CYP1B1-Mediated Ferroptosis Defines a Biomarker and Therapeutic Target in COPD Across Multi-omics and Single-Cell

Background: Chronic obstructive pulmonary disease (COPD) causes progressive airflow limitation and remains without disease-modifying therapy. Ferroptosis—iron-dependent lipid peroxidation—has emerged as a potential mechanism driving epithelial dysfunction and chronic inflammation; however, its upstream regulators in COPD remain incompletely defined. We hypothesized that integrative multi-omics could identify robust biomarkers and pathways, with translational potential for diagnosis and targeted therapy. Objective Primary: discover and validate biomarkers/pathways underlying COPD via bulk transcriptomics, single-cell analyses, machine learning (ML), and experimental validation. Secondary: evaluate CYP1B1 as a diagnostic biomarker and explore druggability through molecular docking. Methods: Public cohorts (GSE47460, GSE76925, GSE37768; total discovery/validation samples reported) were integrated with ComBat batch correction. Single-cell RNA-seq datasets (GSE196341, GSE135893; 12 COPD vs 12 controls) profiled cell-type localization. Differential expression, WGCNA, and enrichment (GO/KEGG/GSEA) were performed. A 12-algorithm ML framework (113 combinations) plus ANN modeling assessed diagnostic performance (ROC/AUC, confusion matrices, calibration). CIBERSORT estimated immune infiltration. A cigarette-smoke mouse model and 16HBE cell assays provided experimental validation; small-n proteomics were deposited (PXD068247; 3 vs 3). Molecular docking screened candidate CYP1B1-binding compounds and recorded binding energies. Statistical reporting included n, effect sizes, 95% CIs, and multiple-testing control. Results: Twenty-four hub genes were initially identified; four (BHLHE22, DPP6, DHRS9, CYP1B1) were prioritized by ML across 113 model combinations. In the training set, the combined model achieved an AUC of 0.996 (95% CI, 0.991–0.999), with an external validation AUC of 0.834 (95% CI, 0.755–0.906). Single-gene ROC in an external cohort yielded AUCs of 0.764–0.795, with CYP1B1 being the most consistent. Single-cell analysis localized CYP1B1 upregulation to airway secretory cells (ASCs) and linked high CYP1B1 expression to the activation of the ferroptosis pathway. In mouse and 16HBE models, cigarette smoke increased lung inflammation/fibrosis and upregulated CYP1B1; proteomics corroborated expression changes. Docking identified α-/β-naphthoflavone, chrysin, and naringenin as CYP1B1 binders (best energies ≈ −7.11 to −5.45 kcal/mol). Immune deconvolution associated CYP1B1 with macrophage and plasma-cell signals. Conclusions: Integrative multi-omics implicates CYP1B1-mediated ferroptosis in ASCs as a central pathway in COPD, with diagnostic promise and a tractable chemistry space. Prospective validation in larger cohorts, causal perturbation of CYP1B1–ferroptosis in vitro/in vivo, and pharmacology against prioritized ligands are warranted to translate these findings.