Abstract Radiation therapy is commonly used to treat cancers in the definitive setting, but the local control often remains suboptimal, thereby motivating us to search for novel radiosensitizers. Recently a series of novel quinacrines based upon a previously reported dimeric chloroquine has been synthesized. We studied one of these agents, DQ660. By itself, the drug upregulates both p53 protein and p53 transcriptional targets and also increases apoptosis in p53-wildtype H460 lung cancer cells. Furthermore, we found that DQ660 is a potent radiosensitizer in vitro. This radiosensitization is not contingent upon wildtype p53 as it occurs in cells null for p53 and containing mutant p53. The combination of radiation and DQ660 proded significant growth retardation of human xenografts grown in vivo compared to radiation or DQ660 alone. We saw higher levels of γ-H2AX foci with combined treatment compared to either alone, suggesting that the drug increases DNA damage induction. Using agarose gel electrophoresis and kinetoplast DNA (kDNA) as a substrate, we found that DQ660 can inhibit topoisomerase II activity. DQ660 did not potentiate the phosphorylation of ATM at S1981 seen after radiation. However, the drug did lead to an increase in Ser428 phosphorylation of ATR. This phosphorylation was not seen in response to radiation alone, suggesting that DQ660 and radiation activate different signaling pathways in response to DNA damage. We saw similar results with another dimeric quinacrine, DQ550, suggesting that this class of agents may be radiosensitizers. Our findings have uncovered a novel class of radiosensitizers that work at nanomolar concentrations and will spur investigations for its use in combination therapy for tumors treated with radiation Citation Format: Ilias V. Karagounis, Jayashree Karar, Christoforos Thomas, Constantinos Koumenis, Jeffrey Winkler, Ravi Amaravadi, Amit Maity. A novel class of dimeric quinacrine compounds induce DNA damage and increase the anticancer activity of radiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 217.