Figure 4 from Combining Brigatinib with mTOR Inhibition to Effectively Treat NF2-SWN–Associated and Sporadic NF2-Deficient Meningiomas

The brigatinib + INK128 combination elicited a superior and more durable suppression of AKT and 4EBP1 phosphorylation compared with the individual drugs. A, AG-NF2-Men cells were treated for 1 and 3 days with DMSO vehicle or 1 x IC50 dose of brigatinib (1.1 μmol/L) or INK128 (20 nmol/L) as single agents or in combination. Treated cell lysates were subjected to Western blot analysis for total and phosphorylated AKT and the mTORC1 target and protein translation repressor 4EBP1. GAPDH served as the loading control. B–D, AG-NF2-Men cells were serum-starved for 24 hours to induce quiescence and then treated with brigatinib, INK128, or brigatinib + INK128 in serum-free medium for 2 hours, followed by growth stimulation for 5 minutes with 50 ng/mL EGF (B), Hrg (C), or IGF-1 (D). Lysates from these cells were analyzed by Western blotting for ligand-mediated phosphorylation of their cognate RTKs and downstream phosphorylation and activation of AKT/PRAS40 and ERK1/2. GAPDH serves as the loading control. kD, kilodalton of molecular weight.