Figure 1. Development of acquired trametinib resistant HNSCC models.

(A) Seven HNSCC lines were treated with various concentrations of trametinib for 96 hours. Cell Titer Glo cell metabolic assay IC50 and maximal growth inhibition results are shown. Data shown are representative of at least three independent experiments. (B) Immunoblot analysis of YAP1, p-Erk1/2 and total Erk levels in seven HNSCC cell lines treated with vehicle control or 100 nM trametinib for 24 hours (Upper panel). Fold-change in the ratio of p-Erk1/2 to total Erk with trametinib was quantified and normalized for each cell line to the ratio of p-Erk1/2 to total Erk with vehicle control treatment. Graphed data were obtained from the quantification of three independent immunoblots. Normalized p-Erk1/2 level following trametinib treatment was compared to vehicle treatment level for each cell line (n=3, **** p<0.0001, ***<0.0005, and ** p<0.01, unpaired t-test) (C) Immunoblot analysis of CAL27, HSC3 and SCC25 parental for p-Erk1/2 and total Erk1/2 with increasing trametinib concentrations. The figure is representative of three independent experiments. (D) Cell ATP concentration, measured by Cell Titer Glo, IC50 and maximal inhibition for HSC3-C and HSC3-TR (left panel) and CAL27-C, and CAL-27 TR (right panel) (n=3). Cell lines were treated with increasing concentrations of drug for 96 hours. Treatment with increasing concentration of trametinib over time led to the emergence of trametinib-resistant HSC3 (HSC3-TR) and CAL27 (CAL27-TR) with indicated IC50s. (E) Cell doubling times did not differ between HSC3-C and HSC3-TR nor between CAL27-C and CAL27-TR.