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. Author manuscript; available in PMC: 2022 Sep 16.
Published in final edited form as: Mol Cell. 2021 Jul 20;81(18):3833–3847.e11. doi: 10.1016/j.molcel.2021.06.027

Figure 1. Mutant IDH2 activity is restricted in human AML cells by FLT3 through controlling inhibitory K413-acetylation of mIDH2, which, however, is sufficient to produce comparable levels of 2-HG as cells expressing mIDH1.

Figure 1.

(A) Human primary AML cells expressing diverse IDH1 or IDH2 mutants were treated with or without FLT3 inhibitor quizartinib (200nM) for 72 hours, followed by NMR analysis to measure the intracellular levels of 2-HG.

(B) Human primary AML cells expressing IDH2 R140Q mutant were treated with or without FLT3 inhibitor quizartinib, followed by mIDH2 enzyme activity assay.

(C) Purified recombinant IDH2 R140Q protein was incubated with recombinant active form of rFLT3 in an in vitro kinase assay, followed by mIDH2 enzyme activity assay (upper); tyrosine phosphorylation of mIDH2 was detected by Western blotting (lower).

(D) Human primary AML cells expressing IDH2 R140Q mutant were treated with or without deacetylase inhibitors NAM or TSA (left panels) or mitochondrial acetyltransferase ACAT1 inhibitor arecoline hydrobromide (AH; right panels), followed by mIDH2 enzyme activity assay; lysine acetylation of mIDH2 was detected by Western blotting (lower panels).

(E) Purified IDH2 R140Q protein was pre-treated with cobB protein deacetylase to remove intrinsic lysine acetylation obtained in bacteria prior to treatment with recombinant acetyltransferases including mitochondrial rACAT1 or cytosolic rACAT2 or rDLAT, followed by mutant IDH2 catalytic activity assay (left) and Western blot to detect lysine acetylation (right).

(F) Purified IDH2 R140Q variants with representative individual K⭢R mutation were incubated with acetyltransferase rACAT1, followed by mIDH2 catalytic activity assay (upper) and Western blot to detect lysine acetylation (lower).

(G) Human primary AML cells expressing IDH2 R140Q mutant were treated with or without FLT3 inhibitor quizartinib, followed by Western blot to detect phosphorylation of ACAT1 (Y407) and mIDH2, and K413-acetylation of mIDH2 by a specific acetyl-IDH2 antibody (K413-Ac).

The error bars represent mean values ±SD from three replicates of each sample (*: 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 t-test.