Gois, M. M.; Bonafe, L.; Silva, L. G. S.; Mancini, M. C. S.; Kampen, R. A.; Pavan, I. B.; Severino, M. B.; Quintero-Ruiz, N.; Noordermeer, S. M.; Simabuco, F. M.

Recent studies have suggested that the S6 kinase 1 (S6K1) contributes to DNA repair. However, questions regarding the specific pathways and mechanisms underlying this regulation remain unanswered. Moreover, it has not yet been investigated whether S6K2, a functional homologue of S6K1, also contributes to DNA repair. Here, we demonstrate that both S6K1 and S6K2 (S6K1/2) are important for efficient homologous recombination (HR) DNA repair. Double knockout of S6K1/2 prevented the formation of BRCA1 and RAD51 foci, increased sensitivity to DNA damage, and increased markers of genomic instability, while single knockout had little effect on HR markers. Our findings suggest that S6K1/2 present overlapping and compensatory functions regulating HR. Mechanistically, we demonstrated that S6K1/2 are important for maintaining BRCA1 protein stability, limiting its proteasomal degradation and promoting genome stability. Finally, pharmacological inhibition of S6K1/2 sensitised HR-proficient breast cancer cells to olaparib, highlighting a potential therapeutic strategy to enhance PARP1 inhibitor efficacy in HR-proficient tumours.