1 5789 128 SRSF2(P95H/+) CO-OPERATES WITH LOSS OF TET2 TO PROMOTE MYELOID BIAS AND INITIATE A CHRONIC MYELOMONOCYTIC LEUKEMIA-LIKE DISEASE IN MICE. RECURRENT MUTATIONS IN RNA SPLICING PROTEINS AND EPIGENETIC REGULATORS CONTRIBUTE TO THE DEVELOPMENT OF MYELODYSPLASTIC SYNDROME (MDS) AND RELATED MYELOID NEOPLASMS. IN CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML), SRSF2 MUTATIONS OCCUR IN ~50% OF PATIENTS AND TET2 MUTATIONS IN ~60%. CLONAL ANALYSIS INDICATES THAT EITHER MUTATION CAN ARISE AS THE FOUNDER LESION. BASED ON HUMAN CANCER GENETICS WE CROSSED AN INDUCIBLE SRSF2(P95H/+) MUTANT MODEL WITH TET2(FL/FL) MICE TO MUTATE BOTH CONCOMITANTLY IN HEMATOPOIETIC STEM CELLS. AT 20-24 WEEKS POST MUTATION INDUCTION, WE OBSERVED SUBTLE DIFFERENCES IN THE SRSF2/TET2 MUTANTS COMPARED TO EITHER SINGLE MUTANT. UNDER CONDITIONS OF NATIVE HEMATOPOIESIS WITH AGING, WE SEE A DISTINCT MYELOID BIAS AND MONOCYTOSIS IN THE SRSF2/TET2 MUTANTS. A SUBSET OF THE COMPOUND SRSF2/TET2 MUTANTS DISPLAY AN INCREASED GRANULOCYTIC AND DISTINCTIVE MONOCYTIC PROLIFERATION (MYELOMONOCYTIC HYPERPLASIA), WITH INCREASED IMMATURE PROMONOCYTES AND MONOBLASTS AND BINUCLEATE PROMONOCYTES. EXOME ANALYSIS OF PROGRESSED DISEASE DEMONSTRATED MUTATIONS IN GENES AND PATHWAYS SIMILAR TO THOSE REPORTED IN HUMAN CMML. UPON TRANSPLANTATION, RECIPIENTS DEVELOPED LEUKOCYTOSIS, MONOCYTOSIS, AND SPLENOMEGALY. WE REPRODUCE SRSF2/TET2 CO-OPERATIVITY IN VIVO, YIELDING A DISEASE WITH CORE CHARACTERISTICS OF CMML, UNLIKE SINGLE SRSF2 OR TET2 MUTATION. THIS MODEL REPRESENTS A SIGNIFICANT STEP TOWARD BUILDING HIGH FIDELITY AND GENETICALLY TRACTABLE MODELS OF CMML. 2022 2 1070 34 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 3 4549 33 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 4837 27 ONCOGENIC GENE EXPRESSION AND EPIGENETIC REMODELING OF CIS-REGULATORY ELEMENTS IN ASXL1-MUTANT CHRONIC MYELOMONOCYTIC LEUKEMIA. MYELOID NEOPLASMS ARE CLONAL HEMATOPOIETIC STEM CELL DISORDERS DRIVEN BY THE SEQUENTIAL ACQUISITION OF RECURRENT GENETIC LESIONS. TRUNCATING MUTATIONS IN THE CHROMATIN REMODELER ASXL1 (ASXL1(MT)) ARE ASSOCIATED WITH A HIGH-RISK DISEASE PHENOTYPE WITH INCREASED PROLIFERATION, EPIGENETIC THERAPEUTIC RESISTANCE, AND POOR SURVIVAL OUTCOMES. WE PERFORMED A MULTI-OMICS INTERROGATION TO DEFINE GENE EXPRESSION AND CHROMATIN REMODELING ASSOCIATED WITH ASXL1(MT) IN CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML). ASXL1(MT) ARE ASSOCIATED WITH A LOSS OF REPRESSIVE HISTONE METHYLATION AND INCREASE IN PERMISSIVE HISTONE METHYLATION AND ACETYLATION IN PROMOTER REGIONS. ASXL1(MT) ARE FURTHER ASSOCIATED WITH DE NOVO ACCESSIBILITY OF DISTAL ENHANCERS BINDING ETS TRANSCRIPTION FACTORS, TARGETING IMPORTANT LEUKEMOGENIC DRIVER GENES. CHROMATIN REMODELING OF PROMOTERS AND ENHANCERS IS STRONGLY ASSOCIATED WITH GENE EXPRESSION AND HETEROGENOUS AMONG OVEREXPRESSED GENES. THESE RESULTS PROVIDE A COMPREHENSIVE MAP OF THE TRANSCRIPTOME AND CHROMATIN LANDSCAPE OF ASXL1(MT) CMML, FORMING AN IMPORTANT FRAMEWORK FOR THE DEVELOPMENT OF NOVEL THERAPEUTIC STRATEGIES TARGETING ONCOGENIC CIS INTERACTIONS. 2022 5 956 37 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 6 1043 37 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 7 2462 32 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 8 4571 27 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 9 5983 38 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 10 5249 29 PROGRAMMED CELL DEATH-1 PATHWAY INHIBITION IN MYELOID MALIGNANCIES: IMPLICATIONS FOR MYELOPROLIFERATIVE NEOPLASMS. MYELOPROLIFERATIVE NEOPLASMS (MPNS) ARE CLONAL HEMATOPOIETIC DISEASES THAT BELONG TO THE SPECTRUM OF MYELOID MALIGNANCIES (MYMS), WHICH ALSO INCLUDE MYELODYSPLASTIC SYNDROMES (MDS), ACUTE MYELOID LEUKEMIA (AML), AND CHRONIC MYELOGENOUS LEUKEMIA (CML). WHILE HEMATOPOIETIC STEM CELL TRANSPLANTATION (HSCT) IS A POTENTIALLY CURATIVE THERAPEUTIC APPROACH TO MANY MYMS, THE ASSOCIATED MORBIDITY AND MORTALITY HAVE NECESSITATED THE DEVELOPMENT OF NON-HSCT THERAPEUTICS FOR SYMPTOM MANAGEMENT AND DISEASE COURSE MODIFICATION. IMMUNE CHECKPOINT INHIBITION, IN PARTICULAR ALONG THE PROGRAMMED CELL DEATH PROTEIN 1 (PD-1)/B7-H1 (PD-L1) AXIS, IS AN ESTABLISHED STRATEGY IN SOLID TUMORS WITH POTENTIAL AS AN ADJUNCTIVE THERAPY IN HEMATOLOGIC MALIGNANCIES. SEMINAL STUDIES SUGGEST THAT THE PRO-INFLAMMATORY MICROENVIRONMENT OF MYMS CAN SUPPRESS T LYMPHOCYTE-MEDIATED IMMUNITY VIA PD-1 SIGNALING AND THAT RESPONSE TO MAINSTAY EPIGENETIC THERAPIES FOR MYMS MAY BE GOVERNED BY PD-1 GENE REGULATION. ALTHOUGH THE ROLE OF PD-1 SIGNALING IN MPN PATHOGENESIS AND PROGRESSION IS AS YET UNCLEAR, RESEARCH IN MPN PATIENTS HAS REVEALED EXPANSION OF MYELOID-DERIVED SUPPRESSOR CELLS (MDSCS), WHICH MAY EFFECT HOST IMMUNE TOLERANCE OF TUMOR VIA TEMPORALLY AND SPATIALLY SPECIFIC ACTIVATION OF PD-1/PD-L1 SIGNALING. THE CURRENT UNDERSTANDING OF IMMUNE DYSFUNCTION IN MPNS AND ANALOGOUS MYMS OFFERS A COMPELLING RATIONALE TO STUDY PD-1/PD-L1 INHIBITION IN PATIENTS AS A NOVEL TREATMENT OPTION. 2017 11 4265 32 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 12 409 20 ANALYSIS OF GENES ENCODING EPIGENETIC REGULATORS IN MYELOPROLIFERATIVE NEOPLASMS: COEXISTENCE OF A NOVEL SETBP1 MUTATION IN A PATIENT WITH A P.V617F JAK2 POSITIVE MYELOFIBROSIS. IN RECENT YEARS IT HAS BEEN SHOWN THAT THE CAUSES OF CHRONIC MYELOPROLIFERATIVE NEOPLASMS (MPNS) ARE MORE COMPLEX THAN A SIMPLE SIGNALING ABERRATION AND MANY OTHER MUTATED GENES AFFECTING DIFFERENT CELL PROCESSES HAVE BEEN DESCRIBED. FOR INSTANCE, MUTATIONS IN GENES ENCODING EPIGENETIC REGULATORS ARE MORE FREQUENT THAN EXPECTED. ONE OF THE LATEST GENES DESCRIBED AS MUTATED IS SET BINDING PROTEIN 1 (SETBP1). IN SILICO TOOLS HAVE REVEALED THAT THERE ARE SEVERAL HUMAN SETBP1 PARALOGOUS TO NUCLEAR RECEPTOR BINDING SET DOMAIN PROTEIN 1 (NSD1), NSD2 AND NSD3, FOR EXAMPLE, WHICH ARE ALSO INVOLVED IN THE DEVELOPMENT OF OTHER HEMATOLOGICAL MALIGNANCIES. THEREFORE, THE PRESENT STUDY ANALYZED THE MUTATIONAL STATUS OF NSD1, NSD2, NSD3 AND SETBP1 IN BCR-ABL1 NEGATIVE MPNS WITH OR WITHOUT JANUS KINASE 2 (JAK2) P.V617F MUTATION. THE PRESENT STUDY REVEALED THAT THE NSD GENES ARE NOT FREQUENTLY MUTATED IN MPNS. HOWEVER, A NOVEL SETBP1 MUTATION WAS IDENTIFIED IN A PATIENT WITH P.V617F JAK2 POSITIVE PRIMARY MYELOFIBROSIS. THESE RESULTS PROVIDE FURTHER INSIGHT INTO THE GENETIC COMPLEXITY OF MPNS. 2019 13 2911 40 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 14 1629 29 DNMT3A ARG882 MUTATION DRIVES CHRONIC MYELOMONOCYTIC LEUKEMIA THROUGH DISTURBING GENE EXPRESSION/DNA METHYLATION IN HEMATOPOIETIC CELLS. THE GENE ENCODING DNA METHYLTRANSFERASE 3A (DNMT3A) IS MUTATED IN APPROXIMATELY 20% OF ACUTE MYELOID LEUKEMIA CASES, WITH ARG882 (R882) AS THE HOTSPOT. HERE, WE ADDRESSED THE TRANSFORMATION ABILITY OF THE DNMT3A-ARG882HIS (R882H) MUTANT BY USING A RETROVIRAL TRANSDUCTION AND BONE MARROW TRANSPLANTATION (BMT) APPROACH AND FOUND THAT THE MUTANT GENE CAN INDUCE ABERRANT PROLIFERATION OF HEMATOPOIETIC STEM/PROGENITOR CELLS. AT 12 MO POST-BMT, ALL MICE DEVELOPED CHRONIC MYELOMONOCYTIC LEUKEMIA WITH THROMBOCYTOSIS. RNA MICROARRAY ANALYSIS REVEALED ABNORMAL EXPRESSIONS OF SOME HEMATOPOIESIS-RELATED GENES, AND THE DNA METHYLATION ASSAY IDENTIFIED CORRESPONDING CHANGES IN METHYLATION PATTERNS IN GENE BODY REGIONS. MOREOVER, DNMT3A-R882H INCREASED THE CDK1 PROTEIN LEVEL AND ENHANCED CELL-CYCLE ACTIVITY, THEREBY CONTRIBUTING TO LEUKEMOGENESIS. 2014 15 4547 21 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 16 2956 37 GENETIC AND EPIGENETIC FACTORS INTERACTING WITH CLONAL HEMATOPOIESIS RESULTING IN CHRONIC MYELOMONOCYTIC LEUKEMIA. PURPOSE OF REVIEW: SINCE 2016, THE WHO HAS RECOGNIZED THE SIGNIFICANT PHENOTYPIC HETEROGENEITY OF CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML) AS A MYELODYSPLASTIC SYNDROME/MYELOPROLIFERATIVE NEOPLASM (MDS/MPN) OVERLAP DISEASE. ALTHOUGH SHARING MANY SOMATIC MUTATIONS WITH MDS AND MPN, THE PURPOSE OF THIS REVIEW IS TO PUT RECENT BIOLOGICAL FINDINGS OF CMML IN THE CONTEXT OF EVOLUTIONARY THEORY, HIGHLIGHTING IT AS A DISTINCT EVOLUTIONARY TRAJECTORY OCCURRING IN THE CONTEXT OF CLONAL HEMATOPOIESIS. RECENT FINDINGS: CLONAL HEMATOPOIESIS OF INDETERMINATE POTENTIAL (CHIP), WITH A MUTATIONAL SPECTRUM AND PREVALENCE CORRELATED WITH AGE, HAS BEEN DEFINED. ENRICHED IN DNMT3A, TET2, AND ASXL1 MUTATIONS, CLONAL EVOLUTION CAN PROGRESS INTO VARIOUS EVOLUTIONARY TRAJECTORIES INCLUDING CMML. IMPACT OF FOUNDER MUTATIONS (PRIMARILY TET2) ON INCREASED HEMATOPOIETIC STEM CELL FITNESS HAS BEEN WELL CHARACTERIZED. EPISTATIC INTERACTIONS BETWEEN MUTATIONS AND EPIGENETIC EVENTS HAVE BEEN EXPLORED, BOTH IN CMML AND ITS PEDIATRIC COUNTERPART JUVENILE MYELOMONOCYTIC LEUKEMIA, INCLUDING CMML TRANSFORMATION TO ACUTE MYELOID LEUKEMIA. TOGETHER, THESE FINDINGS HAVE CONTRIBUTED SIGNIFICANTLY TOWARD CMML EVOLUTIONARY DYNAMICS. SUMMARY: DESPITE RELATIVELY FEW 'DRIVER' MUTATIONS IN CMML, EVOLUTIONARY DEVELOPMENT OF CHRONIC LEUKEMIA REMAINS INCOMPLETELY UNDERSTOOD. RECENT STUDIES HAVE SHED LIGHT ON THE IMPORTANCE OF STUDYING EPIGENETIC CONSEQUENCES OF MUTATIONS AND EPISTASIS BETWEEN KEY MUTATIONS TO BETTER UNDERSTAND CLONAL ARCHITECTURE AND EVOLUTIONARY DYNAMICS. 2020 17 6885 35 [RNA SPLICING DYSREGULATION IN HEMATOLOGICAL MALIGNANCIES]. RECURRENT MUTATIONS IN GENES ENCODING KEY SPLICING FACTORS, SF3B1, SRSF2, U2AF1, AND ZRSR2 HAVE BEEN FOUND IN A VARIETY OF CANCERS, PARTICULARLY IN HEMATOLOGIC MALIGNANCIES, INCLUDING MYELODYSPLASTIC SYNDROMES, CHRONIC MYELOMONOCYTIC LEUKEMIA, ACUTE MYELOID LEUKEMIA, AND CHRONIC LYMPHOCYTIC LEUKEMIA. GLOBAL MIS-SPLICING OF MRNAS TARGETED BY ABERRANT SPLICING FACTORS PARTLY CONTRIBUTES TO LEUKEMOGENESIS THROUGH DECREASE PROTEIN EXPRESSION OF TUMOR SUPPRESSORS AND EPIGENETIC MODIFIERS, CAUSED BY MRNAS DEGRADATION OF ABERRANTLY SPLICED. SOME OF THE MIS-SPLICED MRNAS INFLUENCE INTRACELLULAR ONCOGENIC PATHWAYS AND CELLULAR PROCESSES THROUGH A DYSREGULATED EXPRESSION OF ASSOCIATED PROTEINS, WHEREAS OTHERS INFLUENCE THE FUNCTION OF CO-MUTATED GENES SUCH AS ABERRANT TRANSCRIPTIONAL REGULATORS. SPLICEOSOMAL DISRUPTION IS COMMON IN MANY CANCERS, MAKING SPLICEOSOME AN APPEALING THERAPEUTIC TARGET. THE FINDINGS THAT SPLICEOSOMAL MUTANT CELLS RELY ON WILD-TYPE SPLICING MACHINERY FOR SURVIVAL AND THAT SPLICING FACTOR MUTATIONS OCCUR IN A MUTUALLY EXCLUSIVE MANNER STRONGLY SUGGEST THAT INHIBITING WILD-TYPE SPLICING MACHINERY CAUSES SYNTHETIC LETHALITY IN CANCER CELLS WITH THESE MUTATIONS. WE DISCUSS THE CHARACTERISTICS AND ONCOGENIC MECHANISMS OF SPLICING FACTOR MUTATIONS, AS WELL AS THE DEVELOPMENT OF NOVEL TREATMENT STRATEGIES TARGETING ABERRANT SPLICING FACTORS IN HEMATOLOGIC MALIGNANCIES. 2023 18 535 26 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 19 2088 28 EPIGENETIC DYSREGULATION OF SECRETED FRIZZLED-RELATED PROTEINS IN MYELOPROLIFERATIVE NEOPLASMS COMPLEMENTS THE JAK2V617F-MUTATION. BACKGROUND: SECRETED FRIZZLED-RELATED PROTEINS (SFRPS) ARE ANTAGONISTS OF THE WNT SIGNALING PATHWAY, WHICH PLAYS A CENTRAL ROLE IN STEM CELL MAINTENANCE AND DIFFERENTIATION OF STEM CELLS AND HEMATOPOIETIC PROGENITORS. EPIGENETIC DOWNREGULATION OF SFRPS BY PROMOTER HYPERMETHYLATION HAS BEEN DESCRIBED TO BE INVOLVED IN THE PATHOGENESIS OF HEMATOPOIETIC MALIGNANCIES. THERE IS AN ASSOCIATION BETWEEN ABERRANT WNT SIGNALING AND THE ESTABLISHED CANCER STEM CELL CONCEPT. IN CONTRAST TO BCR-ABL1-POSITIVE CHRONIC MYELOID LEUKEMIA CML, BCR-ABL1-NEGATIVE MYELOPROLIFERATIVE NEOPLASMS (PH-MPN) ARE CHARACTERIZED BY THE FREQUENT OCCURRENCE OF AN AUTOACTIVATING MUTATION IN THE JAK2 TYROSINE KINASE (JAK2V617F) OR OTHER MUTATIONS IN THE JAK-STAT PATHWAY. HOWEVER, PATHOGENETIC MECHANISMS OF JAK2 MUTATED OR UNMUTATED PH-MPN REMAIN NOT COMPLETELY UNDERSTOOD. WE DETERMINED THE PROMOTER METHYLATION STATUS OF SFRP-1, -2, -4, AND -5 IN 57 MPN PATIENT SAMPLES BY METHYLATION-SPECIFIC POLYMERASE CHAIN REACTION (PCR) (MSP). JAK2V617F WAS ASSESSED BY ALLELE-SPECIFIC PCR. RESULTS: ABERRANT METHYLATION AMONG PRIMARY MPN SAMPLES WAS 4% FOR SFRP-1, 25% FOR SFRP-2, 2% FOR SFRP-4, AND 0% FOR SFRP-5. HYPERMETHYLATION OF SFRP-2, WHICH WAS THE MOST FREQUENTLY HYPERMETHYLATED GENE IN OUR STUDY, COULD NOT BE CORRELATED TO ANY SPECIFIC MPN SUBTYPE. HOWEVER, WE DETECTED A SIGNIFICANT CORRELATION BETWEEN SFRP-2 METHYLATION AND PRESENCE OF A JAK2V617F MUTATION (P = 0.008). NONE OF THE 10 CML SAMPLES SHOWED ANY SFRP-METHYLATION. CONCLUSIONS: OUR DATA INDICATE THAT EPIGENETIC DYSREGULATION OF THE WNT SIGNALING PATHWAY IS A COMMON EVENT IN MPN WITH ABERRANT METHYLATION OF AT LEAST ONE SFRP BEING DETECTED IN 25% OF THE PRIMARY PATIENT SAMPLES AND IN 30% IF ONLY ACCOUNTING FOR PH-MPN. A SIGNIFICANT CORRELATION BETWEEN SFRP-2 METHYLATION AND PRESENCE OF JAK2V617F IN OUR DATA SUPPORTS THE HYPOTHESIS THAT EPIGENETIC DYSREGULATION MAY BE A COMPLEMENTARY MECHANISM TO GENETIC ABERRATIONS. ABERRANT METHYLATION OF CRUCIAL STEM CELL MAINTENANCE GENES SEEMS TO CONTRIBUTE TO DISEASE PATHOGENESIS IN PH-MPN. 2012 20 5982 29 TET2 REGULATES IMMUNE TOLERANCE IN CHRONICALLY ACTIVATED MAST CELLS. MUTATION OF THE TET2 DNA-HYDROXYMETHYLASE HAS BEEN ASSOCIATED WITH A NUMBER OF IMMUNE PATHOLOGIES. THE DISPARITY IN PHENOTYPE AND CLINICAL PRESENTATION AMONG THESE PATHOLOGIES LEADS TO QUESTIONS REGARDING THE ROLE OF TET2 MUTATION IN PROMOTING DISEASE EVOLUTION IN DIFFERENT IMMUNE CELL TYPES. HERE WE SHOW THAT, IN PRIMARY MAST CELLS, TET2 EXPRESSION IS INDUCED IN RESPONSE TO CHRONIC AND ACUTE ACTIVATION SIGNALS. IN TET2-DEFICIENT MAST CELLS, CHRONIC ACTIVATION VIA THE ONCOGENIC KITD816V ALLELE ASSOCIATED WITH MASTOCYTOSIS, SELECTS FOR A SPECIFIC EPIGENETIC SIGNATURE CHARACTERIZED BY HYPERMETHYLATED DNA REGIONS (HMR) AT IMMUNE RESPONSE GENES. H3K27AC AND TRANSCRIPTION FACTOR BINDING IS CONSISTENT WITH PRIMING OR MORE OPEN CHROMATIN AT BOTH HMR AND NON-HMR IN PROXIMITY TO IMMUNE GENES IN THESE CELLS, AND THIS SIGNATURE COINCIDES WITH INCREASED PATHOLOGICAL INFLAMMATION SIGNALS. HMR ARE ALSO ASSOCIATED WITH A SUBSET OF IMMUNE GENES THAT ARE DIRECT TARGETS OF TET2 AND REPRESSED IN TET2-DEFICIENT CELLS. REPRESSION OF THESE GENES RESULTS IN IMMUNE TOLERANCE TO ACUTE STIMULATION THAT CAN BE RESCUED WITH VITAMIN C TREATMENT OR REITERATED WITH A TET INHIBITOR. OVERALL, OUR DATA SUPPORT A MODEL WHERE TET2 PLAYS A DIRECT ROLE IN PREVENTING IMMUNE TOLERANCE IN CHRONICALLY ACTIVATED MAST CELLS, SUPPORTING TET2 AS A VIABLE TARGET TO REPROGRAM THE INNATE IMMUNE RESPONSE FOR INNOVATIVE THERAPIES. 2022