Consensus subtypes and a generalizable prognostic signature defined by an R-loop score in hepatocellular carcinoma: Multi-omics analyses pinpoint KIF2A-positive proliferative tumor cells.

BACKGROUND: Hepatocellular carcinoma (HCC) has a poor prognosis and lacks robust, generalizable stratification markers and interpretable therapeutic targets. R-loop dysregulation can induce replication stress and DNA damage, but its clinical relevance and driver cell states in HCC remain unclear. METHODS: Multi-cohort public datasets (n = 2031) integrating bulk, immunotherapy, single-cell, spatial, and proteomic resources were analyzed. R-loop scores, consensus subtypes, and a WGCNA/machine-learning prognostic signature were established and interpreted by SHAP, with experimental validation of KIF2A and MK-1775 sensitivity. RESULTS: The R-loop score was significantly elevated in HCC tumors, increased with clinical stage, and consistently predicted poorer overall survival across cohorts. Three reproducible R-loop molecular subtypes showed distinct prognostic and clinicopathological patterns; the unfavorable subtype was enriched for cell-cycle, DNA replication, and repair programs. A seven-gene R-loop risk signature showed stable external prognostic performance and improved prediction beyond TNM stage and AFP. High R-loop activity was associated with immune checkpoint expression, T-cell exhaustion, and reduced benefit from immune checkpoint blockade. Multi-scale analyses localized the high-risk signal to a reproducible KIF2A-positive proliferative tumor-cell state that correlated with R-loop activity, hypoxic malignant spatial niches, immune-exhausted features, and poor prognosis. KIF2A was the top SHAP-ranked contributor and was consistently upregulated across transcriptomic, proteomic, single-cell, spatial, and histological layers. Stable KIF2A knockdown reduced CCNB2/CDC20 expression, suppressed colony formation, and inhibited xenograft growth. KIF2A-high HCC cells showed greater MK-1775 sensitivity. CONCLUSIONS: R-loop-associated programs define a clinically relevant prognostic axis in HCC, with KIF2A-positive proliferative tumor cells representing a key high-risk cellular source and potential therapeutic vulnerability.