Research On The Correlation Between EXO1 And Tumor Immune Microenvironment Under Replication Stress And Its Regulation Machanisms

Background: Aberrant activation of oncogenes can disturb the normal growth process of the cell,which can promote genomic instability.This series of processes accelerate the mutation frequency of cells,which eventually leads to tumorigenesis.Excessive activation of oncogenes first induces replication stress(RS).The oncogene-induced replication stress causes abnormal replication initiation,replication and transcription conflicts,and defective nucleic acid metabolism,which leads to genomic dysfunction.The persistence of RS in tumor cells affects genes involved in the replication fork process,such as the nuclease EXO1,which leads to excessive cleavage of genomic DNA and reshaping the tumor immune microenvironment.However,the clinical evidence for the role of oncogene-induced RS in modulating tumor mutational load and TME remains scarce.The mechanism by which the important nuclease EXO1 is regulated under replication stress is still not fully understood.Therefore,we aim to investigate the effects of oncogene-induced RS in patient prognosis,tumor mutation frequency and tumor immune microenvironment through bioinformatics methods and molecular biology techniques.Additionally,we aim to investigate the mechanism by which EXO1 is regulated under replication stress.The results of this study will provide clinical prognostic methods,as well as strategies for drug treatment.Methods:(1)TCGA database was used to investigate the genetic alterations,m RNA expression differences,and prognostic impacts of 14 known fork processing factors and 30 replication stress inducing oncogenes in pan-cancer based on the TCGA database.Single-cell sequencing results from CHARTS database was used to clarify whether oncogenes that induce replication stress and fork processing factors are enriched in the same tumor cells with high RS level.(2)R language tool was used to calculate the tumor mutation load,study the relationship between m RNA level of fork processing factors,RS inducing oncogenes and tumor mutational burden.The relationship between the m RNA levels of the RS inducing oncogenes,fork processing factors and immune cell infiltration was explored using R software and the TIMER2 database,demonstrating the impact of replication stress on the tumor immune microenvironment.(3)In tumor cells,DNA fiber spreading experiments confirmed that overexpression of E2F1 can induce replication stress,while immunofluorescence experiments demonstrated that replication stress induces the production of cyto DNA in tumor cells.Using a whole-genome wide CRISPR screen,we identified potential replication fork protection gene that inhibits excessive activation of EXO1 under replication stress.This study explored the mechanisms by which replication fork protection gene inhibits the excessive cleavage of DNA by EXO1 under replication stress using techniques such as immunoblotting,immunofluorescence,immunoprecipitation,DNA fiber spreading,and detection of cytosolic and endoplasmic reticulum calcium ions.Results:(1)Genomic alterations of 14 fork processing factors and 30 replication stress inducing oncogenes are commonly present in pan-cancer,and their genomic alterations lead to poor prognosis.Among them,the m RNA levels of five fork processing factors(EXO1,DNA2,EME1,BLM,and GEN1)and replication stress inducing oncogenes(E2F1,CDCA5,CDC6,AURKA,MYBL2,and CCNE1)are generally overexpressed in tumor tissues.Compared to normal tissues in pan-cancer,and patients with high expression of the 11 genes,including EXO1,have a poor prognosis.Single-cell sequencing results showed that the m RNA of the five fork processing factors(EXO1,DNA2,EME1,BLM,and GEN1)and six replication stress inducing genes(E2F1,CDCA5,CDC6,AURKA,MYBL2,and CCNE1)were enriched in the same tumor cells and in cells with more DNA damage repair.(2)The m RNA of the five fork processing factors(EXO1,DNA2,EME1,BLM,and GEN1)and replication stress inducing oncogenes(E2F1,CDCA5,CDC6,AURKA,MYBL2,and CCNE1),is positively correlated with tumor mutation burden,and tumor immune suppressed cells CD4+Th2 cells and MDSCs in most tumors.(3)The experimental results of DNA fiber spreading showed that the overexpression of E2F1 in tumor cells could decrease replication speed and induce DNA replication stress.The immunofluorescence assay demonstrated that under DNA replication stress,tumor cells produced cyto DNA.The results of a whole-genome CRISPR screen indicated that GADD45 A is a replication fork protection gene that inhibits excessive cleavage of DNA by EXO1 under replication stress.The promotion of AMPK T172 phosphorylation by GADD45 A under replication stress was validated by immunoblotting and immunofluorescence assays.The immunoprecipitation assay confirmed that GADD45 A can bind to the catalytic subunit of phosphatase PP2 A under replication stress.The inhibitory effect of GADD45 A on PP2 A was validated by immunoblotting,which resulted in the phosphorylation of AMPK and EXO1 S746.DNA fiber spreading and Non-denaturing Brd U assay validated that GADD45 A inhibits excessive cleavage of genomic DNA by EXO1.Additionally,immunoblotting revealed that a new small-molecule compound SMG1 inhibitor can stabilize exogenous GADD45 A.Conclusion: This study demonstrates that the m RNA of the 11 genes,including EXO1,is generally overexpressed in pan-cancer and is associated with poor clinical outcomes.The m RNA levels of these 11 genes in pan-cancer are positively correlated with tumor mutation burden,tumor immune suppressed CD4+Th2 cells and MDSCs,indicating that they can be used as markers to evaluate the degree of replication stress,mutation,and tumor immune suppressed cell infiltration and prognosis in patients’ tumor tissues.In terms of regulating EXO1 mechanisms,GADD45 A was found to be a protective factor that inhibits excessive cleavage of genomic DNA by EXO1 under DNA replication stress.The novel small molecule SMG1 inhibitor can stabilize exogenous GADD45 A and can potentially be used as a targeted drug for the treatment of tumors with high replication stress.