Combination Of PARP Inhibitor Niraparib And PD-L1 Blockade And Underlying Molecular Mechanism Of The Synergistic Effect In Epithelial Ovarian Cancer

BackgroundOvarian cancer is the seventh most common cancer in women worldwide,and while its mortality rate has declined slightly over the past 40 years,the mortality rate still ranked eighth in women.The GLOBOCAN study predicts that by 2035,the incidence and death rate of ovarian cancer will increase by 55%and 67%respectively,based on global population increase.Ovarian cancer has a high risk of recurrence and death due to its insidious clinical manifestations and intriguing origins and molecular biological characteristics.In the past decade,two classes of targeted agents for maintenance therapy of ovarian cancer have been approved for clinical use:the antiangiogenic agent Bevacizumab and the PARP inhibitor Olaparib and Niraparib.Although targeted drugs have made some achievements in the maintenance treatment of ovarian cancer,quite a few patients in the patient population receiving treatment that still are "non-responsive" to drugs due to various reasons.Therefore,how to reduce drug resistance so that more ovarian cancer patients can benefit from treatment and achieve more accurate and individualized treatment is the key issue that needs to be focused on.Poly-ADP ribose polymerase(PARP)is a DNA damage repair enzyme,which can repair single strand damage(SSB)via base excision repair(BER)pathway.PARP consists of three zinc finger structures,BRCT,WGR and a catalytic domain including two subdomains(helix domain and ADP ribosyl-transferase domain--ART).In the non-DNA-binding region,the Helical Domain(HD)inhibited the binding of β-NAD to the ART region.When DNA is damaged,the change of helical structure will cause the zinc finger structure in PARP1 to combine with DNA,forming PARP 1/DNA complex,resulting in the conformational change of HD and the release of inhibitory effect,thus activating the catalytic function of ART,and recruiting related damage-repair proteins to play the role of DNA damage repair.PARP inhibitors targeted PARP1 and PARP2,can combine with catalytic domains of PARP1,and prevent the separation of PARP1 from DNA,persistent PARP1/DNA complexes can inhibit DNA replication forks,and can further cause double-stranded DNA damage,at this time if there is a homologous recombination repair defects(HRD)such as BRCA mutations,will produce a "synthesis lethality".PARP inhibitors such as Olaparib、Niraparib showed good antitumor effect in a wide variety of tumors,but also there are literatures revealed some patients showed no response or secondary drug resistance to PARP inhibitors,so it is possible that combination of PARP inhibitors and other drugs is an effective way to solve the drug resistance in ovarian cancer patients.Programmed Cell Death Ligand 1(PD-L1)is a transmembrane glycoprotein with a molecular weight of 33kD.PD-L1 and PD-1 recognize each other in the extracellular domain,which can activate the immunoreceptor and inhibit the activation of T cells.In addition,PD-L1 also interacts with CD80,providing an inhibitory signal to activated T cells and weakening the host’s immune response.It has been reported that immune checkpoint blockers,such as PD-L1/PD-1 blockade,can exert synergistic effect with PARP inhibitors.However,the combination is still in the stage of preclinical researches or clinical trials,and there are few studies in ovarian cancer,so a lot of basic experimental evidence is needed before the real clinical transformation can be achieved.PurposeTo explore the synergistic effect and potential mechanism between PARP inhibitor Niraparib and PD-L1 blockade in epithelial ovarian cancer.Methods1.To detect the changes of PD-L1 in epithelial ovarian cancer cell lines after Niraparib treatment:WB and flow cytometry were used to determine the change of PD-L1 in tumor cells after different PARP inhibitors and different concentrations of Niraparib treatment.2.To detect the effect of Niraparib on PD-L1 in tumor tissues in vivo:WB and immunohistochemistry were used to detect the changes of PD-L1 in tumor tissues after Niraparib treatment.3.To detect whether Niraparib has the synergistic effect with PD-L1 blockade:The previously cultured ID8 cells were resuspended in 100μl of phosphate-buffered saline(PBS)at a volume of 1X107 cells and injected subcutaneously to the right flank of C57BL/6 mice.After 2 weeks,mice were randomized to four groups(n=5 per group),control group,Niraparib group,PD-L1 blockade group,Niraparib and PD-L1 blockade group.The mice were administrated Niraparib orally,and injected PD-L1 blockade twice or three times a week intraperitoneally.The body weight and tumor volume of the mice were measured every other day to detect the difference in the inhibitory effects of different treatment groups on tumors.4.To detect the correlation between PARP1 and clinicopathological features,PD-L1 and CD8 in epithelial ovarian cancer:Immunohistochemical assay was used to investigate the correlation between PARP1 and clinicopathological features and the expression of PD-L1 and CD8 in 72 epithelial ovarian cancer samples.5.To detect the alteration of immune cells and cytokines in high-grade serous ovarian cancer patients with Niraparib treatment:the alterations of PBMC,CD3+T cells,CD4+T cells,CD8+T cells and cytokines IFN-y were investigated by flow cytometry,and the immune cells and cytokines were from peripheral blood of high-grade serous ovarian cancer patients before and after the administration of Niraparib.6.To detect the alteration of immune cells and cytokines in vivo and vitro:tumor cells with the control group,Niraparib treatment,PD-L1 blockade treatment,the combination treatment separately was co-cultured with CD8+T cells;Tumor burdened mice were treated with PBS,Niraparib,PD-L1 blockade and the combination of Niraparib and PD-L1 blockade.Immunohistochemical experiment and flow cytometry were used to detect the changes of TIL,cytokines,immune cells and cytokines.7.To explore the mechanism of the synergistic effect of Niraparib and PD-L1 blockade:WB and flow cytometry were used to detect the changes of cGAS/STING related pathway proteins such as STING,P-STING,TBK1,P-TBK1,IRF3,P-IRF3 and downstream chemokines CCL5 and CXCL10 after Niraparib treatment.Results1.PARP inhibitors upregulated PD-L1 expression in epithelial ovarian tumor cell lines in vitro experiments:BRCA-deficient UWB 1.289 and BRCA-wild SKOV3 ovarian cancer cell lines were treated with different PARP inhibitors--Olaparib,Niraparib,Fluzoparib,and control group(10μM)respectively for 48h.Flow cytometry and WB detection revealed that PD-L1 expression on the tumor cell were upregulated.Among the different PARP inhibitors,Niraparib showed the most obvious and stable up-regulation of PD-L1.Different concentrations of Niraparib(0nM,500nM,1μm,5μm,10μm,and 15μm)treated tumor cells for 48h showed a concentration-dependent upregulation of PD-L1.2.Niraparib upregulated the upregulation of PD-L1 in tumor tissue in vivo:Black mice C57/BL6 were subcutaneously inoculated with mouse ovarian cancer cell line ID8,and after two weeks tumors were formed.The mice were randomly divided into two groups and treated with control group and Niraparib to detect the alteration of PD-L1:WB and IHC results showed that the level of PD-L1 in transplanted tumor tissue was significantly up-regulated after Niraparib drug treatment.3.Niraparib has the synergistic effect with PD-L1 blockade in vivo:While single-agent had an effect on the growth of the tumors,the combination of niraparib and PD-L1 blockade treatment can hamper the tumor growth effectively than the control group.4.Expression of PARP1 in epithelial ovarian cancer and its correlation with clinical indicators:Immunohistochemistry(IHC)was used to detect the expression of PARP1 in 72 cases of ovarian cancer and 12 cases of normal ovarian paraffin-embedded tissues,and analyzing the correlation of PARP1 expression in ovarian cancer and patient clinical indicators such as CA125,ascites,prognosis,CD8 and PD-L1 expression.The results showed that ovarian cancer tissues(mainly nuclei)highly expressed PARP1.The high expression of PARP1 in ovarian cancer tissues was associated with a poor prognosis(P=0.027).However,there is no direct correlation between PARP1,PD-L1,and CD8 considering the limited samples.5.The effect of Niraparib on immune cells and cytokines in peripheral blood of patients with high-grade serous ovarian cancer:Flow cytometry was used to analyze the changes of immune cells and cytokines in clinical ovarian cancer patients before and after Niraparib treatment.CD8+T cells and cytokine IFN-γ were significantly increased in the peripheral blood of clinical patients after the administration of Niraparib,while TNF-α was not significantly changed.6.The effects of Niraparib and Niraparib combined with PD-L1 blockade on immune cells and cytokines in vivo and in vitro:Collect the peripheral blood of healthy volunteers,separate the mononuclear cells(Peripheral Blood Mononuclear Cell,PBMC),and further use the magnetic bead sorting method to extract and sort the CD8+T cells.After verification of the sorting efficiency,the immune cells were co-cultured with tumor cells in accordance with an effect-to-target ratio of 10:1,and treated with different treatments:control group,Niraparib group,PD-L1 blockade group,and the combination group.After 48h of co-culture,ELISA and flow cytometry.were used to investigate the alteration of cytokines.Then ID8 cells were subcutaneously inoculated in C57BL/6 mice to construct an ovarian cancer xenograft model and divided into control group,Niraparib group,PD-L1 blockade group,and the combination group.The changes of immune cells and cytokines in the systemic and local tumor tissues were detected by flow cytometry and IHC.The results showed that the local and systemic immune cells and cytokines were significantly upregulated in the Niraparib-treated group and the combination group,and the differences were statistically significant compared with the PBS group.7.Study on the mechanism of the synergistic effect of Niraparib and PD-L1 blockade:The role of cGAS-STING pathway in this process was explored.Tumor cells were treated with different concentrations of Niraparib for 48 h,and the changes of cGAS-STING pathway proteins and downstream chemokines were verified by WB and ELISA.The results showed that cGAS/STING related pathway proteins,such as p-STING,p-TBK1 and p-IRF3,were significantly up-regulated in a concentration-dependent manner after Niraparib treatment.Secondly,the secretion of downstream cytokines such as IFN-β,CCL5 and CXCL10 was also significantly increased.Conclusion1.Compared with normal ovarian tissues,high-grade serous ovarian cancer tissues expressed PARP1 highly,and its high expression is related to the poor prognosis of patients.2.In epithelial ovarian cancer,Niraparib can induce up-regulation of PD-L1 by activating cGAS-STING signaling pathway,and PD-L1 blockade can enhance the anti-tumor effect of PARP inhibitor.3.Niraparib can up-regulate the proportion of immune cells and the production of cytokines to promote the killing effect of immune cells.Niraparib combined with PD-L1 blockade can significantly inhibit tumor growth in animal models of epithelial ovarian cancer,which is more effective than monotherapy.Innovations and limitationsInnovations:1.The immunoregulatory effect of Niraparib,a PARP inhibitor,on epithelial ovarian cancer was studied.Niraparib elevated PD-L1 expression of tumor cells and could up-regulate immune cells and cytokines.2.The immunoregulatory effect of Niraparib was mediated by the activation of cGAS/STING signaling pathway in vivo and in vitro.3.In vivo and in vitro studies have confirmed that Niraparib combined with PD-L1 blockade can play a synergistic role in epithelial ovarian cancer.Limitations1.Because BRCA mutant mouse ovarian cancer cell lines and mouse models are difficult to obtain,this study did not differentiate the BRCA mutant group and the BRCA wild group for the mouse experiment,which is slightly less persuasive.2.The research on cGAS/STING pathway is planned to be continued in the future experiments,and part of the experiments did not carry out rescue verification,which is a little lack of logic.