Crosstalk of Splicing and Signaling in HSPC fate choices

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Crosstalk of Splicing and Signaling during HSPC Fate Choices Project Abstract Myelodysplastic syndrome (MDS) is a spectrum of disorders arising from hematopoietic stem and progenitor cell (HSPC) dysfunction resulting in ineffective hematopoiesis and cytopenias. Spliceosomal components are mutated in greater than 60% of all MDS cases, yet it remains a puzzle how dysfunction in general splicing factors can mediate the specific outcomes observed in MDS. The slow progress comes, in part, due to our poor understanding of splicing regulation of HSPC fate choices. Indeed, as RNA splicing is mostly studied in yeast and cell culture, tissue-specific regulation of splicing in vivo is largely unexplored. Here, using HSPC development as a paradigm, we seek to illuminate how the molecular regulation of RNA splicing impacts HSPC fate choices. By understanding this fundamental process, we strive to identify potentially novel ways to target splicing factor-defective HSPCs in MDS. We recently reported a severe HSPC formation defect in zebrafish loss-of-function mutants for the spliceosomal component, splicing factor 3b, subunit 1 (sf3b1), which is the most commonly mutated splicing factor in MDS. We uncovered that Sf3b1 regulates stat3 (signal transducer and activator of transcription 3) pre-mRNA splicing and demonstrated that mis-splicing of stat3 results in diminished pathway activation. We determined that overexpression of an active form of Stat3 can suppress the HSPC defects in sf3b1 mutants, demonstrating that Sf3b1 regulation of STAT3 signaling is important for HSPC formation. In preliminary studies, we determined that STAT3 inhibition serves as a conserved synthetic lethality with SF3B1 heterozygosity in both zebrafish and human MDS HSPCs. Based on these findings, we performed a chemical modifier screen in sf3b1 mutant zebrafish and identified other Sf3b1- regulated signaling pathways critical for HSPCs. These studies establish zebrafish as an excellent model for discovery of HSPC splicing regulation and novel MDS vulnerabilities. Here, we will test the hypothesis that the splicing factor Sf3b1 is a key director of the choice of splice isoforms for signaling pathway components essential for HSPC formation. We will use zebrafish genetic and screening capabilities coupled with studies in human MDS cells to explore the role of specific splice isoforms on HSPC fate decisions, determine the function of cis-regulatory sequences on Sf3b1-regulated splicing choices in vivo, and identify novel vulnerabilities for SF3B1-mutated HSPCs. Accomplishing these aims will inform our basic understanding of the poorly understood role of splicing in HSPC fate decisions and lead to the identification of novel approaches for selective targeting of SF3B1-mutated MDS HSPCs.