USP8 Inhibition Reshapes The Tumor Microenvironment That Potentiates The Immunotherapy

Blocking antibodies targeting the immune checkpoint programmed death receptor 1/ programmed death ligand 1(PD-1 / PD-L1)have been used in treatment of a variety of tumors.However,only a small proportion of patients(～ 30%)respond to PD-1/PD-L1 blockade,and the majority fails to respond to these therapies or emerges resistance,the molecular mechanisms of which remain undefined.By dissecting the mechanisms underlying the non-responsiveness or resistance,therapeutic efficiency will be improved and more patients will benefit from this therapeutic strategies.It is one of the urgent and important issues to be solved in current cancer research.Studies have shown that the expression of PD-L1 affect the response rate to PD-1 /PD-L1 blockade,and that ubiquitination modification is critical for intracellular protein stability.The deubiquitinating proteases USP8 has strong catalytic activity for removing ubiquitination of proteins and regulates the stability of proteins.It is important in several cellular processes,including cancer,immunity and inflammation.However,It is unknown whether USP8 regulates the polyubiquitination modification and degradation of PD-L1 and the impact on the tumor immune microenvironment.In this study,we found that inhibition of the deubiquitinating proteases USP8 can reshape tumor microenvironment and significantly improve the therapeutic efficacy of PD-1 / PD-L1 blockade in vivo.At molecular level,USP8 interacts with PD-L1 to remove its K63 linked ubiquitination which is mediated by ubiquitin E3 ligase TRAF6 and promotes PD-L1 degradation.Knocking down/knocking out USP8 or inhibiting USP8 enhanced PD-L1 protein stability and increased PD-L1 protein abundance in tumor cells.Moreover,USP8 removed the K63 linked ubiquitination of TRAF6 itself,which in turn inhibited activation of NF-κ B signaling pathway and downregulated innate and adaptive immune pathway at gene level.According to this research,inhibition of USP8 activates the TRAF6-NF-κ B pathway to upregulate interferon(IFN)as well as surface major histocompatibility complex-I(MHC-I)expression in tumor cells.Based on those exploration on mechanism,this study further established a murine tumor model and demonstrated that the combination of USP8 inhibition and PD-1/PD-L1 blockade significantly reduce tumor growth by upregulating PD-L1 and MHC-I protein abundance on tumor cells and simultaneously raising the number of cytotoxic CD8 + T lymphocytes in tumor microenvironment.Lung cancer patients tissue immunohistochemical staining(IHC)showed that USP8 and PD-L1 are negatively correlated,while TRAF6 and PD-L1 are positively correlated at protein level.Bioinformatics analysis further revealed that MHC-I signal pathway related genes are significantly upregulated in patients with low USP8 expression,and showed that patients expressing low USP8 level concomitantly with high infiltrating killer T lymphocytes demonstrate prolonged survival.In conclusion,this study found that by inhibiting USP8,we can reshape the tumor immune microenvironment and turn " cold tumor " into " hot tumor ",therefore,improve the therapeutic effect by tumor immunotherapies.This study provides a mechanism and theoretical support for the novel combinational therapy which utilizes a USP8 inhibitor and PD-1 / PD-L1 blockade for enhancing anti-tumor efficacy in clinic.