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

RATIONAL APPROACHES TO MELANOMA THERAPY

NOVEL MOLECULAR THERAPIES OF PROSTATE CANCER

IMMUNE RESPONSES TO AAV-MEDIATED FIX GENE TRANSFER

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.

TRAINING PROGRAM IN BASIC CANCER RESEARCH

SPORE IN SKIN CANCER

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

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.

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

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.

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

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.

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.

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

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.

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.

EPIGENETIC REGULATION THROUGH THE FORMATION AND RESOLUTION OF R LOOPS

THE MULTIFACETED ROLE OF ACETATE IN CANCER

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.

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

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.

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

ADVANCING CANCER RESEARCH THROUGH COMPREHENSIVE PROTEOMICS AND METABOLOMICS ANALYSES

PROMOTION OF TUMOR INVASION AND PSEUDOSENESCENCE BY THE AGING MICROENVIRONMENT

PERIPHERAL MEMBRANE PROTEINS AND DISEASE: TOOL DEVELOPMENT, BASIC INVESTIGATIONS, AND INHIBITOR DESIGN - ABSTRACT PERIPHERAL MEMBRANE PROTEINS (PMPS) REPRESENT CRUCIAL MEDIATORS OF BIOLOGICAL AND DISEASE PROCESSES. THIS CLASS OF PROTEINS EXISTS IN A WATER-SOLUBLE STATE UNTIL TARGETED TO ADHERE ON MEMBRANES, ENABLING THEM TO PERFORM THEIR FUNCTION. AS WITH ANY MEMBRANE ASSOCIATED PROTEIN, PMPS ARE CHALLENGING TO STUDY, PARTICULARLY IN THEIR MEMBRANE BOUND STATE, LEAVING MANY BASIC QUESTIONS ABOUT FUNCTION UNRESOLVED. ADDITIONALLY, UTILIZING PMPS AS DRUG TARGETS IS DIFFICULT SINCE CURRENT METHODS ARE DESIGNED FOR WATER-SOLUBLE PROTEINS AND DO NOT WORK WELL FOR MEMBRANE ASSOCIATED PROTEINS. THUS, THERE IS A GREAT NEED FOR NOVEL TOOLS AND PROCEDURES TO ILLUMINATE DETAILS OF PMP FUNCTION AND TO CREATE PMP INHIBITORS FOR USE IN CHEMICAL BIOLOGY STUDY AND AS DRUG LEADS. THE GOAL OF OUR RESEARCH IS TO ENABLE HIGH-RESOLUTION, QUANTITATIVE STUDY OF PMP INTERACTIONS AND ALLOW INHIBITOR DESIGN FOR THIS ELUSIVE TYPE OF PROTEIN. WE WILL INITIALLY FOCUS OUR EFFORTS ON THREE PMPS OF EXTRAORDINARY BIOMEDICAL INTEREST: GLUTATHIONE PEROXIDASE 4 (GPX4) AND PHOX HOMOLOGY (PX) DOMAINS IN THE NADPH OXIDASE FAMILY (P47PHOX-PX AND NOXO1-PX). WE HAVE INITIATED DEVELOPMENT OF A NOVEL MEMBRANE MODEL, MEMBRANE- MIMICKING REVERSE MICELLES (MMRMS), WHICH IS BASED ON THE CHEMISTRY OF CELLULAR MEMBRANES. PMPS EMBED INTO MMRMS AS THEY DO WITH CELLULAR MEMBRANES, ENABLING HIGH-RESOLUTION STUDY USING NMR SPECTROSCOPY AND OTHER TECHNIQUES. MMRMS HAVE A NUMBER OF ADVANTAGES OVER CURRENT MODELS, INCLUDING GREATLY ENHANCED STABILITY, OUTSTANDING SPECTROSCOPIC PROPERTIES, AND AN ABILITY TO HOUSE HIGH CONCENTRATIONS OF ANALYTE ALONG WITH THE PROTEIN. WE WILL MODIFY OUR MMRM SYSTEM TO BETTER REFLECT A VARIETY OF CELLULAR AND ORGANELLE MEMBRANES, ALLOWING THE SYSTEM TO BE TUNED ACCORDING TO THE NATURAL PMP ENVIRONMENT. OUR FOCUS WILL THEN BE TO HARNESS THE UNIQUE PROPERTIES OF OUR MMRMS TO ENABLE UNRIVALED DETAIL IN STUDY AND QUANTIFICATION OF PMP INTERACTIONS WITH MEMBRANES AND LIPID SUBSTRATES. TO FACILITATE INHIBITOR DESIGN, WE WILL DEVELOP A NOVEL METHOD THAT ALLOWS FRAGMENT SCREENING OF MEMBRANE-EMBEDDED PMPS, FOR WHICH CURRENT METHODS ARE NOT SUITABLE. USING THESE TOOLS, WE WILL INITIATE AN INHIBITOR DESIGN CAMPAIGN FOR OUR IMPORTANT PMP TARGETS. OVERALL, OUR GOAL IS TO DEVELOP TOOLS THAT WILL ENABLE BREAKTHROUGHS IN DETAILED STUDY OF PMP BIOLOGY AS WELL AS INHIBITOR AND DRUG DEVELOPMENT FOR THIS LARGELY UNTAPPED CLASS OF PROTEINS.

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. .

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

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

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

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

GRANT/INVESTIGATING METABOLIC CONTROL OF IMMUNE COLD CCNE1-DRIVEN OVARIAN CANCERS

A TRANSGENIC MODEL FOR B CELL TOLERANCE AND AUTOIMMUNITY

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 MODIFICATION P00003 INCORPORATES A REFUND CHECK FROM UNIVERSITY OF PITTSBURGH IN THE AMOUNT OF $198.52. REDUCES THE OBLIGATED AND INDIRECT COSTS AMOUNTS. 18SEPTEMBER26.

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

ENHANCED NK CELL-BASED TARGETING OF HIV RESERVOIRS THROUGH ENGAGERS AND ADCC ENGINEERING - SUMMARY ACTIVELY REPLICATING HIV INFECTED CELLS ARE THE SOURCE OF PROGENY VIRIONS AND THE BASIS OF PERSISTENT RESERVOIRS. WE WILL DEVELOP AND TEST INNOVATIVE STRATEGIES TO ELIMINATE ACTIVELY REPLICATING CELLS BY ENHANCING NK-MEDIATED ANTIBODY DEPENDENT CELLULAR CYTOTOXICITY (ADCC) RESPONSES AGAINST HIV. RECENT EVIDENCE SHOWING THE ABILITY OF BROADLY NEUTRALIZING ANTIBODIES (BNABS) TO CONTROL VIREMIA HIGHLIGHTS THE POTENTIAL TO COMPLEMENT THE ADCC RESPONSE WITH EXOGENOUS BNABS ABLE TO TARGET DIVERSE HIV ISOLATES. BI-SPECIFIC ANTIBODIES ALSO ENHANCE HIV-1 SPECIFIC ADCC BY HIGH AFFINITY BRIDGING BETWEEN EFFECTOR NK CELLS (THROUGH CD16 TARGETING) AND HIV INFECTED CELLS (THROUGH ENVELOPE TARGETING). TRI-SPECIFIC NK CELL ENGAGER MOLECULES (TRIKES) OFFER EVEN MORE ADVANTAGES FOR MEDIATING ADCC AGAINST HIV-1 INFECTED CELLS VIA THE ADDITION OF AN IL-15 CYTOKINE MOIETY THAT STIMULATES NK CELL ACTIVITY WHILE STRENGTHENING TARGET CELL ENGAGEMENT. HERE, WE WILL INCREASE THE HALF-LIFE AND ANTI-VIRAL EFFICACY OF HIV-SPECIFIC TRIKES VIA POLY ANTIGEN CYTOKINE COMPLEX (PACC) ENHANCEMENT AND/OR BY DIRECTLY LOADING SIMILAR IGGFC KILLER ENGAGERS EX VIVO ONTO CD64 GENE-MODIFIED NK CELLS THAT SUSTAIN FC BINDING FOR DAYS AND ARE NOT SUBJECT TO ADAM17-MEDIATED NEGATIVE REGULATION LIKE CD16. TAKEN TOGETHER, WE HYPOTHESIZE THAT CD4/HSA TRIKE-PACC-IL-15 WITH AN FC COMPONENT LOADED ONTO CD64 EXPRESSING NK CELLS ADMINISTERED DURING EARLY ART WILL BE SUPERIOR TO SOLUBLE TRIKES IN TARGETING HIV INFECTED CELLS, REDUCING PERSISTENT HIV LEVELS AND VIRAL REBOUND UPON ART INTERRUPTION IN HUMANIZED MICE. TWO SPECIFIC AIMS WILL TEST THIS HYPOTHESIS: AIM 1: GENERATION OF CD4 ECTO-DOMAIN 1 (CD4ECD1) TRIKE AND CD4/HSA TRIKE/PACC FOR HALF-LIFE AND ANTI- VIRAL EFFICACY ASSESSMENT IN THE IL-15 TRANSGENIC NSG HUMANIZED MOUSE MODEL OF HIV. TWO VERSIONS OF AN HIV-SPECIFIC CD4 ECTO-DOMAIN 1 (CD4ECD1) TARGETING TRIKE WITH IL-15 CYTOKINE MOIETY AS WELL AS A SINGLE DOMAIN ANTIBODY (SDAB/VHH) AGAINST CD16 WILL BE CREATED WITH AND WITHOUT A PACC COMPLEX CONTAINING THE IL- 15 RECEPTOR ALPHA CHAIN AND HUMAN SERUM ALBUMIN (HSA) TO ENHANCE NK CELL SIGNALING AND INCREASE THE MOLECULE HALF-LIFE, RESPECTIVELY. ACTIVITY WILL BE TESTED IN VITRO AND IN VIVO IN AN ART-SUPPRESSED HIV-INFECTED IL- 15 TRANSGENIC NSG HUMANIZED MOUSE MODEL. AIM 2: DEMONSTRATE DURABLE RETENTION AND ANTI-VIRAL EFFICACY OF A IGGFC KILLER ENGAGERS PRE-LOADED EX VIVO ONTO CD64 EXPRESSING NK CELLS IN VITRO AND IN VIVO. NK CELLS WILL BE GENETICALLY MODIFIED USING MRNA LNPS TO EXPRESS THE HIGH-AFFINITY FC RECEPTOR CD64 AND THEN PRE-LOADED EX VIVO WITH “IGGFC KILLER ENGAGERS” CONTAINING A HIV-SPECIFIC CD4ECD1 DOMAIN ALONG WITH IL-15 AND AN FC COMPONENT. ACTIVITY TESTED AS IN AIM 1. THE CULMINATION OF BOTH AIMS WILL ALLOW US TO COMPARE AND IDENTIFY THE BEST STRATEGY BETWEEN THE CD4 TRIKE, CD4/HSA TRIKE-PACC, OR CD64 NK PRELOADED WITH IGGFC KILLER ENGAGERS FOR ANTI-VIRAL POTENCY ON HIV RESERVOIR DYNAMICS IN THE HUMANIZED MICE. THE BEST STRATEGY IDENTIFIED WILL FACILITATE PRIORITIZING A COMPREHENSIVE SINGLE NK CELL THERAPY STRATEGY TO ADVANCE INTO HUMAN PHASE I STUDIES.

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