Effect of GNF-7 on the RAS signaling pathways, and identification of AKT signaling as a functional target. (A) Ba/F3-NRAS-G12D cells were treated with various concentrations of GNF-7 for 2 hours. Changes in phosphorylation of major kinases mediating the RAS signaling pathway were investigated by immunoblot analysis. (B) IC50 of GNF-7 tested against Ba/F3-NRAS-G12D cells and Ba/F3-NRAS-myrAKT cells. MK-2206, an allosteric AKT inhibitor, was also tested against Ba/F3-NRAS-G12D cells. Data are represented as mean ± standard error of the mean (SEM). (C) In vitro kinase assays for kinases related to the RAS signaling pathway were performed as described in “Methods”. Residual kinase activity after treatment with 1.0 μM of GNF-7 of each kinase is shown. Data are represented as mean ± SEM. (D) Ba/F3-NRAS-G12D cells were treated with indicated concentrations of AIM-100 and change of phosphorylation of AKT was immunoblotted. (E) Validation of the KD efficiency of ACK1 by shRNAs and its effect on phospho-AKT. (F) Proliferation of AML cell lines characterized by KD of ACK1 by shRNA. Data are represented as mean ± SEM.
