The DNA-Binding Polyamine Moiety in the Vectorized DNA Topoisomerase II Inhibitor F14512 Alters Reparability of the Consequent Enzyme-Linked DNA Double-Strand Breaks

Abstract

Poisons of topoisomerase II (TOP2) kill cancer cells by preventing religation of intermediate DNA breaks during the enzymatic process and thus by accumulating enzyme-drug-DNA complexes called TOP2 cleavage-complex (TOP2cc). F14512 is a highly cytotoxic polyamine-vectorized TOP2 inhibitor derived from etoposide and currently in clinical trials. It was shown that F14512 has acquired DNA-binding properties and that the stability of TOP2cc was strongly increased. Paradoxically, at equitoxic concentrations in cells, F14512 induced less DNA breaks than etoposide. Here, we directly compared etoposide and F14512 for their rates of TOP2cc production and resolution in human cells. We report that targeting of TOP2α and not TOP2β impacts cell killing by F14512, contrary to etoposide that kills cells through targeting both isoforms. Then, we show that despite being more cytotoxic, F14512 is less efficient than etoposide at producing TOP2α cleavage-complex (TOP2αcc) in cells. Finally, we report that compared with TOP2αcc mediated by etoposide, those generated by F14512 persist longer in the genome, are not dependent on TDP2 for cleaning break ends from TOP2α, are channeled to a larger extent to resection-based repair processes relying on CtIP and BRCA1 and promote RAD51 recruitment to damaged chromatin. In addition to the addressing of F14512 to the polyamine transport system, the properties uncovered here would be particularly valuable for a therapeutic usage of this new anticancer compound. More generally, the concept of increasing drug cytotoxicity by switching the repair mode of the induced DNA lesions via addition of a DNA-binding moiety deserves further developments..

Transcription-associated topoisomerase activities control DNA-breaks production by G-quadruplex ligands

“…In parallel, using the same genetic approach, we isolated F14R HAP1clones resistant to a lethal concentration of 30 nM F14512, a potent and selective TOP2A poison (49). Targeted sequencing of TOP2A cDNA in seven F14R clones revealed TOP2A mutations for five of them, confirming that TOP2A is the main mediator of F14512 cytotoxicity.…”

“…Clonogenic assay was performed as described by (49). Briefly, after transfection with siRNA, HeLa cells were seeded at low density (250 cells/well) the day before treatment, pre-incubated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) or dimethylsulfoxide (DMSO)…”

DNA topoisomerases as molecular targets for anticancer drugs

“…The presence of a spermine moiety in the compound molecule led to an increase in the degree of DNA binding and consequently to an enhancement of the inhibition of Top II activity 143 . Moreover, the overexpression of polyamine transport system observed in cancer tumours was used to intensify selective drug uptake by cancer cells 144,145 . In vitro activity of F14512 was evaluated in 29 human cancer cell lines (i.a.…”

Polyamine-containing etoposide derivatives as poisons of human type II topoisomerases: Differential effects on topoisomerase IIα and IIβ

“…19, 23 In contrast, at least in A549 lung cancer cells, it has been suggested that topoisomerase IIα is the primary cytotoxic target of the drug. 27 However, the level of topoisomerase IIα in the A549 cells used in the study was considerably higher than that of topoisomerase IIβ. 27 Consequently, the relative roles of the two enzyme isoforms in the actions of F14512 in lung and other cancer cells remain an open question.…”