1 3024 149 GENETICS AND PATHOGENESIS OF DIFFUSE LARGE B-CELL LYMPHOMA. BACKGROUND: DIFFUSE LARGE B-CELL LYMPHOMAS (DLBCLS) ARE PHENOTYPICALLY AND GENETICALLY HETEROGENEOUS. GENE-EXPRESSION PROFILING HAS IDENTIFIED SUBGROUPS OF DLBCL (ACTIVATED B-CELL-LIKE [ABC], GERMINAL-CENTER B-CELL-LIKE [GCB], AND UNCLASSIFIED) ACCORDING TO CELL OF ORIGIN THAT ARE ASSOCIATED WITH A DIFFERENTIAL RESPONSE TO CHEMOTHERAPY AND TARGETED AGENTS. WE SOUGHT TO EXTEND THESE FINDINGS BY IDENTIFYING GENETIC SUBTYPES OF DLBCL BASED ON SHARED GENOMIC ABNORMALITIES AND TO UNCOVER THERAPEUTIC VULNERABILITIES BASED ON TUMOR GENETICS. METHODS: WE STUDIED 574 DLBCL BIOPSY SAMPLES USING EXOME AND TRANSCRIPTOME SEQUENCING, ARRAY-BASED DNA COPY-NUMBER ANALYSIS, AND TARGETED AMPLICON RESEQUENCING OF 372 GENES TO IDENTIFY GENES WITH RECURRENT ABERRATIONS. WE DEVELOPED AND IMPLEMENTED AN ALGORITHM TO DISCOVER GENETIC SUBTYPES BASED ON THE CO-OCCURRENCE OF GENETIC ALTERATIONS. RESULTS: WE IDENTIFIED FOUR PROMINENT GENETIC SUBTYPES IN DLBCL, TERMED MCD (BASED ON THE CO-OCCURRENCE OF MYD88(L265P) AND CD79B MUTATIONS), BN2 (BASED ON BCL6 FUSIONS AND NOTCH2 MUTATIONS), N1 (BASED ON NOTCH1 MUTATIONS), AND EZB (BASED ON EZH2 MUTATIONS AND BCL2 TRANSLOCATIONS). GENETIC ABERRATIONS IN MULTIPLE GENES DISTINGUISHED EACH GENETIC SUBTYPE FROM OTHER DLBCLS. THESE SUBTYPES DIFFERED PHENOTYPICALLY, AS JUDGED BY DIFFERENCES IN GENE-EXPRESSION SIGNATURES AND RESPONSES TO IMMUNOCHEMOTHERAPY, WITH FAVORABLE SURVIVAL IN THE BN2 AND EZB SUBTYPES AND INFERIOR OUTCOMES IN THE MCD AND N1 SUBTYPES. ANALYSIS OF GENETIC PATHWAYS SUGGESTED THAT MCD AND BN2 DLBCLS RELY ON "CHRONIC ACTIVE" B-CELL RECEPTOR SIGNALING THAT IS AMENABLE TO THERAPEUTIC INHIBITION. CONCLUSIONS: WE UNCOVERED GENETIC SUBTYPES OF DLBCL WITH DISTINCT GENOTYPIC, EPIGENETIC, AND CLINICAL CHARACTERISTICS, PROVIDING A POTENTIAL NOSOLOGY FOR PRECISION-MEDICINE STRATEGIES IN DLBCL. (FUNDED BY THE INTRAMURAL RESEARCH PROGRAM OF THE NATIONAL INSTITUTES OF HEALTH AND OTHERS.). 2018 2 1693 40 DUSP4 DEFICIENCY CAUSED BY PROMOTER HYPERMETHYLATION DRIVES JNK SIGNALING AND TUMOR CELL SURVIVAL IN DIFFUSE LARGE B CELL LYMPHOMA. THE EPIGENETIC DYSREGULATION OF TUMOR SUPPRESSOR GENES IS AN IMPORTANT DRIVER OF HUMAN CARCINOGENESIS. WE HAVE COMBINED GENOME-WIDE DNA METHYLATION ANALYSES AND GENE EXPRESSION PROFILING AFTER PHARMACOLOGICAL DNA DEMETHYLATION WITH FUNCTIONAL SCREENING TO IDENTIFY NOVEL TUMOR SUPPRESSORS IN DIFFUSE LARGE B CELL LYMPHOMA (DLBCL). WE FIND THAT A CPG ISLAND IN THE PROMOTER OF THE DUAL-SPECIFICITY PHOSPHATASE DUSP4 IS ABERRANTLY METHYLATED IN NODAL AND EXTRANODAL DLBCL, IRRESPECTIVE OF ABC OR GCB SUBTYPE, RESULTING IN LOSS OF DUSP4 EXPRESSION IN 75% OF >200 EXAMINED CASES. THE DUSP4 GENOMIC LOCUS IS FURTHER DELETED IN UP TO 13% OF AGGRESSIVE B CELL LYMPHOMAS, AND THE LACK OF DUSP4 IS A NEGATIVE PROGNOSTIC FACTOR IN THREE INDEPENDENT COHORTS OF DLBCL PATIENTS. ECTOPIC EXPRESSION OF WILD-TYPE DUSP4, BUT NOT OF A PHOSPHATASE-DEFICIENT MUTANT, DEPHOSPHORYLATES C-JUN N-TERMINAL KINASE (JNK) AND INDUCES APOPTOSIS IN DLBCL CELLS. PHARMACOLOGICAL OR DOMINANT-NEGATIVE JNK INHIBITION RESTRICTS DLBCL SURVIVAL IN VITRO AND IN VIVO AND SYNERGIZES STRONGLY WITH THE BRUTON'S TYROSINE KINASE INHIBITOR IBRUTINIB. OUR RESULTS INDICATE THAT DLBCL CELLS DEPEND ON JNK SIGNALING FOR SURVIVAL. THIS FINDING PROVIDES A MECHANISTIC BASIS FOR THE CLINICAL DEVELOPMENT OF JNK INHIBITORS IN DLBCL, IDEALLY IN SYNTHETIC LETHAL COMBINATIONS WITH INHIBITORS OF CHRONIC ACTIVE B CELL RECEPTOR SIGNALING. 2015 3 597 31 BET-BROMODOMAIN AND EZH2 INHIBITOR-TREATED CHRONIC GVHD MICE HAVE BLUNTED GERMINAL CENTERS WITH DISTINCT TRANSCRIPTOMES. DESPITE ADVANCES IN THE FIELD, CHRONIC GRAFT-VERSUS-HOST-DISEASE (CGVHD) REMAINS A LEADING CAUSE OF MORBIDITY AND MORTALITY FOLLOWING ALLOGENIC HEMATOPOIETIC STEM CELL TRANSPLANT. BECAUSE TREATMENT OPTIONS REMAIN LIMITED, WE TESTED EFFICACY OF ANTICANCER, CHROMATIN-MODIFYING ENZYME INHIBITORS IN A CLINICALLY RELEVANT MURINE MODEL OF CGVHD WITH BRONCHIOLITIS OBLITERANS (BO). WE OBSERVED THAT THE NOVEL ENHANCER OF ZESTE HOMOLOG 2 (EZH2) INHIBITOR JQ5 AND THE BET-BROMODOMAIN INHIBITOR JQ1 EACH IMPROVED PULMONARY FUNCTION; IMPAIRED THE GERMINAL CENTER (GC) REACTION, A PREREQUISITE IN CGVHD/BO PATHOGENESIS; AND JQ5 REDUCED EZH2-MEDIATED H3K27ME3 IN DONOR T CELLS. USING CONDITIONAL EZH2 KNOCKOUT DONOR CELLS, WE DEMONSTRATED THAT EZH2 IS OBLIGATORY FOR THE INITIATION OF CGVHD/BO. IN A SCLERODERMATOUS CGVHD MODEL, JQ5 REDUCED THE SEVERITY OF CUTANEOUS LESIONS. TO DETERMINE HOW THE 2 DRUGS COULD LEAD TO THE SAME PHYSIOLOGICAL IMPROVEMENTS WHILE TARGETING UNIQUE EPIGENETIC PROCESSES, WE ANALYZED THE TRANSCRIPTOMES OF SPLENIC GCB CELLS (GCBS) FROM TRANSPLANTED MICE TREATED WITH EITHER DRUG. MULTIPLE INFLAMMATORY AND SIGNALING PATHWAYS ENRICHED IN CGVHD/BO GCBS WERE REDUCED BY EACH DRUG. GCBS FROM JQ5- BUT NOT JQ1-TREATED MICE WERE ENRICHED FOR PROPROLIFERATIVE PATHWAYS ALSO SEEN IN GCBS FROM BONE MARROW-ONLY TRANSPLANTED MICE, LIKELY REFLECTING THEIR UNDERLYING BIOLOGY IN THE UNPERTURBED STATE. IN CONJUNCTION WITH IN VIVO DATA, THESE INSIGHTS LED US TO CONCLUDE THAT EPIGENETIC TARGETING OF THE GC IS A VIABLE CLINICAL APPROACH FOR THE TREATMENT OF CGVHD, AND THAT THE EZH2 INHIBITOR JQ5 AND THE BET-BROMODOMAIN INHIBITOR JQ1 DEMONSTRATED CLINICAL POTENTIAL FOR EZH2I AND BETI IN PATIENTS WITH CGVHD/BO. 2022 4 4432 34 MOLECULAR CHARACTERIZATION OF RICHTER SYNDROME IDENTIFIES DE NOVO DIFFUSE LARGE B-CELL LYMPHOMAS WITH POOR PROGNOSIS. RICHTER SYNDROME (RS) IS THE TRANSFORMATION OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) INTO AGGRESSIVE LYMPHOMA, MOST COMMONLY DIFFUSE LARGE B-CELL LYMPHOMA (DLBCL). WE CHARACTERIZE 58 PRIMARY HUMAN RS SAMPLES BY GENOME-WIDE DNA METHYLATION AND WHOLE-TRANSCRIPTOME PROFILING. OUR COMPREHENSIVE APPROACH DETERMINES RS DNA METHYLATION PROFILE AND UNRAVELS A CLL EPIGENETIC IMPRINT, ALLOWING CLL-RS CLONAL RELATIONSHIP ASSESSMENT WITHOUT THE NEED OF THE INITIAL CLL TUMOR DNA. DNA METHYLATION- AND TRANSCRIPTOMIC-BASED CLASSIFIERS WERE DEVELOPED, AND TESTING ON LANDMARK DLBCL DATASETS IDENTIFIES A POOR-PROGNOSIS, ACTIVATED B-CELL-LIKE DLBCL SUBSET IN 111/1772 SAMPLES. THE CLASSIFICATION ROBUSTLY IDENTIFIES PHENOTYPES VERY SIMILAR TO RS WITH A SPECIFIC GENOMIC PROFILE, ACCOUNTING FOR 4.3-8.3% OF DE NOVO DLBCLS. IN THIS WORK, RS MULTI-OMICS CHARACTERIZATION DETERMINES ONCOGENIC MECHANISMS, ESTABLISHES A SURROGATE MARKER FOR CLL-RS CLONAL RELATIONSHIP, AND PROVIDES A CLINICALLY RELEVANT CLASSIFIER FOR A SUBSET OF PRIMARY "RS-TYPE DLBCL" WITH UNFAVORABLE PROGNOSIS. 2023 5 1107 32 COMBINING CYTOGENETIC AND EPIGENETIC APPROACHES IN CHRONIC LYMPHOCYTIC LEUKEMIA IMPROVES PROGNOSIS PREDICTION FOR PATIENTS WITH ISOLATED 13Q DELETION. BACKGROUND: BOTH DEFECTIVE DNA METHYLATION AND ACTIVE DNA DEMETHYLATION PROCESSES ARE EMERGING AS IMPORTANT RISK FACTORS IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). HOWEVER, ASSOCIATIONS BETWEEN 5-CYTOSINE EPIGENETIC MARKERS AND THE MOST FREQUENT CHROMOSOMAL ABNORMALITIES DETECTED IN CLL REMAIN TO BE ESTABLISHED. METHODS: CLL PATIENTS WERE RETROSPECTIVELY CLASSIFIED INTO A CYTOGENETIC LOW-RISK GROUP (ISOLATED 13Q DELETION), AN INTERMEDIATE-RISK GROUP (NORMAL KARYOTYPE OR TRISOMY 12), AND A HIGH-RISK GROUP (11Q DELETION, 17P DELETION, OR COMPLEX KARYOTYPE [>/= 3 BREAKPOINTS]). THE TWO 5-CYTOSINE DERIVATIVES, 5-METHYLCYTOSINE (5-MCYT) AND 5-HYDROXYMETHYLCYTOSINE (5-HMCYT), WERE TESTED BY ELISA (N = 60), WHILE REAL-TIME QUANTITATIVE PCR WAS USED FOR DETERMINING TRANSCRIPTIONAL EXPRESSION LEVELS OF DNMT AND TET (N = 24). RESULTS: BY USING GLOBAL DNA METHYLATION/DEMETHYLATION LEVELS, IN THE LOW-RISK DISEASE GROUP, TWO SUBGROUPS WITH SIGNIFICANTLY DIFFERENT CLINICAL OUTCOMES HAVE BEEN IDENTIFIED (MEDIAN TREATMENT-FREE SURVIVAL [TFS] 45 VERSUS > 120 MONTHS FOR 5-MCYT, P = 0.0008, AND 63 VERSUS > 120 MONTHS FOR 5-HMCYT, P = 0.04). A DEFECTIVE 5-MCYT STATUS WAS FURTHER ASSOCIATED WITH A HIGHER PERCENTAGE OF 13Q DELETED NUCLEI (> 80%), THUS SUGGESTING AN ACQUIRED PROCESS. WHEN CONSIDERING THE CYTOGENETIC INTERMEDIATE/HIGH-RISK DISEASE GROUPS, AN ASSOCIATION OF 5-MCYT STATUS WITH LYMPHOCYTOSIS (P = 0.0008) AND THE LYMPHOCYTE DOUBLING TIME (P = 0.04) BUT NOT WITH TFS WAS OBSERVED, AS WELL AS A REDUCTION OF DNMT3A, TET1, AND TET2 TRANSCRIPTS. CONCLUSIONS: COMBINING CYTOGENETIC STUDIES WITH 5-MCYT ASSESSMENT ADDS ACCURACY TO CLL PATIENTS' PROGNOSES AND PARTICULARLY FOR THOSE WITH 13Q DELETION AS A SOLE CYTOGENETIC ABNORMALITY. 2017 6 2402 28 EPIGENETIC REPROGRAMMING SENSITIZES CML STEM CELLS TO COMBINED EZH2 AND TYROSINE KINASE INHIBITION. A MAJOR OBSTACLE TO CURING CHRONIC MYELOID LEUKEMIA (CML) IS RESIDUAL DISEASE MAINTAINED BY TYROSINE KINASE INHIBITOR (TKI)-PERSISTENT LEUKEMIC STEM CELLS (LSC). THESE ARE BCR-ABL1 KINASE INDEPENDENT, REFRACTORY TO APOPTOSIS, AND SERVE AS A RESERVOIR TO DRIVE RELAPSE OR TKI RESISTANCE. WE DEMONSTRATE THAT POLYCOMB REPRESSIVE COMPLEX 2 IS MISREGULATED IN CHRONIC PHASE CML LSCS. THIS IS ASSOCIATED WITH EXTENSIVE REPROGRAMMING OF H3K27ME3 TARGETS IN LSCS, THUS SENSITIZING THEM TO APOPTOSIS UPON TREATMENT WITH AN EZH2-SPECIFIC INHIBITOR (EZH2I). EZH2I DOES NOT IMPAIR NORMAL HEMATOPOIETIC STEM CELL SURVIVAL. STRIKINGLY, TREATMENT OF PRIMARY CML CELLS WITH EITHER EZH2I OR TKI ALONE CAUSED SIGNIFICANT UPREGULATION OF H3K27ME3 TARGETS, AND COMBINED TREATMENT FURTHER POTENTIATED THESE EFFECTS AND RESULTED IN SIGNIFICANT LOSS OF LSCS COMPARED TO TKI ALONE, IN VITRO, AND IN LONG-TERM BONE MARROW MURINE XENOGRAFTS. OUR FINDINGS POINT TO A PROMISING EPIGENETIC-BASED THERAPEUTIC STRATEGY TO MORE EFFECTIVELY TARGET LSCS IN PATIENTS WITH CML RECEIVING TKIS. SIGNIFICANCE: IN CML, TKI-PERSISTENT LSCS REMAIN AN OBSTACLE TO CURE, AND APPROACHES TO ERADICATE THEM REMAIN A SIGNIFICANT UNMET CLINICAL NEED. WE DEMONSTRATE THAT EZH2 AND H3K27ME3 REPROGRAMMING IS IMPORTANT FOR LSC SURVIVAL, BUT RENDERS LSCS SENSITIVE TO THE COMBINED EFFECTS OF EZH2I AND TKI. THIS REPRESENTS A NOVEL APPROACH TO MORE EFFECTIVELY TARGET LSCS IN PATIENTS RECEIVING TKI TREATMENT. CANCER DISCOV; 6(11); 1248-57. (C)2016 AACR.SEE RELATED ARTICLE BY XIE ET AL., P. 1237THIS ARTICLE IS HIGHLIGHTED IN THE IN THIS ISSUE FEATURE, P. 1197. 2016 7 4231 32 METHYLATION OF PROTOCADHERIN 10, A NOVEL TUMOR SUPPRESSOR, IS ASSOCIATED WITH POOR PROGNOSIS IN PATIENTS WITH GASTRIC CANCER. BACKGROUND & AIMS: BY USING METHYLATION-SENSITIVE REPRESENTATIONAL DIFFERENCE ANALYSIS, WE IDENTIFIED PROTOCADHERIN 10 (PCDH10), A GENE THAT ENCODES A PROTOCADHERIN AND IS SILENCED IN A TUMOR-SPECIFIC MANNER. WE ANALYZED ITS EPIGENETIC INACTIVATION, BIOLOGICAL EFFECTS, AND PROGNOSTIC SIGNIFICANCE IN GASTRIC CANCER. METHODS: METHYLATION STATUS WAS EVALUATED BY COMBINED BISULFITE RESTRICTION ANALYSIS AND BISULFITE SEQUENCING. THE EFFECTS OF PCDH10 RE-EXPRESSION WERE DETERMINED IN GROWTH, APOPTOSIS, PROLIFERATION, AND INVASION ASSAYS. PCDH10 TARGET GENES WERE IDENTIFIED BY COMPLEMENTARY DNA MICROARRAY ANALYSIS. RESULTS: PCDH10 WAS SILENCED OR DOWN-REGULATED IN 94% (16 OF 17) OF GASTRIC CANCER CELL LINES; EXPRESSION LEVELS WERE RESTORED BY EXPOSURE TO DEMETHYLATING AGENTS. RE-EXPRESSION OF PCDH10 IN MKN45 GASTRIC CANCER CELLS REDUCED COLONY FORMATION IN VITRO AND TUMOR GROWTH IN MICE; IT ALSO INHIBITED CELL PROLIFERATION (P < .01), INDUCED CELL APOPTOSIS (P < .001), AND REPRESSED CELL INVASION (P < .05), UP-REGULATING THE PRO-APOPTOSIS GENES FAS, CASPASE 8, JUN, AND CDKN1A; THE ANTIPROLIFERATION GENE FGFR; AND THE ANTI-INVASION GENE HTATIP2. PCDH10 METHYLATION WAS DETECTED IN 82% (85 OF 104) OF GASTRIC TUMORS COMPARED WITH 37% (38 OF 104) OF PAIRED NONTUMOR TISSUES (P < .0001). IN THE LATTER, PCDH10 METHYLATION WAS HIGHER IN PRECANCEROUS LESIONS (27 OF 45; 60%) THAN IN CHRONIC GASTRITIS SAMPLES (11 OF 59; 19%) (P < .0001). AFTER A MEDIAN FOLLOW-UP PERIOD OF 16.8 MONTHS, MULTIVARIATE ANALYSIS REVEALED THAT PATIENTS WITH PCDH10 METHYLATION IN ADJACENT NONTUMOR AREAS HAD A SIGNIFICANT DECREASE IN OVERALL SURVIVAL. KAPLAN-MEIER SURVIVAL CURVES SHOWED THAT PCDH10 METHYLATION WAS ASSOCIATED SIGNIFICANTLY WITH SHORTENED SURVIVAL IN STAGE I-III GASTRIC CANCER PATIENTS. CONCLUSIONS: PCDH10 IS A GASTRIC TUMOR SUPPRESSOR; ITS METHYLATION AT EARLY STAGES OF GASTRIC CARCINOGENESIS IS AN INDEPENDENT PROGNOSTIC FACTOR. 2009 8 1102 28 COMBINED CHEMOSENSITIVITY AND CHROMATIN PROFILING PRIORITIZES DRUG COMBINATIONS IN CLL. THE BRUTON TYROSINE KINASE (BTK) INHIBITOR IBRUTINIB HAS SUBSTANTIALLY IMPROVED THERAPEUTIC OPTIONS FOR CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). ALTHOUGH IBRUTINIB IS NOT CURATIVE, IT HAS A PROFOUND EFFECT ON CLL CELLS AND MAY CREATE NEW PHARMACOLOGICALLY EXPLOITABLE VULNERABILITIES. TO IDENTIFY SUCH VULNERABILITIES, WE DEVELOPED A SYSTEMATIC APPROACH THAT COMBINES EPIGENOME PROFILING (CHARTING THE GENE-REGULATORY BASIS OF CELL STATE) WITH SINGLE-CELL CHEMOSENSITIVITY PROFILING (QUANTIFYING CELL-TYPE-SPECIFIC DRUG RESPONSE) AND BIOINFORMATIC DATA INTEGRATION. BY APPLYING OUR METHOD TO A COHORT OF MATCHED PATIENT SAMPLES COLLECTED BEFORE AND DURING IBRUTINIB THERAPY, WE IDENTIFIED CHARACTERISTIC IBRUTINIB-INDUCED CHANGES THAT PROVIDE A STARTING POINT FOR THE RATIONAL DESIGN OF IBRUTINIB COMBINATION THERAPIES. SPECIFICALLY, WE OBSERVED AND VALIDATED PREFERENTIAL SENSITIVITY TO PROTEASOME, PLK1, AND MTOR INHIBITORS DURING IBRUTINIB TREATMENT. MORE GENERALLY, OUR STUDY ESTABLISHES A BROADLY APPLICABLE METHOD FOR INVESTIGATING TREATMENT-SPECIFIC VULNERABILITIES BY INTEGRATING THE COMPLEMENTARY PERSPECTIVES OF EPIGENETIC CELL STATES AND PHENOTYPIC DRUG RESPONSES IN PRIMARY PATIENT SAMPLES. 2019 9 2678 26 EVALUATION OF A PROGNOSTIC EPIGENETIC CLASSIFICATION SYSTEM IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS. BACKGROUND: METHYLATION AT 5 CPG SITES WAS PREVIOUSLY SHOWN TO CLASSIFY CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) INTO 3 PROGNOSTIC SUBGROUPS. HERE, WE AIMED TO VALIDATE THE MARKER SET IN AN ADDITIONAL COHORT AND TO EVALUATE ITS CLINICAL UTILITY FOR CLL PATIENT STRATIFICATION. METHODS: WE EVALUATED THIS EPIGENETIC MARKER SET IN 79 GERMAN PATIENTS USING BISULFITE TREATMENT FOLLOWED BY PYROSEQUENCING AND CLASSIFICATION USING A SUPPORT VECTOR MACHINE-LEARNING TOOL. RESULTS: THE N-CLL, I-CLL, AND M-CLL CLASSIFICATION WAS DETECTED IN 28 (35%), 10 (13%), AND 41 (51%) PATIENTS, RESPECTIVELY. EPIGENETIC GROUPING WAS ASSOCIATED WITH IGHV MUTATIONAL STATUS (P = 2 X 10(-12)), ISOLATED DEL13Q (P = 9 X 10(-6)), DEL17P (P = .015), COMPLEX KARYOTYPE (P = .005), VH-USAGE, AND CLINICAL OUTCOME AS TIME TO FIRST TREATMENT (P = 1.4 X 10(-12)) AND OVERALL SURVIVAL (P = .003). MULTIVARIATE COX REGRESSION ANALYSIS IDENTIFIED N-CLL AS A FACTOR FOR EARLIER TREATMENT HAZARD RATIO (HR), 6.3 (95% CONFIDENCE INTERVAL [CI] 2.4-16.4; P = .0002) COMPARED TO IGHV MUTATIONAL STATUS (HR 4.6, 95% CI 1.9-11.3, P = .0008). IN ADDITION, WHEN COMPARING THE PROGNOSTIC VALUE OF THE EPIGENETIC CLASSIFICATION SYSTEM WITH THE IGHV CLASSIFICATION, EPIGENETIC GROUPING PERFORMED BETTER COMPARED TO IGHV MUTATIONAL STATUS USING KAPLAN-MEIER ESTIMATION AND ALLOWED THE IDENTIFICATION OF A THIRD, INTERMEDIATE (I-CLL) GROUP. THUS, OUR STUDY CONFIRMED THE PROGNOSTIC VALUE OF THE EPIGENETIC MARKER SET FOR PATIENT STRATIFICATION IN ROUTINE CLINICAL DIAGNOSTICS. 2022 10 2426 31 EPIGENETIC SILENCING OF LONG NON-CODING RNA BM742401 IN MULTIPLE MYELOMA: IMPACT ON PROGNOSIS AND MYELOMA DISSEMINATION. BACKGROUND: LONG NON-CODING RNA (LNCRNA) BM742401 IS A TUMOR SUPPRESSOR IN GASTRIC CANCER AND CHRONIC LYMPHOCYTIC LEUKEMIA. AS THE PROMOTER AND CODING REGION OF BM742401 ARE FULLY EMBEDDED IN A CPG ISLAND, WE HYPOTHESIZED THAT BM742401 IS A TUMOR SUPPRESSOR LNCRNA EPIGENETICALLY SILENCED BY PROMOTER DNA METHYLATION IN MULTIPLE MYELOMA. METHODS: METHYLATION-SPECIFIC PCR AND QUANTITATIVE BISULFITE PYROSEQUENCING WERE PERFORMED TO DETECT THE METHYLATION OF BM742401 IN NORMAL PLASMA CELLS, MYELOMA CELL LINES AND PRIMARY MYELOMA SAMPLES. THE EXPRESSION OF BM742401 WAS MEASURED BY QRT-PCR. THE FUNCTION OF BM742401 IN MULTIPLE MYELOMA CELLS WAS ANALYZED BY LENTIVIRUS TRANSDUCTION FOLLOWED BY MIGRATION ASSAY. RESULTS: BM742401 METHYLATION WAS DETECTED IN 10 (66.7%) MYELOMA CELL LINES BUT NOT NORMAL PLASMA CELLS, AND INVERSELY CORRELATED WITH EXPRESSION OF BM742401. IN PRIMARY SAMPLES, BM742401 METHYLATION WAS DETECTED IN 3 (12.5%) MONOCLONAL GAMMOPATHY OF UNDETERMINED SIGNIFICANCE, 9 (15.8%) MYELOMA AT DIAGNOSIS AND 8 (17.0%) MYELOMA AT RELAPSE/PROGRESSION. MOREOVER, BM742401 METHYLATION AT DIAGNOSIS WAS ASSOCIATED WITH INFERIOR OVERALL SURVIVAL (MEDIAN OS: 25 VS. 39 MONTHS; P = 0.0496). IN MYELOMA CELL LINE JJN-3, STABLE OVEREXPRESSION OF BM742401 BY LENTIVIRUS TRANSDUCTION RESULTED IN REDUCED CELL MIGRATION (P = 0.0001) BUT NOT IMPACTING CELL DEATH OR PROLIFERATION. CONCLUSIONS: THIS IS THE FIRST REPORT OF TUMOR-SPECIFIC METHYLATION-MEDIATED SILENCING OF BM742401 IN MYELOMA, WHICH IS LIKELY AN EARLY EVENT IN MYELOMAGENESIS WITH ADVERSE IMPACT ON OVERALL SURVIVAL. MOREOVER, BM742401 IS A TUMOR SUPPRESSOR LNCRNA BY INHIBITING MYELOMA CELL MIGRATION, HENCE IMPLICATED IN MYELOMA PLASMA CELL HOMING, METASTASIS AND DISEASE PROGRESSION. 2020 11 2440 28 EPIGENETIC SILENCING OF TUMOR SUPPRESSOR LONG NON-CODING RNA BM742401 IN CHRONIC LYMPHOCYTIC LEUKEMIA. BM742401 IS A TUMOR SUPPRESSOR LNCRNA DOWNREGULATED IN GASTRIC CANCER. AS THE PROMOTER REGION AND THE ENTIRE TRANSCRIPT ARE EMBEDDED IN A CPG ISLAND, WE POSTULATED THAT BM742401 IS A TUMOR SUPPRESSOR LNCRNA INACTIVATED BY DNA METHYLATION IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). THE PROMOTER OF BM742401 WAS UNMETHYLATED IN NORMAL CONTROLS INCLUDING THREE EACH OF NORMAL BONE MARROW, PERIPHERAL BLOOD BUFFY COATS, AND CD19-SORTED PERIPHERAL B-CELLS, BUT METHYLATED IN FOUR (57.1%) CLL CELL LINES. METHYLATION OF BM742401 CORRELATED INVERSELY WITH EXPRESSION. IN THE COMPLETELY METHYLATED WAC3CD5+ CLL CELLS, 5-AZA-2'-DEOXYCYTIDINE TREATMENT LED TO PROMOTER DEMETHYLATION AND RE-EXPRESSION OF BM742401 TRANSCRIPT. FUNCTIONALLY, STABLE OVEREXPRESSION OF BM742401 RESULTED IN INHIBITION OF CELLULAR PROLIFERATION AND ENHANCED APOPTOSIS THROUGH CASPASE-9-DEPENDENT INTRINSIC BUT NOT CASPASE-8-DEPENDENT EXTRINSIC APOPTOSIS PATHWAY, SUGGESTING A TUMOR SUPPRESSOR ROLE OF BM742401 IN CLL. IN PRIMARY CLL SAMPLES, METHYLATION OF BM742401 WAS DETECTED IN 43/98 (43.9%) OF PATIENTS. MOREOVER, AMONG CLL PATIENTS WITH STANDARD-RISK CYTOGENETIC ABERRATIONS, METHYLATION OF BM742401 CORRELATED WITH ADVANCED RAI STAGE (>/= STAGE 2)(P = 0.002). FURTHERMORE, BM742401 METHYLATION WAS ASSOCIATED WITH MIR-129-2 METHYLATION (P = 0.05). BM742401 IS A TUMOR SUPPRESSOR LNCRNA FREQUENTLY METHYLATED IN CLL. THE MECHANISM OF BM742401 AS A TUMOR SUPPRESSOR WARRANTS FURTHER STUDIES. 2016 12 5243 29 PROGNOSTIC IMPACT OF EPIGENETIC CLASSIFICATION IN CHRONIC LYMPHOCYTIC LEUKEMIA: THE CASE OF SUBSET #2. BASED ON THE METHYLATION STATUS OF 5 SINGLE CPG SITES, A NOVEL EPIGENETIC CLASSIFICATION OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) WAS RECENTLY PROPOSED, CLASSIFYING CLL PATIENTS INTO 3 CLINICO-BIOLOGICAL SUBGROUPS WITH DIFFERENT OUTCOME, TERMED MEMORY LIKE CLL (M-CLL), NAIVE LIKE CLL (N-CLL), AND A THIRD INTERMEDIATE CLL SUBGROUP (I-CLL). WHILE M-CLL AND N-CLL PATIENTS AT LARGE CORRESPONDED TO PATIENTS CARRYING MUTATED AND UNMUTATED IGHV GENES, RESPECTIVELY, LIMITED INFORMATION EXISTS REGARDING THE LESS DEFINED I-CLL GROUP. USING PYROSEQUENCING, WE INVESTIGATED THE PROGNOSTIC IMPACT OF THE PROPOSED 5 CPG SIGNATURE IN A WELL-CHARACTERIZED CLL COHORT (135 CASES), INCLUDING IGHV-MUTATED AND UNMUTATED PATIENTS AS WELL AS CLINICALLY AGGRESSIVE STEREOTYPED SUBSET #2 PATIENTS. OVERALL, WE CONFIRMED THE SIGNATURE'S ASSOCIATION WITH ESTABLISHED PROGNOSTIC MARKERS. MOREOVER, IN THE PRESENCE OF THE IGHV MUTATIONAL STATUS, THE EPIGENETIC SIGNATURE REMAINED INDEPENDENTLY ASSOCIATED WITH BOTH TIME-TO-FIRST-TREATMENT AND OVERALL SURVIVAL IN MULTIVARIATE ANALYSES. AS A PRIME FINDING, WE OBSERVED THAT SUBSET #2 PATIENTS WERE PREDOMINANTLY CLASSIFIED AS I-CLL, PROBABLY REFLECTING THEIR BORDERLINE IGHV MUTATIONAL STATUS (97-99% GERMLINE IDENTITY), THOUGH HAVING A SIMILARLY POOR PROGNOSIS AS N-CLL PATIENTS. IN SUMMARY, WE VALIDATED THE EPIGENETIC CLASSIFIER AS AN INDEPENDENT FACTOR IN CLL PROGNOSTICATION AND PROVIDE FURTHER EVIDENCE THAT SUBSET #2 IS A MEMBER OF THE I-CLL GROUP, HENCE SUPPORTING THE EXISTENCE OF A THIRD, INTERMEDIATE EPIGENETIC SUBGROUP. 2016 13 3877 30 KDM6A PROMOTES IMATINIB RESISTANCE THROUGH YY1-MEDIATED TRANSCRIPTIONAL UPREGULATION OF TRKA INDEPENDENTLY OF ITS DEMETHYLASE ACTIVITY IN CHRONIC MYELOGENOUS LEUKEMIA. RATIONALE: DESPITE LANDMARK THERAPY OF CHRONIC MYELOGENOUS LEUKEMIA (CML) WITH TYROSINE KINASE INHIBITORS (TKIS), DRUG RESISTANCE REMAINS PROBLEMATIC. CANCER PATHOGENESIS INVOLVES EPIGENETIC DYSREGULATION AND IN PARTICULAR, HISTONE LYSINE DEMETHYLASES (KDMS) HAVE BEEN IMPLICATED IN TKI RESISTANCE. WE SOUGHT TO IDENTIFY KDMS WITH ALTERED EXPRESSION IN CML AND DEFINE THEIR CONTRIBUTION TO IMATINIB RESISTANCE. METHODS: BIOINFORMATICS SCREENING COMPARED KDM EXPRESSION IN CML VERSUS NORMAL BONE MARROW WITH SHRNA KNOCKDOWN AND FLOW CYTOMETRY USED TO MEASURE EFFECTS ON IMATINIB-INDUCED APOPTOSIS IN K562 CELLS. TRANSCRIPTOMIC ANALYSES WERE PERFORMED AGAINST KDM6A CRISPR KNOCKOUT/SHRNA KNOCKDOWN K562 CELLS ALONG WITH GENE RESCUE EXPERIMENTS USING WILDTYPE AND MUTANT DEMETHYLASE-DEAD KDM6A CONSTRUCTS. CO-IMMUNOPRECIPITATION, LUCIFERASE REPORTER AND CHIP WERE EMPLOYED TO ELUCIDATE MECHANISMS OF KDM6A-DEPENDENT RESISTANCE. RESULTS: AMONGST FIVE KDMS UPREGULATED IN CML, ONLY KDM6A DEPLETION SENSITIZED CML CELLS TO IMATINIB-INDUCED APOPTOSIS. RE-INTRODUCTION OF DEMETHYLASE-DEAD KDM6A AS WELL AS WILD-TYPE KDM6A RESTORED IMATINIB RESISTANCE. RNA-SEQ IDENTIFIED NTRK1 GENE DOWNREGULATION AFTER DEPLETION OF KDM6A. MOREOVER, NTRK1 EXPRESSION POSITIVELY CORRELATED WITH KDM6A IN A SUBSET OF CLINICAL CML SAMPLES AND KDM6A KNOCKDOWN IN FRESH CML ISOLATES DECREASED NTRK1 ENCODED PROTEIN (TRKA) EXPRESSION. MECHANISTICALLY, KDM6A WAS RECRUITED TO THE NTRK1 PROMOTER BY THE TRANSCRIPTION FACTOR YY1 WITH SUBSEQUENT TRKA UPREGULATION ACTIVATING DOWN-STREAM SURVIVAL PATHWAYS TO INVOKE IMATINIB RESISTANCE. CONCLUSION: CONTRARY TO ITS REPORTED ROLE AS A TUMOR SUPPRESSOR AND INDEPENDENT OF ITS DEMETHYLASE FUNCTION, KDM6A PROMOTES IMATINIB-RESISTANCE IN CML CELLS. THE IDENTIFICATION OF THE KDM6A/YY1/TRKA AXIS AS A NOVEL IMATINIB-RESISTANCE MECHANISM REPRESENTS AN UNEXPLORED AVENUE TO OVERCOME TKI RESISTANCE IN CML. 2021 14 5319 25 PTEN IS FUNDAMENTAL FOR ELIMINATION OF LEUKEMIA STEM CELLS MEDIATED BY GSK126 TARGETING EZH2 IN CHRONIC MYELOGENOUS LEUKEMIA. PURPOSE: LEUKEMIA STEM CELLS (LSCS) ARE AN IMPORTANT SOURCE OF TYROSINE KINASE INHIBITOR RESISTANCE AND DISEASE RELAPSE IN PATIENTS WITH CHRONIC MYELOGENOUS LEUKEMIA (CML). TARGETING LSCS MAY BE AN ATTRACTIVE STRATEGY TO OVERRIDE THIS THORNY PROBLEM. GIVEN THAT EZH2 WAS OVEREXPRESSED IN PRIMARY CML CD34(+) CELLS, OUR PURPOSE IN THIS STUDY WAS TO EVALUATE THE EFFECTS OF TARGETING EZH2 ON CML LSCS AND CLARIFY ITS UNDERLYING MECHANISM.EXPERIMENTAL DESIGN: HUMAN PRIMARY CML CD34(+) CELLS AND RETROVIRALLY BCR-ABL-DRIVEN CML MOUSE MODELS WERE EMPLOYED TO EVALUATE THE EFFECTS OF SUPPRESSION OF EZH2 BY GSK126- OR EZH2-SPECIFIC SHRNA IN VITRO AND IN VIVO RECRUITMENT OF EZH2 AND H3K27ME3 ON THE PROMOTER OF TUMOR-SUPPRESSOR GENE PTEN IN CML CELLS WAS MEASURED BY CHROMATIN IMMUNOPRECIPITATION ASSAY.RESULTS: OUR RESULTS SHOWED THAT PHARMACOLOGIC INHIBITION OF EZH2 BY GSK126 NOT ONLY ELICITED APOPTOSIS AND RESTRICTED CELL GROWTH IN CML BULK LEUKEMIA CELLS, BUT ALSO DECREASED LSCS IN CML CD34(+) CELLS WHILE SPARING THOSE FROM NORMAL BONE MARROW CD34(+) CELLS. SUPPRESSION OF EZH2 BY GSK126 OR SPECIFIC SHRNA PROLONGED SURVIVAL OF CML MICE AND REDUCED THE NUMBER OF LSCS IN MICE. EZH2 KNOCKDOWN RESULTED IN ELEVATION OF PTEN AND LED TO IMPAIRED RECRUITMENT OF EZH2 AND H3K27ME3 ON THE PROMOTER OF PTEN GENE. THE EFFECT OF EZH2 KNOCKDOWN IN THE CML MICE WAS AT LEAST PARTIALLY REVERSED BY PTEN KNOCKDOWN.CONCLUSIONS: THESE FINDINGS IMPROVE THE UNDERSTANDING OF THE EPIGENETIC REGULATION OF STEMNESS IN CML LSCS AND WARRANT CLINICAL TRIAL OF GSK126 IN REFRACTORY PATIENTS WITH CML. CLIN CANCER RES; 24(1); 145-57. (C)2017 AACR. 2018 15 66 34 A KEY ROLE FOR EZH2 IN EPIGENETIC SILENCING OF HOX GENES IN MANTLE CELL LYMPHOMA. THE CHROMATIN MODIFIER EZH2 IS OVEREXPRESSED AND ASSOCIATED WITH INFERIOR OUTCOME IN MANTLE CELL LYMPHOMA (MCL). RECENTLY, WE DEMONSTRATED PREFERENTIAL DNA METHYLATION OF HOX GENES IN MCL COMPARED WITH CHRONIC LYMPHOCYTIC LEUKEMIA (CLL), DESPITE THESE GENES NOT BEING EXPRESSED IN EITHER ENTITY. SINCE EZH2 HAS BEEN SHOWN TO REGULATE HOX GENE EXPRESSION, TO GAIN FURTHER INSIGHT INTO ITS POSSIBLE ROLE IN DIFFERENTIAL SILENCING OF HOX GENES IN MCL VS. CLL, WE PERFORMED DETAILED EPIGENETIC CHARACTERIZATION USING REPRESENTATIVE CELL LINES AND PRIMARY SAMPLES. WE OBSERVED SIGNIFICANT OVEREXPRESSION OF EZH2 IN MCL VS. CLL. CHROMATIN IMMUNE PRECIPITATION (CHIP) ASSAYS REVEALED THAT EZH2 CATALYZED REPRESSIVE H3 LYSINE 27 TRIMETHYLATION (H3K27ME3), WHICH WAS SUFFICIENT TO SILENCE HOX GENES IN CLL, WHEREAS IN MCL H3K27ME3 IS ACCOMPANIED BY DNA METHYLATION FOR A MORE STABLE REPRESSION. MORE IMPORTANTLY, HYPERMETHYLATION OF THE HOX GENES IN MCL RESULTED FROM EZH2 OVEREXPRESSION AND SUBSEQUENT RECRUITMENT OF THE DNA METHYLATION MACHINERY ONTO HOX GENE PROMOTERS. THE IMPORTANCE OF EZH2 UPREGULATION IN THIS PROCESS WAS FURTHER UNDERSCORED BY SIRNA TRANSFECTION AND EZH2 INHIBITOR EXPERIMENTS. ALTOGETHER, THESE OBSERVATIONS IMPLICATE EZH2 IN THE LONG-TERM SILENCING OF HOX GENES IN MCL, AND ALLUDE TO ITS POTENTIAL AS A THERAPEUTIC TARGET WITH CLINICAL IMPACT. 2013 16 1469 32 DISTINCT EVOLUTIONARY PATHS IN CHRONIC LYMPHOCYTIC LEUKEMIA DURING RESISTANCE TO THE GRAFT-VERSUS-LEUKEMIA EFFECT. LEUKEMIC RELAPSE REMAINS A MAJOR BARRIER TO SUCCESSFUL ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION (ALLO-HSCT) FOR AGGRESSIVE HEMATOLOGIC MALIGNANCIES. THE BASIS FOR RELAPSE OF ADVANCED LYMPHOID MALIGNANCIES REMAINS INCOMPLETELY UNDERSTOOD AND MAY INVOLVE ESCAPE FROM THE GRAFT-VERSUS-LEUKEMIA (GVL) EFFECT. WE HYPOTHESIZED THAT FOR PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) TREATED WITH ALLO-HSCT, LEUKEMIC CELL-INTRINSIC FEATURES INFLUENCE TRANSPLANT OUTCOMES BY DIRECTING THE EVOLUTIONARY TRAJECTORIES OF CLL CELLS. INTEGRATED GENETIC, TRANSCRIPTOMIC, AND EPIGENETIC ANALYSES OF CLL CELLS FROM 10 PATIENTS REVEALED THAT THE CLINICAL KINETICS OF POST-HSCT RELAPSE ARE SHAPED BY DISTINCT MOLECULAR DYNAMICS. EARLY RELAPSES AFTER ALLO-HSCT EXHIBITED NOTABLE GENETIC STABILITY; SINGLE CLL CELL TRANSCRIPTIONAL ANALYSIS DEMONSTRATED A CELLULAR HETEROGENEITY THAT WAS STATIC OVER TIME. IN CONTRAST, CLL CELLS RELAPSING LATE AFTER ALLO-HSCT DISPLAYED NOTABLE GENETIC EVOLUTION AND EVIDENCE OF NEOANTIGEN DEPLETION, CONSISTENT WITH MARKED SINGLE-CELL TRANSCRIPTIONAL SHIFTS THAT WERE UNIQUE TO EACH PATIENT. WE OBSERVED A GREATER RATE OF EPIGENETIC CHANGE FOR LATE RELAPSES NOT SEEN IN EARLY RELAPSES OR RELAPSES AFTER CHEMOTHERAPY ALONE, SUGGESTING THAT THE SELECTION PRESSURES OF THE GVL BOTTLENECK ARE UNLIKE THOSE IMPOSED BY CHEMOTHERAPY. NO SELECTIVE ADVANTAGE FOR HUMAN LEUKOCYTE ANTIGEN (HLA) LOSS WAS OBSERVED, EVEN WHEN PRESENT IN PRETRANSPLANT SUBPOPULATIONS. GAIN OF STEM CELL MODULES WAS A COMMON SIGNATURE ASSOCIATED WITH LEUKEMIA RELAPSE REGARDLESS OF POSTTRANSPLANT RELAPSE KINETICS. THESE DATA ELUCIDATE THE BIOLOGICAL PATHWAYS THAT UNDERLIE GVL RESISTANCE AND POSTTRANSPLANT RELAPSE. 2020 17 5979 30 TET2 MUTATIONS ARE ASSOCIATED WITH SPECIFIC 5-METHYLCYTOSINE AND 5-HYDROXYMETHYLCYTOSINE PROFILES IN PATIENTS WITH CHRONIC MYELOMONOCYTIC LEUKEMIA. CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML) HAS RECENTLY BEEN ASSOCIATED WITH A HIGH INCIDENCE OF DIVERSE MUTATIONS IN GENES SUCH AS TET2 OR EZH2 THAT ARE IMPLICATED IN EPIGENETIC MECHANISMS. WE HAVE PERFORMED GENOME-WIDE DNA METHYLATION ARRAYS AND MUTATIONAL ANALYSIS OF TET2, IDH1, IDH2, EZH2 AND JAK2 IN A GROUP OF 24 PATIENTS WITH CMML. 249 GENES WERE DIFFERENTIALLY METHYLATED BETWEEN CMML PATIENTS AND CONTROLS. USING INGENUITY PATHWAY ANALYSIS, WE IDENTIFIED ENRICHMENT IN A GENE NETWORK CENTERED AROUND PLC, JNK AND ERK SUGGESTING THAT THESE PATHWAYS, WHOSE DEREGULATION HAS BEEN RECENTLY DESCRIBED IN CMML, ARE AFFECTED BY EPIGENETIC MECHANISMS. MUTATIONS OF TET2, JAK2 AND EZH2 WERE FOUND IN 15 PATIENTS (65%), 4 PATIENTS (17%) AND 1 PATIENT (4%) RESPECTIVELY WHILE NO MUTATIONS IN THE IDH1 AND IDH2 GENES WERE IDENTIFIED. INTERESTINGLY, PATIENTS WITH WILD TYPE TET2 CLUSTERED SEPARATELY FROM PATIENTS WITH TET2 MUTATIONS, SHOWED A HIGHER DEGREE OF HYPERMETHYLATION AND WERE ASSOCIATED WITH HIGHER RISK KARYOTYPES. OUR RESULTS DEMONSTRATE THE PRESENCE OF ABERRANT DNA METHYLATION IN CMML AND IDENTIFIES TET2 MUTANT CMML AS A BIOLOGICALLY DISTINCT DISEASE SUBTYPE WITH A DIFFERENT EPIGENETIC PROFILE. 2012 18 557 37 B-CELL ANTIGEN RECEPTOR SIGNALING IN CHRONIC LYMPHOCYTIC LEUKEMIA: THERAPEUTIC TARGETS AND TRANSLATIONAL OPPORTUNITIES. B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) IS CHARACTERIZED BY CLONALLY EXPANDED AND MOLECULARLY HETEROGENEOUS POPULATIONS OF B LYMPHOCYTES WITH IMPAIRED APOPTOTIC MECHANISMS. THIS OCCURS AS A RESULT OF MULTIPLE GENETIC AND EPIGENETIC ABNORMALITIES, INCLUDING CHROMOSOMAL ABERRATIONS AND ENHANCER REGION HYPOMETHYLATION, OFTEN IMPINGING ON INTRACELLULAR SIGNALING PATHWAYS THAT ARE ESSENTIAL TO NORMAL B-CELL ACTIVATION, PROLIFERATION, AND SURVIVAL. THE B-CELL ANTIGEN RECEPTOR (BCR) SIGNALING IS ONE SUCH PATHWAY USURPED BY MALIGNANT B CELLS, AS EXEMPLIFIED BY THE EARLY PHASE CLINICAL SUCCESS ACHIEVED BY SMALL-MOLECULE AGENTS TARGETING KEY PLAYERS INVOLVED IN THE PATHWAY. SUCH NEW TARGETED AGENTS, INCLUDING THOSE THAT INHIBIT THE FUNCTION OF SPLEEN TYROSINE KINASE (SYK), BRUTON'S TYROSINE KINASE (BTK), PHOSPHATIDYLINOSITOL 3-KINASES (PI3K), AND B-CELL LYMPHOMA 2 (BCL-2), ALONG WITH THE CURRENT STANDARD THERAPY COMPRISING CHEMO-IMMUNOTHERAPIES WITH OR WITHOUT B-CELL DEPLETING BIOLOGIC AGENT RITUXIMAB (ANTI-CD20 MONOCLONAL ANTIBODY), SHOULD EXPAND THE ARMAMENTARIUM FOR CLL THERAPY. WE REVIEW THE THERAPEUTIC AGENTS CURRENTLY IN CLINICAL DEVELOPMENT WHICH TARGET DIFFERENT EFFECTORS OF THE MALIGNANT BCR SIGNALING, AND DISCUSS THEIR OVERLAPPING AND DISCRIMINATING TRANSLATIONAL OPPORTUNITIES IN THE CONTEXT OF CLL TREATMENT. 2013 19 693 32 BREAKING BOUNDARIES: PAN BETI DISRUPT 3D CHROMATIN STRUCTURE, BD2-SELECTIVE BETI ARE STRICTLY EPIGENETIC TRANSCRIPTIONAL REGULATORS. BACKGROUND: BROMODOMAIN AND EXTRATERMINAL PROTEINS (BETS) ARE MORE THAN JUST EPIGENETIC REGULATORS OF TRANSCRIPTION. HERE WE HIGHLIGHT A NEW ROLE FOR THE BET PROTEIN BRD4 IN THE MAINTENANCE OF HIGHER ORDER CHROMATIN STRUCTURE AT TOPOLOGICALLY ASSOCIATING DOMAIN BOUNDARIES (TADBS). BD2-SELECTIVE AND PAN (NON-SELECTIVE) BET INHIBITORS (BETI) DIFFERENTIALLY SUPPORT CHROMATIN STRUCTURE, SELECTIVELY AFFECTING TRANSCRIPTION AND CELL VIABILITY. METHODS: USING RNA-SEQ AND BRD4 CHIP-SEQ, THE DIFFERENTIAL EFFECT OF BETI TREATMENT ON THE TRANSCRIPTOME AND BRD4 CHROMATIN OCCUPANCY OF HUMAN AORTIC ENDOTHELIAL CELLS FROM DIABETIC PATIENTS (DHAECS) STIMULATED WITH TNFALPHA WAS EVALUATED. CHROMATIN DECONDENSATION AND DNA FRAGMENTATION WAS ASSESSED BY IMMUNOFLUORESCENCE IMAGING AND QUANTIFICATION. KEY DHAEC FINDINGS WERE VERIFIED IN PROLIFERATING MONOCYTE-LIKE THP-1 CELLS USING REAL TIME-PCR, BRD4 CO-IMMUNOPRECIPITATION STUDIES, WESTERN BLOTS, PROLIFERATION AND APOPTOSIS ASSAYS. FINDINGS: WE DISCOVERED THAT 1) BRD4 CO-LOCALIZES WITH YING-YANG 1 (YY1) AT TADBS, CRITICAL CHROMATIN STRUCTURE COMPLEXES PROXIMAL TO MANY DNA REPAIR GENES. 2) BD2-SELECTIVE BETI ENRICH BRD4/YY1 ASSOCIATIONS, WHILE PAN-BETI DO NOT. 3) FAILURE TO SUPPORT CHROMATIN STRUCTURES THROUGH BRD4/YY1 ENRICHMENT INHIBITS DNA REPAIR GENE TRANSCRIPTION, WHICH INDUCES DNA DAMAGE RESPONSES, AND CAUSES WIDESPREAD CHROMATIN DECONDENSATION, DNA FRAGMENTATION, AND APOPTOSIS. 4) BD2-SELECTIVE BETI MAINTAIN HIGH ORDER CHROMATIN STRUCTURE AND CELL VIABILITY, WHILE REDUCING DELETERIOUS PRO-INFLAMMATORY TRANSCRIPTION. INTERPRETATION: BRD4 PLAYS A PREVIOUSLY UNRECOGNIZED ROLE AT TADBS. BETI DIFFERENTIALLY IMPACT TADB STABILITY. OUR RESULTS PROVIDE TRANSLATIONAL INSIGHT FOR THE DEVELOPMENT OF BETI AS THERAPEUTICS FOR A RANGE OF DISEASES INCLUDING CVD, CHRONIC KIDNEY DISEASE, CANCER, AND COVID-19. 2022 20 1433 32 DIFFERENTIAL GENOME-WIDE ARRAY-BASED METHYLATION PROFILES IN PROGNOSTIC SUBSETS OF CHRONIC LYMPHOCYTIC LEUKEMIA. GLOBAL HYPOMETHYLATION AND REGIONAL HYPERMETHYLATION ARE WELL-KNOWN EPIGENETIC FEATURES OF CANCER; HOWEVER, IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL), STUDIES ON GENOME-WIDE EPIGENETIC MODIFICATIONS ARE LIMITED. HERE, WE ANALYZED THE GLOBAL METHYLATION PROFILES IN CLL, BY APPLYING HIGH-RESOLUTION METHYLATION MICROARRAYS (27,578 CPG SITES) TO 23 CLL SAMPLES, BELONGING TO THE IMMUNOGLOBULIN HEAVY-CHAIN VARIABLE (IGHV) MUTATED (FAVORABLE) AND IGHV UNMUTATED/IGHV3-21 (POOR-PROGNOSTIC) SUBSETS. OVERALL, RESULTS DEMONSTRATED SIGNIFICANT DIFFERENCES IN METHYLATION PATTERNS BETWEEN THESE SUBGROUPS. SPECIFICALLY, IN IGHV UNMUTATED CLL, WE IDENTIFIED METHYLATION OF 7 KNOWN OR CANDIDATE TUMOR SUPPRESSOR GENES (EG, VHL, ABI3, AND IGSF4) AS WELL AS 8 UNMETHYLATED GENES INVOLVED IN CELL PROLIFERATION AND TUMOR PROGRESSION (EG, ADORA3 AND PRF1 ENHANCING THE NUCLEAR FACTOR-KAPPAB AND MITOGEN-ACTIVATED PROTEIN KINASE PATHWAYS, RESPECTIVELY). IN CONTRAST, THESE LATTER GENES WERE SILENCED BY METHYLATION IN IGHV MUTATED PATIENTS. THE ARRAY DATA WERE VALIDATED FOR SELECTED GENES USING METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION, QUANTITATIVE REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION, AND BISULFITE SEQUENCING. FINALLY, THE SIGNIFICANCE OF DNA METHYLATION IN REGULATING GENE PROMOTERS WAS SHOWN BY REINDUCING 4 METHYLATED TUMOR SUPPRESSOR GENES (EG, VHL AND ABI3) IN IGHV UNMUTATED SAMPLES USING THE METHYL-INHIBITOR 5-AZA-2'-DEOXYCYTIDINE. TAKEN TOGETHER, OUR DATA FOR THE FIRST TIME REVEAL DIFFERENCES IN GLOBAL METHYLATION PROFILES BETWEEN PROGNOSTIC SUBSETS OF CLL, WHICH MAY UNFOLD EPIGENETIC SILENCING MECHANISMS INVOLVED IN CLL PATHOGENESIS. 2010