Abstract 5527: A panel of in vitro kinase assays developed using a novel detection technology to predict and monitor EGFR inhibitor efficacy.
Authors: Dickerson W. Matthew , Beausang Lee Anne , Saab Ashley , Leong Kristen , Alderman Edward 
Published: August 2014

The efficacy of kinase inhibitors, a highly studied class of drugs used to treat many forms of cancer, remains difficult to predict or tailor to individual specific carcinomas. Monitoring multiple biomarkers in the course of drug development may lead to more successful therapeutic design and patient treatment regimens. Here we discuss the generation and use of assays to measure a panel of kinase biomarkers using Acoustic Membrane Microparticle (AMMP) technology on the VIBE workstation to efficiently monitor both on- and off-target effects of kinase inhibitors directed against the EGFR family of tyrosine kinase receptors. These results may be used to better predict the roots of drug resistance.

Two cell lines were selected for use in this study on the basis of EGFR expression. The epidermoid carcinoma A431 cell line is a high expressor of the endothelial growth factor class of receptors and, therefore, subject to EGFR inhibition. This line was used to evaluate ‘on-target’ effects of the selected EGFR inhibitors. In contrast, the MCF-7 breast cancer cell line, a low expressor of EGFR class of receptors, should be much less susceptible to EGFR inhibition, and was used to better understand the ‘off-target’ effects of the EGFR inhibitors.

Cells were preincubated at reported IC50 concentrations of EGFR inhibitors gefitinib and neratinib, then stimulated by EGF, lysed, and assessed for cell surface receptor (EGFR and HER3) and cytosolic kinase (MEK, ERK, p38, JNK and AKT) activity. As expected, EGF stimulated a large phosphorylation event of EGFR, HER3, MEK and ERK in the A431 cell line, with little observable change for the MCF-7 cell line. A significant increase in the phosphorylation of p38 but little change in JNK was seen in both lines.

The phosphorylation of EGFR and HER3 in the A431 cells appeared unaffected by gefitinib, but completely inhibited by neratinib. Unexpectedly, no downstream inhibition of EGF-stimulated MEK or ERK phosphorylation was observed in the A431 cell line for either inhibitor. In contrast, EGF-induced p38 phosphorylation was significantly inhibited in both the A431 and the MCF-7 cell lines.

Here we have constructed a panel of assays that may be used to better predict biological activity, not only at the target site of a kinase drug, but also downstream of that site. This panel demonstrated that EGF was still able to activate both MEK and ERK despite failing to phosphorylate either the EGFR or HER3 receptors, revealing that EGF may be acting through other receptors. Consequently, blocking of this class of receptors may only be partially effective in preventing EGF-induced activity. Further, preventing the phosphorylation of the EGF class of receptors may not be sufficient to completely inhibit the ligand induced phosphorylation of the Mitogen Activated Kinase pathway.

Citation Format: W. Matthew Dickerson, Lee Anne Beausang, Ashley Saab, Kristen Leong, Edward Alderman. A panel of in vitro kinase assays developed using a novel detection technology to predict and monitor EGFR inhibitor efficacy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5527. doi:10.1158/1538-7445.AM2013-5527