CRISPR-Mediated CDKN2B Knockout in NNK-Induced Bronchial Epithelial Transformation: Functional and Transcriptomic Characterization

Abstract

Lung cancer remains the leading cause of cancer-related mortality worldwide, with tobacco smoking representing its primary etiological factor. Among the tobacco-derived carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is classified as one of the most potent inducers and promoters of lung carcinogenesis. While previous studies substantially advanced our understanding of the molecular mechanisms governing NNK-induced carcinogenesis, they have primarily focused on assessing established biological targets. Addressing this gap, a genome-wide CRISPR/Cas9 screen identified the cyclin-dependent kinase inhibitor (CDKN2B) gene, located at the 9p21.3 locus, as a top candidate tumor suppressor gene whose loss sensitizes bronchial epithelial cells (BEAS-2B) to NNK-induced transformation. Despite the frequent alterations of this locus in multiple human cancers, the independent role of CDKN2B gene in the early molecular events of lung carcinogenesis, especially those underlying NNK-induced lung epithelial transformation, remains poorly understood. This study investigated the functional and transcriptomic consequences of CDKN2B loss in BEAS-2B under basal conditions and following NNK exposure. Using CRISPR/Cas9 gene editing and a lentiviral transduction delivery system, we generated, for the first time, a CDKN2B/KO model in non-malignant BEAS-2B cell line, which was further characterized by using cell viability and proliferation assays, cell-cycle analysis, DNA damage assessment, western blotting, and RNA sequencing. Our findings revealed that CDKN2B deficiency altered cellular morphology, enhanced metabolic activity and proliferative capacity, and promoted accumulation of DNA damage. Transcriptomic profiling revealed widespread gene expression alterations affecting pathways involved in epithelial organization, metabolism, cell-cycle regulation, and cellular stress. Under chronic NNK exposure, CDKN2B deficient cells exhibited enhanced phenotypic alterations mainly characterized by increased DNA damage accumulation, as well as a distinct adaptive transcriptional program characterized by increased activation of stress-response pathways, antioxidant and metabolic survival pathways, and suppression of cell-cycle-related pathways. Collectively, these findings demonstrate that CDKN2B functions beyond its canonical role in cell-cycle regulation by maintaining epithelial homeostasis and genomic stability during the early stages of NNK-induced transformation, providing new insights into the early molecular mechanisms underlying tobacco-induced carcinogenesis and identifying CDKN2B as an important determinant of cellular susceptibility to NNK.