Topoisomerase I Inhibition in ETV4‐overexpressed Non‐Small Cell Lung Cancer Promotes Replication and Transcription Mediated R‐Loop Accumulation and DNA Damage

Abstract Coordinating transcription and replication via transcription factors (TFs) is a conserved mechanism in higher eukaryotes. The role of TFs in regulating these processes in cancers remains unclear. Here, it is shown that oncogenetic ETS transcription factor ETV4 controls DNA replication through both transcriptional and non‐transcriptional mechanisms in non‐small cell lung cancer (NSCLC). ETV4 localizes to specific DNA replication origins and interacts with the origin recognition complex subunits ORC1 and ORC6 during the G1/S phase, facilitating origin formation. Using quantitative in situ analysis of protein interactions at DNA replication forks (SIRF) assays, it is shown that ETV4 transiently localizes to replication forks in the S phase. It interacts with replicative helicase MCM2 N‐terminal, histone H3, and histone‐chaperone FACT and is involved in histone processing during replication. Additionally, ETV4 transcriptionally regulates key replisome genes MCM2, MCM4, MCM5, MCM10, and ORC1, influencing their expression and recruitment to chromatin. Due to its binding at the origin‐promoter locus like the MCM4 gene, ETV4 overexpression increases R‐loop formation, DNA damage, and cell death under external replication stress induced by topoisomerase I (TOP1) inhibitor. These findings highlight the dual role of ETV4 in replication and transcription and suggest that targeting TOP1 could be a synthetic‐lethal approach in ETV4‐overexpressed lung cancer.