1 2376 143 EPIGENETIC REGULATION OF TUMOR NECROSIS FACTOR ALPHA (TNFALPHA) RELEASE IN HUMAN MACROPHAGES BY HIV-1 SINGLE-STRANDED RNA (SSRNA) IS DEPENDENT ON TLR8 SIGNALING. HUMAN MACROPHAGES AT MUCOSAL SITES ARE ESSENTIAL TARGETS FOR ACUTE HIV INFECTION. DURING THE CHRONIC PHASE OF INFECTION, THEY ARE PERSISTENT RESERVOIRS FOR THE AIDS VIRUS. HIV VIRIONS GAIN ENTRY INTO MACROPHAGES FOLLOWING LIGATION OF SURFACE CD4-CCR5 CO-RECEPTORS, WHICH LEADS TO THE RELEASE OF TWO COPIES OF HIV SSRNA. THESE EVENTS LEAD TO REVERSE TRANSCRIPTION AND VIRAL REPLICATION INITIATION. TOLL-LIKE RECEPTORS TLR7 AND TLR8 RECOGNIZE SPECIFIC INTRACELLULAR VIRAL SSRNA SEQUENCES, BUT IN HUMAN ALVEOLAR MACROPHAGES, THEIR INDIVIDUAL ROLES IN TLR-MEDIATED HIV SSRNA RECOGNITION ARE UNCLEAR. IN THE CURRENT STUDY, HIV-1 SSRNA INDUCED TNFALPHA RELEASE IN A DOSE-DEPENDENT MANNER IN ADHERENT HUMAN MACROPHAGES EXPRESSING BOTH INTRACELLULAR TLR7 AND TLR8. THIS RESPONSE WAS REDUCED BY INHIBITING EITHER ENDOCYTOSIS (50 MUM DYNASORE) OR ENDOSOMAL ACIDIFICATION (1 MUG/ML CHLOROQUINE). EITHER MYD88 OR TLR8 GENE KNOCKDOWN WITH RELEVANT SIRNA REDUCED HIV-1 SSRNA-MEDIATED TNFALPHA RELEASE, BUT SILENCING TLR7 HAD NO EFFECT ON THIS RESPONSE. FURTHERMORE, HIV-1 SSRNA INDUCED HISTONE 4 ACETYLATION AT THE TNFALPHA PROMOTER AS WELL AS TRIMETHYLATION OF HISTONE 3 AT LYSINE 4, WHEREAS TLR8 GENE KNOCKDOWN REDUCED THESE EFFECTS. TAKEN TOGETHER IN HUMAN MACROPHAGES, TLR8 BINDS AND INTERNALIZES HIV SSRNA, LEADING TO ENDOSOMAL ACIDIFICATION, CHROMATIN REMODELING, AND INCREASES IN TNFALPHA RELEASE. DRUGS TARGETING MACROPHAGE TLR8-LINKED SIGNALING PATHWAYS MAY MODULATE THE INNATE IMMUNE RESPONSE TO ACUTE HIV INFECTION BY REDUCING VIRAL REPLICATION. 2012 2 6470 31 TLR7 AND TLR8 EXPRESSION INCREASES TUMOR CELL PROLIFERATION AND PROMOTES CHEMORESISTANCE IN HUMAN PANCREATIC CANCER. CHRONIC INFLAMMATION AS AN IMPORTANT EPIGENETIC AND ENVIRONMENTAL FACTOR FOR PUTATIVE TUMORIGENESIS AND TUMOR PROGRESSION MAY BE ASSOCIATED WITH SPECIFIC ACTIVATION OF TOLL-LIKE RECEPTORS (TLR). RECENTLY, CARCINOGENESIS HAS BEEN SUGGESTED TO BE DEPENDENT ON TLR7 SIGNALING. IN THE PRESENT STUDY, WE DETERMINED THE ROLE OF BOTH TLR7 AND TLR8 EXPRESSION AND SIGNALING IN TUMOR CELL PROLIFERATION AND CHEMORESISTANCE IN PANCREATIC CANCER. EXPRESSION OF TLR7/TLR8 IN UICC STAGE I-IV PANCREATIC CANCER, CHRONIC PANCREATITIS, NORMAL PANCREATIC TISSUE AND HUMAN PANCREATIC (PANC1) CANCER CELL LINE WAS EXAMINED. FOR IN VITRO/IN VIVO STUDIES TLR7/TLR8 OVEREXPRESSING PANC1 CELL LINES WERE GENERATED AND ANALYZED FOR EFFECTS OF (UN-)STIMULATED TLR EXPRESSION ON TUMOR CELL PROLIFERATION AND CHEMORESISTANCE. TLR EXPRESSION WAS INCREASED IN PANCREATIC CANCER, WITH STAGE-DEPENDENT UPREGULATION IN ADVANCED TUMORS, COMPARED TO EARLIER STAGES AND CHRONIC PANCREATITIS. STIMULATION OF TLR7/TLR8 OVEREXPRESSING PANC1 CELLS RESULTED IN ELEVATED NF-KAPPAB AND COX-2 EXPRESSION, INCREASED CANCER CELL PROLIFERATION AND REDUCED CHEMOSENSITIVITY. MORE IMPORTANTLY, TLR7/TLR8 EXPRESSION INCREASED TUMOR GROWTH IN VIVO. OUR DATA DEMONSTRATE A STAGE-DEPENDENT UPREGULATION OF BOTH TLR7 AND TLR8 EXPRESSION IN PANCREATIC CANCER. FUNCTIONAL ANALYSIS IN HUMAN PANCREATIC CANCER CELLS POINT TO A SIGNIFICANT ROLE OF BOTH TLRS IN CHRONIC INFLAMMATION-MEDIATED TLR7/TLR8 SIGNALING LEADING TO TUMOR CELL PROLIFERATION AND CHEMORESISTANCE. 2015 3 6294 34 THE PROINFLAMMATORY CYTOKINE TNFALPHA INDUCES DNA DEMETHYLATION-DEPENDENT AND -INDEPENDENT ACTIVATION OF INTERLEUKIN-32 EXPRESSION. IL-32 IS A CYTOKINE INVOLVED IN PROINFLAMMATORY IMMUNE RESPONSES TO BACTERIAL AND VIRAL INFECTIONS. HOWEVER, THE ROLE OF EPIGENETIC EVENTS IN THE REGULATION OF IL-32 GENE EXPRESSION IS UNDERSTUDIED. HERE WE SHOW THAT IL-32 IS REPRESSED BY DNA METHYLATION IN HEK293 CELLS. USING CHIP SEQUENCING, LOCUS-SPECIFIC METHYLATION ANALYSIS, CRISPR/CAS9-MEDIATED GENOME EDITING, AND RT-QPCR (QUANTITATIVE RT-PCR) AND IMMUNOBLOT ASSAYS, WE FOUND THAT SHORT-TERM TREATMENT (A FEW HOURS) WITH THE PROINFLAMMATORY CYTOKINE TUMOR NECROSIS FACTOR ALPHA (TNFALPHA) ACTIVATES IL-32 IN A DNA DEMETHYLATION-INDEPENDENT MANNER. IN CONTRAST, PROLONGED TNFALPHA TREATMENT (SEVERAL DAYS) INDUCED DNA DEMETHYLATION AT THE PROMOTER AND A CPG ISLAND IN THE IL-32 GENE IN A TET (TEN-ELEVEN TRANSLOCATION) FAMILY ENZYME- AND NF-KAPPAB-DEPENDENT MANNER. NOTABLY, THE HYPOMETHYLATION STATUS OF TRANSCRIPTIONAL REGULATORY ELEMENTS IN IL-32 WAS MAINTAINED FOR A LONG TIME (SEVERAL WEEKS), CAUSING ELEVATED IL-32 EXPRESSION EVEN IN THE ABSENCE OF TNFALPHA. CONSIDERING THAT IL-32 CAN, IN TURN, INDUCE TNFALPHA EXPRESSION, WE SPECULATE THAT SUCH FEEDFORWARD EVENTS MAY CONTRIBUTE TO THE TRANSITION FROM AN ACUTE INFLAMMATORY RESPONSE TO CHRONIC INFLAMMATION. 2019 4 6077 35 THE EFFECT OF CD4 RECEPTOR DOWNREGULATION AND ITS DOWNSTREAM SIGNALING MOLECULES ON HIV-1 LATENCY. HIV-1 CAN ESTABLISH A LATENT INFECTION IN MEMORY CD4+T CELLS TO EVADE THE HOST IMMUNE RESPONSE. CD4 MOLECULES CAN ACT NOT ONLY AS THE HIV-1 RECEPTOR FOR ENTRY BUT ALSO AS THE TRIGGER IN AN INTRACELLULAR SIGNALING CASCADE FOR T-CELL ACTIVATION AND PROLIFERATION VIA PROTEIN TYROSINE KINASES. NOVEL CHRONIC HIV-1-INFECTED A3.01-DERIVED (NCHA) CELLS WERE USED TO EXAMINE THE INVOLVEMENT OF CD4 DOWNSTREAM SIGNALING IN HIV-1 LATENCY. CD4 RECEPTORS IN NCHA CELLS WERE DRAMATICALLY DOWNREGULATED ON ITS SURFACE BUT WERE SLIGHTLY DECREASED IN WHOLE-CELL LYSATES. THE EXPRESSION LEVELS OF CD4 DOWNSTREAM SIGNALING MOLECULES, INCLUDING P56(LCK), ZAP-70, LAT, AND C-JUN, WERE SHARPLY DECREASED IN NCHA CELLS. THE LOWERED HISTONE MODIFICATIONS OF H3K4ME3 AND H3K9AC CORRELATED WITH THE DOWNREGULATION OF P56(LCK), ZAP-70, AND LAT IN NCHA CELLS. AP-1 BINDING ACTIVITY WAS ALSO REDUCED IN NCHA CELLS. LAT AND C-JUN SUPPRESSED IN NCHA CELLS WERE HIGHLY INDUCED AFTER PMA TREATMENT. IN EPIGENETIC ANALYSIS, OTHER SIGNAL TRANSDUCTION MOLECULES WHICH ARE ASSOCIATED WITH ACTIVE AND/OR LATENT HIV-1 INFECTION SHOWED NORMAL STATES IN HIV-1 LATENTLY INFECTED CELLS COMPARED TO A3.01 CELLS. IN CONCLUSION, WE DEMONSTRATED THAT THE HIV-1 LATENT STATE IS SUSTAINED BY THE REDUCTION OF DOWNSTREAM SIGNALING MOLECULES VIA THE DOWNREGULATION OF CD4 AND THE ATTENUATED ACTIVITY OF TRANSCRIPTION FACTOR AS AP-1. THE HIV-1 LATENCY MODEL VIA T-CELL DEACTIVATION MAY PROVIDE SOME CLUES FOR THE DEVELOPMENT OF THE NEW ANTIRESERVOIR THERAPY. 2011 5 1966 32 EPIGENETIC ALTERATION OF PRKCDBP IN COLORECTAL CANCERS AND ITS IMPLICATION IN TUMOR CELL RESISTANCE TO TNFALPHA-INDUCED APOPTOSIS. PURPOSE: PRKCDBP IS A PUTATIVE TUMOR SUPPRESSOR IN WHICH ALTERATION HAS BEEN OBSERVED IN SEVERAL HUMAN CANCERS. WE INVESTIGATED EXPRESSION AND FUNCTION OF PRKCDBP IN COLORECTAL CELLS AND TISSUES TO EXPLORE ITS CANDIDACY AS A SUPPRESSOR IN COLORECTAL TUMORIGENESIS. EXPERIMENTAL DESIGN: EXPRESSION AND METHYLATION STATUS OF PRKCDBP AND ITS EFFECT ON TUMOR GROWTH WERE EVALUATED. TRANSCRIPTIONAL REGULATION BY NF-KAPPAB SIGNALING WAS DEFINED BY LUCIFERASE REPORTER AND CHROMATIN IMMUNOPRECIPITATION ASSAYS. RESULTS: PRKCDBP EXPRESSION WAS HARDLY DETECTABLE IN 29 OF 80 (36%) PRIMARY TUMORS AND 11 OF 19 (58%) CELL LINES, AND ITS ALTERATION CORRELATED WITH TUMOR STAGE AND GRADE. PROMOTER HYPERMETHYLATION WAS COMMONLY FOUND IN CANCERS. PRKCDBP EXPRESSION INDUCED THE G(1) CELL-CYCLE ARREST AND INCREASED CELLULAR SENSITIVITY TO VARIOUS APOPTOTIC STRESSES. PRKCDBP WAS INDUCED BY TNFALPHA, AND ITS LEVEL CORRELATED WITH TUMOR CELL SENSITIVITY TO TNFALPHA-INDUCED APOPTOSIS. PRKCDBP INDUCTION BY TNFALPHA WAS DISRUPTED BY BLOCKING NF-KAPPAB SIGNALING WHILE IT WAS ENHANCED BY RELA TRANSFECTION. THE PRKCDBP PROMOTER ACTIVITY WAS INCREASED IN RESPONSE TO TNFALPHA, AND THIS RESPONSE WAS ABOLISHED BY DISRUPTION OF A KAPPAB SITE IN THE PROMOTER. PRKCDBP DELAYED THE FORMATION AND GROWTH OF XENOGRAFT TUMORS AND IMPROVED TUMOR RESPONSE TO TNFALPHA-INDUCED APOPTOSIS. CONCLUSIONS: PRKCDBP IS A PROAPOPTOTIC TUMOR SUPPRESSOR WHICH IS COMMONLY ALTERED IN COLORECTAL CANCER BY PROMOTER HYPERMETHYLATION, AND ITS GENE TRANSCRIPTION IS DIRECTLY ACTIVATED BY NF-KAPPAB IN RESPONSE TO TNFALPHA. THIS SUGGESTS THAT PRKCDBP INACTIVATION MAY CONTRIBUTE TO TUMOR PROGRESSION BY REDUCING CELLULAR SENSITIVITY TO TNFALPHA AND OTHER STRESSES, PARTICULARLY UNDER CHRONIC INFLAMMATORY MICROENVIRONMENT. 2011 6 1806 43 EFFECT OF TRANSCRIPTION INHIBITION AND GENERATION OF SUPPRESSIVE VIRAL NON-CODING RNAS. BACKGROUND: HIV-1 PATIENTS RECEIVING COMBINATION ANTIRETROVIRAL THERAPY (CART) SURVIVE INFECTION BUT REQUIRE LIFE-LONG ADHERENCE AT HIGH EXPENSE. IN CHRONIC CART-TREATED PATIENTS WITH UNDETECTABLE VIRAL TITERS, CELL-ASSOCIATED VIRAL RNA IS STILL DETECTABLE, POINTING TO LOW-LEVEL VIRAL TRANSCRIPTIONAL LEAKINESS. TO DATE, THERE ARE NO FDA-APPROVED DRUGS AGAINST HIV-1 TRANSCRIPTION. WE HAVE PREVIOUSLY SHOWN THAT F07#13, A THIRD GENERATION TAT PEPTIDE MIMETIC WITH COMPETITIVE ACTIVITY AGAINST CDK9/T1-TAT BINDING SITES, INHIBITS HIV-1 TRANSCRIPTION IN VITRO AND IN VIVO. RESULTS: HERE, WE DEMONSTRATE THAT INCREASING CONCENTRATIONS OF F07#13 (0.01, 0.1, 1 MICROM) CAUSE A DECREASE IN TAT LEVELS IN A DOSE-DEPENDENT MANNER BY INHIBITING THE CDK9/T1-TAT COMPLEX FORMATION AND SUBSEQUENT UBIQUITIN-MEDIATED TAT SEQUESTRATION AND DEGRADATION. OUR DATA INDICATE THAT COMPLEXES I AND IV CONTAIN DISTINCT PATTERNS OF UBIQUITINATED TAT AND THAT TRANSCRIPTIONAL INHIBITION INDUCED BY F07#13 CAUSES AN OVERALL REDUCTION IN TAT LEVELS. THIS REDUCTION MAY BE TRIGGERED BY F07#13 BUT ULTIMATELY IS MEDIATED BY TAR-GAG VIRAL RNAS THAT BIND SUPPRESSIVE TRANSCRIPTION FACTORS (SIMILAR TO 7SK, NRON, HOTAIR, AND XIST LNCRNAS) TO ENHANCE TRANSCRIPTIONAL GENE SILENCING AND LATENCY. THESE RNAS COMPLEX WITH PRC2, SIN3A, AND CUL4B, RESULTING IN EPIGENETIC MODIFICATIONS. FINALLY, WE OBSERVED AN F07#13-MEDIATED DECREASE OF VIRAL BURDEN BY TARGETING THE R REGION OF THE LONG TERMINAL REPEAT (HIV-1 PROMOTER REGION, LTR), PROMOTING BOTH PAUSED POLYMERASES AND INCREASED EFFICIENCY OF CRISPR/CAS9 EDITING IN INFECTED CELLS. THIS IMPLIES THAT GENE EDITING MAY BE BEST PERFORMED UNDER A REPRESSED TRANSCRIPTIONAL STATE. CONCLUSIONS: COLLECTIVELY, OUR RESULTS INDICATE THAT F07#13, WHICH CAN TERMINATE RNA POLYMERASE II AT DISTINCT SITES, CAN GENERATE SCAFFOLD RNAS, WHICH MAY ASSEMBLE INTO SPECIFIC SETS OF "RNA MACHINES" THAT CONTRIBUTE TO GENE REGULATION. IT REMAINS TO BE SEEN WHETHER THESE EFFECTS CAN ALSO BE SEEN IN VARIOUS CLADES THAT HAVE VARYING PROMOTER STRENGTH, MUTANT LTRS, AND IN PATIENT SAMPLES. 2019 7 4696 32 NF-KAPPAB REPRESSES RETINOIC ACID RECEPTOR-MEDIATED GPRC5A TRANSACTIVATION IN LUNG EPITHELIAL CELLS TO PROMOTE NEOPLASIA. CHRONIC INFLAMMATION IS ASSOCIATED WITH LUNG TUMORIGENESIS, IN WHICH NF-KAPPAB-MEDIATED EPIGENETIC REGULATION PLAYS A CRITICAL ROLE. LUNG TUMOR SUPPRESSOR G PROTEIN-COUPLED RECEPTOR, FAMILY C, MEMBER 5A (GPRC5A), IS REPRESSED IN MOST NON-SMALL CELL LUNG CANCER (NSCLC); HOWEVER, THE MECHANISMS REMAIN UNCLEAR. HERE, WE SHOW THAT NF-KAPPAB ACTS AS A TRANSCRIPTIONAL REPRESSOR IN SUPPRESSION OF GPRC5A. NF-KAPPAB INDUCED GPRC5A REPRESSION BOTH IN VITRO AND IN VIVO. INTRIGUINGLY, TRANSACTIVATION OF NF-KAPPAB DOWNSTREAM TARGETS WAS NOT REQUIRED, BUT THE TRANSACTIVATION DOMAIN OF RELA/P65 WAS REQUIRED FOR GPRC5A REPRESSION. NF-KAPPAB DID NOT BIND TO ANY POTENTIAL CIS-ELEMENT IN THE GPRC5A PROMOTER. INSTEAD, P65 WAS COMPLEXED WITH RETINOIC ACID RECEPTOR ALPHA/BETA (RARALPHA/BETA) AND RECRUITED TO THE RA RESPONSE ELEMENT SITE AT THE GPRC5A PROMOTER, RESULTING IN DISRUPTED RNA POLYMERASE II COMPLEXING AND SUPPRESSED TRANSCRIPTION. NOTABLY, PHOSPHORYLATION ON SERINE 276 OF P65 WAS REQUIRED FOR INTERACTION WITH RARALPHA/BETA AND REPRESSION OF GPRC5A. MOREOVER, NF-KAPPAB-MEDIATED EPIGENETIC REPRESSION WAS THROUGH SUPPRESSION OF ACETYLATED HISTONE H3K9 (H3K9AC), BUT NOT DNA METHYLATION OF THE CPG ISLANDS, AT THE GPRC5A PROMOTER. CONSISTENTLY, A HISTONE DEACETYLASE INHIBITOR, BUT NOT DNA METHYLATION INHIBITOR, RESTORED GPRC5A EXPRESSION IN NSCLC CELLS. THUS, NF-KAPPAB INDUCES TRANSCRIPTIONAL REPRESSION OF GPRC5A VIA A COMPLEX WITH RARALPHA/BETA AND MEDIATES EPIGENETIC REPRESSION VIA SUPPRESSION OF H3K9AC. 2023 8 3128 41 GIPC-REGULATED IGFBP-3 PROMOTES HSC MIGRATION IN VITRO AND PORTAL HYPERTENSION IN VIVO THROUGH A BETA1-INTEGRIN PATHWAY. BACKGROUND & AIMS: TRANSFORMING GROWTH FACTOR (TGF-BETA)-INDUCED ACTIVATION OF QUIESCENT HEPATIC STELLATE CELLS (HSCS) AND THEIR TRANSFORMATION TO MYOFIBROBLASTS IS A KEY EVENT IN LIVER FIBROSIS AND PORTAL HYPERTENSION. GIPC (ALSO REFERRED TO AS SYNECTIN) IS A DOWNSTREAM SIGNAL ACTIVATION MOLECULE OF TGF-BETA AND OTHER RECEPTORS. IN THIS STUDY, WE SOUGHT TO IDENTIFY NOVEL GENES TARGETED BY TGF-BETA AND GIPC AND ELUCIDATE IF AND HOW THEY MAY CONTRIBUTE TO LIVER FIBROSIS. METHODS: WE PERFORMED SEQUENTIAL MESSENGER RNA SEQUENCING ANALYSIS ON TGF-BETA-STIMULATED HSCS AND THEN ON TGF-BETA-STIMULATED HSCS IN THE PRESENCE AND ABSENCE OF GIPC ALSO REFERRED TO AS SYNECTIN (GIPC) KNOCKDOWN. INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-3 (IGFBP-3) TRANSPORT PROTEIN EMERGED AS A TOP ACTIVATION TARGET OF BOTH TGF-BETA AND GIPC. QUANTITATIVE POLYMERASE CHAIN REACTION, ENZYME-LINKED IMMUNOSORBENT ASSAY, TARGETED CHROMATIN IMMUNOPRECIPITATION, AND WESTERN BLOT ANALYSIS WERE DONE FOR FURTHER CONFIRMATION. RESULTS: IGFBP-3, AN INSULIN GROWTH FACTOR TRANSPORT PROTEIN, EMERGED AS A TOP ACTIVATION TARGET OF BOTH TGF-BETA AND GIPC, WHICH WAS CONFIRMED BY QUANTITATIVE POLYMERASE CHAIN REACTION, ENZYME-LINKED IMMUNOSORBENT ASSAY, AND WESTERN BLOT ANALYSIS. TARGETED CHROMATIN IMMUNOPRECIPITATION SHOWED THAT GIPC INCREASES THE HISTONE 3 LYSINE 27 (H3K27) ACETYLATION ACTIVATING MARK AND CONCURRENTLY DECREASES THE H3K27 INHIBITORY TRIMETHYLATION (H3K27M3) MARK, PROVIDING AN EPIGENETIC CORRELATE TO THE GENE REGULATION CHANGES. IN VIVO, GLOBAL KNOCKOUT OF IGFBP-3 MICE RESULTED IN ATTENUATION OF HSC ACTIVATION MARKERS AND ATTENUATION OF PORTAL PRESSURE IN RESPONSE TO CHRONIC LIVER INJURY MODELS. ANALYSIS OF SERUM LEVELS FROM CIRRHOTIC PATIENTS ALSO SHOWED AN IGFBP-3 INCREASE OF MORE THAN 2-FOLD COMPARED WITH HEALTHY CONTROLS. FINALLY, IN VITRO MECHANISM STUDIES SHOWED THAT IGFBP-3 PROMOTES HSC MIGRATION THROUGH INTEGRIN-DEPENDENT PHOSPHORYLATION OF PROTEIN KINASE B. CONCLUSIONS: TGF-BETA UP-REGULATES IGFBP-3 THROUGH GIPC, LEADING TO INCREASED HSC MIGRATION IN VITRO AND PROMOTES PORTAL HYPERTENSION IN VIVO. THESE STUDIES SUPPORT THE ROLE OF IGFBP-3 AS A POTENTIAL PATHOPHYSIOLOGIC TARGET OR BIOMARKER IN CHRONIC LIVER DISEASE. 2020 9 5009 21 PERK IS A CRITICAL METABOLIC HUB FOR IMMUNOSUPPRESSIVE FUNCTION IN MACROPHAGES. CHRONIC INFLAMMATION TRIGGERS COMPENSATORY IMMUNOSUPPRESSION TO STOP INFLAMMATION AND MINIMIZE TISSUE DAMAGE. STUDIES HAVE DEMONSTRATED THAT ENDOPLASMIC RETICULUM (ER) STRESS AUGMENTS THE SUPPRESSIVE PHENOTYPES OF IMMUNE CELLS; HOWEVER, THE MOLECULAR MECHANISMS UNDERPINNING THIS PROCESS AND HOW IT LINKS TO THE METABOLIC REPROGRAMMING OF IMMUNOSUPPRESSIVE MACROPHAGES REMAIN ELUSIVE. IN THE PRESENT STUDY, WE REPORT THAT THE HELPER T CELL 2 CYTOKINE INTERLEUKIN-4 AND THE TUMOR MICROENVIRONMENT INCREASE THE ACTIVITY OF A PROTEIN KINASE RNA-LIKE ER KINASE (PERK)-SIGNALING CASCADE IN MACROPHAGES AND PROMOTE IMMUNOSUPPRESSIVE M2 ACTIVATION AND PROLIFERATION. LOSS OF PERK SIGNALING IMPEDED MITOCHONDRIAL RESPIRATION AND LIPID OXIDATION CRITICAL FOR M2 MACROPHAGES. PERK ACTIVATION MEDIATED THE UPREGULATION OF PHOSPHOSERINE AMINOTRANSFERASE 1 (PSAT1) AND SERINE BIOSYNTHESIS VIA THE DOWNSTREAM TRANSCRIPTION FACTOR ATF-4. INCREASED SERINE BIOSYNTHESIS RESULTED IN ENHANCED MITOCHONDRIAL FUNCTION AND ALPHA-KETOGLUTARATE PRODUCTION REQUIRED FOR JMJD3-DEPENDENT EPIGENETIC MODIFICATION. INHIBITION OF PERK SUPPRESSED MACROPHAGE IMMUNOSUPPRESSIVE ACTIVITY AND COULD ENHANCE THE EFFICACY OF IMMUNE CHECKPOINT PROGRAMMED CELL DEATH PROTEIN 1 INHIBITION IN MELANOMA. OUR FINDINGS DELINEATE A PREVIOUSLY UNDESCRIBED CONNECTION BETWEEN PERK SIGNALING AND PSAT1-MEDIATED SERINE METABOLISM CRITICAL FOR PROMOTING IMMUNOSUPPRESSIVE FUNCTION IN M2 MACROPHAGES. 2022 10 3891 43 KSHV VIL-6 ENHANCES INFLAMMATORY RESPONSES BY EPIGENETIC REPROGRAMMING. KAPOSI SARCOMA-ASSOCIATED HERPESVIRUS (KSHV) INFLAMMATORY CYTOKINE SYNDROME (KICS) IS A NEWLY DESCRIBED CHRONIC INFLAMMATORY DISEASE CONDITION CAUSED BY KSHV INFECTION AND IS CHARACTERIZED BY HIGH KSHV VIRAL LOAD AND SUSTAINED ELEVATIONS OF SERUM KSHV-ENCODED IL-6 (VIL-6) AND HUMAN IL-6 (HIL-6). KICS HAS SIGNIFICANT IMMORTALITY AND POSSESSES GREATER RISKS OF HAVING OTHER COMPLICATIONS, WHICH INCLUDE MALIGNANCIES. ALTHOUGH PROLONGED INFLAMMATORY VIL-6 EXPOSURE BY PERSISTENT KSHV INFECTION IS EXPECTED TO HAVE KEY ROLES IN SUBSEQUENT DISEASE DEVELOPMENT, THE BIOLOGICAL EFFECTS OF PROLONGED VIL-6 EXPOSURE REMAIN ELUSIVE. USING THIOL-LINKED ALKYLATION FOR THE METABOLIC SEQUENCING AND CLEAVAGE UNDER TARGET & RELEASE USING NUCLEASE ANALYSIS, WE STUDIED THE EFFECT OF PROLONGED VIL-6 EXPOSURE IN CHROMATIN LANDSCAPE AND RESULTING CYTOKINE PRODUCTION. THE STUDIES SHOWED THAT PROLONGED VIL-6 EXPOSURE INCREASED BROMODOMAIN CONTAINING 4 (BRD4) AND HISTONE H3 LYSINE 27 ACETYLATION CO-OCCUPANCIES ON CHROMATIN, AND THE RECRUITMENT SITES WERE FREQUENTLY CO-LOCALIZED WITH POISED RNAPII WITH ASSOCIATED ENZYMES. INCREASED BRD4 RECRUITMENT ON PROMOTERS WAS ASSOCIATED WITH INCREASED AND PROLONGED NF-KB P65 BINDING AFTER THE LIPOPOLYSACCHARIDE STIMULATION. THE P65 BINDING RESULTED IN QUICKER AND SUSTAINED TRANSCRIPTION BURSTS FROM THE PROMOTERS; THIS MECHANISM INCREASED TOTAL AMOUNTS OF HIL-6 AND IL-10 IN TISSUE CULTURE. PRETREATMENT WITH THE BRD4 INHIBITOR, OTX015, ELIMINATED THE ENHANCED INFLAMMATORY CYTOKINE PRODUCTION. THESE FINDINGS SUGGEST THAT PERSISTENT VIL-6 EXPOSURE MAY ESTABLISH A CHROMATIN LANDSCAPE FAVORABLE FOR THE REACTIVATION OF INFLAMMATORY RESPONSES IN MONOCYTES. THIS EPIGENETIC MEMORY MAY EXPLAIN THE GREATER RISK OF CHRONIC INFLAMMATORY DISEASE DEVELOPMENT IN KSHV-INFECTED INDIVIDUALS. AUTHOR SUMMARY: COMBINED AND CONTINUOUS CYTOKINE STIMULATION TRIGGERS TRANSCRIPTION REPROGRAMMING AND IS OFTEN USED FOR SPECIFIC TISSUE DEVELOPMENT. CONTINUOUS VIL-6 EXPOSURE OCCURS IN KSHV-INFECTED PATIENTS AND LEADS TO INFLAMMATORY CYTOKINE STORM WITH HIGH MORTALITY. HOWEVER, POSSIBLE EPIGENETIC REPROGRAMMING BY THE VIL-6 AND ITS ASSOCIATION WITH PATHOGENESIS REMAIN UNCLEAR. HERE WE DEMONSTRATE THE ESTABLISHMENT OF A NEW CHROMATIN LANDSCAPE MEDIATED BY BRD4 THROUGH PROLONGED VIL-6 EXPOSURE WHICH CONTRIBUTES TO MORE ROBUST AND RAPID TRANSCRIPTION AND INCREASED CYTOKINES PRODUCTION. INHIBITION OF BRD4 SUPPRESSED THIS INFLAMMATORY RESPONSE. OUR RESULTS INDICATE THAT TARGETING THE EPIGENETIC EFFECT OF VIRAL CYTOKINES MAY LEAD TO NOVEL THERAPIES FOR KSHV-INDUCED INFLAMMATORY CYTOKINE STORMS. 2023 11 1461 33 DISRUPTION OF RCAN1.4 EXPRESSION MEDIATED BY YY1/HDAC2 MODULATES CHRONIC RENAL ALLOGRAFT INTERSTITIAL FIBROSIS. CHRONIC ALLOGRAFT DYSFUNCTION (CAD) IS A MAJOR FACTOR THAT HINDERS KIDNEY TRANSPLANT SURVIVAL IN THE LONG RUN. EPITHELIAL-MESENCHYMAL TRANSITION (EMT) HAS BEEN CONFIRMED TO SIGNIFICANTLY CONTRIBUTE TO INTERSTITIAL FIBROSIS/TUBULAR ATROPHY (IF/TA), WHICH IS THE MAIN HISTOPATHOLOGICAL FEATURE OF CAD. ABERRANT EXPRESSION OF THE REGULATOR OF CALCINEURIN 1 (RCAN1), RECOGNIZED AS AN ENDOGENOUS INHIBITOR OF THE CALCINEURIN PHOSPHATASE, HAS BEEN SHOWN TO BE EXTENSIVELY INVOLVED IN VARIOUS KIDNEY DISEASES. HOWEVER, IT REMAINS UNCLEAR HOW RCAN1.4 REGULATES IF/TA FORMATION IN CAD PATIENTS. HEREIN, AN IN VIVO MOUSE RENAL TRANSPLANTATION MODEL AND AN IN VITRO MODEL OF HUMAN RENAL TUBULAR EPITHELIAL CELLS (HK-2) TREATED WITH TUMOR NECROSIS FACTOR-ALPHA (TNF-ALPHA) WERE EMPLOYED. OUR RESULTS PROVED THAT RCAN1.4 EXPRESSION WAS DECREASED IN VIVO AND IN VITRO, IN ADDITION TO THE UP-REGULATION OF YIN YANG 1 (YY1), A TRANSCRIPTION FACTOR THAT HAS BEEN REPORTED TO CONVEY MULTIPLE FUNCTIONS IN CHRONIC KIDNEY DISEASE (CKD). KNOCKING IN OF RCAN1.4 EFFICIENTLY ATTENUATED CHRONIC RENAL ALLOGRAFT INTERSTITIAL FIBROSIS IN VIVO AND INHIBITED TNF-ALPHA-INDUCED EMT IN VITRO THROUGH REGULATING ANTI-OXIDATIVE STRESS AND THE CALCINEURIN/NUCLEAR FACTOR OF ACTIVATED T CELLS CYTOPLASMIC 1 (NFATC1) SIGNALING PATHWAY. IN ADDITION, SUPPRESSION OF YY1 MEDIATED BY SHRNA OR SIRNA ALLEVIATED TNF-ALPHA-INDUCED EMT THROUGH ABOLISHING REACTIVE SPECIES PARTLY IN AN RCAN1.4-DEPENDENT MANNER. NOTABLY, WE CONFIRMED THAT YY1 NEGATIVELY REGULATED RCAN1.4 TRANSCRIPTION BY DIRECTLY INTERACTING WITH THE RCAN1.4 PROMOTER. IN ADDITION, HISTONE DEACETYLASE 2 (HDAC2) INTERACTED WITH YY1 TO FORM A MULTI-MOLECULAR COMPLEX, WHICH WAS INVOLVED IN TNF-ALPHA-INDUCED RCAN1.4 TRANSCRIPTIONAL REPRESSION. THEREFORE, RCAN1.4 IS SUGGESTED TO BE MODULATED BY THE YY1/HDAC2 TRANSCRIPTION REPRESSOR COMPLEX IN AN EPIGENETIC MANNER, WHICH IS A MEDIATED NEPHROPROTECTIVE EFFECT PARTLY THROUGH MODULATING O2?- GENERATION AND THE CALCINEURIN/NFATC1 SIGNALING PATHWAY. THUS, THE YY1-RCAN1.4 AXIS CONSTITUTES AN INNOVATIVE TARGET FOR IF/TA TREATMENT IN CAD PATIENTS. 2023 12 1334 30 DEREGULATION OF AIOLOS EXPRESSION IN CHRONIC LYMPHOCYTIC LEUKEMIA IS ASSOCIATED WITH EPIGENETIC MODIFICATIONS. CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) IS CHARACTERIZED BY A CLONAL ACCUMULATION OF MATURE NEOPLASTIC B CELLS THAT ARE RESISTANT TO APOPTOSIS. AIOLOS, A MEMBER OF THE IKAROS FAMILY OF ZINC-FINGER TRANSCRIPTION FACTORS, PLAYS AN IMPORTANT ROLE IN THE CONTROL OF MATURE B LYMPHOCYTE DIFFERENTIATION AND MATURATION. IN THIS STUDY, WE SHOWED THAT AIOLOS EXPRESSION IS UP-REGULATED IN B-CLL CELLS. THIS OVEREXPRESSION DOES NOT IMPLICATE ISOFORM IMBALANCE OR DISTURB AIOLOS SUBCELLULAR LOCALIZATION. THE CHROMATIN STATUS AT THE AIOLOS PROMOTER IN CLL IS DEFINED BY THE DEMETHYLATION OF DNA AND AN ENRICHMENT OF EUCHROMATIN ASSOCIATED HISTONE MARKERS, SUCH AS THE DIMETHYLATION OF THE LYSINE 4 ON HISTONE H3. THESE EPIGENETIC MODIFICATIONS SHOULD ALLOW ITS UPSTREAM EFFECTORS, SUCH AS NUCLEAR FACTOR-KAPPAB, CONSTITUTIVELY ACTIVATED IN CLL, TO GAIN ACCESS TO PROMOTER, RESULTING UP-REGULATION OF AIOLOS. TO DETERMINE THE CONSEQUENCES OF AIOLOS DEREGULATION IN CLL, WE ANALYZED THE EFFECTS OF AIOLOS OVEREXPRESSION OR DOWN-REGULATION ON APOPTOSIS. AIOLOS IS INVOLVED IN CELL SURVIVAL BY REGULATING THE EXPRESSION OF SOME BCL-2 FAMILY MEMBERS. OUR RESULTS STRONGLY SUGGEST THAT AIOLOS DEREGULATION BY EPIGENETIC MODIFICATIONS MAY BE A HALLMARK OF CLL. 2011 13 2432 29 EPIGENETIC SILENCING OF MIR-708 ENHANCES NF-KAPPAB SIGNALING IN CHRONIC LYMPHOCYTIC LEUKEMIA. MICRORNAS (MIRNAS) ARE POST-TRANSCRIPTIONAL REGULATORS OF GENE EXPRESSION AND THEIR DEREGULATION IS INVOLVED IN TUMOR DEVELOPMENT. EPIGENETIC GENE SILENCING IN CANCER BY DNA METHYLATION CONTRIBUTES TO THE SILENCING OF TUMOR-SUPPRESSOR GENES, INCLUDING MIRNAS. WE HAVE RECENTLY SHOWN THAT THE PROMOTER OF MIR-708 IS ABERRANTLY METHYLATED IN CHRONIC LYMPHOCYTIC LEUKEMIA (CLL). TO CHARACTERIZE THE MOLECULAR SIGNALING NETWORKS THAT ARE INFLUENCED BY MIR-708, WE PERFORMED A LUCIFERASE-BASED SCREEN EVALUATING THE EFFECTS OF ECTOPIC MIR-708 EXPRESSION ON LEUKEMIA-RELEVANT SIGNALING PATHWAYS. WE FOUND THAT MIR-708 STRONGLY REPRESSED NF-KAPPAB SIGNALING, A PATHWAY KNOWN TO BE DEREGULATED IN CLL. AMONG THE PREDICTED MIR-708 TARGETS WAS IKKBETA (INHIBITOR OF KAPPA LIGHT POLYPEPTIDE GENE ENHANCER IN B CELLS, KINASE-BETA/IKBKB), A KEY KINASE FACILITATING NF-KAPPAB SIGNALING. WE VALIDATED THE INTERACTION OF MIR-708 WITH THE 3'-UNTRANSLATED REGION OF IKKBETA AND FOUND THAT MIR-708 OVEREXPRESSION REPRESSES ENDOGENOUS IKKBETA. PHOSPHORYLATION OF THE IKKBETA TARGET IKAPPABALPHA AND EXPRESSION OF KNOWN NF-KAPPAB TARGET GENES WERE IMPAIRED BY MIR-708. FURTHERMORE, WE IDENTIFIED AN ENHANCER REGION DOWNSTREAM OF THE MIR-708 PROMOTER THAT DISPLAYS A DISTINCT DNA METHYLATION STATUS IN CLL. HIGH ENHANCER METHYLATION IS SIGNIFICANTLY CORRELATED WITH LOWER MIR-708 EXPRESSION AND IS PREDOMINANTLY FOUND IN PATIENTS WITH POOR PROGNOSIS AND SHORTER TIME TO TREATMENT. THESE RESULTS DEMONSTRATE THAT MIR-708 REGULATES THE NF-KAPPAB PATHWAY BY TARGETING IKKBETA, AND THAT METHYLATION OF A KEY ENHANCER REGION CONTRIBUTES TO ITS SUPPRESSION IN CLL. 2015 14 984 34 CHRONIC PSYCHOLOGICAL STRESS ALTERS GENE EXPRESSION IN RAT COLON EPITHELIAL CELLS PROMOTING CHROMATIN REMODELING, BARRIER DYSFUNCTION AND INFLAMMATION. CHRONIC STRESS IS COMMONLY ASSOCIATED WITH ENHANCED ABDOMINAL PAIN (VISCERAL HYPERSENSITIVITY), BUT THE CELLULAR MECHANISMS UNDERLYING HOW CHRONIC STRESS INDUCES VISCERAL HYPERSENSITIVITY ARE POORLY UNDERSTOOD. IN THIS STUDY, WE EXAMINED CHANGES IN GENE EXPRESSION IN COLON EPITHELIAL CELLS FROM A RAT MODEL USING RNA-SEQUENCING TO EXAMINE STRESS-INDUCED CHANGES TO THE TRANSCRIPTOME. FOLLOWING CHRONIC STRESS, THE MOST SIGNIFICANTLY UP-REGULATED GENES INCLUDED ATG16L1, COQ10B, DCAF13, NAT2, PTBP2, RRAS2, SPINK4 AND DOWN-REGULATED GENES INCLUDING ABAT, CITED2, CNNM2, DAB2IP, PLEKHM1, SCD2, AND TAB2. THE PRIMARY ALTERED BIOLOGICAL PROCESSES REVEALED BY NETWORK ENRICHMENT ANALYSIS WERE INFLAMMATION/IMMUNE RESPONSE, TISSUE MORPHOGENESIS AND DEVELOPMENT, AND NUCLEOSOME/CHROMATIN ASSEMBLY. THE MOST SIGNIFICANTLY DOWN-REGULATED PROCESS WAS THE DIGESTIVE SYSTEM DEVELOPMENT/FUNCTION, WHEREAS THE MOST SIGNIFICANTLY UP-REGULATED PROCESSES WERE INFLAMMATORY RESPONSE, ORGANISMAL INJURY, AND CHROMATIN REMODELING MEDIATED BY H3K9 METHYLATION. FURTHERMORE, A SUBPOPULATION OF STRESSED RATS DEMONSTRATED VERY SIGNIFICANTLY ALTERED GENE EXPRESSION AND TRANSCRIPT ISOFORMS, ENRICHED FOR THE DIFFERENTIAL EXPRESSION OF GENES INVOLVED IN THE INFLAMMATORY RESPONSE, INCLUDING UPREGULATION OF CYTOKINE AND CHEMOKINE RECEPTOR GENE EXPRESSION COUPLED WITH DOWNREGULATION OF EPITHELIAL ADHERENS AND TIGHT JUNCTION MRNAS. IN SUMMARY, THESE FINDINGS SUPPORT THAT CHRONIC STRESS IS ASSOCIATED WITH INCREASED LEVELS OF CYTOKINES AND CHEMOKINES, THEIR DOWNSTREAM SIGNALING PATHWAYS COUPLED TO DYSREGULATION OF INTESTINAL CELL DEVELOPMENT AND FUNCTION. EPIGENETIC REGULATION OF CHROMATIN REMODELING LIKELY PLAYS A PROMINENT ROLE IN THIS PROCESS. RESULTS ALSO SUGGEST THAT SUPER ENHANCERS PLAY A PRIMARY ROLE IN CHRONIC STRESS-ASSOCIATED INTESTINAL BARRIER DYSFUNCTION. 2022 15 26 31 A 6-ALKYLSALICYLATE HISTONE ACETYLTRANSFERASE INHIBITOR INHIBITS HISTONE ACETYLATION AND PRO-INFLAMMATORY GENE EXPRESSION IN MURINE PRECISION-CUT LUNG SLICES. LYSINE ACETYLATIONS ARE POST-TRANSLATIONAL MODIFICATIONS OF CELLULAR PROTEINS, THAT ARE CRUCIAL IN THE REGULATION OF MANY CELLULAR PROCESSES. LYSINE ACETYLATIONS ON HISTONE PROTEINS ARE PART OF THE EPIGENETIC CODE REGULATING GENE EXPRESSION AND ARE INSTALLED BY HISTONE ACETYLTRANSFERASES. OBSERVATIONS THAT INFLAMMATORY LUNG DISEASES, SUCH AS ASTHMA AND CHRONIC OBSTRUCTIVE PULMONARY DISEASE, ARE CHARACTERIZED BY INCREASED HISTONE ACETYLTRANSFERASE ACTIVITY INDICATE THAT DEVELOPMENT OF SMALL MOLECULE INHIBITORS FOR THESE ENZYMES MIGHT BE A VALUABLE APPROACH TOWARDS NEW THERAPIES FOR THESE DISEASES. THE 6-ALKYLSALICYLATE MG149 IS A CANDIDATE TO EXPLORE THIS HYPOTHESIS BECAUSE IT HAS BEEN DEMONSTRATED TO INHIBIT THE MYST TYPE HISTONE ACETYLTRANSFERASES. IN THIS STUDY, WE DETERMINED THE K(I) VALUE FOR INHIBITION OF THE MYST TYPE HISTONE ACETYLTRANSFERASE KAT8 BY MG149 TO BE 39 +/- 7.7 MUM. UPON INVESTIGATING WHETHER THE INHIBITION OF HISTONE ACETYLTRANSFERASES BY MG149 CORRELATES WITH INHIBITION OF HISTONE ACETYLATION IN MURINE PRECISION-CUT LUNG SLICES, INHIBITION OF ACETYLATION WAS OBSERVED USING AN LC-MS/MS BASED ASSAY ON HISTONE H4 RES 4-17, WHICH CONTAINS THE TARGET LYSINE OF KAT8. FOLLOWING UP ON THIS, UPON TREATMENT WITH MG149, REDUCED PRO-INFLAMMATORY GENE EXPRESSION WAS OBSERVED IN LIPOPOLYSACCHARIDE AND INTERFERON GAMMA STIMULATED MURINE PRECISION-CUT LUNG SLICES. BASED ON THIS, WE PROPOSE THAT 6-ALKYLSALICYLATES SUCH AS MG149 HAVE POTENTIAL FOR DEVELOPMENT TOWARDS APPLICATIONS IN THE TREATMENT OF INFLAMMATORY LUNG DISEASES. 2017 16 3049 25 GENOME-WIDE ANALYSIS REVEALS ZINC TRANSPORTER ZIP9 REGULATED BY DNA METHYLATION PROMOTES RADIATION-INDUCED SKIN FIBROSIS VIA THE TGF-BETA SIGNALING PATHWAY. RADIATION-INDUCED SKIN FIBROSIS IS A DETRIMENTAL AND CHRONIC DISORDER THAT OCCURS AFTER RADIATION EXPOSURE. DNA METHYLATION HAS BEEN CHARACTERIZED AS AN IMPORTANT REGULATORY MECHANISM OF MULTIPLE PATHOLOGICAL PROCESSES. IN THIS STUDY, WE COMPARED THE GENOME-WIDE DNA METHYLATION STATUS IN RADIATION-INDUCED FIBROTIC SKIN AND ADJACENT NORMAL TISSUES OF RATS BY METHYLATED DNA IMMUNOPRECIPITATION SEQUENCING. RADIATION-INDUCED FIBROTIC SKIN SHOWED DIFFERENTIALLY METHYLATED REGIONS ASSOCIATED WITH 3,650 PROTEIN-CODING GENES, 72 MICRORNAS, 5,836 LONG NONCODING RNAS AND 3 PIWI-INTERACTING RNAS. BY INTEGRATING THE MRNA AND METHYLATION PROFILES, THE ZINC TRANSPORTER SLC39A9/ZIP9 WAS INVESTIGATED IN GREATER DETAIL. THE PROTEIN LEVEL OF ZIP9 WAS INCREASED IN IRRADIATED SKIN TISSUES OF HUMANS, MONKEYS, AND RATS, ESPECIALLY IN RADIOGENIC FIBROTIC SKIN TISSUES. RADIATION INDUCED THE DEMETHYLATION OF A CPG DINUCLEOTIDE IN EXON 1 OF ZIP9 THAT RESULTED IN RECRUITMENT OF THE TRANSCRIPTIONAL FACTOR SP1 AND INCREASED ZIP9 EXPRESSION. OVEREXPRESSION OF ZIP9 RESULTED IN ACTIVATION OF THE PROFIBROTIC TRANSFORMING GROWTH FACTOR-BETA SIGNALING PATHWAY THROUGH PROTEIN KINASE B IN HUMAN FIBROBLASTS. IN ADDITION, RADIATION-INDUCED SKIN FIBROSIS WAS ASSOCIATED WITH INCREASED ZINC ACCUMULATION. THE ZINC CHELATOR N,N,N',N'-TETRAKIS(2-PYRIDYLMETHYL)-1,2-ETHYLENEDIAMINE ABROGATED ZIP9-INDUCED ACTIVATION OF THE TRANSFORMING GROWTH FACTOR-BETA SIGNALING PATHWAY AND ATTENUATED RADIATION-INDUCED SKIN FIBROSIS IN A RAT MODEL. IN SUMMARY, OUR FINDINGS ILLUSTRATE EPIGENETIC REGULATION OF ZIP9 AND ITS CRITICAL ROLE IN PROMOTING RADIATION-INDUCED SKIN FIBROSIS. 2020 17 1293 31 DECREASED ERK AND JNK SIGNALING CONTRIBUTE TO GENE OVEREXPRESSION IN "SENESCENT" CD4+CD28- T CELLS THROUGH EPIGENETIC MECHANISMS. AN INFLAMMATORY AND CYTOTOXIC CD4+CD28- T CELL SUBSET INFILTRATES ATHEROSCLEROTIC PLAQUES AND IS IMPLICATED IN PLAQUE RUPTURE AND MYOCARDIAL INFARCTIONS. THIS PATHOLOGIC SUBSET DEVELOPS WITH REPLICATIVE STRESS AND IS FOUND IN PATIENTS WITH CHRONIC INFLAMMATORY DISEASES SUCH AS RA AS WELL AS WITH AGING. CD4+CD28- CELLS OVEREXPRESS GENES NORMALLY SUPPRESSED BY DNA METHYLATION IN CD4+CD28+ T CELLS, SUCH AS KIR, PERFORIN, AND CD70. HOW THIS SUBSET OVER EXPRESSES METHYLATION-SENSITIVE GENES IS UNKNOWN. DNA METHYLATION PATTERNS ARE MAINTAINED IN PROLIFERATING CELLS BY DNMTS, WHICH ARE UP-REGULATED DURING MITOSIS BY THE ERK AND JNK SIGNALING PATHWAYS. WE HYPOTHESIZED THAT DEFECTS IN THESE SIGNALING PATHWAYS CONTRIBUTE TO ALTERED GENE EXPRESSION IN HUMAN CD4+CD28- CELLS THROUGH EFFECTS ON DNA METHYLATION. WE REPORT THAT SIGNALING THROUGH THE ERK AND JNK PATHWAYS IS DECREASED IN CD4+CD28- RELATIVE TO CD4+CD28+ CELLS FROM THE SAME INDIVIDUALS AND THAT ERK AND JNK PATHWAY INHIBITION DECREASES DNMT1 AND -3A LEVELS, WHICH IN TURN, CAUSES DEMETHYLATION AND OVEREXPRESSION OF THE TNFSF7 (CD70) GENE. WE ALSO REPORT THAT CD4+CD28- T CELLS OVEREXPRESS PP5, A STRESS-INDUCED INHIBITOR OF THE ERK AND JNK SIGNALING PATHWAYS THAT MAY CONTRIBUTE TO THE SIGNALING DEFECTS. WE CONCLUDE THAT DECREASED ERK AND JNK SIGNALING IN THE CD4+CD28- SUBSET, ARISING WITH REPLICATIVE STRESS, CAN LEAD TO THE OVEREXPRESSION OF NORMALLY SUPPRESSED GENES THROUGH EFFECTS ON DNMTS AND CONSEQUENTLY, CHROMATIN STRUCTURE. 2010 18 6659 25 UPREGULATION OF AKT3 CONFERS RESISTANCE TO THE AKT INHIBITOR MK2206 IN BREAST CANCER. ACQUIRED RESISTANCE TO MOLECULAR TARGETED THERAPY REPRESENTS A MAJOR CHALLENGE FOR THE EFFECTIVE TREATMENT OF CANCER. HYPERACTIVATION OF THE PI3K/AKT PATHWAY IS FREQUENTLY OBSERVED IN VIRTUALLY ALL HUMAN MALIGNANCIES, AND NUMEROUS PI3K AND AKT INHIBITORS ARE CURRENTLY UNDER CLINICAL EVALUATION. HOWEVER, MECHANISMS OF ACQUIRED RESISTANCE TO AKT INHIBITORS HAVE YET TO BE DESCRIBED. HERE, WE USE A BREAST CANCER PRECLINICAL MODEL TO IDENTIFY RESISTANCE MECHANISMS TO A SMALL MOLECULE ALLOSTERIC AKT INHIBITOR, MK2206. USING A STEP-WISE AND CHRONIC HIGH-DOSE EXPOSURE, BREAST CANCER CELL LINES HARBORING ONCOGENIC PI3K RESISTANT TO MK2206 WERE ESTABLISHED. USING THIS MODEL, WE REVEAL THAT AKT3 EXPRESSION IS MARKEDLY UPREGULATED IN AKT INHIBITOR-RESISTANT CELLS. INDUCTION OF AKT3 IS REGULATED EPIGENETICALLY BY THE BROMODOMAIN AND EXTRA TERMINAL DOMAIN PROTEINS. IMPORTANTLY, KNOCKDOWN OF AKT3, BUT NOT AKT1 OR AKT2, IN RESISTANT CELLS RESTORES SENSITIVITY TO MK2206. AKT INHIBITOR-RESISTANT CELLS ALSO DISPLAY AN EPITHELIAL TO MESENCHYMAL TRANSITION PHENOTYPE AS ASSESSED BY ALTERATIONS IN THE LEVELS OF E-CADHERIN, N-CADHERIN, AND VIMENTIN, AS WELL AS ENHANCED INVASIVENESS OF TUMOR SPHEROIDS. NOTABLY, THE INVASIVE MORPHOLOGY OF RESISTANT SPHEROIDS IS DIMINISHED UPON AKT3 DEPLETION. WE ALSO SHOW THAT RESISTANCE TO MK2206 IS REVERSIBLE BECAUSE UPON DRUG REMOVAL RESISTANT CELLS REGAIN SENSITIVITY TO AKT INHIBITION, ACCOMPANIED BY REEXPRESSION OF EPITHELIAL MARKERS AND REDUCTION OF AKT3 EXPRESSION, IMPLYING THAT EPIGENETIC REPROGRAMMING CONTRIBUTES TO ACQUISITION OF RESISTANCE. THESE FINDINGS PROVIDE A RATIONALE FOR DEVELOPING THERAPEUTICS TARGETING AKT3 TO CIRCUMVENT ACQUIRED RESISTANCE IN BREAST CANCER. MOL CANCER THER; 15(8); 1964-74. (C)2016 AACR. 2016 19 5101 21 POLYCOMB FACTOR PHF19 CONTROLS CELL GROWTH AND DIFFERENTIATION TOWARD ERYTHROID PATHWAY IN CHRONIC MYELOID LEUKEMIA CELLS. POLYCOMB GROUP (PCG) OF PROTEINS ARE A GROUP OF HIGHLY CONSERVED EPIGENETIC REGULATORS INVOLVED IN MANY BIOLOGICAL FUNCTIONS, SUCH AS EMBRYONIC DEVELOPMENT, CELL PROLIFERATION, AND ADULT STEM CELL DETERMINATION. PHD FINGER PROTEIN 19 (PHF19) IS AN ASSOCIATED FACTOR OF POLYCOMB REPRESSOR COMPLEX 2 (PRC2), OFTEN UPREGULATED IN HUMAN CANCERS. IN PARTICULAR, MYELOID LEUKEMIA CELL LINES SHOW INCREASED LEVELS OF PHF19, YET LITTLE IS KNOWN ABOUT ITS FUNCTION. HERE, WE HAVE CHARACTERIZED THE ROLE OF PHF19 IN MYELOID LEUKEMIA CELLS. WE DEMONSTRATED THAT PHF19 DEPLETION DECREASES CELL PROLIFERATION AND PROMOTES CHRONIC MYELOID LEUKEMIA (CML) DIFFERENTIATION. MECHANISTICALLY, WE HAVE SHOWN HOW PHF19 REGULATES THE PROLIFERATION OF CML THROUGH A DIRECT REGULATION OF THE CELL CYCLE INHIBITOR P21. FURTHERMORE, WE OBSERVED THAT MTF2, A PHF19 HOMOLOG, PARTIALLY COMPENSATES FOR PHF19 DEPLETION IN A SUBSET OF TARGET GENES, INSTRUCTING SPECIFIC ERYTHROID DIFFERENTIATION. TAKEN TOGETHER, OUR RESULTS SHOW THAT PHF19 IS A KEY TRANSCRIPTIONAL REGULATOR FOR CELL FATE DETERMINATION AND COULD BE A POTENTIAL THERAPEUTIC TARGET FOR MYELOID LEUKEMIA TREATMENT. 2021 20 3249 28 HEPATITIS B VIRUS HIJACKS CTHRC1 TO EVADE HOST IMMUNITY AND MAINTAIN REPLICATION. HEPATITIS B VIRUS (HBV) INFECTION CAUSES ACUTE AND CHRONIC LIVER DISEASES, BUT IS NOT DIRECTLY CYTOPATHIC. LIVER INJURY RESULTS FROM REPEATED ATTEMPTS OF THE CELLULAR IMMUNE RESPONSE SYSTEM TO CONTROL THE VIRAL INFECTION. HERE, WE INVESTIGATE THE ROLES OF CELLULAR FACTORS AND SIGNALING PATHWAYS INVOLVED IN THE REGULATION OF HBV REPLICATION TO REVEAL THE MECHANISM UNDERLYING HBV INFECTION AND PATHOGENESIS. WE SHOW THAT COLLAGEN TRIPLE HELIX REPEAT CONTAINING 1 (CTHRC1) EXPRESSION IS ELEVATED IN HBV-INFECTED PATIENTS AND IN HBV-TRANSFECTED CELLS THROUGH EPIGENETIC MODIFICATION AND TRANSCRIPTIONAL REGULATION. CTHRC1 FACILITATES HBV REPLICATION IN CULTURED CELLS AND BALB/C MICE BY ACTIVATING THE PKCALPHA/ERK/JNK/C-JUN CASCADE TO REPRESS THE IFN/JAK/STAT PATHWAY. HBV-ACTIVATED CTHRC1 DOWNREGULATES THE ACTIVITY OF TYPE I INTERFERON (IFN), THE PRODUCTION OF IFN-STIMULATED GENES (ISGS), AND THE PHOSPHORYLATION OF SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 1/2 (STAT1/2), WHEREAS IT UPREGULATES THE PHOSPHORYLATION AND UBIQUITINATION OF TYPE I IFN RECEPTORS (IFNARALPHA/BETA). THUS, OUR RESULTS SHOW THAT HBV USES A NOVEL MECHANISM TO HIJACK CELLULAR FACTORS AND SIGNAL CASCADES IN ORDER TO EVADE HOST ANTIVIRAL IMMUNITY AND MAINTAIN PERSISTENT INFECTION. WE ALSO DEMONSTRATE THAT CTHRC1 HAS A NOVEL ROLE IN VIRAL INFECTION. 2015