Targeting R-Loop–Driven Replicative Stress in Fanconi Anemia– Deficient Tumors

Replicative stress (RS) is a major source of DNA damage in cancer cells, acting both as a driver of genomic instability and as a therapeutic vulnerability. One of the main causes of RS is transcription-replication conflicts (TRCs), which are particularly frequent in tumor cells with high transcriptional activity. These conflicts are closely associated with the formation of R-loops, three-stranded structures composed of an RNA:DNA hybrid and a displaced single-stranded DNA, which can interfere with replication fork progression and induce DNA breaks if not properly resolved. The resolution of R-loops and TRCs relies on specialized enzymes, including Senataxin (SETX), an RNA:DNA helicase that plays a critical role in maintaining genome stability. We have recently identified a synthetic lethal interaction between SETX and the Fanconi anemia (FA) pathway, which is essential for tolerating replicative stress, particularly that arising from TRCs. Inhibition of SETX in FA-deficient cells leads to massive accumulation of unresolved R-loops, incomplete DNA replication, persistence of DNA damage into mitosis, and mitotic catastrophe resulting in cell death. Taking advantage of this vulnerability, we sought to exploit this synthetic lethality by targeting SETX in FA-deficient cancers, for which no effective targeted therapies currently exist. Results obtained using siRNA-mediated knockdown of SETX in 3D spheroid models demonstrate selective impairment of tumor growth in FA-deficient cells, recapitulating the synthetic lethal effect. Using an advanced virtual screening approach, we have identified candidate small-molecule SETX inhibitors that are currently undergoing biophysical and cellular validation. Beyond FA-deficient cancers, we aim to extend this strategy to other tumors characterized by high RS and DNA repair defects, including ovarian cancers. In 3D ovarian cancer spheroid models, SETX inhibition also restricts tumor growth, highlighting the importance of R-loop regulation in the cellular response to RS. Ongoing work seeks to evaluate SETX inhibitors alone or in combination with PARP inhibitors, with the goal of exploiting DNA damage response dependencies and overcoming resistance to PARP inhibition. Overall, these findings identify R-loop regulation as a key vulnerability that can be exploited for targeted therapies in FAdeficient and other RS-high cancers.