STING Signaling Pathway-based Molecular Subtyping And Its Immune Regulation In Small Cell Lung Cancer

Small cell lung cancer(SCLC)is a highly aggressive neuroendocrine tumor,accounting for about 15% of lung cancer,and is characterized by rapid cell growth,high metastasis and recurrence rates,and dismal 5-year survival rates.In the past 40 years,chemotherapy with or without radiation therapy remain the standard treatment for first-and second-line management of SCLC.Despite highly sensitive to first-line chemotherapy and radiotherapy,most SCLC patients will experience tumor relapse within 8-12 months.Recently,the addition of immunotherapy to first-line chemotherapy has been approved for the first-line treatment of extensive-stage SCLC,the absolute improvement in overall survival is modest.The activation of the stimulator of interferon genes(STING)pathway induces production of type I interferons(IFNs)and chemokines,arouses T-cell responses,and thus facilitates antitumor immune responses.Previous studies have shown that targeting DNA damage response promoted antitumor immunity through STING-mediated T-cell activation in SCLC.However,the function and mechanism of STING signaling pathway and its immune regulation in SCLC remain elusive.In this study,we aimed to investigate the intrinsic expression of STING pathwayrelated genes and their epigenetic mechanism in SCLC,and to evaluate the effect of combining ATR and TOP1 inhibitors in enhancing antitumor immunity.Methods:(1)We assessed the m RNA and protein expression of STING pathwayrelated genes in SCLC and normal lung tissue.And the relationship between the methylation levels of STING and c GAS promoters and their m RNA expressions was assessed in SCLC cell lines.(2)Using an unsupervised clustering method to identify SCLC molecular subtypes based on the expression levels of 62 STING pathway-related genes(STING signature).Using ss GSEA,x Cell,and quan TIseq methods to assess subtype-specific patterns of infiltrating immune cells.Pathway enrichment analysis was subsequently conducted.Whole-genome sequencing data was used to identify subtype-specific mutational profiles.(3)In vitro experiments were performed to evaluate the antitumor effect of the combination of ATR and TOP1 inhibitors in SCLC cell lines.We explored the global changes in gene expression in SCLC cells following ATR and TOP1 inhibitors treatment using RNA sequencing.Results:(1)The expression levels of STING pathway-related genes were significantly reduced in SCLC compared with normal lung tissue and lung adenocarcinoma tissue.(2)Hypermethylation of STING and c GAS promoter regions repress their m RNA expression in SCLC.(3)We identified three STING subtypes,in which the STING-high subtype exhibited non-neuroendocrine differentiation,abundant immune cell infiltration,high expression of genes related to MHC and immune checkpoints.Whereas the STING-low subtype exhibited high neuroendocrine differentiation and enrichment for pathways associated with DNA damage response and cell-cycle progression.(4)The combination of ATR and TOP1 inhibitors can yield strongly synergistic anti-tumor effects in SCLC.(5)Concurrent inhibition of ATR and TOP1 triggers IFN signaling response and induces chemokines production,and downregulates the expression of genes related to neuroendocrine differentiation,cell adhesion,and extracellular matrix-receptor interaction.Conclusions:(1)The expression of STING pathway-related genes is suppressed in SCLC,and epigenetic silence of the STING or c GAS promoters represses expression of the corresponding gene.(2)STING signature can help to distinguish SCLC with different immunogenicity,which can be used as a classifier to distinguish SCLC “cold” tumors from“hot” tumors.(3)Combination of ATR and TOP1 inhibitors can effectively suppress cell proliferation,and enhance the immunogenicity of SCLC through triggering IFN signaling response and inducing chemokines production,which provides experimental evidence supporting the development of cancer immunotherapy in SCLC.