ARTICLE ABSTRACTPurpose: Poly(ADP-ribose) polymerases (PARP) and the Mre11, Rad50, and Nbs1 (MRN) complex are key regulators of DNA repair, and have been recently shown to independently regulate telomere length. Sensitivity of cancers to PARPi is largely dependent on the BRCAness of the cells. Unfortunately, the vast majority of cancers are BRCA-proficient. In this study, therefore, we investigated whether a targeted molecular “hit” on the MRN complex, which is upstream of BRCA, can effectively sensitize BRCA-proficient head and neck squamous cell carcinoma (HNSCC) to PARP inhibitor (PARPi).Experimental Design: Human HNSCC cell lines and a mouse model with HNSCC xenografts were used in this study. In vitro and in vivo studies were conducted to evaluate the effects and underlying mechanisms of dual molecular disruption of PARP and the MRN complex, using a pharmacologic inhibitor and a dominant-negative Nbs1 expression vector, respectively.Results: Our findings demonstrate that downregulation of the MRN complex disrupts homologous recombination, and, when combined with PARPi, leads to accumulation of lethal DNA double-strand breaks. Moreover, we show that PARPi and MRN complex disruption induces significantly shortening telomere length. Together, our results demonstrate that dual disruption of these pathways causes significant cell death in BRCA-proficient tumor cells both in vitro and in vivo.Conclusion: Our study, for the first time, elucidates a novel mechanism for MRN complex and PARP inhibition beyond DNA repair, demonstrating the feasibility of a dual disruption approach that extends the utility of PARPi to the treatment of BRCA-proficient cancers. Clin Cancer Res; 20(24); 6465–78. ©2014 AACR.