ALKBH5 Promotes PD-L1-mediated Immune Escape Through M6A Modification Of ZDHHC3 In Glioma

Background:Glioma is the most common and fatal malignant tumor of the intracranial nervous system.The conventional therapeutic strategy is surgical resection supplemented by radiotherapy and chemotherapy,which does not improve the survival prognosis of glioma patients.New therapeutic strategies are urgently needed in the clinical treatment of glioma.Recently,tumor immunotherapy,especially immune checkpoint inhibitors,gain much clinic benefit in various types of tumors.However,immunotherapy has limited effect in glioma patients.Previous studies have found that epigenetic modification of tumor cells can suppress the effect of immunotherapy by modifying the tumor immune microenviroment,but it is not clear whether epigenetic modification affects the effect of immunotherapy in glioma.In the previous study of our research group,it was found that glioma had higher expression of m6A demethylase ALKBH5 than adjacent normal tissues;High expression of ALKBH5 is associated with poor prognosis in glioma patients.Therefore,we speculate that ALKBH5 regulates glioma immunotherapy resistance through epigenetic modification.Objectives:(1)To clarify the role of ALKBH5 in glioma immunotherapy resistance and explore its potential molecular mechanism;(2)Explore the combination treatment scheme according to the specific molecular mechanism of ALKBH5 regulating the immunotherapy resistance of glioma,and provide a new strategy for the clinical treatment of glioma.Methods:(1)The glioma cell line with knockout of ALKBH5 was constructed by cas9 system,and the growth rate of glioma and the effect of immunity on its regulation were analyzed by in situ tumorigenesis in mice;(2)The changes of immune microenvironment of glioma were analyzed by flow cytometry,immunohistochemical staining and ELISA.(3)M6A methylation sequencing and whole transcriptome sequencing to screen potential downstream target genes of ALKBH5;(4)QRT PCR,Western blot,RNA pulldown,rip-pcr,Luciferase Report and other experiments confirmed the specific molecular mechanism of ALKBH5 regulating PD-L1.Result:(1)After knockout of ALKBH5,the growth rate of glioma cells in the brain of mice decreased,and the effect was more significant in the immune complete mice;(2)After knockdown of ALKBH5,the number of infiltrating CD8 T cells in glioma increased and the expression of PD-L1 decreased;(3)After knockdown of ALKBH5,the therapeutic effect of PD-1 monoclonal antibody on glioma mouse models was significantly improved;(4)After knockdown of ALKBH5 in glioma cells,m6A methylation sequencing and whole transcriptome sequencing showed that ZDHHC3 was a potential downstream target of ALKBH5 epigenetic modification.RIP experiments and gene reporter experiments confirmed that ALKBH5 methylation modified the specific site of ZDHHC3 mRNA,and knockdown of ZDHHC3 deprived the regulatory effect of ALKBH5 on PD-L1;(5)ALKBH5 affects the stability of ZDHHC3 mRNA and regulates the expression of ZDHHC3 through m6A methylation modification.Knockdown of m6A methylation reader protein YTHDF2 deprives the regulatory effect of ALKBH5 on ZDHHC3 mRNA;(6)Knockdown of ZDHHC3 did not affect the mRNA level of PD-L1,but significantly increased the degradation rate of PD-L1 protein,thus affecting the protein expression level of PD-L1;(7)The therapeutic regimen of ALKBH5 inhibitor ioxl combined with PD-1 monoclonal antibody can improve the therapeutic effect of PD-1 monoclonal antibody in glioma mouse models.Conclusion:(1)ALKBH5 regulates the expression level of PD-L1 by regulating the level of ZDHHC3 through epigenetic modification,thus affecting the immune microenvironment and immunotherapeutic effect of glioma.(2)The therapeutic regimen of ALKBH5 inhibitor IOX1 combined with PD-1 monoclonal antibody can improve the therapeutic resistance of immune checkpoint inhibitors in glioma.