PROJECT SUMMARY/ABSTRACT Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, peripheral blood cytopenias and increased risk of progression to acute leukemia. Therapeutic options and development of new drugs for patients with MDS are currently very limited and there is a great need for new therapeutic targets. A major recent discovery from large-scale sequencing studies was that over half of MDS patients harbor mutations in genes encoding splicing factors (SFs), with the 3 most commonly mutated being: splicing factor 3B, subunit 1 (SF3B1), serine/arginine-rich splicing factor 2 (SRSF2) and U2 small nuclear RNA auxiliary factor 1 (U2AF1). SF mutations are the most common class of mutations in MDS and occur early in the course of the disease. These strongly suggest that SF mutations are key to the pathogenesis of MDS and can likely provide new therapeutic opportunities. However the mechanisms by which they drive the disease are not understood. These mutations are heterozygous “hotspot” mutations, which strongly suggests a gain or alteration of function mechanism – corroborated by recent biochemical studies showing altered RNA binding specificities of the mutant SFs. Their mutual exclusivity provides yet another clue, as it suggests convergence in one or a few downstream targets. Identifying those targets could have tremendous implications for MDS, but presents a big challenge due to the cellular and genetic heterogeneity of primary patient samples and differences in gene isoforms among species. We (Papapetrou laboratory) have developed the first induced pluripotent stem cell (iPSC) models of MDS and provided proof-of-principle of their use for studying genetic mechanisms of the disease. For the current proposal, we have derived isogenic iPSC lines with the SRSF2 P95L mutation and shown that hematopoietic cells derived from them capture disease-relevant phenotypes and the altered RNA-binding affinity of mutant SRSF2. We (Yeo laboratory) have developed an enhanced CLIP-seq method (eCLIP) and used it to characterize for the first time in preliminary experiments the direct RNA binding of mutant SRSF2. In this multiple PI application we will join forces to identify common downstream effects of SF mutations that may constitute promising therapeutic targets. The proposed studies, which leverage the unique expertise of the Papapetrou lab in iPSC modeling of MDS, combined with the extensive expertise of the Yeo lab in RNA biology and genomics, will generate new insights into the pathogenesis of MDS with SF mutations and identify new therapeutic targets for drug development.