Abstract B28: Mitogen-activated protein kinase-driven Sprouty2 expression mediates resistance to receptor tyrosine kinase-targeted therapeutics in glioblastoma cells

The therapeutic efficacy of inhibitors of oncogenic kinases in glioblastoma multiforme (GBM) has been disappointing to date. In glioblastoma and other malignancies, resistance to targeted therapeutics can arise through dynamic rewiring of cell signaling pathways responsible for tumorigenesis and survival. Here, we identify one such signaling rewiring process in glioblastoma cells that promotes expression of Sprouty2 (SPRY2). SPRY2 expression is of interest because we recently identified SPRY2 as a driver of glioblastoma cell and tumor proliferation and resistance to kinase inhibitors. Specifically, we found that SPRY2 depletion reduced the ability of glioblastoma 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 glioblastoma cell lines, SPRY2 expression was initially reduced in response to inhibition of EGFR and MET. However, at later time points (24-48 hr after receptor inhibition) resurgent SPRY2 expression was observed even as EGFR and MET phosphorylation remained suppressed. To identify regulators responsible for resurgent SPRY2 expression, we undertook a quantitative systems biology approach based on partial least squares regression (PLSR) modeling wherein the phosphorylation states of multiple signaling pathways known to be regulated by EGFR and MET were measured at six time points during the 48 hours after treatment of cells with EGFR and MET inhibitors. The PLSR model identified late stage (48 hr) phosphorylation of extracellular signal-regulated kinase (ERK) as having a role in cellular resistance to the inhibitors distinct from ERK phosphorylation at all other times. Inspection of the experimental data revealed that phosphorylated ERK, which was initially suppressed in response to EGFR and MET inhibition, also displayed a resurgence that paralleled the trend in SPRY2 expression. This correlation between ERK phosphorylation and SPRY2 expression is consistent with the documented ability of ERK to regulate SPRY2 expression transcriptionally. The importance of resurgent ERK phosphorylation was confirmed in experiments showing that its suppression through the use of a MEK inhibitor prevented resurgent SPRY2 expression and augmented cell death in response to EGFR and MET inhibitors. We hypothesize that resurgent ERK phosphorylation and resultant SPRY2 expression occur due to the ability of glioblastoma cells to activate receptor tyrosine kinases other than EGFR and MET in response to inhibition of those receptors. Antibody microarray experiments have provided some initial clues about the identity of those receptors, and experiments are ongoing to validate those results. Citation Format: Evan K. Day, Matthew J. Lazzara. Mitogen-activated protein kinase-driven Sprouty2 expression mediates resistance to receptor tyrosine kinase-targeted therapeutics in glioblastoma cells. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B28.