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

Abstract

G-quadruplexes (G4), non-canonical DNA structures, are involved in several essential processes. Stabilization of G4 structures by small compounds (G4 ligands) affects almost all DNA transactions, including telomere maintenance and genomic stability. Here, thanks to a powerful and unbiased genetic approach, we identify topoisomerase 2-alpha (TOP2A) as the main effector of cell cytotoxicity induced by CX5461, a G4 ligand currently undergoing phase I/II clinical trials. This approach also allowed to identify new point mutations affecting TOP2A activity without compromising cell viability. Moreover, based on cross-resistance studies and siRNA-based protein depletion we report that TOP2A plays a major role in cell cytotoxicity induced by two unrelated clastogenic G4 ligands, CX5461 and pyridostatin (PDS). We also report that cytotoxic effects induced by both compounds are associated with topoisomerase 2mediated DNA breaks production. Finally, we show that TOP2-mediated DNA breaks production is strongly associated with RNA Pol II-dependent transcription and is countered by topoisomerase 1 (TOP1). Altogether our results indicate that clastogenic G4 ligands act as DNA structure-driven TOP2-poisons at transcribed regions bearing G-quadruplex structures.

The RNA polymerase I transcription inhibitor CX-5461 cooperates with topoisomerase 1 inhibition by enhancing the DNA damage response in homologous recombination-proficient high-grade serous ovarian cancer

“…1c). 16,31,32 We have identified that TOP1 inhibition synergises with CX-5461 to inhibit HR-proficient HGSC cell proliferation, a critical finding supported by other studies demonstrating decreased survival upon treatment with pyridostatin in HeLa cells 32 or with CX-5461 in Eμ-Myc lymphoma 31 depleted of TOP1. Both TOP1 and TOP2 can localise to rDNA and are involved in regulating torsional stress during transcription and DNA replication.…”

“…44 In addition, G-quadruplex stabilisers such as pyridostatin can induce TOP2-dependent DNA DSBs that are countered by TOP1, likely through TOP1's ability to regulate negative supercoiling behind RNA Pol I. 32 With the combination of CX-5461 and the TOP1 inhibitor topotecan in HR-proficient HGSC cells, we found robust nucleolar recruitment of phosphorylated RPA (Ser33) and ATR. We previously found that CX-5461 enhances rDNA chromatin accessibility to MNase.…”

“…Recently, several studies have identified that CX-5461 has a similar sensitivity profile to DNA TOP2 poisons 16,31,32 and requires TOP2 for cytotoxicity. 31,32 TOP2α is a component of the RNA polymerase I pre-initiation complex 42 and it is possible that CX-5461's ability to trap TOP2 can contribute to its 200-fold selectivity for Pol I transcription inhibition over the other RNA polymerases 10,43 as well as its therapeutic efficacy across the genome. In contrast, through an independent approach utilising a genome-wide RNAi screen, we show CX-5461 has a different sensitivity profile to those recently reported for TOP2 poisons (Fig.…”

“…2c). Given recent reports identifying CX-5461 as a potential TOP2 poison, 16,31,32 we also compared the response to the treatments with that of the TOP2 inhibitor doxorubicin. We observed increased phospho-CHK1 and phospho-CHK2 within 3 h upon treatment with CX-5461, topotecan or the combination compared to vehicle.…”

DNA folds threaten genetic stability and can be leveraged for chemotherapy

“…3 DSBs can be generated directly, such as those generated by TOP2 poisoning, ionising radiation, or as a result of the conversion of a primary lesion or blocking structure by transcription or DNA replication. 91,92 DSBs are repaired by two principal mechanisms ( Fig. 3): the non-homologous end joining (NHEJ) pathway, which joins two DNA ends without a DNA template; and the homologous recombination (HR), in which nucleases strip away a stretch of nucleotides at broken ends, exposing a 3 0 overhang of ssDNA in a process called DNA end resection, then use the corresponding sister chromatid as a template to repair the damaged strand.…”