Purpose: To identify new therapeutic targets for non-small cell lung cancer (NSCLC), we systematically searched two cancer cell line databases for sensitivity data on a broad range of drugs. We identified polo-like kinase 1 (PLK1) as the most promising target for further investigation based on a subset of sensitive NSCLC cell lines and inhibitors that were in advanced clinical development.;-;Experimental Design: To identify potential biomarkers of response of NSCLC to PLK1 inhibition and mechanisms of PLK1 inhibitor-induced apoptosis, integrated analysis of gene and protein expression, gene mutations, and drug sensitivity was performed using three PLK1 inhibitors (volasertib, BI2536, and GSK461364) with a large panel of NSCLC cell lines.;-;Results: The NSCLC cell lines had different sensitivities to PLK1 inhibition, with a minority demonstrating sensitivity to all three inhibitors. PLK1 inhibition led to G(2)-M arrest, but only treatment-sensitive cell lines underwent substantial apoptosis following PLK1 inhibition. NSCLC lines with high epithelial-mesenchymal transition (EMT) gene signature scores (mesenchymal cell lines) were more sensitive to PLK1 inhibition than epithelial lines (P < 0.02). Likewise, proteomic profiling demonstrated that E-cadherin expression was higher in the resistant cell lines than in the sensitive ones (P < 0.01). Induction of an epithelial phenotype by expression of the miRNA miR-200 increased cellular resistance to PLK1 inhibition. Also, KRAS mutation and alterations in the tight-junction, ErbB, and Rho signaling pathways correlated with drug response of NSCLC.;-;Conclusions: In this first reported large-scale integrated analysis of PLK1 inhibitor sensitivity, we demonstrated that EMT leads to PLK1 inhibition sensitivity of NSCLC cells. Our findings have important clinical implications for mesenchymal NSCLC, a significant subtype of the disease that is associated with resistance to currently approved targeted therapies. (C) 2015 AACR.