ADVANCED MOUSE MODELS OF DDX41-MUTATED MYELODYSPLASTIC SYNDROMES - PROJECT SUMMARY MYELODYSPLASTIC SYNDROMES (MDS) ARE BLOOD DISORDERS CAUSED BY DEFECTIVE HEMATOPOIETIC STEM CELLS (HSC) THAT CLONALLY EXPAND AND FAIL TO PRODUCE BLOOD CELLS OF SUFFICIENT QUALITY AND QUANTITY. GERMLINE HETEROZYGOUS MUTATIONS IN DDX41, AN ESSENTIAL RNA HELICASE, ARE THE MOST COMMON CAUSE OF INHERITED PREDISPOSITION TO MDS. THESE MUTATIONS ARE TYPICALLY FRAMESHIFTS AND CAUSE LOSS OF FULL-LENGTH PROTEIN. PATIENTS WITH THESE MUTATIONS HAVE NORMAL HEALTH INTO ADULTHOOD BUT HAVE AN ELEVATED RISK OF DEVELOPING MDS WITH A MEDIAN AGE OF 69 YEARS. THE CELLULAR AND MOLECULAR MECHANISMS BY WHICH THESE MUTATIONS CONTRIBUTE TO MDS PATHOGENESIS REMAIN POORLY DEFINED. THE MOST COMMON CO-MUTATION IN THESE PATIENTS IS ACQUIRED MISSENSE MUTATION OF THE OTHER DDX41 ALLELE, OFTEN CAUSING THE AMINO ACID SUBSTITUTION R525H. WE DEVELOPED TWO CONDITIONAL MUTANT DDX41 ALLELES TO MODEL THE GERMLINE LOSS-OF-EXPRESSION MUTATION AND THE ACQUIRED R525H MUTATION IN MICE. WE FOUND THAT MICE WITH HEMATOPOIETIC-SPECIFIC, HETEROZYGOUS LOSS OF DDX41 LIVE NORMAL LENGTH LIVES AND HAVE PREDOMINANTLY NORMAL HEMATOPOIESIS WITH ONLY A MODEST REDUCTION IN RED BLOOD CELL NUMBER. IN CONTRAST, THE COMBINATION OF ONE LOSS- OF-EXPRESSION ALLELE AND THE R525H-MUTANT ALLELE CAUSES PROFOUND CELL CYCLE ARREST AND APOPTOSIS IN PROLIFERATIVE HEMATOPOIETIC PROGENITOR CELLS. THIS OBSERVATION CALLS INTO QUESTION WHY THE R525H MUTATION ARISES AND HOW THE MUTANT CLONES THAT ACQUIRE IT CAN EXPAND TO CONTRIBUTE TO DISEASE. IN PATIENTS, THE ACQUIRED MUTATION HAS A MEDIAN VARIANT ALLELE FREQUENCY OF JUST 10%, INDICATING IT OCCURS IN A NON-DOMINANT CLONE, CONSISTENT WITH REDUCED PROLIFERATIVE CAPACITY. IN THIS PROJECT, WE PLAN TO DEVELOP MODELS OF DDX41-MUTATED MDS THAT ACCOUNT FOR THE CELLULAR AND TEMPORAL CONTEXT OF MUTATION ACQUISITION, INCLUDING DDX41-HETEROZYGOSITY IN HEMATOPOIETIC AND NON- HEMATOPOIETIC CELLS DURING NATURAL AGING AND THE ACQUISITION OF THE R525H MUTATION IN ONLY A SUBSET OF HSC AT AN ADVANCED AGE. TO DO THIS, WE WILL AGE LARGE COHORTS OF CONSTITUTIVE DDX41+/- MICE AND DDX41+/FLOX;VAV-CRE (HEMATOPOIETIC-SPECIFIC) MICE TO AT LEAST 24 MONTHS OF AGE. WE WILL ALSO CROSS THESE MODELS WITH A GENETIC MODEL OF ACCELERATED AGING TO DRIVE MORE RAPID DISEASE DEVELOPMENT. FINALLY, WE WILL INDUCE EXPRESSION OF THE R525H MUTATION IN A SUBSET OF HSC IN THE CONTEXT OF DDX41+/- BONE MARROW TO MIMIC THE MIXED CLONALITY OF MDS PATIENT BONE MARROW. WE HYPOTHESIZE THAT THESE GENETIC MODELS, WHICH ACCURATELY FOLLOW THE NATURAL PROGRESSION OF THE DISEASE IN VIVO, WILL YIELD MDS-LIKE DISEASE STATES THAT CAN BE FURTHER STUDIED TO ELUCIDATE THE CELLULAR AND MOLECULAR MECHANISMS OF MDS PATHOGENESIS. THE ULTIMATE GOAL OF THESE STUDIES IS TO FULLY UNDERSTAND THE CAUSE OF MDS PREDISPOSITION IN PATIENTS WITH DDX41 MUTATIONS SUCH THAT WE CAN RATIONALLY DESIGN STRATEGIES TO PREVENT OR TREAT THE DISEASE.