Figure 7.

FLT3 WT and ITD mutant enhance activation of WT and mutant IDH1 through direct phosphorylation of Y391 and indirect phosphorylation of Y42 by JAK2 activation in primary AML cells. A, Endogenous IDH1 protein was immunoprecipitated from primary leukemia cells of representative AML patients (left panel) and AML PDX samples (right panel) with IDH1 R132H mutation. Tyrosine phosphorylation of Y42 and Y391 were detected by Western blotting. Peripheral blood samples from four healthy donors were included as controls. B, Endogenous IDH1 protein was immunoprecipitated from primary leukemia cells of representative AML patients with IDH1 WT. Tyrosine phosphorylation of Y42 and Y391 were detected by Western blotting. Peripheral blood samples from four healthy donors were included as controls. C, Primary leukemia cell lysates with IDH1 R132H/FLT3-ITD or IDH1 WT/FLT3 WT from AML patients (left and right panels, respectively) were treated with PTP1B, followed by endogenous IDH1 protein immunoprecipitation and IDH1 R132H and IDH1 WT enzyme activity assay, respectively (upper panels). Tyrosine phosphorylation of Y42 and Y391 of IDH1 was detected by Western blotting (middle panels). Dimeric and monomeric IDH1 protein levels were measured by Western blotting using native PAGE (lower panels). D, Primary leukemia cells from representative AML patients harboring IDH1 R132H and FLT3-ITD mutations were treated with quizartinib, followed by mutant IDH1 enzyme activity assay (upper) and Western blotting to detect dimers and monomers of IDH1 R132H (lower). E, Primary leukemia cells from representative mutant IDH1-expressing AML patients expressing FLT3 WT (left one) or FLT3-ITD (right three) were treated with quizartinib, followed by detection of cellular 2-HG levels by NMR analysis. F, Primary leukemia cells from representative IDH1 R132H AML patients with FLT3-ITD (left) and FLT3 WT (middle) were treated with quizartinib and/or AG120, followed by IDH1 R132H pull down. Right: Primary leukemia cells from representative IDH1 R132H AML patients with FLT3-ITD were treated with JAK2 inhibitor ruxolitinib or FLT3 inhibitor quizartinib, followed by IDH1 R132H immunoprecipitation. Y42 and Y391 phosphorylation of IDH1 R132H was detected by Western blotting. G, Primary leukemia cells from representative wild type IDH1 AML patients with FLT3-ITD (left) and FLT3 WT (middle) were treated with quizartinib, followed by IDH1 immunoprecipitation. Right: Primary leukemia cells from a wild type IDH1 AML patient with FLT3-ITD were treated with JAK2 inhibitor ruxolitinib or FLT3 inhibitor quizartinib, followed by IDH1 pull down. Y42 and Y391 phosphorylation of IDH1 was detected by Western blotting. H, IHC staining of phosphorylated IDH1 (p-Y42) and total IDH1 in normal or tumor tissues in diverse tumor tissue arrays. Relative intensities of p-IDH1 Y42/IDH1 were calculated. I, Working model (detailed description is included in Discussion).

The error bars represent mean values ±SD from three replicates of each sample except (H) (*: 0.01<p<0.05; **: 0.01<p<0.001; ***: p<0.001; ns: not significant); Data are mean ± SD; p values were obtained by a two-tailed Student’s test.