PROJECT SUMMARY Cancer cells co-opt autophagy - an evolutionarily conserved cellular recycling pathway - to maintain metabolic fitness. Prior studies have shown that Pancreatic Ductal Adenocarcinoma (PDA) up-regulates autophagy and lysosomes – acidic organelles where autophagic cargo is degraded – to dramatically increase the bulk breakdown and recycling of diverse intracellular substrates. An additional, less well understood function of autophagy is the selective removal of specific proteins in order to endow PDA cells with enhanced fitness. We now show that the autophagy-lysosome pathway selectively targets major histocompatibility complex class I (MHC-I) protein for degradation as a mechanism of immune evasion. Through affinity- based capture of intact lysosomes (LysoIP) from normal and PDA cell lines coupled with proteomics-based analysis, we identified MHC-I as a significantly enriched lysosomal substrate in PDA cells. Consistent with this finding, immuno-fluorescence staining demonstrates that, unlike normal cells where MHC-I localizes to the plasma membrane (PM), in PDA cell lines and primary patient PDA specimens, MHC-I is trapped inside autophagosomes and lysosomes. Importantly, tumor-specific suppression of autophagy is sufficient to 1) stabilize and re-localize MHC-I to the PM, 2) increase antigen presentation and 3) boost T cell mediated tumor killing in vitro and in vivo. Building on these findings the goal of this study is to determine the molecular mechanisms underlying aberrant MHC-I trafficking, and to identify targetable nodes that can be manipulated to restore MHC-I on the cell surface of PDA cells. Our revised application will leverage the combined power of biochemistry, genetics, organelle purification and proteomics, a novel mouse model and patient PDA samples to address the following specific aims: 1) determine how post-translational modifications of MHC-I cooperate with the autophagy machinery to facilitate capture by autophagosomes, 2) identify where along its trafficking route is MHC-I diverted to autophagosomes and 3) determine when during the course of PDA evolution does MHC-I dysregulation occur and can we exploit this information to establish more effective strategies to enhance antigen presentation and immune mediated tumor killing. In summary, our discovery of autophagy dependent degradation of MHC-I highlights an important new paradigm for immune evasion in PDA and potentially other cancers. Findings from our proposed studies will determine key molecular mechanisms underlying MHC-I dysregulation and establish new nodes that can be targeted to restore antigen presentation in PDA.