Figure 2. MNK kinases are frequently activated in MPNSTs, and genetic ablation triggers cell death when combined with MEK inhibitors.

(A) (Left) Immunoblot using a phospho-specific (S209) eIF4E antibody of lysates from normal human fibroblasts (IMR90) and MPNST cells (S462) and (Right) mouse MPNST cell lines (1A50 and 2629_C). (B) eIF4E phosphorylation levels in lysates from primary human MPNSTs. (C) Levels of eIF4E phosphorylation in primary mouse MPNSTs. (D) (Left) MNK1 and p-eIF4E levels following expression of shMKNK2, transfection with siRNA against MKNK1 (siMKNK1), or combined shMKNK2 expression and siMKNK1 transfection in S462 cells. (Right) Because existing MNK2 antibodies are not specific, mRNA levels of MKNK2 in shMKNK2-expressing S462 cells are shown relative to nontargeting controls and normalized to GAPDH (mean ± SD, n = 3). (E) (Top) Change in cell number of S462 expressing shCNT or shMKNK2 transfected with siMKNK1 or siCNT and treated with 750 nM PD901 or a vehicle control (DMSO). Graph represents the average log₂ of fold change in cell number 72 hours after treatment with PD901 relative to time 0 (mean ± SD, n = 3, 1-way ANOVA followed by Bonferroni’s multiple comparisons test). (Bottom) Levels of p-ERK in the corresponding cell lines following 24 hours of treatment with 750 nM PD901. Experiments repeated at least 3 times for validation.