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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Touw IP, Palande K, Beekman R. Granulocyte colony-stimulating factor receptor signaling: implications for G-CSF responses and leukemic progression in severe congenital neutropenia. Hematol Oncol Clin North Am. 2013;27:61–73.
Dwivedi P, Greis KD. Granulocyte colony-stimulating factor receptor signaling in severe congenital neutropenia, chronic neutrophilic leukemia, and related malignancies. Exp Hematol. 2017;46:9–20.
Beekman R, Touw IP. G-CSF and its receptor in myeloid malignancy. Blood. 2010;115:5131–6.
Rosenberg PS, Alter BP, Bolyard AA, Bonilla MA, Boxer LA, Chem B, et al. The incidence of leukemia and mortality from sepsis in patients with severe congenital neutropenia receiving long-term G-CSF therapy. Blood. 2006;107:4628–35.
Gotlib J, Maxson JE, George TI, Tyner JW. The new genetics of chronic neutrophilic leukemia and atypical CML: implications for diagnosis and treatment. Blood. 2013;122:1707–11.
Touw IP, Beekman R. Severe congenital neutropenia and chronic neutrophilic leukemia: an intriguing molecular connection unveiled by oncogenic mutations in CSF3R. Haematologica. 2013;98:1490–2.
Zhang H, Nguyen-Jackson H, Panopoulos AD, Li HS, Murray PJ, Watowich SS. STAT3 controls myeloid progenitor growth during emergency granulopoiesis. Blood. 2010;116:2462–71.
Beekman R, Valkhof MG, Sanders MA, van Strien PM, Haanstra JR, Broeders L, et al. Sequential gain of mutations in severe congenital neutropenia progressing to acute myeloid leukemia. Blood. 2012;119:5071–7.
Mehta HM, Glaubach T, Long A, Lu H, Przychodzen B, Makishima H, et al. Granulocyte colony stimulating factor receptor T595I (T618I) mutation confers ligand independence and enhanced signaling. Leukemia. 2013;27:2407–10.
Maxson JE, Gotlib J, Pollyea DA, Fleischman AG, Agarwal A, Eide CA, et al. Oncogenic CSF3R mutations in chronic neutrophilic leukemia and atypical CML. N Engl J Med. 2013;368:1781–90.
Liu F, Kunter G, Krem MM, Eades WC, Cain JA, Tomasson MH, et al. Csf3r mutations in mice confer a strong clonal HSC advantage via activation of Stat5. J Clin Invest. 2008;118:946–55.
Pardanani A, Lasho TL, Laborde RR, Elliott M, Hanson CA, Knudson RA, et al. CSF3R T618I is a highly prevalent and specific mutation in chronic neutrophilic leukemia. Leukemia. 2013;27:1870–3.
Dao KH, Solti MB, Maxson JE. Significant clinical response to JAK1/2 inhibition in a patient with CSF3R-T618I-positive atypical chronic myeloid leukemia. Leuk Res Rep. 2014;3:67–9.
Rohrabaugh S, Kesarwani M, Kincaid Z, Huber E, Leddonne J, Siddiqui Z, et al. Enhanced MAPK signaling is essential for CSF3R-induced leukemia. Leukemia. 2017;31:1770–8.
Tony Hunter. Tyrosine phosphorylation: thirty years and counting. Curr Opin Cell Biol. 2009;21:140–6.
Ong SE, Mann M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat Protoc. 2006;1:2650–60.
Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ, et al. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat Biotechnol. 2005;23:94–101.
Szklarczyk D, Franceschini A, Wyder S, Forslund K, Heller D, Huerta-Cepas J, et al. STRING v10: protein–protein interaction networks, integrated over the tree of life. Nucleic Acid Res. 2015; 43:D447–52
Lachmann A, Ma’ayan A. KEA: kinase enrichment analysis. Bioinformatics. 2009;25:684–6.
Maxson JE, Luty SB, MacManiman JD, Abel ML, Druker BJ, Tyner JW. Ligand independence of the T618I mutation in the granulocyte colony-stimulating factor 3 receptor (CSF3R) protein results from loss of O-linked glycosylation and increased receptor dimerization. J Biol Chem. 2014;289:5820–7.
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 leukemiaderived mutation in the G-CSF receptor gene. J Exp Med. 1999;189:683–92.
Aarts LH, Roovers O, Ward AC, Touw IP. Receptor activation and 2 distinct COOHterminal motifs control G-CSF receptor distribution and internalization kinetics. Blood. 2004;104:571–9.
Wilson WH, Young RM, Schmitz RS, Yang Y, Pittaluga S, Wright G, et al. Targeting B cell receptor signaling with ibrutinib in diffuse large B cell lymphoma. Nat Med. 2015;21:922–6.
Rushworth SA, Murray MY, Zaitseva L, Bowles KM, MacEwan DJ. Identification of Bruton’s tyrosine kinase as a therapeutic target in acute myeloid leukemia. Blood. 2014;123:1229–38.
Oellerich T, Mohr S, Corso J, Beck J, Dobele C, Braun H, et al. FLT3-ITD and TLR9 use Bruton Tyrosine kinase to activate distinct transcriptional programs mediating AML cell survival and proliferation. Blood. 2015;125:1936–47.
Wilson TR, Fridlyand J, Yan Y, Penuel E, Burton L, Chan E, et al. Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors. Nature. 2012;487:505–9.
Stuible M, Abella JV, Feldhammer M, Nossov M, Sangwan V, Blagoev B, et al. Ptpb1b targets the endosomal sorting machinery: dephosphorylation of regulatory sites in the endosomal sorting complex required for transport component STAM2. J Biol Chem. 2010;285:23899–907.
Wang L, Rudert WA, Loutaev I, Roginskaya V, Corey SJ. Repression of c-Cbl leads to enhanced G-CSF Jak-STAT signaling without increased cell proliferation. Oncogene. 2002;21:5346–55.
Joffre C, Barrow R, Menard L, Calleja V, Hart IP, Kermogrant S. A direct role for Met endocytosis in tumorigenesis. Nat Cell Biol. 2011;13:827–37.
Weinstein IB. Addiction to oncogenes-the Achilles heal of Cancer. Science. 2002;297:63–4.
Kesharwani M, Kincaid Z, Gomaa A, Huber E, Rohrabaugh S, Siddiqui Z, et al. Trageting c-Fos and DUSP1 abrogates intrinsic resistance to tyrosine-kinase inhibitor therapy in BCR-ABL-induced leukemia. Nat Med. 2017;23:472–82.
Khandanpour C, Phelan JD, Vassen L, Schutte J, Chen R, Horman SR, et al. Growth factor independence 1 antagonizes a p53-induced DNA damage response pathway in lymphoblastic leukemia. Cancer Cell. 2013;23:200–14.
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.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Rights and permissions
About this article
Cite this article
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
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41375-018-0188-8
This article is cited by
-
Insights into pulmonary phosphate homeostasis and osteoclastogenesis emerge from the study of pulmonary alveolar microlithiasis
Nature Communications (2023)
-
Construction of TSC2 knockout cell line using CRISPR/Cas9 system and demonstration of its effects on NIH-3T3 cells
Cell Biochemistry and Biophysics (2022)
-
Discovery and biological evaluation of N-(3-(7-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-4-methyl-2-oxo-2H-pyrimido[4,5-d][1,3]oxazin-1(4H)-yl)phenyl)acrylamide as potent Bruton’s tyrosine kinase inhibitors
Acta Pharmacologica Sinica (2020)
-
Phospho serine and threonine analysis of normal and mutated granulocyte colony stimulating factor receptors
Scientific Data (2019)