Project Grant F31HL188855

STRUCTURE-FUNCTION ANALYSIS OF DDX41'S ROLE IN HEMATOPOIESIS - PROJECT SUMMARY/ABSTRACT MYELODYSPLASTIC SYNDROMES (MDS) ARE GENETIC DISORDERS OF THE BONE MARROW CHARACTERIZED BY INEFFECTIVE HEMATOPOIESIS AND AN INCREASED RISK OF PROGRESSION TO ACUTE MYELOID LEUKEMIA. MDS IS ASSOCIATED WITH DYSREGULATED INFLAMMATORY SIGNALING, WHICH CONTRIBUTES TO HEMATOPOIETIC STEM CELL EXHAUSTION. GERMLINE MUTATIONS IN DDX41 ARE THE MOST PREVALENT CAUSE OF INHERITED PREDISPOSITION TO MDS, AND MANY PATIENTS ACQUIRE A SECOND-HIT DDX41 MUTATION PREDICTED TO CAUSE LOSS OR REDUCED PROTEIN FUNCTION. HOWEVER, THE MOLECULAR MECHANISM BY WHICH DDX41 DEFICIENCY PROMOTES DISEASE IS POORLY UNDERSTOOD. DDX41 HAS BEEN IMPLICATED IN DIVERSE FUNCTIONS, INCLUDING ROLES IN INNATE IMMUNE SENSING AND RNA SPLICING, YET THE MOLECULAR MECHANISMS OF THESE VARIED FUNCTIONS REMAIN POORLY DEFINED. THE WELL-CONSERVED CORE DOMAINS OF DDX HELICASES FUNCTION IN THE BINDING AND ATPASE-DEPENDENT UNWINDING OF NUCLEIC ACID SUBSTRATES WHEREAS THE VARIABLE N- AND C-TERMINAL DOMAINS ARE THOUGHT TO CONFER PARALOG-SPECIFIC ACTIVITIES. THE FUNCTIONS OF THESE DOMAINS IN DDX41 ARE LARGELY UNKNOWN. THE MAJOR STRUCTURAL FEATURE OF THE DDX41 N-TERMINAL DOMAIN (NTD) IS A COILED COIL (CC). PRELIMINARY FINDINGS DEMONSTRATE THAT THE CC IS ESSENTIAL FOR PROTEIN FUNCTION. CC DOMAINS TYPICALLY FACILITATE PROTEIN-PROTEIN INTERACTIONS (PPI), AND HELICASE ENZYMES ARE KNOWN TO FUNCTION AS DIMERS FOR ENHANCED NUCLEIC ACID RECOGNITION AND UNWINDING. SUPPORTIVE OF THIS, PPI ASSAYS SUGGEST DDX41 CAN FORM HOMODIMERS, AND MOLECULAR MODELING SUGGESTS THE CC IS CRITICAL FOR THIS INTERACTION. RECURRENT MUTATIONS IN THE CC HAVE BEEN IDENTIFIED IN MDS PATIENTS, INCLUDING SOMATIC P.L87F, SUGGESTING CC DISRUPTION CONTRIBUTES TO DISEASE, YET ITS FUNCTIONAL IMPACT IS UNKNOWN. THE PROPOSED STUDIES WILL DETERMINE THE DEPENDENCE ON THE CC DOMAIN FOR DDX41 DIMER FORMATION AND DEFINE HOW CC DOMAIN LOSS IMPAIRS DDX41'S ABILITY TO BIND AND UNWIND NUCLEIC ACID SUBSTRATES (AIM 1). THE FUNCTIONAL IMPACT OF DISEASE-ASSOCIATED CC DOMAIN MUTATIONS ON HEMATOPOIESIS AND THE MECHANISM BY WHICH BIALLELIC DDX41 MUTATIONS PROMOTE A HYPERINFLAMMORY ENVIRONMENT WILL BE EXPLORED IN NOVEL MOUSE MODELS (AIM 2). THE OVERALL GOAL OF THIS STUDY IS TO DETERMINE THE FUNCTIONAL CONTRIBUTION OF ESSENTIAL DDX41 DOMAINS AND DEFINE HOW DISRUPTION OF THESE DOMAINS CONTRIBUTES TO MYELODYSPLASTIC SYNDROME (MDS) PATHOGENESIS. THIS PROJECT WILL ADVANCE SCIENTIFIC UNDERSTANDING BY ELUCIDATING THE MOLECULAR DETAILS OF DDX41 FUNCTION, A CRITICAL SUPPRESSOR OF MDS. BY DETERMINING THE PRECISE ACTIVITY OF THE CC AND REVEALING HOW THIS UNIQUE STRUCTURAL FEATURE IMPARTS THE SPECIFIC FUNCTIONS OF DDX41, THIS STUDY WILL CLARIFY ESSENTIAL MECHANISMS OF DDX41 THAT REMAIN POORLY DEFINED. THE ANALYSIS OF IN VIVO MODELS WILL REVEAL HOW BIALLELIC DDX41 MUTATIONS ALTER HSC HOMEOSTASIS AND THE ACTIVATION OF INFLAMMATORY SIGNALING IN HSC AND INNATE IMMUNE EFFECTOR CELLS. OVERALL, COMPLETION OF THE PROPOSED STUDY WILL ELUCIDATE DDX41-SPECIFIC PATHOGENIC MECHANISMS THAT WILL INFORM THE DEVELOPMENT OF DISEASE PREVENTION OR TREATMENT STRATEGIES, CONTRIBUTING TO IMPROVED CLINICAL OUTCOMES FOR HEMATOLOGIC MALIGNANCY PATIENTS.