Abstract 938: Silencing the elephant in the room: therapeutic KRAS targeting with siRNA.

Abstract

Abstract Introduction: Despite being one of the first known oncogenes in human cancer, to date there are no effective therapeutic options for inhibiting KRAS. We investigated whether systemically delivered KRAS siRNA using biocompatible nanoparticles has therapeutic potential in lung cancer models. Methods: We identified two KRAS siRNA sequences with notable potency in knocking-down KRAS expression, which were combined for the in vivo experiments. Using a lung adenocarcinoma cell line (A549) with a KRAS mutation (G12S), we assessed anti-proliferative affects of KRAS silencing with MTT assays. Using luciferase-labeled A549 cells in athymic nude mice, we created an orthotopic lung cancer model in the left lower lung and performed weekly IVIS imaging beginning one week after injection. We used a nanoliposomal delivery platform, DOPC, for systemic delivery of siRNAs. Mice were randomly distributed into the following treatment groups (n=10 mice per group): 1) Control (NC) siRNA, 2) KRAS siRNA, 3) NC siRNA+cisplatin (CDDP), 4) KRAS siRNA +CDDP. One week after cell line injection, treatment began and continued for 4 weeks. At the end of the experiment mice were sacrificed, necropsied, and tumors were stained and evaluated for Ki-67 and caspase-3 by immunohistochemistry. Results: For both KRAS siRNA sequences, greater than 90% knock-down of KRAS was seen by western blot; which individually resulted in a 38% and 61% reduction in A549 cell viability (p=0.001 and p<0.001, respectively). Following four weeks of systemic therapy, mice in the KRAS siRNA treatment group had an 81% reduction in luminescence (p=0.008), while the addition of CDDP did not lead to significant reductions in the NC siRNA+CDDP (0% reduction, p=0.42) or KRAS siRNA+CDDP (51% reduction, p=0.22) groups. As compared with control siRNA, significant reductions in primary tumor size (KRAS siR: 50%, p=0.003; NC siR+CDDP: 6%, p=0.24; KRAS siR+CDDP: 73%, p=0.008), number of distant metastases (KRAS siR: 71%, p=0.07; NC siR+CDDP: 0%, p=0.49; KRAS siR+CDDP: 81%, p=0.02) and aggregate mass of distant metastases (KRAS siR: 77%, p=0.0002; NC siR+CDDP: 11%, p=0.85; KRAS siR+CDDP: 74%, p=0.009) was only observed in the KRAS siRNA treatment groups. KRAS siRNA significantly reduced the frequency of mediastinal metastases (NC siR: 100% versus KRAS siR: 25%, p=0.002). Compared with control siRNA, significant reductions in Ki-67 indices were seen in all treatment groups (KRAS siR: 34%, NC siR+CDDP: 52%, KRAS siR+CDDP: 36%; all p<0.0001), while significant increases in caspase-3 activity was only seen in the CDDP treatment groups (KRAS siR: 0%, p=0.12; NC siR+CDDP: 223%, p=0.03; KRAS siR+CDDP: 545%, p=0.0007). Discussion: Here, we demonstrate a proof-of-concept approach to therapeutic KRAS targeting using nanoparticle delivery of siRNA. This study highlights the potential translational impact of therapeutic RNA interference, which may have broad applications in oncology. Citation Format: Chad V. Pecot, Anshumaan Maharaj, Sherry Wu, Cristian Rodriguez-Aguayo, Vianey Gonzalez-Villasana, Rajesh Rupaimoole, Behrouz Zand, Thiru Arumugam, Gabriel Lopez, Anil Sood. Silencing the elephant in the room: therapeutic KRAS targeting with siRNA. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 938. doi:10.1158/1538-7445.AM2013-938