Integrative single-cell eQTL and multi-omics analyses reveal AIM1 and ANXA1 as immune-related hub genes and potential therapeutic targets in head and neck cancer.

Introduction: Head and neck cancer (HNC) is characterized by substantial immune heterogeneity and limited availability of clinically actionable molecular targets. Here, we developed an integrative single-cell eQTL-driven multi-omics framework to identify immune cell-specific causal genes and prioritize drug-repurposing candidates for HNC. Methods: Single-cell eQTL data from the OneK1K cohort were integrated with European HNC GWAS summary statistics through two-sample Mendelian randomization, followed by transcriptomic differential expression analysis and weighted gene co-expression network analysis. Candidate targets were further evaluated using diagnostic modeling, immune infiltration analysis, single-cell and spatial transcriptomics, Bayesian colocalization, Western blot validation, molecular docking, molecular dynamics simulations, and FAERS-based safety profiling. Results: We identified 494 immune cell-specific eGenes causally associated with HNC risk. Multi-layered target prioritization highlighted AIM1 and ANXA1 as protective immune-related genes, both of which were downregulated in HNC tissues and showed cell-type-preferential expression in T cells and monocytes, respectively. A dual-gene diagnostic model achieved strong discrimination performance with an AUC of 0.917. Colocalization analysis supported shared genetic signals between AIM1 or ANXA1 loci and HNC susceptibility, while Western blotting confirmed reduced AIM1 and ANXA1 protein expression in SCC-9 cells compared with normal HOK cells. Drug screening and molecular docking identified topotecan as a candidate ligand for AIM1 and terbutaline as a candidate ligand for ANXA1. Subsequent molecular dynamics simulations demonstrated stable drug-target complexes with favorable binding free energies. FAERS analysis further characterized the adverse-event spectrum and potential safety considerations for both compounds. Discussion: Collectively, this study provides a single-cell genetic and pharmacological framework for defining immune-related causal targets in HNC and supports AIM1 and ANXA1 as promising biomarkers and therapeutic entry points for precision drug development.