Abstract
Mitotic inhibitors are widely utilized chemotherapeutic agents that take advantage of mitotic defects in cancer cells. We have identified a novel class of piperazine-based mitotic inhibitors, of which AK301 is the most potent derivative identified to date (EC50 < 200 nM). Colon cancer cells arrested in mitosis with AK301 readily underwent a p53-dependent apoptosis following compound withdrawal and arrest release. This apoptotic response was significantly higher for AK301 than for other mitotic inhibitors tested (colchicine, vincristine, and BI 2536). AK301-treated cells exhibited a robust mitosis-associated DNA damage response, including ATM activation, γH2AX phosphorylation and p53 stabilization. The association between mitotic signaling and the DNA damage response was supported by the finding that Aurora B inhibition reduced the level of γH2AX staining. Confocal imaging of AK301-treated cells revealed multiple γ-tubulin microtubule organizing centers attached to microtubules, but with limited centrosome migration, raising the possibility that aberrant microtubule pulling may underlie DNA breakage. AK301 selectively targeted APC-mutant colonocytes and promoted TNF-induced apoptosis in p53-mutant colon cancer cells. Our findings indicate that AK301 induces a mitotic arrest state with a highly active DNA damage response. Together with a reversible arrest state, AK301 is a potent promoter of a mitosis-to-apoptosis transition that can target cancer cells with mitotic defects.