Abstract PR010: Genome-wide CRISPR screens in CD8+ T cells identify cullin-RING E3 ubiquitin ligase complex as a negative regulator of long-term effector function

Abstract The benefit of adoptive cell transfer (ACT) therapies with T cells engineered to express chimeric antigen receptors (CAR) or T cell receptors (TCR) is limited by T cell dysfunction upon repetitive antigen exposure. We integrated a pooled genome-wide CRISPR knockout screen with in vitro repetitive antigen exposure and phenotypic selection to identify gene knockouts that improved the ability of NY-ESO-1 TCR transgenic T cells to repeatedly kill the antigen- and HLA-matched melanoma cell line M407. In two replicate screens, knocking out of the genes CUL5, RNF7 or ARIH2, which are required for proper cullin 5-RING ligase complex (CRL5) assembly, significantly improved long-term tumor cell killing ability of T cells past five rechallenges (one-way ANOVA p<0.0001). Mechanistically, we discovered that CRL5 interacts with SOCS-box containing proteins to regulate TCR stimulation and cytokine signaling through ubiquitination and proteasomal degradation. Ablation of CRL5 in T cells leads to an increase in polyfunctionality with augmented production and release of cytokines and cytolytic molecules across multiple rounds of killing when compared to control T cells (IFNg/TNFa producing T cells; Challenge 1: 18% versus 10%, Stimulation 3: 6% versus 3%, unpaired t-test p<0.0001). We demonstrated through both CRISPR knockout, and pharmacological inhibition of CRL5 using the neddylation inhibitor MLN4924, the negative regulatory mechanism of CRL5 in CD8+ T cells. These results highlight the previously unrecognized regulation of T cell function through E3 ligase-mediated proteasomal degradation, and that CRL5 may be a target to endow lasting effector function to antigen-specific T cells for cancer immunotherapies. Citation Format: Justin Saco, Daniel Karin, Nataly Naser Al Deen, Katie Campbell, Justin Patten, Yichen Fan, Egmidio Medina, Antoni Ribas. Genome-wide CRISPR screens in CD8+ T cells identify cullin-RING E3 ubiquitin ligase complex as a negative regulator of long-term effector function [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr PR010.