PRKX-mediated stabilization of PD-L1 characterizes an immunosuppressive gastric cancer subtype.

BACKGROUND: Gastric cancer (GC) derives limited benefit from immunotherapy, with clinical responses observed in only a minority of patients. Increasing evidence suggests that heterogeneity within the tumor immune microenvironment (TME) is a critical determinant of immunotherapeutic efficacy, highlighting the need for precise immune stratification and the identification of molecular biomarkers that shape the TME. METHODS: We integrated single-cell transcriptomic data from our cohort and public datasets to characterize immune microenvironment heterogeneity in GC. Functional experiments were performed using in vitro assays and in vivo mouse models to investigate the molecular mechanisms regulating immune exhaustion. Clinical relevance was evaluated using tumor specimens from patients with GC receiving anti-programmed cell death protein 1 (PD-1) therapy. Survival analyses and biomarker evaluation were conducted to assess the prognostic and predictive value of candidate markers. RESULTS: We identified two distinct GC immune microenvironment subtypes: the immunosuppressive (GC1) and the immune-activated (GC2). PRKX was identified as a key regulator associated with immune heterogeneity and exhaustion. Clinically, a high density of PanCK+ PRKX+ PD-L1+ tumor cells was significantly associated with poor prognosis and served as a robust biomarker predicting 5-year survival in patients treated with anti-PD-1 therapy. Mechanistically, PRKX phosphorylates programmed death-ligand 1 (PD-L1) at T285, promoting YWHAE recruitment and preventing UBE2M-mediated ubiquitination and degradation, thereby stabilizing PD-L1 protein. Through this phosphorylation-dependent regulation of PD-L1 stability, PRKX suppresses CD8+ T-cell cytotoxicity, promotes immune exhaustion, and limits the efficacy of anti-PD-1 therapy in vivo. Therapeutically, lipid nanoparticle-mediated delivery of PRKX-targeting siRNA effectively suppressed PRKX expression and synergized with anti-PD-1 therapy to enhance antitumor efficacy. CONCLUSION: PRKX drives immune exhaustion in the GC1 subtype by stabilizing PD-L1 through phosphorylation-dependent inhibition of ubiquitination, thereby promoting immune evasion. Targeting PRKX represents a potential strategy to overcome resistance to anti-PD-1 therapy, and PRKX expression may serve as a prognostic biomarker to guide immunotherapy in GC.