Abstract When a lung epithelial cell acquires a driver mutation (e.g. in KRAS) it often needs to acquire additional ‘permissive’ genetic alterations to enable the cell to tolerate the oncogenic change. These ‘permissive' alterations represent an ‘acquired vulnerability' and consequently a novel therapeutic target in lung cancers driven by known, but currently un-targetable, driver mutations. Immortalized Human Bronchial Epithelial Cells (HBECs) represent a cell appropriate in vitro model to study the origin and pathogenesis of lung cancer following introduction of defined oncogenic changes. We have previously transformed HBECs to full malignancy by introducing five genetic manipulations (to CDK4, TERT, TP53, KRAS, MYC, BCL2, PTEN and/or STK11). While malignant transformation will be largely driven by the exogenous oncogenotype -i.e. the introduced oncogenic alterations- we hypothesize the cells also must undergo additional somatic ‘permissive' changes to bypass anti-tumor mechanisms. While lung cancers are often highly mutated (mainly due to carcinogens in cigarette smoke), manipulated HBECs should exhibit fewer somatic mutations, providing greater power to identify important somatically acquired mutations that facilitate tumorigenic progression of lung epithelial cells. We have sequenced the exomes of a series of 22 genetically manipulated HBECs derived from two individuals and representing 15 unique oncogenotypes (combinations of 3-5 introduced manipulations). Paired-end sequencing was performed with an Illumina HiSeq 2000 using a VCRome 2.1 liquid capture design ‘exome chip’. The median fold coverage was 114X, resulting in at least 90% of targeted bases are covered at >20-fold. Somatic mutations were called by comparing manipulated HBECs to their immortalized parental cell line. Manipulated HBECs had a median somatic mutation rate of 0.92 mutations/Mb, 7-9-fold lower than observed in lung cancers and tumor cell lines. The median mutation rate increased with the number of introduced manipulations (HBECs with three, four and five manipulations had 0.47, 0.97 and 0.92 mutations/Mb, respectively). Non-synonymous somatic mutations were identified in 470 genes. Thirty-nine genes were mutated in ≥3 HBECs (recurrent mutations) and of these, 35 genes (none currently regarded as ‘driver’ oncogenes and 5 potentially ‘druggable' [DGIdb]) were also mutated in primary lung adenocarcinoma and squamous cell carcinomas (TCGA data). We found the introduction of defined oncogenic alterations in HBECs is accompanied by the acquisition of somatic mutations. Leveraging against public somatic mutation data for lung tumors has allowed us to prioritize mutations for follow up to better identify potential acquired vulnerabilities in lung cancer cells and therefore, novel therapeutic targets. Citation Format: Jill E. Larsen, Caleb F. Davis, Kenneth Huffman, Luc Girard, David A. Wheeler, Richard A. Gibbs, John D. Minna. Exome sequencing to identify permissive mutations representing acquired vulnerabilities in lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1525. doi:10.1158/1538-7445.AM2014-1525