Pancreatic ductal adenocarcinoma (PDAC), a malignancy refractory to most therapies including immune checkpoint blockade (ICB) therapy, utilizes diverse mechanisms to evade immune clearance. One mechanism involves reduced presentation of tumor specific antigens by Major Histocompatibility Complex Class I (MHC-I) to immune cells. Many cancers alter MHC-I expression via genetic or epigenetic silencing, however changes in MHC-I trafficking can also profoundly influence antigen presentation at the cell surface and is a previously underappreciated mechanism of MHC-I regulation in cancer.

My dissertation uncovers a role for enhanced autophagy/lysosome function in immune evasion through selective targeting of MHC-I molecules for degradation. Prior studies have shown that highly aggressive PDAC cells and tumors upregulate autophagy, an evolutionarily conserved self-recycling pathway that is hijacked by cancer cells to sustain metabolic fitness. In addition to the metabolic benefits tumor cells receive, autophagy and lysosomal activity have utilized these processes to gain a growth advantage by facilitating degradation and recycling of diverse intracellular materials. My data demonstrates that MHC-I molecules are selectively targeted for lysosomal degradation through an autophagy-dependent mechanism that involves the autophagy cargo receptor NBR1. PDAC cells display reduced MHC-I cell surface expression and instead demonstrate predominant localization within autophagosomes and lysosomes. Notably, autophagy inhibition restores cell surface MHC-I expression, enhances anti-tumor CD8+ T cell responses in vitro and in vivo, and importantly, sensitizes PDAC tumors to dual immune checkpoint blockade (ICB). Our data on immune evasion adds to the growing list of cell- autonomous functions of the autophagy/lysosome system in supporting PDAC tumorigenesis.

To identify the distinct molecular mechanisms of MHC-I regulation in PDAC, we combined a whole-genome CRISPRi screen and Turbo-ID proximity-dependent proteomics to determine regulators and interactors of MHC-I, respectively. From these two datasets, 101 overlapping candidates were identified, many of which were related to post-translational modification (PTM), trafficking machinery, and kinase regulation. The gene candidates likely control MHC-I by diverting plasma membrane localization to degradative organelles. Several gene candidates associated with E3 ubiquitin ligase and kinase signaling were validated and show increased plasma membrane when knocked down or pharmacologically inhibited. Using these two datasets as a resource of MHC-I regulation can help identify PDAC-specific mechanisms that facilitate altered trafficking of MHC-I.Findings from this study will lead to a better understanding of PDA pathophysiology and have the potential to inform the development of rational combination therapies that restore MHC-I cell surface localization, thereby rendering PDA cells more susceptible to immunotherapy. In particular, results from this research project will help to determine the causes of aberrant intracellular localization of MHC-I and can help explain MHC-I is unable to traffic normally. These studies also lay the foundation for mechanisms of immune evasion in other aggressive cancers.