Tubeimoside-1 Induces TFEB-dependent Lysosomal Degradation Of PD-L1 And Promotes Antitumor Immunity

Background:Malignant tumor is one of the life-threatening diseases in the world,its effective treatments is a great challenge for cancer research.In the last decade,immunotherapy has become the hotspot of oncology research,and it is reforming the clinical therapeutic model of tumor.Different from traditional radiotherapy,chemotherapy and surgical resection,tumor immunotherapy not only inhibit the growth of primary tumor,but also prevent tumor recurrence and metastasis.Accumulating evidences show that tumor immunotherapies,including PD-L1/PD-1 immune checkpoints blockage and CAR-T cell therapy,have achieved great clinical breakthrough in multiple cancer.However,there are also some problems,such as low response rate and serious immune related side effects.With the development of smallmolecule immunotherapy drugs,its combination with the PD-L1/PD-1 antibodies would provide a new opportunity for cancer treatment.Therefore,targeting PD-L1/PD-1 axis by small-molecule drug is an attractive approach to enhance antitumor immunity and considered as the next generation of tumor immunotherapy.Tubeimoside I(TBM-1)is a triterpenoid saponide isolated from traditional Chinese medicine Tuple,which is used to treat lymphatic tuberculosis,mastitis,breast fibroma,etc.clinically.Currently,the anti-tumor mechanism of TBM-1 is only limited to the cytotoxic pathway,but the antitumor immunity of TBM-1 remains unknown.In this study,we used a variety of experimental methods to evaluate the expression of PD-L1 in tumor cells from in vitro cell level and the animal in vivo by TBM1 lowering expression of PD-Ll,thereby reshaping the role of anti-tumor in tumor immunochemical environment.The mechanism of TBM-1 downregulating PD-L1 was further discussed.Methods:Natural active compounds specifically reducing tumor cell surface PD-L1 are screened using flow cytometry in the natural active small molecule compounds.Western blot was used to detect the effect of TBM-1 on PD-L1 protein expression,TFEB phosphorylation in human lung cancer cells H157,H460,and human melanocytes A375,A2058,mTOR substrate(p70S6K and 4EBP1)phosphorylations was also detected by western blot.The tests of immunofluorescence and cell co-culture were used to detect the T cell activation by TBM-1.The Fluorescence Quantitative PCR method was used to detect the expression level of PD-L1 mRNA.Laser confocal experiment was performed to detect the position of PD-L1 in lysosomalization under the action of TBM-1.Lyso-Traker Dyeing experiment was carried out to detect the lysosome biosynthesis under the effect of TBM1.Surface plasma resonance techniques and cellular thermal transformation analysis(CETSA)techniques were used to analyze the direct interaction between TBM-1 and MTOR.Experiments in the mouse were carried out,and after TBM-1 was used for LLC and B16 xenograft,tumor volume,tumor weight,mouse main organ,biochemical indicators,and changes in various indicators(CD4+T,CD8+T,IFN-y,Granzyme B,Tregs and MDSCs)in tumor immune microenvironment were detected.In the LLC subcutaneous tumor-bearing model,TBM-1 and CTLA-4 mAb were combined to detect the sensitizing effect of TBM-1 on CTLA-4 mAb.Results:It was detected in the process of screening 282 natural active small molecular compounds with flow cytometry that the TBM-1 can significantly reduce the level of PDL1 in tumor cells.TBM-1 can significantly inhibit the expression of PD-L1 in lung cancer and melanoma cells,and enhance co-cultured T cells to kill tumor cells.In the experiment of LLC and B16 subcutaneous homogenous,TBM-1 can significantly inhibit the growth of tumors transplanted in C57BL/6 mice,while activating the killing activity of tumor mice tumor invasive CD8+ T cells.However,there was no obvious anti-tumor effect in the nude mice which verifies that TBM-1 plays anti-tumor activity through an immune system.It was found in mechanism research that TBM-1 can directly bind directly to the mTOR protein FRB region and inhibit its activity,transfer the transcription factor TFEB to the cellular nucleus,enhance the biological synthesis and function of the lycosomes,and promote the lysosomas pathway degradation of PD-L1 proteins.It leads to the activation of CD8+T cells,the accumulation of inhibitory cells(MDSCs)and regulatory T cells(Tregs)which inhibit the source of bone marrow,the release of immunosuppressive state in tumor immunization microenvironment to play anti-tumor activity.LLC tumor transplanted mouse experiments further confirmed that the combination of TBM-1 and CTLA-4 antibody can further increase the number and activity of CD8+T cells,and reduce the infiltration of MDSCs and Tregs,thereby increasing the anti-tumor effect of CTLA-4 antibody.Conclusion:Our research reveals that by direct binding and inhibiting the activity of mTOR proteins,TBM-1 activates the biosynthesis and function of TFEB dependent lysosomes,promotes the degradation of lysosomal pathways in PD-L1,and thereby activates the new mechanism of T cells to play tumor immunotherapy.This study laid the foundation for the potential application of TBM-1 in tumor immunotherapy drug targeting PD-1/PD-L1.