Inhibition of topoisomerase IIα and G₂ cell cycle arrest by NK314, a novel benzo[c]phenanthridine currently in clinical trials

NK314 is a novel synthetic benzo[c]phenanthridine alkaloid that has recently entered clinical trials as an antitumor compound, based on impressive activities in preclinical models. The present investigations were directed at determining the mechanism of action of this agent. NK314 induced significant G₂ cell cycle arrest in several cell lines, independent of p53 status, suggesting the existence of a common mechanism of checkpoint activation. The Chk1-Cdc25C-Cdk1 G₂ checkpoint pathway was activated in response to 100 nmol/L NK314 in ML-1 human acute myeloid leukemia cells. This was associated with the phosphorylation of the histone variant H2AX, an action that was predominant in the G₂ population, suggesting that double-strand DNA breaks caused cells to activate the checkpoint pathway. Double-strand DNA breaks were visualized as chromosomal aberrations when the G₂ checkpoint was abrogated by 7-hydroxystaurosporine. In vitro assays showed that NK314 inhibited the ability of topoisomerase IIα to relax supercoiled DNA and trapped topoisomerase IIα in its cleavage complex intermediate. CEM/VM1 cells, which are resistant to etoposide due to mutations in topoisomerase IIα, were cross-resistant to NK314. However, CEM/C2 cells, which are resistant to camptothecin due to mutations in topoisomerase I, retained sensitivity. These findings support the conclusion that the major mechanism of NK314 is to inhibit topoisomerase IIα, an action that leads to the generation of double-strand DNA breaks, which activate the G₂ DNA damage checkpoint pathway.