isocitrate dehydrogenase 1 and 2
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Malignant transformation in gliomas is frequently supplemented by somatic mutations in isocitrate dehydrogenase 1 and isocitrate dehydrogenase 2 genes. It has recently emerged that mutations in these genes are associated with prolonged survival and should be used as prognostic factor in management of brain cancer patients. There are several approaches in use for the detection of isocitrate dehydrogenase 1 and 2 mutations; however, these often exhibit shortcomings such as convoluted protocols with long processing time, complex (and costly) dedicated fluorescent probes, and/or demand on amounts of input DNA. Therefore, a simple and rapid method would be highly desired. Here, we present development and validation of simple and reliable isocitrate dehydrogenase 1 and 2 mutation detection assay using denaturing capillary electrophoresis. The detection sensitivity in terms of the limiting mutated allele fraction detectable estimated from a series of dilution runs was 2.9%. The method was validated by comparing to results obtained by a widely accepted detection technique, the multiplex ligation-dependent probe amplification, on a set of 85 brain tumors. The concordance of both methods was 100%, but denaturing capillary electrophoresis assay required fivefold lower input of DNA (1 versus 5 μL of DNA at concentrations typically between 10 and 30 ng/μL).
- MeSH
- alely MeSH
- elektroforéza kapilární metody MeSH
- isocitrátdehydrogenasa genetika MeSH
- lidé MeSH
- mutace * MeSH
- nádory mozku diagnóza enzymologie genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
Over the past few years, new biomarkers have allowed a deeper insight into gliomagenesis and facilitated the identification of possible targets for glioma therapy. Isocitrate dehydrogenase (IDH) 1 and IDH2 mutations have been shown to be promising biomarkers for monitoring disease prognosis and predicting the response to treatment. This review summarizes recent findings in this field. Web of Science, Science Direct, and PubMed online databases were used to search for publications investigating the role of IDH in glioma. References were identified by searching for the keywords "IDH1 or IDH2 and glioma and diagnostic or predictive or prognostic" in papers published from January, 2008, to April, 2014. Only papers in English were reviewed. Publications available only as an abstract were not included. IDH1/2 mutations are tightly associated with grade II and III gliomas and secondary glioblastomas, with better prognosis and production of a recently described oncometabolite, 2-hydroxyglutarate (2HG). Although the contradictory positive effect of IDH mutation on prognosis and negative role of 2HG in tumor transformation remain unresolved, the future direction of personalized treatment strategies targeted to glioma development is likely to focus on IDH1/2 mutations.
Many types of tumors harbor a mutation in the gene for the metabolic enzyme isocitrate dehydrogenase result-ing in the production of the oncometabolite d-2-hydroxy glutarate with a pleiotropic effect on the cell. The paper provides an overview of the latest knowledge of the me-chanism of carcinogenesis, of specific drugs suppressingd-2-hydroxy glutarate production and of the causes of resistance complicating treatment based on specific inhibi-tors of oncogenic forms of isocitate dehydrogenase.
Wild type mitochondrial isocitrate dehydrogenase (IDH2) was previously reported to produce oncometabolite 2-hydroxyglutarate (2HG). Besides, mitochondrial deacetylase SIRT3 has been shown to regulate the oxidative function of IDH2. However, regulation of 2HG formation by SIRT3-mediated deacetylation was not investigated yet. We aimed to study mitochondrial IDH2 function in response to acetylation and deacetylation, and focus specifically on 2HG production by IDH2. We used acetylation surrogate mutant of IDH2 K413Q and assayed enzyme kinetics of oxidative decarboxylation of isocitrate, 2HG production by the enzyme, and 2HG production in cells. The purified IDH2 K413Q exhibited lower oxidative reaction rates than IDH2 WT. 2HG production by IDH2 K413Q was largely diminished at the enzymatic and cellular level, and knockdown of SIRT3 also inhibited 2HG production by IDH2. Contrary, the expression of putative mitochondrial acetylase GCN5L likely does not target IDH2. Using mass spectroscopy, we further identified lysine residues within IDH2, which are the substrates of SIRT3. In summary, we demonstrate that 2HG levels arise from non-mutant IDH2 reductive function and decrease with increasing acetylation level. The newly identified lysine residues might apply in regulation of IDH2 function in response to metabolic perturbations occurring in cancer cells, such as glucose-free conditions.
- MeSH
- acetylace MeSH
- glutaráty metabolismus MeSH
- isocitrátdehydrogenasa genetika metabolismus MeSH
- isocitráty chemie MeSH
- lidé MeSH
- mitochondrie metabolismus MeSH
- nádorové buněčné linie MeSH
- NADP metabolismus MeSH
- oxidace-redukce MeSH
- proteiny nervové tkáně metabolismus MeSH
- sirtuin 3 metabolismus MeSH
- umlčování genů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
V roce 2008 byly mutace u isocitrát dehydrogenázy 1 a 2 (mIDH 1 a 2) poprvé z celé řady typů solidních nádorů zjištěny u multiformního glioblastomu (GBM). Rok nato, tedy v roce 2009, byla prokázána přítomnost takové mutace u akutní myeloidní leukemie (AML). Blokátor mIDH 2 enasidenib se dostal na trh poprvé jako lék pro relabující/refrakterní AML u dospělých pacientů v roce 2017. V roce 2019 byl však pro léčbu relabující/refrakterní AML schválen blokátor mIDH 1 ivosidenib (AG-120). Koncem roku 2019 byl schválen i pro neléčené dospělé pacienty s AML. Již v průběhu časné fáze klinického hodnocení dostal vzhledem ke slibným výsledkům možnost využití v rámci programu rozšířeného přístupu. Zajímavé je, jak molekula léčiva procházela preklinickou fází a regulačním procesem. Stojí za povšimnutí, že obdržela rozhodnutí o registraci, i když ještě nebyla ukončena třetí fáze klinického hodnocení. Klinická hodnocení GBM a AML začala v březnu 2014. Rovněž probíráme okolnosti schválení ivosidenibu, a to nejdříve v případě dospělých pacientů s relabující/refrakterní AML a nikoli gliomu, kde byla mutace poprvé objevena.
In 2008, mutations in isocitric dehydrogenase 1 & 2 (mIDH 1 & 2) was first detected in glioblastoma multiforme (GBM) from among many types of solid tumour. Next year i.e. in 2009 acute myelogenous leukemia (AML) showed presence such mutation. mIDH 2 blocker enasidenib came into market for the first time for adult recurrent and relapsed AML in 2017. But mIDH 1 blocker ivosidenib (AG-120) is approved in 2019 in recurrent and relapsed AML. In the end 2019 it was approved for untreated adult AML. When it was in early phase trial it became eligible for special expanded access program as it showed encouraging results. Drug molecule's progress through preclinical and regulatory path is interesting. It is worthwhile to look into how it received market authorization while phase III trial was not over. Trial of both GBM and AML started in March 2014. Circumstances behind ivosidenib being first approved in adult recurrent and relapsed AML rather than in glioma where the mutation was first found are also discussed.
- Klíčová slova
- Ivosidenib, mIDH1,
- MeSH
- akutní myeloidní leukemie * farmakoterapie metabolismus MeSH
- cholangiokarcinom farmakoterapie MeSH
- glioblastom farmakoterapie MeSH
- glycin analogy a deriváty MeSH
- inhibitory enzymů terapeutické užití MeSH
- isocitrátdehydrogenasa antagonisté a inhibitory genetika účinky léků MeSH
- protinádorové látky terapeutické užití MeSH
- pyridiny terapeutické užití MeSH
Targeting mutations that trigger acute myeloid leukaemia (AML) has emerged as a refined therapeutic approach in recent years. Enasidenib (Idhifa) is the first selective inhibitor of mutated forms of isocitrate dehydrogenase 2 (IDH2) approved against relapsed/refractory AML. In addition to its use as monotherapy, a combination trial of enasidenib with standard intensive induction therapy (daunorubicin + cytarabine) is being evaluated. This study aimed to decipher enasidenib off-target molecular mechanisms involved in anthracycline resistance, such as reduction by carbonyl reducing enzymes (CREs) and drug efflux by ATP-binding cassette (ABC) transporters. We analysed the effect of enasidenib on daunorubicin (Daun) reduction by several recombinant CREs and different human cell lines expressing aldo-keto reductase 1C3 (AKR1C3) exogenously (HCT116) or endogenously (A549 and KG1a). Additionally, A431 cell models overexpressing ABCB1, ABCG2, or ABCC1 were employed to evaluate enasidenib modulation of Daun efflux. Furthermore, the potential synergism of enasidenib over Daun cytotoxicity was quantified amongst all the cell models. Enasidenib selectively inhibited AKR1C3-mediated inactivation of Daun in vitro and in cell lines expressing AKR1C3, as well as its extrusion by ABCB1, ABCG2, and ABCC1 transporters, thus synergizing Daun cytotoxicity to overcome resistance. This work provides in vitro evidence on enasidenib-mediated targeting of the anthracycline resistance actors AKR1C3 and ABC transporters under clinically achievable concentrations. Our findings may encourage its combination with intensive chemotherapy and even suggest that the effectiveness of enasidenib as monotherapy against AML could lie beyond the targeting of mIDH2.
- MeSH
- ABC transportéry metabolismus MeSH
- adenosintrifosfát MeSH
- akutní myeloidní leukemie * farmakoterapie genetika MeSH
- antracykliny MeSH
- cytarabin terapeutické užití MeSH
- daunomycin * farmakologie MeSH
- isocitrátdehydrogenasa genetika metabolismus terapeutické užití MeSH
- lidé MeSH
- protinádorová antibiotika terapeutické užití MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Methyltriphenylphosphonium (TPMP) salts have been widely used to measure the mitochondrial membrane potential and the triphenylphosphonium (TPP+) moiety has been attached to many bioactive compounds including antioxidants to target them into mitochondria thanks to their high affinity to accumulate in the mitochondrial matrix. The adverse effects of these compounds on cellular metabolism have been insufficiently studied and are still poorly understood. Micromolar concentrations of TPMP cause a progressive inhibition of cellular respiration in adherent cells without a marked effect on mitochondrial coupling. In permeabilized cells the inhibition was limited to NADH-linked respiration. We found a mixed inhibition of the Krebs cycle enzyme 2-oxoglutarate dehydrogenase complex (OGDHC) with an estimated IC50 3.93 [3.70-4.17] mM, which is pharmacologically plausible since it corresponds to micromolar extracellular concentrations. Increasing the lipophilic character of the used TPP+ compound further potentiates the inhibition of OGDHC activity. This effect of TPMP on the Krebs cycle ought to be taken into account when interpreting observations on cells and mitochondria in the presence of TPP+ derivatives. Compounds based on or similar to TPP+ derivatives may also be used to alter OGDHC activity for experimental or therapeutic purposes.
- MeSH
- buněčné linie MeSH
- citrátový cyklus účinky léků MeSH
- citrátsynthasa účinky léků metabolismus MeSH
- glutamátdehydrogenasa účinky léků metabolismus MeSH
- isocitrátdehydrogenasa účinky léků metabolismus MeSH
- ketoglutarátdehydrogenasový komplex antagonisté a inhibitory metabolismus MeSH
- kosterní svaly enzymologie MeSH
- krysa rodu rattus MeSH
- malátdehydrogenasa účinky léků metabolismus MeSH
- membránový potenciál mitochondrií účinky léků MeSH
- oniové sloučeniny farmakologie MeSH
- potkani Wistar MeSH
- pyruvátdehydrogenasový komplex účinky léků metabolismus MeSH
- svalové mitochondrie účinky léků enzymologie MeSH
- tritylové sloučeniny farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Východiska: Mutace isocitrátdehydrogenázy 1 a 2 (IDH1/2) jsou slibným prognostickým biomarkerem gliálních nádorů. Cílem naší studie bylo ověřit prognostický efekt IDH1/2 mutací na skupině pacientů s gliálními nádory z České republiky při použití jednoduché a spolehlivé IDH genotypizace. Materiál a metody: U 145 pacientů s gliálními nádory bylo provedeno vyšetření tří nejčastějších IDH mutací IDH1 R132H, IDH1 R132C a IDH2 R172K pomocí kompetitivní polymerazové řetězové reakce (PCR) amplifikace amplikonů s odlišnou teplotou tání (competitive amplification of differentially melting amplicons – CADMA PCR). Dále byla stanovena metylace promotoru O6-metylguanine-DNA metyltransferáza (MGMT), počet kopií genů EGFR, p53, RB1, MDM2, CDKN2A a chromozomálních regionů 1p, 19q a 10p. Výsledky byly korelovány s klinickými charakteristikami pacientů. Výsledky: IDH mutace byly pozitivně asociovány s MGMT metylací (OR 3,08, 95% CI 1,387–7,282; p = 0,007), 1p/19q kodelecí (OR 8,85, 95% CI 2,367–42,786; p = 0,002) a negativně asociovány s EGFR amplifikací (OR 0,12, 95% CI 0,019–0,437; p = 0,006) a ztrátou 10p (OR 0,09, 95% CI 0,005–0,436; p = 0,019). Celkové přežívání ve skupině IDH-mutovaných glioblastomů bylo 25 měsíců, zatímco u IDH-wild-type glioblastomů pouze 9 měsíců (p = 0,035) a současně se přežívání pacientů s metylovaným vs. nemetylovaným promotorem MGMT významně nelišilo (p = 0,166). Závěr: Navzdory tomu, že IDH1/2 mutace jsou úzce asociovány s MGMT metylací u pacientů s gliálními nádory, ve skupině glioblastomů se IDH1/2 mutace jeví jako silnější prognostický marker než MGMT metylace a měly by být biomarkerem první volby pro určení prognózy gliálního nádoru, zvláště při použití genotypizační metody CADMA PCR.
Background: Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are a promising prognostic biomarker of gliomas. The purpose of our study was to examine the clinical prognostic properties of IDH1/2 mutations in a glioma patient cohort from the Czech Republic using an improved platform for simple and reliable IDH genotyping. Material and Methods: We retrospectively analyzed a group of 145 glioma patients by testing for the three most frequent IDH mutations, IDH1 R132H, IDH1 R132C, and IDH2 R172K, through the competitive amplification of differentially melting amplicons (CADMA) polymerase chain reaction (PCR). O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation, copy number of EGFR, p53, RB1, MDM2, CDKN2A genes, and deletions in 1p, 19q and 10p chromosomal regions were also analyzed and correlated with clinical characteristics. Results: Of 145 gliomas, 36 harbored IDH1 R132H mutation and 1 IDH1 R132C mutation. We did not detect any IDH2 R172K mutation. IDH1 mutations were positively associated with MGMT methylation (OR 3.08, 95% CI 1.387–7.282; p = 0.007), 1p/19q co-loss (OR 8.85, 95% CI 2.367–42.786; p = 0.002) and negatively associated with epidermal growth factor receptor amplification (OR 0.12, 95% CI 0.019–0.437; p = 0.006) and 10p loss (OR 0.09, 95% CI 0.005–0.436; p = 0.019). The overall survival of IDH-mutant was 25 months, but only 9 months in IDH-wild type gliomas (p = 0.035); at the same time, survival associated with methylated vs. unmethylated MGMT promoter did not significantly differ (p = 0.166). Conclusion: Despite IDH1 mutations being closely associated with MGMT methylation in glioma patients, IDH1 mutations in glioblastoma patients are stronger marker of overall survival than MGMT methylation and should be the marker of choice, especially when using genotyping by CADMA PCR.
- MeSH
- dospělí MeSH
- glioblastom * diagnóza genetika MeSH
- gliom * diagnóza genetika MeSH
- isocitrátdehydrogenasa MeSH
- klinická studie jako téma MeSH
- lidé středního věku MeSH
- lidé MeSH
- mutace MeSH
- nádorové biomarkery * MeSH
- polymerázová řetězová reakce MeSH
- senioři MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- senioři MeSH
Significance: Cancer cells are stabilized in an undifferentiated state similar to stem cells. This leads to profound modifications of their metabolism, which further modifies their genetics and epigenetics as malignancy progresses. Specific metabolites and enzymes may serve as clinical markers of cancer progression. Recent Advances: Both 2-hydroxyglutarate (2HG) enantiomers are associated with reprogrammed metabolism, in grade III/IV glioma, glioblastoma, and acute myeloid leukemia cells, and numerous other cancer types, while acting also in the cross talk of tumors with immune cells. 2HG contributes to specific alternations in cancer metabolism and developed oxidative stress, while also inducing decisions on the differentiation of naive T lymphocytes, and serves as a signal messenger in immune cells. Moreover, 2HG inhibits chromatin-modifying enzymes, namely 2-oxoglutarate-dependent dioxygenases, and interferes with hypoxia-inducible factor (HIF) transcriptome reprogramming and mammalian target of rapamycin (mTOR) pathway, thus dysregulating gene expression and further promoting cancerogenesis. Critical Issues: Typically, heterozygous mutations within the active sites of isocitrate dehydrogenase isoform 1 (IDH1)R132H and mitochondrial isocitrate dehydrogenase isoform 2 (IDH2)R140Q provide cells with millimolar r-2-hydroxyglutarate (r-2HG) concentrations, whereas side activities of lactate and malate dehydrogenase form submillimolar s-2-hydroxyglutarate (s-2HG). However, even wild-type IDH1 and IDH2, notably under shifts toward reductive carboxylation glutaminolysis or changes in other enzymes, lead to "intermediate" 0.01-0.1 mM 2HG levels, for example, in breast carcinoma compared with 10-8M in noncancer cells. Future Directions: Uncovering further molecular metabolism details specific for given cancer cell types and sequence-specific epigenetic alternations will lead to the design of diagnostic approaches, not only for predicting patients' prognosis or uncovering metastases and tumor remissions but also for early diagnostics.
- MeSH
- energetický metabolismus MeSH
- epigeneze genetická MeSH
- glutaráty metabolismus MeSH
- imunomodulace MeSH
- isocitrátdehydrogenasa genetika metabolismus MeSH
- lidé MeSH
- mutace MeSH
- náchylnost k nemoci * MeSH
- nádorové kmenové buňky metabolismus MeSH
- nádory etiologie metabolismus patologie MeSH
- oxidace-redukce MeSH
- progrese nemoci MeSH
- regulace genové exprese u nádorů MeSH
- signální transdukce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Mitochondrial NADPH-dependent isocitrate dehydrogenase, IDH2, and cytosolic IDH1, catalyze reductive carboxylation of 2-oxoglutarate. Both idh2 and idh1 monoallelic mutations are harbored in grade 2/3 gliomas, secondary glioblastomas and acute myeloid leukemia. Mutant IDH1/IDH2 enzymes were reported to form an oncometabolite r-2-hydroxyglutarate (2HG), further strengthening malignancy. We quantified CO2-dependent reductive carboxylation glutaminolysis (RCG) and CO2-independent 2HG production in HTB-126 and MDA-MB-231 breast carcinoma cells by measuring (13)C incorporation from 1-(13)C-glutamine into citrate, malate, and 2HG. For HTB-126 cells, (13)C-citrate, (13)C-malate, and (13)C-2-hydroxyglutarate were enriched by 2-, 5-, and 15-fold at 5mM glucose (2-, 2.5-, and 13-fold at 25 mM glucose), respectively, after 6 h. Such enrichment decreased by 6% with IDH1 silencing, but by 30-50% upon IDH2 silencing while cell respiration and ATP levels rose up to 150%. Unlike 2HG production RCG declined at decreasing CO2. At hypoxia (5% O2), IDH2-related and unrelated (13)C-accumulation into citrate and malate increased 1.5-2.5-fold with unchanged IDH2 expression; whereas hypoxic 2HG formation did not. (13)C-2HG originated by ∼50% from other than IDH2 or IDH1 reactions, substantiating remaining activity in IDH1&2-silenced cells. Relatively high basal (12)C-2HG levels existed (5-fold higher vs. non-tumor HTB-125 cells) and (13)C-2HG was formed despite the absence of any idh2 and idh1 mutations in HTB-126 cells. Since RCG is enhanced at hypoxia (frequent in solid tumors) and 2HG can be formed without idh1/2 mutations, we suggest 2HG as an analytic marker (in serum, urine, or biopsies) predicting malignancy of breast cancer in all patients.
- MeSH
- glutaráty metabolismus MeSH
- hypoxie buňky fyziologie MeSH
- isocitrátdehydrogenasa genetika metabolismus MeSH
- kyslík metabolismus MeSH
- lidé MeSH
- nádorové biomarkery genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- nádory prsu enzymologie genetika metabolismus MeSH
- parciální tlak MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH