Abstract A182: Identifying biomarkers of sensitivity to polo-like kinase inhibitors (Plki) in non small cell lung cancer (NSCLC).
Authors: Ferrarotto Renata , Suryavanshi Manasi , Young Yoo Suk , Wang Jing , Byers Lauren , Glisson Bonnie S. , Heymach John , Johnson Faye M. 
Published: August 2014

Abstract
Background: NSCLC is a deadly disease. Targeted therapy is very effective; however, only ∼ 20% of patients are candidates for this approach. In a search for new therapeutic targets in NSCLC, we have identified Polo like kinase (Plk). Plk1 regulates centrosomes at the G2/M transition and orchestrates cell cycle progression. Clinical trials have shown that a subgroup of refractory NSCLC patients responded to Plki as single agents. Our aim is to validate and identify biomarkers that predict sensitivity to Plki.
Methods: To identify therapeutic targets, we evaluated 2 databases with 123 NSCLC cell lines tested with 130 drugs incorporating genomic and gene expression data seeking agents that were markedly effective in a subgroup of cell lines. To identify association between sensitivity to Plki and gene expression, we acquired the datasets from the databases and tested the correlation between IC50 and gene expression. Spearman rank was used to test the association between mutation in key genes and IC50. MTT assay was used to test drug sensitivity in NSCLC cell lines in our laboratory. Cell cycle was evaluated by flow cytometry using BrdU incorporation. Immunofluorescence with anti-phospho-H2yAX antibody was used to identify mitotic catastrophe.
Results: In the databases, 12/19 NSCLC lines had IC50 &lt; Cmax of 1.6 µM for the PLKi BI2536 and 6/19 had IC50 &lt; 30 nM. The correlation analysis found mRNA overexpression of 20 genes to be significantly associated with sensitivity to both Plki. Based upon the likelihood of their expression influencing Plk1 function, 4 genes were selected for further study: CEP250, which encodes a core centrosomal protein, and TGFA, RHBDF1 and FOSL2, which are involved with epithelial cell proliferation. KRAS mutation was also correlated with sensitivity to Plki. Our preliminary cell line screening found that 10/37 NSCLC cell lines are exquisitely sensitive to BI2536 with IC70 &lt; 40 nM. The Plki's BI2536 and BI6727 cause complete Plk substrate (TCTP) inhibition with 25 and 100 nM respectively at 12 h as measured by Western blotting. However, in a resistant cell line, TCTP reactivation was observed following 24 h Plk inhibition, while in a sensitive cell line, there was sustained TCTP inhibition up to 120 h. Cell cycle analysis of sensitive cell lines showed a significant arrest in G2/M. Drug treatment caused mitotic catastrophe in sensitive lines.
Conclusion: Our statistical analysis utilizing data from 2 databases demonstrated an association between CEP250, TGFA, RHBDF1 and FOSL2 overexpression and KRAS mutation and sensitivity to Plki. These results are being validated in a larger cell line screen that may identify additional biomarkers. Substrate reactivation was observed in one resistant cell line following 24h of Plk inhibition, demonstrating one potential mechanism for resistance. Screening of additional cell lines and mechanistic studies are ongoing to extend these findings.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A182.
Citation Format: Renata Ferrarotto, Manasi Suryavanshi, Suk Young Yoo, Jing Wang, Lauren Byers, Bonnie S. Glisson, John Heymach, Faye M. Johnson. Identifying biomarkers of sensitivity to polo-like kinase inhibitors (Plki) in non small cell lung cancer (NSCLC). [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 A182.