Abstract Lung cancer is a devastating disease; it is the second most diagnosed cancer type in both men and women, but accounts for more deaths than breast, prostate, and colon cancer combined. The 5-year survival for lung cancer is ~17% due to the late stage of progression many patients present with and the aggressive nature of this disease. Novel targeted approaches are necessary to improve the prognosis of patients that present with stage III and IV disease, and our group proposes to accomplish this by assessing the efficacy of radiosensitization of lung tumors with targeted HDAC6 inhibition. HDAC6 is a class IIb histone deacetylase, a largely cytoplasmic HDAC whose substrates include α-tubulin, cortactin and HSP90, and has been implicated in regulating cell migration and motility. This HDAC is unique in that it contains two deacetylation domains, termed DAC1 and DAC2, and DAC1 has been demonstrated by our lab to possess E3 ubiquitin ligase activity. Unpublished tissue microarray data reveals that HDAC6 is upregulated across all three subtypes of non-small cell lung cancer, suggesting its role as an oncogene that can be specifically targeted by inhibitors currently in clinical trials. Here, we extend our HDAC6 findings into the realm of ionizing radiation (IR) and demonstrate that HDAC6 knockdown enhances radiation-induced damage. Specifically, we find that the induction and persistence of γ-H2AX foci is exacerbated in HDAC6 KD lung cancer cells when compared to control knockdown cells, and PARP-1 cleavage is subsequently enhanced. Our previous work has revealed that nuclear HDAC6 can IP a ~2,000kDa complex including the MRN complex, one of the first responders to DSBs. Further work has confirmed that HDAC6 interacts with the MRN complex and imparts posttranslational modifications on MRN component NBS1, indicating that this interaction could impact the ability of the MRN complex to promote resolution of double-stranded breaks via homologous recombination. In addition, protein half-life assays have indicated that HDAC6 knockdown dramatically increases protein stability of Chk1, the gatekeeper of the G2/M checkpoint whose timely degradation is essential for the progression of the cell cycle. Specifically, accumulating Chk1 post-IR in our lung cancer cell lines is specifically phosphorylated on Ser317, indicating that this population was activated by ATM phosphorylation in an NBS1-dependent manner. Previous reports have indicated that cells arrested in G2/M are sensitive to a variety of genotoxic insults, thus Chk1 persistence could also be leading to enhanced sensitivity to radiation-induced damage in our lung cancer cells. Here, we propose that HDAC6 inhibition will lead to increased tumor radiosensitivity, and the mechanism behind this sensitivity involves a regulatory role for HDAC6 in the function of DNA damage response proteins with particular emphasis on the MRN complex and Chk1. Citation Format: Niko Moses, Mu Zhang, Xiaohong Zhang. HDAC6's functional regulation of Chk1, and its implications on the radiosensitivity of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 663.