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Acute myeloid leukemia

Time resolved quantitative phospho-tyrosine analysis reveals Bruton’s Tyrosine kinase mediated signaling downstream of the mutated granulocyte-colony stimulating factor receptors

Leukemia volume 33, pages 75–87 (2019)Cite this article

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Abstract

Granulocyte-colony stimulating factor receptor (G-CSFR) controls myeloid progenitor proliferation and differentiation to neutrophils. Mutations in CSF3R (encoding G-CSFR) have been reported in patients with chronic neutrophilic leukemia (CNL) and acute myeloid leukemia (AML); however, despite years of research, the malignant downstream signaling of the mutated G-CSFRs is not well understood. Here, we used a quantitative phospho-tyrosine analysis to generate a comprehensive signaling map of G-CSF induced tyrosine phosphorylation in the normal versus mutated (proximal: T618I and truncated: Q741x) G-CSFRs. Unbiased clustering and kinase enrichment analysis identified rapid induction of phospho-proteins associated with endocytosis by the wild type G-CSFR only; while G-CSFR mutants showed abnormal kinetics of canonical Stat3, Stat5, and Mapk phosphorylation, and aberrant activation of Bruton’s Tyrosine Kinase (Btk). Mutant-G-CSFR-expressing cells displayed enhanced sensitivity (3–5-fold lower IC50) for ibrutinib-based chemical inhibition of Btk. Primary murine progenitor cells from G-CSFR-Q741x knock-in mice validated activation of Btk by the mutant receptor and retrovirally transduced human CD34+ umbilical cord blood cells expressing mutant receptors displayed enhanced sensitivity to Ibrutinib. A significantly lower clonogenic potential was displayed by both murine and human primary cells expressing mutated receptors upon ibrutinib treatment. Finally, a dramatic synergy was observed between ibrutinib and ruxolinitib at lower dose of the individual drug. Altogether, these data demonstrate the strength of unsupervised proteomics analyses in dissecting oncogenic pathways, and suggest repositioning Ibrutinib for therapy of myeloid leukemia bearing CSF3R mutations. Phospho-tyrosine data are available via ProteomeXchange with identifier PXD009662.

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Acknowledgements

We thank Dr. Fan Dong (University of Toledo, Ohio) for providing 32D cell line and Dr. Dan Link (University of Washington, St. Louis, MO) for providing the truncation mutation G-CSFR mice. We are also grateful to Drs. Julia Maxson (Oregon Health Science University, Portland, OR) and D Ivo Touw (Erasmus Medical Center, Rotterdam, The Netherlands) for insightful suggestions during the study and Mr. Glenn Doermann for his expertize in graphic design for the figures. This article and our research work associated with G-CSFR are supported by several sources, including National Institutes of Health (NIH) Grant 1S10 RR027015-01 (KDG), the University of Cincinnati Millennium Scholars Fund (KDG), and the Cincinnati Children’s Hospital Research Foundation (KDG), Graduate Student Governance Association (GSGA) funding resources (PD), National Institutes of Health T32 ES007250-06 (DEM), as well as R01 CA196658 (HLG) and a grant from CancerFree Kids (HLG).

Author contributions

PD, DEM, MA, HLG, and KDG designed, performed, and analyzed all of the experiments. MA, HLG, and KDG provided funding, intellectual direction, and overall progression of the study. MW played an instrumental role in the data sorting and processing.

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Author notes

  1. These authors contributed equally: Pankaj Dwivedi, David E. Muench.

Authors and Affiliations

  1. Department of Cancer Biology, University of Cincinnati, Cincinnati, OH, USA

    Pankaj Dwivedi & Kenneth D. Greis

  2. Division of Immunobiology and Center for Systems Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

    David E. Muench & H. Leighton Grimes

  3. Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA

    David E. Muench, Michael Wagner, Mohammad Azam & H. Leighton Grimes

  4. Division of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

    Michael Wagner

  5. Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

    Mohammad Azam & H. Leighton Grimes

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  1. Pankaj Dwivedi
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Correspondence to H. Leighton Grimes or Kenneth D. Greis.

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Dwivedi, P., Muench, D.E., Wagner, M. et al. Time resolved quantitative phospho-tyrosine analysis reveals Bruton’s Tyrosine kinase mediated signaling downstream of the mutated granulocyte-colony stimulating factor receptors. Leukemia 33, 75–87 (2019). https://doi.org/10.1038/s41375-018-0188-8

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