Versiti Blood Health Inc

MOLECULAR & CELLULAR MECHANISMS IN TRANSFUSION MEDICINE

MOLECULAR AND CLINICAL BIOLOGY OF VWD

ZIMMERMAN PROGRAM ON THE BIOLOGY OF VWD

MOLECULAR AND CLINICAL GLYCOBIOLOGY OF THE BONE MARROW ENVIRONMENT - THE LIFE-LONG PRODUCTION OF BLOOD CELLS REQUIRES HEMATOPOIETIC DEVELOPMENTAL PROGRAMS GUIDED BY SYSTEMIC AND LOCAL SIGNALS TO CONVEY THE DYNAMIC NEED FOR THESE CELLS. HOW THESE SIGNALS ARE DELIVERED WITHIN THE INTRA-MEDULLAR NICHES REMAIN POORLY UNDERSTOOD. EXTENSIVE PUBLISHED AND TO-BE-PUBLISHED INFORMATION BY THE PIS OF THIS PROGRAM, COLLABORATIVE AND INDIVIDUALLY, CLEARLY ESTABLISHED THE INVOLVEMENT OF GLYCANS IN THE GUIDANCE OF HEMATOPOIETIC PROGENITOR CELL FATE, FUNCTION, AND ESPECIALLY IN THROMBOPOIESIS AND PLATELET FUNCTIONALITY. THE OVERARCHING HYPOTHESIS IS “CELL-INTRINSIC AND EXTRINSIC GLYCAN-MEDIATED MECHANISMS REGULATE MAINTENANCE, DIFFERENTIATION, AND FUNCTION OF HEMATOPOIETIC CELLS.” PROJECT 1 WILL INVESTIGATE THE ROLES OF THE GALACTOSYLTRANSFERASE SS4GALT1, SS1 INTEGRIN, AND GLYCOSAMINOGLYCANS (GAGS)/HEPARAN SULFATE PROTEOGLYCANS (HSPGS) IN THROMBOPOIESIS AT STEADY-STATE AND FOLLOWING MYELOABLATIVE STRESS USING NOVEL COMBINED SHARED “OMICS” AND STANDARD APPROACHES WITH PROJECT 3. A PREVIOUSLY UNKNOWN ROLE OF SS4GALT1 TO REGULATE MEGAKARYOCYTE (MK) EXPRESSION OF HSPGS WILL ALSO BE INVESTIGATED. A FUNCTIONALLY DEFINED MK-BIASED HEMATOPOIETIC STEM CELL WILL BE INVESTIGATED TOGETHER WITH PROJECT 2, ESPECIALLY WITH RESPECT TO THE HEAVILY A2,6- SIALYLATED CELL SURFACE DESPITE THE ABSENCE OF ST6GAL1 EXPRESSION NECESSARY TO GENERATE THIS STRUCTURE. PROJECT 2 WILL INVESTIGATE THE ROLE OF EXTRACELLULAR GLYCOSYLATION, ESPECIALLY THAT MEDIATED BY EXTRINSIC ST6GAL1 USING THE COMBINED “OMICS” APPROACH (PROJECT 1), HOW EXTRINSIC SIALYLATION IN THE HEMATOPOIETIC NICHE IS REGULATED, IDENTIFYING THE CELL SURFACE TARGETS OF SIALYLATION; AND WITH PROJECT 3, UNDERSTANDING HOW THE NEWLY DISCOVERED GAG COFACTOR MODULATES EXTRINSIC ST6GAL1 ACTIVITY. CLINICAL MYELODYSPLASTIC SYNDROMES (MDS) AND MYELOPROLIFERATIVE NEOPLASMS (MPN) AND PRECLINICAL MOUSE MODELS WILL BE USED IN A FIRST-TIME ASSESSMENT INTO THE GLYCOBIOLOGY OF THESE MARROW DISEASES OF HIGHLY HETEROGENOUS PRESENTATIONS BUT WITH THE COMMONALITY OF DYSPLASTIC MKS AND ALTERED PLATELET NUMBERS AND FUNCTION, AND ANALYSIS OF THESE CLINICAL DISEASES IS SHARED ACROSS ALL THREE PROJECTS. PROJECT 3 WILL INVESTIGATE THE STRUCTURE–FUNCTION RELATIONSHIPS OF GAGS WITH PROTEINS WITHIN THE MARROW MICROENVIRONMENT, SUCH AS GROWTH FACTORS AND THEIR RECEPTORS, AND GLYCOSYLTRANSFERASES. THEIR ROLES IN PROMOTING THROMBOPOIESIS AND CELL FATE DECISIONS WILL BE INTERROGATED USING A MULTI-DIMENSIONAL APPROACH TO IDENTIFYING DISTINCT GAG SEQUENCES. PROJECT 3 WILL DISCOVER SYNTHETIC GAG MIMETICS AS MODULATORS OF HEMATOPOIESIS/THROMBOPOIESIS FOR THERAPEUTIC USE. CORE A WILL OVERSEE THE ADMINISTRATION OF THE PROGRAM, CORE B WILL PROVIDE GENERATION AND SEQUENCING OF CDNA LIBRARIES DERIVED FROM BULK RNA SAMPLES AND SINGLE CELLS, AND CORE C WILL PERFORM COMPARATIVE STRUCTURAL ANALYSIS OF GAGS, PROTEOMICS AND PROTEIN-GAG INTERACTIONS, AND QUANTITATIVE PROTEOMICS OF PROTEIN EXPRESSION. THE THREE PROJECTS ARE INTIMATELY INTERTWINED AND WILL USE ALL CORES. THIS PROGRAM WILL UNCOVER NOVEL INFORMATION TO INCREASE PLATELET PRODUCTION AND HELP UNDERSTAND CLINICAL CONDITIONS CHARACTERIZED BY MK ABNORMALITIES.

CARBOHYDRATE-MEDIATED PLATELET CLEARANCE

ASSOCIATION OF TISSUE FACTOR PATHWAY INHIBITOR WITH ENDOTHELIUM

BASIC INVESTIGATION AND TRANSLATIONAL APPLICATIONS CONCERNING THE CELL AND MOLECULAR BIOLOGY OF BLOOD AND VASCULAR CELLS

STRUCTURAL TRANSITION OF CELLULAR INTEGRINS AND APPLICATIONS THEREOF

PULMONARY ARTERIOLE OCCLUSION BY PLATELET-NEUTROPHIL MICRO-EMBOLI IN ACUTE CHEST SYNDROME

HEMORRHAGIC DISEASES

MOLECULAR MECHANISMS OF IMMUNE THROMBOCYTOPENIA IN TRANSFUSION MEDICINE - OVERALL COMPONENT – PROJECT SUMMARY/ABSTRACT THIS REVISED APPLICATION FOR A PROGRAM PROJECT SEEKS SUPPORT FOR A COORDINATED, MULTI-DISCIPLINARY INVESTIGATION OF MOLECULAR MECHANISMS THAT UNDERLY CLINICALLY IMPORTANT IMMUNE-MEDIATED THROMBOCYTOPENIC AND THROMBOTIC DISORDERS. THE PROGRAM IS HOUSED AT VERSITI'S BLOOD RESEARCH INSTITUTE AND CHILDREN'S HOSPITAL OF PHILADELPHIA – BOTH NATIONALLY RENOWNED FACILITIES WITH LONG-STANDING COMMITMENTS TO BASIC, TRANSLATIONAL, AND CLINICAL BLOOD-RELA- TED RESEARCH, PARTICULARLY IN THE AREAS OF PLATELET IMMUNOLOGY, PLATELET ACTIVATION, THROMBOSIS, AND B CELL IMMUNO- BIOLOGY. THESE DISCIPLINES ARE OF PROGRAMMATIC SIGNIFICANCE TO THE NHLBI, AND OF CENTRAL IMPORTANCE TO TRANS- FUSION MEDICINE AND CLASSICAL HEMATOLOGY - DISCIPLINES THAT, DESPITE THEIR IMPACT ON THE NATION'S HEALTH, ARE CUR- RENTLY UNDERREPRESENTED IN BOTH HUMAN AND FINANCIAL RESOURCES. OUR APPLICATION REFLECTS ONGOING, CLOSE COLLABOR- ATIVE TIES AMONGST THE PROJECT LEADERS, SOME FOR MORE THAN THREE DECADES, AND IS BOLSTERED BY A STRONG HISTORY OF INTERDEPENDENCE THAT HAS ALLOWED US TO TACKLE THEMATICALLY RELATED ISSUES OF CLINICAL RELEVANCE AND IMPORTANCE. THE CENTRAL UNIFYING THEME OF OUR APPLICATION IS TO BUILD ON AND DEVELOP A DEEPER UNDERSTANDING OF CLINICALLY RELE- VANT IMMUNOLOGICAL INSULTS THAT LEAD TO THROMBOCYTOPENIA AND DOWNSTREAM PATHOPHYSIOLOGICAL SEQUELAE IN THE FETUS, NEWBORN, AND ADULT. THE PROGRAM COMPRISES THREE HIGHLY SYNERGISTIC, INTERDEPENDENT PROJECTS, EACH OF WHICH IS CHARACTERIZED BY (1) EXAMINATION OF AN UNPREDICTABLE PATHOLOGY ATTRIBUTABLE TO A SUBSET OF ANTIGEN-SPE- CIFIC ANTIBODIES THAT, IN ADDITION TO CAUSING THROMBOCYTOPENIA, ALSO ADVERSELY AFFECT OTHER CELL TYPES, AND (2) JUDI- CIOUS USE OF PRECLINICAL MOUSE MODELS THAT ARE SPECIFICALLY DESIGNED TO INVESTIGATE THE ETIOLOGY OF DIFFERENT FORMS OF IMMUNE THROMBOCYTOPENIA. PROJECT 1: THE IMMUNOBIOLOGY OF FETAL/NEONATAL ALLOIMMUNE THROMBOCYTOPENIA (FNAIT) EMPLOYS A NOVEL ANTIGEN-SPECIFIC MOUSE MODEL AND A BATTERY OF CLINICALLY RELEVANT MONOCLONAL ANTIBODIES TO DEFINE THE FUNCTIONAL SIGNIFICANCE OF SPECIFIC MATERNAL ANTIBODY SUBPOPULATIONS THAT CONTRIBUTE MECHANISTICALLY TO, AND MAY BE DIAGNOSTIC OF, THE SEVERITY OF FNAIT. THE ETIOLOGY OF THIS DISORDER IS EXAMINED BY EXPLORING ROUTES OF ANTIGEN EXPOSURE THAT CAUSE MATERNAL ALLOIMMUNIZATION TO OCCUR UNPREDICTABLY IN FIRST PREGNANCIES. PROJECT 2: THE IMMUNOBIOLOGY OF HEPARIN-INDUCED THROMBOCYTOPENIA (HIT) USES SOPHISTICATED HUMAN ANTIBODY CLONING TECHNIQUES AND ANALYSIS TO IDENTIFY AND CHARACTERIZE THE SPECIFIC PATHOGENIC ANTIBODY SUBPOPULATIONS THAT CAUSE HIT, AND EXPLORES AN INTRIGUING ROLE FOR THE GUT MICROBIOME IN THE ETIOLOGY OF THIS MAN-MADE IMMUNOLOGICAL DIS- ORDER. PROJECT 3: THE IMMUNOBIOLOGY OF VACCINE-INDUCED THROMBOTIC THROMBOCYTOPENIA (VITT) EXAMINES NOVEL MECHANISMS BY WHICH PLATELET FACTOR 4, PLATELET BASIC PROTEIN, AND NEUTROPHIL ACTIVATING PEPTIDE 2 CONTRIBUTE TO THE ETIOLOGY OF THIS RARE, BUT LIFE-THREATENING, IMMUNOLOGICALLY MEDIATED DISORDER. TAKEN TOGETHER, THERE IS STRONG SCIENTIFIC AND PROGRAMMATIC RATIONALE FOR THIS COMPREHENSIVE EFFORT TO EXAMINE MOLECULAR AND CELLULAR MECHAN- ISMS THAT UNDERLY THE IMMUNE-MEDIATED THROMBOCYTOPENIAS. INSIGHTS AND CONCEPTS DERIVED FROM THESE BASIC STUDIES SHOULD ENHANCE THE DEVELOPMENT OF NOVEL, CLINICALLY RELEVANT DIAGNOSTIC AND THERAPEUTIC APPLICATIONS.

GLYCANS IN BLOOD HOMEOSTASIS AND DISEASE

B CELL RESPONSES IN HEPARIN-INDUCED THROMBOCYTOPENIA

PLC?S IN B CELL BIOLOGY AND AUTOIMMUNITY

MOLECULAR BIOLOGY AND FUNCTION OF PECAM-1

COMPARATIVE EFFECTIVENESS IN THE DIAGNOSIS OF VWD

MOLECULAR BASIS OF THE HUMORAL IMMUNE RESPONSE IN HEPARIN-INDUCED THROMBOCYTOPENIA

DEVELOPMENT OF A B CELL THERAPEUTIC

RAP1 IN VEGF SIGNALING IN ENDOTHELIAL CELLS

CARING FOR THOSE WHO SHARE: MITIGATING IRON DEFICIENCY IN REGULAR BLOOD DONORS

ENDOTHELIAL RAP1 IN THE CONTROL OF HEART FUNCTION - CARDIOVASCULAR DISEASE (CVD) IS A GLOBAL PANDEMIC WITH OVER 26 MILLION PEOPLE AFFECTED WORLDWIDE. CRITICAL FOR REGULATION OF HEART OXYGENATION AND METABOLISM IS THE CROSS-TALK BETWEEN HEART ENDOTHELIAL CELLS (ECS) AND CARDIOMYOCYTES (CMS) AND SMOOTH MUSCLE CELLS (SMCS). THIS CROSS-TALK, MEDIATED BY LOCALLY ACTING, BIOACTIVE SUBSTANCES RELEASED BY CARDIAC ECS (PARACRINE FUNCTION), IN PARTICULAR NITRIC OXIDE (NO), CONTROLS BLOOD FLOW AND VASCULAR PERMEABILITY, AS WELL AS CMS' GROWTH, CONTRACTILITY AND RHYTHMICITY. HOWEVER, THE MECHANISMS UNDERLYING THE FUNCTIONAL INTERACTION BETWEEN CARDIAC ECS AND CMS AND SMCS ARE STILL POORLY UNDERSTOOD. OUR PIONEERING STUDIES ON ENDOTHELIAL FUNCTIONS OF THE SMALL GTPASE (RAS ASSOCIATION PROXIMATE) RAP1 HIGHLIGHT ITS ROLE AS NOVEL REGULATOR OF VASCULAR HOMEOSTASIS. RAP1 IS CRITICALLY REQUIRED FOR NITRIC OXIDE (NO) PRODUCTION AND BIOAVAILABILITY, AS TISSUE-SPECIFIC DELETION OF BOTH RAP1 ISOFORMS (RAP1A AND RAP1B) LEADS TO SEVERE ENDOTHELIAL DYSFUNCTION. EMERGING DATA FROM OUR COLLABORATION STRONGLY SUGGEST THAT THE TWO RAP1 ISOFORMS IN BOTH CORONARY (VASCULAR) AND HEART MICROCAPILLARY (CARDIAC) ECS MAY BE ESSENTIAL TO PRESERVING NORMAL CONTRACTILE FUNCTION OF THE HEART. OUR DATA DEMONSTRATE THAT EC-SPECIFIC DELETION OF RAP1 LEADS TO DECREASED CARDIAC CONTRACTILITY AND IMPENDING HEART FAILURE. MECHANISTICALLY, OUR PRELIMINARY DATA STRONGLY SUGGEST THAT, VIA DISCRETE YET COMPLEMENTARY MECHANISMS, TWO RAP1 ISOFORMS ARE ESSENTIAL FOR ENDOTHELIAL CA2+HANDLING AND ENDOTHELIAL FUNCTION (NO PRODUCTION). THE GOAL OF THIS PROPOSAL IS TO EXAMINE THE ROLE OF THE TWO RAP1 ISOFORMS IN CORONARY AND CARDIAC ECS REQUIRED FOR MAINTENANCE OF CARDIAC CONTRACTILE FUNCTION. WE HYPOTHESIZE THAT RAP1-DEPENDENT EC FUNCTIONS FORM THE NEXUS FOR EC-SMC AND EC- CM COMMUNICATION REQUIRED FOR NORMAL CARDIAC FUNCTION. CONVERSELY, RAP1 DEFICIENCY-DRIVEN EC DYSFUNCTION (IMPAIRED NO RELEASE, CA2+ OVERLOAD) IS THE COMMON CULPRIT IN EC–SMC AND EC–CM MISCOMMUNICATION THAT LEADS TO HEART FAILURE. TO TEST THIS HYPOTHESIS, WE WILL: (1) DETERMINE HOW RAP1 CONTROLS CA2+ HOMEOSTASIS IN ECS; WE WILL UTILIZE PATCH CLAMP ELECTROPHYSIOLOGY AND CA2+ MEASUREMENTS IN VITRO TO EXAMINE THE EFFECT OF RAP1 DEFICIENCY ON CA2+ INFLUX CHANNELS. WE WILL EXAMINE THE EFFECT OF IMPAIRED CA2+ HOMEOSTASIS IN RAP1A KO ECS ON CELLULAR PROCESSES CONTROLLING PARACRINE FUNCTION. (2) EXAMINE A NOVEL SIGNALING PATHWAY INVOLVING CALDAG-GEFIII-MEDIATED RAP1B ACTIVATION IN NO RELEASE. EX VIVO, WE WILL TEST THE EFFECT OF RAP1 SIGNALING AND ION CHANNEL INHIBITION ON MOUSE AND HUMAN CORONARY VESSEL DILATION, TO DETERMINE THE INFLUENCE OF EC RAP1A AND RAP1B IN THE CONTROL OF CORONARY VESSEL BLOOD FLOW. (3) EXAMINE VASCULAR AND CARDIAC FUNCTION IN EC-RAP1 KNOCKOUT MICE EX VIVO AND PARACRINE FUNCTION IN EC-CM CO-CULTURE IN VITRO TO DETERMINE HOW CARDIAC EC RAP1 ISOFORMS CONTROL HEART CONTRACTILE FUNCTION. PROPOSED STUDIES WILL UNCOVER NOVEL, PREVIOUSLY UNEXPECTED MECHANISMS GOVERNING HEART ENDOTHELIUM AND MAY LEAD TO A NEW DIRECTION IN RESTORING CARDIAC FUNCTION BY CONTROLLING RAP1 SIGNALING IN ENDOTHELIUM.

INITIATIVE IN STEM CELL BIOLOGY

TARGETING THE METABOLIC REGULATOR SIRT5 IN ACUTE MYELOID LEUKEMIA

TFPI, PROTEIN S, AND PLASMA FIXA IN HORMONE-INDUCED HYPERCOAGULABILITY - VENOUS THROMBOEMBOLISM (VTE) IN WOMEN TAKING ORAL CONTRACEPTIVES (OC) IS A PROBLEM OF BROAD CLINICAL SIGNIFICANCE THAT IS MEDIATED, AT LEAST IN PART, BY ESTROGEN EFFECTS ON THE EXPRESSION OF BLOOD COAGULATION PROTEINS BY THE LIVER OR VASCULATURE. OUR OVERALL GOAL IS TO IDENTIFY AND CHARACTERIZE THE UNDERLYING COAGULATION PATHWAYS LEADING TO OC-INDUCED HYPERCOAGULABILITY AND TO IDENTIFY GENETIC FACTORS PREVALENT IN INDIVIDUALS AT HIGHEST RISK FOR VTE. OC USE DECREASES THE PLASMA CONCENTRATION OF TWO ENDOGENOUS ANTICOAGULANT PROTEINS, TISSUE FACTOR PATHWAY INHIBITOR (TFPI) AND PROTEIN S (PS) THAT MODULATE PLASMA FIXA PRODUCTION AND ACTIVITY. WE HAVE DEVELOPED A HIGHLY SENSITIVE AND SPECIFIC (<10 PMOL/L) TECHNIQUE FOR MEASUREMENT OF PLASMA FIXA ACTIVITY USING AN ENHANCED THROMBIN GENERATION ASSAY, WHICH AMPLIFIES THE FIXA SIGNAL FROM SUBJECT PLASMA VIA THROMBIN GENERATION IN FIX-DEFICIENT PLASMA. USING THIS ASSAY IN PLASMA SAMPLES FROM BLOOD DONORS WE FOUND THAT OC-INDUCED CHANGES IN TFPI AND PS ARE ASSOCIATED WITH ELEVATED PLASMA FACTOR IXA (FIXA) ACTIVITY IN ~30% OF WOMEN TAKING OC. OUR CENTRAL HYPOTHESIS IS THAT OC USE ALTERS THE INHIBITION OF PROCOAGULANT RESPONSES BY THE NATURAL ANTICOAGULANTS TFPI AND PS, PRODUCING LARGE INCREASES IN PLASMA FACTOR IXA (FIXA) ACTIVITY AND A SYSTEMIC HYPERCOAGULABLE STATE. THIS HYPOTHESIS WILL BE PROBED TO IDENTIFY BIOCHEMICAL AND GENETIC CORRELATES OF OC-INDUCED HYPERCOAGULABILITY. BIOCHEMICAL STUDIES USING RECOMBINANT FIX VARIANTS RESISTANT TO ANTITHROMBIN OR PS BINDING WILL BE USED TO DEFINE HOW TFPI AND PS MODULATE FIXA GENERATION IN HUMAN PLASMA-BASED SYSTEMS. THE CLINICAL IMPACT OF THE BIOCHEMICAL FINDINGS WILL BE EXAMINED BY MEASUREMENT OF PLASMA LEVELS OF TFPI, PS, AND FIXA, GWAS ANALYSIS, AND A THROMBOSIS HISTORY QUESTIONNAIRE IN TWO COHORTS OF PRE-MENOPAUSAL WOMEN: 1) A CROSS-SECTIONAL COHORT OF ~1000 PRE-MENOPAUSAL BLOOD DONORS; AND 2) A LONGITUDINAL COHORT OF ~45 ADOLESCENT GIRLS TO BE FOLLOWED FOR ONE YEAR AFTER INITIATION OF OC-THERAPY. THE DATA COLLECTED IN THE CROSS-SECTIONAL COHORT WILL BE MODELED TO IDENTIFY NON-GENETIC AND GENETIC FACTORS THAT CORRELATE WITH OC-ASSOCIATED DIFFERENCES IN PLASMA TFPI, PS AND FIXA AND THROMBOSIS HISTORY. THE DATA COLLECTED IN THE LONGITUDINAL COHORT WILL BE USED TO EXAMINE THE TIME COURSE AND STABILITY OF THE OC-INDUCED PLASMA HYPERCOAGULABLE STATE WITHIN INDIVIDUALS USING REPEATED MEASURES ANALYSIS. THESE FINDINGS WILL BE USED TO VALIDATE PLASMA PROTEIN CHANGES AND THE EFFECT OF SNPS IDENTIFIED BY GWAS IN THE CROSS-SECTIONAL COHORTS. THE PROPOSED EXPERIMENTS WILL DEFINE THE BIOCHEMICAL AND GENETIC UNDERPINNINGS OF THE OC-INDUCED HYPER- COAGUABLE STATE FOCUSED ON OUR FINDING OF ELEVATED AMOUNTS OF AN ACTIVATED CLOTTING FACTOR, FIXA, IN THE PLASMA OF ABOUT 30% OF WOMEN USING OC. THESE RESULTS WILL ALSO ESTABLISH RATIONALE FOR FURTHER CLINICAL STUDIES TO DEFINE THE PREDICTIVE VALUE OF PLASMA FIXA AND ASSOCIATED GENETIC MARKERS FOR OC-INDUCED THROMBOSIS AND FACILITATE EVIDENCE-BASED DECISION-MAKING FOR WOMEN CONSIDERING USE OF OC AND RELATED HORMONAL THERAPIES.

B-CELL RESPONSE AND THROMBOTIC COMPLICATIONS IN COVID-19 - ABSTRACT COVID-19 SEVERITY/LETHALITY IS ASSOCIATED WITH A DYSFUNCTIONAL INFLAMMATORY IMMUNE RESPONSE AND A HYPER- ENGAGEMENT OF PATHWAYS DRIVING HEMOSTASIS AND THROMBOSIS, BUT THE LINK BETWEEN THE TWO MANIFESTATIONS IS NOT UNDERSTOOD. COMPARED TO PATIENTS WITH MILD SYMPTOMS, SEVERE COVID-19 PATIENTS HAVE STRONGER IGG REACTIVITY TO SARS-COV-2 VIRUS AND ITS SPIKE PROTEIN RECEPTOR BINDING DOMAIN (RBD) AND A ROBUST B-CELL RESPONSE WITH A MARKED INCREASE OF THE CD11C+CD21- B CELLS AND PLASMABLAST COMPARTMENTS. THE INCIDENCE OF THROMBOSIS AND INFLAMMATORY DISEASE IN SEVERE COVID-19 IS UNPRECEDENTED, MANIFESTED BY INCREASED PLASMA INFLAMMATORY MARKERS, SUCH AS IL-6, TUMOR NECROSIS FACTOR ALPHA, C-REACTIVE PROTEIN, AND ACTIVATION OF COMPLIMENT PATHWAY ETC., AND DYSREGULATED ACTIVATION OF CELLULAR COMPONENTS PARTICIPATING IN INFLAMMATORY AND COAGULATION RESPONSES THAT INCLUDE PLATELETS, ENDOTHELIAL, MONOCYTES, AND NEUTROPHILS. THE THROMBOTIC MANIFESTATION RANGES FROM ARTERIAL, VENOUS, AND TISSUE MICRO THROMBOSIS TO THROMBOEMBOLISM AND IS PREDOMINATED BY VENOUS THROMBOEMBOLISM. SIMILAR MANIFESTATIONS ARE OBSERVED IN CATASTROPHIC THROMBOSIS ASSOCIATED WITH HEPARIN- INDUCED THROMBOCYTOPENIA AND THROMBOSIS (HIT). PATIENTS WITH CATASTROPHIC HIT HAVE THE SUDDEN ONSET OF MULTIPLE ARTERIAL AND VENOUS THROMBI WITH, BUT SOMETIME WITHOUT, HEPARIN EXPOSURE, DUE TO PROTHROMBOTIC PLATELET- ACTIVATING IGGS THAT RECOGNIZE PLATELET FACTOR 4 COMPLEXED WITH HEPARIN POLYSACCHARIDE (PF4/H). USING METHODS EMPLOYED IN PREVIOUS STUDIES OF HIT, WE STUDIED PATIENTS WITH SEVERE COVID-19 PATIENTS AND IDENTIFIED PF4/H- REACTIVE, PRO-THROMBOTIC IGG ANTIBODIES THAT CLOSELY RESEMBLE PATHOGENIC ANTIBODIES FOUND IN PATIENTS WITH HIT IN THEIR ABILITY TO ACTIVATE PLATELETS. SURPRISINGLY, LEVELS OF PF4/H ANTIBODIES IN THE PATIENT PLASMA CORRELATED WITH LEVELS OF ANTIBODIES SPECIFIC FOR THE RECEPTOR BINDING DOMAIN (RBD) OF THE SARS-COV-2 SPIKE PROTEIN. WE CLONED RBD-SPECIFIC ANTIBODIES THAT ARE ABLE TO ACTIVATE PLATELETS. COMPARED TO THOSE THAT RECOGNIZE RBD ALONE, SIGNIFICANTLY MORE B CELLS RECOGNIZING BOTH RBD AND PF4/H WERE CD11C+, CD21- AND CXCR3+, WHICH MARK A SUBSET OF EXTRAFOLLICULAR B CELLS ROBUSTLY EXPANDED IN SEVERE COVID-19. BASED ON THESE FINDINGS, WE HYPOTHESIZE THAT SARS-COV-2 INFECTION DRIVES A SUBSET OF RBD-SPECIFIC B CELLS TO RESPOND VIA AN EXTRAFOLLICULAR PATHWAY AND GENERATE PLATELET-ACTIVATING ANTIBODIES THAT CONTRIBUTE TO THROMBOTIC COMPLICATIONS BUT NOT VIRUS NEUTRALIZATION IN SEVERE COVID-19. TO TEST OUR HYPOTHESIS, WE WILL 1) INVESTIGATE THE PROTHROMBOTIC ACTIVITY OF RBD-SPECIFIC ANTIBODIES IN THE PLASMA OF HOSPITALIZED COVID-19 PATIENTS; 2) INVESTIGATE THE EXPANSION OF B CELLS THAT MAKE RBD-SPECIFIC PLATELET-ACTIVATING ANTIBODIES IN SEVERE COVID-19; 3) INVESTIGATE THE DEVELOPMENTAL PATHWAY THAT GOVERNS AFFINITY MATURATION OF RBD AND PF4/H CROSS-REACTIVE B CELLS. OUR PROPOSAL STUDIES A NOVEL B-CELL/PLATELET AXIS IN THROMBOTIC COMPLICATIONS IN COVID-19 AND SHOULD UNRAVEL A LARGE PORTION OF THE COMPLEX PATHOGENESIS OF MORBIDITY/MORTALITY IN THIS DISEASE AND SUGGEST NEW TREATMENT.

MOLECULAR INTERACTIONS OF FVIII AND VWF

CD39-CARRYING EXTRACELLULAR VESICLES REGULATE PULMONARY THROMBOSIS IN SICKLE CELL DISEASE - PROJECT SUMMARY SICKLE CELL DISEASE (SCD) IS THE MOST COMMON HEMOLYTIC DISORDER AFFECTING AFRICAN AMERICANS. IN SITU (DE NOVO) ACUTE PULMONARY THROMBOSIS IS THE UNDERLYING CAUSE IN ~20% OF SCD PATIENTS HOSPITALIZED WITH RESPIRATORY FAILURE. THE CURRENT THERAPY FOR PULMONARY THROMBOSIS IN SCD IS PRIMARILY SUPPORTIVE AND A PREVENTIVE THERAPY DOES NOT EXIST. AUTOPSY AND COMPUTED TOMOGRAPHY STUDIES HAVE IDENTIFIED THAT OCCLUSION OF PULMONARY ARTERIOLES BY PLATELET-RICH THROMBI CONTRIBUTES TO THE DEVELOPMENT OF PULMONARY THROMBOSIS IN SCD PATIENTS. ADENOSINE DIPHOSPHATE (ADP) RELEASED FROM LYSED ERYTHROCYTES ACTIVATES PLATELETS BY STIMULATING PURINERGIC P2Y1 AND P2Y12 RECEPTORS. WE RECENTLY DISCOVERED THAT THIS PATHWAY PROMOTES PULMONARY ARTERIOLE THROMBOSIS FOLLOWING ACUTE-HEMOLYSIS IN WILD-TYPE MICE, SUGGESTING THAT ADP-INDUCED PURINERGIC SIGNALING MAY ALSO PROMOTE PULMONARY THROMBOSIS IN A HEMOLYTIC DISORDER SUCH AS SCD. HOWEVER, P2Y12 RECEPTOR ANTAGONISTS HAVE SHOWN NO BENEFIT TO SCD PATIENTS IN RECENT CLINICAL TRIALS, AND IT REMAINS TO BE IDENTIFIED WHY PULMONARY THROMBOSIS DEVELOPS ONLY IN A SUB-SET BUT NOT ALL SCD PATIENTS. IDENTIFYING MOLECULAR AND GENETIC MECHANISMS THAT TRIGGER PULMONARY THROMBOSIS IN SCD, WOULD ENABLE THE DEVELOPMENT OF PRECISION MEDICINE DIAGNOSTIC AND THERAPEUTIC APPROACHES FOR THESE AT-RISK SCD PATIENTS. BASED ON OUR NEW PRELIMINARY FINDINGS, WE HYPOTHESIZE THAT CD39 (ECTO-NUCLEOTIDASE) PRESENT IN CIRCULATING EXTRACELLULAR VESICLES (CD39+-EVS) DEGRADES EXCESS ADP TO PREVENT PULMONARY THROMBOSIS IN SCD, HOWEVER, SINGLE-NUCLEOTIDE-POLYMORPHISM (SNP) RS3176891G IN THE CD39- ENCODING GENE (ENTPD1) ATTENUATES THIS PROTECTION AND IDENTIFIES SCD PATIENTS WHO CAN BENEFIT FROM ANTI- PURINERGIC THERAPY. WE WILL TEST THIS HYPOTHESIS USING OUR NEWLY DEVELOPED MOUSE MODEL OF PULMONARY THROMBOSIS TRIGGERED BY INTRAVENOUS (IV) ADMINISTRATION OF ADP, IN VIVO IMAGING OF LUNG IN LIVE MICE, IN VITRO MICROFLUIDIC STUDIES WITH SCD PATIENT BLOOD, ISOLATION AND CHARACTERIZATION OF EVS, SCD MICE GENETICALLY DEFICIENT IN CD39, AND GENETIC ANALYSES IN SCD PATIENTS WITH VS WITHOUT SNP RS3176891G. IN AIM 1, WE WILL DETERMINE WHETHER ADP-INDUCED PLATELET AGGREGATION AND PULMONARY THROMBOSIS IS IMPAIRED IN SCD. IN AIM 2, WE WILL DETERMINE WHETHER CIRCULATING CD39+-EVS DEGRADE EXCESS ADP TO PREVENT PULMONARY THROMBOSIS IN SCD. IN AIM 3, WE WILL DETERMINE WHETHER SNP RS3176891G PROMOTES PULMONARY THROMBOSIS EVENT IN SCD PATIENTS BY ATTENUATING CD39+-EVS, LEADING TO INCREASED ADP-INDUCED PLATELET AGGREGATION. THESE STUDIES WILL INTRODUCE A NOVEL PARADIGM THAT CD39+-EVS PREVENT PULMONARY THROMBOSIS IN SCD, AND ESTABLISH THE PREMISE FOR FIRST-EVER PRECISION MEDICINE IN SCD BY IDENTIFYING RS3176891G AS A RISK FOR PULMONARY THROMBOSIS.

UNDERSTANDING AND CONTROLLING THE CONTRIBUTION OF FIBRINOLYSIS TO BLEEDING USING A LONG-ACTING ANTIFIBRINOLYTIC RNA THERAPY - SUMMARY BACKGROUND: THE BALANCE BETWEEN CLOT FORMATION (COAGULATION) AND DEGRADATION (FIBRINOLYSIS) IS DISRUPTED IN PATIENTS WITH BLEEDING DISORDERS OR SEVERE HEMORRHAGE, RESULTING IN INCREASED BLEEDING. THE EXTENT TO WHICH FIBRINOLYSIS CONTRIBUTES TO BLEEDING FROM THE ONSET OF ACUTE BLEEDING IN THESE CONTEXTS IS UNCLEAR, AND THIS GAP IN KNOWLEDGE LIMITS THE DEVELOPMENT OF THERAPIES THAT INHIBIT FIBRINOLYSIS. CURRENT THERAPEUTICS ARE OFTEN NOT SUFFICIENT FOR PATIENTS WITH BLEEDING DISORDERS; FOR EXAMPLE, ANTIFIBRINOLYTIC DRUGS ARE WIDELY USED TO MANAGE ACUTE BLEEDING IN THESE PATIENTS, BUT ARE UNSUITABLE FOR LONG-TERM PROPHYLAXIS DUE TO SHORT HALF-LIVES THAT DECREASE EFFICACY. RATIONALE: WE HYPOTHESIZE THAT FIBRINOLYSIS CONTRIBUTES TO THE INCIDENCE AND SEVERITY OF BLEEDING STARTING FROM THE ONSET OF BLEEDING, AND THAT LONG-ACTING RNA AGENTS TARGETING PLASMINOGEN, THE ZYMOGEN PRECURSOR OF THE MAIN FIBRINOLYTIC ENZYME PLASMIN, CAN DECREASE FIBRINOLYSIS AND HELP MANAGE BLEEDING LONG-TERM FOR PEOPLE ACROSS ALL CLASSES OF BLEEDING DISORDERS, INCLUDING DIAGNOSED AND UNDIAGNOSED DISORDERS. THIS KNOWLEDGE WILL PROVIDE INSIGHTS INTO THE MECHANISMS THAT IMPACT OUTCOMES IN TRAUMATIC HEMORRHAGE. THESE RNA AGENTS WILL BE DELIVERED USING LIPID NANOPARTICLES (LNPS), A CLINICALLY APPROVED DELIVERY PLATFORM WE HAVE UNIQUE EXPERTISE IN. SPECIFIC AIMS: WE WILL DEVELOP AN RNA THERAPEUTIC TARGETING PLASMINOGEN, USING SMALL INTERFERING RNA (SIRNA)-MEDIATED GENE SILENCING (SIPLG). THIS THERAPY IS LONG-ACTING OVER SEVERAL WEEKS PER DOSE AND HIGHLY SPECIFIC FOR THE TARGET PROTEIN, WHICH DECREASES THE BURDEN OF FREQUENT ADMINISTRATION. THE SPECIFIC AIMS OF THIS PROPOSAL ARE TO (1) DEVELOP APPROACHES FOR KNOCKDOWN OF PLASMINOGEN AND INHIBITION OF FIBRINOLYSIS IN MICE, PIGS, AND DOGS, AND CHARACTERIZE THE SAFETY OF THIS APPROACH; (2) DETERMINE THE CONTRIBUTION OF FIBRINOLYSIS TO BLOOD LOSS AT THE ONSET AND IN EARLY STAGES OF HEMORRHAGE IN A TRAUMATIC INJURY MODEL IN SWINE; AND (3) INVESTIGATE THE ROLE OF FIBRINOLYSIS IN BLEEDING DISORDERS BY TESTING THE EFFECT OF PLASMINOGEN KNOCKDOWN IN MOUSE AND CANINE MODELS OF HEMOPHILIA A, AND MOUSE MODELS OF HEMOPHILIA B AND VON WILLEBRAND DISEASE. INNOVATION: THIS WORK HAS BOTH MECHANISTIC AND THERAPEUTIC INNOVATION. IT WILL IMMEDIATELY DETERMINE THE CONTRIBUTION OF FIBRINOLYSIS TO BLEEDING IN EARLY STAGES OF SEVERE HEMORRHAGE AND IN BLEEDING DISORDERS. THIS WILL LIKELY BE THE FIRST DEVELOPMENT OF RNA-LNP THERAPIES TARGETING CIRCULATING PROTEINS IN LARGE ANIMAL MODELS OF BLEEDING. THESE FINDINGS WILL ADDRESS A FUNDAMENTAL QUESTION IN TRAUMA AND HEMOSTASIS RESEARCH. SEPARATELY, IT WILL AID THE DEVELOPMENT OF IMPROVED, LONG-ACTING THERAPIES FOR CONTROLLING BLEEDING IN BLEEDING DISORDERS. THESE THERAPIES WOULD BE PARTICULARLY BENEFICIAL FOR WOMEN WITH MENORRHAGIA CAUSED BY BLEEDING DISORDERS. EXPECTED OUTCOMES: WE EXPECT TO (1) DEVELOP SPECIES-SPECIFIC SIPLG THAT HAVE MINIMAL ADVERSE HEALTH EFFECTS; AND (2-3) DEMONSTRATE THAT KNOCKING DOWN PLASMINOGEN INHIBITS FIBRINOLYSIS AND DECREASES BLOOD LOSS IN SWINE MODELS OF TRAUMATIC HEMORRHAGE, AND IN MURINE AND CANINE MODELS OF BLEEDING DISORDERS.

NON-CONVENTIONAL SIGNALING BY Α5 INTEGRIN IN BLOOD AND ENDOTHELIAL CELLS - SUMMARY – INTEGRINS INTERACT WITH THEIR LIGANDS TO INDUCE INTRACELLULAR SIGNALS THAT MEDIATE CELLULAR ACTIVITIES SUCH AS CELL ADHESION, SPREADING, AND MIGRATION, WHICH ARE THE ESSENTIAL CELLULAR ACTIVITIES FOR THE FUNCTION OF BLOOD AND ENDOTHELIAL CELLS. THESE SIGNALING EVENTS ARE TYPICALLY MEDIATED BY THE CYTOPLASMIC TAIL OF INTEGRIN Β SUBUNIT, WHILE THE ROLE OF Α INTEGRIN CYTOPLASMIC TAIL IN INTEGRIN FUNCTION REMAINS RELATIVELY UNDEREXPLORED. OUR RESEARCH FOUND THAT THE FIBRONECTIN RECEPTOR INTEGRIN Α5Β1, VIA ITS Α CYTOPLASMIC TAIL, IS INVOLVED IN THE FORMATION OF TUNNELING NANOTUBES (TNTS), A NOVEL TYPE OF CELLULAR STRUCTURE FOR CELL-TO-CELL COMMUNICATION. REMARKABLY, OUR RESEARCH FOUND THAT THE Α5Β1-MEDIATED TNT FORMATION CAN BE INDUCED BY THE SPIKE PROTEIN OF CORONAVIRUS SARS- COV-2. WE OBSERVED THE SPIKE-INDUCED AND Α5-DEPENDENT TNT FORMATION IN MODEL CELL LINES AND PRIMARY HUMAN BLOOD AND ENDOTHELIAL CELLS. FURTHERMORE, WE FOUND THAT Α5Β1 INTEGRIN CAN MEDIATE THE SPIKE-INDUCED PROINFLAMMATORY RESPONSE IN HUMAN BLOOD AND ENDOTHELIAL CELLS, WHICH MAY CONTRIBUTE TO THE THROMBOTIC EVENTS IN COVID-19. TNTS ARE LONG ACTIN-RICH CELL MEMBRANE PROTRUSIONS THAT HAVE BEEN INCREASINGLY RECOGNIZED AS FUNCTIONAL SUBCELLULAR STRUCTURES FOR LONG-DISTANCE DYNAMIC INTERCELLULAR CONNECTION. FUNCTIONING AS CONDUITS BETWEEN CONNECTED CELLS, TNTS CAN TRANSPORT CYTOPLASMIC COMPONENTS LIKE SMALL MOLECULES, PROTEINS, VESICLES, AND MITOCHONDRIA INTERCELLULARLY. ACCUMULATING EVIDENCE SUGGESTS THAT TNTS ARE INVOLVED IN THE PROGRESS OF MANY PATHOLOGICAL CONDITIONS SUCH AS CANCER, INFLAMMATION, AND NEURODEGENERATIVE DISEASES. BACTERIA AND VIRUS PATHOGENS ALSO EXPLOIT TNTS FOR CELL-TO-CELL TRANSMISSION. TNTS CAN FORM UNDER CELL STRESS CONDITIONS SUCH AS INFLAMMATION AND VIRUS INFECTION. HOWEVER, THE CELLULAR MECHANISMS REGULATING TNT FORMATION REMAIN LARGELY UNKNOWN. THIS IS MAINLY BECAUSE LITTLE TO NOTHING IS KNOWN ABOUT THE CELL SURFACE RECEPTORS DIRECTLY RESPONSIBLE FOR TNT BIOGENESIS. OUR DISCOVERY OF Α5Β1 INTEGRIN AS A FUNCTIONAL SIGNALING RECEPTOR FOR TNT FORMATION PROVIDES A POWERFUL PLATFORM FOR INVESTIGATING TNT BIOLOGY. MECHANISTICALLY, WE FOUND THAT BOTH Α5Β1-MEDIATED TNT FORMATION AND PROINFLAMMATORY RESPONSE REQUIRE THE PARTICIPATION OF Α5 CYTOPLASMIC TAIL, SUGGESTING THAT THESE TWO PROCESSES ARE INTERCONNECTED EVENTS. MOREOVER, OUR PROTEIN INTERACTION DATA SUGGEST A DIRECT BINDING BETWEEN Α5Β1 AND THE SPIKE PROTEIN, WHICH IS INDEPENDENT OF THE CLASSICAL INTEGRIN RECOGNITION ARG-GLY-ASP (RGD) MOTIF. BASED ON THESE PROMISING DATA, THIS APPLICATION AIMS TO ELUCIDATE THE CELLULAR MECHANISMS GOVERNING THE NON-CONVENTIONAL SIGNALING FUNCTION OF Α5Β1 INTEGRIN IN TNT FORMATION AND INFLAMMATION IN BLOOD AND ENDOTHELIAL CELLS. MULTIFACED BIOCHEMICAL, BIOPHYSICAL, STRUCTURAL AND CELL BIOLOGY APPROACHES WILL BE USED TO IDENTIFY INTRACELLULAR MOLECULES AND SIGNALING PATHWAYS INVOLVED IN THE Α5Β1-MEDIATED TNT FORMATION (AIM 1) AND INFLAMMATION (AIM 2) AND TO CHARACTERIZE THE NON-RGD DEPENDENT Α5Β1 AND LIGAND INTERACTION (AIM 3). THE OUTCOME OF THIS STUDY WILL ADVANCE BOTH INTEGRIN AND TNT BIOLOGY AND UNCOVER POTENTIAL THERAPEUTIC TARGETS FOR MODULATING TNTS AND INFLAMMATION IN VARIOUS DISEASES.

THE CELLULAR AND TRANSCRIPTIONAL CONTROL OF CD8 T CELL FUNCTIONAL ADAPTATION TO CHRONIC VIRUSES

INVESTIGATING THE REGULATION AND SUBSTRATE PROCESSING OF ADAMTS13 AND ADAMTS7 METALLOPROTEASES. - PROJECT SUMMARY THIS MIRA PROPOSAL AIMS TO UNCOVER THE REGULATORY MECHANISMS AND SUBSTRATES FOR METALLOPROTEASES IN THE ADAMTS (A DISINTEGRIN AND METALLOPROTEASE WITH THROMBOSPONDIN MOTIFS) FAMILY. THESE PROTEASES ARE THOUGHT TO BE INVOLVED IN MANY BIOLOGICAL PROCESSES AND SOME MEMBERS ARE KNOWN TO CONTRIBUTE TO IMPORTANT HUMAN DISEASES. THE CURRENT STUDY FOCUSES MAINLY ON TWO MEMBERS OF THE ADAMTS FAMILY, ADAMTS13 AND ADAMTS7. PROJECT 1 WILL INVESTIGATE THE ALLOSTERIC REGULATION OF ADAMTS13, AN IMPORTANT REGULATOR OF BLOOD CLOTTING. I RECENTLY DISCOVERED THAT ADAMTS13 ADOPTS A QUIESCENT CLOSED CONFORMATION AND BECOMES ALLOSTERICALLY ACTIVATED BY ITS SUBSTRATE, VON WILLEBRAND FACTOR (VWF). THIS PROPOSAL OUTLINES A MECHANISTIC APPROACH TO STUDY HOW ALLOSTERIC REGULATION IS MAINTAINED WITHIN ADAMTS13 AND DISRUPTED BY ITS INTERACTION WITH VWF. LEVERAGING FUNCTIONAL, KINETIC, STRUCTURAL AND BIOINFORMATIC ANALYSES, THIS PROJECT SETS TO IDENTIFY THE KEY STRUCTURAL ELEMENTS OF ALLOSTERIC REGULATION IN ADAMTS13. THE OUTCOMES FROM THIS WORK WILL IMPROVE OUR UNDERSTANDING OF INHERITED AND ACQUIRED BLOOD CLOTTING DISORDERS CAUSED BY DEFECTS IN ADAMTS13. I ALSO ANTICIPATE THAT OUR INVESTIGATION OF ADAMTS13 WILL SERVE AS A MODEL TO STUDY THE REGULATION OF OTHER ADAMTS PROTEASES. PROJECT 2 FOCUSES ON ADAMTS7, WHICH IS KNOWN TO CONTRIBUTE TO CORONARY ARTERY DISEASE BY PROMOTING ATHEROSCLEROSIS AND INFLAMMATION. HOWEVER, THE SUBSTRATE TARGETS OF ADAMTS7 AND ITS BIOCHEMICAL PROPERTIES REMAIN POORLY UNDERSTOOD. PREVIOUS ATTEMPTS TO ADDRESS THESE RESEARCH QUESTIONS LED TO CONFLICTING REPORTS IN THE LITERATURE, LIKELY DUE TO THE POOR QUALITY OF REAGENTS AND TOOLS AVAILABLE TO STUDY THIS NOVEL PROTEASE. TO CIRCUMVENT THESE CHALLENGES, WE WILL UTILIZE NOVEL METHODS IN PROTEOMICS, ENZYMOLOGY, AND PROTEIN-PROTEIN INTERACTIONS TO DELINEATE BIOCHEMICAL PROPERTIES AND IDEAL SUBSTRATES FOR ADAMTS7. NEWLY IDENTIFIED SUBSTRATES WILL FORM THE BASIS OF NOVEL BIOCHEMICAL TOOLS TO INVESTIGATE ADAMTS7 ACTIVITY AND ARE NECESSARY FOR FUTURE RESEARCH GOALS TO SCREEN FOR SMALL MOLECULE INHIBITORS OF ADAMTS7 THAT MAY HAVE TRANSLATIONAL APPLICATIONS. SUMMARILY, THE STUDY OF ADAMTS13 AND ADAMTS7 ENZYME CATALYSIS AND REGULATION LAY A FOUNDATION FOR OUR UNDERSTANDING OF THROMBOTIC AND BLEEDING DISORDERS AND CARDIOVASCULAR AND INFLAMMATORY DISEASES, AND THESE OUTCOMES CAN BE APPLIED TO STUDY OTHER ADAMTS PROTEASES. MORE IMPORTANTLY, THE MIRA OFFERS THE PRINCIPAL INVESTIGATOR AN INCREDIBLE OPPORTUNITY TO RECRUIT AND MENTOR STUDENTS AND OTHER TRAINEES FROM DIVERSE BACKGROUNDS.

THE ROLE OF B CELLS IN REGULATING AUTOIMMUNITY

MINIMALLY INVASIVE DEVICE AND BIOABSORBABLE HEMOSTATIC POWDER FOR MANAGING NONCOMPRESSIBLE TORSO HEMORRHAGE

CONTROLLING THROMBOEMBOLISM AFTER POLYTRAUMA WITH AN RNA THERAPY AGAINST EXCESS FIBRINOGEN

RESEARCH TRAINING IN TRANSFUSION MEDICINE

COHESIN MUTATIONS IN ACUTE MYELOGENOUS LEUKEMIA

CYCLIN E REGULATION IN NORMAL AND NEOPLASTIC HEMATOPOIESIS

STRATEGIES TO TARGET BCR-ABL1 COMPOUND MUTANTS IN CML AND PH+ ALL - ABSTRACT: PHILADELPHIA CHROMOSOME-POSITIVE (PH+) LEUKEMIA IS CAUSED BY BCR-ABL1, A CONSTITUTIVELY ACTIVE FUSION KINASE. TYROSINE KINASE INHIBITORS (TKIS) TARGETING THE ATP SITE OF BCR-ABL1 ARE EFFECTIVE IN TREATING CHRONIC-PHASE CHRONIC MYELOID LEUKEMIA (CP-CML) YET MINIMALLY EFFECTIVE AT TREATING BLAST-PHASE CML AND PH+ ACUTE LYMPHOBLASTIC LEUKEMIA. IN THE 20 YEARS SINCE THE APPROVAL OF THE FIRST TKI IN ALL OF MEDICINE, IMATINIB, TKIS HAVE DRAMATICALLY IMPROVED SURVIVAL OF PATIENTS WITH CP-CML, RESULTING IN A PROJECTED INCREASE OF CML PREVALENCE FROM 70,000 AMERICANS IN 2010 TO 180,000 IN 2050. DESPITE THIS PROGRESS, TKI-RESISTANT CML REMAINS A CHALLENGE, WITH >1,000 DEATHS ANNUALLY IN THE U.S. AT LEAST 50% OF TKI TREATMENT FAILURE ARISES THROUGH MUTATIONS IN BCR-ABL1. LABORATORY STUDIES ON THE FIVE FDA-APPROVED BCR-ABL1 TKIS HAVE ESTABLISHED THEIR MUTATIONAL PROFILES AGAINST THE >30 MUTATIONS OBSERVED IN PATIENTS. IN AGGREGATE, THESE TKIS COVER THE CLINICAL SPECTRUM OF BCR-ABL1 SINGLE POINT MUTANTS. PONATINIB IS THE ONLY TKI THAT IS CLINICALLY EFFECTIVE AGAINST THE T315I GATEKEEPER MUTANT. HOWEVER, BCR-ABL1 COMPOUND MUTANTS, DEFINED AS 2 MUTATIONS IN THE SAME BCR-ABL1 ALLELE, THAT INCLUDE T315I WITH ANY SECOND MUTATION ARE RESISTANT TO ALL APPROVED TKIS, INCLUDING PONATINIB, LEAVING THESE PATIENTS WITH NO FURTHER TREATMENT OPTIONS. ASCIMINIB IS THE FIRST INHIBITOR IN CLINICAL DEVELOPMENT THAT BINDS THE BCR-ABL1 MYRISTOYL SITE, AN ALLOSTERIC SITE DISTANT FROM THE ATP SITE, TO ENFORCE AN AUTOINHIBITED, INACTIVE CONFORMATION. WE ESTABLISHED THAT ASCIMINIB, LIKE PONATINIB, IS NOT EFFECTIVE AGAINST T315I-INCLUSIVE COMPOUND MUTANTS, YET COMBINING PONATINIB (BUT NOT NILOTINIB OR DASATINIB) WITH ASCIMINIB IS EXTREMELY EFFECTIVE AT INHIBITING MANY T315I-INCLUSIVE COMPOUND MUTANT FORMS OF BCR-ABL1. THIS DISCOVERY PROVIDES THE BASIS FOR A NOVEL THERAPEUTIC STRATEGY TO ADDRESS AN ENTIRELY UNMET MEDICAL NEED AND IS THE FOUNDATION OF THIS PROPOSAL. IN AIM 1, WE WILL USE COMPUTATIONAL, BIOPHYSICAL AND CRYSTALLOGRAPHIC METHODS TO DECIPHER HOW PONATINIB RE-SENSITIZES COMPOUND MUTANT BCR-ABL1 TO ASCIMINIB. WE WILL TEST THE COMBINATION IN RELEVANT MOUSE MODELS AND IN PRIMARY LEUKEMIA SAMPLES. IN AIM 2A, WE WILL DEVELOP A THERAPEUTIC STRATEGY FOR CLINICALLY RESISTANT BCR-ABL1 COMPOUND MUTANTS THAT ARE NOT INHIBITED BY THE COMBINATION OF PONATINIB WITH ASCIMINIB. INSTEAD, WE WILL TARGET THESE MUTANTS FOR PROTEASOMAL DEGRADATION USING AN ASCIMINIB PROTEOLYSIS TARGETING CHIMERA (PROTAC) STRATEGY. UNLIKE TKIS, PROTACS ARE EFFECTIVE EVEN UPON TRANSIENT OR WEAK BINDING. WE WILL TEST THE HYPOTHESIS THAT PONATINIB-INDUCED STABILIZATION OF THE MYRISTOYL SITE IS THE INITIATING EVENT THAT ALLOWS SUBSEQUENT BINDING OF AN ASCIMINIB-PROTAC AND PROTEASOMAL DEGRADATION OF COMPOUND MUTANT BCR-ABL1. IN AIM 2B, WE WILL DEVELOP A PONATINIB-PROTAC STRATEGY FOR COMPOUND MUTANTS CARRYING A MYRISTOYL SITE RESISTANCE MUTATION. OUR WORK WILL PROVIDE A RATIONALE FOR CLINICAL EVALUATION OF PONATINIB COMBINED WITH ASCIMINIB AS A THERAPY FOR CURRENTLY UNTREATABLE BCR-ABL1 COMPOUND MUTANT LEUKEMIA. COMPOUND MUTATIONS ARE ALSO A MAJOR CAUSE OF RESISTANCE IN ACUTE MYELOID LEUKEMIA, MELANOMA, AND LUNG CANCER, AND OUR STUDY WILL PROVIDE A BLUEPRINT FOR TREATING THESE MALIGNANCIES.

KINDLIN-3 SIGNALING IN NEUTROPHILS - PROJECT SUMMARY/ABSTRACT INFLAMMATION PLAYS A PIVOTAL ROLE IN THE RAPID REMOVAL OF HARMFUL STIMULI, EITHER STERILE OR INFECTIOUS; HOWEVER, UNCONTROLLED INFLAMMATION CAN LEAD TO A VARIETY OF CHRONIC INFLAMMATORY DISORDERS. NEUTROPHILS CONSTITUTE THE FRONT LINE OF THE INNATE IMMUNE RESPONSE, AND SWIFTLY UNDERGO A CAREFULLY CHOREOGRAPHED PROCESS TO LOCATE AND DESTROY POTENTIALLY PATHOLOGICAL THREATS. NEUTROPHIL RECRUITMENT IS AN INTEGRIN-DEPENDENT, CHEMOTAXIS-DIRECTED PROCESS THAT IS FOLLOWED BY PRODUCTION OF REACTIVE OXYGEN SPECIES (ROS) AND RELEASE OF NEUTROPHIL EXTRACELLULAR TRAPS (NETS), TOGETHER WHICH SERVE TO REMOVE OR KILL THE OFFENDING AGENTS. TO MINIMIZE COLLATERAL DAMAGE TO HOST TISSUES, HOWEVER, THE KILLING FUNCTIONS OF NEUTROPHILS MUST BE CAREFULLY REGULATED IN BOTH A TEMPORALLY AND SPATIALLY CONTROLLED MANNER; THE MECHANISMS AT PLAY TO REGULATE NEUTROPHIL ACTIVATION, HOWEVER, REMAIN POORLY UNDERSTOOD. KINDLIN-3, AN INTEGRIN SS CYTOPLASMIC DOMAIN BINDING PARTNER, AND AN ESSENTIAL INTEGRIN ACTIVATOR IN CELLS OF HEMATOPOIETIC ORIGIN, IS KNOWN TO PLAY AN IMPORTANT ROLE IN PROMOTING INTEGRIN-MEDIATED NEUTROPHIL RECRUITMENT TO THE SITES OF INFLAMMATION. PARADOXICALLY, HOWEVER, WE HAVE RECENTLY DISCOVERED THAT NEUTROPHIL KINDLIN-3 ALSO ACTS TO SUPPRESS ROS PRODUCTION AND NET RELEASE IN AN INTEGRIN-BINDING INDEPENDENT MANNER. THUS, KINDLIN-3 IN NEUTROPHILS POSSESSES BOTH PRO- AND ANTI-INFLAMMATORY PROPERTIES, INDICATING THAT IT MAY FUNCTION AS A BIFUNCTIONAL MODULATOR OF NEUTROPHIL ACTIVATION. INTERESTINGLY, OUR PRELIMINARY STUDIES HAVE REVEALED THAT NEUTROPHIL KINDLIN-3 UNEXPECTEDLY BECOMES DEGRADED UPON INFLAMMATORY CHALLENGES. BASED ON THESE FINDINGS, WE PROPOSE THAT DEGRADATION OF NEUTROPHIL KINDLIN-3 IS A KEY STEP IN THE CELLULAR DECISION TREE REGULATING NEUTROPHIL ACTIVATION UNDER INFLAMMATORY CONDITIONS. THE PURPOSE OF THIS APPLICATION, THEREFORE, IS TO EXPLORE NOVEL, HERETOFORE UNEXPLORED MECHANISMS REGULATING KINDLIN-3 DEGRADATION AND TO EXAMINE ITS FUNCTIONAL SIGNIFICANCE IN INFLAMMATORY RESPONSES. TWO SPECIFIC AIMS ARE PROPOSED. IN SPECIFIC AIM 1, WE WILL DETERMINE THE MECHANISM BY WHICH KINDLIN-3 UNDERGOES DEGRADATION IN STIMULATED NEUTROPHILS, INCLUDING BOTH HUMAN AND MOUSE NEUTROPHILS. IN SPECIFIC AIM 2, WE WILL DETERMINE THE FUNCTIONAL SIGNIFICANCE OF KINDLIN-3 DEGRADATION IN NEUTROPHILS IN RESPONSE TO INFLAMMATORY CHALLENGES, SUCH AS UNDER PATHOLOGICAL CONDITIONS OF ENDOTOXEMIA AND ACUTE LUNG INJURY. THESE TIMELY AND COMPLEMENTARY STUDIES WILL ESTABLISH AN IMPORTANT AND NOVEL ROLE FOR KINDLIN-3 IN NEUTROPHILS IN MODULATING INFLAMMATORY RESPONSES. FINDINGS MADE WILL FILL IMPORTANT GAPS IN OUR UNDERSTANDING OF NEUTROPHIL BIOLOGY, AND MAY LEAD TO NOVEL OPPORTUNITIES FOR DEVELOPING MORE SPECIFIC AND SAFER ANTI-INFLAMMATORY STRATEGIES FOR TREATING COMPLICATIONS IN A WHOLE HOST OF INFLAMMATORY DISEASES, INCLUDING THE RECENTLY DESCRIBED INFLAMMATORY COMPLICATIONS PRESENT IN COVID-19.

KINDLIN-3 SIGNALING IN PLATELETS - INTEGRIN AIIBSS3 ACTIVATION IN PLATELETS IS AN IMPORTANT STEP ON THE ROAD TO FIBRINOGEN BINDING, PLATELET AGGREGATION, AND PRIMARY HEMOSTASIS. OVERREACTION OF PLATELET ACCUMULATION, HOWEVER, CAN LEAD TO LIFE-THREATENING ARTERIAL THROMBOSIS, A HALLMARK OF END-STAGE CARDIOVASCULAR DISEASE. CAREFULLY CONTROLLED REGULATION OF THE ACTIVATION STATE OF AIIBSS3, THEREFORE, IS CRUCIAL FOR THE ABILITY OF PLATELETS TO FULFILL THEIR HEMOSTATIC FUNCTION WHILE AT THE SAME TIME LIMITING THROMBOTIC RISK. THE ACTIVATION OF AIIBSS3, LIKE OTHER MEMBERS OF THE INTEGRIN FAMILY OF CELL ADHESION AND SIGNALING RECEPTORS, IS CAREFULLY REGULATED BY THE ASSOCIATION OF CYTOSOLIC PROTEINS THAT BIND IN A CELLULAR ACTIVATION-DEPENDENT MANNER TO THE INTEGRIN SS3 SUBUNIT CYTOPLASMIC DOMAIN. KINDLIN-3 IS A FERM DOMAIN- CONTAINING PROTEIN THAT IS PRIMARILY EXPRESSED IN CELLS OF HEMATOPOIETIC ORIGIN. PREVIOUS STUDIES HAVE SHOWN THAT MEMBERS OF THE KINDLIN FAMILY PROMOTE INTEGRIN ACTIVATION PRIMARILY BY BINDING TO THE INTEGRIN SS CYTOPLASMIC TAIL AND MEDIATING INTEGRIN CLUSTERING VIA A PROCESS KNOWN AS AVIDITY MODULATION. KINDLIN-3 HAS BEEN SHOWN TO BE ESSENTIAL FOR INTEGRIN ACTIVATION, AS EXEMPLIFIED BY THE FINDING THAT PATIENTS WITH LEUKOCYTE ADHESION DEFICIENCY III, A CONGENITAL INHERITED DISORDER CHARACTERIZED BY LEUKOCYTE AND PLATELET DYSFUNCTION AND SEVERE BLEEDING, HAVE CAUSATIVE LOSS-OF-FUNCTION MUTATIONS IN THE KINDLIN-3 GENE. PRECISELY HOW KINDLIN-3 ACTS MECHANISTICALLY TO SUPPORT INTEGRIN AIIBSS3 ACTIVATION, HOWEVER, REMAINS LARGELY UNKNOWN. CONSISTENT WITH THE INTEGRIN CLUSTERING HYPOTHESIS, A RELATED KINDLIN, KINDLIN-2, HAS BEEN SHOWN TO FORM A FERM DOMAIN-SWAPPED DIMER, AND WE HAVE OBTAINED PRELIMINARY DATA SHOWING THAT DIMERIZATION IS ALSO REQUIRED FOR KINDLIN-3 TO PROMOTE AVIDITY MODULATION OF INTEGRIN AIIBSS3. INTERESTINGLY, HOWEVER, DIMERIC FORMS OF HIGHLY PURIFIED KINDLIN-3 HAVE NOT BEEN OBSERVED IN EITHER CRYSTALS OR IN SOLUTION, SUGGESTING THAT EXTRA COMPONENTS MAY BE REQUIRED TO FACILITATE KINDLIN-3 DIMERIZATION AND INTEGRIN CLUSTERING. IN THIS REGARD, WE HAVE RECENTLY FOUND THAT KINDLIN-3 DIRECTLY INTERACTS WITH THE MYOSIN ESSENTIAL LIGHT CHAIN (ELC), A KEY COMPONENT OF MYOSIN THAT EXISTS AS A DIMER BY ATTACHING TO THE PROXIMAL NECK REGIONS OF MYOSIN. IMPORTANTLY, PRELIMINARY DATA OBTAINED IN OUR LAB HAS SHOWN THAT THE MYOSIN ELC ALSO CONTRIBUTES TO AVIDITY MODULATION OF INTEGRIN AIIBSS3 IN PLATELETS. BASED ON THESE FINDINGS, THE PURPOSE OF THIS APPLICATION IS TO EXAMINE THE HYPOTHESIS THAT THE ABILITY OF KINDLIN-3 TO DIMERIZE AND MODULATE AIIBSS3 ACTIVATION INVOLVES A HERETOFORE UNDESCRIBED INTERACTION WITH THE MYOSIN ECL. IN SPECIFIC AIM 1, WE WILL DETERMINE THE MOLECULAR BASIS OF KINDLIN-3 IN INTERACTING WITH THE MYOSIN ECL BY EMPLOYING MULTIPLE BIOCHEMICAL AND STRUCTURAL APPROACHES. IN SPECIFIC AIM 2, WE WILL DETERMINE THE ROLE OF KINDLIN-3/ECL INTERACTIONS IN REGULATING THE AFFINITY AND AVIDITY MODULATIONS OF INTEGRIN AIIBSS3 IN BOTH MOUSE AND HUMAN PLATELETS. TOGETHER, WE BELIEVE THAT THE FINDING FROM THIS STUDY WILL ESTABLISH THE DETAILED MOLECULAR MECHANISM BY WHICH KINDLIN-3 FINE-TUNES INTEGRIN AIIBSS3 ACTIVATION IN PLATELETS, WHICH MAY LEAD TO NOVEL OPPORTUNITIES FOR DEVELOPING SAFER AND MORE SPECIFIC ANTI-THROMBOTIC STRATEGIES.

A COUNTERMEASURE FOR MICROTHROMBOSIS AND TISSUE INJURY IN FREEZING COLD INJURY THAT PREVENTS PATHOLOGIC FIBRINOGEN ELEVATION.

VWF - MECHANISMS OF REGULATION

REGULATION OF INNATE IMMUNITY BY COAGULATION RECEPTORS

KINDLIN-3 SIGNALING IN BLOOD CELLS

ENDOCYTOSIS IN PLATELET AND MEGAKARYOCYTE BIOLOGY

PROTEIN C PATHWAY FUNCTION IN HEMATOPOIESIS

MECHANISM OF ACTIVATED PROTEIN C ACTION IN SEPSIS THERAPY

THE FUNCTION OF MS4A3 IN NORMAL AND MALIGNANT HEMATOPOIESIS - CHRONIC MYELOID LEUKEMIA (CML) IS CAUSED BY BCR-ABL1, A CONSTITUTIVELY ACTIVE TYROSINE KINASE GENERATED FROM THE PHILADELPHIA CHROMOSOME. IN THE CHRONIC PHASE OF CML (CP-CML), MYELOID CELLS ARE EXPANDED, BUT MAINTAIN TERMINAL DIFFERENTIATION. MOST CP-CML PATIENTS ACHIEVE DURABLE RESPONSES TO BCR-ABL1 TYROSINE KINASE INHIBITORS (TKIS), AND THEIR EXTENDED SURVIVAL IS REFLECTED BY A STEEP RISE IN CML PREVALENCE. UNFORTUNATELY, TKIS FAIL TO ELIMINATE QUIESCENT CML STEM CELLS (LSCS) WHOSE SURVIVAL IS INDEPENDENT OF BCR-ABL1, NECESSITATING LIFELONG TKI THERAPY TO PREVENT CML RECURRENCE. IN 5-10% OF PATIENTS, A DIFFERENTIATION BLOCK CONVERTS CP-CML INTO BLAST PHASE CML (BP-CML), AN AGGRESSIVE ACUTE LEUKEMIA THAT IS OFTEN BCR-ABL1-INDEPENDENT AND TKI RESISTANT. OUR OVERARCHING HYPOTHESIS IS THAT BLOCKED DIFFERENTIATION IS CENTRAL TO THE BCR-ABL1 INDEPENDENCE THAT CHARACTERIZES THE EXTREMES OF THE CLINICAL CML SPECTRUM: PERSISTENCE OF RESIDUAL LEUKEMIA DESPITE TKI THERAPY AND TKI-RESISTANT BP-CML. WE HAVE DISCOVERED THAT EXPRESSION OF MS4A3, A MEMBER OF THE MS4A (MEMBRANE-SPANNING FOUR A) FAMILY OF SIGNALING PROTEINS, IS PROFOUNDLY REDUCED IN QUIESCENT, TKI RESISTANT AND BP CML CELLS, AND THAT LOW MS4A3 CORRELATES WITH SHORTER SURVIVAL. MS4A3 KNOCKDOWN (KD) IN CML CD34+ CELLS INHIBITS MYELOID DIFFERENTIATION, AND PROMOTES TKI RESISTANCE, WHILE ECTOPIC MS4A3 EXPRESSION HAS OPPOSITE EFFECTS (ZHAO ET AL. BLOOD. 2021;EPUB AHEAD OF PRINT. PMID: 34780648). OUR PRELIMINARY DATA SUGGEST THAT MS4A3 PROMOTES IL-3 AND GM-CSF SIGNALING IN CML CD34+ CELLS BY PROMOTING ENDOCYTOSIS OF THEIR COGNATE SS COMMON CHAIN (SSC) RECEPTORS. WE HYPOTHESIZE THAT MS4A3 PROMOTES MYELOID DIFFERENTIATION BY ENHANCING RESPONSE TO SSC CYTOKINES IN LEUKEMIC STEM AND PROGENITOR CELLS (LSPCS). TKI RESISTANT CML CELLS DOWNREGULATE MS4A3 TO BLUNT RESPONSE TO DIFFERENTIATION-INDUCING CYTOKINES, THEREBY MAINTAINING A PRIMITIVE, THERAPY-RESISTANT STATE. RE- ESTABLISHMENT OF MS4A3 EXPRESSION WILL ENFORCE DIFFERENTIATION AND ENHANCE DRUG SENSITIVITY. IN AIM 1, WE WILL DELINEATE HOW MS4A3 REGULATES ENDOCYTOSIS AND SIGNALING OF SSC CYTOKINE RECEPTORS. WE WILL TRACK ENDOCYTOSIS BY HIGH-THROUGHPUT IMMUNOFLUORESCENT AND CONFOCAL LIVE CELL IMAGING, IDENTIFY MS4A3 COFACTORS BY MASS SPECTROMETRY, AND MS4A3-REGULATED SIGNALING PATHWAYS BY REVERSE PHASE PROTEIN ARRAY. IN AIM 2, WE WILL DETERMINE THE FUNCTION OF MS4A3 IN NORMAL HEMATOPOIESIS. WE WILL GENERATE MOUSE STRAINS WITH HEMATOPOIETIC- SPECIFIC CONDITIONAL MS4A3 KNOCKOUT OR INDUCIBLE OVEREXPRESSION AND CHARACTERIZE THEIR HEMATOPOIETIC SYSTEM AT STEADY STATE AND UNDER STRESS. IN AIM 3, WE WILL DELINEATE THE ROLE OF MS4A3 IN CML HEMATOPOIESIS AND AS A THERAPEUTIC AGENT IN CML. WE WILL TEST WHETHER MODULATING MS4A3 EXPRESSION IN LSPCS AFFECTS LEUKEMOGENESIS AND TKI RESPONSE, AND WHETHER MS4A3-LOADED NANOPARTICLES ATTENUATE BP-CML IN XENOGRAFTS. IF SUCCESSFUL, WE WILL ESTABLISH MS4A3 AS A NOVEL MASTER REGULATOR OF SSC CYTOKINE SIGNALING THAT GOVERNS SIGNAL STRENGTH BY MODULATING ENDOCYTOSIS. BP-CML REMAINS MOSTLY INCURABLE, AND MOST CP-CML PATIENTS REQUIRE LIFELONG TKI THERAPY. OUR WORK MAY PROVIDE PROOF OF CONCEPT FOR USING FORCED MS4A3 EXPRESSION TO OVERCOME TKI RESISTANCE.

STRUCTURAL MECHANISMS UNDERLYING THE ACTIVITY REGULATION OF THE RECEPTOR-LIKE PROTEIN TYROSINE PHOSPHATASE, CD148/PTPRJ

INTERROGATING CLINICALLY RELEVANT ATTRIBUTES OF MATERNAL ALLOIMMUNITY IN FETAL/NEONATAL ALLOIMMUNE THROMBOCYTOPENIA - ABSTRACT IMMUNE RESPONSES TO PLATELET ALLOANTIGENS CAUSE PATHOLOGY IN THE SETTINGS OF TRANSFUSION AND PREGNANCY, THE LATTER RESULTING IN THE RELATIVELY COMMON AND MORE SERIOUS NON-MALIGNANT HEMATOLOGIC BLEEDING DISORDER, FETAL/ NEONATAL ALLOIMMUNE THROMBOCYTOPENIA (FNAIT). FNAIT OCCURS WHEN MATERNAL ANTIBODIES, SPECIFIC FOR PATERNAL PLATELET ALLOANTIGENS INHERITED BY THE FETUS, CROSS THE PLACENTA AND CLEAR PLATELETS FROM THE FETAL AND/OR NEONATAL CIRCULATION, RESULTING IN THROMBOCYTOPENIA AND BLEEDING THAT ARE OFTEN SERIOUS ENOUGH TO REQUIRE TRANSFUSION IN THE NEONATAL PERIOD. FNAIT ARISES IN SOME BUT NOT ALL AT-RISK PREGNANCIES. IN AN UNPREDICTABLE SUBSET OF SEVERELY THROMBOCYTOPENIC CASES, MAJOR ORGAN BLEEDS SUCH AS INTRACRANIAL HEMORRHAGE OCCUR IN THE FETAL PERIOD, PLACING SUCH INFANTS AT RISK FOR IRREVERSIBLE BRAIN DAMAGE, LIFELONG DISABILITY AND DEATH. OFF-LABEL THERAPIES ARE USED TO MANAGE THROMBOCYTOPENIA IN SUBSEQUENT PREGNANCIES OF A MOTHER WHO HAS DELIVERED AN AFFECTED INFANT. IT IS CURRENTLY NOT POSSIBLE TO IDENTIFY PREGNANCIES AT HIGH RISK FOR SEVERE BLEEDING SO THAT THEY CAN BE SPECIFICALLY TARGETED FOR THERAPY OR TO PREVENT DEVELOPMENT OF FNAIT. RESEARCH NEEDED TO DEVELOP DIAGNOSTIC TESTS FOR SEVERE FORMS OF FNAIT, TO BETTER UNDERSTAND DISEASE ETIOLOGY, AND TO TEST STRATEGIES FOR DISEASE PREVENTION ARE DIFFICULT, IF NOT IMPOSSIBLE, TO PERFORM IN THE PREGNANT WOMEN AND NEONATES WHO CONSTITUTE THE FNAIT POPULATION. THERE IS A COMPELLING NEED FOR TRANSFORMATIVE ANIMAL MODELS THAT CAN NARROW THE EXISTING INFORMATION GAP AND IMPROVE DIAGNOSIS, TREATMENT, AND PREVENTION OF THIS NOTABLE CAUSE OF MORBIDITY AND MORTALITY IN HUMAN NEONATES. WE HAVE DEVELOPED A UNIQUE, ALLOANTIGEN-SPECIFIC, PRECLINICAL MOUSE MODEL OF FNAIT THAT RECAPITULATES CLINICALLY IMPORTANT ASPECTS OF HUMAN DISEASE. HEREIN, WE APPLY EXTENSIVE IMMUNOLOGICAL EXPERTISE TO THIS MODEL TO 1) DETERMINE WHETHER THE SEVERE BLEEDING AND OTHER PREGNANCY COMPLICATIONS THAT ACCOMPANY FNAIT ARE CAUSED BY SUBPOPULATIONS OF ALLOANTIGEN-SPECIFIC ANTIBODIES THAT BIND TO AND IMPAIR THE FUNCTIONS OF FETAL PLATELETS, SYNCYTIOTROPHOBLASTS AND ENDOTHELIAL CELLS AND 2) IDENTIFY EVENTS CAPABLE OF CAUSING DEVELOPMENT OF FNAIT DURING PREGNANCY AND PROPHYLACTIC STRATEGIES NEEDED TO PREVENT IT. OUR STUDIES ARE MADE FEASIBLE BY ESTABLISHED IMMUNIZATION SCHEMES, BREEDING PROTOCOLS, ANTIBODY INFUSION STRATEGIES, AND METHODS FOR CLASSIFYING PREGNANCY OUTCOMES AND ALLOANTIBODY EPITOPE SPECIFICITIES AND FUNCTION BLOCKING ACTIVITIES. OUR STUDIES WILL HAVE DIAGNOSTIC AND THERAPEUTIC SIGNIFICANCE IN THAT THEY WILL LAY THE GROUNDWORK FOR DEVELOPMENT OF DIAGNOSTIC TESTS THAT CAN SAFELY DISCRIMINATE BETWEEN PREGNANCIES AT RISK FOR MILD VS. SEVERE FNAIT, THEREBY PROVIDING A MECHANISTIC BASIS FOR RATIONAL THERAPEUTIC INTERVENTION, AND WILL IDENTIFY POTENTIAL CAUSES OF FNAIT THAT CAN BE VALIDATED IN HUMAN STUDIES AND INFORM EFFORTS TO PREVENT DEVELOPMENT OF FNAIT DURING PREGNANCY. THIS CUTTING-EDGE WORK AT THE INTERFACE OF MOLECULAR BIOLOGY, IMMUNOLOGY, AND HEMATOLOGY WILL PROVIDE NOVEL AND INFORMATIVE INSIGHTS TO TRANS- FORM OUR UNDERSTANDING OF ANTI-PLATELET ALLOIMMUNE RESPONSES TO BENEFIT THE FIELD OF NON-MALIGNANT HEMATOLOGY.

ACTIVATED PROTEIN C FOR TREATMENT OF RADIATION COMBINED INJURY

MEMBRANE-CYTOSKELETON INTERACTIONS IN MEGAKARYOCYTES AND PLATELETS - PROJECT ABSTRACT ABNORMAL PLATELET PRODUCTION AND FUNCTION, DUE TO GENETIC FACTORS, CANCER THERAPY, OR UNKNOWN CAUSES, POSE SIGNIFICANT CLINICAL RISKS, INCLUDING BLEEDING AND THROMBOTIC EVENTS. PLATELETS ARE PRODUCED PRIMARILY IN THE BONE MARROW BY MEGAKARYOCYTES THROUGH INTRICATE PROCESSES INVOLVING POLYPLOIDIZATION AND EXTENSIVE MEMBRANE AND CYTOSKELETAL REARRANGEMENTS. THESE INCLUDE THE FORMATION OF THE DEMARCATION MEMBRANE SYSTEM (DMS), THE SURFACE-CONNECTED MEMBRANE RESERVOIR NECESSARY FOR PROPLATELET EXTENSION AND RELEASE WITHIN BONE MARROW SINUSOIDS. MEGAKARYOCYTES ALSO FORM PODOSOMES, WHICH SERVE AS MECHANOSENSING STRUCTURES TO IDENTIFY THE MOST CONDUCIVE SITES FOR INITIATING TRANS-ENDOTHELIAL PROPLATELET EXTENSION. THE PRECISE MOLECULAR MECHANISMS RESPONSIBLE FOR THESE UNIQUE MEMBRANE AND CYTOSKELETAL REARRANGEMENTS REMAIN POORLY UNDERSTOOD. THE MEMBRANE-SHAPING F-BAR PROTEIN PACSIN2 STANDS CENTRAL IN THE INTERPLAY BETWEEN MEMBRANES AND THE CYTOSKELETON. PACSIN2 CONTAINS AN N-TERMINAL F-BAR DOMAIN TUBULATING MEMBRANES AND A C-TERMINAL SH3 DOMAIN INTERACTING WITH THE ENDOCYTIC GTPASE DYNAMIN 2 (DNM2) AND ACTIN-NUCLEATION-PROMOTING FACTOR WASP. THROUGH ITS F-BAR DOMAIN, PACSIN2 ALSO INTERACTS WITH THE CYTOSKELETAL AND SCAFFOLDING PROTEIN FILAMIN A (FLNA), A CRITICAL REGULATOR OF PLATELET PRODUCTION AND FUNCTION. SINGLE NUCLEOTIDE POLYMORPHISMS IN PACSIN2 HAVE BEEN ASSOCIATED WITH KEY PLATELET PARAMETERS IN HUMANS. OUR RECENT DATA HAVE DISCOVERED PIVOTAL INSIGHTS INTO THE ROLE OF PACSIN2 IN MEGAKARYOCYTE AND PLATELET BIOLOGY. PACSIN2 IS AN INTERNAL COMPONENT OF THE INITIATING DMS IN MEGAKARYOCYTES, WHERE ITS MEMBRANE TUBULATION ACTIVITY IS REGULATED BY FLNA (PMCID: PMC4492198). PACSIN2–/– MICE DISPLAY A MILD THROMBOCYTOPENIA WITH SLIGHTLY ENLARGED PLATELETS AND MARKED PLATELET-INTRINSIC THROMBUS FORMATION DEFECTS (PMCID: PMC10841284). PACSIN2–/– MEGAKARYOCYTES HAVE A MILDLY DEFECTIVE DMS, REDUCED PLOIDY, AND IMPAIRED PODOSOME AND PROPLATELET FORMATION. PACSIN2–/– PLATELETS DISPLAY ELEVATED INTEGRIN Β1 ACTIVITY. DELETION OF INTEGRIN Β1 WITHIN MEGAKARYOCYTES EFFECTIVELY NORMALIZES THE THROMBOCYTOPENIA AND THROMBUS FORMATION DEFECTS. WE HYPOTHESIZE THAT PACSIN2 REGULATES MEMBRANE-CYTOSKELETAL INTERACTIONS AND INTEGRIN Β1 ACTIVITY TO GOVERN THE FORMATION AND ORGANIZATION OF THE DMS AND PODOSOMES DURING MEGAKARYOCYTE MATURATION. WE PROPOSE THREE AIMS TO INVESTIGATE THE MOLECULAR MECHANISMS UNDERLYING HOW PACSIN2 MODULATES PLATELET PRODUCTION AND FUNCTION. IN AIM 1, WE WILL CHARACTERIZE MEGAKARYOCYTE MATURATION AND PLATELET PRODUCTION IN THE PRESENCE OR ABSENCE OF PACSIN2 AND INTEGRIN Β1. IN AIM 2, WE WILL IDENTIFY THE PACSIN2 EFFECTOR PROTEINS MODULATING ACTIN ASSEMBLY/REMODELING AT SITES OF PODOSOME FORMATION IN MEGAKARYOCYTES BY PROTEOMICS AND OVEREXPRESSION METHODOLOGIES. IN AIM 3, WE WILL DEFINE THE MECHANISMS BY WHICH PACSIN2 MODULATES INTEGRIN Β1 ACTIVITY TO REGULATE THROMBUS FORMATION AND PLATELET HEMOSTATIC FUNCTION. WE ANTICIPATE THAT OUR STUDIES WILL YIELD BASIC INFORMATION ON HOW PACSIN2 CONTRIBUTES TO MEGAKARYOCYTE AND PLATELET BIOLOGY.

ENDOTHELIAL RAP1 RESTRICTS INFLAMMATION IN THE RETINA - PROJECT SUMMARY ENDOTHELIAL-LEUKOCYTE INTERACTIONS ARE PIVOTAL IN DIABETIC RETINOPATHY (DR), A MAJOR CAUSE OF GLOBAL VISION LOSS. PROINFLAMMATORY FACTORS LIKE TUMOR NECROSIS FACTOR ALPHA (TNF-Α) DRIVE HYPERACTIVITY IN RETINAL ENDOTHELIAL CELLS (RECS), LEADING TO INFLAMMATION, INCREASED LEUKOCYTE ADHESION, AND ULTIMATELY PATHOLOGICAL NEOVASCULARIZATION. UNDERSTANDING REC-LEUKOCYTE DYNAMICS IS CRUCIAL FOR EARLY INTERVENTION. VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF), IS A KEY PLAYER IN DR INFLUENCING REC BEHAVIOR AND SERVING AS THERAPY TARGET. HOWEVER, IT ALSO MAINTAINS CELLULAR HOMEOSTASIS DURING INFLAMMATION. DECIPHERING RETINAL MECHANISMS BALANCING PROINFLAMMATORY AND HOMEOSTATIC RESPONSES IS CRUCIAL FOR OPTIMIZING DR TREATMENTS. REC RESPONSIVENESS HINGES ON LIPID RAFTS AND SPECIALIZED PLASMA MEMBRANES DOMAINS, CAVEOLAE. UNDERSTANDING HOW HOMEOSTATIC AND PROINFLAMMATORY SIGNALS ARE BALANCED IN THESE STRUCTURES REMAINS A CRITICAL RESEARCH GOAL. OUR PUBLISHED DATA INDICATE THAT ENDOTHELIAL RAP1B (RAS ASSOCIATION PROXIMATE 1B), A SIGNALING MOLECULE IN ECS, ENHANCES NITRIC OXIDE RELEASE AND LIMITS PROINFLAMMATORY SIGNALING IN ECS, AND IS ESSENTIAL FOR VEGFR2 ACTIVATION AND SIGNALING. IN THE RETINA, RAP1B IS CRITICAL FOR PHYSIOLOGICAL RETINAL DEVELOPMENT YET ENDOTHELIAL RAP1B KNOCKOUT (KO) REDUCES VEGF-DRIVEN PATHOLOGICAL HYPER-PERMEABILITY IN STREPTOZOTOCIN-INDUCED DIABETES. OUR PRELIMINARY DATA INDICATE RAP1B LOCALIZES TO CHOLESTEROL-RICH LIPID RAFTS TO ORCHESTRATE VEGFR2 SIGNALING. RESTORING CHOLESTEROL IN RAP1B-DEFICIENT ECS REINSTATES DEFICIENT VEGF-TRIGGERED NO SIGNALING. THESE RESULTS LED TO OUR HYPOTHESIS: RAP1B GOVERNS THE EQUILIBRIUM BETWEEN HOMEOSTASIS AND INFLAMMATION IN RECS DURING DR PROGRESSION BY MODULATING THE COMPOSITION AND ORGANIZATION OF LIPID RAFTS. TO INVESTIGATE THE REGULATORY ROLE OF RAP1B IN RECS IN PROGRESSION OF DR, WE PROPOSE TO: (AIM1) ASSESS RAP1B'S ROLE IN REGULATING VEGF- VEGFR2 LIPID RAFTS. EMPLOY A TWO-TIER GENETIC STRATEGY (RAP1B KO AND FUNCTIONAL MUTANTS IN RECS), COMPLEMENTED BY ADVANCED MICROSCOPY, OMICS, AND SIGNAL TRANSDUCTION ANALYSES, TO ELUCIDATE RAP1B'S INFLUENCE ON VEGFR2 LIPID RAFT STRUCTURE, COMPOSITION, AND SIGNALING. (AIM 2) EXAMINE HOW RAP1B CONTROLS THE BALANCE BETWEEN VEGFR2 AND TNFR1 SIGNALING. APPLY METHODS FROM AIM 1 TO DISCERN RAP1B'S EFFECT ON TNFR1 LIPID RAFT ORGANIZATION, COMPOSITION, AND REDOX SIGNALING. EVALUATE RAP1B'S ROLE IN ROS AND NO-MEDIATED LEUKOSTASIS AND EXPLORE HYPERGLYCEMIA'S IMPACT ON RAP1B SIGNALING. (AIM 3) ESTABLISH THE IMPACT OF EC RAP1B ON EC-LEUKOCYTE DYNAMICS IN VIVO. UTILIZE ADVANCED MICROSCOPY AND TRANSCRIPTOMICS TO DETERMINE RAP1B'S ROLE IN RAP1B ON LEUKOCYTE-DEPENDENT VASCULAR OBLITERATION, PERMEABILITY AND LEUKOSTASIS IN DR AND USING A RETINOPATHY OF PREMATURITY MODEL, ON NEOVASCULARIZATION. THESE STUDIES WILL DEFINE MOLECULAR ROLES OF RAP1 IN RECS AND PROVIDE CRITICAL INSIGHTS INTO THE MECHANISMS CONTROLLING THE BALANCE OF REC RESPONSES TO VEGF AND PROINFLAMMATORY STIMULI IN DR, INFORMATION CRITICAL TO IMPROVE THERAPEUTIC STRATEGIES FOR RETINAL ISCHEMIC DISEASES.

ROLE OF CARBOXYTERMINAL HYPERVARIABLE REGION IN RAP1B FUNCTION

ENHANCING TRANSFUSABLE PLATELETS FOR HEMORRHAGE CONTROL USING LIPOSOMAL TRANSFECTION

NEGATIVE REGULATION OF PLATELET ACTIVITY

THERAPEUTIC TARGETING OF AN ONCOGENIC TRANSLATIONAL PROGRAM IN AML - PROJECT SUMMARY CBFΒ-SMMHC IS THE DRIVER ONCOGENE IN ACUTE MYELOID LEUKEMIA (AML) WITH THE CHROMOSOME INVERSION INV(16)(P13Q22). CBFΒ-SMMHC INDUCES AML BY SEQUESTERING RUNX1, THE TRANSCRIPTION FACTOR THAT PLAYS AN IMPORTANT ROLE IN NORMAL HEMATOPOIESIS. ADULT INV(16) AML PATIENTS PRESENT POOR CLINICAL OUTCOMES - 30-40% OF THESE PATIENTS RELAPSE AND DIE FROM THE DISEASE. THIS SUGGESTS DEVELOPING BETTER THERAPEUTIC STRATEGIES FOR TREATING INV(16) AML IS CRITICAL. WE RECENTLY DEVELOPED A SMALL MOLECULE INHIBITOR, AI-10-49, WHICH SPECIFICALLY DISRUPTS CBFΒ-SMMHC BINDING TO RUNX1. AI-10-49 TREATMENT INDUCED LEUKEMIC CELL APOPTOSIS IN HUMAN PRIMARY INV(16) AML CELLS AND ENHANCED SURVIVAL IN AN INV(16) MOUSE MODEL. AI-10-49 IS THE FIRST TARGETED THERAPY FOR INV(16) AML AND IS UNDER DEVELOPMENT AS AN ANTI-LEUKEMIC DRUG. IN A RECENT STUDY, WE EXPLORED THE MECHANISM OF ACTION OF AI-10-49. WE FOUND THAT AI-10-49-INDUCED CELL DEATH IS PARTLY MEDIATED BY INHIBITION OF THE EUKARYOTIC TRANSLATION INITIATION FACTOR 4 GAMMA 1 (EIF4G1) IN INV(16) AML CELLS. EIF4G1 INHIBITION LEADS TO APOPTOSIS IN INV(16) AML CELLS, INDICATING EIF4G1 AS A THERAPEUTIC TARGET. WE ALSO FOUND THAT REACTIVATION OF EIF4G1 IS A MAJOR SOURCE OF AI-10-49 RESISTANCE IN INV(16) AML CELLS, AND EIF4G1 INHIBITION BY THE SMALL MOLECULE INHIBITOR SBI- 756 CAN OVERCOME DRUG RESISTANCE IN INV(16) AML CELLS. OUR RESULTS PROVIDE PROOF-OF-CONCEPT FOR TESTING EIF4G1 INHIBITORS IN COMBINATION WITH AI-10-49 TO OVERCOME DRUG RESISTANCE DURING TREATMENT OF INV(16) AML. OUR DATA SUGGEST THAT OVEREXPRESSION OF EIF4G1 BY CBFΒ-SMMHC IS A CRITICAL STEP IN INV(16) AML TRANSFORMATION. WE HYPOTHESIZE THAT CBFΒ-SMMHC ACTIVATES EIF4G1 IN INV(16) AML, ALTERING PROTEIN SYNTHESIS AND REWIRING THE AML PROTEOME, WHICH, IN TURN, PROMOTES LEUKEMOGENESIS AND REACTIVATION OF EIF4G1, CONTRIBUTING TO AI-10-49 RESISTANCE. WE WILL TEST THIS HYPOTHESIS BY I) CHARACTERIZING THE THERAPEUTIC VALUE OF SBI-756, AN EIF4G1 SMALL MOLECULE INHIBITOR, IN INV(16) AML CELL SURVIVAL USING A CBFΒ-SMMHC GENETIC MOUSE MODEL AND AN INV(16) AML PATIENT-DERIVED XENOGRAFT MODEL, AND II) DEFINING THE MECHANISMS OF ACTION OF EIF4G1 IN INV(16) AML.

DECODING KINDLIN-3 SIGNALING IN PLATELETS AND NEUTROPHILS - PROJECT SUMMARY THE STUDIES PROPOSED IN THIS APPLICATION REFLECT THE PRINCIPAL INVESTIGATOR’S LONG-STANDING COMMITMENT TO UNRAVELING THE REGULATORY ROLE OF KINDLIN-3 SIGNALING IN THROMBOSIS AND INFLAMMATION. THESE INVESTIGATIONS AIM TO ADDRESS CRITICAL CONCEPTUAL GAPS RELEVANT TO THE NHLBI’S MISSION, AS THROMBOSIS AND INFLAMMATION ARE INTERRELATED PATHOPHYSIOLOGICAL PROCESSES CONTRIBUTING TO NUMEROUS DISEASES, INCLUDING CARDIOVASCULAR DISEASE, ACUTE AND CHRONIC LUNG INJURY, VENOUS THROMBOEMBOLISM, AUTOIMMUNE DISORDERS, AND SEPSIS. CENTRAL TO THESE PROCESSES ARE PLATELETS AND NEUTROPHILS, WHOSE DYSREGULATION CAN LEAD TO SEVERE COMPLICATIONS, SUCH AS EXCESSIVE CLOTTING OR TISSUE DAMAGE. OUR RESEARCH FOCUSES ON ELUCIDATING THE MOLECULAR MECHANISMS BY WHICH KINDLIN-3, A CRITICAL SIGNALING ADAPTOR EXPRESSED IN CELLS OF HEMATOPOIETIC ORIGIN, REGULATES PLATELET ACTIVATION DURING THROMBOSIS AND NEUTROPHIL ACTIVITY DURING INFLAMMATION. KINDLIN-3 IS ESSENTIAL FOR SUPPORTING INTEGRIN ΑIIBΒ3 ACTIVATION IN PLATELETS, YET ITS PRECISE ROLE IN REGULATING INTEGRIN BIDIRECTIONAL SIGNALING REMAINS POORLY UNDERSTOOD. BASED ON OUR NOVEL DISCOVERY THAT KINDLIN-3 INTERACTS WITH MYOSIN IN PLATELETS, WE HYPOTHESIZE THAT THE CROSSTALK BETWEEN KINDLIN-3 AND THE MYOSIN COMPLEX FACILITATES INTEGRIN ΑIIBΒ3 ACTIVATION DURING INSIDE-OUT SIGNALING AND PROMOTES MYOSIN ACTIVATION DURING OUTSIDE-IN SIGNALING. TO TEST THIS HYPOTHESIS, WE WILL INVESTIGATE THE STRUCTURAL BASIS OF THE KINDLIN-3-MYOSIN COMPLEX AND ASSESS THE FUNCTIONAL IMPACT ON PLATELET ACTIVATION AND ACTIVITY USING DESIGNED KINDLIN-3 KNOCK-IN MOUSE MODELS. KINDLIN-3 EXHIBITS DUAL ROLES IN NEUTROPHILS, ACTING AS A PRO-INFLAMMATORY AND ANTI-INFLAMMATORY REGULATOR. WHILE KINDLIN-3 PROMOTES Β2-INTEGRIN- MEDIATED NEUTROPHIL RECRUITMENT, OUR RECENT FINDINGS INDICATE THAT IT ALSO SUPPRESSES REACTIVE OXYGEN SPECIES (ROS) PRODUCTION AND NEUTROPHIL EXTRACELLULAR TRAP (NET) RELEASE IN AN INTEGRIN-INDEPENDENT MANNER. WE PROPOSE THAT KINDLIN-3 LEVELS IN NEUTROPHILS ARE DYNAMICALLY REGULATED ACROSS DIFFERENT STAGES OF INFLAMMATION TO BALANCE THESE OPPOSING FUNCTIONS. SPECIFICALLY, WE AIM TO ELUCIDATE HOW KINDLIN-3 IS DOWNREGULATED IN EXTRAVASATED NEUTROPHILS AT THE LATE STAGE OF INFLAMMATION AND THE MECHANISM BY WHICH IT MODULATES ROS AND NET SIGNALING IN CELLULAR AND MOUSE MODELS. COLLECTIVELY, THIS PROGRAM WILL LEVERAGE CUTTING-EDGE TECHNOLOGY BY INTEGRATING STRUCTURAL AND CELLULAR BIOLOGY WITH EX VIVO AND IN VIVO MODELS TO DECODE THE CRITICAL ROLE OF KINDLIN-3 SIGNALING IN PLATELETS AND NEUTROPHILS. THESE INSIGHTS WILL LAY THE GROUNDWORK FOR ADVANCES IN UNDERSTANDING AND TREATING BLOOD AND VASCULAR DISORDERS, ALIGNING WELL WITH THE MISSION OF THE NHLBI.

GENERATION OF ALLOANTIGEN-SPECIFIC DESIGNER PLATELETS FOR DIAGNOSTIC AND INVESTIGATIVE USE

THE ROLE IN INFLAMMATION IN PLATELET ACTIVATION AND THROMBOSIS

DEFINING THE MECHANISMS OF HEMOGLOBIN SWITCHING AND GENOTOXICITIES ASSOCIATED WITH ITS MANIPULATION - PROJECT SUMMARY INDUCTION OF FETAL HEMOGLOBIN (HBF, A22) BY GENOME EDITING IS A PROMISING THERAPEUTIC STRATEGY FOR SS- HEMOGLOBINOPATHIES. THE FOCUS OF MY WORK IS TO BETTER UNDERSTAND THE DEVELOPMENTAL REGULATION OF -GLOBIN EXPRESSION AND INVESTIGATE THE GENOTOXICITIES ASSOCIATED WITH GENOME EDITING OF CD34+ HEMATOPOIETIC STEM AND PROGENITOR CELLS (HSPCS) TO INDUCE HBF THERAPEUTICALLY. MY RECENT STUDIES HAVE UTILIZED FUNCTIONAL GENOMICS TO IDENTIFY KEY DNA REGULATORY MOTIFS IN THE -GLOBIN PROMOTER THAT ARE ESSENTIAL FOR GENE EXPRESSION FOLLOWING THERAPEUTIC GENOME EDITING OR IN NON-DELETIONAL HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN (HPFH). HPFH IS A BENIGN, GENETIC CONDITION IN WHICH POINT MUTATIONS OR SMALL DELETIONS CAUSE SUSTAINED -GLOBIN EXPRESSION IN ADULT RED BLOOD CELLS. HOWEVER, THE REGULATION OF -GLOBIN EXPRESSION NORMALLY, AND IN SOME FORMS OF HPFH, REMAIN INCOMPLETELY DEFINED. IN PARALLEL RELATED STUDIES, I HAVE SHOWN IN HSPCS THAT CAS9-INDUCED DOUBLE-STRANDED DNA BREAKS (DSBS) RESULTING FROM THERAPEUTIC GENOME EDITING TO INDUCE HBF CAN CAUSE CHROMOSOME SEGREGATION ERRORS DURING CELL DIVISION, LEADING TO MICRONUCLEUS FORMATION AND COPY NUMBER ABNORMALITIES OF THE TELOMERIC CHROMOSOMAL SEGMENT. MOST CELLS WITH THESE ABNORMALITIES SHOULD BE ELIMINATED BY ENDOGENOUS DNA DAMAGE SURVEILLANCE MECHANISMS. HOWEVER, MICRONUCLEI RESULTING FROM DSBS CAN ALSO LEAD TO STABLE CHROMOSOMAL REARRANGEMENTS, CHROMOTHRIPSIS, AND MALIGNANT TRANSFORMATION. HENCE, IT IS IMPORTANT TO DETERMINE WHETHER THESE ABNORMALITIES PERSIST AFTER EDITING OF HSPCS. FOR THIS K01 PROPOSAL, I WILL CONTINUE MY TWO SEPARATE BUT RELATED LINES OF INVESTIGATION TO BETTER UNDERSTAND THE REGULATION OF -GLOBIN TRANSCRIPTION AND THE GENOTOXICITIES ASSOCIATED WITH THERAPEUTIC GENOME EDITING TO INDUCE HBF. SPECIFICALLY, I WILL MAP A NEWLY DISCOVERED REGULATORY ELEMENT IN THE -GLOBIN LOCUS AND DEFINE THE EPIGENETIC CHANGES AND TRANSCRIPTION FACTORS IMPORTANT FOR DELETIONAL HPFH, WHICH IS CAUSED BY KILOBASE-SCALE DELETIONS OF THE EXTENDED SS-GLOBIN LOCUS, USING POPULATION AND SINGLE- CELL GENOMICS (AIM 1). IN PARALLEL, I WILL INVESTIGATE WHETHER MICRONUCLEI AND CHROMOSOMAL ABNORMALITIES PERSIST AFTER DSBS IN HSPCS. THROUGH WHOLE GENOME SEQUENCING, LIVE-, AND FIXED-CELL IMMUNOFLUORESCENCE, I WILL STUDY CAS9-INDUCED CHROMOSOME INSTABILITY, STRUCTURAL VARIATIONS, AND DNA DAMAGE SENSING PATHWAYS IN HSPCS IN VITRO WITH THE LONG-TERM GOAL OF STUDYING THE PERSISTENCE OF CHROMOSOMAL ABNORMALITIES IN VIVO (AIM 2). THE SUCCESSFUL COMPLETION OF THIS K01 CAREER DEVELOPMENT AWARD WILL FORM THE FOUNDATION FOR MY LONG-TERM CAREER GOAL OF ESTABLISHING AN INDEPENDENT RESEARCH PROGRAM THAT INVESTIGATES THE MECHANISMS OF GENE REGULATION AND DNA DAMAGE SENSING TO LEVERAGE THIS INFORMATION FOR IMPROVED GENETIC THERAPIES. THE PROPOSED RESEARCH AND TRAINING PLANS WITHIN THE ACADEMIC ENVIRONMENT WILL ENSURE A SUCCESSFUL PATH FOR INDEPENDENCE.

UNDERSTANDING CELLULAR VARIATION IN FETAL HEMOGLOBIN DISTRIBUTION - PROJECT SUMMARY SICKLE CELL DISEASE (SCD) IS A SEVERE INHERITED BLOOD DISORDER AFFECTING OVER 100,000 INDIVIDUALS IN THE UNITED STATES AND MILLIONS GLOBALLY. IT IS CAUSED BY A MISSENSE VARIANT IN THE -GLOBIN GENE THAT LEADS TO HEMOGLOBIN S (HBS) POLYMERIZATION, RED BLOOD CELL (RBC) SICKLING, HEMOLYSIS, AND DOWNSTREAM COMPLICATIONS INCLUDING VASO-OCCLUSIVE CRISES (VOCS), STROKE, AND PROGRESSIVE ORGAN DAMAGE. FETAL HEMOGLOBIN (HBF, ₂₂) INHIBITS HBS POLYMERIZATION AND IS A WELL-ESTABLISHED MODIFIER OF DISEASE SEVERITY. PHARMACOLOGIC AGENTS SUCH AS HYDROXYUREA (HU), AND NATURALLY OCCURRING DELETIONS IN THE -GLOBIN LOCUS, SUCH AS THOSE FOUND IN HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN (HPFH), INCREASE HBF AND REDUCE CLINICAL COMPLICATIONS. HOWEVER, MANY PATIENTS REMAIN AT RISK FOR COMPLICATIONS DESPITE ACHIEVING HIGH TOTAL HBF LEVELS. EMERGING DATA INDICATE THAT THE DISTRIBUTION OF HBF ACROSS INDIVIDUAL RBCS, WHETHER UNIFORM (PANCELLULAR) OR UNEVEN (HETEROCELLULAR), IS A STRONGER DETERMINANT OF CLINICAL BENEFIT THAN TOTAL HBF ALONE. THE MOLECULAR MECHANISMS THAT GOVERN THIS DISTRIBUTION REMAIN POORLY DEFINED. THIS PROJECT WILL DEFINE HOW GENETIC AND EPIGENETIC REGULATION OF -GLOBIN EXPRESSION SHAPES HBF DISTRIBUTION AND MODIFIES DISEASE SEVERITY IN SCD. THE CENTRAL HYPOTHESIS IS THAT RED CELL–INTRINSIC REGULATION OF -GLOBIN DETERMINES HBF DISTRIBUTION, AND THAT VARIATION IN THIS REGULATION UNDERLIES PERSISTENT CLINICAL HETEROGENEITY AMONG PATIENTS WITH ELEVATED HBF. AIM 1 WILL USE SINGLE-CELL TRANSCRIPTOMICS, CHROMATIN ACCESSIBILITY PROFILING, AND GENOME EDITING TO IDENTIFY AND FUNCTIONALLY VALIDATE REGULATORY ELEMENTS THAT CONTROL HBF DISTRIBUTION IN INDIVIDUALS WITH NATURALLY OCCURRING -GLOBIN LOCUS DELETIONS, INCLUDING HPFH AND A-THALASSEMIA. AIM 2 WILL STUDY HU-TREATED PATIENTS WITH SUSTAINED HIGH HBF LEVELS BUT DIVERGENT CLINICAL OUTCOMES. BY INTEGRATING SINGLE-CELL RNA-SEQ, ATAC-SEQ, WHOLE GENOME SEQUENCING, AND CLINICAL PHENOTYPING FROM MULTICENTER COHORTS, THIS AIM WILL IDENTIFY CELL-INTRINSIC REGULATORS ASSOCIATED WITH DIFFERENTIAL CLINICAL OUTCOMES. FUNCTIONAL STUDIES IN PATIENT-DERIVED ERYTHROID CELLS WILL TEST WHETHER CANDIDATE VARIANTS AND PATHWAYS ALTER HBF DISTRIBUTION. THIS WORK ADDRESSES A CRITICAL GAP IN SCD MANAGEMENT BY IDENTIFYING MECHANISMS OF INCOMPLETE THERAPEUTIC RESPONSE AND BIOMARKERS OF RESIDUAL RISK. FINDINGS WILL SUPPORT PRECISION HBF–INDUCING STRATEGIES AND THE RATIONAL DEVELOPMENT OF COMBINATION THERAPIES TO REDUCE DISEASE BURDEN IN CHILDREN AND ADULTS WITH SCD.

HIGH PARAMETER FLUORESCENCE ACTIVATED CELL SORTER - PROJECT SUMMARY/ABSTRACT THE VERSITI BLOOD RESEARCH INSTITUTE (VBRI) IS AN ACADEMIC HEMATOLOGY RESEARCH CENTER LOCATED IN MILWAUKEE, WI. THE VBRI FLOW CYTOMETRY CORE SERVES THE NEEDS OF SCIENTISTS AT THE VBRI, THE MEDICAL COLLEGE OF WISCONSIN (MCW), AND THE CHILDREN’S RESEARCH INSTITUTE (CRI), THREE RESEARCH-HEAVY INSTITUTIONS ASSEMBLED ON THE MILWAUKEE REGIONAL MEDICAL CAMPUS (MRMC). CURRENTLY, A HEAVILY OVERSUBSCRIBED CYTEK AURORA IN THE CRI IS THE ONLY HIGH PARAMETER CELL SORTER ON THE MRMC. THE VBRI’S 15-YEAR OLD FACS ARIA CAN NO LONGER SUPPORT THE SOPHISTICATED SORTING NEEDS OF MANY MRMC USERS AND WILL NEED TO BE REPLACED IN ANTICIPATION OF BD’S DISCONTINUATION OF SERVICING THE PRODUCT LINE. TO ADDRESS THESE DEFICIENCIES, WE ARE REQUESTING FUNDS TO PURCHASE A BECTON DICKINSON SYMPHONY S6 SE, A 5-LASER, 49-DETECTOR CELL SORTER. THE SYMPHONY S6 MATCHES THE SYMPHONY A5 SE ANALYZER PURCHASED BY VBRI IN 2022, ALLOWING FOR SEAMLESS TRANSFER OF MULTI-COLOR PANELS FROM ANALYSIS TO SORTING. THE RESEARCH PROJECTS OF OUR NINE MAJOR AND EIGHT MINOR NIH-FUNDED USERS REQUIRE HIGH PARAMETER CELL SORTING TO ACCOMPLISH THEIR GOALS. USING THE SYMPHONY A5 SE ANALYZER, THESE INVESTIGATORS HAVE ACQUIRED VALUABLE EXPERTISE IN SPECTRAL FLOW CYTOMETRY, AND THE ACQUISITION OF A SYMPHONY S6 WILL ENABLE THEM TO OVERCOME CRITICAL ROADBLOCKS IN THEIR RESEARCH. VBRI RESEARCH WILL BROADLY BENEFIT FROM THIS NEW INSTRUMENT. IN IMMUNOLOGY, DEVELOPMENT OF A B CELL THERAPEUTIC FOR MULTIPLE SCLEROSIS, THE DELINEATION OF B CELL INVOLVEMENT IN HEPARIN-INDUCED THROMBOCYTOPENIA AS WELL AS CLARIFYING THE MECHANISM OF THROMBOTIC COMPLICATIONS IN COVID- 19 ALL REQUIRE ACCESS TO HIGH PARAMETER SORTING, USING PANELS ALREADY ESTABLISHED ON THE SYMPHONY A5 SE. MAJOR USERS FOCUSED ON HEMATOLOGIC MALIGNANCIES REQUIRE RAPID ACCESS TO A MULTI-PARAMETER CELL SORTER TO ISOLATE RARE CELL POPULATIONS FROM PRIMARY LEUKEMIA SAMPLES THAT CAN ARRIVE ANY TIME. A SIGNATURE PROGRAM STUDYING THE ROLE OF THE GLYCOME IN HEMATOPOIESIS IS CRITICALLY DEPENDENT ON THE SYMPHONY S6’S INCREASED NUMBER OF CHANNELS. IN THROMBOSIS AND HEMOSTASIS, STUDIES ON CELLULAR INTEGRINS, KINDLIN SIGNALING IN NEUTROPHILS AND PLATELETS, AND THE PATHOPHYSIOLOGY OF FETAL/NEONATAL ALLOIMMUNE THROMBOCYTOPENIA WILL ALL HEAVILY USE THE SYMPHONY S6. MOREOVER, THE SYMPHONY S6 WILL OVERCOME CRITICAL BOTTLENECKS FOR MCW RESEARCHERS STUDYING THE ROLE OF EXOSOMES IN OVARIAN CANCER, CHRONIC PAIN IN FABRY AND SICKLE CELL DISEASE, GAMMA HERPESVIRUS ASSOCIATED B-CELL LYMPHOMAS, IMMUNE RESPONSE TO RADIATION EXPOSURE AND TYPE I DIABETES. FACS IS AN INTEGRAL PART OF THE WORKFLOW OF MOST VBRI FACULTY AND MANY RESEARCHERS AT MCW AND CRI. CURRENTLY AVAILABLE HIGH PARAMETER (>20) SORTING CAPACITY IS EXTREMELY LIMITED, SLOWING PROGRESS ON NIH-FUNDED RESEARCH. EVEN LOWER PARAMETER SORTING CAPACITY IS BECOMING LIMITED ON THE MRMC AS BD FACS ARIA INSTRUMENTS ARE APPROACHING END OF LIFESPAN. THE SYMPHONY S6 WILL PROVIDE ESSENTIAL HIGH PARAMETER SORTING CAPACITY FOR SEVERAL NIH-FUNDED PROJECTS, AND DRAMATICALLY ACCELERATE PROGRESS ON OTHERS. EASE OF PANEL TRANSFER, ECONOMIC USE OF PRECIOUS SAMPLES, AND COMPLEMENTARITY TO EXISTING INSTRUMENTATION WILL EXPEDITE RESEARCH PROGRESS TO ASCERTAIN A HIGH RETURN ON INVESTMENT.

THE ROLE OF JMJD1C IN NORMAL AND LEUKEMIC HEMATOPOIESIS

THE BIOCHEMISTRY AND PHYSIOLOGY OF PLATELET TFPI

A BIOLUMINESCENT ASSAY FOR DIRECT MEASUREMENT OF SIRTUIN ACTIVITY IN CANCER CELLS - A BIOLUMINESCENT ASSAY FOR DIRECT MEASUREMENT OF SIRTUIN ACTIVITY IN CANCER CELLS SIRTUINS ARE NICOTINAMIDE ADENINE DINUCLEOTIDE (NAD+)-DEPENDENT LYSINE DEACYLASES THAT ACT ON HISTONES, TRANSCRIPTION FACTORS, EPIGENETIC REGULATORS AND METABOLIC ENZYMES TO REGULATE KEY CELLULAR FUNCTIONS SUCH AS APOPTOSIS, SENESCENCE AND ENERGY METABOLISM. DEPENDING ON THE CELLULAR CONTEXT, SIRTUINS CAN PROMOTE OR INHIBIT ONCOGENESIS, DEFINING A CRITICAL NEED FOR METHODS CAPABLE OF INTERROGATING SIRTUIN ACTIVITY IN INTACT CELLS AND ANIMAL MODELS. HOWEVER CURRENTLY AVAILABLE SIRTUIN ASSAYS ARE CONFINED TO CELL FREE SYSTEMS, ARE PRONE TO ARTIFACTS, AND HAVE LIMITED SENSITIVITY AND DYNAMIC RANGE. THE LACK OF ASSAYS THAT PROVIDE A RELIABLE, DIRECT AND REAL-TIME READOUT OF SIRTUIN ACTIVITY IN VITRO AND IN VIVO POSES A MAJOR BARRIER TO DRUG DEVELOPMENT AND BIOLOGICAL STUDIES. TO OVERCOME THE LIMITATIONS OF CURRENT ASSAYS WE PROPOSE LUCIFERASE-BASED ASSAY THAT REPORTS IN REAL- TIME SIRTUIN ACTIVITY IN CELL FREE AND CELLULAR ASSAYS THROUGH GENERATION OF A BIOLUMINESCENCE SIGNAL BY CONVERSION OF FURIMAZINE TO FURIMAMIDE. OUR DESIGN IS BASED ON A SPLIT-NANOLUC COMPLEMENTATION SYSTEM THAT CONSISTS OF A TRUNCATED CATALYTICALLY INACTIVE N-TERMINAL MOIETY (TERMED 11S), WHICH IS ACTIVATED BY COMPLEMENTATION WITH A HIGH AFFINITY 11-AA C-TERMINAL PEPTIDE CONTAINING TWO LYSINE RESIDUES (K8,K9). OUR PREMISE IS THAT ACYLATION OR OTHER MODIFICATIONS OF THE LYSINE RESIDUES WITHIN THE HIGH AFFINITY C-TERMINAL COMPLEMENTATION PEPTIDE INHIBIT ITS BINDING TO THE N-TERMINAL NANOLUC MOIETY, PREVENTING ACTIVATION; ACTIVE SIRTUIN ENZYME REMOVES THE ACYL GROUPS, ALLOWING FOR LUCIFERASE COMPLEMENTATION AND GENERATION OF A LUMINESCENCE SIGNAL. OUR PRELIMINARY DATA USING SIRT5 ENZYME DEMONSTRATE A ROBUST REPORTER SIGNAL IN CELL FREE ASSAYS, AND SUGGEST THAT MEASUREMENT OF INTRACELLULAR SIRTUIN ACTIVITY IS FEASIBLE. IN AIM 1 WE WILL ADAPT THE SPLIT-NANOLUC ASSAY FOR MEASURING SIRTUIN DEACYLASE ACTIVITY IN CELL FREE SYSTEMS AND DETERMINE WHICH LYSINE MODIFICATIONS WITHIN THE C-TERMINAL COMPLEMENTATION PEPTIDE BLOCK BINDING TO THE LARGE NANOLUC FRAGMENT, AND ARE REMOVED BY SIRTUINS 1 – 7. WE WILL OPTIMIZE ASSAY CONDITIONS, AND DELINEATE PERFORMANCE PARAMETERS (SENSITIVITY, SELECTIVITY, DYNAMIC RANGE, COEFFICIENT OF VARIATION). IN AIM 2 WE WILL ADAPT THE SPLIT-NANOLUC ASSAY FOR MEASUREMENT OF SIRT5 ACTIVITY IN LIVE CELLS AND IN VIVO. WE WILL GENERATE SIRT5 REPORTER CELL LINES, TARGETING THE TRUNCATED NANOLUC TO SPECIFIC CELLULAR COMPARTMENTS. WE WILL OPTIMIZE CONDITIONS FOR QUANTITATIVE BIOLUMINESCENCE MEASUREMENT IN LIVE CANCER CELLS AND IN VIVO, USING CRISPR/CAS9, SHRNA AND INHIBITORS TO MODULATE SIRT5 ACTIVITY. OUR DESIGN HAS CRITICAL ADVANTAGES OVER CURRENT APPROACHES: (I) POTENTIAL ADAPTABILITY TO ANY TYPE OF ACYL MODIFICATION; (II) HIGH SIGNAL- TO-BACKGROUND RATIO; (III) AVOIDANCE OF ARTIFACT-PRONE CHEMICALLY MODIFIED PROBES; (IV) ADAPTABILITY TO MEASUREMENT OF ENZYME ACTIVITY IN LIVE CELLS AND ANIMAL MODELS. SUCCESSFUL COMPLETION OF THIS PROJECT WILL RESULT IN THE ESTABLISHMENT OF A VERSATILE AND SENSITIVE ASSAY FOR DETECTION OF SIRTUIN DEACYLASE ENZYMATIC ACTIVITY IN CELL FREE SYSTEMS, LIVE CANCER CELLS AND ANIMAL MODELS OF CANCER, OVERCOMING A MAJOR BARRIER IN THE FIELD.

EPITOPE SELECTION BY HLA-DR

TMH CLINICAL TRIALS NETWORK-WISCONSIN CORE CLINICAL CENTER

EXPLORING THE TRANSCRIPTIONAL NETWORK REGULATING ES CELL PLURIPOTENCY

MOLECULAR BASIS OF TRANSFUSION-INDUCED AUTO-IMMUNITY

A NOVEL HUMAN REGULATORY B CELL SUBSET

PREVALENCE AND IMMUNOGENICITY OF HNA-3A AND -3B ANTIBODIES AND ANTIGENS

B CELL-MEDIATED IMMUNE REGULATION

IDENTIFICATION OF A PROTEIN THAT ELICITS IMMUNE-MEDIATED NEURONAL DYSFUNCTION

ACTIVATED PROTEIN C FOR TREATMENT OF RADIATION COMBINED INJURY

SPINNING DISK CONFOCAL MICROSCOPE SYSTEM

CONFORMATIONAL REGULATION IN INTEGRIN BIDIRECTIONAL TRANSMEMBRANE SIGNALING

MECHANISMS OF A NOVEL REGULATORY B CELL SUBSET

FUNCTIONS OF BCL10 IN LYMPHOCYTES

MECHANISMS OF REGULATORY B CELL FUNCTION

PERMISSIVE SPECIFICITY IN PEPTIDE BINDING TO HLA-DR

TRAINING IN TRANSFUSION MEDICINE AND CLASSICAL HEMATOLOGY - PROJECT SUMMARY/ABSTRACT WE PROPOSE A NEW T32 PROGRAM TO TRAIN POST-DOCTORAL SCIENTISTS IN CLASSICAL HEMATOLOGY, TRANSFUSION MEDICINE, AND GLYCOBIOLOGY RESEARCH AT THE VERSITI BLOOD RESEARCH INSTITUTE (VBRI). AS INTIMATELY LINKED DISCIPLINES, CLASSICAL HEMATOLOGY AND TRANSFUSION MEDICINE FORM A CORNERSTONE OF RESEARCH IN BLOOD DISEASES BUT NEED HELP ATTRACTING NEW TALENT. WHILE FOUNDED IN HEMATOLOGY, GLYCOBIOLOGY REMAINS A SPECIALIZED RESEARCH DOMAIN WITH FEW EXPERTS TO TRANSLATE BASIC FINDINGS INTO CLINICAL PRACTICE. THE VBRI, ENHANCED BY SELECTED FACULTY FROM THE MEDICAL COLLEGE OF WISCONSIN, IS HOME TO A UNIQUE CLUSTER OF EXPERTS IN THESE AREAS WHO WILL SERVE AS PRECEPTORS IN THIS T32 PROGRAM TO CLOSE THE SUBSTANTIAL GAP IN THIS REQUIRED WORKFORCE. TRAINEES FOR THIS PROGRAM WILL BE ACCEPTED INTO ONE OF TWO TRAINING TRACKS; THROMBOSIS, HEMOSTASIS, AND VASCULAR BIOLOGY (TRACK I); AND GLYCOBIOLOGY IN HEMATOLOGY, TRANSFUSION MEDICINE, AND METABOLISM (TRACK II). TRAINEES WILL GAIN AN IN- DEPTH SCIENTIFIC UNDERSTANDING OF THEIR FIELD, ACQUIRE COMPETENCY IN PERTINENT RESEARCH TECHNOLOGIES, AND LEARN TO CONDUCT AND PRESENT INDEPENDENT RESEARCH, INCLUDING THE SKILLS NEEDED TO FUND, MANAGE, AND SUSTAIN A LABORATORY. TRAINEES WILL UNDERGO A RIGOROUS SELECTION PROCESS BY AN INTERNAL EXECUTIVE COMMITTEE, EMPHASIZING RECRUITING AND RETAINING TRAINEES FROM UNDERREPRESENTED MINORITY GROUPS. SELECTED TRAINEES WILL PARTICIPATE IN A RESEARCH-INTENSIVE PROGRAM BASED ON AN INDIVIDUAL DEVELOPMENT PLAN (IDP)-DRIVEN FORMAT WITH A MENTORED RESEARCH PROJECT AT ITS CORE. IN ADDITION TO THEIR BASIC SCIENCE PRECEPTOR, EACH TRAINEE WILL HAVE A CLINICAL CO-MENTOR TO LEARN ABOUT TRANSLATIONAL AND CLINICAL ASPECTS OF THEIR RESEARCH. WE WILL IMPLEMENT BENCH SCIENCE AND SUPPLEMENT IT WITH A DIDACTIC LECTURE MODULE DESIGNED FOR EACH TRAINING TRACK, A FOCUSED TECHNOLOGY WORKSHOP, REGULAR WEEKLY SCIENTIFIC CONFERENCES, AND PARTICIPATION IN NATIONAL MEETINGS. EACH TRAINEE WILL RECEIVE GROUP MENTORING FROM A SCHOLARSHIP OVERSIGHT COMMITTEE, CONSISTING OF THE TRAINEE'S MENTOR, ONE PROGRAM CO-DIRECTOR, AND ONE MEMBER OF THE EXECUTIVE COMMITTEE, WHO WILL ESTABLISH THE IDP, ENSURE PROJECT COMPLETION AND ACTIVITY PROGRESS, AND PROVIDE REGULAR FEEDBACK TO THE CO-DIRECTORS. PRECEPTORS WILL BE REQUIRED TO UNDERGO MENTOR-THE-MENTOR TRAINING. THE EXECUTIVE COMMITTEE (CO-DIRECTORS DRS. MAST, HOFFMEISTER, AND PRECEPTOR FACULTY DRS. KASTRUP, MA, AND DAHMS) WILL MEET BI-ANNUALLY TO 1) PRIORITIZE PROGRAM GOALS, 2) OVERSEE PROGRAM POLICIES, ACTIVITIES, AND GOVERNANCE PRACTICES TO PROVIDE PROGRAM OVERSIGHT. AN EXTERNAL ADVISORY BOARD WILL MEET ANNUALLY WITH PROGRAM ADMINISTRATION AND TRAINEES TO PROVIDE FEEDBACK. WE REQUEST SUPPORT FOR THREE POST-DOCTORAL FELLOWS TO APPOINT ONE ANNUALLY OVER THE FIRST THREE YEARS. THUS, WE PLAN FOR A SMALL BUT HIGHLY EFFECTIVE PROGRAM WHERE INITIAL TRAINEES SET A HIGH STANDARD FOR THOSE FOLLOWING. AS SUCH, WE EXPECT THE TRAINEES TO PURSUE MENTORED RESEARCH AWARDS FROM THE NIH OR OTHER FUNDING AGENCIES AS THEIR CAREERS PROGRESS FROM POST-DOCTORAL FELLOWS TO JUNIOR FACULTY AND INDEPENDENT RESEARCH CAREERS, STRENGTHENING AND EXPANDING THE US SCIENTIFIC WORKFORCE IN CLASSICAL HEMATOLOGY, TRANSFUSION MEDICINE, AND GLYCOBIOLOGY.

ASSOCIATION OF TFPI WITH ANTEMORTEM DEMENTIA AND POSTMORTEM MICRO BRAIN INFARCTS

DNA DAMAGE RESPONSES TO GENOME EDITING IN HEMATOPOIETIC STEM CELLS - PROJECT SUMMARY SEVERAL GENOME EDITING STRATEGIES HAVE BEEN DEVELOPED FOR THE TREATMENT OF GENETIC DISEASES THAT AFFECT THE HEMATOPOIETIC SYSTEM, SUCH AS SICKLE CELL DISEASE AND BETA-THALASSEMIA. ALTHOUGH THERE HAS BEEN EXTENSIVE RESEARCH ON DETECTING OFF-TARGET EFFECTS OF GENOME EDITORS, THERE IS LIMITED INFORMATION ON THE GENOTOXICITIES OF ON-TARGET EFFECTS. THE UNDERSTANDING OF HOW HEMATOPOIETIC STEM AND PROGENITOR CELLS (HSPCS) RESOLVE ERRORS OF MITOSIS FOLLOWING DNA DAMAGE TO PREVENT PERSISTENT CHROMOSOMAL ABNORMALITIES IS INCOMPLETE. FOR SAFER CLINICAL GENOME EDITING APPLICATIONS, UNDERSTANDING THE FREQUENCY OF SUCH EVENTS IS VITAL. WE HAVE SHOWN IN HSPCS THAT CAS9-INDUCED DOUBLE-STRANDED DNA BREAKS (DSBS) RESULTING FROM GENOME EDITING OF THERAPEUTIC LOCI CAN CAUSE CHROMOSOME SEGREGATION ERRORS DURING CELL DIVISION, LEADING TO MICRONUCLEUS FORMATION AND COPY NUMBER ABNORMALITIES OF THE TELOMERIC CHROMOSOMAL SEGMENT. MICRONUCLEI (MN) CAN CONTRIBUTE TO CHROMOTHRIPSIS, A GENOMIC REARRANGEMENT PHENOMENON OBSERVED IN CANCER AND OTHER DISEASES. THIS PROPOSAL EXPLORES THE POTENTIAL FOR PERSISTENT ANEUPLOIDIES, INCLUDING CHROMOTHRIPSIS, IN HSPCS RESULTING FROM GENOME EDITING AND THE MECHANISMS OF DNA REPAIR IN RESPONSE TO GENOTOXICITIES. PRELIMINARY DATA GENERATED FROM K01-SUPPORTED RESEARCH DEMONSTRATES THAT GENOME EDITING OF HSPCS AT SPECIFIC TARGETS ALLOWS FOR THE ENRICHMENT OF EDITED CELLS USING PHARMACOLOGIC AND FLOW CYTOMETRIC METHODS. AIM 1 WILL EXTEND THIS OBSERVATION BOTH IN VITRO AND IN VIVO BY PERFORMING GENOME EDITING OF THERAPEUTICALLY RELEVANT GENES CENTROMERIC OF THE SELECTABLE GENE. ISOLATED CELLS WILL BE SUBJECT TO WHOLE GENOME SEQUENCING TO REVEAL THE NATURE OF CHROMOSOMAL REARRANGEMENTS. THIS ANALYSIS WILL REVEAL THE CAPACITY OF ANEUPLOID HSPCS TO SURVIVE AND THE HERITABILITY OF REARRANGEMENTS DURING THE PROCESS OF HEMATOPOIESIS. SECONDLY, OUR PRELIMINARY DATA SHOW THE INHIBITION OF P53-MEDIATED RESPONSES TO DNA DAMAGE OR CASPASE INHIBITION REDUCES THE FREQUENCY OF MN. AIM 2 WILL STUDY HOW P53 AND CASPASES MAY CONTRIBUTE TO GENOME INSTABILITY AS A MEANS OF PREVENTING PERSISTENT ERRORS OF DNA REPAIR, ALLOWING FOR THE ELIMINATION OF HSPCS WITH SOMATIC MUTATIONS. TOGETHER, THE PROPOSED STUDIES WILL PROVIDE A DEEPER UNDERSTANDING OF ON-TARGET ADVERSE EVENTS RELATED TO GENOME EDITING TO BETTER INFORM FUTURE GENETIC THERAPIES. DATA GENERATED THROUGH THIS R03 AWARD WILL PROVIDE PRELIMINARY DATA FOR FUTURE R01 APPLICATIONS ON MECHANISMS OF DNA REPAIR IN HUMAN HSPCS WHILE SUPPORTING MY TRANSITION FROM K01 RECIPIENT TO INDEPENDENT INVESTIGATOR.

PRE-MRNA PROCESSING AND FUNCTION OF ALTERNATIVELY SPLICED ISOFORMS OF TFPI - HEMOSTASIS IS A CONSTANT BALANCING ACT BETWEEN PRO- AND ANTICOAGULANT FACTORS, PLATELETS, AND THE VASCULATURE THAT IS REQUIRED TO PREVENT EXCESSIVE BLEEDING OR PATHOLOGICAL CLOTTING. THE ANTICOAGULANT, TISSUE FACTOR PATHWAY INHIBITOR (TFPI), IS A VITAL FACTOR IN THIS BALANCE AND MODULATES A BROAD RANGE OF BLEEDING AND CLOTTING DISORDERS THROUGH INHIBITION OF TF-FVIIA, FXA, AND PROTHROMBINASE (FXA-FVA). THE TFPI GENE IS EVOLUTIONARILY CONSERVED AND DUE TO ALTERNATIVE SPLICING, DIFFERENT TFPI ISOFORMS ARE PREDOMINANT WITHIN DISTINCT POOLS. WHILE THE SPECIFIC INHIBITORY FUNCTION OF EACH TFPI ISOFORM HAS BEEN CHARACTERIZED, LITTLE IS KNOWN REGARDING DIFFERENCES IN ISOFORM- SPECIFIC CONTRIBUTIONS UNDER PROTHROMBOTIC DISEASE CONDITIONS SUCH AS FACTOR V LEIDEN (FVL) AND DURING EMBRYONIC DEVELOPMENT. FURTHER, THE PRE-MRNA SPLICING AND PROCESSING MECHANISMS DICTATING EXPRESSION OF EACH ISOFORM ARE UNKNOWN. AS A CAUSAL RELATIONSHIP EXISTS BETWEEN ABERRANT SPLICING OF FV AND TFPI ISOFORM- SPECIFIC FUNCTION IN HUMAN BLEEDING DISORDERS, THESE MECHANISMS, COORDINATED BY PRECISE CUES DIRECTED AT MAINTAINING THE HEMOSTATIC BALANCE, ARE HIGHLY RELEVANT. THUS, THE LONG-TERM OBJECTIVE OF THIS PROPOSAL IS TO DIFFERENTIATE THE PHYSIOLOGICAL, SITE-SPECIFIC PRODUCTION OF EACH TFPI ISOFORM AT A MOLECULAR LEVEL AND DEFINE THEIR ANTICOAGULANT FUNCTION IN EMBRYONIC DEVELOPMENT AND DISEASE. TFPIA IS THE ONLY ISOFORM PRESENT IN PLATELETS AND THE ONLY ISOFORM THAT INHIBITS PROTHROMBINASE DURING THE INITIATION OF BLOOD COAGULATION. ADDITIONALLY, GLOBAL TFPI DEFICIENCY RESULTS IN PROTHROMBOTIC PERINATAL LETHALITY IN FVL MICE, AND TFPIA PROTHROMBINASE INHIBITORY ACTIVITY IS REDUCED IN THE PRESENCE OF FVL. TO THIS END, K99 PHASE STUDIES PROBE THE PHYSIOLOGICAL ROLE OF TFPIA AS A REGULATOR OF FV/FVL, PARTICULARLY IN PROTHROMBINASE ASSEMBLY ON PLATELET SURFACES DURING DEVELOPMENT (AIM 1) AND CHARACTERIZES BIOLOGICAL ACTIVITY OF NEW PLATELET-SPECIFIC TFPIA SPLICE VARIANTS IDENTIFIED IN MICE AND HUMANS (AIM 2). THE CANDIDATE WILL ACQUIRE TECHNICAL EXPERTISE TO DEFINE TFPIA ANTICOAGULANT FUNCTION BOTH IN VIVO USING TWO UNIQUE ISOFORM- AND SITE-SPECIFIC TFPIA MUTANT MOUSE MODELS AND EX VIVO USING HUMAN AND MOUSE PLATELETS. IN AIM 3 (R00 PHASE), THE CANDIDATE WILL TAKE ADVANTAGE OF THE EVOLUTIONARY CONSERVATION OF ALTERNATIVE TFPI SPLICE FORMS AND SPLICING SIGNALS EMBEDDED IN HIGHLY CONSERVED SEQUENCES TO DETERMINE CIS-RNA ELEMENT AND TRANS-ACTING SPLICING FACTOR INTERACTIONS REGULATING TFPI ISOFORM DIVERSITY IN MICE AND HUMANS. DECIPHERING THE PRE-MRNA PROCESSING MECHANISMS THAT REGULATE SITE-SPECIFIC TFPI ISOFORM EXPRESSION WILL DELINEATE HOW ALTERNATIVE SPLICING CONTRIBUTES TO THE PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL HEMOSTATIC BALANCE DURING EMBRYONIC DEVELOPMENT AND IN ADULTHOOD. AS THERE ARE MANY PATIENTS WITH BLEEDING AND CLOTTING DISORDERS OF UNKNOWN CAUSE, THE RELATION OF ABERRANT SPLICING TO THESE DISEASES REPRESENTS A RELATIVELY NEW AND UNEXPLORED AREA WITH GREAT POTENTIAL FOR LAUNCHING A SUCCESSFUL INDEPENDENT CAREER. THIS PROPOSAL ALSO OUTLINES AN INTENSIVE TRAINING PLAN OF COURSES, SEMINARS, AND HANDS-ON TRAINING FOR TRANSITIONING THE CANDIDATE INTO A WELL-EQUIPPED INDEPENDENT INVESTIGATOR WITH A UNIQUE COMBINATION OF RESEARCH SKILLS AND A HIGHLY PROMISING BASIC RESEARCH PIPELINE.

DEFINING THE TRANSCRIPTIONAL, PHENOTYPIC, AND FUNCTIONAL HETEROGENEITY OF VIRUS-SPECIFIC CD4 T CELLS DURING CHRONIC VIRAL INFECTION

CYTOKINE REGULATION OF RECIRCULATING AND TISSUE-RESIDENT MEMORY CD8+ T CELLS IN HOMEOSTASIS AND INFLAMMATION - PROJECT SUMMARY/ABSTRACT MEMORY CD8+ T CELLS ARE SEEDED THROUGHOUT LYMPHOID AND NON-LYMPHOID TISSUES IN THE AFTERMATH OF ACUTE INFECTION. DURING THIS PROCESS, DIFFERENTIATION OCCURS INTO FUNCTIONALLY DISTINCT CIRCULATING AND TISSUE-RESIDENT SUBSETS WHICH CAN THEN PERSIST FOR YEARS, DECADES, OR EVEN LIFE. HOWEVER, OUR UNDERSTANDING OF THIS REMARKABLE LONGEVITY IS LARGELY CONFINED TO STUDYING CIRCULATING POPULATIONS IN HOMEOSTASIS, NEGLECTING THE PREPONDERANCE OF TISSUE-RESIDENT MEMORY T CELLS IN NON-LYMPHOID TISSUES AND FREQUENT PERTURBATIONS CAUSED BY INFECTION, INFLAMMATION, PHYSIOLOGICAL AND METABOLIC CHANGES, ETC. THROUGH THE PROPOSED STUDIES, THE APPLICANT WILL BEGIN TO ADDRESS THIS KNOWLEDGE GAP BY ESTABLISHING HOW PREEXISTING BYSTANDER MEMORY CD8+ T CELL SUBSETS ARE MAINTAINED DURING HOMEOSTASIS AND INFECTION VIA FLEXIBLE USE OF CYTOKINES, AS ADDRESSED WITH INFECTION MODELS AND CYTOKINE THERAPY. DESPITE VARIABLE REQUIREMENTS FOR IL-15 BETWEEN DIFFERENT MEMORY CD8+ T CELL POPULATIONS, THE APPLICANT RECENTLY PUBLISHED THAT IL-15 SENSITIVITY IS A SHARED FEATURE ACROSS MEMORY SUBSETS THAT OPERATES WITHIN SUBSET- AND TISSUE-SPECIFIC CONSTRAINTS. DR. JARJOUR PROPOSES THAT AS PART OF THE MEMORY CD8+ T CELL PROGRAM, MEMORY CELLS GAIN THE CAPACITY TO PROMISCUOUSLY UTILIZE MANY DIFFERENT CYTOKINES, THEREBY GAINING RESILIENCE TO STARK ALTERATIONS IN AVAILABILITY DURING INFECTION AND OTHER PERTURBATIONS. THIS FLEXIBILITY APPEARS TO OPERATE WITHIN THE CONFINES OF TISSUE- AND SUBSET- SPECIFIC REGULATION, WHICH THE APPLICANT WILL ELUCIDATE USING HIS EXPERTISE IN STUDYING RESIDENT IMMUNE CELLS. BEYOND REVEALING A KEY MECHANISM SUPPORTING DURABILITY OF MEMORY CD8+ T CELLS, THESE STUDIES HAVE IMPLICATIONS FOR IMMUNOTHERAPIES TO EXPAND MEMORY CD8+ T CELL POPULATIONS, INCLUDING DURING VACCINATION AND CANCER. THE DATA GENERATED THROUGH THIS PROPOSAL WILL FORM THE BASIS FOR AN R01 APPLICATION AND WILL POSITION DR. JARJOUR FOR AN INDEPENDENT CAREER IN BASIC IMMUNOLOGY RESEARCH, WITH LONG-TERM REPERCUSSIONS FOR HUMAN THERAPIES.

REPROGRAMMING TUMOR-REACTIVE CD8 T CELLS BY TARGETING THE IL-21-BATF PATHWAY TO TREAT MELANOMA

COHESIN MUTATIONS IN CORE-BINDING FACTOR ACUTE MYELOID LEUKEMIA

BATF-IRF4 COMPLEX CONTROLS TRANSCRIPTIONAL REGULATION AND EFFECTOR FUNCTION OF AUTOREACTIVE CD8 T CELLS IN TYPE 1 DIABETES

PROTEIN S ANTICOAGULANT ACTIVITY: BIOCHEMICAL MECHANISMS AND STRUCTURAL STUDIES

NUCLEAR ARCHITECTURE-DRIVEN CELLULAR HETEROGENEITY IN STEM CELL AND HEMATOPOIETIC DIFFERENTIATION

ENHANCER TRANSCRIBED RNAS: FUNCTION AND MECHANISM IN HEMATOPOIESIS

TARGETING MS4A3 TO TREAT CHRONIC MYELOMONOCYTIC LEUKEMIA (CMML)

DETERMINING THE RAP1B SIGNALING PATHWAY TO INTEGRIN ACTIVATION IN PLATELETS

ROLE OF FORCE-DIRECTED LIPID METABOLISM IN THE ENDOTHELIAL-TO-HEMATOPOIETIC TRANSITION - PROJECT SUMMARY/ABSTRACT IN VERTEBRATES, SELF-RENEWING HEMATOPOIETIC STEM CELLS (HSCS) ARE PRODUCED FROM A DEVELOPMENTAL EVENT CALLED ENDOTHELIAL-TO-HEMATOPOIETIC TRANSITION (EHT). EHT CONSISTS OF A CELLULAR AND TRANSCRIPTIONAL REPROGRAMMING THAT ALLOWS HEMOGENIC ENDOTHELIAL CELLS (HECS) FROM A SUBSET OF EMBRYONIC ARTERIES TO LEAVE THE VESSEL AND BECOME BLOOD STEM CELLS. HSCS HAVE THE CAPACITY TO REPLACE AND RESTORE THE COMPLETE BLOOD SYSTEM UPON TRANSPLANT, MAKING HSC TRANSPLANT THE ONLY CURATIVE THERAPY AVAILABLE FOR BLOOD DISEASES LIKE LEUKEMIA AND LYMPHOMA. GIVEN THIS THERAPEUTIC NEED, GREAT EFFORT HAS FOCUSED ON THE DEVELOPMENT OF IN VITRO PROTOCOLS THAT ATTEMPT TO RECAPITULATE THE CONDITIONS OF EHT FOR CLINICAL EXPANSION OR DE NOVO PRODUCTION OF STEM CELLS IN THE DISH. TO DATE NONE EFFICIENTLY PRODUCE LONG-LIVED MULTIPOTENT HSCS, SUGGESTING THAT ONE OR MORE DEVELOPMENTAL SIGNALS FOR THIS PROCESS REMAIN TO BE DEFINED. MECHANICAL FORCES FROM BLOOD FLOW ARE AN ESSENTIAL CUE FOR HSC PRODUCTION VIA EHT, AND THE ZEBRAFISH DANIO RERIO PROVIDES AN EXCELLENT ANIMAL MODEL IN WHICH TO STUDY THIS CONTRIBUTION TO HEMATOPOIESIS DUE TO CONSERVED MOLECULAR GENETICS OF EHT IN THIS SPECIES AND THE ABILITY TO OBSERVE LIVE EMBRYOS WITH ACTIVE CIRCULATION. FLOW-DRIVEN EHT IS MEDIATED IN PART BY THE YES-ASSOCIATED PROTEIN (YAP) TRANSCRIPTION FACTOR (TF), A TRANSCRIPTIONAL COREGULATOR THAT HAS ROLES IN ORGAN GROWTH, NUTRIENT REGULATION AND CELL FATE SPECIFICATION. YAP CAN BE DIRECTED TO THE NUCLEUS AS A DIRECT RESULT OF PHYSICAL FORCES ACTING ON THE CELL, BUT THE MOLECULAR MECHANISMS BY WHICH THIS PROMOTES EHT AND HSC PRODUCTION ARE UNCLEAR. IN PRELIMINARY DATA GENERATED UNDER K01 SUPPORT, SINGLE-CELL TRANSCRIPTIONAL ANALYSIS OF WILDTYPE, YAP -/- AND YAP-OVEREXPRESSING HECS FROM ZEBRAFISH POINT TO A ROLE FOR YAP IN REGULATING A BATTERY OF SELF-RENEWAL HEMATOPOIETIC TFS, CELL CYCLING AND METABOLIC PROCESSES. IN EXAMINING THESE YAP GAIN- AND LOSS-OF-FUNCTION (GOF/LOF) TRANSCRIPTOMES, GENE MODULE SCORES SUGGEST AN IMPAIRED GLYCOLYSIS-TO-OXIDATIVE PHOSPHORYLATION REWIRING IN HECS. GENES RELATED TO LIPID METABOLISM ARE ALSO DYSREGULATED BY YAP PERTURBATION AND CAN BE IDENTIFIED IN ‘NO FLOW’ DATASETS FROM MOUSE MODELS. THIS R03 APPLICATION WILL INVESTIGATE THE ROLE OF FORCE-DIRECTED LIPID METABOLISM IN DEVELOPMENTAL EHT USING ZEBRAFISH AS A MODEL. WE HYPOTHESIZE THAT HEMODYNAMIC FORCES ALTER LIPID USAGE IN HE TO DRIVE THE METABOLICALLY INTENSIVE PROCESS OF EHT. IN THE FIRST AIM, AN UNBIASED APPROACH OF MASS SPECTROMETRY-BASED LIPIDOMIC PROFILING WILL BE USED TO QUANTIFY THE ABUNDANCE OF LIPID SPECIES IN WILDTYPE AND YAP GAIN OR LOSS OF FUNCTION (GOF/LOF) WHOLE-EMBRYO AND SORTED ENDOTHELIAL CELL POPULATIONS TO DETERMINE THOSE METABOLITES THAT ARE YAP-REGULATED (AS A PROXY FOR A MAJOR CELLULAR TRANSDUCER OF MECHANICAL FORCE). IN THE SECOND AIM A CANDIDATE PATHWAY, THE SECRETED SPHINGOSINE-1-PHOSPHATE LIPID MEDIATOR, WILL BE STUDIED FOR ITS ROLE IN EHT BY LIVE-IMAGING, CHEMICAL PERTURBATION AND STATE-OF-THE-ART GENOME EDITING TECHNOLOGIES TO CREATE TISSUE-SPECIFIC LOF ZEBRAFISH LINES. FINDINGS FROM THIS PROPOSAL WILL UNCOVER FORCE-DRIVEN METABOLIC RESPONSES THAT MIGHT ENHANCE PRODUCTION OF HSCS VIA EHT AND GENERATE CRITICAL PRELIMINARY DATA TO SUPPORT R01 APPLICATIONS.

THE TRANSLATIONAL GLYCOMICS SYMPOSIUM - PROJECT SUMMARY THE 2024 TRANSLATIONAL GLYCOMICS SYMPOSIUM, ORGANIZED BY THE TRANSLATIONAL GLYCOMICS CENTER AT VERSITI BLOOD RESEARCH INSTITUTE IN MILWAUKEE, WISCONSIN, IS SET FOR SEPTEMBER 26-27, 2024. THIS ANNUAL EVENT IS DEDICATED TO ADVANCING TRANSLATIONAL GLYCOBIOLOGY AND NURTURING EMERGING RESEARCHERS. THE SYMPOSIUM'S PRIMARY GOALS ARE TO GIVE TRAINEES AND EARLY-STAGE INVESTIGATORS OPPORTUNITIES TO DEVELOP COLLABORATIVE RESEARCH AND INCREASE THE VISIBILITY OF GLYCOSCIENCE IN BASIC, TRANSLATIONAL, AND CLINICAL DISCIPLINES. AS A COMPLIMENTARY EVENT, IT WELCOMES DIVERSE EXPERTS TO PRESENT THEIR FINDINGS TO THE BROADER GLYCOSCIENCE AND CLINICAL-TRANSLATIONAL COMMUNITY, INCLUDING THOSE AT THE BEGINNING OF THEIR CAREERS. THE SYMPOSIUM COVERS GLYCOBIOLOGY RELATED TOPICS IN AGING, INFLAMMATION, IMMUNOLOGY, CLASSICAL AND MALIGNANT HEMATOLOGY, SOLID CANCER, AND REGENERATIVE MEDICINE, OFFERED IN A HYBRID FORMAT FOR BOTH IN-PERSON AND ONLINE PARTICIPANTS. IT SERVES AS AN ESSENTIAL RESOURCE FOR RESEARCHERS AND CLINICIANS IN GLYCOSCIENCE, OFFERING OPPORTUNITIES FOR NETWORKING, COLLABORATION, AND LEARNING ABOUT THE LATEST DEVELOPMENTS IN THE FIELD. WITH A STRONG COMMITMENT TO SUPPORTING TRAINEES AND EARLY-STAGE INVESTIGATORS, PARTICULARLY FROM UNDERREPRESENTED BACKGROUNDS, THE SYMPOSIUM ENSURES ACCESSIBILITY AND INCLUSIVITY FOR ALL ATTENDEES. THE SPECIFIC AIMS OF THE PROGRAM ARE: 1. EMPOWER TRAINEES AND EARLY-STAGE INVESTIGATORS: PROVIDE A DYNAMIC PLATFORM FOR TRAINEES AND EARLY- STAGE INVESTIGATORS, PARTICULARLY THOSE FROM UNDERREPRESENTED GROUPS, TO PRESENT THEIR RESEARCH, RECEIVE CONSTRUCTIVE FEEDBACK, AND FORGE FUTURE COLLABORATIONS, THEREBY NURTURING THE NEXT GENERATION OF GLYCOSCIENCE RESEARCHERS. 2. FOSTER COLLABORATIVE RESEARCH IN TRANSLATIONAL GLYCOSCIENCE: CREATE OPPORTUNITIES FOR COLLABORATION AMONG GLYCOSCIENCE RESEARCHERS, INTERDISCIPLINARY RESEARCHERS, AND PHYSICIAN-SCIENTISTS TO ENHANCE RESEARCH OUTCOMES AND INNOVATIONS IN THE FIELD. 3. ENHANCE GLYCOSCIENCE VISIBILITY: HIGHLIGHT THE SIGNIFICANCE AND CLINICAL IMPLICATIONS OF GLYCOSCIENCE, ENCOURAGING ITS INTEGRATION INTO BROADER SCIENTIFIC DISCUSSIONS AND ANALYSES. 4. PROMOTE SPECIALIZED TRAINING: ADDRESS THE DEFICIENCY OF GLYCOSCIENCE EXPERTISE IN BIOMEDICAL INSTITUTIONS BY OFFERING SPECIALIZED TRAINING AND EDUCATIONAL RESOURCES. FACILITATE THE INTEGRATION OF CLINICAL TRAINEES INTO GLYCOSCIENCES AND BASIC SCIENCE TRAINEES INTO TRANSLATIONAL MEDICINE, ENSURING THE EFFICIENT TRANSLATION OF NOVEL GLYCO-CONCEPTS FROM LABORATORY RESEARCH TO ENHANCED PATIENT CARE. THROUGH THESE AIMS, THE TRANSLATIONAL GLYCOMICS SYMPOSIUM ASPIRES TO BE A CATALYST FOR GROUNDBREAKING RESEARCH AND TRANSFORMATIVE DEVELOPMENTS IN GLYCOSCIENCE, ULTIMATELY CONTRIBUTING TO IMPROVING PATIENT CARE AND TREATMENT METHODOLOGIES.

BLOOD DYSCRASIAS AND TRANSFUSION MEDICINE