EGFR Regulates PD-L1 Stability And PD-1/PD-L1 Interaction In Cancer Immunity

Malignant tumor is one of major diseases threatening human health and life.In addition to the traditional therapies,tumor immunotherapy which promotes CD8+T cell-response has considered to be an effective way.The activation of T cell depends on the regnization of antigen peptide-MHC complex and the synergistic effect of costimulatory molecules.Among the costimulatory molecules,immune checkpoints suppress immune system by inhibiting the T cell-activity.PD-1(Programmed Death-1)is the most established immune checkpoint.It belongs to the immunoglobulin superfamily and is mainly expressed on the surface of T cells.PD-L1(Programmed Death-Ligand 1),which is modified with different glycosylation forms,is mainly expressed on the surface of APCs or tumor cells.Extracellular interaction between PD-1 and PD-L1 leads to tumor-associated immune escape.PD-1/PD-L1 have been remarkable targets for the clinical treatment on different types of cancers in recent decades.In this study,we found PD-L1 was glycosylated on the membrane of tumor cells and TAMs.Using a high throughput drug screening model for the protein level of membrane PD-L1,we found ES-072,a new generation of EGFR inhibitor,effectively induced PD-L1 degradation and impaired the interaction of PD-1/PD-L1.Further experiments showed EGFR participated in the regulation of PD-L1 degradation through proteasome signaling process.To detect how EGFR regulates the stability and function of membrane PD-L1,we performed a mass spectrometry(MS)analysis on membrane PD-L1,which highlighted phosphorylation sites S279 and S283 locating in intracellular domain of PD-L1.Dephosphorylated experiments verified the phosphorylation on S279/S283 was responsible for the ubiquitin-dependant degradation of membrane PD-L1 and its interaction with PD-1.MS results also suggested GSK3a was involved in the ES-072-dependent PD-L1 degradation.Using a kinase assay in vitro we confirmed that GSK3a phosphorylated PD-L1 directly,and the phosphorylated sites were S279 and S283.We established a xenograft model with 4T1 breast cancer cell line on BALB/c mice and intervened with ES-072.This in vivo data showed that ES-072 supressed tumor growth and M2-macrophages in the microenvironment.This study reveals the mechanism of PD-L1 degradation and the interaction between PD-1/PD-L1 on tumor cells and TAM are regulated by EGFR,which provides experimental basis for the immuno-and targeted therapy for cancer in clinical application.