(172a) ERK1/2-Driven Sprouty2 Expression Mediates Resistance to Receptor Tyrosine Kinase-Targeted Therapeutics in Glioblastoma

The efficacy of inhibitors of oncogenic kinases in glioblastoma multiforme (GBM) has been disappointing to date. In GBM and other malignancies, resistance to targeted therapeutics can arise through dynamic rewiring of cell signaling pathways responsible for tumorigenesis and cell survival. Here, we describe a signaling rewiring process in glioblastoma cells that promotes expression of Sprouty2 (SPRY2), which we recently identified as a driver of GBM tumor growth and resistance to kinase inhibitors. Specifically, we found that SPRY2 depletion reduced the ability of GBM cells to form colonies in soft agar, to form subcutaneous tumors in mice, and to resist co-inhibition of the EGFR and MET receptor tyrosine kinases. In the present study we found that, in a panel of GBM cell lines, SPRY2 expression was initially reduced in response to inhibition of EGFR and MET. However, 24-48 hours after receptor inhibition, resurgent SPRY2 expression was observed while EGFR and MET phosphorylation remained suppressed. Multivariate measurements of several kinases downstream of EGFR and MET revealed that phosphorylation of extracellular regulated kinase 1/2 (ERK1/2), which was initially suppressed in response to EGFR and MET inhibition, also displayed a resurgence that paralleled SPRY2 expression. This correlation between ERK phosphorylation and SPRY2 expression is consistent with the documented ability of ERK to regulate SPRY2 transcription. Antibody array analysis suggested that ERK reactivation is mediated by other RTKs activated in response to EGFR and MET inhibition. This analysis was supported further by measuring transcriptional differences in ligands responsible for activating these receptors. Suppression of the alternate RTKs’ activity through inhibitors or siRNA-mediated knockdown augmented cell death response to EGFR and MET inhibitors. In addition to population-level studies, this effect was demonstrated with single-cell resolution using a fluorescence-based reporter of ERK activity and RNA FISH. Thus, our data support a model wherein resurgent ERK phosphorylation and resultant SPRY2 expression occur due to the ability of glioblastoma cells to activate alternative RTKs, suggesting that targeting multiple RTKs could be a way to overcome resistance to single RTK inhibitor treatments. To explore this hypothesis in tumor xenograft models, we recently engineered a luminescence reporter of ERK activity that enables intravital imaging of ERK activity. Using this new tool, we have demonstrated the improved ability of specific combinations of RTK inhibitors to maintain ERK activity suppression within the tumor.