Reply to: The chemotherapeutic drug CX-5461 is a potent mutagen in cultured human cells

Abstract Replying to GCC Koh et al., Nature Genetics ( www.nature.com/articles/s41588-023-01602-9 ) and the comments by Simon Boulton in Nature Genetics ( www.nature.com/articles/s41588-023-01611-8 ). The drug CX-5461 [Pidnarulex] is an inhibitor of RNA polymerase I and topoisomerase II and a DNA G-quadruplex stabilizer. Koh et al. recently reported that CX-5461 induces extensive, non-selective collateral mutagenesis in vitro at magnitudes surpassing known environmental carcinogens, raising concerns about its potential to promote secondary cancers. Mindful that the report of Koh et al. was exclusively an in vitro study, we applied the same ultra-sensitive, error-corrected TwinStrand Duplex Sequencing approach and analyses (methods) to a longitudinal series of clinical specimens from four patients who participated in the Phase I dose-escalation trial of CX-5461 in advanced haematologic malignancies [ACTRN12613001061729]. In contrast to the findings of Koh et al. we found no evidence that clinical administration of CX-5461 significantly increased mutational burden in patient samples, nor did we observe the reported mutational signature. These results suggest that the mutagenic effects described in vitro do not translate to the clinical setting. This is particularly important given that several clinical trials, including a Phase Ib study of CX-5461 in patients with solid tumours [ NCT04890613 ] and a National Cancer Institute sponsored Phase I trial [ NCT06606990 ] are currently enrolling patients. These ongoing and planned clinical efforts highlight both the therapeutic promise of CX-5461 and the importance of evaluating this agent within rigorous and clinically relevant frameworks.