In vivo veritas: Finding novel genes involved in liver cancer through in vivo genetic screens

Treatment of liver cancer remains challenging, due to a paucity of drugs that target critical dependencies. In two recent publications in vivo genetic screens are employed in genetically sensitized mouse strains to find genes that accelerate liver cancer development, providing new insights into the critical oncogenic driver pathways. Liver cancer is one of the most frequent malignancies and the second leading cause of cancer-related deaths worldwide, with an estimated 850,000 diagnoses and 800,000 deaths per year. More than 80% of primary liver cancers are hepatocellular carcinoma (HCC). Approximately only 30%-40% of HCC patients are candidates for curative treatment at the time of diagnosis. During the past decades, our understanding of the genetic landscape of HCC has improved significantly through an integrated view of the genetic abnormalities in HCC obtained by wholeexome sequencing studies. As a consequence, we now know that several signaling pathways are implicated in HCC progression: telomere maintenance (TERT promoter mutation 54%-60%), WNT-b-catenin pathway (CTNNB1 11-37%, AXIN1 5%-15%), cell cycle (TP53 12%-48%, CDKN2A 2%-12%, RB1 3%-8%), epigenetic regulators (ARID1A 4%-17%, ARID2 3%18%), AKT/mammalian target of rapamycin (PIK3CA 0%-2%, TSC1/2 3%-8%), and mitogen-activated protein kinase pathway (RAS 0%-2%, RP6SKA3 6%9%). However, unlike epidermal growth factor receptor mutation in lung cancer and B-type Raf kinase mutation in melanoma, the most frequent mutations in HCC are currently undruggable. Sorafenib is a multikinase inhibitor that is approved as the standard therapy for advanced HCC patients. However, sorafenib only provides 2.8-month survival benefit for patients with advanced HCC. Lenvatinib is also a multikinase inhibitor, targeting vascular endothelial growth factor receptors 1-3, fibroblast growth factor receptors 1-4, RET, KIT, and platelet-derived growth factor receptor. In a phase 3 trial, it was observed that progression-free survival, time to progression, and objective response rate with lenvatinib were significantly improved compared to sorafenib. However, overall survival outcomes with lenvatinib were nonsuperior to those with sorafenib. It is therefore urgent to further unravel the mechanism underlying tumorigenesis and identify potential therapeutic targets for HCC. In recent decades, transposon and RNA interference–based screens have been used to identify cancer genes and potential drug targets in liver cancer. Transposon-based screens have nucleotide insertion preference, but they mobilize randomly. Also, transposon-based screens can generate both loss of function and gain of function based on the mutagenic transposon design. For in vivo RNA interference– based screens, focused libraries have been applied to identify tumor suppressors in liver cancer. However, it is technically challenging to use these screens with genome-wide coverage. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a powerful gene-editing technology that has been widely employed for genomescale functional screens. In a recent study, we compared genetic screening systems based on CRISPR/ Cas9 or CRISPR interference with a traditional short hairpin RNA–based system for performing lethality screens. CRISPR technology performed best, with low noise, minimal off-target effects and high consistent activity. Chen et al. reported that CRISPR/Cas9 screens are also feasible in vivo and can be used to shed light on cancer metastasis and tumor evolution. For liver cancer, in an elegant study published in Gastroenterology, Song et al. used an integrative genomics approach to identify drivers of oncogenicity in liver cancer. To ask which genes are relevant to liver carcinogenesis, the authors performed a genomewide CRISPR/ Cas9-based knockout screen in mouse embryonic liver progenitor cells immortalized by loss of the tumor suppressor p53 and overexpression of the c-Myc oncogene (two important events that frequently occur in human HCC). The authors sequentially transduced these cells with a construct expressing Cas9 and then with a genome-wide library of 67,000 single-guide RNAs (sgRNAs) targeting 20,611 mouse genes. These liver progenitor cells were subcutaneously injected into