P1.02-084 Polo-Like Kinase 1 (PLK1) Inhibition Decreases Mutational Activity in Bronchial Epithelial Cells Exposed to Tobacco Carcinogens

Abstract

Persistence of bronchial dysplasia (BD) is associated with increased risk for the development of squamous cell carcinoma (SCC) of the lung. A comparison of persistent and regressive BD demonstrated that alterations in gene expression can distinguish these types of lesions. Using the genes that differentiate persistent and regressive BD we have identified PLK1 as a key mediator of persistence and have hypothesized that the role it plays in abrogating G2-M cell cycle arrest in the presence of mutational alterations may be central to progression of premalignant airway lesions to invasive SCC. Cultures of the bronchial epithelium derived BEAS-2B (B2B) cell line were exposed to airway mutagens (cigarette smoke condensate [CSC, Kentucky Reference Cigarette 3R4F] or Benzo[A]pyrene, [BaP]) alone or in the presence of 100 nM PLK1 inhibitor Volasertib. Following treatment, clones were selected by ring isolation and DNA was collected to assess mutational events as compared to untreated controls using the TruSightOne targeted next generation sequencing panel (12 megabase coverage, >4800 genes, Illumina, San Diego, CA). Modal distribution of mutation frequencies indicated that ring clones were composed of between 1 - 3 distinct clones. Mutation rates were calculated using these adjusted clone counts for standardization. Using the untreated B2B cultures as reference sequence, CSC or BaP alone induced mean incidences of mutations of 15.7 and 60.9 per clone, respectively, although the induction of fewer mutations by CSC made accurate estimates of clonal numbers more difficult. B2B cultures treated with the same concentration and duration of mutagen in the presence of Volasertib showed mean mutational incidences of 13 and 17 per clone, respectively. The ratio of mutation rate for the CSC and BaP treated groups with versus without Volasertib were 0.83 and 0.28, respectively. All mutations detected were single nucleotide variants and characteristic patterns of base changes were noted between mutagen groups with C>A and G>T transversions being more prominent in BaP treated cultures. Ongoing analyses using whole genome sequencing will allow for more accurate enumeration of clones represented and a richer assessment of mutation rates including rearrangements and copy number variants in mutagen treated cells in the presence and absence of PLK1 inhibition. Preliminary analyses of bronchial epithelial cell cultures treated with mutagens show reduction of BaP induced mutations in the presence of PLK1 inhibition. The results suggest PLK1 overexpression in BD may promote genomic instability.