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.
DNA is intrinsically dynamic and folds transiently into alternative higher-order structures such as G-quadruplexes (G4s) and three-way DNA junctions (TWJs). G4s and TWJs can be stabilised by small molecules (ligands) that have high chemotherapeutic potential, either as standalone DNA damaging agents or combined in synthetic lethality strategies. While previous approaches have claimed to use ligands that specifically target either G4s or TWJs, we report here on a new approach in which ligands targeting both TWJs and G4s in vitro demonstrate cellular effects distinct from that of G4 ligands, and attributable to TWJ targeting. The DNA binding modes of these new, dual TWJ-/G4-ligands were studied by a panel of in vitro methods and theoretical simulations, and their cellular properties by extensive cell-based assays. We show here that cytotoxic activity of TWJ-/G4-ligands is mitigated by the DNA damage response (DDR) and DNA topoisomerase 2 (TOP2), making them different from typical G4-ligands, and implying a pivotal role of TWJs in cells. We designed and used a clickable ligand, TrisNP-α, to provide unique insights into the TWJ landscape in cells and its modulation upon co-treatments. This wealth of data was exploited to design an efficient synthetic lethality strategy combining dual ligands with clinically relevant DDR inhibitors.
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.
DNA mimicry has been the subject of intensive research and resulted in the development of DNA analogues such as PNAs and LNAs. There are also examples of proteins with structural and/or charge distribution analogies with respect to the DNA double helix which allow them to interfere with other DNA-binding proteins and modulate the biological processes in which they are involved. We previously characterized a new class of DNA-surface mimic molecules constituted by repetitions of dimeric units of 8-amino-2-quinolinecarboxylic acid (Q) and 8-aminomethyl-2-quinolinecarboxylic acid (mQ). The helical folding of these entities can mimic a B-DNA molecule, displaying a minor and a major groove that can be modulated depending on the dimers and of the nature of their side chains. In vitro, these DNA mimics could inhibit, in a relative selective manner, the catalytic activity of DNA topoisomerase I (Top1), whereas they had no effect on the activity of DNA polymerases or DNAses. Inhibition of Top1-mediated relaxation of supercoiled DNA plasmid increased with the length of the DNA mimics. Here, we further characterized the mechanism of Top1 inhibition by these DNA mimics. We found that, conversely to camptothecin (CPT) and its derivatives that poison Top1 via the inhibition or the re-ligation step of the reaction, DNA mimics inhibited Top1-mediated DNA cleavage in vitro by preventing the binding of the enzyme to its substrate, a mechanism of Top1 competitive inhibition that was formerly referred to as catalytic inhibition. We also found that co-incubation of DNA mimics with CPT had an additive effect on the inhibition of Top1-mediated relaxation of supercoiled DNA, further suggesting a mechanism that is different from CPT. Because transfection of DNA mimics could inhibit the growth of various cancer cell lines, we further investigated whether Top1 could play a role in this cytotoxicity. We found that Top1 knock-down in OVCAR4 ovarian cancer cells resulted in decreased sensitivity to the (mQQ4)8 DNA mimic as compared to OVCAR4 control cells, suggesting that Top1 is a target of DNA mimics in cells and is involved in their cytotoxic effects. Conversely to CPT, transfection of HCT116 cells with the (mQQ4)8 DNA mimic was not associated with an increase in γH2AX, suggesting that DNA mimics do not induce DNA breakage. We are currently analyzing whether the (mQQ4)8 can have an impact on CPT-induced Top1-DNA complexes formation. Interestingly, we also showed that several SN38-resistant HCT116 cell clones characterized by specific Top1 mutations were still sensitive to the (mQQ4)8 DNA mimic. Together our results demonstrate that DNA mimics can be considered as a new class of competitive inhibitors of Top1. Further studies are ongoing to identify the structural features that are essential for Top1 inhibition in order to generate more potent derivatives that could be used to counteract resistance to CPT derivatives used in the clinic. Citation Format: Aurélie Garcin, Valentina Corvaglia, Madeleine Bossaert, Marie-Jeanne Pillaire, Ivan Huc, Sébastien Britton, Vincent Parissi, Philippe Pourquier. Foldamers mimicking the B-DNA surface as a new class of DNA topoisomerase I inhibitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4930.
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