Understanding genome instability: from immunogenic potential of micronuclei to the dynamic of transcription–replication conflict stress

Genomic instability is a hallmark of cancer. It arises from defects in DNA replication, repair, transcription, and chromatin regulation, generating genetic diversity. In this thesis, I explore the dual nature of genome instability as it is not only capable of fuelling tumour initiation and progression but can also expose danger signals that may elicit innate immune pathways, creating opportunities for therapeutic intervention. In the first part, I investigated whether micronuclei generated by the stabilization of G-quadruplexes, elicit cGAS-STING pathway and IFN related responses. Using equitoxic doses of distinct G4 binders, I found that all agents increased the MNi production; however, only PDS and CX5461, drove robust IFN-β secretion and an interferon-related upregulated transcriptome, whereas RHPS4 and Braco-19 remained immune-silent. Purifying MNi and profiling them by quantitative proteomics and DNA sequencing revealed that immunogenic MNi are enriched for proteins related to translation, immune autophagy and lysosome programs, while RHPS4-derived MNi show a strong mitochondrial and metabolic signature. Functionally, we discovered autophagy blockade abolished PDS-driven cGAMP and IFN-β production without affecting cGAS recruitment, indicating that macroautophagy enables cGAS activation. Conversely, inhibiting mitophagy relieved RHPS4-induced suppression and restored IFN-β, positioning mitophagy as a tuneable brake on cytosolic DNA sensing. The second part addresses to validate transcription-replication conflicts. First, I performed END-seq in CPT-treated human H1. The results confirmed a time-resolved maps of Top1-dependent DSBs and single-ended DSBs enriched at highly transcribed in early-replicating regions. Secondly, I performed Chromstretch to visualize directly collisions and their geometry. Chromstretch results displayed that R-loops concentrate at replication-bubble boundaries where elongating RNAPolII accumulates, and some local chromatin marks such as H3K9me3 and H2BK120ub intensify at these sites. In conclusion this work contributes to a more integrated view of genome maintenance, unveiling new opportunities to harness genome stress as a therapeutic target rather than an obstacle.