Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

MYELODYSPLASTIC NEOPLASM

Alternatively spliced CSF3R isoforms in SRSF2 P95H mutated myeloid neoplasms

Abstract

Alternatively spliced colony stimulating factor 3 receptor (CSF3R) isoforms Class III and Class IV are observed in myelodysplastic syndromes (MDS), but their roles in disease remain unclear. We report that the MDS-associated splicing factor SRSF2 affects the expression of Class III and Class IV isoforms and perturbs granulopoiesis. Add-back of the Class IV isoform in Csf3r-null mouse progenitor cells increased granulocyte progenitors with impaired neutrophil differentiation, while add-back of the Class III produced dysmorphic neutrophils in fewer numbers. These CSF3R isoforms were elevated in patients with myeloid neoplasms harboring SRSF2 mutations. Using in vitro splicing assays, we confirmed increased Class III and Class IV transcripts when SRSF2 P95 mutations were co-expressed with the CSF3R minigene in K562 cells. Since SRSF2 regulates splicing partly by recognizing exonic splicing enhancer (ESE) sequences on pre-mRNA, deletion of either ESE motifs within CSF3R exon 17 decreased Class IV transcript levels without affecting Class III. CD34+ cells expressing SRSF2 P95H showed impaired neutrophil differentiation in response to G-CSF and was accompanied by increased levels of Class IV. Our findings suggest that SRSF2 P95H promotes Class IV splicing by binding to key ESE sequences in CSF3R exon 17, and that SRSF2, when mutated, contributes to dysgranulopoiesis.

This is a preview of subscription content, access via your institution

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Comparison of CSF3R isoforms and their contributions to colony growth.
Fig. 2: CSF3R minigene splicing assay with MDS-related splicing factors.
Fig. 3: Correlation of CSF3R Class III and IV expression with recurrent mutations of splicing factors found in myeloid neoplasia.
Fig. 4: SRSF2 P95 mutations increase Class III and Class IV splicing.
Fig. 5: Splicing assay with ESE deletions in CSF3R minigene.
Fig. 6: Differentiation of CD34+ cells with expression of WT or mutant SRSF2.

References

  1. Akbarzadeh S, Ward AC, McPhee DO, Alexander WS, Lieschke GJ, Layton JE. Tyrosine residues of the granulocyte colony-stimulating factor receptor transmit proliferation and differentiation signals in murine bone marrow cells. Blood 2002;99:879–87.

    CAS  PubMed  Google Scholar 

  2. Hermans MHA. Signaling mechanisms coupled to tyrosines in the granulocyte colony-stimulating factor receptor orchestrate G-CSF-induced expansion of myeloid progenitor cells. Blood 2002;101:2584–90.

    PubMed  Google Scholar 

  3. Fukunaga R, Ishizaka-Ikeda E, Nagata S. Growth and differentiation signals mediated by different regions in the cytoplasmic domain of granulocyte colony-stimulating factor receptor. Cell 1993;74:1079–87.

    CAS  PubMed  Google Scholar 

  4. Gits J, van Leeuwen D, Carroll HP, Touw IP, Ward AC. Multiple pathways contribute to the hyperproliferative responses from truncated granulocyte colony-stimulating factor receptors. Leukemia 2006;20:2111–8.

    CAS  PubMed  Google Scholar 

  5. Kendrick TS, Lipscombe RJ, Rausch O, Nicholson SE, Layton JE, Goldie-Cregan LC, et al. Contribution of the Membrane-distal Tyrosine in Intracellular Signaling by the Granulocyte Colony-stimulating Factor Receptor. J Biol Chem. 2004;279:326–40.

    CAS  PubMed  Google Scholar 

  6. de Koning JP, Soede-Bobok AA, Schelen AM, Smith L, van Leeuwen D, Santini V, et al. Proliferation Signaling and Activation of Shc, p21Ras, and Myc Via Tyrosine 764 of Human Granulocyte Colony-Stimulating Factor Receptor. Blood 1998;91:1924–33.

    PubMed  Google Scholar 

  7. Tian SS, Tapley P, Sincich C, Stein RB, Rosen J, Lamb P. Multiple signaling pathways induced by granulocyte colony-stimulating factor involving activation of JAKs, STAT5, and/or STAT3 are required for regulation of three distinct classes of immediate early genes. Blood 1996;88:4435–44.

    CAS  PubMed  Google Scholar 

  8. Tian SS, Lamb P, Seidel HM, Stein RB, Rosen J. Rapid activation of the STAT3 transcription factor by granulocyte colony-stimulating factor. Blood 1994;84:1760–4.

    CAS  PubMed  Google Scholar 

  9. Lance A, Druhan LJ, Vestal CG, Steuerwald NM, Hamilton A, Smith M, et al. Altered expression of CSF3R splice variants impacts signal response and is associated with SRSF2 mutations. Leukemia 2019;34:369–79.

    PubMed  Google Scholar 

  10. Fukunaga R, Seto Y, Mizushima S, Nagata S. Three different mRNAs encoding human granulocyte colony-stimulating factor receptor. Proc Natl Acad Sci USA. 1990;87:8702–6.

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Ward AC, van Aesch YM, Schelen AM, Touw IP. Defective internalization and sustained activation of truncated granulocyte colony-stimulating factor receptor found in severe congenital neutropenia/acute myeloid leukemia. Blood 1999;93:447–58.

    CAS  PubMed  Google Scholar 

  12. Hermans MH, Antonissen C, Ward AC, Mayen AE, Ploemacher RE, Touw IP. Sustained receptor activation and hyperproliferation in response to granulocyte colony-stimulating factor (G-CSF) in mice with a severe congenital neutropenia/acute myeloid leukemia-derived mutation in the G-CSF receptor gene. J Exp Med. 1999;189:683–92.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. White SM, Alarcon MH, Tweardy DJ. Inhibition of granulocyte colony-stimulating factor-mediated myeloid maturation by low level expression of the differentiation-defective class IV granulocyte colony-stimulating factor receptor isoform. Blood 2000;95:3335–40.

    CAS  PubMed  Google Scholar 

  14. Mehta HM, Futami M, Glaubach T, Lee DW, Andolina JR, Yang Q, et al. Alternatively spliced, truncated GCSF receptor promotes leukemogenic properties and sensitivity to JAK inhibition. Leukemia 2013;28:1041–51.

    PubMed  PubMed Central  Google Scholar 

  15. White SM, Ball ED, Ehmann WC, Rao AS, Tweardy DJ. Increased expression of the differentiation-defective granulocyte colony-stimulating factor receptor mRNA isoform in acute myelogenous leukemia. Leukemia 1998;12:899–906.

    CAS  PubMed  Google Scholar 

  16. Komeno Y, Huang YJ, Qiu J, Lin L, Xu Y, Zhou Y, et al. SRSF2 is essential for hematopoiesis, and its myelodysplastic syndrome-related mutations dysregulate alternative pre-mRNA splicing. Mol Cell Biol. 2015;35:3071–82.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Xiao R, Sun Y, Ding J-H, Lin S, Rose DW, Rosenfeld MG, et al. Splicing regulator SC35 is essential for genomic stability and cell proliferation during mammalian organogenesis. Mol Cell Biol. 2007;27:5393–402.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Bapat A, Keita N, Martelly W, Kang P, Seet C, Jacobsen JR, et al. Myeloid disease mutations of splicing factor SRSF2 cause G2-M arrest and skewed differentiation of human hematopoietic stem and progenitor cells. Stem Cells. 2018;36:1663–75.

    CAS  PubMed  Google Scholar 

  19. Yoshida K, Sanada M, Shiraishi Y, Nowak D, Nagata Y, Yamamoto R, et al. Frequent pathway mutations of splicing machinery in myelodysplasia. Nature 2011;478:64–9.

    CAS  PubMed  Google Scholar 

  20. 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.

    CAS  PubMed  Google Scholar 

  21. Meggendorfer M, Roller A, Haferlach T, Eder C, Dicker F, Grossmann V, et al. SRSF2 mutations in 275 cases with chronic myelomonocytic leukemia (CMML). Blood 2012;120:3080–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Wang X, Song X, Yan X. Effect of RNA splicing machinery gene mutations on prognosis of patients with MDS: A meta-analysis. Med (Baltim). 2019;98:e15743.

    CAS  Google Scholar 

  23. Papaemmanuil E, Gerstung M, Malcovati L, Tauro S, Gundem G, Van Loo P, et al. Clinical and biological implications of driver mutations in myelodysplastic syndromes. Blood 2013;122:3616–27.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Federmann B, Abele M, Rosero Cuesta DS, Vogel W, Boiocchi L, Kanz L, et al. The detection of SRSF2 mutations in routinely processed bone marrow biopsies is useful in the diagnosis of chronic myelomonocytic leukemia. Hum Pathol. 2014;45:2471–9.

    CAS  PubMed  Google Scholar 

  25. 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.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Kim E, Ilagan JO, Liang Y, Daubner GM, Lee SC, Ramakrishnan A, et al. SRSF2 mutations contribute to myelodysplasia by mutant-specific effects on exon recognition. Cancer Cell. 2015;27:617–30.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Zhang J, Lieu YK, Ali AM, Penson A, Reggio KS, Rabadan R, et al. Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities. Proc Natl Acad Sci USA. 2015;112:E4726–34.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Masaki S, Ikeda S, Hata A, Shiozawa Y, Kon A, Ogawa S, et al. Myelodysplastic syndrome-associated SRSF2 mutations cause splicing changes by altering binding motif sequences. Front Genet. 2019;10:338.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Chen S, Benbarche S, Abdel-Wahab O. Splicing factor mutations in hematologic malignancies. Blood 2021;138:599–612.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Kon A, Yamazaki S, Nannya Y, Kataoka K, Ota Y, Nakagawa MM, et al. Physiological Srsf2 P95H expression causes impaired hematopoietic stem cell functions and aberrant RNA splicing in mice. Blood 2018;131:621–35.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Sloand EM, Yong ASM, Ramkissoon S, Solomou E, Bruno TC, Kim S, et al. Granulocyte colony-stimulating factor preferentially stimulates proliferation of monosomy 7 cells bearing the isoform IV receptor. Proc Natl Acad Sci USA. 2006;103:14483–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Harvey SE, Lyu J, Cheng C. Methods for characterization of alternative RNA splicing. Methods Mol Biol. 2021;2372:209–22.

    PubMed  PubMed Central  Google Scholar 

  33. Hershberger CE, Moyer DC, Adema V, Kerr CM, Walter W, Hutter S, et al. Complex landscape of alternative splicing in myeloid neoplasms. Leukemia 2021;35:1108–20.

    CAS  PubMed  Google Scholar 

  34. Liu M, Wang F, Zhang Y, Chen X, Cao P, Nie D, et al. Gene mutation spectrum of patients with myelodysplastic syndrome and progression to acute myeloid leukemia. Int J Hematol Oncol. 2021;10:IJH34.

    PubMed  PubMed Central  Google Scholar 

  35. Smith PJ, Zhang C, Wang J, Chew SL, Zhang MQ, Krainer AR. An increased specificity score matrix for the prediction of SF2/ASF-specific exonic splicing enhancers. Hum Mol Genet. 2006;15:2490–508.

    CAS  PubMed  Google Scholar 

  36. Cartegni L, Wang J, Zhu Z, Zhang MQ, Krainer AR. ESEfinder: A web resource to identify exonic splicing enhancers. Nucleic Acids Res. 2003;31:3568–71.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Yun JW, Yoon J, Jung CW, Lee K-O, Kim JW, Kim S-H, et al. Next-generation sequencing reveals unique combination of mutations in cis of CSF3R in atypical chronic myeloid leukemia. J Clin Lab Anal 2020;34:e23064.

    CAS  PubMed  Google Scholar 

  38. Trottier AM, Druhan LJ, Kraft IL, Lance A, Feurstein S, Helgeson M, et al. Heterozygous germ line CSF3R variants as risk alleles for development of hematologic malignancies. Blood Adv. 2020;4:5269–84.

    PubMed  PubMed Central  Google Scholar 

  39. Zhang H, Reister Schultz A, Luty S, Rofelty A, Su Y, Means S, et al. Characterization of the leukemogenic potential of distal cytoplasmic CSF3R truncation and missense mutations. Leukemia 2017;31:2752–60.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Wolfler A, Erkeland SJ, Bodner C, Valkhof M, Renner W, Leitner C, et al. A functional single-nucleotide polymorphism of the G-CSF receptor gene predisposes individuals to high-risk myelodysplastic syndrome. Blood 2005;105:3731–6.

    PubMed  Google Scholar 

  41. Fukunaga R, Ishizaka-Ikeda E, Pan CX, Seto Y, Nagata S. Functional domains of the granulocyte colony-stimulating factor receptor. EMBO J. 1991;10:2855–65.

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Gallego ME, Gattoni R, Stevenin J, Marie J, Expert-Bezancon A. The SR splicing factors ASF/SF2 and SC35 have antagonistic effects on intronic enhancer-dependent splicing of the beta-tropomyosin alternative exon 6A. EMBO J. 1997;16:1772–84.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Lareau LF, Inada M, Green RE, Wengrod JC, Brenner SE. Unproductive splicing of SR genes associated with highly conserved and ultraconserved DNA elements. Nature 2007;446:926–9.

    CAS  PubMed  Google Scholar 

  44. Dreumont N, Hardy S, Behm-Ansmant I, Kister L, Branlant C, Stévenin J, et al. Antagonistic factors control the unproductive splicing of SC35 terminal intron. Nucleic Acids Res. 2010;38:1353–66.

    CAS  PubMed  Google Scholar 

  45. Sureau A, Gattoni R, Dooghe Y, Stévenin J, Soret J. SC35 autoregulates its expression by promoting splicing events that destabilize its mRNAs. EMBO J. 2001;20:1785–96.

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by DOD Bone Marrow Failure Grant W81XWH-15-1-0153 (SJC), NIH R01-HL128173 (SJC, MK), and VeloSano Award (HMM). MK was supported by the National Science Centre (Poland) grant 2018/29/B/ST7/02550. Department of Systems Biology and Engineering, Silesian University of Technology, Poland. Juan Sabín, co-founder of Affinimeter, assisted the authors in curve fitting the ITC data. The data set used from Hershberger et al. [32] was supported by funds from “Torsten Haferlach Leukämiediagnostik Stiftung” (https://www.torsten-haferlach-leukaemiediagnostik-stiftung.de/en/).

Author information

Authors and Affiliations

Authors

Contributions

Design of experiments (BAW, HMM, SRP, CC, BST, SJC), the performance of experiments (BAW, HMM, SRP, TH), analysis of data (BAW, HMM, SRP, BST, MK, JPM, SJC), and writing of manuscript (BAW, SJC).

Corresponding author

Correspondence to Seth J. Corey.

Ethics declarations

Competing interests

TH serves on the management board of the Munich Leukemia Laboratory. All of the other authors declare no conflict of interest.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wang, B.A., Mehta, H.M., Penumutchu, S.R. et al. Alternatively spliced CSF3R isoforms in SRSF2 P95H mutated myeloid neoplasms. Leukemia 36, 2499–2508 (2022). https://doi.org/10.1038/s41375-022-01672-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41375-022-01672-4

Search

Quick links