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

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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.", "claims": [{"id": "claim-method", "statement": "Based on Pearson coefficient values, a high reproducibility was observed between replicates (Supplementary Figure 1b).", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:29977015", "locator": "sections[7] \"SILAC- based quantitative phospho-tyrosine analysis of the normal and mutated G-CSFR signaling\" paragraph 3", "quote": "Based on Pearson coefficient values, a high reproducibility was observed between replicates (Supplementary Figure 1b)."}], "state": "complete"}, {"id": "claim-data-access", "statement": "The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD009662", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:29977015", "locator": "sections[15] \"Phosphoproteomics data deposition\" paragraph 2", "quote": "The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD009662"}], "state": "complete"}], "gaps": []}, "experiment": {"state": "complete", "objective": null, "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:29977015"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "complete", "blocked_on": null, "description": "The GEO series accession resolves and its sample accessions are listed. 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Primary murine progenitor cells from G-CSFR-Q741x knock-in", "r clonogenic potential was displayed by both murine and human primary cells expressing mutated r", "ine progenitor cells from G-CSFR-Q741x knock-in mice validated activation of Btk by the mutant re"], "order_basis": "paper_text_order:1200", "id": "assay:1"}, {"role": "data_generation", "definition_id": "mass_spectrometry", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["ata demonstrate the strength of unsupervised proteomics analyses in dissecting oncogenic pathways, a", "tion was confirmed to be greater than 95% by mass spectrometry, the G-CSF induction was initiated for colle", "ntary Figure 1b). To further verify that the mass spectrometry readout was consistent with the timing of th", "o-Stat5 (pY694) peptides from the individual mass spectrometry data sets were examined (Figure 2b). As expe"], "order_basis": "paper_text_order:1775", "id": "assay:2"}, {"role": "data_generation", "definition_id": "western_blot", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["m on the cellular machinery, phospho-Stat3/5 immunoblots on the SILAC lysates were evaluated. As expe", "ing of the Stat3/Stat5 signaling seen by the immunoblot experiments, extracted ion chromatograms (XI", "tion of pStat5 and pStat3 as was detected by immunoblot (Figures 1c, 2a). Overall, the quantitative"], "order_basis": "paper_text_order:4192", "id": "assay:3"}, {"role": "data_generation", "definition_id": "flow_cytometry", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["neration of G-CSFR expressing 32D cells. (b) Flow cytometry plots of transduced 32D cells validating the", "e expected levels of G-CSFR expression after FACS-sorting. 5Figure 3s. 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