Membrane-Associated RING-CH 8 Functions as a Novel PD-L1 E3 Ligase to Mediate PD-L1 Degradation Induced by EGFR Inhibitors

Abstract

◥Expression of programmed death-ligand 1 (PD-L1) on cancer cells is a critical mechanism contributing to immunosuppression and immune escape. PD-L1 expression may also affect therapeutic outcomes of epidermal growth factor receptor (EGFR)targeted therapy (e.g., with osimertinib/AZD9291) against EGFR-mutant non-small cell lung cancers (NSCLC) and can even be altered during the treatment albeit with largely undefined mechanisms. This study primarily focuses on elucidating the mechanism by which osimertinib induces PD-L1 degradation in addition to validating osimertinib's effect on decreasing PD-L1 expression in EGFR-mutant NSCLC cells and tumors. Osimertinib and other EGFR inhibitors effectively decreased PD-L1 levels primarily in EGFR-mutant NSCLCs and xenografted tumors. Osimertinib not only decreased PD-L1 mRNA expression, but also prompted proteasomal degradation of PD-L1 protein, indicating both transcriptional and posttranslational mechanisms accounting for osimertinib-induced reduction of PD-L1. Knockdown of b-TrCP or inhibition of GSK3 failed to prevent PD-L1 reduction induced by osimertinib. Rather, knockdown of membrane-associated RING-CH 8 (MARCH8) that encodes a membrane-bound E3 ubiquitin ligase rescued osimertinib-induced PD-L1 reduction. Furthermore, manipulation of MARCH8 expression accordingly altered PD-L1 degradation rate. Critically, MARCH8 interacted with PD-L1 through its N-terminal region and also ubiquitinated PD-L1 in cells. Collectively, these results strongly suggest that MARCH8 is a previously undiscovered E3 ubiquitin ligase responsible for PD-L1 degradation including osimertinib-induced PD-L1 degradation, establishing a novel connection between MARCH8 and PD-L1 regulation.Implications: This study has demonstrated a previously undiscovered function of MARCH8 in mediating PD-L1 degradation induced by EGFR inhibitors in EGFR-mutant NSCLC cells, establishing a novel connection between MARCH8 and PD-L1 regulation.

Changes of tumor microenvironment in non-small cell lung cancer after TKI treatments

“…This reduction was found to be associated with the NF-kB signaling pathway (15,58,100) and its downstream pathway including pAKT, pERK, and pSTAT3 (59). Osimertinib (55,58,101) could downregulate PD-L1 not only at the mRNA level but also at the protein level. For example, proteasomal degradation of PD-L1 protein was prompted by osimertinib via a novel PD-L1 E3 ligase (MARCH8) (102).…”

“…Osimertinib ( 55 , 58 , 101 ) could downregulate PD-L1 not only at the mRNA level but also at the protein level. For example, proteasomal degradation of PD-L1 protein was prompted by osimertinib via a novel PD-L1 E3 ligase (MARCH8) ( 102 ). However, an increased trend of PD-L1 was observed in other studies.…”

Strategies targeting PD-L1 expression and associated opportunities for cancer combination therapy

“…At translational level, PD-L1 expression is primarily regulated via PI3K-AKT-mTOR and RAS-MEK-ERK (MAPK) pathways 58 , 59 . Several post-translational modifications, including phosphorylation 60 , glycosylation 61 , acetylation 62 , ubiquitination 63 and palmitoylation 64 contribute to regulate PD- L1 degradation and activity.…”

PD-L1 as a Novel Mediator of Lung Fibroblast to Myofibroblast Transition

“…The transcriptional regulation of PD-L1 was demonstrated through an array of assays in this manuscript. It is well known that multiple mechanisms control the level of PD-L1 including manipulating its protein stability [20], e.g., the identification of March8 as a novel E3 ligase for PD-L1 degradation [21]. Whether TGF-β signaling also affects the stability of PD-L1 remains an interesting question and warrants further experimental testing.…”