Downregulation of TREM-like transcript (TLT)-1 and collagen receptor α2 subunit, two novel RUNX1-targets, contributes to platelet dysfunction in familial platelet disorder with predisposition to acute myelogenous leukemia

Abstract

Germline RUNX1 mutations lead to thrombocytopenia and platelet dysfunction in familialplatelet disorder with predisposition to acute myelogenous leukemia. Multiple aspects ofplatelet function are impaired in these patients, associated with altered expression of genesregulated by RUNX1. We aimed to identify RUNX1-targets involved in platelet functionby combining transcriptome analysis of patient and shRUNX1-transduced megakaryocytes.Downregulated genes included TREM-like transcript (TLT)-1 (TREML1) and the integrinsubunit α2 (ITGA2) of collagen receptor α2β1, which are involved in platelet aggregationand adhesion, respectively. RUNX1 binding to regions enriched for H3K27Ac marks wasdemonstrated for both genes using chromatin immunoprecipitation. Cloning of theseregions upstream of the respective promoters in lentivirus allowing mCherry reporterexpression showed that RUNX1 positively regulates TREML1 and ITGA2 and thisregulation was abrogated after deletion of RUNX1 sites. TLT-1 content was reduced inpatient megakaryocytes and platelets. A blocking anti-TLT-1 antibody was able to blockaggregation of normal but not patient platelets, whereas recombinant soluble TLT-1potentiated fibrinogen binding to patient platelets, pointing to a role for TLT-1 deficiencyin the platelet function defect. Low levels of α2 integrin subunit were demonstrated inpatient platelets and megakaryocytes, coupled with reduced platelet and megakaryocyteadhesion to collagen, both under static and flow conditions. In conclusion, we show thatgene expression profiling of RUNX1 knock-down or mutated megakaryocytes provides asuitable approach to identify novel RUNX1-targets, among which downregulation ofTREML1 and ITGA2 clearly contribute to the platelet phenotype of familial plateletdisorder with predisposition to acute myelogenous leukemia.