1 1069 166 CLOFARABINE?PHYTOCHEMICAL COMBINATION EXPOSURES IN CML CELLS INHIBIT DNA METHYLATION MACHINERY, UPREGULATE TUMOR SUPPRESSOR GENES AND PROMOTE CASPASE?DEPENDENT APOPTOSIS. CLOFARABINE (2?CHLORO?2'?FLUORO?2'?DEOXYARABINOSYLADENINE, CIF), A SECOND?GENERATION 2'?DEOXYADENOSINE ANALOG, POSSESSES A VARIETY OF ANTI?CANCER ACTIVITIES, INCLUDING THE CAPACITY TO MODULATE DNA METHYLATION MARKS. BIOACTIVE NUTRIENTS, INCLUDING RESVERATROL (RSV) AND ALL?TRANS RETINOIC ACID (ATRA) HAVE BEEN INDICATED TO REGULATE EPIGENETIC MACHINERY IN MALIGNANT CELLS. THE PURPOSE OF THE CURRENT STUDY WAS TO EVALUATE WHETHER THE TESTED PHYTOCHEMICALS, RSV OR ATRA, CAN IMPROVE THE THERAPEUTIC EPIGENETIC EFFECTS OF CIF IN CHRONIC MYELOID LEUKEMIA (CML) CELLS. THE PRESENT STUDY INVESTIGATES, TO THE BEST OF OUR KNOWLEDGE, FOR THE FIRST TIME, THE INFLUENCE OF CIF IN COMBINATION WITH RSV OR ATRA ON THE EXPRESSION OF RELEVANT MODIFIERS OF DNA METHYLATION MACHINERY, INCLUDING DNA METHYLTRANSFERASE 1 (DNMT1) AND CYCLIN DEPENDENT KINASE INHIBITOR 1A (CDKN1A) IN CML CELLS. SUBSEQUENTLY, THE COMBINATORIAL EFFECTS ON PROMOTER METHYLATION AND TRANSCRIPT LEVELS OF METHYLATION?SILENCED TUMOR SUPPRESSOR GENES (TSGS), INCLUDING PHOSPHATASE AND TENSIN HOMOLOGUE (PTEN) AND RETINOIC ACID RECEPTOR BETA (RARB), WERE ESTIMATED USING MSRA AND QPCR, RESPECTIVELY. THE TESTED TSGS WERE CHOSEN ACCORDING TO BIOINFORMATICAL ANALYSIS OF PUBLICLY AVAILABLE CLINICAL DATA OF HUMAN DNA METHYLATION AND GENE EXPRESSION ARRAYS IN LEUKEMIA PATIENTS. THE K562 CELL LINE WAS USED AS AN EXPERIMENTAL CML IN VITRO MODEL. FOLLOWING A PERIOD OF 72 H EXPOSURE OF K562 CELLS, THE TESTED COMBINATIONS LED TO SIGNIFICANT CELL GROWTH INHIBITION AND INDUCTION OF CASPASE?3?DEPENDENT APOPTOSIS. THESE OBSERVATIONS WERE ACCOMPANIED BY DNMT1 DOWNREGULATION AND CDKN1A UPREGULATION, WITH A CONCOMITANT ENHANCED DECREASE IN DNMT1 PROTEIN LEVEL, ESPECIALLY AFTER ATRA TREATMENT WITH CIF. CONCURRENT METHYLATION?MEDIATED RARB AND PTEN REACTIVATION WAS DETECTED. THE RESULTS OF THE CURRENT STUDY DEMONSTRATED THAT CIF THAT WAS USED IN COMBINATION WITH THE TESTED PHYTOCHEMICALS, RSV OR ATRA, EXHIBITED A GREATER ABILITY TO REMODEL DNA METHYLATION MARKS AND PROMOTE CELL DEATH IN CML CELLS. THESE RESULTS MAY SUPPORT THE APPLICATION OF CIF COMBINATIONS WITH NATURAL BIOACTIVE AGENTS IN ANTI?LEUKEMIC EPIGENETIC THERAPY. 2019 2 3532 44 IMATINIB INDEPENDENT ABERRANT METHYLATION OF NOV/CCN3 IN CHRONIC MYELOGENOUS LEUKEMIA PATIENTS: A MECHANISM UPSTREAM OF BCR-ABL1 FUNCTION? BACKGROUND: THE NOV GENE PRODUCT, CCN3, HAS BEEN REPORTED IN A DIVERSE RANGE OF TUMORS TO SERVE AS A NEGATIVE GROWTH REGULATOR, WHILE ACTING AS A TUMOR SUPPRESSOR IN CHRONIC MYELOGENOUS LEUKEMIA (CML). HOWEVER, THE PRECISE MECHANISM OF ITS SILENCING IN CML IS POORLY UNDERSTOOD. IN THE CURRENT STUDY, WE AIMED TO QUERY IF THE GENE REGULATION OF CCN3 IS MEDIATED BY THE PROMOTER METHYLATION IN THE PATIENTS WITH CML. IN ADDITION, TO CLARIFY WHETHER THE EPIGENETIC SILENCING IS AFFECTED BY BCR-ABL1 INHIBITION, WE ASSESSED THE METHYLATION STATUS IN THE PATIENTS AT DIFFERENT TIME INTERVALS FOLLOWING THE TYROSINE KINASE INHIBITION USING IMATINIB THERAPY, AS THE FIRST-LINE TREATMENT FOR THIS TYPE OF LEUKEMIA. METHODS: TO ADDRESS THIS ISSUE, WE APPLIED BISULFITE-SEQUENCING TECHNIQUE AS A HIGH-RESOLUTION METHOD TO STUDY THE REGULATORY SEGMENT OF THE CCN3 GENE. THE RESULTS WERE ANALYZED IN NEWLY DIAGNOSED CML PATIENTS AS WELL AS FOLLOWING IMATINIB THERAPY. WE ALSO EVALUATED THE CORRELATION OF CCN3 PROMOTER METHYLATION WITH BCR-ABL1 LEVELS. RESULTS: OUR FINDINGS REVEALED THAT THE METHYLATION OCCURS FREQUENTLY IN THE PROMOTER REGION OF CML PATIENTS SHOWING A SIGNIFICANT INCREASE OF THE METHYLATED PERCENTAGE AT THE CPG SITES COMPARED TO NORMAL INDIVIDUALS. INTERESTINGLY, THIS HYPERMETHYLATION WAS INDICATED TO BE INDEPENDENT OF BCR-ABL1 TITERS IN BOTH GROUPS, WHICH MIGHT SUGGEST A MECHANISM BEYOND THE BCR-ABL1 FUNCTION. CONCLUSION: DESPITE SUGGESTING THAT THE CCN3 HYPERMETHYLATION ACTS AS A MOLECULAR MECHANISM INDEPENDENT OF BCR-ABL1 FUNCTION IN CML PATIENTS, THIS SCENARIO REQUIRES FURTHER VALIDATION BY COMPLEMENTARY EXPERIMENTS. IN THE CASE OF ACTING UPSTREAM OF BCR-ABL1 SIGNALING, THE METHYLATION MARKER CAN PROVIDE EARLY DETECTION AND A NOVEL PLATFORM FOR TARGETED EPIGENETIC MODIFIERS FOR EFFICIENT TREATMENT IN IMATINIB RESISTANT PATIENTS. 2019 3 4877 50 OVEREXPRESSION OF MIR-4433 BY SUBEROYLANILIDE HYDROXAMIC ACID SUPPRESSES GROWTH OF CML CELLS AND INDUCES APOPTOSIS THROUGH TARGETING BCR-ABL. BACKGROUND: TARGETING BCR-ABL IS THE KEY FOR THE TREATMENT OF CML. ALTHOUGH GREAT PROGRESS HAS BEEN ACHIEVED FOR THE TREATMENT OF CML PATIENTS IN CHRONIC STAGE, EFFECTIVE DRUGS WITH GOOD SAFETY ARE NOT AVAILABLE FOR THOSE IN ADVANCED STAGES OF CML PATIENTS. IN PRESENT STUDY, A HISTONE DEACETYLASE INHIBITOR, SUBEROYLANILIDE HYDROXAMIC ACID (SAHA), WAS USED TO SCREEN FOR MICRORNA THAT CAN TARGET BCR-ABL. METHODS: RT-QPCR WAS USED TO DETERMINE BCR-ABL AND MIR-4433 TRANSCRIPTION LEVEL IN CML CELLS. IN CML CELLS, PROTEINS INCLUDING PARP, CASPASE-3, ACETYL-HISTONE 3, HISTONE 3 AND BCR-ABL, AS WELL AS BCR-ABL DOWNSTREAM PROTEINS WERE DETECTED USING WESTERN BLOT. CELL VIABILITY AND APOPTOSIS WERE MONITORED RESPECTIVELY BY MTS ASSAY AND FLOW CYTOMETRY. THE CORRELATION BETWEEN MIR-4433 AND BCR-ABL WAS DETERMINED BY LUCIFERASE REPORTER ASSAY. THE ANTI-TUMOR EFFECT OF MIR-4433 TO K562 CELLS WAS EVALUATED BY NUDE MOUSE XENOGRAFT MODEL IN VIVO. RESULTS: SAHA UP-REGULATED THE ACETYLATION LEVEL OF HISTONE 3, AND EFFECTIVELY INHIBITED BCR-ABL MRNA LEVEL AND ITS DOWNSTREAM SIGNAL TRANSDUCTION PATHWAY, WHILE INHIBITING THE GROWTH OF CML CELLS AND INDUCING APOPTOSIS. FURTHERMORE, BIOINFORMATICS TOOLS PREDICTED THAT MIR-4433 IS A PUTATIVE MICRORNA TARGETING BCR-ABL AND THAT THE EXPRESSION LEVEL OF MIR-4433 WAS SIGNIFICANTLY INCREASED AFTER SAHA TREATMENT IN K562 CELLS. LUCIFERASE ACTIVITY ANALYSIS REVEALED THAT MIR-4433 DIRECTLY TARGETS BCR-ABL. ADDITIONALLY, TRANSIENT EXPRESSION OF MIR-4433 ABROGATED BCR-ABL ACTIVITY AND ITS DOWNSTREAM SIGNALING PATHWAYS WHILE INDUCING APOPTOSIS IN K562 CELLS. MOREOVER, STABLE EXPRESSION OF MIR-4433 SUPPRESSED BCR-ABL AND ITS DOWNSTREAM SIGNALING PATHWAY, AND INHIBITED THE GROWTH OF K562 CELLS IN VITRO AND THE GROWTH OF K562-XENOGRAFTS IN NUDE MICE. CONCLUSION: MIR-4433 WAS IDENTIFIED AS A MICRORNA TARGETING BCR-ABL, WHICH MAY BE SUBJECT TO EPIGENETIC REGULATION OF SAHA, A HISTONE DEACETYLASE INHIBITOR THAT HAS BEEN APPROVED BY THE US FDA FOR THE TREATMENT OF CUTANEOUS T-CELL LYMPHOMA. THE FINDINGS OF THIS STUDY PROVIDE A MOLECULAR BASIS FROM ANOTHER ANGLE FOR THE USE OF SAHA IN THE TREATMENT OF CML. 2019 4 402 38 ANALYSIS OF APOPTOSOME DYSREGULATION IN PANCREATIC CANCER AND OF ITS ROLE IN CHEMORESISTANCE. THE APOPTOSOME IS A MULTIPROTEIN COMPLEX MEDIATING THE MITOCHONDRIAL PATHWAY OF CELL DEATH. ITS IMPORTANCE DURING DEVELOPMENT HAS BEEN CLEARLY DEMONSTRATED BY KNOCKING OUT KEY GENES IN MOUSE. APAF1 IS THE CORE PROTEIN OF THE APOPTOSOME AND ITS DOSAGE IS ALSO CRITICAL IN VARIOUS CANCER TYPES, I.E., MELANOMA, GERM LINE TUMOR, GASTROINTESTINAL CANCER AND B-TYPE CHRONIC LYMPHOCYTIC LEUKEMIA. THIS IS GENERALLY DUE TO INACTIVATION OF THE APAF1 LOCUS BY EPIGENETIC PHENOMENA OR BY ACTIVITY OF PROMOTER REGULATORS. WE INVESTIGATED THE PUTATIVE ROLES OF THE APOPTOSOME IN PANCREATIC DUCTAL ADENOCARCINOMA (PDAC). WE FOUND THAT BOTH APAF1 MRNA AND PROTEIN ARE DYSREGULATED IN HUMAN PDAC SAMPLES. SIMILARLY, SEVERAL PDAC CELL LINES EXHIBITED VARIABLE LEVELS OF BOTH APAF1 PROTEIN AND MRNA. THE RESPONSE TO CELL DEATH INDUCTION AND ITS BIOCHEMICAL FEATURES WERE ASSESSED BY TREATMENT OF EACH LINE WITH COMMONLY USED CHEMOTHERAPEUTIC AGENTS. WE FOUND THAT THE APOPTOSOME PATHWAY WAS NOT FUNCTIONAL IN MOST CELL LINES UPON CYTOCHROME C RELEASE FROM MITOCHONDRIA. IN ADDITION, WE RESTORED APAF1 AND CASPASE-9 DOSAGE IN PANC-1 CELLS, WHERE THE APOPTOSOME IS DOWNREGULATED, BY OVEREXPRESSING THE MURINE CDNA OF THE TWO MOLECULES, AND WE IMPROVED THE DEATH RESPONSE TO CHEMOTHERAPEUTIC AGENTS. 2007 5 1144 45 CONCOMITANT HETEROCHROMATINISATION AND DOWN-REGULATION OF GENE EXPRESSION UNVEILS EPIGENETIC SILENCING OF RELB IN AN AGGRESSIVE SUBSET OF CHRONIC LYMPHOCYTIC LEUKEMIA IN MALES. BACKGROUND: THE SENSITIVITY OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) CELLS TO CURRENT TREATMENTS, BOTH IN VITRO AND IN VIVO, RELIES ON THEIR ABILITY TO ACTIVATE APOPTOTIC DEATH. CLL CELLS RESISTANT TO DNA DAMAGE-INDUCED APOPTOSIS DISPLAY DEREGULATION OF A SPECIFIC SET OF GENES. METHODS: MICROARRAY HYBRIDIZATION (HUMAN GENECHIP, AFFYMETRIX), IMMUNOFLUORESCENT IN SITU LABELING COUPLED WITH VIDEO-MICROSCOPY RECORDING/ANALYSES, CHROMATIN-IMMUNOPRECIPITATION (CHIP), POLYMERASE CHAIN REACTIONS (PCR), REAL-TIME QUANTITATIVE PCR (RT-QPCR) AND BISULFITE GENOME SEQUENCING WERE THE MAIN METHODS APPLIED. STATISTICAL ANALYSES WERE PERFORMED BY APPLYING GCRMA AND SAM ANALYSIS (MICROARRAY DATA) AND STUDENT'S T-TEST OR MANN & WHITNEY'S U-TEST. RESULTS: HEREIN WE SHOW THAT, REMARKABLY, IN A RESISTANT MALE CLL CELLS THE VAST MAJORITY OF GENES WERE DOWN-REGULATED COMPARED WITH SENSITIVE CELLS, WHEREAS THIS WAS NOT THE CASE IN CELLS DERIVED FROM FEMALES. THIS GENE DOWN-REGULATION WAS FOUND TO BE ASSOCIATED WITH AN OVERALL GAIN OF HETEROCHROMATIN AS EVIDENCED BY IMMUNOFLUORESCENT LABELING OF HETEROCHROMATIN PROTEIN 1ALPHA (HP-1), TRIMETHYLATED HISTONE 3 LYSINE 9 (3METH3K9), AND 5-METHYLCYTIDINE (5METC). NOTABLY, 17 GENES WERE FOUND TO BE COMMONLY DEREGULATED IN RESISTANT MALE AND FEMALE CELL SAMPLES. AMONG THESE, RELB WAS IDENTIFIED AS A DISCRIMINATORY CANDIDATE GENE REPRESSED IN THE MALE AND UPREGULATED IN THE FEMALE RESISTANT CELLS. CONCLUSION: THE MOLECULAR DEFECTS IN THE SILENCING OF RELB INVOLVE AN INCREASE IN H3K9- BUT NOT CPG-ISLAND METHYLATION IN THE PROMOTER REGIONS. INCREASE IN ACETYL-H3 IN RESISTANT FEMALE BUT NOT MALE CLL SAMPLES AS WELL AS A DECREASE OF TOTAL CELLULAR LEVEL OF RELB AFTER AN INHIBITION OF HISTONE DEACETYLASE (HDAC) BY TRICHOSTATIN A (TSA), FURTHER EMPHASIZE THE ROLE OF EPIGENETIC MODIFICATIONS WHICH COULD DISCRIMINATE TWO CLL SUBSETS. TOGETHER, THESE RESULTS HIGHLIGHTED THE EPIGENETIC RELB SILENCING AS A NEW MARKER OF THE PROGRESSIVE DISEASE IN MALES. 2010 6 5972 28 TET REPRESSION AND INCREASED DNMT ACTIVITY SYNERGISTICALLY INDUCE ABERRANT DNA METHYLATION. CHRONIC INFLAMMATION IS DEEPLY INVOLVED IN VARIOUS HUMAN DISORDERS, SUCH AS CANCER, NEURODEGENERATIVE DISORDERS, AND METABOLIC DISORDERS. INDUCTION OF EPIGENETIC ALTERATIONS, ESPECIALLY ABERRANT DNA METHYLATION, IS ONE OF THE MAJOR MECHANISMS, BUT HOW IT IS INDUCED IS STILL UNCLEAR. HERE, WE FOUND THAT EXPRESSION OF TET GENES, METHYLATION ERASERS, WAS DOWNREGULATED IN INFLAMED MOUSE AND HUMAN TISSUES, AND THAT THIS WAS CAUSED BY UPREGULATION OF TET-TARGETING MIRNAS SUCH AS MIR20A, MIR26B, AND MIR29C, LIKELY DUE TO ACTIVATION OF NF-KAPPAB SIGNALING DOWNSTREAM OF IL-1BETA AND TNF-ALPHA. HOWEVER, TET KNOCKDOWN INDUCED ONLY MILD ABERRANT METHYLATION. NITRIC OXIDE (NO), PRODUCED BY NOS2, ENHANCED ENZYMATIC ACTIVITY OF DNA METHYLTRANSFERASES (DNMTS), METHYLATION WRITERS, AND NO EXPOSURE INDUCED MINIMAL ABERRANT METHYLATION. IN CONTRAST, A COMBINATION OF TET KNOCKDOWN AND NO EXPOSURE SYNERGISTICALLY INDUCED ABERRANT METHYLATION, INVOLVING GENOMIC REGIONS NOT METHYLATED BY EITHER ALONE. THE RESULTS SHOWED THAT A VICIOUS COMBINATION OF TET REPRESSION, DUE TO NF-KAPPAB ACTIVATION, AND DNMT ACTIVATION, DUE TO NO PRODUCTION, IS RESPONSIBLE FOR ABERRANT METHYLATION INDUCTION IN HUMAN TISSUES. 2020 7 3415 42 HSP90 INHIBITION INCREASES SOCS3 TRANSCRIPT AND REGULATES MIGRATION AND CELL DEATH IN CHRONIC LYMPHOCYTIC LEUKEMIA. EPIGENETIC OR TRANSCRIPTIONAL SILENCING OF IMPORTANT TUMOR SUPPRESSORS HAS BEEN DESCRIBED TO CONTRIBUTE TO CELL SURVIVAL AND TUMORIGENESIS IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). USING GENE EXPRESSION MICROARRAY ANALYSIS, WE FOUND THAT THOUSANDS OF GENES ARE REPRESSED MORE THAN 2-FOLD IN CLL COMPARED TO NORMAL B CELLS; HOWEVER THERAPEUTIC APPROACHES TO REVERSE THIS HAVE BEEN LIMITED IN CLL. FOLLOWING TREATMENT WITH THE HSP90 INHIBITOR 17-DMAG, A SIGNIFICANT NUMBER OF THESE REPRESSED GENES WERE SIGNIFICANTLY RE-EXPRESSED. ONE OF THE GENES SIGNIFICANTLY REPRESSED IN CLL AND UP-REGULATED BY 17-DMAG WAS SUPPRESSOR OF CYTOKINE SIGNALING 3, (SOCS3). SOCS3 HAS BEEN SHOWN TO BE SILENCED IN SOLID TUMORS AS WELL AS MYELOID LEUKEMIA; HOWEVER LITTLE IS KNOWN ABOUT THE REGULATION IN CLL. WE FOUND THAT 17-DMAG INDUCES EXPRESSION OF SOCS3 BY VIA THE ACTIVATION OF P38 SIGNALING, AND SUBSEQUENTLY INHIBITS AKT AND STAT3 PHOSPHORYLATION RESULTING IN DOWNSTREAM EFFECTS ON CELL MIGRATION AND SURVIVAL. WE THEREFORE SUGGEST THAT SOCS3 IS AN IMPORTANT SIGNALING PROTEIN IN CLL, AND HSP90 INHIBITORS REPRESENT A NOVEL APPROACH TO TARGET TRANSCRIPTIONAL REPRESSION IN B CELL LYMPHOPROLIFERATIVE DISORDERS WHICH EXHIBIT A SUBSTANTIAL DEGREE OF GENE REPRESSION. 2016 8 3147 45 GLP OVEREXPRESSION IS ASSOCIATED WITH POOR PROGNOSIS IN CHRONIC LYMPHOCYTIC LEUKEMIA AND ITS INHIBITION INDUCES LEUKEMIC CELL DEATH. BACKGROUND HETERODIMERIC METHYLTRANSFERASES GLP (EHMT1/KMT1D) AND G9A (EHMT2/KMT1C) ARE TWO CLOSELY RELATED ENZYMES THAT PROMOTE THE MONOMETHYLATION AND DIMETHYLATION OF HISTONE H3 LYSINE 9. DYSREGULATION OF THEIR ACTIVITY HAS BEEN IMPLICATED IN SEVERAL TYPES OF HUMAN CANCER. PATIENTS AND METHODS HERE, IN ORDER TO INVESTIGATE WHETHER GLP/G9A EXERTS ANY IMPACT ON CHRONIC LYMPHOCYTIC LEUKEMIA (CLL), GLP/G9A EXPRESSION LEVELS WERE ASSESSED IN A COHORT OF 50 PATIENTS AND THE EFFECTS OF THEIR INHIBITION WERE VERIFIED FOR THE VIABILITY OF CLL CELLS. ALSO, QRT-PCR WAS USED TO INVESTIGATE THE TRANSCRIPTIONAL LEVELS OF GLP/G9A IN CLL PATIENTS. IN ADDITION, PATIENT SAMPLES WERE CLASSIFIED ACCORDING TO ZAP-70 PROTEIN EXPRESSION BY FLOW CYTOMETRY AND ACCORDING TO KARYOTYPE INTEGRITY BY CYTOGENETICS ANALYSIS. FINALLY, A SELECTIVE SMALL MOLECULE INHIBITOR FOR GLP/G9A WAS USED TO ASCERTAIN WHETHER THESE METHYLTRANSFERASES INFLUENCED THE VIABILITY OF MEC-1 CLL CELL LINEAGE. RESULTS MRNA ANALYSIS REVEALED THAT CLL SAMPLES HAD HIGHER LEVELS OF GLP, BUT NOT G9A, WHEN COMPARED TO NON-LEUKEMIC CONTROLS. INTERESTINGLY, PATIENTS WITH UNFAVORABLE CYTOGENETICS SHOWED HIGHER EXPRESSION LEVELS OF GLP COMPARED TO PATIENTS WITH FAVORABLE KARYOTYPES. MORE IMPORTANTLY, GLP/G9A INHIBITION MARKEDLY INDUCED CELL DEATH IN CLL CELLS. CONCLUSION TAKEN TOGETHER, THESE RESULTS INDICATE THAT GLP IS ASSOCIATED WITH A WORSE PROGNOSIS IN CLL, AND THAT THE INHIBITION OF GLP/G9A INFLUENCES CLL CELL VIABILITY. ALTOGETHER, THE PRESENT DATA DEMONSTRATE THAT THESE METHYLTRANSFERASES CAN BE POTENTIAL MARKERS FOR DISEASE PROGRESSION, AS WELL AS A PROMISING EPIGENETIC TARGET FOR CLL TREATMENT AND THE PREVENTION OF DISEASE EVOLUTION. 2018 9 2025 35 EPIGENETIC CHANGES DURING DISEASE PROGRESSION IN A MURINE MODEL OF HUMAN CHRONIC LYMPHOCYTIC LEUKEMIA. EPIGENETIC ALTERATIONS, INCLUDING GAIN OR LOSS OF DNA METHYLATION, ARE A HALLMARK OF NEARLY EVERY MALIGNANCY. CHANGES IN DNA METHYLATION CAN IMPACT EXPRESSION OF CANCER-RELATED GENES INCLUDING APOPTOSIS REGULATORS AND TUMOR SUPPRESSORS. BECAUSE SUCH EPIGENETIC CHANGES ARE REVERSIBLE, THEY ARE BEING AGGRESSIVELY INVESTIGATED AS POTENTIAL THERAPEUTIC TARGETS. HERE WE USE THE EMU-TCL1 TRANSGENIC MOUSE MODEL OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) TO DETERMINE THE TIMING AND PATTERNS OF ABERRANT DNA METHYLATION, AND TO INVESTIGATE THE MECHANISMS THAT LEAD TO ABERRANT DNA METHYLATION. WE SHOW THAT CLL CELLS FROM EMU-TCL1 MICE AT VARIOUS STAGES RECAPITULATE EPIGENETIC ALTERATIONS SEEN IN HUMAN CLL. ABERRANT METHYLATION OF PROMOTER SEQUENCES IS OBSERVED AS EARLY AS 3 MONTHS OF AGE IN THESE ANIMALS, WELL BEFORE DISEASE ONSET. ABNORMALLY METHYLATED PROMOTER REGIONS INCLUDE BINDING SITES FOR THE TRANSCRIPTION FACTOR FOXD3. WE SHOW THAT LOSS OF FOXD3 EXPRESSION DUE TO AN NF-KAPPAB P50/P50:HDAC1 REPRESSOR COMPLEX OCCURS IN TCL1-POSITIVE B CELLS BEFORE METHYLATION. THEREFORE, SPECIFIC TRANSCRIPTIONAL REPRESSION IS AN EARLY EVENT LEADING TO EPIGENETIC SILENCING OF TARGET GENES IN MURINE AND HUMAN CLL. THESE RESULTS PROVIDE STRONG RATIONALE FOR THE DEVELOPMENT OF STRATEGIES TO TARGET NF-KAPPAB COMPONENTS IN CLL AND POTENTIALLY OTHER B-CELL MALIGNANCIES. 2009 10 3527 37 IL-6 ENHANCES THE NUCLEAR TRANSLOCATION OF DNA CYTOSINE-5-METHYLTRANSFERASE 1 (DNMT1) VIA PHOSPHORYLATION OF THE NUCLEAR LOCALIZATION SEQUENCE BY THE AKT KINASE. THE EPIGENETIC PROGRAMMING OF GENOMIC DNA IS ACCOMPLISHED, IN PART, BY SEVERAL DNA CYTOSINE-5-METHYLTRANSFERASES THAT ACT BY COVALENTLY MODIFYING CYTOSINES WITH THE ADDITION OF A METHYL GROUP. THIS COVALENT MODIFICATION IS MAINTAINED BY THE DNA CYTOSINE-5-METHYLTRANSFERASE-1 ENZYME (DNMT1), WHICH IS CAPABLE OF ACTING IN CONCERT WITH OTHER SIMILAR ENZYMES TO SILENCE IMPORTANT TUMOR SUPPRESSOR GENES. IL-6 IS A MULTIFUNCTIONAL MEDIATOR OF INFLAMMATION, ACTING THROUGH SEVERAL MAJOR SIGNALING CASCADES, INCLUDING THE PHOSPHATIDYLINOSITOL-3-KINASE PATHWAY (PI-3-K), WHICH ACTIVATES PROTEIN KINASE B (AKT/PKB) DOWNSTREAM. HERE, WE SHOW THAT THE SUBCELLULAR LOCALIZATION OF DNMT1 CAN BE ALTERED BY THE ADDITION OF IL-6, INCREASING THE RATE OF NUCLEAR TRANSLOCATION OF THE ENZYME FROM THE CYTOSOLIC COMPARTMENT. THE MECHANISM OF NUCLEAR TRANSLOCATION OF DNMT1 IS GREATLY ENHANCED BY PHOSPHORYLATION OF THE DNMT1 NUCLEAR LOCALIZATION SIGNAL (NLS) BY PKB/AKT KINASE. MUTAGENIC ALTERATION OF THE TWO AKT TARGET AMINO ACIDS WITHIN THE NLS RESULTS IN A MAJOR LOSS OF DNMT1 NUCLEAR TRANSLOCATION, WHILE THE CREATION OF A "PHOSPHO-MIMIC" AMINO ACID (MUTATION TO ACIDIC RESIDUES) RESTORES THIS COMPARTMENTATION ABILITY. THESE OBSERVATIONS SUGGEST AN INTERESTING HYPOTHESIS REGARDING HOW MEDIATORS OF CHRONIC INFLAMMATION MAY DISTURB THE DELICATE BALANCE OF CELLULAR COMPARTMENTALIZATION OF IMPORTANT PROTEINS, AND REVEALS A POTENTIAL MECHANISM FOR THE INDUCTION OR ENHANCEMENT OF TUMOR GROWTH VIA ALTERATION OF THE COMPONENTS INVOLVED IN THE EPIGENETIC PROGRAMMING OF A CELL. 2007 11 1272 40 CYTOTOXIC ACTIVITY OF VALPROIC ACID ON PRIMARY CHRONIC LYMPHOCYTIC LEUKEMIA CELLS. BACKGROUND: CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) IS THE MOST COMMON ADULT LEUKEMIA IN WESTERN CIVILIZATION. THE ACCUMULATION OF CD5+CD19+ B LYMPHOCYTES IN PERIPHERAL BLOOD IS DUE TO A DEFECT IN THE APOPTOTIC PATHWAY RATHER THAN EXCESSIVE PROLIFERATION IN THE BONE MARROW AND LYMPH NODES. DESPITE A NUMBER OF TREATMENTS, CLL REMAINS AN INCURABLE DISEASE. VALPROIC ACID (VPA) ACTIVITY, AS A HISTONE DEACETYLASE INHIBITOR, COULD RESTORE THE EPIGENETIC CHANGES UNDERLYING THE PATHOGENESIS OF CLL AND THUS INDUCE CELL DEATH. OBJECTIVES: IN THE PRESENT STUDY WE HYPOTHESIZED THAT VPA COULD INDUCE CLL PRIMARY CELLS DEATH THROUGH ACTIVATION OF APOPTOSIS. MATERIAL AND METHODS: PERIPHERAL BLOOD SAMPLES WERE OBTAINED FROM 53 CLL PATIENTS. PERIPHERAL BLOOD MONONUCLEAR CELLS WERE ISOLATED THROUGH DENSITY GRADIENT CENTRIFUGATION AND WERE THE SUBJECT OF A 24-HOUR CELL CULTURE WITH 10 MM OF VPA. THE CYTOTOXIC EFFECT OF VPA WAS EVALUATED WITH AN XTT TEST AND THEREAFTER CONFIRMED USING ANNEXIN V-FITC/PI STAINING AND FLOW CYTOMETRY TECHNIQUES. RESULTS: IN THIS STUDY, A MEDIAN VPA CYTOTOXICITY OF 13.88% WITH A RANGE OF 0-54.65% WAS OBSERVED. ANNEXIN V/PI STAINING CONFIRMED THAT THE DEMONSTRATED CYTOTOXICITY WAS CAUSED BY INCREASED APOPTOSIS IN THE VPA TREATED CELLS AS COMPARED TO CONTROL CELLS. STATISTICAL ANALYSIS SHOWED THAT VPA'S EFFECT ON CLL CELLS DEPENDS ON LACTATE DEHYDROGENASE SERUM LEVELS, BUT IS INDEPENDENT OF ALL OTHER PROGNOSTIC MARKERS. CONCLUSIONS: THE RESULTS OF THE PRESENT EXPERIMENTS FOUND THAT VPA AT A CLINICALLY APPLICABLE CONCENTRATION SIGNIFICANTLY INDUCES APOPTOSIS INDEPENDENTLY OF THE DISEASE STAGE AND MIGHT BE A VALUABLE THERAPEUTIC AGENT FOR ALL CLL PATIENTS. 2015 12 1117 38 COMPARATIVE AND EXPERIMENTAL STUDIES ON THE GENES ALTERED BY CHRONIC HYPOXIA IN HUMAN BRAIN MICROENDOTHELIAL CELLS. BACKGROUND : HYPOXIA INDUCIBLE FACTOR 1 ALPHA (HIF1A) IS A MASTER REGULATOR OF ACUTE HYPOXIA; HOWEVER, WITH CHRONIC HYPOXIA, HIF1A LEVELS RETURN TO THE NORMOXIC LEVELS. IMPORTANTLY, THE GENES THAT ARE INVOLVED IN THE CELL SURVIVAL AND VIABILITY UNDER CHRONIC HYPOXIA ARE NOT KNOWN. THEREFORE, WE TESTED THE HYPOTHESIS THAT CHRONIC HYPOXIA LEADS TO THE UPREGULATION OF A CORE GROUP OF GENES WITH ASSOCIATED CHANGES IN THE PROMOTER DNA METHYLATION THAT MEDIATES THE CELL SURVIVAL UNDER HYPOXIA. RESULTS : WE EXAMINED THE EFFECT OF CHRONIC HYPOXIA (3 DAYS; 0.5% OXYGEN) ON HUMAN BRAIN MICRO ENDOTHELIAL CELLS (HBMEC) VIABILITY AND APOPTOSIS. HYPOXIA CAUSED A SIGNIFICANT REDUCTION IN CELL VIABILITY AND AN INCREASE IN APOPTOSIS. NEXT, WE EXAMINED CHRONIC HYPOXIA ASSOCIATED CHANGES IN TRANSCRIPTOME AND GENOME-WIDE PROMOTER METHYLATION. THE DATA OBTAINED WAS COMPARED WITH 16 OTHER MICROARRAY STUDIES ON CHRONIC HYPOXIA. NINE GENES WERE ALTERED IN RESPONSE TO CHRONIC HYPOXIA IN ALL 17 STUDIES. INTERESTINGLY, HIF1A WAS NOT ALTERED WITH CHRONIC HYPOXIA IN ANY OF THE STUDIES. FURTHERMORE, WE COMPARED OUR DATA TO THREE OTHER STUDIES THAT IDENTIFIED HIF-RESPONSIVE GENES BY VARIOUS APPROACHES. ONLY TWO GENES WERE FOUND TO BE HIF DEPENDENT. WE SILENCED EACH OF THESE 9 GENES USING CRISPR/CAS9 SYSTEM. DOWNREGULATION OF EGLN3 SIGNIFICANTLY INCREASED THE CELL DEATH UNDER CHRONIC HYPOXIA, WHEREAS DOWNREGULATION OF ERO1L, ENO2, ADRENOMEDULLIN, AND SPAG4 REDUCED THE CELL DEATH UNDER HYPOXIA. CONCLUSIONS : WE PROVIDE A CORE GROUP OF GENES THAT REGULATES CELLULAR ACCLIMATIZATION UNDER CHRONIC HYPOXIC STRESS, AND MOST OF THEM ARE HIF INDEPENDENT. 2017 13 4221 50 METHYLATION AND SILENCING OF PROTEIN TYROSINE PHOSPHATASE RECEPTOR TYPE O IN CHRONIC LYMPHOCYTIC LEUKEMIA. PURPOSE: PREVIOUS STUDIES IN OUR LABORATORY HAVE SHOWN THE PROGRESSIVE METHYLATION AND SUPPRESSION OF THE GENE ENCODING PROTEIN TYROSINE PHOSPHATASE, PTPRO, IN THE LIVERS OF RATS FED A METHYL-DEFICIENT DIET THAT INDUCES HEPATOCARCINOGENESIS. SUBSEQUENTLY, WE OBSERVED THE METHYLATION OF PTPRO IN PRIMARY HUMAN LUNG TUMORS AND ALSO SHOWED ITS POTENTIAL TUMOR SUPPRESSOR CHARACTERISTICS. THE PRESENT STUDY WAS UNDERTAKEN TO INVESTIGATE WHETHER THE TRUNCATED FORM OF PTPRO (PTPROT), SPECIFICALLY EXPRESSED IN NAIVE B LYMPHOCYTES, WAS ALSO METHYLATED AND SUPPRESSED IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL), A DISEASE GENERALLY AFFECTING B LYMPHOCYTES. EXPERIMENTAL DESIGN AND RESULTS: INITIAL SCREENING SHOWED THAT 60% OF THE 52 CLL SAMPLES ANALYZED USING METHYLATION-SPECIFIC PCR ASSAY WERE METHYLATED COMPARED WITH B LYMPHOCYTES FROM NORMAL INDIVIDUALS, WHICH WERE NOT METHYLATED. THE EXPRESSION OF PTPROT, AS MEASURED BY SEMIQUANTITATIVE REVERSE TRANSCRIPTION-PCR, INVERSELY CORRELATED WITH METHYLATION IN THE FEW SAMPLES TESTED. ANALYSIS OF ADDITIONAL SAMPLES (N = 50) BY COMBINED BISULFITE RESTRICTION ANALYSIS SHOWED THAT THE PTPRO CPG ISLAND WAS METHYLATED IN 82% OF PATIENTS WITH CLL COMPARED WITH B LYMPHOCYTES FROM NORMAL INDIVIDUALS. FURTHERMORE, OVERALL EXPRESSION OF PTPRO WAS REDUCED IN CLL RELATIVE TO NORMAL LYMPHOCYTES. THE PTPRO GENE WAS ALSO SUPPRESSED BY METHYLATION IN THE CLL CELL LINE WAC3CD5, WHERE IT COULD BE REACTIVATED UPON TREATMENT WITH THE DNA HYPOMETHYLATING AGENT 5-AZAC. ECTOPIC EXPRESSION OF PTPROT IN A NONEXPRESSING CELL LINE INCREASED GROWTH INHIBITION WITH FLUDARABINE TREATMENT, A THERAPY COMMONLY USED FOR CLL. CONCLUSION: THIS STUDY REVEALS THE POTENTIAL ROLE OF PTPRO METHYLATION AND SILENCING IN CLL TUMORIGENESIS AND ALSO PROVIDES A NOVEL MOLECULAR TARGET IN THE EPIGENETIC THERAPY. 2007 14 171 45 ABROGATION OF HISTONE DEACETYLASES (HDACS) DECREASES SURVIVAL OF CHRONIC MYELOID LEUKEMIA CELLS: NEW INSIGHT INTO ATTENUATING EFFECTS OF THE PI3K/C-MYC AXIS ON PANOBINOSTAT CYTOTOXICITY. ALTHOUGH THE IDENTIFICATION OF TYROSINE KINASE INHIBITORS (TKIS) HAS CHANGED THE TREATMENT PARADIGM OF MANY CANCER TYPES INCLUDING CHRONIC MYELOID LEUKEMIA (CML), STILL ADJUSTMENT OF NEOPLASTIC CELLS TO CYTOTOXIC EFFECTS OF ANTICANCER DRUGS IS A SERIOUS CHALLENGE. IN THE AREA OF DRUG RESISTANCE, EPIGENETIC ALTERATIONS ARE AT THE CENTER OF ATTENTION AND THE PRESENT STUDY AIMED TO EVALUATE WHETHER BLOCKAGE OF EPIGENETICS MECHANISMS USING A PAN-HISTONE DEACETYLASE (HDAC) INHIBITOR INDUCES CELL DEATH IN CML-DERIVED K562 CELLS. WE FOUND THAT THE ABROGATION OF HDACS USING PANOBINOSTAT RESULTED IN A REDUCTION IN SURVIVAL OF THE K562 CELL LINE THROUGH P27-MEDIATED CELL CYCLE ARREST. NOTEWORTHY, THE RESULTS OF THE SYNERGISTIC EXPERIMENTS REVEALED THAT HDAC SUPPRESSION COULD BE RECRUITED AS A WAY TO POTENTIATE CYTOTOXICITY OF IMATINIB AND TO ENHANCE THE THERAPEUTIC EFFICACY OF CML. HERE, WE PROPOSED FOR THE FIRST TIME THAT THE INHIBITORY EFFECT OF PANOBINOSTAT WAS OVERSHADOWED, AT LEAST PARTIALLY, THROUGH THE ABERRANT ACTIVATION OF THE PHOSPHOINOSITIDE 3-KINASE (PI3K)/C-MYC AXIS. MEANWHILE, WE FOUND THAT UPON BLOCKAGE OF AUTOPHAGY AND THE PROTEASOME PATHWAY, AS THE MAIN AXIS INVOLVED IN THE ACTIVATION OF AUTOPHAGY, THE ANTI-LEUKEMIC PROPERTY OF THE HDAC INHIBITOR WAS POTENTIATED. TAKEN TOGETHER, OUR STUDY SUGGESTS THE BENEFICIAL APPLICATION OF HDAC INHIBITION IN THE TREATMENT STRATEGIES OF CML; HOWEVER, FURTHER IN VIVO STUDIES ARE NEEDED TO DETERMINE THE EFFICACY OF THIS INHIBITOR, EITHER AS A SINGLE AGENT OR IN COMBINATION WITH SMALL MOLECULE INHIBITORS OF PI3K AND/OR C-MYC IN THIS MALIGNANCY. 2021 15 139 35 ABERRANT DNA METHYLATION IS ASSOCIATED WITH DISEASE PROGRESSION, RESISTANCE TO IMATINIB AND SHORTENED SURVIVAL IN CHRONIC MYELOGENOUS LEUKEMIA. THE EPIGENETIC IMPACT OF DNA METHYLATION IN CHRONIC MYELOGENOUS LEUKEMIA (CML) IS NOT COMPLETELY UNDERSTOOD. TO ELUCIDATE ITS ROLE WE ANALYZED 120 PATIENTS WITH CML FOR METHYLATION OF PROMOTER-ASSOCIATED CPG ISLANDS OF 10 GENES. FIVE GENES WERE IDENTIFIED BY DNA METHYLATION SCREENING IN THE K562 CELL LINE AND 3 GENES IN PATIENTS WITH MYELOPROLIFERATIVE NEOPLASMS. THE CDKN2B GENE WAS SELECTED FOR ITS FREQUENT METHYLATION IN MYELOID MALIGNANCIES AND ABL1 AS THE TARGET OF BCR-ABL TRANSLOCATION. THIRTY PATIENTS WERE IMATINIB-NAIVE (MOSTLY TREATED BY INTERFERON-ALPHA BEFORE THE IMATINIB ERA), 30 WERE IMATINIB-RESPONSIVE, 50 WERE IMATINIB-RESISTANT, AND 10 WERE IMATINIB-INTOLERANT. WE QUANTIFIED DNA METHYLATION BY BISULFITE PYROSEQUENCING. THE AVERAGE NUMBER OF METHYLATED GENES WAS 4.5 PER PATIENT IN THE CHRONIC PHASE, INCREASING SIGNIFICANTLY TO 6.2 IN THE ACCELERATED AND 6.4 IN THE BLASTIC PHASE. HIGHER NUMBERS OF METHYLATED GENES WERE ALSO OBSERVED IN PATIENTS RESISTANT OR INTOLERANT TO IMATINIB. THESE PATIENTS ALSO SHOWED ALMOST EXCLUSIVE METHYLATION OF A PUTATIVE TRANSPORTER OSCP1. ABNORMAL METHYLATION OF A SRC SUPPRESSOR GENE PDLIM4 WAS ASSOCIATED WITH SHORTENED SURVIVAL INDEPENDENTLY OF CML STAGE AND IMATINIB RESPONSIVENESS. WE CONCLUDE THAT ABERRANT DNA METHYLATION IS ASSOCIATED WITH CML PROGRESSION AND THAT DNA METHYLATION COULD BE A MARKER ASSOCIATED WITH IMATINIB RESISTANCE. FINALLY, DNA METHYLATION OF PDLIM4 MAY HELP IDENTIFY A SUBSET OF CML PATIENTS THAT WOULD BENEFIT FROM TREATMENT WITH SRC/ABL INHIBITORS. 2011 16 5871 29 SUSTAINED NF-KAPPAB ACTIVITY IN CHRONIC LYMPHOCYTIC LEUKEMIA IS INDEPENDENT OF GENETIC AND EPIGENETIC ALTERATIONS IN THE TNFAIP3 (A20) LOCUS. INAPPROPRIATE NUCLEAR FACTOR (NF) KAPPAB ACTIVITY IS ONE MAJOR HALLMARK OF B-CELL MALIGNANCIES AND CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). NFKAPPAB-DEPENDENT GENES ARE INVOLVED IN ANTIAPOPTOSIS, CELL PROLIFERATION AND METASTASIS AND ARE RESPONSIBLE FOR SURVIVAL AND PROLIFERATION OF TUMORS. HOWEVER, THE MECHANISMS OF NFKAPPAB ACTIVITY IN CLL STILL NEED TO BE ELUCIDATED. PREVIOUSLY, WE IDENTIFIED TRANSLOCATIONS IN A REGION ON CHROMOSOME 6Q THAT ENCODES TUMOR NECROSIS FACTOR ALPHA-INDUCED PROTEIN 3, WHICH IS A KEY PLAYER IN NEGATIVE FEEDBACK LOOP REGULATION OF NFKAPPAB. INACTIVATION OF THIS UBIQUITIN-EDITING ENZYME IS INVOLVED IN IMMUNOPATHOLOGIES AND IN TUMORIGENESIS. FREQUENT MUTATIONS IN THE A20 LOCUS--LEADING TO SUSTAINED NFKAPPAB ACTIVITY--COULD BE SHOWN TO PLAY A DOMINANT ROLE IN DEVELOPMENT OF DIFFERENT B-CELL MALIGNANCIES. TO CHECK IF A20 IS INVOLVED IN UPREGULATION OF NFKAPPAB ACTIVITY IN CLL, WE SEQUENCED EXONS 2-9 OF THE A20 GENE IN 55 CLL DNA SAMPLES. FURTHERMORE, WE DETERMINED THE METHYLATION STATUS OF THE PROMOTER REGION IN 63 CLL DNA SAMPLES AND COMPARED TO 10 CONTROL DNAS OF B CELLS FROM HEALTHY DONORS. CONTRARY TO REPORTS FROM OTHER B-CELL MALIGNANCIES, THE A20 REGION SHOWED NEITHER MUTATIONS NOR ABERRANT DNA METHYLATION. MOREOVER, ITS EXPRESSION COULD BE CONFIRMED BY IMMUNOBLOTTING AND SHOWING COMPARABLE RESULTS TO HEALTHY B CELLS. THESE RESULTS INDICATE THAT MALIGNANT DEVELOPMENT IN CLL DIFFERS FROM MOST OF OTHER B-CELL MALIGNANCIES, WHICH SHOW FREQUENT INACTIVATION OF A20. 2011 17 1669 41 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 18 206 38 ACTIVATION OF NOTCH AND MYC SIGNALING VIA B-CELL-RESTRICTED DEPLETION OF DNMT3A GENERATES A CONSISTENT MURINE MODEL OF CHRONIC LYMPHOCYTIC LEUKEMIA. CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) IS CHARACTERIZED BY DISORDERED DNA METHYLATION, SUGGESTING THESE EPIGENETIC CHANGES MIGHT PLAY A CRITICAL ROLE IN DISEASE ONSET AND PROGRESSION. THE METHYLTRANSFERASE DNMT3A IS A KEY REGULATOR OF DNA METHYLATION. ALTHOUGH DNMT3A SOMATIC MUTATIONS IN CLL ARE RARE, WE FOUND THAT LOW DNMT3A EXPRESSION IS ASSOCIATED WITH MORE AGGRESSIVE DISEASE. A CONDITIONAL KNOCKOUT MOUSE MODEL SHOWED THAT HOMOZYGOUS DEPLETION OF DNMT3A FROM B CELLS RESULTS IN THE DEVELOPMENT OF CLL WITH 100% PENETRANCE AT A MEDIAN AGE OF ONSET OF 5.3 MONTHS, AND HETEROZYGOUS DNMT3A DEPLETION YIELDS A DISEASE PENETRANCE OF 89% WITH A MEDIAN ONSET AT 18.5 MONTHS, CONFIRMING ITS ROLE AS A HAPLOINSUFFICIENT TUMOR SUPPRESSOR. B1A CELLS WERE CONFIRMED AS THE CELL OF ORIGIN OF DISEASE IN THIS MODEL, AND DNMT3A DEPLETION RESULTED IN FOCAL HYPOMETHYLATION AND ACTIVATION OF NOTCH AND MYC SIGNALING. AMPLIFICATION OF CHROMOSOME 15 CONTAINING THE MYC GENE WAS DETECTED IN ALL CLL MICE TESTED, AND INFILTRATION OF HIGH-MYC-EXPRESSING CLL CELLS IN THE SPLEEN WAS OBSERVED. NOTABLY, HYPERACTIVATION OF NOTCH AND MYC SIGNALING WAS EXCLUSIVELY OBSERVED IN THE DNMT3A CLL MICE, BUT NOT IN THREE OTHER CLL MOUSE MODELS TESTED (SF3B1-ATM, IKZF3, AND MDR), AND DNMT3A-DEPLETED CLL WERE SENSITIVE TO PHARMACOLOGIC INHIBITION OF NOTCH SIGNALING IN VITRO AND IN VIVO. CONSISTENT WITH THESE FINDINGS, HUMAN CLL SAMPLES WITH LOWER DNMT3A EXPRESSION WERE MORE SENSITIVE TO NOTCH INHIBITION THAN THOSE WITH HIGHER DNMT3A EXPRESSION. ALTOGETHER, THESE RESULTS SUGGEST THAT DNMT3A DEPLETION INDUCES CLL THAT IS HIGHLY DEPENDENT ON ACTIVATION OF NOTCH AND MYC SIGNALING. SIGNIFICANCE: LOSS OF DNMT3A EXPRESSION IS A DRIVING EVENT IN CLL AND IS ASSOCIATED WITH AGGRESSIVE DISEASE, ACTIVATION OF NOTCH AND MYC SIGNALING, AND ENHANCED SENSITIVITY TO NOTCH INHIBITION. 2021 19 6404 38 THE RS1001179 SNP AND CPG METHYLATION REGULATE CATALASE EXPRESSION IN CHRONIC LYMPHOCYTIC LEUKEMIA. CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) IS AN INCURABLE DISEASE CHARACTERIZED BY AN EXTREMELY VARIABLE CLINICAL COURSE. WE HAVE RECENTLY SHOWN THAT HIGH CATALASE (CAT) EXPRESSION IDENTIFIES PATIENTS WITH AN AGGRESSIVE CLINICAL COURSE. ELUCIDATING MECHANISMS REGULATING CAT EXPRESSION IN CLL IS PREEMINENT TO UNDERSTAND DISEASE MECHANISMS AND DEVELOP STRATEGIES FOR IMPROVING ITS CLINICAL MANAGEMENT. IN THIS STUDY, WE INVESTIGATED THE ROLE OF THE CAT PROMOTER RS1001179 SINGLE NUCLEOTIDE POLYMORPHISM (SNP) AND OF THE CPG ISLAND II METHYLATION ENCOMPASSING THIS SNP IN THE REGULATION OF CAT EXPRESSION IN CLL. LEUKEMIC CELLS HARBORING THE RS1001179 SNP T ALLELE EXHIBITED A SIGNIFICANTLY HIGHER CAT EXPRESSION COMPARED WITH CELLS BEARING THE CC GENOTYPE. CAT PROMOTER HARBORING THE T -BUT NOT C- ALLELE WAS ACCESSIBLE TO ETS-1 AND GR-BETA TRANSCRIPTION FACTORS. MOREOVER, CLL CELLS EXHIBITED LOWER METHYLATION LEVELS THAN NORMAL B CELLS, IN LINE WITH THE HIGHER CAT MRNA AND PROTEIN EXPRESSED BY CLL IN COMPARISON WITH NORMAL B CELLS. METHYLATION LEVELS AT SPECIFIC CPG SITES NEGATIVELY CORRELATED WITH CAT LEVELS IN CLL CELLS. INHIBITION OF METHYLTRANSFERASE ACTIVITY INDUCED A SIGNIFICANT INCREASE IN CAT LEVELS, THUS FUNCTIONALLY VALIDATING THE ROLE OF CPG METHYLATION IN REGULATING CAT EXPRESSION IN CLL. FINALLY, THE CT/TT GENOTYPES WERE ASSOCIATED WITH LOWER METHYLATION AND HIGHER CAT LEVELS, SUGGESTING THAT THE RS1001179 T ALLELE AND CPG METHYLATION MAY INTERACT IN REGULATING CAT EXPRESSION IN CLL. THIS STUDY IDENTIFIES GENETIC AND EPIGENETIC MECHANISMS UNDERLYING DIFFERENTIAL EXPRESSION OF CAT, WHICH COULD BE OF CRUCIAL RELEVANCE FOR THE DEVELOPMENT OF THERAPIES TARGETING REDOX REGULATORY PATHWAYS IN CLL. 2022 20 3658 38 INDUCTION OF ABERRANT TRIMETHYLATION OF HISTONE H3 LYSINE 27 BY INFLAMMATION IN MOUSE COLONIC EPITHELIAL CELLS. A FIELD FOR CANCERIZATION (FIELD DEFECT), WHERE GENETIC AND EPIGENETIC ALTERATIONS ARE ACCUMULATED IN NORMAL-APPEARING TISSUES, IS INVOLVED IN HUMAN CARCINOGENESIS, ESPECIALLY CANCERS ASSOCIATED WITH CHRONIC INFLAMMATION. ALTHOUGH ABERRANT DNA METHYLATION IS INVOLVED IN THE FIELD DEFECT AND INDUCED BY CHRONIC INFLAMMATION, IT IS STILL UNCLEAR FOR TRIMETHYLATION OF HISTONE H3 LYSINE 27 (H3K27ME3), WHICH IS INVOLVED IN GENE REPRESSION INDEPENDENT OF DNA METHYLATION AND FUNCTIONS AS A PRE-MARK FOR ABERRANT DNA METHYLATION. IN THIS STUDY, USING A MOUSE COLITIS MODEL INDUCED BY DEXTRAN SULFATE SODIUM (DSS), WE AIMED TO CLARIFY WHETHER ABERRANT H3K27ME3 IS INDUCED BY INFLAMMATION AND INVOLVED IN A FIELD DEFECT. CHIP-ON-CHIP ANALYSIS OF COLONIC EPITHELIAL CELLS REVEALED THAT H3K27ME3 LEVELS WERE INCREASED OR DECREASED FOR 266 GENOMIC REGIONS BY AGING, AND MORE EXTENSIVELY (23 INCREASED AND 3574 DECREASED REGIONS) BY COLITIS. SUCH INCREASE OR DECREASE OF H3K27ME3 WAS INDUCED AS EARLY AS 2 WEEKS AFTER THE INITIATION OF DSS TREATMENT, AND PERSISTED AT LEAST FOR 16 WEEKS EVEN AFTER THE INFLAMMATION DISAPPEARED. SOME OF THE ABERRANT H3K27ME3 IN COLONIC EPITHELIAL CELLS WAS CARRIED OVER INTO COLON TUMORS. FURTHERMORE, H3K27ME3 ACQUIRED AT DAPK1 BY COLITIS WAS FOLLOWED BY INCREASED DNA METHYLATION, SUPPORTING ITS FUNCTION AS A PRE-MARK FOR ABERRANT DNA METHYLATION. THESE RESULTS DEMONSTRATED THAT ABERRANT H3K27ME3 CAN BE INDUCED BY EXPOSURE TO A SPECIFIC ENVIRONMENT, SUCH AS COLITIS, AND SUGGESTED THAT ABERRANT HISTONE MODIFICATION, IN ADDITION TO ABERRANT DNA METHYLATION, IS INVOLVED IN THE FORMATION OF A FIELD DEFECT. 2012