NK314, a topoisomerase II inhibitor that specifically targets the α isoform

Topoisomerase II ( Top2) is a ubiquitous nuclear enzyme that relieves torsional stress in chromosomal DNA during various cellular processes. Agents that target Top2, involving etoposide, doxorubicin, and mitoxantrone, are among the most effective anticancer drugs used in the clinic. Mammalian cells possess two genetically distinct Top2 isoforms, both of which are the target of these agents. Top2 beta is essential for cell proliferation and is highly expressed in vigorously growing cells, whereas Top2 beta is nonessential for growth and has recently been implicated in treatment- associated secondary malignancies, highlighting the validity of a Top2 beta-specific drug for future cancer treatment; however, no such agent has been hitherto reported. Here we show that NK314, a novel synthetic benzo[c] phenanthridine alkaloid, targets Top2 beta and not Top2 beta in vivo. Unlike other Top2 inhibitors, NK314 induces Top2- DNA complexes and double- strand breaks ( DSBs) in an beta isoform- specific manner. Heterozygous disruption of the humanTOP2 beta gene confers increased NK314 resistance, whereas TOP2 beta homozygous knock- out cells display increased NK314 sensitivity, indicating that the beta isoform is the cellular target. Wefurther show that the absence of Top2 beta does not alleviate NK314 hypersensitivity of cells deficient in non- homologous end- joining, a critical pathway for repairing Top2- mediated DSBs. Our results indicate that NK314 acts as a Top2 beta- specific poison in mammalian cells, with excellent potential as an efficacious and safe chemotherapeutic agent. We also suggest that a series of human knock- out cell lines are useful in assessingDNAdamage and repair induced by potential topoisomerase- targeting agents.