Fig. 4: The regulatory role of NRAS in kinase activity signatures reveals dual treatment regimen with synergistic potential. | Communications Medicine

Fig. 4: The regulatory role of NRAS in kinase activity signatures reveals dual treatment regimen with synergistic potential.

From: Suppression of NRAS-mutant melanoma growth with NRAS-targeting Antisense Oligonucleotide treatment reveals therapeutically relevant kinase co-dependencies

Fig. 4

a Schematic illustration of HT-KAM analysis of the phosphor-catalytic activity of kinases. D04 and MM415 cells were either treated with NRAS or Control ASOs (50 nM, 1 day). Cells were lysed, and protein lysate was investigated for peptide-associated phosphorylation activity of kinases. b Comparison of kinase activity in treatment groups (NRAS ASO VS. Control ASO) showed that kinase activity of several kinases was significantly upregulated in the D04 and MM415 cell lines upon NRAS ASO treatment. Kinases are ranked by their relative increase of activity (from bottom to top). The top 3 kinases with strongest shift in activity increase are MAP2K1 (MEK1), FGFR2, and CDK4. The RET kinase activity shift is shown as a representative example for kinases that were downregulated in activity. c QRT-PCR analysis showing elevated NRAS-mRNA levels in D04 and MM415 cells after three days of drug-induced Inhibition of MEK (MEKi), using the small molecule inhibitor Trametinib (20 nM or 40 nM), when compared to control, treated with DMSO (n = 3). d QRT-PCR analysis showing elevated NRAS-mRNA levels in the MEKi resistant cell lines D04RM and MM415RM, which were chronically exposed to Trametinib, when compared to their paternal treatment naïve cell lines D04 and MM415 (n = 3). Error bars in panel (c, d) represent s.e.m. e Treatment with NRAS ASO-1 caused significant inhibition of cell growth in the MEKi resistant NRAS mutant melanoma cell lines D04RM (p = 0.011), MM415RM (p = 0.001), WM3629RM (p = 0.0002), and Sk-Mel-2RM (p = 0.015). Data were normalized to treatment with non-targeting Control ASO; treatment period was 5 days, final oligonucleotide concentration was 50 nM, and error bars represent s.d. (n = 3). f–i Dual treatment with 20 nM of NRAS ASO and Trametinib (Tram, 0.5 nM −25 nM) caused robust synergistic effects in D04 (f, g) and MM415 (h, i) cells after 3 (f, h) and 5 (g, i) days of treatment (n = 2). Dose response curves show NRAS ASO treatment (blue), trametinib treatment (yellow) and dual treatment (red). Synergism of dual cell growth inhibition is shown as bar graphs and determined by the HSA synergy score.

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