1 6529 123 TRANSCRIPTIONAL DOWN-REGULATION OF THE WNT ANTAGONIST SFRP1 IN HAEMATOPOIETIC CELLS OF PATIENTS WITH DIFFERENT RISK TYPES OF MDS. SECRETED FRIZZLED RELATED PROTEIN 1 (SFRP1) IS AN EXTRACELLULAR ANTAGONIST OF THE WNT SIGNALLING PATHWAY THAT PLAYS AN IMPORTANT ROLE IN THE PATHOGENESIS OF SOLID TUMOURS AND HAEMATOPOIETIC MALIGNANCIES. SFRP1 HAS BEEN OBSERVED TO BE TRANSCRIPTIONALLY DOWN-REGULATED DUE TO HYPERMETHYLATION IN ACUTE AND CHRONIC LEUKAEMIA, BUT SO FAR NOT IN MYELODYSPLASTIC SYNDROME (MDS). MOREOVER, IT HAS BEEN SHOWN THAT THE EPIGENETIC INACTIVATION OF SFRP1 CORRELATES WITH AN OVEREXPRESSION OF THE WNT RECEPTOR FRIZZLED 3 (FZD3) IN ACUTE LEUKAEMIA. USING REAL-TIME QUANTITATIVE REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION (RT-PCR) WE EXAMINED MRNA EXPRESSION OF SFRP1 AND FZD3 IN BONE MARROW CELLS DERIVED FROM 121 PATIENTS WITH DIFFERENT RISK TYPES OF MDS, ACUTE MYELOID LEUKAEMIA (AML) AND ACUTE LYMPHOBLASTIC LEUKAEMIA (ALL). WE EMPLOYED PYROSEQUENCING TO QUANTIFY PROMOTER DNA METHYLATION IN MDS AND ACUTE LEUKAEMIA. WE DETECTED SIGNIFICANT LOWER MRNA TRANSCRIPTION OF SFRP1 IN MDS COMPARED TO HEALTHY INDIVIDUALS. HOWEVER, DNA SEQUENCE MUTATIONS OR FREQUENT ELEVATED DNA METHYLATION LEVELS OF THE SFRP1 PROMOTER COULD NOT BE OBSERVED IN MDS BUT IN AML AND ALL AS PREVIOUSLY REPORTED. THE EXPRESSION LEVELS OF FZD3 WERE UP-REGULATED IN BOTH ACUTE LEUKAEMIA AND MDS. OUR DATA SHOW A SIGNIFICANT TRANSCRIPTIONAL DOWN-REGULATION OF SFRP1 AS A COMMON EVENT IN AML, ALL AND - AS DEMONSTRATED FOR THE FIRST TIME - IN MDS. AN INACTIVATION OF SFRP1 AND THE TRANSCRIPTIONAL UP-REGULATION OF FZD3 SEEM TO BE ASSOCIATED WITH AN ACTIVATION OF THE WNT SIGNALLING PATHWAY IN THESE HAEMATOPOIETIC DISEASES. 2010 2 1211 35 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 3 3444 39 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 4 18 31 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 5 2090 38 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 6 1968 38 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 7 2435 40 EPIGENETIC SILENCING OF SFRP1 ACTIVATES THE CANONICAL WNT PATHWAY AND CONTRIBUTES TO INCREASED CELL GROWTH AND PROLIFERATION IN HEPATOCELLULAR CARCINOMA. THE WNT PATHWAY IS A KEY REGULATOR OF EMBRYONIC DEVELOPMENT AND STEM CELLS, AND ITS ABERRANT ACTIVATION IS ASSOCIATED WITH HUMAN MALIGNANCIES, MOST NOTABLY HEPATOCELLULAR CARCINOMA (HCC). EPIGENETIC DEREGULATION OF THE GENES ENCODING THE SECRETED FRIZZLED-RELATED PROTEINS (SFRPS), THE WNT SIGNALLING ANTAGONISTS, HAS BEEN LINKED WITH ABERRANT HYPERACTIVATION OF THE WNT SIGNALLING IN HCC CELLS; HOWEVER, THE PRECISE UNDERLYING MECHANISM REMAINS ELUSIVE. WE INVESTIGATED THE METHYLATION PROFILES OF WNT ANTAGONISTS IN LIVER SAMPLES OF DIFFERENT STAGES OF HCC DEVELOPMENT AND LIVER CANCER CELL LINES AND STUDIED THE FUNCTIONAL IMPACT OF ABERRANT EPIGENETIC SILENCING OF SFRPS ON THE CANONICAL WNT PATHWAY AND CELL VIABILITY. WE FOUND THAT THE SFRP1 GENE ENCODING THE SUBUNIT IS A FREQUENT TARGET OF ABERRANT DNA HYPERMETHYLATION AND SILENCING IN HCC TUMOURS, WHEREAS OTHER EXTRACELLULAR WNT ANTAGONISTS, WIF1 AND DKK3, EXHIBITED NO METHYLATION IN TUMOUR CELLS, CONSISTENT WITH THE NOTION THAT ABERRANT METHYLATION EVENTS IN CANCER CELLS ARE NON-RANDOMLY DISTRIBUTED AMONG THE GENES AND THAT THERE IS A STRONG PREFERENCE FOR HYPERMETHYLATION OF SPECIFIC GENES IN HCC. IN ADDITION, BY COMPARING SFRP1 METHYLATION STATUS IN HCC TUMOURS WITH NORMAL, CIRRHOTIC AND CHRONIC HEPATITIS LIVER TISSUES, WE IDENTIFIED SFRP1 GENE AS A POTENTIAL EARLY MARKER OF HCC. THE RESTORATION OF SFRP1 EXPRESSION IN CANCER CELLS BY ECTOPIC EXPRESSION INHIBITED WNT ACTIVITY ACCOMPANIED WITH DESTABILIZATION OF BETA-CATENIN AND DOWNREGULATION OF C-MYC AND CYCLIN D1, THE KNOWN DOWNSTREAM TARGETS OF WNT PATHWAY. IMPORTANTLY, RESTORING SFRP1 LEVELS IN CANCER CELLS INHIBITED CELL GROWTH AND INDUCED APOPTOTIC CELL DEATH. THIS STUDY SUPPORTS THE CRITICAL ROLE FOR SFRP1 SILENCING IN HEPATOCELLULAR CARCINOMA AND REINFORCES THE IMPORTANCE OF THE WNT ANTAGONISTS IN PREVENTING ONCOGENIC STABILIZATION OF BETA-CATENIN AND CHRONIC ACTIVATION OF THE CANONICAL WNT PATHWAY, SUGGESTING THAT SFRP1 MAY BE AN ATTRACTIVE TARGET FOR EARLY CANCER DETECTION AND THERAPEUTIC INTERVENTION. 2012 8 5669 33 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 9 1146 47 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 10 149 36 ABERRANT HYDROXYMETHYLATION IN PROMOTER CPG REGIONS OF GENES RELATED TO THE CELL CYCLE AND APOPTOSIS CHARACTERIZES ADVANCED CHRONIC MYELOID LEUKEMIA DISEASE, POOR IMATINIB RESPONDENTS AND POOR SURVIVAL. BACKGROUND: THERE IS STRONG EVIDENCE THAT DISEASE PROGRESSION, DRUG RESPONSE AND OVERALL CLINICAL OUTCOMES OF CML DISEASE ARE NOT ONLY DECIDED BY BCR/ABL1 ONCOPROTEIN BUT DEPEND ON ACCUMULATION OF ADDITIONAL GENETIC AND EPIGENETIC ABERRATIONS. DNA HYDROXYMETHYLATION IS IMPLICATED IN THE DEVELOPMENT OF VARIETY OF DISEASES. DNA HYDROXYMETHYLATION IN GENE PROMOTERS PLAYS IMPORTANT ROLES IN DISEASE PROGRESSION, DRUG RESPONSE AND CLINICAL OUTCOME OF VARIOUS DISEASES. THEREFORE IN THIS STUDY, WE AIMED TO EXPLORE THE ROLE OF ABERRANT HYDROXYMETHYLATION IN PROMOTER REGIONS OF DIFFERENT TUMOR SUPPRESSOR GENES IN RELATION TO CML DISEASE PROGRESSION, RESPONSE TO IMATINIB THERAPY AND CLINICAL OUTCOME. METHODS: WE RECRUITED 150 CML PATIENTS AT DIFFERENT CLINICAL STAGES OF THE DISEASE. PATIENTS WERE FOLLOWED UP FOR 48 MONTHS AND HAEMATOLOGICAL/MOLECULAR RESPONSES WERE ANALYSED. HAEMATOLOGICAL RESPONSE WAS ANALYSED BY PERIPHERAL BLOOD SMEAR. BCR/ABL1 SPECIFIC TAQMAN PROBE BASED QRT-PCR WAS USED FOR ASSESSING THE MOLECULAR RESPONSE OF CML PATIENTS ON IMATINIB THERAPY. PROMOTER HYDROXYMETHYLATION OF THE GENES WAS CHARACTERIZED USING MS-PCR. RESULTS: WE OBSERVED THAT PROMOTER HYDROXYMETHYLATION OF DAPK1, RIZ1, P16INK4A, RASSF1A AND P14ARF(ARF) GENES CHARACTERIZE ADVANCED CML DISEASE AND POOR IMATINIB RESPONDENTS. ALTHOUGH, CYTOKINE SIGNALLING (SOCS1) GENE WAS HYPERMETHYLATED IN ADVANCED STAGES OF CML AND ACCUMULATED IN PATIENTS WITH POOR IMATINIB RESPONSE, BUT THE DIFFERENCES WERE NOT STATISTICALLY SIGNIFICANT. MOREOVER, WE FOUND HYPERMETHYLATION OF P14(ARF), RASSF1 AND P16(INK4A) GENES AND CYTOKINE SIGNALLING GENE (SOCS1) SIGNIFICANTLY ASSOCIATED WITH POOR OVERALL SURVIVAL OF CML PATIENTS ON IMATINIB THERAPY. THE RESULTS OF THIS STUDY ARE IN AGREEMENT OF THE ROLE OF ABERRANT DNA METHYLATION OF DIFFERENT TUMOR SUPPRESSOR GENES AS POTENTIAL BIOMARKERS OF CML DISEASE PROGRESSION, POOR IMATINIB RESPONSE AND OVERALL CLINICAL OUTCOME. CONCLUSION: IN THIS STUDY, WE REPORT THAT PROMOTER HYDROXYMETHYLATION OF DAPK1, RIZ1, P16INK4A, RASSF1A AND P14ARF(ARF) GENES IS A CHARACTERISTIC FEATURE OF CML DISEASE PROGRESSIONS, DEFINES POOR IMATINIB RESPONDENTS AND POOR OVERALL SURVIVAL OF CML PATIENTS TO IMATINIB THERAPY. 2022 11 206 32 ACTIVATION OF NOTCH AND MYC SIGNALING VIA B-CELL-RESTRICTED DEPLETION OF DNMT3A GENERATES A CONSISTENT MURINE MODEL OF CHRONIC LYMPHOCYTIC LEUKEMIA. CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) IS CHARACTERIZED BY DISORDERED DNA METHYLATION, SUGGESTING THESE EPIGENETIC CHANGES MIGHT PLAY A CRITICAL ROLE IN DISEASE ONSET AND PROGRESSION. THE METHYLTRANSFERASE DNMT3A IS A KEY REGULATOR OF DNA METHYLATION. ALTHOUGH DNMT3A SOMATIC MUTATIONS IN CLL ARE RARE, WE FOUND THAT LOW DNMT3A EXPRESSION IS ASSOCIATED WITH MORE AGGRESSIVE DISEASE. A CONDITIONAL KNOCKOUT MOUSE MODEL SHOWED THAT HOMOZYGOUS DEPLETION OF DNMT3A FROM B CELLS RESULTS IN THE DEVELOPMENT OF CLL WITH 100% PENETRANCE AT A MEDIAN AGE OF ONSET OF 5.3 MONTHS, AND HETEROZYGOUS DNMT3A DEPLETION YIELDS A DISEASE PENETRANCE OF 89% WITH A MEDIAN ONSET AT 18.5 MONTHS, CONFIRMING ITS ROLE AS A HAPLOINSUFFICIENT TUMOR SUPPRESSOR. B1A CELLS WERE CONFIRMED AS THE CELL OF ORIGIN OF DISEASE IN THIS MODEL, AND DNMT3A DEPLETION RESULTED IN FOCAL HYPOMETHYLATION AND ACTIVATION OF NOTCH AND MYC SIGNALING. AMPLIFICATION OF CHROMOSOME 15 CONTAINING THE MYC GENE WAS DETECTED IN ALL CLL MICE TESTED, AND INFILTRATION OF HIGH-MYC-EXPRESSING CLL CELLS IN THE SPLEEN WAS OBSERVED. NOTABLY, HYPERACTIVATION OF NOTCH AND MYC SIGNALING WAS EXCLUSIVELY OBSERVED IN THE DNMT3A CLL MICE, BUT NOT IN THREE OTHER CLL MOUSE MODELS TESTED (SF3B1-ATM, IKZF3, AND MDR), AND DNMT3A-DEPLETED CLL WERE SENSITIVE TO PHARMACOLOGIC INHIBITION OF NOTCH SIGNALING IN VITRO AND IN VIVO. CONSISTENT WITH THESE FINDINGS, HUMAN CLL SAMPLES WITH LOWER DNMT3A EXPRESSION WERE MORE SENSITIVE TO NOTCH INHIBITION THAN THOSE WITH HIGHER DNMT3A EXPRESSION. ALTOGETHER, THESE RESULTS SUGGEST THAT DNMT3A DEPLETION INDUCES CLL THAT IS HIGHLY DEPENDENT ON ACTIVATION OF NOTCH AND MYC SIGNALING. SIGNIFICANCE: LOSS OF DNMT3A EXPRESSION IS A DRIVING EVENT IN CLL AND IS ASSOCIATED WITH AGGRESSIVE DISEASE, ACTIVATION OF NOTCH AND MYC SIGNALING, AND ENHANCED SENSITIVITY TO NOTCH INHIBITION. 2021 12 5668 35 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 13 3098 38 GENOMIC DISRUPTION OF THE HISTONE METHYLTRANSFERASE SETD2 IN CHRONIC LYMPHOCYTIC LEUKAEMIA. HISTONE METHYLTRANSFERASES (HMTS) ARE IMPORTANT EPIGENETIC REGULATORS OF GENE TRANSCRIPTION AND ARE DISRUPTED AT THE GENOMIC LEVEL IN A SPECTRUM OF HUMAN TUMOURS INCLUDING HAEMATOLOGICAL MALIGNANCIES. USING HIGH-RESOLUTION SINGLE NUCLEOTIDE POLYMORPHISM (SNP) ARRAYS, WE IDENTIFIED RECURRENT DELETIONS OF THE SETD2 LOCUS IN 3% (8/261) OF CHRONIC LYMPHOCYTIC LEUKAEMIA (CLL) PATIENTS. FURTHER VALIDATION IN TWO INDEPENDENT COHORTS SHOWED THAT SETD2 DELETIONS WERE ASSOCIATED WITH LOSS OF TP53, GENOMIC COMPLEXITY AND CHROMOTHRIPSIS. WITH NEXT-GENERATION SEQUENCING WE DETECTED MUTATIONS OF SETD2 IN AN ADDITIONAL 3.8% OF PATIENTS (23/602). IN MOST CASES, SETD2 DELETIONS OR MUTATIONS WERE OFTEN OBSERVED AS A CLONAL EVENT AND ALWAYS AS A MONO-ALLELIC LESION, LEADING TO REDUCED MRNA EXPRESSION IN SETD2-DISRUPTED CASES. PATIENTS WITH SETD2 ABNORMALITIES AND WILD-TYPE TP53 AND ATM FROM FIVE CLINICAL TRIALS EMPLOYING CHEMOTHERAPY OR CHEMO-IMMUNOTHERAPY HAD REDUCED PROGRESSION-FREE AND OVERALL SURVIVAL COMPARED WITH CASES WILD TYPE FOR ALL THREE GENES. CONSISTENT WITH ITS POSTULATED ROLE AS A TUMOUR SUPPRESSOR, OUR DATA HIGHLIGHT SETD2 ABERRATION AS A RECURRENT, EARLY LOSS-OF-FUNCTION EVENT IN CLL PATHOBIOLOGY LINKED TO AGGRESSIVE DISEASE. 2016 14 4243 40 METHYLATION STATUS OF CEBPA GENE PROMOTER IN CHRONIC MYELOID LEUKEMIA. CCAAT/ENHANCER BINDING PROTEIN ALPHA IS ONE OF THE CRUCIAL TRANSCRIPTION FACTORS FOR MYELOID CELL DEVELOPMENT THAT HAS BEEN FOUND TO BE INVOLVED IN HEMATOPOIETIC DIFFERENTIATION AND LEUKEMIOGENESIS. RECENTLY, EPIGENETIC REGULATION OF CEBPA EXPRESSION THROUGH DNA METHYLATION HAS BEEN DEMONSTRATED IN LEUKEMIA. THE AIM OF THIS STUDY WAS TO INVESTIGATE THE METHYLATION STATUS OF CEBPA GENE IN CHRONIC MYELOID LEUKEMIA (CML) PATIENTS. THE METHYLATION STATUS OF CEBPA PROMOTER WAS STUDIED IN 100 PATIENTS WITH CML AND 98 NORMAL HEALTHY INDIVIDUALS FROM HYDERABAD, INDIA, USING METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION. THE ABERRANT METHYLATION OF CEBPA GENE PROMOTER WAS FOUND IN 32 OF THE 100 CML CASES. A HIGHLY SIGNIFICANT ASSOCIATION WAS FOUND BETWEEN THE FREQUENCY OF CEBPA GENE PROMOTER HYPERMETHYLATION AND THE CML STAGES (P = 0.017), BUT ASSOCIATION WITH RESPECT TO AGE AND GENDER OF THE PATIENT WAS NOT FOUND. THE RESULTS SUGGEST THAT ABERRANT METHYLATION IN THE CPG ISLAND OF THE PROMOTER REGION OF THIS GENE MIGHT BE A COMMON EVENT IN CML, AND SYSTEMIC EXPRESSION STUDIES WILL BE NEEDED TO UNFOLD THE ROLE OF CEBPA PROMOTER METHYLATION IN THE DEVELOPMENT, PROGRESSION, AND PROGNOSIS OF CML. 2014 15 59 40 A GENOME-WIDE SCREEN IDENTIFIES FREQUENTLY METHYLATED GENES IN HAEMATOLOGICAL AND EPITHELIAL CANCERS. BACKGROUND: GENETIC AS WELL AS EPIGENETIC ALTERATIONS ARE A HALLMARK OF BOTH EPITHELIAL AND HAEMATOLOGICAL MALIGNANCIES. HIGH THROUGHPUT SCREENS ARE REQUIRED TO IDENTIFY EPIGENETIC MARKERS THAT CAN BE USEFUL FOR DIAGNOSTIC AND PROGNOSTIC PURPOSES ACROSS MALIGNANCIES. RESULTS: HERE WE REPORT FOR THE FIRST TIME THE USE OF THE MIRA ASSAY (METHYLATED CPG ISLAND RECOVERY ASSAY) IN COMBINATION WITH GENOME-WIDE CPG ISLAND ARRAYS TO IDENTIFY EPIGENETIC MOLECULAR MARKERS IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) ON A GENOME-WIDE SCALE. WE IDENTIFIED 30 GENES DEMONSTRATING METHYLATION FREQUENCIES OF > OR =25% IN CHILDHOOD ALL, NINE GENES SHOWED SIGNIFICANTLY DIFFERENT METHYLATION FREQUENCIES IN B VS T-ALL. FOR MAJORITY OF THE GENES EXPRESSION COULD BE RESTORED IN METHYLATED LEUKEMIA LINES AFTER TREATMENT WITH 5-AZADC. FORTY-FOUR PERCENT OF THE GENES REPRESENT TARGETS OF THE POLYCOMB COMPLEX. IN CHRONIC MYELOID LEUKEMIA (CML) TWO OF THE GENES, (TFAP2A AND EBF2), DEMONSTRATED INCREASED METHYLATION IN BLAST CRISIS COMPARED TO CHRONIC PHASE (P < 0.05). FURTHERMORE HYPERMETHYLATION OF AN AUTOPHAGY RELATED GENE ATG16L2 WAS ASSOCIATED WITH POORER PROGNOSIS IN TERMS OF MOLECULAR RESPONSE TO IMATINIB TREATMENT. LASTLY WE DEMONSTRATED THAT TEN OF THESE GENES WERE ALSO FREQUENTLY METHYLATED IN COMMON EPITHELIAL CANCERS. CONCLUSION: IN SUMMARY WE HAVE IDENTIFIED A LARGE NUMBER OF GENES SHOWING FREQUENT METHYLATION IN CHILDHOOD ALL, METHYLATION STATUS OF TWO OF THESE GENES IS ASSOCIATED WITH ADVANCED DISEASE IN CML AND METHYLATION STATUS OF ANOTHER GENE IS ASSOCIATED WITH PROGNOSIS. IN ADDITION A SUBSET OF THESE GENES MAY ACT AS EPIGENETIC MARKERS ACROSS HEMATOLOGICAL MALIGNANCIES AS WELL AS COMMON EPITHELIAL CANCERS. 2010 16 825 31 CHARACTERIZATION OF FUNCTIONAL TRANSPOSABLE ELEMENT ENHANCERS IN ACUTE MYELOID LEUKEMIA. TRANSPOSABLE ELEMENTS (TES) HAVE BEEN SHOWN TO HAVE IMPORTANT GENE REGULATORY FUNCTIONS AND THEIR ALTERATION COULD LEAD TO DISEASE PHENOTYPES. ACUTE MYELOID LEUKEMIA (AML) DEVELOPS AS A CONSEQUENCE OF A SERIES OF GENETIC CHANGES IN HEMATOPOIETIC PRECURSOR CELLS, INCLUDING MUTATIONS IN EPIGENETIC FACTORS. HERE, WE SET OUT TO STUDY THE GENE REGULATORY ROLE OF TES IN AML. WE FIRST EXPLORED THE EPIGENETIC LANDSCAPE OF TES IN AML PATIENTS USING ATAC-SEQ DATA. WE SHOW THAT A LARGE NUMBER OF TES IN GENERAL, AND MORE SPECIFICALLY MAMMALIAN-WIDE INTERSPERSED REPEATS (MIRS), ARE MORE ENRICHED IN AML CELLS THAN IN NORMAL BLOOD CELLS. WE OBTAINED A SIMILAR FINDING WHEN ANALYZING HISTONE MODIFICATION DATA IN AML PATIENTS. GENE ONTOLOGY ENRICHMENT ANALYSIS SHOWED THAT GENES NEAR MIRS IN OPEN CHROMATIN REGIONS ARE INVOLVED IN LEUKEMOGENESIS. TO FUNCTIONALLY VALIDATE THEIR REGULATORY ROLE, WE SELECTED 19 MIR REGIONS IN AML CELLS, AND TESTED THEM FOR ENHANCER ACTIVITY IN AN AML CELL LINE (KASUMI-1) AND A CHRONIC MYELOID LEUKEMIA (CML) CELL LINE (K562); THE RESULTS REVEALED SEVERAL MIRS TO BE FUNCTIONAL ENHANCERS. TAKEN TOGETHER, OUR RESULTS SUGGEST THAT TES ARE POTENTIALLY INVOLVED IN MYELOID LEUKEMOGENESIS AND HIGHLIGHT THESE SEQUENCES AS POTENTIAL CANDIDATES HARBORING AML-ASSOCIATED VARIATION. 2020 17 1669 37 DOWNREGULATION OF THE HISTONE METHYLTRANSFERASE SETD2 PROMOTES IMATINIB RESISTANCE IN CHRONIC MYELOID LEUKAEMIA CELLS. OBJECTIVES: EPIGENETIC MODIFIERS WERE IMPORTANT PLAYERS IN THE DEVELOPMENT OF HAEMATOLOGICAL MALIGNANCIES AND SENSITIVITY TO THERAPY. MUTATIONS OF SET DOMAIN-CONTAINING 2 (SETD2), A METHYLTRANSFERASE THAT CATALYSES THE TRIMETHYLATION OF HISTONE 3 ON LYSINE 36 (H3K36ME3), WERE FOUND IN VARIOUS MYELOID MALIGNANCIES. HOWEVER, THE DETAILED MECHANISMS THROUGH WHICH SETD2 CONFERS CHRONIC MYELOID LEUKAEMIA PROGRESSION AND RESISTANCE TO THERAPY TARGETING ON BCR-ABL REMAIN UNCLEAR. MATERIALS AND METHODS: THE LEVEL OF SETD2 IN IMATINIB-SENSITIVE AND IMATINIB-RESISTANT CHRONIC MYELOID LEUKAEMIA (CML) CELLS WAS EXAMINED BY IMMUNOBLOTTING AND QUANTITATIVE REAL-TIME PCR. WE ANALYSED CD34(+) CD38(-) LEUKAEMIC STEM CELLS BY FLOW CYTOMETRY AND COLONY FORMATION ASSAYS UPON SETD2 KNOCKDOWN OR OVEREXPRESSION. THE IMPACT OF SETD2 EXPRESSION ALTERATIONS OR SMALL-MOLECULE INHIBITOR JIB-04 TARGETING H3K36ME3 LOSS ON IMATINIB SENSITIVITY WAS ASSESSED BY IC50, CELL APOPTOSIS AND PROLIFERATION ASSAYS. FINALLY, RNA SEQUENCING AND CHIP-QUANTITATIVE PCR WERE PERFORMED TO VERIFY PUTATIVE DOWNSTREAM TARGETS. RESULTS: SETD2 WAS FOUND TO ACT AS A TUMOUR SUPPRESSOR IN CML. THE NOVEL ONCOGENIC TARGETS MYCN AND ERG WERE SHOWN TO BE THE DIRECT DOWNSTREAM TARGETS OF SETD2, WHERE THEIR OVEREXPRESSION INDUCED BY SETD2 KNOCKDOWN CAUSED IMATINIB INSENSITIVITY AND LEUKAEMIC STEM CELL ENRICHMENT IN CML CELL LINES. TREATMENT WITH JIB-04, AN INHIBITOR THAT RESTORES H3K36ME3 LEVELS THROUGH BLOCKADE OF ITS DEMETHYLATION, SUCCESSFULLY IMPROVED THE CELL IMATINIB SENSITIVITY AND ENHANCED THE CHEMOTHERAPEUTIC EFFECT. CONCLUSIONS: OUR STUDY NOT ONLY EMPHASIZES THE REGULATORY MECHANISM OF SETD2 IN CML, BUT ALSO PROVIDES PROMISING THERAPEUTIC STRATEGIES FOR OVERCOMING THE IMATINIB RESISTANCE IN PATIENTS WITH CML. 2019 18 535 30 ASXL1 MUTATION CORRECTION BY CRISPR/CAS9 RESTORES GENE FUNCTION IN LEUKEMIA CELLS AND INCREASES SURVIVAL IN MOUSE XENOGRAFTS. RECURRENT SOMATIC MUTATIONS OF THE EPIGENETIC MODIFIER AND TUMOR SUPPRESSOR ASXL1 ARE COMMON IN MYELOID MALIGNANCIES, INCLUDING CHRONIC MYELOID LEUKEMIA (CML), AND ARE ASSOCIATED WITH POOR CLINICAL OUTCOME. CRISPR/CAS9 HAS RECENTLY EMERGED AS A POWERFUL AND VERSATILE GENOME EDITING TOOL FOR GENOME ENGINEERING IN VARIOUS SPECIES. WE HAVE USED THE CRISPR/CAS9 SYSTEM TO CORRECT THE ASXL1 HOMOZYGOUS NONSENSE MUTATION PRESENT IN THE CML CELL LINE KBM5, WHICH LACKS ASXL1 PROTEIN EXPRESSION. CRISPR/CAS9-MEDIATED ASXL1 HOMOZYGOUS CORRECTION RESULTED IN PROTEIN RE-EXPRESSION WITH RESTORED NORMAL FUNCTION, INCLUDING DOWN-REGULATION OF POLYCOMB REPRESSIVE COMPLEX 2 TARGET GENES. SIGNIFICANTLY REDUCED CELL GROWTH AND INCREASED MYELOID DIFFERENTIATION WERE OBSERVED IN ASXL1 MUTATION-CORRECTED CELLS, PROVIDING NEW INSIGHTS INTO THE ROLE OF ASXL1 IN HUMAN MYELOID CELL DIFFERENTIATION. MICE XENOGRAFTED WITH MUTATION-CORRECTED KBM5 CELLS SHOWED SIGNIFICANTLY LONGER SURVIVAL THAN UNCORRECTED XENOGRAFTS. THESE RESULTS SHOW THAT THE SOLE CORRECTION OF A DRIVER MUTATION IN LEUKEMIA CELLS INCREASES SURVIVAL IN VIVO IN MICE. THIS STUDY PROVIDES PROOF-OF-CONCEPT FOR DRIVER GENE MUTATION CORRECTION VIA CRISPR/CAS9 TECHNOLOGY IN HUMAN LEUKEMIA CELLS AND PRESENTS A STRATEGY TO ILLUMINATE THE IMPACT OF ONCOGENIC MUTATIONS ON CELLULAR FUNCTION AND SURVIVAL. 2015 19 160 34 ABERRANT PROMOTER HYPOMETHYLATION IN CLL: DOES IT MATTER FOR DISEASE DEVELOPMENT? OVER THE LAST 30 YEARS, STUDIES OF ABERRANT DNA METHYLATION IN HEMATOLOGIC MALIGNANCIES HAVE BEEN DOMINATED BY THE PRIMARY FOCUS OF UNDERSTANDING PROMOTER HYPERMETHYLATION. THESE EFFORTS NOT ONLY RESULTED IN A BETTER UNDERSTANDING OF THE BASIS OF EPIGENETIC SILENCING OF TUMOR SUPPRESSOR GENES BUT ALSO RESULTED IN APPROVAL OF HYPOMETHYLATING AGENTS FOR THE TREATMENT OF SEVERAL MALIGNANCIES, SUCH AS MYELODYSPLASTIC SYNDROME AND ACUTE MYELOID LEUKEMIA. RECENT ADVANCES IN GLOBAL METHYLATION PROFILING COUPLED WITH THE USE OF MOUSE MODELS SUGGEST THAT ABERRANT PROMOTER HYPOMETHYLATION IS ALSO A FREQUENT EVENT IN HEMATOLOGIC MALIGNANCIES, PARTICULARLY IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). PROMOTER HYPOMETHYLATION AFFECTS GENE EXPRESSION AND, THEREFORE, MAY PLAY AN IMPORTANT ROLE IN DISEASE PATHOGENESIS. HERE, WE REVIEW RECENT FINDINGS AND DISCUSS THE POTENTIAL INVOLVEMENT OF ABERRANT PROMOTER HYPOMETHYLATION IN CLL. 2016 20 151 40 ABERRANT METHYLATION AND IMPAIRED EXPRESSION OF THE P15(INK4B) CELL CYCLE REGULATORY GENE IN CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML). THE IMPORTANT CELL CYCLE REGULATORY GENE P15(INK4B) HAS BEEN SHOWN TO BE INACTIVATED IN ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROME. LITTLE IS KNOWN ABOUT THE EXPRESSION AND EPIGENETIC MODIFICATION OF THIS GENE IN CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML) THAT BELONGS TO THE MYELODYSPLASTIC/MYELOPROLIFERATIVE DISORDERS (MDS/MPD) WITH A HIGH PROPORTION OF BLASTIC TRANSFORMATION. ANALYSIS OF BONE MARROW TREPHINES IN A SERIES OF 33 CMML CASES SHOWED AN ABERRANT P15(INK4B) GENE METHYLATION IN UP TO 58% OF CASES. METHYLATION WAS ANALYZED EMPLOYING DIFFERENT METHYLATION-SPECIFIC PCR AND GENOMIC SEQUENCING PROTOCOLS. IT TURNED OUT TO BE SPREAD OVER A BROAD AREA OF THE 5' REGION AND EXHIBITED SUBSTANTIAL HETEROGENEITY BETWEEN CASES AND EVEN IN INDIVIDUAL PATIENTS. THE DEGREE OF ABERRANT METHYLATION WAS CORRELATED WITH A REDUCED MRNA AS WELL AS REDUCED PROTEIN EXPRESSION, AND WAS ASSOCIATED WITH A HIGHER EXPRESSION OF DNA METHYLTRANSFERASE DNMT 3A. WE CONCLUDE THAT ABERRANT GENE METHYLATION IS A FREQUENT EVENT IN CMML THAT MIGHT CONTRIBUTE TO THE PATHOGENESIS OF THIS MDS/MPD. 2003