Abstract LB080: iOTarg screening platform reveals novel mechanisms of colorectal cancer evasion from antigen-specific tumor cell killing by T cells

Abstract While immune checkpoint therapies have revolutionized cancer treatment, their efficacy remains limited in colorectal cancer (CRC) and other malignancies. Tumors employ diverse strategies to evade immune-mediated destruction, hindering the success of immunotherapies. We introduce iOTarg, a robust high-throughput screening platform designed to uncover resistance pathways against T cell-mediated tumor killing within the tumor microenvironment. Focusing on CRC, we utilized iOTarg to dissect evasion mechanisms against antigen-specific T cell (TC) killing. In a representative CRC cell line co-culture system (SW480, HCT116 or COLO-678) with various TC sources, we gained key insights of tumor resistance towards killing. Survivin-specific TCs induce strong tumor lysis while tumor-infiltrating lymphocyte 412 (TIL412) represent a weaker basal killing representative of the situation in the tumor. Knockout of β₂-microglobulin abrogated TC-mediated killing, while c-FLIP knockout significantly enhanced tumor lysis. Intriguingly, PD-L1 blockade had no impact on TC activation or killing, underlining its lack of efficacy in CRC. Screening over 6000 druggable genes revealed several critical candidates, including anti-apoptotic BIRC family members (BIRC2, 3), apoptosis regulators (Bcl-xL, XIAP), TNF signaling regulators (TRAF2, MAP3K7), and genes recently described as sensitization mediators (B3GNT3, CHMP4B). Caspase-8 knockout prevented TC-mediated killing of CRC cells, confirming its crucial role in immune-mediated tumor cell clearance. Further validation in secondary screenings reinforced a subset of genes as strong regulators of tumor cell killing, uncovering an array of CRC resistance mechanisms. Our findings present a pioneering platform delineating resistance mechanisms against T cell-mediated colorectal tumor killing. We identify established and novel targets, offering prospects for novel therapeutics and expanding immune oncology treatment options. This platform holds promise for developing first-in-class therapies targeting the tumor microenvironment, potentially transforming CRC treatment paradigms. Citation Format: Tillmann Michels, Valentina Volpin, Carmen Amerhauser, Claudia Tschulik, Leonie Majunke, Aleksandra Weglarz, Kilian Baritz, Kritika Sudan, Szilvia Bak-Kiss, Alina Huth, Carolin Strobl, Adriana Turqueti-Neves, Nisit Khandelwal. iOTarg screening platform reveals novel mechanisms of colorectal cancer evasion from antigen-specific tumor cell killing by T cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (7Suppl): Abstract nr LB080.