The potential of Topoisomerase II as an anticancer drug target

By Yawen (Angela) Yang

Topoisomerase II (TOP2) is an enzyme engaged in DNA replication, transcription and chromosomal segregation.1 During DNA replication and transcription, DNA helicase separates double-stranded DNA into single strands. However, helicase cannot unwind DNA indefinitely because DNA ahead of the replication fork becomes overwound and forms supercoils. The tension built up makes DNA too tight to allow for further unwinding, preventing further DNA replication or transcription. As DNA replication takes place in the S-phase of cell division, its failure will also disrupt the cell cycle, resulting in sister chromatid separation failure. TOP2 is responsible for overcoming this topological stress. It uses the energy from ATP hydrolysis to generate a transient double-stranded break in one DNA duplex and passes a segment of another DNA duplex across the break. Each time, this relaxes 2 superhelical twists, removing tension gradually. After relaxing supercoils, TOP2 re-ligates the damaged DNA strands to restore DNA integrity.

Because of the significance of TOP2 in cell division, TOP2 is a prospective target in chemotherapy. Through TOP2 paralysis in cancer cells, normal cell division is disrupted thus preventing the uncontrolled growth of cancer cells. Although many anti-cancer drugs such as signal transduction inhibitors, gene expression modulators, and toxin delivery molecules have already been approved, TOP2-targeting chemotherapies are especially of interest due to some cancer cells developing resistance to these drugs. Additionally, the identification of a new target such as TOP2 increases the likelihood of successfully combating cancer cells. In addition, TOP2-targeting anti-cancer drugs are successful in treating both solid tumours and haematological malignancies, indicating they have considerable therapeutic promise.2

TOP2-targeting anti-cancer drugs fall into two categories, one of them being TOP2 poisons. TOP2 poisons bind at the interface of the TOP2 enzyme-DNA complex and form a stable drug-TOP2-DNA ternary complexes.3 It is worth noting, however, that under normal conditions, there should only be short-lived TOP2-DNA cleavage complexes. The stable ternary complex blocks the release of DNA segments, thereby inhibiting the re-ligation step during replication and transcription. This leaves the DNA double-stranded breaks unsealed and thus disturbs the genomic structure of cells. Consequently, cell cycle may arrest in the S-phase and apoptosis may be triggered if the DNA damage cannot be repaired. 

TOP-2 poisons are widely applied in clinics. Etoposide, an epipodophyllotoxin used in combination with other chemotherapy agents to treat refractory testicular tumours as well as small cell lung cancer, and doxorubicin, an anthracycline used in combination with other chemotherapy to treat breast cancer, various leukaemia, lymphoma, and sarcomas, are two effective anti-cancer drugs that contain TOP-2 poisons.4 Unfortunately, these poisons also have several possible side-effects such as inducing DNA or protein perturbations, resulting in continuous cleavage cycles. It can also cause the formation of secondary malignancies due to certain chromosomal translocations, which is the most common and worrying adverse effect. 5These secondary malignancies are thought to be caused by TOP2β, a TOP2 isoform expressed in almost all tissues. The results of a study conducted found that mice treated with etoposide were substantially more likely to develop melanomas in the skin when TOP2β was expressed, compared to mice that had the TOP2β gene removed, thus supporting this theory.6 An additional side effect of TOP-2 poisons is that they may induce cardiotoxicity. Again, this is related to TOP2β since cardiomyocytes missing TOP2β in mice were shown to be protected against doxorubicin-induced cardiomyocytes damage and progression of heart failure.7 There are also limitations with anti-cancer drugs containing TOP-2 poisons, perhaps the most significant one being cell resistance. Some cancer cells are resistant to TOP-2 poisons, either because they express such a low level of TOP2 that they are insensitive to TOP-2 poisons, or because they have efflux pumps on their plasma membrane that transport drugs out of the intracellular space. 

  The other category of TOP2-targeting anti-cancer drugs is TOP2 catalytic inhibitors. This class is far less prevalent in clinics when compared to the prevalence of TOP-2 poisons. Its cytotoxic effects are achieved by inhibiting the catalytic function of TOP2 and unlike TOP-2 poisons, it does not enable DNA damage.  There are 3 main types of TOP2 catalytic inhibitors, each of which acts via a different mechanism, such as blocking TOP2’s DNA-binding domain to prevent the binding of TOP2 to DNA, thus stabilising the non-covalent DNA-TOP2 complex to prevent DNA cleavage by TOP2, and inhibiting the ATP binding site in the N-terminal domain of TOP2 so that the ATP-driving catalytic ability of TOP2 is lost.8 The clinically authorised drugs that correspond to these 3 mechanisms respectively are aclarubicin, merbarone and novobiocin.

  TOP2 catalytic inhibitors are far safer than TOP2 poisons, not only because they do not introduce DNA breaks that might harm normal cells, but also because they do not induce secondary malignancies or cardiotoxicity as TOP2 poisons do. Anti-cancer drugs containing TOP2 catalytic inhibitors, on the other hand, have relatively lower specificity and potency than those containing TOP2 poisons and so their clinical application is greatly limited. No new topoisomerase II inhibitor has been approved in clinical trials over the last two decades.

   In a nutshell, anti-cancer drugs that target TOP2 have significant promise. They can be used as parts of drug cocktails to overcome cancer cells resistance to other chemotherapy. They also raise possibility of developing innovative dual-target inhibitors that target TOP2 and another protein with a similar ligand. This might have synergistic effects, boosting chemotherapeutic effectiveness significantly. For example, since heat shock proteins such as Hsp90 are similarly overexpressed in proliferating cancer cells, inhibitors that target TOP2 and Hsp90 simultaneously improve the anti-cancer effects while also slowing the emergence of resistance. Moreover, Hsp90 contributes to DNA repair, meaning that apoptosis of normal cells due to DNA damage caused by TOP2 poisons may be prevented.

   The present hurdles in designing TOP2-targeting chemotherapy are mostly around selectivity. It is critical that the drugs targeting TOP2 solely target cancer cells for safety reason, highlighting the need for molecules that can attach to drugs and deliver them precisely to TOP2 on cancer cells exclusively. Considering the β isoform of TOP2 

is associated with the side-effects of TOP2 poisons, developing drugs that target TOP2α specifically will greatly reduce the risk although this could pose as challenging as TOP2β has a higher affinity for DNA than TOP2α. Additionally, TOP2β is expressed in all cells whereas TOP2α is only expressed in proliferating cancer cells. 

   To conclude, the field of TOP2-targeting anti-cancer drugs is thriving and here will undoubtedly be more novel TOP2-targeting anti-cancer drugs revealed in the future. A possible shift in focus from TOP2 poisons to TOP2 catalytic inhibitors may also be seen.


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