Decatenation: fixing your knots

Topoisomerase II (Topo II) performs essential DNA topologic changes in proliferating eukaryotic cells, mostly notably the decatenation (unknotting) of the newly replicated sister DNA molecules, needed for chromatid separation in anaphase of mitosis (see figure). Because inhibiting this enzyme selectively kills proliferating cells, Topo II is the target of widely used cancer drugs. Despite their prevalence as chemotherapeutics, resistance to such drugs arises, for example, through up-regulation of ABC transporters that pump the drugs out of cells. Understanding all of the mechanisms of drug resistance is of great importance for the design of improved cancer treatment strategies.

In this issue of Blood, Wray et al compared the effects of a Topo II inhibitor, ICRF-193, on normal human CD34⁺ hematopoietic cells and a number of acute leukemia cell lines, finding that the leukemia cells were able to proliferate.¹  Like many cell types, the CD34⁺ cells arrested cell-cycle progression in the presence of ICRF-193. This normal cellular response is mediated by checkpoint controls that respond to perturbed Topo II by inducing cell-cycle arrest in G₂ phase as well as in metaphase of mitosis.²  The Topo II checkpoints aim to prevent failed attempts at chromosome segregation when the cell has insufficient decatenatory activity. The leukemia cells lacked both the G₂ phase and metaphase checkpoint responses and continued to proliferate regardless of ICRF-193 treatment.

There are several plausible explanations for this behavior that relate directly to Topo II: (1) a deficient checkpoint signal, allowing unperturbed mitotic progression despite the inability to decatenate DNA; (2) a mutation in Topo II such that ICRF-193 could not bind; (3) an elevated level of Topo II protein, which compensated for the presence of drug; (4) a stimulation of Topo II activity such that catenations were processed faster than ICRF-193 could act; and (5) a factor that interfered with drug-enzyme interaction. Wray et al took significant steps in their attempts to clarify this mechanism of drug resistance.

Based on previous work, they asked if Metnase is required for ICRF-193 resistance in the leukemia cells.^3,4  Metnase binds directly to Topo II and stimulates its ability to decatenate DNA in vitro.³  Assuming Metnase stimulates decatenation in vivo, the continued proliferation of the leukemia cells treated with ICRF-193 ought to be accompanied by relatively normal chromosome segregation. This would explain the lack of checkpoint induction. Remarkably, they found that depletion of Metnase from the leukemia cells rendered them sensitized to ICRF-193, as measured by an increased propensity to arrest in mitosis. This effect seemed to be specific to the metaphase Topo II checkpoint, since the cells did not accumulate in G₂ phase. Most strikingly, the absence of Metnase similarly sensitized to an unrelated Topo II inhibitor, VP-16. The leukemia cells were able to proliferate in the presence of VP-16, but rapidly succumbed to apoptosis if Metnase was depleted before VP-16 treatment.

The significance of these data is that some leukemia cells may be killed by combination therapies that target Topo II and Metnase. But before this is possible, a detailed understanding of the mechanism of Metnase action is needed. One confusing aspect of the current state of knowledge is that Metnase stimulates Topo II–mediated decatenation in vitro in the presence of several different Topo II inhibitors, despite these drugs having varied modes of action and distinct binding sites on Topo II. This may rule out simple interference of drug binding. An alternative is that Metnase stimulates or somehow modifies the Topo II enzyme cycle such that it is resistant to drug action. Perhaps investigating whether Metnase can stimulate Topo II in the presence of all classes of inhibitor would be revealing.

A second key issue is the nature of the enhanced Metnase activity in leukemia cells. This is important because Metnase activity could help predict the efficacy of Topo II inhibition as a cancer treatment. Metnase protein levels were apparently not altered in the leukemia cells, although they did have in common a phosphorylated isoform not abundant in CD34⁺ cells. It is possible that phosphorylation activates Metnase-stimulated decatenation. There also is evidence that methylation of Metnase regulates its activity.³ 

Whatever the mechanism of Metnase action and the predictive power of Metnase activity toward strategizing appropriate cancer therapies, these initial studies warrant rigorous further investigation. In the long term, small molecule inhibitors that disrupt the Metnase–Topo II interaction could be valuable in the clinic.