Abstract In a search for novel agents that boost the anti-neoplastic effects of polo-like kinase 1(PLK1) inhibitor volasertib, we found that a sepantronium and volasertib combination at the nano mole concentration potently inhibited growth of various non-small cell lung cancer (NSCLC) cell lines than either drug alone in vitro. Combination use of volasertib with sepantronium inhibited adaptation of cells to polo arrest. Addition of sepantronium to volasertib prevented accumulation of survivin and cyclin B protein at a concentration causing no appreciable survivin down regulation. Sepantronium, was originally developed as a survivin inhibitor. However, sepantronium induced cell cycle arrest from G1 or G2/M phase. Further studies demonstrated DNA damage of cancer cells when they are treated with sepantronium, which is evidenced by induction of phospho-gammaH2AX. In line with induction of a DNA damage response in cancer cells, known DNA damage response sensors and transducers ATM, ATR, CHK1, CHK2, p53 are phosphorylated following drug treatment. Meanwhile, expression of CDKN1A, BAX and XRCC5 are induced at the mRNA level as determined by quantitative real time PCR. A single cell electrophoresis assay (Comet assay) of cells treated with sepantronium revealed severe DNA strand breaks. M-phase arrest does not increase the lethality of DNA damage by sepantronium as compared to G1 phase arrest. Knock down of survivin did not cause DNA damage. Hence, sepantronium is a DNA damaging agent that synergizes with volasertib and down- regulation of survivin is likely the consequence of DNA damage induced by sepantronium. Citation Format: Mei Hong, Mingqiang Ren, Jeane Silva, Zhonglin Hao. Sepantronium is a DNA damaging agent that potentiates PLK1 inhibitor volasertib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 817. doi:10.1158/1538-7445.AM2014-817