1 4839 164 ONCOGENIC ROLE AND TARGET PROPERTIES OF THE LYSINE-SPECIFIC DEMETHYLASE KDM1A IN CHRONIC LYMPHOCYTIC LEUKEMIA. IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL), EPIGENETIC ALTERATIONS ARE CONSIDERED TO CENTRALLY SHAPE THE TRANSCRIPTIONAL SIGNATURES THAT DRIVE DISEASE EVOLUTION AND UNDERLIE ITS BIOLOGICAL AND CLINICAL SUBSETS. CHARACTERIZATIONS OF EPIGENETIC REGULATORS, PARTICULARLY HISTONE-MODIFYING ENZYMES, ARE VERY RUDIMENTARY IN CLL. IN EFFORTS TO ESTABLISH EFFECTORS OF THE CLL-ASSOCIATED ONCOGENE T-CELL LEUKEMIA 1A (TCL1A), WE IDENTIFIED HERE THE LYSINE-SPECIFIC HISTONE DEMETHYLASE KDM1A TO INTERACT WITH THE TCL1A PROTEIN IN B CELLS IN CONJUNCTION WITH AN INCREASED CATALYTIC ACTIVITY OF KDM1A. WE DEMONSTRATE THAT KDM1A IS UPREGULATED IN MALIGNANT B CELLS. ELEVATED KDM1A AND ASSOCIATED GENE EXPRESSION SIGNATURES CORRELATED WITH AGGRESSIVE DISEASE FEATURES AND ADVERSE CLINICAL OUTCOMES IN A LARGE PROSPECTIVE CLL TRIAL COHORT. GENETIC KDM1A KNOCKDOWN IN EMU-TCL1A MICE REDUCED LEUKEMIC BURDEN AND PROLONGED ANIMAL SURVIVAL, ACCOMPANIED BY UPREGULATED P53 AND PROAPOPTOTIC PATHWAYS. GENETIC KDM1A DEPLETION ALSO AFFECTED MILIEU COMPONENTS (T, STROMAL, AND MONOCYTIC CELLS), RESULTING IN SIGNIFICANT REDUCTIONS IN THEIR CAPACITY TO SUPPORT CLL-CELL SURVIVAL AND PROLIFERATION. INTEGRATED ANALYSES OF DIFFERENTIAL GLOBAL TRANSCRIPTOMES (RNA SEQUENCING) AND H3K4ME3 MARKS (CHROMATIN IMMUNOPRECIPITATION SEQUENCING) IN EMU-TCL1A VS IKDM1AKD;EMU-TCL1A MICE (CONFIRMED IN HUMAN CLL) IMPLICATE KDM1A AS AN ONCOGENIC TRANSCRIPTIONAL REPRESSOR IN CLL WHICH ALTERS HISTONE METHYLATION PATTERNS WITH PRONOUNCED EFFECTS ON DEFINED CELL DEATH AND MOTILITY PATHWAYS. FINALLY, PHARMACOLOGIC KDM1A INHIBITION ALTERED H3K4/9 TARGET METHYLATION AND REVEALED MARKED ANTI-B-CELL LEUKEMIC SYNERGISMS. OVERALL, WE ESTABLISHED THE PATHOGENIC ROLE AND EFFECTOR NETWORKS OF KDM1A IN CLL VIA TUMOR-CELL INTRINSIC MECHANISMS AND ITS IMPACTS IN CELLS OF THE MICROENVIRONMENT. OUR DATA ALSO PROVIDE RATIONALES TO FURTHER INVESTIGATE THERAPEUTIC KDM1A TARGETING IN CLL. 2023 2 4728 37 NOTCH SIGNALING PROMOTES DISEASE INITIATION AND PROGRESSION IN MURINE CHRONIC LYMPHOCYTIC LEUKEMIA. NOTCH1 GAIN-OF-FUNCTION MUTATIONS ARE RECURRENT IN B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA (B-CLL), WHERE THEY ARE ASSOCIATED WITH ACCELERATED DISEASE PROGRESSION AND REFRACTORINESS TO CHEMOTHERAPY. THE SPECIFIC ROLE OF NOTCH1 IN THE DEVELOPMENT AND PROGRESSION OF THIS MALIGNANCY IS UNCLEAR. HERE, WE ASSESS THE IMPACT OF LOSS OF NOTCH SIGNALING AND PATHWAY HYPERACTIVATION IN AN IN VIVO MOUSE MODEL OF CLL (IGH.TEMU) THAT FAITHFULLY REPLICATES MANY FEATURES OF THE HUMAN PATHOLOGY. ABLATION OF CANONICAL NOTCH SIGNALING USING CONDITIONAL GENE INACTIVATION OF RBP-J IN IMMATURE HEMATOPOIETIC OR B-CELL PROGENITORS DELAYED CLL INDUCTION AND REDUCED INCIDENCE OF MICE DEVELOPING DISEASE. IN CONTRAST, FORCED EXPRESSION OF A DOMINANT ACTIVE FORM OF NOTCH RESULTED IN MORE ANIMALS DEVELOPING CLL WITH EARLY DISEASE ONSET. COMPARATIVE ANALYSIS OF GENE EXPRESSION AND EPIGENETIC FEATURES OF NOTCH GAIN-OF-FUNCTION AND CONTROL CLL CELLS REVEALED DIRECT AND INDIRECT REGULATION OF CELL CYCLE-ASSOCIATED GENES, WHICH LED TO INCREASED PROLIFERATION OF NOTCH GAIN-OF-FUNCTION CLL CELLS IN VIVO. THESE RESULTS DEMONSTRATE THAT NOTCH SIGNALING FACILITATES DISEASE INITIATION AND PROMOTES CLL CELL PROLIFERATION AND DISEASE PROGRESSION. 2021 3 2025 40 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 4 3415 41 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 5 5691 41 SILENCING OF HDAC6 AS A THERAPEUTIC TARGET IN CHRONIC LYMPHOCYTIC LEUKEMIA. ALTHOUGH THE TREATMENT PARADIGM FOR CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) IS RAPIDLY CHANGING, THE DISEASE REMAINS INCURABLE, EXCEPT WITH ALLOGENEIC BONE MARROW TRANSPLANTATION, AND RESISTANCE, RELAPSED DISEASE, AND PARTIAL RESPONSES PERSIST AS SIGNIFICANT CHALLENGES. RECENT STUDIES HAVE UNCOVERED ROLES FOR EPIGENETIC MODIFICATION IN THE REGULATION OF MECHANISMS CONTRIBUTING TO MALIGNANT PROGRESSION OF CLL B CELLS. HOWEVER, THE EXTENT TO WHICH EPIGENETIC MODIFIERS CAN BE TARGETED FOR THERAPEUTIC BENEFIT IN CLL PATIENTS REMAINS POORLY EXPLORED. WE REPORT FOR THE FIRST TIME THAT EXPRESSION OF EPIGENETIC MODIFIER HISTONE DEACETYLASE 6 (HDAC6) IS UPREGULATED IN CLL PATIENT SAMPLES, CELL LINES, AND EUTCL1 TRANSGENIC MOUSE MODELS COMPARED WITH HDAC6 IN NORMAL CONTROLS. GENETIC SILENCING OF HDAC6 CONFERRED SURVIVAL BENEFIT IN EUTCL1 MICE. ADMINISTRATION OF ISOFORM-SPECIFIC HDAC6 INHIBITOR ACY738 IN THE EUTCL1 AGING AND ADOPTIVE TRANSFER MODELS DETERRED PROLIFERATION OF CLL B CELLS, DELAYED DISEASE ONSET VIA DISRUPTION OF B-CELL RECEPTOR SIGNALING, AND SENSITIZED CLL B CELLS TO APOPTOSIS. FURTHERMORE, COADMINISTRATION OF ACY738 AND IBRUTINIB DISPLAYED SYNERGISTIC CELL KILL AGAINST CLL CELL LINES AND IMPROVED OVERALL SURVIVAL COMPARED WITH EITHER SINGLE AGENT IN VIVO. THESE RESULTS DEMONSTRATE FOR THE FIRST TIME THE THERAPEUTIC EFFICACY OF SELECTIVE HDAC6 INHIBITION IN PRECLINICAL CLL MODELS AND SUGGEST A RATIONALE FOR THE CLINICAL DEVELOPMENT OF HDAC6 INHIBITORS FOR CLL TREATMENT, EITHER ALONE OR IN COMBINATION WITH BRUTON TYROSINE KINASE INHIBITION. 2018 6 5475 38 RESTORING MLL REACTIVATES LATENT TUMOR SUPPRESSION-MEDIATED VULNERABILITY TO PROTEASOME INHIBITORS. MLL UNDERGOES MULTIPLE DISTINCT CHROMOSOMAL TRANSLOCATIONS TO YIELD AGGRESSIVE LEUKEMIA WITH DISMAL OUTCOMES. BESIDES THEIR WELL-ESTABLISHED ROLE IN LEUKEMOGENESIS, MLL FUSIONS ALSO POSSESS LATENT TUMOR-SUPPRESSIVE ACTIVITY, WHICH CAN BE EXPLOITED AS EFFECTIVE CANCER TREATMENT STRATEGIES USING PHARMACOLOGICAL MEANS SUCH AS PROTEASOME INHIBITORS (PIS). HERE, USING MLL-REARRANGED XENOGRAFTS AND MLL LEUKEMIC CELLS AS MODELS, WE SHOW THAT WILD-TYPE MLL IS INDISPENSABLE FOR THE LATENT TUMOR-SUPPRESSIVE ACTIVITY OF MLL FUSIONS. MLL DYSFUNCTION, SHOWN AS LOSS OF THE CHROMATIN ACCUMULATION AND SUBSEQUENT DEGRADATION OF MLL, COMPROMISES THE LATENT TUMOR SUPPRESSION OF MLL-AF4 AND IS INSTRUMENTAL FOR THE ACQUIRED PI RESISTANCE. MECHANISTICALLY, MLL DYSFUNCTION IS CAUSED BY CHRONIC PI TREATMENT-INDUCED EPIGENETIC REPROGRAMMING THROUGH THE H2BUB-ASH2L-MLL AXIS AND CAN BE SPECIFICALLY RESTORED BY HISTONE DEACETYLASE (HDAC) INHIBITORS, WHICH INDUCE HISTONE ACETYLATION AND RECRUITS MLL ON CHROMATIN TO PROMOTE CELL CYCLE GENE EXPRESSION. OUR FINDINGS NOT ONLY DEMONSTRATE THE MECHANISM UNDERLYING THE INEVITABLE ACQUISITION OF PI RESISTANCE IN MLL LEUKEMIC CELLS, BUT ALSO ILLUSTRATE THAT PREVENTING THE EMERGENCE OF PI-RESISTANT CELLS CONSTITUTES A NOVEL RATIONALE FOR COMBINATION THERAPY WITH PIS AND HDAC INHIBITORS IN MLL LEUKEMIAS. 2020 7 1976 33 EPIGENETIC ALTERATIONS IN A MURINE MODEL FOR CHRONIC LYMPHOCYTIC LEUKEMIA. EARLY STAGES IN THE DEVELOPMENT OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) HAVE NOT BEEN EXPLORED MAINLY DUE TO THE INABILITY TO STUDY NORMAL B-CELLS EN ROUTE TO TRANSFORMATION. IN ORDER TO DETERMINE SUCH EARLY EVENTS OF LEUKEMOGENESIS, WE HAVE USED A WELL ESTABLISHED MOUSE MODEL FOR CLL. OVER-EXPRESSION OF HUMAN TCL1, A KNOWN CLL ONCOGENE IN MURINE B-CELLS LEADS TO THE DEVELOPMENT OF MATURE CD19+/CD5+/IGM+ CLONAL LEUKEMIA WITH A DISEASE PHENOTYPE SIMILAR TO THAT SEEN IN HUMAN CLL. HEREIN, WE REVIEW OUR RECENT STUDY USING THIS TCL1-DRIVEN MOUSE MODEL FOR CLL AND CORRESPONDING HUMAN CLL SAMPLES IN A CROSS-SPECIES EPIGENOMICS APPROACH TO ADDRESS THE TIMING AND RELEVANCE OF EPIGENETIC EVENTS OCCURRING DURING LEUKEMOGENESIS. WE DEMONSTRATED THAT THE MOUSE MODEL RECAPITULATES THE EPIGENETIC EVENTS THAT HAVE BEEN REPORTED FOR HUMAN CLL, AFFIRMING THE POWER AND VALIDITY OF THIS MOUSE MODEL TO STUDY EARLY EPIGENETIC EVENTS IN CANCER PROGRESSION. EPIGENETIC ALTERATIONS ARE DETECTED AS EARLY AS THREE MONTHS AFTER BIRTH, FAR BEFORE DISEASE MANIFESTS AT ABOUT 11 MONTHS OF AGE. THESE MICE UNDERGO NFKAPPAB REPRESSOR COMPLEX MEDIATED INACTIVATION OF THE TRANSCRIPTION FACTOR FOXD3, WHOSE TARGETS BECOME ABERRANTLY METHYLATED AND SILENCED IN MOUSE AND HUMAN CLL. OVERALL, OUR DATA SUGGEST THE ACCUMULATED EPIGENETIC ALTERATIONS DURING CLL PATHOGENESIS AS A CONSEQUENCE OF GENE SILENCING THROUGH TCL1 AND NFKAPPAB REPRESSOR COMPLEX, SUGGESTING THE RELEVANCE FOR NFKAPPAB AS A THERAPEUTIC TARGET IN CLL. 2009 8 206 40 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 9 5871 41 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 10 3098 37 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 11 1568 27 DNA METHYLATION OF TUMOR-SUPPRESSOR MIRNA GENES IN CHRONIC LYMPHOCYTIC LEUKEMIA. DNA METHYLATION IS ONE OF THE MOST IMPORTANT EPIGENETIC MODIFICATIONS OF THE GENOME INVOLVED IN THE REGULATION OF NUMEROUS CELLULAR PROCESSES THROUGH GENE SILENCING WITHOUT ALTERING DNA SEQUENCES. MIRNAS, A CLASS OF SINGLE-STRANDED NONCODING RNAS OF 19-25 NUCLEOTIDES IN LENGTH, FUNCTION AS POST-TRANSCRIPTIONAL REGULATORS OF GENE EXPRESSION LEADING TO MRNA CLEAVAGE OR TRANSLATIONAL REPRESSION OF THEIR CORRESPONDING TARGET PROTEIN-CODING GENES. RECENTLY, DYSREGULATION OF TUMOR SUPPRESSOR MIRNAS MEDIATED BY PROMOTER DNA HYPERMETHYLATION IS IMPLICATED IN HUMAN CANCERS, INCLUDING B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). MOREOVER, IT APPEARS THAT METHYLATED MIRNA GENES COULD BE POTENTIAL BIOMARKERS FOR CLL DIAGNOSIS OR THERAPY. THIS REVIEW WILL HIGHLIGHT THE ROLE OF ABERRANT METHYLATION OF MIRNA GENES IN THE PATHOGENESIS OF CLL. 2015 12 851 26 CHIP-SEQ ANALYSIS OF HUMAN CHRONIC MYELOID LEUKEMIA CELLS. MANY TRANSCRIPTION FACTORS, CHROMATIN-ASSOCIATED PROTEINS AND REGULATORY DNA ELEMENTS ARE GENETICALLY AND/OR EPIGENETICALLY ALTERED IN CANCER, INCLUDING CHRONIC MYELOID LEUKEMIA (CML). THIS LEADS TO DEREGULATION OF TRANSCRIPTION THAT IS OFTEN CAUSALLY LINKED TO THE TUMORIGENIC STATE. CHROMATIN-IMMUNOPRECIPITATION COUPLED WITH MASSIVELY PARALLEL DNA SEQUENCING (CHIP-SEQ) IS THE KEY TECHNOLOGY TO STUDY TRANSCRIPTION AS IT ALLOWS IN VIVO WHOLE-GENOME MAPPING OF EPIGENETIC MODIFICATIONS AND INTERACTIONS OF PROTEINS WITH DNA OR CHROMATIN. HOWEVER, NUMEROUS DNA/CHROMATIN-BINDING PROTEINS, INCLUDING EZH2, REMAIN DIFFICULT TO "CHIP," THUS YIELDING GENOME-WIDE BINDING MAPS OF ONLY SUBOPTIMAL QUALITY. HERE, WE DESCRIBE A CHIP-SEQ PROTOCOL OPTIMIZED FOR HIGH-QUALITY PROTEIN-GENOME BINDING MAPS THAT HAVE PROVEN ESPECIALLY USEFUL FOR STUDYING DIFFICULT TO 'CHIP' TRANSCRIPTION REGULATORY FACTORS IN CHRONIC MYELOID LEUKEMIA (CML) AND RELATED MALIGNANCIES. 2016 13 825 34 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 14 2389 43 EPIGENETIC REPOLARIZATION OF T LYMPHOCYTES FROM CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS USING 5-AZA-2'-DEOXYCYTIDINE. T CELL IMMUNE DYSFUNCTION HAS AN IMPORTANT ROLE IN THE PROFOUND IMMUNE SUPPRESSION THAT CHARACTERIZES CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). IMPROPER POLARIZATION OF T CELLS HAS BEEN PROPOSED AS ONE OF THE MECHANISM INVOLVED. MOUNTING DATA IMPLICATES CHROMATIN REGULATION, NAMELY PROMOTER METHYLATION, IN THE PLASTICITY OF NAIVE HUMAN T CELLS. RECENT IN VITRO EVIDENCE INDICATES THAT THIS PLASTICITY MAY BE PHENOTYPICALLY ALTERED BY USING METHYLATION INHIBITORS WHICH ARE APPROVED FOR CLINICAL USE IN CERTAIN TYPES OF CANCER. THESE RESULTS BEG THE QUESTION: CAN THE INEFFECTIVE POLARIZATION OF T LYMPHOCYTES IN THE CONTEXT OF CLL BE EFFECTIVELY MODULATED USING METHYLATION INHIBITORS IN A SUSTAINABLE THERAPEUTIC FASHION? TO ANSWER THIS QUESTION OUR LABORATORY HAS STUDIED THE EFFECTS OF 5-AZA-2'-DEOXYCYTIDINE (5A2) IN HELPER AND CYTOTOXIC T LYMPHOCYTES FROM HEALTHY DONORS AND CLL PATIENTS IN WELL CHARACTERIZED MOLECULAR AND EPIGENETIC SIGNALING PATHWAYS INVOLVED IN EFFECTIVE POLARIZATION. MOREOVER, WE SOUGHT TO INVESTIGATE THE CONSEQUENCES OF METHYLATION INHIBITOR TREATMENT ON LYMPHOCYTE SURVIVAL, ACTIVATION INTENSITY, AND NAIVE CELL POLARIZATION. OUR DATA INDICATES THAT 5A2 TREATMENT CAN DEPOLARIZE TH2 CELLS TO EFFECTIVELY SECRETE INTERFERON GAMMA, SIGNAL VIA T-BET, AND ACHIEVE DEMETHYLATION OF CRITICAL TH1 SPECIFIC PROMOTERS. MOREOVER, WE DEMONSTRATE THAT 5A2 CAN FORCE TH1 POLARIZATION OF NAIVE T CELLS DESPITE A STRONG IL-4 STIMULI AND A LACK OF IL-12. IN CONCLUSION OUR DATA SEEKS TO DEFINE A MODALITY IN WHICH IMPROPER OR INEFFECTIVE T CELL POLARIZATION CAN BE ALTERED BY 5AZA AND COULD BE INCORPORATED IN FUTURE THERAPEUTIC INTERVENTIONS. 2011 15 3740 35 INSIGHT INTO GENETIC PREDISPOSITION TO CHRONIC LYMPHOCYTIC LEUKEMIA FROM INTEGRATIVE EPIGENOMICS. GENOME-WIDE ASSOCIATION STUDIES HAVE PROVIDED EVIDENCE FOR INHERITED GENETIC PREDISPOSITION TO CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). TO GAIN INSIGHT INTO THE MECHANISMS UNDERLYING CLL RISK WE ANALYZE CHROMATIN ACCESSIBILITY, ACTIVE REGULATORY ELEMENTS MARKED BY H3K27AC, AND DNA METHYLATION AT 42 RISK LOCI IN UP TO 486 PRIMARY CLLS. WE IDENTIFY THAT RISK LOCI ARE SIGNIFICANTLY ENRICHED FOR ACTIVE CHROMATIN IN CLL WITH EVIDENCE OF BEING CLL-SPECIFIC OR DIFFERENTIALLY REGULATED IN NORMAL B-CELL DEVELOPMENT. WE THEN USE IN SITU PROMOTER CAPTURE HI-C, IN CONJUNCTION WITH GENE EXPRESSION DATA TO REVEAL LIKELY TARGET GENES OF THE RISK LOCI. CANDIDATE TARGET GENES ARE ENRICHED FOR PATHWAYS RELATED TO B-CELL DEVELOPMENT SUCH AS MYC AND BCL2 SIGNALLING. AT 14 LOCI THE ANALYSIS HIGHLIGHTS 63 VARIANTS AS THE PROBABLE FUNCTIONAL BASIS OF CLL RISK. BY INTEGRATING GENETIC AND EPIGENETIC INFORMATION OUR ANALYSIS REVEALS NOVEL INSIGHTS INTO THE RELATIONSHIP BETWEEN INHERITED PREDISPOSITION AND THE REGULATORY CHROMATIN LANDSCAPE OF CLL. 2019 16 3918 42 LINKING ABERRANT CHROMATIN FEATURES IN CHRONIC LYMPHOCYTIC LEUKEMIA TO TRANSCRIPTION FACTOR NETWORKS. IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL), A DIVERSE SET OF GENETIC MUTATIONS IS EMBEDDED IN A DEREGULATED EPIGENETIC LANDSCAPE THAT DRIVES CANCEROGENESIS. TO ELUCIDATE THE ROLE OF ABERRANT CHROMATIN FEATURES, WE MAPPED DNA METHYLATION, SEVEN HISTONE MODIFICATIONS, NUCLEOSOME POSITIONS, CHROMATIN ACCESSIBILITY, BINDING OF EBF1 AND CTCF, AS WELL AS THE TRANSCRIPTOME OF B CELLS FROM CLL PATIENTS AND HEALTHY DONORS. A GLOBALLY INCREASED HISTONE DEACETYLASE ACTIVITY WAS DETECTED AND HALF OF THE GENOME COMPRISED TRANSCRIPTIONALLY DOWNREGULATED PARTIALLY DNA METHYLATED DOMAINS DEMARCATED BY CTCF CLL SAMPLES DISPLAYED A H3K4ME3 REDISTRIBUTION AND NUCLEOSOME GAIN AT PROMOTERS AS WELL AS CHANGES OF ENHANCER ACTIVITY AND ENHANCER LINKAGE TO TARGET GENES. A DNA BINDING MOTIF ANALYSIS IDENTIFIED TRANSCRIPTION FACTORS THAT GAINED OR LOST BINDING IN CLL AT SITES WITH ABERRANT CHROMATIN FEATURES. THESE FINDINGS WERE INTEGRATED INTO A GENE REGULATORY ENHANCER CONTAINING NETWORK ENRICHED FOR B-CELL RECEPTOR SIGNALING PATHWAY COMPONENTS. OUR STUDY PREDICTS NOVEL MOLECULAR LINKS TO TARGETS OF CLL THERAPIES AND PROVIDES A VALUABLE RESOURCE FOR FURTHER STUDIES ON THE EPIGENETIC CONTRIBUTION TO THE DISEASE. 2019 17 942 30 CHRONIC LYMPHOCYTIC LEUKEMIA PRESENCE IMPAIRS ANTIGEN-SPECIFIC CD8(+) T-CELL RESPONSES THROUGH EPIGENETIC REPROGRAMMING TOWARDS SHORT-LIVED EFFECTORS. T-CELL DYSREGULATION IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) ASSOCIATES WITH LOW RESPONSE RATES TO AUTOLOGOUS T CELL-BASED THERAPIES. HOW CLL AFFECTS ANTIGEN-SPECIFIC T-CELL RESPONSES REMAINS LARGELY UNKNOWN. WE INVESTIGATED (EPI)GENETIC AND FUNCTIONAL CONSEQUENCES OF ANTIGEN-SPECIFIC T-CELL RESPONSES IN PRESENCE OF CLL IN VITRO AND IN AN ADOPTIVE-TRANSFER MURINE MODEL. ALREADY AT STEADY-STATE, ANTIGEN-EXPERIENCED PATIENT-DERIVED T CELLS WERE SKEWED TOWARDS SHORT-LIVED EFFECTOR CELLS (SLEC) AT THE EXPENSE OF MEMORY-PRECURSOR EFFECTOR CELLS (MPEC). STIMULATION OF THESE T CELLS IN VITRO SHOWED RAPID INDUCTION OF EFFECTOR GENES AND SUPPRESSION OF KEY MEMORY TRANSCRIPTION FACTORS ONLY IN PRESENCE OF CLL CELLS, INDICATING EPIGENETIC REGULATION. THIS WAS INVESTIGATED IN VIVO BY FOLLOWING ANTIGEN-SPECIFIC RESPONSES OF NAIVE OT-I CD8(+) CELLS TO MCMV-OVA IN PRESENCE/ABSENCE OF TCL1 B-CELL LEUKEMIA. PRESENCE OF LEUKEMIA RESULTED IN INCREASED SLEC FORMATION, WITH DISTURBED INFLAMMATORY CYTOKINE PRODUCTION. CHROMATIN AND TRANSCRIPTOME PROFILING REVEALED STRONG EPIGENETIC MODIFICATIONS, LEADING TO ACTIVATION OF AN EFFECTOR AND SILENCING OF A MEMORY PROFILE THROUGH PRESENCE OF CLL CELLS. SECONDARY CHALLENGE IN VIVO CONFIRMED DYSFUNCTIONAL MEMORY RESPONSES BY ANTIGEN-EXPERIENCED OT-I CELLS GENERATED IN PRESENCE OF CLL. ALTOGETHER, WE SHOW THAT PRESENCE OF CLL INDUCES A SHORT-LIVED EFFECTOR PHENOTYPE AND IMPAIRED MEMORY RESPONSES BY EPIGENETIC REPROGRAMMING DURING PRIMARY RESPONSES. 2023 18 5210 35 PRENEOPLASTIC ALTERATIONS DEFINE CLL DNA METHYLOME AND PERSIST THROUGH DISEASE PROGRESSION AND THERAPY. MOST HUMAN CANCERS CONVERGE TO A DEREGULATED METHYLOME WITH REDUCED GLOBAL LEVELS AND ELEVATED METHYLATION AT SELECT CPG ISLANDS. TO INVESTIGATE THE EMERGENCE AND DYNAMICS OF THE CANCER METHYLOME, WE CHARACTERIZED GENOME-WIDE DNA METHYLATION IN PRE-NEOPLASTIC MONOCLONAL B CELL LYMPHOCYTOSIS (MBL) AND CHRONIC LYMPHOCYTIC LEUKEMIA (CLL), INCLUDING SERIAL SAMPLES COLLECTED ACROSS DISEASE COURSE. WE DETECTED THE ABERRANT TUMOR-ASSOCIATED METHYLATION LANDSCAPE AT CLL DIAGNOSIS AND FOUND NO SIGNIFICANTLY DIFFERENTIALLY METHYLATED REGIONS IN THE HIGH-COUNT MBL-TO-CLL TRANSITION. PATIENT METHYLOMES SHOWED REMARKABLE STABILITY WITH NATURAL DISEASE AND POST-THERAPY PROGRESSION. SINGLE CLL CELLS WERE CONSISTENTLY ABERRANTLY METHYLATED, INDICATING A HOMOGENEOUS TRANSITION TO THE ALTERED EPIGENETIC STATE, AND A DISTINCT EXPRESSION PROFILE TOGETHER WITH MBL CELLS COMPARED TO NORMAL B CELLS. OUR LONGITUDINAL ANALYSIS REVEALS THE CANCER METHYLOME TO EMERGE EARLY, WHICH MAY PROVIDE A PLATFORM FOR SUBSEQUENT GENETICALLY-DRIVEN GROWTH DYNAMICS AND TOGETHER WITH ITS PERSISTENT PRESENCE SUGGESTS A CENTRAL ROLE IN THE NORMAL-TO-CANCER TRANSITION. 2021 19 230 41 ADAPTIVE AND INNATE CYTOTOXIC EFFECTORS IN CHRONIC LYMPHOCYTIC LEUKAEMIA (CLL) SUBJECTS WITH STABLE DISEASE. CHRONIC LYMPHOCYTIC LEUKAEMIA (CLL) IS CHARACTERISED BY THE EXPANSION OF A NEOPLASTIC MATURE B CELL CLONE. CLL CLINICAL OUTCOME IS VERY HETEROGENEOUS, WITH SOME SUBJECTS NEVER REQUIRING THERAPY AND SOME SHOWING AN AGGRESSIVE DISEASE. GENETIC AND EPIGENETIC ALTERATIONS AND PRO-INFLAMMATORY MICROENVIRONMENT INFLUENCE CLL PROGRESSION AND PROGNOSIS. THE INVOLVEMENT OF IMMUNE-MEDIATED MECHANISMS IN CLL CONTROL NEEDS TO BE INVESTIGATED. WE ANALYSE THE ACTIVATION PROFILE OF INNATE AND ADAPTIVE CYTOTOXIC IMMUNE EFFECTORS IN A COHORT OF 26 CLL PATIENTS WITH STABLE DISEASE, AS KEY ELEMENTS FOR IMMUNE-MEDIATED CONTROL OF CANCER PROGRESSION. WE OBSERVED AN INCREASE IN CD54 EXPRESSION AND INTERFERON (IFN)-GAMMA PRODUCTION BY CYTOTOXIC T CELLS (CTL). CTL ABILITY TO RECOGNISE TUMOUR-TARGETS DEPENDS ON HUMAN LEUKOCYTE ANTIGENS (HLA)-CLASS I EXPRESSION. WE OBSERVED A DECREASED EXPRESSION OF HLA-A AND HLA-BC ON B CELLS OF CLL SUBJECTS, ASSOCIATED WITH A SIGNIFICANT REDUCTION IN INTRACELLULAR CALNEXIN THAT IS RELEVANT FOR HLA SURFACE EXPRESSION. NATURAL KILLER (NK) CELLS AND CTL FROM CLL SUBJECTS SHOW AN INCREASED EXPRESSION OF THE ACTIVATING RECEPTOR KIR2DS2 AND A REDUCTION OF 3DL1 AND NKG2A INHIBITING MOLECULES. THEREFORE, AN ACTIVATION PROFILE CHARACTERISES CTL AND NK CELLS OF CLL SUBJECTS WITH STABLE DISEASE. THIS PROFILE IS CONCEIVABLE WITH THE FUNCTIONAL INVOLVEMENT OF CYTOTOXIC EFFECTORS IN CLL CONTROL. 2023 20 2966 28 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