Abstract 3967: CRISPR/Cas9 screening identified novel membrane targets sensitizing hepatocellular carcinoma cells to natural killer cell-mediated cytotoxicity

Abstract

Abstract Natural killer cells (NK) play a central role in cancer immune surveillance by directly killing cancer cells and producing pro-inflammatory cytokines to stimulate and recruit other immune cells to elaborate long-lasting anti-tumor responses. However, little is known about the immune escape mechanisms and regulatory proteins, particularly those for NK, involved in hepatocellular carcinoma (HCC), one of the most common and lethal cancers. To identify the determinants of NK killing function, we performed an HCC cell line-based CRISPR/Cas9 loss-of-function genetic screen, using a human whole membrane protein library containing ~7000 genes curated from 3 public membrane protein datasets. The human SNU449 was chosen as a representative HCC cell line for CRISPR/Cas9 editing based on the genetic profile analysis, representing one HCC patient subpopulation (> 30%) in the TCGA cohort. The cell line was incubated with NK92 cells at ratios of 2:1, 1:1, and 0.5:1. Bioinformatics methods for prioritizing genes (e.g., MAGeCK) were applied on 3 tested groups with different ratios. Single guide RNAs targeting genes in the antigen presentation machinery were depleted, including HLA-C, B2M, and TAP1/2, whereas components of the tumor-immune synapse, such as NCR3LG1 and ICAM1, were enriched, confirming the validity of the screen. SLC33A1 (an endosomal acetylCoA transporter) and SLC31A1 (the major copper influx transporter) were also depleted, previously unknown as NK regulators. The effects of SLC33A1 and SLC31A1 were validated in individual gene knockout cells generated respectively by lentiviral sgRNA expression. Furthermore, knockout of either transporter in SNU449 cells increased IFNγ secretion, confirming NK92 activation. Our results revealed that CRISPR/Cas9-based functional genomics screens in HCC cell lines could identify novel targets modulating NK-mediated killing. Further analysis of mechanisms of action, and particularly functional validation in in vivo systems, will potentially provide novel targets for the treatment of HCC. Citation Format: Shuo Li, Lin Han, Siyao Zhang, Guangfeng Geng, Lei Huo, Dawei Huang, Zhaoren He, Shuoran Li, Zhaoshi Jiang, James X. Rong. CRISPR/Cas9 screening identified novel membrane targets sensitizing hepatocellular carcinoma cells to natural killer cell-mediated cytotoxicity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3967.