Abstract

Clinical efficacy of PD-1/PD-L1 blockade in Epstein-Barr virus-associated gastric cancer (EBVaGC) remains limited by adaptive immune resistance, despite robust lymphocyte infiltration. Through integrated bioinformatics, we identified malignant cell-intrinsic JAK2 as a central upstream regulator of CD274 (encoding PD-L1) overexpression in EBVaGC, with an exceptionally strong correlation (r = 0.9, P < 10-10) distinguishing this subtype from others. Single-nucleus RNA sequencing confirmed that JAK2 is predominantly expressed in malignant cells and drives CD8+ T-cell exhaustion via the PD-L1-PD-1 axis. Spatial transcriptomics revealed that JAK2-high niches orchestrate a "recruitment-exhaustion trap", characterized by elevated CXCL9/CXCL10, enhanced CD8+ T-cell infiltration, and a spatial gradient of functional exhaustion. To enable translational validation, we established a humanized EBVaGC model that recapitulates the clinical "recruitment-exhaustion" paradox and hyperactivated IFN-γ-JAK2-STAT1-PD-L1 signaling. Using this platform, we characterized strebloside as a natural cardenolide that induces proteasome-dependent JAK2 protein degradation. Strebloside (3 mg/kg) outperformed AG490 and achieved efficacy equivalent to 5-fluorouracil (5-FU; 30 mg/kg) at one-tenth of the dose (n = 5 per group, P < 0.0001). Unlike AG490, which only blocks kinase activity, and 5-FU, which triggers compensatory p-JAK2 upregulation, strebloside induces complete JAK2 depletion with a favorable cardiac safety profile at the therapeutic dose. These findings establish JAK2 as a therapeutic vulnerability and position strebloside-mediated JAK2 degradation as a promising strategy to overcome immune evasion in EBV-associated malignancies.