herein refers to cysteine and/or cystine. Depletion of extracellular cystine (i.e., cystine starvation) leads to a loss of intracellular glutathione supply and the accumulation of oxidative damage to membrane lipids. Uncontrolled lipid peroxidation propagates by reacting with ferrous iron and producing hydroxyl radicals leading to a form of cell death known as ferroptosis.

[0094] Pyruvate kinase (PK) is an enzyme that converts phosphoenolpyruvate to pyruvate and produces adenosine triphosphate (ATP). The pyruvate kinase muscle (PKM) gene may have 70%, 71%, 72%, 73%, 74%, 76%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to nucleic acid SEQ ID NO: 1 and may express pyruvate kinase muscle isoform 1 (PMK1) or pyruvate kinase muscle isoform 2 (PKM2). Cancer cells can switch from PKM1 isoform expression to PKM2 isoform expression. The PKM1 isoform may have 70%, 71%, 72%, 73%, 74%, 76%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to amino acid SEQ ID NO: 9. The PKM2 isoform may have 70%, 71%, 72%, 73%, 74%, 76%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to amino acid SEQ ID NO: 2.

[0095] The term “agent” as used herein refers to a drug, protein, peptide, gene, compound, or other active pharmaceutical ingredient. Agents may be used to treat, reduce, and/or prevent diseases and conditions. An agent may be an activating agent or an inhibiting agent. Exemplary agents are cysteine/cystine metabolism inhibiting agents, pyruvate kinase muscle isoform 2 (PKM2) activating agents, and/or anti-cancer agents provided herein.

[0096] An “activating agent” or “enzyme activating agent” as referred to herein is a molecule that binds to an enzyme and increases its activity. Activating agents may be involved in the allosteric regulation of enzymes in the control of metabolism. Activating agents may be the opposite of inhibiting agents. An activating agent can also be defined as an “agonist.” Agonists can be full, partial, or inverse. A full agonist has high efficacy and produces the desired response (e.g., activation of an enzyme) by binding few receptors. A partial agonist has lower efficacy (does not produce as much of the desired response). An inverse agonist produces the opposite effect to an agonist in the absence of a full/partial agonist (e.g., targets the basal constitutive activity of an enzy me/receptor/etc . ) .

[0097] Thus, as referred to herein, an “inhibiting agent” or an “enzyme inhibiting agent” is a molecule that binds an enzyme and decreases its activity. Inhibitors can also be called “antagonists.” There are competitive and non-competitive antagonists. A competitive antagonist binds at an active site where an agonist would. A non-competitive antagonist binds at an allosteric site. Antagonists can also be reversible (bind non-covalently to the enzy me/receptor/etc.) or irreversible (bind covalently to the enzyme/receptor/etc.).

[0098] As referred to herein, an “allosteric agent” may change their conformational ensemble upon binding of an effector (allosteric modulator) which results in an apparent change in binding affinity at a different ligand binding site.

[0099] Activating agents and inhibiting agents may specifically bind to an enzyme. Activating agents may turn enzyme activity “up” by specifically binding an enzyme, while inhibiting agents may turn enzyme activity “down” by specifically binding an enzyme.

[00100] The term “reduce” as used herein refers to a negative alteration of at least 1%, 5%, 10%, 15%, 20%, 25%, 50%, 75%, or 100%.

[00101] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. In particular, this disclosure utilizes routine techniques in the field of cysteine/cy stine metabolism and cancer therapy.

C. Therapeutic Combinations and Pharmaceutical Compositions

[00102] Provided herein is a therapeutic combination that includes one or more cysteine/cystine metabolism inhibiting agent(s) and one or more pyruvate kinase isoform 2 (PKM2) activating agent(s). Also provided herein is a therapeutic combination that includes a means for inhibiting cysteine/cystine metabolism, a means for activating PKM2, and a pharmaceutically acceptable carrier.

[00103] In some embodiments, the therapeutic combination may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cysteine/cystine metabolism inhibiting agents. In certain embodiments, the therapeutic combination may include one cysteine/cystine metabolism inhibiting agent. In some embodiments, the means for inhibiting cysteine/cystine metabolism may be one or more cysteine/cystine metabolism inhibiting agents as described herein. [00104] In some embodiments, the therapeutic combination may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 PKM2 activating agents. In certain embodiments, the therapeutic combination may include one PKM2 activating agent. In specific embodiments, the therapeutic combination may include one cysteine/cy stine metabolism inhibiting agent and one PKM2 activating agent. In some embodiments, the means for activating PKM2 may be one or more PKM2 activating agents as described herein.

[00105] In some embodiments, the cysteine/cystine metabolism inhibiting agent may be any agent that inhibits cysteine/cystine metabolism. In some embodiments, the cysteine/cystine metabolism inhibiting agent may be imidazole ketone erastin (IKE), erastin, sulfasalazine, (s)-4- carboxyphenylglycine, cyst(e)inase, sorafenib, IKE-linked nanoparticles, or a combination thereof. In certain embodiments, the cysteine/cystine metabolism inhibiting agent is IKE. In some embodiments, the cysteine/cystine metabolism inhibiting agent may be referred to as a cysteine/cystine metabolism antagonist. In some embodiments, the cysteine/cystine metabolism inhibiting agent may be an allosteric antagonist or a non-allosteric antagonist. In certain embodiments, the cysteine/cystine metabolism inhibiting agent may be an allosteric antagonist. In some embodiments, the cysteine/cystine metabolism inhibiting agent may be a competitive or a non-competitive antagonist. In some embodiments, the cysteine/cystine metabolism inhibiting agent may be reversible or irreversible.

[00106] In some embodiments, the PKM2 activating agent may be any agent that activates PKM2. In some embodiments, the PKM2 activating agent may be TEPP-46, DASA-58, TP-1454, l-(sulfonyl)-5-(arylsulfonyl)indoline, micheliolide, diarylsulfonamides, thieno[3,2-b]pyrrole[3,2- d]pyridazinones, 4-(2,3-dichlorobenzoyl)-l-methyl-pyrrole-2-carboxamide, 2-((lH- benzo[d]imidazole-l-yl)methyl)-4H-pyrido[l,2-a]pyrimidin-4-ones, l-(sulfonyl)-5-

(arylsulfonyl)indoline, or a combination thereof. In certain embodiments, the PKM2 activating agent may be TEPP-46. In some embodiments, the PKM2 activating agent may be referred to as a PKM2 agonist. In some embodiments, the PKM2 activating agent may be an allosteric agonist or a non-allosteric agonist. In certain embodiments, the PKM2 activating agent may be an allosteric agonist. In some embodiments, the PKM2 activating agent may be a full, partial, or inverse agonist. In some embodiments, the PKM2 activating agent may be TEPP-46, and TEPP- 46 may be an allosteric agonist. TEPP-46 has molecular formula: C17H16N4O2S2 and may also be known as ML265, CID-44246499, and NCGC00186528 (CAS Number: 1221186-53-3).

[00107] In some embodiments, the cysteine/cystine metabolism inhibiting agent may be IKE and the PKM2 activating agent may be TEPP-46. [00108] In some embodiments, the therapeutic combination may include an additional agent. In some embodiments, the additional agent may be one or more anti-cancer agent (also known as an antineoplastic agent). In some embodiments, the therapeutic combination may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional active agents. In some embodiments, the therapeutic combination may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional anti-cancer agents.

[00109] In some embodiments, the anti-cancer agent may be a chemotherapy agent, a hormone therapy agent, a targeted therapy agent, and immunotherapy agent, or a combination thereof. In some embodiments, the anti-cancer agent may be folfirinox, 5-fluorouracil, Gemcitabine, Capecitabine, Oxaliplatin, Cisplatin, Carboplatin, Irinotecan, Leucovorin, Paclitaxel, albumin bound paclitaxel, docetaxel, Erlotinib, Olaparib, rucaparib, Pembrolizumab, Larotrectinib, Entrectinib, Vinorelbine, Etoposide, Pemetrexed, Trifluridine, Tipiracil, bevacizumab, ramuciriumab, cetuximab, panitumumab, trastuzumab, pertuzumab, Sacituzumab govitecan-hziy, tucatinib, lapatinib, fam-trastuzumab deruxtecan, abiraterone, enzalutamide, apalutamide, darolutamide, imatinib, sunitinib, everolimus, temsirolimus, vemurafenib, neratinib, tamoxifen, or a combination thereof.

[00110] In some embodiments, the anti-cancer agent may have one or more primary mechanism of action. In some embodiments, the primary mechanism of action of an anti-cancer agent may be chemotherapy, hormone therapy, targeted therapy, or immunotherapy.

[00111] In some embodiments, the primary mechanism of action of any one of anti-cancer agents folfirinox, 5-fluorouracil, Gemcitabine, Capecitabine, Oxaliplatin, Cisplatin, Carboplatin, Irinotecan, Leucovorin, Paclitaxel, albumin bound paclitaxel, docetaxel, Vinorelbine, Etoposide, Pemetrexed, Trifluridine, and Tipiracil may be chemotherapy.

[00112] In some embodiments, the primary mechanism of action of any one of anti-cancer agents abiraterone, enzalutamide, apalutamide, darolutamide, and tamoxifen may be hormone therapy.

[00113] In some embodiments, the primary mechanism of action of any one of anti-cancer agents Erlotinib, Olaparib, rucaparib, Larotrectinib, Entrectinib, bevacizumab, ramuciriumab, cetuximab, panitumumab, trastuzumab, pertuzumab, Sacituzumab govitecan-hziy, tucatinib, lapatinib, fam-trastuzumab deruxtecan, imatinib, sunitinib, everolimus, temsirolimus, vemurafenib, and neratinib may be targeted therapy.

[00114] In some embodiments, the primary mechanism of action of anti-cancer agent Pembrolizumab may be immunotherapy. [00115] In some embodiments, the anti-cancer agent may be an alkylating agent, platinum compound, anti-metabolite, mitotic spindle inhibitor, topoisomerase inhibitor, tyrosine kinase inhibitor, proteasome inhibitor, poly ADP ribose polymerase (PARP) inhibitor, CdK inhibitor, monoclonal antibody, immunotoxin, antiandrogen, other anti-cancer agent, or a combination thereof.

[00116] In some embodiments, the therapeutic combination may be used to treat, prevent, and/or reduce the occurrence of one or more cancers. In some embodiments, the therapeutic combination may be used to inhibit survival of one or more cancer cells. In some embodiments, the therapeutic combination may induce tumor cell death. In some embodiments, the therapeutic combination may induce ferroptosis in cancer cells. In some embodiments, the therapeutic combination may be used for a combination of any one of: treating, preventing, and/or reducing the occurrence of cancer; inhibiting survival of cancer cells; inducing tumor cell death, or inducing ferroptosis in cancer cells.

[00117] Ferroptosis, also known as oxytosis, is a type of programmed cell death that may be dependent on iron and characterized by the accumulation of lipid peroxides. In some embodiments, ferroptosis suppresses tumor growth. In some embodiments, ferroptosis is cystine deprivation/starvation-induced ferroptosis. In some embodiments, cystine starvation may impede the generation of glutathione (GSH). Thus, in some embodiments, ferroptosis may be achieved by intracellular GSH depletion. In some embodiments, ferroptosis may be achieved by decreased glutathione peroxidase 4 (GPX4) activity. In some embodiments, inhibition of GPX4 may lead to ferroptosis with low PK activity. For example, in some embodiments, excessive lipid peroxidases may not all be metabolized by the GPX4-catalyzed reduction reaction, leading to the accumulation of reactive oxygen species (ROS), and thus leading to ferroptosis. In some embodiments, inhibition of MEI may promote ferroptosis and/or circumvent PDAC metabolic defense strategies for surviving low cystine conditions.

[00118] In some embodiments, the therapeutic combination may be included in a pharmaceutical composition. Thus, the present disclosure provides a pharmaceutical composition including a therapeutic composition as described herein. In some embodiments, the pharmaceutical composition may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 pharmaceutically acceptable carriers. In certain embodiments, the pharmaceutical composition may include at least one pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may include a means for inducing ferroptosis in one or more cancer cells. [00119] Also described herein is a pharmaceutical composition including a means for inducing ferroptosis in one or more cancer cells and a pharmaceutically acceptable carrier. In some embodiments, the means for inducing ferroptosis may be a therapeutic combination as described herein. In some embodiments, the means for inducing ferroptosis may be one or more cysteine/cystine metabolism inhibiting agents. In some embodiments, the means for inducing ferroptosis may be one or more PKM2 activating agents. In some embodiments, the means for inducing ferroptosis may be a combination of one or more cysteine/cystine metabolism inhibiting agents and one or more PKM2 activating agents. In some embodiments, the means for inducing ferroptosis may be any agent that induced ferroptosis. In some embodiments, the means for inducing ferroptosis may be a combination of two or more agents that induces ferroptosis.

[00120] In some embodiments, it may be beneficial to include one or more excipients in a pharmaceutical composition. One of skill in the art would appreciate that the choice of any one excipient may influence the choice of any other excipient. For example, the choice of a particular excipient may preclude the use of one or more additional excipients because the combination of excipients would produce undesirable effects. One of skill in the art would be able to determine empirically which excipients, if any, to include in the formulations or compositions disclosed herein. Excipients may include, but are not limited to, co-solvents, solubilizing agents, buffers, pH adjusting agents, bulking agents, surfactants, encapsulating agents, tonicity-adjusting agents, stabilizing agents, protectants, and viscosity modifiers. In some embodiments, it may be beneficial to include a pharmaceutically acceptable carrier.

[00121] In some embodiments, it may be beneficial to include a solubilizing agent. Solubilizing agents may be useful for increasing the solubility of any of the components of the therapeutic combination provided herein, such as a cysteine/cystine metabolism inhibiting agent and/or a pyruvate kinase isoform 2 (PKM2) activating agent. The solubilizing agents described herein are not intended to constitute an exhaustive list but are provided merely as exemplary solubilizing agents that may be used. In certain embodiments, solubilizing agents include, but are not limited to, ethyl alcohol, tert-butyl alcohol, polyethylene glycol, glycerol, methylparaben, propylparaben, polyethylene glycol, polyvinyl pyrrolidone, and any pharmaceutically acceptable salts and/or combinations thereof.

[00122] The pH may be any pH that provides desirable properties for the composition. Desirable properties may include, for example, agent stability, increased agent retention as compared to compositions at other pHs, and improved filtration efficiency. [00123] In some embodiments, it may be beneficial to include a tonicity-adjusting agent. The tonicity of a liquid composition is an important consideration when administering the composition to a patient, for example, by parenteral administration. Tonicity-adjusting agents, thus, may be used to help make a composition suitable for administration. Tonicity-adjusting agents are well known in the art. Accordingly, the tonicity-adjusting agents described herein are not intended to constitute an exhaustive list but are provided merely as exemplary tonicity-adjusting agents that may be used. Tonicity-adjusting agents may be ionic or non-ionic and include, but are not limited to, inorganic salts, amino acids, carbohydrates, sugars, sugar alcohols, and carbohydrates. Exemplary inorganic salts may include sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate. An exemplary amino acid is glycine. Exemplary sugars may include sugar alcohols such as glycerol, propylene glycol, glucose, sucrose, lactose, and mannitol.

[00124] In some embodiments, it may be beneficial to include a stabilizing agent. Stabilizing agents help increase the stability of agents in compositions of the disclosure.

[00125] In some embodiments, it may be beneficial to include a protectant. Protectants protect agents such as cysteine/cystine metabolism inhibiting agents, pyruvate kinase muscle isoform 2 (PKM2) activating agents, and/or anti-cancer agents as disclosed herein from an undesirable condition (e.g., instability caused by freezing or lyophilization, or oxidation). Protectants can include, for example, cryoprotectants, lyoprotectants, and antioxidants. Cryoprotectants are useful in preventing loss of potency of an active pharmaceutical ingredient when a formulation is exposed to a temperature below its freezing point. For example, a cryoprotectant could be included in a reconstituted lyophilized formulation so that the formulation could be frozen before dilution for intravenous (IV) administration. Cryoprotectants are well known in the art. Accordingly, the cryoprotectants described herein are not intended to constitute an exhaustive list but are provided merely as exemplary cryoprotectants that may be used. Cryoprotectants include, but are not limited to, solvents, surfactants, encapsulating agents, stabilizing agents, viscosity modifiers, and combinations thereof. Cryoprotectants may include, for example, disaccharides (e.g., sucrose, lactose, maltose, and trehalose), polyols (e.g., glycerol, mannitol, sorbitol, and dulcitol), glycols (e.g., ethylene glycol, polyethylene glycol, propylene glycol).

[00126] Lyoprotectants are useful in stabilizing the components of a lyophilized formulation or composition. For example, an agent as disclosed herein could be lyophilized with a lyoprotectant prior to reconstitution. Lyoprotectants are well known in the art. Accordingly, the lyoprotectants described herein are not intended to constitute an exhaustive list but are provided merely as exemplary lyoprotectants that may be used. Lyoprotectants include, but are not limited to, solvents, surfactants, encapsulating agents, stabilizing agents, viscosity modifiers, and combinations thereof. Exemplary lyoprotectants may be, for example, sugars and polyols, trehalose, sucrose, dextran, and hydroxypropylbeta-cyclodextrin are non-limiting examples of lyoprotectants.

[00127] Antioxidants are useful in preventing oxidation of the components of a composition. Oxidation may result in aggregation of a drug product or other detrimental effects to the purity of the drug product or its potency. Antioxidants are well known in the art. Accordingly, the antioxidants described herein are not intended to constitute an exhaustive list but are provided merely as exemplary antioxidants that may be used. Antioxidants may be, for example, sodium ascorbate, citrate, thiols, metabisulfite, and combinations thereof.

[00128] Also provided herein is a vaccine including a therapeutic combination as described herein and/or a pharmaceutical composition as described herein.

D. Methods of Use

Method, of treating, preventing, or reducing the occurrence of cancer

[00129] Provided herein is a method of treating, preventing, or reducing the occurrence of cancer in a subject in need thereof. In some embodiments, the method may include administering to the subject an effective amount of a therapeutic combination as described herein, a pharmaceutical composition as described herein, and/or a vaccine as described herein. In some embodiments, the method may include administering to the subject a combination of a means for inhibiting cysteine/cystine metabolism, a means for activating PKM2, and a pharmaceutically acceptable carrier.

[00130] Thus, in some embodiments, as described herein, the therapeutic combination, pharmaceutical composition, and/or vaccine may include one or more means for inhibiting cysteine/cystine metabolism, one or more means for activating PKM2, and one or more pharmaceutically acceptable carrier. In some embodiments, the means for inhibiting cysteine/cystine metabolism may be one or more cysteine/cystine metabolism inhibiting agents, such as IKE. In some embodiments, the means for activating PKM2 may be one or more PKM2 activating agent, such as TEPP-46.

[00131] In some embodiments, the therapeutic combination, pharmaceutical composition, and/or vaccine may include one or more means for inducing ferroptosis in a cancer cell and a pharmaceutically acceptable carrier. In some embodiments, the means for inducing ferroptosis may be the combination of a cysteine/cystine metabolism inhibiting agent and a PKM2 activating agent.

[00132] In some embodiments, as described herein, the therapeutic combination, pharmaceutical composition, and/or vaccine may include one or more cysteine/cystine metabolism inhibiting agent as described herein and/or one or more PKM2 activating agent as described herein.

[00133] In some embodiments, the one or more cysteine/cystine metabolism inhibiting agent and the one or more PKM2 activating agent may be administered by intravenous, intratumoral, or intraperitoneal injection. In some embodiments, the one or more cysteine/cystine metabolism inhibiting agent and the one or more PKM2 activating agent may be independently administered by intravenous, intratumoral, or intraperitoneal injection. In some embodiments, the one or more cysteine/cystine metabolism inhibiting agent and the one or more PKM2 activating agent may be administered by the same route of administration. In some embodiments, the one or more cysteine/cystine metabolism inhibiting agent may be administered to the subject prior to, after, simultaneously, or sequentially with the one or more PKM2 activating agent. In certain embodiments, the one or more cysteine/cystine metabolism inhibiting agent and the one or more PKM2 activating agent may be administered by intraperitoneal injection.

[00134] In some embodiments, the one or more cysteine/cystine metabolism inhibiting agent and the one or more PKM2 activating agent may be formulated in the same pharmaceutical composition. Alternatively, in some embodiments, the one or more cysteine/cystine metabolism inhibiting agent and the one or more PKM2 activating agent may be formulated in separate pharmaceutical compositions.

[00135] “Subject,” ‘ ‘individual,” and “patient” interchangeably refer to a mammal, preferably a human or a non-human primate, but also domesticated mammals (e.g., canine or feline), laboratory mammals (e.g., mouse, rat, rabbit, hamster, guinea pig), and agricultural mammals (e.g., equine, bovine, porcine, ovine). In some embodiments, the subject may be human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child). Alternatively, in some embodiments, the subject may be a non-human animal. For example, in some embodiments, the non-human animal may be a canine or a feline. In certain embodiments, the subject can be under the care of a physician or other health worker. In certain embodiments the subject may not be under the care of a physician or other health worker.

[00136] In some embodiments, administration is preferably in an “effective amount” or a “therapeutically effective amount” (as the case may be), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, may depend on the nature and severity of what is being treated. For injection, the pharmaceutical composition according to the present invention may be provided, for example, in a pre-filled syringe.

[00137] In some embodiments, the cancer may be one or more types of cancer. In some embodiments, the cancer may be any PKM2-expressing cancer. The pyruvate kinase muscle (PKM) gene may have 80%, 85%, 90%, 95%, or 100% sequence identity to nucleic acid SEQ ID NO: 1 and may express pyruvate kinase muscle isoform 1 (PMK1) or pyruvate kinase muscle isoform 2 (PKM2). In some embodiments, the cancer cells may switch from PKM1 isoform expression to PKM2 isoform expression. The PKM1 isoform may have 80%, 85%, 90%, 95%, or 100% sequence identity to amino acid SEQ ID NO: 9. The PKM2 isoform may have 80%, 85%, 90%, 95%, or 100% sequence identity to amino acid SEQ ID NO: 2. In some embodiments, expression of PKM2 may cause an increase in glucose consumption and lactate secretion in cancer cells. In some embodiments, cancer cells expressing PKM2 may have rapid proliferation and survival in low nutrient (e.g., glucose) conditions. This may be, for example, because the PKM2- expressing cancer cells alter glucose metabolism. In some embodiments, the cancer may be particularly vulnerable to cystine starvation induced ferroptosis. In some embodiments, the cancer cells exhibit increased ROS production.

[00138] In some embodiments, the cancer may be pancreatic cancer, prostate cancer, breast cancer, bladder cancer, endometrial cancer, cholangiocarcinoma, ovarian cancer, kidney cancer, renal cell carcinoma, leukemia, liver cancer, intrahepatic bile duct cancer, lymphoma (e.g., nonHodgkin lymphoma), thyroid cancer (e.g., papillary thyroid cancer), cervical cancer, gallbladder cancer, gastric cancer, skin cancer, bronchus cancer, lung cancer, colon cancer, rectal cancer, melanoma, multiple myeloma, urothelial carcinoma, osteosarcoma, head and neck cancers, colorectal cancer, hepatocellular carcinoma, glioma, medulloblastoma, testicular cancer, or a combination thereof. In certain embodiments, the cancer may be pancreatic cancer. In some embodiments, the pancreatic cancer may be pancreatic ductal adenocarcinoma (PDAC).

[00139] In some embodiments, “treat” and “treatment” refer to an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results may include one or more of the following: a) inhibiting cancer, (e.g., decreasing one or more symptoms resulting from the cancer, and/or diminishing the extent of the cancer); b) slowing or arresting the development of one or more clinical symptoms associated with the cancer (e.g., stabilizing the cancer, preventing or delaying the worsening or progression of the cancer, and/or preventing or delaying the spread (e.g., metastasis) of the cancer); and/or c) relieving the cancer, that is, causing the regression of clinical symptoms (e.g., ameliorating the cancer state, providing partial or total remission of the cancer, enhancing effect of another medication, delaying the progression of the cancer, increasing the quality of life, and/or prolonging survival).

[00140] In some embodiments, the subject in need of treatment may include a subject diagnosed as having, or suspected to have, cancer. In a particular embodiment, treatment may include administering a therapeutic combination or pharmaceutical composition as described herein to a subject having, diagnosed as having, or suspected of having cancer, such as pancreatic cancer. In some embodiments, the subject may be asymptomatic.

[00141] In some embodiments, prevention includes treatment of a cancer that causes the clinical symptoms of the cancer not to develop or progress.

[00142] In some embodiments, co-action activity exists between the cysteine/cystine metabolism inhibiting agent and the PKM2 activating agent. In some embodiments, co-action activity exists between the means for inhibiting cysteine/cystine metabolism and the means for activating PKM2. For example, co-action activity may exist between IKE (cysteine/cystine metabolism inhibiting agent) and TEPP-46 (PKM2 activating agent). The therapeutic combination and/or pharmaceutical composition may have an enhanced beneficial (i.e. more than additive) effect for the subject compared to the therapeutic combination and/or pharmaceutical composition only containing one of the two components. For example, statistical analysis demonstrates that the composition containing both IKE and TEPP-46 has an enhanced effect to reduce tumor volume compared to the oral composition with just IKE or just TEPP-46 (see, Example 9).

[00143] Also described herein is a combination therapy for treating, preventing, and/or reducing the occurrence of cancer in a subject in need thereof. In some embodiments, the combination therapy may include administering a first component including one or more cysteine/cystine metabolism inhibiting agents and a second component including one or more PKM2 activating agents. In some embodiments, the combination therapy may include administering a first component including a means for inhibiting cysteine/cystine metabolism and a second component including a means for activating PKM2. In some embodiments, the combination therapy includes administration of a third component including an anti-cancer agent.

[00144] In some embodiments, treating, preventing, or reducing the occurrence of cancer may include inducing ferroptosis in one or more cancer cell in a subject.

Method of inducing ferroptosis in a cancer cell [00145] Also provided herein is a method of inducing ferroptosis in one or more cancer cell. The method includes inhibiting cysteine/cystine metabolism in the one or more cancer cell and activating PKM2 in the one or more cancer cell.

[00146] In some embodiments, the one or more cancer cells includes one or more cancer cell type. In some embodiments, the cancer may be any cancer type, for example those as described herein. Thus, in some embodiments, the cancer may be pancreatic cancer. In some embodiments, the pancreatic cancer may be pancreatic ductal adenocarcinoma (PDAC).

[00147] In some embodiments, inhibiting cysteine/cystine metabolism in the cancer cell may include one or more of: contacting the cancer cell with one or more cysteine/cystine metabolism inhibiting agent; inhibiting a cystine transporter system of the cancer cell; knocking down cystine transporter system X_c’ in the cancer cell; knocking out cystine transporter system X_c’ in the cancer cell; withholding cystine and/or cysteine from the cancer cell; inhibiting glutathione (GSH) synthesis in the cancer cell; and inhibiting CoA synthesis from cysteine (phosphopantothenoylcysteine synthetase) in the cancer cell. In some embodiments either one or both of SLC7A11 and SLC3A2 can be knocked down and/or out to result in knocking down and/or out cystine transporter system Xc-. In some embodiments, either one or both of the GLS subunits (GCLC and GCLM) can be inhibited.

[00148] In some embodiments, knocking down cystine transporter system X_c’ in the cancer cell may include using a gene silencing technique, such as RNA interference (RNAi), epigenetic modification, CRISPR, or small interfering RNA (siRNA). In some embodiments, knocking out cystine transporter system X_c’ in the cancer cell may include using a gene editing technique, such as CRISPR, restriction enzymes, zinc finger nucleases, or transcription activator-like effector nucleases (TALENS).

[00149] In some embodiments, the method includes contacting the one or more cancer cells with one or more cysteine/cystine metabolism inhibiting agent as described herein and/or one or more PKM2 activating agent as described herein. As mentioned herein, the one or more cysteine/cystine metabolism inhibiting agent may be imidazole ketone erastin (IKE), erastin, sulfasalazine, (s)-4-carboxyphenylglycine, cyst(e)inase, sorafenib, IKE-linked nanoparticles, or a combination thereof. In certain embodiments, the cysteine/cystine metabolism inhibiting agent may be IKE. As mentioned herein, the one or more PKM2 activating agent may be TEPP-46, DASA-58, TP- 1454, l-(sulfonyl)-5-(arylsulfonyl)indoline, micheliolide, diarylsulfonamides, thieno[3,2-b]pyrrole[3,2-d]pyridazinones, 4-(2,3-dichlorobenzoyl)-l-methyl-pyrrole-2- carboxamide, 2-((lH-benzo[d]imidazole-l-yl)methyl)-4H-pyrido[l,2-a]pyrimidin-4-ones, 1- (sulfonyl)-5-(arylsulfonyl)indoline, or a combination thereof. In certain embodiments, the PKM2 activating agent may be TEPP-46.

[00150] In some embodiments, the method is performed in vitro. Alternatively, the method may be performed in vivo.

EXAMPLES

[00151] The following examples are merely illustrative, and do not limit this disclosure in any way.

EXAMPLE 1: General procedures for EXAMPLES 2-9

Statistical analysis

[00152] All data are expressed as means +/- standard deviation. Each experiment was conducted with three to six replicates and was reproduced in at least two independent experiments unless otherwise indicated. The figures shown are the results of one independent experiment representative of the results reproducible in each independent experiment. Statistical analysis and graphing were performed in Graph Pad vlO using Student’s T-Test, one-Way Analysis of Variance (ANOVA), or two-Way ANOVA and corrected for multiple hypothesis testing using Tukey’s multiple comparison test or Sidak’s multiple comparison test where appropriate. A p- value of < 0.05 was used as the cutoff for determining significance. In figures * or # = p-val <0.05, ** = p-val < 0.01, *** = p-val < 0.001, **** = p-val <0.0001.

Cell culture

[00153] The human pancreatic cancer cell lines, AsPCl, Panel, BxPC3, and MiaPaCa2 were acquired as a gift from Dr. Nouri Neamati at University of Michigan. Cells were routinely cultured in Dulbecco’s modified eagle medium (DMEM) (MT10013CV, Fisher Scientific®) with sodium pyruvate, supplemented with 10% fetal bovine serum (FBS) (13206C, Sigma- Aldrich®), 1% penicillin and streptomycin (P/S) (15140122, Fisher Scientific®), 5 pg/mE plasmocin (ant-mpp, Invivogen®), and cultured in a humidified incubator with 5% CO2 at 37°C. During experimental procedures, cells were incubated in a humidified incubator with 5% CO2 at 37 °C in DMEM without glucose, glutamine, pyruvate, or cystine (D9815, US Biological) supplemented with 5 mM glucose, 1 mM pyruvate, and varying amounts of glutamine or cystine as indicated, 10% dialyzed FBS (F0392, Sigma- Aldrich®), and 1% P/S. Cells were routinely tested for mycoplasma detection with the kit (rep-mysnc-100, Invivogen®).

Cell culture reagents [00154] The following is a list of chemical compounds used in cell culture experiments: 1 mM N-acetyl-L-cysteine (NAC) (A9615, Sigma- Aldrich®), 100 pM Trolox (TRO) (238813, Sigma- Aldrich®), 100 pM deferoxamine (DFO) (D9533, Sigma- Aldrich®), 5 pM Ferrostatin-1 (SML0583, Sigma-Aldrich®), 50 pM Z-VAD-FMK (ZVAD) (14463, Cayman Chemical®), 10 pM Necrostatin-lS (20924, Cayman Chemical®), 0.16 - 50 pM imidazole ketone erastin (IKE) (27088, Cayman Chemical®), 0.16 - 10 pM ras selective lethal 3 (RSL3) (SML2234, Sigma- Aldrich®), 25-100 pM TEPP-46 (HY-18657, MedChem Express®), 10 pM compound 3k (36815, Cayman Chemical®), 50 pM Malic enzyme inhibitor (MEli) (HY-124861, MedChem Express®), 8 and 32 mM DM-Malate (DM-Mal) (374318, Sigma- Aldrich®), 8 mM DM-Succinate (DM-Suc) (73605, Sigma- Aldrich®), 8 mM DM-a-ketoglutarate (DM-akg) (28394, Cayman Chemical®), 5 pM CB-839 (22038, Cayman Chemical®), 300 pM buthionine sulfoximine (BSO) (14484, Cayman Chemical®), 1 mM Glutathione ethyl ester (GSH-EE) (14953, Cayman Chemical®), and 0.1-0.5% Dimethyl sulfoxide (DMSO) (D4540, Sigma- Aldrich®).

Western blot analysis

[00155] Cells were plated in 10 cm plates and grown until about 60-70% confluent. Plates were washed twice with phosphate buffered saline (PBS) without calcium or magnesium (D8537, Sigma- Aldrich®) and the media was replaced with experimental media. Panel cells were incubated for 12 hours, AspCl were incubated for 24 hours as described above. Cell lysis for protein extraction was completed using cell lysis buffer (9803, Cell Signaling Technology®). Western blot analysis was carried out using standard protocols. Briefly, protein samples were diluted to 1 pg/mL, reduced with 6x Laemmli loading dye, and boiled for 5 minutes. 30 pg of protein was loaded to precast Bolt 10- or 17-well 4-12% polyacrylamide, Bis-Tris, 1.0 mm gels (NW04122BOX, Fisher Scientific®). Gel electrophoresis was carried out in 2-(N- morpholino)ethanesulfonic acid (MES) running buffer at 200V for 22 minutes. Protein was transferred to nitrocellulose membranes, which were then stained with Ponceau stain to confirm total protein equivalence between samples and were then cut for appropriate targets of interest. The following dilutions of primary commercial antibodies in 5% bovine serum albumin (BSA) in tris buffered saline with 0.1% TWEEN™ 20 (TBST) were used as probes: 1:1000 dilution of antipyruvate kinase muscle isoform 1 (PKM1) (7067S, Cell Signaling Technology®), 1:1000 dilution of anti-pyruvate kinase muscle isoform 2 (PKM2) (4053S, Cell Signaling Technology®), 1:1000 dilution of anti-glutathione peroxidase 4 (GPX4) (52455, Cell Signaling Technology®), 1:500 dilution of anti-xCT (SLC7A11, component of system X_c” cystine/glutamate antiporter) (D2M7A, Cell Signaling Technology®), 1:1000 dilution of anti-P-actin (49070S, Cell Signaling Technology®), and 1:1000 dilution of anti-vinculin (13901, Cell Signaling Technology®). Primary antibodies were diluted in 5% BSA in TBST and incubated overnight at 4°C. Horseradish peroxidase (HRP)-linked secondary anti-rabbit antibodies (7074S, Cell Signaling Technology®) were diluted in 5% non-fat milk at a dilution of 1:5000 and incubated at room temperature for 1 hour. Clarity™ Western ECL Substrate Kit (1705060, BIO-RAD®) was used for detection. Images were captured on the BIO-RAD® CHEMIDOC® XRS+ imager and Image Lab 5.2.1 was used for image processing.

Cell proliferation and viability analysis

[00156] Cells were plated on 96-well plates at 2000 cells/well for Panel and 4000 cells/well for all other cell lines under standard conditions described above. Cells were allowed to seed overnight (approximately 18 hours). Media was aspirated and cells were washed twice with PBS without calcium or magnesium (D8537, Sigma- Aldrich®) before adding 100 pL of experimental media containing compounds and nutrients at indicated concentrations to each well. Proliferation was measured using the Incucyte® SX1 HD/2CLR System (Sartorius®). Images were captured every 2-4 hours and automatically counted using the Incucyte® Cell-by-Cell software analysis package (Sartorius®). Proliferation counts were normalized to the first cell count obtained immediately after beginning experimental conditions. Viability was assessed at 48-hour time points for AsPCl and BxPC3 cells and 24 hour timepoints for Panel and MiaPaCa2 cells using almarBlue™ HS Cell Viability Reagent (Fisher Scientific®, A50100) according to the manufacturer's instructions. 10 pL of almarBlue™ HS Cell Viability Reagent was added to each well containing 100 pL of experimental media. Plates were gently agitated to promote mixing, then incubated under standard conditions. Fluorescence was measured using 545 nm excitation and 590 nm emission using a BioTek Synergy Hl Multimode Reader (Agilent®). For the trypan blue viability assay, 50,000 cells/well were plated on 6-well plates overnight and switched to experimental media as described previously. At the indicated time point cells were washed twice with 1 mL of PBS without calcium or magnesium (D8537, Sigma- Aldrich®) and removed from the plate using 200 pL of Trypsin-Ethylenediaminetetraacetic acid (EDTA) (0.25%) (2520056, Fisher Scientific®) incubated for 5 minutes, and quenched with 200 pL of cell culture media. The suspended cell solution was mixed with equal parts of trypan blue 0.4% (15250061, Fisher Scientific®) and counted using the Cellometer® AutoT4 (Nexcelom Bioscience) for cell counting and viability measurement.

Lipid and general reactive oxygen species (ROS) quantification

[00157] Cells were seeded at 25,000 cells/well in 24 well plates overnight. Cells were then washed with PBS twice and incubated in the experimental media for 24 hours. Cells were then stained for 30 minutes with either 10 pM chloromethyl-2', 7'-dichlorodihydrofuorescein diacetate (CM-H2DCFDA) (C6827, Fisher Scientific®) for general reactive oxygen species (ROS) quantification or 10 pM BODIPY™ 581/591 Cll reagent (D3861, Invitrogen™) for lipid peroxidation according to the manufacturers protocol. Cells were also stained for 30 minutes simultaneously with 1 pM 2'-[4-ethoxyphenyl]-5-[4-methyl-l-piperazinyl]-2,5'-bi-lH- benzimidazoletrihydrochloride trihydrate (Hoechst 33342, Fisher Scientific®) for nuclear visualization and cell localization. Cells were then washed two times with PBS and placed in live imaging solution (A14281DJ, Fisher Scientific®) for image capture. Brightfield images and fluorescence were measured using a Leica® DMi8 microscope, a PE4000 LED light source, 20x and 40x objective, a DFC9000GT camera, 4',6-diamidino-2-phenylindole (DAPI), green fluorescent protein (GFP), or Texas Red® filter set, and LAS X imaging software (Leica®). Three images for each fluorescent channel were captured for each well. For image processing and quantification, the images were imported into the Fiji version of ImageJ (fiji.sc). Cells in each image were selected, background selected and normalized to cell area. Fluorescence of cells for each well was determined by the average fluorescent signal for the three images for that well. Relative fluorescence is shown in the figures relative to control media conditions. Images shown are representative of the images captured for each condition.

Metabolomic profiling and stable isotope labeling

[00158] To quantify metabolites, each cell line was seeded at 200,000 cells/well in triplicate (n=3) in 6-well plates with media as described in Example 1 until achieving approximately 80% confluency (approximately 24 hours). For stable isotope labeling, media was refreshed on the plates and incubated for two hours. Plates were then washed with PBS then switched to labeling media containing either 1 mM ¹³C5-glutamine (CLM-1822-H-PK, Cambridge Isotope Laboratories) or 5 mM ¹³Ci,2-glucose (CLM-504-PK, Cambridge Isotope Laboratories) and 10% dialyzed FBS (F0392, Sigma-Aldrich®). Samples were collected at zero minutes (unlabeled control) and four hours for ¹³Ci,2-glucose and ¹³C5-glutamine labeling for the Panel cells. AsPCl samples were collected identically with the exception that ¹³C-glutamine labeling was also tested at 24 hours. Metabolite extraction was performed as described previously. Briefly, each well was washed with 0.9% saline (16005-092, VWR), then 500 pL of high performance liquid chromatography (HPLC)-grade methanol was added followed by 300 pL of HPLC-grade water containing 0.5 pM camphorsulfonic acid as an internal control. Cells are scraped from the plate and the solution was transferred to a 1.5 mL Eppendorf® tube containing 500 pL of HPLC-grade chloroform. Samples were vortexed for ten minutes, then centrifuged at 4°C 16,000xg for 15 minutes. The polar layer was then removed and dried by lyophilization. In addition to intracellular metabolites, 100 pL media samples were also taken from each well at the same time points and extracted using the same procedure outline above. Protein extracted from the cells was dissolved in 0.2 M potassium hydroxide aqueous solution overnight and quantified using Pierce™ BCA Protein Assay Kit (PI23225, Fisher Scientific®). Extracted metabolites were then resuspended in HPLC-grade water containing 5 pM 1,4-piperazinedi ethanesulfonic acid (PIPES; P6757, Sigma- Aldrich®) as an internal standard.

[00159] Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed with ion-pairing reverse phase chromatography using an Ascentis® Express column (C18, 5 cm * 2.1 mm, 2.7 pm, Sigma-Aldrich®) for separation and a Waters™ Xevo TQ-XS triple quadrupole mass spectrometer. Metabolite peak processing was performed in MAVEN™. Total metabolite abundance (labeled and unlabeled) was scaled by the camphorsulphonic acid internal standard and protein content. The entire dataset for each independent experiment was then normalized by Probabilistic Quotient Normalization. For isotope labeled samples, the data was corrected for the natural ¹³C abundance using IsoCor (Isotope Correction software) and reported as the abundance of labeled metabolite relative to the control cell under control media conditions.

EXAMPLE 2: Pyruvate kinase muscle isoform 2 knock-out (PKM2KO) enhances pancreatic ductal adenocarcinoma (PDAC) survival during cystine starvation

[00160] To evaluate the impact of PKM2 on cysteine/cystine metabolism, human pancreatic ductal adenocarcinoma (PDAC) cell lines that lack PKM2 expression were generated. PKM isoform selection occurs through mutually exclusive alternative splicing of the PKM gene (SEQ ID NO: 1). Inclusion of exon 9 leads to PKM1 expression and inclusion of exon 10 leads to PKM2 expression. Human PDAC typically expresses PKM2. Two human PDAC cell lines, AsPCl and Panel, were selected due to their sensitivity to cystine starvation.

[00161] Using a lentiviral clustered regularly interspaced short palindromic repeats (CRISPR)/Caspase 9 (Cas9) gene editing technology to target exon 10, PKM2 was selectively knocked out (KO) (FIG. 1). Guide RNAs targeting exon 10 of the PKM gene (region determinative of PKM2 expression) were designed and set just before the protospacer adjacent motif (PAM), a DNA sequence immediately following the Cas9-targeted DNA sequence. Lentiviral vectors were used, each expressing one single guide RNA (sgRNA), human caspase 9, and an antibiotic selection marker. The sgRNA sequence for PKM2 knockout plasmid vector is 5'-GTTCTTCAAACAGCTTGCGG-3' (SEQ ID NO: 3) along with puromycin resistance. The sequence for scramble control plasmid with blasticidin selection maker is 5'- GCACTACCAGAGCTAACTCA-3' (SEQ ID NO: 4). CRISPR gene editing plasmid vectors with guide RNA (gRNA) and Cas9 co-expression were acquired from VectorBuilder (en.vectorbuilder.com). The Vesicular Stomatitis Virus G (VSVG) plasmid was a gift from Bob Weinberg (Addgene® plasmid #8454; n2t.net/addgene: 8454; RRID: Addgene® 8454). The 2nd generation packaging plasmid (psPAX2) was a gift from Didier Trono (Addgene® plasmid #12260; n2t.net/addgene: 12260; RRID: Addgene® 12260). Vectors were amplified by transforming Stbl3 bacterial cells grown in LB broth under 100 pg/mL ampicillin for antibiotic selection. Plasmids were harvested by Midi-prep (12243, Qiagen®). To produce lentivirus, HEK293T cells were seeded in 10-cm plates containing Opti-MEM™ (110580221, Fisher Scientific®) with 4% FBS. When the HEK293T cells reached about 50% confluency, they were transfected using Lipofectamine™ 3000 (L3000001, Fisher Scientific®) according to the manufacturer’s instructions with 10.0 pg lentivirus plasmids, 0.5 pg VSVG, and 5.0 pg psPAX2 plasmids. After 24 hours, fresh DMEM with 15% FBS and 1% P/S was added, and cells were grown for another 48 hours to generate virus. For transduction with lentivirus, the AsPCl and Panel cells (1 x 10⁵ cells) were seeded in 10-cm plates. The supernatant of transfected HEK293T was collected and passed through a 0.45 micron polyvinylidene fluoride (PVDF) syringe filter. Five mL of the viral supernatant and 5 mL of fresh media were added to recipient AsPCl and Panel cell plates with polybrene (TR1003G, Fisher Scientific®) at final concentration of 4 pg/ml. The cells were cultured for 24 hours followed by adding fresh DMEM medium supplemented with 10% FBS and treated for 12 days with 2 pg/mE puromycin (A1113803, Fisher Scientific®) for selection. The selected cells were then expanded and analyzed for successful gene knockout by sequencing and western blot analysis. Genomic DNA was extracted using DNeasy® Blood and Tissue Kit (69506, Qiagen®) to check for successful gene editing. PCR primers used to amplify the targeted region around exon 10 of PKM consisted of a forward primer 5’- GCACTTGGTGAAGGACTGGT-3’ (SEQ ID NO: 5) and reverse primer 5’-

AATGGACTGCTCCCAGGAC-3’ (SEQ ID NO: 6). A nested primer 5’-

GTGACTCTTCCCCTCCCTCT-3’ (SEQ ID NO: 7) was used for sequencing. Sequencing was completed by ACTG DNA Sequencing Services. Individual clones were selected by diluting the population to single cells plated in a 96-well plate and then expanding the population. PKM2 deletion for isolated clones was determined by western blot.

[00162] The expression of PKM isoforms was characterized within targeted cells and successful Pyruvate kinase muscle isoform 2 knock-out (PKM2K0) clones for AsPCl and Panel were obtained. Deletion of exon 10 led to complete loss of PKM2 expression and low level reexpression of PKM 1 in all generated clones (FIG. 2).

[00163] The tolerance of PKM2K0 cells to low nutrient stress was evaluated by assessing the viability of PKM2K0 cells in response to depleting each of the individual amino acids typically included in DMEM. This produced significant differences in survival between controls and PKM2K0 clones under starvation of several amino acids (FIG. 3A-B). The most dramatic and consistent difference was the increased viability of all PKM2K0 clones under cystine starvation, the most restrictive condition for the respective control cell lines. Morphologic examination of these cells revealed distinct cell volume shrinkage and membrane blistering in the control cells, characteristic of ferroptosis morphology, and retention of normal morphology in the PKM2K0 cells (FIG. 4A-B). This experiment was repeated using DMEM containing physiologic levels of glucose (5 mM) and glutamine (1 mM) and either 200 pM cystine (supraphysiologic) or 0 pM cystine (starvation). Under cystine starvation, the PKM2KO cells had significantly higher viability compared to the PKM2 expressing controls when evaluated using the almarBlue™ viability assay (FIG. 5A-B). Given that the almarBlue™ viability assay relies on metabolic reducing power to determine viability, viability was also measured using trypan blue exclusion, which also showed significantly increased viability of PKM2KO cells under cystine starvation, confirming the effect is not due only to changes in reducing power production (FIG. 6). To further ensure that the enhanced viability of PKM2KO cells was not simply an artifact of the vector introduced in the control cells, the parental wild-type (WT) AsPCl and Panel cells were evaluated, and nearly identical sensitivity to cystine starvation as the control cells and significantly lower viability compared to the PKM2KO cells was observed (FIG. 7A-B). Next, the proliferative capacity of the control and PKM2K0 cells under cystine starvation was evaluated. Cystine starvation resulted in dramatic inhibition of cell proliferation in the AsPCl and Panel control and PKM2K0 cell lines tested (FIG. 8A-B), indicating that despite the enhanced viability of PKM2K0 cells, they are not actively dividing in this environment. Then, the AsPCl PKM2K0 cells were exposed to a range of cystine concentrations, and it was found that the significant increase in viability in PKM2K0 expressing cells occurs as cystine concentrations reach 10 pM or lower (FIG. 9A-B). Together, these results suggest that cells that are not expressing PKM2 may represent a subpopulation of cells able to persist under stressful low nutrient conditions and allow for tumor survival.

EXAMPLE 3: PKM2KO improves defense against cystine depletion induced ferroptosis in PDAC

[00164] Next, the mechanism of cell death occurring in the control cells under cystine starvation was examined. Cystine starvation is known to induce ferroptosis, a non-apoptotic and oxidative damage-related form of cell death driven by iron accumulation and lipid peroxidation. Under cystine starvation, co-treatment of the AsPCl and Panel cells with the ferroptosis inhibitors Ferrostatin-1 (a lipid peroxide inhibitor, FER), Trolox (a vitamin E derivative and lipophilic antioxidant; TRO), and deferoxamine (an iron chelator; DFO) significantly restored viability in control cells (FIG. 10A-B) with little effect on the PKM2K0 clones. These results indicate that the PKM2 expressing control cells are undergoing ferroptosis while the PKM2K0 clones resist this process. Importantly, co-treatment with Z-VAD-FMK (an apoptosis inhibitor; ZVAD) and Necrostatin-lS (a necroptosis inhibitor; NEC) had no significant effect on viability indicating that cell death is not occurring through either apoptosis or necroptosis (FIG. 10A and FIG. 11A). Given that ferroptosis is characterized by an accumulation of unchecked lipid peroxidation, the ratio of oxidized to reduced lipids was measured using the BODIPY™ 581/591 Cll probe (D3861, Invitrogen™), which detects lipid peroxides by shifting from red (about 590 nm) to green (about 510 nm) emission when oxidized. Consistently, PKM2K0 cells have nearly no increase in lipid peroxidation compared to the significantly elevated levels in the respective control cells (FIG. 11A-B and FIG. 12A-B). The difference in lipid peroxidation can be eliminated by Ferrostatin-1 co-treatment (FIG. 11A and FIG. 12A). AsPCl and Panel PKM2KO cells also have significantly lower general ROS accumulation as measured by the 2’,7’- dichlorodihydrofluorescein diacetate (H2DCFDA) probe, a general detector of ROS (FIG. 13A- B). These observations support the conclusion that PKM2KO in PDAC cells enhances their resistance to cystine starvation induced ferroptosis.

EXAMPLE 4; Pyruvate kinase activity dictates response to cystine starvation induced ferroptosis

[00165] The inventors sought to identify the specificity of the pyruvate kinase (PK) isoform to determine how PKM2K0 cells have enhanced survival under cystine starvation. Given that the PKM2K0 cells re-express PKM1, it could be either the loss of PKM2 or the gain of PKM1 that is driving the difference in viability.

[00166] To address these possibilities, lentiviral vectors were used to overexpress PKM1 in the control cells and PKM2 expression was re-introduced in the PKM2K0 cells for both AsPCl and Panel (FIG. 14). Overexpression of PKM1 and PKM2 was achieved using a lentiviral gene expression purchased from VectorBuilder (en.vectorbuilder.com). The vector contained the blasticidin resistance gene and either the PKM1 or PKM2 protein coding sequence under the EFl A promoter. Lentivirus was produced and used to transduce target cells in an identical manner as described in the previous section. Transduced cells were selected using 10 pg/ml blasticidin (Al 113903, Fisher Scientific®). The selected cells were then expanded and analyzed for successful gene knockout by western blot analysis, using the methods described in Example 2.

[00167] Evaluation of the response to cystine starvation revealed that neither re-expression of PKM1 nor PKM2 altered the viability response (FIG. 15A-D). However, it was observed that the control cells in which PKM1 was overexpressed continued to express PKM2 and similarly the PKM2K0 cells with PKM2 re-expressed continue to also express PKM1 at a low level indicating that the re-expression of either PK isoform does not restore cells to their original state (FIG. 14 and FIG. 15A). Thus, the total dosage of PK and activity of PK is likely different with respect to their parental cell lines.

[00168] To determine PK activity, cells were plated 200,000 cells/well in 6-well plates and incubated overnight. Plates were washed twice with PBS without calcium or magnesium (D8537, Sigma-Aldrich®) and the media was replaced with experimental media in triplicate for each condition as indicated. Panel cells were incubated for 12 hours, AspCl were incubated for 24 hours as described above. Pyruvate kinase activity kit (MAK072, Sigma-Aldrich®) was used according to the manufacturer’s protocol to evaluate pyruvate kinase activity. Briefly cells were lysed and scraped from the plate and centrifuged at 16,000xg for 5 minutes to clear debris. 5 pL of supernatant was used for each sample for the assay. The Pierce™ BCA Protein Assay Kit (PI23225, Fisher Scientific®) was used to quantify protein extracted from the cells to normalize activity. After quantifying PK activity in nmole/min/mL, activity was normalized to the control cell line under normal media conditions to show relative PK activity.

[00169] Overexpression of PKM1 in the control cells does significantly increase PK activity but did not further inhibit viability likely because they are already maximally inhibited by cystine starvation (FIG. 16A-B). PKM2 re-expression significantly increased PK activity under cystine replete conditions, but not under cystine starvation (FIG. 16C-D). It was hypothesized that the difference in cystine starvation survival is due to differences in total PK activity, not isoform specificity. The hypothesis was tested by evaluating the activity of PK and found that the AsPCl PKM2K0 cells, despite expressing PKM1 at low levels, had consistently lower overall PK activity regardless of cystine availability, whereas the control cells decreased their PK activity in response to 0 pM cystine (FIG. 17A-B), consistent with PKM2’s response to oxidative stress. It was then tested whether modulation of PK activity would influence viability under 0 pM cystine. By treating cells with compound 3k (36815, Cayman Chemical®), a potent and specific PKM2 antagonist (FIG. 18), the inventors were able to dramatically restore viability in four different WT PDAC cell lines (AsPCl, Panel, MiaPaCa2, and BxPC3) under 0 pM cystine stress (FIG. 19A- D). Additionally, co-treatment of imidazole ketone erastin (IKE), a potent inhibitor of xCT, with compound 3k restored viability in the majority of WT PDAC cell lines tested (FIG. 20A-D), demonstrating that decreased PK activity can improve resistance to cystine starvation induced ferroptosis. To further confirm the hypothesis, IKE treatment was combined with TEPP-46, a potent and selective PKM2 allosteric agonist (FIG. 18). The combination treatment synergistically and significantly decreased viability in a concentration dependent manner in each cell line tested except BxPC3 (FIG. 21A-D and FIG. 22A-D). Interestingly, BxPC3 showed strong response to IKE but limited synergistic response when co-treated with TEPP-46 (FIG. 21D and FIG. 22D). Of the four WT PDAC cell lines tested, BxPC3 retained residual PKM1 expression suggesting that this cell line likely has reprogrammed glycolytic activity relative to the other WT cells (FIG. 23). The mechanism of cell death caused by the co-treatment of IKE and TEPP-46 was evaluated, and it was found that ferrostatin-1 but not Z-VAD-FMK nor Necrostatin- 1S was able to restore viability, indicating that TEPP-46 is working with IKE to induce ferroptosis and not apoptosis or necroptosis (FIG. 24A-D). Together, these observations present evidence that PK activity dictates the response to cystine starvation induced ferroptosis.

EXAMPLE 5: PKM2KO enhances defense against ferroptosis induction specific to cystine starvation

[00170] Two key defensive proteins against ferroptosis are xCT (SLC7A11, component of system X_c” cystine/glutamate antiporter) and glutathione peroxidase 4 (GPX4). xCT is a component of the cystine/glutamate antiporter and is the transporter by which cancer cells acquire nearly all of their cystine. GPX4 is primarily responsible for quenching lipid peroxides. Two of the drugs known to potently induce ferroptosis, IKE and ras selective lethal 3 (RSL3), target xCT and GPX4 respectively (FIG. 25). The expression of these proteins was evaluated, and it was found that under cystine starvation, AsPCl control cells increase xCT to a greater extent than PKM2K0 cells, while Panel control and PKM2K0 cells have similar expression (FIG. 26A-B). Both AsPCl and Panel control cells have decreased GPX4 expression under cystine starvation, but PKM2K0 cells retain equivocal expression of GPX4 (FIG. 26A-B). This suggests that the presence of PKM2 may directly or indirectly modulate expression of these genes through a yet to be determined mechanism.

[00171] The difference in ferroptosis defense between control and PKM2K0 cells was further explored by evaluating their dose response to IKE and RSL3. Fascinatingly, both AsPCl and Panel PKM2K0 cells demonstrate a significantly high degree of resistance to IKE compared to controls; however, these same cells have equivalent or enhanced sensitivity to RSL3 (FIG. 27A- D). While the control cells treated with IKE were significantly rescued by co-treatment with Ferrostatin-1, Trolox, or deferoxamine, the PKM2K0 clones largely showed little to no response to these rescue agents (FIG. 28A-B). Additionally, there were no rescue effects from Z-VAD- FMK nor Necrostatin-lS indicating that the mechanism of cell death induced by IKE is specific to ferroptosis (FIG. 28A-B). AsPCl PKM2K0 cells show decreased lipid peroxidation under IKE and equivalent lipid peroxidation under RSL3 compared to control cells (FIG. 29). Additionally, all AsPCl cells show strong sensitivity to RSL3 that can only be rescued by ferroptosis inhibitors, while Panel cells show limited sensitivity to RSL3 (FIG. 30). This suggests that PK is not uniformly responsible for providing defense against all routes to ferroptosis, but rather specifically influences the metabolic reprogramming that occurs under cystine starvation and the subsequent propensity towards ferroptosis.

[00172] Next, it was investigated whether this difference in IKE sensitivity would have the same effect on control and PKM2K0 cells in vivo. Xenograft tumors were generated in NSG mice using AsPCl control and PKM2K0 clone #1 cell lines.

[00173] To inoculate mice and produce xenograft tumors in mice, AsPCl Control and PKM2K0 Clone #1 cells were grown and prepared into a solution of 5xl0⁷ cells/mL in PBS without calcium or magnesium (D8537, Sigma- Aldrich®). 100 pL of each solution (5xl0⁶ cells) was injected subcutaneously into the right flank of eight week old male NOD scid gamma (NSG) mice. Twelve mice received the control cells, and twelve mice received the PKM2K0 cells. After one month the two groups of tumor bearing mice were each divided into two groups of six mice to receive intraperitoneal injections of either 200 pL of 50 mg/kg IKE in 65% DW5 (5% Dextrose in water), 5% TWEEN™-80, and 30% PEG-400 or 200 pL of vehicle control. Treatment was delivered 6 days a week for two total weeks. Tumor volume and mouse body weight were measured four times a week. Tumor volume was estimated by caliper measurement across the long and short axes of the tumor using the equation length * width² * 0.5 = volume (mm³). After two weeks, the mice were euthanized, and tumors were excised.

[00174] To evaluate the combination of IKE and TEPP-46 in vivo. Panel WT cells were grown and prepared into a solution of 5xl0⁷ cells/mL in PBS without calcium or magnesium (D8537, Sigma- Aldrich®). 100 pLof each solution (5xl0⁶ cells) was injected subcutaneously into the right flank of 6 week old female NSG mice. After about three weeks the tumor bearing mice were divided randomly into four groups (n=6 for each group except the IKE and TEPP46 combination which had n=4). Mice received daily intraperitoneal injections for two weeks of either 50 mg/kg IKE, 30 mg/kg TEPP-46, the combination of 50 mg/kg IKE with 30 mg/kg TEPP-46, or vehicle control. IKE treatment was delivered in 200 pL volumes and was formulated in 65% DW5 (5% Dextrose in water), 5% TWEEN™-80, and 30% PEG300. TEPP-46 was delivered in 200 pL volumes and was formulated in 5% DMSO, 40% PEG300, 5% TWEEN™-80, and 50% saline (0.9%). Tumor volume and mouse body weight were measured 4 times a week. Tumor volume was estimated by caliper measurement across the long and short axes of the tumor using the equation length * width² * 0.5 = volume (mm³). After two weeks, the mice were euthanized, and tumors were excised.

[00175] PKM2KO cell tumors had significantly lower tumor growth compared to the control (FIG. 31). IKE treatment produced a significant decrease in tumor volume in the PKM2 expressing control tumors in contrast to the limited response in the PKM2KO tumors (FIG. 31). Therefore, PKM2KO in PDAC provides resistance to cystine starvation induced cell death in vitro and in vivo.

EXAMPLE 6: PKM2KO PDACs exhibit increased glutamine anaplerosis and decreased glucose metabolism under cystine starvation

[00176] PKM isoform selection is known to cause reprogramming in the overall metabolic activity within cancer cells, including both glucose and glutamine metabolism. To evaluate changes in metabolic pathways under low cystine conditions that may explain the survival advantage in PKM2KO cells, targeted mass-spectrometry and stable isotope tracing were performed. Using ¹³Ci.2-glucose, the flow of glucose-derived carbon was traced through glycolysis and into the tricarboxylic acid (TCA) cycle. The abundance of labeled hexosephosphate is equivalent between control and PKM2KO cells under high and low cystine conditions (FIG. 32A). Downstream lactate production is significantly higher in control cells, which is consistent with PKM2’s role in mediating increased glucose fermentation to lactate, even in the presence of oxygen (Warburg effect). However, labeled lactate production significantly decreases under cystine starvation, suggesting cystine starvation may limit the Warburg effect (FIG. 32B). Glucose-derived carbon entry into the TCA cycle intermediates is also lower under cystine starvation, consistent with the observation that PK activity is decreased under these conditions (FIG. 32C-F).

[00177] Given the decrease in glucose metabolism, it was hypothesized that the cells were turning to alternative sources for metabolic fuel. Glutamine is a candidate, as Kirsten rat sarcoma virus (KRAS) mutated pancreatic cancer cells rely on this metabolite, and mitochondrial glutaminolysis contributes to ferroptosis. Given this connection, it was hypothesized that PKM2 expression alters glutamine metabolism and promotes ferroptosis. Thus, the cells were supplied with uniformly (U) labeled U-¹³Cs-glutamine and its utilization was observed in downstream pathways. It was first found that the breakdown of glutamine into glutamate is slightly decreased in PKM2KO cells and under cystine starvation (FIG. 32G). PKM2KO cells also secrete significantly lower glutamate than the PKM2 expressing controls (FIG. 32H). Additionally, PKM2KO cells have significantly lower production of a-ketoglutarate from glutamine while having largely equivalent amount of malate and succinate production downstream in the TCA cycle (FIG. 321- J). It was also observed that PKM2K0 cells have increased glutamine contribution to glutathione (FIG. 32L) and amino acid synthesis, including aspartate and proline (FIG. 32K, FIG. 33A and FIG. 33C). Asparagine synthesis from glutamine is significantly lower in PKM2K0 cells under high cystine (FIG. 33B). However, asparagine synthesis from glutamine dramatically increases under cystine starvation, suggesting that asparagine synthesis may be involved in an important metabolic defense system against cystine starvation induced ferroptosis (FIG. 33B). Importantly, increased glucose contributions to aspartate or alanine in the PKM2K0 cells was not observed (FIG. 33D-E), suggesting that there is not a global increase in amino acid synthesis, but rather a specific utilization of glutamine for production of these amino acids. Together, these observations show that under cystine starvation, there is metabolic reprogramming away from glucose metabolism with a persistent reliance on glutamine metabolism, and that the absence of PKM2 influences how glucose and glutamine are metabolized.

[00178] Additionally, these metabolic tracer studies were used to address other hypotheses that might explain the increased viability of PKM2KO cells under cystine starvation. Decreased PK activity has been linked to increased flux through the pentose phosphate pathway (PPP), an important pathway for generating NADPH by glucose-6-phospate dehydrogenase. Since NADPH is a critical reducing agent for reducing glutathione (GSH) and defending against lipid peroxidation, this raises the possibility that utilization of the PPP is more active in the PKM2KO cells under low cystine stress. To address this, the ¹³Ci,2-glucose tracer was used to take advantage of the fact that flux through the PPP results in the loss of the first carbon. Subsequent re-entry into glycolysis would result in Ml labeling, while flux directly through glycolysis will produce M2 labeling downstream. It was observed that Ml labeling in ribose- 5 -phosphate and ribulose-5- phosphate is actually decreased under cystine starvation, though the result was not significant (FIG. 34A-D). Further, there were no significant differences between control and PKM2K0 cells. Significantly more Ml isotopologue hexose-phosphate was observed in one AsPCl PKM2KO clone, but no consistent or significant trend in the other cells (FIG. 34E-F). This suggests that differential utilization of the PPP is likely not a major driver for explaining the difference in defense against ferroptosis between PKM2K0 cells and PKM2 expressing cells.

[00179] GSH is a critical antioxidant defense molecule, synthesized from cysteine, glycine, and glutamate. Given the difference in glutamine metabolism between control and PKM2KO cells under cystine starvation, it was hypothesized that the PKM2KO cells would have higher GSH synthesis. U-¹³C5-glutamine was used to trace the flow of glutamine derived carbon into GSH, to produce the M5 isotopologue of GSH. GSH pool size plummets under cystine starvation, likely due to rapid consumption for quenching lipid peroxides and the lack of new cysteine to synthesize more (FIG. 35A-B). Although the pool of GSH is extremely low, a significant increase in M5 GSH in two of the AsPCl PKM2K0 clones was observed under cystine starvation, but this effect was not seen in the Panel cells (FIG. 32L). To further explore potential differences in GSH synthesis, the control and PKM2K0 cells were treated with BSO (an inhibitor of glutathione synthesis that does not induce ferroptosis), glutathione ethyl ester (GSH-EE; a cell permeable analog of glutathione), and/or Ferrostatin-1 (a selective ferroptosis inhibitor). In the presence of cystine, BSO treatment caused a decrease in viability that could not be restored with GSH-EE addition but can be restored by co-treatment with Ferrostatin-1 in AsPCl cells but not Panel cells (FIG. 36A-B). Under cystine starvation, BSO treatment does not alter viability of either control or PKM2K0 cells, indicating that inhibition of GSH synthesis cannot further alter ferroptosis when environmental cystine is removed. Together, these results suggest that there are some minor differences in GSH synthetic capabilities between control and PKM2K0 cells, but the ability to synthesize GSH is likely not a major contributor to the difference in ability to defend against ferroptosis.

[00180] Lastly, it was hypothesized that the increased production of amino acids from glutamine in the PKM2K0 cells was providing the cells with additional resources to maintain viability under cystine starvation. Environmental cystine was removed and the cells were supplemented with supraphysiologic concentrations of aspartate, asparagine, glutamate, and proline individually. However, this produced no change in viability compared to cystine starvation alone and did not alter the difference in viability between control and PKM2K0 cells (FIG. 37). This suggests that the ability of the PKM2K0 cells to increase production of amino acids from glutamine is not essential for improving cellular defense against ferroptosis. However, it should be noted that directly supplying the end products (amino acids) is not equivalent to synthesizing them, as the reactions to produce these products also affect redox cofactors and stoichiometries of the reactants. Altogether, this data suggests a complex utilization of glutamine metabolism by PKM2K0 cells to support defense against cystine starvation induced ferroptosis.

EXAMPLE ?: Glutamine is required for PKM2KO PDAC defense against cystine starvation induced ferroptosis

[00181] Given the apparent importance of glutamine metabolism in ferroptosis, the role of environmental glutamine in PKM2K0 mediated defense against ferroptosis was investigated. Given the difference in glutamine metabolism in the PKM2K0 cells, whether environmental glutamine availability would influence cell survival was tested. The difference in viability under cystine starvation between PKM2K0 and control cells is present with glutamine levels as low as 250 pM, but complete removal of glutamine significantly decreases viability of the PKM2KO cells and restores viability to control cells (FIG. 38). This is consistent with the observation that PKM2K0 cells have elevated glutamine metabolism, and therefore increased dependence, on glutamine. It was found that complete removal of glutamine significantly enhances the viability of the control cells under cystine starvation. In contrast, the PKM2KO cells have reduced viability when glutamine is removed under cystine starvation induced by IKE treatment (FIG. 39A-B). Additionally, the same effect was observed when the cells are grown under 0 pM cystine starvation mediated ferroptosis (FIG. 40). Glutamine metabolism can also be inhibited by the glutaminase inhibitor CB-839. Treatment with CB-839 significantly decreased the viability of PKM2K0 cells but had a more muted effect on the control cells (FIG. 41). In contrast, under cystine starvation, CB-839 had no effect on viability in the PKM2KO cells, while it restored viability in the control cells (FIG. 41). It was further observed that removal of glutamine eliminated the increased lipid peroxidation observed in control cells under 0 pM cystine conditions and that glutamine removal caused no significant increase in lipid ROS in either control or PKM2KO cells compared to replete media conditions (FIG. 42). Next, whether simultaneous glutamine and cystine starvation would alter expression of the ferroptosis defense proteins, xCT and GPX4, was evaluated. Removal of cystine caused an increase in xCT expression and decrease in GPX4 expression in the AsPCl and Panel control cells, but only modest changes in the PKM2KO cells (FIG. 43A-B). When both environmental glutamine and cystine are removed, the increase in xCT or decrease in GPX4 was no longer observed, indicating that either the stressful stimuli promoting their expression is absent or the presence of glutamine is required to promote expression (FIG. 43A-B). Together, these results suggest that altered glutamine metabolism in PKM2KO cells is in part responsible for the differential ferroptosis response under cystine starvation conditions.

[00182] Glutamine anaplerosis is an important metabolic process for filling the resources required for the TCA cycle to function in the mitochondria of cells in the low nutrient conditions of the PDAC tumor microenvironment and is influenced by PK enzyme activity. To address whether metabolites downstream of glutaminolysis would influence ferroptosis, the AsPCl and Panel PKM2KO cells were grown in the absence of cystine and co-treated with cell permeable dimethyl a-ketoglutarate (DM-akg), dimethyl succinate (DM-Suc), and dimethyl malate (DM- Mal) at concentrations shown to increase ferroptosis. In the AsPCl cells, the addition of DM-akg or DM-Suc did not change viability. Fascinatingly, the addition of malate significantly restored viability to these cells (FIG. 44A-D). Even more surprisingly, when environmental glutamine was removed, the same trend was observed where DM-akg and DM-Suc did not affect viability, and treatment with DM-Mal further increased viability (FIG. 44A-D). The AsPCl PKM2K0 cells had worse viability when environmental glutamine is removed, yet the addition of DM-akg or DM-Mal, but not DM-Suc, rescued viability loss caused by glutamine removal (FIG. 44A-D). Treatment with DM-Mal also restored viability when glutamine metabolism was blocked by CB- 839 (FIG. 41). While this effect is prominent in the AsPCl cells, the same effect was not observed in Panel. The viability of Panel cells under cystine starvation was restored by DM-akg, but further decreased when DM-Suc and DM-Mal were supplemented (FIG. 45A-C). To confirm that this is not an artifact of higher concentration of malate, it was also found that using a lower concentration of DM-Mal (8 mM) was sufficient to cause the same changes in the AsPCl and Panel PKM2K0 cells (FIG. 46A-B). Supplementation with DM-akg and DM-Suc produced little change under cystine and glutamine replete conditions, but DM-Mal supplementation consistently increased viability (FIG. 47A-G). Collectively, this data presents strong evidence that PK plays a key role in altering ferroptosis by reprogramming glutamine metabolism and suggests that under low PK activity, cells utilize enhanced glutamine metabolism under low cystine conditions for survival.

EXAMPLE 8: Malic enzyme enables survival of PKM2KO PDAC under cystine starvation [00183] The next aim was to identify the metabolic pathway that the PKM2KO cells rely on to alter glutamine metabolism and promote defense against ferroptosis. KRAS mutant PDAC upregulates several metabolic enzymes, including glutamate oxaloacetate transaminase 1 (GOT1), glutamate oxaloacetate transaminase 2 (GOT2), and malic enzyme 1 (MEI), to alter glutamine metabolism and promote defense against ferroptosis. Additionally, inhibition of GOT1 promotes ferroptosis in pancreatic cancer. The fact that malate promotes increased survival of cystine starvation and that the U-¹³Cs-glutamine labeling data demonstrates high levels of glutamine flux into malate and aspartate led to the hypothesis that malic enzyme and the malate aspartate shuttle are important for providing the metabolic advantage in PKM2KO cells under cystine starvation. To address this possibility, the expression of MEI in the AsPCl and Panel PKM2K0 was evaluated. Surprisingly, the PKM2K0 clones demonstrated elevated MEI expression compared to their respective controls; however, expression was minimally impacted by the presence or absence of cystine (FIG. 48A-B). Next, MEI was blocked by using malic enzyme inhibitor (MEli) to test whether this would influence ferroptosis. It was found that inhibition of MEI significantly decreased viability of control and PKM2K0 cells under cystine starvation (FIG. 49A-G). Co-treatment with Ferrostatin-1 was able to significantly restore viability in the Panel cells (FIG. 49A-C). In AsPCl cells, only N-acetyl-L-cysteine (NAC), an antioxidant, was able to rescue this effect consistently (FIG. 49D-E). This indicates that the decrease in viability induced by cystine starvation and MEI inhibition in AsPCl cells is likely specific to oxidative stress but may not be entirely explained by ferroptosis and speaks to the metabolic heterogeneity that has been observed in pancreatic cancer. In AsPCl cells, supplementation with malate in addition to MEI inhibition ablated the viability advantage from malate supplementation alone (FIG. 50A- G). Because MEI is important for generating the antioxidant NADPH, NADPH levels were evaluated in the PKM2K0 cells.

[00184] To quantify NADPH, cells were plated in a white 96-well plate with clear bottoms at 8,000 cells/well for each cell line tested. Cells were allowed to seed overnight before switching the media to experimental conditions for a 24-hour incubation in Panel and a 48 hour incubation in AsPCl. The media was aspirated from the wells, the cells were washed twice with PBS, and 50 pL of PBS was added to each well. The manufacturer’s protocol was then followed for the NADP/NADPH-Glo™ assay (G9081, Promega®). Briefly, 50 pL of a 1% dodecyltrimethylammonium bromide (DTAB) and bicarbonate base buffer were added to each sample and 50 pL of sample were moved to a new set of empty wells. The plate is then heated at 60°C for 15 minutes. After the plate has been heated, the plate incubates at room temperature for 10 minutes. To the base-treated samples, 50 pL of a solution containing equal parts of 0.4N hydrochloric acid (HC1) and Trizma base were added to each well. Luciferase detection reagent was prepared and 100 pL were added to each well. The plate was briefly mixed on a plate shaker and covered in aluminum foil to keep light from entering the reaction. The plate was incubated for 30-60 minutes at room temperature before the luminescence was read on a BioTek Synergy Hl Multimode Reader (Agilent®).

[00185] It was observed that NADPH levels were consistently higher in the PKM2K0 cells under zero pM cystine, consistent with increased defense against ferroptosis (FIG. 51A-B). Based on these results, a model was developed in which PK influences the expression of MEI and that under low PKM2 activity, PDAC cells increase glutamine metabolism and utilization of MEI to promote NADPH production and defense against ferroptosis (FIG. 52A). Furthermore, activation of PKM2 increases glycolysis and de-emphasizes utilization of glutamine for NAPDH generation and promotes metabolic conditions more susceptible to ferroptosis (FIG. 52B).

EXAMPLE 9: The combination of pyruvate activation and cystine starvation is an efficacious treatment for PDAC in vivo

[00186] Finally, the impact of increasing pyruvate kinase activity and inhibiting xCT on PDAC growth in vivo was tested using NOD scid gamma (NSG) mice injected with Panel WT cells. Mice were divided into four treatment groups to receive either 50 mg/kg IKE, 30 mg/kg TEPP- 46, combination of 50 mg/kg IKE and 30 mg/kg TEPP-46, or vehicle control (FIG. 53). After two weeks of daily intraperitoneal injections, tumor volume was significantly lower in the TEPP-46 and combination treatment groups compared to the vehicle control group (FIG. 54). Tumor weight was also significantly lower in the combination treatment group compared to the control and TEPP-46 treatment groups, with a trend towards decreased weight compared to IKE treatment group (FIG. 55 and FIG. 56). No significant differences in body weight were observed between each of the groups over the duration of the treatment (FIG. 57). This demonstrates that activating pyruvate kinase and inducing cystine starvation is a viable, novel treatment strategy for PDAC.

EXAMPLE 10: General procedures for EXAMPLES 11-12

Statistical analysis

[00187] All experiments were conducted with three replicates and were reproduced in at least two independent experiments. Statistical analysis and graphing were performed in Graph Pad v9 using Student’s T-Test, one-Way Analysis of Variance (ANOVA), or two-Way ANOVA where appropriate. A - value of < 0.05 was used as the cutoff for determining significance. In figures * = p-val <0.05, ** = p-val < 0.01, *** = p-val < 0.001. Multiple comparison correction was completed using either Sidak’s multiple comparison test or Tukey’s multiple comparison test for one-way ANOVA or two-way ANOVA, respectively.

Cell culture

[00188] The human prostate cancer cell lines, C4-2B, PC3, and LNCaP were acquired as a gift from Dr. Hani Goodarzi (University of California, San Francisco). Cells were routinely cultured in Dulbecco’s modified eagle medium (DMEM) (MT10013CV, Fisher Scientific®) with sodium pyruvate, supplemented with 10% fetal bovine serum (FBS) (13206C, Sigma- Aldrich®), 1% penicillin and streptomycin (P/S) (15140122, Fisher Scientific®), and cultured in a humidified incubator with 5% CO2 at 37°C. During experimental procedures, cells were incubated in a humidified incubator with 5% CO2 at 37°C in DMEM without glucose, glutamine, pyruvate, or cystine (D9815, US Biological) supplemented with 5 mM glucose, 1 mM glutamine, 1 mM pyruvate, either 0 or 50 pM cystine, and 10% dialyzed FBS (F0392, Sigma- Aldrich®) and 1% P/S.

Cell culture reagents

[00189] The following is a list of chemical compounds used in cell culture experiments: 1 rnM N-acetyl-L-cysteine (A9615, Sigma-Aldrich®), 100 pM Trolox (238813, Sigma-Aldrich®), deroxamine (D9533, Sigma-Aldrich®), and Dimethyl sulfoxide (DMSO) (D4540, Sigma- Aldrich®).

Western blot analysis

[00190] Cell lysis for protein extraction was completed using cell lysis buffer (9803, Cell Signaling Technology®). Western blot analysis was carried out using standard protocols. Briefly, protein samples were diluted to 1 pg/mL, reduced with 6x Laemmli loading dye, and boiled for 5 minutes. 20 pg of protein was loaded to precast Bolt 17-well 4-12% polyacrylamide, Bis-Tris, 1.0 mm gels (NW04122BOX, Fisher Scientific®). Gel electrophoresis was carried out in 2-(N- morpholino)ethanesulfonic acid (MES) running buffer at 200V for 22 minutes. Protein was transferred to nitrocellulose membranes, which were then cut for appropriate targets of interest. The following dilutions of primary commercial antibodies were used as probes: 1:1000 dilution of anti-pyruvate kinase muscle isoform 1 (PKM1) (7067S, Cell Signaling Technology®), 1:1000 dilution of anti-pyruvate kinase muscle isoform 2 (PKM2) (4053S, Cell Signaling Technology®), 1:1000 dilution of P-actin (13E5) (4970S, Cell Signaling Technology®). Primary antibodies were diluted in 5% BSA in TBST and incubated overnight at 4°C. Horseradish peroxidase (HRP)-linked secondary antibodies (7074S, Cell Signaling Technology®) were diluted in 5% non-fat milk at a dilution of 1 : 5000 and incubated at room temperature for 1 hour.

Cell proliferation and viability analysis

[00191] Cells were plated on 96-well plates at 4000 cells/well under standard conditions described above. Cells were allowed to seed overnight (approximately 18 hours). Media was aspirated and cells were washed twice with PBS without calcium or magnesium (D8537, Sigma- Aldrich®) before adding 100 pL of experimental media containing compounds and nutrients at indicated concentrations to each well. Proliferation was measured using the Incucyte® SX1 HD/2CLR System (Sartorius®). Images were captured every 2 hours and automatically counted using the Incucyte® Cell-by-Cell software analysis package (Sartorius®). Proliferation counts were normalized to the first cell count obtained immediately after beginning experimental conditions. Viability was assessed using almarBlue™ HS Cell Viability Reagent (A50100, Fisher Scientific®) according to the manufacturer's instructions. 10 pLof almarBlue™ HS Cell Viability Reagent was added to each well containing 100 pL of experimental media. Plates were gently agitated to promote mixing, then incubated under standard conditions for 4 hours. Fluorescence at 570 nm and 600 nm was recorded on a BioTek Synergy Hl Multimode Reader (Agilent®).

EXAMPLE 11: Deletion of PKM2 in prostate cancer cell lines

[00192] The inventors discovered deletion of PKM2 produces low re-expression of PKM1 and a profound resistance to cystine starvation induced ferroptosis. To explore if this effect would also be found in prostate cancer, 2 human prostate cancer cell lines, C4-2B and LNCaP, were selected for further analysis. LNCaP is reported to be responsive to androgens, whereas the C4-2B line was developed from LNCaP cells to be metastatic and castrate resistant through sequential inoculation in castrated mice and isolation of bone metastases (FIG. 58).

[00193] Using a lentiviral clustered regularly interspaced short palindromic repeats (CRISPR)/Caspase 9 (Cas9) system, PKM2 expression was deleted (knocked out) by selectively targeting exon 10 of the PKM gene (FIG. 59). Dual-guide RNAs targeting exon 10 of the PKM gene (region determinative of PKM2 expression) were designed and set just before the protospacer adjacent motif (PAM), a DNA sequence immediately following the Cas9-targeted DNA sequence. Two lentiviral vectors were used, each expressing one single guide RNA (sgRNA), human caspase 9, and an antibiotic selection marker. The sgRNA sequences for PKM2 knockout plasmid vector are 5'-TTGATAGTTCTGACGGAGTC-3' (SEQ ID NO: 8) along with blasticidin resistance and 5'-GTTCTTCAAACAGCTTGCGG-3' (SEQ ID NO: 3) along with puromycin resistance. The sequence for scramble control plasmid with blasticidin selection maker is 5'- GCACTACCAGAGCTAACTCA-3' (SEQ ID NO: 4). CRISPR gene editing plasmid vectors with guide RNA (gRNA) and Cas9 co-expression were acquired from VectorBuilder. The Vesicular Stomatitis Virus G (VSVG) plasmid was a gift from Bob Weinberg (Addgene® plasmid #8454; n2t.net/addgene: 8454; RRID: Addgene® 8454). The 2nd generation packaging plasmid (psPAX2) was a gift from Didier Trono (Addgene® plasmid #12260; n2t.net/addgene: 12260; RRID: Addgene® 12260). To produce lentivirus, HEK293T cells seeded in 10-cm plates containing Opti- MEM™ (110580221, Fisher Scientific®) with 4% FBS. When the HEK293T cells reached about 50% confluency, they were transfected using Lipofectamine™ 3000 (L3000001, Fisher Scientific®) according to the manufacturer’s instructions with 10.0 pg lentivirus plasmids, 0.5 pg VSVG, and 5.0 pg psPAX2 plasmids. After 24 hours, fresh DMEM with 15% FBS and 1% P/S was added, and cells were grown for another 48 hours to generate virus. For transduction with lentivirus, the LNCaP and C4-2B cells (l x 10⁵ cells) were seeded in 10-cm plates. The supernatant of transfected HEK293T was collected and passed through a 0.45 micron polyvinylidene fluoride (PVDF) syringe filter. Five mL of the viral supernatant and 5 mL of fresh media were added to recipient Al 3M 13 cell plates with polybrene (TR1003G, Fisher Scientific®) at final concentration of 4 pg/ml. The cells were cultured for 24 hours followed by adding fresh DMEM medium supplemented with 10% FBS and treated for 12 days with 10 pg/mL blasticidin (A1113903, Fisher Scientific®) and 2 pg/mL puromycin (Al 113803, Fisher Scientific®) for selection. The selected cells were then expanded and analyzed for successful gene knockout by sequencing and western blot analysis. Genomic DNA was extracted using DNeasy® Blood and Tissue Kit (69506, Qiagen®) to check for successful gene editing. PCR primers used to amplify the targeted region around exon 10 of PKM consisted of a forward primer 5’-GCACTTGGTGAAGGACTGGT-3’ (SEQ ID NO: 5) and reverse primer 5’-AATGGACTGCTCCCAGGAC-3’ (SEQ ID NO: 6). A nested primer 5’-GTGACTCTTCCCCTCCCTCT-3’ (SEQ ID NO: 7) was used for sequencing. Sequencing was completed by ACTG DNA Sequencing Services.

[00194] After antibiotic selection, the population of modified cells was evaluated for expression of PKM1 and PKM2. The scramble control cells retained high levels of PKM2 expression, but the PKM2K0 cell population had complete loss of PKM2. Interestingly, these cells reverted to a low level re-expression of PKM1 (FIG. 60). This observation is consistent with other published studies investigating the deletion of PKM2 from cancer cells and points to reliance of cancer cells on PKM independent of isoform. Establishment of these cell lines now allows for characterizing the metabolic effects of PKM2 in the context of hormone sensitive and advanced castration resistant cancer cells.

EXAMPLE 12: PKM2KO influence on cystine starvation survival

[00195] Having established PKM2K0 prostate cancer cells, it was first sought to evaluate the effects of PKM2 deletion on proliferation under normal and cystine starvation conditions. LNCaP and C4-2B cells were grown in DMEM containing 5 mM glucose, 1 mM glutamine, and 50 pM cystine characteristic of physiologic conditions in contrast to traditional cell culture media that contains high levels of these nutrients that create artificial metabolic conditions. PKM2K0 in LNCaP cells produced a significant decrease in proliferation (FIG. 61A) whereas in the PKM2K0 C4-2B cells there was a slight increase but non-significant change in proliferation (FIG. 61B). When these cells were challenged with cystine starvation, the LNCaP cells continued to proliferate well and the difference between control and PKM2K0 cells observed under 50 pM was eliminated (FIG. 61C). In contrast, cystine starvation produced a dramatic decrease in proliferation in the C4-2B. PKM2K0 C4-2B cells also had decreased proliferation but were significantly more proliferative than the control cells (FIG. 61D). These results indicate that advanced castrate resistant prostate cancer cells may be more dependent on cystine and the expression of PKM2 may promote enhanced vulnerability to cystine starvation.

[00196] To further explore this effect, a viability assay was conducted in each cell line at 24 and 48 hours of growth. Consistent with the proliferation data, there was no significant decrease in viability in LNCaP control or PKM2KO cells when starved of cystine. Additionally, there was no difference in viability between control and PKM2KO cells in either cystine condition (FIG. 62A-B). In contrast, the C4-2B cells demonstrated a significant decrease in viability when starved of cystine, especially after 48 hours of starvation. PKM2K0 in the C4-2B resulted in significantly higher viability compared to control under cystine starvation at both time points indicated that the expression of PKM2 is associated with a metabolic state that is vulnerable to cystine starvation (FIG. 62C-D).

[00197] Cystine starvation induced death is known to induce a specific type of cell death known as ferroptosis. Ferroptosis is a non- apop to tic, non-necrotic, type of cell death characterized by accumulation of oxidation of polyunsaturated phospholipids propagated by iron and loss of lipid peroxide repair. To assess if C4-2B cells are indeed undergoing ferroptosis when starved of cystine specific inhibitors of ferroptosis were used in an attempt to rescue them from cell death. Three well characterized ferroptosis inhibitors were selected, specifically: N-acetyl-cysteine (NAC) which is a cell permeable analog of cysteine and antioxidant, Trolox (TRO) which is a vitamin E analog and antioxidant, and deferoxamine (DFO) which is an iron chelating agent. When both control and PKM2K0 cells in cystine starvation conditions were treated with these three compounds independently, there was significant restoration of cell viability (FIG. 62E-F). This result indicates that ferroptosis is the specific mechanism of cell death in the C4-2B and that the PKM2K0 results in decreased sensitivity to ferroptosis induced by cystine starvation.

EXAMPLE 13: The combination of pyruvate activation and inhibiting cysteine/cystine metabolism is an efficacious treatment for breast, skin, lung, and colon cancer cell lines in vitro

Statistical Analysis

[00198] All experiments were conducted with three replicates and were reproduced in at least 3 independent experiments. Statistical analysis and graphing were performed in Graph Pad v9 using Student’s T-Test, one-Way Analysis of Variance (ANOVA), or two-Way ANOVA where appropriate. A p-value of < 0.05 was used as cutoff for determining significance. In figures * = p- val <0.05, ** = p-val < .01, *** = p-val < 0.001. Multiple comparison correction was completed using either Sidak’s multiple comparison test or Tukey’s multiple comparison test for one-way ANOVA or two-way ANOVA respectively. Cell culture

[00199] MDA-MB-231 (breast tumor), A375 (skin tumor), A549 (lung tumor) and HCT-116 (colon tumor) cell lines were acquired as a gift from Dr. Hani Goodarzi (University of California, San Francisco). Cells were routinely cultured in DMEM (MT100013CV, Fisher Scientific®) with sodium pyruvate, supplemented with 10% fetal bovine serum (FBS) (13206C, Sigma-Aldrich®), 1% penicillin and streptomycin (P/S) (15140122, Fisher Scientific®), and cultured in a humidified incubator with 5% CO2 at 37°C. Cells were incubated in a humidified incubator with 5% CO2 at 37°C in DMEM.

Cell culture reagents

[00200] The following is a list of chemical compounds used in cell culture experiments: 10 pM Imidazole ketone erastin (IKE), inhibitor of the cystine-glutamate antiporter (27088, Cayman Chemical®), 100 pM TEPP-46, PKM2 Activator (HY- 18657, MedChem Express®) and Dimethyl sulfoxide (DMSO) (D4540, Sigma- Aldrich®).

Cell proliferation and viability analysis

[00201] Cells were plated on 96-well plates at 5000 cells/well under standard conditions described above. Cells were allowed to seed overnight (approximately 18 hours). Media was aspirated and cells were washed twice with PBS w/o calcium or magnesium (D8537, Sigma- Aldrich®) before 100 pL of experimental media containing compounds and nutrients at indicated concentrations was added to each well. Proliferation was measured using the Incucyte® SX1 HD/2CLR System (Sartorius®). Briefly, images were captured every 2-3 hours and automatically counted using the Incucyte® Cell-by-Cell software analysis package. Proliferation counts were normalized to the first cell count obtained immediately after beginning experimental conditions. Viability was assessed using almarBlue™ HS Cell Viability Reagent (Fisher Scientific®, A50100) according to the manufacturer's instructions. Briefly, 10 pL of almarBlue™ HS Cell Viability Reagent was added to each well containing 100 pL of experimental media. Plates were gently agitated to promote mixing, then incubated under standard conditions for 4 hours. Fluorescence at 570 nm and 600 nm was recorded on a BioTek Synergy Hl Multimode Reader (Agilent®).

Results

[00202] The inventors discovered combination treatment with IKE and TEPP-46 synergistically decreases cancer cell viability in various cancer types, including HCT-116 human colon cancer cells (FIG. 63A), A375 human skin cancer cells (FIG. 63B), A549 human lung cancer cells (FIG. 63C), and MDA-MD-231 human breast cancer cells (FIG. 63D). Consistently, combination treatment with IKE and TEPP-46 also synergistically decreases cancer cell proliferation in various cancer types, including HCT-116 human colon cancer cells (FIG. 64A), A375 human skin cancer cells (FIG. 64B), A549 human lung cancer cells (FIG. 64C), and MDA- MD-231 human breast cancer cells (FIG. 64D).

Claims

WHAT IS CLAIMED IS:

1. A therapeutic combination comprising: one or more cysteine/cystine metabolism inhibiting agent; and one or more pyruvate kinase muscle isoform 2 (PKM2) activating agent.

2. The therapeutic combination of claim 1, wherein the one or more cysteine/cystine metabolism inhibiting agent comprises imidazole ketone erastin (IKE), erastin, sulfasalazine, (s)-4- carboxyphenylglycine, cyst(e)inase, sorafenib, IKE-linked nanoparticles, or a combination thereof, preferably IKE.

3. The therapeutic combination of any one of the previous claims, wherein the one or more

PKM2 activating agent comprises TEPP-46, DASA-58, TP-1454, l-(sulfonyl)-5- (arylsulfonyl)indoline, micheliolide, diarylsulfonamides, thieno[3,2-b]pyrrole[3,2- d]pyridazinones, 4-(2,3-dichlorobenzoyl)- l-methyl-pyrrole-2-carboxamide, 2-((lH- benzo[d]imidazole-l-yl)methyl)-4H-pyrido[l,2-a]pyrimidin-4-ones, l-(sulfonyl)-5-

(arylsulfonyl)indoline, or a combination thereof, preferably TEPP-46.

4. The therapeutic combination of any one of the previous claims, wherein the therapeutic combination further comprises an additional agent comprising one or more anti-cancer agent.

5. The therapeutic combination of claim 4, wherein the anti-cancer agent comprises a chemotherapy agent, a hormone therapy agent, a targeted therapy agent, an immunotherapy agent, or a combination thereof.

6. The therapeutic combination of claim 4 or claim 5, wherein the anti-cancer agent comprises folfirinox, 5 -fluorouracil, Gemcitabine, Capecitabine, Oxaliplatin, Cisplatin, Carboplatin, Irinotecan, Leucovorin, Paclitaxel, albumin bound paclitaxel, docetaxel, Erlotinib, Olaparib, rucaparib, Pembrolizumab, Larotrectinib, Entrectinib, Vinorelbine, Etoposide, Pemetrexed, Trifluridine, Tipiracil, bevacizumab, ramuciriumab, cetuximab, panitumumab, trastuzumab, pertuzumab, Sacituzumab govitecan-hziy, tucatinib, lapatinib, fam-trastuzumab deruxtecan, abiraterone, enzalutamide, apalutamide, darolutamide, imatinib, sunitinib, everolimus, temsirolimus, vemurafenib, neratinib, tamoxifen, or a combination thereof.

7. The therapeutic combination of any one of the previous claims, wherein the therapeutic combination is for use in treating, preventing, and/or reducing the occurrence of cancer; inhibiting survival of cancer cells; inducing tumor cell death, inducing ferroptosis in cancer cells, or a combination thereof.

8. A pharmaceutical composition comprising the therapeutic combination of any one of claims 1-7 and at least one pharmaceutically acceptable carrier.

9. A method of treating, preventing, or reducing the occurrence of cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the therapeutic combination of any one of claims 1-7 or the pharmaceutical composition of claim 8.

10. The method of claim 9, wherein the one or more cysteine/cystine metabolism inhibiting agent and the one or more pyruvate kinase muscle isoform 2 (PKM2) activating agent are independently administered by intravenous, intratumoral, or intraperitoneal injection.

11. The method of claim 9 or claim 10, wherein the one or more cysteine/cystine metabolism inhibiting agent and the one or more pyruvate kinase muscle isoform 2 (PKM2) activating agent are administered by the same route of administration.

12. The method of any one of claims 9-11, wherein the one or more cysteine/cystine metabolism inhibiting agent and the one or more pyruvate kinase muscle isoform 2 (PKM2) activating agent are formulated in the same pharmaceutical composition.

13. The method of any one of claims 9-11, wherein the one or more cysteine/cystine metabolism inhibiting agent and the one or more pyruvate kinase muscle isoform 2 (PKM2) activating agent are formulated in separate pharmaceutical compositions.

14. The method of any one of claims 9-13 wherein the subject is human.

15. The method of any one of claims 9-13, wherein the subject is a non-human animal, such as a canine or a feline.

16. The method of any one of claims 9-15, wherein the cancer is a PKM2-expressing cancer.

17. The method of any one of claims 9-16, wherein the cancer is pancreatic cancer, prostate cancer, breast cancer, bladder cancer, endometrial cancer, cholangiocarcinomas, ovarian cancer, kidney cancer, leukemia, liver cancer, intrahepatic bile duct cancer, non-Hodgkin lymphoma, thyroid cancer, cervical cancer, gallbladder cancer, gastric cancer, skin cancer, lung cancer, colon cancer, rectal cancer, or a combination thereof, preferably pancreatic cancer.

18. The method of claim 17, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

19. A vaccine comprising the therapeutic combination of any one of claims 1-7 or the pharmaceutical composition of claim 8.

20. A method of inducing ferroptosis in a cancer cell, the method comprising: inhibiting cysteine/cystine metabolism in the cancer cell; and activating PKM2 in the cancer cell.

21. The method of claim 20, wherein the inhibiting cysteine/cystine metabolism in the cancer cell step comprises: contacting the cancer cell with one or more cysteine/cystine metabolism inhibiting agent; inhibiting a cystine transporter system of the cancer cell; knocking down cystine transporter system X_c’ in the cancer cell; knocking out cystine transporter system X_c’ in the cancer cell; withholding cystine and/or cysteine from the cancer cell; inhibiting glutathione (GSH) synthesis in the cancer cell; inhibiting CoA synthesis from cysteine (phosphopantothenoylcysteine synthetase) in the cancer cell; or a combination thereof.

22. The method of claim 21, wherein knocking down cystine transporter system X_c’ in the cancer cell comprises using a gene silencing technique, such as RNA interference (RNAi), epigenetic modification, clustered regularly interspaced short palindromic repeats (CRISPR), or small interfering RNA (siRNA).

23. The method of claim 21, wherein knocking out cystine transporter system X_c’ in the cancer cell comprises using a gene editing technique, such as CRISPR, one or more restriction enzymes, one or more zinc finger nucleases, or one or more transcription activator-like effector nucleases (TALENS).

24. The method of claim 21, comprising contacting the cancer cell with one or more cysteine/cystine metabolism inhibiting agent; and one or more pyruvate kinase muscle isoform 2 (PKM2) activating agent.

25. The method of claim 21 or claim 24, wherein the one or more cysteine/cystine metabolism inhibiting agent comprises imidazole ketone erastin (IKE), erastin, sulfasalazine, (s)-4- carboxyphenylglycine, cyst(e)inase, sorafenib, IKE-linked nanoparticles, or a combination thereof, preferably IKE.

26. The method of claim 24 or claim 25, wherein the one or more PKM2 activating agent comprises TEPP-46, DASA-58, TP- 1454, l-(sulfonyl)-5-(arylsulfonyl)indoline, micheliolide, diarylsulfonamides, thieno[3,2-b]pyrrole[3,2-d]pyridazinones, 4-(2,3-dichlorobenzoyl)- 1- methyl-pyrrole-2-carboxamide, 2-((lH-benzo[d] imidazole- 1 -yl)methyl)-4H-pyrido[ 1 ,2- a]pyrimidin-4-ones, l-(sulfonyl)-5-(arylsulfonyl)indoline, or a combination thereof, preferably TEPP-46.

27. The method of any one of claims 20-26, wherein the method is performed in vitro.

28. The method of any one of claims 20-26, wherein the method is performed in vivo.

29. A therapeutic combination comprising: a means for inhibiting cysteine/cystine metabolism; a means for activating PKM2; and a pharmaceutically acceptable carrier.

30. A pharmaceutical composition comprising: a means for inducing ferroptosis in a cancer cell; and a pharmaceutically acceptable carrier.

31. A method for treating, preventing, and/or reducing the occurrence of cancer in a subject in need thereof, the method comprising administering a combination of: a means for inhibiting cysteine/cystine metabolism; a means for activating PKM2; and a pharmaceutically acceptable carrier.