AZD1775 Upregulates PD-L1 In Ovarian Cancer Cells And Its Potential Value In Combination With αPD-L1 In The Treatment Of Ovarian Cancer

Objectives: AZD1775 promotes mitosis and propagates genomic instability by forcing the cell through successive replication cycles,ultimately resulting in apoptosis from mitotic catastrophe.However,there is no study of the effect of AZD1775 on the immune microenvironment.The main purpose of this study is to elucidate the mechanism of AZD1775 up-regulating PD-L1 in ovarian cancer cells and its potential value in combination with αPD-L1 in the treatment of ovarian cancer.Methods: The cytotoxicity of MK1775 was examined in a panel of tumor cells using CCK8 in vitro.The effect of AZD1775 on cell cycle was detected by flow cytometry.Immunofluorescence,comet assay and clone formation assay were used for detection of DNA damage in tumor cells.The expression of PD-L1 in ID8 cells,OV2008 cells and C13 cells treated with AZD1775 was detected by quantitative real time polymerase chain reaction(q PCR)and Western blot.The RNAseq data of ID8 cells treated with AZD1775 was used for mechanism investigation.Interferon expression in ID8 cells,OV2008 cells and C13 cells after AZD1775 treatment was detected by enzyme-linked immunosorbent assay(ELISA).DNMT1 and interferon signaling pathway-related genes were detected by q PCR and WB.ds RNA expression was detected by immunofluorescence to confirm the mechanism of PD-L1 expression upregulated by AZD1775.The ID8 tumor model was used to evaluate the efficacy of AZD1775 in combination with αPD-L1 in the treatment of ovarian cancer.Flow cytometry was used to detect the changes of immune cell components in mice tumor tissue.The Cancer Genome Atlas(TCGA)database was used to verify the accuracy of the signature in prediction of response to immune checkpoints inhibitors.Results: We confirmed the effect of AZD1775 on the cycle regulation and DNA damage in ovarian cancer cells.Moreover,we found that AZD1775 significantly up-regulated the expression of PD-L1 in ovarian cancer cells.GSEA analysis of RNAseq data revealed that interferon-signaling pathways were significantly enriched.After AZD1775 treatment,all types of interferon had a certain degree of secretion,among which the most significant change was type III interferon.AZD1775 lead to increased secretion of interferon by ds RNA.We also found that AZD1775 down-regulated the expression of DNMT1 and mediated the expression of endogenous retrovirus(ERVs)genes,which in turn affect the expression of ds RNA.Down-regulation of E2 F pathway by AZD1775 decreased DNMT1 expression.Animal experiments showed that the combination of AZD1775 and αPD-L1 was better than AZD1775 alone in the treatment of ovarian cancer.In addition,the immune cell components in the tumor tissues were detected by flow cytometry.It was found that the proportion of T lymphocytes in tumor tissues increased after treatment with AZD1775,mainly CD8+ T cells,the proportion of Treg cells decreased,and the ratio of CD8+/Treg increased significantly.We used TCGA database to analyze the correlation of WEE1 gene with CD3 e,CD4,CD8 a,CD68,DNMT1,STAT1.We found that WEE1 expression was negatively correlated with CD3 e,CD8a,and CD68 expression,and positively correlated with DNMT1 expression.Our signature based on the virus defense response genes was compared with the ovarian cancer data in the TCGA database and it was found that the signature was very consistent with the "immunoreactive" subtype of ovarian cancer.Conclusion: Our study illustrates the mechanism by which AZD1775 up-regulates PD-L1 expression in ovarian cancer cells and describes its therapeutic utility in combination withαPD-L1.It not only complements the role of AZD1775 in the immune microenvironment of tumors,but more importantly,it helps to explore more precise therapeutic models for small molecule targeted therapy combined with immunotherapy,which will open a new direction for improving the prognosis of ovarian cancer.