PMC

A: Summary of phosphoproteomic data from the mass spectrometry based pY analysis. Hierarchical clustering using the Pearson correlation lead to a clear separation of BRAF- and NRAS-mutant melanoma cell lines, Pearson correlation between BRAF- and NRAS-mutant melanoma cell lines shown on heatmap (deeper clustering not shown). B: Western blot showing expression of amuvatinib targets in BRAF- and NRAS-mutant melanoma cell lines. C: Amuvatinib has growth inhibitory effects in NRAS but not BRAF-mutant melanoma cell lines. Melanoma cell lines were treated with increasing concentrations of amuvatinib for 72 hrs before being analyzed by the MTT assay. D: Amuvatinib (3 µM, 72 hrs), leads to decreased pAXL, pAKT and pERK expression by Western blot.

A: Cell cycle effects of amuvatinib upon WM1366 NRAS-mutant melanoma cells. 72 hr treatment with amuvatinib leads to a decrease in G1-phase cells and an increase in G2/M and S-phase. B: Amuvatinib induces apoptosis in NRAS-mutant but not BRAF-mutant melanoma cell lines. Cells were treated with amuvatinib (3 µM, 72 hrs) before being analyzed for Annexin-V binding/loss of TMRM by flow cytometry. C: Amuvatinib is cytotoxic in a 3D collagen implanted spheroid model of NRAS-mutant melanoma. Spheroids from WM1366 and WM1346 melanoma cells were treated with amuvatinib for 7 days before being stained with calcein-AM (green, live cells) and propidium iodide (red, dead cells). D: Amuvatinib leads to long-term growth suppression of NRAS-mutant melanoma cells. Data shows colony formation assays performed over 14 days.