The efficacy of molecularly targeted therapeutics in cancer is limited by the ability of cancer cells to rewire downstream cell signaling pathways for survival. Here, we describe a signaling rewiring process we observed in glioblastoma cells. In response to inhibitors of epidermal growth factor receptor (EGFR) and MET (the receptor for hepatocyte growth factor), the extracellular regulated kinase (ERK) pathway was initially inhibited (for up to eight hours), but subsequently reactivated due to bypass signaling driven by fibroblast growth factor receptor (FGFR). ERK reactivation rescued cells from death by driving the expression of Sprouty2 (SPRY2), a protein we recently identified as a tumor promoter in glioblastoma. To enable the real-time investigation of this mechanism in vivo without sacrificing animals at multiple time points, we developed a novel bioluminescence-based reporter of ERK activity. The reporter design borrows from a previously described fluorescence-based reporter of ERK activity and includes click beetle luciferase fused to a domain of FRA1 (a substrate of ERK) and a nuclear localization sequence. Phosphorylation of the FRA1 domain by ERK slows turnover and therefore stabilizes fusion protein expression, creating an ERK activity-dependent luminescence signal. Using this reporter, we demonstrated that the combination of inhibitors against EGFR, MET, and FGFR more durably suppressed ERK activity in glioblastoma xenograft tumors than the combination of EGFR and MET inhibitors alone. Enhanced ERK suppression correlated with reduced SPRY2 expression and smaller tumors, demonstrating the in vivo relevance of the mechanism we first identified using in vitro cell culture experiments. In addition to new understanding of tumor biology, our study has yielded a new technical advance. For in vivo studies of the activity of ERK, one of the most commonly dysregulated pathways in cancer, our reporter enables real-time quantification during longitudinal studies, obviating the need for sacrificing animals at multiple time points and drastically reducing the total number of animals needed per study. This method also eliminates concerns about artifacts introduced into measurements of pathway activity arising due to ex vivo manipulation of tumors.

Engineering a bioluminescence-based protein kinase reporter for in vivo, longitudinal studies of receptor tyrosine kinase inhibitor response

2019 Spring National Meeting

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