(96g) Cell Signaling Perturbations Leading to Increased Response to EGFR Kinase Inhibitors

The majority of non-small-cell lung cancers involve elevated expression of the epidermal growth factor receptor (EGFR), but clinical response to EGFR kinase inhibitors is limited to the small fraction of tumors expressing recently identified EGFR mutants. Compared to WT EGFR counterparts, lung cancer cells with mutant EGFR display increased basal activity of the receptor, prolonged ligand-induced receptor phosphorylation, and altered downstream activation of several key signaling intermediates. We previously demonstrated that these effects result partially from impaired ligand-induced endocytosis of EGFR mutants. Here, we extend that analysis and demonstrate that EGFR mutation and the associated alterations to endocytosis impact specific downstream signaling pathways in fundamentally different ways. While the activities of some pathways are enhanced as a result of mutation, the activities of others are impaired. By over-expressing EGFR mutants in cell lines of interest and working with cell lines with an inducible switch for the expression of a key protein involved in EGFR endocytosis, we have demonstrated that the alterations observed in human lung cancer cell lines can be recapitulated in predictable ways and that they are tightly tied to receptor trafficking. In addition, RNAi-mediated knockdown of signaling intermediates impaired downstream of EGFR mutation in cells with WT EGFR increases cellular sensitivity to kinase inhibitors. Conversely, reconstitution of those pathways in kinase-inhibitor-sensitive cells reverses sensitivity. In total, our results demonstrate that the EGFR mutations under consideration create a quantitative bifurcation of downstream signaling pathways, and that the integrated effect of these signals determines cellular response to EGFR kinase inhibition.