NQO1/p65/CXCL12 Axis-Recruited Tregs Mediate Resistance to Anti-PD-1 Plus Lenvatinib Therapy in PIVKA-II-Positive Hepatocellular Carcinoma.

Immune checkpoint inhibitors (ICIs) combined with anti-angiogenic agents manifest improved survival in advanced hepatocellular carcinoma (HCC), but responses remain heterogeneous. Although high PIVKA-II expression correlates with advanced disease stage, early recurrence, shorter survival, and may predict resistance to anti-PD-1 plus lenvatinib therapy, the tumor microenvironment (TME) and resistance mechanisms in HCC with high PIVKA-II expression remain unclear. Clinical data from 156 resected HCC patients and 104 patients treated with anti-PD-1 plus lenvatinib are analyzed to correlate PIVKA-II expression with clinical features and outcomes. Single-cell RNA sequencing (scRNA-seq) is performed on tumors from 15 untreated and 7 treated patients. Mechanistic findings are validated in vitro and in vivo. High PIVKA-II expression is associated with advanced disease stage, increased microvascular invasion (MVI), early recurrence, and poor response to therapy. ScRNA-seq revealed an immunosuppressive TME enriched with regulatory T cells (Tregs), exhausted CD8⁺ T cells, and SPP1⁺ tumor-associated macrophages (TAMs). Mechanistically, tumors with high PIVKA-II expression upregulated NQO1, which stabilized p65 by inhibiting ubiquitination, activating the NF-κB/CXCL12 axis, and recruiting Tregs. This pathway mediated therapeutic resistance. Plerixafor, a CXCL12 inhibitor, disrupted this axis and significantly enhanced anti-tumor efficacy when combined with anti-PD-1 plus lenvatinib in vivo. PIVKA-II is a potentially effective biomarker for predicting resistance to anti-PD-1 plus lenvatinib therapy. Its high expression denotes an immunosuppressive TME. Targeting the NQO1/CXCL12/Tregs axis with Plerixafor may restore sensitivity and improve outcomes.