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. Top2alpha is essential for cell proliferation and is highly expressed in vigorously growing cells, whereas Top2beta is nonessential for growth and has recently been implicated in treatment-associated secondary
malignancies, highlighting the validity of a Top2alpha-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 Top2alpha and not Top2beta in vivo. Unlike other Top2 inhibitors,
NK314 induces Top2-DNA complexes and double-strand breaks (DSBs) in an alpha
isoform-specific manner. Heterozygous disruption of the human TOP2alpha gene confers increased
NK314 resistance, whereas TOP2beta homozygous knock-out cells display increased
NK314 sensitivity, indicating that the alpha
isoform is the cellular target. We further show that the absence of Top2beta 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 Top2alpha-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 assessing DNA damage and repair induced by potential topoisomerase-targeting agents.