U2AF1-S34F and U2AF1-Q157R induce distinct RNA splicing and hematopoietic phenotypes in vivo

However, each mutant protein predominantly affects a distinct set of alternatively spliced downstream target genes suggesting that global mRNA splicing alterations, rather than specific splicing targets, may be responsible for MDS phenotypes, including dysplasia, ineffective hematopoiesis, and cytopenias. U2AF1 provides a unique opportunity to address this question because it has two hotspot positions (serine 34 [S34] and glutamine 157 [Q157]) that are each commonly mutated in MDS and are associated with unique mRNA splicing consequences. In addition, U2AF1 S34 and Q157 codon mutations co-occur with mutations in different genes (e.g., BCOR and ASXL1, respectively) and patients with these mutations may have different hematopoietic phenotypes—highlighting that these mutations may induce distinct phenotypes. We asked if the splicing differences resulting from S34F and Q157R mutations were thus associated with different or similar effects on hematopoiesis. To do so, we engineered a new Cre/lox conditional mouse model with the Q157R mutation knocked-in to the endogenous U2af1 locus based on our established conditional S34F knock-in mouse model and utilized both models to directly study the hematopoietic phenotype, transcriptional, and mRNA splicing consequences of individual U2AF1 gene mutations in vivo. Our results indicate that the two mutations induce distinct hematopoietic phenotypes in mice, suggesting that the U2af1S34F and U2af1Q157R mutations should not be conflated as they may impact disease pathogenesis differently in patients. Mice expressing U2af1S34F have a more severe reduction in their PB and BM cell counts, and reduced HSPCs repopulating ability, compared to mice expressing U2af1Q157R. The expression and splicing of the majority of target genes are unique between the mutations, in both mouse and human samples, potentially driving the phenotypic differences induced by the two mutations. The two mutations co-occur with different gene mutations and are not equally represented in various myeloid neoplasms, suggesting that multiple mechanisms are likely to drive the pathogenesis of U2AF1 mutant myeloid diseases. Collectively, our results support that U2AF1S34F and U2AF1Q157R mutations induce distinct hematopoietic, gene expression, and RNA splicing phenotypes in vivo, suggesting that larger prospective studies are needed to understand if patients with these mutations should be classified differently.