Compositions and methods of targeting pyruvate kinase isoform m2 (PKM2)

Abstract

In certain embodiments, the present invention provides a method of treating a hyperproliferative disorder in a patient in need thereof, comprising administering an agent that induces tetrameric pyruvate kinase isoform M2 (tet-PKM2) to the patient. In certain embodiments, the present invention provides a method of suppressing tumor growth comprising administering an agent that induces tetrameric pyruvate kinase isoform M2 (tet-PKM2) to the patient. In certain embodiments, the present invention provides a method of inducing tetramerization of PKM2 in a cell comprising contacting the cell with TEPP46.

Description

[0001]VHPM Ref.09531.584WO1 / Client Ref.2022-294 COMPOSITIONS AND METHODS OF TARGETING PYRUVATE KINASE ISOFORM M2 (PKM2) CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to United States Provisional Application Number 63 / 613,418 that was filed on December 21, 2023. The entire content of the applications referenced above is hereby incorporated by reference herein. BACKGROUND Immunotherapy has markedly transformed the landscape of cancer treatment, with a significant number of patients experiencing durable anti-tumor effects. Nonetheless, many patients, especially with solid tumors, do not respond adequately to immunotherapy. The microenvironment of solid tumors is complex and has a profound effect on immune cell activation and function. Signals generated within the tumor microenvironment (TME) can induce exhaustion in immune cells in part by depleting their nutrition and suppressing their mitochondrial function. Mitochondria are independent organelles that play a crucial role in energy generation and signaling within cells. Mitochondria are involved in multiple roles including oxidative phosphorylation (OXPHOS), calcium buffering, generation of reactive oxygen species, regulation of metabolism, transcription in T cells, and homeostatic regulation of cytotoxicity. Most of these processes are influenced by the optimum structural organization of mitochondria. During T cell differentiation, mitochondrial fission and fusion modify mitochondrial morphology and function. Mitochondrial fusion supports greater OXPHOS capacity and enhanced metabolism leading to appropriate cell activation compared with smaller fissed mitochondria, which are associated with exhausted T lymphocytes. Restructuring mitochondria is also important for T cell development, as demonstrated by the disrupted development of naïve T cells after deletion of genes encoding proteins required for mitochondrial fusion and fission. Deletion of the inner mitochondrial membrane fusion protein OPA1 (required for proper cristae formation) disrupts early T cell development. Similarly, impairing mitochondrial transcription by mitochondrial transcription factor A (TFAM) deletion affects T cell development and reduces the number of circulating T cells. In addition to T cell development, mitochondrial dynamics are critical for the transformation of T cells from a naïve state to an effector phase able to eliminate cancer cells. Naïve non-activated cells have small mitochondria that are sufficient to support cell energetics via basal OXPHOS. Upon antigen encounter, cells upregulate their effector functions that require substantial metabolic VHPM Ref.09531.584WO1 / Client Ref.2022-294 rearrangement, lack of which blocks cell activation and drives the cells towards an exhausted phenotype. Accordingly, development of strategies that can improve the mitochondrial architecture and metabolism are of paramount importance. Summary In certain aspects, provided herein is a method of treating a hyperproliferative disorder in a patient in need thereof, comprising administering to the patient an agent that induces tetrameric pyruvate kinase isoform M2 (tet-PKM2). In certain aspects, provided herein is a method of suppressing tumor growth comprising administering an agent that induces tetrameric pyruvate kinase isoform M2 (tet-PKM2) to the patient. In certain aspects, provided herein is a method of inducing tetramerization of PKM2 in a cell comprising contacting the cell with TEPP46 or DASA58. In certain aspects, provided herein is a method of enhancing mitochondrial structural organization and / or metabolism in CD8 T cells in a patient comprising administering to the patient an agent that induces tetrameric pyruvate kinase isoform M2 (tet-PKM2). In certain aspects, provided herein is a method of inducing apoptosis of tumor cells in a patient, comprising administering to the patient an agent that induces tetrameric pyruvate kinase isoform M2 (tet-PKM2). BRIEF DESCRIPTION OF DRAWINGS Figures 1A-1L. PKM2 agonist induces CD8 T cell dependent anti-tumor immunity. (Figs.1A, 1B) Tumor growth profiles and associated mice survival plots when B16-F10 breast cancer and CT26 colon cancer bearing mice were treated with TEPP46 for total of 10 doses (4-on 2- off starting at day 5 after tumor inoculation) (n=5 mice per group). (Fig.1C) Percentage of total CD8 T cells and (Fig.1D) Percentage of dextramer gp100 specific+CD8 T cells in the TME of B16-F10 bearing mice after TEPP46 treatment (n=5 mice per group). (Figs.1E-1H) FACS micrographs and associated statistical analysis of IFNγ (Fig.1E), Gzm B (Fig.1F), PD1 (Fig.1G), and LAG3 (Fig.1H), in CD8 T cells in the TME of B16-F10 bearing mice (n=5 mice per group). (Fig.1I) Estimation of tumor growth rates and mice survival when CD8 T cells were depleted before TEPP46 treatment of B16-F10 bearing mice (n= 5 mice). Anti-CD8 antibody was injected twice on day 5 and 6 post-tumor induction followed by TEPP46 treatment started at day 7. (Fig.1J) CD8 T cells infiltration in the TME of CT26 bearing mice after TEPP46 treatment (untreated; n= 5 mice, treated; n= 6 mice). (Figs.1K, 1L) FACS micrographs and associated statistical analysis of VHPM Ref.09531.584WO1 / Client Ref.2022-294 IFNγ and TNFα in CD8 T cells in the TME of CT26 bearing mice. Each dot represents one mouse in each treatment group. Measurements were taken from distinct samples and statistical analysis was done by unpaired t-test. Error bars represent Standard error of mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figures 2A-2K. PKM2 activation alters cell transcriptome augmenting effector functions in CD8 T cells. (Fig.2A) Levels of tetrameric (tet-) and dimeric (di-) PKM2 in OT1- CD8 T cells activated with Ova peptide in the presence of TEPP46 at 40 and 60 µM for 48hours. (Figs.2B, 2C) The principal component analysis (PCA) and Venn diagram showing gene expression pattern comparison between Ova and Ova+TEPP46 (60 µM) treated OT1-CD8 T cells at 24 and 48 hours. (Fig.2D) Pathways upregulated in TEPP46 treated CD8 T cells at 48 hours post activation with Ova peptide using RNA-seq data. (Fig.2E) Gene expression profiles associated with T cell activation in CD8 T cells treated with TEPP46 for 48 hours. (Fig.2F) FACS micrographs and associated statistical analyses of IFNγ, TNFα, and Gzm B in CD8 T cell activated for 48 hours in the presence of TEPP46. (Figs.2G, 2H) FACS micrographs (Fig.2G) and associated fractions of CD62L-CD44+(TEM) (H) cells after TEPP46 treatment of CD8 T cells for 48 hours. (Fig.2I) Fractions of IFNγ+and TNFα+cells in TEMpopulations in TEPP46 activated CD8 T cells at 48 hours post activation. (Fig.2J) FACS micrographs and associated fractions of TNFα+IFNɣ+and (Fig.2K) CD44+CD45RA- (effector cells) in human CD8 T cells activated by CD3 / CD28 in the presence or absence of TEPP46. Each dot represents one replicate in each treatment group. Number of repeats (n) =4, measurements were taken from distinct samples and statistical analysis was done by one-way ANOVA. Error bars represent Standard error of mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figures 3A-3J PKM2 activation facilitates mitochondrial generation and structural reorganization. (Fig.3A) RNA-seq analysis of genes associated with mitochondrial biogenesis of Ova+ / -TEPP46 treated CD8 T cells (n=4). (Figs.3B, 3C) Analysis of expression of PGC1α by Western blot (numbers representing the densitometry) (Fig.3B) and qRTPCR (Fig.3C) in CD8 T cells activated with Ova + / - TEPP46 at 48 hours post activation (n=4, statistical analysis was done by unpaired t-test). (d-g) FACS micrograph and statistical analysis of mitochondrial mass (MitoFM) (Fig.3D), mitochondrial membrane potential (TMRM) (Fig.3E), mitochondrial ATP production (Fig.3F), and reactive oxygen species (ROS) production (Fig.3G) in CD8 T cells activated with Ova + / - TEPP46 for 48 hours (n=4, statistical analysis was done by one-way ANOVA). (Figs.3H, 3I) Scanning electron microscopic images. Yellow arrows highlight the mitochondria:mitochondria contact sites (number of analyzed cells for IL2 = 4 and Ova and TEPP46 treated cells = 15) (Fig. 3H), and statistical analysis of variously activated CD8 T cells showing mitochondrial numbers (each dot represents one cell), cristae numbers (each dot represents the number of cristae in one VHPM Ref.09531.584WO1 / Client Ref.2022-294 mitochondria) and cristae density (each dot represents one ratio value of cristae area / mitochondrial area) (Fig.3I). (Fig.3J) Statistical analysis of mitochondria endoplasmic reticulum contact site (MERCS) index obtained from electron microscopy of CD8 T cells activated with Ova + / - TEPP46 for 48 hours (each dot represents one MERCS score in mitochondria). Measurements were taken from distinct samples. Error bars represent Standard error of mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figures 4A-4J. PKM2 activation reduces mtDNA methylation and enhances mitochondrial translation supporting effector functions in CD8 T cells. (Fig.4A) PCA analysis based on WGBS regarding CpG methylation showing distinct clustering of Ova and Ova+TEPP46 treated CD8 T cells (n=3). (Fig.4B) Methylation ratio of chromosome M (mtDNA) in CD8 T cells activated with Ova + / - TEPP46 at 48 hours post activation. (Fig.4C) Methylation levels of various genes associated with respiratory complexes on chromosome M in CD8 T cells activated with Ova + / - TEPP46 at 48 hours post activation. (Fig.4D) Expression profiles of mitochondrial-complexes related genes (n=4). (Figs.4E, 4F) Western blot analysis of puromycin incorporation in total cell lysate (Fig.4E) and purified mitochondrial lysate (Fig.4F) from CD8 T cells activated with Ova + / - TEPP46 in the presence or absence of doxycycline, an inhibitor of mitochondrial translation (n=3). (Fig.4G) Western blot analysis of various respiratory complexes in CD8 T cells activated with Ova + / - TEPP46 in the presence or absence of doxycycline (n=3). (Fig.4H) FACS analysis of IFNγ and TNFα production in CD8 T cells activated by ova + / - TEPP46 in the presence of doxycycline (n=5). (Fig.4I, 4J) FACS analysis of IFNγ and TNFα production in CD8 T cells activated by ova + / - TEPP46 in the presence of inhibitors of respiratory complex I (Rotenon) (I) or complex II (TTFA) (n=4) (J). Each dot represents one replicate in each treatment group. Statistical analysis was done by one-way ANOVA. Measurements were taken from distinct samples. Error bars represent Standard error of mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figures 5A-5K. Activated PKM2 alters cell metabolism supporting pentose phosphate pathway and one-carbon pathway in CD8 T cells. (Fig.5A) PCA analysis of whole cell metabolome in CD8 T cells activated with Ova peptide + / - TEPP46 at 24- and 48-hours post activation. (Fig.5B) Volcano plot representation of differentially altered metabolites in TEPP46 treated CD8 T cells compared to Ova-alone activated CD8 T cells at 48 hours post-activation. (Fig. 5C) Analysis of various metabolites belonging to glycolytic pathway, TCA cycle, pentose phosphate pathway (PPP), and one carbon metabolism in CD8 T cells activated with Ova + / - TEPP46 at 48 hours post activation. (Fig.5D) Normalized levels of D-Ribose 5-Phosphate and D- Ribulose 5-Phosphate in Ova + / - TEPP46 activated CD8 T cells. (Figs.5E-5H) Fraction of Fructose 1,6biphosphate (F1,6BP) (Fig.5E), glucose 6 phosphogluconate (6PGL) (Fig.5F), Ribose 5 phosphate (R5P) (Fig.5G), and Glyceraldehyde 3 phosphate (G3P) (Fig.5H) incorporating VHPM Ref.09531.584WO1 / Client Ref.2022-29413Carbon in Ova+ / -TEPP46 treated CD8 T cells. (Fig.5I) Analysis of differential incorporation of13Carbon from13C-Glucose into one carbon metabolism in TEPP46 treated CD8 T cells 48 hours post activation. (Fig.5J) Normalized levels of metabolites from one carbon metabolism including folate, methylene-THF, serine, glycine, betaine, dimethylglycine, homocysteic acid, methionine, and nicotinic acid in TEPP46 treated CD8 T cells. (Fig.5K) The ratio of homocysteic acid / methionine in ova+ / -TEPP46 activated CD8 T cells. Each dot represents one technical replicate in each treatment group (n=4-5). Each experiment was repeated at least twice at different time points. Statistical analysis was done by one-way ANOVA. Measurements were taken from distinct samples. Error bars represent Standard error of mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figures 6A-6N. PKM2 activation enhances antigen recall responses and anti-tumor efficacy of adoptive cell therapy. (Fig.6A) Schematic diagram for antigen recall-response experiment including resting-rechallenge assay and chronic stimulation of CD8 T cells pre-treated with Ova + / - TEPP46. (Figs.6B-6D) Percentages of IFNg+(Fig.6B), Gzm B+(Fig.6C), and 41BB+(Fig.6D) CD8 T cells activated by Ova + / - TEPP46 for 2 days and daily stimulated by Ova for 4 days (chronic stimulation) (n=4). (Figs.6E-6G) Evaluation of CD62L-CD44+(TEM), KLRG- CD127+(MPEC), and KLRG+CD127- (SLEC) populations during chronic stimulation of CD8 T cells activated by Ova + / - TEPP46 (n=4). (Fig.6H) Fraction of viable B16F10-Ova tumor cells incubated with CD8 T cells activated by Ova + / - TEPP46 for 48 hours (n=4). (I) Fraction of viable B16-EGFRvIII tumor cells incubated with Thy1.1+CART cells that were treated with TEPP46 for 24 hours (n=4). (Figs.6J, 6K) Percentage of IFNg+OT1-CD8 T cells (Fig.6J) and Thy1.1+CART cells (K) after co-incubation with tumor cells (n=4) as described in H&I. (Fig.6L) Schematic diagram for adoptive cell therapy. C57BL6 mice were inoculated with B16F10-Ova tumors followed by treatment with OT1-CD8 T cells on day 6. (Figs.6M, 6N) B16F10-OVA tumor volumes (Fig.6M) and mice survival (Fig.6N) in groups treated with OT1-CD8 T cells activated by Ova+ / -TEPP46 (n=4-6). Statistical analysis was done by unpaired t-test. Mice survival was analyzed by Kaplan-Meier plots. Error bars represent Standard error of mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figures 7A-7K. PKM2 agonist enhances the anti-tumor efficacy of anti-PD1 therapy. (Fig.7A) Schematic of mice treatment using TEPP46 and anti-PD1 antibody. B16-F10 tumor bearing mice were treated with TEPP46 for a total of ten doses (4-days-on-2-days-off) starting day 5. Mice in various groups were treated with anti-PD1 starting day 5. Anti-PD1 was given twice / week for a maximum of 4-5 doses. (Fig.7B) Tumor growth profiles and associated mice survival plots after treatment of B16-F10 tumor bearing mice with TEPP46 and anti-PD1 as described in A. (Figs.7C, 7D) Percentage of CD3+T cells in total live tumor cells (Fig.7D) and CD8+T cells in CD3+T cells in TME (Gating strategy in Figure S8A). (E-I) FACS micrographs and VHPM Ref.09531.584WO1 / Client Ref.2022-294 statistical analysis of IFNg+(Fig.7E), TNFa+(Fig.7F) Gzm B+(Fig.7G), Ki67+(Fig.7H), and GP100-dextramer+Fig.7 (I) in CD8 T cells isolated from tumor tissues of mice treated with αPD1, TEPP46 or αPD1+TEPP46. (J,K) FACS analysis of the percentage of IFNg+(Fig.7J) and Gzm B+(Fig.7K) cells in tumor specific gp100+CD8 T cells. Statistical analysis was done by one way ANOVA. Measurements were taken from distinct samples. Each dot represents one mouse in each treatment group (n=5). The experiment was repeated two times with similar outcomes. Error bars represent Standard error of mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figure 8: PKM2 agonist shows immune modulatory effects resulting in CD8 T cell dependent anti-tumor effects. (Fig.8A) Body weight measured in untreated and TEPP46 treated B16-F10 and CT26 tumor bearing mice. (Figs.8B-8E) Percentage of CD3+cells in live cell population in tumors (Fig.8B), Percentage of CD4+T cells in CD3+T cells (Fig.8C), and IFNg+and TNFα+CD4+T cells (Figs.8D, 8E) in TME of untreated and TEPP46 treated B16- F10 tumor bearing mice. (Figs.8F, 8G) Expression of PD1 and LAG on CD4+T cells in TME variously treated mice. (Fig.8H) FACS micrograph and percentage of FOXP3+cells (Treg cells) in the TME of untreated and TEPP46 treated B16-F10 tumor bearing mice. (Fig.8I) Analysis of CD8+T cell depletion in the blood of mice treated with anti-CD8 antibody on day 5 and 6. (Figs.8J, 8K) Expression PD1 and LAG on CD8 (J) and CD4 T cells (K) in tumors of CT26 bearing mice (n=6-8). (Figs.8L, 8M) Percentage of TIGIT+CD8 and CD4 cells in B16F10 (n=5) (Fig.8L) and CT26 bearing mice (n=6-8) (Fig.8M). Each dot represents one mouse in each treatment group. Measurements were taken from distinct samples and statistical analysis was done by unpaired t-test. Error bars represent Standard error of mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figure 9A-9J: PKM2 activation alters gene expression and induces effector phenotype in OT1-CD8 T cells. (Fig.9A) transcriptome and functional analysis were performed in CD8+T cells derived from OT1 mice that activated with Ova peptide with or without TEPP46 for 24 and 48 hours. (Fig.9B) volcano plot of RNA seq. results from untreated and TEPP46 treated CD8 T cells after 48 hours. (Fig.9C) IFNɣ, TNFα and Gzm B expression analyzed by qRT-PCR in Ova activated CD8 T cells in the presence and absence of TEPP46 after 48 hours (n=3). (Fig.9D) the percentage of CD62L+CD44+cells (TCMpopulation) in OT1-CD8 T cells after activation with Ova + / - TEPP46 (n=4). (Fig.9E) FACS micrographs for IFNɣ and TNFα expression in TCMpopulation of untreated and TEPP treated cells (n=4). (Fig.9F) FACS analysis of the percentage of CD127+KLRG- (MPEC) and CD127-KLRG+(SLEC) cells in CD8 T cells activated by Ova + / - TEPP46 (n=4). (Fig.9G) FACS micrograph of cell proliferation by VCT (violet cell trace) staining in untreated and TEPP46 treated cells (n=4). (H) the percentage of CD8+T cells in every peak of VCT (n=4). (Figs.9I, 9J) Percentage of IFNɣ+and TNFα+CD8 T cells in different peaks of VHPM Ref.09531.584WO1 / Client Ref.2022-294 proliferation (VCT1, VCT2 and VCT3) (n=4). Each dot represents one technical repeat in each group. Measurements were taken from distinct samples and statistical analysis was done by unpaired one-way ANOVA. Error bars represent Standard error of mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figures 10A-10K: PKM2 activation augments effector functions in CD8 T cells of mouse and human origin. (Figs.10A, 10B) the effects of PKM2 activation on effector function in already activated CD8 T cells (n=4). (Figs.10C, 10D) IFNg and TNFα production in CD8 T cells derived from wild type (Fig.10C) and PMEL (Fig.10D) mice that were activated in the presence and absence of TEPP46 (n=4). (Figs.10E-10G) Proliferation levels and IFNg and TNFα production in untreated and TEPP46 treated CD4 T cells isolated from wild type mice (n=3). (Figs.10H, 10I) the percentage of IFNγ+TNFα+CD8+cells (H) and TEM population (CD62L-CD44+) generation (I) in OT1-CD8 T cells that were activated by Ova + / - DASA58 (n=4). (Figs.10J, 10K) Percentage of IFNg+TNFα+CD8+cells (Fig.10J) and effector population (CD44+CD45RA-CD8+) generation (Fig.10K) in human CD8 T cells that were activated with antiCD3 / 28 in the presence and absence of DASA58 (n=4). Each dot represents one repeat in each group. Measurements were taken from distinct samples and statistical analysis was done by t-test for comparison between two groups and unpaired one-way ANOVA for more than two groups. Error bars represent Standard error of mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figures 11A-11F. PKM2 activation augments mitochondrial mass in CD8 T cells of mouse and human origin. (Figs.11A, 11B) MitoFM incorporation in activated OT1-CD8 T cells after DASA58 treatment in vitro (n=4) (Fig.11A) or TEPP46 treatment in vivo in B16-F10 (n=5) and CT26 tumors (n=5-8) bearing mice (Fig.11B). (Fig.11C) MitoFM incorporation in activated human CD8 T cells in the presence of TEPP46 and DASA58 (n=4). (Fig.11D) mitochondrial area measured in OT1-CD8 T cells activated by Ova+ / - TEPP46 (mitochondrial structure were analyzed from n=15 cells in Ova and Ova+TEPP46 and n=4 cells from IL2 treated cells). (Figs.11E, 11F) qRTPCR analysis of genes associated with mitochondrial dynamic regulation (Mfn1, Mfn2 and Opa1) (Figs.11E) and Minos1, Immt and Apool (Figs. 11F) in Ova or Ova+TEPP46 treated CD8 T cells (n=3). Each dot represents one repeat in each group. Measurements were taken from distinct samples and statistical analysis was done by t- test for comparison between two groups and unpaired one-way ANOVA for more than two groups. Error bars represent Standard error of mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figures 12. Activated PKM2 reduces DNA methylation and enhances respiratory complex generation in CD8 T cell. (Fig.12A) Differentially methylated regions (DMRs) in Ova VHPM Ref.09531.584WO1 / Client Ref.2022-294 and Ova+TEPP46 treated CD8 T cells. (Fig.12B) Mapping of DMRs of nuclear and mitochondrial chromosomes (ChrM) in CD8 T cells treated with Ova+TEPP46. (Fig.12C) Gene Set Enrichment Analysis (GSEA) analysis of transcriptome and correlation with different mitochondrial pathways. (Fig.12D) Expression profiles of mitochondrial ribosomal protein encoded genes depicting status of mitochondrial translation. (Figs.12E, 12F) Statistical analysis of Western blot shown in Figure 4E and F depicting the level of puromycin incorporation in total (Fig.12E) and mitochondrial translated proteins (Fig.12F) in CD8 T cells activated by Ova + / - TEPP46 in the presence of doxycycline (Doxy). (Figs.12G, 12H) Statistical analysis of Western blot shown in Figure 4G (Fig. 12G) and analysis of effect of fold-change in the expression of various respiratory complex proteins in CD8 T cells treated with TEPP46+ / -doxycycline (Fig.12H). (Figs.12I, 12J) IFNg and TNFα production in CD8 T cells activated by Ova + / - TEPP46 in the presence of antimycin (complex III inhibitor) and oligomycin (ATP synthase inhibitor) (n=4). Each dot represents one repeat in each group. Measurements were taken from distinct samples and statistical analysis was done by one-way ANOVA. Error bars represent Standard error of mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figures 13. Effects of PKM2 activation on metabolomics in CD8 T cells. (Fig.13A) Analysis of whole intracellular targeted metabolome in OT1-CD8 T cells activated with Ova + / - TEPP46 for 24h and 48h. (Fig.13B) Enrichment of lactate, citrate and isocitrate metabolites in Ova+ / -TEPP46 treated OT1-CD8 T cells (n=5). Each dot represents one repeat in each group. Measurements were taken from distinct samples and statistical analysis was done by t-test. Error bars represent Standard error of mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figures 14A-14N. PKM2 activation enhances antigen recall responses after acute and chronic stimulation. (Figs.14A-14C) Mean fluorescent intensity of IFNγ (Fig.14A), Gzm B (Fig. 14B), and 4-1BB (Fig.14C) production in CD8 T cells upon chronic antigenic stimulation after 2, 4 and 6 days (n=4). (Fig.14D) FACS micrographs showing the percentage of CD44+CD62L- (TEMpopulation) and CD44+CD62L+(TCMpopulation) in CD8 T cells upon chronic antigenic stimulation (n=4). (Figs.14E-14G) Percentage of IFNg (Fig.14E), TNFα (Fig.14F), and 4-1BB (Fig.14G) producing CD8 T cells in TEMpopulation over the course of chronic stimulation at different time points (day 2, 4, 6) (n=4). Fig.14 (H) Enrichment of KLRG1-CD127+memory precursor effector cells (MPEC) in TEPP46 treated CD8 T cell cultures in chronic antigen stimulation (n=4). (Figs. 14I-14K) IFNg ( Fig.14I), 41-BB (Fig.14J), and Gzm B (Fig.14K) production in Ova+ / -TEPP46 activated CD8 T cells under acute (resting) restimulation (n=4). (Fig.14L) Schematic diagram for the cancer cell killing experiment using activated OT1-CD8 T cells or CAR-T cells treated with TEPP46 for 48 hours. (Fig.14M) Schematic of the EGFRvIII directed Thy1.1+CAR-T cell construct. (Fig.14N) FACS micrograph and the percentage of CAR-T cell induction compared to VHPM Ref.09531.584WO1 / Client Ref.2022-294 untraduced cells. Each dot represents one repeat in each group. Measurements were taken from distinct samples and statistical analysis was done by t-test. Error bars represent Standard error of mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figures 15A-15E: PKM2 agonist enhances the anti-tumor efficacy of anti-PD1 therapy. (Fig.15A) gating strategy for FACS analysis of tumor samples from B16-F10 tumor bearing mice untreated or treated with TEPP46. (Figs.15B-15E) mean fluorescent intensity of IFNg (Fig.15B), TNFα (Fig.15C), Gzm B (Fig.15D) and Ki67 (Fig.15E) in CD8 T cells in the TME of B16-F10 tumor bearing mice untreated or treated with TEPP46, αPD1 or combination of TEPP46 and αPD1 (n=5). Each dot represents one mouse in each group. Measurements were taken from distinct samples and statistical analysis was done by one-way ANOVA. Error bars represent Standard error of mean. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figures 16A-16P: Tumor cell demonstrate mitochondrial malformation. (Fig.16A) qRTPCR analysis of expression of PGC1a in normal skin and colon tissue from mice and B16 and CT26 tumor cells. (Figs.16B-16I) qRTPCR analysis of OPA1 (Fig.16B), Mfn1 (Fig.16C), Mfn2 (Fig.16D), Fis1 (Fig.16E), Mief2 (Fig.16F), Apool (Fig.16G), Immt (Fig.16H), Minos1 (Fig.16I), various genes associated with mitochondrial structural organization. (Fig. 16J) Expression levels of PGC1a in human melanoma cell line A375 vs normal human melanocytes (NHEM). (Fig.16K) Relative mitochondrial DNA content calculated from mtDNA vs nuclear DNA content in human melanoma cells (A375) compared to normal melanocytes (NHEM). (Figs.16L-16N) Gene expression levels of OPA1 (Fig.16L), Apool (Fig.16M), Minos1 (Fig.16N) in human melanoma cells (A375) compared to normal melanocytes (NHEM). (Fig.16O and 16P) FACS micrographs and associated bar graphs depicting MitoFM incorporation (Fig.16O), and mitochondrial ATP production (Fig.16P) in human melanoma cells compared to normal melanocytes. All experiments were done at least two times with similar outcomes. Representative experiments are shown. None of the replicates were censored. Error bars represent mean + standard error of mean (SEM). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figures 17A-17H: Tumor cell demonstrate mitochondrial malformation. (Fig.17A) Relative mitochondrial DNA content calculated from mtDNA vs nuclear DNA content in mouse B16 and CT26 cancer cells compared to normal mouse skin and colon tissue. (B&C) MFI of MitoFM (Fig.17B) and ATP (Fig.17C) in A375 cells. (Fig.17D) We compared normal-tumor pair (n = 41 in each group) in TCGA COAD RNAseqdataset. Volcano plot depicted all genes that were significantly different between normal and tumor sample.5613 genes were significantly up regulated (fdr< 0.001, logFC> 1) in tumor group denoted using red color, while VHPM Ref.09531.584WO1 / Client Ref.2022-294 4028 genes were significantly down regulated (fdr< 0.001, logFC< -1) in tumor group denoted by blue color. Top differentially expressed genes that overlapped with Human Mitocarta3.0 datasets were also color labeled in the volcano plot. (Figs.17E-17H) Survival analysis on each color labeled genes using all tumor samples (n = 428) in TCGA COAD RNAseqdataset. Higher expression of TOMM34, TRAP1 (Fig.17E), SLC25A5, SLC25A34 (Fig.17F), and TOP1MT (Fig.17G) indicated better overall survival while PINK1 and SLC25A27 (Fig.17H) indicated worst survival at HR = 0.56 (0.31-1), log rank p = 0.048 and HR = 0.59 (0.35-1), log rank p = 0.047, respectively. Figures 18A-18N: TEPP46 mediated PKM2 activation enhances mitochondrial organization and functions leading to halted tumor growth. (Figs.18A&18B) FACS micrographs and associated statistical analysis of MitoFM incorporation (Fig.18A) and mitochondrial ATP production (Fig.18B) in B16 cells with / without TAEPP46 treatment. (Fig.18C) qRTPCR analysis of PGC1a expression in TEPP46 treated B16 cells. (Fig.18D) Electron micrographs of untreated and TEPP46 treated B16 cells. Red satellites are highlighting mitochondria in untreated cells while green satellite is for TEPP46 treated B16 cells. (Figs.18E-18G) Statical analysis of electron micrographs shown in Fig.18D for cristae numbers (Fig.18E), cristae density (Fig.18F) and endoplasmic reticulum to mitochondria contact sites (ERMICC) (Fig.18G) in B16 cells. (Fig.18H) Estimation of mitochondria related pathways from RNA-seq analysis of B16 cells after TEPP46 treatment. (Fig.18I) Heat map showing the expression of genes associated with mitochondrial structural regulation for the RNA-seq of B16 after TEPP46 treatment. (Fig.18J) Growth pattern analysis of B16 cells with and without TEPP46 treatment using Incucyte. (Fig.18K) FACS analysis of B16 cell proliferation by estimating Ki67 expression 48 hours after TEPP46 treatment. (Fig.18L) B16 tumor growth profiles in mice after TEPP46 treatment (40mg / Kg) of mice as shown in Supplementary Fig2F. (Figs.18M&18N) FACS analysis for the expression of Ki67 (Fig.18M) and MitoFM (Fig.18N) on CD3- tumor cells from B16 tumors grown in mice in L. All experiments were repeated two times with similar outcomes. Representative experiments are shown. None of the replicates were censored. Error bars represent mean + standard error of mean (SEM). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figures 19A-19F: TEPP46 mediated PKM2 activation enhances mitochondrial organization and functions leading to halted tumor growth. (Fig.19A) Western blot analysis of PKM2 activation after TEPP46 treatment of B16 cells. (Fig.19B&19C) FACS micrographs and associated bar graphs depicting MitoFM incorporation (Fig.19B), and mitochondrial ATP production (Fig.19C) in TEPP46 treated B16 cells. (Fig.19D) Statistical analysis of electron micrographs shown in Fig 2D to estimate mitochondrial numbers and mitochondrial area in VHPM Ref.09531.584WO1 / Client Ref.2022-294 TEPP46 treated B16 cells. (Fig.19E) qRTPCR analysis for the expression of Minos, Mfn1, Mfn2, Mief2, and Apool in TEPP46 treated B16 cells. (Fig.19F) Schematic for B16 tumor induction and mice treatment using TEPP46. Figures 20A-20K: TEPP46 mediated enhances glucose utilization through mitochondria in tumor cells. (Fig.20A) Glucose uptake rates measured by FACS analysis of 2-NBDG uptake in TEPP46 treated B16 cells. (Fig.20B-20D) Metabolome analysis showing levels of glucose-6- phosphate and fructose-6-phosphate (Fig.20B), acetyl-coA (Fig.20C), and lactate (Fig.20D) in TEPP46 treated B16 cells. (Fig.20E) Expression levels of LDHA gene estimated by qRTPCR Analysis of TEPP46 treated B16 cells. (Fig.20F&20G) LDHA protein levels estimated by western blot analysis (Fig.20F) in TEPP46 treated B16 cells. Fig.20G represents the statistical analysis of densitometry of LDHA expression from two experiments. (Fig.20H&20I) Expression of LDHA proteins (Fig.20H) and bar graphs of the densitometry (Fig.20I) from the tumor tissue of untreated or TEPP46 treated B16 bearing mice. (Fig.20J) 13-C Glucose Tracer experiment evaluating the movement of various carbon molecules in various metabolites belonging to TCA cycle. (Fig.20K) Western blot analysis of expression of phosphorylated (S293) and total PDH protein and PDK1 protein from in vitro TEPP46 treated B16 cells. All experiments were repeated two times with similar outcomes. Representative experiments are shown. None of the replicates were censored. Error bars represent mean + standard error of mean (SEM). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figures 21A-21B: TEPP46 mediated enhances glucose utilization through mitochondria in tumor cells. (Fig.21A) MFI of 2-NBDG uptake in B16 cells treated with TEPP46. (Fig.21B) Lactate concentration in TEPP46 treated B16 cells. Figures 22A-22F: TEPP46 mediated enhanced mitochondrial metabolism is associated with increased tumor cell apoptosis and MHCI expression. (Fig.22A&22B) FACS micrographs and statistical analysis for the expression of annexin V in B16 (Fig.22A) and CT26 (Fig.22B) cells after TEPP46 treatment. (Fig.22C&D) FACS micrographs and statistical analysis for the expression of MHCI in B16 (Fig.22C) and CT26 (Fig.22D) cells after TEPP46 treatment. (Fig. 22E&22F) FACS micrographs and statistical analysis for the expression of MHCII in B16 (Fig. 22E) and CT26 (Fig.22F) cells after TEPP46 treatment. All experiments were repeated two times with similar outcomes. Representative experiments are shown. None of the replicates were censored. Error bars represent mean + standard error of mean (SEM). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. VHPM Ref.09531.584WO1 / Client Ref.2022-294 DETAILED DESCRIPTION Certain aspects utilize PKM2 as a putative target in CD8 T cells for enhancement of their effector functions, and certain aspects employ targeting PKM2 in CD8 T cells. Pharmacological agents that target PKM2 in CD8 T cells are used for enhancement of their effector functions. Certain aspects include methods to enhance the mitochondrial fitness in CD8 T cell using PKM2 as a target. The present invention addresses three problems: (1) Differential targeting of the tumor and the immune compartment. It is extremely difficult to find a target that can be used to prevent the growth of tumor cells, while simultaneously enhancing the effector functions in immune cells. As described herein, it is shown that tetramerization of pyruvate kinase mouse 2 (PKM2) is inhibitory for the tumor cells while it enhances the effector functions of CD8 T cells. (2) It is difficult to maintain effector functions while preventing exhaustion in CD8 T cells. This is especially important in environments such as tumor microenvironment where too much antigenic stimulation along with other tumor derived suppressive factors render the CD8 T cells exhausted. Hence, devising strategies that prevent exhaustion are useful. As described herein, it is shown that the PKM2 enzyme is a putative target for enhancing the effector functions and preventing the exhaustion in CD8 T cells. (3) Metabolism is a crucial factor that decides the functional states of the immune cells and not many metabolism-related targets have been developed that can be used to enhance the effector functions in CD8 T cells. Using PKM2, an enzyme that controls the last step of glycolysis in cells, it has been found that metabolism can be effectively targeted to enhance the effector functions in CD8 T cells. The problem of differentially targeting the tumor and the immune cells has not been addressed. As described herein, it was found that while enhancing the tetrameric-PKM2 is inhibitory for the tumor cells, tetramerization of PKM2 in CD8 T cells leads to their activation. The problem of exhaustion in CD8 T cells is currently addressed by preventing over-activation by using agents that induce memory in CD8 T cells. These strategies are based on the assumption that the CD8 T cell with a memory phenotype will give rise to well activated effector T cells when they encounter the antigen. However, none of the strategies actually maintain the effector functions as a result of direct therapeutic intervention. Described herein is a methodology to maintain the effector functions is CD8 T cells that have been activated with their cognate antigen. Moreover, previously-known techniques to target the metabolic pathways in CD8 T cells were not explained. Here, using a target directly involved in metabolism, a method is described to target metabolism and enhance effector functions in CD8 T cells. In certain aspects, described herein is a commercially significant method where tetramerization of PKM2 leads to inhibition of tumor while enhancing CD8 T cells. The VHPM Ref.09531.584WO1 / Client Ref.2022-294 development of the pharmacological agents that enhance the tetrameric-PKM2 have direct patient-centric implications. Further, the methods are used for ex vivo enhancement of effector functions and metabolic fitness in chimeric antigen receptor (CAR)-T cells. THERAPEUTIC AGENTS TEPP46 In certain embodiments, the therapeutic treating agent is TEPP46, which has the chemical name 6-[(3-Aminophenyl)methyl]-4,6-dihydro-4-methyl-2-(methylsulfinyl)-5H- thieno[2',3':4,5]pyrrolo[2,3-d]pyridazin-5-one. TEPP46 has the following chemical structure: DASA58 In certain embodiments, the therapeutic agent is DASA58, which is a specific and potent Pyruvate kinase M2 (PKM2) activator. DASA58 has the chemical name 3-[[4-[(2,3-dihydro- 1,4-benzodioxin-6-yl)sulfonyl]hexahydro-1H-1,4-diazepin-1-yl]sulfonyl]-benzenamine. DASA58 has the following chemical structure: VHPM Ref.09531.584WO1 / Client Ref.2022-294 Hyperproliferative Disorders Highly proliferative cancer cells that produce copious amounts of lactate because of their heightened cytoplasmic metabolism and diminished mitochondrial metabolism are targeted by TEPP46 or DASA58 because TEPP46 and decrease lactate production and divert glucose to mitochondrial metabolism. In certain aspects, the cancers are melanoma, colon carcinoma, ovarian cancer, non-small cell lung carcinoma, and renal cell carcinoma. In certain aspects, tumors with a high tendency to spread (metastasize) such as melanoma, colon cancer, ovarian cancer and gliomas benefit from this strategy. Melanoma Melanoma is a dangerous type of skin cancer that develops in cells that produce melanin (melanocytes), usually presenting as an irregular spot / mole on the skin. Causes of melanoma include UV radiation and a genetic predisposition to this type of cancer. Unlike other cancers, prevalence of melanoma is increasing, with the highest occurrence among individuals 25-29 years old. The overall lifetime risk of developing melanoma is 2.4%. In 2015, 73,870 new invasive melanomas are expected to be diagnosed, with 9,940 people expected to die of melanoma. With early treatment, survival rate is 97%. Melanoma can migrate to other parts of the body (metastatic melanoma), and one year survival rate drastically decreases with metastasis—15-20% for Stage IV. Current types of treatment include surgery, immunotherapy (Immune checkpoint inhibitors for advanced melanoma), chemotherapy, radiation therapy, targeted therapy (target cells with gene changes) and BRAF Inhibitors. BRAF is a protein kinase of the mitogen-activated protein kinase (MAPK) pathway, and it regulates cell growth, proliferation, and differentiation. Research suggests a BRAFV600Emutation causes the BRAF protein (produced through the MAPK pathway) to become oncogenic. The mutation may lead to increased and uncontrolled cell proliferation, and resistance to apoptosis. The BRAF mutation is observed in about 50% of melanoma tumors. Its presence is associated with poor prognosis in metastatic melanoma. Melanoma is the fastest growing cancer incidence in the United States. Surgery is curative for melanoma confined to the skin, but metastatic melanoma is lethal. Current FDA approved therapies for metastatic melanoma (e.g., Vemurafenib, Ipilimumab), have increased life expectancy by months, however resistance develops rapidly. The exact mechanism by which drug resistance develops is unclear; however, autophagy is known to play a major role. Autophagy is a self-degradative response of the cell towards nutrient stress. Conversely, autophagy also plays a housekeeping role by removing mis-folded or aggregated proteins and clearing damaged organelles by forming autophagosomes. Thus, autophagy is believed to play an important role in tumor progression and developing drug resistance during later stages of VHPM Ref.09531.584WO1 / Client Ref.2022-294 cancer. The Unfolded Protein Response (UPR) mediated by GRP78 ER associated protein degradation is one of the pathways that initiate autophagy in stressed cells. UPR involves the activation of three signaling pathways mediated by IRE-1, PERK and ATF6. These pathways work towards decreasing the protein load of ER by increasing the expression of molecular chaperons, activation of ERAD (ER associated protein degradation) and autophagy. However if the damage caused by the stress is extensive UPR signaling pathways initiate apoptosis. Amy S. Lee, Cancer Res (2007); 77:3496-3499. Emerging evidence shows that in malignant cells ER stress can be pro-survival and contribute to the development of drug resistance by initiating autophagy. In cerain embodiments, the melanoma is metastatic melanoma. In cerain embodiments, the melanoma is drug-resistant (e.g., vemurafenib-resistant) metastatic melanoma. Compositions and Methods of Administration In certain aspects, provided herein is a method of treating a hyperproliferative disorder in a patient in need thereof, comprising administering to the patient an agent that induces tetrameric pyruvate kinase isoform M2 (tet-PKM2). In certain aspects, the induction (i.e., activation) of tet-PKM2 enhances mitochondrial structural organization and metabolism in CD8 T cells, which increases antitumor functions of CD8 T cells. In certain aspects, the activation of tet-PKM2 suppresses growth of tumor cells, induces apoptosis and enhances immunogenicity leading to better recognition of the tumor cells by CD8 T cells. In certain aspects, the tet-PKM2 is TEPP46 or DASA58. In certain aspects, the tet- PKM2 is TEPP46. In certain aspects, the tet-PKM2 is DASA58. In certain aspects, the hyperproliferative disorder is cancer. In certain aspects, the cancer is a solid tumor. In certain aspects, the cancer is melanoma or colon cancer. In certain aspects, the cancer is melanoma. In certain aspects, the cancer is colon cancer. In certain aspects, the tet-PKM2 is administered orally or parenterally. In certain aspects, provided herein is a method of suppressing tumor growth in a patient comprising administering an agent that induces tetrameric pyruvate kinase isoform M2 (tet- PKM2) to the patient. In certain aspects, the tet-PKM2 is TEPP46 or DASA58. In certain aspects, the tet- PKM2 is TEPP46. In certain aspects, the tet-PKM2 is DASA58. In certain aspects, the hyperproliferative disorder is cancer. VHPM Ref.09531.584WO1 / Client Ref.2022-294 In certain aspects, the cancer is a solid tumor. In certain aspects, the cancer is melanoma or colon cancer. In certain aspects, the cancer is melanoma. In certain aspects, the cancer is colon cancer. In certain aspects, the tumor growth is suppressed by at least 10%. In certain aspects, the growth is suppressed by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% 90% or 100%. In certain aspects, provided herein is a method of inducing tetramerization of PKM2 in a cell comprising contacting the cell with TEPP46 or DASA58. In certain aspects, the tet-PKM2 is TEPP46. In certain aspects, the tet-PKM2 is DASA58. In certain aspects, the cells are CD8 T-cells. In certain aspects, provided herein are methods of treating a hyperproliferative disorder in a patient in need thereof, comprising administering a therapeutically effective amount of TEPP46 to the patient. The term "therapeutically effective amount" or "effective amount" is an amount sufficient to generate beneficial or desired clinical results. An effective amount can be administered in one or more administrations. An effective amount is typically sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state. The present invention provides a “substantially pure compound”. The term “substantially pure compound” is used herein to describe a molecule, such as a polypeptide (e.g., a polypeptide that binds MC1R, or a fragment thereof) that is substantially free of other proteins, lipids, carbohydrates, nucleic acids, and other biological materials with which it is naturally associated. For example, a substantially pure molecule, such as a polypeptide, can be at least 60%, by dry weight, the molecule of interest. The purity of the polypeptides can be determined using standard methods including, e.g., polyacrylamide gel electrophoresis (e.g., SDS-PAGE), column chromatography (e.g., high performance liquid chromatography (HPLC)), and amino-terminal amino acid sequence analysis. “Treatment”, “treating”, “treat” or “therapy” as used herein refers to administering, to a mammal, agents that are capable of eliciting a prophylactic, curative or other beneficial effect in the individual. Treatment may additionally result in attenuating or ameliorating a disease or symptoms of a disease in a subject. In certain embodiments, the conjugate is administered along with an additional conventional cancer therapy modality. In certain embodiments, the additional cancer therapy is chemotherapy and / or radiation. In certain embodiments, the agent that increases expression of VHPM Ref.09531.584WO1 / Client Ref.2022-294 MCR1 and an MCR1 ligand are administered sequentially to a mammal rather than in a single composition. In certain embodiments, the mammal is a human. In certain embodiments of the methods described above, agent that increases expression of MCR1 does not significantly inhibit viability of comparable non-cancerous cells. In certain embodiments of the methods described above, the tumor is reduced in volume by at least 10%. In certain embodiments, the tumor is reduced by any amount between 1-100%. In certain embodiments, the tumor uptake of molecular imaging agents, such as fluorine-18 deoxyglucose, fluorine-18 thymidine or other suitable molecular imaging agent, is reduced by any amount between 1-100%. In certain embodiments the imaging agent is fluorine-18 deoxyglucose, fluorine-18 thymidine or other suitable molecular imaging agent. In certain embodiments, the mammal’s symptoms (such as flushing, nausea, fever, or other maladies associated with cancerous disease) are alleviated. Administration of a compound as a pharmaceutically acceptable acid or base salt may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, α- ketoglutarate, and α-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts. Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made. The agent that increases expression of MCR1 and the MCR1 ligand can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes. Thus, the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of VHPM Ref.09531.584WO1 / Client Ref.2022-294 course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained. The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices. The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for VHPM Ref.09531.584WO1 / Client Ref.2022-294 example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions. For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids. However, it may be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid. Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol / glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers. Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user. Examples of useful dermatological compositions which can be used to deliver the compounds of the present invention to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No.4,608,392), Geria (U.S. Pat. No.4,992,478), Smith et al. (U.S. Pat. No.4,559,157) and Wortzman (U.S. Pat. No.4,820,508). The dosage of the agent that increases expression of MCR1 and the MCR1 ligand varies depending on age, weight, and condition of the subject. Treatment may be initiated with small dosages containing less than optimal doses, and increased until a desired, or even an optimal effect under the circumstances, is reached. In general, the dosage is about 450 – 600 mg / kg / day in patients weighing less than 20 kg, or 9.9 – 13.0 g / m² / day in larger patients. Higher or lower doses, however, are also contemplated and are, therefore, within the confines of this invention. VHPM Ref.09531.584WO1 / Client Ref.2022-294 A medical practitioner may prescribe a small dose and observe the effect on the subject's symptoms. Thereafter, he / she may increase the dose if suitable. In general, agent that increases expression of MCR1 and the MCR1 ligand are administered at a concentration that affords effective results without causing any unduly harmful or deleterious side effects, and may be administered either as a single unit dose, or if desired in convenient subunits administered at suitable times. A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. For example, the therapeutic agent may be introduced directly into the cancer of interest via direct injection. Additionally, examples of routes of administration include oral, parenteral, e.g., intravenous, slow infusion, intradermal, subcutaneous, oral (e.g., ingestion or inhalation), transdermal (topical), transmucosal, and rectal administration. Such compositions typically comprise the agent that increases expression of MCR1 and the MCR1 ligand and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration, and a dietary food-based form. The use of such media and agents for pharmaceutically active substances is well known in the art and food as a vehicle for administration is well known in the art. Solutions or suspensions can include the following components: a sterile diluent such as water for injection, saline solution (e.g., phosphate buffered saline (PBS)), fixed oils, a polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), glycerine, or other synthetic solvents; antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Prolonged administration of the injectable compositions can be brought about by including an agent that delays absorption. Such agents include, for example, aluminum monostearate and gelatin. The parenteral preparation can be enclosed in ampules, disposable syringes, or multiple dose vials made of glass or plastic. It may be advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically VHPM Ref.09531.584WO1 / Client Ref.2022-294 discrete units suited as unitary dosages for an individual to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The dosage unit forms of the invention are dependent upon the amount of a compound necessary to produce the desired effect(s). The amount of a compound necessary can be formulated in a single dose, or can be formulated in multiple dosage units. Treatment may require a one-time dose, or may require repeated doses. “Systemic delivery,” as used herein, refers to delivery of an agent or composition that leads to a broad biodistribution of an active agent within an organism. Some techniques of administration can lead to the systemic delivery of certain agents, but not others. Systemic delivery means that a useful, preferably therapeutic, amount of an agent is exposed to most parts of the body. To obtain broad biodistribution generally requires a blood lifetime such that the agent is not rapidly degraded or cleared (such as by first pass organs (liver, lung, etc.) or by rapid, nonspecific cell binding) before reaching a disease site distal to the site of administration. Systemic delivery of lipid particles can be by any means known in the art including, for example, intravenous, subcutaneous, and intraperitoneal. In a preferred embodiment, systemic delivery of lipid particles is by intravenous delivery. “Local delivery,” as used herein, refers to delivery of an active agent directly to a target site within an organism. For example, an agent can be locally delivered by direct injection into a disease site, other target site, or a target organ such as the skin. The term “mammal” refers to any mammalian species such as a human, mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock, and the like. The terms "treat" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or decrease an undesired physiological change or disorder, such as the development or spread of cancer. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented. The invention has been described as “comprising” certain steps and / or elements, which those of skill in the art also “consist of” or “consist essentially of” those steps and / or elements. As used herein, the transitional term “comprising” is synonymous with “including,” VHPM Ref.09531.584WO1 / Client Ref.2022-294 “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. Where the invention is intended to be more narrowly defined, the terms “consisting of” or “consisting essentially of” also are used to describe the invention. As used herein, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified elements or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. As used herein, a claim reciting “consisting essentially of” occupies a middle ground between closed claims reciting a “consisting of” format and fully open claims that recite “comprising.” The invention will now be illustrated by the following non-limiting Examples. EXAMPLE 1 PKM2 mediated mitochondrial reprogramming supports enhanced effector functions in CD8 T cells increasing the anti-tumor efficacy of anti-PD1 therapy Abstract Mitochondria regulate T cell functions and response to cancer immunotherapy. We show that pharmacologically induced PKM2 activation enhances mitochondria-dependent effector functions in mouse and human CD8 T cells and CAR-T cells. Multi-omics analysis and13C-glucose tracer experiments showed that PKM2 activation alters one-carbon metabolism with decreased levels of methionine leading to hypomethylation of mitochondrial and nuclear DNA and enhanced expression of genes supporting mitochondrial biogenesis and respiratory complex formation in CD8 T cells. Functionally, PKM2 activation increased the recall responses and anti-tumor functions of CD8 T cells after adoptive cell therapy. Using preclinical melanoma and colon cancer models, we found that PKM2-agonist induced CD8 T cell-dependent anti-tumor responses that synergized with PD1 blockade therapy. Immunologically, PKM2 agonists resulted in enhanced activation and decreased exhaustion of effector T cells while reducing the numbers of suppressive FoxP3+Treg cells in the tumor microenvironment. Anti-PD1 combination further enhanced the frequency and activation of tumor specific CD8 T cells in the tumors. Together, we show that PKM2 agonism modulates mitochondrial structure and function, leading to enhanced effector functions of cytotoxic lymphocytes. Hence, targeting PKM2 via pharmacologically available small molecules can be a clinically viable strategy for the enhancement of therapeutic potential of in-situ immune responses, adoptive cell therapy, and immune checkpoint blockade therapy. VHPM Ref.09531.584WO1 / Client Ref.2022-294 Introduction Immunotherapy has markedly transformed the landscape of cancer treatment, with a significant number of patients experiencing durable anti-tumor effects. Nonetheless, many patients, especially with solid tumors, do not respond adequately to immunotherapy. The microenvironment of solid tumors is complex and has a profound effect on immune cell activation and function. Signals generated within the tumor microenvironment (TME) can induce exhaustion in immune cells in part by depleting their nutrition and suppressing their mitochondrial function. Mitochondria are independent organelles that play a crucial role in energy generation and signaling within cells. Mitochondria are involved in multiple roles including oxidative phosphorylation (OXPHOS), calcium buffering, generation of reactive oxygen species, regulation of metabolism, transcription in T cells, and homeostatic regulation of cytotoxicity. Most of these processes are influenced by the optimum structural organization of mitochondria as mitochondrial dynamics have been shown to control T cell functionality through metabolic reprogramming. Hence, restructuring mitochondria is important for development of appropriate cell activation and immune responses. Mitochondrial dynamics are also critical for the transformation of T cells from naïve to effector phase. Naïve non- activated cells contain small mitochondria that are sufficient to support cell energetics via basal OXPHOS. Upon antigen encounter, cells upregulate their effector functions that require substantial metabolic rearrangement, lack of which blocks cell activation and drives the cells towards an exhausted phenotype. Hence, development of strategies that can improve the mitochondrial architecture and metabolism are of paramount importance. Pyruvate kinase isoform M2 (PKM2) is an enzyme that catalyzes the last step of cytoplasmic glycolysis, facilitating the production of pyruvate from phosphoenol pyruvate. PKM2 exists in two isomeric forms: the transcriptionally active dimeric form (di-PKM2) and enzymatically active tetrameric form (tet-PKM2). PKM2 has both gene regulatory and metabolic effects. In tumor cells, di-PKM2 enhances growth and survival by modulating the HIF1α / mTORC1 / AKT pathway. On the contrary, tet-PKM2 suppresses tumor cell growth by promoting the flux of glucose-derived carbon into mitochondrial OXPHOS thus limiting the cytoplasmic aerobic glycolysis. In addition to its tumor associated functions, a limited number of studies have shown the immune modulatory effects of PKM2. Pharmacological stabilization of tet-PKM2 has been shown to inhibit proliferation and activation of CD4 T cells. In addition, tet-PKM2 also suppressed the generation of Th17 cells. In macrophages, tet-PKM2 prevents lipopolysaccharide (LPS) induced cell activation, preventing autoimmune disorders in pre-clinical models. However, the immune modulatory effects of PKM2 in CD8 T cells have not been well studied. In a recent study, CD28 driven upregulation of arsenic resistance protein 2 (ARS2) has been found to enhance effector functions in CD8 T cells by alternate splicing of PKM2, hinting towards immune enhancing effects of PKM2 in CD8 T cells. VHPM Ref.09531.584WO1 / Client Ref.2022-294 Here, we have deciphered the primary roles of PKM2 in CD8 T cells. We show that pharmacological activation of PKM2 is associated with enhanced effector functions in CD8 T cells that are critically dependent on mitochondrial organization and metabolism. We show that PKM2 activation alters the cell transcriptome and metabolome which is supportive of enhanced effector functions in CD8 T cells. Interestingly, PKM2 activation shunts glucose derived Carbon atoms into pentose phosphate pathway and alters one-Carbon metabolism with a significant decline in methionine levels which leads to decreased methylation of cellular DNA. Whole genome bisulfite sequencing (WGBS) revealed mitochondrial DNA to be most differentially methylated in the whole genome with an increased expression of genes associated with mitochondrial biogenesis and respiratory complex formation. This was accompanied by enhanced mitochondrial translation, increased cristae organization, and increased mitochondria: endoplasmic reticulum (ER) interaction. Importantly, enhancement of effector functions in CD8 T cells after PKM2 activation was regulated by mitochondrial translation as an inhibition of translation prevented the induction of IFNγ and TNFα. Finally, we report that CD8 T cells with activated PKM2 have higher antigen recall response and stronger anti-tumor activity after adoptive cell therapy. Moreover, in-situ PKM2 activation by pharmacological agonist TEPP46 in tumor bearing mice resulted in CD8 T cell dependent anti- tumor activity that synergized well with anti-PD1 based immunotherapy. Thus, this study demonstrates hitherto unexplored roles of PKM2 in CD8 T cell activation and cancer immunotherapy enhancement. Results PKM2 agonist induces CD8 T cell dependent anti-tumor immunity Pyruvate kinase M2 (PKM2) activation using pharmacological agonists has been shown to suppress tumor cell growth. However, its impact on tumor immune microenvironment remains unexplored. To address this, we established B16-F10 tumors in mice followed by TEPP46 treatment for a total of 10 doses (50 mg / Kg; once per day; 4 days-on two-days off). In accordance with previous reports, we found that TEPP46 treatment significantly suppressed tumor growth and enhanced mice survival (Figure 1A). In the untreated group, 100% of the mice succumbed to the growing tumor by day 20, while 50% of the mice were alive after TEPP46 treatment at day 30 post- tumor inoculation (Figure 1A). We also tested the anti-tumor effects of TEPP46 on growth profiles of subcutaneously inoculated CT26-colon cancer cells in mice. Reflecting the data obtained from melanoma model, TEPP46 treatment of CT26 bearing mice led to a significant inhibition of tumor growth and enhancement of mice survival as 40% of TEPP46 treated mice were still alive by day 35 while all mice in untreated group succumbed to growing tumor by day 20 (Figure 1B). No significant difference in body weight was observed between untreated and TEPP46 treated mice in both B16-F10 and CT26 tumor bearing mice (Figure 8A). VHPM Ref.09531.584WO1 / Client Ref.2022-294 Next, we inquired if TEPP46 demonstrated immune modulatory effects in tumors in mice. For this, B16-F10 melanoma tumors from TEPP46 treated and untreated mice were probed for lymphocytic cell populations. We found a significant increase in the fractions of CD3+T cells in the tumors of TEPP46 treated mice (Figure 8B). Within lymphocytic compartment, we found a significant increase in the percentage of total CD8 T cells (Figure 1C) and CD4 T cells (Figure 8C) in the TME of TEPP46 treated mice. Within the CD8 T cell compartment, the frequency of tumor specific gp100-dextramer+CD8 T cells (Figure 1D) were significantly increased in the tumors of TEPP46 treated mice. Gp100 is a tumor intrinsic antigen expressed on the surface of B16-F10 cells; an increase in the number of gp100 specific CD8 T cells indicates de novo generation of antigen specific cytotoxic cell responses. Together, these data show that TEPP46 treatment enhances the infiltration and expansion of CD8 T cells in the TME that was coupled with increased tendency of CD4 T cell infiltration in the TME. Next, we set out to establish the activation and exhaustion status of T cells in tumors of TEPP46 treated mice. CD8 T cells in the tumors of TEPP46 treated mice showed a significant increase in the production of IFNγ and Granzyme (Gzm) B (Figures 1E, 1F), denoting robust effector functions. Along with enhanced effector cytokines, the expression level of exhaustion markers PD1 and LAG3 was significantly downregulated on CD8 T cells in the tumors of TEPP46 treated mice (Figures 1G, 1H). In contrast to CD8 T cells, the levels of IFNγ and TNFα in CD4 T cells were unaltered between TEPP46 treated and untreated mice (Figures 8D, 8E) though expression of PD1 and LAG3 was significantly downregulated in CD4 T cells from tumors of TEPP46 treated mice (Figures 8F, 8G). Within the CD4 lymphocytes, FoxP3+cells represent an immune regulatory cell population that suppresses the functions of other effector cells. TEPP46 treatment has been shown to prevent the induction of a regulatory phenotype in CD4 T cells in vitro. In line with these findings, our in vivo data showed a significant decrease in the frequencies of FoxP3+CD4+T regulatory cells in tumors of TEPP46 treated mice (Figure 8H). Together, these data show that TEPP46 treatment enhances the frequencies, and effector functions and decreases exhaustion of CD8 T cells. While within CD4 T cell population, TEPP46 treatment does not seem to enhance the secretion of effector cytokines, but the treatment decreases cell exhaustion and reduces frequency of suppressive Treg cells. Since we saw an increase in the effector functions in CD8 T cells, next we established the role of these cells in TEPP46 mediated anti-tumor effects. For this, using specific antibodies we depleted CD8 T cells in a group of B16-F10 tumor bearing mice followed by TEPP46 treatment (Figure 8I). In accordance with their increased activation and effector functions in the tumors of TEPP46 treated mice, we observed a partial though significant decline in TEPP46 mediated anti- tumor effects after CD8 T cell depletion (Figure 1I). Hence these data show that CD8 T cells play a significant role in TEPP46 induced antitumor responses. However, given the effect of PKM2 VHPM Ref.09531.584WO1 / Client Ref.2022-294 agonism on other immune cell populations including natural killer cells and dendritic cells, it is possible that these cells may also be contributing to the anti-tumor effects observed after TEPP46 treatment of tumor bearing mice. Nonetheless, these data clearly demonstrate that TEPP46 treatment leads to enhancement of CD8 T cell activation that contributes significantly to observed TEPP46 mediated anti-tumor activity. These findings were also confirmed in the CT26 tumor model. In agreement with B16-F10 tumors, TEPP46 treatment of CT26 tumor bearing mice led to an increase in the frequency of total CD8 T cells (Figure 1J) as well as IFNγ and TNFα secreting CD8 T cells (Figures 1K, 1L). Moreover, in line with B16-F10 tumors, we found a downregulation in the frequencies of PD1+and LAG3+CD8 and CD4 T cells (Figures 8J, 8K). Interestingly, expression levels of TIGIT remained unaltered in CD8 and CD4 T cells in both B16-F10 and CT26 tumors after TEPP46 treatment (Figures 8L, 8M). Together these data show that TEPP46 treatment resulted in robust cell activation, increased effector functions and decreased exhaustion of CD8 T cells in the TME while having minimal effects on activation in CD4 T cells. Hence, these data show that effects were primarily mediated through CD8 T cell modulation as TEPP46 enhanced activation and reduced exhaustion in effector T cells in the TME. PKM2 activation alters cell transcriptome augmenting effector functions in CD8 T cells Our in vivo data demonstrated that PKM2 agonist inhibited the tumor growth in a CD8 T cell dependent manner. Next, we set out to define the effects of PKM2 activation and its role in anti- tumor functions of CD8 T cells. As expected, PKM2 agonist (TEPP46) significantly increased the levels of enzymatically active tetrameric (tet)-PKM2 while decreased the levels of transcriptionally active dimeric (di)-PKM2 in OT1-CD8 T cells (Figure 2A). Hence, we next checked the transcriptional profiles of CD8 T cells after activation with Ova + / - TEPP46 for 24 and 48 hours (Figure 9A). Principal Component Analysis (PCA) of cell transcriptome showed a distinct clustering pattern in TEPP46 treated CD8 T cells compared to Ova-alone activated cells at 48 hours post- activation (Figure 2B). Fewer changes were observed between TEPP46 treated and Ova-alone treated CD8 T cells (Figure 2B) at 24 hours post-activation. Comparing the gene expression profiles between Ova and Ova+TEPP46 treated cells at 24 and 48 hours showed that at 24 hours a total of 386 genes were differentially expressed between two groups (Ova vs Ova+TEPP46) while at 48 hours, the two groups showed a total of 1325 differentially expressed genes (Figure 2C). Of these 1325 genes, 1144 genes were altered uniquely at 48 hours between Ova and Ova+TEPP46 treated cells, while 181 genes exhibited common changes in their expression between two time points (Figure 2C). We focused on differentially expressed genes and found that out of the 1144 differentially regulated genes between TEPP46 treated and untreated CD8 T cells at 48 hours, 415 genes were upregulated while 729 genes were downregulated (Figure 9B). Upon mapping the upregulated genes into various pathways, proteasome, spliceosome, and RNA-polymerase pathways VHPM Ref.09531.584WO1 / Client Ref.2022-294 were found to be most prominently upregulated in TEPP46 treated CD8 T cells (Figure 2D). Interestingly, these pathways have been shown to be associated with enhanced effector functions, metabolism, and fate decisions. In fact, spliceosome mediated alternate splicing of RNA is associated with upregulation of many transcriptional pathways including JAK-STAT, mTOR, and NF-kB that contribute to effector cytokine production (IFN, TNF, and IL2) and clonal expansion. In line with these observations, we found an enrichment in genes associated with effector functions and cell activation (Ifng, Tnf, Jun, Il2ra and Gzmb) in TEPP46 treated CD8 T cells at 48 hours post- incubation (Figure 2E). The transcriptional profiles were validated by qRTPCR where we found increased transcripts in TEPP46 treated CD8 T cells for IFNγ, TNFα and GzmB (Figure 9C). We also confirmed the secretion of these cytokines at protein level by FACS analysis where Ova+TEPP46 treated CD8 T cells were found to produce significantly higher levels of IFNγ, TNFα and Gzm B compared to Ova alone treated cells (Figure 2F), which was in line with the enhanced effector functions observed in CD8 T cells in vivo (Figures 1E,1F). These data demonstrate that TEPP46 mediated PKM2 activation in CD8 T cells enhances their effector functions both in vivo and in vitro. Next, we checked the state of CD8 T cell differentiation after PKM2 activation by TEPP46 by estimating the expression of CD62L and CD44. TEPP46 treatment significantly increased the numbers of CD62L-CD44+effector memory (TEM) CD8 T cells (Figures 2G, 2H) with an corresponding decrease in the numbers of CD62L+CD44+central memory (TCM) cells (Figure 9D). The levels of IFNγ and TNFα in TEM(Figure 2I) and TCM(Figure 9E) of TEPP46 treated cells remained significantly higher than the respective populations of Ova-alone treated CD8 T cells. Next, we asked if TEPP46 mediated PKM2 activation induces KLRG+CD127- short lived effector cells (SLECs) that represent an exhausted phenotype or KLRG1-CD127+memory precursor effector cells (MPECs) that tend to be long-lived. We found a significant increase in long-lived KLRG1- CD127+MPEC population while a reduction in KLRG+CD127- SLEC population in CD8 T cells (Figure 9F). These data show that TEPP46 treatment modulated the transcriptional and translational profiles of CD8 T cells supporting the cell activation and induction of long-lived memory state. TCR mediated cell activation is closely associated with proliferation of effector T cells. We next measured the effects of TEPP46 treatment on CD8 T cell proliferation using violet cell trace (VCT) dilution in activated CD8 T cells. After 48 hours of cell activation, TEPP46 treated CD8 T cells proliferated robustly and showed at least 3 peaks of cell divisions (VCT1, 2 and 3) though compared to Ova-alone treated cells, proliferating cells in TEPP46 treatment showed a significant accumulation in earlier peaks (VCT1 and VCT2) (Figures 9G, 9H) indicating a delay in cell division. To ascertain if delayed cell division affected the effector functions in these cells, we measured the production of effector cytokines in individual peaks of cell division and found higher VHPM Ref.09531.584WO1 / Client Ref.2022-294 levels of IFNγ and TNFα within each peak of proliferation in TEPP46 treated CD8 T cells (Figures 9I, 9J). Thus, these observations indicate that though TEPP46 treatment delays cell proliferation but does not disrupt the ability to activate cells after TCR engagement supporting robust activation and effector cytokine production by CD8 T cells. Next, we tested if TEPP46 induced PKM2 activation can enhance effector function in already activated CD8 T cells. For this OT1-CD8 T cells were activated for 24 hours with Ova peptide followed by TEPP46 treatment (Figure 10A). Under these conditions we did not see any enhancement of effector functions as the fraction of IFNγ+, TNFα+and Gzm B+CD8 T cells remained similar in TEPP46 treated and untreated cell cultures (Figure 10B) showing pharmacological PKM2 activation enhances effector functions in CD8 T cells during TCR engagement. In addition to its effects on OT1-CD8 T cells, we confirmed the effects of TEPP46 treatment on the activation profile of CD8 T cells isolated from pMEL and wild type (WT) mice. pMEL-CD8 T cells were activated using cognate gp100 peptide, while WT CD8 T cells were activated with anti- CD3 / 28 antibody coated beads in the presence or absence of TEPP46. Similar to OT1-CD8 T cells, expression levels of IFNγ and TNFα in WT (Figure 10C) and pMEL-CD8 T cells (Figure 10D) at 48 hours post-incubation remained significantly higher in TEPP46 treated group compared to the activation-alone group. Thus, TEPP46 enhances effector functions in CD8 T cells from diverse backgrounds and with varied antigenic specificity. We also studied the effects of TEPP46 treatment on activation potential of CD4 T cells. Consistent with earlier observations, we found a significant decrease in proliferation (VCT) and effector functions (IFNγ and TNFα) in CD4 T cells that were activated in the presence of TEPP46 (Figures 10E-10G). These observations are also consistent with our in vivo data where we did not see an enhancement of effector functions in CD4 T cells in the tumors of TEPP46 treated mice (Figures 8D, 8E). We also checked the induction of effector functions in OT1-CD8 T cells using DASA58, another agonist of PKM2. In line with TEPP46, we saw an increase in the production of IFNγ and TNFα (Figure 10H) and an increase in the numbers of CD62L-CD44+TEMCD8 T cells (Figure 10I) after their activation in the presence of DASA58. Finally, we checked the effects of PKM2 activation on human CD8 T cells. For this, purified CD8 T cells from healthy donors were activated using anti-CD3 / CD28 antibodies for 72 hours with TEPP46 or DASA58. PKM2 activation in human CD8 T cells was found to significantly increase the production of IFNγ and TNFα (Figure 2J and Figure 10J) and CD44+CD45RA- cells representing the effector T cell phenotype (Figure 2K and Figure 10K). Together, these data show that PKM2 activation influences global gene expression favoring the activation and effector functions in CD8 T cells of both murine and human origine. These data also indicate the universality of TEPP46 mediated enhancement of effector functions in cytotoxic CD8 T cells. VHPM Ref.09531.584WO1 / Client Ref.2022-294 PKM2 activation facilitates mitochondrial generation and structural reorganization Mitochondria play a crucial role in regulating T cell adaptive immune responses by supplying ATP and other signaling molecules. Accordingly, accumulation of damaged mitochondria results in decreased adaptive immune responses to cancer and infections. Since we found increased effector functions in CD8 T cells with activated PKM2, we next inquired about mitochondrial characteristics including its generation and structural organization. Mitochondrial biogenesis involves a complex coordination of both nuclear and mitochondria associated genes. The RNA-seq analysis revealed a gene expression pattern indicative of mitochondrial biogenesis with a strong upregulation in the expression of Tfam, Tfb1m, and Tfb2m, Pprc1 and Ppargc1 genes (Figure 3A). The expression of PPARG coactivator 1 alpha (PGC-1α), the nuclear DNA encoded gene required for mitochondrial biogenesis was strongly upregulated both at mRNA and protein levels in TEPP46 treated CD8 T cells (Figures 3B, 3C) indicating a greater mitochondrial biogenesis. We next measured key mitochondrial characteristics including mass, ATP production and membrane associated potential which are indicators of enhanced cell functions. We observed an increased mitochondrial biomass, estimated by MitoFM incorporation in CD8 T cells after TEPP46 (Figure 3D) or DASA58 treatment in vitro (Figure 11A) or TEPP46 treatment in vivo in B16-F10 and CT26 tumors bearing mice (Figure 11B). Human CD8 T cells also showed an increase in MitoFM incorporation after PKM2 activation (Figure 11C). We also found an increase in the membrane potential and mitochondrial ATP production in TEPP46 treated CD8 T cells (Figures 3E, 3F). These data show that TEPP46 treatment upregulated mitochondrial biogenesis, ATP production and membrane potential, characteristics associated with mitochondria-supported activation in immune cells. Mitochondria utilize oxidizable substrates to produce an electrochemical proton gradient across the mitochondrial membrane that is used for ATP production. Mitochondrial OXPHOS is not completely coupled, and 0.2-2% of electrons leak out of the electron transport chain (ETC) and interact with oxygen to produce superoxide and hydrogen peroxide, collectively called reactive oxygen species (ROS). Though ROS is required for optimal functioning of the cells, when the production is high, lipid peroxidation, DNA damage, protein oxidation, and damage to mitochondria are increased. Since we observed a very significant increase in mitochondrial membrane potential (Figure 3E), next we quantified ROS levels in TEPP46 treated CD8 T cells. Surprisingly, despite high mitochondrial potential, the ROS levels in TEPP46 treated CD8 T cells remained significantly below those in Ova-activated control cells (Figure 3G), indicating tightly organized mitochondria that do not permit electron leak. Hence, we next ascertained the mitochondrial morphology and organization in CD8 T cells using electron microscopy (Figure 3H). TEPP46 treated CD8 T cells had significantly higher numbers of mitochondria (Figures 3H, 3I) with a strong trend for increased VHPM Ref.09531.584WO1 / Client Ref.2022-294 mitochondrial area (Figure 11D), a significant increase in the numbers of cristae per mitochondria, and an increase in cristae density compared to Ova-alone treated cells (Figures 3H, 3I). Electron microscopy of TEPP46 treated CD8 T cells also revealed greater mitochondrion: mitochondria (Figure 3H; arrows) interaction indicating that indeed mitochondria undergo structural reorganization after TEPP46 treatment. Additionally, we observed increase in mitochondria endoplasmic reticulum contact sites (MERS) after TEPP46 treatment of CD8 T cells (Figure 3J) which are associated with increased effector functions and metabolic fitness in cytotoxic T cells. Extending the observations of increased cristae numbers and density, we measured the expression of mitochondrial contact site and cristae organizing system (MICOS), which plays a critical role in regulating the formation of cristae junction and mitochondrial organization. We observed a significant increase in the expression of MICOS complex genes Mic60(Immt) / Mic23, Apool / Mic19 / Mic10(Minos1) in TEPP46 treated CD8 T cells (Figure 11E). In addition, expression of genes associated with dynamic regulation of mitochondrial architecture such as mfn1, mfn2, and opa1 was significantly increased in CD8 T cells after TEPP46 treatment (Figure 11F). Taken together, these data show that TEPP treatment leads to increased mitochondrial biogenesis, enhanced structural organization and improved functional characteristics in TEPP46 treated CD8 T cells. Activated PKM2 reduces DNA methylation and enhances mitochondrial translation supporting effector functions in CD8 T cells Mitochondrial biogenesis and structural organization require a coordinated expression of mitochondria- and nuclear-encoded genes to generate respiratory complexes. Epigenetic regulation via CpG methylation is an important mechanism of regulating the gene expression that helps cells to react on acute basis to external stimuli such as TCR mediated activation. Hence, using whole genome bisulfite sequencing (WGBS) we looked at the methylation profiles of Ova + / - TEPP46 treated CD8 T cells. PCA analysis showed a distinct clustering pattern between Ova and Ova+TEPP46 samples (Figure 4A) that resulted in >2000 differentially methylated regions (DMRs) between the two cell types (Figure 12A). Mapping DMRs onto chromosomes showed that the mitochondrial chromosome (ChrM) was most differentially methylated in Ova+TEPP46 treated CD8 T cells (Figure 12B) with a significantly reduced methylation of mtDNA in Ova+TEPP46 treated CD8 T cells compared to Ova-alone treated cells (Figure 4B). Specifically, the CpG islands in the promoter region (1000 bp before and 50 bp after transcription start site) of mitochondria encoded genes were differentially hypomethylated in TEPP46 treated CD8 T cells (Figure 4C) indicating an upregulated expression of mitochondrial genes. Indeed, a Gene Set Enrichment Analysis (GSEA) of transcriptome showed a significant upregulation of mitochondria related pathways including mitochondrial translation, mitochondrial ribosomal subunits and respiratory VHPM Ref.09531.584WO1 / Client Ref.2022-294 chain complex assembly in TEPP46 treated CD8 T cells (Figure 12C). Accordingly, we found an increased expression of mitochondrial ribosomal subunit (mrpl) genes (Figure 12D) and mitochondria complex related genes (Figure 4D) in TEPP46 treated CD8 T cells. These data indicate that in addition to cytoplasmic translation, mitochondrial translation contributes to the enhanced mitochondrial functions seen in TEPP46 treated CD8 T cells. We next established the status of total cellular and mitochondrial translation by puromycin incorporation. We observed an increased puromycin incorporation in total cellular and purified mitochondrial proteins in TEPP46 treated CD8 T cells compared to control Ova treated cells (Figure 4E,F). Inhibition of mitochondrial protein translation by doxycycline reduced cellular puromycin incorporation in both TEPP46 treated and Ova-alone treated CD8 T cells though the level of reduction was greater in TEPP46 treated cells than in Ova treated cells (Figure 4E and Figure 12E). On the other hand, doxycycline treatment significantly reduced the mitochondrial translation by 3- fold in TEPP46 treated CD8 T cells, however the reduction was marginal in Ova-alone treated cells (Figure 4F and Figure 12F), indicating that TEPP46 differentially enhances the mitochondrial translation in CD8 T cells. We measured the expression of proteins associated with individual mitochondrial respiratory complexes (NDUFB9, SDHB, UQRC2, Mt-Cox1, ATP5A1) in TEPP46 treated CD8 T cells. Consistent with decreased methylation of mtDNA, increased expression of mitochondrial-complex related genes, and increased puromycin incorporation, we observed an increased expression of mitochondrial proteins associated with respiratory complexes in TEPP46 treated CD8 T cells with most profound upregulation in NDUFB9, SDHB, UQRC2, and ATP5A1 (Figure 4G and Figure 12G). In the same experiment, we also studied the effect of inhibiting mitochondrial translation by doxycycline on different respiratory complex proteins in TEPP46 treated CD8 T cells. The most drastic decrease was observed in SDHB (Complex II) and UQRC2 (Complex III) proteins (Figure 4G and Figures 12G, 12H). NDUFB9 (Complex I), Cox1 (Complex IV) and ATP5A1 (ATP synthase subunit) expression was inhibited in both TEPP46 and control CD8 T cells after doxycycline treatment (Figure 4G and Figuress 12G, 12H); the extent of inhibition was more pronounced in TEPP46 treated samples (Figure 12H). These data show that TEPP46 treatment leads to enhanced translation of proteins associated with various respiratory complexes in mitochondria in CD8 T cells. We next asked if TEPP46 mediated increased mitochondrial translation was responsible for enhanced effector functions observed in TEPP46 treated CD8 T cells (Figures 1E, 1F and 2F). Activating CD8 T cells in the presence of TEPP46 and the mitochondrial translation inhibitor doxycycline significantly abrogated IFNγ and TNFα production (Figure 4H), strongly suggesting that TEPP46 mediated increased mitochondrial translation is crucial for the generation of effector functions in CD8 T cells. We also confirmed the involvement of various respiratory complexes in VHPM Ref.09531.584WO1 / Client Ref.2022-294 effector functions in TEPP46-treated CD8 T cells by inhibiting the respiratory complexes using specific inhibitors followed by estimation of IFNγ and TNFα production. Consistent with a significantly increased expression of NDUFB9 and SDHB, inhibition of complex I and II using rotenone and TTFA respectively significantly reduced the secretion of IFNγ in TEPP46 treated CD8 T cells compared to Ova activated cells (Figure 4I). Interestingly, Ova-alone activated cells upon treatment with doxycycline or TTFA showed a significant upregulation of IFNγ production that could be because of stress induced cytokine production in CD8 T cells. On the other hand, production of TNFα was significantly reduced by rotenone in TEPP46 treated and untreated cells, though the reduction was far stronger in TEPP46 treated CD8 T cells compared to control CD8 T cells (Figure 4J). Interestingly TTFA treatment of Ova treated cells led to a marginal increase in IFNγ an TNFα production in Ova alone cells (Figures 4I, 4J), which could be due to stress response to CII blockade in these cells. Together, these data show that in TEPP46 treated CD8 T cells CI and CII significantly contributed to enhancement of effector function. Moreover, CD8 T cell treatment with antimycin inhibiting CIII resulted in equal inhibition of IFNγ an TNFα in both, TEPP46 treated and control CD8 T cells (Figures 12I, 12J). On the other hand, inhibition of ATP synthase by oligomycin reduced effector cytokines more significantly in TEPP46 treated CD8 T cells indicating greater reliance of these cells on mitochondrial ATP generation (Figures 12I, 12J). Taken together, these data show that TEPP46 prevents methylation of mitochondrial DNA, resulting in enhanced translation of mitochondrial proteins. These data also show that TEPP46 treatment increases translation of mitochondrial respiratory complexes that supports enhanced effector functions in in CD8 T cells. Activated PKM2 alters cell metabolism supporting pentose phosphate pathway and one-carbon pathway in CD8 T cells Metabolic status of immune cells is a critical regulator of their effector functions. Next, to have a comprehensive view of the metabolic status, we performed a targeted metabolome analysis of OT1-CD8 cells that were activated with Ova peptide in the presence or absence of TEPP46. Consistent with the gene expression data (Figure 2B), PCA analysis of whole intracellular targeted metabolome showed a distinct clustering pattern in TEPP46 treated CD8 T cells compared to Ova- alone activated cells at 48 hours post-activation (Figure 5A) while only fewer changes were observed between the two cell types at 24 hours post-activation (Figure 5A and Figure 13A). Using this method, we identified 98 metabolites significantly enriched (p < 0.05) in TEPP46 treated CD8 T cells while 515 metabolites enriched in control CD8 T cells (FDR <0.1) (Figure 5B). Pathway analysis of enriched metabolites in TEPP46 treated CD8 T cells revealed differential regulation of several pathways including glycolysis, TCA cycle, pentose phosphate pathway and one-Carbon metabolism pathway (Figure 5C). We observed a decrease in metabolites within the glycolytic VHPM Ref.09531.584WO1 / Client Ref.2022-294 pathway including diphosphoglycerates and lactate, while dihydroxyacetone and fructose 1,6- biphosphate (F-1,6BP) were found to be upregulated (Figure 5C and Figure 13B). In TCA cycle, we observed an increase in the levels of citrate and isocitrate while fumarate, oxaloacetate, and malate were found to be decreased in TEPP46 treated CD8 T cells (Figure 5C). In line with enhanced nucleotide requirement of activated effector cells the pentose phosphate pathway (PPP) with ribose 5-phosphate (R5P), ribulose 5-phosphate, phosphogluconate and F,1,6-BP was found to be upregulated in CD8 T cells activated in the presence of TEPP46 (Figures 5C, 5D). Reflecting the metabolomics results, tracer experiment using13C-glucose showed a similar pattern of cellular metabolism with an upregulation of F-1,6BP, 6-phosphoglucolactone (6PGL), R5P, and glyceraldehyde 3-phosphate (G3P) (Figures 5E-H), components of the PPP pathway involved in carbon homeostasis, generation of metabolic intermediates for nucleotide synthesis and cell anabolism. These data show that TEPP46 treatment has system wide effects on CD8 T cell metabolism with a preferential increase in PPP pathway. One-Carbon metabolism plays a crucial role in regulating CD8 T cells functions, especially by altering the methionine mediated DNA methylation status. Since, we observed a prominent change in DNA methylation in TEPP46 treated CD8 T cells, next we assessed the status of one-Carbon metabolism including methionine cycle where one-carbon units from the folate cycle are used to methylate homocysteine to form methionine (Figure 5I). We found a prominent alteration in the components of methionine metabolism cycle, largely due to increased levels of homocysteine and folate, and decreased levels of methionine, serine, glycine, betaine, and dimethylglycine in TEPP46 treated CD8 T cells (Figure 5J). We observed a two- fold decrease in the levels of methionine, that was coupled with an over two-fold increase in the levels of homocysteic acid (Figure 5J). Moreover, the capacity of methylation reactions, reflected in the ratio of homocysteine to methionine, was downregulated in TEPP46 activated CD8 T cells (Figure 5K). Thus, these data show that TEPP46 treatment induces a wide array of metabolic reprogramming in CD8 T cells with a pronounced increase in PPP generating essential nucleotide for cell proliferation and downregulation in methionine cycle with a potential effect on methylation status in mtDNA. CD8 T cells and CAR-T cells with activated PKM2 have higher recall responses and superior anti-tumor efficacy after adoptive cell therapy One functional implication of enhanced mitochondrial metabolism is the ability of activated CD8 T cells to have stronger recall responses and increased anti-tumor activity. Since we observed robust mitochondrial reorganization in CD8 T cells after TEPP46 treatment, we estimated the ability of these cells to reactivate upon antigenic rechallenge. For this TEPP46-treated CD8 T cells were treated with Ova peptide under chronic and acute (resting) conditions, followed by estimation of cell VHPM Ref.09531.584WO1 / Client Ref.2022-294 phenotype and effector cytokines (Figure 6A). TEPP46 treated CD8 T cells upon chronic antigenic stimulation for 5-6 days demonstrated continued significantly higher levels (p<0.0001) of IFNγ and Gzm B, and higher expression of 4-1BB, the antigen mediated activation and proliferation marker at all time points tested with maximum 6-7-fold increase at day4 in TEPP46 treated CD8 T cells compared to Ova treated cells (Figures 6B-6D and Figures 14A-14C). In addition to effector cytokines, over the course of antigenic stimulation, TEPP46 treated CD8 cells demonstrated higher numbers of CD62L-CD44+TEMcells (Figure 6E and Figure 14D). The TEMcells induced after TEPP46 treatment compared to those generated after Ova treatment demonstrated sustained higher expression of IFNγ, Gzm B and 4-1BB over the course of chronic antigenic stimulation (Figures 14E-14G). These data showed that TEPP46 treatment led to generation of well activated cells that were able to sustain their effector functions upon chronic antigenic stimulation. In line with this, we also observed a significant enrichment of KLRG1-CD127+memory precursor effector cells (MPEC) in TEPP46 treated CD8 T cell cultures at all time points of chronic antigen stimulation (Figure 6F) while CD8 T cells that were activated without TEPP46 enriched KLRG1+CD127- short lived effector cells (SLEC) upon antigenic rechallenge (Figure 6G and Figure 14H). In addition to chronic stimulation, acute (resting) restimulation (Figure 6A) of TEPP46 activated CD8 T cells also demonstrated enhanced IFNγ production and 4-1BB expression at all time points tested (Figures 14I, 14J) while the levels of Gzm B remained higher in TEPP46 activated CD8 T cells at earlier time points and became similar to Ova alone activated cells at later time points tested (Figure 14K) which could be due to different signaling pathways engaged in acute antigenic stimulation compared to chronic antigenic stimulation. Nonetheless, these data show that TEPP46 treated CD8 T cells mount a stronger adaptive response when rechallenged with a cognate antigen and maintained their effector functions upon repeated antigenic stimulation that was associated with induction of increased numbers of long-lived memory precursor cells. We next asked if TEPP46 activated CD8 T cells with upregulated effector functions also demonstrated enhanced cytotoxic functions. For this, first in vitro, OT1-CD8 T cells were activated with Ova or Ova+TEPP46 for 48 hours followed by their co-incubation with antigen B16-F10-OVA tumor cells (Figure 14L). After 24 hours of co-incubation, the percentage of live tumor cells were estimated. OT1-CD8 T cells that were activated in the presence of TEPP46 resulted in significantly higher 70-80% killing of B16-F10-OVA cells compared to 30-40% killing by control CD8 T cells (Figure 6H). We also tested the ability of TEPP46 in enhancing the effector functions of CAR-T cells in vitro. For this, third generation EGFRvIII targeting Thy1.1+CAR-T cells were generated (Figure 14M) as reported earlier, followed by TEPP46 treatment for 24 hours. TEPP46-treated CAR-T cells were co-incubated with B16-EGFRvIII+tumor cells and tumor cell viability was noted 24 hours later. TEPP46 treated CAR-T cells demonstrated significantly higher killing of EGFR+B16 VHPM Ref.09531.584WO1 / Client Ref.2022-294 tumor cell compared to non-TEPP46 treated CAR-T cells (Figure 6I). Importantly, TEPP46 treated OT1-CD8 T cells as well as CAR-T cells continued to have higher expression of IFNγ after co- incubation with tumor cells (Figures 6J, 6K). These data show that TEPP46 treatment enhances the effector functions in CD8 and CAR-T cells in vitro. We also tested the anti-tumor ability of TEPP46 treated CD8 T cell in vivo for which TEPP46 treated OT1-CD8 T cells were adoptively transferred into B16-F10-OVA bearing mice on day 6 after tumor inoculation (Figure 6L). We found that compared to untreated tumor bearing mice both TEPP46 and Ova-alone activated CD8 T cells significantly inhibited the tumor growth. However, TEP46 treated cells suppressed tumor growth significantly and enhanced survival compared with Ova alone treated CD8 T cell (Figures 6M, 6N). Thus, these data demonstrate that TEPP46 treatment enhances the activation and effector functions of CD8 T cells and CAR-T cells leading to their greater anti-tumor activity after adoptive transfer in vivo. PKM2 agonist enhances the anti-tumor efficacy of anti-PD1 therapy Immune checkpoint blockade is the cornerstone of anti-cancer immunotherapy. PD1 signaling has been found to disarm effector T cells by mitochondrial dysfunction. Accordingly, anti- PD1 therapy has been shown to synergize with mitochondrial activation chemicals leading to enhanced T cell dependent antitumor activity. Since we saw a robust increase in mitochondria mediated effector functions and a decrease in exhaustion in PKM2 activated CD8 T cells in vivo and in vitro, we next checked if TEPP46 treatment would enhance the anti-tumor efficacy of anti-PD1 therapy. For this we established B16-F10 tumors in mice followed by TEPP46 and anti-PD1 treatment (Figure 7A). As expected, TEPP46 alone resulted in a significant reduction in tumor growth rates and enhanced mice survival compared to untreated mice (Figure 7B). Anti-PD1 alone resulted in a marginal decrease in tumor growth that was in line with earlier reports showing poor sensitivity of B16-F10 tumors to this therapy regimen. However, combination of TEPP46 significantly enhanced the anti-tumor efficacy of anti-PD1 therapy. Mice in anti-PD1 alone succumbed to the growing tumors by day 24, combination of TEPP46 and anti-PD1 resulted in enhanced survival of tumor bearing mice till day 40 (p<0.01). Thus, these data show the ability of TEPP46 mediated PKM2 activation in reversing the resistance to anti-PD1 therapy. Next, we probed the immune modulatory effects of TEPP46+anti-PD1 treatment in the TME (gating strategy in Figure 15A). Compared to TEPP46 and anti-PD1 treatment alone, combination of two agents resulted in a significant increase in the numbers of CD3+lymphocytes (Figure 7C) that was accompanied with a significant increase in the numbers of CD8 T cells (Figure 7D). Importantly, compared to single treatments using TEPP46 and anti-PD1, combination treatment resulted in a significant increase in the frequencies of IFNγ, TNFα and Gzm B secreting CD8 T cells (Figures 7E-7G and Figures 15B, 15C) along with an enhanced proliferative capacity depicted by increased VHPM Ref.09531.584WO1 / Client Ref.2022-294 Ki67+CD8 T cells (Figure 7H and Figure 15E). In addition to enhanced effector functions, combination treatment using TEPP46+anti-PD1 resulted in an increased frequency of tumor antigen specific gp100-dextramer+CD8 T cells (Figure 7I). Importantly, combination treatment not only increased the numbers of antigen specific cells but also their functionality as we observed a significant increase in the frequency of dextramer+CD8 T cells that were secreting higher amounts of IFNγ and Gzm B (Figures 7J, 7K). These data also show that TEPP46 enhanced the expansion and activation of antigen specific CD8 T cells when combined with anti-PD1 therapy. Together, these data show that TEPP46 works synergistically along with anti-PD1 therapy resulting in enhanced activation of effector T cells in the TME which led to stronger anti-tumor activity highlighting the potential of this treatment strategy in reversing the resistance to immune checkpoint blockade therapy. Discussion Here we report the unexplored effects of activated PKM2 on transcription, translation, DNA methylation, metabolism, and effector functions in CD8 T cells. We show that activation of PKM2 using two different pharmacological agonists lead to enhanced effector functions in mouse and human CD8 T cells. We report that PKM2 agonism in CD8 T cells is associated with reduced exhaustion, increased mitochondrial reorganization, and enhanced metabolic fitness in CD8 T cells both in vitro and in vivo. TEPP46 mediated activation of PKM2 alters cell metabolism with favored pentose phosphate pathway and reduced methionine levels through 1-C metabolism which is associated with reduced methylation and increased translation of mtDNA. Functionally we see that PKM2 agonism enhances the tumor cell killing ability of CAR-T cells and CD8 T cells after adoptive cell therapy. We also show that in-situ PKM2 activation using TEPP46 in tumor bearing mice enhances CD8 T cell mediated anti-tumor effects that boosts the efficacy of anti-PD1 therapy. Metabolic reprogramming can influence the differentiation and function of distinct T cell subsets, including effector, memory, and regulatory T cells, thus exerting a profound impact on immune responses and immunopathology. PKM2 exerts multifaceted effects on cell activation through its regulatory actions on metabolic pathways. PKM2-targeting under in vitro conditions in CD4 T cells has been shown to prevent cell activation, proliferation, and cytokine production by reducing signaling through HIF1a, cMYC and mTORC1 while PKM2 activation in macrophages under in vitro conditions has been shown to reduce LPS induced activation and cytokine production by downregulating HIF1a and IL-1b induction. In contrast to these data, our data from in vitro and in vivo models show that activating PKM2 in CD8 T cells results in enhanced effector functions characterized by increased IFNγ, TNFα and Gzm B, and induction of effector cell phenotype. These findings are in line with recently published observations showing that a knockdown of PKM2 in CD8 T cells results in decreased IFNγ production and reduced effector functions. These VHPM Ref.09531.584WO1 / Client Ref.2022-294 observations support our data showing that activation of PKM2 enhances effector functions in both mice and human CD8 T cells. Moreover, our data in conjugation with previous observations draws a contrast between the role of PKM2 in CD8 and CD4 T cells wherein an PKM2 agonism in CD4 T cells, at least under in vitro conditions seems to prevent T cell activation, which could be due to differential activation kinetics and involvement of disparate signaling pathways in two cell populations. Nonetheless, our data along with recent observations clearly show that PKM2 activation supports enhanced effector functions in CD8 T cells. Mitochondria play a vital role in maintaining metabolic agility required for T cell activation and effector functions after TCR engagement. In CD8 T cells with activated PKM2 we see an increase in expression of PGC1α which regulates mitochondrial biogenesis. In addition to its generation, mitochondrial structural organization is closely associated with cell function as cells with disorganized mitochondria engage in cytoplasmic glycolysis and demonstrate an exhausted phenotype. We see a clear enhancement of the mitochondrial architecture with an increase in the numbers and density of mitochondria after PKM2 activation. The interplay between PKM2, metabolic enzymes and signaling pathways has been shown to prevent mitochondrial dysfunction, impacting ATP generation, ROS production, and mitochondrial membrane potential. Compact mitochondria with densely packed cristae tend to support polarized inner membrane which is required for appropriate cell activation while simultaneously reducing the electron leakage, preventing the upregulation of cellular ROS. Compact packing of mitochondria is facilitated by enhanced translation and appropriate organization of proteins involved in respiratory complex generation which are arranged on inner mitochondrial membrane. We see an enhanced translation of mitochondrial proteins in CD8 T cells with activated PKM2. Particularly, PKM2 mediated enhancement of complex I and III in mitochondria seem to be crucial for increased effector functions in CD8 T cells. The mitochondrial genome encodes 13 subunits that are components of complexes I, III, IV and V of the electron transport chain (ETC). The silencing of mitochondrial transcription factor A (Tfam) has been shown to induce profound ETC dysfunction, and this nuclear-encoded mitochondrial protein along with PGC1α regulates replication, transcription, and the stability of mtDNA. Accordingly, dysfunctional complex I, II and III impairs T cell proliferation. Remodeling of mitochondrial ultrastructure, passing from punctuate mitochondria to one single elongated mitochondria of greater mass tends to support prolonged effector functions in immune cells. Indeed, PKM2 may have a role in regulating mitochondrial dynamics as depicted by enhanced cristae density in CD8 T cells with activated PKM2. Interestingly, PKM2 mediated mitochondrial translation seems to be critical in regulating effector functions in CD8 T cells as an inhibition of translation using doxycycline prevented the expression of IFNγ and TNFα which is in line with observations showing that increased mitochondrial translation supports enhanced VHPM Ref.09531.584WO1 / Client Ref.2022-294 mitochondrial mass and effector functions. Hence, PKM2 activation seems to play a major role in mitochondrial biogenesis, translation, and structural organization leading to enhanced effector functions in CD8 T cells. However, the exact mechanisms through which PKM2 regulates mitochondrial structural organization need further exploration. It is possible that different isoforms of PKM2 may get associated with various gene elements either in nucleus or in mitochondria regulating gene transcription and translation. Another possible explanation is that PKM2 may facilitate bidirectional crosstalk between nuclear and mitochondrial genomes which is crucial for appropriate CD8 T cell functions, though establishment of these observations would need further investigation. Coordinated regulation of nuclear and mitochondrial genomes is required for the production of respiratory complexes that are composed of protein subunits encoded in both genomes. In addition to the bidirectional transcriptional regulation of mitochondrial and nuclear genome, environmental changes and cellular metabolism modify gene expression by regulating the epigenome. As key epigenetic elements that control gene expression, methylation marks are enriched in the promoter region of genes at CpG islands with reduced expression. However, there are contradictory reports regarding the process of methylation of mtDNA. Shock et al have shown that mammalian mitochondria have DNA cytosine methylation sites that are regulated by nuclear respiratory factor 1 and PGC1α-controlled mitochondrial DNA methyl transferase (mtDNMT1). In contrast, Lacopo et al showed the absence of CpG methylation in mammalian mtDNA. Our studies show the presence of CpG methylation in CD8 T cells, and that this methylation is significantly downregulated upon PKM2 activation. Hence our data support the findings from Shock et al showing the presence of altered CpG methylation of mitochondrial DNA. Our data also indicates that PKM2 mediated epigenetic regulation in CD8 T cells is not limited to mtDNA; nuclear genes that regulate mitochondrial dynamics acutely during T cell activation may also be regulated epigenetically. However, the kinetics of PKM2 interaction with mitochondrial and nuclear DNA, especially if PKM2 interacts directly with the gene elements or regulates their expression through altering pathways such as HIF1α and c-MYC need further investigation. PKM2 plays a central role in regulating metabolic intermediates, influencing cellular processes, and affecting cell growth. In CD8 T cells, using metabolome and13C-glucose tracer studies, we found that PKM2 activation and a corresponding decrease in non-activated PKM2 shunts glucose-6-phosphate into pentose phosphate pathway, which is in line with previous observations, resulting in increased precursors for dinucleotide synthesis. In addition to enhanced PPP, we find that PKM2 activation regulated 1-Carbon metabolism which has been known to drive mitochondrial biogenesis and remodeling. One carbon metabolism is comprised of interlinking metabolic pathways including the methionine and folate cycles, where VHPM Ref.09531.584WO1 / Client Ref.2022-294 methionine acts as a major methyl donor for regulating gene expression by DNA methylation. The observed significant decrease in methionine levels could partly explain the decreased genome methylation in CD8 T cells with activated PKM2. The accompanying increase in homocysteic acid has also been shown to contribute to the enhanced effector functions partly by supporting increase in mitochondria:ER coupling, as observed in CD8 T cells with activated PKM2. In fact, enhanced mitochondria endoplasmic reticulum contact sites (MERS), as observed after PKM2 activation in CD8 T cells, have been found to support enhanced metabolic fitness and increased rapid recall responses in CD8 T cells. Moreover, enhanced MERS has also been associated with increased calcium signaling which further supports mitochondrial energetics and CD8 T cell effector functions. Thus, PKM2 has major influences on multiple pathways that, likely acting in a synergistic manner, regulate mitochondrial architecture, metabolism, and effector functions in CD8 T cells. Additionally, by enhancing the structural integrity of mitochondria, activated PKM2 may prevent the release of mtDNA linked to activation of a STING-mediated increase in tumor promoting inflammation. Glycolysis is critical for effector differentiation as it is required for the post-transcriptional regulation of interferon (IFN)-γ production. However, persistently heightened glycolysis limits the capacity of effector T cells to establish immunological memory making them short lived, while moderately dampened glycolysis supports generation of long-lived memory CD8 T cells. In line with these observations, we see an increase in the numbers of long lived MPEC compared to short lived effector cells in CD8 T cells with activated PKM2 which contributes to the enhanced and prolonged anti-tumor effects of CD8 T cells after adoptive cell therapy. Our data also provide new insights into the immune modulatory effects of PKM2 in the TME. Pharmacological activation of PKM2 resulted in the generation and infiltration of tumor specific activated CD8 T cells with reduced exhaustion and enhanced metabolic fitness. In fact, our data showed that PKM2 mediated anti-tumor effects were partially though significantly dependent on CD8 T cells. In addition to CD8 T cells, a decrease in the numbers of immune suppressive Treg cells and prevention of exhaustion in CD4 T cells might also be contributory factors for observed anti-tumor effects after TEPP46 treatment of tumor bearing mice. Interestingly, decreased levels of glucosamine-6-phopsphate (G6P) in CD4 T cells have been reported to destabilize Foxp3 by decreased O-linked β-N-acetylglucosamine (O-GlcNac) post- translational modifications. Hence, PKM2 in the TME seems to have differential effects on multiple cell populations. Moreover, we observe that PKM2 activation can enhance the anti- tumor efficacy of anti-PD1 therapy which is critically limited by the exhaustion status of the immune cells. Interestingly, PD1 signaling in immune cells has been shown to induce mitochondrial disorganization and the enhanced effects seen in the current study after VHPM Ref.09531.584WO1 / Client Ref.2022-294 combination of PKM2 activation and anti-PD1 Ab may be a synergistic effect on mitochondrial metabolism. Nonetheless, these data establish PKM2 targeting as a viable combination strategy to enhance efficacy of anti-PD1 therapy in solid tumors. In conclusion, our results suggest that pharmacological activation of PKM2 induces a complex regulation of transcription, translation, epigenome structure, and metabolism in CD8 T cells; these changes determine cell activation, proliferation, and generation of effector functions. We also demonstrate the unexplored roles of PKM2 in immune modulation in solid tumors. Importantly, through this study we establish PKM2 as a viable pharmacological target that can be used for enhancement of mitochondrial metabolism in cytotoxic cells, ex vivo and in vivo. Material and methods Mice and cell culture Four-to-six-week-old C57BL / 6J or Balb / c wild type mice were purchased from Jackson Laboratory. In-house-bred pMel-1 mice [B6.Cg-Thy1a / Cy Tg (TcraTcrb)8Rest / J], which carry a rearranged TCR transgene (Vβ13) specific for the mouse homolog (pmel-17) of human gp100, with transgenic CD8 T cells having a melanoma-gp100 peptide-specific TCR were used as outlined in various experiments. Similarly, OT-1 transgenic mice (Jax-Strain #003831) with specific anti- Ova257-264T cell receptor (TCR) in CD8+T cells were bred in-house and used in various experiments as listed. Animals had free access to water and food. All experiments were performed under protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Minnesota. Cancer cell lines used in the present study included B16-F10, B16-F10-OVA melanoma and CT26 colorectal carcinoma that were obtained from the American Type Culture Collection (ATCC). B16-EGFRvIII cells were kindly provided by Dr. Richard Vile at Mayo Clinic, Rochester, MN. Melanoma (B16-F10, B16-F10-OVA, B16-EGFRvIII cells) and CT26 Cells were respectively cultured in DMEM (Gibco) and RPMI (Gibco) media containing 10% FBS and 1% Penicillin- Streptomycin. Cell lines were routinely checked for the absence of mycoplasma, by microscopic evaluation and PCR-based methods. Primary murine CD8+T and CD4+T cells, were isolated from mice spleen by negative selection using magnetic beads (Stemcell, Cat# 19853A) and cultured in T cell media contains RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine, 1% Penicillin-Streptomycin, and 55 µM β-mercaptoethanol. Purity (>95%) of the cell populations was determined via flowcytometry. Human CD8 T cells were purchased from Stemcell Inc. (Cat# 200- 0164). VHPM Ref.09531.584WO1 / Client Ref.2022-294 Antibodies and reagents The Live / Dead detector dye, eBioscience™ Fixable Viability Dye eFluor™ 450, was purchased from ThermoFisher Scientific (Invitrogen, Cat# 65-0865-14). CellTrace™ Violet Cell Proliferation Kit, for flow cytometry (Invitrogen, Cat# C34557) were used for cell proliferation evaluation TEPP46 (Cat#S7302) and DASA58 (Cat#S7928) were purchased from Selleckchem Inc. The fluorochrome-labeled anti-mouse antibodies used for flow cytometry measurements in vitro and in vivo were: Alexa Fluor 700-CD45 (1:200, clone 30-F11, Cat# 56045182; eBioscience); TxRd- CD3 (1:200, clone 145–2c11, Cat# 562286; BD Biosciences); Alexa Fluor 700-CD8a (1:200, clone 53-6-7, Cat# 566985; BD Horizon); FITC-IFNγ (1:100, clone XMG1.2, Cat# 11-7311-82; Invitrogen), APC-TNFα (clone MP6-XT22, Cat#17-7321-82, Invitrogen), FITC-TNFα (clone TN3- 19.12, Cat#11-7423-82; eBioscience), Buv395-CD62L (0.2 mg / ml, 1:200, clone MEL-14, Cat# 363- 0621-82; eBiosciences); TxRed-CD44 (1:200, clone IM7, Cat# 562464; BD Horizon); PE-PD-1 (1:200, clone J43, Cat# 12-9985; Invitrogen); V450-Ki-67 (1:200, clone SolA15, Cat# 48-5698-82; Invitrogen); BV421-Granzyme B (1:100, clone GB11, Cat# 563389; BD Horizon); APC-LAG3 (1:200, clone C9B7W, Cat# 562346; Invitrogen); V450-LY-6G / C (clone RB6-8C5; Cat# 560454; BD Horizon), BUV395-CD4 (clone: RM4-5, Cat# 363-0042-80; Invitrogen), BV421-TIGIT (clone TX99, Cat# 749781; BD Biosciences), V450-Foxp3 (clone MF23, Cat# 561293; BD Horizon), APC-CD11b (clone: M1 / 70, Cat# 17-0112-82; Invitrogen), CD127, APC-KLRG (clone: 2F1, Cat# 17-589-81; Invitrogen), BV421-CD127 (clone: A7R34, Cat# 404-1271-80; Invitrogen). APC-gp100 (1:50, Cat# WB2158- APC) dextramers were obtained from Immudex Inc. The fluorochrome- labeled anti-human antibodies used for flow cytometry measurements were Alexa Fluor700-CD8 (clone: RPA-T8, Cat# 56-0088-42; Invitrogen), APC-IFNɣ (clone: 4S.B3, Cat# 17-7319-82; Invitrogen), BUV395-CD45RA (clone: HI100, Cat# 363-0458-41; Invitrogen), PE-CD44 (clone: IM7, Cat# 61-0441-82, Invitrogen). CD8 enrichment kits were purchased from STEMCELL Technologies, Cat# 19725A). Antibodies used for western blot assay were: PKM2 (Cat#4053T), Glut1 (Cat#12939), PGC1α (Cat# 2178), COX1 (Cat#55159), ATP5A1 (Cat#18023), SDHB (Cat#92649), NDUFB9 (Cat#99235), VDAC (Cat# 4661T), HIF-1α (Cat#14179), phospho-mTOR (Ser2448; Cat#5536), mTOR (Cat# 2983), all from Cell Signaling, UQCRC2 (Cat#A305-414A-T; Invitrogen), phospho-c-Myc (Cat#AP0989; ABclonal), beta-actin (Cat#66009-1-Ig; proteintech), anti-mouse IgG HRP-linked (Cat# 7076P2; Cell Signaling), anti-rabbit IgG HRP-linked (Cat# 7074S; Cell Signaling). Ova (257-264) (Cat# AS-60193-1) and gp100 peptides (Cat# AS-64752) were purchased from Anaspec Inc. Cell activation and drug treatment Isolated CD8 T cells from OT1 mice (1.5 ×106cells per ml) were activated with Ova peptide (0.5 µM) either alone or in combination with TEPP46 at final concentration of 40 and 60 µM or VHPM Ref.09531.584WO1 / Client Ref.2022-294 DASA58 (30uM) in T cell medium supplemented with 30 international units (IU) of IL-2 for 24h and 48h. PKM2 protein tetramer level was detected by western blot. Isolated CD8 T cells from spleen of pMel-1 mice (1.5 ×106cells per ml) were activated by gp100 (1µM) in the presence and absence of TEPP46 (60 µM) for 48h. WT CD8 T or CD4 T cells were isolated from C57BL / 6J mice spleen and activated by Dynabeads™ Mouse T-Activator CD3 / CD28 (ThermoFisher Scientific; Cat#11456D) either alone or in combination with TEPP46 for 48h. CD4 T cells were isolated EasyStepTM Mouse CD4 T cell isolation kit (STEMCELL, Cat# 19812A). Human CD8 T cells (1.5 ×106cells per ml) were activated by plate bound anti-CD3 (clone: OKT3, Cat#567118; 10 µg / ml) and soluble anti-CD28 (clone: CD28.2, Cat#567117; 5 ug / ml) both from BD Pharmingen, in the presence or absence of IL2 (100 IU / ml) and TEPP46 (60uM) or DASA58 (30uM) for 72 hours followed by FACS analysis. Gene expression evaluation by RNA sequencing and comparative analysis with metabolome data Total RNA was extracted from cells using RNA isolation kit (Cat# 74104; QIAGEN).2µg of total RNA was used for RNA sequencing. RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). NEBNext Ultra II RNA Library Prep Kit (Illumina) was utilized for making RNA libraries and samples were sequenced by a 2x150bp Paired End (PE) configuration. RNAseq raw data (fastq or fasq.gz files) quality was checked using FastQC (v0.11.9), and adapter trimming on raw data was performed using Cutadapt (v3.5). Reads with low quality (quality score < 33, error rate > 10%) or short length (<25 bp) after trimming were removed before alignment. We used the reference genome downloaded from ENSEMBL hg38 release 108, and the reference index was built using Star (v2.7.9a) software. Paired end trimmed read alignment and raw read count calculation were performed using RSEM software (v1.3.1). DESeq2 package (v1.36.0) was used for group wise statistical analysis. We considered the genes with fdr <0.05 differentially expressed. Principal component analysis plots were generated using plotly (v4.10.2). All genes with expression levels were used as input for Gene Set Enrichment Analysis (GSEA) (v4.2.3, Broad Institute). Both KEGG and HALLMARK gene sets were selected for enrichment score calculation. We made comparisons between Ova-48h and T60-48h, significantly different (p value < 0.05) genes and metabolites from RNAseq and Metabolomics data were integrated using the STITCH database. Visualization was generated in Cytoscape (v 3.9.0). Cell proliferation assay The cell proliferation was analyzed by CellTrace™ Violet dye (VCT) labeling of cells before activation and TEPP46 treatment. Briefly, isolated CD8+T cells were labeled with VCT by VHPM Ref.09531.584WO1 / Client Ref.2022-294 the incubation of cells in 37°C for 10 min in PBS containing VCT based on manufacture’s instruction. Labeled cells were activated with Ova peptide alone or in combination with TEPP46. After 24h and 48h of cell activation, cells division was estimated by checking VCT dilution via FACS analysis. Flowcytometry analysis Activation markers producing CD8+T cells were detected by flowcytometry. In this method isolate cells were plated at a density of 1.5 × 105cells per well in a 96-well plate and incubated in T cell medium containing Ova (1µM) in the presence and absence of TEPP46 (40 and 60 µM) for 24h and 48h, then cells were stained with Live / Dead Fixable Near-IR Dead Cell Stain Kit and fixed by formaldehyde (1×). To detect intracellular protein markers for instance IFN-γ, TNFα and Granzyme B (Gzm B), cells were permeabilized followed by antibody staining and FACS analysis. Data acquisition was performed on a BD LSR Fortessa flow cytometer using the software FACS Diva v.9.0. Results were analyzed using FlowJo software v10.8.1. For evaluation of different subsets of T cells (TCM and TEM) in CD8+T cells that treated with or without TEPP46, CD44 and CD62L expression levels were evaluated by flowcytometry. Activator markers were also evaluated in CD8+T cells isolated from pMel-1 and wild type mice. These cells were stained similarly for detection of IFNγ, TNFα, CD44 and CD62L. Glucose uptake was assayed using 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4- yl)Amino)-2-Deoxyglucose; Cat# N13195, Invitrogen) staining and Glucose Uptake-Glo™ Assay kit (Cat# J1341,Promega). For NBDG staining, briefly, after 48h of CD8 T cells activation with Ova+ / - TEPP46, cells were stained by Live / Dead dye, then incubated in 37 °C / 5% CO2for 30 min in T cell media:PBS (1:1) containing 2-NBDG (20 µM) for 30 minutes and NBDG cells were detected by flowcytometry. To evaluate ATP production in mitochondria, cells were stained by BioTrackerTMATP-Red Live Cell Dye (Millipore, Cat# SCT045) (5 µM) as described by the manufacturer. Mitochondrial mass was evaluated by MitoFM (MitoTracker™ Green FM, Cat# M7514, Invitrogen) staining. Briefly, activated cells were stained by Live / Dead dye and then incubated in PBS containing 50nM MitoFM at 37 °C / 5% CO2for 30 min and MitoFM positive cells were detected by flowcytometry. For mitochondrial membrane potential estimation TMRM (Cat# M20036, Invitrogen) staining. Resting-Rechallenge assay CD8 T cells from OT1 mice were activated by Ova peptide (0.5 µM) in the presence or absence of TEPP46 (60µM) for 2 days, followed by resting the cells in T cell medium with IL2 (30 IU / ml) for 2 days, and then re-stimulation with IL2+Ova peptide (0.5 µM) for 2 or 3 days. During re-stimulation, cells were harvested at various time points as depicted in Figure 6A for detection of VHPM Ref.09531.584WO1 / Client Ref.2022-294 CD8 T cells effector function (IFNγ, Gzm B, 4-1BB, CD44, CD62L, CD127 and KLRG production) by FACS analysis as explained above. Chronic stimulation CD8 T cells from OT1 mice were activated by Ova peptide (0.5 µM) in the presence or absence of TEPP46 (60µM) for 2 days, then cells were washed and counted and restimulated by Ova peptide (0.5 µM) every other day for 4 days without any resting period. After re-stimulation, cells were harvested at various time points as depicted in Figure 6A for analysis of effector function (IFNγ, GB, 4-1BB, CD44, CD62L, CD127 and KLRG production) by FACS analysis as explained above. Feeder layer (non-purified spleen without mitomycin treatment, 1-5% of total CD8 T cell numbers) was added to purified CD8 T cells culture only during 1stphase of T cell activation using cognate antigen. During antigenic restimulation feeder layer was not used. CAR-T cell generation EGFRvIII targeting third generation MSGV1-retroviral CAR construct containing CD28, 4- 1BB, and CD3-zeta moieties, in tandem with the scFv derived from the human monoclonal Ab and the marker Thy1.1 were prepared. Briefly, splenocytes from C57BL / 6 mice were activated with concanavalin A (2.5 µg / ml) for 48 hours in T cell medium supplemented with IL-2 (50 IU / ml) followed by retrovirus transduction for 24 hours. Retrovirus was produced from 293T cells cotransfected with the MSGV1 retroviral plasmid and the helper plasmid pCL Eco (Imgenex). T cells were transduced on RetroNectin-coated plates 2 days after stimulation. Retroviral plasmids were kindly provided by Dr. Richard Vile, Mayo Clinic, Rochester, MN. Typically, we achieve a CAR-T induction of 50-70 %. For all experiments, Thy1.1+CAR-T cells were purified either by FACS sorting or using Thy1.1 purification kit from Stem cell Inc. (Cat#18958). Kit purified CAR-T cells were assessed for purity by FACS and were used when >90% pure. Cell Killing assay and adoptive cell transfer assay For in vitro evaluation of effect of TEPP46 on anti-tumor activity of CD8 T cells, TEPP46 treated OT1-CD8 or CAR-T cells were co-cultured with target cells (B16-F10-OVA for OT1-CD8 and B16-EGFRvIII cells for CAR-T cells) for 24 hours followed by estimation of tumor cell viability by gating on negative population for fixable near infrared dye. From the same experiments cells were also probed for production of IFNγ by gating upon CD3+CD8 T cells or Thy1.1+CD3+CAR-T cells. For adoptive cell transfer assay, B16-F10-OVA tumor cells were subcutaneously injected into the right flank of WT C57BL / 6 mice. CD8 T cells were purified from the spleen and activated with Ova + / -TEPP46 as detailed above. Tumor bearing mice were treated with one dose of cyclophosphamide (100 mg / Kg; at day 5-6) followed by intravenous injection of activated cells (2- 3x106per mouse) at day 7-8 post-tumor inoculation. Variously treated mice were observed for tumor growth. VHPM Ref.09531.584WO1 / Client Ref.2022-294 Targeted cell metabolomics Targeted metabolomics method was used to quantitate >500 endogenous metabolites using QTRAP® 7500 LC-MS / MS System (Sciex, MA, USA). For the purpose, cell pellet was suspended in 50 μL of PBS and heat shock was done by keeping samples on ice for 30s and 37 °C for 90s, then samples were sonicated for 1 minute and 100 μL of extraction buffer (methanol / water 50 / 50) containing 200 ng / mL of debrisoquine (DBQ) as internal standard for positive mode and 200 ng / mL of 4-nitrobenzoic acid as internal standard for negative mode was added. Proteins were precipitated by incubation at -20 °C and samples were centrifuged at 13,000 rpm for 20 minutes at 4 °C. The supernatant was used for LC-MS analysis.2 μL of the prepared sample was injected onto a Kinetex 2.6 μm polar C18100 Å 100 × 3.0 mm (Phenomenex, CA, USA) using SIL-30 AC auto sampler (Shimazdu, Kytoto, Japan) connected with a high flow LC-30AD solvent delivery unit (Shimazdu, Kytoto, Japan) and CBM-20A communication bus module (Shimazdu, Kytoto, Japan) online with QTRAP 7500 (Sciex, MA, USA) operating in positive and negative ion mode. A binary solvent comprising of water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B) was used. The extracted metabolites were resolved at 0.2 mL / min flow rate. The LC gradient conditions were as follows: Initial – 100% A, 0% B for 2.1 minutes; 14 minutes – 5% A, 95% B till 15 minutes; 15.1 minutes – 100% A, 0% B till 20 minutes. The auto sampler and oven were kept at 15 °C and 30 °C, respectively. Source and gas setting for the method were as follows: curtain gas = 40, CAD gas = 9, ion spray voltage = 1700 V in positive mode and ion spray voltage = 1600 V in negative mode, temperature = 350 °C, ion source gas 1 = 30 and ion source gas 2 = 50. The data were normalized to respective internal standard area and processed using MultiQuant 3.0.3 (Sciex). The quality and reproducibility of LC-MS data was ensured using a number of measures. The column was conditioned using the pooled QC samples initially and were also injected periodically to monitor shifts in signal intensities and retention time as measures of reproducibility and data quality of the LC-MS data. Metabolic Flux To evaluate cellular glucose flux, CD8+T cells were activated by Ova alone or Ova+TEPP46 for 24 and 48 h. Cells were washed with PBS and cultured in no-Glu / no-Phenol Red media (Agilent Technology; Cat# 10357-100) containing C13D-Glucose (11 mM) (U13C6, 99%, Cambridge Isotype), IL2 (30 IU / ml), Glutamine (2mM), Arginine (1mM), Lysin (1mM), and FBS (10%) for 4 h followed by harvesting the cells for estimation of13C-Glucose by LC / MS. All LC-MS grade solvents including acetonitrile and water were purchased from Fisher Optima grade, Fisher Scientific. High purity formic acid (99%) was purchased from Thermo-Scientific. Debrisoquine and 4-nitrobenzoic acid were purchased from Sigma- Aldrich. VHPM Ref.09531.584WO1 / Client Ref.2022-294 Cell fluxomics using QTRAP 7500 Targeted fluxomics analysis method, developed in-house, was used to quantitate 13C labelled (fully or partially) small endogenous metabolites using QTRAP® 7500 LC-MS / MS System (Sciex, MA, USA). For the purpose, 25 μL of PBS was added to the cell pellet and sample tube was plunged into dry ice for 30 sec and 37 °C water bath for 90 sec. This cycle was repeated for two more times and then samples were sonicated for 1 minute and 100 μL of extraction buffer (methanol / water 50 / 50) containing 200 ng / mL of debrisoquine (DBQ) as internal standard for positive mode and 200 ng / mL of 4-nitrobenzoic acid as internal standard for negative mode was added. The samples were vortexed for 1 min and kept on ice for 20 minutes followed by incubation at -20 °C for 20 minutes for protein precipitation. The samples were centrifuged at 13,000 rpm for 20 minutes at 4 °C. The supernatant was transferred to MS vial for LC-MS analysis.20 μL of each prepared sample was mixed to generate the pooled QC sample. One microliter of the prepared sample was injected onto a Kinetex F5, 2.6 μm 100 Å 150 × 2.1 mm (Phenomenex, CA, USA) using SIL-30 AC auto sampler (Shimazdu) connected with a high flow LC-30AD solvent delivery unit (Shimazdu) and Exion 30AD communication bus module (Shimazdu) online with QTRAP 7500 (Sciex, MA, USA) operating in positive and negative ion mode. A binary solvent comprising of water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B) was used. The extracted metabolites were resolved at 0.2 mL / min flow rate. The LC gradient conditions were as follows: Initial – 100% A, 0% B for 2.1 minutes; 14 minutes – 5% A, 95% B till 15 minutes; 15.1 minutes – 100% A, 0% B till 20 minutes. The auto sampler and oven were kept at 15 °C and 30 °C, respectively. Source and gas setting for the method were as follows: curtain gas = 45, CAD gas = 10, ion spray voltage = 2000 V in positive mode and ion spray voltage = 4500 V in negative mode, temperature = 500 °C, ion source gas 1 = 45 and ion source gas 2 = 70. Data Processing The data were normalized to internal standard area and processed using Sciex OS software. Internal Standard normalized data was further QC-RLSC normalized. The quality and reproducibility of LC-MS data was ensured using a number of measures. The column was conditioned using the pooled QC samples initially and were also injected periodically to monitor shifts in signal intensities and retention time as measures of reproducibility and data quality of the LC-MS data. We also have blank solvent runs between set of samples to minimize carry-over effects. Whole-genome bisulfite sequencing (WGBS) Briefly, genomic DNA was isolated from CD8 T cells after activation in the presence or absence of TEPP46 and treated with bisulfite by the EZ DNA Methylation gold kit (Zymo VHPM Ref.09531.584WO1 / Client Ref.2022-294 Research). To detect methylated cytosines and unmethylated cytosines which are deaminated to uracils, routine sequencing was performed after bisulfate treatment. The library was generated by the EpiGnome kit (Epicentre) and Illumina HiSeq system was used for sequencing of Bisulfite- modified DNA. WGBS raw data (fastq or fasq.gz files) quality was checked using FastQC (v0.11.9), and adapter trimming on raw data was performed using Trimmomatic (v0.39). Reads with low quality (quality score < 33, error rate > 10%) or length are too short (<35 bp) after trimming were removed before alignment. We used the reference genome downloaded from UCSC mm10, a bowtie2 (v2.4.2) genome indexing was produced for the next step. Paired end trimmed read alignment, deduplication and methylation extraction were performed using bismark (v0.24.0). Statistical analysis of DMRs were conducted using DMRichR (v1.7.8) R package, DMRs were annotated using ChIPseeker (v1.36.0) R package. Mitochondrial translation analysis Total cell translation and mitochondria translation level were analyzed by incorporation of puromycin in neosynthesized proteins in cytoplasm and mitochondria. In this method CD8 T cells were activated by Ova+ / -TEPP46 for 48h, followed by puromycin (1µg / ml) (gibco) treatment for 15 min at 37°C / 5% CO2. Total and mitochondrial proteins were isolated from these cells (Mitochondrial isolation kit, termo scientific, Cat# 89874) and puromycin incorporation in proteins were detected by western blot with anti-puromycin antibody (Millipore Sigma, Cat# MABE-343). In a few experiments, CD8 T cells from OT1 mice were activated by Ova with or without TEPP46 in the presence or absence of doxycycline (10 µg / ml) or chloramphenicol (100 µg / ml) for 48h. The translation of mitochondrial proteins including NDUFB9, SDHB, UQCRC2, Cox1, and ATP5A1 was analyzed by western blot. The percentage of CD8 T cells which produce activation markers IFNγ and TNFα was also determined by flowcytometry. To check the effect of PKM2 tetramerization by TEPP46 on mitochondria respiratory complexes, OT1 isolated CD8 T cells were activated with Ova or Ova+TEPP46 in the presence or absence of mitochondrial respiratory complex inhibitors. Rotenone (0.25 µM), TTFA (100 µM), antimycin (0.5 µM) and oligomycin (0.025 µM) were used as mitochondria complex I, II, III and V inhibitors respectively. After 48h cells were harvested and activation markers in CD8 T cells including IFNγ and TNFα were analyzed by flowcytometry. Quantitative PCR with reverse transcription analysis Total RNA was extracted from Ova or Ova+TEPP46-activated CD8 T cells using RNeasy Kit (QIAGEN) and dissolved in RNase-free water. One μg total RNA was subjected to single-strand complementary DNA synthesis using High-Capacity RNA-to-cDNA Kit (Appliedbiosystems). Primer sequences and respective genes are listed in Table 1. VHPM Ref.09531.584WO1 / Client Ref.2022-294 Data was obtained using QuantStudio 12K Flex Real-Time PCR System from Applied Biosystems. Housekeeping gene β-actin was utilized for data normalization. Western blot OT1-CD8 T cells were activated with Ova in the presence and absence of TEPP46 as described above. Protein isolation was performed by making lysis of cells with RIPA buffer containing 1% protease inhibitor. Protein concentrations were estimated using nanodrop (ThermoFisher Scientific). Proteins (20-40 µg) were loaded onto NuPAGETM4-12% Bis-Tris Gel (ThermoFisher Scientific, Cat# NP0322BOX) and transferred onto nitrocellulose membranes by semi-dry method using iBlot 2NC Regular Stacks kit (Invitrogen, Cat# IB23001). Membranes were blocked with 4% BSA in TBST buffer (containing 1X Tris-Buffered Saline, 0.1% Tween 20) followed by overnight shacking in 4⁰C in the presence of primary antibodies against PKM2, GLUT1, PGC1α, Cox1, NDUFB9, UQRC2, ATP5A1, SDHB, HIF1α, p-mTOR / t-mTOR, and cMYC. The blots were detected with appropriate (anti-rabbit or anti-mouse) HRP-conjugated secondary antibodies. The level of β-actin was used as a control. Densitometric of the bands was analyzed via ImageJ software. VHPM Ref.09531.584WO1 / Client Ref.2022-294 Transmission electron microscopy To analyze ultrastructural mitochondrial changes, variously activated CD8+T cells from OT- 1 mice (TEPP46, OVA, and IL2), were fixed with Karnovsky’s Fixative 15720 kit (Electron Microscopy Science). Fixated cell pellets were embedded in low melting point agarose and sectioned into 1-2 mm pieces, followed by incubation in 1% OsO 4 in 0.12 M sodium cacodylate buffer for 1 hr. After rinsing with 0.12M (3x10 min), cells were placed in 1% uranyl acetate in 0.1M sodium acetate buffer (overnight / RT). Next day, samples were rinsed and dehydrated through graded ethanol series, infiltrated with epoxy resin (EMbed-812; Electron Microscopy Science, Hatfield, PA, USA), and embedded in beam capsules. After polymerization in the oven (60oC / 48 hr), blocks containing cells were sectioned using an automated ultramicrotome (UC7 Leica Microsystem). The resulting ultrathin sections (120 nm) were placed in silicon wafers, dried, and carbon-taped in aluminum stubs. We performed SEM imaging in a Helios NanoLab 660 dual-beam microscope (ThermoFisher), using an insertable concentric backscatter detector in 2kV, with a beam current of 0.40 nA, and in immersion mode at 4 um working distance. Overviews of the entire sample were performed in low magnification to identify the cells. Then, high-resolution tile SEM images (80.000x magnification, pixel size 1.76 nm) were performed per cell to obtain the resolution to undoubtfully identify mitochondrial ultrastructural alterations. ImageJ software was used for determining the number of mitochondria per cell, mitochondrial area, cristae number and area, and ER–mitochondria contact coefficient (MERS) contact index. MERS was calculated using the formula: In this formula L = Interface length (um), Per= Mitochondria perimeter (um) and Dist = distance ER-mitochondria (um) Tumor establishment, mice treatment and immune response analysis in tumor samples C57BL / 6J mice were injected with 1 × 106B16-F10 or CT26 cells per mouse subcutaneously in the right flank. Treatment in respective groups started when tumors reached an average size of approximately 0.075 cm3. TEPP46 (dissolved in 50% PEG in PBS) treatment was performed by oral gavage for 10 days (4 days on and 2 days off) at a dose of 40mg / Kg, once daily. Tumors were measured every 2 days using a digital Vernier caliper, and the tumor volume was calculated using the formula, L × W2 / 2, where L is the long axis of the tumor and W is the short axis of the tumor). Mice were monitored for tumor growth and survival. Mice were sacrificed when the tumor volume reached 1.5 cm3. In a few experiments, B16-F10 bearing mice were inoculated with anti-CD8 monoclonal antibody (100 µg / mouse; BioXcell, Cat# BE0061), intraperitoneally on day 4 VHPM Ref.09531.584WO1 / Client Ref.2022-294 and 5, post-tumor inoculation. Blood (100 µl) was collected from each mouse in K2EDTA-treated vials on day 6 for confirmation of CD8 T cell depletion. Flowcytometry was used to assess the depletion of CD8 T cells. Mice were treated with TEPP46 using the same protocol explained above. Tumor volume and survival were monitored as mentioned before. For α-PD1 and TEPP46 combination therapy analysisC57BL / 6J mice were injected with 1×106B16-F10. Treatment was started when tumors reached an average size of approximately 0.075 cm3. TEPP46 treatment method was same as explained before and α-PD1 was injected to the tumor bearing mice with the concentration of 2.5mg / kg, twice a week for 4-5 doses. Tumor growth was recorded by Vernier caliper using the formula L*W2 / 2000 where L is the long axis and W is the short axis of tumor. In separate immune experiments, mice were sacrificed two days after last dosing and tumor tissues were harvested and filtered through 40 µm nylon mesh cell strainer (Fisherbrand). To evaluate mitochondrial mass in CD8 T cells, first tumor cell suspension was stained with Live / Dead dye containing appropriately labeled anti-CD3 and anti-CD8 antibodies, followed by incubation for 30 min in 37°C, 5% CO2in the presence of MitoFM dye as described before. To determine the immune response, single cell suspension from tumor tissue was stained with Live / Dead dye, followed by fixation with formaldehyde. The level of activated T cells (IFNγ+, TNFα+, and Granzyme B+cells) and T reg cells (Foxp3+cells) were analyzed by flowcytometry. EXAMPLE 2 PKM2 regulates mitochondria dependent anti-tumor functions in CD8 T cells by modulating cell transcriptome, metabolome and methylome Loss of mitochondrial structure and function is associated with exhaustion and therapeutic failure of lymphocytes. Using the pharmacological agent TEPP46, we explored the mitochondrial and immune-modulatory effects of PKM2 in CD8 T cells. PKM2 differentially regulates the transcriptome, epigenome, metabolome, and effector functions in CD8 T cells. TEPP46 treatment differentially regulated the transcriptome, resulting in enhanced TCR- mediated effector functions along with PGC1a-directed mitochondrial biogenesis, OXPHOS, and gene expression supporting mitochondrial fusion and the MICOS complex. Whole-genome bisulfite sequencing showed hypomethylation of mtDNA and increased mitochondrial translation, enhancing mitochondrial mass and cristae density in TEPP46-treated lymphocytes. Metabolome analysis revealed differential regulation of the glycolytic and pentose phosphate pathways. Antigenic re-challenge of TEPP46-treated CD8 T cells showed stronger recall responses with sustained mitochondrial metabolism. TEPP46 treatment of B16 tumors in mice showed CD8 T cell-dependent anti-tumor responses and an increased infiltration, activation, and metabolic fitness of CD8 T cells with fewer Treg and MDSCs in tumors. In conclusion, we VHPM Ref.09531.584WO1 / Client Ref.2022-294 establish the unexplored roles of PKM2 in CD8 T cells, showing that PKM2 enhances effector functions and mitochondrial metabolism in cytotoxic cells, with potential applications in adoptive cell therapies and in-situ enhancement of CD8 T cell functions. EXAMPLE 3 PKM2 agonism enhances mitochondrial organization and anti-tumor functions of CD8 T cells in solid tumors by modulating cell transcriptome, metabolome and methylome Background: Impaired mitochondrial functions are associated with increased T cell exhaustion and reduced therapeutic efficacy of lymphocytes. Proper organization of mitochondrial structures is critical for optimal mitochondrial functions. However, pharmacological strategies to preserve mitochondrial organization are not well-developed. Pyruvate kinase (PK)M2 is intricately involved in regulating cell metabolism and gene expression. Tetrameric form of PKM2 (tet-PKM2) catalyzes the conversion of phosphoenolpyruvate to pyruvate in cytoplasm, and dimeric form (di-PKM2) controls the gene expression in nucleus. In tumor cells di-PKM2 enhances while tet-PKM2 suppresses proliferation and growth. In immune cells tet-PKM2 was shown to suppress the generation of Th17 cells and LPS induced mature macrophages. However, the effects of PKM2 on CD8 T cells have not been studied. Methods: Using pharmacological agent TEPP46, mitochondria-directed immune- modulatory effects of tet-PKM2 were explored in CD8 T cells. In vitro, OT1-CD8 T cells were activated with Ova-peptide+TEPP46 followed by transcriptome, methylome, metabolome, and functional analysis by FACS, Seahorse and electron microscopy. In vivo, B16-melanoma bearing mice were treated with TEPP46 followed by estimation of tumor growth rates and tumor immune-microenvironment. Results: TEPP46-induced tet-PKM2 leads to differential regulation of transcription, mitochondrial epigenome, metabolome, and effector functions in CD8 T cells. TEPP46 treatment differentially regulated HIF1a, MYC and mTORC regulated genes, resulting in enhanced TCR-mediated effector functions (IFNg, TNFa, Granzyme B). TEPP46 treatment upregulated PGC1a, mitochondrial biogenesis, OXPHOS and expression of genes associated with mitochondrial fusion (Opa1, Mif1 / 2, Immt) and MICOS complex. Whole genome bisulfite sequencing showed hypomethylation of mtDNA and increased translation of components of oxidative phosphorylation generating mitochondria with higher mass, increased cristae density, and enhanced mitochondria:ER interaction in TEPP46-treated CD8 T cells. Metabolomic analysis revealed differential regulation of glycolytic and pentose phosphate pathway in TEPP46-treated CD8 T cells. Antigenic re-challenge of TEPP46-treated CD8 T cells showed VHPM Ref.09531.584WO1 / Client Ref.2022-294 stronger recall responses with sustained mitochondrial metabolism. Treatment of B16-F10 tumor bearing mice with TEPP46 (n=7-8 / group) showed CD8 T cell-dependent anti-tumor responses (p<0.05), accompanied with significantly increased infiltration of activated, metabolically fit effector CD8 T cells, decreased expression of exhaustion markers and reduced fraction of immune-suppressive Treg and MDSCs in tumors. Conclusions: The unexplored roles of PKM2 in CD8 T cells were establish and it was shown that PKM2 is a clinically viable target for enhancing the functions and mitochondrial metabolism of cytotoxic cells, with potential applications in adoptive and CAR-T cell therapy, and in-situ enhancement of CD8 T cell functions. EXAMPLE 4 PKM2 mediated enhanced mitochondrial metabolism is a checkpoint for tumor growth and immune suppression. Introduction The Pasteur effect, which is the ability of oxidative phosphorylation (OXPHOS) to suppress glucose fermentation, was first described over 150 years ago in yeast. The Pasteur effect implies that if oxidative phosphorylation is enhanced cytoplasmic glucose fermentation and the production of lactate can be inhibited that should further inhibit the growth of tumor cells. Mitochondria, the organelles responsible for (OXPHOS) in cells, have been found to play a significant role in cancer development and progression. While mutations in mitochondrial genes are common in cancer cells, they do not generally inactivate mitochondrial energy metabolism but rather alter the mitochondrial bioenergetic and biosynthetic state. Distorted mitochondria and altered mitochondrial metabolism reduce the efficiency of OXPHOS while increasing the production of mitochondrial reactive oxygen species (ROS) and change the cellular redox state. Excessive ROS production can promote DNA damage and genomic instability, contributing to cancer development. Given the central role of mitochondria in cancer cell metabolism and survival, targeting mitochondria has emerged as a potential strategy for cancer therapy. Various approaches, such as targeting mitochondrial metabolism or inducing mitochondrial apoptosis, have been explored to enhance the killing of cancer cells. In current study we show that stabilizing mitochondrial architecture using PKM2 targeted pharmacological approach leads to enhanced mitochondrial metabolism that acts as a metabolic checkpoint for tumor cells. We show that compared to their normal cell counterparts, tumor cells tend to have distorted mitochondria and stabilization of mitochondrial cristae structures and TCA cycle can halt the tumor growth profiles, induce cell apoptosis and increase immunogenic cell death with a potential enhancement of anti-tumor immune responses. VHPM Ref.09531.584WO1 / Client Ref.2022-294 Results Tumor cells are associated with mitochondrial malformation. Tumorigenic transformation of normal cells coincides with profound changes in metabolism, particularly the mitochondrial content, structural dynamics, and functions. We first checked if tumor cells compared to their normal cell counterparts, have impaired mitochondrial characteristics. For this we first determined the mitochondrial numbers in mouse B16-melanoma and CT26-colon cancer cells and compared it to the normal mice epithelial and colon cells respectively. In agreement with our hypothesis that tumor cells have diminished mitochondria we observed a significant downregulation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), which is essential for mitochondrial biogenesis, in tumor cells compared to normal cells (Fig.16A). Also, we saw a significant decrease in the ratio of mtDNA / nDNA in CT26 colon cancer cells indicating a decrease in mitochondrial numbers compared to their normal cell counterparts (Fig.17A). Surprisingly the numbers of mitochondria in B16 cells were similar to mouse normal epithelium (Fig.17A). However, the genes associated with dynamic regulation of mitochondrial architecture were significantly downregulated in both B16 and CT26 cells (Figs.16C-16I). Particularly, the genes associated with mitochondrial fusion (OPA1, mfn1, mfn2) (Figs.16B-16D) and fission (Fis1, Mief2) were downregulated in tumor cells compared to normal cells counterparts (Figs.16E&16F). Also, the expression of genes associated with mitochondrial contact sites and cristae organizing system (MICOS) (Apo, Immt and Minos) that play a significant role in dynamic regulation of mitochondrial organization were downregulated in tumor cells compared to the normal cells (Figs.16G-16I). Next, we checked the expression of mitochondria related genes in human melanoma cells and normal melanocytes. In agreement with mouse data, human melanoma cells demonstrated a decreased expression of PGC1α, (Fig.16J) and a decrease in the numbers of mitochondria (Fig.16K) compared to normal melanocytes. Additionally, the expression of OPA1, Apool, and Minos10 genes expression which are associated with mitochondrial fusion and MICOS complex formation were significantly reduced in melanoma cells (A375) compared to normal human melanocytes (NHEM) (Figs.16L-16N). We also estimated the functional characteristics of mitochondria and found that in accordance with altered gene expression, the mitochondrial mass (Fig. 16O and Fig.17B) and mitochondrial ATP (Fig.16P and Fig.17C) production were downregulated in A375 melanoma-cells compared to NHEM. These data show that tumor cells have reduced mitochondrial content and diminished dynamism in structural organization compared to normal cells. We also estimated the gene expression patterns in normal and colon cancer cells in human patients from the cancer genome atlas (TCGA) and correlated it to the patient survival. The two cell types (tumor and normal cells) showed a distinct gene expression pattern with a major alteration in expression of mitochondria related genes (Fig.17D). Further analysis showed that a higher expression of genes associated with mitochondrial structural components (TOMM34, TRAP1) (Fig.17E) and VHPM Ref.09531.584WO1 / Client Ref.2022-294 transporters / carrier proteins (SLC25A5, SLC25A34) (Fig.17F) in tumors resulted in better survival of colon cancer patients compared to patient with lower expression. TOP1MT, a gene encoding type IB topoisomerase protein required for mitochondrial DNA (mtDNA) replication, transcription, and translation was also positively correlated with patient survival (Fig.16G). On the other hand, increased expression of PTEN-induced kinase 1 (PINK1) and SLC25A27 genes respectively encoding mitochondrial serine / threonine-protein kinase and mitochondrial uncoupling protein 4 (UCP4) that separate OXPHOS from ATP synthesis with energy dissipation as heat, are associated with poor prognosis in colon cancer patients (Fig.16H). Together, these data demonstrate that compared to normal cells tumor cells show downregulation of genes associated with dynamic regulation of mitochondrial organization and that decreased expression of mitochondria related genes are associated with diminished survival in cancer patients. TEPP46 mediated enhancement of mitochondrial biogenesis and functions suppresses tumor cell growth and lactate production. Next, we intended to check if enhancement of mitochondrial biogenesis could alter the tumor cell characteristics including growth patterns and cell metabolism. For this we targeted pyruvate kinase M2 using the agonist TEPP464that has been shown to promote mitochondrial fusion and prevent mitochondrial dysfunction in kidney cells protecting against diabetic glomerular pathology. We first confirmed the enhancement of mitochondrial characteristics after TEPP46 treatment of tumor cells. TEPP46 mediated PKM2 activation (Fig.19A) was found to enhance the mitochondrial mass estimated by MitoFM incorporation in mice and human melanoma cells (Fig.18A and Fig.19A) and mitochondrial ATP production (Fig.18B and Fig.19C). TEPP46 mediated PKM2 activation also resulted in enhanced expression of PGC1α gene in tumor cells (Fig.18C). To ascertain the enhanced mitochondrial organization, using electron microscopy we looked at mitochondrial morphology in TEPP46 treated B16-melanoma cells. In line with the mtDNA / nDNA data (Fig.17A) we did not find an increase in the numbers of mitochondria or mitochondrial area in TEPP46treated B16 cells (Fig 19D). However, we observed an increase in the cristae numbers and cristae density after TEPP46 treatment (Fig.18D-18F). A closer observation of tumor cells revealed that while untreated cells had loose widely spaced cristae TEPP46 treatment led to enhanced packing and well-organized cristae in tumor cells (Fig.18D). Electron microscopy data also revealed enhancement of mitochondrial-endoplasmic reticulum interaction (ERMICC) in TEPP46 treated cells (Fig.18G). Thus, these data show that TEPP46 treatment of B16 cells led to an increase in mitochondrial generation, enhanced structure organization and improved function as depicted by increased MitoFM, electron microscopy and ATP production respectively. Next, we checked the effects of TEPP46 treatment on transcriptional profiles of B16 cells. For this we performed total RNA-seq on TEPPP46 treated B16 cells and found a distinct pattern of gene expression in TEPP46 treated and untreated cells with 3499 genes upregulated and 3760 VHPM Ref.09531.584WO1 / Client Ref.2022-294 genes downregulated. Pathway analysis showed a major enrichment in mitochondria related pathways in TEPP46 treated tumor cells (Fig.18H) with an enhanced expression of genes associated with mitochondrial reorganization (Fig.18I) which was confirmed by qRTPCR of various genes in TEPP46 treated B16 (Fig.19E). Together these data show that TEPP46 treatment enhances dynamic regulation of mitochondrial biogenesis and function in B16 cells. Next, we estimated the impact of TEPP46 mediated enhanced mitochondrial characteristics in B16 cells. First under, in vitro conditions, we found that TEPP46 treatment decreased the proliferation of B16 cells, as assessed by incucyte mediated growth pattern estimation (Fig.18J) and by decreased expression of Ki67, a marker of cell proliferation, by FACS analysis (Fig.18K). We also checked the effects of TEPP46 treatment on tumor cells in vivo. For this B16-F10 tumors were induced in mice followed by TEPP46 treatment of tumor bearing mice (40 mg / kg, 4-day-on-2-day-off, ten doses) (Fig. 19F). We noted that TEPP46 treatment significantly reduced the tumor growth rates compared to untreated mice (Fig.18L). Importantly, CD45-CD3- tumor cells in the TME were found to have reduced proliferation estimated by Ki67 expression (Fig.18N) that was associated with increased mitochondrial mass depicted by higher MitoFM incorporation (Fig.18N) in tumor cells. These data show that TEPP46 mediated pharmacological intervention leads to enhancement of mitochondrial biogenesis resulting in reduced cell proliferation and diminished tumor growth rates. TEPP46 mediated enhanced mitochondrial organization enables utilization of glucose through mitochondria in tumor cells. Since tumor cells having malformed mitochondria rely on aerobic glycolysis to sustain their proliferation, we next inquired if TEPP46 mediated enhancement of mitochondrial biogenesis and organization was associated with altered cell metabolism. We first tested the glucose uptake rates and found that TEPP46 treated B16-melanoma cells demonstrated higher glucose uptake (Fig.20A and Fig. 21A). We next performed targeted metabolome analysis in TEPP46 treated B16 cells and found a downregulation of metabolites associated with glycolytic pathway including glucose 6 phosphate and fructose 6 phosphate (Fig.20B) and a corresponding increase in acetyl-CoA (Fig.20C) indicating a shift in metabolism from cytoplasmic glycolysis to mitochondrial oxidative phosphorylation (OXPHOS). In line with this, metabolome analysis revealed a significant decrease in intracellular concentrations of lactate in B16 cells after TEPP46 treatment (Fig.20D) that was also confirmed by a kit-based method (Fig.21B). Production of lactate from glucose is regulated by the levels of lactate dehydrogenase (ldha) enzyme. In line with reduced lactate levels, we found a downregulation of ldha expression at gene and protein levels (Figs.20E-20G) in TEPP46 treated tumor cells. Importantly, the levels of ldha were also downregulated in tumor tissue of TEPP46 treated mice (Figs.20H&20I). Thus, these data show that TEPP46 mediated enhancement of mitochondria results in skewed metabolism with a decrease in lactate and an increase in acetyl-CoA. To ascertain if TEPP46 mediated enhanced VHPM Ref.09531.584WO1 / Client Ref.2022-294 mitochondrial organization supports glucose utilization through TCA cycle, we performed tracer experiments using uniformly labeled13C-glucose. In line with metabolomic analysis, we found a significant enrichment of13Carbons into pyruvate and acetyl-CoA (Fig.20J). Further, metabolites in the TCA cycle including citrate, isocitrate, a-ketoglutarate, succinate, fumarate and oxaloacetate were significantly enriched for glucose derived Carbons showing that TEPP46 mediated activation of PKM2 in tumor cells leads to enhancement of mitochondrial utilization of glucose (Fig.20J). These data show that indeed TEPP46 mediated PKM2 activation favors glucose utilization through mitochondrial TCA cycle. These data also prompted us to examine the activity of pyruvate dehydrogenase complex (PDH) complex, since it depicts the rate-limiting step for pyruvate entry into the TCA cycle. PDH activity is regulated by the levels of inhibitory phosphorylation on the E1a subunit at three serine residues: S232, S293 and S300. We found a downregulation of the inhibitory phosphorylation at S293 in TEPP46 treated B16 cells which was associated with a significant decrease in the expression of pyruvate dehydrogenase kinase 1 (PDK1) (Fig.20K) that facilitates phosphorylation of inhibitory serine residues on PDH. Thus, these data show that TEPP46 mediated PKM2 activation regulates pyruvate metabolism in B16-melanoma cells by regulating PDH complex resulting in enhanced utilization of glucse through mitochondria. TEPP46 and DASA58 mediated enhanced mitochondrial metabolism is associated with increased tumor cell apoptosis and MHCI expression. Alteration of mitochondrial dynamics is associated with the regulation of major histocompatibility complex (MHC)-I antigen expression by cancer cells and their immunogenicity. Mitochondrial fission induces immunoescape in solid tumors through decreasing MHC-I surface expression. Since, we found a dynamic mitochondrial regulation in TEPP46 treated B16 cells (Fig18D, Fig.19E), along with decreased cell proliferation after treatment of tumor cells (Figs.18J and 18L), we next asked if PKM2 activation would induce cell death in tumor cells. For this, B16-melanoma and CT26 cells were treated with two agonists of PKM2, TEPP46 or DASA58. Both the agonists resulted in increased apoptosis in B16-mlenoma (Fig.22A) CT26-colon cancer cells (Fig.22B) that was associated with increased expression of MHCI in both B16 and CT26 tumor cells (Figs.22C&22D). In addition to upregulated MHCI expression, we found a significant upregulation in the expression of MHCII which is associated with CD4 T cell mediated immune responses (Figs.22E&22F). Thus, these data show that PKM2 activation using pharmacologically active small molecules induce cell death in tumor cells which is associated with increased immunogenicity of tumor cells possibly resulting in enhanced immune mediated anti-tumor effects. VHPM Ref.09531.584WO1 / Client Ref.2022-294 Materials and methods Mice and cell culture Four-to-six-week-old C57BL / 6J or Balb / c wild type mice were purchased from Jackson Laboratory. Animals had free access to water and food. All experiments were performed under protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Minnesota. Cancer cell lines used in the present study, the mouse B16F10 and CT26 colorectal carcinoma cell line and human malignant melanoma A375, were obtained from the American Type Culture Collection (ATCC). B16F10, CT26 and A375 cells were cultured in DMEM (Gibco) and RPMI (Gibco) media containing 10% FBS and 1% Penicillin-Streptomycin. Human normal melanocyte cell line (NHEM) (PromoCell; Cat# C-12400) were grown and maintained in melanocyte growth medium M3 as per the providers instructions. Cell lines were routinely checked for the absence of mycoplasma, by microscopic evaluation and PCR-based methods. Antibodies and reagents The Live / Dead detector dye, eBioscience™ Fixable Viability Dye eFluor™ 450, was purchased from ThermoFisher Scientific (Invitrogen, Cat# 65-0865-14). TEPP46 (Cat#S7302) and DASA58 (Cat#S7928) were purchased from Selleckchem Inc. The fluorochrome-labeled antibodies used for flow cytometry measurements in vitro and in vivo were: APC-Ki67, FITC-MHCI, FITC MHCII, FITC-Annexin V. Antibodies used for western blot assay were: PKM2 (CST Cat#4053T), LDHA (Abconal Cat#A0861), p-PDH(S293) (CST Cat#37115), PDH (CST Cat#2784), PDK1 (Cat#3820), beta-actin (Cat#66009-1-Ig; proteintech), anti-mouse IgG HRP-linked (Cat# 7076P2; Cell Signaling), anti-rabbit IgG HRP-linked (Cat# 7074S; Cell Signaling). Cell activation and drug treatment and Incucyte growth estimation Various tumor cell lines were plated (20K cells / well per ml) in 24 well plate overnight followed by treatment with TEPP46 (80 µM) or DASA58 (40uM) in cRPMI or DMEM as recommended for cell lines. PKM2 protein tetramer level was detected by western blot. Various cell characteristics such as mitochondrial mass, ATP concentrations were estimated by FACS analysis. For estimation of growth patterns using Incucyte (PHCBI-SX-5 equipped with G / R module and its compatible lamps as prescribed by the manufacturer) 20K cells / well were plated in 24 well plate followed by treatment with TEPP46. Growth patterns were estimated over a period of 5 days. Gene expression evaluation by RNA sequencing and comparative analysis with metabolome data Total RNA was extracted from cells using RNA isolation kit (Cat# 74104; QIAGEN).2µg of total RNA was used for RNA sequencing. RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). NEBNext Ultra II RNA Library VHPM Ref.09531.584WO1 / Client Ref.2022-294 Prep Kit (Illumina) was utilized for making RNA libraries and samples were sequenced by a 2x150bp Paired End (PE) configuration. RNAseq raw data (fastq or fasq.gz files) quality was checked using FastQC (v0.11.9), and adapter trimming on raw data was performed using Cutadapt (v3.5). Reads with low quality (quality score < 33, error rate > 10%) or short length (<25 bp) after trimming were removed before alignment. We used the reference genome downloaded from ENSEMBL hg38 release 108, and the reference index was built using Star (v2.7.9a) software. Paired end trimmed read alignment and raw read count calculation were performed using RSEM software (v1.3.1). DESeq2 package (v1.36.0) was used for group wise statistical analysis. We considered the genes with fdr <0.05 differentially expressed. Principal component analysis plots were generated using plotly (v4.10.2). All genes with expression levels were used as input for Gene Set Enrichment Analysis (GSEA) (v4.2.3, Broad Institute). Both KEGG and HALLMARK gene sets were selected for enrichment score calculation. We made comparisons between Ova-48h and T60-48h, significantly different (p value < 0.05) genes and metabolites from RNAseq and Metabolomics data were integrated using the STITCH database. Visualization was generated in Cytoscape (v 3.9.0). Flowcytometry analysis Glucose uptake was assayed using 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)- 2-Deoxyglucose; Cat# N13195, Invitrogen) staining by flowcytometry. Briefly, after 48h of CD8 T cells activation with Ova+ / - TEPP46, cells were stained by Live / Dead dye, then incubated in 37 °C / 5% CO2for 30 min in T cell media:PBS (1:1) containing 2-NBDG (20 µM) for 30 minutes and NBDG cells were detected by flowcytometry. To evaluate ATP production in mitochondria, cells were stained by BioTrackerTMATP-Red Live Cell Dye (Millipore, Cat# SCT045) (5 µM) as described by the manufacturer. Mitochondrial mass was evaluated by MitoFM (MitoTracker™ Green FM, Cat# M7514, Invitrogen) staining. Briefly, activated cells were stained by Live / Dead dye and then incubated in PBS containing 50nM MitoFM at 37 °C / 5% CO2for 30 min and MitoFM positive cells were detected by flowcytometry. Targeted cell metabolomics Targeted metabolomics method was used to quantitate >500 endogenous metabolites using QTRAP® 7500 LC-MS / MS System (Sciex, MA, USA). For the purpose, cell pellet was suspended in 50 μL of PBS and heat shock was done by keeping samples on ice for 30s and 37 °C for 90s, then samples were sonicated for 1 minute and 100 μL of extraction buffer (methanol / water 50 / 50) containing 200 ng / mL of debrisoquine (DBQ) as internal standard for positive mode and 200 ng / mL of 4-nitrobenzoic acid as internal standard for negative mode was added. Proteins were precipitated by incubation at -20 °C and samples were centrifuged at 13,000 rpm for 20 minutes at 4 °C. The supernatant was used for LC-MS analysis.2 μL of the prepared sample was injected onto a Kinetex VHPM Ref.09531.584WO1 / Client Ref.2022-294 2.6 μm polar C18100 Å 100 × 3.0 mm (Phenomenex, CA, USA) using SIL-30 AC auto sampler (Shimazdu, Kytoto, Japan) connected with a high flow LC-30AD solvent delivery unit (Shimazdu, Kytoto, Japan) and CBM-20A communication bus module (Shimazdu, Kytoto, Japan) online with QTRAP 7500 (Sciex, MA, USA) operating in positive and negative ion mode. A binary solvent comprising of water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B) was used. The extracted metabolites were resolved at 0.2 mL / min flow rate. The LC gradient conditions were as follows: Initial – 100% A, 0% B for 2.1 minutes; 14 minutes – 5% A, 95% B till 15 minutes; 15.1 minutes – 100% A, 0% B till 20 minutes. The auto sampler and oven were kept at 15 °C and 30 °C, respectively. Source and gas setting for the method were as follows: curtain gas = 40, CAD gas = 9, ion spray voltage = 1700 V in positive mode and ion spray voltage = 1600 V in negative mode, temperature = 350 °C, ion source gas 1 = 30 and ion source gas 2 = 50. The data were normalized to respective internal standard area and processed using MultiQuant 3.0.3 (Sciex). The quality and reproducibility of LC-MS data was ensured using a number of measures. The column was conditioned using the pooled QC samples initially and were also injected periodically to monitor shifts in signal intensities and retention time as measures of reproducibility and data quality of the LC-MS data. Metabolic Flux To evaluate cellular glucose flux, B16-F10 cells were treated with TEPP46 as explained above followed by a washing in PBS and culturing in no-Glu / no-Phenol Red media (Agilent Technology; Cat# 10357-100) containing C13D-Glucose (11 mM) (U13C6, 99%, Cambridge Isotype) Glutamine (2mM), Arginine (1mM), Lysin (1mM), and FBS (10%) for 4 h followed by harvesting the cells for estimation of13C-Glucose by LC / MS. All LC-MS grade solvents including acetonitrile and water were purchased from Fisher Optima grade, Fisher Scientific. High purity formic acid (99%) was purchased from Thermo-Scientific. Debrisoquine and 4-nitrobenzoic acid were purchased from Sigma- Aldrich. Cell fluxomics using QTRAP 7500 Targeted fluxomics analysis method, developed in-house, was used to quantitate 13C labelled (fully or partially) small endogenous metabolites using QTRAP® 7500 LC-MS / MS System (Sciex, MA, USA). For the purpose, 25 μL of PBS was added to the cell pellet and sample tube was plunged into dry ice for 30 sec and 37 °C water bath for 90 sec. This cycle was repeated for two more times and then samples were sonicated for 1 minute and 100 μL of extraction buffer (methanol / water 50 / 50) containing 200 ng / mL of debrisoquine (DBQ) as internal standard for positive mode and 200 ng / mL of 4-nitrobenzoic acid as internal standard for negative mode was added. The samples were vortexed for 1 min and kept on ice for 20 minutes followed by incubation at -20 °C for 20 minutes for protein precipitation. The samples were centrifuged at 13,000 rpm for VHPM Ref.09531.584WO1 / Client Ref.2022-294 20 minutes at 4 °C. The supernatant was transferred to MS vial for LC-MS analysis.20 μL of each prepared sample was mixed to generate the pooled QC sample. One microliter of the prepared sample was injected onto a Kinetex F5, 2.6 μm 100 Å 150 × 2.1 mm (Phenomenex, CA, USA) using SIL-30 AC auto sampler (Shimazdu) connected with a high flow LC-30AD solvent delivery unit (Shimazdu) and Exion 30AD communication bus module (Shimazdu) online with QTRAP 7500 (Sciex, MA, USA) operating in positive and negative ion mode. A binary solvent comprising of water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B) was used. The extracted metabolites were resolved at 0.2 mL / min flow rate. The LC gradient conditions were as follows: Initial – 100% A, 0% B for 2.1 minutes; 14 minutes – 5% A, 95% B till 15 minutes; 15.1 minutes – 100% A, 0% B till 20 minutes. The auto sampler and oven were kept at 15 °C and 30 °C, respectively. Source and gas setting for the method were as follows: curtain gas = 45, CAD gas = 10, ion spray voltage = 2000 V in positive mode and ion spray voltage = 4500 V in negative mode, temperature = 500 °C, ion source gas 1 = 45 and ion source gas 2 = 70. Data Processing The data were normalized to internal standard area and processed using Sciex OS software. Internal Standard normalized data was further QC-RLSC normalized. The quality and reproducibility of LC-MS data was ensured using a number of measures. The column was conditioned using the pooled QC samples initially and were also injected periodically to monitor shifts in signal intensities and retention time as measures of reproducibility and data quality of the LC-MS data. We also have blank solvent runs between set of samples to minimize carry-over effects. Quantitative PCR with reverse transcription analysis Total RNA was extracted from Ova or Ova plus TEPP46-activated CD8 T cells using RNeasy Kit (QIAGEN) and dissolved in RNase-free water. One microgram total RNA was subjected to single-strand complementary DNA synthesis using High-Capacity RNA-to-cDNA Kit (Appliedbiosystems). Primer sequences are listed in Table 2. VHPM Ref.09531.584WO1 / Client Ref.2022-294 Data was obtained using QuantStudio 12K Flex Real-Time PCR System from Applied Biosystems. Housekeeping gene β-actin was utilized for data normalization. Western blot Protein was isolated from variously treated tumor cells by making cell lysates in RIPA buffer containing 1% protease inhibitor. Protein concentrations were estimated using nanodrop (ThermoFisher Scientific). Proteins (20-40 µg) were loaded onto NuPAGETM4-12% Bis-Tris Gel (ThermoFisher Scientific, Cat# NP0322BOX) and transferred onto nitrocellulose membranes by semi-dry method using iBlot 2NC Regular Stacks kit (Invitrogen, Cat# IB23001). Membranes were blocked with 4% BSA in TBST buffer (containing 1X Tris-Buffered Saline, 0.1% Tween 20) followed by overnight shaking in 4⁰C in the presence of primary antibodies against PKM2, LDHA, p-PDH, PDH, and PDK1. The blots were detected with appropriate (anti-rabbit or anti-mouse) HRP- conjugated secondary antibodies. The level of β-actin was used as a control. Densitometric of the bands was analyzed via ImageJ software. Transmission electron microscopy To analyze ultrastructural mitochondrial changes, TEPP46 treated B16-F10 cells were fixed with Karnovsky’s Fixative 15720 kit (Electron Microscopy Science). Fixated cell pellets were embedded in low melting point agarose and sectioned into 1-2 mm pieces, followed by incubation in 1% OsO4in 0.12 M sodium cacodylate buffer for 1 hr. After rinsing with 0.12M (3x10 min), cells were placed in 1% uranyl acetate in 0.1M sodium acetate buffer (overnight / RT). Next day, samples VHPM Ref.09531.584WO1 / Client Ref.2022-294 were rinsed and dehydrated through graded ethanol series, infiltrated with epoxy resin (EMbed-812; Electron Microscopy Science, Hatfield, PA, USA), and embedded in beam capsules. After polymerization in the oven (60oC / 48 hr), blocks containing cells were sectioned using an automated ultramicrotome (UC7 Leica Microsystem). The resulting ultrathin sections (120 nm) were placed in silicon wafers, dried, and carbon-taped in aluminum stubs. We performed SEM imaging in a Helios NanoLab 660 dual-beam microscope (ThermoFisher), using an insertable concentric backscatter detector in 2kV, with a beam current of 0.40 nA, and in immersion mode at 4 um working distance. Overviews of the entire sample were performed in low magnification to identify the cells. Then, high-resolution tile SEM images (80.000x magnification, pixel size 1.76 nm) were performed per cell to obtain the resolution to undoubtfully identify mitochondrial ultrastructural alterations. ImageJ software was used for determining the number of mitochondria per cell, mitochondrial area, cristae number and area, and ER–mitochondria contact coefficient (ERMICC) contact index. ERMICC was calculated using the formula: In this formula L = Interface length (um), Per= Mitochondria perimeter (um) and Dist = distance ER-mitochondria (um) Tumor establishment, mice treatment and immune response analysis in tumor samples C57BL / 6J mice were injected with 1 × 106B16F10 or CT26 cells per mouse subcutaneously in the right flank. Treatment in respective groups was started when tumors reached an average size of approximately 0.075 cm3. TEPP46 (dissolved in 50% PEG in PBS) treatment was performed by oral gavage for 10 days (4 days on and 2 days off) at a dose of 40mg / Kg, once daily. Tumors were measured every 2 days using a digital Vernier caliper, and the tumor volume was calculated using the formula, L × W2 / 2, where L is the long axis of the tumor and W is the short axis of the tumor). Mice were monitored for tumor growth and survival. Mice were sacrificed when the tumor volume reached 1.5 cm3. Although the foregoing specification and examples fully disclose and enable the present invention, they are not intended to limit the scope of the invention, which is defined by the claims appended hereto. All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments VHPM Ref.09531.584WO1 / Client Ref.2022-294 and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention. The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. When used in this specification and the claims as an adverb rather than a preposition, "about" means "approximately" and comprises the stated value and every non-negative value within 10% of that value; in other words, "about 100%" includes 90% and 110% and every value in between. Unless stated otherwise, every range or interval includes both endpoints and every value in between. The invention has been described as “comprising” certain steps and / or elements, which those of skill in the art also “consist of” or “consist essentially of” those steps and / or elements. As used herein, the transitional term “comprising” is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. Where the invention is intended to be more narrowly defined, the terms “consisting of” or “consisting essentially of” also are used to describe the invention. As used herein, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified elements or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. As used herein, a claim reciting “consisting essentially of” occupies a middle ground between closed claims reciting a “consisting of” format and fully open claims that recite “comprising.” VHPM Ref.09531.584WO1 / Client Ref.2022-294 Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

VHPM Ref.09531.584WO1 / Client Ref.2022-294 WHAT IS CLAIMED IS:

1. A method of treating a hyperproliferative disorder in a patient in need thereof, comprising administering to the patient an agent that induces tetrameric pyruvate kinase isoform M2 (tet-PKM2).

2. The method of claim 1, wherein the tet-PKM2 is TEPP46 or DASA58.

3. The method of claim 2, wherein the tet-PKM2 is TEPP46.

4. The method of claim 2, wherein the tet-PKM2 is DASA58.

5. The method of any one of claims 1-4, wherein the hyperproliferative disorder is cancer.

6. The method of claim 5, wherein the cancer is a solid tumor.

7. The method of claim 5, wherein the cancer is melanoma or colon cancer.

8. The method of claim 6, wherein the cancer is melanoma.

9. The method of claim 6, wherein the cancer is colon cancer.

10. The method of any one of claims 1-9, wherein the tet-PKM2 is administered orally or parenterally.

11. A method of suppressing tumor growth in a patient comprising administering to the patient an agent that induces tetrameric pyruvate kinase isoform M2 (tet-PKM2).

12. The method of claim 9, wherein the tet-PKM2 is TEPP46 or DASA58.

13. The method of claim 12, wherein the tet-PKM2 is TEPP46.

14. The method of claim 12, wherein the tet-PKM2 is DASA58.VHPM Ref.09531.584WO1 / Client Ref.2022-294 15. The method of any one of claims 11-14, wherein the hyperproliferative disorder is cancer.

16. The method of claim 15, wherein the cancer is a solid tumor.

17. The method of claim 15, wherein the cancer is melanoma or colon cancer.

18. The method of claim 16, wherein the cancer is melanoma.

19. The method of claim 16, wherein the cancer is colon cancer.

20. The method of any one of claims 11-19, wherein the tumor growth is suppressed by at least 10%.

21. A method of inducing tetramerization of PKM2 in a cell comprising contacting the cell with TEPP46 or DASA58.

22. The method of claim 21, wherein the cell is contacted with TEPP46.

23. The method of claim 22, wherein the cell is contacted with DASA58.

24. The method of any one of claims 21-23, wherein the cells are CD8 T-cells.

25. A method of enhancing mitochondrial structural organization and / or metabolism in CD8 T cells in a patient comprising administering to the patient an agent that induces tetrameric pyruvate kinase isoform M2 (tet-PKM2).

26. A method of inducing apoptosis of tumor cells in a patient, comprising administering to the patient an agent that induces tetrameric pyruvate kinase isoform M2 (tet- PKM2).

27. The method of claim 25 or 26, wherein the tet-PKM2 is TEPP46.

28. The method of claim 25 or 26, wherein the tet-PKM2 is DASA58.

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