Extended Data Fig. 5: Second-site IDH2 mutations in cis can confer resistance to enasidenib.
From: Acquired resistance to IDH inhibition through trans or cis dimer-interface mutations
a, Western blot showing IDH2 expression in Ba/F3 cells transduced with the indicated constructs. Vinculin serves as a loading control. The same membrane was probed for both IDH2 and vinculin. These are the same cells as in Fig. 4a. b–g, Purification and enzymatic activity of IDH2 WT–R140Q dimers with or without in cis second-site mutations. b, Schematic of experimental approach: 293T cells were co-transfected with HA-tagged wild-type IDH2 plus Flag-tagged IDH2(R140Q), in cis double-mutant IDH2 R140Q/Q316E (RQ/QE) or in cis double-mutant IDH2 R140Q/I319M (RQ/IM). After 2 days, cells were lysed and IDH2 enzyme complexes were purified by HA-immunoprecipitation. c–e, Purity and dimerization of HA-precipitated enzymes were assessed by denatured SDS–PAGE with Coomassie staining (c), denatured SDS–PAGE with western blotting with the indicated antibodies (d), or native PAGE with Coomassie staining (e). Separate membranes were used for Western blots. f, g, In vitro enzyme assays measuring relative activity (f) and rate of NADPH consumption (g) by HA-precipitated IDH2 dimers. Reactions contained purified enzyme (7.5 μg ml−1), NADPH (0.3 mM), αKG (5 mM), and vehicle or increasing doses of AG-221 (0.1, 0.3, 1, 3, 10 or 30 μM). Data are mean ± 95% confidence interval for triplicate reactions (duplicate reactions for WT–RQ/QE AG-221 3 μM and 30 μM). Results are representative of ≥3 independent experiments. For gel source data, see Supplementary Fig. 3.
