Characterization of the Platelet Phenotype Caused by a Germline RUNX1 Variant in a CRISPR/Cas9-Generated Murine Model

Ana Marín-Quílez; Ignacio García-Tuñón; Cristina Fernández-Infante; Luis Hernández-Cano; Verónica Palma-Barqueros; Elena Vuelta; Manuel Sánchez-Martín; José Ramón González-Porras; Carmen Guerrero; Rocío Benito; José Rivera; Jesús María Hernández-Rivas; José María Bastida

doi:10.1055/s-0041-1723987

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2021; 121(09): 1193-1205
DOI: 10.1055/s-0041-1723987

Cellular Haemostasis and Platelets

Characterization of the Platelet Phenotype Caused by a Germline RUNX1 Variant in a CRISPR/Cas9-Generated Murine Model

Ana Marín-Quílez

¹Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain

,

Ignacio García-Tuñón

¹Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain

,

Cristina Fernández-Infante

¹Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain

,

Luis Hernández-Cano

¹Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain

,

Verónica Palma-Barqueros

²Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, University of Murcia, Murcia, Spain

,

Elena Vuelta

¹Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain

³Transgenic Facility, Nucleus, University of Salamanca, Salamanca, Spain

,

Manuel Sánchez-Martín

¹Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain

³Transgenic Facility, Nucleus, University of Salamanca, Salamanca, Spain

⁴Department of Medicine, University of Salamanca, Salamanca, Spain

,

José Ramón González-Porras

⁴Department of Medicine, University of Salamanca, Salamanca, Spain

⁵Department of Hematology, University Hospital of Salamanca - IBSAL, Salamanca, Spain

,

Carmen Guerrero

¹Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain

⁴Department of Medicine, University of Salamanca, Salamanca, Spain

,

Rocío Benito

¹Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain

,

José Rivera^*

²Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, University of Murcia, Murcia, Spain

⁶On behalf of the “Grupo Español de Alteraciones Plaquetarias Congénitas (GEAPC)”, Hemorrhagic Diathesis Working Group, SETH

,

Jesús María Hernández-Rivas^*

¹Cancer Research Center - CSIC, Instituto de Investigación Biomédica de Salamanca, University of Salamanca, Salamanca, Spain

⁴Department of Medicine, University of Salamanca, Salamanca, Spain

⁵Department of Hematology, University Hospital of Salamanca - IBSAL, Salamanca, Spain

,

José María Bastida ^*

⁴Department of Medicine, University of Salamanca, Salamanca, Spain

⁵Department of Hematology, University Hospital of Salamanca - IBSAL, Salamanca, Spain

⁶On behalf of the “Grupo Español de Alteraciones Plaquetarias Congénitas (GEAPC)”, Hemorrhagic Diathesis Working Group, SETH

› Institutsangaben

Abstract

RUNX1-related disorder (RUNX1-RD) is caused by germline variants affecting the RUNX1 gene. This rare, heterogeneous disorder has no specific clinical or laboratory phenotype, making genetic diagnosis necessary. Although international recommendations have been established to classify the pathogenicity of variants, identifying the causative alteration remains a challenge in RUNX1-RD. Murine models may be useful not only for definitively settling the controversy about the pathogenicity of certain RUNX1 variants, but also for elucidating the mechanisms of molecular pathogenesis. Therefore, we developed a knock-in murine model, using the CRISPR/Cas9 system, carrying the RUNX1 p.Leu43Ser variant (mimicking human p.Leu56Ser) to study its pathogenic potential and mechanisms of platelet dysfunction. A total number of 75 mice were generated; 25 per genotype (RUNX1^WT/WT, RUNX1^WT/L43S, and RUNX1^L43S/L43S). Platelet phenotype was assessed by flow cytometry and confocal microscopy. On average, RUNX1^L43S/L43S and RUNX1^WT/L43S mice had a significantly longer tail-bleeding time than RUNX1^WT/WT mice, indicating the variant's involvement in hemostasis. However, only homozygous mice displayed mild thrombocytopenia. RUNX1^L43S/L43S and RUNX1^WT/L43S displayed impaired agonist-induced spreading and α-granule release, with no differences in δ-granule secretion. Levels of integrin α_IIbβ₃ activation, fibrinogen binding, and aggregation were significantly lower in platelets from RUNX1^L43S/L43S and RUNX1^WT/L43S using phorbol 12-myristate 13-acetate (PMA), adenosine diphosphate (ADP), and high thrombin doses. Lower levels of PKC phosphorylation in RUNX1^L43S/L43S and RUNX1^WT/L43S suggested that the PKC-signaling pathway was impaired. Overall, we demonstrated the deleterious effect of the RUNX1 p.Leu56Ser variant in mice via the impairment of integrin α_IIbβ₃ activation, aggregation, α-granule secretion, and platelet spreading, mimicking the phenotype associated with RUNX1 variants in the clinical setting.

Keywords

RUNX1 - inherited platelet disorders - platelet function - mouse model - CRISPR/Cas9

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Referenzen

References
1 Kurokawa M. AML1/Runx1 as a versatile regulator of hematopoiesis: regulation of its function and a role in adult hematopoiesis. Int J Hematol 2006; 84 (02) 136-142
2 Ichikawa M, Asai T, Saito T. et al. AML-1 is required for megakaryocytic maturation and lymphocytic differentiation, but not for maintenance of hematopoietic stem cells in adult hematopoiesis. Nat Med 2004; 10 (03) 299-304
3 Growney JD, Shigematsu H, Li Z. et al. Loss of Runx1 perturbs adult hematopoiesis and is associated with a myeloproliferative phenotype. Blood 2005; 106 (02) 494-504
4 Wang Q, Stacy T, Binder M, Marin-Padilla M, Sharpe AH, Speck NA. Disruption of the Cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis. Proc Natl Acad Sci U S A 1996; 93 (08) 3444-3449
5 Arber DA, Orazi A, Hasserjian R. et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood 2016; 127 (20) 2391-2405
6 Song WJ, Sullivan MG, Legare RD. et al. Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia. Nat Genet 1999; 23 (02) 166-175
7 Bastida JM, Benito R, Lozano ML. et al. Molecular diagnosis of inherited coagulation and bleeding disorders. Semin Thromb Hemost 2019; 45 (07) 695-707
8 Stockley J, Morgan NV, Bem D. et al; UK Genotyping and Phenotyping of Platelets Study Group. Enrichment of FLI1 and RUNX1 mutations in families with excessive bleeding and platelet dense granule secretion defects. Blood 2013; 122 (25) 4090-4093
9 Lentaigne C, Freson K, Laffan MA, Turro E, Ouwehand WH. BRIDGE-BPD Consortium and the ThromboGenomics Consortium. Inherited platelet disorders: toward DNA-based diagnosis. Blood 2016; 127 (23) 2814-2823
10 Bastida JM, Lozano ML, Benito R. et al. Introducing high-throughput sequencing into mainstream genetic diagnosis practice in inherited platelet disorders. Haematologica 2018; 103 (01) 148-162
11 Brown AL, Arts P, Carmichael CL. et al. RUNX1-mutated families show phenotype heterogeneity and a somatic mutation profile unique to germline predisposed AML. Blood Adv 2020; 4 (06) 1131-1144
12 Richards S, Aziz N, Bale S. et al; ACMG Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015; 17 (05) 405-424
13 Garcia JS, Madzo J, Cooper D. et al. Pre-Donor evaluation of an HLA matched sibling identifies a novel inherited RUNX1 mutation encoding a missense mutation found outside of the RUNT domain in familial platelet disorder. Blood 2010; 116 (21) 2709
14 Koh CP, Wang CQ, Ng CEL. et al. RUNX1 meets MLL: epigenetic regulation of hematopoiesis by two leukemia genes. Leukemia 2013; 27 (09) 1793-1802
15 Prieto-Conde MI, Labrador J, Hermida G. et al. Genomic analysis of a familial myelodysplasia/acute myeloid leukemia and inherited RUNX1 mutations without a pre-existing platelet disorder. Leuk Lymphoma 2020; 61 (01) 181-184
16 Brown AL, Hahn C, Hiwase D, Godley LA, Scott HS. Correct application of variant classification guidelines in germline RUNX1 mutated disorders to assist clinical diagnosis. Leuk Lymphoma 2020; 61 (01) 246-247
17 Duployez N, Fenwarth L. Controversies about germline RUNX1 missense variants. Leuk Lymphoma 2020; 61 (02) 497-499
18 Sakurai M, Kunimoto H, Watanabe N. et al. Impaired hematopoietic differentiation of RUNX1-mutated induced pluripotent stem cells derived from FPD/AML patients. Leukemia 2014; 28 (12) 2344-2354
19 Lamolda M, Montes R, Simón I. et al. GENYOi005-A: an induced pluripotent stem cells (iPSCs) line generated from a patient with familial platelet disorder with associated myeloid malignancy (FPDMM) carrying a p.Thr196Ala variant. Stem Cell Res (Amst) 2019; 41: 101603
20 Liu Y, Jennings NL, Dart AM, Du XJ. Standardizing a simpler, more sensitive and accurate tail bleeding assay in mice. World J Exp Med 2012; 2 (02) 30-36
21 Gutiérrez-Herrero S, Fernández-Infante C, Hernández-Cano L. et al. C3G contributes to platelet activation and aggregation by regulating major signaling pathways. Signal Transduct Target Ther 2020; 5 (01) 29
22 Lood C, Tydén H, Gullstrand B. et al. Decreased platelet size is associated with platelet activation and anti-phospholipid syndrome in systemic lupus erythematosus. Rheumatology (Oxford) 2017; 56 (03) 408-416
23 Hardy AT, Palma-Barqueros V, Watson SK. et al. Significant hypo-responsiveness to GPVI and CLEC-2 agonists in pre-term and full-term neonatal platelets and following immune thrombocytopenia. Thromb Haemost 2018; 118 (06) 1009-1020
24 Gutiérrez-Herrero S, Maia V, Gutiérrez-Berzal J. et al. C3G transgenic mouse models with specific expression in platelets reveal a new role for C3G in platelet clotting through its GEF activity. Biochim Biophys Acta 2012; 1823 (08) 1366-1377
25 De Cuyper IM, Meinders M, van de Vijver E. et al. A novel flow cytometry-based platelet aggregation assay. Blood 2013; 121 (10) e70-e80
26 Tucker KL, Sage T, Gibbins JM. Clot retraction. Methods Mol Biol 2012; 788: 101-107
27 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Δ Δ C(T)) method. Methods 2001; 25 (04) 402-408
28 Ortiz-Rivero S, Baquero C, Hernández-Cano L. et al. C3G, through its GEF activity, induces megakaryocytic differentiation and proplatelet formation. Cell Commun Signal 2018; 16 (01) 101
29 Trevino AE, Zhang F. Genome editing using Cas9 nickases. Methods Enzymol 2014; 546: 161-174
30 Cifuni SM, Wagner DD, Bergmeier W. CalDAG-GEFI and protein kinase C represent alternative pathways leading to activation of integrin alphaIIbbeta3 in platelets. Blood 2008; 112 (05) 1696-1703
31 Hayashi Y, Harada Y, Huang G, Harada H. Myeloid neoplasms with germ line RUNX1 mutation. Int J Hematol 2017; 106 (02) 183-188
32 Duarte BKL, Yamaguti-Hayakawa GG, Medina SS. et al. Longitudinal sequencing of RUNX1 familial platelet disorder: new insights into genetic mechanisms of transformation to myeloid malignancies. Br J Haematol 2019; 186 (05) 724-734
33 Owen CJ, Toze CL, Koochin A. et al. Five new pedigrees with inherited RUNX1 mutations causing familial platelet disorder with propensity to myeloid malignancy. Blood 2008; 112 (12) 4639-4645
34 Rao AK, Poncz M. Defective acid hydrolase secretion in RUNX1 haplodeficiency: evidence for a global platelet secretory defect. Haemophilia 2017; 23 (05) 784-792
35 Glembotsky AC, Bluteau D, Espasandin YR. et al. Mechanisms underlying platelet function defect in a pedigree with familial platelet disorder with a predisposition to acute myelogenous leukemia: potential role for candidate RUNX1 targets. J Thromb Haemost 2014; 12 (05) 761-772
36 Antony-Debré I, Manchev VT, Balayn N. et al. Level of RUNX1 activity is critical for leukemic predisposition but not for thrombocytopenia. Blood 2015; 125 (06) 930-940
37 Morowski M, Vögtle T, Kraft P, Kleinschnitz C, Stoll G, Nieswandt B. Only severe thrombocytopenia results in bleeding and defective thrombus formation in mice. Blood 2013; 121 (24) 4938-4947
38 Heijnen H, van der Sluijs P. Platelet secretory behaviour: as diverse as the granules … or not?. J Thromb Haemost 2015; 13 (12) 2141-2151
39 Gabbeta J, Yang X, Sun L, McLane MA, Niewiarowski S, Rao AK. Abnormal inside-out signal transduction-dependent activation of glycoprotein IIb-IIIa in a patient with impaired pleckstrin phosphorylation. Blood 1996; 87 (04) 1368-1376
40 Sood R, Kamikubo Y, Liu P. Role of RUNX1 in hematological malignancies. Blood 2017; 129 (15) 2070-2082
41 Franke B, van Triest M, de Bruijn KMT. et al. Sequential regulation of the small GTPase Rap1 in human platelets. Mol Cell Biol 2000; 20 (03) 779-785
42 Harper MT, Poole AW. Diverse functions of protein kinase C isoforms in platelet activation and thrombus formation. J Thromb Haemost 2010; 8 (03) 454-462
43 Jalagadugula G, Mao G, Kaur G, Dhanasekaran DN, Rao AK. Platelet protein kinase C-θ deficiency with human RUNX1 mutation: PRKCQ is a transcriptional target of RUNX1. Arterioscler Thromb Vasc Biol 2011; 31 (04) 921-927
44 Hug BA, Ahmed N, Robbins JA, Lazar MA. A chromatin immunoprecipitation screen reveals protein kinase Cbeta as a direct RUNX1 target gene. J Biol Chem 2004; 279 (02) 825-830
45 Sun L, Mao G, Rao AK. Association of CBFA2 mutation with decreased platelet PKC-θ and impaired receptor-mediated activation of GPIIb-IIIa and pleckstrin phosphorylation: proteins regulated by CBFA2 play a role in GPIIb-IIIa activation. Blood 2004; 103 (03) 948-954
46 Bynagari-Settipalli YS, Cornelissen I, Palmer D. et al. Redundancy and interaction of thrombin- and collagen-mediated platelet activation in tail bleeding and carotid thrombosis in mice. Arterioscler Thromb Vasc Biol 2014; 34 (12) 2563-2569
47 Schlegelberger B, Heller PG. RUNX1 deficiency (familial platelet disorder with predisposition to myeloid leukemia, FPDMM). Semin Hematol 2017; 54 (02) 75-80
48 Jagadeeswaran P, Cooley BC, Gross PL, Mackman N. Animal models of thrombosis from zebrafish to nonhuman primates. Circ Res 2016; 118 (09) 1363-1379
49 Lin Q, Zhang Y, Zhou R. et al. Establishment of a congenital amegakaryocytic thrombocytopenia model and a thrombocyte-specific reporter line in zebrafish. Leukemia 2017; 31 (05) 1206-1216
50 Marconi C, Di Buduo CA, LeVine K. et al. Loss-of-function mutations in PTPRJ cause a new form of inherited thrombocytopenia. Blood 2019; 133 (12) 1346-1357

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