The Role Of YAP Phase Separation In Tumor Resistance To Immunotherapy

Background:Anti-PD-1/PD-L1 immunotherapies have achieved durable responses in the treatment of many types of cancer,but only a minority of patients benefit and resistance is commonly observed.Immunotherapy reinvigorate T cells to secrete interferon-?(IFN-?)and granzyme which induce tumor cell death.Despite its role as major tumor killing cytokine,IFN-?has also been reported to mediate tumor adaptive resistance to immunotherapy.Hippo signaling pathway is an evolutionarily conserved pathway controlled by cascades of kinases,of which core transcriptional coactivator YAP controls cell proliferation,cell fate,tumorigenesis and tumor immune evasion.Recent study shows that phase separation plays key role in transcriptional regulation.Whether YAP regulate gene transcription through phase separation to promote tumor survival and immune evasion is unclear.In this study,YAP phase separation was observed in lung cancer mice model,which is resistant to anti-PD-1therapy,suggesting that phase separation of YAP is the underlying mechanism of immunotherapy resistance.Objective:Study the trigger of YAP phase separation,the liquid property of YAP phase separation and the mechanism by which YAP phase separation regulate immunotherapy resistance.Methods:(1)Lung adenocarcinoma mice model were established followed by anti-PD-1 treatment,the correlation between YAP condensation and immunotherapy resistance was studied by Flowcytometry(FC)and Immunofluorescence(IF).(2)The expression level and YAP puncta were observed by IF after IFN-?treatment.IFN-?signal was blocked to determine whether YAP condensation is IFN-?dependent.(3)Study the phase separation properties of YAP droplets through in vitro droplets assay,Opto-Droplets assay,and overexpression of full length YAP.(4)The structural basis of YAP phase separation was studied through overexpression of YAP mutants and in vitro droplets test.(5)Multiple components were identified by IF in YAP phase separated condensates.(6)The role of YAP phase separation in transcription was studied by DNA Fluorescence in situ Hybridization(FISH),H3K27ac staining,Ch IP-qPCR and target gene qPCR.(7)TDCL with YAP phase separation defect was used as a model to study whether YAP phase separation was involved in tumor proliferation and immune regulation.(8)RNA-seq,qPCR and luciferase assay were performed to study the mechanism by which YAP phase separation mediate immunotherapy resistance.Results:(1)Tumor showed adaptive resistance to anti-PD-1 therapy.In resistant stage,tumor YAP showed punctate distribution in CD8~+T cells infiltrated region.(2)YAP formed droplets in IFN-?stimulated cells,and anti-PD-1 treatment cannot induce tumor YAP condensation after blocking IFN-?signal.(3)YAP condensates exhibited phase separation properties in cells and solution.(4)Coiled-coil domain is required for YAP phase separation.Mutation of four indicated Leucine to glutamic acid in CC completely blocked phase separation of YAP.(5)YAP puncta colocalized with TAZ,EP300,TEAD4 and MED1 in tumor cells.(6)YAP puncta colocalized with H3K27AC and target gene CYR61 and MYC loci.In addition,a defect in YAP phase separation reduced Myc enhancer H3K27ac level and target gene expression.(7)Disruption of YAP phase separation inhibited tumor growth and sensitized tumor to immunotherapy.(8)RNA-seq and qPCR showed YAP target genes,but not ISGs,were down-regulated owing to YAP phase separation defection.Luciferase assay showed CD155 is YAP direct target,in addition,allograft of TDCL with CD155 RNAi was sensitive to anti-PD-1 therapy.Conclusion:Anti-PD-1 therapy reinvigorate CD8~+T cells and lead to IFN-?accumulation in TME,therefore inducing tumor cell YAP activation and phase separation,which induces tumor adaptive resistance to anti-PD-1 therapy.YAP form transcriptional hub by recruit TAZ,TEAD4,MED1 and EP300,maximizing immunosuppressive gene expression.Interfering with YAP phase separation of tumor cells or knocking down CD155 sensitize tumors to anti-PD-1 therapy.