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G-quadruplex DNA reprograms the photochemistry of CX-5461 toward radical-driven anticancer activity

Aug 12, 2026

Abstract
CX-5461 (pidnarulex) is the most clinically advanced small-molecule ligand associated with G-quadruplex (G4) targeting and shows activity in DNA repair–deficient tumors, yet its clinical use is associated with dose-limiting phototoxicity. Here, we repurpose this intrinsic photoreactivity to investigate CX-5461 as a G4-associated, light-activated antitumor scaffold. In solution, CX-5461 produces both Type I and Type II reactive oxygen species (ROS), whereas complexation with G4 DNA markedly decreases detectable singlet oxygen (1O2) formation and favors radical-associated Type I oxidation. This G4-modulated photochemical shift drives oxidative remodeling and destabilization of G4-containing DNA. In cancer cells, photoactivation enhances cytotoxicity by up to two orders of magnitude relative to the dark state and is accompanied by elevated intracellular ROS, increased 8-oxoG formation, γ-H2AX accumulation, and changes in BG4-detected nuclear G4 structures. In vivo, light-activated CX-5461 suppresses tumor growth and extends survival in immunocompetent syngeneic models, eliciting hallmarks consistent with immunogenic genome stress. Collectively, these findings reposition CX-5461 as a clinically relevant scaffold for G4-associated photogenomic cancer therapy, while supporting a model in which broader oxidative DNA damage may also contribute to the biological response.

Introduction
G-quadruplexes (G4s) are guanine-rich, noncanonical nucleic acid secondary structures enriched in gene promoters, telomeres, and replication origins, where they regulate transcription, replication-fork progression, and genome stability [1, 2]. Their prevalence in highly transcribed and replication-stressed genomic regions has positioned G4s as compelling therapeutic targets, particularly in cancers with defective DNA damage response (DDR) pathways [2–6]. Beyond their regulatory roles, the exceptional guanine density of G4 motifs renders them intrinsically susceptible to oxidative modification, marking G4s as privileged substrates for spatially confined oxidative genome damage [7–9]. Despite this convergence of structure, function, and chemistry, clinically actionable strategies that directly exploit G4 reactivity remain scarce [10–15].

CX-5461 (pidnarulex) is arguably the most clinically advanced small molecule associated with G4 engagement [16]. Initially developed as an inhibitor of RNA polymerase I–driven ribosomal DNA transcription, CX-5461 was later shown to stabilize G4 DNA, induce replication-associated lesions, and activate ATM/ATR-mediated checkpoint signaling [17–21]. Its high therapeutic activity in homologous recombination–deficient malignancies, including BRCA1/2-mutant breast and ovarian cancers, underscores a synthetic-lethal interaction between G4 stabilization, replication stress, and impaired DDR [17, 22–24]. These properties have motivated clinical evaluation of CX-5461 in DNA repair–defective solid tumors, positioning it as a rare example of a genome-structure-directed therapeutic agent in advanced clinical development.

Notably, clinical studies also reported dose-limiting phototoxicity upon sun exposure, implicating intrinsic photoreactivity of the CX-5461 chemotype rather than its canonical synthetic-lethal mechanism [22]. This observation places CX-5461 at an unusual and largely unexplored crossroad of G4-directed genome stress and photoinduced chemical reactivity. Whether such photoreactivity represents a liability, or a structure-guided therapeutic opportunity, has remained unresolved.

Here we repurpose CX-5461 as a G4-directed, light-activated antitumor agent. Because CX-5461 is an established G4-binding ligand, we reasoned that its clinically observed photoreactivity could be exploited to generate oxidative genome stress in proximity to G4-containing regions rather than relying solely on its canonical dark activity. We show that while CX-5461 generates both Type I and Type II reactive oxygen species (ROS) in solution, complexation with G4 DNA reprograms its photoactivity toward predominantly Type I oxidation. Our data support a model in which photoactivated CX-5461 induces oxidative alteration of G4-containing DNA, leading to changes in G4 stability, polymerase progression, and cellular markers of genome stress. In cancer cells, photoactivation induces up to a two-orders-of-magnitude enhancement in cytotoxicity, which correlates with elevated intracellular ROS levels, increased 8-oxoG formation, and γ-H2AX accumulation, ultimately leading to pronounced nuclear G4 remodeling. In immunocompetent syngeneic models, light-activated CX-5461 suppresses tumor growth and extends individual survival while inducing oxidative genome damage associated with immunogenic signaling, including the release of damage-associated molecular patterns (DAMPs) and enhanced CD8⁺ T-cell infiltration. Together, these findings support a G4-associated photogenomic mechanism, while recognizing that broader oxidative DNA damage may also contribute to the cellular and in vivo responses.