Two-step forward genetic screen in mice identifies driving forces of hepatocellular carcinoma and its new drug target

Background & Aims: High throughput sequencing technologies have identified thousands of infrequently mutated genes in hepatocellular carcinoma (HCC). However, tumor heterogeneity and passenger mutations have made it difficult to identify true drivers of HCC. Here we use transposon mutagenesis and shRNA library screening to identify HCC drivers. Methods: Whole-genome transposon mutagenesis in mice was used to generate an unbiased catalogue of HCC candidate cancer genes (CCGs). Pooled shRNA libraries targeting 250 selected CCGs were introduced into immortalized mouse liver cells and their tumor-forming ability were monitored. Results: Transposon mutagenesis identified 1917 HCC CCGs and highlighted the importance of Ras signaling in HCC. Subsequent pooled shRNA library screening validated 27 new HCC tumor suppressor genes (TSGs). Individual knockdown of four TSGs, including Acaa2, Hbs1l, Ralgapa2 and Ubr2, confirmed their role as anti-proliferative tumor suppressor. We also show that the tumor suppressive function of Ralgapa2 is mediated through Rala and Ralb inhibition, revealing an oncogenic role for the Ral pathway in HCC. Lastly, we show that dual inhibition of the Ras downstream pathways, Ral and Raf, by RBC8 and Sorafenib, synergistically suppress HCC growth in vivo. Conclusion: Two-step forward genetic screens provide a rich resource for identifying new drivers of HCC and therapeutic targets.