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.
Our proposed investigations will yield a comprehensive understanding of how autophagy is used by HSCs to preserve themselves and maintain blood production throughout life. Moreover, they will uncover how corruption of this essential stress-response mechanism in transformed HSCs contributes to the development of myeloid malignancies and resistance to current targeted therapies. Collectively, they will provide unique insights into the mechanisms regulating the survival of normal and leukemic HSCs, and stand to make critical contributions to the identification of molecular targets that could be engaged to destroy therapy-resistant LSCs in human myeloid malignancies.
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|Warr, Matthew R; Kohli, Latika; PasseguÃ©, Emmanuelle (2013) Born to survive: autophagy in hematopoietic stem cell maintenance. Cell Cycle 12:1979-80|
|Warr, Matthew R; Binnewies, Mikhail; Flach, Johanna et al. (2013) FOXO3A directs a protective autophagy program in haematopoietic stem cells. Nature 494:323-7|
|Bakker, Sietske T; PasseguÃ©, Emmanuelle (2013) Resilient and resourceful: genome maintenance strategies in hematopoietic stem cells. Exp Hematol 41:915-23|