Fig. 4.

eEF2K silencing by pharmacological or genetic means reverses the antiproliferative effects of PI3Ki plus MEKi cotreatment in cell models that responded synergistically to cotreatment. A, scheme applied for NTERA2 cells. The signaling pathways PI3K/mTOR and MAPK negatively regulate eEF2K, thereby allowing for eEF2 activity to promote protein translation. With the individual addition of MEKi or PI3Ki into the cell culture, eEF2K still remains inactive, facilitating translation elongation. Only the combined therapy (B) leads to a full eEF2K activation and further eEF2 phosphorylation, slowing down the elongation stage of protein synthesis. B, phosphorylation of pathway activity markers in cells pretreated with an eEF2Ki for 24 h or transfected with nontarget siRNA or siRNA against eEF2K for 3 days. C and D, reduction in cell numbers (measured using a Guava assay) in cells in which eEF2K was silenced as in (A) and (B), followed by treatment with PI3Ki or MEKi individually or in combination for further 3 days at the concentrations shown. Values are mean ± SEM (n = 3 independent experiments). Statistical significance was calculated by two-way ANOVA, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. DMSO, dimethyl sulfoxide; eEF2, eukaryotic elongation factor 2; eEF2K, eukaryotic elongation factor 2 kinase; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin.