Role of ThPOK in HSC Maintenance and Leukemogenesis

 描述（由申请人提供）：造血干细胞（HSC）功能和血液癌症中涉及的基因之间存在大量重叠，表明HSC功能失调如何可能导致转化。事实上，某些形式的白血病对化疗的抗性归因于一小部分白血病干细胞（LSC），其能够像HSC一样自我更新和分化，但缺乏适当的控制机制。我们有令人信服的数据表明，重要的新功能的转录因子ThPOK既作为一个正常的调节HSC的维持/分化，并作为一个调解人的白血病。我们提出根据两个具体目标来定义这些功能：目标1：定义ThPOK在HSC分化/维持中的作用。HSC的功能取决于HSC自我更新和分化之间的平衡。这在ThPOK缺陷小鼠中被破坏，因为长期自我更新的HSC减少，而髓样祖细胞扩增。为了评估ThPOK在控制HSC自我更新和分化中的作用，我们将测试ThPOK消融对以下各项的影响：a）通过有限稀释骨髓移植（BMT）测定的HSC频率，B）通过非竞争性系列BMT测定的长期HSC重建，c）通过竞争性BMT测定相对于wt HSC竞争性重建造血的能力，d）HSC和祖细胞群体在体内和体外的分化能力，以及e）鉴定HSC中ThPOK的靶基因，以开始定义ThPOK调节HSC平衡的潜在机制。 目的2：阐明ThPOK在白血病发生中的作用。白血病起始细胞（LIC）或白血病干细胞（LSC）被认为具有自我更新和分化的同时能力，并代表耐药白血病的关键介质。我们的初步结果表明，ThPOK缺陷的Lin-BM细胞在体外Hoxa 9/Meis 1依赖性急性髓性白血病（AML）模型中部分地受到保护，可能是由于LIC生成减少。相反，我们表明，ThPOK在T细胞系中的过度表达促进了长寿的淋巴瘤祖细胞群的出现。因此，在当前的目标中，我们提出解决以下关键问题：a）在AML的良好建立的体内MLL-AF 9和AML-ETO模型中，ThPOK是启动AML所必需的吗？B）ThPOK对于AML的体内维持是必需的吗？c）ThPOK是否促进L-HSC和L-GMP的白血病起始活性？d）抑制人AML细胞系中的ThPOK是否减少白血病细胞生长并诱导分化？

 DESCRIPTION (provided by applicant): There is substantial overlap between genes involved in hematopoietic stem cell (HSC) function and in hematological cancers, demonstrating how disregulation of HSC function may predispose to transformation. Indeed, resistance of some forms of leukemia to chemotherapy has been attributed to a small fraction of leukemic-stem cells (LSCs), which are capable of self-renewal and differentiation like HSCs, but lack proper control mechanisms. We have compelling data to suggest important novel functions for the transcription factor ThPOK both as a normal regulator of HSC maintenance/differentiation, and as a mediator of leukemogenesis. We propose to define these functions according to two specific aims: Aim 1: Defining the role of ThPOK in HSC differentiation/maintenance. HSC function depends on a careful balance between HSC self-renewal and differentiation. This is disrupted in ThPOK-deficient mice, since long-term self-renewing HSCs are diminished while myeloid progenitors are expanded. To assess the role of ThPOK in control of HSC self renewal and differentiation, we will test the effect of ThPOK ablation on: a) HSC frequency by limited dilution bone marrow transplantation (BMT) assay, b) Long-term HSC reconstitution by non-competitive serial BMT assays, c) Ability to competitively reconstitute hematopoiesis versus wt HSCs by competitive BMT assay, d) Proliferative capacity of HSCs and progenitor populations in vivo and in vitro, and also e) Identify target genes of ThPOK in HSCs, to begin to define the underlying mechanism/s by which ThPOK regulates HSC balance. Aim 2: Elucidating the role of ThPOK in leukemogenesis. Leukemia-initiating cells (LICs) or leukemic- stem-cells (LSCs) are postulated to have simultaneous capacity for self-renewal and differentiation and to represent critical mediators of drug-resistant leukemia. Our preliminary results indicate that ThPOK-deficient Lin- BM cells are partly protected from leukemogenesis in an in vitro Hoxa9/Meis1-dependent acute myeloid leukemia (AML) model, potentially due to diminished generation of LICs. Conversely, we show that overexpression of ThPOK in the T cell lineage promotes appearance of a long-lived lymphoma progenitor cell population. Therefore, in the current aim, we propose to address the following key questions: a) Is ThPOK required for initiation of AML in the well-established in vivo MLL-AF9 and AML-ETO models of AML? b) Is ThPOK necessary for the maintenance of AML in vivo? c) Does ThPOK promote the leukemia-initiating activity of L-HSCs and L-GMPs? d) Does inhibition of ThPOK in human AML cell lines reduce leukemia cell growth and induce differentiation?