Simultaneous Sessions I: Molecular Implications in MDS
Splicing factor (SF) mutations are characteristic of myelodysplastic syndromes (MDS) and related myeloid neoplasms and implicated in their pathogenesis. Among these, U2AF1 mutations are more prevalent in AML with myelodysplasia-related changes and are associated with a poor prognosis. U2AF1 mutations exclusively involved two highly conserved amino acid positions (S34 or Q157) within the two zinc finger motifs flanking the U2AF homology motif domain. The molecular mechanism by which U2AF1 mutations lead to myelodysplasia remains largely unknown.
This study aimed to clarify the molecular pathogenesis of SF-mutated myelodysplasia through the analysis of U2af1 S34F conditional knock-in mice.
We first generated a heterozygous conditional knock-in mouse model of U2af1 S34F mutation. We then investigated the hematological phenotype of these mice with RNA sequencing of hematopoietic cells, to clarify the molecular mechanism underlying the phenotypes.
Vav1-Cre mediated U2af1 S34F knock-in mice exhibited macrocytic anemia and leukopenia at 8-20 weeks after birth, which was not observed in controls. Flow cytometry of U2af1 S34F bone marrow (BM) cells showed a significant decrease in the number of hematopoietic stem and progenitor cells (HSPCs), including Kit+Sca-1+Linlow cells (KSLs), common myeloid progenitors (CMPs) and megakaryocyte/erythrocyte lineage-restricted progenitors (MEPs), and an increase of the granulocyte/macrophage lineage-restricted progenitors (GMPs) compared to controls. In non-competitive BM transplantation settings, multilineage cytopenia and dysplasia, accompanied by strong myeloid skewing were observed in the mutant mice. Surprisingly, all of the U2af1 mutant-transplanted mice died within two months after transplantation due to severe bone marrow failure. These observations suggest that the U2af1 mutation leads to ineffective hematopoiesis and morphological abnormalities, which recapitulate, at least partly, the phenotype of MDS in transplantation settings.
In competitive BM transplantation experiments, the donor chimerism of U2af1 S34F-derived cells in the peripheral blood was remarkably reduced compared to that of wild-type cells, suggesting the impaired reconstitution capacity of U2af1 mutant stem cells.
RNA sequencing analysis of KSLs and CMPs from the mutant mice showed significant changes in alternative splicing, involving those genes implicated in the pathogenesis of hematopoietic malignancies, such as Bcor, Gnas, Csf3r, and Hnrnpa2b1. Most commonly observed abnormal splicing events were enhanced inclusion or exclusion of cassette exons. Interestingly, there was a significant overlap between genes abnormally spliced in U2af1 S34F HSPCs and genes we reported to be aberrantly spliced in Srsf2 P95H HSPCs (Kon et al., Blood 2018). Gene set enrichment analysis revealed a significant enrichment of several pathways, including enhanced DNA repair, cell cycle and RNA processing, as well as, downregulation of the inflammatory response genes. Further, the gene sets significantly upregulated or downregulated in Srsf2 P95H HSPCs were significantly upregulated or downregulated in U2af1 S34F HSPCs, respectively.
Our results demonstrated that U2af1 S34F mutation led to impaired HSC functions that was evident from reduced competitive repopulation and deregulated hematopoietic differentiation, which were augmented in transplantation settings. Transcriptional analysis suggested that U2af1 S34F and Srsf2 P95H mutations induce similar transcriptional changes and have several common targets of abnormal splicing.