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. 2016 Mar 2;8(328):328ra30.
doi: 10.1126/scitranslmed.aad7666. Epub 2016 Mar 2.

A Dominant Gain-Of-Function Mutation in Universal Tyrosine Kinase SRC Causes Thrombocytopenia, Myelofibrosis, Bleeding, and Bone Pathologies

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Free PMC article

A Dominant Gain-Of-Function Mutation in Universal Tyrosine Kinase SRC Causes Thrombocytopenia, Myelofibrosis, Bleeding, and Bone Pathologies

Ernest Turro et al. Sci Transl Med. .
Free PMC article

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.

Conflict of interest statement

Competing interests: The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1. Selection of the c.1579G>A mutation in SRC as a candidate pathogenic variant.
(A) Pedigree showing male (square) and female (circle) members who have macrothrombocytopenia (blue), are unaffected (empty), or are without clinical information (gray), some of whom are deceased (slash). Cases carry the c.1579G>A mutation in SRC (M) in variant calling done by whole-genome sequencing (WGS) (#), whole-exome sequencing (WES) (*), and/or Sanger sequencing (all genotyped subjects). (B) Bar plot showing the mean phenotypic similarity of cases 13, 31, and 35 to OMIM/Mouse Phenotype Ontology (MPO) phenotypes associated with each gene, truncated at the top 20 genes, with novel variants absent from control data indicated by * and SRC highlighted in blue [as in (D) and (E)]. (C) The numbers inside each node indicate which cases were coded with the corresponding HPO term. Terms in green are also present in the OMIM/MPO entries for SRC/Src. The size of each node is determined by its contribution to the mean phenotype similarity score between the three cases and the OMIM/MPO terms for SRC/Src. See Materials and Methods for abbreviations. The cases of this pedigree were the only ones enrolled in the BRIDGE Bleeding and Platelet Disorders (BRIDGE-BPD) study coded with “Thrombocytopenia,” “Myelofibrosis,” and “Abnormality of the skeletal system.” (D) Bar plot showing the CADD Phred score of the rare variant for each candidate gene. (E) Probability that each candidate gene is specifically overexpressed in MKs compared to blood stem cells and six other hematopoietic progenitors.
Fig. 2
Fig. 2. Platelet phenotype related to SRC E527K gain-of-function mutation.
(A) Blood smears from cases 31, 35, and 19 showing large platelets with a grayish appearance (black arrows). EM images of platelets for the three cases show (i) round platelets that rarely have a normal discoid shape, (ii) some dysmorphic platelets (DP), (iii) platelets with many open canalicular system (OCS)–forming vacuoles, (iv) a reduced level of microtubules (MT), and (v) a reduced number of α-granules with a subpopulation of platelets exhibiting small granules (SG) or a near absence of all types of granules, reduced levels of internal membranes, and a cytoplasm that appears amorphous. Scale bars, 1 μm. See fig. S2 for EM images of normal platelets. (B and C) The platelet area and number of total α-granules/platelet were quantified. Values are the means and SEM as quantified for 50 randomly selected platelets for a healthy control (C) and cases 31, 35, and 19. ****P < 0.0001, one-way analysis of variance (ANOVA) with Bonferroni’s multiple test. (D) Western blot shows VWF, TSP1, and integrin β3 (ITGB3 as loading control) expression for platelets from a healthy control and cases 31, 35, and 19. (E) Left: Flow cytometric analysis of P-selectin (CD62P) on the outer membrane of platelets from case 35 (blue) and an unrelated healthy control [red, wild type (WT)] after activation with collagen. Right: Summarized mean fluorescence intensities (MFIs) for two healthy controls (red) and cases 19, 31, and 35 (blue) (see the Supplementary Materials for analysis and fig. S3 for representative image of convulxin activation). (F) Crystallography-based models of the SRC structures show the interaction of the C-terminal regulatory tail with SH2 domain residues (fig. S5). Left: Zoom-in of the surroundings of the atomic environment of phosphorylated Y530 in WT SRC. Residues 158, 159, 178, and 181 of the SH2 domain involved in the interaction with residues 520, 521, 527, and 530 of the kinase domain (Velcro strap) are in stick mode. Electrostatic interactions are shown with orange dotted lines, and the temperature factor of the Velcro strap is shown with a blue to red gradient (from lower and higher flexibility, respectively). Right: Similar structure for the SRC-K527 mutant protein but note the reduced predicted flexibility of the Velcro strap (residues are blue), leading to a rearrangement between the Velcro strap and SH2 domain. (G) Left: SRC kinase activity was measured with a colorimetric enzyme-linked immunosorbent assay (ELISA) assay using different concentrations of ATP. The optical density at 450 nm (OD450) was measured for glutathione S-transferase (GST)–tagged WT (GST-WT) and E527K (GST-527K) SRC protein with and without the addition of the SRC inhibitor. Right: Total (Pan), active (Y419), and inactive (Y530) SRC protein levels were assayed via immunoblot analysis of GST-WT and GST-527K fusion proteins used for the kinase assay.
Fig. 3
Fig. 3. SRC expression studies in platelets and transfected COS-7 cells.
(A) Triplicate Western blots for total (Pan), active (Y419), and inactive (Y530) SRC were performed for platelet lysates obtained from nonstimulated and collagen-stimulated platelets for 30, 60, and 300 s for two controls and three cases (19, 31, 34). Left: All blots were quantified, and linear mixed model was fitted to the log-normalized mean pixel intensity data of the SRC protein levels in platelets for carriers of the WT or mutant allele. The residuals after adjusting for genotype and antibody fixed effects are shown for the Y419- and Y530-targeting antibodies. Right: Representative blot. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as normalization control. The unaffected relative 21 has normal levels of total, active, and inactive SRC in nonstimulated platelets. (B) Immunoblot analysis showing whole-platelet tyrosine phosphorylation (4G10 antibody) stimulated by collagen or ADP from 30 to 300 s in platelets from a control and cases 19 and 35. The same platelet lysates were used as above. (C) Western blots were performed for lysates obtained from COS-7 cells transfected with empty (V), SRC-WT, and SRC-K527 vectors from a triplicate transfection experiment. Left: Residuals obtained after fitting a model without an interaction term between the presence of the mutant allele and the level of binding of anti-Y530 antibody. Such a term is required to obtain a good model fit due to the marked decrease of Y530 in cells carrying the mutant rather than the WT allele (see the Supplementary Materials). Right: Representative blot. (D) Western blots were performed for lysates obtained from COS-7 cells transfected with the same SRC vectors as in (C) with or without addition of SRC inhibitor-1 (inh) in a triplicate transfection experiment. Left: Residuals obtained after fitting a model without a fixed inhibitor term. Such a term is required to obtain a good model fit (see the Supplementary Materials). Right: Representative blot.
Fig. 4
Fig. 4. Effect of the SRC E527K gain-of-function mutation on megakaryopoiesis.
(A) Plasma TPO levels (pg/ml) and platelet count (PLT) (1 × 109/liter) for a patient with proven CAMT (black square), three SRC cases (blue symbols), and five unrelated healthy controls (red circles). The assay sensitivity limit is 25 pg/ml (indicated by red line). Squares and circles in blue represent samples collected in 2004 and 2014, respectively, for SRC cases. (B) Total amount of CFU (colony-forming units)–MK (upper panel) and CFU-GEMM (lower panel) colonies derived from peripheral blood CD34+ mononuclear cells from a control (C1) and case 31 counted at days 7 and 12 of culture. Values are means and SEM as quantified for a triplicate plating experiment. Upper panel: day 7, **P = 0.0059; day 12, ***P = 0.0006; lower panel: day 7, **P = 0.0015; day 12, **P = 0.0048, one-way ANOVA with Bonferroni’s multiple test. (C) Left: Representative light microscopy images of cultured MK showing formation of proplatelet extensions (white arrows) for the controls (C1 and C2). Proplatelet-forming MKs are almost absent for the cases. Scale bars, 20 μm. Right: Proplatelet formation was quantified in MK suspension triplicated liquid cultures (for each condition) performed on two separate occasions (first, two controls and three cases, and second, a control, case 31, and unaffected relative 21 with and without SRC inhibitor-1). The proportion of proplatelet formation was lower in the cultures from cases compared to controls but normal for the unaffected case. Addition of the inhibitor resulted in a rescue of the proplatelet formation defect. Values are means and SEM as counted for MKs present in 10 randomly selected slides for each condition. ****P < 0.0001, *P = 0.049, one-way ANOVA with Bonferroni’s correction. (D) Representative immunofluorescence confocal microscopy images of differentiated fibrinogen-adhered MKs at day 12 of culture visualized for active SRC (green, Y419) and 4′,6-diamidino-2-phenylindole (blue), showing rosette-like structures in MKs from case 31, whereas control MK (C3) only stained weakly positive for active SRC. Scale bars, 5 μm. Quantification of MKs positive for active SRC staining showed a significant difference between controls and cases. Values are means and SEM as counted for 10 randomly selected images for each condition. P = 0.0003, t test. (E) Western blots for total (Pan), active (Y419), inactive (Y530) SRC, green fluorescent protein (GFP), and overall tyrosine phosphorylation (4G10) were performed for lentiviral-transduced cells differentiated for 12 days to MKs. GFP only generates a signal for the control vector. WT-transduced cells overexpress inactive SRC, whereas E527K-transduced cells express active SRC. The overexpression of SRC in WT- and E527K-transduced cells is also visible in the 4G10 blots, although E527K-transduced MKs also display increased tyrosine phosphorylation of other proteins. (F) Quantification of the ploidy analysis of two independent transduction experiments showed a significant left shift for E527K transduced MKs (blue) indicative of more immature diploid (2N DNA) MKs compared to WT (red). For the interaction between ploidy and genotype: ****P < 0.0001, two-way ANOVA. (G) Proplatelet formation was quantified in MK suspension cultures from a duplicated transduction experiment in a blinded fashion. The proportion of proplatelet formation was lower in the cultures from E527K-transduced cells compared to WT conditions. Values are means and SEM as counted for MKs present in 10 randomly selected slides for each condition. For both WT-1 versus E527K-1 and WT-2 versus E527K-2: ****P < 0.0001, one-way ANOVA with Bonferroni’s correction. (H) Immunofluorescence confocal microscopy images of differentiated fibrinogenadhered and lentiviral-transduced MKs at day 12 of culture visualized for active SRC (green, Y419) and phalloidin (red, F-actin), showing colocalization in MKs from E527K-transduced MKs but not in WT-transduced cells. Scale bars, 20 μm.
Fig. 5
Fig. 5. Phenotype analysis of zebrafish injected with buffer, src-E525K, or src-WT mRNA in the absence or presence of SRC inhibitor-1.
(A) Left: Stereomicroscope images of the CHT region in the tail at 3 dpf to visualize the GFP-labeled thrombocytes using Tg(cd41:EGFP) zebrafish. Middle: Immunoblot analysis of 15 lysed embryos at 3 dpf for expression of total Src, GFP, and total tyrosine phosphorylation using 4G10 antibody. Right: Blot quantification of a triplicate injection experiment showed a trend for reduced GFP levels for E525K-injected embryos that is corrected when the inhibitor is added to the fish water. (B) Alcian blue cartilage staining at 5 dpf showing cartilage defects in src-E525K overexpression embryos that can be rescued by SRC inhibition. pq, palatoquadrate; mc, Meckel’s cartilage; cb, ceratobranchials; cl, cranial length (as specified in fig. S15). Left: Representative images. Middle: Graph of length ratio of palatoquadrate, Meckel’s cartilage, and ceratobranchials cartilages standardized by cranial length. Right: Graph of area ratio of palatoquadrate, Meckel’s cartilage, and ceratobranchials cartilages standardized by cranial length. Values are means and SEM as quantified for six randomly selected embryos for each condition. Length: ***P = 0.0002, ****P < 0.0001, *P = 0.0196. Area: ****P < 0.0001, ***P = 0.0006, one-way ANOVA with Bonferroni’s correction.

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