Multi-omics dissection of R-loop dynamics in tumorigenesis: From transcription-replication conflict to therapeutic targets.

R-loops, three-stranded nucleic acid structures comprising an RNA-DNA hybrid and a displaced single-stranded DNA strand, play context-dependent roles in cancer-serving essential physiological functions while also driving tumorigenesis when dysregulated. Their pathological effects are mediated through replication stress, genomic instability, transcriptional disruption, and defective RNA processing. This review highlights the emerging potential of targeting R-loops as a therapeutic strategy in oncology. We survey advanced methodologies for R-loop mapping, addressing technical limitations of current approaches, and advocate for multi-omics integration to elucidate R-loop dynamics and functional networks in cancers such as lung, bladder, and prostate malignancies. Critically, we explore two promising therapeutic avenues: (1) direct inhibition of R-loop resolvers to trigger excessive R-loop accumulation and replication catastrophe, and (2) synthetic lethality strategies that capitalize on cancer-specific R-loop handling defects. Clinical evidence supporting these approaches is discussed, along with challenges including tumor heterogeneity, detection limitations, and adaptive resistance. We argue that a multi-omics-driven understanding of R-loop biology will accelerate the translation of R-loop-directed therapies into precision oncology.