G-quadruplexes (G4s) are noncanonical DNA structures that promote genomic instability, particularly in α-thalassemia/mental retardation X-linked (ATRX)-deficient gliomas. Although TOP2B has been implicated in chromatin remodeling, its role in G4 resolution remains poorly understood. Here, we identify TOP2B as a previously unrecognized regulator of G4 homeostasis and show that it functionally cooperates with ATRX to facilitate G4 resolution during DNA replication. Disruption of this pathway by CX-5461, a small molecule originally developed as an RNA polymerase I inhibitor, leads to G4 accumulation, replication stress, and DNA damage. Mechanistically, CX-5461 acts as a TOP2B poison that selectively impairs TOP2B binding at G4 sites, alters replication fork dynamics, and induces MRE11-dependent degradation of stalled forks. These effects are strongly enhanced in ATRX-deficient glioma cells, where TOP2B plays a dominant role in G4 regulation. While etoposide similarly induces G4-related DNA damage, it does not affect the ATRX-TOP2B interaction, highlighting CX-5461's unique mechanism. Our findings establish TOP2B as a critical player in G4 resolution, reveal CX-5461's dual function as a TOP2B poison and G4 stabilizer, and propose G4-associated replication stress as a potential therapeutic target in ATRX-deficient gliomas.
© The Author(s) 2025. Published by Oxford University Press.