Abstract DNA-dependent protein kinase (DNA-PK) plays a critical role in non-homologous end joining (NHEJ), the predominant pathway that repairs DNA double-strand breaks (DSB) in response to ionizing radiation (IR) to govern genome integrity. How DNA-PK is activated in response to DSBs has remained elusive. Here, we show that the SIRT2 sirtuin deacetylase and tumor suppressor directs the activation of DNA-PK through deacetylation of its catalytic subunit (DNA-PKcs). SIRT2 deacetylase activity governs cellular resistance to DSB-inducing agents and promotes NHEJ. SIRT2 furthermore interacts with and deacetylates DNA-PKcs in response to IR, which facilitates its interaction with Ku and recruitment to DSBs, thereby leading to DNA-PKcs autophosphorylation and DNA-PK signaling to downstream NHEJ substrates. Moreover, SIRT2 inhibitor sensitizes resistant cancer cells and tumors to IR. Our findings define a mechanism for DNA-PK activation by SIRT2-mediated deacetylation, elucidating a critical upstream signaling event initiating the repair of DSBs by NHEJ to promote genome integrity and govern IR resistance, which can be exploited for improvements in cancer therapy. Citation Format: PamelaSara E. Head, Nagaraju P. Ganji, Shi-Ya Wang, Duc Duong, Hui Zhang, Waaqo Daddacha, Shuyi Li, Nicholas T. Seyfried, David M. Smalley, Ya Wang, Xingming Deng, William S. Dynan, Bassel El-Rayes, Anthony J. Davis, David S. Yu. DNA-PKCS deacetylation by SIRT2 promotes DNA double-strand break repair by non-homologous end joining [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2561.