Abstract PR06: Ovarian cancers harbor defects in nonhomologous end joining resulting in error prone repair and resistance to rucaparib

Abstract

Abstract Introduction: The most toxic forms of DNA damage, double strand breaks (DSBs), are repaired by two main pathways - homologous recombination repair (HRR) and non-homologous end joining (NHEJ). Fifty percent of ovarian cancers have defects in HRR resulting in hypersensitivity to platinum and PARP inhibitors. When NHEJ is defective, DSBs with mismatched ends can be joined by an error-prone mechanism, involving joining between regions of nucleotide microhomology, but this is highly mutagenic. Defects in the NHEJ pathway may therefore contribute to genomic instability and have been associated with resistance development. In this study we demonstrate that NHEJ is defective, resulting in error prone repair, in ~50% of ovarian cancers. Methods: In a panel of ovarian cancer cell lines (OVCAR3, SKOV3, IGROV-1, A2780, CP70 and MDAH) and 30 primary ovarian cancer cultures generated from ascites collected at the time of surgery, we assessed NHEJ using an assay measuring the ability of cell extracts to end-join linearized plasmid monomers into multimers. We determined the effect of inhibition of DNA-PK by concurrent treatment with a DNA-PK inhibitor (NU7441) on the cytotoxicity of the PARP inhibitor, rucaparib, using the sulforhodamine B (SRB) assay. mRNA expression of DNA-PK, XRCC4, LIG4, KU70 and KU80 was determined using qRT-PCR normalised to GAPDH expression. DNA-PK function was determined by immunofluorescence assessment of auto-phosphorylation of DNA-PK one hour after 2Gy irradiation. Furthermore, we assessed the effect of NU7441 on the homologous recombination function using the γH2AX / RAD51 foci formation assay in a BRCA1 defective cell line. Results: We demonstrated that many cell lines and primary ovarian cancer cultures were able to rejoin compatible DNA-ends accurately, however the amount of rejoining of mismatched ends varied significantly between cultures and four out of six cell lines and 14 of 30 primary cultures assessed were unable to accurately rejoin DSBs with mismatched ends resulting in large deletions and error prone repair. This process was not dependent on expression of DNA-PK, XRCC4 or LIG4. Also, whilst in cell lines the autophosphorylation of DNA-PK correlated with error free rejoining, accurate rejoining in primary cultures was independent of DNA-PK auto-phosphorylation. Error-prone repair in ovarian cancer extracts correlated with reduced expression of KU70 (p=0.006) and KU80 (p=0.009). We found that defects in the NHEJ pathway are associated with resistance to rucaparib in primary ovarian cultures (p=0.02). Furthermore concurrent treatment with the DNA-PK inhibitor was cytostatic and induced resistance to rucaparib in the majority of ovarian cancer cultures. These findings are supported by the finding of HR function recovery in a BRCA1 defective cell line treated with DNA-PK inhibitor. Conclusion: This study has shown, for the first time, that some ovarian cancers are unable to perform accurate end joining, such that error-prone joining predominates. Error prone repair is associated with resistance to PARP inhibitors in vitro. Error prone rejoining is highly mutagenic and may contribute to the genomic instability and development of resistance that is observed in ovarian cancer. This abstract is also presented as Poster A31. Citation Format: Aiste McCormick, Michelle Dixon, Rachel ODonnell, Nicola J. Curtin, Richard J. Edmondson. Ovarian cancers harbor defects in nonhomologous end joining resulting in error prone repair and resistance to rucaparib. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr PR06.