In vitro co-culture CRISPR screen for kinase potentiating 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 also 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 MHC-I-mediated antigen presentation, while 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 associated with infiltration of CD8+ T cells 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.