Wild-type IDH2 is a therapeutic target for triple-negative breast cancer.

Mutations in isocitrate dehydrogenases (IDH) are oncogenic events due to the generation of oncogenic metabolite 2-hydroxyglutarate. However, the role of wild-type IDH in cancer development remains elusive. Here we show that wild-type IDH2 is highly expressed in triple negative breast cancer (TNBC) cells and promotes their proliferation in vitro and tumor growth in vivo. Genetic silencing or pharmacological inhibition of wt-IDH2 causes a significant increase in α-ketoglutarate (α-KG), indicating a suppression of reductive tricarboxylic acid (TCA) cycle. The aberrant accumulation of α-KG due to IDH2 abrogation inhibits mitochondrial ATP synthesis and promotes HIF-1α degradation, leading to suppression of glycolysis. Such metabolic double-hit results in ATP depletion and suppression of tumor growth, and renders TNBC cells more sensitive to doxorubicin treatment. Our study reveals a metabolic property of TNBC cells with active utilization of glutamine via reductive TCA metabolism, and suggests that wild-type IDH2 plays an important role in this metabolic process and could be a potential therapeutic target for TNBC.

Distribution and Functional Analysis of Isocitrate Dehydrogenases across Kinetoplastids.

Isocitrate dehydrogenase is an enzyme converting isocitrate to α-ketoglutarate in the canonical tricarboxylic acid (TCA) cycle. There are three different types of isocitrate dehydrogenase documented in eukaryotes. Our study points out the complex evolutionary history of isocitrate dehydrogenases across kinetoplastids, where the common ancestor of Trypanosomatidae and Bodonidae was equipped with two isoforms of the isocitrate dehydrogenase enzyme: the NADP+-dependent isocitrate dehydrogenase 1 with possibly dual localization in the cytosol and mitochondrion and NADP+-dependent mitochondrial isocitrate dehydrogenase 2. In the extant trypanosomatids, isocitrate dehydrogenase 1 is present only in a few species suggesting that it was lost upon separation of Trypanosoma spp. and replaced by the mainly NADP+-dependent cytosolic isocitrate dehydrogenase 3 of bacterial origin in all the derived lineages. In this study, we experimentally demonstrate that the omnipresent isocitrate dehydrogenase 2 has a dual localization in both mitochondrion and cytosol in at least four species that possess only this isoform. The apparent lack of the NAD+-dependent isocitrate dehydrogenase activity in trypanosomatid mitochondrion provides further support to the existence of the noncanonical TCA cycle across trypanosomatids and the bidirectional activity of isocitrate dehydrogenase 3 when operating with NADP+ cofactor instead of NAD+. This observation can be extended to all 17 species analyzed in this study, except for Leishmania mexicana, which showed only low isocitrate dehydrogenase activity in the cytosol. The variability in isocitrate oxidation capacity among species may reflect the distinct metabolic strategies and needs for reduced cofactors in particular environments.

The diagnostic efficiency of integration of 2HG MRS and IVIM versus individual parameters for predicting IDH mutation status in gliomas in clinical scenarios: A retrospective study.

PURPOSE: Currently, there remains a scarcity of established preoperative tests to accurately predict the isocitrate dehydrogenase (IDH) mutation status in clinical scenarios, with limited research has explored the potential synergistic diagnostic performance among metabolite, perfusion, and diffusion parameters. To address this issue, we aimed to develop an imaging protocol that integrated 2-hydroxyglutarate (2HG) magnetic resonance spectroscopy (MRS) and intravoxel incoherent motion (IVIM) by comprehensively assessing metabolic, cellular, and angiogenic changes caused by IDH mutations, and explored the diagnostic efficiency of this imaging protocol for predicting IDH mutation status in clinical scenarios. METHODS: Patients who met the inclusion criteria were categorized into two groups: IDH-wild type (IDH-WT) group and IDH-mutant (IDH-MT) group. Subsequently, we quantified the 2HG concentration, the relative apparent diffusion coefficient (rADC), the relative true diffusion coefficient value (rD), the relative pseudo-diffusion coefficient (rD*) and the relative perfusion fraction value (rf). Intergroup differences were estimated using t-test and Mann-Whitney U test. Finally, we performed receiver operating characteristic (ROC) curve and DeLong's test to evaluate and compare the diagnostic performance of individual parameters and their combinations. RESULTS: 64 patients (female, 21; male, 43; age, 47.0 ± 13.7 years) were enrolled. Compared with IDH-WT gliomas, IDH-MT gliomas had higher 2HG concentration, rADC and rD (P < 0.001), and lower rD* (P = 0.013). The ROC curve demonstrated that 2HG + rD + rD* exhibited the highest areas under curve (AUC) value (0.967, 95%CI 0.889-0.996) for discriminating IDH mutation status. Compared with each individual parameter, the predictive efficiency of 2HG + rADC + rD* and 2HG + rD + rD* shows a statistically significant enhancement (DeLong's test: P < 0.05). CONCLUSIONS: The integration of 2HG MRS and IVIM significantly improves the diagnostic efficiency for predicting IDH mutation status in clinical scenarios.

Distinguishing IDH mutation status in gliomas using FTIR-ATR spectra of peripheral blood plasma indicating clear traces of protein amyloid aggregation.

BACKGROUND: Glioma is a primary brain tumor and the assessment of its molecular profile in a minimally invasive manner is important in determining treatment strategies. Among the molecular abnormalities of gliomas, mutations in the isocitrate dehydrogenase (IDH) gene are strong predictors of treatment sensitivity and prognosis. In this study, we attempted to non-invasively diagnose glioma development and the presence of IDH mutations using multivariate analysis of the plasma mid-infrared absorption spectra for a comprehensive and sensitive view of changes in blood components associated with the disease and genetic mutations. These component changes are discussed in terms of absorption wavenumbers that contribute to differentiation. METHODS: Plasma samples were collected at our institutes from 84 patients with glioma (13 oligodendrogliomas, 17 IDH-mutant astrocytoma, 7 IDH wild-type diffuse glioma, and 47 glioblastomas) before treatment initiation and 72 healthy participants. FTIR-ATR spectra were obtained for each plasma sample, and PLS discriminant analysis was performed using the absorbance of each wavenumber in the fingerprint region of biomolecules as the explanatory variable. This data was used to distinguish patients with glioma from healthy participants and diagnose the presence of IDH mutations. RESULTS: The derived classification algorithm distinguished the patients with glioma from healthy participants with 83% accuracy (area under the curve (AUC) in receiver operating characteristic (ROC) = 0.908) and diagnosed the presence of IDH mutation with 75% accuracy (AUC = 0.752 in ROC) in cross-validation using 30% of the total test data. The characteristic changes in the absorption spectra suggest an increase in the ratio of ß-sheet structures in the conformational composition of blood proteins of patients with glioma. Furthermore, these changes were more pronounced in patients with IDH-mutant gliomas. CONCLUSIONS: The plasma infrared absorption spectra could be used to diagnose gliomas and the presence of IDH mutations in gliomas with a high degree of accuracy. The spectral shape of the protein absorption band showed that the ratio of ß-sheet structures in blood proteins was significantly higher in patients with glioma than in healthy participants, and protein aggregation was a distinct feature in patients with glioma with IDH mutations.

IDH1/MDH1 deacetylation promotes acute liver failure by regulating NETosis.

BACKGROUND: Acute liver failure (ALF) is a life-threatening disease, but its pathogenesis is not fully understood. NETosis is a novel mode of cell death. Although the formation of neutrophil extracellular traps (NETs) has been found in various liver diseases, the specific mechanism by which NETosis regulates the development of ALF is unclear. In this article, we explore the role and mechanism of NETosis in the pathogenesis of ALF. METHODS: Clinically, we evaluated NETs-related markers in the liver and peripheral neutrophils of patients with ALF. In in vitro experiments, HL-60 cells were first induced to differentiate into neutrophil-like cells (dHL-60 cells) with dimethyl sulfoxide (DMSO). NETs were formed by inducing dHL-60 cells with PMA. In in vivo experiments, the ALF model in mice was established with LPS/D-gal, and the release of NETs was detected by immunofluorescence staining and western blotting. Finally, the acetylation levels of IDH1 and MDH1 were detected in dHL-60 cells and liver samples by immunoprecipitation. RESULTS: Clinically, increased release of NETs in liver tissue was observed in patients with ALF, and NETs formation was detected in neutrophils from patients with liver failure. In dHL-60 cells, mutations at IDH1-K93 and MDH1-K118 deacetylate IDH1 and MDH1, which promotes the formation of NETs. In a mouse model of ALF, deacetylation of IDH1 and MDH1 resulted in NETosis and promoted the progression of acute liver failure. CONCLUSIONS: Deacetylation of IDH1 and MDH1 reduces their activity and promotes the formation of NETs. This change aggravates the progression of acute liver failure.

NADP+-dependent isocitrate dehydrogenase as a novel target for altering carbon flux to lipid accumulation and enhancing antioxidant capacity in Tetradesmus obliquus.

Regulatory complexities in lipogenesis hinder the harmonization of metabolic carbon precursors towards lipid synthesis. Exploring regulatory complexities in lipogenesis, this study identifies NADP+-dependent isocitrate dehydrogenase (IDH) in Tetradesmus obliquus as a key factor. Overexpression IDH in strains ToIDH-1 and ToIDH-2 resulted in a 1.69 and 1.64-fold increase in neutral lipids, respectively, compared to the wild type, with lipid yield reaching 234.56 and 227.17 mg/L. Notably, despite slower growth, the cellular biomass augmented to 790.67 mg/L. Metabolite analysis indicated a shift in carbon precursors from protein to lipid and carbohydrate synthesis. Morphological observations revealed increases in the volume and number of lipid droplets, alongside a change in the fatty acid profile favoring monounsaturated and saturated fatty acids. Furthermore, IDH overexpression enhanced NADPH production and antioxidant activity, thereby further boosting lipid accumulation when combined with salt stress. This study suggests a pathway for improved lipogenesis and algal growth via metabolic engineering.

Evaluation of Immunohistochemical Expression of ALK-1 in Gliomas, WHO Grade 4 and Its Correlation with IDH1-R132H Mutation Status.

BACKGROUND: Glioblastoma (GB), a grade 4 glioma is the most common primary malignant brain tumor in adults. Recently, the mutation status of isocitrate dehydrogenase (IDH) has been crucial in the treatment of GB. IDH mutant cases display a more favorable prognosis than IDH-wild type ones. The anaplastic lymphoma kinase (ALK) is expressed as a receptor tyrosine kinase in both the developing central and peripheral nervous systems. Increasing lines of evidence suggest that ALK is over-expressed in GB and represents a potential therapeutic target. OBJECTIVES: The goal of the current study was to investigate ALK-1 immunohistochemical expression in gliomas, grade 4, besides its correlation with IDH1-R132H mutation status and the clinicopathological parameters of the tumors. MATERIAL AND METHODS: Seventy cases of gliomas, grade 4 were tested for immunohistochemical expression of ALK-1 & IDH1-R132H in the tumor cells. RESULTS: ALK-1 immunoexpression was detected in 22.9% of our cases and IDH1-R132H mutation was detected in 12.9% of them. ALK-1 expression (100%) was only detected in the more aggressive IDH R132H-negative GBs. ALK-1 expression was also noted in the larger-sized tumors, more in males and patients older than the mean age.  Conclusion: Our results suggest that mutations in ALK-1 may predict a more dismal prognosis since ALK expression was only noted in IDH-R132H negative GBs known to have a considerably poorer outcome compared to IDH-R132H mutant cases. GBs with detectable ALK-protein expression could potentially experience substantial clinical advantages through the utilization of newly introduced ALK inhibitors allowing personalized treatment to a subset of patients. Hence, future studies targeting ALK in IDH wildtype Glioblastomas including clinical trials on larger scales are recommended.

Interplay between ATRX and IDH1 mutations governs innate immune responses in diffuse gliomas.

Stimulating the innate immune system has been explored as a therapeutic option for the treatment of gliomas. Inactivating mutations in ATRX, defining molecular alterations in IDH-mutant astrocytomas, have been implicated in dysfunctional immune signaling. However, little is known about the interplay between ATRX loss and IDH mutation on innate immunity. To explore this, we generated ATRX-deficient glioma models in the presence and absence of the IDH1R132H mutation. ATRX-deficient glioma cells are sensitive to dsRNA-based innate immune agonism and exhibit impaired lethality and increased T-cell infiltration in vivo. However, the presence of IDH1R132H dampens baseline expression of key innate immune genes and cytokines in a manner restored by genetic and pharmacological IDH1R132H inhibition. IDH1R132H co-expression does not interfere with the ATRX deficiency-mediated sensitivity to dsRNA. Thus, ATRX loss primes cells for recognition of dsRNA, while IDH1R132H reversibly masks this priming. This work reveals innate immunity as a therapeutic vulnerability of astrocytomas.

Phospholipid metabolic adaptation promotes survival of IDH2 mutant acute myeloid leukemia cells.

Genetic mutations in the isocitrate dehydrogenase (IDH) gene that result in a pathological enzymatic activity to produce oncometabolite have been detected in acute myeloid leukemia (AML) patients. While specific inhibitors that target mutant IDH enzymes and normalize intracellular oncometabolite level have been developed, refractoriness and resistance has been reported. Since acquisition of pathological enzymatic activity is accompanied by the abrogation of the crucial WT IDH enzymatic activity in IDH mutant cells, aberrant metabolism in IDH mutant cells can potentially persist even after the normalization of intracellular oncometabolite level. Comparisons of isogenic AML cell lines with and without IDH2 gene mutations revealed two mutually exclusive signalings for growth advantage of IDH2 mutant cells, STAT phosphorylation associated with intracellular oncometabolite level and phospholipid metabolic adaptation. The latter came to light after the oncometabolite normalization and increased the resistance of IDH2 mutant cells to arachidonic acid-mediated apoptosis. The release of this metabolic adaptation by FDA-approved anti-inflammatory drugs targeting the metabolism of arachidonic acid could sensitize IDH2 mutant cells to apoptosis, resulting in their eradication in vitro and in vivo. Our findings will contribute to the development of alternative therapeutic options for IDH2 mutant AML patients who do not tolerate currently available therapies.

The Epigenetic Evolution of Glioma Is Determined by the IDH1 Mutation Status and Treatment Regimen.

Tumor adaptation or selection is thought to underlie therapy resistance in glioma. To investigate longitudinal epigenetic evolution of gliomas in response to therapeutic pressure, we performed an epigenomic analysis of 132 matched initial and recurrent tumors from patients with IDH-wildtype (IDHwt) and IDH-mutant (IDHmut) glioma. IDHwt gliomas showed a stable epigenome over time with relatively low levels of global methylation. The epigenome of IDHmut gliomas showed initial high levels of genome-wide DNA methylation that was progressively reduced to levels similar to those of IDHwt tumors. Integration of epigenomics, gene expression, and functional genomics identified HOXD13 as a master regulator of IDHmut astrocytoma evolution. Furthermore, relapse of IDHmut tumors was accompanied by histologic progression that was associated with survival, as validated in an independent cohort. Finally, the initial cell composition of the tumor microenvironment varied between IDHwt and IDHmut tumors and changed differentially following treatment, suggesting increased neoangiogenesis and T-cell infiltration upon treatment of IDHmut gliomas. This study provides one of the largest cohorts of paired longitudinal glioma samples with epigenomic, transcriptomic, and genomic profiling and suggests that treatment of IDHmut glioma is associated with epigenomic evolution toward an IDHwt-like phenotype. SIGNIFICANCE: Standard treatments are related to loss of DNA methylation in IDHmut glioma, resulting in epigenetic activation of genes associated with tumor progression and alterations in the microenvironment that resemble treatment-naïve IDHwt glioma.

A biosensor for D-2-hydroxyglutarate in frozen sections and intraoperative assessment of IDH mutation status.

The oncometabolite D-2-hydroxyglutarate (D-2-HG) has emerged as a valuable biomarker in tumors with isocitrate dehydrogenase (IDH) mutations. Efficient detection methods are required and rapid intraoperative determination of D-2-HG remains a huge challenge. Herein, D-2-HG dehydrogenase from Achromobacter xylosoxidans (AX-D2HGDH) was found to have high substrate specificity. AX-D2HGDH dehydrogenizes D-2-HG and reduces flavin adenine dinucleotide (FAD) bound to the enzyme. Interestingly, the dye resazurin can be taken as another substrate to restore FAD. AX-D2HGDH thus catalyzes a bisubstrate and biproduct reaction: the dehydrogenation of D-2-HG to 2-ketoglutarate and simultaneous reduction of non-fluorescent resazurin to highly fluorescent resorufin. According to steady-state analysis, a ping-pong bi-bi mechanism has been concluded. The Km values for resazurin and D-2-HG were determined as 0.56 µM and 10.93 µM, respectively, suggesting high affinity to both substrates. On the basis, taking AX-D2HGDH and resazurin as recognition and fluorescence transducing element, a D-2-HG biosensor (HGAXR) has been constructed. HGAXR exhibits high sensitivity, accuracy and specificity for D-2-HG in different biological samples. With the aid of HGAXR and the matched low-cost palm-size detecting device, D-2-HG levels in frozen sections of resected brain tumor tissues can be measured in a direct, simple and accurate manner with a fast detection (1-3 min). As the technique of frozen section is familiar to surgeons and pathologists, HGAXR and the portable device can be easily integrated into the current workflow, having potential to provide rapid intraoperative pathology for IDH mutation status and guide decision-making during surgery.

Wild-type IDH1 maintains NSCLC stemness and chemoresistance through activation of the serine biosynthetic pathway.

Tumor-initiating cells (TICs) reprogram their metabolic features to meet their bioenergetic, biosynthetic, and redox demands. Our previous study established a role for wild-type isocitrate dehydrogenase 1 (IDH1WT) as a potential diagnostic and prognostic biomarker for non-small cell lung cancer (NSCLC), but how IDH1WT modulates NSCLC progression remains elusive. Here, we report that IDH1WT activates serine biosynthesis by enhancing the expression of phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase 1 (PSAT1), the first and second enzymes of de novo serine synthetic pathway. Augmented serine synthesis leads to GSH/ROS imbalance and supports pyrimidine biosynthesis, maintaining tumor initiation capacity and enhancing gemcitabine chemoresistance. Mechanistically, we identify that IDH1WT interacts with and stabilizes PHGDH and fragile X-related protein-1 (FXR1) by impeding their association with the E3 ubiquitin ligase parkin by coimmunoprecipitation assay and proximity ligation assay. Subsequently, stabilized FXR1 supports PSAT1 mRNA stability and translation, as determined by actinomycin D chase experiment and in vitro translation assay. Disrupting IDH1WT-PHGDH and IDH1WT-FXR1 interactions synergistically reduces NSCLC stemness and sensitizes NSCLC cells to gemcitabine and serine/glycine-depleted diet therapy in lung cancer xenograft models. Collectively, our findings offer insights into the role of IDH1WT in serine metabolism, highlighting IDH1WT as a potential therapeutic target for eradicating TICs and overcoming gemcitabine chemoresistance in NSCLC.

IDH1R132H mutation increases radiotherapy efficacy and a 4-gene radiotherapy-related signature of WHO grade 4 gliomas.

The prognosis for the WHO grade 4 IDH-mutant astrocytoma is better than IDH-wildtype glioblastoma (GBM) patients. The purpose of this study is to explore the potential mechanism of how IDH1 mutation can increase the efficacy of radiotherapy and to establish a risk-score model to predict the efficacy of radiotherapy in WHO grade 4 gliomas. First, we conducted experimental study on the effect of IDH1R132H mutation on glioma cells in vitro. Radiosensitivity of glioma cells was detected by γ-H2AX after 5 Gy radiation. Cell proliferation, migration and invasion were determined respectively by CCK-8, EDU, monolayer cell migration scratch assay and Transwell assay. Then we analyzed IDH1 gene status and the survival of WHO grade 4 glioma patients received radiotherapy in our center and verified our results by analyzing CGGA and TCGA database. For the risk-score model, we use CGGA data to find genetic differences between WHO grade 4 IDH-mutant astrocytoma and IDH-wildtype GBM patients, and determined a 4-gene radiotherapy-related signature through survival analysis by R software. Evaluation and verification through different glioma validation sets and different statistical methods. For in vitro experiments, we established glioma cells stably overexpressing IDH1 wild-type and IDH1-mutant proteins. γ-H2AX assay showed that IDH1-mutant glioma cells had higher radiosensitivity than wild-type. CCK-8 and EDU assay showed that proliferation capacity of IDH1-mutant glioma cells declined. Transwell assay and monolayer cell migration scratch assay also showed that IDH1-mutant glioma cells reduced migration and invasion capabilities. Among the 83 WHO grade 4 glioma patients who received radiotherapy in our center, WHO grade 4 IDH-mutant astrocytoma patients had longer OS and PFS versus IDH-wildtype GBM (P = 0.0336, P = 0.0324, respectively). TCGA and CGGA database analysis had the similar results. Through complex analysis of CGGA and TCGA databases, we established a risk-model that can predict the efficacy of radiotherapy for WHO grade 4 glioma patients. The 4-gene radiotherapy-related signature including ADD3, GRHPR, RHBDL1 and SLC9A9. Patients in the high-risk group had worse OS compared to low-risk group (P = 0.0001). High- and low-risk groups of patients receiving radiotherapy have significant survival differences, while patients who did not receive radiotherapy have no survival difference both in CGGA and TCGA databases. WHO grade 4 IDH-mutant astrocytoma is more radiosensitive than IDH-wildtype GBM patients. Our 4-gene radiotherapy-related signature can predict the radiation efficacy of WHO grade 4 glioma patients, and it may provide some reference for clinical treatment options.

Novel, clinically relevant genomic patterns identified by comprehensive genomic profiling in ATRX-deficient IDH-wildtype adult high-grade gliomas.

Glioblastomas are the most common IDH-wildtype adult high-grade gliomas, frequently harboring mutations in the TERT gene promoter (pTERT) and utilizing the subsequent telomerase overexpression for telomere length maintenance. However, some rare cases show loss of ATRX and use alternative mechanisms of telomere lengthening. In this study, we performed the first complex genomic analysis specifically concentrating on the latter subgroup. Comprehensive genomic profiling of 12 ATRX-deficient and 13 ATRX-intact IDH-wildtype adult high-grade gliomas revealed that ATRX and pTERT mutations are mutually exclusive. DNMT3A alterations were confined to ATRX-deficient, while PTEN mutations to ATRX-intact cases. RAS-MAPK pathway alterations, including NF1 mutations, were more characteristic in the ATRX-deficient group. Variants of genes related to homologous recombination repair showed different patterns of affected genes. Two ATRX-deficient tumors with high tumor mutational burden and mismatch repair deficiency were found. One of these contained a novel fusion involving the NTRK2 and LRRFIP2 genes, while the other showed loss of MSH2 and MSH6 without genetic alterations in the encoding genes suggesting an epigenetic background. Genetic characteristics of ATRX-deficient IDH-wildtype adult high-grade gliomas suggest that these tumors are particularly intriguing targets of potential future therapeutic interventions including immunotherapies combined with MAPK pathway inhibition and DNA repair inhibitors.

IDH1 mutation impairs antiviral response and potentiates oncolytic virotherapy in glioma.

IDH1 mutations frequently occur early in human glioma. While IDH1 mutation has been shown to promote gliomagenesis via DNA and histone methylation, little is known regarding its regulation in antiviral immunity. Here, we discover that IDH1 mutation inhibits virus-induced interferon (IFN) antiviral responses in glioma cells. Mechanistically, D2HG produced by mutant IDH1 enhances the binding of DNMT1 to IRF3/7 promoters such that IRF3/7 are downregulated, leading to impaired type I IFN response in glioma cells, which enhances the susceptibility of gliomas to viral infection. Furthermore, we identify DNMT1 as a potential biomarker predicting which IDH1mut gliomas are most likely to respond to oncolytic virus. Finally, both D2HG and ectopic mutant IDH1 can potentiate the replication and oncolytic efficacy of VSVΔ51 in female mouse models. These findings reveal a pivotal role for IDH1 mutation in regulating antiviral response and demonstrate that IDH1 mutation confers sensitivity to oncolytic virotherapy.

N-Acetylglutamate and N-acetylmethionine compromise mitochondrial bioenergetics homeostasis and glutamate oxidation in brain of developing rats: Potential implications for the pathogenesis of ACY1 deficiency.

Aminoacylase 1 (ACY1) deficiency is an inherited metabolic disorder biochemically characterized by high urinary concentrations of aliphatic N-acetylated amino acids and associated with a broad clinical spectrum with predominant neurological signs. Considering that the pathogenesis of ACY1 is practically unknown and the brain is highly dependent on energy production, the in vitro effects of N-acetylglutamate (NAG) and N-acetylmethionine (NAM), major metabolites accumulating in ACY1 deficiency, on the enzyme activities of the citric acid cycle (CAC), of the respiratory chain complexes and glutamate dehydrogenase (GDH), as well as on ATP synthesis were evaluated in brain mitochondrial preparations of developing rats. NAG mildly inhibited mitochondrial isocitrate dehydrogenase 2 (IDH2) activity, moderately inhibited the activities of isocitrate dehydrogenase 3 (IDH3) and complex II-III of the respiratory chain and markedly suppressed the activities of complex IV and GDH. Of note, the NAG-induced inhibitory effect on IDH3 was competitive, whereas that on GDH was mixed. On the other hand, NAM moderately inhibited the activity of respiratory complexes II-III and GDH activities and strongly decreased complex IV activity. Furthermore, NAM was unable to modify any of the CAC enzyme activities, indicating a selective effect of NAG toward IDH mitochondrial isoforms. In contrast, the activities of citrate synthase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and of the respiratory chain complexes I and II were not changed by these N-acetylated amino acids. Finally, NAG and NAM strongly decreased mitochondrial ATP synthesis. Taken together, the data indicate that NAG and NAM impair mitochondrial brain energy homeostasis.

Two Different Isocitrate Dehydrogenases from Pseudomonas aeruginosa: Enzymology and Coenzyme-Evolutionary Implications.

Pseudomonas aeruginosa PAO1, as an experimental model for Gram-negative bacteria, harbors two NADP+-dependent isocitrate dehydrogenases (NADP-IDHs) that were evolved from its ancient counterpart NAD-IDHs. For a better understanding of PaIDH1 and PaIDH2, we cloned the genes, overexpressed them in Escherichia coli and purified them to homogeneity. PaIDH1 displayed higher affinity to NADP+ and isocitrate, with lower Km values when compared to PaIDH2. Moreover, PaIDH1 possessed higher temperature tolerance (50 °C) and wider pH range tolerance (7.2-8.5) and could be phosphorylated. After treatment with the bifunctional PaIDH kinase/phosphatase (PaIDH K/P), PaIDH1 lost 80% of its enzymatic activity in one hour due to the phosphorylation of Ser115. Small-molecule compounds like glyoxylic acid and oxaloacetate can effectively inhibit the activity of PaIDHs. The mutant PaIDH1-D346I347A353K393 exhibited enhanced affinity for NAD+ while it lost activity towards NADP+, and the Km value (7770.67 µM) of the mutant PaIDH2-L589 I600 for NADP+ was higher than that observed for NAD+ (5824.33 µM), indicating a shift in coenzyme specificity from NADP+ to NAD+ for both PaIDHs. The experiments demonstrated that the mutation did not alter the oligomeric state of either protein. This study provides a foundation for the elucidation of the evolution and function of two NADP-IDHs in the pathogenic bacterium P. aeruginosa.

D-2-hydroxyglutarate regulates human brain vascular endothelial cell proliferation and barrier function.

Gain-of-function mutations in isocitrate dehydrogenase (IDH) genes result in excessive production of (D)-2-hydroxyglutarate (D-2HG) which intrinsically modifies tumor cell epigenetics and impacts surrounding noncancerous cells through nonepigenetic pathways. However, whether D-2HG has a paracrine effect on endothelial cells in the tumor microenvironment needs further clarification. We quantified microvessel density by immunohistochemistry using tissue sections from 60 high-grade astrocytic gliomas with or without IDH mutation. Microvessel density was found to be reduced in tumors carrying an IDH mutation. Ex vivo experiments showed that D-2HG inhibited endothelial cell migration, wound healing, and tube formation by suppressing cell proliferation but not viability, possibly through reduced activation of the mTOR/STAT3 pathway. Further, D-2HG reduced fluorescent dextran permeability and decreased paracellular T-cell transendothelial migration by augmenting expression of junctional proteins thereby collectively increasing endothelial barrier function. These results indicate that D-2HG may influence the tumor vascular microenvironment by reducing the intratumoral vasculature density and by inhibiting the transport of metabolites and extravasation of circulating cells into the astrocytoma microenvironment. These observations provide a rationale for combining IDH inhibition with antitumor immunological/angiogenic approaches and suggest a molecular basis for resistance to antiangiogenic drugs in patients whose tumors express a mutant IDH allele.

Clinico-Genomic Profiling of Conventional and Dedifferentiated Chondrosarcomas Reveals TP53 Mutation to Be Associated with Worse Outcomes.

PURPOSE: Chondrosarcomas are the most common primary bone tumor in adults. Isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations are prevalent. We aimed to assess the clinico-genomic properties of IDH mutant versus IDH wild-type (WT) chondrosarcomas as well as alterations in other genes. EXPERIMENTAL DESIGN: We included 93 patients with conventional and dedifferentiated chondrosarcoma for which there were available clinical next-generation sequencing data. Clinical and genomic data were extracted and compared between IDH mutant and IDH WT chondrosarcomas and between TP53 mutant and TP53 WT chondrosarcomas. RESULTS: IDH1 and IDH2 mutations are prevalent in chondrosarcoma (50.5%), more common in chondrosarcomas arising in the extremities, associated with higher age at diagnosis, and more common in dedifferentiated chondrosarcomas compared with grades 1-3 conventional chondrosarcoma. There was no difference in survival based on IDH mutation in univariate and multivariate analyses. TP53 mutation was the next most prevalent (41.9%) and is associated with worse overall survival and metastasis-free survival in both univariate and multivariate analyses. TP53 mutation was also associated with higher risk of recurrence following curative-intent surgery and worse survival among patients that presented with de novo metastatic disease. CONCLUSIONS: IDH mutations are prevalent in chondrosarcoma though were not associated with survival outcomes in this cohort. TP53 mutations were the next most common alteration and were associated with worse outcomes.