Abstract C017: Unravelling tumor-intrinsic resistance mechanisms in pancreatic cancers to T-cell mediated cytotoxicity
Authors: Diaz Judit , Levy Pierre , Gonzalez Jonatan , Palazon Asis , Guedan Sonia , Gros Alena 
Published: November 2022

Abstract
The vast majority of human Gastrointestinal Cancers (GI) including colon, rectal and pancreatic cancers are resistant to immune checkpoint inhibitors (ICB), an immunotherapy strategy approved for the treatment of melanoma, non-small cell lung cancer, renal cancer and others. Although the lack of response has been largely attributed to their low tumor mutational burden, recent articles have demonstrated that neoantigen-specific tumor-infiltrating lymphocyte (TIL) can be successfully isolated in pancreatic cancer. This suggests that other factors, both tumor intrinsic and suppressive tumor microenvironmental, likely contribute to the lack of response of ICB. In this study we investigated the interplay between T cells and pancreatic tumors in vitro. To this aim we co-cultured a patient-derived pancreatic cell line, TC-4177, with autologous PBLs capable of recognizing the MMP14p.R158C neoantigen expressed by these cancer cells. Strikingly and in contrast to melanoma, this cancer cell line was not effectively recognized nor killed. The same results were obtained when co-culturing TC-4177 with MMPP14p.R158C specific TCR-transduced CD8+ T cells, suggesting that the lack of recognition was not due to T cell dysfunction but, rather, tumor intrinsic. The TC-4177 tumor cell line expressed the neoantigen at RNA level, did not present mutations in antigen presentation machinery, in the IFNg pathway nor in Caspase 8, suggesting none of the widely described resistance mechanisms to immune cell attack were limiting factors underpinning the poor recognition of this cell line. Furthermore, tumor cell recognition was restored upon exogenous neoantigen overexpression. Altogether, these results suggest that additional tumor-intrinsic mechanisms in pancreatic cancer cell line 4177 dampen T cell-mediated recognition and lysis that could be shared with other pancreatic tumors. To unravel the unknown resistance mechanism, a Genome-wide CRIPSR/Cas9 coculture-based screen was performed using this cell line. As a result, a number of genes known to be essential for T cell-mediated tumor immunity were identified, including key genes in the antigen presentation, IFN-g and TNF-a pathways. Further illustrating the robustness of the screen, sgRNAs targeting the antigen itself (MMP14) and the MMP14 HLA-restriction (HLA-B) conferred resistance to T cell killing. Moreover, in this screen we identified genes involved in known pathways that render susceptibility to T cell killing in pancreatic cancers such as autophagy pathway and vesicle trafficking. In addition to these pathways, genes involved in ubiquitination, chromatin remodeling and maintenance of gastrointestinal epithelium appeared to sensitize pancreatic cells to immune cell attack. Further experiments need to be performed to independently validate these candidates in the TC-4177 cell line as well as other pancreatic cell lines. Overall, these results open a new avenue to render pancreatic cancers susceptible to T cell killing and thus respond to immunotherapy.
Citation Format: Judit Diaz, Pierre Levy, Jonatan Gonzalez, Asis Palazon, Sonia Guedan, Alena Gros. Unravelling tumor-intrinsic resistance mechanisms in pancreatic cancers to T-cell mediated cytotoxicity [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C017.