Mechanisms of Hematopoietic Stem Cell Maintenance

描述(申请人提供)：本申请的总体目标是揭示细胞凋亡是如何被造血干细胞(HSC)用来保存自己和产生髓系细胞的，并解决了细胞凋亡调节的破坏如何促进髓系恶性肿瘤的发展。虽然目前已有大量关于细胞凋亡机制特定成分的机制作用的信息，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学活性的全面了解。此外，我们仍然不完全了解凋亡调控的破坏是如何赋予转化的HSCs异常的生存特性和对治疗的抵抗的。在这里，我们将使用一系列互补的方法来剖析在正常和疾病条件下内源性线粒体和外源性死亡受体(DR)凋亡通路在HSC功能中的调节和意义。具体目标1的研究将侧重于了解线粒体内部途径在细胞凋亡中的作用。我们将扩大我们对造血特异性BakBaxcKO小鼠的研究，以描绘这种死亡机制对体内血液动态平衡的确切贡献。我们还将直接测试Bcl2蛋白的比例如何控制HSCs和粒/巨噬细胞前体细胞(GMP)的存活和消除之间的平衡。我们将在体内进行shRNA筛选，以了解促凋亡基因Bcl2的作用，并将使用稳定的Bcl2结构域的α螺旋(SAHB)来探索促生存家族成员的功能含义。这些实验将产生详细的分子和细胞方面的了解，即通过内在线粒体途径介导的凋亡信号如何有助于维持HSC的功能并调节髓系细胞的产生。在特定的目标2中，我们将讨论外源性DR途径在细胞凋亡中的作用。我们将使用我们的新的原位可视化方法来研究骨髓腔中DR配体的局部表达如何激活HSCs和GMP中的DR途径。我们还将结合分子图谱、体外分析和体内实验在互补基因小鼠模型(即Faslpr/LPR、造血特异性Caspase-8cKO、p50-/-小鼠)中分析DR激活在HSCs和GMPs中的调节和功能结果。这些实验将对外源性DR途径如何单独或与内在线粒体途径共同作用于HSC的维持和调节髓系细胞的产生提供独特的理解。在特定目标3中的研究将解决如何破坏凋亡调节赋予转化的HSCs异常的生存特性，并有助于骨髓增生性肿瘤(MPN)的发展。我们将使用我们已建立的人类MPN的小鼠模型(即JunB缺陷和可诱导的TTA-BCR/ABL小鼠)来识别转化的HSCs和GMP在调节凋亡机制方面发生的变化，并了解这些放松调控在为这些人群提供异常生存特性方面的功能含义。我们还将评估针对这些异常的凋亡调节功能是否可以用于特异性地杀死具有白血病启动干细胞(LSC)特性的转化的HSC。这些实验将揭示HSCs通常用来维持血液平衡的细胞保存机制的破坏如何导致转化的HSCs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.