Cold Spring Harbor Laboratory

CSHL CANCER CENTER SUPPORT GRANT

COLD SPRING HARBOR LABORATORY CANCER RESEARCH CENTER

A COMPREHENSIVE CENTER FOR MOUSE BRAIN CELL ATLAS

COMPREHENSIVE CHARACTERIZATION AND CLASSIFICATION OF THE HUMAN TRANSCRIPTOME

COLLABORATORY FOR ATLASING CELL TYPE ANATOMY IN THE FEMALE AND MALE MOUSE BRAIN

INITIATION OF DNA REPLICATION AT CELL ORIGINS IN YEAST

IPGA: GRAMENE - EXPLORING FUNCTION THROUGH COMPARATIVE GENOMICS AND NETWORK ANALYSIS

PROTEIN TYROSINE DEPHOSPHORYLATION & SIGNAL TRANSDUCTION

LANDSCAPE OF TRANSCRIPTION IN HUMAN AND MOUSE

STRUCTURE AND FUNCTION OF NMDA RECEPTORS

SPATIAL ORGANIZATION OF GENE EXPRESSOIN

BIOCHEMISTRY OF PRE-MRNA SPLICING

COMPUTATIONAL AND FUNCTIONAL APPROACHES TO VALIDATING CANCER GENOME TARGETS

GRAMENE: A PLATFORM FOR COMPARATIVE PLANT GENOMICS

THE BASAL GANGLIA HABENULA CIRCUITRY IN REWARD PROCESSING

THE CENTRAL AMYGDALA CIRCUITS IN FEAR LEARNING AND FEAR EXPRESSION

MAIZECODE - AN INITIAL ANALYSIS OF FUNCTIONAL ELEMENTS IN THE MAIZE GENOME

TRPGR: MAIZE CELL GENOMICS: RESOURCES FOR VISUALIZING PROMOTER ACTIVITY AND PROTEIN DYNAMICS USING FLUORESCENT PROTEIN LINES

BET BROMODOMAIN INHIBITION AS TARGETED THERAPY IN ACUTE MYELOID LEUKEMIA

PROBING THE FUNCTION OF LONG NON-CODING RNAS IN DIFFERENTIATION

A HIGH-THROUGHPUT SEQUENCING AND IMAGING APPROACH TO UNDERSTAND THE FUNCTIONAL BASIS OF OLFACTION

A 3D MULTIMODAL MICRON-SCALE HUMAN BRAIN ATLAS BRIDGING SINGLE CELL DATA, NEUROPATHOLOGY AND NEURORADIOLOGY - DIGITIZED REFERENCE BRAINS, ALSO REFERRED TO AS COMMON COORDINATE FRAMEWORKS (CCFS), TOGETHER WITH SUPERPOSED ATLAS ANNOTATIONS, ARE OF CENTRAL IMPORTANCE TO NEUROSCIENCE. THEY BEAR THE SAME RELATION TO NEUROSCIENCE AS DO REFERENCE GENOMES AND GENOME ANNOTATIONS TO CELLULAR AND MOLECULAR BIOLOGY. STRIKINGLY, HOWEVER, SUCH REFERENCE BRAINS FOR HUMANS LAG FAR BEHIND THE CORRESPONDING CCFS FOR NON-HUMAN MODEL ORGANISMS SUCH AS THE LABORATORY MOUSE. EXISTING DATA SETS EITHER HAVE SECTIONS SPACED RELATIVELY FAR APART OR LACK IN-PLANE RESOLUTION DOWN TO THE MICRON SCALE. CRUCIALLY, EXISTING DATA SETS ARE NOT WELL CONNECTED TO THE MAJOR AREAS IN MEDICINE THAT DEAL WITH THE HUMAN BRAIN, NAMELY NEURORADIOLOGY AND NEUROPATHOLOGY. WE WILL MEET THIS NEED BY CREATING AN UNPRECEDENTED MICRON-SCALE, 3D ATLAS THAT COMBINES MULTIPLE MRI MODALITIES AS WELL AS CONTINUOUS SERIAL SECTION HISTOLOGY. IN PARTICULAR, THE REFERENCE ATLAS WILL CONSIST OF NISSL, MYELIN AND H&E STAINS, WITH 20 MICRON CONTIGUOUS SERIAL SECTIONS, AND APPROXIMATELY ~8000 SECTIONS/BRAIN. WE WILL DO SO USING THE TAPE-TRANSFER METHOD, WHICH PRESERVES TISSUE GEOMETRY EVEN IN THE PRESENCE OF DISCONNECTED PIECES TO THE BRAIN BEING SECTIONED, AND PERMITS 3D REASSEMBLY OF THE SECTIONS INTO A 3D VOLUME. WE WILL UTILIZE DIFFEOMORPHIC MAPPING METHODS TO CO-REGISTER THE MRI AND HISTOLOGICAL DATA, AND WILL CREATE A HUMAN BRAIN CCF IN WHICH SINGLE-CELL TRANSCRIPTOMIC AND EPIGENOMIC DATA CAN BE PINNED IN ORDER TO CREATE A HUMAN BRAIN CELL ATLAS. WE WILL USE MACHINE LEARNING APPROACHES TO SEGMENT CELLS AND PROCESSES IN THESE IMAGES AND TO ALGORITHMICALLY DETECT CYTOARCHITECTONIC BOUNDARIES; SUCH MACHINE LEARNING METHODS WILL ALSO BE USED TO PREDICT HISTOLOGY AND CYTOARCHITECTURE FROM MRI DATA, WITH OUR COLLECTED DATA AS A TRAINING SET. WE WILL MAKE OUR DATA FREELY AVAILABLE TO SCIENTISTS AS WELL AS MEDICAL PROFESSIONALS THROUGH AN ONLINE DATA PORTAL WITH A MULTI-RESOLUTION VIEWER FOR ZOOMING AND PANNING THROUGH TERAPIXEL IMAGE DATA, AND ALSO DEPOSIT THE DATA IN A SHARED DATA REPOSITORY TO MAKE IT EASILY ACCESSIBLE TO OTHER RESEARCHERS. WE WILL CONNECT OUR DATA TO A UNIQUE ON-LINE NEUROPATHOLOGY RESOURCE CONTAINING OVER A PETABYTE OF NEUROPATHOLOGICAL IMAGES, INCLUDING H&E STAINED SECTIONS FROM THE CORONAL PLANE. WE EXPECT THAT THE REFERENCE BRAIN DATA WE PRODUCE WILL BECOME THE DE- FACTO STANDARD FOR A HIGH-RESOLUTION REFERENCE ATLAS FOR THE HUMAN BRAIN.

MAKING HYPOMORPHIC TUMOR SUPRESSORS IN VIVO USING RNAI

COMPUTATIONAL AND GENOMIC RESOURCES TO SUPPORT PLANT GENOME FUNCTION - TO GENERATE RESOURCES AND ACCELERATE OUR UNDERSTANDING OF GENOME FUNCTION.

BIOCHEMISTRY OF PRE-MRNA SPLICING

EPIGENETIC REGULATION OF SEX DIFFERENCES IN THE BRAIN

RESEARCH-PGR: DISSECTING THE DYNAMIC EVOLUTION OF PARALOGS IN SHAPING TRAIT VARIATION ACROSS THE SOLANUM PAN-GENOME -THE GROWING POPULATION AND CLIMATE EXTREMES ARE THREATENING FOOD SECURITY. AGRICULTURE IS LARGELY BASED ON A FEW MAJOR CROPS, AND REVOLUTIONARY TECHNOLOGIES IN GENOME SEQUENCING AND CRISPR GENOME ENGINEERING ARE ACCELERATING THEIR IMPROVEMENT. THESE TECHNOLOGIES CAN ALSO IMPROVE ?ORPHAN? CROPS, WHICH ARE NOT WIDELY CULTIVATED OR STUDIED BUT HAVE THE POTENTIAL TO INCREASE THE DIVERSITY AND RESILIENCE OF FOOD PRODUCTION. ORPHAN CROPS ARE RELATED TO MAJOR CROPS, ALLOWING TRANSLATION OF KNOWLEDGE BETWEEN THEM. HOWEVER, ORPHAN CROPS LACK RESEARCH TOOLS, AND AN EVEN GREATER CHALLENGE IS DETERMINING WHETHER SPECIFIC GENETIC MUTATIONS THAT BENEFITTED MAJOR CROPS CAN BE ENGINEERED TO IMPROVE TRAITS SIMILARLY IN ORPHAN CROPS. THIS IS BECAUSE GENE SEQUENCE AND FUNCTION CHANGE AS SPECIES EVOLVE, ESPECIALLY AMONG GENES THAT BECOME DUPLICATED, WHICH IS COMMON IN PLANTS. THIS PROJECT WILL TAKE ADVANTAGE OF THE NIGHTSHADE FAMILY ? A SOURCE OF MANY MAJOR AND ORPHAN CROPS, SUCH AS EGGPLANT, PEPINO, AND TOMATO ? TO STUDY HOW DUPLICATED GENES EVOLVE AND AFFECT AGRICULTURAL TRAITS IN RELATED SPECIES. COMBINING GENOME SEQUENCING AND CRISPR WILL REVEAL SEQUENCE DIVERSITY AMONG THOUSANDS OF DUPLICATED GENES AND ENABLE IMPROVED PREDICTABILITY IN ENGINEERING GENES AND TRAITS ACROSS SPECIES. THIS PROJECT WILL TRAIN YOUNG SCIENTISTS WITH A FOCUS ON DIVERSITY AND INCLUSION, AS WELL AS PROMOTE PUBLIC UNDERSTANDING OF GENOME ENGINEERING IN PLANT BIOLOGY THROUGH A COMMUNITY SCIENCE PROGRAM ON ORPHAN CROPS. FINALLY, NEW CURRICULA AND RESEARCH OPPORTUNITIES FOR UNDERGRADUATE STUDENTS AT A SMALL LIBERAL ARTS COLLEGE WILL BROADEN PARTICIPATION AND TRAINING OF UNDERREPRESENTED GROUPS IN THE PLANT SCIENCES. THIS PROJECT WILL EXPLOIT ADVANCES IN LARGE-SCALE REFERENCE GENOME SEQUENCING, GENE CO-EXPRESSION ANALYSES, AND CRISPR GENOME EDITING TO DISSECT HOW PARALOG DIVERSIFICATION IMPACTS SPECIES-SPECIFIC PHENOTYPES IN A GENUS OF BOTH FUNDAMENTAL AND APPLIED IMPORTANCE. FIFTY SOLANUM SPECIES, INCLUDING 16 ORPHAN CROPS, WILL BE SEQUENCED TO ESTABLISH A SOLANUM PAN-GENOME WITH TELOMERE-TO-TELOMERE REFERENCE ASSEMBLIES, PROVIDING A FOUNDATION FOR GENUS-WIDE COMPARATIVE GENOMICS AND FUNCTIONAL GENETICS. COMPUTATIONAL APPROACHES BASED ON GENOMICS DATA WILL BE DEVELOPED FOR PRECISE ASSEMBLY AND COMPARISON OF COMPLEX GENOMES, AND IDENTIFICATION AND CLASSIFICATION OF PARALOGS AND THEIR RELATIONSHIPS BASED ON THEIR VARIANTS AND EXPRESSION PATTERNS. SIMULTANEOUSLY, TRANSFORMATION PROTOCOLS AND GENOME EDITING WILL BE DEVELOPED AND DEPLOYED FOR AN ARRAY OF SOLANUM TO TEST HOW PARALOGS IMPACT GENOTYPE-TO-PHENOTYPE RELATIONSHIPS WITHIN AND BETWEEN SPECIES. BY FOCUSING ON MAJOR DOMESTICATION GENE FAMILIES AND THE ADAPTATION AND PRODUCTIVITY TRAITS THEY CONTROL, THIS SYNERGISTIC WORK WILL PROVIDE BOTH A NEW UNDERSTANDING OF PARALOG DIVERSIFICATION IN EVOLUTION AND A MORE ROBUST TRANSLATION OF AGRICULTURALLY RELEVANT GENOTYPE-TO-PHENOTYPE RELATIONSHIPS TO ORPHAN CROPS. BEYOND A VALUABLE COMMUNITY RESOURCE OF SOLANUM REFERENCE GENOMES, EXPRESSION DATA, AND CRISPR LINES FOR PLANT RESEARCHERS AND BREEDERS, THIS MULTIDISCIPLINARY PROJECT WILL RESULT IN NEW TOOLS, RESOURCES, AND PRINCIPLES THAT WILL ENABLE THE STUDY AND ENGINEERING OF OTHER TAXA AND TRAITS OF SIGNIFICANCE TO BOTH PLANT BIOLOGY AND CROP IMPROVEMENT. 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.

CSHL MOLECULAR TARGET DISCOVERY AND DEVELOPMENT CENTER

RESEARCH PGR: STRUCTURAL VARIANT LANDSCAPES IN TOMATO GENOMES AND THEIR ROLE IN NATURAL VARIATION, DOMESTICATION AND CROP IMPROVEMENT

EVOLUTIONARY HUMAN GENOMICS: DEMOGRAPHY, NATURAL SELECTION, AND TRANSCRIPTIONAL REGULATION

DOCK7 SIGNALING IN NEURONAL DEVELOPMENT

RESEARCH-PGR: DISSECTING THE GENOMIC ARCHITECTURE OF FUNCTIONAL REDUNDANCY TO MODULATE MERISTEM HOMEOSTASIS AND CROP YIELDS

RNAI, HISTONE MODIFICATION AND THE DDB1/CPSF-LIKE COMPLEX RIK1

UNDERSTANDING THE LOGIC OF THE BRAIN-WIDE OLFACTORY BULB PROJECTOME

COLD SPRING HARBOR LABORATORY CANCER WORKSHOPS

TARGETING THE TUMOR MICROENVIRONMENT TO TERMINATE DRUG RESISTANT BREAST CANCER

BIOLOGICAL DESIGN OF LEMNACEAE AGUATIC PLANTS FOR BIODIESEL PRODUCTION

THE MISSING CIRCUIT: THE FIRST BRAINWIDE CONNECTIVITY MAP FOR MOUSE

THE ROLE OF CRYPTOCHROMES IN ENVIRONMENTAL REGULATION OF GROWTH

STRUCTURE AND FUNCTION OF HETERO-MULTIMERIC LIGAND-GATED ION CHANNELS

CSHL-JAX PATIENT-DERIVED MODELS OF PANCREATIC CANCER AS SYSTEMS FOR INVESTIGATING TUMOR HETEROGENEITY

A UNIFIED QUANTITATIVE MODELING STRATEGY FOR MULTIPLEX ASSAYS OF VARIANT EFFECT - PROJECT SUMMARY / ABSTRACT A CENTRAL GOAL OF GENOMICS IS TO UNDERSTAND THE RELATIONSHIP BETWEEN GENOTYPE AND PHENOTYPE. IN RECENT YEARS, THE ABILITY TO QUANTITATIVELY STUDY GENOTYPE-PHENOTYPE MAPS HAS BEEN REVOLUTIONIZED BY THE DEVELOPMENT OF MULTIPLEX ASSAYS OF VARIANT EFFECT (MAVES), WHICH MEASURE MOLECULAR PHENOTYPES FOR THOUSANDS TO MILLIONS OF GENOTYPIC VARIANTS IN PARALLEL. MAVE IS AN UMBRELLA TERM THAT INCLUDES MASSIVELY PARALLEL REPORTER ASSAYS FOR STUDIES OF DNA OR RNA REGULATORY SEQUENCES, AS WELL AS DEEP MUTATIONAL SCANNING ASSAYS OF PROTEINS OR STRUCTURAL RNAS. THE RAPID ADOPTION OF MAVE TECHNIQUES ACROSS MULTIPLE GENOMIC DISCIPLINES HAS CREATED AN ACUTE NEED FOR COMPUTATIONAL METHODS THAT CAN ROBUSTLY AND REPRODUCIBLY INFER QUANTITATIVE GENOTYPE- PHENOTYPE (G-P) MAPS FROM THE LARGE DATASETS THAT MAVES PRODUCE. HERE WE PROPOSE A UNIFIED CONCEPTUAL AND COMPUTATIONAL FRAMEWORK FOR QUANTITATIVELY MODELING G-P MAPS FROM MAVE DATA. THIS PROPOSAL IS MOTIVATED BY OUR REALIZATION THAT ACCOUNTING FOR THE NOISE AND NONLINEARITIES THAT ARE OMNIPRESENT IN MAVE EXPERIMENTS REQUIRES EXPLICIT MODELING OF BOTH THE MAVE MEASUREMENT PROCESS AND THE G-P MAP OF INTEREST. THIS JOINT INFERENCE STRATEGY IS MORE COMPUTATIONALLY DEMANDING THAN MOST MAVE ANALYSIS METHODS, BUT IT IS FEASIBLE USING MODERN DEEP LEARNING FRAMEWORKS. OUR EXTENSIVE PRELIMINARY DATA SHOW THAT THIS MODELING STRATEGY IS ABLE TO RECOVER HIGH-PRECISION G-P MAPS EVEN IN THE PRESENCE OF MAJOR CONFOUNDING EFFECTS, AND THUS HAS THE POTENTIAL TO BENEFIT MAVE STUDIES IN MULTIPLE AREAS OF GENOMICS. AIM 1 WILL DEVELOP METHODS FOR MODELING THE MEASUREMENT PROCESSES THAT ARISE IN DIVERSE MAVE EXPERIMENTAL DESIGNS. AIM 2 WILL DEVELOP GENERAL METHODS FOR MODELING GENETIC INTERACTIONS WITHIN G-P MAPS, AND WILL USE THESE METHODS IN CONJUNCTION WITH NEW EXPERIMENTS TO ELUCIDATE THE MOLECULAR MECHANISM OF A RECENTLY APPROVED DRUG THAT TARGETS ALTERNATIVE MRNA SPLICING. AIM 3 WILL DEVELOP METHODS FOR INFERRING G-P MAPS THAT REFLECT BIOPHYSICAL MODELS OF GENE REGULATION, INCLUDING BOTH THERMODYNAMIC (I.E., QUASI-EQUILIBRIUM) AND KINETIC (I.E., NON-EQUILIBRIUM STEADY-STATE) MODELS. THESE METHODS WILL THEN BE USED, IN CONJUNCTION WITH NEW MAVE EXPERIMENTS, TO DEVELOP A BIOPHYSICAL MODEL FOR HOW A PLEIOTROPIC TRANSCRIPTION FACTOR REGULATES GENE EXPRESSION THROUGHOUT THE ESCHERICHIA COLI GENOME. AIM 4 WILL STUDY AND DEVELOP METHODS FOR TREATING GAUGE FREEDOMS AND SLOPPY MODES IN THE ABOVE CLASSES OF MODELS, THEREBY FACILITATING THE COMPARISON, INTERPRETATION, AND EXPLORATION OF INFERRED G-P MAPS. ALL OF THE COMPUTATIONAL TECHNIQUES WE DEVELOP WILL BE INCORPORATED INTO A ROBUST AND EASY- TO-USE PYTHON PACKAGE CALLED MAVE-NN. WE WILL BENCHMARK MAVE-NN ON A DIVERSE ARRAY OF MAVE DATASETS, INCLUDING PUBLISHED DATASETS AND DATA GENERATED AS PART OF THIS PROJECT. IN ALL, THIS WORK WILL FILL A MAJOR NEED IN THE ANALYSIS OF MAVE EXPERIMENTS, YIELDING A ROBUST, FLEXIBLE, AND SCALABLE COMPUTATIONAL PLATFORM THAT WILL HELP ACCELERATE THE USE OF MAVES FOR UNDERSTANDING THE EFFECTS OF HUMAN GENETIC VARIATION AT THE GENOMIC SCALE.

RESEARCH-PGR/NSF-BSF: IDENTIFICATION AND FUNCTIONAL DISSECTION OF SHARED CIS-REGULATORY ELEMENTS CONTROLLING QUANTITATIVE TRAIT VARIATION ACROSS ANGIOSPERMS

OSCILLATORY GENE EXPRESSION AND THE MAINTENANCE OF TEMPORAL PATTERNING

2/2-RARE BIPOLAR LOCI IDENTIFICATION THROUGH SYNAPTOME SEQUENCING.

RESEARCH-PGR: LIVING FOSSILS: APPLYING ADVANCES IN SINGLE MOLECULE SEQUENCING TO DECODE LARGE AND COMPLEX GENOMES OF ANCIENT PLANT LINEAGES

FIBROBLAST HETEROGENEITY IN PANCREATIC CANCER - ABSTRACT PANCREATIC DUCTAL ADENOCARCINOMA (PDA) IS CHARACTERIZED BY AN EXTENSIVE DESMOPLASTIC STROMA THAT REPRESENTS A MAJOR CHALLENGE FOR ITS EFFECTIVE TREATMENT AND IMPROVED SURVIVAL OF PATIENTS. WE SEEK TO DEFINE THE COMPOSITION AND ROLES OF CANCER-ASSOCIATED FIBROBLASTS (CAFS), THE MOST ABUNDANT CELL POPULATION IN THE PDA STROMA, AS A POTENTIAL AVENUE FOR THE DEVELOPMENT OF NEW THERAPEUTIC STRATEGIES. ALTHOUGH CAFS HAVE BEEN HISTORICALLY CONSIDERED TUMOR-PROMOTING COMPONENTS, THEIR ABLATION IN PRE-CLINICAL AND CLINICAL STUDIES HAVE LED TO MIXED OUTCOMES, INDICATING THE POORLY UNDERSTOOD COMPLEXITY OF CAFS. TO INVESTIGATE CAF BIOLOGY, WE PREVIOUSLY ESTABLISHED A PANCREATIC TUMOR ORGANOID/FIBROBLAST CO-CULTURE MODEL. IN ADDITION, WE PERFORMED SINGLE CELL RNA- SEQUENCING (SCRNA-SEQ) OF MURINE AND HUMAN PDA SPECIMENS TO CHARACTERIZE CAF COMPOSITION AT SINGLE-CELL RESOLUTION. THESE ANALYSES HAVE REVEALED THAT FIBROBLASTS ARE HETEROGENEOUS AND COMPRISED OF AT LEAST THREE DISTINCT SUBTYPES WITH UNIQUE TRANSCRIPTIONAL AND FUNCTIONAL FEATURES. MORE SO, THIS HETEROGENEITY EMPHASIZES THE NEED TO DESIGN THERAPIES THAT SELECTIVELY TARGET THE TUMOR-PROMOTING CAF POPULATIONS. ALTHOUGH OUR WORK HAS STARTED TO REVEAL THE COMPLEX HETEROGENEITY OF FIBROBLASTS IN PRIMARY OR METASTATIC PDA TISSUES, ITERATIVE DEVELOPMENTS IN SCRNA-SEQ AND ANALYSIS METHODS HAVE REVEALED FOUR ADDITIONAL CAF SUBTYPES IN PRIMARY AND METASTATIC PDA. TO COMPREHENSIVELY DEFINE THE CAF REPERTOIRE IN PRIMARY AND METASTATIC PDA, WE WILL SYSTEMATICALLY DIFFERENTIATE CAF SUBTYPES AND THEIR REGULATORY ELEMENTS USING SCRNA-SEQ, SCATAC-SEQ, MACHINE-LEARNING COMPUTATIONAL METHODS, AND IN SITU TISSUE ANALYTIC METHODS THAT DETECT RNA AND PROTEIN SPECIES, SUCH AS IMAGING MASS CYTOMETRY (AIM 1). WE HYPOTHESIZE THAT A DEEPER UNDERSTANDING OF THE DYNAMIC CAF STATES THAT OCCURS IN PRIMARY AND METASTATIC PDA WILL GUIDE THE SELECTION OF SPECIFIC THERAPEUTIC REGIMENS. TO COMPLEMENT THIS ANALYSIS, WE WILL IDENTIFY NEW CAF SUBTYPES AND STUDY THEIR DYNAMICS IN A NOVEL MURINE MODEL WITH A REVERSIBLE MUTANT KRAS ALLELE (AIM 2). FURTHERMORE, WE WILL STUDY CAF-ACTIVATING PATHWAYS BY INVESTIGATING SEVERAL GENES IMPLICATED IN STROMAL ACTIVATION THAT WERE REVEALED USING OUR RECENTLY DEVELOPED INTRADUCTAL TRANSPLANTATION MODEL OF PDA (AIM 2). THESE NEW MEDIATORS APPEAR TO HAVE ROLES IN PDA PROGRESSION, IMMUNOSUPPRESSION, AND STROMAL ACTIVATION, AND MAY REPRESENT NEW PDA THERAPEUTIC TARGETS. FINALLY, WE HAVE DEMONSTRATED DIFFERENT IMMUNOMODULATORY FUNCTIONS OF DISTINCT CAF SUBTYPES. WE WILL TEST COMBINATORIAL STRATEGIES TO TARGET DISTINCT CAF SUBTYPES IN THE PDA MICROENVIRONMENT, AND STUDY THE EFFECT OF THESE STRATEGIES ON TUMOR PROGRESSION IN A MURINE PDA MODEL (AIM 3). IN ADDITION, WE WILL ASSESS THE ROLE OF MACROPHAGE-CAF CROSSTALK IN PROMOTING AND MAINTAINING CAF IDENTIFY. OVERALL, THIS PROJECT WILL CLARIFY THE DIVERSITY OF PDA CAFS AND THE ROLE OF CANCER CELLS IN REGULATING CAF SUBTYPES. OUR RESULTS WILL PROVIDE NEW AVENUES FOR CAF-TARGETING THAT WE INTEND TO TRANSLATE TOWARDS IMPROVED THERAPIES FOR PATIENTS AFFLICTED BY THIS LETHAL DISEASE.

TOWARDS QUANTITATIVE CELL TYPE-BASED MAPPING OF THE WHOLE MOUSE BRAIN

DISSEMINATION OF MAPSEQ AND BARSEQ FOR HIGH-THROUGHPUT BRAIN MAPPING - THE GOAL OF THIS PROJECT IS TO DISSEMINATE MAPSEQ AND BARSEQ TO THE BROADER NEUROSCIENCE COMMUNITY. THESE ARE NOVEL METHODS DEVELOPED IN MY LABORATORY BASED ON HIGH-THROUGHPUT DNA SEQUENCING FOR DETERMINING NEURONAL CIRCUITRY. NEURONS TRANSMIT INFORMATION TO DISTANT BRAIN REGIONS VIA LONG-RANGE AXONAL PROJECTIONS. IN SOME CASES, FUNCTIONALLY DISTINCT POPULATIONS OF NEURONS ARE INTERMINGLED WITHIN A SMALL REGION. DISRUPTIONS OF CONNECTIVITY MAY UNDERLIE MANY NEUROPSYCHIATRIC DISORDERS INCLUDING AUTISM AND SCHIZOPHRENIA. AT PRESENT, NEUROANATOMICAL TECHNIQUES—PARTICULARLY THOSE WITH SINGLE NEURON RESOLUTION—ARE EXPENSIVE AND LABOR INTENSIVE. MAPSEQ AND BARSEQ CONVERT NEUROANATOMY INTO A PROBLEM OF DNA SEQUENCING, AND THEREBY ALLOW US TO EXPLOIT THE TREMENDOUS BREAKTHROUGHS IN NEXT- GENERATION SEQUENCING THROUGHPUT. THE DISSEMINATION OF THESE HIGH-THROUGHPUT METHODS FOR DETERMINING NEURONAL PROJECTIONS WILL HAVE IMPORTANT IMPLICATIONS FOR UNDERSTANDING NORMAL NEURONAL CIRCUITRY, AND HOW THIS CIRCUITRY IS DISRUPTED IN ANIMAL MODELS OF NEUROPSYCHIATRIC DISORDERS LIKE AUTISM AND SCHIZOPHRENIA.

GENETIC TARGETING OF CORTICAL PYRAMIDAL NEURON SUBTYPES

CELL-BASED GENOMIC ANALYSIS OF MOLECULAR PATHOLOGY IN MOUSE MODELS OF RETT SYNDRO

RECEPTOR PTPS, CELL CONTRACT AND SIGNAL TRANSDUCTION

COMPUTATIONAL AND CIRCUIT MECHANISMS FOR INFORMATION TRANSMISSION IN THE BRAIN

GENE DISCOVERY IN THE CEREALS

CSHL WATSON SCHOOL OF BIOLOGICAL SCIENCES

COMPUTATIONAL ANALYSIS OF COMPLEX GENETIC INTERACTIONS

MOLECULAR AND CELLULAR MECHANISMS GOVERNING INTERNEURON DEVELOPMENT AND CONNECTIVITY

HIGH-THROUGHPUT APPROACHES TO LOCAL AND LONG-RANGE SYNAPTIC CONNECTIVITY

TAS::89 0321::TAS LONG ISLAND BIOFUELS ALLIANCE

RNAI AS A ROUTINE ROUTE TOWARD BREAST CANCER THERAPY

THE ROLE OF CHD5 IN CHROMATIN-MEDIATED REGULATION OF NEURAL STEM CELLS AND GLIOMA

CHARACTERIZING SUB-CELLULAR COMPARTMENTS IN MAIZE USING FLUORESCENT PROTEIN TAGGED LINES

MECHANISMS AND TREATMENT OF PTEN MUTANT PROSTATE TUMORIGENESIS

ELECTROPHYSIOLOGICAL ANALYSIS OF OLFACTORY REPRESENTATIONS IN DROSOPHILA

EPIGENETIC DYNAMICS OF DEVELOPING GERM CELLS AND EARLY EMBRYOS

NRF2 REGULATION OF DUCTAL PANCREATIC CANCER ETIOLOGY AND TREATMENT RESPONSE

THE MOLECULAR BASIS OF PREGNANCY-ASSOCIATED PROTECTION FROM BREAST CANCER

TARGETING ABERRANT ENHANCER LANDSCAPES IN PANCREATIC CANCER

NEURODEVELOPMENTAL DISORDER-ASSOCIATED RHO REGULATORS IN NEOCORTICAL DEVELOPMENT

DEVELOPMENT OF PAN-GENOMIC & COMPUTATIONAL RESOURCES FOR AGRICULTURE

NEUROIMMUNOLOGICAL INSIGHTS INTO BRAIN DEVELOPMENT AND DYSFUNCTION: AN INTEGRATIVE APPROACH FOCUSED ON MICROGLIAL DYNAMICS - PROJECT SUMMARY: NEURODEVELOPMENTAL DISORDERS SUCH AS AUTISM AND INTELLECTUAL DISABILITY AFFECT 14% OF AMERICAN CHILDREN AND OVER 50 MILLION PEOPLE WORLD-WIDE. PHARMACOLOGICAL THERAPIES FOR TREATING THESE DISORDERS ARE VIRTUALLY NON- EXISTENT IN LARGE PART DUE TO OUR LIMITED UNDERSTANDING OF THE NEURAL CIRCUIT WIRING DEFICITS THAT UNDERLIE THEIR DIVERSE AND NUANCED SYMPTOMS. WHILE IT HAS BEEN WELL-ESTABLISHED THAT NEURODEVELOPMENTAL DISORDERS EMERGE THROUGH A COMPLEX INTERPLAY OF GENETICS (NATURE) AND ENVIRONMENTAL FACTORS (NURTURE), THE MAJORITY OF RESEARCH INTO THESE CONDITIONS HAS FOCUSED ON SPECIFIC GENETIC MUTATIONS UNDERLYING RARE SUBSETS OF DISORDERS, LEAVING THE ENVIRONMENTAL FACTORS THAT AFFECT A MUCH WIDER ARRAY OF THESE CONDITIONS POORLY UNDERSTOOD. TO ALLEVIATE THE SUFFERING OF AS MANY INDIVIDUALS AS POSSIBLE, WE WILL ADDRESS THIS MAJOR GAP IN KNOWLEDGE BY DEFINING THE ENVIRONMENTAL FACTORS THAT EXACERBATE NEURODEVELOPMENTAL DYSFUNCTION. OUR APPROACH IS BASED UPON MOUNTING CLINICAL AND EXPERIMENTAL EVIDENCE THAT IMPAIRMENTS IN INTERACTIONS BETWEEN THE IMMUNE SYSTEM AND THE BRAIN DRIVE NEUROPATHOLOGY, AN UNEXPECTED FINDING GIVEN THAT THE IMMUNE AND NERVOUS SYSTEMS WERE CLASSICALLY CONSIDERED TO BE DISTINCT BIOLOGICAL DOMAINS. THE CENTRAL HYPOTHESIS UNDERLYING THE PROPOSED WORK IS THAT A SPECIALIZED CLASS OF BRAIN-RESIDENT IMMUNE CELLS CALLED MICROGLIA PLAY A CRITICAL ROLE IN COORDINATING A LATE STAGE OF CIRCUIT DEVELOPMENT THAT IS THOUGHT TO GO AWRY IN NEUROPATHOLOGICAL STATES: THE SENSORY EXPERIENCE-DEPENDENT REFINEMENT OF DEVELOPING SYNAPSES. IF SO, THE DISRUPTION OF MICROGLIAL FUNCTION IN EXPERIENCE-DEPENDENT REFINEMENT MAY BE A CORE FEATURE OF NEURODEVELOPMENTAL DISORDERS RENDERING MICROGLIA A PROMISING TARGET FOR TREATMENT, PARTICULARLY BECAUSE EXPERIENCE-DEPENDENT REFINEMENT REPRESENTS A STATE OF HEIGHTENED PLASTICITY WHEN THE BRAIN MAY BE PARTICULARLY RECEPTIVE TO THERAPEUTIC INTERVENTION. HARNESSING THE UNIQUE ADVANTAGES OF THE VISUAL CIRCUITRY OF THE MOUSE AS A MODEL SYSTEM, WE WILL MERGE TWO-PHOTON IMAGING OF MICROGLIA AND SYNAPSES IN THE BRAINS OF LIVE, AWAKE MICE WITH SINGLE-CELL GENOMICS AND CRISPR-BASED SCREENS TO DEFINE THE ROLES OF MICROGLIA IN EXPERIENCE-DEPENDENT REFINEMENT. IN PARALLEL, WE WILL UTILIZE THE MATERNAL IMMUNE ACTIVATION MOUSE MODEL TO IDENTIFY SPECIFIC MECHANISMS OF REFINEMENT THAT ARE LIKELY TO BE DISRUPTED IN NEURODEVELOPMENTAL DISORDERS. GIVEN THAT MALES ARE AT LEAST THREE TIMES MORE LIKELY TO HAVE AUTISM THAN FEMALES, OUR WORK WILL ALSO ASSESS HOW THESE MICROGLIAL PROCESSES DIFFER DEPENDING UPON SEX. IN THE COURSE OF THIS WORK, WE WILL DEVELOP MUCH-NEEDED VIRAL TOOLS FOR STUDYING MICROGLIA IN THE BRAIN. OUR OVERARCHING GOAL IS TO LAY THE FOUNDATION FOR THE DEVELOPMENT OF NEW PHARMACOLOGICAL STRATEGIES FOR TREATING NEURODEVELOPMENTAL DISORDERS BY RESTORING HEALTHY MICROGLIAL FUNCTION DURING POSTNATAL BRAIN DEVELOPMENT. IN ADDITION TO THE HIGH-RISK/HIGH-REWARD NATURE OF THE PROPOSAL, THIS PROJECT IS PARTICULARLY WELL-SUITED FOR THE NIH DIRECTOR’S NEW INNOVATOR AWARD BASED UPON CONCEPTUAL NOVELTY, PROMISE TO ACHIEVE KEY TECHNOLOGICAL ADVANCES, AND POTENTIAL TO ALLEVIATE A MAJOR HEALTHCARE BURDEN AFFECTING THE UNITED STATES AND THE WORLD.

SYNPLA: A SCALEABLE METHOD FOR MONITORING CIRCUIT-SPECIFIC LEARNING-INDUCED CHANGES IN SYNAPTIC STRENGTH

GENES AND NETWORKS REGULATING SHOOT MATURATION AND FLOWER PRODUCTION IN TOMATO AND RELATED NIGHTSHADES

IDENTIFICATION AND CHARACTERIZATION OF GENOMIC FEATURES AFFECTING SURVIVAL DURATION IN CANCER

TRANSPOSONS, RNA INTERFERENCE AND HETEROCHROMATIN

DEEP SEQUENCING OF AUTISM CANDIDATE GENES IN 2000 FAMILIES FROM THE SIMONS SIMPLE

SOCIAL ODOR MODULATION OF AUDITORY CORTEX VIA THE BASAL AMYGDALA - PROJECT SUMMARY OLFACTION IS UNIQUE AMONG THE SENSES FOR ITS DIRECT AND INTIMATE CONNECTIONS TO THE LIMBIC SYSTEM. THIS MAY EXPLAIN THE WIDESPREAD SUBJECTIVE EXPERIENCE THAT ODORS CAN TRIGGER POWERFUL AFFECTIVE RESPONSES, ESPECIALLY ODORS FROM SOCIAL PARTNERS. THIS IS A FUNDAMENTAL MODE OF SOCIAL COMMUNICATION IN MANY SPECIES, INCLUDING HUMANS, YET THE NEURAL CIRCUITS THAT LINK ODOR-EVOKED EMOTIONAL STATES TO CHANGES IN PERCEPTION AND SOCIAL INTERACTION REMAIN UNCLEAR. OUR GOAL IS TO DETERMINE HOW ODOR CUES FROM OFFSPRING INFLUENCE NEURAL AND BEHAVIORAL RESPONSES TO INFANT VOCALIZATIONS IN MICE. WE WILL USE A ROBUST AND ESSENTIAL MATERNAL BEHAVIOR (PUP RETRIEVAL) THAT REQUIRES BOTH OLFACTION AND AUDITION TO ASK: IF AND UNDER WHAT CONDITIONS DOES RETRIEVAL ENGAGE DIRECT CROSS TALK BETWEEN THE TWO MODALITIES? AND, WHAT IS THE FUNCTIONAL SIGNIFICANCE OF THIS CROSS TALK? ONE IMPORTANT ASPECT OF MATERNAL CARE IN MICE IS THE NEED TO RETRIEVE THEIR PUPS WHEN THEY ARE AWAY FROM THE NEST AND EMITTING ULTRASONIC DISTRESS VOCALIZATIONS (USVS). FIRST TIME MOTHERS AND NULLIPAROUS FEMALES (‘SURROGATES’) HOUSED WITH A DAM AND HER LITTER RAPIDLY LEARN TO RECOGNIZE THESE CALLS AND RETURN THE YOUNG TO THE NEST. THERE ARE CONTEMPORANEOUS CHANGES IN NEURAL RESPONSES TO USVS IN THE AUDITORY CORTEX. DIRECT INTERACTION WITH PUPS IS HIGHLY REWARDING, SUGGESTING THAT SENSORY EXPERIENCE WITH OFFSPRING MAY BE A CRUCIAL TRIGGER FOR NEURAL AND BEHAVIORAL PLASTICITY. ACCORDINGLY, BOTH THE SMELL OF PUPS AND THE SOUND OF THEIR VOCALIZATIONS ARE REQUIRED FOR PERFORMANCE OF PUP RETRIEVAL. INDEED, PREVIOUS WORK SHOWED THAT PUP ODOR MODULATES NEURONAL RESPONSES TO USVS IN THE AUDITORY CORTEX OF MATERNALLY-EXPERIENCED FEMALES. OUR PRELIMINARY DATA SUGGEST THAT PUP ODOR MAY INFLUENCE RESPONSES TO USVS VIA A PATHWAY TO THE AUDITORY CORTEX (AC) FROM THE BASAL AMYGDALA (BA), A PART OF THE BRAIN THAT HAS BEEN LINKED TO MATERNAL BEHAVIOR, AND TO MOTIVATED BEHAVIOR BROADLY. THE FACT THAT THE PROJECTION TO THE AC ARISES FROM THE BA, A STRUCTURE CRITICAL FOR MOTIVATED BEHAVIOR, RAISES THE POSSIBILITY THAT THE ODOR SIGNAL CONVEYED TO THE AC ENCODES AFFECTIVE STATE INFORMATION SUCH AS SALIENCE OR REWARD. WE HYPOTHESIZE THAT THE BA IS A CONDUIT FOR PUP ODORS TO TRIGGER AFFECTIVE STATES THAT DYNAMICALLY MODULATE THE NEURAL REPRESENTATION AND SALIENCE OF USVS. HERE, WE PROPOSE TO TEST THIS HYPOTHESIS BY COMBINING A NOVEL STRUCTURED RETRIEVAL TASK, SPATIOTEMPORALLY CONTROLLED EXPOSURE TO PUP ODOR, AND NEURONAL RECORDINGS IN FREELY BEHAVING MICE. WE WILL IDENTIFY THE TEMPORAL STRUCTURE AND BEHAVIORAL DETERMINANTS OF BAACN ACTIVITY DURING RETRIEVAL (AIM 1). WE WILL DETERMINE THE RELATIONSHIP BETWEEN BAACN FIRING AND AUDITORY ACTIVITY (AIM 2). FINALLY, WE WILL TEST THE NECESSITY OF BAACN ACTIVITY DURING DISTINCT BEHAVIORAL COMPONENTS OF PUP RETRIEVAL (AIM 3).

HIGH THROUGHPUT MAPPING OF NEURONAL CIRCUITRY USING DNA SEQUENCING

THE SYNAPTIC CIRCUITRY OF THE LATERAL HABENULA AND BEHAVIORAL DEPRESSION

METASTASIS OF PTEN MUTANT PROSTATE CANCER - WITH AN ESTIMATED 190,000 NEW DIAGNOSES IN 2020, PROSTATE CANCER (PC) IS THE SECOND MOST FREQUENT TUMOR DIAGNOSED IN MEN AND THE SECOND LEADING CAUSE OF MALE CANCER DEATHS IN AMERICA. APPROXIMATELY ONE IN THREE MEN OVER THE AGE OF 50 SHOWS HISTOLOGICAL EVIDENCE OF THIS TUMOR, HOWEVER ONLY ONE IN TEN WILL BE DIAGNOSED WITH CLINICALLY SIGNICANT PC. THERAPEUTIC OPTIONS FOR LOCALIZED PC ARE EFFECTIVE, HOWEVER, METASTATIC PC IS A YET INCURABLE DISEASE BECAUSE STANDARD OF CARE ANTI-ANDROGEN THERAPY INVARIABLY RESULTS IN INCURABLE DISEASE RELAPSE. PTEN-TP53 LOSS IS A MOST SIGNICANT EVENT OF HUMAN LETHAL METASTATIC PC AS SUMMARIZED BY THE PCF/SU2C INTERNATIONAL PROSTATE CANCER DREAM TEAM (ARMENIA ET AL., 2018) AND CONRMED BY TWO DECADES OF FUNCTIONAL PC MODELING IN MOUSE. THE CENTRAL GOAL OF THIS PROJECT IS TO UNDERSTAND WHAT CAUSES PTEN-MUTANT INDOLENT PROSTATE CANCER (PC) TO METASTASIZE TO DIFFERENT PARTS OF THE BODY AND KILL THE PATIENT. WHILE THE PCF/SU2C REPORT SIGNICANT ASSOCIATION OF PTEN LOSS WITH LOSS OF TP53, THE DENITION OF FURTHER GENES THAT ARE SIGNICANTLY CO-MUTATED WITH PTEN HAS REMAINED A PROBLEM. IMPORTANTLY, SUCH GENES COULD POINT TO PATHWAYS AND PRINCIPLES THAT DRIVE METASTASIS AND/ OR THERAPY RESISTANCE. WE AIM TO SOLVE THIS PROBLEM BY COMBINING SINGLE CELL ANALYSIS OF PATIENT METASTATIC RAPID AUTOPSY SAMPLES WITH FUNCTIONAL GENETICS AND 3D WHOLE ORGAN IMAGING OF LETHAL METASTASIS IN MOUSE. WE USE A HIGHLY EXIBLE GENETICALLY ENGINEERED MOUSE (GEM) MODEL OF LETHAL, ENDOGENOUS METASTATIC PROSTATE CANCER, TERMED RAPIDCAP. SPONTANEOUS PROGRESSION TO LETHAL METASTASIS IS SEEN IN ~70% OF PTEN/TRP53-MUTANT RAPIDCAP, SUGGESTING A CRITICAL STOCHASTIC PIONEER EVENT THAT SWITCHES A PTEN/ TRP53 NULL TUMOR CELL FROM INDOLENCE TO METASTATIC ESCAPE, SIMILAR TO A CRITICAL PIONEERING EVENT THAT CAUSES RELAPSE AFTER CASTRATION THERAPY. HOWEVER, CLASSICAL APPROACHES CAN ONLY CAPTURE AND REVEAL THE TIME AND PHYSICAL NATURE OF THESE PIONEERING EVENTS WHEN ALREADY THOUSANDS OR MILLIONS OF CELLS ARE INVOLVED. IN AIM 1, WE REVEAL, ISOLATE, AND ANALYZE SINGLE PIONEER CELLS IN SPACE AND TIME. WE COMBINE RAPIDCAP WITH SERIAL TWO PHOTON TOMOGRAPHY (STPT), WHICH ALLOWS US TO IMAGE WHOLE ORGANS AT SINGLE CELL RESOLUTION AND CONSTRUCT A 3D MAP OF THE TIME COURSE OF ESCAPE FROM INDOLENCE AND OF METASTATIC RELAPSE AFTER THERAPY. THIS GUIDES OUR ISOLATION OF CELL TYPES THAT THEN SERVE AS VALIDATED PROXIES FOR PIONEER CELLS OF ESCAPE OR RELAPSE. THROUGH AIM 2, WE SOLVE THE PTEN CO-MUTATION IMPASSE AND DENE GENES CO-DELETED WITH PTEN THROUGH SINGLE CELL WHOLE GENOME ANALYSIS OF HUMAN RAPID AUTOPSY SAMPLES. WE PRIORITIZE CANDIDATES BASED ON COMPREHENSIVE FUNCTIONAL MOLECULAR PROBING OF ALREADY ISOLATED PIONEER CELLS IN VITRO. THROUGH AIM 3, WE DEVELOP TWO NEW MODELING PLATFORMS FOR MORE EXIBLE VALIDATION OF CANDIDATE GENES AND PRINCIPLES BEHIND ESCAPE FROM INDOLENCE AND RELAPSE FROM CASTRATION THERAPY.

EMPOWERING NEXTGEN ADVANCED BIOMEDICAL LEADERSHIP (ENABL)

DISRUPTED AUDITORY CORTICAL PLASTICITY AND BEHAVIOR IN A MODEL OF RETT SYNDROME

DETECTION OF GENETIC AND EPIGENETIC CHANGES IN PRIMARY BREAST AND OVARIAN CANCER GENOMES UTILIZING GENOMIC MICROARRAYS

DYNAMIC REGULATION OF THE EPIGENOME DURING HEMATOPOIETIC DIFFERNTIATION

COLLABORATIVE RESEARCH: ARECIBO C3 - CENTER FOR CULTURALLY RELEVANT AND INCLUSIVE SCIENCE EDUCATION, COMPUTATIONAL SKILLS, AND COMMUNITY ENGAGEMENT -THIS AWARD ESTABLISHES A SCIENCE, TECHNOLOGY, ENGINEERING, AND MATHEMATICS (STEM) EDUCATION AND RESEARCH CENTER AT THE FEDERALLY OWNED SITE IN ARECIBO, PUERTO RICO AND LOCATION OF THE ARECIBO OBSERVATORY. ARECIBO CENTER FOR CULTURALLY RELEVANT AND INCLUSIVE SCIENCE EDUCATION, COMPUTATIONAL SKILLS, AND COMMUNITY ENGAGEMENT (ARECIBO C3) WILL INTEGRATE SCIENCE (CIENCIA), COMPUTATION (COMPUTACI?N), AND COMMUNITY (COMUNIDAD)?GEOGRAPHICAL, DISCIPLINARY, AND CULTURAL?WITH THE OVERALL GOAL OF PROMOTING CULTURALLY-RELEVANT AND INCLUSIVE TEACHING WITH SCIENCE EDUCATION RESEARCH, OUTREACH, AND WORKFORCE DEVELOPMENT. ARECIBO C3 IS A COLLABORATION OF: COLD SPRING HARBOR LABORATORY, UNIVERSITY OF PUERTO RICO AT R?O PIEDRAS, UNIVERSITY OF MARYLAND BALTIMORE COUNTY, AND UNIVERSIDAD DEL SAGRADO CORAZ?N. ARECIBO C3 (A.K.A. THE CENTER) IS PURPOSE-BUILT TO ADVANCE STEM-EQUITY AND PROMOTE FULL PARTICIPATION IN THE BENEFITS OF STEM. THE SCHOLARLY COMMUNITY OF STEM EDUCATION RESEARCHERS WILL BENEFIT FROM THE OUTPUTS OF THE CENTER?S EDUCATION RESEARCH PROGRAMS. STEM STUDENTS, PROFESSIONAL EDUCATORS, AND THE PUBLIC WILL BENEFIT FROM THE CENTER?S SKILL BUILDING AND WORKFORCE DEVELOPMENT ACTIVITIES. ARECIBO C3 WILL SERVE AS A COLLABORATIVE HUB FOR STEM DISCOVERY AND EXPLORATION BY BUILDING UPON EXISTING PROGRAMS AND OPPORTUNITIES ESTABLISHED AT THE ARECIBO SITE BY PREVIOUS NSF PROGRAMS, WHILE ALSO CREATING NEW STEM EDUCATION, RESEARCH, AND OUTREACH PROGRAMS AND INITIATIVES. THE GOALS FOR THE CENTER ARE TO (1) PROMOTE STEM EDUCATION, LEARNING, AND TEACHING; (2) SUPPORT FUNDAMENTAL AND APPLIED STEM AND STEM EDUCATION RESEARCH; (3) BROADEN PARTICIPATION IN STEM; AND (4) BUILD AND STRENGTHEN COLLABORATIONS AND PARTNERSHIPS. THE PROPOSED SUITE OF EXPERIENCES AND RESEARCH WILL BE INNOVATIVE AND DIVERSE. RESEARCH ON STEM EDUCATION AND LEARNING WILL EXAMINE THE IMPACT OF MULTISENSORY TEACHING STRATEGIES IN SECONDARY AND POST-SECONDARY EDUCATION, AS WELL AS APPROACHES FOR ADULT LEARNING IN INFORMAL CONTEXTS, ULTIMATELY CONTRIBUTING TO INCREASING PARTICIPATION OF ALL PEOPLE ?WITH AND WITHOUT DISABILITIES? IN STEM. A COMPLEMENTARY PRIORITY WILL BE INVESTIGATING HOW CULTURALLY SUSTAINING PEDAGOGY SUPPORTS STUDENT STEM IDENTITY DEVELOPMENT. INTRODUCTORY RESEARCH EXPERIENCES FOR ALL WILL BE OFFERED THROUGH DNA BARCODE PUERTO RICO, AN EXPERIENCE USING DNA TO STUDY THE BIODIVERSITY OF THE ISLAND. MODELED ON COLD SPRING HARBOR LABORATORY DNA LEARNING CENTER BARCODING PROGRAMS, COLLABORATIONS WITH HIGH SCHOOLS AND SCIENCE CENTERS ACROSS PUERTO RICO WILL BE SERVED BY NEW ARECIBO C3 MOLECULAR AND COMPUTER LABORATORY SPACES AND EQUIPMENT LOANS. VISITOR GROUPS WILL ALSO ACCESS DNA LEARNING CENTER FIELD TRIPS AND SUMMER COURSES. VISITORS WILL ALSO EXPERIENCE A MULTISENSORY COURSE AND EXHIBITION FEATURING SENSORY EXPERIENCES OF DATA, SUCH AS THE MERGING OF BLACK HOLES OR BEHAVIOR OF BIOLUMINESCENT ORGANISMS. TEACHERS AND FACULTY WORKING WITH EXPERTS IN SCIENTIFIC AND EDUCATION RESEARCH WILL BE MENTORED TO CREATE, IMPLEMENT, AND ASSESS CURRICULA OF COURSE-BASED CONVERGENT RESEARCH EXPERIENCES FOR HIGH SCHOOL AND UNDERGRADUATES. ARECIBO C3 WILL CREATE WORKFORCE DEVELOPMENT OPPORTUNITIES FOCUSING ON DATA SCIENCE, COMPUTATION, AND BIOTECHNOLOGY. COMPUTATIONAL TRAINING WILL ALSO REACH INTO COMMUNITIES EMPOWERING WOMEN AND GIRLS IN PUBLIC HOUSING WITH DATA SCIENCE SKILLS AND CAREER OPPORTUNITIES. BIOTECHNOLOGY TRAINING AND CREDENTIALS WILL TIE TO LOCAL INDUSTRIES AND JOBS. ARECIBO C3 WILL FEATURE A MEETINGS PROGRAM THAT CONVENES RESEARCHERS, EDUCATORS, POLICY MAKERS, INDUSTRY PARTNERS, AND OTHER STAKEHOLDERS FOR THINK TANK STYLE CONFERENCES ON PRESSING STEM TOPICS. ADDITIONALLY, THE CENTER WILL PILOT A FELLOWSHIP PROGRAM IN WHICH PARTICIPANTS ARE TRAINED AND CERTIFIED IN SCIENTIFIC COMMUNITY MANAGEMENT. ARECIBO C3 FELLOWS WILL THEN SERVE AS AMBASSADORS USING RECENTLY ACQUIRED COLLABORATIVE STRATEGIES TO INVOLVE, ORGANIZE, TRAIN, SUPPORT THEIR PEERS IN COMMUNITY SCIENCE EXPERIENCES, AND BUILD COMMUNITIES OF PRACTICE. ULTIMATELY, ARECIBO C3 WILL FOSTER EQUITY-ENABLING STEM RESEARCH AND EDUCATION, ENRICH PUERTO RICO?S VIBRANT STEM COMMUNITIES, AND SERVE AS A CATALYST FOR INCLUSIVE STEM EDUCATION GLOBALLY. ARECIBO C3 IS JOINTLY FUNDED BY THE FOLLOWING PROGRAMS AT NSF: ADVANCING INFORMAL STEM LEARNING (AISL), THE ESTABLISHED PROGRAM TO STIMULATE COMPETITIVE RESEARCH (EPSCOR), DISCOVERY RESEARCH PREK-12 (DRK-12), INNOVATIVE TECHNOLOGY EXPERIENCES FOR STUDENTS AND TEACHERS (ITEST), HISPANIC-SERVING INSTITUTIONS (HSI), AND THE LOUIS STOKES ALLIANCES FOR MINORITY PARTICIPATION (LSAMP) PROGRAMS. 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.

SYNAPSE ENGULFMENT BY OLIGODENDROCYTE PRECURSOR CELLS: A NEW MECHANISM OF CIRCUIT REFINEMENT IN THE DEVELOPING BRAIN - PROJECT SUMMARY THE ESTABLISHMENT OF SYNAPTIC CONNECTIVITY DURING BRAIN DEVELOPMENT INVOLVES THE INITIAL FORMATION OF AN OVERABUNDANCE OF SYNAPSES FOLLOWED BY A REFINEMENT PROCESS IN WHICH SOME OF THESE SYNAPSES ARE MAINTAINED WHILE OTHERS ARE ELIMINATED. THE PRECISE ELIMINATION OF EXCESS SYNAPSES IS DRIVEN BY SENSORY EXPERIENCE DURING CRITICAL PERIODS OF EARLY POSTNATAL LIFE. IMPAIRMENTS IN SENSORY-DEPENDENT SYNAPSE ELIMINATION CONTRIBUTE TO NEURODEVELOPMENTAL DISORDERS SUCH AS AUTISM, UNDERSCORING THE IMPORTANCE OF THIS PROCESS FOR THE PROPER DEVELOPMENT AND FUNCTION OF NEURAL CIRCUITS. HOWEVER, ALTHOUGH NEURODEVELOPMENTAL DISORDERS ARE GROWING IN PREVALENCE AT AN ALARMING RATE, THERAPEUTIC STRATEGIES FOR TREATING THEM ARE SCARCE IN PART DUE TO A LACK OF INSIGHT INTO THE FACTORS THAT CONTROL SYNAPSE ELIMINATION IN THE HEALTHY BRAIN. A KEY GOAL OF THIS PROPOSAL IS TO UNCOVER NOVEL CELLULAR AND MOLECULAR MECHANISMS UNDERLYING THE ELIMINATION OF SYNAPSES DOWNSTREAM OF EXPERIENCE, THEREBY LAYING THE GROUNDWORK FOR NEW THERAPEUTIC APPROACHES TO TREAT DISORDERS OF POSTNATAL BRAIN DEVELOPMENT. WORK IN THE VISUAL SYSTEM OF THE MOUSE HAS REVEALED THAT NON-NEURONAL BRAIN CELLS, PREDOMINANTLY MICROGLIA AND ASTROCYTES, COORDINATE SYNAPSE ELIMINATION BEFORE THE ONSET OF VISUAL EXPERIENCE BY PHAGOCYTOSING EXCESS SYNAPSES. HOWEVER, DATA SUGGEST THAT THESE CELLS MAY NOT BE MAJOR REGULATORS OF SYNAPSE ELIMINATION DURING LATE PHASES OF DEVELOPMENT THAT ARE COORDINATED BY VISUAL EXPERIENCE. ON THE CONTRARY, WE RECENTLY DISCOVERED A KEY ROLE FOR A LESS WELL UNDERSTOOD CLASS OF GLIA, OLIGODENDROCYTE PRECURSOR CELLS (OPCS), IN ELIMINATING SYNAPSES IN RESPONSE TO EXPERIENCE THROUGH SYNAPTIC PHAGOCYTOSIS. THIS RESULT IS CONSISTENT WITH WIDE-SPREAD SPECULATION THAT OPCS, WHILE PREDOMINANTLY APPRECIATED FOR THEIR DIFFERENTIATION INTO MATURE OLIGODENDROCYTES, PLAY KEY ROLES IN THE BRAIN BEYOND MYELINATION. IN LINE WITH THIS POSSIBILITY, OUR DATA SUGGEST THAT OPCS ARE ESSENTIAL FOR SHAPING FUNCTIONAL NEURAL CIRCUITS DURING THE MATURATION OF THE BRAIN. IN THIS APPLICATION, WE PROPOSE A MULTI-DISCIPLINARY STRATEGY TO TEST THE HYPOTHESIS THAT THE ENGULFMENT OF SYNAPSES BY OPCS IS A CORE MECHANISM UNDERLYING THE SENSORY-DEPENDENT ELIMINATION OF FUNCTIONAL SYNAPSES IN THE DEVELOPING BRAIN. IN AIM 1, WE WILL EMPLOY VIRAL AND TRANSGENIC TOOLS TO CHARACTERIZE THE SPATIO-TEMPORAL DYNAMICS AND ACTIVITY-DEPENDENT BASIS OF SYNAPTIC ENGULFMENT BY OPCS USING IN VIVO TWO-PHOTON MICROSCOPY. IN AIM 2, WE WILL APPLY PHYSIOLOGICAL AND BEHAVIORAL ASSAYS TO DETERMINE THE CONSEQUENCES OF SYNAPTIC ENGULFMENT BY OPCS ON BRAIN FUNCTION IN THE INTACT ANIMAL. IN AIM 3, WE WILL MERGE UNBIASED TRANSCRIPTOMIC AND CRISPR- BASED SCREENING TECHNIQUES WITH A CANDIDATE-BASED APPROACH FOCUSED ON THE PHAGOCYTIC RECEPTOR LRP1 TO REVEAL MOLECULAR PATHWAYS UNDERLYING THE ENGULFMENT OF SYNAPSES BY OPCS. ALTOGETHER, WE EXPECT THESE STUDIES TO ESTABLISH SYNAPSE ENGULFMENT BY OPCS AS A NEW MECHANISM LINKING EXPERIENCE TO NEURAL CIRCUIT DEVELOPMENT, AND TO LAY THE FOUNDATION FOR FUTURE STUDIES GEARED TOWARD MODIFYING OPC FUNCTION AS A POTENTIAL THERAPEUTIC STRATEGY FOR TREATING NEURODEVELOPMENTAL DISORDERS.

CELL ADHESION MOLECULES IN AUTISM: A WHOLE-BRAIN STUDY OF GENETIC MOUSE MODELS

RHO REGULATOR-MEDIATED SIGNALING IN INTERNEURON DEVELOPMENT

BIOPHYSICAL MODELING OF CIS-REGULATORY COMPLEXES IN TRANSCRIPTION AND SPLICING USING MASSIVELY PARALLEL REPORTER ASSAYS

QUANTITATIVE ANALYSIS OF THE BIOLOGICAL FORCES AND THEIR CONTEXT IN TISSUE PATTERNING

EMOTIONAL MODULATION OF NEURAL AND BEHAVIORAL RESPONSES TO SOCIAL CUES BY LOCUS COERULEUS

NOVEL ALGORITHM DEVELOPMENT, USER SUPPORT AND MAINTENANCE FOR STAR

DECONSTRUCTING AGING WITH SENOLYTIC CAR T CELLS - SUMMARY GAINING A BETTER UNDERSTANDING OF AGING BIOLOGY IS A PRESSING HEALTH AND SOCIOECONOMIC ISSUE. IN THIS REGARD, CELLULAR SENESCENCE IS A KEY, BUT POORLY UNDERSTOOD DETERMINANT OF ORGANISMAL AGING. SENESCENCE IS A STRESS RESPONSE PROGRAM CHARACTERIZED BY STABLE CELL CYCLE ARREST. INITIALLY DESCRIBED AS A TUMOR SUPPRESSOR MECHANISM, IT INVOLVES THE INTERPLAY BETWEEN THE TUMOR SUPPRESSORS P53 AND RB LEADING TO A TRANSCRIPTIONAL PROGRAM OF GENE REPRESSION THAT SILENCES PROLIFERATION ASSOCIATED GENES. BEYOND THEIR ARREST, SENESCENT CELLS SECRETE THE SENESCENCE ASSOCIATED SECRETORY PHENOTYPE (SASP) COMPOSED OF CYTOKINES AND MATRIX REMODELING ENZYMES. THE SASP CONTRIBUTES TO THE INDUCTION OF PARACRINE SENESCENCE AS WELL AS TO THE RECRUITMENT OF IMMUNE CELLS, WHOSE ROLE IS TO CLEAR THE SENESCENT CELLS AND RESTORE TISSUE HOMEOSTASIS. INDEED, THIS IS WHAT HAPPENS IN THE CONTEXT OF WOUND HEALING OR TUMOR SUPPRESSION. HOWEVER, FOR MECHANISMS THAT REMAIN UNCLEAR, IN CERTAIN SETTINGS SUCH AS AGING OR AGE-RELATED DISEASES, SENESCENT CELLS ACCUMULATE OVER TIME GENERATING A PATHOLOGICAL CHRONIC PRO-INFLAMMATORY MILIEU THAT PLAYS A KEY ROLE IN THE PATHOPHYSIOLOGY OF THESE CONDITIONS. OUR GOALS ARE TO IDENTIFY CELL SURFACE MARKERS THAT ARE SELECTIVELY EXPRESSED IN SENESCENT CELLS IN AGING FOR THE PURPOSE OF BETTER IDENTIFYING, ISOLATING AND CHARACTERIZING THE SENESCENCE PROGRAM IN AGING. IN PARALLEL, WE WILL EXPLOIT THE SELECTIVE FEATURES OF THESE MOLECULES WITH THE AIM OF DEVELOPING BETTER IMAGING STRATEGIES TO MONITOR SENESCENT CELLS IN VIVO AS WELL AS OF DEVELOPING CAR T CELLS ABLE TO SELECTIVELY ELIMINATE SENESCENT CELLS IN AGING. WE WILL USE THESE SENOLYTIC CAR T CELLS TO FUNCTIONALLY INTERROGATE SENESCENCE BIOLOGY IN VIVO AS WELL AS TO STUDY THEIR THERAPEUTIC POTENTIAL IN PRECLINICAL MODELS OF AGE-RELATED DISEASES. PRELIMINARY DATA STRONGLY SUPPORTS THE FEASIBILITY OF THE PROPOSED WORK: WE HAVE ALREADY IDENTIFIED ONE CELL SURFACE MOLECULE EXPRESSED ON SENESCENT CELLS IN AGING AND VALIDATED THE POTENTIAL OF CAR T CELLS TARGETING IT AS EFFECTIVE AND SAFE SENOLYTICS IN AGING. IN OUR APPLICATION, WE CONTINUE TO IDENTIFY AND VALIDATE ADDITIONAL MARKERS, CHARACTERIZE THE MOLECULAR PROGRAM OF SENESCENCE IN AGING AND OPTIMIZE CELLULAR THERAPY DESIGNS FOR AGING CONDITIONS. COLD SPRING HARBOR LABORATORY HAS A LONGSTANDING HISTORY OF NURTURING SUCCESSFUL EARLY SCIENTIST THROUGH THEIR INDEPENDENT FELLOWS PROGRAM. THIS HIGHLY SUPPORTIVE ENVIRONMENT IS AN IDEAL NICHE TO DEVELOP THIS PROJECT AND LAUNCH MY OWN RESEARCH PROGRAM AS AN INDEPENDENT FELLOW. COMPLETION OF THE PROPOSED WORK WILL BETTER DEFINE THE SENESCENCE STATE IN AGING, PRODUCE NEW INSIGHTS INTO THE BIOLOGY OF SENESCENCE IMMUNE SURVEILLANCE AND GENERATE A NEW MODALITY TO BOTH INTERROGATE SENESCENCE AND TREAT AGE- RELATED DISEASES.

SHORT AXON CELLS IMPLEMENT GAIN CONTROL IN THE MOUSE OLFACTORY BULB

CENTRAL MECHANISMS OF GLUCOCORTICOID CIRCADIAN RHYTHM DYSFUNCTION IN BREAST CANCER - PROJECT SUMMARY BREAST CANCER IS STILL THE MOST COMMON MALIGNANCY IN WOMEN WORLDWIDE. DESPITE THE INCREDIBLE ADVANCES MADE IN THE PAST DECADES TOWARDS ELIMINATING BREAST CANCER, NEARLY ALL WOMEN WITH METASTATIC BREAST CANCER WILL SUCCUMB TO THE DISEASE. THEREFORE, WE DESPERATELY NEED NEW AND IMPROVED APPROACHES TOWARDS ELIMINATING BREAST CANCER. THE PROPOSED WORK FOCUSES ON A WIDELY DOCUMENTED CLINICAL FINDING WITH NO KNOWN UNDERLYING MECHANISM: BLUNTED CIRCULATING GLUCOCORTICOID RHYTHMS IN PATIENTS WITH BREAST CANCER. CRITICALLY, PATIENTS WITH ‘FLAT’ OR ‘BLUNTED’ CIRCADIAN RHYTHMS IN CIRCULATING GLUCOCORTICOIDS (E.G., CORTISOL) REPORT LOWER QUALITY OF LIFE AND DIE FROM CANCER EARLIER THAN PATIENTS WITH NORMAL ‘ROBUST’ CIRCADIAN RHYTHMS. GLUCOCORTICOID RHYTHMS ARE LARGELY CONTROLLED BY CONCERTED INTERACTIONS AMONG THE HYPOTHALAMUS, PITUITARY, AND ADRENAL GLANDS (I.E., HPA-AXIS), AND ACT TO REGULATE DIVERSE PHYSIOLOGICAL PROCESSES ESSENTIAL FOR HEALTH INCLUDING IMMUNE CELL TRAFFICKING/POLARIZATION, ENERGY MOBILIZATION, AROUSAL, AND SYNCHRONIZATION OF PERIPHERAL CIRCADIAN CLOCKS. OUR PRELIMINARY DATA DEMONSTRATE THAT PRECLINICAL MOUSE MODELS OF BREAST CANCER ALSO EXHIBIT THIS ‘BLUNTING’ OF THE GLUCOCORTICOID RHYTHM, AND THIS IS ACCOMPANIED BY ABERRANT ACTIVITY WITHIN HYPOTHALAMIC NEURONS THAT REGULATE THE HPA-AXIS (I.E., PVNCRH NEURONS). ADDITIONALLY, BY RE-ESTABLISHING A ROBUST GLUCOCORTICOID RHYTHM VIA CHEMOGENETIC STIMULATION OF THESE NEURONS, WE WERE ABLE TO SIGNIFICANTLY IMPEDE PRIMARY BREAST CANCER GROWTH IN MOUSE MODELS. IN THE PRESENT PROPOSAL, WE WILL SYSTEMATICALLY DETERMINE WHERE ALONG HPA-AXIS DYSFUNCTION OCCURS IN RESPONSE TO BREAST CANCER, IDENTIFY THE MECHANISM(S) DRIVING THIS DYSFUNCTION, AND THEN USE ORTHOGONAL APPROACHES TO MANIPULATE THESE MECHANISMS TO CONFER BENEFITS (REDUCED TUMOR AND METASTATIC BURDEN, ENHANCED RESPONSES TO CHECKPOINT BLOCKADE IMMUNOTHERAPY) IN MOUSE MODELS OF BREAST CANCER. THESE STUDIES REPRESENT A SIGNIFICANT STEP FORWARD IN OUR UNDERSTANDING OF HOW MALIGNANT PROCESSES INFLUENCE THE HOST, AND HOW HOST RESPONSES DICTATE CANCER PROGRESSION AND THERAPEUTIC EFFICACY. THESE DATA WILL FURTHER INFORM THE DESIGN OF NOVEL THERAPEUTIC APPROACHES TOWARDS ELIMINATING BREAST CANCER BY TARGETING THE HPA-AXIS.

STRUCTURE AND FUNCTION OF NEURONAL HEMICHANNELS

POSTNATAL TRANSIENT CONNECTIVITY IN BRAIN DEVELOPMENT AND IMPLICATIONS IN FRAGILE X SYNDROME - ABSTRACT FRAGILE X SYNDROME (FXS) IS THE MOST COMMON GENETIC CAUSE OF AUTISM SPECTRUM DISORDERS. AUTISM SYMPTOMS IN FXS PATIENTS TYPICALLY MANIFEST DURING EARLY INFANCY WHEN CHILDREN ACTIVELY INTERACT WITH THEIR ENVIRONMENT AND FORM SENSORY-COGNITIVE ASSOCIATIONS. DURING THIS STAGE, THE NEOCORTEX UNDERGOES EXTENSIVE EXPERIENCE-DEPENDENT SYNAPTOGENESIS AND SYNAPTIC PRUNING TO EITHER MAINTAIN OR ELIMINATE, SO-CALLED TRANSIENT, NEURONAL CIRCUITS. SEVERAL STUDIES SUGGEST THAT BOTH SENSORY AND NEUROMODULATORY INPUTS TO THE CORTEX CAN ESTABLISH TRANSIENT CONNECTIONS WITH SPECIFIC CELL-TYPES, AND SUBSEQUENTLY CONTRIBUTE TO THE MATURATION OF PROPER ADULT CORTICAL CIRCUITS. GIVEN THAT EARLY BEHAVIORAL THERAPY IS WIDELY CONSIDERED TO BE THE MOST EFFECTIVE TREATMENT FOR CHILDREN WITH AUTISM, PHARMACOLOGICAL MANIPULATIONS OF SENSORY AND NEUROMODULATORY TRANSIENT CONNECTIVITY AT THE EARLY ONSET OF THE DISORDERS COULD POTENTIALLY MIMIC OR SYNERGIZE WITH THE EFFECTS OF SUCH BENEFICIAL BEHAVIORAL THERAPIES. HOWEVER, TO DATE, THE ROLE AND MECHANISMS OF TRANSIENT CONNECTIVITY IN NORMAL AND FXS- ASSOCIATED CORTICAL MATURATION REMAIN LARGELY UNKNOWN. TO ADDRESS THIS KNOWLEDGE GAP, WE PROPOSE TO INVESTIGATE THE MECHANISMS OF TRANSIENT CONNECTIVITY UNDERLYING SENSORY- AND NEUROMODULATORY-DEPENDENT CORTICAL MATURATION IN CONTROL MICE AND IN A MOUSE MODEL OF FXS. SENSORY EXPERIENCES ARE ENCODED IN THE CORTEX BY THALAMOCORTICAL (TC) NEURONS FORMING TRANSIENT CONNECTIONS WITH A SUBSET OF INHIBITORY NEURONS, THE SOMATOSTATIN (SST) CELLS. THESE TRANSIENT CONNECTIONS ARE BELIEVED TO CONTROL MATURATION OF EXCITATION/INHIBITION (E/I) BALANCE IN THE CORTEX, WHICH IS ALTERED IN THE MATURE BRAIN OF FXS MICE OR PATIENTS. OUR MOUSE DATA INDICATE THAT, IN PARALLEL TO SENSORY TC INPUTS, SST NEURONS RECEIVE CHOLINERGIC MODULATORY INPUTS ASSOCIATED WITH THE EARLY DEVELOPMENT OF TC PATHWAYS. THEREFORE, WE HYPOTHESIZE THAT THE SYNERGY BETWEEN TC AND CHOLINERGIC TRANSIENT CONNECTIVITY TO SST NEURONS IS AN INSTRUCTIVE MECHANISM THAT TRIGGERS CORTICAL MATURATION AND THAT THIS MECHANISM IS IMPAIRED IN FXS. OUR PREVIOUS RESEARCH USING COMBINATORIAL MOUSE GENETICS AND VIRAL CHEMOGENETIC APPROACHES SHOWED THAT METABOTROPIC SIGNALING CONTROLS THE FORMATION OF TRANSIENT TC INPUTS TO SST NEURONS. BASED ON THIS DATA, WE PROPOSE THAT CORTICAL E/I MATURATION RESULTS FROM METABOTROPIC SIGNALING THAT TRANSIENTLY TRANSDUCES SENSORY CUES AND NEUROMODULATION ONTO SST NEURONS. TO TEST THIS HYPOTHESIS, WE WILL EMPLOY MULTIDISCIPLINARY APPROACHES TO INVESTIGATE WHETHER TRANSIENT TC CONNECTIVITY GOVERNS HEALTHY AND FXS CORTICAL MATURATION (AIM 1), ELUCIDATE THE ROLE OF CHOLINERGIC INPUTS IN THE MATURATION OF HEALTHY AND FXS CORTICAL CIRCUITS (AIM 2), AND CHARACTERIZE METABOTROPIC MOLECULAR PATHWAYS UNDERLYING TRANSIENT CONNECTIVITY (AIM 3). OVERALL, OUR WORK WILL PROVIDE NOVEL INSIGHTS INTO THE ROLE OF TRANSIENT CONNECTIVITY ON SENSORY AND COGNITIVE EXPERIENCE-DEPENDENT CORTICAL MATURATION AND LAY THE FOUNDATIONS FOR EXPLORING APPROACHES TARGETING THE ONSET OF CIRCUIT DYSFUNCTIONS IN FXS.

REVEALING THE TRANSCRIPTOMIC BASIS OF NEURONAL IDENTITY THROUGH FUNCTIONAL META-ANALYSIS

HIGH-THROUGHPUT DNA SEQUENCING METHOD FOR PROBING THE CONNECTIVITY OF NEURAL CIRC

UNDERSTANDING THE NEURONAL MECHANISMS OF CLOSED-LOOP OLFACTION - PROJECT SUMMARY IN NATURE, SENSORY PERCEPTION AND MOTOR PROCESSING OPERATE IN CLOSED-LOOP. SELF-GENERATED MOVEMENTS IMPACT SENSORY INPUT, AND SENSORY INPUTS GUIDE FUTURE MOTOR COMMANDS. THROUGH EXPERIENCE, THE BRAIN MAY LEARN THE RECIPROCAL RELATIONSHIP BETWEEN SENSORY INPUTS AND MOVEMENTS IN THE FORM OF GENERATIVE SENSORIMOTOR MODELS THAT PREDICT THE SENSORY CONSEQUENCES OF UPCOMING ACTIONS. IN VERTEBRATES, OLFACTION IS INTRINSICALLY LINKED TO MOTOR ACTION THROUGH SNIFFING, AND JUST AS FOR OTHER SENSORY MODALITIES, VIA HEAD AND BODY MOVEMENTS. DUE TO TECHNICAL CHALLENGES, HOWEVER, MOST STUDIES IN LABORATORY SETTINGS HAVE PROBED OLFACTORY PROCESSING DURING PASSIVE ODOR SAMPLING. EVEN WHEN INVESTIGATING ODOR-DRIVEN NAVIGATION, THE EFFECT OF MOVEMENTS ON ODOR RESPONSES HAS RARELY BEEN ANALYZED. HERE WE WILL TEST THE CENTRAL HYPOTHESIS THAT, IN CLOSED-LOOP OLFACTION, MICE GENERATE OLFACTO-MOTOR PREDICTIONS ON THE SENSORY CONSEQUENCES OF THEIR ACTIONS, WHICH FURTHER GUIDE ODOR SAMPLING MOVEMENTS. AT THE CIRCUIT LEVEL, WE HYPOTHESIZE THAT SPECIFIC OLFACTORY CORTEX CIRCUITS REPRESENT OLFACTO- MOTOR PREDICTION ERRORS, COMPUTED BY COMPARING ODOR INPUT AND MOVEMENT-RELATED PREDICTIONS OF THE EXPECTED ODOR INPUT. WE PLAN TO TEST THESE HYPOTHESES USING A NOVEL CLOSED-LOOP ODOR LOCALIZATION TASK (SMELLOCATOR) DEVELOPED IN OUR GROUP, TOGETHER WITH A RICH REPERTOIRE OF SENSORIMOTOR PERTURBATIONS, STATE-OF-THE-ART RECORDINGS AND CELL-TYPE CIRCUIT ANALYSIS TOOLS WITH INCREASING LEVELS OF SPECIFICITY. TO THIS END, WE WILL FIRST INVESTIGATE WHETHER UNDER CLOSED-LOOP COUPLING OF MOVEMENTS AND ODOR SENSING, MICE DETECT OLFACTO-MOTOR ERRORS, AND FURTHER COMPENSATE FOR THEM. IN THE SMELLOCATOR TASK, HEAD-FIXED MICE LEARN TO STEER A LIGHTWEIGHT LEVER WITH THEIR PAWS TO CONTROL THE LATERAL LOCATION OF AN ODOR SOURCE ACCORDING TO A FIXED-GAIN SENSORIMOTOR MAPPING RULE. IN CATCH TRIALS, WE WILL TRANSIENTLY ALTER THE RELATIONSHIP BETWEEN LEVER MOVEMENT AND ODOR DISPLACEMENT VIA A RANGE OF PRECISE, UNEXPECTED SENSORIMOTOR PERTURBATIONS. PRELIMINARY DATA INDICATE THAT EXPERT MICE SUCCESSFULLY COMPUTE SENSORIMOTOR PREDICTION ERRORS, AND QUICKLY ENGAGE IN FINE CORRECTIVE MOVEMENTS TRIGGERED BY THESE PERTURBATIONS IN AN INDIVIDUAL SPECIFIC MANNER. - THEN, WE WILL INVESTIGATE WHETHER THE OLFACTORY CORTEX (PIRIFORM VS. ANTERIOR OLFACTORY NUCLEUS) REPRESENTS OLFACTO- MOTOR PREDICTION ERRORS IN FACE OF TRANSIENT SURPRISES. WE WILL CHECK WHETHER BRIEF SENSORIMOTOR PERTURBATIONS TRIGGER SUDDEN CHANGES IN CORTICAL ACTIVITY (MISMATCH RESPONSES). WE WILL REFINE OUR ANALYSIS TO DETERMINE IF DIFFERENT SEMILUNAR AND PYRAMIDAL CELLS TYPES (E.G. NETRIN+, CUX1+, TBR1+, TLE4+) REPRESENT PRIMARILY SENSORY INPUTS VS. OLFACTO-MOTOR ERRORS BY COMBINING DISTRIBUTED RECORDINGS AND MODERN GENETIC LABELING STRATEGIES. - FINALLY, WE WILL INVESTIGATE WHETHER THE OLFACTORY CORTEX ENABLES ADAPTATION IN THE PRESENCE OF PERSISTENT OLFACTO- MOTOR ERRORS. WE WILL CHANGE THE SENSORIMOTOR MAPPING RULES IN BLOCKS OF TRIALS, AND ACROSS BEHAVIORAL SESSIONS, AND COMPARE THE ROLES OF SPECIFIC CELL TYPES IN SUPPORTING SENSORIMOTOR ADAPTATION TAKING ADVANTAGE OF FLEXIBLE OPTOGENETIC SUPPRESSION STRATEGIES.

(PQA-4) ORGANOID OMICS TO DETECT AND DEFEAT DUCTAL PANCREATIC CANCER

GENOME-WIDE REGULATORY NETWORK GOVERNING NEURONAL MRNA TRANSLATION.

EXPLOITING POU2F3 ADDICTION IN THE TUFT CELL VARIANT OF SMALL CELL LUNG CANCER

FUNCTIONS OF DISTINCT ORBITOFRONTAL CELL-TYPES AND PATHWAYS IN DECISION MAKING

CRCNS: REWARD AND MOTIVATION IN NEURAL NETWORKS

PROBING THE ROLE OF EPIGENOMICS IN BRCA1-DEFICIENT MAMMARY TUMORS.

INTERPRETABLE COMPUTATIONAL MODELS OF FUNCTIONAL GENOMICS DATA - PROJECT SUMMARY UNDERSTANDING HOW THE COORDINATION OF CIS-REGULATORY ELEMENTS (CRES) INFLUENCES BIOLOGICAL PROCESSES, SUCH AS TRANSCRIPTION AND ALTERNATIVE SPLICING, IS A MAJOR GOAL IN COMPUTATIONAL GENOMICS. THIS REMAINS A CHALLENGE BECAUSE CRE ACTIVITY AT ANY GIVEN LOCUS MAY DEPEND ON A HOST OF OTHER FACTORS, INCLUDING SEQUENCE CONTEXT AND/OR THE PRESENCE OF OTHER CRES NEARBY. RECENT DEVELOPMENTS IN DEEP CONVOLUTIONAL NEURAL NETWORKS (CNNS) HAVE REVOLUTIONIZED OUR ABILITY TO PREDICT REGULATORY FUNCTIONS FROM DNA SEQUENCE. UNLIKE PREVIOUS COMPUTATIONAL METHODS BASED ON POSITION-WEIGHT MATRICES, WHICH CAPTURE AN ADDITIVE MODEL OF CRES, CNNS CAN, IN PRINCIPLE, ALSO LEARN HIGHER-ORDER DEPENDENCIES WITHIN THE CRE, WITH OTHER CRES, AND WITH THE BROADER SEQUENCE CONTEXT. HOWEVER, CNNS ARE ESSENTIALLY BLACK BOX MODELS, WITH PARAMETERS THAT DON’T HAVE CLEAR BIOLOGICAL MEANING. HENCE IT REMAINS A CHALLENGE TO TRANSLATE THE IMPROVED PREDICTIONS OF A CNN TO NEW BIOLOGICAL INSIGHTS. HERE WE PROPOSE TO DEVELOP THREE DIFFERENT COMPUTATIONAL METHODS THAT CAN COMPREHENSIVELY CHARACTERIZE HIGHER-ORDER INTERACTIONS WITHIN CRES AND ACROSS DIFFERENT CRES FROM FUNCTIONAL GENOMICS DATA, SPECIFICALLY CHIP-SEQ AND CLIP-SEQ DATA PUBLICLY AVAILABLE THROUGH ENCODE. EACH METHOD SERVES AS ITS OWN SEPARATE AIM AND WILL BE DEVELOPED IN PARALLEL. IN AIM 1, WE WILL DEVELOP A NEW POST HOC MODEL INTERPRETABILITY METHOD BASED ON EMPLOYING INTERPRETABLE QUANTITATIVE MODELS ORIGINALLY DEVELOPED TO UNDERSTAND COMPLEX GENETIC INTERACTIONS IN LABORATORY- BASED COMPREHENSIVE MUTAGENESIS (E.G. MULTIPLEX ASSAYS OF VARIANT EFFECTS) TO CHARACTERIZE CRE DEPENDENCIES LEARNED BY A CNN, USING SYNTHETIC SEQUENCES TO TARGET SPECIFIC BIOLOGICAL HYPOTHESES. IN AIM 2, WE WILL DEVELOP NEW CNN ARCHITECTURES WHERE THE LEARNED PARAMETERS WILL EXPRESS HIGHER-ORDER INTERACTIONS THAT HAVE DIRECT BIOLOGICAL INTERPRETATIONS. IN AIM 3, WE WILL COMBINE A BAYESIAN NONPARAMETRIC FRAMEWORK FOR MODELING CRES WITH CNN-BASED CRE ANNOTATIONS AND GPU ACCELERATION TO DEVELOP NEW METHODS FOR UNDERSTANDING HOW CRES ARE SPECIFIED IN THE GENOME. SUCCESSFUL COMPLETION OF THESE AIMS WILL PROVIDE A LEAP FORWARD IN OUR UNDERSTANDING OF HIGHER-ORDER CRE DEPENDENCIES THAT ARE EXPLOITED BUT HAVE NOT YET BEEN FULLY REVEALED BY CNNS. THIS WORK WILL PROVIDE THE COMMUNITY WITH: (1) A NEW SUITE OF OPEN-SOURCE COMPUTATIONAL TOOLS THAT ADDRESS THE PROBLEM OF MODELING CRES AND THEIR DEPENDENCIES IN FUNCTIONAL GENOMICS DATA; AND (2) A COMPREHENSIVE GENOME-WIDE CATALOGUE OF CRE SYNTAX FOR TRANSCRIPTION FACTORS AND RNA-BINDING PROTEINS THAT WILL BE HOSTED ON A USER-FRIENDLY WEBSERVER.

HIGH RESOLUTION ANALYSIS OF COPY NUMBER MUTATION IN BREAST CANCER

CRCNS: THEORY AND EXPERIMENT OF NEURAL CIRCUIT MAPPING BY DNA SEQUENCING

ELUCIDATING THE SCP4 PATHWAY AS A MULTI-CATALYTIC SIGNALING DEPENDENCY IN ACUTE MYELOID LEUKEMIA - PROJECT SUMMARY PHOSPHATASES AND KINASES ARE ESTABLISHED CLASSES OF DRUG TARGETS IN ONCOLOGY DUE TO THE PRESENCE OF STRUCTURED CATALYTIC POCKETS OR ALLOSTERIC SITES THAT CAN BE TARGETED WITH SMALL MOLECULE INHIBITORS. TO EXPAND OUR KNOWLEDGE OF PHOSPHATASE AND KINASE AND DEPENDENCIES IN AML, WE RECENTLY COMPLETED DOMAIN-FOCUSED CRISPR SCREENING STUDIES IN SEARCH OF AML-SPECIFIC DEPENDENCIES. THESE EFFORTS REVEALED A POORLY STUDIED PHOSPHATASE SCP4 AS A DEPENDENCY UNIQUE TO AML, WHEREAS NORMAL HUMAN HEMATOPOIETIC CELLS CAN TOLERATE LOSS OF THIS ENZYME. OUR BIOCHEMICAL INVESTIGATION REVEALED AN INTIMATE LINKAGE BETWEEN SCP4 AND THE POORLY STUDIED KINASE PARALOGS STK35 AND PDIK1L. WE HAVE SHOWN THAT SCP4 INTERACTS WITH, STABILIZES, AND REMOVES INHIBITORY PHOSPHORYLATION FROM THE ACTIVATION LOOPS OF STK35 AND PDIK1L. WHILE THE NOVELTY AND THE THERAPEUTIC POTENTIAL OF OUR WORK IS CLEAR, MECHANISTIC GAPS STILL REMAIN IN OUR KNOWLEDGE OF THIS SIGNALING COMPLEX. THEREFORE, WE NOW SEEK TO ELUCIDATE THE GENETIC, BIOCHEMICAL, AND STRUCTURAL MECHANISMS OF SCP4-STK35-PDIK1L FUNCTION IN AML WITH THE INTENT OF ESTABLISHING INSIGHTS AND ASSAYS THAT CAN REVEAL THE THERAPEUTIC POTENTIAL OF TARGETING THIS PATHWAY. THE TWO PRINCIPAL INVESTIGATORS OF THIS PROJECT ARE DR. CHRIS VAKOC (CSHL) AND DR. YAN JESSIE ZHANG (UT AUSTIN), WHO BRING COMPLEMENTARY EXPERTISE IN EPIGENOMICS AND STRUCTURAL BIOLOGY, RESPECTIVELY. THE FIRST AIM OF THIS PROJECT WILL BE TO EVALUATE SCP4 DEPENDENCY IN PRIMARY HUMAN AML PATIENT SAMPLES AND IN NORMAL HUMAN HEMATOPOIETIC STEM AND PROGENITOR CELLS (HSPCS). THESE EXPERIMENTS WILL RELY ON NEW CRISPR VECTORS DEVELOPED BY DR. JUNWEI SHI (UPENN, CO-INVESTIGATOR) THAT ALLOW FOR THE EFFICIENT GENERATION OF GENETIC KNOCKOUTS IN PRIMARY AML PATIENT SAMPLES. BY TARGETING SCP4 IN DIVERSE AML PATIENT SAMPLES AND IN NORMAL HUMAN HEMATOPOIETIC CELLS (IN VITRO AND IN VIVO), WE SEEK TO UNDERSTAND WHICH AML SUBTYPE IS MOST DEPENDENT ON SCP4 FOR DISEASE MAINTENANCE. THE SECOND AIM WILL SEEK TO APPLY STRUCTURAL BIOLOGY APPROACHES TO SCP4, STK35, AND PDIK1L, WITH A FOCUS ON X-RAY CRYSTALLOGRAPHY. A MAJOR OBJECTIVE WILL BE TO UNDERSTAND THE CATALYTIC MECHANISM AND THE STRUCTURAL BASIS OF SUBSTRATE RECOGNITION, AND A SUBSTANTIAL BODY OF PRELIMINARY DATA SUPPORTS THE FEASIBILITY OF THESE EFFORTS. THE THIRD AIM WILL PERFORM GENOMESCALE EPISTASIS SCREENS, IN SEARCH OF MODIFIERS AND DETERMINANTS OF SCP4 DEPENDENCY. THIS STRATEGY WILL EVALUATE SCP4 AS A CRITICAL COMPONENT OF A LARGER SIGNALING NETWORK, WITH A GOAL OF REVEALING NOVEL COMPONENTS OF THE PATHWAY. FINALLY, THE FOURTH AIM WILL SEEK TO APPLY BIOCHEMICAL AND EPIGENOMIC METHODS TO SCP4, STK35, AND PDIK1L IN SEARCH OF DOWNSTREAM EFFECTORS, WITH A PARTICULAR FOCUS ON TRANSCRIPTION FACTORS WHOSE FUNCTION IS DIRECTLY OR INDIRECTLY REGULATED BY THIS SIGNALING COMPLEX. IN SUMMARY, OUR COLLABORATIVE TEAM WILL APPLY RIGOROUS AND INNOVATIVE GENETIC, BIOCHEMICAL, AND STRUCTURAL APPROACHES TO REVEAL FUNDAMENTAL INSIGHTS INTO AN ENTIRELY NOVEL SIGNALING PATHWAY AND TO PROVIDE A RICH RESOURCE OF INSIGHTS AND ASSAYS THAT CAN DRIVE THE DEVELOPMENT NOVEL TARGETED THERAPIES FOR AML.

NEURAL MECHANISMS FOR FLEXIBLE VOCAL COMMUNICATION - PROJECT SUMMARY: WHETHER TO LAUGH AT A JOKE OR TO ENGAGE IN A LIVELY DEBATE, WE FLEXIBLY MODIFY OUR VOCALIZATIONS BASED UPON SOCIAL CONTEXTS. SUCH ADAPTIVE BEHAVIOR REQUIRES REAL-TIME ADJUSTMENTS OF MOTOR OUTPUTS IN RESPONSE TO RAPIDLY CHANGING SENSORY INPUTS. HOW DOES THE BRAIN ACCOMPLISH THIS SENSORIMOTOR FEAT? PIONEERING STUDIES HAVE CHARACTERIZED THE BRAIN AREAS RESPONSIBLE FOR SOUND PRODUCTION IN MANY SPECIES (E.G., DROSOPHILA, ZEBRA FINCHES, MARMOSETS, MICE), BUT THE NEURAL CIRCUITS THAT GENERATE VOCAL FLEXIBILITY REMAIN POORLY UNDERSTOOD. VOCAL FLEXIBILITY, SUCH AS DURING A CONVERSATION, REQUIRES VOLUNTARY, CONTEXT-DEPENDENT CONTROL OVER SOUND PRODUCTION. IN MAMMALS, BASED ON HUMAN BRAIN LESIONS, GENE EXPRESSION PROFILES, AND NEUROPHYSIOLOGY DATA IN PRIMATES, CORTICAL CONTROL HAS BEEN PROPOSED TO EXERT VOLITIONAL CONTROL OVER SOUND PRODUCTION. HOWEVER, DIRECT EVIDENCE FOR THIS IDEA IS SCARCE AND THE NEURAL CIRCUIT-LEVEL MECHANISMS UNDERLYING VOCAL FLEXIBILITY, ESPECIALLY IN MAMMALS, REMAIN LARGELY UNKNOWN. FINDING AN APPROPRIATE RODENT MODEL WOULD COMPLEMENT PRIOR WORK IN THE PRIMATES AND WOULD PERMIT CIRCUIT-LEVEL MECHANISMS TO BE DECIPHERED. ALSTON’S SINGING MICE (S. TEGUINA), A HIGHLY VOCAL RODENT FROM THE CLOUD FORESTS OF CENTRAL AMERICA, ARE IDEALLY SUITED TO STUDY FLEXIBLE VOCAL BEHAVIORS. SINGING MICE SHOW REMARKABLE VOCAL FLEXIBILITY, SWITCHING BETWEEN VARIABLE, ULTRASONIC VOCALIZATIONS (USVS) AND STEREOTYPED, HUMAN-AUDIBLE SONGS DEPENDING UPON SOCIAL CONTEXT. IN CONTRAST, MOST RODENTS INCLUDING LAB MICE (M. MUSCULUS) PRODUCE ONLY USVS AND ARE NOT KNOWN TO PARTICIPATE IN VOCAL INTERACTIONS. SINGING MICE AND LAB MICE ARE ROUGHLY THE SAME BODY SIZE, AND BRAIN SLICES OF S. TEGUINA AT A FIRST GLIMPSE IS INDISTINGUISHABLE FROM THOSE OF M. MUSCULUS. NEURAL CIRCUIT DIFFERENCES UNDERLYING SUCH DRASTIC BEHAVIORAL DIVERGENCE ARE UNKNOWN. HERE WE PROPOSE TO TEST WHETHER THE ABILITY OF THE SINGING MICE TO APPLY VOCALIZATIONS FLEXIBLY WITHIN A SOCIAL CONTEXT, AND LACK THEREOF IN MOST OTHER RODENT SPECIES, IS DEPENDENT UPON MOTOR CORTICAL FUNCTION, ACTING VIA DOWNSTREAM VOCAL PRODUCTION CIRCUITS. USING CHRONIC ELECTROPHYSIOLOGY (AIM 1), SINGLE-CELL COMPARATIVE CONNECTOMICS (AIM 2), WE WILL DETERMINE THE ROLE OF MOTOR CORTEX DURING NATURAL VOCAL BEHAVIORS AND COMPARE CORTICAL CONNECTIVITY AND FUNCTION BETWEEN TWO SPECIES. IN AIM 3, WE WILL MANIPULATE THE CIRCUIT TO DETERMINE THEIR CAUSAL ROLE IN VARIOUS VOCAL BEHAVIORS IN EACH SPECIES. BY MAPPING, MEASURING AND MANIPULATING CORTICAL CIRCUITS, WE WILL LEARN HOW MOTOR CORTEX MODULATES BEHAVIORAL FLEXIBILITY IN SERVICE OF SOCIAL COMMUNICATION. MORE BROADLY, THESE EXPERIMENTS WILL PROVIDE A SYSTEMS-LEVEL FRAMEWORK TO STUDY HIERARCHICAL MOTOR CONTROL CIRCUITS – FOR E.G., HOW HIGH-LEVEL (CORTICAL) CONTROL CAN INFORM LOW-LEVEL CONTROLLERS (SUBCORTICAL PATTERN-GENERATORS) TO GENERATE APPROPRIATE MOTOR COMMANDS – A CHALLENGE FACED BY BIOLOGICAL AND ARTIFICIAL AGENTS MOVING THROUGH THE WORLD.

PRECLINICAL MODELS FOR CANCER THERAPEUTIC DEVELOPMENT

DISSECTING DYNAMIC GENETIC EFFECTS FROM THYMUS DEVELOPMENT TO IMMUNE-MEDIATED DISEASE - PROJECT SUMMARY: IMBALANCE OF IMMUNE AND INAMMATORY ACTIVITY IS A HALLMARK OF IMMUNE-MEDIATED DIS- EASES. GENOME-WIDE ASSOCIATION STUDIES HAVE IDENTIED HUNDREDS OF LOCI THAT INCREASE SUSCEPTIBILITY TO IMMUNE- MEDIATED DISEASES. FUNCTIONAL EFFECTS OF THESE GENETIC VARIANTS ARE DIFCULT TO INFER AS THEY MIGHT ONLY IMPACT A PARTICULAR CELL TYPE AND THEIR EFFECTS MAY BE RESTRICTED TO SPECIC POINTS IN DEVELOPMENT. STUDIES ON ADULT IMMUNE CELLS HAVE SHED LIGHT ON MEDIATOR CELLS AND MECHANISMS OF DISEASE-ASSOCIATED VARIANTS, HOWEVER, CHILDHOOD IS A CRITICAL YET UNDERSTUDIED STAGE IN THE DEVELOPMENT OF THE IMMUNE SYSTEM. GENETIC DETERMINANTS OF T CELL DEVELOP- MENT AND EFFECTOR FUNCTION DURING CHILDHOOD HAVE NOT BEEN STUDIED AND THEIR ROLE IN IMMUNE-MEDIATED DISEASES IS UNKNOWN. WE PROPOSE TO LL GAPS THAT LINK THE GENETICS OF IMMUNE-MEDIATED DISEASES TO THEIR EFFECTOR CELLS, DEVELOPMEN- TAL STAGES, AND MECHANISMS. OUR GOALS ARE 1) TO UNDERSTAND THE IMPORTANCE OF GENETICALLY MEDIATED T CELL DEVELOPMENT AND CENTRAL TOLERANCE INDUCTION ON IMMUNE-MEDIATED DISEASES AND 2) TO IDENTIFY THE IMPORTANCE OF CHILDHOOD IMMUNE PHENOTYPES ON DISEASE IN ADULTHOOD. TO REACH THESE GOALS, WE PROPOSE STUDYING THE DYNAMIC GENETIC INUENCES OF HUMAN T CELL DEVELOPMENT USING SINGLE-CELL GENE EXPRESSION QUANTITATIVE TRAIT MAPPING IN HUMAN PEDIATRIC THYMUS SAMPLES. TO STUDY THE GENETICS OF THE PROGRESSION FROM DEVELOPMENT TO PERIPHERAL EFFECTOR FUNCTION, WE WILL COLLECT PERIPHERAL IMMUNE CELLS FROM THE SAME PEDIATRIC PATIENTS. FURTHERMORE, WE WILL STUDY GENETIC EFFECTS ON PEDIATRIC IMMUNE CELLS IN DIFFERENT ACTIVATION STATES TO PINPOINT GENETIC EFFECTS ACTING UPON STIMULATION. TO INVESTIGATE GENETIC EFFECTS ON IMMUNE CELLS AT DIFFERENT AGES, WE WILL INTEGRATE THE GENETIC EFFECTS WE IDENTIFY IN PEDIATRIC IMMUNE CELLS WITH THOSE DERIVED FROM ADULT AND NEONATAL CELLS FROM OTHER STUDIES. LASTLY, WE WILL EXAMINE GENETIC EFFECTS ON CENTRAL AND PERIPHERAL IMMUNE PHENOTYPES IN THE CONTEXT OF IMMUNE-MEDIATED DISEASES. WE WILL GENERATE PROLES OF CHROMATIN ACCESSI- BILITY IN THYMUS AND PERIPHERAL IMMUNE CELLS TO IDENTIFY REGULATORY MECHANISMS IN CHROMATIN. THIS WILL ALLOW US TO CONDUCT COLOCALIZATION STUDIES OF IMMUNE-MEDIATED DISEASE VARIANTS, IMMUNE CELL-ASSOCIATED VARIANTS AND OPEN CHROMATIN REGIONS. WE WILL THEN ESTIMATE CAUSALITY OF IMMUNE PHENOTYPES ON IMMUNE-MEDIATED DISEASES USING A MENDELIAN RANDOMIZATION FRAMEWORK. IN SUMMARY, OUR RESEARCH OFFERS AN INNOVATIVE APPROACH TO STUDY COMPLEX DISEASE MECHANISMS: IT COMBINES QUANTITATIVE GENETICS AND IMMUNOLOGY USING CLINICAL SPECIMENS TO GENERATE NEW INSIGHTS INTO THE GENETICS OF IMMUNE-MEDIATED DISEASES, THEIR EFFECTOR CELLS AND MOLECULAR MECHANISMS. THIS MECHANISTIC UNDERSTANDING OF DISEASE-ASSOCIATED VARIANTS IS FUNDAMENTAL FOR ADVANCING TOWARDS NOVEL TREATMENTS OF IMMUNE-MEDIATED DIS- EASES.

THE ROLES OF DISTINCT NUCLEUS BASALIS PROJECTIONS IN COGNITION

CIRCUIT PLASTICITY UNDERLYING ACQUISITION OF SENSORY DECISION TASK

CONTROL OF E1 OLIGOMERIZATION REGULATES E1 FUNCTION IN PAPILLOMAVIRUS DNA REPLICATION

HARNESSING CAR T CELLS TO DECONSTRUCT THE INTERCONNECTIVITY AMONG HALLMARKS OF AGING - SUMMARY TO DATE NINE HALLMARKS OF THE AGING PROCESS HAVE BEEN DESCRIBED, WHICH CAN BE GROUPED INTO THREE MAJOR CATEGORIES: PRIMARY HALLMARKS OR CAUSE OF DAMAGE (GENOMIC INSTABILITY, TELOMERE ATTRITION, EPIGENETIC ALTERATIONS AND LOSS OF PROTEOSTASIS), ANTAGONIST HALLMARKS OR RESPONSES TO DAMAGE (CELLULAR SENESCENCE, MITOCHONDRIAL DYSFUNCTION AND DEREGULATED NUTRIENT SENSING) AND INTEGRATIVE HALLMARKS OR MANIFESTATIONS OF THE AGING PHENOTYPE (STEM CELL EXHAUSTION AND ALTERED INTERCELLULAR COMMUNICATION). DESPITE INTENSIVE RESEARCH ON EACH INDIVIDUAL HALLMARK, LITTLE IS KNOWN ABOUT THEIR INTERCONNECTIVITY AND THE HIERARCHIES GOVERNING THOSE INTERACTIONS. A LIMITING FACTOR IN THESE STUDIES IS THE TIME AND RESOURCES NEEDED FOR DEVELOPING, BREEDING AND AGING MOUSE MODELS THAT WOULD ALLOW TO UNTANGLE THE INTERACTIONS AS WELL AS THE LACK OF HIGHLY SELECTIVE SMALL MOLECULES THAT WOULD ENABLE SOMATIC APPROACHES. IN THIS REGARD, OUR RECENT WORK SHOWED FOR THE FIRST TIME THE FEASIBILITY OF EMPLOYING CELLULAR THERAPY TO ELIMINATE CELLS THAT HAVE ENTERED A SPECIFIC CELLULAR PROGRAM SUCH AS CELLULAR SENESCENCE. IN A PROOF- OF-CONCEPT STUDY WE IDENTIFIED UPAR AS A SURFACE MOLECULE UPREGULATED IN CELLULAR SENESCENCE AND DEVELOPED CAR T CELLS ABLE TO ELIMINATE SENESCENT CELLS WITH HIGH POTENCY AND SPECIFICITY. CAR T CELLS HAVE THE ADVANTAGE THAT THEY CAN BE ADMINISTERED SOMATICALLY AND ARE HIGHLY EFFECTIVE AT EXCLUSIVELY TARGETING CELLS EXPRESSING A SPECIFIC ANTIGEN. IN ADDITION, THEY OFFER THE POSSIBILITY OF SPATIAL AND TEMPORAL CONTROL MAKING THEM HIGHLY VERSATILE TOOLS. HEREIN WE LEVERAGE OUR TEAM’S EXPERTISE IN SENESCENCE BIOLOGY AND CELL ENGINEERING TECHNOLOGY TO SHED LIGHT INTO THE INTERACTIONS AMONG THE HALLMARKS OF AGING. OUR GOALS ARE TO CHARACTERIZE IN DEPTH THE IMPACT OF SENOLYTIC CAR T CELLS ON THE ANTAGONISTIC AND INTEGRATIVE HALLMARKS OF AGING; DEVELOP TISSUE SPECIFIC SENOLYTIC CAR T CELLS THAT WOULD ALLOW US TO UNTANGLE HIERARCHIES AMONG THE HALLMARKS ACROSS PHYSIOLOGICAL LIFESPAN AND STUDY THE THERAPEUTIC POTENTIAL OF MODULATING THE INTERACTIONS FOR THE TREATMENT OF AGE-RELATED PATHOLOGIES. PRELIMINARY DATA STRONGLY SUPPORTS THE FEASIBILITY OF THE PROPOSED WORK: WE HAVE SUCCESSFULLY AND SAFELY EMPLOYED SENOLYTIC CAR T CELLS IN NATURALLY AGED MICE AND SHOWED THEIR HIGH POTENCY AT ELIMINATING TARGET POSITIVE CELLS IN THE TISSUES OVER LONG PERIODS OF TIME. IMPORTANTLY, TREATMENT WITH SENOLYTIC CAR T CELLS RESULTS IN SIGNIFICANT IMPROVEMENTS IN HEALTHSPAN. IN OUR APPLICATION, WE CONTINUE TO CHARACTERIZE IN A SYSTEMATIC WAY THE EFFECTS ACROSS HALLMARKS AND HARNESS THE FLEXIBILITY OF CELLULAR THERAPY TO UNTANGLE THE HIERARCHIES GOVERNING THE INTERACTIONS. OVERALL, WE EXPECT OUR STUDIES WILL LEAD TO VERSATILE TOOLS TO INTERROGATE AGING BIOLOGY AS WELL AS TO THE DEVELOPMENT OF NEW THERAPIES FOR A RANGE OF AGE-RELATED PATHOLOGIES.

COMBINED TOPOLOGICAL AND MACHINE LEARNING TOOLS FOR NEUROSCIENCE

UNDERSTANDING THE ROLES OF CORTICO-BULBAR FEEDBACK IN ODOR IDENTIFICATION

STRUCTURE AND FUNCTION OF HETERO-MULTIMERIC GLUTAMATE RECEPTORS

FUNCTIONAL MICROCIRCUITRY UNDERLYING SOUND PROCESSING IN THE PRIMARY AUDITORY COR

TRNA-FRAGMENTS IN TRANSPOSON CONTROL

BIOLOGY AND VULNERABILITIES OF POU2F3+ TUFT CELL-LIKE CARCINOMAS - PROJECT SUMMARY HUMAN CANCERS CAN BE CLASSIFIED ACCORDING TO THEIR CELLULAR LINEAGE, WHICH IS OFTEN DEFINED BY THE PRESENCE OF MASTER REGULATOR TRANSCRIPTION FACTORS. IN SEVERAL CLINICAL CONTEXTS, OPPORTUNITIES EXIST TO TARGET MASTER REGULATORS AS A THERAPEUTIC STRATEGY (E.G. ANTAGONISTS OF THE ANDROGEN RECEPTOR OR DEGRADERS OF IKAROS/AIOLOS). HOWEVER, SCENARIOS ALSO EXIST IN WHICH THE ABERRANT EXPRESSION OF A MASTER REGULATOR CAN PROMOTE LINEAGE PLASTICITY, WHICH CAN ALLOW TUMOR CELLS TO EVADE TARGETED THERAPIES. THESE CLINICAL OBSERVATIONS HIGHLIGHT THE IMPORTANCE OF INVESTIGATING THE BIOLOGY AND MECHANISMS OF LINEAGE MASTER REGULATORS IN ONCOLOGY. OUR LAB RECENTLY REPORTED THE EXISTENCE OF A TUFT CELL-LIKE LINEAGE OF HUMAN CARCINOMA, WHICH IS A TUMOR DEFINED BY THE EXPRESSION OF POU2F3. WE DESCRIBED TUFT CELL-LIKE CARCINOMAS AS A NON-NEUROENDOCRINE FORM OF SMALL CELL LUNG CANCER (SCLC), HOWEVER SUBSEQUENT STUDIES HAVE SHOWN THAT POU2F3+ CARCINOMAS EXIST IN SEVERAL OTHER ORGANS AS WELL. IN THIS RESEARCH PROPOSAL, WE SEEK TO DEFINE THE UNIQUE BIOLOGY AND MOLECULAR MECHANISMS THAT DISTINGUISH POU2F3+ CARCINOMAS FROM OTHER FORMS OF HUMAN CANCER, WITH A LONG-TERM OBJECTIVE OF ADVANCING THERAPEUTIC SOLUTIONS FOR THIS NEWLY IDENTIFIED TUMOR LINEAGE. THE FIRST AIM OF THIS PROJECT WILL FOCUS ON REVEALING MOLECULAR MECHANISMS, BUILDING UPON OUR RECENT DISCOVERY OF OCA-T1/OCA-T2 AS OBLIGATE COACTIVATORS OF POU2F3 IN THE TUFT CELL LINEAGE. FOR THIS WORK, WE WILL EMPLOY OUR LATEST INNOVATIONS IN GENOME EDITING TECHNOLOGY AND EPIGENOMICS TO DEFINE THE EFFECTORS OF THE POU2F3/OCA-T COMPLEX IN POU2F3+ SCLC. THESE EFFORTS WILL HAVE POTENTIAL TO REVEAL BIOCHEMICAL OPPORTUNITIES THAT ALLOW FOR PHARMACOLOGICAL MODULATION OF POU2F3 IN CANCER. THE SECOND AIM WILL EVALUATE THE UNIQUE BIOLOGICAL PROPERTIES OF NEOPLASTIC TUFT CELLS IN VIVO, WHICH BUILDS UPON OUR PRELIMINARY DATA SHOWING THAT ENGINEERING OF CANCER GENE MUTATIONS INTO PULMONARY TUFT CELLS LEADS TO THE GENERATION OF SCLC- LIKE TUMORS THAT EXPRESS POU2F3. THESE UNIQUE GENETIC MODELS WILL ALLOW US TO EXPLORE BIOLOGICAL QUESTIONS THAT WOULD OTHERWISE BE IMPOSSIBLE USING HUMAN CANCER CELL LINES. SPECIFICALLY, WE WILL INVESTIGATE A) WHICH SPECIFIC TUMOR GENOTYPES ARE PERMISSIVE FOR FORMING TUFT CELL CARCINOMAS B) WHICH TUMOR GENOTYPES DRIVE LINEAGE PLASTICITY IN THIS CONTEXT, PARTICULARLY TRANSITIONS BETWEEN TUFT AND NEUROENDOCRINE FATES AND C) HOW CYTOTOXIC CHEMOTHERAPY SELECTS FOR SPECIFIC CELLULAR STATES IN THE SETTING OF A TUFT CELL CARCINOMA. THE THIRD AIM OF THIS PROPOSAL SEEKS TO UNDERSTAND TUFT CELL IDENTITY AS AN ACQUIRED STATE AND AS A DRUG RESISTANCE MECHANISM IN PROSTATE ADENOCARCINOMA. WE HAVE DEVELOPED METHODS FOR REPROGRAMMING CARCINOMAS INTO A TUFT CELL STATE USING POU2F3/OCA-T CO-EXPRESSION, WHICH WE WILL INCORPORATE INTO THE RAPIDCAP MODEL OF PROSTATE CANCER TO STUDY ITS IMPACT ON DISEASE PROGRESSION AND CASTRATION RESISTANCE. COLLECTIVELY, THE RESEARCH OUTLINED IN THIS PROPOSAL WILL ADVANCE OUR FUNDAMENTAL UNDERSTANDING OF THE BIOLOGY AND THERAPEUTIC VULNERABILITIES OF POU2F3+ HUMAN CARCINOMAS.

INTEGRATED TRANSLATIONAL CANCER SCIENCE CENTER

COMMUNITY PROJECT FUNDING/CONGRESSIONALLY DIRECTED SPENDING - CONSTRUCTION

RESEARCH-PGR: UNLOCKING THE GENETIC AND EPIGENETIC BASIS OF CEREAL CROP ADAPTATION TO ACIDIC SOIL REGIONS -IN A CLIMATE THAT IS RAPIDLY TRANSITIONING INTO MUCH HARSHER CROP CULTIVATION CONDITIONS, RESILIENCE BECOMES VITAL FOR SUSTAINABLE FOOD PRODUCTION AND SECURITY ACROSS THE GLOBE. ACIDIC SOILS WITH LOW PH ARE WIDESPREAD GLOBALLY AND COMMON IN TROPICAL AND SUB-TROPICAL REGIONS, WHERE FOOD SECURITY IS A CHALLENGE. ALUMINUM (AL) TOXICITY, A SIGNIFICANT ABIOTIC STRESS ON ACIDIC SOILS, DAMAGES ROOT SYSTEMS AND ENHANCES CROP SUSCEPTIBILITY TO DROUGHT AND MINERAL DEFICIENCIES. PREVIOUS STUDIES HAVE SHOWN THAT GENES IN THE MULTIDRUG AND TOXIC COMPOUND EXTRUSION (MATE) FAMILY OF MEMBRANE TRANSPORTERS PLAY AN IMPORTANT ROLE IN AL TOLERANCE IN SORGHUM AND MAIZE WHILE OTHERS STRONGLY IMPLICATE EPIGENETIC VARIATION IN DNA METHYLATION AND HISTONE MODIFICATION IN AL TOLERANCE IN VARIOUS PLANT SPECIES. THIS PROJECT WILL TAKE A MULTI-OMICS APPROACH TO IDENTIFY AND EXPLORE VARIATION IN AL TOLERANCE AT BOTH THE GENETIC AND EPIGENETIC LEVELS, USING AL TOLERANCE AS A PARADIGM TO CONNECT GENE REGULATION TO ABIOTIC STRESS TOLERANCE IN CEREALS. IT IS EXPECTED THAT THIS PROJECT WILL HAVE SIGNIFICANT IMPACTS FOR FACILITATING TRANSLATION OF FINDINGS FROM MAIZE AND SORGHUM GENETICS/EPIGENETICS AND GENOMICS RESEARCH TO DEVELOP MORE EFFECTIVE BREEDING STRATEGIES TARGETING ADAPTATION TO BROADER ABIOTIC STRESS CONDITIONS. WITH RESPECT TO TRAINING AND OUTREACH, IN ADDITION TO THE TRAINING OF PROJECT STUDENTS AND POSTDOCTORAL SCIENTISTS, THE PROJECT WILL DEVELOP A MODULE ON TRANSLATIONAL GENOMICS FOCUSING ON AL TOLERANCE THAT WILL BE INCLUDED IN THE LONG-RUNNING CEREAL GENOMICS WORKSHOP HELD AT COLD SPRING HARBOR LAB. HOMOLOGOUS GENES IN THE MATE FAMILY OF MEMBRANE TRANSPORTERS PLAY AN IMPORTANT ROLE IN AL TOLERANCE IN SORGHUM (SBMATE) AND MAIZE (ZMMATE1) BY MEDIATING RELEASE OF AL DETOXIFYING CITRATE INTO THE RHIZOSPHERE. STUDIES HAVE SHOWN THAT AL-INDUCED EXPRESSION OF SBMATE IS NOT JUST LOCALIZED TO THE ROOT APEX, WHICH IS THE SITE OF AL TOXICITY AND TOLERANCE, BUT ALSO VERY SPECIFICALLY TO THE EPIDERMAL AND CORTICAL CELLS IN THE DISTAL TRANSITION ZONE. THIS PROJECT WILL LEVERAGE SINGLE-CELL TRANSCRIPTOMICS TECHNOLOGIES THAT WILL PROVIDE THE SPATIAL RESOLUTION NECESSARY TO DISCOVER NOVEL AL TOLERANCE GENES BY ASSOCIATING EPIGENETIC RESPONSES WITH CELL-SPECIFIC GENE EXPRESSION. CANDIDATE REGULATORY ELEMENTS WILL BE FUNCTIONALLY VALIDATED USING GENE EDITING AND MUTANT ANALYSES AND AL TOLERANCE PHENOTYPES WILL BE EVALUATED IN CONTROLLED CONDITIONS AND IN THE FIELD ON WELL-CHARACTERIZED ACIDIC SOILS. ALL PROJECT OUTCOMES WILL BE MADE AVAILABLE AND BROADLY DISTRIBUTED THROUGH DEPOSITION AT THE APPROPRIATE LONG-TERM DATA REPOSITORIES AND STOCK CENTERS. 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 PLANNED FOR THIS AWARD.

CIRCUIT MECHANISMS OF AUDITORY CATEGORIZATION

IMPLEMENTING DNA BARCODING FOR COURSE-BASED UNDERGRADUATE RESEARCH EXPERIENCES

RECEPTOR PTPS, CELL CONTACT AND SIGNAL TRANSDUCTION

TESTING THE DISC1 PATHWAY FOR FUNCTIONAL GENETIC VARIATION AND EPISTASIS IN MAJOR

EARLY EVENTS OF CARCINOMA INDUCED BY ERBB RECEPTORS

ROLE OF DOK-1 IN ONCOGENIC TYROSINE KINASE SIGNALING

DEFINING THE CELLULAR AND MOLECULAR EFFECTS OF AGING AND AGE OF PREGNANCY ON BREAST TISSUE HOMEOSTASIS AND CANCER INITIATION

THE TUMOR SUPPRESSIVE ROLE OF CHD5

MECHANISMS OF DSRNA-INDUCED GENE SILENCING

PRINCIPLES OF SPECKLE-BASED GENE REGULATION - THE CELL NUCLEUS HOUSES DIFFERENT TYPES OF NON-DNA SUBSTRUCTURES, CALLED NUCLEAR BODIES. AMONG THESE, NUCLEAR SPECKLES PREDOMINATE. SPECKLES OCCUPY ~15-30% OF THE NUCLEAR VOLUME AND ARE PRESENT IN CELLS THROUGHOUT OUR BODIES. PROTEINS INVOLVED IN DIFFERENT STAGES OF RNA PRODUCTION ARE HIGHLY CONCENTRATED WITHIN SPECKLES, AND SPECKLES ARE THE KEY NUCLEAR BODY THAT ASSOCIATES WITH ACTIVE EUCHROMATIN. HOW SPECKLES REGULATE GENE EXPRESSION HAS HISTORICALLY BEEN MURKY DUE TO LACK OF MECHANISTIC INSIGHT. MY PAST AND CURRENT RESEARCH CENTERS ON THE VIEW THAT NUCLEAR SPECKLES REGULATE GENE EXPRESSION IN UNIQUE AND DIVERSE WAYS, AND THAT UNDERSTANDING HOW THIS WORKS WILL ILLUMINATE DISEASE MECHANISMS AND NEW THERAPEUTIC PATHS. MY PREVIOUS WORK FINDS TWO MAJOR WAYS THAT GENE REGULATION CAN BE IMPACTED BY SPECKLES. FIRST, TRANSCRIPTION FACTORS HELP SPECIFIC GENOMIC REGIONS LOCALIZE AT SPECKLES, BOOSTING EXPRESSION OF MODESTLY-SIZED GENE NEIGHBORHOODS. IN THE PROPOSED RESEARCH, WE SEEK TO IDENTIFY NEW TRANSCRIPTION FACTORS THAT DRIVE DNA-SPECKLE ASSOCIATION, DETERMINE THE IMPORTANCE OF THIS NOVEL TRANSCRIPTION FACTOR FUNCTION FOR CANCER BIOLOGY, AND DEVELOP WAYS TO INHIBIT IT USING COVALENT LIGANDS. WE APPLY THESE OBJECTIVES TO CLEAR CELL RENAL CELL CARCINOMA, NEUROBLASTOMA, AND MACROPHAGE INFLAMMATORY SIGNALING, BUT HOPE THAT THE PRINCIPLES UNCOVERED WILL BE APPLICABLE ACROSS DISEASE STATES AND MODEL SYSTEMS. SECOND, SPECKLES THEMSELVES CAN ADOPT TWO MAJOR STATES, ALTERING EXPRESSION OF LARGE GENE NEIGHBORHOODS. WE PROPOSE HERE THAT THIS IS CONTROLLED AT THE MOLECULAR LEVEL BY FACTORS THAT LINK TWO STRUCTURAL SUB-COMPARTMENTS OF THE SPECKLE. WE ALSO PROPOSE THAT SPECKLE STATES ARE HIGHLY DYNAMIC AND RESPONSIVE TO EXTRACELLULAR CUES VIA KEY WELL-KNOWN SIGNALING PATHWAYS. WE WILL EXAMINE THIS IN CELL CULTURE MODELS AND IN LIVING ANIMALS, TESTING THE HYPOTHESIS THAT THE SIGNALS SECRETED AFTER A MEAL DYNAMICALLY REGULATE SPECKLE FORM AND GENE-ACTIVATING FUNCTION TO COORDINATE EXPRESSION OF HUNDREDS OF GENES WITHIN LARGE SPECKLE-ASSOCIATING GENOMIC NEIGHBORHOODS. IN THE LONG TERM, WE ANTICIPATE THAT UNDERSTANDING HOW SPECKLES ARE DYNAMICALLY REGULATED WILL PROVIDE NEW TOOLS THAT COULD SENSITIZE TUMORS TO EXISTING TREATMENTS. OVER THE COURSE OF THE PROPOSED RESEARCH, WE WILL INTEGRATE IMAGING, GENOMIC, BIOINFORMATIC, PROTEOMIC, AND FUNCTIONAL APPROACHES TO UNDERSTANDING THE PRINCIPLES OF 1) REGULATED DNA POSITIONING AT SPECKLES AND 2) REGULATED SPECKLE STATES. ULTIMATELY, WE STRIVE TO CREATE TEXTBOOK MODELS FOR SPECKLE-BASED GENE REGULATION THAT CAN THEN BE APPLIED ACROSS DISEASE CONDITIONS TO IMPROVE PATIENT OUTCOMES AND QUALITY-OF-LIFE.

RNA INTERFERENCE AND HETEROCHROMATIC SILENCING IN REPLICATION AND QUIESCENCE - GENE REGULATION BY RNA INTERFERENCE IS USUALLY ATTRIBUTED TO MICRORNA, BUT RNAI HAS A MORE ANCIENT AND FUNDAMENTAL ROLE IN HETEROCHROMATIC SILENCING AND GENOME STABILITY. HETEROCHROMATIN COMPRISES CONDENSED REPETITIVE REGIONS OF EUKARYOTIC CHROMOSOMES, AND MEDIATES TRANSCRIPTIONAL SILENCING, CHROMOSOME SEGREGATION AND GENOME INTEGRITY. WE HAVE FOUND THAT HETEROCHROMATIN IS UNEXPECTEDLY TRANSCRIBED, AND THAT SUBSEQUENT RNAI GUIDES HISTONE MODIFICATION. IN THE FISSION YEAST S. POMBE, “CO- TRANSCRIPTIONAL” SILENCING OCCURS DURING THE S PHASE OF THE CELL CYCLE, FOLLOWED BY TRANSCRIPTIONAL SILENCING THEREAFTER. RELEASE OF RNA POLYMERASE II (POL II) DURING REPLICATION PREVENTS DNA DAMAGE, BUT WITHOUT RNAI, REPLICATION FORKS STALL AND ARE REPAIRED BY HOMOLOGOUS RECOMBINATION (HR), CAUSING GENOME INSTABILITY. WE HAVE FOUND THAT RNAI REGULATES GENOME STABILITY THROUGH R-LOOPS, RNA-DNA HYBRID STRUCTURES AT TRANSCRIPTION-REPLICATION COLLISIONS THAT PROMOTE HR. SILENCING DEPENDS ON HISTONE MODIFICATION, BUT RECENT STUDIES SHOW THIS MAY BE AN OVERSIMPLIFICATION. FIRST, HISTONE METHYLATION RECRUITS HETEROCHROMATIN PROTEIN 1, WHICH MEDIATES LIQUID-LIQUID PHASE SEPARATION (LLPS) AND MAY LIMIT ACCESS TO POL II. SECOND, RNAI ALSO RECRUITS UBIQUITIN LIGASE, AND WE RECENTLY FOUND THAT UBIQUITIN PROMOTES THESE PHASE TRANSITIONS. RNAI GUIDES HISTONE MODIFICATION IN C. ELEGANS AND DROSOPHILA, BUT CONSERVATION IN MAMMALIAN SYSTEMS HAS BEEN CONTROVERSIAL. FOR EXAMPLE, PIRNAS MEDIATE HISTONE MODIFICATION IN THE GERMLINE BUT DO NOT DEPEND ON DICER, WHILE GENOME INSTABILITY IN DICER MUTANT MOUSE EMBRYONIC STEM CELLS (MESC) DEPENDS ON TRANSCRIPTION OF SATELLITE REPEATS. STRIKINGLY, WE HAVE FOUND GENOME INSTABILITY IN DICER-/- MESC DEPENDS ON THE TRANSCRIPTIONAL CO- ACTIVATOR BRD4, AND IDENTICAL IN-FRAME BROMODOMAIN DELETIONS RESCUE DICER MUTANTS IN FISSION YEAST. S. POMBE IS AN OUTSTANDING MODEL SYSTEM FOR CELL CYCLE RESEARCH, AND WE WERE THE FIRST TO SHOW THAT RNAI IS ESSENTIAL FOR QUIESCENCE (G0). GENETIC SCREENS HAVE REVEALED THAT NUCLEOLAR RNA SILENCING AND HISTONE MODIFICATIONS MEDIATE THIS NOVEL FUNCTION. OUR GOALS IN THE NEXT FIVE YEARS ARE TO DETERMINE THE ELUSIVE MECHANISM BY WHICH RNAI GUIDES EACH ASPECT OF HETEROCHROMATIN FROM REPEAT INSTABILITY, TO SILENCING, CHROMOSOME SEGREGATION AND DNA REPAIR AS WELL AS SURVIVAL IN QUIESCENCE. OUR RECENT WORK SUGGESTS A CENTRAL ROLE FOR LONG NON-CODING RNA, R-LOOPS AND THE ACTIVITY OF RNASE H IN THE UPSTREAM STEPS IN THIS PATHWAY. DOWNSTREAM EVENTS INCLUDE HISTONE MODIFICATION AND LLPS THAT MAY UNDERLIE THE CLASSICALLY “CONDENSED” PROPERTIES OF HETEROCHROMATIN. WE WILL USE OUR STEM CELL MODEL TO ASSESS THE CONSERVATION AND RELEVANCE OF THESE MECHANISMS IN HEALTH AND DISEASE, ESPECIALLY IN CANCER.

CSHL CANCER GENE DISCOVER AND CANCER BIOLOGY POSTDOCTORAL RESEARCH TRAINING

CHARACTERIZING VITAMIN B6 PATHWAY DEPENDENCY IN ACUTE MYELOID LEUKEMIA - PROJECT SUMMARY/ABSTRACT ACUTE MYELOID LEUKEMIA (AML) IS ONE OF THE MOST DEVASTATING BLOOD CANCERS, AND IT AFFECTS AROUND ONE MILLION PEOPLE AND RESULTS IN 147,000 DEATHS PER YEAR WORLDWIDE. AML IS CHARACTERIZED BY THE ABNORMAL PRODUCTION OF MYELOID LINEAGE OF BLOOD CELLS AND THE RAPID GROWTH OF LEUKEMIA BLASTS IN BONE MARROW AND PERIPHERAL BLOOD. AML IS A VERY AGGRESSIVE CANCER, AND IT PROGRESSES RAPIDLY AND AML PATIENTS BECOME FATAL WITHIN WEEKS TO MONTHS. CURRENTLY, THERAPEUTIC OPTIONS FOR AML ARE VERY LIMITED. ONLY ABOUT 35% OF AML PATIENTS UNDER 60 YEARS-OLD AND 10% OVER 60 YEARS-OLD BENEFIT FROM STANDARD CHEMOTHERAPY. THUS, THERE IS AN URGENT AND UNMET MEDICAL NEED TO DEVELOP NEW THERAPEUTIC APPROACHES PARTICULARLY COMBINATION THERAPIES WITH ADVANCED EFFICACY AND REDUCED TOXICITY FOR AML PATIENTS. OUR LONG-TERM GOALS OF THE PROPOSED RESEARCH ARE TO STUDY ABNORMAL VITAMIN PATHWAYS AS METABOLIC DEPENDENCIES IN AML, TO INVESTIGATE CRITICAL REGULATORS OF VITAMIN PATHWAYS IN SUPPORTING AML CELL ABERRANT PROLIFERATION, AND TO IDENTIFY DRUGGABLE THERAPEUTIC TARGETS IN AML. WE HAVE RECENTLY UNCOVERED VITAMIN B6 PATHWAY AS A NOVEL DEPENDENCY IN AML. BASED ON THIS FINDING, WE HYPOTHESIZE THAT VITAMIN B6 PATHWAY IS A SPECIFIC ANTI-LEUKEMIA TARGET AND HAS PROMISING THERAPEUTIC POTENTIAL TO TREAT AML. TO ASSESS THE HYPOTHESIS, WE HAVE IDENTIFIED MULTIPLE KEY ENZYMES IN VITAMIN B6 DEPENDENT METABOLIC PATHWAYS ESSENTIAL FOR AML CELL, BUT NOT FOR NORMAL BONE MARROW HEMATOPOIETIC STEM AND PROGENITOR CELL AND IMMUNE CELL, PROLIFERATION. DEPLETION OF THESE KEY ENZYMES EXHIBITED ROBUST INHIBITION ON AML PROLIFERATION. WE HAVE ALSO VALIDATED VITAMIN B6 PATHWAY AS A PHARMACOLOGICALLY ACTIONABLE PATHWAY AND SHOWED THAT VITAMIN B6 PATHWAY INHIBITORS EXHIBIT PROMISING THERAPEUTIC EFFICACY IN MULTIPLE GENETICALLY ENGINEERED AML MODELS IN VIVO. THESE ROBUST PRELIMINARY DATA ENCOURAGE US TO PURSUE THREE SPECIFIC AIMS TO FURTHER CHARACTERIZE VITAMIN B6 PATHWAY: 1) TO CHARACTERIZE BOTH UPSTREAM AND DOWNSTREAM REGULATORS OF VITAMIN B6 PATHWAY IN AML; 2) TO EVALUATE THE SYNERGY BETWEEN VITAMIN B6 PATHWAY AND BCL-2 IN AML THERAPEUTICS TO FURTHER ENHANCE ITS EFFICACY; AND 3) TO PERFORM FUNCTIONAL GENOMICS SCREENS TO IDENTIFY VITAMIN B6 PATHWAY SYNERGISTIC EFFECTORS IN AML. WE PROPOSE TO STUDY AML FROM THE ASPECT OF VITAMIN B6 PATHWAY AND METABOLIC PROGRAMMING, A PROCESS WHICH IS NOVEL AND ESSENTIAL FOR AML CELL PROLIFERATION. THE PROPOSED RESEARCH WILL BE FOCUSING ON BOTH MECHANISTIC UNDERSTANDING AND THERAPEUTIC TARGETING OF VITAMIN B6 PATHWAY IN ASSOCIATION WITH DIFFERENT SYNERGISTIC EFFECTORS IN AML. THE MAJOR GOALS OF THIS PROPOSAL ARE TO IDENTIFY THE UNIQUE VITAMIN B6 UPSTREAM REGULATORS AND DOWNSTREAM EFFECTORS AS WELL AS ITS SYNERGISTIC EFFECTORS TO TREAT AML. THE PROPOSAL IS SIGNIFICANT BECAUSE IT WILL NOT ONLY UNCOVER THE UNDERLYING MECHANISM BUT ALSO PINPOINT THERAPEUTIC TARGETS TO SYNERGISTICALLY ENHANCE THE ANTI-LEUKEMIA EFFECTS IN AML.

COMPUTATIONAL METHODS FOR HUMAN GENOMIC DATA INTEGRATION: DEMOGRAPHY, SELECTION,

AN INTEGRATIVE APPROACH TO CANCER GENE DISCOVERY IN HEPATOCELLULAR CARCINOMA

QUANTITIATIVE MODEL FOR THE DEVELOPMENT OF NEURAL TOPOGRAPHIC MAPS

RELIABLE POST HOC INTERPRETATIONS OF DEEP LEARNING IN GENOMICS - PROJECT SUMMARY UNDERSTANDING HOW THE COORDINATION OF TRANSCRIPTION FACTORS BIND TO NON-CODING DNA PROVIDES MECHANISTIC INSIGHTS INTO TRANSCRIPTIONAL REGULATION. RECENT DEVELOPMENTS IN DEEP NEURAL NETWORKS (DNNS) HAVE REVOLUTIONIZED OUR ABILITY TO STUDY REGULATORY GENOMICS. WHILE THEY HAVE DEMONSTRATED IMPROVED PREDICTIONS COMPARED TO PREVIOUS METHODS BASED ON TRADITIONAL COMPUTATIONAL GENOMICS, THEIR LOW INTERPRETABILITY HAS EARNED THEM A REPUTATION AS A BLACK BOX. TO ADDRESS THIS GAP, POST HOC MODEL INTERPRETABILITY METHODS HAVE EMERGED TO INTERROGATE IMPORTANT FEATURES THAT THE NETWORK HAS LEARNED. OF THESE, ATTRIBUTION MAPS HAVE DEMONSTRATED PROMISE, PROVIDING IMPORTANCE SCORES FOR EACH NUCLEOTIDE IN A GIVEN SEQUENCE; THESE HAVE A NATURAL INTERPRETATION AS SINGLE-NUCLEOTIDE VARIANT EFFECTS. IN PRINCIPLE, ATTRIBUTION MAPS SHOULD CONTAIN INFORMATION TO IDENTIFY MOTIFS THAT ARE IMPORTANT FOR CELL-TYPE SPECIFIC REGULATORY FUNCTIONS AND ANNOTATE THEIR POSITIONS AT BASE- RESOLUTION. HOWEVER, ATTRIBUTION MAPS ARE OFTEN NOISY IN PRACTICE; IN ADDITION TO MOTIFS, THEY CONTAIN SPURIOUS IMPORTANCE SCORES FOR ARBITRARY NUCLEOTIDES FOR REASONS THAT ARE NOT WELL ESTABLISHED. DESPITE THEIR PROMISE, INTERPRETING A DNN THROUGH ATTRIBUTION MAPS REMAINS CHALLENGING. HERE WE PROPOSE THREE COMPLEMENTARY AIMS THAT SERVE TO MAXIMIZE THE BIOLOGICAL INSIGHTS THAT WE CAN ACHIEVE FROM ATTRIBUTION MAPS FOR GENOMIC DNNS. IN AIM 1, WE WILL DEVELOP A MODEL SELECTION FRAMEWORK TO IDENTIFY THE OPTIMAL DNN FROM A SET OF CANDIDATE DNNS THAT YIELDS HIGH GENERALIZATION PERFORMANCE AND INTERPRETABLE ATTRIBUTION MAPS. IN AIM 2, WE WILL DEVELOP ROBUST TRAINING STRATEGIES BASED ON REGULARIZATION AND DATA AUGMENTATIONS TAILORED FOR GENOMICS, WITH THE BROADER AIM OF ENSURING THAT DNNS YIELD HIGH-QUALITY ATTRIBUTION MAPS AND HIGH GENERALIZATION. IN AIM 3, WE WILL DEVELOP AND EMPLOY INTERPRETABLE COMPUTATIONAL METHODS TO DIRECTLY ANALYZE ATTRIBUTION MAPS TO FACILITATE DISCOVERY OF FUNCTIONAL MOTIFS AND ANNOTATE THEIR POSITIONS. EACH AIM WILL BE IMPLEMENTED AS OPEN-SOURCE SOFTWARE IN TENSORFLOW AND PYTORCH. AS THE NUMBER OF DEEP LEARNING APPLICATIONS IN GENOMICS IS RISING QUICKLY, THE BIOMEDICAL COMMUNITY WILL GREATLY BENEFIT FROM THESE USER-FRIENDLY COMPUTATIONAL TOOLS BY ENABLING THE DEPLOYMENT OF ROBUST TRAINING AND INTERPRETABILITY ANALYSIS FOR ANY DNN TRAINED ON FUNCTIONAL GENOMICS ASSAYS. THIS, IN TURN, WILL DRIVE NEW DISCOVERIES IN CIS-REGULATORY BIOLOGY ACROSS THE MANY BIOLOGICAL SYSTEMS THAT DEEP LEARNING HAS ALREADY BEEN APPLIED TO AND THE NEW APPLICATIONS THAT WILL CONTINUE TO EMERGE IN THE FUTURE.

HEURISTICS TO EVALUATE BIOMEDICAL AND GENOMIC KNOWLEDGE BASES FOR VALIDITY

TARGETING NEUTROPHIL EXTRACELLULAR TRAPS TO PREVENT BREAST CANCER METASTASIS CAUSED BY STRESS

MECHANISM BY WHICH KETOGENIC DIET UNCOUPLES TUMOR GROWTH AND OVERALL SURVIVAL - THE OVERARCHING GOAL OF THIS PROPOSAL IS TO ADD TO THE UNDERSTANDING OF THE COMPLEX INTERACTION BETWEEN THE TUMOR AND THE HOST ORGANISM TO FACILITATE NEW THERAPEUTIC APPROACHES IN CANCER MEDICINE. SPECIFICALLY, WE AIM TO ESTABLISH THE MECHANISTIC EXPLANATION FOR THE OBSERVATION IN TWO IL-6 ASSOCIATED MURINE MODELS OF CANCER CACHEXIA THAT KD DELAYS TUMOR GROWTH BUT ACCELERATES CANCER CACHEXIA AND SHORTENS SURVIVAL. WE DISCOVERED THAT THIS UNCOUPLING MAY BE A CONSEQUENCE OF THE BIOCHEMICAL INTERACTION OF TWO SIMULTANEOUSLY OCCURRING NADPH-DEPENDENT PATHWAYS. WITHIN THE TUMOR, INCREASED PRODUCTION OF LPPS AND, CONSEQUENTLY, SATURATION OF THE GLUTATHIONE SYSTEM LEADS TO FERROPTOTIC DEATH OF CANCER CELLS. SYSTEMICALLY, REDOX IMBALANCE AND NADPH DEPLETION IMPAIRS THE BIOSYNTHESIS OF CORTICOSTERONE, THE MAIN REGULATOR OF METABOLIC STRESS, IN THE ADRENAL GLANDS. THE PROPOSAL SEEKS TO BUILD ON THESE DISCOVERIES IN THREE WAYS. FIRST, WE WILL DETERMINE SPECIFIC POLYUNSATURATED FAT ENRICHED DIETS AND FERROPTOSIS INDUCING DRUGS, INCLUDING LICENSED MEDICATIONS, THAT EXACERBATE THE ANTI-CANCER EFFECT. SECOND, AFTER VALIDATING THAT GLUCOCORTICOID DEFICIENCY IS A CRITICAL COMPONENT OF SURVIVAL REDUCTION OF THE HOST ORGANISM, USING PLASMA, LIVER, AND ADRENAL FOCUSED ASSAYS, WE WILL USE SYSTEMATIC PHARMACOLOGICAL RESCUE EXPERIMENTS TO ENABLE A THERAPEUTIC WINDOW FOR KD INTERVENTION. THIRD, WE WILL TRACK HOW OXIDATIVE STRESS INDUCED GDF-15 ELEVATIONS MAY CONSPIRE WITH IL-6 ELEVATIONS TO DRIVE CACHEXIA ASSOCIATED ANOREXIA VIA NEURONAL CIRCUITS AND THEREBY CAUSE THE NEGATIVE ENERGY BALANCE THAT ACCELERATES CACHEXIA ONSET IN KD FED MICE. THE RESULTS OF THIS RESEARCH MAY BE OF INTEREST TO SCIENTISTS AND MEDICAL PROFESSIONALS. A COMBINATION OF ADMINISTRATION OF SYNTHETIC GCS MAY IMPROVE FOOD INTAKE, NORMALIZES GLUCOSE HOMEOSTASIS AND UTILIZATION OF NUTRITIONAL SUBSTANCES, DELAY THE ONSET OF CANCER CACHEXIA AND EXTENDS SURVIVAL OF TUMOR-BEARING MICE FED KD, WHILE NOT COUNTERACTING THE REDUCED TUMOR GROWTH INDUCED BY THE KD-INDUCED FERROPTOSIS. THESE STUDIES WILL EMPHASIZE THE IMPORTANCE OF CONSIDERING THE IMPACT OF CANCER TREATMENTS ON THE HOST AND THE TUMOR.

DEVELOPMENT OF RECOMBINASE-EXPRESSING MOUSE LINES FOR STUDYING THE DEVELOPMENT

DEFINING THE ROLE OF PHOSPHATIDIC ACID AS AN ALLOSTERIC REGULATOR OF MITOCHONDRIAL GLUTAMINASE - PROJECT SUMMARY/ABSTRACT MITOCHONDRIAL GLUTAMINASES (GLS AND GLS2) CATALYZE THE HYDROLYSIS OF GLUTAMINE TO GLUTAMATE AND AMMONIUM, A METABOLIC REACTION THAT IS CRITICAL FOR NUMEROUS ASPECTS OF MAMMALIAN PHYSIOLOGY. WHEREAS GLS2 EXPRESSION IS LARGELY RESTRICTED TO THE LIVER, GLS IS UBIQUITOUSLY EXPRESSED, WITH PARTICULARLY HIGH LEVELS IN THE KIDNEY, DIGESTIVE TRACT, AND BRAIN. ITS ROLES INCLUDE THE REGULATION OF SYSTEMIC ACID-BASE HOMEOSTASIS AND THE BIOSYNTHESIS OF EXCITATORY AND INHIBITORY NEUROTRANSMITTERS. ACCORDINGLY, HUMAN INBORN ERRORS OF METABOLISM INVOLVING MUTATION OF THE GLS GENE HAVE SEVERE PHENOTYPES, INCLUDING NEURAL EXCITOTOXICITY AND LETHAL NEONATAL ENCEPHALOPATHY. IN ADDITION TO ITS FUNCTIONS IN ORGANISMAL HEALTH, DYSREGULATED GLS ACTIVITY HAS BEEN IMPLICATED IN A SPECTRUM OF HUMAN PATHOLOGIES, RANGING FROM NEURODEGENERATION TO CANCER. CONSEQUENTLY, THERE HAVE BEEN INTENSIVE EFFORTS TO DEVELOP SELECTIVE ALLOSTERIC INHIBITORS OF GLS, ONE OF WHICH IS NOW BEING EVALUATED IN CLINICAL TRIALS FOR THE TREATMENT OF SOLID AND HEMATOLOGICAL MALIGNANCIES. HOWEVER, RESULTS TO DATE INDICATE THAT THE RAPID DEVELOPMENT OF RESISTANCE LIMITS THE EFFICACY OF THIS THERAPEUTIC STRATEGY. REMARKABLY, DESPITE THE LONG HISTORY OF GLS RESEARCH, IT IS NOT UNDERSTOOD HOW THE ACTIVITY OF THIS ENZYME IS REGULATED IN CELLS. PURIFIED RECOMBINANT GLS HAS MINIMAL CATALYTIC ACTIVITY, AND VASTLY SUPRAPHYSIOLOGICAL CONCENTRATIONS OF INORGANIC PHOSPHATE (100-150 MM) ARE REQUIRED TO ACTIVATE GLS IN VITRO. WE HAVE RECENTLY DISCOVERED THAT THAT THE BIOACTIVE PHOSPHOLIPID PHOSPHATIDIC ACID (PA) IS AN EXTREMELY POTENT ACTIVATOR OF GLS, APPROXIMATELY 107-FOLD MORE POTENT THAN INORGANIC PHOSPHATE. IMPORTANTLY, WE HAVE FOUND THAT PA CAN ACTIVATE GLS EVEN IN THE PRESENCE OF CLINICAL GLS INHIBITORS, THUS RENDERING THESE DRUGS INEFFECTIVE. IN THIS PROJECT, WE PROPOSE TO DEFINE THE MOLECULAR MECHANISM BY WHICH PA ACTIVATES GLS, AND THEN TO PROBE THE UPSTREAM REGULATION OF MITOCHONDRIAL PA SIGNALING, WITH THE ULTIMATE GOAL OF PERTURBING THIS PROCESS TO OVERCOME RESISTANCE TO GLS INHIBITORS. IN THE COURSE OF THESE STUDIES, WE WILL DEVELOP NEW CHEMICAL PROBES FOR INVESTIGATING THE PA-GLS INTERACTION, INCLUDING PHOSPHORUS-BASED COVALENT CAPTURE PROBES TO DETERMINE THE BINDING SITE OF THE PA PHOSPHOMONOESTER HEAD GROUP. WE WILL THEN CONDUCT A SCREEN TO IDENTIFY ADDITIONAL POTENTIAL METABOLITE-GLS INTERACTIONS, AND FUNCTIONALLY VALIDATE ANY ‘HITS’ OBTAINED. IN THE SECOND AIM, WE WILL BUILD ON PRELIMINARY DATA THAT STRONGLY IMPLICATE THE PA-GENERATING ENZYME PHOSPHOLIPASE D2 (PLD2), AS WELL AS THE UNCHARACTERIZED PROTEIN PLD5, IN THE REGULATION OF THE MITOCHONDRIAL PA-GLS AXIS. WE WILL ATTEMPT TO DETERMINE THE FUNCTION OF PLD5, FOCUSING ON WHETHER ITS REPORTED INTERACTION WITH PLD2 REGULATES EITHER THE ACTIVITY OR SUBCELLULAR LOCALIZATION OF THE LATTER. FINALLY, SINCE OUR PRELIMINARY DATA INDICATE THAT PLD2/5 MEDIATE RESISTANCE TO CLINICAL GLS INHIBITORS, WE WILL TEST WHETHER PHARMACOLOGICAL OR GENETIC BLOCKADE OF PLD2 OR PLD5 CAN OVERCOME THIS RESISTANCE MECHANISM. THUS, OUR PROPOSED STUDIES WILL DEFINE THE PHYSIOLOGICAL PROCESSES THAT REGULATE GLS ACTIVITY, AND WILL THEN APPLY THIS KNOWLEDGE TO IDENTIFY OPPORTUNITIES FOR ENHANCING THE EFFICACY OF THERAPEUTIC STRATEGIES TARGETING GLUTAMINE METABOLISM.

OPTIMIZING ANTI-NMDAR ANTIBODIES FOR TRIPLE-NEGATIVE BREAST CANCER THERAPY - PROJECT SUMMARY/ABSTRACT TRIPLE-NEGATIVE BREAST CANCER (TNBC), WHICH LACKS HORMONE RECEPTOR AND HER2 EXPRESSION, IS ASSOCIATED WITH A HIGH RISK OF METASTATIC RECURRENCE AND HIGH MORTALITY, NECESSITATING NOVEL THERAPEUTIC STRATEGIES. A HIGH PERCENTAGE OF TNBC (>70%) EXHIBIT UPREGULATION OF N-METHYL-D-ASPARTATE (NMDA) RECEPTOR (NMDAR), WHICH ARE NORMALLY EXCLUSIVELY EXPRESSED IN THE CENTRAL NERVOUS SYSTEM, AND HIGH NMDAR EXPRESSION IS ASSOCIATED WITH PERINEURAL INVASION AND POOR PROGNOSIS. DESPITE THE POTENTIAL OF ANTI-NMDAR ANTIBODIES TO KILL NMDAR- POSITIVE CELLS, CANCER PATIENTS WITH SPONTANEOUS ANTI-NMDAR PRODUCTION OFTEN EXPERIENCE SEVERE NEUROLOGICAL TOXICITY MANIFESTING AS PSYCHOSIS AND SEIZURES (TERMED ANTI-NMDAR ENCEPHALITIS). HOWEVER, IT REMAINS UNKNOWN WHETHER IT IS POSSIBLE TO UNCOUPLE THE ANTI-TUMOR EFFECTS OF ANTI-NMDAR ENCEPHALITIS FROM THEIR TOXICITY, THEREBY ESTABLISHING A SAFE AND EFFECTIVE THERAPEUTIC STRATEGY FOR TNBC. A KEY INNOVATION OF THIS PROPOSAL IS THE USE OF SINGLE-PARTICLE CRYO-EM TO SCREEN FOR ANTIBODY-INDUCED STRUCTURAL ALTERATIONS IN NMDAR, WHICH ARE NECESSARY AND SUFFICIENT FOR NEUROLOGICAL TOXICITY. WE PROPOSE A WORKFLOW FOR THE IDENTIFICATION, CHARACTERIZATION, AND VALIDATION OF ANTI-NMDAR ANTIBODY THERAPIES. FIRST, WE WILL ESTABLISH AN ORTHOTOPIC MOUSE MODEL BASED ON INOCULATION OF 4T1 (TRIPLE-NEGATIVE BREAST CANCER) CELLS, ENGINEERED TO HAVE DOXYCYCLINE-INDUCIBLE EXPRESSION OF NMDAR IN THE MAMMARY FAT PAD (AIM 1). THE NMDAR-POSITIVE MODELS WILL FIRST BE USED TO IMMUNIZE MICE WITH NMDAR AND ISOLATE NMDAR-REACTIVE ANTIBODIES BY SINGLE-CELL RNA SEQUENCING OF INTRATUMORAL B CELLS. RECOMBINANT EXPRESSION OF CANDIDATE NMDAR-REACTIVE ANTIBODIES WILL BE USED TO GENERATE CRYO-EM MODELS OF ANTIBODY-RECEPTOR COMPLEXES, THEREBY SCREENING FOR ANTIBODY-INDUCED STRUCTURAL CHANGES (AIM 2). FINALLY, CANDIDATE ANTI-NMDAR ANTIBODIES WITH LOW PREDICTED PATHOGENICITY DUE TO MINIMAL STRUCTURAL EFFECTS WILL BE USED TO TREAT MICE WITH NMDAR-POSITIVE ORTHOTOPIC TUMORS TO ELUCIDATE MAXIMAL THERAPEUTIC EFFICACY AND MINIMAL TOXICITY (AIM 3). IN PRELIMINARY DATA GENERATED FOR THIS PROPOSAL, WE HAVE PERFORMED A PROOF-OF-PRINCIPLE EXPERIMENTAL SEQUENCE DEMONSTRATING THAT DOXYCYCLINE TREATMENT IN VIVO IS SUFFICIENT TO INDUCE NMDAR EXPRESSION IN 4T1-NMDAR TUMORS AND ELEVATED ANTI-NMDAR ANTIBODY TITERS IN PLASMA. SCRNA-SEQ OF INTRATUMORAL B CELLS, WAS ENRICHED FOR NMDAR BINDERS AND WE HAVE IDENTIFIED MULTIPLE SEQUENCES WITH HIGH- AFFINITY FOR NMDAR, TWO OF WHICH WERE CHARACTERIZED BY CRYO-EM DISPLAYING DISTINCT EPITOPES. ALTOGETHER, THIS PROPOSAL WILL LEVERAGE ANTI-NMDAR ANTIBODIES AS AN EFFECTIVE AND NON-TOXIC THERAPY FOR NMDAR-POSITIVE TNBC, ADDRESSING A KEY UNMET NEED FOR THE 40,000 PATIENTS PER YEAR DIAGNOSED WITH TNBC IN THE USA.

SOAT1 AS A TARGETABLE DEPENDENCY IN PANCREATIC ADENOCARCINOMA - ABSTRACT PANCREATIC DUCTAL ADENOCARCINOMA (PDAC) IS A DEVASTATING DISEASE THAT IS NOTORIOUSLY REFRACTORY TO TREATMENT. HIGH MORTALITY IS DRIVEN BY LATE DIAGNOSIS, AGGRESSIVE METASTASIS, AND RESISTANCE TO BOTH CYTOTOXIC AND TARGETED THERAPIES, MAKING THE DEVELOPMENT OF NEW TREATMENT STRATEGIES ESSENTIAL. TO INTERROGATE THE MOLECULAR DRIVERS OF AGGRESSIVE PDAC BIOLOGY AND IDENTIFY NEW THERAPEUTIC TARGETS, WE DEVELOPED MOUSE AND PATIENT-DERIVED ORGANOID MODELS OF PROGRESSIVE DISEASE. IN RECENT WORK, WE LEVERAGED THESE MODELS TO FIND THAT THE NON-ESSENTIAL ENZYME SOAT1 (STEROL-O-ACYL TRANSFERASE 1) IS ENRICHED IN INVASIVE, METASTATIC ORGANOIDS WHERE IT PROMOTES THE HYPERACTIVATION OF THE MEVALONATE OR CHOLESTEROL SYNTHESIS PATHWAY. SOAT1 ESTERIFIES FREE CHOLESTEROL FOR STORAGE, DISRUPTING FEEDBACK INHIBITION AND LEADING TO INCREASED PRODUCTION OF IMPORTANT METABOLIC BYPRODUCTS INCLUDING ISOPRENOIDS, WHICH ARE REQUIRED FOR KRAS PRENYLATION, MEMBRANE LOCALIZATION, AND ACTIVATION. GENETIC ABLATION OF SOAT1 MARKEDLY SUPPRESSED TUMOR GROWTH IN THE TRANSPLANT SETTING, DESIGNATING SOAT1 AS A ROBUST THERAPEUTIC TARGET IN PDAC. SOAT1-DEFICIENT PDAC CELLS ALSO HAD AN IMPAIRED ABILITY TO MIGRATE, INVADE, SURVIVE AND METASTASIZE. THUS, WE HYPOTHESIZE THAT SOAT1-MEDIATED MVA PATHWAY HYPERACTIVATION STIMULATES MULTIPLE PATHWAYS THAT COLLECTIVELY ENABLE THE INVASION OF PRENEOPLASTIC PANIN CELLS DURING DISEASE INITIATION, AND PROMOTE THE METASTASIS OF ESTABLISHED PDAC CELLS. HERE, WE WILL USE GENETICALLY ENGINEERED MOUSE MODELS TO TEST THE EFFECT OF SOAT1 DELETION ON PDAC DEVELOPMENT AND METASTASIS. FURTHER, WE WILL LEVERAGE INDUCIBLE DEGRON MODELS TO CONDUCT IN-DEPTH CHARACTERIZATION OF THE METABOLOMIC IMPACTS OF SOAT1 ABLATION (AIM 1). OUR ULTIMATE GOAL IS TO TRANSLATE THESE BIOLOGICAL FINDINGS INTO NEW PDAC TREATMENT STRATEGIES. TO THIS END, WE WILL USE CLICK CHEMISTRY AND MOLECULAR DYNAMICS SIMULATION TO DEVELOP FIRST-IN-CLASS SMALL MOLECULES THAT FUNCTION AS POTENT AND SELECTIVE COVALENT-BINDING ANTAGONISTS OF SOAT1 (AIM 2). USING ORTHOTOPIC AND METASTATIC TRANSPLANTS OF PATIENT-DERIVED ORGANOIDS IN PARALLEL WITH AUTOCHTHONOUS MOUSE MODELS, WE WILL COMPREHENSIVELY EVALUATE THE EFFICACY OF LEAD SOAT1-INHIBITORY COMPOUNDS AS MONOTHERAPY OR COMBINATION THERAPEUTIC AGENTS IN THE PREVENTION OF BOTH EARLY PDAC DEVELOPMENT AND METASTASIS IN VIVO (AIM 3). TOGETHER, THE STUDIES OUTLINED HERE WILL PROVIDE AN EXHAUSTIVE ASSESSMENT OF THE BIOLOGICAL ROLE OF SOAT1 AND THE MEVALONATE PATHWAY ACROSS THE STAGES OF PDAC DEVELOPMENT, USING BOTH DEFINITIVE GENETIC MODELS AND PHARMACOLOGICAL APPROACHES. THESE RESULTS WILL DEFINE THE STAGE-SPECIFIC UTILITY OF SOAT1 ANTAGONISM AS AN EXCITING NEW THERAPEUTIC AVENUE IN PDAC, AND ULTIMATELY YIELD POTENT AND SELECTIVE COVALENT SOAT1 INHIBITORS THAT MAY HAVE DIRECT TRANSLATIONAL RELEVANCE FOR PDAC TREATMENT.

BIOCHEMICAL ANALYSIS OF PAPILLOMAVIRUS REPLICATION

JOINT NSF/ERA-CAPS: INTREPID - INVESTIGATING TRITICEAE EPIGENOMES FOR DOMESTICATION

TARGETING HYPOTHALAMIC NEURONS TO ABROGATE STRESS-INDUCED BREAST CANCER GROWTH

BLOCKADE OF CMYC ONCOGENIC FUNCTION BY PREGNANCY-INDUCED ALTERATIONS AND REMODELING OF THE MAMMARY GLAND - ABSTRACT/PROJECT SUMMARY THE GOAL OF THIS PROPOSAL IS TO UNDERSTAND HOW TRANSITIONS THROUGH PREGNANCY INFLUENCE THE MOLECULAR AND CELLULAR STATE OF MAMMARY EPITHELIAL CELLS AND ONCOGENESIS. NUMEROUS EPIDEMIOLOGICAL STUDIES HAVE FOUND THAT PREGNANCY ALTERS THE RISK OF BREAST CANCER. THESE STUDIES HAVE SHOWN CONSISTENTLY THAT A FULL-TERM PREGNANCY PRIOR TO THE AGE OF 25 IS ASSOCIATED WITH A ONE-THIRD DECREASE IN THE LIFE-TIME INCIDENCE OF BREAST CANCER. WHILE A PROTECTIVE EFFECT OF PREGNANCY HAS BEEN VALIDATED IN RODENT MODELS OF CANCER, THE UNDERLYING MOLECULAR BASIS FOR THIS EFFECT REMAINS UNCLEAR. WE HAVE FOUND THAT PREGNANCY LEADS TO PERSISTENT CHANGES IN THE EPIGENOME AND ENHANCER LANDSCAPE OF MECS. USING AN INDUCIBLE C-MYC MOUSE MODEL, WE ALSO FIND THAT ONCOGENESIS AND THE TRANSCRIPTIONAL OUTPUT DOWNSTREAM OF THIS ONCOGENE ARE REDUCED IN POST-PREGNANCY MECS. IN THIS PROPOSAL WE WILL INVESTIGATE THE MOLECULAR BASIS UNDERLYING THESE ROBUST PHENOTYPES. WE WILL USE EPIGENOMICS, EX VIVO, IN VIVO AND BIOCHEMICAL METHODS TO DEFINE HOW THE TRANSCRIPTIONAL FUNCTION OF C-MYC IS MODULATED BY PREGNANCY. WE HYPOTHESIZE THAT C- MYC INTERACTIONS WITH COFACTORS AND WITH SPECIFIC CIS-REGULATORY ELEMENTS ARE ALTERED IN THIS SYSTEM. WE WILL ALSO STUDY HOW SPECIFIC CHROMATIN REGULATORS CONTRIBUTE TO PREGNANCY-MEDIATED EPIGENOMIC REWIRING. THESE EFFORTS WILL BUILD UPON OUR RECENT DATA SUGGESTING THAT EZH2 SUPPORTS THE POST-PREGNANCY EPIGENOME IN WILD-TYPE MICE. COLLECTIVELY, THIS RESEARCH WILL PROVIDE FUNDAMENTAL INSIGHTS INTO THE CELL BIOLOGY OF THE MAMMARY GLAND AND CARRY THE POTENTIAL FOR DISCOVERIES THAT COULD BE HARNESSED TO DEVELOP PREVENTATIVE TREATMENTS THAT MODULATE BREAST CANCER RISK IN HUMANS.

STRUCTURAL AND FUNCTIONAL INSIGHTS INTO LARGE-PORE CHANNELS - PROJECT SUMMARY THE PRIMARY GOAL OF THIS PROJECT IS TO ENHANCE OUR UNDERSTANDING OF THE STRUCTURES AND FUNCTIONS OF CALCIUM HOMEOSTASIS MODULATOR (CALHM) PROTEINS. THESE PROTEINS PLAY PIVOTAL ROLES IN DIVERSE BIOLOGICAL PROCESSES, RANGING FROM NEUROMODULATION AND NEUROINFLAMMATION TO TASTE PERCEPTION AND NEURODEGENERATION. CALHMS ARE LARGE-PORE CHANNELS FACILITATING THE PASSAGE OF IONS AND LARGE MOLECULES LIKE ATP. WITHIN THE BROADER FAMILY OF LARGE-PORE CHANNELS, CALHMS SHARE CLASSIFICATION WITH CONNEXIN, INNEXIN, PANNEXIN, AND LEUCINE-RICH REPEAT- CONTAINING 8 (LRRC8). DYSFUNCTIONAL CALHMS ARE IMPLICATED IN NEUROLOGICAL DISORDERS, INCLUDING ALZHEIMER'S DISEASE, DEPRESSION, AND NEUROINFLAMMATION. AMONG THE SIX CALHM MEMBERS (1-6), CALHM1 HAS BEEN UNEQUIVOCALLY DEMONSTRATED TO FORM VOLTAGE-SENSITIVE CHANNELS PERMEABLE TO IONS AND ATP. A POLYMORPHISM IN CALHM1 RESULTS IN THE PRO86LEU MUTATION, IDENTIFIED AS A RISK FACTOR FOR ALZHEIMER'S DISEASE IN VARIOUS POPULATION GROUPS. GENETIC KNOCKOUT STUDIES IN MICE REVEALED THAT THE ABSENCE OF CALHM1 LEADS TO A SIGNIFICANT DECLINE IN LEARNING AND MEMORY, UNDERSCORING ITS CRITICAL ROLE IN CEREBRAL NEURONAL ACTIVITIES. OUR RESEARCH GROUP HAS, THUS FAR, ACCOMPLISHED THE FOLLOWING MILESTONES TO PROMOTE THE FIELD OF LARGE-PORE CHANNELS: (1) DEVELOPED THE INSECT CELL EXPRESSION TECHNOLOGY, EARLYBAC, FOR EFFICIENT EXPRESSION OF CALHM AND OTHER MULTI-HETEROMERIC MEMBRANE PROTEINS; (2) ELUCIDATED THE STRUCTURES OF OCTAMERIC CHICKEN CALHM1 AND UNDECAMERIC HUMAN CALHM2 USING SINGLE-PARTICLE ELECTRON CRYOMICROSCOPY (CRYO-EM); (3) DELINEATED THE STRUCTURE OF HUMAN CALHM1, UNCOVERING THE INTERPLAY BETWEEN THE CONSERVED LIPID-BINDING POCKET AND CHANNEL ACTIVITY; (4) DISCLOSED THE BINDING SITE AND MODE OF AN INHIBITOR, RUTHENIUM RED, AT THE CALHM1 CHANNEL PORE; AND (5) REVEALED THE INAUGURAL STRUCTURE OF THE PANNEXIN1 CHANNEL, ELUCIDATING THE MECHANISM GOVERNING CL- SELECTIVITY IN THE HEPTAMERIC CHANNEL ASSEMBLY, DISTINCT FROM CALHM. HOWEVER, THE FOLLOWING SPECIFIC GAPS PERSIST: (A) THE ABSENCE OF A CLEAR STRUCTURAL BASIS FOR OLIGOMERIC STATES AND FUNCTION OF CALHMS, (B) THE DEARTH OF STRUCTURAL AND FUNCTIONAL INSIGHTS INTO ATP PERMEATION IN THE CALHM1 CHANNEL; AND (C) THE LACK OF STRUCTURAL UNDERSTANDING REGARDING CALHM1/3 HETEROMERS, KNOWN TO EXIST PHYSIOLOGICALLY IN TYPE-II TASTE CELLS AND EXHIBIT DISTINCT VOLTAGE-SENSITIVITY COMPARED TO CALHM1 HOMOMERS. HERE WE AIM TO ADDRESS THESE SHORTCOMINGS. AIM 1 WILL CORRELATE OLIGOMERIC STATES OF CALHM TO CHANNEL FUNCTIONS BY ANALYZING THE STRUCTURES AND FUNCTIONS OF THE CALHM1-2 CHIMERIC CONSTRUCTS. AIM 2 WILL UNCOVER CRITICAL ELEMENTS FOR ATP PERMEATION IN CALHM1. AIM 3 WILL REVEAL CALHM1/3 HETEROMER STRUCTURES. THESE AIMS WILL BE ACHIEVED THROUGH SINGLE-PARTICLE CRYO-EM, AND MECHANISTIC HYPOTHESES WILL BE TESTED USING PATCH-CLAMP ELECTROPHYSIOLOGY AND ATP FLUX ASSAYS. SUCCESSFUL COMPLETION PROMISES SIGNIFICANT STRIDES IN UNDERSTANDING CALHM STRUCTURES AND FUNCTIONS, PAVING THE WAY FOR THERAPEUTIC STRATEGIES IN NEUROLOGICAL DISORDERS.

TARGETING TUMOR-SPECIFIC ISOFORMS OF AURKA IN PANCREATIC CANCER WITH SPLICE-SWITCHING ANTISENSE OLIGONUCLEOTIDES

NEURONAL FUNCTION AND INCURABLE PROSTATE CANCER

COMPUTATIONAL TOOLS FOR ACCURATE INFERENCE OF GENETIC ANCESTRY FROM CANCER-DERIVED MOLECULAR DATA - PROJECT SUMMARY/ABSTRACT FOR MULTIPLE CANCER TYPES, EPIDEMIOLOGICAL DATA EXHIBIT STRONG CORRELATIONS BETWEEN, ON THE ONE HAND, THE INCIDENCE OF THE DISEASE, ITS SEVERITY WHEN DIAGNOSED, AND ITS CLINICAL OUTCOME, AND, ON THE OTHER HAND, THE ANCESTRAL BACKGROUND OF THE PATIENT. THIS WELL-DOCUMENTED PHENOMENON STRONGLY SUGGESTS A LINK BETWEEN THE BIOLOGY AND GENETICS OF CANCER IN AN INDIVIDUAL AND THE INDIVIDUAL'S GENETIC ANCESTRY. INDEED, RECENT RESEARCH IN CANCER GENOMICS, BOTH PAN-CANCER AND CANCER TYPE-SPECIFIC, POINTS TO GENETIC AND PHENOTYPIC DIFFERENCES BETWEEN TUMORS OCCURRING IN PATIENT POPULATIONS WITH DIFFERING GENETIC ANCESTRIES, AND TO THE NEED FOR MORE DATA COLLECTION TO POWER FURTHER STUDY IN THIS AREA. IT IS THE PURPOSE OF THIS PROPOSAL TO FACILITATE SUCH DATA ANALYSIS ON A MUCH GREATER SCALE, BY ENABLING GENETIC ANCESTRY INFERENCE DIRECTLY FROM CANCER-DERIVED MOLECULAR DATA, WITHOUT THE NEED FOR THE PATIENT'S CANCER-FREE GENOTYPE OR SELF-DECLARED RACE OR ETHNICITY. SUCCESSFUL COMPLETION OF THIS PROJECT WILL UNLOCK VAST AMOUNTS OF SUCH DATA FOR ANCESTRY- ORIENTED STUDIES OF CANCER FROM TWO MAJOR SOURCES. ONE IS THE BODY OF DATA STORED BY THE SEQUENCE READ ARCHIVE AND SIMILAR MASSIVE DIGITAL REPOSITORIES, ON THE ORDER OF 106 CANCER- DERIVED MOLECULAR PROFILES. THE OTHER IS THE BODY OF ARCHIVAL TUMOR TISSUES ACROSS MULTIPLE MEDICAL CENTERS, FROM MILLIONS OF WHICH MOLECULAR DATA MAY BE GENERATED. WE WILL DEVELOP SOFTWARE TOOLS FOR GENETIC ANCESTRY INFERENCE FROM MULTIPLE TYPES OF CANCER-DERIVED DATA, NAMELY, DNA SEQUENCE DATA FROM WHOLE EXOMES, WHOLE GENOMES AT LOW COVERAGE AND TARGETED SEQUENCE PANELS; RNA SEQUENCE DATA; ATAC-SEQ AND BISULFITE-CONVERTED SEQUENCE DATA. THE TOOLS TO BE DEVELOPED WILL DELIVER INFERENCE OF GLOBAL GENETIC ANCESTRY AT A SUB-CONTINENTAL LEVEL OF RESOLUTION, OF ANCESTRAL ADMIXTURES AND OF LOCAL ANCESTRY. THESE TOOLS WILL BE ADAPTIVE, ENDOWED WITH THE ABILITY TO OPTIMIZE THEIR PERFORMANCE FOR EACH INPUT CANCER-DERIVED MOLECULAR PROFILE. THIS ADAPTABILITY WILL BE ACHIEVED USING SIMULATED DATA, COMBINING THE INPUT CANCER-DERIVED PROFILE WITH ANCESTRAL BACKGROUNDS REPRESENTING WELL-DEFINED POPULATION GROUPS. AS A RESULT, THESE INFERENCE METHODS WILL PERFORM CONSISTENTLY, AND WITH QUANTIFIABLE ACCURACY, ACROSS A RANGE OF PROFILING DEPTHS AND QUALITIES, AND MITIGATE CANCER-RELATED DAMAGE TO THE GENOME. AN OPEN-SOURCE, USER-FRIENDLY AND FAIR-COMPLIANT SOFTWARE IMPLEMENTATION OF THESE METHODS WILL BE MADE AVAILABLE TO THE RESEARCH COMMUNITY THROUGH A NUMBER OF CHANNELS, INCLUDING GITHUB, BIOCONDUCTOR AND GALAXY. TRAINING AND COMMUNITY OUTREACH FOR THIS SOFTWARE WILL BE PROVIDED IN COLLABORATION WITH ITCR TRAINING NETWORK.

THE LET-7 REGULATORY NETWORK

CRCNS: COMPUTATIONAL MODEL FOR NEURAL STEM CELL DIVISIONS IN THE ADULT BRAIN

CSHL ADVANCED TECHNIQUES IN MOLECULAR NEUROSCIENCE COURSE

EPIGENETIC RE-ESTABLISHMENT OF TUMOR SUPPRESSION IN GLIOBLASTOMA - PROJECT SUMMARY THIS PROJECT FOCUSES ON A NEW EPIGENETIC VULNERABILITY OF HUMAN GLIOBLASTOMA (GBM), THE MOST COMMON AND DEADLY ADULT PRIMARY BRAIN MALIGNANCY. WE DISCOVERED THE BROMODOMAIN (BD)-CONTAINING CHROMATIN REGULATOR BRD8 AS ESSENTIAL TO GBM LACKING P53 MUTATIONS (TP53WT), WHICH MAKE UP ~71% OF CASES (SUN ET AL., NATURE 2023) (1). BRD8 MAINTAINS MALIGNANCY BY CRIPPLING P53-MEDIATED TUMOR SUPPRESSION IN A WAY DISTINCT FROM PREVIOUSLY DESCRIBED MECHANISMS: IT REPROGRAMS THE P53 NETWORK THROUGH THE EP400 HISTONE ACETYLTRANSFERASE COMPLEX AND BY BD-DIRECTED OCCUPANCY OF THE HISTONE VARIANT H2AZ AT P53-INDUCED TARGETS, ENFORCING A REPRESSIVE CHROMATIN STATE THAT PREVENTS P53-MEDIATED TRANSACTIVATION. CENTRAL TO THIS APPLICATION IS OUR FINDING THAT TARGETING BRD8 IN TP53WT GBM REMODELS CHROMATIN BY EVICTING H2AZ AND ENHANCING CHROMATIN ACCESSIBILITY, ENABLING P53 TO BIND AND TRANSACTIVATE ITS TARGETS. THIS CHROMATIN REMODELING CASCADE (REFERRED TO AS THE “BRD8/P53 EPIGENETIC SWITCH” IN THIS PROPOSAL) RE-ESTABLISHES P53 ACTIVITY IN TP53WT GBM, NORMALIZING GENE EXPRESSION, EVOKING CELL CYCLE ARREST, INHIBITING GLIOMAGENESIS, AND PROLONGING SURVIVAL IN XENOGRAFT MODELS OF TP53WT GBM (1). WE ADVANCE THIS WORK BY: (I) REVEALING THAT BRD8 OPPOSES P53 FUNCTION IN NON-MALIGNANT BRAIN CELLS; (II) SETTING UP A SINGLE-CELL GENE EXPRESSION PIPELINE; (III) SHOWING THAT THE BRD8 BD IS THE ONLY BD THAT SELECTIVELY REPROGRAMS THE P53 NETWORK; (IV) ENGINEERING A GBM-PRONE MOUSE MODEL WITH INTACT P53 IN WHICH WE CAN ABLATE BRD8; AND (V) ESTABLISHING TP53WT GBM ORGANOIDS FROM PATIENTS. THE ABOVE FINDINGS SUPPORT THE HYPOTHESIS THAT TARGETING BRD8 IN TP53WT GBM EVOKES THE BRD8/P53 EPIGENETIC SWITCH THAT RE- ESTABLISHES P53-MEDIATED TUMOR SUPPRESSION. DESPITE THESE ADVANCES, HOW THE BRD8/P53 EPIGENETIC SWITCH WORKS, AND WHETHER IT WILL BE USEFUL FOR TREATING PATIENTS WITH TP53WT GBM, REMAIN OBSCURE. THEREFORE, THE GOAL OF AIM 1 IS TO DEFINE THE EPIGENETIC MECHANISM BY WHICH TARGETING BRD8 RE-ESTABLISHES P53-MEDIATED TUMOR SUPPRESSION IN TP53WT GBM ORGANOIDS. THIS WILL BE ACCOMPLISHED BY: (AIM 1.1) DEFINING THE SET OF GENES TRANSACTIVATED BY THE BRD8/P53 EPIGENETIC SWITCH AT SINGLE-CELL RESOLUTION AND CHARACTERIZING THE GENOMIC AND EPIGENOMIC FEATURES OF THESE LOCI; (AIM 1.2) IDENTIFYING PATHWAYS INDUCED BY THE BRD8/P53 EPIGENETIC SWITCH THAT FACILITATE P53-MEDIATED CELL CYCLE ARREST; AND (AIM 1.3) ELUCIDATING THE DYNAMICS OF THE BRD8/P53 EPIGENETIC SWITCH AND HOW IT IS MODULATED BY EP400. THE GOAL OF AIM 2 IS TO DEMONSTRATE THE PRECLINICAL RELEVANCE OF TARGETING BRD8 TO RE-ESTABLISH P53-MEDIATED TUMOR SUPPRESSION IN VIVO. THIS WILL BE ACCOMPLISHED BY: (AIM 2.1) ASSESSING THE EFFECTIVENESS OF TARGETING BRD8 IN ENGINEERED GBM-PRONE MICE; (AIM 2.2) ESTABLISHING THE EFFECTIVENESS OF TARGETING BRD8 IN PATIENT-DERIVED XENOGRAFT (PDX) MODELS OF TP53WT GBM; AND (AIM 2.3) EVALUATING THE HETEROGENEITY OF P53-INDUCED TARGETS IN SINGLE CELLS OF GBM FROM SPONTANEOUS AND PDX MODELS AND DEFINING THE CELL POPULATIONS IMPACTED BY THE BRD8/P53 EPIGENETIC SWITCH.

ENHANCING IMMUNITY IN COLORECTAL CANCER LIVER METASTASES

CITIZEN DNA BARCODE NETWORK: A COMMUNITY-BASED INFRASTRUCTURE FOR MONITORING BIODIVERSITY AND DISEASE VECTORS

THE REPLICATION INITIATOR PROTEIN E1 AND ITS INTERACTION WITH DNA

DISCOVERING DYNAMIC COMPUTATIONS FROM LARGE-SCALE NEURAL ACTIVITY RECORDINGS

MOLECULAR TIMEKEEPING AND TEMPORAL CELL FATE SPECIFICATION IN C. ELEGANS - ABSTRACT/SUMMARY DEVELOPMENT IS AN INHERENTLY DYNAMIC PROCESS THAT OPERATES ON DIVERSE TIMESCALES. COORDINATING GENE EXPRESSION, CELL FATE DETERMINATION, AND TISSUE GROWTH IS CRUCIAL IN DEVELOPMENT, BUT OUR UNDERSTANDING IS LIMITED. THROUGH GENETIC ANALYSES OF C. ELEGANS TEMPORAL PATTERNING, WE HAVE DISCOVERED A MOLECULAR CLOCK, COMPOSED OF CIRCADIAN CLOCK ORTHOLOGS, THAT COORDINATES CELL FATE SPECIFICATION TO DEVELOPMENTAL PACE. IT ACCOMPLISHES THIS TASK BY GENERATING OSCILLATORY PATTERNS OF TRANSCRIPTION THAT EXHIBIT HIGHLY REPRODUCIBLE ONSET PHASES, DURATIONS, AND AMPLITUDES. THIS CLOCK REGULATES THE TRANSCRIPTIONAL DOSAGE OF KEY MICRORNA (MIRNA) GENES THAT PROGRAM TEMPORAL CELL FATE TRANSITIONS DURING DEVELOPMENT. THE EXPERIMENTS OUTLINED IN THIS PROPOSAL AIM TO UNDERSTAND THE MOLECULAR UNDERPINNINGS OF THIS CLOCK BY DETERMINING THE STRUCTURES OF THREE SEPARATE COMPLEXES (PROTEIN::PROTEIN AND PROTEIN::DNA) THAT ASSEMBLE AND DISASSEMBLE IN A SPECIFIC ORDER TO TAILOR DYNAMIC MIRNA TRANSCRIPTIONAL PATTERNS. WE WILL THEN USE THIS KNOWLEDGE TO ENGINEER CLOCK COMPONENT MUTATIONS THAT ALTER FEATURES OF THESE DYNAMIC INTERACTIONS. WE WILL VALIDATE HOW THESE NOVEL MUTANTS AFFECT COMPLEX ASSEMBLY IN VITRO AND HOW THEY DISTURB DEVELOPMENT IN VIVO. SPECIFICALLY, WE HYPOTHESIZE THAT THESE TAILORED MUTATIONS, BOTH LOSS-OF- AND GAIN-OF-FUNCTION, WILL ALTER MIRNA TRANSCRIPTIONAL OUTPUT BY DISRUPTING SPECIFIC FEATURES OF OSCILLATORY TRANSCRIPTION (PHASE, AMPLITUDE, OR DURATION). WE WILL QUANTIFY CHANGES IN MIRNA EXPRESSION IN THESE MUTANTS BY DIRECTLY MONITORING MIRNA TRANSCRIPTIONAL DYNAMICS IN MUTANT ANIMALS USING A NEW STATE-OF-THE-ART IMAGING PLATFORM. THIS IMAGING SYSTEM WILL ALSO ENABLE US TO DIRECTLY CORRELATE CHANGES IN TRANSCRIPTIONAL DYNAMICS TO ALTERATIONS IN TEMPORAL STEM CELL DIVISION PATTERNS DURING DEVELOPMENT. IN ADDITION, WE AIM TO USE BIOCHEMICAL STRATEGIES TO IDENTIFY THE LIGANDS THAT COORDINATE CLOCK TRANSCRIPTION FACTORS. IDENTIFYING THESE LIGANDS WILL REVEAL HOW MIRNA EXPRESSION PATTERNS SYNC ACROSS TISSUES TO ORGANIZE TEMPORAL PATTERNING. NOTABLY, MANY OF THE PHYSICAL INTERACTIONS BETWEEN THE CLOCK COMPONENTS WE AIM TO STUDY IN THE NEMATODE SYSTEM ARE CONSERVED BETWEEN THE MAMMALIAN ORTHOLOGS. THEREFORE, THESE STUDIES WILL NOT ONLY SHED LIGHT ON HOW THE TIMING OF GENE EXPRESSION CONTRIBUTES TO DEVELOPMENTAL ROBUSTNESS BUT ALSO MAY ILLUMINATE HOW TRANSCRIPTION IS COORDINATED WITH METABOLIC CHANGES DURING DEVELOPMENT.

"METHODS FROM COMPUTATIONAL TOPOLOGY AND GEOMETRY FOR ANALYSING NEURONAL TREE AND GRAPH DATA"

METABOLIC REPROGRAMMING TO BOOST THE FITNESS OF ANTI-TUMOR IMMUNITY AGAINST METASTATIC COLON CANCER - ABSTRACT THE PRIMARY GOAL OF THIS PROJECT IS TO ENHANCE THE EFFICACY OF IMMUNOTHERAPY AGAINST METASTATIC COLORECTAL CANCER (MCRC) BY ACTIVATING THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR DELTA (PPARΔ)-CARNITINE PALMITOYLTRANSFERASE 1A (CPT1A) AXIS IN CD8+ T CELLS. COLORECTAL CANCER (CRC) IS AMONG THE DEADLIEST CANCERS WORLDWIDE, WITH RISING INCIDENCE AND MORTALITY IN YOUNGER POPULATIONS. PARTICULARLY, MCRC PATIENTS, PREDOMINANTLY OF THE MICROSATELLITE STABLE (MSS) SUBTYPE, SHOW POOR RESPONSIVENESS TO CURRENT IMMUNOTHERAPIES DUE TO THE IMMUNOSUPPRESSIVE TUMOR MICROENVIRONMENT AND LOW MUTATION BURDEN THAT ASSOCIATES WITH LIMITED IMMUNOGENICITY. THIS CREATES AN URGENT NEED FOR INNOVATIVE APPROACHES TO BOOST THE IMMUNOGENICITY AND IMPROVE TREATMENT OUTCOMES. OUR PRELIMINARY STUDIES HIGHLIGHT THE ROLE OF PPARΔ, A LIPID-SENSING TRANSCRIPTION FACTOR, IN MODULATING THE METABOLIC FITNESS OF CD8+ T CELLS WITHIN THE TUMOR MICROENVIRONMENT, THEREBY ENHANCING THEIR IMMUNOGENIC POTENTIAL AGAINST MCRC. WE FOUND THAT BY PROMOTING MITOCHONDRIAL FATTY ACID OXIDATION (FAO) THROUGH THE PPARΔ-CPT1A AXIS, WE COULD SIGNIFICANTLY AUGMENT THE EFFICACY OF EXISTING IMMUNOTHERAPIES. THE ROLES OF PPARΔ AND FAO IN TUMORIGENESIS AND IMMUNITY ARE CONTROVERSIAL DUE TO LACK OF CELL TYPE-SPECIFIC GENETIC LOSS-OF-FUNCTION AND GAIN-OF-FUNCTION MODELS. WE HYPOTHESIZE THAT THE METABOLIC COMPETITION BETWEEN CANCER CELLS AND THE IMMUNE SYSTEM IS A CRITICAL DETERMINANT OF IMMUNE CELL FITNESS AGAINST CANCER. BY ACTIVATING PPARΔ IN CD8+ T CELLS, WE AIM TO TIP THIS BALANCE TO ENHANCE IMMUNE CELL FUNCTION AND THUS IMPROVE THE ANTI-TUMOR EFFICACY OF IMMUNOTHERAPIES IN MCRC. TO ACHIEVE OUR GOAL, WE PROPOSE THREE SYNERGISTIC AIMS. FIRST, WE WILL DELINEATE THE SPECIFIC MECHANISMS BY WHICH PPARΔ ACTIVATION ENHANCES THE ANTI-TUMOR ACTIVITY OF CD8+ T CELLS IN THE CONTEXT OF MSS MCRC, FOCUSING ON THEIR ANTI-METASTATIC FUNCTION AND THE UNDERLYING EPIGENETIC ALTERATIONS. SECOND, WE WILL ELUCIDATE THE ROLE OF CPT1A-MEDIATED FATTY ACID OXIDATION IN THE CONTEXT OF PPARΔ-INDUCED MODIFICATIONS IN CD8+ T CELLS, ASSESSING HOW THESE METABOLIC CHANGES IMPACT THEIR EFFECTIVENESS AGAINST CANCER. FINALLY, WE WILL EVALUATE THE THERAPEUTIC POTENTIAL OF ENHANCING THE PPARΔ-CPT1A AXIS WITHIN CD8+ T CELLS ON THE EFFICACY OF IMMUNOTHERAPEUTIC APPROACHES AGAINST MCRC, USING BOTH MURINE MODELS AND HUMAN PATIENT-DERIVED ORGANOIDS TO ENSURE CLINICAL RELEVANCE. COLLECTIVELY, THIS PROJECT IS POISED TO TRANSFORM MCRC TREATMENT BY DEMONSTRATING THAT MODULATING THE METABOLIC DYNAMICS WITHIN THE TUMOR MICROENVIRONMENT CAN SUBSTANTIALLY IMPROVE IMMUNOTHERAPEUTIC OUTCOMES. SUCCESSFULLY HARNESSING THE PPARΔ-CPT1A AXIS IN IMMUNE CELLS HOLDS THE PROMISE OF DEVELOPING NEW THERAPEUTIC MODALITIES THAT SIGNIFICANTLY EXTEND SURVIVAL AND ENHANCE THE QUALITY OF LIFE FOR MCRC PATIENTS, WHO SUFFER FROM LACK OF EFFECTIVE TREATMENT OPTIONS.

TARGETING NEUTROPHIL EXTRACELLULAR TRAPS TO PREVENT BREAST CANCER METASTASIS CAUSED BY STRESS

CRCNS: COMMON ALGORITHMIC STRATEGIES USED BY THE BRAIN FOR LABELING POINTS IN HIGH-DIMENSIONAL SPACE

COLD SPRING HARBOR LABORATORY FACULTY RECRUITMENT IN DEVELOPMENTAL NEUROBIOLOGY

CSHL CELL & DEVELOPMENTAL BIOLOGY OF XENOPUS COURSE

ENHANCING IMMUNITY IN COLORECTAL CANCER LIVER METASTASES

THE ONLINE BRAIN ATLAS RECONCILIATION TOOL

TARGETING PROSTATE CANCER STEMLIKE CELLS THROUGH CELL SURFACE-EXPRESSED GRP78

A UNIFIED PROBABILISTIC MODEL AND SOFTWARE IMPLEMENTATION FOR ANALYSIS OF NASCENT RNA SEQUENCING DATA - PROJECT SUMMARY THE PROCESS BY WHICH RNA MOLECULES ARE ASSEMBLED FROM DNA TEMPLATES, CALLED TRANSCRIPTION, IS FUNDAMENTAL TO ALL LIFE AND DYSREGULATED IN MANY HUMAN DISEASES. OVER THE PAST 15 YEARS, STUDIES OF THE MECHANISMS AND DYNAMICS OF TRANSCRIPTION HAVE INCREASINGLY RELIED ON A FAMILY OF TECHNIQUES FOR ISOLATING AND SEQUENCING NEWLY TRANSCRIBED, OR “NASCENT” RNAS. IN CONTRAST TO STANDARD RNA-SEQ, THESE NASCENT RNA SEQUENCING (NRS) METHODS ENABLE TRANSCRIPTION TO BE MEASURED SEPARATELY FROM RNA DEGRADATION, RESPOND RAPIDLY TO CHANGES IN TRANSCRIPTION, AND REVEAL THE POSITIONS OF RNA POLYMERASES ALONG A DNA TEMPLATE. HOWEVER, NRS DATA REQUIRE SOPHISTICATED COMPUTATIONAL AND STATISTICAL METHODS FOR ANALYSIS, WHICH ARE ONLY BEGINNING TO EMERGE. HERE, WE PROPOSE TO DEVELOP A POWERFUL AND FLEXIBLE PROBABILISTIC MODELING FRAMEWORK FOR THE ANALYSIS OF NRS DATA. OUR FRAMEWORK IS BASED ON A HIGHLY GENERAL “UNIFIED MODEL” THAT MATHEMATICALLY DESCRIBES BOTH THE KINETICS OF TRANSCRIPTION INITIATION, ELONGATION, AND PROMOTER-PROXIMAL PAUSE RELEASE, AND THE GENERATION OF SEQUENCING READ COUNTS. IT CAN BE USED TO ESTIMATE TRANSCRIPTIONAL RATES DIRECTLY FROM NRS DATA, IN EITHER A STEADY-STATE OR NONEQUILIBRIUM SETTING. OUR PROPOSAL INCLUDES THREE SPECIFIC AIMS, FOCUSED ON THE DEVELOPMENT OF (1) A SERIES OF STATISTICAL TESTS AND MACHINE-LEARNING METHODS FOR DIFFERENTIAL ANALYSIS OF TRANSCRIPTION-ASSOCIATED RATES; (2) A STATISTICAL AND MACHINE-LEARNING FRAMEWORK AND NEW EXPERIMENTAL METHODS FOR CHARACTERIZING VARIATION IN ELONGATION RATE AND ITS DEPENDENCY ON GENOMIC AND EPIGENOMIC COVARIATES; AND (3) AN OPEN-SOURCE SOFTWARE PACKAGE IMPLEMENTING THESE NEW METHODS IN THE R PROGRAMMING ENVIRONMENT (STADYUM), INTEGRATED WITH THE BIOCONDUCTOR, ANVIL, AND PYTORCH ENVIRONMENTS. SUCCESSFUL COMPLETION OF THESE AIMS WILL RESULT IN POWERFUL, VERSATILE, AND HIGHLY ACCESSIBLE NEW COMPUTATIONAL TOOLS THAT WILL ACCELERATE PROGRESS IN TRANSCRIPTIONAL RESEARCH.

DEVELOPMENT OF GENOMIC AND COMPUTATIONAL RESOURCES TO SUPPORT AGRICULTURE - THE OBJECTIVES OF THIS COOPERATIVE RESEARCH PROJECT ARE TO GENERATE RESOURCES TO SUPPORT OUR UNDERSTANDING OF GENOME FUNCTION.

BARCODE LONG ISLAND: EXPLORING BIODIVERSITY IN A UNIQUE URBAN LANDSCAPE

DROSOPHILA MODELS OF HUMAN COGNITIVE DISORDERS: NF1 AND NOONAN SYNDROME

CAREER: ALGORITHMS IN NATURE: UNCOVERING PRINCIPLES OF PLANT STRUCTURE, GROWTH, AND ADAPTATION

CSHL PREP - ABSTRACT THE GOAL OF THE COLD SPRING HARBOR LABORATORY (CSHL) POSTBACCALAUREATE RESEARCH EDUCATION PROGRAM (PREP) IS TO INCREASE THE NUMBER OF UNDERREPRESENTED MINORITY (URM) STUDENTS IN THE BIOMEDICAL SCIENCES BY PREPARING TRAINEES FOR MATRICULATION INTO HIGHLY COMPETITIVE AND RIGOROUS PH.D. GRADUATE PROGRAMS AND FUTURE SUCCESS IN SCIENCE-RELATED FIELDS. CSHL PREP WILL PROVIDE WORLD-CLASS RESEARCH EXPERIENCE, PROFESSIONAL DEVELOPMENT, AND DEVELOPMENT OF SCIENTIFIC IDENTITY AND COMMUNITY TO ITS PARTICIPANTS. FUNDING IS REQUESTED FOR FOUR PREP SCHOLARS TO CONDUCT A YEAR OF RESEARCH IN CANCER AND MOLECULAR BIOLOGY, NEUROSCIENCE, GENETICS AND GENOMICS, QUANTITATIVE BIOLOGY, AND PLANT BIOLOGY, UNDER THE MENTORSHIP OF A CSHL PREP FACULTY MEMBER. SCHOLARS WILL CREATE AN INDIVIDUAL DEVELOPMENT PLAN THAT DEFINES THEIR RESEARCH INTERESTS, IDENTIFIES RELEVANT COURSEWORK, AND ESTABLISHES GOALS AND TIMELINES FOR THEIR TIME IN THE PROGRAM AND THEIR FUTURE CAREERS. THEY WILL PARTICIPATE IN THE SKILLS DEVELOPMENT COURSE, DESIGNED EXCLUSIVELY FOR THEM, THAT INCLUDES WRITING AND ORAL PRESENTATION WORKSHOPS, AND GUIDANCE IN PREPARING FOR GRADUATE SCHOOL APPLICATIONS. THEY WILL ALSO ATTEND THE PREP BOOTCAMP COURSE TO BROADEN THEIR KNOWLEDGE BASE IN THE BIOSCIENCES. SCHOLARS WILL HAVE NUMEROUS OPPORTUNITIES TO DEVELOP THEIR SCIENTIFIC IDENTITY AND COMMUNITY. THEY WILL PARTICIPATE IN CAMPUS-WIDE SEMINARS, MEETINGS AND JOURNAL CLUBS WITH THEIR RESEARCH LABORATORIES, NATIONAL CONFERENCES, AND THE GRADUATE STUDENT RUN GRADUATE SEMINAR SERIES. THEY WILL BENEFIT FROM HAVING A RESEARCH MENTOR, A DIRECT MENTOR (A GRADUATE STUDENT OR POSTDOC IN THEIR RESEARCH LABORATORIES) AND A NEAR-PEER MENTOR (A GRADUATE STUDENT). THESE MENTORS WILL PLAY AN ACTIVE ROLE IN INTEGRATING THE SCHOLARS INTO THE CSHL COMMUNITY, AND HELP THEM BUILD A SCIENTIFIC NETWORK, SUCH AS WITH THE APPROXIMATELY 12,000 SCIENTISTS THAT VISIT CSHL ANNUALLY TO ATTEND THE MEETINGS AND COURSES PROGRAM. THE PREP COMMITTEE WILL HAVE OVERSIGHT OF CSHL PREP. THE COMMITTEE WILL MEET MONTHLY WITH THE SCHOLARS AT PREP TEAS, AND WITH THE SCHOLAR AND THEIR RESEARCH MENTOR IN QUARTERLY PROGRESS MEETINGS. MOREOVER, THE PROGRAM DIRECTOR AND PROGRAM COORDINATOR WILL HAVE CHECK-IN MEETINGS WITH INDIVIDUAL SCHOLARS MONTHLY. EFFECTIVENESS OF CSHL PREP WILL BE EVALUATED INTERNALLY BY CSHL’S SCHOOL ADMINISTRATORS AND EXTERNALLY BY BARONE RESEARCH SERVICES THROUGH A COMBINATION OF SURVEY RESULTS, CONFIDENTIAL REPORTS BY RESEARCH MENTORS, AND EXIT INTERVIEWS.

GENOMES TO NETWORKS: UNDERSTANDING FUNCTION UTILIZING GENOMIC SEQUENCING, COMPARATIVE AND NETWORK BASED APPROACHES

CELL-BASED GENOMIC ANALYSIS IN MOUSE MODELS OF RETT SYNDROME

GENOMIC APPROACHES IN BIOTECHNOLOGY

2008 THE BIOLOGY OF GENOMES CONFERENCE

TEMPERATURE SCALING OF ANIMAL DEVELOPMENT -THE DEVELOPMENT OF ANIMALS FROM A SINGLE CELL TO ADULTHOOD IS A COMPLEX OPERATION REQUIRING EXTREME COORDINATION OF BILLIONS OF MOLECULAR EVENTS. REMARKABLY, DEVELOPMENT OF MOST ANIMAL SPECIES OCCURS NORMALLY DESPITE RADICAL CHANGES THAT MAY OCCUR IN THE ENVIRONMENT THAT WOULD INDIVIDUALLY IMPACT THESE MOLECULAR REACTIONS. THIS PROPERTY SUGGESTS THAT EVOLUTION HAS SELECTED FOR AND CAN ENCODE GENE REGULATORY NETWORKS THAT GENERATE SIMILAR DEVELOPMENTAL OUTCOMES DESPITE CHANGING CONDITIONS. THIS PROPOSAL AIMS TO UNDERSTAND HOW ANIMALS CAN MAINTAIN THE CORRECT TIMING OF GENE EXPRESSION WHEN DEVELOPING AT DIFFERENT TEMPERATURES. SPECIFICALLY, THE INVESTIGATORS HAVE DEVELOPED TOOLS TO DIRECTLY MEASURE HOW AND WHEN ANIMALS TURN ON AND OFF KEY DEVELOPMENTAL GENES. THEY WILL USE THIS SYSTEM TO DISSECT HOW CHANGES IN GLOBAL GENE EXPRESSION ARE MAINTAINED ACROSS DIFFERENT CELLS AND TISSUES DURING DEVELOPMENT AT VARIABLE TEMPERATURES. THIS WILL ALLOW THE INVESTIGATORS TO DETERMINE THE UNKNOWN ARCHITECTURES OF GENE EXPRESSION THAT ARE IN PLACE TO MAKE SURE DEVELOPMENT IN VARYING ENVIRONMENTS GENERATES SIMILAR OUTCOMES. AS PART OF THIS PROJECT THEY WILL ALSO PROVIDE RESEARCH EXPERIENCES FOR AN UNDERGRADUATE STUDENT, AND THEY WILL PERFORM OUTREACH TO MINORITY-SERVING HIGH SCHOOLS IN NEW YORK CITY. TEMPERATURE IMPACTS ALL BIOCHEMICAL REACTIONS. FOR MULTICELLULAR DEVELOPMENTAL SYSTEMS, TEMPERATURE FLUCTUATIONS POSE PARTICULAR CHALLENGES BECAUSE THE ORDERLY MORPHOLOGICAL PROGRESSION DEPENDS ON BOTH SPATIALLY AND TEMPORALLY COORDINATED CELLULAR DECISIONS. IT HAS BEEN SHOWN THAT OVERALL DEVELOPMENTAL RATES OF POIKILOTHERMIC ANIMALS OBEY ARRHENIUS SCALING, THOUGH SCALING PARAMETERS ARE GENUS AND EVEN SPECIES DEPENDENT. DESPITE THE MYRIAD DIVERSE CHEMICAL REACTIONS INVOLVED, THIS TEMPERATURE SCALING IS UNIFORM, LEAVING THE RELATIVE TIMING OF KEY DEVELOPMENTAL MILESTONES UNCHANGED. IT REMAINS UNKNOWN HOW THIS IS ACHIEVED AND, MORE GENERALLY, HOW INDIVIDUAL MOLECULAR DECISIONS THAT CONTROL DEVELOPMENTAL PROCESSES ARE BUFFERED AGAINST TEMPERATURE AND SYNCHRONIZED WITHIN AND ACROSS TISSUES TO MAINTAIN DEVELOPMENTAL PRECISION. THE GOAL OF THIS PROJECT IS TO UNCOVER THE ORGANIZATIONAL STRATEGY AND MOLECULAR MECHANISMS THAT CONFER THIS ROBUSTNESS. THE INVESTIGATORS HAVE DEVELOPED A UNIQUE MICROFLUIDICS METHODOLOGY THAT ALLOWS HIGH-RESOLUTION IMAGING OF GENE EXPRESSION IN MULTIPLE C. ELEGANS LARVAE SIMULTANEOUSLY THROUGHOUT POST-EMBRYONIC DEVELOPMENT. THIS TECHNOLOGY WILL ENABLE THE INVESTIGATORS TO DETERMINE HOW TRANSCRIPTIONAL PATTERNS OF KEY REGULATORY MOLECULES ARE ORGANIZED AT DIFFERENT TEMPERATURES AND ENABLE THEM TO MODEL HOW GENE REGULATORY NETWORKS (GRNS) THAT GENERATE THESE PATTERNS WORK. THE STUDIES PUT THE INVESTIGATORS IN A POSITION TO NOW PIONEER QUANTITATIVE IN-VIVO STUDIES OF THE TEMPORAL PATTERNING GRN. 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.

CONFERENCE: CSHL ADVANCED COURSES FOR MODEL GENETIC SYSTEMS (2023-2025) -THE COLD SPRING HARBOR LABORATORY (CSHL) ADVANCED COURSES IN MODEL GENETIC SYSTEMS SERVE A CRITICAL ROLE IN THE TRAINING OF INVESTIGATORS NEW TO THE USE OF GENETIC AND GENOMIC APPROACHES IN BACTERIA, YEAST AND PLANTS. THESE HIGHLY INTENSIVE THREE-WEEK COURSES COVER THE FUNDAMENTALS OF GENETICS IN THE RESPECTIVE SYSTEM, WHICH THEN ESTABLISHES THE CONCEPTUAL BACKBONE FOR THE MORE SOPHISTICATED GENOMIC METHODS THAT ARE COVERED AS THE COURSES PROGRESS. THE HANDS-ON EXPERIMENTAL PORTION OF THE COURSES, OFTEN INCLUDING MULTI-DAY MODULES, ARE COMPLEMENTED BY DAILY THEORETICAL LECTURES BY INSTRUCTORS CHOSEN FOR THEIR PROVEN EXPERTISE IN THE SYSTEM AND A WORLD CLASS SEMINAR SERIES FROM RENOWNED INVESTIGATORS THAT UTILIZE THE RESPECTIVE SYSTEM. AS A RESULT, EVERY STUDENT IS EXPOSED TO THE UNDERLYING PRINCIPLES OF THE METHODS THEY ARE BEING TAUGHT AND THEIR STATE-OF-THE-ART APPLICATION BY THE EXPERTS IN THE FIELD. THE COURSES HAVE A WELL-ESTABLISHED REPUTATION FOR SERVING A DIVERSE COMMUNITY OF BIOLOGISTS AS WELL AS TRAINING RESEARCHERS FROM THE PHYSICAL SCIENCES. THE STUDENTS RANGE FROM EARLY-STAGE GRADUATE STUDENTS TO TENURED PROFESSORS, WITH BACKGROUNDS IN AREAS INCLUDING GENETICS, MOLECULAR AND CELLULAR BIOLOGY, BIOINFORMATICS, ENGINEERING, AND PHYSICS. SINCE THEIR FOUNDATION IN THE 1970S AND EARLY 1980S, THE CSHL SUMMER COURSES ON ADVANCED BACTERIAL GENETICS, YEAST GENETICS & GENOMICS AND FRONTIERS & TECHNIQUES IN PLANT SCIENCE (COLLECTIVELY, THE MODEL GENETICS SYSTEMS COURSES) HAVE EACH PLAYED A CRITICAL ROLE IN DISSEMINATING NEW APPROACHES AND TECHNIQUES TO THE BROAD SCIENTIFIC COMMUNITY INTERESTED IN THE USE OF MODEL SYSTEMS IN GENETICS AND GENOMICS. THE PROPOSED COURSES (2023-2025) WILL CONTINUE THIS TRADITION BY PROVIDING ADVANCED TRAINING IN THE FUNDAMENTALS OF WORKING IN THESE MODEL SYSTEMS MAKING USE OF CLASSICAL GENETIC AND MOLECULAR BIOLOGICAL APPROACHES, ANALYSIS OF GENETIC INTERACTIONS, AND GENOMICS. THE COURSES EACH CONSIST OF HANDS-ON LABORATORY MODULES, A VIGOROUS LECTURE SERIES, TECHNICAL PRESENTATIONS AND DEMONSTRATIONS, AND INFORMAL DISCUSSIONS. THE INSTRUCTORS AND INVITED SPEAKERS, ACKNOWLEDGED LEADERS IN THEIR FIELDS, PRESENT UP-TO-THE-MOMENT RESEARCH ON A WIDE RANGE OF TOPICS. THE INFORMAL, HIGHLY INTERACTIVE COURSE FORMAT ALLOWS STUDENTS AND TEACHING APPRENTICES TO LEARN FROM EACH OTHER AS WELL AS FROM THE INSTRUCTORS AND INVITED SPEAKERS. IN SUM, THE COURSES PROVIDE FOR INTENSIVE THREE-WEEK IMMERSION INTO CROSS-DISCIPLINARY AND CUTTING-EDGE APPROACHES, HELPING PREPARE THE NEXT GENERATION OF MICROBIOLOGISTS AND PLANT SCIENTISTS TO APPLY THE LATEST GENETIC/GENOMIC TOOLS AND APPROACHES TO THEIR OWN RESEARCH, OFTEN IN NON-MODEL AND ?REAL WORLD? APPLICATIONS. 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.

MRI: ACQUISITION OF NEXT GENERATION SEQUENCERS

THE FIRST COMPREHENSIVE NEURAL CONNECTIVITY MAP OF MOUSE

INSPIRE TRACK 1: ZERO-ONE LAWS AT THE INTERFACE BETWEEN PHYSICS, ENGINEERING AND BIOLOGY

CSHL STRUCTURAL BIOLOGY SHORT COURSES IN CRYSTALLOGRAPHY & CRYO-ELECTRON MICROSCOPY - ABSTRACT COLD SPRING HARBOR LABORATORY (CSHL)’S WORLD-RENOWNED POSTGRADUATE COURSE AND WORKSHOP PROGRAM HAS TRAINED INVESTIGATORS AND INFLUENCED RESEARCH IN FIELDS RANGING FROM PROTEOMICS TO YEAST BIOLOGY, FROM ION CHANNEL ANALYSIS TO HIGH THROUGHPUT NEUROANATOMY. THIS PROPOSAL SEEKS SUPPORT TO CONTINUE TWO ADVANCED POSTGRADUATE ~TWO-WEEK RESIDENTIAL COURSES ON COMPLEMENTARY TECHNIQUES IN STRUCTURAL BIOLOGY, X-RAY CRYSTALLOGRAPHY AND CRYO-ELECTRON MICROSCOPY, WHICH TOGETHER REPRESENT TWIN METHODOLOGICAL PILLARS IN THE ATOMIC AND NEAR-ATOMIC RESOLUTION OF MACROMOLECULAR STRUCTURES AND COMPLEXES. X-RAY CRYSTALLOGRAPHY AND RELATED METHODS HAVE BEEN THE CORNERSTONE OF STRUCTURAL BIOLOGY FOR HALF A CENTURY, AND REMAIN THE TECHNIQUES OF CHOICE FOR ATOMIC RESOLUTION UNDERSTANDING OF MACROMOLECULES AND FOR STRUCTURE GUIDED DRUG DISCOVERY. CSHL’S MACROMOLECULAR CRYSTALLOGRAPHY COURSE WAS INITIATED IN 1988 AND COMBINES LABORATORY AND COMPUTATIONAL INSTRUCTION TO TRAIN COURSE PARTICIPANTS IN THE MAJOR TECHNIQUES USED TO DETERMINE THREE-DIMENSIONAL STRUCTURES. IT IS DESIGNED FOR SCIENTISTS WITH A WORKING KNOWLEDGE OF PROTEIN STRUCTURE AND FUNCTION, BUT WHO ARE NEW TO MACROMOLECULAR CRYSTALLOGRAPHY OR WHO WISH TO INCREASE THEIR IN- DEPTH KNOWLEDGE OF MACROMOLECULAR CRYSTALLOGRAPHY. ALUMNI OF THE COURSE, INCLUDING TWO ALUMNI WHO WENT ON TO WIN NOBEL PRIZES, HAVE GONE ON TO MAKE MAJOR CONTRIBUTIONS IN THEIR FIELDS. CRYO-ELECTRON MICROSCOPY (CRYO-EM) IS AN EMERGING TECHNIQUE IN STRUCTURAL BIOLOGY WHERE THE BIOLOGICAL SAMPLE OF INTEREST IS PREPARED UNDER CRYOGENIC (DEEP-FREEZE) CONDITIONS. THE UTILITY OF CRYO-EM STEMS FROM THE FACT THAT IT ALLOWS THE OBSERVATION OF SPECIMENS THAT HAVE NOT BEEN STAINED OR FIXED IN ANY WAY, SHOWING THEM IN THEIR "NATIVE" ENVIRONMENT. THE RESOLUTION OF CRYO-EM MAPS IS IMPROVING STEADILY, AND IN RECENT YEARS NEAR- ATOMIC RESOLUTION OF SOME STRUCTURES HAD BEEN OBTAINED, INCLUDING THOSE OF VIRUSES, RIBOSOMES, MITOCHONDRIAL PROTEIN COMPLEXES, ION CHANNELS, AND ENZYME COMPLEXES. CSHL’S COURSE ON CRYO-ELECTRON MICROSCOPY WAS INITIATED IN 2018 AND COVERS THE THEORY, PRACTICE, AND APPLICATION OF SINGLE PARTICLE CRYO-EM. PARTICIPANTS IN THE COURSE WILL LEARN TO PERFORM ALL STEPS INVOLVED IN SOLVING HIGH RESOLUTION CRYO-EM STRUCTURES, INCLUDING SAMPLE PREPARATION, MICROSCOPE ALIGNMENT, DATA COLLECTION, IMAGE PROCESSING, AND MODEL BUILDING. BOTH COURSES WILL TEACH PARTICIPANTS THROUGH EXTENSIVE HANDS-ON EXPERIMENTS ALONG WITH LECTURES AND INFORMAL DISCUSSIONS ON THE THEORY BEHIND THE TECHNIQUES AND THE LATEST DEVELOPMENTS IN AUTOMATING EXPERIMENTAL AND COMPUTATIONAL PROCEDURES, FROM WORLD LEADERS IN METHODS DEVELOPMENT, TAKING ADVANTAGE OF THE OUTSTANDING LEARNING ENVIRONMENT OF THE LABORATORY’S COURSE PROGRAM. APPLICATIONS ARE OPEN TO A WIDE RANGE OF STUDENTS INCLUDING SCIENTISTS FROM UNIVERSITIES AND MEDICAL SCHOOLS, AS WELL AS PH.D. AND M.D. SCIENTISTS FROM INDUSTRY, AIMING TO ALLOW STUDENTS TO LEARN TECHNIQUES THAT CAN BE IMMEDIATELY INCORPORATED INTO THEIR OWN RESEARCH, AND ALSO ENABLING TRANSLATIONAL APPROACHES INCLUDING RATIONAL DRUG DESIGN. TRAINEES ACCEPTED INTO THE COURSE PROGRAM WILL INCLUDE ADVANCED GRADUATE STUDENTS, POSTDOCTORAL TRAINEES, SENIOR SCIENTISTS AND PRINCIPAL INVESTIGATORS; ACROSS THE PROGRAM, TRAINEES ARE DIVERSE AND BALANCED WITH RESPECT TO GENDER, WITH 70% COMING FROM US LABS, OF WHOM 15-20% ARE FROM UNDER-REPRESENTED MINORITIES.

COLLABORATIVE RESEARCH: PLANTTRANSFORM: MORPHOGENIC-BASED MECHANISMS OF MAIZE REGENERATION -INEFFICIENT METHODS FOR TRANSFORMATION AND REGENERATION OF RECALCITRANT PLANT SPECIES PREVENT WIDESPREAD APPLICATIONS OF GENOME EDITING TECHNOLOGIES FOR BOTH BASIC AND APPLIED RESEARCH IN ESTABLISHED AND EMERGING CROP SPECIES. OVERCOMING THESE LIMITATIONS IS PARTICULARLY RELEVANT IN MONOCOTYLEDONOUS CROPS, SUCH AS MAIZE, WHICH ALONE PROVIDE MOST OF THE CALORIES CONSUMED BY HUMANS. IN THIS PROJECT, MAIZE LINES EXPRESSING GENES THAT PROMOTE REGENERATION, ALSO KNOWN AS MORPHOGENIC FACTORS, WILL BE USED TO PROVIDE A THOROUGH UNDERSTANDING OF THE MOLECULAR EVENTS LEADING TO THE SUCCESSFUL FORMATION OF NEW PLANTS STARTING FROM DIFFERENTIATED TISSUE. THIS KNOWLEDGE WILL BE INSTRUMENTAL IN DEVELOPING NEW STRATEGIES FOR IMPROVING TRANSFORMATION OF MAIZE AND OTHER PLANT SPECIES, AND WILL BE INTEGRATED INTO COURSE-BASED UNDERGRADUATE RESEARCH EXPERIENCES (CURES) AS WELL AS HANDS-ON TRANSFORMATION WORKSHOPS. THE PROPOSED RESEARCH WILL EXPLOIT A MORPHOGENIC-BASED SYSTEM CALLED ?GGB? TO UNDERSTAND HOW CERTAIN MORPHOGENIC REGULATORS REPROGRAM SOMATIC CELLS TO DEVELOP INTO EMBRYOS AND IDENTIFY KEY REGENERATION GENES THAT COULD BE TARGETED TO IMPROVE TRANSFORMATION EFFICIENCY IN RECALCITRANT GENETIC BACKGROUNDS. THIS WILL BE ACCOMPLISHED BY THE IDENTIFICATION OF DIRECT TARGETS OF REGULATION OF THE GGB COMPONENTS VIA SINGLE-CELL TRANSCRIPTOMIC AND DNA-BINDING APPROACHES, AND BY THE DEVELOPMENT OF A DIVERSE PANEL OF MAIZE INBRED LINES EXPRESSING THE GGB MORPHOGENIC REGULATORS. BY EXPLOITING THE REGENERATIVE CAPACITY OF THIS SYSTEM, PROTOPLAST REGENERATION, A CHALLENGING BUT ADVANTAGEOUS SYSTEM FOR THE RAPID GENERATION OF NON-GMO EDITED PLANTS, WILL ALSO BE REVISITED. THIS RESEARCH WILL PROVIDE INSIGHTS INTO THE MOLECULAR BASIS OF TISSUE- AND GENOTYPE-DEPENDENT REGENERATION, HELPING TO IDENTIFY AND EVENTUALLY BYPASS ROADBLOCKS TO REGENERATION, AND WILL FACILITATE THE DEVELOPMENT OF HIGH-THROUGHPUT SYSTEMS FOR GENOME-EDITING AND TRANSGENIC LINE GENERATION IN DIVERSE GENETIC BACKGROUNDS. THIS PROJECT IS JOINTLY FUNDED BY GENETIC MECHANISMS (BIO-MCB). EMERGING FRONTIERS (BIO), AND THE PLANT GENOME RESEARCH PROGRAM (BIO-IOS). 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.

CSHL COURSE ON ADVANCED SEQUENCING TECHNOLOGIES & APPLICATIONS

CSHL PROGRAMMING FOR BIOLOGY COURSE

COLD SPRING HARBOR LABORATORY COURSE ON PROTEOMICS

COMPUTATIONAL FRAMEWORK FOR SINGLE-CELL GENOMICS OF TUMORS

STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF AUTOIMMUNE ANTIBODIES AGAINST NMDAR - PROJECT SUMMARY. THE GOAL OF THIS PROJECT IS TO UNDERSTAND THE ORIGINS AND MOLECULAR MECHANISMS UNDERLYING THE ANTI-CANCER AUTOIMMUNE RESPONSE AGAINST THE N-METHYL-D-ASPARTATE RECEPTOR (NMDAR) AND ITS CORRELATION WITH ANTI-N-METHYL-D-ASPARTATE RECEPTOR AUTOIMMUNE ENCEPHALITIS (NMDARAE). WHILE ANTI-CANCER IMMUNE RESPONSES CAN PROMOTE TUMOR ELIMINATION, THEY MAY ALSO LEAD TO THE PRODUCTION OF SELF-REACTIVE ANTIBODIES THAT TRIGGER AUTOIMMUNE DISEASES. NMDARAE IS THE MOST COMMON FORM OF IMMUNE-MEDIATED ENCEPHALITIS, WHICH RESULTS IN PROMINENT NEUROPSYCHIATRIC SYMPTOMS, INCLUDING SEIZURES, PSYCHOSIS, AND MEMORY DEFICITS. NMDARS BELONG TO A FAMILY OF LIGAND-GATED ION CHANNELS EXPRESSED EXCLUSIVELY IN THE CENTRAL NERVOUS SYSTEM. THEY ARE INVOLVED IN VARIOUS ASPECTS OF BRAIN DEVELOPMENT AND FUNCTION, INCLUDING LEARNING AND MEMORY. THEY RESPOND TO THE NEUROTRANSMITTER GLUTAMATE AND A CO-AGONIST, GLYCINE OR D-SERINE, TO MEDIATE EXCITATORY NEUROTRANSMISSION, WHICH PLAYS A CENTRAL ROLE IN SYNAPTIC PLASTICITY. NMDARAE IS ASSOCIATED WITH OVARIAN TERATOMAS, WHERE ABERRANT NMDAR EXPRESSION IS BELIEVED TO TRIGGER AN AUTOIMMUNE RESPONSE. IN NMDARAE, ANTI-NMDAR ANTIBODIES, AS WELL AS B CELLS AND ANTIBODY-SECRETING CELLS, CROSS THE BLOOD-BRAIN BARRIER VIA UNKNOWN MECHANISMS, RESULTING IN THE PRESENCE OF ANTI-NMDAR ANTIBODIES AT HIGH TITERS WITHIN THE BRAIN AND CEREBROSPINAL FLUID (CSF). THESE ANTIBODIES TARGET NMDARS, MODULATING THEIR FUNCTION AND CONTRIBUTING TO DISEASE PATHOLOGY. EMERGING EVIDENCE, SUPPORTED BY OUR PRELIMINARY DATA, SUGGESTS THAT NMDARS ARE ALSO EXPRESSED IN TRIPLE-NEGATIVE BREAST CANCER (TNBC), EXTENDING THE RELEVANCE OF ANTI-NMDAR AUTOIMMUNITY BEYOND OVARIAN TERATOMAS. IN OUR TNBC MOUSE MODEL, WHICH ECTOPICALLY EXPRESSES NMDARS (TNBC-NMDAR), WE OBSERVED THE ONSET OF ANTI-NMDAR AUTOIMMUNITY, WHERE THE PRODUCED ANTIBODIES CAUSE BOTH ANTI-TUMOR ACTIVITY AND SYMPTOMS SUCH AS LOWERED SEIZURE THRESHOLD, MIRRORING KEY FEATURES OF NMDARAE. HERE, WE WILL ESTABLISH THIS TNBC MOUSE MODEL AS WE DEVELOP MOLECULAR METHODS TO CHARACTERIZE IT. AIM 1 WILL FOCUS ON ESTABLISHING AND CHARACTERIZING THE TNBC- NMDAR MOUSE MODEL. WE WILL DEVELOP A DETECTION METHOD UTILIZING THE INTACT TETRAMERIC NMDAR CHANNEL PROTEINS AND A METHOD TO ISOLATE B CELLS EXPRESSING B CELL RECEPTORS AGAINST NMDAR FROM BIOLOGICAL SAMPLES BY USING FLUORESCENTLY LABELED INTACT NMDAR PROTEINS, FOLLOWED BY SINGLE-CELL RNA SEQUENCING. AIM 2 WILL UTILIZE SINGLE-PARTICLE CRYO-ELECTRON MICROSCOPY (CRYO-EM) TO INVESTIGATE THE INTERACTIONS BETWEEN NMDAR AND THE CLONED ANTIBODIES, PROVIDING INSIGHTS INTO EPITOPE RECOGNITION, NMDAR SUBTYPE SPECIFICITY, AND CONFORMATIONAL CHANGES INDUCED BY ANTIBODY BINDING. AIM 3 WILL ASSESS THE IMPACT OF THE CLONED ANTIBODIES ON NMDAR CHANNEL ACTIVITY USING ELECTROPHYSIOLOGY. WE WILL ALSO ASSESS ANTI-TUMOR ACTIVITY AND NMDARAE ONSET BY EACH ANTIBODY CLONE. TOGETHER, THE PROPOSED RESEARCH WILL GAIN INSIGHTS INTO THE LINK BETWEEN ANTI-CANCER ANTI-NMDAR AUTOIMMUNITY AND NMDARAE. IT WILL ALSO ELUCIDATE WHICH FUNCTIONAL PROPERTIES OF THE CLONED ANTIBODIES PROMOTE ANTI-TUMOR ACTIVITY WHILE CONTRIBUTING TO NMDARAE, THEREBY INFORMING POTENTIAL THERAPEUTIC STRATEGIES.

VARIATION IN SMALL RNA PATHWAYS ACROSS MAIZE TISSUES AND INBREDS

A FRAMEWORK FOR ANALYZING CONVERGING FEEDFORWARD AND CORTICAL-BULBAR FEEDBACK DYNAMICS IN TARGET DETECTION FROM COMPLEX ODOR SCENES