1 5458 124 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	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
2 5668  38 SFRP1 EXPRESSION REGULATES WNT SIGNALING IN CHRONIC MYELOID LEUKEMIA K562 CELLS. BACKGROUND: WNT SIGNALING CASCADES PLAY IMPORTANT ROLES IN CELL FATE DECISIONS AND THEIR DEREGULATION HAS BEEN DOCUMENTED IN MANY DISEASES, INCLUDING MALIGNANT TUMORS AND LEUKEMIA. ONE MECHANISM OF ABERRANT WNT SIGNALING IS THE SILENCING OF WNT INHIBITORS THROUGH EPIGENETIC MECHANISMS. THE SFRPS ARE ONE OF THE MOST STUDIED WNT INHIBITORS; AND THE SFRP1 LOSS IS KNOWN IN MANY HEMATOLOGICAL MALIGNANCIES. THEREFORE, WE AIMED TO COMPARE THE EXPRESSION OF WNT RELATED GENES IN THE PRESENCE AND ABSENCE OF SFRP1 IN A CHRONIC MYELOID LEUKEMIA (CML) CELL LINE. OBJECTIVE: IT IS IMPORTANT TO UNDERSTAND HOW SFRP1 AND SFRP1 PERFORM THEIR EFFECTS ON CML TO DESIGN NEW AGENTS AND STRATEGIES FOR RESISTANT AND ADVANCED FORMS OF CML. MATERIALS AND METHODS: WE USED K562 CELLS, WHICH NORMALLY DO NOT EXPRESS SFRP1 AND ITS SFRP1 EXPRESSING SUBCLONE K562S. TOTAL RNA WAS ISOLATED FROM K562 AND K562S CELL LINES AND CONVERTED TO CDNA. PCR ARRAY EXPERIMENTS WERE PERFORMED USING HUMAN WNT SIGNALING PATHWAY PLUS RT2 PROFILER KIT. WNT SIGNALING PATHWAY ACTIVATION WAS STUDIED BY WESTERN BLOT FOR DOWNSTREAM SIGNALING TARGETS. RESULTS: THE WNT3, LRP6, PRICKLE1 AND BTRC EXPRESSIONS WERE SIGNIFICANTLY DECREASED IN THE PRESENCE OF SFRP1; WHILE WNT5B INCREASED. THE SFRP1 EXPRESSION INHIBITED STABILIZATION OF TOTAL BETA-CATENIN PROTEIN AND DOWNSTREAM EFFECTOR PHOSPHORYLATION OF NONCANONICAL WNT/PCP SIGNALING; WHEREAS CA2+/PKC SIGNALING REMAINED ACTIVE. CONCLUSION: THE RESULTS SUGGEST THAT SFRP1 COULD BE A PROMISING THERAPEUTIC ANTICANCER AGENT. DEFINING THESE PATHWAY INTERACTIONS IS CRUCIAL FOR DESIGNING NEW AGENTS RESISTANT AND ADVANCED FORMS OF CML.	2022	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          
3 5669  28 SFRP1 PROMOTER METHYLATION IS ASSOCIATED WITH PERSISTENT PHILADELPHIA CHROMOSOME IN CHRONIC MYELOID LEUKEMIA. EPIGENETIC SILENCING OF SFRP GENES HAS BEEN SHOWN TO LEAD TO CONSTITUTIVE ACTIVATION OF THE CANONICAL WNT-SIGNALING PATHWAY. THE FIRST DESCRIPTION OF DEREGULATED WNT-SIGNALING ACTIVATION IN A HEMATOLOGICAL MALIGNANCY WAS REPORTED IN CHRONIC MYELOID LEUKEMIA (CML). TO INVESTIGATE WHETHER EPIGENETIC SILENCING OF SFRP IS RESPONSIBLE FOR THE OBSERVED WNT ACTIVATION IN CML, WE STUDIED THE METHYLATION AND MUTATIONAL STATUS OF THE SFRP1 PROMOTER IN 48 CHRONIC PHASE CML PATIENTS. OF THE 48 CML PATIENTS 41 WERE SHOWN TO BE UNMETHYLATED, 6 PATIENTS HEMI-METHYLATED AND 1 PATIENT FULLY METHYLATED AT THE SFRP1 PROMOTER. ALBEIT OBSERVED INFREQUENTLY IN CHRONIC PHASE CML, SFRP1 PROMOTER METHYLATION CORRELATED WITH PRIMARY CYTOGENETIC RESISTANCE TO IMATINIB MESYLATE. SFRP1 PROMOTER METHYLATION MAY INDICATE A GENETICALLY MORE UNSTABLE FORM OF DISEASE RESISTANT TO THERAPY AND PROVIDE A KEY BIOLOGICAL DIFFERENCE IN THERAPY RESISTANT PATIENTS, IN ADDITION TO A POSSIBLE MECHANISM FOR THE OBSERVED ACTIVATION OF CANONICAL WNT SIGNALING IN CML.	2009	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       
4 1733  45 E-CADHERIN GENE RE-EXPRESSION IN CHRONIC LYMPHOCYTIC LEUKEMIA CELLS BY HDAC INHIBITORS. BACKGROUND: THE TUMOR SUPPRESSOR GENE E-CADHERIN GENE IS FREQUENTLY SILENCED IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) CELLS AND RESULTS IN WNT-PATHWAY ACTIVATION. WE ANALYZED THE ROLE OF HISTONE EPIGENETIC MODIFICATIONS IN E-CADHERIN GENE SILENCING. METHODS: CLL SPECIMENS WERE TREATED WITH HISTONE DEACETYLASE INHIBITOR (HDACI) MS-275 AND ANALYZED FOR E-CADHERIN EXPRESSION WITH WESTERN BLOT AND RT-PCR ANALYSIS. THE DOWNSTREAM EFFECTS OF HDACI TREATED LEUKEMIC CELLS WERE STUDIED BY ANALYZING THE EFFECT ON WNT-PATHWAY SIGNALING. HDACI INDUCED ALTERATIONS IN E-CADHERIN SPLICING WERE INVESTIGATED BY TRANSCRIPT SPECIFIC REAL TIME PCR ANALYSIS. RESULTS: TREATMENT OF CLL SPECIMENS WITH HISTONE DEACETYLASE INHIBITORS (HDACI) TREATMENT RESULTED IN AN INCREASE OF THE E-CADHERIN RNA TRANSCRIPT (5 TO 119 FOLD INCREASE, N=10) IN EIGHT OUT OF TEN CLL SPECIMENS INDICATING THAT THIS GENE IS DOWN REGULATED BY HISTONE HYPOACETYLATION IN A MAJORITY OF CLL SPECIMENS. THE E-CADHERIN RE-EXPRESSION IN CLL SPECIMENS WAS NOTED BY WESTERN BLOT ANALYSIS AS WELL. BESIDES EPIGENETIC SILENCING ANOTHER MECHANISM OF E-CADHERIN INACTIVATION IS ABERRANT EXON 11 SPLICING RESULTING IN AN ALTERNATIVELY SPLICED TRANSCRIPT THAT LACKS EXON 11 AND IS DEGRADED BY THE NON-SENSE MEDIATED DECAY (NMD) PATHWAY. OUR CHROMATIN IMMUNOPRECIPITATION EXPERIMENTS SHOW THAT HDACI INCREASED THE ACETYLATION OF HISTONES H3 AND H4 IN THE E-CADHERIN PROMOTER REGION. THIS ALSO AFFECTED THE E-CADHERIN EXON 11 SPLICING PATTERN AS HDACI TREATED CLL SPECIMENS PREFERENTIALLY EXPRESSED THE CORRECTLY SPLICED TRANSCRIPT AND NOT THE EXON 11 SKIPPED ABERRANT TRANSCRIPT. THE RE-EXPRESSED E- CADHERIN BINDS TO BETA-CATENIN WITH INHIBITION OF THE ACTIVE WNT-BETA-CATENIN PATHWAY IN THESE CELLS. THIS RESULTED IN A DOWN REGULATION OF TWO WNT TARGET GENES, LEF AND CYCLIND1 AND THE WNT PATHWAY REPORTER. CONCLUSION: THE E-CADHERIN GENE IS EPIGENETICALLY MODIFIED AND HYPOACETYLATED IN CLL LEUKEMIC CELLS. TREATMENT OF CLL SPECIMENS WITH HDACI MS-275 ACTIVATES TRANSCRIPTION FROM THIS SILENT GENE WITH EXPRESSION OF MORE CORRECTLY SPLICED E-CADHERIN TRANSCRIPTS AS COMPARED TO THE ABERRANT EXON11 SKIPPED TRANSCRIPTS THAT IN TURN INHIBITS THE WNT SIGNALING PATHWAY. THE DATA HIGHLIGHTS THE ROLE OF EPIGENETIC MODIFICATIONS IN ALTERING GENE SPLICING PATTERNS.	2013	
                                                                                                                       
5 1669  42 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	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              
6 4877  42 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	
                                                                                                                                                                                                                            
7 6238  39 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	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                
8  139  32 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	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              
9 3531  35 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	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     
10 1146  36 CONCURRENT EPIGENETIC SILENCING OF WNT/BETA-CATENIN PATHWAY INHIBITOR GENES IN B CELL CHRONIC LYMPHOCYTIC LEUKAEMIA. BACKGROUND: THE WNT/BETA-CATENIN SIGNALLING IS ABERRANTLY ACTIVATED IN PRIMARY B CELL CHRONIC LYMPHOCYTIC LEUKAEMIA (CLL). EPIGENETIC SILENCING OF PATHWAY INHIBITOR GENES MAY BE A MECHANISM FOR ITS ACTIVATION. IN THIS STUDY, WE INVESTIGATED SYSTEMATICALLY AND QUANTITATIVELY THE METHYLATION STATUS OF 12 WNT/BETA-CATENIN PATHWAY INHIBITOR GENES - CDH1, DACT1, DKK1, DKK2, DKK3, DKK4, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5 AND WIF1 - IN THE CELL LINES EHEB AND MEC-1 AS WELL AS PATIENT SAMPLES. METHODS: QUANTIFICATION OF DNA METHYLATION WAS PERFORMED BY MEANS OF BISULPHITE PYROSEQUENCING AND CONFIRMED BY BISULPHITE SANGER SEQUENCING. GENE EXPRESSION WAS ANALYSED BY QPCR USING GAPDH AS INTERNAL CONTROL. E-CADHERIN AND BETA-CATENIN PROTEIN QUANTIFICATION WAS CARRIED OUT BY MICROSPHERE-BASED IMMUNOASSAYS. METHYLATION DIFFERENCES OBSERVED BETWEEN THE PATIENT AND CONTROL GROUPS WERE TESTED USING GENERALISED LEAST SQUARES MODELS. RESULTS: FOR 10 GENES, A HIGHER METHYLATION LEVEL WAS OBSERVED IN TUMOUR MATERIAL. ONLY DKK4 EXHIBITED SIMILARLY HIGH METHYLATION LEVELS IN BOTH TUMOUR AND NORMAL SPECIMENS, WHILE DACT1 WAS ALWAYS ESSENTIALLY UNMETHYLATED. HOWEVER, ALSO FOR THESE INHIBITORS, TREATMENT OF CELLS WITH THE DEMETHYLATING AGENT 5-AZA-2 -DEOXYCYTIDINE RESULTED IN AN INDUCTION OF THEIR EXPRESSION, AS SHOWN BY QUANTITATIVE PCR, SUGGESTING AN INDIRECT EPIGENETIC CONTROL OF ACTIVITY. WHILE THE DEGREE OF DEMETHYLATION AND ITS TRANSCRIPTIONAL CONSEQUENCES DIFFERED BETWEEN THE GENES, THERE WAS AN OVERALL HIGH CORRELATION OF DEMETHYLATION AND INCREASED ACTIVITY. PROTEIN EXPRESSION STUDIES REVEALED THAT NO CONSTITUTIVE WNT/BETA-CATENIN SIGNALLING OCCURRED IN THE CELL LINES, WHICH IS IN DISCREPANCY WITH RESULTS FROM PRIMARY CLL. HOWEVER, TREATMENT WITH 5-AZA-2 -DEOXYCYTIDINE CAUSED ACCUMULATION OF BETA-CATENIN. SIMULTANEOUSLY, E-CADHERIN EXPRESSION WAS STRONGLY INDUCED, LEADING TO THE FORMATION OF A COMPLEX WITH BETA-CATENIN AND THUS DEMONSTRATING ITS EPIGENETICALLY REGULATED INHIBITION EFFECT. CONCLUSIONS: THE RESULTS SUGGEST AN EPIGENETIC SILENCING MECHANISM OF THE WNT/BETA-CATENIN PATHWAY INHIBITOR GENES IN CLL. HYPERMETHYLATION AND SILENCING OF FUNCTIONALLY RELATED GENES MAY NOT BE COMPLETELY STOCHASTIC BUT RESULT FROM THE TUMOUR EPIGENOME REPROGRAMMING ORCHESTRATED BY POLYCOMB-GROUP REPRESSIVE COMPLEXES. THE DATA ARE OF INTEREST IN THE CONTEXT OF EPIGENETIC-BASED THERAPY.	2012	

11 3461  37 HYPOMETHYLATION-MEDIATED H19 OVEREXPRESSION INCREASES THE RISK OF DISEASE EVOLUTION THROUGH THE ASSOCIATION WITH BCR-ABL TRANSCRIPT IN CHRONIC MYELOID LEUKEMIA. PREVIOUS STUDY HAS REVEALED THAT H19 EXPRESSION IS REQUIRED FOR EFFICIENT TUMOR GROWTH INDUCED BY BCR-ABL IN CHRONIC MYELOID LEUKEMIA (CML). HEREIN, WE FURTHER DETERMINED H19 EXPRESSION AND ITS CLINICAL IMPLICATION IN PATIENTS WITH CML. H19 EXPRESSION AND METHYLATION WERE DETECTED BY REAL-TIME QUANTITATIVE PCR AND REAL-TIME QUANTITATIVE METHYLATION-SPECIFIC PCR, AND THEN CLINICAL IMPLICATION OF H19 EXPRESSION WAS FURTHER ANALYZED. H19 EXPRESSION WAS SIGNIFICANTLY UP-REGULATED IN CML PATIENTS (P < 0.001). H19 EXPRESSION WITH AN AREA UNDER RECEIVER OPERATING CHARACTERISTIC CURVE VALUE OF 0.824 MIGHT SERVE AS A PROMISING BIOMARKER IN DISTINGUISHING CML PATIENTS FROM CONTROLS. THE PATIENTS WITH HIGH H19 EXPRESSION HAD A TENDENCY OF HIGHER WHITE BLOOD CELLS AND BCR-ABL TRANSCRIPT THAN THOSE WITH LOW H19 EXPRESSION. H19 OVEREXPRESSION OCCURRED WITH THE HIGHER FREQUENCY IN BLAST CRISIS STAGE (11/11, 100%), LOWER IN ACCELERATED PHASE (3/5, 60%), AND CHRONIC PHASE (42/62, 66%) STAGES. MOREOVER, PAIRED PATIENTS DURING DISEASE PROGRESSION WITH INCREASED BCR-ABL TRANSCRIPT ALSO SHOWED A SIGNIFICANT UPREGULATION OF H19 EXPRESSION. MEANWHILE, H19 EXPRESSION WAS DECREASED IN FOLLOW-UP PATIENTS WHO ACHIEVED COMPLETE MOLECULAR REMISSION AFTER TYROSINE KINASE INHIBITORS-BASED THERAPY. EPIGENETIC STUDIES SHOWED THAT H19 DIFFERENTIALLY METHYLATED REGION/IMPRINTING CONTROL REGION (DMR/ICR) WAS HYPOMETHYLATED AND ASSOCIATED WITH H19 EXPRESSION IN CML PATIENTS. MOREOVER, DEMETHYLATION OF H19 DMR/ICR REACTIVATED H19 EXPRESSION IN K562 CELLS. COLLECTIVELY, H19 OVEREXPRESSION, A FREQUENT EVENT IN CML, WAS ASSOCIATED WITH HIGHER BCR-ABL TRANSCRIPT INVOLVING IN DISEASE PROGRESSION. MOREOVER, H19 DMR/ICR HYPOMETHYLATION IN CML MAY BE ONE OF THE MECHANISMS MEDIATING H19 OVEREXPRESSION.	2018	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     
12 2090  30 EPIGENETIC DYSREGULATION OF THE WNT SIGNALLING PATHWAY IN CHRONIC LYMPHOCYTIC LEUKAEMIA. BACKGROUND: WNT SIGNALLING HAS RECENTLY BEEN IMPLICATED IN THE PATHOGENESIS OF CANCER. METHODS: THIS STUDY INVESTIGATED THE ACTIVITY OF WNT SIGNALLING IN PERIPHERAL BLOOD CHRONIC LYMPHOCYTIC LEUKAEMIA (CLL) LYMPHOCYTES, AND THE METHYLATION STATUS OF SEVEN SOLUBLE WNT ANTAGONIST GENES, INCLUDING WIF1, DKK3, APC, SFRP1, SFRP2, SFRP4 AND SFRP5, BY USING METHYLATION-SPECIFIC PCR IN THE PERIPHERAL BLOOD CLL LYMPHOCYTES AND BONE MARROW SAMPLES OF PATIENTS WITH CLL AT DIAGNOSIS. RESULTS: IN THE PERIPHERAL BLOOD CLL LYMPHOCYTES, CONSTITUTIVE ACTIVATION OF WNT SIGNALLING WAS DETECTED, ASSOCIATED WITH HYPERMETHYLATION OF THE SOLUBLE WNT INHIBITOR GENES. IN THE DIAGNOSTIC CLL MARROW SAMPLES, METHYLATION OF THE SEVEN GENES WAS DETECTED IN UP TO 36.4% OF SAMPLES. MOREOVER, 23 (52.3%) PATIENTS HAD METHYLATION OF AT LEAST ONE OF THE SEVEN GENES, OF WHOM 14 (60.8%) HAD METHYLATION OF TWO OR MORE WNT INHIBITOR GENES. APART FROM AN ASSOCIATION OF ADVANCED AGE WITH DKK3 METHYLATION, THERE WAS NO ASSOCIATION OF GENE HYPERMETHYLATION WITH EITHER CLINICAL CHARACTERISTICS (INCLUDING AGE, GENDER, LYMPHOCYTE COUNT AT DIAGNOSIS, RAI STAGE AND POOR-RISK KARYOTYPE) OR SURVIVAL. CONCLUSION: WNT SIGNALLING IS CONSTITUTIVELY ACTIVATED IN CLL B LYMPHOCYTES IN ASSOCIATION WITH METHYLATION OF MULTIPLE SOLUBLE WNT ANTAGONIST GENES. METHYLATION OF THESE SOLUBLE WNT ANTAGONIST GENES, OCCASIONALLY MULTIPLE GENES, IN PRIMARY CLL MARROW SAMPLES SUGGESTS AN IMPORTANT ROLE IN CLL PATHOGENESIS. MOREOVER, THIS STUDY UNDERSCORED THE IMPORTANCE OF STUDYING METHYLATION OF A PANEL OF, BUT NOT INDIVIDUAL, GENES REGULATING A CELLULAR PATHWAY.	2008	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   
13 1211  37 CPG ISLAND METHYLATION AND EXPRESSION OF THE SECRETED FRIZZLED-RELATED PROTEIN GENE FAMILY IN CHRONIC LYMPHOCYTIC LEUKEMIA. B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) IS CHARACTERIZED BY A CLONAL ACCUMULATION OF MATURE NEOPLASTIC B CELLS INDICATING DISRUPTION OF APOPTOSIS. RESTRICTION LANDMARK GENOME SCANNING WAS DONE TO IDENTIFY NOVEL TARGET GENES SILENCED BY CPG ISLAND METHYLATION IN CLL. SECRETED FRIZZLED-RELATED PROTEIN 4 (SFRP4), A NEGATIVE REGULATOR OF THE WNT SIGNALING PATHWAY, WAS FOUND TO BE FREQUENTLY METHYLATED IN CLL SAMPLES. WNT SIGNALING HAS BEEN SHOWN TO CONTROL NORMAL APOPTOTIC BEHAVIOR AND IS REQUIRED FOR NORMAL B-CELL DEVELOPMENT WHEREAS ABERRANT ACTIVATION OF THIS PATHWAY HAS BEEN OBSERVED IN CLL. WE SHOW ABERRANT DNA METHYLATION AND SILENCING OF SFRP4, AS WELL AS OF ADDITIONAL SFRP FAMILY MEMBERS, IN PRIMARY CLL SAMPLES. INDUCTION OF THEIR EXPRESSION IN A DOSE-DEPENDENT MANNER FOLLOWING TREATMENT WITH A DEMETHYLATING AGENT, 5-AZA-2'-DEOXYCYTIDINE, WAS SHOWN. OF THE FIVE SFRP FAMILY MEMBERS STUDIED IN DETAIL, SFRP1 WAS HYPERMETHYLATED AND DOWN-REGULATED IN ALL CLL PATIENT SAMPLES STUDIED, SUGGESTING THAT THIS EPIGENETIC EVENT IS A CRITICAL STEP DURING LEUKEMOGENESIS. OUR RESULTS SUGGEST THAT SILENCING OF SFRPS BY CPG ISLAND METHYLATION IS ONE POSSIBLE MECHANISM CONTRIBUTING TO ABERRANT ACTIVATION OF WNT SIGNALING PATHWAY IN CLL.	2006	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        
14 2379  35 EPIGENETIC REGULATION OF WNT PATHWAY ANTAGONISTS IN HUMAN GLIOBLASTOMA MULTIFORME. EPIGENETIC INACTIVATION OF TUMOR SUPPRESSOR GENES IS COMMON IN HUMAN CANCER. USING A LARGE-SCALE WHOLE-GENOME APPROACH IN AN EARLIER STUDY, THE AUTHORS IDENTIFIED EPIGENETICALLY SILENCED GENES WITH POTENTIAL TUMOR SUPPRESSOR FUNCTION IN GLIOBLASTOMA (GBM). THREE GENES IDENTIFIED IN THIS ANALYSIS-DKK1, SFRP1, AND WIF1-ARE POTENT INHIBITORS OF THE WNT SIGNAL TRANSDUCTION PATHWAY. HERE, THE AUTHORS CONFIRM DECREASED EXPRESSION OF THESE GENES IN GBM TUMOR TISSUE SAMPLES RELATIVE TO NONTUMOR BRAIN TISSUE SAMPLES USING REAL-TIME PCR. THEY THEN SHOW THAT EXPRESSION OF ALL 3 GENES IS RESTORED IN T98 GBM CELLS BY TREATMENT WITH THE HISTONE DEACETYLASE INHIBITOR TRICHOSTATIN A (TSA), BUT ONLY DKK1 EXPRESSION IS RESTORED BY TREATMENT WITH THE DEMETHYLATING AGENT 5-AZACYTIDINE. BISULFITE SEQUENCING DID NOT REVEAL SIGNIFICANT METHYLATION IN THE PROMOTER REGION OF DKK1, WHEREAS HISTONE ACETYLATION AND CHROMATIN ACCESSIBILITY INCREASED SIGNIFICANTLY FOR ALL 3 GENES AFTER TSA TREATMENT. ECTOPIC EXPRESSION OF DKK1 SIGNIFICANTLY REDUCES COLONY FORMATION AND INCREASES CHEMOTHERAPY-INDUCED APOPTOSIS IN T98 CELLS. ECTOPIC EXPRESSION OF THE CANONICAL WNT PATHWAY INHIBITORS WIF1 AND SFRP1 SHOWS A RELATIVE LACK OF RESPONSE. CHRONIC WNT3A STIMULATION ONLY PARTIALLY REVERSES GROWTH SUPPRESSION AFTER DKK1 REEXPRESSION, WHEREAS A SPECIFIC INHIBITOR OF THE JNK PATHWAY SIGNIFICANTLY REVERSES THE EFFECT OF DKK1 REEXPRESSION ON COLONY FORMATION AND APOPTOSIS IN T98 CELLS. THESE RESULTS SUPPORT A POTENTIAL GROWTH-SUPPRESSIVE FUNCTION FOR EPIGENETICALLY SILENCED DKK1 IN GBM AND SUGGEST THAT DKK1 RESTORATION COULD MODULATE WNT SIGNALING THROUGH BOTH CANONICAL AND NONCANONICAL PATHWAYS.	2010	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              
15 1613  40 DNA METHYLTRANSFERASE 1 MEDIATED ABERRANT METHYLATION AND SILENCING OF SHP-1 GENE IN CHRONIC MYELOGENOUS LEUKEMIA CELLS. INTRODUCTION: EXTENSIVE STUDIES ON SHP-1 PROTEIN AND SHP-1 MRNA REVEALED THAT THE DIMINISHMENT OR ABOLISHMENT OF THE EXPRESSION OF SHP-1 IN LEUKEMIAS/LYMPHOMAS WAS DUE TO ABERRANT PROMOTER METHYLATION. THUS FAR, THE MECHANISM OF EPIGENETIC SILENCING OF THE SHP-1 TYROSINE PHOSPHATASE GENE THAT OCCURS IN CHRONIC MYELOGENOUS LEUKEMIA CELLS REMAINS POORLY UNDERSTOOD. METHODS: THE EXPRESSIONS OF THE TARGET MOLECULES WERE DETERMINED BY QUANTITATIVE REAL TIME PCR AND WESTERN BLOT, RESPECTIVELY. BISULFITE SEQUENCING PCR WAS USED TO DETECT METHYLATION STATUS OF DNA CPG. THE LENTIVIRAL VECTORS WERE APPLIED TO MODIFY GENE EXPRESSION. RESULTS: IN THE PRESENT STUDY, WE FOUND THAT THE PROMOTER 2 OF SHP-1 GENE IS LOCATED BETWEEN POSITIONS FROM -577BP TO +300BP, AND 22 CPG SITES CONTAINED IN POSITIONS -353BP APPROXIMATELY +182BP ARE ABERRANTLY METHYLATED IN K562 CELLS. IN VITRO, WE DEMONSTRATED THAT DNMT1 SILENCING INDUCED DEMETHYLATION OF THE 22 CPG SITES LOCATED IN THE SHP-1 PROMOTER AND RE-EXPRESSION OF SHP-1 GENE IN K562 CELLS. MOREOVER, WE PROVED THAT THE EXPRESSION LEVELS OF DNMT1 AND SHP-1 MRNA AND PROTEIN WERE NEGATIVELY CORRELATED IN K562 CELLS AND BM ASPIRATES MONONUCLEAR CELLS FROM CML PATIENTS. CONCLUSION: COLLECTIVELY, THESE RESULTS INDICATE THAT DNMT1 MEDIATES ABERRANT METHYLATION AND SILENCING OF SHP-1 GENE IN CHRONIC MYELOGENOUS LEUKEMIA CELLS, AND PROVIDE A NOVEL THERAPEUTIC TARGET FOR CML.	2017	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 
16 3532  37 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	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      
17 1968  31 EPIGENETIC ALTERATION OF THE SOCS1 GENE IN CHRONIC MYELOID LEUKAEMIA. THE EXPRESSION OF THE SUPPRESSOR OF CYTOKINE SIGNALLING-1 (SOCS1) PROTEIN IS INDUCED IN RESPONSE TO STIMULATION BY SEVERAL CYTOKINES. THE INDUCED SOCS1 INHIBITS THE SIGNALLING PATHWAY THROUGH THE ASSOCIATION WITH A VARIETY OF TYROSINE KINASE PROTEINS. IN THIS STUDY, THE MUTATION ANALYSES, CPG ISLAND METHYLATION STATUS, AND THE EXPRESSION OF THE SOCS1 GENE IN 112 CHRONIC MYELOID LEUKAEMIA (CML) SAMPLES, FIVE LEUKAEMIA CELL LINES, AND 30 NORMAL CONTROLS WERE ANALYSED. NO GENETIC MUTATIONS OF SOCS1 GENE WERE NOTED IN THE CML SAMPLES. THE SOCS1 GENE WAS HYPERMETHYLATED IN 67% AND 46% OF THE BLASTIC AND CHRONIC PHASE CML SAMPLES RESPECTIVELY (P < 0.0001). HOWEVER, THERE WAS NO METHYLATION OF THE SOCS1 GENE IN NORMAL CONTROLS OR CML IN MOLECULAR REMISSION. THE METHYLATION STATUS OF THE SOCS1 GENE IS CONSISTENT WITH THE RESULTS OF THE REAL-TIME QUANTITATIVE REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION AND IMMUNOCYTOCHEMISTRY STAINING. OUR RESULTS DEMONSTRATE THAT THE SOCS1 GENE SILENCING IS CAUSED BY THE METHYLATION OF CPG ISLANDS IN CML AND IS REVERSED TO AN UNMETHYLATED STATUS IN MOLECULAR REMISSION. AS SOCS1 HAS UNIVERSAL ACTIVITY TO NEGATIVELY REGULATE SEVERAL CYTOKINE SIGNALLING PATHWAYS, THE LOSS OF THE NEGATIVE REGULATION OF CYTOKINE SIGNALLING BY THE SOCS1 MAY PLAY A ROLE IN THE PATHOGENESIS OF CML PROGRESSION.	2003	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  
18 2081  42 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	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 
19 3147  33 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	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             
20 1272  43 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