| In the first part of our study,we concluded that immune evasion was recognized as a major feature of carcinogenesis,overexpression of inhibitory immune checkpoints,such as PD-L1/PD-1,is one of the crucial steps.PD-L1 are known to be upregulated in multiple types of cancer cells,it switches off T cell effector function by interacting with its co-inhibitory receptor,PD-1,at the surface of tumor-infiltrating lymphocytes.Blockade of PD-L1/PD-1 axis can reactivate these exhausted T cells.Several monoclonal antibodies targeting PD-L1/PD-1 have been approved and achieved great success in multiple types of cancer,especially advanced and refractory cancer.However,they have limitations,such as immunogenicity,unstability,high production cost and low penetration into tissue,highlighting the need for developing small molecular inhibitors.In order to develop effective PD-L1/PD-1 inhibitors,we first established an invitro assay to evaluate PD-L1/PD-1 interaction,which incorporated with two stable strains,PD-L1-expressing PC-9/PD-L1 tumor cells and PD-1-expressing Jurkat/PD-1 T cells.We found that coculture of PC-9/PD-L1 and Jurkat/PD-1 cells accelerated the lysosomal degradation of PD-1,which reflected the extent of PD-L1/PD-1 interaction.Variation of PD-1 level after inhibitors treatment in the coculture assay was an useful indicator of PD-L1/PD-1 blockade.Based on this,we have screened and found a lead compound,007,which effectively counteracting PD-L1/PD-1 interaction but acted in a different way from blocking antibodies.Further research showed that PD-L1 was the primary target of 007.It induced aggregation of nascent PD-L1 s so that they were stuck in ER.Instead of being properly modified and transported through the secretory pathway to membrane,the aggregate PD-L1 s were degraded by ERAD pathway,which impeded PD-L1/PD-1 interaction and PD-L1 glycosylation.We have confirmed 014,a derivative of 007,effectively inhibited PD-L1 glycosylation in mouse model.Our study will help to guide further structural optimization of PD-L1/PD-1 molecular inhibitors and improve the immunotherapy.In the second part of our study,we understood that overexpression or activation of Axl has been reported as a prevalent mechanism of cancer progression and metastasis,and Axl has been considered as a promising drug target for cancer drug development.R428 is the first specific Axl inhibitor entering clinical trials,which marks an important milestone.However,though R428 inhibits the phosphorylation of Axl and induces apoptosis of many types of cancer cells,the relationship between the two has not been well established.Our study verified that R428 was a transient Axl inhibitor and its anti-tumor effects was independent of its Axl inhibition.To clarify its mode of action,we started with R428-induced cytoplasmic vacuolation and found that R428 blocked lysosomal acidification and recycling,which lead to expansion of lysosomal volume.Besides,R428 stimulated autophagic induction by accelerating the transcriptions of the autophagy-related genes,while blocking autophagic degradation,which resulted in accumulation of autophagosomes and lysosomes.Inhibition of autophagy by autophagic inhibitors or gene-knockout alleviated the R428-induced vacuoles formation and cell apoptosis.Our study uncovered a novel function and mechanism of R428 in addition to its ability to inhibit Axl.These data will help to better direct the application of R428 as an anti-cancer reagent.It also adds new knowledge to understand the regulation of autophagy and apoptosis. |
