The Moonlighting Function of Glutaminase 2 Promotes Immune Evasion of Pancreatic Ductal Adenocarcinoma by Tubulin Tyrosine Ligase-like 1-Mediated Yes1 Associated Transcriptional Regulator Glutamylation.

Objective: Elevated programmed cell death-ligand 1 (PD-L1) expression in tumor cells facilitates immune evasion. However, the mechanism via which PD-L1 expression is regulated in pancreatic ductal adenocarcinoma (PDAC) cells remains inadequately elucidated.

Methods: Immunoprecipitation, pull-down assays, and mass spectrometry were used to identify glutaminase 2 (GLS2) and yes1 associated transcriptional regulator (YAP1) binding proteins and modification sites. Immunoblotting, immunofluorescence, chromatin immunoprecipitation, and luciferase reporter assays were used to analyze YAP1 activation. Protein expression levels were assessed using immunoblotting, immunoprecipitation, immunofluorescence, and immunohistochemistry. RNA expression levels were analyzed using real-time quantitative polymerase chain reaction.

Results: Hypoxia-induced general control nondepressible 5 (GCN5)-mediated acetylation of GLS2 at K151, which enhanced GLS2 interaction with YAP1. Subsequently, tubulin tyrosine ligase-like 1 mediated YAP1 glutamylation at E100 and promoted its nuclear translocation and the activation-dependent transcriptional up-regulation of PD-L1 expression. The expression of GLS2-K151R or YAP1-E100A mutants in PDAC cells blocked hypoxia-induced PD-L1 expression and enhanced CD4+ and CD8+ T-cell activation and tumor infiltration, thereby suppressing PDAC tumor growth. Simultaneous administration of MB-3, a GCN5 inhibitor, and an anti-programmed cell death 1 (PD-1) antibody abolished tumor immune evasion, boosting the anti-tumor efficacy of immune checkpoint blockade. Furthermore, GLS2-K151 acetylation and YAP1 E100 glutamylation levels correlated positively with PD-L1 expression and poor prognosis in PDAC patients.

Conclusions: The present study revealed a novel mechanism by which hypoxia up-regulates PD-L1 expression and highlighted the involvement of GLS2 in noncanonical metabolic pathways involved in tumor immune evasion, with implications for PDAC treatment.