The Application Of DNA Damage Response In Innovative Therapeutic Strategies In Glioma

BackgroundGlioma is the most common and malignant central nervous system tumor.Even after surgical resection,standardized radiotherapy,and chemotherapy,patients still have poor prognoses and extremely low five-year survival rates.Temozolomide(TMZ)is a first-line chemotherapy agent for the treatment of glioma,but the acquired drug resistance and the consequent recurrence of tumors remain to be urgent problems to be solved.Specific defects of the DNA damage response(DDR)pathway are ubiquitous in tumor cells,however,during tumor genome evolution,cells selectively utilize alternative repair pathways to maintain the efficiency of DNA replication and genomic stability,promoting tumor proliferation and leading to therapeutic resistance,which also emphasizes the need for joint interventing DDR pathways for tumor therapy.Recently,accumulating evidence has also suggested the determining role of DDR in anticancer immunity.DDR-targeted treatments are considered to promote tumor immunogenicity by boosting antigenicity through accumulated tumor neoantigen burden(TNB),promoting adjuvanticity through cytosolic immunity activation,and enhancing reactogenicity through the induction of immune checkpoint.Here we propose that a comprehensive investigation of the DDR landscape and validation by experiments can facilitate exploring novel targets to sensitize clinical treatment,thereby optimizing glioma management,especially the development of innovative treatment strategies such as synthetic lethality and immunotherapy.MethodsWe established the TMZ-resistant U87 MG cell lines by increasing drug concentrations and identified the pronounced up-regulation of DDR in TMZ-resistant glioma cells by RNA sequencing.DDR score was constructed based on differentially expressed genes between TMZ-sensitive/resistant glioma cells using lasso regression analysis,then the patients were divided into high and low DDR score groups based on the DDR score cut-off value.The Kaplan-Meier curves,ROC,univariate and multivariate cox analyses were used to evaluate the predictive ability of DDR score.GO,KEGG,GSEA,and ss GSEA were employed to investigate the functional enrichment of DDR subgroups.Multiple bioinformatic algorithms were applied to infer the immune infiltrating patterns and tumor immunogenicity.Immunohistochemistry was used to determine the protein levels of PD-L1 and TGFβin primary and recurrent glioma specimens.The maftools package was applied to visualize the mutational landscape and calculate the tumor mutational burden(TMB).The onco Predict package was employed to predict the candidate chemotherapy agents of two DDR score groups.Clonal formation and CCK-8 were used to verify the correlation of FEN1 and TMZ cytotoxicity in glioma cells.Proximity ligation assay and immunoprecipitation were employed to analyze the binding status of FEN1 and other proteins.Immunofluorescence staining was applied to label proteins such as Brd U,γ-H2 AX,and 53BP1 to assess cell proliferation capacity or double-strand break(DSB)accumulation.DNA fiber spreading analysis was utilized to analyze the effect of inhibiting FEN1/DNA-PKcs on DNA regeneration strands in glioma cells.Comet analysis was used to analyze the effects of collaborative interventions in FEN1/DNAPKcs on DSB accumulation in glioma cells.Flow cytometry assays were employed to analyze the effects of FEN1 or/and DNA-PKcs inhibition on the cell cycle.The CCK-8 and Transwell experiments were used to evaluate the effects of co-inhibition of FEN1/DNA-PKcs on cell proliferation,invasion,or migration.Intracranial tumor models were used to demonstrate the effects of combined deficiency of FEN1/DNAPKcs on tumor size and mice survival in vivo.ResultsHigh DDR score gliomas exhibited significantly worse clinical outcomes and shorter median overall survival.The DDR scores were higher in high-grade tumors,and lower DDR scores were observed in 1p19 q co-deletion and IDH mutant gliomas.Among other clinicopathological parameters,DDR score independently predicted the prognosis of patients with glioma.Functional enrichment analyses revealed that high and low DDR score groups were characterized by distinct immune activity and metabolic processes.Decreased tumor purity and elevated levels of infiltrating immune cells were found in the high DDR score glioma.Further,high DDR scores correlated with increased mutation burden,up-regulated immune checkpoints,and tumor immunity activation,indicating enhanced glioma immunogenicity.PD-L1 and TGFβ were overexpressed in recurrent glioma specimens compared with primary ones.Then we estimated that PI3 K inhibitors may serve as latent regimens for high DDR score patients.Finally,our analysis demonstrated that DDR potentially influenced the response rate of PD-L1 blockade therapy in a urothelial cancer cohort.In another study on synergistic interventions of DDR,we found that FEN1 promoted glioma cell proliferation and maintained replication fork stability when coping with replication pressure,DNA-PKcs-deficient cells selectively highly express FEN1 to enhance their stability in binding to replicates.Compared with the inhibition of FEN1 or DNA-PKcs alone,the joint intervention of FEN1/DNA-PKcs not only promoted DSBs accumulation and genomic instability under replication stress,but also weakened the ability of glioma cells to proliferate,invade,and migrate,ultimately leading to cell death.Mice treated with FEN1 and DNA-PKcs-specific small molecule inhibitors were significantly smaller in tumor size than the single inhibitor treatment groups and had significantly longer survival.ConclusionDDR score can predict the prognosis of glioma patients and assess the immunogenicity of tumors.Co-inhibition of FEN1 and DNA-PKcs,a pair of DDR compensatory pathways,synergistically kills glioma cells.Comprehensive analysis of the DDR status of glioma patients can provide novel insights to promote personalized immunotherapy,synthetic lethality,and innovative combination of multiple therapeutics.