CLONAL DOMINANCE OF HEMATOPOIETIC STEM CELLS EXPRESSING MUTANT CSF3R

描述（由申请人提供）：白血病干细胞（LSCs）的一个基本特性是克隆优势。克隆优势是指LSCs在骨髓微环境中以牺牲正常造血细胞为代价进行克隆扩增。在白血病发生过程中积累的突变赋予克隆优势的机制在很大程度上是未知的。严重先天性中性粒细胞减少症（SCN）是一种骨髓衰竭综合征，其特征是发展为急性髓性白血病的显著倾向。编码G-CSF受体（G-CSFR）的CSF3R的截断突变是与SCN患者白血病进展相关的常见早期突变。我们之前描述了携带Csf3r敲入突变的转基因小鼠（称为d715 G-CSFR），该突变复制了SCN中发现的突变。我们的初步数据表明，d715 G-CSFR在造血干细胞（HSC）水平上具有很强的克隆优势。重要的是，克隆性HSC的优势依赖于G-CSF的给药，这为研究克隆优势提供了一种新的、生理的、“可诱导的”模型。我们建议使用该模型来表征表达突变体CSF3R的造血干细胞获得克隆优势的分子机制。更好地了解这些机制可能为开发特异性靶向LSCs的分子疗法提供新的策略。这是高度相关的，因为有证据表明LSCs对大多数当前的化疗具有固有的抗性。初步数据表明，d715 G-CSFR增强STAT5激活是介导克隆性HSC优势的近端信号。这一假设将通过有条件地删除Stat5和评估对HSC功能的影响来验证。在特定目标2中，我们将确定d715 G-CSFR造血干细胞中G-CSF失调的基因，并优先考虑它们进行生物学验证。初步的mRNA表达谱研究已经确定了G-CSF在d715 G-CSFR HSC中差异诱导的两个候选基因：Cdkn1a （p21cip1/waf1）和Enah。我们假设d715 G-CSFR诱导Cdkn1a表达允许HSC增殖而不丧失自我更新。我们还假设Enah表达的增加有利于改变造血干细胞与骨髓基质的相互作用。提出以下具体目标。目的1。我们将确定Stat5是否介导表达d715 G-CSFR的造血干细胞的克隆优势。目标2。我们将在d715个G-CSFR造血干细胞中发现导致克隆优势的G-CSF失调基因。目标3。我们将明确Cdkn1a和Enah在表达d715 G-CSFR的造血干细胞克隆优势中的作用。公共卫生相关性：克隆优势是所有白血病的一个基本但鲜为人知的特性，它允许白血病干细胞以牺牲正常血细胞为代价扩张。本研究的目的是提高我们对介导克隆优势的途径的理解。我们相信这项研究将带来新的策略，专门针对白血病干细胞，并最终提高白血病患者的治愈率。

DESCRIPTION (provided by applicant): A fundamental property of leukemic stem cells (LSCs) is clonal dominance. Clonal dominance refers to the clonal expansion of LSCs within the bone marrow microenvironment at the expense of normal hematopoietic cells. The mechanisms by which mutations that accumulate during leukemogenesis confer clonal dominance are largely unknown. Severe congenital neutropenia (SCN) is a bone marrow failure syndrome characterized by a marked propensity to develop acute myeloid leukemia. Truncation mutations of CSF3R, encoding the G-CSF receptor (G-CSFR), are common early mutations associated with leukemic progression in patients with SCN. We previously described transgenic mice (termed d715 G-CSFR) carrying a knock-in mutation of Csf3r that reproduces a mutation found in SCN. Our preliminary data show that the d715 G-CSFR confers a strong clonal advantage at the hematopoietic stem cell (HSC) level. Importantly, the clonal HSC advantage is dependent upon G-CSF administration, providing a novel, physiological, and "inducible" model to study clonal dominance. We propose to use this model to characterize the molecular mechanisms by which HSCs expressing mutant CSF3R gain clonal dominance. A better understanding of these mechanisms may provide novel strategies to develop molecular therapies that specifically target LSCs. This is highly relevant, since there is evidence that LSCs are inherently resistant to most current chemotherapy. Preliminary data suggest that accentuated STAT5 activation by the d715 G-CSFR is a proximal signal mediating the clonal HSC advantage. This hypothesis will be tested by conditionally deleting Stat5 and assessing the effect on HSC function. In specific aim 2, we will identify genes that are dysregulated by G-CSF in d715 G-CSFR HSCs and prioritize them for biological validation. Preliminary mRNA expression profiling studies have identified two candidate genes that are differentially induced by G-CSF in d715 G-CSFR HSC: Cdkn1a (p21cip1/waf1) and Enah. We hypothesize that induction of Cdkn1a expression by the d715 G-CSFR allows for HSC proliferation without loss of self-renewal. We also hypothesize that increased Enah expression favorably alters the interaction of HSCs with bone marrow stromal. The following specific aims are proposed. Aim 1. We will determine whether Stat5 mediates the clonal advantage of HSCs expressing the d715 G-CSFR. Aim 2. We will identify genes dysregulated by G-CSF in d715 G-CSFR HSCs that contribute to clonal dominance. Aim 3. We will define the role of Cdkn1a and Enah in the clonal advantage of HSCs expressing the d715 G-CSFR. PUBLIC HEALTH RELEVANCE: Clonal dominance is a fundamental but poorly understood property of all leukemias that allows the leukemic stem cell to expand at the expense of normal blood cells. The goal of this research is to improve our understanding of the pathways that mediate clonal dominance. We believe this research will lead to novel strategies to specifically target the leukemic stem cell and ultimately lead to improved cure rates in individuals with leukemia.