Biological Characterization of the U2af1 S34F Mutation in the Pathogenesis of Myelodysplasia
Authors: Kon Ayana , Nannya Yasuhito , Nakagawa Masahiro , Kataoka Keisuke , Sanada Masashi , Nakayama Manabu , Koseki Haruhiko , Ogawa Seishi 
Published: October 2019

Recent genetic studies have revealed frequent and specific pathway mutations involving multiple components of the RNA splicing machinery in myelodysplasia. Among these, U2AF1 mutations are more prevalent in MDS without increased ring sideroblasts and AML with myelodysplasia-related changes and are associated with a poor prognosis. Also found in approximately 4% of lung adenocarcinoma, U2AF1 mutations exclusively involved two highly conserved amino acid positions (S34 or Q157) within the amino- and the carboxyl-terminal zinc finger motifs flanking the U2AF homology motif (UHM) domain. Comprehensive analysis in a large cohort of MDS showed that U2AF1 mutations showed a significant trend to coexist with ASXL1. The molecular mechanism by which U2AF1 mutations lead to myelodysplasia have not fully been elucidated.

To elucidate the role of U2AF1 mutations in the development of myelodysplasia, we generated heterozygous conditional knock-in mice for the U2af1 S34F mutation, which were crossed them with Vav1-Cre transgenic mice. Vav1-Cre mediated U2af1 S34F knock-in mice exhibited severe leukopenia and macrocytic anemia at 8-20 weeks after birth. Although there was no significant difference in blood cell morphology between wild-type and mutant mice in bone marrow (BM) and peripheral blood (PB) cells, there was strong myeloid skewing in lineage composition both in U2af1 mutant BM and PB cells compared to wild-type controls. Flow cytometry of U2af1 S34F BM cells showed a significant decrease in the number of hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs) and megakaryocyte/erythrocyte lineage-restricted progenitors (MEPs), and an increase of the granulocyte/macrophage lineage-restricted progenitors (GMPs) compared to wild-type BM cells. These observations suggest that heterozygous U2af1 mutation leads to differentiation defects of HSCs, which however, is not sufficient for the development of MDS.

We next assessed the phenotype of U2af1-mutated BM cells in transplantation settings to evaluate the effect of increasing replicative stress, which has been shown to substantially affect the behavior of normal and abnormal stem cells. In PB, progressive leukopenia, macrocytic anemia and decreased platelet counts were observed in mutant mice in transplantation settings. Surprisingly, all of the U2af1 mutant-transplanted mice died within two months after transplantation due to severe bone marrow failure. Cytological analysis of BM cells revealed morphological abnormalities in U2af1 mutant-transplanted mice, including hypersegmentation in neutrophils and erythroid dysplasia. Flow cytometrical analysis revealed decreased numbers of HSCs, CMPs and MEPs, and increased number of GMPs. These observations suggest that the U2af1 mutation leads to ineffective hematopoiesis and morphological abnormalities, which seems to recapitulate the phenotype of MDS in transplantation settings.

Subsequently, we assessed the reconstitution capacity of whole BM cells from U2af1 mutant mice in competitive transplantation experiments. The donor chimerism of U2af1 S34F-derived cells in PB was remarkably reduced compared to that of wild-type cells. At four months post transplantation, the chimerism of U2af1 S34F-derived cells was markedly lower than that of wild-type cells in the fractions of HSCs, CMPs, MEPs, GMPs and common lymphoid progenitors (CLPs) in BM.

RNA sequencing analysis of HSCs defined as Kit+Sca-1+Linlow (KSL) cells and CMPs from the mutant mice showed significant changes in alternative splicing and expression levels in many genes, including several potential targets implicated in the pathogenesis of hematopoietic malignancies.

In summary, our results demonstrated that heterozygous 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. Our mice model provides a valuable tool to understand the molecular pathogenesis of U2af1-mutated myeloid neoplasms.

Disclosures

Nakagawa: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding.