1 1445 122 DIFFUSE PEDIATRIC-TYPE HIGH-GRADE GLIOMA ARISING IN AN OVARIAN MATURE CYSTIC TERATOMA. IMMATURE NEUROECTODERMAL TISSUE CAN BE FOUND IN THE OVARY AS PART OF AN IMMATURE TERATOMA OR AS PART OF A TERATOMA WITH MALIGNANT NEUROECTODERMAL TRANSFORMATION. SUCH LESIONS MAY CLOSELY RESEMBLE CENTRAL NERVOUS SYSTEM TUMORS, BUT THEIR BIOLOGIC SIMILARITY IS UNCLEAR. WE DESCRIBE AN 18-YR-OLD FEMALE WHO PRESENTED WITH ABDOMINAL PAIN CAUSED BY AN OVARIAN MASS WITH WIDESPREAD METASTASES. HISTOLOGY SHOWED A PRIMITIVE, HIGH-GRADE TUMOR ARISING IN THE BACKGROUND OF A MATURE TERATOMA. THE TUMOR WAS SOX10 POSITIVE, WITH FOCAL EXPRESSION OF GFAP, S100, NSE, AND SYNAPTOPHYSIN. MOLECULAR ANALYSIS DEMONSTRATED CO-AMPLIFICATION OF PDGFRA AND KIT, ALTERATIONS COMMON IN HIGH-GRADE GLIOMAS. BY WHOLE-GENOME METHYLATION PROFILING, IT CLUSTERED INTO THE "DIFFUSE PEDIATRIC-TYPE HIGH-GRADE GLIOMA, RTK1 SUBTYPE, SUBCLASS C" GROUP. DESPITE PROGRESSING THROUGH 2 LINES OF CHEMOTHERAPY WITH WIDESPREAD METASTATIC DISEASE, SHE ACHIEVED AN EXCELLENT RESPONSE TO CHEMOTHERAPY DIRECTED TOWARD AGGRESSIVE GERM CELL TUMORS. THIS CASE EMPHASIZES THE IMPORTANCE OF IMMUNOHISTOCHEMICAL, GENOMIC, AND EPIGENETIC ANALYSES TO ACCURATELY CLASSIFY THESE EXCEEDINGLY RARE TUMORS AND DETERMINE THE OPTIMAL THERAPY. 2023 2 5453 24 REPROGRAMMING OF COPD LUNG FIBROBLASTS THROUGH FORMATION OF INDUCED PLURIPOTENT STEM CELLS. REPROGRAMMING SOMATIC CELLS TO INDUCED PLURIPOTENT STEM CELLS (IPSCS) ELIMINATES MANY EPIGENETIC MODIFICATIONS THAT CHARACTERIZE DIFFERENTIATED CELLS. IN THIS STUDY, WE TESTED WHETHER FUNCTIONAL DIFFERENCES BETWEEN CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) AND NON-COPD FIBROBLASTS COULD BE REDUCED UTILIZING THIS APPROACH. PRIMARY FIBROBLASTS FROM NON-COPD AND COPD PATIENTS WERE REPROGRAMMED TO IPSCS. REPROGRAMMED IPSCS WERE POSITIVE FOR OCT3/4, NANOG, AND SOX2, FORMED EMBRYOID BODIES IN VITRO, AND INDUCED TERATOMAS IN NONOBESE DIABETIC/SEVERE COMBINED IMMUNODEFICIENT MICE. REPROGRAMMED IPSCS WERE THEN DIFFERENTIATED INTO FIBROBLASTS (NON-COPD-I AND COPD-I) AND WERE ASSESSED EITHER FUNCTIONALLY BY CHEMOTAXIS AND GEL CONTRACTION OR FOR GENE EXPRESSION BY MICROARRAYS AND COMPARED WITH THEIR CORRESPONDING PRIMARY FIBROBLASTS. PRIMARY COPD FIBROBLASTS CONTRACTED THREE-DIMENSIONAL COLLAGEN GELS AND MIGRATED TOWARD FIBRONECTIN LESS ROBUSTLY THAN NON-COPD FIBROBLASTS. IN CONTRAST, REDIFFERENTIATED FIBROBLASTS FROM IPSCS DERIVED FROM THE NON-COPD AND COPD FIBROBLASTS WERE SIMILAR IN RESPONSE IN BOTH FUNCTIONAL ASSAYS. MICROARRAY ANALYSIS IDENTIFIED 1,881 GENES THAT WERE DIFFERENTIALLY EXPRESSED BETWEEN PRIMARY COPD AND NON-COPD FIBROBLASTS, WITH 605 GENES DIFFERING BY MORE THAN TWOFOLD. AFTER REDIFFERENTIATION, 112 GENES WERE DIFFERENTIALLY EXPRESSED BETWEEN COPD-I AND NON-COPD-I WITH ONLY THREE GENES BY MORE THAN TWOFOLD. SIMILAR FINDINGS WERE OBSERVED WITH MICRORNA (MIRNA) EXPRESSION: 56 MIRNAS WERE DIFFERENTIALLY EXPRESSED BETWEEN NON-COPD AND COPD PRIMARY CELLS; AFTER REDIFFERENTIATION, ONLY 3 MIRNAS WERE DIFFERENTIALLY EXPRESSED BETWEEN NON-COPD-I AND COPD-I FIBROBLASTS. INTERESTINGLY, OF THE 605 GENES THAT WERE DIFFERENTIALLY EXPRESSED BETWEEN COPD AND NON-COPD FIBROBLASTS, 293 GENES WERE CHANGED TOWARD CONTROL AFTER REDIFFERENTIATION. IN CONCLUSION, FUNCTIONAL AND EPIGENETIC ALTERATIONS OF COPD FIBROBLASTS CAN BE REPROGRAMMED THROUGH FORMATION OF IPSCS. 2014 3 1359 18 DEVELOPMENT REFRACTORINESS OF MLL-REARRANGED HUMAN B CELL ACUTE LEUKEMIAS TO REPROGRAMMING INTO PLURIPOTENCY. INDUCED PLURIPOTENT STEM CELLS (IPSCS) ARE A POWERFUL TOOL FOR DISEASE MODELING. THEY ARE ROUTINELY GENERATED FROM HEALTHY DONORS AND PATIENTS FROM MULTIPLE CELL TYPES AT DIFFERENT DEVELOPMENTAL STAGES. HOWEVER, REPROGRAMMING LEUKEMIAS IS AN EXTREMELY INEFFICIENT PROCESS. FEW STUDIES GENERATED IPSCS FROM PRIMARY CHRONIC MYELOID LEUKEMIAS, BUT IPSC GENERATION FROM ACUTE MYELOID OR LYMPHOID LEUKEMIAS (ALL) HAS NOT BEEN ACHIEVED. WE ATTEMPTED TO GENERATE IPSCS FROM DIFFERENT SUBTYPES OF B-ALL TO ADDRESS THE DEVELOPMENTAL IMPACT OF LEUKEMIC FUSION GENES. OKSM(L)-EXPRESSING MONO/POLYCISTRONIC-, RETROVIRAL/LENTIVIRAL/EPISOMAL-, AND SENDAI VIRUS VECTOR-BASED REPROGRAMMING STRATEGIES FAILED TO RENDER IPSCS IN VITRO AND IN VIVO. ADDITION OF TRANSCRIPTOMIC-EPIGENETIC REPROGRAMMING "BOOSTERS" ALSO FAILED TO GENERATE IPSCS FROM B CELL BLASTS AND B-ALL LINES, AND WHEN IPSCS EMERGED THEY LACKED LEUKEMIC FUSION GENES, DEMONSTRATING NON-LEUKEMIC MYELOID ORIGIN. CONVERSELY, MLL-AF4-OVEREXPRESSING HEMATOPOIETIC STEM CELLS/B PROGENITORS WERE SUCCESSFULLY REPROGRAMMED, INDICATING THAT B CELL ORIGIN AND LEUKEMIC FUSION GENE WERE NOT REPROGRAMMING BARRIERS. GLOBAL TRANSCRIPTOME/DNA METHYLOME PROFILING SUGGESTED A DEVELOPMENTAL/DIFFERENTIATION REFRACTORINESS OF MLL-REARRANGED B-ALL TO REPROGRAMMING INTO PLURIPOTENCY. 2016 4 2928 29 GENERATION OF IPSCS FROM CULTURED HUMAN MALIGNANT CELLS. INDUCED PLURIPOTENT STEM CELLS (IPSCS) CAN BE GENERATED FROM VARIOUS DIFFERENTIATED CELL TYPES BY THE EXPRESSION OF A SET OF DEFINED TRANSCRIPTION FACTORS. SO FAR, IPSCS HAVE BEEN GENERATED FROM PRIMARY CELLS, BUT IT IS UNCLEAR WHETHER HUMAN CANCER CELL LINES CAN BE REPROGRAMMED. HERE WE DESCRIBE THE GENERATION AND CHARACTERIZATION OF IPSCS DERIVED FROM HUMAN CHRONIC MYELOID LEUKEMIA CELLS. WE SHOW THAT, DESPITE THE PRESENCE OF ONCOGENIC MUTATIONS, THESE CELLS ACQUIRED PLURIPOTENCY BY THE EXPRESSION OF 4 TRANSCRIPTION FACTORS AND UNDERWENT DIFFERENTIATION INTO CELL TYPES DERIVED OF ALL 3 GERM LAYERS DURING TERATOMA FORMATION. INTERESTINGLY, ALTHOUGH THE PARENTAL CELL LINE WAS STRICTLY DEPENDENT ON CONTINUOUS SIGNALING OF THE BCR-ABL ONCOGENE, ALSO TERMED ONCOGENE ADDICTION, REPROGRAMMED CELLS LOST THIS DEPENDENCY AND BECAME RESISTANT TO THE BCR-ABL INHIBITOR IMATINIB. THIS FINDING INDICATES THAT THE THERAPEUTIC AGENT IMATINIB TARGETS CELLS IN A SPECIFIC EPIGENETIC DIFFERENTIATED CELL STATE, AND THIS MAY CONTRIBUTE TO ITS INABILITY TO FULLY ERADICATE DISEASE IN CHRONIC MYELOID LEUKEMIA PATIENTS. 2010 5 5523 19 RISKS AND MECHANISMS OF ONCOLOGICAL DISEASE FOLLOWING STEM CELL TRANSPLANTATION. UNIQUE BIOLOGICAL PROPERTIES OF STEM CELLS MAKE THEM A PRECIOUS SOURCE OF CELL MATERIAL FOR TREATMENT OF A NUMBER OF PATHOLOGICAL CONDITIONS. AMONG ISSUES INHIBITING TRANSITION OF STEM CELL TECHNOLOGIES TO THE CLINICS, THE RISK OF ONCOLOGICAL COMPLICATIONS OF STEM CELL-BASED THERAPIES IS THE MOST CRITICAL. A MASSIVE AMOUNT OF CLINICAL AND EXPERIMENTAL DATA DEMONSTRATES THAT BOTH HEMATOLOGICAL (INCLUDING ACUTE AND CHRONIC MYELOID LEUKEMIA) AND NON-HEMATOLOGICAL (INCLUDING TERATOMA AND NON-TERATOMA TUMORS) MALIGNANCIES COULD ARISE FROM DONOR STEM CELLS OF DIFFERENT TYPES. A WIDE SPECTRUM OF MECHANISMS COULD UNDERLIE THE DEVELOPMENT OF ONCOLOGICAL DISEASE IN RECIPIENTS, INCLUDING: I) BLAST TRANSFORMATION OF PROLIFERATING DONOR STEM CELLS UNDER PERSISTENT ACTION OF CERTAIN FACTORS IN THE RECIPIENT, THUS CAUSING DE NOVO MALIGNANCIES; II) CONTAMINATION OF DONOR CELL MATERIAL WITH MALIGNANT CELLS; III) TRANSMISSION OF PARTICULAR VIRAL SUBTYPES WITH DONOR STEM CELLS, COMBINED WITH IMMUNOSUPPRESSION THERAPY EFFECTS; IV) UNCONTROLLABLE PROLIFERATION OF RESIDUAL UNDIFFERENTIATED STEM CELLS OF VARIOUS PLASTICITY; AND V) KARYOTYPIC INSTABILITY IN STEM CELLS FOLLOWING PROLONGED CULTURING/EXPANSION IN VITRO. POTENTIAL PREVENTIVE STRATEGIES ARE DIVERSE AND INCLUDE I) HIGH-THROUGHPUT CELL SORTING-BASED STRATEGIES; II) INTRODUCTION OF SUICIDE GENES INTO THE DONOR STEM CELL GENOME; III) APPLICATION OF APOPTOSIS-INDUCING EPIGENETIC FACTORS; AND SOME OTHER OPTIONS. 2010 6 1116 27 COMPARATIVE ANALYSIS OF NEUROECTODERMAL DIFFERENTIATION CAPACITY OF HUMAN BONE MARROW STROMAL CELLS USING VARIOUS CONVERSION PROTOCOLS. HUMAN ADULT BONE MARROW-DERIVED MESODERMAL STROMAL CELLS (HMSCS) ARE ABLE TO DIFFERENTIATE INTO MULTIPLE MESODERMAL TISSUES, INCLUDING BONE AND CARTILAGE. THERE IS EVIDENCE THAT THESE CELLS ARE ABLE TO BREAK GERM LAYER COMMITMENT AND DIFFERENTIATE INTO CELLS EXPRESSING NEUROECTODERMAL PROPERTIES. THERE IS STILL DEBATE ABOUT WHETHER THIS RESULTS FROM CELL FUSION, ABERRANT MARKER GENE EXPRESSION OR REAL NEUROECTODERMAL DIFFERENTIATION. HERE WE EXTEND OUR WORK ON NEUROECTODERMAL CONVERSION OF ADULT HMSCS IN VITRO BY EVALUATING VARIOUS EPIGENETIC CONVERSION PROTOCOLS USING QUANTITATIVE RT-PCR AND IMMUNOCYTOCHEMISTRY. UNDIFFERENTIATED HMSCS EXPRESSED HIGH LEVELS OF FIBRONECTIN AS WELL AS SEVERAL NEUROECTODERMAL GENES COMMONLY USED TO CHARACTERIZE NEURAL CELL TYPES, SUCH AS NESTIN, BETA-TUBULIN III, AND GFAP, SUGGESTING THAT HMSCS RETAIN THE ABILITY TO DIFFERENTIATE INTO NEUROECTODERMAL CELL TYPES. PROTOCOLS USING A DIRECT DIFFERENTIATION OF HMSCS INTO A NEURAL PHENOTYPE FAILED TO INDUCE SIGNIFICANT CHANGES IN MORPHOLOGY AND/OR EXPRESSION OF MARKERS OF EARLY AND MATURE GLIAL/NEURONAL CELLS TYPES. IN CONTRAST, A MULTISTEP PROTOCOL WITH CONVERSION OF HMSCS INTO A NEURAL STEM CELL-LIKE POPULATION AND SUBSEQUENT TERMINAL DIFFERENTIATION IN MATURE GLIA AND NEURONS GENERATED RELEVANT MORPHOLOGICAL CHANGES AS WELL AS SIGNIFICANT INCREASE OF EXPRESSION LEVELS OF MARKER GENES FOR EARLY AND LATE NEURAL CELL TYPES, SUCH AS NESTIN, NEUROGENIN2, MBP, AND MAP2AB, ACCOMPANIED BY A LOSS OF THEIR MESENCHYMAL PROPERTIES. OUR DATA PROVIDE AN IMPETUS FOR DIFFERENTIATING HMSCS IN VITRO INTO MATURE NEUROECTODERMAL CELLS. NEUROECTODERMALLY CONVERTED HMSCS MAY THEREFORE ULTIMATELY HELP IN TREATING ACUTE AND CHRONIC NEURODEGENERATIVE DISEASES. ANALYSIS OF MARKER GENE EXPRESSION FOR CHARACTERIZATION OF NEURAL CELLS DERIVED FROM MSCS HAS TO TAKE INTO ACCOUNT THAT SEVERAL EARLY AND LATE NEUROECTODERMAL GENES ARE ALREADY EXPRESSED IN UNDIFFERENTIATED MSCS. 2006 7 1668 27 DOWNREGULATION OF SOCS1 INCREASES INTERFERON-INDUCED ISGYLATION DURING DIFFERENTIATION OF INDUCED-PLURIPOTENT STEM CELLS TO HEPATOCYTES. BACKGROUND & AIMS: INCREASED EXPRESSION OF IFN-STIMULATED GENE 15 (ISG15) AND SUBSEQUENTLY INCREASED ISGYLATION ARE KEY FACTORS IN THE HOST RESPONSE TO VIRAL INFECTION. IN THIS STUDY, WE SOUGHT TO CHARACTERIZE THE EXPRESSION OF ISG15, ISGYLATION, AND ASSOCIATED ENZYMES AT EACH STAGE OF DIFFERENTIATION FROM INDUCED PLURIPOTENT STEM CELLS (IPSCS) TO HEPATOCYTES. METHODS: TO STUDY THE REGULATION OF ISGYLATION, WE UTILIZED PATIENT SAMPLES AND IN VITRO CELL CULTURE MODELS INCLUDING IPSCS, HEPATOCYTES-LIKE CELLS, IMMORTALIZED CELL LINES, AND PRIMARY HUMAN HEPATOCYTES. PROTEIN/MRNA EXPRESSION WERE MEASURED FOLLOWING TREATMENT WITH POLY(I:C), IFNALPHA AND HCV INFECTION. RESULTS: WHEN COMPARED TO HLCS, WE OBSERVED SEVERAL NOVEL ASPECTS OF THE ISGYLATION PATHWAY IN IPSCS. THESE INCLUDE A LOWER BASELINE EXPRESSION OF THE ISGYLATION-ACTIVATING ENZYME, UBE1L, A LACK OF IFN-INDUCED EXPRESSION OF THE ISGYLATION-CONJUGATION ENZYME UBE2L6, AN ATTENUATED ACTIVATION OF THE TRANSCRIPTION FACTOR STAT1 AND CONSTITUTIVE EXPRESSION OF SOCS1. ISGYLATION WAS OBSERVED IN IPSCS FOLLOWING DOWNREGULATION OF SOCS1, WHICH FACILITATED STAT1 ACTIVATION AND SUBSEQUENTLY INCREASED EXPRESSION OF UBE2L6. INTRIGUINGLY, HCV PERMISSIVE TRANSFORMED HEPATOMA CELL LINES DEMONSTRATED HIGHER INTRINSIC EXPRESSION OF SOCS1 AND WEAKER ISGYLATION FOLLOWING IFN TREATMENT. SOCS1 DOWNREGULATION IN HCV-INFECTED HUH 7.5.1 CELLS LED TO INCREASED ISGYLATION. CONCLUSIONS: HEREIN, WE SHOW THAT HIGH BASAL LEVELS OF SOCS1 INHIBIT STAT1 ACTIVATION AND SUBSEQUENTLY IFN-INDUCED UBE2L6 AND ISGYLATION IN IPSCS. FURTHERMORE, AS IPSCS DIFFERENTIATE INTO HEPATOCYTES, EPIGENETIC MECHANISMS REGULATE ISGYLATION BY MODIFYING UBE1L AND SOCS1 EXPRESSION LEVELS. OVERALL, THIS STUDY DEMONSTRATES THAT THE DEVELOPMENT OF CELL-INTRINSIC INNATE IMMUNITY DURING THE DIFFERENTIATION OF IPSCS TO HEPATOCYTES PROVIDES INSIGHT INTO CELL TYPE-SPECIFIC REGULATION OF HOST DEFENSE RESPONSES AND RELATED ONCOGENIC PROCESSES. IMPACT AND IMPLICATIONS: TO ELUCIDATE THE MECHANISM UNDERLYING REGULATION OF ISGYLATION, A KEY PROCESS IN THE INNATE IMMUNE RESPONSE, WE STUDIED CHANGES IN ISGYLATION-ASSOCIATED GENES AT THE DIFFERENT STAGES OF DIFFERENTIATION FROM IPSCS TO HEPATOCYTES. WE FOUND THAT HIGH BASAL LEVELS OF SOCS1 INHIBIT STAT1 ACTIVATION AND SUBSEQUENTLY IFN-INDUCED UBE2L6 AND ISGYLATION IN IPSCS. IMPORTANTLY, EPIGENETIC REGULATION OF SOCS1 AND SUBSEQUENTLY ISGYLATION MAY BE IMPORTANT FACTORS IN THE DEVELOPMENT OF CELL TYPE-SPECIFIC HOST DEFENSE RESPONSES IN HEPATOCYTES THAT SHOULD BE CONSIDERED WHEN STUDYING CHRONIC INFECTIONS AND ONCOGENIC PROCESSES IN THE LIVER. 2022 8 5924 19 TARGETING DNMT1 BY DEMETHYLATING AGENT OR-2100 INCREASES TYROSINE KINASE INHIBITORS-SENSITIVITY AND DEPLETES LEUKEMIC STEM CELLS IN CHRONIC MYELOID LEUKEMIA. ABL1 TYROSINE KINASE INHIBITORS (TKIS) DRAMATICALLY IMPROVE THE PROGNOSIS OF CHRONIC MYELOID LEUKEMIA (CML), BUT 10-20% OF PATIENTS ACHIEVE SUBOPTIMAL RESPONSES WITH LOW TKIS SENSITIVITY. FURTHERMORE, RESIDUAL LEUKEMIC STEM CELLS (LSCS) ARE INVOLVED IN THE MOLECULAR RELAPSE AFTER TKIS DISCONTINUATION. ABERRANT DNA HYPERMETHYLATION CONTRIBUTES TO LOW TKIS SENSITIVITY AND THE PERSISTENCE OF LSCS IN CML. DNMT1 IS A KEY REGULATOR OF HEMATOPOIETIC STEM CELLS, SUGGESTING THAT ABERRANT DNA HYPERMETHYLATION TARGETING DNMT1 REPRESENTS A POTENTIAL THERAPEUTIC TARGET FOR CML. WE INVESTIGATED THE EFFICACY OF OR-2100 (OR21), THE FIRST ORALLY AVAILABLE SINGLE-COMPOUND PRODRUG OF DECITABINE. OR21 EXHIBITED ANTI-TUMOR EFFECTS AS A MONOTHERAPY, AND IN COMBINATION THERAPY IT INCREASED TKI-INDUCED APOPTOSIS AND INDUCTION OF TUMOR SUPPRESSOR GENES INCLUDING PTPN6 ENCODING SHP-1 IN CML CELLS. OR21 IN COMBINATION WITH IMATINIB SIGNIFICANTLY SUPPRESSED TUMOR GROWTH IN A XENOTRANSPLANT MODEL. OR21 AND COMBINATION THERAPY DECREASED THE ABUNDANCE OF LSCS AND INHIBITED ENGRAFTMENT IN A BCR-ABL1-TRANSDUCED MOUSE MODEL. THESE RESULTS DEMONSTRATE THAT TARGETING DNMT1 USING OR21 EXERTS ANTI-TUMOR EFFECTS AND IMPAIRS LSCS IN CML. THEREFORE, COMBINATION TREATMENT OF TKIS AND OR21 REPRESENTS A PROMISING TREATMENT STRATEGY IN CML. 2022 9 2072 26 EPIGENETIC CONVERSION OF HUMAN ADULT BONE MESODERMAL STROMAL CELLS INTO NEUROECTODERMAL CELL TYPES FOR REPLACEMENT THERAPY OF NEURODEGENERATIVE DISORDERS. TISSUE-SPECIFIC STEM CELLS, SUCH AS BONE MARROW-DERIVED MESODERMAL STROMAL CELLS (MSCS), ARE THOUGHT TO BE LINEAGE RESTRICTED AND, THEREFORE, COULD ONLY BE DIFFERENTIATED INTO CELL TYPES OF THE TISSUE OF ORIGIN. SEVERAL RECENT STUDIES, HOWEVER, SUGGEST THAT THESE TYPES OF STEM CELLS MIGHT BE ABLE TO BREAK BARRIERS OF GERM LAYER COMMITMENT AND DIFFERENTIATE IN VITRO AND/OR IN VIVO INTO CELLS OF DIFFERENT TISSUES, SUCH AS NEUROECTODERMAL CELL TYPES. RECENTLY, PROTOCOLS FOR HIGH-YIELD GENERATION OF UNDIFFERENTIATED NEURAL STEM CELL (NSC)-LIKE CELLS FROM MSCS OF PRIMATE AND HUMAN ORIGIN WERE REPORTED. UNDIFFERENTIATED NSCS ARE COMMONLY USED AND ARE MORE SUITABLE FOR NEUROTRANSPLANTATION COMPARED WITH FULLY DIFFERENTIATED NEURAL CELLS, AS DIFFERENTIATED NEURAL CELLS ARE WELL KNOWN TO POORLY SURVIVE DETACHMENT AND SUBSEQUENT TRANSPLANTATION PROCEDURES. THESE HUMAN MSC-DERIVED NSC-LIKE CELLS (MSC-NSCS) GROW IN NEUROSPHERE-LIKE STRUCTURES AND EXPRESS HIGH LEVELS OF EARLY NEUROECTODERMAL MARKERS, BUT LOSE CHARACTERISTICS OF MSCS. IN THE PRESENCE OF SELECTED GROWTH FACTORS, HUMAN MSC-NSCS CAN BE DIFFERENTIATED INTO THE THREE MAIN NEURAL PHENOTYPES: ASTROGLIA, OLIGODENDROGLIA AND NEURONS. COMPARED WITH DIRECT DIFFERENTIATION OF HUMAN MSCS INTO MATURE NEURAL CELLS, THE CONVERSION STEP SEEMS TO BE ESSENTIAL TO GENERATE MATURE FUNCTIONAL NEUROECTODERMAL CELLS. THIS REVIEW DESCRIBES THE TECHNIQUES FOR THE CONVERSION OF HUMAN MSCS INTO NSCS AND SUMMARISES THE DATA ON EPIGENETIC CONVERSION OF HUMAN MSCS INTO IMMATURE NEUROECTODERMAL CELLS. THESE CELLS PROVIDE A POWERFUL TOOL FOR INVESTIGATING THE MOLECULAR MECHANISMS OF NEURAL DIFFERENTIATION, AND MIGHT SERVE AS AN AUTOLOGOUS CELL SOURCE TO TREAT ACUTE AND CHRONIC NEURODEGENERATIVE DISEASES. 2006 10 2888 24 GAIN-OF-FUNCTION MUTATION OF GATA-2 IN ACUTE MYELOID TRANSFORMATION OF CHRONIC MYELOID LEUKEMIA. ACQUISITION OF ADDITIONAL GENETIC AND/OR EPIGENETIC ABNORMALITIES OTHER THAN THE BCR/ABL FUSION GENE IS BELIEVED TO CAUSE DISEASE PROGRESSION IN CHRONIC MYELOID LEUKEMIA (CML) FROM CHRONIC PHASE TO BLAST CRISIS (BC). TO GAIN INSIGHTS INTO THE UNDERLYING MECHANISMS OF PROGRESSION TO BC, WE SCREENED DNA SAMPLES FROM CML PATIENTS DURING BLAST TRANSFORMATION FOR MUTATIONS IN A NUMBER OF TRANSCRIPTION FACTOR GENES THAT ARE CRITICAL FOR MYELOID-LYMPHOID DEVELOPMENT. IN 85 CASES OF CML BLAST TRANSFORMATION, WE IDENTIFIED TWO NEW MUTATIONS IN THE CODING REGION OF GATA-2, A NEGATIVE REGULATOR OF HEMATOPOIETIC STEM/PROGENITOR CELL DIFFERENTIATION. A L359V SUBSTITUTION WITHIN ZINC FINGER DOMAIN (ZF) 2 OF GATA-2 WAS FOUND IN EIGHT CASES WITH MYELOMONOBLASTIC FEATURES, WHEREAS AN IN-FRAME DELETION OF 6 AA (DELTA341-346) SPANNING THE C-TERMINAL BORDER OF ZF1 WAS DETECTED IN ONE PATIENT AT MYELOID BC WITH EOSINOPHILIA. FURTHER STUDIES INDICATED THAT L359V NOT ONLY INCREASED TRANSACTIVATION ACTIVITY OF GATA-2 BUT ALSO ENHANCED ITS INHIBITORY EFFECTS ON THE ACTIVITY OF PU.1, A MAJOR REGULATOR OF MYELOPOIESIS. CONSISTENT WITH THE MYELOMONOBLASTIC FEATURES OF CML TRANSFORMATION WITH THE GATA-2 L359V MUTANT, TRANSDUCTION OF THE GATA-2 L359V MUTANT INTO HL-60 CELLS OR BCR/ABL-HARBORING MURINE CELLS DISTURBED MYELOMONOCYTIC DIFFERENTIATION/PROLIFERATION IN VITRO AND IN VIVO, RESPECTIVELY. THESE DATA STRONGLY SUGGEST THAT GATA-2 MUTATIONS MAY PLAY A ROLE IN ACUTE MYELOID TRANSFORMATION IN A SUBSET OF CML PATIENTS. 2008 11 3158 24 GLYCOGEN SYNTHASE KINASE 3BETA MISSPLICING CONTRIBUTES TO LEUKEMIA STEM CELL GENERATION. RECENT EVIDENCE SUGGESTS THAT A RARE POPULATION OF SELF-RENEWING CANCER STEM CELLS (CSC) IS RESPONSIBLE FOR CANCER PROGRESSION AND THERAPEUTIC RESISTANCE. CHRONIC MYELOID LEUKEMIA (CML) REPRESENTS AN IMPORTANT PARADIGM FOR UNDERSTANDING THE GENETIC AND EPIGENETIC EVENTS INVOLVED IN CSC PRODUCTION. CML PROGRESSES FROM A CHRONIC PHASE (CP) IN HEMATOPOIETIC STEM CELLS (HSC) THAT HARBOR THE BCR-ABL TRANSLOCATION, TO BLAST CRISIS (BC), CHARACTERIZED BY ABERRANT ACTIVATION OF BETA-CATENIN WITHIN GRANULOCYTE-MACROPHAGE PROGENITORS (GMP). A MAJOR BARRIER TO PREDICTING AND INHIBITING BLAST CRISIS TRANSFORMATION HAS BEEN THE IDENTIFICATION OF MECHANISMS DRIVING BETA-CATENIN ACTIVATION. HERE WE SHOW THAT BC CML MYELOID PROGENITORS, IN PARTICULAR GMP, SERIALLY TRANSPLANT LEUKEMIA IN IMMUNOCOMPROMISED MICE AND THUS ARE ENRICHED FOR LEUKEMIA STEM CELLS (LSC). NOTABLY, CDNA SEQUENCING OF WNT/BETA-CATENIN PATHWAY REGULATORY GENES, INCLUDING ADENOMATOUS POLYPOSIS COLI, GSK3BETA, AXIN 1, BETA-CATENIN, LYMPHOID ENHANCER FACTOR-1, CYCLIN D1, AND C-MYC, REVEALED A NOVEL IN-FRAME SPLICE DELETION OF THE GSK3BETA KINASE DOMAIN IN THE GMP OF BC SAMPLES THAT WAS NOT DETECTABLE BY SEQUENCING IN BLASTS OR NORMAL PROGENITORS. MOREOVER, BC CML PROGENITORS WITH MISSPLICED GSK3BETA HAVE ENHANCED BETA-CATENIN EXPRESSION AS WELL AS SERIAL ENGRAFTMENT POTENTIAL WHILE REINTRODUCTION OF FULL-LENGTH GSK3BETA REDUCES BOTH IN VITRO REPLATING AND LEUKEMIC ENGRAFTMENT. WE PROPOSE THAT CP CML IS INITIATED BY BCR-ABL EXPRESSION IN AN HSC CLONE BUT THAT PROGRESSION TO BC MAY INCLUDE MISSPLICING OF GSK3BETA IN GMP LSC, ENABLING UNPHOSPHORYLATED BETA-CATENIN TO PARTICIPATE IN LSC SELF-RENEWAL. MISSPLICING OF GSK3BETA REPRESENTS A UNIQUE MECHANISM FOR THE EMERGENCE OF BC CML LSC AND MIGHT PROVIDE A NOVEL DIAGNOSTIC AND THERAPEUTIC TARGET. 2009 12 4741 23 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 13 2402 23 EPIGENETIC REPROGRAMMING SENSITIZES CML STEM CELLS TO COMBINED EZH2 AND TYROSINE KINASE INHIBITION. A MAJOR OBSTACLE TO CURING CHRONIC MYELOID LEUKEMIA (CML) IS RESIDUAL DISEASE MAINTAINED BY TYROSINE KINASE INHIBITOR (TKI)-PERSISTENT LEUKEMIC STEM CELLS (LSC). THESE ARE BCR-ABL1 KINASE INDEPENDENT, REFRACTORY TO APOPTOSIS, AND SERVE AS A RESERVOIR TO DRIVE RELAPSE OR TKI RESISTANCE. WE DEMONSTRATE THAT POLYCOMB REPRESSIVE COMPLEX 2 IS MISREGULATED IN CHRONIC PHASE CML LSCS. THIS IS ASSOCIATED WITH EXTENSIVE REPROGRAMMING OF H3K27ME3 TARGETS IN LSCS, THUS SENSITIZING THEM TO APOPTOSIS UPON TREATMENT WITH AN EZH2-SPECIFIC INHIBITOR (EZH2I). EZH2I DOES NOT IMPAIR NORMAL HEMATOPOIETIC STEM CELL SURVIVAL. STRIKINGLY, TREATMENT OF PRIMARY CML CELLS WITH EITHER EZH2I OR TKI ALONE CAUSED SIGNIFICANT UPREGULATION OF H3K27ME3 TARGETS, AND COMBINED TREATMENT FURTHER POTENTIATED THESE EFFECTS AND RESULTED IN SIGNIFICANT LOSS OF LSCS COMPARED TO TKI ALONE, IN VITRO, AND IN LONG-TERM BONE MARROW MURINE XENOGRAFTS. OUR FINDINGS POINT TO A PROMISING EPIGENETIC-BASED THERAPEUTIC STRATEGY TO MORE EFFECTIVELY TARGET LSCS IN PATIENTS WITH CML RECEIVING TKIS. SIGNIFICANCE: IN CML, TKI-PERSISTENT LSCS REMAIN AN OBSTACLE TO CURE, AND APPROACHES TO ERADICATE THEM REMAIN A SIGNIFICANT UNMET CLINICAL NEED. WE DEMONSTRATE THAT EZH2 AND H3K27ME3 REPROGRAMMING IS IMPORTANT FOR LSC SURVIVAL, BUT RENDERS LSCS SENSITIVE TO THE COMBINED EFFECTS OF EZH2I AND TKI. THIS REPRESENTS A NOVEL APPROACH TO MORE EFFECTIVELY TARGET LSCS IN PATIENTS RECEIVING TKI TREATMENT. CANCER DISCOV; 6(11); 1248-57. (C)2016 AACR.SEE RELATED ARTICLE BY XIE ET AL., P. 1237THIS ARTICLE IS HIGHLIGHTED IN THE IN THIS ISSUE FEATURE, P. 1197. 2016 14 573 27 BCR-ABL1-INDUCED DOWNREGULATION OF WASP IN CHRONIC MYELOID LEUKEMIA INVOLVES EPIGENETIC MODIFICATION AND CONTRIBUTES TO MALIGNANCY. CHRONIC MYELOID LEUKEMIA (CML) IS A MYELOPROLIFERATIVE DISEASE CAUSED BY THE BCR-ABL1 TYROSINE KINASE (TK). THE DEVELOPMENT OF TK INHIBITORS (TKIS) REVOLUTIONIZED THE TREATMENT OF CML PATIENTS. HOWEVER, TKIS ARE NOT EFFECTIVE TO THOSE AT ADVANCED PHASES WHEN AMPLIFIED BCR-ABL1 LEVELS AND INCREASED GENOMIC INSTABILITY LEAD TO SECONDARY ONCOGENIC MODIFICATIONS. WISKOTT-ALDRICH SYNDROME PROTEIN (WASP) IS AN IMPORTANT REGULATOR OF SIGNALING TRANSDUCTION IN HEMATOPOIETIC CELLS AND WAS SHOWN TO BE AN ENDOGENOUS INHIBITOR OF THE C-ABL TK. HERE, WE SHOW THAT THE EXPRESSION OF WASP DECREASES WITH THE PROGRESSION OF CML, INVERSELY CORRELATES WITH THE EXPRESSION OF BCR-ABL1 AND IS PARTICULARLY LOW IN BLAST CRISIS. ENFORCED EXPRESSION OF BCR-ABL1 NEGATIVELY REGULATES THE EXPRESSION OF WASP. DECREASED EXPRESSION OF WASP IS PARTIALLY DUE TO DNA METHYLATION OF THE PROXIMAL WASP PROMOTER. IMPORTANTLY, LOWER LEVELS OF WASP IN CML ADVANCED PHASE PATIENTS CORRELATE WITH POORER OVERALL SURVIVAL (OS) AND IS ASSOCIATED WITH TKI RESPONSE. INTERESTINGLY, ENFORCED EXPRESSION OF WASP IN BCR-ABL1-POSITIVE K562 CELLS INCREASES THE SUSCEPTIBILITY TO APOPTOSIS INDUCED BY TRAIL OR CHEMOTHERAPEUTIC DRUGS AND NEGATIVELY MODULATES BCR-ABL1-INDUCED TUMORIGENESIS IN VITRO AND IN VIVO. TAKEN TOGETHER, OUR DATA REVEAL A NOVEL MOLECULAR MECHANISM THAT OPERATES IN BCR-ABL1-INDUCED TUMORIGENESIS THAT CAN BE USED TO DEVELOP NEW STRATEGIES TO HELP TKI-RESISTANT, CML PATIENTS IN BLAST CRISIS (BC). 2017 15 4436 20 MOLECULAR EVOLUTION OF CHRONIC MYELOID LEUKAEMIA. CHRONIC MYELOID LEUKAEMIA (CML) IS A CLONAL DISORDER OF THE PLURIPOTENT HAEMATOPOIETIC STEM CELL. THE TYPICAL TRIPHASIC COURSE OF CML STARTS WITH THE PREMALIGNANT CHRONIC PHASE INITIATED BY BCR-ABL HYBRID ONCOGENE FORMATION. SECONDARY GENETIC AND EPIGENETIC ABERRATIONS ACCOMPANY THE PROGRESSION TO THE ACCELERATED PHASE AND FATAL BLASTIC CRISIS. PROPERLY TIMED BONE MARROW TRANSPLANTATION IN ELIGIBLE PATIENTS CAN RESULT IN DURABLE REMISSIONS OR CURE. BOTH OF THESE STATES ARE OFTEN ACCOMPANIED BY A LONG-TERM PERSISTENCE OF QUIESCENT LEUKAEMIC CELLS. ACCORDINGLY, A "FUNCTIONAL CURE" (I.E. TUMOUR DORMANCY INDUCTION), RATHER THAN COMPLETE ERADICATION OF THE MALIGNANT CELLS, IS AN ADEQUATE THERAPEUTICAL GOAL. THE LEVEL OF THE RESIDUAL BCR-ABL-POSITIVE CLONES SHOULD BE MONITORED AND SALVAGE TREATMENT INITIATED WHENEVER THESE QUIESCENT LEUKAEMIC CELLS EXIT THEIR DORMANT STATE. 2001 16 2697 19 EX VIVO MODELS OF CHRONIC GRANULOMATOUS DISEASE. INDUCED PLURIPOTENT STEM CELLS (IPSCS) ARE PLURIPOTENT STEM CELLS THAT CAN BE ESTABLISHED FROM DEDIFFERENTIATION OF ALL SOMATIC CELL TYPES BY EPIGENETIC PHENOMENA. IPSCS CAN BE DIFFERENTIATED INTO ANY MATURE CELLS LIKE NEURONS, HEPATOCYTES, OR PANCREATIC CELLS THAT HAVE NOT BEEN EASILY AVAILABLE TO DATE. THUS, IPSCS ARE WIDELY USED FOR DISEASE MODELING, DRUG DISCOVERY, AND CELL THERAPY DEVELOPMENT. HERE, WE DESCRIBE A PROTOCOL TO OBTAIN HUMAN MATURE AND FUNCTIONAL NEUTROPHILS AND MACROPHAGES AS EX VIVO MODELS OF X-LINKED CHRONIC GRANULOMATOUS DISEASE (X-CGD). THIS METHOD CAN BE APPLIED TO MODEL THE OTHER GENETIC FORMS OF CGD. WE ALSO DESCRIBE METHODS FOR TESTING THE CHARACTERISTICS AND FUNCTIONS OF NEUTROPHILS AND MACROPHAGES BY MORPHOLOGY, PHAGOCYTOSIS ASSAY, RELEASE OF GRANULE MARKERS OR CYTOKINES, CELL SURFACE MARKERS, AND NADPH OXIDASE ACTIVITY. 2019 17 3530 25 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 18 5608 24 RUNX1-EVI1 DISRUPTS LINEAGE DETERMINATION AND THE CELL CYCLE BY INTERFERING WITH RUNX1 AND EVI1 DRIVEN GENE REGULATORY NETWORKS. HEMATOLOGICAL MALIGNANCIES ARE CHARACTERISED BY A BLOCK IN DIFFERENTIATION, WHICH IN MANY CASES IS CAUSED BY RECURRENT MUTATIONS AFFECTING THE ACTIVITY OF HEMATOPOIETIC TRANSCRIPTION FACTORS. RUNX1-EVI1 IS A FUSION PROTEIN FORMED BY THE T(3;21) TRANSLOCATION LINKING TWO TRANSCRIPTION FACTORS REQUIRED FOR NORMAL HEMATOPOIESIS. RUNX1-EVI1 EXPRESSION IS FOUND IN MYELODYSPLASTIC SYNDROME, SECONDARY ACUTE MYELOID LEUKEMIA, AND BLAST CRISIS OF CHRONIC MYELOID LEUKEMIA; WITH CLINICAL OUTCOMES BEING WORSE THAN IN PATIENTS WITH RUNX1-ETO, RUNX1 OR EVI1 MUTATIONS ALONE. RUNX1-EVI1 IS USUALLY FOUND AS A SECONDARY MUTATION, THEREFORE THE MOLECULAR MECHANISMS UNDERLYING HOW RUNX1-EVI1 ALONE CONTRIBUTES TO POOR PROGNOSIS ARE UNKNOWN. TO ADDRESS THIS QUESTION, WE INDUCED EXPRESSION OF RUNX1-EVI1 IN HEMATOPOIETIC CELLS DERIVED FROM AN EMBRYONIC STEM CELL DIFFERENTIATION MODEL. INDUCTION RESULTED IN DISRUPTION OF THE RUNX1-DEPENDENT ENDOTHELIAL-HEMATOPOIETIC TRANSITION, BLOCKED THE CELL CYCLE AND UNDERMINED CELL FATE DECISIONS IN MULTIPOTENT HEMATOPOIETIC PROGENITOR CELLS. INTEGRATIVE ANALYSES OF GENE EXPRESSION WITH CHROMATIN AND TRANSCRIPTION FACTOR BINDING DATA DEMONSTRATED THAT RUNX1-EVI1 BINDING CAUSED THE RE-DISTRIBUTION OF ENDOGENOUS RUNX1 WITHIN THE GENOME AND INTERFERED WITH BOTH RUNX1 AND EVI1 REGULATED GENE EXPRESSION PROGRAMS. IN SUMMARY, RUNX1-EVI1 EXPRESSION ALONE LEADS TO EXTENSIVE EPIGENETIC REPROGRAMMING WHICH IS INCOMPATIBLE WITH HEALTHY BLOOD PRODUCTION. 2021 19 671 23 BOTHROPS MOOJENI L-AMINO ACID OXIDASE INDUCES APOPTOSIS AND EPIGENETIC MODULATION ON BCR-ABL(+) CELLS. BACKGROUND: RESISTANCE TO APOPTOSIS IN CHRONIC MYELOID LEUKEMIA (CML) IS ASSOCIATED WITH CONSTITUTIVE TYROSINE KINASE ACTIVITY OF THE BCR-ABL ONCOPROTEIN. THE DEREGULATED EXPRESSION OF APOPTOSIS-RELATED GENES AND ALTERATION IN EPIGENETIC MACHINERY MAY ALSO CONTRIBUTE TO APOPTOSIS RESISTANCE IN CML. TYROSINE KINASE INHIBITORS TARGET THE BCR-ABL ONCOPROTEIN AND ARE USED IN CML TREATMENT. THE RESISTANCE OF CML PATIENTS TO TYROSINE KINASE INHIBITORS HAS GUIDED THE SEARCH FOR NEW COMPOUNDS THAT MAY INDUCE APOPTOSIS IN BCR-ABL(+) LEUKEMIC CELLS AND IMPROVE THE DISEASE TREATMENT. METHODS: IN THE PRESENT STUDY, WE INVESTIGATED WHETHER THE L-AMINO ACID OXIDASE ISOLATED FROM BOTHROPS MOOJENI SNAKE VENOM (BMOOLAAO-I) (I) WAS CYTOTOXIC TO BCR-ABL(+) CELL LINES (HL-60.BCR-ABL, K562-S, AND K562-R), HL-60 (ACUTE PROMYELOCYTIC LEUKEMIA) CELLS, THE NON-TUMOR CELL LINE HEK-293, AND PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC); AND (II) AFFECTED EPIGENETIC MECHANISMS, INCLUDING DNA METHYLATION AND MICRORNAS EXPRESSION IN VITRO. RESULTS: BMOOLAAO-I INDUCED ROS PRODUCTION, APOPTOSIS, AND DIFFERENTIAL DNA METHYLATION PATTERN OF REGULATORY APOPTOSIS GENES. THE TOXIN UPREGULATED EXPRESSION OF THE PRO-APOPTOTIC GENES BID AND FADD AND DOWNREGULATED DFFA EXPRESSION IN LEUKEMIC CELL LINES, AS WELL AS INCREASED MIR-16 EXPRESSION - WHOSE MAJOR PREDICTED TARGET IS THE ANTI-APOPTOTIC GENE BCL2 - IN BCR-ABL(+) CELLS. CONCLUSION: BMOOLAAO-I EXERTS SELECTIVE ANTITUMOR ACTION MEDIATED BY H(2)O(2) RELEASE AND INDUCES APOPTOSIS, AND ALTERATIONS IN EPIGENETIC MECHANISMS. THESE RESULTS SUPPORT FUTURE INVESTIGATIONS ON THE EFFECT OF BMOOLAAO-I ON IN VIVO MODELS TO DETERMINE ITS POTENTIAL IN CML THERAPY. 2020 20 5870 26 SUSTAINED KNOCKDOWN OF A DISEASE-CAUSING GENE IN PATIENT-SPECIFIC INDUCED PLURIPOTENT STEM CELLS USING LENTIVIRAL VECTOR-BASED GENE THERAPY. PATIENT-SPECIFIC INDUCED PLURIPOTENT STEM CELLS (IPSCS) HOLD GREAT PROMISE FOR STUDIES ON DISEASE-RELATED DEVELOPMENTAL PROCESSES AND MAY SERVE AS AN AUTOLOGOUS CELL SOURCE FOR FUTURE TREATMENT OF MANY HEREDITARY DISEASES. NEW GENETIC ENGINEERING TOOLS SUCH AS ZINC FINGER NUCLEASES AND TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE ALLOW TARGETED CORRECTION OF MONOGENETIC DISORDERS BUT ARE VERY CUMBERSOME TO ESTABLISH. AIMING AT STUDIES ON THE KNOCKDOWN OF A DISEASE-CAUSING GENE, LENTIVIRAL VECTOR-MEDIATED EXPRESSION OF SHORT HAIRPIN RNAS (SHRNAS) IS A VALUABLE OPTION, BUT IT IS LIMITED BY SILENCING OF THE KNOCKDOWN CONSTRUCT UPON EPIGENETIC REMODELING DURING DIFFERENTIATION. HERE, WE PROPOSE AN APPROACH FOR THE EXPRESSION OF A THERAPEUTIC SHRNA IN DISEASE-SPECIFIC IPSCS USING THIRD-GENERATION LENTIVIRAL VECTORS. TARGETING SEVERE ALPHA-1-ANTITRYPSIN (A1AT) DEFICIENCY, WE OVEREXPRESSED A HUMAN MICRORNA 30 (MIR30)-STYLED SHRNA DIRECTED AGAINST THE PIZ VARIANT OF A1AT, WHICH IS KNOWN TO CAUSE CHRONIC LIVER DAMAGE IN AFFECTED PATIENTS. THIS KNOCKDOWN CASSETTE IS TRACEABLE FROM CLONAL IPSC LINES TO DIFFERENTIATED HEPATIC PROGENY VIA AN ENHANCED GREEN FLUORESCENCE PROTEIN REPORTER EXPRESSED FROM THE SAME RNA-POLYMERASE II PROMOTER. IMPORTANTLY, THE CYTOMEGALOVIRUS I/E ENHANCER CHICKEN BETA ACTIN (CAG) PROMOTER-DRIVEN EXPRESSION OF THIS CONSTRUCT IS SUSTAINED WITHOUT TRANSGENE SILENCING DURING HEPATIC DIFFERENTIATION IN VITRO AND IN VIVO. AT LOW LENTIVIRAL COPY NUMBERS PER GENOME WE CONFIRMED A FUNCTIONAL RELEVANT REDUCTION (-66%) OF INTRACELLULAR PIZ PROTEIN IN HEPATIC CELLS AFTER DIFFERENTIATION OF PATIENT-SPECIFIC IPSCS. IN CONCLUSION, WE HAVE DEMONSTRATED THAT LENTIVIRAL VECTOR-MEDIATED EXPRESSION OF SHRNAS CAN BE EFFICIENTLY USED TO KNOCK DOWN AND FUNCTIONALLY EVALUATE DISEASE-RELATED GENES IN PATIENT-SPECIFIC IPSCS. 2013