Regulation of PD-L1 Protein Expression by the E3 Ubiquitin Ligase GP78

Immune checkpoint inhibitors (ICIs), including PD-L1 inhibitors, have been approved by the FDA for the treatment of cancers; however, only a small number of cancer patients benefit from these ICIs. Furthermore, the development of drug resistance to this type of treatment is often inevitable. The mechanisms of resistance to PD-L1 inhibitors can be attributed, in part, to an incomplete understanding of the regulation of PD-L1 protein expression. In this study, we identified the role of the E3 ligase GP78, also known as the Autocrine Motility Factor Receptor (AMFR), in the regulation of PD-L1 protein levels. We show that GP78 physically interacts with PD-L1, which is confirmed by IP and Western blotting and is supported by molecular modelling using AlphaFold2. Our modeling studies predict that the interface amino acids of the Ig1 domain of PD-L1 interact with the RING domain and a β-hairpin preceding the CUE domain of GP78. The crystal structure of the PD-1/PD-L1 complex reveals that the interaction with PD-1 is mediated by the Ig1 domain of PD-L1. Furthermore, proteasomal degradation of PD-L1 has been observed via GP78-mediated K48-linked ubiquitination, indicating a key regulatory role for GP78 in the downregulation of PD-L1. Because GP78 expression is inversely correlated with PD-L1 levels in cancer, these findings may have clinical implications for predicting tumor immune evasion and patient response to PD-1/PD-L1 blockade therapies. Taken together, these findings identify a previously unknown mechanism by which GP78 targets PD-L1 for ubiquitination and subsequent degradation in cancer cells, and suggest that blocking the interaction between PD-L1 and PD-1 by an E3 ligase is a novel strategy to improve immunotherapies for cancer patients.