The Wistar Institute Of Anatomy And Biology

CONSOLIDATED BASIC CANCER RESEARCH PROGRAM

TARGETED THERAPIES IN MELANOMA

BEAT-HIV: DELANEY COLLABORATORY TO CURE HIV-1 INFECTION BY COMBINATION IMMUNOTHERAPY - SUMMARY CURRENT HIV CURATIVE STRATEGIES HAVE PROVEN INSUFFICIENT TO ERADICATE VIRAL RESERVOIRS OR PREVENT VIRAL REBOUND AFTER ANTIRETROVIRAL THERAPY (ART) CESSATION. THE UNIFYING HYPOTHESIS FOR THE BEAT-HIV COLLABORATORY IS THAT THROUGH A BETTER MECHANISTIC UNDERSTANDING OF HIV LATENT RESERVOIRS AND HOST FACTORS GOVERNING VIRAL CONTROL AND REACTIVATION, LONG-TERM VIRAL REMISSION OR ERADICATION OF HIV WILL BE ACHIEVED BY COMBINATION IMMUNOTHERAPY INCLUSIVE OF BNABS, ADOPTIVELY TRANSFERRED IMMUNE CELLS, AND NANOPARTICLE THERAPIES. WE WILL TEST THIS HYPOTHESIS BY PURSUING THREE HIGHLY INTERCONNECTED RESEARCH FOCUS AREAS. THE FIRST AIM WILL SEEK TO UNDERSTAND EPIGENETIC STATUS OF INTACT PROVIRUSES, EXTRINSIC/INTRINSIC FACTORS AFFECTING PROVIRAL REACTIVATION AND EXPRESSION, AND NOVEL HOST MECHANISMS FOR POST-TREATMENT CONTROL OF HIV. THE SECOND AIM WILL DEVELOP STRATEGIES FOR LONG-TERM CONTROL IN THE ABSENCE OF ART BY USE OF DNA-DELIVERED ANTI-HIV BNABS AND ECD4IG IN COMBINATION WITH OPTIMIZED TISSUE-BASED CD8 T-CELL- AND NK CELL-MEDIATED RESPONSES. THE THIRD AIM WILL DEVELOP A COMBINATION NANOTHERAPY AND IMMUNOTHERAPY STRATEGY TO ERADICATE VIRAL RESERVOIRS. ALL AIMS WILL BE SUPPORTED BY A CLINICAL BIOREPOSITORY (BLOOD AND TISSUE), CD34+ OR PATIENT-DERIVED XENOGRAFT HUMANIZED MICE, NON-HUMAN PRIMATE (NHP) MODELS, AND A CLINICAL TRIAL DEVELOPMENT GROUP AS A LINK TO THE ACTG. COMMUNITY ENGAGEMENT WILL ADVANCE EDUCATION AND A SOCIO-BEHAVIORAL SCIENCES AND ETHICS FOCUS BY LEVERAGING A >25-YEAR RELATIONSHIP WITH THE LOCAL HIV COMMUNITY THEREBY ENSURING PARTNERSHIP WITH STAKEHOLDERS. CENTRAL ADMINISTRATION OF RESOURCES WILL ENSURE ACHIEVEMENT OF HIGH IMPACT MILESTONES, STUDY TEAM COMMUNICATIONS, AND YEARLY GOAL- ORIENTED RESOURCE ALLOCATION AND/OR REDISTRIBUTION AS INFORMED BY ADVANCES IN THE FIELD. AS AN ESTABLISHED COLLABORATORY, WE BRING TOGETHER DIVERSE EXPERTISE, INNOVATION, AND INDUSTRY PARTNERS TO DEVELOP AND TEST NOVEL STRATEGIES TO ADVANCE AN HIV CURE AND/OR DURABLE VIRAL CONTROL IN THE ABSENCE OF ART UNDER A SINGLE COMMON MULTI-INVESTIGATOR, MULTI-INDUSTRY TEAM. STUDIES WITHIN THE THREE INTERCONNECTED AIMS TOGETHER WITH A STRONG COMMUNITY ENGAGEMENT PLAN WILL LAY THE GROUNDWORK FOR FUTURE CLINICAL TRIALS THAT WILL INTEGRATE NEW KNOWLEDGE GAINED BY THE BEAT HIV-1 COLLABORATORY TO ERADICATE OR FUNCTIONALLY CURE HIV INFECTION.

BEAT-HIV: DELANEY COLLABORATORY TO CURE HIV-1 INFECTION BY COMBINATION IMMUNOTHERAPY

SYNTHETIC DNA-LAUNCHED AND ADJUVANTED ENV IMMUNOGENS FOR HIV - OVERALL SUMMARY A PROTECTIVE VACCINE FOR HIV IS ARGUABLY THE MOST IMPORTANT PREVENTION STRATEGY TO END THE GLOBAL HIV PANDEMIC. THE RV144 TRIAL DEMONSTRATED A CORRELATION BETWEEN PROTECTION AND ENVELOPE (ENV) SPECIFIC SERUM IGG ANTIBODIES. HOWEVER, THERE WAS A NOTABLE LACK OF HIV-SPECIFIC NEUTRALIZING ANTIBODIES (NABS) AND T CELL RESPONSES. THE DNA PLATFORM HAS SEVERAL ADVANTAGES, IT IS (1) SIMPLE TO DESIGN, (2) CAN CO-DELIVER MOLECULAR ADJUVANTS, (3) CAN DELIVER COMPLEX STRUCTURAL ANTIGENS, AND (4) IS SAFE AND WELL TOLERATED, EVEN AFTER NUMEROUS BOOSTS. IN OUR PREVIOUS IPCAVD PROGRAM WE DEVELOPED DNA-LAUNCHED NANOPARTICLE (DLNP) VACCINES TARGETING ENV WHICH INDUCED POTENT AND NEUTRALIZING IMMUNITY IN VIVO. WE HAVE ALSO DEVELOPED DNA-LAUNCHED NATIVE-LIKE TRIMER (DL-NLT) IMMUNOGENS INCLUDING THE CLINICALLY RELEVANT BG505 MD39 TRIMER (XU ET AL. NAT. COMM 2022). PROTEIN NANOPARTICLES ARE DIFFICULT TO MANUFACTURE AND POORLY STIMULATE CD8+ T CELLS. WE REPORTED THAT IN VIVO ASSEMBLED NANOPARTICLES DRIVE EXTREMELY RAPID AND STRONG B AND T CELL IMMUNITY. FUNCTIONAL ANTIBODY RESPONSES REQUIRE HELP FROM GERMINAL CENTER (GC) FOLLICULAR HELPER T CELLS (TFH). IN PREVIOUS CLINICAL TRIALS WITH HIV ANTIGENS, WE OBSERVED INCREASED B AND T CELL RESPONSES IN THE PRESENCE OF PLASMID-ENCODED IL-12 (PIL-12). WORK FROM MEMBERS OF THIS TEAM HAS DEMONSTRATED THAT CO-DELIVERY OF ADENOSINE DEAMINASE (ADA-1) AND PLASMID-ENCODED IL-21 (PIL-21) CAN ENHANCE ANTIBODY INDUCTION IN MICE IN A GC-DEPENDENT FASHION. HERE, WE COMBINE THE DLNP AND DL-NLT PLATFORMS TO GENERATE DNLPS BEARING STABILIZED NLTS FOCUSING IMMUNE RESPONSES ON APEX AND CD4BS B CELL LINEAGE-TARGETING ENVS (DNLP-ACE). WE WILL CHARACTERIZE HUMORAL AND CELLULAR IMMUNITY TO THESE CONSTRUCTS IN THE PRESENCE OF MOLECULAR IL-12 WITH AND WITHOUT ADDITIONAL GC-TARGETING ADJUVANTS, IN MOUSE AND NON-HUMAN PRIMATE VACCINATION STUDIES. THE OVERARCHING HYPOTHESIS OF THIS PROJECT IS THAT DLNP-ACE IMMUNOGEN TECHNOLOGY COMBINED WITH THE CO-DELIVERY OF GENETIC ADJUVANTS IS A NOVEL APPROACH FOR THE DEVELOPMENT OF HIV-1 VACCINES THAT PROMOTES ROBUST, DURABLE, BROAD, AND NEUTRALIZING ANTIBODY RESPONSES, AND SUPPORTS EFFECTOR T CELL FUNCTION IN VIVO A KEY INNOVATION OF THIS PROJECT WILL BE TO INTEGRATE THE SELECTED DNLP-ACE HIV VACCINE CONSTRUCTS IN A DUAL EXPRESSING PLASMID CONSTRUCT WITH NEXT GENERATION NONINVASIVE INTRADERMAL SKIN ELECTROPORATION (EP) DEVICES WHICH PROMOTE FUNCTIONAL CTL AND HUMORAL IMMUNE RESPONSES. THE CLINICAL DEVELOPMENT PLAN FOR THIS IPCAVD IS DIRECTED BY A LEADING HIV VACCINE DEVELOPMENT ORGANIZATION, INOVIO PHARMACEUTICALS (INO), WHICH HAS EXPERTISE IN DEVELOPMENT OF SYNTHETIC DNA VACCINES AND IN VIVO ELECTROPORATION DELIVERY. INOVIO WILL OVERSEE CLINICAL GRADE PRODUCTION OF 2 PLASMID CONSTRUCTS AT A STATE- OF-THE-ART CGMP PLASMID MANUFACTURING FACILITY. INOVIO’S PROCESSES HAVE PASSED RIGOROUS INTERNATIONAL REGULATORY REVIEWS AND HAVE BEEN USED IN DOZENS OF HUMAN CLINICAL TRIALS IN THE U.S., EUROPE AND ASIA, INCLUDING MULTIPLE PHASE II AND IN PHASE III STUDIES.

SYNTHETIC DNA & NOVEL ENV VACCINE FOR HIV

HIV-1 VACCINE BASED ON CHIMP SEROTYPES OF ADENOVIRUS

NOVEL MOLECULAR THERAPIES OF PROSTATE CANCER

IMMUNE RESPONSES TO AAV-MEDIATED FIX GENE TRANSFER

RATIONAL APPROACHES TO MELANOMA THERAPY

SPORE IN SKIN CANCER - PROJECT SUMMARY – OVERALL THIS WISTAR/UPENN SKIN SPORE REPRESENTS A HIGHLY SUCCESSFUL AND LONGSTANDING COLLABORATION. IMMUNE CHECKPOINT INHIBITION HAS REVOLUTIONIZED MELANOMA THERAPY TO THE POINT WHERE EVERY HIGH-RISK MELANOMA PATIENT WILL BE TREATED AT SOME POINT WITH THESE AGENTS. HOWEVER, MANY MAJOR QUESTIONS REMAIN ON HOW BEST TO USE THESE IMMUNE THERAPEUTICS. PROJECT 1 WILL ADDRESS THE UNMET NEED TO FIND AN EFFECTIVE BIOMARKER TO SELECT PATIENTS FOR SINGLE AGENT VERSUS COMBINATION IMMUNOTHERAPY. MANY PATIENTS START TREATMENT WITH IPILIMUMAB AND NIVOLUMAB, WHEN THEY MAY HAVE RESPONDED TO ANTI-PD-1 ANTIBODY (AB) ALONE, EXPOSING THESE PATIENTS UNNECESSARILY TO THE TOXICITY OF COMBINATION CHECKPOINT INHIBITION. PROJECT 1 BUILDS ON A FUNDAMENTAL DISCOVERY MADE THROUGH OUR DEVELOPMENTAL RESEARCH PROGRAM (DRP) THAT EXOSOMAL PD-L1 IS AN IMMUNOSUPPRESSIVE FACTOR SECRETED BY MELANOMAS. WE PROPOSE RIGOROUS CLINICAL UTILITY STUDIES DESIGNED TO DEMONSTRATE THIS BLOOD- BASED MEASUREMENT AS A HIGHLY SENSITIVE AND SPECIFIC PREDICTIVE BIOMARKER FOR ANTI-PD-1 ANTIBODY (AB)-BASED THERAPY. PROJECT 2 WILL ADDRESS A SECOND UNMET NEED FOR A SAFER AND EFFECTIVE COMBINATION REGIMEN THAT PROMISES TO BE EFFECTIVE IN ANTI-PD-1 AB REFRACTORY PATIENTS. BASED ON EXTENSIVE PRECLINICAL DATA AND A NEW MOLECULAR TARGET IN THE AUTOPHAGY PATHWAY, WE HAVE DEVELOPED A CLINICAL TRIAL OF COMBINED ANTI-PD1 AB AND AUTOPHAGY INHIBITION, A NEW STRATEGY FOR REPROGRAMMING TUMOR-ASSOCIATED MACROPHAGES TO ENHANCE THE EFFICACY OF T CELL KILLING. PROJECT 3 FILLS A MAJOR GAP IN THE TREATMENT OF EARLY DISEASE BY CONDUCTING A CLINICAL TRIAL WITH ANTI-PD1 AB IN STAGE IIB/C MELANOMA PATIENTS. BESIDES IN-DEPTH CHARACTERIZATION OF THE IMMUNE RESPONSE, THE PROJECT’S PRECLINICAL STUDIES WILL LEAD TO NEW STRATEGIES FOR ENHANCING THE IMMUNE STIMULATORY CAPACITY OF DENDRITIC CELLS IN THE TUMOR MICROENVIRONMENT. THESE THREE HIGHLY TRANSLATIONAL PROJECTS ARE SUPPORTED BY LONGSTANDING CORES THAT HAVE A PROVEN TRACK RECORD OF ADAPTING TO THE RAPIDLY CHANGING NEEDS OF MELANOMA AND NON-MELANOMA SKIN CANCER RESEARCHERS. EACH PROJECT WAS CHOSEN BY THE CURRENT SPORE LEADERSHIP FOR ITS POTENTIAL FOR SIGNIFICANCE, IMPACT AND INNOVATION. TOGETHER, THEY HAVE THE POTENTIAL TO ADVANCE THERAPEUTICALLY EXPLOITABLE BIOLOGICAL INSIGHTS INTO NEW, CLINICALLY IMPORTANT THERAPIES OF PATIENTS WITH MELANOMA. FUNDING FROM THE SPORE HAS PROVIDED US WITH IMPORTANT ADVANTAGES, INCLUDING A MATURE, COLLECTIVE, TRANSLATIONAL MINDSET, AN EFFICIENTLY FUNCTIONING TUMOR BANK, AND A HIGHLY EVOLVED FRAMEWORK OF COLLABORATION BETWEEN THE WISTAR INSTITUTE AND UPENN. THE SPORE HAS ALLOWED US TO BOLSTER HORIZONTAL AND VERTICAL COLLABORATIONS WITH ACADEMIC AND INDUSTRY PARTNERS THROUGHOUT THE WORLD. THE CAREER ENHANCEMENT PROGRAM AND DRP HAVE ENABLED TRANSITION TO NEW LEADERSHIP, HAVE FORMED THE THREE PROJECTS PROPOSED, AND HAVE ALLOWED OUR RESEARCH TO REACH INTO OTHER CANCERS OF THE SKIN INCLUDING SCC, CTCL AND MERKEL CELL CARCINOMA. THESE PROGRAMS WILL CONTINUE TO BE SUPPORTED ROBUSTLY BY STRONG INSTITUTIONAL SUPPORT FROM BOTH WISTAR AND UPENN. FUNDING OF THIS SPORE WILL BRING NEW ADVANCES FROM THE BENCH TO THE BEDSIDE AND FULFILL OUR OVERALL MISSION OF IMPROVING SURVIVAL FOR SKIN CANCER PATIENTS.

SPORE IN SKIN CANCER

TRAINING PROGRAM IN BASIC CANCER RESEARCH

HUMAN MELANOMA - ETIOLOGY, PROGRESSION AND THERAPY

TARGETING THE EPIGENETIC AND METABOLIC CONTROL OF EBV-EPITHELIAL CANCERS - OVERALL – PROJECT SUMMARY THE OVERALL AIM OF THIS NEW PROGRAM PROJECT (P01) PROPOSAL IS TO GENERATE HIGHLY COLLABORATIVE AND INTEGRATED BASIC AND TRANSLATIONAL RESEARCH ON THE URGENT, UNMET MEDICAL NEED OF EPITHELIAL CANCERS CAUSED BY EPSTEIN-BARR VIRUS (EBV). EBV LATENT INFECTION IS CAUSALLY LINKED TO OVER 200,000 NEW CANCER CASES PER YEAR. EBV EPITHELIAL CANCERS REPRESENT OVER 75% OF ALL EBV CANCERS WITH HIGHEST MORTALITY RATES AND TREATMENT FAILURES. EBV- ASSOCIATED GASTRIC CARCINOMA (EBVAGC) AND NASOPHARYNGEAL CARCINOMA (NPC) HAVE MANY SIMILARITIES WITH RESPECT TO VIRAL LATENCY AND ONCOGENIC TRANSFORMATION. THE MECHANISMS THROUGH WHICH EBV CONTRIBUTES TO THESE EPITHELIAL CANCERS REMAINS ELUSIVE, AND TO DATE, THERE ARE NO VIRAL-SPECIFIC THERAPIES THAT ARE FDA APPROVED TO TREAT CANCERS INFECTED WITH EBV. WE HAVE ASSEMBLED A TEAM OF EBV INVESTIGATORS WITH EXPERTISE IN COMPLEMENTARY ASPECTS OF TUMOR VIROLOGY AND CANCER BIOLOGY, WITH SPECIFIC AREAS OF INTEREST IN VIRAL GENETICS, EPIGENETICS, METABOLISM, DRUG DISCOVERY, AND MODELS OF EBV CARCINOGENESIS. THE PROGRAM TEAM WILL COLLABORATE IN A COORDINATED STRATEGY TO IDENTIFY KEY VIRAL AND CELLULAR VULNERABILITIES IN EBV EPITHELIAL CANCERS THAT CAN BE TARGETED FOR THERAPEUTIC INTERVENTION. THE PROGRAM WILL TEST THE CENTRAL HYPOTHESIS THAT EBV CANCERS ARISE IN THE CONTEXT OF SOMATIC MUTATIONS IN METABOLIC AND EPIGENETIC PATHWAYS THAT ALTER EBV LATENCY AND ONCOGENICITY, AND HOW THIS VIRAL-HOST CO-DEPENDENCY PROVIDES THERAPEUTIC OPPORTUNITIES. TO ACHIEVE THESE GOALS FOR THE PROGRAM WE PROPOSE THREE PROJECTS AND THREE SCIENTIFIC CORES TO ADDRESS THE FOLLOWING: (1) DETERMINE HOW EBV ESTABLISHES A LATENT AND ONCOGENIC INFECTION IN EPITHELIAL CANCER CELLS (2) DETERMINE HOW EBV LATENT INFECTION DRIVES EPIGENETIC AND METABOLIC SHIFTS INCLUDING THE FORMATION OF CPG ISLAND METHYLATOR PHENOTYPE (CIMP) TO PROMOTE EPITHELIAL CELL ONCOGENESIS. (3) LEVERAGE MECHANISTIC INSIGHTS TO DEVELOP NEW THERAPEUTIC STRATEGIES TO TREAT EBV EPITHELIAL CANCERS. THE 3 PROJECTS FOCUS BROADLY ON EBV LATENCY AND EPIGENOME (LIEBERMAN), PARP, NAD AND DNA DAMAGE METABOLISM (TEMPERA), AND DNA METHYLATION AND METHIONINE METABOLISM (GEWURZ). THE 3 SCIENTIFIC CORES SUPPORT BIOINFORMATICS, DRUG DISCOVERY, AND MODELS OF EBV CANCERS TO SUPPORT EACH OF THE 3 PROJECTS AS RESEARCH ENHANCERS. TOGETHER, THIS TEAM AND PROGRAM WILL INVESTIGATE KEY FEATURES OF EBV CANCER MECHANISMS, BUILD NEW TOOLS TO STUDY EBV CANCERS, AND DEVELOP NEW THERAPEUTIC STRATEGIES THAT ARE VIRAL-SPECIFIC AND PRECISION-BASED MEDICINE.

EFFECTS OF MU-OPIATE RECEPTOR ENGAGEMENT ON MICROBIAL TRANSLOCATION AND RESIDUAL IMMUNE ACTIVATION IN HIV-INFECTED, ART SUPPRESSED OPIOID USE DISORDER PATENTS INITIATING MEDICATION-ASSISTED TREATMENT

TUMOR PLASTICITY

TOWARDS ERADICATION: REDUCING PROVIRAL HIV DNA WITH INTERFERON-A IMMUNOTHERAPY

ANALYSIS OF MRNA POLYADENYLATION ACROSS SPECIES AND TISSUES

FUNCTIONAL ANALYSIS OF P53 POLYMORPHIC VARIANTS

STRUCTURE AND FUNCTION OF DSRNA ADENOSINE DEAMINASE

CHROMATIN REGULATION OF THE EPSTEIN-BARR VIRUS LATENCY

CONTROL OF TELOMERE HETEROCHROMATIN BY TERRA

CHROMATIN DYNAMICS OF KSHV LATENCY

CORRELATES OF PROTECTION AGAINST SIV/SHIV CHALLENGE

ROLE OF UVRAG-MEDIATED AUTOPHAGY IN TUMOR SUPPRESSION

REGULATION OF EPSTEIN-BARR VIRUS LATENCY

NOVEL DNA ENCODED MONOCLONAL ANTIBODIES (DMABS) FOR CONTROL OF ANTIMICROBIAL RESISTANT (AMR) PSEUDOMONAS AERUGINOSA INFECTION

GLYCOMIC MODULATION OF GUT MICROBIOME DURING HIV INFECTION

EFFECTS OF MU-OPIATE RECEPTOR ENGAGEMENT ON THE PERSISTENCE OF HIV-ASSOCIATED ACTIVATION AND VIRAL RESERVOIRS IN INDIVIDUALS RECEIVING MEDICATION ASSISTED TREATMENT FOR OPIOID USE DISORDER

IDENTIFICATION OF OVARIAN CANCER PLASMA BIOMARKERS

INTEGRATION OF BIOMARKER SIGNATURES FROM PERIPHERAL BLOOD FOR DIAGNOSIS, PROGNOSIS, REMISSION AND RECURRENCE OF LUNG CANCER

THE ARF TUMOR SUPPRESSOR

REGULATION OF VIRAL CHROMATIN ARCHITECTURE DURING EBV LATENCY

LIPIDS AND MYELOID CELL FUNCTION IN CANCER

EARLY INNATE/IGA ANTI-HIV/SIV RESPONSE IN EXPOSED UNINFECTED

INVESTIGATION INTO THE ACTIVATION OF MULTIPLE BNAB PRECURSORS USING STRUCTURE-DESIGNED IMMUNOGENS AND IG KNOCK-IN MICE - PROJECT SUMMARY HIV-1 VACCINES THAT CAN ELICIT BROADLY NEUTRALIZING ANTIBODIES (BNABS) ARE A PRIMARY GOAL. TO DATE, IT HAS BEEN DEMONSTRATED THAT A SINGLE BNAB LINEAGE (VRC01-CLASS) CAN BE SPECIFICALLY ACTIVATED IN HUMAN TRIALS. ANTIBODIES FROM THIS TRIAL DO NOT NEUTRALIZE HIV-1 AND HETEROLOGOUS SEQUENTIAL IMMUNIZATION IS THOUGHT TO BE REQUIRED TO DEVELOP BNABS. HETEROLOGOUS SEQUENTIAL IMMUNIZATION HAS BEEN EMPLOYED TO GENERATE BNABS IN PRE-CLINICAL MODELS. WHILE VRC01-CLASS ANTIBODIES ISOLATED FROM INFECTED INDIVIDUALS CAN BE QUITE BROAD, THE LIMIT OF BREADTH FOR VRC01-CLASS ANTIBODIES INDUCED BY THESE TYPES OF VACCINATION STRATEGIES IN PEOPLE IS UNKNOWN. THEREFORE, WE ARE DEVELOPING AN ALTERNATIVE BNAB LINEAGE TARGETING VACCINE (VH1-46 CLASS) THROUGH ADVANCED PROTEIN ENGINEERING APPROACHES AND ASSESSMENT IN NEWLY GENERATED HUMAN IMMUNOGLOBULIN KNOCK-IN MICE HARBORING VH1-46 GERMLINE ANTIBODIES. FURTHER, WE ARE PURSUING INNOVATIVE APPROACHES TO DEVELOP A DUAL BNAB LINEAGE TARGETING VACCINE WHICH COULD SYNERGIZE WITH CURRENT VRC01-CLASS VACCINES. INDIVIDUAL LINEAGE TARGETING VACCINES MAY NOT SUCCEED AT FULLY MATURATING THESE LINEAGES, THUS SEVERELY LIMITING THE NEUTRALIZATION BREADTH AND ULTIMATE EFFECTIVENESS OF THESE VACCINES. DUAL LINEAGE TARGETING MAY BE CRITICAL FOR SUCCESS OF THE FIRST BNAB- ELICITING HIV-1 VACCINE.

SPORE IN SKIN CANCER

CORRECTION OF DENDRITIC CELLS DEFECTS IN CANCER

INDIVIDUALIZED HIV CURATIVE COMBINATION STRATEGY AGAINST PERSISTENT HIV ON ART - SUMMARY WE HAVE DEMONSTRATED MULTIPLE LINES OF EVIDENCE SUPPORTING THE ROLE FOR AUTOLOGOUS NEUTRALIZING ANTIBODIES (ANABS) TO RESTRICT VIRUS REBOUND. THE POSITIVE RELATIONSHIP BETWEEN ANAB SUPPRESSION OF VIRUS REACTIVATION EX VIVO AND TIME TO REBOUND IN VIVO INDICATES THAT ANABS ARE A STRONG HOST DETERMINANT IN THE SUPPRESSION OF THE HIV VIRAL RESERVOIR. THIS OFFERS A UNIQUE OPPORTUNITY TO DEVELOP AN INDIVIDUALIZED HIV CURE STRATEGY TARGETING THE ANAB-RESISTANT COMPONENT OF THE HIV RESERVOIR THAT IS NOT CONTROLLED BY THE HOST UPON REACTIVATION. TAKING ADVANTAGE OF THE FULL COMPLEMENT OF IMMUNE RESPONSES (I.E., A COMBINATION OF INNATE, HUMORAL AND CELL-MEDIATED COMPONENTS), THIS PROPOSAL SEEKS TO DEVELOP AN INDIVIDUALLY TAILORED STRATEGY THAT WILL BRING TOGETHER A) NEUTRALIZING ANTIBODY RESPONSES ENHANCED VIA MRNA-LIPID NANOPARTICLE (LNP) TECHNOLOGY, B) ANAB-RESISTANT HIV ENV HLA-E RESTRICTED ADAPTIVE NK CELLS OR CREATION OF MULTI-SPECIFIC MOLECULES TARGETING THE HIV LOADED HLA-E MHC COMPLEX, AND C) ENGINEERED CELL-MEDIATED EFFECTOR STRATEGIES, CONSISTING OF MULTIVALENT CAR T CELLS AND CD64-TRANSDUCED NK CELLS WITH A MEMBRANE-BOUND COMBINATIONS OF BROADLY NEUTRALIZING ANTIBODIES OPTIMIZED AGAINST THE ANAB-RESISTANT RESERVOIR OF EACH PARTICIPANT. OUR PRELIMINARY DATA INDICATES THAT EACH OF THE STRATEGIES PROPOSED HAS POTENTIAL FOR ANTIVIRAL CONTROL, SUPPORTING THEIR INCLUSION IN THE COMBINED APPROACH PROPOSED. OUR CENTRAL HYPOTHESIS IS THAT CHARACTERIZATION OF THE ANAB-RESISTANT HIV RESERVOIR WILL PERMIT DEVELOPMENT OF A TARGETED, PERSONALIZED COMBINATION STRATEGY OF ANTIVIRAL INNATE, HUMORAL AND CELL-MEDIATED RESPONSES, WHICH TOGETHER WILL ELICIT SUSTAINED VIRAL CONTROL AND/OR VIRAL ERADICATION. TO TEST THIS HYPOTHESIS, WE WILL FIRST CONDUCT COMPREHENSIVE RESERVOIR SCREENS IN PERSONS WITH HIV SUPPRESSED ON ART AFTER EARLY OR LATE ART INITIATION (TO EVALUATE STRATEGIES AGAINST DISTINCT LEVELS OF RESERVOIR DIVERSITY) TO IDENTIFY SEQUENCES OF ANAB-RESISTANT RESERVOIR VIRUSES WHICH WILL BE USED TO GENERATE INDIVIDUAL TAILORED ANABS (INDUCED BY MRNA-LNP-BASED VACCINATION) AND, CONCURRENTLY, TO IDENTIFY THE BEST COMBINATION OF EXISTING BROADLY NEUTRALIZING ANTIBODIES (BNABS) THAT CAN ACHIEVE VIRUS CONTROL. SECOND, WE WILL BUILD PERSONALIZED CELL-MEDIATED RESPONSES TO CLEAR INFECTED CELLS BY (I) HLA-E RESPONSIVE ENV PEPTIDES FOR ADAPTIVE NK CELL GENERATION, (II) PHAGE DISPLAY TARGETING FOR BI/TRI-SPECIFIC HLA-E- ENV PEPTIDE COMPLEX ENGAGERS, AND (III) INDIVIDUALLY DEFINED BNAB COMBINATIONS LOADED ON TO CD64-NK OR EXPRESSED AS TRI-BNAB CONSTRUCTS ON CAR T CELLS. TO FACILITATE FUTURE CLINICAL DEPLOYMENT OF THIS COMBINED IMMUNE STRATEGY, WE WILL DEVELOP PERSONALIZED VIRUS REACTIVATION STRATEGIES, WHEREIN INDIVIDUAL’S CD4+ CELLS BEARING INTEGRATED HIV WILL BE CHARACTERIZED TO DESIGN PERSONALIZED LRA APPROACHES OPTIMIZED TO MAXIMIZE HIV REACTIVATION. TOGETHER WITH SIGNIFICANT INSTITUTIONAL AND INDUSTRY COMMITMENT, OUR PROPOSAL BRINGS TOGETHER COLLABORATIVE GROUPS FROM ACCELEVIR, MERCK, ACUITAS THERAPEUTICS, BIONTECH, BLUEWHALE BIO, CYTOIMMUNE THERAPEUTICS, GEORGE WASHINGTON UNIV., UNIV. OF NEBRASKA, RAGON INSTITUTE, MASSACHUSETTS INSTITUTE OF TECHNOLOGY, DUKE UNIV., PHILADELPHIA FIGHT JOHNS HOPKINS UNIV., UNIV. OF PENNSYLVANIA, AND WISTAR INSTITUTE.

STRUCTURAL VACCINOLOGY GUIDED DEVELOPMENT OF A UNIVERSAL COV VACCINE UTILIZING NUCLEIC ACID DELIVERED NANOPARTICLES - PROJECT SUMMARY IN DECEMBER 2019 A NOVEL CORONAVIRUS, NAMED SUDDEN ACUTE RESPIRATORY SYNDROME CORONAVIRUS-2 (SARS-COV- 2). SARS-COV-2 RAPIDLY SPREAD AROUND THE GLOBE CAUSING A PANDEMIC DISEASE TERMED CORONAVIRUS DISEASE OF 2019 (COVID-19). THERE HAVE BEEN MORE THAN 80 MILLION INFECTIONS AND CLOSE TO TWO MILLION DEATHS FROM COVID- 19 TO DATE. SARS-COV-2 IS THE THIRD BETA CORONAVIRUS OF ZOONOTIC ORIGIN TO CAUSE HUMAN EPIDEMICS. IT IS SIMILAR TO, BUT DISTINCT FROM, MIDDLE EAST RESPIRATORY SYNDROME CORONAVIRUS (MERS-COV) AND SUDDEN ACUTE RESPIRATORY SYNDROME VIRUS-1 (SARS-COV-1), BOTH OF WHICH HAVE CAUSED OUTBREAKS THIS CENTURY. WHILE SEVERAL CANDIDATE VACCINES FOR SARS-COV-2 HAVE RECENTLY RECEIVED EMERGENCY USE AUTHORIZATION, THE LONGEVITY OF VACCINE-INDUCED RESPONSES, THE CONTINUED EMERGENCY OF MUTATION WITHIN SARS-COV-2 STRAINS, AND THE DISPROPORTIONATE MORBIDITY AND MORTALITY AMONG ELDERLY PATIENT POPULATIONS PRESENT CONTINUED CHALLENGES TO CONTROL OF SARS-COV-2. THUS, VACCINE MODALITIES WHICH CAN ADDRESS THESE CHALLENGES FOR SARS-COV-2 VACCINES AND ALLOW FOR TARGETING OF MULTIPLE POTENTIALLY PANDEMIC CORONAVIRUSES SIMULTANEOUSLY ARE GREATLY NEEDED. INNOVATIVE VACCINES WHICH CAN DEVELOP BROAD IMMUNITY AGAINST KNOWN AND NEWLY EMERGENT HUMAN CORONAVIRUS IS A KEY GOAL IN THE FIELD. THE EFFECTS OF ANTIGEN EPITOPE DIVERSITY, DENSITY, VALENCY, DURATION OF ANTIGEN AVAILABILITY, AND ADJUVANT- INDUCED CYTOKINE ENVIRONMENT ON THE POTENCY AND BREADTH OF VACCINE-INDUCED REPONSES REMAINS UNCLEAR. NANOPARTICLE VACCINE FORMULATIONS ALLOW THE ABILITY TO MANIPULATE THESE VARIABLES. WE HAVE GENERATED SELF- ASSEMBLING SYNTHETIC DNA-LAUNCHED NANOPARTICLE VACCINES (DLNPS) WHICH DISPLAYED INCREASED IMMUNOGENICITY COMPARED TO MATCHED SYNTHETIC DNA LAUNCHED MONOMER VACCINES OR PROTEIN-IN-ADJUVANT FORMULATIONS. WE DETERMINED THAT SYNTHETIC DNA LAUNCHED NANOPARTICLES INCREASED BOTH CELLULAR AND HUMORAL RESPONSES. RECOMBINANT NANOPARTICLE VACCINES ARE THOUGHT TO MEDIATE THEIR INCREASED IMMUNOGENICITY BY PERSISTING IN THE LYMPH NODES FOR EXTENDED PERIODS COMPARED TO PROTEIN ANTIGENS, PROMOTING ENHANCED ANTIGEN PRESENTATION BY FOLLICULAR DENDRITIC CELLS AND INCREASING GERMINAL CENTER FORMATION AND HUMORAL IMMUNITY. CELL-MEDIATED RESPONSES TO NANOPARTICLE VACCINES ARE LESS WELL UNDERSTOOD BUT SIMILAR MECHANISMS MAY BE AT PLAY. WE WILL CAPITALIZE ON THE NOVEL IN VIVO ASSEMBLING SYNDLNP PLATFORM WE HAVE CREATED TO MANIPULATE THESE VARIABLES AND DETERMINE THEIR EFFECTS ON ACUTE AND LONG-TERM RESPONSES TO COV ANTIGENS IN YOUNG AND AGED MODELS.

THERAPEUTIC TARGETING MDSC-MEDIATED IMMUNE SUPPRESSION IN CANCER - PROJECT SUMMARY THE PROMINENT CHANGE IN THE MYELOID COMPARTMENT IN CANCER IS THE EXPANSION OF PATHOLOGICALLY ACTIVATED IMMATURE MYELOID CELLS WITH THE POTENT ABILITY TO SUPPRESS IMMUNE RESPONSES – MYELOID-DERIVED SUPPRESSOR CELLS (MDSC). IN TUMOR-BEARING MICE, THE TOTAL POPULATION OF MDSC CONSISTS OF THREE GROUPS OF CELLS: THE MOST ABUNDANT (>75%) IMMATURE, PATHOLOGICALLY ACTIVATED NEUTROPHILS (PMN-MDSC); LESS ABUNDANT (<20%) POPULATION OF PATHOLOGICALLY ACTIVATED MONOCYTES (M-MDSC); AND SMALL (<5%) POPULATION OF EARLY MYELOID PRECURSORS. IN THE TUMOR MICROENVIRONMENT MDSC ARE MORE IMMUNOSUPPRESSIVE THAN IN PERIPHERAL LYMPHOID ORGAN. HOWEVER, THE MECHANISM OF THIS PHENOMENON REMAINS RATHER ELUSIVE. THE GAPS IN OUR KNOWLEDGE IS IN UNDERSTANDING THE MECHANISMS REGULATING THE FUNCTION OF MDSC IN TUMORS AND SPECIFIC REQUIREMENTS FOR THEIR TARGETING. IN THIS PROPOSAL WE WILL TEST THE HYPOTHESIS THAT THERE ARE DISTINCT POPULATIONS OF MDSC IN TUMORS. THESE POPULATIONS CAN BE DEFINED BY SPECIFIC MARKERS AND MOST IMPORTANTLY, HAVE DIFFERENT SENSITIVITY TO FERROPTOTIC CELL DEATH WHICH DETERMINES THEIR FUNCTIONAL ACTIVITY. WE WILL TEST THE CONCEPT THAT TARGETING FERROPTOSIS IN PMN-MDSC IN CANCER MAY HAVE FUNCTIONAL CONSEQUENCES FOR IMMUNE RESPONSES. THE GOAL OF THIS PROJECT IS TO UNCOVER THE MECHANISMS REGULATING MYELOID CELL FUNCTION IN TUMORS AND TO DEVELOP NOVEL APPROACHES TO THE REGULATION OF IMMUNE RESPONSES IN CANCER. WE PROPOSE THE FOLLOWING SPECIFIC AIMS: (1) TO IDENTIFY THE MECHANISM OF FERROPTOSIS-MEDIATED IMMUNE SUPPRESSION INDUCED BY PMN-MDSC IN TUMORS; AND (2) TO INVESTIGATE THERAPEUTIC POTENTIAL OF TARGETING FERROPTOSIS IN PMN-MDSC.

HOST GLYCOMIC MODULATION OF HIV-ASSOCIATED NEURO-INFLAMMATION DURING VIRAL SUPPRESSION

CONTROL OF APOPTOSIS IN CANCER BY SURVIVIN

NK CELL ACTIVATION AND FUNCTION IN HIV-1 EXPOSED UNINFECTED IV DRUG USERS

DRUGGING EBNA1 TO TREAT EBV-ASSOCIATED CANCERS - PROJECT SUMMARY EBV LATENT INFECTION IS RESPONSIBLE FOR ~200,000 NEW CANCERS PER YEAR. TO DATE, THERE ARE NO EBV- SPECIFIC THERAPEUTIC AGENTS THAT SELECTIVELY AND EFFICACIOUSLY TREAT EBV-POSITIVE TUMORS. ALL KNOWN EBV TUMORS CONSISTENTLY EXPRESS ONE VIRAL NUCLEAR PROTEIN, EBNA1, THAT IS REQUIRED FOR MAINTAINING THE EBV GENOME AND PROMOTING INFECTED CELL SURVIVAL. WE HAVE DEVELOPED HIGHLY SELECTIVE, DRUG-LIKE SMALL MOLECULES THAT BIND EBNA1 AND BLOCK ITS ABILITY TO BIND DNA, MAINTAIN EBV GENOMES, AND PROMOTE HOST-CELL SURVIVAL. HERE WE PROPOSE TO BETTER UNDERSTAND THE MECHANISM THROUGH WHICH DISRUPTION OF EBNA1 DNA BINDING LEADS TO TUMOR GROWTH INHIBITION, AND USE THIS INFORMATION TO IDENTIFY RATIONAL COMBINATORIAL AGENTS TO ENHANCE CHEMOTHERAPEUTIC EFFICACY. WE PROPOSE TO ENHANCE THE POTENCY OF THE FIRST GENERATION EBNA1 INHIBITORS BY ATTACHING PROTEASOME TARGETING MOLECULES (PROTACS) TO SELECTIVELY TARGET EBNA1 FOR DEGRADATION. FINALLY, WE WILL TAKE ADVANTAGE OF NEW MECHANISTIC DATA REVEALING THAT EBNA1 FUNCTIONS AS AN ORIP-SPECIFIC ENDONUCLEASE AND RESOLVASE. WE PROPOSE TO DEVELOP NEW STRUCTURE AND MECHANISM-BASED INHIBITORS OF EBNA1 THAT CAN INCREASE POTENCY NECESSARY FOR HIGHLY EFFICACIOUS CANCER THERAPY. BY INTEGRATING THESE STRATEGIES TO UNDERSTAND THE GROWTH ARREST RESPONSE OF EBNA1 INHIBITION (AIM 1) TO BETTER DEVELOP RATIONAL APPROACHES FOR COMBINATORIAL THERAPIES (AIM 2) AND DEVELOP NEXT GENERATION MOLECULE WITH STRUCTURE/MECHANISM BASED DRUG DESIGN PRINCIPLES (AIM 3), WE WILL ADVANCE EBNA1 INHIBITORS FOR THE TREATMENT OF EBV-ASSOCIATED MALIGNANCIES AND RELATED-DISEASES. WE WILL TEST THE OVERARCHING HYPOTHESIS THAT EBNA1 IS AN EFFECTIVE TARGET FOR SMALL MOLECULE INHIBITORS TO TREAT EBV CANCERS. THE MAJOR GOAL OF THIS PROPOSAL IS TO UNDERSTAND THE TUMOR CELL RESPONSE TO EBNA1 INHIBITION AND TO ENHANCE EFFICACY OF EBNA1 INHIBITORS TO TREAT EBV-ASSOCIATED CANCERS MORE EFFICACIOUSLY. THE TEAM ASSOCIATED WITH THIS PROPOSAL HAS THE UNIQUE EXPERTISE AND STRONG COLLABORATIVE HISTORY TO EXECUTE THE AIMS OF THIS PROPOSAL. COLLECTIVELY, THESE INVESTIGATIONS WILL PROVIDE FUNDAMENTAL INSIGHTS INTO HOW EBNA1 FUNCTIONS AT THE MOLECULAR LEVEL AND WILL LAY THE FOUNDATION FOR THE DEVELOPMENT OF NEW STRATEGIES TO TREAT EBV CANCERS.

MULTIVALENT DNA VACCINE-MEDIATED PROTECTION AGAINST TUBERCULOSIS

DEVELOPMENT OF A UNIVERSAL INFLUENZA SEASONAL VACCINE

A NEW METHODOLOGY TO DECIPHER THREE-DIMENSIONAL GENOME STRUCTURE

ROLE OF AFRICAN-CENTRIC TP53 VARIANT IN HIGHER H. PYLORI PREVALENCE IN AFRICAN AMERICANS - PROJECT SUMMARY AFRICAN AMERICANS HAVE A 6-FOLD HIGHER H. PYLORI (HP) INFECTION THAN THE NON-HISPANIC WHITE POPULATION; HOWEVER, UNDERLYING GENE(S) FOR THIS DISPARITY REMAIN ELUSIVE. WE DISCOVERED THAT A HUMAN TP53 GENE VARIANT AT AMINO ACID 47 EXISTS PREDOMINANTLY IN PEOPLE OF AFRICAN DESCENT. MACROPHAGES CONTAINING THE S47 VARIANT ARE DEFECTIVE IN FERROPTOSIS, M2 POLARIZED AND SHOW MORE PRODUCTIVE INFECTIONS BY HP AND OTHER BACTERIA. MACROPHAGE ADAPTIVE RESPONSE IS ESSENTIAL FOR ELIMINATING HP INFECTION AND IS SUPPRESSED IN CHRONIC INFECTIONS THAT LEAD TO GASTRITIS. OUR COLLABORATOR, DR. KEITH WILSON, REPORTED THE ROLE OF ARG2 ACTIVATION BY HP IN SUPPRESSING THE MACROPHAGE RESPONSE. UNBIASED WT AND S47 MACROPHAGE PROTEOMICS REVEALED MARKED DIFFERENCES IN LIVER X RECEPTOR ACTIVATION, ARGINASE II ACTIVITY, INFLAMMATION, IRON TRANSPORT AND ANTIBACTERIAL DEFENSE MACHINERY WHICH REGULATE IMMUNE RESPONSE AND DIRECTLY AFFECT OUTCOMES OF BACTERIAL INFECTIONS. ALTHOUGH P53 IS KNOWN TO REGULATE IMMUNE RESPONSE, WE ARE THE FIRST TO DISCOVER THE EXACT GENETIC VARIANT CAUSING THE DISPARITIES. WE WILL REVERSE THE HP INFECTION DISPARITIES IN AFRICAN AMERICANS WITH THE S47 SNP BY IN-DEPTH MECHANISTIC AND THERAPEUTIC STUDY. OUR HUMAN STUDIES WILL GIVE TRANSLATIONAL RELEVANCE TO THIS PROJECT. WE WILL TEST ~500 HP SEROPOSITIVE AFRICAN AMERICAN SAMPLES FOR THE PREVALENCE OF S47 AND OTHER SNPS. THEN WE WILL FOCUS ON IMPROVING MACROPHAGE RESPONSE, HP CLEARANCE AND REDUCE CHRONIC GASTRIC DYSPLASIA IN S47 MICE USING LIVER X RECEPTOR (LXR) AGONISTS. WE WILL TEST IF LXR ACTIVATION IMPROVES THESE OUTCOMES FOR MULTIPLE HP STRAINS WITH DIFFERING VIRULENCE. MOREOVER, WE WILL GENERATE A GENOME-WIDE MAP OF MACROPHAGE BINDING OF LXR TO ‘FINE TUNE’ ITS ACTIVITY ON MACROPHAGE ACTIVATION AND ELIMINATE UNDESIRED EFFECT OF HEPATIC STEATOSIS. SINCE HP IS FOUND BOTH EXTRACELLULARLY AND WITHIN MACROPHAGES, SPECIFIC DEPLETION OF IMMUNE CELLS WILL BE USED TO DISTINGUISH THE ROLE OF MACROPHAGES FROM OTHER TISSUE ENVIRONMENT IN H. PYLORI PATHOGENESIS. THESE STUDIES WILL BE VALIDATED BY BONE MARROW SWAPS BETWEEN WT AND S47 MICE. WE WILL TEST MACROPHAGE REGULATION AND TISSUE ENVIRONMENT FOR MORE. WE WILL PRIORITIZE AND TEST PATHWAYS ASSOCIATED WITH LXR SUCH AS ARGINASE PATHWAY (ARG2, SLC7A2), IRON TRANSPORT (TFRC, SLC40A1, FTH1) AND INNATE IMMUNE PROTEINS (TLR3 AND TLR7) AS NOVEL THERAPEUTIC TARGETS. LASTLY, WE WILL TEST MACROPHAGE REGULATORY FACTORS (NOTCH1 AND MTOR), ENERGY METABOLISM (SWITCHING FROM OX-PHOS TO GLYCOLYSIS BY METFORMIN) AND POLYAMINE PATHWAY (DFMO-POLYAMINE SYNTHESIS INHIBITOR) IN FINE TUNING LXR AGONIST INDUCED REVERSAL OF ANTI-INFLAMMATORY POLARIZATION IN S47 MICE, CLEARANCE OF HP INFECTION AND REDUCTION OF GASTRIC DYSPLASIA. THIS PROPOSAL WILL A) IMPROVE OUR UNDERSTANDING OF THE BIOLOGICAL MECHANISM FOR THE DISPARITY IN HP INFECTION IN AFRICAN AMERICANS AND B) PROVIDE SEVERAL THERAPEUTIC AVENUES TO IMPROVE CLEARANCE OF HP INFECTION AND GASTRITIS. THIS PROJECT IS A STEPPING STONE FOR HUMAN STUDIES IN PERSONALIZED MEDICINE TO ADDRESS THIS HEALTH DISPARITY. HP INFECTION HAS BEEN DEEMED OF CRITICAL IMPORTANCE IN THE AFRICAN AMERICAN MINORITY, BY THE NIDDK.

NK/DC CROSS-TALK AND ANTIVIRAL RESPONSE

SIALIC ACID MODULATION OF HIV-ASSOCIATED CHRONIC INFLAMMAGING

TRANSCRIPTIONAL REPROGRAMMING IN MELANOMA PLASTICITY - PROJECT SUMMARY/ABSTRACT THIS APPLICATION FOCUSES ON ADVANCED MELANOMA, A HIGHLY AGGRESSIVE TYPE OF SKIN CANCER THAT ARISES FROM THE PIGMENT-PRODUCING MELANOCYTES OF THE BODY. DESPITE RECENT ADVANCES IN MOLECULARLY TARGETED THERAPY AND IMMUNOTHERAPEUTIC AGENTS WITH IMPRESSIVE RESPONSE RATES, THERE REMAIN PATIENTS WHO EITHER DO NOT RESPOND TO SUCH THERAPIES OR WHO EVENTUALLY RELAPSE. THE 5-YEAR OVERALL SURVIVAL RATE FOR METASTATIC MELANOMA IS BELOW 20%. PROGRESSIVE DEDIFFERENTIATION AND PHENOTYPIC SWITCHING OF MELANOMA CELLS UNDER CELLULAR STRESS ARE CONSIDERED TO BE A MAJOR DRIVER FOR BOTH TUMOR PROGRESSION AND THERAPY RESISTANCE. HOWEVER, THE MOLECULAR MECHANISMS THAT GOVERN THIS PROCESS, AND THEIR INTERPLAY WITH GENETIC LESIONS AND THE TUMOR MICROENVIRONMENT ARE POORLY UNDERSTOOD. TFEB IS A MEMBER OF THE MIT/TFE FAMILY OF TRANSCRIPTION FACTORS AND MASTER REGULATORS OF CELL DIFFERENTIATION PATHWAYS. OUR PRELIMINARY STUDIES IDENTIFIED TFEB REPRESSION AS A NOVEL DEPENDENCY OF ONCOGENE-DRIVEN MELANOMA PROGRESSION. WE SHOWED THAT TFEB ACTIVATION GLOBALLY RE-INVIGORATES TRANSCRIPTIONAL DIFFERENTIATION OF MELANOCYTES, WHILE ITS INACTIVATION PROVOKES PHENOTYPIC TRANSITION TOWARDS THE INVASIVE AND DRUG-RESISTANT STATES THAT IS ASSOCIATED WITH ABERRANT TGF-B UPREGULATION. FURTHERMORE, TRANSCRIPTOMIC AND IMMUNE CELL PROFILING OF TUMORS FROM PRIMARY MELANOMA PATIENTS CONFIRMED DAMPENED TFEB EXPRESSION AND FUNCTION THAT ALSO CORRELATES WITH TUMORS’ IMMUNE EVASIVE MICROENVIRONMENT. THESE FINDINGS LEAD US TO HYPOTHESIZE THAT TFEB-MEDIATED TRANSCRIPTIONAL REPROGRAMMING OF MELANOMA CELL DIFFERENTIATION STATES REPRESENTS A KEY MECHANISM DISABLING MELANOMA PROGRESSION, WHICH ALSO RESHAPES TUMOR IMMUNE MICROENVIRONMENT FOR ENHANCED ANTI-TUMOR IMMUNITY. WE WILL TEST THE HYPOTHESIS BY (AIM 1) DEFINING THE MOLECULAR MECHANISMS OF TFEB-MEDIATED TRANSCRIPTIONAL REPROGRAMMING OF MELANOMA CELL PLASTICITY AND PHENOTYPE SWITCHING; AND BY (AIM 2) INVESTIGATING IN DETAIL THE IN VIVO IMPACT OF TFEB ALTERATION IN MELANOMA PROGRESSION AND IMMUNE EVASION. SUCCESSFUL COMPLETION OF THIS STUDY WILL PROVIDE MECHANISTIC INSIGHTS INTO TUMOR CELL-SWITCHING PROCESSES AND HOLD PROMISE FOR THE DEVELOPMENT OF NOVEL THERAPEUTIC STRATEGIES TO REVERSE THIS PROCESS FOR THE PREVENTION AND ELIMINATION OF TUMOR METASTASIS AND RECURRENCE.

MOLECULAR MECHANISM OF UV PROTECTION IN CUTANEOUS MELANOMA

MECHANISM OF DENDRITIC CELL DIFFERENTIATION IN CANCER

METABOLIC AND EPIGENETIC REPROGRAMMING IN CYCLIN E HIGH OVARIAN CANCER - PROJECT SUMMARY/ABSTRACT THE ULTIMATE GOAL OF THIS MPI PROPOSAL IS TO ADDRESS A FUNDAMENTAL GAP IN KNOWLEDGE ON THE ROLE OF ACETYL-COA METABOLIC REPROGRAMMING IN REGULATING CYCLIN E-HIGH OVARIAN CANCER DNA DAMAGE RESPONSE, TRANSFORMATION, AND RESPONSE TO THERAPY. THE RESULTS FROM THESE STUDIES COULD HAVE A SIGNIFICANT IMPACT ON THE TREATMENT OF THE ~20% OF HIGH GRADE SEROUS OVARIAN CANCER (HGSOC) PATIENTS WITH HIGH CYCLIN E EXPRESSION, WHICH ARE RESISTANT TO EMERGING PARP INHIBITOR THERAPIES DUE TO PROFICIENCY IN HOMOLOGOUS RECOMBINATION (HR)-MEDIATED DNA REPAIR. THIS RESEARCH PLAN FOCUSES ON ASSESSING THE EXPERIMENTALLY AND MECHANISTICALLY DETERMINING THE SPACIOTEMPORAL METABOLIC REPROGRAMMING OF ACETYL-COA ON HISTONE HYPERACETYLATION AND ENHANCEMENT OF HR-MEDIATED DNA REPAIR AND WHETHER THIS PATHWAY CAN BE TARGETED IN CYCLIN E-HIGH HGSOC PATIENTS IN COMBINATION WITH EMERGING PARP INHIBITOR THERAPIES TO OBTAIN A SYNTHETIC LETHALITY AND SUSTAINED THERAPEUTIC RESPONSE. THE PROPOSED STUDIES ARE BASED ON OUR PRELIMINARY FINDINGS THAT GLUCOSE-DERIVED ACETYL-COA IS UPREGULATED IN CYCLIN E-HIGH CELLS, ACETYL-COA IS SPATIALLY REGULATED IN THE CYTOPLASM AND NUCLEUS, AND CYCLIN E-HIGH CELLS DISPLAY HYPERACETYLATION OF HISTONES KNOWN TO BE INVOLVED IN HR REPAIR. IN LINE WITH THESE DATA, WE WILL EXPLORE TWO OVERARCHING SCIENTIFIC AIMS: 1) QUANTITATIVELY DISSECT ACETYL-COA METABOLIC REPROGRAMMING IN CYCLIN E-HIGH HGSOC AND ITS CONTRIBUTION TO HR-MEDIATED DNA REPAIR; AND 2) TO DETERMINE WHETHER ACETYL-COA MEDIATED EPIGENETIC CHANGES CONTRIBUTES TO OVARIAN TUMORIGENESIS AND THERAPEUTIC RESPONSE. THE COMPLETION OF THE SCIENTIFIC AIMS OF THIS PROPOSAL WILL NOT ONLY PROVIDE NEW MECHANISTIC INSIGHTS INTO THE INTERPLAY BETWEEN THE ACETYL-COA-MEDIATED METABOLIC-EPIGENETIC AXIS DURING OVARIAN TUMORIGENESIS, BUT WILL ALSO ESTABLISH TARGETING THIS AXIS AS A STRATEGY TO IMPROVE THERAPEUTIC OUTCOME FOR HGSOC PATIENTS WITH HIGH CYCLIN E. THE PROPOSED RESEARCH IS OF HIGH IMPACT BECAUSE THE MECHANISTIC UNDERPINNING OF THESE PATHWAYS HAS THE POTENTIAL TO TRANSFORM THE MANAGEMENT OF HGSOC PATIENTS WITH HIGH CYCLIN E. AS PARP INHIBITORS ARE BEING DEVELOPED FOR MANY CANCER TYPES, STUDIES WILL HAVE FAR-REACHING IMPLICATIONS FOR IDENTIFYING NOVEL STRATEGIES TO INHIBIT HR-MEDIATED DNA REPAIR AND DEVELOP FUTURE CANCER THERAPEUTICS STRATEGIES FOR A WIDE RANGE OF PATIENTS.

A CANCER-DERIVED TRUNCATING MUTATION IN DISEASE PENETRANCE AND PROGRESSION OF MSI CRC

ROLE OF AN INTEGRATOR-EGR AXIS IN THE REGULATION OF MYELOID ENHANCERS

MANIPULATING EPITOPE IMMUNODOMINANCE AND TRACKING B-CELL-ANTIGEN INTERACTIONS FOR VACCINE DESIGN. - PROJECT SUMMARY/ ABSTRACT INFECTIOUS DISEASES ARE SERIOUS AND RECURRENT HEALTH THREATS. PARTICULARLY CONCERNING ARE VIRUSES WITH THE CAPACITY TO MUTATE AND GENERATE DE NOVO DIVERSITY IN SHORT PERIODS OF TIME. THESE VIRUSES ADAPT TO NEW HOSTS AND ENVIRONMENTS, AND CONTINUOUSLY ESCAPE FROM THE HOST ANTI-VIRAL IMMUNE RESPONSE. PREVENTATIVE VACCINES ARE HIGHLY DESIRABLE; HOWEVER, NO DEFINED GUIDELINES EXIST FOR THE DESIGN OF EFFICACIOUS VACCINES AGAINST RAPIDLY MUTATING VIRUSES SUCH AS HIV-1, INFLUENZA, OR THE CURRENT SARS-COV2, WITH MULTIPLE DIFFERENT CIRCULATING VARIANTS. DESPITE THEIR HIGH DIVERSITY, VIRAL VARIANTS PRESENT CONSERVED REGIONS THAT ARE ESSENTIAL FOR VIRAL FITNESS AND INFECTIVITY. THESE CONSERVED EPITOPES ARE THEIR ACHILLES HEELS, AND THE FOCUS OF ANTIBODY-BASED VACCINE DESIGN EFFORTS. A VACCINE AGAINST A HIGHLY MUTATING VIRUS SHOULD ELICIT AN ANTIBODY RESPONSE THAT SPECIFICALLY TARGETS THE CONSERVED REGIONS OF THE VIRUS, AS IT WOULD RECOGNIZE AND NEUTRALIZE THE BROAD DIVERSITY OF ITS VARIANTS. SIGNIFICANT EFFORTS IN THE FIELD HAVE FOCUSED ON ENGINEERING VIRAL IMMUNOGENS TO MAKE THEIR CONSERVED EPITOPES MORE AVAILABLE FOR ANTIBODY RECOGNITION. UNFORTUNATELY, TARGETING ANTIBODY RESPONSES TO SPECIFIC CONSERVED EPITOPES OF INTEREST IS INCREDIBLY CHALLENGING. COMPLEX ANTIGENS, SUCH AS VIRAL SPIKE PROTEINS, ELICIT POLYCLONAL RESPONSES DOMINATED BY ANTIBODIES TO NON-CONSERVED EPITOPES. THESE ANTIBODIES HAVE NO POTENTIAL TO BROADLY NEUTRALIZE THE VIRUS, AND ALSO INTERFERE WITH THE MATURATION OF BROADLY PROTECTIVE ANTIBODIES IN THE GERMINAL CENTERS. AIMING TO ELICIT BROADLY NEUTRALIZING ANTIBODIES (BNABS) AGAINST A CONSERVED EPITOPE OF HIV-1, WE RECENTLY DESIGNED AND EVALUATED A NEW HIV-1 ENVELOPE (ENV)-BASED PRIMING IMMUNOGEN, WHICH ELICITED BNAB-LIKE ANTIBODIES AGAINST A CONSERVED EPITOPE OF ENV IN WILD TYPE MICE AND MACAQUES; DESPITE THIS ACHIEVEMENT, THESE ANTIBODIES SHOWED NO NEUTRALIZATION ACTIVITY AGAINST HIV-1, SUGGESTING THAT ADDITIONAL IMMUNIZATION WOULD BE REQUIRED TO INDUCE BNABS. NEVERTHELESS, FURTHER IMMUNIZATION IN MACAQUES ELICITED A POLYCLONAL ANTIBODY RESPONSE OF ONLY LIMITED POTENCY AND BREADTH, DOMINATED BY ANTIBODIES TO NON-CONSERVED EPITOPES OF ENV. BASED ON THESE OBSERVATIONS, I HYPOTHESIZE THAT REDUCING INTERFERING ANTIBODY RESPONSES TO NON-CONSERVED VIRAL EPITOPES, AND TRACKING THE ANTIBODY RESPONSES WITH POTENTIAL TO BECOME BNABS, WILL PAVE THE PATH TOWARDS BNAB DEVELOPMENT AND INFORM VACCINE DESIGN EFFORTS. IN THIS PROPOSAL, WE WILL DESIGN AND EVALUATE A NOVEL STRATEGY TO MODULATE EPITOPE IMMUNODOMINANCE, WHICH IN ADDITION, WILL ALLOW US TO RECORD AND TRACK THE HISTORY OF ANTIGEN-B-CELL INTERACTIONS IN VIVO. THE PROPOSED TECHNOLOGY WILL BE USED TO CUSTOMIZE THE IMMUNODOMINANCE PROPERTIES OF COMPLEX ANTIGENS IN ORDER TO DIRECT THE ANTIBODY RESPONSE TO THE EPITOPES OF INTEREST. IN ADDITION, WE WILL USE OUR NEW TECHNOLOGY TO BARCODE B CELLS RESPONDING TO MULTIPLE IMMUNIZATIONS, TRACK THEIR FATES AND RECORD THEIR HISTORY OF ANTIGEN ENCOUNTERS. THIS GROUNDBREAKING TECHNOLOGY WILL PROVIDE VERY VALUABLE INFORMATION TO ELUCIDATE THE MECHANISMS GOVERNING THE B CELL RESPONSES TO VACCINATION AND INFECTION, AND WILL SIGNIFICANTLY CONTRIBUTE TO ESTABLISH GUIDELINES FOR VACCINE DESIGN.

EPIGENETIC REGULATION THROUGH THE FORMATION AND RESOLUTION OF R LOOPS

THE MULTIFACETED ROLE OF ACETATE IN CANCER

NEW CONTROL OF ONCOGENE ACTIVATION IN T-CELL LEUKEMIA - PROJECT SUMMARY/ABSTRACT NOTCH1 SIGNALING IS AN IMPORTANT MEDIATOR OF STEM CELL SELF-RENEWAL AND THERAPEUTIC RESISTANCE, AND THE MOST PREVALENT ONCOGENE (~60%) IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA (T-ALL) - AN AGGRESSIVE NEOPLASM OF T CELL PROGENITORS THAT AFFECTS BOTH CHILDREN AND ADULTS. ALTHOUGH CURRENT INTENSIVE CHEMOTHERAPIES CAN SUPPRESS THE DISEASE, THEY COME AT THE COST OF SERIOUS SIDE EFFECTS AND ARE INSUFFICIENT TO ELIMINATE NOTCH1-DRIVEN LEUKEMIC CELLS. ONE IN FIVE CHILDREN AND ONE IN TWO ADULTS WITH T-ALL DO NOT SURVIVE DUE TO EITHER UNRESPONSIVE OR RELAPSED DISEASE. EFFORTS TO TARGET ONCOGENIC NOTCH1 WITH SMALL-MOLECULE INHIBITORS HAVE BEEN HAMPERED BY THEIR INHERENT CYTOTOXICITY. OVERCOMING THESE DIFFICULTIES WILL REQUIRE IMPROVED UNDERSTANDING OF THE ONCOGENIC MECHANISMS CONTROLLED BY NOTCH1 AND A BETTER APPRECIATION OF THE GENES AND PATHWAYS THAT REGULATE NOTCH1- DRIVEN LEUKEMOGENESIS AS POTENTIAL TARGETS OF T-ALL THERAPY. THROUGH DROSOPHILA STUDIES AND THE GENERATION OF MOUSE MODELS FOR T-ALL, WE HAVE DISCOVERED THAT, T-ALL-ASSOCIATED NOTCH COULD BE DEGRADED BY AN UNCONVENTIONAL ENDO-LYSOSOMAL MODULE THROUGH A PHYSICAL INTERACTION WITH THE AUTOPHAGIC TUMOR SUPPRESSOR UVRAG, WHICH RESHAPES NOTCH ACTIVITY AND RESULTANT NOTCH-DEPENDENT CELLULAR RESPONSE. THUS, THE CENTRAL HYPOTHESIS OF THIS PROPOSAL IS THAT THE ENDO-LYSOSOMAL TITRATION OF NOTCH ACTIVITY BY UVRAG REPRESENTS A UNIQUE MECHANISM GOVERNING NOTCH1 BEFORE PROTEOLYTIC PROCESSING, AND THAT DISRUPTION OF THIS REGULATORY MODULE IMPACTS T-CELL HOMEOSTASIS AND CONTRIBUTES TO T-ALL. SPECIFICALLY, WE PROPOSE EXPERIMENTS TO COMPREHENSIVELY DISSECT THE MOLECULAR MECHANISM OF UVRAG-MEDIATED ENDO-LYSOSOMAL INHIBITION OF NOTCH1 IN T-ALL. FURTHERMORE, WE WILL ELUCIDATE THE UNEQUIVOCAL IMPACT OF THIS MECHANISM ON THE SELF-RENEWAL AND STEMNESS OF LEUKEMIA-INITIATING CELL FUNCTION IN HUMAN T-ALL PRIMARY SAMPLES. FINALLY, WE WILL USE THE MOUSE MODELS TO TEST THE CONCEPT THAT BOOSTING THIS MECHANISM COULD RESTORE NOTCH HOMEOSTASIS AND ACHIEVE SUSTAINED T-ALL REMISSION. THESE AIMS WILL BE ADDRESSED USING MULTIDISCIPLINARY INNOVATIVE APPROACHES THAT INTEGRATE STATE-OF-THE-ART GENETIC, BIOCHEMISTRY, HIGH-RESOLUTION IMAGING, AND PHYSIOLOGICAL ASSAYS IN CELLS AND TRANSGENIC MOUSE MODELS. WE NOW BRING WITHIN THIS PROPOSAL A COLLABORATION OF WORLD-WIDE LEADERS IN T-ALL PATHOLOGY AND MOLECULAR BIOLOGY ALONG WITH CLINICIANS AND PATHOLOGISTS. OUR USE OF PATIENT-DERIVED T-ALL SAMPLES WILL MAXIMIZE THE RELEVANCE OF OUR FINDINGS FOR EVENTUAL TRANSLATION TO T-ALL PATIENTS IN THE CLINIC. OVERALL, THIS PROJECT WILL LEAD TO AN IN-DEPTH UNDERSTANDING OF NOTCH1-DRIVEN LEUKEMOGENESIS, AND PROVIDES A CRITICAL TRAJECTORY FOR THE DEVELOPMENT OF OPTIMAL ANTI-LEUKEMIA STRATEGIES AGAINST THIS AGGRESSIVE LYMPHOID MALIGNANCY.

SPECIFICITY AND FUNCTION OF CD25+ REGULATORY T CELLS

REGULATION OF NEONATAL INFLAMMATION BY MYELOID-DERIVED SUPPRESSOR CELLS

NEGATIVE REGULATION OF MYELOID-DERIVED SUPPRESSIVE CELLS IN CANCER

TARGETING TERT IN MELANOMA

TARGETING S6K2 TO OVERCOME DRUG RESISTANCE IN NRAS-MUTANT MELANOMA - PROJECT SUMMARY WHILE SEVERAL THERAPIES HAVE BEEN APPROVED FOR MELANOMA, THERE ARE LIMITED TREATMENT OPTIONS FOR NRAS MUTANT (NRASMUT) TUMORS, WHICH ACCOUNT FOR ~30% OF ALL MELANOMAS. NRASMUT TUMORS ARE EXTREMELY AGGRESSIVE AND ARE ASSOCIATED WITH POOR PATIENT SURVIVAL. THESE TYPES OF TUMORS ARE HIGHLY RESISTANT TO AVAILABLE TARGETED THERAPIES AND ARE POORLY RESPONSIVE TO IMMUNOTHERAPIES. THEREFORE, THERE IS AN URGENT UNMET NEED TO IDENTIFY NOVEL TARGETS AND EFFECTIVE THERAPIES TO HELP THIS LARGE POPULATION OF MELANOMA PATIENTS WHO DO NOT RESPOND TO CURRENTLY AVAILABLE TREATMENTS. OUR GOAL IS TO IDENTIFY CRITICAL VULNERABILITIES THAT CAN BE TARGETED TO OFFSET DRUG RESISTANCE IN NRAS MUTANT MELANOMA. ONCOGENIC NRAS ACTIVATES BOTH THE MAPK AND PI3K PATHWAYS. HOWEVER, INHIBITING EITHER PATHWAY ALONE IS BARELY EFFECTIVE IN PATIENTS AND CO-TARGETING BOTH PATHWAYS LEADS TO UNACCEPTABLE TOXICITIES DUE TO THE BROAD SPECTRUM OF CELL FUNCTIONS THAT THESE SIGNALING NETWORKS CONTROL. WE PREVIOUSLY REPORTED THAT BRAF-MUTANT MELANOMAS RESISTANT TO BRAF AND MEK INHIBITORS (MAPKI-R) HAVE SUSTAINED ACTIVATION OF THE RIBOSOMAL PROTEIN S6 KINASE. WE HAVE NOW DISCOVERED THAT MAPKI-R NRASMUT MELANOMAS RELY SPECIFICALLY ON THE S6K2 ISOFORM FOR SURVIVAL. SELECTIVE S6K2 BLOCKADE, IN THE CONTEXT OF ACTIVE S6K1, PERTURBS REDOX AND LIPID METABOLISM, TRIGGERING LETHAL LIPID PEROXIDATION EXCLUSIVELY IN NRASMUT MELANOMA CELLS THAT ARE RESISTANT TO MAPK INHIBITION. BASED ON OUR PRELIMINARY FINDINGS, WE POSTULATE THAT S6K2 CONTROLS METABOLIC AND REDOX HOMEOSTASIS, AND MELANOMA CELL SURVIVAL, THEREBY CONSTITUTING A NOVEL AND PROMISING THERAPEUTIC TARGET. AS S6K IS A NODE OF CONVERGENCE OF THE MAPK AND PI3K/MTOR PATHWAYS, WE FURTHER POSIT THAT SELECTIVELY BLOCKING S6K2 CAN OVERCOME RESISTANCE TO MAPKI MEDIATED BY BROAD MOLECULAR MECHANISMS THAT RELY ON THESE PATHWAYS. IN THIS PROJECT WE WILL DEFINE THE MECHANISMS WHEREBY S6K2-DEPENDENT LIPID AND REDOX HOMEOSTASIS CONTRIBUTES TO DRUG RESISTANCE AND PROMOTES SURVIVAL OF MAPKI-R MELANOMA (AIM 1). FURTHERMORE, WE WILL OPTIMIZE NOVEL S6K2 INHIBITORS AND EXPLOIT THE DEPENDENCY OF MELANOMA ON S6K2 TO OFFSET MAPKI RESISTANCE (AIM 2). OUR PROPOSED STRATEGY, WHICH IS SIGNIFICANTLY DIFFERENT FROM MAPK/PI3K INHIBITION, WILL ENABLE FUNCTIONAL PRECISION BY TARGETING A CONVERGENT SUBNETWORK REPRESENTING A VULNERABILITY SELECTIVELY IN TUMOR CELLS. WE ANTICIPATE THAT OUR STUDIES WILL PROVIDE A MECHANISTIC FRAMEWORK TO INFORM THE DESIGN OF THERAPEUTIC STRATEGIES TARGETING S6K2 DIRECTLY, OR ALTERNATIVELY, S6K2 SPECIFIC EFFECTOR PATHWAYS AND IMPROVE THE OUTCOMES OF NRASMUT MELANOMA PATIENTS AND POSSIBLE OTHER TYPES OF RAS-MUTANT TUMORS.

TARGETING TELOMERASE FOR MELANOMA THERAPEUTICS

MEMBRANE PROTEINS OF NORMAL AND ABNORMAL RED CELLS

REGULATION OF EBV LATENCY BY PURINE METABOLISM AND SIGNALING - EPSTEIN-BARR VIRUS (EBV) REPROGRAMS HOST CELL GENE EXPRESSION AND METABOLISM DURING THE ESTABLISHMENT OF LATENCY AND THE IMMORTALIZATION OF B-LYMPHOCYTES. THE REGULATORY MECHANISMS COORDINATING THIS REPROGRAMMING WITH EBV LATENCY REFLECT IMPORTANT EVENTS IN VIRAL ONCOGENESIS, YET REMAIN POORLY UNDERSTOOD. WE HAVE FOUND THAT KEY VIRAL REGULATORS OF EBV LATENCY, INCLUDING THE MAJOR TEGUMENT PROTEIN BNRF1 AND THE EBV NUCLEAR ANTIGEN EBNA1 COORDINATE KEY ASPECTS OF PURINE METABOLISM DURING ESTABLISHMENT OF LATENCY AND IMMORTALIZATION OF PRIMARY B-CELLS. IN THIS R01, WE FOCUS ON HOW EBV REPROGRAMS PURINE METABOLIC GENE EXPRESSION, AND HOW PURINE METABOLITES CONTRIBUTE DIRECTLY TO EBV TUMORIGENESIS. ONE CLUE TO THIS COORDINATE REGULATION IS PROVIDED BY THE VIRAL-ENCODED TEGUMENT PROTEIN BNRF1 THAT SHARES EXTENSIVE STRUCTURAL SIMILARITY TO THE PURINE BIOSYNTHETIC ENZYME FGARAT (ALSO CALLED PFAS) AND FUNCTIONS IN VIRAL CHROMATIN ASSEMBLY DURING PRIMARY INFECTION. ORTHOLOGUES OF BNRF1 ARE FOUND IN ALL GAMMA HERPESVIRUSES, INCLUDING KSHV ORF75, AND SHARE THE COMMON FUNCTION OF DISARMING COMPONENTS OF THE PML-NUCLEAR BODY (PML-NB) AND ITS ANTI-VIRAL FUNCTIONS. WE HAVE PREVIOUSLY SHOWN THAT BNRF1 INTERACTS WITH THE HISTONE H3.3 CHAPERONE DAXX AND DISPLACES ITS INTERACTION WITH THE ATP-DEPENDENT SNF2-LIKE HELICASE ATRX TO ENABLE SELECTIVE EXPRESSION OF LATENCY-SPECIFIC VIRAL GENES DURING PRIMARY INFECTION. HOWEVER, IT HAS NOT YET BEEN SHOWN HOW THE VIRAL FGARAT HOMOLOGY DOMAIN IS LINKED TO CELLULAR PURINE BIOSYNTHESIS AND/OR SIGNALING. USING METABOLOMICS MASS SPECTROMETRY, WE PROVIDE NEW PRELIMINARY DATA INDICATING THAT THE PURINE BIOSYNTHETIC PATHWAY IS AMONG THE MOST SIGNIFICANTLY PERTURBED BY EBV DURING B-CELL IMMORTALIZATION. INTEGRATING GENE EXPRESSION (RNA-SEQ), CHROMATIN ACCESSIBILITY (ATAC-SEQ), AND EBNA1-DNA BINDING TO HOST CHROMOSOME (CHIP-SEQ), WE IDENTIFIED CELLULAR METABOLIC GENES, INCLUDING ADENINE DEAMINASE (ADA), ADENOSINE KINASE 4 (AK4), AND PURINERGIC RECEPTORS P2RY8 AND P2RX5 AS DIRECT TARGETS OF EBNA1 TRANSCRIPTIONAL REGULATION DURING EBV IMMORTALIZATION. WE NOW PROPOSE TO INVESTIGATE THE MECHANISMS BY WHICH EBV SENSES AND REPROGRAMS PURINE METABOLISM AND HOW PURINERGIC SIGNALING REGULATES ESTABLISHMENT OF EBV LATENCY AND HOST CELL TRANSFORMATION. WE WILL TEST THE CENTRAL HYPOTHESIS THAT EBV COORDINATELY REGULATES CELLULAR PURINE METABOLISM WITH VIRAL AND CELLULAR GENE EXPRESSION DURING THE B-CELL IMMORTALIZATION PROCESS, AND THAT PURINERGIC SIGNALING IS CRITICAL FOR VIRAL LATENCY AND ONCOGENESIS. SPECIFICALLY, WE WILL INVESTIGATE HOW EBV REGULATES EXPRESSION OF PURINE METABOLIC GENES DURING PRIMARY INFECTION (AIM 1), ELUCIDATE HOW PURINE METABOLISM IMPACTS THE ESTABLISHMENT OF EBV LATENCY (AIM 2), AND INVESTIGATE THE ROLE OF PURINE METABOLISM AND SIGNALING IN B-CELL IMMORTALIZATION, IMMUNE SIGNALING, AND EBV-INDUCED TUMORIGENESIS (AIM 3). THESE STUDIES WILL ADVANCE OUR UNDERSTANDING OF BASIC MECHANISMS COORDINATING GENE EXPRESSION WITH METABOLISM AND IDENTIFY NEW TARGETS FOR THERAPEUTIC INTERVENTION IN VIRAL LATENCY AND VIRAL-ASSOCIATED CANCERS.

THE IMPACT OF CODING REGION VARIANTS ON MUTANT P53 BIOLOGY

P53 VARIANTS IN CANCER RISK AND THERAPY

CELL-CELL COMMUNICATION DURING MELANOMA DEVELOPMENT

EXTRACELLULAR DNA IN REGULATION OF MULTIPLE MYELOMA

DESIGN OF THE FIRST MDM2 TARGETING PROTACS FOR TREATMENT OF P53 MUTANT OR DEFICIENT CANCERS - PROJECT SUMMARY / ABSTRACT OUR PROPOSAL FOCUSES ON THE DEVELOPMENT OF NOVEL MDM2-TARGETED PROTEOLYSIS TARGETING CHIMERAS (PROTACS) THAT EFFICIENTLY DEGRADE MDM2 IN P53 MUTANT AND DEFICIENT CANCER CELLS AND ARE EXPECTED TO TARGET P53- INDEPENDENT FUNCTIONS OF MDM2. OUR LEAD COMPOUNDS BIND WITH HIGH AFFINITY, DEGRADE MDM2, KILL CANCER CELLS THAT LACK FUNCTIONAL P53, AND ARE EFFICACIOUS IN VIVO. WE HAVE ALSO DEMONSTRATED SAFETY AND TOLERABILITY IN VIVO, SYNTHETIC TRACTABILITY, METABOLIC STABILITY, AND SUITABLE IN VIVO EXPOSURE IN MOUSE PHARMACOKINETIC STUDIES FOR IN VIVO EFFICACY EVALUATION. THE TUMOR SUPPRESSOR P53, A TRANSCRIPTION FACTOR, HAS AN ESSENTIAL ROLE IN THE PREVENTION OF HUMAN CANCER. IN THE ABSENCE OF CELLULAR STRESS, THE P53 PROTEIN IS MAINTAINED AT LOW LEVELS DUE TO ITS BINDING TO MDM2, AN E3 UBIQUITIN LIGASE. HOWEVER, HALF OF ALL HUMAN CANCERS HAVE INACTIVATED P53 BY MUTATION OR DELETION. TO TEST IF MDM2 IS REQUIRED FOR THE SURVIVAL OF CANCER CELLS THAT LACK P53 WE INDUCIBLY DELETED MDM2 IN PRIMARY MURINE P53-NULL LYMPHOMA AND SARCOMA CELLS. MDM2 LOSS RESULTED IN APOPTOSIS IN VITRO AND IN VIVO IN P53-NULL CANCERS, WITH SIGNIFICANTLY REDUCED TUMOR BURDEN AND INCREASED SURVIVAL BENEFIT. WE AND OTHERS HAVE REPORTED THAT MDM2 HAS P53-INDEPENDENT FUNCTIONS BY BINDING AND REGULATING OTHER PROTEINS, SUCH AS THE P53 FAMILY MEMBER, P73, AND NBS1 IN THE MRE11-RAD50-NBS1 DNA BREAK REPAIR COMPLEX. P73 HAS A HOMOLOGOUS N-TERMINAL TRANSACTIVATION DOMAIN TO P53 WHICH BINDS IN THE SAME SITE ON MDM2. ADDITIONALLY, UNLIKE P53, P73 IS RARELY INACTIVATED IN HUMAN CANCERS. SINCE MDM2-P53 INHIBITORS ARE NOT RESPONSIVE TO TUMORS WITH INACTIVATED P53, WE PURSUED A PROTAC APPROACH AND PROVIDE PRELIMINARY DATA TO CONFIRM THEIR ABILITY TO KILL P53 MUTATED OR DEFICIENT CANCER CELLS. OUR HYPOTHESIS IS THAT THE DEGRADATION OF MDM2 VIA MDM2-TARGETED PROTACS WILL KILL CANCERS WITH MUTANT OR DELETED P53 BY ACTIVATING THE P53-INDEPENDENT ACTIVITIES OF MDM2. WE WILL TEST THIS HYPOTHESIS WITH TWO SPECIFIC AIMS. AIM 1 WILL FOCUS ON EXPANDING THE CHARACTERIZATION OF OUR LEAD MDM2 TARGETING COMPOUNDS AND EVALUATE THESE IN IN VIVO XENOGRAFT MODELS. AIM 2 WILL BE LEAD OPTIMIZATION OF TWO MDM2 PROTACS THAT KILLED P53 MUTANT AND DELETED CANCERS. THE GOAL OF THESE AIMS IS TO HAVE A CHARACTERIZED, EFFECTIVE, AND POTENT MDM2 PROTAC FOR CLINICAL EVALUATION FOR THE TREATMENT OF P53-INACTIVATED CANCERS.

PROMOTION OF TUMOR INVASION AND PSEUDOSENESCENCE BY THE AGING MICROENVIRONMENT

CTCF-DEPENDENT MECHANISMS OF ATRX IN NEURONAL DIFFERENTIATION - PROJECT SUMMARY ATRX IS AN EPIGENETIC FACTOR THAT IS MUTATED IN ATRX SYNDROME. ATRX IS REQUIRED FOR THE MAINTENANCE OF MULTIPOTENT NEUROPROGENITOR CELLS (NPCS), PARTICULARLY AS THESE CELLS INITIATE DIFFERENTIATION PROGRAMS IN THE BRAIN. MUTATIONS THAT CAUSE ATRX SYNDROME CLUSTER WITHIN TWO DOMAINS: THE ATRX PHD FINGER DOMAIN, AND THE HELICASE DOMAIN. THESE MEDIATE INTERACTIONS WITH HISTONES, AND REMODEL CHROMATIN, RESPECTIVELY. INTERESTINGLY, MUTATIONS IN THE PHD FINGER ARE ASSOCIATED WITH SEVERE INTELLECTUAL DISABILITY AND PSYCHOMOTOR IMPAIRMENT, WHILE MUTATIONS IN THE HELICASE DOMAIN OFTEN MANIFEST WITH MILDER NEURODEVELOPMENTAL DELAYS BUT MORE SEVERE GENITAL ABNORMALITIES13. THE BASIS FOR THIS GENOTYPE-PHENOTYPE CORRELATION HAS NEVER BEEN INVESTIGATED. ATRX HAS WELL- ESTABLISHED ROLES IN MOLECULAR PROCESSES THAT ARE CRUCIAL FOR NORMAL BRAIN DEVELOPMENT INCLUDING HISTONE VARIANT H3.3 DEPOSITION AND POLYCOMB REPRESSIVE COMPLEX 2 TARGETING FOR EPIGENETIC SILENCING. IT ALSO HAS A POORLY UNDERSTOOD ROLE IN REGULATING CTCF, A CRITICAL GENOME ARCHITECTURAL PROTEIN THAT IS ESSENTIAL FOR DEVELOPMENT. OUR PRELIMINARY DATA INDICATE PHD FINGER AND HELICASE MUTATIONS OF ATRX DIFFERENTIALLY REGULATE CTCF LOCALIZATION AND MAY UNDERLIE GENOTYPE-PHENOTYPE CORRELATIONS. OUR PRELIMINARY DATA HAS UNCOVERED A GENOME-WIDE ROLE FOR ATRX IN CTCF LOCALIZATION. WE FOUND THAT ATRX KNOCK DOWN (KD) IN MOUSE EMBRYONIC STEM CELLS (MESCS) RESULTS IN CTCF ACCUMULATION AT MANY GENOMIC SITES, INCLUDING BOTH IMPRINTED AND NON-IMPRINTED LOCI. WE ALSO DISCOVERED THAT ATRX INTERACTS WITH ADNP AND DNMT3L, BOTH OF WHICH CAN PREVENT CTCF BINDING. USING CRISPR/CAS9, WE HAVE GENERATED ISOGENIC ESCS WHERE THE ENDOGENOUS ATRX ALLELE HAS BEEN REPLACED BY POINT MUTANTS FOUND IN ATRX SYNDROME PATIENTS. WE SHOW THAT MUTATIONS IN THE PHD FINGER CAUSE SIGNIFICANT IMPAIRMENT OF NPC DIFFERENTIATION, WHILE MUTATIONS IN THE HELICASE DOMAIN CAUSE MORE SUBTLE DIFFERENTIATION DELAYS. OUR PRELIMINARY FINDINGS AND NOVEL REAGENTS UNIQUELY POSITION US TO INTERROGATE THE CONSEQUENCE OF ATRX MUTATIONS, BOTH FOR GENOME ORGANIZATION THROUGH CTCF, AND FOR THE ABILITY OF ESCS TO DIFFERENTIATE INTO NPCS. BASED ON OUR PRELIMINARY DATA, WE PROPOSE TO TEST THE HYPOTHESIS THAT DISTINCT ATRX DOMAINS REGULATE SPECIFIC CHROMATIN PROCESSES THAT IMPINGE TO DIFFERENT EXTENTS ON THE FUNCTION OF CTCF. IN AIM 1, WE WILL INVESTIGATE THE CONSEQUENCE OF ATRX SYNDROME MUTATIONS TO GENE EXPRESSION AND GENOME ORGANIZATION DURING NEURONAL DIFFERENTIATION. IN AIM 2, WE WILL DECIPHER THE MECHANISMS BY WHICH ATRX REGULATES CTCF LOCALIZATION.

RECEPTOR DIVERSITY IN RECOGNITION OF INFLUENZA HA

THE ROLE OF GENETIC SUSCEPTIBILITY IN MELANOMA DEVELOPMENT

REGULATION OF GENE EXPRESSION BY ALTERNATIVE POLYADENYLATION - PROJECT SUMMARY MOST MAMMALIAN GENES HARBOR MULTIPLE CLEAVAGE AND POLYADENYLATION SITES, OR PASS, ACROSS THE GENE BODY, RESULTING IN MRNA ISOFORMS WITH DIFFERENT CODING SEQUENCES (CDS) AND/OR 3’ UNTRANSLATED REGIONS (3’UTRS). ALTERNATIVE CLEAVAGE AND POLYADENYLATION (APA) IS AN IMPORTANT LAYER OF GENE REGULATION, AFFECTING GENE EXPRESSION LEVELS, PROTEIN DIVERSITY, AND MRNA METABOLISM. THE APA ISOFORM EXPRESSION VARIES ACROSS CELL TYPES AND IS DYNAMICALLY REGULATED IN A GROWING NUMBER OF CELL CONDITIONS, SUCH AS CELL PROLIFERATION AND DIFFERENTIATION, CHANGE OF METABOLIC STATES, AND CELLULAR STRESS. OUR LAB EMPLOYS INTERDISCIPLINARY APPROACHES TO STUDY APA, INVOLVING MOLECULAR BIOLOGY, FUNCTIONAL GENOMICS, AND COMPUTATIONAL BIOLOGY. OUR LONG-TERM GOAL IS TO UNDERSTAND THE FUNCTIONAL GENOMICS OF APA ACROSS SPECIES AS WELL AS MOLECULAR MECHANISMS AND CELLULAR CONSEQUENCES OF GENE REGULATION BY APA IN DIFFERENT CELL CONTEXTS AND PATHOLOGICAL CONDITIONS. IN THE NEXT FIVE YEARS, WE PLAN TO ADDRESS A FEW KEY GAPS IN THE FIELD: FIRST, WE PLAN TO EXAMINE REGULATORY RULES GOVERNING INTRONIC POLYADENYLATION THAT LEADS TO EARLY TERMINATION OF TRANSCRIPTION. SECOND, WE WILL EXAMINE THE ROLE OF 3’UTR ISOFORM REGULATION IN CELL METABOLIC REPROGRAMMING, SUCH AS GROWTH AND AUTOPHAGY. THIRD, WE WILL INVESTIGATE THE MECHANISMS AND CONSEQUENCES OF THE UNIQUE APA ISOFORM PROFILE IN SECRETORY CELLS.

HDAC INHIBITORS AND CTCL

UNDERSTANDING PPARGAMMA SIGNALING IN MELANOMA BRAIN METASTASIS

DEVELOPMENT OF A SMALL MOLECULE INHIBITOR FOR KSHV LANA

CONTROL OF BREAST CANCER METASTASIS BY EPSTEIN-BARR VIRUS MICRORNA

MEPCS (MULTI-EPITOPE PROTEASE CLEAVAGE SITES) VACCINE FOR PROTECTING AGAINST SIV RECTAL TRANSMISSION - SUMMARY NOVEL VACCINE STRATEGIES IS NEEDED TO DEVELOP A SAFE, EFFECTIVE, AND BROADLY ACCESSIBLE HIV VACCINE TO END THE HIV PANDEMIC. THIS PROPOSAL IS BUILT UPON SEVERAL INNOVATIVE ADVANCEMENTS. FIRST, LEARNED FROM NATURAL IMMUNITY OBSERVED IN A GROUP OF HIV RESISTANT KENYAN FEMALE SEX WORKERS, WE FOUND 12 PROTEASE CLEAVAGE SITES (PCS) IN HIV ARE CRITICAL FOR VIRUS MATURATION DUE TO THEIR ESSENTIAL FUNCTION IN TIGHTLY CONTROLLED CLEAVAGE OF GAG, GAG-POL, AND NEF PRECURSOR PROTEINS. SEQUENCES SURROUNDING THE HIV PCS ARE IMMUNOGENIC AND HIGHLY CONSERVED ACROSS GLOBAL HIV SUBTYPES, THEREBY A VACCINE BASED ON 12 PCS IMMUNOGENS WILL BE GLOBALLY ACCESSIBLE AND LIKELY TO PREVENT VIRUS ESCAPE MUTATIONS. FURTHER, OUR RECENT STUDIES DEMONSTRATED THAT A PCS VACCINE DELIVERED BY RECOMBINANT VESICULAR STOMATITIS VIRUS (RVSV) VECTOR AND NANOFORMULATION PROTECTED MORE THAN 80% OF VACCINATED FEMALE MAURITIAN CYNOMOLGUS MACAQUES (MCMS) AGAINST SIVMAC251 INTRAVAGINAL CHALLENGES, WHICH IS ASSOCIATED WITH VACCINE ELICITED POTENT PCS-SPECIFIC CD8 T CELL RESPONSES. HOWEVER, IN PREVIOUS STUDIES, 12 PCS IMMUNOGENS WERE INDIVIDUALLY EXPRESSED IN THE RVSV. TO IMPROVE VACCINE IMMUNOGEN PRESENTATION AND FACILITATE THE VACCINE PREPARATION, 12 PCS IMMUNOGENS SHOULD BE EXPRESSED IN A SINGLE CASSETTE, I.E., MULTI-EPITOPE PCS (MEPCS). I.E., MULTI-EPITOPE PCS (MEPCS). TO THAT END, WE HAVE DEVELOPED A COLD-CHAIN FRIENDLY AND LONG-TERM STABLE MEPCS-MRNA-LNP VACCINE. WE FOUND THAT SIV MEPCS-MRNA-LNP VACCINE INDUCED A POTENT PCS-SPECIFIC CD8 T-CELL IMMUNITY WITHOUT INDUCING GENERALIZED INFLAMMATION AND CD4 T-CELL ACTIVATION. BASED ON OUR STRONG PRELIMINARY DATE, WE HYPOTHESIZE THAT MEPCS VACCINES WOULD PROTECT RHESUS MACAQUES AGAINST SIVMAC251 RECTAL TRANSMISSION. THE REASON WE WILL USE A RHESUS MACAQUE (RM)-SIV RECTAL CHALLENGE MODEL IS THAT THE EFFECTIVENESS OF MEPCS VACCINE NEEDS TO BE VALIDATED IN THE MOST RIGOROUS RHESUS MACAQUES AND ANAL SEX IS THE MOST COMMON HIV TRANSMISSION IN THE UNITED STATES OF AMERICA. THE PRIMARY OBJECTIVE OF THIS STUDY IS TO EVALUATE THE EFFECTIVENESS OF COLD- CHAIN FRIENDLY AND LONG-TERM STABLE MEPCS-MRNA-LNP VACCINE IN COMPARISON WITH RVSV-MEPCS VACCINES IN PROTECTING RMS AGAINST SIVMAC251 INTRARECTAL CHALLENGE AND TO BETTER UNDERSTAND THE IMMUNE CORRELATED PROTECTION. IN THIS PROPOSED STUDY WE WILL COMPARATIVELY EVALUATE THE EFFECTIVENESS OF MEPCS-MRNA-LNP WITH RVSV-MEPCS VACCINES AGAINST SIVMAC251 INTRARECTAL CHALLENGE IN RMS. THE PROPOSED STUDY HAS THE GREAT POTENTIAL TO DEVELOP A NEW HIV/SIV VACCINE TO PREVENT HIV/SIV RECTAL TRANSMISSION. THE NOVEL MEPCS IMMUNOGENS PLUS THE COLD-CHAIN FRIENDLY AND LONG-TERM STABLE MRNA- LNP FORMULATION MAKES THIS PROPOSED STUDY HIGHLY INNOVATIVE AND SIGNIFICANT. .

ADVANCING CANCER RESEARCH THROUGH COMPREHENSIVE PROTEOMICS AND METABOLOMICS ANALYSES

CLINICAL DEVELOPMENT OF GAMITRINIB, A NOVEL MITOCHONDRIAL-TARGETED SMALL MOLECULE HSP90 INHIBITOR

BIOLOGY OF MELANOMA MATASTASIS

FUNCTIONAL ANALYSIS OF THE BAP1 METASTASIS SUPPRESSOR GENE IN UVEAL MELANOMA

EBNA1 INHIBITOR FOR TREATMENT OF EBV-POSITIVE DLBCL - PROJECT SUMMARY THE EPSTEIN-BARR VIRUS (EBV) IS RESPONSIBLE FOR APPROXIMATELY 200,000 NEW CANCER CASES EACH YEAR WORLDWIDE. AMONG THESE, EBV+ DIFFUSE LARGE B-CELL LYMPHOMA (DLBCL) IS AN EMERGENT GLOBAL CANCER THREAT IN PATIENTS WITHOUT OVERT IMMUNOSUPPRESSION, IRRESPECTIVE OF AGE, AND REPRESENTS A GROWING UNMET MEDICAL NEED. NEW THERAPEUTIC APPROACHES ARE NEEDED TO TREAT EBV+ DLBCL. ONLY ONE VIRAL-ENCODED PROTEIN, EBNA1, IS CONSISTENTLY EXPRESSED IN ALL KNOWN EBV-ASSOCIATED MALIGNANCIES AND IS A VALIDATED TARGET FOR INHIBITION OF EBV- DEPENDENT TRANSFORMATION AND CARCINOGENESIS. INVESTIGATORS AT THE WISTAR INSTITUTE HAVE DEVELOPED VK-2019, A FIRST-IN-CLASS EBNA1 INHIBITOR AS A THERAPEUTIC AGENT, SELECTING IT FROM OVER 2500 CANDIDATE INHIBITOR COMPOUNDS DURING THE HIT-TO-LEAD AND LEAD OPTIMIZATION PHASES. VK-2019 MEETS OR EXCEEDS INDUSTRY-ACCEPTED CRITERIA FOR POTENCY, SELECTIVITY, METABOLIC STABILITY, DRUG SUITABILITY, DRUG SAFETY, TOXICOLOGY AND BIOAVAILABILITY. WE ANTICIPATED THAT VK-2019 WOULD HAVE A FAVORABLE SAFETY PROFILE BECAUSE THERE ARE NO HUMAN ORTHOLOGS OF EBNA1. BASED ON PRECLINICAL EVIDENCE AND A FIRST-IN-HUMAN PHASE I CLINICAL STUDY IN PATIENTS WITH ADVANCED NASOPHARYNGEAL CARCINOMA (NPC), VK-2019 MET ALL SAFETY, TOLERABILITY AND PHARMACOKINETIC ENDPOINTS WITH FEW DOCUMENTED ADVERSE EVENTS (AES) OR SEVERE ADVERSE EVENTS (SAES). IN THIS EARLY STUDY, WE OBSERVED STABLE DISEASE IN MORE THAN A THIRD AND A SIGNIFICANT DECREASE IN EBV PLASMA LEVELS, A KNOWN BIOMARKER OF NPC PROGRESSION, IN MORE THAN HALF OF THE PATIENTS, THAT CORRELATED WITH PHARMACOKINETIC EXPOSURE. WE BELIEVE THAT DATA FROM THIS PHASE I STUDY ARE ENCOURAGING IN TERMS OF BOTH ON TARGET EFFECT AND CLINICAL BENEFIT, AND SUPPORTS A FOLLOW-ON PROOF-OF-CONCEPT STUDY IN PATIENTS WITH EBV-POSITIVE DLBCL. THE PURPOSE OF THIS GRANT IS TO FUND A PHASE IB CLINICAL TRIAL OF DAILY ORAL ADMINISTRATION OF VK-2019 TO (1) FURTHER CONFIRM THE SAFETY PROFILE AND DETERMINE ANY DOSE-LIMITING TOXICITIES (DLT) IN ADVANCED EBV+ DLBCL PATIENT POPULATIONS; (2) UNDERSTAND THE EFFECTS OF TREATMENT ON EBV-SPECIFIC BIOMARKERS, INCLUDING EBV AND CELLULAR GENE EXPRESSION AND (3) STUDY THE EFFECTS OF TREATMENT ON THE TUMOR MICROENVIRONMENT AND IMMUNE RESPONSE. THE CLINICAL TRIAL INFRASTRUCTURE NECESSARY FOR THE CONDUCT OF THIS STUDY IS ALREADY IN PLACE AT THE SIDNEY KIMMEL CANCER CENTER AT THOMAS JEFFERSON UNIVERSITY. THIS CLINICAL TRIAL WILL PROVIDE CRITICAL INFORMATION ON THE SAFETY, TOLERABILITY, AND PRELIMINARY EFFICACY OF VK-2019 IN A EBV+ DLBCL PATIENT POPULATION. THIS APPLICATION BRINGS TOGETHER BASIC AND TRANSLATIONAL INVESTIGATORS TO UNDERSTAND WHETHER EBNA1 INHIBITORS CAN BE A THERAPEUTIC OPTION FOR LATENT EBV INFECTION AND CANCER AND EXAMINES THE MECHANISM OF ACTION.

REGULATION OF PRC2 FUNCTIONS BY PARP1

OPTIMIZING ACTIVE IMMUNOTHERAPY OF MELANOMA THROUGH METABOLIC REPROGRAMMING OF MELANOMA ANTIGEN-SPECIFIC CD8+ T CELLS COMBINED WITH CHECKPOINT BLOCKADE

DEFINING THE MOLECULAR MECHANISMS OF KLF17 REGULATION AND FUNCTION IN EMT

CHARACTERIZING THE SOURCES, MECHANISMS, AND TRANSLATIONAL RELEVANCE OF MICROBIAL TMAO IN DRIVING ANTI-TUMOR IMMUNITY IN PANCREATIC CANCER. - PROJECT SUMMARY THE TREATMENT OF PANCREATIC DUCTAL ADENOCARCINOMA (PDAC) REMAINS A MAJOR HURDLE WITH 5-YEAR SURVIVAL OF 12%. PDAC RESISTANCE TO CHEMOTHERAPY AND IMMUNOTHERAPY IS THOUGHT TO ARISE FROM THE IMMUNOSUPPRESSIVE TUMOR MICROENVIRONMENT (TME), CHARACTERIZED BY A FIBROTIC STROMA AND HIGH INFILTRATES OF IMMUNOSUPPRESSIVE CELLS, INCLUDING TUMOR ASSOCIATED MACROPHAGES (TAMS). TAMS BLOCK ANTI-TUMOR EFFECTOR T CELL FUNCTION AND TRIGGER EXHAUSTION. IT HAS BEEN POSTULATED THAT REPROGRAMMING TAMS TO AN IMMUNOSTIMULATORY PHENOTYPE COULD IMPROVE THERAPY RESPONSE. RECENT STUDIES SUGGEST THAT METABOLITES ORIGINATING FROM THE GUT MICROBIOME INFLUENCE PHENOTYPE OF IMMUNE CELLS, INCLUDING TAMS. HOWEVER, LITTLE IS UNDERSTOOD ABOUT SUCH METABOLITES, INCLUDING THEIR IDENTITY, THE SIGNALING PATHWAYS BY WHICH THEY ALTER TAM PHENOTYPE, AND WHETHER THESE METABOLITES INFLUENCE CANCER DEVELOPMENT. WE ARE IDENTIFYING MICROBIAL METABOLITES THAT MODULATE TAMS IN THE PDAC TME. USING UNBIASED, GLOBAL, LC-MS/MS METABOLOMIC SCREENS, WE FOUND A GUT MICROBE-DERIVED METABOLITE, TRIMETHYLAMINE N-OXIDE (TMAO) THAT INDUCED SIGNIFICANT ANTI-TUMOR EFFECTS IN THE PDAC TME. SPECIFICALLY, DELIVERY OF TMAO INTRAPERITONEALLY, OR BY SUPPLEMENTING DIET WITH THE TMAO PRECURSOR CHOLINE, TO PDAC-BEARING MICE REDUCED TUMOR GROWTH AND WAS ASSOCIATED WITH AN IMMUNOSTIMULATORY TAM PHENOTYPE AND ACTIVATED EFFECTOR T CELL RESPONSE IN THE TME. THE IMMUNOSTIMULATORY MACROPHAGE PHENOTYPE WAS DUE TO A DIRECT EFFECT OF TMAO AND THE TMAO-CONDITIONED MACROPHAGES COULD ACTIVATE CD8+ T CELLS. COMBINING TMAO WITH IMMUNE CHECKPOINT BLOCKADE REDUCED TUMOR BURDEN AND IMPROVED ANTI-TUMOR IMMUNE RESPONSES. THESE DATA SUPPORT OUR HYPOTHESIS THAT THE DIET AND MICROBIOME-DERIVED METABOLITES SHAPE ANTI-TUMOR IMMUNITY AND TREATMENT RESPONSE IN PDAC. AIM 1 WILL CHARACTERIZE DIETARY AND MICROBIAL SOURCES OF TMAO FOR ANTI-TUMOR RESPONSES IN PDAC. WE WILL CHARACTERIZE THE (I) DIETARY (L-CARNITINE OR BETAINE), AND (II) THE MICROBIOME (E.G., ENTEROCOCCUS ASINI, ENGINEERED E. COLICUTC/D) SOURCES OF TMAO FOR THEIR ANTI-TUMOR EFFECTS. AIM 2 WILL TEST THE HYPOTHESIS THAT TMAO INDUCES ITS ANTI-TUMOR EFFECTS BY POTENTIATING THE TYPE I IFN AND/OR STING SIGNALING IN TAMS. WE WILL DETERMINE (I) THE REQUIREMENT OF TYPE-I IFN AND/OR STING SPECIFICALLY IN MACROPHAGES FOR THE ANTI-TUMOR EFFECTS, AND (II) THE IMPACT OF TMAO ON THE TRANSCRIPTIONAL ACTIVITY OF TYPE-I IFN RESPONSIVE STAT1 AND/OR STAT3. AIM 3 WILL TEST THE EFFICACY OF TMAO TO IMPROVE TREATMENT RESPONSE IN PRE-CLINICAL MODELS OF PDAC. WE WILL EVALUATE THE TRANSLATIONAL RELEVANCE OF TMAO USING (I) A GENETICALLY ENGINEERED MOUSE MODEL OF PDAC AND PATIENT DERIVED PDAC ORGANOID CULTURES, AND (II) A TREATMENT STRATEGY COMBINING TMAO WITH STING AGONISTS. OUR STUDIES WILL CHARACTERIZE THE SOURCES, MECHANISM OF ACTION, AND TRANSLATIONAL RELEVANCE OF A NOVEL, MINIMALLY UNDERSTOOD, HIGH IMPACT MICROBIAL METABOLITE, TMAO, IN BOOSTING ANTI-TUMOR IMMUNE RESPONSES IN THE PDAC TME AND RENDERING PDAC RESPONSIVE TO CHEMO-IMMUNOTHERAPY. IN THE LONGER TERM, THIS WORK MAY LAY THE GROUNDWORK FOR NEW DIET/MICROBIOME-BASED TREATMENTS FOR PDAC.

ADNP MECHANISMS IN R-LOOP REGULATION DURING DIFFERENTIATION - PROJECT SUMMARY ABERRATIONS IN CHROMATIN STRUCTURE CAN BE A CAUSAL MECHANISM OF NUMEROUS HUMAN DISEASE AND DEVELOPMENTAL SYNDROMES. R-LOOPS ARE RNA CONTAINING CHROMATIN STRUCTURES THAT ARE DEREGULATED IN NUMEROUS DEVELOPMENTAL DISORDERS AND CANCERS. IN MOST CASES THE UNDERLYING MECHANISMS AND THE FUNCTIONAL SIGNIFICANCE OF R-LOOP DEREGULATION AND WHETHER THEY ARE CAUSAL TO SPECIFIC DISEASE IS NOT CLEAR. ACTIVITY DEPENDENT NEUROPROTECTIVE PROTEIN (ADNP) IS A HOMEODOMAIN CONTAINING TRANSCRIPTIONAL REPRESSOR THAT IS CRITICAL FOR NEURONAL DIFFERENTIATION AND NEURODEVELOPMENT. ADNP MUTATIONS CAUSE ADNP SYNDROME, A CONDITION CHARACTERIZED BY INTELLECTUAL DISABILITY AND FEATURES OF AUTISM SPECTRUM DISORDER. ADNP IS UPREGULATED DURING NEURODEVELOPMENT. ADNP CONTAINS ZINC FINGER MOTIFS AND A HOMEODOMAIN, BOTH OF WHICH CAN BIND NUCLEIC ACIDS. ADNP SYNDROME MUTATIONS RESULT IN PROTEIN PRODUCTS THAT LACK THE HOMEODOMAIN, UNDERSCORING THE IMPORTANCE OF THIS REGION TO ADNP FUNCTION. HOWEVER, THE PRECISE CONTRIBUTIONS OF THE ZINC FINGERS AND HOMEODOMAIN TO ADNP LOCALIZATION AND FUNCTION IN GENE EXPRESSION DURING NEURONAL DIFFERENTIATION IS NOT WELL UNDERSTOOD. OUR PRELIMINARY DATA HAVE UNCOVERED A NOVEL ROLE FOR ADNP IN THE RESOLUTION OF R-LOOPS. BIOCHEMICAL ASSAYS REVEALED THAT ADNP RESOLVES R-LOOPS – IN A HOMEODOMAIN DEPENDENT MANNER. ADNP ALSO SUPPRESSES R-LOOPS IN VIVO, AS DELETION OF ADNP IN MOUSE EMBRYONIC STEM CELLS (MESCS) RESULTED IN R-LOOP ACCUMULATION SPECIFICALLY AT ADNP TARGET SITES, BUT NOT AT SITES NOT BOUND BY ADNP. LAST, ADNP-DEFICIENT MESCS AND MESCS EXCLUSIVELY EXPRESSING AN ADNP MUTANT LACKING THE HOMEODOMAIN FAIL TO DIFFERENTIATE INTO NEURAL PROGENITOR CELLS (NPCS), INDICATING AN ESSENTIAL ROLE FOR ADNP AND ITS HOMEODOMAIN IN NEURONAL DIFFERENTIATION. THESE RESULTS SUGGEST A POTENTIALLY NOVEL MECHANISM – R-LOOP RESOLUTION – FOR ADNP, AND POSSIBLY OTHER HOMEODOMAIN-CONTAINING PROTEINS, IN GENE REGULATION, WHICH CAN LINK R-LOOP DYSFUNCTION TO NUMEROUS DISORDERS AND DISEASES CAUSED BY MUTATIONS IN HOMEODOMAIN PROTEINS. BASED ON OUR PRELIMINARY DATA, WE HYPOTHESIZE THAT ADNP DRIVES NEURONAL DIFFERENTIATION BY SUPPRESSING R-LOOPS; AS A COROLLARY, WE PROPOSE THAT RESOLVING R-LOOPS THAT ACCUMULATE UPON ADNP LOSS MAY RESCUE GENE EXPRESSION PROGRAMS TO ENABLE NEURONAL DIFFERENTIATION. WE PROPOSE TWO AIMS TO TEST OUR CENTRAL HYPOTHESIS. IN AIM 1, WE WILL ELUCIDATE THE MOLECULAR BASIS OF R-LOOP RESOLUTION BY ADNP BY IDENTIFYING ROLES OF THE ADNP PROTEIN DOMAINS AND THEIR INTERACTIONS WITH NUCLEIC ACIDS. IN AIM 2, WE WILL ELUCIDATE HOW ADNP SUPPRESSION OF R-LOOPS INFLUENCES NEURONAL DIFFERENTIATION.

RATIONAL DESIGN AND CLINICAL DEVELOPMENT OF SHEPHERDIN: A NOVEL ANTI-CANCER AGENT

DEVELOPMENT OF A NOVEL INDUCER FOR EBV LYTIC THERAPY

PEDIATRIC IMMUNE CORRELATES OF EARLY ANTI-HIV THERAPY

THE GENETICS OF TUMOR SUPPRESSION BY P53 - PROJECT SUMMARY THIS SUBMISSION REPRESENTS A COLLABORATIVE APPLICATION FROM MAUREEN MURPHY PHD AT THE WISTAR INSTITUTE IN PHILADELPHIA AND KARA MAXWELL MD/PHD FROM THE UNIVERSITY OF PENNSYLVANIA PERELMAN SCHOOL OF MEDICINE. THE GOAL IS TO STUDY THE FUNCTION OF A NOVEL P53 MUTATION THAT WAS FIRST DISCOVERED IN SEVERAL HIGHLY CANCER- PRONE FAMILIES SEEN IN DR. MAXWELL’S CLINIC AT THE HOSPITAL OF THE UNIVERSITY OF PENNSYLVANIA. THIS VARIANT, G334R IN P53 PROTEIN (G331R IN MOUSE P53) WAS FOUND IN NINE DIFFERENT FAMILIES WITH MULTIPLE CANCERS IN MULTIPLE GENERATIONS, INCLUDING COUSINS WITH PEDIATRIC ADRENAL TUMORS; THE LATTER ARE A HALLMARK OF CANCER- PREDISPOSING MUTATIONS IN TP53. MOREOVER, WE FIND THAT SEVERAL TUMORS FROM THESE PATIENTS SHOW LOSS OF HETEROZYGOSITY FOR P53. WE SHOW THAT THE G334R VARIANT IS IMPAIRED FOR OLIGOMERIZATION, AND FOR THE INDUCTION OF A SMALL SUBSET OF P53 TARGET GENES, SEVERAL OF WHICH ARE THEMSELVES TUMOR SUPPRESSORS (PCLO, PLXNB3, AND OTHERS). OUR FUNCTIONAL ANALYSES OF THE G334R VARIANT SUGGEST THAT THIS IS NOT A TRADITIONAL LI FRAUMENI MUTANT; THAT IS, A MUTATION THAT COMPLETELY INACTIVATES P53 FUNCTION. RATHER, OUR FUNCTIONAL DATA ARE MOST CONSISTENT WITH G334R BEING A P53 “HYPOMORPH”, OR A GENETIC VARIANT THAT SHOWS IMPAIRED, BUT NOT COMPLETELY INACTIVE, FUNCTION. SPECIFICALLY, 1) G334R POSSESSES SOME ABILITY TO SUPPRESS COLONY FORMATION IN TUMOR CELLS, THOUGH IT SHOWS LESS ABILITY THAN WILD TYPE P53; 2) G334R IS FULLY CAPABLE OF TRANSACTIVATING THE OVERWHELMING MAJORITY OF P53-REGULATED GENES, BUT IS DEFECTIVE IN THE TRANSACTIVATION OF APPROXIMATELY TWO DOZEN P53 TARGET GENES; 3) THE PEAK INCIDENCE FOR BREAST CANCER IN G334R INDIVIDUALS IS BETWEEN THE AGES OF 35-55, WHILE MOST CASES OF LI FRAUMENI OCCUR BETWEEN THE AGES OF 20-40, AND SPORADIC BREAST CANCER PEAKS BETWEEN THE AGES OF 55- 75. THE OVERARCHING GOAL OF THIS PROPOSAL IS TO IDENTIFY THE MECHANISM(S) WHEREBY G334R IS IMPAIRED FOR FUNCTION. IN A COMPLETELY UNEXPECTED FINDING, WE SHOW THAT THERE ARE SOME ACTIVITIES OF THE G334R HYPOMORPH THAT ARE SHARED WITH TWO OTHER CANCER-PREDISPOSING P53 HYPOMORPHS, P47S AND Y107H. THESE ACTIVITIES INCLUDE A HEIGHTENED PROPENSITY TO MISFOLD INTO A CONFORMATION THAT IS SPECIFIC FOR MUTANT P53, AS WELL AS INCREASED SENSITIVITY TO GLUTAMINASE INHIBITORS. WE ALSO SHOW THAT USING RNA SEQUENCING DATA AND MACHINE LEARNING APPROACHES, WE HAVE IDENTIFIED A 42-GENE SIGNATURE THAT IS PREDICTIVE OF A P53 HYPOMORPH, AND CAN DISTINGUISH A P53 HYPOMORPH FROM WT P53 OR A BENIGN VARIANT WITH 100% ACCURACY. THE PROPOSED RESEARCH WILL HELP US BETTER UNDERSTAND TUMOR SUPPRESSION BY P53, AND TO IDENTIFY OTHER CARRIERS OF HYPOMORPHIC GENETIC VARIANTS OF P53.

DEVELOPING POTENT HIV-SPECIFIC CAR T CELLS THROUGH OPTIMIZING ANTIGEN SENSITIVITY - PROJECT SUMMARY GIVEN THE INABILITY OF THE IMMUNE RESPONSE TO CONSISTENTLY SUPPRESS HIV WITHOUT ANTIRETROVIRAL THERAPY AND THE INSUFFICIENT POTENCY OF CURRENT CURE APPROACHES, GENETIC ENGINEERING MODALITIES MAY OFFER A POTENT ALTERNATIVE TO INTRINSIC IMMUNITY. CHIMERIC ANTIGEN RECEPTOR (CAR) T CELLS HAVE SHOWN IMPRESSIVE EFFICACY IN ELIMINATING BLOOD CELL CANCERS IN THE CLINIC, AND CAR T CELLS RE-ENGINEERED TO TARGET HIV USING THE CD4 ECTODOMAIN REPRESENT A POTENT ESCAPE-RESISTANT CELLULAR THERAPY DEMONSTRATED TO HAVE ENHANCED CYTOLYTIC FUNCTION. OUR PRIOR WORK HAS DEMONSTRATED THAT WHEN ENGINEERED TO EXPRESS BOTH THE 4-1BB AND CD28 COSTIMULATORY DOMAINS AND PROTECTED FROM HIV INFECTION, HIV-SPECIFIC CD4 ECTODOMAIN CAR T CELLS CAN REDUCE ACUTE VIREMIA, PREVENT CD4+ T CELL LOSS, AND REDUCE VIRAL BURDEN IN THE TISSUES OF HIV-INFECTED HUMANIZED MICE. HOWEVER, THE OF REDUCTION PLASMA VIRAL LOADS WAS ULTIMATELY TRANSIENT, SUGGESTING THAT THE POTENCY OF HIV-SPECIFIC CAR T CELLS SHOULD BE FURTHER OPTIMIZED FOR CLINICAL TRANSLATION. WE PROPOSE THAT LOW EXPRESSION OF HIV ENVELOPE (ENV) ON HIV-INFECTED CELLS YIELDS TARGETS WITH LOW ANTIGEN DENSITY. OUR PRELIMINARY DATA DEMONSTRATES THAT THE POTENCY OF CD4 ECTODOMAIN CARS IS SEVERELY ATTENUATED IN INSTANCES OF LOW ANTIGEN DENSITY. WE REASON THAT THIS NECESSITATES THE DESIGN OF HIV-SPECIFIC CAR T CELLS WITH ENHANCED ANTIGEN SENSITIVITY. AS THE CHARACTERISTICS DICTATING ANTIGEN SENSITIVITY ARE NOT YET FULLY DESCRIBED FOR CAR T CELLS, WE GENERATED A DIVERSE PANEL OF BROADLY NEUTRALIZING ANTIBODY-BASED CAR T CELLS, SPECIFICALLY TO (1) DETERMINE THE RELATIONSHIP BETWEEN EPITOPE ACCESSIBILITY, CAR AVIDITY, AND ANTIGEN SENSITIVITY ON HIV-SPECIFIC CAR T CELL POTENCY AND (2) IDENTIFY ORTHOGONAL CAR-DRIVEN ESCAPE PATTERNS INFORMING OPTIMAL MULTI-SPECIFICITY CAR PRODUCTS THAT RESTRICT HIV ESCAPE IN VIVO. THE ULTIMATE GOAL IS TO GENERATE AN ENHANCED HIV-SPECIFIC CAR T CELL PRODUCT WITH THE ABILITY TO KILL HIV-INFECTED CELLS EXPRESSING LOW LEVELS OF ANTIGEN IN ORDER TO IMPROVE THE TRANSLATIONAL POTENTIAL OF A CAR T CELL APPROACH FOR A FUNCTIONAL HIV CURE.

REMODELING OF THE HOST EPIGENOME DURING EBV INFECTION - PROJECT SUMMARY IN EBV+ LYMPHOMAS VIRAL PROTEIN EXPRESSION IS THE ONCOGENIC DRIVER AND PROGRESSIVE FORCE. HOWEVER, THE MECHANISMS BY WHICH EBV INFECTION ALTERS B CELL BIOLOGY TO CONTRIBUTE TO THE DEVELOPMENT OF EBV+ LYMPHOMAS ARE NOT WELL UNDERSTOOD. EBV INFECTION OF PRIMARY B LYMPHOCYTES RESULTS IN CELL ACTIVATION, PROLIFERATION, AND IMMORTALIZATION. THESE PROGRAMS ARE INDUCED BY EBV MIMICKING THE NORMAL ACTIVATION OF B CELLS BY ANTIGENS OR T CELLS. SINCE LMP1 IS NEEDED FOR EBV TO TRANSFORM NAÏVE B CELLS INTO IMMORTALIZED B CELLS, CONSIDERABLE WORK HAS BEEN ATTEMPTED TO PHARMACOLOGICALLY TARGET THE LMP1-ACTIVATED ONCOGENIC PATHWAYS. HOWEVER, IN VIVO ATTEMPTS TO TARGET THESE PATHWAYS HAVE FAILED TO ABOLISH EBV-DRIVEN ONCOGENESIS COMPLETELY. THIS LACK OF EFFICACY OF THE TARGETED APPROACHES SUGGESTED TO US THAT ANOTHER, UNDISCOVERED OR UNAPPRECIATED, ONCOGENIC MECHANISM DOWNSTREAM OF LMP1 MIGHT BE AT WORK. THIS PROPOSAL AIMS TO IDENTIFY THE MECHANISM BY WHICH EBV INFECTION CONTRIBUTES TO DEVELOPING B CELL MALIGNANCIES. OUR RECENT, PUBLISHED WORK SHOWED THAT THE EBV ONCOPROTEIN LMP1 ACTIVATES THE CHROMATIN-ASSOCIATED FACTOR PARP1 TO EPIGENETICALLY REGULATE GENES THAT ARE IMPORTANT FOR SUSTAINING CELL PROLIFERATION. EXPANDING UPON THESE FINDINGS, OUR PRELIMINARY DATA INDICATE THAT THE REORGANIZATION OF THE 3D STRUCTURE AND FUNCTION OF CHROMATIN IN B CELLS IS A NOVEL AND UNDERAPPRECIATED MECHANISM TARGETED BY EBV THROUGH LMP1 TO ALTER GENE EXPRESSION AND REPROGRAMMING IN EBV INFECTED CELLS. WE DISCOVERED THAT: 1) EXPRESSION OF LMP1 ALONE IS SUFFICIENT TO ALTER THE 3D STRUCTURE OF THE B CELL GENOME; 2) THE EBV/LMP1-INDUCED CHANGES IN CHROMATIN STRUCTURE ARE ASSOCIATED WITH PARP1 ACTIVITY, AND WITH OCCUPANCY BY THE CHROMATIN ORGANIZERS CTCF AND YY1; 3) EBV ACTIVATION OF PARP1 REQUIRES ERK ACTIVATION BY LMP1; AND 4) PARP INHIBITORS ELICIT CYTOTOXICITY IN EBV+ LYMPHOMAS IN VIVO. BASED ON THESE PRELIMINARY DATA AND OUR PREVIOUS WORK WE HYPOTHESIZE THAT IN EBV+ B CELLS, LMP1 EXPRESSION ACTIVATES, VIA THE ERK PATHWAY, PARP1, PROMOTING CONFORMATIONAL CHANGES IN THE HOST GENOME ARCHITECTURE THAT DRIVE ONCOGENESIS. WE WILL ALSO TEST THE HYPOTHESIS THAT PARP1 IS AN ATTRACTIVE THERAPEUTIC TARGET FOR EBV LYMPHOMAS.

HIV THERAPY & INTERRUPTION RCT IN RESOURCE POOR CLINIC

STRUCTURAL AND BIOCHEMICAL ANALYSIS OF TELOMERASE FUNCTION

REGULATION OF SMOOTH MUSCLE CELL FUNCTION BY CD44 IN CARDIOVASCULAR DISEASE

METABOLIC AND MOLECULAR REGULATION OF MYELOID CELL FUNCTIONS IN BRAIN CANCER - GLIOBLASTOMA (GBM), THE MOST AGGRESSIVE AND LETHAL FORM OF BRAIN CANCER, IS CHARACTERIZED BY A PROFOUND IMMUNOSUPPRESSIVE MICROENVIRONMENT (TME) THAT RESTRICTS THE EFFECTS OF PROMISING IMMUNOTHERAPIES. THEREFORE, THERE IS A PRESSING NEED TO DEVELOP MORE EFFECTIVE INTERVENTIONS TO OVERCOME THIS MECHANISM OF RESISTANCE. TUMOR ASSOCIATED MACROPHAGES (TAMS) ARE A MIXTURE OF MONOCYTE-DERIVED MACROPHAGES (MDM) AND MICROGLIA (MG), AND THEY ARE INSTRUMENTAL FOR THE MAINTENANCE OF THE IMMUNOSUPPRESSIVE STATE OF GBM. HOWEVER, THERE ARE NO EFFECTIVE APPROACHES TO OVERCOME THE IMMUNOSUPPRESSIVE ACTIVITY OF TAMS IN GBM, MAINLY DUE TO AN INCOMPLETE UNDERSTANDING OF TAM REGULATORY FUNCTIONS. OUR LONG TERM-GOAL IS TO DISSECT TARGETABLE METABOLIC AND MOLECULAR MECHANISMS REGULATING TAM FUNCTIONS IN THE CONTEXT OF GBM; AS THESE DISCOVERIES WILL FACILITATE NOVEL THERAPIES TO TARGET IMMUNOSUPPRESSION AND IMPROVE THE DISMAYING OUTCOME OF GBM PATIENTS. A RECENT STUDY DEMONSTRATED THAT TAM ARE MAJOR CONSUMERS OF GLUCOSE AND MAINTAIN A ROBUST GLUCOSE METABOLISM IN THE TME. HOWEVER, IT HAS NOT YET BEEN DETERMINED HOW GBM SUPPORTS THE ADAPTATION TO GLUCOSE METABOLISM IN TAMS AND THE FUNCTIONAL CONSEQUENCES OF THIS ADAPTATION ALSO REMAIN ELUSIVE. ENDOPLASMIC RETICULUM (ER) STRESS ACTIVATION IS ASSOCIATED WITH THE MALIGNANT PROGRESSION OF GLIOMA AND WITH THE INFILTRATION OF ANTI-INFLAMMATORY MACROPHAGES. PKR-LIKE ER KINASE (PERK), A CRITICAL ER STRESS SENSOR, WAS FOUND TO BE SIGNIFICANTLY ACTIVATED IN HUMAN GLIOMA TISSUES, AND ITS INHIBITION ALTERED ATP/LACTATE PRODUCTION BY GLIOMA CELLS. OUR PRELIMINARY DATA EXPANDED THESE FINDINGS INDICATING THAT MDM DEMONSTRATED HIGHEST GLUCOSE AVIDITY AMONG MG AND NEOPLASTIC CELLS IN GBM TUMORS, AND PERK WAS STRONGLY ACTIVATED IN GBM INFILTRATING GLUT1+MDM. CONTRARY TO MG, MDM EXHIBITED POTENT IMMUNOSUPPRESSIVE ACTIVITY. GLUT1+MDM WERE THE ONLY CONTRIBUTORS TO THE SUPPRESSIVE ACTIVITY ASSOCIATED WITH MDM IN GBM TUMORS. GBM-DERIVED FACTORS PRIMED ACTIVATION OF PERK SIGNALING IN MDM, WHICH CORRELATED WITH METABOLIC REPROGRAMMING RESULTING IN HIGH GLYCOLYSIS, IMMUNOSUPPRESSIVE FUNCTIONS, HISTONE LACTYLATION, AND NO CHANGE IN HISTONE ACETYLATION. BASED ON OUR CRUCIAL OBSERVATIONS, WE HYPOTHESIZE THAT A PERK-DRIVEN PERTURBATION OF GLUCOSE METABOLISM IN MDM GOVERNS THEIR IMMUNOSUPPRESSIVE FUNCTIONS VIA LACTATE-DERIVED LACTYLATION OF HISTONE LYSINE RESIDUES. WE WILL TEST THIS HYPOTHESIS THROUGH THE FOLLOWING AIMS: AIM1: TO ELUCIDATE UNDERLYING MECHANISMS OF HOW PERK GOVERNS GLYCOLYSIS IN MDM IN GBM TUMORS; AIM2: TO DEFINE GLUCOSE-DRIVEN EPIGENETIC MODIFICATIONS THAT REGULATES IMMUNOSUPPRESSIVE PROGRAMS IN MDM; AIM3: TO INVESTIGATE THE THERAPEUTIC POTENTIAL OF AN EPIGENETIC TARGETING APPROACH TO MODULATE THE FUNCTIONS OF TAMS IN GBM. THE PROPOSED STUDIES ARE HIGHLY INNOVATIVE BECAUSE THEY WILL ELUCIDATE A PREVIOUSLY UNCHARACTERIZED LINK BETWEEN ER STRESS AND GLUCOSE METABOLISM THAT REGULATES THE ACTIVITY OF TAMS VIA EPIGENETIC MECHANISMS. OUR PROPOSAL WILL PROVIDE A MECHANISTIC RATIONALE FOR THE DEVELOPMENT OF NOVEL THERAPIES TO TARGET IMMUNOSUPPRESSIVE TAMS AND ENHANCE THE EFFICACY OF IMMUNOTHERAPY IN GBM PATIENTS.

CHARACTERIZING MECHANISMS OF TRANSCRIPTIONAL ACTIVATION USING LIVE CELL IMAGING

CELLULAR INNATE ACTIVATION AS A TACTIC TO PREVENT HIV-1 TRANSMISSION

MOLECULAR NETWORK REGULATING DENDRITIC CELL DIFFERENTIATION IN CANCER

REGULATION AND FUNCTIONS OF 3'UTRS IN CELLULAR STRESS

MOLECULAR PATHOLOGY OF VASCULAR INJURY

R-LOOP FUNCTIONS IN NEURONAL GENE EXPRESSION AND GENOME ORGANIZATION - PROJECT SUMMARY THE GOAL OF THIS PROPOSAL IS TO UNCOVER THE MECHANISTIC CONNECTION BETWEEN R-LOOPS AND GENE EXPRESSION THROUGH EFFECTS ON GENOME ARCHITECTURE TO UNDERSTAND HOW R-LOOP DEREGULATION CAN CONTRIBUTE TO NEURODEVELOPMENTAL DISORDERS. R-LOOPS ARE POORLY UNDERSTOOD RNA-CONTAINING CHROMATIN STRUCTURES THAT ACCUMULATE IN, AND CONTRIBUTE TO THE ETIOLOGY OF, SEVERAL NEURODEVELOPMENTAL DISORDERS. THIS INCLUDES ACTIVITY-DEPENDENT NEUROPROTECTIVE PROTEIN (ADNP) SYNDROME, ALSO KNOWN AS HELSMOORTEL-VAN DER AA SYNDROME, WHICH IS A RARE CONDITION IN CHILDREN THAT EXHIBIT SIGNS OF AUTISM. AT PRESENT, HOW R-LOOPS CONTRIBUTE TO NEUROLOGICAL DISORDERS LIKE ADNP SYNDROME IS UNCLEAR. ACCUMULATED R LOOPS LEAD TO AN INCREASED DNA DAMAGE RESPONSE AND CAN ALSO ALTER TRANSCRIPTION OF NEIGHBORING GENES. HOWEVER, THE MECHANISTIC BASIS FOR R LOOPS-MEDIATED CHANGES IN GENE EXPRESSION ARE UNKNOWN AND THE FUNCTIONAL RELEVANCE OF THIS PROCESS TO DISORDERS LIKE ADNP SYNDROME IS UNEXPLORED. WE HAVE MADE THE SURPRISING FINDING THAT R-LOOPS ARE HIGHLY ENRICHED AT A SUBSET OF BINDING SITES FOR CTCF. PRELIMINARY DATA SHOW THAT R-LOOPS STRENGTHEN CTCF INTERACTIONS WITH CHROMATIN. WE FOUND THAT CONDITIONS LEADING TO LOSS OR GAIN OF R-LOOPS CAN DECREASE OR INCREASE CTCF RECRUITMENT, RESPECTIVELY, AND AFFECT LONG- RANGE GENOME INTERACTIONS. WE RECENTLY DEMONSTRATED THAT THE ACTIVITY-DEPENDENT NEUROPROTECTIVE PROTEIN (ADNP), A CRITICAL PROTEIN FOR BRAIN DEVELOPMENT, IS A SITE-SPECIFIC R-LOOP RESOLVER. ADNP HETEROZYGOUS MISSENSE OR FRAMESHIFT MUTATIONS CAUSE ADNP SYNDROME, A SEVERE NEURODEVELOPMENTAL DISORDER. HUMAN INDUCED PLURIPOTENT STEM CELLS (HIPSCS) DERIVED FROM PATIENTS WITH ADNP SYNDROME SHOW INCREASED R-LOOPS AND CTCF ACCUMULATION AT ADNP BINDING SITES. WE FIND THAT ADNP BINDING SITES ARE ENRICHED FOR SEQUENCES THAT ARE RECOGNIZED BY THE GENOME ARCHITECTURAL PROTEIN YY1, WHICH HAS IMPORTANT FUNCTIONS IN REGULATING ENHANCER- PROMOTER INTERACTIONS ESPECIALLY IN THE NEURAL LINEAGE. ADNP AND YY1 CO-LOCALIZES AT ACTIVE ENHANCERS. OUR PRELIMINARY DATA ALSO IDENTIFIED CHANGES IN DNA METHYLATION IN ADNP SYNDROME HIPSCS THAT CAN POTENTIALLY IMPACT CTCF AND YY1 BINDING TO CAUSE PATHOGENIC GENOME MISFOLDING AND ABERRANT NEURAL GENE EXPRESSION IN ADNP SYNDROME. WE HYPOTHESIZE THAT R-LOOPS HAVE A REGULATORY FUNCTION, AND THAT THEY TARGET CTCF AND YY1 TO SPECIFIC GENOMIC SITES DURING NEURODIFFERENTIATION. WE POSIT THAT THEY MAY BE CRITICAL FOR LONG-RANGE GENOME INTERACTIONS THAT REINFORCE NEURAL LINEAGE SPECIFIC GENE EXPRESSION PROGRAMS. WE PROPOSE TO DECIPHER THE IMPACT OF R-LOOP DEREGULATION ON GENOME ORGANIZATION AND GENE EXPRESSION IN THE NEURAL LINEAGE THROUGH THE LENS OF ADNP SYNDROME. IN AIM 1, WE WILL EVALUATE R-LOOP MEDIATED REGULATION OF CTCF AND YY1 LOCALIZATION DURING NEURODIFFERENTIATION. IN AIM 2, WE WILL EXAMINE THE EPIGENETIC CONSEQUENCES OF DISTINCT ADNP SYNDROME MUTATIONS AND THEIR IMPACT ON GENOME REGULATORY INTERACTIONS.

STRESS RESPONSE FUNCTIONS OF ADAR1 REGULATED BY MAP KINASES

INTEGRATIVE APPROACH TO COMPREHENSIVE ANALYSIS OF HIGH THROUGHPUT DATA ON A CANCER CENTER LEVEL

CELL INTRINSIC AND EXTRINSIC EFFECTS OF ZINC METABOLISM IN THERAPY RESISTANT MELANOMA - PROJECT SUMMARY/ABSTRACT THE ULTIMATE GOAL OF THIS PROPOSAL IS TO ADDRESS THE FUNDAMENTAL GAP IN KNOWLEDGE ON THE MECHANISMS DRIVING RESISTANCE TO IMMUNE CHECKPOINT BLOCKADE (ICB), PARTICULARLY IN MELANOMA PATIENTS WHO EXHIBIT DOWNREGULATION OR DELETION OF P16 (~40% OF CASES). THE RESEARCH PLAN FOCUSES ON ELUCIDATING THE MECHANISTIC ROLE OF P16 LOSS IN REGULATING ZINC (ZN) HOMEOSTASIS THROUGH THE ZN TRANSPORTER SLC39A9. THE GOAL IS TO EXPLORE WHETHER MANIPU- LATING ZN LEVELS ENHANCE THE EFFICACY OF CURRENT ICB IN P16LOW MELANOMAS. DRAWING ON A COMPELLING IN VIVO CRISPR KNOCKOUT SCREEN, OUR DATA REVEAL THAT THE KNOCKDOWN OF SLC39A9 SENSITIZES P16LOW MELANOMAS TO ANTI- PD1 ICB. ADDITIONALLY, INCREASED SLC39A9 EXPRESSION CORRELATES WITH UNFAVORABLE OUTCOMES IN MELANOMA PA- TIENTS UNDERGOING ICB. NOTABLY, LOSS OF P16 EXPRESSION LEADS TO AN INCREASE IN PLASMA MEMBRANE SLC39A9, RESULTING IN THE SEQUESTRATION OF ZN FROM THE MICROENVIRONMENT AND CREATING A ZN-DEPLETED TUMOR MICROENVIRON- MENT. WE PROPOSE TWO MAJOR CONSEQUENCES OF THIS REPROGRAMED ZN HOMEOSTASIS IN P16LOW TUMORS THAT CONTRIBUTE TO IMPAIRED ICB RESPONSE. FIRSTLY, INCREASED INTRACELLULAR ZN IN P16LOW CANCER CELLS DECREASES CGAS-STING SIG- NALING AND DOWNREGULATES CXCL10, A T CELL CHEMOKINE. SECONDLY, LIMITED ACCESS TO MICROENVIRONMENTAL ZN COMPROMISES CD8 T CELL EFFECTOR FUNCTION, GIVEN ITS CRUCIAL ROLE IN EARLY TCR ACTIVATION. THIS RESEARCH PLAN IS STRUCTURED AROUND TWO OVERARCHING SCIENTIFIC AIMS: 1) INTERROGATE THE MECHANISM UNDERLYING ZN-DEPENDENT ATTEN- UATION OF CGAS-STING SIGNALING AS A CONSEQUENCE OF SLC39A9 AND 2) DETERMINE THE CONTRIBUTION OF LOW TME ZN ON CD8 T CELL RECRUITMENT AND DIFFERENTIATION AND ICB RESPONSE. SUCCESSFUL COMPLETION OF THESE AIMS WILL NOT ONLY YIELD NEW INSIGHTS INTO THE MECHANISTIC ROLE OF P16 IN ZN METABOLISM BUT WILL ALSO POSITION THE MODULATION OF ZN HOMEOSTASIS AS A PROMISING STRATEGY TO ENHANCE THERAPEUTIC OUTCOMES FOR MELANOMA PATIENTS WITH LOW P16 EXPRESSION. GIVEN THE PREVALENCE OF P16 ALTERATIONS IN APPROXIMATELY 50% OF ALL HUMAN CANCERS, THE IMPLICATIONS OF THESE STUDIES EXTEND FAR BEYOND MELANOMA, OFFERING A FOUNDATION FOR IDENTIFYING METABOLIC VULNERABILITIES AND INFORMING FUTURE CANCER THERAPEUTIC STRATEGIES ON A BROADER SCALE.

NOTCH AS A NEW THERAPEUTIC TARGET IN HEMATOLOGICAL MALIGNANCIES

ROLE OF GENE SILENCING IN THE DNA DAMAGE RESPONSE

UNDERSTANDING THE IMMUNE BIOLOGY OF CHECKPOINT INHIBITORS TO DEVELOP NEW STRATEGIES FOR THERAPY

TARGETING RESIDENT MACROPHAGES TO TREAT METASTATIC OVARIAN CANCER

DOES INFLAMMATION ENHANCE RENGENERATION?

DETERMINE THE ROLE OF CANONICAL WNT SIGNALING IN OVARIAN TUMORIGENESIS

COMPUTATIONAL METHODS FOR DISCOVERY OF DISEASE-MODULATING MICROBIAL GENES - ABSTRACT MICROBIAL GENES IN GUT AND DISEASED TISSUES WERE RECENTLY LINKED WITH PROGRESSION AND OUTCOMES OF DIFFERENT HUMAN DISEASES, INCLUDING CANCER AND IMMUNE DISEASES. DECIPHERING THE PATHOGENIC ROLES OF DIFFERENT MICROBIAL GENES CAN IMPROVE DIAGNOSIS, PROGNOSTICATION, AND TREATMENT OF HUMAN DISEASES. YET, CURRENT METHODS FOR RNA AND SHOTGUN METAGENOMIC SEQUENCING FOCUS ON MICROBIAL SPECIES, AND DO NOT ALLOW A SYSTEMATIC DETECTION OR QUANTIFICATION OF MICROBIAL GENES. THEREFORE, CURRENT METHODS FOR DISEASE PROGNOSTICATION AND BIOMARKER DISCOVERY ARE UNABLE TO CONSIDER MICROBIAL GENES THAT INFLUENCE HUMAN DISEASES. OUR OVERARCHING HYPOTHESIS IS THAT THERE ARE UNKNOWN ASSOCIATIONS BETWEEN HUMAN DISEASES AND THE MICROBIAL GENES IN DISEASED TISSUES AND IN THE GUT. OUR LONG-TERM GOAL IS TO UNRAVEL THESE ASSOCIATIONS USING NOVEL COMPUTATIONAL APPROACHES THAT WILL ALLOW DETECTION AND QUANTIFICATION OF MICROBIAL GENES IN DISEASES. IN AIM 1 WE WILL DEVELOP METHODS THAT HARNESS RNA SEQUENCING TO DETECT MICROBIAL GENE EXPRESSION IN DISEASED TISSUES. THIS WILL ALLOW MICROBIAL BIOMARKER DISCOVERY AND PROVIDE A COMPREHENSIVE DATABASE OF THE MICROBIAL GENES THAT ARE EXPRESSED IN VARIOUS HUMAN TISSUES AND CONDITIONS. IN AIM 2 WE WILL DEVELOP METHODS TO QUANTIFY GUT MICROBIAL GENE CAPACITY IN HUMAN DISEASES. THIS WILL ALLOW IDENTIFICATION OF GUT MICROBIAL PROTEINS, PEPTIDES, AND DOMAINS THAT ARE IMPORTANT IN HUMAN DISEASES, TO ULTIMATELY YIELD NEW DIAGNOSTIC AND TREATMENT STRATEGIES BASED ON GUT MICROBIOMES. OVERALL, THIS PROJECT WILL PROVIDE INNOVATIVE METHODS TO ALLOW DETECTION AND QUANTIFICATION OF MICROBIAL GENES FROM ABUNDANTLY USED SEQUENCING TECHNOLOGIES. WE WILL ESTABLISH USER FRIENDLY SOFTWARE AND DATABASES, ALLOWING NEW DISCOVERIES WITH EXISTING SEQUENCING PLATFORMS. WE EXPECT THAT THE METHODS DEVELOPED THROUGH THIS PROJECT WILL BE EXTENSIVELY ADOPTED BY THE RELEVANT RESEARCH COMMUNITIES, IMPROVING OUR UNDERSTANDING OF THE ROLES OF MICROBES IN HUMAN DISEASES AND ULTIMATELY ALLOWING THE DEVELOPMENT OF NEW DISEASE DETECTION AND INTERVENTION STRATEGIES BASED ON MICROBIAL GENES.

ANALYSIS OF HUMAN TELOMERIC STRUCTURAL VARIATION

DEVELOPING RATIONAL APPROACHES FOR ACRAL MELANOMA

(PQC2) PLASTICITY OF THE.PI3K NETWORK IN EARLY DORMANCY

DISCOVERY AND VALIDATION OF NOVEL SEROLOGICAL BIOMARKERS OF COLON CANCER

REGULATION OF TUMOR MICROENVIRONMENT IN CANCER

OVERCOMING PLATINUM RESISTANCE IN OVARIAN CANCER THROUGH BET INHIBITION

IMMUNOTHERAPEUTIC TARGETING OF SLC39A9 IN CDKN2A-LOW MELANOMA

REGULATION OF EBV REACTIVATION

ANTIVIRAL ACTIVITY OF PEG-IFN ALPHA IN CHRONIC HIV-1

A TRANSGENIC MODEL FOR B CELL TOLERANCE AND AUTOIMMUNITY

PARKIN TUMOR SUPPRESSION - PROJECT SUMMARY THE LOSS OF ENDOGENOUS TUMOR SUPPRESSORS IS AN OBLIGATORY STEP IN TUMOR ONSET AND PROGRESSION, BUT THE UNDERLYING MECHANISMS ARE ELUSIVE AND THE BREADTH OF THEIR TARGETS MOSTLY UNKNOWN. WE HAVE NOW DISCOVERED THAT PARKIN, A MITOCHONDRIA-ASSOCIATED E3 UBIQUITIN LIGASE BIALLELICALLY ALTERED IN EARLY-ONSET PARKINSON’S DISEASE, FUNCTIONS AS A NOVEL, DUAL MODE TUMOR SUPPRESSOR. THIS INVOLVES INHIBITION OF INTRINSIC TUMOR TRAITS OF CELL MOTILITY AND METABOLISM BUT ALSO REPROGRAMMING OF AN ANTITUMOR IMMUNE MICROENVIRONMENT. IN THIS PATHWAY, PARKIN RECRUITMENT TO MITOCHONDRIA “PRIMES” IMMUNOGENIC CELL DEATH, CONTROLS THE RELEASE OF DAMAGE- ASSOCIATED MOLECULAR PATTERN (DAMP), AND ACTIVATES KINASE CASCADES FOR TRANSCRIPTION OF INTERFERON GENES AND INFLAMMATORY CYTOKINES, INDEPENDENTLY OF MITOPHAGY. THE RESULT IS ENHANCED INTRATUMORAL INFILTRATION OF CD8+ T CELLS, REDUCED MYELOID IMMUNOSUPPRESSION, AND INHIBITION OF PRIMARY AND METASTATIC TUMOR GROWTH. BECAUSE PARKIN IS EPIGENETICALLY SILENCED IN ALL HUMAN CANCERS, DUAL MODE TUMOR SUPPRESSION IS A UNIVERSAL BARRIER AGAINST MALIGNANCY. THEREFORE, THE HYPOTHESIS THAT PARKIN TUMOR SUPPRESSION BRIDGES MITOCHONDRIAL ACTIVATION OF INTERFERON SIGNALING TO IMMUNE REPROGRAMMING OF THE MICROENVIRONMENT CAN BE FORMULATED AND WILL CONSTITUTE THE FOCUS OF THE PRESENT APPLICATION. THREE COMPLEMENTARY AND MULTIDISCIPLINARY SPECIFIC AIMS WILL ELUCIDATE THE ROLE OF PARKIN INNATE IMMUNITY IN TUMOR SUPPRESSION. IN THE FIRST SPECIFIC AIM, WE WILL CHARACTERIZE HOW PARKIN “PRIMES” IMMUNOGENIC CELL DEATH, ELUCIDATE THE ROLE OF ER STRESS AND AUTOPHAGY IN MITOCHONDRIAL NECROPTOSIS, AND MAP THE CELLULAR AND BIOCHEMICAL REQUIREMENTS OF DAMP RELEASE. THE SECOND SPECIFIC AIM WILL DISSECT THE SIGNALING REQUIREMENTS OF PARKIN ACTIVATION OF INNATE IMMUNITY WITH RESPECT TO CYTOSOLIC CGAS-STING AND INFLAMMASOME ACTIVATION, MITOCHONDRIAL RIG-I-MAVS SENSING AND ASSEMBLY OF A STAT1-REGULATED ISGF3 TRANSCRIPTIONAL COMPLEX FOR T CELL ACTIVATION AND DENDRITIC CELL FUNCTION. IN THE THIRD SPECIFIC AIM, WE WILL TEST THE IMPACT OF PARKIN INNATE IMMUNITY IN PRECLINICAL ORTHOTOPIC AND TRANSGENIC MODELS OF PRIMARY AND METASTATIC TUMOR GROWTH, IN VIVO, MODULATION OF ANTITUMOR VACCINATION STRATEGIES, AND DIFFERENTIAL SENSITIVITY TO CONVENTIONAL AND IMMUNE THERAPIES. THE APPLICATION BUILDS ON EXPANSIVE PRELIMINARY DATA, A NOVEL CONCEPT OF DUAL MODE PARKIN TUMOR SUPPRESSION, AND THE REDIRECTION OF MITOCHONDRIAL INNATE IMMUNITY, PREVIOUSLY KNOWN IN VIRAL INFECTIONS, FOR ANTITUMOR RESPONSES. THE RESULTS MAY BE PRACTICE CHANGING. AS PROPOSED HERE, THE ELUCIDATION OF PARKIN INNATE IMMUNITY WILL IDENTIFY NEW STRATEGIES TO RESTORE AN ANTITUMOR IMMUNE MICROENVIRONMENT, DAMPEN MYELOID IMMUNOSUPPRESSION AND ENABLE BROADER, MORE DURABLE PATIENT RESPONSES TO CONVENTIONAL AND IMMUNE THERAPY, INCLUDING IN LATE-STAGE DISEASE.

EPSTEIN-BARR VIRUS-DRIVEN B-CELL INFLAMMATION AND CNS TRAFFICKING IN MS

A THERAPEUTIC VACCINE FOR EBV-ASSOCIATED MALIGNANCIES

CONTROL OF CARDIOGENESIS BY MICRORNA EDITING

RECEPTOR DIVERSITY IN RECOGNITION OF INFLUENZA HA

3D HUMAN SKIN MODELS

EPIGENETIC REPROGRAMMING TO TARGET SENESCENT OVARIAN CANCER CELLS AND OVERCOME THERAPEUTIC RESISTANCE FEB 4, 2026, MOD 1 TRANSFERRED AWARD TO WISTAR INSTITUTE

STRUCTURE AND FUNCTION OF DNA REGULATORY PROTEINS

A FIRST IN HUMAN, PHASE I CLINICAL TRIAL OF MITOCHONDRIAL-TARGETED HSP90 INHIBITOR, GAMITRINIB IN ADVANCED AND METASTATIC PROSTATE CANCER

INVESTIGATING N332-GLYCAN INDEPENDENT V3-GLYCAN ANTIBODIES AS NOVEL TARGETS FOR HIV-1 VACCINE DESIGN - PROJECT SUMMARY / ABSTRACT A VACCINE THAT ELICITS BROADLY NEUTRALIZING ANTIBODIES (BNABS) AGAINST THE ENVELOPE (ENV) PROTEIN OF HIV-1 WOULD BE THE BEST APPROACH TO END THE AIDS EPIDEMIC; HOWEVER, NO EFFICACIOUS VACCINE HAS BEEN DEVELOPED TO DATE. BNABS TARGETING THE CONSERVED V3-GLYCAN EPITOPE OF ENV SUCH AS PGT121, 10-1074 AND BG18 BIND TO THE CONSERVED GDIR MOTIF OF ENV AND TO A KEY GLYCAN AT POSITION N332 (N332 GLYCAN). THE ABSENCE OF THE N332 GLYCAN IN MULTIPLE HIV-1 STRAINS LIMITS THE NEUTRALIZATION BREADTH OF THESE BNABS TO ~60-70%. BASED ON OBSERVATIONS BY US AND OTHERS, WE HYPOTHESIZED THAT V3-GLYCAN ANTIBODIES THAT DO NOT REQUIRE THE N332-GLYCAN FOR BINDING CAN BE ELICITED AND MATURED INTO BNABS BY VACCINATION. WE FURTHER HYPOTHESIZED THAT N332-GLYCAN INDEPENDENT V3-GLYCAN ANTIBODIES WILL HAVE GREATER NEUTRALIZATION BREADTH AND THAT IMMUNOTHERAPIES COMBINING N332-GLYCAN DEPENDENT AND INDEPENDENT BNABS WILL IMPOSE STRONG RESTRICTIONS TO VIRAL ESCAPE, RESULTING IN LONGER PERIODS OF SUPPRESSED VIREMIA. TO TEST THESE HYPOTHESES, WE DEVELOPED WIN332, AN ENV-BASED IMMUNOGEN THAT LACKS THE N332-GLYCAN. IMMUNIZATION WITH WIN332 IN NONHUMAN PRIMATES (NHPS) INDUCED A NEW CLASS OF N332-GLYCAN INDEPENDENT V3-GLYCAN ANTIBODIES. MOST IMPORTANTLY, WIN332 ELICITED N332-GLYCAN INDEPENDENT NEUTRALIZING ANTIBODIES TO THE V3-GLYCAN EPITOPE AS EARLY AS THREE WEEKS AFTER A SINGLE IMMUNIZATION. OUR STUDIES ESTABLISHED A NEW CLASSIFICATION OF V3-GLYCAN ANTIBODIES INTO TYPE-I (N332-GLYCAN DEPENDENT) AND TYPE-II (N332-GLYCAN INDEPENDENT) AND PRESENTED WIN332 AS A PROMISING VACCINE CANDIDATE TO STREAMLINE BNAB DEVELOPMENT (RELANO ET AL, NAT.IMMUNOL. IN PRESS). FURTHER SUPPORTING OUR OVERARCHING HYPOTHESIS AND THIS PROPOSAL, NOW TWO TYPE-II HUMAN BNABS THAT TARGET THE V3-GLYCAN EPITOPE IN A N332-GLYCAN INDEPENDENT MANNER, EPTC112 AND 007, HAVE BEEN REPORTED, DEMONSTRATING THAT TYPE-II LINEAGES CAN DEVELOP IN HUMANS AND ARE A SIGNIFICANT TARGET FOR VACCINE DESIGN. IN THIS PROPOSAL, WE WILL BUILD ON THESE STUDIES AND LEVERAGE WIN332 TO INVESTIGATE THE NEW CLASS OF TYPE-II V3-GLYCAN ANTIBODIES AND ITS POTENTIAL AS A NOVEL TARGET FOR VACCINE AND IMMUNOTHERAPY DESIGN. IN AIM 1, WE WILL LEVERAGE WIN332 AS A PROBE TO ISOLATE NEW TYPE-II BNABS AND BNAB PRECURSORS FROM INFANTS AND ADULTS LIVING WITH HIV-1 AND FROM HEALTHY DONORS RESPECTIVELY. WE WILL CHARACTERIZE THE NEWLY ISOLATED TYPE-II BNABS AND EVALUATE THEIR CAPACITY TO INCREASE NEUTRALIZATION COVERAGE AND RESTRICT VIRAL ESCAPE. IN AIM 2, WE WILL USE OUR STATE-OF-THE-ART METHODOLOGY TO PRODUCE NEW IMMUNOGLOBULIN KNOCKIN (IG KI) MICE THAT EXPRESS UNMUTATED PRECURSORS OF HUMAN AND NHP TYPE-II BNABS AND USE THESE MICE TO INVESTIGATE THE MATURATION PATHWAYS OF THESE BNABS. THROUGH ADVANCED PROTEIN ENGINEERING APPROACHES, STRUCTURAL STUDIES AND ASSESSMENT IN HUMANIZED MICE, WE WILL DESIGN NOVEL VACCINATION STRATEGIES TO MATURE N332-GLYCAN INDEPENDENT LINEAGES. WE EXPECT OUR INNOVATIVE VISION OF THE V3-GLYCAN EPITOPE WILL ENABLE THE IDENTIFICATION AND CHARACTERIZATION OF PREVIOUSLY OVERLOOKED BNAB LINEAGES OF POTENTIAL CLINICAL VALUE AND WILL RESULT IN NEW IMMUNIZATION PROTOCOLS TO STREAMLINE BNAB DEVELOPMENT.

REDIRECTING ANTIBODY EPITOPE SPECIFICITY WITH HIV-1 ENV-BASED IMMUNIZATION - PROJECT SUMMARY THE MAIN BARRIER TO DEVELOPING AN EFFICACIOUS HIV-1 VACCINE IS THE REQUIREMENT OF ELICITING BROADLY NEUTRALIZING ANTIBODIES (BNABS) TARGETING NEUTRALIZATION-SENSITIVE EPITOPES OF THE ENVELOPE (ENV) GLYCOPROTEIN. A MAJOR PROBLEM IS THE FREQUENT AND OVERWHELMING INDUCTION OF NON-NEUTRALIZING ANTIBODY RESPONSES DIRECTED TO THE GP41 BASE OF ENV WHEN USING SOLUBLE ENV TRIMERS AS IMMUNOGENS. THESE OFF-TARGET ANTIBODIES FAIL TO NEUTRALIZE HIV- 1, AS THE ENV BASE IS OCCLUDED BY THE VIRAL SURFACE AND NOT ACCESSIBLE FOR ANTIBODY RECOGNITION IN HIV-1 VIRIONS. THE BASE OF SOLUBLE ENV IMMUNOGENS HAS BEEN SHOWN TO BE IMMUNODOMINANT IN PRECLINICAL ANIMAL MODELS AND HUMANS, WHERE BASE ANTIBODIES HAVE DERAILED MULTIPLE CLINICAL TRIALS. DESPITE NUMEROUS ATTEMPTS TO OCCLUDE THE ENV BASE USING GLYCANS, MEMBRANE-BOUND ENV AND NANOPARTICLE SCAFFOLDS, THE IMMUNODOMINANCE OF THE BASE PERSISTS. INTERESTINGLY, OUR PRELIMINARY STUDIES SHOW THAT THE SPECIFICITY OF ANTI-ENV ANTIBODY LINEAGES IS NOT FIXED, INSTEAD, ANTIBODY LINEAGES CAN SWITCH THEIR EPITOPE SPECIFICITY THROUGH SOMATIC HYPERMUTATION IN RESPONSE TO IMMUNIZATION WITH ENV. IN PARTICULAR, WE SHOW THAT V3-GLYCAN BNAB LINEAGES CAN LOSE TRACK TO BECOME BASE ANTIBODIES AND THAT BASE ANTIBODIES CAN BE REDIRECTED TO TARGET THE NEUTRALIZING V3-GLYCAN EPITOPE. THUS, WE HYPOTHESIZE THAT BNAB PRECURSORS CAN BE DERAILED FROM MATURING INTO BNABS AND THAT NON-NEUTRALIZING ANTIBODIES CAN BE RESCUED TO MATURE INTO BNABS THROUGH IMMUNIZATION. HERE, WE WILL INVESTIGATE THIS PHENOMENON OF EPITOPE SWITCHING FROM ON-TARGET TO OFF-TARGET EPITOPES AND VICE VERSA THROUGH CHARACTERIZATION OF “DERAILED” AND “RESCUED” ANTIBODIES. WE WILL INVESTIGATE THE ON-TARGET TO OFF- TARGET SWITCH TO INFORM VACCINE DESIGN STRATEGIES THAT PREVENT ANTIBODY SWITCHING TOWARDS THE ENV BASE. WE WILL INVESTIGATE THE OFF-TARGET TO ON-TARGET SWITCH TO DESIGN VACCINE APPROACHES THAT RECYCLE BASE RESPONSES TO INDUCE BNABS. IN AIM 1, WE WILL INVESTIGATE THE MECHANISMS OF EPITOPE SWITCHING FROM THE NEUTRALIZING V3-GLYCAN EPITOPE OF ENV TO THE BASE USING MOUSE MODELS. IN AIM 2, WE WILL INVESTIGATE THE MECHANISM OF EPITOPE SWITCHING FROM THE BASE TO THE NEUTRALIZING V3-GLYCAN EPITOPE AND THE POTENTIAL OF “RESCUED” ANTIBODIES TO MATURE INTO BNABS IN MOUSE MODELS. IN AIM 3, WE WILL INVESTIGATE WHETHER OTHER BNAB LINEAGES AND OTHER OFF-TARGET ANTIBODIES CAN UNDERGO EPITOPE SWITCHING TOO. WE HAVE PRODUCED NINE NEW HUMANIZED AND RHESUS-IZED MOUSE MODELS AND INNOVATIVE AI-GENERATED IMMUNOGENS THAT WE WILL LEVERAGE FOR THESE SPECIFIC AIMS. STRUCTURAL APPROACHES WILL BE USED TO CHARACTERIZE THE MOLECULAR MECHANISMS OF EPITOPE SWITCHING. THESE AIMS WILL INFORM IMMUNOGEN DESIGN STRATEGIES TO KEEP BNAB LINEAGES ON TRACK AFTER INITIAL ACTIVATION AND TO “RESCUE” OFF-TARGET RESPONSES. COLLECTIVELY, THIS PROPOSAL WILL INVESTIGATE PREVIOUSLY OVERLOOKED CHALLENGES FOR HIV-VACCINE DESIGN AND WILL BRING NEW OPPORTUNITIES NOT ONLY FOR HIV VACCINES BUT FOR VACCINE DESIGN IN GENERAL.

EARLY INTERACTIONS OF DNA VIRUSES AND HOST

FUNCTIONAL ELUCIDATION OF BRCA1-CONTAINING COMPLEXES

SYNTHETIC LETHAL THERAPEUTIC APPROACHES FOR ARIDIA-MUTATED OVARIAN CANCER

MAPPING MURINE REGENEATION GENES

METABOLIC APPROACHES FOR ARID1A-MUTATED OVARIAN CANCER

IMMUNOLOGICAL APPROACHES FOR ARID1A-MUTATED OVARIAN CANCER

CHARACTERIZATION OF NOVEL VACCINE TARGETING FOLLICLE-STIMULATING HORMONE RECEPTOR IN OVARIAN CANCER

INVESTIGATING BCAA OXIDATION AND SIGNALING IN MACROPHAGES TO DRIVE ANTI-TUMOR IMMUNITY IN PDAC - PROJECT SUMMARY THE TREATMENT OF PANCREATIC DUCTAL ADENOCARCINOMA (PDAC) REMAINS A SIGNIFICANT CHALLENGE, WITH A 5-YEAR SURVIVAL RATE OF JUST 13%. RESISTANCE TO THERAPIES IS LARGELY ATTRIBUTED TO THE IMMUNO-SUPPRESSIVE TUMOR MICROENVIRONMENT (TME), WHICH IS CHARACTERIZED BY A FIBROTIC STROMA AND HIGH INFILTRATION OF IMMUNO- SUPPRESSIVE CELLS, INCLUDING TUMOR-ASSOCIATED MACROPHAGES (TAMS), THAT HINDER EFFECTOR T CELL RESPONSES. SHIFTING THE IMMUNO-SUPPRESSIVE TAM PHENOTYPE TOWARD AN IMMUNE-ACTIVATED STATE PRESENTS A PROMISING THERAPEUTIC STRATEGY. PREVIOUS STUDIES, INCLUDING OUR OWN, SUGGEST THAT TAM PHENOTYPES ARE SHAPED BY METABOLIC PATHWAYS. NOTABLY, THE METABOLISM OF BRANCHED-CHAIN AMINO ACIDS (BCAAS) HAS GAINED ATTENTION, AS ELEVATED BCAA LEVELS HAVE BEEN ASSOCIATED WITH MORE THAN A TWOFOLD INCREASED RISK OF DEVELOPING PDAC. WHILE PRIOR RESEARCH HAS FOCUSED ON BCAA METABOLISM IN TUMOR CELLS, PARTICULARLY THROUGH THE STUDY OF BCAA METABOLISM-RELATED KINASES AND ENZYMES, THE ROLE OF BCAA METABOLISM IN TAMS AND ITS IMPACT ON TAM PHENOTYPE AND ANTI-TUMOR IMMUNITY REMAINS UNEXPLORED. CONSEQUENTLY, IT REMAINS UNKNOWN WHETHER, AND HOW, THIS PATHWAY COULD BE THERAPEUTICALLY TARGETED. OUR FINDINGS INDICATED THAT IMMUNO-SUPPRESSIVE TAMS EXHIBIT REDUCED BCAA OXIDATION AND REDUCED INCORPORATION OF 13C-LABELED BCAAS INTO THE TCA CYCLE. INCREASING BCAA OXIDATION IN TAMS (VIA BCKDK DELETION) ENHANCED THEIR IMMUNO-STIMULATORY PHENOTYPE, ACTIVATED CD8+ T CELLS, AND REDUCED PDAC GROWTH. CONVERSELY, BCAA SUPPLEMENTATION INDUCED AN IMMUNO- SUPPRESSIVE TAM PHENOTYPE AND ACTIVATED THE PROTEIN KINASE RNA-LIKE ENDOPLASMIC RETICULUM KINASE (PERK) PATHWAY, A KEY ARM OF THE UNFOLDED PROTEIN RESPONSE. PERK DELETION IN TAMS REVERSED THIS EFFECT, PROMOTING IMMUNE ACTIVATION. PHARMACOLOGICALLY TARGETING BCAA METABOLISM—EITHER BY INCREASING BCAA OXIDATION OR INHIBITING PERK SIGNALING—ENHANCED ANTI-TUMOR IMMUNITY AND REDUCED PDAC GROWTH. LASTLY, IN PATIENTS WITH SOLID TUMORS, HIGH BCAA OXIDATION AND LOW PERK SIGNALING CORRELATED WITH BETTER RESPONSES TO ANTI-PD1 THERAPY. WE PROPOSE THAT REDUCED BCAA OXIDATION AND INCREASED BCAA SIGNALING IN TAMS DRIVE IMMUNO- SUPPRESSION IN PDAC. AIM 1 WILL INVESTIGATE THE MECHANISMS BY WHICH BCAA OXIDATION AND BCAA SIGNALING IN TAMS DIFFERENTIALLY INFLUENCE IMMUNE RESPONSES IN PDAC, FOCUSING ON TAM-SPECIFIC (I) BCAA OXIDATION VIA THE TCA CYCLE AND OXPHOS, AND (II) BCAA SIGNALING VIA THE PERK PATHWAY. AIM 2 WILL TEST WHETHER PHARMACOLOGICALLY TARGETING BCAA METABOLISM IMPROVES CHEMO-IMMUNOTHERAPY RESPONSES IN AGGRESSIVE PDAC MOUSE MODELS AND PATIENT-DERIVED ORGANOIDS. SUCCESSFUL COMPLETION OF THESE OBJECTIVES WILL ELUCIDATE HOW TAM-SPECIFIC BCAA OXIDATION AND BCAA SIGNALING INFLUENCE IMMUNO-SUPPRESSIVE TAM PHENOTYPE. IN THE LONG TERM, THIS WORK COULD LEAD TO NOVEL THERAPIES TARGETING TAM-SPECIFIC METABOLISM FOR PDAC TREATMENT.

MOLECULAR APPROACHES TO OVERCOME INTRINSIC DRUG RESISTANCE IN BRAF AND NRAS-MUTAN

DEVELOPMENT OF NOVEL BIOLOGICS TARGETING SIGLEC-7/9 FOR ACTIVATING AND ENGAGING NK CELLS AND MACROPHAGES FOR TREATMENT OF ADVANCED BREAST CANCER

INTEGRATED ANALYSES OF THE EPIGENOME TO UNDERSTAND THE MOLECULAR BASIS OF HEMATOPOIETIC MALIGNANCIES - PROJECT SUMMARY RESEARCH PLAN: AN IMPAIRED HEMATOPOIETIC DIFFERENTIATION PROCESS UNDERLIES BONE MARROW MALIGNANCIES LIKE LEUKEMIA, BUT WE STILL LACK THE MECHANISTIC UNDERSTANDING OF THE SEQUENCE OF REGULATORY EVENTS THAT MISLEADS THE DIFFERENTIATION PROCESS. SINCE EPIGENOMIC REGULATORY PATTERNS ARE MAJOR FEATURES OF LEUKEMIC DEVELOPMENT, UNDERSTANDING THE CHROMATIN DYNAMICS OF A FAILED (MALIGNANT) HEMATOPOIETIC DIFFERENTIATION PROCESS CAN HELP DEFINE THE MOLECULAR BASIS OF LEUKEMIA. A PREREQUISITE TO SUCH AN UNDERSTANDING IS A FRAMEWORK THAT ALLOWS INVESTIGATION OF THE PROGRESSIVE CHANGES IN THE ACTIVITY OF THE REGULATORY ELEMENTS (RE) DURING HEMATOPOIETIC DIFFERENTIATION. SINGLE-CELL CUT&TAG (SCCUT&TAG) TECHNOLOGY IS WELL-SUITED FOR SUCH STUDIES AS RE ACTIVITY THROUGH HISTONE MODIFICATION PROFILES CAN BE INVESTIGATED IN A LINEAGE-SPECIFIC MANNER. HOWEVER, POOR UNDERSTANDING OF THE CELL-TYPE-SPECIFIC HISTONE MODIFICATION PATTERNS MAKES THE TASK CHALLENGING. TO OVERCOME THIS CHALLENGE, WE DESIGNED SCCUT&TAG-PRO WHICH ALLOWS SIMULTANEOUS MEASUREMENT OF CELL-SURFACE PROTEIN AND IN-SILICO INTEGRATION OF GENE-EXPRESSION AND CHROMATIN ACCESSIBILITY. I WILL LEVERAGE THIS NOVEL MULTIMODAL FRAMEWORK TO INVESTIGATE THE RE AND PROGRESSIVE CHANGES IN THEIR ACTIVITY DURING HEMATOPOIESIS. FIRST, I WILL DEFINE A MULTIMODAL REFERENCE MAPPING FRAMEWORK FOR MOUSE HEMATOPOIESIS. THIS FRAMEWORK WILL ALLOW ME TO INTEGRATE MULTIPLE HISTONE MODIFICATION PROFILES ONTO ONE REFERENCE AND COMPARE THE CHROMATIN STATES OF THE RE BETWEEN A WILD TYPE (WT) AND MOUSE MODEL WITH LOSS OF FUNCTION IN HISTONE METHYL TRANSFERASE (HMT) (AIM 1). SECOND, SINCE HMTS REGULATE TRANSCRIPTION THROUGH THE INTERACTION NETWORK OF RE. I WILL DEFINE A CHROMATIN STATE AWARE MAP THAT DYNAMICALLY LINKS RES ACROSS DEVELOPMENTAL TRAJECTORIES. I WILL USE THIS FRAMEWORK TO INVESTIGATE THE CHANGES IN THE INTERACTION OF RES DUE TO HMT LOSS (AIM 2). THIRD, SINCE THE TRANSCRIPTIONAL STATE OF A CELL EMERGES FROM THE UNDERLYING GENE REGULATORY NETWORK (GRN), I WILL INTEGRATE SINGLE-CELL GENE EXPRESSION DATA WITH HISTONE MODIFICATION PROFILES AND EXTEND IT TO DEFINE A CHROMATIN STATE AWARE MODEL OF GRN. I WILL COMPARE THE WT AND HMT LOSS EXPERIMENTS AND DEFINE THE DIFFERENTIAL GRN (AIM 3). ALTOGETHER, THIS RESEARCH PROPOSAL SEEKS TO PIONEER THE COMPUTATIONAL METHODS FOR THE INTEGRATED ANALYSES OF MULTIMODAL SINGLE-CELL HISTONE MODIFICATIONS AND SYSTEMATICALLY DISSECT PROGRESSIVE CHANGES IN THE SYSTEM-LEVEL FUNCTION OF THE REGULATORY CIRCUITS THAT MISLEADS HEMATOPOIETIC DIFFERENTIATION USING MOUSE MODELS WITH CONDITIONAL HMT LOSS OF FUNCTION IN THE HEMATOPOIETIC COMPARTMENT. I HAVE DEVELOPED A 5-YEAR CAREER DEVELOPMENT PLAN TO MEET MY GOAL OF BECOMING AN INDEPENDENT INVESTIGATOR IN THE MULTI-DISCIPLINARY FIELD OF COMPUTATIONAL CANCER BIOLOGY. THE MENTORSHIP COMMITTEE WILL ALSO PROVIDE ME THE GUIDANCE IN MY RESEARCH AND ACADEMIC JOB SEARCH. GIVEN THE EXCELLENT THE OUTSTANDING RECORD OF TRAINING MULTIPLE INDEPENDENT SCIENTISTS, NEW YORK GENOME CENTER PROVIDES ME AN IDEAL ENVIRONMENT TO ATTAIN MY SCIENTIFIC CAREER GOALS.

MYELOID-DERIVED SUPPRESSOR CELLS IN CHECKPOINT PROTEIN INHIBITION FOR MELANOMA

MYELOID-DERIVED SUPPRESSOR CELLS IN CHECKPOINT PROTEIN INHIBITION FOR MELANOMA

A FIRST-IN-HUMAN PHASE I CLINICAL TRIAL OF MITOCHONDRIAL-TARGETED HSP90 INHIBITOR, GAMITRINIB - PROJECT SUMMARY  TARGETING A SINGLE ONCOGENIC PATHWAY FOR CANCER THERAPY IS FEASIBLE BUT GENERALLY NOT VERY EFFECTIVE, AS PATIENT  RESPONSES ARE SHORT-­LIVED, HAMPERED BY TOXICITY AND INVARIABLY SUPPLANTED BY PROGRESSIVE DISEASE. AN  ALTERNATIVE STRATEGY IS TO TARGET GLOBAL CANCER NETWORKS. THIS IS EXPECTED TO DISABLE MULTIPLE MECHANISMS OF  TUMOR GROWTH AT ONCE, CIRCUMVENT THE EMERGENCE OF DRUG RESISTANCE AND BE EFFECTIVE IN DISPARATE  MALIGNANCIES, REGARDLESS OF GENETIC OR MOLECULAR HETEROGENEITY. A POOL OF HEAT SHOCK PROTEIN-­90 (HSP90)  CHAPERONES LOCALIZED IN MITOCHONDRIA ORCHESTRATES ONE SUCH CANCER NETWORK. MITOCHONDRIAL HSP90S ARE  OVEREXPRESSED IN CANCER, COMPARED TO NORMAL TISSUES, SUPPORT MULTIPLE MECHANISMS OF TUMOR GROWTH THROUGH  HEIGHTENED PROTEIN FOLDING, AND CONFER WORSE DISEASE OUTCOME IN THE CLINIC. UNEXPECTEDLY, THIS PATHWAY COULD  NOT BE TARGETED PHARMACOLOGICALLY, AS NONE OF THE HSP90 ANTAGONISTS DEVELOPED SO FAR HAS THE ABILITY TO  ACCUMULATE IN MITOCHONDRIA. FOR THIS REASON, WE DEVELOPED GAMITRINIB (GA MITOCHONDRIAL MATRIX INHIBITOR), THE  FIRST-­IN-­CLASS, MITOCHONDRIAL-­TARGETED, SMALL MOLECULE HSP90 INHIBITOR. WITH A UNIQUE COMBINATORIAL STRUCTURE,  GAMITRINIB SELECTIVELY ACCUMULATES IN MITOCHONDRIA, DISRUPTS THE ORGANELLE PROTEIN FOLDING ENVIRONMENT, AND  SHUTS DOWN MULTIPLE PATHWAYS OF BIOENERGETICS, METABOLISM, AND CELL SURVIVAL REQUIRED FOR TUMOR GROWTH. IN  TURN, THIS TRANSLATES IN POTENT CYTOTOXIC ACTIVITY AGAINST HETEROGENEOUS TUMORS AS MONOTHERAPY OR IN  COMBINATION, AND INHIBITION OF PRIMARY AND METASTATIC TUMOR GROWTH IN XENOGRAFT AND TRANSGENIC DISEASE  MODELS. ADVANCED SOLELY THROUGH PUBLIC FUNDING, THE PRECLINICAL DEVELOPMENT OF GAMITRINIB IS NOW COMPLETE  (PIND #132453), SHOWING FAVORABLE DRUG-­LIKE PROPERTIES, ENCOURAGING SAFETY IN TWO ANIMAL SPECIES, AND A  UNIQUE SIGNATURE OF “CELLULAR STARVATION” AS BIOMARKER OF TARGET INHIBITION, IN VIVO. THEREFORE, THE HYPOTHESIS  THAT GAMITRINIB PROVIDES THE FIRST SUBCELLULARLY-­DIRECTED CANCER THERAPY TARGETING A MITOCHONDRIAL  NETWORK OF TUMOR MAINTENANCE CAN BE FORMULATED, AND WILL CONSTITUTE THE FOCUS OF THE PRESENT APPLICATION.  THE FIRST SPECIFIC AIM WILL SUPPORT A FIRST-­IN-­HUMAN, PHASE I CLINICAL TRIAL OF WEEKLY INTRAVENOUS INFUSION OF  GAMITRINIB IN PATIENTS WITH ADVANCED CANCER. THESE STUDIES WILL DETERMINE THE MAXIMUM TOLERATED DOSE (MTD),  DOSE-­LIMITING TOXICITIES (DLT) AND PHARMACOKINETICS OF GAMITRINIB USING AN ACCELERATED DOSE-­ESCALATION  PROTOCOL WITH EXPANSION COHORT AT MTD. THE SECOND SPECIFIC AIM WILL CHARACTERIZE THE PHARMACODYNAMICS OF  GAMITRINIB IN PRE-­ AND POST-­TREATMENT TUMOR BIOPSIES AND PERIPHERAL BLOOD MONONUCLEAR CELLS HARVESTED FROM  THE PATIENT EXPANSION COHORT. THESE STUDIES WILL PROFILE THE METABOLIC DEFECTS OF GAMITRINIB THERAPY AND  EVALUATE A “CELLULAR STARVATION” SIGNATURE COMPRISING INHIBITION OF AMPK SIGNALING, INDUCTION OF AUTOPHAGY,  MODULATION OF PROTEOTOXIC STRESS AND SUPPRESSION OF MTOR SIGNALING. OVERALL, THE PROPOSAL IS DESIGNED TO BRING  TO THE CLINIC A NOVEL ANTICANCER AGENT WITH A UNIQUE MECHANISM OF ACTION AND BROAD, “TUMOR-­AGNOSTIC” EFFICACY.

TECHNOLOGY FOR DETECTION AND QUANTITATION OF TELOMERIC DNA ABERRATIONS IN CANCER

MODELING CANCER EVOLUTION FOR PREDICTION WITH NEURAL NETWORKS: METHODS AND APPLICATIONS - PROJECT SUMMARY/ABSTRACT THE STUDY OF TUMOR EVOLUTION CAN UNCOVER EVENTS AND INTERACTIONS THAT DRIVE TUMOR DEVELOPMENT THROUGH ALTERNATIVE ROUTES, REVEAL DIFFERENCES IN THERAPEUTIC VULNERABILITIES AND IMPROVE CLINICAL DECISION MAKING. YET, STUDYING TUMOR EVOLUTION IS CHALLENGING, HINDERED BY THE DIFFICULTY TO INTERPRET NOISY GENOMIC DATA AND THE LACK OF TEMPORAL ORDERING OF MAJOR GENETIC EVENTS. THERE IS THEREFORE A CRITICAL NEED FOR THE DEVELOPMENT OF COMPUTATIONAL APPROACHES THAT CAN FACILITATE EFFICIENT INVESTIGATION OF CANCER DATA UNDER AN EVOLUTIONARY, TEMPORAL PERSPECTIVE. THE LONG-TERM GOAL OF THIS PROJECT IS TO DEVELOP COMPUTATIONAL TOOLS COMBINING ADVANCED MACHINE LEARNING WITH MOLECULAR EVOLUTION TECHNIQUES AND PROVIDE NOVEL STRATEGIES TO INVESTIGATE TUMOR EVOLUTION. THE OVERALL OBJECTIVE IS TO ESTABLISH A DEEP-LEARNING FRAMEWORK TO STUDY TUMOR DEVELOPMENT THAT WILL BE USED TO DISTINGUISH EARLY AND LATE GENETIC EVENTS THAT ARE ASSOCIATED WITH TUMOR CHARACTERISTICS, SURVIVAL AND THERAPEUTIC VULNERABILITIES. THE RATIONALE OF THE PROPOSED RESEARCH IS THAT THE STUDY OF TUMOR EVOLUTION THROUGH INTEGRATION OF MACHINE LEARNING WITH MOLECULAR EVOLUTION TECHNIQUES COULD ENHANCE THE PERFORMANCE OF OTHERWISE DIFFICULT CLINICAL CLASSIFICATION TASKS. THE SPECIFIC AIMS OF THIS PROJECT ARE TO (1) CHARACTERIZE THE INTERPLAY BETWEEN DRIVER MUTATIONS AND ANEUPLOIDY IN TUMOR EVOLUTION AND IDENTIFY DETERMINANTS OF CLINICAL OUTCOME AND THERAPEUTIC VULNERABILITIES (2) INTRODUCE COMPUTATIONAL APPROACHES TO REPRESENT SNAPSHOT GENOMIC DATA THROUGH TEMPORAL AND FUNCTIONAL ORDERING OF GENETIC EVENTS (3) DEVELOP A RECURRENT NEURAL NETWORK APPROACH TO LEARN DIFFERENT DYNAMICS IN TUMOR EVOLUTION FROM ORDERED GENOMIC DATA, AND PREDICT PHENOTYPIC FEATURES AND CLINICAL OUTCOME. THE PROPOSED RESEARCH IS INNOVATIVE BECAUSE IT WILL COMBINE RECENT ADVANCES IN MACHINE LEARNING WITH EVOLUTIONARY TECHNIQUES INTO A SINGLE FRAMEWORK, ESTABLISHING NOVEL COMPUTATIONAL TOOLS THAT WILL FACILITATE A COMPREHENSIVE INVESTIGATION OF CANCER DEVELOPMENT. THE PROPOSED FRAMEWORK IS SIGNIFICANT BECAUSE IT WILL ENABLE APPLICATION OF TEMPORAL MODELING WITH MACHINE LEARNING TO CANCER DATA, FOR PREDICTION OF CLINICAL FEATURES. TO ACHIEVE THE PROPOSED GOALS THE CANDIDATE, DR. NOAM AUSLANDER, REQUIRES ADDITIONAL TRAINING AND MENTORING IN EVOLUTIONARY RESEARCH, COMPARATIVE GENOMICS AND MATHEMATICAL MODELING. DURING THE K99 PHASE, DR. AUSLANDER WILL CONDUCT THIS RESEARCH AS A POSTDOCTORAL FELLOW AT THE NATIONAL CENTER FOR BIOTECHNOLOGY INFORMATION. SHE WILL BE MENTORED BY DR. EUGENE KOONIN, A RECOGNIZED EXPERT IN THE FIELDS OF MOLECULAR EVOLUTION AND COMPUTATIONAL BIOLOGY, AND ADDITIONAL MENTORING FROM SENIOR MEMBERS FROM THE KOONIN LAB. TOGETHER WITH HER PREVIOUS TRAINING IN MACHINE LEARNING AND CANCER DATA SCIENCE, THIS APPLICATION FOR THE NIH PATHWAY TO INDEPENDENCE AWARD (K99/R00) DESCRIBES A CAREER DEVELOPMENT PLAN THAT WILL ALLOW DR. AUSLANDER TO ACHIEVE HER CAREER GOALS AND BECOME AN INDEPENDENT INVESTIGATOR AND LEADER IN COMPUTATIONAL RESEARCH OF CANCER EVOLUTION.

TB-RELATED IMMUNE RECONSTITUTION SYNDROME IN HIV-1 INFECTED SOUTH AFRICANS

REGULATION OF MYELOMA PROGRESSION BY PROTEIN CITRULLINATION

GENOMEWIDE DISCOVERY & ANALYSIS OF ALTERNATIVE PROMOTERS

REGULATION OF ADIPOSE TISSUE INFLAMMATION AND INSULIN RESISTANCE BY T CELL SUBSET

ELUCIDATION OF TARGETABLE MECHANISMS SUSTAINING IMMUNOSUPPRESSIVE NEUTROPHILS IN THE BRAIN TUMOR MICROENVIRONMENT - PROJECT SUMMARY GLIOBLASTOMA (GBM), THE MOST AGGRESSIVE AND LETHAL FORM OF BRAIN CANCER IN ADULTS, IS HIGHLY RESISTANT TO PROMISING IMMUNOTHERAPIES, LARGELY DUE TO A MYELOID CELL-DRIVEN IMMUNOSUPPRESSIVE MICROENVIRONMENT (TME). THE EMERGING KEY CONTRIBUTION OF NEUTROPHILS TO THIS IMMUNOSUPPRESSIVE MILIEU THAT RESTRICTS ANTI- CANCER IMMUNITY, THEREFORE PROMOTING TUMOR GROWTH AND RESISTANCE TO IMMUNOTHERAPIES, HAS SPARKED INTEREST IN THERAPEUTICALLY TARGETING THESE CELLS IN BRAIN TUMORS. DESPITE RECENT ADVANCES IN UNDERSTANDING THE PHENOTYPIC AND FUNCTIONAL HETEROGENEITY OF NEUTROPHILS IN TUMOR BEDS, THERE ARE NO EFFECTIVE APPROACHES TO SPECIFICALLY TARGET IMMUNOSUPPRESSIVE SUBSETS. THUS, THE GOAL OF THIS STUDY IS TO DISSECT TARGETABLE TUMOR- DRIVEN MECHANISMS SUSTAINING PRO-TUMORAL AND IMMUNOSUPPRESSIVE NEUTROPHILS IN THE TME; AS THIS KNOWLEDGE IS THE KEY TO DEVELOPING IMPACTFUL, NEW, AND SELECTIVE NEUTROPHIL-TARGETING THERAPIES TO UNLEASH ANTI-TUMOR IMMUNITY AND SENSITIZE RESISTANT TUMORS, LIKE GBM, TO DIFFERENT FORMS OF IMMUNOTHERAPY. OUR PRELIMINARY DATA SHOW THAT THE GBM TME SPECIFICALLY REPROGRAMMED A SUBSET OF IMMUNOSUPPRESSIVE NEUTROPHILS INTO CELLS WITH PROLONGED LIFESPANS, PROVIDING ABNORMALLY HIGH LEVELS OF LONG-LASTING IMMUNOSUPPRESSIVE CELLS IN TUMOR BEDS. IMMUNOSUPPRESSIVE NEUTROPHILS SHOWED BOOSTED GLUCOSE METABOLISM, WHICH CORRELATED WITH HIGH RESISTANCE TO FERROPTOSIS AND HIGH LEVELS OF HISTONE LACTYLATION (KLA). SINCE NEUTROPHILS ARE GENERALLY CONSIDERED SHORT-LIFE CELLS, IT BECOMES IMPORTANT TO UNDERSTAND IF AND HOW LOCAL TUMORAL CUES ARE LINKED TO ADAPTIVE CHANGES ENABLING SURVIVAL OF SPECIFIC NEUTROPHIL SUBSETS. BASED ON OUR PRELIMINARY KEY OBSERVATIONS, WE HYPOTHESIZE THAT ACTIONABLE HISTONE LACTYLATION-DRIVEN RESISTANCE TO FERROPTOSIS SELECTIVELY MAINTAINS THE POOL OF IMMUNOSUPPRESSIVE NEUTROPHILS IN HYPOXIC NICHES OF TUMORS. WE WILL TEST THIS HYPOTHESIS THROUGH THE FOLLOWING AIMS: AIM1. TO DEFINE METABOLIC CUES THAT SUSTAIN IMMUNOSUPPRESSIVE NEUTROPHILS IN TUMOR BEDS; AIM2. TO DISSECT THE GLUCOSE CATABOLISM-DRIVEN EPIGENETIC MECHANISM DETERMINING RESISTANCE TO CELL DEATH OF IMMUNOSUPPRESSIVE NEUTROPHILS; AIM3. TO ESTABLISH THE THERAPEUTIC POTENTIAL OF TARGETING SUPPRESSIVE NEUTROPHILS. THUS, THE PROPOSED STUDIES WILL ELUCIDATE TUMOR- INDUCED METABOLIC AND EPIGENETIC DETERMINANTS THAT SUSTAIN THE POOL OF IMMUNOSUPPRESSIVE NEUTROPHILS IN TUMOR BEDS. OUR WORK WILL EXERT A PROFOUND EFFECT IN THE FIELD BY PROVIDING A MECHANISTIC RATIONALE TO SPECIFICALLY TARGET A MAJOR IMMUNOSUPPRESSIVE, TUMOR-PROMOTING COMPONENT OF THE GBM TME, THEREFORE CONTRIBUTING TO THE REFINEMENT AND EXPANSION OF THERAPEUTIC APPROACHES TO OVERCOME IMMUNOSUPPRESSION AND ENHANCE THE EFFICACY OF IMMUNOTHERAPY.

TRI-STATE EXPANSION, CURRICULUM EVOLUTION, AND EQUITY DURING BIOTECHNICIAN TRAINING (TRI EXCEED BTT) -THE GROWTH OF THE LIFE SCIENCE INDUSTRY, FUELED BY CELL AND GENE THERAPY (CGT) IN THE GREATER PHILADELPHIA REGION, HAS LED TO A HIGH DEMAND FOR LABORATORY TECHNICIANS IN BOTH ACADEMIC AND INDUSTRY LABS. BOTH AN INCREASE IN CLINICAL TRIALS AND APPROVED CGT PRODUCTS WILL BRING INCREASED DEMANDS FOR LABORATORY TECHNICIAN POSITIONS IN BIOPROCESSING, INCREASING OPPORTUNITIES FOR INDIVIDUALS WITH LESS THAN A BACHELOR?S DEGREE. THE WISTAR INSTITUTE, DEVELOPED THE BIOMEDICAL TECHNICIAN TRAINING (BTT) PROGRAM TO PREPARE STUDENTS FROM THE COMMUNITY COLLEGE OF PHILADELPHIA (CCP) FOR POSITIONS AS BIOLOGICAL LABORATORY TECHNICIANS AND RESEARCH ASSISTANTS. THE BTT PROGRAM HAS SERVED AS BOTH RESEARCH EXPERIENCE AND WORKFORCE DEVELOPMENT FOR COMMUNITY COLLEGE STUDENTS, AND THE BTT PROGRAM WAS REGISTERED AS A PRE-APPRENTICESHIP PROGRAM IN 2019. THIS PROJECT WILL WORK WITH NEW COMMUNITY COLLEGE PARTNERS TO IDENTIFY APPROPRIATE PRE-REQUISITE COURSES AND ALLOW STUDENTS TO EARN CREDITS FOR THEIR EXPERIENCE IN THE BTT PRE-APPRENTICESHIP PROGRAM THAT INCLUDES A LABORATORY ORIENTATION AND TWO LAB EXPERIENCES, ONE IN AN ACADEMIC RESEARCH LAB AND THE OTHER IN AN INDUSTRY LAB OR CORE FACILITY. THE PROJECT WILL EXPAND THE NUMBER AND GEOGRAPHICAL LOCATION OF EMPLOYER PARTNERS TO NEW JERSEY AND DELAWARE. THE PROJECT WILL EXPAND RECRUITMENT EFFORTS IN THE GREATER PHILADELPHIA REGION AND INCREASE NUMBERS OF APPLICANTS BY INCLUDING 3 ADDITIONAL COMMUNITY COLLEGE PARTNERS, BRINGING A TOTAL OF 8 COMMUNITY COLLEGES COVERING AT LEAST 10 COUNTIES IN 3 STATES INVOLVED IN THE BTT PRE-APPRENTICESHIP PROGRAM. THE PROJECT WILL PROVIDE A BLUEPRINT FOR INCORPORATION OF A BIOTECHNICIAN PRE-APPRENTICESHIP PROGRAM INTO VARIOUS BIOTECHNOLOGY AND SCIENCE CURRICULA AT COMMUNITY COLLEGES AND SERVE AS A MODEL FOR REGIONAL PROGRAM EXPANSION. NEW EMPLOYER PARTNERS IN 2 NEIGHBORING STATES WILL BE RECRUITED, ALLOWING THE REGISTRATION OF THE APPRENTICESHIP FOR USE NATIONALLY. PROJECT-BASED LABORATORY CURRICULUM WILL BE DEVELOPED AND MAPPED TO NEWLY DEVELOPED STANDARDS IN CGT. BY USING RESEARCH FROM WISTAR SPECIFICALLY CHOSEN TO ENGAGE UNDERSERVED STUDENTS AND PAYING PARTICIPANTS DURING THE PROGRAM, WE WILL INVESTIGATE WHETHER THIS PROGRAM PROVIDES THE EQUITY NEEDED TO CLOSE COMPLETION GAPS. FACULTY LIAISONS AT EACH NEW COMMUNITY COLLEGE PARTNER WILL BE IDENTIFIED AND THEY WILL BE EXPOSED TO CURRENT RESEARCH AND CGT. THESE EXPERIENCES WILL ALLOW THEM TO MAKE CONNECTIONS TO ONE ANOTHER, INDUSTRY PARTNERS, AND THE ATE COMMUNITY. ADDING EMPLOYER PARTNERS TO OFFER LAB EXPERIENCES IS EXPECTED TO RESULT IN MORE HIRING OF EMPLOYEES WITH AN ASSOCIATE DEGREE. THIS PROJECT IS FUNDED BY THE ADVANCED TECHNOLOGICAL EDUCATION PROGRAM THAT FOCUSES ON THE EDUCATION OF TECHNICIANS FOR THE ADVANCED-TECHNOLOGY FIELDS THAT DRIVE THE NATION'S ECONOMY. 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.

PURCHASE OF A SARRP 200 PLATFORM FOR IRRADIATION - PROJECT SUMMARY THIS APPLICATION REQUESTS FUNDS FOR A STAND-ALONE PRECISION X-RAY IRRADIATOR AND IMAGING INSTRUMENT WITH INTEGRATED CONE BEAM CT (CBCT): THE XSTRAHL SMALL ANIMAL RADIATION RESEARCH PLATFORM-200 (SARRP- 200). THIS INSTRUMENT IS REQUESTED BY THE FACULTY AT THE WISTAR INSTITUTE IN PHILADELPHIA. THE WISTAR INSTITUTE IS THE NATION’S OLDEST INDEPENDENT BIOMEDICAL RESEARCH INSTITUTE, WITH AN ENERGIZED AND HIGHLY COLLABORATIVE FACULTY WHO PERFORM CUTTING-EDGE RESEARCH ON CANCER BIOLOGY, IMMUNOLOGY AND VACCINES. THE INSTITUTE BOASTS A LONG HISTORY OF RESEARCH SUCCESS IN MONOCLONAL ANTIBODY THERAPY AND CANCER BIOLOGY (STELARA®) AND VACCINES (ROTATEQ©), INCLUDING THE SECOND COVID-19 VACCINE TO MOVE INTO CLINICAL TESTING IN THE UNITED STATES. THE INSTRUMENT SELECTED, THE SARRP-200, INTRODUCES CRITICAL NEW CAPABILITIES FOR WISTAR INVESTIGATORS, WHO ARE CURRENTLY UNABLE TO PERFORM IMAGE-GUIDED RADIOTHERAPY IN SMALL ANIMALS (MICE), OR TO USE WHOLE-BODY RADIATION OF MICE FOR MYELO-ABLATION USING OUR CURRENT SYSTEM. THE LATTER TECHNIQUE IS URGENTLY NEEDED FOR OUR ABILITY TO CREATE AND CHARACTERIZE HUMANIZED MICE (MICE WITH HUMAN IMMUNE SYSTEMS). OUR STUDIES ON HUMANIZED MICE IS A PIONEERING EFFORT SHARED BY SEVERAL INVESTIGATORS AT THE INSTITUTE, AND FOR THIS AND OTHER PROJECTS IT IS EXPECTED THAT THE SARRP-200 WILL BE A CRITICAL SYNERGY PLATFORM FOR WISTAR RESEARCH EFFORTS. BECAUSE OF IACUC RESTRICTIONS REGARDING THE TRANSPORT OF IMMUNO- COMPROMISED MICE, WISTAR FACULTY ARE UNABLE TO USE EITHER OF THE TWO SARRPS LOCATED AT THE NEARBY UNIVERSITY OF PENNSYLVANIA. HOWEVER, WE HAVE TAKEN ADVANTAGE OF THE EXPERTISE OF OUR NEARBY COLLEAGUES, AND THEY ARE LISTED ON OUR ADVISORY BOARD AND PROGRAM MANAGEMENT TEAM. INSTALLATION AND IMMEDIATE USAGE OF THE SARRP-200 WILL BE FACILITATED BY OUR ALREADY-TRAINED PERSONNEL, OUR INTERNAL ADVISORY COMMITTEE, AND BY OUR EXPERIENCED PROGRAM MANAGEMENT TEAM, WHICH INCLUDES RADIATION ONCOLOGY AND MEDICAL PHYSICS PERSONNEL FROM THE UNIVERSITY OF PENNSYLVANIA. OUR PLANNED PURCHASE OF A SARRP-200 WILL REPLACE A THIRTY YEAR OLD CESIUM IRRADIATOR WITH A FRAGILE MOTORIZED PLATFORM THAT IS SUBJECT TO FREQUENT DISREPAIR. THE REQUESTED INSTRUMENT WILL BE PLACED WITHIN THE CELL AND ANIMAL IRRADIATION CORE, IN DESIGNATED SPACE ON THE GROUND FLOOR OF THE EAST BUILDING AT THE WISTAR INSTITUTE IN PHILADELPHIA PA, WITH DIRECT ACCESS TO A DESIGNATED PROCEDURE ROOM AND THE LABORATORY ANIMAL FACILITY. THE PLANNED PURCHASE OF THE SARRP-200 IS EXPECTED TO OFFER SIGNIFICANT NEW CAPABILITIES TO WISTAR RESEARCHERS, AND TO ENERGIZE THE RESEARCH OF AN ALREADY HIGHLY COLLABORATIVE FACULTY.

PURCHASE OF A Q EXACTIVE HF MASS SPECTROMETER SYSTEM FOR METABOLOMICS

EXPANSION, CURRICULUM EVOLUTION, AND ENHANCEMENT OF BIOTECHNICIAN TRAINING

PURCHASE A BECKMAN COULTER MOFLO ASTRIOS FLOW CYTOMETER

CONTROL OF TRANSCRIPTIONAL INTEGRITY BY INTEGRATOR AND PP2A - PROJECT SUMMARY OBJECTIVE: THE OVERALL GOALS OF THIS APPLICATION ARE UNDERSTANDING THE MOLECULAR BASIS OF RNA POLYMERASE II (RNAPII) PAUSING AND PAUSE-RELEASE AND DETERMINING HOW DISEASE-ASSOCIATED MUTATIONS IN THE PP2A PHOSPHATASE REVERSE THE PAUSING BALANCE TO PROMOTE TRANSCRIPTIONAL ELONGATION. PROPOSED RESEARCH: THE MAMMALIAN RNAPII TRANSCRIPTION CYCLE IS REGULATED BY CYCLIN-DEPENDENT KINASES (CDKS) THAT TARGET THE CATALYTIC CORE OF RNAPII AND MANY OF ITS CO-FACTORS. WE RECENTLY IDENTIFIED INT-PP2A, A MODULE OF THE RNAPII-ASSOCIATED INTEGRATOR COMPLEX THAT RECRUITS THE PP2A PHOSPHATASE TO ACTIVE GENES TO OPPOSE CDK9 ACTIVITY. WE DEMONSTRATED THAT INT-PP2A TARGETS SER-2 RESIDUES AT THE C TERMINAL DOMAIN (CTD) OF RNAPII AS WELL AS DSIF/SPT5. WHILE OUR WORK REVEALED THAT PHOSPHATASES ARE RECRUITED CO-TRANSCRIPTIONALLY IN MAMMALIAN CELLS TO PROMOTE PAUSING, IT REMAINS UNKNOWN HOW INT-PP2A’S ACTIVITY IS ESTABLISHED AT THE PROXIMAL PROMOTER AND HOW IT IS MODULATED. HERE WE PROPOSE TO: A) DETERMINE HOW INT-PP2A ESTABLISHES PAUSED RNAPII VIA THE INTS3 SUBUNIT; B) DETERMINE THE ROLE OF PP2A CARBOXY-METHYLATION IN TRANSCRIPTIONAL REGULATION; C) DETERMINE THE EFFECT OF MUTANT PP2A ON TRANSCRIPTION IN MODELS OF NEURONAL DIFFERENTIATION.

JUXTACRINE REGULATION OF METASTASIS FROM BRAIN MICROENVIRONMENTS

THE SNAIL-INDUCED SULFONATION PATHWAY IN BREAST CANCER METASTASIS

PURCHASE OF A PERKINELMER IVIS SPECTRUMCT IMAGING SYSTEM

CHARACTERIZATION OF TRBP-CONTAINING COMPLEXES

PURCHASE OF AN ECHO 650 ACOUSTIC LIQUID HANDLER WITH ACCESS WORKSTATION - PROJECT SUMMARY SUPPORT IS REQUESTED FOR THE PURCHASE OF AN ECHO 650 ACOUSTIC LIQUID HANDLER WITH ACCESS WORKSTATION TO REPLACE THE JANUS NANOHEAD, THE EXISTING AND AGING LOW VOLUME LIQUID HANDLER PURCHASED IN 2008 WITHIN THE MOLECULAR SCREENING AND PROTEIN EXPRESSION FACILITY AT THE WISTAR INSTITUTE IN PHILADELPHIA. THIS INSTRUMENT UPGRADE IS CRITICAL FOR THE FACILITY TO CONTINUE TO PROVIDE STATE-OF-THE-ART SUPPORT FOR THE DEVELOPMENT OF ROBUST, HIGH-THROUGHPUT BIOCHEMICAL AND CELL-BASED ASSAYS REQUIRED FOR DRUG DISCOVERY. INDEED, WISTAR HAS MADE A COMMITMENT TO ACADEMIC DRUG DISCOVERY BY RECRUITING ITS FIRST MEDICINAL CHEMIST, DR. JOSEPH M. SALVINO, WHO IS THE PRINCIPAL INVESTIGATOR OF THIS APPLICATION. DR. SALVINO AND NINE OTHER MAJOR AND MINOR USERS WHO HOLD A COMBINED 17 UNIQUE NIH GRANTS (R01/R35/R61/P01/UM1/U54/DP2) HAVE REQUESTED THE ECHO 650 WITH ACCESS WORKSTATION TO DEVELOP IMPROVED BIOCHEMICAL AND CELL-BASED ASSAYS THAT GENERATE BETTER DATA WHICH WILL ENABLE MORE INFORMED DECISION MAKING REGARDING NOVEL STRUCTURE-ACTIVITY RELATIONSHIPS. THERE ARE CURRENTLY NO SIMILAR INSTRUMENTS THAT ARE ACCESSIBLE TO WISTAR INVESTIGATORS. THE ECHO 650 IS A HIGHLY ACCURATE, LOW VOLUME, NON-CONTACT LIQUID HANDLER THAT MOVES NANOLITER VOLUMES OF LIQUID FROM ONE MICROPLATE TO ANOTHER USING SOUND WAVES. USING DIRECT DILUTION, IT CAN DETERMINE THE POTENCIES OF SMALL MOLECULES WITH BETTER PRECISION AS OPPOSED TO TRADITIONAL SERIAL DILUTION. THE MINIATURIZATION OF SUCH ASSAYS ALSO REDUCES RESEARCH COSTS. THE ACCESS WORKSTATION IS THE STACKER UNIT INTEGRATED WITH THE ECHO 650 WHICH ALLOWS FOR AUTOMATED PLATE HANDLING NECESSARY FOR HIGH-THROUGHPUT SCREENING AND HIT PICKING OF ACTIVE COMPOUNDS. BECAUSE THERE IS NO CONTACT BETWEEN SOURCE PLATES, THIS TECHNOLOGY ELIMINATES COMPOUND CARRY-OVER AND PROTECTS THE INTEGRITY OF EXISTING COMPOUNDS IN STORAGE PLATES. THE PRELIMINARY DATA DESCRIBED IN THIS APPLICATION DEMONSTRATE THAT THE ECHO 650 IS A SUPERIOR TECHNOLOGY TO OUR EXISTING JANUS NANOHEAD. IN ADDITION, WE ALSO SHOW THAT WE HAVE THE NECESSARY INFRASTRUCTURE TO MINIATURIZE OUR ASSAYS TO 1536-WELL FORMAT. THIS INSTRUMENT WILL SIGNIFICANTLY UPGRADE OUR CAPABILITIES REGARDING ASSAY MINIATURIZATION WHILE SIMULTANEOUSLY INCREASING THE QUALITY OF OUR DATA. THE UNIT WILL BE HOUSED IN THE MOLECULAR SCREENING AND PROTEIN EXPRESSION FACILITY, OVERSEEN BY DR. SALVINO AND MANAGED BY MR. JOEL CASSEL. BOTH DR. SALVINO AND MR. CASSEL HAVE OVER 40 YEARS AGGREGATE EXPERIENCE IN THE PHARMACEUTICAL INDUSTRY. THE FACILITY IS WELL REGARDED AND CUMULATIVELY RANKED WITH A SCORE OF “EXCEPTIONAL” AT THE 2018 NIH/NCI CANCER CENTER SUPPORT GRANT (CCSG) REVIEW. WE ARE SEEKING TO UPGRADE OUR EXISTING LOW VOLUME LIQUID HANDLING TECHNOLOGY TO A SUPERIOR STATE OF THE ART, NON-CONTACT NANOLITER LIQUID HANDLING TECHNOLOGY IN THE ECHO 650 WITH ACCESS WORKSTATION IN ORDER TO OPTIMALLY SUPPORT THE ACADEMIC DRUG DISCOVERY INITIATIVES OF THE WISTAR INSTITUTE.

CONTROL OF COLON CANCER PROGRESSION BY THE COLON MICROBIOME

TARGETING ACRAL MELANOMA BY INDUCING TERT DEGRADATION (ME190011). NEW AWARD.

SINGLE-CELL ANALYSIS OF GLYCOMIC AND PROTEOMIC FEATURES OF THE HIV RESERVOIR - PROJECT SUMMARY: IT HAS EMERGED IN RECENT YEARS THAT MULTIPLE TYPES OF HIV RESERVOIR CELLS CAN PERSIST IN THE FACE OF ANTIRETROVIRAL THERAPY (ART). HIV RESERVOIR CELLS INCLUDE THOSE THAT ARE “LATENT” (TRANSCRIPTIONALLY- INACTIVE) AND THOSE THAT ARE TRANSCRIPTIONALLY-ACTIVE. THE TRANSCRIPTIONALLY-ACTIVE RESERVOIR CELLS MAY BE A MAJOR CONTRIBUTOR TO HIV REBOUND AFTER ART INTERRUPTION, AS SHOWN IN ANALYTIC TREATMENT INTERRUPTION STUDIES. IN ADDITION, SOME RESERVOIR CELLS ARE ABLE TO PRODUCE VIRAL PROTEINS UPON STIMULATION (I.E., INDUCIBLE RESERVOIR CELLS), AND THESE RESERVOIR CELLS MAY ALSO BE IMPORTANT FOR REBOUND AS THEY ARE ENRICHED FOR GENOME-INTACT AND REPLICATION- COMPETENT HIV PROVIRUS. UNFORTUNATELY, THE LACK OF A DETAILED UNDERSTANDING OF THE CELL SURFACE PHENOTYPES OF THESE HIV RESERVOIR CELLS HAS PRECLUDED A FULL UNDERSTANDING OF THE BIOLOGY OF HIV PERSISTENCE AND HAMPERED THE DEVELOPMENT OF A CURE. IMPORTANTLY, PRIOR EFFORTS TO CHARACTERIZE RESERVOIR PHENOTYPES ALL FOCUSED ON CELL SURFACE PROTEINS AND IGNORED CELL SURFACE GLYCANS, DESPITE THE FACT THAT GLYCANS PLAY A CRITICAL ROLE IN REGULATING MULTIPLE KEY CELLULAR PROCESSES AND IMMUNOLOGICAL FUNCTIONS AND HAVE SERVED AS USEFUL BIOMARKERS IN MANY DISEASES. WE RECENTLY DEMONSTRATED THAT PERIPHERAL CD4+ T CELLS HARBORING HIGH LEVELS OF FUCOSYLATED CARBOHYDRATES ARE ENRICHED FOR HIV+ TRANSCRIPTIONALLY-ACTIVE CELLS AND DEFICIENT IN HIV+ TRANSCRIPTIONALLY-INACTIVE ONES, AND THAT THIS FUCOSYLATION IS DIRECTLY INDUCED BY HIV INFECTION. THESE STUDIES SUGGEST THAT GLYCANS MAY HAVE UTILITY AS NOVEL BIOMARKERS OF SPECIFIC TYPES OF RESERVOIR CELLS. IN THIS STUDY, WE COMBINE NEWLY DEVELOPED SINGLE-CELL SURFACE GLYCOMIC + PROTEOMIC TECHNOLOGIES WITH BIOINFORMATIC ANALYSES TO ANALYZE CELLS FROM AN HIV LATENCY MODEL AND ART-SUPPRESSED HIV+ INDIVIDUALS IN ORDER BETTER UNDERSTANDING THE MAKEUP OF THESE CELLS, WITH THE ULTIMATE GOAL OF UNDERSTAND HOW THEY CAN PERSIST IN THE FACE OF ART AND WHETHER THEY EXPRESS A UNIQUE PROFILE OF THERAPEUTICALLY TARGETABLE BIOMARKERS. IN AIM 1, WE WILL TEST THE HYPOTHESIS THAT CELL-SURFACE GLYCOSYLATION PATTERNS, INCLUDING FUCOSYLATION, DISTINGUISH TRANSCRIPTIONALLY-ACTIVE AND INACTIVE HIV RESERVOIR CELLS IN VITRO AND IN VIVO. THESE STUDIES WILL TAKE ADVANTAGE OF OUR RECENTLY ESTABLISHED TECHNIQUE CYTOF-LEC, WHICH PAIRS IN-DEPTH CYTOF PHENOTYPING WITH SURFACE GLYCAN CHARACTERIZATION AT THE SINGLE-CELL LEVEL. IN AIM 2, WE WILL EXAMINE THE GLYCAN FEATURES OF IN VIVO INDUCIBLE RESERVOIR CELLS. TO DO THIS, WE WILL ADAPT OUR RECENTLY ESTABLISHED AND VALIDATED PP-SLIDE (PREDICTED PRECURSOR SINGLE-CELL LINKAGE USING DISTANCE ESTIMATION) APPROACH TO CHARACTERIZE THE GLYCAN + PROTEIN FEATURES OF PATIENT-DERIVED RESERVOIR CELLS INDUCIBLE BY EX VIVO STIMULATION. TOGETHER, THESE ANALYSES WILL ALLOW US TO TEST OUR CENTRAL HYPOTHESIS THAT SPECIFIC CELL-SURFACE GLYCOSYLATION PATTERNS, INCLUDING FUCOSYLATION, CAN DISTINGUISH HIV RESERVOIR CELLS FROM UNINFECTED ONES, AND FURTHERMORE DISTINGUISH DIFFERENT TYPES OF RESERVOIR CELLS (E.G., LATENT, TRANSCRIPTIONALLY-ACTIVE, AND INDUCIBLE).

MOLECULAR AND NEURODEVELOPMENTAL CONSEQUENCES OF ADNP MUTATION - PROJECT SUMMARY THE ABUNDANCE OF MUTATIONS IN CHROMATIN REGULATORY PROTEINS IN AUTISM SPECTRUM DISORDERS (ASD) HIGHLIGHTS THE SIGNIFICANCE OF CHROMATIN STRUCTURE IN NORMAL BRAIN DEVELOPMENT. A RELATIVELY NEW AND LITTLE UNDERSTOOD CHROMATIN STRUCTURE THAT HAS BEEN IMPLICATED IN SEVERAL NEURODEVELOPMENTAL DISORDERS IS THE R-LOOP. R-LOOPS ARE THREE- STRANDED, NUCLEIC ACID STRUCTURES CONTAINING A DNA:RNA HYBRID AND A DISPLACED SINGLE STRANDED DNA, THAT FORM AND RESOLVE CONTINUALLY. HOWEVER, WHEN R-LOOPS ACCUMULATE, THEY CAN IMPEDE GENE TRANSCRIPTION, PROMOTE DNA DAMAGE, AND RESULT IN DISEASE. R-LOOPS ARE IMPLICATED IN SEVERAL NEURODEGENERATIVE DISORDERS, SUCH AS FRONTOTEMPORAL DEMENTIA AND AMYOTROPHIC LATERAL SCLEROSIS, AND IN NEURODEVELOPMENTAL DISORDERS SUCH AS FRAGILE X SYNDROME. WHETHER R-LOOP DEREGULATION CONTRIBUTES TO THE ETIOLOGY OF ASD IS NOT CLEAR. THE CENTRAL CHALLENGE IN LINKING R-LOOP DEREGULATION TO ASDS HAS BEEN PINPOINTING FACTORS THAT ARE MUTATED IN ASD THAT ARE ALSO R-LOOP REGULATORS. WE HAVE IDENTIFIED FACTORS THAT LOCALIZE TO R-LOOPS USING A PROXIMITY LABELING PROTEOMIC APPROACH. OUR APPROACH YIELDED CHROMATIN REMODELERS, HOMEOBOX TRANSCRIPTION FACTORS, AND HELICASES. A NUMBER OF THESE PROTEINS HAD ALSO BEEN REPORTED TO BE MUTATED IN ASD, E.G. ADNP, POGZ, CHD2, AND DHX30. THESE PRELIMINARY RESULTS PROVIDE THE FIRST INDICATION THAT R-LOOP DYSFUNCTION MAY CONTRIBUTE TO ASD. WE FOCUS OUR STUDY ON THE ANALYSIS OF ADNP, AN R-LOOP REGULATOR THAT IS FREQUENTLY MUTATED IN ASD AND IS CAUSAL IN ADNP SYNDROME. OUR PRELIMINARY STUDIES SHOW THAT ADNP LOSS IN MOUSE EMBRYONIC STEM CELLS (MESCS) RESULTS IN R-LOOP ACCUMULATION SPECIFICALLY AT ADNP BINDING SITES. IMPORTANTLY, OUR DATA SHOW THAT THE HOMEODOMAIN OF ADNP IS CRITICAL FOR R-LOOP SUPPRESSION AND THAT DELETION OF THE HOMEODOMAIN COMPROMISES THE ABILITY OF MESCS TO DIFFERENTIATE INTO NEURAL PROGENITOR CELLS (NPCS). WE FOUND THAT HUMAN INDUCED PLURIPOTENT STEM CELLS (HIPSCS) WITH AN ADNP TYROSINE 719* MUTATION (ADNP TYR719*) ALSO SHOW R-LOOP DEREGULATION. BASED ON THESE DATA, WE HYPOTHESIZE THAT ACCUMULATION OF R-LOOPS AT SPECIFIC GENOMIC REGIONS CHANGES NORMAL GENE EXPRESSION PROGRAMS LEADING TO IMPROPER NEURONAL DIFFERENTIATION, WHICH CAN ULTIMATELY CONTRIBUTE TO ASD. HERE WE WILL DETERMINE HOW R-LOOP ACCUMULATION IN ADNP TYR719* HIPSCS AFFECTS GENE EXPRESSION IN THE PLURIPOTENT STATE AND UPON NEURONAL DIFFERENTIATION. WE WILL EVALUATE THE NEURODIFFERENTIATION POTENTIAL OF ADNP TYR719* USING A CEREBRAL ORGANOID MODEL AND DETERMINE IF R-LOOP ATTENUATION CAN AMELIORATE THE NEURODIFFERENTIATION DEFECTS OBSERVED IN ADNP MUTANTS.

MECHANISMS OF RESISTANCE IN HIV-1 EXPOSED SERO-NEGATIVE IV-DRUG USERS: INDUCTION OF IFN-MEDIATED FACTORS AND S100 PROTEINS AS DETERMINANTS OF NK CELL

EXPLORING CELL-FREE GLYCOMIC INTERACTIONS IN HIV-ASSOCIATED NEUROLOGICAL DISORDERS

NEUTRALIZING PERSISTENT IFN-I TO IMPROVE HIV-SPECIFIC CAR T CELL THERAPY - PROJECT SUMMARY A CRITICAL HURDLE TO FURTHER IMPROVING THE QUALITY OF LIFE FOR PEOPLE LIVING WITH HIV (PLWH) IS THE NEED TO RESOLVE THE RESIDUAL IMMUNE ACTIVATION AND INFLAMMATION THAT PERSISTS EVEN IN THOSE TAKING EFFECTIVE ANTIRETROVIRAL THERAPY (ART), WHICH SUPPRESSES HIV REPLICATION. THIS UNRESOLVED AND PERSISTENT IMMUNE ACTIVATION IS ASSOCIATED WITH INCREASED TYPE-I INTERFERON (IFN-I) SIGNALING, AND INCREASED INCIDENCE OF COMORBIDITIES. ENCOURAGINGLY, REPORTS DEMONSTRATE THAT BLOCKING IFN-I SIGNALING IN ANIMAL MODELS OF HIV INFECTION CAN REDUCE HIV RESERVOIRS AND RESTORE T CELL IMMUNE FUNCTION. WE HYPOTHESIZE THAT BLOCKING IFN-I WOULD LIKEWISE AUGMENT ENGINEERED T CELL-BASED THERAPIES AGAINST HIV, SUCH AS CHIMERIC ANTIGEN RECEPTOR (CAR) T CELLS. OUR PRIOR WORK HAS DEMONSTRATED THAT WHEN ENGINEERED TO EXPRESS BOTH THE 4-1BB AND CD28 COSTIMULATORY DOMAINS AND PROTECTED FROM HIV INFECTION, HIV-SPECIFIC CD4 ECTODOMAIN CAR T CELLS CAN REDUCE ACUTE VIREMIA, PREVENT CD4+ T CELL LOSS, AND REDUCE VIRAL BURDEN IN THE TISSUES OF HIV-INFECTED HUMANIZED MICE. HOWEVER, THE REDUCTION OF PLASMA VIRAL LOADS WAS ULTIMATELY TRANSIENT, SUGGESTING THAT THE POTENCY OF HIV-SPECIFIC CAR T CELLS SHOULD BE FURTHER OPTIMIZED FOR CLINICAL TRANSLATION. OUR PRELIMINARY DATA HIGHLIGHTS INTERFERON-BETA (IFNB) AS A KEY IMMUNOSUPPRESSIVE IFN-I NEGATIVELY REGULATING CAR T CELL PROLIFERATION, AND WE DEMONSTRATE THAT NEUTRALIZING IFNB IN VIVO ENHANCED THE ENGRAFTMENT AND PERSISTENCE OF HIV-SPECIFIC CAR T CELLS ADOPTIVELY TRANSFERRED INTO HIV-INFECTED ART- SUPPRESSED HUMANIZED MICE. THIS PROPOSAL WILL INTERROGATE WHETHER IFNB NEUTRALIZATION AUGMENTS CAR T CELL THERAPY THROUGH 1) IDENTIFYING THE MECHANISM(S) BY WHICH CHRONIC IFNB EXPOSURE MEDIATES HIV-SPECIFIC CAR T CELL DYSFUNCTION, AND 2) DETERMINING THE EFFECT OF NEUTRALIZING IFNB ON CAR T CELL FUNCTION AND PERSISTENCE IN HIV INFECTION IN VIVO. THE PROPOSED AIMS SEEK TO DEVELOP THE NEUTRALIZATION OF IFNB AS A NOVEL IMMUNOTHERAPY APPROACH TO MAXIMIZE THE POTENCY OF HIV-SPECIFIC CAR T CELLS AIMED AT ACHIEVING A FUNCTIONAL HIV CURE.

CROSS-SECTIONAL STUDY TO IDENTIFY HOST PLASMA BIOMARKERS AND SOMALOGIC DISEASE PROGNOSIS SCORES ASSOCIATED WITH CELLULAR HIV-1 RNA EXPRESSION IN ART SUPPRESSED PERSONS - PROJECT SUMMARY ANTIRETROVIRAL THERAPY (ART) RESULTS IN SUPPRESSION OF HUMAN IMMUNODEFICIENCY VIRUS (HIV) REPLICATION, INCREASE IN CD4+ T CELL COUNT, AND PARTIAL RESTORATION OF IMMUNE RESPONSES. YET DESPITE SUPPRESSIVE ART, BOTH TRANSCRIPTIONALLY SILENT AND TRANSCRIPTIONALLY ACTIVE [I.E. CELL-ASSOCIATED RNA (CA-RNA)] HIV VIRUSES REMAIN IN VARIOUS CD4+ T CELL SUBSETS IN PERSONS LIVING WITH HIV (PLWH) AND ARE PROPOSED TO CONTRIBUTE TO RESIDUAL IMMUNE ACTIVATION, LOWER IMMUNE RECONSTITUTION AND DEVELOPMENT OF COMORBIDITIES. MUCH ATTENTION HAS BEEN GIVEN IN OUR FIELD TO THE CLINICAL SIGNIFICANCE OF CONTINUED HIV EXPRESSION AFTER ART, BUT WE STILL LACK PLASMA HOST BIOMARKERS THAT COULD ILLUSTRATE THE IMPACT OF CA-RNA OR COULD LINK CA-RNA TO FUTURE RISK FOR COMORBIDITIES. PART OF THE LIMITATION TO ADDRESS THIS GAP HAS BEEN THAT STUDIES ATTEMPTING TO FIND A CORRELATION BETWEEN HIV RESERVOIR LEVELS AND PLASMA MARKERS OF IMMUNE ACTIVATION HAVE INCLUDED ONLY A LIMITED NUMBER HOST CELLULAR OR PLASMA VARIABLES, HAVE NOT STARTED WITH A PRE-DEFINED COHORT WITH KNOWN RESERVOIR LEVELS, HAVE NOT LINKED VARIABLES TO PREDEFINED PROGNOSTIC BIOMARKERS, AND/OR HAVE YIELDED INCONSISTENT RESULTS (SEE BACKGROUND). THIS R21 NOW ADDRESSES THESE LIMITATIONS BY STUDYING A COHORT WITH KNOWN HIV RESERVOIR LEVELS AND BY INTRODUCING RECENT ADVANCES IN SOMALOGIC-BASED TECHNOLOGY INCLUSIVE OF (A) MEASURING A LARGE SET (AT LEAST 7000) OF PLASMA PROTEINS AND (B) DETERMINING LINKS OF PREDEFINED HOST BIOMARKER SIGNATURES TO RISK SCORES FOR COMORBIDITY OUTCOMES AS SHOWN BY OUR PRELIMINARY DATA AND OTHER STUDIES. THIS R21 PROPOSAL IS POSSIBLE AS A RESULT OF THE COLLECTION THROUGH THE BEAT-HIV MARTIN COLLABORATORY PROGRAM OF PLASMA AND PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) FROM 94 ART SUPPRESSED PLWH WITH KNOWN DISTRIBUTION OF HIV PROVIRAL DNA MEASURED BY THE INTACT PROVIRUS ASSAY (IPDA) WHICH IN TURN IS ASSOCIATED WITH CA-RNA. SPECIFICALLY, WE HYPOTHESIZE THAT IN PLWH ON SUPPRESSIVE ART THE LEVELS OF CELL-ASSOCIATED HIV RNA WILL BE ASSOCIATED WITH A) PLASMA HOST PROTEOMIC BIOMARKERS, AND B) PRE-DEFINED CLINICAL COMORBIDITY RISK SCORES FOR CARDIOVASCULAR, LIVER/KIDNEY, AND METABOLIC DISEASE FOLLOWING AGE ADJUSTMENT. WE WILL TEST THIS HYPOTHESIS BY THE FOLLOWING SPECIFIC AIMS: (1) IDENTIFY HOST PLASMA BIOMARKERS AMONG 7000 PROTEIN MEASURES THAT WILL BEST PREDICT PLWH ON ART WITH HIGHER CA-RNA INDEPENDENTLY OF AGE, AND (2) DETERMINE IF PLASMA CLINICAL PROGNOSTIC SCORES FOR COMORBIDITY RISKS OF CARDIOVASCULAR, LIVER/KIDNEY, AND METABOLIC DISEASE ARE HIGHER IN PLWH ON ART WITH HIGH CA-RNA WHEN ADJUSTED FOR AGE. COMPLETION OF THIS R21 PROPOSAL WILL PROVIDE FOUNDATIONAL DATA TO FURTHER EVALUATE THE USAGE OF BIOMARKER CHANGES IN FUTURE RO1S DESCRIBING STRATEGIES TARGETING TRANSCRIPTIONALLY ACTIVE RESERVOIRS ON ART. THE LONG-TERM IMPACT OF THIS PROPOSAL IS ITS POTENTIAL TO ADVANCE CURE-DIRECTED STRATEGIES TARGETING HIV EXPRESSING CELLS (I.E., IMMUNE-BASED, GAG- POL/CARD8 ACTIVATING, ETC.) AND TO MAKE POSSIBLE EARLY DETERMINATIONS IN BIOMARKER CHANGES OF SIGNIFICANCE TO PROGNOSIS RISK PROFILES UPON A REDUCTION OF PERSISTENT HIV RESERVOIRS.

INVESTIGATING MECHANISMS CONTROLLING THE DEVELOPMENT OF TIM4+ MACROPHAGES - PROJECT SUMMARY 1. OVERVIEW OF RESEARCH IN THE LABORATORY THERE ARE CURRENTLY TWO FOCUSES OF MY LAB: ONE FOCUS IS ON MECHANISMS CONTROLLING THE DEVELOPMENT OF MACROPHAGES AFTER DIFFERENTIATING FROM MONOCYTES IN THE STEADY STATE AND DISEASE SETTINGS. THIS IS AN IMPORTANT BUT POORLY UNDERSTOOD BIOLOGICAL PROCESS. MOST STUDIES IN THE PAST HAVE BEEN FOCUSING ON THE DIFFERENTIATION PROCESS FROM MONOCYTES TO MACROPHAGES, NOT MUCH ON THEIR CONTINUED DEVELOPMENT AFTERWARDS. AS DESCRIBED IN THE APPLICATION, WE PROPOSE A NOVEL CONCEPT THAT IN ORDER TO DEVELOP INTO PROFESSIONAL PHAGOCYTES THAT HANDLE APOPTOTIC CELLS EFFICIENTLY AND “SILENTLY”, THE TIM4- MACROPHAGES NEED TO BE “TRAINED AND TESTED” CONTINUOUSLY TO BECOME QUALIFIED TO DEVELOP INTO TIM4+ MACROPHAGES. FURTHERMORE, WE AIM TO REVEAL NOVEL EPIGENETIC AND METABOLIC REGULATORS FOR THIS DEVELOPMENTAL PROCESS. THE OTHER FOCUS OF MY LAB IS TO LEVERAGE MYELOID CELL ACTIVATION TO TREAT METASTATIC OVARIAN CANCER. THE FIRST MANUSCRIPT OF MY LAB ON THIS TOPIC WAS RECENTLY PUBLISHED IN THE JOURNAL OF EXPERIMENTAL MEDICINE AND HAS RECEIVED TOP 2-3% ATTENTION SCORES. 2. GOALS FOR THE NEXT FIVE YEARS THE GOALS INCLUDE THE FOLLOWING: (1) TO TEST HOW EFFEROCYTOSIS AND IL4 SIGNALING SYNERGISTICALLY PROMOTE THE DEVELOPMENT OF TIM4+ MACROPHAGES, (2) TO PROFILE THE EPIGENETIC AND METABOLIC CHANGES OF MACROPHAGES DURING THEIR DEVELOPMENT INTO TIM4+ MACROPHAGES, AND (3) TO IDENTIFY NOVEL EPIGENETIC AND METABOLIC REGULATORS FOR THE DEVELOPMENT OF TIM4+ MACROPHAGES. 3. OVERALL VISION OF THE RESEARCH PROGRAM THIS RESEARCH PROGRAM IS DESIGNED TO ADDRESS THE FUNDAMENTAL QUESTION: “WHAT FACTORS DICTATE THE DEVELOPMENT TIM4+ MACROPHAGES FROM TIM4- MACROPHAGES AFTER THEY FINISH DIFFERENTIATION FROM MONOCYTES?”. THIS QUESTION IS OF PARAMOUNT SIGNIFICANCE FOR GENERAL MEDICINE BECAUSE OF THE BROAD PRESENCE AND KEY ROLE OF TIM4+ MACROPHAGES IN VIRTUALLY ALL TISSUES AS WELL AS THEM BEING THE BEST EFFEROCYTES, I.E., PHAGOCYTOSING APOPTOTIC CELLS SILENTLY AND EFFICIENTLY, IN THE BODY. DYSREGULATION OF TIM4+ MACROPHAGES COULD AFFECT A WIDE SPECTRUM OF DISEASES, FROM AUTOIMMUNE DISEASES TO SEVERE INFECTIONS, TO CANCER, AND TO TISSUE INJURY. BASED ON PUBLISHED RESULTS FROM OTHERS AND PROMISING DATA FULLY GENERATED IN MY OWN LABORATORY AT THE WISTAR INSTITUTE IN THE PAST YEAR, WE PROPOSE A NEW MODEL FOR THE DEVELOPMENT OF TIM4+ MACROPHAGES: “CONTINUOUS “TRAINING AND TESTING” OF SILENT AND EFFICIENT EFFEROCYTOSIS BY MACROPHAGES EVENTUALLY PRODUCES TIM4+ MACROPHAGES”. COMPLETION OF THIS RESEARCH PROGRAM WILL PROVIDE CONVINCING EVIDENCE FOR THIS NEW MODEL AND REVEAL NOVEL EPIGENETIC AND METABOLIC REGULATORS FOR THIS PROCESS. THIS WILL OPEN A NEW DIRECTION FOR MODULATING MACROPHAGE HETEROGENEITY. CONSIDERING ITS SIGNIFICANCE IN ALMOST ALL DISEASES, FUTURE WORK WILL FOCUS ON MANIPULATING THE NEWLY IDENTIFIED REGULATORS TO MODULATE MACROPHAGE SUBSETS FOR BETTER DISEASE OUTCOMES.

COMPARTMENTALIZATION AND DISCRIMINATION OF DSRNA IN THE NUCLEUS - PROJECT SUMMARY THE LONG-TERM GOAL OF OUR LAB IS TO UNDERSTAND THE FUNDAMENTAL MECHANISMS BY WHICH RNA STRUCTURES AND RNA MODIFICATIONS CAN HELP CELLS DISTINGUISH BETWEEN SELF AND NON-SELF MOLECULES. ONE OF THESE POTENT NON-SELF MOLECULES IS DOUBLE-STRANDED RNA (DSRNA), A PARTICULAR STRUCTURE OF RNA THAT IS NOT PRESENT AT HIGH LEVELS IN UNSTRESSED CELLS, BUT CAN BE SEEN AS A HALLMARK OF VIRAL INFECTION. UNFORTUNATELY, TRANSCRIPTION OF ENDOGENOUS REPETITIVE ELEMENTS WITHIN OUR OWN GENOMES CAN LEAD TO THE FORMATION OF DSRNA AND ACTIVATION OF RNA SENSORS AND INAPPROPRIATE DESTRUCTIVE CELL SIGNALING PATHWAYS EVEN IN THE ABSENCE OF INFECTION. AS SUCH, THE BALANCE BETWEEN SENSING PATHOGENIC DSRNA AND TOLERATING SELF DSRNA MUST BE FINELY TUNED BY NEGATIVE REGULATORS OF RNA SENSING. IN ADDITION, RNA SENSING IS ALSO COMPARTMENTALIZED, WITH SENSORS IN THE CYTOPLASM KEPT AWAY FROM THE NUCLEUS WHERE RNA TRANSCRIPTION TAKES PLACE. IMPORTANT FOR HUMAN HEALTH, LOSS OF THIS BALANCE IN EITHER DIRECTION IS HARMFUL, WITH NEGATIVE REGULATOR LOSS-OF-FUNCTION LEADING TO INFLAMMATORY DISEASE OR AUTOIMMUNITY WHILE GAIN-OF-FUNCTION HAS BEEN ASSOCIATED WITH CANCER. OUR RESEARCH HAS SHOWN THAT HUMAN VIRUSES CAN ACTIVELY PREVENT THE FORMATION OF DSRNA BY REGULATION OF RNA SPLICING AND REMOVAL OF COMPLEMENTARY INTRONS. HOWEVER, PERTURBATION OF VIRAL SPLICING EFFICIENCY LEADS TO PRODUCTION OF NUCLEAR DSRNA THAT IS SENSED BY CYTOPLASMIC SENSORS THAT SUBSEQUENTLY MOVE TO THE NUCLEUS. THIS TOOL PROVIDES A UNIQUE OPPORTUNITY TO ASSESS HOW CELLS RESPOND TO DSRNA DERIVED FROM THE NUCLEAR COMPARTMENT, AND HOW TOLERANCE OF THIS NON-SELF MOLECULE CAN BE BROKEN. THIS PROPOSAL WILL EXPLORE THE BASIC BIOLOGICAL PRINCIPLES GOVERNING THE INTERACTIONS BETWEEN NUCLEAR DSRNA, RNA PROCESSING, AND CYTOPLASMIC RNA SENSORS. WE WILL ASK FUNDAMENTAL QUESTIONS ABOUT HOW RNA STRUCTURES, INCLUDING INTRAMOLECULAR HAIRPINS AND INTERMOLECULAR TWO-STRANDED HELICES, INFLUENCE RNA SENSOR ACTIVATION AND THE INTERPLAY BETWEEN REDUNDANT SENSING PATHWAYS. ADDITIONALLY, WE WILL INVESTIGATE WHETHER NUCLEAR DSRNA CARRIES SPECIFIC CHEMICAL MODIFICATIONS THAT MODULATE ITS STRUCTURE, RECOGNITION BY SENSORS, OR SENSOR ACTIVITY. FINALLY, WE WILL DETERMINE RNA BINDING PROTEINS THAT SPECIFICALLY BIND OR REGULATE NUCLEAR DSRNA USING QUANTITATIVE PROTEOMICS. THE RESULTS WILL SHED LIGHT ON HOW HEALTHY CELLS PREVENT INAPPROPRIATE ACTIVATION OF RNA SENSING RESPONSES TO SELF-DERIVED RNA. WE WILL ALSO LEARN HOW STRESSFUL SITUATIONS, SUCH AS VIRAL INFECTION, CAN REWIRE EXISTING SIGNALING PATHWAYS TO ALLOW NUCLEIC ACID SURVEILLANCE OF THE NUCLEUS. THE COMPLETION OF THIS PROJECT WILL ULTIMATELY LEAD TO A GREATER MECHANISTIC UNDERSTANDING OF NUCLEIC ACID SENSING IN CELLULAR HEALTH AND HOMEOSTASIS.

DEVELOPMENT OF BROAD CORONAVIRUS IMMUNITY TARGETING THE FUSION PEPTIDE - PROJECT SUMMARY THIS REVISED APPLICATION WAS ORIGINALLY RESPONDING TO THE EMERGENCY AWARD ‘RAPID INVESTIGATION OF SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 (SARS-COV-2) AND CORONAVIRUS DISEASE 2019 (COVID-19)’ SUBMITTED THROUGH PAR-20-177. HOWEVER, WE ARE SUBMITTING THE REVISED APPLICATION, UPON ADVICE FROM NIH, TO THE PARENT PA-20-195 WITH A DUE DATE OF NOV 16TH 2020. THE APPLICATION IS TITLED ‘DEVELOPMENT OF BROAD CORONAVIRUS IMMUNITY TARGETING THE FUSION PEPTIDE’. THE ASSEMBLED TEAM BRINGS TOGETHER HIGHLY COMPLEMENTARY EXPERTISE IN ORDER TO DEVELOP A UNIQUE CORONAVIRUS VACCINE APPROACH TO PROVIDE MORE EFFECTIVE, FOCUSED AND BROADENED HUMORAL IMMUNITY. THIS PROPOSAL FOCUSES ON THE FUSION PEPTIDE SITE OF VULNERABILITY AS IT HAS BEEN UNDERSTUDIED, IT IS HIGHLY CONSERVED AND RECENTLY BEEN IDENTIFIED AS AN EPITOPE OF NEUTRALIZING ANTIBODIES IN HUMAN CONVALESCENT SERA. THE PROJECT AIMS ARE: (1) DEVELOP PAN- CORONAVIRUS FUSION PEPTIDE NANOPARTICLE AND TRIMER IMMUNOGENS (2) DEVELOP IMMUNIZATION REGIMES TO INDUCE BROADLY NEUTRALIZING ANTIBODIES USING NUCLEIC ACID DELIVERY.

DEVELOPING A GENOMIC TOOLKIT TO IDENTIFY RNAS WITHIN NON-CANONICAL DNA STRUCTURES - PROJECT SUMMARY THE GOAL OF THIS RESEARCH PROPOSAL IS TO DEVELOP NEW TECHNOLOGIES FOR THE IDENTIFICATION OF REGIONS WITHIN LONG NON-CODING RNAS THAT ENGAGE IN R-LOOP AND RNA-TRIPLEX FORMATION. LONG NON-CODING RNAS (LNCRNAS) CAN LOCALIZE TO CHROMATIN AND REGULATE IMPORTANT NUCLEAR PROCESSES SUCH AS TRANSCRIPTION, RECOMBINATION, AND REPAIR. LNCRNAS ARE TARGETED TO CHROMATIN THROUGH THEIR ASSOCIATION WITH SPECIFIC PROTEINS OR THEY CAN INTERACT DIRECTLY WITH THE DNA IN CHROMATIN. THE EXTENT TO WHICH LNCRNAS DIRECTLY CONTACT DNA AND THE RNA REGIONS RESPONSIBLE FOR THESE CONNECTIONS ARE LARGELY UNKNOWN. WE WILL BRIDGE THIS GAP IN KNOWLEDGE BY DEVELOPING NEW GENOMICS TECHNOLOGIES THAT WILL YIELD HIGH-RESOLUTION MAPS OF REGIONS WITHIN ALL NUCLEAR RNAS THAT INTERACT WITH DNA THROUGH R-LOOP AND TRIPLEX STRUCTURES. RNA-DNA INTERACTIONS ON CHROMATIN OCCUR THROUGH THE FORMATION OF TWO DISTINCT STRUCTURES: 1) R-LOOPS THAT FORM THROUGH WATSON-CRICK BASE PAIRING BETWEEN RNA AND ITS COMPLEMENTARY DNA STRAND, AND 2) RNA-TRIPLEXES THAT FORM BECAUSE AN RNA OCCUPIES THE MAJOR GROOVE OF THE DOUBLE HELIX AND FORMS HOOGSTEEN OR REVERSE HOOGSTEEN HYDROGEN BONDS WITH THE PURINES IN THE WATSON-CRICK DNA STRANDS. ANTIBODY- DEPENDENT AND -INDEPENDENT STRATEGIES EXIST TO IDENTIFY THE GENOME-WIDE DISTRIBUTION OF R-LOOP STRUCTURES. HOWEVER, MOST OF THESE METHODS SEQUENCE THE DNA COMPONENT OF R-LOOPS. WHILE INFORMATION FROM THE PERSPECTIVE OF DNA PROVIDES A VIEW OF WHERE R-LOOPS FORM ACROSS THE GENOME, IT CANNOT ANSWER WHETHER R- LOOPS AT SPECIFIC GENOMIC REGIONS FORM AS A BY-PRODUCT OF TRANSCRIPTION OR BECAUSE OF THE PRESENCE OF REGULATORY NON-CODING RNAS. MOREOVER, THE EXISTING ANTIBODY-DEPENDENT APPROACH TO RECOVER THE RNA COMPONENT OF R- LOOPS LACKS SENSITIVITY, SPECIFICITY, AND RESOLUTION. KNOWLEDGE OF THE RNA COMPONENT OF R-LOOPS WILL ALLOW US TO DISTINGUISH REGULATORY R-LOOPS, WHICH MAY INVOLVE LNCRNAS, FROM CO-TRANSCRIPTIONAL R-LOOPS AND DISCOVER NEW R-LOOP BASED MECHANISMS IN GENOME REGULATION. THE NEED FOR NEW TOOLS TO IDENTIFY RNA-TRIPLEXES IS MORE URGENT. WHILE COMPUTATIONAL APPROACHES TO PREDICT TRIPLEX FORMATION HAVE BEEN DEVELOPED, GENOMIC METHODS TO DETECT ENDOGENOUS TRIPLEXES IN VIVO DO NOT EXIST. NEW METHODS TO DETECT RNAS WITHIN R-LOOPS AND RNA-TRIPLEXES WILL ENHANCE OUR UNDERSTANDING OF THESE STRUCTURES AND IDENTIFY THE EXTENT OF THEIR FUNCTION IN GENE REGULATION. THESE TOOLS CAN BE APPLIED TO EXAMINE HOW CODING AND NON-CODING RNAS DIFFER IN THEIR INTERACTIONS WITH CHROMATIN AND CAN HELP DEFINE NEW PRINCIPLES FOR GENE REGULATION THROUGH RNA INTERACTIONS. HERE, WE PROPOSE TO DEVELOP NEW ANTIBODY-INDEPENDENT STRATEGIES THAT EMPLOY EPITRANSCRIPTOMICS TO ENRICH FOR AND IDENTIFY THE RNA COMPONENT OF R-LOOPS AND RNA-TRIPLEXES.

A MACHINE LEARNING ALGORITHM TO ACCELERATE DEVELOPMENT OF IN VIVO DNA-VECTORED ANTIBODY COUNTERMEASURES FOR THE WARFIGHTER

ENGINEERING HIV-RESISTANT CAR T CELLS FOR A FUNCTIONAL HIV CURE - PROJECT SUMMARY/ABSTRACT THE HUMAN IMMUNODEFICIENCY VIRUS (HIV) PROMPTLY SUBVERTS THE HOST CELLULAR IMMUNE RESPONSE THROUGH RAPID VIRAL ESCAPE, AS WELL AS HIGH ANTIGEN LOADS LEADING TO CHRONIC IMMUNE ACTIVATION, T CELL EXHAUSTION, AND IMMUNE DYSFUNCTION. THE ADVENT OF POTENT ANTIRETROVIRAL THERAPY (ART) CAPABLE OF FULLY SUPPRESSING VIRAL REPLICATION HAS DRASTICALLY REDUCED THE MORBIDITY AND MORTALITY OF HIV INFECTION. HOWEVER, ART MUST BE TAKEN INDEFINITELY AS HIV PERSISTS IN LONG-LIVED STABLE RESERVOIRS AND THUS PRESENTS A SIGNIFICANT PUBLIC HEALTH BURDEN THAT CAN ONLY BE ALLEVIATED WITH A PREVENTATIVE VACCINE AND MORE POTENT CURE APPROACHES. GIVEN THAT HIV EFFECTIVELY EVADES THE CELLULAR IMMUNE RESPONSE, AND THE LATENT HIV RESERVOIR IS PREFERENTIALLY SEEDED WITH VIRUS HARBORING RELEVANT CYTOTOXIC T LYMPHOCYTE (CTL)-ESCAPE MUTATIONS, GENETIC ENGINEERING MODALITIES MAY OFFER A POTENT ALTERNATIVE TO INTRINSIC IMMUNITY. CHIMERIC ANTIGEN RECEPTOR (CAR) T CELLS HAVE SHOWN IMPRESSIVE EFFICACY IN ELIMINATING BLOOD CELL CANCERS IN THE CLINIC, AND CAR T CELLS RE-ENGINEERED TO TARGET HIV USING THE CD4 ECTODOMAIN (CD4-CAR T) REPRESENT A POTENT ESCAPE-RESISTANT CELLULAR THERAPY DEMONSTRATED TO HAVE ENHANCED CYTOTOXIC FUNCTION OVER TRADITIONAL CYTOTOXIC T LYMPHOCYTES. WE HAVE RECENTLY DESCRIBED THE CREATION OF A SIGNIFICANTLY ENHANCED DUAL COSTIMULATORY DOMAIN CAR T CELL PRODUCT (DUAL CARS), WHICH SIGNIFICANTLY OUTPERFORMED 3RD GENERATION CAR T CELLS IN VIVO. IMPORTANTLY, THESE STUDIES IDENTIFIED AT LEAST TWO ADDITIONAL HURDLES TO CD4-CAR T CELL EFFICACY IN VIVO, WHICH LIKELY INFORMS TRANSLATION TO THE CLINIC. FIRST, CD4-CAR T CELLS RAPIDLY UPREGULATE MULTIPLE INHIBITORY RECEPTORS, EXPRESS TRANSCRIPTION FACTORS ASSOCIATED WITH EXHAUSTION, AND DISPLAY ATTENUATED FUNCTION EX VIVO. SECONDLY, OUR STUDIES DEFINITIVELY SHOW THAT SUPPRESSION OF PLASMA VIRAL LOAD REQUIRES PROTECTION OF THE CAR T CELL PRODUCT FROM HIV INFECTION. THIS PROPOSAL SEEKS TO ADDRESS THESE DEFICITS IN CD4-ECTODOMAIN CAR T CELL THERAPY BY 1) DEFINING THE MECHANISM(S) BY WHICH CHRONIC HIV EXPOSURE ATTENUATES T CELL FUNCTION, AND 2) DEVELOPING NOVEL COMBINATORIAL STRATEGIES TO FULLY PROTECT CD4-CAR T CELLS FROM HIV INFECTION, WITH THE ULTIMATE GOAL OF CREATING A T CELL IMMUNOTHERAPY EXHIBITING ENHANCED EFFICACY IN THE CLINIC.

DSRNA PRODUCTION AND SENSING DURING DNA VIRUS INFECTION - PROJECT SUMMARY VIRAL INFECTIONS ARE KNOWN TO PRODUCE DOUBLE-STRANDED RNA (DSRNA), A MOLECULE THAT IS NOT PRESENT AT HIGH LEVELS IN UNINFECTED HOST CELLS. THIS PROPERTY OF DSRNA IS EXPLOITED BY CELLS TO SENSE VIRAL INFECTION AND DEPLOY ANTI-VIRAL COUNTERMEASURES. WHILE DNA VIRUSES PRODUCE VIRAL MRNA MOLECULES THAT LOOK IDENTICAL TO CELLULAR RNA, MANY DNA VIRUSES ARE THOUGHT TO PRODUCE DSRNA DUE TO THE PROCESS OF SYMMETRICAL GENE TRANSCRIPTION OF BOTH STRANDS OF DNA. WHEN WE LOOKED FOR THE PRESENCE OF DSRNA DURING ADENOVIRUS (ADV) INFECTION USING MODERN ANTIBODY-BASED TECHNIQUES WE FOUND NO EVIDENCE OF DSRNA PRODUCTION, DIRECTLY COUNTERING THE EXISTING DOGMA. CONSIDERING MANY DNA VIRUSES ENCODE ANTAGONISTS OF CELLULAR DSRNA-SENSING PATHWAYS, THIS DIRECTLY CALLS INTO QUESTION THE RELEVANCE OF DSRNA SENSING DURING DNA VIRUS INFECTION. WHILE WILDTYPE ADV DID NOT PRODUCE DETECTABLE DSRNA, VIRAL MUTANTS WHICH CAN NO LONGER SPLICE THEIR OWN TRANSCRIPTS EFFICIENTLY SAW ROBUST ACCUMULATION OF DSRNA WITHIN THE NUCLEUS. FURTHERMORE, THESE DSRNA-PRODUCING MUTANTS ACTIVATED CYTOPLASMIC SENSORS OF DSRNA SUCH AS PKR AND RNASEL. THE USE OF MUTANT VIRUSES PROVIDES A UNIQUE OPPORTUNITY TO ASSESS HOST RESPONSES TO DSRNAS DERIVED FROM DNA VIRUS INFECTION. STILL, THE QUESTION OF HOW THESE NUCLEAR DSRNAS ARE DETECTED BY CYTOPLASMIC SENSORS REMAINS UNANSWERED. BY COMPLETION OF THIS MENTORED CAREER DEVELOPMENT AWARD I WILL GAIN TRAINING IN RNA SEQUENCING, QUANTITATIVE MASS SPECTROMETRY, AND THE BIOINFORMATICS APPROACHES TO ANALYZE BOTH. IN THE MENTORED PHASE I WILL CONTINUE MY TRAINING WITH ADV, A RELATIVELY SIMPLE VIRUS THAT PROVIDES POWERFUL TOOLS TO UNDERSTAND REGULATION AND SENSING OF DNA VIRUS DERIVED NUCLEAR DSRNA. IN THE INDEPENDENT PHASE I WILL UTILIZE HERPES SIMPLEX VIRUS (HSV- 1), A COMPLEX VIRUS ABLE TO EXERT CONTROL OVER DSRNA-SENSING PATHWAYS, AS A MODEL VIRUS TO STUDY EXPLOITATION OF DSRNA FOR VIRAL GENE REGULATION. THIS PROPOSAL WILL REVEAL THE BINDING PARTNERS AND LOCALIZATIONS OF DNA VIRUS DERIVED DSRNA AS WELL AS NEW STRATEGIES IN WHICH VIRUSES EXPLOIT HOST CELL GENE REGULATORY MACHINERY. IN AIM 1 I WILL DETERMINE THE LOCALIZATION AND BINDING PARTNERS OF VIRAL DSRNA USING IMMUNOPRECIPITATION COUPLED TO NEXT GENERATION SEQUENCING AND MASS SPECTROMETRY. THESE EXPERIMENTS WILL DETERMINE HOW NUCLEAR DSRNA LEADS TO ACTIVATION OF CYTOPLASMIC SENSORS, AS WELL AS HOW ADV INTERACTS WITH AND BLOCKS THESE NOVEL PATHWAYS. IN AIM 2 I WILL DETERMINE HOW HSV-1 REGULATES ITS OWN VIRAL GENE EXPRESSION USING THE NUCLEAR RETENTION OF OVERLAPPING VIRAL TRANSCRIPT PAIRS THAT FORM DSRNA. THE OUTCOME OF THESE EXPERIMENTS WILL REVEAL A NEW MECHANISM FOR VIRAL GENE REGULATION WITH BROAD IMPLICATIONS FOR ALL HERPESVIRUSES. THE OUTSTANDING TRAINING ENVIRONMENT AT CHOP AND THE UNIVERSITY OF PENNSYLVANIA, COUPLED WITH THE EXCELLENT ADVISORY COMMITTEE I HAVE ASSEMBLED, WILL GREATLY FACILITATE MY RESEARCH DURING THE MENTORED PHASE AS WELL AS LAUNCH MY CAREER WITH THE SKILLS NECESSARY TO TRANSITION TO AN INDEPENDENT FACULTY POSITION STUDYING HOW HOST CELLS SENSE THE RNAS GENERATED BY DNA VIRUSES.

REPROGRAMMING T CELL FUNCTION WITH MULTIPLEXED GENOME ENGINEERING TO DEVELOP NEXT-GENERATION IMMUNOTHERAPY - ADOPTIVE CELLULAR IMMUNOTHERAPY HAS REVOLUTIONIZED CANCER TREATMENT, WITH ENGINEERED T CELLS ACHIEVING REMARKABLE SUCCESS IN HEMATOLOGIC MALIGNANCIES. HOWEVER, CHALLENGES SUCH AS RELAPSE, LIMITED EFFICACY IN SOLID TUMORS, AND THE IMMUNOSUPPRESSIVE TUMOR MICROENVIRONMENT (TME) REMAIN. THIS PROPOSAL ADDRESSES CRITICAL GAPS IN THE FIELD BY DEVELOPING TWO INNOVATIVE STRATEGIES: (1) INTRINSIC REGULATIONS, THROUGH SELF-REGULATABLE THERAPEUTIC T CELLS THAT DYNAMICALLY ADJUST CHIMERIC ANTIGEN RECEPTOR (CAR) EXPRESSION IN RESPONSE TO ANTIGEN STIMULATION, ENABLING PRECISE CONTROL OVER T CELL ACTIVITY AND MINIMIZING TOXICITY; AND (2) EXTRINSIC REGULATIONS, DEVELOPING NOVEL PROTEIN CHIMERAS THAT EXPLOIT ENDOGENOUS CELLULAR MACHINERY TO DEGRADE IMMUNOSUPPRESSIVE PROTEINS IN THE TME, RESTORING ANTI-TUMOR IMMUNITY. BY INTEGRATING THESE APPROACHES, THIS RESEARCH AIMS TO TRANSFORM CANCER IMMUNOTHERAPY, OFFERING SAFER AND MORE EFFECTIVE TREATMENTS FOR SOLID TUMORS AND OTHER CHALLENGING MALIGNANCIES. THE PROPOSED WORK HAS THE POTENTIAL TO IMPROVE THE SAFETY AND EFFICACY OF CAR-T CELL THERAPIES, PROVIDE NEW INSIGHTS INTO RECEPTOR DYNAMICS AND IMMUNE MODULATION, AND ALIGN WITH THE NCI’S MISSION TO ADVANCE INNOVATIVE CANCER TREATMENTS.

DESIGN OF VACCINATION STRATEGIES TO ELICIT BROADLY NEUTRALIZING ANTIBODIES AGAINST HIV-1 - PROJECT SUMMARY/ ABSTRACT AIDS IS A PREVENTABLE DISEASE, NEVERTHELESS MILLIONS OF NEW INFECTIONS OCCUR EVERY YEAR ACCORDING TO UNAIDS. A VACCINE THAT ELICITS BROADLY NEUTRALIZING ANTIBODIES (BNABS) AGAINST HIV-1 WOULD BE THE BEST WAY TO PREVENT THE SPREADING OF THE AIDS PANDEMIC, HOWEVER, NO EFFICACIOUS VACCINE HAS BEEN DEVELOPED TO DATE. PREVIOUS EFFORTS TO DESIGN AN ANTIBODY-BASED VACCINE HAVE BEEN UNSUCCESSFUL, IN PART DUE TO THE LIMITED INFORMATION AVAILABLE AT THE TIME ON THE HIV-1 PARTICLE, ITS MECHANISM OF INFECTION AND THE ANTI HIV-1 ANTIBODY RESPONSES ELICITED IN INFECTED INDIVIDUALS. RECENT ADVANCES IN THE FIELD HAVE OPENED NEW AVENUES FOR VACCINE DESIGN THAT WE WILL INVESTIGATE AS PART OF THE RESEARCH OF THIS PROPOSAL. OUR WORK RECENTLY SHOWED THAT COMMON VACCINATION STRATEGIES USING SINGULAR ENVELOPE (ENV)-BASED IMMUNOGENS WERE NOT SUITABLE TO ELICIT ANTI HIV-1 BNABS; INSTEAD, NOVEL SEQUENTIAL IMMUNIZATION STRATEGIES ELICITED NEUTRALIZING ANTIBODIES OF REMARKABLE POTENCY AND BREADTH IN KNOCK-IN MOUSE MODELS WITH A RESTRICTED ANTIBODY REPERTOIRE. IN THIS PROPOSAL, WE AIM TO DESIGN AND EVALUATE NEW IMMUNOGENS AND SEQUENTIAL IMMUNIZATION STRATEGIES TO ELICIT BNABS IN ANIMAL MODELS THAT HAVE A COMPLETE IMMUNOGLOBULIN (IG) REPERTOIRE. IN PARTICULAR WE PLAN TO: 1) TEST NEW IMMUNOGENS AND SEQUENTIAL IMMUNIZATION REGIMENS TO ELICIT BNABS AGAINST THE V3-N332 EPITOPE OF ENV IN A) WILD TYPE MICE, B) ALIVAMAB MICE CARRYING HUMAN IG LOCI AND C) MICE EXPRESSING THE HUMAN TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE (TDT) ENZYME AS MODELS THAT MORE CLOSELY RESEMBLE THE HUMAN IG REPERTOIRE; AND 2) DOCUMENT THE REQUIREMENT OF GLYCANS ON THE V3-N332 EPITOPE AT THE EARLY STAGES OF V3-N332 BNAB DEVELOPMENT. THE RESULTS OF THE PROPOSED RESEARCH WILL PROVIDE VALUABLE INFORMATION FOR THE DESIGN OF A VACCINE AGAINST HIV-1. THIS WORK WILL ALSO INFORM ABOUT GENERAL RULES GOVERNING THE ANTIBODY MATURATION PROCESS UPON SEQUENTIAL IMMUNIZATION THAT COULD FACILITATE THE DESIGN OF VACCINES AGAINST OTHER UNRELATED PATHOGENS. THE K99 PHASE OF THIS PROPOSAL WILL TAKE PLACE IN THE NUSSENZWEIG LABORATORY AT THE ROCKEFELLER UNIVERSITY IN COLLABORATION WITH THE BJORKMAN LABORATORY IN CALTECH. BOTH GROUPS AND INSTITUTIONS WILL OFFER AN OUTSTANDING ENVIRONMENT AND ALL NECESSARY RESOURCES TO CARRY OUT THE RESEARCH OF THIS PROPOSAL. THE NUSSENZWEIG LABORATORY HAS A LONG AND CONSOLIDATED TRAJECTORY STUDYING B CELLS AND HIV-1. THE BJORKMAN LABORATORY SPECIALIZES IN STRUCTURAL BIOLOGY AND IS PARTICULARLY INTERESTED IN THE IMMUNE RECOGNITION OF VIRAL PATHOGENS SUCH AS HIV-1. THE WORK OF THE BJORKMAN GROUP PERFECTLY COMPLEMENTS THE AREAS OF EXPERTISE OF THE NUSSENZWEIG LABORATORY AND PROVIDES AN OPTIMAL SCENARIO FOR THE RESEARCH OF THIS PROPOSAL. THE TRAINING RECEIVED DURING THE K99 PHASE IN THIS TERRIFIC ENVIRONMENT WILL UNDOUBTEDLY PROPEL AND FACILITATE MY TRANSITION TO INDEPENDENCE.

HOST GLYCOMIC DETERMINANTS OF HIV PERSISTENCE IN VIVO

ROLE OF HOST GLYCOSYLATION IN THE GALECTIN-9-MEDIATED REVERSAL OF HIV LATENCY

THE ROLE OF GUT MICROBE-DERIVED CHOLINE METABOLITES IN DRIVING THE PRO-INFLAMMATORY MACROPHAGE PHENOTYPE AND RESTRICTING PANCREATIC CANCER - PROJECT SUMMARY THE TREATMENT OF PANCREATIC DUCTAL ADENOCARCINOMA (PDAC) REMAINS A MAJOR HURDLE, WITH A 5-YEAR SURVIVAL RATE OF ONLY 9%. RESISTANCE OF PDAC TO CHEMOTHERAPY AND IMMUNOTHERAPY IS THOUGHT TO ARISE PRIMARILY DUE THE IMMUNOSUPPRESSIVE TUMOR MICROENVIRONMENT (TME), CHARACTERIZED BY A DENSE FIBROTIC STROMA AND HIGH INFILTRATES OF IMMUNOSUPPRESSIVE CELLS, INCLUDING TUMOR ASSOCIATED MACROPHAGES (TAM). TAMS BLOCK EFFECTOR T CELL FUNCTION AND TRIGGER EXHAUSTION. IMMUNE RESPONSES IN THE TME ARE LIKELY CONTROLLED BY MULTIPLE MECHANISMS, INCLUDING HOST INTRINSIC FACTORS AND, MORE RECENTLY APPRECIATED, CHANGES IN THE GUT MICROBIOME. HOWEVER, THE MOLECULAR MECHANISMS AND PATHWAYS BY WHICH THE GUT MICROBIOME IMPACT THE IMMUNE RESPONSES IN THE PDAC TME REMAIN POORLY UNDERSTOOD. OUR NEW PRELIMINARY WORK INDICATES THAT THE GUT MICROBIOTA CAN INFLUENCE THE IMMUNE RESPONSE THROUGH MICROBIAL METABOLITES THAT ENTER THE BLOOD STREAM TO MODULATE TAM ACTIVITY. IN AN UNTARGETED METABOLOMIC SCREEN ON CIRCULATING METABOLITES, WE DISCOVERED THE MOST SIGNIFICANT CHANGE WAS A DRAMATIC REDUCTION (>80-FOLD) IN TRIMETHYLAMINE N-OXIDE (TMAO) LEVELS IN PDAC-BEARING MICE TREATED WITH ANTIBIOTIC METRONIDAZOLE. TMAO IS FORMED IN TWO STEPS: THE GUT MICROBIAL TMA LYASE DEGRADES CHOLINE TO TRIMETHYLAMINE (TMA), WHICH THEN UNDERGOES OXIDATION IN THE LIVER TO FORM TMAO. INTRAPERITONEAL ADMINISTRATION OF PHYSIOLOGICALLY RELEVANT AMOUNTS OF TMAO TO PDAC-BEARING MICE SIGNIFICANTLY SUPPRESSED TUMOR GROWTH. THIS WAS ACCOMPANIED BY A SIGNIFICANT DECREASE IN TUMOR-INFILTRATING, IMMUNOSUPPRESSIVE MYELOID CELLS, AN INCREASE IN A PRO-INFLAMMATORY TAM PHENOTYPE, AND A STRIKING INCREASE IN INFILTRATES OF ACTIVATED CD8+ T CELLS IN THE PDAC TME. THESE AND OTHER DATA SUPPORT OUR HYPOTHESIS THAT DIETARY CHOLINE AND THE GUT MICROBIAL TMA LYASE ARE KEY FACTORS CONTRIBUTING TO CIRCULATING TMAO LEVELS AND THE PRO-INFLAMMATORY TAM PHENOTYPE. WE PROPOSE THAT SUPPLEMENTING TMAO OR ENACTING STRATEGIES THAT INCREASE IT WILL IMPROVE RESPONSE TO IMMUNOTHERAPY IN PDAC. AIM 1 TESTS THE HYPOTHESIS THAT DIETARY CHOLINE AND THE GUT MICROBIAL TMA PATHWAY CONTRIBUTE TO THE ACQUISITION OF A PRO-INFLAMMATORY TAM PHENOTYPE AND INDUCTION OF ANTI-TUMOR IMMUNE RESPONSE. WE WILL EMPLOY SUPPLEMENTATION OF CHOLINE DIET, DEPLETION OF TMA- PRODUCING BACTERIA, AND INHIBITION OF TMA LYASE TO EVALUATE THE MECHANISTIC LINKS BETWEEN DIETARY PRECURSORS OF TMAO, THE GUT MICROBIAL TMA PATHWAY, AND IMMUNE RESPONSES IN THE PDAC TME. AIM 2 TESTS THE HYPOTHESIS THAT INCREASING TMAO SENSITIZES PDAC TUMORS TO IMMUNOTHERAPY. WE WILL EVALUATE DIRECT ADMINISTRATION OF TMAO OR ENRICHMENT OF TMA-PRODUCING GUT BACTERIA SUCH AS CLOSTRIDIUM SPOROGENES IN COMBINATION WITH IMMUNE CHECKPOINT BLOCKADE. THIS AIM WILL ALSO EVALUATE THE CLINICAL SIGNIFICANCE OF THE TMAO BIOLOGY BY DETERMINING THE ASSOCIATION BETWEEN PLASMA TMAO LEVELS AND CLINICAL OUTCOME OF SURVIVAL AND DISEASE PROGRESSION IN PDAC. IN THE LONGER TERM, THIS WORK MAY FORM THE BASIS FOR DEVELOPING NEW, MICROBIOME-BASED THERAPIES FOR THE AGGRESSIVE AND HARD TO TREAT PDAC.

TWIST MAINTAINS STEMNESS IN LATENT BREAST CANCER METASTASES THROUGH A NOVEL NON-DNA-BINDING FUNCTION TARGETABLE WITH SMALL MOLECULES

MYELOID CELL-DERIVED INTERLEUKIN 1B PROMOTES CHEMORESISTANCE IN METASTATIC OVARIAN CANCER - PROJECT SUMMARY MOST OVARIAN CANCER (OC) PATIENTS RESPOND TO THE FRONT-LINE CHEMOTHERAPY BUT SUCCUMB TO CHEMORESISTANT RELAPSE WITH PERITONEAL METASTASES. HOW CHEMORESISTANCE DEVELOPS IN OC PATIENTS AFTER THE FRONT-LINE CHEMOTHERAPY REMAINS POORLY UNDERSTOOD. RECENT UNDERSTANDING OF THE TUMOR IMMUNE MICROENVIRONMENT (TIME) SUGGESTS THAT INFLAMMATORY MEDIATORS FROM MYELOID CELLS PLAY KEY ROLES IN DEVELOPING CHEMORESISTANCE. THIS PROPOSAL SEEKS TO UNDERSTAND WHETHER AND HOW A CLASSICAL INFLAMMATORY MEDIATOR FROM MYELOID CELLS, INTERLEUKIN 1Β (IL1Β), PROMOTES OC CHEMORESISTANCE, AIMING TO ERADICATE METASTATIC OC BY OVERCOMING IL1Β-MEDIATED CHEMORESISTANCE. BASED ON PUBLISHED AND OUR PRELIMINARY RESULTS, WE HYPOTHESIZE THAT MYELOID CELL-DERIVED IL1Β PROMOTES CHEMORESISTANCE IN METASTATIC OC BY MODIFYING TIME VIA ACTIVATION OF OC CELLS AND FIBROBLASTS; COMBINING IL1Β NEUTRALIZATION AND CHEMOTHERAPY ELIMINATES CHEMORESISTANT OC. THIS WILL BE TESTED WITH THE FOLLOWING AIMS. AIM 1: TEST THE HYPOTHESIS THAT THE COMBINATION OF CHEMOTHERAPY AND IL1Β NEUTRALIZATION OVERCOMES OC CHEMORESISTANCE. AIM 2: TEST THE HYPOTHESIS THAT MYELOID CELL-DERIVED IL1Β DRIVES OC CHEMORESISTANCE BY MODIFYING TIME. AIM 3: TEST THE HYPOTHESIS THAT MYELOID-DERIVED IL1Β PROMOTES FORMATION OF CHEMORESISTANT SPHEROIDS.

REGULATION OF MULTIPLE MYELOMA BY S100A9 PROTEIN

INNATE EFFECTOR FUNCTION AND HIV-1 CONTROL

FLOWCORE UPGRADE: BD FACSARIA II HIGH-SPEED CELL SORTER

ELUCIDATING NEURAL CREST-LIKE REPROGRAMMING IN MELANOMA