Abstract 1583: Proteasome inhibition as a strategy for non-small cell lung carcinoma via inhibition of DNA double strand break repair.

Abstract

Abstract Background: Each year, ∼60,000 US patients are diagnosed with locally advanced non-small cell lung carcinoma (NSCLC). Despite optimal radiation therapy (RT), chemotherapy +/- surgery, over 30% of patients fails locoregionally. The addition of concurrent chemotherapy to RT adds only ∼5% absolute survival benefit. Novel radiosensitizers may improve the therapeutic index. To identify the best potential targets, we performed whole genome RNAi screens that identified several proteasome subunits among top genes whose knockdown increased NSCLC cytotoxicity. Materials and Methods: Cytotoxicity was assessed by luminescent cell viability and clonogenic assays. Homologous recombination (HR) and non-homologous end-joining (NHEJ) were assessed using NSCLC cell lines transduced with reporter constructs that express GFP upon repair of I-SceI-induced DNA double strand breaks (DSBs). DNA damage-induced focus formation was assessed by immunofluorescence, scoring % of cells showing ≥5 foci. In vivo radiosensitization by proteasome gene knockdown was assessed using NCr nude mice injected with 1x106 NCI-H460 cells stably transfected with inducible PSMA1 shRNA. Once tumors reached ≥3 mm, knockdown was induced with doxycycline, and then one week later, RT to a dose of 20 Gy in 5 fractions was initiated. Results: Radiation and proteasome inhibition showed synergistic cytotoxicity. Irradiation of A549 cells with 1 Gy x 3 decreased clonogenic survival by 58% compared to control, while ionizing radiation (IR) plus 10 nM bortezomib decreased survival by 74% compared to bortezomib alone. Similar results were seen in NCI-H460. Proteasome inhibition via bortezomib or PSMA1 siRNA knockdown resulted in 80-90% decreased HR and NHEJ. Additionally, bortezomib or PSMA1 shRNA knockdown resulted in ≥50% decreases in BRCA1, FANCD2 or RAD51 IR-induced focus formation. Treatment of NCI-H460 xenografts with RT in the setting of PSMA1 knockdown showed marked improvements in survival; at 100 days post treatment initiation, only 20% of RT-only treated mice and 30% of doxycycline-treated mice (for PSMA1 shRNA knockdown in tumors) survived, compared to 100% following dual treatment (n = 10 per arm). There was a statistically significant difference in survival between mice with tumors treated with RT alone vs. RT + PSMA1 knockdown, with median survivals of 43 days vs. not reached, P = .0003 by the log-rank test. Conclusions: Proteasome inhibition has emerged as a promising target in NSCLC radiosensitization with evidence indicating the mechanism is through inhibition of DNA DSB repair pathways HR and NHEJ. Citation Format: Kyle R. Cron, Kaya Zhu, Deepa Kushwaha, Grace Hsieh, Dmitry Merzon, Jack Monahan, Clark C. Chen, Alan D'Andrea, David Kozono. Proteasome inhibition as a strategy for non-small cell lung carcinoma via inhibition of DNA double strand break repair. [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 1583. doi:10.1158/1538-7445.AM2013-1583