1 1107 150 COMBINING CYTOGENETIC AND EPIGENETIC APPROACHES IN CHRONIC LYMPHOCYTIC LEUKEMIA IMPROVES PROGNOSIS PREDICTION FOR PATIENTS WITH ISOLATED 13Q DELETION. BACKGROUND: BOTH DEFECTIVE DNA METHYLATION AND ACTIVE DNA DEMETHYLATION PROCESSES ARE EMERGING AS IMPORTANT RISK FACTORS IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). HOWEVER, ASSOCIATIONS BETWEEN 5-CYTOSINE EPIGENETIC MARKERS AND THE MOST FREQUENT CHROMOSOMAL ABNORMALITIES DETECTED IN CLL REMAIN TO BE ESTABLISHED. METHODS: CLL PATIENTS WERE RETROSPECTIVELY CLASSIFIED INTO A CYTOGENETIC LOW-RISK GROUP (ISOLATED 13Q DELETION), AN INTERMEDIATE-RISK GROUP (NORMAL KARYOTYPE OR TRISOMY 12), AND A HIGH-RISK GROUP (11Q DELETION, 17P DELETION, OR COMPLEX KARYOTYPE [>/= 3 BREAKPOINTS]). THE TWO 5-CYTOSINE DERIVATIVES, 5-METHYLCYTOSINE (5-MCYT) AND 5-HYDROXYMETHYLCYTOSINE (5-HMCYT), WERE TESTED BY ELISA (N = 60), WHILE REAL-TIME QUANTITATIVE PCR WAS USED FOR DETERMINING TRANSCRIPTIONAL EXPRESSION LEVELS OF DNMT AND TET (N = 24). RESULTS: BY USING GLOBAL DNA METHYLATION/DEMETHYLATION LEVELS, IN THE LOW-RISK DISEASE GROUP, TWO SUBGROUPS WITH SIGNIFICANTLY DIFFERENT CLINICAL OUTCOMES HAVE BEEN IDENTIFIED (MEDIAN TREATMENT-FREE SURVIVAL [TFS] 45 VERSUS > 120 MONTHS FOR 5-MCYT, P = 0.0008, AND 63 VERSUS > 120 MONTHS FOR 5-HMCYT, P = 0.04). A DEFECTIVE 5-MCYT STATUS WAS FURTHER ASSOCIATED WITH A HIGHER PERCENTAGE OF 13Q DELETED NUCLEI (> 80%), THUS SUGGESTING AN ACQUIRED PROCESS. WHEN CONSIDERING THE CYTOGENETIC INTERMEDIATE/HIGH-RISK DISEASE GROUPS, AN ASSOCIATION OF 5-MCYT STATUS WITH LYMPHOCYTOSIS (P = 0.0008) AND THE LYMPHOCYTE DOUBLING TIME (P = 0.04) BUT NOT WITH TFS WAS OBSERVED, AS WELL AS A REDUCTION OF DNMT3A, TET1, AND TET2 TRANSCRIPTS. CONCLUSIONS: COMBINING CYTOGENETIC STUDIES WITH 5-MCYT ASSESSMENT ADDS ACCURACY TO CLL PATIENTS' PROGNOSES AND PARTICULARLY FOR THOSE WITH 13Q DELETION AS A SOLE CYTOGENETIC ABNORMALITY. 2017 2 4741 34 NOVEL HDAC INHIBITOR MAKV-8 AND IMATINIB SYNERGISTICALLY KILL CHRONIC MYELOID LEUKEMIA CELLS VIA INHIBITION OF BCR-ABL/MYC-SIGNALING: EFFECT ON IMATINIB RESISTANCE AND STEM CELLS. BACKGROUND: CHRONIC MYELOID LEUKEMIA (CML) PATHOGENESIS IS MAINLY DRIVEN BY THE ONCOGENIC BREAKPOINT CLUSTER REGION-ABELSON MURINE LEUKEMIA VIRAL ONCOGENE HOMOLOG 1 (BCR-ABL) FUSION PROTEIN. SINCE BCR-ABL DISPLAYS ABNORMAL CONSTITUTIVE TYROSINE KINASE ACTIVITY, THERAPIES USING TYROSINE KINASE INHIBITORS (TKIS) SUCH AS IMATINIB REPRESENT A MAJOR BREAKTHROUGH FOR THE OUTCOME OF CML PATIENTS. NEVERTHELESS, THE DEVELOPMENT OF TKI RESISTANCE AND THE PERSISTENCE OF LEUKEMIA STEM CELLS (LSCS) REMAIN BARRIERS TO CURE THE DISEASE, JUSTIFYING THE DEVELOPMENT OF NOVEL THERAPEUTIC APPROACHES. SINCE THE ACTIVITY OF HISTONE DEACETYLASE (HDAC) IS DEREGULATED IN NUMEROUS CANCERS INCLUDING CML, PAN-HDAC INHIBITORS MAY REPRESENT PROMISING THERAPEUTIC REGIMENS FOR THE TREATMENT OF CML CELLS IN COMBINATION WITH TKI. RESULTS: WE ASSESSED THE ANTI-LEUKEMIC ACTIVITY OF A NOVEL HYDROXAMATE-BASED PAN-HDAC INHIBITOR MAKV-8, WHICH COMPLIED WITH THE LIPINSKI'S "RULE OF FIVE," IN VARIOUS CML CELLS ALONE OR IN COMBINATION WITH IMATINIB. WE VALIDATED THE IN VITRO HDAC-INHIBITORY POTENTIAL OF MAKV-8 AND DEMONSTRATED EFFICIENT BINDING TO THE LIGAND-BINDING POCKET OF HDAC ISOENZYMES. IN CELLULO, MAKV-8 SIGNIFICANTLY INDUCED TARGET PROTEIN ACETYLATION, DISPLAYED CYTOSTATIC AND CYTOTOXIC PROPERTIES, AND TRIGGERED CONCOMITANT ER STRESS/PROTECTIVE AUTOPHAGY LEADING TO CANONICAL CASPASE-DEPENDENT APOPTOSIS. CONSIDERING THE SPECIFIC UPREGULATION OF SELECTED HDACS IN LSCS FROM CML PATIENTS, WE INVESTIGATED THE DIFFERENTIAL TOXICITY OF A CO-TREATMENT WITH MAKV-8 AND IMATINIB IN CML VERSUS HEALTHY CELLS. WE ALSO SHOWED THAT BECLIN-1 KNOCKDOWN PREVENTED MAKV-8-IMATINIB COMBINATION-INDUCED APOPTOSIS. MOREOVER, MAKV-8 AND IMATINIB CO-TREATMENT SYNERGISTICALLY REDUCED BCR-ABL-RELATED SIGNALING PATHWAYS INVOLVED IN CML CELL GROWTH AND SURVIVAL. SINCE OUR RESULTS SHOWED THAT LSCS FROM CML PATIENTS OVEREXPRESSED C-MYC, IMPORTANTLY MAKV-8-IMATINIB CO-TREATMENT REDUCED C-MYC LEVELS AND THE LSC POPULATION. IN VIVO, TUMOR GROWTH OF XENOGRAFTED K-562 CELLS IN ZEBRAFISH WAS COMPLETELY ABROGATED UPON COMBINED TREATMENT WITH MAKV-8 AND IMATINIB. CONCLUSIONS: COLLECTIVELY, THE PRESENT FINDINGS SHOW THAT COMBINATIONS HDAC INHIBITOR-IMATINIB ARE LIKELY TO OVERCOME DRUG RESISTANCE IN CML PATHOLOGY. 2020 3 6793 39 [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 4 32 35 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 5 2719 33 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 6 337 49 ALTERATIONS IN DNA METHYLATION/DEMETHYLATION INTERMEDIATES PREDICT CLINICAL OUTCOME IN CHRONIC LYMPHOCYTIC LEUKEMIA. CYTOSINE DERIVATIVE DYSREGULATIONS REPRESENT IMPORTANT EPIGENETIC MODIFICATIONS WHOSE IMPACT ON THE CLINICAL OUTCOME IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) IS INCOMPLETELY UNDERSTOOD. HENCE, GLOBAL LEVELS OF 5-METHYLCYTOSINE (5-MCYT), 5-HYDROXYMETHYLCYTOSINE (5-HMCYT), 5-CARBOXYLCYTOSINE (5-CACYT) AND 5-HYDROXYMETHYLURACIL WERE TESTED IN PURIFIED B CELLS FROM CLL PATIENTS (N = 55) AND CONTROLS (N = 17). THE DNA METHYLATION 'WRITERS' (DNA METHYLTRANSFERASES [DNMT1/3A/3B]), 'READERS' (METHYL-CPG-BINDING DOMAIN [MBD2/4]), 'EDITORS' (TEN-ELEVEN TRANSLOCATION [TET1/2/3]) AND 'MODULATORS' (SAT1) WERE ALSO EVALUATED. ACCORDINGLY, PATIENTS WERE STRATIFIED INTO THREE SUBGROUPS. FIRST, A SUBGROUP WITH A GLOBAL DEFICIT IN CYTOSINE DERIVATIVES CHARACTERIZED BY HYPERLYMPHOCYTOSIS, REDUCED MEDIAN PROGRESSION FREE SURVIVAL (PFS = 52 MONTHS) AND SHORTER TREATMENT FREE SURVIVAL (TFS = 112 MONTHS) WAS IDENTIFIED. IN THIS SUBGROUP, MAJOR EPIGENETIC MODIFICATIONS WERE HIGHLIGHTED INCLUDING A REDUCTION OF 5-MCYT, 5-HMCYT, 5-CACYT ASSOCIATED WITH DNMT3A, MBD2/4 AND TET1/2 DOWNREGULATION. SECOND, THE CYTOSINE DERIVATIVE ANALYSIS REVEALED A SUBGROUP WITH A PARTIAL DEFICIT (PFS = 84, TFS = 120 MONTHS), MAINLY AFFECTING DNA DEMETHYLATION (5-HMCYT REDUCTION, SAT1 INDUCTION). THIRD, A SUBGROUP EPIGENETICALLY SIMILAR TO CONTROLS WAS IDENTIFIED (PFS AND TFS > 120 MONTHS). THE PROGNOSTIC IMPACT OF STRATIFYING CLL PATIENTS WITHIN THREE EPIGENETIC SUBGROUPS WAS CONFIRMED IN A VALIDATION COHORT. IN CONCLUSION, OUR RESULTS SUGGEST THAT DYSREGULATIONS OF CYTOSINE DERIVATIVE REGULATORS REPRESENT MAJOR EVENTS ACQUIRED DURING CLL PROGRESSION AND ARE INDEPENDENT FROM IGHV MUTATIONAL STATUS. 2017 7 2848 27 FREQUENT SOMATIC MUTATIONS IN EPIGENETIC REGULATORS IN NEWLY DIAGNOSED CHRONIC MYELOID LEUKEMIA. ALTHOUGH TYROSINE KINASE INHIBITORS (TKIS) HAVE SIGNIFICANTLY IMPROVED THE PROGNOSIS OF CHRONIC MYELOID LEUKEMIA (CML), THE ABILITY OF TKIS TO ERADICATE CML REMAINS UNCERTAIN AND PATIENTS MUST CONTINUE TKI THERAPY FOR INDEFINITE PERIODS. IN THIS STUDY, WE PERFORMED WHOLE-EXOME SEQUENCING TO IDENTIFY SOMATIC MUTATIONS IN 24 PATIENTS WITH NEWLY DIAGNOSED CHRONIC PHASE CML WHO WERE REGISTERED IN THE JALSG CML212 STUDY. WE IDENTIFIED 191 SOMATIC MUTATIONS OTHER THAN THE BCR-ABL1 FUSION GENE (MEDIAN 8, RANGE 1-17). AGE, HEMOGLOBIN CONCENTRATION AND WHITE BLOOD CELL COUNTS WERE CORRELATED WITH THE NUMBER OF MUTATIONS. PATIENTS WITH MUTATIONS ?6 SHOWED HIGHER RATE OF ACHIEVING MAJOR MOLECULAR RESPONSE THAN THOSE<6 (P=0.0381). MUTATIONS IN EPIGENETIC REGULATOR, ASXL1, TET2, TET3, KDM1A AND MSH6 WERE FOUND IN 25% OF PATIENTS. TET2 OR TET3, AKT1 AND RUNX1 WERE MUTATED IN ONE PATIENT EACH. ASXL1 WAS MUTATED WITHIN EXON 12 IN THREE CASES. MUTATED GENES WERE SIGNIFICANTLY ENRICHED WITH CELL SIGNALING AND CELL DIVISION PATHWAYS. FURTHERMORE, DNA COPY NUMBER ANALYSIS SHOWED THAT 2 OF 24 PATIENTS HAD UNIPARENTAL DISOMY OF CHROMOSOME 1P OR 3Q, WHICH DISAPPEARED MAJOR MOLECULAR RESPONSE WAS ACHIEVED. THESE MUTATIONS MAY PLAY SIGNIFICANT ROLES IN CML PATHOGENESIS IN ADDITION TO THE STRONG DRIVER MUTATION BCR-ABL1. 2017 8 3530 29 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 9 6249 31 THE METHYLATION STATUS OF THE MAJOR BREAKPOINT CLUSTER REGION IN HUMAN LEUKEMIA CELLS, INCLUDING PHILADELPHIA CHROMOSOME-POSITIVE CELLS, IS LINKED TO THE LINEAGE OF HEMATOPOIETIC CELLS. THE PHILADELPHIA (PH) TRANSLOCATION [T(9;22)(Q34;Q11)] IS THE MOST COMMON GENETIC ABNORMALITY IN HUMAN LEUKEMIA; A TRANSPOSITION OF THE ABL GENE TO THE MAJOR-BREAKPOINT CLUSTER REGION (M-BCR) IS ASSOCIATED WITH THE PATHOGENESIS IN PH+ CHRONIC MYELOGENOUS LEUKEMIA (PH+ CML) AND IN SOME CASES OF PH+ ACUTE LEUKEMIA (PH+ AL). OUR CURRENT UNDERSTANDING OF THE METHYLATION OF HUMAN GENOMES ALLOWS US TO CONSIDER THE ASSOCIATION BETWEEN THE EPIGENETIC PHENOMENON AND THE CONTROL OF DIFFERENTIATION AND PROLIFERATION IN MAMMALIAN CELLS. IN ORDER TO DETERMINE WHETHER THE METHYLATION STATUS OF THE M-BCR IS ASSOCIATED WITH BREAKPOINT-LOCALIZATION IN THIS REGION AND WITH THE LINEAGE OF HEMATOPOIETIC CELLS, WE HAVE EXAMINED 28 PATIENTS WITH PH+ LEUKEMIAS, INCLUDING NINE WITH PH+ AL, SIX PATIENTS WITH ACUTE MYELOBLASTIC LEUKEMIA WITHOUT PH (PH- AML), AND FIVE PATIENTS WITH PH- ACUTE LYMPHOBLASTIC LEUKEMIA (PH- ALL); USING THE RESTRICTION ENDONUCLEASE ISOCHIZOMERS, MSPI AND HPAII. IN CML PATIENTS IN THE CHRONIC PHASE, THE HYPOMETHYLATED STATUS WITHIN THE NORMAL M-BCR ALLELE IS HETEROGENEOUS. IN CONTRAST, PATIENTS WITH PH+ CML IN THE LYMPHOID BLAST CRISIS PHASE EXHIBITED A 2.5/2.7 KB BAND WITH A COMPLETE DISAPPEARANCE OF THE GERMLINE M-BCR FRAGMENT (TYPE L). THIS PATTERN IS CONSISTENTLY NOTED IN PH- ALL CELLS, AND THE PATTERN IS QUITE DIFFERENT FROM THAT FOUND IN MYELOID BLAST CRISIS OR PH- AML (TYPE M). IN PATIENTS WITH M-BCR-NONREARRANGED PH+ ALL, IT IS SUGGESTED THAT THE M-BCR METHYLATION PATTERNS ARE CELL-LINEAGE SPECIFIC BUT SOME PH+ ALL CELLS HAD A HYPOMETHYLATION PATTERN THAT WAS IDENTICAL TO THAT OBSERVED IN PH- AML, SUGGESTING A DISTINCTION OF GENETIC DIVERSITY OF LEUKEMIA CELLS WITH THE PH CHROMOSOME, ESPECIALLY PH+ AL. 1993 10 4555 28 MUTATIONAL SPECTRUM ANALYSIS OF CHRONIC MYELOMONOCYTIC LEUKEMIA INCLUDES GENES ASSOCIATED WITH EPIGENETIC REGULATION: UTX, EZH2, AND DNMT3A. CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML), A MYELODYSPLASTIC/MYELOPROLIFERATIVE NEOPLASM, IS CHARACTERIZED BY MONOCYTIC PROLIFERATION, DYSPLASIA, AND PROGRESSION TO ACUTE MYELOID LEUKEMIA. CMML HAS BEEN ASSOCIATED WITH SOMATIC MUTATIONS IN DIVERSE RECENTLY IDENTIFIED GENES. WE ANALYZED 72 WELL-CHARACTERIZED PATIENTS WITH CMML (N = 52) AND CMML-DERIVED ACUTE MYELOID LEUKEMIA (N = 20) FOR RECURRENT CHROMOSOMAL ABNORMALITIES WITH THE USE OF ROUTINE CYTOGENETICS AND SINGLE NUCLEOTIDE POLYMORPHISM ARRAYS ALONG WITH COMPREHENSIVE MUTATIONAL SCREENING. CYTOGENETIC ABERRATIONS WERE PRESENT IN 46% OF CASES, WHEREAS SINGLE NUCLEOTIDE POLYMORPHISM ARRAY INCREASED THE DIAGNOSTIC YIELD TO 60%. AT LEAST 1 MUTATION WAS FOUND IN 86% OF ALL CASES; NOVEL UTX, DNMT3A, AND EZH2 MUTATIONS WERE FOUND IN 8%, 10%, AND 5.5% OF PATIENTS, RESPECTIVELY. TET2 MUTATIONS WERE PRESENT IN 49%, ASXL1 IN 43%, CBL IN 14%, IDH1/2 IN 4%, KRAS IN 7%, NRAS IN 4%, AND JAK2 V617F IN 1% OF PATIENTS. VARIOUS MUTANT GENOTYPE COMBINATIONS WERE OBSERVED, INDICATING MOLECULAR HETEROGENEITY IN CMML. OUR RESULTS SUGGEST THAT MOLECULAR DEFECTS AFFECTING DISTINCT PATHWAYS CAN LEAD TO SIMILAR CLINICAL PHENOTYPES. 2011 11 2326 32 EPIGENETIC REGULATION OF HOTAIR IN ADVANCED CHRONIC MYELOID LEUKEMIA. PURPOSE: CHRONIC MYELOID LEUKEMIA (CML) ACCOUNTS FOR ~10% OF LEUKEMIA CASES, AND ITS PROGRESSION INVOLVES EPIGENETIC GENE REGULATION. THIS STUDY INVESTIGATED EPIGENETIC REGULATION OF HOTAIR AND ITS TARGET MICRORNA, MIR-143, IN ADVANCED CML. PATIENTS AND METHODS: WE FIRST ISOLATED BONE MARROW MONONUCLEAR CELLS FROM 70 PATIENTS WITH DIFFERENT PHASES OF CML AND FROM HEALTHY DONORS AS NORMAL CONTROL; WE ALSO CULTURED K562 AND KCL22 CELLS, TREATED WITH DEMETHYLATION DRUG; MTT ASSAY, FLOW CYTOMETRY, QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION (QPCR), METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION (MSP), WESTERN BLOT, LUCIFERASE ASSAY, RNA PULL-DOWN ASSAY AND RNA-BINDING PROTEIN IMMUNOPRECIPITATION (RIP) ASSAY WERE PERFORMED. RESULT: AS MEASURED BY QPCR, HOTAIR EXPRESSION IN K562 CELLS, KCL22 CELLS, AND SAMPLES FROM CASES OF ADVANCED-STAGE CML INCREASED WITH LEVELS OF SEVERAL DNA METHYLTRANSFERASES AND HISTONE DEACETYLATES, INCLUDING DNMT1, DNMT3A, HDAC1, EZH2, AND LSD1, AND MIR-143 LEVELS WERE DECREASED AND HOTAIR LEVELS WERE INCREASED. TREATMENT WITH 5-AZACYTIDINE, A DNA METHYLATION INHIBITOR, DECREASED DNMT1, DNMT3A, HDAC1, EZH2, LSD1 MRNA, PROTEIN LEVELS, AND HOTAIR MRNA LEVELS BUT INCREASED MIR-143 LEVELS. HOTAIR KNOCKDOWN AND MIR-143 OVEREXPRESSION BOTH INHIBITED PROLIFERATION AND PROMOTED APOPTOSIS IN KCL22 AND K562 CELLS THROUGH THE PI3K/AKT PATHWAY. RNA PULL-DOWN, MASS SPECTROMETRY, AND RIP ASSAYS SHOWED THAT HOTAIR INTERACTED WITH EZH2 AND LSD1. A DUAL-LUCIFERASE ASSAY DEMONSTRATED THAT HOTAIR INTERACTED WITH MIR-143. CONCLUSION: OUR FINDINGS DEMONSTRATE THE KEY EPIGENETIC INTERACTIONS OF HOTAIR RELATED TO CML PROGRESSION AND SUGGEST HOTAIR AS A POTENTIAL THERAPEUTIC TARGET FOR ADVANCED CML. FURTHERMORE, OUR RESULTS SUPPORT THE USE OF DEMETHYLATION DRUGS AS A CML TREATMENT STRATEGY. 2018 12 690 24 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 13 791 24 CELLULAR AND MOLECULAR NETWORKS IN CHRONIC MYELOID LEUKEMIA: THE LEUKEMIC STEM, PROGENITOR AND STROMAL CELL INTERPLAY. THE USE OF IMATINIB, SECOND AND THIRD GENERATION ABL TYROSINE KINASE INHIBITORS (TKI) (I.E. DASATINIB, NILOTINIB, BOSUTINIB AND PONATINIB) MADE CML A CLINICALLY MANAGEABLE AND, IN A SMALL PERCENTAGE OF CASES, A CURED DISEASE. TKI THERAPY ALSO TURNED CML BLASTIC TRANSFORMATION INTO A RARE EVENT; HOWEVER, DISEASE PROGRESSION STILL OCCURS IN THOSE PATIENTS WHO ARE REFRACTORY, NOT COMPLIANT WITH TKI THERAPY OR DEVELOP RESISTANCE TO MULTIPLE TKIS. IN THE PAST FEW YEARS, IT BECAME CLEAR THAT THE BCRABL1 ONCOGENE DOES NOT OPERATE ALONE TO DRIVE DISEASE EMERGENCE, MAINTENANCE AND PROGRESSION. INDEED, IT SEEMS THAT BONE MARROW (BM) MICROENVIRONMENT-GENERATED SIGNALS AND CELL AUTONOMOUS BCRABL1 KINASE-INDEPENDENT GENETIC AND EPIGENETIC ALTERATIONS ALL CONTRIBUTE TO: I. PERSISTENCE OF A QUIESCENT LEUKEMIC STEM CELL (LSC) RESERVOIR, II. INNATE OR ACQUIRED RESISTANCE TO TKIS, AND III. PROGRESSION INTO THE FATAL BLAST CRISIS STAGE. HEREIN, WE REVIEW THE INTRICATE LEUKEMIC NETWORK IN WHICH ABERRANT, BUT FINELY TUNED, SURVIVAL, MITOGENIC AND SELF-RENEWAL SIGNALS ARE GENERATED BY LEUKEMIC PROGENITORS, STROMAL CELLS, IMMUNE CELLS AND METABOLIC MICROENVIRONMENTAL CONDITIONS (E.G. HYPOXIA) TO PROMOTE LSC MAINTENANCE AND BLASTIC TRANSFORMATION. 2017 14 826 30 CHARACTERIZATION OF K562 CELLS: UNCOVERING NOVEL CHROMOSOMES, ASSESSING TRANSFERRIN RECEPTOR EXPRESSION, AND PROBING PHARMACOLOGICAL THERAPIES. HUMAN ERYTHROLEUKEMIC K562 CELLS REPRESENT THE PROTOTYPICAL CELL CULTURE MODEL OF CHRONIC MYELOID LEUKEMIA (CML). THE CELLS ARE PSEUDO-TRIPLOID AND POSITIVE FOR THE PHILADELPHIA CHROMOSOME. THEREFORE, K562 CELLS HAVE BEEN WIDELY USED FOR INVESTIGATING THE BCR/ABL1 ONCOGENE AND THE TYROSINE KINASE INHIBITOR, IMATINIB-MESYLATE. FURTHER, K562 CELLS OVEREXPRESS TRANSFERRIN RECEPTORS (TFR) AND HAVE BEEN USED AS A MODEL FOR TARGETING CYTOTOXIC THERAPIES, VIA RECEPTOR-MEDIATED ENDOCYTOSIS. HERE, WE HAVE CHARACTERIZED K562 CELLS FOCUSING ON THE KARYOTYPE OF CELLS IN PROLONGED CULTURE, REGULATION OF EXPRESSION OF TFR IN WILDTYPE (WT) AND DOXORUBICIN-RESISTANT CELLS, AND RESPONSES TO HISTONE DEACETYLASE INHIBITION (HDACI). KARYOTYPE ANALYSIS INDICATES NOVEL CHROMOSOMES AND GENE EXPRESSION ANALYSIS SUGGESTS A SHIFT OF CULTURED K562 CELLS AWAY FROM PATIENT-DERIVED LEUKEMIC CELLS. WE CONFIRM THE HIGH EXPRESSION OF TFR ON K562 CELLS USING IMMUNOFLUORESCENCE AND CELL-SURFACE RECEPTOR BINDING RADIOASSAYS. IMPORTANTLY, HIGH TFR EXPRESSION IS OBSERVED IN PATIENT-DERIVED CELLS, AND WE HIGHLIGHT THE PERSISTENT EXPRESSION OF TFR FOLLOWING DOXORUBICIN ACQUIRED RESISTANCE. EPIGENETIC ANALYSIS INDICATES THAT PERMISSIVE HISTONE ACETYLATION AND METHYLATION AT THE PROMOTER REGION REGULATES THE TRANSCRIPTION OF TFR IN K562 CELLS. FINALLY, WE SHOW RELATIVELY HIGH EXPRESSION OF HDAC ENZYMES IN K562 CELLS AND DEMONSTRATE THE CHEMOTOXIC EFFECTS OF HDACI, USING THE FDA-APPROVED HYDROXAMIC ACID, VORINOSTAT. TOGETHER WITH A DESCRIPTION OF MORPHOLOGY, INFRARED SPECTRAL ANALYSIS, AND EXAMINATION OF METABOLIC PROPERTIES, WE PROVIDE A COMPREHENSIVE CHARACTERIZATION OF K562 CELLS. OVERALL, K562 CELL CULTURE SYSTEMS REMAIN WIDELY USED FOR THE INVESTIGATION OF NOVEL THERAPEUTICS FOR CML, WHICH IS PARTICULARLY IMPORTANT IN CASES OF IMATINIB-MESYLATE RESISTANCE. 2023 15 955 20 CHRONIC MYELOID LEUKEMIA: MECHANISMS OF BLASTIC TRANSFORMATION. THE BCR-ABL1 ONCOPROTEIN TRANSFORMS PLURIPOTENT HSCS AND INITIATES CHRONIC MYELOID LEUKEMIA (CML). PATIENTS WITH EARLY PHASE (ALSO KNOWN AS CHRONIC PHASE [CP]) DISEASE USUALLY RESPOND TO TREATMENT WITH ABL TYROSINE KINASE INHIBITORS (TKIS), ALTHOUGH SOME PATIENTS WHO RESPOND INITIALLY LATER BECOME RESISTANT. IN MOST PATIENTS, TKIS REDUCE THE LEUKEMIA CELL LOAD SUBSTANTIALLY, BUT THE CELLS FROM WHICH THE LEUKEMIA CELLS ARE DERIVED DURING CP (SO-CALLED LEUKEMIA STEM CELLS [LSCS]) ARE INTRINSICALLY INSENSITIVE TO TKIS AND SURVIVE LONG TERM. LSCS OR THEIR PROGENY CAN ACQUIRE ADDITIONAL GENETIC AND/OR EPIGENETIC CHANGES THAT CAUSE THE LEUKEMIA TO TRANSFORM FROM CP TO A MORE ADVANCED PHASE, WHICH HAS BEEN SUBCLASSIFIED AS EITHER ACCELERATED PHASE OR BLASTIC PHASE DISEASE. THE LATTER RESPONDS POORLY TO TREATMENT AND IS USUALLY FATAL. HERE, WE DISCUSS WHAT IS KNOWN ABOUT THE MOLECULAR MECHANISMS LEADING TO BLASTIC TRANSFORMATION OF CML AND PROPOSE SOME NOVEL THERAPEUTIC APPROACHES. 2010 16 6198 31 THE IMPLICATION OF CANCER PROGENITOR CELLS AND THE ROLE OF EPIGENETICS IN THE DEVELOPMENT OF NOVEL THERAPEUTIC STRATEGIES FOR CHRONIC MYELOID LEUKEMIA. SIGNIFICANCE: CHRONIC MYELOID LEUKEMIA (CML) INVOLVES THE MALIGNANT TRANSFORMATION OF HEMATOPOIETIC STEM CELLS, DEFINED LARGELY BY THE PHILADELPHIA CHROMOSOME AND EXPRESSION OF THE BREAKPOINT CLUSTER REGION-ABELSON (BCR-ABL) ONCOPROTEIN. PHARMACOLOGICAL TYROSINE KINASE INHIBITORS (TKIS), INCLUDING IMATINIB MESYLATE, HAVE OVERCOME LIMITATIONS IN CONVENTIONAL TREATMENT FOR THE IMPROVED CLINICAL MANAGEMENT OF CML. RECENT ADVANCES: ACCUMULATED EVIDENCE HAS LED TO THE IDENTIFICATION OF A SUBPOPULATION OF QUIESCENT LEUKEMIA PROGENITOR CELLS WITH STEM-LIKE SELF RENEWAL PROPERTIES THAT MAY INITIATE LEUKEMOGENESIS, WHICH ARE ALSO SHOWN TO BE PRESENT IN RESIDUAL DISEASE DUE TO THEIR INSENSITIVITY TO TYROSINE KINASE INHIBITION. CRITICAL ISSUES: THE CHARACTERIZATION OF QUIESCENT LEUKEMIA PROGENITOR CELLS AS A UNIQUE CELL POPULATION IN CML PATHOGENESIS HAS BECOME CRITICAL WITH THE COMPLETE ELUCIDATION OF MECHANISMS INVOLVED IN THEIR SURVIVAL INDEPENDENT OF BCR-ABL THAT IS IMPORTANT IN THE DEVELOPMENT OF NOVEL ANTICANCER STRATEGIES. UNDERSTANDING OF THESE FUNCTIONAL PATHWAYS IN CML PROGENITOR CELLS WILL ALLOW FOR THEIR SELECTIVE THERAPEUTIC TARGETING. IN ADDITION, DISEASE PATHOGENESIS AND DRUG RESPONSIVENESS IS ALSO THOUGHT TO BE MODULATED BY EPIGENETIC REGULATORY MECHANISMS SUCH AS DNA METHYLATION, HISTONE ACETYLATION, AND MICRORNA EXPRESSION, WITH A CAPACITY TO CONTROL CML-ASSOCIATED GENE TRANSCRIPTION. FUTURE DIRECTIONS: A NUMBER OF COMPOUNDS IN COMBINATION WITH TKIS ARE UNDER PRECLINICAL AND CLINICAL INVESTIGATION TO ASSESS THEIR SYNERGISTIC POTENTIAL IN TARGETING LEUKEMIC PROGENITOR CELLS AND/OR THE EPIGENOME IN CML. DESPITE THE COLLECTIVE PROMISE, FURTHER RESEARCH IS REQUIRED IN ORDER TO REFINE UNDERSTANDING, AND, ULTIMATELY, ADVANCE ANTILEUKEMIC THERAPEUTIC STRATEGIES. 2015 17 4748 29 NOVEL MUTATIONS AND THEIR FUNCTIONAL AND CLINICAL RELEVANCE IN MYELOPROLIFERATIVE NEOPLASMS: JAK2, MPL, TET2, ASXL1, CBL, IDH AND IKZF1. MYELOPROLIFERATIVE NEOPLASMS (MPNS) ORIGINATE FROM GENETICALLY TRANSFORMED HEMATOPOIETIC STEM CELLS THAT RETAIN THE CAPACITY FOR MULTILINEAGE DIFFERENTIATION AND EFFECTIVE MYELOPOIESIS. BEGINNING IN EARLY 2005, A NUMBER OF NOVEL MUTATIONS INVOLVING JANUS KINASE 2 (JAK2), MYELOPROLIFERATIVE LEUKEMIA VIRUS (MPL), TET ONCOGENE FAMILY MEMBER 2 (TET2), ADDITIONAL SEX COMBS-LIKE 1 (ASXL1), CASITAS B-LINEAGE LYMPHOMA PROTO-ONCOGENE (CBL), ISOCITRATE DEHYDROGENASE (IDH) AND IKAROS FAMILY ZINC FINGER 1 (IKZF1) HAVE BEEN DESCRIBED IN BCR-ABL1-NEGATIVE MPNS. HOWEVER, NONE OF THESE MUTATIONS WERE MPN SPECIFIC, DISPLAYED MUTUAL EXCLUSIVITY OR COULD BE TRACED BACK TO A COMMON ANCESTRAL CLONE. JAK2 AND MPL MUTATIONS APPEAR TO EXERT A PHENOTYPE-MODIFYING EFFECT AND ARE DISTINCTLY ASSOCIATED WITH POLYCYTHEMIA VERA, ESSENTIAL THROMBOCYTHEMIA AND PRIMARY MYELOFIBROSIS; THE CORRESPONDING MUTATIONAL FREQUENCIES ARE APPROXIMATELY 99, 55 AND 65% FOR JAK2 AND 0, 3 AND 10% FOR MPL MUTATIONS. THE INCIDENCE OF TET2, ASXL1, CBL, IDH OR IKZF1 MUTATIONS IN THESE DISORDERS RANGES FROM 0 TO 17%; THESE LATTER MUTATIONS ARE MORE COMMON IN CHRONIC (TET2, ASXL1, CBL) OR JUVENILE (CBL) MYELOMONOCYTIC LEUKEMIAS, MASTOCYTOSIS (TET2), MYELODYSPLASTIC SYNDROMES (TET2, ASXL1) AND SECONDARY ACUTE MYELOID LEUKEMIA, INCLUDING BLAST-PHASE MPN (IDH, ASXL1, IKZF1). THE FUNCTIONAL CONSEQUENCES OF MPN-ASSOCIATED MUTATIONS INCLUDE UNREGULATED JAK-STAT (JANUS KINASE/SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION) SIGNALING, EPIGENETIC MODULATION OF TRANSCRIPTION AND ABNORMAL ACCUMULATION OF ONCOPROTEINS. HOWEVER, IT IS NOT CLEAR AS TO WHETHER AND HOW THESE ABNORMALITIES CONTRIBUTE TO DISEASE INITIATION, CLONAL EVOLUTION OR BLASTIC TRANSFORMATION. 2010 18 138 42 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 19 5979 36 TET2 MUTATIONS ARE ASSOCIATED WITH SPECIFIC 5-METHYLCYTOSINE AND 5-HYDROXYMETHYLCYTOSINE PROFILES IN PATIENTS WITH CHRONIC MYELOMONOCYTIC LEUKEMIA. CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML) HAS RECENTLY BEEN ASSOCIATED WITH A HIGH INCIDENCE OF DIVERSE MUTATIONS IN GENES SUCH AS TET2 OR EZH2 THAT ARE IMPLICATED IN EPIGENETIC MECHANISMS. WE HAVE PERFORMED GENOME-WIDE DNA METHYLATION ARRAYS AND MUTATIONAL ANALYSIS OF TET2, IDH1, IDH2, EZH2 AND JAK2 IN A GROUP OF 24 PATIENTS WITH CMML. 249 GENES WERE DIFFERENTIALLY METHYLATED BETWEEN CMML PATIENTS AND CONTROLS. USING INGENUITY PATHWAY ANALYSIS, WE IDENTIFIED ENRICHMENT IN A GENE NETWORK CENTERED AROUND PLC, JNK AND ERK SUGGESTING THAT THESE PATHWAYS, WHOSE DEREGULATION HAS BEEN RECENTLY DESCRIBED IN CMML, ARE AFFECTED BY EPIGENETIC MECHANISMS. MUTATIONS OF TET2, JAK2 AND EZH2 WERE FOUND IN 15 PATIENTS (65%), 4 PATIENTS (17%) AND 1 PATIENT (4%) RESPECTIVELY WHILE NO MUTATIONS IN THE IDH1 AND IDH2 GENES WERE IDENTIFIED. INTERESTINGLY, PATIENTS WITH WILD TYPE TET2 CLUSTERED SEPARATELY FROM PATIENTS WITH TET2 MUTATIONS, SHOWED A HIGHER DEGREE OF HYPERMETHYLATION AND WERE ASSOCIATED WITH HIGHER RISK KARYOTYPES. OUR RESULTS DEMONSTRATE THE PRESENCE OF ABERRANT DNA METHYLATION IN CMML AND IDENTIFIES TET2 MUTANT CMML AS A BIOLOGICALLY DISTINCT DISEASE SUBTYPE WITH A DIFFERENT EPIGENETIC PROFILE. 2012 20 4557 19 MUTATIONS IN ASXL1 ARE ASSOCIATED WITH POOR PROGNOSIS ACROSS THE SPECTRUM OF MALIGNANT MYELOID DISEASES. THE ASXL1 GENE IS ONE OF THE MOST FREQUENTLY MUTATED GENES IN MALIGNANT MYELOID DISEASES. THE ASXL1 PROTEIN BELONGS TO PROTEIN COMPLEXES INVOLVED IN THE EPIGENETIC REGULATION OF GENE EXPRESSION. ASXL1 MUTATIONS ARE FOUND IN MYELOPROLIFERATIVE NEOPLASMS (MPN), MYELODYSPLASTIC SYNDROMES (MDS), CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML) AND ACUTE MYELOID LEUKEMIA (AML). THEY ARE GENERALLY ASSOCIATED WITH SIGNS OF AGGRESSIVENESS AND POOR CLINICAL OUTCOME. BECAUSE OF THIS, A SYSTEMATIC DETERMINATION OF ASXL1 MUTATIONAL STATUS IN MYELOID MALIGNANCIES SHOULD HELP IN PROGNOSIS ASSESSMENT. 2012