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

Abstract 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.