In vivo CRISPR-Cas9 library screen for liver cancer therapeutic targets essential for T cell killing

T-cell checkpoint inhibition by antibodies blocking programmed cell death protein 1 (PD1) has been a major breakthrough in cancer therapeutic development. However, anti-PD1 drugs approved by FDA are ineffective in treating all cancer types or in all patients in a responsive cancer type. Therefore, it is important to stratify responsive patient population and identify synergistically effective combination therapies. Primary hepatocellular carcinoma (HCC) is a human malignancy with a high incidence, mortality and recurrence rate worldwide. We established a syngeneic mouse HCC model using a murine hepatoma cell line, Hepa1-6, which, interestingly, shrunk in volume shortly after reaching up to 400 mm3. The tumor failed to resume growth unless treated with anti-CD4/CD8 antibodies to deplete endogenous T cells, suggesting that an adaptive T cell immunity suppresses the late-stage tumor growth. To identify the genetic regulators of the cancer elimination, we employed CRISPR-Cas9 based genome-wide knockout screening strategy to identify the potential targets essential for cancer cell sensitivity to the T cell immunity. Mouse xenograft models derived from hepa1-6 cells infected with the library were treated with anti-CD4/CD8 antibodies or anti-mPD1 antibody. Deep sequencing of the tumors revealed T cell-dependent enrichment of guide RNAs targeting 241 genes with variable functions. We have performed a second round of in vivo screen with a CRISPR library focused on these candidate genes. We will discuss the technical challenges of the in vivo screen as well as the broad application of the technology for discovery of immune oncology targets. Citation Format: Haixin Zhao, Chenlu Geng, Wenrong Zhou, Zhengang Peng, Qunsheng Ji, Yong Cang. In vivo CRISPR-Cas9 library screen for liver cancer therapeutic targets essential for T cell killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3974.