Targeting Topoisomerase II Inhibitors
Topoisomerases are critical enzymes during DNA replication and transcription, as they are responsible for untangling the knots and twists that can form as DNA is unwound and rewound during these processes. Type II topoisomerases accomplish this task by making a temporary double stranded break in one segment of DNA and then passing another DNA segment through the break before repairing it. The enzyme stabilizes the break by covalently binding the 5´-ends of the two DNA strands to tyrosine residues in its active site, forming a cleavage complex. An important class of anti-cancer drugs, the topoisomerase inhibitors, acts by stabilizing the cleavage complex so that the DNA strands cannot be reconnected. Unresolved cleavage complexes eventually lead to permanent DNA double strand breaks that, if not repaired, can result in cell death. Cancer cells are especially sensitive to the toxic effects of topoisomerase poisons, but these drugs can also affect normal cells, leading to adverse effects. Among these are secondary cancers in some patients who survive their original disease. This led Vanderbilt Basic Sciences investigator Neil Osheroff and his graduate student Lorena Infante Lara to design topoisomerase inhibitors specifically targeted to cancer cells. The researchers used an X-ray crystal structure of a topoisomerase II cleavage complex that had been stabilized by the inhibitor etoposide and molecular modeling to design their oligonucleotide-linked topoisomerase inhibitors (OTIs). Their hypothesis was that by joining the active core of etoposide to a single-stranded oligonucleotide using a linker of just the right length, they could induce selective topoisomerase II-mediated cleavage of DNA that is complementary to the oligonucleotide in the OTI. This would enable them to target topoisomerase to a sequence of DNA found only in a cancer cell. They initially tested this hypothesis using an OTI designed to bind to a sequence in the promyelocytic leukemia (PML) gene known to have a cleavage hotspot for topoisomerase II. This OTI induced topoisomerase-mediated cleavage of DNA carrying the sequence of the PML gene hotspot. They next showed that by moving the point where the etoposide core and linker were attached to the oligonucleotide in the OTI, they could alter the cleavage site on the target DNA so that it occurred at points other than the known hotspot. Finally, they designed an OTI bearing an oligonucleotide complementary to the sequence of a translocation found only in promyelocytic leukemia cells. This OTI induced cleavage of the DNA sequence found in the translocation, but not of sequences found only in the individual genes that were part of the translocation. This finding demonstrated that OTIs can target DNA sequences specific to cancer cells. Much further work is required before this discovery can be translated to the clinic, but it provides an important proof-of-concept for a new way to obtain more effective and less toxic anti-cancer agents. The work is published in the journal Nucleic Acids Research [L. Infante Lara, et al. Nucl. Acids Res., (2018) published online February 16, DOI:10.1093/nar/gky072/4852808]