1 5301 101 PROTEIN PHOSPHATASE 2A CATALYTIC SUBUNIT ALPHA PLAYS A MYD88-DEPENDENT, CENTRAL ROLE IN THE GENE-SPECIFIC REGULATION OF ENDOTOXIN TOLERANCE. MYD88, THE INTRACELLULAR ADAPTOR OF MOST TLRS, MEDIATES EITHER PROINFLAMMATORY OR IMMUNOSUPPRESSIVE SIGNALING THAT CONTRIBUTES TO CHRONIC INFLAMMATION-ASSOCIATED DISEASES. ALTHOUGH GENE-SPECIFIC CHROMATIN MODIFICATIONS REGULATE INFLAMMATION, THE ROLE OF MYD88 SIGNALING IN ESTABLISHING SUCH EPIGENETIC LANDSCAPES UNDER DIFFERENT INFLAMMATORY STATES REMAINS ELUSIVE. USING QUANTITATIVE PROTEOMICS TO ENUMERATE THE INFLAMMATION-PHENOTYPIC CONSTITUENTS OF THE MYD88 INTERACTOME, WE FOUND THAT IN ENDOTOXIN-TOLERANT MACROPHAGES, PROTEIN PHOSPHATASE 2A CATALYTIC SUBUNIT ALPHA (PP2AC) ENHANCES ITS ASSOCIATION WITH MYD88 AND IS CONSTITUTIVELY ACTIVATED. KNOCKDOWN OF PP2AC PREVENTS SUPPRESSION OF PROINFLAMMATORY GENES AND RESISTANCE TO APOPTOSIS. THROUGH SITE-SPECIFIC DEPHOSPHORYLATION, CONSTITUTIVELY ACTIVE PP2AC DISRUPTS THE SIGNAL-PROMOTING TLR4-MYD88 COMPLEX AND BROADLY SUPPRESSES THE ACTIVITIES OF MULTIPLE PROINFLAMMATORY/PROAPOPTOTIC PATHWAYS AS WELL, SHIFTING PROINFLAMMATORY MYD88 SIGNALING TO A PROSURVIVAL MODE. CONSTITUTIVELY ACTIVE PP2AC TRANSLOCATED WITH MYD88 INTO THE NUCLEI OF TOLERANT MACROPHAGES ESTABLISHES THE IMMUNOSUPPRESSIVE PATTERN OF CHROMATIN MODIFICATIONS AND REPRESSES CHROMATIN REMODELING TO SELECTIVELY SILENCE PROINFLAMMATORY GENES, COORDINATING THE MYD88-DEPENDENT INFLAMMATION CONTROL AT BOTH SIGNALING AND EPIGENETIC LEVELS UNDER ENDOTOXIN-TOLERANT CONDITIONS. 2013 2 392 24 AN ORALLY BIOAVAILABLE CHEMICAL PROBE OF THE LYSINE METHYLTRANSFERASES EZH2 AND EZH1. EZH2 OR EZH1 IS THE CATALYTIC SUBUNIT OF THE POLYCOMB REPRESSIVE COMPLEX 2 THAT CATALYZES METHYLATION OF HISTONE H3 LYSINE 27 (H3K27). THE TRIMETHYLATION OF H3K27 (H3K27ME3) IS A TRANSCRIPTIONALLY REPRESSIVE POST-TRANSLATIONAL MODIFICATION. OVEREXPRESSION OF EZH2 AND HYPERTRIMETHYLATION OF H3K27 HAVE BEEN IMPLICATED IN A NUMBER OF CANCERS. SEVERAL SELECTIVE INHIBITORS OF EZH2 HAVE BEEN REPORTED RECENTLY. HEREIN WE DISCLOSE UNC1999, THE FIRST ORALLY BIOAVAILABLE INHIBITOR THAT HAS HIGH IN VITRO POTENCY FOR WILD-TYPE AND MUTANT EZH2 AS WELL AS EZH1, A CLOSELY RELATED H3K27 METHYLTRANSFERASE THAT SHARES 96% SEQUENCE IDENTITY WITH EZH2 IN THEIR RESPECTIVE CATALYTIC DOMAINS. UNC1999 WAS HIGHLY SELECTIVE FOR EZH2 AND EZH1 OVER A BROAD RANGE OF EPIGENETIC AND NON-EPIGENETIC TARGETS, COMPETITIVE WITH THE COFACTOR SAM AND NON-COMPETITIVE WITH THE PEPTIDE SUBSTRATE. THIS INHIBITOR POTENTLY REDUCED H3K27ME3 LEVELS IN CELLS AND SELECTIVELY KILLED DIFFUSED LARGE B CELL LYMPHOMA CELL LINES HARBORING THE EZH2(Y641N) MUTANT. IMPORTANTLY, UNC1999 WAS ORALLY BIOAVAILABLE IN MICE, MAKING THIS INHIBITOR A VALUABLE TOOL FOR INVESTIGATING THE ROLE OF EZH2 AND EZH1 IN CHRONIC ANIMAL STUDIES. WE ALSO DESIGNED AND SYNTHESIZED UNC2400, A CLOSE ANALOGUE OF UNC1999 WITH POTENCY >1,000-FOLD LOWER THAN THAT OF UNC1999 AS A NEGATIVE CONTROL FOR CELL-BASED STUDIES. FINALLY, WE CREATED A BIOTIN-TAGGED UNC1999 (UNC2399), WHICH ENRICHED EZH2 IN PULL-DOWN STUDIES, AND A UNC1999-DYE CONJUGATE (UNC2239) FOR CO-LOCALIZATION STUDIES WITH EZH2 IN LIVE CELLS. TAKEN TOGETHER, THESE COMPOUNDS REPRESENT A SET OF USEFUL TOOLS FOR THE BIOMEDICAL COMMUNITY TO INVESTIGATE THE ROLE OF EZH2 AND EZH1 IN HEALTH AND DISEASE. 2013 3 2825 32 FLOW-DEPENDENT EPIGENETIC REGULATION OF IGFBP5 EXPRESSION BY H3K27ME3 CONTRIBUTES TO ENDOTHELIAL ANTI-INFLAMMATORY EFFECTS. RATIONALE: ATHEROSCLEROSIS IS A CHRONIC INFLAMMATORY AND EPIGENETIC DISEASE THAT IS INFLUENCED BY DIFFERENT PATTERNS OF BLOOD FLOW. HOWEVER, THE EPIGENETIC MECHANISM WHEREBY ATHEROPROTECTIVE FLOW CONTROLS ENDOTHELIAL GENE PROGRAMMING REMAINS ELUSIVE. HERE, WE INVESTIGATED THE POSSIBILITY THAT FLOW ALTERS ENDOTHELIAL GENE EXPRESSION THROUGH EPIGENETIC MECHANISMS. METHODS: EN FACE STAINING AND WESTERN BLOT WERE USED TO DETECT PROTEIN EXPRESSION. REAL-TIME PCR WAS USED TO DETERMINE RELATIVE GENE EXPRESSION. RNA-SEQUENCING OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS TREATED WITH SIRNA OF ENHANCER OF ZESTE HOMOLOG 2 (EZH2) OR LAMINAR FLOW WAS USED FOR TRANSCRIPTIONAL PROFILING. RESULTS: WE FOUND THAT TRIMETHYLATION OF HISTONE 3 LYSINE 27 (H3K27ME3), A REPRESSIVE EPIGENETIC MARK THAT ORCHESTRATES GENE REPRESSION, WAS REDUCED IN LAMINAR FLOW AREAS OF MOUSE AORTA AND FLOW-TREATED HUMAN ENDOTHELIAL CELLS. THE DECREASE OF H3K27ME3 PARALLELED A REDUCTION IN THE EPIGENETIC "WRITER"-EZH2, THE CATALYTIC SUBUNIT OF THE POLYCOMB REPRESSIVE COMPLEX 2 (PRC2). MOREOVER, LAMINAR FLOW DECREASED EXPRESSION OF EZH2 VIA MECHANOSENSITIVE MIR101. GENOME-WIDE TRANSCRIPTOME PROFILING STUDIES IN ENDOTHELIAL CELLS TREATED WITH EZH2 SIRNA AND FLOW REVEALED THE UPREGULATION OF NOVEL MECHANOSENSITIVE GENE IGFBP5 (INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN 5), WHICH IS EPIGENETICALLY SILENCED BY H3K27ME3. FUNCTIONALLY, INHIBITION OF H3K27ME3 BY EZH2 SIRNA OR GSK126 (A SPECIFIC EZH2 INHIBITOR) REDUCED H3K27ME3 LEVELS AND MONOCYTE ADHESION TO ENDOTHELIAL CELLS. ADENOVIRAL OVEREXPRESSION OF IGFBP5 ALSO RECAPITULATED THE ANTI-INFLAMMATORY EFFECTS OF H3K27ME3 INHIBITION. MORE IMPORTANTLY, WE OBSERVED EZH2 UPREGULATION, AND IGFBP5 DOWNREGULATION, IN ADVANCED ATHEROSCLEROTIC PLAQUES FROM HUMAN PATIENTS. CONCLUSION: TAKEN TOGETHER, OUR FINDINGS REVEAL THAT ATHEROPROTECTIVE FLOW REDUCES H3K27ME3 AS A CHROMATIN-BASED MECHANISM TO AUGMENT THE EXPRESSION OF GENES THAT CONFER AN ANTI-INFLAMMATORY RESPONSE IN THE ENDOTHELIUM. OUR STUDY EXEMPLIFIES FLOW-DEPENDENT EPIGENETIC REGULATION OF ENDOTHELIAL GENE EXPRESSION, AND ALSO SUGGESTS THAT TARGETING THE EZH2/H3K27ME3/IGFBP5 PATHWAY MAY OFFER NOVEL THERAPEUTICS FOR INFLAMMATORY DISORDERS SUCH AS ATHEROSCLEROSIS. 2018 4 2425 24 EPIGENETIC SILENCING OF IRF1 DYSREGULATES TYPE III INTERFERON RESPONSES TO RESPIRATORY VIRUS INFECTION IN EPITHELIAL TO MESENCHYMAL TRANSITION. CHRONIC OXIDATIVE INJURY PRODUCED BY AIRWAY DISEASE TRIGGERS A TRANSFORMING GROWTH FACTOR-BETA (TGF-BETA)-MEDIATED EPIGENETIC REPROGRAMMING KNOWN AS THE EPITHELIAL-MESENCHYMAL TRANSITION (EMT). WE OBSERVE THAT EMT SILENCES PROTECTIVE MUCOSAL INTERFERON (IFN)-I AND III PRODUCTION ASSOCIATED WITH ENHANCED RHINOVIRUS (RV) AND RESPIRATORY SYNCYTIAL VIRUS (RSV) REPLICATION. MESENCHYMAL TRANSITIONED CELLS ARE DEFECTIVE IN INDUCIBLE INTERFERON REGULATORY FACTOR 1 (IRF1) EXPRESSION BY OCCLUDING RELA AND IRF3 ACCESS TO THE PROMOTER. IRF1 IS NECESSARY FOR THE EXPRESSION OF TYPE III IFNS (IFNLS 1 AND 2/3). INDUCED BY THE EMT, ZINC FINGER E-BOX BINDING HOMEOBOX 1 (ZEB1) BINDS AND SILENCES IRF1. ECTOPIC ZEB1 IS SUFFICIENT FOR IRF1 SILENCING, WHEREAS ZEB1 KNOCKDOWN PARTIALLY RESTORES IRF1-IFNL UPREGULATION. ZEB1 SILENCES IRF1 THROUGH THE CATALYTIC ACTIVITY OF THE ENHANCER OF ZESTE 2 POLYCOMB REPRESSIVE COMPLEX 2 SUBUNIT (EZH2), FORMING REPRESSIVE H3K27(ME3) MARKS. WE OBSERVE THAT IRF1 EXPRESSION IS MEDIATED BY ZEB1 DE-REPRESSION, AND OUR STUDY DEMONSTRATES HOW AIRWAY REMODELLING/FIBROSIS IS ASSOCIATED WITH A DEFECTIVE MUCOSAL ANTIVIRAL RESPONSE THROUGH ZEB1-INITIATED EPIGENETIC SILENCING. 2017 5 1906 27 ENHANCER OF ZESTE HOMOLOG 2-CATALYSED H3K27 TRIMETHYLATION PLAYS A KEY ROLE IN ACUTE-ON-CHRONIC LIVER FAILURE VIA TNF-MEDIATED PATHWAY. ACUTE-ON-CHRONIC LIVER FAILURE IS MAINLY DUE TO HOST IMMUNITY SELF-DESTRUCTION. THE HISTONE H3 LYSINE 27 (H3K27) TRIMETHYLATING ENZYME, ENHANCER OF ZESTE HOMOLOG 2 (EZH2) MEDIATES EPIGENETIC SILENCING OF GENE EXPRESSION AND REGULATES IMMUNITY, ALSO INVOLVES PATHOGENESIS OF SEVERAL LIVER DISEASES. THE CURRENT STUDY WAS TO DETERMINE THE ROLE OF METHYLTRANSFERASE EZH2 AND ITS CATALYSED H3K27 TRIMETHYLATION (H3K27ME3) IN LIVER FAILURE, AND TO FURTHER INVESTIGATE THE POTENTIAL TARGET FOR LIVER FAILURE TREATMENT. EZH2 AND ITS CATALYSED H3K27ME3 WERE DETERMINED IN PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) FROM LIVER FAILURE PATIENTS AND KUPFFER CELLS FROM EXPERIMENTAL MICE. FURTHERMORE, GSK126 (AN INHIBITOR FOR EZH2 TRIMETHYLATION FUNCTION) WAS APPLIED IN LIVER FAILURE MICE IN VIVO, AND LIPOPOLYSACCHARIDE-STIMULATED MONONUCLEAR CELLS IN VITRO. EZH2 AND H3K27ME3 WERE SIGNIFICANTLY UPREGULATED IN HUMAN PBMC FROM LIVER FAILURE PATIENTS OR MURINE KUPFFER CELLS FROM THE LIVER FAILURE ANIMALS, RESPECTIVELY. GSK126 AMELIORATED DISEASE SEVERITY IN LIVER FAILURE MICE, WHICH MAYBE ATTRIBUTE TO DOWN-REGULATE CIRCULATING AND HEPATIC PROINFLAMMATORY CYTOKINES, ESPECIALLY TNF VIA REDUCING H3K27ME3. IN-DEPTH CHROMATIN IMMUNOPRECIPITATION ANALYSIS UNRAVELLED THAT DECREASED ENRICHMENT OF H3K27ME3 ON TNF PROMOTOR, RESULTING IN TNF ELEVATION IN KUPFFER CELLS FROM LIVER FAILURE MICE. NUCLEAR FACTOR KAPPA B (NF-KAPPAB) AND PROTEIN KINASE B (AKT) SIGNALLING PATHWAYS WERE ACTIVATED UPON LIPOPOLYSACCHARIDE STIMULATION, BUT ATTENUATED BY USING GSK126, ACCOMPANIED WITH DECREASED TNF IN VITRO. IN CONCLUSION, EZH2 AND H3K27ME3 CONTRIBUTED TO THE PATHOGENESIS OF LIVER FAILURE VIA TRIGGERING TNF AND OTHER INDISPENSABLE PROINFLAMMATORY CYTOKINES. EZH2 WAS TO MODIFY H3K27ME3 ENRICHMENT, AS WELL AS, ACTIVATION OF THE DOWNSTREAM NF-KAPPAB AND AKT SIGNALLING PATHWAYS. 2018 6 3175 23 H2AX PHOSPHORYLATION REGULATED BY P38 IS INVOLVED IN BIM EXPRESSION AND APOPTOSIS IN CHRONIC MYELOGENOUS LEUKEMIA CELLS INDUCED BY IMATINIB. INCREASING EVIDENCE SUGGESTS THAT HISTONE H2AX PLAYS A CRITICAL ROLE IN REGULATION OF TUMOR CELL APOPTOSIS AND ACTS AS A NOVEL HUMAN TUMOR SUPPRESSOR PROTEIN. HOWEVER, THE ACTION OF H2AX IN CHRONIC MYELOGENOUS LEUKEMIA (CML) CELLS IS UNKNOWN. THE DETAILED MECHANISM AND EPIGENETIC REGULATION BY H2AX REMAIN ELUSIVE IN CANCER CELLS. HERE, WE REPORT THAT H2AX WAS INVOLVED IN APOPTOSIS OF CML CELLS. OVEREXPRESSION OF H2AX INCREASED APOPTOTIC SENSITIVITY OF CML CELLS (K562) INDUCED BY IMATINIB. HOWEVER, OVEREXPRESSION OF SER139-MUTATED H2AX (BLOCKING PHOSPHORYLATION) DECREASED SENSITIVITY OF K562 CELLS TO APOPTOSIS. SIMILARLY, KNOCKDOWN OF H2AX MADE K562 CELLS RESISTANT TO APOPTOTIC INDUCTION. THESE RESULTS REVEALED THAT THE FUNCTION OF H2AX INVOLVED IN APOPTOSIS IS STRICTLY RELATED TO ITS PHOSPHORYLATION (SER139). OUR DATA FURTHER INDICATED THAT IMATINIB MAY STIMULATE MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) FAMILY MEMBER P38, AND H2AX PHOSPHORYLATION FOLLOWED A SIMILAR TIME COURSE, SUGGESTING A PARALLEL RESPONSE. H2AX PHOSPHORYLATION CAN BE BLOCKED BY P38 SIRNA OR ITS INHIBITOR. THESE DATA DEMONSTRATED THAT H2AX PHOSPHORYLATION WAS REGULATED BY P38 MAPK PATHWAY IN K562 CELLS. HOWEVER, THE P38 MAPK DOWNSTREAM, MITOGEN- AND STRESS-ACTIVATED PROTEIN KINASE-1 AND -2, WHICH PHOSPHORYLATED HISTONE H3, WERE NOT REQUIRED FOR H2AX PHOSPHORYLATION DURING APOPTOSIS. FINALLY, WE PROVIDED EPIGENETIC EVIDENCE THAT H2AX PHOSPHORYLATION REGULATED APOPTOSIS-RELATED GENE BIM EXPRESSION. BLOCKING OF H2AX PHOSPHORYLATION INHIBITED BIM GENE EXPRESSION. TAKEN TOGETHER, THESE DATA DEMONSTRATED THAT H2AX PHOSPHORYLATION REGULATED BY P38 IS INVOLVED IN BIM EXPRESSION AND APOPTOSIS IN CML CELLS INDUCED BY IMATINIB. 2014 7 3261 28 HEPATITIS C VIRUS-INDUCED UP-REGULATION OF PROTEIN PHOSPHATASE 2A INHIBITS HISTONE MODIFICATION AND DNA DAMAGE REPAIR. THE MOLECULAR MECHANISMS UNDERLYING HEPATOCARCINOGENESIS IN CHRONIC VIRAL HEPATITIS ARE POORLY UNDERSTOOD. A POTENTIAL TUMORIGENIC PATHWAY COULD INVOLVE PROTEIN PHOSPHATASE 2A (PP2A) AND PROTEIN ARGININE METHYLTRANSFERASE 1 (PRMT1), BECAUSE BOTH ENZYMES ARE DYSREGULATED IN CHRONIC HEPATITIS C, AND BOTH ENZYMES HAVE BEEN INVOLVED IN CHROMATIN REMODELING AND DNA DAMAGE REPAIR. WE USED CELL LINES THAT ALLOW THE INDUCIBLE EXPRESSION OF HEPATITIS C VIRUS PROTEINS (UHCV57.3) AND OF THE CATALYTIC SUBUNIT OF PP2A (UPP2A-C8) AS WELL AS HUH7.5 CELLS INFECTED WITH RECOMBINANT CELL CULTURE-DERIVED HEPATITIS C VIRUS (HCVCC) TO STUDY EPIGENETIC HISTONE MODIFICATIONS AND DNA DAMAGE REPAIR. THE INDUCTION OF VIRAL PROTEINS, THE OVEREXPRESSION OF PP2AC, OR THE INFECTION OF HUH7.5 CELLS WITH HCVCC RESULTED IN AN INHIBITION OF HISTONE H4 METHYLATION/ACETYLATION AND HISTONE H2AX PHOSPHORYLATION, IN A SIGNIFICANTLY CHANGED EXPRESSION OF GENES IMPORTANT FOR HEPATOCARCINOGENESIS, AND INHIBITED DNA DAMAGE REPAIR. OVEREXPRESSION OF PP2AC IN NIH-3T3 CELLS INCREASED ANCHORAGE-INDEPENDENT GROWTH. THESE CHANGES WERE PARTIALLY REVERSED BY THE TREATMENT OF CELLS WITH THE METHYL-GROUP DONOR S-ADENOSYL-L-METHIONINE (SAME). CONCLUSION: HEPATITIS C VIRUS-INDUCED OVEREXPRESSION OF PP2AC CONTRIBUTES TO HEPATOCARCINOGENESIS THROUGH DYSREGULATION OF EPIGENETIC HISTONE MODIFICATIONS. THE CORRECTION OF DEFECTIVE HISTONE MODIFICATIONS BY S-ADENOSYL-L-METHIONINE MAKES THIS DRUG A CANDIDATE FOR CHEMOPREVENTIVE THERAPIES IN PATIENTS WITH CHRONIC HEPATITIS C WHO ARE AT RISK FOR DEVELOPING HEPATOCELLULAR CARCINOMA. 2010 8 4546 29 MUTANT P53 REGULATES ENHANCER-ASSOCIATED H3K4 MONOMETHYLATION THROUGH INTERACTIONS WITH THE METHYLTRANSFERASE MLL4. MONOMETHYLATION OF HISTONE H3 LYSINE 4 (H3K4ME1) IS ENRICHED AT ENHANCERS THAT ARE PRIMED FOR ACTIVATION AND THE LEVELS OF THIS HISTONE MARK ARE FREQUENTLY ALTERED IN VARIOUS HUMAN CANCERS. YET, HOW ALTERATIONS IN H3K4ME1 ARE ESTABLISHED AND THE CONSEQUENCES OF THESE EPIGENETIC CHANGES IN TUMORIGENESIS ARE NOT WELL UNDERSTOOD. USING CHIP-SEQ IN HUMAN COLON CANCER CELLS, WE DEMONSTRATE THAT MUTANT P53 DEPLETION RESULTS IN DECREASED H3K4ME1 LEVELS AT ACTIVE ENHANCERS THAT REVEAL A STRIKING COLOCALIZATION OF MUTANT P53 AND THE H3K4 MONOMETHYLTRANSFERASE MLL4 FOLLOWING CHRONIC TUMOR NECROSIS FACTOR ALPHA (TNFALPHA) SIGNALING. WE FURTHER REVEAL THAT MUTANT P53 FORMS PHYSIOLOGICAL ASSOCIATIONS AND DIRECT INTERACTIONS WITH MLL4 AND PROMOTES THE ENHANCER BINDING OF MLL4, WHICH IS REQUIRED FOR TNFALPHA-INDUCIBLE H3K4ME1 AND HISTONE H3 LYSINE 27 ACETYLATION (H3K27AC) LEVELS, ENHANCER-DERIVED TRANSCRIPT (ERNA) SYNTHESIS, AND MUTANT P53-DEPENDENT TARGET GENE ACTIVATION. COMPLEMENTARY IN VITRO STUDIES WITH RECOMBINANT CHROMATIN AND PURIFIED PROTEINS DEMONSTRATE THAT BINDING OF THE MLL3/4 COMPLEX AND H3K4ME1 DEPOSITION IS ENHANCED BY MUTANT P53 AND P300-MEDIATED ACETYLATION, WHICH IN TURN REFLECTS A MLL3/4-DEPENDENT ENHANCEMENT OF MUTANT P53 AND P300-DEPENDENT TRANSCRIPTIONAL ACTIVATION. COLLECTIVELY, OUR FINDINGS ESTABLISH A MECHANISM IN WHICH MUTANT P53 COOPERATES WITH MLL4 TO REGULATE ABERRANT ENHANCER ACTIVITY AND TUMOR-PROMOTING GENE EXPRESSION IN RESPONSE TO CHRONIC IMMUNE SIGNALING. 2018 9 5965 23 TEN-ELEVEN-TRANSLOCATION 2 (TET2) NEGATIVELY REGULATES HOMEOSTASIS AND DIFFERENTIATION OF HEMATOPOIETIC STEM CELLS IN MICE. THE TEN-ELEVEN-TRANSLOCATION 2 (TET2) GENE ENCODES A MEMBER OF TET FAMILY ENZYMES THAT ALTERS THE EPIGENETIC STATUS OF DNA BY OXIDIZING 5-METHYLCYTOSINE TO 5-HYDROXYMETHYLCYTOSINE (5HMC). SOMATIC LOSS-OF-FUNCTION MUTATIONS OF TET2 ARE FREQUENTLY OBSERVED IN PATIENTS WITH DIVERSE MYELOID MALIGNANCIES, INCLUDING MYELODYSPLASTIC SYNDROMES, MYELOPROLIFERATIVE NEOPLASMS, AND CHRONIC MYELOMONOCYTIC LEUKEMIA. BY ANALYZING MICE WITH TARGETED DISRUPTION OF THE TET2 CATALYTIC DOMAIN, WE SHOW HERE THAT TET2 IS A CRITICAL REGULATOR OF SELF-RENEWAL AND DIFFERENTIATION OF HEMATOPOIETIC STEM CELLS (HSCS). TET2 DEFICIENCY LED TO DECREASED GENOMIC LEVELS OF 5HMC AND AUGMENTED THE SIZE OF THE HEMATOPOIETIC STEM/PROGENITOR CELL POOL IN A CELL-AUTONOMOUS MANNER. IN COMPETITIVE TRANSPLANTATION ASSAYS, TET2-DEFICIENT HSCS WERE CAPABLE OF MULTILINEAGE RECONSTITUTION AND POSSESSED A COMPETITIVE ADVANTAGE OVER WILD-TYPE HSCS, RESULTING IN ENHANCED HEMATOPOIESIS INTO BOTH LYMPHOID AND MYELOID LINEAGES. IN VITRO, TET2 DEFICIENCY DELAYED HSC DIFFERENTIATION AND SKEWED DEVELOPMENT TOWARD THE MONOCYTE/MACROPHAGE LINEAGE. OUR DATA INDICATE THAT TET2 HAS A CRITICAL ROLE IN REGULATING THE EXPANSION AND FUNCTION OF HSCS, PRESUMABLY BY CONTROLLING 5HMC LEVELS AT GENES IMPORTANT FOR THE SELF-RENEWAL, PROLIFERATION, AND DIFFERENTIATION OF HSCS. 2011 10 5319 20 PTEN IS FUNDAMENTAL FOR ELIMINATION OF LEUKEMIA STEM CELLS MEDIATED BY GSK126 TARGETING EZH2 IN CHRONIC MYELOGENOUS LEUKEMIA. PURPOSE: LEUKEMIA STEM CELLS (LSCS) ARE AN IMPORTANT SOURCE OF TYROSINE KINASE INHIBITOR RESISTANCE AND DISEASE RELAPSE IN PATIENTS WITH CHRONIC MYELOGENOUS LEUKEMIA (CML). TARGETING LSCS MAY BE AN ATTRACTIVE STRATEGY TO OVERRIDE THIS THORNY PROBLEM. GIVEN THAT EZH2 WAS OVEREXPRESSED IN PRIMARY CML CD34(+) CELLS, OUR PURPOSE IN THIS STUDY WAS TO EVALUATE THE EFFECTS OF TARGETING EZH2 ON CML LSCS AND CLARIFY ITS UNDERLYING MECHANISM.EXPERIMENTAL DESIGN: HUMAN PRIMARY CML CD34(+) CELLS AND RETROVIRALLY BCR-ABL-DRIVEN CML MOUSE MODELS WERE EMPLOYED TO EVALUATE THE EFFECTS OF SUPPRESSION OF EZH2 BY GSK126- OR EZH2-SPECIFIC SHRNA IN VITRO AND IN VIVO RECRUITMENT OF EZH2 AND H3K27ME3 ON THE PROMOTER OF TUMOR-SUPPRESSOR GENE PTEN IN CML CELLS WAS MEASURED BY CHROMATIN IMMUNOPRECIPITATION ASSAY.RESULTS: OUR RESULTS SHOWED THAT PHARMACOLOGIC INHIBITION OF EZH2 BY GSK126 NOT ONLY ELICITED APOPTOSIS AND RESTRICTED CELL GROWTH IN CML BULK LEUKEMIA CELLS, BUT ALSO DECREASED LSCS IN CML CD34(+) CELLS WHILE SPARING THOSE FROM NORMAL BONE MARROW CD34(+) CELLS. SUPPRESSION OF EZH2 BY GSK126 OR SPECIFIC SHRNA PROLONGED SURVIVAL OF CML MICE AND REDUCED THE NUMBER OF LSCS IN MICE. EZH2 KNOCKDOWN RESULTED IN ELEVATION OF PTEN AND LED TO IMPAIRED RECRUITMENT OF EZH2 AND H3K27ME3 ON THE PROMOTER OF PTEN GENE. THE EFFECT OF EZH2 KNOCKDOWN IN THE CML MICE WAS AT LEAST PARTIALLY REVERSED BY PTEN KNOCKDOWN.CONCLUSIONS: THESE FINDINGS IMPROVE THE UNDERSTANDING OF THE EPIGENETIC REGULATION OF STEMNESS IN CML LSCS AND WARRANT CLINICAL TRIAL OF GSK126 IN REFRACTORY PATIENTS WITH CML. CLIN CANCER RES; 24(1); 145-57. (C)2017 AACR. 2018 11 5116 18 POSITIVE REGULATION OF HUMAN TELOMERASE REVERSE TRANSCRIPTASE GENE EXPRESSION AND TELOMERASE ACTIVITY BY DNA METHYLATION IN PANCREATIC CANCER. AIM: WE SOUGHT TO DETERMINE THE ROLE OF TELOMERASE AND ITS CATALYTIC SUBUNIT HTERT IN PANCREATIC CANCER AND EVALUATE THE EPIGENETIC REGULATION OF HTERT BY PROMOTER METHYLATION. METHODS: THIRTY PAIRED SAMPLES OF PANCREATIC DUCTAL ADENOCARCINOMAS AND ADJACENT NORMAL TISSUE AND 12 CHRONIC PANCREATITIS SAMPLES WERE STUDIED. REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION, TELOMERIC REPEAT AMPLIFICATION PROTOCOL ASSAY, AND METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION WERE PERFORMED TO ANALYZE HTERT EXPRESSION, TELOMERASE ACTIVITY, AND METHYLATION STATUS OF GENE PROMOTERS, RESPECTIVELY. RESULT: HTERT AND TELOMERASE ACTIVITY WERE UPREGULATED IN PANCREATIC CANCER COMPARED WITH PAIRED NORMAL TISSUES AND SAMPLES OF PANCREATITIS. HTERT EXPRESSION CORRELATED WITH TELOMERASE ACTIVITY (P \ .05) AND IN TURN CORRELATED POSITIVELY WITH HTERT PROMOTER METHYLATION (P \ .001) AND P16 PROMOTER METHYLATION. HTERT TRANSCRIPT EXPRESSION AND TELOMERASE ACTIVITY BOTH CONFERRED A WORSE OUTCOME BY UNIVARIATE AND MULTIVARIATE ANALYSIS (P \ .05). CONCLUSION: HTERT EXPRESSION AND TELOMERASE ACTIVITY ARE PREDICTORS OF POOR OUTCOME IN PANCREATIC CANCER. HTERT GENE EXPRESSION IS POSITIVELY REGULATED BY PROMOTER METHYLATION. 2009 12 2326 24 EPIGENETIC REGULATION OF HOTAIR IN ADVANCED CHRONIC MYELOID LEUKEMIA. PURPOSE: CHRONIC MYELOID LEUKEMIA (CML) ACCOUNTS FOR ~10% OF LEUKEMIA CASES, AND ITS PROGRESSION INVOLVES EPIGENETIC GENE REGULATION. THIS STUDY INVESTIGATED EPIGENETIC REGULATION OF HOTAIR AND ITS TARGET MICRORNA, MIR-143, IN ADVANCED CML. PATIENTS AND METHODS: WE FIRST ISOLATED BONE MARROW MONONUCLEAR CELLS FROM 70 PATIENTS WITH DIFFERENT PHASES OF CML AND FROM HEALTHY DONORS AS NORMAL CONTROL; WE ALSO CULTURED K562 AND KCL22 CELLS, TREATED WITH DEMETHYLATION DRUG; MTT ASSAY, FLOW CYTOMETRY, QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION (QPCR), METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION (MSP), WESTERN BLOT, LUCIFERASE ASSAY, RNA PULL-DOWN ASSAY AND RNA-BINDING PROTEIN IMMUNOPRECIPITATION (RIP) ASSAY WERE PERFORMED. RESULT: AS MEASURED BY QPCR, HOTAIR EXPRESSION IN K562 CELLS, KCL22 CELLS, AND SAMPLES FROM CASES OF ADVANCED-STAGE CML INCREASED WITH LEVELS OF SEVERAL DNA METHYLTRANSFERASES AND HISTONE DEACETYLATES, INCLUDING DNMT1, DNMT3A, HDAC1, EZH2, AND LSD1, AND MIR-143 LEVELS WERE DECREASED AND HOTAIR LEVELS WERE INCREASED. TREATMENT WITH 5-AZACYTIDINE, A DNA METHYLATION INHIBITOR, DECREASED DNMT1, DNMT3A, HDAC1, EZH2, LSD1 MRNA, PROTEIN LEVELS, AND HOTAIR MRNA LEVELS BUT INCREASED MIR-143 LEVELS. HOTAIR KNOCKDOWN AND MIR-143 OVEREXPRESSION BOTH INHIBITED PROLIFERATION AND PROMOTED APOPTOSIS IN KCL22 AND K562 CELLS THROUGH THE PI3K/AKT PATHWAY. RNA PULL-DOWN, MASS SPECTROMETRY, AND RIP ASSAYS SHOWED THAT HOTAIR INTERACTED WITH EZH2 AND LSD1. A DUAL-LUCIFERASE ASSAY DEMONSTRATED THAT HOTAIR INTERACTED WITH MIR-143. CONCLUSION: OUR FINDINGS DEMONSTRATE THE KEY EPIGENETIC INTERACTIONS OF HOTAIR RELATED TO CML PROGRESSION AND SUGGEST HOTAIR AS A POTENTIAL THERAPEUTIC TARGET FOR ADVANCED CML. FURTHERMORE, OUR RESULTS SUPPORT THE USE OF DEMETHYLATION DRUGS AS A CML TREATMENT STRATEGY. 2018 13 3877 26 KDM6A PROMOTES IMATINIB RESISTANCE THROUGH YY1-MEDIATED TRANSCRIPTIONAL UPREGULATION OF TRKA INDEPENDENTLY OF ITS DEMETHYLASE ACTIVITY IN CHRONIC MYELOGENOUS LEUKEMIA. RATIONALE: DESPITE LANDMARK THERAPY OF CHRONIC MYELOGENOUS LEUKEMIA (CML) WITH TYROSINE KINASE INHIBITORS (TKIS), DRUG RESISTANCE REMAINS PROBLEMATIC. CANCER PATHOGENESIS INVOLVES EPIGENETIC DYSREGULATION AND IN PARTICULAR, HISTONE LYSINE DEMETHYLASES (KDMS) HAVE BEEN IMPLICATED IN TKI RESISTANCE. WE SOUGHT TO IDENTIFY KDMS WITH ALTERED EXPRESSION IN CML AND DEFINE THEIR CONTRIBUTION TO IMATINIB RESISTANCE. METHODS: BIOINFORMATICS SCREENING COMPARED KDM EXPRESSION IN CML VERSUS NORMAL BONE MARROW WITH SHRNA KNOCKDOWN AND FLOW CYTOMETRY USED TO MEASURE EFFECTS ON IMATINIB-INDUCED APOPTOSIS IN K562 CELLS. TRANSCRIPTOMIC ANALYSES WERE PERFORMED AGAINST KDM6A CRISPR KNOCKOUT/SHRNA KNOCKDOWN K562 CELLS ALONG WITH GENE RESCUE EXPERIMENTS USING WILDTYPE AND MUTANT DEMETHYLASE-DEAD KDM6A CONSTRUCTS. CO-IMMUNOPRECIPITATION, LUCIFERASE REPORTER AND CHIP WERE EMPLOYED TO ELUCIDATE MECHANISMS OF KDM6A-DEPENDENT RESISTANCE. RESULTS: AMONGST FIVE KDMS UPREGULATED IN CML, ONLY KDM6A DEPLETION SENSITIZED CML CELLS TO IMATINIB-INDUCED APOPTOSIS. RE-INTRODUCTION OF DEMETHYLASE-DEAD KDM6A AS WELL AS WILD-TYPE KDM6A RESTORED IMATINIB RESISTANCE. RNA-SEQ IDENTIFIED NTRK1 GENE DOWNREGULATION AFTER DEPLETION OF KDM6A. MOREOVER, NTRK1 EXPRESSION POSITIVELY CORRELATED WITH KDM6A IN A SUBSET OF CLINICAL CML SAMPLES AND KDM6A KNOCKDOWN IN FRESH CML ISOLATES DECREASED NTRK1 ENCODED PROTEIN (TRKA) EXPRESSION. MECHANISTICALLY, KDM6A WAS RECRUITED TO THE NTRK1 PROMOTER BY THE TRANSCRIPTION FACTOR YY1 WITH SUBSEQUENT TRKA UPREGULATION ACTIVATING DOWN-STREAM SURVIVAL PATHWAYS TO INVOKE IMATINIB RESISTANCE. CONCLUSION: CONTRARY TO ITS REPORTED ROLE AS A TUMOR SUPPRESSOR AND INDEPENDENT OF ITS DEMETHYLASE FUNCTION, KDM6A PROMOTES IMATINIB-RESISTANCE IN CML CELLS. THE IDENTIFICATION OF THE KDM6A/YY1/TRKA AXIS AS A NOVEL IMATINIB-RESISTANCE MECHANISM REPRESENTS AN UNEXPLORED AVENUE TO OVERCOME TKI RESISTANCE IN CML. 2021 14 1212 21 CPG ISLAND METHYLATION OF THE HTERT PROMOTER IS ASSOCIATED WITH LOWER TELOMERASE ACTIVITY IN B-CELL LYMPHOCYTIC LEUKEMIA. OBJECTIVE: EXPRESSION OF THE CATALYTIC SUBUNIT OF THE TELOMERASE ENZYME HTERT IS ESSENTIAL FOR PROLONGING THE REPLICATIVE LIFESPAN AND IS THE RATE-LIMITING STEP IN CELLULAR IMMORTALIZATION AND CARCINOGENESIS. BECAUSE HTERT EXPRESSION IS POSITIVELY CORRELATED WITH TELOMERASE ACTIVITY, ITS REGULATION IS SUGGESTED AS THE MAJOR DETERMINANT OF ENZYMATIC ACTIVITY. THE HTERT PROMOTER REGION CONTAINS TWO CPG ISLANDS, WHICH ARE KNOWN TO BE TARGET SITES FOR DE NOVO DNA METHYLATION. TO ELUCIDATE THE IMPACT OF THIS EPIGENETIC MECHANISM ON TELOMERASE ACTIVITY, WE ANALYZED THE DEGREE OF HTERT PROMOTER METHYLATION IN 30 PATIENTS WITH B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA. MATERIALS AND METHODS: HTERT PROMOTER METHYLATION WAS ASSESSED USING A METHYLATION-SPECIFIC COMPETITIVE POLYMERASE CHAIN REACTION ASSAY. THE ASSAY IS BASED ON DIGESTION OF GENOMIC DNA WITH A METHYLATION-SENSITIVE RESTRICTION ENZYME BEFORE AMPLIFICATION WITH AN INTERNAL STANDARD. RESULTS: PATIENTS EXHIBITING HIGH TELOMERASE ACTIVITY SHOWED SIGNIFICANTLY LESS METHYLATION OF THE HTERT PROMOTER CORE DOMAIN THAN PATIENTS WITH LOW ENZYME ACTIVITY. IN ADDITION, TELOMERASE ACTIVITY WAS SIGNIFICANTLY ASSOCIATED WITH TELOMERE LENGTH AND OVERALL SURVIVAL. CONCLUSIONS: OUR DATA SHOW THAT THE DEGREE OF CPG ISLAND METHYLATION OF THE HTERT PROMOTER EXHIBITS AN IMPACT ON TELOMERASE ACTIVITY IN A SUBGROUP OF PATIENTS WITH B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA AND THEREFORE IS ASSUMED TO PLAY A ROLE IN REGULATING HTERT GENE EXPRESSION IN THESE PATIENTS. 2002 15 1945 27 EPIGALLOCATECHIN-3-GALLATE, A HISTONE ACETYLTRANSFERASE INHIBITOR, INHIBITS EBV-INDUCED B LYMPHOCYTE TRANSFORMATION VIA SUPPRESSION OF RELA ACETYLATION. BECAUSE THE P300/CBP-MEDIATED HYPERACETYLATION OF RELA (P65) IS CRITICAL FOR NUCLEAR FACTOR-KAPPAB (NF-KAPPAB) ACTIVATION, THE ATTENUATION OF P65 ACETYLATION IS A POTENTIAL MOLECULAR TARGET FOR THE PREVENTION OF CHRONIC INFLAMMATION. DURING OUR ONGOING SCREENING STUDY TO IDENTIFY NATURAL COMPOUNDS WITH HISTONE ACETYLTRANSFERASE INHIBITOR (HATI) ACTIVITY, WE IDENTIFIED EPIGALLOCATECHIN-3-GALLATE (EGCG) AS A NOVEL HATI WITH GLOBAL SPECIFICITY FOR THE MAJORITY OF HAT ENZYMES BUT WITH NO ACTIVITY TOWARD EPIGENETIC ENZYMES INCLUDING HDAC, SIRT1, AND HMTASE. AT A DOSE OF 100 MICROMOL/L, EGCG ABROGATES P300-INDUCED P65 ACETYLATION IN VITRO AND IN VIVO, INCREASES THE LEVEL OF CYTOSOLIC IKAPPABALPHA, AND SUPPRESSES TUMOR NECROSIS FACTOR ALPHA (TNFALPHA)-INDUCED NF-KAPPAB ACTIVATION. WE ALSO SHOWED THAT EGCG PREVENTS TNFALPHA-INDUCED P65 TRANSLOCATION TO THE NUCLEUS, CONFIRMING THAT HYPERACETYLATION IS CRITICAL FOR NF-KAPPAB TRANSLOCATION AS WELL AS ACTIVITY. FURTHERMORE, EGCG TREATMENT INHIBITED THE ACETYLATION OF P65 AND THE EXPRESSION OF NF-KAPPAB TARGET GENES IN RESPONSE TO DIVERSE STIMULI. FINALLY, EGCG REDUCED THE BINDING OF P300 TO THE PROMOTER REGION OF INTERLEUKIN-6 GENE WITH AN INCREASED RECRUITMENT OF HDAC3, WHICH HIGHLIGHTS THE IMPORTANCE OF THE BALANCE BETWEEN HATS AND HISTONE DEACETYLASES IN THE NF-KAPPAB-MEDIATED INFLAMMATORY SIGNALING PATHWAY. IMPORTANTLY, EGCG AT 50 MICROMOL/L DOSE COMPLETELY BLOCKS EBV INFECTION-INDUCED CYTOKINE EXPRESSION AND SUBSEQUENTLY THE EBV-INDUCED B LYMPHOCYTE TRANSFORMATION. THESE RESULTS SHOW THE CRUCIAL ROLE OF ACETYLATION IN THE DEVELOPMENT OF INFLAMMATORY-RELATED DISEASES. 2009 16 3531 21 IMATINIB CAUSES EPIGENETIC ALTERATIONS OF PTEN GENE VIA UPREGULATION OF DNA METHYLTRANSFERASES AND POLYCOMB GROUP PROTEINS. WE HAVE RECENTLY REPORTED THE POSSIBLE IMATINIB-RESISTANT MECHANISM; LONG-TERM EXPOSURE OF LEUKEMIA CELLS TO IMATINIB DOWNREGULATED LEVELS OF PHOSPHATASE AND TENSIN HOMOLOG DELETED ON CHROMOSOME 10 (PTEN) VIA HYPERMETHYLATION OF ITS PROMOTER REGION (LEUKEMIA 2010; 24: 1631). THE PRESENT STUDY EXPLORED THE MOLECULAR MECHANISMS BY WHICH IMATINIB CAUSED METHYLATION ON THE PROMOTER REGION OF THIS TUMOR SUPPRESSOR GENE IN LEUKEMIA CELLS. REAL-TIME REVERSE TRANSCRIPTION PCR FOUND THAT LONG-TERM EXPOSURE OF CHRONIC EOSINOPHILIC LEUKEMIA EOL-1 CELLS EXPRESSING FIP1L1/PLATELET-DERIVED GROWTH FACTOR RECEPTOR-ALPHA TO IMATINIB INDUCED EXPRESSION OF DNA METHYLTRANSFERASE 3A (DNMT3A) AND HISTONE-METHYLTRANSFERASE ENHANCER OF ZESTE HOMOLOG 2 (EZH2), A FAMILY OF POLYCOMB GROUP, THEREBY INCREASING METHYLATION OF THE GENE. IMMUNOPRECIPITATION ASSAY FOUND THE INCREASED COMPLEX FORMATION OF DNMT3A AND EZH2 PROTEINS IN THESE CELLS. MOREOVER, CHROMATIN IMMUNOPRECIPITATION ASSAY SHOWED THAT AMOUNTS OF BOTH DNMT3A AND EZH2 PROTEINS BOUND AROUND THE PROMOTER REGION OF PTEN GENE WERE INCREASED IN EOL-1 CELLS AFTER EXPOSURE TO IMATINIB. FURTHERMORE, WE FOUND THAT LEVELS OF DNMT3A AND EZH2 WERE STRIKINGLY INCREASED IN LEUKEMIA CELLS ISOLATED FROM INDIVIDUALS WITH CHRONIC MYELOGENOUS LEUKEMIA (N=1) AND PHILADELPHIA CHROMOSOME-POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA (N=2), WHO RELAPSED AFTER TREATMENT WITH IMATINIB COMPARED WITH THOSE ISOLATED AT THEIR INITIAL PRESENTATION. TAKEN TOGETHER, IMATINIB COULD CAUSE DRUG-RESISTANCE VIA RECRUITMENT OF POLYCOMB GENE COMPLEX TO THE PROMOTER REGION OF THE PTEN AND DOWNREGULATION OF THIS GENE'S TRANSCRIPTS IN LEUKEMIA PATIENTS. 2011 17 1669 25 DOWNREGULATION OF THE HISTONE METHYLTRANSFERASE SETD2 PROMOTES IMATINIB RESISTANCE IN CHRONIC MYELOID LEUKAEMIA CELLS. OBJECTIVES: EPIGENETIC MODIFIERS WERE IMPORTANT PLAYERS IN THE DEVELOPMENT OF HAEMATOLOGICAL MALIGNANCIES AND SENSITIVITY TO THERAPY. MUTATIONS OF SET DOMAIN-CONTAINING 2 (SETD2), A METHYLTRANSFERASE THAT CATALYSES THE TRIMETHYLATION OF HISTONE 3 ON LYSINE 36 (H3K36ME3), WERE FOUND IN VARIOUS MYELOID MALIGNANCIES. HOWEVER, THE DETAILED MECHANISMS THROUGH WHICH SETD2 CONFERS CHRONIC MYELOID LEUKAEMIA PROGRESSION AND RESISTANCE TO THERAPY TARGETING ON BCR-ABL REMAIN UNCLEAR. MATERIALS AND METHODS: THE LEVEL OF SETD2 IN IMATINIB-SENSITIVE AND IMATINIB-RESISTANT CHRONIC MYELOID LEUKAEMIA (CML) CELLS WAS EXAMINED BY IMMUNOBLOTTING AND QUANTITATIVE REAL-TIME PCR. WE ANALYSED CD34(+) CD38(-) LEUKAEMIC STEM CELLS BY FLOW CYTOMETRY AND COLONY FORMATION ASSAYS UPON SETD2 KNOCKDOWN OR OVEREXPRESSION. THE IMPACT OF SETD2 EXPRESSION ALTERATIONS OR SMALL-MOLECULE INHIBITOR JIB-04 TARGETING H3K36ME3 LOSS ON IMATINIB SENSITIVITY WAS ASSESSED BY IC50, CELL APOPTOSIS AND PROLIFERATION ASSAYS. FINALLY, RNA SEQUENCING AND CHIP-QUANTITATIVE PCR WERE PERFORMED TO VERIFY PUTATIVE DOWNSTREAM TARGETS. RESULTS: SETD2 WAS FOUND TO ACT AS A TUMOUR SUPPRESSOR IN CML. THE NOVEL ONCOGENIC TARGETS MYCN AND ERG WERE SHOWN TO BE THE DIRECT DOWNSTREAM TARGETS OF SETD2, WHERE THEIR OVEREXPRESSION INDUCED BY SETD2 KNOCKDOWN CAUSED IMATINIB INSENSITIVITY AND LEUKAEMIC STEM CELL ENRICHMENT IN CML CELL LINES. TREATMENT WITH JIB-04, AN INHIBITOR THAT RESTORES H3K36ME3 LEVELS THROUGH BLOCKADE OF ITS DEMETHYLATION, SUCCESSFULLY IMPROVED THE CELL IMATINIB SENSITIVITY AND ENHANCED THE CHEMOTHERAPEUTIC EFFECT. CONCLUSIONS: OUR STUDY NOT ONLY EMPHASIZES THE REGULATORY MECHANISM OF SETD2 IN CML, BUT ALSO PROVIDES PROMISING THERAPEUTIC STRATEGIES FOR OVERCOMING THE IMATINIB RESISTANCE IN PATIENTS WITH CML. 2019 18 590 21 BET BROMODOMAIN PROTEIN INHIBITION REVERSES CHIMERIC ANTIGEN RECEPTOR EXTINCTION AND REINVIGORATES EXHAUSTED T CELLS IN CHRONIC LYMPHOCYTIC LEUKEMIA. CHIMERIC ANTIGEN RECEPTOR (CAR) T CELLS HAVE INDUCED REMARKABLE ANTITUMOR RESPONSES IN B CELL MALIGNANCIES. SOME PATIENTS DO NOT RESPOND BECAUSE OF T CELL DEFICIENCIES THAT HAMPER THE EXPANSION, PERSISTENCE, AND EFFECTOR FUNCTION OF THESE CELLS. WE USED LONGITUDINAL IMMUNE PROFILING TO IDENTIFY PHENOTYPIC AND PHARMACODYNAMIC CHANGES IN CD19-DIRECTED CAR T CELLS IN PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). CAR EXPRESSION MAINTENANCE WAS ALSO INVESTIGATED BECAUSE THIS CAN AFFECT RESPONSE DURABILITY. CAR T CELL FAILURE WAS ACCOMPANIED BY PREEXISTING T CELL-INTRINSIC DEFECTS OR DYSFUNCTION ACQUIRED AFTER INFUSION. IN A SMALL SUBSET OF PATIENTS, CAR SILENCING WAS OBSERVED COINCIDENT WITH LEUKEMIA RELAPSE. USING A SMALL MOLECULE INHIBITOR, WE DEMONSTRATED THAT THE BROMODOMAIN AND EXTRA-TERMINAL (BET) FAMILY OF CHROMATIN ADAPTERS PLAYS A ROLE IN DOWNREGULATING CAR EXPRESSION. BET PROTEIN BLOCKADE ALSO AMELIORATED CAR T CELL EXHAUSTION AS MANIFESTED BY INHIBITORY RECEPTOR REDUCTION, ENHANCED METABOLIC FITNESS, INCREASED PROLIFERATIVE CAPACITY, AND ENRICHED TRANSCRIPTOMIC SIGNATURES OF T CELL REINVIGORATION. BET INHIBITION DECREASED LEVELS OF THE TET2 METHYLCYTOSINE DIOXYGENASE, AND FORCED EXPRESSION OF THE TET2 CATALYTIC DOMAIN ELIMINATED THE POTENCY-ENHANCING EFFECTS OF BET PROTEIN TARGETING IN CAR T CELLS, PROVIDING A MECHANISM LINKING BET PROTEINS AND T CELL DYSFUNCTION. THUS, MODULATING BET EPIGENETIC READERS MAY IMPROVE THE EFFICACY OF CELL-BASED IMMUNOTHERAPIES. 2021 19 2782 21 EZH2 INHIBITION CONFERS PIK3CA-DRIVEN LUNG TUMORS ENHANCED SENSITIVITY TO PI3K INHIBITION. MEMBERS OF THE PI3K SIGNALING PATHWAY, ESPECIALLY PIK3CA, THE GENE ENCODING THE CATALYTIC SUBUNIT OF THE PI3K COMPLEX, ARE HIGHLY MUTATED AND AMPLIFIED IN VARIOUS CANCER TYPES, INCLUDING NON-SMALL CELL LUNG CANCER. ALTHOUGH PI3K INHIBITORS HAVE BEEN USED IN CLINICS FOR FOLLICULAR LYMPHOMA AND CHRONIC LYMPHOCYTIC LEUKEMIA, NO AGENTS TARGETING PI3K ABERRATIONS IN LUNG CANCER HAVE BEEN APPROVED BY THE FDA SO FAR. IN THIS STUDY, WE OBSERVED THAT PIK3CA-E545K, THE MOST COMMON MUTATION IN LUNG CANCER, HARBORED A MODEST INDUCTION OF STEM-LIKE PROPERTIES IN LUNG EPITHELIAL CELLS, AND DROVE DEVELOPMENT OF ADENOCARCINOMA AUTOCHTHONOUSLY WHEN PAIRED WITH P53 LOSS IN A MURINE MOUSE MODEL. WE ALSO FOUND THAT PIK3CA-MUTANT OF AMPLIFIED LUNG CANCER CELLS WERE SENSITIVE TO EZH2 INHIBITION. EZH2 INHIBITION SYNERGIZED WITH PI3K INHIBITION IN HUMAN CANCER CELLS IN VITRO AND WORKED TOGETHER EFFICIENTLY IN VIVO. MECHANISTICALLY, EZH2 INHIBITION COOPERATED WITH PI3K INHIBITION TO PRODUCE A MORE POTENT SUPPRESSION OF PHOSPHO-AKT DOWNSTREAM OF PI3K. THIS STUDY SUGGESTS A PROMISING COMBINATION THERAPY TO COMBAT LUNG CANCERS WITH PIK3CA MUTATION OR AMPLIFICATION. BOTH COPANLISIB, THE PI3K INHIBITOR, AND TAZEMETOSTAT, THE EZH2 INHIBITOR, ARE FDA-APPROVED, WHICH SHOULD ENHANCE THE CLINICAL TRANSLATION OF THIS WORK. 2022 20 66 25 A KEY ROLE FOR EZH2 IN EPIGENETIC SILENCING OF HOX GENES IN MANTLE CELL LYMPHOMA. THE CHROMATIN MODIFIER EZH2 IS OVEREXPRESSED AND ASSOCIATED WITH INFERIOR OUTCOME IN MANTLE CELL LYMPHOMA (MCL). RECENTLY, WE DEMONSTRATED PREFERENTIAL DNA METHYLATION OF HOX GENES IN MCL COMPARED WITH CHRONIC LYMPHOCYTIC LEUKEMIA (CLL), DESPITE THESE GENES NOT BEING EXPRESSED IN EITHER ENTITY. SINCE EZH2 HAS BEEN SHOWN TO REGULATE HOX GENE EXPRESSION, TO GAIN FURTHER INSIGHT INTO ITS POSSIBLE ROLE IN DIFFERENTIAL SILENCING OF HOX GENES IN MCL VS. CLL, WE PERFORMED DETAILED EPIGENETIC CHARACTERIZATION USING REPRESENTATIVE CELL LINES AND PRIMARY SAMPLES. WE OBSERVED SIGNIFICANT OVEREXPRESSION OF EZH2 IN MCL VS. CLL. CHROMATIN IMMUNE PRECIPITATION (CHIP) ASSAYS REVEALED THAT EZH2 CATALYZED REPRESSIVE H3 LYSINE 27 TRIMETHYLATION (H3K27ME3), WHICH WAS SUFFICIENT TO SILENCE HOX GENES IN CLL, WHEREAS IN MCL H3K27ME3 IS ACCOMPANIED BY DNA METHYLATION FOR A MORE STABLE REPRESSION. MORE IMPORTANTLY, HYPERMETHYLATION OF THE HOX GENES IN MCL RESULTED FROM EZH2 OVEREXPRESSION AND SUBSEQUENT RECRUITMENT OF THE DNA METHYLATION MACHINERY ONTO HOX GENE PROMOTERS. THE IMPORTANCE OF EZH2 UPREGULATION IN THIS PROCESS WAS FURTHER UNDERSCORED BY SIRNA TRANSFECTION AND EZH2 INHIBITOR EXPERIMENTS. ALTOGETHER, THESE OBSERVATIONS IMPLICATE EZH2 IN THE LONG-TERM SILENCING OF HOX GENES IN MCL, AND ALLUDE TO ITS POTENTIAL AS A THERAPEUTIC TARGET WITH CLINICAL IMPACT. 2013