1 2888 138 GAIN-OF-FUNCTION MUTATION OF GATA-2 IN ACUTE MYELOID TRANSFORMATION OF CHRONIC MYELOID LEUKEMIA. ACQUISITION OF ADDITIONAL GENETIC AND/OR EPIGENETIC ABNORMALITIES OTHER THAN THE BCR/ABL FUSION GENE IS BELIEVED TO CAUSE DISEASE PROGRESSION IN CHRONIC MYELOID LEUKEMIA (CML) FROM CHRONIC PHASE TO BLAST CRISIS (BC). TO GAIN INSIGHTS INTO THE UNDERLYING MECHANISMS OF PROGRESSION TO BC, WE SCREENED DNA SAMPLES FROM CML PATIENTS DURING BLAST TRANSFORMATION FOR MUTATIONS IN A NUMBER OF TRANSCRIPTION FACTOR GENES THAT ARE CRITICAL FOR MYELOID-LYMPHOID DEVELOPMENT. IN 85 CASES OF CML BLAST TRANSFORMATION, WE IDENTIFIED TWO NEW MUTATIONS IN THE CODING REGION OF GATA-2, A NEGATIVE REGULATOR OF HEMATOPOIETIC STEM/PROGENITOR CELL DIFFERENTIATION. A L359V SUBSTITUTION WITHIN ZINC FINGER DOMAIN (ZF) 2 OF GATA-2 WAS FOUND IN EIGHT CASES WITH MYELOMONOBLASTIC FEATURES, WHEREAS AN IN-FRAME DELETION OF 6 AA (DELTA341-346) SPANNING THE C-TERMINAL BORDER OF ZF1 WAS DETECTED IN ONE PATIENT AT MYELOID BC WITH EOSINOPHILIA. FURTHER STUDIES INDICATED THAT L359V NOT ONLY INCREASED TRANSACTIVATION ACTIVITY OF GATA-2 BUT ALSO ENHANCED ITS INHIBITORY EFFECTS ON THE ACTIVITY OF PU.1, A MAJOR REGULATOR OF MYELOPOIESIS. CONSISTENT WITH THE MYELOMONOBLASTIC FEATURES OF CML TRANSFORMATION WITH THE GATA-2 L359V MUTANT, TRANSDUCTION OF THE GATA-2 L359V MUTANT INTO HL-60 CELLS OR BCR/ABL-HARBORING MURINE CELLS DISTURBED MYELOMONOCYTIC DIFFERENTIATION/PROLIFERATION IN VITRO AND IN VIVO, RESPECTIVELY. THESE DATA STRONGLY SUGGEST THAT GATA-2 MUTATIONS MAY PLAY A ROLE IN ACUTE MYELOID TRANSFORMATION IN A SUBSET OF CML PATIENTS. 2008 2 1629 36 DNMT3A ARG882 MUTATION DRIVES CHRONIC MYELOMONOCYTIC LEUKEMIA THROUGH DISTURBING GENE EXPRESSION/DNA METHYLATION IN HEMATOPOIETIC CELLS. THE GENE ENCODING DNA METHYLTRANSFERASE 3A (DNMT3A) IS MUTATED IN APPROXIMATELY 20% OF ACUTE MYELOID LEUKEMIA CASES, WITH ARG882 (R882) AS THE HOTSPOT. HERE, WE ADDRESSED THE TRANSFORMATION ABILITY OF THE DNMT3A-ARG882HIS (R882H) MUTANT BY USING A RETROVIRAL TRANSDUCTION AND BONE MARROW TRANSPLANTATION (BMT) APPROACH AND FOUND THAT THE MUTANT GENE CAN INDUCE ABERRANT PROLIFERATION OF HEMATOPOIETIC STEM/PROGENITOR CELLS. AT 12 MO POST-BMT, ALL MICE DEVELOPED CHRONIC MYELOMONOCYTIC LEUKEMIA WITH THROMBOCYTOSIS. RNA MICROARRAY ANALYSIS REVEALED ABNORMAL EXPRESSIONS OF SOME HEMATOPOIESIS-RELATED GENES, AND THE DNA METHYLATION ASSAY IDENTIFIED CORRESPONDING CHANGES IN METHYLATION PATTERNS IN GENE BODY REGIONS. MOREOVER, DNMT3A-R882H INCREASED THE CDK1 PROTEIN LEVEL AND ENHANCED CELL-CYCLE ACTIVITY, THEREBY CONTRIBUTING TO LEUKEMOGENESIS. 2014 3 2763 36 EXPRESSION OF THE LEUKEMIC PROGNOSTIC MARKER CD7 IS LINKED TO EPIGENETIC MODIFICATIONS IN CHRONIC MYELOID LEUKEMIA. BACKGROUND: EXPRESSION LEVELS OF THE CELL SURFACE GLYCOPROTEIN, CD7, AND THE SERINE PROTEASE, ELASTASE 2 (ELA2), IN THE LEUKEMIC CELLS OF PATIENTS WITH CHRONIC MYELOID LEUKEMIA (CML) HAVE BEEN ASSOCIATED WITH CLINICAL OUTCOME. HOWEVER, LITTLE IS KNOWN ABOUT THE MECHANISMS THAT UNDERLIE THE VARIABLE EXPRESSION OF THESE GENES IN THE LEUKEMIC CELLS. RESULTS: TO ADDRESS THIS QUESTION, WE COMPARED THE LEVEL OF THEIR EXPRESSION WITH THE DNA METHYLATION AND HISTONE ACETYLATION STATUS OF 5' SEQUENCES OF BOTH GENES IN LEUKEMIC CELL LINES AND PRIMITIVE (LIN-CD34+) LEUKEMIC CELLS FROM CHRONIC PHASE CML PATIENTS. DNA METHYLATION OF THE ELA2 GENE PROMOTER DID NOT CORRELATE WITH ITS EXPRESSION PATTERN IN LIN-CD34+ CELLS FROM CHRONIC PHASE CML PATIENT SAMPLES EVEN THOUGH THERE WAS CLEAR DIFFERENTIAL DNA METHYLATION OF THIS LOCUS IN ELA2-EXPRESSING AND NON-EXPRESSING CELL LINES. IN CONTRAST, WE FOUND A STRONG RELATION BETWEEN CD7 EXPRESSION AND TRANSCRIPTION-PERMISSIVE CHROMATIN MODIFICATIONS, BOTH AT THE LEVEL OF DNA METHYLATION AND HISTONE ACETYLATION WITH EVIDENCE OF HYPOMETHYLATION OF THE CD7 PROMOTER REGION IN THE LIN-CD34+ CELLS FROM CML PATIENTS WITH HIGH CD7 EXPRESSION. CONCLUSION: THESE FINDINGS INDICATE A LINK BETWEEN EPIGENETIC MODIFICATIONS AND CD7 EXPRESSION IN PRIMITIVE CML CELLS. 2010 4 5608 43 RUNX1-EVI1 DISRUPTS LINEAGE DETERMINATION AND THE CELL CYCLE BY INTERFERING WITH RUNX1 AND EVI1 DRIVEN GENE REGULATORY NETWORKS. HEMATOLOGICAL MALIGNANCIES ARE CHARACTERISED BY A BLOCK IN DIFFERENTIATION, WHICH IN MANY CASES IS CAUSED BY RECURRENT MUTATIONS AFFECTING THE ACTIVITY OF HEMATOPOIETIC TRANSCRIPTION FACTORS. RUNX1-EVI1 IS A FUSION PROTEIN FORMED BY THE T(3;21) TRANSLOCATION LINKING TWO TRANSCRIPTION FACTORS REQUIRED FOR NORMAL HEMATOPOIESIS. RUNX1-EVI1 EXPRESSION IS FOUND IN MYELODYSPLASTIC SYNDROME, SECONDARY ACUTE MYELOID LEUKEMIA, AND BLAST CRISIS OF CHRONIC MYELOID LEUKEMIA; WITH CLINICAL OUTCOMES BEING WORSE THAN IN PATIENTS WITH RUNX1-ETO, RUNX1 OR EVI1 MUTATIONS ALONE. RUNX1-EVI1 IS USUALLY FOUND AS A SECONDARY MUTATION, THEREFORE THE MOLECULAR MECHANISMS UNDERLYING HOW RUNX1-EVI1 ALONE CONTRIBUTES TO POOR PROGNOSIS ARE UNKNOWN. TO ADDRESS THIS QUESTION, WE INDUCED EXPRESSION OF RUNX1-EVI1 IN HEMATOPOIETIC CELLS DERIVED FROM AN EMBRYONIC STEM CELL DIFFERENTIATION MODEL. INDUCTION RESULTED IN DISRUPTION OF THE RUNX1-DEPENDENT ENDOTHELIAL-HEMATOPOIETIC TRANSITION, BLOCKED THE CELL CYCLE AND UNDERMINED CELL FATE DECISIONS IN MULTIPOTENT HEMATOPOIETIC PROGENITOR CELLS. INTEGRATIVE ANALYSES OF GENE EXPRESSION WITH CHROMATIN AND TRANSCRIPTION FACTOR BINDING DATA DEMONSTRATED THAT RUNX1-EVI1 BINDING CAUSED THE RE-DISTRIBUTION OF ENDOGENOUS RUNX1 WITHIN THE GENOME AND INTERFERED WITH BOTH RUNX1 AND EVI1 REGULATED GENE EXPRESSION PROGRAMS. IN SUMMARY, RUNX1-EVI1 EXPRESSION ALONE LEADS TO EXTENSIVE EPIGENETIC REPROGRAMMING WHICH IS INCOMPATIBLE WITH HEALTHY BLOOD PRODUCTION. 2021 5 3898 35 LARGE-SCALE TOPOLOGICAL DISRUPTION OF CHROMOSOME TERRITORIES 9 AND 22 IS ASSOCIATED WITH NONRESPONSE TO TREATMENT IN CML. CHRONIC MYELOID LEUKEMIA (CML) IS A MYELOPROLIFERATIVE NEOPLASM DEFINED BY THE PRESENCE OF T(9;22) TRANSLOCATION WHOSE ORIGIN HAS BEEN ASSOCIATED WITH THE TRIDIMENSIONAL GENOME ORGANIZATION. THIS REARRANGEMENT LEADS TO THE FUSION OF BCR AND ABL1 GENES GIVING RISE TO A CHIMERIC PROTEIN WITH CONSTITUTIVE KINASE ACTIVITY. IMATINIB, A TYROSINE KINASE INHIBITOR (TKI), IS USED AS A FIRST-LINE TREATMENT FOR CML, THOUGH ~40% OF CML PATIENTS DO NOT RESPOND. HERE, USING STRUCTURED ILLUMINATION MICROSCOPY (SIM) AND 3D RECONSTRUCTION, WE STUDIED THE 3D ORGANIZATION PATTERNS OF THE ABL1 AND BCR GENES, AND THEIR CHROMOSOME TERRITORIES (CTS) CT9 AND CT22, IN CD34+ CELLS FROM CML PATIENTS THAT RESPONDED OR NOT TO TKI. WE FOUND THAT TKI RESISTANCE IN CML IS ASSOCIATED WITH HIGH LEVELS OF STRUCTURAL DISRUPTION OF CT9 AND CT22 IN CD34+ CELLS, INCREASED CT VOLUMES (ESPECIALLY FOR CT22), INTERMINGLING BETWEEN CT9 AND CT22, AND AN OPEN-CHROMATIN EPIGENETIC MARK IN CT22. ALTOGETHER OUR RESULTS SUGGEST THAT LARGE-SCALE DISRUPTION OF CT9 AND CT22 CORRELATES WITH THE CLINICAL RESPONSE OF CML PATIENTS, WHICH COULD BE TRANSLATED INTO A POTENTIAL PROGNOSTIC MARKER OF RESPONSE TO TREATMENT IN THIS DISEASE AND PROVIDE NOVEL INSIGHTS INTO THE MECHANISMS UNDERLYING RESISTANCE TO TKI IN CML. 2022 6 5101 32 POLYCOMB FACTOR PHF19 CONTROLS CELL GROWTH AND DIFFERENTIATION TOWARD ERYTHROID PATHWAY IN CHRONIC MYELOID LEUKEMIA CELLS. POLYCOMB GROUP (PCG) OF PROTEINS ARE A GROUP OF HIGHLY CONSERVED EPIGENETIC REGULATORS INVOLVED IN MANY BIOLOGICAL FUNCTIONS, SUCH AS EMBRYONIC DEVELOPMENT, CELL PROLIFERATION, AND ADULT STEM CELL DETERMINATION. PHD FINGER PROTEIN 19 (PHF19) IS AN ASSOCIATED FACTOR OF POLYCOMB REPRESSOR COMPLEX 2 (PRC2), OFTEN UPREGULATED IN HUMAN CANCERS. IN PARTICULAR, MYELOID LEUKEMIA CELL LINES SHOW INCREASED LEVELS OF PHF19, YET LITTLE IS KNOWN ABOUT ITS FUNCTION. HERE, WE HAVE CHARACTERIZED THE ROLE OF PHF19 IN MYELOID LEUKEMIA CELLS. WE DEMONSTRATED THAT PHF19 DEPLETION DECREASES CELL PROLIFERATION AND PROMOTES CHRONIC MYELOID LEUKEMIA (CML) DIFFERENTIATION. MECHANISTICALLY, WE HAVE SHOWN HOW PHF19 REGULATES THE PROLIFERATION OF CML THROUGH A DIRECT REGULATION OF THE CELL CYCLE INHIBITOR P21. FURTHERMORE, WE OBSERVED THAT MTF2, A PHF19 HOMOLOG, PARTIALLY COMPENSATES FOR PHF19 DEPLETION IN A SUBSET OF TARGET GENES, INSTRUCTING SPECIFIC ERYTHROID DIFFERENTIATION. TAKEN TOGETHER, OUR RESULTS SHOW THAT PHF19 IS A KEY TRANSCRIPTIONAL REGULATOR FOR CELL FATE DETERMINATION AND COULD BE A POTENTIAL THERAPEUTIC TARGET FOR MYELOID LEUKEMIA TREATMENT. 2021 7 4556 28 MUTATIONAL SPECTRUM OF MYELOID MALIGNANCIES WITH INV(3)/T(3;3) REVEALS A PREDOMINANT INVOLVEMENT OF RAS/RTK SIGNALING PATHWAYS. MYELOID MALIGNANCIES BEARING CHROMOSOMAL INV(3)/T(3;3) ABNORMALITIES ARE AMONG THE MOST THERAPY-RESISTANT LEUKEMIAS. DEREGULATED EXPRESSION OF EVI1 IS THE MOLECULAR HALLMARK OF THIS DISEASE; HOWEVER, THE GENOME-WIDE SPECTRUM OF COOPERATING MUTATIONS IN THIS DISEASE SUBSET HAS NOT BEEN SYSTEMATICALLY ELUCIDATED. HERE, WE SHOW THAT 98% OF INV(3)/T(3;3) MYELOID MALIGNANCIES HARBOR MUTATIONS IN GENES ACTIVATING RAS/RECEPTOR TYROSINE KINASE (RTK) SIGNALING PATHWAYS. IN ADDITION, HEMIZYGOUS MUTATIONS IN GATA2, AS WELL AS HETEROZYGOUS ALTERATIONS IN RUNX1, SF3B1, AND GENES ENCODING EPIGENETIC MODIFIERS, FREQUENTLY CO-OCCUR WITH THE INV(3)/T(3;3) ABERRATION. NOTABLY, NEITHER MUTATIONAL PATTERNS NOR GENE EXPRESSION PROFILES DIFFER ACROSS INV(3)/T(3;3) ACUTE MYELOID LEUKEMIA, CHRONIC MYELOID LEUKEMIA, AND MYELODYSPLASTIC SYNDROME CASES, SUGGESTING RECOGNITION OF INV(3)/T(3;3) MYELOID MALIGNANCIES AS A SINGLE DISEASE ENTITY IRRESPECTIVE OF BLAST COUNT. THE HIGH INCIDENCE OF ACTIVATING RAS/RTK SIGNALING MUTATIONS MAY PROVIDE A TARGET FOR A RATIONAL TREATMENT STRATEGY IN THIS HIGH-RISK PATIENT GROUP. 2015 8 671 44 BOTHROPS MOOJENI L-AMINO ACID OXIDASE INDUCES APOPTOSIS AND EPIGENETIC MODULATION ON BCR-ABL(+) CELLS. BACKGROUND: RESISTANCE TO APOPTOSIS IN CHRONIC MYELOID LEUKEMIA (CML) IS ASSOCIATED WITH CONSTITUTIVE TYROSINE KINASE ACTIVITY OF THE BCR-ABL ONCOPROTEIN. THE DEREGULATED EXPRESSION OF APOPTOSIS-RELATED GENES AND ALTERATION IN EPIGENETIC MACHINERY MAY ALSO CONTRIBUTE TO APOPTOSIS RESISTANCE IN CML. TYROSINE KINASE INHIBITORS TARGET THE BCR-ABL ONCOPROTEIN AND ARE USED IN CML TREATMENT. THE RESISTANCE OF CML PATIENTS TO TYROSINE KINASE INHIBITORS HAS GUIDED THE SEARCH FOR NEW COMPOUNDS THAT MAY INDUCE APOPTOSIS IN BCR-ABL(+) LEUKEMIC CELLS AND IMPROVE THE DISEASE TREATMENT. METHODS: IN THE PRESENT STUDY, WE INVESTIGATED WHETHER THE L-AMINO ACID OXIDASE ISOLATED FROM BOTHROPS MOOJENI SNAKE VENOM (BMOOLAAO-I) (I) WAS CYTOTOXIC TO BCR-ABL(+) CELL LINES (HL-60.BCR-ABL, K562-S, AND K562-R), HL-60 (ACUTE PROMYELOCYTIC LEUKEMIA) CELLS, THE NON-TUMOR CELL LINE HEK-293, AND PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC); AND (II) AFFECTED EPIGENETIC MECHANISMS, INCLUDING DNA METHYLATION AND MICRORNAS EXPRESSION IN VITRO. RESULTS: BMOOLAAO-I INDUCED ROS PRODUCTION, APOPTOSIS, AND DIFFERENTIAL DNA METHYLATION PATTERN OF REGULATORY APOPTOSIS GENES. THE TOXIN UPREGULATED EXPRESSION OF THE PRO-APOPTOTIC GENES BID AND FADD AND DOWNREGULATED DFFA EXPRESSION IN LEUKEMIC CELL LINES, AS WELL AS INCREASED MIR-16 EXPRESSION - WHOSE MAJOR PREDICTED TARGET IS THE ANTI-APOPTOTIC GENE BCL2 - IN BCR-ABL(+) CELLS. CONCLUSION: BMOOLAAO-I EXERTS SELECTIVE ANTITUMOR ACTION MEDIATED BY H(2)O(2) RELEASE AND INDUCES APOPTOSIS, AND ALTERATIONS IN EPIGENETIC MECHANISMS. THESE RESULTS SUPPORT FUTURE INVESTIGATIONS ON THE EFFECT OF BMOOLAAO-I ON IN VIVO MODELS TO DETERMINE ITS POTENTIAL IN CML THERAPY. 2020 9 2088 44 EPIGENETIC DYSREGULATION OF SECRETED FRIZZLED-RELATED PROTEINS IN MYELOPROLIFERATIVE NEOPLASMS COMPLEMENTS THE JAK2V617F-MUTATION. BACKGROUND: SECRETED FRIZZLED-RELATED PROTEINS (SFRPS) ARE ANTAGONISTS OF THE WNT SIGNALING PATHWAY, WHICH PLAYS A CENTRAL ROLE IN STEM CELL MAINTENANCE AND DIFFERENTIATION OF STEM CELLS AND HEMATOPOIETIC PROGENITORS. EPIGENETIC DOWNREGULATION OF SFRPS BY PROMOTER HYPERMETHYLATION HAS BEEN DESCRIBED TO BE INVOLVED IN THE PATHOGENESIS OF HEMATOPOIETIC MALIGNANCIES. THERE IS AN ASSOCIATION BETWEEN ABERRANT WNT SIGNALING AND THE ESTABLISHED CANCER STEM CELL CONCEPT. IN CONTRAST TO BCR-ABL1-POSITIVE CHRONIC MYELOID LEUKEMIA CML, BCR-ABL1-NEGATIVE MYELOPROLIFERATIVE NEOPLASMS (PH-MPN) ARE CHARACTERIZED BY THE FREQUENT OCCURRENCE OF AN AUTOACTIVATING MUTATION IN THE JAK2 TYROSINE KINASE (JAK2V617F) OR OTHER MUTATIONS IN THE JAK-STAT PATHWAY. HOWEVER, PATHOGENETIC MECHANISMS OF JAK2 MUTATED OR UNMUTATED PH-MPN REMAIN NOT COMPLETELY UNDERSTOOD. WE DETERMINED THE PROMOTER METHYLATION STATUS OF SFRP-1, -2, -4, AND -5 IN 57 MPN PATIENT SAMPLES BY METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION (PCR) (MSP). JAK2V617F WAS ASSESSED BY ALLELE-SPECIFIC PCR. RESULTS: ABERRANT METHYLATION AMONG PRIMARY MPN SAMPLES WAS 4% FOR SFRP-1, 25% FOR SFRP-2, 2% FOR SFRP-4, AND 0% FOR SFRP-5. HYPERMETHYLATION OF SFRP-2, WHICH WAS THE MOST FREQUENTLY HYPERMETHYLATED GENE IN OUR STUDY, COULD NOT BE CORRELATED TO ANY SPECIFIC MPN SUBTYPE. HOWEVER, WE DETECTED A SIGNIFICANT CORRELATION BETWEEN SFRP-2 METHYLATION AND PRESENCE OF A JAK2V617F MUTATION (P = 0.008). NONE OF THE 10 CML SAMPLES SHOWED ANY SFRP-METHYLATION. CONCLUSIONS: OUR DATA INDICATE THAT EPIGENETIC DYSREGULATION OF THE WNT SIGNALING PATHWAY IS A COMMON EVENT IN MPN WITH ABERRANT METHYLATION OF AT LEAST ONE SFRP BEING DETECTED IN 25% OF THE PRIMARY PATIENT SAMPLES AND IN 30% IF ONLY ACCOUNTING FOR PH-MPN. A SIGNIFICANT CORRELATION BETWEEN SFRP-2 METHYLATION AND PRESENCE OF JAK2V617F IN OUR DATA SUPPORTS THE HYPOTHESIS THAT EPIGENETIC DYSREGULATION MAY BE A COMPLEMENTARY MECHANISM TO GENETIC ABERRATIONS. ABERRANT METHYLATION OF CRUCIAL STEM CELL MAINTENANCE GENES SEEMS TO CONTRIBUTE TO DISEASE PATHOGENESIS IN PH-MPN. 2012 10 1334 34 DEREGULATION OF AIOLOS EXPRESSION IN CHRONIC LYMPHOCYTIC LEUKEMIA IS ASSOCIATED WITH EPIGENETIC MODIFICATIONS. CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) IS CHARACTERIZED BY A CLONAL ACCUMULATION OF MATURE NEOPLASTIC B CELLS THAT ARE RESISTANT TO APOPTOSIS. AIOLOS, A MEMBER OF THE IKAROS FAMILY OF ZINC-FINGER TRANSCRIPTION FACTORS, PLAYS AN IMPORTANT ROLE IN THE CONTROL OF MATURE B LYMPHOCYTE DIFFERENTIATION AND MATURATION. IN THIS STUDY, WE SHOWED THAT AIOLOS EXPRESSION IS UP-REGULATED IN B-CLL CELLS. THIS OVEREXPRESSION DOES NOT IMPLICATE ISOFORM IMBALANCE OR DISTURB AIOLOS SUBCELLULAR LOCALIZATION. THE CHROMATIN STATUS AT THE AIOLOS PROMOTER IN CLL IS DEFINED BY THE DEMETHYLATION OF DNA AND AN ENRICHMENT OF EUCHROMATIN ASSOCIATED HISTONE MARKERS, SUCH AS THE DIMETHYLATION OF THE LYSINE 4 ON HISTONE H3. THESE EPIGENETIC MODIFICATIONS SHOULD ALLOW ITS UPSTREAM EFFECTORS, SUCH AS NUCLEAR FACTOR-KAPPAB, CONSTITUTIVELY ACTIVATED IN CLL, TO GAIN ACCESS TO PROMOTER, RESULTING UP-REGULATION OF AIOLOS. TO DETERMINE THE CONSEQUENCES OF AIOLOS DEREGULATION IN CLL, WE ANALYZED THE EFFECTS OF AIOLOS OVEREXPRESSION OR DOWN-REGULATION ON APOPTOSIS. AIOLOS IS INVOLVED IN CELL SURVIVAL BY REGULATING THE EXPRESSION OF SOME BCL-2 FAMILY MEMBERS. OUR RESULTS STRONGLY SUGGEST THAT AIOLOS DEREGULATION BY EPIGENETIC MODIFICATIONS MAY BE A HALLMARK OF CLL. 2011 11 5775 37 SPERMIDINE/SPERMINE N(1)-ACETYLTRANSFERASE ACTIVITY ASSOCIATES WITH WHITE BLOOD CELL COUNT IN MYELOID LEUKEMIAS. THE METABOLISM OF POLYAMINES, THE CATIONIC SMALL MOLECULES ESSENTIAL FOR CELL PROLIFERATION AND DIFFERENTIATION, IS ALTERED IN CANCER CELLS AND CAN BE EXPLOITED IN CANCER DIAGNOSIS AND THERAPY. SPERMIDINE/SPERMINE N(1)-ACETYLTRANSFERASE (SSAT), WHICH REGULATES INTRACELLULAR LEVELS OF POLYAMINES BY CATABOLIZING SPERMIDINE AND SPERMINE, HAS A CONTROVERSIAL ROLE IN THE DEVELOPMENT OF CANCERS. IN THIS STUDY, THE POLYAMINE METABOLISM AND FUNCTION OF SSAT WERE CHARACTERIZED IN ACUTE MYELOID LEUKEMIA (AML), CHRONIC MYELOID LEUKEMIA (CML), AND ACUTE LYMPHOID LEUKEMIA PATIENT SAMPLES. ALSO, MICE OVEREXPRESSING SSAT AND HAVING A MYELOPROLIFERATIVE PHENOTYPE WERE ANALYZED FOR THEIR RESPONSE TO DECITABINE AND HISTONE DEACETYLASE INHIBITOR TRICHOSTATIN A. THE PRESENCE OF EPIGENETIC FACTORS IN THE BONE MARROW CELLS OF SSAT MICE WAS ANALYZED. ELEVATED LEVELS OF SPERMIDINE AND SPERMINE, AS WELL AS INCREASED ACTIVITY OF SSAT, WERE DETECTED IN AML, CML, AND ACUTE LYMPHOID LEUKEMIA PATIENTS COMPARED WITH THE CONTROLS. HOWEVER, WE FOUND SSAT ACTIVITY TO BE ASSOCIATED WITH WHITE BLOOD CELL COUNT ONLY IN AML AND CML PATIENTS. DECITABINE TREATMENT BROUGHT THE PERIPHERAL BLOOD AND BONE MARROW CELL COUNTS OF SSAT MICE TO THE LEVEL OF WILD-TYPE MICE. SPERMIDINE/SPERMINE N(1)-ACETYLTRANSFERASE MICE HAD INCREASED HISTONE METHYLATION AND AN INCREASED LEVEL OF HISTONE DEACETYLASE 1 IN THEIR BONE MARROW CELLS. THE STUDY SUGGESTS THAT SSAT INFLUENCES THE DEVELOPMENT OF MYELOID MALIGNANCIES, AND EPIGENETIC FACTORS PARTLY CONTRIBUTE TO THE SSAT OVEREXPRESSION-INDUCED MYELOPROLIFERATIVE DISEASE IN MICE. 2014 12 4837 33 ONCOGENIC GENE EXPRESSION AND EPIGENETIC REMODELING OF CIS-REGULATORY ELEMENTS IN ASXL1-MUTANT CHRONIC MYELOMONOCYTIC LEUKEMIA. MYELOID NEOPLASMS ARE CLONAL HEMATOPOIETIC STEM CELL DISORDERS DRIVEN BY THE SEQUENTIAL ACQUISITION OF RECURRENT GENETIC LESIONS. TRUNCATING MUTATIONS IN THE CHROMATIN REMODELER ASXL1 (ASXL1(MT)) ARE ASSOCIATED WITH A HIGH-RISK DISEASE PHENOTYPE WITH INCREASED PROLIFERATION, EPIGENETIC THERAPEUTIC RESISTANCE, AND POOR SURVIVAL OUTCOMES. WE PERFORMED A MULTI-OMICS INTERROGATION TO DEFINE GENE EXPRESSION AND CHROMATIN REMODELING ASSOCIATED WITH ASXL1(MT) IN CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML). ASXL1(MT) ARE ASSOCIATED WITH A LOSS OF REPRESSIVE HISTONE METHYLATION AND INCREASE IN PERMISSIVE HISTONE METHYLATION AND ACETYLATION IN PROMOTER REGIONS. ASXL1(MT) ARE FURTHER ASSOCIATED WITH DE NOVO ACCESSIBILITY OF DISTAL ENHANCERS BINDING ETS TRANSCRIPTION FACTORS, TARGETING IMPORTANT LEUKEMOGENIC DRIVER GENES. CHROMATIN REMODELING OF PROMOTERS AND ENHANCERS IS STRONGLY ASSOCIATED WITH GENE EXPRESSION AND HETEROGENOUS AMONG OVEREXPRESSED GENES. THESE RESULTS PROVIDE A COMPREHENSIVE MAP OF THE TRANSCRIPTOME AND CHROMATIN LANDSCAPE OF ASXL1(MT) CMML, FORMING AN IMPORTANT FRAMEWORK FOR THE DEVELOPMENT OF NOVEL THERAPEUTIC STRATEGIES TARGETING ONCOGENIC CIS INTERACTIONS. 2022 13 3484 45 IDENTIFICATION OF CHROMATIN REMODELING GENES ARID4A AND ARID4B AS LEUKEMIA SUPPRESSOR GENES. BACKGROUND: LEUKEMIA EVOLVES THROUGH A MULTISTEP PROCESS FROM PREMALIGNANCY TO MALIGNANCY. EPIGENETIC ALTERATIONS, INCLUDING HISTONE MODIFICATIONS, HAVE BEEN PROPOSED TO PLAY AN IMPORTANT ROLE IN TUMORIGENESIS. THE INVOLVEMENT OF TWO CHROMATIN REMODELING GENES, RETINOBLASTOMA-BINDING PROTEIN 1 (RBBP1/ARID4A) AND RBBP1-LIKE 1 (RBBP1L1/ARID4B), IN LEUKEMOGENESIS WAS NOT CHARACTERIZED. METHODS: THE LEUKEMIC PHENOTYPE OF MICE DEFICIENT FOR ARID4A WITH OR WITHOUT HAPLOINSUFFICIENCY FOR ARID4B WAS INVESTIGATED BY SERIALLY MONITORING COMPLETE BLOOD COUNTS TOGETHER WITH MICROSCOPIC HISTOLOGIC ANALYSIS AND FLOW CYTOMETRIC ANALYSIS OF BONE MARROW AND SPLEEN FROM THE ARID4A(-/-) MICE OR ARID4A(-/-)ARID4B(+/-) MICE. REGULATION IN BONE MARROW CELLS OF DOWNSTREAM GENES IMPORTANT FOR NORMAL HEMATOPOIESIS WAS ANALYZED BY REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION. GENOTYPIC EFFECTS ON HISTONE MODIFICATIONS WERE EXAMINED BY WESTERN BLOTTING AND IMMUNOFLUORESCENCE ANALYSIS. ALL STATISTICAL TESTS WERE TWO-SIDED. RESULTS: YOUNG (2-5 MONTHS OLD) ARID4A-DEFICIENT MICE HAD INEFFECTIVE BLOOD CELL PRODUCTION IN ALL HEMATOPOIETIC LINEAGES. BEYOND 5 MONTHS OF AGE, THE ARID4A(-/-) MICE MANIFESTED MONOCYTOSIS, ACCOMPANIED BY SEVERE ANEMIA AND THROMBOCYTOPENIA. THESE SICK ARID4A(-/-) MICE SHOWED BONE MARROW FAILURE WITH MYELOFIBROSIS ASSOCIATED WITH SPLENOMEGALY AND HEPATOMEGALY. FIVE OF 42 ARID4A(-/-) MICE AND 10 OF 12 ARID4A(-/-)ARID4B(+/-) MICE PROGRESSED TO ACUTE MYELOID LEUKEMIA (AML) AND HAD RAPID FURTHER INCREASES OF LEUKOCYTE COUNTS. EXPRESSION OF HOX GENES (HOXB3, HOXB5, HOXB6, AND HOXB8) WAS DECREASED IN ARID4A-DEFICIENT BONE MARROW CELLS WITH OR WITHOUT ARID4B HAPLOINSUFFICIENCY, AND FOXP3 EXPRESSION WAS REDUCED IN ARID4A(-/-)ARID4B(+/-) BONE MARROW. INCREASES OF HISTONE TRIMETHYLATION OF H3K4, H3K9, AND H4K20 (FOLD INCREASES IN TRIMETHYLATION = 32, 95% CONFIDENCE INTERVAL [CI] = 27 TO 32; 45, 95% CI = 41 TO 49; AND 2.2, 95% CI = 1.7 TO 2.7, RESPECTIVELY) WERE OBSERVED IN THE BONE MARROW OF ARID4A-DEFICIENT MICE. CONCLUSIONS: ARID4A-DEFICIENT MICE INITIALLY DISPLAY INEFFECTIVE HEMATOPOIESIS, FOLLOWED BY TRANSITION TO CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML)-LIKE MYELODYSPLASTIC/MYELOPROLIFERATIVE DISORDER, AND THEN TRANSFORMATION TO AML. THE DISEASE PROCESSES IN THE ARID4A-DEFICIENT MICE ARE VERY SIMILAR TO THE COURSE OF EVENTS IN HUMANS WITH CMML AND AML. THIS MOUSE MODEL HAS THE POTENTIAL TO FURNISH ADDITIONAL INSIGHTS INTO THE ROLE OF EPIGENETIC ALTERATIONS IN LEUKEMOGENESIS, AND IT MAY BE USEFUL IN DEVELOPING NOVEL PHARMACOLOGICAL APPROACHES TO TREATMENT OF PRELEUKEMIC AND LEUKEMIC STATES. 2008 14 4388 51 MLL2/KMT2D AND MLL3/KMT2C EXPRESSION CORRELATES WITH DISEASE PROGRESSION AND RESPONSE TO IMATINIB MESYLATE IN CHRONIC MYELOID LEUKEMIA. BACKGROUND: CHRONIC MYELOID LEUKEMIA (CML) IS A CLONAL MYELOPROLIFERATIVE NEOPLASM WHOSE PATHOGENESIS IS LINKED TO THE PHILADELPHIA CHROMOSOME PRESENCE THAT GENERATES THE BCR-ABL1 FUSION ONCOGENE. TYROSINE KINASE INHIBITORS (TKI) SUCH AS IMATINIB MESYLATE (IM) DRAMATICALLY IMPROVED THE TREATMENT EFFICIENCY AND SURVIVAL OF CML PATIENTS BY TARGETING BCR-ABL TYROSINE KINASE. THE DISEASE SHOWS THREE DISTINCT CLINICAL-LABORATORY STAGES: CHRONIC PHASE, ACCELERATED PHASE AND BLAST CRISIS. ALTHOUGH PATIENTS IN THE CHRONIC PHASE RESPOND WELL TO TREATMENT, PATIENTS IN THE ACCELERATED PHASE OR BLAST CRISIS USUALLY SHOW THERAPY RESISTANCE AND CML RELAPSE. IT IS CRUCIAL, THEREFORE, TO IDENTIFY BIOMARKERS TO PREDICT CML GENETIC EVOLUTION AND RESISTANCE TO TKI THERAPY, CONSIDERING NOT ONLY THE EFFECTS OF GENETIC ABERRATIONS BUT ALSO THE ROLE OF EPIGENETIC ALTERATIONS DURING THE DISEASE. ALTHOUGH DYSREGULATIONS IN EPIGENETIC MODULATORS SUCH AS HISTONE METHYLTRASNFERASES HAVE ALREADY BEEN DESCRIBED FOR SOME HEMATOLOGIC MALIGNANCIES, TO DATE VERY LIMITED DATA IS AVAILABLE FOR CML, ESPECIALLY WHEN CONSIDERING THE LYSINE METHYLTRANSFERASE MLL2/KMT2D AND MLL3/KMT2C. METHODS: HERE WE INVESTIGATED THE EXPRESSION PROFILE OF BOTH GENES IN CML PATIENTS IN DIFFERENT STAGES OF THE DISEASE, IN PATIENTS SHOWING DIFFERENT RESPONSES TO THERAPY WITH IM AND IN NON-NEOPLASTIC CONTROL SAMPLES. IMATINIB SENSITIVE AND RESISTANT CML CELL LINES WERE ALSO USED TO INVESTIGATE WHETHER TREATMENT WITH OTHER TYROSINE KINASE INHIBITORS INTERFERED IN THEIR EXPRESSION. RESULTS: IN PATIENTS, BOTH METHYLTRANSFERASES WERE EITHER UPREGULATED OR WITH BASAL EXPRESSION LEVEL DURING THE CHRONIC PHASE COMPARED TO CONTROLS. INTERESTINGLY, MLL3/KMT2C AND SPECIALLY MLL2/KMT2D LEVELS DECREASED DURING DISEASE PROGRESSION CORRELATING WITH DISTINCT CLINICAL STAGES. FURTHERMORE, MLL2/KMT2D WAS DECREASED IN PATIENTS RESISTANT TO IM TREATMENT. A RESCUE IN THE EXPRESSION OF BOTH MLL GENES WAS OBSERVED IN KCL22S, A CML CELL LINE SENSITIVE TO IM, AFTER TREATMENT WITH DASATINIB OR NILOTINIB WHICH WAS ASSOCIATED WITH A HIGHER RATE OF APOPTOSIS, AN ENHANCED EXPRESSION OF P21 (CDKN1A) AND A CONCOMITANT DECREASE IN THE EXPRESSION OF CDK2, CDK4 AND CYCLIN B1 (CCNB1) IN COMPARISON TO UNTREATED KCL22S CONTROL OR IM RESISTANT KCL22R CELL LINE, WHICH SUGGESTS INVOLVEMENT OF P53 REGULATED PATHWAY. CONCLUSION: OUR RESULTS ESTABLISHED A NEW ASSOCIATION BETWEEN MLL2/KMT2D AND MLL3/KMT2C GENES WITH CML AND SUGGEST THAT MLL2/KMT2D IS ASSOCIATED WITH DISEASE EVOLUTION AND MAY BE A POTENTIAL MARKER TO PREDICT THE DEVELOPMENT OF THERAPY RESISTANCE. 2018 15 3352 40 HISTONE DEMETHYLASE RBP2 MEDIATES THE BLAST CRISIS OF CHRONIC MYELOID LEUKEMIA THROUGH AN RBP2/PTEN/BCR-ABL CASCADE. EPIGENETIC DISORDERS PLAY A KEY ROLE IN TUMORIGENESIS AND DEVELOPMENT, AMONG WHICH HISTONE METHYLATION ABNORMALITIES ARE COMMON. WHILE PATIENTS LIVING WITH CHRONIC MYELOID LEUKEMIA IN THE CHRONIC PHASE (CML-CP) HAVE A GOOD RESPONSE TO TKI, BLASTIC PHASE (CML-BP) PATIENTS DEMONSTRATE POOR EFFICACY AND HIGH FATALITY RATES. HOWEVER, WHILE THE MECHANISM OF BLAST CRISIS OF CHRONIC MYELOID LEUKEMIA REMAINS UNCLEAR, HIGH EXPRESSION AND ACTIVATION OF BCR-ABL ARE USUALLY RELATED TO CML BLAST CRISIS TRANSITION. WE FOUND THAT HISTONE H3 LYSINE 4 (H3K4) DEMETHYLASE RBP2 EXPRESSION IS NEGATIVELY CORRELATED WITH BCR-ABL EXPRESSION, WHICH SUGGESTS A REGULATORY LINK BETWEEN THESE TWO GENES. WE ALSO DISCOVERED THAT RBP2 MEDIATES THE DEPHOSPHORYLATION OF BCR-ABL BY DIRECTLY DOWNREGULATING PTEN EXPRESSION, DEPENDING ON HISTONE DEMETHYLASE ACTIVITY, WHILE PTEN TARGETS PROTEIN PHOSPHATASE ACTIVITY OF BCR-ABL, A PHOSPHATASE WHICH DIRECTLY DEPHOSPHORYLATES BCR-ABL. IN CLINICAL SPECIMENS, THE MRNA EXPRESSION OF RBP2 WAS FOUND TO BE POSITIVELY CORRELATED WITH THAT OF PTEN. THESE DATA SUGGEST THAT THE UNDER-EXPRESSION OF RBP2 PROMOTES BLAST CRISIS TRANSITION BY ACTIVATING AN RBP2/PTEN/BCR-ABL CASCADE. 2019 16 573 43 BCR-ABL1-INDUCED DOWNREGULATION OF WASP IN CHRONIC MYELOID LEUKEMIA INVOLVES EPIGENETIC MODIFICATION AND CONTRIBUTES TO MALIGNANCY. CHRONIC MYELOID LEUKEMIA (CML) IS A MYELOPROLIFERATIVE DISEASE CAUSED BY THE BCR-ABL1 TYROSINE KINASE (TK). THE DEVELOPMENT OF TK INHIBITORS (TKIS) REVOLUTIONIZED THE TREATMENT OF CML PATIENTS. HOWEVER, TKIS ARE NOT EFFECTIVE TO THOSE AT ADVANCED PHASES WHEN AMPLIFIED BCR-ABL1 LEVELS AND INCREASED GENOMIC INSTABILITY LEAD TO SECONDARY ONCOGENIC MODIFICATIONS. WISKOTT-ALDRICH SYNDROME PROTEIN (WASP) IS AN IMPORTANT REGULATOR OF SIGNALING TRANSDUCTION IN HEMATOPOIETIC CELLS AND WAS SHOWN TO BE AN ENDOGENOUS INHIBITOR OF THE C-ABL TK. HERE, WE SHOW THAT THE EXPRESSION OF WASP DECREASES WITH THE PROGRESSION OF CML, INVERSELY CORRELATES WITH THE EXPRESSION OF BCR-ABL1 AND IS PARTICULARLY LOW IN BLAST CRISIS. ENFORCED EXPRESSION OF BCR-ABL1 NEGATIVELY REGULATES THE EXPRESSION OF WASP. DECREASED EXPRESSION OF WASP IS PARTIALLY DUE TO DNA METHYLATION OF THE PROXIMAL WASP PROMOTER. IMPORTANTLY, LOWER LEVELS OF WASP IN CML ADVANCED PHASE PATIENTS CORRELATE WITH POORER OVERALL SURVIVAL (OS) AND IS ASSOCIATED WITH TKI RESPONSE. INTERESTINGLY, ENFORCED EXPRESSION OF WASP IN BCR-ABL1-POSITIVE K562 CELLS INCREASES THE SUSCEPTIBILITY TO APOPTOSIS INDUCED BY TRAIL OR CHEMOTHERAPEUTIC DRUGS AND NEGATIVELY MODULATES BCR-ABL1-INDUCED TUMORIGENESIS IN VITRO AND IN VIVO. TAKEN TOGETHER, OUR DATA REVEAL A NOVEL MOLECULAR MECHANISM THAT OPERATES IN BCR-ABL1-INDUCED TUMORIGENESIS THAT CAN BE USED TO DEVELOP NEW STRATEGIES TO HELP TKI-RESISTANT, CML PATIENTS IN BLAST CRISIS (BC). 2017 17 5934 31 TARGETING FEATURES OF CURAXIN CBL0137 ON HEMATOLOGICAL MALIGNANCIES IN VITRO AND IN VIVO. THE ANTICANCER ACTIVITY OF CURAXIN CBL0137, A DNA-BINDING SMALL MOLECULE WITH CHROMATIN REMODULATING EFFECT, HAS BEEN DEMONSTRATED IN DIFFERENT CANCERS. HEREIN, A COMPARATIVE EVALUATION OF CBL0137 ACTIVITY WAS PERFORMED IN RESPECT TO ACUTE MYELOID LEUKEMIA (AML), ACUTE LYMPHOBLASTIC LEUKEMIA (ALL), CHRONIC MYELOID LEUKEMIA AND MULTIPLE MYELOMA (MM) CULTURED IN VITRO. MTT ASSAY SHOWED AML AND MM HIGHER SENSITIVITY TO CBL0137'S CYTOSTATIC EFFECT COMPARATIVELY TO OTHER HEMATOLOGICAL MALIGNANCY CELLS. FLOW CYTOMETRY CELL CYCLE ANALYSIS REVEALED AN INCREASE IN SUBG1 AND G2/M POPULATIONS AFTER CBL0137 CELL TREATMENT, BUT THE PREVALENT TYPE OF ARREST VARIED. APOPTOSIS ACTIVATION BY CBL0137 MEASURED BY ANNEXIN-V/PI DUAL STAINING WAS MORE ACTIVE IN AML AND MM CELLS. RT2 PCR ARRAY SHOWED THAT CHANGES CAUSED BY CBL0137 IN SIGNALING PATHWAYS INVOLVED IN CANCER PATHOGENESIS WERE MORE INTENSIVE IN AML AND MM CELLS. ON THE MURINE MODEL OF AML WEHI-3, CBL0137 SHOWED SIGNIFICANT ANTICANCER EFFECTS IN VIVO, WHICH WERE EVALUATED BY CORRESPONDING CHANGES IN SPLEEN AND LIVER. THUS, MORE PRONOUNCED ANTICANCER EFFECTS OF CBL0137 IN VITRO WERE OBSERVED IN RESPECT TO AML AND MM. EXPERIMENTS IN VIVO ALSO INDICATED THE PERSPECTIVE OF CBL0137 USE FOR AML TREATMENT. THIS IN ACCORDANCE WITH THE FRONTLINE TREATMENT APPROACH IN AML USING EPIGENETIC DRUGS. 2023 18 3168 37 GTPASE REGULATOR ASSOCIATED WITH THE FOCAL ADHESION KINASE (GRAF) TRANSCRIPT WAS DOWN-REGULATED IN PATIENTS WITH MYELOID MALIGNANCIES. BACKGROUND: GTPASE REGULATOR ASSOCIATED WITH THE FOCAL ADHESION KINASE (GRAF), A PUTATIVE TUMOR SUPPRESSOR GENE, IS FOUND INACTIVATED IN HEMATOPOIETIC MALIGNANCIES BY EITHER GENETIC OR EPIGENETIC ABNORMALITIES. HOWEVER, THE EXPRESSION LEVEL OF GRAF GENE HAS NOT YET BEEN STUDIED IN LEUKEMIA. THE AIM OF THIS STUDY WAS TO INVESTIGATE THE EXPRESSION LEVEL OF GRAF GENE IN THOSE PATIENTS WITH MYELOID MALIGNANCIES INCLUDING ACUTE MYELOID LEUKEMIA (AML), MYELODYSPLASTIC SYNDROME (MDS) AND CHRONIC MYELOID LEUKEMIA (CML). METHODS: THE EXPRESSION LEVELS OF GRAF TRANSCRIPT WERE DETERMINED IN 94 PATIENTS USING REAL-TIME QUANTITATIVE PCR (RQ-PCR). CLINICAL AND LABORATORY DATA OF THESE PATIENTS WERE COLLECTED AND ANALYZED. RESULTS: THE SIGNIFICANTLY DECREASED LEVEL OF GRAF TRANSCRIPT WAS OBSERVED IN THREE MYELOID MALIGNANCIES COMPARED TO CONTROLS. WITHIN AML, THERE WAS NO DIFFERENCE IN THE LEVEL OF GRAF TRANSCRIPT AMONG DIFFERENT FAB SUBTYPES (P > 0.05). DIFFERENCE WAS NOT OBSERVED IN THE AMOUNT OF GRAF MRNA BETWEEN CML AT CHRONIC PHASE AND CONTROLS. AS CML PROGRESSED, GRAF TRANSCRIPT SIGNIFICANTLY DECREASED. IN MDS, THREE CASES WITH 5Q DELETION HAD LOWER GRAF TRANSCRIPT THAN FOUR WITHOUT 5Q DELETION (MEDIAN 0.76 VS 2.99) (P > 0.05). CONCLUSION: OUR RESULTS DEMONSTRATE THAT THE GRAF TRANSCRIPT IS DECREASED IN MYELOID MALIGNANCIES. 2010 19 102 27 A REGULATORY ROLE FOR CHD2 IN MYELOPOIESIS. THE TRANSCRIPTIONAL PROGRAM THAT DICTATES HAEMATOPOIETIC CELL FATE AND DIFFERENTIATION REQUIRES AN EPIGENETIC REGULATORY AND MEMORY FUNCTION, PROVIDED BY A NETWORK OF EPIGENETIC FACTORS THAT REGULATE DNA METHYLATION, POST-TRANSLATIONAL HISTONE MODIFICATIONS AND CHROMATIN STRUCTURE. DISTURBED EPIGENETIC REGULATION CAUSES PERTURBATIONS IN THE BLOOD CELL DIFFERENTIATION PROGRAM THAT RESULTS IN VARIOUS TYPES OF HAEMATOPOIETIC DISORDERS. THUS, ACCURATE EPIGENETIC REGULATION IS ESSENTIAL FOR FUNCTIONAL HAEMATOPOIESIS. IN THIS STUDY, WE USED A CRISPR-CAS9 SCREENING APPROACH TO IDENTIFY NEW EPIGENETIC REGULATORS IN MYELOID DIFFERENTIATION. WE DESIGNED A CHROMATIN-UMI CRISPR GUIDE LIBRARY TARGETING 1092 EPIGENETIC REGULATORS. PHORBOL 12-MYRISTATE 13-ACETATE (PMA) TREATMENT OF THE CHRONIC MYELOID LEUKAEMIA CELL LINE K-562 WAS USED AS A MEGAKARYOCYTIC MYELOID DIFFERENTIATION MODEL. BOTH PREVIOUSLY DESCRIBED DEVELOPMENTAL EPIGENETIC REGULATORS AND NOVEL FACTORS WERE IDENTIFIED IN OUR SCREEN. IN THIS STUDY, WE VALIDATED AND CHARACTERIZED A ROLE FOR THE CHROMATIN REMODELLER CHD2 IN MYELOID PROLIFERATION AND MEGAKARYOCYTIC DIFFERENTIATION. 2020 20 493 38 ASSESSMENT OF P53 AND ATM FUNCTIONALITY IN CHRONIC LYMPHOCYTIC LEUKEMIA BY MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION. THE ATM-P53 DNA-DAMAGE RESPONSE (DDR) PATHWAY HAS A CRUCIAL ROLE IN CHEMORESISTANCE IN CLL, AS INDICATED BY THE ADVERSE PROGNOSTIC IMPACT OF GENETIC ABERRATIONS OF TP53 AND ATM. IDENTIFYING AND DISTINGUISHING TP53 AND ATM FUNCTIONAL DEFECTS HAS BECOME RELEVANT AS EPIGENETIC AND POSTTRANSCRIPTIONAL DYSREGULATION OF THE ATM/P53 AXIS IS INCREASINGLY BEING RECOGNIZED AS THE UNDERLYING CAUSE OF CHEMORESISTANCE. ALSO, SPECIFIC TREATMENTS SENSITIZING TP53- OR ATM-DEFICIENT CLL CELLS ARE EMERGING. WE THEREFORE DEVELOPED A NEW ATM-P53 FUNCTIONAL ASSAY WITH THE AIM TO (I) IDENTIFY AND (II) DISTINGUISH ABNORMALITIES OF TP53 VERSUS ATM AND (III) ENABLE THE IDENTIFICATION OF ADDITIONAL DEFECTS IN THE ATM-P53 PATHWAY. REVERSED TRANSCRIPTASE MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION (RT-MLPA) WAS USED TO MEASURE ATM AND/OR P53-DEPENDENT GENES AT THE RNA LEVEL FOLLOWING DNA DAMAGE USING IRRADIATION. HERE, WE SHOWED THAT THIS ASSAY IS ABLE TO IDENTIFY AND DISTINGUISH THREE SUBGROUPS OF CLL TUMORS (I.E., TP53-DEFECTIVE, ATM-DEFECTIVE AND WT) AND IS ALSO ABLE TO DETECT ADDITIONAL SAMPLES WITH A DEFECTIVE DDR, WITHOUT MOLECULAR ABERRATIONS IN TP53 AND/OR ATM. THESE FINDINGS MAKE THE ATM-P53 RT-MLPA FUNCTIONAL ASSAY A PROMISING PROGNOSTIC TOOL FOR PREDICTING TREATMENT RESPONSES IN CLL. 2015