Investigating PDS and CPT-induced genome instability in human cancer cells

Genome instability is a nearly ubiquitous hallmark of human cancer cell lines and results from defective coordination of essential biological processes. It encompasses diverse genetic alterations and drives tumorigenesis and therapy resistance. High levels of genome instability can also compromise viability, creating opportunities for selective therapeutic intervention. Among its hallmarks, micronuclei (MNi) can directly trigger a Type I Interferon β (IFN-β) innate immune response. Accordingly, antineoplastic compounds such as the G4 binder pyridostatin (PDS) and the Top1 poison camptothecin (CPT) induce high genome instability, increasing MNi formation and activating IFN-β signaling. Thus, my PhD work investigates the molecular mechanisms underlying genome instability in human cancer cells using these two complementary models. In the first part, we characterized the molecular mechanisms of MNi formation induced by G-quadruplex stabilization. By combining immunofluorescence approaches with labeling of active replication, we showed that PDS-induced MNi formation occurs via two complementary pathways determined by the genomic context. During the G1–S transition, R-loop-dependent replication stress drives MNi formation, with DNA polymerase η facilitating replication restart at stalled forks. In contrast, in late S phase, MNi formation occurs independently of R-loops and depends on PrimPol-mediated repriming, with Pol η supporting fork progression. In the second part, we characterized the mechanisms of genome instability activated by Top1 cleavage complex stabilization. Combining immunofluorescence and genome-wide approaches, we found that CPT induces MNi formation specifically during the G1–S transition and early S phase by promoting R-loop accumulation. Impairment of TFIIS, which resolves RNAPII backtracking, further enhanced transcription–replication conflicts and MNi formation. Indeed, Top1cc stabilization led to RNAPII stalling and backtracking at specific genomic loci, thereby stabilizing R-loops and promoting double-strand break formation. Collectively, these results reveal conserved mechanisms of genome instability in human cancer cells and point to potential targets for improving anticancer therapies.