Identification of GCK as a functionally relevant target of GNF-7. OCI-AML3 cells (NRAS mutant) were treated with DMSO, 0.3 μM or 1 μM of GNF-7, or 1.0 μM of YHJ0557 for 2 hours and in situ kinase profiling by Kinativ method was performed as described in “Methods”. (A) Shown are the leading targets as revealed by the in situ kinase selectivity profiling. The difference between GNF-7 (1.0 μM) and inactive YHJ0557 in terms of inhibitory activity displayed against each target kinase was calculated by subtraction. (B) Venn diagram showing 3 groups of target kinases. a. Kinases inhibited more than 85% by 0.3 μM of GNF-7. b. Kinases inhibited more than 95% by 1.0 μM of GNF-7. c. Kinases that lost inhibitory activity more than 50% compared with group b after treatment with 1.0 μM of YHJ0557. (C) Proliferation of MK-2206-treated Ba/F3-NRAS-G12D cells in the absence or presence of IL-3 after KD of Gck by shRNA. Data are represented as mean ± SEM. (D) Proliferation of Ba/F3-NRAS-G12D cells treated with MK-2206 and the MAP4K2 inhibitor, NG25, in the absence or presence of IL-3. Data are represented as mean ± standard error of the mean. (E) Effect of overexpression of GCK-G96V in Ba/F3-NRAS-G12D on phosphor JNK, which is downstream of GCK, and phospho p38 as assessed by immunoblotting. (F) Rescue of growth suppression of MK-2206- and NG25-treated Ba/F3-NRAS-G12D cells by overexpression of GCK-G96V. Data are represented as mean ± SEM. Lys1, conserved Lysine 1; Lys2, conserved Lysine 2; activation loop, activation loop near DFG (Asp-Phe-Gly) motif peptide sequences detected are shown in supplemental Figure 3A. *P < .05; **P < .01; ***P < .001.