1 6404 123 THE RS1001179 SNP AND CPG METHYLATION REGULATE CATALASE EXPRESSION IN CHRONIC LYMPHOCYTIC LEUKEMIA. CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) IS AN INCURABLE DISEASE CHARACTERIZED BY AN EXTREMELY VARIABLE CLINICAL COURSE. WE HAVE RECENTLY SHOWN THAT HIGH CATALASE (CAT) EXPRESSION IDENTIFIES PATIENTS WITH AN AGGRESSIVE CLINICAL COURSE. ELUCIDATING MECHANISMS REGULATING CAT EXPRESSION IN CLL IS PREEMINENT TO UNDERSTAND DISEASE MECHANISMS AND DEVELOP STRATEGIES FOR IMPROVING ITS CLINICAL MANAGEMENT. IN THIS STUDY, WE INVESTIGATED THE ROLE OF THE CAT PROMOTER RS1001179 SINGLE NUCLEOTIDE POLYMORPHISM (SNP) AND OF THE CPG ISLAND II METHYLATION ENCOMPASSING THIS SNP IN THE REGULATION OF CAT EXPRESSION IN CLL. LEUKEMIC CELLS HARBORING THE RS1001179 SNP T ALLELE EXHIBITED A SIGNIFICANTLY HIGHER CAT EXPRESSION COMPARED WITH CELLS BEARING THE CC GENOTYPE. CAT PROMOTER HARBORING THE T -BUT NOT C- ALLELE WAS ACCESSIBLE TO ETS-1 AND GR-BETA TRANSCRIPTION FACTORS. MOREOVER, CLL CELLS EXHIBITED LOWER METHYLATION LEVELS THAN NORMAL B CELLS, IN LINE WITH THE HIGHER CAT MRNA AND PROTEIN EXPRESSED BY CLL IN COMPARISON WITH NORMAL B CELLS. METHYLATION LEVELS AT SPECIFIC CPG SITES NEGATIVELY CORRELATED WITH CAT LEVELS IN CLL CELLS. INHIBITION OF METHYLTRANSFERASE ACTIVITY INDUCED A SIGNIFICANT INCREASE IN CAT LEVELS, THUS FUNCTIONALLY VALIDATING THE ROLE OF CPG METHYLATION IN REGULATING CAT EXPRESSION IN CLL. FINALLY, THE CT/TT GENOTYPES WERE ASSOCIATED WITH LOWER METHYLATION AND HIGHER CAT LEVELS, SUGGESTING THAT THE RS1001179 T ALLELE AND CPG METHYLATION MAY INTERACT IN REGULATING CAT EXPRESSION IN CLL. THIS STUDY IDENTIFIES GENETIC AND EPIGENETIC MECHANISMS UNDERLYING DIFFERENTIAL EXPRESSION OF CAT, WHICH COULD BE OF CRUCIAL RELEVANCE FOR THE DEVELOPMENT OF THERAPIES TARGETING REDOX REGULATORY PATHWAYS IN CLL.	2022	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                
2 2753  36 EXPRESSION OF BCL2L12 IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS: ASSOCIATION WITH CLINICAL AND MOLECULAR PROGNOSTIC MARKERS. DYSREGULATION OF APOPTOSIS IS A DISTINCTIVE FEATURE OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL), ALTHOUGH A UNIQUE MECHANISM UNDERLYING APOPTOSIS RESISTANCE OF CLL B LYMPHOCYTES HAS NOT BEEN IDENTIFIED YET. ABERRANT EXPRESSION AS WELL AS GENETIC AND EPIGENETIC ALTERATIONS OF NUMEROUS GENES INVOLVED IN DIFFERENT PATHWAYS OF APOPTOSIS REGULATION HAS BEEN DESCRIBED IN CLL. HERE, WE REPORT THE EXPRESSION ANALYSIS OF BCL2L12 (BCL2-LIKE 12), A NOVEL APOPTOTIC GENE BELONGING TO BCL2 FAMILY, IN 58 SERBIAN CLL PATIENTS. QUANTITATIVE REVERSE-TRANSCRIPTASE POLYMERASE CHAIN REACTION (QRT-PCR) ANALYSIS REVEALED A SIGNIFICANT OVEREXPRESSION OF BCL2L12 MRNA IN CLL SAMPLES COMPARED TO NON-LEUKEMIC SAMPLES, IMPLYING ITS ROLE IN THE PATHOGENESIS OF THE DISEASE. RECEIVER OPERATING CHARACTERISTIC (ROC) ANALYSIS SHOWED THAT BCL2L12 EXPRESSION EFFICIENTLY DISCRIMINATES CLL CASES FROM HEALTHY CONTROLS. HOWEVER, RELATIVELY HOMOGENOUS BCL2L12 MRNA EXPRESSION AMONG PATIENTS DID NOT REFLECT THEIR CLINICAL CHARACTERISTICS (WITH THE EXCEPTION OF LACTATE DEHYDROGENASE STATUS AND TIME FROM DIAGNOSIS TO TREATMENT) AND FAILED TO SHOW ASSOCIATION WITH THE MOST INFORMATIVE PROGNOSTIC MARKERS, NAMELY THE MUTATIONAL STATUS OF REARRANGED IMMUNOGLOBULIN HEAVY CHAIN VARIABLE REGION GENES, CD38 AND LIPOPROTEIN LIPASE GENE (LPL) EXPRESSION.	2013	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         
3 5871  40 SUSTAINED NF-KAPPAB ACTIVITY IN CHRONIC LYMPHOCYTIC LEUKEMIA IS INDEPENDENT OF GENETIC AND EPIGENETIC ALTERATIONS IN THE TNFAIP3 (A20) LOCUS. INAPPROPRIATE NUCLEAR FACTOR (NF) KAPPAB ACTIVITY IS ONE MAJOR HALLMARK OF B-CELL MALIGNANCIES AND CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). NFKAPPAB-DEPENDENT GENES ARE INVOLVED IN ANTIAPOPTOSIS, CELL PROLIFERATION AND METASTASIS AND ARE RESPONSIBLE FOR SURVIVAL AND PROLIFERATION OF TUMORS. HOWEVER, THE MECHANISMS OF NFKAPPAB ACTIVITY IN CLL STILL NEED TO BE ELUCIDATED. PREVIOUSLY, WE IDENTIFIED TRANSLOCATIONS IN A REGION ON CHROMOSOME 6Q THAT ENCODES TUMOR NECROSIS FACTOR ALPHA-INDUCED PROTEIN 3, WHICH IS A KEY PLAYER IN NEGATIVE FEEDBACK LOOP REGULATION OF NFKAPPAB. INACTIVATION OF THIS UBIQUITIN-EDITING ENZYME IS INVOLVED IN IMMUNOPATHOLOGIES AND IN TUMORIGENESIS. FREQUENT MUTATIONS IN THE A20 LOCUS--LEADING TO SUSTAINED NFKAPPAB ACTIVITY--COULD BE SHOWN TO PLAY A DOMINANT ROLE IN DEVELOPMENT OF DIFFERENT B-CELL MALIGNANCIES. TO CHECK IF A20 IS INVOLVED IN UPREGULATION OF NFKAPPAB ACTIVITY IN CLL, WE SEQUENCED EXONS 2-9 OF THE A20 GENE IN 55 CLL DNA SAMPLES. FURTHERMORE, WE DETERMINED THE METHYLATION STATUS OF THE PROMOTER REGION IN 63 CLL DNA SAMPLES AND COMPARED TO 10 CONTROL DNAS OF B CELLS FROM HEALTHY DONORS. CONTRARY TO REPORTS FROM OTHER B-CELL MALIGNANCIES, THE A20 REGION SHOWED NEITHER MUTATIONS NOR ABERRANT DNA METHYLATION. MOREOVER, ITS EXPRESSION COULD BE CONFIRMED BY IMMUNOBLOTTING AND SHOWING COMPARABLE RESULTS TO HEALTHY B CELLS. THESE RESULTS INDICATE THAT MALIGNANT DEVELOPMENT IN CLL DIFFERS FROM MOST OF OTHER B-CELL MALIGNANCIES, WHICH SHOW FREQUENT INACTIVATION OF A20.	2011	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                   
4 2025  33 EPIGENETIC CHANGES DURING DISEASE PROGRESSION IN A MURINE MODEL OF HUMAN CHRONIC LYMPHOCYTIC LEUKEMIA. EPIGENETIC ALTERATIONS, INCLUDING GAIN OR LOSS OF DNA METHYLATION, ARE A HALLMARK OF NEARLY EVERY MALIGNANCY. CHANGES IN DNA METHYLATION CAN IMPACT EXPRESSION OF CANCER-RELATED GENES INCLUDING APOPTOSIS REGULATORS AND TUMOR SUPPRESSORS. BECAUSE SUCH EPIGENETIC CHANGES ARE REVERSIBLE, THEY ARE BEING AGGRESSIVELY INVESTIGATED AS POTENTIAL THERAPEUTIC TARGETS. HERE WE USE THE EMU-TCL1 TRANSGENIC MOUSE MODEL OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) TO DETERMINE THE TIMING AND PATTERNS OF ABERRANT DNA METHYLATION, AND TO INVESTIGATE THE MECHANISMS THAT LEAD TO ABERRANT DNA METHYLATION. WE SHOW THAT CLL CELLS FROM EMU-TCL1 MICE AT VARIOUS STAGES RECAPITULATE EPIGENETIC ALTERATIONS SEEN IN HUMAN CLL. ABERRANT METHYLATION OF PROMOTER SEQUENCES IS OBSERVED AS EARLY AS 3 MONTHS OF AGE IN THESE ANIMALS, WELL BEFORE DISEASE ONSET. ABNORMALLY METHYLATED PROMOTER REGIONS INCLUDE BINDING SITES FOR THE TRANSCRIPTION FACTOR FOXD3. WE SHOW THAT LOSS OF FOXD3 EXPRESSION DUE TO AN NF-KAPPAB P50/P50:HDAC1 REPRESSOR COMPLEX OCCURS IN TCL1-POSITIVE B CELLS BEFORE METHYLATION. THEREFORE, SPECIFIC TRANSCRIPTIONAL REPRESSION IS AN EARLY EVENT LEADING TO EPIGENETIC SILENCING OF TARGET GENES IN MURINE AND HUMAN CLL. THESE RESULTS PROVIDE STRONG RATIONALE FOR THE DEVELOPMENT OF STRATEGIES TO TARGET NF-KAPPAB COMPONENTS IN CLL AND POTENTIALLY OTHER B-CELL MALIGNANCIES.	2009	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  
5 1995  42 EPIGENETIC AND GENETIC ALTERATIONS AND THEIR INFLUENCE ON GENE REGULATION IN CHRONIC LYMPHOCYTIC LEUKEMIA. BACKGROUND: TO UNDERSTAND THE CHANGES OF GENE REGULATION IN CARCINOGENESIS, WE EXPLORED SIGNALS OF DNA METHYLATION - A STABLE EPIGENETIC MARK OF GENE REGULATORY ELEMENTS - AND DESIGNED A COMPUTATIONAL MODEL TO PROFILE LOSS AND GAIN OF REGULATORY ELEMENTS (RES) DURING CARCINOGENESIS. WE ALSO UTILIZED SEQUENCING DATA TO ANALYZE THE ALLELE FREQUENCY OF SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS) AND DETECTED THE CANCER-ASSOCIATED SNPS, I.E., THE SNPS DISPLAYING THE SIGNIFICANT ALLELE FREQUENCY DIFFERENCE BETWEEN CANCER AND NORMAL SAMPLES. RESULTS: AFTER APPLYING THIS MODEL TO CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) DATA, WE IDENTIFIED RES DIFFERENTIALLY ACTIVATED (DRES) BETWEEN NORMAL AND CLL CELLS, CONSISTING OF 6,802 DRES GAINED AND 4,606 DRES LOST IN CLL. THE IDENTIFIED REGULATORY PERTURBATIONS COINCIDE WITH CHANGES IN THE EXPRESSION OF TARGET GENES. IN PARTICULAR, THE GENES ENCODING DNA METHYLTRANSFERASES HARBOR MULTIPLE LOST-IN-CANCER DRES AND ZERO GAINED-IN-CANCER DRES, INDICATING THAT THE DAMAGED REGULATION OF THESE GENES MIGHT BE ONE OF THE KEY CAUSES OF TUMOR FORMATION. DRES DISPLAY A SIGNIFICANTLY ELEVATED DENSITY OF THE GENOME-WIDE ASSOCIATION STUDY (GWAS) SNPS ASSOCIATED WITH CLL AND CLL-RELATED TRAITS. WE OBSERVED THAT MOST OF DRE GWAS SNPS ASSOCIATED WITH CLL AND CLL-RELATED TRAITS (83%) DISPLAY A SIGNIFICANT HAPLOTYPE ASSOCIATION AMONG THE IDENTIFIED CANCER-ASSOCIATED ALLELES AND THE RISK ALLELES THAT HAVE BEEN REPORTED IN GWAS. ALSO DRES ARE ENRICHED FOR THE BINDING SITES OF THE WELL-ESTABLISHED B-CELL AND CLL TRANSCRIPTION FACTORS (TFS) NF-KB, AP2, P53, E2F1, PAX5, AND SP1. WE ALSO IDENTIFIED CLL-ASSOCIATED SNPS AND DEMONSTRATED THAT THE MUTATIONS AT THESE SNPS CHANGE THE BINDING SITES OF KEY TFS MUCH MORE FREQUENTLY THAN EXPECTED. CONCLUSIONS: THROUGH EXPLORING SEQUENCING DATA MEASURING DNA METHYLATION, WE IDENTIFIED THE EPIGENETIC ALTERATIONS (MORE SPECIFICALLY, DNA METHYLATION) AND GENETIC MUTATIONS ALONG NON-CODING GENOMIC REGIONS CLL, AND DEMONSTRATED THAT THESE CHANGES PLAY A CRITICAL ROLE IN CARCINOGENESIS THROUGH DAMAGING THE REGULATION OF KEY GENES AND ALTERNATING THE BINDING OF KEY TFS IN B AND CLL CELLS.	2017	
                                                                                                                                                                                                                                                                                                                                                                                                                                                
6 3098  36 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	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                
7 3147  39 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	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    
8 1144  38 CONCOMITANT HETEROCHROMATINISATION AND DOWN-REGULATION OF GENE EXPRESSION UNVEILS EPIGENETIC SILENCING OF RELB IN AN AGGRESSIVE SUBSET OF CHRONIC LYMPHOCYTIC LEUKEMIA IN MALES. BACKGROUND: THE SENSITIVITY OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) CELLS TO CURRENT TREATMENTS, BOTH IN VITRO AND IN VIVO, RELIES ON THEIR ABILITY TO ACTIVATE APOPTOTIC DEATH. CLL CELLS RESISTANT TO DNA DAMAGE-INDUCED APOPTOSIS DISPLAY DEREGULATION OF A SPECIFIC SET OF GENES. METHODS: MICROARRAY HYBRIDIZATION (HUMAN GENECHIP, AFFYMETRIX), IMMUNOFLUORESCENT IN SITU LABELING COUPLED WITH VIDEO-MICROSCOPY RECORDING/ANALYSES, CHROMATIN-IMMUNOPRECIPITATION (CHIP), POLYMERASE CHAIN REACTIONS (PCR), REAL-TIME QUANTITATIVE PCR (RT-QPCR) AND BISULFITE GENOME SEQUENCING WERE THE MAIN METHODS APPLIED. STATISTICAL ANALYSES WERE PERFORMED BY APPLYING GCRMA AND SAM ANALYSIS (MICROARRAY DATA) AND STUDENT'S T-TEST OR MANN & WHITNEY'S U-TEST. RESULTS: HEREIN WE SHOW THAT, REMARKABLY, IN A RESISTANT MALE CLL CELLS THE VAST MAJORITY OF GENES WERE DOWN-REGULATED COMPARED WITH SENSITIVE CELLS, WHEREAS THIS WAS NOT THE CASE IN CELLS DERIVED FROM FEMALES. THIS GENE DOWN-REGULATION WAS FOUND TO BE ASSOCIATED WITH AN OVERALL GAIN OF HETEROCHROMATIN AS EVIDENCED BY IMMUNOFLUORESCENT LABELING OF HETEROCHROMATIN PROTEIN 1ALPHA (HP-1), TRIMETHYLATED HISTONE 3 LYSINE 9 (3METH3K9), AND 5-METHYLCYTIDINE (5METC). NOTABLY, 17 GENES WERE FOUND TO BE COMMONLY DEREGULATED IN RESISTANT MALE AND FEMALE CELL SAMPLES. AMONG THESE, RELB WAS IDENTIFIED AS A DISCRIMINATORY CANDIDATE GENE REPRESSED IN THE MALE AND UPREGULATED IN THE FEMALE RESISTANT CELLS. CONCLUSION: THE MOLECULAR DEFECTS IN THE SILENCING OF RELB INVOLVE AN INCREASE IN H3K9- BUT NOT CPG-ISLAND METHYLATION IN THE PROMOTER REGIONS. INCREASE IN ACETYL-H3 IN RESISTANT FEMALE BUT NOT MALE CLL SAMPLES AS WELL AS A DECREASE OF TOTAL CELLULAR LEVEL OF RELB AFTER AN INHIBITION OF HISTONE DEACETYLASE (HDAC) BY TRICHOSTATIN A (TSA), FURTHER EMPHASIZE THE ROLE OF EPIGENETIC MODIFICATIONS WHICH COULD DISCRIMINATE TWO CLL SUBSETS. TOGETHER, THESE RESULTS HIGHLIGHTED THE EPIGENETIC RELB SILENCING AS A NEW MARKER OF THE PROGRESSIVE DISEASE IN MALES.	2010	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                
9 4221  44 METHYLATION AND SILENCING OF PROTEIN TYROSINE PHOSPHATASE RECEPTOR TYPE O IN CHRONIC LYMPHOCYTIC LEUKEMIA. PURPOSE: PREVIOUS STUDIES IN OUR LABORATORY HAVE SHOWN THE PROGRESSIVE METHYLATION AND SUPPRESSION OF THE GENE ENCODING PROTEIN TYROSINE PHOSPHATASE, PTPRO, IN THE LIVERS OF RATS FED A METHYL-DEFICIENT DIET THAT INDUCES HEPATOCARCINOGENESIS. SUBSEQUENTLY, WE OBSERVED THE METHYLATION OF PTPRO IN PRIMARY HUMAN LUNG TUMORS AND ALSO SHOWED ITS POTENTIAL TUMOR SUPPRESSOR CHARACTERISTICS. THE PRESENT STUDY WAS UNDERTAKEN TO INVESTIGATE WHETHER THE TRUNCATED FORM OF PTPRO (PTPROT), SPECIFICALLY EXPRESSED IN NAIVE B LYMPHOCYTES, WAS ALSO METHYLATED AND SUPPRESSED IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL), A DISEASE GENERALLY AFFECTING B LYMPHOCYTES. EXPERIMENTAL DESIGN AND RESULTS: INITIAL SCREENING SHOWED THAT 60% OF THE 52 CLL SAMPLES ANALYZED USING METHYLATION-SPECIFIC PCR ASSAY WERE METHYLATED COMPARED WITH B LYMPHOCYTES FROM NORMAL INDIVIDUALS, WHICH WERE NOT METHYLATED. THE EXPRESSION OF PTPROT, AS MEASURED BY SEMIQUANTITATIVE REVERSE TRANSCRIPTION-PCR, INVERSELY CORRELATED WITH METHYLATION IN THE FEW SAMPLES TESTED. ANALYSIS OF ADDITIONAL SAMPLES (N = 50) BY COMBINED BISULFITE RESTRICTION ANALYSIS SHOWED THAT THE PTPRO CPG ISLAND WAS METHYLATED IN 82% OF PATIENTS WITH CLL COMPARED WITH B LYMPHOCYTES FROM NORMAL INDIVIDUALS. FURTHERMORE, OVERALL EXPRESSION OF PTPRO WAS REDUCED IN CLL RELATIVE TO NORMAL LYMPHOCYTES. THE PTPRO GENE WAS ALSO SUPPRESSED BY METHYLATION IN THE CLL CELL LINE WAC3CD5, WHERE IT COULD BE REACTIVATED UPON TREATMENT WITH THE DNA HYPOMETHYLATING AGENT 5-AZAC. ECTOPIC EXPRESSION OF PTPROT IN A NONEXPRESSING CELL LINE INCREASED GROWTH INHIBITION WITH FLUDARABINE TREATMENT, A THERAPY COMMONLY USED FOR CLL. CONCLUSION: THIS STUDY REVEALS THE POTENTIAL ROLE OF PTPRO METHYLATION AND SILENCING IN CLL TUMORIGENESIS AND ALSO PROVIDES A NOVEL MOLECULAR TARGET IN THE EPIGENETIC THERAPY.	2007	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               
10 3062  42 GENOME-WIDE DNA METHYLATION ANALYSIS REVEALS NOVEL EPIGENETIC CHANGES IN CHRONIC LYMPHOCYTIC LEUKEMIA. WE CONDUCTED A GENOME-WIDE DNA METHYLATION ANALYSIS IN CD19 (+) B-CELLS FROM CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) PATIENTS AND NORMAL CONTROL SAMPLES USING REDUCED REPRESENTATION BISULFITE SEQUENCING (RRBS). THE METHYLATION STATUS OF 1.8-2.3 MILLION CPGS IN THE CLL GENOME WAS DETERMINED; ABOUT 45% OF THESE CPGS WERE LOCATED IN MORE THAN 23,000 CPG ISLANDS (CGIS). WHILE GLOBAL CPG METHYLATION WAS SIMILAR BETWEEN CLL AND NORMAL B-CELLS, 1764 GENE PROMOTERS WERE IDENTIFIED AS BEING DIFFERENTIALLY METHYLATED IN AT LEAST ONE CLL SAMPLE WHEN COMPARED WITH NORMAL B-CELL SAMPLES. NINETEEN PERCENT OF THE DIFFERENTIALLY METHYLATED GENES WERE INVOLVED IN TRANSCRIPTIONAL REGULATION. ABERRANT HYPERMETHYLATION WAS FOUND IN ALL HOX GENE CLUSTERS AND A SIGNIFICANT NUMBER OF WNT SIGNALING PATHWAY GENES. HYPOMETHYLATION OCCURRED MORE FREQUENTLY IN THE GENE BODY INCLUDING INTRONS, EXONS, AND 3'-UTRS IN CLL. THE NFATC1 P2 PROMOTER AND FIRST INTRON WAS FOUND TO BE HYPOMETHYLATED AND CORRELATED WITH UPREGULATION OF BOTH NFATC1 RNA AND PROTEIN EXPRESSION LEVELS IN CLL SUGGESTING THAT AN EPIGENETIC MECHANISM IS INVOLVED IN THE CONSTITUTIVE ACTIVATION OF NFAT ACTIVITY IN CLL CELLS. THIS COMPREHENSIVE DNA METHYLATION ANALYSIS WILL FURTHER OUR UNDERSTANDING OF THE EPIGENETIC CONTRIBUTION TO CELLULAR DYSFUNCTION IN CLL.	2012	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                
11 6684  33 VALIDATION OF AN LC-MS BASED APPROACH FOR PROFILING HISTONES IN CHRONIC LYMPHOCYTIC LEUKEMIA. THE IN VITRO EVALUATION OF HISTONES AND THEIR PTMS HAS DRAWN SUBSTANTIAL INTEREST IN THE DEVELOPMENT OF EPIGENETIC THERAPIES. THE DIFFERENTIAL EXPRESSION OF HISTONE ISOFORMS MAY SERVE AS A POTENTIAL MARKER IN THE CLASSIFICATION OF DISEASES AFFECTED BY CHROMATIN ABNORMALITIES. IN THIS STUDY, PROTEIN PROFILING BY LC AND MS WAS USED TO EXPLORE DIFFERENCES IN HISTONE COMPOSITION IN PRIMARY CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) CELLS. EXTENSIVE METHOD VALIDATIONS WERE PERFORMED TO DETERMINE THE EXPERIMENTAL VARIANCES THAT WOULD IMPACT HISTONE RELATIVE ABUNDANCE. THE RESULTING DATA DEMONSTRATED THAT THE PROPOSED METHODOLOGY WAS SUITABLE FOR THE ANALYSIS OF HISTONE PROFILES. IN 4 NORMAL INDIVIDUALS AND 40 CLL PATIENTS, A SIGNIFICANT DECREASE IN THE RELATIVE ABUNDANCE OF HISTONE H2A VARIANTS (H2AFL AND H2AFA/M*) WAS OBSERVED IN PRIMARY CLL CELLS AS COMPARED TO NORMAL B CELLS. PROTEIN IDENTITIES WERE DETERMINED USING HIGH MASS ACCURACY MS AND SHOTGUN PROTEOMICS.	2009	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    
12 3532  43 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	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            
13 3455  47 HYPOMETHYLATION COORDINATES ANTAGONISTICALLY WITH HYPERMETHYLATION IN CANCER DEVELOPMENT: A CASE STUDY OF LEUKEMIA. BACKGROUND: METHYLATION CHANGES ARE FREQUENT IN CANCERS, BUT UNDERSTANDING HOW HYPER- AND HYPOMETHYLATED REGION CHANGES COORDINATE, ASSOCIATE WITH GENOMIC FEATURES, AND AFFECT GENE EXPRESSION IS NEEDED TO BETTER UNDERSTAND THEIR BIOLOGICAL SIGNIFICANCE. THE FUNCTIONAL SIGNIFICANCE OF HYPERMETHYLATION IS WELL STUDIED, BUT THAT OF HYPOMETHYLATION REMAINS LIMITED. HERE, WITH PAIRED EXPRESSION AND METHYLATION SAMPLES GATHERED FROM A PATIENT/CONTROL COHORT, WE ATTEMPT TO BETTER CHARACTERIZE THE GENE EXPRESSION AND METHYLATION CHANGES THAT TAKE PLACE IN CANCER FROM B CELL CHRONIC LYMPHOCYTE LEUKEMIA (B-CLL) SAMPLES. RESULTS: ACROSS THE DATASET, WE FOUND THAT CONSISTENT DIFFERENTIALLY HYPOMETHYLATED REGIONS (C-DMRS) ACROSS SAMPLES WERE RELATIVELY FEW COMPARED TO THE MANY POORLY CONSISTENT HYPO- AND HIGHLY CONSERVED HYPER-DMRS. HOWEVER, GENES IN THE HYPO-C-DMRS TENDED TO BE ASSOCIATED WITH FUNCTIONS ANTAGONISTIC TO THOSE IN THE HYPER-C-DMRS, LIKE DIFFERENTIATION, CELL-CYCLE REGULATION AND PROLIFERATION, SUGGESTING COORDINATED REGULATION OF METHYLATION CHANGES. HYPO-C-DMRS IN B-CLL WERE FOUND ENRICHED IN KEY SIGNALING PATHWAYS LIKE B CELL RECEPTOR AND P53 PATHWAYS AND GENES/MOTIFS ESSENTIAL FOR B LYMPHOPOIESIS. HYPO-C-DMRS TENDED TO BE PROXIMAL TO GENES WITH ELEVATED EXPRESSION IN CONTRAST TO THE TRANSCRIPTION SILENCING-MECHANISM IMPOSED BY HYPERMETHYLATION. HYPO-C-DMRS TENDED TO BE ENRICHED IN THE REGIONS OF ACTIVATING H4K4ME1/2/3, H3K79ME2, AND H3K27AC HISTONE MODIFICATIONS. IN COMPARISON, THE POLYCOMB REPRESSIVE COMPLEX 2 (PRC2) SIGNATURE, MARKED BY EZH2, SUZ12, CTCF BINDING-SITES, REPRESSIVE H3K27ME3 MARKS, AND "REPRESSED/POISED PROMOTER" STATES WERE ASSOCIATED WITH HYPER-C-DMRS. MOST HYPO-C-DMRS WERE FOUND IN INTRONS (36 %), 3' UNTRANSLATED REGIONS (29 %), AND INTERGENIC REGIONS (24 %). MANY OF THESE GENIC REGIONS ALSO OVERLAPPED WITH ENHANCERS. THE METHYLATION OF CPGS FROM 3'UTR EXONS WAS FOUND TO HAVE WEAK BUT POSITIVE CORRELATION WITH GENE EXPRESSION. IN CONTRAST, METHYLATION IN THE 5'UTR WAS NEGATIVELY CORRELATED WITH EXPRESSION. TO BETTER CHARACTERIZE THE OVERLAP BETWEEN METHYLATION AND EXPRESSION CHANGES, WE IDENTIFIED CORRELATION MODULES THAT ASSOCIATE WITH "APOPTOSIS" AND "LEUKOCYTE ACTIVATION". CONCLUSIONS: DESPITE CLINICAL HETEROGENEITY IN DISEASE PRESENTATION, A NUMBER OF METHYLATION CHANGES, BOTH HYPO AND HYPER, APPEAR TO BE COMMON IN B-CLL. HYPOMETHYLATION APPEARS TO PLAY AN ACTIVE, TARGETED, AND COMPLEMENTARY ROLE IN CANCER PROGRESSION, AND IT INTERPLAYS WITH HYPERMETHYLATION IN A COORDINATED FASHION IN THE CANCER PROCESS.	2016	

14 2747  39 EXPRESSION ANALYSIS OF THE EPIGENETIC METHYLTRANSFERASES AND METHYL-CPG BINDING PROTEIN FAMILIES IN THE NORMAL B-CELL AND B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). THE IMPORTANCE OF EPIGENETIC MODIFICATIONS IN CARCINOGENESIS HAS BEEN A SOURCE OF CONTROVERSY FOR SOME TIME. THERE IS LITTLE DOUBT THAT CHANGES IN GENOMIC HYPERMETHYLATION CONTRIBUTE TO THE SILENCING OF TUMOR SUPPRESSOR GENES. FURTHERMORE, RECENT STUDIES HAVE ALSO IDENTIFIED THE SIGNIFICANCE OF GENOMIC HYPOMETHYLATION ASSOCIATED WITH CHROMOSOMAL INSTABILITY AND TUMORIGENESIS. ONE OF THE MOST PERPLEXING QUESTIONS REGARDING EPIGENETIC MODIFICATIONS AND LEUKEMOGENESIS IS THE RELATIONSHIP WITH DNA METHYLTRANSFERASES (DNMT'S). THE PRIMARY FUNCTION OF THE DNMT ENZYMES IS TO METHYLATE GENOMIC DNA, WHEREAS THE METHYL-CPG BINDING DOMAIN PROTEINS (MBD) INTERPRET THIS METHYLATION SIGNAL AND REGULATE GENE EXPRESSION AND CHROMATIN BEHAVIOR. IN THIS STUDY WE ANALYSE THESE GENE FAMILIES BY QUANTITATIVE REAL-TIME PCR TO INVESTIGATE WHETHER EXPRESSION LEVELS AND THE B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA (B-CLL) PHENOTYPE ARE ASSOCIATED. FURTHERMORE, GIVEN THE EPIGENETIC CROSSTALK BETWEEN GENOME STABILITY AND THE HISTONE CHROMATIN CODE WE HAVE ANALYSED EUKARYOTIC HISTONE METHYLTRANSFERASE (EU-HMTASEI). SURPRISINGLY, WE DID NOT OBSERVE SIGNIFICANT CHANGES IN DNMT1 EXPRESSION IN B-CLL CASES WHEN COMPARED TO NORMAL LYMPHOCYTES, REGARDLESS OF WHETHER WE NORMALISE AGAINST GAPDH OR PCNA AS REFERENCE STANDARDS. INDEED, EXPRESSION OF THE MAINTENANCE AND DE NOVO METHYLASES WERE INDEPENDENTLY REGULATED. OF PARTICULAR NOTE WAS THE SIGNIFICANT DOWN REGULATION OF DNMT3B. FURTHERMORE, WE OBSERVED A POSITIVE CORRELATION BETWEEN HMTASEI EXPRESSION LEVELS AND STAGE OF LEUKEMIA SUGGESTING THAT CHANGES IN THE METHYLATION PATTERNS IN B-CLL MAY REPRESENT DEREGULATION OF THE EPIGENETIC REPERTOIRE THAT ALSO INCLUDE THE METHYLATION DEPENDENT BINDING PROTEINS, MBD2 AND MECP2. WE ENVISAGE CHANGES IN THE EPIGENETIC PROGRAM ARE MULTIFACTORIAL IN NATURE AND POSTULATE THAT THE PREVALENT GENOMIC METHYLASES JUST ONE COMPONENT OF A LARGER EPIGENETIC REPERTOIRE.	2004	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           
15 2262  39 EPIGENETIC PROFILING IN CHRONIC LYMPHOCYTIC LEUKEMIA REVEALS NOVEL METHYLATION TARGETS. CPG ISLAND METHYLATION IS AN EPIGENETIC ALTERATION THAT CONTRIBUTES TO TUMORIGENESIS BY TRANSCRIPTIONAL INACTIVATION OF GENES. LITTLE IS KNOWN ABOUT THE OVERALL LEVELS OF CPG ISLAND METHYLATION IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). TO PROVIDE A BASELINE ESTIMATE OF GLOBAL ABERRANT METHYLATION AND IDENTIFY TARGET SEQUENCES FOR ADDITIONAL INVESTIGATION, WE PERFORMED RESTRICTION LANDMARK GENOMIC SCANNING ON 10 CLL SAMPLES. TWO METHYLATION-SENSITIVE LANDMARK ENZYMES WERE USED (NOTI AND ASCI), ALLOWING ASSESSMENT OF OVER 3000 CPG ISLANDS IN EACH SAMPLE. TUMOR-DERIVED RESTRICTION LANDMARK GENOMIC SCANNING PROFILES WERE COMPARED WITH PROFILES FROM CD19-SELECTED B CELLS FROM NORMAL VOLUNTEERS AND MATCHED NORMAL NEUTROPHILS FROM 4 CLL PATIENTS. WE FOUND 2.5-8.1% (MEAN 4.8%) OF THE CPG ISLANDS IN CLL SAMPLES WERE ABERRANTLY METHYLATED COMPARED WITH CONTROLS, AND THE METHYLATION EVENTS HAD A NONRANDOM DISTRIBUTION (P < 0.0001). FURTHERMORE, WE IDENTIFIED 193 ABERRANTLY METHYLATED SEQUENCES, OF WHICH 93% HAVE CPG ISLAND CHARACTERISTICS AND 90% HAVE HOMOLOGY TO GENES OR EXPRESSED SEQUENCES. ONE SUCH GENE, THE G PROTEIN-COUPLED METABOTROPIC GLUTAMATE RECEPTOR 7 (GRM7), POSSIBLY INHIBITS CYCLIC AMP SIGNALING IN THE INDUCTION OF APOPTOSIS. BISULFITE SEQUENCING OF GRM7 CONFIRMED EXTENSIVE CPG ISLAND METHYLATION, AND TREATMENT WITH 5-AZA-2'-DEOXYCYTIDINE (DECITABINE) RESULTED IN UP-REGULATED EXPRESSION OF SEVERAL GENES IN VITRO WITH CONCURRENT CELLULAR DEPLETION OF DNMT1 PROTEIN. OUR DUAL-ENZYME GLOBAL METHYLATION STUDY SHOWS THAT CLL IS CHARACTERIZED BY WIDESPREAD NONRANDOM CPG ISLAND METHYLATION SIMILAR TO OTHER TUMORS AND PROVIDES A PANEL OF NOVEL METHYLATION TARGETS THAT CAN BE USED IN LARGER STUDIES DESIGNED TO ASSESS IMPACT ON DISEASE PROGRESSION AND SURVIVAL.	2004	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           
16 1424  27 DIFFERENTIAL DNA METHYLATION OF GENE PROMOTERS IN SMALL B-CELL LYMPHOMAS. IMPROVED CARE OF PATIENTS WITH SMALL B-CELL LYMPHOMAS (SBCLS) IS LIKELY TO RESULT FROM THE ONGOING DISCOVERY OF MOLECULAR MARKERS THAT BETTER DEFINE THESE MALIGNANT NEOPLASMS. WE IDENTIFIED MULTIPLE GENE LOCI WHOSE DNA METHYLATION PATTERNS DIFFERED BETWEEN 3 TYPES OF SBCL: B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA/SMALL LYMPHOCYTIC LYMPHOMA, MANTLE CELL LYMPHOMA, AND GRADES I AND II FOLLICULAR LYMPHOMA. THIS ANALYSIS WAS PERFORMED USING AN OLIGONUCLEOTIDE MICROARRAY THAT ALLOWED DETERMINATION OF THE DNA METHYLATION STATUS OF 156 LOCI IN 38 GENES. COMBINED BISULFITE RESTRICTION ANALYSIS AND METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION WERE USED TO VALIDATE THE DIFFERENTIAL METHYLATION OF 6 OF THESE GENES. BY USING NON-HODGKIN LYMPHOMA CELL LINES AS MODELS, THESE GENES WERE EXAMINED FURTHER FOR METHYLATION AND GENE EXPRESSION RELATIONSHIPS. THIS STUDY ILLUSTRATES NONRANDOM EPIGENETIC ALTERATIONS IN SBCLS THAT SEEM TO PREFERENTIALLY INVOLVE LYMPHOMAS OF GERMINAL CENTER DERIVATION.	2005	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                
17  206  41 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	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           
18   59  35 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	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              
19 2966  36 GENETIC AND EPIGENETIC PROFILING OF CLL DISEASE PROGRESSION REVEALS LIMITED SOMATIC EVOLUTION AND SUGGESTS A RELATIONSHIP TO MEMORY-CELL DEVELOPMENT. WE EXAMINED GENETIC AND EPIGENETIC CHANGES THAT OCCUR DURING DISEASE PROGRESSION FROM INDOLENT TO AGGRESSIVE FORMS OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) USING SERIAL SAMPLES FROM 27 PATIENTS. ANALYSIS OF DNA MUTATIONS GROUPED THE LEUKEMIA CASES INTO THREE CATEGORIES: EVOLVING (26%), EXPANDING (26%) AND STATIC (47%). THUS, APPROXIMATELY THREE-QUARTERS OF THE CLL CASES HAD LITTLE TO NO GENETIC SUBCLONAL EVOLUTION. HOWEVER, WE IDENTIFIED SIGNIFICANT RECURRENT DNA METHYLATION CHANGES DURING PROGRESSION AT 4752 CPGS ENRICHED FOR REGIONS NEAR POLYCOMB 2 REPRESSIVE COMPLEX (PRC2) TARGETS. PROGRESSION-ASSOCIATED CPGS NEAR THE PRC2 TARGETS UNDERGO METHYLATION CHANGES IN THE SAME DIRECTION DURING DISEASE PROGRESSION AS DURING NORMAL DEVELOPMENT FROM NAIVE TO MEMORY B CELLS. OUR STUDY SHOWS THAT CLL PROGRESSION DOES NOT TYPICALLY OCCUR VIA SUBCLONAL EVOLUTION, BUT THAT CERTAIN CPG SITES UNDERGO RECURRENT METHYLATION CHANGES. OUR RESULTS SUGGEST CLL PROGRESSION MAY INVOLVE DEVELOPMENTAL PROCESSES SHARED IN COMMON WITH THE GENERATION OF NORMAL MEMORY B CELLS.	2015	
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          
20 3415  36 HSP90 INHIBITION INCREASES SOCS3 TRANSCRIPT AND REGULATES MIGRATION AND CELL DEATH IN CHRONIC LYMPHOCYTIC LEUKEMIA. EPIGENETIC OR TRANSCRIPTIONAL SILENCING OF IMPORTANT TUMOR SUPPRESSORS HAS BEEN DESCRIBED TO CONTRIBUTE TO CELL SURVIVAL AND TUMORIGENESIS IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). USING GENE EXPRESSION MICROARRAY ANALYSIS, WE FOUND THAT THOUSANDS OF GENES ARE REPRESSED MORE THAN 2-FOLD IN CLL COMPARED TO NORMAL B CELLS; HOWEVER THERAPEUTIC APPROACHES TO REVERSE THIS HAVE BEEN LIMITED IN CLL. FOLLOWING TREATMENT WITH THE HSP90 INHIBITOR 17-DMAG, A SIGNIFICANT NUMBER OF THESE REPRESSED GENES WERE SIGNIFICANTLY RE-EXPRESSED. ONE OF THE GENES SIGNIFICANTLY REPRESSED IN CLL AND UP-REGULATED BY 17-DMAG WAS SUPPRESSOR OF CYTOKINE SIGNALING 3, (SOCS3). SOCS3 HAS BEEN SHOWN TO BE SILENCED IN SOLID TUMORS AS WELL AS MYELOID LEUKEMIA; HOWEVER LITTLE IS KNOWN ABOUT THE REGULATION IN CLL. WE FOUND THAT 17-DMAG INDUCES EXPRESSION OF SOCS3 BY VIA THE ACTIVATION OF P38 SIGNALING, AND SUBSEQUENTLY INHIBITS AKT AND STAT3 PHOSPHORYLATION RESULTING IN DOWNSTREAM EFFECTS ON CELL MIGRATION AND SURVIVAL. WE THEREFORE SUGGEST THAT SOCS3 IS AN IMPORTANT SIGNALING PROTEIN IN CLL, AND HSP90 INHIBITORS REPRESENT A NOVEL APPROACH TO TARGET TRANSCRIPTIONAL REPRESSION IN B CELL LYMPHOPROLIFERATIVE DISORDERS WHICH EXHIBIT A SUBSTANTIAL DEGREE OF GENE REPRESSION.	2016