Abstract
Colorectal cancer is one of the most common cancers and the second deadliest in France. Due to their altered metabolism and rapid proliferation, cancer cells experience increased replicative stress compared to normal cells. Consequently, cancer cells rely more heavily on replicative stress response pathways, such as the ATR/Chk1 signaling pathway, for their development. Furthermore, conventional chemotherapies damage DNA and exacerbate the replicative stress already inherently high in cancer cells. However, resistance often develops, constituting the primary cause of therapeutic failure. Therefore, a promising option is to combine conventional chemotherapies with inhibitors of proteins on which cancer cells depend for survival. For instance, ATR inhibitors are very promising and are undergoing clinical trials, but resistance and toxicity issues still hinder their use in patients. It appears urgent to find alternative therapeutic strategies.TopBP1 is the main activator of ATR and forms nuclear condensates that act as molecular switches to amplify ATR activity. It is a scaffolding protein involved in the initiation of DNA replication, DNA repair, transcription regulation, and checkpoint activation. TopBP1 has been implicated in oxaliplatin resistance in gastric cancers and has been proposed as a novel biomarker for advanced-stage colorectal cancers. The weak and site-specific interactions within TopBP1 condensates are highly sensitive to changes in cellular properties, suggesting that small molecules could alter the formation of TopBP1 condensates. To test this hypothesis, we developed a high-throughput screening system for TopBP1 condensation modulators. We identified FDA-approved drugs, Quinacrine and Thimerosal, that inhibit TopBP1 condensation, block ATR/Chk1 signaling activation, and sensitize colon cancer cells to chemotherapeutic agents in vitro and in vivo. This initial study provided proof of concept that TopBP1 is a promising therapeutic target for treating advanced-stage colorectal cancer.These findings enabled us to conduct a second screen containing anti-cancer molecules in clinical and pre-clinical studies. We identified a potent inhibitor of TopBP1 condensates, AZD2858, which specifically inhibits the ATR/Chk1 pathway. It strongly synergizes with the combination of conventional chemotherapies FOLFIRI on a panel of human and murine colorectal cancer cell lines cultured as spheroids, including lines resistant to SN38 (the active metabolite of Irinotecan, a topoisomerase I inhibitor). In the long term, this inhibitor enhances apoptosis induction in tumor cells.Lastly, this doctoral project included a study of the ATR activation domain (AAD) of TopBP1. We mutated three conserved sites of TopBP1’s AAD (R1113A, R1116A, R1118A), which resulted in a significant reduction in Chk1 phosphorylation. Unexpectedly, ATR remained active and phosphorylated other substrates like RPA32 S33, and this TopBP1 mutant retained its ability to form biomolecular condensates. This mutant thus demonstrates, for the first time, a functional separation between ATR and Chk1. This project has enabled the study of TopBP1 from both fundamental and applied perspectives, positioning this protein as a novel and promising target for the treatment of advanced-stage colorectal cancer.