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NORMAL HEMATOPOIESIS

U2af1 is required for survival and function of hematopoietic stem/progenitor cells

Leukemia volume 35, pages 2382–2398 (2021)Cite this article

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Abstract

U2AF1 is involved in the recognition of the 3′ splice site during pre-mRNA splicing. Mutations in U2AF1 are frequently observed in myelodysplastic syndromes. However, the role of wild-type U2AF1 in normal hematopoiesis has remained elusive. Using a novel conditional U2af1 knockout allele, we have found that deletion of U2af1 results in profound defects in hematopoiesis characterized by pancytopenia, ablation of hematopoietic stem/progenitor cells (HSPC) leading to bone marrow failure and early lethality in mice. U2af1 deletion impairs HSPC function and repopulation capacity. U2af1 deletion also causes increased DNA damage and reduced survival in hematopoietic progenitors. RNA sequencing analysis reveals significant alterations in the expression of genes related to HSC maintenance, cell proliferation, and DNA damage response-related pathways in U2af1-deficient HSPC. U2af1 deficiency also induces splicing alterations in genes important for HSPC function. This includes altered splicing and perturbed expression of Nfya and Pbx1 transcription factors in U2af1-deficient HSPC. Collectively, these results suggest an important role for U2af1 in the maintenance and function of HSPC in normal hematopoiesis. A better understanding of the normal function of U2AF1 in hematopoiesis is important for development of appropriate therapeutic approaches for U2AF1 mutant induced hematologic malignancies.

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Fig. 1: Conditional deletion of U2af1 in mouse hematopoietic compartments results in fatal BM failure.
Fig. 2: Effects of U2af1 deletion on hematopoietic stem/progenitor cells.
Fig. 3: BM failure observed in U2af1 deficient mice is cell autonomous.
Fig. 4: Defective stem cell function in U2af1-deficient mice.
Fig. 5: Deletion of U2af1 leads to DNA damage and apoptosis in hematopoietic cells.
Fig. 6: Effect of U2af1 deletion on gene expression profile in HSPC.
Fig. 7: Effect of U2af1 deletion on RNA splicing.

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Data availability

The RNA-Sequencing datasets generated in this study are deposited to NCBI GEO database (GSE162888).

References

  1. Yoshida K, Sanada M, Shiraishi Y, Nowak D, Nagata Y, Yamamoto R, et al. Frequent Pathway Mutations of Splicing Machinery in Myelodysplasia. Nature. 2011;478:64–69.

    Article  CAS  Google Scholar 

  2. Papaemmanuil E, Cazzola M, Boultwood J, Malcovati L, Vyas P, Bowen D, et al. Somatic SF3B1 Mutation in Myelodysplasia With Ring Sideroblasts. N Engl J Med. 2011;365:1384–95.

    Article  CAS  Google Scholar 

  3. Graubert TA, Shen D, Ding L, Okeyo-Owuor T, Lunn CL, Shao J, et al. Recurrent mutations in the U2AF1 splicing factor in myelodysplastic syndromes. Nat Genet. 2012;44:53–57.

    Article  CAS  Google Scholar 

  4. Thol F, Kade S, Schlarmann C, Löffeld P, Morgan M, Krauter J, et al. Frequency and Prognostic Impact of Mutations in SRSF2, U2AF1, and ZRSR2 in Patients With Myelodysplastic Syndromes. Blood. 2012;119:3578–84.

    Article  CAS  Google Scholar 

  5. Damm F, Kosmider O, Gelsi-Boyer V, Renneville A, Carbuccia N, Hidalgo-Curtis C, et al. Groupe Francophone des Myélodysplasies., Mutations Affecting mRNA Splicing Define Distinct Clinical Phenotypes and Correlate With Patient Outcome in Myelodysplastic Syndromes. Blood. 2012;119:3211–18.

    Article  CAS  Google Scholar 

  6. Ogawa S. Genetics of MDS. Blood. 2019;133:1049–59.

    Article  CAS  Google Scholar 

  7. Inoue D, Bradley R, Abdel-Wahab O. Spliceosomal Gene Mutations in Myelodysplasia: molecular Links to Clonal Abnormalities of Hematopoiesis. Genes Dev. 2016;30:989–1001.

    Article  CAS  Google Scholar 

  8. Wu S, Romfo CM, Nilsen TW, Green MR. Functional Recognition of the 3’ Splice Site AG by the Splicing Factor U2AF35. Nature. 1999;402:832–35.

    Article  CAS  Google Scholar 

  9. Makishima H, Visconte V, Sakaguchi H, Jankowska AM, Abu Kar S, Jerez A, et al. Mutations in the spliceosome machinery, a novel and ubiquitous pathway in leukemogenesis. Blood. 2012;119:3203–10.

    Article  CAS  Google Scholar 

  10. Przychodzen B, Jerez A, Guinta K, Sekeres MA, Padgett R, Maciejewski JP, et al. Patterns of Missplicing Due to Somatic U2AF1 Mutations in Myeloid Neoplasms. Blood. 2013;122:999–1006.

    Article  CAS  Google Scholar 

  11. Ilagan JO, Ramakrishnan A, Hayes B, Murphy ME, Zebari AS, Bradley P, et al. U2AF1 Mutations Alter Splice Site Recognition in Hematological Malignancies. Genome Res. 2015;25:14–26.

    Article  CAS  Google Scholar 

  12. Brooks AN, Choi P, de Waal L, Sharifnia T, Imielinski M, Saksena G, et al. A Pan-Cancer Analysis of Transcriptome Changes Associated With Somatic Mutations in U2AF1 Reveals Commonly Altered Splicing Events. PLoS ONE. 2014;9:e87361.

    Article  Google Scholar 

  13. Okeyo-Owuor T, White BS, Chatrikhi R, Mohan DR, Kim S, Griffith M, et al. U2AF1 mutations alter sequence specificity of pre-mRNA binding and splicing. Leukemia. 2015;29:909–17.

    Article  CAS  Google Scholar 

  14. Yip BH, Steeples V, Repapi E, Armstrong RN, Llorian M, Roy S, et al. The U2AF1S34F Mutation Induces Lineage-Specific Splicing Alterations in Myelodysplastic Syndromes. J Clin Investig. 2017;127:2206–21.

    Article  Google Scholar 

  15. Shirai CL, Ley JN, White BS, Kim S, Tibbitts J, Shao J, et al. Mutant U2AF1 Expression Alters Hematopoiesis and Pre-mRNA Splicing In Vivo. Cancer Cell. 2015;27:631–43.

    Article  CAS  Google Scholar 

  16. Fei DL, Zhen T, Durham B, Ferrarone J, Zhang T, Garrett L, et al. Impaired hematopoiesis and leukemia development in mice with a conditional knock-in allele of a mutant splicing factor gene U2af1. Proc Natl Acad Sci USA. 2018;115:E10437–46.

    Article  CAS  Google Scholar 

  17. Kühn R, Schwenk F, Aguet M, Rajewsky K. Inducible gene targeting in mice. Science. 1995;269:1427–29.

    Article  Google Scholar 

  18. Lee Y, Wang Q, Shuryak I, Brenner DJ, Turner HC. Development of a High-Throughput γ-H2AX Assay Based on Imaging Flow Cytometry. Radiat Oncol. 2019;14:150.

    Article  Google Scholar 

  19. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 2005;102:15545–50.

    Article  CAS  Google Scholar 

  20. Shen S, Park JW, Lu ZX, Lin L, Henry MD, Wu YN, et al. rMATS: robust and flexible detection of differential alternative splicing from replicate RNA-Seq data. Proc Natl Acad Sci USA 2014;111:E5593–601.

    Article  CAS  Google Scholar 

  21. Chen L, Chen J, Huang YJ, Gu Y, Qiu J, Qian H, et al. The Augmented R-Loop Is a Unifying Mechanism for Myelodysplastic Syndromes Induced by High-Risk Splicing Factor Mutations. Mol Cell. 2018;69:412–25.

    Article  CAS  Google Scholar 

  22. Nguyen HD, Leon W, Li W, Reddy PNG, Sullivan JD, Walter MJ, et al. Spliceosome Mutations Induce R Loop-Associated Sensitivity to ATR Inhibition in Myelodysplastic Syndromes. Cancer Res. 2018;78:5363–74.

    Article  CAS  Google Scholar 

  23. Zhu J, Zhang Y, Joe GJ, Pompetti R, Emerson SG. NF-Ya Activates Multiple Hematopoietic Stem Cell (HSC) Regulatory Genes and Promotes HSC Self-Renewal. Proc Natl Acad Sci USA 2005;102:11728–33.

    Article  CAS  Google Scholar 

  24. Bungartz G, Land H, Scadden DT, Emerson SG. NF-Y Is Necessary for Hematopoietic Stem Cell Proliferation and Survival. Blood. 2012;119:1380–89.

    Article  CAS  Google Scholar 

  25. Ficara F, Murphy MJ, Lin M, Cleary ML. Pbx1 regulates self-renewal of long-term hematopoietic stem cells by maintaining their quiescence. Cell Stem Cell. 2008;2:484–96.

    Article  CAS  Google Scholar 

  26. Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 1999;13:1501–12.

    Article  CAS  Google Scholar 

  27. Mackarehtschian K, Hardin JD, Moore KA, Boast S, Goff SP, Lemischka IR. Targeted Disruption of the flk2/flt3 Gene Leads to Deficiencies in Primitive Hematopoietic Progenitors. Immunity. 1995;3:147–61.

    Article  CAS  Google Scholar 

  28. Kuo YH, Zaidi SK, Gornostaeva S, Komori T, Stein GS, Castilla LH. Runx2 Induces Acute Myeloid Leukemia in Cooperation With Cbfbeta-SMMHC in Mice. Blood. 2009;113:3323–32.

    Article  CAS  Google Scholar 

  29. Unnisa Z, Clark JP, Roychoudhury J, Thomas E, Tessarollo L, Copeland NG, et al. Meis1 Preserves Hematopoietic Stem Cells in Mice by Limiting Oxidative Stress. Blood. 2012;120:4973–81.

    Article  CAS  Google Scholar 

  30. Ariki R, Morikawa S, Mabuchi Y, Suzuki S, Nakatake M, Yoshioka K, et al. Homeodomain Transcription Factor Meis1 Is a Critical Regulator of Adult Bone Marrow Hematopoiesis. PLoS ONE. 2014;9:e87646.

    Article  Google Scholar 

  31. Folco EG, Coli KE, Reed R. The Anti-Tumor Drug E7107 Reveals an Essential Role for SF3b in Remodeling U2 snRNP to Expose the Branch Point-Binding Region. Genes Dev. 2011;25:440–44.

    Article  CAS  Google Scholar 

  32. Eskens FA, Ramos F, Burger H, O’Brien JP, Piera A, de Jonge MJ, et al. Phase I Pharmacokinetic and Pharmacodynamic Study of the First-In-Class Spliceosome Inhibitor E7107 in Patients With Advanced Solid Tumors. Clin Cancer Res. 2013;19:6296–04.

    Article  CAS  Google Scholar 

  33. Lee SC-W, Abdel-Wahab O. Therapeutic Targeting of Splicing in Cancer. Nat Med. 2016;22:976–86.

    Article  CAS  Google Scholar 

  34. Shirai CL, White BS, Tripathi M, Tapia R, Ley JN, Ndonwi M, et al. Mutant U2AF1-expressing Cells Are Sensitive to Pharmacological Modulation of the Spliceosome. Nat Commun. 2017;8:14060.

    Article  CAS  Google Scholar 

  35. Seiler M, Yoshimi A, Darman R, Chan B, Keaney G, Thomas M, et al. H3B-8800, an Orally Available Small-Molecule Splicing Modulator, Induces Lethality in Spliceosome-Mutant Cancers. Nat Med. 2018;24:497–504.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr Roberto Mantovani (University of Milan) and Dr Licia Selleri (University of California San Francisco) for NF-Ya and Pbx1 expression constructs, and Ms Julia Dreksler for assistance with generating U2AF1 knockdown cell lines. We also thank the flow cytometry and microscopy core facilities at the University of Virginia for assistance with FACS sorting and confocal microscopy. Flow cytometry and microscopy cores are supported by the UVA Cancer Center through NCI P30CA044578 grant. This work was supported in part by US National Institute of Health (NIH) grants R01 HL095685 (GM), R35 GM133712 (CZ) and a start-up fund from the Department of Biochemistry and Molecular Genetics of the University of Virginia (GM).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA

    Avik Dutta, Yue Yang, Bao T. Le, Chongzhi Zang & Golam Mohi

  2. Center for Public Health Genomics, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA

    Yifan Zhang & Chongzhi Zang

  3. Department of Biomedical Engineering, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA

    Yifan Zhang & Chongzhi Zang

  4. Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA

    Omar Abdel-Wahab

  5. Leukemia Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA

    Omar Abdel-Wahab

  6. Department of Public Health Sciences, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA

    Chongzhi Zang

  7. University of Virginia Cancer Center, Charlottesville, VA, 22908, USA

    Chongzhi Zang & Golam Mohi

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Contributions

AD performed research, analyzed data and wrote the paper; YY performed research; BTL performed data analysis; YZ performed data analysis; OAW provided critical advice on genomic data analysis and revised the paper; CZ performed data analysis; GM designed the research, analyzed data, and prepared the paper with the help from co-authors.

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Correspondence to Golam Mohi.

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Dutta, A., Yang, Y., Le, B.T. et al. U2af1 is required for survival and function of hematopoietic stem/progenitor cells. Leukemia 35, 2382–2398 (2021). https://doi.org/10.1038/s41375-020-01116-x

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