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Targeting Topoisomerase II Inhibitors to Specific DNA Sequences


By: Carol A. Rouzer, VICB Communications
Published:  February 26, 2018


Linking the topoisomerase II inhibitor etoposide to a targeting oligonucleotide directs DNA cleavage to a specific site.


Type II topoisomerases play critical roles during DNA replication and transcription by relieving torsional stress and removing knots and tangles from the genetic material. They accomplish these tasks by binding a DNA duplex, cleaving both strands, and then stabilizing the cleaved DNA by covalently joining the 5´-ends of each strand to two active site tyrosine residues. The resulting structure is called a cleavage complex. The enzyme then binds a second DNA duplex and passes it through the cleaved site of the first duplex using energy provided by ATP. Finally, the two cleaved strands are reunited (Figure 1). Topoisomerase II inhibitors, including etoposide, mitoxantrone, and doxorubicin, stabilize the cleavage complex by intercalating into the cleaved DNA bonds, thereby preventing their re-ligation. Failure to resolve the cleavage complex ultimately leads to permanent double-stranded breaks, which if not repaired, can lead to cell death. Cancer cells are particularly susceptible to the toxic effects of topoisomerase II inhibitors. Thus, these inhibitors have been used effectively as anticancer agents for a wide array of malignancies. However the compounds are not without their toxicity, leading Vanderbilt Institute of Chemical Biology member Neil Osheroff and his doctoral student Lorena Infante Lara to design etoposide-based inhibitors targeted specifically to cancer cells  [L. Infante Lara et al., (2018) Nuc. Acids Res., published online February 16, DOI:10.1093/nar/gky072/4852808].


FIGURE 1. Structure and mechanism of topoisomerase II. The enzyme cleaves both strands of one DNA duplex (green) and retains the cut ends by binding them to two active site tyrosine residues. The enzyme then uses energy from ATP hydrolysis to pass a second DNA duplex (pink) through the break in the first DNA segment. The ends of the first double helix are then ligated. To the right is shown the structure of the ATPase and DNA cleavage domains of topoisomerase II from S. cerevisiae). Image reproduced by permission from Springer Nature, from S. M. Vos, et al., (2011) Nat. Rev. Mol. Cell Biol, 12, 827. Copyright 2011.



A particularly devastating negative side effect of the use of topoisomerase II inhibitors is the development of secondary cancers in some patients who survive their original disease. The most common secondary cancers are acute myelogenous leukemia, which results from rearrangements in the mixed lineage leukemia (MLL) gene, and acute promyelocytic leukemia, which results from translocations between the promyelocytic leukemia (PML) gene and the retinoic acid receptor alpha (RARA) gene. The frequency of these rearrangements in patients treated with topoisomerase II inhibitors indicates that sites in these genes are highly susceptible to topoisomerase II-mediated DNA cleavage. Thus, the investigators chose a topoisomerase II cleavage hotspot in the PML gene as their initial target site for study. They then used the crystal structure of a topoisomerase IIβ cleavage complex stabilized with etoposide (Figure 2A) and molecular modeling to design an oligonucleotide-linked topoisomerase inhibitor (OTI). The inhibitor comprised the active core of etoposide (demethylepipodophyllotoxin, DEPT) attached to a single-stranded oligonucleotide complementary to the target PML cleavage hotspot. The drug was joined to the oligonucleotide by a linker that provided the needed length and flexibility to allow topoisomerase II-mediated cleavage of the target strand following binding of the OTI (Figure 2B). The first OTI that was investigated, OTI28, placed the attached linker at the cytosine in position 28 of the OTI oligonucleotide (Figure 3). When bound to the target sequence in the PML gene, this cytosine lies opposite the guanosine at position 23, and molecular modeling predicted that topoisomerase II-mediated cleavage should occur between bases 24 and 25. To test this prediction, the investigators incubated OIT28 with the complementary target oligonucleotide in the presence of topoisomerase IIα or topoisomerase Iiβ (the two human topoisomerase II isoforms). They found that, as predicted, cleavage occurred between positions 24 and 25 of the target oligonucleotide, as well as between positions 23 and 24. Results were similar with both enzyme isoforms. Molecular modeling explained the ability of OTI28 to induce topoisomerase-mediated cleavage at positions on both sides of cytosine 24 (Figure 4). As a control, the researchers also incubated the target oligonucleotide duplexed with an unmodified complementary strand in the presence of etoposide and each topoisomerase isoform. In this case, cleavage also occurred between positions 24 and 25, the known topoisomerase II hotspot, but the secondary site of cleavage was between positions 19 and 20 rather than between 23 and 24.




FIGURE 2. (A) Details from the crystal structure of a topoisomerase IIβ cleavage complex stabilized by etoposide (orange). The tyrosine residues binding the 5´ ends of the DNA strands are shown in blue and gray. The DNA strands are shown in dark and light green. Numbers of the nucleotides on each strand are shown. Cleavage occurs between -1 and +1, and the two strands are differentiated by the presence or absence of an asterisk. (B) Model based on the structure shown in (A) showing OTI28 in orange with the modified cytosine in the +5* position and the scission site at position 23-24 (-1 to +1) on the target strand (green). Figure reproduced under the Creative Commons Attribution License 4.0 from L. Infante Lara et al., (2018) Nuc. Acids Res., published online February 16, DOI:10.1093/nar/gky072/4852808.


FIGURE 3. Structure of OTI28, showing the DEPT nucleus (left), the linker, and the modified cytosine residue at position 28 of the targeting oligonucleotide. Figure reproduced under the Creative Commons Attribution License 4.0 from L. Infante Lara et al., (2018) Nuc. Acids Res., published online February 16, DOI:10.1093/nar/gky072/4852808.




FIGURE 4. Molecular models of DNA cleavage complexes formed by OTI28 (orange strand) with the point of cleavage between positions 24 and 25 (A) or between 23 and 24 (B) of the target strand (green). Figure reproduced under the Creative Commons Attribution License 4.0 from L. Infante Lara et al., (2018) Nuc. Acids Res., published online February 16, DOI:10.1093/nar/gky072/4852808.


The initial results confirmed that OTI28 could target topoisomerase II-mediated cleavage of an oligonucleotide to a specific known hotspot. The investigators next constructed OTI29, OTI33, and OTI23, by attaching the DEPT-bearing linker to the corresponding positions 29, 33, and 23 of the targeting oligonucleotide, respectively. They found that each of these OTIs induced topoisomerase IIα- or IIβ-dependent cleavage of the target oligonucleotide at sites within 2 bases of the modified nucleotide on the OTI. As some of these sites were not known topoisomerase II-DNA cleavage hotspots, the findings demonstrated the ability of an OTI to guide topoisomerase II to cleave DNA at sites that would not otherwise be favored.

The goal of this research was to target topoisomerase II-mediated cleavage to chromosomal sites that would only be present in a cancer cell. One such site is a translocation between PML and RARA that leads to acute promyelocytic leukemia. The researchers designed an OTI bearing a 50 nucleotide-long oligonucleotide complementary to a sequence at the site of the PML/RARA translocation. They found that this OTI induced strong cleavage of the complementary target sequence that spanned the translocation, but not of sequences derived only from the parental PML or RARA genes. Strong cleavage also resulted using an OTI bearing a 30 nucleotide-long oligonucleotide, but not one bearing a nucleotide of only 20 bases in length. These findings were consistent with the requirement of topoisomerase II for a DNA substrate of >20 nucleotides.


The ability of a targeted OTI to selectively induce topoisomerase II-mediated cleavage of DNA corresponding to a cancer-related translocation provides proof-of-concept that topoisomerase II inhibition can be directed to more specifically kill cancer cells. A similar approach might be used in the case of any cancer bearing a known mutation in a driver gene. Although much work remains before this approach can be translated to the clinic, it is an important first step toward developing more effective and less toxic topoisomerase II inhibitors


View Nucleic Acids Res.article: Coupling the core of the anticancer drug etoposide to an oligonucleotide induces topoisomerase II-mediated cleavage at specific DNA sequences










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