Abstract B47: Therapeutic KRAS silencing in lung and colon cancer models

Abstract

Abstract Introduction: Despite being amongst the most common oncogenes in human cancer, to date there are no effective clinical options for inhibiting KRAS activity. We investigated whether systemically delivered KRAS siRNAs using biocompatible nanoparticles has therapeutic potential in KRAS mutated lung and colorectal cancer models. Methods: We identified two KRAS siRNA sequences with notable potency in knocking-down KRAS expression independent of nucleotide substitution. Using human lung (A549; KRASG12S) and colon (HCP1; KRASG12D) adenocarcinoma cell lines, we assessed anti-proliferative effects of KRAS silencing with MTT assays in vitro. For in vivo experiments, we used a nano-liposomal delivery platform, DOPC, for systemic delivery of siRNAs. We used luciferase-labeled A549 cells to create an orthotopic lung cancer model in athymic nude mice. One week after cell injection, we began weekly IVIS imaging and treatments to assess efficacy of systemic KRAS siRNA with and without cisplatin (CDDP) chemotherapy. Proliferation and apoptosis indices were assessed using Ki-67 and caspase-3 IHC, respectively. Using a patient-derived xenograft colon cancer cell line, HCP1, we performed several in vivo experiments to assess KRAS siRNA effects on down-stream signaling, primary tumor growth, and experimental metastasis. Results: For both KRAS siRNA sequences, greater than 90% knock-down of KRAS was observed by western blot; ∼40-60% reductions in cell viability was observed in both A549 and HCP1 (p<0.001). In the lung cancer model, following four weeks of systemic therapy, the KRAS siRNA treatment group demonstrated an 81% reduction in luminescence compared to mice in the NC siRNA group (p=0.008). In addition, only in the KRAS siRNA treatment groups did we observe 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). 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 (NC siR+CDDP: 2-fold increase, p=0.03; KRAS siR+CDDP: 5.5-fold increase, p=0.0007). Using the HCP1 colon cancer model, KRAS siRNA treatment reduced tumor KRAS and pERK expression levels at 48 and 96 hours after systemic delivery. Consistent with these effects, as compared with NC siRNA, treatment with KRAS siRNAs reduced HCP1 subcutaneous tumor growth by 69% (p<0.05). To assess effects of KRAS siRNA on liver metastases, HCP1 cells were delivered by intra-splenic injection followed by splenectomy the following day. Following 3 weeks of treatment with KRAS siRNAs, we observed reduced aggregate liver metastatic tumor burden compared to controls (73%, p=0.014). Notably, no mice in any of the therapeutic experiments exhibited any overt signs of toxicity from KRAS siRNA treatment, such as decreased feeding habits, activity, or total body weight. Discussion: Our studies 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, especially for traditional “undruggable” targets. Citation Format: Chad Pecot, Sherry Wu, Seth Bellister, Rajat Bhattacharya, Anshumaan Maharaj, Cristian Rodriguez-Aguayo, Vianey Gonzalez-Villasana, Rajesha Rupaimoole, Gabriel Lopez-Berestein, Lee M. Ellis, Anil Sood. Therapeutic KRAS silencing in lung and colon cancer models. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B47. doi: 10.1158/1557-3125.RASONC14-B47