LAUSR

Hepatocellular Carcinoma (HCC) is the most prevalent type of liver cancer and the third leading cause of cancer deaths worldwide. Drugs approved for first- and second-line therapy extend survival by only several months. Hence, there is still a pressing need for new and effective treatments. Sequencing technologies applied to human HCC tissues have identified hundreds of genes that undergo amplification and that may be correlated with mortality. Nevertheless, correlation does not equate to functionality. We aim to annotate gene function by employing an innovative approach with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and catalytically dead version of CRISPR-associated endonuclease (dCas9) in a CRISPR/dCas9 activation (CRISPRa) screen to identify driver genes of HCC tumorigenesis in mice. We deliver to the liver’s hepatocytes a gRNA library targeting genes of interest on plasmids that contain: (1) a transposon system for stable integration of DNA sequences into the cell genome, (2) Myc to drive hyperproliferation, as MYC is frequently over-expressed in human HCC, (3) synergistic activation mediator complex sequences for transcriptional activation of target genes, and (4) the gRNA sequence, the only variable sequence in the library. Using datasets from the Cancer Genome Atlas, we identified 51 genes that are both enriched and amplified in HCC patients. We included gRNAs targeting Tert, Vegfa, and Ccnd1, as control genes with known roles in driving HCC. In total, the screening library contained 290 gRNAs targeting 51 genes and 3 controls. After delivery of the plasmid library to the liver, the Myc gene provided a baseline level of oncogenic hyperproliferation, and the readout was gRNA prevalence pre- and post-proliferation via next-generation sequencing. We found that the combination of Myc and CRISPRa gRNA library led to a large number of expansive tumors in the liver as compared to Myc or the CRISPRa gRNA library only, which contained few to no tumors. gRNAs targeting control genes Ccnd1 and Vegfa were enriched as expected, validating our approach to annotate gene function during tumorigenesis. Additionally, we identified enrichment in gRNAs targeting genes Zbtb7b, Vps72, and Gba in multiple tumors, implicating a role in liver tumorigenesis. ZBTB7B and GBA expression levels are increased in patients with HCC recurrence as compared to patients in remission (data from BIOSTORM trial), while VPS72 is an unfavorable prognostic marker for HCC (data from The Human Protein Atlas). In this study, we have established a novel model of liver cancer that combines the Myc oncogene with a CRISPRa gRNA library to annotate driver genes in human HCC. Additionally, we identified several promising targets for the treatment of HCC. Ultimately, this study has the potential to impact on the care of patients with HCC in the US and worldwide. Citation Format: Alexandra Mariel Vázquez Salgado, Shirui He, Kirk J. Wangensteen. Identifying drivers of liver cancer using CRISPR activation screening in vivo [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 250.