Leveraging cancer-evolved resistance mechanisms to enhance EZH2 activity in adoptive t cells

Abstract

The present invention provides constructs comprising activating mutations in EZH1 and or EZH2 and constructs for overexpressing wild-type EZH1 or EZH2 or polypeptides with 95% identity to EZH1 or EZH2 and comprising at least one mutations. Also provided are lymphocytes comprising the constructs and method of making and using the same.

Description

[0001]LEVERAGING CANCER-EVOLVED RESISTANCE MECHANISMS TO ENHANCE EZH2 ACTIVITY IN ADOPTIVE T CELLS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application Nos.63 / 549,888 filed on February 5, 2024, and 63 / 715,248 filed on November 1, 2024, the contents of which are incorporated by reference in their entireties. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under grant number R01CA236209 and 1DP5OD031863-01 awarded by the National Institutes of Health. The government has certain rights in this invention. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (16985200153.xml; Size: 16,739 bytes; and Date of Creation: February 04, 2025) is herein incorporated by reference in its entirety. BACKGROUND The clinical landscape of cancer has been revolutionized by immune checkpoint blockade (ICB); however, despite these advancements, the effectiveness of ICB for solid tumors is limited to approximately half of patients. The development of acquired resistance, in part through the loss of MHC-I antigen presentation, leaves most patients to experience disease progression. Recent advancements have highlighted the use of adoptive cell therapy (ACT) for treatment of ICB resistant cancers (e.g., CAR T cells). However, the solid tumor microenvironment (TME) presents a critical barrier for success of T cell-based therapies largely due to the direct competition between tumor-infiltrating lymphocytes (TILs) and cancer cells for metabolic resources, rendering T cells dysfunctional and exhausted. There is an urgent clinical need for the development of new strategies to engineer adoptive T cells with the ability to sustain function in the harsh solid TME, particularly if these approaches can be combined with other therapies (e.g., small molecule targeted therapy) to improve clinical outcomes. SUMMARY Disclosed herein are constructs comprising activating mutations in EZH1 and / or EZH2. Also provided are lymphocytes comprising the constructs and method of making and using the same. In some embodiments, a lymphocyte comprising a first heterologous promoter operably linked to a first polynucleotide encoding a EZH1 polypeptide or a EZH2 polypeptide, and a second heterologous promoter operably linked to a second polynucleotide encoding a CAR is provided. The EZH1 or EZH2 polypeptide has at least 95% sequence identity to at least one of SEQ ID NOs:1-9 and comprises at least one mutation at a position corresponding to SEQ ID NO: 1 (EZH2 isoform A, see alignment in Figure 10) selected from the group consisting of S21, Y111, W113, F120, T283, T344, T350, S368, S371, T372, S380, T421, T492, Y646, Y666, A682, A692 and combinations thereof. In some embodiments, the lymphocyte is resistant to a EZH2 inhibitor. Another aspect of the invention provides a method of treating cancer in a subject comprising administering a therapeutically effective amount of the lymphocyte described herein and a pharmaceutically acceptable excipient, carrier and / or diluent. Another aspect of the invention provides a method of enhancing a CAR-T cell therapy. The CAR-T comprises a heterologous promoter operably linked to a polynucleotide encoding a EZH1 polypeptide or a EZH2 polypeptide. The EZH1 or EZH2 polypeptide has at least 95% identity to at least one of SEQ ID NOs: 1-9 and comprises at least one mutation at a position corresponding to SEQ ID NO: 1 (EZH2 A, see alignment in figure 10) selected from the group consisting of S21, Y111, W113, F120, T283, T344, T350, S368, S371, T372, S380, T421, T492, Y646, Y666, A682, A692 and combinations thereof. The resulting CAR T cell has an enhanced therapeutic effect as compared to a control CAR T cell. BRIEF DESCRIPTION OF THE DRAWINGS The present technology can be better understood by reference to the following drawings. The drawings are merely exemplary to illustrate certain features that may be used singularly or in combination with other features and the present technology should not be limited to the embodiments shown. Figure 1. Schematic of EZH2 expression in TILs. Activated cytotoxic T cells in the tumor periphery have elevated EZH2. The metabolically hostile solid TME reduces EZH2 expression, promoting T cell exhaustion. Armoring CAR T cells with improved EZH2 activity could promote enhanced infiltration and persistence. Combination with EZH2i treatment can simultaneously impair Tregs and kill cancer cells. Figure 2. Loss of EZH2 is a driver of T cell dysfunction and exhaustion. A. Expression of EZH2 during human peripheral T cell activation with anti-CD3 / 28 antibodies. B. IFN-γ, TNF, and granzyme B triple positive (polyfunctional) ovarian cancer infiltrating CD8+T cells were analyzed based on EZH2 expression. n=5 patients, * P<0.01. C. Immunoblot of TDLN and B16F10 TILs in C57BL / 6 mice. D. Immunoblot of in vitro preactivated, purified primary CD8+T cells treated with EZH2i (EPZ6438 or GSK-126). E. Subset of inhibitory receptors and exhaustion markers from RNA-seq analysis of EZH2i-treated preactivated CD8+T cells. F. Glycolytic dependency measured by ECAR / OCR, * P<0.01. G. Tumor growth curves depict average tumor volume in immunocompetent (C57BL / 6; WT) or immune incompetent (Rag1- / -) mice treated with vehicle or EZH2i (n=5-6 per group). Error bars, SEM, * P<0.05. H. Kaplan Meier survival of recipient mice from Panel G (tumor size> 500 mm3). P value log rank test. I. Genetic deletion of EZH2 in T regulatory cells treated with anti-CTLA-4 therapy. MB49 tumor-bearing EZH2fl / +and FoxP3CreEZH2fl / flmice treated with anti-CTLA-4 on days 7 and 9. One-way ANOVA, ** P < 0.01. Adapted from: A,B30(Fig.S2A,1F); C-H11(Fig.1A,B,D, Fig.2B, Fig.1F); and I32(Fig.3D). Figure 3. EZH2 GoF mutation enhances T cell function. A. Immunoblot of in vitro-activated CD8+EZH2WTand EZH2Y641Fmutant T cells. B. In vitro killing was used to determine CD8+T cell function. MC38SIINFEKLkilling shown as the percentage of viable tumor cells remaining after 10 h coculture of target. C. C57BL / 6 mice inoculated with 5x106MC38SIINFEKLreceived adoptively transferred preactivated CD8+T cells (WT, OT-1, or OT-1 EZH2Y641F) at day 9. D. Tumor growth curves depict average tumor volume in each group (n=8-10), error bars, SEM, * P< 0.01. Figure 4. Heathy subject CAR T cell comparison reveal EZH2 expression correlates with cytotoxicity. A. CD8+T cells were isolated, activated, and transduced with CD19 CAR (28z, CD3z, or 4-1BBz) lentivirus at MOI of 10 from 40 healthy subjects. B. Graph of T cell killing data of CD1928z CARs on CD19+ NALM6 cells from 40 donors. C. Heatmap of killing data from each PBMC donor for all three signaling domains, where dark indicates poor killing and light indicates good killing. CAR T cells generated from 24 healthy donors were subjected to proteomic analysis and CAR T cell killing was found to correlate with HDAC2 (D) and EZH2 (E) expression and inversely correlate with KAT2B (F) expression. Figure 5. EZH2DRmutant is resistant to EZH2i. Stably expressed EZH2WT, EZH2Y111L, or EZH2DRactivated CD8+T cells were treated with 2.5 µM EZH2i (EPZ6438) for 48 h and subjected to immunoblot. Figure 6. EZH2 mutant plasmid construction for PMEL-1 murine system. Figure 7. Construction of CAR EZH2 mutants for human and murine ACT experiments. Figure 8. EZH2 and EZH1 alignment. EZH2 isoform a cartoon with included domains and mutation locations. Domains are highlighted and mutations are listed above. For clarity and ease of reading, the cartoon has been split into two halves. Figure 9. EZH2 isoform A amino acid sequence (SEQ ID NO: 1) with highlighted domains and mutations. Where domains are highlighted below the sequences in various boxes. Boxes with vertical lines indicates phospho mutants, boxes with horizontal lines indicates drug resistant mutants, and boxes with a checkerboard pattern indicates gain of function mutants. Murine mutants are also identified. Figure 10. Sequence alignment of EZH2 and EZH1 in homo sapiens, with each isoform of EZH2 and EZH1 depicted. The top sequence is the consensus sequence (SEQ ID NO: 10) genes listed to the left of the alignment in descending order are: EZH2 isoform B (SEQ ID NO: 2) , EZH2 isoform A (SEQ ID NO: 1) , EZH2 isoform E (SEQ ID NO: 5) , EZH2 isoform D (SEQ ID NO: 4) , EZH2 isoform C (SEQ ID NO: 3) , EZH1 isoform 1 (SEQ ID NO: 6) , EZH1 isoform 3 (SEQ ID NO: 8), EZH1 isoform 4 (SEQ ID NO: 9), EZH1 isoform 2 (SEQ ID NO: 7). For clarity, and ease of reading, the sequence alignment is distributed continuously onto 6 pages as evident by the position numbering to the right of the sequences. All sequences are human. Figure 11. Loss of EZH2 results in T cell exhaustion and mitochondrial dysfunction. A. Western blot analysis of tumor draining lymph node and B16F10 melanoma TIL populations. FACS was used to purify CD4+ and CD8+ lymphocyte populations and in vitro-activated (CD3e / CD28) CD8+ T cells were used as a positive control. B. C57BL / 6 (immune competent) or Rag1- / - (immune compromised) mice were injected subcutaneously with 1x106 B16SIY cells. Starting on day 5, mice were injected (orally) with vehicle (0.5% w / v methyl cellulose and 0.1% Tween-80) or 250 mg / kg EHZ2i (EPZ6438) twice daily for 5 days. C. Tumor growth curves depict an average tumor volume in each group (n=5-6). Error bars, SEM. D. Kaplan-Meier survival of recipient mice (tumor size > 500 mm3). P value denotes statistical significance by log-rank test. E. Representative transmission electron microscopy of activated CD8+ T cells + / - EZH2i. Relative mitochondrial mass FACS analysis (MitoTracker FM) from T cells + / - EZH2i. Data represent mean (n=3), and error bars represent the SEM. P value was determined by unpaired t test. Fig.12. EZH2i treatment abrogates ICB responsive (ICBR) melanoma cell line. A. Cartoon of treatment scheme which consisted of subcutaneous injection of 1x106 B16F10OVA cells into C57BL / 6 mice. Starting on day 2, mice were injected (orally) with vehicle (0.5% w / v methyl cellulose and 0.1% Tween-80) or 250 mg / kg EZH2i (EPZ6438) twice daily for 4 days. Mice underwent intraperitoneal ICB injections of a-PD1 (250 µg) and a-CTLA4 (100 µg) from days 7- 20. B. Detailed treatment timeline of oral EZH2i injections as well as a-PD1 and a-CTLA4 intraperitoneal injections. C. Kaplan-Meier survival of recipient mice (tumor size > 1,000 mm3). D. Tumor growth curves depict an average tumor volume per group (n=10; 5 female, 5 male). Error bars, SEM. Fig. 13. EZH2i treatment diminishes ACT efficacy. A. Cartoon of treatment scheme which consisted of subcutaneous injection of 1x106B16F10OVA cells into C57BL / 6 mice. Starting on day 2, mice were injected (orally) with vehicle (0.5% w / v methyl cellulose and 0.1% Tween-80) or 250 mg / kg EZH2i (EPZ6438) twice daily for 4 days. Simultaneously, OT-1 CD8+ T cells were isolated via magnetic sorting and activated. Activated CD8+ OT-1 T cells were adoptively transferred (4x106) via tail vein injection on day 7. B. Detailed treatment timeline of oral EZH2i injections as well as adoptive transfer of CD8+ OT-1 T cells. C. Kaplan-Meier survival of recipient mice (tumor size > 1,000 mm3). Fig.14. EZH2i results in diminished persistence and T cell killing in both mouse and human T cell models. A. and B. Western blot analysis of mouse (A) and human (B) CD8+ T cells treated with 2.5 µM EZH2i for 72 h. C. Representative T cell killing images of B16F10OVA cells which express IncuCyte NucLightTM Red, where the top image represents T cell killing after 48 h and the bottom image represents no T cell killing. D.20,000 B16F10OVA cells were co-cultured with murine CD8+ OT-1 T cells at a 4:1 effector:target ratio in the presence or absence of 2.5 µM EZH2i. At 48 h, half of the CD8+ OT-1 T cells were removed and re-cultured with 20,000 B16F10OVA cells for a total of two rounds of T cell killing. E. 20,000 CD19 positive NALM6 cells were co-cultured with CD19 BBz CAR T cells at an effector:target ratio of 4:1 in the presence or absence of 2.5 µM EZH2i. As in D., at 48 h co-culture, half of the CD19 BBz CAR T cells were removed and co- cultured with 20,000 NALM6 cells for 48 h for a total of 5 rounds of T cell killing. For both D. and E. images were automatically acquired every 4 h with an ImageExpress confocal HT.AI microscope (Molecular Devices) at 4X resolution. Images were quantified and plotted as percentage of B16F10OVA or NALM6 cells at each time point. F. Percentage of NALM6 cells remaining after four rounds of T cell killing (described previously) with CD8+ T cells from four patients treated with or without 2.5 µM EZH2i. Fig.15. EZH2 overexpression (EZH2OE) results in enhanced T cell persistence and killing. Murine OT-1 CD8+ T cells were isolated from OT-1 splenocytes, activated and transduced 24 h after activation with EZH2OEretrovirus at an MOI of 10. At day 4 post transduction, cells were sorted and pure populations of either CD8+ OT-1 EZH2OEor CD8+ OT-1 T cells were co-cultured with 20,000 B16F10OVAcells at an effector:target ratio of 4:1. At 48 h co-culture, half of the CD8+ OT-1, or CD8+ EZH2OET cells were removed and reintroduced to 20,000 B16F10OVAcells for 48 h for a total of 7 rounds of T cell killing. Images were automatically acquired every 4 h with an ImageExpress confocal HT.AI microscope (Molecular Devices) at 4X resolution. Images were quantified and plotted as percentage of B16F10OVAcells at each time point. Fig.16. Human CD8+ T cells overexpressing EZH2 mutants. A. Human T cells were activated and transduced with the following mutants: EZH2WT, EZH2Y111L, EZH2Y646F, EZH2S21A, and a triple mutant containing all three point mutations (EZH2DR). Flow cytometry showing transduction of EZH2 mutants into human CD8+ T cells. B. Western blot of sorted EZH2 mutant T cells showing EZH2 overexpression. This western blot demonstrates that we can generate EZH2 mutant overexpressing human T cells using a viral transduction method. Fig. 17. A375 cells containing particular EZH2 mutants are resistant to EZH2 inhibitors. A375 cells which have stable expression of EZH2WT, EZH2Y111L, EZH2S21A, EZH2Y646F, or the triple mutant EZH2DR were treated with EZH2 inhibitor EPZ 6438 (0.5 and 2.5 uM) for 48h. Histones were extracted and western blotting for the repressive mark H3K27me3 shows that after EPZ treatment in cells that over express Y111L (drug resistant mutant) or DR (a triple point mutation of EZH2 containing Y111L mutant) are resistant to EPZ6438 treatment. Fig.18. Human T cells with CRISPR knock-in of EZH2Y111L. To genetically modify EZH2 in human T cells, we isolated CD8+ T cells, which will be activated and transduced with lentiviral particles containing chimeric antigen receptors (CARs). At 24 hours post human T cell activation, nucleofection (Lonza 4D Nucleofector System) will be used to introduce recombinant Cas9 bound to the EZH2 mutant guide RNA. For selection purposes, we will simultaneously target TRAC, a surface protein routinely used to select for successful transfer of Cas9 complexes into T cells. On Day 7 post-activation, T cells will be selected for RFP+ (CAR), GFP+ (EZH2 mutant), and CD3- (TRAC-KO). This approach will allow us to replace endogenous EZH2 with the desired mutant EZH2 (all mutants described in the patent application previously can be generated using this method). CAR T cells will be expanded until Day 10 and used in the in vitro and in vivo experiments. Amplicon sequencing was used to determine efficiency of mutagenesis. Successful introduction of EZH2 point mutation Y111L 59% in the pie chart, non-mutated allele 7% in the pie chart, and insertions / deletions 26% in the pie chart. Using this CRISPR method we can introduce EZH2 mutations into human T cells with a 59% efficiency. DETAILED DESCRIPTION The present invention provides constructs comprising activating mutations in EZH1 and or EZH2. Also provided are lymphocytes comprising the constructs and method of making and using the same. As shown herein, the inventors describe leveraging cancer-evolved resistance mechanisms to enhance EZH2 activity in adoptive T cells. The inventors have found that tumor microenvironment (TME)-induced environmental stress leads to epigenome remodeling events within TILs, including loss of the histone methyltransferase EZH2. EZH2, a key component of the Polycomb Repressive Complex 2 (PRC2), induces the repression of gene transcription via methylation of lysine 27 of Histone H3 (H3K27me3). Loss of EZH2, and thereby H3K27me3, in activated T cells leads to transcriptional reprogramming that results in an exhausted T cell phenotype. The inventor’s data suggests that protecting EZH2 activity in activated T cells circumvents this exhaustion and produces a T cell with durable activity in the harsh TME. Data disclosed herein support the premise that protection of T cell EZH2 expression and / or activity will create a more durable and potent adoptive cell therapy (ACT). One embodiment the present invention provides a lymphocyte comprising a first heterologous promoter operably linked to a first polynucleotide encoding a EZH1 polypeptide or a EZH2 polypeptide. Enhancer of zeste homolog 2 (EZH2) is a histone-lysine N-methyltransferase enzyme encoded by the EZH2 gene, that participates in histone methylation and, ultimately, transcriptional repression. EZH2 catalyzes the addition of methyl groups to histone H3 at lysine 27 (H3K27), by using the cofactor S-adenosyl-L-methionine. Methylation activity of EZH2 facilitates heterochromatin formation and thereby silences gene function. EZH2 is the functional enzymatic component of the Polycomb Repressive Complex 2 (PRC2), which is responsible for healthy embryonic development through the epigenetic maintenance of genes responsible for regulating development and differentiation. Mutation or over-expression of EZH2 has been linked to many forms of cancer. EZH2 inhibits genes responsible for suppressing tumor development, and blocking EZH2 activity may slow tumor growth. EZH2 has been targeted for inhibition because it is upregulated in multiple cancers including, but not limited to, breast, prostate, melanoma, and bladder cancer. Mutations in EZH2 are also associate with some neurodegenerative diseases and congenital disorders. EZH2 can also methylate non-histone targets or directly interact with other proteins to activate downstream genes in a PRC2-independent matter. Thus, when mutation or abnormal expression happens, EZH2 is related to cancer initiation, metastasis, immunity, metabolism, and drug resistance. EZH1 (Enhancer of zeste homolog 1), a close homolog of EZH2, contains a SET domain, forms an alternative PRC2 complex and catalyzes H3K27 methylation. EZH1 has traditionally been considered the minor catalytic subunit of PRC2, in part because its histone lysine methyltransferase activity is weaker than that of EZH2. However, it is now recognized that EZH1 is important during differentiation and for postmitotic cells, repression of multipotency of hematopoietic stem cells in favor of more lineage restricted embryonic progenitors and neuronal synaptic development and protection. As used herein, a “lymphocyte” is a circulating cell that is part of the immune system. Lymphocytes include B cells and T cells. In some embodiments, the lymphocyte described herein comprises a CD8+ T lymphocyte and or a CD4+ T lymphocyte. The term “promoter” refers to a nucleic acid sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleic acid segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity. Heterologous promoters useful in the practice of the present invention include, but are not limited to, constitutive, inducible, temporally-regulated, developmentally regulated, chemically regulated, tissue-preferred, tissue-specific promoters and cell- type specific. The heterologous promoter may be a plant, animal, bacterial, fungal, or synthetic promoter. Suitable promoters are known and described in the art. In mammalian cells, typical promoters include, without limitation, promoters for Rous sarcoma virus (RSV), human immunodeficiency virus (HIV-1), cytomegalovirus (CMV), SV40 virus, as well as the translational elongation factor EF-lα promoter or ubiquitin promoter. The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. "Operably linked” refers to a linkage of polynucleotide (or polypeptide) elements in a functional relationship. As used herein, the terms “polynucleotide,” “polynucleotide sequence,” “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide (which terms may be used interchangeably), or any fragment thereof. These phrases also refer to DNA or RNA of natural or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or the antisense strand). The polynucleotides may be cDNA or genomic DNA. Polynucleotides homologous to the polynucleotides described herein are also provided. Those of skill in the art understand the degeneracy of the genetic code and that a variety of polynucleotides can encode the same polypeptide. The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. A protein may comprise different domains, for example, a nucleic acid binding domain and a nucleic acid cleavage domain. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain. One embodiment of the present invention provides a lymphocyte comprising a first heterologous promoter operably linked to a first polynucleotide encoding a EZH1 polypeptide or a EZH2 polypeptide, and a second heterologous promoter operably linked to a second polynucleotide encoding a Chimeric Antigen Receptor (CAR). The first heterologous promoter operably linked to the polynucleotide encoding the EZH1 polypeptide or the EZH2 polypeptide may be part of a construct. The construct comprising the polynucleotide encoding the CAR and the construct comprising the polynucleotide encoding EZH1 or EZH2 maybe on the same construct or multiple constructs and the constructs may be in a single vector, such as a gene delivery or viral vector or may be in more than one gene delivery or viral vector. The term "chimeric antigen receptor" or “chimeric receptor” or "CAR" or "CARs" as used herein refers to a polypeptide having a pre-defined binding specificity to a desired target and operably connected to (e.g., as a fusion or as separate chains linked by one or more disulfide bonds, etc.) the intracellular part of a T-cell activation domain. More particularly, CAR are engineered receptors, which, when expressed graft an antigen specificity onto a cytotoxic cell, for example T cells, NK cells or macrophages. For example, CAR proteins are engineered to give T cells the new ability to target a specific protein. The CARs of the present invention may comprise an extracellular domain with at least one antigen specific targeting region, a transmembrane domain (TM), and an intracellular domain (ID) including one or more co-stimulatory domains (CSD) in a combination that is not naturally found together on a single protein. This particularly includes receptors wherein the extracellular domain and the cytoplasmic domain are not naturally found together on a single receptor protein. Further, the chimeric receptor is different from the TCR expressed in the native T cell lymphocyte. An extracellular domain is external to the cell or organelle and functions to recognize and respond to a ligand. A transmembrane domain spans the membrane of a cell, and an intracellular domain is situated inside a cell. Intracellular co-stimulatory domains provide secondary signals to the cell. They can recruit signaling molecules, cytoskeletal mobilization or induce cell proliferation, differentiation or survival. In the present disclosure a CAR may include an antigen specific extracellular domain, a transmembrane domain and one or more intracellular domains with one or more co-stimulatory domains. The antigen binding domain of a CAR may bind to a single target, or multiple targets. The antigen binding domain of a CAR may bind to a tumor specific antigen. In some embodiments, the tumor specific antigen is a solid tumor antigen. As used herein, the term “construct” refers to recombinant polynucleotides including, without limitation, DNA and RNA, which may be single-stranded or double-stranded and may represent the sense or the antisense strand. Recombinant polynucleotides are polynucleotides formed by laboratory methods that include polynucleotide sequences derived from at least two different natural sources or they may be synthetic. Constructs thus may include new modifications to endogenous genes introduced by, for example, genome editing technologies. Constructs may also include recombinant polynucleotides created using, for example, recombinant DNA methodologies. A construct is a polynucleotide which allows the encoded sequence to be replicated and / or expressed in the target cell. A construct may contain an exogenous promoter, operably linked to any one of the polynucleotides described herein. As used herein, a polynucleotide is “operably connected” or “operably linked” when it is placed into a functional relationship with a second polynucleotide sequence. As used herein, the terms “heterologous promoter,” “promoter,” “promoter region,” or “promoter sequence” refer generally to transcriptional regulatory regions of a gene, which may be found at the 5’ or 3’ side of a polynucleotides described herein, or within the coding region of said polynucleotides. Typically, a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3’ direction) coding sequence. The typical 5’ promoter sequence is bounded at its 3’ terminus by the transcription initiation site and extends upstream (5’ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. In some embodiments, the construct is an expression construct, a vector or a viral vector. A vector is any particle used as a vehicle to artificially carry a foreign nucleic sequence, typically DNA into another cell, where it can be replicated and / or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Expression constructs comprise a heterologous promoter and the nucleic acid sequence encoding protein of interest (e.g., EZH1 or EZH2) which is capable of expression in the cell in which it is introduced. The expression constructs include vectors which are capable of directing the expression of exogenous genes to which they are operatively linked. Such vectors are referred to herein as "recombinant constructs," "expression constructs," "recombinant expression vectors" (or simply, "expression vectors" or "vectors") and may be used interchangeably. Suitable vectors are known in the art and contain the necessary elements in order for the gene encoded within the vector to be expressed as a protein in the host cell. The term "vector'' refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated, specifically exogenous DNA segments encoding the mutant α-gal protein. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Viral vectors are incorporated into viral particles that are then used to transport the viral polynucleotide encoding the protein of interest into the target cells. Certain vectors are capable of autonomous replication in a host cell into which they are introduced. Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., lentiviral vectors). Moreover, certain vectors are capable of directing the expression of exogenous genes to which they are operatively linked. In general, vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification "vector" include expression vectors, such as viral vectors (e.g., replication defective retroviruses (including lentiviruses), adenoviruses and adeno-associated viruses (AAV)), which serve equivalent functions. The vectors are heterogeneous exogenous constructs containing sequences from two or more different sources. Suitable vectors include, but are not limited to, plasmids, expression vectors, lentiviruses (lentiviral vectors), adeno-associated viral vectors (rAAV), among others and includes constructs that are able to express the protein of interest in lymphocytes. A preferred vector is a lentiviral vector or adeno-associated vector. Suitable methods of making viral particles are known in the art to be able to transform cells in order to express the protein of interest in lymphocytes described herein. In some embodiments, the EZH1 or EZH2 polypeptide has at least 95% sequence identity to at least one of SEQ ID NO: 1-9. SEQ ID NO: 1-9, and their alignments and consensus sequence are shown in Figure 10. In some embodiments, the EZH2 polypeptide has at least 95% sequence identity to SEQ ID NO: 1. The EZH2 polynucleotide may be used to generate multiple EZH2 isoforms, for example SEQ ID NOs: 2-5 and those in Figure 10. In some embodiments, the EZH1 polypeptide has at least 95% sequence identity to SEQ ID NO: 6 and the EZH1 polynucleotide may be used to generate multiple EZH1 isoforms, for example SEQ ID NOs: 7-9 and those in Fig. 10. In some embodiments, the polypeptide optionally comprises at least one mutation. The mutations may alter the expression, function or localization of EZH1 or EZH2. In some embodiments, the mutation is an activating mutation. Activating mutations can mimic the proteins physiological activation mechanism. An activating mutation may, without limitation, increase expression, prevent degradation, enhance function, increase signaling or signal strength, or cause the loss of a mechanism of inhibition, for example render a protein resistant to an inhibitor. Without limitation the mutations may comprise those listed in Table 1. A protein “isoform”, or “protein variant", is a member of a set of highly similar proteins that originate from a single gene or gene family and are the result of genetic differences. While many perform the same or similar biological roles, some isoforms have unique functions. A set of protein isoforms may be formed from alternative splicing, variable promoter usage, or other post- transcriptional modifications of a single gene. Through RNA splicing mechanisms, mRNA has the ability to select different protein-coding segments (exons) of a gene, or even different parts of exons from RNA to form different mRNA sequences. Each unique sequence produces a specific form of a protein. In some embodiments, a mutation is described in EZH2 isoform A. EZH1 and its isoforms as well as other EZH2 isoforms may be genetically aligned to the EZH2 isoform A sequence, wherein regions of similarity are found between the sequences to be aligned. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Following alignment, a mutation in EZH2 isoform A may align to a homologous mutation in other EZH2 isoforms or EZH1. These may be referred to as corresponding mutations. Figure 10 is an alignment of several EZH2 and EZH1 isoforms. Mutations in EZH2 isoform A and corresponding mutations in other EZH2 isoforms and EZH1 are shown in Table 1. In some embodiments, the EZH1 or EZH2 polypeptide may comprise at least one mutation at a position corresponding to SEQ ID NO: 1, which is EZH2 isoform A. The alignments and the corresponding mutations are shown in Figure 10 and Table 1. For example, S21 in EZH2 isoform A corresponds to S21 in EZH2 isoform B, E, and D and EZH1 isoform 1, 2, and 4 and to position S27 in EZH1 isoform 2. The Y111 position in EZH2 isoform A corresponds to Y102 in EZH2 isoform E and D, Y111 in EZH2 isoform C, Y112 in EZH1 isoform 1, Y103 in EZH1 isoform 4 and Y118 in EZH1 isoform 2. Thus, the reference sequence used herein is with reference to SEQ ID NO: 1, but the alignment in Figure 10 is meant to allow the references to the position in SEQ ID NO: 1 to be translated into the corresponding positions in the other exemplified sequences. Without limitation EZH2 mutations may comprise S21, Y111, W113, F120, T283, T344, T350A, S368, S371, T372, S380, T421, T492, Y646, Y666, A682, and A692 and combinations thereof and those listed in Table 1. In some embodiments, the EZH2 mutations may comprise S21A, Y111L, W113C, F120L, T283A, T344A, T350A, S368A, S371A, T372A, S380A, T421A, T492A, Y646F or N, Y666D, A682G, and A692V and combinations thereof and those listed in Table 1. Human EZH2 Isoform A corresponds to SEQ ID NO: 1 and NCBI Reference sequence NP_004447.2. Human EZH2 Isoform B corresponds to SEQ ID NO: 2 and NCBI Reference sequence NP_694543.1. Human EZH2 Isoform C corresponds to SEQ ID NO: 3 and NCBI Reference sequence NP_001190176.1. Human EZH2 Isoform D corresponds to SEQ ID NO: 4 and NCBI Reference sequence NP_001190177.1. Human EZH2 Isoform E corresponds to SEQ ID NO: 5 and NCBI Reference sequence NP_001190178.1. Human EZH1 Isoform 1 corresponds to SEQ ID NO: 6 and NCBI Reference sequence NP_001982.2. Human EZH1 Isoform 2 corresponds to SEQ ID NO: 7 and NCBI Reference sequence NP_001308008.1. Human EZH1 Isoform 3 corresponds to SEQ ID NO: 8 and NCBI Reference sequence NP_001308011.1. Human EZH1 Isoform 4 corresponds to SEQ ID NO: 9 and NCBI Reference sequence NP_001308010.1. The term “mutation” as used herein indicates any modification of a nucleic acid and / or polypeptide which results in an altered nucleic acid or polypeptide (i.e., relative to the wild-type nucleic acid or polypeptide sequence). Mutations include, for example, point mutations, substitutions, deletions, or insertions of single or multiple residues in a polynucleotide (or the encoded polypeptide), which includes alterations arising within a protein-encoding region of a gene as well as alterations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences. A genetic alteration may be a mutation of any type. In some embodiments, the mutations are naturally- occurring. In other embodiments, the mutations are the results of artificial selection pressure. Mutations may be made by any means known in the art. Methods for introducing mutations include, but are not limited to site-directed mutagenesis, chemical mutagenesis, radiation mutagenesis, transposon insertion, or viral vector-based mutagenesis. In some embodies gene editing is used to introduce a mutation. In some embodiments, the gene edition comprises homology-directed repair using CRISPR-Cas9 system. In some embodiments, the CRISPR editing may be used in primary T lymphocytes. An example of CRISPR based editing of T lymphocytes can be seen in Figure 18. In some embodiments, a construct including an EZH1 or EZH2 polynucleotide may be provided. The construct may provide one or more copies of EZH1 or EZH2 in addition to endogenous EZH1 to EZH2. In some embodiments, the construct causes overexpression of EZH1 or EZH2. Overexpression of EZH1 or EZH2 results in additional copies of EZH1 or EZH2 being generated. In some embodiments, both EZH1 and EZH2 are overexpressed. EZH1 and EZH2 inhibitors may be suppressed or removed with the addition of EZH1 or EZH2 polypeptides. In some embodiments, a construct including an EZH1 or EZH2 polynucleotide encoding the EZH1 or EZH2 polypeptide may comprise a mutation or a combination of mutations resulting in a change of an amino acid present in at least one position of the polypeptide. By way of example, and not limitation combination of mutations in EZH2 isoform A may comprise S21A, Y111L and / or Y646F. Additional combinations may include one or more mutations that confer drug resistance, such as W113C, F120L and Y666D or mutations in or near the SET domain. Combinations of mutations may have additive results or confer more than one change to the protein. For example, a combination of mutations may make the EZH2 protein more resistant to inhibitors than one mutation by itself. In some embodiments, a lymphocyte may comprise a construct described herein. A lymphocyte may be a T lymphocyte, for example a CD4+ or CD8+ T lymphocyte. The lymphocyte may be an ex-vivo lymphocyte. In some embodiments a construct described herein or an expression vector comprising the construct is introduced into a cell for purposes of expressing the polypeptide of the expression vector. In particular embodiments the cell is a T lymphocyte. The expression vector can be introduced into the cell by any means known in the art. These means may include transfection or transduction. Transfection is the process of introducing nucleic acids into cells by non-viral methods. Transduction is the process whereby foreign DNA is introduced into another cell via a viral vector. These are common tools to introduce a foreign gene into host cells. In some embodiments the lymphocyte is an “engineered lymphocyte”. An engineered lymphocyte is a lymphocyte which has been genetically modified to (a) express one or more exogenous polynucleotides, (b) over-express one or more endogenous and / or one or more exogenous polynucleotides, such as those included in a vector, or which have an alteration in expression of an endogenous gene or (c) knock-out or down-regulate an endogenous gene. In addition, certain genes may be physically removed from the genome (e.g., knock-outs) or they may be engineered to have reduced, altered or enhanced activity. In some embodiments, the engineered lymphocyte may comprise one more constructs described herein, for example comprising a CAR and / or a EZH1 or 2 polynucleotide. In some embodiments, a lymphocyte may comprise one or more copies of EZH1 and or EZH2 in addition to endogenous EZH1 to EZH2. The construct may cause the overexpression of EZH1 and or EZH2. In some embodiments the construct provides a mutated EZH1 and or EZH2 while endogenous EZH1 and or EZH2 are not modified. In some embodiments, overexpression of EZH1 or EZH2 may increase lymphocyte persistence and or increase lymphocyte cell killing. In some embodiments, a lymphocyte described herein may be used therapeutically, for example in adoptive cell therapy. In some embodiments, a lymphocyte comprising a construct described herein, or an engineered lymphocyte described herein may be resistant or more resistant to an EZH1 or EZH2 inhibitor. As used herein, an “EZH1 or EZH2 inhibitor” refers to any compound or molecule that is capable of inhibiting the action of, and or reducing the expression and or function of EZH1 or EZH2. In some embodiments, an EZH1 or EZH2 inhibitor may comprise an antibody, small molecules, a miRNA, siRNA, oligonucleotides, cytokines, agonists, a dominant negative EZH1 or EZH2, and combination thereof. By way of example, and not limitation, EZH1 or EZH2 inhibitors comprise, Tazemetostat (EPZ-6438), Tulmimetostat, GSK126, GSK2816126, CPI-1205, SHR2554, and PF-06821497, 3-deazaneplanocin A (DZNeP), El1. Methods: Another aspect of the present disclosure provides methods of generating a lymphocyte comprising introducing the construct described herein into an ex-vivo lymphocyte. The expression vector can be introduced into a lymphocyte by any means known in the art. These means may include transfection or transduction. Transfection is the process of introducing nucleic acids into cells by non-viral methods. Transduction is the process whereby foreign DNA is introduced into another cell via a viral vector. These are common tools to introduce a foreign gene into host cells. Among others, additional means include transformation, and conjugation. In some embodiments lymphocytes may be transduced with retroviral particles containing a bicistronic expression cassette coding for a CAR and an EZH1 or EZH2 polypeptide described herein. In some embodiments a tag or marker may also be comprised in the vector. In some embodiments separate viral vectors may be used to deliver a CAR and an EZH1 / 2 polynucleotide. In some embodiments, a method of treating cancer is provided. The method comprising administering a therapeutically effective amount of a lymphocyte described herein and a pharmaceutically acceptable excipient, carrier and / or diluent to a subject in need. A “subject in need thereof” as utilized herein may refer to a subject in need of treatment for a disease or disorder associated with a cancer. A subject in need thereof may include a subject having a cancer that is characterized by gross abnormality visible by X-ray, computerized tomography (CT), or magnetic resonance imaging (MRI), but which has not been diagnosed as a central nervous system (CNS) tumor by histology or immunofluorescence. The term “subject” may be used interchangeably with the terms “individual” and “patient” and includes human and non-human mammalian subjects. As used herein, “subject” or "patient" refers to both mammals and non-mammals. “Mammals” include any member of the class Mammalia, such as humans, non-human primates (e.g., chimpanzees, other apes and monkey species), farm animals (e.g., cattle, horses, sheep, goats, and swine), domestic animals (e.g., rabbits, dogs, and cats), and laboratory animals (e.g., rats, mice, and guinea pigs). The term “subject” does not denote a particular age or sex. In one embodiment, the subject is a human. Cancer is a group of disease involving abnormal cell growth with the potential to invade or spread to other parts of the body. Treating cancer in a subject includes the reducing, repressing, delaying or preventing cancer growth, reduction of tumor volume, and / or preventing, repressing, delaying or reducing metastasis of the tumor. Treating cancer in a subject also includes the reduction of the number of tumor cells within the subject. The term "treatment" can be characterized by at least one of the following: (a) reducing, slowing or inhibiting growth of cancer and cancer cells, including slowing or inhibiting the growth of metastatic cancer cells; (b) preventing further growth of tumors; (c) reducing or preventing metastasis of cancer cells within a subject; and (d) reducing or ameliorating at least one symptom of cancer. In some embodiments, the optimum effective amount can be readily determined by one skilled in the art using routine experimentation. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer comprises a cancer associated with hyperactive or overexpression of EZH1 or 2. In some embodiments, the cancer is being treated with an immunotherapy, for example CAR therapy or immune checkpoint inhibitors. Such cancers may include, but are not limited to melanoma, breast cancer, prostate cancer, hepatocellular carcinoma, lymphoma (including lymphoma sub-types such as germinal center diffuse large B cell lymphoma, activated B cell-like diffuse large B cell lymphoma, follicular lymphoma, cutaneous T cell lymphoma, mantel cell lymphoma, adult T cell leukemia / lymphoma), leukemias, myeloma, ovarian cancer, lung cancer, colorectal cancer, esophageal cancer, bladder cancer, sarcoma, endometrial cancer, glioblastoma (type of brain cancer), and kidney cancer. As used herein, the terms “administering” and “administration” refer to any method of providing a pharmaceutical preparation or composition to a subject described herein. Such methods are well known to those skilled in the art and include, but are not limited to, transdermal administration, administration by inhalation, nasal administration, and parenteral administration, including injectable such as intramuscular administration, intradermal administration, and subcutaneous administration. In some embodiments, the lymphocyte may be administered intratumorally or systemically. In some embodiments, the subject may be administered an EZH1 or 2 inhibitor or other methods of standard of care, such other immunotherapies, chemotherapy, radiation or surgery. As used herein, the terms “treating” or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and / or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder. In some embodiments, the subject is responsive to therapy with the engineered HSC cells disclosed herein and include use in combination with one or more additional therapeutic agents. The term "treat" further include the reduction in one or more symptom associated with cancer. As used herein the term “effective amount” refers to the amount or dose of the compound that provides the desired effect. In some embodiments, the effective amount is the amount or dose of the compound, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment. Suitably the desired effect may be reducing tumor size, volume, or number. As used herein, the term “carrier” refers to a pharmaceutically acceptable solid or liquid filler, diluent or encapsulating material. A water-containing liquid carrier can contain pharmaceutically acceptable additives such as acidifying agents, alkalizing agents, antimicrobial preservatives, antioxidants, buffering agents, chelating agents, complexing agents, solubilizing agents, humectants, solvents, suspending and / or viscosity-increasing agents, tonicity agents, wetting agents or other biocompatible materials. A tabulation of ingredients listed by the above categories, may be found in the U.S. Pharmacopeia National Formulary, 1857-1859, (1990). Some examples of the materials which can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen free water; isotonic saline; Ringer's solution, ethyl alcohol and phosphate buffer solutions, as well as other nontoxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions, according to the desires of the formulator. Examples of pharmaceutically acceptable antioxidants include water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and metal-chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like. In another embodiment, the present formulation may also comprise other suitable agents such as a stabilizing delivery vehicle, carrier, support or complex-forming species. The coordinate administration methods and combinatorial formulations of the instant invention may optionally incorporate effective carriers, processing agents, or delivery vehicles, to provide improved formulations for delivery of the construct or lymphocyte comprising the construct described herein. The lymphocyte composition my additionally include a biologically acceptable buffer to maintain a pH close to neutral (7.0-7.3). Such buffers preferably used are typically phosphates, carboxylates, and bicarbonates. More preferred buffering agents are sodium phosphate, potassium phosphate, sodium citrate, calcium lactate, sodium succinate, sodium glutamate, sodium bicarbonate, and potassium bicarbonate. The buffer may comprise about 0.0001-5% (w / v) of the vaccine formulation, more preferably about 0.001-1% (w / v). Other excipients, if desired, may be included as part of the final lymphocyte composition. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g. , aluminum hydroxide); and preservative. Another aspect of the present invention provides a method of enhancing CAR-T cell therapy. In some embodiments, the CAR-T cells comprise a construct described herein. For example, the construct may encode an EZH1 or EZH2 polypeptide with an activating mutation including those described in Table 1. Without wishing to be bound by any theory, data described herein demonstrate that CAR-T cells comprising an EZH1 or EZH2 activating mutation may survive longer, be more functional or have improved infiltration in a tumor microenvironment. Enhancing CAR-T cell therapy can comprise increasing CAR-T cell survival, increasing CAR-T cell tumor infiltration, and / or increased tumor cell killing. In some embodiments, the CAR-T cells may have increased global or site specific H3K27me3. In some embodiments, a method for enhancing EZH2 inhibitor therapy is provided. The method comprises administering any of the lymphocytes described herein prior to, or together with EZH2 inhibitor therapy. EZH2 inhibitor therapy may comprise treatment with Tazemetostat. In some embodiments, a method for enhancing immune checkpoint blockade (ICB) therapy is provided. ICB therapy includes immune checkpoint inhibitors. ICB drugs block checkpoint proteins, which are molecules that regulate the immune system and prevent it from becoming too strong. When these checkpoints are blocked, the immune system can better recognize and destroy cancer cells. Immune checkpoint inhibitors include but are not limited to anti-PD-1 antibody, anti- PD-L1 antibody, anti-CTLA4 antibody, anti- LAG-3 antibody, and / or anti-TIM-3 antibody. Approved checkpoint inhibitors in the U.S. include atezolizumab, ipimilumab, pembrolizumab, and nivolumab, and tislelizumab. The inhibitor need not be an antibody but can be a small molecule or other polymer. If the inhibitor is an antibody it can be a polyclonal, monoclonal, fragment, single chain, or other antibody variant construct. Inhibitors may target any immune checkpoint known in the art, including but not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and the B-7 family of ligands. Combinations of inhibitors for a single target immune checkpoint or different inhibitors for different immune checkpoints may be used. In some embodiments, the cancer is ICB resistant. In some embodiments, the method comprises administering any of the lymphocytes described herein prior to, or together with EZH2 inhibitor therapy. In some embodiments, the method comprises administering any of the lymphocytes described herein prior to, or together with ICB therapy. In some embodiments, the method comprises administering any of the lymphocytes described herein prior to, or together with EZH2 inhibitor therapy and ICB therapy. By way of example, and not limitation a lymphocyte described herein may be administered together with an EZH2 inhibitor therapy, followed by a ICB therapy. The lymphocyte, EZH2 inhibitor therapy or ICB therapy may be administered one or more times separated by days, weeks or months. Additional definitions The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and / or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps. 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 facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter. "Percentage of sequence identity'' or "percent similarity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or peptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. The term "substantial identity'' or "substantial similarity" of polynucleotide or peptide sequences means that a polynucleotide or peptide comprises a sequence that has at least 75% sequence identity to the reference sequence. Alternatively, percent identity can be any integer from 75% to 100%. More preferred embodiments include at least: 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described. These values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. "Substantial identity" of amino acid sequences for purposes of this invention normally means polypeptide sequence identity of at least 75%. Preferred percent identity of polypeptides can be any integer from 75% to 100%. More preferred embodiments include at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.7%, or 99%. Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.” As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus ≤10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term. As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter. 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. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise. In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.” No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references. Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art 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. The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims. EXAMPLES Example 1: In the following example, the inventors describe leveraging cancer-evolved resistance mechanisms to enhance EZH2 activity in adoptive T cells. The inventors have found that tumor microenvironment (TME)-induced environmental stress leads to epigenome remodeling events within TILs, including loss of the histone methyltransferase EZH211. EZH2, a key component of the Polycomb Repressive Complex 2 (PRC2), induces the repression of gene transcription via methylation of lysine 27 of Histone H3 (H3K27me3)12. Loss of EZH2, and thereby H3K27me3, in activated T cells leads to transcriptional reprogramming that results in an exhausted T cell phenotype. The inventors data suggests that protecting EZH2 activity in activated T cells circumvents this exhaustion and produces a T cell with durable activity in the harsh TME. Data disclosed herein support the premise that protection of T cell EZH2 expression and / or activity will create a more durable and potent adoptive cell therapy (Figure 1). Background The evolving landscape of metastatic melanoma therapy has witnessed a paradigm shift through the introduction of immune checkpoint blockade (ICB) therapeutics, especially with monoclonal antibodies targeting CTLA-4 and PD-1, which are quickly becoming a standard-of- care treatment of patients with unresectable or metastatic melanoma1,13,14. The introduction of ICB greatly improved upon the clinical outcomes over traditional chemotherapy. However, durable response is experienced for only a minority of patients and of those ICB responders, only half survive 6.5 years after treatment discontinuation2,15–20. In a wide range of cancers, efficacy of ICB remains constrained by low initial response rates and acquired resistance, which is driven in large part through loss of MHC-I presentation and immune recognition of tumor cells21–26. There is a critical need for development of new treatment strategies that bypass limitations to current ICB, such as decreased antigen presentation by tumor cells correlating with poor outcomes. One such promising strategy is adoptive cell therapy (ACT) which can leverage engineering of T cells to overcome challenges like loss of tumor antigen presentation. Cytotoxic T cells play a vital role in cancer prevention during normal homeostasis and have been harnessed for robust therapeutic intervention27,28. While ACTs utilizing cytotoxic T cells have shown success in hematological malignancies, tumor-intrinsic factors in metastatic melanoma, as well as in other solid tumors, presents a critical barrier to the successful application of this therapy for solid malignancies29. Investigations have underscored the histone methyltransferase EZH2 as a critical regulator of T cell biology11,30,31. EZH2 expression is associated with multiple effector, triple positive, cytokine-producing T cells (IFN-y, TNF, granzyme B). Zhao et al., found that EZH2 is robustly upregulated during T cell activation in human peripheral T cells stimulated with anti-CD3 / 28 antibodies (Fig. 2A), and T cell polyfunctionality is lost in EZH2-deficient lymphocytes during solid tumor infiltration (Fig.2B)30. Further work implicates the hypoglycemic conditions of the solid TME in the reduction of EZH2 expression in TILs and dampened T cell function11,30. Additionally, the inventors have shown that the product of EZH2 enzymatic activity, the H3K27me3 mark, is reduced in TILs relative to tumor-draining lymph node (TDLN) lymphocytes in a mouse model of melanoma (Fig. 2C)11. The inventors have previously shown that pharmacological inhibition of EZH2 leads to a functionally exhausted T cell phenotype including an increase in inhibitory receptors (e.g., PD-1) and transcription factors known to drive exhaustion (e.g., Tox2) (Fig.2D&E)11. This EZH2i-induced exhausted phenotype coincided with an increased dependence on glycolytic metabolism in T cells, indicated by the ratio of basal ECAR / OCR (Glycolysis / OxPhos; Fig.2F), suggesting that EZH2 activity is necessary for T cell polyfunctionality in the hypoglycemic solid TME11. In vivo inhibition of EZH2 negatively impacts the immune control of the B16.SIY cell line, a variant of B16F10 that expresses a highly immunogenic model antigen, further underscoring its importance in mediating antitumor immunity (Fig.2G&H)11. The role for EZH2 is not limited to CD8+T cells and recent findings by Goswami et al. have shown that genetic deletion of EZH2 specifically in T regulatory cells enhances anti-CTLA4 therapy (Fig.2I)32. Collectively, these data support a critical role for EZH2 in T cell biology and the ability of the immune system to control solid tumor growth. Given the pivotal role of cytotoxic T cells in shaping cancer outcomes, including in metastatic melanoma, comprehensive evaluation of strategies to enhance EZH2 activity, and understanding how these changes impact cytotoxic T cell physiology, is paramount to gauging the clinical potential of armoring T cells with EZH2. Several gain-of-function (GoF) mutations in EZH2 have been reported in neoplastic cells that result in immune evasion either through hyperactive methylation of H3K27me3, repression of EZH2 inactivation signals, or resistance to EZH2 inhibitors (EZH2i)39–43. Thus, understanding how to leverage these EZH2 mutations to improve T cell function will offer additional therapeutic avenues for EZH2i. Several EZH2i have been developed and are currently in use either in the clinic or under clinical trials (Tazemetostat, GSK126, CPI-1205, SHR2554, and 06821497) for use as a monotherapy or in combination with ICBs32. While EZH2i have been used in several clinical trials and shown modest success for hematological malignancies, durable response in solid tumors was minimal44,45. The inventors predict this low response to EZH2i for solid tumors is in large part due to loss of EZH2 activity in cytotoxic T cells and induction of an exhausted TIL phenotype, as supported by their previous work using EZH2i in immune-competent mice11. Therefore, the inventors note that using cancer- inspired, GoF EZH2 mutations to armor cytotoxic T cells will enhance durability of CAR T therapy and increase EZH2i efficacy. Exogenous expression of a GoF EZH2 mutant improves tumor control EZH2 GoF mutations have been observed in many forms of cancer and are correlated with metastasis and poor prognosis40–42,46. One such GoF mutant (murine EZH2Y641F, human EZH2Y646F) functions by increasing substrate specificity, preferentially increasing methylation of H3K27me2 over mono-methylation sites, resulting in increased gene silencing43,47,48. To examine if this type of GoF mutant could enhance CD8+T cell function, we engineered murine T cells with the EZH2Y641Fvariant which resulted in elevated H3K27me3 (Fig. 3A). Tumor-specific T cells were generated from OT-I and OT-I-Lck-EZH2Y641Fmice; activated CD8+T cells expressing the EZH2Y641Fmutant were superior at killing of MC38SIINFEKLcells in vitro (Fig. 3B). In vivo experiments showed MC38SIINFEKLtumor-bearing mice that received adoptive transfer of either OT-I or OT-I EZH2Y641F-positive T cells were able to control tumor growth significantly better than mice that received either tumor-specific (OT-I) and nonspecific (wt) control cells (Fig. 3C&D). These findings are consistent with the concept that EZH2 is necessary to protect TILs from TME induced metabolic stress, and manipulation of EZH2 could result in further advancement of cellular therapies against solid tumors. EZH2 expression impacts CAR T cell function CAR T cells have been used clinically for patients with leukemias and lymphomas since 201749. While this form of treatment works well for hematological malignancies, factors innate to solid tumors such as the hypoglycemic conditions, blunt the successful implementation of ACT29,50. To illuminate factors which contribute to CAR T cell response, 40 unique healthy patient PBMCs were obtained and transformed into CAR T cells (Fig. 4A). CAR T cell killing assays were performed and a range of responses were observed, where some patient T cells made better CARs than others (Fig.4B). For each patient sample, CAR T cells were generated which contained three separate co-stimulatory domains (28z, 4-1BBz, and CD3z) and killing assays were performed with NALM6 cells. Interestingly, it was found that killing capacity of a patient CAR T cell had no correlation to a given signaling domain (Fig. 4C), suggesting universal T cell intrinsic factors impacted CAR T cell functionality. Proteomics (>7000 protein IDs) was performed on these patient CAR T cells which revealed a correlation between expression of histone modifying enzymes (HDAC2 and EZH2) with improved T cell killing, where patients who had higher EZH2 expression had improved CAR T cell killing (Fig.4D-F). EZH2i resistant CAR T cells By screening a combination of transduced GoF EZH2 mutants in activated CD8+T cells, we uncovered a triple mutant that exhibited robust drug resistance (DR) to EZH2i (EZH2S21A,Y111L,Y646F, or herein EZH2DR). This triple mutant contains a mutation which prevents proteasomal degradation through phosphorylation by AKT (S21A), which enhances methylation of H3K27me2 to H3K27me3 (Y646F), and that was previously reported to be associated with EZH2i resistance (Y111L)39,43,51,52. Figure 5 shows how EZH2DRis significantly more EZH2i resistant than the reported EZH2Y111L. Prophetic Data Demonstrate enhanced EZH2 activity can protect T cell function in the solid tumor microenvironment The inventors will leverage cancer cell evolved GoF resistance mechanisms to protect EZH2 function in T cells, which they predict will increase tumor killing and infiltration, while decreasing exhaustion. It is established that EZH2 function can be enhanced by single amino acid mutations that stabilize EZH2 protein levels by preventing AKT-mediated phosphorylation and proteasomal degradation (EZH2S21A), and that increase EZH2 methyltransferase activity and thereby H3K27me3 levels (EZH2Y646F). Here, GoF EZH2 mutant CD8+T cells will be used in a series of in vitro and in vivo tumor killing experiments. Ex vivo tumor analysis will be used to determine if modulation of EZH2 in T cells results in increased persistence and infiltration into the tumor microenvironment. Mutant T cells will also be functionally characterized. Experimental Design Expression of EZH2 mutants. Activated T cells will be transduced with retroviral particles containing a bicistronic expression cassette coding for GFP and one of three versions of EZH2: EZH2 wild type (EZH2WT), EZH2S21A, or EZH2Y646F(Fig.6). For these studies, the inventors will use the PMEL-1 mouse model system, which is a more biologically relevant T cell receptor engineered system compared to the OT-I model system. This system was established to model the treatment of melanoma using ACT, where PMEL-1 mouse lymphocytes specifically recognize the melanoma antigen gp10053,54. Prior to viral transduction, naïve lymphocytes will be isolated from a single cell suspension of splenocytes (from spleens of PMEL-1 mice) by negative selection using the CD8+T cell isolation kit (Miltenyi). Purified CD8+T cells will then be activated the same day using plate-bound anti-CD3 and soluble anti-CD28 antibodies in media containing recombinant murine IL-2 (100U / mL). T cells will then be transduced with retroviral vectors 24 h after activation and maintained at 1x106 / mL in media containing IL-2. In this way, we can monitor if overexpressing exogenous EZH2 alone is enough to impact T cell function or if some of the common hyper trimethylating EZH2 mutations are required for increased T cell activity. Day 4 T cells will be sorted for GFP+T cells which contain the EZH2 mutations described in Figure 6 and re-stimulated (anti-CD3, anti-CD28). Day 5 T cells will be used for in vitro and in vivo T cell killing experiments. In vitro T cell mediated tumor killing. Purified and activated PMEL-1 CD8+T cells will be transduced with EZH2 GFP (EZH2WT, EZH2S21A, or EZH2Y646F) or vector GFP retrovirus at 24 h post activation and sorted at Day 4 post activation for GFP+T cells as described previously. Target cells expressing gp100+, specifically murine melanoma B16 (H-2b) cells containing nuclear RFP (IncuCyte® NucLight™ Red), will be used for PMEL-1 killing assays54. Cells will be plated for 16 h prior to culturing with T cells. The co-cultures will be conducted at target: effector (T:E) ratios of 8:1 down to 0.5:1 in 96 well live cell imaging plates. Plates will then be automatically imaged every 2 h with an ImageExpress confocal HT.AI microscope (Molecular Devices) in the RFP channel. At each timepoint, nine unique sites will be imaged, and the total number of RFP+(target) cells quantified. Plates are imaged for up to 72 h, and the values will be normalized to target cell counts at t = 0. Experiments will be performed in 3 independent biological replicates. The statistical analysis will consist of pairwise ANOVA post-hoc tests or their equivalent Mann- Whitney tests. All tests will employ 2-sided α=0.01 significance levels to adjust for multiple comparisons. In vivo tumor-specific killing. For in vivo T cell killing experiments, purified and activated PMEL-1 CD8+T cells will be transduced with EZH2 GFP (EZH2WT, EZH2S21A, or EZH2Y646F) or vector GFP retrovirus at 24 h post activation and sorted at Day 4 post activation for GFP+T cells as described above. T cells will be adoptively transferred (4 million / mouse) into mice bearing subcutaneous B16 (H-2b) tumors (N=20 per condition, 10F / 10M). Tumor volume will be measured daily using caliper measurements until tumor endpoint (>1000 mm3). Additionally, the percent of CD8+ / GFP+vs. CD8+ / GFP- tumor infiltrating lymphocytes (TILs) will be determined from tumors at endpoint by flow cytometry. Analysis plan. Time in days to reach endpoint will be plotted as Kaplan-Meier curves and compared for differences with the 2-sided log-rank test at α=0.05. To estimate the power achieved by the log-rank test with 20 recipient mice / group, we assume that the number of days until tumor sizes exceed threshold follow log normal distributions that differ in their group means while maintaining equal group CVs [coefficients of variation] of 35%. Under this assumption, a simulation study with 10,000 simulated tests using SAS v9.4 software indicates that the 2-sided log-rank test at α=0.5 will achieve estimated power ±standard error of 92.77% ±0.26% to detect a 1.5-fold change between groups in the average number of days until tumor sizes exceed threshold. Quantities of tumor-infiltrating CD8+cells extracted from harvested tumors will be expressed as number of cells per mm3of tumor, summarized by donor genotype as means and SDs, and compared for genotype differences at α=0.05 with Poisson regression, negative-binomial regression, or Wilcoxon’s rank-sum test as warranted by the degree of adherence to distributional assumptions. Functional characterization. Transduced murine PMEL-1 CD8+T cells will be obtained before and after in vitro T cell killing assays and from in vivo TILs as described previously. Previously established flow cytometry panels will be used to monitor changes in cytokine production as well as T cell exhaustion in EZH2 mutant CD8+T cells. Flow cytometry panels contain cytokine production and T cell exhaustion markers: CD4, CD8, CD45RA, CCR7, TIGIT, CTLA4, CD223, Lag3, PD-1, Tim3, IFN-^, IL-2, and Granzyme. TILs will be assayed for EZH2 stability and activity by immunoblot for EZH2 and H3K27me3 (as in Fig.2C, 5). Furthermore, the inventors seek to understand how mutant versions of EZH2 impact intracellular T cell signaling because a pathway level understanding of EZH2-mediated dysregulation may identify new vulnerabilities for future therapeutic development or uncover novel targets for further engineering T cells for ACT. Thus, transduced PMEL-1 CD8+T cells will also be subjected epigenomic, transcriptomic, and proteomics analyses. For epigenomics, the inventors will use cleavage under targets & release using nuclease (CUT&RUN) to genomically localize wild-type and mutant versions of EZH2. CUT&RUN will be performed in accordance to EpiCypher methodology. The inventors will affinity enrich using either α-EZH2, α-H3K27me3, or IgG control antibodies. We will use qPCR-ChIP to validate the top 20 genomic sites. For transcriptomics, RNAseq will be performed and the inventors will use qPCR to validate the top 50 dysregulated transcripts. Epigenomic and transcriptomic data integration will be used to determine direct or indirect impacts of EZH2 mutant activity at a given locus. In brief, normalized data sets will be integrated by using various techniques, such as correlation analysis. Multi-omics gene-set analysis (MOGSA) (integrative single sample gene-set analysis) will be used to investigate data integration at the pathway / gene set level. For proteomics, the inventors will measure proteins and protein phosphorylations levels altered by the EZH2 mutations. To identify proteins and site-specific protein phosphorylation, we will use a quantitative mass spectrometric workflow based on Thermo Tandem Mass Tags. Significant proteins and phosphopeptides will be imported into pathway analysis pipelines using Ensemble of Gene Set Enrichment Analyses (EGSEA), Ingenuity Pathway Analysis (Qiagen), and PTM Enrichment Analysis (PTM-SEA). Expected outcomes The inventors predict that elevated EZH2 itself or expression of EZH2 GoF mutations proposed herein will decrease T cell exhaustion and positively impact T cell killing both in vitro and in vivo. Furthermore, we anticipate that T cells with EZH2WT, EZH2S21A, or EZH2Y646Fwill more effectively infiltrate tumors and show less exhaustion. The inventors anticipate that epigenomic, transcriptomic, and proteomics studies will highlight known T cell signaling pathways and uncover new cellular pathways dysregulated with the EZH2 GoF mutations. Proteins in these pathways will be prioritized for future therapeutic development or engineering of T cells for ACT. Engineered EZH2i resistant CAR T cells for combinatorial treatment of metastatic melanoma EZH2 plays a prominent role in melanoma cell survival. Accordingly, EZH2i have been explored as single agents and in combination with ICB for treatment of melanoma. However, EZH2i also targets T cells and creates an exhausted phenotype. The inventors will leverage cancer cell-evolved resistance mechanisms to engineer CAR T cells with EZH2i resistant mutations, thereby creating an ACT with durable adoptive CD8+T cells that can be used in combination with EZH2i. Engineered CAR T cells will be functionally characterized in vitro and then in vivo using combination treatment with EZH2i. The anticipated outcome of these experiments is a CAR T cell therapy that has increased efficacy and durability in combination with EZH2i treatment. Experimental Design Characterization of EZH2DRT cells. The inventors will first characterize the EZH2i resistant EZH2DRtriple mutant (see Fig.5) using the in vitro and immunocompetent in vivo PMEL- 1 model of CD8+T cells, with functional characterization studies, each as specifically outlined perviously (here ±EZH2i). For in vitro studies with EZH2i (EPZ6438), the inhibitor will be added while cancer cells are seeding at a concentration of 2.5 µM. For in vivo studies, EPZ6438 will be orally administered twice daily starting on Day 5 for 5 days at a previously established dose of 250 mg / kg. Expression of EZH2 CAR T cells. Activated T cells will be transduced with lentiviral particles containing a tricistronic expression cassette coding for GFP, one of two versions of EZH2 (EZH2 wild type (EZH2WT) or EZH2i resistant (EZH2DR)), a CAR sequence (which contains one of the following signaling domains: 28z, CD3z, or 4-1BB), and an antigen recognition domain (CD19 or HER2) (Fig.7). Prior to viral transduction, cytotoxic T cells will be isolated by negative selection from human peripheral blood mononuclear cells (PBMCs) purchased from Stemcell Technologies using the CD8+T cell isolation kit (Stemcell Technologies). Purified CD8+T cells will then be activated and transduced the same day using VivoX15 culture media containing activating cytokines IL-2 (100U / mL), CD3 / CD28 / CD2 activation cocktail and Vectofusion-1 (1mg / mL) viral transduction additive (Miltenyi). T cells will be maintained at 1x106 / mL in media containing IL-2. Day 4 T cells will be sorted for GFP+T cells (CAR+T cells) which contain the EZH2 versions described above. CAR+T cells will be cultured and used for in vitro and in vivo T cell killing experiments detailed below. CAR T cell in vitro killing. Exogenous expression of (CD19 or HER2) CAR will provide the specificity required for T cell–mediated killing of NALM6 (CD19+) and HS-695 (HER2+) tumor cells56,57. Nuclear RFP+NALM6 and HS-695 cells (5 x 104) will be co-cultured with CAR T cells in triplicate at 0:1, 0.5:1, 1:1, 1:2, or 1:4 Effector:Target ratio in T cell medium. For experiments where cells are treated with EZH2i (EPZ6438), the inhibitor will be added while cancer cells are seeding at a concentration of 2.5 µM. Imaging and analysis will be performed as previously described. In vivo CAR tumor killing. CD8+HER2 CAREZH2(WT or DR)T cells will be adoptively transferred (4 million / mouse) into NOD / SCID / IL2Rgc-KO (NSG) mice bearing subcutaneous HER2+tumors (HS-695)58. This experiment will be performed with and without pretreatment with EZH2i (EPZ6438) to determine if a synergistic effect exists in vivo (N=20 per condition, 10F / 10M). EPZ6438 will be orally administered twice daily starting on Day 5 for 5 days at a previously established dose of 250 mg / kg. T cells will be intravenously injected 10 days after tumor injections when tumors are palpable, where selection of CAR co-stimulatory domain will be determined by results of our in vitro studies. Tumor volume will be measured daily using caliper measurements, and tumor endpoints (>1000 mm3) will be used to generate Kaplan Meyer survival curves. The percent of CD45.2+TILs will be determined from tumors at endpoint by flow cytometry. To achieve the desired power of 0.8, and assuming a coefficient of variance (CV) of 35%, our power analysis indicates 20 mice / group, which will be done 2 independent times with 10 mice / group, 5 male and 5 female. Adoptive transfer of CD8+HER2 CAREZH2(WT or DR)T cells, EZH2i studies, endpoint analysis, and power are as described in the above section. Functional characterization of mutant T cells. Functional characterization experiments will be performed as detailed previously described in the functional characterization section, including flow cytometry, epigenomics, transcriptomics, and proteomics analysis. Analysis plan. As detailed previously. Expected outcomes The inventors predict that the EZH2DRT cells and CAR T cells will show increased tumor killing and infiltration with EZH2i treatment relative to control and EZH2WT, while markers of T cell exhaustion will decrease. The inventors expect to see this observation across the in vitro and in vivo model systems. Table 1. Position of mutations in human EZH2 Isoform A, as well as the corresponding mutations in other EZH2 and EZH1 isoforms. EZH2  EZH2  EZH2  EZH2  EZH2  EZH1  EZH1  EZH1  EZH1  isoform  isoform  isoform  isoform  isoform  isoform  isoform  isoform  isoform   L C L A A A A A A A A F D G V  References: 1. Carlino, M. S., Larkin, J. & Long, G. V. Immune checkpoint inhibitors in melanoma. Lancet 398, 1002–1014 (2021). 2. Larkin, J. et al. Five-Year Survival with Combined Nivolumab and Ipilimumab in Advanced Melanoma. N Engl J Med 381, 1535–1546 (2019). 3. Ribas, A. et al. 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Example 2: Protecting EZH2 activity in T cells to increase melanoma therapy efficacy Background The clinical landscape of metastatic melanoma has been revolutionized by immune checkpoint blockade (ICB); however, despite these advancements, the effectiveness of ICB for metastatic melanoma is limited to approximately half of patients. The solid tumor microenvironment (TME) still presents a critical barrier for success of T cell-based therapies largely due to the direct competition between tumor infiltrating lymphocytes (TILs) and cancer cells for metabolic resources, rendering T cells dysfunctional and exhausted. There is an urgent clinical need for the development of new strategies to engineer ACT with the ability to sustain function in the harsh solid TME, particularly if these approaches can be combined with other therapies to improve clinical response. Recent publications have underscored the importance of the histone methyltransferase EZH2 in protecting TILs from TME-induced exhaustion. EZH2 is the catalytic component of the Polycomb Repressive Complex 2 (PRC2), and functions as a transcriptional repressor via methylation of lysine 27 of Histone H3 (H3K27me3). Methods To examine the impact of EZH2i on T cell function we generated several in vivo and in vitro tumor models where cells were treated with or without EZH2i (EPZ6438). To further study the impact of EZH2 expression on T cells, we developed retroviral particles containing wild-type EZH2. Viral particles were transduced into T cells to examine the impact of EZH2 overexpression (EZH2OE) on T cell function. Functional characterization of T cells were performed using an ImageExpress confocal HT.AI microscope for live cell imaging of T cell killing, where images were automatically collected every 2 or 4 hours for 48 hours. Serial T cell killing assays were conducted to examine impact of EZH2i or EZH2OE on T cell persistence. Results Preliminary data indicate that EZH2i treatment results in T cell dysfunction and exhaustion in both mouse and human T cell models in vivo and in vitro (Figures 11-14). Engineering EZH2 overexpression into these T cells increases T cell persistence and killing in vitro (Figure 15). These results support a scientific premise that engineering EZH2 into T cells could create a more durable ACT which could be utilized to treat melanoma. Conclusion The metabolically stressful solid TME is a critical barrier to overcome for successful implementation of ACT. In an attempt to overcome this barrier, we have explored the concept of armoring T cells with enhanced EZH2 activity to generate T cells that are more capable of persisting and infiltrating into a more nutrient depleted microenvironment. EZH2 plays a prominent survival role in melanoma cells by promoting proliferation, EMT, and invasion. To that end, EZH2 inhibitors (EZH2i) have shown promise as single agents and in combination with immune-based therapies for treatment of neoplasms. However, in addition to targeting melanoma cells, EZH2i also targets T cells, generating an exhausted phenotype, which limits clinical utility of EZH2i. Our preliminary data indicates that arming T cells with protected EZH2 function will greatly improve the ability of a T cell to persist in the melanoma solid tumor microenvironment.

Claims

CLAIMS What is claimed:

1. A lymphocyte comprising a first heterologous promoter operably linked to a first polynucleotide encoding a EZH1 polypeptide or a EZH2 polypeptide, and a second heterologous promoter operably linked to a second polynucleotide encoding a Chimeric Antigen Receptor (CAR), wherein the EZH1 or EZH2 polypeptide has at least 95% sequence identity to at least one of SEQ ID NOs:1-9 and optionally comprises at least one mutation at a position corresponding to SEQ ID NO: 1 (EZH2 isoform A, see alignment in Figure 10) selected from the group consisting of S21, Y111, W113, F120, T283, T344, T350, S368, S371, T372, S380, T421, T492, Y646, Y666, A682, A692 and combinations thereof.

2. The lymphocyte of claim 1, wherein the first polynucleotide encodes the EZH2 polypeptide, wherein the EZH2 polypeptide has at least 95% sequence identity to SEQ ID NO: 1 (EZH2-A) and optionally comprises at least one mutation selected from the group consisting of S21, Y111, Y646, W113, F120, Y666 and combinations thereof, optionally wherein the mutation is S21A, Y111L, Y646F, W113C, F120L or Y666D.

3. The lymphocyte of claim 1 or 2 wherein the lymphocyte is selected from the group consisting of CD8+ T lymphocyte and a CD4+ T lymphocyte.

4. The lymphocyte of any one of the preceding claims, wherein the lymphocyte is resistant to an EZH2 inhibitor.

5. The lymphocyte of claim 4, wherein the EZH2 inhibitor is selected from the group consisting of Tazemetostat, GSK126, CPI-1205, SHR2554, and 06821497.

6. The lymphocyte of any one of the preceding claims, wherein the endogenous EZH1 or EZH2 is unmodified.

7. A construct comprising a first heterologous promoter operably linked to a first polynucleotide encoding a EZH1 polypeptide or a EZH2 polypeptide and a second heterologouspromoter operably linked to a second polynucleotide encoding a CAR, wherein the EZH1 or EZH2 polypeptide has at least 95% identity to at least one of SEQ ID NOs: 1-9 and optionally comprises at least one mutation at a position corresponding to SEQ ID NO: 1(EZH2 A, see alignment in figure 10) selected from the group consisting of S21, Y111, W113, F120, T283, T344, T350, S368, S371, T372, S380, T421, T492, Y646, T666, A682, A692 and combinations thereof.

8. The construct of claim 7, wherein the first polynucleotide encodes a EZH2 polypeptide, wherein the EZH2 polypeptide has at least 95% sequence identity to SEQ ID NO: 1 and optionally comprises at least one mutation selected from the group consisting of S21, Y111, Y646, W113, F120L, Y666 and combinations thereof, optionally wherein the mutation is S21A, Y111L, Y646F, W113C, F120L or Y666D.

9. The construct of claim 7 or 8, wherein the construct is included in a lentiviral, retroviral or AAV vector.

10. An engineered lymphocyte comprising the construct of any one of claims 7-9.

11. The engineered lymphocyte of claim 10, wherein the lymphocyte is selected from the group consisting of CD8+ T lymphocyte and a CD4+ T lymphocyte.

12. A method of generating an engineered lymphocyte comprising introducing the construct of any one of claims 7-9 into an ex-vivo lymphocyte.

13. The method of claim 12, wherein the construct is introduced via transduction into the ex- vivo lymphocyte.

14. A method of treating cancer in a subject, the method comprising administering a therapeutically effective amount of the lymphocyte of any one of claims 1-6 or 10-11 and a pharmaceutically acceptable excipient, carrier and / or diluent.

15. The method of claim 14, wherein at least one cancer cell in the cancer overexpresses EZH1 or EZH2.

16. The method of claim 14 or 15, wherein the cancer is a solid tumor.

17. The method of any one of claims 14-16, wherein the cancer is selected from the group consisting of melanoma, breast cancer, prostate cancer, hepatocellular carcinoma, lymphoma, leukemias, myeloma, ovarian cancer, lung cancer, colorectal cancer, esophageal cancer and kidney cancer.

18. The method of any one of claims 14-17, wherein the lymphocyte is administered systemically or intratumorally.

19. The method of any one of claims 14-18, wherein the subject is further administered an EZH2 inhibitor.

20. The method of claim 19, wherein the EZH2 inhibitor selected from the group consisting of Tazemetostat, GSK126, CPI-1205, SHR2554, and 06821497.

21. A method of enhancing a CAR-T cell therapy, wherein the CAR-T comprises a heterologous promoter operably linked to a polynucleotide encoding a EZH1 polypeptide or a EZH2 polypeptide, wherein the EZH1 or EZH2 polypeptide has at least 95% identity to at least one of SEQ ID NOs: 1-9 and optionally comprises at least one mutation at a position corresponding to SEQ ID NO: 1 (EZH2 A, see alignment in figure 10) selected from the group consisting of S21, Y111, W113, F120, T283, T344, T350, S368, S371, T372, S380, T421, T492, Y646, Y666, A682, A692 and combinations thereof and wherein the CAR T cell has an enhanced therapeutic effect as compared to a control CAR T cell.

22. The method of claim 21, wherein the CAR-T cells have increased H3K27me3.

23. A method for enhancing the efficacy of EZH2 inhibitor therapy, the method comprising administering the lymphocyte of any one of claims 1-6 or 10-11 prior to or with an EZH2 inhibitor.

24. A lymphocyte comprising a heterologous promoter operably linked to a polynucleotide encoding a EZH1 polypeptide or a EZH2 polypeptide, wherein the EZH1 or EZH2 polypeptide has at least 95% sequence identity to at least one of SEQ ID NOs:1-9.

25. The lymphocyte of claim 24, additionally comprising a second heterologous promoter operably linked to a second polynucleotide encoding a Chimeric Antigen Receptor (CAR).

26. A construct comprising a first heterologous promoter operably linked to a first polynucleotide encoding a EZH1 polypeptide or a EZH2 polypeptide and a second heterologous promoter operably linked to a second polynucleotide encoding a CAR, wherein the EZH1 or EZH2 polypeptide has at least 95% identity to at least one of SEQ ID Nos: 1-9.

27. The construct of claim 26, wherein the construct comprises a lentiviral, retroviral or AAV vector.

28. An engineered lymphocyte comprising the construct of claim 27.

29. The engineered lymphocyte of claim 28, wherein the lymphocyte is selected from the group consisting of CD8+ T lymphocyte and CD4+ T lymphocyte.

30. A method of generating an engineered lymphocyte comprising introducing the construct of any one of claims 26 or 27 into an ex-vivo lymphocyte.

31. The method of claim 30, wherein the construct is introduced via transduction into the ex- vivo lymphocyte.

32. A method of treating cancer in a subject, the method comprising administering a therapeutically effective amount of the lymphocyte of claim 28 and a pharmaceutically acceptable excipient, carrier and / or diluent.

33. A method of enhancing immune checkpoint blockade therapy responsiveness, the method comprising administering a therapeutically effective amount of the lymphocyte of any one of claims 1-6, 10-11, 24-25 or 28-29 and a pharmaceutically acceptable excipient, carrier and / or diluent.

34. The method of claim 33, additionally comprising administering an immune checkpoint inhibitor.