Integration of single-cell transcriptomics and genomic mutation analysis identifies an immunotherapy-resistant tumor subcluster and validates ARNTL2 as a malignant driver in lung adenocarcinoma

Background Immunotherapy resistance in lung adenocarcinoma (LUAD) remains a critical clinical challenge, and the mechanisms underlying resistance-associated intratumoral heterogeneity are poorly characterized. Methods We performed single-cell RNA sequencing of LUAD patients receiving neoadjuvant immunotherapy (responders vs. non-responders), integrating inferCNV, GSVA, and differential expression analyses. Cluster-specific genes were validated across seven independent cohorts (TCGA-LUAD, GSE13213, GSE26939, GSE29016, GSE30219, GSE31210, GSE42127). A multi-algorithm machine learning framework was used to construct a prognostic model, and the immune microenvironment was characterized using TCIA scoring, seven infiltration algorithms, and ESTIMATE. ARNTL2 function was assessed by CCK-8 and Transwell assays in A549 and H1299 cells. Results Non-responders showed significant enrichment of epithelial cells, depletion of cytotoxic T/NK cells, and elevated copy number variation burden versus responders ( p < 0.0001). A resistance-enriched malignant subcluster (Cluster 2) exhibited hyperproliferative and metabolic reprogramming signatures with upregulated KRT17, S100A2, and CST6, which showed tumor-specific overexpression, adverse prognostic value, and genomic amplification across cohorts. CoxBoost combined with survivalSVM achieved optimal predictive performance (C-index = 0.686), yielding robust risk stratification (HR: 2.54–10.51, all p < 0.05). Low-risk patients showed greater immune infiltration and higher TCIA immunophenoscores. ARNTL2 was an independent prognostic factor (HR: 2.07–4.64) strongly correlated with risk score ( r = 0.69), and its knockdown suppressed proliferation and invasion in both LUAD cell lines (all p < 0.05). Conclusion This study identifies a resistance-associated malignant subcluster in LUAD, constructs a validated CoxBoost + survivalSVM prognostic model with robust immune stratification, and establishes ARNTL2 as a core oncogenic driver and therapeutic target.