Genetic and molecular basis for SRSF2 mutations in myelodysplasia

Fred Hutchinson Cancer Center, Seattle WA

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Abstract

Mutations in genes encoding RNA splicing factors are the single most common class of genetic alterations in patients with myelodysplastic syndromes (MDS). Recurrent mutations affecting SF3B1, SRSF2, and U2AF1 are the most common and occur as heterozygous point mutations at specific amino acid residues. In the first two periods of funding of this award, we identified that mutations in SRSF2 confer an alteration of function distinct from loss-of-function, discovered the molecular impacts of mutations in SRSF2 on RNA splicing and binding, and identified potential therapeutic vulnerabilities of SRSF2-mutant cells. Much of our prior work focused on identification of novel, unannotated RNA splicing events created by mutations in SRSF2. In this proposal, we will leverage this knowledge to develop two novel approaches to understand the mechanistic basis of and develop new therapies for SRSF2-mutant MDS. Firstly, we will utilize our recently developed technology – termed synthetic introns – to harness the neomorphic change in splicing created by mutations in RNA splicing factors to drive selective gene expression in SRSF2-mutant cells. These synthetic RNA sequences are efficiently spliced in cells bearing mutant SRSF2, but unspliced in wild-type cells, allowing for mutation-dependent protein production only in SRSF2-mutant cells. These synthetic introns will be utilized to generate a comprehensive map of the RNA regulatory environment that modulates mis-splicing in SRSF2-mutant MDS. This will include assessment of cis elements in mRNA as well as identification of specific trans-acting factors that are required for mutant SRSF2-dependent mis-splicing. Additionally, the development of optimized SRSF2 mutation-responsive synthetic introns here will enable future therapeutic approaches reliant upon selective protein expression in SRSF2-mutant cells. In addition to the above, we will develop novel immunotherapeutic approaches that harness the stereotypical patterns of mis-splicing that characterize SRSF2-mutant MDS. Our prior work demonstrates that mutations in SRSF2 generate novel, unannotated mRNAs which are not otherwise created in normal tissues. We have therefore used these splicing changes to identify novel antigens arising from mutant SRSF2- dependent mis-splicing which are presented by MHC class I on the mutant cell surface as well as identify corresponding antigen-specific CD8+ T cells in MDS patients and characterize their phenotypes and T cell receptors (TCRs). We now aim to comprehensively identify and functionally characterize endogenous antigen- specific CD8+ T cells in SRSF2-mutant MDS patients and test the therapeutic potential of T cells engineered to express a panel of patient-derived, SRSF2-mutant peptide-specific TCRs. Ultimately, completing these aims will increase our mechanistic understanding of how MDS-associated mutations in SRSF2 result in mis-splicing to drive MDS development as well as develop mechanism-based therapeutic approaches that are highly selective for SRSF2-mutant cells.