Purpose: DNA damage defects are common in ovarian cancer and can be used to 
stratify treatment. Although most work has focused on homologous recombination 
(HR), DNA double-strand breaks are repaired primarily by nonhomologous end 
joining (NHEJ). Defects in NHEJ have been shown to contribute to genomic 
instability and have been associated with the development of 
chemoresistance.Experimental Design: NHEJ was assessed in a panel of ovarian 
cancer cell lines and 47 primary ascetic-derived ovarian cancer cultures, by 
measuring the ability of cell extracts to end-join linearized plasmid monomers 
into multimers. mRNA and protein expression of components of NHEJ was determined 
using RT-qPCR and Western blotting. Cytotoxicities of cisplatin and the PARP 
inhibitor rucaparib were assessed using sulforhodamine B (SRB) assays. HR 
function was assessed using γH2AX/RAD51 foci assay.Results: NHEJ was defective 
(D) in four of six cell lines and 20 of 47 primary cultures. NHEJ function was 
independent of HR competence (C). NHEJD cultures were resistant to rucaparib (P 
= 0.0022). When HR and NHEJ functions were taken into account, only NHEJC/HRD 
cultures were sensitive to rucaparib (compared with NHEJC/HRC P = 0.034, 
NHEJD/HRC P = 0.0002, and NHEJD/HRD P = 0.0045). The DNA-PK inhibitor, NU7441, 
induced resistance to rucaparib (P = 0.014) and HR function recovery in a 
BRCA1-defective cell line.Conclusions: This study has shown that NHEJ is 
defective in 40% of ovarian cancers, which is independent of HR function and 
associated with resistance to PARP inhibitors in ex vivo primary cultures. Clin 
Cancer Res; 23(8); 2050-60. ©2016 AACR.