Neddylation Inhibitor MLN4924 Upregulates PD-L1 Expression In Glioblastoma And Its Mechanism

BackgroundGlioma is a common disease in neurosurgery.It originates from nerve exomorphic tissue and is highly invasive.The treatment of glioma is limited and cannot be cured completely at present.After decades of exploration and development,as well as the improvement of diagnosis and treatment level and the application of new drugs,the treatment technology of glioma has made great progress,and the survival rate of patients has been improved.But the overall survival rate is still low,bringing heavy burden to patients,families and society.The current two-year survival rate for low-grade astrocytomas is about 66%,for anaplastic astrocytomas is about 45%,and for glioblastoma multiforme is only 9%.Despite the application of surgery,chemoradiotherapy and temozolomide,the treatment effect and prognosis of patients with glioblastoma are not ideal at present.Therefore,it is urgently needed to develop new drugs and develop new treatment strategies.With the continuous research of immunology,tumor immunotherapy has developed rapidly in recent years,and some research results and clinical trials have achieved good results,significantly improving the prognosis of some tumor patients.In particular,human monoclonal antibodies blocking immune checkpoints PD-1/PD-L1and CTLA-4 have been approved for the treatment of lung cancer and advanced melanoma and other solid tumors,respectively.More importantly,PD-1 antibody drugs have also been approved for marketing in China.Although tumor immunotherapy has achieved great success,clinical trials of glioma immunotherapy using immune checkpoint inhibitors(anti-PD-1/PD-L1)have not achieved satisfactory results.In view of the current status of immunotherapy and with the gradual elucidation of tumor immunological mechanism,the treatment strategy of tumor patients has gradually changed.In the past,cancer has been the focus of research,but now researchers are slowly shifting their focus and focus to patients,including the immune system.Among them,combination therapy has become one of the very important means,mainly through the interaction between drugs to achieve synergistic effect.The combination of immune checkpoint inhibitors and other antitumor drugs has a broad clinical prospect,which is also a hot spot in the immunotherapy of glioma.NEDD8,as a ubiquittin protein,is currently the focus of researchers in tumor and other fields.NEDD8 is similar to ubiquitin.Under the cascade catalysis of activase,binding enzyme and ligase,NEDD8 binds with substrate,which is Neddylation modification.Neddylation is one of the post-translational modifications of proteins,which is a new type of post-translational modifications widely involved in the regulation of various signal transduction pathways.Recently,a growing body of experimental evidence strongly suggests that protein ubiquitination processes are overexpressed in various human cancers.These findings suggest that inhibition of protein ubiquitination may be an important strategy for cancer treatment.MLN4924,as a newly screened small molecule drug,is a selective small molecule inhibitor of NEDD8 activating enzyme NAE.Multiple experimental studies have shown that MLN4924 can inhibit a variety of tumor-related signaling pathways by inhibiting the activity of NAE and blocking subsequent ubiquitination modification of proteins.MLN4924 has been studied in a variety of tumor cells.When MLN4924 was added to tumor cells,changes in multiple biological processes were observed.According to the characteristics of different tumor cells,MLN4924 can induce different biological reactions such as cell cycle arrest,apoptosis,senescence and autophagy.More importantly,MLN4924 has shown good tumor suppressive effect in various preclinical tumor animal models,and has entered several phaseⅠ/Ⅱclinical trials.Previous study found that both NEDD8 and PD-L1 were highly expressed in glioma,and showed a significant positive correlation.Therefore,we speculated that the use of MLN4924 in glioma could block NEDD8 and PD-L1 pathways and show stronger anti-tumor effects combinating with anti-PD-L1,but the specific effects and mechanisms need to be further studied.In order to explore the role of MLN4924 in glioma,the regulation of PD-L1 and the therapeutic effect of combined therapy with immune checkpoint blockers on glioblastoma,this study intended to analyze the relationship between Neddylation and the occurrence and development of glioma and its influence on prognosis through TCGA database and clinical data of glioma patients.The regulation mechanism of MLN4924 on immune cells and its synergistic inhibitory effect on glioblastoma combined with immune checkpoint inhibitors were further analyzed through U87 and T98G cell lines and GL261 transplanted tumor model.Part1.The Role of NEDD8 in The Occurrence and Development of Glioma and Its Effect on The Prognosis of PatientsObjectiveTo explore the relationship between neddylation modification of ubiquitin-like protein NEDD8 and the development of glioma,the correlation of pathological level and the influence on the prognosis of patients.Methods(1)Based on the data of NEDD8 m RNA in glioma patients from the public database TCGA,the differential expression of NEDD8 in all levels of glioma was analyzed,and the patient survival analysis was performed to draw the Kaplan-Meier(K-M)survival curve;(2)Tumor tissue samples from 80 patients with glioma of different pathological grades and normal brain tissue control samples from patients with craniocerebral trauma or cerebral hemorrhage were screened in the biological sample bank of International Joint Experiment on Glioma Metabolism and Microenvironment in Henan Province to detect the expression of NEDD8 protein in glioma tissue and control brain tissue.The relationship between NEDD8 expression and pathological grade in glioma tissue was analyzed.(3)The survival time of patients was calculated according to the follow-up results.Kaplan-meier method was used to analyze the relationship between the expression of NEDD8 in glioma and the survival time of patients,and the effect of Neddylation on the prognosis of patients with glioma.(4)In clinical studies and basic experiments,SPSS 25.0 was used for statistical analysis of the data.The shapiro-Wilk normality test was used for quantitative data.Quantitative data were expressed by T test,quantitative data are expressed by(?)±SD.The mean difference between the two groups was described by relative number,and the difference between groups was analyzed byχ~2test.Survival analysis was performed by Kaplan-Meier method and Cox regression model to analyze the effect of NEDD8 expression on prognosis.Test levelα=0.05(double-tailed).In terms of bioinformatics,two independent sample T tests were used to analyze the expression differences of NEDD8 in patients with glioma at all levels.The prognosis of NEDD8 was analyzed by Kaplan-Meier survival curve.P<0.05 was considered statistically significant.Results(1)The expression of NEDD8 m RNA in TCGA database was positively correlated with the pathological grade of glioma,and patients with high expression of NEDD8m RNA had worse prognosis;(2)NEDD8 expression was low in normal brain tissue,but significantly increased in glioma tissue,especially in high-grade glioma tissue.(3)NEDD8 is closely related to the pathological grade and prognosis of gliomapatients,and can be used as a new biomarker to evaluate the grade and prognosis of glioma.ConclusionNeddylation is involved in the development of glioma.NEDD8 is closely related to the pathological grade of glioma and the prognosis of patients.Blocking Neddylation is a new target for the treatment of glioma.Part2.MLN4924 Inhibited The Growth of Glioblastoma and Up-regulated The Expression of PD-L1ObjectiveTo explore the effect of MLN4924 on glioblastoma and the change of PD-L1 protein level in glioblastoma after inhibition of neddylation pathway.Methods(1)The effect of MLN4924 on colony formation of U87 cells was studied by clonal formation experiment;(2)The effect of MLN4924 on Ki-67 protein expression in U87 cells was detected by immunohistochemistry;(3)The expression of PD-L1 in U87 and T98 G cells was detected after knockdown of ubiquitin pathway enzymes NAE1,UBA3 or UBC12;(4)After inhibiting Neddylation with MLN4924 or proteasome inhibitor MG132,the expression of PD-L1 protein in U87 and T98 G cells was determined;(5)The changes of PD-L1 protein in mice carrying U87 and T98 G cells were detected after MLN4924 treatment.(6)All data are represented by (?)±SD.Student T test or univariate variance was used to assess the statistical significance of differences between groups using Graph Pad RISM6 software.Unpaired two-tailed T test was used to compare the parameters between the two groups.Three significance levels(*P < 0.05,**P < 0.01,and ***P <0.001)were used for all tests.Results(1)MLN4924 inhibited the cloning of glioblastoma U87 cells;(2)MLN4924 can reduce the expression level of Ki-67 in glioblastoma;(3)Inhibition of Neddylation pathway up-regulated the expression of PD-L1 in glioblastoma in vitro and in vivo;ConclusionNeddylation inhibitor MLN4924 inhibited the growth of glioblastoma and up-regulated the expression of PD-L1 in tumor cells at the same time.Part3.The Effect and Mechanism of MLN4924 Up-regulating PD-L1 Expression in GlioblastomaObjectiveTo explore the role and molecular mechanism of MLN4924 up-regulating PD-L1 in the treatment of glioblastoma,and provide theoretical support for the development of new and reasonable anti-glioblastoma programs.Methods(1)The up-regulation of PD-L1 by MLN4924 in the treatment of glioblastoma was studied by T cell killing experiment;(2)The specific molecular mechanism of MLN4924 up-regulating PD-L1 expression in glioblastoma was investigated by chromatin immunoprecipitation,gene knockout and in vivo ubiquitination.(3)The synergistic inhibitory effect of MLN4924 and anti-PD-L1 on glioma growth was verified by establishing GL261 glioma transplant model.(4)All data are represented by (?)±SD.Student T test or univariate variance was used to assess the statistical significance of differences between groups using Graph Pad RISM6 software.Unpaired two-tailed T test was used to compare the parameters between the two groups.Three significance levels(*P < 0.05,**P < 0.01,and ***P <0.001)were used for all tests.Results(1)MLN4924 can attenuate the killing effect of T cells on glioblastoma by up-regulating the expression of PD-L1 protein in glioblastoma;(2)PD-L1/PD-1 immune checkpoint inhibitors can reverse ml N4924-induced immune resistance;(3)PD-L1 expression was related to Cullin1 and Cullin3,but was more closely related to Cullin1.(4)FBXW7 knockdown resulted in the accumulation of c-MYC oncoprotein and increased PD-L1 m RNA and protein levels in U87 and T98 G cells;(5)Cullin1-FBXW7/ c-MYC axis is mainly involved in the regulation of PD-L1 in glioblastoma cells;(6)The combination of MLN4924 and immunotest blocker has a synergistic inhibitory effect on glioblastoma.Conclusion(1)MLN4924 induced c-MYC accumulation through Cullin1-FBXW7,which up-regulated PD-L1 expression and attenuated the killing effect of T cells;(2)MLN4924 combined with PD-L1 inhibitor can eliminate immunosuppression and synergistically inhibit the growth of glioblastoma.Conclusions of this paperThis project mainly studied the effect of Neddylation modification on glioma and its effect on prognosis of patients,and explored the inhibition of glioblastoma by Neddylation small molecule inhibitor MLN4924 and the up-regulation of PD-L1 expression as well as the molecular mechanism,which was verified by the mouse glioma transplant model.Finally,the following conclusions are drawn:(1)Neddylation was involved in the development of glioma and affected the prognosis of patients;(2)MLN4924 could inhibit the proliferation of glioblastoma as an inhibitor of Neddylation;(3)MLN4924 inhibited glioblastoma and up-regulated THE expression of PD-L1 and attenuated the killing effect of T cells;(4)MLN4924 mainly induces c-MYC accumulation through Cullin1-FBXW7,leading to up-regulation of PD-L1 expression;(5)The combination of MLN4924 and PD-L1 inhibitor can synergistically inhibit the growth of glioblastoma.