1 1901 114 ENGRAFTMENT OF CHRONIC MYELOMONOCYTIC LEUKEMIA CELLS IN IMMUNOCOMPROMISED MICE SUPPORTS DISEASE DEPENDENCY ON CYTOKINES. CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML) IS A CLONAL HEMATOPOIETIC STEM CELL DISORDER THAT TYPICALLY ASSOCIATES WITH MUTATIONS IN EPIGENETIC, SPLICING, AND SIGNALING GENES. GENETICALLY MODIFIED MOUSE MODELS ONLY PARTIALLY RECAPITULATE THE DISEASE PHENOTYPE, WHEREAS XENOTRANSPLANTATION OF CMML CELLS IN IMMUNOCOMPROMISED MICE HAS BEEN RARELY SUCCESSFUL SO FAR. HERE, CMML CD34(+) CELLS SORTED FROM PATIENT BONE MARROW (BM) OR PERIPHERAL BLOOD (PB) WERE INJECTED INTRAVENOUSLY INTO NSG (NOD/LTSZ-SCID IL2RGAMMANULL) MICE AND NSG MICE ENGINEERED TO EXPRESS HUMAN GRANULO-MONOCYTE COLONY-STIMULATING FACTOR, STEM CELL FACTOR, AND INTERLEUKIN-3 (NSGS MICE). FIFTEEN OUT OF 16 PATIENT SAMPLES (94%) SUCCESSFULLY ENGRAFTED INTO NSG OR NSGS OR BOTH MOUSE STRAINS. THE EXPANSION OF HUMAN CELLS, PREDOMINANT IN THE BM, WAS ALSO OBSERVED IN THE SPLEEN AND THE PB AND WAS GREATLY ENHANCED IN MICE PRODUCING THE 3 HUMAN CYTOKINES. GENE MUTATIONS IDENTIFIED IN ENGRAFTED CELLS WERE MOSTLY SIMILAR TO THOSE IDENTIFIED IN PATIENT CELLS BEFORE INJECTION. SUCCESSFUL SECONDARY ENGRAFTMENT WAS OBTAINED IN NSGS MICE IN 3 OUT OF 10 ATTEMPTS. THUS, PRIMARY CMML LEUKEMIC CELLS EXPAND MUCH BETTER IN NSGS COMPARED WITH NSG MICE WITH LIMITED EFFICACY OF SECONDARY TRANSPLANT. 2017 2 2462 28 EPIGENETIC THERAPY OF MYELODYSPLASTIC SYNDROMES CONNECTS TO CELLULAR DIFFERENTIATION INDEPENDENTLY OF ENDOGENOUS RETROELEMENT DEREPRESSION. BACKGROUND: MYELODYSPLASTIC SYNDROMES (MDS) AND ACUTE MYELOID LEUKAEMIA (AML) ARE CHARACTERISED BY ABNORMAL EPIGENETIC REPRESSION AND DIFFERENTIATION OF BONE MARROW HAEMATOPOIETIC STEM CELLS (HSCS). DRUGS THAT REVERSE EPIGENETIC REPRESSION, SUCH AS 5-AZACYTIDINE (5-AZA), INDUCE HAEMATOLOGICAL IMPROVEMENT IN HALF OF TREATED PATIENTS. ALTHOUGH THE MECHANISMS UNDERLYING THERAPY SUCCESS ARE NOT YET CLEAR, INDUCTION OF ENDOGENOUS RETROELEMENTS (ERES) HAS BEEN HYPOTHESISED. METHODS: USING RNA SEQUENCING (RNA-SEQ), WE COMPARED THE TRANSCRIPTION OF ERES IN BONE MARROW HSCS FROM A NEW COHORT OF MDS AND CHRONIC MYELOMONOCYTIC LEUKAEMIA (CMML) PATIENTS BEFORE AND AFTER 5-AZA TREATMENT WITH HSCS FROM HEALTHY DONORS AND AML PATIENTS. WE FURTHER EXAMINED ERE TRANSCRIPTION USING THE MOST COMPREHENSIVE ANNOTATION OF ERE-OVERLAPPING TRANSCRIPTS EXPRESSED IN HSCS, GENERATED HERE BY DE NOVO TRANSCRIPT ASSEMBLY AND SUPPORTED BY FULL-LENGTH RNA-SEQ. RESULTS: CONSISTENT WITH PRIOR REPORTS, WE FOUND THAT TREATMENT WITH 5-AZA INCREASED THE REPRESENTATION OF ERE-DERIVED RNA-SEQ READS IN THE TRANSCRIPTOME. HOWEVER, SUCH INCREASES WERE COMPARABLE BETWEEN TREATMENT RESPONSES AND FAILURES. THE EXTENDED VIEW OF HSC TRANSCRIPTIONAL DIVERSITY OFFERED BY DE NOVO TRANSCRIPT ASSEMBLY ARGUED AGAINST 5-AZA-RESPONSIVE ERES AS DETERMINANTS OF THE OUTCOME OF THERAPY. INSTEAD, IT UNCOVERED PRE-TREATMENT EXPRESSION AND ALTERNATIVE SPLICING OF DEVELOPMENTALLY REGULATED GENE TRANSCRIPTS AS PREDICTORS OF THE RESPONSE OF MDS AND CMML PATIENTS TO 5-AZA TREATMENT. CONCLUSIONS: OUR STUDY IDENTIFIES THE DEVELOPMENTALLY REGULATED TRANSCRIPTIONAL SIGNATURES OF PROTEIN-CODING AND NON-CODING GENES, RATHER THAN ERES, AS CORRELATES OF A FAVOURABLE RESPONSE OF MDS AND CMML PATIENTS TO 5-AZA TREATMENT AND OFFERS NOVEL CANDIDATES FOR FURTHER EVALUATION. 2019 3 4549 30 MUTATION ANALYSIS OF THERAPY-RELATED MYELOID NEOPLASMS. WE ANALYZED THE GENETIC MUTATION STATUS OF 13 PATIENTS WITH THERAPY-RELATED MYELOID NEOPLASMS (T-MN). CONSISTENT WITH PREVIOUS REPORTS, T-MN CELLS PREFERENTIALLY ACQUIRED MUTATIONS IN TP53 AND EPIGENETIC MODIFYING GENES, INSTEAD OF MUTATIONS IN TYROSINE KINASE AND SPLICEOSOME GENES. FURTHERMORE, WE COMPARED THE MUTATION STATUS OF THREE T-MN CELLS WITH EACH OF THE INITIAL LYMPHOID MALIGNANT CELLS, AND IDENTIFIED COMMON MUTATIONS AMONG T-MN AND THE INITIAL MALIGNANT CELLS IN TWO PATIENTS. IN A PATIENT WHO DEVELOPED CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML) AFTER FOLLICULAR LYMPHOMA (FL), TET2 MUTATION WAS IDENTIFIED IN BOTH CMML AND FL CELLS. NOTABLY, THE TET2 MUTATION WAS ALSO IDENTIFIED IN PERIPHERAL BLOOD CELLS IN THE DISEASE-FREE PERIOD WITH THE SAME ALLELIC FREQUENCY AS CMML AND FL CELLS, BUT NOT IN A GERM-LINE CONTROL, INDICATING THAT THE TET2 MUTATION OCCURRED SOMATICALLY IN THE INITIATING CLONE FOR BOTH MALIGNANT CELLS. ON THE OTHER HAND, A GERM-LINE MYB MUTATION WAS IDENTIFIED IN A PATIENT WHO DEVELOPED MYELODYSPLASTIC SYNDROMES (MDS) AFTER FL. THESE RESULTS SUGGEST THAT GERM-LINE DEPOSITION AND CLONAL HEMATOPOIESIS ARE CLOSELY ASSOCIATED WITH T-MN SUSCEPTIBILITY; HOWEVER, FURTHER ANALYSIS IS NECESSARY TO CLARIFY THE MECHANISM REQUIRED TO PROVIDE THE INITIATING CLONE WITH LINEAGE COMMITMENT AND CLONAL EXPANSION. 2018 4 1070 27 CLONAL ARCHITECTURE OF CHRONIC MYELOMONOCYTIC LEUKEMIAS. GENOMIC STUDIES IN CHRONIC MYELOID MALIGNANCIES, INCLUDING MYELOPROLIFERATIVE NEOPLASMS (MPN), MYELODYSPLASTIC SYNDROMES (MDS), AND MPN/MDS, HAVE IDENTIFIED COMMON MUTATIONS IN GENES ENCODING SIGNALING, EPIGENETIC, TRANSCRIPTION, AND SPLICING FACTORS. IN THE PRESENT STUDY, WE INTERROGATED THE CLONAL ARCHITECTURE BY MUTATION-SPECIFIC DISCRIMINATION ANALYSIS OF SINGLE-CELL-DERIVED COLONIES IN 28 PATIENTS WITH CHRONIC MYELOMONOCYTIC LEUKEMIAS (CMML), THE MOST FREQUENT MPN/MDS. THIS ANALYSIS REVEALS A LINEAR ACQUISITION OF THE STUDIED MUTATIONS WITH LIMITED BRANCHING THROUGH LOSS OF HETEROZYGOSITY. SERIAL ANALYSIS OF UNTREATED AND TREATED SAMPLES DEMONSTRATES A DYNAMIC ARCHITECTURE ON WHICH MOST CURRENT THERAPEUTIC APPROACHES HAVE LIMITED EFFECTS. THE MAIN DISEASE CHARACTERISTICS ARE EARLY CLONAL DOMINANCE, ARISING AT THE CD34(+)/CD38(-) STAGE OF HEMATOPOIESIS, AND GRANULOMONOCYTIC DIFFERENTIATION SKEWING OF MULTIPOTENT AND COMMON MYELOID PROGENITORS. COMPARISON OF CLONAL EXPANSIONS OF TET2 MUTATIONS IN MDS, MPN, AND CMML, TOGETHER WITH FUNCTIONAL INVALIDATION OF TET2 IN SORTED PROGENITORS, SUGGESTS A CAUSATIVE LINK BETWEEN EARLY CLONAL DOMINANCE AND SKEWED GRANULOMONOCYTIC DIFFERENTIATION. ALTOGETHER, EARLY CLONAL DOMINANCE MAY DISTINGUISH CMML FROM OTHER CHRONIC MYELOID NEOPLASMS WITH SIMILAR GENE MUTATIONS. 2013 5 4265 25 MICRO-RNA-125A MEDIATES THE EFFECTS OF HYPOMETHYLATING AGENTS IN CHRONIC MYELOMONOCYTIC LEUKEMIA. BACKGROUND: CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML) IS AN AGGRESSIVE HEMATOPOIETIC MALIGNANCY THAT ARISES FROM HEMATOPOIETIC STEM AND PROGENITOR CELLS (HSPCS). PATIENTS WITH CMML ARE FREQUENTLY TREATED WITH EPIGENETIC THERAPEUTIC APPROACHES, IN PARTICULAR THE HYPOMETHYLATING AGENTS (HMAS), AZACITIDINE (AZA) AND DECITABINE (DEC). ALTHOUGH HMAS ARE BELIEVED TO MEDIATE THEIR EFFICACY VIA RE-EXPRESSION OF HYPERMETHYLATED TUMOR SUPPRESSORS, KNOWLEDGE ABOUT RELEVANT HMA TARGETS IS SCARCE. AS SILENCING OF TUMOR-SUPPRESSIVE MICRO-RNAS (MIRS) BY PROMOTER HYPERMETHYLATION IS A CRUCIAL STEP IN MALIGNANT TRANSFORMATION, WE ASKED FOR A ROLE OF MIRS IN HMA EFFICACY IN CMML. RESULTS: INITIALLY, WE PERFORMED GENOME-WIDE MIR-EXPRESSION PROFILING IN A KRAS(G12D)-INDUCED CMML MOUSE MODEL. SELECTED CANDIDATES WITH PROMINENTLY DECREASED EXPRESSION WERE VALIDATED BY QPCR IN CMML MICE AND HUMAN CMML PATIENTS. THESE EXPERIMENTS REVEALED THE CONSISTENT DECREASE IN MIR-125A, A MIR WITH PREVIOUSLY DESCRIBED TUMOR-SUPPRESSIVE FUNCTION IN MYELOID NEOPLASIAS. FURTHERMORE, WE SHOW THAT MIR-125A DOWNREGULATION IS CAUSED BY HYPERMETHYLATION OF ITS UPSTREAM REGION AND CAN BE REVERSED BY HMA TREATMENT. BY EMPLOYING BOTH LENTIVIRAL AND CRISPR/CAS9-BASED MIR-125A MODIFICATION, WE DEMONSTRATE THAT HMA-INDUCED MIR-125A UPREGULATION INDEED CONTRIBUTES TO MEDIATING THE ANTI-LEUKEMIC EFFECTS OF THESE DRUGS. THESE DATA WERE VALIDATED IN A CLINICAL CONTEXT, AS MIR-125A EXPRESSION INCREASED AFTER HMA TREATMENT IN CMML PATIENTS, A PHENOMENON THAT WAS PARTICULARLY PRONOUNCED IN CASES SHOWING CLINICAL RESPONSE TO THESE DRUGS. CONCLUSIONS: TAKEN TOGETHER, WE REPORT DECREASED EXPRESSION OF MIR-125A IN CMML AND DELINEATE ITS RELEVANCE AS MEDIATOR OF HMA EFFICACY WITHIN THIS NEOPLASIA. 2021 6 5983 32 TET2 RESTRAINS INFLAMMATORY GENE EXPRESSION IN MACROPHAGES. TET METHYLCYTOSINE DIOXYGENASE 2 (TET2) IS ONE OF THE EARLIEST AND MOST FREQUENTLY MUTATED GENES IN CLONAL HEMATOPOIESIS OF INDETERMINATE POTENTIAL (CHIP) AND MYELOID CANCERS, INCLUDING MYELODYSPLASTIC SYNDROMES (MDS) AND CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML). TET2 CATALYZES THE OXIDATION OF 5-METHYLCYTOSINE TO 5-HYDROXYMETHYLCYTOSINE, LEADING TO DNA DEMETHYLATION, AND ALSO AFFECTS TRANSCRIPTION BY RECRUITING HISTONE MODIFIERS. INACTIVATING TET2 MUTATIONS CAUSE EPIGENETIC DYSREGULATION, CLONAL HEMATOPOIETIC STEM CELL (HSC) DOMINANCE, AND MONOCYTIC LINEAGE SKEWING. HERE, WE FOUND THAT TET2 WAS THE MOST HIGHLY EXPRESSED TET ENZYME IN MURINE MACROPHAGE (MPHI) DIFFERENTIATION. TET2 TRANSCRIPTION WAS FURTHER INDUCED BY LIPOPOLYSACCHARIDE (LPS), BUT NOT INTERLEUKIN (IL)-4, STIMULATION, POTENTIALLY IN A NUCLEAR FACTOR KAPPABETA-DEPENDENT MANNER. TET2 LOSS DID NOT AFFECT EARLY LPS GENE RESPONSES IN VITRO, BUT INCREASED IL-1B, IL-6, AND ARGINASE 1 (ARG1) MRNA EXPRESSION AT LATER STAGES OF STIMULATION IN BONE-MARROW-DERIVED MPHIS (BMMPHIS). TET2-DEFICIENT PERITONEAL MPHIS, HOWEVER, DEMONSTRATED PROFOUND, CONSTITUTIVE EXPRESSION OF LPS-INDUCED GENES ASSOCIATED WITH AN INFLAMMATORY STATE IN VIVO. IN CONTRAST, TET2 DEFICIENCY DID NOT AFFECT ALTERNATIVE MPHI GENE EXPRESSION SIGNIFICANTLY IN RESPONSE TO IL-4. THESE RESULTS SUGGESTED IMPAIRED RESOLUTION OF INFLAMMATION IN THE ABSENCE OF TET2 BOTH IN VITRO AND IN VIVO. FOR THE FIRST TIME, WE ALSO DETECTED TET2 MUTATIONS IN BMMPHIS FROM MDS AND CMML PATIENTS AND ASSAYED THEIR EFFECTS ON LPS RESPONSES, INCLUDING THEIR POTENTIAL INFLUENCE ON HUMAN IL-6 EXPRESSION. OUR RESULTS SHOW THAT TET2 RESTRAINS INFLAMMATION IN MURINE MPHIS AND MICE, RAISING THE POSSIBILITY THAT LOSS OF TET2 FUNCTION IN MPHIS MAY ALTER THE IMMUNE ENVIRONMENT IN THE LARGE ELDERLY POPULATION WITH TET2-MUTANT CHIP AND IN TET2-MUTANT MYELOID CANCER PATIENTS. 2017 7 4547 20 MUTATION ALLELE BURDEN REMAINS UNCHANGED IN CHRONIC MYELOMONOCYTIC LEUKAEMIA RESPONDING TO HYPOMETHYLATING AGENTS. THE CYTIDINE ANALOGUES AZACYTIDINE AND 5-AZA-2'-DEOXYCYTIDINE (DECITABINE) ARE COMMONLY USED TO TREAT MYELODYSPLASTIC SYNDROMES, WITH OR WITHOUT A MYELOPROLIFERATIVE COMPONENT. IT REMAINS UNCLEAR WHETHER THE RESPONSE TO THESE HYPOMETHYLATING AGENTS RESULTS FROM A CYTOTOXIC OR AN EPIGENETIC EFFECT. IN THIS STUDY, WE ADDRESS THIS QUESTION IN CHRONIC MYELOMONOCYTIC LEUKAEMIA. WE DESCRIBE A COMPREHENSIVE ANALYSIS OF THE MUTATIONAL LANDSCAPE OF THESE TUMOURS, COMBINING WHOLE-EXOME AND WHOLE-GENOME SEQUENCING. WE IDENTIFY AN AVERAGE OF 14+/-5 SOMATIC MUTATIONS IN CODING SEQUENCES OF SORTED MONOCYTE DNA AND THE SIGNATURES OF THREE MUTATIONAL PROCESSES. SERIAL SEQUENCING DEMONSTRATES THAT THE RESPONSE TO HYPOMETHYLATING AGENTS IS ASSOCIATED WITH CHANGES IN DNA METHYLATION AND GENE EXPRESSION, WITHOUT ANY DECREASE IN THE MUTATION ALLELE BURDEN, NOR PREVENTION OF NEW GENETIC ALTERATION OCCURENCE. OUR FINDINGS INDICATE THAT CYTOSINE ANALOGUES RESTORE A BALANCED HAEMATOPOIESIS WITHOUT DECREASING THE SIZE OF THE MUTATED CLONE, ARGUING FOR A PREDOMINANTLY EPIGENETIC EFFECT. 2016 8 1593 29 DNA METHYLATION PROFILING REVEALS A PATHOLOGICAL SIGNATURE THAT CONTRIBUTES TO TRANSCRIPTIONAL DEFECTS OF CD34(+) CD15(-) CELLS IN EARLY CHRONIC-PHASE CHRONIC MYELOID LEUKEMIA. DESPITE THE HIGH EFFICIENCY OF TYROSINE KINASE INHIBITORS (TKI), SOME PATIENTS WITH CHRONIC MYELOID LEUKEMIA (CML) WILL DISPLAY RESIDUAL DISEASE THAT CAN BECOME RESISTANT TO TREATMENT, INDICATING INTRACLONAL HETEROGENEITY IN CHRONIC-PHASE CML (CP-CML). TO DETERMINE THE BASIS OF THIS HETEROGENEITY, WE CONDUCTED THE FIRST EXHAUSTIVE CHARACTERIZATION OF THE DNA METHYLATION PATTERN OF SORTED CP-CML CD34(+) CD15(-) (IMMATURE) AND CD34(-) CD15(+) (MATURE) CELLS AT DIAGNOSIS (PRIOR TO ANY TREATMENT) AND COMPARED IT TO THAT OF CD34(+) CD15(-) AND CD34(-) CD15(+) CELLS ISOLATED FROM HEALTHY DONORS (HD). IN BOTH CELL TYPES, WE IDENTIFIED SEVERAL HUNDREDS OF DIFFERENTIALLY METHYLATED REGIONS (DMRS) SHOWING DNA METHYLATION CHANGES BETWEEN CP-CML AND HD SAMPLES, WITH ONLY A SUBSET OF THEM IN COMMON BETWEEN CD34(+) CD15(-) AND CD34(-) CD15(+) CELLS. THIS SUGGESTED DNA METHYLATION VARIABILITY WITHIN THE SAME CML CLONE. WE ALSO IDENTIFIED 70 GENES THAT COULD BE ABERRANTLY REPRESSED UPON HYPERMETHYLATION AND 171 GENES THAT COULD BE ABERRANTLY EXPRESSED UPON HYPOMETHYLATION OF SOME OF THESE DMRS IN CP-CML CELLS, AMONG WHICH 18 AND 81, RESPECTIVELY, WERE IN CP-CML CD34(+) CD15(-) CELLS ONLY. WE THEN VALIDATED THE DNA METHYLATION AND EXPRESSION DEFECTS OF SELECTED CANDIDATE GENES. SPECIFICALLY, WE IDENTIFIED GAS2, A CANDIDATE ONCOGENE, AS A NEW EXAMPLE OF GENE THE HYPOMETHYLATION OF WHICH IS ASSOCIATED WITH ROBUST OVEREXPRESSION IN CP-CML CELLS. ALTOGETHER, WE DEMONSTRATED THAT DNA METHYLATION ABNORMALITIES EXIST AT EARLY STAGES OF CML AND CAN AFFECT THE TRANSCRIPTIONAL LANDSCAPE OF MALIGNANT CELLS. THESE OBSERVATIONS COULD LEAD TO THE DEVELOPMENT OF COMBINATION TREATMENTS WITH EPIGENETIC DRUGS AND TKI FOR CP-CML. 2018 9 2911 36 GENE EXPRESSION PROFILING OF LOSS OF TET2 AND/OR JAK2V617F MUTANT HEMATOPOIETIC STEM CELLS FROM MOUSE MODELS OF MYELOPROLIFERATIVE NEOPLASMS. MYELOPROLIFERATIVE NEOPLASMS (MPNS) ARE CLINICALLY CHARACTERIZED BY THE CHRONIC OVERPRODUCTION OF DIFFERENTIATED PERIPHERAL BLOOD CELLS AND THE GRADUAL EXPANSION OF MALIGNANT INTRAMEDULLARY/EXTRAMEDULLARY HEMATOPOIESIS. IN MPNS MUTATIONS IN JAK2 MPL OR CALR ARE DETECTED MUTUALLY EXCLUSIVE IN MORE THAN 90% OF CASES [1,2]. MUTATIONS IN THEM LEAD TO THE ABNORMAL ACTIVATION OF JAK/STAT SIGNALING AND THE AUTONOMOUS GROWTH OF DIFFERENTIATED CELLS THEREFORE THEY ARE CONSIDERED AS "DRIVER" GENE MUTATIONS. IN ADDITION TO THE ABOVE DRIVER GENE MUTATIONS MUTATIONS IN EPIGENETIC REGULATORS SUCH AS TET2 DNMT3A ASXL1 EZH2 OR IDH1/2 ARE DETECTED IN ABOUT 5%-30% OF CASES RESPECTIVELY [3]. MUTATIONS IN TET2 DNMT3A EZH2 OR IDH1/2 COMMONLY CONFER THE INCREASED SELF-RENEWAL CAPACITY ON NORMAL HEMATOPOIETIC STEM CELLS (HSCS) BUT THEY DO NOT LEAD TO THE AUTONOMOUS GROWTH OF DIFFERENTIATED CELLS AND ONLY EXHIBIT SUBTLE CLINICAL PHENOTYPES [4,6-8,5]. IT WAS UNCLEAR HOW MUTATIONS IN SUCH EPIGENETIC REGULATORS INFLUENCED ABNORMAL HSCS WITH DRIVER GENE MUTATIONS HOW THEY INFLUENCED THE DISEASE PHENOTYPE OR WHETHER A SINGLE DRIVER GENE MUTATION WAS SUFFICIENT FOR THE INITIATION OF HUMAN MPNS. THEREFORE WE FOCUSED ON JAK2V617F AND LOSS OF TET2-THE FORMER AS A REPRESENTATIVE OF DRIVER GENE MUTATIONS AND THE LATTER AS A REPRESENTATIVE OF MUTATIONS IN EPIGENETIC REGULATORS-AND EXAMINED THE INFLUENCE OF SINGLE OR DOUBLE MUTATIONS ON HSCS (LINEAGE(-)SCA-1(+)C-KIT(+) CELLS (LSKS)) BY FUNCTIONAL ANALYSES AND MICROARRAY WHOLE-GENOME EXPRESSION ANALYSES [9]. GENE EXPRESSION PROFILING SHOWED THAT THE HSC FINGERPRINT GENES [10] WAS STATISTICALLY EQUALLY ENRICHED IN TET2-KNOCKDOWN-LSKS BUT NEGATIVELY ENRICHED IN JAK2V617F-LSKS COMPARED TO THAT IN WILD-TYPE-LSKS. DOUBLE-MUTANT-LSKS SHOWED THE SAME TENDENCY AS JAK2V617F-LSKS IN TERMS OF THEIR HSC FINGERPRINT GENES BUT THE EXPRESSION OF INDIVIDUAL GENES DIFFERED BETWEEN THE TWO GROUPS. AMONG 245 HSC FINGERPRINT GENES 100 WERE MORE HIGHLY EXPRESSED IN DOUBLE-MUTANT-LSKS THAN IN JAK2V617F-LSKS. THESE ALTERED GENE EXPRESSIONS MIGHT PARTLY EXPLAIN THE MECHANISMS OF INITIATION AND PROGRESSION OF MPNS WHICH WAS OBSERVED IN THE FUNCTIONAL ANALYSES [9]. HERE WE DESCRIBE GENE EXPRESSION PROFILES DEPOSITED AT THE GENE EXPRESSION OMNIBUS (GEO) UNDER THE ACCESSION NUMBER GSE62302 INCLUDING EXPERIMENTAL METHODS AND QUALITY CONTROL ANALYSES. 2015 10 6249 22 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 11 535 27 ASXL1 MUTATION CORRECTION BY CRISPR/CAS9 RESTORES GENE FUNCTION IN LEUKEMIA CELLS AND INCREASES SURVIVAL IN MOUSE XENOGRAFTS. RECURRENT SOMATIC MUTATIONS OF THE EPIGENETIC MODIFIER AND TUMOR SUPPRESSOR ASXL1 ARE COMMON IN MYELOID MALIGNANCIES, INCLUDING CHRONIC MYELOID LEUKEMIA (CML), AND ARE ASSOCIATED WITH POOR CLINICAL OUTCOME. CRISPR/CAS9 HAS RECENTLY EMERGED AS A POWERFUL AND VERSATILE GENOME EDITING TOOL FOR GENOME ENGINEERING IN VARIOUS SPECIES. WE HAVE USED THE CRISPR/CAS9 SYSTEM TO CORRECT THE ASXL1 HOMOZYGOUS NONSENSE MUTATION PRESENT IN THE CML CELL LINE KBM5, WHICH LACKS ASXL1 PROTEIN EXPRESSION. CRISPR/CAS9-MEDIATED ASXL1 HOMOZYGOUS CORRECTION RESULTED IN PROTEIN RE-EXPRESSION WITH RESTORED NORMAL FUNCTION, INCLUDING DOWN-REGULATION OF POLYCOMB REPRESSIVE COMPLEX 2 TARGET GENES. SIGNIFICANTLY REDUCED CELL GROWTH AND INCREASED MYELOID DIFFERENTIATION WERE OBSERVED IN ASXL1 MUTATION-CORRECTED CELLS, PROVIDING NEW INSIGHTS INTO THE ROLE OF ASXL1 IN HUMAN MYELOID CELL DIFFERENTIATION. MICE XENOGRAFTED WITH MUTATION-CORRECTED KBM5 CELLS SHOWED SIGNIFICANTLY LONGER SURVIVAL THAN UNCORRECTED XENOGRAFTS. THESE RESULTS SHOW THAT THE SOLE CORRECTION OF A DRIVER MUTATION IN LEUKEMIA CELLS INCREASES SURVIVAL IN VIVO IN MICE. THIS STUDY PROVIDES PROOF-OF-CONCEPT FOR DRIVER GENE MUTATION CORRECTION VIA CRISPR/CAS9 TECHNOLOGY IN HUMAN LEUKEMIA CELLS AND PRESENTS A STRATEGY TO ILLUMINATE THE IMPACT OF ONCOGENIC MUTATIONS ON CELLULAR FUNCTION AND SURVIVAL. 2015 12 956 35 CHRONIC MYELOMONOCYTIC LEUKAEMIA STEM CELL TRANSCRIPTOMES ANTICIPATE DISEASE MORPHOLOGY AND OUTCOME. BACKGROUND: CHRONIC MYELOMONOCYTIC LEUKAEMIA (CMML) IS A CLINICALLY HETEROGENEOUS STEM CELL MALIGNANCY WITH OVERLAPPING FEATURES OF MYELODYSPLASIA AND MYELOPROLIFERATION. OVER 90% OF PATIENTS CARRY MUTATIONS IN EPIGENETIC AND/OR SPLICING GENES, TYPICALLY DETECTABLE IN THE LIN(-)CD34(+)CD38(-) IMMUNOPHENOTYPIC STEM CELL COMPARTMENT IN WHICH THE LEUKAEMIA-INITIATING CELLS RESIDE. TRANSCRIPTIONAL DYSREGULATION AT THE STEM CELL LEVEL IS LIKELY FUNDAMENTAL TO DISEASE ONSET AND PROGRESSION. METHODS: WE PERFORMED SINGLE-CELL RNA SEQUENCING ON 6826 LIN(-)CD34(+)CD38(-)STEM CELLS FROM CMML PATIENTS AND HEALTHY CONTROLS USING THE DROPLET-BASED, ULTRA-HIGH-THROUGHPUT 10X PLATFORM. FINDINGS: WE FOUND SUBSTANTIAL INTER- AND INTRA-PATIENT HETEROGENEITY, WITH CMML STEM CELLS DISPLAYING DISTINCTIVE TRANSCRIPTIONAL PROGRAMS. COMPARED WITH NORMAL CONTROLS, CMML STEM CELLS EXHIBITED TRANSCRIPTOMES CHARACTERIZED BY INCREASED EXPRESSION OF MYELOID-LINEAGE AND CELL CYCLE GENES, AND LOWER EXPRESSION OF GENES SELECTIVELY EXPRESSED BY NORMAL HAEMATOPOIETIC STEM CELLS. NEUTROPHIL-PRIMED PROGENITOR GENES AND A MYC TRANSCRIPTION FACTOR REGULOME WERE PROMINENT IN STEM CELLS FROM CMML-1 PATIENTS, WHEREAS CMML-2 STEM CELLS EXHIBITED STRONG EXPRESSION OF INTERFERON-REGULATORY FACTOR REGULOMES, INCLUDING THOSE ASSOCIATED WITH IRF1, IRF7 AND IRF8. CMML-1 AND CMML-2 STEM CELLS (STAGES DISTINGUISHED BY PROPORTION OF DOWNSTREAM BLASTS AND PROMONOCYTES) DIFFERED SUBSTANTIALLY IN BOTH TRANSCRIPTOME AND PSEUDOTIME, INDICATING FUNDAMENTALLY DIFFERENT BIOLOGY UNDERPINNING THESE DISEASE STATES. GENE EXPRESSION AND PATHWAY ANALYSES HIGHLIGHTED POTENTIALLY TRACTABLE THERAPEUTIC VULNERABILITIES FOR DOWNSTREAM INVESTIGATION. IMPORTANTLY, CMML PATIENTS HARBOURED VARIABLY-SIZED SUBPOPULATIONS OF TRANSCRIPTIONALLY NORMAL STEM CELLS, INDICATING A POTENTIAL RESERVOIR TO RESTORE FUNCTIONAL HAEMATOPOIESIS. INTERPRETATION: OUR FINDINGS PROVIDE NOVEL INSIGHTS INTO THE CMML STEM CELL COMPARTMENT, REVEALING AN UNEXPECTED DEGREE OF HETEROGENEITY AND DEMONSTRATING THAT CMML STEM CELL TRANSCRIPTOMES ANTICIPATE DISEASE MORPHOLOGY, AND THEREFORE OUTCOME. FUNDING: PROJECT FUNDING WAS SUPPORTED BY OGLESBY CHARITABLE TRUST, CANCER RESEARCH UK, BLOOD CANCER UK, AND UK MEDICAL RESEARCH COUNCIL. 2020 13 151 23 ABERRANT METHYLATION AND IMPAIRED EXPRESSION OF THE P15(INK4B) CELL CYCLE REGULATORY GENE IN CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML). THE IMPORTANT CELL CYCLE REGULATORY GENE P15(INK4B) HAS BEEN SHOWN TO BE INACTIVATED IN ACUTE MYELOID LEUKEMIA AND MYELODYSPLASTIC SYNDROME. LITTLE IS KNOWN ABOUT THE EXPRESSION AND EPIGENETIC MODIFICATION OF THIS GENE IN CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML) THAT BELONGS TO THE MYELODYSPLASTIC/MYELOPROLIFERATIVE DISORDERS (MDS/MPD) WITH A HIGH PROPORTION OF BLASTIC TRANSFORMATION. ANALYSIS OF BONE MARROW TREPHINES IN A SERIES OF 33 CMML CASES SHOWED AN ABERRANT P15(INK4B) GENE METHYLATION IN UP TO 58% OF CASES. METHYLATION WAS ANALYZED EMPLOYING DIFFERENT METHYLATION-SPECIFIC PCR AND GENOMIC SEQUENCING PROTOCOLS. IT TURNED OUT TO BE SPREAD OVER A BROAD AREA OF THE 5' REGION AND EXHIBITED SUBSTANTIAL HETEROGENEITY BETWEEN CASES AND EVEN IN INDIVIDUAL PATIENTS. THE DEGREE OF ABERRANT METHYLATION WAS CORRELATED WITH A REDUCED MRNA AS WELL AS REDUCED PROTEIN EXPRESSION, AND WAS ASSOCIATED WITH A HIGHER EXPRESSION OF DNA METHYLTRANSFERASE DNMT 3A. WE CONCLUDE THAT ABERRANT GENE METHYLATION IS A FREQUENT EVENT IN CMML THAT MIGHT CONTRIBUTE TO THE PATHOGENESIS OF THIS MDS/MPD. 2003 14 5691 28 SILENCING OF HDAC6 AS A THERAPEUTIC TARGET IN CHRONIC LYMPHOCYTIC LEUKEMIA. ALTHOUGH THE TREATMENT PARADIGM FOR CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) IS RAPIDLY CHANGING, THE DISEASE REMAINS INCURABLE, EXCEPT WITH ALLOGENEIC BONE MARROW TRANSPLANTATION, AND RESISTANCE, RELAPSED DISEASE, AND PARTIAL RESPONSES PERSIST AS SIGNIFICANT CHALLENGES. RECENT STUDIES HAVE UNCOVERED ROLES FOR EPIGENETIC MODIFICATION IN THE REGULATION OF MECHANISMS CONTRIBUTING TO MALIGNANT PROGRESSION OF CLL B CELLS. HOWEVER, THE EXTENT TO WHICH EPIGENETIC MODIFIERS CAN BE TARGETED FOR THERAPEUTIC BENEFIT IN CLL PATIENTS REMAINS POORLY EXPLORED. WE REPORT FOR THE FIRST TIME THAT EXPRESSION OF EPIGENETIC MODIFIER HISTONE DEACETYLASE 6 (HDAC6) IS UPREGULATED IN CLL PATIENT SAMPLES, CELL LINES, AND EUTCL1 TRANSGENIC MOUSE MODELS COMPARED WITH HDAC6 IN NORMAL CONTROLS. GENETIC SILENCING OF HDAC6 CONFERRED SURVIVAL BENEFIT IN EUTCL1 MICE. ADMINISTRATION OF ISOFORM-SPECIFIC HDAC6 INHIBITOR ACY738 IN THE EUTCL1 AGING AND ADOPTIVE TRANSFER MODELS DETERRED PROLIFERATION OF CLL B CELLS, DELAYED DISEASE ONSET VIA DISRUPTION OF B-CELL RECEPTOR SIGNALING, AND SENSITIZED CLL B CELLS TO APOPTOSIS. FURTHERMORE, COADMINISTRATION OF ACY738 AND IBRUTINIB DISPLAYED SYNERGISTIC CELL KILL AGAINST CLL CELL LINES AND IMPROVED OVERALL SURVIVAL COMPARED WITH EITHER SINGLE AGENT IN VIVO. THESE RESULTS DEMONSTRATE FOR THE FIRST TIME THE THERAPEUTIC EFFICACY OF SELECTIVE HDAC6 INHIBITION IN PRECLINICAL CLL MODELS AND SUGGEST A RATIONALE FOR THE CLINICAL DEVELOPMENT OF HDAC6 INHIBITORS FOR CLL TREATMENT, EITHER ALONE OR IN COMBINATION WITH BRUTON TYROSINE KINASE INHIBITION. 2018 15 3532 21 IMATINIB INDEPENDENT ABERRANT METHYLATION OF NOV/CCN3 IN CHRONIC MYELOGENOUS LEUKEMIA PATIENTS: A MECHANISM UPSTREAM OF BCR-ABL1 FUNCTION? BACKGROUND: THE NOV GENE PRODUCT, CCN3, HAS BEEN REPORTED IN A DIVERSE RANGE OF TUMORS TO SERVE AS A NEGATIVE GROWTH REGULATOR, WHILE ACTING AS A TUMOR SUPPRESSOR IN CHRONIC MYELOGENOUS LEUKEMIA (CML). HOWEVER, THE PRECISE MECHANISM OF ITS SILENCING IN CML IS POORLY UNDERSTOOD. IN THE CURRENT STUDY, WE AIMED TO QUERY IF THE GENE REGULATION OF CCN3 IS MEDIATED BY THE PROMOTER METHYLATION IN THE PATIENTS WITH CML. IN ADDITION, TO CLARIFY WHETHER THE EPIGENETIC SILENCING IS AFFECTED BY BCR-ABL1 INHIBITION, WE ASSESSED THE METHYLATION STATUS IN THE PATIENTS AT DIFFERENT TIME INTERVALS FOLLOWING THE TYROSINE KINASE INHIBITION USING IMATINIB THERAPY, AS THE FIRST-LINE TREATMENT FOR THIS TYPE OF LEUKEMIA. METHODS: TO ADDRESS THIS ISSUE, WE APPLIED BISULFITE-SEQUENCING TECHNIQUE AS A HIGH-RESOLUTION METHOD TO STUDY THE REGULATORY SEGMENT OF THE CCN3 GENE. THE RESULTS WERE ANALYZED IN NEWLY DIAGNOSED CML PATIENTS AS WELL AS FOLLOWING IMATINIB THERAPY. WE ALSO EVALUATED THE CORRELATION OF CCN3 PROMOTER METHYLATION WITH BCR-ABL1 LEVELS. RESULTS: OUR FINDINGS REVEALED THAT THE METHYLATION OCCURS FREQUENTLY IN THE PROMOTER REGION OF CML PATIENTS SHOWING A SIGNIFICANT INCREASE OF THE METHYLATED PERCENTAGE AT THE CPG SITES COMPARED TO NORMAL INDIVIDUALS. INTERESTINGLY, THIS HYPERMETHYLATION WAS INDICATED TO BE INDEPENDENT OF BCR-ABL1 TITERS IN BOTH GROUPS, WHICH MIGHT SUGGEST A MECHANISM BEYOND THE BCR-ABL1 FUNCTION. CONCLUSION: DESPITE SUGGESTING THAT THE CCN3 HYPERMETHYLATION ACTS AS A MOLECULAR MECHANISM INDEPENDENT OF BCR-ABL1 FUNCTION IN CML PATIENTS, THIS SCENARIO REQUIRES FURTHER VALIDATION BY COMPLEMENTARY EXPERIMENTS. IN THE CASE OF ACTING UPSTREAM OF BCR-ABL1 SIGNALING, THE METHYLATION MARKER CAN PROVIDE EARLY DETECTION AND A NOVEL PLATFORM FOR TARGETED EPIGENETIC MODIFIERS FOR EFFICIENT TREATMENT IN IMATINIB RESISTANT PATIENTS. 2019 16 4388 31 MLL2/KMT2D AND MLL3/KMT2C EXPRESSION CORRELATES WITH DISEASE PROGRESSION AND RESPONSE TO IMATINIB MESYLATE IN CHRONIC MYELOID LEUKEMIA. BACKGROUND: CHRONIC MYELOID LEUKEMIA (CML) IS A CLONAL MYELOPROLIFERATIVE NEOPLASM WHOSE PATHOGENESIS IS LINKED TO THE PHILADELPHIA CHROMOSOME PRESENCE THAT GENERATES THE BCR-ABL1 FUSION ONCOGENE. TYROSINE KINASE INHIBITORS (TKI) SUCH AS IMATINIB MESYLATE (IM) DRAMATICALLY IMPROVED THE TREATMENT EFFICIENCY AND SURVIVAL OF CML PATIENTS BY TARGETING BCR-ABL TYROSINE KINASE. THE DISEASE SHOWS THREE DISTINCT CLINICAL-LABORATORY STAGES: CHRONIC PHASE, ACCELERATED PHASE AND BLAST CRISIS. ALTHOUGH PATIENTS IN THE CHRONIC PHASE RESPOND WELL TO TREATMENT, PATIENTS IN THE ACCELERATED PHASE OR BLAST CRISIS USUALLY SHOW THERAPY RESISTANCE AND CML RELAPSE. IT IS CRUCIAL, THEREFORE, TO IDENTIFY BIOMARKERS TO PREDICT CML GENETIC EVOLUTION AND RESISTANCE TO TKI THERAPY, CONSIDERING NOT ONLY THE EFFECTS OF GENETIC ABERRATIONS BUT ALSO THE ROLE OF EPIGENETIC ALTERATIONS DURING THE DISEASE. ALTHOUGH DYSREGULATIONS IN EPIGENETIC MODULATORS SUCH AS HISTONE METHYLTRASNFERASES HAVE ALREADY BEEN DESCRIBED FOR SOME HEMATOLOGIC MALIGNANCIES, TO DATE VERY LIMITED DATA IS AVAILABLE FOR CML, ESPECIALLY WHEN CONSIDERING THE LYSINE METHYLTRANSFERASE MLL2/KMT2D AND MLL3/KMT2C. METHODS: HERE WE INVESTIGATED THE EXPRESSION PROFILE OF BOTH GENES IN CML PATIENTS IN DIFFERENT STAGES OF THE DISEASE, IN PATIENTS SHOWING DIFFERENT RESPONSES TO THERAPY WITH IM AND IN NON-NEOPLASTIC CONTROL SAMPLES. IMATINIB SENSITIVE AND RESISTANT CML CELL LINES WERE ALSO USED TO INVESTIGATE WHETHER TREATMENT WITH OTHER TYROSINE KINASE INHIBITORS INTERFERED IN THEIR EXPRESSION. RESULTS: IN PATIENTS, BOTH METHYLTRANSFERASES WERE EITHER UPREGULATED OR WITH BASAL EXPRESSION LEVEL DURING THE CHRONIC PHASE COMPARED TO CONTROLS. INTERESTINGLY, MLL3/KMT2C AND SPECIALLY MLL2/KMT2D LEVELS DECREASED DURING DISEASE PROGRESSION CORRELATING WITH DISTINCT CLINICAL STAGES. FURTHERMORE, MLL2/KMT2D WAS DECREASED IN PATIENTS RESISTANT TO IM TREATMENT. A RESCUE IN THE EXPRESSION OF BOTH MLL GENES WAS OBSERVED IN KCL22S, A CML CELL LINE SENSITIVE TO IM, AFTER TREATMENT WITH DASATINIB OR NILOTINIB WHICH WAS ASSOCIATED WITH A HIGHER RATE OF APOPTOSIS, AN ENHANCED EXPRESSION OF P21 (CDKN1A) AND A CONCOMITANT DECREASE IN THE EXPRESSION OF CDK2, CDK4 AND CYCLIN B1 (CCNB1) IN COMPARISON TO UNTREATED KCL22S CONTROL OR IM RESISTANT KCL22R CELL LINE, WHICH SUGGESTS INVOLVEMENT OF P53 REGULATED PATHWAY. CONCLUSION: OUR RESULTS ESTABLISHED A NEW ASSOCIATION BETWEEN MLL2/KMT2D AND MLL3/KMT2C GENES WITH CML AND SUGGEST THAT MLL2/KMT2D IS ASSOCIATED WITH DISEASE EVOLUTION AND MAY BE A POTENTIAL MARKER TO PREDICT THE DEVELOPMENT OF THERAPY RESISTANCE. 2018 17 4571 24 MYELOMONOCYTIC SKEWING IN CHRONIC MYELOMONOCYTIC LEUKEMIA: PHENOTYPIC, MOLECULAR AND BIOLOGIC FEATURES AND IMPACT ON SURVIVAL. BACKGROUND: MYELOMONOCYTIC SKEWING IS CONSIDERED AS A KEY PATHOPHYSIOLOGIC PHENOMENON IN CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML), BUT ITS PREVALENCE AND POTENTIAL CORRELATION WITH PHENOTYPIC, GENOTYPIC, AND CLINICAL FEATURES ARE POORLY DEFINED. METHODS: SKEWED DIFFERENTIATION TOWARD THE MYELOMONOCYTIC OVER ERYTHROID COMMITMENT AS INDICATED BY AN INVERSE RATIO OF MYELOMONOCYTIC/ERYTHROID COLONIES WAS INVESTIGATED IN 146 PATIENTS WITH CMML BY SEMISOLID IN VITRO CULTURES. RESULTS: THERE WAS A HIGH PREVALENCE OF MYELOMONOCYTIC SKEWING IN PATIENTS WITH CMML (120/146, 82%); WHEREAS, THIS PHENOMENON WAS RARE IN NORMAL INDIVIDUALS (1/98, 1%). PATIENTS WITH CMML WITH MYELOMONOCYTIC SKEWING HAD HIGHER WHITE BLOOD CELL AND PERIPHERAL BLAST CELL COUNTS, AND LOWER PLATELET VALUES. THE NUMBER OF MUTATIONS IN GENES OF THE EPIGENETIC AND/OR SPLICING CATEGORY WAS HIGHER IN CMML PATIENTS WITH AS COMPARED WITH PATIENTS WITHOUT SKEWING. PATIENTS WITH MYELOMONOCYTIC SKEWING HAD MORE FREQUENTLY MUTATIONS IN RASOPATHY GENES AND HIGHER GROWTH FACTOR INDEPENDENT MYELOID COLONY FORMATION. INTERESTINGLY, THE LACK OF MYELOMONOCYTIC SKEWING DISCRIMINATED PATIENTS WITH CMML WITH A PARTICULARLY FAVORABLE PROGNOSIS (60 VS 19 MONTHS, P = .003) AND A MINIMAL RISK OF TRANSFORMATION. CONCLUSION: MYELOMONOCYTIC SKEWING AS DETERMINED BY SEMISOLID CULTURES CAN DISCRIMINATE SUBGROUPS OF PATIENTS WITH CMML WITH A DIFFERENT PHENOTYPE, A DIFFERENT GENOTYPE, AND A DIFFERENT PROGNOSIS. 2021 18 1043 29 CLINICAL CHARACTERISTICS AND WHOLE EXOME/TRANSCRIPTOME SEQUENCING OF COEXISTING CHRONIC MYELOID LEUKEMIA AND MYELOFIBROSIS. MYELOPROLIFERATIVE NEOPLASMS (MPNS) ARE CLONAL HEMATOPOIETIC STEM CELL (HSC) DISORDERS THAT CAN BE CLASSIFIED ON THE BASIS OF GENETIC, CLINICAL, PHENOTYPIC FEATURES. GENETIC LESIONS SUCH AS JAK2 MUTATIONS AND BCR-ABL TRANSLOCATION ARE OFTEN MUTUALLY EXCLUSIVE IN MPN PATIENTS AND LEAD TO ESSENTIAL THROMBOCYTHEMIA, POLYCYTHEMIA VERA, OR MYELOFIBROSIS OR CHRONIC MYELOID LEUKEMIA, RESPECTIVELY. NEVERTHELESS, COEXISTENCE OF THESE GENETIC ABERRATIONS IN THE SAME PATIENT HAS BEEN REPORTED. WHETHER THESE ABERRATIONS OCCUR IN THE SAME STEM CELL OR A DIFFERENT CELL IS UNCLEAR, BUT AN UNSTABLE GENOME IN THE HSCS SEEMS TO BE THE COMMON ANTECEDENT. IN AN EFFORT TO CHARACTERIZE THE UNDERLYING GENETIC EVENTS THAT MIGHT CONTRIBUTE TO THE APPEARANCE OF MORE THAN ONE MPN IN A PATIENT, WE STUDIED NEOPLASTIC CELLS FROM PATIENTS WITH DUAL MPNS BY NEXT-GENERATION SEQUENCING. WE OBSERVED THAT MOST PATIENTS WITH TWO MPNS HARBORED MUTATIONS IN GENES KNOWN TO CONTRIBUTE TO CLONAL HEMATOPOIESIS THROUGH ALTERED EPIGENETIC REGULATION SUCH AS TET2, ASXL1/2, SRSF2, AND IDH2 AT VARYING FREQUENCIES (1%-47%). IN ADDITION, WE FOUND THAT SOME PATIENTS ALSO HARBORED ONCOGENIC MUTATIONS IN N/KRAS, TP53, BRAF, EZH2, AND GNAS AT LOW FREQUENCIES, WHICH PROBABLY REPRESENT CLONAL EVOLUTION. THESE FINDINGS SUPPORT THE HYPOTHESIS THAT HEMATOPOIETIC CELLS FROM MPN PATIENTS HARBOR MULTIPLE GENETIC ABERRATIONS, SOME OF WHICH CAN CONTRIBUTE TO CLONAL DOMINANCE. ACQUIRING MUTATIONS IN JAK2/CALR/MPL OR THE BCR-ABL TRANSLOCATION PROBABLY DRIVE THE ONCOGENIC PHENOTYPE TOWARDS A SPECIFIC MPN. FURTHER, WE PROPOSE THAT THE ACQUISITION OF BCR-ABL IN THESE PATIENTS IS FREQUENTLY A SECONDARY EVENT RESULTING FROM AN UNSTABLE GENOME. 2017 19 2237 33 EPIGENETIC MODIFIERS IN MYELOID MALIGNANCIES: THE ROLE OF HISTONE DEACETYLASE INHIBITORS. MYELOID HEMATOLOGICAL MALIGNANCIES ARE CLONAL BONE MARROW NEOPLASMS, COMPRISING OF ACUTE MYELOID LEUKEMIA (AML), THE MYELODYSPLASTIC SYNDROMES (MDS), CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML), THE MYELOPROLIFERATIVE NEOPLASMS (MPN) AND SYSTEMIC MASTOCYTOSIS (SM). THE FIELD OF EPIGENETIC REGULATION OF NORMAL AND MALIGNANT HEMATOPOIESIS IS RAPIDLY GROWING. IN RECENT YEARS, HETEROZYGOUS SOMATIC MUTATIONS IN GENES ENCODING EPIGENETIC REGULATORS HAVE BEEN FOUND IN ALL SUBTYPES OF MYELOID MALIGNANCIES, SUPPORTING THE RATIONALE FOR TREATMENT WITH EPIGENETIC MODIFIERS. HISTONE DEACETYLASE INHIBITORS (HDACI) ARE EPIGENETIC MODIFIERS THAT, IN VITRO, HAVE BEEN SHOWN TO INDUCE GROWTH ARREST, APOPTOTIC OR AUTOPHAGIC CELL DEATH, AND TERMINAL DIFFERENTIATION OF MYELOID TUMOR CELLS. THESE EFFECTS WERE OBSERVED BOTH AT THE BULK TUMOR LEVEL AND IN THE MOST IMMATURE CD34(+)38(-) CELL COMPARTMENTS CONTAINING THE LEUKEMIC STEM CELLS. THUS, THERE IS A STRONG RATIONALE SUPPORTING HDACI THERAPY IN MYELOID MALIGNANCIES. HOWEVER, DESPITE INITIAL PROMISING RESULTS IN PHASE I TRIALS, HDACI IN MONOTHERAPY AS WELL AS IN COMBINATION WITH OTHER DRUGS, HAVE FAILED TO IMPROVE RESPONSES OR SURVIVAL. THIS REVIEW PROVIDES AN OVERVIEW OF THE RATIONALE FOR HDACI IN MYELOID MALIGNANCIES, CLINICAL RESULTS AND SPECULATIONS ON WHY CLINICAL TRIALS HAVE THUS FAR NOT MET THE EXPECTATIONS, AND HOW THIS MAY BE IMPROVED IN THE FUTURE. 2018 20 3234 25 HEMATOPOIETIC AND CHRONIC MYELOID LEUKEMIA STEM CELLS: MULTI-STABILITY VERSUS LINEAGE RESTRICTION. THERE IS COMPELLING EVIDENCE TO SUPPORT THE VIEW THAT THE CELL-OF-ORIGIN FOR CHRONIC MYELOID LEUKEMIA IS A HEMATOPOIETIC STEM CELL. UNLIKE NORMAL HEMATOPOIETIC STEM CELLS, THE PROGENY OF THE LEUKEMIA STEM CELLS ARE PREDOMINANTLY NEUTROPHILS DURING THE DISEASE CHRONIC PHASE AND THERE IS A MILD ANEMIA. THE HALLMARK ONCOGENE FOR CHRONIC MYELOID LEUKEMIA IS THE BCR-ABLP210 FUSION GENE. VARIOUS STUDIES HAVE EXCLUDED A ROLE FOR BCR-ABLP210 EXPRESSION IN MAINTAINING THE POPULATION OF LEUKEMIA STEM CELLS. STUDIES OF BCR-ABLP210 EXPRESSION IN EMBRYONAL STEM CELLS THAT WERE DIFFERENTIATED INTO HEMATOPOIETIC STEM CELLS AND OF THE EXPRESSION IN TRANSGENIC MICE HAVE REVEALED THAT BCR-ABLP210 IS ABLE TO VEER HEMATOPOIETIC STEM AND PROGENITOR CELLS TOWARDS A MYELOID FATE. FOR THE TRANSGENIC MICE, GLOBAL CHANGES TO THE EPIGENETIC LANDSCAPE WERE OBSERVED. IN CHRONIC MYELOID LEUKEMIA, THE ABILITY OF THE LEUKEMIA STEM CELLS TO CHOOSE FROM THE MANY FATES THAT ARE AVAILABLE TO NORMAL HEMATOPOIETIC STEM CELLS APPEARS TO BE DEREGULATED BY BCR-ABLP210 AND CHANGES TO THE EPIGENOME ARE ALSO IMPORTANT. EVEN SO, WE STILL DO NOT HAVE A PRECISE PICTURE AS TO WHY NEUTROPHILS ARE ABUNDANTLY PRODUCED IN CHRONIC MYELOID LEUKEMIA. 2022