Cleyrat Cédric, Girard Romain, Choi Eun H, Jeziorski Éric, Lavabre-Bertrand Thierry, Hermouet Sylvie, Carillo Serge, Wilson Bridget S
Department of Pathology and Comprehensive Cancer Center, University of New Mexico Health Sciences Center, Albuquerque, NM.
Service de Pédiatrie III, Hôpital Arnaud de Villeneuve, Montpellier, France.
Blood Adv. 2017 Sep 22;1(21):1815-1826. doi: 10.1182/bloodadvances.2016002915. eCollection 2017 Sep 26.
Thrombopoietin (Tpo) and its receptor (Mpl) are the principal regulators of early and late thrombopoiesis and hematopoietic stem cell maintenance. Mutations in can drastically impair its function and be a contributing factor in multiple hematologic malignancies, including congenital amegakaryocytic thrombocytopenia (CAMT). CAMT is characterized by severe thrombocytopenia at birth, which progresses to bone marrow failure and pancytopenia. Here we report unique familial cases of CAMT that presented with a previously unreported mutation: T814C (W272R) in the background of the activating G117T (K39N or Baltimore) mutation. Confocal microscopy, proliferation and surface biotinylation assays, co-immunoprecipitation, and western blotting analysis were used to elucidate the function and trafficking of Mpl mutants. Results showed that Mpl protein bearing the W272R mutation, alone or together with the K39N mutation, lacks detectable surface expression while being strongly colocalized with the endoplasmic reticulum (ER) marker calreticulin. Both WT and K39N-mutated Mpl were found to be signaling competent, but single or double mutants bearing W272R were unresponsive to Tpo. Function of the deficient Mpl receptor could be rescued by using 2 separate approaches: (1) GRASP55 overexpression, which partially restored Tpo-induced signaling of mutant Mpl by activating an autophagy-dependent secretory pathway and thus forcing ER-trapped immature receptors to traffic to the cell surface; and (2) CRISPR-Cas9 gene editing used to repair T814C mutation in transfected cell lines and primary umbilical cord blood-derived CD34 cells. We demonstrate proof of principle for rescue of mutant Mpl function by using gene editing of primary hematopoietic stem cells, which indicates direct therapeutic applications for CAMT patients.
血小板生成素（Tpo）及其受体（Mpl）是早期和晚期血小板生成以及造血干细胞维持的主要调节因子。[基因名称]中的突变可严重损害其功能，并成为包括先天性无巨核细胞血小板减少症（CAMT）在内的多种血液系统恶性肿瘤的一个促成因素。CAMT的特征是出生时严重血小板减少，随后发展为骨髓衰竭和全血细胞减少。在此，我们报告了CAMT的独特家族病例，这些病例呈现出一种先前未报道的[基因名称]突变：在激活的G117T（K39N或巴尔的摩）突变背景下的T814C（W272R）。利用共聚焦显微镜、增殖和表面生物素化分析、免疫共沉淀以及蛋白质印迹分析来阐明Mpl突变体的功能和运输。结果显示，携带W272R突变的Mpl蛋白，单独或与K39N突变一起，缺乏可检测到的表面表达，同时与内质网（ER）标记钙网蛋白强烈共定位。发现野生型和K39N突变的Mpl均具有信号传导能力，但携带W272R的单突变或双突变体对Tpo无反应。可通过两种不同方法挽救缺陷型Mpl受体的功能：（1）GRASP55过表达，通过激活自噬依赖性分泌途径部分恢复突变型Mpl的Tpo诱导信号，从而迫使内质网滞留的未成熟受体运输到细胞表面；（2）CRISPR-Cas9基因编辑用于修复转染细胞系和原代脐带血来源的CD34细胞中的T814C突变。我们通过对原代造血干细胞进行基因编辑证明了挽救突变型Mpl功能的原理，这表明对CAMT患者有直接的治疗应用。
