1 3877 151 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 2 4708 22 NO ASSOCIATION OF POLYMORPHISMS IN NAV1.7 OR NERVE GROWTH FACTOR RECEPTOR GENES WITH TRIGEMINAL NEURALGIA. OBJECTIVE: TRIGEMINAL NEURALGIA IS DEFINED AS A SUDDEN SEVERE SHOCK-LIKE PAIN WITHIN THE DISTRIBUTION OF THE TRIGEMINAL NERVE. PAIN IS A SUBJECTIVE EXPERIENCE THAT IS INFLUENCED BY GENDER, CULTURE, ENVIRONMENT, PSYCHOLOGICAL TRAITS, AND GENES. SODIUM CHANNELS AND NERVE GROWTH FACTOR PLAY IMPORTANT ROLES IN THE TRANSMISSION OF NOCICEPTIVE SIGNALS AND PAIN. THE AIM OF THIS STUDY WAS TO INVESTIGATE THE OCCURRENCE OF NAV1.7 SODIUM CHANNEL AND NERVE GROWTH FACTOR RECEPTOR TRKA GENE POLYMORPHISMS (SCN9A/RS6746030 AND NTRK1/RS633, RESPECTIVELY) IN TRIGEMINAL NEURALGIA PATIENTS. METHODS: NINETY-SIX SUBJECTS FROM PAIN SPECIALTY CENTERS IN THE SOUTHEASTERN REGION OF BRAZIL WERE DIVIDED INTO 2 GROUPS: 48 WITH CLASSICAL TRIGEMINAL NEURALGIA DIAGNOSIS AND 48 CONTROLS. PAIN WAS EVALUATED USING THE VISUAL ANALOG SCALE AND MULTIDIMENSIONAL MCGILL PAIN QUESTIONNAIRE. GENOMIC DNA WAS OBTAINED FROM ORAL SWABS IN ALL INDIVIDUALS AND WAS ANALYZED BY REAL-TIME POLYMERASE CHAIN REACTION. RESULTS: NO ASSOCIATION WAS OBSERVED BETWEEN EVALUATED POLYMORPHISMS AND TRIGEMINAL NEURALGIA. FOR ALLELE ANALYSES, PATIENTS AND CONTROLS HAD SIMILAR FREQUENCIES FOR BOTH GENES. GENOTYPE DISTRIBUTION OR ALLELE FREQUENCIES OF POLYMORPHISMS ANALYZED HERE DID NOT CORRELATE TO PAIN SCORES. CONCLUSIONS: ALTHOUGH NO ASSOCIATION OF EVALUATED POLYMORPHISMS AND TRIGEMINAL NEURALGIA DIAGNOSIS OR PAIN SEVERITY WAS OBSERVED, OUR DATA DO NOT EXCLUDE THE POSSIBILITY THAT OTHER GENOTYPES AFFECTING THE EXPRESSION OF NAV1.7 OR TRKA ARE ASSOCIATED WITH THE DISEASE. FURTHER STUDIES SHOULD INVESTIGATE DISTINCT GENETIC POLYMORPHISMS AND EPIGENETIC FACTORS THAT MAY BE IMPORTANT IN EXPRESSION OF THESE MOLECULES. 2019 3 2326 43 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 4 5402 39 REDUCTION OF SIRT1-MEDIATED EPIGENETIC UPREGULATION OF NAV1.7 CONTRIBUTES TO OXALIPLATIN-INDUCED NEUROPATHIC PAIN. BACKGROUND: CLINICALLY, NEUROPATHIC PAIN IS A SEVERE SIDE EFFECT OF OXALIPLATIN CHEMOTHERAPY, WHICH USUALLY LEADS TO DOSE REDUCTION OR CESSATION OF TREATMENT. DUE TO THE UNAWARENESS OF DETAILED MECHANISMS OF OXALIPLATIN-INDUCED NEUROPATHIC PAIN, IT IS DIFFICULT TO DEVELOP AN EFFECTIVE THERAPY AND LIMITS ITS CLINICAL USE. OBJECTIVES: THE AIM OF THE PRESENT STUDY WAS TO IDENTIFY THE ROLE OF SIRTUIN 1 (SIRT1) REDUCTION IN EPIGENETIC REGULATION OF THE EXPRESSION OF VOLTAGE-GATED SODIUM CHANNELS 1.7 (NAV1.7) IN THE DORSAL ROOT GANGLION (DRG) DURING OXALIPLATIN-INDUCED NEUROPATHIC PAIN. STUDY DESIGN: CONTROLLED ANIMAL STUDY. SETTING: UNIVERSITY LABORATORY. METHODS: THE VON FREY TEST WAS PERFORMED TO EVALUATE PAIN BEHAVIOR IN RATS. REAL-TIME QUANTITATIVE POLYMERASE CHAIN REACTION, WESTERN BLOTTING, ELECTROPHYSIOLOGICAL RECORDING, CHROMATIN IMMUNOPRECIPITATION, AND SMALL INTERFERING RNA (SIRNA) WERE USED TO ILLUSTRATE THE MECHANISMS. RESULTS: IN THE PRESENT STUDY, WE FOUND THAT BOTH THE ACTIVITY AND EXPRESSION OF SIRT1 WERE SIGNIFICANTLY DECREASED IN RAT DRG FOLLOWING OXALIPLATIN TREATMENT. THE ACTIVATOR OF SIRT1, RESVERATROL, NOT ONLY INCREASED THE ACTIVITY AND EXPRESSION OF SIRT1, BUT ALSO ATTENUATED THE MECHANICAL ALLODYNIA FOLLOWING OXALIPLATIN TREATMENT. IN ADDITION, LOCAL KNOCKDOWN OF SIRT1 BY INTRATHECAL INJECTION OF SIRT1 SIRNA CAUSED MECHANICAL ALLODYNIA IN NAIVE RATS. BESIDES, OXALIPLATIN TREATMENT ENHANCED THE ACTION POTENTIAL FIRING FREQUENCY OF DRG NEURONS AND THE EXPRESSION OF NAV1.7 IN DRG AND ACTIVATION OF SIRT1 BY RESVERATROL REVERSED THIS EFFECT. FURTHERMORE, BLOCKING NAV1.7 BY PROTX II (A SELECTIVE NAV1.7 CHANNEL BLOCKER) REVERSED OXALIPLATIN-INDUCED MECHANICAL ALLODYNIA. IN ADDITION, HISTONE H3 HYPERACETYLATION AT THE NAV1.7 PROMOTER IN DRG OF RATS FOLLOWING OXALIPLATIN TREATMENT WAS SIGNIFICANTLY SUPPRESSED BY ACTIVATION OF SIRT1 WITH RESVERATROL. MOREOVER, BOTH THE EXPRESSION OF NAV1.7 AND HISTONE H3 ACETYLATION AT THE NAV1.7 PROMOTER WERE UPREGULATED IN THE DRG BY LOCAL KNOCKDOWN OF SIRT1 WITH SIRT1 SIRNA IN NAIVE RATS. LIMITATIONS: MORE UNDERLYING MECHANISM(S) OF SIRT1 REDUCTION AFTER OXALIPLATIN TREATMENT NEEDS TO BE EXPLORED IN FUTURE RESEARCH. CONCLUSIONS: THESE FINDINGS SUGGEST THAT REDUCTION OF SIRT1-MEDIATED EPIGENETIC UPREGULATION OF NAV1.7 IN THE DRG CONTRIBUTES TO THE DEVELOPMENT OF OXALIPLATIN-INDUCED NEUROPATHIC PAIN IN RATS. THE INTRATHECAL DRUG DELIVERY TREATMENT OF ACTIVATING SIRT1 MIGHT BE A NOVEL THERAPEUTIC OPTION FOR OXALIPLATIN-INDUCED NEUROPATHIC PAIN. 2023 5 5459 35 RESEARCH ON THE EPIGENETIC REGULATION MECHANISM OF THE PTPN6 GENE IN ADVANCED CHRONIC MYELOID LEUKAEMIA. PTPN6, A TYROSINE PHOSPHATASE PROTEIN, PLAYS A NEGATIVE ROLE IN CELL SIGNAL TRANSDUCTION AND IS NEGATIVELY CORRELATED WITH TUMOUR FORMATION AND GROWTH. HOWEVER, EPIGENETIC REGULATION MECHANISM OF THE PTPN6 GENE IN ADVANCED CHRONIC MYELOID LEUKAEMIA (CML) REMAINS UNCLEAR. THIS STUDY INVESTIGATED BONE MARROW OR BLOOD SAMPLES FROM 44 CML PATIENTS AND 10 HEALTHY VOLUNTEERS. KCL22 AND K562 CELLS WERE CULTURED AND TREATED WITH DEMETHYLATION DRUGS AND HISTONE DEACETYLASE INHIBITORS. REAL TIME QUANTITATIVE POLYMERASE CHAIN REACTION (QPCR), METHYLATION-SPECIFIC PCR, BISULFITE SEQUENCING PCR, WESTERN BLOTTING, CO-IMMUNOPRECIPITATION AND CHROMATIN IMMUNOPRECIPITATION (CHIP) WAS PERFORMED. PTPN6 WAS DOWN-REGULATED IN CELL LINES AND PATIENTS WITH ADVANCED PHASE CML, WHEREAS DNMT1, DNMT3A, MECP2, MBD2 AND HDAC1 WERE UP-REGULATED. TREATMENT WITH 5-AZACYTIDINE, DECITABINE, SODIUM VALPROATE AND LBH589 INCREASED PTPN6 EXPRESSION, BUT DECREASED THAT OF DNMT1, DNMT3A, MECP2, MBD2 AND HDAC1. IMMUNOPRECIPITATION AND MASS SPECTROMETRY SHOWED THAT HDAC1 COMBINED DIRECTLY WITH PTPN6. CHIP-SEQ SHOWED THAT HDAC1 DID NOT COMBINE WITH THE PROMOTER REGION OF PTPN6, WHILE MAPK, AKT, STAT5, JAK2 AND MYC PROMOTER REGIONS ALL COMBINED WITH HDAC1. PTPN6 IS ASSOCIATED WITH PROGRESSION OF CML. LOW EXPRESSION LEVEL OF PTPN6 WAS ASSOCIATED WITH DNA METHYLATION AND REGULATED BY HISTONE ACETYLATION. HDAC1 PARTICIPATES IN THE REGULATION OF PTPN6. 2017 6 5458 39 RESEARCH ON EPIGENETIC MECHANISM OF SFRP2 IN ADVANCED CHRONIC MYELOID LEUKEMIA. SECRETED FRIZZLED-RELATED PROTEIN 2 (SFRP2) HAS BEEN REPORTED TO ACT AS A TUMOR SUPPRESSORS. THIS STUDY AIMS TO DETECT THE BIOLOGICAL ROLE OF SFRP2 IN ADVANCED CHRONIC MYELOID LEUKEMIA (CML). IN THIS STUDY WE EXAMINED BONE MARROW SAMPLES FROM 45 CML PATIENTS AND 10 HEALTHY DONORS. K562 AND KCL22 CELLS WERE CULTURED AND TREATED WITH DEMETHYLATION DRUG AND HISTONE DEACETYLASE INHIBITOR (HDACI). KCL22 AND K562 CELLS WERE TRANSFECTED WITH LENTIVIRAL VECTOR (LV)-SFRP2, LV-CONTROL. THE CELLS WERE THEN SUBJECTED TO PROLIFERATION AND APOPTOSIS ASSAYS, REAL TIME POLYMERASE CHAIN REACTION (PCR), METHYLATION-SPECIFIC PCR (MSP), WESTERN BLOTTING, CO-IMMUNOPRECIPITATION (COIP) AND CHROMATIN IMMUNOPRECIPITATION (CHIP), WE FOUND THAT SFRP2 WAS DOWN-REGULATED IN THE ACCELERATED AND BLAST PHASE OF CML, WHEREAS, THE LEVELS OF WNT1, WNT3 AND WNT5A WERE UP-REGULATED IN THE ACCELERATED AND BLAST PHASE OF CML. OVEREXPRESSION SFRP2 INHIBITED PROLIFERATION, PROMOTED APOPTOSIS AND ACTIVATED THE WNT PATHWAY. COIP-MS RESULTS SHOWED THAT SFRP2 INTERACTED WITH WNT1 AND WNT5A. CHIP-SEQ RESULT INDICATED THAT THE PROMOTER OF H3K4ME3 AND H3K27ME3 WERE ABLE TO INTERACT WITH SFRP2. IN CONCLUSION, OUR FINDINGS DEMONSTRATED THE SFRP2 ACT AS A POTENTIAL THERAPEUTIC TARGET FOR ADVANCED CML. FURTHERMORE, OUR RESULTS SUPPORT THE USE OF DEMETHYLATION DRUGS AND HDACI AS A POTENTIAL CML TREATMENT STRATEGY. 2018 7 4601 46 NDRG2 MRNA LEVELS AND MIR-28-5P AND MIR-650 ACTIVITY IN CHRONIC LYMPHOCYTIC LEUKEMIA. BACKGROUND: NDRG2 IS IDENTIFIED AS A TUMOR SUPPRESSOR GENE IN MANY TUMORS, AND FUNCTIONS IN CELL PROLIFERATION, DIFFERENTIATION AND APOPTOSIS. RECENT DATA INDICATE THAT NDRG2 EXPRESSION IS UP-REGULATED BY TP53. MOREOVER, PROPOSED MECHANISMS OF NDRG2 INACTIVATION INCLUDE EPIGENETIC SILENCING OF THE NDRG2 PROMOTER AND DOWN-REGULATION BY MICRORNAS (MIRNAS). HOWEVER, FEW STUDIES HAVE EVER BEEN DONE ON THE ROLE OF NDRG2 AND THE NDRG2-REGULATING MIRNAS INTERFERENCE IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). METHODS: NDRG2 AND MICRORNAS MRNA LEVELS IN CLL SUBJECTS WERE ASSESSED BY QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION (QRT-PCR). THE DUAL-LUCIFERASE REPORTER ASSAY WAS PERFORMED TO DETERMINE NDRG2-RELATED MIRNAS. LOW EXPRESSION OF MATURE EXOGENOUS MIRNAS IN CLL CELLS WAS ESTABLISHED BY TRANSIENT TRANSFECTION. NDRG2 PROTEIN LEVELS IN CLL CELLS WERE DETECTED BY WESTERN BLOT. IN ADDITION, FLOW CYTOMETRY WAS CONDUCTED TO EXAMINE THE APOPTOSIS OF CLL CELLS. RESULTS: LOWER EXPRESSION OF NDRG2 WAS FOUND IN THE B-CELLS FROM 102 CLL PATIENTS COMPARED THE 40 NORMAL SUBJECTS (P < 0.001). PATIENTS WITH ADVANCED BINET STAGE (P = 0.001), HIGH LACTATE DEHYDROGENASE (LDH) LEVEL (P = 0.036), UN-MUTATED IMMUNOGLOBULIN HEAVY CHAIN VARIABLE REGION GENE (IGHV) (P = 0.004) AND THOSE WITH P53 ABERRATIONS (P < 0.001) HAD A MARKEDLY LOWER LEVELS OF NDRG2 MRNA. THIS DECREASE WAS ASSOCIATED WITH BRIEFER TIME-TO-TREATMENT (P = 0.001) AND POORER SURVIVAL (P < 0.001). HIGH EXPRESSION OF MIR-28-5P AND MIR-650 WAS ASSOCIATED WITH BINET B/C STAGE (P = 0.044) AND IGHV UN-MUTATED (P = 0.011), AS WELL AS BINET B/C STAGE (P = 0.013) AND P53 ABERRATIONS (P = 0.037), RESPECTIVELY. INHIBITION OF MIR-28-5P OR MIR-650 COULD INDUCE MORE APOPTOSIS IN CLL CELLS WITH GERMLINE TP53. CONCLUSIONS: NDRG2 MRNA LEVELS MIGHT BE A USEFUL PROGNOSTIC VARIABLE FOR PATIENTS OF CLL AND UP-REGULATING NDRG2 TRANSCRIPTION MAY BE A THERAPY APPROACH IN CLL WITHOUT P53 ABERRATIONS. 2018 8 6793 36 [DOWN-REGULATION OF TRANSCRIPTION FACTOR PU.1 VIA ABNORMAL EPIGENETIC MODIFICATION IN CHRONIC MYELOID LEUKEMIA]. OBJECTIVE: TO INVESTIGATE THE UNDERLYING MECHANISM AND CLINICAL SIGNIFICANCE OF PU.1 DOWN-EXPRESSION IN CHRONIC MYELOID LEUKEMIA (CML) PATIENTS. METHODS: DIFFERENT METHYLATION STATUS OF PU.1 PROMOTER REGION CONTAINING 20 CPG ISLANDS IN NORMAL INDIVIDUALS, CML CHRONIC PHASE AND BLAST CRISIS PATIENTS, COMPLETE CYTOGENETIC REMISSION PATIENTS AFTER IMATINIB TREATMENT, AND BLAST CRISIS BONE MARROW K562 CML CELLS WAS DETECTED BY BISULFITE SEQUENCING. SEMI-QUANTITATIVE PCR WAS USED TO DETECT THE PU.1 MRNA EXPRESSION IN NORMAL CONTROLS, CML CHRONIC PHASE AND BLAST CRISIS PATIENTS, AND BLAST CRISIS BONE MARROW K562 CML CELLS. INDIRECT IMMUNE FLUORESCENCE AND WESTERN BLOT WERE USED TO ANALYZE THE EXPRTESSION OF PU.1 PROTEIN IN NORMAL INDIVIDUALS, CML CHRONIC PHASE AND BLAST CRISIS PATIENTS, AND BLAST CRISIS BONE MARROW K562 CML CELLS. RESULTS: ABERRANT METHYLATION IN THE PROMOTER REGION OF TRANSCRIPTION FACTOR PU.1 WAS FOUND IN BOTH CML CHRONIC PHASE AND BLAST CRISIS PHASE BONE MARROW CELLS, AS WELL AS IN CML BLAST K562 CELLS. DOWN-EXPRESSION OF PU.1 MRNA AND PROTEIN LEVELS WAS FOUND IN ABOVE CELLS. NO METHYLATION IN THE PROMOTER REGION OF PU.1 WAS OBSERVED IN NORMAL INDIVIDUALS, AND THE PU.1 MRNA AND PROTEIN EXPRESSIONS WERE NOT REDUCED AT ALL. FURTHERMORE, HIGH METHYLATION STATUS OF BONE MARROW CELLS WAS EVEN OBSERVED IN THE CML PATIENTS WHO ACQUIRED COMPLETE CYTOGENETIC REMISSION. CONCLUSIONS: THE RESULTS OF OUR STUDY INDICATE THAT THE EPIGENETIC MODIFICATION OF PU.1 IN CML PATIENTS AND K562 CELL LINE MIGHT BE RESPONSIBLE FOR THE DOWN-EXPRESSION OF PU.1. THE DATA SUGGEST THAT ABERRANT METHYLATION OF PU.1 PLAYS A ROLE IN CML PATHOGENESIS, THEREFORE, IT MIGHT SERVE AS A USEFUL BIOMARKER AND POTENTIAL TARGET IN THERAPY FOR CHRONIC MYELOID LEUKEMIA. 2012 9 3409 33 HOXA4 GENE PROMOTER HYPERMETHYLATION AS AN EPIGENETIC MECHANISM MEDIATING RESISTANCE TO IMATINIB MESYLATE IN CHRONIC MYELOID LEUKEMIA PATIENTS. DEVELOPMENT OF RESISTANCE TO IMATINIB MESYLATE (IM) IN CHRONIC MYELOID LEUKEMIA (CML) PATIENTS HAS EMERGED AS A SIGNIFICANT CLINICAL PROBLEM. THE OBSERVATION THAT INCREASED EPIGENETIC SILENCING OF POTENTIAL TUMOR SUPPRESSOR GENES CORRELATES WITH DISEASE PROGRESSION IN SOME CML PATIENTS TREATED WITH IM SUGGESTS A RELATIONSHIP BETWEEN EPIGENETIC SILENCING AND RESISTANCE DEVELOPMENT. WE HYPOTHESIZE THAT PROMOTER HYPERMETHYLATION OF HOXA4 COULD BE AN EPIGENETIC MECHANISM MEDIATING IM RESISTANCE IN CML PATIENTS. THUS A STUDY WAS UNDERTAKEN TO INVESTIGATE THE PROMOTER HYPERMETHYLATION STATUS OF HOXA4 IN CML PATIENTS ON IM TREATMENT AND TO DETERMINE ITS ROLE IN MEDIATING RESISTANCE TO IM. GENOMIC DNA WAS EXTRACTED FROM PERIPHERAL BLOOD SAMPLES OF 95 CML PATIENTS (38 GOOD RESPONDERS AND 57 RESISTANT) AND 12 NORMAL CONTROLS. ALL SAMPLES WERE BISULFITE TREATED AND ANALYSED BY METHYLATION-SPECIFIC HIGH-RESOLUTION MELT ANALYSIS. COMPARED TO THE GOOD RESPONDERS, THE HOXA4 HYPERMETHYLATION LEVEL WAS SIGNIFICANTLY HIGHER (P = 0.002) IN IM-RESISTANT CML PATIENTS. ON COMPARING THE RISK, HOXA4 HYPERMETHYLATION WAS ASSOCIATED WITH A HIGHER RISK FOR IM RESISTANCE (OR 4.658; 95% CI, 1.673-12.971; P = 0.003). THUS, IT IS REASONABLE TO SUGGEST THAT PROMOTER HYPERMETHYLATION OF HOXA4 GENE COULD BE AN EPIGENETIC MECHANISM MEDIATING IM RESISTANCE IN CML PATIENTS. 2013 10 6238 32 THE MALIGNANCY SUPPRESSION ROLE OF MIR-23A BY TARGETING THE BCR/ABL ONCOGENE IN CHROMIC MYELOID LEUKEMIA. THE AIM OF THIS STUDY WAS TO INVESTIGATE THE ROLE AND MECHANISM OF MIR-23A IN THE REGULATION OF BCR/ABL AND TO PROVIDE A NEW PROGNOSTIC BIOMARKER FOR CHRONIC MYELOID LEUKEMIA (CML). THE EXPRESSION LEVELS OF MIR-23A AND BCR/ABL WERE ASSESSED IN 42 NEWLY DIAGNOSED CML PATIENTS, 37 CML PATIENTS IN FIRST COMPLETE REMISSION AND 25 HEALTHY CONTROLS. QUANTITATIVE REAL-TIME PCR, WESTERN BLOT ANALYSIS AND COLONY FORMATION ASSAY WERE USED TO EVALUATE CHANGES INDUCED BY OVEREXPRESSION OR INHIBITION OF MIR-23A OR BCR/ABL. MIR-23A MIMIC OR NEGATIVE CONTROL MIMIC WAS TRANSFECTED INTO A CML CELL LINE (K562) AND TWO LUNG CANCER CELL LINES (H157 AND SKMES1) USING LIPOFECTAMINE 2000, AND THE CELLS WERE USED FOR REAL-TIME REVERSE TRANSCRIPTION-PCR (RT-PCR) AND WESTERN BLOT ANALYSIS. WE FOUND THAT THE DOWNREGULATION OF MIR-23A EXPRESSION WAS A FREQUENT EVENT IN BOTH LEUKEMIA CELL LINES AND PRIMARY LEUKEMIC CELLS FROM PATIENTS WITH DE NOVO CML. THE MICROARRAY RESULTS SHOWED THAT MOST OF THE CML PATIENTS EXPRESSED HIGH LEVELS OF BCR/ABL AND LOW LEVELS OF MIR-23A. REAL-TIME RT-PCR AND WESTERN BLOT ANALYSIS SHOWED THAT THE BCR/ABL LEVELS IN MIR-23A-TRANSFECTED CELLS WERE LOWER THAN THOSE IN THE CONTROL GROUPS. ECTOPIC EXPRESSION OF MIR-23A IN K562 CELLS LED TO CELLULAR SENESCENCE. MOREOVER, WHEN K562 CELLS WERE TREATED WITH 5-AZA-2'-DEOXYCYTIDINE, A DNA METHYLATION INHIBITOR, BCR/ABL EXPRESSION WAS UPREGULATED, WHICH INDICATES EPIGENETIC SILENCING OF MIR-23A IN LEUKEMIC CELLS. BCR/ABL AND MIR-23A EXPRESSIONS WERE INVERSELY RELATED TO CML, AND BCR/ABL EXPRESSION WAS REGULATED BY MIR-23A IN LEUKEMIC CELLS. THE EPIGENETIC SILENCING OF MIR-23A LED TO DEREPRESSION OF BCR/ABL EXPRESSION, AND CONSEQUENTLY CONTRIBUTES TO CML DEVELOPMENT AND PROGRESSION. 2014 11 2081 41 EPIGENETIC DOWN-REGULATION OF BIM EXPRESSION IS ASSOCIATED WITH REDUCED OPTIMAL RESPONSES TO IMATINIB TREATMENT IN CHRONIC MYELOID LEUKAEMIA. BACKGROUND: EXPRESSION OF THE PRO-APOPTOTIC BCL-2-INTERACTING MEDIATOR (BIM) HAS RECENTLY BEEN IMPLICATED IN IMATINIB-INDUCED APOPTOSIS OF BCR-ABL1(+) CELLS. HOWEVER, THE MECHANISMS INVOLVED IN THE REGULATION OF BIM IN CML AND ITS ROLE IN THE CLINICAL SETTING HAVE NOT BEEN ESTABLISHED. DESIGN AND METHODS: WE ANALYSED THE MRNA EXPRESSION OF BIM IN 100 NEWLY DIAGNOSED PATIENTS WITH CML IN CHRONIC PHASE BY Q-RT-PCR AND THE PROTEIN LEVELS BY WESTERN BLOT ANALYSIS. METHYLATION STATUS WAS ANALYSED BY BISULPHITE GENOMIC SEQUENCING AND MSP. CML CELL LINES WERE TREATED WITH IMATINIB AND 5-AZA-2'-DEOXYCYTIDINE, AND WERE TRANSFECTED WITH TWO DIFFERENT SIRNAS AGAINST BIM AND CELL PROLIFERATION AND APOPTOSIS WERE ANALYSED. RESULTS: WE DEMONSTRATED THAT DOWN-REGULATION OF BIM EXPRESSION WAS PRESENT IN 36% OF THE PATIENTS AND WAS SIGNIFICANTLY ASSOCIATED WITH A LACK OF OPTIMAL RESPONSE TO IMATINIB AS INDICATED BY THE DECREASE IN CYTOGENETIC AND MOLECULAR RESPONSES AT 6, 12 AND 18 MONTHS IN COMPARISON WITH PATIENTS WITH NORMAL BIM EXPRESSION (P<0.05). EXPRESSION OF BIM WAS MEDIATED BY PROMOTER HYPERMETHYLATION AS DEMONSTRATED BY RESTORATION OF BIM EXPRESSION AFTER TREATMENT OF CML CELLS WITH 5-AZA-2'-DEOXYCYTIDINE. USING CML CELL LINES WITH LOW AND NORMAL EXPRESSION OF BIM WE FURTHER DEMONSTRATED THAT THE EXPRESSION OF BIM IS REQUIRED FOR IMATINIB-INDUCED CML APOPTOSIS. CONCLUSION: OUR DATA INDICATE THAT DOWN-REGULATION OF BIM IS EPIGENETICALLY CONTROLLED BY METHYLATION IN A PERCENTAGE OF CML PATIENTS AND HAS AN UNFAVOURABLE PROGNOSTIC IMPACT, AND THAT THE COMBINATION OF IMATINIB WITH A DE-METHYLATING AGENT MAY RESULT IN IMPROVED RESPONSES IN PATIENTS WITH DECREASED EXPRESSION OF BIM. 2009 12 138 36 ABERRANT DNA METHYLATION AT HOXA4 AND HOXA5 GENES ARE ASSOCIATED WITH RESISTANCE TO IMATINIB MESYLATE AMONG CHRONIC MYELOID LEUKEMIA PATIENTS. BACKGROUND: IMATINIB MESYLATE IS A MOLECULARLY TARGETED TYROSINE KINASE INHIBITOR DRUG. IT IS EFFECTIVELY USED IN THE TREATMENT OF CHRONIC MYELOID LEUKEMIA (CML) PATIENTS. HOWEVER, DEVELOPMENT OF RESISTANCE TO IMATINIB MESYLATE AS A RESULT OF BCR-ABL DEPENDENT AND BCR-ABL INDEPENDENT MECHANISMS HAS EMERGED AS A DAUNTING PROBLEM IN THE MANAGEMENT OF CML PATIENTS. BETWEEN THESE MECHANISMS, BCR-ABL INDEPENDENT MECHANISMS ARE STILL NOT ROBUSTLY UNDERSTOOD. AIM: TO INVESTIGATE THE CORRELATION OF HOXA4 AND HOXA5 PROMOTER DNA HYPERMETHYLATION WITH IMATINIB RESISTANCE AMONG CML PATIENTS. METHODS AND RESULTS: SAMPLES FROM 175 PHILADELPHIA POSITIVE CML PATIENTS (83 GOOD RESPONSE AND 92 BCR-ABL NON-MUTATED IMATINIB RESISTANT PATIENTS) WERE SUBJECTED TO METHYLATION SPECIFIC HIGH RESOLUTION MELT ANALYSIS FOR METHYLATION LEVELS QUANTIFICATION OF THE HOXA4 AND HOXA5 PROMOTER REGIONS. RECEIVER OPERATING CHARACTERISTIC CURVE ANALYSIS WAS DONE TO ELUCIDATE THE OPTIMAL METHYLATION CUT-OFF POINT FOLLOWED BY MULTIPLE LOGISTIC REGRESSION ANALYSIS. LOG-RANK ANALYSIS WAS DONE TO MEASURE THE OVERALL SURVIVAL DIFFERENCE BETWEEN CML GROUPS. THE OPTIMAL METHYLATION CUT-OFF POINT WAS FOUND TO BE AT 62.5% FOR BOTH HOXA4 AND HOXA5. CHRONIC MYELOID LEUKEMIA PATIENTS WITH >/=63% HOXA4 AND HOXA5 METHYLATION LEVEL WERE SHOWN TO HAVE 3.78 AND 3.95 TIMES THE ODDS, RESPECTIVELY, TO ACQUIRE RESISTANCE TO IMATINIB. HOWEVER, OVERALL SURVIVAL OF CML PATIENTS THAT HAVE /= 63% METHYLATION LEVELS OF HOXA4 AND HOXA5 GENES WERE FOUND TO BE NOT SIGNIFICANT (P-VALUE = 0.126 FOR HOXA4; P-VALUE = 0.217 FOR HOXA5). CONCLUSION: HYPERMETHYLATION OF THE HOXA4 AND HOXA5 PROMOTER IS CORRELATED WITH IMATINIB RESISTANCE AND WITH FURTHER INVESTIGATION, IT COULD BE A POTENTIAL EPIGENETIC BIOMARKER IN SUPPLEMENT TO THE BCR-ABL GENE MUTATION IN PREDICTING IMATINIB TREATMENT RESPONSE AMONG CML PATIENTS BUT COULD NOT BE CONSIDERED AS A PROGNOSTIC MARKER. 2018 13 4245 35 METHYLATION STATUS OF DDIT3 GENE IN CHRONIC MYELOID LEUKEMIA. BACKGROUND: DNA-DAMAGE-INDUCIBLE TRANSCRIPT 3 (DDIT3), A CANDIDATE TUMOR SUPPRESSOR GENE (TSG), HAS BEEN FOUND INVOLVED IN THE REGULATION OF CELLULAR GROWTH AND DIFFERENTIATION. THE EPIGENETIC CHANGES OF TSGS ARE RECENTLY RECOGNIZED AS AN ABNORMAL MECHANISM CONTRIBUTING TO THE DEVELOPMENT OF CHRONIC MYELOID LEUKEMIA (CML). THE AIM OF THIS STUDY WAS TO INVESTIGATE THE METHYLATION STATUS OF DDIT3 GENE IN CML PATIENTS. METHODS: THE METHYLATION STATUS OF DDIT3 PROMOTER WAS DETECTED IN THE BONE MARROW MONONUCLEAR CELLS FROM 53 PATIENTS WITH CML USING METHYLATION-SPECIFIC PCR (MSP). THE EXPRESSION LEVELS OF DDIT3 AND BCR/ABL TRANSCRIPT WERE DETERMINED BY REAL-TIME QUANTITATIVE PCR (RQ-PCR). CLINICAL DATA OF THESE PATIENTS WERE COLLECTED AND ANALYZED. RESULTS: THE ABERRANT METHYLATION OF DDIT3 GENE PROMOTER WAS FOUND IN 35 OF 53 (66%) CML CASES. CORRELATION WAS NOT FOUND BETWEEN DDIT3 PROMOTER HYPERMETHYLATION AND THE AGE, SEX, HEMOGLOBIN CONCENTRATION, PLATELET COUNTS, CHROMOSOMAL ABNORMALITIES, BCR/ABL TRANSCRIPT, AND STAGING OF CML PATIENTS (P > 0.05), BUT FOUND BETWEEN DDIT3 PROMOTER HYPERMETHYLATION AND WBC COUNTS OF CML CASES (R = 0.781, P < 0.001). THE LEVEL OF DDIT3 TRANSCRIPT IN CML PATIENTS WAS SIGNIFICANTLY LOWER THAN THAT IN CONTROLS (MEDIAN 3.28 VS 19.69, P < 0.001), HOWEVER, THERE WAS NO DIFFERENCE IN THE LEVEL OF DDIT3 TRANSCRIPT BETWEEN METHYLATION-POSITIVE CML CASES (0.05-65.32, MEDIAN 2.13) AND METHYLATION- NEGATIVE CML CASES (0.12-126.04, MEDIAN 3.92) (P > 0.05). CONCLUSION: OUR RESULTS DEMONSTRATE THAT ABERRANT METHYLATION OF DDIT3 OCCURS IN CML FREQUENTLY. 2010 14 5319 40 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 15 1194 28 CORRELATION OF EPIGENETIC CHANGE AND IDENTIFICATION OF RISK FACTORS FOR ORAL SUBMUCOUS FIBROSIS. BACKGROUND: DNA METHYLATION OF CERTAIN GENES IS AN EPIGENETIC CHANGE THAT IS ESSENTIAL FOR TUMORIGENESIS. ORAL SUBMUCOUS FIBROSIS (OSF) IS A PRECANCEROUS CONDITION OF ORAL MUCOSA WITH INFLAMMATION AND PROGRESSIVE FIBROSIS OF THE LAMINA PROPRIA AND DEEPER CONNECTIVE TISSUE. THE HYPERMETHYLATION OF E-CADHERIN AND CYCLOOXYGENASE 2 (COX-2) IN CHRONIC INFLAMMATION MAY DEMONSTRATE A MILD LESION/MUTATION AT EPIGENETIC LEVELS. THIS STUDY COMPARES THE HYPERMETHYLATION STATUS OF E-CADHERIN AND COX-2 GENES IN PATIENTS WITH ORAL CANCER AND PATIENTS WITH OSF AND ALSO AIMS TO IDENTIFY RISK FACTORS FOR THE DEVELOPMENT OF OSF. METHODS: DNA WAS EXTRACTED FROM BLOOD SAMPLES OF 50 HEALTHY SUBJECTS, 50 PATIENTS WITH OSF AND 60 PATIENTS WITH ORAL CANCER. METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION FOR E-CADHERIN AND COX-2 WAS PERFORMED ON THESE SAMPLES AND THE PRODUCTS WERE ANALYZED ON 2% AGAROSE GEL. SURVEYS ABOUT ORAL HEALTH HABITS AND CLINICAL PERIODONTAL EXAMINATIONS IN PATIENTS WITH OSF AND HEALTHY SUBJECTS WERE ALSO CONDUCTED BY WELL-TRAINED DENTISTS, AND LOGISTIC REGRESSION WAS PERFORMED TO IDENTIFY RISK FACTORS FOR OSF. RESULTS: HYPERMETHYLATION OF E-CADHERIN AND COX-2 WAS OBSERVED IN 36% AND 22% OF ORAL CANCER SAMPLES, RESPECTIVELY. IN PATIENTS WITH OSF, THE RATES WERE 52% AND 30%, AND IN HEALTHY CONTROLS THE RATES WERE 4% AND 6%. HYPERMETHYLATION WAS SHOWN TO BE CORRELATED BETWEEN THE 3 GROUPS WITH STATISTICAL SIGNIFICANCE (P<0.01). METHYLATION OF CPG ISLANDS IN E-CADHERIN AND COX-2 OCCURRED MORE FREQUENTLY IN PATIENTS WITH OSF THAN IN THE CONTROL GROUP, BUT LESS FREQUENTLY THAN IN PATIENTS WITH ORAL CANCER. IN THE LOGISTIC REGRESSION ANALYSIS, SMOKING, BRUSHING MORE THAN TWICE DAILY, PERIODONTAL PROBING DEPTH AND PLAQUE INDEX WERE IDENTIFIED AS 4 MAJOR RISK FACTORS FOR OSF. CONCLUSIONS: THESE DATA CONFIRM THAT E-CADHERIN AND COX-2 EXPRESSIONS ARE RELATED TO OSF. THE EPIGENETIC CHANGES PRESENTED IN PATIENTS WITH CHRONIC INFLAMMATION MIGHT DEMONSTRATE AN IRREVERSIBLE DESTRUCTION IN THE TISSUES OR ORGANS SIMILAR TO THE EFFECTS OF CANCER. CHRONIC OSF WAS SIGNIFICANTLY ASSOCIATED WITH HYPERMETHYLATION, A CANCER RISK FACTOR. 2012 16 3531 36 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 2845 35 FREQUENT EPIGENETIC INACTIVATION OF THE SLIT2 GENE IN CHRONIC AND ACUTE LYMPHOCYTIC LEUKEMIA. RECENTLY A MOUSE MODEL OF T/NATURAL KILLER ACUTE LYMPHOBLASTIC LEUKEMIA WAS USED TO ASSESS GLOBAL PROMOTER METHYLATION ACROSS THE MOUSE GENOME USING THE RESTRICTION LANDMARK GENOMIC SCANNING TECHNIQUE. ONE OF THE METHYLATED MOUSE GENES IDENTIFIED IN THIS WAY WAS SLIT2. THERE ARE THREE MAMMALIAN SLIT GENES (SLIT1, SLIT2, SLIT3), THAT BELONG TO A HIGHLY CONSERVED FAMILY OF AXON GUIDANCE MOLECULES. WE HAVE PREVIOUSLY DEMONSTRATED THAT SLIT2 IS FREQUENTLY INACTIVATED IN LUNG, BREAST, COLORECTAL AND GLIOMA TUMORS BY HYPERMETHYLATION OF A CPG ISLAND IN ITS PROMOTER REGION, WHILST INACTIVATING SOMATIC MUTATIONS ARE RARE. FURTHERMORE, WE DEMONSTRATED THAT SLIT2 ACTS AS A TUMOR SUPPRESSOR GENE IN BREAST AND COLORECTAL CANCER CELLS. IN THIS REPORT WE DETERMINED THE METHYLATION STATUS OF THE SLIT2 GENE IN LEUKEMIAS (CLL AND ALL). SLIT2 WAS METHYLATED IN ALL TEN LEUKEMIA CELL LINES ANALYZED (EIGHT COMPLETELY AND TWO PARTIALLY METHYLATED). SLIT2 EXPRESSION WAS RESTORED AFTER TREATING ALL LINES WITH 5-AZA-2DC. IN PRIMARY ALL AND CLL SAMPLES, SLIT2 WAS ALSO FREQUENTLY METHYLATED, 58% (30/52) B-ALL; 83% (10/12) T-ALL AND IN 80% (24/30) CLL. WHILST DNA FROM PERIPHERAL BLOOD AND BONE MARROW FROM HEALTHY CONTROL SAMPLES SHOWED NO SLIT2 METHYLATION. METHYLATION RESULTS IN LEUKEMIA CELL LINES AND ALL AND CLL PRIMARY SAMPLES WERE CONFIRMED BY DIRECT SEQUENCING OF BISULFITE MODIFIED DNA. OUR RESULTS DEMONSTRATE THAT METHYLATION OF THE SLIT2 5' CPG ISLAND IS CONSERVED BETWEEN MICE AND HUMANS, AND THEREFORE IS LIKELY TO BE OF FUNCTIONAL IMPORTANCE. 2009 18 2374 38 EPIGENETIC REGULATION OF TNFA EXPRESSION IN PERIODONTAL DISEASE. BACKGROUND: TUMOR NECROSIS FACTOR-ALPHA (TNF-ALPHA) PLAYS A CENTRAL ROLE IN THE MOLECULAR PATHOGENESIS OF PERIODONTAL DISEASE. HOWEVER, THE EPIGENETIC REGULATION ATTRIBUTABLE TO MICROBIAL AND INFLAMMATORY SIGNALS AT THE BIOFILM-GINGIVAL INTERFACE ARE POORLY UNDERSTOOD. IN THIS STUDY, THE DNA METHYLATION ALTERATION WITHIN THE TNFA PROMOTER IN HUMAN GINGIVAL BIOPSIES FROM DIFFERENT STAGES OF PERIODONTAL DISEASE IS INVESTIGATED AND THE REGULATORY MECHANISM OF TNFA TRANSCRIPTION BY DNA METHYLATION IS EXPLORED. METHODS: GINGIVAL BIOPSIES WERE OBTAINED FROM 17 PATIENTS WITH CHRONIC PERIODONTITIS (CP) AND 18 PERIODONTALLY HEALTHY INDIVIDUALS. ANOTHER 11 INDIVIDUALS PARTICIPATED IN AN EXPERIMENTALLY INDUCED GINGIVITIS STUDY, AND GINGIVAL BIOPSIES WERE COLLECTED AT THE BASELINE, INDUCTION, AND RESOLUTION PHASE. TO CONFIRM THAT TNFA PROMOTER METHYLATION MODULATED TNFA TRANSCRIPTION, THP.1 CELLS WERE TREATED WITH A DNA METHYLTRANSFERASE INHIBITOR, 5-AZA-2-DEOXYCYTIDINE (5-AZA-2DC), AND AN RAW294.7 CELL LINE TRANSFECTED WITH A TNFA PROMOTER-SPECIFIC LUCIFERASE REPORTER SYSTEM WITH OR WITHOUT METHYLATION WAS USED. RESULTS: IN GINGIVAL BIOPSIES FROM INDIVIDUALS WITH SEVERE CP, TWO INDIVIDUAL CYTOSINE-GUANINE DINUCLEOTIDES (CPG SITES) WITHIN THE TNFA PROMOTER (AT -163 AND -161 BP) DISPLAYED INCREASED METHYLATION IN CP SAMPLES COMPARED TO THOSE WITH GINGIVAL HEALTH (16.1% +/- 5.1% VERSUS 11.0% +/- 4.6%, P = 0.02 AND 19.8% +/- 4.1% VERSUS 15.4% +/- 3.6%, P = 0.04, RESPECTIVELY). THE METHYLATION LEVEL AT -163 BP WAS INVERSELY ASSOCIATED WITH THE TRANSCRIPTION LEVEL OF TNFA (P = 0.018). HOWEVER, NO SIGNIFICANT DIFFERENCE IN THE TNFA PROMOTER METHYLATION PATTERN WAS OBSERVED IN SAMPLES BIOPSIED DURING THE INDUCTION OR RESOLUTION PHASE OF EXPERIMENTALLY INDUCED GINGIVITIS, WHICH REPRESENTED A REVERSIBLE PERIODONTAL LESION. THP.1 CELLS TREATED WITH 5-AZA-2DC DEMONSTRATED A TIME-DEPENDENT INCREASE IN TNFA MESSENGER LEVEL. IT WAS ALSO FOUND THAT THE LUCIFERASE ACTIVITY DECREASED 2.6-FOLD IN A CONSTRUCT CONTAINING AN IN VITRO METHYLATED TNFA PROMOTER WHEN COMPARED TO THE UNMETHYLATED INSERT (P = 0.03). CONCLUSION: ALTHOUGH THE BIOPSY SAMPLES REPRESENTED A MIXED CELL POPULATION, THE CHANGE IN PROMOTER METHYLATION STATUS IN CHRONIC PERIODONTAL DISEASE SUGGESTED THAT DNA METHYLATION MAY BE AN IMPORTANT REGULATORY MECHANISM IN CONTROLLING TNFA TRANSCRIPTIONAL EXPRESSION IN PERIODONTAL DISEASE. 2013 19 1669 47 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 20 5086 25 PILOT STUDY OF DNA METHYLATION IN THE PATHOGENESIS OF CHRONIC RHINOSINUSITIS WITH NASAL POLYPS. BACKGROUND: DNA METHYLATION HAS BEEN IMPLICATED IN THE PATHOGENESIS OF ALLERGY AND ATOPY. THIS STUDY AIMED TO IDENTIFY WHETHER DNA METHYLATION ALSO PLAYS AN IMPORTANT ROLE IN THE PATHOGENESIS OF NASAL POLYPS (NP). METHODOLOGY: NP TISSUES WERE OBTAINED FROM 32 PATIENTS WITH CHRONIC RHINOSINUSITIS WITH BILATERAL NP. BIOPSIES OF INFERIOR TURBINATE MUCOSA (ITM) WERE TAKEN FROM 18 PATIENTS WHO UNDERWENT RHINOSEPTOPLASTY (CONTROL GROUP). THE METHYLATED GENES, WHICH WERE DETECTED BY DNA METHYLATION MICROARRAY, WERE VALIDATED BY METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION, BISULPHITE SEQUENCING, REAL-TIME POLYMERASE CHAIN REACTION AND IMMUNOHISTOCHEMISTRY. RESULTS: DNA METHYLATION MICROARRAY IDENTIFIED 8,008 CPG ISLANDS IN 2,848 GENES. ONE HUNDRED AND NINETY-EIGHT GENES WERE FOUND TO HAVE A METHYLATED SIGNAL IN THE PROMOTER REGION IN NP SAMPLES COMPARED WITH ITM SAMPLES. THE FOUR TOP GENES THAT CHANGED, COL18A1, EP300, GNAS AND SMURF1, WERE SELECTED FOR FURTHER STUDY. THE METHYLATION FREQUENCY OF COL18A1 WAS SIGNIFICANTLY HIGHER IN NP SAMPLES THAN IN ITM SAMPLES. CONCLUSIONS: DNA METHYLATION MIGHT PLAY AN IMPORTANT ROLE IN THE PATHOGENESIS OF NP. PROMOTER METHYLATION OF COL18A1 WAS FOUND TO BE SIGNIFICANTLY INCREASED IN NP TISSUES, FURTHER STUDIES ARE NECESSARY TO CONFIRM THE SIGNIFICANCE OF THESE EPIGENETIC FACTORS IN THE MECHANISMS UNDERLYING THE DEVELOPMENT OR PERSISTENCE OF NP. 2015