Mechanism Of Ailanthone Improving The Efficacy Of Anti-PD-L1 In Melanoma By Targeting C-Jun

Background and objective:Currently,the main treatment options for melanoma include surgery,actinotheraphy,chemotherapy,targeted therapy and immunotherapy.Despite the continuous precise optimization of various treatment options,the 5-year survival rate of advanced melanoma patients in China is still less than 5%.Therefore,the treatment of melanoma remains a hot spot and a difficult research area in the global medical field.Surgery is the main method to eradicate early-stage melanoma,but the survival rate of patients with advanced melanoma is often poor due to the lack of effective therapeutic drugs.Although targeted therapy has a high initial response rate and significantly improved patient survival,it also has the drawbacks of a short median duration of response and a high rate of recurrence.Immune checkpoint inhibitors have ushered in a new era of melanoma treatment,but only a small number of patients have benefited from them and there are more immune-related side effects.Therefore,the search for effective melanoma therapeutic targets and the combination of immunotherapy and targeted therapeutic regimens to improve treatment response rates and reduce recurrence rates are popular strategies for melanoma treatment.c-Jun,an important component of the AP-1 transcription factor complex,shows abnormally high expression in melanoma and is considered an important target for melanoma treatment.However,there are no effective c-Jun inhibitors approved for clinical use.In recent years,traditional Chinese medicine(TCM)monomer has been considered as an important source of novel antitumor drugs that combine the advantages of structural diversity,biocompatibility,clear pharmacological activity,less toxic side effects and multiple targets.At present,the research on TCM monomer is getting more and more advanced and has become one of the important avenues for innovative drug research in China,and the rate of related new drug development is increasing.This study aims to screen out the TCM that can target c-Jun inhibition and investigate the combined effect of c-Jun inhibitor and anti-programmed death-ligand 1(PD-L1),and then elucidate the related molecular mechanisms to provide new ideas for the clinical treatment of melanoma.Methods:1.A combinatorial screening strategy of molecular docking and quantitative structure-activity relationship(QSAR)modeling was adopted to filter out c-Jun inhibitors from 1616 TCM monomers;2.Cell proliferation and cytotoxicity assay,wound healing and transwell assays were performed to detect the effects of Ailanthone(AIL)on the proliferation,migration and invasive ability of melanoma cells;3.C57BL/6 mice subcutaneously transplanted tumor model was constructed to evaluate the efficacy of AIL in the treatment of melanoma;4.Pull down assay was applicated to validate the binding effect of AIL and c-Jun;5.Western blot and protein half-life assay were used to detect the effect of AIL on c-Jun protein level;6.C57BL/6 mice subcutaneously transplanted tumor model was constructed to evaluate the efficacy of AIL combined with PD-L1monoclonal antibody in the treatment of melanoma;7.Multi-color flow cytometry was performed to analyze the changes of immune cell populations in the tumor microenvironment;8.Western blot,RT-PCR and ELISA assays were used to investigate the effect of AIL on PD-L1;9.Luciferase reporter gene assay and Chromatin immunoprecipitation(Ch IP)assay were employed to reveal the transcriptional regulation of c-Jun on PD-L1;10.CD4~+T-cell differentiation assay was applied to investigate the effect of AIL on Tregs differentiation in vitro.Results1.AIL was screened out as a c-Jun inhibitor by molecular docking combined with QSAR integrated modeling on a step-by-step basis;2.The primary potential target of AIL was c-Jun;3.AIL significantly inhibited melanoma cell proliferation,migration and invasion,and suppresses tumor growth in a mouse transplantation tumor model of melanoma;4.AIL interacted with c-Jun and induced its degradation;5.AIL enhanced the efficacy of PD-L1 monoclonal antibody against melanoma by reducing Tregs infiltration in the TME;6.AIL significantly inhibited the expression and secretion of PD-L1;7.AIL regulated PD-L1 transcription through c-Jun;8.AIL suppressed the differentiation of Tregs by targeting the c-Jun/PD-L1 signaling pathway.ConclusionAIL is a novel c-Jun inhibitor that interacts with c-Jun to promote its degradation and inhibits melanoma progression in vitro and in vivo;meanwhile,AIL can target the c-Jun/PD-L1 signaling pathway to suppress Tregs differentiation in the TME and enhance the efficacy of PD-L1monoclonal antibody.This study is the first to identify a TCM monomer that can inhibit the progression of melanoma by targeting c-Jun,which provides a solid scientific basis for breaking through the bottleneck of clinical drug development of this potential therapeutic target,and also provides a new research direction for the clinical combination therapy of PD-L1 monoclonal antibody.There are totally 34 figures,24 tables and 145 references.