Role of autophagy in normal and transformed hematopoietic stem cells

描述（由申请人提供）：本申请的总体目标是了解自噬如何支持造血干细胞（HSC）的维持和功能，以及转化的HSC中这种应激反应机制的破坏如何有助于骨髓恶性肿瘤如慢性髓性白血病（CML）的发展。我们最近证明，HSC通过诱导强有力的保护性自噬反应而在代谢应激中存活（Warr et al.，2013年）。特别是，我们发现转录因子FoxO 3A对于维持促自噬基因程序是必不可少的，该程序使HSC能够快速诱导自噬。然而，HSC如何感知代谢应激并激活自噬仍然是未知的，并且关于自噬在正常和转化的HSC中的作用还有很多有待了解。我们将使用药理学和遗传学方法来剖析自噬对HSC生物学的贡献，以及我们建立的人慢性期CML的Scl-tTA：TRE-BCR/ABL（tTA-BA）小鼠模型（Eschaud et al.，2011）以探测自噬在白血病起始干细胞（LSC）活性和CML发展中的功能。在具体目标1中，我们将确定HSC激活自噬的机制。我们将使用我们建立的方案，在细胞因子撤出后离体诱导HSC中的代谢应激，在食物剥夺后体内诱导HSC中的代谢应激，并将利用现有的遗传小鼠模型和化学抑制剂来鉴定HSC如何感知代谢应激并触发自噬诱导。这些方法将确定HSC如何在代谢挑战时引发保护性自噬反应。在具体目标2中，我们将讨论自噬的丧失如何影响体内HSC功能和基因组稳定性，并研究蛋白质和细胞器周转的替代形式是否可以支持自噬缺陷HSC的长期维持。这些方法将描述自噬在体内通常如何被HSC利用，以及其废除如何改变正常的造血。在具体目标3中，我们将探索自噬在转化的BCR/ABL表达HSC中的功能，并将利用我们的诱导型tTA-BA小鼠模型来研究自噬对CML发病机制的贡献以及CML LSC对酪氨酸激酶抑制剂（TKI）治疗的反应。这些方法将为血液系统恶性转化的机制提供重要的新见解。他们将阐明自噬在HSC转化和CML发展中的作用，并确定如何操纵自噬机制以实现治疗益处。总之，这些研究将揭示通常由HSC用于维持血液稳态的细胞保存的基本机制的腐败如何有助于转化的HSC的异常功能和血液疾病的发展。

DESCRIPTION (provided by applicant): The overall goal of this application is to understand how autophagy supports the maintenance and function of blood-forming hematopoietic stem cells (HSC), and how corruption of this stress-response mechanism in transformed HSCs contributes to the development of myeloid malignancies such as chronic myelogenous leukemia (CML). We recently demonstrated that HSCs survive metabolic stress by inducing a robust protective autophagy response (Warr et al., 2013). In particular, we showed that the transcription factor FoxO3A is essential to maintain a pro-autophagy gene program that poises HSCs for rapid autophagy induction. However, how HSCs sense metabolic stress and activate autophagy is still unknown, and much remains to be understood about the role of autophagy in normal and transformed HSCs. We will use both pharmacological and genetic approaches to dissect the contribution of autophagy to HSC biology, and our established Scl- tTA:TRE-BCR/ABL (tTA-BA) mouse model of human chronic phase CML (Reynaud et al., 2011) to probe the function of autophagy in leukemia-initiating stem cell (LSC) activity and CML development. In Specific Aim 1, we will determine the mechanisms by which HSCs activate autophagy. We will use our established protocols to induce metabolic stress in HSCs ex vivo upon cytokine withdrawal and in vivo upon food deprivation, and will take advantage of existing genetic mouse models and chemical inhibitors to identify how HSCs sense metabolic stress and trigger autophagy induction. These approaches will establish how HSCs elicit a protective autophagy response upon metabolic challenges. In Specific Aim 2, we will address how loss of autophagy affects HSC function and genomic stability in vivo, and investigate whether alternative forms of protein and organelle turnover can support the long-term maintenance of autophagy-deficient HSCs. These approaches will delineate how autophagy is normally utilized by HSCs in vivo, and how its abrogation alters normal hematopoiesis. In Specific Aim 3, we will probe the function of autophagy in transformed BCR/ABL- expressing HSCs, and will take advantage of our inducible tTA-BA mouse model to investigate the contribution of autophagy to CML pathogenesis and response of CML LSCs to tyrosine kinase inhibitor (TKI) treatments. These approaches will provide important new insights into the mechanisms of malignant transformation in the blood system. They will elucidate the contribution of autophagy in HSC transformation and CML development, and determine how the autophagy machinery can be manipulated to achieve a therapeutic benefit. Taken together, these studies will uncover how corruption of an essential mechanism of cell preservation normally used by HSCs to maintain blood homeostasis contributes to the aberrant function of transformed HSCs and the development of blood diseases.