Abstract 5720: RK-33, a novel small molecule for lung cancer therapy via inhibition of NHEJ
Authors: Bol Guus M. , Tran Phuoc T. , Vesuna Farhad , Xie Min , van Diest Paul J. , Raman Venu 
Published: August 2012

Lung cancer is the leading cause of cancer mortality worldwide. More importantly, only about 30% of non-small-cell lung cancer patients respond to current combination therapy, which includes radiation and platinum based compounds. Currently, there are limited molecular targets that can be drugable for lung cancer treatment. Our investigation in cancer biogenesis has identified an RNA helicase, DDX3, being dysregulated in many cancer types including lung cancer. DDX3 is a member of the DEAD-box RNA helicase family and contributes to cancer by promoting proliferation, cell growth, transformation and inhibition of apoptosis. Importantly, knockdown of DDX3 in the highly aggressive lung cancer cell line, A549, abrogated its colony forming abilities. In our efforts to abrogate DDX3 functions in vivo, we synthesized a tricyclic 5:7:5-fused diimidazodiazepine ring (RK-33) to fit into the ATP binding domain of DDX3. Initial results confirmed the binding of RK-33 to DDX3 using a streptavidin pull down assay. Subsequently, we demonstrated the effects of RK-33 in vitro and found a synergy between radiation and RK-33 in lung cancer cells. Also, treatment with RK-33 caused a cell cycle arrest in G1 phase and decreased Cyclin D1 levels in A549 and H1299 lung cancer cells. Furthermore, treatment with RK-33 resulted in decreased radiation induced DNA break repair demonstrated by immunofluorescent staining of A549 lung cancer cells with γH2AX and 53BP1 antibodies. In addition, we showed that the non-homologous end joining (NHEJ) activity in cancer cells was decreased by treatment with RK-33. However, homologues recombination activity was not affected by RK-33. Impaired NHEJ is known to increase radiation sensitivity hence inhibiting NHEJ by RK-33 could improve radiation therapy. Following these encouraging results observed in vitro, we initiated in vivo studies, using a lung cancer model with immune competent mice by expressing Twist1 and Kras selectively in the alveolar type II pneumocytes, which confers lung tissue specificity. Following tumor development, confirmed by micro-CT, the animals were treated with RK-33 (20 mg/kg) and different doses of stereotactic radiation (15 Gy) using a small animal radiation platform. Four weeks following treatment, tumor volumes were determined by micro-CT and H&E staining after necroscopy. A 72% reduction of tumor load was accomplished after treatment with RK-33 and radiation (15 Gy) compared to 28% tumor volume reduction in the radiation alone group. Interestingly, there was less tumor reduction when RK-33 was used in combination with 3 Gy x 10 radiation treatment. No evident toxicity became apparent during the treatment with RK-33. All experiments were done in replicates. Conclusion: These in vitro and in vivo results indicate that RK-33 is a promising and safe radiosensitizer, via inhibition of NHEJ, and could be a promising new drug in combination with stereotactic radiotherapy for lung cancer treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5720. doi:1538-7445.AM2012-5720