Résumé
Despite the recent advances achieved in the treatment of colon cancer, tumor resistance is a frequent cause of chemotherapy failure. The aim of this project is thus to identify new targets involved in resistance to oxaliplatin using a phenotypic high-throughput screening. We established oxaliplatin-resistant cellular clones from the colon carcinoma cell line HCT-116. Using a clone displaying mild resistance, HCT116-R1 (i.e., with an IC50 10-fold higher than the parental line), we performed a genetic screening based on short hairpin RNA (ShRNA) targeting genes of human kinome. The final aim of this procedure was to reveal genes whose silencing restores drug sensitivity, so displaying a synthetic lethal interaction with the drug. We identified ATR (ataxia-telangiectasia mutated and rad3 related), a protein playing a key role in DNA repair that is activated in response to persistent single-stranded DNA (ssDNA) indirectly induced by various DNA-damaging agents, as a determinant of oxaliplatin resistance. We showed that ShRNA-mediated repression of ATR in HCT 116-R1 sensitize them to the drug. Then, we evaluated the effect of combining oxalipaltin with VE-822, an ATR inhibitor. The analysis of interaction between oxaliplatin and VE-822 was addressed by mean of concentration matrix cytotoxic test and the Bliss equation for additivity. We demonstrated that coincubation of oxaliplatin and VE-822 led to a dramatic synergistic effect in oxaliplatin-sensitive CRC cell lines (HCT116 and SW48) and in oxaliplatin-resistant correlative clones (HCT116-R1, HCT116-R2, SW48-R1, and SW48-R2) both in 2D and 3D. We also showed that the synergistic effect of oxaliplatin and VE-822 was accompanied by an increase of ssDNA followed by DNA double-strand breaks, growth arrest, and apoptosis induction. The appearance of these DNA damages is correlated with activation of ATM pathway, p53, and inhibition of CDK2 activity. Finally, Ve-822 + oxaliplatin association is also efficient in vivo, on immunodepressed mice xenografted with oxaliplatin-resistant cells as well as on immunocompetent mice, with a higher synergistic effect indicating that immune cell death (ICD) is part of the mechanism of this drug combination. In conclusion, all these data confirm the results of our screen by demonstrating for the first time the functional role of ATR in sensitivity to oxaliplatin. Our results highlight the strong potential of ATR inhibition combined to oxaliplatin in vitro but also in vivo to sensitize cells to chemotherapy, creating dramatic DNA damages and stimulating ICD.
Citation Format: Eve Combes, Augusto Faria Andrade, Diego Tosi, Flavie Coquel, Delphine Désigaud, Veronique Garambois, Arnaud Coquelle, Pierre Martineau, Maguy Del Rio, Philippe Pasero, Jerome Moreaux, Roderick Beijersbergen, Nadia Vie, Céline Gongora. Synthetic lethality screening reveals ATR as responsible for oxaliplatin resistance in colorectal cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A147.