title: 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 α-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.
authors.affiliation: Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. National Health Service (NHS) Blood and Transplant, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. Medical Research Council Biostatistics Unit, Cambridge Institute of Public Health, Cambridge Biomedical Campus, Cambridge CB2 0SR, UK. National Institute for Health Research (NIHR) BioResource-Rare Diseases, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK.
authors.forename: Ernest
authors.initials: E
authors.lastname: Turro
authors.affiliation: Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. Medical Research Council Biostatistics Unit, Cambridge Institute of Public Health, Cambridge Biomedical Campus, Cambridge CB2 0SR, UK. National Institute for Health Research (NIHR) BioResource-Rare Diseases, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK.
authors.forename: Daniel
authors.initials: D
authors.lastname: Greene
authors.affiliation: Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, 3000 Leuven, Belgium.
authors.forename: Anouck
authors.initials: A
authors.lastname: Wijgaerts
authors.affiliation: Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, 3000 Leuven, Belgium.
authors.forename: Chantal
authors.initials: C
authors.lastname: Thys
authors.affiliation: Centre for Haematology, Hammersmith Campus, Imperial College Academic Health Sciences Centre, Imperial College London, London W12 0HS, UK. Imperial College Healthcare NHS Trust, Du Cane Road, London W12 0HS, UK.
authors.forename: Claire
authors.initials: C
authors.lastname: Lentaigne
authors.affiliation: Department of Haematology, University College London Cancer Institute, London WC1E 6BT, UK. Katharine Dormandy Haemophilia Centre and Thrombosis Unit, Royal Free London NHS Foundation Trust, London NW3 2QG, UK.
authors.forename: Tadbir K
authors.initials: TK
authors.lastname: Bariana
authors.affiliation: School of Clinical Sciences, University of Bristol, Bristol BS2 8DZ, UK.
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Westbury
authors.affiliation: Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. National Health Service (NHS) Blood and Transplant, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Kelly
authors.affiliation: Academisch Ziekenhuis Sint-Jan Brugge-Oostende, 8000 Brugge, Belgium.
authors.forename: Dominik
authors.initials: D
authors.lastname: Selleslag
authors.affiliation: Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. National Health Service (NHS) Blood and Transplant, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. National Institute for Health Research (NIHR) BioResource-Rare Diseases, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK.
authors.forename: Jonathan C
authors.initials: JC
authors.lastname: Stephens
authors.affiliation: Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. National Institute for Health Research (NIHR) BioResource-Rare Diseases, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK.
authors.forename: Sofia
authors.initials: S
authors.lastname: Papadia
authors.affiliation: Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. National Institute for Health Research (NIHR) BioResource-Rare Diseases, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK.
authors.forename: Ilenia
authors.initials: I
authors.lastname: Simeoni
authors.affiliation: Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. National Institute for Health Research (NIHR) BioResource-Rare Diseases, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Penkett
authors.affiliation: Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. National Institute for Health Research (NIHR) BioResource-Rare Diseases, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK.
authors.forename: Sofie
authors.initials: S
authors.lastname: Ashford
authors.affiliation: Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. National Health Service (NHS) Blood and Transplant, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. National Institute for Health Research (NIHR) BioResource-Rare Diseases, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK.
authors.forename: Antony
authors.initials: A
authors.lastname: Attwood
authors.affiliation: Department of Haematology, Guy's and St Thomas' NHS Foundation Trust, London SE1 7EH, UK.
authors.forename: Steve
authors.initials: S
authors.lastname: Austin
authors.affiliation: Institute for Immunology and Transfusion Medicine, Universitätsmedizin Greifswald, 17475 Greifswald, Germany.
authors.forename: Tamam
authors.initials: T
authors.lastname: Bakchoul
authors.affiliation: Arthur Bloom Haemophilia Centre, Institute of Infection and Immunity, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK.
authors.forename: Peter
authors.initials: P
authors.lastname: Collins
authors.affiliation: Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. National Institute for Health Research (NIHR) BioResource-Rare Diseases, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK.
authors.forename: Sri V V
authors.initials: SV
authors.lastname: Deevi
authors.affiliation: Assistance Publique-Hôpitaux de Paris, Armand Trousseau Children Hospital, 75012 Paris, France. INSERM U1170, 94805 Villejuif, France.
authors.forename: Rémi
authors.initials: R
authors.lastname: Favier
authors.affiliation: Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. National Health Service (NHS) Blood and Transplant, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK.
authors.forename: Myrto
authors.initials: M
authors.lastname: Kostadima
authors.affiliation: Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Michele P
authors.initials: MP
authors.lastname: Lambert
authors.affiliation: Department of Haematology, Great Ormond Street Hospital for Children NHS Foundation Trust, London WC1N 3JH, UK.
authors.forename: Mary
authors.initials: M
authors.lastname: Mathias
authors.affiliation: Centre for Haematology, Hammersmith Campus, Imperial College Academic Health Sciences Centre, Imperial College London, London W12 0HS, UK. Imperial College Healthcare NHS Trust, Du Cane Road, London W12 0HS, UK.
authors.forename: Carolyn M
authors.initials: CM
authors.lastname: Millar
authors.affiliation: Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, 3000 Leuven, Belgium.
authors.forename: Kathelijne
authors.initials: K
authors.lastname: Peerlinck
authors.affiliation: Department of Haematology, Addenbrooke's Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge Biomedical Campus, Cambridge CB2 0QQ, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Perry
authors.affiliation: Beth Israel Deaconess Medical Centre, Harvard Medical School, Boston, MA 02215, USA.
authors.forename: Sol
authors.initials: S
authors.lastname: Schulman
authors.affiliation: Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. National Health Service (NHS) Blood and Transplant, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK.
authors.forename: Deborah
authors.initials: D
authors.lastname: Whitehorn
authors.affiliation: Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, 3000 Leuven, Belgium.
authors.forename: Christine
authors.initials: C
authors.lastname: Wittevrongel
authors.affiliation: Biochemistry, Molecular and Structural Biology Section, University of Leuven, 3001 Leuven, Belgium.
authors.forename: Marc
authors.initials: M
authors.lastname: De Maeyer
authors.affiliation: Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. Genomics England Ltd., London EC1M 6BQ, UK.
authors.forename: Augusto
authors.initials: A
authors.lastname: Rendon
authors.affiliation: Department of Haematology, University College London Cancer Institute, London WC1E 6BT, UK. Katharine Dormandy Haemophilia Centre and Thrombosis Unit, Royal Free London NHS Foundation Trust, London NW3 2QG, UK.
authors.forename: Keith
authors.initials: K
authors.lastname: Gomez
authors.affiliation: Pathology and Laboratory Medicine, University of Western Australia, Crawley, Western Australia WA 6009, Australia.
authors.forename: Wendy N
authors.initials: WN
authors.lastname: Erber
authors.affiliation: School of Clinical Sciences, University of Bristol, Bristol BS2 8DZ, UK. School of Cellular and Molecular Medicine, University of Bristol, Bristol BS8 1TD, UK.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Mumford
authors.affiliation: Institut Hospitalo-Universitaire LIRYC, PTIB, Hôpital Xavier Arnozan, 33600 Pessac, France.
authors.forename: Paquita
authors.initials: P
authors.lastname: Nurden
authors.affiliation: Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. National Institute for Health Research (NIHR) BioResource-Rare Diseases, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Stirrups
authors.affiliation: National Institute for Health Research (NIHR) BioResource-Rare Diseases, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. Research and Development, Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 0QQ, UK.
authors.forename: John R
authors.initials: JR
authors.lastname: Bradley
authors.affiliation: National Institute for Health Research (NIHR) BioResource-Rare Diseases, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, UK.
authors.forename: F Lucy
authors.initials: FL
authors.lastname: Raymond
authors.affiliation: Centre for Haematology, Hammersmith Campus, Imperial College Academic Health Sciences Centre, Imperial College London, London W12 0HS, UK. Imperial College Healthcare NHS Trust, Du Cane Road, London W12 0HS, UK.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Laffan
authors.affiliation: Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, 3000 Leuven, Belgium.
authors.forename: Chris
authors.initials: C
authors.lastname: Van Geet
authors.affiliation: Medical Research Council Biostatistics Unit, Cambridge Institute of Public Health, Cambridge Biomedical Campus, Cambridge CB2 0SR, UK.
authors.forename: Sylvia
authors.initials: S
authors.lastname: Richardson
authors.affiliation: Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, 3000 Leuven, Belgium. kathleen.freson@med.kuleuven.be.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Freson
authors.affiliation: Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. National Health Service (NHS) Blood and Transplant, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. National Institute for Health Research (NIHR) BioResource-Rare Diseases, Cambridge University Hospitals, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK. Human Genetics, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
authors.forename: Willem H
authors.initials: WH
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mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000473:pathology; D001842:Bone and Bones / Q000473:pathology*; D019556:COS Cells; D002522:Chlorocebus aethiops; D005260:Female; D006410:Hematopoiesis; D006470:Hemorrhage / Q000150:complications / Q000235:genetics*; D006801:Humans; D008297:Male; D009154:Mutation / Q000235:genetics*; D010375:Pedigree; D010641:Phenotype; D055728:Primary Myelofibrosis / Q000150:complications / Q000235:genetics*; D013921:Thrombocytopenia / Q000150:complications / Q000235:genetics*; D014162:Transfection; D015027:Zebrafish; D019061:src-Family Kinases / Q000235:genetics*
chemical_list: D019061:src-Family Kinases