Project Summary/Abstract Notch1 signaling is an important mediator of stem cell self-renewal and therapeutic resistance, and the most prevalent oncogene (~60%) in T-cell acute lymphoblastic leukemia (T-ALL) - an aggressive neoplasm of T cell progenitors that affects both children and adults. Although current intensive chemotherapies can suppress the disease, they come at the cost of serious side effects and are insufficient to eliminate Notch1-driven leukemic cells. One in five children and one in two adults with T-ALL do not survive due to either unresponsive or relapsed disease. Efforts to target oncogenic Notch1 with small-molecule inhibitors have been hampered by their inherent cytotoxicity. Overcoming these difficulties will require improved understanding of the oncogenic mechanisms controlled by Notch1 and a better appreciation of the genes and pathways that regulate Notch1- driven leukemogenesis as potential targets of T-ALL therapy. Through Drosophila studies and the generation of mouse models for T-ALL, we have discovered that, T-ALL-associated Notch could be degraded by an unconventional endo-lysosomal module through a physical interaction with the autophagic tumor suppressor UVRAG, which reshapes Notch activity and resultant Notch-dependent cellular response. Thus, the central hypothesis of this proposal is that the endo-lysosomal titration of Notch activity by UVRAG represents a unique mechanism governing Notch1 before proteolytic processing, and that disruption of this regulatory module impacts T-cell homeostasis and contributes to T-ALL. Specifically, we propose experiments to comprehensively dissect the molecular mechanism of UVRAG-mediated endo-lysosomal inhibition of Notch1 in T-ALL. Furthermore, we will elucidate the unequivocal impact of this mechanism on the self-renewal and stemness of leukemia-initiating cell function in human T-ALL primary samples. Finally, we will use the mouse models to test the concept that boosting this mechanism could restore Notch homeostasis and achieve sustained T-ALL remission. These aims will be addressed using multidisciplinary innovative approaches that integrate state-of-the-art genetic, biochemistry, high-resolution imaging, and physiological assays in cells and transgenic mouse models. We now bring within this proposal a collaboration of world-wide leaders in T-ALL pathology and molecular biology along with clinicians and pathologists. Our use of patient-derived T-ALL samples will maximize the relevance of our findings for eventual translation to T-ALL patients in the clinic. Overall, this project will lead to an in-depth understanding of Notch1-driven leukemogenesis, and provides a critical trajectory for the development of optimal anti-leukemia strategies against this aggressive lymphoid malignancy.