Abstract 2590: Selective disruption of Rb-Raf-1 kinase interaction is a suitable therapeutic option for pancreatic adenocarcinoma

Abstract Retinoblastoma tumor suppressor binding protein, Rb, is a major regulator of the mammalian cell cycle progression. Inactivation of Rb by a cascade of phosphorylation events leads to its inactivation, facilitating release of transcriptionally active E2F and S-phase entry. Rb phosphorylaiton is mediated mainly by CDK, but it was observed Raf-1 could bind and phosphorylate Rb early in the cell cycle, facilitating phosphorylation events. Although previous reports have suggested that K-Ras, which is mutated in over 80% of pancreatic cancers, activates MAPK through Raf/Mek signaling, inhibition of this pathway have proven clinically unsuccessful. We demonstrated that increased binding of Raf-1 to Rb, independent of the Mek/MAPK pathway, has contributed to tumor progression and inhibition of this interaction might be a suitable strategy for cancer therapies. Therefore, we examined whether targeting Rb-Raf-1 interaction with a small molecule inhibitor RRD-251 is a viable strategy against pancreatic cancer. To test our hypothesis, we initially probed a panel of pancreatic cancer cell lines (PANC-1, MiaPaCa-2, CD18/HPAF, L3.6pl) and demonstrated the disruption of Rb-Raf-1 interaction by RRD-251, using immunoprecipitation western blot experiments. To fully define the anti-tumor effects of RRD-251, cell cycle analyses, proliferation/cell viability (MTT), cell migration (wound healing assay), and invasion (Boyden chamber assay) experiments were performed. Animal studies were performed in nude mice with subcutaneous and orthotopic pancreas models with intraperitoneal injections of RRD-251 at 50 mpk/daily. RRD-251 significantly reduced proliferation by 3-fold and significantly reduced the ability of metastatic variant, L3.6pl, and other pancreatic cancer cell lines to migrate; there was also a a 5-fold decrease in invasion (p<0.05). Prolonged exposure of RRD-251 (48 hours) demonstrated that disruption of the Rb-Raf-1 interaction would induce apoptosis as defined by cleavage of PARP protein, decreased anti-apoptotic proteins Mcl-1 and Bcl-2, and an increase in pro-apoptotic protein Bax. Additionally, combination of RRD-251 with gemcitabine (125ng/ml) induced a synergistic effect on induction of apoptosis, proliferation/cell viability (7.5 fold decrease), migration, and almost complete loss of the invasive capacity of pancreatic cancer cells (p<0.05). In vivo, RRD-251 significantly abrogated primary tumor growth after subcutaneous and orthotopic injections of metastatic variant, L3.6pl. Additionally, there was a significant reduction (>80%) of liver metastasis in the RRD-251 treated group. In this study, we demonstrate a disruption of the Rb-Raf-1 kinase interaction with RRD-251 significantly affects the malignant properties of pancreatic cancer cells. Treatment with combination RRD-251 and gemcitabine appears to be a viable strategy against pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2590. doi:10.1158/1538-7445.AM2011-2590