A small molecule inhibitor of mutant IDH2 rescues cardiomyopathy in a D-2-hydroxyglutaric aciduria type II mouse model - PubMed (original) (raw)

. 2016 Nov;39(6):807-820.

doi: 10.1007/s10545-016-9960-y. Epub 2016 Jul 28.

Jeremy Travins 1, Zhizhong Lin 2, Yaguang Si 1, Yue Chen 1, Josh Powe 1, Stuart Murray 1, Dongwei Zhu 1, Erin Artin 1, Stefan Gross 1, Stephanie Santiago 1, Mya Steadman 1, Andrew Kernytsky 1, Kimberly Straley 1, Chenming Lu 2, Ana Pop 3, Eduard A Struys 3, Erwin E W Jansen 3, Gajja S Salomons 3, Muriel D David 4 5, Cyril Quivoron 4 5, Virginie Penard-Lacronique 4 5, Karen S Regan 6, Wei Liu 1, Lenny Dang 1, Hua Yang 1, Lee Silverman 1, Samuel Agresta 1, Marion Dorsch 1, Scott Biller 1, Katharine Yen 1, Yong Cang 2, Shin-San Michael Su 1, Shengfang Jin 7

Affiliations

A small molecule inhibitor of mutant IDH2 rescues cardiomyopathy in a D-2-hydroxyglutaric aciduria type II mouse model

Fang Wang et al. J Inherit Metab Dis. 2016 Nov.

Abstract

D-2-hydroxyglutaric aciduria (D2HGA) type II is a rare neurometabolic disorder caused by germline gain-of-function mutations in isocitrate dehydrogenase 2 (IDH2), resulting in accumulation of D-2-hydroxyglutarate (D2HG). Patients exhibit a wide spectrum of symptoms including cardiomyopathy, epilepsy, developmental delay and limited life span. Currently, there are no effective therapeutic interventions. We generated a D2HGA type II mouse model by introducing the Idh2R140Q mutation at the native chromosomal locus. Idh2R140Q mice displayed significantly elevated 2HG levels and recapitulated multiple defects seen in patients. AGI-026, a potent, selective inhibitor of the human IDH2R140Q-mutant enzyme, suppressed 2HG production, rescued cardiomyopathy, and provided a survival benefit in Idh2R140Q mice; treatment withdrawal resulted in deterioration of cardiac function. We observed differential expression of multiple genes and metabolites that are associated with cardiomyopathy, which were largely reversed by AGI-026. These findings demonstrate the potential therapeutic benefit of an IDH2R140Q inhibitor in patients with D2HGA type II.

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Conflict of interest statement

None. Animal rights All institutional and national guidelines for the care and use of laboratory animals were followed. This article does not contain studies with human subjects performed by any of the authors. Funding This work was funded by Agios. Writing assistance was provided by Helen Varley, PhD, CMPP, Excel Scientific Solutions, Horsham, UK and funded by Agios.

Figures

Fig. 1

Fig. 1

Characteristics of Idh2R140Q KI mice. (a) 2HG levels in plasma (nmol/mL), bone marrow, brain, spleen (all n = 4), and heart (n = 7 for Idh2wt and n = 5 for Idh2R140Q) in Idh2R140Q and Idh2wt mice. Black horizontal bars indicate mean. Statistical significance (p) calculated with t tests using the Holm-Sidak method (p values shown above brackets). (b) Kaplan-Meier survival curves in Idh2R140Q and Idh2wt mice from week 3. Statistical significance (p) tested using the log-rank (Mantel-Cox) test. (c) Abnormal phenotypes (red arrow) in Idh2R140Q mice: runting, facial dysmorphism, and abnormal shape of the head. (d) Histology of the heart, brain, and kidney in Idh2R140Q mice at approximately 12 weeks of age (hematoxylin and eosin stained). Longitudinal sections of the whole heart demonstrate ventricle wall thickening, and cardiomyocytes show hypertrophy. Transverse sections of the mid-brain showed vacuoles were consistently present in the lateral septal nuclei, various strata of the CA1 and CA3 areas of the hippocampus, the dentate gyrus, and the anterior cingulate and retrosplenial cortices, but were also observed laterally and ventrally in the cerebrum to involve the motor (shown), somatosensory and piriform cortices, particularly in the deeper neuronal layers (3 − 6), and were also sometimes present in the amygdala and medulla. Transverse sections of the whole kidney illustrate hydronephrosis, showing thinning of the cortex and almost total loss of the medulla. Histology is representative of six mice per group. (e) Echocardiographs demonstrating cardiac functional defects in 12-week old Idh2R140Q mice

Fig. 2

Fig. 2

AGI-026, a brain-penetrant, potent and selective IDH2R140Q mutant inhibitor. Structure and biochemical properties of AGI-026. IC50 for 2HG production was measured in vitro for IDH2R140Q homodimer and IDH2wt/R140Q heterodimer recombinant purified enzymes. IC50 for αKG production was measured in vitro for IDH2wt homodimer recombinant purified enzyme. IC50 for 2HG production was measured in: the engineered glioblastoma U87MG cell line overexpressing IDH2R140Q or IDH1R132H; mouse Idh2R140Q embryonic stem cells; lymphoblast cells obtained from three unrelated patients with D2HGA type II. Mouse Idh2R140Q and human IDH2R140Q show 95.13 % identity

Fig. 3

Fig. 3

Efficacy of AGI-026 in reversing the phenotype observed in Idh2R140Q mice. Treatment was administered twice daily for 13 weeks. (a) Inhibition of 2HG in plasma and heart following 6 weeks’ treatment with AGI-026 10 mg/kg compared with vehicle (n = 8 for plasma, n = 7 for heart Idh2wt-veh, and n = 5 for other groups). (b) Kaplan-Meier survival curves in Idh2R140Q mice treated with 10 mg/kg AGI-026 or vehicle and Idh2wt mice receiving vehicle. Statistical significance (p) tested using the log-rank (Mantel-Cox) test. (c) Histopathological analysis of heart tissue in Idh2wt mice and Idh2R140Q mice with and without AGI-026 treatment, showing rescue of cardiac hypertrophy (cytoplasmic and nuclear enlargement) with treatment (scale bar denotes 100 μm). (d) Inhibition of 2HG in plasma following 6 weeks’ treatment with AGI-026 compared with vehicle in the second efficacy study (n = 20 for Idh2wt-veh, n = 21 for Idh2R140Q-veh, n = 14 for Idh2R140Q-AGI 2 mg/kg, and n = 10 for Idh2R140Q-AGI 10 mg/kg); % inhibition calculated as 100 % in Idh2wt-veh and 0 % in Idh2R140Q-veh. (e) Kaplan-Meier survival curves in Idh2R140Q mice treated with 2 mg/kg AGI-026, 10 mg/kg AGI-026 or vehicle and Idh2wt mice receiving vehicle. Statistical significance (p) tested using the log-rank (Mantel-Cox) test. (f) Left ventricular mass/body weight ratio and (g) ejection fraction assessed by echocardiogram at week 13 (n = 20 for Idh2wt-veh, n = 16 for Idh2R140Q-veh, n = 13 for Idh2R140Q-AGI 2 mg/kg, and n = 9 for Idh2R140Q-AGI 10 mg/kg). Error bars represent mean ± standard deviation; statistical significance (p) was tested with one-way ANOVA with Sidak’s multiple comparison test

Fig. 4

Fig. 4

Cross-over study to determine effects of withdrawal of AGI-026 treatment. (a) Cross-over study design. (b) Left ventricular mass/body weight and (c) ejection fraction before and after cross-over study treatment (n = 9 in Idh2wt, n = 7 in Idh2R140Q-veh/AGI, and n = 4 in Idh2R140Q-AGI/veh). Error bars represent mean ± standard deviation. Statistical significance (p) tested with one-way ANOVA with Sidak’s multiple comparison test

Fig. 5

Fig. 5

5hmC and 5mC levels in heart tissue of mice. Percentage of 5hmc (a) or 5mc (b) in hearts of Idh2wt mice receiving vehicle (n = 10) and Idh2R140Q mice treated for 13 weeks with vehicle (n = 5) or AGI-026 10 mg/kg (n = 10). Error bars represent mean ± standard deviation. Statistical significance (p) was tested by one-way ANOVA with Sidak’s multiple comparison test

Fig. 6

Fig. 6

Differences in metabolite levels and gene expression in Idh2R140Q and Idh2wt heart tissue and effect of AGI-026. For heat maps (a) and (c), red indicates an increase, blue a decrease, darker colors indicate greater change, and pale colors indicate less change. Left column shows difference for Idh2R140Q versus Idh2wt, central column shows difference for AGI-026 versus vehicle treatment in Idh2R140Q mice, and right column shows difference for AGI-026-treated Idh2R140Q versus Idh2wt mice (pale colors illustrate correction of alterations by AGI-026 and shift to Idh2wt status). (a) Expression changes in 52 candidate genes with ≥ 2-fold difference between Idh2R140Q (n = 6) and Idh2wt (n = 5); (Idh2R140Q-AGI n = 6). (b) Quantitative RT-PCR validation of expression changes in two candidate genes, shown as relative expression normalized to one Idh2wt sample. Each data point represents the mean of a technical triplicate from each of the five animals per group. P tested using Kruskal-Wallis tests with Dunn’s multiple comparison test. For all figures horizontal black bars indicate group means. (c) The metabolites (excluding 2HG) that were significantly changed (p < 0.05) in heart tissue of Idh2R140Q versus Idh2wt mice, and effects of AGI-026. (d) Levels of cAMP and metabolites in the glycogenolysis and glycolysis pathways that are corrected by AGI-026 treatment. Statistical significance (p) was tested with one-way ANOVA with Sidak’s multiple comparison test (n = 10 Idh2wt-veh and Idh2R140Q-AGI, n = 5 Idh2R140Q-veh). (e) Correlation of fold-changes for Idh2R140Q-AGI versus Idh2R140Q-veh against Idh2R140Q-veh versus Idh2wt-veh for the metabolites in heart tissue significantly altered by Idh2R140Q expression (excluding 2HG). Red dots indicate metabolites significantly changed on both axes (n = 32). R2 denotes coefficient of determination

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