Integrative RNA-omics discovers GNAS alternative splicing as a phenotypic driver of splicing factor mutant neoplasms

Mutations in splicing factors (SFs) are the predominant class of mutations in myelodysplastic syndrome (MDS), but convergent downstream disease drivers remain elusive. To identify common direct targets of mis-splicing by mutant U2AF1 and SRSF2, we performed RNA-Seq and eCLIP in human hematopoietic stem/progenitor cells (HSPCs) derived from isogenic induced pluripotent stem cell (iPSC) models. Integrative analyses of alternative splicing and differential binding converged on a long isoform of GNAS (GNAS-L), promoted by both mutant factors. MDS population genetics, functional and biochemical analyses support that GNAS-L is a driver of MDS and encodes a hyperactive long form of the stimulatory G protein alpha subunit, Gas-L, that activates ERK/MAPK signaling. SF-mutant MDS cells have activated ERK signaling and consequently are sensitive to MEK inhibitors. Our findings highlight an unexpected and unifying mechanism by which SF mutations drive oncogenesis with potential therapeutic implications for MDS and other SF-mutant neoplasms. Overall design: Isogenic iPSCs derived with CRISPR genome editing to insert heterozygous point mutations in SRSF2 and U2AF1 and insertion of a FLAG tag a the endogeneous were differentiated into heatopoietic stem and progenitor cells (HSPCs). eCLIP-Seq of each splicing factor and RNA-Seq were performed on the HSPCs.