Abstract 3122: The DNA-PK inhibitor NU7441 inhibits non-homologous end joining, enhances radio- and chemosensitivity and increases dependence on homologous recombination in hepatocellular carcinoma cell lines
Background DNA double-strand breaks (DSBs), the most cytotoxic lesions induced by ionizing radiation (IR) and anticancer drugs such as topoisomerase II poisons (e.g., doxorubicin), are repaired by non-homologous end joining (NHEJ) and homologous recombination (HR). DNA-dependent protein kinase (DNA-PK), which initiates NHEJ, is up-regulated in hepatocellular carcinoma (HCC) (GEO profiles), possibly contributing to anticancer therapy resistance. To assess DNA-PK as a potential therapeutic target for chemo- and radio-sensitisation in HCC we determined the effect of the DNA-PK inhibitor, NU7441, on DSB repair and cytotoxicity in HCC cells. Methods DNA-PK protein levels and activation by IR (Western blot), DSB levels (y-H2AX foci), HR (RAD51 foci), cell growth (DAPI fluorescence) and cytotoxicity (colony formation) following exposure to IR or doxorubicin was determined in a panel of 6 hepatoma cell lines (HepG2, Hep3B, Huh7, SNU-182, SNU475 and PLC/PRF/5). Results DNA-PK protein concentration and activity did not vary significantly across the panel (±24% and ±37%, respectively) but there were cell-specific sensitivities to IR and doxorubicin (e.g. HepG2 2-fold more resistant than Hep3B). NU7441 significantly sensitised all cells to both doxorubicin (average PF50 4.3±3.0) and IR (average PF50 3.9±1.1) in growth inhibition assays and significantly reduced survival (4.8 to 3.3-fold) in colony forming assays. Following exposure to IR, NU7441 significantly delayed yH2AX focus clearance in all cell lines (e.g. only 13% cleared at 4 hr compared to 50% in control), but increased RAD51 focus formation (3-fold). Conclusion While chemo- and radio-sensitivity in HCC cells was not dependent on DNA-PK expression or activity, NU7441 causes greater than 2-fold chemo- and radiosensitisation in all cells. This was accompanied by a substantial reduction in the rapid phase of DNA repair (NHEJ-dependent) and a shift to a greater reliance on the slower HR repair. DNA-PK inhibitors may have potential as chemo- and radio-sensitisors in hepatoma patients. 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 3122. doi:1538-7445.AM2012-3122