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.

Chronic myelogenous leukemia

SRSF1 mediates cytokine-induced impaired imatinib sensitivity in chronic myeloid leukemia

Abstract

Patients with chronic myeloid leukemia (CML) who are treated with tyrosine kinase inhibitors (TKIs) experience significant heterogeneity regarding depth and speed of responses. Factors intrinsic and extrinsic to CML cells contribute to response heterogeneity and TKI resistance. Among extrinsic factors, cytokine-mediated TKI resistance has been demonstrated in CML progenitors, but the underlying mechanisms remain obscure. Using RNA-sequencing, we identified differentially expressed splicing factors in primary CD34+ chronic phase (CP) CML progenitors and controls. We found SRSF1 expression to be increased as a result of both BCR-ABL1- and cytokine-mediated signaling. SRSF1 overexpression conferred cytokine independence to untransformed hematopoietic cells and impaired imatinib sensitivity in CML cells, while SRSF1 depletion in CD34+ CP CML cells prevented the ability of extrinsic cytokines to decrease imatinib sensitivity. Mechanistically, PRKCH and PLCH1 were upregulated by elevated SRSF1 levels, and contributed to impaired imatinib sensitivity. Importantly, very high SRSF1 levels in the bone marrow of CML patients at presentation correlated with poorer clinical TKI responses. In summary, we find SRSF1 levels to be maintained in CD34+ CP CML progenitors by cytokines despite effective BCR-ABL1 inhibition, and that elevated levels promote impaired imatinib responses. Together, our data support an SRSF1/PRKCH/PLCH1 axis in contributing to cytokine-induced impaired imatinib sensitivity in CML.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: SRSF1 is upregulated in CD34+ CML progenitors.
Fig. 2: SRSF1 is upregulated by BCR-ABL1 and extrinsic cytokine signaling.
Fig. 3: Elevated SRSF1 levels confer cytokine independence in hematopoietic cells and impaired imatinib sensitivity in CML cells.
Fig. 4: Upregulated SRSF1 levels antagonize imatinib-related gene expression via a nonsplicing function of SRSF1.
Fig. 5: Schematic depicting genes in the PLC signaling pathway.
Fig. 6: Targeting PKC signaling rescues SRSF1-mediated impaired imatinib responses.
Fig. 7: SRSF1 levels correlate with imatinib response in CP CML patients.
Fig. 8

References

  1. 1.

    Arrigoni E, Del ReM, Galimberti S, Restante G, Rofi E, Crucitta S, et al. Concise review: chronic myeloid leukemia: stem cell niche and response to pharmacologic treatment. Stem Cells Transl Med. 2018;7:305–14.

    PubMed  PubMed Central  Google Scholar 

  2. 2.

    Mahon F-X, Réa D, Guilhot J, Guilhot F, Huguet F, Nicolini F, et al. Discontinuation of imatinib in patients with chronic myeloid leukaemia who have maintained complete molecular remission for at least 2 years: the prospective, multicentre Stop Imatinib (STIM) trial. Lancet Oncol. 2010;11:1029–35.

    CAS  PubMed  Google Scholar 

  3. 3.

    Rea D, Nicolini FE, Tulliez M, Guilhot F, Guilhot J, Guerci-Bresler A, et al. Discontinuation of dasatinib or nilotinib in chronic myeloid leukemia: interim analysis of the STOP 2G-TKI study. Blood. 2017;129:846–54.

    CAS  PubMed  Google Scholar 

  4. 4.

    Copland M, Hamilton A, Elrick LJ, Baird JW, Allan EK, Jordanides N, et al. Dasatinib (BMS-354825) targets an earlier progenitor population than imatinib in primary CML but does not eliminate the quiescent fraction. Blood. 2006;107:4532–9.

    CAS  PubMed  Google Scholar 

  5. 5.

    Graham SM, Jørgensen HG, Allan E, Pearson C, Alcorn MJ, Richmond L, et al. Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro. Blood. 2002;99:319–25.

    CAS  PubMed  Google Scholar 

  6. 6.

    Corbin AS, Agarwal A, Loriaux M, Cortes J, Deininger MW, Druker BJ. Human chronic myeloid leukemia stem cells are insensitive to imatinib despite inhibition of BCR-ABL activity. J Clin Investig. 2011;121:396–409.

    CAS  PubMed  Google Scholar 

  7. 7.

    Graf L, Iwata M, Torok-Storb B. Gene expression profiling of the functionally distinct human bone marrow stromal cell lines HS-5 and HS-27a. Blood. 2002;100:1509–11.

    CAS  PubMed  Google Scholar 

  8. 8.

    Ng KP, Manjeri A, Lee KL, Huang W, Tan SY, Chuah CT, et al. Physiologic hypoxia promotes maintenance of CML stem cells despite effective BCR-ABL1 inhibition. Blood, J Am Soc Hematol. 2014;123:3316–26.

  9. 9.

    Jiang X, Lopez A, Holyoake T, Eaves A, Eaves C. Autocrine production and action of IL-3 and granulocyte colony-stimulating factor in chronic myeloid leukemia. Proc Natl Acad Sci. 1999;96:12804–9.

    CAS  PubMed  Google Scholar 

  10. 10.

    Traer E, MacKenzie R, Snead J, Agarwal A, Eiring AM, O’Hare T, et al. Blockade of JAK2-mediated extrinsic survival signals restores sensitivity of CML cells to ABL inhibitors. Leukemia. 2012;26:1140.

    CAS  PubMed  Google Scholar 

  11. 11.

    Bewry NN, Nair RR, Emmons MF, Boulware D, Pinilla-Ibarz J, Hazlehurst LA. Stat3 contributes to resistance toward BCR-ABL inhibitors in a bone marrow microenvironment model of drug resistance. Mol Cancer Ther. 2008;7:3169–75.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Wang Y, Cai D, Brendel C, Barett C, Erben P, Manley PW, et al. Adaptive secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates imatinib and nilotinib resistance in BCR/ABL+ progenitors via JAK-2/STAT-5 pathway activation. Blood. 2007;109:2147–55.

    CAS  PubMed  Google Scholar 

  13. 13.

    Chang JS, Santhanam R, Trotta R, Neviani P, Eiring AM, Briercheck E, et al. High levels of the BCR/ABL oncoprotein are required for the MAPK-hnRNP-E2–dependent suppression of C/EBPα-driven myeloid differentiation. Blood. 2007;110:994–1003.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Holm F, Hellqvist E, Mason CN, Ali SA, Delos-Santos N, Barrett CL, et al. Reversion to an embryonic alternative splicing program enhances leukemia stem cell self-renewal. Proc Natl Acad Sci. 2015;112:15444–9.

    CAS  PubMed  Google Scholar 

  15. 15.

    Salesse S, Dylla SJ, Verfaillie CM. p210BCR/ABL-induced alteration of pre-mRNA splicing in primary human CD34+ hematopoietic progenitor cells. Leukemia. 2004;18:727–33.

    CAS  PubMed  Google Scholar 

  16. 16.

    Iervolino A, Santilli G, Trotta R, Guerzoni C, Cesi V, Bergamaschi A, et al. hnRNP A1 nucleocytoplasmic shuttling activity is required for normal myelopoiesis and BCR/ABL leukemogenesis. Mol Cell Biol. 2002;22:2255–66.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Das S, Krainer AR. Emerging functions of SRSF1, splicing factor and oncoprotein, in RNA metabolism and cancer. Mol Cancer Res. 2014;12:1195–204.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    de Miguel FJ, Sharma RD, Pajares MJ, Montuenga LM, Rubio A, Pio R. Identification of alternative splicing events regulated by the oncogenic factor SRSF1 in lung cancer. Cancer Res. 2014;74:1105–15.

    PubMed  Google Scholar 

  19. 19.

    Anczukow O, Rosenberg AZ, Akerman M, Das S, Zhan L, Karni R, et al. The splicing factor SRSF1 regulates apoptosis and proliferation to promote mammary epithelial cell transformation. Nat Struct Mol Biol. 2012;19:220–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Sheng J, Zhao J, Xu Q, Wang L, Zhang W, Zhang Y. Bioinformatics analysis of SRSF1‑controlled gene networks in colorectal cancer. Oncol Lett. 2017;14:5393–9.

    PubMed  PubMed Central  Google Scholar 

  21. 21.

    Karni R, Hippo Y, Lowe SW, Krainer AR. The splicing-factor oncoprotein SF2/ASF activates mTORC1. Proc Natl Acad Sci USA. 2008;105:15323–7.

    CAS  PubMed  Google Scholar 

  22. 22.

    Shultz JC, Goehe RW, Murudkar CS, Wijesinghe DS, Mayton EK, Massiello A, et al. SRSF1 regulates the alternative splicing of caspase 9 via a novel intronic splicing enhancer affecting the chemotherapeutic sensitivity of non-small cell lung cancer cells. Mol Cancer Res. 2011;9:889–900.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Adesso L, Calabretta S, Barbagallo F, Capurso G, Pilozzi E, Geremia R, et al. Gemcitabine triggers a pro-survival response in pancreatic cancer cells through activation of the MNK2/eIF4E pathway. Oncogene. 2013;32:2848–57.

    CAS  PubMed  Google Scholar 

  24. 24.

    Gout S, Brambilla E, Boudria A, Drissi R, Lantuejoul S, Gazzeri S, et al. Abnormal expression of the pre-mRNA splicing regulators SRSF1, SRSF2, SRPK1 and SRPK2 in non small cell lung carcinoma. PloS One. 2012;7:e46539.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    van der Werf I, Cloos J. Involvement of SRSF1 in alternative splicing of FPGS and methotrexate resistance in children with acute lymphoblastic leukemia. Student Undergrad Res E-J 2015;1.

  26. 26.

    Zou L, Zhang H, Du C, Liu X, Zhu S, Zhang W, et al. Correlation of SRSF1 and PRMT1 expression with clinical status of pediatric acute lymphoblastic leukemia. J Hematol Oncol. 2012;5:42.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Jiang L, Huang J, Higgs BW, Hu Z, Xiao Z, Yao X, et al. Genomic landscape survey identifies SRSF1 as a key oncodriver in small cell lung cancer. PLoS Genet. 2016;12:e1005895.

    PubMed  PubMed Central  Google Scholar 

  28. 28.

    Bhatia R, McGlave PB, Dewald GW, Blazar B, Verfaillie C. Abnormal function of the bone marrow microenvironment in chronic myelogenous leukemia: role of malignant stromal macrophages. Blood. 1995;85:3636–45.

    CAS  PubMed  Google Scholar 

  29. 29.

    Chu S, Holtz M, Gupta M, Bhatia R. BCR/ABL kinase inhibition by imatinib mesylate enhances MAP kinase activity in chronic myelogenous leukemia CD34+ cells. Blood. 2004;103:3167–74.

    CAS  PubMed  Google Scholar 

  30. 30.

    Anders S, Pyl PT, Huber W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics. 2015;31:166–9.

    CAS  PubMed  Google Scholar 

  31. 31.

    Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:550.

    PubMed  PubMed Central  Google Scholar 

  32. 32.

    Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic acids Res. 2015;43:e47–e.

    PubMed  PubMed Central  Google Scholar 

  33. 33.

    Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Baccarani M, Deininger MW, Rosti G, Hochhaus A, Soverini S, Apperley JF, et al. European LeukemiaNet recommendations for the management of chronic myeloid leukemia: 2013. Blood. 2013;122:872–84.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Giulietti M, Piva F, D’Antonio M, D’Onorio De Meo P, Paoletti D, Castrignano T, et al. SpliceAid-F: a database of human splicing factors and their RNA-binding sites. Nucleic Acids Res. 2012;41(D1):D125–31.

    PubMed  PubMed Central  Google Scholar 

  36. 36.

    Skorski T, Kanakaraj P, Nieborowska-Skorska M, Ratajczak M, Wen S-C, Zon G, et al. Phosphatidylinositol-3 kinase activity is regulated by BCR/ABL and is required for the growth of Philadelphia chromosome-positive cells. Blood 1995;86:726–36.

    CAS  PubMed  Google Scholar 

  37. 37.

    Cortez D, Reuther G, Pendergast AM. The Bcr-Abl tyrosine kinase activates mitogenic signaling pathways and stimulates G1-to-S phase transition in hematopoietic cells. Oncogene. 1997;15:2333.

    CAS  PubMed  Google Scholar 

  38. 38.

    Tago K, Kaziro Y, Satoh T. Functional involvement of mSos in interleukin-3 and thrombin stimulation of the Ras, mitogen-activated protein kinase pathway in BaF3 murine hematopoietic cells. J Biochem. 1998;123:659–67.

    CAS  PubMed  Google Scholar 

  39. 39.

    Valacca C, Bonomi S, Buratti E, Pedrotti S, Baralle FE, Sette C, et al. Sam68 regulates EMT through alternative splicing–activated nonsense-mediated mRNA decay of the SF2/ASF proto-oncogene. J Cell Biol. 2010;191:87–99.

  40. 40.

    Lee CR, Kang JA, Kim HE, Choi Y, Yang T, Park SG. Secretion of IL‐1β from imatinib‐resistant chronic myeloid leukemia cells contributes to BCR–ABL mutation‐independent imatinib resistance. FEBS Lett. 2016;590:358–68.

    CAS  PubMed  Google Scholar 

  41. 41.

    Levescot A, Flamant S, Basbous S, Jacomet F, Féraud O, Bourgeois EA, et al. BCR-ABL–induced deregulation of the IL-33/ST2 pathway in CD34 (+) progenitors from chronic myeloid leukemia patients. Cancer Res. 2014;74:2669–76.

    CAS  PubMed  Google Scholar 

  42. 42.

    Zhang B, Ho YW, Huang Q, Maeda T, Lin A, Lee SU, et al. Altered microenvironmental regulation of leukemic and normal stem cells in chronic myelogenous leukemia. Cancer Cell. 2012;21:577–92.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Quentmeier H, Eberth S, Romani J, Zaborski M, Drexler HG. BCR-ABL1-independent PI3Kinase activation causing imatinib-resistance. J Hematol Oncol. 2011;4:6.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Wöhrle FU, Halbach S, Aumann K, Schwemmers S, Braun S, Auberger P, et al. Gab2 signaling in chronic myeloid leukemia cells confers resistance to multiple Bcr-Abl inhibitors. Leukemia. 2013;27:118.

    PubMed  Google Scholar 

  45. 45.

    Wagle M, Eiring A, Wongchenko M, Lu S, Guan Y, Wang Y, et al. A role for FOXO1 in BCR–ABL1-independent tyrosine kinase inhibitor resistance in chronic myeloid leukemia. Leukemia. 2016;30:1493.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Mitchell R, Hopcroft LE, Baquero P, Allan EK, Hewit K, James D, et al. Targeting BCR-ABL-independent TKI resistance in chronic myeloid leukemia by mTOR and autophagy inhibition. J Natl Cancer Inst. 2017;110:467–78.

    PubMed Central  Google Scholar 

  47. 47.

    Ly C, Arechiga AF, Melo JV, Walsh CM, Ong ST. Bcr-Abl kinase modulates the translation regulators ribosomal protein S6 and 4E-BP1 in chronic myelogenous leukemia cells via the mammalian target of rapamycin. Cancer Res. 2003;63:5716–22.

    CAS  PubMed  Google Scholar 

  48. 48.

    Nakamura Y, Fukami K. Regulation and physiological functions of mammalian phospholipase C. J Biochem. 2017;161:315–21.

    CAS  PubMed  Google Scholar 

  49. 49.

    Deka SJ, Trivedi V. Potentials of PKC in cancer progression and anticancer drug development. Curr Drug Discov Technol. 2019;16:135–47.

    CAS  PubMed  Google Scholar 

  50. 50.

    Holyoake TL, Vetrie D. The chronic myeloid leukemia stem cell: stemming the tide of persistence. Blood. 2017;129:1595–606.

    CAS  PubMed  Google Scholar 

  51. 51.

    Shah M, Bhatia R. Preservation of quiescent chronic myelogenous leukemia stem cells by the bone marrow microenvironment. In: Biological mechanisms of minimal residual disease and systemic cancer. Springer International Publishing, 2018. p. 97–110.

  52. 52.

    Ma L, Shan Y, Bai R, Xue L, Eide CA, Ou J, et al. A therapeutically targetable mechanism of BCR-ABL–independent imatinib resistance in chronic myeloid leukemia. Sci Transl Med. 2014;6:252ra121–252ra121.

    PubMed  PubMed Central  Google Scholar 

  53. 53.

    Wilkinson SE, Parker P, Nixon J. Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C. Biochemical J. 1993;294:335–7.

    CAS  Google Scholar 

  54. 54.

    Gschwendt M, Muller H, Kielbassa K, Zang R, Kittstein W, Rincke G, et al. Rottlerin, a novel protein kinase inhibitor. Biochem Biophys Res Commun. 1994;199:93–8.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the National Medical Council Singapore (NMRC/CSA/0051/2013 and NMRC/CIRG/1404/2014).

Author information

Affiliations

Authors

Contributions

JRS designed and performed experiments for cloning, western blots, quantitative PCR, and colony formation. MGY and SPT performed experiments for cloning, western blots, and qPCR. KLL performed the Fluidigm experiments for validation of alternative splicing isoforms. SC, JL, KLL, SR, and HY analyzed the RNA-sequencing data for gene expression and ASEs. CC and TH provided the primary CML samples. CO and JI performed the immunohistochemistry (IHC) staining and scoring. JH performed statistical analysis on the IHC scoring by CO and JI. OA-C, XR, and ARK provided experimental advice and essential reagents. STO supervised the project, and JRS prepared figures and wrote the manuscript which was approved by all authors.

Corresponding author

Correspondence to Sin Tiong Ong.

Ethics declarations

Conflict of interest

The authors declare that they have 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

Supp Table 1: Short hair pin sequences

Supp Table 2: Primer list

Supp Table 3: SRSF1 IHC Clinical data

Supp Table 4: Statistical comparison between good responders and poor responders

Supp Table 5: Normalized log counts from cultured chronic phase and normal bone marrow from RNA-sequencing analysis

41375_2020_732_MOESM6_ESM.xlsx

Supp Table 6: List of detected alternative splicing events from K562 cells with SRSF1 overexpression vs vector in DMSO and IM conditions

41375_2020_732_MOESM7_ESM.xlsx

Supp Table 7: Differential alternative splicing event analysis of K562 cells with SRSF1 overexpression vs vector control in DMSO

41375_2020_732_MOESM8_ESM.xlsx

Supp Table 8: Differential alternative splicing event analysis of K562 cells with SRSF1 overexpression vs vector control in IM

Supp Table 9: Fluidigm validation for K562 cells with SRSF1 overexpression vs vector control in DMSO and IM conditions

Supp Table 10: Normalized log counts from K562 overexpressing SRSF1 and treated with IM from RNA-sequencing analysis

41375_2020_732_MOESM11_ESM.xlsx

Supp Table 11: List of significant and differential genes from analysis of K562 cells with SRSF1 overexpression vs vector control in DMSO

41375_2020_732_MOESM12_ESM.xlsx

Supp Table 12: List of significant and differential genes from analysis of K562 cells with SRSF1 overexpression vs vector control in IM

Supp Table 13: Genes in IPA biological processes from K562 cells with SRSF1 overexpression vs vector control in DMSO

Supp Table 14: Genes in IPA biological processes from K562 cells with SRSF1 overexpression vs vector control in IM

Supplementary Figures

Supplementary Figure Legends

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Sinnakannu, J.R., Lee, K.L., Cheng, S. et al. SRSF1 mediates cytokine-induced impaired imatinib sensitivity in chronic myeloid leukemia. Leukemia 34, 1787–1798 (2020). https://doi.org/10.1038/s41375-020-0732-1

Download citation

Further reading

Search

Quick links