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Low TLT-1 and collagen receptor α2 in FPD/AML
Introduction
The transcription factor RUNX1 is a key regulator of the megakaryocytic lineage, where it participates in a complex transcriptional network co-ordinating platelet biogenesis and function.1,2 RUNX1 co-operates with other transcriptional regulators, including GATA-1, FLI-1 and SCL at megakaryocyte (MK)-specific promoters,1 whereas co-occupancy of RUNX1 with FLI-1 and NF-E2 has been shown to prime the late MK program.3 Conditional RUNX1 inactivation in mice leads to MK maturation arrest and a substantial decline in platelet counts, high- lighting the key role of RUNX1 as a master regulator of the MK lineage.4 Germline RUNX1 mutations in humans underlie familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML), which is char- acterized by thrombocytopenia, platelet dysfunction, and a lifelong 30-50% predisposition to hematologic malig- nancies, including myeloid and lymphoid neoplasms.5 RUNX1 mutations exerting dominant-negative effects over the wild-type (WT) protein are associated with a higher leukemic rate than those acting via haploinsuffi- ciency,6 whereas no differences in the severity of the platelet phenotype are seen between both types of muta- tions.7 Although once considered a rare condition, FPD/AML is now diagnosed at increasing frequency due to heightened diagnostic awareness during the workup of individuals presenting with thrombocytopenia of uncer- tain etiology or hereditary myeloid malignancies.
The platelet defect in FPD/AML is complex and includes abnormalities in platelet number and function, which lead to a bleeding diathesis of variable severity, ranging from mild or asymptomatic cases to a severe bleeding tendency. Thrombocytopenia is usually mild to moderate and is caused by impaired platelet production secondary to defects in multiple steps of MK develop- ment, including MK differentiation, maturation, poly- ploidization and proplatelet formation.2 While marked dysmegakaryopoiesis with a severe defect in proplatelet formation is observed in vitro, the presence of only mild thrombocytopenia, often at the lower limit of normal range, suggests a yet unknown compensatory mechanism in vivo. The platelet function defect is present in most, if not all, patients with FPD/AML and involves multiple abnormalities in platelet structure and activation path- ways, including defective platelet aggregation and release, dense granule deficiency, associated in some pedigrees with partial alpha (α)-granule defect, and impaired αIIb-beta (b)-3 (GPIIbIIIa) activation and out- side-in signaling.8,9 These abnormalities are likely due to altered expression of RUNX1-targets involved in platelet biology. The study of FPD/AML platelet samples has revealed downregulation of several RUNX1-regulated genes. Transcriptome analysis of platelets from one patient showed reduced levels of MYL9, ALOX12, PKCθ, RAB1B and PLDN,10 whereas dysregulated expression of MPL,11 MYH1012, RAB27B8 and NF-E28 has been identified by a candidate-gene approach in other pedigrees. However, the mechanisms underlying FPD/AML platelet function defect and the effects of RUNX1 mutations on the expression of other potential genes are still not com- pletely understood. In this study, we combined expres- sion profiling of mature shRUNX1-transduced and FPD/AML MK to gain further insight into RUNX1-regu- lated genes involved in platelet function. Using this
approach, we identified triggering receptor expressed on myeloid cells (TREM)-like transcript (TLT)-1 and integrin subunit α2 of collagen receptor α2b1 as two novel RUNX-1 targets, whose expression was decreased in FPD/AML MK and platelets.
Methods
Human samples
Patients from three previously described FPD/AML pedi- grees2,8,11 (Table 1 and Online Supplementary Table S1), healthy subjects, and individuals after stem cell mobilization were included. At the time of the study, patients had thrombocytope- nia and/or platelet dysfunction with no evidence of myelodys- plastic or leukemic transformation. Details on experiments per- formed on each patient are provided in Online Supplementary Table S2. The study was approved by the Ethics Committee of INSERM RBM 01-14 for the project “Network on the inherited diseases of platelet function and platelet production” in France and the Ethics Committee of the Instituto de Investigaciones Médicas “Dr. Alfredo Lanari” in Argentina. Patients and controls gave signed informed consent.
Megakaryocyte culture and transcriptome analysis
CD34+ cells were isolated from cord blood, leukapheresis samples or peripheral blood of patients and healthy subjects using a magnetic cell-sorting system (AutoMACS or MiniMACS, Miltenyi Biotec SAS, Paris, France) and grown in serum free medium2 or Stem Span medium (StemCell Technologies, Vancouver, BC, Canada), supplemented with 10 ng/mL thrombopoietin (TPO) (Kirin Brewery, Tokyo, Japan or Miltenyi Biotec) and 25 ng/mL Stem Cell Factor (SCF) (Biovitrum AB, Stockholm, Sweden or Miltenyi Biotec). For cul- ture of patient MK, 10 ng/mL IL-6 (Tebu or Miltenyi Biotec), 100 U/mL IL-3 (Novartis or R&D Systems, MN, USA) and 1 ng/mL fetal liver tyrosine kinase 3 ligand (FLT3-L) (Celldex Therapeutics or R&D Systems) were added.
For transcriptome analysis, patient and control MK were cul- tured as detailed above, stained on day 10 of culture with allo- phycocyanin (APC)-conjugated anti-CD41 and phycoerythrin (PE)-anti-CD42 antibodies (BD Biosciences, Le Pont de Claix, France), and CD41+CD42+ were sorted by flow cytometry. CD34+ cells from leukapheresis samples were transduced on days 6 and 7 of culture with lentiviruses encoding shRUNX1_1, shRUNX1_2 and shSCR (control shRNA), and CD41+CD42+GFP+ cells were sorted on day 10 by flow cytome- try, as previously described.2,13 RNA was extracted using the RNeasy Micro Kit (Qiagen, France) according to the manufactur- er’s instructions. Transcriptome analysis was performed using the Agilent Whole Human Genome Microarray (see Online Supplementary Methods).
Statistical analysis
For comparison between patients and controls, Mann- Whitney test or Wilcoxon matched pairs test were applied. For promoter activity and chromatin immunoprecipitation-poly- merase chain reaction (ChIP-PCR) assays, paired t-test was used. For assessment of the effect of a blocking anti-TLT-1 antibody on proplatelet formation, repeated measures ANOVA was used. All statistical analyses were two-sided; P<0.05 was considered significant. The GraphPad Prism 6.01 (La Jolla, CA, USA) soft- ware was used for analysis.
Other methods are described in the Online Supplementary Appendix.
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