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早幼粒细胞阶段的骨髓细胞成熟停滞。在大多数 SCN 患者中已检测到编码中性粒细胞弹性蛋白酶 (NE) 的 ELANE 杂合突变。髓样细胞中 NE 突变体的表达诱导过早凋亡。 SCN 患者患急性髓系白血病 (AML) 的风险增加，从 SCN 进展为 AML 与编码 G-CSF 受体 (G-CSFR) 的 CSF3R 突变有关，这种突变会导致受体 C 末端截短。截短的G-CSFR介导增强的细胞增殖和存活。关于 ELANE 突变导致细胞凋亡的机制以及为什么 CSF3R 突变与 SCN 特异性相关，人们仍然知之甚少。我们观察到野生型（WT）NE的共表达加速了NE突变体诱导的细胞凋亡，并且G-CSFR的C端截短阻断了Elane启动子响应G-CSF的激活。我们的初步数据还确定转录因子 Stat5a 和 Stat5b 是 NE 的潜在靶标，因为 NE 的异位表达会抑制造血细胞中 Stat5 蛋白水平和 Stat5 依赖性增殖和存活。我们假设WT NE的蛋白酶活性在NE突变体诱导细胞凋亡中发挥重要作用，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促细胞凋亡作用来消除NE突变体诱导的细胞凋亡。提出了三个具体目标来检验这些假设。在目标 1 中，我们将评估 WT NE 在 NE 突变体诱导细胞凋亡中的作用。我们将评估NE敲低和过表达对NE突变体诱导细胞凋亡的影响，并确定NE蛋白酶活性是否是NE突变体诱导细胞凋亡所必需的。在目标 2 中，我们将研究截短的 G-CSFR 对原代人骨髓细胞中 NE 表达和 NE 突变体诱导凋亡的影响。在目标3中，我们将进一步探讨NE在Stat5表达/功能负调控中的作用，并探讨NE和NE突变体的共表达是否对Stat5依赖性增殖和存活表现出协同抑制作用。拟议的研究可能为 SCN 的病理生理学及其进展为 AML 提供重要的见解，并可能导致开发 SCN 治疗的新治疗策略。公共卫生相关性：中性粒细胞弹性蛋白酶是一种蛋白酶，在中性粒细胞早期发育过程中产生极高水平，中性粒细胞是一种参与宿主防御细菌感染的白细胞。在本提案中，我们将研究中性粒细胞弹性蛋白酶基因突变如何导致严重先天性中性粒细胞减少症（SCN）（一种血液中中性粒细胞数量异常减少的疾病），以及为什么 SCN 患者患白血病的风险增加。拟议的研究将帮助我们了解 SCN 的潜在致病机制，并可能确定 SCN 治疗干预的潜在靶点。