Acquired resistance to IDH inhibition through trans or cis dimer-interface mutations

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)1-8. 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.

Conflict of interest statement

Competing Financial Interests

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.

Figures

Extended Data Figure 1. Acquired clinical resistance to the mutant IDH2 inhibitor enasidenib (AG-221)

(a, b) Hematoxylin 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 leukemic blasts and increased myeloid differentiation which 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 Figure 2. Structures illustrating potential interactions between IDH2 second-site mutations and enasidenib

(a–h) Detailed view of the interactions between wildtype IDH2 Q316 (a, e) and Q316’ (c, g) or mutant IDH2 Q316E (b, f) and 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 wildtype IDH2 I319 (i, m) and I319’ (k, o) or mutant IDH2 I319M (j, n) and 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 gray. 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. Unfavorable steric interactions between AG-221 and these atoms are depicted in red. Throughout the figure, the IDH2 subunit is depicted in blue-gray, the IDH2’ subunit in purple, and AG-221 in teal. Non-polar hydrogens 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 ID 5I96 retrieved from the RCSB (see Methods).

Extended Data Figure 3. Expression and activity of in trans IDH2 second-site mutations in hematopoietic cells

(a) Allele-specific quantitative RT-PCR (qPCR) showing similar expression of constructs in Ba/F3 cells co-transduced with IDH2 R140Q (RQ) plus IDH2 wildtype (WT), Q316E (QE), or I319M (IM) in trans. Control from IDH2 WT 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 (‘Veh’) 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 hematopoietic stem/progenitor cells (HSPC) from Idh2 R140Q/Flt3 ITD mice transduced with WT, QE, or IM and untransduced control cells for comparison (‘None’). GAPDH serves as a loading control. The same membrane was stripped and reprobed for Western blots. (d) Intracellular 2HG levels in primary HSPC from Idh2 R140Q/Flt3 ITD mice transduced with WT, QE, or IM and harvested 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 Extended Data Figure 6.

Extended Data Figure 4. Purification and activity of IDH2 R140Q dimers with IDH2 wildtype, Q316E, or I319M in trans

(a) Schematic of experimental approach: 293T cells were co-transfected with HA-tagged IDH2 R140Q (RQ) plus FLAG-tagged wildtype (WT), Q316E (QE) or I319M (IM). After 2 days, cells were lysed and enzyme complexes were purified by HA-immunoprecipitation. Reactions were performed with purified enzyme, NADPH, alpha-ketoglutarate (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), NADPH (0.3 mM), alpha-ketoglutarate (αKG; 5 mM), and AG-221 at indicated concentrations. Data are mean ± 95% c.i. for triplicate reactions. Results are representative of ≥3 (b–d, f) or 2 (e) independent experiments. For gel source data, see Extended Data Figure 7.

Extended Data Figure 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 IDH2 wildtype (WT) plus FLAG-tagged IDH2 R140Q (RQ), 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), NADPH (0.3 mM), alpha-ketoglutarate (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% c.i. 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 Extended Data Figure 8.

Extended Data Figure 6. Full blots for Extended Data Figure 3c

k.D., kiloDalton.

Extended Data Figure 7. Full blots for Extended Data Figure 4

(a) Full blots for Extended Data Figure 4c. (b) Full blots for Extended Data Figure 4e. k.D., kiloDalton.

Extended Data Figure 8. Full blots for Extended Data Figure 5

(a) Full blots for Extended Data Figure 5a. (b) Full blots for Extended Data Figure 5d. k.D., kiloDalton.

Figure 1. Acquired resistance to the mutant IDH2 inhibitor enasidenib (AG-221) associated with emergence of second-site mutations in IDH2

(a–d) Clinical, laboratory, and pathologic features for Patient A in relation to enasidenib (AG-221) treatment (blue box) and decitabine (DAC; gray box), including bone marrow blast percentage (a), blood absolute neutrophil count (ANC; b), plasma 2HG concentration (c), and variant allele frequency (VAF) for mutations identified by targeted next-generation sequencing of bone marrow cells (d). (e–h) Clinical, laboratory, and pathologic features for Patient B in relation to enasidenib (AG-221) treatment (blue box) and other treatments (gray boxes), including blood absolute blast count (e), blood ANC (f), plasma 2HG concentration (g), and VAF for mutations identified by targeted next-generation sequencing of bone marrow cells (h). Also see Extended Data Figure 1.

Figure 2. Second-site mutations in IDH2 occur on the allele without the neomorphic R140Q mutation

(a) Schematic of the IDH2 locus (ENSG00000182054|CCDS10359), highlighting the nucleotides encoding arginine 140 (R140), glutamine 316 (Q316), and isoleucine 319 (I319). Positions of sequencing primers are indicated by half-arrows. (b, c) Examples of Sanger sequencing in the forward (‘For’) and reverse (‘Rev’) direction from two clones (‘Cl’) for Patient A (b) and Patient B (c). Magenta boxes highlight the somatic mutations. (d, e) Summary of Sanger sequencing results for Patient A (d) and Patient B (e), demonstrating that the R140Q mutations and the Q316E (d) or I319M (e) mutations do not occur on the same allele.

Figure 3. Second-site mutations in IDH2 confer resistance to enasidenib in trans

(a) Structure of the IDH2 dimer highlighting the binding pocket for enasidenib (AG-221; teal) at the dimer interface and the amino acids affected by second-site resistance mutations (Q316, I319; modeled from PDB ID 5I96, see Methods). Second-site mutations are structurally distant from the catalytic active site containing the neomorphic R140Q mutation and NADP/H cofactor. (b) Detailed view of the Q316E’ mutation showing loss of a hydrogen bond (H bond) that normally forms between the amino side chain of Q316’ and a nitrogen in the diaminotriazine ring of AG-221. (c) Detailed view of the I319M mutation demonstrating steric effects from the bulky side chain of methionine predicted to hinder binding by AG-221. (d) Intracellular 2HG levels in Ba/F3 cells that express IDH2 R140Q (RQ), Q316E (QE) or I319M (IM) via retroviral transduction. Values are relative to untransduced parental Ba/F3 cells. Data are mean ± s.e.m. for n=5 cultures. (e) Intracellular 2HG levels in Ba/F3 cells co-expressing IDH2 RQ plus IDH2 wildtype (WT), QE, or IM in trans and treated with vehicle (‘Veh’) or increasing doses of AG-221 (1, 10, or 100 nM). Data are mean ± s.e.m. for triplicate cultures. (f, g) Serial-replating of primary hematopoietic stem/progenitor cells (HSPC) from Idh2 R140Q (f) or Idh2 R140Q/Flt3 ITD (g) mice expressing IDH2 WT, QE, or IM in trans and cultured in methylcellulose containing AG-221 at 50 nM. c.f.u., colony forming unit. * indicates value of 0. Data are mean ± s.e.m. for triplicate cultures. (h) Serial-replating of primary HSPC from Idh2 R140Q/Flt3 ITD mice cultured in methylcellulose containing either vehicle, AG-221 (50 nM), or AG-221 (50 nM) plus cell-permeable 2HG (‘Octyl-2HG’; 0.5 mM). Data are mean ± s.e.m. for duplicate (CFU1) or triplicate (CFU2/3) cultures. * indicates value of 0. (i, j) Mice reconstituted with Idh2 R140Q bone marrow HSPC transduced with IDH2 WT or QE were subjected to 2 (i) or 4 (j) weeks of treatment with enasidenib (40 mg/kg twice daily) and assessed for WT or QE allele frequencies before and after treatment (i) or intracellular 2HG levels in bone marrow mononuclear cells (j). See Methods. Data are mean ± s.e.m. for n=5 WT and n=8 QE mice. p=0.008 (i) or p=4×10−7 (j) by two-tailed t-test. (k–l) In vitro enzyme assays measuring absolute velocity (k) and relative activity (l) of NADPH-dependent reduction of alpha-ketoglutarate (αKG) by HA-precipitated IDH2 dimers purified from cells co-expressing IDH2 HA-RQ + FLAG-WT, HA-RQ + FLAG-QE, or HA-RQ + FLAG-IM (see Methods). Reactions contained purified enzyme (10 µg/ml), NADPH (0.3 mM), αKG (5 mM), and AG-221 at 0.1, 0.3, 1, 3, 10, and 30 µM (k) or indicated concentrations (l). Data for (k–l) are mean ± 95% c.i. for triplicate reactions. Results are representative of ≥3 (d, e, k–l), 2 (f, g, h), or 1 (i, j) independent experiments. Also see Extended Data Figures 2–4.

Figure 4. Second-site mutations in cis can confer resistance to IDH inhibitors

(a) Intracellular 2HG levels in Ba/F3 cells transduced with IDH2 R140Q (RQ), in cis double-mutant IDH2 R140Q/Q316E (RQ/QE) or in cis double-mutant IDH2 R140Q/I319M (RQ/IM) and treated with vehicle (‘Veh’) or increasing doses of AG-221 (10, 50, 100, 500, or 1000 nM). Data are mean ± s.e.m. for triplicate cultures. (b) In vitro enzyme assays measuring absolute velocity of NADPH-dependent reduction of alpha-ketoglutarate (αKG) by HA-precipitated IDH2 WT dimerized with RQ alone or RQ plus in cis second-site mutations (RQ/QE or RQ/IM). See Extended Data Fig. 5. Reactions contained purified enzyme (7.5 µg/ml), NADPH (0.3 mM), αKG (5 mM), and vehicle (‘Veh’) or increasing doses of AG-221 (0.1, 0.3, 1, 3, 10, or 30 µM). Data are mean ± 95% c.i. for triplicate reactions (duplicate reactions for WT:RQ/QE AG-221 3 µM and 30 µM). (c–e) Clinical and laboratory features for Patient × in relation to mutant IDH1 inhibitor ivosidenib (AG-120) treatment (blue box), including bone marrow blast percentage (c), plasma 2HG concentration (d), and variant allele frequency (VAF) for mutations identified by targeted next-generation sequencing of bone marrow cells (e). (f) Alignment of IDH1 and IDH2 protein sequences demonstrating that serine 280 (S280) of IDH1 corresponds to isoleucine 319 (I319) of IDH2. (g) Summary of Sanger sequencing results from Patient × demonstrating that the IDH1 R132C neomorphic mutation and the S280F mutation occur in cis on the same allele.