The Moonlighting Function of GLS2 Promotes Immune Evasion of Pancreatic Ductal Adenocarcinoma by TTLL1-mediated YAP1 Glutamylation

Abstract

Background & Aims

Elevated PD-L1 expression in tumour 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 glutamine synthase 2 (GLS2) and yes1 associated transcriptional regulator (YAP1) binding proteins and modification sites. Immunoblotting, immunofluorescence, chromatin immunoprecipitation (ChIP), and luciferase reporter assays were used to analyze YAP1 activation. Protein expression levels were assessed by immunoblotting, immunoprecipitation, immunofluorescence, and immunohistochemistry. RNA expression levels were analyzed using RT-qPCR.

Results

Hypoxia-induced GCN5-mediated acetylation of GLS2 at K151, which enhanced GLS2 interaction with YAP1. Subsequently, tubulin tyrosine ligase-like 1 (TTLL1) mediated YAP1 glutamylation at E100 and promoted its nuclear translocation and the activation-dependent transcriptional upregulation of PD-L1 expression. The expression of GLS2-K151R or YAP1-E100A mutants in PDAC cells blocked hypoxia-induced PD-L1 expression, enhanced CD4⁺ and CD8⁺ T cell activation and tumour infiltration, thereby suppressing PDAC tumour growth. Simultaneous administration of MB-3, a GCN5 inhibitor, and an anti-PD-1 antibody abolished tumour immune evasion, boosting the anti-tumour 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 upregulates PD-L1 expression and highlighted the involvement of GLS2 in non-canonical metabolic pathways involved in tumour immune evasion, with implications for PDAC treatment.