Integrative network toxicology and virtual knockout analysis suggest TSNA-associated molecular features in large cell neuroendocrine carcinoma.

OBJECTIVE: Electronic nicotine delivery systems (ENDS) may contain or generate low levels of tobacco-specific nitrosamines (TSNAs), including N-nitrosoanatabine (NAT) and N-nitrosoanabasine (NAB), under certain thermal conditions. Given the uncertain long-term carcinogenic effects of ENDS, this study explored potential molecular associations between TSNA-related exposure signatures and LCNEC using an integrative computational framework. METHODS: We integrated network toxicology, clinical transcriptomics (GSE1037), and single-cell RNA sequencing (GSE269942) to identify key targets. Protein-protein interaction (PPI) networks and hub genes were established. Interactions between TSNAs and hub targets were validated using molecular docking and 100-ns molecular dynamics (MD) simulations. Potential network-level perturbation effects were explored via scTenifoldKnk-based virtual knockouts (vKO), diagnostic ROC analysis, and CIBERSORT-based immune infiltration analysis. RESULTS: Network analysis identified EGFR, CASP3, CCND1, STAT3, and SRC as central toxicological sensors, with MD simulations suggesting stable predicted interactions with NAT and NAB.The PI3K-Akt pathway emerged as a recurrently enriched pathway linking predicted TSNA targets with LCNEC-associated transcriptomic alterations. Single-cell profiling localized the transcriptomic reprogramming-characterized by IGFBP2 upregulation and EDNRB silencing-specifically to malignant LCNEC clusters. EDNRB (AUC=0.954) and IGFBP2 (AUC=0.855) demonstrated high diagnostic accuracy. vKO simulations suggested that IGFBP2 and EDNRB may be associated with network-level perturbations involving growth-related and immune-related genes.Drug screening anchored this signature to nicotine and prioritized EGFR-related inhibitors and natural compounds as hypothesis-generating candidates for further validation. CONCLUSION: This study proposes a hypothesis-generating computational model in which TSNA-associated target networks converge on PI3K-Akt-related signaling and the IGFBP2/EDNRB expression axis in LCNEC.These findings provide exploratory molecular evidence that may inform future experimental studies on TSNA-related lung cancer biology and potential biomarker discovery.