Abstract
Somatic mutations in the isocitrate dehydrogenase 2 gene (IDH2) contribute to the pathogenesis of acute myeloid leukaemia (AML) through the production of the oncometabolite 2-hydroxyglutarate (2HG)1,2,3,4,5,6,7,8. Enasidenib (AG-221) is an allosteric inhibitor that binds to the IDH2 dimer interface and blocks the production of 2HG by IDH2 mutants9,10. In a phase I/II clinical trial, enasidenib inhibited the production of 2HG and induced clinical responses in relapsed or refractory IDH2-mutant AML11. Here we describe two patients with IDH2-mutant AML who had a clinical response to enasidenib followed by clinical resistance, disease progression, and a recurrent increase in circulating levels of 2HG. We show that therapeutic resistance is associated with the emergence of second-site IDH2 mutations in trans, such that the resistance mutations occurred in the IDH2 allele without the neomorphic R140Q mutation. The in trans mutations occurred at glutamine 316 (Q316E) and isoleucine 319 (I319M), which are at the interface where enasidenib binds to the IDH2 dimer. The expression of either of these mutant disease alleles alone did not induce the production of 2HG; however, the expression of the Q316E or I319M mutation together with the R140Q mutation in trans allowed 2HG production that was resistant to inhibition by enasidenib. Biochemical studies predicted that resistance to allosteric IDH inhibitors could also occur via IDH dimer-interface mutations in cis, which was confirmed in a patient with acquired resistance to the IDH1 inhibitor ivosidenib (AG-120). Our observations uncover a mechanism of acquired resistance to a targeted therapy and underscore the importance of 2HG production in the pathogenesis of IDH-mutant malignancies.
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Acknowledgements
We thank members of the Levine and Thompson laboratories for discussions. We thank W.K. Chatila and N. Schultz for assistance with data deposition. A.M.I. is supported by the NIH/NCI (K08 CA201483), Leukemia & Lymphoma Society (3356-16), Burroughs Wellcome Fund (1015584), Susan & Peter Solomon Divisional Genomics Program, Steven A. Greenberg Fund, and Cycle for Survival. A.H.S. is supported by the NIH/NCI (K08 CA181507) and Leukemia & Lymphoma Society. The work was also supported, in part, by the Conquer Cancer Foundation of ASCO (A.M.I., A.H.S. and J.T.), the Leukemia & Lymphoma Society Specialized Center of Research Program (7011-16; A.M.I. and C.B.T.), a Translational and Integrative Medicine Research Fund (TIMRF) grant (A.H.S. and E.M.S.), the American Association for Cancer Research (J.T.), the American Society of Hematology/Robert Woods Johnson Foundation (J.T.), and grants from the NIH, including R01 CA168802-02 (C.B.T.), R35 CA197594-01A1 (R.L.L.), U54 OD020355 (R.L.L.), and the Memorial Sloan Kettering Cancer Center Support Grant (NIH P30 CA008748) including a supplement to R.L.L., C.B.T. and A.H.S. We acknowledge the use of the Integrated Genomics Operation Core, funded by the Memorial Sloan Kettering Cancer Center Support Grant (NIH P30 CA008748), Cycle for Survival, and the Marie-Josée and Henry R. Kravis Center for Molecular Oncology.
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Nature thanks J. Cortes and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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Contributions
A.M.I., A.H.S., C.B.T., R.L.L. and E.M.S. conceived the project, designed the experiments, analysed the data, and wrote the manuscript. A.M.I., A.H.S., B.W. and A.N. performed the experiments with technical assistance from F.M.C., N.T., V.D., H.L. and J.R.C. M.P., C.F., J.T. and M.S.T. assisted with management of clinical data and specimens. M.E.A. and M.R. assisted with pathological assessment of biospecimens. A.S.R., S.K.A., G.A.P. and J.D.C. performed the structural modelling. N.F. and E.P. performed the mutational analysis. B.W., S.C., Z.D.K. and S.A.B. assisted with identification and analysis of the IDH1-mutant leukaemia. All authors read and approved the manuscript.
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C.B.T. is a founder of Agios Pharmaceuticals and a member of its scientific advisory board. He also serves on the board of directors of Merck and Charles River Laboratories. R.L.L. is on the Supervisory Board of Qiagen. J.D.C. is a member of the scientific advisory board of Schrödinger. B.W., S.C., Z.D.K. and S.A.B. are employees of Agios Pharmaceuticals, Inc.
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Extended data figures and tables
Extended Data Fig. 1 Acquired clinical resistance to the mutant IDH2 inhibitor enasidenib (AG-221).
a, b, Haematoxylin and eosin staining of bone marrow cells aspirated from patient A (a) and patient B (b) at indicated points in relation to treatment with AG-221. Remission images demonstrate decreased leukaemic blasts and increased myeloid differentiation that are reversed at the time of relapse. Images show 100× magnification. Images are representative fields of a single bone marrow aspiration performed at each time point.
Extended Data Fig. 2 Structures illustrating potential interactions between IDH2 second-site mutations and enasidenib.
a–h, Detailed view of the interactions between wild-type IDH2 Q316 (a, e) and Q316’ (c, g) or mutant IDH2(Q316E) (b, f) and IDH2(Q316E’) (d, h) with AG-221 in the predicted dominant conformation (a–d) or a minor conformation (e–h). Hydrogen bonds are depicted in light green. Note the disrupted hydrogen bond (depicted as orange bar) in d resulting from the Q316E mutation in the IDH2’ subunit. i–p, Detailed view of the interactions between wild-type IDH2 I319 (i, m) and I319’ (k, o) or mutant IDH2(I319M) (j, n) and IDH2(I319M’) (l, p) with AG-221 in the predicted dominant conformation (i–l) or a predicted minor conformation (m–p). The solvent-excluded surface of AG-221 is shown transparently in grey. The van der Waals radius of the Cδ1 and Cγ2 atoms of I319/I319’ or the Sδ and Cε atoms of I319M/I319M’ are depicted as spheres. Unfavourable steric interactions between AG-221 and these atoms are depicted in red. Throughout the figure, the IDH2 subunit is depicted in blue-grey, the IDH2’ subunit in purple, and AG-221 in teal. Non-polar hydrogen atoms are not shown. White, red, blue and yellow portions of stick structures indicate hydrogen, oxygen, nitrogen and sulfur atoms, respectively. All models were based on the AG-221–IDH2 structure (PDB code 5I96)9 (see Methods).
Extended Data Fig. 3 Expression and activity of in trans IDH2 second-site mutations in haematopoietic cells.
a, Allele-specific quantitative PCR (qPCR) showing similar expression of constructs in Ba/F3 cells co-transduced with IDH2R140Q (RQ) plus IDH2WT (WT), IDH2Q316E (QE), or IDH2I319M (IM) in trans. Control from IDH2WT human cell line (293T). Data are mean ± s.e.m. for triplicate reactions. b, Intracellular 2HG levels in Ba/F3 cells co-expressing RQ plus WT, QE or IM in trans and treated with vehicle or increasing doses of AG-221 (1 nM, 10 nM, 100 nM, 1 μM or 10 μM). Data are mean ± s.e.m. for triplicate cultures. c, Western blot showing IDH2 protein levels in primary HSPCs from Idh2R140Q/Flt3ITD mice transduced with WT, QE or IM and untransduced control cells for comparison. GAPDH serves as a loading control. The same membrane was stripped and reprobed for western blots. d, Intracellular 2HG levels in primary HSPCs from Idh2R140Q/Flt3ITD mice transduced with WT, QE or IM and collected from the first passage of methylcellulose cultures containing AG-221 at 50 nM. Data for are mean ± s.e.m. for triplicate cultures. e, Flow cytometry gating strategy for Fig. 3i. SSC-A, side scatter area; FSC-A, forward scatter area. DAPI is a viability dye. mCherry identifies retrovirally transduced cells. Results are representative of ≥2 (a–d) or 1 (e) independent experiments. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 4 Purification and activity of IDH2(R140Q) dimers with wild-type IDH2 or mutants Q316E or I319M in trans.
a, Schematic of experimental approach: 293T cells were co-transfected with HA-tagged IDH2(R140Q) plus Flag-tagged wild-type, Q316E or I319M. After 2 days, cells were lysed and enzyme complexes were purified by HA-immunoprecipitation. Reactions were performed with purified enzyme, NADPH, αKG and varying doses of AG-221 as detailed in Fig. 3. b–e, Purity and dimerization of HA-precipitated enzymes were assessed by denatured SDS–PAGE with Coomassie staining (b), denatured SDS–PAGE with western blotting (c), native PAGE with Coomassie staining (d), or native PAGE with western blotting for the indicated proteins (e). Separate membranes were used for western blots. f, In vitro enzyme assays measuring rate of NADPH consumption of IDH2 dimers purified as in b–e. Reactions contained purified enzyme (10 μg ml−1), NADPH (0.3 mM), αKG (5 mM) and AG-221 at indicated concentrations. Data are mean ± 95% confidence intervals for triplicate reactions. Results are representative of ≥3 (b–d, f) or 2 (e) independent experiments. For gel source data, see Supplementary Fig. 2.
Extended Data Fig. 5 Second-site IDH2 mutations in cis can confer resistance to enasidenib.
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.
Supplementary Information
Supplementary Figures 1-3
Supplementary Figure 1 contains full blots for Extended Data Figure 3c, Supplementary Figure 2 contains full blots for Extended Data Figure 4c and 4e and Supplementary Figure 3 contains full blots for Extended Data Figure 5a and 5d.
Supplementary Tables 1-5
Supplementary Table 1 contains RainDance microdroplet-based PCR sequencing results for IDH2 in Patient A (Q316E mutation on enasidenib). Supplementary Table 2 contains RainDance microdroplet-based PCR sequencing results for IDH2 in Patient B (I319M mutation on enasidenib). Supplementary Table 3 contains FoundationOne Heme hybrid-capture sequencing results for IDH1 in Patient X (S280F mutation on ivosidenib). Supplementary Table 4 contains RainDance microdroplet-based PCR sequencing results for IDH2 in patients treated with enasidenib, including those with primary or acquired resistance. Supplementary Table 5 contains MSK-IMPACT hybrid-capture sequencing results for IDH2 (R140, Q316, I319) in patients treated with enasidenib, including those with primary or acquired resistance.
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Intlekofer, A.M., Shih, A.H., Wang, B. et al. Acquired resistance to IDH inhibition through trans or cis dimer-interface mutations. Nature 559, 125–129 (2018). https://doi.org/10.1038/s41586-018-0251-7
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DOI: https://doi.org/10.1038/s41586-018-0251-7
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