Abstract B276: A live-cell apoptosis assay identifies novel combination approaches that selectively kill EGFR mutant NSCLC cells.

Abstract

Abstract Introduction: Non-small cell lung cancer (NSCLC) patients with EGFR mutation positive (EGFR M+) tumours initially respond well to EGFR targeted monotherapy (e.g. gefitinib), but the benefit is usually limited due to the emergence of drug resistance. A major challenge in the field is to identify the most promising combination approaches that inhibit or prevent this resistance and then to prioritise these options for clinical testing in order to produce more durable patient benefits. Our aim in this study was to identify compounds that selectively and rapidly induced cell death but only when combined with gefitinib in EGFR M+ cells. Methods: We developed a high-throughput 96-well live-cell phenotypic assay (based on an acute apoptosis end point) to assess the combination potential of probe inhibitors targeting kinases (n=402), as well as compounds that are approved or in clinical development for the treatment of cancer (n=326). Screens were carried out using EGFR M+ (PC-9, HCC-827, HCC-2935, H1975) and EGFR WT (A549, H1993) NSCLC cells, and a non-tumor immortalized lung epithelial cell line (BEAS2B). Cells were treated with gefitinib (0-1 µM) and test compounds (0-10 µM) for 18 h. Hoechst 33258 (25 µM) was added and 30 min later cells with condensed nuclei and a high nuclear intensity were scored as apoptotic, and the percentage of apoptotic cells was calculated. SYTOX green DNA staining confirmed that apoptotic cells were scored positive using our simple Hoechst staining method. Results: The screening assay was robust and reproducible with Pearson correlation coefficients ranging from 0.66-0.92 when comparing inter-assay apoptosis scores. Using the compound library we identified 21 positive hits (Z-score≥10) that selectively induced apoptosis in the presence of gefitinib in EGFR M+ cells, but not in EGFR WT cells. Positive hits included compounds in four major target categories; BCL inhibitors (n=3), anti-mitotic agents (n=11), HDAC inhibitors (n=4), and AKT/PI3K inhibitors (n=5). Several of these targets have been suggested in the literature using different methodologies to our own, thereby validating our overall screening approach. Importantly, we have also identified several previously unreported combinations that induced marked apoptosis within 18 h of gefitinib treatment, and which we are currently evaluating in in vivo studies. In addition, we observed that combinations of EGFR TKIs with gefitinib were antagonistic in EGFR M+ cells. Finally, in the probe inhibitor library set, we have identified 20 diverse chemical structures that are strongly positive in our combination assay, suggesting new targets for selective killing of EGFR M+ NSCLC cells. Unexpectedly, screening the chemical probe library also identified 7 compounds that selectively induced cell death in combination with gefitinib in EGFR wild-type KRAS mutant A549 cells. Conclusion: We have developed a robust and reproducible high-throughput live cell apoptosis assay to screen compound libraries in combination with gefitinib in NSCLC cells. We have identified several novel combinations that are highly selective for EGFR M+ cells in the presence of gefitinib. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B276. Citation Format: Sivan M. Bokobza, Aoife M. Devery, Anika M. Weber, Anderson J. Ryan. A live-cell apoptosis assay identifies novel combination approaches that selectively kill EGFR mutant NSCLC cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B276.