Abstract 2276: Sorafenib/Refametinib potently inhibits Wnt/β-catenin in vitro and patient-derived xenograft models of human hepatocellular carcinoma

Background: Mutations affecting the Wnt/β-catenin pathway have been found in 26-40% of HCC samples. Aberrant constitutive activation of this pathway leads to uncontrolled cell proliferation growth and survival of tumor cells. Thus, identification of Wnt/β-catenin pathway inhibitors might lead to a new treatment option for a large subset of HCC patients. In the present study, we aim to investigate the effects of Sorafenib plus the allosteric MEK1/2 inhibitor Refametinib (Sorafenib/Refametinib) on Wnt/β-catenin signaling in vitro and in PDX models of HCC. Methods: We treated 15 patient-derived HCC xenograft models with i) 10 mg/kg Sorafenib QD, ii) 15 mg/kg Refametinib QD, and iii) Sorafenib/Refametinib. Western blotting was employed to determine pharmacodynamic changes in biomarkers relevant Wnt/β-catenin signaling. Apoptosis, cell proliferation and localization of β-catenin were analyzed by immunohistochemistry. In-vitro explant culture from 3 patient-derived HCC lines was also tested. Results: In both wild-type and activated-mutated β-catenin HCC primary cells, Refametinib and Sorafenib/Refametinib potently inhibited cell proliferation and caused cell death, which were correlated with downregulation of Dvl3 and β-catenin target genes (Axin2, c-myc, cyclin D1 and survivin). Inhibition of p-LRP6 at Ser1490 and p-β-catenin at Tyr142 and elevation of E-cadherin were also observed. These changes were not observed in Sorafenib-treated samples. In addition, the Sorafenib/Refametinib combination also suppressed Wnt3a-induced p-LRP6 at Ser1490 and β-catenin accumulation in cells. In vivo, significant reductions in p-LRP6 at Ser1490 and p-β-catenin at Tyr142 were observed in tumor samples from the Sorafenib/Refametinib treatment group and to a lesser extent, samples from the Refametinib. Although the overall protein levels of β-catenin were not significantly changed, non-phosphorylated (active) β-catenin Ser33/37/Thr41 and WNT/β-catenin target genes in Refametinib- and Sorafenib/Refametinib-treated tumors were significantly reduced. While vehicle-treated β-catenin-mutated tumors showed strong cytoplasmatic and nuclear β-catenin localization, most of β-catenin in Sorafenib/Refametinib- and, to a lesser extent, Refametinib-treated tumors was accumulated at the membrane. Conclusion: Sorafenib/Refametinib and, to a lesser extent, Refametinib inhibit the growth of patient derived explant cultures and PDX models. They also inactivate β-catenin signaling. Since activating