Targeting EP300 Induces Anti-tumor Immunity By Activating ERV-dsRNA Pathway

Objective As one of the main causes of death in the world,cancer seriously threatens human health.Immunotherapy has become the fifth largest treatment for malignant tumors along with surgery,radiotherapy,chemotherapy and targeted therapy.However,due to the high heterogeneity of tumors,only a subset of patients benefit from it.Therefore,it is urgent to identify new therapeutic target for sensitizing immunotherapy.Studies have shown that epigenetic modification plays an important role in tumor immune regulation.In this study,we used breast cancer as a model to identify the role of E1A Binding Protein P300(EP300)antitumor immunity.Through multi-omics sequencing technology combined with bioinformatics analysis,which revealed that EP300 regulates the immune activity of tumor cells and reshapes the immune microenvironment by regulating tumor endogenous immune signaling pathways.Using mouse breast cancer EMT6 cell line and mouse spleen primary culture lymphocyte coculture cell model and mouse subcutaneous tumor-bearing breast cancer model to verify the function of EP300 in regulating immunity.Finally,bioinformatics methods were used to verify the relationship between EP300 expression and immune pathway activity and immune efficacy in multiple cancer species.The purpose of this study is to clarify the role of EP300 in tumor immune regulation,and preliminarily reveal the mechanism of EP300 regulating tumor immunogenicity,so as to provide experimental basis and theoretical basis for the development of epigenetic inhibitors and sensitizing clinical anti-tumor immunotherapy targets.Methods(1)In this study,breast cancer as a model,EP300 small molecule inhibitor A485 was used as a tool to explore the regulatory function of EP300 through transcriptomics and proteomics sequencing in human breast cancer MDA-MB-453 cell line.Immunofluorescence(IF)was used to investigate the formation of double-stranded RNA(dsRNA)following EP300 activity inhibition in breast cancer cells MDA-MB-453 and SUM159.Western Blot(WB)and Realtime Quantitative polymerase chain reaction(qRT-PCR)were applied to confirm the activation of the cytoplasmic RNA sensing signaling pathway and the IFN pathway after EP300 inhibition.Furthermore,EP300 knockdown in MDA-MB-453 and SUM159 cell lines was established to verify that EP300 inhibition increased the function of tumor immunogenicity.(2)Based on the transcriptomics dataset,bioinformatics methods were used to analyze the expression of endogenous retrovirus elements(ERVs)in genome annotation after treatment with EP300 inhibitor A485,and expression difference and classification of ERVs were performed to explore the cause of dsRNA formation after EP300 inhibition.(3)The cytotoxic effect of lymphocytes on tumor cells after inhibition of EP300 was evaluated to reveal the function of inhibitor A485 to enhance tumor immunogenicity through the co-culture model comprising mouse-derived breast cancer EMT6 cell line and mouse spleen primary cultured lymphocyte.Furthermore,EP300 inhibitor A485 inhibitory effect on tumor growth was evaluated by tumor regression.IFN mRNA expression in tumor tissues was detected by qRT-PCR.A485 toxicity was evaluated by tumor pathological examination(H&E staining)and organ index.(4)Based on the TCGA database,the correlation between EP300 mRNA expression and immune-related pathways in breast cancer patients was explored to further verify the potential of EP300 in regulating tumor immunity.(5)To further demonstrate the potential of EP300 as a target in the regulation of anti-tumor immunity in other cancer types,the expression level of EP300 mRNA in pan-cancer was examined through bioinformatics based on the TCGA database.Moreover,gastric cancer,which has been proven to be a highly malignant disease,was applied to verify the broadspectrum effect of EP300 in regulating anti-tumor immunity.The correlation between the expression level of EP300 mRNA in gastric cancer patients and immune-related pathways was assessed by bioinformatics in the TCGA database.Furthermore,the association between the expression of EP300 mRNA in patients and the response to immunotherapy was employed to evaluate the potential of EP300 inhibition to sensitize anti-tumor immunotherapy based on the clinical data originating from gastric cancer immunotherapy.qRT-PCR to verify the expression of IFN-related mRNA and IF,WB,and qPCR to confirm the formation of dsRNA and the activation of cytoplasmic RNA sensing signaling pathway after EP300 inhibition in gastric cancer cell lines AGS and SNU1.Results(1)The transcriptomic and proteomic analysis demonstrated that EP300 inhibitor A485 activates the cytosolic RNA pathway,typeⅠinterferon pathway,virus defense reactions,and endogenous immune response in breast cancer compared with the DMSO control group,leading to the accumulation of dsRNA and the elevation of IFIH1、DDX58、MAVS、ISG15、IFNα/γ mRNA expression level and MDA5、RIG-Ⅰ、TBK1 protein expression level.(2)The increase in ERVs expression of long terminal repeats(LTR)and non-LTR transcripts of long interspersed nuclear elements(LINEs)in the genome is the cause of dsRNA accumulation after EP300 inhibition.(3)The co-culture model showed that EP300 inhibitor A485 promoted breast cancer cell apoptosis and strengthen the killing efficacy of lymphocytes on cancer cells.(4)Compared with the control group,EP300 inhibitor A485 significantly inhibits tumor growth,elevates the expression of IFN gene at mRNA level.Additionally,A485 shows no obvious adverse reactions in mice according to pathological examination and lung,spleen,liver,kidney,and thymus indexes.(5)The results of bioinformatics analysis revealed that EP300 mRNA expression was negatively associated with immune-related pathways in breast cancer patients based on the TCGA database.(6)EP300 mRNA is highly expressed in breast cancer,gastric cancer,and many other cancer types.Further analysis illustrates that the expression level of EP300 in gastric cancer was negatively correlated with immune-related pathways.The gastric cancer patients with a higher expression level of EP300 mRNA showed worse responses to immunotherapy.Furthermore,inhibition of EP300 in gastric cancer cells leads to the accumulation of dsRNA and stimulates the mRNA expression of IFIH1、DDX58、MAVS、ISG15,and protein expression of MDA5、RIG-I,promoting endogenous immune response via elevating the IFNα/γ mRNA expression level.Conclusion(1)EP300 inhibition enhanced immunogenicity.(2)Inhibition of EP300 prompted the transcription of the ERVs,which results in the accumulation of cytosolic dsRNA,leading to the upregulation of the IFN signaling pathway via stimulating the dsRNA sensors MDA5 and RIG-Ⅰ,and further activation of the endogenous viral immune response.(3)EP300 inhibition increased tumor immunogenicity,which has the potential to turn"cold" tumors with low immunogenicity into "hot" tumors by enhancing the cytotoxic effect of lymphocytes on tumor cells and improving the tumor immune environment.