Van Andel Research Institute

TARGETING DNA METHYLATION AND THE CANCER EPIGENOME

MASTER REGULATORS OF UNEXPLAINED VARIATION IN DISEASE RISK - PROJECT SUMMARY PRECISION MEDICINE REQUIRES AN UNDERSTANDING OF THE ORIGINS AND MOLECULAR CONTROL OVER COMPLEX TRAITS AND DISEASE. THE FIELD IS LARGELY DRIVEN BY HUMAN GENETICS, WHICH ADHERES TO A 1918 DOGMA THAT PHENOTYPE IS DETERMINED SOLELY BY GENETICS AND THE ENVIRONMENT. YET, EVIDENCE FROM MONOZYGOTIC TWINS AND ISOGENIC ANIMAL MODELS INDICATE THAT UP TO 50% OF PHENOTYPIC VARIATION ACROSS DIVERSE PHYSIOLOGICAL TRAITS AND DISEASES CANNOT BE EXPLAINED BY GENETICS OR ENVIRONMENT – THERE IS SOMETHING `MORE' THAT IS UNIQUE TO EACH INDIVIDUAL, AND THAT CANNOT BE DETERMINED BY ANALYZING POPULATION-LEVEL MEAN EFFECTS. THESE FINDINGS ALSO INDICATE THAT EVEN IF WE DID HAVE `COMPLETE' GENETIC AND ENVIRONMENTAL KNOWLEDGE, A SUBSTANTIAL PORTION OF DISEASE HETEROGENEITY WOULD REMAIN UNACCOUNTED FOR. THE OPERATING HYPOTHESIS FOR THIS PROJECT IS THAT A SUBSTANTIAL FRACTION OF UNEXPLAINED DISEASE HETEROGENEITY REFLECTS INHERENTLY PROBABILISTIC PROPERTIES OF THE BIOLOGICAL SYSTEM THAT LEAD TO FIXED, DETERMINISTIC, REAL BIOLOGICAL VARIATION. THERE IS COMPELLING EVIDENCE FOR AN EVOLVED MOLECULAR CIRCUITRY THAT CONTROLS PHENOTYPIC VARIABILITY AS A QUANTITATIVE TRAIT. THUS, UNDERSTANDING VARIABILITY AS A QUANTITATIVE TRAIT IS ESSENTIAL TO UNDERSTANDING THE ETIOLOGY OF PHENOTYPIC DIVERSITY (IN GENERAL) AND AN INDIVIDUAL'S DISEASE POTENTIAL (IN PARTICULAR). HERE, WE WILL BEGIN TO FINALLY ANSWER THE PRECISION MEDICINE QUESTIONS OF: WHAT IS THE NORMAL OR EXPECTED DISEASE POTENTIAL FOR ME? AND, WHAT ARE THE ORIGINS AND REGULATORY CONTROLS OF NON-GENETIC, NON- ENVIRONMENTAL PHENOTYPIC AND DISEASE VARIABILITY IN HUMANS? THE FIRST STEPS TOWARDS ADDRESSING THESE QUESTIONS AND IDENTIFYING MECHANISMS THROUGH WHICH PROBABILISTIC PROCESSES LEAD TO DISEASE HETEROGENEITY IS TO CREATE A CATALOGUE OF PUTATIVE VARIANCE REGULATORS AND GENES; A PHENOTYPIC, EPIGENETIC, AND CELLULAR VARIANCE ATLAS CHARTING THE LANDSCAPE OF PROBABILISTIC VARIATION IN AN ISOGENIC MODEL SYSTEM (MICE); AND, TO DEMONSTRATE THAT THE REGULATORY ARCHITECTURE OF VARIANCE CONTROL IS CONSERVED BETWEEN MOUSE AND HUMANS. IF IT IS TRUE THAT A SIGNIFICANT PORTION OF UNEXPLAINED DISEASE HETEROGENEITY IS DUE TO THE MOLECULAR CONTROL OF VARIABILITY ITSELF, THEN WE WILL HAVE UNCOVERED AN ENTIRELY NEW AREA OF DISEASE ETIOLOGY THAT CAN BE HARNESSED BY THE COMMUNITY TO DEVELOP FUNDAMENTALLY NEW PREDICTIVE, DIAGNOSTIC, AND THERAPEUTIC INTERVENTIONS, IRRESPECTIVE OF THE DISEASE OF INTEREST.

STRUCTURAL STUDIES OF THE M. TUBERCULOSIS PROTEASOME AND PROTEASOMAL ATPASE

CRYO-EM HIGH PERFORMANCE DATA STORAGE

DETECTION OF PRE-INVASIVE PANCREATIC CYSTS USING PROTEIN AND GLYCAN BIOMARKERS

TO INVESTIGATE THE CONTRIBUTIONS OF LIPID MEMBRANES TO PRION DISEASE

STRUCTURAL GENOMICS OF ORPHAN NUCLEAR RECEPTORS

THE STRUCTURE AND FUNCTION OF EUKARYOTIC PROTEIN GLYCOSYLATION ENZYMES

INFLAMMATORY FACTORS AND KYNURENINE METABOLITES TRACKING SUICIDAL BEHAVIOR

CELLULAR EPIGENETIC HETEROGENEITY AS A PREDETERMINANT OF MALIGNANT TRANSFORMATION POTENTIAL

ANALYZING THE ROLE OF WNT SIGNALING IN BONE DEVELOPMENT

HIGH-THROUGHPUT EPIGENOMIC MAPPING OF REGULATORY ELEMENTS IN OVARIAN CANCER AT BASEPAIR RESOLUTION

MOLECULAR MECHANISMS OF IAS-MEDIATED CARCINOGENESIS THROUGH THE LENS OF HISTONE H2B VARIANTS - ESTABLISHING THE INFLUENCE OF POLLUTANTS ON GENOME FUNCTION IS ESSENTIAL IN DEFINING THEIR IMPACT ON HUMAN HEALTH. ARSENIC IS A UBIQUITOUS ENVIRONMENTAL TOXIC METALLOID THAT LEADS TO CARCINOGENESIS. THE WORLD HEALTH ORGANIZATION ESTIMATES THAT OVER 100 MILLION PEOPLE WORLDWIDE ARE AT RISK TO DRINKING ARSENIC CONTAMINATED WATER. RECENT STUDIES INDICATE THAT ARSENIC ALTERS GENE EXPRESSION LEADING TO TUMORIGENESIS. PROPER GENE REGULATION IS ESSENTIAL FOR NORMAL GROWTH, DEVELOPMENT AND ETIOLOGY OF DISEASES SUCH AS CANCER. EUKARYOTIC DNA STORED AS CHROMATIN WHOSE BASIC REPEATING UNIT IS THE NUCLEOSOME, PLAYS AN INTEGRAL ROLE IN GENE REGULATION. PREVIOUSLY, WE (AND OTHERS) SHOWED THAT NUCLEOSOME LOCATIONS WITHIN PROMOTERS PLAY CRITICAL ROLES IN CHROMATIN ACCESSIBILITY, THUS CONTROLLING GENE ACTIVITY. CONSEQUENTLY, CHROMATIN ACCESSIBILITY IS AN ESSENTIAL COMPONENT IN GENE REGULATION YET IS NOT FULLY UNDERSTOOD. CHROMATIN ACCESSIBILITY IS MODULATED BY SEVERAL KEY EPIGENETIC FACTORS: HISTONE POST-TRANSLATIONAL MODIFICATIONS (PTMS), DNA METHYLATION, NUCLEOSOME POSITION/OCCUPANCY, TRANSCRIPTION FACTORS AND CHROMATIN ARCHITECTURAL PROTEINS (CAPS). WE SHOWED RECENTLY THAT IN ADDITION TO THE ABOVE, INCORPORATION OF HISTONE VARIANTS (THE ELUSIVE H2B), INTO CHROMATIN PLAY A SIGNIFICANT ROLE IN ARSENIC-MEDIATED DISEASES PATHOLOGY, YET A MECHANISTIC UNDERSTANDING OF THEIR IMPACT IS UNKNOWN. THESE HISTONE VARIANTS DIFFER BY JUST ONE TO MAXIMALLY THREE AMINO ACIDS FROM THE CANONICAL H2B HISTONE AND ARE HIGHLY EXPRESSED IN CANCERS, SUGGESTING THAT THEY COULD ACT AS `ONCOHISTONES'. THUS IT IS CRITICALLY IMPORTANT TO UNDERSTAND HOW AND WHY THESE HISTONE VARIANTS GET EXPRESSED (AIM 1); HOW THEIR CHROMATIN INTEGRATION IMPACTS CHROMATIN STRUCTURAL DYNAMICS (AIM 2) AND INTEGRATION REGULATE THE CHROMATIN STATE AND GENE EXPRESSION DURING ARSENIC EXPOSURE TO DRIVE CARCINOGENESIS (AIM3). OUR INTERDISCIPLINARY, BROAD APPROACH WILL ESTABLISH UNIQUE COMPREHENSIVE FUNCTIONAL AND MECHANISTIC INSIGHT INTO HISTONE H2B VARIANT EXPRESSION, CHROMATIN INTEGRATION AND DISEASE PATHOLOGY. FURTHER IT WILL PROVIDE A DETAILED UNDERSTANDING OF THE INTERPLAY BETWEEN ARSENIC-INDUCED EPIGENETIC CHANGES AND CHROMATIN IN THE MAMMALIAN CELL. WE HAVE DEVELOPED NOVEL SYSTEMS THAT WILL PROVIDE AN UNPRECEDENTED AND UNIQUE OPPORTUNITY TO DISCOVER THE FUNCTIONAL AND MECHANISTIC ROLES OF THE EPIGENOME IN TOXICANT-INDUCED DISEASES.

KETONE BODY METABOLISM IN CD8+ T CELL RESPONSES - ABSTRACT IT HAS BEEN BELIEVED THAT INFECTION-FIGHTING T CELLS PRIMARILY USE GLUCOSE TO FUEL HOST DEFENSE AGAINST VIRUSES AND BACTERIA; HOWEVER, USING NEW IN VIVO METABOLITE PROFILING TECHNIQUES, IT HAS RECENTLY BEEN FOUND THAT GLUCOSE IS NOT THE PRIMARY FUEL FOR MITOCHONDRIAL ENERGY (ATP) PRODUCTION IN CD8+ T CELLS RESPONDING TO INFECTION IN VIVO, PARTICULARLY IN LATER PHASES OF INFECTION. THIS PRESENTS THE IMMUNOMETABOLISM COMMUNITY WITH A CRITICAL NEED TO DEFINE AND UNDERSTAND THE METABOLITES THAT SUSTAIN INFECTION-FIGHTING CD8+ T CELLS. THIS KNOWLEDGE IS CRUCIAL FOR DEVELOPING EFFECTIVE IMMUNO-METABOLIC INTERVENTIONS THAT STRENGTHEN HOST DEFENSE AND/OR ENHANCE IMMUNIZATION STRATEGIES. PRELIMINARY DATA POINT TO KETONE BODIES—A METABOLITE CLASS ENRICHED IN THE BLOOD IN RESPONSE TO INFECTION, FASTING, AND CERTAIN DIETS—AS A KEY FUEL FOR CD8+ T CELL EFFECTOR FUNCTION, WITH DATA SUGGESTING THAT KETONE BODY AVAILABILITY INFLUENCES T CELL-MEDIATED ADAPTIVE IMMUNITY BY REGULATING EFFECTOR FUNCTION (I.E., CYTOKINE PRODUCTION) AT THE EPIGENETIC LEVEL. HOWEVER, THE FULL IMPACT OF T CELL-INTRINSIC KETONE BODY METABOLISM ON CD8+ T CELL RESPONSES REMAINS UNCHARACTERIZED. OVERALL OBJECTIVE: TO CHARACTERIZE THE MECHANISMS BY WHICH T CELL-INTRINSIC KETONE BODY METABOLISM (“KETOLYSIS”) IMPACTS CD8+ T CELL METABOLISM AND FUNCTION DURING IMMUNE RESPONSES TO INFECTION, AND TO DETERMINE THE ROLE OF SYSTEMIC KETONE BODY METABOLISM IN CD8+ T CELL FUNCTION AND HOST DEFENSE. HYPOTHESIS: T CELL-INTRINSIC KETONE BODY METABOLISM IS A NON-REDUNDANT PATHWAY SUPPORTING HOST DEFENSE BY FUELING AND DIRECTING CD8+ T CELL BIOENERGETICS AND EFFECTOR FUNCTION IN VIVO. SPECIFIC AIMS: (1) CHARACTERIZE KETONE BODY METABOLISM IN CD8+ T CELLS AND ITS IMPACT ON CD8+ T CELL-MEDIATED IMMUNE RESPONSES, (2) DECONSTRUCT THE MECHANISMS BY WHICH KETONE BODY METABOLISM IMPACTS T CELL EFFECTOR FUNCTION, (3) DISSECT THE IMPACT OF SYSTEMIC KETONE BODY METABOLISM ON CD8+ T CELL RESPONSES. IMPACT: UPON COMPLETION, THIS PROPOSED RESEARCH WILL HAVE DETERMINED HOW ACTIVATED CD8+ T CELLS UTILIZE KETONE BODIES, DISSECTED THE IMPACT OF KETONE BODIES ON CD8+ T CELL EFFECTOR FUNCTION (PRIMARY INFECTION CLEARANCE) AND MEMORY RESPONSES (LONG-TERM IMMUNITY), AND DEFINED HOW SYSTEMIC KETONE BODY AVAILABILITY IS CONTROLLED AND IMPACTS CD8+ T CELL-MEDIATED HOST DEFENSE. BY DEFINING THE ROLE OF KETONE BODY METABOLISM IN PROTECTIVE CD8+ T CELL-MEDIATED IMMUNE RESPONSES, WE WILL HAVE LAID THE GROUNDWORK FOR DEFINING DIETARY AND PHARMACOLOGICAL INTERVENTIONS TO OPTIMIZE ENDOGENOUS HOST DEFENSES TO BOOST CLEARANCE OF INFECTIONS AS WELL AS ENHANCE IMMUNIZATION STRATEGIES.

SUBPOPULATIONS OF PANCREATIC CANCER CELLS DEFINED BY GLYCAN MARKERS

ACCELERATED DNA METHYLATION ALTERATIONS IN HUTCHINSON-GILFORD PROGERIA SYNDROME - PROJECT SUMMARY / ABSTRACT HUTCHINSON-GILFORD PROGERIA SYNDROME (HGPS) IS A RARE GENETIC DISORDER CHARACTERIZED BY THE RAPID ONSET OF PREMATURE AGING BEGINNING IN CHILDHOOD. THE CONDITION IS CAUSED BY A MUTATION IN THE NUCLEAR LAMIN LMNA GENE, WHICH LEADS TO THE PRODUCTION OF A DOMINANT GAIN-OF-FUNCTION ISOFORM OF THE PROTEIN, CALLED PROGERIN. PROGERIN CAUSES MAJOR DISRUPTIONS TO NUCLEAR MORPHOLOGY AND FUNCTION, INCLUDING NUCLEAR PROTEIN EXPORT AND MITOSIS, REPLICATION STRESS, AND CONTRIBUTES TO INCREASED DNA DAMAGE AS WELL AS THE LOSS OF HETEROCHROMATIN AND DYSREGYLATION OF OTHER EPIGENETIC MARKS, INCLUDING CYTOSINE-5 DNA METHYLATION. WE HAVE RECENTLY PROVIDED DIRECT EXPERIMENTAL EVIDENCE THAT AGING-ASSOCIATED LOSS OF DNA METHYLATION AT NUCLEAR LAMINA-ATTACHED REGIONS OF THE GENOME IS A DIRECT CONSEQUENCE OF CELL DIVISION-ASSOCIATED DNA REPLICATION. WE USED THIS PRINCIPLE TO DEVELOP AN EPIGENETIC MITOTIC CLOCK, CALLED REPLITALI, WHICH PROVIDES A RELIABLE ESTIMATE OF CELLULAR REPLICATIVE HISTORY. WE HYPOTHESIZE THAT ALTERED DNA METHYLATION PATTERNS IN PATIENTS WITH HGPS MAY CONTRIBUTE ACTIVELY TO THE SEVERELY ACCELERATED AGING OBSERVED IN PATIENTS WITH HGPS. HERE, WE PROPOSE TO CONDUCT HIGH- RESOLUTION ANALYSES OF DNA METHYLATION ALTERATIONS IN SERIALLY CULTURED HGPS CELLS AND TO INVESTIGATE WHETHER WE CAN EXTEND THE LIFESPAN OF HGPS MODEL SYSTEMS BY MANIPULATING DNA METHYLATION PATTERNS. IN SPECIFIC AIM 1 WE WILL DEFINE DNA METHYLATION DYNAMICS IN SERIALLY CULTURED HGPS FIBROBLASTS FROM EARLY PASSAGE THROUGH REPLICATIVE SENESCENCE USING COST-EFFECTIVE INFINIUM DNA METHYLATION ARRAYS. IN SPECIFIC AIM 2 WE WILL CONDUCT HIGH-RESOLUTION SINGLE-CELL METHYLOME ANALYSES AT KEY STAGES OF HGPS FIBROBLAST CULTURE TO DETECT ARISING ABERRATIONS AND DELINEATE POPULATION TRANSITIONS. WE HAVE DEVELOPED A SINGLE-CELL WHOLE-GENOME BISULFITE SEQUENCING (SC-WGBS) METHOD THAT DELIVERS GENOMIC COVERAGE FAR SUPERIOR TO ANY OTHER PUBLISHED SC-WGBS METHODS. IN SPECIFIC AIM 3 WE WILL INVESTIGATE WHETHER DNA METHYLATION MANIPULATION CAN EXTEND LIFESPAN IN HGPS MODELS. IN AIM 3A WE WILL TEST WHETHER OVEREXPRESSION OF DNA METHYLATION WRITERS AND ERASERS INCREASES THE REPLICATIVE LIFESPAN OF HGPS FIBROBLASTS. IN AIM 3B, WE WILL TARGET DNA METHYLTRANSFERASE OVEREXPRESSION TO ARTERIAL SMOOTH MUSCLE CELLS IN AN HGPS MOUSE MODEL TO INVESTIGATE WHETHER THIS REDUCES ARTERIAL SMOOTH MUSCLE LOSS AND EXTENDS LIFESPAN. WE WILL MONITOR DNA METHYLATION CHANGES IN THIS MOUSE MODEL USING A NEW COST-EFFECTIVE DNA METHYLATION ARRAY. WE PRESENT EXTENSIVE AND COMPELLING PRELIMINARY DATA THAT DEMONSTRATES BOTH THE FEASIBILITY AND RELEVANCE OF THE PROPOSED AIMS. THE OUTCOME OF THIS PROPOSED RESEARCH COULD HAVE IMPORTANT IMPACTS ON OUR UNDERSTANDING OF THE CONTRIBUTION OF DNA METHYLATION ALTERATIONS TO HGPS PHENOTYPES, POTENTIALLY OPENING AVENUES FOR NEW THERAPEUTIC APPROACHES TO TREAT PROGERIA. IN ADDITION, THIS STUDY COULD SHED LIGHT ON SIMILAR MECHANISMS OPERATING AT A LONGER TIMESCALE IN NORMAL AGING.

PROGRESSIVE DNA HYPOMETHYLATION AS A MEASURE OF MITOTIC HISTORY AND POTENTIAL CONTRIBUTOR TO REPLICATIVE SENESCENCE.

LINKING SYNUCLEINOPATHY AND DYSFUNCTION OF OLFACTORY PATHWAYS

MOLECULAR UNDERPINNINGS IN THE ESTABLISHMENT OF AN ONCOGENIC 3D GENOME IN RESPONSE TO ENVIRONMENTAL ARSENIC EXPOSURE

STRUCTURAL AND FUNCTIONAL STUDIES OF THE TRPM2 CHANNEL

UNDERSTANDING WNT9A/FZD9 TRAFFICKING AND SIGNALING - CELL TO CELL SIGNALING IS FUNDAMENTAL TO ALL MULTICELLULAR LIFE. SECRETED SIGNALING MOLECULES FROM ONE CELL ARE ABLE TO COMMUNICATE WITH OTHER CELLS THROUGH INTERACTION WITH RECEPTORS ON THE SURFACE, WHICH INTERPRET AND TRANSDUCE THESE SIGNALS. ONE SUCH CLASS OF SECRETED SIGNALING MOLECULES IS ENCODED BY THE WNT GENE FAMILY. THESE PROTEIN PRODUCTS DRIVE A PLETHORA OF CELLULAR PROCESSES, INCLUDING CELLULAR SYMMETRY, PROLIFERATION, TISSUE POLARITY AND STEM CELL MAINTENANCE. THIS VARIETY OF PROCESSES OCCURS USING CONSERVED INTRACELLULAR MACHINERY TO PROGRAM THESE OUTPUTS, INDICATING A NEED FOR COMPLEX CONTROL AT THE LEVEL OF THE CELL MEMBRANE. AT LEAST PART OF THE REGULATION OF THIS SIGNALING COMPLEXITY LIES IN THE MULTIPLE GENES ENCODING SPECIFIC LIGANDS (19 IN MAMMALS) AND RECEPTORS (10 IN MAMMALS). HOWEVER, THE SPECIFIC MECHANISMS THAT START WITH THE ASSOCIATION OF VARIOUS LIGANDS AND RECEPTORS AT THE MEMBRANE, AND LEAD TO NUCLEAR TRANSCRIPTIONAL ACTIVITY ARE INCOMPLETELY UNDERSTOOD. ONE OF THE MISSING LINKS IN THIS ASPECT IS TO DECIPHER HOW THE RECEPTOR COMPLEX IS INTERNALIZED BY THE CELL, WHERE IT IS TRAFFICKED TO, AND HOW THESE EVENTS LEAD TO DIVERSE TRANSCRIPTIONAL OUTPUTS. THIS SYSTEM OF LIGAND-RECEPTOR INPUTS LEADING TO DIVERSE OUTPUTS IS LIKELY DEPENDENT ON THE COMPOSITION OF THE RECEPTOR COMPLEX, AND THE CELLULAR CONTEXT OF THE SIGNAL. DECIPHERING THIS CODE WILL BE CRUCIAL TO OUR UNDERSTANDING OF HOW CELLS RECEIVE AND TRANSDUCE SPECIFIC SIGNALS. THIS PROPOSAL AIMS TO CHARACTERIZE THE COMMUNICATION BETWEEN TRAFFICKING OF A SPECIFIC WNT SIGNALING RECEPTOR COMPLEX, AND ACTIVATION OF TRANSCRIPTION IN THE NUCLEUS. ALTHOUGH IT IS KNOWN THAT PAIRING A WNT LIGAND AND FRIZZLED (FZD) RECEPTOR IS REQUIRED FOR THE TRANSDUCTION OF MANY (IF NOT MOST) WNT SIGNALS, OUR UNDERSTANDING OF THE MECHANISMS THROUGH WHICH SPECIFIC WNT/FZD PAIRINGS ALLOW THE TRANSCRIPTIONAL ACTIVATOR SS-CATENIN (SS-CAT) TO ENTER THE NUCLEUS IS LACKING. WE HAVE DEVELOPED A NOVEL SYSTEM TO STUDY SPECIFIC SIGNAL TRANSDUCTION OF THIS NATURE BY FLUORESCENTLY LABELED THE WNT LIGAND, FZD RECEPTOR, AND THE TRANSCRIPTIONAL ACTIVATOR SS-CAT. THIS IS BASED ON OUR PREVIOUS STUDIES WHICH HAVE INDICATED THAT THERE IS AN EXQUISITELY SPECIFIC PAIRING OF THE LIGAND WNT9A WITH THE RECEPTOR FRIZZLED (FZD)9B, AND THAT SPECIFICITY OF THIS SIGNAL IS CONFERRED BY THE EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR). THIS PROPOSAL AIMS TO FURTHER DEFINE THE MECHANISM OF WNT9A/FZD9B SIGNAL TRANSDUCTION BY STUDYING THE INTRACELLULAR TRAFFICKING OF THIS RECEPTOR COMPLEX, AND COUPLING THIS TO SS-CAT MOVEMENTS. BY FIRST UNDERSTANDING HOW ONE SPECIFIC WNT/FZD PAIRING ESTABLISHES A SPECIFIC SIGNAL, WE CAN GENERATE A MODEL FOR FURTHER TESTING OTHER WNT/FZD COMPLEXES IN DIFFERENT CELLULAR CONTEXTS. FURTHERMORE, CELL SIGNALING EVENTS OFTEN CROSS-OVER TO OTHER PATHWAYS; THESE FINDINGS COULD CERTAINLY IMPACT OUR UNDERSTANDING OF HOW OTHER RECEPTOR-LIGAND COMPLEXES LEAD TO DIVERSE OUTPUTS IN CELLS. THE PI HAS EXPERTISE IN WNT SIGNALING AND CELL BIOLOGY, AS WELL AS EXPERIENCE WITH MENTORING STUDENTS TO PUBLICATIONS IN THESE AREAS. SUCCESSFUL COMPLETION OF THESE PROJECTS SET THE PI AND HER LAB UP TO BE LEADERS IN THE FIELD OF WNT SIGNALING, AND FOR LONG-TERM SUCCESS IN THE FIELD.

INTEGRATIVE CANCER EPIGENOMIC DATA ANALYSIS CENTER (ICE-DAC)

ROLE OF DESUMOYLASE SENP6 IN JOINT AGING AND OSTEOARTHRITIS DEVELOPMENT

MECHANISMS OF GENOMIC IMPRINTING

EPIGENETIC DRIVERS OF CANCER (PQ 10)

DIET-MEDIATED REGULATION OF FATTY ACID SATURATION IMPACTS CANCER PROGRESSION AND THERAPY RESPONSES. - PROJECT SUMMARY/ABSTRACT MANY “ANTI-CANCER” DIETS ARE PROMOTED IN POPULAR CULTURE FOR CANCER PREVENTION AND TREATMENT, REFLECTING THE ENTHUSIASM OF PATIENTS, CLINICIANS, AND RESEARCHERS ALIKE FOR USING DIET TO IMPROVE PATIENT PROGNOSIS. MANY OF THESE DIETARY RECOMMENDATIONS, HOWEVER, ARE POORLY SUPPORTED BY SCIENTIFIC EVIDENCE. WHILE DIET IS A KNOWN CONTRIBUTOR TO CANCER RISK, HOW DIET INFLUENCES SURVIVAL IN PATIENTS WHO ALREADY HAVE CANCER IS POORLY UNDERSTOOD. THIS IS A KEY DISTINCTION BECAUSE MANY PATIENTS ARE ONLY MOTIVATED TO MAKE DIETARY CHANGES AFTER RECEIVING A CANCER DIAGNOSIS. MOREOVER, BECAUSE DIETARY ADVICE FOR PATIENTS MUST BE GIVEN IN THE CONTEXT OF OTHER TREATMENT STRATEGIES, IT IS IMPORTANT TO DETERMINE HOW DIET MIGHT ENHANCE OR ANTAGONIZE STANDARD-OF-CARE AND EMERGING CANCER THERAPIES. THEREFORE, THERE IS A CRITICAL NEED TO DEFINE THE MOLECULAR MECHANISMS THAT UNDERLIE DIETARY EFFECTS ON CANCER PROGRESSION AND THERAPY BEFORE IT WILL BE POSSIBLE TO INTEGRATE DIET EFFECTIVELY INTO CANCER TREATMENT. UNCOVERING THESE MECHANISMS WILL TRANSFORM PRECISION NUTRITION THERAPEUTIC APPROACHES, WHICH AIM TO TAILOR DIETARY CHANGES WITH PARTICULAR THERAPIES TO TARGET SPECIFIC TUMOR TYPES. RECENT WORK IN CANCER METABOLISM HAS SHOWN THAT CHANGES TO NUTRIENT LEVELS IN THE TUMOR MICROENVIRONMENT ARE KEY TO DETERMINING THE IMPACTS OF DIETARY INTERVENTIONS ON TUMOR METABOLISM, GROWTH, AND RESPONSES TO THERAPY. INTERACTIONS BETWEEN WHOLE-BODY AND TUMOR LIPID METABOLISM HAVE A CENTRAL ROLE IN LINKING DIET WITH CANCER PROGRESSION. LIPIDS ARE SYNTHESIZED FROM FATTY ACIDS, WHICH CAN BE SATURATED, MONOUNSATURATED, OR POLYUNSATURATED, DEPENDING ON THE NUMBER OF DOUBLE BONDS THEY CONTAIN. CELLULAR FATTY ACID SATURATION IS TIGHTLY REGULATED TO MAINTAIN MEMBRANE STRUCTURE AND CELLULAR FUNCTION. PREVIOUS DATA SHOWS THAT DIETARY MANIPULATIONS CAN ALTER THE BALANCE BETWEEN SATURATED AND UNSATURATED FATTY ACIDS WITHIN TUMORS. THE PROPOSED RESEARCH WILL DEFINE THE MECHANISMS BY WHICH DIET DYSREGULATES TUMOR FATTY ACID SATURATION STATES TO INFLUENCE TUMOR PROGRESSION AND THERAPY RESPONSES. MOUSE PANCREATIC AND LUNG CANCER MODELS WILL BE USED TO EXAMINE: (1) HOW A CALORIC RESTRICTION DIET ALTERS TUMOR-INTRINSIC POLYUNSATURATED FATTY ACID METABOLISM, THEREBY DEFINING DIETARY CONTEXTS UNDER WHICH THIS PATHWAY CAN BE THERAPEUTICALLY TARGETED; (2) HOW EXCESSIVE SATURATED FATTY ACID CONSUMPTION INDUCES LIPOTOXIC STRESS IN TUMORS AND GENERATES RESISTANCE TO CANCER THERAPY, THE REVERSAL OF WHICH COULD BE A STRATEGY TO ENHANCE THERAPY RESPONSES; AND (3) HOW MANIPULATING THE FAT TYPE IN DIETS ALTERS TUMOR METABOLISM AND GROWTH IN A PREDICTABLE MANNER BY PROVIDING TUMORS WITH DIFFERENT RELATIVE AMOUNTS OF SATURATED, MONOUNSATURATED, AND POLYUNSATURATED FATTY ACIDS, LEADING TO DIETARY FAT TYPE-ASSOCIATED METABOLIC DEPENDENCIES THAT CAN BE THERAPEUTICALLY EXPLOITED. THESE STUDIES WILL DEFINE THE METABOLIC MECHANISMS UNDERLYING HOW DIET-INDUCED CHANGES TO WHOLE-BODY AND TUMOR LIPID METABOLISM INTERACT WITH CANCER THERAPIES. THE EXPECTED OUTCOMES WILL SET THE FOUNDATION FOR THE IMPROVED DESIGN OF FUTURE TRANSLATIONAL STUDIES THAT USE DIETARY INTERVENTIONS AS AN ASSISTIVE THERAPY FOR CANCER TREATMENT.

ENDOGENOUS RETROVIRUS IN JOINT AGING AND OSTEOARTHRITIS DEVELOPMENT - PROJECT SUMMARY/ABSTRACT OSTEOARTHRITIS (OA) IS A DEGENERATIVE DISEASE OF THE JOINTS, THAT AFFECTS OVER 16% OF PEOPLE OVER 60 IN THE US. AGE AND INJURY ARE THE PRIMARY RISK FACTORS FOR OA. THERE IS A CRITICAL NEED TO DEVELOP THERAPIES FOR OA TREATMENT, AS CURRENT TREATMENT OPTIONS RELY PRIMARILY ON PAIN MANAGEMENT AND JOINT REPLACEMENT, WHILE OA IS ONLY BECOMING INCREASINGLY PREVALENT IN OUR AGING POPULATION. CHRONIC EXPOSURE TO CELLULAR STRESSORS PROFOUNDLY DISRUPTS THE HOMEOSTASIS OF ARTICULAR CHONDROCYTES (ACS), THE PRIMARY CELL TYPE COVERING THE SURFACE OF THE JOINT, LEADING TO AC SENESCENCE, APOPTOSIS, AND DEGENERATION OF THE SMOOTH SURFACE OF THE JOINT. WE KNOW THAT NUCLEAR CHANGES TO CHROMATIN STRUCTURE IMPACTS THE BIOLOGICAL AGE OF MANY TISSUES AND CONTRIBUTES TO THE MANIFESTATION OF AGING PHENOTYPES AND WANTED TO INVESTIGATE WHETHER THIS IS ALSO TRUE IN OA. OUR PRELIMINARY DATA SUGGESTS THAT CHROMATIN STRUCTURE IS DISRUPTED IN ACS IN OA AND MAY BE A CONTRIBUTING FACTOR TO OA DISEASE PROGRESSION. THE OVERALL OBJECTIVE OF THIS PROPOSAL IS TO DISSECT THE MECHANISM(S) OF HOW AGING AND STRESSORS INFLUENCE CHROMATIN STRUCTURE TO DISRUPT AC HOMEOSTASIS TO ULTIMATELY GIVE RISE TO OA. OUR PRELIMINARY FINDINGS SUGGEST THAT ABNORMAL ACTIVATION OF TRANSPOSABLE ELEMENTS (TES) OCCURS PREFERENTIALLY IN OA TISSUE. WE HYPOTHESIZE THAT CELLULAR STRESSES ACCUMULATE WITH AGE TO IMPAIR TE SILENCING IN ACS, THEREBY TRIGGERING INFLAMMATION AND EVENTUALLY, OA. WE WILL TEST OUR HYPOTHESIS: BY DETERMINING IF CHROMATIN ACCESSIBILITY AND TE ACTIVATION INCREASE WITH AGE AND PRESENCE OF OSTEOARTHRITIS IN ACS; BY DETERMINING IF TE ACTIVATION IN OA MODELS CAN AUGMENT OA PROGRESSION; AND BY EVALUATING IF STRESS CAUSES TE ACTIVATION AND OSTEOARTHRITIS. IMPACT: THIS PROJECT WILL YIELD A MECHANISTIC UNDERSTANDING OF THE CONNECTION BETWEEN AGING, STRESS CONDITIONS, ERV ACTIVATION, AND OA PATHOGENESIS. IMPORTANTLY, THIS PROPOSAL HAS THE POTENTIAL TO IMPACT JOINT HEALTH AND MOBILITY OF OUR AGING POPULATION.

MOLECULAR MECHANISMS OF CHROMATIN AND EPIGENETIC REGULATION

DNA HYPERMETHYLATION IN LUNG TUMORS

MECHANISMS OF MUTANT MITOCHONDRIAL GENOME MODULATION - PROJECT SUMMARY/ABSTRACT EVER SINCE FORMING A SYMBIOTIC RELATIONSHIP WITH THE EUKARYOTIC HOST 2 BILLION YEARS AGO, MITOCHONDRIA HAVE BECOME INTEGRAL COMPONENTS OF THE CELLULAR NETWORK. THANKS TO THE RESURGENCE OF INTEREST IN MITOCHONDRIA, THERE IS A GREATER APPRECIATION FOR THE WIDE VARIETY OF CELLULAR AND PHYSIOLOGICAL FUNCTIONS FOR THESE ORGANELLES BEYOND THEIR ROLE IN ENERGY GENERATION. DESPITE THIS RENEWED INTEREST, HOWEVER, RESEARCH INTO THE MITOCHONDRIAL GENOME (MTDNA) HAS LAGGED. MULTIPLE FACTORS LIKELY CONTRIBUTE TO THIS UNDERAPPRECIATION FOR MTDNA, INCLUDING ITS DIMINUTIVE SIZE AND SMALL GENETIC CONTENT, THE DIFFICULTY OF GENETICALLY MANIPULATING IT IN METAZOANS, AND ITS ‘MESSY’ GENETICS, WHICH STANDS IN STARK CONTRAST TO THE SIMPLE AND ELEGANT MENDELIAN GENETICS OF THE NUCLEAR GENOME. CONSEQUENTLY, FUNDAMENTAL QUESTIONS PERTAINING TO MTDNA BIOLOGY REMAIN UNADDRESSED. IT IS A MISTAKE, HOWEVER, TO VIEW MTDNA AS SIMPLE AND INCONSEQUENTIAL VESTIGES OF MITOCHONDRIA’S ANCESTRAL PAST. DEVASTATING DISEASES CAUSED BY MTDNA MUTATIONS SERVE AS IMPORTANT REMINDERS OF ITS FUNCTIONAL RELEVANCE. THE OVERARCHING GOAL OF RESEARCH IN MY LAB OVER THE COMING FIVE YEARS IS TO STUDY MTDNA GENETICS, AND MTDNA BIOLOGY MORE BROADLY. REPLICATION OF MTDNA IS NOT CONFINED TO THE CELL CYCLE AND CAN OCCUR CONTINUOUSLY THROUGHOUT THE LIFE OF THE CELLS. CONSEQUENTLY, NEW MUTATIONS CAN ARISE FREQUENTLY, CONTRIBUTING TO A STATE OF HETEROPLASMY, IN WHICH TWO OR MORE SEQUENCE VARIANTS OF MTDNA COEXIST. BECAUSE CELLS ARE POLYPLOID FOR MTDNA, CONTAINING HUNDREDS TO THOUSANDS OF COPIES, MUTATIONS ARE SCORED AS THE PERCENTAGE OF MTDNA COPIES THAT ARE MUTANT (I.E. HETEROPLASMY LEVELS). EVEN HIGHLY DELETERIOUS MTDNA MUTATIONS ARE BENIGN AT LOW HETEROPLASMY LEVELS. HOWEVER, THEY BECOME PATHOGENIC WHEN THEIR LEVELS RISE BEYOND A CRITICAL THRESHOLD AND THERE ARE INSUFFICIENT COPIES OF THE NORMAL MTDNA TO SUPPORT CELLULAR FUNCTION. THE CELLULAR AND MOLECULAR MECHANISMS THAT IMPACT HETEROPLASMY LEVELS ARE POORLY UNDERSTOOD. CONSEQUENTLY, IT IS DIFFICULT TO PREDICT WHO WILL INHERIT OR DEVELOP MTDNA-ASSOCIATED DISEASES. THE FIRST MAJOR GOAL OF MY LAB IS TO IDENTIFY THE CELLULAR AND MOLECULAR MECHANISMS THAT MODULATE HETEROPLASMY DYNAMICS. WE HAVE ADAPTED C. ELEGANS AS A MODEL SYSTEM OF CHOICE TO STUDY THESE MECHANISMS. C. ELEGANS ALLOWS US TO TRACK HETEROPLASMY LEVELS ACROSS INDIVIDUALS AND GENERATIONS WITH UNPRECEDENTED FEASIBILITY. THE ABILITY OF MTDNA MUTANTS TO RISE IN HETEROPLASMY LEVELS BEYOND THE CRITICAL THRESHOLD ARE MUTANT- SPECIFIC. HOWEVER, THE MECHANISTIC BASIS FOR THIS SPECIFICITY IS NOT WELL UNDERSTOOD. CONSEQUENTLY, THE SECOND MAJOR GOAL OF MY LAB IS TO UTILIZE A DIVERSE PANEL OF MUTATIONS TO EXPLAIN THE DIFFERENCES IN THEIR HETEROPLASMY LEVELS. TAKEN TOGETHER, OUR RESEARCH PROGRAM TACKLES LONG-STANDING BUT FUNDAMENTAL QUESTIONS IN THE FIELD OF MITOCHONDRIAL GENETICS. IN THE PROCESS, WE WILL ALSO GAIN INSIGHTS INTO BROADER ASPECTS OF MTDNA BIOLOGY SUCH AS ITS REPLICATION AND HOW CELLS COUNT MTDNA COPIES.

MOLECULAR MECHANISMS FOR SORTING LYSOSOMAL PROTEINS - PROJECT SUMMARY THE OBJECTIVE OF THIS PROPOSAL IS TO GAIN MECHANISTIC AND PATHOLOGICAL UNDERSTANDING OF HUMAN GLCNAC-1- PHOSPHOTRANSFERASE (PTASE), AN ENZYME WHICH MODIFIES LYSOSOMAL HYDROLASES. LYSOSOMES CONTAIN SEVERAL DOZEN ACID HYDROLASES THAT BREAK DOWN UNWANTED PROTEINS AND LIPIDS. LYSOSOMAL ENZYMES ACQUIRE A MANNOSE- 6-P MOIETY IN THE GOLGI APPARATUS BY THE ACTION OF PTASE, WHICH USES UDP-GLCNAC AS A DONOR SUBSTRATE. THE MANNOSE-6-P FUNCTIONS LIKE A ZIP CODE THAT GUIDES THE DELIVERY OF THESE ENZYMES TO THE LYSOSOMES. PTASE IS ENCODED BY THE GNPTAB AND GNPTG GENES AND ASSEMBLES A 340-KDA HETEROHEXAMER OF A2SS22. PTASE SELECTIVELY MODIFIES LYSOSOMAL (BUT NOT NON-LYSOSOMAL) GLYCOPROTEINS TRANSITING THE ER–GOLGI SYSTEM. IT HAS BEEN UNCLEAR HOW PTASE CATALYZES THE GLCNAC-P TRANSFER REACTION AND HOW IT DISTINGUISHES LYSOSOMAL FROM NON-LYSOSOMAL GLYCOPROTEINS. MUTATIONS IN THE GENES ENCODING PTASE CAUSE MUCOLIPIDOSIS II AND III AND LEAD TO ABNORMAL BODY STRUCTURE, MENTAL RETARDATION, AND SEVERAL NEUROLOGICAL DISEASES. IN PRELIMINARY STUDIES, WE HAVE DETERMINED THE ATOMIC STRUCTURE OF THE CATALYTIC CORE OF THE DIMERIC A2SS2 SUBCOMPLEX IN THE APO FORM. WE HAVE IDENTIFIED AN INHIBITORY HOCKEY STICK MOTIF THAT MOVES IN AND OUT OF THE CATALYTIC POCKET IN THE ABSENCE OF SUBSTRATES. WE HAVE ALSO DERIVED A NM-RESOLUTION CRYO-EM 3D MAP OF THE HOLOENZYME. BUILDING ON THESE STRONG PRELIMINARY STUDIES, WE PROPOSE TO DETERMINE THE STRUCTURES OF THE CATALYTIC CORE BOUND TO THE DONOR SUBSTRATE UDP-GLCNAC AND THE ACCEPTOR SUBSTRATE MIMIC A-METHYLMANNOSIDE, AND TO PERFORM STRUCTURE-GUIDED MUTAGENESIS AND IN VITRO ACTIVITY ASSAYS. THESE STRUCTURE-FUNCTION STUDIES OF THE CATALYTIC CORE WILL ENABLE US TO FORMULATE THE UNIQUE PHOSPHO-GLYCOSYL TRANSFER REACTION MECHANISM. WE ALSO PROPOSE TO CARRY OUT SYSTEMATIC MUTATIONAL AND FUNCTIONAL ASSAYS TO EXAMINE THE MANY REPORTED DISEASE-CAUSING MUTATIONS. FINALLY, WE PROPOSE TO STUDY THE STRUCTURES OF A LARGE LYSOSOMAL HYDROLASE-BINDING PERIPHERAL REGION AND THE -SUBUNIT OF THE PTASE HOLOENZYME AND TO EXPLORE HOW THEY INTERACTS WITH SELECTED SUBSTRATE LYSOSOMAL HYDROLASES. THE PROPOSED RESEARCH WILL ADDRESS THE MOLECULAR MECHANISM OF A KEY SIGNALING PATHAY IN GLYCOBIOLOGY AND WILL PROVIDE MOLECULAR INSIGHTS INTO HOW MUTATIONS IN THE PTASE CAUSE THE ASSOCIATED HUMAN DISEASES.

LRRK2 ENZYMATIC MECHANISMS OF NEURODEGENERATION IN PARKINSON'S DISEASE - PROJECT SUMMARY PARKINSON'S DISEASE (PD) IS A PROGRESSIVE NEURODEGENERATIVE MOVEMENT DISORDER CAUSED PRIMARILY BY THE DEGENERATION OF DOPAMINERGIC NEURONS IN THE SUBSTANTIA NIGRA. MUTATIONS IN THE LEUCINE-RICH REPEAT KINASE 2 (LRRK2) GENE CAUSE LATE-ONSET, AUTOSOMAL DOMINANT PD, AND LRRK2 CODING AND NON-CODING VARIANTS ARE ASSOCIATED WITH RISK OF SPORADIC PD. LRRK2 HAS EMERGED HAS AN IMPORTANT THERAPEUTIC TARGET FOR TREATING PD AND THEREFORE IT IS CRITICAL TO UNDERSTAND THE KEY MECHANISMS UNDERLYING DISEASE-LINKED MUTATIONS. LRRK2 IS A LARGE MULTI-DOMAIN PROTEIN CONTAINING RAS-OF-COMPLEX (ROC) GTPASE AND PROTEIN KINASE ENZYMATIC DOMAINS SEPARATED BY A C-TERMINAL-OF-ROC (COR) DOMAIN. GTP-BINDING VIA THE ROC DOMAIN IS CRITICAL FOR NORMAL KINASE ACTIVITY. FAMILIAL LRRK2 MUTATIONS CLUSTER WITHIN THE ROC (R1441C/G/H), COR (Y1699C) AND KINASE (G2019S, I2020T) DOMAINS WHERE THEY COMMONLY ENHANCE THE PHOSPHORYLATION OF A SUBSET OF RAB GTPASE SUBSTRATES IN CELLS. ROC-COR DOMAIN MUTATIONS ACT INDIRECTLY ON KINASE ACTIVITY BY IMPAIRING GTP HYDROLYSIS AND PROMOTING THE GTP-BOUND `ON' STATE. THE GTPASE AND KINASE DOMAINS REPRESENT PROMISING TARGETS FOR INHIBITING LRRK2. WHILE FAMILIAL LRRK2 MUTATIONS SHARE THE CAPACITY TO INDUCE NEURONAL DAMAGE IN CULTURED CELLS, THEIR EFFECTS IN ANIMAL MODELS ARE LESS CERTAIN DUE TO A LACK OF ROBUST NEURODEGENERATIVE PHENOTYPES. IN ADDITION, HOW THE TWO ENZYMATIC ACTIVITIES CONTRIBUTE TO NEURONAL DAMAGE IN VIVO INDUCED BY FAMILIAL LRRK2 MUTATIONS IS POORLY UNDERSTOOD. WE HAVE RECENTLY DEVELOPED AN ADENOVIRAL-MEDIATED GENE TRANSFER MODEL IN ADULT RATS WHERE G2019S LRRK2 INDUCES NIGROSTRIATAL PATHWAY DOPAMINERGIC NEURODEGENERATION THROUGH A KINASE-DEPENDENT MECHANISM. IN THE PRESENT APPLICATION, WE PROPOSE TO EXPLOIT THIS ROBUST AND RAPID RODENT MODEL TO DETERMINE WHETHER KINASE ACTIVITY IS COMMONLY REQUIRED FOR NEURODEGENERATION INDUCED BY FAMILIAL MUTATIONS (R1441C, Y1699C AND G2019S) OR PD RISK VARIANTS (G2385R) IN LRRK2 BY GENETIC AND PHARMACOLOGICAL KINASE INHIBITION (AIM 1.1). THE CONTRIBUTION OF RAB PHOSPHORYLATION TO NEURONAL DAMAGE INDUCED BY MUTANT LRRK2 IS NOT KNOWN. WE WILL DETERMINE THE NEUROPROTECTIVE EFFECTS OF GLOBALLY REDUCING RAB PHOSPHORYLATION IN MUTANT LRRK2 NEURONAL AND ADENOVIRAL RAT MODELS BY OVEREXPRESSING A NOVEL RAB-SPECIFIC PHOSPHATASE, PPM1H (AIM 1.2). KNOCKDOWN OF PPM1H WILL EXPLORE WHETHER INCREASING RAB PHOSPHORYLATION IS SUFFICIENT TO PHENOCOPY THE NEUROTOXIC EFFECTS OF MUTANT LRRK2. OUR STUDIES WILL FURTHER EXPLORE WHETHER GENETICALLY MODULATING GTPASE ACTIVITY CAN PROVIDE A COMMON NEUROPROTECTIVE MECHANISM AGAINST DIFFERENT FAMILIAL LRRK2 MUTATIONS IN RODENTS (AIM 2.1). IN PARTICULAR, WE WILL EVALUATE HYPOTHESIS-TESTING MUTATIONS THAT INCREASE GTP HYDROLYSIS AND PROMOTE THE GDP-BOUND `OFF' STATE OF LRRK2. FINALLY, WE WILL EXPLORE HOW THE NATIVE INTERACTOME OF LRRK2 IN NEURONS IS REGULATED BY THE GTPASE CYCLE TO IDENTIFY NOVEL PROTEIN TARGETS THAT INTERACT WITH LRRK2 IN ITS GDP- OR GTP- BOUND STATES (AIM 2.2). OUR STUDIES WILL PROVIDE CRITICAL MECHANISTIC INSIGHT INTO HOW GTPASE AND KINASE ACTIVITY REGULATE LRRK2-MEDIATED NEURODEGENERATION, AND WILL BE IMPORTANT FOR THERAPEUTIC DISCOVERY EFFORTS FOR PD.

ELUCIDATING STRUCTURES AND MOLECULAR MECHANISMS OF PANNEXIN CHANNELS

TARGETED GLYCOMICS AND AFFINITY REAGENTS FOR CANCER BIOMARKER DEVELOPMENT

NETWORK AND CELLULAR VULNERABILITY TO PATHOLOGICAL PROTEIN PROGRESSION - PROJECT SUMMARY/ABSTRACT OVER 6.5 MILLION PEOPLE IN THE UNITED STATES CURRENTLY LIVE WITH PROGRESSIVE NEURODEGENERATIVE ALZHEIMER’S DISEASE (AD) AND RELATED DEMENTIAS INCLUDING DEMENTIA WITH LEWY BODIES (DLB), YET NO TREATMENTS TO STOP THEIR PROGRESSION ARE AVAILABLE. THE PRESENCE OF AGGREGATED PROTEINS IN THE BRAIN (TAU, AMYLOID SS (ASS), AND A- SYNUCLEIN) IS THOUGHT TO UNDERLIE DISEASE, AND CLINICAL OVERLAP IN PRESENTATION OF THESE DISEASES ASSOCIATES WITH THE PRESENCE OF MORE THAN ONE TYPE OF PATHOLOGY. THE FACTORS INFLUENCING CELLULAR AND NETWORK VULNERABILITY TO EACH INDIVIDUAL PATHOLOGY OR CO-OCCURRING PATHOLOGIES ARE NOT WELL-UNDERSTOOD. A PROPER UNDERSTANDING OF VULNERABILITY IN THE CONTEXT OF CO-PATHOLOGIES IS NECESSARY FOR THE DEVELOPMENT AND EVALUATION OF NOVEL DISEASE- MODIFYING TREATMENTS. THIS PROJECT WILL TEST THE HYPOTHESIS THAT PROGRESSION OF INTRACELLULAR A-SYNUCLEIN AND TAU PATHOLOGIES ARE BOUND BY NEUROANATOMICAL CONNECTIVITY BUT INFLUENCED BY NETWORK AND CELL-LEVEL VULNERABILITY. NETWORK AND CELL-LEVEL VULNERABILITY MAY ALSO BE INFLUENCED BY ADDITIONAL FACTORS SUCH AS THE PRESENCE OF CO-PATHOLOGIES, INCLUDING EXTRACELLULAR AMYLOID SS (ASS) PLAQUES. TO TEST THIS HYPOTHESIS, THE FOLLOWING AIMS WILL BE PURSUED: (I) PROGRESSION OF A-SYNUCLEIN AND TAU PATHOLOGIES WILL BE MAPPED IN WILDTYPE MICE BEARING SINGLE OR MULTIPLE PATHOLOGIES AT HIGH RESOLUTION USING REGISTRATION TO A COMMON NEUROANATOMICAL ATLAS, AND NETWORK PARAMETERS OF PROGRESSION WILL BE ASSESSED WITH COMPUTATIONAL MODELING (SPECIFIC AIM 1); QUANTITATIVE PATHOLOGY AND NETWORK ANALYSIS WILL BE UTILIZED TO ASSESS THE IMPACT OF ASS PLAQUES ON THE DEVELOPMENT, SPREAD, AND NETWORK VULNERABILITY TO A-SYNUCLEIN AND TAU CO-PATHOLOGY IN KNOCK-IN MICE THAT EXPRESS HUMAN TAU AND DEVELOP ASS PLAQUES AND IN A TIME-DEPENDENT MANNER (SPECIFIC AIM 2); SPATIAL TRANSCRIPTOMICS OF HUMAN AD BRAIN, DLB BRAIN, AND MOUSE CO-PATHOLOGY BRAIN WILL BE USED TO DETERMINE UNIQUE MOLECULAR SIGNATURES OF NEURONS VULNERABLE TO A-SYNUCLEIN OR TAU PATHOLOGIES. VULNERABILITY SIGNATURES WILL BE VALIDATED WITH COMBINED IN SITU HYBRIDIZATION-IMMUNOFLUORESCENCE AND INTEGRATED WITH NETWORK VULNERABILITY AND REGIONAL GENE EXPRESSION TO DEVELOP AND EVALUATE A MODEL OF HUMAN CO-PATHOLOGY PROGRESSION AS A RESOURCE FOR UNDERSTANDING DISEASE PROGRESSION AND EVALUATING THERAPEUTIC EFFICACY (SPECIFIC AIM 3). THE PROPOSED RESEARCH IS EXPECTED TO IMPROVE OUR UNDERSTANDING OF THE SPATIOTEMPORAL PROGRESSION OF CO- PATHOLOGIES AND TO DEVELOP IMPROVED TOOLS TO EVALUATE EFFICACY OF NOVEL THERAPEUTIC STRATEGIES THAT COULD SLOW THE PROGRESSION OF NEURODEGENERATIVE DISEASES.

INTEGRATIVE CANCER EPIGENOMIC DATA ANALYSIS CENTER (ICE-DAC) - PROJECT SUMMARY / ABSTRACT THE WIDESPREAD NATURE OF EPIGENETIC ABNORMALITIES IN HUMAN CANCERS HAS BECOME INCREASINGLY APPRECIATED IN THE PAST DECADE, WITH CLINICAL IMPACTS IN CANCER DETECTION, CANCER CLASSIFICATION, CHEMORESISTANCE PREDICTION, AND THERAPY. LARGE-SCALE CANCER GENOMIC SCREENS HAVE REVEALED A PREVIOUSLY UNRECOGNIZED PREVALENCE OF SOMATIC MUTATIONS AMONG EPIGENETIC REGULATORS IN HUMAN CANCERS, INCLUDING CHROMATIN REMODELERS, AS WELL AS HISTONE OR DNA METHYLATION READERS, WRITERS, AND ERASERS. EPIGENETIC ALTERATIONS CAN SERVE AS DRIVER EVENTS IN CANCER BY INACTIVATING TUMOR-SUPPRESSOR GENES. THE FINDING THAT THESE SILENCING EVENTS ARE MUTUALLY EXCLUSIVE WITH STRUCTURAL OR MUTATIONAL INACTIVATION OF THE SAME GENE REINFORCES THE FUNCTIONAL SIGNIFICANCE OF EPIGENETIC SILENCING. THE MAJORITY OF CASES OF MICROSATELLITE INSTABILITY IN SPORADIC HUMAN TUMORS CAN BE ATTRIBUTED TO EPIGENETIC SILENCING OF THE MLH1 MISMATCH REPAIR GENE. CLEARLY, EPIGENETIC MECHANISMS PLAY A KEY ROLE IN HUMAN CANCER, AND A COMPREHENSIVE MOLECULAR CHARACTERIZATION OF CANCER SHOULD INCLUDE EPIGENOMIC PROFILING. IN 2015 WE ESTABLISHED AN INTEGRATIVE CANCER EPIGENOMIC DATA ANALYSIS CENTER (ICE-DAC) TO PROVIDE SPECIALIZED EXPERTISE IN EPIGENOMIC DATA ANALYSIS AS PART OF THE GENOME DATA ANALYSIS NETWORK (GDAN). WE HAVE MADE MAJOR CONTRIBUTIONS TO THE GDAN IN THE PAST FIVE YEARS, DEVELOPING CUTTING-EDGE DNA METHYLATION BIOINFORMATICS TOOLS, LEADING ANALYSIS TEAMS IN PANCANATLAS, ATAC-SEQ, PAN-GASTROINTESTINAL (PAN-GI) CANCERS AND TUMOR MOLECULAR PATHOLOGY (TMP), AND MAKING MAJOR CONTRIBUTIONS TO THE EXCEPTIONAL RESPONDERS (ER) STUDY, TESTICULAR GERMCELL TUMORS (TGCT), DATA HARMONIZATION (QC), CCG ANCESTRY INFORMATIVE MARKERS (AIM), AND MANY OTHER PROJECTS. HERE WE PROPOSE TO SUSTAIN THIS PRODUCTIVE ACTIVITY IN A CONTINUATION OF THE ICE-DAC, LENDING OUR DEEP EXPERTISE IN EPIGENOMIC DATA ANALYSIS TO COLLABORATIVE, INTEGRATIVE GENOMIC AND EPIGENOMIC ANALYSES OF CLINICAL SPECIMENS WITHIN THE NCI GDAN. IN SPECIFIC AIM 1, WE WILL PROVIDE ADVANCED SPECIALIZED ANALYSIS OF BULK AND SINGLE-CELL CANCER EPIGENOMIC DATA GENERATED BY PROGRAMS WITHIN THE NCI CENTER FOR CANCER GENOMICS. WE HAVE DEVELOPED VARIOUS TOOLS FOR EPIGENETIC ANALYSIS AND IMPLEMENTED AN AUTOMATED WORKFLOW TO PROVIDE TIMELY PRIMARY DATA ANALYSIS FOR AWGS. IN SPECIFIC AIM 2 WE WILL IMPLEMENT INNOVATIVE TOOLS TO EXTRACT ADDITIONAL INFORMATION FROM SPECIALIZED DATA TYPES. THIS AIM MAXIMIZES THE UTILITY OF THE DATA GENERATED, AND ADDS TO THE RIGOR OF ANALYSIS BY PROVIDING ORTHOGONAL VALIDATION. THESE ANALYSES WILL INCLUDE PREDICTION OF COMMON COVARIATES (SUCH AS SEX, AGE AND RACE) FOR THE SAMPLES, ANALYSIS OF TUMOR PURITY AND COMPOSITION, INFERENCES OF GENETIC INFORMATION (INCLUDING GENETIC MUTATION, COPY NUMBER AND LARGE STRUCTURAL VARIANTS), ALL FROM THE DNA METHYLATION ASSAYS. IN SPECIFIC AIM 3 WE WILL INTEGRATE EPIGENOMIC DATA WITH OTHER GENOMIC, TRANSCRIPTOMIC, PROTEOMIC, AND CLINICAL DATA TO DERIVE BIOLOGICALLY AND CLINICALLY RELEVANT NOVEL INSIGHTS. OUR DEEP EXPERTISE IN SPECIALIZED EPIGENOMICS WILL ADDRESS A CORE COMPETENCY REQUIRED IN GDAN, AND COMPLEMENT OTHER GENOMIC ANALYSES OF CLINICAL TRIAL SAMPLES.

THE CONTRIBUTION OF THE VERMIFORM APPENDIX TO PARKINSON'S DISEASE

EXPLORING MECHANISMS OF PARKINSON'S DISEASE-LINKED D620N VPS35 IN RAT MODELS

EPIGENETIC CONTRIBUTIONS TO SYMPTOM ASYMMETRY IN PARKINSON'S DISEASE

VISUALIZATION AND EPIGENOMIC CHARACTERIZATION OF EARLY INTESTINAL TUMORIGENESIS IN MICE

BREAST CANCER RISK ENHANCERS

A NOVEL PROCESS SAFEGUARDS GENOME INTEGRITY IN THE MAMMALIAN GERM LINE - PROJECT SUMMARY SIMPLE PURINE–PYRIMIDINE (PU/PY) REPEATS (PPRS) ARE KNOWN TO FORM THE LEFT-HANDED, FRAGILE Z-DNA STRUCTURE. SUCH REPEATS ARE ALSO KNOWN TO BE HIGHLY MUTAGENIC, INDUCING LARGE CHROMOSOMAL DELETIONS AND REARRANGEMENTS IN THE CELLS OF HIGHER ORGANISMS. THE MUTAGENIC EFFECTS OF Z-DNA WOULD BE THE MOST DETRIMENTAL TO A SPECIES IF BREAKS OCCURRED UNCONTROLLABLY IN THE GERM LINE AND MUTATIONS OCCURRING AT UNREPAIRED BREAKS WERE PASSED ON TO THE NEXT GENERATION. WE DISCOVERED A NOVEL BIOLOGICAL PROCESS IN THE MAMMALIAN GERM LINE THAT CONTROLS Z- DNA STRUCTURE AT PPRS. USING THE ZBTB43 MUTANT MOUSE GENETIC MODEL AND OUR TRANSGENIC MOUSE LINE THAT ALLOWS US TO ISOLATE GERM CELLS, WE FOUND THAT A PREVIOUSLY UNCHARACTERIZED DNA BINDING PROTEIN, ZBTB43 REMODELS Z-DNA STRUCTURE AND PROTECTS FROM DOUBLE-STRAND BREAKS IN FETAL MALE GERM CELLS IN VIVO. BY BIOCHEMICAL ASSAYS WE FOUND THAT ZBTB43 BINDS TO PPR-RICH DNA SITES IN THE GENOME IN VITRO. ZBTB43 BINDING SITES FORM Z-DNA AND CAUSE LARGE GENOMIC REARRANGEMENTS IN MAMMALIAN CELLS. BY IN VIVO EPIGENOME MAPPING WE DETECTED Z-DNA IN MUTANT GERM CELLS AT THE LOCATIONS WHERE ZBTB43 BINDING OCCURS IN WILD-TYPE PROSPERMATOGONIA. WE HYPOTHESIZE, THEREFORE, THAT ZBTB43 SAFEGUARDS GENOME INTEGRITY IN THE GERM LINE BY BINDING AND ELIMINATING Z-DNA AT PPRS. IN ADDITION, WE FOUND THAT BY ELIMINATING Z-DNA, ZBTB43 PROMOTES DE NOVO METHYLATION AT PPRS DURING THE TIME OF GLOBAL EPIGENETIC REMODELING. WE PROPOSE TO PURSUE THE FOLLOWING AIMS, USING A COMBINATION OF GENETIC, CELL BIOLOGY, BIOCHEMISTRY, AND EPIGENOMIC APPROACHES. IN AIM 1, WE WILL TEST THE WORKING HYPOTHESIS THAT ZBTB43 ELIMINATES Z-DNA STRUCTURE IN VIVO BY DIRECTLY BINDING TO PPRS IN FETAL MALE GERM CELLS. WE WILL DETERMINE 1) THE SPATIAL AND TEMPORAL CHANGES OF THE Z-DNA STRUCTURE IN FETAL MALE GERM CELLS IN THE PRESENCE OR ABSENCE OF ZBTB43 PROTEIN IN VIVO; 2) THE DEPENDENCE OF THE Z-DNA REMODELING PROCESS ON THE DIRECT BINDING OF ZBTB43 TO THE Z-DNA STRUCTURE IN VIVO; AND 3) THE MOLECULAR REQUIREMENTS OF ZBTB43 ACTION ON Z-DNA. IN AIM 2, WE WILL TEST THE WORKING HYPOTHESIS THAT ZBTB43 FACILITATES DE NOVO DNA METHYLATION IN PROSPERMATOGONIA INDIRECTLY BY ELIMINATING Z-DNA, THUS REVEALING THE SEQUENCES AS SUBSTRATES FOR DE NOVO METHYLTRANSFERASES. WE WILL MAP DNA METHYLATION IN THE PRESENCE AND ABSENCE OF ZBTB43 DURING THE EPIGENOME REMODELING PROCESS IN FETAL MALE GERM CELLS. WE WILL TEST WHETHER DE NOVO DNMTS METHYLATE Z- DNA SUBSTRATES IN VITRO. WE WILL TEST WHETHER ZBTB43 AFFECTS NUCLEOSOME OCCUPANCY AT PPRS. IN AIM 3, WE WILL TEST THE HYPOTHESIS THAT Z-DNA IS MUTAGENIC IN THE GERM LINE AND THAT ZBTB43 HAS EVOLVED TO MANAGE THAT BURDEN. WE WILL MAP DOUBLE-STRAND BREAKS IN MUTANT FETAL GERM CELLS, TEST THE ANTI-MUTAGENIC EFFECT OF ZBTB43 IN CELL CULTURE, AND SEARCH FOR GENOMIC REARRANGEMENTS IN SPERM OF ZBTB43 MUTANT MALES. BY THE END OF THE GRANT PERIOD, WE WILL HAVE IDENTIFIED AND CHARACTERIZED THE FIRST EXAMPLE OF HOW A DNA BINDING PROTEIN PROTECTS GENOME INTEGRITY AND TARGETS DE NOVO DNA METHYLATION BY CONTROLLING Z-DNA STRUCTURE IN THE MAMMALIAN GERM LINE.

MECHANISMS OF VPS35-DEPENDENT NEURODEGENERATION IN PARKINSON'S DISEASE

NOVEL MECHANISMS OF LRRK2-DEPENDENT NEURODEGENERATION IN PARKINSON'S DISEASE

ELUCIDATING THE STRUCTURAL MECHANISMS OF NAIP RECEPTORS IN BACTERIAL DETECTION AND INFLAMMASOME ACTIVATION - PROJECT SUMMARY INFLAMMASOMES ARE MEGA-DALTON PROTEIN COMPLEXES THAT INITIATE INFLAMMATION RESPONSES AND PLAY IMPORTANT ROLES IN THE INNATE IMMUNE SYSTEM. UPON ACTIVATION, INFLAMMASOMES RECRUIT AND ACTIVATE THE EFFECTOR PROTEIN CASPASE-1. CASPASE-1 IN TURN CLEAVES THE GASDERMIN D (GSDMD) PROTEIN TO RELEASE ITS N-TERMINAL DOMAIN, WHICH INSERTS INTO THE CELL MEMBRANE TO PUNCH HOLES ON THE CELL SURFACE. AS A RESULT, THE HOST CELL WILL UNDERGO PYROPTOTIC CELL DEATH (PYROPTOSIS) AND RELEASE THE CELL CONTENTS INTO THE EXTRACELLULAR ENVIRONMENT. PRO-INFLAMMATORY CYTOKINES IL-1 AND IL-18, BOTH ACTIVATED BY CASPASE-1, WILL ALSO BE RELEASED TO ACTIVATE THE DOWNSTREAM INFLAMMATION REACTIONS. NAIP IS A FAMILY OF CYTOSOLIC IMMUNOLOGICAL RECEPTORS THAT ACTIVATE THE NAIP/NLRC4 INFLAMMASOMES IN RESPONSE TO GRAM-NEGATIVE BACTERIAL INFECTIONS. THERE ARE SEVEN NAIP PROTEINS IN MOUSE, EACH SENSE A SPECIFIC LIGAND SUCH AS FLAGELLIN, NEEDLE PROTEIN OR INNER ROD PROTEIN IN THE TYPE III SECRETION SYSTEM. IN PREVIOUS STUDIES, WE HAVE SHOWN ACTIVE NAIPS ACTIVATE NLRC4 THROUGH THE NUCLEATED POLYMERIZATION MECHANISM. HOWEVER, IT IS STILL LARGELY UNKNOWN ABOUT HOW NAIPS REMAIN INACTIVE IN THE RESTING CELLS, AND HOW IS THE LIGAND SPECIFICITY IS DETERMINED AMONG DIFFERENT NAIPS. UNLIKE IN MOUSE, HUMANS ONLY HAVE ONE NAIP AND IT WAS SHOWN TO BE ACTIVATED BY ALL THREE BACTERIAL LIGANDS. THIS LEADS TO MANY QUESTIONS INCLUDING WHAT IS THE STRUCTURAL BASIS OF THE BROAD DETECTION OF BACTERIAL LIGANDS BY HUMAN NAIP AND WHAT DOES IT TELL US ABOUT THE HUMAN INFLAMMASOMAL BACTERIAL DETECTION MECHANISM? TO ADDRESS THESE QUESTIONS, WE PROPOSE TO COMBINE ELECTRON CRYO-MICROSCOPY (CRYOEM) WITH BIOCHEMICAL AND FUNCTIONAL STUDIES TO ELUCIDATE THE STRUCTURAL MECHANISMS UNDERLYING THE SIGNAL TRANSDUCTION BY NAIP PROTEINS. DYSREGULATION OF NAIP/NLRC4 INFLAMMASOMES CAUSES MACROPHAGE ACTIVATION SYNDROME (MAS) AND AUTOINFLAMMATION, AND MUTATIONS OF NAIP PROTEIN ARE HIGHLY CORRELATED WITH SPINAL MUSCULAR ATROPHY (SMA). THE SUCCESSFUL EXECUTION OF THIS WORK WILL BROADLY ADVANCE THE DEVELOPMENT OF EFFECTIVE THERAPIES TO TREAT INFECTIOUS DISEASES, AUTOINFLAMMATORY DISEASES, SMA, AND CANCER THROUGH TARGETING THE INFLAMMASOME PATHWAY.

5-METHYLCYTOSINE OXIDATION IN DEVELOPMENT AND DISEASE - PROJECT SUMMARY: 5-METHYLCYTOSINE OXIDATION IN DEVELOPMENT AND DISEASE DNA METHYLATION AT GENE PROMOTERS AND ENHANCERS IS USUALLY AN INDICATION THAT GENES ARE SILENCED, WHEREAS LOSS OF METHYLATION THROUGH THE TET-MEDIATED 5-METHYLCYTOSINE (5MC) OXIDATION PATHWAY CAN LEAD TO GENE ACTIVATION. TET PROTEINS AND THE 5MC OXIDATION PATHWAY ARE KNOWN, PIVOTAL ACTORS IN EMBRYONIC DEVELOPMENT AND DISEASE, YET THE MECHANISMS BY WHICH TET-MEDIATED 5MC OXIDATION IS REGULATED ARE COMPLETELY UNKNOWN. WE RECENTLY DISCOVERED THAT THE PROTEIN SMCHD1 IS A POTENTIAL NEGATIVE REGULATOR OF TET ACTIVITY. IN HUMANS, SMCHD1 DEPLETION ACTIVATES THE DUX4 GENE(S) THAT CAUSE(S) CELL TOXICITY, MUSCLE DEGENERATION, AND FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY (FSHD). WE HYPOTHESIZE THAT ONE KEY FUNCTION OF SMCHD1 IS TO CONTROL 5MC OXIDASE ACTIVITIES AND THAT LACK OF SMCHD1 LEADS TO AN OVER-ACTIVATION OF TET-MEDIATED EPIGENETIC FUNCTION, AND THE ACTIVATION OF DUX4. THE OBJECTIVE OF THIS PROJECT IS TO TEST THIS HYPOTHESIS AND OBTAIN A FUNDAMENTAL UNDERSTANDING OF HOW SMCHD1 INTERACTS WITH AND CONTROLS TET-MEDIATED EPIGENETIC FUNCTION IN NORMAL EMBRYONIC DEVELOPMENT AND DISEASE. IN AIM 1, WE WILL USE MOUSE EMBRYONIC STEM CELLS AND HUMAN INDUCED PLURIPOTENT STEM CELLS TO DETERMINE THE BASIC MECHANISMS OF SMCHD1-REGULATED TET FUNCTION IN NORMAL DEVELOPMENT. WE WILL EMPLOY SMCHD1-KNOCKOUT CELL AND MOUSE LINES AND DETERMINE HOW TET PROTEIN DISTRIBUTION IN THE GENOME IS ALTERED; WHICH LOCI ARE AFFECTED; IF THE DISTRIBUTION OF OXIDIZED 5-METHYLCYTOSINE BASES (5HMC) IS SPECIFIC TO SMCHD1 BINDING SITES; WHICH REGIONS OF THE (ZYGOTIC) GENOME ARE ACTIVATED UPON SMCHD1 LOSS; AND IF THERE ARE SHARED SMCHD1-MEDIATED REGULATORY PATHWAYS BETWEEN MOUSE AND HUMAN PLURIPOTENT CELLS. THE RESULTS OF THESE EXPERIMENTS WILL ESTABLISH THE NEGATIVE REGULATORY FUNCTION OF SMCHD1 IN NORMAL DEVELOPMENT AND SET THE STAGE FOR DISEASE-SPECIFIC STUDIES IN AIM 2. IN AIM 2, WE WILL INACTIVATE SMCHD1 IN HUMAN MUSCLE CELLS AND DETERMINE THE EPIGENETIC ROLE OF SMCHD1 IN CELL DIFFERENTIATION AND DISEASE BY DETERMINING GENE EXPRESSION CHANGES, CHROMATIN STRUCTURE, AND GENOME-WIDE DNA METHYLATION STATUS. WE EXPECT THAT SMCHD1-MEDIATED TET INHIBITION AND REGULATION OF GENE EXPRESSION WILL BE SIMILAR IN HUMAN AND MOUSE CELLS, AND THAT DEPLETING TET ACTIVITY IN SMCHD1 KNOCKOUT CELLS WILL AT LEAST PARTIALLY REVERSE THE EFFECTS OF SMCHD1 LOSS-OF-FUNCTION PHENOTYPES. IN AIM 3, WE WILL PERFORM BIOCHEMICAL STUDIES OF SMCHD1-TET PROTEIN-PROTEIN INTERACTIONS TO IDENTIFY KEY BINDING DOMAINS, AND DETERMINE IF TET INHIBITORS CAN SILENCE DUX4 EXPRESSION IN CELL LINES LACKING FUNCTIONAL SMCHD1, AND IN MUSCLE CELLS DERIVED FROM FSHD PATIENTS. IF SUCCESSFUL, THESE DATA WILL PROVIDE PROOF OF PRINCIPLE THAT TET INHIBITORS MAY HAVE THERAPEUTIC BENEFIT FOR FSHD PATIENTS. TOGETHER, THE RESULTS OF THIS PROJECT WILL GENERATE THE FIRST MECHANISTIC, REGULATORY MODEL OF THE TET- MEDIATED 5MC OXIDATION PATHWAY IN MOUSE AND MAN, AND THE ROLE OF THE NOVEL TET-SMCHD1 AXIS IN NORMAL DEVELOPMENT AND DISEASE.

STRUCTURAL BASIS OF NOCICEPTOR CHANNEL TRPM3 GATING AND PHARMACOLOGY - PROJECT SUMMARY THE DETECTION AND RAPID AVOIDANCE OF HARMFUL EXTERNAL STIMULI VIA PAIN PERCEPTION IS CRUCIAL FOR SURVIVAL. THE SENSORS OF PAIN PATHWAYS ARE NOCICEPTORS, SPECIALIZED PERIPHERAL SENSORY NEURONS. NOCICEPTORS DETECT EXTERNAL EXTREME STIMULI SUCH AS TEMPERATURE, PRESSURE AND CHEMICALS ASSOCIATED WITH INJURY VIA SENSORY ION CHANNELS EXPRESSED ON THE PLASMA MEMBRANE. IN RESPONSE TO SUCH STIMULI, THEY FIRE ACTION POTENTIALS AND CONVEY THE INFORMATION TO THE BRAIN, THUS ALERTING US TO POTENTIAL INJURY AND ALLOWING US TO TAKE PROPER ACTIONS. TRANSIENT RECEPTOR POTENTIAL MELASTATIN 3 (TRPM3) IS ONE OF THREE MOLECULAR PAIN SENSORS THAT, TOGETHER WITH TRPV1 AND TRPA1, DETECT NOXIOUS HEAT. TRPM3 IS A CALCIUM-PERMEABLE NONSELECTIVE CATION CHANNEL BELONGING TO THE MELASTATIN SUBFAMILY OF THE TRP SUPERFAMILY. IT IS EXPRESSED IN A LARGE SUBSET OF SOMATOSENSORY NEURONS AND IS ACTIVATED BY HEAT AND BY A VARIETY OF CHEMICAL LIGANDS, INCLUDING THE ENDOGENOUS NEUROSTEROID PREGNENOLONE SULFATE (PS). BOTH HEAT AND PS-INDUCED ACTIVATION OF TRPM3 EVOKE PAIN. TRPM3-DEFICIENT MICE SHOW A DEFICIT IN THE DEVELOPMENT OF INFLAMMATORY THERMAL HYPERALGESIA. INHIBITION OF TRPM3 BY LOW DOSES OF PRIMIDONE IN PS- AND HEAT-INDUCED PAIN MODELS SHOWED ANALGESIC EFFECTS IN MICE. THE ACTIVITY OF TRPM3 IN SOMATOSENSORY NEURONS IS ALSO NEGATIVELY MODULATED BY GSS PROTEIN WHICH IS RELEASED UPON ACTIVATION OF SEVERAL G-PROTEIN- COUPLED RECEPTORS, THEREBY REDUCING TRPM3-MEDIATED PAIN. BECAUSE PAIN IS A MAJOR UNRESOLVED MEDICAL PROBLEM AND BECAUSE PHARMACOTHERAPIES TARGETING GPCR HAVE MANY AND SEVERE UNWANTED EFFECTS, PHARMACOLOGICAL TARGETING OF THE DOWNSTREAM TRPM3 MAY HAVE GREAT POTENTIAL TO INFLUENCE SEVERAL SIGNALING PATHWAYS RELEVANT IN PAIN SENSATION AND IN THE DEVELOPMENT OF NEW AND ALTERNATIVE ANALGESIC DRUGS. TRPM3 IS ALSO HIGHLY EXPRESSED IN PANCREATIC BETA CELLS AND HAS APPLICATION IN GLUCOSE-INDUCED INSULIN RELEASE. MOREOVER, TRPM3 IS IMPLICATED IN DEVELOPMENTAL AND EPILEPTIC ENCEPHALOPATHIES WHICH ARE A HETEROGENEOUS GROUP OF DISORDERS CHARACTERIZED BY EPILEPSY WITH COMORBID INTELLECTUAL DISABILITY. AT PRESENT, THE PHYSIOLOGICAL AND PATHOLOGICAL PROPERTIES OF TRPM3 ARE STILL POORLY UNDERSTOOD. THE FOCUS OF THIS PROPOSAL IS TO DETERMINE THE FIRST TRPM3 STRUCTURES IN DIFFERENT FUNCTIONAL STATES AT HIGH-RESOLUTION, AND TO UNDERSTAND THE MOLECULAR ARCHITECTURE, GATING MECHANISM, AND PHARMACOLOGY OF TRPM3 BY COMBINING SINGLE-PARTICLE CRYO ELECTRON MICROSCOPY (CRYO-EM) AND PATCH-CLAMP ELECTROPHYSIOLOGY. WE WILL ALSO INITIATE THE PURIFICATION OF TRPM3 FROM NATIVE TISSUES USING MONOCLONAL ANTIBODIES THAT RECOGNIZE THREE-DIMENSIONAL EPITOPES OF TRPM3 AS WELL AS KNOCKED-IN AFFINITY TAG TO TRPM3 GENE IN MICE AS THERE IS GROWING EVIDENCE THAT PROTEINS PURIFIED FROM NATIVE SOURCES MAY DISPLAY DIFFERENT ASSEMBLY, COMPLEX COMPOSITION, AND FUNCTIONAL PROPERTIES RELATIVE TO PROTEINS FROM RECOMBINANT SYSTEMS. THE STRONG PRELIMINARY DATA WE PROVIDE WILL ENSURE THE SUCCESS OF THIS PROPOSAL, AND THE OUTCOME OF THIS PROPOSAL WILL PAVE A SOLID FOUNDATION FOR THE DEVELOPMENT OF NOVEL ANALGESICS.

ROLE OF GAS41 IN HUMAN CANCER

ANALYZING THE ROLE OF TXNIP AS A UNIVERSAL REGULATOR OF GLUCOSE TRANSPORT

MECHANISMS OF DE NOVO METHYLATION IN CANCER

ALZHEIMER'S DISEASE GENETIC ARCHITECTURE IN THE PORTUGUESE POPULATION

THE ROLE OF DNA METHYLATION IN TUMOR MAINTENANCE

MECHANISMS OF CANCER MUTATIONS - PROJECT SUMMARY: CANCER GENOME SEQUENCING HAS IDENTIFIED THOUSANDS OF SOMATIC MUTATIONS IN DIFFERENT TYPES OF HUMAN CANCER. THESE MUTATIONS PROVIDE A VALUABLE SOURCE OF INFORMATION AS TO THE POTENTIAL MUTAGENIC EVENTS THAT HAVE OCCURRED AND CREATED THE MUTATIONS, SOMETIMES DECADES BEFORE THE TUMOR DEVELOPS. FOR CERTAIN CANCERS, CONVINCING LINKS BETWEEN AN EXPOSURE AND CANCER MUTATIONS HAVE BEEN ESTABLISHED, BEST EXEMPLIFIED BY TYPICAL SUNLIGHT-INDUCED MUTATIONS (C TO T MUTATIONS AT DIPYRIMIDINE SEQUENCES) THAT ARE PREVALENT IN MELANOMA AND IN NON-MELANOMA SKIN CANCERS AND BY SMOKING- RELATED G TO T TRANSVERSION MUTATIONS THAT ARE COMMON IN LUNG CANCERS FROM SMOKERS BUT NOT NONSMOKERS. THE TYPES AND FREQUENCIES OF MUTATIONS IN DIFFERENT TUMOR TYPES IS CALLED THE MUTATIONAL SIGNATURE. SEVERAL SIGNATURES ALSO HAVE A CHARACTERISTIC DNA STRAND BIAS RELATED TO, FOR EXAMPLE, THE DIRECTION OF TRANSCRIPTION. IN SEVERAL HUMAN CANCERS, THERE ARE UNUSUAL AND CHARACTERISTIC MUTATIONAL SIGNATURES OF UNKNOWN ORIGIN. THESE SIGNATURES ARE THOUGHT TO ARISE FROM SPECIFIC DNA DAMAGE, DEFECTIVE DNA REPAIR OR FROM ENDOGENOUS PROCESSES. WE HYPOTHESIZE THAT WE CAN IDENTIFY AND RECREATE MUTAGENIC SIGNATURES USING DNA DAMAGE MAPPING, A PROCEDURE IN WHICH WE DETERMINE DNA LESIONS OF A SPECIFIC TYPE AT ALL SEQUENCE POSITIONS OF THE HUMAN GENOME, COMBINED WITH MUTATIONAL ANALYSIS, WITH THE GOAL OF ASSIGNING MUTATIONAL SIGNATURES TO ETIOLOGICAL AGENTS RELEVANT FOR SPECIFIC HUMAN CANCERS. ONE KEY CHALLENGE IN THIS FIELD IS METHOD DEVELOPMENT. THE METHOD NEEDS TO BE SUFFICIENTLY SENSITIVE TO DETECT RARE DNA LESIONS AND MUST BE CAPABLE OF DOING SO AT ALL POSITIONS OF THE GENOME. WE HAVE DEVELOPED SUCH A METHOD, WHICH WE NAMED CIRCLE-DAMAGE-SEQUENCING (CD-SEQ). WE PROPOSE THREE SPECIFIC AIMS. IN THE FIRST AIM, WE WILL USE OUR NEW METHOD TO CHARACTERIZE AND MAP REACTIVE ALDEHYDE-DERIVED DNA ADDUCTS THAT WE HYPOTHESIZE PLAY A CRUCIAL ROLE IN LIVER CANCER. IN THE SECOND AIM, WE WILL TEST THE HYPOTHESIS THAT 8-OXO-DG INCORPORATED FROM THE NUCLEOTIDE POOL IS RESPONSIBLE FOR SEQUENCE-SPECIFIC A TO C TRANSVERSION MUTATIONS OBSERVED AS A DOMINANT MUTATION TYPE IN HUMAN ESOPHAGEAL ADENOCARCINOMAS. THE LAST AIM WILL HAVE THE GOAL OF UNDERSTANDING THE MECHANISMS OF TARGETED MUTAGENESIS BY TOBACCO SMOKE CARCINOGENS OF THE PAH CLASS AND HOW THIS TARGETING RELATES TO LUNG AND ORAL SQUAMOUS CELL CARCINOMAS IN TOBACCO USERS. OUR DNA DAMAGE STUDIES WILL BE COMPLEMENTED BY MUTATION ASSAYS TO CONFIRM THAT THE RELEVANT PATHWAYS INDUCE THE EXPECTED TYPES OF MUTATIONS. OUR PROPOSED WORK WILL PROVIDE MECHANISTIC INSIGHTS INTO THE POTENTIAL ORIGIN OF HUMAN CANCER MUTATIONS. WE ANTICIPATE THAT OUR METHODS WILL AID IN MANY FUTURE STUDIES OF DNA DAMAGE AND REPAIR AND WILL HELP IDENTIFY OTHER MUTAGENIC MECHANISMS.

SEXUALLY DIMORPHIC REGULATION OF BONE MASS BY THE X-LINKED HISTONE MODIFIER KDM5C - PROJECT SUMMARY/ABSTRACT WOMEN ARE DISPROPORTIONALLY SUSCEPTIBLE TO OSTEOPOROSIS WITH GENERALLY POORER TREATMENT OUTCOMES. FOR DECADES, RESEARCH REGARDING THIS DIFFERENCE WAS FOCUSED ON THE ACTION OF SEX HORMONES. MORE RECENTLY, GENETIC, AND EPIGENETIC MECHANISMS HAVE BEEN RECOGNIZED TO SIGNIFICANTLY CONTRIBUTE TO SEX DIFFERENCES IN BONE MASS. THERE IS A CRITICAL NEED TO UNDERSTAND THE CONTRIBUTION OF GENETIC DIFFERENCES TO SEX-BIAS IN BONE MASS REGULATION, ESPECIALLY THOSE LINKED TO SEX CHROMOSOMES. WE OBSERVED THAT LOSS OF THE X-LINKED CHROMATIN MODIFYING ENZYME, KDM5C, INCREASES BONE MASS EXCLUSIVELY IN FEMALES. THE OBJECTIVE OF THIS PROPOSAL IS TO DETERMINE THE MECHANISMS BY WHICH KDM5C REGULATES FEMALE BONE MASS. WE HYPOTHESIZE THAT KDM5C TRANSCRIPTIONALLY PROGRAMS A WIDE SPECTRUM OF GENES THAT ACT SYNERGISTICALLY TO REGULATE OSTEOCLAST (OC) DIFFERENTIATION AND FUNCTION, AND THAT INHIBITION OF KDM5C WILL PREVENT AGE AND PATHOLOGY-INDUCED BONE LOSS. WE, DR. TAO YANG (MPI, EXPERT IN SKELETAL BIOLOGY) AND DR. CONNIE KRAWCZYK (MPI, EXPERT IN IMMUNOLOGY AND METABOLISM), HAVE ASSEMBLED A MOTIVATED TEAM TO ADDRESS THIS HYPOTHESIS. WE WILL FIRST INVESTIGATE HOW LOSS OF KDM5C AFFECTS OC FUNCTION, HOW OCS COMMUNICATE WITH OSTEOBLASTS (OB) VIA CROSS-TALK, AND OC ENERGY METABOLISM. WE WILL ALSO DEFINE HOW KDM5C REGULATES EPIGENETIC PROGRAMMING IN OC PROGENITORS, USING GENOMIC AND EPIGENETIC PROFILING ANALYSES. FINALLY, AS A FIRST STEP TOWARD THE DEVELOPMENT OF THERAPEUTICS, WE WILL EXAMINE THE IMPACT OF KDM5C DELETION AND PHARMACOLOGICAL INHIBITION ON ESTROGEN-DEFICIENCY INDUCED, INFLAMMATION INDUCED, OR AGE-RELATED MOUSE MODELS OF OSTEOPOROSIS. IMPACT: IN THIS STUDY, WE WILL DEFINE HOW KDM5C-MEDIATED EPIGENETIC PROGRAMMING OF OC REGULATES BONE MASS IN FEMALES. THESE RESULTS WILL HELP TO ESTABLISH A MULTIFACETED MOLECULAR NETWORK INTEGRATING EPIGENETIC STATUS, GENE EXPRESSION, METABOLISM, AND BONE MICROENVIRONMENT FOR A BETTER UNDERSTANDING OF OC PROGRAMMING AND BONE MASS REGULATION BETWEEN SEXES. THIS IS AN IMPORTANT STEP TOWARD OUR LONG-TERM GOAL TO UNCOVER THE GENETIC FACTORS AND THEIR MECHANISMS THAT CAN BE TARGETED TO IMPROVE WOMEN’S BONE HEALTH.

REGULATION OF MITOCHONDRIAL FATTY ACID SYNTHESIS IN THE CONTROL OF MITOCHONDRIAL METABOLISM. - PROJECT SUMMARY LIPIDS ARE IMPORTANT CELLULAR MACROMOLECULES THAT FORM MEMBRANES, ARE A MAJOR FORM OF ENERGY STORAGE, PLAY CRUCIAL ROLES IN CELL SIGNALING, AND ARE FREQUENTLY DYSREGULATED IN DISEASE. CELLS OBTAIN LIPIDS IN TWO MAIN WAYS: EITHER THROUGH UPTAKE FROM EXOGENOUS SOURCES OR ELSE VIA SYNTHESIS FROM ACETYL-COA IN THE CYTOPLASM BY FATTY ACID SYNTHASE (FASN). HOWEVER, CELLS HAVE EVOLUTIONARILY MAINTAINED A SECOND, COMPLETELY DISTINCT FATTY ACID SYNTHESIS PATHWAY IN THE MITOCHONDRIA (MTFAS). MITOCHONDRIA ARE WELL KNOWN AS THE “POWERHOUSE OF THE CELL.” IN DIRECT CONTRAST TO THIS WELL-KNOWN FUNCTION, THE MTFAS PATHWAY USES ACETYL-COA TO BUILD FATTY ACIDS INSTEAD OF BREAKING THEM DOWN. WHY MITOCHONDRIA HARBOR A PATHWAY THAT BUILDS FATTY ACIDS, ESPECIALLY GIVEN THAT MITOCHONDRIA TAKE UP LIPIDS FROM OTHER AREAS OF THE CELL, IS INCOMPLETELY UNDERSTOOD. IN FACT, MTFAS IS SO UNDERSTUDIED THAT THE GENES ENCODING SEVERAL STEPS IN THE PATHWAY ARE STILL UNANNOTATED. THE MAJOR GOAL OF MY RESEARCH GROUP IS TO DEFINE THE BASIC BIOLOGY OF THE MTFAS PATHWAY. I PREVIOUSLY DEFINED A MECHANISM WHEREBY MTFAS COORDINATES THREE MAJOR FUNCTIONS OF THE MITOCHONDRIA: THE TCA CYCLE, ELECTRON TRANSPORT CHAIN ASSEMBLY, AND IRON-SULFUR CLUSTER BIOGENESIS. WE HYPOTHESIZE THAT THIS COORDINATING FUNCTION OF MTFAS PROVIDES CELLS WITH A MEANS TO MONITOR MITOCHONDRIAL ACETYL-COA – THE SUBSTRATE OF THE PATHWAY – AND TO ADJUST MITOCHONDRIAL METABOLISM ACCORDINGLY. THE MECHANISMS THAT REGULATE THIS NUTRIENT SENSING FUNCTION AND MTFAS ACTIVITY IN GENERAL ARE UNDESCRIBED. THE STUDIES PLANNED IN THIS PROPOSAL WILL ADDRESS CRUCIAL GAPS IN OUR UNDERSTANDING OF HOW GENES AND METABOLITES CONTROL MTFAS PATHWAY ACTIVITY. WE WILL ALSO EXPLORE CELL-TYPE SPECIFICITY OF MTFAS PATHWAY ACTION, AND THE ROLE OF MTFAS IN CELLULAR DIFFERENTIATION SIGNALING AND CELL FATE DECISIONS. DEFINING HOW MTFAS REGULATES MITOCHONDRIAL FUNCTION IN RESPONSE TO CHANGES IN METABOLITE AVAILABILITY WILL REWRITE THE FIELD’S WORKING MODEL OF HOW MITOCHONDRIA SENSE NUTRIENTS AND ADAPT TO CHANGING NUTRITIONAL ENVIRONMENTS. I AM WELL SUITED TO ACHIEVE THESE GOALS DUE TO MY EXPERTISE IN MTFAS PATHWAY FUNCTION AND MY PRIOR RESEARCH EXPERIENCE IN MITOCHONDRIAL BIOLOGY, WHICH LIES AT THE INTERSECTION OF BIOENERGETICS, CELL SIGNALING, AND METABOLIC REGULATION. SUCCESSFUL COMPLETION OF THESE STUDIES WILL PLACE ME AND MY TRAINEES AT THE FOREFRONT OF THE FIELD OF MITOCHONDRIAL METABOLISM, AND WELL-POSITION MY LABORATORY FOR LONG-TERM SUCCESS.

EPIGENETIC MECHANISMS OF RETROTRANSPOSON SILENCING - PROJECT SUMMARY/ABSTRACT RETROTRANSPOSONS ARE ANCIENT COMPONENTS OF OUR GENETIC CODE THAT HAVE SELF-REPLICATED OVER MILLIONS OF YEARS AND NOW MAKE UP 45% OF OUR GENOME. RETROTRANSPOSON ACTIVATION HAS SERIOUS IMPLICATIONS FOR CELL VIABILITY – RETROTRANSPOSITION IS MUTAGENIC BY ITS NATURE AND EVEN EXPRESSION OF “DEAD” RETROTRANSPOSONS CAN TRIGGER INNATE IMMUNE RESPONSES THAT CAUSE INFLAMMATION. THEREFORE, THE CELL HAS EVOLVED INTRICATE EPIGENETIC MECHANISMS TO SILENCE EXPRESSION OF RETROTRANSPOSONS BY SEQUESTERING THEM IN CONSTITUTIVE HETEROCHROMATIN, WHICH IS MARKED THE EPIGENETIC MODIFICATIONS OF H3K9 METHYLATION AND DNA CPG METHYLATION. DYSREGULATION OF H3K9 METHYLATION AND CPG METHYLATION CAUSES WIDESPREAD RE-ACTIVATION OF RETROTRANSPOSONS AND OCCURS IN MULTIPLE HUMAN DISEASES. HOWEVER, VERY LITTLE IS KNOWN ABOUT THE PRECISE MOLECULAR MECHANISMS THAT INITIATE RETROTRANSPOSON SILENCING OR MAINTAIN RETROTRANSPOSONS IN A SILENT STATE. THE BROAD GOAL OF THIS RESEARCH PROGRAM IS TO DEFINE THE PRECISE MOLECULAR MECHANISMS THE CELL USES TO SUPPRESS RETROTRANSPOSON EXPRESSION. A KEY COMPONENT OF THE CELLS RETROTRANSPOSON SILENCING MACHINERY IS THE HISTONE LYSINE METHYLTRANSFERASE SETDB-1, WHICH DEPOSITS H3K9 METHYL MARKS AT RETROTRANSPOSONS. BECAUSE OF ITS CENTRAL ROLE, SETDB-1 ACTIVITY IS TIGHTLY REGULATED BY A COMPLEX CAST OF MOLECULAR PLAYERS THAT CONTROL ITS CATALYTIC ACTIVATION, RECRUITMENT TO RETROTRANSPOSON SEQUENCES AND ITS ABILITY TO SPREAD HISTONE METHYLATION ACROSS THE LENGTH OF RETROTRANSPOSONS. APPROPRIATE CONTROL OF SETDB-1 ACTIVITY IS CRITICAL FOR RETROTRANSPOSON SILENCING, BUT MANY ASPECTS OF SETDB-1 REGULATION REMAIN ENIGMATIC. WE WILL USE IN VITRO BIOCHEMICAL RECONSTITUTIONS, CRYO-EM AND GENOMICS TO DECIPHER THE PRECISE MECHANISMS THAT UNDERLY REGULATION OF SETBD-1. THE WORK OUTLINED IN THIS PROPOSAL WILL EXPLAIN HOW SETDB-1 IS CATALYTICALLY ACTIVATED FOR RETROTRANSPOSON SILENCING, HOW SETDB-1 READS THE EPIGENETIC CODE ON RETROTRANSPOSONS AND HOW SETDB-1 IS RECRUITED TO RETROTRANSPOSON SEQUENCES TO ESTABLISH H3K9 METHYLATION PATTERNS.

CANCER EPIGENETICS TRAINING (CET) PROGRAM - PROJECT SUMMARY THIS PROPOSAL SEEKS TO DEVELOP A DEDICATED CANCER EPIGENETICS TRAINING (CET) PROGRAM AT VAN ANDEL RESEARCH INSTITUTE (VARI) TO TRAIN TWO NEW POSTDOCTORAL FELLOWS PER YEAR FOR THREE YEARS EACH. THE OBJECTIVE OF THE CET IS TO PREPARE 10 POSTDOCTORAL FELLOWS TO BECOME INDEPENDENT ACADEMIC AND INDUSTRY RESEARCH LEADERS IN THE FIELD OF CANCER EPIGENETICS. THE CET PROGRAM WILL BUILD UPON THE CENTER FOR EPIGENETICS THAT WAS ESTABLISHED IN 2014 BY PETER JONES, PH.D., D.SC. (HON) AND HAS GROWN INTO A WORLD-CLASS, INTERNATIONALLY RECOGNIZED CENTER OF EXCELLENCE FOR BASIC AND TRANSLATIONAL EPIGENETICS RESEARCH. TO LAUNCH THE CET PROGRAM, WE HAVE ASSEMBLED A TEAM OF 9 MENTORS AND 6 CO-MENTORS WHO COLLECTIVELY HOLD ~$5.7M OF FUNDING PER YEAR AND HAVE HIGHLY COMPLEMENTARY AND INTERDISCIPLINARY CANCER AND EPIGENETICS EXPERTISE. CET FACULTY INCLUDE SEVERAL EPIGENETICS PIONEERS; TWO MEMBERS OF THE NATIONAL ACADEMY OF SCIENCES; THE VAN ANDEL INSTITUTE STAND UP TO CANCER EPIGENETICS DREAM TEAM (VAI-SU2C); TWO LEADERS OF THE CANCER GENOME ATLAS (TCGA) PROJECT; AND MENTORS/CO-MENTORS FROM ALL CAREER STAGES. TRAINEES WILL BE ABLE TO PURSUE BASIC AND TRANSLATIONAL RESEARCH PROJECTS IN CANCER BIOLOGY; TUMORIGENESIS, CELLULAR DEVELOPMENT AND DIFFERENTIATION; MECHANISMS GOVERNING DNA METHYLATION AND CHROMATIN REGULATION; EPIGENOMICS AND ADVANCED TOOL DEVELOPMENT; GWAS AND EWAS; TRANSGENERATIONAL INHERITANCE; STRUCTURAL BIOLOGY; METABOLISM; AND EPIGENETIC THERAPIES. THE CET PROGRAM IS OVERSEEN BY A DEDICATED ADVISORY COMMITTEE COMPOSED OF DOMAIN-LEVEL EXPERTS FROM ACADEMIC, INDUSTRIAL, AND CLINICAL CAREERS. WE HAVE DEVELOPED A TARGETED DIVERSITY AND RECRUITMENT PLAN TO INCREASE THE T32-ELIGIBLE POOL SIZE AND ATTRACT AND RETAIN INDIVIDUALS FROM UNDERREPRESENTED GROUPS. UPON ACCEPTANCE, EACH TRAINEE IS ASSIGNED AN INDIVIDUAL DEVELOPMENT TEAM (IDT) TO COACH, MENTOR, AND GUIDE THE TRAINEE THROUGH THE SCIENTIFIC AND NON-SCIENTIFIC DIMENSIONS OF CAREER DEVELOPMENT. THE FACULTY AND CET TRAINEES ARE SUPPORTED BY A DEDICATED OFFICE OF POSTDOCTORAL AFFAIRS THAT IS DIRECTED BY A PHD-LEVEL BIOMEDICAL CAREER DEVELOPMENT PROFESSIONAL. EACH TRAINEE IS REQUIRED TO COMPLETE A COMPREHENSIVE CURRICULUM OF SCIENTIFIC AND PROFESSIONAL DEVELOPMENT TRAINING. KEY DELIVERABLES AND MILESTONES FOR T32 TRAINEES INCLUDE ANNUAL SEMINARS (INTERNAL AND EXTERNAL); AT LEAST ONE SEMESTER OF TEACHING OR MENTORING EXPERIENCE; ONE FIRST-AUTHORED PUBLICATION; AND AT LEAST ONE SUBMITTED F32 (OR EQUIVALENT) AND K99/R00 APPLICATION. THE TRAINING REQUIREMENTS AND EXPECTATIONS ARE FORMALIZED WITHIN INDIVIDUAL DEVELOPMENT PLANS (IDPS) THAT ARE REVIEWED ANNUALLY BY THE ADVISORY COMMITTEE. RESEARCH-RELATED, INDIVIDUALIZED TRAINING NOT OTHERWISE DEFINED IN THE CET TRAINING PROGRAM IS PROVIDED BY VARI. THE CET WILL BE RIGOROUSLY EVALUATED THROUGH ENTRANCE, ANNUAL, EXIT, AND ALUMNI ASSESSMENTS. WITH THE SUPERLATIVE FACULTY, RESOURCES, AND GUIDANCE AFFORDED BY THE CET, POSTDOCTORAL FELLOWS WILL EMERGE WITH A COMPREHENSIVE, MULTI-DISCIPLINARY SKILL SET, PERSPECTIVE, AND FOUNDATION FROM WHICH TO ESTABLISH THEIR INDEPENDENT RESEARCH CAREERS IN CANCER EPIGENETICS.

VISUALIZING THE EFFECT OF EPIGENETIC MODIFICATIONS ON TRANSCRIPTION AND MUTATION

CIRCSATB2, A NOVEL REGULATOR OF ARSENIC-INDUCED CARCINOGENESIS - ABSTRACT INORGANIC ARSENIC (IAS) IS AN ENVIRONMENTALLY UBIQUITOUS TOXIN THAT AFFECTS >100 MILLION PEOPLE WORLDWIDE, WITH HEALTH CONSEQUENCES RANGING FROM ACUTE TOXICITY TO CANCER. WHILE IAS EXPOSURE CAN GENERATE REACTIVE OXYGEN SPECIES THAT DAMAGE DNA, A SIGNIFICANT FRACTION OF IAS-INDUCED DISEASE CANNOT BE ATTRIBUTED TO DNA LESIONS. WE PREVIOUSLY SHOWED THAT IAS EXPOSURE REMODELS DNA AND HISTONE EPIGENETIC MARKS IN IAS-INDUCED ONCOGENESIS, AND NOW KNOW THAT IAS EXPOSURE ALSO ALTERS HIGHER-ORDER (3-DIMENSIONAL, OR 3D) CHROMATIN STRUCTURE. ONE OF THOSE STRUCTURES INVOLVES A DYNAMIC LINK BETWEEN CHROMATIN AND THE INNER NUCLEAR MATRIX, WHICH BRINGS DISTAL GENES INTO PROXIMITY WITH ONE ANOTHER. THIS LINK IS MAINTAINED BY THE SATB PROTEINS; IS NECESSARY FOR PROPER GENE REGULATION DURING CELLULAR DIFFERENTIATION; AND IS DISRUPTED BY IAS EXPOSURE. SATB1 IS A KNOWN ONCOGENE, WHEREAS SATB2 CAN EITHER ACT AS AN ONCOGENE OR TUMOR SUPPRESSOR DEPENDING ON THE CELL TYPE AND GENE ENVIRONMENT. WE RECENTLY DISCOVERED THAT DURING IAS-INDUCED ONCOGENESIS, SATB2 IS UPREGULATED AND PRODUCES A NOVEL, CIRCULAR RNA (CIRCSATB2) THAT “PROTECTS” LINEAR SATB2 MRNA FROM DEGRADATION, POSSIBLY BY INTERFERING WITH A MICRORNA (MIR31). THE CIRCSATB2 RNA IS TRANSLATED INTO A PEPTIDE (CIRCSATB2P), AND THIS PEPTIDE ALTERS THE NATIVE SATB2 INTERACTOME AND GENE EXPRESSION PROFILE. THESE DATA SUPPORT THE IDEA THAT TRANSCRIPTIONAL AND POST-TRANSLATIONAL, TEMPORAL CONTROL OVER SATB2 AND CHROMATIN HIGHER-ORDER STRUCTURES ARE CRITICAL FOR COORDINATING NORMAL CELLULAR DIFFERENTIATION. THEY SUGGEST THAT SATB2 IS REGULATED BY A NOVEL CIRCRNA-MIRNA- MRNA REGULATORY AXIS. THE OBJECTIVE OF THIS PROJECT IS THEREFORE TO DETERMINE EXACTLY HOW IAS EXPOSURE REGULATES SATB2 EXPRESSION AND ACTIVITY TO PROMOTE ONCOGENESIS. IN AIM 1, WE WILL TEST HOW SATB2 IS REGULATED TRANSCRIPTIONALLY (VIA THE CIRCRNA-MIR-MRNA PATHWAY) AND POST-TRANSCRIONALLY (VIA REGULATION OF THE PROTEIN). IN AIM 2, WE WILL TEST HOW CIRCSATB2P AND SATB2 DIFFERENTIALLY REGULATE THE 3D GENOME ORGANIZATION AND NUCLEAR INTEGRITY, AND IN AIM 3, WE WILL TEST THE ROLE OF CIRCSATB2 IN METASTASIS AND TUMORIGENESIS. THESE STUDIES WILL IDENTIFY THE CIRCSATB2-MIRRNA-SATB2 GENE REGULATORY AXIS, AND ESTABLISH THAT A NOVEL SATB2 ISOFORM SWITCH DRIVES IAS-INDUCED ONCOGENESIS. THE MECHANISTIC UNDERSTANDING OF HOW IAS AFFECTS 3D CHROMATIN STRUCTURE AND NUCLEAR INTEGRITY WILL ELUCIDATE KEY (AND NOVEL) PROCESSES IN CHROMATIN BIOLOGY, AND CONTRIBUTE TO A FRAMEWORK FOR DEVELOPING DIAGNOSTICS AND TARGETED THERAPEUTICS FOR IAS-INDUCED DISEASE.

MOLECULAR MECHANISM OF PRION STRAIN

HARNESSING THE THERAPEUTIC POTENTIAL OF HISTOTRIPSY FOCUSED ULTRASOUND-INDUCED IMMUNOGENIC CANCER CELL DEATH - PROJECT SUMMARY/ABSTRACT IMMUNOTHERAPY CAN BE HIGHLY EFFECTIVE AGAINST CANCERS THAT ELICIT SOME RECOGNITION FROM THE IMMUNE SYSTEM (E.G., MELANOMA), BUT IT REMAINS INEFFECTUAL AGAINST CANCERS THAT ARE LARGELY INVISIBLE TO IMMUNE DETECTION (E.G., PANCREATIC CANCER). ADVANCES IN CANCER IMMUNOTHERAPY WILL REQUIRE INTERVENTIONS THAT CAN MAKE CANCERS MORE APPARENT TO THE IMMUNE SYSTEM. A GROWING BODY OF EVIDENCE SUGGESTS THAT FOCUSED ULTRASOUND (FUS) TUMOR ABLATION COULD BE THAT INTERVENTION. BY NON-INVASIVELY DISRUPTING CANCER CELLS, THERMAL AND MECHANICAL MODES OF FUS HAVE BEEN SHOWN TO TRIGGER SURPRISINGLY POTENT IMMUNE RESPONSES AGAINST TUMORS. WE HAVE FOUND THAT HISTOTRIPSY, A NON-THERMAL MODE OF MECHANICAL FUS, STIMULATES A POWERFUL AND SYSTEMIC ANTI-TUMOR IMMUNE RESPONSE STRONG ENOUGH TO CAUSE ABSCOPAL REGRESSION OF DISTANT, NON-ABLATED TUMOR SITES – EFFECTS NOT GENERALLY SEEN WITH TRADITIONAL THERAPIES LIKE RADIATION OR THERMAL ABLATION. HISTOTRIPSY IS A TECHNOLOGY THAT IS PRESENTLY BEING INTRODUCED INTO CLINICAL USE; THEREFORE, IT WILL BE IMPERATIVE TO UNDERSTAND THE MECHANISTIC UNDERPINNINGS OF HISTOTRIPSY IMMUNOSTIMULATION. OUR PRELIMINARY STUDIES POINT TO A STEPWISE SERIES OF EVENTS THAT MAY EXPLAIN THIS PHENOMENON. FIRST, HISTOTRIPSY CAUSES THE RELEASE OF SUBCELLULAR CANCER CELL ANTIGENS IN A MANNER THAT PRESERVES THEIR IMMUNOGENIC INTEGRITY. SECOND, HISTOTRIPSY INDUCES CANCER CELLS TO UNDERGO A SPECIFIC PATHWAY OF CELLULAR SUICIDE CALLED NECROPTOSIS – A DEATH PATHWAY THAT ATTRACTS INFLAMMATION AND IMMUNE ATTENTION, EFFECTIVELY PRIMING THE IMMUNE SYSTEM TO RECOGNIZE CANCER ANTIGENS. WHAT FOLLOWS IS A PROGRESSIVE INFILTRATION OF CD8+ T CELLS INTO DISTANT TUMORS THAT IS ACCOMPANIED BY ANOTHER PATHWAY OF CANCER CELL DEATH CALLED FERROPTOSIS – A DEATH PATHWAY RECENTLY DISCOVERED TO BE THE CRITICAL MECHANISM BY WHICH IMMUNOTHERAPY-PRIMED CD8+ T CELLS KILL CANCER CELLS. IN THIS PROPOSAL, WE WILL RETRACE THESE STEPS TO UNDERSTAND HOW HISTOTRIPSY EXERTS ITS UNUSUALLY POTENT IMMUNE EFFECTS. FIRST, WE WILL QUANTITATIVELY FINE-TUNE THE PARAMETERS OF HISTOTRIPSY TISSUE CAVITATION THAT CAUSE MAXIMALLY IMMUNOGENIC TUMOR ANTIGEN RELEASE. NEXT, WE WILL FOCUS ON THE EARLY INDUCTION OF NECROPTOSIS TO DETERMINE IF THIS IS A NECESSARY LOCAL PRECURSOR EVENT ON WHICH LATER MANIFESTATIONS OF HISTOTRIPSY IMMUNOSTIMULATION DEPEND. THEN, WE WILL EXAMINE THE LATER PROCESS OF CD8+ T CELL-DRIVEN FERROPTOSIS TO DETERMINE IF THIS IS THE MECHANISM BY WHICH THE DISTANT, ABSCOPAL EFFECTS OF HISTOTRIPSY ARE MEDIATED. FINALLY, WE WILL LEVERAGE MECHANISTIC INSIGHTS GAINED FROM THESE INVESTIGATIONS TO DEVELOP AND TEST POTENTIAL PRECLINICAL STRATEGIES WITH WHICH THE EFFECTS OF HISTOTRIPSY ON TUMOR ANTIGEN RELEASE, NECROPTOSIS, AND FERROPTOSIS COULD BE MAXIMIZED FOR CANCER IMMUNOTHERAPY. WE HAVE ASSEMBLED A MULTIDISCIPLINARY TEAM WITH EXPERTISE IN FUS, IMMUNOLOGY AND CANCER IMMUNOTHERAPY TO PURSUE THIS WORK, WHICH PROMISES TO REVEAL INSIGHTS AND STRATEGIES TO BRING THE IMPACT OF CANCER IMMUNOTHERAPY TO A WIDER RANGE OF PATIENTS IN NEED OF CURE.

5-HYDROXYMETHYLCYTOSINE IN HUMAN CANCER

MECHANISMS AND APPLICATIONS OF RNA-MEDIATED ENZYMES IN TRANSLATION AND IMMUNITY - PROJECT SUMMARY DESCRIPTION: RNA-GUIDED, OR RIBONUCLEOPROTEIN PARTICLE (RNP), ENZYMES COMPRISE AN EMERGING CLASS OF MOLECULES THAT FUNCTION IN A WIDE RANGE OF BIOLOGICAL PATHWAYS. UNLIKE MOST OF THE PROTEIN-BASED ENZYMES, RNP ENZYMES RELY ON THE GUIDE RNA TO SECURE THE SUBSTRATES AND ON THE PARTNER PROTEINS TO CATALYZE THE CHEMICAL REACTIONS. THE RNA-GUIDED TARGETING MECHANISM HAS CREATED UNPRECEDENTED OPPORTUNITIES IN GENOME MANIPULATION, GENE REGULATION AND NUCLEIC ACID DETECTION. RESEARCH IN THE LI LABORATORY EMPLOYS STRUCTURAL BIOLOGY AND COMPLEMENTARY METHODS TO UNCOVER THE THEORETICAL BASIS FOR THEIR BIOLOGICAL FUNCTION AND EFFECTIVE USE AS RESEARCH, DIAGNOSTIC AND THERAPEUTIC TOOLS. IN THE PROPOSED FUNDING PERIOD, THE LI LABORATORY WILL TACKLE THE MECHANISMS OF THE RNP ENZYMES TO ENABLE THEIR APPLICATIONS. THESE INCLUDE THE METHYLATION- SENSITIVE CLUSTERED, REGULARLY INTERSPACED, SHORT PALINDROMIC REPEATS (CRISPR)-CAS9, THE MULTI-FUNCTIONAL TYPE III CRISPR EFFECTORS AND THEIR ASSOCIATED SIGNALING PROCESSES, AND THE SMALL NUCLEOLAR RNPS (SNORNPS) RESPONSIBLE FOR CHEMICAL MODIFICATIONS OF RIBOSOMAL RNA. THOUGH UNRELATED IN ENZYME COMPOSITION OR BIOLOGICAL PATHWAY, THESE RNP ENZYMES ARE GOVERNED BY A SIMILAR SET OF PHYSICOCHEMICAL PRINCIPLES AND SHARE THE GREAT POTENTIAL IN THERAPEUTIC AND DIAGNOSTIC INNOVATIONS BY REPROGRAMMING THE CHEMISTRY OF CELLULAR NUCLEIC ACIDS. THE PROPOSED RESEARCH WILL ASSESS THE FEASIBILITY OF ENGINEERING AND APPLYING THE EPIGENETIC DNA- SPECIFIC CAS9, THE UTILITY OF RAPID VIRUS DETECTION WITH THE CRISPR TYPE III EFFECTORS, AND THE POSSIBILITY OF TUNNING CELLULAR FUNCTIONS THROUGH CHEMICAL MODIFICATIONS OF RNA. THE LI LABORATORY HAS ASSEMBLED A TEAM OF SCIENTISTS WITH COMPLEMENTARY EXPERTISE IN MICROBIOLOGY, MAMMALIAN GENOME BIOLOGY, YEAST GENETICS, SINGLE MOLECULE BIOPHYSICS, VIROLOGY, VIRUS DETECTION, X-RAY CRYSTALLOGRAPHY, AND HIGH-THROUGHPUT CRYOGENIC ELECTRON MICROSCOPY TO MAXIMIZE THE IMPACT WHILE MITIGATING THE RISKS OF THE RESEARCH. THE GOAL OF THIS TRANSFORMATIVE PROPOSAL IS TO DEMONSTRATE AND ENABLE THE APPLICABILITY OF THE RNA-MEDIATED ENZYMES IN A WIDE RANGE OF BIOMEDICAL FIELDS.

A TRIM28-ERV AXIS DRIVES PHENOTYPIC VARIATION IN OBESITY - MODIFIED PROJECT SUMMARY/ABSTRACT SECTION ESTIMATES FROM TWIN AND INBRED ANIMAL MODELS SUGGEST THAT UP TO 50% OF METABOLIC DISEASE HETEROGENEITY IS DUE TO SOMETHING OTHER THAN GENETICS OR THE ENVIRONMENT. WE PREVIOUSLY DISCOVERED THAT TRIM28 IS A MASTER REGULATOR OF NON-GENETIC, NON-ENVIRONMENTAL PHENOTYPIC HETEROGENEITY; DEMONSTRATED THAT TRIM28 BUFFERS AGAINST A UNIQUE (BISTABLE) EPIGENETIC OBESITY IN ISOGENIC TRIM28+/D9 MICE; AND IDENTIFIED A COMPARABLE TRIM28-ASSOCIATED MRNA EXPRESSION SIGNATURE IN HUMAN OBESITY. INDIVIDUALS AT THE OBESE, EPIGENETICALLY “ON” END OF THE SPECTRUM ARE CHARACTERIZED BY DYSREGULATION OF TRIM28-SENSITIVE LOCI, INCLUDING ENDOGENOUS RETROVIRUSES (ERVS). TRIM28-NORMALLY INTERACTS WITH KZFPS TO KEEP ERVS IN A STABLE, FULLY SILENCED STATE THROUGHOUT DEVELOPMENT. HOWEVER, A SUBSET OF ERVS PARTIALLY ESCAPE THIS SILENCING AND BECOME VARIABLY METHYLATED (VM-ERVS), WHICH TRANSLATES INTO VARIABLE LEVELS OF REPRESSION/ACTIVATION, SUBSEQUENT VARIABILITY IN NEIGHBORING GENE EXPRESSION, AND ULTIMATELY PHENOTYPIC VARIATION. OUR PRELIMINARY DATA INDICATE THAT VM-ERVS HAVE SEQUENCE CONTENT ALLOWING THEM TO BE BOUND BY ZINC-FINGER-CXXC (ZF-CXXC) DOMAIN CONTAINING PROTEINS, THEREBY PROTECTING THESE LOCI FROM METHYLATION. WE HYPOTHESIZE THAT ACTIVATION VS SILENCING AT VM-ERV LOCI IS CONTROLLED BY A COMPETITION BETWEEN KZFP/TRIM28 AND ZF-CXXC BINDING, AND THAT TRIM28-SENSITIVE VM-ERV EXPRESSION IS A SIGNIFICANT DRIVER OF UNEXPLAINED PHENOTYPIC VARIATION AND METABOLIC DISEASE HETEROGENEITY. WE WILL TEST THESE HYPOTHESES THROUGH THREE INDEPENDENT AIMS. IN AIM 1, WE WILL DEFINE THE EPIGENOMIC CHARACTERISTICS OF ERVS IN HEPATOCYTES AND ADIPOCYTES TO IDENTIFY THE EPIGENOMIC AND GENOMIC FEATURES THAT DEFINE VM-ERVS. IN AIM 2, WE WILL PROFILE BINDING OF SPECIFIC ZF-CXXC PROTEINS (CFP1 AND TET1) TO PROVE THAT THESE PROTEINS GAIN A COMPETITIVE BINDING ADVANTAGE AS TRIM28 LEVELS ARE REDUCED AND DEMONSTRATE THAT ZF-CXXC – KZFP/TRIM28 COMPETITION TRIGGERS EPIGENETIC VARIABILITY AT VM-ERVS. IN AIM 3, WE WILL DETERMINE IF VM-ERV ACTIVATION IS COUPLED TO METABOLIC DISEASE HETEROGENEITY. WE EXPECT THIS PROJECT WILL IDENTIFY TRIM28/ZF-CXXC COMPETITION AS A NOVEL MECHANISM GOVERNING THE VARIABLE SILENCING AT ERVS AND PROVIDE THE RESEARCH COMMUNITY WITH A COMPREHENSIVE LIST OF TARGET PROTEINS AND PATHWAYS TO STUDY AND BEGIN UNDERSTANDING HOW VM-ERV DYSREGULATION TRIGGERS AND/OR INFLUENCES COMPLEX DISEASE ETIOLOGY.

DISCOVERY OF SMALL MOLECULES TARGETING THE HISTONE ACETYLATION READER ENL - PROJECT SUMMARY THE MIXED-LINEAGE LEUKEMIA (MLL) GENE REARRANGEMENTS ACCOUNT FOR APPROXIMATELY 80% OF INFANT ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) AND 35-50% OF INFANT ACUTE MYELOID LEUKEMIA (AML). PATIENTS BEARING REARRANGEMENTS OF THE MLL GENE ARE ASSOCIATED WITH DISMAL PROGNOSIS AND PARTICULARLY POOR RESPONSE TO STANDARD TREATMENTS. UP-TO-DATE, EFFECTIVE THERAPIES FOR THIS SUBTYPE OF FATAL DISEASE ARE STILL LACKING. MOLECULARLY, INTER-CHROMOSOMAL TRANSLOCATIONS OF MLL LEAD TO IN FRAME FUSIONS OF THE N-TERMINUS OF MLL TO THE C-TERMINUS OF VARIOUS FUSION PARTNERS, WHICH ARE KNOWN AS THE “DRIVER” LESIONS OF THE DISEASES. AMONG MORE THAN 70 MLL FUSION PARTNERS, A SMALL SUBSET OF FUSIONS ACCOUNT FOR MOST LEUKEMOGENIC CASES. IN ALL, OVER 90% MLL REARRANGEMENTS INVOLVE ONLY FOUR FUSION PARTNERS: AFF1, AF9, ENL, AND AF10, ALL ARE COMPONENTS OF THE SUPER ELONGATION COMPLEX (SEC) OR THE COMPLEX OF THE HISTONE H3K79 METHYLTRANSFERASE DOT1L. IT IS BELIEVED THAT THESE MLL FUSIONS SHARE A COMMON PATHWAY BY “HIJACKING” SEC OR THE DOT1L COMPLEX TO PROMOTE ABERRANT ACTIVATION OF THE TARGET GENES OF MLL FUSIONS, LEADING TO THE PATHOGENESIS OF LEUKEMIAS. STUDIES FROM THE APPLICANT AND OTHERS HAVE DEMONSTRATED THAT ENL, A STOICHIOMETRIC COMPONENT OF SEC AND THE DOT1L COMPLEX, IS CRITICAL FOR THE ONCOGENIC FUNCTION OF THE MLL-FUSIONS. ENL CONTAINS AN EVOLUTIONALLY CONSERVED YEATS DOMAIN. THE APPLICANT FOUND THAT THE YEATS DOMAIN OF ENL FUNCTIONS AS A READER OF HISTONE ACETYLATION (CELL, 2014, 159:558-71). IMPORTANTLY, THE ACETYLATION READING FUNCTION OF THE YEATS DOMAIN IS ESSENTIAL FOR GROWTH AND SURVIVAL OF THE MLL-REARRANGED LEUKEMIC CELLS (NATURE, 2017, 543:265-269). THESE KEY FINDINGS PROVIDE THE PROOF OF CONCEPT THAT TARGETING THE YEATS DOMAIN OF ENL IS A POTENTIALLY VALUABLE THERAPEUTIC OPTION IN TREATMENT OF MLL-REARRANGED LEUKEMIAS. THE GOAL OF THIS PROPOSAL WITHIN THE SCOPE OF THIS FOA IS TO DEVELOP POTENT AND SELECTIVE INHIBITORS OF THE ENL YEATS DOMAIN. THE SPECIFIC AIMS OF THE PROPOSAL ARE TO (1) CONDUCT A HIGH-THROUGHPUT SCREENING FOR ENL SMALL-MOLECULE INHIBITORS; (2) VALIDATE AND EVALUATE CANDIDATE HITS BY ORTHOGONAL ASSAYS; AND (3) CHARACTERIZE HITS IN CELL-BASED ASSAYS. THE INHIBITORS OBTAINED FROM THE PROPOSED STUDY WILL SERVE AS TOOL COMPOUNDS TO STUDY THE FUNCTIONS AND MECHANISMS OF ENL IN PROMOTING MLL-FUSION PROTEINS IN GENE REGULATION AND DISEASE MAINTENANCE. THESE COMPOUNDS WILL ALSO PROVIDE THE BASIS FOR FURTHER DEVELOPMENT OF SMALL MOLECULES FOR TARGETED THERAPIES OF MLL-TRANSLOCATED LEUKEMIAS. PRECLINICAL STUDIES SUGGEST THAT BET INHIBITORS EXHIBIT LIMITED EFFICACY AS SINGLE AGENTS. SELECTIVE AND POTENT ENL INHIBITORS PROVIDED BY THIS PROJECT WILL BE AN IMPORTANT TOOL TO TEST THE SYNERGISTIC EFFECT OF ENL AND BET INHIBITIONS, PROVIDING AN INNOVATIVE THERAPEUTIC STRATEGY FOR THE TREATMENT OF MLL-REARRANGED LEUKEMIAS.

MK-STYX: A REQUISITE GATEKEEPER TO MITOCHONDRIAL FUNCTION AND DEATH

HIGH THROUGHPUT SCREEN FOR INHIBITORS OF THE YEATS2 HISTONE ACYLATION READER - PROJECT SUMMARY LUNG CANCER IS THE LEADING CAUSE OF CANCER-RELATED DEATHS IN BOTH MEN AND WOMEN IN THE U.S AND WORLDWIDE. THERE ARE MORE THAN 400,000 DEATHS OF LUNG CANCER WORLDWIDE EACH YEAR, AMONG WHICH LUNG SQUAMOUS CELL CARCINOMA (LUSC) ACCOUNTS FOR ABOUT 30%. HOWEVER, NO EFFECTIVE TREATMENTS FOR LUSC ARE AVAILABLE. LUSC IS ONE TYPE OF NON-SMALL CELL LUNG CANCER (NSCLC) CHARACTERIZED BY NUMEROUS DNA ALTERATIONS, INCLUDING FREQUENT AMPLIFICATION OF THE 3Q26 CHROMOSOMAL SEGMENT. THE 3Q26 SEGMENT IS NOTEWORTHY BECAUSE IT CONTAINS THE YEATS DOMAIN CONTAINING 2 (YEATS2) GENE, A GENE THAT IS FREQUENTLY AMPLIFIED IN A NUMBER OF HUMAN CANCERS, INCLUDING LUSC (~50%), OVARIAN (28%), HEAD AND NECK (25%), AND ESOPHAGUS CANCERS (25%). HIGH YEATS2 MRNA EXPRESSION IS ASSOCIATED WITH A POOR PROGNOSIS OF NSCLC PATIENTS, INDICATING THAT YEATS2 MAY HAVE A TUMOR-PROMOTING ROLE. YEATS2 IS A STOICHIOMETRIC SUBUNIT OF THE ADA-TWO-A-CONTAINING (ATAC) COMPLEX, A CONSERVED METAZOAN HISTONE ACETYLTRANSFERASE (HAT) COMPLEX. YEATS2 CONTAINS AN EVOLUTIONALLY CONSERVED YEATS DOMAIN. WE PREVIOUSLY SHOWED THAT THE YEATS DOMAIN OF YEATS2 FUNCTIONS AS A READER OF HISTONE ACETYLATION AND OTHER TYPES OF HISTONE ACYLATION SUCH AS CROTONYLATION. IMPORTANTLY, DISRUPTING THE YEATS HISTONE READING ACTIVITY IMPAIRS THE NORMAL FUNCTIONS OF YEATS2 AND THE ATAC COMPLEX, RESULTING IN REDUCED HISTONE ACETYLATION, DECREASED TARGET GENE EXPRESSION, AND INHIBITION OF CELL GROWTH AND SURVIVAL OF NSCLC. THESE DATA DEMONSTRATE THAT THE YEATS DOMAIN OF YEATS2 IS A POTENTIAL DRUG TARGET, AND THAT TARGETING YEATS2 MAY PROVIDE A THERAPEUTIC APPROACH FOR TREATING NSCLC AND OTHER TYPES OF CANCER CHARACTERIZED BY YEATS2 AMPLIFICATION. THE OBJECTIVE OF THIS PROPOSAL IS TO DEVELOP POTENT AND SPECIFIC INHIBITORS TARGETING THE HISTONE ACYLATION BINDING ACTIVITY OF THE YEATS DOMAIN OF YEATS2. FOR THIS, WE WILL (1) CONDUCT A HIGH-THROUGHPUT SCREEN TO IDENTIFY YEATS2 SMALL-MOLECULE INHIBITORS, AND (2) EVALUATE AND CHARACTERIZE HIT COMPOUNDS IN IN VITRO AND CELL-BASED ASSAYS. THROUGH THE PROPOSED STUDIES, WE EXPECT TO IDENTIFY POTENT, SPECIFIC YEATS2 YEATS DOMAIN CHEMICAL PROBES OF DIFFERENT CHEMOTYPES. THESE COMPOUNDS WILL PROVIDE THE BASIS FOR FURTHER DEVELOPMENT OF SMALL MOLECULES FOR TARGETED THERAPIES. LIKEWISE, THE RESEARCH COMMUNITY WILL BE ABLE TO USE THESE NEW INHIBITORS AS IMPORTANT TOOLS TO UNDERSTAND THE FUNCTIONS AND MECHANISMS OF YEATS2 IN HUMAN CANCERS.

IDENTIFYING MICROBIAL MECHANISMS THAT REGULATE ANIMAL INSULIN SIGNALING - PROJECT SUMMARY INSULIN RESISTANCE AND TYPE 2 DIABETES AFFECT NEARLY 10% OF THE POPULATION, AND ARE ON THE RISE. RECENT EVIDENCE SUGGESTS THAT HUMAN INTESTINAL BACTERIA CAN REGULATE INSULIN RELEASE, WHICH IMPLIES THAT MICROORGANISMS HARBOR NOVEL MECHANISMS OR FACTORS THAT INFLUENCE INSULIN SIGNALING. IDENTIFYING MICROORGANISMS AND NOVEL BACTERIAL MECHANISMS OF MODULATING ANIMAL INSULIN RELEASE IN VIVO IS VERY CHALLENGING IN MAMMALS BECAUSE OF THE EXTENSIVE MICROBIAL DIVERSITY IN THE MAMMALIAN INTESTINAL MICROBIOTA, AND BECAUSE RESIDENT MICROORGANISMS ARE RESISTANT TO COLONIZATION BY ARTIFICIALLY ADMINISTERED ISOLATES. I HAVE OVERCOME THESE LIMITATIONS BY DEVELOPING A NOVEL, HIGH-THROUGHPUT C. ELEGANS MODEL FOR IDENTIFYING BACTERIAL ISOLATES THAT REGULATE IN VIVO INSULIN SIGNALING, AND DEMONSTRATED (FOR THE FIRST TIME) THAT ENVIRONMENTAL BACTERIA CONTAIN PREVIOUSLY UNDISCOVERED, NOVEL MECHANISMS OR FACTORS FOR REGULATING ANIMAL INSULIN RESISTANCE. IN THIS TRANSFORMATIVE PROJECT, I WILL PERFORM THE FIRST-EVER, LARGE-SCALE SCREEN FOR BIOACTIVE BACTERIA THAT MODIFY ANIMAL INSULIN SIGNALING; USE TRANSPOSON MUTAGENESIS TO IDENTIFY THOSE BACTERIAL GENES OR FACTORS RESPONSIBLE FOR REGULATING INSULIN SIGNALING; AND ENGINEER AT LEAST ONE HUMAN PROBIOTIC STRAIN TO EXPRESS A BACTERIAL INSULIN-REGULATING SYSTEM OR FACTOR. TAKEN TOGETHER, THIS PROJECT WILL GENERATE NEW INSIGHTS INTO THE TYPES OF BACTERIA THAT MODIFY ANIMAL INSULIN SIGNALING; THE MECHANISMS BACTERIA HAVE EVOLVED TO DO SO; AND PROVIDE THE FIELD WITH FUNDAMENTALLY NEW DIRECTIONS AND STRATEGIES FOR ALLEVIATING ONE OF THE MOST COMMON AND IMPACTFUL HUMAN PATHOLOGIES IN EXISTENCE.

THE IMMUNOBIOLOGICAL MECHANISMS OF DEPRESSION IN PERIMENOPAUSE - PROJECT SUMMARY/ABSTRACT THIS PROJECT AIMS TO IDENTIFY IMMUNOBIOLOGICAL MECHANISMS UNDERLYING THE DEVELOPMENT OF DEPRESSION DURING THE PERIMENOPAUSE, IN RESPONSE TO THE NIMH FUNDING OPPORTUNITY “MOOD AND PSYCHOSIS SYMPTOMS DURING THE MENOPAUSE TRANSITION”. DURING THE PERIMENOPAUSE, THE RISK FOR RECURRENCE OF DEPRESSION IS INCREASED 13-FOLD AMONG WOMEN WHO PREVIOUSLY HAD DEPRESSION. MOREOVER, PERIMENOPAUSE OVERLAPS WITH THE AGE SPAN EXHIBITING THE HIGHEST SUICIDE RATE FOR WOMEN. THE PERIMENOPAUSE CAN LAST FOR SEVERAL YEARS AND STARTS WHEN ESTROGEN AND PROGESTERONE LEVELS BEGIN TO FLUCTUATE, GRADUALLY DECLINING. SEX HORMONES ARE POWERFUL MODULATORS OF THE IMMUNE RESPONSE AND WE THEREFORE PROPOSE THAT CHANGES IN LYMPHOCYTES PLAY A CENTRAL ROLE IN DEPRESSION DURING THIS TIME. NOTABLY, OUR PREVIOUS WORK HAS DEMONSTRATED DEFICIENT REGULATORY T-CELL RESPONSES IN WOMEN WITH POST- PARTUM DEPRESSION AND IDENTIFIED A SPECIFIC PRO-INFLAMMATORY PROFILE (COMPOSED OF CYTOKINES AND TRYPTOPHAN METABOLITES) THAT PREDICTS DEPRESSION DURING PREGNANCY. IMPORTANTLY, OUR NEW PILOT DATA DERIVED FROM WOMEN WITH SEVERE DEPRESSION IN THE PERIMENOPAUSAL AGE SPAN REVEALS ALTERATIONS IN TRYPTOPHAN METABOLITES THAT CONTROL T- CELL RESPONSES AS WELL AS IN THE METHYLATION OF GENES INVOLVED IN T-CELL FATE AND CHROMATIN REMODELING. TAKEN TOGETHER, THESE DATA SUPPORT OUR HYPOTHESIS THAT THE PERIMENOPAUSAL HORMONAL SHIFTS INDUCE A PRO-INFLAMMATORY STATE, REGULATED BY CHROMATIN MODIFICATIONS IN T-CELLS, THAT TRIGGERS DEPRESSION IN VULNERABLE WOMEN. TO TEST THIS HYPOTHESIS, WE WILL ENROLL 150 PERIMENOPAUSAL WOMEN IN A MULTI-SITE STUDY TO: 1) DETERMINE PLASMA TRYPTOPHAN METABOLITE AND CYTOKINE PATTERNS IN PERIMENOPAUSAL DEPRESSION; 2) DEFINE EPIGENETIC MECHANISMS IN LYMPHOCYTES THAT CONFER VULNERABILITY TO DEVELOPING DEPRESSION IN PERIMENOPAUSE; AND 3) IDENTIFY INFLAMMATORY AND METABOLIC MARKERS THAT NORMALIZE IN A SUBSET OF 60 WOMEN RECOVERING FROM DEPRESSION (RE-ASSESSED 8 WEEKS AFTER THEIR INITIAL VISIT), EITHER TREATED WITH HORMONAL SUBSTITUTION THERAPY OR UNTREATED. WE WILL UNDERTAKE CAREFUL CLINICAL DIAGNOSTICS AND CONFIRM PERIMENOPAUSAL STATUS USING ESTABLISHED CRITERIA. IN ALL PARTICIPANTS, WE WILL QUANTIFY PLASMA TRYPTOPHAN METABOLITES, CYTOKINES, AND SEX HORMONES. WE WILL ALSO DEFINE PERIPHERAL IMMUNE CELL POPULATIONS AND DETERMINE CHANGES IN DNA-METHYLATION, CHROMATIN ACCESSIBILITY AND GENE EXPRESSION IN NAÏVE T-CELLS. FINALLY, WE WILL TEST THE FUNCTIONAL RESPONSE TO INFLAMMATORY STIMULATION IN CULTURED LYMPHOCYTES DERIVED FROM THE SAME WOMEN. WE EXPECT THAT WOMEN WITH PERIMENOPAUSAL DEPRESSION WILL EXHIBIT REDUCED LEVELS OF IMMUNOREGULATORY TRYPTOPHAN METABOLITES AND SIGNS OF PERIPHERAL INFLAMMATION AS WELL AS DIFFERENCES IN TRANSCRIPTIONAL REGULATION IN LYMPHOCYTES. WE PREDICT THAT THESE DISTINCT INFLAMMATORY RESPONSES WILL BE LINKED TO THE DEVELOPMENT OF DEPRESSION. WE ALSO EXPECT IMPROVEMENT OF DEPRESSIVE SYMPTOMS AND REVERSAL OF IMMUNOBIOLOGICAL MARKERS IN WOMEN RECEIVING HORMONAL THERAPY. WE ENVISION THAT OUR PROJECT WILL LEAD TO AN IMPROVED UNDERSTANDING OF THE MOLECULAR UNDERPINNINGS OF DEPRESSION DURING PERIMENOPAUSE, AIDING THE FUTURE IDENTIFICATION OF BIOMARKERS OF WOMEN AT RISK AS WELL AS THE DEVELOPMENT OF MORE EFFECTIVE TREATMENTS.

STRUCTURAL BASIS OF EUKARYOTIC REPLICATION INITIATION BY CRYO-EM

MITOCHONDRIAL OXIDATIVE METABOLISM IS A CRITICAL DETERMINANT OF MUSCLE SATELLITE CELL FATE & FUNCTION - MUSCLE STEM CELLS (MUSCS) GIVE SKELETAL MUSCLE THE REMARKABLE ABILITY TO COMPLETELY REGENERATE FOLLOWING INJURY AND PLAY AN IMPORTANT ROLE IN TISSUE MAINTENANCE OVER TIME UNDER NORMAL CONDITIONS. DECREASES IN MUSC NUMBER AND FUNCTIONALITY HAVE BEEN IMPLICATED IN AGING-ASSOCIATED MUSCLE WASTING AND SEVERAL FORMS OF MUSCULAR DYSTROPHY. IN RESPONSE TO INJURY, QUIESCENT MUSCS ARE ACTIVATED, PROLIFERATE TO REFILL THE MUSCLE NICHE, AND DIFFERENTIATE INTO TERMINALLY DIFFERENTIATED, CONTRACTILE MYOFIBERS. ACROSS THIS PROCESS, DIFFERENTIATING MUSCS UNDERGO EXTENSIVE METABOLIC REPROGRAMMING THAT ENTAILS A SWITCH TO OXIDATIVE PHOSPHORYLATION AND SUBSTANTIAL MITOCHONDRIAL BIOGENESIS, AN EFFECT THAT HAS LONG BEEN ASSUMED TO BE NECESSARY BECAUSE OF THE MASSIVE ATP DEMAND OF MUSCLE CONTRACTION. ACCORDINGLY, MITOCHONDRIAL IMPAIRMENTS BLOCK MYOGENIC DIFFERENTIATION, THOUGH THE MOLECULAR MECHANISM(S) UNDERLYING THIS BLOCKADE ARE NOT UNDERSTOOD. MITOCHONDRIA ARE HUBS OF METABOLIC ACTIVITY IN CELLS, AND AMONG MANY CATABOLIC AND BIOSYNTHETIC PATHWAYS THEY HARBOR AN EVOLUTIONARILY CONSERVED PATHWAY THAT SYNTHESIZES FATTY ACIDS IN THE MITOCHONDRIAL MATRIX (MTFAS). MTFAS PATHWAY ACTIVITY IS CRUCIAL FOR TCA CYCLE ACTIVITY, THROUGH THE GENERATION OF THE ENZYMATIC CO-FACTOR LIPOIC ACID, AS WELL AS FOR ELECTRON TRANSPORT CHAIN (ETC) COMPLEX ASSEMBLY AND FUNCTION. WE FOUND THAT ERRORS IN THE MTFAS PATHWAY BLOCK THE DIFFERENTIATION OF C2C12 MYOBLASTS IN VITRO AND IMPEDE MUSCLE REGENERATION FOLLOWING BARIUM CHLORIDE INJECTION-INDUCED INJURY IN VIVO. OUR PRELIMINARY DATA SUGGEST THAT THIS BLOCK IS EARLY IN DIFFERENTIATION, AND THEREFORE CHALLENGE THE MODEL THAT ATP FOR CONTRACTION IS THE CRUCIAL OUTPUT OF MITOCHONDRIAL METABOLISM DURING DIFFERENTIATION. THE EXPERIMENTS IN THIS PROPOSAL WILL DEFINITIVELY DETERMINE THE CRITICAL METABOLIC REQUIREMENTS OF MYOGENIC DIFFERENTIATION AND UNCOVER THE MOLECULAR MECHANISMS BY WHICH THESE METABOLIC PATHWAYS IMPINGE ON DIFFERENTIATION SIGNALING. IN AIM 1, WE WILL USE MTFAS KNOCKOUT AS A WINDOW THROUGH WHICH TO EXAMINE THE EFFECT OF MITOCHONDRIAL IMPAIRMENT ON MYOGENIC REGULATORY FACTOR (MRF) SIGNALING, FOCUSING ON PROMOTER BINDING, RECRUITMENT OF CO-ACTIVATORS, AND EPIGENETIC MODIFICATIONS AT MRF-TARGET PROMOTERS. IN AIM 2, WE WILL USE TARGETED METABOLIC STRATEGIES TO RESCUE INDIVIDUAL ASPECT(S) OF MITOCHONDRIAL FUNCTION DOWNSTREAM OF MTFAS TO TEST THEIR REQUIREMENT FOR DIFFERENTIATION, AND EMPLOY AN UNBIASED SCREENING METHOD TO IDENTIFY WAYS TO RESCUE DIFFERENTIATION IN MTFAS-DEFICIENT CELLS. UNDERSTANDING THE METABOLIC REGULATION OF MYOGENESIS WILL BE CRUCIAL TO DEVELOPING THE NEXT GENERATION OF THERAPIES FOR DISEASES OF MUSC LOSS OR DYSFUNCTION.

MOLECULAR MECHANISMS OF CHROMATIN SIGNALING AND EPIGENETIC REGULATION - PROJECT SUMMARY DNA METHYLATION AND HISTONE POST-TRANSLATIONAL MODIFICATIONS (PTMS) ARE KEY EPIGENETIC REGULATORS OF CHROMATIN ACCESSIBILITY, INTERACTION, AND FUNCTION. CHANGES IN THE COMPOSITION, ABUNDANCE, AND DISTRIBUTION OF DNA METHYLATION AND HISTONE PTMS, AND ASSOCIATED REARRANGEMENTS IN CHROMATIN STRUCTURE, ARE DEFINING FEATURES OF HUMAN CANCER AND OTHER DISEASES. WHILE MUCH PROGRESS HAS BEEN MADE CONNECTING EPIGENETIC REGULATION TO CELLULAR FUNCTION AND DISEASE, IT IS STILL NOT CLEAR HOW THESE REGULATORY FUNCTIONS ARE ACCOMPLISHED WITH SPATIAL AND TEMPORAL PRECISION, HOW THESE EPIGENETIC SIGNALS TRANSLATE TO FUNCTIONAL OUTCOMES IN CHROMATIN REGULATION, AND HOW THESE CHANGES CONTRIBUTE TO DISEASE STATES. OUR LONG-TERM GOAL IS TO TRANSLATE BASIC MECHANISTIC UNDERSTANDING OF CHROMATIN REGULATORY FUNCTION INTO NEW STRATEGIES FOR THE TREATMENT OF HUMAN DISEASES. TOWARD THIS GOAL, WE DEVELOP AND USE CUTTING EDGE BIOCHEMICAL, GENOMIC, AND PROTEOMIC TECHNOLOGIES TO UNDERSTAND THE INTERCONNECTED MOLECULAR ACTIVITIES OF CHROMATIN REGULATORY PROTEINS THAT “READ,” “WRITE,” AND “ERASE” DNA METHYLATION AND HISTONE PTMS. OUR PAST WORK FOCUSED ON DEFINING MOLECULAR MECHANISMS REGULATING THE EPIGENETIC INHERITANCE OF DNA METHYLATION THROUGH CELL DIVISIONS. WE REVEALED COMPLEX MECHANISMS INVOLVING MULTIVALENT DNA AND HISTONE ASSOCIATIONS AND ALLOSTERIC REGULATION THAT INFLUENCE DNA METHYLTRANSFERASE CHROMATIN TARGETING AND SUBSTRATE SPECIFICITY. THESE STUDIES ALSO CONTRIBUTED TO THE APPRECIATION THAT DNA METHYLTRANSFERASES UTILIZE UBIQUITIN-DEPENDENT PROTEIN INTERACTIONS TO FACILITATE THEIR ENZYMATIC ACTIVITIES, BUT THE MECHANISTIC DETAILS OF HOW DNA METHYLATION AND UBIQUITIN SIGNALING INTERFACE ARE UNCLEAR. OUR GOALS IN THE NEXT FIVE YEARS ARE TO DEFINE REGULATORY AND PATHOLOGIC MECHANISMS THAT CONNECT UBIQUITIN SIGNALING TO THE EPIGENETIC INHERITANCE OF DNA METHYLATION. TOWARD ACCOMPLISHING THESE GOALS, WE WILL STUDY ROLES FOR PROTEIN UBIQUITINATION IN BIG PICTURE QUESTIONS OF: 1) HOW, WHEN, AND WHERE DNA METHYLATION REGULATORS ARE TARGETED IN THE GENOME TO CARRY OUT THEIR ENZYMATIC FUNCTIONS; 2) HOW ABNORMAL 5MC PATTERNS ARISE IN CANCER AND AGING CELLS; AND 3) HOW THESE CHANGES CONTRIBUTE TO OTHER MOLECULAR HALLMARKS OF THESE DISEASE AND DISEASE-ASSOCIATED STATES. TO FACILITATE THESE STUDIES, WE WILL ALSO 4) DEVELOP A NEW CLASS OF UBIQUITINATED HISTONE AFFINITY REAGENTS THAT IS GENERALIZABLE FOR THE ENRICHMENT OF SINGLY AND COMBINATORIALLY MODIFIED HISTONE PTM STATES. COLLECTIVELY, OUR STUDIES WILL CONTINUE TO DEFINE FUNDAMENTAL MECHANISMS REGULATING DNA METHYLATION INHERITANCE, REVEAL PATHOLOGIC MECHANISMS ASSOCIATED WITH ABNORMAL DNA METHYLATION SIGNALING, AND INTRODUCE NEW REAGENTS AND METHODS FOR THIS FIELD.

DIETARY REGULATION OF ANTI-TUMOR IMMUNITY THROUGH CALORIC RESTRICTION - ABSTRACT OVERALL OBJECTIVE: THE FOOD WE EAT CAN AFFECT TUMOR GROWTH. YET, IT IS UNCLEAR WHETHER DIETS THAT ALTER NUTRIENT LEVELS IN THE TUMOR MICROENVIRONMENT (TME) PRIMARILY IMPACT CANCER CELLS OR INSTEAD IMPACT IMMUNE CELLS THAT PROVIDE CRITICAL DEFENSE AGAINST TUMOR PROGRESSION. WE HAVE FOUND THAT REDUCING OVERALL CALORIE INTAKE BY 30–50%, A DIETARY REGIMEN KNOWN AS CALORIC RESTRICTION, ANTAGONIZES TUMOR GROWTH BY BOOSTING ANTI-TUMOR IMMUNITY. CALORIC RESTRICTION PROFOUNDLY IMPACTS THE FATE AND FUNCTION OF CD8 T CELLS IN THE TME, FAVORING THE ACTIVITY OF TUMOR-CONTROLLING T EFFECTOR (TEFF) CELLS WHILE MINIMIZING THE ACCUMULATION OF DYSFUNCTIONAL OR “EXHAUSTED” T (TEX) CELLS. BY STUDYING HOW CALORIC RESTRICTION STEERS CD8 T CELLS BETWEEN EFFECTOR AND EXHAUSTED FATES, WE WILL IDENTIFY NOVEL DIET-REGULATED MECHANISMS THAT UNDERPIN CD8 T CELL- MEDIATED ANTI-TUMOR IMMUNITY AND THUS GAIN MECHANISTIC INSIGHT INTO THE EPIGENETIC, TRANSCRIPTIONAL, AND METABOLIC NETWORKS THAT LINK DIET, LIPID METABOLISM, AND CANCER PROGRESSION. HYPOTHESIS: THAT THE AVAILABILITY OF KETONE BODIES VERSUS FATTY ACIDS IN THE TME IS A CRITICAL DETERMINANT OF ANTI-TUMOR IMMUNITY. OUR PRELIMINARY DATA INDICATE THAT CALORIC RESTRICTION ANTAGONIZES TUMOR GROWTH BY BOOSTING ANTI-TUMOR CD8 T CELL RESPONSES. THESE EFFECTS ARE LOST WHEN CD8 T CELLS CANNOT PROCESS KETONE BODIES, PROVIDING A MECHANISTIC LINK BETWEEN DIET-REGULATED NUTRIENTS (KETONE BODIES) AND ANTI-TUMOR T CELL FUNCTION. WHILE CALORIC RESTRICTION AND OTHER DIETS CAN INCREASE CIRCULATING LEVELS OF KETONE BODIES, SOME OF THESE DIETS ALSO INCREASE CIRCULATING LEVELS OF FATTY ACIDS, WHICH WE HYPOTHESIZE CAN NEGATE OR DIMINISH THE POSITIVE IMPACT OF KETONE BODIES ON ANTI-TUMOR IMMUNITY. OUR WORKING MODEL IS THAT THE BALANCE BETWEEN CIRCULATING LEVELS OF KETONE BODIES AND FATTY ACIDS IMPACTS KETONE BODY-DEPENDENT METABOLISM AND EPIGENETIC PROGRAMMING IN CD8 T CELLS AND, BY PROXY, THE BALANCE BETWEEN FUNCTIONAL TEFF CELLS (PROVIDING ANTI-TUMOR IMMUNITY) AND DYSFUNCTIONAL TEX CELLS (DAMPENING ANTI-TUMOR IMMUNITY) IN THE TME. SPECIFIC AIMS: (1) DETERMINE HOW DIETARY FATS INFLUENCE ANTI-TUMOR T CELL RESPONSES IN THE CONTEXT OF CALORIC RESTRICTION; (2) DETERMINE HOW CALORIC RESTRICTION DIFFERENTIALLY CONTROLS T CELL METABOLISM IN THE TME; AND (3) DEFINE HOW CALORIC RESTRICTION INFLUENCES CD8 T CELL FATE DECISIONS IN THE TME. IMPACT: UPON COMPLETION, THIS PROPOSED RESEARCH WILL HAVE DETERMINED THE MECHANISMS BY WHICH CALORIC RESTRICTION BOOSTS CD8 T CELL-MEDIATED ANTI-TUMOR IMMUNITY, NOT ONLY INCREASING THE FUNCTION OF TEFF CELLS BUT ALSO DECREASING THE ACCUMULATION OF DYSFUNCTIONAL (OR “EXHAUSTED”) TEX CELLS IN THE TME. OUR WORK WILL HAVE LAID THE GROUNDWORK FOR DEFINING DIETARY INTERVENTIONS WITH THE POWER TO BOOST THE EFFICACY OF IMMUNE CHECKPOINT INHIBITORS (ICI) AND DIMINISH AND/OR REVERSE CANCER PROGRESSION.

PPM: FUNCTIONAL ASYMMETRIES OF TRANS-HISTONE AND HISTONE-DNA CROSSTALK IN DEVELOPMENT -UNDERSTANDING HOW CELLS CONTROL GENE EXPRESSION IS A CENTRAL CHALLENGE IN BIOLOGY. GENES ARE PACKAGED IN CHROMATIN AND THEIR EXPRESSION IS CONTROLLED BY CHEMICAL MODIFICATIONS ON HISTONE PROTEINS AND DNA IN NUCLEOSOMES, THE FUNDAMENTAL BUILDING BLOCKS OF CHROMATIN. THIS PROJECT EXAMINES HOW COMPLEX COMBINATIONS OF HISTONE OR DNA MODIFICATIONS THAT OCCUR ASYMMETRICALLY WITHIN INDIVIDUAL NUCLEOSOMES WORK TOGETHER TO DETERMINE GENE EXPRESSION DURING CELL TYPE SPECIFICATION. THE MOLECULAR RULES FOR HOW THESE PATTERNS ARE ESTABLISHED AND INTERPRETED REMAIN POORLY UNDERSTOOD BECAUSE OF LONGSTANDING TECHNICAL BARRIERS. THIS PROJECT WILL USE NEW EXPERIMENTAL TOOLS THAT ALLOW PRECISE CONTROL OF THE GEOMETRY OF HISTONE AND DNA MODIFICATIONS IN INDIVIDUAL NUCLEOSOMES, AND THEREBY ENABLE FUNDAMENTAL RESEARCH INTO HOW DIFFERENT COMBINATIONS OF THESE MODIFICATIONS ELICIT DISTINCT OUTCOMES. THE FINDINGS WILL ADVANCE UNDERSTANDING OF EPIGENETIC REGULATION, DEVELOPMENTAL BIOLOGY, AND GENOME ORGANIZATION THAT CONSTITUTE FOUNDATIONAL KNOWLEDGE TO ADVANCE BIOTECHNOLOGY AND BIOMEDICINE. BROADER IMPACTS INCLUDE INTERDISCIPLINARY TRAINING FOR STUDENTS AND POSTDOCTORAL RESEARCHERS IN STRUCTURAL BIOLOGY AND EPIGENETICS, OUTREACH ACTIVITIES FOR HIGH SCHOOL BIOTECHNOLOGY EDUCATION PROGRAMS, AND PUBLIC ENGAGEMENT EFFORTS AIMED AT INCREASING UNDERSTANDING OF GENE REGULATION. PROPER CELLULAR DIFFERENTIATION DEPENDS ON PRECISELY PATTERNED HISTONE AND DNA MODIFICATIONS THAT REGULATE DEVELOPMENTAL GENE EXPRESSION. MOUNTING EVIDENCE INDICATES THAT THESE MODIFICATIONS ARE OFTEN DEPOSITED ASYMMETRICALLY WITHIN SINGLE NUCLEOSOMES, WITH DISTINCT MARKS POSITIONED ON OPPOSITE HISTONE TAILS. THIS PROJECT WILL DEFINE HOW THESE ASYMMETRIES ARE GENERATED AND INTERPRETED THROUGH TRANS-HISTONE AND HISTONE-DNA CROSSTALK. THE INVESTIGATORS WILL BUILD ON THEIR TECHNOLOGICAL BREAKTHROUGHS TO ASSEMBLE FULLY ASYMMETRIC NUCLEOSOMES WITH PRECISELY DEFINED HISTONE AND DNA GEOMETRIES. THESE ASYMMETRIC NUCLEOSOMES WILL BE USED TO (I) ESTABLISH THE GEOMETRIC RULES BY WHICH H2BK120-UB, H2BK34-UB, AND H4K16AC DIRECT DOT1L METHYLTRANSFERASE TO METHYLATE H3K79 ON ONE SIDE OF THE NUCLEOSOME OR THE OTHER, (II) DETERMINE HOW H2BK120-UB-DEPENDENT CONFORMATIONAL SWITCHING BIASES MLL2 METHYLTRANSFERASE TOWARD A SPECIFIC H3 TAIL, AND (III) DEFINE WHETHER H3K4 AND H3K36 METHYLATION DIRECT DNMT3A2/3B3 METHYLTRANSFERASE TO METHYLATE DNA IN A DIRECTIONAL MANNER. IN ADDITION, THE PROJECT WILL DETERMINE STRUCTURES OF THE ACTIVE COMPLEX FORMED BETWEEN THE NUCLEOSOME AND DNMT3A2/3B3. THIS WORK INTEGRATES ASYMMETRIC CHROMATIN ENGINEERING BIOCHEMISTRY AND CRYO-ELECTRON MICROSCOPY TO DETERMINE HOW MODIFICATION ASYMMETRIES WITHIN NUCLEOSOMES MODULATE HISTONE AND DNA CROSSTALK INTERACTIONS. BY REVEALING HOW THE SPATIAL ORGANIZATION OF CHROMATIN MODIFICATIONS ENCODES REGULATORY INFORMATION, THIS RESEARCH WILL ESTABLISH FUNDAMENTAL PRINCIPLES LINKING THE GEOMETRY OF EPIGENETIC MODIFICATIONS TO GENE REGULATION. THIS AWARD REFLECTS NSF'S STATUTORY MISSION AND HAS BEEN DEEMED WORTHY OF SUPPORT THROUGH EVALUATION USING THE FOUNDATION'S INTELLECTUAL MERIT AND BROADER IMPACTS REVIEW CRITERIA.- SUBAWARDS ARE NOT PLANNED FOR THIS AWARD.

THE ROLES OF GENETICS, HORMONES, AND GENDER IN SEXUALLY DIMORPHIC IMMUNE RESPONSE - ABSTRACT SEX AND GENDER CONTRIBUTE TO THE IMMUNE SYSTEM AND HOW WE AS HUMANS RESPOND TO FOREIGN INFECTIONS. MALES TRADITIONALLY HAVE AN ELEVATED RISK OF HOSPITALIZATIONS DUE TO INFECTIONS FROM BIRTH TO OLD AGE. FEMALES, HOWEVER, HAVE A MORE PROTECTIVE IMMUNE SYSTEM, YET HAVE AN ELEVATED RISK OF AUTOIMMUNITY. THUS, THE BALANCE BETWEEN FIGHTING INFECTIONS AND LIMITING IMMUNE RESPONSE IS STRATIFIED. ANIMAL MODELS HAVE ALLOWED US TO LARGELY TEASE OUT THE ROLE OF SEX CHROMOSOMES AND HORMONES (SUCH AS ESTROGEN/TESTOSTERONE), BUT THEY LEND LITTLE INSIGHT INTO THE ROLE OF HUMAN GENDER ON THE IMMUNE RESPONSE. DISPARITIES BETWEEN TRANS- AND CIS-GENDER WOMEN ARE OBSERVED IN MULTIPLE INFLAMMATORY AND IMMUNE PHENOTYPES, FROM HIV RISK TO RHEUMATOLOGICAL CONDITIONS. AS GENDER IS A NON-BINARY SPECTRUM, MANY PRECISION MEDICINE TOOLS CAN BE APPLIED TO TEASE OUT INDIVIDUALS’ IMMUNE SYSTEM MODULATORS FOCUSED ON INDIVIDUAL ASSESSMENTS OF SEX CHROMOSOMES, SEX HORMONES, AND ENVIRONMENTAL FACTORS. TO DO THIS, WE HAVE COMPILED A MULTIPLE PI TEAM THAT BRINGS STRENGTHS FROM EPIGENETICS/EPIDEMIOLOGY (DR. TRICHE), INFECTIOUS DISEASE MODELING (DR. KRAWCZYK), AND SEX CHROMOSOME/HORMONE GENETICS (DR. PROKOP). WE HIGHLIGHT IN THIS WORK HOW THE HISTONE METHYLATION REGULATOR CHROMOSOME PAIR, KDM5C (CHRX) AND KDM5D (CHRY), RESULTS IN AN IMMUNE MODULATION THROUGH GENOME-WIDE EPIGENETIC ALTERATION. WE HAVE DEVELOPED THREE COMPLEMENTARY AIMS TO BUILD AN INTEGRATED MODEL OF KDM5C/KDM5D INFLUENCE ON INDIVIDUALS’ IMMUNE RESPONSES. AIM1) GENETICS OF KDM5C HEMATOPOIESIS CONDITIONAL KNOCKOUT ALTERATION AT THE CELLULAR RESOLUTION OF THE IMMUNE SYSTEM FOLLOWING A CHRONIC VIRAL INFECTION, ALLOWING US TO DETERMINE CELL TYPES AND GLOBAL EPIGENETIC ALTERATIONS REGULATED BY THE PATHWAY. AIM2) PRECISION INSIGHTS FOR KDM5C/KDM5D OF ~20,000 HUMAN BLOOD SAMPLES FOLLOWING VARIOUS IMMUNE CHALLENGES AND THE SEPARATION OF HORMONE AND SEX CHROMOSOME USING THE FOUR-CORE GENOTYPES RODENT MODEL BLOOD SINGLE-CELL DATA GENERATION. AIM3) INTEGRATING OUR KNOWLEDGE TO INDIVIDUALS FOR HUMAN VARIANTS, GENE REGULATION, ENVIRONMENTAL MODULATION, AND PHENOTYPIC ASSOCIATIONS FOR KDM5C/KDM5D. THE COMBINATION OF THE THREE AIMS REPRESENTS A PRECISION MEDICINE APPROACH, ALLOWING US INSIGHTS INTO THE CELLS AND PATHWAYS IMPACTED BY THE SEX CHROMOSOME GENES DURING INFECTION (AIM1), DECONVOLUTION OF HOW HORMONES AND SEX CHROMOSOMES SYNERGISTICALLY CONTRIBUTE TO IMMUNE CELLS (AIM2), DEFINING THE BROAD INFECTIONS AND SIGNATURES THAT MODULATE THE GENES (AIM2), AND INTEGRATING THIS KNOWLEDGE TO BE APPLIED TO ANY INDIVIDUAL IN A NON-BINARY APPROACH (AIM3). AS GENDER CAN NEVER BE BINNED INTO GROUPS, WITH AN UNDEFINED SPECTRUM OF INDIVIDUALS WITHIN, WE LAY FORTH A NOVEL, INNOVATIVE WAY FOR THINKING ABOUT HOW SEX AND GENDER BOTH CONTRIBUTE TO IMMUNE SYSTEMS WITHOUT THE NEED FOR DEFINING GENDER BUT ALLOWING FOR INDIVIDUALIZED ASSESSMENTS.

STRUCTURAL INTERROGATION OF ALLOSTERIC AMPK REGULATION

UNDERSTANDING PUTATIVE BETA-CELL SUBTYPES - PROJECT SUMMARY TYPE 2 DIABETES (T2D) IS A DISEASE THAT RESULTS FROM RELATIVE PANCREATIC Β-CELL DYSFUNCTION, AND AFFECTS >400 MILLION PEOPLE. WE (RUTTER) PREVIOUSLY IDENTIFIED FUNCTIONAL Β-CELL SUBSETS BASED UPON ANALYSIS OF REAL-TIME CA2+ RESPONSES AND CELL-CELL CONNECTIVITY. A “ΒLEADER” POPULATION (~5% OF Β-CELLS) EXHIBITS FAST AND ROBUST CA2+ INFLUX THAT CONTROLS THE TIMING OF ISLET-WIDE GLUCOSE RESPONSES, AND A SEPARATE POPULATION OF HIGHLY INTERCONNECTED ΒHUB -CELLS (5-10% OF Β-CELLS) THAT SYNCHRONIZES GLUCOSE RESPONSES, ISLET-WIDE. MORE RECENTLY, WE (POSPISILIK) DISCOVERED THAT H3K27ME3 DOSAGE IS A NOVEL REGULATOR OF Β-CELL LINEAGE DIFFERENTIATION, STABILITY, AND FUNCTIONAL SPECIFICATION. WE IDENTIFIED TWO EPIGENETICALLY DISTINCT Β-CELL POPULATIONS (ΒHI AND ΒLO) THAT ARE CONSERVED ACROSS MICE AND HUMANS AND COMPRISE >90% OF THE Β-CELL COMPARTMENT. THEY ARE DISTINGUISHED BY DISTINCT NUCLEAR AND CELL SURFACE PROTEIN EXPRESSION, BY DISTINCT EPIGENOMES AND TRANSCRIPTOMES, AND BY A SERIES OF MORPHOLOGICAL AND FUNCTIONAL DIFFERENCES. IMPORTANTLY, ΒHI AND ΒLO CELLS CAN BE PURIFIED BY FACS, AND THEIR RELATIVE RATIOS ARE ALTERED IN MOUSE MODELS AND HUMAN T2D. OUR PRELIMINARY DATA NOW SUGGEST THAT ΒLEADER AND ΒHUB CELLS TOGETHER COMPRISE THE EPIGENETICALLY-DEFINED ΒHI CELLS, AND THAT ΒFOLLOWER CELLS ARE SYNONYMOUS WITH ΒLO CELLS. THE OBJECTIVE OF THIS PROJECT IS TO TEST THIS HYPOTHESIS, AND DEFINE THE FUNCTIONAL AND DISEASE RELEVANCE OF THESE PUTATIVE Β-CELL SUBTYPES. IN AIM 1, WE WILL USE NOVEL SINGLE-CELL EPIGENOMIC TOOLS TO DETERMINE THE EXTENT TO WHICH FUNCTIONALLY- AND EPIGENETICALLY-DEFINED Β-CELL SUBSETS ARE THE SAME. IN AIM 2, WE WILL USE CONDITIONAL DELETION MOUSE MODELS TO DETERMINE WHEN AND HOW H3K27ME3 LEVELS CONTROL THE EMERGENCE OF EACH Β-CELL SUBSET, AND THEIR RELATIVE RATIOS. IN AIM 3, WE WILL USE THE SAME MOUSE MODELS TO DETERMINE IF AND HOW Β-CELL SUBTYPE RATIOS RELATE TO OBESITY-ASSOCIATED DIABETES, AND IF SUBTYPE PROPORTIONS CAN BE REVERSED BY DIET, SGLT2I, OR GLP1R AGONIST TREATMENTS. WE WILL PERFORM PARALLEL STUDIES IN HUMAN SC-ISLETS TO DETERMINE IF HNF1A MUTANTS (A CAUSAL T2D GENETIC VARIANT) ALTER Β-CELL SUBTYPE RATIO AND FUNCTION, AND IF THOSE EFFECTS ARE REVERSIBLE. FINALLY, WE WILL SCREEN BIOBANKED NPOD AND ADI PANCREATIC SAMPLES FOR THE RELATIVE PROPORTIONS OF THESE FUNCTIONAL SUBSETS ACROSS METABOLIC DISEASES. THUS, AIM 3 WILL ESTABLISH THE EXTENT AND IMPACT OF SUBTYPE-SPECIFIC Β-CELL DYSFUNCTION IN T2D. BY USING THIS UNIFIED THEORETICAL AND EXPERIMENTAL FRAMEWORK, WE WILL ESTABLISH AN EPIGENETIC BASIS FOR UNDERSTANDING AND MANIPULATING PUTATIVE Β-CELL SUBTYPE NUMBER AND FUNCTION IN VITRO AND IN VIVO, AND NOVEL METHODS FOR GENERATING SC-ISLETS WITH PREFERENTIAL SKEWS IN Β-CELL SUBTYPES.

MECHANISMS OF DNA METHYLATION SIGNALING AND DYSREGULATION IN COLON CANCER - PROJECT SUMMARY COLON CANCER IS A LEADING CAUSE OF CANCER-RELATED DEATH FOR BOTH MEN AND WOMEN IN THE UNITED STATES. ABNORMAL DNA METHYLATION PATTERNING IS AN EPIGENETIC HALLMARK OF COLON AND OTHER CANCER TYPES AND IS A MAJOR CONTRIBUTING FACTOR TO GENOME INSTABILITY AND TUMOR SUPPRESSOR GENE SILENCING IN THIS DISEASE. DESPITE A DEEP APPRECIATION OF ROLES FOR DNA METHYLATION IN DEVELOPMENT AND CANCER, CAUSAL MECHANISMS THAT CONTRIBUTE TO THE EVOLUTION OF DNA METHYLATION ABNORMALITIES IN COLON AND OTHER CANCER TYPES, AND CONSEQUENCES OF THIS HALLMARK EPIGENETIC REMODELING ON CANCER DEVELOPMENT, REMAIN POORLY UNDERSTOOD. THIS PROPOSAL SEEKS TO ADDRESS THESE FUNDAMENTAL KNOWLEDGE GAPS. OUR PRELIMINARY DATA IMPLICATE PROTEIN UBIQUITINATION AS AN ESSENTIAL POST- TRANSLATIONAL MODIFICATION (PTM) REGULATING THE ACTIVITY OF DNMT1, THE PRIMARY ENZYME RESPONSIBLE FOR THE EPIGENETIC INHERITANCE OF DNA METHYLATION PATTERNS THROUGH MITOTIC CELL DIVISIONS AND A MAJOR TARGET FOR EPIGENETIC CANCER THERAPY. BUILDING FROM THESE DATA, STUDIES IN AIM 1 WILL CHARACTERIZE THE UBIQUITIN SIGNALING NETWORK THAT INTERFACES WITH DNA METHYLATION INHERITANCE. THE THERAPEUTIC POTENTIAL OF TARGETING UBIQUITIN SIGNALING REGULATORS IN THIS PATHWAY WILL ALSO BE CONSIDERED. IN AIM 2, WE WILL TEST A MECHANISTIC HYPOTHESIS CONNECTING OVEREXPRESSION OF THE E3 UBIQUITIN LIGASE UHRF1 TO DNA METHYLATION DYSREGULATION AND COLON TUMORIGENESIS. AIM 3 WILL INVESTIGATE A NON-CANONICAL FUNCTION FOR UHRF1 IN THE REGULATION OF HETEROCHROMATIC HISTONE PTM SIGNALING AND ITS IMPLICATIONS FOR DNA HYPOMETHYLATION THERAPY. OVERALL, OUR STUDIES AT THE INTERFACE OF UBIQUITIN SIGNALING AND DNA METHYLATION INHERITANCE WILL REVEAL FUNDAMENTAL CAUSES AND CONSEQUENCES OF ABNORMAL DNA METHYLATION SIGNALING IN COLON CANCER AND IDENTIFY NOVEL APPROACHES TO ENHANCE THE EFFICACY OF DNA HYPOMETHYLATION THERAPY AS A TREATMENT STRATEGY TO MANAGE THIS DEADLY DISEASE.

ROLE OF KYNURENINE PATHWAY METABOLITES IN PERINATAL DEPRESSION AND SUICIDALITY

STRUCTURAL MECHANISM OF ENERGY CONSERVATION IN HYPERTHERMOPHILES

DEFINING THE EFFECTS OF A-SYNUCLEIN PATHOLOGY ON THE AMYGDALA CIRCUITRY: RELEVANCE TO NEUROPSYCHIATRIC DYSFUNCTION IN PARKINSON'S DISEASE

AIRBORNE POLLUTANTS AS TRIGGERS OF PARKINSONS DISEASE VIA THE OLFACTORY SYSTEM

MECHANISMS OF DEVELOPMENTAL PLASTICITY -WHILE DEVELOPMENTAL EVENTS ARE GENERALLY TIGHTLY CONTROLLED DURING EMBRYOGENESIS, A CERTAIN LEVEL OF FLEXIBILITY EXISTS. THIS SO-CALLED DEVELOPMENTAL PLASTICITY ALLOWS AN ORGANISM TO ADJUST IN RESPONSE TO ENVIRONMENTAL FACTORS EXPERIENCED DURING EARLY STAGES OF DEVELOPMENT OR EVEN TO THE PARENTAL ENVIRONMENT. FOR EXAMPLE, IT HAS BEEN SHOWN THAT THE METABOLIC STATE OF THE PARENTS AT CONCEPTION CAN INFLUENCE EMBRYO DEVELOPMENT AND SUBSEQUENTLY AFFECT THE CHARACTERISTICS OF THEIR OFFSPRING. THE MECHANISMS BY WHICH THE NEXT GENERATION RETAINS A MEMORY OF THE PARENTAL ENVIRONMENT, HOWEVER, ARE NOT YET CLEAR. THE AIM OF THIS AWARD IS TO GAIN AN UNDERSTANDING OF HOW MATERNAL METABOLIC STATE - EXCESS, NORMAL, AND REDUCED LONG TERM CALORIE INTAKE - ALTERS THE DEVELOPMENT AND TRAITS OF THEIR CHILDREN. IN THIS STUDY, THE RESEARCHERS USE FRUIT FLIES AS A MODEL. FRUIT FLIES ARE IDEAL FOR THIS RESEARCH BECAUSE, ONCE THE MOTHER LAYS THE EGGS, THEY DEVELOP ON THEIR OWN WITHOUT ANY FURTHER INPUT FROM THE MOTHER, UNLIKE IN MAMMALS. THIS ALLOWS THE RESEARCHERS TO DIRECTLY ADDRESS THE IMPACT OF THE MATERNAL ENVIRONMENT BEFORE CONCEPTION ON EMBRYO DEVELOPMENT. THEY WILL UTILIZE A NOVEL METHODOLOGY TO DETERMINE HOW EMBRYONIC METABOLISM AND GENE EXPRESSION CHANGES IN RESPONSE TO MATERNAL METABOLIC STATE AND PERFORM EXPERIMENTS TO UNDERSTAND HOW THESE MOLECULAR CHANGES INFLUENCE DEVELOPMENTAL OUTCOMES TO TRIGGER LIFE-LONG CHANGES. ADDITIONALLY, A NEW HANDS-ON TEACHING MODULE FOR A K-12 AFTERSCHOOL PROGRAM WILL BE DEVELOPED AND TURNED INTO DIGITAL LESSONS TO BE DISTRIBUTED TO EDUCATORS ACROSS THE COUNTRY IN COLLABORATION WITH THE VAN ANDEL EDUCATION INSTITUTE. HOW PARENTAL METABOLIC STATES TRANSLATE INTO DISTINCT DEVELOPMENTAL TRAJECTORIES AND ADULT PHENOTYPES REMAINS UNCLEAR. THIS STUDY WILL INVESTIGATE THE MATRILINEAL EFFECTS OF METABOLIC STATE ON OFFSPRING DEVELOPMENT. THE OBJECTIVES ARE TO DETERMINE THE IMPACT OF MATERNAL METABOLIC STATE ON METABOLIC AND TRANSCRIPTIONAL PROCESSES AND EPIGENOMIC PROFILES IN OFFSPRING EMBRYOS AND LINK THE MATERNAL INTERGENERATIONAL SIGNAL TO OFFSPRING REPROGRAMMING EVENTS AND PHENOTYPES. TECHNICAL LIMITATIONS, DUE TO THE FAST PROGRESSION OF DEVELOPMENT AND SMALL AMOUNT OF MATERIAL AVAILABLE DURING THE EARLY EMBRYONIC TIME WINDOW, HAVE BEEN BARRIERS TO STUDYING DEVELOPMENTAL PLASTICITY. THIS GROUP RECENTLY REPORTED A NEW SINGLE EMBRYO RNA-SEQUENCING METHODOLOGY THAT OVERCOMES THESE LIMITATIONS. USING THIS METHODOLOGY THEY WILL GENERATE A TIME-RESOLVED, INTEGRATED TRANSCRIPTOMIC AND METABOLIC DATASET OF REPROGRAMMING EVENTS, TRIGGERED BY MATERNAL METABOLIC STATE, DURING EARLY (10 MIN?3H) DROSOPHILA EMBRYOGENESIS. THEY WILL DETERMINE HOW THESE METABOLITE AND TRANSCRIPTOME CHANGES TRANSLATE INTO DIFFERENCES IN CHROMATIN ASSOCIATED HISTONE MARKS BY ASSESSING THEIR GENOME WIDE DISTRIBUTION AT DIFFERENT DEVELOPMENTAL TIMEPOINTS. TO TEST IF SUBSTRATE AVAILABILITY AND/OR ENZYMES INVOLVED IN HISTONE MODIFICATIONS ARE A DRIVER OF THE INTERGENERATIONAL PHENOTYPE, TWO DIFFERENT APPROACHES WILL BE EMPLOYED 1) SHRNA-KNOCKDOWN (KD) EXPERIMENTS IN THE OOCYTE AND ZYGOTE AND 2) DIETARY SUPPLEMENTATION. THE COMBINATION OF NEWLY DEVELOPED TOOLS WITH TRADITIONAL EPIGENOMIC AND SHRNA KNOCK-DOWN ANALYSIS WILL FACILITATE THE IDENTIFICATION OF THE EARLIEST REPROGRAMMING SIGNATURES AND ALLOW THE TRACKING OF THOSE SIGNATURES THROUGH TO SPECIFIC DEVELOPMENTAL PROGRAMS AND ADULT PHENOTYPES. THE BROADER IMPACT ACTIVITIES INCLUDE DEVELOPMENT OF AN EDUCATIONAL MODULE ON EPIGENETICS AND THE IMPACT OF EXTERNAL FORCES ON EMBRYONIC DEVELOPMENT FOR K-12 STUDENTS. THIS AWARD REFLECTS NSF'S STATUTORY MISSION AND HAS BEEN DEEMED WORTHY OF SUPPORT THROUGH EVALUATION USING THE FOUNDATION'S INTELLECTUAL MERIT AND BROADER IMPACTS REVIEW CRITERIA.- SUBAWARDS ARE NOT PLANNED FOR THIS AWARD.

STRUCTURAL AND FUNCTIONAL ANALYSIS OF A DYNAMIC ABA SIGNALING COMPLEX

REVEALING THE MOLECULAR BASIS OF MICROTUBULE-INTERMEDIATE FILAMENT INTERACTIONS. - PROJECT SUMMARY MICROTUBULES (MTS) AND INTERMEDIATE FILAMENTS (IFS) ARE TWO MAIN COMPONENTS OF THE CYTOSKELETON IN EUKARYOTIC CELLS, EACH WITH UNIQUE STRUCTURAL AND REGULATORY ROLES. INTERACTIONS BETWEEN MTS AND IFS ARE CRUCIAL FOR MAINTAINING CELLULAR SHAPE, STABILITY, AND ORGANIZATION. DESPITE THEIR WIDE IMPORTANCE IN BIOLOGY, EXACTLY HOW MTS AND IFS INTERACT AT THE MOLECULAR LEVEL REMAINS UNCLEAR. A MAJOR BARRIER TO ELUCIDATING THESE INTERACTIONS IS THE STRONG CONTEXT-DEPENDENCY OF CYTOSKELETAL ARRANGEMENT AND FUNCTION ACROSS TISSUES, CELL TYPES, AND SUBCELLULAR LOCATIONS. MTS AND IFS ARE SPECIALIZED BY SUBUNIT COMPOSITION, BOUND PROTEINS, AND POST- TRANSLATIONAL MODIFICATIONS TO CREATE POLYMERS WITH DISTINCT PHYSICAL PROPERTIES, INTERACTION NETWORKS, AND REGULATORY MECHANISMS. UNDERSTANDING HOW MTS AND IFS ARE ADAPTED TO FORM MORPHOLOGICALLY AND FUNCTIONALLY DISTINCT CYTOSKELETAL ARRAYS WILL HAVE BROAD APPLICATIONS IN DEVELOPING THERAPEUTIC STRATEGIES, IMPROVING TISSUE ENGINEERING, AND ADVANCING OUR KNOWLEDGE OF CELLULAR MECHANICS. THE GOAL OF THIS PROPOSAL IS TO REVEAL THE MOLECULAR ADAPTATIONS OF MTS AND IFS THAT REGULATE CYTOSKELETAL ARRAY CONFIGURATION AND FUNCTION SPECIFICALLY IN MYELINATED AXONS, WHICH ARE IMPORTANT FOR RAPID, LONG DISTANCE NEURONAL COMMUNICATION. ALL AXONS CONTAIN A STAGGERED, PARALLEL ARRAY OF HIGHLY DECORATED MTS AND NEURON-SPECIFIC IFS CALLED NEUROFILAMENTS (NFS) THAT LOCALLY REGULATE CELL SHAPE AND COMPOSITION. MYELINATED AXONS REGULATE THEIR CYTOSKELETAL ARRAY THROUGH A PHOSPHORYLATION CASCADE THAT DIRECTLY TARGETS MTS AND NFS TO CHANGE THEIR ABUNDANCE AND REARRANGE THEIR 3D ORGANIZATION. MY RESEARCH PLAN BUILDS ON THE IMPLEMENTATION OF A NOVEL METHOD FOR ISOLATING MYELINATED AXONS FROM THE BRAIN THAT PRESERVES THE INTERNAL CONFIGURATION OF MTS AND NFS AND WILL FOR THE FIRST TIME ENABLE THE STUDY OF TISSUE-DERIVED MATERIAL IN ITS NATIVE FORM. THIS MULTIFACETED WORK COMBINES STRUCTURAL DETERMINATION, BIOCHEMICAL ASSAYS, COMPARATIVE PROTEOMICS, AND IMMUNOFLUORESCENCE AIMING TO REVEAL HOW MTS AND NFS ARE ADAPTED AND INTERACT INSIDE MYELINATED AXONS. ATOMIC STRUCTURES OF NATIVE MTS AND NFS DETERMINED BY CRYO-ELECTRON MICROSCOPY (CRYO-EM) WILL REVEAL THE MOLECULAR MECHANISMS BEHIND THEIR EXCEPTIONAL STABILITY INSIDE THE AXON, WHICH IS IMPORTANT FOR MAINTAINING CELL INTEGRITY. COMPLEMENTARY BIOCHEMICAL ANALYSES WILL INVESTIGATE HOW MT AND NF TURNOVER IS REGULATED TO INFLUENCE CYTOSKELETAL ARRAY COMPOSITION. TO FURTHER IDENTIFY REGULATORS OF THE MT-NF ARRAY, FOCUSED ION BEAM MILLING (FIB- MILLING) AND CRYO-ELECTRON TOMOGRAPHY (CRYO-ET) WILL RECONSTRUCT ENTIRE CYTOSKELETAL NETWORKS WITHIN INTACT AXONS TO QUANTIFY MT-NF NETWORK ORGANIZATION AND ASSOCIATION WITH CELLULAR CARGO. ALTOGETHER, SUPPORT OF THIS RESEARCH BY THE R35 MIRA AWARD WOULD ESTABLISH A FRAMEWORK FOR FUTURE STUDIES ON CYTOSKELETAL ADAPTATIONS IN DIFFERENT BIOLOGICAL CONTEXTS AND DISEASE STATES THROUGHOUT MY CAREER.

A NOVEL GENE THERAPY APPROACH TO REDUCE NEUROTOXICITY IN PARKINSON'S DISEASE - PROJECT SUMMARY/ABSTRACT PARKINSON’S DISEASE (PD) IS A COMMON NEURODEGENERATIVE DISORDER, WITH ANNUAL SOCIETAL COSTS EXCEEDING $52 BILLION IN THE US. PD IS A COMPLEX, MULTIFACTORIAL DISEASE THAT EXHIBITS A HIGH DEGREE OF HETEROGENEITY AMONG AFFECTED INDIVIDUALS, WHO DEVELOP SEVERAL DEBILITATING MOTOR AND NON-MOTOR SYMPTOMS. CURRENT TREATMENTS MAINLY TARGET MOTOR SYMPTOMS AND CANNOT SLOW OR STOP DISEASE PROGRESSION. RECENT BIOMARKER STUDIES, INCLUDING TWO CARRIED OUT BY OUR TEAM, SHOW PD-SPECIFIC ALTERATIONS IN KYNURENINE METABOLITES THAT STRONGLY ASSOCIATE WITH SYMPTOM SEVERITY, POSITIONING THE KYNURENINE PATHWAY AS A POTENTIAL THERAPEUTIC TARGET IN PD. THE KYNURENINE PATHWAY PRODUCES SEVERAL METABOLITES WITH IMMUNOLOGICAL AND NEUROACTIVE PROPERTIES, INCLUDING QUINOLINIC ACID (QUIN), A WELL-KNOWN PRO-INFLAMMATORY AND EXCITOTOXIC METABOLITE. IN THIS PROJECT WE WILL CONDUCT PHARMACODYNAMIC, PHARMACOKINETIC, AND IN VIVO EFFICACY STUDIES TO AID THE DEVELOPMENT OF A NOVEL GENE THERAPY APPROACH AIMED AT PREVENTING OR SLOWING THE PROGRESSION OF NEURODEGENERATION OF PD. OUR STRATEGY AIMS TO INCREASE THE ACTIVITY OF THE ENZYME AMINOCARBOXY-MUCONATE SEMIALDEHYDE DECARBOXYLASE (ACMSD), A PIVOTAL ENZYME THAT CONTROLS QUIN LEVELS. ENHANCING ACMSD ACTIVITY RESULTS IN A METABOLIC SHIFT WITH LESS QUIN PRODUCTION, WHILST PRODUCING MORE OF ANOTHER METABOLITE, PICOLINIC ACID (PIC). BECAUSE PIC COUNTERACTS QUIN-INDUCED TOXICITY AND CAN AVERT OXIDATIVE STRESS THROUGH ITS CHELATING PROPERTIES, ENHANCING ACMSD ACTIVITY IS EXPECTED TO HAVE A PROTECTIVE EFFECT ON THE NEURONAL ENVIRONMENT. OUR RIGOROUS BIOMARKER STUDIES SHOW PD-ALTERATIONS CORRESPONDING TO A COMPROMISED ACMSD ACTIVITY, PROVIDING RATIONALE, WHICH IS FURTHER SUPPORTED BY STRONG GENETIC AND EXPERIMENTAL DATA. OUR PRELIMINARY DATA SHOW THAT (I) ADENO-ASSOCIATED VIRUS (AAV)-MEDIATED OVEREXPRESSION OF ACMSD REDUCES NIGRAL NEURODEGENERATION AND NEUROINFLAMMATION AS WELL AS QUIN LEVELS (IN CEREBROSPINAL FLUID) IN A RAT MODEL OF PD, AND (II) ITS ABLATION LEADS TO PROGRESSIVE NIGRAL NEURODEGENERATION IN AGING MICE. IN THE R61 PHASE, WE WILL TEST TWO AAV-VECTORS (ENGINEERED TO TARGET EITHER NEURONS OR GLIA) SIDE BY SIDE, EVALUATING TARGET ENGAGEMENT AND TOXICITY OVER TIME. AT THE END OF THIS PHASE, WE WILL IDENTIFY >1 DOSE THAT SUSTAINS TARGET ENGAGEMENT (≥20% DECREASE IN QUIN/PIC RATIO) BEFORE PROCEEDING TO THE R33 PHASE. IN THE R33 PHASE WE WILL DEMONSTRATE THAT INCREASING ACMSD ACTIVITY WILL MITIGATE PROGRESSIVE NIGRAL DEGENERATION AND BEHAVIORAL DEFICITS IN TWO RAT MODELS OF PD (TOXIN- AND Α-SYNUCLEIN-BASED). IMPORTANTLY, OUR STUDIES WILL BE INTERVENTIONAL AS WE WILL TEST THE EFFECTS OF AAV-MEDIATED ACMSD OVEREXPRESSION AFTER TRIGGERING NEUROPATHOLOGY. ALTOGETHER, THIS PROJECT WILL SYSTEMATICALLY DEFINE THE PARAMETERS REQUIRED TO DEMONSTRATE THAT OUR APPROACH RESULTS IN NEUROPROTECTION AND AMELIORATION OF MOTOR DEFICITS IN TWO PROGRESSIVE MODELS OF PD-LIKE NEURODEGENERATION, SERVING AS A FOUNDATION FOR FUTURE CLINICAL TRIALS.

THE ROLE OF NF1 IN MAMMARY DEVELOPMENT, BREAST CANCER, AND ENDOCRINE RESISTANCE

ESTABLISHING THE NEUROPROTECTIVE POTENTIAL OF C9ORF72 HYPERMETHYLATION IN REPEAT EXPANSION-CAUSED ALS

THE ROLE OF WNT SIGNALING IN NORMAL AND ABNORMAL HEMATOPOIESIS

COMBINING SYNUCLEINOPATHY AND MITOCHONDRIAL DEFICITS IN A NOVEL MOUSE MODEL OF PARKINSONS DISEASE

INVESTIGATING KDM5C AS A REGULATORY SWITCH BETWEEN ANABOLIC AND CATABOLIC METABOLISM - ABSTRACT CLONAL EXPANSION OF T CELLS IS A UNIQUE PHYSIOLOGICAL PROCESS AND A CRITICAL COMPONENT OF THE ADAPTIVE IMMUNE RESPONSE NECESSARY FOR COMBATING A DIVERSE RANGE OF PATHOGENS AND CANCER. T CELL EXPANSION IS HIGHLY CONTROLLED BY METABOLIC REPROGRAMMING TO ANABOLIC METABOLISM TO MEET THE BIOSYNTHETIC DEMANDS OF RAPID PROLIFERATION, AND METABOLIC FLEXIBILITY TO ENGAGE CATABOLIC METABOLISM WHEN ENERGY LEVELS ARE LOW (SUCH AS IN TISSUES OR TUMORS). THE PRECISE MECHANISMS BY WHICH THESE PROGRAMS INTEGRATE REMAIN LARGELY UNCHARACTERIZED. IN OUR WORK, WE DISCOVERED THAT T CELL EXPANSION LEADS TO SIGNIFICANT UPREGULATION OF HISTONE LYSINE (K) DEMETHYLASE KDM5C. KDM5C REMOVES METHYL GROUPS FROM LYSINE 4 ON HISTONE 3 (H3K4), RESULTING IN REPRESSION OF GENES AND THUS IS BEST KNOWN AS A TRANSCRIPTIONAL REPRESSOR. WE FOUND THAT KDM5C IS INTRINSICALLY REQUIRED FOR ANTIGEN-SPECIFIC EXPANSION OF CD8 T CELLS IN RESPONSE TO VIRAL INFECTION (LCMV). WE FOUND THAT KDM5C-DEFICIENT CD8 T CELLS HAVE DECREASED EXPRESSION OF KEY ANABOLIC GENES AND DECREASED CELLULAR LEVELS OF ANABOLIC METABOLITES. OUR FINDINGS WERE SURPRISING SINCE KDM5C IS CONSIDERED A TRANSCRIPTIONAL REPRESSOR. FURTHER, THE FUNCTION OF KDM5C IS THOUGHT TO BE PREDOMINANTLY NUCLEAR; HOWEVER, WE FIND SIGNIFICANT KDM5C PROTEIN IN THE CYTOSOL, ESPECIALLY FOLLOWING T CELL ACTIVATION, SUGGESTING THAT KDM5C HAS FUNCTIONS INDEPENDENT OF ITS HISTONE DEMETHYLASE ACTIVITY. WE ALSO FOUND THAT KDM5C IS INVOLVED IN METABOLIC SIGNALING: KDM5C IS A DIRECT TARGET OF AMPK, A KINASE THAT SHIFTS METABOLISM AWAY FROM ENERGY-CONSUMING ANABOLIC METABOLISM IN FAVOR OF ENERGY-GENERATING CATABOLIC METABOLISM, AND AMPK ACTIVATION LEADS TO DECREASED LEVELS OF KDM5C. OUR DATA IMPLIES THAT KDM5C HAS NON-CANONICAL FUNCTIONS IN MODULATING CELLULAR METABOLISM IN ACTIVATED T CELLS AND THAT THIS FUNCTION DIRECTLY PROMOTES T CELL EXPANSION. THEREFORE, THE OBJECTIVE OF THIS PROPOSAL IS TO ANSWER HOW KDM5C PROMOTES ANABOLIC METABOLISM TO SUPPORT POST-ACTIVATION EXPANSION IN CD8 T CELLS. WE HYPOTHESIZE THAT KDM5C IS A METABOLIC SWITCH BETWEEN ANABOLIC AND CATABOLIC METABOLISM THAT SUPPORTS T CELL EXPANSION. IN AIM 1 WE WILL DETERMINE HOW KDM5C REGULATES ANABOLIC METABOLISM THROUGH DIRECT TRANSCRIPTIONAL PROGRAMMING USING CUT&TAG AND IF THIS REGULATION IS INDEPENDENT OF ITS HISTONE DEMETHYLASE ACTIVITY. WE WILL ALSO INVESTIGATE IF KDM5C REGULATES ANABOLIC METABOLISM THROUGH METABOLITE PARTITIONING. IN AIM 2 WE WILL DETERMINE IF AMPK ACTIVATION NEUTRALIZES KDM5C PRO-ANABOLIC FUNCTION THROUGH DEGRADATION AND ALTERED FUNCTION. WE WILL ALSO INVESTIGATE WHETHER KDM5C FUNCTIONS DOWNSTREAM OF AMPK IS REQUIRED TO SUPPORT ENERGY PRODUCTION FROM ALTERNATE PATHWAYS SUCH AS GLUTAMINOLYSIS. OUR WORK IN BOTH AIMS WILL REVEAL NOVEL CYTOPLASMIC FUNCTIONS OF KDM5C. AT COMPLETION, WE WILL DEFINE AN ENTIRELY NOVEL, NON-CANONICAL FUNCTION OF KDM5C, AND THE NEW REGULATORY PATHWAY THAT PROMOTES EXPANSION OF ACTIVATED T CELLS. THIS MECHANISM COULD SERVE AS THE BASIS FOR FUTURE THERAPEUTIC DEVELOPMENT MODULATING T CELL EXPANSION FOR CHRONIC INFECTIONS, CANCER, OR AUTOIMMUNE DISEASES.

TARGETING P53-ASSOCIATED THERAPY RESISTANCE INNF1-RELATED MPNSTS.

REDUCING TOXICITY DRIVEN BY C9ORF72 REPEAT EXPANSIONS IN ALS

METABOLIC TARGETING OF CD4 T CELLS IN CNS AUTOIMMUNITY - PROJECT SUMMARY/ABSTRACT INFLAMMATORY CD4 T HELPER (TH) CELLS ARE CENTRAL TO THE PATHOGENESIS OF MANY AUTOIMMUNE DISEASES— INCLUDING MULTIPLE SCLEROSIS (MS)—WHILE FOXP3+ REGULATORY T (TREG) CELLS PROVIDE PROTECTION. YET, MAJOR GAPS REMAIN IN OUR UNDERSTANDING OF HOW TO SELECTIVELY TARGET PATHOGENIC TH CELLS IN AUTOIMMUNE DISEASE WHILE LIMITING IMPACT ON TREG CELLS AND OTHER IMMUNE CELLS THAT MEDIATE PROTECTIVE IMMUNITY AND TISSUE REPAIR. METABOLIC REPROGRAMMING IS A FUNDAMENTAL PROCESS UNDERLYING THE FATE AND FUNCTION OF ACTIVATED T CELLS. WE HAVE FOUND THAT ALTERING THE METABOLISM OF THE AMINO ACID METHIONINE BY LOWERING METHIONINE LEVELS IN THE DIET SLOWS THE ONSET AND PROGRESSION OF A MOUSE MODEL OF MS (EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS, EAE), IN PART BY LIMITING THE PRESENCE AND FUNCTION OF PATHOGENIC CD4 TH CELLS IN THE CENTRAL NERVOUS SYSTEM (CNS). PRELIMINARY DATA FROM OUR LABORATORY HAS IDENTIFIED METHIONINE ADENOSYLTRANSFERASE 2A (MAT2A)THE ENZYME THAT SYNTHESIZES S-ADENOSYLMETHIONINE (SAM) FROM METHIONINE—AS THE CRITICAL MEDIATOR DOWNSTREAM OF METHIONINE REGULATING CD4 T CELL FUNCTION. HERE, WE WILL APPLY GENETIC AND PHARMACOLOGIC TOOLS (MAT2A CONDITIONAL KNOCKOUT MICE AND MAT2A INHIBITORS) TO CELL CULTURE AND ANIMAL MODELS TO DETERMINE HOW MAT2A CONTROLS CD4 T CELL FATE AND FUNCTION IN THE CONTEXT OF CNS AUTOIMMUNITY. DEFINING HOW MAT2A DIFFERENTIALLY REGULATES PATHOGENIC VERSUS REGULATORY CD4 T CELL FUNCTION IS CRUCIAL TO UNDERSTANDING THE METABOLIC REGULATION OF CNS AUTOIMMUNITY, AND OPENS NEW AVENUES FOR TREATING AUTOIMMUNITY BY TARGETING BIOSYNTHESIS OF SAM. OBJECTIVE: TO DEFINE THE MECHANISMS BY WHICH MAT2A REGULATES THE DIFFERENTIATION AND FUNCTION OF PATHOGENIC TH CELLS VERSUS PROTECTIVE TREG CELLS TO MODULATE NEUROINFLAMMATION IN THE CNS. HYPOTHESIS: INHIBITING MAT2A ALTERS CD4 T CELL FUNCTION THROUGH EFFECTS ON EPIGENETIC PROGRAMMING, RESULTING IN (A) REDUCED TH CELL PATHOGENICITY AND NEUROINFLAMMATION IN EAE, AS WELL AS (B) ENHANCED DEVELOPMENT OF SUPPRESSIVE TREG SUBSETS THAT PROVIDE PROTECTION. SPECIFIC AIMS: (1) DETERMINE HOW MAT2A DIFFERENTIALLY CONTROLS TH VERSUS TREG CELL DIFFERENTIATION, (2) DETERMINE HOW MAT2A INHIBITION COUNTERS T CELL-MEDIATED CNS INFLAMMATION, (3) ESTABLISH HOW MAT2A EPIGENETICALLY PROMOTES TH VERSUS TREG CELL IDENTITY. IMPACT: THE SIGNIFICANCE OF THIS PROPOSAL IS UNDERSTANDING HOW SAM DIRECTS CD4 T CELLS BETWEEN PATHOGENIC AND PROTECTIVE STATES, WITH THE POTENTIAL TO DETERMINE HOW INHIBITING MAT2A CAN BLOCK OR “RETRAIN” PATHOGENIC T CELL RESPONSES AND, THUS, REDUCE CNS AUTOIMMUNITY. WE ALSO EXPECT TO GAIN NEW MECHANISTIC INSIGHT INTO HOW MAT2A INFLUENCES TH AND TREG CELL PROGRAMMING BY REGULATING CHROMATIN MODIFICATIONS. THIS PROJECT WILL LAY THE ESSENTIAL GROUNDWORK FOR FUTURE BASIC DISCOVERY AND TRANSLATIONAL STUDIES WITH NOVEL THERAPEUTICS TARGETING SAM METABOLISM TO TREAT AUTOIMMUNE DISEASES SUCH AS MS.

RESEARCH SPECIALIST SUPPORT-TARGETING DNA METHYLATION AND THE CANCER EPIGENOME

STRUCTURAL MECHANISMS OF AMPK REGULATION

UNDERSTANDING THE EFFECTS OF DIETARY INTERVENTIONS ON PANCREATIC DUCTAL ADENOCARCINOMA THERAPY CANCER - PROJECT SUMMARY/ABSTRACT PANCREATIC DUCTAL ADENOCARCINOMA (PDAC) IS A HIGHLY AGGRESSIVE CANCER WITH LIMITED TREATMENT OPTIONS THAT HAS A FIVE-YEAR SURVIVAL RATE OF <10%. PDAC PROGRESSION IS ASSOCIATED WITH DYSREGULATED TUMOR AND WHOLE- BODY METABOLISM THAT IMPACTS THERAPY AND QUALITY OF LIFE, WHICH HAS MOTIVATED RESEARCH ON HOW TO BEST EXPLOIT METABOLIC DEPENDENCIES IN PDAC FOR BETTER CANCER TREATMENT. NUTRIENT UTILIZATION BY CANCER CELLS IS REGULATED IN PART BY THE AVAILABILITY OF METABOLITES IN THE ENVIRONMENT, AND THE PDAC TUMOR MICROENVIRONMENT IN PARTICULAR IS HIGHLY DESMOPLASTIC, CONSISTING OF STROMAL CELLS, EXTRACELLULAR MATRIX, AND NUTRIENT-POOR INTERSTITIAL FLUID. THESE MICROENVIRONMENTAL FACTORS CAN IMPACT THERAPY RESPONSE, SUGGESTING THAT THE EFFICACIES OF METABOLISM-TARGETED DRUGS CAN BE IMPROVED BY MANIPULATING COMPONENTS OF THE PDAC TUMOR MICROENVIRONMENT. ONE DETERMINANT OF METABOLITE LEVELS IN THE TUMOR MICROENVIRONMENT IS DIET, AND HOW DIET AFFECTS CANCER PROGRESSION AND TREATMENT IS AN IMPORTANT QUESTION FOR MANY PATIENTS. SINCE DIETARY RECOMMENDATIONS TO PATIENTS MUST BE MADE IN THE CONTEXT OF THERAPIES BEING RECEIVED, THE VALUE OF ANY DIETARY INTERVENTION LIKELY LIES IN ITS ABILITY TO ENHANCE TUMOR RESPONSES TO CANCER THERAPIES. UNDERSTANDING THE MOLECULAR MECHANISMS THAT DRIVE SYNERGISTIC INTERACTIONS BETWEEN DIET AND CANCER THERAPIES IS CRITICAL FOR THE TRANSLATION OF DIETARY RECOMMENDATIONS INTO PATIENT CARE. THE MAIN OBJECTIVE OF THIS PROPOSAL IS TO IDENTIFY DIETARY INTERVENTIONS THAT SYNERGIZE WITH CANCER THERAPIES TO IMPAIR PDAC PROGRESSION. MOUSE PDAC MODELS WILL BE USED TO EXAMINE HOW DIFFERENT DIETS ENHANCE THE EFFICACIES OF STANDARD-OF-CARE FOLFIRINOX CHEMOTHERAPY (AIM 1), LIPID METABOLISM INHIBITORS IN DEVELOPMENT FOR CANCER TREATMENT (AIM 2), AND INDUCERS OF FERROPTOSIS, A NON-APOPTOTIC FORM OF CELL DEATH BEING EXPLORED FOR PDAC TREATMENT (AIM 3). MASS SPECTROMETRY-BASED METABOLOMICS AND LIPIDOMICS, STABLE ISOTOPE NUTRIENT TRACING, AND RNA SEQUENCING WILL BE USED TO DETERMINE HOW DIET-MEDIATED CHANGES TO NUTRIENT LEVELS IN THE TUMOR MICROENVIRONMENT ALTER THE METABOLISM OF PDAC TUMORS TO SHAPE THEIR RESPONSES TO THESE THERAPIES. ELUCIDATING THE METABOLIC MECHANISMS THAT UNDERLIE SYNERGISTIC DIET-DRUG COMBINATIONS WILL PROVIDE SCIENTIFIC EVIDENCE THAT CAN BENEFIT PATIENTS WITH GUIDANCE ON HOW TO BEST INCORPORATE DIET AND NUTRITION INTO CANCER THERAPY. THE PROPOSED TRAINING PLAN WILL HELP ME TRANSITION INTO AN INDEPENDENT ACADEMIC POSITION. A TEAM OF OUTSTANDING SCIENTISTS WILL MENTOR ME TO HELP ME ACHIEVE THIS GOAL: DR. TYLER JACKS, A LEADER IN MOUSE CANCER MODELS; DR. OMER YILMAZ, A LEADER IN DIETARY EFFECTS ON CANCER PROGRESSION; DR. CAROLINE LEWIS, A LEADER IN MASS SPECTROMETRY-BASED METABOLOMICS AND LIPIDOMICS TECHNOLOGIES; AND DR. BRIAN WOLPIN, A LEADER IN PDAC EPIDEMIOLOGY. MY TRAINING PLAN ALSO OUTLINES ACTIVITIES THAT WILL HELP ME CULTIVATE MENTORS, IMPROVE MY SCIENTIFIC SKILLSET, IMPROVE SCIENCE COMMUNICATION SKILLS, DEVELOP TEACHING AND MENTORING SKILLS, BUILD MY NETWORK, AND LEARN LAB MANAGEMENT SKILLS. TOGETHER, THE RESEARCH PROPOSAL AND CAREER DEVELOPMENT PLAN WILL PROVIDE ME WITH THE EXPERTISE NEEDED TO BECOME A SUCCESSFUL INDEPENDENT INVESTIGATOR AND EDUCATOR IN THE CANCER BIOLOGY FIELD.

MECHANISMS REGULATING DNA METHYLATION MAINTENANCE IN CHROMATIN

CRYO-EM OF THE EUKARYOTIC REPLISOME

STRUCTURAL AND FUNCTIONAL STUDIES OF RHODOPSIN AND G-PROTEIN COUPLED RECEPTOR KINASES

EPIGENETIC CONTROL

ON-CHIP GLYCAN ANALYSIS OF CLINICAL SPECIMENS

MOLECULAR MECHANISMS UNDERLYING METABOLIC REPROGRAMMING BY PATERNAL BENZENE EXPOSURE - ABSTRACT THERE IS A MAJOR GAP IN OUR UNDERSTANDING OF HOW PATERNAL EXPERIENCES LEADS TO THE INCREASED RISK OF METABOLIC DISEASE IN OFFSPRING. HERE WE FOCUS ON THE HERITABLE METABOLIC EFFECTS OF BENZENE. USING A NOVEL MOUSE MODEL FOR PATERNAL BENZENE EXPOSURE, WE PROVIDE CONVINCING EVIDENCE FOR A CAUSAL RELATIONSHIP BETWEEN PATERNAL BENZENE EXPOSURE AND A SEX SPECIFIC OFFSPRING PHENOTYPE OF IMPAIRED GLYCEMIC CONTROL WHEN UNDER METABOLIC STRESS. WE PROVIDE EVIDENCE FOR CLEAR TRANSCRIPTIONAL SIGNATURES OF METABOLIC DISEASE, SPECIFICALLY IN MALE OFFSPRING. BASED ON THESE DATA WE HYPOTHESIZE THAT PATERNAL BENZENE EXPOSURE TRIGGERS A SPECIFIC SIGNAL IN SPERM, WHICH WHEN TRANSFERRED LEADS TO PREDISPOSITION TO METABOLIC DISEASE. TO TEST THE HYPOTHESIS, WE WILL UTILIZE STATE OF THE ART EXPOSURE SYSTEM, A NOVEL MOUSE MODEL OF INTERGENERATIONAL BENZENE EXPOSURE, AND PHARMACOLOGICAL INTERVENTION TO CRITICALLY ADDRESS THE MECHANISM AND PATHOPHYSIOLOGICAL SIGNIFICANCE UNDERLYING METABOLIC PROGRAMMING TRIGGERED BY BENZENE EXPOSURE. FIRST WE WILL PERFORM IN DEPTH METABOLIC AND ENERGY HOMEOSTASIS PHENOTYPING TO IDENTIFY THE PHYSIOLOGICAL CONSEQUENCES OF PATERNAL BENZENE EXPOSURE IN UNEXPOSED OFFSPRING. SECOND, WE WILL IDENTIFY THE TRANSCRIPTIONAL SIGNATURES OF INTERGENERATIONAL BENZENE EXPOSURE IN INSULIN SENSITIVE TISSUES FROM OFFSPRING. FINALLY WE WILL TEST IF TREATMENT OF PATERNAL METABOLIC DISEASE WITH A WELL CHARACTERIZED DIABETES DRUG PREVENTS THE OFFSPRING PHENOTYPE; TO DEFINE THE MECHANISTIC BASIS FOR BENZENE-INDUCED INTERGENERATIONAL REPROGRAMING, WE WILL IDENTIFY GERMLINE AND EPIGENETIC SIGNATURES OF BENZENE EXPOSURE, AND MECHANISTICALLY CONNECT THE EPIGENETIC CHANGES IN THE GERMLINE TO OFFSPRING TRANSCRIPTIONAL REWIRING. THE PROPOSED RESEARCH WILL, FOR THE FIRST TIME, DEFINE THE CAUSAL RELATIONSHIP BETWEEN PATERNAL EXPOSURE TO BENZENE, AND OFFSPRING METABOLIC DISEASE SUSCEPTIBILITY. SUCH A STUDY WILL BE OF IMPORTANCE TO UNDERSTAND HOW PATERNAL EXPOSURE DRIVES NON-GENETIC, METABOLIC REPROGRAMMING ACROSS GENERATIONS.

NEURODEGENERATIVE MECHANISMS OF ATP13A2-LINKED PARKINSON'S DISEASE AND RELATED DISORDERS - PROJECT SUMMARY PARKINSON’S DISEASE (PD) AND KUFOR-RAKEB SYNDROME (KRS) ARE RELATED PROGRESSIVE NEURODEGENERATIVE MOVEMENT DISORDERS CAUSED PRIMARILY BY THE DEGENERATION OF DOPAMINERGIC NEURONS IN THE SUBSTANTIA NIGRA. CURRENT THERAPIES FOR PD AND KRS ARE PALLIATIVE BUT NO DISEASE-MODIFYING THERAPIES EXIST TODAY. LOSS-OF-FUNCTION MUTATIONS IN THE ATP13A2 (PARK9) GENE HAVE BEEN IDENTIFIED AS A CAUSE OF AUTOSOMAL RECESSIVE JUVENILE-ONSET KRS AND EARLY-ONSET PD, AS WELL AS RARE FAMILIAL FORMS OF HEREDITARY SPASTIC PARAPLEGIA, NEURONAL CEROID LIPOFUSCINOSIS, AND ALS. HOW THE LOSS OF ATP13A2 PRECIPITATES DOPAMINERGIC NEURODEGENERATION IN PD AND KRS REMAINS OBSCURE. IT IS CRITICAL TO IDENTIFY THE MOLECULAR AND CELLULAR MECHANISMS THAT LEAD TO NEURODEGENERATION DUE TO ATP13A2 MUTATIONS IN ORDER TO BETTER UNDERSTAND THE PATHOPHYSIOLOGY OF PD, KRS AND RELATED DISORDERS, AND FOR THE DEVELOPMENT OF NOVEL THERAPEUTIC STRATEGIES. ATP13A2 IS A LYSOSOMAL TRANSMEMBRANE P5B-TYPE ATPASE THAT HAS RECENTLY BEEN SHOWN TO PLAY A ROLE IN THE POLYAMINE TRANSPORT SYSTEM. ATP13A2 MEDIATES THE LYSOSOMAL EFFLUX OF THE POLYAMINES, SPERMIDINE AND SPERMINE, INTO THE CYTOSOL AND MAY ALSO REGULATE THE EXTRACELLULAR UPTAKE OF POLYAMINES INTO CELLS. IMPAIRED POLYAMINE TRANSPORT CAN LEAD TO LYSOSOMAL DYSFUNCTION AND IMPAIRED MITOCHONDRIAL HOMEOSTASIS, AT LEAST IN CULTURED CELLS. HOW ATP13A2 LOSS-OF-FUNCTION MUTATIONS COMPROMISE POLYAMINE TRANSPORT AND LYSOSOMAL FUNCTION IN PD-RELEVANT NEURONAL POPULATIONS AND ANIMAL MODELS IS NOT YET KNOWN. WE HAVE RECENTLY DEVELOPED AN ADULT-ONSET ATP13A2 CONDITIONAL KNOCKOUT (CKO) MOUSE MODEL OF PD/KRS THAT FOR THE FIRST TIME EXHIBITS THE ROBUST AND PROGRESSIVE DEGENERATION OF SUBSTANTIA NIGRA DOPAMINERGIC NEURONS TOGETHER WITH LYSOSOMAL ABNORMALITIES. IN THE PRESENT APPLICATION, WE PROPOSE TO EXPLOIT THIS NEW MOUSE MODEL OF PD/KRS TO ELUCIDATE THE PATHOGENIC MECHANISMS RESULTING FROM ATP13A2 LOSS-OF- FUNCTION MUTATIONS, WITH A KEY FOCUS ON POLYAMINES AND LYSOSOMAL DAMAGE. IN AIM 1, WE WILL EVALUATE LYSOSOMAL DAMAGE, MITOCHONDRIAL FUNCTION, AND POLYAMINE LEVELS OCCURRING IN THE NIGROSTRIATAL PATHWAY AND IN DOPAMINERGIC NEURONS OF ATP13A2 CKO MICE WITH ADVANCING AGE, AND THE EXTENT, ONSET AND PROGRESSION OF MOTOR, NEUROTRANSMITTER AND NEURODEGENERATIVE PHENOTYPES. LYSOSOMAL ABNORMALITIES WILL BE FURTHER DEFINED BY PROTEOMIC PROFILING OF PURIFIED INTACT LYSOSOMES FROM DOPAMINERGIC NEURONS, AND BY SINGLE-NUCLEI RNA SEQUENCING OF THE SUBSTANTIA NIGRA TO IDENTIFY LYSOSOMAL-RELATED GENE SIGNATURES. IN AIM 2, WE WILL EXPLORE THE NEUROPROTECTIVE EFFECTS OF MODULATING POLYAMINE METABOLISM IN THE ATP13A2 CKO MICE. WE WILL USE COMPLEMENTARY PHARMACOLOGICAL AND GENETIC APPROACHES TO EITHER ACTIVATE OR INHIBIT THE POLYAMINE SYNTHESIS PATHWAY VIA THE RATE-LIMITING ENZYME ORNITHINE DECARBOXYLASE 1, OR TO SPECIFICALLY TARGET SPERMIDINE/SPERMINE METABOLISM. THE IMPACT OF ALTERING POLYAMINE METABOLISM ON THE ONSET AND PROGRESSION OF PD/KRS-RELATED NEURODEGENERATION AND LYSOSOMAL DAMAGE IN THE ATP13A2 CKO MICE WILL BE EVALUATED. OUR PROPOSAL IS NOVEL, INNOVATIVE AND TIMELY AND WILL PROVIDE CRITICAL INSIGHT INTO THE MECHANISMS DRIVING ATP13A2-LINKED PD AND KRS.

ROLE OF POLY(ADP-RIBOSE) POLYMERASE 1 IN REGULATING RNA POLYMERASE II ELONGATION AND MRNA SPLICING -PARP1 (POLY-ADP-RIBOSE POLYMERASE), A CHROMATIN-BINDING PROTEIN, IS CRUCIAL FOR MAINTAINING GENOMIC INTEGRITY IN MAMMALS THROUGH REGULATION OF PROLIFERATION, DIFFERENTIATION AND AGING. CONTROL OF GENE EXPRESSION OCCURS AT DIFFERENT LEVELS ? IMPORTANTLY, AT TRANSCRIPTION INITIATION AND MRNA SPLICING. RECENT STUDIES SHOW THAT TRANSCRIPTION AND SPLICING ARE CO-REGULATED AND THAT CHROMATIN STRUCTURE PLAYS A KEY ROLE IN THIS PROCESS. IN AN EARLIER STUDY, THE RESEARCH GROUP FOUND THAT PARP1, WHICH IS WELL KNOWN FOR DNA REPAIR, ALSO REGULATES ALTERNATIVE SPLICING. THIS PROJECT AIMS TO UNDERSTAND HOW PARP1-CHROMATIN COMPLEX COORDINATES RNA POLYMERASE II (RNAPII) ELONGATION, RNA, AND SPLICING FACTORS TO REGULATE CO-TRANSCRIPTIONAL SPLICING. THE OUTCOMES WILL PROVIDE INSIGHTS INTO PARP1 FUNCTION, ESPECIALLY IN RNA BIOGENESIS. THE PROJECT WILL OFFER TRAINING OPPORTUNITIES FOR UNDERREPRESENTED HIGH SCHOOL AND UNDERGRADUATE STUDENTS DURING SUMMER MONTHS. OUTREACH ACTIVITIES WILL INCLUDE MENTORING GRADUATE STUDENTS AT LOCAL AND REGIONAL SMALL COLLEGES, AS WELL AS EARLY CAREER FACULTY AT US MINORITY-SERVING INSTITUTIONS AS PART OF A UNIQUE UNIVERSITY OF KENTUCKY MENTORING PROGRAM. MECHANISTIC DETAILS OF HOW PARP1 REGULATES CO-TRANSCRIPTIONAL SPLICING ARE LACKING. THIS RESEARCH PROJECT WILL TEST THE HYPOTHESES THAT PARP1 REGULATES CO-TRANSCRIPTIONAL SPLICING IN TWO NON-MUTUALLY EXCLUSIVE WAYS: I) BY ACTING AS AN ADAPTER TO RECRUIT SPLICING FACTORS TO RNA; AND/OR II) BY MODULATING CHROMATIN STRUCTURE IN WAYS THAT AFFECT RNAPII ELONGATION AND KINETICS. LARGE-SCALE GENOMICS AND GENE-SPECIFIC APPROACHES WILL BE APPLIED TO MAP THE GENETIC AND BIOCHEMICAL INTERACTIONS OF PARP1-RNA-CHROMATIN. THESE STUDIES WILL PROVIDE SIGNIFICANT NEW KNOWLEDGE ABOUT THIS NEWLY IDENTIFIED ACTIVITY OF PARP1 IN ALTERNATIVE SPLICING. FURTHERMORE, DECIPHERING THE MECHANISM(S) BY WHICH PARP1 MODULATES CHROMATIN TO REGULATE ALTERNATIVE SPLICING AND RNA PROCESSING WILL PROVIDE FUNDAMENTAL INSIGHTS INTO THE COMPLEX ROLE OF CHROMATIN STRUCTURE IN EUKARYOTIC GENE REGULATION. THIS PROJECT IS JOINTLY FUNDED BY THE GENETIC MECHANISMS PROGRAM IN THE MOLECULAR AND CELLULAR BIOSCIENCES DIVISION OF THE BIOLOGICAL SCIENCES DIRECTORATE AND THE ESTABLISHED PROGRAM TO STIMULATE COMPETITIVE RESEARCH (EPSCOR). THIS AWARD REFLECTS NSF'S STATUTORY MISSION AND HAS BEEN DEEMED WORTHY OF SUPPORT THROUGH EVALUATION USING THE FOUNDATION'S INTELLECTUAL MERIT AND BROADER IMPACTS REVIEW CRITERIA.

ONCOPROTEIN-REGULATED LINEAGE PLASTICITY IN ACUTE LEUKEMIA - ABSTRACT CHROMOSOMAL TRANSLOCATIONS INVOLVING THE LYSINE METHYLTRANSFERASE 2A (KMT2A) GENE ARE COMMONLY FOUND IN HIGH-RISK SUBTYPES OF ACUTE LEUKEMIA AND PRODUCE POTENT ONCOPROTEINS THAT OCCUPY THE CHROMATIN AND CAUSE MIS-REGULATION OF DEVELOPMENTAL GENES INVOLVED IN HEMATOPOIESIS. IMMUNOTHERAPIES TARGETING THE CELL SURFACE PROTEIN CD19 HAVE REVOLUTIONIZED THE TREATMENT OF B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA (B-ALL). HOWEVER, KMT2A-REARANGED (KMT2AR) B-ALLS FREQUENTLY CONVERT FROM A B-ALL TO AN ACUTE MYELOID LEUKEMIA (AML) PHENOTYPE TO DOWNREGULATE THE EXPRESSION OF CD19 AND EVADE TARGETED THERAPY. GENOME-WIDE SEQUENCING INDICATES THESE B-ALL-TO-AML-LINEAGE-SWITCHING EVENTS CAN OCCUR IN THE ABSENCE OF ADDITIONAL MUTATIONS, SUGGESTING THAT NON-GENETIC CHANGES IN TUMOR REGULATION CONTRIBUTE TO THIS PHENOTYPIC SHIFT. WE IDENTIFIED GROUPS OF KMT2A ONCOPROTEIN TARGET GENES THAT SHOW DIVERGENT PATTERNS OF TRANSCRIPTIONALLY ACTIVE AND DEVELOPMENTALLY REPRESSED CHROMATIN IN SINGLE CELLS OF THE SAME LEUKEMIA (JANSSENS DH ET AL NATURE GENETICS 2021). MORE RECENTLY, I DEVELOPED SINGLE-CELL COMBINATORIAL INDEXING CUT&TAG (SCICUT&TAG) AND APPLIED THIS METHOD TO PROFILE THE CHROMATIN LANDSCAPES OF THOUSANDS OF CELLS FROM A PRIMARY INFANT KMT2AR B-ALL AS WELL AS THE INFANT KMT2AR B-ALL DERIVED CELL LINE, KOPN-8. I IDENTIFIED A COLLECTION OF 18 ONCOPROTEIN TARGET GENES THAT SHOW THE SAME DIVERGENT REGULATORY PROGRAMS ACROSS HETEROGENEOUS CELL TYPES IN BOTH THE PRIMARY INFANT KMT2AR B-ALL AND THE KOPN-8 CELL LINE. THESE PATTERNS SUGGEST THE KMT2A ONCOPROTEINS ACTIVATE DISTINCT GROUPS OF TARGET GENES IN RELATED LEUKEMIA CELLS, PROVIDING THE FIRST GLIMPSE INTO HOW THESE ONCOPROTEINS CONTRIBUTE TO THE NON-GENETIC HETEROGENEITY OF KMT2AR LEUKEMIA. THIS PROPOSAL WILL (1) ADDRESS HOW KMT2A ONCOPROTEINS CONTRIBUTE TO B-ALL-TO-AML-LINEAGE-SWITCHING EVENTS AND (2) IDENTIFY NOVEL THERAPEUTIC APPROACHES TO EFFECTIVELY IDENTIFY AND TREAT KMT2AR B-ALLS WITH LINEAGE-SWITCHING POTENTIAL. IN THE FIRST AIM, I LEVERAGE THE KOPN-8 CELL LINE TO IDENTIFY ONCOPROTEIN-REGULATED GENES THAT PROMOTE INTRA-TUMORAL HETEROGENEITY OF HIGH-RISK KMT2AR B-ALLS. IN ADDITION, I WILL IDENTIFY CLINICALLY AVAILABLE SMALL MOLECULES WITH THE POTENTIAL TO BE USED IN COMBINATION WITH CD19-DIRECTED IMMUNOTHERAPIES TO PREVENT LINEAGE SWITCHING. IN THE SECOND AND THIRD AIMS, I WILL APPLY SCICUT&TAG TO CHARACTERIZE THE CHROMATIN LANDSCAPES OF A COLLECTION OF PRIMARY KMT2AR B-ALLS AS WELL AS NORMAL HEMATOPOIETIC STEM AND PROGENITOR CELL TYPES. USING COMPARATIVE GENOMICS, I WILL IDENTIFY NOVEL BIOMARKERS THAT LABEL KMT2AR B-ALL CELLS WITH LINEAGE-SWITCHING POTENTIAL AND THAT CAN POTENTIALLY BE USED TO TARGET THIS POPULATION VIA NOVEL IMMUNOTHERAPIES. THIS PROPOSAL APPLIES THE GENOMICS METHODS DR. DEREK JANSSENS DEVELOPED AS A POSTDOC TO IDENTIFY NOVEL TREATMENT STRATEGIES TO TARGET HIGH-RISK KMT2AR B-ALLS AND WILL ESTABLISH LONG-TERM COLLABORATIONS WITH COMPUTATIONAL AND TRANSLATIONAL BIOLOGISTS AS WELL AS CLINICAL RESEARCHERS AS HE TRANSITIONS TO A PRINCIPAL INVESTIGATOR ROLE AND ESTABLISHES AN INDEPENDENT RESEARCH PROGRAM AT A TIER ONE INSTITUTION.

MECHANISMS OF GAIN-OF-REPEAT-TOXICITY AND LOSS-OF-C9ORF72-FUNCTION IN FTD AND ALS - PROJECT SUMMARY/ABSTRACT FRONTOTEMPORAL DEMENTIA (FTD) AND AMYOTROPHIC LATERAL SCLEROSIS (ALS) ARE INCURABLE, ADULT-ONSET NEURODEGENERATIVE DISEASES. OUR UNDERSTANDING OF THEIR ETIOLOGY IS INCOMPLETE, AND NO TREATMENT CAN HALT THEIR PROGRESSION. THIS PROJECT FOCUSES ON THE MOST COMMON GENETIC CAUSE OF FTD AND ALS–THE GGGGCC REPEAT EXPANSION IN THE C9ORF72 GENE (KNOWN AS C9FTD/ALS). THIS EXPANSION CAN MEDIATE BOTH GAIN-OF-TOXICITY (VIA REPEAT RNAS AND ACCUMULATION OF DIPEPTIDE-REPEAT PROTEINS) AND LOSS OF THE NORMAL FUNCTION OF THE C9ORF72 PROTEIN. WHETHER AND HOW GAIN AND LOSS MECHANISMS INTERACT WITH EACH OTHER AND WHETHER THERE ARE CONVERGENT PATHWAYS TO PROVOKE THE ACCELERATED DECLINE THAT CHARACTERIZES THESE TWO DISTINCT DISEASES REMAIN UNRESOLVED QUESTIONS. THE OBJECTIVE OF THIS PROJECT IS THEREFORE TO DEFINE THE CONTRIBUTIONS OF GAIN-OF-REPEAT-TOXICITY AND LOSS- OF-C9ORF72-FUNCTION TO THE COGNITIVE AND MOTOR PHENOTYPES USING MOUSE MODELS (OUR ESTABLISHED MODEL OF FTD AND OUR NEW MOUSE MODEL OF C9ALS, WHICH FOR THE FIRST TIME SUCCESSFULLY RECAPITULATES C9ALS HALLMARKS). OUR PRELIMINARY DATA SHOW THAT (I) REPEAT EXPRESSION BY ITSELF LEADS TO ALS PHENOTYPES, WHICH SHEDS LIGHT INTO A LONG-STANDING QUESTION IN THE FIELD; (II) SUPPRESSING MICROGLIAL ACTIVATION DELAYS THE ONSET OF PARALYSIS IN OUR C9ALS MODEL; AND (III) REDUCTION OF C9ORF72 ACCELERATES MOTOR FUNCTION DECLINE AND PARALYSIS ONSET, INCREASES MICROGLIOSIS, AND ASSOCIATES WITH PROFOUND AND REGIONALIZED TRANSCRIPTIONAL CHANGES IN MICROGLIA IN THE C9ALS MODEL. BECAUSE MICROGLIA HAVE REGIONAL SPECIFICITY IN THE CENTRAL NERVOUS SYSTEM, IT IS POSSIBLE THAT C9ORF72 REDUCTION WOULD AFFECT C9FTD AND C9ALS MICROGLIA DIFFERENTLY. ALTOGETHER, WE HYPOTHESIZE THAT MICROGLIAL OVERACTIVATION MEDIATED BY C9ORF72-REDUCTION ACCELERATES THE DEVELOPMENT OF COGNITIVE AND MOTOR DEFICITS IN FTD AND ALS TRIGGERED BY EXPANDED REPEAT EXPRESSION. TO TEST THIS HYPOTHESIS, WE WILL FIRST IDENTIFY THE NEUROPATHOLOGICAL CHANGES LINKED TO NEURONAL GAIN-OF-TOXICITY IN OUR C9ALS MODEL. NEXT, WE WILL DEFINE THE EFFECTS OF SUPPRESSING MICROGLIAL ACTIVATION TO DISSECT THEIR ROLE (NON- CELL AUTONOMOUS). WE WILL ALSO DETERMINE WHETHER REDUCED C9ORF72 FUNCTION WORSENS THE MOTOR DEFICITS IN C9ALS, AS WE PREVIOUSLY SHOWED IN C9FTD. WE WILL THEN CONFIRM THAT MICROGLIAL REDUCTION OF C9ORF72 IS NECESSARY FOR THE ACCELERATED PHENOTYPE, BY DEFINING THE EFFECTS OF KNOCKING OUT C9ORF72 EXCLUSIVELY IN MICROGLIA ON BOTH COGNITIVE/MOTOR PHENOTYPES AND NEUROPATHOLOGY. LAST, WE WILL IDENTIFY MICROGLIAL TRANSCRIPTIONAL CHANGES AND SUBPOPULATIONS IN BOTH MODELS AS NEURODEGENERATION PROGRESSES, USING SPATIAL TRANSCRIPTOMICS AT SINGLE- CELL LEVEL AND VALIDATION IN C9FTD/ALS POSTMORTEM TISSUE. ONCE COMPLETED, THE PROPOSED RESEARCH IS EXPECTED TO UNCOVER MOLECULAR MECHANISMS UNDERLYING THESE DISORDERS WITH THE POTENTIAL TO BE DEVELOPED INTO NOVEL THERAPEUTIC INTERVENTIONS. SINCE THE C9ORF72 REPEAT EXPANSION IS ALSO IMPLICATED IN SPORADIC CASES OF ALS AND FTD, OUR FINDINGS CAN HAVE BROAD RELEVANCE.

IMPACT OF CHROMATIN STRUCTURE ON DNA REPLICATION

DECIPHERING THE ROLE OF FRIZZLED RECEPTORS IN PALATAL DEVELOPMENT - PROJECT SUMMARY/ABSTRACT CLEFT LIP AND/OR CLEFT PALATE HAS AN INCIDENCE OF APPROXIMATELY 1 IN 700 BIRTHS MAKING IT ONE OF THE MOST COMMON CONGENITAL BIRTH DEFECTS. GAINING A MORE COMPLETE UNDERSTANDING OF THE GENETICS AND SIGNALING MECHANISMS INVOLVED WILL PROVIDE A FOUNDATION FOR IMPROVING TREATMENT OF PATIENTS WITH OROFACIAL CLEFTS, ULTIMATELY REDUCING AN ENORMOUS BURDEN ON THE HEALTHCARE SYSTEM. SEVERAL SIGNALING PATHWAYS INTERSECT TO REGULATE THE PROPER DEVELOPMENT OF THE PALATE WHICH MAKES DEVELOPING TARGETED THERAPIES TO TREAT OR PREVENT CLEFT PALATE CHALLENGING. DISSECTING THE COMPONENTS OF EACH PATHWAY INVOLVED WILL PROVIDE A MORE COMPLETE PICTURE OF PALATOGENESIS. THE WNT SIGNALING PATHWAY IS AN IMPORTANT REGULATOR OF PALATAL DEVELOPMENT AND REGULATES EARLY PATTERNING BY CRANIAL NEURAL CREST CELLS THROUGH REGULATION OF INDUCTION, MIGRATION, AND DIFFERENTIATION OF THESE CELLS. WNT SIGNALING ALSO INTERSECTS MANY MORPHOGENIC PATHWAYS THAT REGULATE PALATAL SHELF ELONGATION, ELEVATION, AND FUSION. FRIZZLEDS (FZDS) ARE TRANSMEMBRANE RECEPTORS FOR WNT LIGANDS AND MUTATIONS IN SOME OF THE 10 FZD GENES HAVE BEEN IDENTIFIED IN PATIENTS WITH BOTH SYNDROMIC AND NON-SYNDROMIC CLEFT LIP AND/OR PALATE. SPECIFICALLY, HETEROZYGOUS NONSENSE MUTATIONS IN FZD2 HAVE BEEN IDENTIFIED IN PATIENT FAMILIES WITH ROBINOW SYNDROME (RS) OR AUTOSOMAL DOMINANT OMODYSPLASIA (ADO), SYNDROMES WHICH ARE CHARACTERIZED BY LIMB REDUCTIONS AND CRANIOFACIAL ANOMALIES INCLUDING CLEFT PALATE. WHILE THESE MUTATIONS IN FZD2 WERE THOUGHT TO CAUSE HAPLOINSUFFICIENCY, HETEROZYGOUS DELETION OF FZD2 IN MICE DOES NOT LEAD TO CLEFT PALATE. WE WILL USE MOUSE MODELS HARBORING RS/ADO-ASSOCIATED MUTATIONS TO BETTER UNDERSTAND HOW FZD2 FUNCTIONS IN THE DEVELOPING PALATE. GIVEN THE ASSOCIATION OF MUTATIONS IN OTHER WNT PATHWAY GENES (WNT5A, ROR2, DVL) IN RS AND ADO, WE HYPOTHESIZE THAT THESE COMPONENTS SIGNAL TOGETHER WITH FZD2 TO REGULATE PALATOGENESIS. WE HYPOTHESIZE THAT FZD2 REGULATES CELL MIGRATION AND PALATAL PATTERNING THROUGH A NON-CANONICAL WNT5A- ROR2 PATHWAY AND FURTHER HYPOTHESIZE THAT HUMAN RS- AND ADO-ASSOCIATED FZD2 MUTATIONS ACT DOMINANTLY TO INTERFERE WITH FZD SIGNALING. TO ACHIEVE THESE RESEARCH GOALS, WE PROPOSE THE FOLLOWING AIMS: 1) DETERMINE THE PHENOTYPIC CONSEQUENCES OF FZD2 DELETION AND RS/ADO-FZD2 MUTATIONS ON PALATAL DEVELOPMENT IN MICE, AND 2) DETERMINE THE MOLECULAR MECHANISMS BEHIND FZD2 REGULATION OF PALATE DEVELOPMENT. DATA GENERATED IN THIS PROPOSAL WILL ULTIMATELY SUPPORT THE DEVELOPMENT OF NOVEL THERAPEUTIC APPROACHES AND INTERVENTIONS IN WNT SIGNALING-RELATED DISEASES. THESE RESEARCH PLAN AND CAREER DEVELOPMENT ACTIVITIES PROPOSED HERE WILL FORM A SOLID BASIS FOR MY FUTURE INDEPENDENT RESEARCH PROGRAM.

REGULATION OF ENDOPLASMIC RETICULUM (ER) QUALITY CONTROL ACROSS CELL TYPES WITH UNIQUE ER MORPHOLOGIES. - PROJECT SUMMARY THE ENDOPLASMIC RETICULUM (ER) PERFORMS MANY VITAL CELLULAR FUNCTIONS, INCLUDING PROTEIN AND LIPID SYNTHESIS AND CALCIUM STORAGE, AND IMPORTANTLY, THE ER IS A CELL HOMEOSTASIS SENSOR AND SIGNALING NETWORK. FOR EXAMPLE, WHEN PROTEIN SYNTHESIS GETS OVERLOADED IN THE ER, THE ER SIGNALS THE INTEGRATED STRESS RESPONSE. THE ER ALSO FORMS CONTACTS WITH OTHER ORGANELLES TO SIGNAL AND TRANSPORT MATERIALS LIKE CALCIUM AND LIPIDS – ER HEALTH IS THEREFORE AT THE FOUNDATION OF CELLULAR HEALTH AND DISEASE PREVENTION. IN ORDER TO PERFORM ER LOCALIZED PROTEIN SYNTHESIS AND SIGNALING TO OTHER ORGANELLES, THE ER NETWORK DYNAMICALLY REARRANGES TO FORM STRUCTURES FOR DEDICATED FUNCTIONS. FOR INSTANCE, SECRETORY CELLS ARE FILLED WITH EXPANSIVE RIBOSOME-STUDDED SHEETS FOR PROTEIN SYNTHESIS, WHEREAS CELLS WHICH ARE CONSTANTLY SIGNALING, LIKE NEURONS, ARE JAM-PACKED WITH BUNDLED TUBULES. SO, WHAT DEFINES THESE FUNCTIONS AND SHAPES? CHANGING ER PROTEIN LEVELS ALTERS BOTH SHAPE AND FUNCTION. I PREVIOUSLY SHOWED THAT ER-PHAGY, A PROCESS WHERE ER MEMBRANE AND ATTACHED PROTEINS ARE SELECTIVELY DEGRADED, IS REQUIRED TO SET UP THE ER NETWORK AS CELLS DIFFERENTIATE. SPECIFICALLY, BY DELETING KEY AUTOPHAGY AND SELECTIVE ER-PHAGY MACHINERIES IN STEM CELLS, I SHOWED ER ACCUMULATION IN AXONS AND ALTERATIONS IN THE ER PROTEOME WHEN THEY CONVERTED TO NEURONS. THESE OBSERVATIONS RAISE A KEY QUESTION: ARE THERE SIMILARITIES AND/OR DIFFERENCES IN HOW THESE MACHINERIES FUNCTION OR ARE REGULATED IN CELL TYPES WITH DIFFERENT FUNCTIONAL OUTPUTS? IN ADDITION, WE ARE MISSING KEY DETAILS ABOUT ER-PHAGY SITE FORMATION AND REGULATION, BOTH UNDER BASAL CONDITIONS IN DIFFERENT NON-DIVIDING CELL TYPES AND IN POST MITOTIC CELLS WHEN STRESSORS LIKE ANTIOXIDANT DAMAGE OR PROTEIN MISFOLDING OCCUR. THUS, I WILL COMBINE QUANTITATIVE METHODS INCLUDING ER PROTEIN TURNOVER ASSAYS AND MASS SPECTROMETRY, WITH TOP-OF-THE-LINE IMAGING TECHNIQUES INCLUDING LIVE-CELL IMAGING AND CRYO-ELECTRON TOMOGRAPHY, AND FUNCTIONAL ASSAYS TO ESTABLISH HOW ER CLEARANCE CONTROLS (I) THE ER PROTEOME LANDSCAPE, (II) ER STRUCTURAL REARRANGEMENTS INTO TUBULES AND SHEETS, AND (III) ER FUNCTIONS VITAL TO CELL-TYPE ESTABLISHMENT AND HOMEOSTASIS. IN ESSENCE, THE ULTIMATE GOAL OF MY RESEARCH PROGRAM IS TO PROVIDE A BLUEPRINT DETAILING CONNECTIONS BETWEEN THE UNDERLYING ER PROTEOME AND HOW ITS REGULATION CONTROLS CELLULAR FUNCTION AS CELLS SPECIALIZE INTO UNIQUE TYPES OR RESPOND TO STRESS CONDITIONS. WITH EXTENSIVE EXPERIENCE STUDYING BOTH ER-PHAGY AND ER DYNAMICS, I AM WELL SUITED TO ACHIEVE THE GOALS OF THE PROPOSED STUDY, WHICH LIES AT THE INTERSECTION OF THE FIELDS OF ORGANELLE BIOGENESIS AND QUALITY CONTROL. DEFINING THE MECHANISMS DRIVING THE CLEARANCE OF REGIONS OF THE ER NETWORK AND THE SPECIFICITY OF THESE ER PROTEINS IN HOMEOSTASIS AND DISEASE IS ESSENTIAL TO THE FIELD. IN CONCLUSION, THE PROPOSED WORK WILL LAY THE FOUNDATION FOR MY LABORATORY, ACCELERATING OUR RESEARCH TO THE FOREFRONT OF OUR FIELD AND DEEPENING OUR UNDERSTANDING OF KEY PROCESSES RELEVANT TO THE AREAS OF CELL AND DEVELOPMENTAL BIOLOGY.

LINEAGE-TRACED SINGLE-CELL ANALYSIS OF EPIGENETIC CLOCKS - PROJECT SUMMARY / ABSTRACT IT HAS LONG BEEN KNOWN THAT DNA METHYLATION PROFILES IN THE GENOME PROGRESSIVELY CHANGE WITH INCREASING AGE. IN THE PAST DECADE, THIS KNOWLEDGE HAS BEEN LEVERAGED TO CREATE EPIGENETIC CLOCK MODELS CALIBRATED TO ORGANISMAL CHRONOLOGICAL AGE OR CELLULAR REPLICATIVE HISTORY. ENVIRONMENTAL EXPOSURES, STRESS CONDITIONS, AND DISEASE STATES HAVE BEEN SHOWN TO ACCELERATE ESTIMATED MODEL AGE BEYOND ACTUAL CHRONOLOGICAL AGE. HOWEVER, THE MECHANISMS UNDERLYING THIS ACCELERATED BIOLOGICAL AGING ARE NOT WELL UNDERSTOOD. THE DISCRETE BINARY NATURE OF DNA METHYLATION AT SELECT CPG SITES IMPLIES THAT QUANTITATIVE SHIFTS IN DNA METHYLATION LEVELS REFLECT CHANGES IN UNDERLYING CELLULAR HETEROGENEITY IN THE SAMPLE. BULK ANALYSIS OF EPIGENETIC CLOCKS CANNOT DISTINGUISH BETWEEN AGE-ASSOCIATED CHANGES IN CELL POPULATIONS, VERSUS PROGRESSIVE DNA METHYLATION CHANGES WITHIN MOST CELLS IN A POPULATION. RESOLVING THIS DISTINCTION, AND UNDERSTANDING THE BIOLOGICAL MECHANISMS UNDERLYING EPIGENETIC CLOCK RESEARCH WOULD BENEFIT FROM THE ABILITY TO STUDY EPIGENETIC CLOCKS AT THE SINGLE-CELL LEVEL. HOWEVER, EXISTING TECHNOLOGY FOR SINGLE-CELL DNA METHYLATION PROFILING PROVIDES TOO SPARSE GENOMIC COVERAGE TO ALLOW DIRECT APPLICATION OF EPIGENETIC CLOCKS. WE HAVE DEVELOPED ADVANCED NEW SINGLE-CELL WHOLE- GENOME BISULFITE SEQUENCING TECHNOLOGY THAT PROVIDES SUFFICIENTLY DEEP GENOMIC COVERAGE TO ALLOW APPLICATION OF EPIGENETIC CLOCKS, AND IN-DEPTH CHARACTERIZATION OF DNA METHYLATION BIOLOGY. LINEAGE TRACING TECHNOLOGY IN MOUSE MODELS HAS YIELDED NEW INSIGHTS INTO STEM-CELL BIOLOGY AND THE PHYLOGENETIC RELATIONSHIP AMONG CELLS IN VIVO. RECENTLY DEVELOPED ADVANCED LINEAGE TRACING TECHNOLOGY EMPLOYING CRISPR/CAS9-TDT CUT AND REPAIR- GENERATED UNIQUE BARCODES HAS FACILITATED UNPRECEDENTED HIGH-RESOLUTION ANALYSIS OF CELL LINEAGES. IN THE ONE SPECIFIC AIM FOR THIS PROJECT, WE PROPOSE TO USE OUR ADVANCED SINGLE-CELL WHOLE-GENOME BISULFITE SEQUENCING TO CHARACTERIZE INTERCELLULAR HETEROGENEITY OF EPIGENETIC CLOCKS IN LINEAGE-TRACED CELLS DERIVED FROM THE MOUSE COLON AND SMALL INTESTINE. WE WILL USE TWO PULSED INDUCTIONS OF BARCODE GENERATION TO BOTH MARK REGIONAL LINEAGES IN EMBRYONIC DEVELOPMENT AND TO SECONDARILY MARK POSTNATAL INTESTINAL CRYPT MORPHOGENESIS. WE WILL COLLECT AND ANALYZE SINGLE CELLS FROM THE MOUSE COLON AND SMALL INTESTINE AT FOUR DIFFERENT AGES TO ASSESS BOTH WITHIN-MOUSE LINEAGE RELATIONSHIPS, AS WELL AS MEASURE LOSS OF BARCODE DIVERSITY AS A MEASURE OF STEM-CELL EXHAUSTION. OUR HIGH-COVERAGE SINGLE-CELL WHOLE-GENOME BISULFITE SEQUENCE ANALYSIS WILL ALLOW US TO BOTH RECOVER THE UNIQUE LINEAGE BARCODES FROM EACH CELL, AS WELL AS ASSESS DNA METHYLATION TO APPLY AVAILABLE MOUSE EPIGENETIC CLOCKS, IDENTIFY CELL TYPE BY THE ANALYSIS OF HYPOMETHYLATION OF CELL-TYPE-SPECIFIC ENHANCERS, ASSESS AGE-ASSOCIATED HYPERMETHYLATION OF POLYCOMB REPRESSIVE COMPLEX TARGET GENES, AND MEASURE REPLICATIVE HISTORY BY THE DEGREE OF HYPOMETHYLATION AT LATE-REPLICATING, LAMINA-ATTACHED REGIONS OF THE GENOME. THIS STUDY WILL PROVIDE A PROOF-OF-PRINCIPLE FOR FUTURE STUDIES THAT WILL EXPLORE THE IMPACTS OF ENVIRONMENTAL INTERVENTIONS AND GENETIC MANIPULATION ON BIOLOGICAL AGING AND EPIGENETIC CLOCKS IN VARIOUS CELL TYPES.

DETECTING ALPHA-SYNUCLEIN AGGREGATES

DOES MICROGLIAL ACTIVATION INFLUENCE PROPAGATION OF ALPHA-SYNUCLEIN PATHOLOGY

EPIGENETIC CONTROL AND PROBABILISTIC DISEASE PROGRAMMING - PROJECT SUMMARY DOGMA TEACHES THAT AN INDIVIDUAL’S PHENOTYPE (AND DISEASE) RESULTS FROM GENETICS, THE ENVIRONMENT, AND THEIR INTERACTIONS. YET NUMEROUS STUDIES OF MONOZYGOTIC HUMAN TWINS AND ISOGENIC ANIMAL MODELS INDICATE THAT SIGNIFICANT PORTIONS OF DISEASE VARIABILITY CANNOT BE EXPLAINED BY GENETIC AND ENVIRONMENTAL INPUTS. FOR EXAMPLE, GENETICS ACCOUNTS FOR ~50% AND ENVIRONMENT ACCOUNTS FOR <1% OF METABOLIC DISEASE IN MONOZYGOTIC TWINS, LEAVING A STRIKING UNEXPLAINED VARIANCE (DISCORDANCE) OF ~50%. SIMILAR RESULTS ARE REPORTED FOR MANY OTHER HUMAN DISEASES AND COMPLEX TRAITS. THE LONG-TERM GOAL OF THIS PROJECT IS TO UNDERSTAND THE ORIGINS AND REGULATORY MECHANISMS UNDERLYING THIS UNEXPLAINED PHENOTYPIC AND DISEASE VARIATION. THE OPERATING HYPOTHESES ARE THAT PHENOTYPIC VARIATION ITSELF IS A QUANTITATIVE TRAIT, AND THERE ARE PROBABILISTIC, INTRACELLULAR PROCESSES REGULATED BY EPIGENETIC MECHANISMS THAT ARE RESPONSIBLE FOR SIGNIFICANT PORTIONS OF UNEXPLAINED PHENOTYPIC VARIATION. THE HYPOTHESIS IS BASED ON PRIOR WORK WITH HAPLOINSUFFICIENT TRIM28+/D9 MICE, WHERE GENETICALLY AND ENVIRONMENTALLY IDENTICAL LITTERMATES EMERGE AS EITHER LEAN OR OBESE, WITH FEW INTERMEDIATES (I.E., AN EPIGENETICALLY DRIVEN OBESITY POLYPHENISM). TRIM28 MRNA EXPRESSION LEVELS ALSO PREDICT OBESITY IN HUMAN CHILDREN. THIS BODY OF WORK SUGGESTS THAT EPIGENETIC SILENCERS ARE MASTER REGULATORS OF PROBABILISTIC PROCESSES IN HUMANS, AND MAY ALSO BE RESPONSIBLE FOR REGULATING PHENOTYPIC VARIATION. HOWEVER, THE EPIGENETIC MECHANISMS AND GENOMIC LOCI RESPONSIBLE FOR THIS REMARKABLE, PROBABILISTIC, AND BI-STABLE DISEASE POTENTIAL ARE UNKNOWN. BEFORE THE FIELD CAN EVEN BEGIN DECIPHERING THE (EPI)GENETIC ARCHITECTURE THAT REGULATES PROBABILISTIC PROCESS AND VARIABILITY, WE NEED TO FIRST DETERMINE WHICH TYPE OF EPIGENETIC SILENCERS ARE INVOLVED IN THE BISTABLE SWITCH FROM ONE DEVELOPMENT TRAJECTORY TO THE OTHER, AND WHICH GENETIC LOCI RESPOND TO THE SWITCH. WE WILL MEET THIS OBJECTIVE BY PERFORMING A FOCUSED GENE-GENE AND GENE-ENVIRONMENT EPISTASIS EXPERIMENT WITH TRIM28+/D9 MICE, AND SCORE THE OFFSPRING FOR STABILITY, SEVERITY, AND FREQUENCY (I.E. THE VARIABILITY) OF BISTABLE METABOLIC DISEASE. FOR CROSSES SHOWING ADDITIVE EFFECTS ON DISEASE VARIABILITY, WE WILL PERFORM TOTAL RNASEQ AND RELACS IN PRECURSOR AND MATURE ADIPOCYTES TO IDENTIFY GENOMIC LOCI ASSOCIATED WITH METABOLIC DISEASE SWITCHES AND VARIATION. WITH THIS KNOWLEDGE, WE WILL BE ABLE TO GENERATE SPECIFIC HYPOTHESES ABOUT THE GENES, PATHWAYS, AND PHYSIOLOGICAL MECHANISMS THAT NOT ONLY REGULATE METABOLIC DISEASE, BUT CONTROL PHENOTYPIC VARIATION AS A QUANTITATIVE TRAIT.

IDENTIFICATION OF ENVIRONMENTAL CHEMICALS CAPABLE OF INDUCING HEALTH IMPAIRMENTS ACUTELY AND ACROSS GENERATIONS - SUMMARY EPIDEMIOLOGICAL EVIDENCE SUGGESTS THAT PARENTAL ENVIRONMENTAL EXPOSURE CORRELATES WITH INCREASED RISK OF CHRONIC DISEASES IN CHILDREN AND EVEN GRANDCHILDREN. ANIMAL STUDIES PROVIDE CLEAR EVIDENCE THAT ANCESTRAL EXPOSURE TO CHEMICALS SUCH AS ENDOCRINE DISRUPTORS, DIOXINS, OR PESTICIDES CAN INDUCE NON-MENDELIAN (BUT HERITABLE) HEALTH IMPAIRMENTS ACROSS GENERATIONS. MOST RESEARCH THUS FAR HAS FOCUSED ON RODENTS, WHICH IMPOSES LIMITS OF A FEW COMPOUNDS AT A TIME IN SMALL NUMBERS OF ANIMALS AND STATISTICALLY SOUND EXPERIMENTS COVERING MULTIPLE GENERATIONS REQUIRE YEARS. THUS, COMPREHENSIVE ATTEMPTS TO IDENTIFY CHEMICALS CAPABLE OF INDUCING INTERGENERATIONAL EFFECTS HAVE BEEN COMPLETELY LACKING. IMPORTANTLY, THE MECHANISMS BY WHICH PARENTAL EXPOSURE LEADS TO HERITABLE HEALTH EFFECTS IN SUBSEQUENT GENERATIONS ARE STILL POORLY UNDERSTOOD. THE OBJECTIVES OF THIS PROJECT ARE TO 1) DEVELOP AND VERIFY A NEW HIGH-THROUGHPUT FRUIT FLY (DROSOPHILA) MODEL FOR CHEMICAL EXPOSURE AND INTERGENERATIONAL HEALTH EFFECTS, AND 2) IDENTIFY REPROGRAMING SIGNATURES TO HELP UNCOVER POTENTIAL BIOLOGICAL MECHANISMS FOR TRANSMITTING THOSE NON-GENETIC EFFECTS FROM PARENT TO OFFSPRING.

MECHANISMS OF HISTONE CROSSTALK WITH BACTERIAL PATHOGENS - PROJECT SUMMARY BACTERIAL PATHOGENS MODULATE THE PHYSIOLOGY OF HUMAN CELLS BY SECRETING EFFECTOR PROTEINS THAT ASSIST PATHOGEN IMMUNE EVASION AND PROLIFERATION. THE DIVERSE REPERTOIRE OF SECRETED PATHOGENIC EFFECTORS RE-WIRE AN ARRAY OF CELLULAR SIGNALING PATHWAYS THAT ARE AT THE CORE OF HOST CELL FUNCTION. ONE CLASS OF THESE EFFECTOR PROTEINS ENTER THE HUMAN CELL NUCLEUS AND DIRECTLY MODULATE TRANSCRIPTIONAL PROGRAMS OF THE HOST BY ALTERING OUR EPIGENETIC CODE. THESE SO-CALLED EPIGENETIC “NUCLEOMODULINS” REPRESENT AN IMPORTANT ASPECT OF HOST-PATHOGEN INTERACTION, HOWEVER VERY LITTLE IS UNDERSTOOD ABOUT HOW THESE EFFECTORS MIGHT INTERACT WITH AND SENSE THE EPIGENETIC LANDSCAPE OF THE HOST CELL DURING INFECTION. ONE OF THE MOST WELL-STUDIED EPIGENETIC NUCLEOMODULINS IS ROMA, A HISTONE LYSINE METHYLTRANSFERASE FROM LEGIONELLA PNEUMOPHILA, THE CAUSATIVE AGENT OF LEGIONNAIRES DISEASE. ROMA MONO-, DI- AND TRI-METHYLATES HISTONE H3K14, SILENCES THE EXPRESSION OF HOST CELL IMMUNE RESPONSE GENES AND ENABLES EFFICIENT REPLICATION OF L. PNEUMOPHILA INSIDE THE HOST CYTOPLASM. ROMA CONTAINS SEVERAL DOMAINS THAT ARE CONSERVED IN EUKARYOTIC CHROMATIN-BINDING PROTEINS, SUGGESTING THAT ITS H3K14 METHYLATION ACTIVITY MAY BE REGULATED BY INTERACTING WITH HISTONE MODIFICATIONS OF THE HOST. IN EUKARYOTES, THE PHENOMENON WHERE AN EXISTING HISTONE MODIFICATION CONTROLS THE RECOGNITION OR DEPOSITION OF ANOTHER IS CALLED HISTONE MODIFICATION CROSSTALK. THESE HISTONE CROSSTALK MECHANISMS UNDERLIE THE COMPLEX REGULATION OF TRANSCRIPTION AND GENOME ARCHITECTURE IN EUKARYOTES. HOWEVER, THERE ARE NO KNOWN EXAMPLES OF A BACTERIAL PATHOGEN THAT CAN INTERPRET HUMAN HISTONE MODIFICATIONS THROUGH HISTONE CROSSTALK AND ALTER THE HUMAN EPIGENOME. SUCH AN OBSERVATION WOULD GREATLY EXPAND OUR UNDERSTANDING OF HOST-PATHOGEN INTERACTIONS DURING INFECTION. IN THIS R21 PROPOSAL, WE WILL USE THE L. PNEUMOPHILA EFFECTOR ROMA AS A MODEL TO ESTABLISH IF PATHOGENIC BACTERIA CAN INTERACT WITH AND INTERPRET EXISTING HISTONE MODIFICATIONS THROUGH CROSSTALK MECHANISMS. IN AIM 1, WE WILL SUBJECT ROMA AND INDIVIDUAL ROMA DOMAINS TO A COMPREHENSIVE THREE-PART SCREEN TO ESTABLISH IF ANY HUMAN HISTONE MODIFICATIONS CAN BIND TO ROMA OR REGULATE ITS CATALYTIC ACTIVITY. THEN, IN AIM 2 WE WILL ESTABLISH A CRYO- EM SCREENING WORKFLOW TO IDENTIFY OPTIMAL SUBSTRATES AND FREEZING CONDITIONS THAT WILL BE NECESSARY TO DETERMINE A STRUCTURE OF ROMA BOUND TO ITS PREFERRED SUBSTRATE NUCLEOSOME.

CHROMATIN ARCHITECTURE AS A DETERMINANT OF DENDRITIC CELL FUNCTION

DEVELOPING NOVEL TREATMENTS FOR PARKINSON'S DISEASE BY TARGETING MOLECULAR PATHWAYS THAT HAVE BEEN SHOWN TO MODULATE AGING

PROMOTING SURVIVAL OF DOPAMINE NEURONS IN MODELS OF PARKINSONS DISEASE USING A NOVEL TRANSCRIPTIONAL REGULATOR

ROLE OF T CELLS IN ALPHA-SYNUCLEIN PATHOLOGY - SUMMARY/ABSTRACT PARKINSON’S DISEASE (PD) IS A PROGRESSIVE NEURODEGENERATIVE DISORDER, AFFECTING OVER 1 MILLION PEOPLE IN THE U.S. ALONE. THERE ARE NO THERAPIES TO STOP ITS PROGRESSION, LIKELY DUE TO OUR LACK OF UNDERSTANDING OF THE MECHANISMS UNDERLYING PD. SOLID EVIDENCE HAS EMERGED INDICATING THAT THE SPREADING OF A-SYNUCLEIN (A-SYN) PATHOLOGY ACROSS THE BRAIN AND THE PRESENCE OF NEUROINFLAMMATION PLAY KEY ROLES. PROPAGATION OF A-SYN AGGREGATES ALONG NEURAL NETWORKS ASSOCIATES WITH THE DEVELOPMENT OF NEUROPATHOLOGY AND SYMPTOM WORSENING. ACCUMULATION OF A-SYN AGGREGATES IS THOUGHT TO INCREASE NEUROINFLAMMATION AND NEUROINFLAMMATION HAS BEEN SHOWN TO PROMOTE A-SYN AGGREGATION, EVENTUALLY LEADING TO NEURONAL LOSS. IT IS ALSO KNOWN THAT THE IMMUNE SYSTEM IS ACTIVATED IN PD, FEATURING ELEVATED PRO-INFLAMMATORY CYTOKINES IN THE BRAIN, AS WELL AS ACTIVATION OF T CELLS AND MICROGLIA. NOTABLY, T CELLS CAN BE FOUND IN THE BRAINS OF PD PATIENTS AND CIRCULATING T CELL SUBSETS ARE REDUCED IN PD, INDICATING DYSREGULATION OF IMMUNE FUNCTION. REPORTS ALSO DESCRIBED CHANGES IN THE RATIO OF SPECIFIC SUBSETS OF CIRCULATING T CELLS IN PD. DESPITE THE GROWING BODY OF EVIDENCE AND THE FACT THAT SUCH CHANGES ARE KEY IN INFLAMMATORY SIGNALING, IT REMAINS UNCLEAR WHETHER THE REPORTED CHANGES IN T CELL POPULATIONS AFFECT THE PROGRESSIVE INCREASE IN A-SYN PATHOLOGY AND NEURODEGENERATION. TO HELP ANSWER THIS QUESTION, WE WILL USE REFINED EXPERIMENTAL MODELS TO DEFINE THE ROLE OF SPECIFIC SUBTYPES OF T CELLS IN THE DEVELOPMENT AND SPREADING OF PD-LIKE A-SYN PATHOLOGY AND ASSOCIATED NEUROPATHOLOGY. IN LINE WITH THE PREMISE THAT T CELLS ARE KEY IN THE PATHOGENESIS OF PD, OUR PRELIMINARY DATA SHOW THAT WHEN A- SYN PATHOLOGY IS TRIGGERED IN IMMUNOCOMPROMISED MICE (LACKING T CELLS, B CELLS AND NATURAL KILLER CELLS) THE ACCUMULATION OF A-SYN PATHOLOGY INCREASES SUBSTANTIALLY. ACCORDINGLY, THIS INCREASE WAS AMELIORATED UPON T CELL RECONSTITUTION. THESE DATA STRONGLY SUPPORT OUR HYPOTHESIS THAT T CELLS ARE INVOLVED IN CONTROLLING THE DEVELOPMENT OF A-SYN PATHOLOGY. THE OBJECTIVE OF THIS PROJECT IS TO ESTABLISH WHICH T CELL POPULATION (CD4+, CD8+ OR TREG) CAN LIMIT THE SPREAD OF A-SYN PATHOLOGY, AND MODULATE PD-LIKE NEUROPATHOLOGY (I.E., MICROGLIOSIS, ASTROGLIOSIS AND DOPAMINE NEURON DEATH) IN A MOUSE MODEL OF PD. THE PROPOSED WORK COMBINES THE EXPERTISE OF PD RESEARCHERS (BRUNDIN LAB) AND IMMUNOLOGISTS (BETTELLI LAB). FIRST, THE CAPACITY OF CD4+ AND CD8+ T CELLS IN ALTERING THE PROPAGATION OF A-SYN PATHOLOGY WILL BE TESTED USING CD4 AND CD8 KNOCK-OUT (KO) MICE COMBINATION WITH ADOPTIVE TRANSFER OF THE INDIVIDUAL POPULATIONS IN TCRB KO MICE (DEFICIENT IN ALPHA/BETA T-CELL RECEPTOR). SECOND, THE SPECIFIC CONTRIBUTION OF TREG CELLS WILL BE ESTABLISHED USING A FOXP3 KNOCK-IN MICE TO DETERMINE CHANGES IN TREG POPULATIONS (E.G., BRAIN VS PERIPHERY). BY DEFINING WHICH T CELL SUBTYPES ARE IMPORTANT FOR THE DEVELOPMENT OF A- SYN PATHOLOGY, STRATEGIES TO CONTROL THEIR FUNCTION COULD POTENTIALLY BE DEVELOPED INTO NOVEL THERAPIES TO STOP PD PROGRESSION.

STRUCTURAL AND FUNCTIONAL STUDIES OF THE HUMAN TRPM4 AND TRPM5 CHANNELS

GENERATION AND INITIAL CHARACTERIZATION OF OSTEOCALCIN-DEFICIENT RATS

IMMUNOLOGICAL APPROACHES TO PRION DISEASE

ETHENO ADDUCTS IN LIVER CANCER

INVESTIGATING THE ROLE OF MARKS IN PARKINSON'S DISEASE PATHOGENESIS

THE ROLE OF AHR IN MODULATINGIMMUNOTHERAPY RESPONSE AND ADVERSE EVENTS IN BLADDER CANCER - PROJECT SUMMARY ONE HALLMARK OF CANCER IS A TUMOR’S ABILITY TO EVADE IMMUNE DETECTION AND ELIMINATION. IMMUNE CHECKPOINT INHIBITORS (ICIS) ARE AN ATTRACTIVE THERAPEUTIC STRATEGY FOR MANY CANCERS. AFTER MANY CLINICAL TRIALS TARGETING VARIOUS SOLID CANCER TYPES, HOWEVER, ONLY 15-40% OF PATIENTS BENEFITED FROM ICI THERAPY. SHOCKINGLY, 4-29% OF NON- RESPONSIVE PATIENTS DEVELOPED HYPERPROGRESSIVE DISEASE (HPD), A CONDITION CHARACTERIZED BY ACCELERATED TUMOR GROWTH AND RAPID CLINICAL DETERIORATION. VERY LITTLE IS KNOWN ABOUT WHAT CAUSES HPD OR HOW TO PREVENT IT, BUT RECENT WORK SUGGESTS THAT TUMOR HYPERPROLIFERATION IS AN IMMUNE-RELATED ADVERSE EVENT (IRAE). AS PART OF THE VAI-SU2C EPIGENETIC DREAM TEAM, WE DISCOVERED THAT ACTIVATING THE ARYL HYDROCARBON RECEPTOR (AHR) PATHWAY AND ITS DOWNSTREAM TARGET GENE CYP1A1 ARE PROGNOSTIC INDICATORS FOR THE HYPERPROLIFERATIVE-IRAE (HP-IRAE), AND THAT HP-IRAE BLADDER TUMORS SHOW A METABOLIC SWITCH TOWARDS FATTY ACID AND XENOBIOTIC METABOLISM. AHR IS KNOWN TO HAVE AN ONCOGENIC ROLE IN TUMORIGENESIS AND IMMUNE DYSFUNCTION, WHICH LEADS TO THE WORKING HYPOTHESIS THAT AHR ACTIVATION BEFORE ICI THERAPY PRECONDITIONS THE CANCER CELLS OR TUMOR MICROENVIRONMENT FOR RAPID PROLIFERATION, WHERE HP-IRAE IS THEN TRIGGERED BY INTRACELLULAR PD-L1 SIGNALING IN IMMUNE CELLS AND/OR CANCER CELLS AFTER ICI THERAPY. TO TEST THIS HYPOTHESIS, WE WILL FIRST USE THE NANOSTRING GEOMX PLATFORM TO SPATIALLY PROFILE RNA AND PROTEIN EXPRESSION IN CYP1A1-POSITIVE BLADDER TUMOR MICROENVIRONMENTS TO RESOLVE THE SPATIAL HETEROGENEITY OF IMMUNE AND CANCER CELL DEPENDENCIES (AIM 1). IN AIM 2, WE WILL DETERMINE HOW ICI TREATMENT AND AHR ACTIVATION TRANSCRIPTIONALLY AND METABOLICALLY REPROGRAMS BLADDER CANCER AND IMMUNE CELLS. THESE EXPERIMENTS WILL SEPARATE THE CANCER-INTRINSIC, IMMUNE-RELATED, AND METABOLIC CONSEQUENCE OF AHR AND PD-L1 SIGNALING, AND PROVIDE POWERFUL MODELS FOR FUTURE GENETIC- OR DRUG-SCREENING STUDIES. FINALLY (AIM 3), WE WILL USE A BLADDER CANCER MOUSE MODELS TO DEFINE HOW AHR AND ICI TREATMENT IMPACTS THE EVOLUTION OF THE TUMOR MICROENVIRONMENT IN VIVO. THESE DATA WILL PROVIDE VALUABLE INSIGHT INTO HOW AHR ACTIVATION MIGHT INFLUENCE ICI EFFICACY AND ADVERSE EVENTS. THE K99 PHASE OF THIS PROJECT WILL PROVIDE VITAL TRAINING IN SPATIAL TRANSCRIPT/PROTEOMICS, METABOLOMICS, AND MULTIMODAL SINGLE-CELL TECHNOLOGIES, AND HOW TO PROCESS AND INTEGRATE HIGH-DIMENSIONAL DATA TO UNDERSTAND (BLADDER) CANCER EVOLUTION AND IRAES. THIS EFFORT WILL BE GUIDED BY AN OUTSTANDING, MULTIDISCIPLINARY ADVISORY COMMITTEE, LED BY DR. PETER JONES AT VAN ANDEL INSTITUTE, THAT AMALGAMATE BASIC AND TRANSLATIONAL SCIENCE ACROSS CANCER BIOLOGY, IMMUNOLOGY, EPIGENETIC, AND METABOLISM FIELDS. THESE TRAINING AND NEW SKILL SETS WILL BE LEVERAGED TO ULTIMATELY TRANSITION INTO AN INDEPENDENT RESEARCH CAREER (R00 PHASE) AND VALIDATE IN VITRO FINDINGS WITH IN VIVO MOUSE MODELS. TOGETHER, THIS PROJECT WILL PROVIDE THE FIRST MECHANISTIC INSIGHTS INTO HOW THE AHR PATHWAY EXACERBATES BLADDER CANCER RESPONSE TO ICI TREATMENT, WHICH WILL PROVIDE NEW OPPORTUNITIES FOR FUTURE DRUG DISCOVERY AND PRECISION MEDICINE EFFORTS TO MINIMIZE HP-IRAE IN ICI-TREATED PATIENTS.

COMBINING SYNUCLEINOPATHY AND MITOCHONDRIAL DEFICITS IN A NOVEL MOUSE MODEL OF PARKINSONS DISEASE - THE LACK OF ANIMAL MODELS RECREATING THE PROGRESSIVE PATHOLOGY CHARACTERISTIC OF PARKINSON’S DISEASE (PD) HINDERS THE DEVELOPMENT OF EFFECTIVE DISEASE-MODIFYING THERAPIES. THUS, THE GOAL OF THIS PROJECT IS TO GENERATE A NEW ANIMAL PD MODEL THAT SUPPORTS THE DEVELOPMENT OF SUCH THERAPIES. CLINICAL DATA REVEALED THAT AXON TERMINAL FAILURE AND “DYING BACK” OF DOPAMINERGIC NEURONS LIKELY PRECEDE LOSS OF SUBSTANTIA NIGRA CELL BODIES BY MANY YEARS IN PD. THIS PROTRACTED PROCESS IS NOT REPLICATED IN THE ACUTE TOXIN-INDUCED ANIMAL MODELS OF PD, PROVIDING ONE POSSIBLE EXPLANATION FOR THE LOW PREDICTIVE POWER OF THESE MODELS. THE HETEROZYGOUS DELETION OF THE ENGRAILED 1 GENE IN MICE (EN1+/–) RESULTS IN AXON TERMINAL DYSFUNCTION AND DEGENERATION EVENTUALLY LEADING TO PROTRACTED LOSS OF NIGRAL DOPAMINERGIC NEURONS. THIS PROCESS CAUSES STRIATAL DOPAMINE DEFICIENCY THAT LEADS TO MOTOR IMPAIRMENT. FURTHERMORE, THESE CHANGES ARE ASSOCIATED WITH MITOCHONDRIAL DEFICITS AKIN TO THOSE OBSERVED IN SOME PD PATIENTS. DESPITE ALL THE ADVANTAGES THAT THE EN1+/– MOUSE MODEL REPRESENTS, IT LACKS A-SYN AGGREGATION. THUS, WE HYPOTHESIZE THAT MITOCHONDRIAL DEFICITS (DUE TO HETEROZYGOUS LOSS OF EN1) COMBINED WITH BILATERAL INJECTIONS OF PATHOGENIC A-SYN FIBRILS (PFFS) WILL SYNERGISTICALLY GENERATE A HIGHLY RELEVANT PD MODEL – EN1/SYN. WE FURTHER PREDICT THAT THE EN1/SYN MODEL WILL EXHIBIT A COMPREHENSIVE SET OF PD-RELEVANT BEHAVIORAL DEFICITS (BOTH MOTOR AND NON-MOTOR) AND WILL MIMIC PD NEUROPATHOLOGY. THIS APPROACH IS INNOVATIVE IN COMBINING, IN MICE, KEY ASPECTS OF PD, THE SUSCEPTIBILITY OF THE DOPAMINERGIC SYSTEM, A-SYN AND THE MULTIFACTORIAL NATURE OF THE ETIOLOGY OF PD. SUPPORTING OUR HYPOTHESIS, OUR PRELIMINARY DATA SHOW THAT PFFS-INDUCED A-SYN PATHOLOGY IS SIGNIFICANTLY EXACERBATED BY THE LOSS OF EN1. THERE ARE TWO MAJOR GOALS IN THIS PROJECT: (1) WE WILL TRIGGER PFFS-INDUCED A-SYN PATHOLOGY BILATERALLY BY INJECTING PATHOGENIC A-SYNUCLEIN INTO BOTH STRIATA. BY TRIGGERING THE PATHOLOGY ON BOTH SIDES OF THE BRAIN THE NEW MOUSE MODEL IS EXPECTED TO INDUCE ROBUST MOTOR, AND MORE IMPORTANTLY COGNITIVE DEFICITS. WE WILL BILATERALLY INJECT RECOMBINANT FIBRILLAR A-SYN (PROVIDED BY DR. JIYAN MA), MEASURE THE DEVELOPMENT OF A-SYN-ASSOCIATED PATHOLOGY, AND THEN ASSESS MOTOR AND NON-MOTOR FUNCTION OF MICE AT DIFFERENT TIME POINTS; (2) WE WILL VALIDATE THE EN1/SYN MODEL AS A MODEL OF PD, BY COMPARING THE FEATURES OF OUR MODEL TO HUMAN PD IN LONG-TERM EXPERIMENTS, AS WELL AS BY TESTING IF THE GOLD-STANDARD PD TREATMENT L-DOPA REVERSES MOTOR DEFICITS INDUCED BY LOSS OF NIGRAL DOPAMINE NEURONS AND WIDESPREAD A-SYN AGGREGATION. OUR RESEARCH IS EXPECTED TO GENERATE A POWERFUL TOOL, WHICH CAN ACCELERATE THE DEVELOPMENT OF SYMPTOMATIC AND/OR DISEASE-MODIFYING THERAPIES TO TREAT MOTOR AND NON-MOTOR PD.

MOLECULAR SIGNATURES OF PARKINSONS DISEASE IN THE GUT AND BRAIN

THE INFLUENCE OF PRIMARY MICROENVIRONMENT ON PROSTATE CANCER OCTEOBLASTIC BONE LESION DEVELOPMENT

DISSECTING THE ROLE OF GERMLINE GENETIC VARIATION AND VIRAL EXPOSURES AS DETERMINANTS OF HEMATOLOGICAL DISEASE PREDISPOSITION - ABSTRACT THE ROLE OF GERMLINE GENETIC VARIATION AND VIRAL INFECTION IN DEVELOPMENT AND PROGRESSION HAS BEEN STUDIED EXTENSIVELY IN ADULT TUMORS AND AUTOIMMUNE DISEASE. LESS ATTENTION HAS BEEN PAID TO THE INTERACTION OF THESE FACTORS WITH BIRTH DEFECTS AND PEDIATRIC MALIGNANCIES, PARTICULARLY ACUTE MYELOID LEUKEMIA (AML), WHICH IN THE YOUNGEST PATIENTS IS DRIVEN ALMOST EXCLUSIVELY BY STRUCTURAL VARIANTS (SVS) WITH POORLY UNDERSTOOD ETIOLOGY. THE PREVALENCE OF GENE FUSION TRANSCRIPTS ASSOCIATED WITH LEUKEMIA AT LIVE BIRTH IS 10X TO 100X GREATER THAN THE INCIDENCE OF CHILDHOOD LEUKEMIA, WHICH SUGGESTS OTHER RISK FACTORS MUST INTERACT WITH SVS. ONE POSSIBLE CANDIDATE FOR INTERACTION IS THE TIMING OF VIRAL INFECTION, EITHER IN PARENTS OR CHILDREN. CLINICAL TRIALS OF GENE THERAPY WITH VIRAL VECTORS FAILED IN PART DUE TO VIRAL INTEGRATIONS ACTIVATING ONCOGENES SUCH AS MECOM. RECENT WORK HAS SHOWN A DIRECT MECHANISM FOR DERIVATIVE CHROMOSOME FORMATION AT THE MOST COMMON BREAKPOINTS IN LEUKEMIA, AND HUMAN HERPESVIRUSES, INCLUDING CMV, ARE ONE OF THE SINGLE GREATEST RISK FACTORS FOR CHROMOSOMAL BIRTH DEFECTS. ELSEWHERE, WE AND OTHERS HAVE DOCUMENTED GERMLINE AND SOMATIC COPY NUMBER AND SHORT SEQUENCE VARIANTS AFFECTING E26 TRANSFORMATION SPECIFIC (ETS) FACTORS, WHICH PARTICIPATE IN HIGH-RISK GENE FUSIONS SEEN IN BOTH SOLID AND LIQUID TUMORS. THESE FACTORS AND THEIR BINDING SITES DETERMINE DEVELOPMENTAL FATES ACROSS TISSUES, YET THEIR MOTIFS ARE SHORT TANDEM REPEATS -- THE SINGLE MOST VARIABLE CLASS OF FEATURES IN THE HUMAN GENOME. SMALL CHANGES IN DOSAGE, AS CREATED BY HAPLOINSUFFICIENCY OR VARIATION IN RAMP SEQUENCES, MAY BE SUFFICIENT TO PREDISPOSE INDIVIDUALS TO DISEASE. THE PRIMARY OBSTACLE TO STUDYING EITHER OF THESE MECHANISMS HAS LONG BEEN THE SMALL SAMPLE SIZES AND BIASED COVERAGE OF COHORTS ASSEMBLED FOR RARE AND CHILDHOOD DISEASES. THE VAST QUANTITY OF WHOLE-GENOME, WHOLE-TRANSCRIPTOME, AND LONG-READ SEQUENCING DATA PROVIDED BY THE GABRIELLA MILLER KIDS FIRST! (GMKF) CONSORTIUM AND THE INCLUDE COHORTS NEGATE THIS OBSTACLE. WHEN COMBINED WITH THE THOUSANDS OF PEDIATRIC CLINICAL TRIAL PARTICIPANTS IN PROJECT: EVERY CHILD, AND THE FORTHCOMING X01 LONG READ PILOT PROJECT FOR OMICS-COLD PEDIATRIC LEUKEMIA PATIENTS, WE POSIT THAT BOTH THE COMPUTATIONAL INFRASTRUCTURE AND THE SAMPLE SIZES REQUIRED FOR PROGRESS ARE NOW IN PLACE. WE PROPOSE TO CHARACTERIZE GERMLINE REGULATORY, SPLICING, AND STRUCTURAL VARIANTS AS CATALYSTS OF RISK FOR LEUKEMIA AND RELATED PRELEUKEMIC CONDITIONS, NOTING THAT TRIPLICATION OF THE ETS FACTOR ERG IS AN INHERENT FEATURE OF DOWN SYNDROME-DRIVEN DISEASE (A 500X MULTIPLIER FOR RISK OVER THE GENERAL POPULATION). WITH COLLEAGUES, WE HAVE IDENTIFIED NON-CODING VARIANTS WITH STRONG EFFECTS IN DOWN SYNDROME. THIS SUGGESTS THAT THE COMBINATION OF SAMPLE SIZE, COHORT DIVERSITY, AND REPRESENTATION OF THE MOST COMMON STRUCTURAL VARIANTS IN HUMAN DISEASE WITHIN THE GMKF! CONSORTIUM PRESENTS AN IDEAL OPPORTUNITY TO ADDRESS THIS URGENT NEED AND DETERMINE IF PREDISPOSITION RISK CAN BE MITIGATED BY SCREENING OR PROPHYLAXIS.

THE ROLE OF AHR IN MODULATINGIMMUNOTHERAPY RESPONSE AND ADVERSE EVENTS IN BLADDER CANCER - PROJECT SUMMARY ONE HALLMARK OF CANCER IS A TUMOR’S ABILITY TO EVADE IMMUNE DETECTION AND ELIMINATION. IMMUNE CHECKPOINT INHIBITORS (ICIS) ARE AN ATTRACTIVE THERAPEUTIC STRATEGY FOR MANY CANCERS. AFTER MANY CLINICAL TRIALS TARGETING VARIOUS SOLID CANCER TYPES, HOWEVER, ONLY 15-40% OF PATIENTS BENEFITED FROM ICI THERAPY. SHOCKINGLY, 4-29% OF NON- RESPONSIVE PATIENTS DEVELOPED HYPERPROGRESSIVE DISEASE (HPD), A CONDITION CHARACTERIZED BY ACCELERATED TUMOR GROWTH AND RAPID CLINICAL DETERIORATION. VERY LITTLE IS KNOWN ABOUT WHAT CAUSES HPD OR HOW TO PREVENT IT, BUT RECENT WORK SUGGESTS THAT TUMOR HYPERPROLIFERATION IS AN IMMUNE-RELATED ADVERSE EVENT (IRAE). AS PART OF THE VAI-SU2C EPIGENETIC DREAM TEAM, WE DISCOVERED THAT ACTIVATING THE ARYL HYDROCARBON RECEPTOR (AHR) PATHWAY AND ITS DOWNSTREAM TARGET GENE CYP1A1 ARE PROGNOSTIC INDICATORS FOR THE HYPERPROLIFERATIVE-IRAE (HP-IRAE), AND THAT HP-IRAE BLADDER TUMORS SHOW A METABOLIC SWITCH TOWARDS FATTY ACID AND XENOBIOTIC METABOLISM. AHR IS KNOWN TO HAVE AN ONCOGENIC ROLE IN TUMORIGENESIS AND IMMUNE DYSFUNCTION, WHICH LEADS TO THE WORKING HYPOTHESIS THAT AHR ACTIVATION BEFORE ICI THERAPY PRECONDITIONS THE CANCER CELLS OR TUMOR MICROENVIRONMENT FOR RAPID PROLIFERATION, WHERE HP-IRAE IS THEN TRIGGERED BY INTRACELLULAR PD-L1 SIGNALING IN IMMUNE CELLS AND/OR CANCER CELLS AFTER ICI THERAPY. TO TEST THIS HYPOTHESIS, WE WILL FIRST USE THE NANOSTRING GEOMX PLATFORM TO SPATIALLY PROFILE RNA AND PROTEIN EXPRESSION IN CYP1A1-POSITIVE BLADDER TUMOR MICROENVIRONMENTS TO RESOLVE THE SPATIAL HETEROGENEITY OF IMMUNE AND CANCER CELL DEPENDENCIES (AIM 1). IN AIM 2, WE WILL DETERMINE HOW ICI TREATMENT AND AHR ACTIVATION TRANSCRIPTIONALLY AND METABOLICALLY REPROGRAMS BLADDER CANCER AND IMMUNE CELLS. THESE EXPERIMENTS WILL SEPARATE THE CANCER-INTRINSIC, IMMUNE-RELATED, AND METABOLIC CONSEQUENCE OF AHR AND PD-L1 SIGNALING, AND PROVIDE POWERFUL MODELS FOR FUTURE GENETIC- OR DRUG-SCREENING STUDIES. FINALLY (AIM 3), WE WILL USE A BLADDER CANCER MOUSE MODELS TO DEFINE HOW AHR AND ICI TREATMENT IMPACTS THE EVOLUTION OF THE TUMOR MICROENVIRONMENT IN VIVO. THESE DATA WILL PROVIDE VALUABLE INSIGHT INTO HOW AHR ACTIVATION MIGHT INFLUENCE ICI EFFICACY AND ADVERSE EVENTS. THE K99 PHASE OF THIS PROJECT WILL PROVIDE VITAL TRAINING IN SPATIAL TRANSCRIPT/PROTEOMICS, METABOLOMICS, AND MULTIMODAL SINGLE-CELL TECHNOLOGIES, AND HOW TO PROCESS AND INTEGRATE HIGH-DIMENSIONAL DATA TO UNDERSTAND (BLADDER) CANCER EVOLUTION AND IRAES. THIS EFFORT WILL BE GUIDED BY AN OUTSTANDING, MULTIDISCIPLINARY ADVISORY COMMITTEE, LED BY DR. PETER JONES AT VAN ANDEL INSTITUTE, THAT AMALGAMATE BASIC AND TRANSLATIONAL SCIENCE ACROSS CANCER BIOLOGY, IMMUNOLOGY, EPIGENETIC, AND METABOLISM FIELDS. THESE TRAINING AND NEW SKILL SETS WILL BE LEVERAGED TO ULTIMATELY TRANSITION INTO AN INDEPENDENT RESEARCH CAREER (R00 PHASE) AND VALIDATE IN VITRO FINDINGS WITH IN VIVO MOUSE MODELS. TOGETHER, THIS PROJECT WILL PROVIDE THE FIRST MECHANISTIC INSIGHTS INTO HOW THE AHR PATHWAY EXACERBATES BLADDER CANCER RESPONSE TO ICI TREATMENT, WHICH WILL PROVIDE NEW OPPORTUNITIES FOR FUTURE DRUG DISCOVERY AND PRECISION MEDICINE EFFORTS TO MINIMIZE HP-IRAE IN ICI-TREATED PATIENTS.

FATTY ACID METABOLISM IN DISEASE MODELS OF MITOCHONDRIAL FATTY ACID SYNTHESIS DEFICIENCY. - TITLE: FATTY ACID METABOLISM IN DISEASE MODELS OF MITOCHONDRIAL FATTY ACID SYNTHESIS DEFICIENCY PROJECT SUMMARY/ABSTRACT: RARE GENETIC DISEASES CAUSE 35% OF DEATHS IN CHILDREN UNDER 1 YEAR OF AGE AND ARE A SIGNIFICANT CAUSE OF PEDIATRIC HOSPITAL ADMISSIONS. PRIMARY MITOCHONDRIAL DISEASES ARE ONE GROUP OF INBORN ERRORS OF METABOLISM (IEM) CAUSED BY GERMLINE MUTATIONS IN GENES INVOLVED IN METABOLISM. WHILE INDIVIDUALLY RARE, IEMS COLLECTIVELY AFFECT 1 IN 5,000 CHILDREN MAKING THEM RELATIVELY COMMON AS A GROUP. DESPITE THEIR PREVALENCE, MITOCHONDRIAL DISORDERS ARE DIFFICULT TO DIAGNOSE AND TREAT, IN PART BECAUSE OF OUR OFTEN-POOR UNDERSTANDING OF THE UNDERLYING MOLECULAR ETIOLOGY. DEFICIENCIES IN THE MITOCHONDRIAL FATTY ACID SYNTHESIS (MTFAS) PATHWAY ARE AN EMERGING GROUP OF CHILDHOOD-ONSET MITOCHONDRIAL DISEASES WITH PARTICULARLY UNDERSTUDIED PATHOPHYSIOLOGY. GENETIC VARIANTS IN MTFAS GENES CAUSE MITOCHONDRIAL ENOYL REDUCTASE PROTEIN ASSOCIATED NEURODEGENERATION (MEPAN) SYNDROME, WHICH PRESENTS WITH SEVERE DEVELOPMENTAL DELAYS, SEIZURES, HYPOTONIA, HEARING AND VISION LOSS, AND A PLETHORA OF SYSTEMIC PROBLEMS. MTFAS PRODUCES BOTH LIPOIC ACID (AN IMPORTANT CO-FACTOR FOR SEVERAL ENZYMES) AND LONG-CHAIN FATTY ACIDS THAT SUPPORT ELECTRON TRANSPORT CHAIN FUNCTION. HOWEVER, THE CELLULAR ROLES OF MTFAS PRODUCTS AND THE PATHWAY’S FUNCTION IN DIVERSE CELLULAR CONTEXTS ARE STILL UNCLEAR, PREVENTING THE DEVELOPMENT OF RATIONALE-BASED THERAPIES FOR MTFAS DEFICIENCY. TO BETTER TREAT MEPAN SYNDROME, WE NEED NEW APPROACHES. TOWARD THIS OVERARCHING GOAL, WE WILL USE TWO COMPLEMENTARY METHODOLOGIES TO IDENTIFY POTENTIAL THERAPEUTIC TARGETS THAT RESCUE GROWTH AND DEVELOPMENT IN MTFAS-DEFICIENT PATIENT MODELS. WE HAVE DEVELOPED A NOVEL, ORGANISMAL MODEL OF MEPAN SYNDROME BY RECREATING PATIENT MUTATIONS IN CAENORHABDITIS ELEGANS, WHICH RESULT IN MARKED DEVELOPMENTAL DELAYS. WE SHOW THAT WE CAN USE THIS MODEL TO PERFORM GENOME-WIDE SCREENS IN WHOLE ORGANISMS, AND SUCCESSFULLY RECOVER GENETIC SUPPRESSORS OF THE PHENOTYPES CAUSED BY MTFAS PATIENT VARIANTS. IN PARALLEL, WE HAVE PERFORMED PRELIMINARY CRISPR/CAS9 SCREENS IN MTFAS-DEFICIENT CELLS AND FOUND THAT LOSS OF FATTY ACID OXIDATION PATHWAY GENES, AS WELL AS GENES ENCODING SUBUNITS OF THE MITOCHONDRIAL ELECTRON TRANSPORT CHAIN, RESCUES GROWTH IN MTFAS-DEFICIENT CELLS. WE PROPOSE TO TEST THE UTILITY OF THESE FINDINGS IN A PANEL OF PRIMARY PATIENT FIBROBLASTS, AND TO EXPAND OUR ANALYSES BY PERFORMING GENOME WIDE LOSS OF FUNCTION AND CRISPRA SCREENS TO IDENTIFY ADDITIONAL PATHWAYS OF SUPPRESSION. TOGETHER, THESE EXPERIMENTS WILL LAY THE GROUNDWORK FOR FUTURE DEVELOPMENT OF THERAPEUTICS FOR MTFAS-DEFICIENT PATIENTS AND THEIR FAMILIES.

ROLE OF THE ENDOLYSOSOMAL PATHWAY IN LEWY BODY DEMENTIA - FROM POPULATION GENOMICS TO SINGLE CELLS - PROJECT SUMMARY/ABSTRACT ONE OF THE MOST COMMON ALZHEIMER’S DISEASE RELATED DEMENTIAS (ADRD) IS LEWY BODY DEMENTIA. THIS UMBRELLA TERM COMPRISES TWO CLINICALLY DISTINCT ADRDS: PARKINSON’S DISEASE DEMENTIA (PD-DEMENTIA) AND DEMENTIA WITH LEWY BODIES (DLB). THERE HAS BEEN A GROWING INTEREST IN THE GENETIC BASES OF DLB, HOWEVER, PD-DEMENTIA HAS NOT YET BEEN STUDIED USING LARGE-SCALE GENETIC ANALYSES. NEVERTHELESS, IT HAS BEEN SHOWN THAT DYSFUNCTION OF THE ENDOLYSOSOMAL PATHWAY PLAYS A KEY ROLE IN ALZHEIMER’S DISEASE AND ADRDS. THIS INVOLVEMENT, HOWEVER, IS NOT FULLY UNDERSTOOD AND THE EXACT FAILURE POINTS FOR EACH DISEASE HAVE YET TO BE IDENTIFIED. THUS, THERE IS A GAP IN KNOWLEDGE REGARDING THE SPECIFIC MOLECULAR MECHANISMS UNDERLYING EACH DISEASE. THIS APPLICATION AIMS TO IDENTIFY GENETIC VARIABILITY THAT MODULATES RISK FOR PD-DEMENTIA AND ITS NEUROPATHOLOGICAL FEATURES, DETERMINING THE OVERLAP WITH OTHER ADRDS AND THE IMMEDIATE DOWNSTREAM EFFECTS ON ENDOLYSOSOMAL FUNCTION. THE HYPOTHESIS, BASED ON PRELIMINARY DATA, IS THAT PD-DEMENTIA HAS A MEASURABLE AND UNIQUE GENETIC ARCHITECTURE AND THAT ITS INTEGRATION WITH CURRENT KNOWLEDGE OF THE MOLECULAR BASES OF OTHER ADRDS, WILL IMPROVE THE UNDERSTANDING OF THE LYSOSOME AS A CENTRAL PATHOLOGICAL HUB IN NEURODEGENERATIVE DISEASES. THIS HYPOTHESIS WILL BE TESTED BY PURSUING THREE SPECIFIC AIMS: 1) IDENTIFY CANDIDATE RISK-MODULATING VARIANTS FOR PD-DEMENTIA; 2) DETERMINE NEUROPATHOLOGICAL CORRELATES OF GENETIC RISK; AND 3) IDENTIFY DOWNSTREAM EFFECTS OF HIGH-RISK VARIABILITY IN NEURONS AND GLIAL CELLS ACROSS ADRDS. ONE COHORT OF CLINICALLY DIAGNOSED PD- DEMENTIA CASES AND ONE COHORT OF NEUROPATHOLOGICAL DIAGNOSED CASES WILL BE TESTED UNDER A GWAS FRAMEWORK TO IDENTIFY RISK MODULATING VARIANTS IN A TWO-STAGE STUDY. THE NEUROPATHOLOGICAL CASES WILL ALSO UNDERGO WHOLE- GENOME SEQUENCING TO IDENTIFY COMMON AND RARE GENETIC VARIABILITY, ALLOWING FOR THE FIRST GENOMICS STUDY WITH GOLD-STANDARD CHARACTERIZATION OF NEUROPATHOLOGY IN A LARGE COHORT OF PD-DEMENTIA CASES. LAST, THE IMMEDIATE CELL-SPECIFIC EFFECTS OF DISEASE-SPECIFIC RISK PROFILE WILL BE DETERMINED BY PERFORMING SINGLE-CELL RNA-SEQ IN BRAIN TISSUE FROM INDIVIDUALS WITH THE HIGHEST POLYGENIC RISK FOR EACH OF THESE TWO ADRDS AND FOR AD. THE PROPOSED RESEARCH FOCUSES FOR THE FIRST TIME ON PD-DEMENTIA CASES, AS A STRICT DIAGNOSIS, TAKING ADVANTAGE OF LARGE-SCALE UNBIASED GENETIC ANALYSES AND INTEGRATING THAT WITH RELATED DISORDERS. THE PROPOSED RESEARCH IS SIGNIFICANT BECAUSE IT WILL PROVIDE A COMPOSITE GENETIC PROFILE SPECIFIC FOR THIS LEWY BODY DEMENTIA. THE PROPOSED STUDY DESIGN HAS THE POTENTIAL TO IDENTIFY TARGETS THAT ARE COMMON TO DIFFERENT FORMS OF ADRDS, AS WELL AS NOVEL, PERSONALIZED, DISEASE-SPECIFIC TARGETS; THIS PROPOSAL IS A DIRECT RESPONSE TO THE NATIONAL ALZHEIMER'S PROJECT ACT (NAPA) PUBLIC LAW 111-375, NAPA 2012 2013, AND NAPA 2016.

EPIGENOMICS WORKSHOP FOR GRADUATE STUDENTS - PROJECT SUMMARY THE FIELD OF EPIGENOMICS IS EXPANDING RAPIDLY, DRIVEN IN LARGE PART BY NEW AND EVOLVING TECHNOLOGY, METHODS, AND TECHNIQUES. IT ALSO IS BECOMING INCREASINGLY INTERDISCIPLINARY. THE RAPID ADVANCE OF OMICS TECHNOLOGIES INTO MULTIPLE SPECIALTY DISCIPLINES HAS CREATED A NEW SERIES OF CHALLENGES FOR SCIENTISTS-IN-TRAINING, PRIMARILY BECAUSE EVERY TECHNIQUE OR SOFTWARE PROGRAM HAS ITS OWN ASSUMPTIONS, BIASES, CAVEATS, AND RELATIVE MERITS THAT DIRECTLY AFFECT (OR LIMIT) THE CONCLUSIONS THAT CAN BE DRAWN FROM SUCH STUDIES. THE OBJECTIVES OF THE “EPIGENOMICS WORKSHOP FOR GRADUATE STUDENTS” ARE TO PROVIDE A BROAD OVERVIEW OF CUTTING-EDGE OMICS TECHNOLOGIES, EPIGENETICS RESEARCH QUESTIONS, AND AN INTIMATE, PROBLEM-BASED LEARNING ENVIRONMENT WITH RESEARCH EXPERTS SO THAT PARTICIPANTS WILL UNDERSTAND INNOVATIVE METHODS FOR STUDYING EPIGENOMICS, APPLY THESE TOOLS TO INTERDISCIPLINARY RESEARCH QUESTIONS IN THE FIELD, AND BUILD MEANINGFUL CONNECTIONS WITH RESEARCH LEADERS AND OTHER GRADUATE STUDENTS IN THE BROADER EPIGENOMICS FIELD. THE ONE-WEEK WORKSHOP AT VAN ANDEL RESEARCH INSTITUTE WILL BE LED BY EPIGENOMICS RESEARCH LEADERS AND HAVE SPACE FOR 15 GRADUATE STUDENTS PER YEAR. IT WILL INCREASE DIVERSITY AND ENHANCE THE TRAINING OF 75 BIOMEDICAL RESEARCHERS TO MEET THE GROWING DEMAND FOR HIGHLY SKILLED EPIGENOMICS RESEARCHERS WITH BROAD FOUNDATIONS, ENABLING THEM TO TACKLE INNOVATIVE, INTERDISCIPLINARY QUESTIONS AND BRING THIS KNOWLEDGE BACK TO THEIR CURRENT LABS. WE ALSO WILL BUILD A COMMUNITY OF EARLY CAREER EPIGENOMICS SCIENTISTS WHO ARE CONNECTED BEYOND THEIR SUBFIELDS TO THE BROADER EPIGENOMICS FIELD.

NOTCH SIGNALING IN PROSTATE CANCER CELLS PROMOTE OSTEOBLASTIC MATASTASIS

STRUCTURAL AND FUNCTIONAL STUDIES ON PROTON-ACTIVATED CHLORIDE (PAC) CHANNEL - ABSTRACT ISCHEMIC STROKE IS ONE OF THE LEADING CAUSES OF DISABILITY AND DEATH IN THE UNITED STATES. ACID ACCUMULATION IN THE BRAIN DURING ISCHEMIC STROKE CAUSES NEUROTOXICITY AND IRREVERSIBLE TISSUE DAMAGE. UNDERSTANDING THE FACTORS THAT CONTRIBUTE TO ACID-INDUCED CELL DEATH DURING ISCHEMIC STROKE IS THUS CRITICAL TO DEFINE THE PATHOLOGICAL PROCESS AND DEVELOP EFFECTIVE TREATMENT STRATEGIES. THE PROTON-ACTIVATED CHLORIDE (PAC) CHANNEL (ALSO KNOWN AS ASOR OR PAORAC) IS A RECENTLY DISCOVERED CELLULAR PH-SENSOR THAT PLAYS A CRITICAL ROLE IN DETERMINING THE OUTCOME OF BRAIN DAMAGE AFTER ISCHEMIC STROKE. UNDER ACIDIC CONDITIONS, THE ACTIVATION OF PAC ALLOWS AN INFLUX OF CHLORIDE CURRENT INTO THE NEURON WHICH FURTHER CAUSES CELL SWELLING AND DEATH. IN 2019, THE PAC GENE WAS CLONED BY TWO INDEPENDENT GROUPS AND WAS FOUND TO BE A NOVEL CHLORIDE CHANNEL. IN 2020, I REVEALED THE FIRST NEAR-ATOMIC CRYO-EM STRUCTURES OF THE HUMAN PAC CHANNEL AT TWO DIFFERENT CONFORMATIONAL STATES, INCLUDING AN APO STATE AND A PROTON-BOUND NON-CONDUCTING STATE. OUR STUDY PROVIDED A WEALTH OF INFORMATION ABOUT CHANNEL STOICHIOMETRY, DOMAIN ARCHITECTURE, AND ANION SELECTIVITY MECHANISMS OF PAC. WHILE PROMISING PROGRESS HAS BEEN MADE TOWARDS UNDERSTANDING THE FUNCTION OF THIS CHANNEL, A COMPLETE PICTURE OF HOW PAC RESPONDS TO ENVIRONMENTAL ACIDIFICATION IS STILL OBSCURE DUE TO THE LIMITED KNOWLEDGE ABOUT THE PH- SENSOR AND THE LACK OF AN OPEN STATE STRUCTURE. LIKEWISE, ALTHOUGH THE PAC CURRENT IS SENSITIVE TO SEVERAL NON- SPECIFIC CHLORIDE CHANNEL BLOCKERS, THEIR INHIBITION MECHANISMS ARE UNEXPLORED. THE LONG-TERM OBJECTIVE OF THIS RESEARCH IS TO UNVEIL THE MOLECULAR PRINCIPLES UNDERLYING PAC CHANNEL FUNCTION IN BOTH PHYSIOLOGICAL AND PATHOLOGICAL CONDITIONS, AND TO DEVELOP SPECIFIC COMPOUNDS THAT COULD BE USED TO MITIGATE THE EFFECT OF ISCHEMIC STROKE IN PATIENTS. IN THIS K99/R00 PROPOSAL, WILL CARRY OUT A COMPREHENSIVE STRUCTURAL AND FUNCTIONAL INVESTIGATION OF PAC BY REVEALING ITS PH-SENSING RESIDUES AND THE ASSOCIATED STRUCTURAL MECHANISMS (AIM 1). I WILL ALSO EXPLORE STRATEGIES TO OBTAIN AN OPEN STATE STRUCTURE OF PAC AND PROVIDE DETAILED MECHANISTIC KNOWLEDGE ABOUT ITS VOLTAGE-DEPENDENT GATING MECHANISMS (AIM 2). I WILL ALSO STUDY THE PAC CHANNEL IN ITS NATIVE STATE BY PURIFYING ENDOGENOUS PAC PROTEIN FROM MOUSE BRAIN (AIM 3). LASTLY, I WILL INVESTIGATE SMALL MOLECULE-MEDIATED INHIBITION MECHANISMS THROUGH COMBINED STRUCTURAL AND FUNCTIONAL APPROACHES (AIM 4). THE MENTORED PHASE OF THE AWARD WILL BE CONDUCTED AT VAN ANDEL INSTITUTE UNDER THE SUPERVISION OF DR. JUAN DU. DURING THIS TIME, I WILL RECEIVE ADDITIONAL TRAINING IN MEMBRANE PROTEIN STRUCTURE DETERMINATION, PATCH-CLAMP ELECTROPHYSIOLOGY EXPERIMENTS, AND ENDOGENOUS PROTEIN PURIFICATION TECHNIQUES. THESE COMPONENTS ARE NOT ONLY ESSENTIAL FOR THE COMPLETION OF THE RESEARCH BUT WILL ALSO PREPARE ME TO BECOME AN INDEPENDENT INVESTIGATOR IN THE NEAR FUTURE.

DEFINING THE REGULATION OF UHRF1 AND DNMT1 FOR MAINTENANCE OF THE EPIGENOME - PROJECT SUMMARY IN MULTICELLULAR ORGANISMS, METHYLATION OF DNA ON THE 5’ CARBON OF CYTOSINE HELPS ENSURE PROPER GENE EXPRESSION. METHYLATION OCCURS SYMMETRICALLY ON BOTH DNA STRANDS, AND THE MAINTENANCE METHYLTRANSFERASE DNMT1 IS RESPONSIBLE FOR COPYING THE METHYLATION PATTERN FROM THE PARENT STRAND TO THE DAUGHTER STRAND DURING DNA REPLICATION. MAINTENANCE METHYLATION BY DNMT1 REQUIRES THE CHROMATIN-ASSOCIATED UBIQUITIN LIGASE UHRF1, WHICH CATALYZES UBIQUITINATION OF HISTONE H3 (H3UB). MANY QUESTIONS REMAIN ABOUT HOW THE VARIOUS DOMAIN FUNCTIONS OF DNMT1 AND UHRF1 ARE REGULATED AND WHICH ARE NECESSARY TO MAINTAIN DNA METHYLATION. THE OBJECTIVE OF THIS PROPOSAL IS TO DEFINE THE MECHANISTIC RELATIONSHIP BETWEEN UHRF1, DNMT1, AND H3 UBIQUITINATION IN THE MAINTENANCE OF DNA METHYLATION. THE FIRST AIM INVESTIGATES THE HYPOTHESIS THAT UBIQUITINATION OF HISTONE H3 BY UHRF1 REGULATES THE CATALYTIC ACTIVITY OF DNMT1 AND THAT MAINTENANCE METHYLATION BY DNMT1 DEPENDS ON BOTH UHRF1 UBIQUITIN LIGASE-DEPENDENT AND INDEPENDENT MECHANISMS. AIM 1 WILL USE IN VITRO BIOCHEMICAL ASSAYS TO CHARACTERIZE POINT MUTATIONS IN DNMT1 AND UHRF1 THAT COMPROMISE INDIVIDUAL DOMAINS OF THESE PROTEINS. TRANSGENES ENCODING THESE MUTANT ENZYMES WILL THEN BE TESTED IN CANCER CELLS USING WESTERN BLOT ANALYSIS OF H3UB AND EPIC ARRAY ANALYSIS OF DNA METHYLATION. THESE EXPERIMENTS WILL DEFINE THE CONTRIBUTION OF INDIVIDUAL FUNCTIONS OF DNMT1 AND UHRF1 TO H3 UBIQUITINATION AND DNA METHYLATION IN VITRO AND DURING DNA MAINTENANCE METHYLATION IN CANCER CELLS. THE SECOND AIM TESTS THE HYPOTHESIS THAT ALLOSTERIC ACTIVATION OF UHRF1’S UBIQUITIN LIGASE ACTIVITY BY HEMIMETHYLATED DNA (HEDNA) OPERATES IN CELLS AND REGULATES GENOME ARCHITECTURE IN COOPERATION WITH CTCF/COHESIN. AIM 2 WILL USE UHRF1 MUTANT TRANSGENES TO TEST WHETHER HEDNA STIMULATES UHRF1’S ENZYMATIC ACTIVITY IN CANCER CELLS USING WESTERN BLOT ANALYSIS OF H3UB AND HAIRPIN BISULFITE SEQUENCING TO DETERMINE LEVELS OF HEDNA. THE CONTRIBUTION OF UHRF1 FUNCTION TO GENOME ARCHITECTURE THROUGH RECENTLY IDENTIFIED STABLE HEDNA AT CTCF/COHESIN SITES WILL ALSO BE DEFINED USING CTCF CHIP-SEQ AND HI-C. THESE EXPERIMENTS WILL DETERMINE IF HEDNA REGULATES THE ACTIVITY OF UHRF1 IN CANCER CELLS AND TEST THE CONTRIBUTION OF STABLE HEDNA AT CTCF/COHESIN SITES TO GENOME ARCHITECTURE THROUGH UHRF1. MISREGULATION OF DNA METHYLATION IS A DRIVING FORCE IN MANY CANCERS. THUS, INSIGHT INTO HOW DNA METHYLATION IS SYNTHESIZED AND PROPAGATED IS CENTRAL TO UNDERSTANDING HOW CANCER BEGINS AND PROGRESSES. THE STUDIES PROPOSED HERE WILL ADVANCE OUR KNOWLEDGE OF THE MECHANISMS INVOLVED IN METHYLATION MAINTENANCE TO ALLOW THE RATIONAL DESIGN OF THERAPIES THAT HALT OR REVERSE THE FORMATION OF ONCOGENIC DNA METHYLATION PATTERNS.

ALTERNATIVE NF1 ISOFORMS IN RAS DEREGULATION AND BREAST CANCER PROGRESSION

THE ROLE OF THE EPIGENETIC REGULATOR UHRF1 IN COLON TUMORIGENESIS

MODELING PARKINSON'S DISEASE BY DELETING FOXO FACTORS FROM DOPAMINERGIC NEURONS

GRADUATE RESEARCH FELLOWSHIP PROGRAM (GRFP) -THE NATIONAL SCIENCE FOUNDATION (NSF) GRADUATE RESEARCH FELLOWSHIP PROGRAM (GRFP) IS A HIGHLY COMPETITIVE, FEDERAL FELLOWSHIP PROGRAM. GRFP HELPS ENSURE THE VITALITY AND DIVERSITY OF THE SCIENTIFIC AND ENGINEERING WORKFORCE OF THE UNITED STATES. THE PROGRAM RECOGNIZES AND SUPPORTS OUTSTANDING GRADUATE STUDENTS WHO ARE PURSUING RESEARCH-BASED MASTER'S AND DOCTORAL DEGREES IN SCIENCE, TECHNOLOGY, ENGINEERING, AND MATHEMATICS (STEM) AND IN STEM EDUCATION. THE GRFP PROVIDES THREE YEARS OF FINANCIAL SUPPORT FOR THE GRADUATE EDUCATION OF INDIVIDUALS WHO HAVE DEMONSTRATED THEIR POTENTIAL FOR SIGNIFICANT RESEARCH ACHIEVEMENTS IN STEM AND STEM EDUCATION. THIS AWARD SUPPORTS THE NSF GRADUATE FELLOWS PURSUING GRADUATE EDUCATION AT THIS GRFP INSTITUTION. THIS AWARD REFLECTS NSF'S STATUTORY MISSION AND HAS BEEN DEEMED WORTHY OF SUPPORT THROUGH EVALUATION USING THE FOUNDATION'S INTELLECTUAL MERIT AND BROADER IMPACTS REVIEW CRITERIA.

UPSTREAM REQUIREMENTS FOR THE REGULATION OF MITOCHONDRIAL BIOENERGETICS BY THE MITOCHONDRIAL ACYL CARRIER PROTEIN - PROJECT SUMMARY RECENT ADVANCES IN CELL BIOLOGY HAVE IMPLICATED METABOLISM IN DISEASES RANGING FROM CANCER TO HEART FAILURE. THE CELLULAR HUBS OF METABOLISM, MITOCHONDRIA, ARE OFTEN CALLED THE “POWERHOUSES OF THE CELL,” AND HAVE BECOME AN ATTRACTIVE THERAPEUTIC TARGET FOR THESE COMMON DISEASES. PATIENTS SUFFERING FROM MITOCHONDRIAL DISEASE ALSO STAND TO BENEFIT FROM MITOCHONDRIALLY-TARGETED THERAPEUTICS. HOWEVER, CURRENTLY, OUR ABILITY TO INFLUENCE MITOCHONDRIAL FUNCTION THERAPEUTICALLY IS LIMITED. UNCOVERING HOW MITOCHONDRIA REGULATE ENERGY PRODUCTION IN NORMAL CONDITIONS WILL PROVIDE THE GROUNDWORK FOR THE DEVELOPMENT OF FUTURE TREATMENT STRATEGIES. THE MITOCHONDRIAL ACYL CARRIER PROTEIN (MTACP) SITS AT THE INTERSECTION OF NUTRIENT INPUTS AND ENERGY EXPENDITURE IN THE MITOCHONDRIA. THE ACYLATED FORM OF MTACP BOTH SERVES AS A SOURCE FOR CITRIC ACID CYCLE (TCA) ENZYME ACTIVATION VIA LIPOYLATION, AS WELL AS A STABILIZATION FACTOR IN ELECTRON TRANSPORT CHAIN ASSEMBLY, CONTROLLING THE TWO MAJOR ARMS OF MITOCHONDRIAL OXIDATIVE METABOLISM. DESPITE ITS CENTRAL ROLE IN OXIDATIVE METABOLISM, HOW ACYL-MTACP IS FORMED IN THE MITOCHONDRIA IS POORLY DEFINED. ACYL-MTACP FORMATION RELIES ON A 4’- PHOSPHOPANTETHEINE (4’-PP) MODIFICATION, FOLLOWED BY THE ADDITION OF 2-CARBON UNITS FROM MALONYL-COA DECARBOXYLATION VIA DE NOVO MITOCHONDRIAL FATTY ACID SYNTHESIS (MTFAS). THE ENZYME RESPONSIBLE FOR MTACP 4’- PHOSPHOPANTETHENYLATION IN VIVO IS NOT KNOWN, AND THE SUBCELLULAR COMPARTMENT WHERE THIS OCCURS IS UNDEFINED YET HAS MAJOR IMPLICATIONS FOR THE REGULATION OF THIS PROCESS. FINALLY, THE CARBON SUBSTRATE OF MTFAS IS HIGHLY DISPUTED. THE CARBON SOURCE FOR MTACP ACYLATION HAS HUGE IMPLICATIONS FOR THE REGULATION OF MTACP AND THEREFORE MITOCHONDRIAL OXIDATIVE METABOLISM. OUR PRELIMINARY ANALYSES INDICATE THAT MTACP IS 4’- PHOSPHOPANTETHEINYLATED IN THE MITOCHONDRIA BY THE ONLY KNOWN MAMMALIAN PPTASE: AASDHPPT, AND THAT MITOCHONDRIAL ACETYL-COA PROVIDES CARBON FOR ACYLATION OF MTACP BY MTFAS. THE EXPERIMENTS IN THIS PROPOSAL WILL DEFINITIVELY EXPLAIN HOW MTACP IS ACTIVATED (4’-PP) AND HOW THE ACYLATION SUBSTRATE (MALONYL-COA) IS GENERATED, THUS PROVIDING A CLEARER PICTURE OF UPSTREAM REGULATION OF MTACP IN MAMMALIAN MITOCHONDRIA. UNDERSTANDING THE KEY ENZYMES IN MTACP ACTIVATION AND THEIR ACTIVITY IN CELLS WILL PROVIDE THE FOUNDATION FOR FUTURE STUDIES THAT SEEK TO MODULATE MTACP ACTIVITY IN PATIENTS WITH MITOCHONDRIAL DYSFUNCTION. THE PROPOSED STUDY WILL BE CONDUCTED AS PART OF THE APPLICANT’S TRAINING IN A JOINT MD-PHD PROGRAM THROUGH MICHIGAN STATE UNIVERSITY COLLEGE OF HUMAN MEDICINE AND THE VAN ANDEL INSITUTE. THE VAN ANDEL INSITUTE PROVIDES PHD TRAINING IN A SMALL AND COLLABORATIVE ENVIRONMENT WITH WORLD-CLASS SCIENTISTS AND STATE-OF-THE ART FACILITIES. THE APPLICANT WILL RECEIVE MENTORSHIP FROM LEADING EXPERTS IN THE FIELD OF METABOLISM, METABOLOMICS, AND CELL BIOLOGY, AS WELL AS COMPLETE SUPPORT FOR CONTINUED CLINICAL ACTIVITY, COLLABORATION ACROSS SCIENTIFIC DISCIPLINES, AND PROFESSIONAL DEVELOPMENT THROUGHOUT THE DEGREE PROGRAM.

PTPMT1 REGULATES CANCER CELL BIOENERGETICS BY SUPPRESSING OXIDATIVE METABOLISM

THE ROLE OF POLYCOMB TARGET GENE DNA METHYLATION IN INTESTINAL TUMORIGENESIS

CD82 AND CELL-CELL ADHESION IN MATASTIC PROSTATE CANCER

TARGETING PTGIR-PROSTACYCLIN SIGNALING TO OVERCOME T CELL EXHAUSTION AND ENHANCE ANTI-TUMOR IMMUNITY - PROJECT SUMMARY CONTINUOUS EXPOSURE TO CANCER ANTIGENS CAUSES CD8 T CELLS TO BECOME INEFFECTIVE; A STATE REFERRED TO AS CD8 T CELL EXHAUSTION (TEX). ONE HALLMARK OF TEX IS THE INCREASE IN CELL SURFACE IMMUNE CHECKPOINT PROTEINS THAT ENGAGE IN PROTEIN-PROTEIN INTERACTIONS WITH THEIR RESPECTIVE LIGANDS ON CANCER CELLS. IMMUNE CHECKPOINT INHIBITORS (ICIS) INTERCEPT THESE INTERACTIONS AND “REINVIGORATE” TEX CELLS, AND ARE NOW A STANDARD TREATMENT FOR MANY CANCERS. UNFORTUNATELY, UP TO 50% OF ICI-TREATED CANCER PATIENTS DO NOT RESPOND TO TREATMENT OR RELAPSE, INDICATING THAT THERE ARE OTHER MECHANISMS THAT ENFORCE T CELL EXHAUSTION. THE LONG-TERM GOAL OF THIS PROJECT IS TO IDENTIFY AND UNDERSTAND THESE ALTERNATIVE MECHANISMS, SO WE CAN DEVELOP COMPLEMENTARY THERAPIES THAT WILL DELIVER MORE DURABLE CLINICAL RESPONSES. TO THAT END, WE RECENTLY FOUND THAT HYPERACTIVATION OF THE TRANSCRIPTION FACTOR NRF2 (NUCLEAR FACTOR ERYTHROID 2-RELATED FACTOR 2) UPREGULATES THE PROSTACYCLIN RECEPTOR (PTGIR) TO DRIVE TEX IN MOUSE CD8 T CELLS. SPECIFICALLY, PTGIR SIGNALING REDUCES THE PERCENTAGE OF CYTOKINE-PRODUCING CD8+ TUMOR INFILTRATING LYMPHOCYTES (TILS), RESULTING IN AGGRESSIVE GROWTH OF MOUSE TUMORS. PRELIMINARY IN VITRO DATA HINT THAT PTGIR SIGNALING IN CD8 T CELLS MAY DAMPEN MITOCHONDRIAL BIOENERGETICS BY REDUCING ACETATE ENTRY INTO THE TCA CYCLE. CONVERSELY, PATIENTS (AND MICE) WHO HAVE ELEVATED LEVELS OF PROSTACYCLIN SYNTHASE, AND HENCE PROSTACYCLIN, IN THEIR TUMORS EXHIBIT POOR OVERALL SURVIVAL. THESE DATA SUGGEST THAT THE PTGIR:PROSTACYCLIN INTERACTION IS A NOVEL IMMUNE CHECKPOINT THAT DRIVES MITOCHONDRIAL DYSFUNCTION TO ENFORCE TEX. IF TRUE, THEN PHARMACOLOGICALLY INTERCEPTING PTGIR-PROSTACYCLIN SIGNALING IN VIVO MIGHT BE A NEW MEANS OF ATTENUATING T CELL EXHAUSTION AND BOOSTING ANTI-TUMOR IMMUNITY. THESE HYPOTHESES WILL BE TESTED IN SEVERAL INTER-RELATED AIMS USING MOUSE TUMOR MODELS. AIM 1 WILL USE A JOINT RNA-SEQ/13C METABOLOMICS METHOD TO DETERMINE HOW PTGIR DRIVES T CELL EXHAUSTION IN VIVO, THUS LINKING PROSTACYCLIN RECEPTOR SIGNALING IN THE TME TO INTRACELLULAR CD8+ TIL MITOCHONDRIAL DYSFUNCTION. AIM 2 WILL LEVERAGE 13C METABOLOMICS TO SHOW THAT CANCER CELLS SECRETE PROSTACYCLIN, AND SCRNA-SEQ TO SHOW THAT PROSTACYCLIN INDUCES TEX, AND AN IMMUNOSUPPRESSIVE TME. THESE DATA WILL LAY A FOUNDATION FOR DEVELOPING PROSTACYCLIN SYNTHASE INHIBITORS AS AN ADJUNCT CANCER THERAPY. AIM 3 WILL TEST WHETHER REPURPOSING OF PTGIR INHIBITORS THAT BLOCK THE PTGIR:PROSTACYCLIN CHECKPOINT, CAN PREVENT TEX, OR REJUVENATE AN IMMUNOSUPPRESSED TME. THIS PROJECT WILL ALSO HELP US DEVELOP NEW SKILLS IN RNA-SEQ, SCRNA-SEQ, AND IN VIVO 13C METABOLOMICS, THAT WILL BE INSTRUMENTAL IN ESTABLISHING AN INDEPENDENT LAB AND CAREER. ACCORDINGLY, OUR FUTURE TEAM WILL BE IN A STRONG POSITION TO DEVELOP AND TEST NEW IMMUNE CHECKPOINT INHIBITORS THAT TARGET PROTEIN:LIPID INTERACTIONS, WHICH MAY COMPLEMENT STANDARD-OF-CARE IMMUNOTHERAPIES TO IMPROVE CANCER PATIENT SURVIVAL RATES.

DETERMINANTS OF SUSCEPTIBILITY TO PEDIATRIC ACUTE MYELOID LEUKEMIA - F99CA294248 PI: LAUREN HARMON ADMINISTRATIVE SUPPLEMENT FOR FELLOWSHIP AWARD – CHILDCARE COSTS PROJECT SUMMARY/ABSTRACT NO CHANGE FROM ORIGINAL SUBMISSION.

MECHANISMS OF DNMT3B-MEDIATED DNA METHYLATION MAINTENANCE - PROJECT SUMMARY DNA METHYLATION (5MC) IS CATALYZED BY THREE DNA METHYLTRANSFERASES (DNMTS): DNMT1, DNMT3A, AND DNMT3B. DNMT3A AND 3B ARE OFTEN REFERRED TO AS DE NOVO DNMTS, FUNCTIONING TO ESTABLISH 5MC PATTERNS DURING EARLY EMBRYONIC DEVELOPMENT. DNMT1 IS REFERRED TO AS THE MAINTENANCE METHYLTRANSFERASE, FUNCTIONING TO COPY ESTABLISHED 5MC PATTERNS ON NEWLY REPLICATED DNA IN DIFFERENTIATED AND SELF-RENEWING STEM CELLS. DESPITE THIS SIMPLIFIED DIVISION OF LABOR, DNMT3A AND 3B ALSO CONTRIBUTE TO 5MC MAINTENANCE, THOUGH THE REGULATORY MECHANISMS INVOLVED ARE INCOMPLETELY UNDERSTOOD. A KEY 5MC REGULATORY MECHANISM IS THE INTERACTION BETWEEN DNMTS AND HISTONE POST-TRANSLATIONAL MODIFICATIONS (PTMS). FOR EXAMPLE, LYSINE METHYLATION ON H3K4 AND H3K36 HAS BEEN REPORTED TO REPEL OR RECRUIT DNMT3B BINDING, RESPECTIVELY. THE FUNCTIONAL OUTCOMES OF THESE INTERACTIONS AID IN THE REGULATION OF GENE EXPRESSION AND TRANSCRIPTION FIDELITY. SPECIFICALLY, DNMT3B-MEDIATED METHYLATION OF ACTIVELY TRANSCRIBED GENE BODIES WAS SHOWN TO PROTECT AGAINST SPURIOUS TRANSCRIPTION, AND THIS FUNCTION IS DEPENDENT ON TRI-METHYLATION OF H3K36 (H3K36ME3). HOWEVER, MY PRELIMINARY DATA, AS WELL AS DATA FROM PRIOR LITERATURE, SHOW THAT H3K36ME3 IS DISPENSABLE FOR GENE BODY 5MC MAINTENANCE. THESE RESULTS CHALLENGE THE ESTABLISHED MODEL AND SUGGEST THAT THERE ARE ALTERNATIVE MECHANISMS WHICH REGULATE DNMT3B-MEDIATED 5MC AT INTRAGENIC REGIONS. THE OVERARCHING GOAL OF THIS FELLOWSHIP PROPOSAL IS TO DEFINE MECHANISMS OF DNMT3B RECRUITMENT AND ACTIVITY IN GENE BODIES OF EMBRYONIC AND DIFFERENTIATED CELLS. IN ADDITION TO HISTONE H3, DNMT3B WAS REPORTED TO DIRECTLY INTERACT WITH THE TRANSCRIPTION ELONGATION-ASSOCIATED HPAF1C COMPLEX IN EMBRYONIC DEVELOPMENT. FURTHER, HPAF1C COMPONENTS ARE REQUIRED FOR DEPOSITION OF H3K36ME3 ACROSS GENE BODIES, AND HPAF1C MUTATION RESULTS IN ABERRANT EXPRESSION OF GENES SILENCED BY DNA HYPERMETHYLATION. TAKEN TOGETHER, THESE DATA LEAD ME TO HYPOTHESIZE THAT DNMT3B IS RECRUITED TO CHROMATIN THROUGH MULTIVALENT ENGAGEMENT OF THE N-TERMINUS OF HISTONE H3 AND HPAF1C TO ESTABLISH AND MAINTAIN GENE BODY 5MC. THIS HYPOTHESIS WILL BE TESTED WITH TWO SPECIFIC AIMS. IN AIM 1, I WILL UTILIZE ONCOHISTONE MUTATIONS, CRISPR/CAS9 GENOME EDITING, AND GENETIC COMPLEMENTATION EXPERIMENTS TO RIGOROUSLY DEFINE THE CONTRIBUTION OF H3K4 AND H3K36 METHYLATION IN THE REGULATION OF DNMT3B-MEDIATED 5MC MAINTENANCE. ADDITIONALLY, I WILL USE PUBLICLY AVAILABLE CANCER GENOME DATA TO UNDERSTAND THE CLINICAL RELEVANCE OF DNMT3B- MEDIATED GENE BODY 5MC. IN AIM 2, I WILL USE CHEMICAL INHIBITORS OF TRANSCRIPTION ELONGATION, PROTEIN-PROTEIN INTERACTION ANALYSIS, AND DEEP RNA-SEQUENCING TO INVESTIGATE THE INTERACTION BETWEEN DNMT3B AND HPAF1C AND ITS ROLE IN CO-TRANSCRIPTIONAL DEPOSITION OF 5MC IN GENE BODIES. COLLECTIVELY, THESE STUDIES WILL LEAD TO A DEEPER UNDERSTANDING OF MECHANISMS OF EPIGENETIC AND TRANSCRIPTIONAL CROSSTALK THAT CONTRIBUTE TO THE PROPAGATION OF ABERRANT 5MC PATTERNING IN HUMAN CANCERS.

SURVIVAL SIGNALING IN PROSTATE CANCER: ROLE OF ANDROGEN RECEPTOR AND INTERGRINS IN REGULATING SURVIVAL

STRUCTURAL BASIS FOR REPRESSOR-ACTIVATOR TRANSITIONS IN RESPONSE TO THE PLANT DEFENSE HORMONE, JASMONIC ACID

IMPACT OF NEUROFIBROMIN ON ER-ALPHA AND NUCLEAR SPECK POST-TRANSCRIPTIONAL GENE REGULATION - PROJECT SUMMARY ALTERNATIVE SPLICING (AS) OF TRANSCRIPTS IS AN ESSENTIAL POST-TRANSCRIPTIONAL PROCESS THAT IS INSTRUMENTAL FOR NUMEROUS CELL FUNCTIONS INCLUDING PROLIFERATION, DIFFERENTIATION, AND SURVIVAL. THE PROCESS OF AS IS REGULATED BY RNA BINDING PROTEINS (RBPS) AND THEIR PROPER NUCLEAR LOCALIZATION AND INTERACTIONS WITH SPLICEOSOME COMPONENTS WHICH FORM VISIBLY DENSE COMPARTMENTS CALLED NUCLEAR SPECKS (NS). THE ABILITY FOR CANCER TO HIJACK AND UTILIZE AS BY TARGETING GENES INVOLVED IN EMT, INVASION, CELL CYCLE REGULATION, AND TRANSCRIPTIONAL CONTROL HAS BEEN DESCRIBED TO INFLUENCE METASTATIC POTENTIAL AND THERAPEUTIC RESISTANCE. MORE RECENTLY, IT HAS BEEN SHOWN THAT 50% OF BREAST CANCERS CONTAIN OVEREXPRESSION OR AMPLIFICATION OF AT LEAST ONE RBP WHICH CONSEQUENTLY TARGETS GENE TRANSCRIPTS FOR AS TO INDUCE INVASIVE CHARACTERISTICS. FURTHERMORE, A NOVEL FUNCTION OF ESTROGEN RECEPTOR ALPHA (ERA) RECENTLY REVEALED NON-CANONICAL RNA BINDING OF ERA TO INFLUENCE POST- TRANSCRIPTIONAL REGULATION AND PROMOTE CELL FITNESS IN RESPONSE TO ENVIRONMENTAL STRESSORS. OUR LABORATORY, ALONG WITH OTHER, RECENTLY ESTABLISHED A CRITICAL LINK BETWEEN NF1 AND ERA IN REGULATION OF ERA SIGNALING. NF1 IS A TUMOR SUPPRESSOR GENE, ENCODING FOR THE PROTEIN NEUROFIBROMIN, THAT WHEN MUTATED DRIVES OF A SUBSET OF BREAST CANCERS WITH POOR PROGNOSIS. THESE STUDIES HAVE SPARKED MOTIVATION TO INVESTIGATE MECHANISMS IN WHICH NF1-DEFICIENT BREAST CANCERS CONTRIBUTE TO METASTASIS AND ENDOCRINE RESISTANCE. THE INTERACTION BETWEEN NEUROFIBROMIN AND ERA DESCRIBES NEUROFIBROMINS ABILITY TO ACT AS A CO- REPRESSOR OF ERA’S TRANSCRIPTIONAL ACTIVITY. HOWEVER, IT STILL REMAINS UNKNOWN WHETHER THIS NEUROFIBROMIN-ERA INTERACTION REGULATES ERA’S POST-TRANSCRIPTIONAL REGULATION OR THE IMPORTANCE OF NEUROFIBROMIN LOCALIZATION FOR THIS REGULATION. THE OVERALL GOAL OF MY F99 TRAINING PHASE (AIM 1) IS TO DETERMINE THE ROLE NEUROFIBROMIN PLAYS IN ERA POST-TRANSCRIPTIONAL REGULATION. SPECIFICALLY, THE AIMS ARE 1.1) TO DEFINE NEUROFIBROMIN’S IMPACT ON ERA DEPENDENT POST-TRANSCRIPTIONAL REGULATION IN NF1 MUTANT ER+ BREAST CANCER MODELS AND 1.2) DETERMINE THE EFFICACY OF SELECTIVE ESTROGEN RECEPTOR MODULATORS (SERMS) TO IMPACT ERA POST-TRANSCRIPTIONAL REGULATION IN NF1-DEFICIENT CELLS. THE TRAINING OBJECTIVES IN THE F99 PHASE WILL EXPAND MY MOLECULAR TECHNIQUES, SEQUENCING, AND DATA ANALYSIS IN CANCER BIOLOGY. DURING MY K00 PHASE (AIM 2) I WILL DEFINE THE IMPACT NUCLEAR NEUROFIBROMIN HAS ON POST-TRANSCRIPTIONAL REGULATION THROUGH NUCLEAR SPECK FORMATION AND FUNCTION. NF1- DEFICIENT BREAST CANCER MODELS WILL BE USED TO DETERMINE NUCLEAR SPECK FORMATION, COMPOSITION, AND POST- TRANSCRIPTIONAL AND GENE REGULATION. THE TRAINING AND DATA RESULTING FROM THIS FELLOWSHIP AWARD WILL ESTABLISH THE FOUNDATION OF SCIENTIFIC AND PROFESSIONAL SKILLS FOR MY CAREER AS AN INDEPENDENT RESEARCHER.

GRADUATE RESEARCH FELLOWSHIP PROGRAM

DEVELOPMENT OF MITHRAMYCIN ANALOGUES TO TARGET TRANSCRIPTIONAL VULNERABILITIES IN RHABDOID TUMOR

IDENTIFYING DETERMINANTS OF TAU PATHOLOGY SEEDING - PROJECT SUMMARY/ABSTRACT ALZHEIMER'S DISEASE (AD) IS THE MOST COMMON NEURODEGENERATIVE DISORDER IN THE UNITED STATES AND IS PROJECTED TO AFFECT 14 MILLION PEOPLE BY 2060. ACCUMULATION OF TAU PATHOLOGY IN AD STRONGLY CORRELATES TO PATIENTS' COGNITIVE DECLINE. CURRENT MODELS OF PROGRESSIVE TAU PATHOLOGY RELY ON EITHER TRANSGENIC MICE OR TAU FIBRILS ISOLATED FROM THE DISEASED HUMAN BRAIN IN WILD-TYPE MICE. ATTEMPTS TO INDUCE TAU PATHOLOGY IN WILD-TYPE MICE USING RECOMBINANT TAU FIBRILS HAVE FAILED. RECENT STUDIES HAVE REVEALED STRUCTURAL AND POST-TRANSLATIONAL DIFFERENCES BETWEEN RECOMBINANT AND HUMAN-DERIVED TAU FIBRILS THAT MAY DRIVE THIS DIFFERENCE IN SEEDING CAPACITY. THE OVERARCHING GOAL OF THIS FELLOWSHIP IS TO USE IN VITRO AND IN VIVO METHODS TO DETERMINE THE CONFORMATIONAL AND ENVIRONMENTAL DRIVERS OF TAU PATHOLOGY SEEDING. RECENT WORK PRODUCED TRUNCATED TAU FIBRILS WITH VARIOUS CORE STRUCTURES, INCLUDING ONE WITH A CORE THAT IS STRUCTURALLY IDENTICAL TO THE CORE REGION OF AD PHFS. YET, THESE RECOMBINANT FIBRILS LACK POST-TRANSLATIONAL MODIFICATIONS ASSOCIATED WITH NEUROFIBRILLARY TANGLES. PROTEOMIC WORK HAS IDENTIFIED POST-TRANSLATIONALLY MODIFIED RESIDUES ON THE TAU PROTEIN ASSOCIATED WITH PATHOLOGY, AND MANY OF THESE DISEASE-ASSOCIATED POST-TRANSLATIONAL MODIFICATIONS CAN BE INTRODUCED BY PRODUCING TAU IN INSECT CELLS. THE PROPOSED RESEARCH WILL TAKE ADVANTAGE OF THESE RECENT INNOVATIONS TO ASSESS THE ROLE OF TAU FIBRIL STRUCTURE AND POST-TRANSLATIONAL MODIFICATIONS IN SEEDING CAPACITY. AIM 1 OF THIS WORK WILL USE TAU FIBRILS WITH VARYING CORE STRUCTURES, INCLUDING THOSE IDENTICAL TO AD TAU, AND POST-TRANSLATIONAL MODIFICATIONS TO EVALUATE THE PATHOLOGICAL SEEDING CAPACITY IN A PRIMARY NEURON CULTURE MODEL. AIM 2 OF THIS WORK WILL UTILIZE A QUANTITATIVE PATHOLOGY MODELING WORKFLOW TO QUANTIFY PROGRESSIVE PATHOLOGICAL SEEDING OF SELECTED TAU FIBRILS IN WILD-TYPE MICE OVER THE COURSE OF NINE MONTHS. THE DATA COLLECTED AND TRAINING ACQUIRED DURING THIS FELLOWSHIP NOT ONLY HAVE THE POTENTIAL TO SHAPE FUTURE MODEL SYSTEMS IN AD BUT WILL ALSO SHAPE AND FOCUS MY TRAJECTORY TOWARDS BECOMING A LEADER IN NEURODEGENERATIVE RESEARCH. UNDER THIS TRAINING PERIOD AT VAN ANDEL RESEARCH INSTITUTE, I WILL DEVELOP TECHNICAL SKILLS IN IMAGE ANALYSIS, MOUSE NEUROANATOMY, AND NEGATIVE STAIN EM. I WILL ALSO HAVE AMPLE OPPORTUNITY TO GAIN CONFIDENCE IN BOTH ORAL AND POSTER PRESENTATIONS. MY COLLABORATIONS WITH THE CRYO-ELECTRON MICROSCOPY CORE AT VAI, A BIOTECHNOLOGY COMPANY, AND A WORLD LEADER IN TAU FIBRIL STRUCTURE WILL ALLOW ME TO GAIN EXPERTISE IN RUNNING COLLABORATIVE PROJECTS IN ADDITION TO RECEIVING ADDITIONAL GUIDANCE ON STRUCTURE-BASED QUESTIONS IN THE FIELD OF NEURODEGENERATIVE DISEASE.

EPIGENETIC REGULATION OF A NON-CODING RNA, NC886

TARGETING MECHANISMS OF CHROMATIN PLASTICITY TO IMPROVE THE EFFICACY OF EPIGENETIC SOLID TUMOR THERAPY - PROJECT SUMMARY ABERRANT DNA METHYLATION IS A HALLMARK OF NEARLY ALL HUMAN CANCERS, DRIVING ONCOGENESIS THROUGH ABNORMAL GENE EXPRESSION. DNA METHYLTRANSFERASE (DNMT) INHIBITORS HOLD PROMISE FOR REVERSING THESE ABNORMAL DNA METHYLATION PATTERNS AND ACT AS “PRIMING AGENTS” TO ENHANCE IMMUNOTHERAPY IN HEMATOLOGICAL CANCERS. HOWEVER, DNMT INHIBITORS AS SINGLE-AGENT THERAPIES HAVE YIELDED LIMITED CLINICAL BENEFIT IN SOLID TUMORS DUE TO INSUFFICIENT DNA DEMETHYLATION AND POORLY UNDERSTOOD RESISTANCE MECHANISMS. THE OVERARCHING GOAL OF MY F99 TRAINING PHASE IS TO POTENTIATE THE EFFICACY OF DNMT INHIBITORS FOR TREATING SOLID TUMORS. PRELIMINARY STUDIES FROM MY DISSERTATION RESEARCH HAVE DEMONSTRATED THAT DNMT INHIBITOR TREATMENT ELEVATES UHRF1-DEPENDENT HISTONE H3 LYSINE 18 UBIQUITINATION (H3K18UB), WHICH STIMULATES THE SUV39H1/H2 METHYLTRANSFERASE ACTIVITY TO ESTABLISH TRANSCRIPTIONALLY REPRESSIVE HISTONE H3 LYSINE 9 TRI-METHYLATION (H3K9ME3) AT TUMOR SUPPRESSOR GENE (TSG) PROMOTERS IN COLON CANCER CELLS. DISRUPTING THIS H3K18UB-DIRECTED SUV39H1 ACTIVITY THROUGH LOSS-OF-FUNCTION MUTATIONS IN SUV39H1 UBIQUITIN INTERACTION MOTIF (UIM) ENHANCED THE DNMT INHIBITOR-MEDIATED TSG REACTIVATIONS, INNATE IMMUNE RESPONSE GENE UPREGULATIONS, AND ANTI-PROLIFERATION EFFECT IN COLON CANCER CELLS. I HYPOTHESIZE THAT THIS H3K18UB-H3K9ME3 CROSSTALK IS A TARGETABLE MECHANISM THAT LIMITS DNMT INHIBITOR EFFICACY IN SOLID TUMORS. IN AIM 1 (F99 PHASE), I WILL DETERMINE THE EFFECTS OF COMBINED SUV39H1 UIM DISRUPTION AND DNMT INHIBITION ON ANTI-TUMOR EFFICACY AND IMMUNOTHERAPY RESPONSES USING SYNGENEIC COLON CANCER MODELS. I WILL ALSO UTILIZE MACHINE LEARNING-BASED PROTEIN DESIGN TOOLS TO DEVELOP SUV39H1 UIM PROTEIN/PEPTIDE ANTAGONISTS THAT MIGHT BECOME LEAD COMPOUNDS FOR POTENTIAL THERAPEUTIC DEVELOPMENT. IN AIM 2 (K00 PHASE), I WILL EXTEND THESE CHROMATIN PLASTICITY INSIGHTS TO IMPROVE CHIMERIC ANTIGEN RECEPTOR (CAR) T CELL THERAPY IN SOLID TUMORS, LEVERAGING EPIGENETIC MODULATION TO ENHANCE T CELL PERSISTENCE AND REDUCE EXHAUSTION. DNMT3A DEPLETION HAS BEEN SHOWN TO ENHANCE CAR T CELL MEMORY AND RESISTANCE TO EXHAUSTION, WHILE IT IS UNCLEAR WHETHER COMPENSATORY CHROMATIN MODIFICATIONS IN DNMT3A-DEPLETED T CELLS MAY LIMIT THEIR THERAPEUTIC EFFICACY IN SOLID TUMORS. USING A CRISPR SCREEN TARGETING EPIGENETIC REGULATORS IN DNMT3A-DEPLETED CAR T CELLS, I WILL IDENTIFY KEY FACTORS THAT SYNERGIZE WITH DNMT3A TO STRENGTHEN CAR T CELL FUNCTION. I WILL VALIDATE TOP CANDIDATES FOR THEIR EFFECTS ON T CELL PERSISTENCE, MEMORY FORMATION, AND ANTITUMOR ACTIVITY IN VITRO, AND I WILL ASSESS THEIR POTENTIAL TO SUSTAIN DURABLE ANTITUMOR RESPONSES IN VIVO. OVERALL, THIS PROJECT AIMS TO OVERCOME KEY BARRIERS TO DNMT INHIBITOR AND CAR T CELL THERAPIES IN SOLID TUMORS BY IDENTIFYING NOVEL EPIGENETIC VULNERABILITIES. BY BRIDGING FUNDAMENTAL EPIGENETIC RESEARCH WITH TRANSLATIONAL CANCER THERAPY, THIS PROJECT WILL ADVANCE THERAPEUTIC STRATEGIES FOR SOLID TUMORS AND SUPPORT MY DEVELOPMENT INTO AN INDEPENDENT TRANSLATIONAL RESEARCH SCIENTIST.

GENOMIC ANALYSIS OF TUMOR CONTEXT VULNERABILITIES IN HUMAN METASTATIC MELANOMA

CORTICAL NEURONAL VULNERABILITY IN PARKINSON'S DISEASE - ABSTRACT PARKINSON’S DISEASE (PD) IS A DEBILITATING, PROGRESSIVE NEUROLOGICAL DISEASE CHARACTERIZED BY ACCUMULATION OF MISFOLDED Α-SYNUCLEIN (REFERRED TO AS LEWY PATHOLOGY) THAT PROGRESSES THROUGHOUT THE BRAIN AND THE LOSS OF PATIENT MOTOR FUNCTION. NO DISEASE MODIFYING TREATMENTS ARE AVAILABLE, AND THEREFORE THESE PATIENTS CONTINUE TO DETERIORATE THROUGHOUT THE DISEASE. THEREFORE, THIS PROJECT INTENDS TO DETERMINE WHICH NEURONS ARE VULNERABLE AND RESILIENT IN PD, AND WHAT CELLULAR DYSFUNCTION(S) THEY EXPERIENCE. THIS TRAINING INTENDS TO 1.) DEVELOP AND REFINE SKILLS IN BIOINFORMATICS, GENOMICS, AND WET-LAB TECHNIQUES 2.) APPLY THESE DEVELOPED SKILLS TO LEAD THE INDEPENDENT RESEARCH PROJECT OUTLINED IN THIS PROPOSAL 3.) ENHANCE COMMUNICATION AND PRESENTATION SKILLS TO ENSURE A SUCCESSFUL TRANSITION TO THE NEXT STEP IN A CAREER FOCUSED ON NEURODEGENERATIVE DISEASE RESEARCH. MY PREVIOUS WORK HAS DETERMINED THAT LEWY PATHOLOGY BEARING CELLS EXPERIENCE SPECIFIC CELLULAR DYSFUNCTIONS WHICH LEADS TO ACTIVATION OF CELL-DEATH PATHWAYS. IN ADDITION, I HAVE IDENTIFIED THAT SPECIFIC NEURON TYPES ARE VULNERABLE (LAYER 5 INTRATELENCEPHALIC NEURONS) AND RESILIENT (LAYER 5 PYRAMIDAL TRACT NEURONS) TO LEWY PATHOLOGY IN THE PD CORTEX. EXPANDING ON MY PREVIOUS WORK, I WILL (AIM 1) IDENTIFY RESILIENT NEURONS AND PATHOLOGIC SIGNATURES IN THE PD CORTEX AT SINGLE CELL RESOLUTION USING SPATIAL TRANSCRIPTOMICS. THROUGH THIS, I WILL DEFINE THE NEURONAL RESPONSE TO AGGREGATES AS THEY MATURE WITHIN INDIVIDUAL NEURONS. ADDITIONALLY, I WILL (AIM 2) DETERMINE THE SPATIOTEMPORAL BASIS OF RESILIENCE TO Α-SYNUCLEIN PATHOLOGY. IN THIS AIM, I WILL DETERMINE WHETHER DISEASE STAGE (TIME) AND/OR SYMPTOM SPECIFIC (BRAIN REGION) TREATMENTS ARE TARGETABLE OR DETERMINE IF MORE UNIVERSAL DISEASE MODIFYING TREATMENTS ARE POSSIBLE DUE TO OVERLAP IN CELLULAR DYSFUNCTIONS ACROSS BRAIN REGIONS AND TIME POINTS. IN ADDRESSING THESE TWO AIMS, WE GAIN UNDERSTANDING OF THE STAGES OF MOLECULAR RESPONSE TO LEWY PATHOLOGY AS AGGREGATES MATURE, AS DISEASE PROGRESSES, AND IN MULTIPLE BRAIN REGIONS AND THEREFORE GAIN UNDERSTANDING OF THE MECHANISMS OF RESILIENCE TO LEWY PATHOLOGY. IN ADDITION, WE IDENTIFY PATHWAYS AND SPECIFIC GENES TO TARGET FOR DISEASE MODIFYING TREATMENT OF PARKINSON’S DISEASE. FINALLY, THE PROPOSED TRAINING GOALS AND OBJECTIVES PROVIDE AN EXCELLENT FOUNDATION IN THE REQUISITE SKILLS AND EXPERTISE NECESSARY FOR A SUCCESSFUL FUTURE CAREER IN NEURODEGENERATIVE DISEASE RESEARCH.

MIDWEST ZEBRAFISH MEETING 2026 - PROJECT SUMMARY THE MIDWEST ZEBRAFISH MEETING (MWZM) IS A BIENNIAL SCIENTIFIC CONFERENCE THAT BRINGS TOGETHER RESEARCHERS USING THE ZEBRAFISH MODEL TO STUDY FUNDAMENTAL QUESTIONS IN DEVELOPMENTAL BIOLOGY, REPRODUCTION, GENETICS, AND DISEASE. THE MEETING SERVES AS A KEY REGIONAL HUB FOR FOSTERING COLLABORATION, TECHNICAL TRAINING, AND EARLY- CAREER DEVELOPMENT IN THE ZEBRAFISH RESEARCH COMMUNITY ACROSS THE MIDWEST, WITH PARTICIPATION FROM RESEARCHERS NATIONWIDE. THIS YEAR, MWZM WILL BE HOSTED AT THE VAN ANDEL RESEARCH INSTITUTE IN GRAND RAPIDS, MICHIGAN. GRAND RAPIDS IS CENTRALLY LOCATED IN THE MIDWEST, AND EASILY ACCESSIBLE VIA PLANE, TRAIN, OR AUTOMOBILE. THE VAN ANDEL RESEARCH INSTITUTE (VARI) IS WELL EQUIPPED TO HOST A MEETING OF THIS SIZE (APPROXIMATELY 175 ATTENDEES), WITH AN AUDITORIUM AND BREAK-OUT ROOMS, AV SPECIALISTS, AND EVENTS TEAM SUPPORT ON SITE. VARI IS LOCATED WITHIN WALKING DISTANCE OF HOTELS AND RESTAURANTS. WE HAVE ARRANGED FOR DISCOUNTED HOTEL BLOCKS AT TWO NEIGHBORING HOTELS. ZEBRAFISH ARE A POWERFUL VERTEBRATE MODEL ORGANISM UNIQUELY SUITED FOR REAL-TIME IMAGING, HIGH-THROUGHPUT GENETIC MANIPULATION, AND MODELING OF HUMAN DEVELOPMENTAL DISORDERS. THE MWZM WILL HIGHLIGHT ZEBRAFISH RESEARCH IN AREAS SUCH AS VISUALIZING DEVELOPMENT, TISSUE PATTERNING, NEURAL DEVELOPMENT AND REGENERATION, GERM AND STEM CELL BIOLOGY, CARDIOVASCULAR AND HEMATOPOIETIC DEVELOPMENT, AND DISEASE MODELING. THIS R13 APPLICATION REQUESTS SUPPORT TO ENHANCE THE MWZM’S ABILITY TO ADVANCE RESEARCH AND TRAINING IN THE AREAS OF DEVELOPMENT AND DISEASE, ESPECIALLY NEUROBIOLOGY AND CARDIOVASCULAR BIOLOGY. THE MEETING WILL FEATURE KEYNOTE LECTURES, INVITED AND CONTRIBUTED TALKS, POSTER SESSIONS, AND TECHNICAL WORKSHOPS THAT HIGHLIGHT ZEBRAFISH-BASED DISCOVERIES FROM RESEARCHERS IN THE MIDWEST. SPECIAL EMPHASIS WILL BE PLACED ON PROVIDING EARLY-CAREER RESEARCHERS WITH OPPORTUNITIES TO PRESENT THEIR WORK AND PARTICIPATE IN PROFESSIONAL DEVELOPMENT SESSIONS. R13 SUPPORT WILL ENSURE THE CONTINUED SUCCESS AND ACCESSIBILITY OF THE MEETING BY HELPING TO OFFSET OPERATIONAL COSTS, PROVIDING TRAVEL ASSISTANCE TO TRAINEES, AND DISSEMINATING EDUCATIONAL RESOURCES BEYOND THE CONFERENCE ITSELF. THROUGH THIS SUPPORT, MWZM WILL CONTINUE TO PROMOTE SCIENTIFIC EXCELLENCE, ACCELERATE BIOMEDICAL DISCOVERY, AND STRENGTHEN THE WORKFORCE.

GRAND CHALLENGES IN PARKINSON'S DISEASE

GRAND CHALLENGES IN PARKINSON'S DISEASE