Abstract 3336: NF-κB inhibition by DMAPT radiosensitizes non-small cell lung carcinoma by impairing DNA double strand break repair

Abstract

Abstract Background: Despite optimal radiation therapy (RT), chemotherapy and/or surgery, non-small cell lung carcinoma (NSCLC) remains the leading cause of cancer-related death in the United States. Previously, we demonstrated that proteasome inhibition blocks the expression of Fanconi anemia/homologous recombination (FA/HR) genes via inhibition of the NF-κB pathway. Dimethylaminoparthenolide (DMAPT) is a water-soluble analog of parthenolide that has been shown to decrease p65/p50 heterodimer DNA binding. We hypothesized that DMAPT may radiosensitize NSCLC cells by impairing NF-κB-mediated expression of DNA double strand break (DSB) repair genes. Materials and Methods: Cytotoxicity and radiosensitivity were assessed using clonogenic assays. IκB kinase (IKK) activity was measured using a substrate phosphorylation assay. HR and canonical non-homologous end joining (cNHEJ) were measured using A549 cells stably expressing reporter constructs that express GFP upon repair of I-SceI-induced DNA DSBs. DNA damage and DSB repair biomarker foci were assessed by immunofluorescence. For in vivo studies, 1×106 A549 cells were injected subcutaneously into the flanks of NCr nude mice. Once tumors reached an average size of 200 mm3, mice were treated concurrently with vehicle or DMAPT (100 mg/kg) ± focal ionizing radiation (4 Gy x 5) using a small animal radiation research platform (SAARP). Tumor growth was followed for three months following treatment. Results: Radiation and NF-κB inhibition with DMAPT showed synergistic cytotoxicity. Treatment of A549 cells with DMAPT resulted in a decrease in constitutive IκBα phosphorylation by IKK. NF-κB inhibition via DMAPT resulted in a 60-70% decrease in HR. In addition, treatment with DMAPT resulted in ≥80% loss of IR-induced BRCA1, FANCD2, and RAD51 foci. NF-κB inhibition with either DMAPT or overexpression of the NF-κB super-repressor IκBα(S32A, S36A) resulted in a 50-70% decrease in cNHEJ. Treatment with DMAPT resulted in the loss of IR-induced phosphoDNA-PKS2056 foci. Further, treatment with DMAPT resulted in the persistence of radiation-induced γH2AX foci over a 24 hour period (100% of DMAPT treated cells showing foci compared to 30% of vehicle treated cells). Compared to either single agent DMAPT or IR alone, combined treatment of A549 xenografts with DMAPT and IR prevented tumor growth over three months (p < 0.0001). Conclusions: DMAPT inhibits NF-κB signaling by inhibiting IKK. This in turn inhibits HR and cNHEJ by preventing the recruitment of DNA repair proteins to sites of IR-induced DNA damage. Both in vitro and in vivo experiments show support for further evaluation of DMAPT as a NSCLC radiosensitizer. Citation Format: Colin O'Leary, Peter Deraska, Christopher Sweeney, Alan D'Andrea, David Kozono. NF-κB inhibition by DMAPT radiosensitizes non-small cell lung carcinoma by impairing DNA double strand break repair. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3336. doi:10.1158/1538-7445.AM2015-3336