Transcription-associated topoisomerase 2α (TOP2A) activity is a major effector of cytotoxicity induced by G-quadruplex ligands

Abstract

G-quadruplexes (G4) are non-canonical DNA structures found in the genome of most species including human. Small molecules stabilizing these structures, called G4 ligands, have been identified and, for some of them, shown to induce cytotoxic DNA double-strand breaks. Through the use of an unbiased genetic approach, we identify here topoisomerase 2-alpha (TOP2A) as a major effector of cytotoxicity induced by two clastogenic G4 ligands, pyridostatin and CX-5461, the latter molecule currently undergoing phase I/II clinical trials in oncology. We show that both TOP2 activity and transcription account for DNA break production following G4 ligand treatments. In contrast, clastogenic activity of these G4 ligands is countered by topoisomerase 1 (TOP1), which limits co-transcriptional G4 formation, and by factors promoting transcriptional elongation. Altogether our results support that clastogenic G4 ligands act as DNA structure-driven TOP2-poisons at transcribed regions bearing G4 structures.

Harnessing the Nucleolar DNA Damage Response in Cancer Therapy

“…CX-5461 was shown to induce DSBs through a TOP2-dependent mechanism, however, its activity seems to differ from F14512, a selective and potent TOP2A poison. Furthermore, DNA breaks induced by CX-5461 were reduced when Pol I activity was inhibited by another Pol I inhibitor BMH-21 [123], indicating the contribution of rDNA transcription to the cellular response to CX-5461. The data suggest that the interaction of CX-5461 with DNA and sensitivity to CX-5461 are facilitated by DNA topological stress provoked by a high level of Pol I transcription [123,124].…”

“…Furthermore, DNA breaks induced by CX-5461 were reduced when Pol I activity was inhibited by another Pol I inhibitor BMH-21 [123], indicating the contribution of rDNA transcription to the cellular response to CX-5461. The data suggest that the interaction of CX-5461 with DNA and sensitivity to CX-5461 are facilitated by DNA topological stress provoked by a high level of Pol I transcription [123,124]. CX-5461-mediated chromatin defects, G4 stabilization or R-loops formation at transcriptionally active loci leads to inhibition of Pol I transcription, mobilizing of TOP2 to resolve topological stresses and subsequent TOP2 poisoning.…”

“…Since CX-5461 has been shown to induce activation of ATR at rDNA indicating nucleolar replication stress [97,122], it is plausible that CX-5461 may carry out its action by potentially trapping TOP2α with high selectivity to rDNA promoters compared to regions across the genome. Indeed, a recent study has shown that CX-5461 may act as a DNA structure-driven TOP2poison at transcribed regions bearing G4 structures [123]. CX-5461 was shown to induce DSBs through a TOP2-dependent mechanism, however, its activity seems to differ from F14512, a selective and potent TOP2A poison.…”

“…tivity to CX-5461 are facilitated by DNA topological stress provoked by a high level of Pol I transcription [123,124]. CX-5461-mediated chromatin defects, G4 stabilization or R-loops formation at transcriptionally active loci leads to inhibition of Pol I transcription, mobilizing of TOP2 to resolve topological stresses and subsequent TOP2 poisoning.…”

Proteins from the DNA Damage Response: Regulation, Dysfunction, and Anticancer Strategies

“…A basal level of DNA damage and telomere deprotection increase their sensitivity in cancer cells [276]. The CX-5461 G4 ligand, a quarfloxin derivative initially identified as a RNA-Pol I inhibitor [277], was recently shown to act at transcribed regions bearing G4 structures as a DNA structure-driven TOP2 poisons [278,279]. CX-5461 induces replication-dependent DNA damage through the stabilization of G4 DNA structures, which has entered phase I/II of clinical trials for patients with BRCA1/2 deficient tumors [280] and hematologic cancers [281].…”