Molecular Mechanism Of SIRT1 Regulating WEE1 Inhibitor Sensitivity In Cancer Cells

Cell cycle checkpoints are highly conserved surveillance mechanisms that regulate the programmed and orderly progression of the cell cycle.Proper activation of cell cycle checkpoints is critical for cell survival and genomic stability,especially when the genomic DNA is damaged.Cancer cells exhibit higher levels of endogenous DNA damage than normal cells due to their rapid proliferation characteristics.Targeting checkpoint kinase activities by disrupting them represents a promising strategy for cancer therapy that force cell cycle progression even in the presence of damaged DNA,and leading to cancer cells death in a mitotic catastrophe.Many cancer cells have defective G1/S cell cycle checkpoint due to the loss of function of the tumor suppressor p53,making these cells highly dependent on intra-S and G2/M cell cycle checkpoints.The WEE1 kinase plays a pivotal role in regulating the intra-S-phase and G2/M cell cycle checkpoints by phosphorylating cyclin-dependent kinases CDK1/2 at Tyr15,thereby inhibiting their kinase activities.Inhibition of WEE1 leads to unscheduled initiation of replication origins,mitotic catastrophe,and ultimately genomic instability and cancer cell death.Targeting WEE1 kinase in cancer cells with increased replication stress and G1/S checkpoint deficiency can lead to more effective cancer treatment without damaging normal cells that have a functional G1/S checkpoint.The WEE1 inhibitor MK-1775 is a potent and selective ATP-competitive small-molecule inhibitor of WEE1 and is currently undergoing Phase Ⅰ and Ⅱ clinical trials with promising results.However,the regulation of WEE1 catalytic activity remains poorly understood,and reliable biomarkers for predicting response to WEE1 inhibitor are yet to be identified.In this study,we discovered a significant negative correlation between the expression level of SIRT1 and the sensitivity of WEE1 inhibitor MK-1775 in cancer cells through bioinformatics analysis,cell experiments and animal experiments.Mechanicaly,we found that SIRT1 mediated the deacetylation of WEE1 at the Lys177 site.A conserved segment surrounding the deacetylated Lys177 residue interacts with the catalytic kinase domain of WEE 1 in the form of a dimer,leading to low activity of WEE1.Therefore,cancer cells with high levels of SIRT1 are sensitive to WEE1 inhibitors.Upon DNA damage,WEE1 is phosphorylated at Ser642 in a CHK1-dependent manner,which disrupts its interaction with SIRT1.The subsequent acetylation of WEE1 at Lys177 by acetyltransferase GCN5 leads to the dissociation of the inhibitory segment from the kinase domain,resulting in the activation of WEE 1 and the cell-cycle checkpoints.Finally,WEE1-mediated inhibitory phosphorylation of CDK1/2 at Tyr15 arrests the cell cycle.Therefore,cancer cells with high levels of acetylation and activity of WEE1 caused by low levels of SIRT1 are resistant to WEE 1 inhibitors.In conclusion,our study identifies a novel self-inhibiting regulatory mechanism for the cell cycle checkpoint kinase WEE1,where SIRT1-mediated deacetylation of WEE1 at Lys177 promotes its inhibitory interaction with the kinase domain,leading to a low kinase activity of WEE 1.Our findings suggest that the expression level of the deacetylase SIRT1 and the abundance of WEE1 Lys177 acetylation can serve as useful biomarkers for predicting the therapeutic effect of WEE 1 inhibitors in cancer therapy,and provide the basis for more precise and effective cancer treatment strategies.