GNAQ/11 Mutant Dependent Uveal Melanoma Sensitivity to MEK and PI3K Inhibitors

Abstract

Purpose: : Activating GNAQ or GNA11 mutations have been identified in approximately 85% of uveal melanoma tissues. The MEK/MAPK and PI3K/AKT pathways mediate GNAQ and GNA11 growth and survival signaling under normal conditions. The purpose of this study was to test the sensitivity of uveal melanoma cell lines to combination treatment with clinically relevant MEK and PI3K small molecule inhibitors.

Methods: : The GNAQ/11 mutant (MUT) and wild-type (WT) status of eight uveal melanoma cell lines was determined using standard sequencing approaches. Concentration and time-course western blot experiments were used to determine the optimal conditions for MEK inhibitor (MEKi) or PI3K inhibitor (PI3Ki) treatment using GSK1120212 and GSK2126458, respectively. Cell proliferation assays were performed to determine the relative sensitivity of MUT vs. WT cells to MEKi or PI3Ki. Cell cycle analysis was performed to determine the relative percentages of cells in each stage before and after MEKi and/or PI3Ki treatment. Annexin/Propidium-iodide staining was determined by flow cytometry to assess the frequency of apoptotic cells before and after MEKi and/or PI3Ki treatment.

Results: : MEKi treatment resulted in early and sustained loss of MAPK phosphorylation beginning at 10 nM and PI3Ki treatment resulted in early and sustained loss of AKT phosphorylation beginning at 1 nM in both MUT and WT uveal melanoma cell lines. Both MEKi and PI3Ki treatment induced G1 cell cycle arrest in both MUT and WT cells. However, neither MEKi nor PI3Ki treatment induced apoptotic cell death in MUT or WT cells. The combination of MEKi + PI3Ki also failed to induce apoptotic death in WT cells. Conversely, the combination of MEKi + PI3Ki induced a marked increase (40-60% of cells) in the subG0 cellular fraction of MUT cells consistent with apoptotic death at 48 hrs. In addition Annexin V staining was markedly increased MUT cells after combination treatment.

Conclusions: : These results suggest that uveal melanoma cells with driver mutations in GNAQ/11 are highly dependent on the MEK/MAPK and PI3K/AKT pathways for growth and survival. Combination MEKi + PI3Ki small molecule treatment may be an effective therapeutic strategy for the majority of metastatic uveal melanoma patients.

Keywords: melanoma • signal transduction: pharmacology/physiology • genetics