SF3B1-mutant mis-splicing ofUBA1confers a targetable therapeutic vulnerability through UBA1 inhibition

Abstract SF3B1 mutation-driven myelodysplastic syndromes (MDS- SF3B1 ) arise due to somatic mutation in the splicing factor SF3B1 gene. SF3B1 mutations induce RNA mis-splicing and loss of expression of critical genes for erythropoiesis, leading to erythroid dysplasia and ultimately refractory anemia. The development of precision medicine approaches for MDS- SF3B1 is hampered by the complexity of the mis-splicing landscape and its evaluation in disease-accurate model systems. To identify novel RNA mis-splicing events, isogenic SF3B1 K700E and SF3B1 WT iPSC lines from an MDS- SF3B1 patient were differentiated into hematopoietic cells in vitro and subjected to unsupervised splicing event analysis using full-length RNA sequencing data. This revealed SF3B1 K700E -specific mis-splicing of ubiquitin-like modifier activating enzyme 1 ( UBA1 ) transcripts, which encode the essential E1 protein at the apex of the ubiquitination cascade. UBA1 mis-splicing ( UBA1 ms ) preserved UBA1 ms mRNA but not protein expression. Consequently, UBA1 ms diminished the pool of functional UBA1, sensitizing SF3B1 K700E cell lines to the small-molecule UBA1 inhibitor TAK-243. Finally, analysis of CD34 + RNA sequencing data from an MDS patient cohort confirmed unique and ubiquitous UBA1 ms in MDS- SF3B1 patients, without detection in other splicing factor-mutated MDS patients, or in healthy individuals. TAK-243 selectively targeted MDS- SF3B1 primary CD34 + cells and reduced mutant cell number in colony-forming unit studies. In contrast, normal hematopoietic progenitor cells were unaffected. Altogether, we here define UBA1 ms as a novel therapeutic vulnerability in SF3B1 -mutant cells, introducing UBA1 inhibition as a potential avenue for future MDS- SF3B1 treatments.