Abstract B095: Discovery of treatment-associated peptide antigens for targeted immunotherapies in pancreatic ductal adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies and is largely refractory to immunotherapies due to its immune-cold tumor microenvironment and reduced antigen presentation. Mutated KRAS, present in most PDAC cases, drives tumor growth and contributes to immune evasion. Though RAS inhibitors have emerged as an extremely promising strategy to treat PDAC, tumor remission due to cancer resistance is virtually inevitable, indicating the need for combination therapies. As several studies have shown that RAS inhibition can resensitize PDAC tumors to anti-tumoral immunity, we hypothesized that RAS inhibition could reshape the PDAC MHC-I immunopeptidome (the cohort of all MHC peptides expressed on the cell surface) and thereby reveal treatment-induced tumor-associated peptide antigens that can be exploited for targeted immunotherapy. To test this hypothesis, we treated human PDAC cell lines with the pan-RAS inhibitor RMC-6236 and profiled the MHC-restricted peptides using an LC-MS/MS-based immunopeptidomics analysis platform. In parallel, we conducted paired global proteomics analysis, and differential peptide expression and pathway enrichment analyses were performed. We found that RAS inhibition increased MHC-I presentation on the PDAC cell surface and induced broad remodeling of the PDAC immunopeptidome. Of the 15706 peptide antigens identified, many that are downregulated upon 72 hours of treatment were sourced from cell cycle and DNA replication and repair pathways, including cyclin A2 and GDF15, suggesting that the cells were responding to the inhibitor and slowing cell proliferation. In contrast, many peptides that were upregulated are derived from pathways known to be involved in responses to or resistance to RAS inhibition, including altered signaling (Ral, PI3K/AKT, YAP-TAZ/Hippo pathways), apical junction, EMT, and ECM remodeling. Many of these pathway changes were corroborated by the global proteomics analysis, but the correlation between the immunopeptidome and proteome was weak, suggesting that the protein expression profile is a poor representative proxy for that of the immunopeptidome. Candidate peptide antigens were further prioritized based on treatment induction fold-change, source-protein expression, PDAC relevance, and tissue-expression patterns, and selected peptides were further validated using a targeted MS-based method, SureQuant MHC, for accurate identification and quantification (copies/cell). Several peptides we have validated are confirmed to be the correct sequence, and they have shown a 2- to 4-fold increase following treatment, consistent with the results from our discovery analysis. Together, these data suggest that RAS inhibition can expose new antigenic vulnerabilities in PDAC by reshaping the antigen presentation landscape. This work provides a framework for identifying actionable treatment-induced peptide antigens and supports rational combination strategies pairing KRAS/RAS inhibitors with T cell-based immunotherapies, including BiTE, CAR-pMHC, TCR-T, and cancer vaccine approaches.