The U2AF1S34F mutation induces lineage-specific splicing alterations in myelodysplastic syndromes
Bon Ham Yip, … , Andrea Pellagatti, Jacqueline Boultwood
Bon Ham Yip, … , Andrea Pellagatti, Jacqueline Boultwood
Published April 24, 2017
Citation Information: J Clin Invest. 2017;127(6):2206-2221. https://doi.org/10.1172/JCI91363.
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Research Article Hematology

The U2AF1S34F mutation induces lineage-specific splicing alterations in myelodysplastic syndromes

Abstract

Mutations of the splicing factor–encoding gene U2AF1 are frequent in the myelodysplastic syndromes (MDS), a myeloid malignancy, and other cancers. Patients with MDS suffer from peripheral blood cytopenias, including anemia, and an increasing percentage of bone marrow myeloblasts. We studied the impact of the common U2AF1S34F mutation on cellular function and mRNA splicing in the main cell lineages affected in MDS. We demonstrated that U2AF1S34F expression in human hematopoietic progenitors impairs erythroid differentiation and skews granulomonocytic differentiation toward granulocytes. RNA sequencing of erythroid and granulomonocytic colonies revealed that U2AF1S34F induced a higher number of cassette exon splicing events in granulomonocytic cells than in erythroid cells. U2AF1S34F altered mRNA splicing of many transcripts that were expressed in both cell types in a lineage-specific manner. In hematopoietic progenitors, the introduction of isoform changes identified in the U2AF1S34F target genes H2AFY, encoding an H2A histone variant, and STRAP, encoding serine/threonine kinase receptor–associated protein, recapitulated phenotypes associated with U2AF1S34F expression in erythroid and granulomonocytic cells, suggesting a causal link. Furthermore, we showed that isoform modulation of H2AFY and STRAP rescues the erythroid differentiation defect in U2AF1S34F MDS cells, suggesting that splicing modulators could be used therapeutically. These data have critical implications for understanding MDS phenotypic heterogeneity and support the development of therapies targeting splicing abnormalities.

Authors

Bon Ham Yip, Violetta Steeples, Emmanouela Repapi, Richard N. Armstrong, Miriam Llorian, Swagata Roy, Jacqueline Shaw, Hamid Dolatshad, Stephen Taylor, Amit Verma, Matthias Bartenstein, Paresh Vyas, Nicholas C.P. Cross, Luca Malcovati, Mario Cazzola, Eva Hellström-Lindberg, Seishi Ogawa, Christopher W.J. Smith, Andrea Pellagatti, Jacqueline Boultwood

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Figure 2

Expression of U2AF1S34F skews granulomonocytic differentiation toward granulocytes.

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Expression of U2AF1S34F skews granulomonocytic differentiation toward gr...
(A) Expression levels of U2AF1S34F and U2AF1WT protein in transduced granulomonocytic cells on day 11. Anti-U2AF1 and anti-FLAG antibodies were used to measure total U2AF1 protein and exogenous U2AF1S34F or U2AF1WT protein produced by the vector, respectively. (B) Median fluorescence intensity (MFI) of forward scatter (an indication of cell size) of granulomonocytic cells on days 11 and 14. (C) Percentage of CD11b+ cells in granulomonocytic cultures on days 11 and 14. (D) Cell counts for U2AF1S34F granulomonocytic cells from day 8 (the day when Geneticin selection was complete) to day 14 compared with EV and U2AF1WT controls. (E) Cell-cycle analysis of granulomonocytic cells on day 11. (F) Percentage of CD11b+ cells in granulomonocytic cultures on day 20. (G and H) Percentages of (G) CD14+CD15– monocytic cells and (H) CD14–CD15+ granulocytic cells in granulomonocytic cultures on day 20. (I) Representative flow cytometric plots on day 20 (n = 7). (J) Representative images of May-Grünwald-Giemsa–stained granulomonocytic cells on day 20 (n = 7). The red arrows indicate eosinophils. Scale bars: 25 μm. (K) Percentage of eosinophils per 100 cells on day 20. (L) Number of CFU granulocytes-macrophages (CFU-GM), CFU granulocytes (CFU-G), and CFU macrophages (CFU-M) obtained from hematopoietic CD34+ progenitors transduced with EV, U2AF1WT, or U2AF1S34F after 14 days in methylcellulose. Results shown in panels B–H, K, and L were obtained from 6, 8, 7, 6, 7, 7, 7, 6, and 7 independent experiments, respectively. Data represent the mean ± SEM. P values in panels B, C, E–H, K and L were calculated by repeated-measures 1-way ANOVA with Tukey’s post-hoc test. P values in panel D were calculated by 2-way ANOVA with Bonferroni’s post test. *P < 0.05, **P < 0.01, and ***P < 0.001.