Z Gastroenterol 2014; 52 - P_4_31
DOI: 10.1055/s-0033-1360984

Multiplex in vivo RNAi screening instructs combination therapies to increase the therapeutic efficacy of the multikinase inhibitor sorafenib

R Rudalska 1, D Dauch 1, K McJunkin 2, TW Kang 1, T Wuestefeld 1, R Geffers 3, P Schirmacher 4, S Lowe 6, T Longerich 4, S Laufer 5, L Zender 1
  • 1University Hospital Tuebingen, & German Center for Translational Cancer Research/German Cancer Research Center (DKFZ), Division of Translational Gastrointestinal Oncology, Dept. of Internal Medicine I, Tuebingen/Heidelberg, Germany
  • 2Cold Spring Harbor Laboratory, Cold Spring Harbor, USA
  • 3Helmholtz Centre for Infection Research, Braunschweig, Germany
  • 4University Hospital Heidelberg, Institute of Pathology, Heidelberg, Germany
  • 5University of Tuebingen, Dept. of Pharmaceutical Chemistry, Tuebingen, Germany
  • 6Memorial Sloan Kettering Cancer Center, New York, USA

Hepatocellular carcinoma represents the third common cause of cancer death and the incidence is steadily increasing. Standard treatments are limited due to frequent tumor recurrence and a poor responsiveness to conventional chemotherapy. The approval of sorafenib, a small multikinase inhibitor as the first active systemic treatment against HCC, increases survival of HCC patients by less than 3 months. Thus, there is a strong need to understand the molecular mechanisms of sorafenib sensitivity and resistance in order to instruct sorafenib basedcombination 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. We found that sorafenib showed moderate but distinct treatment responses in a murine HCC mouse model (NrasG12V driven, p19Arf deficient HCCs), resembling the response rates of sorafenib treated human HCCs.

To identify genes mediating resistance or sensitivity towards sorafenib, an in vivo RNAi screen was conducted. Pools of shRNAs targeting genes found amplified in human hepatocellular carcinomas were introduced into p19Arf-/- mice harboring NrasG12V expressing liver carcinomas with stable expression of shRNA library pools were either treated with sorafenib or carrier. After 5 weeks of treatment, shRNA distribution was quantified in tumors from both cohorts using deep sequencing, whereas depleted shRNAs pinpoint potential resistance genes towards sorafenib treatment.

Validation experiments with single shRNAs found depleted in sorafenib treated mice showed

a significant survival advantage compared to non-targeting control shRNA under sorafenib treatment. Our top-scoring candidate gene represents a kinase (Mapk14) which allowed us to perform a combination therapy with commonly available inhibitors that results in a significant survival advantage.

The identified new combination treatment was also expanded to murine HCC cell lines with additional genetic backgrounds. In these cell lines, therapeutic efficacy as a result of sorafenib and Mapk14 blockade was also found.Additionally, it was shown that the newly identified combination therapy significantly decreased proliferation and induced cell death in a panel of well established human hepatoma cell lines.

To characterize how Mapk14 blockade sensitizes towards sorafenib treatment, microarray based mRNA expression analyses were conducted and analyzed using Ingenuity pathway analysis software. This analysis along with genetic validation experiments identified the Mapk14 downstream target and transcription factor Atf2 as relevant for Mapk14 mediated resistance towards sorafenib treatment.

In summary, our study establishes a new sorafenib based combination therapy for the treatment of hepatocellular carcinoma and highlights the utility of RNAi loss-of-function screening for revealing new therapeutic agents in cancer therapy.