Mechanisms of Hematopoietic Stem Cell Maintenance

描述（由申请人提供）：本申请的总体目标是揭示造血干细胞（HSC）如何利用细胞凋亡来保护自身和产生髓系细胞，并解决细胞凋亡调节的破坏如何促进髓系恶性肿瘤的发展。虽然目前关于凋亡机制的特定组成部分的机制作用有丰富的信息，但我们仍然缺乏对细胞凋亡如何调节早期干细胞和祖细胞的生物活性的全面理解。此外，我们仍然不完全了解凋亡调控的破坏如何赋予转化的造血干细胞异常的生存特性和对治疗的抗性。在这里，我们将使用一系列互补的方法来剖析正常和病变条件下HSC功能中线粒体和外源性死亡受体（DR）凋亡途径的调控和含义。Specific Aim 1的研究将集中在了解细胞凋亡内在线粒体途径的作用。我们将扩展我们对造血特异性BakBaxcKO小鼠的研究，以描述这种死亡机制对体内血液稳态的精确贡献。我们还将直接测试Bcl2蛋白的比例如何控制hsc和粒细胞/巨噬细胞祖细胞（GMP）中生存和消除之间的平衡。我们将进行体内shRNA筛选以了解促凋亡Bcl2基因的作用，并将使用Bcl2结构域的稳定α螺旋（SAHB）来探索促存活家族成员的功能含义。这些实验将产生详细的分子和细胞理解凋亡信号如何通过内在线粒体途径介导，有助于维持功能性HSC室和调节髓细胞生成。在Specific Aim 2中，我们将讨论外源性DR通路在细胞凋亡中的作用。我们将使用新的原位可视化方法来研究BM腔中DR配体的局部表达如何激活hsc和gmp中的DR通路。我们还将在互补遗传小鼠模型（即Faslpr/lpr，造血特异性Caspase-8cKO， p50-/-小鼠）中使用分子分析，离体分析和体内实验的组合来剖析DR激活在造血干细胞和gmp中的调节和功能结果。这些实验将提供一个独特的理解外源性DR途径如何促进HSC维持和调节髓细胞生成，无论是单独还是与内在线粒体途径合作。特异性目标3的研究将探讨细胞凋亡调控的破坏如何赋予转化的造血干细胞异常的生存特性，并促进骨髓增生性肿瘤（MPN）的发展。我们将使用我们建立的人类mpn小鼠模型（即junb缺陷和诱导的tTA-BCR/ABL小鼠）来确定在转化的hsc和gmp中凋亡机制调控发生的变化，并了解这些调控的缺失对这些群体提供异常生存特性的功能影响。我们还将评估靶向这些凋亡调节的异常特征是否可用于特异性杀死具有白血病起始干细胞（LSC）特性的转化造血干细胞。这些实验将揭示造血干细胞通常用于维持血液稳态的细胞保存机制的破坏如何导致转化的造血干细胞功能异常和髓系恶性肿瘤的发展。

DESCRIPTION (provided by applicant): The overall goal of this application is to uncover how apoptosis is used by hematopoietic stem cells (HSC) to preserve themselves and produce myeloid cells, and to address how corruption of apoptosis regulation contributes to the development of myeloid malignancies. While a wealth of information is currently available on the mechanistic role of particular components of the apoptotic machinery, we still lack a comprehensive understanding of how apoptosis regulates the biological activity of early stem and progenitor cells. Furthermore, we still do not fully understand how corruption of apoptosis regulation endows transformed HSCs with aberrant survival properties and resistance to therapy. Here, we will use an array of complementary approaches to dissect the regulation and implication of the intrinsic mitochondrial and extrinsic death receptor (DR) pathways of apoptosis in HSC function under normal and diseased conditions. Studies in Specific Aim 1 will focus on understanding the role of the intrinsic mitochondrial pathway of apoptosis. We will extend our investigations of hematopoietic-specific BakBaxcKO mice to delineate the precise contribution of this death mechanism to blood homeostasis in vivo. We will also directly test how the ratio of Bcl2 proteins controls the balance between survival and elimination in HSCs and granulocyte/macrophage progenitors (GMP). We will perform an in vivo shRNA screen to understand the role of the pro-apoptotic Bcl2 genes and will use stabilized alpha helices of Bcl2 domains (SAHB) to probe the functional implication of the pro-survival family members. These experiments will yield a detailed molecular and cellular understanding of how apoptotic signals mediated through the intrinsic mitochondrial pathway contribute to the maintenance of a functional HSC compartment and regulate myeloid cell production. In Specific Aim 2, we will address the role of the extrinsic DR pathway of apoptosis. We will use our new in situ visualization approach to investigate how local expression of DR ligands in the BM cavity can activate the DR pathway in HSCs and GMPs. We will also use a combination of molecular profiling, ex vivo analyses and in vivo experiments in complementary genetic mouse models (i.e., Faslpr/lpr, hematopoietic-specific Caspase-8cKO, p50-/- mice) to dissect the regulation and functional outcome of DR activation in HSCs and GMPs. These experiments will provide a unique understanding of how the extrinsic DR pathway contributes to HSC maintenance and regulates myeloid cell production, either by itself or in cooperation with the intrinsic mitochondrial pathway. Studies in Specific Aim 3 will address how corruption of apoptosis regulation endows transformed HSCs with aberrant survival properties and contributes to the development of myeloproliferative neoplasms (MPN). We will use our established mouse models of human MPNs (i.e., junB-deficient and inducible tTA-BCR/ABL mice) to identify changes that occur in the regulation of the apoptotic machinery in transformed HSCs and GMPs, and to understand the functional implications of these deregulations in providing aberrant survival properties to these populations. We will also assess whether targeting these aberrant features of apoptosis regulation can be used to specifically kill transformed HSCs with leukemia-initiating stem cell (LSC) properties. These experiments will uncover how corruption of a mechanism of cell preservation normally used by HSCs to maintain blood homeostasis contributes to the aberrant function of transformed HSCs and the development of myeloid malignancies. PUBLIC HEALTH RELEVANCE: Our proposed investigations will yield a comprehensive understanding of how apoptosis is regulated in early hematopoietic stem and progenitor cells, and will provide essential information on how this stress-response mechanism is used by HSCs to preserve them and maintain myeloid cell production throughout life. Moreover, they will uncover how corrupted regulation of apoptosis endows transformed HSCs with aberrant survival properties and contributes to the development of myeloid malignancies. Collectively, these studies will provide unique insights into the mechanisms of leukemogenesis, and stand to make critical contributions to the identification of molecular targets that could be engaged to destroy the therapy-resistant LSC populations in human myeloid leukemia.