Abstract 5249: <i>In vitro</i> co-culture CRISPR screen identifies SMG1 to potentiate T cell-mediated cytotoxicity in hepatocellular carcinoma

Abstract The overall response rate to immune checkpoint blockade (ICB) in hepatocellular carcinoma (HCC) patients remains suboptimal, highlighting the need for immediate efforts to identify resistance factors and develop synergistic strategies to enhance the antitumor activity of ICB. Cytotoxic T lymphocytes (CTLs) are the central effectors of anti-tumor immune response. Understanding the relationship between tumor intrinsic kinases and CTL-mediated cytotoxicity could reveal novel mechanisms in promoting immunotherapy efficacy. Here, we conducted a kinome knockout (KO) CRISPR screen in an in vitro tumor-T cell co-culture system to identify kinases in HCC cells that contribute to immune escape from cytotoxic T cell killing. Within the top depleted candidates, Tumor Immune Dysfunction and Exclusion (TIDE) analysis of The Cancer Genome Atlas (TCGA) HCC dataset revealed that higher suppressor of morphogenesis in genitalia 1 (SMG1) expression in patients predicted ICB resistance. Further bioinformatics analysis showed that SMG1 expression is inversely associated with CTL signature, and patients with low SMG1 expression and high CTL signature have more favorable survival outcomes. In vitro proliferation assay and orthotopic transplantation of murine HCC cells in mice demonstrated that SMG1 depletion elicited immune-dependent tumor suppressive effect. Using single-cell RNA sequencing (scRNA-seq), we observed an increased proportion of effector CD8+ T cells in SMG1-depleted tumors, which exhibited enhanced activation of T cell receptor (TCR) signaling pathway along pseudotime, indicating an enhanced T cell-mediated immune response in tumors following SMG1 inhibition. Additionally, we showed that SMG1 loss promoted transcription of major histocompatibility complex class I expression (MHC-I) genes and enhanced antigen presentation on the surface of tumor cells. On the other hand, further investigation of the transcriptome profile following SMG1 knockout indicated dysregulation of alternative splicing (AS) in HCC cells. We also demonstrated that nonsense-mediated mRNA decay (NMD) inhibition can enhance T cell-mediated cytotoxic response, and NMD signature is inversely associated with CD8+ T cells signature in HCC. This suggested the potential of inhibiting SMG1 to promote antigen presentation, as well as disrupt AS and NMD coupled events to drive the generation of splicing-derived neoantigens, thereby augmenting anti-tumor immunity. Citation Format: Ka Hei Lam, Tin Lok Wong, Kai Yu Ng, Vincent Tano, Jia Jian Loh, Ianto B. Huang, Guang Sheng Ling, Terence KW Lee, Lei Lei Chen, Stephanie KY Ma. In vitro co-culture CRISPR screen identifies SMG1 to potentiate T cell-mediated cytotoxicity in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5249.