Abstract 205: Analysis of resistance to RTK inhibitors using a novel RTK multiplex assay

Abstract

Abstract A novel multiplex immunoassay was developed for examining acquired resistance to Receptor Tyrosine Kinase (RTK) inhibitors. RTKs are transmembrane proteins which act as receptors for growth factors, neurotrophic factors and other extracellular signaling molecules. These cell-surface kinases are activated by extracellular ligands leading to receptor dimerization and tyrosine phosphorylation at specific residues in the cytoplasmic tails to initiate RTK-mediated signal transduction. Of the >500 known protein kinases in the human genome there are approximately 60 RTKs. They are central components of cell signaling networks and play crucial roles in normal physiological processes and disease processes ranging from diabetes to cancer. Many RTKs, such as EGFR, HER2, c-Kit, PDGFR and VEGFR, have been used as targets for drug development and RTK-targeted therapies have illustrated the utility of these treatments for selected cancers. However, in many cases, compensatory RTK signaling enables cancer cells to acquire resistance to RTK inhibitors that selectively target a single RTK. For example, some EGFR and HER2 inhibitors have led to resistance and are associated with increased expression of IGF1R. In order to understand the mechanisms of resistance to RTK inhibitors, we have developed a bead-based multiplex immunoassay capable of simultaneously detecting phosphorylation of 18 different RTKs. First we demonstrate detection of RTK phosphorylation in response to growth factor stimulation using various cell lines. Next we examined the specificity of two inhibitors targeting EGFR and HER2. These inhibitors specifically reduced growth factor-stimulated phosphorylation of EGFR and HER2, without inhibiting the activation of other RTKs. Although we did not observe compensatory activation of other RTKs in response to the two EGFR and HER2 inhibitors we tested, this study demonstrates the feasibility of using this novel 18-plex RTK panel for examining the mechanism of resistance to single RTK inhibitors. In summary, the RTK multiplex panel allowed for simultaneous detection of multiple tyrosine phosphorylated RTKs in a specific, sensitive, and reproducible manner. Citation Format: Lu Chen, Anthony J. Saporita, Wen-Rong Lie, Reeti Maheshwari, Melissa Schluter, Jehangir Mistry, Joseph Hwang. Analysis of resistance to RTK inhibitors using a novel RTK multiplex assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 205.