1 3409 105 HOXA4 GENE PROMOTER HYPERMETHYLATION AS AN EPIGENETIC MECHANISM MEDIATING RESISTANCE TO IMATINIB MESYLATE IN CHRONIC MYELOID LEUKEMIA PATIENTS. DEVELOPMENT OF RESISTANCE TO IMATINIB MESYLATE (IM) IN CHRONIC MYELOID LEUKEMIA (CML) PATIENTS HAS EMERGED AS A SIGNIFICANT CLINICAL PROBLEM. THE OBSERVATION THAT INCREASED EPIGENETIC SILENCING OF POTENTIAL TUMOR SUPPRESSOR GENES CORRELATES WITH DISEASE PROGRESSION IN SOME CML PATIENTS TREATED WITH IM SUGGESTS A RELATIONSHIP BETWEEN EPIGENETIC SILENCING AND RESISTANCE DEVELOPMENT. WE HYPOTHESIZE THAT PROMOTER HYPERMETHYLATION OF HOXA4 COULD BE AN EPIGENETIC MECHANISM MEDIATING IM RESISTANCE IN CML PATIENTS. THUS A STUDY WAS UNDERTAKEN TO INVESTIGATE THE PROMOTER HYPERMETHYLATION STATUS OF HOXA4 IN CML PATIENTS ON IM TREATMENT AND TO DETERMINE ITS ROLE IN MEDIATING RESISTANCE TO IM. GENOMIC DNA WAS EXTRACTED FROM PERIPHERAL BLOOD SAMPLES OF 95 CML PATIENTS (38 GOOD RESPONDERS AND 57 RESISTANT) AND 12 NORMAL CONTROLS. ALL SAMPLES WERE BISULFITE TREATED AND ANALYSED BY METHYLATION-SPECIFIC HIGH-RESOLUTION MELT ANALYSIS. COMPARED TO THE GOOD RESPONDERS, THE HOXA4 HYPERMETHYLATION LEVEL WAS SIGNIFICANTLY HIGHER (P = 0.002) IN IM-RESISTANT CML PATIENTS. ON COMPARING THE RISK, HOXA4 HYPERMETHYLATION WAS ASSOCIATED WITH A HIGHER RISK FOR IM RESISTANCE (OR 4.658; 95% CI, 1.673-12.971; P = 0.003). THUS, IT IS REASONABLE TO SUGGEST THAT PROMOTER HYPERMETHYLATION OF HOXA4 GENE COULD BE AN EPIGENETIC MECHANISM MEDIATING IM RESISTANCE IN CML PATIENTS. 2013 2 138 51 ABERRANT DNA METHYLATION AT HOXA4 AND HOXA5 GENES ARE ASSOCIATED WITH RESISTANCE TO IMATINIB MESYLATE AMONG CHRONIC MYELOID LEUKEMIA PATIENTS. BACKGROUND: IMATINIB MESYLATE IS A MOLECULARLY TARGETED TYROSINE KINASE INHIBITOR DRUG. IT IS EFFECTIVELY USED IN THE TREATMENT OF CHRONIC MYELOID LEUKEMIA (CML) PATIENTS. HOWEVER, DEVELOPMENT OF RESISTANCE TO IMATINIB MESYLATE AS A RESULT OF BCR-ABL DEPENDENT AND BCR-ABL INDEPENDENT MECHANISMS HAS EMERGED AS A DAUNTING PROBLEM IN THE MANAGEMENT OF CML PATIENTS. BETWEEN THESE MECHANISMS, BCR-ABL INDEPENDENT MECHANISMS ARE STILL NOT ROBUSTLY UNDERSTOOD. AIM: TO INVESTIGATE THE CORRELATION OF HOXA4 AND HOXA5 PROMOTER DNA HYPERMETHYLATION WITH IMATINIB RESISTANCE AMONG CML PATIENTS. METHODS AND RESULTS: SAMPLES FROM 175 PHILADELPHIA POSITIVE CML PATIENTS (83 GOOD RESPONSE AND 92 BCR-ABL NON-MUTATED IMATINIB RESISTANT PATIENTS) WERE SUBJECTED TO METHYLATION SPECIFIC HIGH RESOLUTION MELT ANALYSIS FOR METHYLATION LEVELS QUANTIFICATION OF THE HOXA4 AND HOXA5 PROMOTER REGIONS. RECEIVER OPERATING CHARACTERISTIC CURVE ANALYSIS WAS DONE TO ELUCIDATE THE OPTIMAL METHYLATION CUT-OFF POINT FOLLOWED BY MULTIPLE LOGISTIC REGRESSION ANALYSIS. LOG-RANK ANALYSIS WAS DONE TO MEASURE THE OVERALL SURVIVAL DIFFERENCE BETWEEN CML GROUPS. THE OPTIMAL METHYLATION CUT-OFF POINT WAS FOUND TO BE AT 62.5% FOR BOTH HOXA4 AND HOXA5. CHRONIC MYELOID LEUKEMIA PATIENTS WITH >/=63% HOXA4 AND HOXA5 METHYLATION LEVEL WERE SHOWN TO HAVE 3.78 AND 3.95 TIMES THE ODDS, RESPECTIVELY, TO ACQUIRE RESISTANCE TO IMATINIB. HOWEVER, OVERALL SURVIVAL OF CML PATIENTS THAT HAVE /= 63% METHYLATION LEVELS OF HOXA4 AND HOXA5 GENES WERE FOUND TO BE NOT SIGNIFICANT (P-VALUE = 0.126 FOR HOXA4; P-VALUE = 0.217 FOR HOXA5). CONCLUSION: HYPERMETHYLATION OF THE HOXA4 AND HOXA5 PROMOTER IS CORRELATED WITH IMATINIB RESISTANCE AND WITH FURTHER INVESTIGATION, IT COULD BE A POTENTIAL EPIGENETIC BIOMARKER IN SUPPLEMENT TO THE BCR-ABL GENE MUTATION IN PREDICTING IMATINIB TREATMENT RESPONSE AMONG CML PATIENTS BUT COULD NOT BE CONSIDERED AS A PROGNOSTIC MARKER. 2018 3 10 31 14-3-3 LIGAND PREVENTS NUCLEAR IMPORT OF C-ABL PROTEIN IN CHRONIC MYELOID LEUKEMIA. HERE WE DEMONSTRATED THAT THE 'LOSS OF FUNCTION' OF NOT-REARRANGED C-ABL IN CHRONIC MYELOID LEUKEMIA (CML) IS PROMOTED BY ITS CYTOPLASMIC COMPARTMENTALIZATION BOUND TO 14-3-3 SIGMA SCAFFOLDING PROTEIN. IN PARTICULAR, CONSTITUTIVE TYROSINE KINASE (TK) ACTIVITY OF P210 BCR-ABL BLOCKS C-JUN N-TERMINAL KINASE (JNK) PHOSPHORYLATION LEADING TO 14-3-3 SIGMA PHOSPHORYLATION AT A CRITICAL RESIDUE (SER(186)) FOR C-ABL BINDING IN RESPONSE TO DNA DAMAGE. MOREOVER, IT IS ASSOCIATED WITH 14-3-3 SIGMA OVER-EXPRESSION ARISING FROM EPIGENETIC MECHANISMS (PROMOTER HYPER-ACETYLATION). ACCORDINGLY, P210 BCR-ABL TK INHIBITION BY THE TK INHIBITOR IMATINIB MESYLATE (IM) EVOKES MULTIPLE EVENTS, INCLUDING JNK PHOSPHORYLATION AT THR(183), P38 MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) PHOSPHORYLATION AT THR(180), C-ABL DE-PHOSPHORYLATION AT SER RESIDUES INVOLVED IN 14-3-3 BINDING AND REDUCTION OF 14-3-3 SIGMA EXPRESSION, THAT LET C-ABL RELEASE FROM 14-3-3 SIGMA AND NUCLEAR IMPORT, AND ADDRESS BCR-ABL-EXPRESSING CELLS TOWARDS APOPTOTIC DEATH. INFORMATIONAL SPECTRUM METHOD (ISM), A VIRTUAL SPECTROSCOPY METHOD FOR ANALYSIS OF PROTEIN INTERACTIONS BASED ON THEIR STRUCTURE, AND MATHEMATICAL FILTERING IN CROSS SPECTRUM (CS) ANALYSIS IDENTIFIED 14-3-3 SIGMA/C-ABL BINDING SITES. FURTHER INVESTIGATION ON CS PROFILES OF C-ABL- AND P210 BCR-ABL-CONTAINING COMPLEXES REVEALED THE MECHANISM LIKELY INVOLVED 14-3-3 PRECLUDED PHOSPHORYLATION IN CML CELLS. 2009 4 6793 38 [DOWN-REGULATION OF TRANSCRIPTION FACTOR PU.1 VIA ABNORMAL EPIGENETIC MODIFICATION IN CHRONIC MYELOID LEUKEMIA]. OBJECTIVE: TO INVESTIGATE THE UNDERLYING MECHANISM AND CLINICAL SIGNIFICANCE OF PU.1 DOWN-EXPRESSION IN CHRONIC MYELOID LEUKEMIA (CML) PATIENTS. METHODS: DIFFERENT METHYLATION STATUS OF PU.1 PROMOTER REGION CONTAINING 20 CPG ISLANDS IN NORMAL INDIVIDUALS, CML CHRONIC PHASE AND BLAST CRISIS PATIENTS, COMPLETE CYTOGENETIC REMISSION PATIENTS AFTER IMATINIB TREATMENT, AND BLAST CRISIS BONE MARROW K562 CML CELLS WAS DETECTED BY BISULFITE SEQUENCING. SEMI-QUANTITATIVE PCR WAS USED TO DETECT THE PU.1 MRNA EXPRESSION IN NORMAL CONTROLS, CML CHRONIC PHASE AND BLAST CRISIS PATIENTS, AND BLAST CRISIS BONE MARROW K562 CML CELLS. INDIRECT IMMUNE FLUORESCENCE AND WESTERN BLOT WERE USED TO ANALYZE THE EXPRTESSION OF PU.1 PROTEIN IN NORMAL INDIVIDUALS, CML CHRONIC PHASE AND BLAST CRISIS PATIENTS, AND BLAST CRISIS BONE MARROW K562 CML CELLS. RESULTS: ABERRANT METHYLATION IN THE PROMOTER REGION OF TRANSCRIPTION FACTOR PU.1 WAS FOUND IN BOTH CML CHRONIC PHASE AND BLAST CRISIS PHASE BONE MARROW CELLS, AS WELL AS IN CML BLAST K562 CELLS. DOWN-EXPRESSION OF PU.1 MRNA AND PROTEIN LEVELS WAS FOUND IN ABOVE CELLS. NO METHYLATION IN THE PROMOTER REGION OF PU.1 WAS OBSERVED IN NORMAL INDIVIDUALS, AND THE PU.1 MRNA AND PROTEIN EXPRESSIONS WERE NOT REDUCED AT ALL. FURTHERMORE, HIGH METHYLATION STATUS OF BONE MARROW CELLS WAS EVEN OBSERVED IN THE CML PATIENTS WHO ACQUIRED COMPLETE CYTOGENETIC REMISSION. CONCLUSIONS: THE RESULTS OF OUR STUDY INDICATE THAT THE EPIGENETIC MODIFICATION OF PU.1 IN CML PATIENTS AND K562 CELL LINE MIGHT BE RESPONSIBLE FOR THE DOWN-EXPRESSION OF PU.1. THE DATA SUGGEST THAT ABERRANT METHYLATION OF PU.1 PLAYS A ROLE IN CML PATHOGENESIS, THEREFORE, IT MIGHT SERVE AS A USEFUL BIOMARKER AND POTENTIAL TARGET IN THERAPY FOR CHRONIC MYELOID LEUKEMIA. 2012 5 3530 40 IMATINIB (ST1571) PROVIDES ONLY LIMITED SELECTIVITY FOR CML CELLS AND TREATMENT MIGHT BE COMPLICATED BY SILENT BCR-ABL GENES. VERY PROMISING RESULTS HAVE BEEN OBTAINED IN CLINICAL TRIALS ON CHRONIC-PHASE CHRONIC MYELOID LEUKEMIA (CP-CML) PATIENTS TREATED WITH IMATINIB MESYLATE (IM; GLEEVECR, STI571), A BCR-ABL TYROSINE KINASE INHIBITOR. HOWEVER, WE FOUND THAT IM CAUSED CONSIDERABLE INHIBITION OF NORMAL HEMATOPOIETIC PROGENITOR CELLS UPON TREATING CONTROL BONE MARROW (BM) CULTURES. IN VITRO IM TREATMENT GAVE A DECREASE IN THE YIELD AND SIZE OF COLONIES FROM BM OF UNTREATED CP-CML PATIENTS THAT WAS ONLY TWO TO THREE TIMES THAT FROM THE NORMAL SAMPLES. MOREOVER, ABOUT 30% OF MYELOID PROGENITORS (CFU-GM) FROM CML BM STILL FORMED COLONIES IN THE PRESENCE OF IM, MOST OF WHICH HAD BCR-ABL RNA. ABOUT HALF OF THESE TREATED COLONIES ALSO DISPLAYED METHYLATION OF THE INTERNAL ABL PA PROMOTER, A CML-SPECIFIC EPIGENETIC ALTERATION, WHICH WAS USED IN THIS STUDY AS A MARKER FOR BCR-ABL TRANSLOCATION-CONTAINING CELLS. HOWEVER, ~5-8% OF THE TREATED OR THE UNTREATED CML BM-DERIVED COLONIES HAD NO DETECTABLE BCR-ABL RNA BY TWO OR THREE ROUNDS OF RT-PCR DESPITE BEING POSITIVE FOR THE INTERNAL STANDARD RNA AND DISPLAYING HALLMARKS OF CML, EITHER T(9;22)(Q34;QL 1) OR ABL PA METHYLATION. OUR RESULTS INDICATE THAT IM IS ONLY PARTIALLY SPECIFIC FOR CML PROGENITOR CELLS COMPARED TO NORMAL HEMATOPOIETIC PROGENITOR CELLS AND SUGGEST THAT SOME CML CELLS MAY HAVE A SILENT BCR-ABL ONCOGENE THAT COULD INTERFERE WITH THERAPY. 2003 6 690 25 BRD4 DEGRADATION BLOCKS EXPRESSION OF MYC AND MULTIPLE FORMS OF STEM CELL RESISTANCE IN PH(+) CHRONIC MYELOID LEUKEMIA. IN MOST PATIENTS WITH CHRONIC MYELOID LEUKEMIA (CML) CLONAL CELLS CAN BE KEPT UNDER CONTROL BY BCR::ABL1 TYROSINE KINASE INHIBITORS (TKI). HOWEVER, OVERT RESISTANCE OR INTOLERANCE AGAINST THESE TKI MAY OCCUR. WE IDENTIFIED THE EPIGENETIC READER BRD4 AND ITS DOWNSTREAM-EFFECTOR MYC AS GROWTH REGULATORS AND THERAPEUTIC TARGETS IN CML CELLS. BRD4 AND MYC WERE FOUND TO BE EXPRESSED IN PRIMARY CML CELLS, CD34(+) /CD38(-) LEUKEMIC STEM CELLS (LSC), AND IN THE CML CELL LINES KU812, K562, KCL22, AND KCL22(T315I) . THE BRD4-TARGETING DRUG JQ1 WAS FOUND TO SUPPRESS PROLIFERATION IN KU812 CELLS AND PRIMARY LEUKEMIC CELLS IN THE MAJORITY OF PATIENTS WITH CHRONIC PHASE CML. IN THE BLAST PHASE OF CML, JQ1 WAS LESS EFFECTIVE. HOWEVER, THE BRD4 DEGRADER DBET6 WAS FOUND TO BLOCK PROLIFERATION AND/OR SURVIVAL OF PRIMARY CML CELLS IN ALL PATIENTS TESTED, INCLUDING BLAST PHASE CML AND CML CELLS EXHIBITING THE T315I VARIANT OF BCR::ABL1. MOREOVER, DBET6 WAS FOUND TO BLOCK MYC EXPRESSION AND TO SYNERGIZE WITH BCR::ABL1 TKI IN INHIBITING THE PROLIFERATION IN THE JQ1-RESISTANT CELL LINE K562. FURTHERMORE, BRD4 DEGRADATION WAS FOUND TO OVERCOME OSTEOBLAST-INDUCED TKI RESISTANCE OF CML LSC IN A CO-CULTURE SYSTEM AND TO BLOCK INTERFERON-GAMMA-INDUCED UPREGULATION OF THE CHECKPOINT ANTIGEN PD-L1 IN LSC. FINALLY, DBET6 WAS FOUND TO SUPPRESS THE IN VITRO SURVIVAL OF CML LSC AND THEIR ENGRAFTMENT IN NSG MICE. TOGETHER, TARGETING OF BRD4 AND MYC THROUGH BET DEGRADATION SENSITIZES CML CELLS AGAINST BCR::ABL1 TKI AND IS A POTENT APPROACH TO OVERCOME MULTIPLE FORMS OF DRUG RESISTANCE IN CML LSC. 2022 7 5940 32 TARGETING METHYLTRANSFERASE PRMT5 ELIMINATES LEUKEMIA STEM CELLS IN CHRONIC MYELOGENOUS LEUKEMIA. IMATINIB-INSENSITIVE LEUKEMIA STEM CELLS (LSCS) ARE BELIEVED TO BE RESPONSIBLE FOR RESISTANCE TO BCR-ABL TYROSINE KINASE INHIBITORS AND RELAPSE OF CHRONIC MYELOGENOUS LEUKEMIA (CML). IDENTIFYING THERAPEUTIC TARGETS TO ERADICATE CML LSCS MAY BE A STRATEGY TO CURE CML. IN THE PRESENT STUDY, WE DISCOVERED A POSITIVE FEEDBACK LOOP BETWEEN BCR-ABL AND PROTEIN ARGININE METHYLTRANSFERASE 5 (PRMT5) IN CML CELLS. OVEREXPRESSION OF PRMT5 WAS OBSERVED IN HUMAN CML LSCS. SILENCING PRMT5 WITH SHRNA OR BLOCKING PRMT5 METHYLTRANSFERASE ACTIVITY WITH THE SMALL-MOLECULE INHIBITOR PJ-68 REDUCED SURVIVAL, SERIAL REPLATING CAPACITY, AND LONG-TERM CULTURE-INITIATING CELLS (LTC-ICS) IN LSCS FROM CML PATIENTS. FURTHER, PRMT5 KNOCKDOWN OR PJ-68 TREATMENT DRAMATICALLY PROLONGED SURVIVAL IN A MURINE MODEL OF RETROVIRAL BCR-ABL-DRIVEN CML AND IMPAIRED THE IN VIVO SELF-RENEWAL CAPACITY OF TRANSPLANTED CML LSCS. PJ-68 ALSO INHIBITED LONG-TERM ENGRAFTMENT OF HUMAN CML CD34+ CELLS IN IMMUNODEFICIENT MICE. MOREOVER, INHIBITION OF PRMT5 ABROGATED THE WNT/BETA-CATENIN PATHWAY IN CML CD34+ CELLS BY DEPLETING DISHEVELLED HOMOLOG 3 (DVL3). THIS STUDY SUGGESTS THAT EPIGENETIC METHYLATION MODIFICATION ON HISTONE PROTEIN ARGININE RESIDUES IS A REGULATORY MECHANISM TO CONTROL SELF-RENEWAL OF LSCS AND INDICATES THAT PRMT5 MAY REPRESENT A POTENTIAL THERAPEUTIC TARGET AGAINST LSCS. 2016 8 3877 33 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 9 162 34 ABL1 METHYLATION IN PH-POSITIVE ALL IS EXCLUSIVELY ASSOCIATED WITH THE P210 FORM OF BCR-ABL. IN HUMAN PH-POSITIVE LEUKEMIA THERE IS A CLEAR ASSOCIATION OF DIFFERENT FORMS OF THE BCR-ABL ONCOGENE WITH DISTINCT TYPES OF LEUKEMIA. THE P190 FORM OF BCR-ABL IS RARELY OBSERVED IN CHRONIC MYELOID LEUKEMIA (CML) BUT IS PRESENT IN 50% OF PH-POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA (ALL). IN CONTRAST, THE P210 FORM IS OBSERVED BOTH IN CML AND 50% OF PH-POSITIVE ALL. METHYLATION OF THE PROXIMAL PROMOTER OF THE ABL1 GENE HAS BEEN SHOWN TO BE A NEARLY UNIVERSAL EVENT ASSOCIATED WITH CLINICAL PROGRESSION OF CML. THIS RAISES THE QUESTION OF WHETHER METHYLATION OF THE ABL1 PROMOTER IS AN EPIGENETIC MODIFICATION ALSO ASSOCIATED WITH PH-POSITIVE ALL. TO STUDY THIS ISSUE, WE USED METHYLATION-SPECIFIC PCR AND BISULFITE SEQUENCING TO DETERMINE THE METHYLATION STATUS OF THE ABL1 PROMOTER IN 18 PH-POSITIVE ALL SAMPLES. WE REPORT HERE THAT GENE-SPECIFIC ABL1 PROMOTER METHYLATION IS ASSOCIATED MAINLY WITH THE P210 FORM OF BCR-ABL AND NOT THE P190 FORM. WHILE SIX OUT OF THE SEVEN P210-POSITIVE ALL SAMPLES HAD ABL1 PROMOTER METHYLATION, NONE OF THE 11 P190-POSITIVE ALL SAMPLES DEMONSTRATED ABL1 PROMOTER METHYLATION. IN ADDITION, WE ESTIMATED THE EXTENT AND RELATIVE ABUNDANCE OF ABL1 PROMOTER METHYLATION IN SEVERAL PH-POSITIVE ALL SAMPLES AND COMPARED IT TO THE METHYLATION PATTERN IN CHRONIC, ACCELERATED AND BLASTIC CRISIS PHASES OF CML. WE PUT FORTH A MODEL THAT CORRELATES THE DIFFERENT TYPES OF LEUKEMIAS WITH THE DIFFERENT LEVELS OF ABL1 PROMOTER METHYLATION. 2001 10 2719 28 EXOME SEQUENCING REVEALS DNMT3A AND ASXL1 VARIANTS ASSOCIATE WITH PROGRESSION OF CHRONIC MYELOID LEUKEMIA AFTER TYROSINE KINASE INHIBITOR THERAPY. OBJECTIVE: THE DEVELOPMENT OF TYROSINE KINASE INHIBITORS (TKIS) HAS SIGNIFICANTLY IMPROVED THE TREATMENT OF CHRONIC MYELOID LEUKEMIA (CML). HOWEVER, APPROXIMATELY ONE THIRD OF PATIENTS ARE RESISTANT TO TKI AND/OR PROGRESS TO ADVANCED DISEASE STAGES. TKI THERAPY FAILURE HAS A WELL-KNOWN ASSOCIATION WITH ABL1 KINASE DOMAIN (KD) MUTATIONS, BUT ONLY AROUND HALF OF TKI NON-RESPONDERS HAVE DETECTABLE ABL1 KD MUTATIONS. METHOD: WE ATTEMPT TO IDENTIFY GENETIC MARKERS ASSOCIATED WITH TKI THERAPY FAILURE IN 13 PATIENTS (5 RESISTANT, 8 PROGRESSED) WITHOUT ABL1 KD MUTATIONS USING WHOLE-EXOME SEQUENCING. RESULTS: IN 6 PATIENTS, WE DETECTED MUTATIONS IN 6 GENES COMMONLY MUTATED IN OTHER MYELOID NEOPLASMS: ABL1, ASXL1, DNMT3A, IDH1, SETBP1, AND TP63. WE THEN USED TARGETED DEEP SEQUENCING TO VALIDATE OUR FINDING IN AN INDEPENDENT COHORT CONSISTING OF 100 CML PATIENTS WITH VARYING DRUG RESPONSES (74 RESPONSIVE, 18 RESISTANT, AND 8 PROGRESSED PATIENTS). MUTATIONS IN GENES ASSOCIATED WITH EPIGENETIC REGULATIONS SUCH AS DNMT3A AND ASXL1 SEEM TO PLAY AN IMPORTANT ROLE IN THE PATHOGENESIS OF CML PROGRESSION AND TKI-RESISTANCE INDEPENDENT OF ABL1 KD MUTATIONS. CONCLUSION: THIS STUDY SUGGESTS THE INVOLVEMENT OF OTHER SOMATIC MUTATIONS IN THE DEVELOPMENT OF TKI RESISTANT PROGRESSION TO ADVANCED DISEASE STAGES IN CML, PARTICULARLY IN PATIENTS LACKING ABL1 KD MUTATIONS. 2017 11 6366 38 THE ROLE OF METHYLATION IN CML. METHYLATION OF THE PROXIMAL PROMOTER OF THE ABL1 ONCOGENE IS COMMON EPIGENETIC ALTERATION ASSOCIATED WITH CLINICAL PROGRESSION OF CHRONIC MYELOID LEUKEMIA (CML). IN PRESENTED STUDY WE QUERIED WHETHER BOTH THE PH'-ASSOCIATED AND NORMAL ABL1 ALLELES UNDERGO METHYLATION; WHAT MAY BE THE PROPORTION OF HEMATOPOIETIC PROGENITORS BEARING METHYLATED ABL1 PROMOTERS IN CHRONIC VERSUS ACUTE PHASE DISEASE; WHETHER METHYLATION AFFECTS THE PROMOTER UNIFORMLY OR IN PATCHES WITH DISCRETE CLINICAL RELEVANCE; AND, FINALLY WHETHER METHYLATION OF ABL1 REFLECTS A GENERALIZED PROCESS OR IS GENE-SPECIFIC. TO ADDRESS THESE ISSUES, THE TECHNIQUE OF METHYLATION-SPECIFIC PCR AND BISULFITE-SEQUENCING WAS ADAPTED TO STUDY THE REGULATORY REGIONS OF ABL1 AND OTHER GENES. IN CELL LINES ESTABLISHED FROM CML BLAST CRISIS, WHICH ONLY CARRY A SINGLE ABL1 ALLELE NESTED WITHIN THE BCR-ABL FUSION GENE, ABL1 PROMOTERS WERE UNIVERSALLY METHYLATED. IN CLINICAL SAMPLES FROM PATIENTS AT ADVANCED STAGES OF THE DISEASE, BOTH METHYLATED AND UNMETHYLATED PROMOTER ALLELES WERE DETECTABLE. IN COLONIES DERIVED FROM SINGLE HEMATOPOIETIC PROGENITORS METHYLATED AND UNMETHYLATED PROMOTER ALLELES WERE REVEALED AS WELL. ABL1 METHYLATION WAS WAS NOTED IN THE VAST MAJORITY OF COLONIES FROM BLAST CRISIS, BUT NOT CHRONIC-PHASE CML. IT WAS SHOWN FINALLY THAT ABL1 METHYLATION DOES NOT REFLECT A GENERALIZED PROCESS AND MAY BE UNIQUE AMONG DNA REPAIR/GENOTOXIC STRESS RESPONSE GENES. THESE DATA SUGGEST THAT SPECIFIC METHYLATION OF THE PH'-ASSOCIATED ABL1 ALLELE ACCOMPANIES CLONAL EVOLUTION IN CML. 2000 12 2465 34 EPIGENETIC THERAPY WITH HYDRALAZINE AND MAGNESIUM VALPROATE REVERSES IMATINIB RESISTANCE IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA. THE EPIGENETIC DRUGS HYDRALAZINE AND VALPROATE WERE ADMINISTERED IN A COMPASSIONATE MANNER TO 8 PATIENTS WITH CHRONIC MYELOID LEUKEMIA (CML) REFRACTORY TO IMATINIB. TWO PATIENTS HAD A COMPLETE HEMATOLOGIC RESPONSE (25%),1 MAJOR CYTOGENETIC RESPONSE, 1 COMPLETE CYTOGENETIC RESPONSE (25% ANY CYTOGENETIC RESPONSE), AND 3 (37.5%)STABLE DISEASE. NO GRADE 3 OR 4 TOXICITY WAS OBSERVED. THESE RESULTS SHOW THE ABILITY OF EPIGENETIC THERAPY TO REVERT IMATINIB RESISTANCE. BACKGROUND: EPIGENETIC ALTERATIONS PARTICIPATE IN THE DEVELOPMENT OF ACQUIRED RESISTANCE TO IMATINIB, HENCE, THE DNA METHYLATION, AND HISTONE DEACETYLASE INHIBITORS HYDRALAZINE AND VALPROATE, RESPECTIVELY, HAS THE POTENTIAL TO OVERCOME IT. PATIENT AND METHODS: A SERIES OF 8 PATIENTS WITH CHRONIC MYELOID LEUKEMIA (CML) REFRACTORY TO IMATINIB MESYLATE WITH NO ACCESS TO SECOND-GENERATION TYROSINE KINASE INHIBITORS WERE TREATED WITH HYDRALAZINE AND VALPROATE IN A COMPASSIONATE MANNER. CLINICAL EFFICACY AND SAFETY OF THESE DRUGS ADDED TO IMATINIB MESYLATE WERE EVALUATED. RESULTS: TWO PATIENTS WERE IN THE BLAST PHASE, 5 WERE IN THE ACCELERATED PHASE, AND 1 WAS IN THE CHRONIC PHASE. ALL THE PATIENTS CONTINUED WITH THE SAME DOSE OF IMATINIB THAT THEY HAD BEEN RECEIVING AT THE TIME OF DEVELOPMENT OF RESISTANCE, WITH A MEDIAN DOSE OF 600 MG DAILY (RANGE, 400-800 MG). THE MEDIAN TIME FROM DIAGNOSIS OF CML TO THE START OF HYDRALAZINE AND VALPROATE WAS 53.6 MONTHS (RANGE, 19-84 MONTHS). TWO (25%) PATIENTS HAD A COMPLETE HEMATOLOGIC RESPONSE, ONE (12.5%) HAD AN MAJOR CYTOGENETIC RESPONSE, AND ONE (12.5%) HAD A COMPLETE CYTOGENETIC RESPONSE. THREE (37.5%) PATIENTS HAD STABLE DISEASE, AND ONLY ONE (12.5%) PATIENT FAILED TO RESPOND. AT A MEDIAN FOLLOW-UP TIME OF 18 MONTHS (RANGE, 3-18 MONTHS), THE MEDIAN SURVIVAL HAD NOT BEEN REACHED, AND THE PROJECTED OVERALL SURVIVAL WAS 63%. ALL THE PATIENTS HAD MILD NEUROLOGIC TOXICITY, INCLUDING DISTAL TREMOR AND SOMNOLENCE. NO GRADE 3 OR 4 TOXICITY WAS OBSERVED. CONCLUSIONS: OUR RESULTS SUGGEST THAT THE EPIGENETIC DRUGS HYDRALAZINE AND VALPROATE REVERT THE RESISTANCE TO IMATINIB IN PATIENTS WITH CML. 2012 13 5274 21 PROMOTER METHYLATION OF P16 AND EDNRB GENE IN LEUKEMIA PATIENTS IN TAIWAN. BOTH EPIGENETIC AND GENETIC ALTERNATIONS ARE INVOLVED IN CANCER FORMATION. IN THIS STUDY, WE HAVE IDENTIFIED THE METHYLATION FREQUENCY OF P16 AND ENDOTHELIN RECEPTOR TYPE B (EDNRB) OF 26 LEUKEMIA PATIENTS AND 8 RANDOMLY SELECTED NORMAL BLOOD DONORS IN TAIWAN. PROMOTER METHYLATION OF P16 WAS DETECTED IN 85% OF ACUTE LYMPHOCYTIC LEUKEMIA (ALL), 83% IN ACUTE MYELOID LEUKEMIA (AML) WHEREAS NO METHYLATION WAS DETECTED IN CHRONIC MYELOID LEUKEMIA (CML) IN BLAST CRISIS. HYPERMETHYLATION OF EDNRB WAS OBSERVED IN 92% OF ALL, 75% AML AND 100% IN CML IN BLAST CRISIS. NO ABERRANT METHYLATION OF P16 AND EDNRB WAS FOUND IN 8 NORMAL BLOOD DONORS. TAKEN TOGETHER, ABERRANT METHYLATION OF P16 AND EDNRB WAS HIGHLY PREVALENT IN LEUKEMIA PATIENTS IN TAIWAN. 2008 14 571 28 BCR-ABL INDUCES AUTOCRINE IGF-1 SIGNALING. BCR-ABL ONCOGENE IS RESPONSIBLE FOR THE INITIAL PHASE OF CHRONIC MYELOGENOUS LEUKEMIA (CML), WHICH IS EFFECTIVELY TREATED BY THE BCR-ABL INHIBITOR IMATINIB. OVER TIME PATIENTS BECOME RESISTANT TO TREATMENT AND PROGRESS TO BLAST CRISIS, AN EVENT THAT IS DRIVEN BY ADDITIONAL GENETIC AND EPIGENETIC ABERRATIONS. RECENTLY, WE SHOWED THAT RIZ1 EXPRESSION DECREASES IN BLAST CRISIS AND THAT RE-EXPRESSION OF RIZ1 INHIBITS IGF-1 EXPRESSION. IGF-1 SIGNALING IS REQUIRED IN MANY STAGES OF HEMATOPOIESIS AND INAPPROPRIATE ACTIVATION OF AUTOCRINE IGF-1 SIGNALING MAY FACILITATE TRANSFORMATION TO BLAST CRISIS. WE OBSERVED THAT IN 8 OUT OF 11 MATCHED CML PATIENT BIOPSIES THE IGF-1 EXPRESSION IS ELEVATED IN BLAST CRISIS. WE EXAMINED MECHANISMS USED BY CML BLAST CRISIS CELL LINES TO ACTIVATE IGF-1 EXPRESSION. WE FOUND THAT BCR-ABL ACTIVATES AUTOCRINE IGF-1 SIGNALING USING HCK AND STAT5B. INHIBITION OF THESE SIGNALING COMPONENTS USING SMALL MOLECULE DRUGS OR SHRNA DECREASES PROLIFERATION AND ENHANCES APOPTOSIS. TOGETHER, OUR STUDY SUGGESTS THAT ABERRANT IGF-1 SIGNALING IS AN IMPORTANT EVENT IN BLAST CRISIS TRANSFORMATION AND IT PROVIDES A MECHANISM TO EXPLAIN THE ACTIVITY OF IGF-1R AND HCK INHIBITORS IN BLOCKING CML BLAST CRISIS PHENOTYPES. 2008 15 1660 37 DOWN-REGULATION OF HEMATOPOIESIS MASTER REGULATOR PU.1 VIA ABERRANT METHYLATION IN CHRONIC MYELOID LEUKEMIA. THE PU.1 TRANSCRIPTION FACTOR IS A CRUCIAL REGULATOR OF HEMATOPOIESIS, AND ITS EXPRESSION IS ALTERED IN VARIOUS LEUKEMIC PROCESSES. IT HAS BEEN SHOWN THAT EXPRESSION OF PU.1 IS SEVERELY IMPAIRED IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA (CML), BUT THE MECHANISM UNDERLYING THIS EFFECT REMAINS UNKNOWN. THROUGH BISULFITE SEQUENCING, SEMI-QUANTITATIVE PCR, AND INDIRECT IMMUNOFLUORESCENCE AND WESTERN BLOT TECHNIQUES, WE FOUND ABERRANT METHYLATION IN THE PROMOTER REGION OF TRANSCRIPTION FACTOR PU.1 IN CML PATIENTS BOTH IN THE CHRONIC AND BLAST CRISIS PHASES, AS WELL AS IN THE CML BLAST K562 CELL LINE. OF THESE, SEVERAL CPG SITES WERE MORE HIGHLY METHYLATED IN BLAST CRISIS THAN CHRONIC PHASE, WHILE NO METHYLATION OF THESE SITES WAS OBSERVED IN HEALTHY INDIVIDUALS. INTERESTINGLY, CML PATIENTS ACHIEVED COMPLETE CYTOGENETIC REMISSION UNDER IMATINIB MESYLATE TREATMENT, BUT THE ABERRANT METHYLATION STATUS OF PU.1 WAS NOT REVERSED. DOWN-REGULATION OF PU.1 EXPRESSION AT THE MRNA AND PROTEIN LEVELS WAS ALSO OBSERVED IN ASSOCIATION WITH ABERRANT METHYLATION. THUS, FOR THE FIRST TIME, WE HAVE REVEALED A POTENTIAL EPIGENETIC MODIFICATION OF PU.1 IN CML, WHICH MAY BE RESPONSIBLE FOR THE DOWN-REGULATION OF PU.1. THESE DATA SUGGEST THAT ABERRANT METHYLATION OF PU.1 MAY PLAY A ROLE IN CML PATHOGENESIS, AND MAY THEREFORE SERVE AS A USEFUL BIOMARKER AND POTENTIAL TARGET FOR DEMETHYLATING DRUGS. 2012 16 5669 34 SFRP1 PROMOTER METHYLATION IS ASSOCIATED WITH PERSISTENT PHILADELPHIA CHROMOSOME IN CHRONIC MYELOID LEUKEMIA. EPIGENETIC SILENCING OF SFRP GENES HAS BEEN SHOWN TO LEAD TO CONSTITUTIVE ACTIVATION OF THE CANONICAL WNT-SIGNALING PATHWAY. THE FIRST DESCRIPTION OF DEREGULATED WNT-SIGNALING ACTIVATION IN A HEMATOLOGICAL MALIGNANCY WAS REPORTED IN CHRONIC MYELOID LEUKEMIA (CML). TO INVESTIGATE WHETHER EPIGENETIC SILENCING OF SFRP IS RESPONSIBLE FOR THE OBSERVED WNT ACTIVATION IN CML, WE STUDIED THE METHYLATION AND MUTATIONAL STATUS OF THE SFRP1 PROMOTER IN 48 CHRONIC PHASE CML PATIENTS. OF THE 48 CML PATIENTS 41 WERE SHOWN TO BE UNMETHYLATED, 6 PATIENTS HEMI-METHYLATED AND 1 PATIENT FULLY METHYLATED AT THE SFRP1 PROMOTER. ALBEIT OBSERVED INFREQUENTLY IN CHRONIC PHASE CML, SFRP1 PROMOTER METHYLATION CORRELATED WITH PRIMARY CYTOGENETIC RESISTANCE TO IMATINIB MESYLATE. SFRP1 PROMOTER METHYLATION MAY INDICATE A GENETICALLY MORE UNSTABLE FORM OF DISEASE RESISTANT TO THERAPY AND PROVIDE A KEY BIOLOGICAL DIFFERENCE IN THERAPY RESISTANT PATIENTS, IN ADDITION TO A POSSIBLE MECHANISM FOR THE OBSERVED ACTIVATION OF CANONICAL WNT SIGNALING IN CML. 2009 17 4361 43 MIR-96 ACTS AS A TUMOR SUPPRESSOR VIA TARGETING THE BCR-ABL1 ONCOGENE IN CHRONIC MYELOID LEUKEMIA BLASTIC TRANSFORMATION. MICRORNA-MEDIATED POSTTRANSCRIPTIONAL REGULATION IS AN IMPORTANT EPIGENETIC REGULATORY MECHANISM OF GENE EXPRESSION, AND ITS DYSREGULATION IS INVOLVED IN THE DEVELOPMENT AND PROGRESSION OF A VARIETY OF MALIGNANCIES, INCLUDING CHRONIC MYELOID LEUKEMIA (CML). THE BCR-ABL1 FUSION GENE IS NOT ONLY THE INITIATING FACTOR OF CML, BUT IT IS ALSO AN IMPORTANT DRIVING FACTOR FOR BLASTIC TRANSFORMATION. TYROSINE KINASE INHIBITORS (TKIS) TARGETING BCR-ABL1 TYROSINE KINASE ACTIVITY, REPRESENTED BY IMATINIB, ARE CURRENTLY THE FIRST-LINE TREATMENT FOR CML. HOWEVER, DUE TO PRIMARY RESISTANCE OR SECONDARY RESISTANCE CAUSED BY MUTATIONS IN THE BCR-ABL1 KINASE DOMAIN, TKIS CANNOT COMPLETELY PREVENT THE PROGRESSION OF CML; THUS, THE STUDY OF BCR-ABL1 GENE EXPRESSION REGULATION IS OF GREAT SIGNIFICANCE. IN THIS STUDY, BIOINFORMATICS ANALYSIS AND OUR RESULTS SHOWED THAT MIR-96 COULD DIRECTLY BIND TO THE 3'UTR REGION OF BCR-ABL1 TO REGULATE FUSION PROTEIN EXPRESSION, THEREBY REGULATING ITS DOWNSTREAM SIGNALING PATHWAY ACTIVITY. WE ALSO FOUND THAT MIR-96 WAS DOWNREGULATED DURING THE PROGRESSION FROM THE CHRONIC PHASE (CML-CP) TO THE BLAST CRISIS (CML-BC). DOWNREGULATION OF MIR-96 COULD PROMOTE THE PROLIFERATION AND PARTICIPATE IN THE CELL DIFFERENTIATION OF CML-BC CELLS. ADDITIONALLY, WE FOUND THAT THE NOVEL HISTONE DEACETYLASE DRUG CHIDAMIDE AND THE DNA METHYLTRANSFERASE INHIBITOR DECITABINE COULD RESTORE THE LOW EXPRESSION OF MIR-96 IN CML CELLS, AND THERE WERE TWO ABNORMAL HYPERMETHYLATED SITES IN THE PROMOTER REGION OF MIR-96 IN CML, SUGGESTING THAT ITS LOW EXPRESSION MIGHT BE AT LEAST PARTIALLY REGULATED BY EPIGENETIC MECHANISMS. IN ADDITION, RE-EXPRESSION OF MIR-96 COULD INCREASE THE SENSITIVITY OF CML-BC CELLS TO IMATINIB. THUS, MIR-96 FUNCTIONS AS A TUMOR SUPPRESSOR, AND RE-EXPRESSION OF THIS MICRORNA MIGHT HAVE THERAPEUTIC BENEFITS IN CML BLASTIC TRANSFORMATION. 2019 18 163 40 ABL1 METHYLATION IS A DISTINCT MOLECULAR EVENT ASSOCIATED WITH CLONAL EVOLUTION OF CHRONIC MYELOID LEUKEMIA. METHYLATION OF THE PROXIMAL PROMOTER OF THE ABL1 ONCOGENE IS A COMMON EPIGENETIC ALTERATION ASSOCIATED WITH CLINICAL PROGRESSION OF CHRONIC MYELOID LEUKEMIA (CML). IN THIS STUDY WE QUERIED WHETHER BOTH THE PH'-ASSOCIATED AND NORMAL ABL1 ALLELES UNDERGO METHYLATION; WHAT MAY BE THE PROPORTION OF HEMATOPOIETIC PROGENITORS BEARING METHYLATED ABL1 PROMOTERS IN CHRONIC VERSUS ACUTE PHASE DISEASE; WHETHER METHYLATION AFFECTS THE PROMOTER UNIFORMLY OR IN PATCHES WITH DISCRETE CLINICAL RELEVANCE; AND, FINALLY, WHETHER METHYLATION OF ABL1 REFLECTS A GENERALIZED PROCESS OR IS GENE-SPECIFIC. TO ADDRESS THESE ISSUES, WE ADAPTED THE TECHNIQUES OF METHYLATION-SPECIFIC PCR AND BISULFITE-SEQUENCING TO STUDY THE REGULATORY REGIONS OF ABL1 AND OTHER GENES WITH A ROLE IN DNA REPAIR OR GENOTOXIC STRESS RESPONSE. IN CELL LINES ESTABLISHED FROM CML BLAST CRISIS, WHICH ONLY CARRY A SINGLE ABL1 ALLELE NESTED WITHIN THE BCR-ABL FUSION GENE, ABL1 PROMOTERS WERE UNIVERSALLY METHYLATED. BY CONTRAST, IN CLINICAL SAMPLES FROM PATIENTS AT ADVANCED STAGES OF DISEASE, BOTH METHYLATED AND UNMETHYLATED PROMOTER ALLELES WERE DETECTABLE. TO DISTINGUISH BETWEEN ALLELE-SPECIFIC METHYLATION AND A MIXED CELL POPULATION PATTERN, WE STUDIED THE METHYLATION STATUS OF ABL1 IN COLONIES DERIVED FROM SINGLE HEMATOPOIETIC PROGENITORS. OUR RESULTS SHOWED THAT BOTH METHYLATED AND UNMETHYLATED PROMOTER ALLELES COEXISTED IN THE SAME COLONY. FURTHERMORE, ABL1 METHYLATION WAS NOTED IN THE VAST MAJORITY OF COLONIES FROM BLAST CRISIS, BUT NOT CHRONIC-PHASE CML. BOTH CELL LINES AND CLINICAL SAMPLES FROM ACUTE-PHASE CML SHOWED NEARLY UNIFORM HYPERMETHYLATION ALONG THE PROMOTER REGION. FINALLY, WE SHOWED THAT ABL1 METHYLATION DOES NOT REFLECT A GENERALIZED PROCESS AND MAY BE UNIQUE AMONG DNA REPAIR/GENOTOXIC STRESS RESPONSE GENES. OUR DATA SUGGEST THAT SPECIFIC METHYLATION OF THE PH'-ASSOCIATED ABL1 ALLELE ACCOMPANIES CLONAL EVOLUTION IN CML. 1999 19 156 43 ABERRANT METHYLATION OF THE DEATH-ASSOCIATED PROTEIN KINASE 1 (DAPK1) CPG ISLAND IN CHRONIC MYELOID LEUKEMIA. THE DEATH-ASSOCIATED PROTEIN KINASE 1 (DAPK1) GENE IS A CANDIDATE TUMOR SUPPRESSOR (TSG) AND THE ABNORMAL METHYLATION OF DAPK1 GENE HAS BEEN FOUND IN MANY CARCINOMAS. THE EPIGENETIC CHANGES OF TSGS ARE NOW RECOGNIZED AS A MECHANISM CONTRIBUTING TO THE DEVELOPMENT OF CHRONIC MYELOID LEUKEMIA (CML). TO CLARIFY THE ROLE OF DAPK1 IN CML, WE EXAMINED THE METHYLATION STATUS OF DAPK1 IN 49 PATIENTS WITH CML USING METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION. THE ABERRANT METHYLATION OF THE DAPK1 GENE WAS FOUND IN 25 OF 49 (51.0%) CML CASES, NOT IN ALL CONTROLS. NO CORRELATION WAS FOUND BETWEEN DAPK1 GENE METHYLATION AND THE AGE, HEMATOLOGIC PARAMETERS, CHROMOSOMAL ABNORMALITIES, THE TYPES AND LEVELS OF BCR/ABL TRANSCRIPTS OF CML PATIENTS. HOWEVER, CORRELATION COULD BE OBSERVED BETWEEN THE SEX AND THE STATUS OF DAPK1 METHYLATION IN CML PATIENTS (R = 0.374, P = 0.008). FURTHERMORE, THERE WAS A SIGNIFICANT CORRELATION BETWEEN DAPK1 METHYLATION AND THE STAGES OF CML (R = 0.354, P = 0.013). THE CML PATIENTS IN ACCELERATED PHASE (AP) AND BLAST CRISIS (BC) HAD HIGHER FREQUENCY OF DAPK1 METHYLATION THAN THOSE IN CHRONIC PHASE (CP) (75.0% VS. 34.5%) (CHI(2) = 7.776, P = 0.005). IN ONE PATIENT, THE STATUS OF DAPK1 METHYLATION BECAME POSITIVE ON THE TRANSITION FROM CP TO AP AND BC. THESE RESULTS SUGGESTED THAT DAPK1 PROMOTER METHYLATION MIGHT PLAY A SIGNIFICANT ROLE IN THE PROGRESSION OF CML. 2009 20 32 36 A CASE OF TYROSINE KINASE INHIBITOR-RESISTANT CHRONIC MYELOID LEUKEMIA, CHRONIC PHASE WITH ASXL1 MUTATION. HEMATOLOGICAL MALIGNANCIES, INCLUDING CHRONIC MYELOID LEUKEMIA (CML), EXHIBIT ASXL1 MUTATIONS; HOWEVER, THE FUNCTION AND MOLECULAR MECHANISM OF THESE MUTATIONS REMAIN UNCLEAR. ASXL1 WAS ORIGINALLY IDENTIFIED AS TUMOR SUPPRESSOR GENE, IN WHICH LOSS OF FUNCTION CAUSES MYELODYSPLASTIC SYNDROME (MDS). ASXL1 MUTATIONS ARE COMMON AND ASSOCIATED WITH DISEASE PROGRESSION IN MYELOID MALIGNANCIES INCLUDING MDS, ACUTE MYELOID LEUKEMIA, AND SIMILARLY IN CML. IN MDS, ASXL1 MUTATIONS HAVE BEEN ASSOCIATED WITH POOR PROGNOSIS; HOWEVER, THE IMPACT OF ASXL1 MUTATIONS IN CML HAS NOT BEEN WELL DESCRIBED. A 31-YEAR-OLD MALE WAS DIAGNOSED AS CML-CHRONIC PHASE (CP). LABORATORY FINDINGS SHOWED A WHITE BLOOD CELL COUNT OF 187,200/MICROL, WITH ASYMPTOMATIC SPLENOMEGALY. BLAST COUNT WAS 5.0% IN PERIPHERAL BLOOD AND 7.3% IN BONE MARROW. THERE WAS NO ADDITIONAL CHROMOSOMAL ABNORMALITY EXCEPT FOR T(9;22)(Q34;Q11.2) BY CHROMOSOMAL ANALYSIS. AT ONSET, THE SOKAL SCORE WAS 1.4, INDICATING HIGH RISK. THE PATIENT RECEIVED TYROSINE KINASE INHIBITOR (TKI) THERAPY, COMPRISING NILOTINIB APPROXIMATELY 600 MG/DAY, BOSUTINIB APPROXIMATELY 600 MG/DAY, PONATINIB APPROXIMATELY 45 MG/DAY, AND DASATINIB APPROXIMATELY 100 MG/DAY. NEVERTHELESS, AFTER 1.5 YEARS OF CONTINUOUS TKI THERAPY, THE BEST OUTCOME WAS A HEMATOLOGICAL RESPONSE. ALTHOUGH ADDITIONAL CHROMOSOMAL ABERRATIONS AND ABL1 KINASE MUTATIONS WERE ANALYZED REPEATEDLY BEFORE AND DURING TKI THERAPY, KNOWN GENETIC ABNORMALITIES WERE NOT DETECTED. THEREAFTER, THE PATIENT UNDERWENT BONE MARROW TRANSPLANTATION FROM AN HLA 7/8 MATCHED UNRELATED DONOR (HLA-CW 1 LOCUS MISMATCH, GRAFT-VERSUS-HOST DIRECTION). THE PATIENT ACHIEVED NEUTROPHIL ENGRAFTMENT, 18 DAYS AFTER TRANSPLANTATION, LEADING TO COMPLETE REMISSION WITH AN UNDETECTABLE LEVEL OF BCR-ABL1 MRNA. THE PATIENT, HOWEVER, DIED FROM GRAFT-VERSUS-HOST DISEASE AND THROMBOTIC MICROANGIOPATHY AFTER 121 DAYS. GENE SEQUENCE ANALYSIS OF HIS CML CELL BEFORE STEM CELL TRANSPLANTATION REVEALED ASXL1 MUTATIONS. PHYSIOLOGICALLY, ASXL1 CONTRIBUTES TO EPIGENETIC REGULATION. IN THE CML-CP PATIENT IN THIS CASE REPORT, ASXL1 MUTATION CONFERRED RESISTANCE TO TKI THROUGH OBSCURE RESISTANCE MECHANISMS. EVEN THOUGH A MOLECULAR MECHANISM FOR TKI RESISTANCE IN ASXL1 MUTATION IN CML HAS REMAINED OBSCURE, EPIGENETIC MODULATION IS A PLAUSIBLE MODE OF CML DISEASE PROGRESSION. THE CLINICAL IMPACT INCLUDING PROGNOSIS OF ASXL1 FOR CML IS UNDERSCORED. AND THE TREATMENT STRATEGY OF CML WITH ASXL1 MUTATION HAS NOT BEEN ESTABLISHED. A DISCUSSION OF THIS CASE WAS EXPECTED TO FACILITATE TREATMENT OPTIONS. 2020