Abstract 4876: Multiplex in vivo RNAi screening instructs combination therapies to increase the therapeutic efficacy of the multikinase inhibitor sorafenib
Authors: Rudalska Ramona , Dauch Daniel , McJunkin Katherine , Kang Tae-Won , Wuestefeld Torsten , Geffers Robert , Lowe Scott W. , Zender Lars 
Published: August 2012

Abstract
Hepatocellular carcinoma (HCC), the third leading cause of cancer death worldwide, represents a highly chemoresistant tumor and only recently the multikinase inhibitor sorafenib was approved as the first active systemic treatment against HCC. This illustrates that targeted therapies can be effective against HCC and therefore heralds a new era in HCC treatment. However, sorafenib monotherapy increases survival of HCC patients by less than 3 month, thus emphasizing the strong need to understand the molecular mechanisms of sorafenib sensitivity and resistance in order to inform new combination therapies with higher therapeutic efficacy. Activation of the Ras/MAPK signaling pathway was reported to play a major role in liver tumor development and progression. Taking advantage of genetically defined mosaic liver cancer mouse models, we found that sorafenib treatment of NrasG12V driven murine liver carcinomas results in moderate but distinct treatment responses resembling the response rates of sorafenib treated human HCCs. To identify genes mediating resistance or sensitivity towards sorafenib, we conducted an in vivo RNAi screen. Pools of shRNAs targeting genes found amplified in human hepatocellular carcinomas were applied and mice harboring NrasG12V expressing liver carcinomas with stable expression of shRNA library pools were either treated with sorafenib or carrier control. After 5 weeks of treatment, shRNA distribution was quantified in tumors from both cohorts using deep sequencing, whereas enriched shRNAs pinpoint potential resistance genes and depleted shRNAs pinpoint targets that may be exploited as sorafenib sensitizers. Functional validation experiments were performed with multiple independent single shRNAs against each “sensitizing” target and confirmed a significant survival benefit over sorafenib treated tumors that express non-targeting control shRNAs. Importantly, our top-scoring candidate represents a kinase and, taking advantage of readily available pharmacological inhibitors, we show that combination therapy with sorafenib is well tolerated by the mice and results in a significant survival advantage. We also expanded our new combination treatment to murine HCCs of additional genetic backgrounds, overexpressing c-myc or AKT. As therapeutic efficacy was also shown in these models, it is likely that our new combination therapy will be active against a broad range of human HCCs. Furthermore, we found that the newly identified combination therapy significantly decreased proliferation and induced cell death in a panel of well established human hepatoma cell lines. In summary, our study establishes a new sorafenib based combination therapy for the treatment of hepatocellular carcinoma and highlights the potential of in vivo RNAi screens to identify genes that modulate the treatment response of targeted therapies.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4876. doi:1538-7445.AM2012-4876