Discovery Institute

CANCER CENTER SUPPORT GRANT

BURNHAM CENTER FOR CHEMICAL GENOMICS

PROTEIN GLYCOSYLATION IN THE COAGULOPATHY AND INFLAMMATION OF SEPSIS

GLOBAL INNATE IMMUNE RESPONSES TO HIV-1 INFECTION

TARGETING PTEN - AN UPSTREAM DOWNSTREAM AND OFFSTREAM APPROACH

AGING AS A RISK FACTOR AND TARGET FOR PREVENTION OF LIVER CANCER - PROJECT SUMMARY – OVERALL THE INCIDENCES OF LIVER CANCER (PRIMARILY HEPATOCELLULAR CARCINOMA (HCC)) ARE INCREASING AND DISEASE OUTCOME IS POOR. CONSEQUENTLY, THERE IS AN URGENT NEED FOR NEW THERAPIES AND BETTER PREVENTIVE STRATEGIES. AGE IS A MAJOR RISK FACTOR FOR HCC. IN LINE WITH THE GEROSCIENCE HYPOTHESIS, WE HYPOTHESIZE THAT AGING DRIVES A DYSFUNCTIONAL MITOCHONDRIAL, EPIGENETIC AND METABOLIC NETWORK THAT PROMOTES AND EXACERBATES AGE-ASSOCIATED DYSREGULATION OF IMMUNE FUNCTION AND INFLAMMATION IN LIVER. LOSS OF HOMEOSTASIS ACROSS MULTIPLE SYSTEMS IS PERMISSIVE FOR NEOPLASTIC LIVER DISEASE. WE FURTHER HYPOTHESIZE THAT DYSREGULATED CHRONIC INTERFERON SIGNALING IS CENTRAL TO THIS PATHOGENIC NETWORK. WE WILL DISSECT THIS NETWORK AND TEST THE CONSEQUENCE OF CHRONIC INTERFERON SIGNALING, TO UNDERSTAND WHY THE INCIDENCE OF LIVER CANCER INCREASES WITH AGE. WE WILL ALSO INVESTIGATE APPROACHES THAT TARGET THIS NETWORK FOR THEIR ABILITY TO PREVENT AND COMBAT LIVER CANCER. OUR OVERALL SPECIFIC OBJECTIVES ARE: OBJECTIVE 1. INVESTIGATE AGE-ASSOCIATED CHANGES TO MITOCHONDRIA, CHROMATIN, METABOLISM (SPECIFICALLY, BILE ACIDS) AND INNATE AND ADAPTIVE IMMUNITY, THEIR CAUSAL ROLE IN HCC AND UNDERLYING MECHANISMS. OBJECTIVE 2. INVESTIGATE HOW INTERACTIONS BETWEEN THESE DIFFERENT SYSTEMS AND AGE-DEPENDENT DYSREGULATION OF THESE INTERACTIONS CONTRIBUTES TO HCC. OBJECTIVE 3. TEST THE HYPOTHESIS THAT AT LEAST SOME OF THESE AGE-ASSOCIATED ALTERATIONS AND CONSEQUENT PREDISPOSITION TO HCC ARE DEPENDENT ON CHRONIC INTERFERON SIGNALING IN AGED TISSUE. OBJECTIVE 4. INVESTIGATE APPROACHES THAT TARGET AGE DYSREGULATION, FOR EXAMPLE SUPPRESSORS OF CHRONIC INTERFERON ACTIVATION, MITOHORMETIC INTERVENTIONS, RAPAMYCIN, SENOLYTICS, BILE ACID MODULATORS AND IMMUNE-MODULATORS, FOR THEIR ABILITY TO SUPPRESS THE ONSET OF LIVER CANCER AND BETTER COUNTER ESTABLISHED CANCER. SINCE AGE IS THE BIGGEST SINGLE RISK FACTOR FOR HCC, IT FOLLOWS THAT A MOLECULAR UNDERSTANDING OF THE AGE- DEPENDENCE OF HCC CAN LEAD TO IMPROVED DISEASE MANAGEMENT THROUGH RISK ASSESSMENT, EARLY DETECTION, PROGNOSTICATION AND THERAPY. MOREOVER, AN UNDERSTANDING OF HOW HCC DEVELOPS DURING AGING CAN ALSO LEAD TO PREVENTATIVE INTERVENTIONS. THIS PPG WILL DEFINE CRITICAL MOLECULAR MECHANISMS UNDERPINNING AGE-DEPENDENCE OF HCC. WE WILL ALSO PROMOTE APPROACHES FOR IMPROVED RISK ASSESSMENT THROUGH APPLICATION, TESTING AND REFINEMENT OF A TRANSCRIPTOME-BASED “TUMORIGENIC INDEX” TO QUANTITATE THE RISK OF HCC. FINALLY, BASED ON OUR DISCOVERIES, WE WILL TEST A PANEL OF CANDIDATE INTERVENTIONS FOR THOSE THAT CAN PREVENT AND COMBAT HCC.

CLINICAL DEVELOPMENT OF AN MGLU2 POSITIVE ALLOSTERIC MODULATOR TO TREAT NICOTINE ADDICTION

SPATIAL MAPPING SENESCENT CELLS ACROSS THE MOUSE LIFESPAN BY MULTIPLEX TRANSCRIPTOMICS AND EPIGENOMICS - PROJECT SUMMARY CELLULAR SENESCENCE, CHARACTERIZED BY STABLE PROLIFERATION ARREST AND SECRETION OF PRO-INFLAMMATORY FACTORS, IS NOT ONLY A HALLMARK OF AGING, BUT ALSO A KEY CONTRIBUTOR TO AGE-ASSOCIATED DISEASES IN HUMANS. AS THE US POPULATION IS AGING, THERE IS AN ADDED URGENCY TO GAIN A BETTER UNDERSTANDING OF CELLULAR SENESCENCE IN DIFFERENT TISSUES OVER THE LIFESPAN. UNFORTUNATELY, WE STILL LACK THE KNOWLEDGE TO UNAMBIGUOUSLY DEFINE SENESCENCE AT THE MOLECULAR AND CELLULAR LEVELS, DUE TO ITS HETEROGENEOUS PHENOTYPES. TO ADDRESS THIS MAJOR GAP IN KNOWLEDGE, WE PROPOSE TO ESTABLISH A TISSUE MAPPING CENTER THAT FOCUSES ON THE IDENTIFICATION AND CHARACTERIZATION OF SENESCENT CELLS IN HEALTHY MOUSE BRAIN, BONE MARROW, BREAST, COLON AND LIVER. OUR RESEARCH STRATEGY BUILDS ON RECENT ADVANCES IN SINGLE CELL EPIGENOMICS TECHNOLOGIES THAT OUR TEAM DEVELOPED AND THE KNOWLEDGE THAT SENESCENT CELLS EXHIBIT CHARACTERISTIC CHANGES IN THE CHROMATIN LANDSCAPES AND HISTONE MODIFICATIONS ALONG WITH GENE EXPRESSION LEVELS AT MARKER GENES OF CELLULAR SENESCENCE. WE WILL DEPLOY CUTTING-EDGE SINGLE CELL IN SITU AND TISSUE DISSOCIATIVE MULTI-OMIC TOOLS THAT HAVE BEEN WELL ESTABLISHED IN OUR CENTER TO PRODUCE COMPREHENSIVE SINGLE CELL RESOLUTION MAPS OF THE TRANSCRIPTOME AND EPIGENOME IN MALE AND FEMALE MOUSE BRAIN, BONE MARROW, BREAST, COLON AND LIVER, AND TO PROVIDE QUALITATIVE AND QUANTITATIVE SPATIAL MAPS OF THE NORMAL BURDEN OF SENESCENT CELLS IN THESE VITAL ORGANS, ACROSS THE LIFESPAN OF TWO MOUSE STRAINS. WE WILL RIGOROUSLY VALIDATE THE NEWLY DEFINED SENESCENT CELL POPULATIONS USING PHARMACOLOGIC AND GENETIC APPROACHES TO ERADICATE SENESCENT CELLS OR SUPPRESS THEIR INFLAMMATORY PHENOTYPE, AND ORTHOGONAL STATE-OF-THE-ART AND CONVENTIONAL ASSAYS FOR CELLULAR SENESCENCE. WE WILL GENERATE WHOLE GENOME SINGLE CELL DNA METHYLATION DATA TO LINK OUR SPATIAL ATLAS TO MEASUREMENT OF EPIGENETIC AGE, A CANDIDATE PREDICTOR OF BENEFICIAL VERSUS DETRIMENTAL EFFECTS OF SENESCENT CELLS. WE EXPECT THAT COMPREHENSIVE SINGLE CELL ATLASES OF EPIGENOME AND TRANSCRIPTOME WILL ENABLE US TO IDENTIFY AND CHARACTERIZE CELLULAR SENESCENCE IN DIFFERENT TISSUE CONTEXTS AND DURING AGING. WE EXPECT THAT THE PLANNED RESEARCH WILL PROVIDE A REFERENCE FOR FUTURE STUDIES THAT SEEK TO CHARACTERIZE AND TARGET SENESCENT CELLS ASSOCIATED WITH OR PRECEDING DISEASE IN BRAIN, BONE MARROW, BREAST, COLON AND LIVER.

PRECLINICAL STUDIES FOR THE DEVELOPMENT OF SELECTIVE MGLU2 POSITIVE ALLOSTERIC MODULATORS TO TREAT SUBSTANCE USE DISORDERS

SAFETY/TOXICOLOGY, ADME AND CMC ACTIVITIES TO SUPPORT THE ASSESSMENT OF THE MGLU2 PAM SBP-9330 IN A PHASE 2 CLINICAL STUDY IN SMOKERS - PROJECT SUMMARY THIS RESUBMISSION APPLICATION, “SAFETY/TOXICOLOGY, ADME AND CMC ACTIVITIES TO SUPPORT THE ASSESSMENT OF THE MGLU2 PAM SBP-9330 IN A PHASE 2 CLINICAL STUDY IN SMOKERS”, IS IN RESPONSE TO PAR-22-202 AND REPRESENTS THE CONTINUATION OF OUR CURRENT WORK FUNDED THROUGH 07/31/2023 BY U01 DA051077 “CLINICAL DEVELOPMENT OF AN MGLU2 POSITIVE ALLOSTERIC MODULATOR TO TREAT NICOTINE ADDICTION”. CIGARETTE SMOKING REMAINS ONE OF THE LEADING CAUSES OF DEATH AND DISEASE WORLDWIDE. ONLY THREE TYPES OF MEDICATIONS FOR SMOKING CESSATION HAVE BEEN APPROVED BY THE UNITED STATES FOOD AND DRUG ADMINISTRATION (FDA) (BUPROPION, VARENICLINE, AND NICOTINE REPLACEMENT THERAPY), ALL OF WHICH HAVE POOR EFFICACY AND TOLERABILITY. POSITIVE ALLOSTERIC MODULATORS (PAMS) OF THE METABOTROPIC GLUTAMATE RECEPTOR SUBTYPE 2 (MGLU2) DECREASE NICOTINE SELF-ADMINISTRATION AND CUE-INDUCED REINSTATEMENT OF NICOTINE SEEKING IN ANIMAL MODELS, PROVIDING SUPPORT FOR MGLU2 AS A VALID TARGET FOR THE TREATMENT OF NICOTINE ADDICTION. OUR INVESTIGATIONAL DRUG, THE SMALL MOLECULE MGLU2 PAM SBP-9330 (CHARACTERIZED DURING PRIOR GRANT U01 DA041731), HAS RECENTLY BEEN EVALUATED IN HEALTHY NONSMOKERS AND SMOKERS IN A PLACEBO- CONTROLLED, RANDOMIZED, AND DOUBLE-BLIND PHASE 1 CLINICAL STUDY (NCT04948827). TO DATE, THE DATA FOR THE HEALTHY NONSMOKERS HAVE BEEN UNBLINDED AND ANALYZED, REVEALING THAT SBP-9330 WAS WELL TOLERATED, WITH NO SERIOUS ADVERSE EVENTS OR SAFETY CONCERNS. HUMAN PHARMACOKINETIC DATA REVEALED THAT PLASMA EXPOSURES WERE NEARLY DOSE-PROPORTIONAL, ACCUMULATED APPROXIMATELY 2-FOLD OVER 14 DAYS OF DOSING, AND WERE SUFFICIENTLY HIGH FOR POTENTIAL EFFICACY. THESE PROMISING DATA SUPPORT CONTINUATION OF THE CLINICAL DEVELOPMENT OF SBP-9330.THE STUDIES PROPOSED IN THIS GRANT APPLICATION ARE REQUIRED BY THE FDA BEFORE WE CAN DOSE SBP-9330 FOR LONGER DURATION IN TOBACCO SMOKERS IN A PHASE 2 STUDY AND BEYOND. TO THIS END, OUR SPECIFIC AIMS ARE: (1) PERFORM THE PRECLINICAL ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION (ADME) STUDIES NEEDED TO SUPPORT THE PHASE 2 CLINICAL DEVELOPMENT OF SBP-9330 IN SMOKERS; (2) PERFORM THE PRECLINICAL TOXICOLOGY STUDIES NEEDED TO SUPPORT PHASE 2 CLINICAL DEVELOPMENT OF SBP-9330 IN SMOKERS; AND (3) PERFORM THE CHEMISTRY, MANUFACTURING, AND CONTROLS (CMC) ACTIVITIES NEEDED TO SUPPORT PHASE 2 CLINICAL DEVELOPMENT IN SMOKERS. TO ACHIEVE THESE SPECIFIC AIMS, WE HAVE RETAINED THE SAME HIGHLY EXPERIENCED AND QUALIFIED MULTIDISCIPLINARY TEAM OF INVESTIGATORS THAT HAS COLLABORATED PRODUCTIVELY DURING THE PREVIOUS GRANT (U01 DA041731) AND THE CURRENTLY ACTIVE GRANT (U01 DA051077). WE HAVE ALSO COMPILED AN EXTENSIVE DATA PACKAGE TO SUPPORT THE FURTHER CLINICAL DEVELOPMENT OF SBP-9330. ACHIEVEMENT OF THE MILESTONES IN THIS PROPOSAL WILL PROVIDE THE ADDITIONAL DATA AND DRUG SUBSTANCE NEEDED TO ADVANCE SBP-9330 INTO A PHASE 2 PROOF-OF-CONCEPT CLINICAL STUDY IN SMOKERS. OUR TEAM HAS THE DEPTH AND BREADTH OF EXPERTISE AND EXPERIENCE TO EXECUTE THE PROPOSED RESEARCH PLAN, AS EVIDENCED BY THE COMPLETE AND TIMELY ACHIEVEMENT OF THE MILESTONES FOR BOTH U01 DA041731 AND U01 DA051077 IN THE PAST SIX YEARS.

GENETIC CONTROL OF THE HEART BY TINMAN AND NEUROMANCER

LA JOLLA-PARKINSON'S DISEASE CENTER GRANT

ULTRASTRUCTURAL BASIS OF MECHANOTRANSDUCTION IN MATRIX ADHESIONS

REWIRED SIGNALING AT THE NEXUS OF MELANOMA METASTASIS AND RESISTANCE

GLYCOSYLTRANSFERASE FUNCTION IN DEVELOPMENT AND DISEASE

CARBOHYDRATE-DEPENDENT ADHESION IN NORMAL AND TUMOR CELLS

LA JOLLA INTERDISCIPLINARY NEUROSCIENCE CENTER CORES

EPIGENETICS & SIGNALING IN HESC COMMITMENT AND DIFFERENTIATION INTO MUSCLE

TARGETING EPH RECEPTORS IN CANCER

ENHANCEMENT OF A PREDICTIVE TOXICOLOGY TOOLBOX VIA MULTI-PARAMETRIC DATA INFORMED QSAR MODELING OF BENCHMARK TOXINS

MOLECULAR PATHOBIOLOGY OF SOLUBLE TREM2 IN ALZHEIMER'S DISEASE.

CHARACTERIZATION, OPTIMIZATION, AND DEVELOPMENT OF DUAL MGLU2/3 POSITIVE ALLOSTERIC MODULATORS FOR OPIOID USE DISORDER - PROJECT SUMMARY OPIOID USE DISORDER (OUD) IS A SIGNIFICANT PROBLEM WORLDWIDE AND A SURGE IN METHAMPHETAMINE (MA) USE HAS EMERGED IN CHRONIC OPIOID USERS. GIVEN RECENT INCREASES IN CO-ABUSE, THERE IS A DIRE NEED FOR NOVEL TREATMENT STRATEGIES THAT PREVENT RELAPSE TO DRUG USE IN BOTH OUD AND MA USE DISORDER (MUD). LONG-TERM DRUG EXPOSURE INDUCES ENHANCED GLUTAMATE (GLU)-MEDIATED SYNAPTIC PLASTICITY, WHICH UNDERLIES EXCESSIVE PHYSIOLOGICAL AND BEHAVIORAL RESPONSES TO DRUG-RELATED CUES. OPIOID AND MA EXPOSURE ALSO ACTIVATE MICROGLIA AND ASTROCYTES, PROMOTING RELEASE OF PRO-INFLAMMATORY CYTOKINES. ALL THESE FACTORS INCREASE THE RISK OF RELAPSE TO DRUG USE. NORMALIZATION OF ABERRANT GLU ACTIVITY CAUSED BY CHRONIC DRUG USE REPRESENTS A NOVEL THERAPEUTIC STRATEGY TO PREVENT RELAPSE IN OUD/MUD. THE ACTIVATION OF METABOTROPIC GLU RECEPTOR SUBTYPES 2 AND 3 (MGLU2/3) USING AGONISTS OR POSITIVE ALLOSTERIC MODULATORS (PAMS) DECREASES PSYCHOSTIMULANT SELF-ADMINISTRATION (SA) AS WELL AS CUE-INDUCED REINSTATEMENT (RI) IN ANIMALS. MOREOVER, PRECLINICAL STUDIES INDICATE THAT MGLU2/3 ACTIVATION HAS PROMISE FOR TREATING STRESS- AND ANXIETY-RELATED DISORDERS IN HUMANS AND CAN SYSTEMATICALLY AUGMENT SLEEP. HOWEVER, THE RELATIVE CONTRIBUTION OF MGLU2 VERSUS MGLU3 ACTIVATION FOR TREATING OUD/MUD IS NOT YET KNOWN. ACTIVATION OF PRESYNAPTIC MGLU2 AUTORECEPTORS RESULTS IN DOWNSTREAM INHIBITION OF GLU RELEASE IN THE NUCLEUS ACCUMBENS, WHICH IN TURN ATTENUATES DRUG RI. IN ADDITION, ACTIVATION OF MGLU3, WHICH IS HIGHLY EXPRESSED IN ASTROCYTES, LEADS TO THE RELEASE OF THE ANTI-INFLAMMATORY CYTOKINE TRANSFORMING GROWTH FACTOR BETA (TGF-SS). THE SPECIFIC LOCALIZATION AND SIGNAL TRANSDUCTION OF MGLU2 AND MGLU3 RECEPTORS LEAD TO OUR OVERARCHING HYPOTHESIS THAT DUAL ACTIVATION OF BOTH MGLU2 AND MGLU3 WILL PROVIDE A CLEAR ADVANTAGE FOR THE TREATMENT FOR OUD AND MUD OVER MGLU2 ACTIVATION ALONE. WE HAVE RECENTLY SYNTHESIZED AND CHARACTERIZED SBI-0799220, A PAM WITH EQUAL POTENCY FOR MGLU2 AND MGLU3 AND SBI-0801315, A PAM WITH >50-FOLD SELECTIVITY FOR MGLU2 VS MGLU3. PRELIMINARY DATA INDICATE THAT SBI-0801315 ATTENUATES OXY CUE-INDUCED RI AND SA, AND THAT SBI-0799220 ATTENUATES MA SA. HOWEVER, A DIRECT COMPARISON OF MGLU2/3 WITH MGLU2 PAMS IN MODELS OF OUD AND MUD HAS NOT YET BEEN CONDUCTED. THE GOAL OF THIS APPLICATION IS TO ADVANCE MGLU2/3 PAMS AS A NOVEL TREATMENT FOR PREVENTING RELAPSE TO OUD, EXAMINE THEIR POTENTIAL FOR TREATING MUD, AND SIMULTANEOUSLY OPTIMIZE MGLU2/3 PAMS. WE WILL DETERMINE THE IN VIVO EFFICACY OF MGLU2/3 AND MGLU2-PREFERRING PAMS TO ATTENUATE OXY/MA SA AND RI, OXY/MA-INDUCED WITHDRAWAL, MA-INDUCED NEUROINFLAMMATION, AND OPIOID-INDUCED ANTINOCICEPTION IN RATS. SIMULTANEOUSLY, WE WILL OPTIMIZE THE PHARMACOLOGICAL AND PHARMACEUTICAL PROPERTIES OF OUR MGLU2/3 PAM SERIES INCLUDING ABSORPTION, DISTRIBUTION, METABOLISM, EXCRETION, AND PHARMACOKINETICS. WE HAVE ASSEMBLED A MULTIDISCIPLINARY TEAM OF INVESTIGATORS THAT HAS THE KNOWLEDGE AND EXPERIENCE TO ACHIEVE THESE OUTCOMES. SUCCESSFUL COMPLETION OF THESE STUDIES WILL EXPEDITE DEVELOPMENT OF A NOVEL MGLU2/3 PAM TOWARDS INVESTIGATIONAL NEW DRUG (IND)-ENABLING STUDIES AND ULTIMATELY, A NOVEL TREATMENT FOR PREVENTING RELAPSE IN OUD.

ASSEMBLY, DYNAMICS AND EVOLUTION OF CELL-CELL AND CELL-MATRIX ADHESIONS

DEVELOPMENT OF SBI-553, AN ALLOSTERIC MODULATOR OF NTR1, FOR THE TREATMENT OF SUBSTANCE USE DISORDERS - PROJECT SUMMARY OPIOID USE DISORDER (OUD) IS A MAJOR PUBLIC HEALTH CRISIS IN THE UNITED STATES, WITH >50,000 OVERDOSE DEATHS IN 2017 ALONE. ADDICTION TO OPIOIDS IS INTIMATELY RELATED TO THE PHYSIOLOGY OF THE BRAIN’S DOPAMINE-BASED REWARDING SYSTEM. THE NEUROTENSIN 1 RECEPTOR (NTR1) IS FOUND AMONG DOPAMINE PRE AND POST-SYNAPTIC NEURONS IN THE CENTRAL NERVOUS SYSTEM, AND ACTS AS A MODULATOR OF DOPAMINERGIC SYSTEMS. EVEN THOUGH SUBSTANTIAL EVIDENCE POINTS TOWARDS NTR1 AS A MOLECULAR TARGET FOR TREATING ADDICTION DISORDERS, FEW NON- PEPTIDE BRAIN PENETRANT NEUROTENSIN MODULATORS HAVE BEEN IDENTIFIED, AND ORTHOSTERIC NTR1 LIGANDS DISPLAY SIDE EFFECTS INCLUDING HYPOTENSION AND HYPOTHERMIA THAT HAVE LIMITED THEIR CLINICAL DEVELOPMENT. RECENTLY, WE HAVE DISCOVERED A SERIES OF BRAIN-PENETRANT NTR1 MODULATORS, INCLUDING A LEAD COMPOUND SBI-553, WITH A UNIQUE MECHANISM OF ACTION AT NTR1. SBI-553 IS AN ORALLY AVAILABLE AND BRAIN PENETRANT B-ARRESTIN BIASED ALLOSTERIC MODULATOR OF NTR1, WHICH SHOWS EFFICACY IN A RANGE OF ADDICTION MODELS. THE LITERATURE CONTAINS NO DOCUMENTED REPORTS OF SUCH MOLECULES AMONG POSITIVE OR NEGATIVE ALLOSTERIC MODULATORS OF GPCRS. IN ADDITION, THE UNIQUE PROFILE OF SBI-553 APPEARS TO CIRCUMVENT THE CLINICALLY LIMITING SIDE EFFECTS OF HYPOTHERMIA AND HYPOTENSION DISPLAYED BY ORTHOSTERIC NTR1 LIGANDS. WHILE POTENTIALLY HIGH RISK, THE ACTIVITY OF SBI-553 HAS BEEN VALIDATED IN VITRO AND IN VIVO, AND THE INITIAL SAFETY PROFILING INDICATES NO ISSUES THAT WOULD PRECLUDE FURTHER DEVELOPMENT. THE GOAL OF THE APPLICATION IS TO DEVELOP SBI-553 AS A TREATMENT FOR OPIOID USE DISORDERS. A 2-YEAR UG3 PHASE IS PROPOSED WITH THE PRIMARY OBJECTIVE OF DETERMINING THE SUITABILITY OF SBI-553 (OR A BACKUP COMPOUND) ENTERING IND-ENABLING STUDIES FOLLOWED BY A 2-YEAR UH3 PHASE WITH THE PRIMARY OBJECTIVE OF COMPLETING A PHASE 1 SINGLE ASCENDING DOSE STUDY. DURING THE UG3 PHASE, THE TEAM WILL COMPLETE PHARMACOKINETIC (PK) STUDIES AND THE PRECLINICAL SAFETY PROFILE OF SBI-553 IN A RELEVANT SPECIES; IDENTIFY A BACKUP COMPOUND TO SBI-553; AND CONDUCT DEFINITIVE CELLULAR AND IN VIVO PHARMACOLOGY STUDIES WITH SBI-553 (AND/OR POTENTIAL BACKUP) IN MODELS OF ADDICTION TO INFORM BEST CLINICAL INDICATION AND CLINICAL ENDPOINTS (PK/PHARMACODYNAMIC). DURING THE UH3 PHASE, THE TEAM WILL ENSURE COMPLETION OF GMP SYNTHESIS, STABILITY TESTING AND FORMULATION; GLP TOXICOLOGY STUDIES; AND THE PHASE 1 SINGLE ASCENDING DOSE STUDY.

GENETIC ANALYSIS OF DROSOPHILA FUNCTIONAL AGING

NF-KAPPAB AND MITOCHONDRIAL SIGNALS AS POSITIVE AND NEGATIVE REGULATORS OF INFLAMMATION

MOLECULAR PATHOGENESIS AND TREATMENT OF HYPOPHOSPHATASIA

NEW CONGENITAL DISORDERS OF GLYCOSYLATION: THERAPY AND MODELS

NANOTHERAPY FOR VULNERABLE PLAQUE

NEURON-GLIA COMMUNICATION IN DEVELOPMENT

FUNCTION OF CD19 IN B-CELL DEVELOPMENT & DIFFERENTIATION

HEPARAN SULFATE IN SKELETAL DEVELOPMENT AND DISEASES

ALTERED REVERSE TRANSCRIPTASE-DEPENDENT GENE DIVERSIFICATION MECHANISMS IN ALZHEIMER'S DISEASE BRAINS

A NOVEL ROLE FOR THE UPR COMPONENT, ATF6 IN AD-ASSOCIATED NEUROPROTECTIVE PATHWAYS

SREBPS IN REGULATION OF LIPID METABOLISM

REGULATION OF NUTRIENT STRESS-INDUCED MACROPINOCYTOSIS IN PANCREATIC DUCTAL ADENOCARCINOMA

ROLE OF HYPERNUTRITION AND METABOLIC STRESS IN NON-ALCOHOLIC STEATOHEPATITIS (NASH) DRIVEN HEPATOCELLULAR CARCINOMA (HCC) - PROJECT SUMMARY – OVERALL HEPATOCELLULAR CARCINOMA (HCC) IS THE MOST COMMON PRIMARY LIVER CANCER AND A RISING CAUSE OF CANCER MORTALITY. HCC RELATED TO METABOLIC STRESS IS ON THE RISE, WITH NONALCOHOLIC STEATOHEPATITIS (NASH) RAPIDLY BECOMING THE DOMINANT ETIOLOGY. WE HYPOTHESIZE THAT STRESS-INDUCED METABOLIC REPROGRAMMING VIA NRF2 AND ATF6 ALTERS, GLUCOSE, LIPID, AND CHOLESTEROL METABOLISM TO AUGMENT GLYCOLYSIS AND INDUCE MITOCHONDRIAL DYSFUNCTION THROUGH STARD1 AND SAB, WHICH COLLABORATES WITH GENETIC ALTERATIONS TO ACCELERATE NASH PROGRESSION TO HCC THROUGH A SELF-SUSTAINED DOWNWARD SPIRAL. INTERFERENCE WITH KEY COMPONENTS OF THIS NETWORK MAY SUPPRESS HCC PROGRESSION AND CAN YIELD NOVEL INTERCEPTIVE STRATEGIES. OUR PRELIMINARY DATA INDICATE THAT INTERACTIONS BETWEEN THE CYTOSOL AND NUCLEUS (AKT;NRF2), THE ENDOPLASMIC RETICULUM (ER; ATF6) AND MITOCHONDRIA (STARD1+SAB) CONTRIBUTE SYNERGISTICALLY TO NASH-DRIVEN HCC. ELIMINATION OF ANY OF THESE COMPONENTS ATTENUATES HCC AND THEIR ACTIVATION EXACERBATES HCC PROGRESSION IN MURINE MODELS. TOGETHER, OUR OUTSTANDING TEAM OF INVESTIGATORS WILL ADDRESS THE PATHOLOGICAL SIGNIFICANCE AND MECHANISMS UNDERLYING THE CROSSTALK BETWEEN THESE PATHWAYS VIA 3 PROJECTS: PROJECT 1 WILL INTERROGATE HOW THE NEWLY DISCOVERED NRF2- FBP1 TUG-OF-WAR IS REGULATED TO SUPPORT HCC PROGRESSION. IT WILL IDENTIFY THE FIRST CELLS IN WHICH NRF2 AND AKT ARE ACTIVATED TO DRIVE FBP1 DEGRADATION AND THE CUES THAT TRIGGER THIS SWITCH. LINEAGE TRACING WILL DETERMINE WHETHER NRF2-HIGH HCC DIRECTLY ORIGINATES FROM FBP1-HIGH SENESCENT NASH HEPATOCYTES AND THE ROLE OF DIET- INDUCED DNA DAMAGE IN HEPATOCYTE SENESCENCE AND MUTAGENESIS WILL BE ELUCIDATED. PROJECT 2 WILL DEFINE HOW ATF6 DRIVES HCC IN RESPONSE TO METABOLIC STRESS. IT WILL UNCOVER THE MECHANISM BY WHICH ER STRESS AND THE UNFOLDED PROTEIN RESPONSE TRANSDUCER ATF6 PROMOTES NASH PROGRESSION TO HCC, LIKELY THROUGH NRF2 AND FBP1 DEGRADATION, THE REPROGRAMMING OF LIPID AND CHOLESTEROL METABOLISM, AND WILL TEST WHETHER AND HOW ATF6 ABLATION OR INHIBITION ABROGATE NASH-DRIVEN HCC. PROJECT 3 WILL EXPLORE HOW METABOLIC STRESS AND CHOLESTEROL TRIGGER MITOCHONDRIAL DYSFUNCTION THROUGH STARD1 AND SAB TO DRIVE HCC. MITOCHONDRIAL OUTER MEMBRANE PROTEINS STARD1 AND SAB MAY CONTRIBUTE TO NASH AND HCC DEVELOPMENT THROUGH ALTERED CHOLESTEROL TRAFFICKING AND BILE ACID SYNTHESIS, MITOCHONDRIAL DYSFUNCTION THAT TRIGGERS ROS PRODUCTION, RESULTING IN NRF2 AND ATF6 ACTIVATING OXIDATIVE AND/OR ER STRESS. THIS PPG OFFERS AN UNPRECEDENTED OPPORTUNITY FOR ADVANCED UNDERSTANDING OF NASH-DRIVEN HCC VIA PROXIMAL METABOLIC REGULATORS AND DNA DAMAGE CONTROLLED BY NRF2, ATF6, AND STARD1/SAB. EXTENSIVE COLLABORATIVE INTERACTIONS AND COORDINATED ADMINISTRATIVE RESOURCES (CORE A), SHARED USE OF DIETARY AND TRANSGENIC MOUSE MODELS, METABOLIC PROFILING/FLUX STUDIES (CORE 1) AND EXTENSIVE “OMIC” AND BIOINFORMATIC ANALYSES (CORE 2) WILL FURTHER ENHANCE COLLABORATIONS AND ENSURE UNIFORM SUCCESS THROUGHOUT ALL PROJECTS. THIS P01 OFFERS A UNIQUE TRANSLATIONAL OPPORTUNITY TO DEFINE HOW ENERGY RICH DIETS IMPACT REGULATORS THAT PROMOTE NASH PROGRESSION TO HCC, GENERATING NEW PREVENTIVE AND THERAPEUTIC STRATEGIES.

TRANSFORMATIVE RESEARCH ON SOMATIC GENE RECOMBINATION IN THE NORMAL AND ALZHEIMER'S DISEASE-RELATED DEMENTIA BRAIN

ROLE OF THE UNFOLDED PROTEIN RESPONSE IN BETA CELL

REGULATION OF FACTOR VIII SECRETION

A PHARMACOCHAPERONE-BASED STRATEGY FOR IDENTIFYING CHEMICAL PROBES OF BRAIN-DERIVED ORPHAN GPCRS

PRESENILINS IN PROTEIN TRAFFICKING

CENTER ON PROTEOLYTIC PATHWAYS(RMI)

MODIFIERS OF PROINSULIN INFLUENCE T2D SUSCEPTIBILITY

CONTROL OF LIPOGENESIS AND HEPATIC STEATOSIS BY CASPASE-2

COUNTERMEASURES FOR BIOTERROISM TARGETING CELLULAR HOST FACTORS

ROLES OF AN APP-BINDING PRO-APOPTOTIC PROTEIN IN MEDIATING NEURONAL CELL DEATH

DEFINING CHROMOSTASIS - A CANDIDATE REGULATOR OF HEALTHY AGING AND LONGEVITY

NEUROTENSIN 1 ALLOSTERIC MODULATOR FOR THE TREATMENT OF PAIN - PROJECT SUMMARY THE NIH HEAL INITIATIVE AIMS TO TACKLE TWO ONGOING HEALTH CARE CRISES: OPIOID USE DISORDER AND UNCONTROLLED PAIN. PHARMACOLOGICAL APPROACHES TARGETING BOTH CRISES ARE URGENTLY NEEDED. THE NEUROTENSIN RECEPTOR 1 (NTR1) HAS BEEN A SOUGHT-AFTER TARGET FOR THE TREATMENT OF BOTH PAIN AND ADDICTION, BUT DEVELOPMENT OF BALANCED NTR1 AGONISTS IS PRECLUDED BY THEIR SEVERE SIDE EFFECTS. AS A G PROTEIN-COUPLED RECEPTOR (GPCR) NTR1 SIGNALS THROUGH THE ACTIVATION OF G PROTEINS AND Β-ARRESTINS (E.G., ARRB2). ARRB2 ATTENUATES DRUG REWARD AND SUPPRESSES PAIN VIA REGULATION OF BOTH GPCRS AND NON-GPCRS, INCLUDING THE NMDA RECEPTOR. WE HAVE DEMONSTRATED IN RODENTS THAT SBI-553, A NOVEL ARRB2-BIASED ALLOSTERIC MODULATOR (BAM) OF NTR1, ATTENUATES OPIOID REWARD AND THE REINFORCING EFFECTS OF PSYCHOSTIMULANTS WITHOUT THE SIDE EFFECTS CHARACTERISTIC OF BALANCED NTR1 SIGNALING. CRYO- EM STUDIES DEMONSTRATE THAT SBI-553 BINDS AT AN INTRACELLULAR ALLOSTERIC SITE AND ACTS LIKE A MOLECULAR GLUE, DIRECTING SIGNALING TO ARRB2 IN THE PRESENCE OR ABSENCE OF AGONISTS. RECENTLY WE HAVE SHOWN THAT NTR1 BAMS PRODUCE POTENT ANTINOCICEPTION IN RODENT MODELS, A FINDING THAT BUILDS OFF PREVIOUS REPORTS THAT NEUROTENSIN, NTR1’S ENDOGENOUS LIGAND PROVIDED MORE POTENT PAIN RELIEF THAN MORPHINE THROUGH A MECHANISM THAT IS INDEPENDENT OF OPIOID RECEPTORS. NTR1 BAMS RAISE NOCICEPTIVE THRESHOLDS IN WILD-TYPE MICE, BUT NOT IN NTSR1−/−OR ARRB2−/− (KNOCKOUT) MICE, CONFIRMING THE MECHANISTIC REQUIREMENT FOR NTR1 AND ARRB2. LOCAL OR SYSTEMIC DELIVERY OF NTR1 BAMS REDUCED HYPERSENSITIVITY IN MOUSE MODELS OF POSTOPERATIVE AND NEUROPATHIC PAIN AND SUPPRESSED EXCITATORY SYNAPTIC TRANSMISSION AND NMDAR/ERK SIGNALING IN SPINAL CORD NOCICEPTIVE NEURONS. ADDITIONALLY, NTR1 BAMS SUPPRESSED C-FIBER-INDUCED RESPONSES IN VIVO AND ACTION POTENTIAL FIRING IN MOUSE AND HUMAN NOCICEPTIVE SENSORY NEURONS IN VITRO. COLLECTIVELY, OUR FINDINGS INDICATE THAT NTR1 BAMS ARE AN EXCITING AND NOVEL APPROACH TO DEVELOP A FIRST-IN-CLASS NON-OPIOID DRUG FOR THE TREATMENT OF PAIN. IN THIS APPLICATION WE OUTLINE OUR PLAN TO OPTIMIZE AND DEVELOP SUCH A DRUG FOR POSTOPERATIVE PAIN. IN THE UG3 STAGE, WE WILL LEVERAGE THE STRUCTURE ACTIVITY RELATIONSHIPS DEVELOPED AROUND SBI-553 AND, THROUGH A STRUCTURE-BASED DESIGN LEAD OPTIMIZATION PROCESS, REFINE AND IMPROVE THE PROPERTIES OF THE LEADS. CO-STRUCTURES OF LEADS WITH NTR1 WILL BE DETERMINED THROUGH CRYOEM, SUCH THAT THE ITERATIVE MEDICINAL CHEMISTRY, DESIGN-MAKE-TEST CYCLE IS INFORMED BY COMPUTATIONAL PREDICTIONS USING BOTH ARTIFICIAL INTELLIGENCE AND STRUCTURE-BASED DRUG DESIGN. IN THE UH3 PHASE THE OBJECTIVE IS TO DERIVE ADVANCED LEADS WITH EXCELLENT IN VIVO POTENCY IN ACUTE INFLAMMATORY PAIN, SURGICAL PAIN, AND CHRONIC NEUROPATHIC PAIN MODELS, OUTSTANDING DRUG-LIKE PROPERTIES, AND NO LIMITING CV OR CNS SAFETY RISK. IN THE UH3 PHASE, THE SELECTED CLINICAL CANDIDATE WILL BE SUBJECTED TO A STANDARD BATTERY OF REQUIRED IND-ENABLING STUDIES, LEADING TO AN INVESTIGATIONAL NEW DRUG APPLICATION (IND). UPON ACCEPTANCE OF THE IND, WE WILL CONDUCT A PHASE I, SINGLE ASCENDING DOSE STUDY IN HEALTHY VOLUNTEERS TO ASSESS THE SAFETY, TOLERABILITY AND PK OF OUR CLINICAL CANDIDATE.

SURVIVAL MECHANISMS FOR APOPTOTIC CASPASE

LIGAND FUNCTIONAL SELECTIVITY IN EPHA2 RECEPTOR SIGNALING

TARGETED NANOPARTICLES FOR BREAST CANCER DIAGNOSIS AND THERAPY

HVEM-BTLA SYSTEM IN INFLAMMATION

ANTI-HERPESVIRUS SIGNALING BY LTALPHABETA/LIGHT CYTOKINES

COMPARATIVE RESISTOMICS OF GRAM-NEGATIVE BACTERIAL PATHOGENS - ABSTRACT INCREASING ANTIBIOTIC RESISTANCE NECESSITATES EXPANDING RESEARCH INTO THE MECHANISMS BY WHICH BACTERIAL PATHOGENS ACQUIRE AND PERPETUATE DRUG RESISTANCE. DESPITE RAPIDLY EXPANDING GENOMIC MAPPING OF RESISTANCE-CONFERRING MUTATIONS IN CLINICAL ISOLATES AND LABORATORY STUDIES, OUR KNOWLEDGE OF DYNAMICS AND MECHANISMS UNDERLYING EVOLUTION OF ANTIMICROBIAL RESISTANCE IS STILL INSUFFICIENT. TO FILL-IN THIS GAP, THE AUTHORS OF THIS PROPOSAL COMBINE EXPERIMENTAL EVOLUTION IN A CONTINUOUS CULTURING DEVICE, MORBIDOSTAT, WITH TIME- RESOLVED ULTRADEEP GENOMIC SEQUENCING OF EVOLVING BACTERIAL CULTURES. THE UTILITY OF THE DEVELOPED MORBIDOSTAT-BASED WORKFLOW IS SUPPORTED BY PUBLISHED AND ONGOING STUDIES WITH ESTABLISHED ANTIMICROBIALS AND EXPERIMENTAL DRUG CANDIDATES. THE PRELIMINARY RESULTS OF COMPARATIVE RESISTOMICS STUDIES OVER A RANGE OF GRAM-NEGATIVE BACTERIAL SPECIES PROVIDED INITIAL SUPPORT TO A PREMISE THAT EVOLUTION OF DRUG RESISTANCE IN MORBIDOSTAT PROCEEDS VIA A LIMITED SET OF TRAJECTORIES DEFINED BY A COMBINATION OF RESISTANCE AND FITNESS CONSTRAINS APPROXIMATING CLINICAL EVOLUTION, WHICH FAVORS SELECTION OF LOW-FREQUENCY/HIGH-FITNESS OVER HIGH- FREQUENCY/LOW-FITNESS MUTANTS. A COMPARATIVE RESISTOMICS APPROACH ENABLES MAPPING OF BOTH UNIVERSAL AND STRAIN-SPECIFIC MECHANISMS AS DEMONSTRATED IN A RECENT PROOF-OF-CONCEPT STUDY ON EXPERIMENTAL EVOLUTION OF CIPROFLOXACIN RESISTANCE IN THREE GRAM-NEGATIVE BACTERIA. THE PROPOSED 5-YEAR PROJECT WILL TEST THE CENTRAL HYPOTHESIS AND EXTEND EXPLORATION OF ANTIMICROBIAL RESISTOME BY PURSUING THE FOLLOWING SPECIFIC AIMS: (I) IN AIM 1, THE ESTABLISHED MORBIDOSTAT-BASED WORKFLOW WILL BE USED TO DETERMINE MAJOR MECHANISMS DRIVING RESISTANCE TO BROAD-SPECTRUM CLINICAL ANTIBIOTICS, CIPROFLOXACIN, COLISTIN, TIGECYCLINE AND MEROPENEM, IN FOUR DIFFICULT-TO-TREAT GRAM-NEGATIVE BACTERIAL PATHOGENS, ACINETOBACTER BAUMANNII ATCC17978, P. AERUGINOSA ATCC27853, E. COLI ATCC25922, AND K. PNEUMONIAE ATCC13883; (II) IN AIM 2, A REPRESENTATIVE PANEL OF SELECTED CLONES WILL BE SYSTEMATICALLY CHARACTERIZED TO ASSESS THE EFFECTS OF INDIVIDUAL MUTATIONS AND COMBINATIONS THEREOF ON ACQUIRED RESISTANCE AND FITNESS; (III) AIM 3 WILL LEVERAGE A MORIBIDOSTAT-BASED WORKFLOW TO MAKE FIRST STEPS TOWARD EXPERIMENTAL EVOLUTION OF MULTIDRUG RESISTANCE FOCUSING ON A. BAUMANNII AND STARTING FROM CLONES SELECTED IN SINGLE-DRUG EVOLUTION STUDIES. THE RESULTS THAT WILL BE OBTAINED IN ALL PLANNED STUDIES WILL BE A SUBJECT OF IN-DEPTH BIOINFORMATICS ANALYSIS (INCLUDING COMPARISON WITH PUBLIC DATA FOR CLINICAL ISOLATES), PREDICTIVE MODELING, INTEGRATION AND SHARING WITH BROAD RESEARCH COMMUNITY VIA A SPECIALIZED WEB-SITE ON INTEGRATIVE GENOMICS OF EVOLUTION OF ANTIMICROBIAL RESISTANCE (IGEAR). THE PROPOSED STUDY IS EXPECTED TO HAVE TRANSLATIONAL IMPACTS IN ADVANCING METHODOLOGY TO SUPPORT RATIONAL OPTIMIZATION OF ANTIBIOTIC TREATMENT REGIMENS AND DEVELOPMENT OF NEW DRUGS WITH MINIMIZED RESISTIBILITY.

MOLECULAR MECHANISM OF BCL2-DEPENDENT APOPTOSIS

THE NRF2-FBP1 CROSSREGULATORY LOOP AND THE CONTROL OF HEALTHY AND DISEASED LIVER METABOLISM - PROJECT SUMMARY/ABSTRACT FRUCTOSE BISPHOSPHATE PHOSPHATASE (FBP1) IS THE RATE-LIMITING ENZYME IN GLUCONEOGENESIS (GNG), WHEREAS NUCLEAR FACTOR ERYTHROID-RELATED FACTOR 2 ENCODED BY THE NFE2L2 GENE (NRF2) IS A TRANSCRIPTION FACTOR, PREVIOUSLY IDENTIFIED AS THE MASTER ACTIVATOR OF THE ANTIOXIDANT DEFENSE RESPONSE. NRF2 ALSO ACTIVATES THE TRANSCRIPTION OF MANY METABOLIC GENES, ESPECIALLY IN LIVER. WE FOUND THAT NRF2ACT-HEP MICE, IN WHICH NRF2 WAS SELECTIVELY ACTIVATED IN HEPATOCYTES, AND FASTED FBP1HEP MICE, IN WHICH FBP1 WAS CONDITIONALLY DELETED IN HEPATOCYTES, EXHIBIT SIMILAR METABOLIC PHENOTYPES, INCLUDING HYPOGLYCEMIA, HEPATOMEGALY, HEPATOSTEATOSIS AND HYPERTRIGLYCERIDEMIA, WHICH ARE ALSO MANIFESTED BY INSULIN OVERDOSED INDIVIDUALS AND GLUCOSE- OR CARBOHYDRATE-DEPRIVED FBP1-DEFICIENT CHILDREN. GIVEN THESE SIMILARITIES, WE ASKED WHETHER NRF2 AND FBP1 ENGAGE IN BIOCHEMICAL CROSSTALK. SURPRISINGLY, WE FOUND THAT HEPATIC ACTIVATION OF NRF2 INDUCES FBP1 DEGRADATION, MEDIATED BY NRF2 INDUCED EGF AND PDGF EXPRESSION, WHICH THROUGH AN AUTOCRINE SIGNALING MECHANISM LED TO ACTIVATION OF ERK1/2 MAP KINASES THAT PHOSPHORYLATED FBP1 AT SERINE 271 AND TRIGGERED ITS UBIQUITINATION AND PROTEASOMAL DEGRADATION. EVEN MORE SURPRISING WAS THE FINDING THAT FBP1 EXPRESSION LED TO INHIBITION OF AKT, THEREBY RELIEVING INHIBITORY PHOSPHORYLATION OF GSK3 ISOZYMES, WHICH PHOSPHORYLATE A DEGRON EMBEDDED WITHIN THE NRF2 MOLECULE AND THEREBY INDUCE ITS UBIQUITIN-DEPENDENT PROTEOLYSIS. THESE FINDINGS LED US TO HYPOTHESIZE THAT THE NRF2-FBP1 CROSSREGULATORY LOOP IS A KEY REGULATOR OF LIVER METABOLISM AND HOMEOSTASIS, WHOSE ABERRANT FUNCTION CAN PROMOTE LIVER DAMAGE AND CANCER. WE PLAN TO TEST THIS HYPOTHESIS THROUGH THREE SPECIFIC AIMS: 1). INVESTIGATE THE HYPOTHESIS THAT FBP1 INDUCES NRF2 DEGRADATION IN PERIPORTAL HEPATOCYTES BY ACTIVATING GSK3 OR ENHANCING ITS RECRUITMENT TO NRF2; 2). DETERMINE WHETHER NRF2 ACTIVATION ALTERS LIVER ZONATION AND CONTRIBUTES TO THE METABOLIC DEFECTS CAUSED BY FBP1 ABLATION; 3). INVESTIGATE WHETHER NRF2-INDUCED FBP1 DEGRADATION OR NRF2 UPREGULATION CONTROL THE PROGRESSION FROM CHRONIC METABOLIC STRESS TO HEPATOCELLULAR CARCINOMA. PURSUING THESE AIMS VIA NEW MOUSE MODELS, CELL BIOLOGICAL STUDIES AND HIGHLY INNOVATIVE SEQ-SCOPE TECHNOLOGY, WHICH WE HAD DEVELOPED FOR HIGH-CONTENT SPATIAL TRANSCRIPTOMIC AND PROTEOMIC PROFILING OF SINGLE LIVER CELLS, WILL ANSWER SEVERAL CRITICAL QUESTIONS OF GENERAL IMPORTANCE: 1). WHAT ARE THE NON-ENZYMATIC MECHANISMS THROUGH WHICH FBP1 HAS A BROAD EFFECT ON LIVER METABOLISM BEYOND ITS WELL-STUDIED INVOLVEMENT IN GNG? 2). WHAT IS THE ROLE OF NRF2 IN THE METABOLIC ALTERATIONS CAUSED BY FBP1 DEFICIENCY? 3). WHAT IS THE ROLE OF A PREVIOUSLY DESCRIBED FBP1- ALDOLASE B INTERACTION IN AKT INHIBITION AND GSK3-INDUCED NRF2 DEGRADATION? AND 4). WHAT IS THE ROLE OF AKT ACTIVATION IN THE METABOLIC DEFECTS AND INCREASED SUSCEPTIBILITY TO ONCOGENIC TRANSFORMATION EXHIBITED BY THE FBP1-DEFICIENT LIVER?

DEVELOPMENT OF SBI-553, AN ALLOSTERIC MODULATOR OF NTR1, FOR THE TREATMENT OF SUBSTANCE USE DISORDERS

PRECLINICAL DISCOVERY OF NOVEL FARNESYLTRANSFERASE INHIBITORS FOR THE TREATMENT OF ALZHEIMER'S DISEASE AND RELATED TAUOPATHIES - PROJECT SUMMARY ALZHEIMER’S DISEASE (AD) IS A FATAL DISEASE THAT CURRENTLY AFFLICTS ALMOST SIX MILLION AMERICANS. WITH AN AGING POPULATION, WE RISK A PUBLIC HEALTH CRISIS BY 2050, UNLESS EFFECTIVE TREATMENTS ARE IDENTIFIED. DESPITE EXTENSIVE RESEARCH, THERE ARE CURRENTLY NO DRUGS THAT SLOW OR ALTER THE COURSE OF DISEASE. AD IS DEFINED BY THE PRESENCE OF SS-AMYLOID (ASS) PLAQUES AND INTRANEURONAL TAU INCLUSIONS CALLED NEUROFIBRILLARY TANGLES (NFTS) IN THE BRAIN. DRUG CANDIDATES THAT REDUCE ASS PLAQUES HAVE NOT, YET, BEEN SHOWN TO HAVE CLINICAL BENEFIT, AND GROWING DATA SUGGESTS THAT IT MAY BE MORE IMPORTANT TO TARGET NFTS OVER ASS PLAQUES TO PREVENT COGNITIVE DECLINE. RECENTLY, THE MACROAUTOPHAGY-LYSOSOMAL PATHWAY OF PROTEIN DEGRADATION HAS EMERGED AS A COMPELLING TARGET FOR REDUCING PATHOGENIC TAU IN THE BRAIN. OUR HYPOTHESIS IS THAT INCREASING THE RATE OF TAU DEGRADATION WILL REDUCE TAU LEVELS AND STOP, OR GREATLY SLOW, THE RATE OF TAU AGGREGATION. WE RECENTLY DISCOVERED A NOVEL PATHWAY TO ACCOMPLISH THIS OBJECTIVE. INHIBITING THE FARNESYLATION OF RHES, A GTPASE PROTEIN IN THE RAS FAMILY, ACTIVATES THE LYSOSOME AND RESULTS IN THE SELECTIVE DEGRADATION OF PATHOLOGICAL TAU. CONFIRMATION OF THE THERAPEUTIC HYPOTHESIS WAS ACHIEVED BY ADMINISTERING A FARNESYLTRANSFERASE INHIBITOR (FTI) IN A MOUSE MODEL OF TAUOPATHY, WHICH REDUCED TAU PATHOLOGY AND ATTENUATED BEHAVIORAL ABNORMALITIES IN THE MICE. KNOWN FTIS ARE NOT SUITABLE FOR HUMAN DEVELOPMENT AS CNS DRUGS. OPTIMIZED FOR CANCER INDICATIONS, THEY ARE EFFICIENTLY PUMPED OUT OF THE BRAIN BY EFFLUX PROTEINS. WE PROPOSE A THREE-PRONGED APPROACH TO IDENTIFY CHEMICAL MATTER THAT CAN REACH PHARMACOLOGICALLY SIGNIFICANT AND DOSE-PROPORTIONAL BRAIN LEVELS. FOR TWO OF THE KNOWN INHIBITORS, L-778,123 AND LONAFARNIB, WE WILL MAKE STRATEGIC CHANGES TO THE STRUCTURES, ELIMINATING FUNCTIONAL GROUPS THAT SERVE AS RECOGNITION SUBSTRATES FOR THE EFFLUX PUMPS. CONCURRENTLY, WE WILL INITIATE A HIGH- THROUGHPUT SCREEN OF A CHEMICAL LIBRARY WITH CHEMICAL PROPERTIES CONSISTENT WITH KNOWN CNS DRUGS. AS A THIRD STEP, WE WILL ENGAGE IN A MULTI-MILLION COMPOUND ARTIFICIAL INTELLIGENCE-BASED VIRTUAL SCREEN WITH ATOMWISE TO IDENTIFY NOVEL FTIS. BY GENERATING X-RAY CO-CRYSTAL STRUCTURES OF THE MOST PROMISING HITS AND USING COMPUTER- AIDED DRUG DESIGN, WE PLAN TO ACCELERATE THE PROCESS OF HIT VALIDATION, LEAD DISCOVERY, AND OPTIMIZATION TO IDENTIFY SMALL MOLECULE DRUG CANDIDATES. WE WILL ADVANCE INHIBITORS TO AN IN VIVO PHARMACODYNAMIC MODEL AND SELECT COMPOUNDS WITH LINEAR PHARMACOKINETIC/PHARMACODYNAMIC (PK/PD) RELATIONSHIPS THAT CAN BE ADVANCED INTO THE CLINIC. THREE OF THE TOP COMPOUNDS WILL BE TESTED FOR EFFICACY IN A TAUOPATHY ANIMAL MODEL USING DOSES DERIVED FROM THE PK/PD RELATIONSHIP. SHORT-TERM STUDIES WILL IDENTIFY COMPOUNDS THAT REDUCE ALL PATHOGENIC TAU SPECIES. THE MOST EFFICACIOUS COMPOUND WILL BE MOVED INTO LONG-TERM DOSING STUDIES TO EVALUATE LIFE-SPAN EXTENSION AND REDUCTION IN NFT FORMATION.

SIGNAL TRANSDUCTION AND CELL DEATH REGULATION

CYCLIC EPHA4 PEPTIDE ANTAGONISTS FOR NEUROPROTECTION IN ALS

ER STRESS AND UPR IN NON-ALCOHOLIC STEATOHEPATITIS AND HEPATOCELLULAR CARCINOMA

NOVEL ROLES FOR THE ALZHEIMER'S DISEASE (AD) RISK GENE, SORLA IN NEUROPROTECTION IN AD

CONTROL OF MUSCLE GENE EXPRESSION BY SIGNALING PATHWAYS

TARGETING NEUROINFLAMMATION IN AD WITH NOVEL CX3CR1 AGONISTS

DEVELOPMENT OF SMAC MIMETICS AS LATENCY-REVERSING AGENTS

BRAIN PATHOLOGY AND FUNCTION IN A CHRONIC MOUSE MODEL OF ZIKV TRANSMISSION

CHARACTERIZING THE NUCLEAR PORE COMPLEX-T CELL RECEPTOR CONNECTION

PLEKHA7 AND BETA-CATENIN INTERACT TO REGULATE MUTANT KRAS

ROLE OF FBXW7-MEDIATED PROTEASOMAL DEGRADATION IN MYOFIBERS IN DETERMINING MUSCLE STEM CELL POOL SIZE

MECHANISMS OF INITATION OF SKELETAL MINERALIZATION

SOMATIC GENE RECOMBINATION OF MAPT IN SINGLE CELLS FROM FRONTOTEMPORAL LOBAR DEMENTIA (FTLD) BRAINS WITH TAU PATHOLOGY

MECHANISMS THAT COUPLE IRREGULAR DEVELOPMENT OF FETAL MELANOBLASTS TO PREMATURE EXHAUSTION OF ADULT MELANOCYTE STEM CELLS - PROJECT SUMMARY MAINTENANCE OF TISSUE FUNCTION DURING ADULTHOOD, AND HENCE SUPPRESSION OF TISSUE DEGENERATION AND DISEASE, DEPENDS ON MAINTENANCE OF STEM CELL POPULATIONS. ADULT STEM CELLS ARE EPIGENETICALLY PROGRAMMED AND A CULMINATION OF A SERIES OF DEVELOPMENTAL DECISIONS INITIATED IN THE EMBRYO. IN UTERO ENVIRONMENTAL EXPOSURES ON THE EMBRYO CAN INFLUENCE ADULT AND LATE-LIFE DISEASE, LIKELY IN PART VIA EFFECTS ON STEM CELL DEVELOPMENT THAT ARE TRANSMITTED TO MAINTENANCE AND FUNCTION OF STEM CELLS IN THE ADULT. HOWEVER, THE MOLECULAR LINKS BETWEEN EMBRYONIC DEVELOPMENT AND LONG-TERM MAINTENANCE OF STEM CELL FUNCTION AND PHENOTYPE IN ADULTS ARE POORLY DEFINED. WE WILL EMPLOY LINEAGE-SPECIFIC GENETIC INACTIVATION OF A HISTONE CHAPERONE TO UNDERSTAND HOW EMBRYONIC DEVELOPMENTAL INTEGRITY OF MELANOBLASTS (MB) IMPACTS ON MAINTENANCE ADULT MELANOCYTE (MC) STEM CELLS (MCSC). THE HISTONE CHAPERONE HIRA DEPOSITS HISTONE VARIANT H3.3 INTO ACTIVE GENES, PROMOTERS AND ENHANCERS. THROUGH IN VITRO AND IN VIVO STUDIES AND SINGLE CELL RNA-SEQ OF MOUSE EMBRYO MELANOBLASTS (MBS) FROM WILD TYPE MICE AND MICE LACKING EXPRESSION OF HIRA IN EMBRYONIC MBS, WE HAVE UNCOVERED A ROLE FOR HIRA IN SUSTAINING THE PAX3/SOX10-MITF MB SPECIFICATION PATHWAY. INACTIVATION OF HIRA IN MBS DEPLETES THE NUMBER OF MBS IN EARLY/MID STAGE EMBRYOS. HOWEVER, THIS EMBRYONIC DEFECT IS RESCUED BY BIRTH AND YOUNG MICE EXHIBIT NORMAL NUMBERS OF MELANOCYTIC CELLS, AND ONLY A VERY SUBTLE PIGMENTATION DEFECT. NEVERTHELESS, IN NEW-BORN MICE, HIRA KNOCK OUT (KO) MELANOCYTIC CELLS EXHIBIT A HIGHER FREQUENCY OF TELOMERE-ASSOCIATED DNA DAMAGE FOCI, INDICATING THAT HIRA KNOCK OUT MCSC AND/OR MELANOCYTES HARBOR MOLECULAR DAMAGE, EVEN IN NEW-BORN MICE. INDEED, MELANOBLASTS AND MELANOCYTES FROM NEW-BORN HIRA KO MICE RESPOND POORLY TO PRO-PROLIFERATIVE CHALLENGE IN VITRO AND IN VIVO, AND THESE MICE SHOW MARKED ACCELERATED MCSC AND MELANOCYTE DEPLETION AND DRAMATICALLY ACCELERATED HAIR GREYING DURING ADULTHOOD. BUILDING ON THESE EXTENSIVE PRELIMINARY DATA, WE WILL INVESTIGATE THE ROLE OF HIRA IN DIFFERENTIATION AND DEVELOPMENT OF THE MELANOCYTIC LINEAGE, AND INVESTIGATE THE LINKS BETWEEN ABNORMAL EMBRYONIC DEVELOPMENT AND ADULT STEM CELL DEPLETION DURING ADULTHOOD AND AGING. DYSREGULATION OF THE PAX3/SOX10-MITF SIGNALING PATHWAY CONTRIBUTES TO DEVELOPMENTAL DISORDERS AND MELANOMA. THESE STUDIES TO DEFINE HIRA'S ROLE IN THE PAX3/SOX10-MITF AXIS CAN PROMOTE THERAPEUTIC INTERVENTIONS TO COMBAT THESE DEVELOPMENTAL AND NEOPLASTIC DISORDERS. MOREOVER, COMPLETION OF THESE SPECIFIC AIMS WILL ADDRESS HOW THE INTEGRITY OF EMBRYONIC DEVELOPMENT OF TISSUE SPECIFIC STEM CELLS IMPACTS MAINTENANCE OF THOSE STEM CELLS DURING ADULTHOOD.

REGULATION OF ER STRESS-INDUCED CELL DEATH

IMPROVING PROINSULIN FOLDING TO AMELIORATE TYPE II DIABETES - PROJECT SUMMARY DIABETES IS AMONG THE FASTEST GROWING HEALTH CHALLENGES OF THE 21ST CENTURY, AFFECTING >30 MILLION PEOPLE, WITH =80,000 DEATHS ANNUALLY, AND INVOLVING ~15% OF U.S. NATIONAL HEALTH EXPENDITURES. TYPE 2 DIABETES (T2D) IS THE MOST COMMON FORM OF DIABETES, WHICH IS LINKED TO AN INSUFFICIENT AMOUNT OF CIRCULATING INSULIN BECAUSE OF THE BODY’S INSENSITIVITY TO THE HORMONE. MAINTENANCE OF THE INSULIN STORAGE POOL REQUIRES SYNTHESIS OF ~6000 PROINSULIN (PI) MOLECULES/SS-CELL/SECOND, EACH DELIVERED TO THE ENDOPLASMIC RETICULUM (ER) FOR FOLDING. EVEN MORE MOLECULES ARE NEEDED IN STATES OF INSULIN RESISTANCE. SIGNIFICANTLY, WE DISCOVERED THAT PI ENTERS ABERRANT DISULFIDE-LINKED INTERMOLECULAR COMPLEXES, EVEN IN HEALTHY (HUMAN AND MURINE) ISLETS. UNDER CONDITIONS THAT DEMAND INCREASED INSULIN PRODUCTION (EVEN PREDIABETES), THESE COMPLEXES DRAMATICALLY INCREASE, THUS LIMITING INSULIN PRODUCTION. WE SHOW NEW KEY EVIDENCE THAT THESE ABERRANTLY FOLDED PI COMPLEXES CAN BE RESOLVED TO MONOMERIC PI WITHIN THE ER. WE RECENTLY ELUCIDATED THE FIRST MAP OF THE HUMAN PI INTERACTOME IDENTIFYING PI FOLDING MODIFIERS. THE MOST SIGNIFICANT PI INTERACTOR IN HUMAN ISLETS IS THE ER CHAPERONE BIP AND WE PRESENT NEW EVIDENCE (BOTH GAIN OF FUNCTION, AND LOSS OF FUNCTION) THAT THIS INTERACTION, SUPPORTED BY BIP CO- CHAPERONES, IS ABSOLUTELY REQUIRED FOR PRODUCTIVE PROINSULIN FOLDING (AND LIMITING MISFOLDING), LEADING TO SUCCESSFUL ANTEROGRADE TRANSPORT. FOR OUR STUDIES WE GENERATED A NOVEL BIP-TAGGED MOUSE THAT CAN FOR THE FIRST TIME IDENTIFY FUNDAMENTAL STEPS IN PI FOLDING ESSENTIAL FOR INSULIN PRODUCTION. MOREOVER, WE SHOW THAT INCREASED EXPRESSION OF BIP AND ITS CO-CHAPERONE P58IPK DRAMATICALLY REDUCES ACCUMULATION OF THE HIGH MOLECULAR WEIGHT PI COMPLEXES. THUS, OUR DISCOVERIES OPEN THE POSSIBILITY THAT PHARMACOLOGIC INTERVENTION MAY IMPROVE CHAPERONE-DEPENDENT PI FOLDING, AND THIS MAY ATTENUATE T2D. AS WE BEGIN TO ELUCIDATE THE HUMAN PI FOLDING PATHWAY, WE ARE DEVELOPING PARALLEL ANIMAL MODELS TO DETERMINE HOW PI FOLDS/MISFOLDS. HERE WE PROPOSE TO: 1) MECHANISTICALLY DISSECT HOW BIP AND ADDITIONAL PI INTERACTORS IN THE ER ORCHESTRATE SUCCESSFUL PI FOLDING AND DETERMINE WHICH STEP(S) OF PI FOLDING GO AWRY IN T2D; 2) IDENTIFY HOW THE PI INTERACTOME CHANGES IN HUMAN T2D; DETERMINE THE FUNCTION OF ALTERED PI INTERACTIONS IN ISLETS FROM PATIENTS WITH T2D; AND UTILIZE NOVEL ASSAYS TO MEASURE PRODUCTIVE PI FOLDING/TRAFFICKING IN SS-CELLS. WE WILL INTEGRATE PHYSIOLOGIC STUDIES OF HUMAN ISLETS WITH NOVEL GENETIC AND BIOCHEMICAL APPROACHES TO GENERATE A COMPREHENSIVE UNDERSTANDING OF HOW PI FOLDING HOMEOSTASIS IMPACTS SS-CELL FUNCTION IN HEALTH AND DISEASE. WE BELIEVE THAT THIS HYPOTHESIS IS A HIGH-IMPACT IDEA ESSENTIAL TO THE MISSION OF THE NIDDK, AND WE NOW BRING TOOLS, ASSAYS, AND APPROACHES THAT ARE NOT CURRENTLY AVAILABLE ANYWHERE ELSE.

NG2-PG IN TUMOR VASCULARIZATION AND PROGRESSION

OVERRIDING THE IMMUNE EVASION TACTICS OF CORONAVIRUS

DISCOVERY OF SELECTIVE INHIBITORS FOR THE EPHA4 KINASE

VIRULENCE FACTORS AND CELL DEATH

LINKING THE MICROBIOME AND IMMUNE-CHECKPOINT IN MELANOMA BY RNF5

FRAGMENT-BASED DISCOVERY OF STEP MODULATORS IN ALZHEIMER'S DISEASE ADMINISTRATIVE SUPPLEMENT - PROJECT SUMMARY STRIATAL-ENRICHED TYROSINE PHOSPHATASE (STEP) IS A NEURON-SPECIFIC PROTEIN TYROSINE PHOSPHATASE (PTP) AND A NOVEL THERAPEUTIC TARGET FOR ALZHEIMER’S DISEASE (AD), A DEBILITATING NEURODEGENERATIVE DISORDER FOR WHICH NO CURE CURRENTLY EXISTS. MULTIPLE STUDIES INDICATE THAT LEVELS OF STEP ARE ELEVATED IN AD AND OTHER NEURODEGENERATIVE AND NEUROPSYCHIATRIC DISORDERS, INCLUDING PARKINSON’S DISEASE, SCHIZOPHRENIA, AND FRAGILE X SYNDROME. THE DATA SUGGEST THAT INCREASED STEP ACTIVITY INTERFERES WITH SYNAPTIC FUNCTION AND CONTRIBUTES TO THE CHARACTERISTIC COGNITIVE AND BEHAVIORAL DEFICITS IN THESE DEVASTATING DISEASES. CROSSING STEP KO MICE WITH MOUSE MODELS OF AD, SCHIZOPHRENIA, OR FRAGILE X SYNDROME COMPLETELY REVERSED THE COGNITIVE AND BEHAVIORAL DEFICITS, GENERATING PROGENY WITH LOWER STEP LEVELS INDISTINGUISHABLE FROM WT MICE. SIMILAR EFFECTS WERE OBSERVED WHEN STEP WAS PHARMACOLOGICALLY INHIBITED BY OUR TOOL COMPOUND TC-2153. THESE STUDIES VALIDATE STEP AS A NOVEL DRUG TARGET FOR THE TREATMENT OF AD AND OTHER NEURODEGENERATIVE AND NEUROPSYCHIATRIC DISORDERS. TC-2153 IS THE ONLY REPORTED STEP INHIBITOR WITH CELLULAR AND IN VIVO ACTIVITY. HOWEVER, TC-2153 AND SIMILAR COMPOUNDS ARE KNOWN TO REACT WITH CELLULAR THIOLS AND MODIFY DNA, PRECLUDING THIS INHIBITOR FROM FURTHER PRECLINICAL STUDIES. OTHER REPORTED STEP INHIBITORS SUFFER FROM POOR SELECTIVITY FOR STEP AND LACK OF EFFICACY UNDER PHYSIOLOGICAL CONDITIONS. TO OVERCOME THESE TYPICAL CHALLENGES FOR INHIBITORS TARGETING PTPS - ENZYMES THAT HAVE A HIGHLY CONSERVED AND HIGHLY CHARGED ACTIVE SITE - WE SET OUT TO DISCOVER SMALL MOLECULES THAT BIND TO LESS CONSERVED, ALLOSTERIC SITES IN STEP. USING INNOVATIVE FRAGMENT-BASED SCREENING TECHNOLOGIES, WE IDENTIFIED 19 FRAGMENTS, LOW MOLECULAR WEIGHT COMPOUNDS ADHERING TO THE “RULE OF 3”, THAT SELECTIVELY BIND TO NOVEL, LESS CONSERVED SITES IN STEP WITH UP TO SINGLE DIGIT MICROMOLAR AFFINITY. THESE FRAGMENTS ARE IDEAL STARTING POINTS FOR GENERATING STEP-TARGETED DEGRADERS SUCH AS PROTEOLYSIS TARGETING CHIMERAS (PROTACS) WITH PHYSICOCHEMICAL PROPERTIES SUITABLE FOR CROSSING THE BLOOD-BRAIN BARRIER (BBB). IN FACT, IN PRELIMINARY EXPERIMENTS WE HAVE DEMONSTRATED THAT A PROTAC BASED ON OUR BEST FRAGMENT CAN EFFECTIVELY REDUCE STEP LEVELS IN VIVO. BASED ON THESE EXCITING RESULTS, WE PROPOSE A STEP-TARGETED PROTEIN DEGRADATION APPROACH AS A THERAPEUTIC STRATEGY IN AD. IN AIM 1, WE WILL OPTIMIZE OUR FRAGMENT BINDERS FOR LIGAND EFFICIENCY AND STEP SELECTIVITY. IN AIM 2, WE WILL GENERATE STEP PROTACS FROM THE MOST PROMISING BINDERS, ASSESS THEM IN OUR ESTABLISHED TESTING FUNNEL, AND OPTIMIZE THEM FOR DRUG METABOLISM AND PHARMACOKINETICS (DMPK), PHARMACODYNAMICS (PD), AND TOXICITY (TOX) PROPERTIES. THE GOAL IS TO GENERATE NON-TOXIC STEP DEGRADERS WITH EXPOSURE AND EFFICACY IN THE BRAIN. IN AIM 3, WE WILL TEST WHETHER CANDIDATE PROTACS ARE ABLE TO IMPROVE COGNITION IN MOUSE AD MODELS. OUR STUDIES ARE DESIGNED TO PROVIDE PROOF-OF-CONCEPT (POC) FOR STEP DEGRADERS AS NOVEL THERAPEUTICS IN AD. WE EXPECT TO DEVELOP AT LEAST ONE LEAD SERIES AND ONE BACKUP SERIES OF EFFECTIVE PROTACS THAT ARE READY FOR PRECLINICAL DEVELOPMENT TOWARDS A FIRST-IN-CLASS EARLY TREATMENT OPTION FOR AD.

EPHA2 RECEPTOR SIGNALING IN BREAST CANCER MECHANOTRANSDUCTION - SUMMARY MECHANICAL FORCES GENERATED BY A RIGID EXTRACELLULAR MATRIX (ECM) IN THE TUMOR MICROENVIRONMENT PLAY A KEY ROLE IN TUMOR PROGRESSION AND METASTASIS. WE RECENTLY DISCOVERED A CRITICAL ROLE OF EPHA2 NON-CANONICAL SIGNALING IN PROMOTING EPITHELIAL-MESENCHYMAL TRANSITION (EMT) AND TUMOR INVASION AND METASTASIS IN RESPONSE TO INCREASING ECM STIFFNESS IN THE TUMOR MICROENVIRONMENT. FURTHERMORE, WE FOUND THAT ACTIVATION OF EPHA2 CANONICAL SIGNALING BY THE EPHRINA1 LIGAND POTENTLY INHIBITS STIFFNESS-INDUCED BREAST CANCER CELL INVASIVENESS. BASED ON THESE RESULTS, WE HYPOTHESIZE THAT EPHA2 FUNCTIONS AS A KEY RHEOSTAT THAT INTEGRATES BOTH MECHANICAL AND BIOCHEMICAL CUES FROM THE TUMOR MICROENVIRONMENT TO REGULATE MECHANOSIGNALING IN BREAST CANCER PROGRESSION AND METASTASIS. WE PROPOSE TO COMBINE BIOCHEMICAL, 2D AND 3D CELL CULTURE ASSAYS, AND IN VIVO XENOGRAFT MODELS TO ELUCIDATE EPHA2 SIGNALING MECHANISMS IN BREAST CANCER MALIGNANCY INDUCED BY ECM RIGIDITY THROUGH THREE SPECIFIC AIMS. (1) DETERMINE HOW ECM RIGIDITY ACTIVATES EPHA2 NON-CANONICAL SIGNALING TO PROMOTE EMT AND INVASION. (2) DETERMINE HOW EPHA2 CANONICAL SIGNALING BLOCKS EMT AND INVASION INDUCED BY ECM RIGIDITY. (3) EXAMINE THE ROLE OF EPHA2 NON-CANONICAL AND CANONICAL SIGNALING IN BREAST CANCER INVASION AND METASTASIS IN VIVO. TOGETHER, THESE STUDIES WILL INFORM ON THE POTENTIAL USEFULNESS OF ACTIVATING EPHA2 CANONICAL SIGNALING FOR INHIBITION OF BREAST CANCER INVASIVENESS AND METASTASIS.

A GENOMIC/METABOLOMIC STRATEGY TO CHARACTERIZE CARDIAC MITOCHONDRIAL DYSFUNCTION

HOMEOSTATIC ROLE OF IRE1A-XBP1-PDI1 IN HEPATIC LIPID METABOLISM

GENERATION OF RETINOID SIGNALS DURING DEVELOPMENT

MICROGLIAL HEPARAN SULFATE IN THE MODULATION OF APOE FUNCTION AND NEURODEGENERATION - HEPARAN SULFATE (HS), A SULFATED GLYCAN EXPRESSED AT THE CELL SURFACE AND IN EXTRACELLULAR MATRIX, HAS LONG ATTRACTED ATTENTION AS A PUTATIVE FACTOR INVOLVED IN ALZHEIMER'S DISEASE (AD), BASED ON CIRCUMSTANTIAL EVIDENCE FROM IN VITRO AND CLINICOPATHOLOGICAL STUDIES. NEVERTHELESS, THE FUNCTIONAL SIGNIFICANCE OF HS IN AD, ESPECIALLY IN THE LATE-ONSET TYPE (LOAD) THAT COMPRISES MORE THAN 90% OF AD CASES, IS ELUSIVE. RECENT HUMAN GENOMIC STUDIES IMPLICATE APOE AND MICROGLIA AS DOMINANT CONTRIBUTORS TO NEURODEGENERATION IN LOAD. THE APOE GENE IS THE STRONGEST KNOWN GENETIC RISK FACTOR FOR LOAD. ACCUMULATING DATA INDICATE THAT APOE PROTEINS EXERT IMMUNOMODULATORY EFFECTS ON MICROGLIA, AND THAT THIS FUNCTION OF APOE IS AT LEAST PARTLY MEDIATED BY THE TREM2 RECEPTOR, ANOTHER AD RISK FACTOR THAT IS EXPRESSED BY MICROGLIA. AMONG THE THREE HUMAN APOE ALLELES, THE APOE4 ALLELE CONFERS AN INCREASED RISK FOR LOAD, WHILE THE APOE2 ALLELE CONFERS A DECREASED RISK RELATIVE TO THE MORE COMMON APOE3 ALLELE. THE MOLECULAR BASIS OF THESE ALLELE- SPECIFIC RISK VARIATIONS IS ONE OF THE KEY UNANSWERED ISSUES IN AD RESEARCH. IN THIS CONTEXT, IT IS INTERESTING TO NOTE THAT APOE PROTEINS BIND HS IN AN ISOFORM-SPECIFIC MANNER — APOE4 EXHIBITS 2- TO 3-FOLD GREATER AFFINITY FOR HS THAN APOE2 AND APOE3, THUS APPARENTLY CORRELATING WITH THEIR RELATIVE AD RISKS. FURTHERMORE, A RECENT REPORT REGARDING THE UNIQUE CASE OF A COLUMBIAN WOMAN, WHO CARRIES A HIGHLY DETRIMENTAL PSEN1E280A MUTATION, SUGGESTS THAT HER APOE3 "CHRISTCHURCH" MUTATION, WHICH ABOLISHES THE AFFINITY OF APOE3 FOR HS, CONFERS STRONG PROTECTION AGAINST NEURODEGENERATION AND COGNITIVE IMPAIRMENT. ALSO SUGGESTING THE FUNCTIONAL INVOLVEMENT OF HS IN LOAD ARE RECURRENT REPORTS OF GENETIC ASSOCIATION OF HS SULFOTRANSFERASE GENES WITH LOAD. IN A SERIES OF PRELIMINARY STUDIES, WE HAVE OBTAINED MULTIPLE PIECES OF EVIDENCE SUGGESTING THAT THE STRONG INTERACTION OF HS WITH APOE4, RELATIVE TO ITS INTERACTIONS WITH APOE2 AND APOE3, IS THE KEY UNDERLYING MECHANISM BY WHICH APOE4 EXERTS A DETRIMENTAL EFFECT ON THE BRAIN. SIGNIFICANTLY, WE HAVE FOUND THAT HAPLOINSUFFICIENT REDUCTION IN HS EXPRESSION LEADS TO THE MITIGATION OF SYNAPSE LOSS AND MICROGLIAL ACTIVATION IN THE PS19 TAUP310S MOUSE MODEL. WE HYPOTHESIZE THAT HS PLAYS A CRITICAL ROLE IN MEDIATING THE APOE4 EFFECT ON MICROGLIAL RESPONSE AND FUNCTION, AND THAT THE DETRIMENTAL EFFECT OF THE APOE4 ALLELE IS MAINLY DUE TO ITS STRONGER INTERACTION WITH THE 3-O SULFATE-RICH HS SPECIES EXPRESSED IN MICROGLIA. WE WILL: (1) DETERMINE THE ROLE OF HS IN APOE-TREM2 INTERACTION AND SIGNALING IN MICROGLIA; (2) DETERMINE THE ROLE OF MICROGLIAL HS IN APOE/ABCA1-MEDIATED CHOLESTEROL EFFLUX; AND (3) EXAMINE IN VIVO EFFECTS OF MICROGLIAL HS ON BRAIN PATHOLOGY AND FUNCTION IN AD MOUSE MODELS. THIS RESEARCH WILL PROVIDE ENTIRELY NOVEL INSIGHTS INTO THE ROLE OF HS IN MICROGLIA, AND MOREOVER, INTO LONG-TERM QUESTIONS REGARDING THE ISOFORM-SPECIFIC RISK OF APOE VARIANTS. SUCCESSFUL COMPLETION OF THIS PROJECT WILL REVEAL THE APOE4-HS INTERACTION AS A PROMISING THERAPEUTIC TARGET FOR REDUCING AD RISK IN APOE4 CARRIERS.

UNLOCKING REGENERATIVE POTENTIAL THROUGH IN VIVO GENETIC REPROGRAMMING

REGULATION AND FUNCTION OF STROMAL MACROPINOCYTOSIS IN PANCREATIC DUCTAL ADENOCARCINOMA - PROJECT SUMMARY RECENT YEARS HAVE WITNESSED AN APPRECIATION OF THE ROLE THAT METABOLIC ADAPTATION PLAYS IN CONFERRING SURVIVAL ADVANTAGES TO CELLS THAT ENCOUNTER THE HARSH NUTRIENT-POOR CONDITIONS OF THE TUMOR MICROENVIRONMENT. OF PARTICULAR RELEVANCE TO THIS PROPOSAL IS THE NOW WIDELY ACCEPTED NOTION THAT MACROPINOCYTOSIS, AN ENDOCYTIC MECHANISM OF FLUID-PHASE UPTAKE, FUNCTIONS IN TUMORS AS AN AMINO ACID SUPPLY ROUTE. BY STIMULATING THE UPTAKE OF EXTRACELLULAR PROTEIN AND TARGETING IT FOR LYSOSOMAL DEGRADATION, MACROPINOCYTOSIS PROVIDES CELLS WITH A SOURCE OF PROTEIN-DERIVED AMINO ACIDS, ALLOWING TUMORS TO CIRCUMVENT AMINO ACID DEPLETION AND SURVIVE NUTRIENT STRESS. PANCREATIC DUCTAL ADENOCARCINOMA (PDAC) TUMORS ARE DEFICIENT IN GLUTAMINE, A VITAL NUTRIENT THAT SUPPORTS TUMOR GROWTH. OUR PUBLISHED WORK IN PDAC CELLS HAS ESTABLISHED THAT GLUTAMINE DEPLETION HAS THE CAPACITY TO MODULATE MACROPINOCYTOSIS – DIALING THE PROCESS UP OR DOWN AS REQUIRED. INTERESTINGLY, OUR PRELIMINARY DATA PRESENTED IN THIS PROPOSAL DEMONSTRATE FOR THE FIRST TIME THAT GLUTAMINE SCARCITY CAN ALSO STIMULATE MACROPINOCYTOSIS IN CANCER-ASSOCIATED FIBROBLASTS (CAFS). MECHANISTICALLY, WE HAVE ATTRIBUTED GLUTAMINE STRESS-INDUCED MACROPINOCYTIC UPTAKE IN CAFS TO A CAMKK2-AMPK SIGNAL THAT LEADS TO THE RAC1- DEPENDENT ACTIN CYTOSKELETON DYNAMICS THAT ARE REQUIRED FOR MACROPINOCYTOSIS. AMPK IS A BIOENERGETIC STRESS SENSOR THAT IS MOST OFTEN STUDIED IN THE CONTEXT OF GLUCOSE STARVATION, WHICH UNLIKE GLUTAMINE DEPLETION, DOES NOT BOOST CAF MACROPINOCYTOSIS. NOTABLY, NOT MUCH IS KNOWN ABOUT AMPK ACTIVATION AND FUNCTION DURING GLUTAMINE DEPLETION. OUR PRELIMINARY STUDIES SUGGEST THAT MACROPINOCYTOSIS HAS A DUAL PURPOSE IN CAFS – IT CAN SERVE TO SUSTAIN CAF VIABILITY AND FUNCTION, AND IT CAN PROVIDE SECRETED AMINO ACIDS TO NOURISH THE TUMOR CELLS. IMPORTANTLY, OUR IN VIVO AND IN VITRO EXAMINATIONS OF MACROPINOCYTOSIS IN NORMAL FIBROBLASTS, AS WELL AS IN CAFS ORIGINATING FROM OTHER TUMOR TYPES, SUGGEST THAT GLUTAMINE DEPLETION-INDUCED STROMAL UPTAKE IS UNIQUE TO PANCREATIC CAFS. BASED ON THESE DATA, OUR CENTRAL HYPOTHESIS IS THAT GLUTAMINE SCARCITY SELECTIVELY DRIVES CAMKK2-AMPK-DEPENDENT MACROPINOCYTOSIS IN CAFS, AND THAT STROMAL MACROPINOCYTOSIS IS A PROCESS THAT CAN BE HARNESSED IN PDAC THERAPY. IN THIS PROPOSAL, WE WILL 1) EXAMINE THE MOLECULAR MECHANISMS DRIVING THE SELECTIVE ROLE OF GLUTAMINE IN CAF MACROPINOCYTOSIS, 2) FUNCTIONALLY CHARACTERIZE STROMAL MACROPINOCYTOSIS IN PDAC, AND 3) DETERMINE WHETHER THE STROMAL REORGANIZATION THAT OCCURS WITH MACROPINOCYTOSIS INHIBITION CAN BE LEVERAGED FOR PDAC THERAPY. THIS PROJECT WILL BE OF GREAT SIGNIFICANCE, NOVELTY AND IMPACT, AS IT WILL CONSTITUTE THE FIRST EVALUATION OF THE ROLE OF MACROPINOCYTOSIS IN THE PDAC TUMOR STROMA AND THE FIRST ANALYSIS TO SELECTIVELY LINK GLUTAMINE STARVATION TO CAMKK2-AMPK SIGNALING. MOREOVER, BECAUSE MACROPINOCYTOSIS IS IMPORTANT IN BOTH THE TUMOR CELLS AND THE STROMA, OUR WORK COULD HAVE TREMENDOUS IMPACT ON THE DEVELOPMENT OF NOVEL THERAPEUTIC MODALITIES FOR PDAC.

DEVELOPING ER STRESS INHIBITORS FOR TREATING ALS

HYBRID NANOTECHNOLOGIES FOR DETECTION AND SYNERGISTIC THERAPIES FOR BREAST CANCER

ELUCIDATING A MICROGLIAASSOCIATED ROLE FOR SORLA IN MODULATING AD PATHOGENESIS - PROJECT SUMMARY MUTATIONS IN SORLA (ENCODED BY SORL1) IDENTIFIED THROUGH GWAS AND WHOLE EXOME SEQUENCING ANALYSIS HAVE BEEN LINKED TO INCREASED ALZHEIMER’S DISEASE (AD) RISK. ALTHOUGH A NEURONAL ROLE FOR SORLA IN SUPPRESSING AMYLOIDOGENIC APP PROCESSING AND CONSEQUENT ASS GENERATION HAS BEEN ESTABLISHED, SORLA EXPRESSION IS ~8-FOLD HIGHER IN HUMAN MICROGLIA COMPARED TO NEURONS, THUS IMPLICATING A MICROGLIAL ROLE FOR SORLA IN AD PATHOGENESIS. SO FAR, FUNCTIONAL ROLES FOR SORLA IN MICROGLIA HAVE NOT YET BEEN DESCRIBED. HERE, WE USED CRISPR/CAS9 EDITING METHODS TO INTEGRATE AD-ASSOCIATED A528T AND R744X MUTATIONS INTO THE SORL1 (ENCODING SORLA) LOCUS IN HUMAN H9 EMBRYONIC STEM CELLS, WHICH WERE SUBSEQUENTLY DIFFERENTIATED INTO HUMAN MICROGLIA-LIKE (HMGL) CELLS. COMPARING TRANSCRIPTOMIC PROFILES BETWEEN WILDTYPE AND AD-ASSOCIATED SORL1 (A528T, R744X) AND TREM2 (R47H) MUTANT HMGLS REVEALS PATHOGENIC MICROGLIA GENE SIGNATURES SUCH AS INDUCED APOE EXPRESSION PREVIOUSLY DESCRIBED IN AD MOUSE MODELS AND HUMAN AD BRAIN. OUR RESULTS ALSO SHOW THAT CULTURED SORL1R744X AND TREM2R47H HMGLS FEATURE DEFECTS IN ASS UPTAKE IN AN APOE-DEPENDENT MANNER, ALONG WITH IMPAIRED ASS CLEARANCE AND PLAQUE ASSOCIATION IN MOUSE BRAIN XENOTRANSPLANTS BY MICRODIALYSIS/HISTOLOGY. THESE RESULTS PROVIDE PIONEERING EVIDENCE THAT SORLA DYSFUNCTION CAN CONFER PATHOGENIC EXPRESSION SIGNATURES AND IMPAIR MICROGLIAL FUNCTION. WE HYPOTHESIZE THAT MICROGLIAL DYSFUNCTION WILL VARY ACCORDING TO DOMAIN-SPECIFIC MUTATIONS IN THE SORLA EXTRACELLULAR REGION, AND THAT EARLY AND LATE ONSET SORLA MUTATIONS MAY SHOW DIFFERENTIAL EFFECTS ON MICROGLIA DYSREGULATION AND MICROGLIA/NEURON INTERACTION. USING OUR GENE EDITING/HUMAN MICROGLIAL MODELING AND ANALYSIS PLATFORM, WE WILL EXPAND OUR SORLA MUTANT EMBRYONIC STEM CELL (ESC) PANEL TO INCLUDE REPRESENTATIVE EARLY AND LATE AD ONSET MUTATIONS WITHIN EACH OF THE FUNCTIONAL DOMAINS IN THE SORLA EXTRACELLULAR REGION. WE WILL CHARACTERIZE GENE EXPRESSION PROFILES OF WILDTYPE (WT) AND SORLA MUTANT ESC-DERIVED MICROGLIA (“XHMGS”) IN VIVO BY RNASEQ/PROTEOMIC ANALYSIS, AS WELL AS FUNCTIONAL ASPECTS OF MICROGLIAL FUNCTION (ASS UPTAKE, CYTOKINE PROFILES) IN XHMGS XENOTRANSPLANTED IN AD MOUSE BRAIN. AS OUR RESULTS INDICATE THAT SORLA MUTATIONS SUCH AS R744X AND A528T UPREGULATE APOE WHICH MAY TRIGGER CERTAIN ASPECTS OF MICROGLIA FUNCTION, WE WILL ALSO TEST WHETHER APOE AND OTHER POTENTIAL DRIVERS OF MICROGLIA DYSFUNCTION EPISTATICALLY MEDIATE DOWNSTREAM PATHOGENIC BEHAVIOR IN SORLA MUTANT MICROGLIA XENOTRANSPLANTS IN AD MOUSE BRAIN. WE WILL ALSO EXPLORE CELLULAR MECHANISMS ASSOCIATED WITH VARIOUS SORLA MUTATIONS THAT DRIVE CELLULAR DYSFUNCTION IN ESC-DERIVED MICROGLIA AND NEURONS; TO THIS END, WE WILL INVESTIGATE HOW SORLA MUTATIONS IN HUMAN MICROGLIA (XHMGS) INTERACT WITH NEURONS IN MOUSE AD BRAIN XENOTRANSPLANTS, AS WELL AS WT AND SORLA MUTANT HMGLS/NEURONS IN CO-CULTURE. THESE RESULTS WILL PROVIDE US WITH MECHANISTIC INSIGHT INTO HOW VARIOUS MUTATIONS CAN TRIGGER MICROGLIA DYSFUNCTION, AND POTENTIALLY DESCRIBE HOW ASPECTS OF MICROGLIAL AND NEURONAL-RELATED SORLA PATHWAYS ARE AFFECTED TO ALTER AGE-RELATED ONSET IN AD PATHOGENESIS.

MECHANISMS OF NUP210 REGULATION OF MUSCLE DEVELOPMENT AND REGENERATION

BRAIN-PENETRANT GPR88 AGONISTS AS NOVEL THERAPEUTICS FOR OPIOID ABUSE - SUMMARY SUBMITTED IN RESPONSE TO RFA-DA-22-031, THIS APPLICATION DESCRIBES A MULTIDISCIPLINARY EFFORT TO DEVELOP NOVEL, BRAIN-PENETRANT, SMALL-MOLECULE GPR88 AGONISTS TO ATTENUATE ADDICTION-RELEVANT BEHAVIORAL RESPONSES TO OPIOID DRUGS AND AMELIORATE WITHDRAWAL SYNDROME IN OPIOID-DEPENDENT INDIVIDUALS. GPR88 IS AN ORPHAN G-PROTEIN COUPLED RECEPTOR (GPCR) WITH CONCENTRATED EXPRESSION IN STRIATOPALLIDAL (INDIRECT PATHWAY) MEDIUM SPINY NEURONS (MSNS) IN THE STRIATUM. RECENT OBSERVATIONS IN TRANSGENIC MICE DEMONSTRATE THAT GPR88 EXERTS AN INHIBITORY INFLUENCE OVER OPIOID RECEPTOR SIGNALING IN THE STRIATUM. BUILDING ON THIS AND OTHER RECENT PROMISING PRECLINICAL OBSERVATIONS, WE PROPOSE TO LEVERAGE OUR EXPERTISE IN NEUROBIOLOGICAL MECHANISMS OF OPIOID USE DISORDERS AND SMALL-MOLECULE DRUG DISCOVERY TO FORMALLY VALIDATE GPR88 AS A DRUG TARGET FOR OPIOID USE DISORDERS (OUD) AND ESTABLISH A DRUG DEVELOPMENT PATH FORWARD BY DISCOVERING GPR88 AGONIST LEAD COMPOUNDS. WE WILL LEVERAGE AN IN VIVO MURINE MODEL OF OPIOID DEPENDENCE WE HAVE DEVELOPED AND GPR88-/- MICE ALONG WITH AVAILABLE GPR88 PHARMACOLOGICAL TOOLS. UNFORTUNATELY, PUBLISHED GPR88 AGONISTS HAVE LIMITATIONS THAT RESTRICT THEIR UTILITY IN VIVO AND PREVENT THEIR DEVELOPMENT INTO THERAPEUTICS. TO ADDRESS THIS SHORT COMING WE WILL DEVELOP NOVEL, POTENT GPR88 AGONISTS WITH PROPERTIES OPTIMIZED FOR THE TREATMENT OF OPIOID DEPENDENCE. WE ARE WELL POSITIONED TO ACCOMPLISH THIS TASK. WE RECENTLY CONDUCTED A GPR88 HIGH THROUGHPUT SCREEN (HTS), USING AN INNOVATIVE PHARMACOCHAPERONE ASSAY FORMAT, AND IDENTIFIED A NOVEL CHEMICAL SCAFFOLD EXEMPLIFIED BY SBI-‘2037 AS A VALIDATED GPR88 AGONIST. IN ADDITION, WE HAVE ALREADY DEVELOPED ROBUST CELL-BASED ASSAYS (AND APPROPRIATE COUNTER-SCREENS) TO RELIABLY MONITOR GPR88 FUNCTION AND ITS IMPACT ON OPIOID RECEPTORS. LEVERAGING THESE ASSETS, WE WILL CONDUCT A MEDICINAL CHEMISTRY CAMPAIGN TO INCREASE POTENCY AND SELECTIVITY OF THE SBI-‘2037 SCAFFOLD WITH A CRITICAL PATH CONSISTING OF CELLULAR ASSAYS FOCUSED ON THEIR UTILITY IN OUD THAT INCLUDES EARLY ASSESSMENT OF ABSORPTION, DISTRIBUTION, METABOLISM AND EXCRETION (ADME) AND BRAIN PENETRATION PROPERTIES. TO INCREASE THE OVERALL PROBABILITY OF SUCCESS, WE INTEND TO CONDUCT AN ADDITIONAL GPR88 HTS SCREENING CAMPAIGN IN SEARCH OF ONE OR MORE BACK UP SERIES. WE HAVE ESTABLISHED A STRINGENT SET OF CRITERIA FOR GPR88 AGONIST LEADS, AND ONLY COMPOUNDS THAT MATCH THIS PROFILE WILL BE ADVANCED INTO EFFICACY TESTING IN THE INTRAVENOUS OXYCODONE SELF- ADMINISTRATION PROCEDURE IN WILD-TYPE AND GPR88 KNOCKOUT MICE. THIS MULTIDISCIPLINARY RESEARCH PLAN CAPITALIZES ON THE UNIQUELY RELEVANT SCIENTIFIC AND DRUG DISCOVERY EXPERTISE OF OUR TEAM OF COMMITTED INVESTIGATORS AND IS AN INITIAL STEP TOWARDS OUR ULTIMATE GOAL OF DEVELOPING GPR88 AGONISTS AS NOVEL THERAPEUTICS TO FACILITATE ABSTINENCE IN OPIOID-DEPENDENT INDIVIDUALS.

DEVELOPMENT OF AN EFFECTIVE THERAPY AGAINST POST-EXPOSURE ANTHRAX

EPIGENETIC CONTROL OF HEMATOPOIETIC STEM CELLS

LEAD OPTIMIZATION OF NOVEL ML-IAP ANTAGONISTS TO TREAT LUNG CANCER

MAGNETIC RESONANCE OF CARDIAC C13 FLUX & METABOLISM RATE

NEW DOWN SYNDROME BRAIN ORGANIZATION REVEALED BY SINGLE-CELL GENOMICS - PROJECT SUMMARY/ABSTRACT NEARLY 100% OF DOWN SYNDROME (DS; TRISOMY OF HUMAN CHROMOSOME 21) INDIVIDUALS THAT LIVE INTO THEIR 5TH DECADE OF LIFE AND BEYOND, SHOW ALZHEIMER’S DISEASE (AD)-LIKE DEMENTIA AND NEUROPATHOLOGY (DS-AD), REPRESENTING A PROMINENT DS COMORBIDITY THAT HAS RECENTLY BEEN REPORTED AS THE LEADING CAUSE OF DEATH FOR DS ADULTS. BEYOND TRISOMY OF CHROMOSOME 21, WHICH INCLUDES THE KEY AD GENE, AMYLOID PRECURSOR PROTEIN (APP), THE MOLECULAR MECHANISMS UNDERLYING DS-AD HAVE RESISTED IDENTIFICATION. THERE ARE NO DISEASE-MODIFYING THERAPIES (DMTS) TO PREVENT OR TREAT DS-AD, WHICH COULD IMPROVE DS QUALITY OF LIFE AND LIFESPAN. THIS INCLUDE PROPOSAL WILL TRANSFORM OUR UNDERSTANDING OF DS BY REVEALING THE TRANSCRIPTOMIC (RNA) AND GENOMIC (DNA) SINGLE-CELL LANDSCAPE OF THE AGING DS INDIVIDUAL, AND ESPECIALLY THE DS-AD BRAIN, BY CENTERING UPON A NOVEL HUMAN BRAIN MOLECULAR MECHANISM THAT MIGHT UNDERLIE DS-AD: SOMATIC GENE RECOMBINATION (SGR) AND RESULTANT GENOMIC AND TRANSCRIPTOMIC HETEROGENEITY. SGR HAS THE POTENTIAL TO CHANGE THE DNA BLUEPRINT OF DS BRAIN CELLS RESULTING IN FUNCTIONAL CONSEQUENCES FOR BRAIN CELLS THAT COULD EXPLAIN DS-AD ONSET AS WELL AS OTHER DS BRAIN COMORBIDITIES SUCH AS AUTISM AND EPILEPSY. SGR HAS NOT BEEN EXAMINED IN DS BRAINS, WHICH IF OPERATIONAL, WOULD PROVIDE A FUNDAMENTALLY NEW VIEW ON HOW GENES AND GENE DOSAGE ACT TO PROMOTE DS-AD OVER TIME. SGR IS KNOWN TO ACT ON APP IN NORMAL AND SPORADIC AD NEURONS, RESULTING IN THOUSANDS OF NEW APP VARIANTS WITHIN INDIVIDUAL HUMAN BRAINS AND HAS BEEN INDEPENDENTLY CONFIRMED IN THE SCIENTIFIC LITERATURE. THE PROVEN INCREASES IN DS BRAIN GENE EXPRESSION, COMBINED WITH THE IDENTIFIED LINKAGE OF GENE EXPRESSION TO SGR, IMPLICATES GENES TRANSCRIPTIONALLY INCREASED BY TRISOMY 21 AS NEW TARGETS FOR SGR IN DS-AD. APP IS LIKELY THE “TIP OF THE ICEBERG” FOR DS-AFFECTED GENES IN NEURONS AND NON-NEURONAL CELLS, WITH IMPLICATIONS FOR BOTH DS AS WELL AS OTHER STATES OF THE NORMAL AND DISEASED HUMAN BRAIN. THREE INDEPENDENT, BUT DEEPLY-CONNECTED, RESEARCH ELEMENTS (RES) WILL BE COMPLETED BY A PROVEN, COLLABORATIVE TEAM OF MOLECULAR BIOLOGISTS, NEUROSCIENTISTS, NEUROLOGISTS, BIOENGINEERS, AND BIOINFORMATICIANS TO TEST THE HYPOTHESIS THAT SGR CONTRIBUTES TO DS BRAIN DEFICITS AND DS-AD BY ALTERING KNOWN AND UNKNOWN DISEASE GENES AT THE SINGLE-CELL TRANSCRIPTOMIC AND GENOMIC LEVEL WITHIN THE DS BRAIN. RE1 AND RE2 WILL USE CUTTING-EDGE SEQUENCING TECHNOLOGIES TO INTERROGATE THE TRANSCRIPTOMIC AND GENOMIC HETEROGENEITY OF SINGLE NUCLEI FROM DS AND DS-AD BRAINS COMPARED TO CONTROLS, AND WILL INFORMATICALLY INTEGRATE TRANSCRIPTOMIC EXPRESSION, CHROMATIN ACCESSIBILITY, NOVEL ISOFORM DETECTION AND GENOMIC MOSAICISM FORMS, INCLUDING GENCDNAS, WITHIN SINGLE CELLS ACROSS CELL TYPES AND WITH AGE. RE3 WILL EXPLORE THE FUNCTIONAL CONSEQUENCES SGR IN PRIMARY NEURONAL AND INDUCED PLURIPOTENT STEM CELL (IPSC) MODELS TOWARDS UNDERSTANDING THE FUNCTIONAL IMPLICATIONS OF DISEASE ENHANCED SGR, AND THE THERAPEUTIC OPPORTUNITIES THAT SGR UNVEILS.

DEVELOPMENT OF STEP ALLOSTERIC INHIBITORS AS NOVEL THERAPEUTICS FOR ALZHEIMER'S DISEASE

MOLECULAR DETERMINANTS OF TLR TRAFFICKING

NOVEL NEUROPROTECTIVE ROLES FOR THE ALZHEIMER'S DISEASE RISK GENE SORLA IN TAU PATHOLOGY AND PATHOGENESIS - NUMEROUS MUTATIONAL VARIANTS IN THE SORLA (SORL1) GENE ARE ASSOCIATED WITH INCREASED ALZHEIMER’S DISEASE (AD) RISK, AND ALTHOUGH GROWING EVIDENCE FROM OUR GROUP AND OTHERS SUPPORT A NEUROPROTECTIVE ROLE FOR SORLA IN SUPPRESSING EFFECTS ASSOCIATED WITH AΒ, VIRTUALLY NOTHING IS KNOWN WITH RESPECT TO WHETHER SORLA CAN MODULATE PROTEOTOXIC EFFECTS ASSOCIATED WITH TAU. HERE, WE PRESENT EVIDENCE THAT TRANSGENIC UPREGULATION OF SORLA (“SORLA TG”) CAN REDUCE TAU PHOSPHORYLATION, AS WELL AS PATHOLOGICAL VENTRICULAR DILATION IN 9 MONTH- OLD (9MO) PS19 (P301S TAU) MOUSE BRAIN. PROTEOMIC ANALYSIS REVEALS CHANGES IN PROTEOMIC PROFILES RELATED TO SYNAPTIC DYSFUNCTION AND MICROGLIAL ACTIVATION, INCLUDING UPREGULATION OF APOE AND THE COMPLEMENT COMPONENT, C1Q IN PS19 MOUSE HIPPOCAMPUS; THESE CHANGES WERE LARGELY SUPPRESSED IN SORLA TG/PS19 HIPPOCAMPUS, INDICATING THAT SORLA UPREGULATION CAN POTENTIALLY REVERSE PATHOLOGICAL CHANGES ASSOCIATED WITH TAU PROTEOTOXICITY. HISTOLOGICAL ANALYSIS INDICATES THAT C1Q AND ASTROGLIOSIS IS HIGHLY UPREGULATED IN PS19 HIPPOCAMPUS, AND THESE EFFECTS WERE REDUCED IN SORLA TG/PS19 ANIMALS. INTERESTINGLY, C1Q HAS BEEN PREVIOUSLY SHOWN TO LABEL SYNAPSES FOR MICROGLIAL UPTAKE AND ELIMINATION IN BOTH J20 (HAPP) AND PS19 MOUSE BRAIN, WHICH TOGETHER, SUGGESTS THAT SORLA POTENTIALLY MEDIATES NEUROPROTECTIVE EFFECTS THROUGH SUPPRESSION OF DELETERIOUS CHANGES ASSOCIATED WITH SYNAPTIC FACTORS AND GLIOSIS IN PS19 HIPPOCAMPUS. OUR PRIOR WORK ALSO DEMONSTRATES THAT UPREGULATION OF A CLEAVED SOLUBLE SORLA (SSORLA) FORM IN SORLA TG ANIMALS IS ALSO PROTECTIVE IN NEURONS, AND ENHANCES NEURITE REGENERATION WITH NEURONAL INJURY. WE THEREFORE HYPOTHESIZE THAT SORLA CAN MEDIATE NEUROPROTECTIVE EFFECTS TO LIMIT TAU PATHOLOGY/PROTEOTOXICITY AND DETERMINE HERE, WHETHER AND HOW SORLA OVEREXPRESSION, DELETION OR SSORLA CAN AFFECT TAU PATHOLOGY AND PATHOGENESIS. AS OUR RESULTS DEMONSTRATE THAT SORLA TG/PS19 ANIMALS CAN REVERSE PATHOLOGICAL AND PROTEOMIC FEATURES ASSOCIATED WITH LATE-STAGE (9MO) TAU PATHOLOGY, WE WILL ALSO CHARACTERIZE CHANGES AT EARLY AND MID- STAGES OF TAU PATHOLOGY, AND CHARACTERIZE CELL-TYPE SPECIFIC TRANSCRIPTOMIC CHANGES IN 9MO HIPPOCAMPUS IN PS19 MICE THAT MAY BE POTENTIALLY SUPPRESSED IN SORLA TG/PS19 ANIMALS BY SNRNASEQ ANALYSIS. WE WILL ALSO CHARACTERIZE EFFECTS OF SORLA UPREGULATION ON GLIAL-ASSOCIATED SYNAPTIC UPTAKE AND PRUNING IN PS19 AND SORLA TG/PS19 ANIMALS, AND COMPARE EFFECTS OF INTRANEURONAL TAU DISPERSION FROM THE MEC TO DG REGION IN WILDTYPE (WT) VS SORLA TG BRAIN USING AN AAV-TAU PROPAGATION MODEL USED IN OUR LAB (AIM 1) (ZHU ET AL., 2022). WE WILL ALSO DETERMINE EFFECTS OF SORLA DELETION ON TAU PATHOLOGY, GLIOSIS AND SYNPATOTOXICITY (AIM 2), AND CHARACTERIZE EFFECTS OF AD-ASSOCIATED SORLA MUTATIONAL VARIANTS (T2134M, E2055X) THAT POTENTIALLY UPREGULATE SSORLA ON MICROGLIAL FUNCTION AND SORLA/SSORLA EXPRESSION IN AN ESC-BASED HUMAN MICROGLIA CELLS IN VITRO, AND IN HUMAN MICROGLIAL XENOTRANSPLANT MODELS IN PS19/HMCSF MOUSE BRAIN IN VIVO (AIM 3). TOGETHER, RESULTS FROM THIS STUDY WILL PROVIDE INSIGHT INTO A PREVIOUSLY UNDESCRIBED ROLE FOR SORLA AND SSORLA IN TAU PATHOLOGY.

PRENATAL BLOOD-BORNE LIPIDS IN POST-HEMORRHAGIC HYDROCEPHALUS

DISSECTING THE INTRACELLULAR AND EXTRACELLULAR ROLE OF TREM2 IN THE PATHOGENESIS OF NON-ALCOHOLIC STEATOHEPATITIS. - ABSTRACT: NON-ALCOHOLIC STEATOHEPATITIS (NASH) IS THE FASTEST GROWING CAUSE OF LIVER CANCER AND LIVER TRANSPLANT IN THE U.S. THERE IS CURRENTLY NO APPROVED TREATMENT FOR NASH AND THE MECHANISTIC UNDERSTANDING OF WHY SOME PATIENTS WITH FATTY LIVER MAINTAIN A BENIGN CONDITION WHILE OTHERS PROGRESS TO NASH IS NOT CLEAR. MACROPHAGES PLAY A PIVOTAL ROLE IN NASH PATHOGENESIS. ACCUMULATING EVIDENCE SUGGEST THAT ‘TRIGGERING RECEPTOR EXPRESSED ON MYELOID CELLS’ (TREM2) IS OVEREXPRESSED IN NASH ASSOCIATED MACROPHAGES. INTERESTINGLY, EVEN THOUGH TREM2 EXPRESSION IS INCREASED DURING NASH, ITS ABSENCE EXACERBATES NASH AND FIBROSIS DEVELOPMENT. THIS INDICATES THAT TREM2 IS PROTECTIVE IN NASH AND FIBROSIS PROGRESSION. HOWEVER, THE MECHANISM OF TREM2 SIGNALING REMAINS POORLY UNDERSTOOD. MOREOVER, UPON LIGAND ENGAGEMENT, TREM2 GETS CLEAVED FROM THE CELL SURFACE INTO A SOLUBLE FORM (STREM2) THAT CAN BE DETECTED IN THE SERUM. WE FOUND THAT STREM2 LEVELS INCREASE WITH NAFLD AND NASH PROGRESSION, HOWEVER, THE TRANS-SIGNALING AXIS OF STREM2 IS AN AREA THAT HAS NOT BEEN ADDRESSED IN THE FIELD YET. STREM2 HAS THE ABILITY TO SOAK UP INFLAMMATORY LIGANDS SUCH AS LPS AND LIPIDS AS WELL AS BIND TO MACROPHAGES. HOWEVER, WHETHER STREM2 CAN BIND AND INFLUENCE DOWNSTREAM SIGNALING IN OTHER CELLS REMAIN UNKNOWN. WE DISCOVERED THAT STREM2 ITSELF HAS NASH PROTECTIVE AS WELL AS THERAPEUTIC ABILITIES. INTRODUCTION OF STREM2 FRAGMENT INTO MICE THAT ALREADY HAD ADVANCED NASH HAD PROFOUND EFFECTS ON NASH AND FIBROSIS RESOLUTION. THE GOAL OF THIS PROPOSAL IS TO BETTER UNDERSTAND HOW TREM2 EXERTS ITS PROTECTIVE FUNCTIONS AND DISSECT THE INTRACELLULAR AND TRANS-SIGNALING AXES OF TREM2. WE WILL USE MOUSE MODELS OF NASH AND HCC THAT CLOSELY MIMICS DIET INDUCED METABOLIC SYNDROME AND HUMAN NASH GENE SIGNATURE AS WELL AS PRIMARY CULTURES OF BOTH MOUSE AND HUMAN LIVER CELLS IN ORDER TO ASSESS THE FOLLOWING 2 SPECIFIC AIMS: AIM 1 WILL IDENTIFY THE KEY INTRACELLULAR TREM2 SIGNALING PATHWAYS THAT EXERTS ITS PROTECTIVE ACTIONS IN CONTROLLING NASH DEVELOPMENT. WE WILL ALSO IDENTIFY THE TRANSCRIPTION FACTORS RESPONSIBLE FOR TREM2 EXPRESSION IN NASH ASSOCIATED MACROPHAGES. AIM 2 WILL ELUCIDATE THE TRANS-SIGNALING PROPERTIES OF STREM2 AND WHETHER STREM2 IS DIRECTLY INVOLVED IN DISEASE PROGRESSION. THE PROPOSED STUDIES WILL PROVIDE KEY INSIGHTS INTO THE MECHANISMS OF NASH AND HCC DEVELOPMENT. THE FINDINGS FROM THIS STUDY WILL LAY THE FOUNDATION FOR FUTURE DEVELOPMENT AND REFINEMENT OF STREM2 AS A NOVEL THERAPEUTIC AGENT FOR NASH-FIBROSIS AND HCC.

DISCOVERY OF NOVEL INHIBITORS TARGETING TRANS-GOLGI NETWORK ACIDIFICATION IN PANCREATIC CANCER - PROJECT SUMMARY PANCREATIC DUCTAL ADENOCARCINOMA (PDAC) IS PROJECTED TO BE THE SECOND LEADING CAUSE OF CANCER-RELATED DEATHS IN THE UNITED STATES BY 2030. WITH A 5-YEAR RELATIVE SURVIVAL RATE OF JUST 10%, PDAC HAS THE HIGHEST DEATH RATE AMONG THE MOST COMMON CANCERS, UNDERSCORING THE URGENT NEED FOR NEW THERAPEUTIC STRATEGIES THAT IMPROVE CLINICAL OUTCOMES. RECENT YEARS HAVE WITNESSED A GROWING APPRECIATION OF THE ROLE THAT METABOLIC REPROGRAMMING PLAYS IN CONFERRING SURVIVAL ADVANTAGES TO PDAC CELLS, AND THE TARGETING OF METABOLIC PROCESSES IS CONSIDERED A PROMISING AREA FOR THE DEVELOPMENT OF NOVEL THERAPIES. IN CONTRAST TO NORMAL CELLS, CANCER CELLS FAVOR GLYCOLYSIS FOR ENERGY PRODUCTION, A PHENOMENON CALLED THE WARBURG EFFECT. AS A GLYCOLYTIC BYPRODUCT, TRANSFORMED CELLS PRODUCE AN EXCESSIVE AMOUNT OF H+ IONS. THIS CYTOSOLIC ACCUMULATION OF H+ WOULD BE DETRIMENTAL TO CELL FITNESS; THEREFORE, TUMOR CELLS HAVE EVOLVED MECHANISMS TO ACHIEVE PH HOMEOSTASIS. USING PDAC AS A MODEL SYSTEM, WE DISCOVERED THAT CYTOSOLIC PH IN CANCER CELLS IS REGULATED BY ORGANELLAR SEQUESTRATION OF H+ IONS VIA COMPARTMENTAL ION TRANSPORT AND THAT THE TRANS-GOLGI NETWORK (TGN) CAN ACT AS A “SINK” FOR CYTOSOLIC H+. IMPORTANTLY, NORMAL CELLS DO NOT EMPLOY THIS HOMEOSTATIC MECHANISM. USING THE NHE7 NA+/H+ ANTIPORTER, WHICH IS PRIMARILY LOCALIZED TO THE TGN, WE HAVE SHOWN THAT TARGETING NHE7 CAUSES ACIDIFICATION OF THE CYTOSOL AND SUBSEQUENT CELL DEATH IN PDAC CELLS BUT NOT NORMAL CELLS. WE HAVE FURTHER VALIDATED THESE FINDINGS IN MOUSE MODELS OF PDAC. PATIENT-CENTRIC DATA ANALYSES SHOW THAT TGN LOCALIZED TRANSPORTERS ARE FREQUENTLY OVEREXPRESSED IN PDAC AND THEIR ELEVATED EXPRESSION IS CORRELATED WITH WORSE OVERALL SURVIVAL. TO ROBUSTLY QUANTIFY PH IN THE LUMEN OF THE TGN, WE HAVE DEVELOPED AN INNOVATIVE TGN-TARGETED PH BIOSENSOR ASSAY, WHICH HAS BEEN OPTIMIZED FOR HIGH-THROUGHPUT (HT) SCREENING OF SMALL MOLECULE COMPOUND LIBRARIES TO IDENTIFY NOVEL TGN-SELECTIVE ACIDIFICATION INHIBITORS. WE HAVE VALIDATED OUR TGN PH BIOSENSOR HT ASSAY IN A SET OF CLINICAL TRIAL AND APPROVED DRUGS, AS WELL AS KINASE INHIBITORS. TO COMPLEMENT OUR HT SCREEN, WE HAVE DESIGNED A CUTTING- EDGE SECONDARY AND TERTIARY ASSAY PIPELINE TO FURTHER VALIDATE TGN-SELECTIVE COMPOUNDS. WE HYPOTHESIZE THAT NOVEL GOLGI–SPECIFIC SMALL MOLECULE INHIBITORS OF ORGANELLE ACIDIFICATION WILL SELECTIVELY PREVENT CANCER CELLS FROM ACHIEVING PROPER PH HOMEOSTASIS, RESULTING IN ACIDIC CYTOSOLIC PH AND CELL DEATH. IN THIS GRANT WE WILL: 1) PERFORM A LARGE-SCALE HT SCREEN TO IDENTIFY INHIBITORS OF TGN ACIDIFICATION AND CONFIRM THE HITS, 2) VALIDATE THE CONFIRMED HITS USING SECONDARY ASSAYS AND PRIORITIZE HIT SCAFFOLDS, AND 3) MAP COMPOUND EFFECTS TO TGN COMPONENTS AND SELECT AND CHARACTERIZE FINAL PROBE(S). SUCCESSFUL COMPLETION OF THIS WORK WILL PROVIDE NOVEL SMALL MOLECULE CHEMICAL PROBES VALIDATED TO INTERFERE WITH TGN ACIDIFICATION AND CAUSE CANCER-SELECTIVE CELL DEATH, SERVING AS STARTING POINTS TO BE FURTHER DEVELOPED INTO SAFE AND EFFECTIVE THERAPEUTIC AGENTS FOR PDAC.

DRUG DISCOVERY FOR FATTY ACID SYNTHASE IN ONCOLOGY

TAS::97 0130::TAS CELLULAR SENTINELS TOXICITY PLATFORM

FXR SIGNALING PATHWAY IS A VALID TARGET FOR CHEMOPREVENTION IN COLORECTAL CANCER

LEAD OPTIMIZATION OF NOVEL MGLU2 NEGATIVE ALLOSTERIC MODULATORS

MECHANISM OF ER PROTEIN MISFOLDING-INDUCED MITOCHONDRIAL DYSFUNCTION

OPTIMIZATION OF DRUG-LIKE PROPERTIES OF CRFBP-CRF2 NEGATIVE ALLOSTERIC MODULATORS FOR ALCOHOL USE DISORDER - PROJECT SUMMARY THIS APPLICATION, “OPTIMIZATION OF DRUG-LIKE PROPERTIES OF CRFBP-CRF2 NEGATIVE ALLOSTERIC MODULATORS FOR ALCOHOL USE DISORDER”, IS IN RESPONSE TO PAR-22-031 “DRUG DISCOVERY FOR NERVOUS SYSTEM DISORDERS (R01 CLINICAL TRIALS NOT ALLOWED)”. ALCOHOL USE DISORDER (AUD) REMAINS A HUGE CLINICAL AND PUBLIC HEALTH PROBLEM WITH NO EFFECTIVE PHARMACOLOGICAL RECOURSE, AFFECTING 28.3 MILLION ADULTS IN THE UNITED STATES, AND IS THE 3RD LEADING PREVENTABLE CAUSE OF DEATH. THE ONLY FDA-APPROVED MEDICATIONS FOR AUD ARE DISULFIRAM, NALTREXONE, AND ACAMPROSATE, ALL OF WHICH EXHIBIT LIMITED EFFICACY AND HAVE LIMITING CONTRAINDICATIONS. HENCE, THERE IS A CRITICAL NEED TO DEVELOP MORE EFFECTIVE THERAPEUTICS TO TREAT AUD. WHILE MANY FACTORS CONTRIBUTE TO THE DEVELOPMENT AND MAINTENANCE OF AUD, INCREASING ATTENTION IS BEING PAID TO POTENTIALLY DRUGGABLE TARGETS WITHIN THE STRESS SYSTEM. THE PRIMARY REGULATOR OF THE STRESS RESPONSE, CORTICOTROPHIN-RELEASING FACTOR (CRF), EXERTS ITS EFFECTS BY BINDING TO CRF1 AND CRF2 RECEPTORS AND, ALSO, A SECRETED 37-KD CRF-BINDING PROTEIN (CRFBP). IN ADDITION, CRFBP UNDERGOES SPONTANEOUS CLEAVAGE INTO A 27-KD N-TERMINAL FRAGMENT, CRFBP(27KD), THAT BINDS CRF AND A 10- KD C-TERMINAL FRAGMENT, CRFBP(10KD), THAT DOES NOT. WE HYPOTHESIZE THAT CRFBP HAS DUAL EXCITATORY AND INHIBITORY EFFECTS ON CRF FUNCTION AND, THUS, ETHANOL CONSUMPTION, AND THAT THE CRFBP-CRF2 INTERACTION REPRESENTS A NOVEL PHARMACOLOGICAL TARGET FOR THE TREATMENT OF AUD. TO TEST THIS HYPOTHESIS, WE DEVELOPED CHEMICAL PROBES SPECIFIC FOR THE CRFBP(10KD)-CRF2 COMPLEX IN TWO LEAD SERIES THAT ACT AS NEGATIVE ALLOSTERIC MODULATORS (NAMS) OF THE CRFBP-CRF2 COMPLEX ONLY IN THE PRESENCE OF CRFBP(10KD). OUR RECENT STRUCTURE- ACTIVITY RELATIONSHIP (SAR) STUDIES HAVE PROVIDED CRFBP-CRF2 NAMS WITH GOOD ON-TARGET POTENCY AND SELECTIVITY PROFILES IN VITRO, BUT ADDITIONAL CHEMICAL OPTIMIZATION IS REQUIRED TO PRODUCE CHEMICAL PROBES THAT ARE READY FOR COMPREHENSIVE IN VIVO EVALUATION. THESE IN VIVO PROBES WOULD ALLOW US TO ESTABLISH THE ROLE OF CRFBP IN ALCOHOL CONSUMPTION AND FACILITATE THE DEVELOPMENT OF EFFECTIVE TREATMENTS TARGETING CRFBP FOR AUD. THUS, OUR OVERALL OBJECTIVE IS TO DEVELOP SYSTEMICALLY ACTIVE CRFBP-CRF2 NAMS SUITABLE FOR ADVANCED IN VIVO PROOF- OF-CONCEPT STUDIES FOR THE TREATMENT OF AUD. ACCORDINGLY, OUR SPECIFIC AIMS ARE: (1) DESIGN AND SYNTHESIZE NOVEL CRFBP-CRF2 NAMS WITH OPTIMAL DRUG-LIKE PROPERTIES; (2) CHARACTERIZE NOVEL CRFBP-CRF2 NAMS IN ASSAYS MEASURING POTENCY, SELECTIVITY AND DRUG-LIKE PROPERTIES; AND (3) DEMONSTRATE IN VIVO PROOF-OF-CONCEPT FOR SELECT CRFBP-CRF2 NAMS IN RODENT MODELS OF AUD. THE CRFBP-CRF2 NAMS GENERATED WILL PROVIDE POWERFUL IN VIVO PROBES FOR TESTING THE ROLE OF THE CRFBP-CRF2 INTERACTION IN VIVO. WE ARE WELL-POSITIONED TO DEVELOP POTENT AND SELECTIVE SMALL MOLECULE CRFBP-CRF2 NAMS WITH EXCELLENT PHARMACOKINETIC PROPERTIES FOR IN VIVO PROOF-OF- CONCEPT STUDIES IN RODENT MODELS OF AUD. THIS MULTIDISCIPLINARY RESEARCH PROGRAM HAS THE POTENTIAL FOR SIGNIFICANT SCIENTIFIC AND MEDICAL IMPACT BY CONTRIBUTING TO THE DISCOVERY OF NEW MEDICATIONS FOR AUD.

HYALURONAN IN BRAIN EXTRACELLULAR SPACE AS A REGULATOR OF GLYMPHATIC CLEARANCE OF ABETA

CHARACTERIZING NEUROPROTECTIVE ROLES FOR SNX27 SIGNALING PATHWAYS IN AD AND DS

MECHANISMS OF HEMATOPOIETIC ACUTE RADIATION SYNDROME INDUCTION AND RADIOPROTECTION THROUGH SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 SIGNAL MODULATION

LEAD OPTIMIZATION OF NOVEL CRFBP-CRFR2 COMPLEX MODULATORS FOR ALCOHOL USE DISORDER

POLYGENIC BASIS OF CARDIAC FUNCTION IN DROSOPHILA MELANOGASTER

PROTEIN SIALYLATION AND DE-SIALYATION IN CELL SURFACE GLYCOPROTEIN HOMEOSTASIS AND DISEASE - SUMMARY PROTEIN SIALYLATION IS A POST-TRANSLATIONAL MODIFICATION PRODUCED BY MEMBERS OF A FAMILY OF SIALYLTRANSFERASES. WE RECENTLY DISCOVERED THAT THE RESULTING SIALIC ACID LINKAGES ARE INVOLVED IN THE REGULATION OF GLYCOPROTEIN ABUNDANCE AND FUNCTION IN AN INTRINSIC HOMEOSTATIC MECHANISM THAT IS TARGETED BY MULTIPLE PATHOGENS. ENVIRONMENTAL FACTORS ARE DOMINANT IN THE ORIGINS OF THE HUMAN INFLAMMATORY BOWEL DISEASES (IBDS) AND SEASONAL BACTERIAL INFECTIONS HAVE BEEN IMPLICATED. WE THEREFORE DEVELOPED A MOUSE MODEL OF REPEATED HUMAN FOOD POISONING COMPRISED OF RECURRENT LOW-TITER NON-LETHAL GASTRIC INFECTIONS OF THE GRAM-NEGATIVE BACTERIAL PATHOGEN SALMONELLA ENTERICA TYPHIMURIUM (ST), A LEADING CAUSE OF HUMAN FOODBORNE ILLNESS WORLDWIDE. IN THIS UNIQUE MODEL, THE ST PATHOGEN WAS RAPIDLY CLEARED BY THE HOST, HOWEVER A PROGRESSIVELY SEVERE AND PERSISTENT COLITIS DEVELOPED SIMILAR TO ULCERATIVE COLITIS (UC). WE DEMONSTRATED THAT PATHOGENESIS WAS LINKED TO THE DISABLING OF A PROTECTIVE MECHANISM IN THE HOST INVOLVING THE ANTI-INFLAMMATORY INTESTINAL ALKALINE PHOSPHATASE (IAP) GLYCOPROTEIN ENZYME PRODUCED BY ENTEROCYTES. RECURRENT ST INFECTIONS RESULTED IN TOLL-LIKE RECEPTOR-4 (TLR4) INDUCTION OF NEURAMINIDASE (NEU) ACTIVITY AND HOST NEU3 ABUNDANCE WITH NASCENT IAP DE-SIALYLATION AND ENDOCYTIC DEGRADATION, THEREBY REDUCING IAP HALF-LIFE, ABUNDANCE, AND FUNCTION. IAP DEFICIENCY WAS SIMILARLY ACQUIRED IN MICE LACKING THE ST3GAL6 SIALYLTRANSFERASE RESULTING IN SPONTANEOUS COLITIS. THE DISEASE MECHANISM IN BOTH CASES WAS TLR4-DEPENDENT AND LINKED TO REDUCED DEPHOSPHORYLATION AND DETOXIFICATION OF THE LIPOPOLYSACCHARIDE-PHOSPHATE PRODUCED BY COMMENSAL BACTERIA OF THE COLON. IN HUMANS, SIMILAR MODULATION OF IAP AND NEU3 HAVE BEEN REPORTED IN COLITIS, AND GENETIC DEFICIENCY OF IAP CAUSES COLITIS, SUPPORTING THE RATIONALE FOR ONGOING CLINICAL TRIALS OF IAP AUGMENTATION AND NEURAMINIDASE INHIBITION. HOWEVER, THE KEY INVOLVEMENT OF NEU3 REMAINS TO BE ESTABLISHED. RESEARCH PROPOSED HEREIN WILL DIRECTLY TEST THE ROLE OF HOST NEU3 IN THE ONSET AND PROGRESSION OF COLITIS AMONG NEU3-NULL MICE. IN ADDITION, THE POSSIBILITY THAT OTHER ENTERIC GRAM-NEGATIVE PATHOGENS SIMILARLY PROVOKE IAP DEFICIENCY WILL BE INVESTIGATED WITH THE DEVELOPMENT OF OTHER RECURRENT NON-LETHAL GASTRIC INFECTION MODELS USING RELATED GRAM-NEGATIVE ENTERIC PATHOGENS INCLUDING THE HYPERVIRULENT SALMONELLA ENTERICA CHOLERAESUIS SEROVAR. WE HAVE RECENTLY MEASURED ELEVATED NEU ACTIVITY AND NEU3 PROTEIN ABUNDANCE IN THE COLON DURING RECURRENT ST INFECTION ASSOCIATED WITH THE DE-SIALYLATION OF MUCIN– 2 (MUC2), THE MAJOR GLYCOPROTEIN COMPONENT OF THE PROTECTIVE MUCIN BARRIER. THE MECHANISM OF EROSION OF THE MUCIN BARRIER IN COLITIS REMAINS UNKNOWN BUT PLAYS A LARGE ROLE IN PATHOGENESIS. WE HAVE RECENTLY LINKED EROSION OF THE MUCIN BARRIER TO REDUCTIONS OF MUC2 PROTEIN SIALYLATION, LIKELY BY ST3GAL6. REMARKABLY, NEU TREATMENT INCREASES MUC2 PROTEOLYSIS WITH REDUCED MUC2 ABUNDANCE. RESEARCH PROPOSED HEREIN WILL DETERMINE THE ROLE OF MUC2 SIALYLATION BY ST3GAL6 IN GENERATING AND MAINTAINING THE PROTECTIVE MUCIN BARRIER. TOGETHER THESE STUDIES WILL PROVIDE IMPORTANT ADVANCES PERTAINING TO THE PREVENTION AND TREATMENT OF COLITIS.

TRANSFORMATIVE RESEARCH ON THE NORMAL AND ALZHEIMER'S DISEASE BRAIN THROUGH STUDIES OF NEURONAL GENE RECOMBINATION

VIRULENCE MECHANISMS OF VIRAL BCL-2 HOMOLOGS

MATURATION OF NORMAL AND DISEASE-SPECIFIC HUMAN STEM CELL-DERIVED CARDIOMYOCYTES

ANTIGEN-SPECIFIC B CELL TOLERANCE IN THE GUT-ASSOCIATED LYMPHOID TISSUE (GALT)

NLR FAMILY PROTEINS: MECHANISMS AND REGULATION

SIGNALING INTERACTIONS IN CELL SURVIVAL AND INVASION

NEW HYDROCEPHALUS THERAPIES THROUGH INTERRUPTION OF LIPID SIGNALING AND INFLAMMATORY PATHWAYS USING NOVEL DRUG-LIKE COMPOUNDS

MSX2-WNT SIGNALING IN CARDIOVASCULAR CALCIFICATION

DISCOVERY OF GLUTARYL-COA DEHYDROGENASE INHIBITORS FOR MELANOMA AND PANCREATIC CANCER - PROJECT SUMMARY MELANOMA IS AN EXTREMELY AGGRESSIVE CANCER WITH HIGH MORTALITY. ITS PHENOTYPIC PLASTICITY AND HETEROGENEITY ENABLE IT TO ADAPT TO DIVERSE PHYSIOLOGICAL SETTINGS AND DEFEAT TREATMENT APPROACHES. TARGETED THERAPIES AND CHECK POINT INHIBITORS BECAME AVAILABLE IN THE PAST DECADE; HOWEVER, THESE DRUGS WORK IN ONLY A SUBSET OF PATIENTS AND DRUG RESISTANCE EVENTUALLY EMERGES, EVEN IN INITIAL RESPONDERS. THEREFORE, NEW TARGETS AND CLINICAL APPROACHES FOR MELANOMA ARE AN UNMET MEDICAL NEED. WE IDENTIFIED GLUTARYL-COA DEHYDROGENASE (GCDH) AS ONE SUCH TARGET. GCDH EXPRESSION CORRELATES WITH AGGRESSIVE CANCERS AND LOW SURVIVAL IN MELANOMA PATIENTS. OUR DATA DEMONSTRATES THAT GCDH KNOCKDOWN RESULTS IN APOPTOTIC CELL DEATH IN MELANOMA CELLS. MELANOMAS SEEM UNIQUELY SENSITIVE TO TOXIC GLUTARATE METABOLITES RESULTING FROM GCDH DEFICIENCIES, SINCE SUPPRESSION OF THE DEHYDROGENASE E1 AND TRANSKETOLASE DOMAIN CONTAINING 1 (DHTKD1) ENZYME, CATALYZING THE PRECEDING REACTION IN THE CATABOLIC PATHWAY AND CONVERTING 2-OXOADIPATE TO GLUTARYL-COA, RESCUES MELANOMA CELLS FROM APOPTOSIS. PANCREATIC CANCER CELLS, BUT NOT OTHER CANCERS NOR NORMAL CELLS, SHARE THIS OVERRELIANCE ON GCDH AND UNDERGO APOPTOSIS UPON GCDH KNOCKDOWN. WE HYPOTHESIZE THAT SMALL-MOLECULES INTERFERRING WITH GCDH WILL RESULT IN THE OBLITERATION OF MELANOMA CELLS THROUGH APOPTOSIS. WE PROPOSE TO IDENTIFY CHEMICAL PROBES OF GCDH TO FURTHER VALIDATE THE ENZYME AS A MOLECULAR TARGET FOR MELANOMA. IN A PILOT SCREEN, WE ESTABLISHED AND VALIDATED ALL OF THE ASSAYS PROPOSED HEREIN. WE WILL PERFORM LARGE-SCALE HTS, HIT CONFIRMATION AND OPTIMIZATION, AND VALIDATE THE IDENTIFIED HITS IN A PANEL OF DIVERSE CELL LINES FOR MELANOMA AND OTHER CANCERS. COMPOUNDS IDENTIFIED WILL PROVIDE DESIRABLE PHARMACOLOGICAL TOOLS TO STUDY THE PATHOPHYSIOLOGY OF GCDH IN MELANOMA AND OTHER CANCERS, AND THE MOLECULAR MECHANISMS OF LYSINE METABOLISM LIABILITY IN MELANOMA, AS WELL AS PROVIDE POTENTIAL STARTING POINTS FOR FUTURE THERAPEUTIC TREATMENTS. THIS 4-YEAR PROJECT WILL PURSUE THE FOLLOWING SPECIFIC AIMS, CONSISTENT WITH THE EXPECTATIONS IN PAR-20-271: AIM 1 GENERATE GCDH PROTEIN, OPTIMIZE CONDITIONS AND PERFORM SCREENING TO IDENTIFY COMPOUNDS TARGETING GCDH. OPTIMIZE TERTIARY ASSAYS FOR HIT VALIDATION. PRIMARY ASSAYS WILL TARGET BINDING ACTIVITY AND INHIBITION, AND TERTIARY ASSAYS WILL MONITOR CELLULAR TARGET ENGAGEMENT AND PROTEIN LEVEL. AIM 2 PERFORM HIT SELECTION, CONFIRMATION AND PROFILING USING A PANEL OF SECONDARY ASSAYS. FUNCTIONAL HIT PROFILES WILL BE ESTABLISHED USING INHIBITION ASSAYS FOR A REPRESENTATIVE OF ACYL-COA DEHYDROGENASES AND MECHANISM OF ACTION STUDIES BY USING PROTEIN THERMAL SHIFT WITH SUBSTRATE ANALOGS AND ENZYME KINETIC STUDIES TO ESTABLISH COMPETITION PROFILES WITH THE SUBSTRATES. AIM 3 PERFORM HIT VALIDATION AND SCAFFOLD PRIORITIZATION USING BIOCHEMICAL AND CELLULAR ASSAYS. GCDH TARGET ENGAGEMENT WILL BE CONFIRMED IN AN IN-CELL PROTEIN STABILITY ASSAY. EFFECTS ON CELLULAR PROTEIN LEVELS WILL BE EVALUATED USING ENDOGENOUS-TAGGED GCDH IN MELANOMA CELLS. BEST SCAFFOLDS WILL BE VALIDATED IN CELLULAR ASSAYS TO MONITOR APOPTOSIS, CELL VIABILITY, AND SPECIFICITY TOWARDS MELANOMA AND PANCREATIC CANCER.

LYMPHOTOXIN ALPHA BETA AND LIGHT CYTOKINE SYSTEMS

(PQD1) TREATMENT-EMERGENT RESISTANCE TO NEDD8-ACTIVATING ENZYME INHIBITION

IDENTIFYING GENES IN BETA-AMYLOID PRODUCTION

CONTROL OF PROTEIN SYNTHESIS BY THE UPS UNDER STRESS

LEAD OPTIMIZATION OF NOVEL INHIBITORS OF THE THIOESTERASE DOMAIN OF FASN

CYTOPLASMIC CHROMATIN FRAGMENTS IN CELL SENESCENCE - NOVEL MECHANISMS AND INTERVENTIONS - PROJECT SUMMARY CELLULAR SENESCENCE IS A CAUSE OF CELL AND TISSUE AGING. SENESCENCE IS CAUSED BY A RANGE OF CELLULAR STRESSES AND CHARACTERIZED BY AN IRREVERSIBLE PROLIFERATION ARREST AND A POTENT PRO-INFLAMMATORY PHENOTYPE, THE SENESCENCE-ASSOCIATED SECRETORY PHENOTYPE (SASP). SENESCENCE-ASSOCIATED PROLIFERATION ARREST AND SASP COOPERATE IN TUMOR SUPPRESSION, BY ARRESTING PROLIFERATION OF DAMAGED PRE-MALIGNANT CELLS AND PROMOTING IMMUNE CLEARANCE OF THE DAMAGED CELLS. HOWEVER, OVER THE LONGER TERM, AS A SOURCE OF CHRONIC INFLAMMATION, SASP ALSO PROMOTES TISSUE AGING AND DISEASE. CONSEQUENTLY, THERE IS CURRENTLY MUCH EFFORT DEVOTED TO DEVELOPMENT OF PHARMACOLOGIC APPROACHES TO ELIMINATE SENESCENT CELLS TO PROMOTE HEALTHY AGING. HOWEVER, THESE SO-CALLED SENOLYTIC DRUGS TEND TO SHOW UNWANTED TOXICITIES. AN ALTERNATIVE, PERHAPS LESS TOXIC APPROACH, IS TO USE SENOMORPHIC DRUGS TO SPECIFICALLY INHIBIT THE PRO-AGING SASP. IMPORTANTLY, INHIBITION OF SASP DOES NOT NECESSARILY IMPAIR THE TUMOR SUPPRESSIVE ROLE OF SENESCENCE. ON THE CONTRARY, ELIMINATION OF SASP CAN PREVENT CANCER. HENCE, IT IS IMPORTANT TO DEFINE THE MECHANISM OF SASP ACTIVATION, BECAUSE ITS INHIBITION MAY BE AN APPROACH TO COMBAT THE PRO-AGING EFFECTS OF SENESCENT CELLS. RECENTLY, WE SHOWED THAT SENESCENT CELLS SHED FRAGMENTS OF NUCLEAR CHROMATIN INTO THE CYTOPLASM, CYTOPLASMIC CHROMATIN FRAGMENTS (CCF), VIA A NUCLEUS-TO-CYTOPLASMIC BLEBBING PROCESS. CCF ARE VERY STRONGLY POSITIVE FOR A DNA DAMAGE MARKER, PHOSPHORYLATED HISTONE H2AX. FORMATION OF CCF DEPENDS UPON A NOVEL MANIFESTATION OF THE CELLULAR RECYCLING PROCESS AUTOPHAGY, SPECIFICALLY NUCLEAR AUTOPHAGY. CCF SIGNAL THROUGH CYTOPLASMIC DNA SENSORS TO ACTIVATE NFB, THE MAJOR TRANSCRIPTIONAL ACTIVATOR OF SASP. MOST RECENTLY, WE HAVE DEFINED AN UNANTICIPATED UPSTREAM TRIGGER OF CCF AND SASP, NAMELY DYSFUNCTIONAL MITOCHONDRIA IN SENESCENT CELLS. DYSFUNCTIONAL MITOCHONDRIA ARE THEMSELVES ALREADY LINKED TO CHRONIC INFLAMMATION AND AGING. WE HYPOTHESIZE THAT NUCLEAR EXPULSION OF CCF HARBORING DNA DOUBLE STRAND BREAKS IS A MECHANISM FOR SENESCENT CELLS WITH IMPAIRED DNA REPAIR PATHWAYS TO DECREASE THE INTRANUCLEAR LOAD OF TOXIC DNA DOUBLE STRAND BREAKS. WE ALSO HYPOTHESIZE THAT DYSFUNCTIONAL MITOCHONDRIA TRIGGER AUTOPHAGY-DEPENDENT FORMATION OF CCF IN SENESCENT CELLS BY RETROGRADE MITOCHONDRIA-TO-NUCLEUS SIGNALING, INVOLVING JNK KINASE AND THE TRANSCRIPTION FACTORS HYPOXIA INDUCIBLE FACTOR 1A (HIF1A), THE “JNK-CCF” AND “HIF-CCF” PATHWAYS, RESPECTIVELY. DELINEATION OF THE JNK-CCF AND HIF-CCF PATHWAYS AND THEIR INTERACTIONS WILL PROVIDE NEW OPPORTUNITIES TO INTERVENE TO SUPPRESS CHRONIC INFLAMMATION DRIVEN BY SASP IN VIVO, THEREBY POTENTIATING HEALTHY AGING AND LONGEVITY.

SHOTGUN LIPIDOMICS AND ALTERATIONS IN SPHINGOLIPIDOMES IN ALZHEIMER'S DISEASES

FUNCTION AND MECHANISM OF THE HIPPO-PI5P4K AXIS FOR GROWTH CONTROL - PROJECT SUMMARY OUR LONG-TERM GOAL IS TO DECIPHER THE MOLECULAR MECHANISMS UNDERLYING PI5P4K-DRIVEN GROWTH AND TO ELUCIDATE HOW THEIR DYSREGULATION CONTRIBUTES TO BREAST TUMORIGENESIS. IN THIS PROPOSAL, WE WILL SET OUT TO PROVIDE A COMPREHENSIVE UNDERSTANDING OF A NEW SIGNALING NETWORK THAT IS CRITICAL TO INTEGRATE METABOLIC INFORMATION AND DETERMINE HOW THE RELATED PROTEINS AND PATHWAYS INTERACT TO MAINTAIN CELLULAR HOMEOSTASIS. PHOSPHATIDYLINOSITOL-5-PHOSPHATE 4-KINASES (PI5P4KS) ARE A FAMILY OF NON-CANONICAL PHOSPHOINOSITIDE KINASES WHICH WE HAVE SHOWN TO BE CRITICAL FOR PRESERVING NUTRIENT AVAILABILITY AND FUEL GROWTH YET HOW THEY ARE REGULATED THEMSELVES IS LARGELY UNKNOWN. USING A COMPREHENSIVE IN VITRO KINASE SCREEN, WE HAVE DISCOVERED THE CORE KINASES OF THE HIPPO PATHWAY, MST1 AND MST2 AS DIRECT NEGATIVE REGULATORS OF THE PI5P4KS AS WELL AS THE LIPID SUBSTRATE OF THE PI5P4KS, PI-5-P TO INTERACT WITH THE HIPPO ADAPTOR PROTEIN MOB1 WHICH IN TURN EFFECTS THE ACTIVITY OF THE HIPPO DOWNSTREAM EFFECTOR YAP. THE HIPPO PATHWAY IS KNOWN TO BE IMPORTANT FOR DEVELOPMENT, GROWTH AND ORGANOGENESIS, AND DYSREGULATION OF THIS PATHWAY LEADS TO TUMOR PROGRESSION. WHILE PI5P4KS HAVE BEEN LINKED TO TUMOR GROWTH, EXACTLY HOW THE PI5P4KS ARE REGULATED IS POORLY UNDERSTOOD. FURTHERMORE, HOW THE PI5P4KS ACTUALLY COMMAND CELL GROWTH UNDER BOTH PHYSIOLOGICAL CONDITIONS AND PATHOLOGICAL CONTEXTS SUCH AS BREAST CANCER IS INCOMPLETE. TO ANSWER THESE IMPORTANT QUESTIONS, WE WILL PERFORM THE FOLLOWING AIMS: IN AIM 1 WE WILL USE VERY FEASIBLE BIOPHYSICAL AND STRUCTURAL BIOLOGY TO DETERMINE ATOMIC-RESOLUTION DETAILS OF THE INTERFACE BETWEEN PI-5-P AND MOB1 AND QUANTITATE HOW PI-5-P REGULATES MOB1 BINDING TO OTHER HIPPO PATHWAY COMPONENTS IN VITRO. IN AIM 2 WE WILL INVESTIGATE HOW THE ACTIVITY OF THE PI5P4KS CONTROL THE ACTIVATION OF THE MOB-LATS COMPLEX DOWNSTREAM OF MST1/2, EXPLORING ONE POTENTIAL MECHANISM FOR PI5P4K TUMORIGENIC PROPERTIES. IN AIM 3 WE WILL ESTABLISH THE BIOLOGICAL SIGNIFICANCE OF THE PI5P4K-HIPPO AXIS IN BREAST CANCER USING A PANEL OF TNBC CELL LINES, NOVEL MOUSE BREAST TUMOR MODELS, AND PATIENT BREAST TUMOR SAMPLES. THIS PROJECT SEEKS TO COMBINE QUANTITATIVE STRUCTURAL BIOLOGY WITH MOLECULAR CELL GENETICS TO DECIPHER HOW PI-5- P REGULATES THE HIPPO PATHWAY FOR BREAST CANCER CELL GROWTH. GIVEN THAT PI5P4KS ARE EXCITING ‘DRUGGABLE’ TARGETS FOR BREAST CANCER, CHARACTERIZING HOW PI5P4KS CONNECT TO THE HIPPO PATHWAY REVEALS A POSSIBLE STRATEGY FOR FUTURE ANTI-CANCER THERAPIES.

COLLAGEN FIBERS AND FRAGMENTS REGULATE PANCREATIC CANCER METABOLISM AND GROWTH. - PROJECT SUMMARY PANCREATIC DUCTAL ADENOCARCINOMA (PDAC) IS CHARACTERIZED BY EXTENSIVE DESMOPLASIA, RESULTING IN OCCUPANCY OF MOST OF THE TUMOR MASS BY A DENSE STROMA CONSISTING OF FIBROBLASTS, MACROPHAGES, AND EXTRACELLULAR MATRIX (ECM). FIBRILLAR COLLAGENS, SUCH AS TYPE I COLLAGEN (COL I), ARE MAJOR ECM COMPONENTS AND WERE REPORTED TO EXERT POORLY UNDERSTOOD STIMULATORY AND INHIBITORY EFFECTS ON PDAC GROWTH AND MALIGNANCY. IT WAS ALSO OBSERVED THAT PATIENTS WHOSE STROMA IS FIBROLYTIC, WITH A HIGH CONTENT OF CLEAVED COLLAGEN FRAGMENTS GENERATED BY MATRIX METALLOPROTEASES (MMP), SHOW POOR OVERALL SURVIVAL AFTER RESECTION, WHEREAS PATIENTS WITH AN INERT STROMA, RICH IN INTACT COLLAGEN FIBERS, HAVE CONSIDERABLY IMPROVED SURVIVAL. THE MECHANISMS UNDERLYING THESE CLINICAL OBSERVATIONS AND THE TUMOR STIMULATORY OR INHIBITORY EFFECTS OF COL I WERE HERETOFORE UNKNOWN. USING A UNIQUE CULTURE SYSTEM, PRECLINICAL MODELS, AND CLINICAL SPECIMENS, WE DISCOVERED THAT INTACT (I) COL I FIBERS AND MMP- GENERATED CLEAVED (C) COL I FRAGMENTS HAVE DIAMETRICALLY OPPOSED EFFECTS ON PDAC METABOLISM AND GROWTH DUE TO THEIR DIFFERENTIAL INTERACTIONS WITH THE COLLAGEN RECEPTOR DDR1. WHEREAS CCOL I (AND OTHER MMP-CLEAVED COLLAGENS) ACTIVATES DDR1 AND ITS DOWNSTREAM EFFECTORS, WHICH INCLUDE TRANSCRIPTION FACTORS NF-KB AND NRF2, TO STIMULATE PDAC METABOLISM, MITOCHONDRIAL BIOGENESIS, AND RESPIRATION, ICOL I DOES NOT ACTIVATE DDR1 AND INSTEAD INDUCES ITS UBIQUITIN-DEPENDENT PROTEASOMAL DEGRADATION, THEREBY INHIBITING NRF2 ACTIVATION AND PDAC METABOLISM. OUR GOAL IS TO UNDERSTAND HOW MMP CLEAVAGE ENABLES THE BINDING OF CCOL I TO DDR1 AND DETERMINE IF BINDING TO DDR1 IS REQUIRED FOR INDUCTION OF ITS DEGRADATION BY ICOL I. TO FULLY UNDERSTAND HOW CCOL I AND DDR1 SIGNALING CONTROL PDAC METABOLISM AND GROWTH, AND WHY COL I ABLATION IS NOT AS INHIBITORY AS PREVENTION OF COL I CLEAVAGE, WE WILL TEST WHETHER THE DIFFERENTIAL EFFECTS OF CCOL I AND ICOL I ON DDR1 EXPRESSION AND ACTIVATION APPLY TO OTHER FIBRILLARY COLLAGENS (COL III) AND DEFINE THE UNDERLYING BIOCHEMICAL MECHANISMS THAT ENABLE DDR1 BINDING. NEXT, WE WILL INVESTIGATE HOW ICOL I TRIGGERS DDR1 UBIQUITINATION AND PROTEASOMAL DEGRADATION, AND THE ROLE OF THE NEWLY UNCOVERED DDR1 E3 LIGASE FBXW2 IN THIS PROCESS. THESE STUDIES WILL INCLUDE IDENTIFICATION OF THE MECHANISM BY WHICH ICOL I STIMULATES DDR1 UBIQUITINATION BY FBXW2 AND DEVELOPMENT OF LIGASE-RECRUITING TOOL COMPOUNDS THAT INDUCE DDR1 DEGRADATION, WHOSE BIOLOGICAL EFFECTS WILL BE EVALUATED IN CULTURED CELLS AND MOUSE MODELS. THIS WILL BE FOLLOWED BY STUDYING THE ROLE OF INDIVIDUAL MMP ISOZYMES, EXPRESSED IN PDAC CANCER CELLS, CANCER-ASSOCIATED FIBROBLASTS, AND MACROPHAGES, IN COLLAGEN REMODELING AND PDAC METABOLISM AND GROWTH. WE WILL DETERMINE THE SUITABILITY OF INDIVIDUAL COLLAGEN CLEAVING MMPS AS DRUG TARGETS FOR THE TREATMENT OF CCOL I ENRICHED PDAC. WE WILL ALSO DETERMINE THE MECHANISMS RESPONSIBLE FOR MMP INDUCTION IN THE PDAC MICROENVIRONMENT, AS THEY MAY PROVIDE ADDITIONAL THERAPEUTIC TARGETS. THE PROPOSED STUDIES WILL EMPLOY CUTTING- EDGE TECHNOLOGIES, INNOVATIVE ECM BASED PDAC CULTURES, NEW GENETICALLY MODIFIED MOUSE STRAINS THAT ALLOW THE EVALUATION OF BOTH PRIMARY AND LIVER METASTATIC PDAC AND CLINICAL SPECIMENS.

STRUCTURE AND FUNCTION OF BCAR1 AND BCAR3 ASSOCIATION IN BREAST CANCER MALIGNANCY

LEAD OPTIMIZATION OF NOVEL INHIBITORS OF TISSUE NON-SPECIFIC ALKALINE PHOSPHATASE

JIP MIMICS FOR THE TREATMENT OF DIABETES

APOPTOSIS-BASED CANCER DRUG DISCOVERY

NOVEL INSIGHTS IN THE REGULATION OF HIF1ALPHA STABILITY

MOLECULAR PATHOGENESIS OF AF10-REARRANGED LEUKEMIAS - PROJECT SUMMARY THERE HAS BEEN CONSIDERABLE PROGRESS IN THE LAST FEW YEARS IN THE CHARACTERIZATION OF MOLECULAR ALTERATIONS IN CHILDHOOD CANCER. MANY DEFINING GENOMIC ABNORMALITIES AS WELL AS TRANSCRIPTIONAL AND SIGNALING NETWORKS INVOLVED IN SPECIFIC SUB-TYPES OF PEDIATRIC CANCERS HAVE BEEN IDENTIFIED. HOWEVER, DESPITE THESE IMPRESSIVE ADVANCES, SAFE AND EFFECTIVE THERAPIES FOR MOST PEDIATRIC CANCERS ARE LACKING. LEUKEMIAS ARE THE LEADING CAUSE OF CANCER-LINKED MORTALITY IN CHILDREN AND FOR MANY PEDIATRIC LEUKEMIAS, THERAPIES THAT CAN SELECTIVELY ELIMINATE CANCER CELLS WITH FEW UNDESIRABLE SIDE EFFECTS HAVE BEEN ELUSIVE. THEREFORE, THERE IS AN URGENT CLINICALLY UNMET NEED TO DEVELOP NON-TOXIC AND TARGETED THERAPIES FOR PEDIATRIC LEUKEMIA. OUR PROPOSAL IS FOCUSED ON LEUKEMIAS WITH REARRANGEMENTS OF THE AF10 GENE. THESE REARRANGEMENTS ARE OBSERVED IN ACUTE MYELOID LEUKEMIA (AML), T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA (T-ALL) AND OTHER SUB-TYPES OF LEUKEMIA IN CHILDREN AND ADULTS. AF10-REARRANGED (AF10-R) LEUKEMIAS CAN BE CONSIDERED PROTOTYPICAL MODELS OF PEDIATRIC CANCERS IN WHICH CHIMERIC FUSION ONCOPROTEINS OR ALTERED TRANSCRIPTION FACTORS DRIVE EXTENSIVE SELF- RENEWAL AND IMPAIRED DIFFERENTIATION OF CANCER CELLS. OUR RECENT ANALYSIS OF DATA FROM OVER 1,000 PEDIATRIC AML PATIENTS DEMONSTRATES THAT AF10 GENE FUSIONS ARE MUCH MORE COMMON THAN GENERALLY APPRECIATED (REPRESENTING 6.5% OF PEDIATRIC AML) AND CONSTITUTE ONE OF THE HIGHEST RISK-GROUPS IN TERMS OF RESISTANCE TO PRIMARY THERAPIES AND RELAPSE RATES. OUR DETAILED MECHANISTIC STUDIES DEMONSTRATE THAT THE AF10 FUSION PROTEIN CALM-AF10 ACTIVATES A NUMBER OF PATHWAYS LINKED TO STEM CELL SELF-RENEWAL AND INFLAMMATORY SIGNALING, MANY OF WHICH ARE INDEPENDENTLY ASSOCIATED WITH POOR OUTCOMES IN AML. IN THIS PROPOSAL, WE WILL USE A SERIES OF ORTHOGONAL METHODOLOGIES, INCLUDING PROTEOMIC, GENOMIC, EPIGENOMIC AND DETAILED MECHANISTIC STUDIES IN BOTH HUMAN AND MOUSE AML MODELS, TO DETERMINE HOW THE CALM-AF10 FUSION PROTEIN CAUSES LEUKEMOGENESIS. THE SUCCESSFUL EXECUTION OF THE PROPOSED AIMS WILL ADVANCE OUR EFFORTS TOWARDS FINDING NEWER TARGETED THERAPIES FOR AF10-R AML WITH FEWER UNWANTED TOXICITIES. RESULTS FROM OUR STUDIES ALSO HAVE THE POTENTIAL TO SIGNIFICANTLY INFLUENCE TREATMENT STRATEGIES IN SEVERAL OTHER SUB-TYPES OF PEDIATRIC LEUKEMIAS DRIVEN BY RELATED MECHANISMS.

THE P62/MEKK3 COMPLEX IN MTORC1 ACTIVATION

NOVEL PATHWAYS AND TECHNOLOGIES TOWARD BROAD-SPECTRUM THERAPEUTICS

PROTEIN KINASE CZ TARGETS IN INTESTINAL CANCER STEM CELLS

NON-CANONICAL PHOSPHATIDYLINOSITOL KINASES IN TRIPLE NEGATIVE BREAST CANCER

TARGETING ER+ BREAST CANCER THROUGH INDUCED VIRAL MIMICRY - PROJECT SUMMARY REPETITIVE ELEMENTS (RES) COMPOSE ~45% OF THE HUMAN GENOME AND ARE NORMALLY TRANSCRIPTIONALLY SILENCED IN SOMATIC CELLS, ALTHOUGH THE MECHANISM HAD REMAINED ELUSIVE. THROUGH A HIGH-CONTENT RNAI SCREEN, WE IDENTIFIED THE LARGELY UNCHARACTERIZED PROTEIN FBXO44 AS AN ESSENTIAL REPRESSOR OF RES IN BREAST CANCER CELLS. FBXO44 BOUND REPRESSIVE HISTONE H3 LYSINE 9 TRIMETHYLATED (H3K9ME3) NUCLEOSOMES AT THE REPLICATION FORK AND RECRUITED H3K9ME3 METHYLTRANSFERASE SUV39H1, UBIQUITIN LIGASE CRL4RBBP4/7, AND HISTONE DEACETYLASE AND CHROMATIN- REMODELING COMPLEX MI-2/NURD TO TRANSCRIPTIONALLY SILENCE RES POST-DNA REPLICATION. FBXO44/SUV39H1 INHIBITION TRANSCRIPTIONALLY REACTIVATED ENDOGENOUS RETROVIRUSES (ERVS) AND RETROTRANSPOSONS (E.G. ALU, LINE-1) IN BREAST CANCER CELLS, LEADING TO EXTENSIVE DNA REPLICATION STRESS AND STIMULATION OF RIG-I/MDA5-MAVS AND CGAS-STING INTRACELLULAR ANTIVIRAL PATHWAYS TO PROMOTE ENHANCED IMMUNOGENICITY AND DECREASED TUMORIGENICITY. IN SILICO ANALYSIS REVEALED THE FBXO44/SUV39H1 PATHWAY INVERSELY CORRELATED WITH DNA REPLICATION STRESS, ANTIVIRAL PATHWAYS, AND CYTOTOXIC T AND NATURAL KILLER (NK) CELL INFILTRATION IN HUMAN BREAST TUMORS. IMPORTANTLY, FBXO44/SUV39H1 WERE FOUND DISPENSABLE FOR RE SILENCING IN NORMAL CELLS AND THEIR INHIBITION HAD NO EFFECT ON H3K9ME3 LEVELS, DNA REPLICATION STRESS, OR VIABILITY, SUGGESTING A THERAPEUTIC WINDOW. OUR HYPOTHESIS IS THAT FBXO44/SUV39H1-MEDIATED RE SILENCING IS AN EPIGENETIC VULNERABILITY OF BREAST CANCER CELLS THAT COULD BE TARGETED TO INHIBIT TUMOR GROWTH/PROGRESSION AND ENHANCE THE EFFICACY OF CERTAIN ANTITUMOR THERAPIES THROUGH THE UNIQUE MECHANISM OF INDUCED VIRAL MIMICRY. IN THIS PROPOSAL, WE WILL EVALUATE 3 POTENTIAL THERAPEUTIC APPLICATIONS OF FBXO44/SUV39H1 PATHWAY TARGETING IN THE TREATMENT OF ESTROGEN RECEPTOR (ER)+ BREAST CANCERS BASED ON OUR PRELIMINARY DATA: 1) PREVENTION OF BONE METASTASIS RELAPSE THROUGH STIMULATION OF NK CELL RECOGNITION AND KILLING OF DORMANT BREAST CANCER CELLS; 2) ENHANCEMENT OF IMMUNE CHECKPOINT BLOCKADE (ICB) THERAPY THROUGH STIMULATION OF IFN SIGNALING AND INTRATUMORAL INFILTRATION OF CYTOTOXIC T CELLS; AND 3) SYNERGY WITH PARP INHIBITORS THROUGH INDUCTION OF DNA REPLICATION STRESS AND DOUBLE-STRAND BREAKS (DSBS) AT RES. THESE STUDIES COULD LEAD TO THE DEVELOPMENT OF A SAFE AND EFFECTIVE THERAPEUTIC APPROACH THAT SELECTIVELY INDUCES VIRAL MIMICRY IN ER+ BREAST CANCER CELLS TO PREVENT BONE METASTASIS RELAPSE AND ENHANCE THE EFFICACY OF ICB AND PARP INHIBITOR THERAPIES, UNDOUBTEDLY LEADING TO A SIGNIFICANT REDUCTION IN DISEASE MORTALITY.

TARGETING THE FBXO44/SUV39H1 PATHWAY IN CANCER - PROJECT SUMMARY REPETITIVE ELEMENTS (RES) COMPOSE ~45% OF THE HUMAN GENOME AND ARE NORMALLY TRANSCRIPTIONALLY SILENCED, ALTHOUGH THE MECHANISM HAS REMAINED ELUSIVE. THROUGH A HIGH-CONTENT RNAI SCREEN, WE IDENTIFIED FBXO44 AS AN ESSENTIAL REPRESSOR OF RES IN CANCER CELLS. FBXO44 BOUND REPRESSIVE H3K9ME3-MODIFIED NUCLEOSOMES AT THE REPLICATION FORK AND RECRUITED H3K9ME3 METHYLTRANSFERASE SUV39H1, UBIQUITIN LIGASE CRL4RBBP4/7, AND HISTONE DEACETYLASE AND CHROMATIN-REMODELING COMPLEX MI-2/NURD TO TRANSCRIPTIONALLY SILENCE RES POST-DNA REPLICATION. FBXO44/SUV39H1 INHIBITION TRANSCRIPTIONALLY ACTIVATED SATELLITE REPEATS AND ENDOGENOUS RETROVIRUSES AND RETROTRANSPOSONS IN CANCER CELLS, LEADING TO DNA REPLICATION STRESS AND STIMULATION OF MAVS AND STING INTRACELLULAR ANTIVIRAL PATHWAYS TO PROMOTE DECREASED TUMORIGENICITY AND ENHANCED IMMUNOTHERAPY RESPONSE. IN SILICO ANALYSIS REVEALED THAT FBXO44 EXPRESSION INVERSELY CORRELATED WITH DNA REPLICATION STRESS, ANTIVIRAL PATHWAYS, AND CYTOTOXIC T AND NATURAL KILLER (NK) CELL INFILTRATION IN HUMAN CANCERS. IMPORTANTLY, WE FOUND THAT FBXO44/SUV39H1 ARE DISPENSABLE FOR RE SILENCING IN NORMAL CELLS AND THEIR INHIBITION DID NOT AFFECT H3K9ME3 LEVELS AT RES OR CELL VIABILITY. OUR HYPOTHESIS IS THAT FBXO44/SUV39H1-MEDIATED RE ELEMENT SILENCING IS AN EPIGENETIC VULNERABILITY OF CANCER CELLS THAT COULD POTENTIALLY BE TARGETED TO INDUCE VIRAL MIMICRY RESPONSES THAT INHIBIT TUMOR GROWTH AND PROGRESSION AND ENHANCE THE EFFICACY OF CERTAIN CANCER THERAPIES. IN THIS PROPOSAL, WE WILL PERFORM PRECLINICAL STUDIES THAT EVALUATE TWO POTENTIAL THERAPEUTIC APPLICATIONS OF FBXO44/SUV39H1 PATHWAY TARGETING IN CANCER: 1) PREVENTION OF METASTATIC RELAPSE THROUGH STIMULATION OF INTRACELLULAR ANTIVIRAL PATHWAYS AND NK CELL RECOGNITION; AND 2) ENHANCEMENT OF PARP INHIBITOR EFFICACY THROUGH INDUCTION OF EXTENSIVE DNA REPLICATION STRESS AT RES. MOREOVER, WE WILL INVESTIGATE THE ROLE OF THE CRL4RBBP4/7 UBIQUITIN LIGASE IN FBXO44/SUV39H1-MEDIATED RE SILENCING AND EVALUATE ITS TARGETING FOR CANCER TREATMENT. THESE STUDIES COULD UNCOVER A TARGETABLE EPIGENETIC VULNERABILITY OF CANCER CELLS WHOSE INHIBITION INDUCES VIRAL MIMICRY TO PREVENT TUMOR GROWTH AND PROGRESSION AND ENHANCE THE EFFICACY OF CANCER THERAPEUTICS, UNDOUBTEDLY LEADING TO A SIGNIFICANT REDUCTION IN DISEASE MORTALITY.

GENETIC DISSECTION OF THE ROLE OF CHONDROITIN SULFATE IN CARTILAGE

FACTORS REGULATING DEVELOPMENT OF APPENDICULAR SKELETAL PROGENITORS

REGULATION OF MEDULLOBLASTOMA METASTASIS BY EMP1

S1P SIGNALING MACHINERY IN NEUROIMMUNE INTERACTIONS

DENERVATION ACTIVATED SUPER-ENHANCERS OF PATHOGENIC IL6-STAT3 FEEDFORWARD LOOP IN FAPS

MODELING B CELL LYMPHOMA IN THE MOUSE

ROLE OF THE LONG NON-CODING RNA PVT1 IN CANCER

STRUCTURAL BASIS OF ALLOSTERY AND MECHANICAL PROPERTIES OF F-ACTIN

ROLE OF STAT3 IN MUSCLE STEM CELL ACTIVATION

THE MECHANISM OF DISC FORMATION

THE P62/ATYPICAL PKC SIGNALING COMPLEX IN TH2 DIFFERENTIATION AND ASTHMA

DE-ORPHANIZING MMPS IN INTERCELLUAR INTERACTIONS

ROLE OF MELK IN GLIOBLASTOMAS

REGULATION OF FOOD INTAKE VIA THE GLUCAGON-LIKE PEPTIDE-1 RECEPTOR

ATF2 IN MELANOMA DEVELOPMENT AND PROGRESSION

PATIENT-DERIVED STEM CELLS FOR PHOSPHOPROTEOMIC PROFILING NEUROPSYCHOPATHOLOGY

A NANOSYSTEM FOR TUMOR TREATMENT AND IMAGING

MECHANISMS OF IMMUNE MODULATION IN JIA

CYSTEINYL LEUKOTRIENES IN HIV BRAIN INJURY

DISCOVERY OF SMALL MOLECULE PROMOTERS OF CARDIOMYOCYTE PROLIFERATION TO RESTORE CARDIAC PERFORMANCE IN DISEASE

CARBOHYDRATE DEFICIENT GLYCOPROTEIN SYNDROMES

ROLES OF SN27 IN REGULATING GLUTAMATE RECEPTORS DURING NEURODEGENERATION

HVEM-BTLA PATHWAY IN LYMPHOMA

IAP FAMILY PROTEINS AND CANCER

METALLOPROTEINASES IN TUMOR NEOVASCULARIZATION

UBIQUITIN D AS A POTENTIAL THERAPEUTIC TARGET FOR NASH, HCC AND CHRONIC KIDNEY DISEASES - ABSTRACT: NON-ALCOHOLIC STEATOHEPATITIS (NASH), AN ADVANCED FORM OF NON-ALCOHOLIC FATTY LIVER DISEASE (NAFLD), IS ONE OF THE MAJOR CAUSES OF CIRRHOSIS AND HEPATOCELLULAR CARCINOMA (HCC). THERE IS CURRENTLY NO APPROVED TREATMENT FOR NASH AND PATIENTS WITH ADVANCED FIBROSIS ARE AT HIGHEST RISK OF MORTALITY DUE TO CARDIOVASCULAR DISEASES (CVD). FURTHERMORE, PRESENCE OF NAFLD INCREASES THE RISK OF CHRONIC KIDNEY DISEASE (CKD) BY ABOUT 80%. IT IS NOT KNOWN HOW NASH AND CKD INFLUENCE EACH OTHER, BUT TOGETHER THEY CAN INFLUENCE CVD AND PATIENT MORTALITY. THEREFORE, A THERAPEUTIC TARGET THAT CAN IMPROVE NASH AND ITS ASSOCIATED CO-MORBIDITIES SUCH AS CKD AND CVD WILL BE OF GREAT BENEFIT IN CLINICS. WE FOUND THAT UBIQUITIN D (UBD) IS HIGHLY UPREGULATED IN NASH LIVERS (PRIMARILY IN THE HEPATOCYTES), HCC TUMORS AND KIDNEYS OF CKD MICE. INTERESTINGLY, UBD WAS RECENTLY FOUND TO BE ONE OF THE SIX PROGRESSION-RELATED GENES THAT PLAY A VITAL ROLE IN THE PROGRESSION OF NAFLD AND POSITIVELY CORRELATES WITH STEATOSIS, BALLOONING, INFLAMMATION, FIBROSIS AND NAS SCORE. SIMILARLY, MULTIPLE REPORTS LINK RENAL UBD TO PROTEINURIA AND KIDNEY DISEASE. UBD IS KNOWN TO PARTICIPATE IN ALTERNATIVE UBIQUITINATION PATHWAY AND INTERACT WITH KEY CELLULAR PROTEINS SUCH AS P53, P62 AND MAD2 BUT THE PRECISE MECHANISM OF ITS ROLE IN NASH/HCC AND CKD REMAINS UNCLEAR. ABSENCE OF UBD (IN UBD-/- MICE) IMPROVED OVERALL METABOLIC HEALTH AND EXTENDED THE MOUSE LIFESPAN. DESPITE ALL THE COMPELLING EVIDENCES, UBD HAS NOT BEEN TESTED AS A THERAPEUTIC TARGET FOR NASH AND CKD. HERE, WE PROPOSE TO SUPPRESS UBD USING ANTI-SENSE OLIGOS (ASOS) AS A THERAPEUTIC APPROACH TO TREAT NASH AND CKD. WE HAVE ALREADY DEVELOPED A “GALNAC” CONJUGATED ASO THAT IS POTENT IN SUPPRESSING UBD GENE EXPRESSION IN THE HEPATOCYTES. OUR PRELIMINARY DATA INDICATES THAT ANTI-UBD GALNAC-ASO SIGNIFICANTLY IMPROVED NASH AND FIBROSIS. ACCORDING TO OUR KNOWLEDGE, UBD PATHWAY HAS NOT BEEN INVESTIGATED FOR ITS THERAPEUTIC POTENTIAL BEFORE AND ALSO IS NOT A CURRENT FOCUS OF ANY INDUSTRY OR ACADEMIA. THEREFORE, UBD IS A NOVEL TARGET AND ANTI-UBD ASOS PROVIDE NOVEL THERAPEUTIC LEADS WITH A POTENTIAL TO IMPROVE BOTH NASH AND CKD. WE WILL TAKE ADVANTAGE OF A UNIQUE PRE-CLINICAL MOUSE MODEL OF DIET-INDUCED NASH THAT ALSO DEVELOP CKD AND CVD WITH MORTALITY AT LATER STAGES (ESTABLISHED IN OUR LAB). WE WILL ADDRESS WHETHER ANTI-UBD ASOS COULD IMPROVE ALL THREE DISEASE OUTCOME ALONG WITH SURVIVAL BENEFIT. WE WILL SUPPLEMENT THIS MODEL WITH ADDITIONAL PRECLINICAL MODELS OF NASH, HCC AND CKD. THE FOLLOWING 2 SPECIFIC AIMS WILL BE ADDRESSED IN THIS PROPOSAL: AIM 1 WILL IDENTIFY WHETHER UBD SUPPRESSION PREVENTS NASH AND ITS PROGRESSION TO CIRRHOSIS AND HCC. ADDITIONALLY, WE WILL TRANSLATE OUR FINDINGS TO HUMAN USING HUMAN PRIMARY CELL DERIVED 3-D SPHEROID CELL CULTURES. AIM 2 WILL INVESTIGATE THE EFFECT OF UBD SUPPRESSION ON CKD, CVD AND SURVIVAL BENEFIT EITHER IN THE CONTEXT OF NASH AND/OR CKD. SUCCESSFUL COMPLETION OF THE PROPOSED AIMS WILL ESTABLISH UBD AS A NOVEL THERAPEUTIC TARGET FOR NASH AND CKD.

THE DEVELOPMENT OF TGR5 ANTAGONISTS FOR THE TREATMENT OF CHOLANGIOPATHIES

TUMOR PENETRATING PEPTIDES FOR CANCER TARGETING

CHARACTERIZATION OF JNK IN CELL CYCLE CONTROL

ROLE AND MECHANISM OF ACTION OF P62/SQSTM1 IN RAS-INDUCED TUMORIGENESIS IN LUNG

T CELL IMMUNITY TO INFLUENZA VIRUS IN THE AGED NASAL MUCOSA

ROLE OF PEPC, A CIRCULAR RNA ENCODED PROTEIN, IN CASTRATION-RESISTANT PROSTATE CANCER

MOLECULAR MECHANISMS UNDERLYING THE ASSEMBLY OF THE HUMAN PROTEASOME AND ENDOGENOUS PROTEIN COMPLEXES - PROJECT SUMMARY ADVANCES IN STRUCTURAL BIOLOGY TECHNIQUES INCLUDING SINGLE PARTICLE CRYOGENIC ELECTRON MICROSCOPY (CRYO-EM) HAVE ENABLED UNPRECEDENTED MOLECULAR INSIGHTS INTO THE FUNCTION OF BIOLOGICAL MACROMOLECULES. HOWEVER, THE STUDY OF MANY PROTEINS AND RIBONUCLEOPROTEIN COMPLEXES REMAINS CHALLENGING DUE TO CURRENT LIMITATIONS IN SAMPLE PREPARATION APPROACHES. TRADITIONALLY, PROTEINS OF INTEREST ARE PRODUCED IN OVER-EXPRESSION SYSTEMS WITHIN BACTERIAL, INSECT, AND MAMMALIAN CELL LINES. WHILE THIS APPROACH CAN ALLOW THE PRODUCTION AND PURIFICATION OF PROTEINS WITH HIGH YIELDS, IT OFTEN REQUIRES SUBSTANTIAL OPTIMIZATION THAT CAN LIMIT THE STUDY OF BIOMEDICALLY IMPORTANT MEMBRANE PROTEINS AND LARGE PROTEIN COMPLEXES, ESPECIALLY WHERE SPECIFIC CHAPERONES AND CELLULAR CONDITIONS ARE REQUIRED THAT ARE DIFFICULT TO REPLICATE IN VITRO. TO OVERCOME THESE LIMITATIONS, WE ARE DEVELOPING METHODOLOGY TO EFFICIENTLY TAG AND PURIFY ENDOGENOUS PROTEINS BY LEVERAGING ADVANCES IN CRISPR/CAS GENE EDITING. THIS APPROACH ENABLES US TO INVESTIGATE MACROMOLECULAR COMPLEXES AND THEIR INTRICATE ASSEMBLY PATHWAYS UNDER NATIVE AND CONTEXT-SPECIFIC CONDITIONS THAT ARE RELEVANT TO HUMAN HEALTH AND DISEASE. WE ARE INTERESTED IN DEVELOPING AND APPLYING THE APPROACH IN THREE MAJOR AREAS OF STUDY: CONSTITUTIVE PROTEIN COMPLEXES, CELL-TYPE SPECIFIC MACROMOLECULAR ASSEMBLIES, AND CELL STATE DEPENDENT MEMBRANE PROTEIN COMPLEXES. USING THE PROTEASOME AS A MODEL SYSTEM, WE WILL INVESTIGATE THE ASSEMBLY PATHWAY OF PROTEASOMAL COMPLEXES BY CRYO-EM AND MASS SPECTROMETRY. THIS WORK WILL PROVIDE MECHANISTIC INSIGHTS INTO CRITICAL PROTEIN DEGRADATION MACHINERY AND HELP TO ESTABLISH IMPORTANT METHODOLOGY FOR THE STUDY OF ENDOGENOUS PROTEIN ASSEMBLIES. NEXT, WE WILL EXPAND OUR APPROACH TO THE STUDY OF PROTEIN COMPLEXES IN DIFFERENT CELL TYPES, INCLUDING HEMATOPOIETIC AND EPITHELIAL CELLS. THIS GOAL WILL BE ACHIEVED BY DEVELOPING EFFICIENT STRATEGIES TO OPTIMIZE CRISPR/CAS GENE EDITING IN SPECIALIZED CELL TYPES, WHICH WILL CONSTITUTE AN IMPORTANT STEP TOWARDS THE STUDY OF PROTEINS IN THEIR NATIVE STATES. FINALLY, WE WILL EXAMINE THE CONDITIONAL ASSEMBLY OF PROTEIN COMPLEXES AND MEMBRANE PROTEIN ASSEMBLIES. FOR THIS DIRECTION, WE WILL INVESTIGATE PROTEINS INVOLVED IN NUTRIENT SENSING AT THE LYSOSOME IN CONJUNCTION WITH MTOR SIGNALING. THIS WORK WILL PROVIDE MOLECULAR INSIGHTS INTO THE MECHANISMS REGULATING KEY METABOLIC PATHWAYS AND SHED LIGHT ON HOW MTOR INTEGRATES DIFFERENT SIGNALS TO PROMOTE CELL GROWTH AND PROLIFERATION. ADDITIONALLY, WE WILL ESTABLISH PROTOCOLS FOR SCREENING CELLULAR AND BIOCHEMICAL CONDITIONS TO ACQUIRE CONTEXT-SPECIFIC PROTEIN ASSEMBLIES. ALTOGETHER, THESE STUDIES WILL PROVIDE MECHANISTIC INSIGHTS INTO REMARKABLE MOLECULAR MACHINES AND DEVELOP IMPORTANT METHODOLOGY THAT CAN BE APPLIED TO THE STUDY OF OTHER BIOLOGICAL SYSTEMS. THESE METHODS WILL ENABLE US TO UNRAVEL THE MOLECULAR MECHANISMS UNDERLYING THE FUNCTION OF PROTEINS IN SPECIFIC CELLULAR ENVIRONMENTS AND HELP ADVANCE STRUCTURAL BIOLOGY TOWARDS UNDERSTANDING HOW BIOLOGICAL MACROMOLECULES WORK IN THEIR NATIVE CONTEXTS.

REGULATION OF BLOOD GLYCOPROTEINS BY LECTIN RECEPTORS IN HEALTH AND DISEASE - SUMMARY THE COMPOSITION OF THE BLOOD PROTEOME PROVIDES INDICATIONS OF NORMAL HEALTH AND THE PRESENCE OF DISEASE. OVER 90% OF NON-ALBUMIN PROTEINS IN THE BLOOD OF MAMMALS ARE N-GLYCOPROTEINS BEARING N-GLYCAN STRUCTURES PRODUCED IN THE GOLGI APPARATUS PRIOR TO NASCENT GLYCOPROTEIN SECRETION. WE HAVE PREVIOUSLY DISCOVERED AN INTRINSIC MECHANISM CONTROLLING THE HALF-LIVES AND THEREBY ABUNDANCE AND FUNCTION OF CIRCULATING BLOOD GLYCOPROTEINS. THIS MECHANISM IS LINKED TO THE PROGRESSIVE GLYCOSIDIC REMODELING OF NASCENT BLOOD GLYCOPROTEINS BY CIRCULATING EXO-GLYCOSIDASES THEREBY RESULTING IN THE EXPOSURE OF CRYPTIC ENDOCYTIC LECTIN RECEPTOR LIGANDS. ENDOCYTIC LECTIN RECEPTORS ARE HIGHLY CONSERVED AMONG MAMMALS AND ARE EXPRESSED ON THE SURFACE OF VARIOUS VASCULAR AND ORGAN CELL TYPES WHERE THEY RECOGNIZE AND BIND THEIR LIGANDS FROM AMONG CIRCULATING BLOOD COMPONENTS AND GLYCOPROTEINS. WE HAVE FOUND THAT THIS INTRINSIC MECHANISM OF BLOOD GLYCOPROTEIN REMODELING AND CLEARANCE BY LECTIN RECEPTORS IS TARGETED BY PATHOGENS AND THE RESULTING CHANGES IN BLOOD GLYCOPROTEIN ABUNDANCE ARE LINKED TO DISEASE ONSET. LECTIN RECEPTOR LIGANDS INCLUDE GLYCOSIDIC LINKAGES OF GALACTOSE, N-ACETYLGLUCOSAMINE, FUCOSE, OR MANNOSE; HOWEVER, GLYCOPROTEINS BEARING PHYSIOLOGICAL LECTIN RECEPTOR LIGANDS REMAIN MOSTLY UNKNOWN AND THUS THE FUNCTIONS OF LECTIN RECEPTORS ARE ALSO MYSTERIOUS. THIS LABORATORY HAS DEVELOPED AN APPROACH TO IDENTIFY PHYSIOLOGICAL BLOOD GLYCOPROTEIN LIGANDS OF INDIVIDUAL LECTIN RECEPTORS BY CHROMATOGRAPHY AND MASS SPECTROMETRY PROTOCOLS. A SUBSET OF MAMMALIAN LECTIN RECEPTORS BIND TO GLYCOSIDIC LINKAGES BEARING EXPOSED MANNOSE, HEREIN TERMED MANNOSYLATED BLOOD GLYCOPROTEINS. THIS LABORATORY DISCOVERED IN PAST RELATED STUDIES THAT THE ACCUMULATION OF MANNOSYLATED GLYCOPROTEINS DUE TO DEFECTS IN N-GLYCAN SYNTHESIS CAUSES CHRONIC INFLAMMATION, AUTOIMMUNITY, AND DEGENERATIVE DISEASE. NORMALLY HOWEVER, THE RECOGNITION AND CLEARANCE OF MANNOSYLATED BLOOD GLYCOPROTEINS INVOLVES THE EXPRESSION AND FUNCTION OF MANNOSE BINDING LECTINS INCLUDING MRC1. THIS PROPOSAL ADDRESSES THE HYPOTHESIS THAT MRC1 CONTROLS THE LEVELS OF MANNOSYLATED BLOOD GLYCOPROTEINS IN NORMAL PHYSIOLOGY AND PROTECTS AGAINST THE ONSET OF DISEASE. OUR SUPPORTING DATA DEMONSTRATE THAT MRC1 HAS A BLOOD LIGAND REPERTOIRE INCLUDING KEY REGULATORS OF THE VASCULATURE. ABSENCE OF MRC1 RESULTS IN THE ACCUMULATION OF MANNOSYLATED RENIN AND ANGIOTENSIN CONVERTING ENZYME WITH ELEVATED BLOOD PRESSURE. IN ADDITION, ACCUMULATING MANNOSYLATED MYELOPEROXIDASE IN THE BLOOD IS ASSOCIATED WITH VASCULAR INFLAMMATION, BLOOD-BRAIN BARRIER BREAKDOWN, TISSUE DAMAGE, AND AUTISM-LIKE FEATURES. THE ROLES OF MRC1 LIGANDS WILL BE ADDRESSED WITH INHIBITORS. IN THE BLOOD OF HUMANS, WE HAVE FURTHER DISCOVERED A LINK BETWEEN INDIVIDUALS WITH LOW LEVELS OF MANNOSIDASE ACTIVITY AND HIGH LEVELS OF MANNOSYLATED BLOOD PROTEINS. THIS PROPOSAL IN SUMMARY WILL INVESTIGATE HOW THE ACCUMULATION OF MANNOSYLATED BLOOD GLYCOPROTEIN LIGANDS IN MRC1 DEFICIENCY CAUSE DISEASE AND ESTABLISH WHETHER HUMANS BEARING HIGH LEVELS OF MANNOSYLATED BLOOD GLYCOPROTEINS SIMILARLY CONTAIN ELEVATED MARKERS OF INFLAMMATION AND VASCULAR DEFECTS.

GENETIC PATHWAYS IN CERAMIDE-ASSOCIATED LIPOTOXIC CARDIOMYOPATHY AND HEART FAILURE

CELLULAR/MOLECULAR SUBSTRATES LINKING CHANNEL DYSFUNCTION AND PATHOLOGICAL CARDIAC REMODELING

REGULATION OF CHRONIC VIRAL INFECTIONS

DISCOVERY OF SMALL MOLECULE REGULATORS OF ATRIAL CARDIOMYOCYTE ACTION POTENTIAL DURATION TO RESTORE NORMAL CARDIAC RHYTHM IN ATRIAL FIBRILLATION - PROJECT SUMMARY ATRIAL FIBRILLATION (AF) IS THE MOST PREVALENT CARDIAC ARRHYTHMIA, AFFLICTING OVER 33 MILLION PEOPLE WORLDWIDE AND 6 MILLION IN THE US. AF CAUSES REDUCED QUALITY OF LIFE, STROKE AND SYSTEMIC THROMBOEMBOLISM, HEART FAILURE, AND INCREASED MORTALITY. TREATMENT OF AF AND ITS COMPLICATIONS WITH NONSPECIFIC DRUGS OR PROCEDURES IS CHARACTERIZED BY UNSATISFACTORY OUTCOMES AND SIGNIFICANT COST. ACQUIRED HEART DISEASE, CARDIAC REMODELING, NEUROHORMONAL FACTORS, AGING, AND GENETIC TRAITS HAVE ALL BEEN CORRELATED WITH PRESENCE OF AF. RAPID, UNCOORDINATED ATRIAL CHAMBER ACTIVITY IS DUE TO SHORTENED OR PROLONGED CARDIOMYOCYTE ACTION POTENTIAL DURATIONS ACTING WITHIN A VULNERABLE MYOCARDIAL SUBSTRATE, CAUSING PERSISTENT ARRHYTHMIA THAT FEATURES TRIGGERING OR SUSTAINING CIRCUIT RE- ENTRY OR EARLY AND/OR DELAYED AFTER-DEPOLARIZATIONS, RESPECTIVELY. WE HAVE RECENTLY DEVELOPED A NOVEL, HIGH THROUGHPUT KINETIC IMAGING AND ANALYSIS PLATFORM TO CHARACTERIZE CARDIOMYOCYTE ELECTROPHYSIOLOGICAL PROPERTIES AT SINGLE CELL RESOLUTION, WHICH CAN BE USED TO CONDUCT HIGH THROUGHPUT SCREENING (HTS) ON FUNCTIONAL HUMAN ATRIAL CARDIOMYOCYTES DERIVED FROM ID1-PROGRAMMED CARDIAC PROGENITORS CREATED FROM IPS CELLS. OUR INNOVATION IS THE USE OF THIS AND RELATED ASSAYS IN A PHENOTYPIC SCREENING CASCADE DESIGNED TO DISCOVER PREVIOUSLY UNKNOWN, ATRIAL-SPECIFIC MODULATORS OF CARDIOMYOCYTE ELECTRICAL PROPERTIES AND RHYTHM. OUR HYPOTHESIS IS THIS APPROACH WILL ULTIMATELY GENERATE DRUG-LIKE STARTING POINTS FOR FUTURE DISEASE-MODIFYING CARDIOVASCULAR THERAPEUTICS. THE PRIMARY HTS ASSAY HAS BEEN FULLY OPTIMIZED IN A 384-WELL FORMAT, AND AS A DEMONSTRATION OF ASSAY READINESS, 400 COMPOUNDS HAVE BEEN SCREENED (KOLMOGOROV-SMIRNOV D-STATISTIC >0.1). MULTIPLE HITS FROM PILOT SCREENS WERE IDENTIFIED AND WERE CONFIRMED AND VALIDATED IN CONCENTRATION RESPONSE EXPERIMENTS. A BATTERY OF DOWNSTREAM ASSAYS HAS BEEN DEVELOPED AND PILOTED TO ESTABLISH A CRITICAL PATH-TESTING FUNNEL. SEVERAL COMPOUNDS IDENTIFIED FROM THE PILOT SCREEN WERE TESTED TO DETERMINE IF THEY AFFECTED THE ACTION POTENTIAL DURATION OF ATRIAL CARDIOMYOCYTES SENSITIZED BY THE E375X MUTATION IN KCNA5, AND IF THEY HAD EFFECTS ON THE ACTION POTENTIAL DURATIONS OF WILD TYPE AND PRIMARY ATRIAL AND VENTRICULAR CARDIOMYOCYTES. THIS PROPOSAL BUILDS ON DATA FROM THE APPLICANTS, AN ESTABLISHED TEAM FROM SBP (DRS. COLAS AND LARSON) WITH BASIC BIOLOGY AND DRUG DISCOVERY EXPERTISE IN THE FIELD AND ACCESS TO ALL NECESSARY TECHNOLOGIES. THE OVERALL GOAL OF THIS PROPOSAL IS TO GENERATE CHEMICAL BIOLOGY RESEARCH TOOLS AND STARTING POINTS FOR NEW DRUGS. AS THE CRITICAL PATH ASSAYS ARE ALL IN PLACE, WE ANTICIPATE WE CAN RAPIDLY OBTAIN SUCH PROBE MOLECULES AND START TO EXPLORE THEIR ACTIVITY. OUR FUTURE PLANS ARE TO ULTIMATELY DETERMINE HITS’ SUITABILITY FOR HIT-TO-LEAD ACTIVITIES, BEGIN IN VIVO EVALUATION OF LEAD COMPOUNDS IN ANIMAL MODELS AND EVENTUALLY PATIENTS, AND DETERMINE THEIR CELLULAR MECHANISM OF ACTION. THIS GRANT’S WORK PRODUCT WILL SERVE AS PRELIMINARY DATA FOR HIT-TO-LEAD (HTL) GRANT SUBMISSIONS AND PARENT R01 GRANT SUBMISSIONS TO PURSUE UNDERSTANDING OF THEIR BIOLOGICAL MECHANISMS.

NUCLEAR PORE COMPLEXES IN THE MAINTENANCE OF SKELETAL MUSCLE INTEGRITY AND FUNCTION

INVESTIGATION OF ECDNA AS A DRIVER OF INTRATUMORAL HETEROGENEITY AND TREATMENT RESISTANCE IN HIGH-RISK MEDULLOBLASTOMA - THE OVERALL OBJECTIVE OF THIS PROPOSAL IS TO INVESTIGATE CIRCULAR EXTRACHROMOSOMAL DNA (ECDNA) AS A POTENTIAL DRIVER OF INTRATUMORAL HETEROGENEITY AND TREATMENT RESISTANCE IN MEDULLOBLASTOMA, THE MOST COMMON PEDIATRIC MALIGNANT BRAIN TUMOR. INTRATUMORAL HETEROGENEITY IS ONE OF THE LEADING DETERMINANTS OF THERAPEUTIC RESISTANCE AND TREATMENT FAILURE AND ONE OF THE MAIN REASONS FOR POOR OVERALL SURVIVAL IN CANCER PATIENTS. HOWEVER, THE FUNCTIONAL RELEVANCE OF ECDNA AS A DRIVER OF TUMOR HETEROGENEITY AND TREATMENT RESISTANCE IN MEDULLOBLASTOMA HAS HARDLY BEEN STUDIED. TO ANALYZE THE CLINICAL IMPACT OF ECDNA IN THE DIFFERENT MOLECULAR SUBGROUPS OF MEDULLOBLASTOMA, WE ASSEMBLED A MULTI-INSTITUTIONAL COHORT OF WHOLE GENOME SEQUENCING DATA FROM 468 MEDULLOBLASTOMA PATIENT SAMPLES. USING NOVEL COMPUTATIONAL METHODS FOR THE DETECTION AND RECONSTRUCTION OF ECDNA, WE FOUND ECDNA IN 82 PATIENTS (18%) AND OBSERVE THAT THE PRESENCE OF ECDNA IS ASSOCIATED WITH SIGNIFICANTLY POORER OUTCOMES. IN ADDITION, WE FIND THAT INDIVIDUAL MEDULLOBLASTOMA TUMORS OFTEN HARBOR MULTIPLE VARIANTS OF ECDNA, EACH CONTAINING DIFFERENT AMPLIFIED ONCOGENES ALONG WITH CO-AMPLIFIED NON-CODING REGULATORY DNA (‘ENHANCERS’). BASED ON OUR PRELIMINARY RESULTS, WE PROPOSE THE CENTRAL HYPOTHESIS THAT ECDNA DRIVES INTRATUMORAL HETEROGENEITY AND TREATMENT RESISTANCE IN HIGH-RISK MEDULLOBLASTOMA PATIENTS. THE CENTRAL HYPOTHESIS WILL BE TESTED THROUGH THE FOLLOWING THREE SPECIFIC AIMS: TO INVESTIGATE THE MOLECULAR EVOLUTION OF ECDNA AS A POTENTIAL DRIVER OF TREATMENT RESISTANCE (AIM 1); TO EVALUATE COMBINATORIAL THERAPIES TARGETED AGAINST MECHANISMS OF ECDNA FORMATION AND CLUSTERING TO REDUCE TREATMENT RESISTANCE (AIM 2); TO PROBE MEDULLOBLASTOMA TUMOR-DEPENDENCIES BY FUNCTIONAL INHIBITION OF CODING AND NON-CODING REGULATORY DNA CO- AMPLIFIED ON ECDNA (AIM 3). THE RESEARCH PROPOSED IN THIS APPLICATION HAS TECHNICAL, CONCEPTUAL, AND BIOLOGICAL INNOVATIONS, INCLUDING THE ANALYSIS OF ECDNA ON THE SINGLE-CELL LEVEL USING NOVEL IMAGING AND MULTIOME SINGLE-NUCLEUS SEQUENCING METHODS IN MEDULLOBLASTOMA TUMORS AND IN PATIENT-DERIVED XENOGRAFT (PDX) MODELS. THE PROPOSED RESEARCH IS SIGNIFICANT, BECAUSE THE CURRENT STANDARD TREATMENT FOR CHILDREN WITH MEDULLOBLASTOMA CAUSES DEVELOPMENTAL DISORDERS, NEUROLOGICAL DAMAGE, AND SECONDARY METASTASES. NOVEL THERAPEUTIC APPROACHES ARE URGENTLY NEEDED. OUR APPROACH WILL TEST THE IMPACT OF STANDARD-OF-CARE TREATMENTS ON THE MOLECULAR EVOLUTION OF ECDNA AND FUNCTIONALLY TEST NOVEL COMBINATION TREATMENTS TARGETED AGAINST ECDNA GENESIS AND CLUSTERING. THESE PRECLINICAL STUDIES HAVE THE POTENTIAL TO UNCOVER NOVEL MECHANISMS BY WHICH ECDNA CONTRIBUTES TO THE PATHOGENESIS OF MEDULLOBLASTOMA AND TO IDENTIFY NEW SCIENTIFIC LEADS FOR THE DEVELOPMENT OF IMPROVED TREATMENTS. WE EXPECT THAT OUR STUDIES WILL EXPOSE THE CONTRIBUTION OF ECDNA VARIANTS TO THE EMERGENCE OF THERAPY RESISTANCE, REVEAL THEIR SELECTION ADVANTAGES, VALIDATE RECENTLY DESCRIBED PROPERTIES OF ECDNA AND THEIR THERAPEUTIC SUSCEPTIBILITIES, AND IDENTIFY NOVEL TUMOR-DEPENDENCY GENES AMPLIFIED ON ECDNA IN SOME OF THE MOST AGGRESSIVE MEDULLOBLASTOMA TUMORS.

TARGETING APOPTOSIS VIA CHEMICAL DESIGN OF BCL-2 ANTAGONISTS

THE BCL-2-P85/PI3K SIGNALING AXIS

MPO OXIDANTS IN SYNUCLEINOPATHIES

EXPLOITING TUMOR STROMA INTERACTIONS FOR CANCER THERAPY

INHIBITING ONCOGENIC KRAS FOR CANCER THERAPY

ALPHA-CATENIN/F-ACTIN STRUCTURE AT CELL-CELL JUNCTIONS

FACTORS DETERMINING PROTEIN LOSING ENTEROPATHY

MYOCARDIAL ANGIOGENESIS AND CARDIOPROTECTION

ROLE OF AUTOPHAGY AND LIPID METABOLISM IN ORGANISMAL AGING

BIOCHEMISTRY OF MT1-MMP ACTIVATION IN MALIGNANCY

MECHANISMS AND CONSEQUENCES OF CNS ANEUPLOIDIES ALTERED BY FETAL ETHANOL EXPOSURE

INSTRUMENTATION UPGRADE: ACQUISITION OF AN INTERMEDIATE VOLTAGE TEM

ROLE OF POST-TRANSCRIPTIONAL RNA METHYLATION IN THE REGULATION OF RNA ACTIVITY

YEAST-BASED HTS ASSAY TECHNOLOGIES FOR PROTEASES