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）如何使用凋亡来保存自身并产生骨髓细胞，并解决凋亡调节的破坏如何有助于骨髓恶性肿瘤的发展。虽然大量的信息是目前可用的特定组件的凋亡机制的机械作用，我们仍然缺乏一个全面的了解细胞凋亡如何调节早期干细胞和祖细胞的生物活性。此外，我们仍然没有完全理解细胞凋亡调控的破坏如何赋予转化的HSC异常的存活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疾病条件下HSC功能中内在线粒体和外在死亡受体（DR）凋亡途径的调节和影响。在特定目标1的研究将集中在了解细胞凋亡的内在线粒体途径的作用。我们将扩大我们的研究造血特异性BakhycKO小鼠，以描绘这种死亡机制的精确贡献，在体内血液稳态。我们还将直接测试Bcl 2蛋白的比例如何控制HSC和粒细胞/巨噬细胞祖细胞（GMP）中存活和消除之间的平衡。我们将进行体内shRNA筛选以了解促凋亡Bcl 2基因的作用，并将使用Bcl 2结构域的稳定化α螺旋（SAHB）来探测促存活家族成员的功能含义。这些实验将产生一个详细的分子和细胞的理解，如何通过内在线粒体途径介导的凋亡信号有助于维持一个功能性的HSC区室和调节骨髓细胞的生产。在具体目标2中，我们将讨论细胞凋亡的外源性DR途径的作用。我们将使用我们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，造血特异性胱天蛋白酶-8cKO，p50-/-小鼠）来剖析HSC和GMP中DR活化的调节和功能结果。这些实验将提供一个独特的理解如何外在DR途径有助于HSC的维护和调节髓系细胞的生产，无论是通过本身或与内在线粒体途径合作。特定目标3的研究将解决凋亡调控的腐败如何赋予转化的HSC异常的存活特性，并有助于骨髓增生性肿瘤（MPN）的发展。我们将使用我们建立的人MPN的小鼠模型（即，junB缺陷型和诱导型tTA-BCR/ABL小鼠），以鉴定转化的HSC和GMP中凋亡机制调节中发生的变化，并了解这些失调在为这些群体提供异常存活特性中的功能意义。我们还将评估是否可以使用靶向这些凋亡调节的异常特征来特异性地杀死具有白血病起始干细胞（LSC）特性的转化的HSC。这些实验将揭示HSC通常用于维持血液稳态的细胞保存机制的破坏如何有助于转化的HSC的异常功能和骨髓恶性肿瘤的发展。

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.