Abstract A114: SIRT2 directs DNA-PKcs in the DNA damage response

Abstract

Abstract DNA Damage Response (DDR) pathways, including DNA repair and cell-cycle checkpoints, are critical for maintaining genome integrity and preventing disease including cancer. Deficiencies in the DDR result in increased susceptibility to cancer, a leading cause of morbidity and mortality worldwide. However, the mechanisms mediating the activities of the DDR and how their dysregulation leads to genomic instability and a tumor-permissive phenotype are not fully understood. Sirtuin 2 (SIRT2) is a class III NAD+ dependent histone deacetylase implicated in maintaining genomic stability and tumor suppression in that genetic loss of Sirt2 results in both genomic instability and specific murine breast and liver tumors. SIRT2 deficiency in human cells results in hypersensitivity to DNA damage, impaired recovery from replication arrest, and a defect in the G2/M checkpoint in response to ionizing radiation (IR). Preliminary data demonstrate a strong interaction between SIRT2 and DNA-PKcs following IR in human cell lines. DNA-PKcs is a well-established nonhomologous end-joining (NHEJ) mediator kinase that is capable of self-regulation by autophosphorylation at serine 2056 to promote end ligation. SIRT2 knockdown inhibits localization of DNA-PKcs to sites of microirradiation and co-immunoprecipitation with the KU complex in response to IR. Inhibition of localization leads to decreased self-activation of DNA-PKcs at serine 2056 and decreased interaction with KU in SIRT2 knockout lines in response to IR. DNA-PKcs self-phosphorylation and interaction with KU is rescued following damage with wild-type SIRT2 but not with deacetylase dead SIRT2 H187Y, indicating that SIRT2 enzymatic function is necessary for regulation. Overexpression of SIRT2 leads to premature autophosphorylation of DNA-PKcs a serine 2056 without induction of DNA damage. We also demonstrate SIRT2 is capable of deacetylating DNA-PKcs in vitro and in cells. SIRT2 overexpression leads to increased DNA-PKcs self-phosphorylation before and after IR. In addition SIRT2 knockout cell lines exhibit increased sensitivity to IR and CPT. Overall, these data demonstrate that SIRT2 could be a strong potential cancer therapeutic target. Citation Format: Pamelasara E. Head, Hui Zhang, David S. Yu. SIRT2 directs DNA-PKcs in the DNA damage response [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A114.