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A dominant gain-of-function mutation in universal tyrosine kinase SRC causes thrombocytopenia, myelofibrosis, bleeding, and bone pathologies.

ABSTRACT: The Src family kinase (SFK) member SRC is a major target in drug development because it is activated in many human cancers, yet deleterious SRC germline mutations have not been reported. We used genome sequencing and Human Phenotype Ontology patient coding to identify a gain-of-function mutation in SRC causing thrombocytopenia, myelofibrosis, bleeding, and bone pathologies in nine cases. Modeling of the E527K substitution predicts loss of SRC's self-inhibitory capacity, which we confirmed with in vitro studies showing increased SRC kinase activity and enhanced Tyr(419) phosphorylation in COS-7 cells overexpressing E527K SRC. The active form of SRC predominates in patients' platelets, resulting in enhanced overall tyrosine phosphorylation. Patients with myelofibrosis have hypercellular bone marrow with trilineage dysplasia, and their stem cells grown in vitro form more myeloid and megakaryocyte (MK) colonies than control cells. These MKs generate platelets that are dysmorphic, low in number, highly variable in size, and have a paucity of ?-granules. Overactive SRC in patient-derived MKs causes a reduction in proplatelet formation, which can be rescued by SRC kinase inhibition. Stem cells transduced with lentiviral E527K SRC form MKs with a similar defect and enhanced tyrosine phosphorylation levels. Patient-derived and E527K-transduced MKs show Y419 SRC-positive stained podosomes that induce altered actin organization. Expression of mutated src in zebrafish recapitulates patients' blood and bone phenotypes. Similar studies of platelets and MKs may reveal the mechanism underlying the severe bleeding frequently observed in cancer patients treated with next-generation SFK inhibitors.

SUBMITTER: Turro E

PROVIDER: S-EPMC5903547 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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A dominant gain-of-function mutation in universal tyrosine kinase SRC causes thrombocytopenia, myelofibrosis, bleeding, and bone pathologies.

Turro Ernest E Greene Daniel D Wijgaerts Anouck A Thys Chantal C Lentaigne Claire C Bariana Tadbir K TK Westbury Sarah K SK Kelly Anne M AM Selleslag Dominik D Stephens Jonathan C JC Papadia Sofia S Simeoni Ilenia I Penkett Christopher J CJ Ashford Sofie S Attwood Antony A Austin Steve S Bakchoul Tamam T Collins Peter P Deevi Sri V V SV Favier Rémi R Kostadima Myrto M Lambert Michele P MP Mathias Mary M Millar Carolyn M CM Peerlinck Kathelijne K Perry David J DJ Schulman Sol S Whitehorn Deborah D Wittevrongel Christine C De Maeyer Marc M Rendon Augusto A Gomez Keith K Erber Wendy N WN Mumford Andrew D AD Nurden Paquita P Stirrups Kathleen K Bradley John R JR Raymond F Lucy FL Laffan Michael A MA Van Geet Chris C Richardson Sylvia S Freson Kathleen K Ouwehand Willem H WH

Science translational medicine 20160302 328

The Src family kinase (SFK) member SRC is a major target in drug development because it is activated in many human cancers, yet deleterious SRC germline mutations have not been reported. We used genome sequencing and Human Phenotype Ontology patient coding to identify a gain-of-function mutation in SRC causing thrombocytopenia, myelofibrosis, bleeding, and bone pathologies in nine cases. Modeling of the E527K substitution predicts loss of SRC's self-inhibitory capacity, which we confirmed with i ...[more]

PMID: 26936507

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Project description:Severe neonatal thrombocytopenia is a rare disease with multiple etiologies. Severe thrombocytopenia with bleeding is life-threatening and has attracted significant attention from clinicians. However, only a few studies have focused on the association between severe thrombocytopenia and bleeding. Thus, this study aimed to describe the neonates' postnatal age at which severe thrombocytopenia was first recognized, clinical characteristics, bleeding patterns, and outcomes and to evaluate the association between minimum platelet count and bleeding. A single-center retrospective cohort study for neonates with severe thrombocytopenia (platelet count ≤ 50 × 109/L) was conducted. Neonates who were admitted to our neonatal intensive care unit between October 2016 and February 2021 and developed severe thrombocytopenia were analyzed. Data were collected retrospectively until the patients were referred to other hospitals, discharged, or deceased. Among the 5819 neonatal inpatients, 170 with severe thrombocytopenia were included in this study. More than 30% of the patients had severe thrombocytopenia in the first 3 days of life. Among the 118 neonates with bleeding, 47 had more than one type of pathological bleeding. Neonates with very severe thrombocytopenia (point estimate: 53.7%, 95% confidence interval [CI]: 44.2%-63.1%) had a higher incidence rate of cutaneous bleeding than those with severe thrombocytopenia (point estimate: 23.4%, 95% CI: 12.3%-34.4%). The gestational age (median: 36.2 [interquartile range [IQR]: 31.4-39.0] weeks) and birth weight (median: 2310 [IQR: 1213-3210] g) of the major bleeding group were the lowest among no bleeding, minor bleeding, and major bleeding groups. Regression analysis controlled for confounders and confirmed that a lower platelet count (odds ratio [OR]: 2.504 [95% CI: 1.180-5.314], P = 0.017) was associated with a significant increase in the rate of bleeding. Very severe thrombocytopenia (point estimate: 49.1%, 95% CI: 39.6%-58.6%) had a higher rate of platelet transfusion than severe thrombocytopenia (point estimate: 5.7%, 95% CI: 0.7%-10.7%). The mortality rate was higher in neonates with bleeding than in those without bleeding (point estimates with 95% CI: 33.1% [24.4%-41.7%] vs. 7.7% [0.2%-15.2%]). These findings describe the incidence of severe thrombocytopenia and demonstrate that a lower platelet count is associated with an increased bleeding rate in patients with severe thrombocytopenia.

Project description:Protein kinases are enzymes that catalyze the covalent transfer of the γ-phosphate of an adenosine triphosphate (ATP) molecule onto a tyrosine, serine, threonine, or histidine residue in the substrate and thus send a chemical signal to networks of downstream proteins. They are important cellular signaling enzymes that regulate cell growth, proliferation, metabolism, differentiation, and migration. Unregulated protein kinase activity is often associated with a wide range of diseases, therefore making protein kinases major therapeutic targets. A prototypical system of central interest to understand the regulation of kinase activity is provided by tyrosine kinase c-Src, which belongs to the family of Src-related non-receptor tyrosine kinases (SFKs). Although the broad picture of autoinhibition via the regulatory domains and via the phosphorylation of the C-terminal tail is well characterized from a structural point of view, a detailed mechanistic understanding at the atomic-level is lacking. Advanced computational methods based on all-atom molecular dynamics (MD) simulations are employed to advance our understanding of tyrosine kinase activation. The computational studies suggest that the isolated kinase domain (KD) is energetically most favorable in the inactive conformation when the activation loop (A-loop) of the KD is not phosphorylated. The KD makes transient visits to a catalytically competent active-like conformation. The process of bimolecular trans-autophosphorylation of the A-loop eventually locks the KD in the active state. Activating point mutations may act by slightly increasing the population of the active-like conformation, enhancing the availability of the A-loop to be phosphorylated. The Src-homology 2 (SH2) and Src-homology 3 (SH3) regulatory domains, depending upon their configuration, either promote the inactive or the active state of the kinase domain. In addition to the roles played by the SH3, SH2, and KD, the Src-homology 4-Unique domain (SH4-U) region also serves as a key moderator of substrate specificity and kinase function. Thus, a fundamental understanding of the conformational propensity of the SH4-U region and how this affects the association to the membrane surface are likely to lead to the discovery of new intermediate states and alternate strategies for inhibition of kinase activity for drug discovery. The existence of a multitude of KD conformations poses a great challenge aimed at the design of specific inhibitors. One promising computational strategy to explore the conformational flexibility of the KD is to construct Markov state models from aggregated MD data.

Dataset Information

A dominant gain-of-function mutation in universal tyrosine kinase SRC causes thrombocytopenia, myelofibrosis, bleeding, and bone pathologies.

Publications

A dominant gain-of-function mutation in universal tyrosine kinase SRC causes thrombocytopenia, myelofibrosis, bleeding, and bone pathologies.

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