1 5274 63 PROMOTER METHYLATION OF P16 AND EDNRB GENE IN LEUKEMIA PATIENTS IN TAIWAN. BOTH EPIGENETIC AND GENETIC ALTERNATIONS ARE INVOLVED IN CANCER FORMATION. IN THIS STUDY, WE HAVE IDENTIFIED THE METHYLATION FREQUENCY OF P16 AND ENDOTHELIN RECEPTOR TYPE B (EDNRB) OF 26 LEUKEMIA PATIENTS AND 8 RANDOMLY SELECTED NORMAL BLOOD DONORS IN TAIWAN. PROMOTER METHYLATION OF P16 WAS DETECTED IN 85% OF ACUTE LYMPHOCYTIC LEUKEMIA (ALL), 83% IN ACUTE MYELOID LEUKEMIA (AML) WHEREAS NO METHYLATION WAS DETECTED IN CHRONIC MYELOID LEUKEMIA (CML) IN BLAST CRISIS. HYPERMETHYLATION OF EDNRB WAS OBSERVED IN 92% OF ALL, 75% AML AND 100% IN CML IN BLAST CRISIS. NO ABERRANT METHYLATION OF P16 AND EDNRB WAS FOUND IN 8 NORMAL BLOOD DONORS. TAKEN TOGETHER, ABERRANT METHYLATION OF P16 AND EDNRB WAS HIGHLY PREVALENT IN LEUKEMIA PATIENTS IN TAIWAN. 2008 2 2326 20 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 3 2133 23 EPIGENETIC INACTIVATION OF THE MIR-34A IN HEMATOLOGICAL MALIGNANCIES. MIR-34A IS A TRANSCRIPTIONAL TARGET OF P53 AND IMPLICATED IN CARCINOGENESIS. WE STUDIED THE ROLE OF MIR-34A METHYLATION IN A PANEL OF HEMATOLOGICAL MALIGNANCIES INCLUDING ACUTE LEUKEMIA [ACUTE MYELOID LEUKEMIA (AML) AND ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)], CHRONIC LEUKEMIA [CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) AND CHRONIC MYELOID LEUKEMIA (CML)], MULTIPLE MYELOMA (MM) AND NON-HODGKIN'S LYMPHOMA (NHL). THE METHYLATION STATUS OF MIR-34A PROMOTER WAS STUDIED IN 12 CELL LINES AND 188 DIAGNOSTIC SAMPLES BY METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION. MIR-34A PROMOTER WAS UNMETHYLATED IN NORMAL CONTROLS BUT METHYLATED IN 75% LYMPHOMA AND 37% MYELOMA CELL LINES. HYPOMETHYLATING TREATMENT LED TO RE-EXPRESSION OF PRI-MIR-34A TRANSCRIPT IN LYMPHOMA CELLS WITH HOMOZYGOUS MIR-34A METHYLATION. IN PRIMARY SAMPLES AT DIAGNOSIS, MIR-34A METHYLATION WAS DETECTED IN 4% CLL, 5.5% MM SAMPLES AND 18.8% OF NHL AT DIAGNOSIS BUT NONE OF ALL, AML AND CML (P = 0.011). IN MM PATIENTS WITH PAIRED SAMPLES, MIR-34A METHYLATION STATUS REMAINED UNCHANGED AT PROGRESSION. AMONGST LYMPHOID MALIGNANCIES, MIR-34A WAS PREFERENTIALLY METHYLATED IN NHL (P = 0.018), IN PARTICULAR NATURAL KILLER (NK)/T-CELL LYMPHOMA. IN CONCLUSION, AMONGST HEMATOLOGICAL MALIGNANCIES, MIR-34A METHYLATION IS PREFERENTIALLY HYPERMETHYLATED IN NHL, IN PARTICULAR NK/T-CELL LYMPHOMA, IN A TUMOR-SPECIFIC MANNER, THEREFORE THE ROLE OF MIR-34A IN LYMPHOMAGENESIS WARRANTS FURTHER STUDY. 2010 4 6793 24 [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 5 2438 23 EPIGENETIC SILENCING OF THE CIRCADIAN CLOCK GENE CRY1 IS ASSOCIATED WITH AN INDOLENT CLINICAL COURSE IN CHRONIC LYMPHOCYTIC LEUKEMIA. DISRUPTION OF CIRCADIAN RHYTHM IS BELIEVED TO PLAY A CRITICAL ROLE IN CANCER DEVELOPMENT. CRYPTOCHROME 1 (CRY1) IS A CORE COMPONENT OF THE MAMMALIAN CIRCADIAN CLOCK AND WE HAVE PREVIOUSLY SHOWN ITS DEREGULATED EXPRESSION IN A SUBGROUP OF PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). USING REAL-TIME RT-PCR IN A COHORT OF 76 CLL PATIENTS AND 35 NORMAL BLOOD DONORS WE NOW DEMONSTRATE THAT DIFFERENTIAL CRY1 MRNA EXPRESSION IN HIGH-RISK (HR) CD38+/IMMUNOGLOBULIN VARIABLE HEAVY CHAIN GENE (IGVH) UNMUTATED PATIENTS AS COMPARED TO LOW-RISK (LR) CD38-/IGVH MUTATED PATIENTS CAN BE ATTRIBUTED TO DOWN-MODULATION OF CRY1 IN LR CLL CASES. ANALYSIS OF THE DNA METHYLATION PROFILE OF THE CRY1 PROMOTER IN A SUBGROUP OF 57 PATIENTS REVEALED THAT CRY1 EXPRESSION IN LR CLL CELLS IS SILENCED BY ABERRANT PROMOTER CPG ISLAND HYPERMETHYLATION. THE METHYLATION PATTERN OF THE CRY1 PROMOTER PROVED TO HAVE HIGH PROGNOSTIC IMPACT IN CLL WHERE ABERRANT PROMOTER METHYLATION PREDICTED A FAVOURABLE OUTCOME. CRY1 MRNA TRANSCRIPT LEVELS DID NOT CHANGE OVER TIME IN THE MAJORITY OF PATIENTS WHERE SEQUENTIAL SAMPLES WERE AVAILABLE FOR ANALYSIS. WE ALSO COMPARED THE CRY1 EXPRESSION IN CLL WITH OTHER LYMPHOID MALIGNANCIES AND OBSERVED EPIGENETIC SILENCING OF CRY1 IN A PATIENT WITH B CELL ACUTE LYMPHOBLASTIC LEUKEMIA (B-ALL). 2012 6 2132 23 EPIGENETIC INACTIVATION OF THE MIR-124-1 IN HAEMATOLOGICAL MALIGNANCIES. MIR-124-1 IS A TUMOUR SUPPRESSOR MICRORNA (MIR). EPIGENETIC DEREGULATION OF MIRS IS IMPLICATED IN CARCINOGENESIS. PROMOTER DNA METHYLATION AND HISTONE MODIFICATION OF MIR-124-1 WAS STUDIED IN 5 NORMAL MARROW CONTROLS, 4 LYMPHOMA, 8 MULTIPLE MYELOMA (MM) CELL LINES, 230 DIAGNOSTIC PRIMARY SAMPLES OF ACUTE MYELOID LEUKAEMIA (AML), ACUTE LYMPHOBLASTIC LEUKAEMIA (ALL), CHRONIC MYELOID LEUKAEMIA (CML), CHRONIC LYMPHOCYTIC LEUKAEMIA (CLL), MM, AND NON-HODGKIN'S LYMPHOMA (NHL), AND 53 MM SAMPLES AT STABLE DISEASE OR RELAPSE. PROMOTER OF MIR-124-1 WAS UNMETHYLATED IN NORMAL CONTROLS BUT HOMOZYGOUSLY METHYLATED IN 4 OF 4 LYMPHOMA AND 4 OF 8 MYELOMA CELL LINES. TREATMENT OF 5-AZA-2'-DEOXYCYTIDINE LED TO MIR-124-1 DEMETHYLATION AND RE-EXPRESSION OF MATURE MIR-124, WHICH ALSO ASSOCIATED WITH EMERGENCE OF EUCHROMATIC TRIMETHYL H3K4 AND CONSEQUENT DOWNREGULATION OF CDK6 IN MYELOMA CELLS HARBORING HOMOZYGOUS MIR-124-1 METHYLATION. IN PRIMARY SAMPLES AT DIAGNOSIS, MIR-124-1 METHYLATION WAS ABSENT IN CML BUT DETECTED IN 2% EACH OF MM AT DIAGNOSIS AND RELAPSE/PROGRESSION, 5% ALL, 15% AML, 14% CLL AND 58.1% OF NHL (P<0.001). AMONGST LYMPHOID MALIGNANCIES, MIR-124-1 WAS PREFERENTIALLY METHYLATED IN NHL THAN MM, CLL OR ALL. IN PRIMARY LYMPHOMA SAMPLES, MIR-124-1 WAS PREFERENTIALLY HYPERMETHYLATED IN B- OR NK/T-CELL LYMPHOMAS AND ASSOCIATED WITH REDUCED MIR-124 EXPRESSION. IN CONCLUSION, MIR-124-1 WAS HYPERMETHYLATED IN A TUMOUR-SPECIFIC MANNER, WITH A HETEROCHROMATIC HISTONE CONFIGURATION. HYPOMETHYLATION LED TO PARTIAL RESTORATION OF EUCHROMATIC HISTONE CODE AND MIR RE-EXPRESSION. INFREQUENT MIR-124-1 METHYLATION DETECTED IN DIAGNOSTIC AND RELAPSE MM SAMPLES SHOWED AN UNIMPORTANT ROLE IN MM PATHOGENESIS, DESPITE FREQUENT METHYLATION FOUND IN CELL LINES. AMONGST HAEMATOLOGICAL CANCERS, MIR-124-1 WAS MORE FREQUENTLY HYPERMETHYLATED IN NHL, AND HENCE WARRANTS FURTHER STUDY. 2011 7 3444 27 HYPERMETHYLATION OF E-CADHERIN IN LEUKEMIA. E-CADHERIN GENE IS OFTEN TERMED A "METASTASIS SUPPRESSOR" GENE BECAUSE THE E-CADHERIN PROTEIN CAN SUPPRESS TUMOR CELL INVASION AND METASTASIS. INACTIVATION OF THE E-CADHERIN GENE OCCURS IN UNDIFFERENTIATED SOLID TUMORS BY BOTH GENETIC AND EPIGENETIC MECHANISMS; HOWEVER, THE ROLE OF E-CADHERIN IN HEMATOLOGIC MALIGNANCIES IS ONLY NOW BEING RECOGNIZED. E-CADHERIN EXPRESSION IS ESSENTIAL FOR ERYTHROBLAST AND NORMOBLAST MATURATION, YET EXPRESSION IS REDUCED OR ABSENT IN LEUKEMIC BLAST CELLS. THIS STUDY EXAMINED THE MESSENGER RNA (MRNA) AND PROTEIN EXPRESSION OF THE E-CADHERIN GENE IN BONE MARROW AND BLOOD SAMPLES FROM NORMAL DONORS AND PATIENTS WITH LEUKEMIA. WE FOUND THAT ALL NORMAL DONOR SAMPLES EXPRESSED E-CADHERIN MRNA, WHEREAS BOTH SAMPLES OF ACUTE MYELOGENOUS LEUKEMIA AND CHRONIC LYMPHOCYTIC LEUKEMIA HAD A SIGNIFICANT REDUCTION OR ABSENCE OF EXPRESSION. HOWEVER, NORMAL BLAST COUNTERPARTS EXPRESSED ONLY A LOW LEVEL OF E-CADHERIN SURFACE PROTEIN. SODIUM BISULPHITE GENOMIC SEQUENCING WAS USED TO FULLY CHARACTERIZE THE METHYLATION PATTERNS OF THE CPG ISLAND ASSOCIATED WITH THE E-CADHERIN GENE PROMOTER IN THOSE SAMPLES WITH MATCHED DNA. ALL OF THE NORMAL CONTROL SAMPLES WERE ESSENTIALLY UNMETHYLATED; HOWEVER, 14 OF 18 (78%) OF THE LEUKEMIA SAMPLES HAD ABNORMAL HYPERMETHYLATION OF THE E-CADHERIN CPG ISLAND. IN FACT BOTH ALLELES OF THE E-CADHERIN GENE WERE OFTEN HYPERMETHYLATED. WE CONCLUDE THE E-CADHERIN GENE IS A COMMON TARGET FOR HYPERMETHYLATION IN HEMATOLOGIC MALIGNANCIES. 2000 8 5458 20 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 9 6231 21 THE LONG NONCODING RNA MEG3 AND ITS TARGET MIR-147 REGULATE JAK/STAT PATHWAY IN ADVANCED CHRONIC MYELOID LEUKEMIA. BACKGROUND: LONG NON-CODING (LNC) RNAS PLAYS AN IMPORTANT ROLE IN CHRONIC MYELOID LEUKEMIA (CML). IN THIS STUDY, WE AIMED TO UNCOVER THE MECHANISM OF THE LNCRNA MATERNALLY EXPRESSED 3 (MEG3) AND ITS TARGET MICRORNA-147 (MIR-147) IN CML. METHODS: SIXTY CML PATIENTS AND 10 HEALTHY DONORS WERE INCLUDED IN THE STUDY. THE METHYLATION OF MEG3 AND MIR-147 PROMOTER WAS DETERMINED BY METHYLATION-SPECIFIC PCR. THE RELATIONSHIP OF MEG3 AND MIR-147 WAS EXPLORED BY LUCIFERASE ASSAY. THE INTERACTIONS OF PROTEINS WERE STUDIED BY RNA PULL-DOWN ASSAY, RNA IMMUNOPRECIPITATION AND CO-IMMUNOPRECIPITATION. FINDINGS: PATIENTS IN ACCELERATED PHASE CML (CML-AP) AND BLAST PHASE CML (CML-BP) SHOWED LOWER EXPRESSIONS OF MEG3 AND MIR-147 AND HIGHER EXPRESSIONS OF DNMT1, DNMT3B, MBD2, MECP2 AND HDAC1 COMPARED TO THE CONTROLS. THESE PATIENTS ALSO SHOWED A HIGHER DEGREE OF METHYLATION OF MEG3 AND MIR-147 WHILE THERE WAS A REDUCTION AFTER CHIDAMIDE TREATMENT. FURTHERMORE, THE OVEREXPRESSION OF MEG3 AND MIR-147 INHIBITED CELL PROLIFERATION BOTH IN VIVO AND IN VITRO, PROMOTED APOPTOSIS AND DECREASED THE EXPRESSIONS OF DNMT1, DNMT3A, DNMT3B, MBD2, HDAC1 AND MECP2. WE ALSO FOUND MEG3 INTERACTED WITH DNMT1, JAK2, STAT3, HDAC1, AND TYK2, AND JAK2 WAS BOUND TO STAT3, STAT5 AND MYC. MORE INTERESTINGLY, JAK2 WAS BOUND TO TYK2 BY THE BRIDGE OF MEG3. INTERPRETATION: LNCRNA MEG3 AND ITS TARGET MIR-147 MAY SERVE AS A NOVEL THERAPEUTIC TARGET FOR CML BLAST CRISIS, AND CHIDAMIDE MIGHT HAVE A POTENTIAL CLINICAL APPLICATION IN TREATING CML BLAST CRISIS. 2018 10 5459 19 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 11 150 23 ABERRANT HYPOMETHYLATION OF THE CANCER-TESTIS ANTIGEN PRAME CORRELATES WITH PRAME EXPRESSION IN ACUTE MYELOID LEUKEMIA. PRAME IS A TUMOR-ASSOCIATED ANTIGEN, WHICH BELONGS TO THE FAMILY OF CANCER-TESTIS ANTIGENS (CTA). THE EXPRESSION OF CTA IS MAINLY RESTRICTED TO THE TESTIS AND VARIOUS TUMORS. IN CONTRAST TO OTHER CTA, PRAME EXPRESSION IS ALSO FREQUENTLY DETECTED IN ACUTE AND CHRONIC LEUKEMIAS. DUE TO THIS EXPRESSION PATTERN, PRAME HAS ATTRACTED GREAT INTEREST AS A PROGNOSTIC TUMOR MARKER THAT CAN BE USED FOR THE DETECTION OF MINIMAL RESIDUAL DISEASE AND AS A POTENTIAL TARGET FOR IMMUNOTHERAPY. IN ACUTE MYELOID LEUKEMIA (AML), PRAME EXPRESSION HAS BEEN OBSERVED IN 30-64% OF CASES. TO EVALUATE WHETHER EPIGENETIC MECHANISMS CONTRIBUTE TO PRAME ACTIVATION IN AML, WE STUDIED DNA METHYLATION OF 15 CPG DINUCLEOTIDES WITHIN A CPG-RICH REGION LOCATED IN THE INTRON 1 OF THE PRAME GENE. DNA METHYLATION WAS DETERMINED BY SEQUENCE ANALYSIS OF CLONED PCR PRODUCTS GENERATED FROM BISULFITE-TREATED GENOMIC DNA. METHYLATION PATTERNS WERE CORRELATED WITH PRAME MRNA LEVELS AS DETERMINED BY MICROARRAY ANALYSIS AND REAL-TIME PCR. WE FOUND ALMOST COMPLETE METHYLATION IN MONONUCLEAR BLOOD CELLS FROM TWO HEALTHY DONORS AND IN BONE MARROW CELLS OF FOUR PRAME-NEGATIVE AML PATIENTS. IN CONTRAST, THE DEGREE OF PRAME METHYLATION WAS CLEARLY REDUCED IN FOUR PRAME-POSITIVE AML BONE MARROW SAMPLES. IN PARTICULAR, THESE SAMPLES WERE CHARACTERIZED BY THE PRESENCE OF CLONES, WHICH WERE COMPLETELY DEVOID OF METHYLATION. THE SIGNIFICANT INVERSE CORRELATION BETWEEN THE DEGREE OF METHYLATION AND PRAME EXPRESSION SUGGESTS A CAUSAL ROLE OF DNA METHYLATION IN PRAME REGULATION. SUCH A ROLE IS FURTHER SUPPORTED BY THE OBSERVATION THAT TREATMENT OF PRAME-NEGATIVE CELL LINES U-937 AND THP-1 WITH THE DEMETHYLATING AGENT 5'-AZA-2'DC RESULTED IN A DOSE-RELATED UPREGULATION OF PRAME EXPRESSION. 2008 12 2261 16 EPIGENETIC PROFILE IN CHRONIC LYMPHOCYTIC LEUKEMIA USING METHYLATION-SPECIFIC MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION. AIM: TO ANALYZE THE METHYLATION STATUS OF 35 TUMOR SUPPRESSOR GENES USING METHYLATION-SPECIFIC MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION (MS-MLPA) IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). MATERIALS & METHODS: THE DNA OF 37 SAMPLES FROM PATIENTS WITH CLL, SIX HEALTHY DONORS, AND JURKAT AND RAMOS CELL LINES WAS ANALYZED BY MS-MLPA. RESULTS: OUR RESULTS CONFIRM THAT HYPERMETHYLATION IS A COMMON AND NOT RANDOMLY DISTRIBUTED EVENT IN CLL, AND SOME GENES, SUCH AS WT1, CDH13, IGSF4/TSLC1, GATA5, DAPK1 AND RARB, ARE HYPERMETHYLATED IN MORE THAN 25% OF THE ANALYZED SAMPLES. IMPORTANTLY, MS-MLPA ALSO DETECTED HYPERMETHYLATION OF SOME GENES NOT REPORTED PREVIOUSLY IN CLL, AND THEIR METHYLATION STATUS WAS CONFIRMED BY BISULFITE SEQUENCING. CONCLUSION: THESE RESULTS INDICATE THAT MS-MLPA IS A USEFUL TECHNIQUE FOR THE DETECTION OF METHYLATION IN CLL SAMPLES. SELECTING CLL-SPECIFIC METHYLATION TARGETS IN ORDER TO GENERATE A CLL-SPECIFIC MS-MLPA PROBE SET COULD ENHANCE ITS USEFULNESS AS A TOOL IN STUDIES OF RISK STRATIFICATION AND GUIDING THE BEST THERAPEUTIC DECISION. 2012 13 2131 22 EPIGENETIC INACTIVATION OF THE HSA-MIR-203 IN HAEMATOLOGICAL MALIGNANCIES. MIR-203 IS A TUMOUR SUPPRESSOR MICRORNA (MIRNA). WE STUDIED THE METHYLATION OF HSA-MIR-203 IN 150 SAMPLES INCLUDING ACUTE MYELOID LEUKAEMIA (AML), ACUTE LYMPHOBLASTIC LEUKAEMIA (ALL), CHRONIC MYELOID LEUKAEMIA (CML), CHRONIC LYMPHOCYTIC LEUKAEMIA (CLL) AND NON-HODGKIN'S LYMPHOMA (NHL) BY METHYLATION-SPECIFIC PCR, AND MIRNA EXPRESSION BY STEM-LOOP RT-QPCR. HSA-MIR-203 PROMOTER WAS UNMETHYLATED IN NORMAL CONTROLS BUT HOMOZYGOUSLY METHYLATED IN TWO AML AND FOUR LYMPHOMA CELL LINES, IN WHICH 5-AZA-2'-DEOXYCYTIDINE TREATMENT LED TO PROMOTER DEMETHYLATION AND MIR-203 RE-EXPRESSION. RESTORATION OF MIR-203 EXPRESSION IN LYMPHOMA CELLS INHIBITED CELLULAR PROLIFERATION AND INCREASED CELL DEATH, SUGGESTING AN INHERENT TUMOUR SUPPRESSOR ACTIVITY. IN PRIMARY SAMPLES, HSA-MIR-203 METHYLATION WAS ABSENT IN CML BUT DETECTED IN 5.0% ALL, 10.0% AML, 42.0% CLL AND 38.8% OF NHL (INCLUDING SIX [60.0%] NATURAL KILLER-CELL, NINE [40.9%] B-CELL AND FOUR [23.5%] T CELL NHL). MOREOVER, HSA-MIR-203 METHYLATION WAS ASSOCIATED WITH HYPERMETHYLATION OF HSA-MIR-34A, -124A AND -196B IN NHL BUT NOT CLL. IN CLL, HSA-MIR-203 METHYLATION WAS ASSOCIATED WITH A HIGHER PRESENTING HB LEVEL (P = 0.033). THE PROJECTED 10 YEAR OVERALL SURVIVAL OF THE CLL PATIENTS WAS 58.2%, WHICH WAS IMPACTED BY RAI STAGE AND HIGH-RISK KARYOTYPES BUT NOT HSA-MIR-203 METHYLATION. HSA-MIR-203 WAS MORE FREQUENTLY METHYLATED IN LYMPHOID THAN MYELOID MALIGNANCIES (P = 0.002). IN CONCLUSION, MIR-203, A TUMOUR SUPPRESSOR GENE, WAS HYPERMETHYLATED IN A TUMOUR-SPECIFIC MANNER WITH GENE SILENCING. HSA-MIR-203 WAS MORE FREQUENTLY HYPERMETHYLATED IN LYMPHOID THAN MYELOID MALIGNANCIES. IN NHL, HSA-MIR-203 METHYLATION WAS ASSOCIATED WITH CONCOMITANT METHYLATION OF OTHER TUMOUR SUPPRESSOR MIRNAS. THE FREQUENT HSA-MIR-203 METHYLATION IN LYMPHOID MALIGNANCIES SUGGESTED A PATHOGENETIC ROLE OF HSA-MIR-203 METHYLATION. 2011 14 6238 19 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 15 1433 22 DIFFERENTIAL GENOME-WIDE ARRAY-BASED METHYLATION PROFILES IN PROGNOSTIC SUBSETS OF CHRONIC LYMPHOCYTIC LEUKEMIA. GLOBAL HYPOMETHYLATION AND REGIONAL HYPERMETHYLATION ARE WELL-KNOWN EPIGENETIC FEATURES OF CANCER; HOWEVER, IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL), STUDIES ON GENOME-WIDE EPIGENETIC MODIFICATIONS ARE LIMITED. HERE, WE ANALYZED THE GLOBAL METHYLATION PROFILES IN CLL, BY APPLYING HIGH-RESOLUTION METHYLATION MICROARRAYS (27,578 CPG SITES) TO 23 CLL SAMPLES, BELONGING TO THE IMMUNOGLOBULIN HEAVY-CHAIN VARIABLE (IGHV) MUTATED (FAVORABLE) AND IGHV UNMUTATED/IGHV3-21 (POOR-PROGNOSTIC) SUBSETS. OVERALL, RESULTS DEMONSTRATED SIGNIFICANT DIFFERENCES IN METHYLATION PATTERNS BETWEEN THESE SUBGROUPS. SPECIFICALLY, IN IGHV UNMUTATED CLL, WE IDENTIFIED METHYLATION OF 7 KNOWN OR CANDIDATE TUMOR SUPPRESSOR GENES (EG, VHL, ABI3, AND IGSF4) AS WELL AS 8 UNMETHYLATED GENES INVOLVED IN CELL PROLIFERATION AND TUMOR PROGRESSION (EG, ADORA3 AND PRF1 ENHANCING THE NUCLEAR FACTOR-KAPPAB AND MITOGEN-ACTIVATED PROTEIN KINASE PATHWAYS, RESPECTIVELY). IN CONTRAST, THESE LATTER GENES WERE SILENCED BY METHYLATION IN IGHV MUTATED PATIENTS. THE ARRAY DATA WERE VALIDATED FOR SELECTED GENES USING METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION, QUANTITATIVE REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION, AND BISULFITE SEQUENCING. FINALLY, THE SIGNIFICANCE OF DNA METHYLATION IN REGULATING GENE PROMOTERS WAS SHOWN BY REINDUCING 4 METHYLATED TUMOR SUPPRESSOR GENES (EG, VHL AND ABI3) IN IGHV UNMUTATED SAMPLES USING THE METHYL-INHIBITOR 5-AZA-2'-DEOXYCYTIDINE. TAKEN TOGETHER, OUR DATA FOR THE FIRST TIME REVEAL DIFFERENCES IN GLOBAL METHYLATION PROFILES BETWEEN PROGNOSTIC SUBSETS OF CLL, WHICH MAY UNFOLD EPIGENETIC SILENCING MECHANISMS INVOLVED IN CLL PATHOGENESIS. 2010 16 157 22 ABERRANT METHYLATION OF TUMOUR SUPPRESSOR GENE ADAM12 IN CHRONIC LYMPOCYTIC LEUKEMIA PATIENTS: APPLICATION OF METHYLATION SPECIFIC-PCR TECHNIQUE. OBJECTIVE: CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) IS A COMMON LEUKEMIA AMONG CAUCASIANS BUT RARE IN ASIANS POPULATION. WE POSTULATED THAT ABERRANT METHYLATION EITHER HYPERMETHYLATION OR PARTIAL METHYLATION MIGHT BE ONE OF THE SILENCING MECHANISMS THAT INACTIVATES THE TUMOUR SUPPRESSOR GENES IN CLL. THIS STUDY AIMED TO COMPARE THE METHYLATION STATUS OF TUMOUR SUPPRESSOR GENE, ADAM12, AMONG CLL PATIENTS AND NORMAL INDIVIDUALS. WE ALSO EVALUATED THE ASSOCIATION BETWEEN METHYLATION OF ADAM12 AND CLINICAL AND DEMOGRAPHIC CHARACTERISTICS OF THE PARTICIPANTS. METHODS: A TOTAL OF 25 CLL PATIENTS AND 25 NORMAL INDIVIDUALS WERE RECRUITED IN THIS STUDY. THE METHYLATION STATUS OF ADAM12 WAS DETERMINED USING METHYLATION-SPECIFIC PCR (MSP); WHEREAS, DNA SEQUENCING METHOD WAS APPLIED FOR VALIDATION OF THE MSP RESULTS. RESULTS: AMONG CLL PATIENTS, 12 (48%) WERE PARTIALLY METHYLATED AND 13 (52%) WERE UNMETHYLATED. MEANWHILE, 5 (20%) AND 20 (80.6%) OF HEALTHY INDIVIDUALS WERE PARTIALLY METHYLATED AND UNMETHYLATED, RESPECTIVELY. THERE WAS A STATISTICALLY SIGNIFICANT ASSOCIATION BETWEEN THE STATUS OF METHYLATION AT ADAM12 AND THE PRESENCE OF CLL (P=0.037). CONCLUSION: THE ABERRANT METHYLATION OF ADAM12 FOUND IN THIS STUDY USING MSP ASSAY MAY PROVIDE NEW EXPOSURE TO CLL THAT MAY IMPROVE THE GAPS INVOLVED IN GENETIC EPIGENETIC STUDY IN CLL. 2021 17 4601 18 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 18 4364 18 MIRNA DEREGULATION BY EPIGENETIC SILENCING DISRUPTS SUPPRESSION OF THE ONCOGENE PLAG1 IN CHRONIC LYMPHOCYTIC LEUKEMIA. MICRORNAS (MIRNA) PLAY A KEY ROLE IN CELLULAR REGULATION AND, IF DEREGULATED, IN THE DEVELOPMENT OF NEOPLASTIC DISORDERS INCLUDING CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). RNAS FROM PRIMARY CELLS OF 50 TREATMENT-NAIVE CLL PATIENTS AND PERIPHERAL B CELLS OF 14 HEALTHY DONORS WERE APPLIED TO MIRNA EXPRESSION PROFILING USING BEAD CHIP TECHNOLOGY. IN CLL CELLS, A SET OF 7 UP- AND 19 DOWN-REGULATED MIRNAS WAS IDENTIFIED. AMONG THE MIRNAS DOWN-REGULATED IN CLL CELLS, 6 OF 10 MIRNA PROMOTERS EXAMINED SHOWED GAIN OF METHYLATION COMPARED WITH NORMAL B-CELL CONTROLS. SUBSEQUENT TARGET PREDICTION OF DEREGULATED MIRNAS REVEALED A HIGHLY SIGNIFICANT BINDING PREDICTION AT THE 3' UNTRANSLATED REGION OF THE PLEOMORPHIC ADENOMA GENE 1 (PLAG1) ONCOGENE. LUCIFERASE REPORTER ASSAYS INCLUDING SITE-DIRECTED MUTAGENESIS OF BINDING SITES REVEALED A SIGNIFICANT REGULATION OF PLAG1 BY MIR-181A, MIR-181B, MIR-107, AND MIR-424. ALTHOUGH EXPRESSION OF PLAG1 MRNA WAS NOT AFFECTED, PLAG1 PROTEIN EXPRESSION WAS SHOWN TO BE SIGNIFICANTLY ELEVATED IN CLL CELLS COMPARED WITH THE LEVELS IN HEALTHY DONOR B CELLS. IN SUMMARY, WE COULD DEMONSTRATE DISRUPTION OF MIRNA-MEDIATED TRANSLATIONAL CONTROL, PARTLY DUE TO EPIGENETIC TRANSCRIPTIONAL SILENCING OF MIRNAS, WITH SUBSEQUENT OVEREXPRESSION OF THE ONCOGENIC TRANSCRIPTION FACTOR PLAG1 AS A PUTATIVE NOVEL MECHANISM OF CLL PATHOGENESIS. 2009 19 18 16 5-AZACYTIDINE MODULATES CPG METHYLATION LEVELS OF EZH2 AND NOTCH1 IN MYELODYSPLASTIC SYNDROMES. PURPOSE: MOLECULAR MECHANISMS OF RESPONSE TO HYPOMETHYLATING AGENTS IN PATIENTS WITH MYELODYSPLASTIC SYNDROMES (MDS) AND CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML) STILL REMAIN LARGELY UNKNOWN. THEREFORE, THE EFFECTS OF 5-AZACYTIDINE (AZA) ON CLONAL ARCHITECTURE AND DNA METHYLATION WERE INVESTIGATED IN THIS STUDY. METHODS: USING NEXT-GENERATION SEQUENCING (NGS), 30 MYELOID LEUKEMIA-ASSOCIATED GENES WERE ANALYZED IN 15 MDS/CMML PATIENTS WITH EXCELLENT RESPONSE TO AZA. EFFECTS ON METHYLATION LEVELS WERE ANALYZED BY QUANTITATIVE METHYLATION ANALYSIS USING PYROSEQUENCING FOR THE GLOBAL METHYLATION MARKER LINE-1 IN PATIENTS AND MYELOID CELL LINES. VARIOUS MYELOID CELL LINES AND A HEALTHY COHORT WERE SCREENED FOR METHYLATION LEVELS IN 23 GENES. SELECTED TARGETS WERE VERIFIED ON THE MDS/CMML COHORT. RESULTS: THE STUDY PRESENTED HERE SHOWED A STABLE VARIANT ALLELE FREQUENCY AND STABLE GLOBAL METHYLATION LEVELS IN RESPONDING PATIENTS. A SIGNIFICANT DEMETHYLATION OF EZH2 AND NOTCH1 WAS REVEALED IN PATIENTS WITH AZA RESPONSE. CONCLUSIONS: A RESPONSE TO AZA IS NOT ASSOCIATED WITH ERADICATION OF MALIGNANT CLONES, BUT RATHER WITH A STABILIZATION OF THE CLONAL ARCHITECTURE. WE SUGGEST CHANGES IN CPG METHYLATION LEVELS OF EZH2 AND NOTCH1 AS POTENTIAL TARGETS OF EPIGENETIC RESPONSE TO AZA TREATMENT WHICH MAY ALSO SERVE AS USEFUL BIOMARKERS AFTER CLINICAL EVALUATION. 2019 20 1581 17 DNA METHYLATION PROFILES IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS TREATED WITH CHEMOIMMUNOTHERAPY. BACKGROUND: IN ORDER TO GAIN INSIGHT INTO THE CONTRIBUTION OF DNA METHYLATION TO DISEASE PROGRESSION OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL), USING 450K ILLUMINA ARRAYS, WE DETERMINED THE DNA METHYLATION PROFILES IN PAIRED PRE-TREATMENT/RELAPSE SAMPLES FROM 34 CLL PATIENTS TREATED WITH CHEMOIMMUNOTHERAPY, MOSTLY (N = 31) WITH THE FLUDARABINE-CYCLOPHOSPHAMIDE-RITUXIMAB (FCR) REGIMEN. RESULTS: THE EXTENT OF IDENTIFIED CHANGES IN CLL CELLS VERSUS MEMORY B CELLS FROM HEALTHY DONORS WAS TERMED "EPIGENETIC BURDEN" (EB) WHEREAS THE NUMBER OF CHANGES BETWEEN THE PRE-TREATMENT VERSUS THE RELAPSE SAMPLE WAS TERMED "RELAPSE CHANGES" (RC). SIGNIFICANT (P < 0.05) ASSOCIATIONS WERE IDENTIFIED BETWEEN (I) HIGH EB AND SHORT TIME-TO-FIRST-TREATMENT (TTFT); AND, (II) FEW RCS AND SHORT TIME-TO-RELAPSE. BOTH THE EB AND THE RC CLUSTERED IN SPECIFIC GENOMIC REGIONS AND CHROMATIN STATES, INCLUDING REGULATORY REGIONS CONTAINING BINDING SITES OF TRANSCRIPTION FACTORS IMPLICATED IN B CELL AND CLL BIOLOGY. CONCLUSIONS: OVERALL, WE SHOW THAT DNA METHYLATION IN CLL FOLLOWS DIFFERENT DYNAMICS IN RESPONSE TO CHEMOIMMUNOTHERAPY. THESE EPIGENETIC ALTERATIONS WERE LINKED WITH SPECIFIC CLINICAL AND BIOLOGICAL FEATURES. 2019