Mechanism Of WEE1 And ATR Inhibitor Combined With αPD-L1 To Enhance Antitumor Effect

Objectives:WEE1 kinase plays an important role in the regulation of cell cycle G2/M checkpoints and DNA damage response.Inhibiting WEE1 can increase the instability of cell genome and promote tumor cell apoptosis.ATR is the homologous product of mammalian mitotic checkpoint protein 1 and a major member of the DNA damage checkpoint.The current focus of tumor therapy is the synergistic lethality of small molecule inhibitors.Based on this,this study intends to explore the anti-tumor effects and mechanisms by combining WEE 1 inhibitors and ATR inhibitors.Methods:Western blot experiments were used to verify the effect of WEE1 inhibitors on ATR-related pathways.The interference of WEE1 inhibitor combined with ATR inhibitor on tumor cell expansion and apoptosis was analyzed by flow cytometry experiment,CCK8 and clone formation experiments.The ID8 ovarian cancer xenograft mouse model was constructed to evaluate the efficacy.Western blot experiments,dsDNA quantitative experiments and immunofluorescence experiments are used to explore the DNA damage caused by the combination of two inhibitors to tumor cells.And through real-time quantitative PCR(RT-PCR)experiment,immunofluorescence experiment,western blotting experiment and enzyme-linked immunosorbent assay(ELISA)to explore the effect of the combination of two inhibitors on the immune microenvironment of tumor cells.On this basis,ELISA,RT-PCR,western blot assay and immunohistochemical staining were used to detect the effects of the combination of the two drugs on Programmed death-ligand 1(PDL1)expression,and the CT26 colorectal cancer mouse model was constructed to verify the tumor suppressive performance of the two inhibitors combined with PD-L1 blocking therapy.Results:Through experimental analysis,it is found that after WEE1 inhibitor treatment of tumor cells,the ATR pathway is complemented and activated.Blocking ATR significantly increased tumor cell apoptosis induced by WEE1 inhibitors.At the same time,it was also shown in mouse models of ovarian cancer that dual inhibition of WEE1 and ATR can alleviate the tumor burden in vivo.In vitro and in vivo studies found that this combination induced an increase in DNA damage and promoted the accumulation of dsDNA in the cytoplasm,which subsequently activates the stimulator of interferon genes(STING)pathway,triggering the interferon response and the generation of CD8+T cells,thereby inducing antitumor immunity.After using small interfering RNA to silence the expression of STING,it was found that the killing effect of the combination of the two drugs was significantly reduced,which once again proved the necessary role of the STING pathway in it.More importantly,this study also demonstrated that combination therapy can substantially upregulate the expression of PD-L1.Blocking PD-L1 in a mouse model of colorectal cancer further enhanced the effect of the combination therapy.Conclusion:This study elucidates the synergistic lethal effect and mechanism of combined WEE1 inhibitor and ATR inhibitor in tumor cells.The effect of the combination of the two inhibitors on the expression of PD-L1 in tumor cells was described,and the therapeutic effect of the combination of the two inhibitors with PD-L1 blockade was verified.This project contributes a theoretical basis for the subsequent clinical application of WEE1 inhibitors and ATR inhibitors,and provides a new treatment model for cancer treatment.