PROJECT SUMMARY Currently, there is immense interest in modulating autophagy against cancer. Anti-malarials, such as hydroxychloroquine (HCQ), have been aggressively repurposed as autophagy inhibitors in numerous clinical oncology trials. Despite this enthusiasm, we still do not fully understand how autophagy impacts cancer progression and treatment. To date, the primary rationale for targeting autophagy against cancer is because this catabolic pathway promotes tumor cell survival and metabolic adaptation. However, recent findings challenge this prevailing idea in the field. Although traditionally viewed as an ?auto-digestive? pathway, emerging genetic evidence now implicates autophagy as an important regulator of cellular secretion. For example, we recently discovered new roles for autophagy regulators (ATGs) in promoting the coordinate secretion of cytokines required for tumor cell invasion. Nevertheless, our understanding of autophagy- dependent secretion in cancer remains rudimentary because of major conceptual and technical barriers that hamper deciphering how autophagy enables secretion. Most importantly, studies to date have exclusively relied on phenotypic analysis following genetic ablation of specific pathway components, such as ATGs; such loss-of-function approaches are limited because they fail to discern whether secretory defects represent a direct versus indirect consequence of impaired autophagy. To overcome these major obstacles in the field, we have created novel reporters utilizing proximity-based biotinylation (BioID) to specifically label proteins that rely upon the autophagy machinery for their release into the extracellular space. We will now leverage these innovative reporters to decipher the autophagy-dependent secretome and rigorously define the mechanisms by which autophagy promotes secretion. In Aim 1, we will elaborate the repertoire of autophagy-dependent secreted products using quantitative proteomics and assess their utility as biomarkers for monitoring tumor progression and treatment in vivo. For these studies, we will focus on KRAS mutant lung cancer cells exhibiting high levels of basal autophagy and secretion. In Aim 2, we will define the mechanisms by which autophagy controls tumor cell secretion. We will utilize proximity-based biotinylation strategies, combined with genetic approaches, to dissect the intracellular trafficking of autophagy-dependent secreted proteins as they exit the cell, with the long-term goal of identifying targets in these pathways for therapeutic intervention in cancer. Overall, these studies provide timely mechanistic insight into an entirely new role for autophagy in cancer progression and therapeutic response.