Identification and validation of a novel DDR inhibitor provides novel insights into the mechanisms of resistance to Chk1 inhibitors
Résumé
Genomic DNA is continuously challenged by endogenous and exogenous stressors. To maintain genomic integrity, cells have evolved the DNA damage response (DDR), an integrated response which induces a transcriptional program, control the cell cycle progression and repair DNA. Targeting the DDR is a promising strategy to treat cancers as exemplified with the efficacy of PARP1 inhibitors in BRCA1/BRCA2-deficient cancers. However, cancers are prone to develop resistance to these novel targeted therapies and there is a need for novel molecules, targets or biomarkers to exploit some of their vulnerabilities and circumvent their resistance. To identify novel molecules targeting the DDR, we developed a phenotypical screen to identify sensitizers to DNA damaging agents in the French Essential National Chemical Library. This led to identify a novel molecule, that we named Shuri1, as a sensitizer of proliferative cells to camptothecin, an anticancer agent poisoning DNA topoisomerase 1. Shuri1 induces replication stress, leading to RPA and γH2AX foci formation and phosphorylation of Chk1 on Ser345 in replicating cells. Using in vitro kinase assays, we demonstrated that Shuri1 behaves as a Chk1 inhibitor, with a good selectivity as compared to its activity on Chk2. Moreover, the Chk1 autophosphorylation on Ser296 is inhibited upon Shuri1 treatment in human cells validating its function of kinase inhibitor. In addition, Shuri1 and its derivatives, potentiated the toxic effect of hydroxyurea and camptothecin on mammalian cells. Our current efforts aim at improving its activity and solubility through the development of prodrugs. To confirm that the cellular effects of Shuri1 are mediated by its ability to inhibit Chk1 in cells, we used an approach recently implemented in the lab (Bossaert et al, 2021) to isolate from mutagenized human haploid cells, clones resistant to Shuri1 and, as a control, to a commercially available CHk1 inhibitor, AZD-7762. Using Next-Generation Sequencing to identify the mutations conferring the resistance, we discovered that some clones carried mutations in the Chk1 kinase itself, validating our approach, while others were mutated in other essential genes known to control genome stability but not connected to Chk1 yet. I will present these data that identify 1) a novel Chk1 inhibitor and 2) novel determinants of the sensitivity to Chk1 inhibitors. Bossaert M, Pipier A, Riou J-F, Noirot C, Nguyên L-T, Serre R-F, Bouchez O, Defrancq E, Calsou P, Britton S, et al (2021) Transcription-associated topoisomerase 2α (TOP2A) activity is a major effector of cytotoxicity induced by G-quadruplex ligands. eLife 10: e65184