Pharmacodynamics of mutant-IDH1 inhibitors in glioma patients probed by in vivo 3D MRS imaging of 2-hydroxyglutarate.

Inhibitors of the mutant isocitrate dehydrogenase 1 (IDH1) entered recently in clinical trials for glioma treatment. Mutant IDH1 produces high levels of 2-hydroxyglurate (2HG), thought to initiate oncogenesis through epigenetic modifications of gene expression. In this study, we show the initial evidence of the pharmacodynamics of a new mutant IDH1 inhibitor in glioma patients, using non-invasive 3D MR spectroscopic imaging of 2HG. Our results from a Phase 1 clinical trial indicate a rapid decrease of 2HG levels by 70% (CI 13%, P = 0.019) after 1 week of treatment. Importantly, inhibition of mutant IDH1 may lead to the reprogramming of tumor metabolism, suggested by simultaneous changes in glutathione, glutamine, glutamate, and lactate. An inverse correlation between metabolic changes and diffusion MRI indicates an effect on the tumor-cell density. We demonstrate a feasible radiopharmacodynamics approach to support the rapid clinical translation of rationally designed drugs targeting IDH1/2 mutations for personalized and precision medicine of glioma patients.

The role of IDH mutations in acute myeloid leukemia.

Isocitrate dehydrogenases (IDHs) are enzymes involved in multiple metabolic and epigenetic cellular processes. Mutations in IDH1 or IDH2 are detected in approximately 20% of patients with acute myeloid leukemia (AML) and induce amino acid changes in conserved residues resulting in neomorphic enzymatic function and production of an oncometabolite, 2-hydroxyglutarate (R-2-HG). This leads to DNA hypermethylation, aberrant gene expression, cell proliferation and abnormal differentiation. IDH mutations diversely affect prognosis of patients with AML based on the location of the mutation and other co-occurring genomic abnormalities. Recently, novel therapies specifically targeting mutant IDH have opened new avenues of therapy for these patients. In the present review, we will provide an overview of the biological, clinical and therapeutic implications of IDH mutations in AML.

The Burgeoning World of Immunometabolites: Th17 Cells Take Center Stage.

Dysregulation of the Th17/Treg balance is a key driver of autoimmunity. A new study by Xu et al. (2017) has revealed that small-molecule inhibition of 2-hydroxyglutarate synthesis skews Th17 differentiation to the Treg lineage, which is protective against autoimmune inflammation.

Enasidenib induces acute myeloid leukemia cell differentiation to promote clinical response.

Recurrent mutations at R140 and R172 in isocitrate dehydrogenase 2 (IDH2) occur in many cancers, including ∼12% of acute myeloid leukemia (AML). In preclinical models these mutations cause accumulation of the oncogenic metabolite R-2-hydroxyglutarate (2-HG) and induce hematopoietic differentiation block. Single-agent enasidenib (AG-221/CC-90007), a selective mutant IDH2 (mIDH2) inhibitor, produced an overall response rate of 40.3% in relapsed/refractory AML (rrAML) patients with mIDH2 in a phase 1 trial. However, its mechanism of action and biomarkers associated with response remain unclear. Here, we measured 2-HG, mIDH2 allele burden, and co-occurring somatic mutations in sequential patient samples from the clinical trial and correlated these with clinical response. Furthermore, we used flow cytometry to assess inhibition of mIDH2 on hematopoietic differentiation. We observed potent 2-HG suppression in both R140 and R172 mIDH2 AML subtypes, with different kinetics, which preceded clinical response. Suppression of 2-HG alone did not predict response, because most nonresponding patients also exhibited 2-HG suppression. Complete remission (CR) with persistence of mIDH2 and normalization of hematopoietic stem and progenitor compartments with emergence of functional mIDH2 neutrophils were observed. In a subset of CR patients, mIDH2 allele burden was reduced and remained undetectable with response. Co-occurring mutations in NRAS and other MAPK pathway effectors were enriched in nonresponding patients, consistent with RAS signaling contributing to primary therapeutic resistance. Together, these data support differentiation as the main mechanism of enasidenib efficacy in relapsed/refractory AML patients and provide insight into resistance mechanisms to inform future mechanism-based combination treatment studies.

D-2-hydroxyglutaric acid inhibits creatine kinase activity from cardiac and skeletal muscle of young rats.

BACKGROUND: Tissue accumulation of high amounts of D-2-hydroxyglutaric acid (DGA) is the biochemical hallmark of the inherited neurometabolic disorder D-2-hydroxyglutaric aciduria (DHGA). Patients affected by this disease usually present hypotonia, muscular weakness, hypertrophy and cardiomyopathy, besides severe neurological findings. However, the underlying mechanisms of muscle injury in this disorder are virtually unknown. MATERIALS AND METHODS: In the present study we have evaluated the in vitro role of DGA, at concentrations ranging from 0.25 to 5.0 mM, on total, cytosolic and mitochondrial creatine kinase activities from skeletal and cardiac muscle of 30-day-old Wistar rats. We also tested the effects of various antioxidants on the effects elicited by DGA. RESULTS: We first verified that total creatine kinase (CK) activity from homogenates was significantly inhibited by DGA (22-24% inhibition) in skeletal and cardiac muscle, and that this activity was approximately threefold higher in skeletal muscle than in cardiac muscle. We also observed that CK activities from mitochondrial (Mi-CK) and cytosolic (Cy-CK) preparations from skeletal muscle and cardiac muscle were also inhibited (12-35% inhibition) by DGA at concentrations as low as 0.25 mm, with the effect being more pronounced in cardiac muscle preparations. Finally, we verified that the DGA-inhibitory effect was fully prevented by preincubation of the homogenates with reduced glutathione and cysteine, suggesting that this effect is possibly mediated by modification of essential thiol groups of the enzyme. Furthermore, alpha-tocopherol, melatonin and the inhibitor of nitric oxide synthase L-NAME were unable to prevent this effect, indicating that the most common reactive oxygen and nitrogen species were not involved in the inhibition of CK provoked by DGA. CONCLUSION: Considering the importance of creatine kinase activity for cellular energy homeostasis, our results suggest that inhibition of this enzyme by increased levels of DGA might be an important mechanism involved in the myopathy and cardiomyopathy of patients affected by DHGA.

Contribution of reactive oxygen species to 3-hydroxyglutarate neurotoxicity in primary neuronal cultures from chick embryo telencephalons.

Glutaryl-CoA dehydrogenase deficiency is an autosomal recessively inherited neurometabolic disorder with a distinct neuropathology characterized by acute encephalopathic crises during a vulnerable period of brain development. 3-Hydroxyglutarate (3-OH-GA), which accumulates in affected patients, has been identified as an endogenous neurotoxin mediating excitotoxicity via N-methyl-D-aspartate receptors. As increased generation of reactive oxygen species (ROS) and nitric oxide (NO) plays an important role in excitotoxic neuronal damage, we investigated whether ROS and NO contribute to 3-OH-GA neurotoxicity. 3-OH-GA increased mitochondrial ROS generation in primary neuronal cultures from chick embryo telencephalons, which could be prevented by MK-801, confirming the central role of N-methyl-D-aspartate receptor stimulation in 3-OH-GA toxicity. ROS increase was reduced by alpha-tocopherol and--less effectively-by melatonin. alpha-Tocopherol revealed a wider time frame for neuroprotection than melatonin. Creatine also reduced neuronal damage and ROS formation but only if it was administered >or=6 h before 3-OH-GA. NO production revealed only a slight increase after 3-OH-GA incubation. NO synthase inhibitor N(omega)-nitro-L-arginine prevented NO increase but did not protect neurons against 3-OH-GA. The NO donor S-nitroso-N-acetylpenicillamine revealed no effect on 3-OH-GA toxicity at low concentrations (0.5-5 microM), whereas it potentiated neuronal damage at high concentrations (50-500 microM), suggesting that weak endogenous NO production elicited by 3-OH-GA did not affect neuronal viability. We conclude from our results that ROS generation contributes to 3-OH-GA neurotoxicity in vitro and that radical scavenging and stabilization of brain energy metabolism by creatine are hopeful new strategies in glutaryl-CoA dehydrogenase deficiency.

IGF-1 and bFGF reduce glutaric acid and 3-hydroxyglutaric acid toxicity in striatal cultures.

Glutaric acid (GA) and 3-hydroxyglutaric acid (3GA) are thought to contribute to the degeneration of the caudate and putamen that is seen in some children with glutaric acidaemia type I, a metabolic disorder caused by a glutaryl-CoA dehydrogenase deficiency. This study assessed the neurotoxicity of GA and 3GA (0-50 mmol/L) compared to quinolinic acid (QUIN) in striatal and cortical cultures. All three acids were neurotoxic in a dose-dependent manner; however, GA and 3GA were both more toxic than QUIN. The neurotoxic effects of low concentrations of GA or 3GA were additive to QUIN toxicity. A series of hormones and growth factors were tested for protection against GA and 3GA toxicity. Insulin (5-500 microU /ml), basic fibroblast growth factor (bFGF; 10 ng/ml), insulin-like growth factor (IGF-1; 50 ng/ml), brain-derived neurotrophic factor (BDNF; 10 ng/ml), glial-derived neurotrophic factor (GDNF; 10 ng/ml), and two glutamate antagonists were evaluated in brain cultures to which 7 mmol/L GA or 3GA were added. GA and 3GA neurotoxicities were prevented by bFGF. Attenuation of 3GA-induced neurotoxicity was seen with insulin (5 microU/ml) and IGF-1. BDNF and GDNF had no effects on neuronal survival. Glutamate antagonists MK801 (10 micromol/L) and NBQX (10 micromol/L) failed to prevent GA or 3GA neurotoxicity. We conclude that GA and 3GA are neurotoxic in cultures of embryonic rat striatum and cortex. Striatal neurons were rescued from death by bFGF and IGF-1 but not by glutamate antagonist, suggesting that toxicity in this embryonic system is not necessarily mediated by glutamate receptors.

Induction of the mitochondrial permeability transition in vitro by short-chain carboxylic acids.

We recently reported that acrylic acid (AA) induces the MPT in vitro, which we suggested might be a critical event in the acute inflammatory and hyperplastic response of the olfactory epithelium. The purpose of the present investigation was to determine if induction of the MPT is a general response to short-chain carboxylic acids or if there are critical physical chemical parameters for this response. Freshly isolated rat liver mitochondria were incubated in the presence of varying concentrations of selected carboxylic acids. All of the acids that we tested caused a concentration-dependent induction of the MPT, which was blocked by cyclosporine A. Although the C4 carboxylic acids were slightly more potent than the C5 acids, there was no correlation with the degree of saturation, the octanol/water coefficient (log P), or the dissociation constant (pK(a)) of the acids that we tested. We conclude that induction of the MPT in vitro is a general response to short-chain carboxylic acids having a pK(a) of 4 to 5.

Cerebral organic acid disorders induce neuronal damage via excitotoxic organic acids in vitro.

Glutaryl-CoA dehydrogenase deficiency (GDD), which is one of the most frequent organic acid disorders, is characterized by a specific age- and regional-dependent neuropathology. We hypothesized that the distinct brain damage in GDD could be caused by the main pathologic metabolites, the organic acids glutaric (GA) and 3-hydroxyglutaric (3-OH-GA) acids, through an excitotoxic sequence. Therefore, we investigated the effects of 3-OH-GA and GA on primary neuronal cultures from chick embryonic telencephalons. Here we report that 3-OH-GA and GA decreased cell viability concentration- and time-dependently, which could be only totally prevented by preincubation with MK-801, ifenprodil and NR2B antibodies. Furthermore, cell viability decreased in parallel with the increasing expression of NR2B subunit on cultured neurons from 2nd to 6th DIV. We conclude that GA and 3-OH-GA act as excitotoxic organic acids (EOA) specifically through NR1/NR2B and that the extent of induced neurotoxicity is dependent on NR1/NR2B expression during maturation.

Maturation-dependent neurotoxicity of 3-hydroxyglutaric and glutaric acids in vitro: a new pathophysiologic approach to glutaryl-CoA dehydrogenase deficiency.

Glutaryl-CoA dehydrogenase deficiency is a neurometabolic disorder with a specific age- and region-dependent neuropathology. Between 6 and 18 mo of age, unspecific illnesses trigger acute encephalopathic crises resulting in acute striatal and cortical necrosis. We hypothesized that acute brain damage in glutaryl-CoA dehydrogenase deficiency is caused by the main pathologic metabolites 3-hydroxyglutaric and glutaric acids through an excitotoxic sequence. Therefore, we investigated the effect of 3-hydroxyglutaric acid and glutaric acid on primary neuronal cultures from chick embryo telencephalons and mixed neuronal and glial cell cultures from neonatal rat hippocampi. Exposure to glutaric acid and 3-hydroxyglutaric acid decreased cell viability in a concentration- and time-dependent fashion. This neurotoxic effect could be totally prevented by preincubation with an N-methyl-D-aspartate receptor subunit 2B (NR2B)-specific antagonist, NR2B antibodies, and an unspecific N-methyl-D-aspartate receptor blocker and was partially blocked with an NR2A-specific antagonist but not with NR2A antibodies or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor and metabotropic glutamate receptor antagonists. Furthermore, metabolite toxicity increased in parallel with the increasing expression of the NR2B subunit on cultured neurons from second to sixth day in vitro. We conclude from these results that 3-hydroxyglutaric acid and glutaric acid act as false neurotransmitters, in particular through NR1/2B, and that the extent of induced neurotoxicity is dependent on the temporal and spatial expression of NR1/2B in the CNS during maturation. Beyond favorable implications for treatment and long-term prognosis, glutaryl-CoA dehydrogenase deficiency is the first neurologic disease in which specific neuropathology could be experimentally linked to ontogenetic expression of a particular neurotransmitter receptor subtype.

Mechanism of action of glutaryl-CoA and butyryl-CoA dehydrogenases. Purification of glutaryl-CoA dehydrogenase.

Glutaryl-CoA dehydrogenase, a flavoprotein, catalyzes the reaction -OOCCH3CH2--CH2COSR (FAD leads to FADH2) leads to CH3CH = CHCOSR + CO2 (SR = CoA or pantetheine). With the isolated enzyme, a dye serves as the final electron acceptor. The enzyme from Pseudomonas fluorescens (ATCC 11250) has been purified to homogeneity. It was established with appropriate isotopic substitutions that the proton which is added to the gamma position of the product, subsequent to decarboxylation, is not derived from the solvent but is derived from the alpha position of the substrate. Under conditions where no net conversion of substrate occurs, i.e., in the absence of electron acceptor, the enzyme catalyzes the exchange of the beta hydrogen of the substrate with solvent protons. Butyryl-CoA dehydrogenase (M. elsedenii), which catalyzes an analogous reaction, catalyzes the exchange of both the alpha and beta hydrogens with solvent protons in the absence of electron acceptor. Glutaryl-CoA dehydrogenase and butyryl-CoA dehydrogenase are irreversibly inactivated by the substrate analogues 3-butynoylpantetheine and 3-pentynoylpantetheine. These inactivators do not form an adduct with the flavin and probably react with a nucleophile at the active site. Upon inactivation, the spectrum of the enzyme-bound flavin is essentially unchanged, and the flavin can be reduced by Na2S2O4. We suggest that inactivation involves intermediate allene formation. We proposed that these results support an oxidation mechanism for glutaryl-CoA dehydrogenase and butyryl-CoA dehydrogenase which is initiated by proton abstraction. With glutaryl-CoA dehydrogenase, the base, which abstracts the substrate alpha proton, is shielded from the solvent and is then used to protonate the carbanion (CH2--CH--CHCOSCoA) formed after oxidation and decarboxylation.