1 5101 107 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 2 1334 29 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 3 2763 29 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 4 102 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 5 4837 26 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 6 2432 33 EPIGENETIC SILENCING OF MIR-708 ENHANCES NF-KAPPAB SIGNALING IN CHRONIC LYMPHOCYTIC LEUKEMIA. MICRORNAS (MIRNAS) ARE POST-TRANSCRIPTIONAL REGULATORS OF GENE EXPRESSION AND THEIR DEREGULATION IS INVOLVED IN TUMOR DEVELOPMENT. EPIGENETIC GENE SILENCING IN CANCER BY DNA METHYLATION CONTRIBUTES TO THE SILENCING OF TUMOR-SUPPRESSOR GENES, INCLUDING MIRNAS. WE HAVE RECENTLY SHOWN THAT THE PROMOTER OF MIR-708 IS ABERRANTLY METHYLATED IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). TO CHARACTERIZE THE MOLECULAR SIGNALING NETWORKS THAT ARE INFLUENCED BY MIR-708, WE PERFORMED A LUCIFERASE-BASED SCREEN EVALUATING THE EFFECTS OF ECTOPIC MIR-708 EXPRESSION ON LEUKEMIA-RELEVANT SIGNALING PATHWAYS. WE FOUND THAT MIR-708 STRONGLY REPRESSED NF-KAPPAB SIGNALING, A PATHWAY KNOWN TO BE DEREGULATED IN CLL. AMONG THE PREDICTED MIR-708 TARGETS WAS IKKBETA (INHIBITOR OF KAPPA LIGHT POLYPEPTIDE GENE ENHANCER IN B CELLS, KINASE-BETA/IKBKB), A KEY KINASE FACILITATING NF-KAPPAB SIGNALING. WE VALIDATED THE INTERACTION OF MIR-708 WITH THE 3'-UNTRANSLATED REGION OF IKKBETA AND FOUND THAT MIR-708 OVEREXPRESSION REPRESSES ENDOGENOUS IKKBETA. PHOSPHORYLATION OF THE IKKBETA TARGET IKAPPABALPHA AND EXPRESSION OF KNOWN NF-KAPPAB TARGET GENES WERE IMPAIRED BY MIR-708. FURTHERMORE, WE IDENTIFIED AN ENHANCER REGION DOWNSTREAM OF THE MIR-708 PROMOTER THAT DISPLAYS A DISTINCT DNA METHYLATION STATUS IN CLL. HIGH ENHANCER METHYLATION IS SIGNIFICANTLY CORRELATED WITH LOWER MIR-708 EXPRESSION AND IS PREDOMINANTLY FOUND IN PATIENTS WITH POOR PROGNOSIS AND SHORTER TIME TO TREATMENT. THESE RESULTS DEMONSTRATE THAT MIR-708 REGULATES THE NF-KAPPAB PATHWAY BY TARGETING IKKBETA, AND THAT METHYLATION OF A KEY ENHANCER REGION CONTRIBUTES TO ITS SUPPRESSION IN CLL. 2015 7 1629 20 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 8 5608 33 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 9 1966 26 EPIGENETIC ALTERATION OF PRKCDBP IN COLORECTAL CANCERS AND ITS IMPLICATION IN TUMOR CELL RESISTANCE TO TNFALPHA-INDUCED APOPTOSIS. PURPOSE: PRKCDBP IS A PUTATIVE TUMOR SUPPRESSOR IN WHICH ALTERATION HAS BEEN OBSERVED IN SEVERAL HUMAN CANCERS. WE INVESTIGATED EXPRESSION AND FUNCTION OF PRKCDBP IN COLORECTAL CELLS AND TISSUES TO EXPLORE ITS CANDIDACY AS A SUPPRESSOR IN COLORECTAL TUMORIGENESIS. EXPERIMENTAL DESIGN: EXPRESSION AND METHYLATION STATUS OF PRKCDBP AND ITS EFFECT ON TUMOR GROWTH WERE EVALUATED. TRANSCRIPTIONAL REGULATION BY NF-KAPPAB SIGNALING WAS DEFINED BY LUCIFERASE REPORTER AND CHROMATIN IMMUNOPRECIPITATION ASSAYS. RESULTS: PRKCDBP EXPRESSION WAS HARDLY DETECTABLE IN 29 OF 80 (36%) PRIMARY TUMORS AND 11 OF 19 (58%) CELL LINES, AND ITS ALTERATION CORRELATED WITH TUMOR STAGE AND GRADE. PROMOTER HYPERMETHYLATION WAS COMMONLY FOUND IN CANCERS. PRKCDBP EXPRESSION INDUCED THE G(1) CELL-CYCLE ARREST AND INCREASED CELLULAR SENSITIVITY TO VARIOUS APOPTOTIC STRESSES. PRKCDBP WAS INDUCED BY TNFALPHA, AND ITS LEVEL CORRELATED WITH TUMOR CELL SENSITIVITY TO TNFALPHA-INDUCED APOPTOSIS. PRKCDBP INDUCTION BY TNFALPHA WAS DISRUPTED BY BLOCKING NF-KAPPAB SIGNALING WHILE IT WAS ENHANCED BY RELA TRANSFECTION. THE PRKCDBP PROMOTER ACTIVITY WAS INCREASED IN RESPONSE TO TNFALPHA, AND THIS RESPONSE WAS ABOLISHED BY DISRUPTION OF A KAPPAB SITE IN THE PROMOTER. PRKCDBP DELAYED THE FORMATION AND GROWTH OF XENOGRAFT TUMORS AND IMPROVED TUMOR RESPONSE TO TNFALPHA-INDUCED APOPTOSIS. CONCLUSIONS: PRKCDBP IS A PROAPOPTOTIC TUMOR SUPPRESSOR WHICH IS COMMONLY ALTERED IN COLORECTAL CANCER BY PROMOTER HYPERMETHYLATION, AND ITS GENE TRANSCRIPTION IS DIRECTLY ACTIVATED BY NF-KAPPAB IN RESPONSE TO TNFALPHA. THIS SUGGESTS THAT PRKCDBP INACTIVATION MAY CONTRIBUTE TO TUMOR PROGRESSION BY REDUCING CELLULAR SENSITIVITY TO TNFALPHA AND OTHER STRESSES, PARTICULARLY UNDER CHRONIC INFLAMMATORY MICROENVIRONMENT. 2011 10 5433 26 REL/NF-KAPPA B/I KAPPA B SIGNAL TRANSDUCTION IN THE GENERATION AND TREATMENT OF HUMAN CANCER. THE REL/NF-KAPPA B FAMILY IS A GROUP OF STRUCTURALLY-RELATED, TIGHTLY-REGULATED TRANSCRIPTION FACTORS THAT CONTROL THE EXPRESSION OF A MULTITUDE OF GENES INVOLVED IN KEY CELLULAR AND ORGANISMAL PROCESSES. THE REL/NF-KAPPA B SIGNAL TRANSDUCTION PATHWAY IS MISREGULATED IN A VARIETY OF HUMAN CANCERS, ESPECIALLY ONES OF LYMPHOID CELL ORIGIN, DUE EITHER TO GENETIC CHANGES (SUCH AS CHROMOSOMAL REARRANGEMENTS, AMPLIFICATIONS, AND MUTATIONS) OR TO CHRONIC ACTIVATION OF THE PATHWAY BY EPIGENETIC MECHANISMS. CONSTITUTIVE ACTIVATION OF THE REL/NF-KAPPA B PATHWAY CAN CONTRIBUTE TO THE ONCOGENIC STATE IN SEVERAL WAYS, FOR EXAMPLE, BY DRIVING PROLIFERATION, BY ENHANCING CELL SURVIVAL, OR BY PROMOTING ANGIOGENESIS OR METASTASIS. IN MANY CASES, INHIBITION OF REL/NF-KAPPA B ACTIVITY REVERSES ALL OR PART OF THE MALIGNANT STATE. THUS, THE REL/NF-KAPPA B PATHWAY HAS RECEIVED MUCH ATTENTION AS A FOCAL POINT FOR CLINICAL INTERVENTION. 2002 11 493 25 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 12 3049 31 GENOME-WIDE ANALYSIS REVEALS ZINC TRANSPORTER ZIP9 REGULATED BY DNA METHYLATION PROMOTES RADIATION-INDUCED SKIN FIBROSIS VIA THE TGF-BETA SIGNALING PATHWAY. RADIATION-INDUCED SKIN FIBROSIS IS A DETRIMENTAL AND CHRONIC DISORDER THAT OCCURS AFTER RADIATION EXPOSURE. DNA METHYLATION HAS BEEN CHARACTERIZED AS AN IMPORTANT REGULATORY MECHANISM OF MULTIPLE PATHOLOGICAL PROCESSES. IN THIS STUDY, WE COMPARED THE GENOME-WIDE DNA METHYLATION STATUS IN RADIATION-INDUCED FIBROTIC SKIN AND ADJACENT NORMAL TISSUES OF RATS BY METHYLATED DNA IMMUNOPRECIPITATION SEQUENCING. RADIATION-INDUCED FIBROTIC SKIN SHOWED DIFFERENTIALLY METHYLATED REGIONS ASSOCIATED WITH 3,650 PROTEIN-CODING GENES, 72 MICRORNAS, 5,836 LONG NONCODING RNAS AND 3 PIWI-INTERACTING RNAS. BY INTEGRATING THE MRNA AND METHYLATION PROFILES, THE ZINC TRANSPORTER SLC39A9/ZIP9 WAS INVESTIGATED IN GREATER DETAIL. THE PROTEIN LEVEL OF ZIP9 WAS INCREASED IN IRRADIATED SKIN TISSUES OF HUMANS, MONKEYS, AND RATS, ESPECIALLY IN RADIOGENIC FIBROTIC SKIN TISSUES. RADIATION INDUCED THE DEMETHYLATION OF A CPG DINUCLEOTIDE IN EXON 1 OF ZIP9 THAT RESULTED IN RECRUITMENT OF THE TRANSCRIPTIONAL FACTOR SP1 AND INCREASED ZIP9 EXPRESSION. OVEREXPRESSION OF ZIP9 RESULTED IN ACTIVATION OF THE PROFIBROTIC TRANSFORMING GROWTH FACTOR-BETA SIGNALING PATHWAY THROUGH PROTEIN KINASE B IN HUMAN FIBROBLASTS. IN ADDITION, RADIATION-INDUCED SKIN FIBROSIS WAS ASSOCIATED WITH INCREASED ZINC ACCUMULATION. THE ZINC CHELATOR N,N,N',N'-TETRAKIS(2-PYRIDYLMETHYL)-1,2-ETHYLENEDIAMINE ABROGATED ZIP9-INDUCED ACTIVATION OF THE TRANSFORMING GROWTH FACTOR-BETA SIGNALING PATHWAY AND ATTENUATED RADIATION-INDUCED SKIN FIBROSIS IN A RAT MODEL. IN SUMMARY, OUR FINDINGS ILLUSTRATE EPIGENETIC REGULATION OF ZIP9 AND ITS CRITICAL ROLE IN PROMOTING RADIATION-INDUCED SKIN FIBROSIS. 2020 13 2888 32 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 14 136 29 ABERRANT DNA HYPERMETHYLATION PATTERNS LEAD TO TRANSCRIPTIONAL SILENCING OF TUMOR SUPPRESSOR GENES IN UVB-EXPOSED SKIN AND UVB-INDUCED SKIN TUMORS OF MICE. OVEREXPOSURE OF THE HUMAN SKIN TO SOLAR ULTRAVIOLET (UV) RADIATION IS THE MAJOR ETIOLOGIC FACTOR FOR DEVELOPMENT OF SKIN CANCERS. HERE, WE REPORT THE RESULTS OF EPIGENETIC MODIFICATIONS IN UV-EXPOSED SKIN AND SKIN TUMORS IN A SYSTEMATIC MANNER. THE SKIN AND TUMOR SAMPLES WERE COLLECTED AFTER CHRONIC EXPOSURE OF THE SKIN OF SKH-1 HAIRLESS MICE TO UVB RADIATION USING A WELL-ESTABLISHED PHOTOCARCINOGENESIS PROTOCOL. WE FOUND A DISTINCT DNA HYPERMETHYLATION PATTERN IN THE UVB-EXPOSED EPIDERMAL SKIN AND UVB-INDUCED SKIN TUMORS THAT WAS ASSOCIATED WITH THE ELEVATED EXPRESSION AND ACTIVITY OF THE DNA METHYLTRANSFERASES (DNMT) 1, DNMT3A AND DNMT3B. TO EXPLORE THE ROLE OF HYPERMETHYLATION IN SKIN PHOTOCARCINOGENESIS, WE FOCUSED ON THE P16(INK4A) AND RASSF1A TUMOR SUPPRESSOR GENES, WHICH ARE TRANSCRIPTIONALLY SILENCED ON METHYLATION. WE ESTABLISHED THAT THE SILENCING OF THESE GENES IN UVB-EXPOSED EPIDERMIS AND UVB-INDUCED SKIN TUMORS IS ASSOCIATED WITH A NETWORK OF EPIGENETIC MODIFICATIONS, INCLUDING HYPOACETYLATION OF HISTONE H3 AND H4 AND INCREASED HISTONE DEACETYLATION, AS WELL AS RECRUITMENT OF METHYL-BINDING PROTEINS, INCLUDING MECP2 AND MBD1, TO THE METHYLATED CPGS. HIGHER LEVELS OF DNA METHYLATION AND DNMT ACTIVITY IN HUMAN SQUAMOUS CELL CARCINOMA SPECIMENS THAN IN NORMAL HUMAN SKIN SUGGEST THAT THE DATA ARE RELEVANT CLINICALLY. OUR DATA INDICATE FOR THE FIRST TIME THAT UVB-INDUCED DNA HYPERMETHYLATION, ENHANCED DNMT ACTIVITY AND HISTONE MODIFICATIONS OCCUR IN UVB-EXPOSED SKIN AND UVB-INDUCED SKIN TUMORS AND SUGGEST THAT THESE EVENTS ARE INVOLVED IN THE SILENCING OF TUMOR SUPPRESSOR GENES AND IN SKIN TUMOR DEVELOPMENT. 2011 15 5153 26 PPP2R2B HYPERMETHYLATION CAUSES ACQUIRED APOPTOSIS DEFICIENCY IN SYSTEMIC AUTOIMMUNE DISEASES. CHRONIC INFLAMMATION CAUSES TARGET ORGAN DAMAGE IN PATIENTS WITH SYSTEMIC AUTOIMMUNE DISEASES. THE FACTORS THAT ALLOW THIS PROTRACTED RESPONSE ARE POORLY UNDERSTOOD. WE ANALYZED THE TRANSCRIPTIONAL REGULATION OF PPP2R2B (B55SS), A MOLECULE NECESSARY FOR THE TERMINATION OF THE IMMUNE RESPONSE, IN PATIENTS WITH AUTOIMMUNE DISEASES. ALTERED EXPRESSION OF B55SS CONDITIONED RESISTANCE TO CYTOKINE WITHDRAWAL-INDUCED DEATH (CWID) IN PATIENTS WITH AUTOIMMUNE DISEASES. THE IMPAIRED UPREGULATION OF B55SS WAS CAUSED BY INFLAMMATION-DRIVEN HYPERMETHYLATION OF SPECIFIC CYTOSINES LOCATED WITHIN A REGULATORY ELEMENT OF PPP2R2B PREVENTING CTCF BINDING. THIS PHENOTYPE COULD BE INDUCED IN HEALTHY T CELLS BY EXPOSURE TO TNF-ALPHA. OUR RESULTS REVEAL A GENE WHOSE EXPRESSION IS AFFECTED BY AN ACQUIRED DEFECT, THROUGH AN EPIGENETIC MECHANISM, IN THE SETTING OF SYSTEMIC AUTOIMMUNITY. BECAUSE FAILURE TO REMOVE ACTIVATED T CELLS THROUGH CWID COULD CONTRIBUTE TO AUTOIMMUNE PATHOLOGY, THIS MECHANISM ILLUSTRATES A VICIOUS CYCLE THROUGH WHICH AUTOIMMUNE INFLAMMATION CONTRIBUTES TO ITS OWN PERPETUATION. 2019 16 5788 26 SREBP1 DRIVES KERATIN-80-DEPENDENT CYTOSKELETAL CHANGES AND INVASIVE BEHAVIOR IN ENDOCRINE-RESISTANT ERALPHA BREAST CANCER. APPROXIMATELY 30% OF ERALPHA BREAST CANCER PATIENTS RELAPSE WITH METASTATIC DISEASE FOLLOWING ADJUVANT ENDOCRINE THERAPIES. THE CONNECTION BETWEEN ACQUISITION OF DRUG RESISTANCE AND INVASIVE POTENTIAL IS POORLY UNDERSTOOD. IN THIS STUDY, WE DEMONSTRATE THAT THE TYPE II KERATIN TOPOLOGICAL ASSOCIATING DOMAIN UNDERGOES EPIGENETIC REPROGRAMMING IN AROMATASE INHIBITORS (AI)-RESISTANT CELLS, LEADING TO KERATIN-80 (KRT80) UPREGULATION. KRT80 EXPRESSION IS DRIVEN BY DE NOVO ENHANCER ACTIVATION BY STEROL REGULATORY ELEMENT-BINDING PROTEIN 1 (SREBP1). KRT80 UPREGULATION DIRECTLY PROMOTES CYTOSKELETAL REARRANGEMENTS AT THE LEADING EDGE, INCREASED FOCAL ADHESION AND CELLULAR STIFFENING, COLLECTIVELY PROMOTING CANCER CELL INVASION. SHEARWAVE ELASTICITY IMAGING PERFORMED ON PROSPECTIVELY RECRUITED PATIENTS CONFIRMS KRT80 LEVELS CORRELATE WITH STIFFER TUMORS. IMMUNOHISTOCHEMISTRY SHOWED INCREASED KRT80-POSITIVE CELLS AT RELAPSE AND, USING SEVERAL CLINICAL ENDPOINTS, KRT80 EXPRESSION ASSOCIATES WITH POOR SURVIVAL. COLLECTIVELY, OUR DATA UNCOVER AN UNPREDICTED AND POTENTIALLY TARGETABLE DIRECT LINK BETWEEN EPIGENETIC AND CYTOSKELETAL REPROGRAMMING PROMOTING CELL INVASION IN RESPONSE TO CHRONIC AI TREATMENT. 2019 17 4545 27 MUTANT P53 GAIN OF FUNCTION AND CHEMORESISTANCE: THE ROLE OF MUTANT P53 IN RESPONSE TO CLINICAL CHEMOTHERAPY. PURPOSE: TO REVIEW MECHANISMS UNDERLYING MUTANT P53 (MUTP53) GAIN OF FUNCTION (GOF) AND MUTP53-INDUCED CHEMORESISTANCE, AND TO INVESTIGATE THE ROLE OF MUTP53 IN RESPONSE TO CLINICAL CHEMOTHERAPY. METHODS: WE SEARCHED THE PUBMED DATABASE FOR CLINICAL STUDIES FROM THE PAST DECADE, INCLUDING DATA EVALUATING THE IMPACT OF MUTP53 IN CLINICAL CHEMOTHERAPY RESPONSE. RESULTS: INTERACTIONS BETWEEN MUTP53 AND TRANSCRIPTIONAL FACTORS, PROTEINS OR DNA STRUCTURES, AS WELL AS EPIGENETIC REGULATION, CONTRIBUTE TO MUTP53 GOF. MAJOR MECHANISMS OF MUTP53-INDUCED CHEMORESISTANCE INCLUDE ENHANCED DRUG EFFLUX AND METABOLISM, PROMOTING SURVIVAL, INHIBITING APOPTOSIS, UPREGULATING DNA REPAIR, SUPPRESSING AUTOPHAGY, ELEVATING MICROENVIRONMENTAL RESISTANCE AND INDUCING A STEM-LIKE PHENOTYPE. CLINICALLY, MUTP53 PREDICTED RESISTANCE TO CHEMOTHERAPY IN DIFFUSE LARGE B-CELL LYMPHOMA, AND ESOPHAGEAL AND OROPHARYNGEAL CANCERS, BUT ITS IMPACT ON CHRONIC LYMPHOCYTIC LEUKEMIA WAS UNCLEAR. IN BLADDER CANCER, MUTP53 DID NOT PREDICT RESISTANCE, WHEREAS IN SOME BREAST AND OVARIAN CANCERS, IT WAS ASSOCIATED WITH SENSITIVITY TO CERTAIN CHEMOTHERAPEUTIC AGENTS. CONCLUSION: MUTP53 HAS AN INTRICATE ROLE IN THE RESPONSE TO CLINICAL CHEMOTHERAPY AND SHOULD NOT BE INTERPRETED IN ISOLATION. FURTHERMORE, WHEN PREDICTING TUMOR RESPONSE TO CHEMOTHERAPY BASED ON THE P53 STATUS, THE DRUGS USED SHOULD ALSO BE TAKEN INTO CONSIDERATION. THESE CONCEPTS REQUIRE FURTHER INVESTIGATION. 2017 18 6294 23 THE PROINFLAMMATORY CYTOKINE TNFALPHA INDUCES DNA DEMETHYLATION-DEPENDENT AND -INDEPENDENT ACTIVATION OF INTERLEUKIN-32 EXPRESSION. IL-32 IS A CYTOKINE INVOLVED IN PROINFLAMMATORY IMMUNE RESPONSES TO BACTERIAL AND VIRAL INFECTIONS. HOWEVER, THE ROLE OF EPIGENETIC EVENTS IN THE REGULATION OF IL-32 GENE EXPRESSION IS UNDERSTUDIED. HERE WE SHOW THAT IL-32 IS REPRESSED BY DNA METHYLATION IN HEK293 CELLS. USING CHIP SEQUENCING, LOCUS-SPECIFIC METHYLATION ANALYSIS, CRISPR/CAS9-MEDIATED GENOME EDITING, AND RT-QPCR (QUANTITATIVE RT-PCR) AND IMMUNOBLOT ASSAYS, WE FOUND THAT SHORT-TERM TREATMENT (A FEW HOURS) WITH THE PROINFLAMMATORY CYTOKINE TUMOR NECROSIS FACTOR ALPHA (TNFALPHA) ACTIVATES IL-32 IN A DNA DEMETHYLATION-INDEPENDENT MANNER. IN CONTRAST, PROLONGED TNFALPHA TREATMENT (SEVERAL DAYS) INDUCED DNA DEMETHYLATION AT THE PROMOTER AND A CPG ISLAND IN THE IL-32 GENE IN A TET (TEN-ELEVEN TRANSLOCATION) FAMILY ENZYME- AND NF-KAPPAB-DEPENDENT MANNER. NOTABLY, THE HYPOMETHYLATION STATUS OF TRANSCRIPTIONAL REGULATORY ELEMENTS IN IL-32 WAS MAINTAINED FOR A LONG TIME (SEVERAL WEEKS), CAUSING ELEVATED IL-32 EXPRESSION EVEN IN THE ABSENCE OF TNFALPHA. CONSIDERING THAT IL-32 CAN, IN TURN, INDUCE TNFALPHA EXPRESSION, WE SPECULATE THAT SUCH FEEDFORWARD EVENTS MAY CONTRIBUTE TO THE TRANSITION FROM AN ACUTE INFLAMMATORY RESPONSE TO CHRONIC INFLAMMATION. 2019 19 3898 31 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 20 6659 35 UPREGULATION OF AKT3 CONFERS RESISTANCE TO THE AKT INHIBITOR MK2206 IN BREAST CANCER. ACQUIRED RESISTANCE TO MOLECULAR TARGETED THERAPY REPRESENTS A MAJOR CHALLENGE FOR THE EFFECTIVE TREATMENT OF CANCER. HYPERACTIVATION OF THE PI3K/AKT PATHWAY IS FREQUENTLY OBSERVED IN VIRTUALLY ALL HUMAN MALIGNANCIES, AND NUMEROUS PI3K AND AKT INHIBITORS ARE CURRENTLY UNDER CLINICAL EVALUATION. HOWEVER, MECHANISMS OF ACQUIRED RESISTANCE TO AKT INHIBITORS HAVE YET TO BE DESCRIBED. HERE, WE USE A BREAST CANCER PRECLINICAL MODEL TO IDENTIFY RESISTANCE MECHANISMS TO A SMALL MOLECULE ALLOSTERIC AKT INHIBITOR, MK2206. USING A STEP-WISE AND CHRONIC HIGH-DOSE EXPOSURE, BREAST CANCER CELL LINES HARBORING ONCOGENIC PI3K RESISTANT TO MK2206 WERE ESTABLISHED. USING THIS MODEL, WE REVEAL THAT AKT3 EXPRESSION IS MARKEDLY UPREGULATED IN AKT INHIBITOR-RESISTANT CELLS. INDUCTION OF AKT3 IS REGULATED EPIGENETICALLY BY THE BROMODOMAIN AND EXTRA TERMINAL DOMAIN PROTEINS. IMPORTANTLY, KNOCKDOWN OF AKT3, BUT NOT AKT1 OR AKT2, IN RESISTANT CELLS RESTORES SENSITIVITY TO MK2206. AKT INHIBITOR-RESISTANT CELLS ALSO DISPLAY AN EPITHELIAL TO MESENCHYMAL TRANSITION PHENOTYPE AS ASSESSED BY ALTERATIONS IN THE LEVELS OF E-CADHERIN, N-CADHERIN, AND VIMENTIN, AS WELL AS ENHANCED INVASIVENESS OF TUMOR SPHEROIDS. NOTABLY, THE INVASIVE MORPHOLOGY OF RESISTANT SPHEROIDS IS DIMINISHED UPON AKT3 DEPLETION. WE ALSO SHOW THAT RESISTANCE TO MK2206 IS REVERSIBLE BECAUSE UPON DRUG REMOVAL RESISTANT CELLS REGAIN SENSITIVITY TO AKT INHIBITION, ACCOMPANIED BY REEXPRESSION OF EPITHELIAL MARKERS AND REDUCTION OF AKT3 EXPRESSION, IMPLYING THAT EPIGENETIC REPROGRAMMING CONTRIBUTES TO ACQUISITION OF RESISTANCE. THESE FINDINGS PROVIDE A RATIONALE FOR DEVELOPING THERAPEUTICS TARGETING AKT3 TO CIRCUMVENT ACQUIRED RESISTANCE IN BREAST CANCER. MOL CANCER THER; 15(8); 1964-74. (C)2016 AACR. 2016