Effect And Mechanism Of Guanylate Binding Protein 2 (GBP2) On Immune Checkpoint Resistance In Microsatellite Stability Colorectal Cancer

ObjectiveCompared with deficient-mismatch-repair or microsatellite instability(d MMR/MSI)colorectal cancer(CRC),proficient-mismatch-repair or microsatellite stability(p MMR/MSS)CRC has limited efficacy for immune checkpoint blockade(ICB)therapy and its underlying mechanism remains unclear.Previous studies have reported that guanylate-binding protein 2(GBP2),a member of the GTPases family,is crucial to host immunity against pathogens.However,the correlations between GBP2 and immunosurveillance and immunotherapy for p MMR/MSS CRC have not been reported.MethodsFirst,the RNA sequencing data of CRC patients with microsatellite status in Gene Expression Omnibus(GEO)and The Cancer Genome Atlas(TCGA)databases were searched.Unsupervised clustering was employed to classify immune and non-immune class in p MMR/MSS patients.This binary classification was validated using immune cells or response related signatures by single-sample gene set enrichment analysis(ss GSEA).Then,the differentially expressed genes(DEGs)were calculated between two subtypes and intersected with RNA binding proteins(RBPs).Based on the correlation with immune response and prognosis,GBP2 was selected as a gene of interest.The correlation between GBP2 and immune microenvironment was explored using gene set enrichment analysis(GSEA),gene ontology(GO),single-cell RNA sequencing,ss GSEA,multiplex immunohistochemistry(m IHC).Afterwards,knockdown of GBP2 using small interfering RNA(si RNA)were performed in two MSS CRC cell lines(HT29 and SW480).The expression and secretion of chemokines were detected by q-PCR and ELISA.The expression of antigen processing and presentation machinery(APM)were detected by q-PCR and flow cytometry.Western blot and coimmunoprecipitation(Co IP)were used to explore the mechanism by which GBP2 regulates STAT1 phosphorylation.Finally,the relationship between GBP2 and ICB response was predicted by GSEA,Sub Map algorithm and ss GSEA.The murine MSS CRC cell line CT26 was used to construct stable GBP2 knockout(KO)cells.The CT26 tumor-bearing model was used to explore the therapeutic effect of GBP2 combined with ICB.Results1.After screening the GEO and TCGA databases,we included six independent cohorts with a total of 1424 p MMR/MSS and 457 d MMR/MSI CRC patients: GSE39582,TCGACOAD,GSE41258,GSE26682,pooled＿cohort＿1 and pooled＿cohort＿2.Pooled cohort 1consisted of GSE4554,GSE13067,GSE13294,GSE18088 and GSE75316.Pooled cohort 2consisted of GSE35896 and GSE39084.We classified 1424 p MMR/MSS CRC patients into two classes,"immune" and "non-immune".2.By calculating differential genes between subtypes and taking intersections with RBPs,GBP2 was selected as a gene of interest due to its high correlation with prognosis and immune response.We first found that GBP2 expression was significantly decreased in CRC compared with normal samples,and primarily decreased in the MSS CRC.Lower GBP2 expression was correlated with poor prognosis and metastasis.3.We found that GBP2 expression was upregulated in the immune class and highly associated with interferon-γ(IFN-γ)response and CD8+ T-cell infiltration using GSEA,GO analysis,single-cell sequencing and m IHC.4.Moreover,we found that upregulation of GBP2 enhanced the antigen processing and presentation machinery and CXCL10/11 expression in MSS CRC cells upon IFN-γ stimulation.5.Mechanistically,GBP2 promoted STAT1 phosphorylation by competing with SHP1 for binding to STAT1 in MSS CRC cells.6.Finally,Sub Map algorithms showed that p MMR/MSS patients with high GBP2 expression may correlate with a favorable response to anti-PD-1 therapy.We further confirmed that GBP2 knockout abrogated the efficacy of PD-1 blockade in tumor-bearing mice.ConclusionOur study stratified the p MMR/MSS CRC into immune and non-immune classes and identified that GBP2 is a promising target for combinatorial therapy with ICB.