A Dominant Gain-of-Function Mutation in Universal Tyrosine Kinase SRC Causes Enhanced Podosome Formation in a Syndrome with Thrombocytopenia, Myelofibrosis, Bleeding and Bone Pathologies
Authors: Freson Kathleen , Turro Ernest , Ouwehand Willem 
Published: October 2019

Abstract
For the BRIDGE-Bleeding and Platelet Disorder Consortium.
We here present a pedigree with nine cases that present with a new syndrome characterized by early onset myelofibrosis, thrombocytopenia, bleeding and bone pathologies. The syndrome is caused by a dominant gain-of-function mutation in universal tyrosine kinase SRC and it is the first example of a pathogenic germline mutation in this extensively studied kinase. The genetic discovery has been made possible by making use of our collection of 1,115 patients with inherited bleeding and platelet disorders of unknown molecular etiology enrolled by us at 25 tertiary referral centers as part of the NIHR BioResource - Rare Diseases pilot phase of the 100,000 Genomes Project. The analysis of genome sequencing data for 2 affected individuals was empowered by comparison of Human Phenotype Ontology (HPO; Westbury et al., Genome Medicine, 2015) terms of the patients with OMIM and the Mouse Phenotype Ontology terms from 7,541 strains. The selection of the SRC gene as the most likely candidate gene was also informed by the use of our Blueprint blood cell progenitor RNA-seq data (Chen et al., Science 2014).
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 Tyr419 phosphorylation in COS7 cells overexpressing E527K SRC. The active form of SRC predominates in patients’ platelets, resulting in enhanced overall tyrosine phosphorylation and reduced platelet activity. Patients with myelofibrosis have a hypercellular bone marrow with trilineage dysplasia and their stem cells grown in vitro form more myeloid and megakaryocyte (MK) colonies than control cells. Patient MKs generate platelets which are dysmorphic, low in number, highly variable in size and with a paucity of α-granules. Over-active 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 enhanced tyrosine phosphorylation levels and with reduced ploidy compared to cells transduced with wild-type SRC. Patient-derived and E527K-transduced MKs show Y419 SRC positive stained podosomes that induce a strongly altered organization of filamentous actin. Expression of mutated src in zebrafish recapitulates patients’ blood and bone phenotypes. Our findings reveal an unexpected and wide-ranging pathobiology for this universal kinase Src with podosome formation.
In hematopoiesis constitutive active Src primarily affects megakaryopoiesis with ineffective formation of platelets. Its inadequate function is associated with bleeding. The kinase Src is activated frequently in human cancers hence new SFK inhibitors are undergoing clinical trials and side effects with severe bleeding are observed frequently. It remains to be determined whether this new class of SFK inhibitors inadvertently causes inadequate formation of platelets, which do not function properly.

Disclosures:
No relevant conflicts of interest to declare.