ELANE and CSF3R mutations in severe congenital neutropenia

严重先天性中性粒细胞减少症中的 ELANE 和 CSF3R 突变

基本信息

批准号：
8367147
负责人：
FAN DONG
金额：
$ 43.65万
依托单位：
UNIVERSITY OF TOLEDO
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-01 至 2017-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Severe congenital neutropenia (SCN) is a group of heterogeneous disorders characterized by severe absolute neutropenia and a maturation arrest of myeloid cells at the promyelocyte stage. Heterozygous mutations in ELANE encoding neutrophil elastase (NE) have been detected in most SCN patients. Expression of the NE mutants in myeloid cells induces premature apoptosis. Patients with SCN are at increased risk of developing acute myeloid leukemia (AML) and progression from SCN to AML is associated with mutations in CSF3R encoding the G-CSF receptor (G-CSFR), which lead to C-terminal truncation of the receptor. The truncated G- CSFRs mediate augmented cell proliferation and survival. It is still poorly understood about the mechanism by which the ELANE mutations cause apoptosis and why CSF3R mutations are specifically associated with SCN. We have observed that co-expression of the wild type (WT) NE accelerates apoptosis induced by the NE mutant and that the C-terminal truncation of the G-CSFR blocks the activation of the Elane promoter in response to G-CSF. Our preliminary data also identified transcription factors Stat5a and Stat5b as potential targets of NE in that ectopic expression of NE inhibits Stat5 protein levels and Stat5-depedent proliferation and survival in hematopoietic cells. We hypothesize that the protease activity of WT NE plays an important role in apoptosis induction by the NE mutants and that the nonsense mutations in CSF3R abolishes NE mutant- induced apoptosis by suppressing the expression of the NE mutants and directly counteracting their pro- apoptotic action. Three specific aims are proposed to test these hypotheses. In aim 1, we will evaluate the involvement of WT NE in apoptosis induction by the NE mutants. We will assess the effects of NE knockdown and overexpression on apoptosis induction by the NE mutants, and determine whether the protease activity of NE is required for apoptosis induced by the NE mutants. In aim 2, we will investigate the effects of the truncated G-CSFR on NE expression and apoptosis induction by the NE mutant in primary human bone marrow cells. In aim 3, we will further explore the role of NE in the negative regulation of Stat5 expression/function and address whether co-expression of NE and the NE mutants display a synergistic inhibitory effect on Stat5-depdent proliferation and survival. The proposed studies may provide critical insight into the pathophysiology of SCN and its progression to AML, and may lead to the development of new therapeutic strategies for the treatment of SCN. PUBLIC HEALTH RELEVANCE: Neutrophil elastase is a protease that is produced at extremely high levels during the early development of neutrophils, a type of white blood cells involved in host defense against bacterial infections. In this proposal, we will investigate how mutations in neutrophil elastase gene cause severe congenital neutropenia (SCN), a condition with an abnormal decrease in the number of neutrophils in the blood, and why patients with SCN are at increased risk for developing leukemia. The proposed studies will help us understand the underlying pathogenic mechanisms of SCN and may lead to the identification of potential targets for therapeutic intervention in the treatment of SCN.

描述（由申请人提供）：严重的先天性中性粒细胞减少症（SCN）是一组异质性疾病，其特征是严重的绝对中性粒细胞减少症和骨髓细胞阶段的髓样细胞成熟。在大多数SCN患者中，已经检测到编码嗜中性粒细胞弹性酶（NE）的烯烃中的杂合突变。 NE突变体在髓样细胞中的表达诱导过早凋亡。 SCN患者患急性髓样白血病（AML）的风险增加，从SCN到AML的进展与编码G-CSF受体（G-CSFR）的CSF3R突变有关，这导致受体的C末端截断。截短的G-CSFR介导增强的细胞增殖和存活。关于烯烃突变引起凋亡的机制以及CSF3R突变与SCN特别相关的机制，它仍然鲜为人知。我们已经观察到，野生型（WT）NE的共表达会加速由NE突变体诱导的凋亡，而G-CSFR的C末端截断会阻止Elane启动子激活G-CSF。我们的初步数据还将转录因子STAT5A和STAT5B确定为NE的潜在靶标，因为NE的异位表达抑制了造血细胞中STAT5蛋白水平和Stat5蛋白质水平和STAT5蛋白质增殖和存活。我们假设WT NE的蛋白酶活性在NE突变体的凋亡诱导中起重要作用，并且CSF3R中的无义突变通过抑制NE突变体的表达并直接抵消其肢体作用来消除NE突变体诱导的凋亡。提出了三个特定目标来检验这些假设。在AIM 1中，我们将评估NE突变体的WT NE参与凋亡诱导。我们将评估NE敲低和过表达对NE突变体凋亡诱导的影响，并确定NE突变体诱导的凋亡所必需的NE的蛋白酶活性。在AIM 2中，我们将研究截短的G-CSFR对原代人骨髓细胞中NE突变体诱导NE表达和凋亡诱导的影响。在AIM 3中，我们将进一步探讨NE在STAT5表达/功能的负调控中的作用，并解决NE和NE突变体的共表达是否对STAT5 epdent的增殖和存活产生协同抑制作用。拟议的研究可能会对SCN的病理生理及其向AML的发展提供批判性见解，并可能导致开发用于治疗SCN的新治疗策略。公共卫生相关性：中性粒细胞弹性酶是一种蛋白酶，在嗜中性粒细胞的早期发育期间以极高的水平产生，这是一种涉及宿主防御细菌感染的白细胞。在此提案中，我们将研究中性粒细胞弹性酶基因的突变如何引起严重的先天性嗜中性粒细胞减少症（SCN），这种疾病的血液中嗜中性粒细胞数量异常降低，而SCN患者的患者的风险增加了患白血病的风险。拟议的研究将有助于我们了解SCN的潜在致病机制，并可能导致鉴定SCN治疗的潜在靶标。