Isocitrate Dehydrogenase 1 Activity Research Articles

HSP90a promotes the resistance to oxaliplatin in HCC through regulating IDH1-induced cell competition

Though burgeoning research manifests that cell competition, an essential selection and quality control mechanism for maintaining tissue or organ growth and homeostasis in multicellular organisms, is closely related to tumorigenesis and development, the mechanism of cell competition associated with tumor drug resistance remains elusive. In the study, we uncovered that oxaliplatin-resistant hepatocellular carcinoma (HCC) cells exhibit a pronounced competitive advantage against their sensitive counterparts, which is related to lipid takeover of resistant cells from sensitive cells. Of note, such lipid takeover is dependent on the existence of isocitrate dehydrogenase 1 (IDH1) in resistant HCC cells. Mechanistically, IDH1 activity is regulated by heat shock protein 90 alpha (HSP90α) through binding with each other, which orchestrates the expressions of lipid metabolic enzymes and lipid accumulation in resistant HCC cells. Our results suggest that HCC cell competition-driven chemoresistance can be regulated by HSP90α/IDH1-mediated lipid metabolism, which may serve as a promising target for overcoming drug resistance in HCC.

Ag120-Mediated Inhibition of ASCT2-Dependent Glutamine Transport has an Anti-Tumor Effect on Colorectal Cancer Cells.

Metabolic reprogramming is considered to be a hallmark of cancer, and increased glutamine metabolism plays an important role in the progression of many tumors, including colorectal cancer (CRC). Targeting of glutamine uptake via the transporter protein ASCT2/SLC1A5 (solute carrier family 1 member 5) is considered to be an effective strategy for the treatment of malignant tumors. Here, we demonstrate that Ag120 (ivosidenib), a mutant isocitrate dehydrogenase 1 (IDH1) inhibitor approved for the treatment of certain cancers, acts as an ASCT2 inhibitor in CRC cells. Ag120 blocked glutamine uptake and metabolism, leading to reduced cell proliferation, elevated autophagy, and increased oxidative stress in CRC cells in vitro and in vivo, potentially via the ERK and mTOR signaling pathways. These effects occurred independently of mutant IDH1 activity and were supported by experiments with ASCT2-depleted or -overexpressing cells. These data identify a novel mechanism of Ag120 anti-tumor activity and support further exploration of ASCT2 inhibitors for cancer therapy.

Morphine Reduces Tumor Growth in Glioma Mouse Xenograft through Modulation of IDH1 Activity and Metabolic Reprogramming

Background Isocitrate dehydrogenase 1 (IDH1) is a key extra-mitochondrial enzyme, responsible for converting isocitrate to α-ketoglutarate. In low grade gliomas, IDH1 is often mutated (Arg132His), altering the enzymatic product to the oncometabolite 2-hydroxyglutarate (2HG) and inducing drastic metabolic reprogramming1. 2HG also inhibits DNA demethylases thus causing DNA hypermethylation. Contrary to expectation, IDH1 mutation slows tumor growth and increases susceptibility to antineoplastic agents, improving patient outcome2. We previously found that morphine, a commonly used opiate for pain management in cancer patients, is a mixed partial inhibitor of wild type IDH1. We sought to determine if morphine's effect on IDH1 activity replicates the beneficial changes caused by IDH1 mutation. Methods U87 cells (IDH1 wild type) were treated with clinically relevant concentrations of morphine (0.1-10µM) or equipotent hydromorphone for 7 days. Hydromorphone has no effect on IDH1 activity and served as a negative opiate control. DNA methylation was quantified by ELISA assay. Targeted metabolomics was performed using LC/MS. Mitochondrial function was determined using a Seahorse assay. The in vivo effect of morphine was tested using a mouse xenograft model (intraperitoneal U87 injection), with daily subcutaneous injections of morphine (5mg/kg or 10mg/kg) or hydromorphone (2mg/kg). Tumors at 30 days were measured by weight and histopathology. Cooperative capacity of morphine and the antineoplastic drug temozolomide (TMZ) was determined by measuring in vitro cell viability. Results We observed a dose-dependent increase in both DNA methylation (91%, p<0.05) and 2HG oncometabolite levels (75%, p<0.01) relative to vehicle in morphine samples. Levels of 22 metabolites were uniquely affected by morphine (fig. 1). Pathway enrichment analysis highlighted changes to glutamate metabolism, glycolysis, and the pentose phosphate pathway, all previously implicated in the beneficial IDH1 mutant glioma phenotype. Consistent with metabolic reprogramming, morphine reduced basal and ATP-coupled respiration (15%, p<0.01). Tumors from morphine treated mice were significantly smaller than vehicle (3.7-fold, p<0.01). Histological staining of these tumors revealed increased apoptosis (172%, p<0.01) and reduced proliferation (26%, p<0.01) (fig. 2). Morphine potentiated the effect of TMZ, co-treatment exhibited more cell death after 36 hours (18%, p<0.05) than either drug alone. Conclusion Our research has shown that morphine treatment, through its interaction with IDH1, is able to increase 2HG and DNA methylation. The opiate altered pathways significant in glioma metabolism and energy production. Moreover, it significantly reduced glioma tumor growth and enhanced the chemotherapeutic effect of TMZ, much like the known impact of the IDH1 mutation. We conclude that the interaction between morphine and IDH1 mimics the beneficial IDH1 mutation phenotype. Our research highlights the need for personalized medicine pathways, illustrating that that even the choice of opiate for pain management can potentially alter the course of oncologic disease. Sources 1. Dang, L. et al. Nature (2009) 2. Paldor, I. et al. J. Clin. Neurosci. (2016)

Decreased expression of IDH1 by chronic unpredictable stress suppresses proliferation and accelerates senescence of granulosa cells through ROS activated MAPK signaling pathways

Studies suggested that psychosocial stress was associated with female fertility decline, but the underlying mechanisms remained unclear. Granulosa cells (GCs) are important somatic cells to support follicular development and oocyte maturation. Herein, by using a mouse model of chronic unpredictable stress (CUS), we found that CUS induced oxidative stress damage in mouse ovaries, also inhibited GCs proliferation and accelerated GCs senescence. Isocitrate dehydrogenase-1 (IDH1), an antioxidant related gene by generating NADPH, was shown to be downregulated in GCs of CUS mice. Consistently, IDH1 knockdown inhibited cell proliferation and accelerated cellular senescence in KGN cells in vitro. In addition, IDH1 knockdown increased ROS content, induced autophagy activation and triggered cell cycle arrest in S and G2/M phases in KGN cells, which could be rescued by N-acetyl-l-cysteine (NAC), a ROS scavenger in these cells. Besides, IDH1 knockdown activated MAPK signaling pathways, including ERK, JNK and p38 signaling pathways in KGN cells, while NAC could suppress the activation. Through using inhibitors of MAPK signaling pathways, we showed that the activation of ERK pathway participated in autophagy related cell proliferation inhibition and cellular senescence, whereas JNK and p38 MAPK signaling pathways took part in regulation cell cycle arrest associated cell proliferation inhibitory and senescence in IDH1 knockdown KGN cells. Our findings suggested that downregulated expression of IDH1 induced by CUS has a physiological function in GCs proliferation and senescence through ROS activated MAPK signaling pathways, and improvement of IDH1 activity might be a beneficial therapeutic strategy for ovarian dysfunction.

AHA1 upregulates IDH1 and metabolic activity to promote growth and metastasis and predicts prognosis in osteosarcoma

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. Although activator of HSP90 ATPase activity 1 (AHA1) is reported to be a potential oncogene, its role in osteosarcoma progression remains largely unclear. Since metabolism reprogramming is involved in tumorigenesis and cancer metastasis, the relationship between AHA1 and cancer metabolism is unknown. In this study, we found that AHA1 is significantly overexpressed in osteosarcoma and related to the prognosis of osteosarcoma patients. AHA1 promotes the growth and metastasis of osteosarcoma both in vitro and in vivo. Mechanistically, AHA1 upregulates the metabolic activity to meet cellular bioenergetic needs in osteosarcoma. Notably, we identified that isocitrate dehydrogenase 1 (IDH1) is a novel client protein of Hsp90-AHA1. Furthermore, the IDH1 protein level was positively correlated with AHA1 in osteosarcoma. And IDH1 overexpression could partially reverse the effect of AHA1 knockdown on cell growth and migration of osteosarcoma. Moreover, high IDH1 level was also associated with poor prognosis of osteosarcoma patients. This study demonstrates that AHA1 positively regulates IDH1 and metabolic activity to promote osteosarcoma growth and metastasis, which provides novel prognostic biomarkers and promising therapeutic targets for osteosarcoma patients.

An acidic residue buried in the dimer interface of isocitrate dehydrogenase 1 (IDH1) helps regulate catalysis and pH sensitivity.

Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate to α-ketoglutarate (αKG) to provide critical cytosolic substrates and drive NADPH-dependent reactions like lipid biosynthesis and glutathione regeneration. In biochemical studies, the forward reaction is studied at neutral pH, while the reverse reaction is typically characterized in more acidic buffers. This led us to question whether IDH1 catalysis is pH-regulated, which would have functional implications under conditions that alter cellular pH, like apoptosis, hypoxia, cancer, and neurodegenerative diseases. Here, we show evidence of catalytic regulation of IDH1 by pH, identifying a trend of increasing kcat values for αKG production upon increasing pH in the buffers we tested. To understand the molecular determinants of IDH1 pH sensitivity, we used the pHinder algorithm to identify buried ionizable residues predicted to have shifted pKa values. Such residues can serve as pH sensors, with changes in protonation states leading to conformational changes that regulate catalysis. We identified an acidic residue buried at the IDH1 dimer interface, D273, with a predicted pKa value upshifted into the physiological range. D273 point mutations had decreased catalytic efficiency and, importantly, loss of pH-regulated catalysis. Based on these findings, we conclude that IDH1 activity is regulated, at least in part, by pH. We show this regulation is mediated by at least one buried acidic residue ∼12 Å from the IDH1 active site. By establishing mechanisms of regulation of this well-conserved enzyme, we highlight catalytic features that may be susceptible to pH changes caused by cell stress and disease.

Abstract 3732: Characterization of tumor relevant isocitrate dehydrogenase 1 (IDH1)

Abstract Isocitrate dehydrogenase 1 (IDH1) is found commonly mutated in grade II and III gliomas and secondary glioblastomas. Normally, IDH1 catalyzes the conversion of isocitrate and NADP+ to α-ketoglutarate and NADPH. However, in mutated form, IDH1 catalyzes α-ketoglutarate and NADPH into NADP+ and D-2-hydroxyglutarate, an oncometabolite. D-2-hydroxyglutarate is toxic to cells and drives a number of pro-tumorigenic pathways. To understand the molecular mechanisms of how mutated IDH1 can lead to brain cancer, we created point mutations in IDH1 to understand the effects on enzyme activity. Site-directed mutagenesis was used to generate mutations at residue 132, the most common site of mutations in cancer patients. Wild type (WT) IDH1 and mutant IDH1 were heterologously expressed in bacteria . Following purification using affinity column chromatography, steady-state kinetic assays were used to compare the catalytic activity of WT versus mutant IDH1. By elucidating the types of mutations at R132 that facilitate D2HG production, we can have a better understanding of how IDH1 mutations affect prognosis and therapeutic response. This work was funded by a Research Scholar Grant, RSG-19-075-01-TBE, from the American Cancer Society (CDS), National Institutes of Health R00 CA187594 (CDS), National Institutes of Health U54CA132384 (SDSU) &amp; U54CA132379 (UC San Diego), MARC 5T34GM008303 (SDSU), and IMSD 5R25GM058906 (SDSU), and the California Metabolic Research Foundation (SDSU). Citation Format: Nalani J. Coleman, Ella Thornberg, Danielle Caliger, Lucas Luna, Christal D. Sohl. Characterization of tumor relevant isocitrate dehydrogenase 1 (IDH1) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3732.

Abstract 3731: Targeting the isocitrate dehydrogenase 1 (IDH1) metabolic enzyme in prostate cancer

Abstract Prostate cancer cells (PCa) are dependent on the androgen receptor (AR) for their aberrant proliferation and survival. We have recently discovered that AR induces a reprogramming of PCa cell metabolism by controlling the cytoplasmic wild-type enzyme isocitrate dehydrogenase 1 (IDH1), which results in an enhanced proliferation of tumour cells. However, the specific metabolic functions of IDH1 in PCa or how to use the reliance of tumour cells on this enzyme as a therapeutic avenue, is elusive. In in vitro human PCa models, we showed that IDH1 protein levels and activity are increased in an AR-dependent manner. Using pharmacological and genetic tools, we showed that IDH1 is a major contributor to the replenishment of NADPH levels in PCa. This cofactor plays a key role in the synthesis of biomaterials required for cellular division, and our results indicate that IDH1 contribute to 30-40% of total cellular NADPH levels. In that context, blockade of IDH1 was shown to alter the mTOR signaling, a central regulator of cellular anabolism, which is linked to decreased cellular proliferation rates. FDA-approved pharmacological inhibitors of mutant IDH1 significantly inhibited IDH activity and proliferation in PCa cells, suggesting that such inhibitors could be used to treat PCa patients even in absence of IDH1 mutation. Globally, our results demonstrate that IDH1 is a key player in proliferative anabolic pathways in PCa. Importantly, they also support the hypothesis that inhibition of IDH1 using already-approved molecules represents one viable therapeutic solution. Citation Format: Kevin Gonthier, Cindy Weidmann, Lilianne Frégeau-Proulx, Étienne Audet-Walsh. Targeting the isocitrate dehydrogenase 1 (IDH1) metabolic enzyme in prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3731.

In vivo efficacy of mutant IDH1 inhibitor HMS-101 and structural resolution of distinct binding site.

Mutations in isocitrate dehydrogenase 1 (IDH1) are found in 6% of AML patients. Mutant IDH produces R-2-hydroxyglutarate (R2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators, thus linking metabolism to tumorigenesis. Here we report the biochemical characterization, in vivo antileukemic effects, structural binding and molecular mechanism of the inhibitor HMS-101, which inhibits the enzymatic activity of mutant IDH1 (IDH1mut). Treatment of IDH1mut primary AML cells reduced 2-hydroxyglutarate levels (2HG) and induced myeloid differentiation in vitro. Co-crystallization of HMS-101 and mutant IDH1 revealed that HMS-101 binds to the active site of IDH1mut in close proximity to the regulatory segment of the enzyme in contrast to other IDH1 inhibitors. HMS-101 also suppressed 2HG production, induced cellular differentiation and prolonged survival in a syngeneic mutant IDH1 mouse model and a patient-derived human AML xenograft model in vivo. Cells treated with HMS-101 showed a marked upregulation of the differentiation-associated transcription factors CEBPA and PU.1, and a decrease in cell cycle regulator cyclin A2. In addition, the compound attenuated histone hypermethylation. Together, HMS-101 is a unique inhibitor that binds to the active site of IDH1mut directly and is active in IDH1mut preclinical models.

IDH1 fine-tunes cap-dependent translation initiation.

The metabolic enzyme isocitrate dehydrogenase 1 (IDH1) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). Its mutation often leads to aberrant gene expression in cancer. IDH1 was reported to bind thousands of RNA transcripts in a sequence-dependent manner; yet, the functional significance of this RNA-binding activity remains elusive. Here, we report that IDH1 promotes mRNA translation via direct associations with polysome mRNA and translation machinery. Comprehensive proteomic analysis in embryonic stem cells (ESCs) revealed striking enrichment of ribosomal proteins and translation regulators in IDH1-bound protein interactomes. We performed ribosomal profiling and analyzed mRNA transcripts that are associated with actively translating polysomes. Interestingly, knockout of IDH1 in ESCs led to significant downregulation of polysome-bound mRNA in IDH1 targets and subtle upregulation of ribosome densities at the start codon, indicating inefficient translation initiation upon loss of IDH1. Tethering IDH1 to a luciferase mRNA via the MS2-MBP system promotes luciferase translation, independently of the catalytic activity of IDH1. Intriguingly, IDH1 fails to enhance luciferase translation driven by an internal ribosome entry site. Together, these results reveal an unforeseen role of IDH1 in fine-tuning cap-dependent translation via the initiation step.

Abstract LB-274: Identification and characterization of LY3410738, a novel covalent inhibitor of cancer-associated mutant Isocitrate Dehydrogenase 1 (IDH1)

Abstract Somatic gain-of-function mutations in IDH1 have been identified in multiple tumor types including AML, glioma, cholangiocarcinoma and chondrosarcoma. The neo-enzymatic activity of mutant IDH1 results in accumulation of the oncometabolite 2-hydroxyglutarate (2-HG), leading to a hyper-methylation phenotype, a block in cell differentiation, and tumor growth. Using a structure-based drug design approach, we have developed a highly potent covalent inhibitor of mutant IDH1. LY3410738 modifies a single cysteine (Cys269) in the allosteric binding pocket and rapidly inactivates the enzyme with a KI/Kinact = 84,257 M-1sec-1. The compound selectively inhibits the 2-HG in IDH1 mutant tumor cells without depleting the levels of a-ketoglutarate. Using patient-derived primary AML cells, we demonstrated that LY3410738 was more potent than AG120 in reversing the block in differentiation associated with IDH1 mutant activity. In vivo, LY3410738 displayed prolonged pharmacodynamic activity, depleting 2-HG levels in tumors at low circulating exposures and for an extended time after clearance of compound. Importantly, LY3410738 has the ability to cross the blood-brain barrier and can achieve concentrations in the brain that exceed those needed to engage the target. Consistent with this, LY3410738 effectively inhibits 2HG in orthotopic glioma models. Using patient-derived IDH1 mutant orthotopic AML models, we demonstrated that LY3410738 effectively inhibited 2-HG, induced differentiation, and cleared AML from mice. Collectively, LY3410738 represents the first covalent brain-penetrant mutant IDH1 inhibitor with potential for Best-in-Class activity. Citation Format: Nathan Brooks, Robin DeWalt, Serge Boulet, ZhaoHai Lu, Lisa Kays, Rachel cavitt, Sandra Gomez, John Strelow, Paul Milligan, Kenneth Roth, Renato Bauer, Stephen Antonysamy, Patric Hahn, Zoran Rankovic, Denis McCann, Gary Mo, Ramon Tiu, Timothy Burkholder, Sandaruwan Geeganage, Raymond Gilmour. Identification and characterization of LY3410738, a novel covalent inhibitor of cancer-associated mutant Isocitrate Dehydrogenase 1 (IDH1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-274.

R132 mutations in canine isocitrate dehydrogenase 1 (IDH1) lead to functional changes.

Glioma is the second most common intracranial neoplasia in dogs, but the pathogenic mechanisms remain unclear. In humans, isocitrate dehydrogenase 1 (IDH1) is frequently mutated in gliomas. Although almost all human IDH1 mutations have been identified as involving the Arg132 codon, few studies have reported structural, functional, and mutational information for canine IDH1. Therefore, in this study, we cloned the canine IDH1 homologue and used PCR mutagenesis to substitute the wildtype (WT) Arg132 with His (R132H) or Ser (R132S). WT and mutated IDH1 were overexpressed in HeLa cells, and their presence was confirmed by immunoblotting and immunocytochemistry using mutation-specific antibodies. The IDH1 activity between WT, R132H, and R132S transfectants was compared by measuring the production of NADH and NADPH. NADPH production in R132H and R132S transfectants was lower than that in WT, but NADH levels were not significantly different. Finally, we detected increased expression of hypoxia inducible factor 1 alpha (HIF-1α) in the R132H and R132S transfectants. These results indicated that the canine IDH1 Arg132 mutation has the potential to induce carcinogenesis in canine somatic cells.

Radiolabeled inhibitors as probes for imaging mutant IDH1 expression in gliomas: Synthesis and preliminary evaluation of labeled butyl-phenyl sulfonamide analogs

IntroductionMalignant gliomas frequently harbor mutations in the isocitrate dehydrogenase 1 (IDH1) gene. Studies suggest that IDH mutation contributes to tumor pathogenesis through mechanisms that are mediated by the neomorphic metabolite of the mutant IDH1 enzyme, 2-hydroxyglutarate (2-HG). The aim of this work was to synthesize and evaluate radiolabeled compounds that bind to the mutant IDH1 enzyme with the goal of enabling noninvasive imaging of mutant IDH1 expression in gliomas by positron emission tomography (PET). MethodsA small library of nonradioactive analogs were designed and synthesized based on the chemical structure of reported butyl-phenyl sulfonamide inhibitors of mutant IDH1. Enzyme inhibition assays were conducted using purified mutant IDH1 enzyme, IDH1-R132H, to determine the IC50 and the maximal inhibitory efficiency of the synthesized compounds. Selected compounds, 1 and 4, were labeled with radioiodine (125I) and/or 18F using bromo- and phenol precursors, respectively. In vivo behavior of the labeled inhibitors was studied by conducting tissue distribution studies with [125I]1 in normal mice. Cell uptake studies were conducted using an isogenic astrocytoma cell line that carried a native IDH1-R132H mutation to evaluate the potential uptake of the labeled inhibitors in IDH1-mutated tumor cells. ResultsEnzyme inhibition assays showed good inhibitory potency for compounds that have iodine or a fluoroethoxy substituent at the ortho position of the phenyl ring in compounds 1 and 4 with IC50 values of 1.7 μM and 2.3 μM, respectively. Compounds 1 and 4 inhibited mutant IDH1 activity and decreased the production of 2-HG in an IDH1-mutated astrocytoma cell line. Radiolabeling of 1 and 4 was achieved with an average radiochemical yield of 56.6 ± 20.1% for [125I]1 (n = 4) and 67.5 ± 6.6% for [18F]4 (n = 3). [125I]1 exhibited favorable biodistribution characteristics in normal mice, with rapid clearance from the blood and elimination via the hepatobiliary system by 4 h after injection. The uptake of [125I]1 in tumor cells positive for IDH1-R132H was significantly higher compared to isogenic WT-IDH1 controls, with a maximal uptake ratio of 1.67 at 3 h post injection. Co-incubation of the labeled inhibitors with the corresponding nonradioactive analogs, and decreasing the normal concentrations of FBS (10%) in the incubation media substantially increased the uptake of the labeled inhibitors in both the IDH1-mutant and WT-IDH1 tumor cell lines, suggesting significant non-specific binding of the synthesized labeled butyl-phenyl sulfonamide inhibitors. ConclusionsThese data demonstrate the feasibility of developing radiolabeled probes for the mutant IDH1 enzyme based on enzyme inhibitors. Further optimization of the labeled inhibitors by modifying the chemical structure to decrease the lipophilicity and to increase potency may yield compounds with improved characteristics as probes for imaging mutant IDH1 expression in tumors.

Inhibition of Cancer-Associated Mutant Isocitrate Dehydrogenases by 2-Thiohydantoin Compounds.

Somatic mutations of isocitrate dehydrogenase 1 (IDH1) at R132 are frequently found in certain cancers such as glioma. With losing the activity of wild-type IDH1, the R132H and R132C mutant proteins can reduce α-ketoglutaric acid (α-KG) to d-2-hydroxyglutaric acid (D2HG). The resulting high concentration of D2HG inhibits many α-KG-dependent dioxygenases, including histone demethylases, to cause broad histone hypermethylation. These aberrant epigenetic changes are responsible for the initiation of these cancers. We report the synthesis, structure-activity relationships, enzyme kinetics, and binding thermodynamics of a novel series of 2-thiohydantoin and related compounds, among which several compounds are potent inhibitors of mutant IDH1 with Ki as low as 420 nM. X-ray crystal structures of IDH1(R132H) in complex with two inhibitors are reported, showing their inhibitor-protein interactions. These compounds can decrease the cellular concentration of D2HG, reduce the levels of histone methylation, and suppress the proliferation of stem-like cancer cells in BT142 glioma with IDH1 R132H mutation.

IDH1 regulates phospholipid metabolism in developing astrocytes

Isocitrate dehydrogenase 1 (IDH1) is an evolutionarily conserved enzyme that catalyzes the interconversion of isocitrate to α-ketoglutarate with the concomitant reduction of NADP+ to NADPH. IDH1 has previously been shown to participate in lipid biosynthesis in various tissues such as the liver and adipose tissue. We examined the potential role of IDH1 in phospholipid metabolism in the brain. Here we show that IDH1 is highly expressed in the brain and astrocytes during embryonic development and the postnatal period and subsequently declines in adulthood. Silencing of IDH1 expression using siRNA in astrocytes isolated from E18.5 mouse cortices led to increased incorporation of [3H]-palmitate into the phosphatidylcholines (PCs) and decreased the incorporation of [3H]-palmitate into sphingomyelin and the phosphatidylethanolamines (PEs). In pulse-chase experiments, knock-down of IDH1 expression impaired the turnover of PCs and decreased the synthesis of PEs. The decrease in [3H]-palmitate incorporation into PEs when IDH1 was knocked-down in astrocytes was not due to impairments within the CDP-ethanolamine pathway or in the rate of decarboxylation of phosphatidylserine (PS). In conclusion, our results reveal a role for IDH1 in the synthesis/turnover of phospholipids in developing astrocytes and highlight the lipid alterations resulting from the loss of wild-type IDH1 activity.

Non-invasive in vivo assessment of IDH1 mutational status in glioma

Gain-of-function mutations of the isocitrate dehydrogenase 1 (IDH1) gene are among the most prevalent in low-grade gliomas and secondary glioblastoma. They lead to intracellular accumulation of the oncometabolite 2-hydroxyglutarate, represent an early pathogenic event and are considered a therapeutic target. Here we show, in this proof-of-concept study, that [1-(13)C] α-ketoglutarate can serve as a metabolic imaging agent for non-invasive, real-time, in vivo monitoring of mutant IDH1 activity, and can inform on IDH1 status. Using (13)C magnetic resonance spectroscopy in combination with dissolution dynamic nuclear polarization, the metabolic fate of hyperpolarized [1-(13)C] α-ketoglutarate is studied in isogenic glioblastoma cells that differ only in their IDH1 status. In lysates and tumours that express wild-type IDH1, only hyperpolarized [1-(13)C] α-ketoglutarate can be detected. In contrast, in cells that express mutant IDH1, hyperpolarized [1-(13)C] 2-hydroxyglutarate is also observed, both in cell lysates and in vivo in orthotopic tumours.

Renal cell carcinoma with a novel germ-line mutation in isocitrate dehydrogenase 1 (IDH1) and its functional study.

e15568 Background: Recently, omics study such as metabolome analysis and whole genome sequencing has been applied to various disorders. We predicted the dysfunction of isocitrate dehydrogenase 1 (IDH1) in renal cell carcinoma (RCC) using metabolome analysis and identified a novel mutation of IDH1. In addition, we examined its function. Methods: We determined the global-scale metabolome profiling of human RCC by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), and compared the metabolite levels of tumors and paired normal tissues in 10 patients with RCC. We performed the genotyping of IDH1 and von Hippel-Lindau (VHL) gene. We also performed transfection experiments by using Lipofectamine and measured the enzymatic activity of IDH1 in RCC cell lines. All protocols were performed by the Institutional Review Board (IRB) of Hamamatsu University School of Medicine and Keio University. Informed consent was obtained from the patient in accordance with the IRB of Hamamatsu University School of Medicine. Results: The acceleration of glycolysis with low level of NADPH and citrate in one patient led to IDH1 dysfunction followed by identifying a novel missense mutation, V178I. In transfection experiment, the reductive activity of IDH1 variant (V178I) increased 2.5 times than that of IDH1 wild type while the oxidative activity of IDH1 variant oxidative activity was as well as that of IDH1 wild type. Furthermore, in the metabolome analysis using labeling glutamine, RCC cell lines deficient in the von Hippel-Lindau (VHL) protein preferentially use reductive glutamine metabolism. On the other hand, this patient with V178I mutation of IDH1 had no VHL mutation. Conclusions: Metabolome analysis led to the identification a novel IDH1 missense mutation in RCC. This mutation might be involved with acceleration of reductive reaction in tricarboxylic acid cycle. Metabolome analysis using CE-TOFMS could be a powerful tool for the global-scale exploration of disease-specific metabolism.

NADP+-dependent IDH1R132 mutation and its relevance for glioma patient survival

The isocitrate dehydrogenase 1 (IDH1) mutation occurs in high frequency in glioma and secondary glioblastoma (GBM). Mutated IDH1 produces the oncometabolite 2-hydroxyglutarate rather than α-ketoglutarate or isocitrate. The oncometabolite is considered to be the major cause of the association between the IDH1 mutation and gliomagenesis. On the other hand, the IDH1 mutation in GBM is associated with prolonged patient survival. This association is not well understood yet but IDH1 involvement in epigenetic silencing of O-6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme is considered to be an important mechanism. However, it was shown recently that the IDH1 mutation and MGMT silencing are independent prognostic factors. Here, we hypothesize that the IDH1 mutation reduces the capacity to produce NADPH and thus reduces the capacity to scavenge reactive oxygen species that are generated during irradiation and chemotherapy. IDH1 activity is responsible for two-thirds of the NADPH production capacity in normal brain, whereas the IDH1 mutation reduces this capacity by almost 40%. Therefore, we hypothesize that the reduced NADPH production capacity due to the IDH1 mutation renders GBM cells more vulnerable to irradiation and chemotherapy thus prolonging survival of the patients.

Isocitrate dehydrogenase 1 is downregulated during early skin tumorigenesis which can be inhibited by overexpression of manganese superoxide dismutase

Isocitrate dehydrogenase 1 (IDH1), a cytosolic enzyme that converts isocitrate to alpha-ketoglutarate, has been shown to be dysregulated during tumorigenesis. However, at what stage of cancer development IDH1 is dysregulated and how IDH1 may affect cell transformation and tumor promotion during early stages of cancer development are unclear. We used a skin cell transformation model and mouse skin epidermal tissues to study the role of IDH1 in early skin tumorigenesis. Our studies demonstrate that both the tumor promoter TPA and UVC irradiation decreased expression and activity levels of IDH1, not IDH2, in the tumor promotable JB6 P+ cell model. Skin epidermal tissues treated with dimethylbenz[α]anthracene/TPA also showed decreases in IDH1 expression and activity. In non-promotable JB6 P-cells, IDH1 was upregulated upon TPA treatment, whereas IDH2 was maintained at similar levels with TPA treatment. Interestingly, IDH1 knockdown enhanced, whereas IDH1 overexpression suppressed, TPA-induced cell transformation. Finally, manganese superoxide dismutase overexpression suppressed tumor promoter induced decreases in IDH1 expression and mitochondrial respiration, while intracellular alpha-ketoglutarate levels were unchanged. These results suggest that decreased IDH1 expression in early stage skin tumorigenesis is highly correlated with tumor promotion. In addition, oxidative stress might contribute to IDH1 inactivation, because manganese superoxide dismutase, a mitochondrial antioxidant enzyme, blocked decreases in IDH1 expression and activity.

R132H-mutation of isocitrate dehydrogenase-1 is not sufficient for HIF-1α upregulation in adult glioma

R132H-mutation of isocitrate dehydrogenase-1 is not sufficient for HIF-1α upregulation in adult glioma