Increased mitochondrial activity in a novel IDH1-R132H mutant human oligodendroglioma xenograft model: in situ detection of 2-HG and α-KG
- Anna C Navis1,
- Simone P Niclou2,
- Fred Fack2,
- Daniel Stieber2,
- Sanne van Lith1,
- Kiek Verrijp1,
- Alan Wright3,
- Jonathan Stauber4,
- Bastiaan Tops1,
- Irene Otte-Holler1,
- Ron A Wevers5,
- Arno van Rooij5,
- Stefan Pusch6, 7,
- Andreas von Deimling6, 7,
- Wikky Tigchelaar8,
- Cornelis JF van Noorden8,
- Pieter Wesseling†1, 9 and
- William PJ Leenders†1Email author
© Navis et al.; licensee BioMed Central Ltd. 2013
Received: 03 May 2013
Accepted: 09 May 2013
Published: 29 May 2013
Point mutations in genes encoding NADP+-dependent isocitrate dehydrogenases (especially IDH1) are common in lower grade diffuse gliomas and secondary glioblastomas and occur early during tumor development. The contribution of these mutations to gliomagenesis is not completely understood and research is hampered by the lack of relevant tumor models. We previously described the development of the patient-derived high-grade oligodendroglioma xenograft model E478 that carries the commonly occurring IDH1-R132H mutation. We here report on the analyses of E478 xenografts at the genetic, histologic and metabolic level.
LC-MS and in situ mass spectrometric imaging by LESA-nano ESI-FTICR revealed high levels of the proposed oncometabolite D-2-hydroxyglutarate (D-2HG), the product of enzymatic conversion of α-ketoglutarate (α-KG) by IDH1-R132H, in the tumor but not in surrounding brain parenchyma. α-KG levels and total NADP+-dependent IDH activity were similar in IDH1-mutant and -wildtype xenografts, demonstrating that IDH1-mutated cancer cells maintain α-KG levels. Interestingly, IDH1-mutant tumor cells in vivo present with high densities of mitochondria and increased levels of mitochondrial activity as compared to IDH1-wildtype xenografts. It is not yet clear whether this altered mitochondrial activity is a driver or a consequence of tumorigenesis.
The oligodendroglioma model presented here is a valuable model for further functional elucidation of the effects of IDH1 mutations on tumor metabolism and may aid in the rational development of novel therapeutic strategies for the large subgroup of gliomas carrying IDH1 mutations.
KeywordsGlioma IDH mutations Xenograft D-2-hydroxyglutarate α-ketoglutarate Mitochondria: LESA-nano ESI-FTICR
Diffuse gliomas are notoriously difficult to treat, and remain incurable to this date. Based on WHO guidelines, these tumors are categorized in grade II-IV, grade IV (glioblastoma) being the most aggressive subtype . Grade IV gliomas may develop de novo (primary glioblastoma) or evolve from lower grade tumors (secondary glioblastoma) [2–5]. The role of specific tumor suppressor genes and oncogenes in gliomagenesis has rapidly been elucidated in the last decades [6–12]. A recent breakthrough was the discovery of the involvement of mutations in the genes for isocitrate dehydrogenase 1 (IDH1) and, less frequently, IDH2 [13–15] in grade II/III gliomas and secondary glioblastomas. IDH mutations are uncommon in other tumor types, with the exception of acute myeloid leukemia (AML), angioimmunoblastic T-cell lymphomas, intrahepatic cholangiocarcinomas and chondrosarcomas [13, 16–19]. Mutations in glioma almost always involve an arginine-to-histidine conversion at position 132 in the catalytic site of IDH1 [14, 20, 21].
The high frequency of mutations in IDH1 suggests an important role for the mutant protein in early glioma development, but the exact underlying oncogenic mechanism is not completely understood. IDH1 is a cytoplasmic enzyme that converts isocitrate to α-ketoglutarate (α-KG), with simultaneous reduction of NADP+ to NADPH . The other family members IDH2 and −3 reside in mitochondria and are either NADP+- (IDH2) or NAD+-dependent (IDH3). For as yet unknown reasons, the occurrence of IDH mutations in glioma is restricted to the NADP+ dependent variants .
The R132H mutation equips the enzyme with a neomorphic activity resulting in a reduction of α-KG to D-2-hydroxyglutarate (D-2HG) , an NADPH consuming process. D-2HG may be an ‘oncometabolite’, but whether and how it contributes to gliomagenesis is a matter of debate. Patients with D-2-hydroxyglutaric aciduria, a rare metabolic condition in which high levels of D-2HG occur due to mutations in the D-2HG dehydrogenase gene D2HGDH or in IDH2, do not show increased levels of tumor development . Paradoxically, the metabolic condition L-2-hydroxyglutaric aciduria has been suggested to predispose for brain tumorigenesis [26, 27]. Depletion of α-KG, an essential citric acid cycle intermediate, in IDH1 mutant tumor cells may play a role during tumor development  but competitive inhibition by D-2HG of the 60 known α-KG-dependent enzymes appears to be a more important factor . For example, inhibition of α-KG-dependent TET2 and histone demethylase KDM4C (also known as JMJD2C) results in DNA hypermethylation and histone demethylation respectively and, as a result, blocks cell differentiation [30–32]. Furthermore, increased degradation of hypoxia-inducible factor 1α (HIF1α) via inhibition of α-KG dependent EGLN prolyl 4-hydroxylases may play a role . Recently, it was described that this mechanism contributes to leukemogenesis in a reversible manner .
IDH1 mutations nearly always occur in a heterozygous fashion and the presence of a wild type (wt) allele is required for the maximal neomorphic activity of the mutant protein . The enzyme consists of two independently acting subunits. Wild type enzyme subunits, either as homodimer or in mutant/wt heterodimers, convert isocitrate to α-KG which is subsequently processed by the mutant enzyme (subunits) to D-2HG . Whereas wtIDH1 can convert α-KG back to isocitrate in a CO2- and NADPH-dependent manner, this activity is lost by the R132 mutation . The stoichiometry of IDH1 wt and mutant proteins is likely an important parameter which determines α-KG and D-2HG levels and may therefore influence cell metabolism . Therefore, it is not clear to what extent exogenous introduction of recombinant mutant IDH1 in cell lines provides relevant information as overexpression may result in a non-physiological amount of IDH1-mutant homodimers which may differ in activity from heterodimers . Moreover, metabolism of cells under standard culture conditions may significantly differ from in vivo conditions where local areas of hypoxia and hypoglycemia routinely occur. Therefore, it is important to study relevant orthotopic glioma xenograft models that recapitulate the biology of tumors carrying the endogenous mutation. It has been shown by several groups that gliomas with IDH1 mutations are difficult, if not impossible, to culture and propagate in vitro under standard serum-free or serum-containing culture conditions [38–40]. Similarly, in vivo models are difficult to propagate and as a result, preclinical glioma models carrying the IDH1 or IDH2 mutation are scarce.
Here, we report on the genetic, histologic and metabolic characterization of the E478 human oligodendroglioma xenograft line which carries the endogenous heterozygous IDH1-R132H mutation and provide novel insight into the metabolism of these tumors.
Development of IDH1-R132H xenografts
In our institute, we have a long history of developing patient-derived orthotopic glioma xenograft models by direct intracerebral implantation of cancer cell suspensions from surgically-obtained glioma specimens . As long-term in vitro cell cultures are known to be genetically unstable , the development of such direct orthotopic xenografts is important in the context of clinical relevance and reproducibility. Remarkably, from 5 biopsies derived from IDH1-R132H-mutated high-grade oligodendroglioma specimens only one so far gave rise to the stable xenograft line described here (E478). This is in line with a previous report which demonstrated that xenografting of cultured IDH1 mutant glioma cells hardly results in in vivo tumor growth  and is in sharp contrast with our experience with orthotopic xenografting of IDH1wt gliomas in which success rates approach 100%. Attempts to culture E478 cells in vitro using both neurosphere and standard culture conditions were so far unsuccessful (data not shown). Occasionally, we managed to maintain short-term organotypic spheroid cultures  and these were used for genetic analyses (Additional file 1 and Additional file 2: Figure S1).
To increase the versatility of the E478 model, we generated cell suspensions directly from xenografts and cryopreserved these before re-injecting them intracerebrally in mice. This procedure resulted in successful orthotopic engraftment in 100% of the animals, also after re-transplantation.
Genetic analysis of E478 xenografts
To compare the chromosomal aberrations in the E478 xenografts and the parental tumor, we performed array comparative genomic hybridization (aCGH) analyses (Figure 1D). The original tumor showed complete hemizygous loss of chromosomal arms 1p and 19q, a characteristic feature of oligodendroglial tumors. Additionally, hemizygous losses of chromosomes 4, 9 as well as 13q21.33-31.2 were detected. A region in chromosome 4 (59.5-62.2 Mb), which is devoid of any known genes or miRNAs, was homozygously deleted. Furthermore, the tumor was triploid for 7p15.2-qter, 8q12.3-qter and 11 (Figure 1D, upper panel). All aberrations were maintained in late passages (Figure 1D, lower panel). Some additional aberrations were also detected in the xenograft, including loss of 2q22-qter, 3pter-p21.2, chromosomes 10, 12 and 18 as well as gains of chromosome 21 (Additional file 2: Figure S1). Glial tumors show a high level of intratumoral genomic heterogeneity  which might explain the differences observed between the original tumor biopsy and its derived xenograft.
We determined the DNA quantity per cell of the xenograft tumor by flow cytometry of DAPI-labeled nuclei (Additional file 3: Figure S2A). A minor fraction of the cells appeared to be diploid and was considered to consist of stromal or host-derived cells. The majority of cells were aneuploïd with a DNA index of 1.925, which corresponds to 3.85 N. The xenograft tumor thus has a near tetraploïd genome with some xenograft-specific losses. The inferred copy number at the IDH1 locus on 2q34 is 3.
In order to determine the genotype at the IDH1 locus, we set up an allele-specific TaqMan SNP genotyping assay that can discriminate between the wild type and R132H alleles of IDH1. This assay revealed that the R132H allele was twice more abundant than the wt allele in the xenograft (Additional file 1 and Additional file 3: Figure S2B), resulting in an IDH1R132H/R132H/WT genotype in the xenograft.
Phenotype of the E478 xenografts
D-2HG production in E478 xenografts
D-2HG and αKG levels in individual spots in sections of othotopic xenografts carrying the IDH1-R132H mutation (italic) or wild-type IDH
In line with previous observations  we detected similar levels of α-KG in all tumor samples, both IDH1-mutant and -wt. There was a tendency towards higher levels in tumors compared to normal brain, although the levels were not significantly different. These findings suggest that the α-KG pool is maintained at physiological levels despite its depletion by conversion into D-2HG in the IDH1-mutant tumor.
In summary we show that in IDH1-mutant xenografts D-2HG levels are approximately 100-fold higher as compared to normal brain tissue and that these high levels are restricted to the tumor area. Moreover, α-KG levels are maintained at physiological levels in these tumors.
NAD+- and NADP+-dependent dehydrogenase activity in E478 tumors
We here present a detailed histologic and metabolic characterization of the E478 IDH1-R132H mutant oligodendroglioma xenograft line, the development of which has been described before . This line was established already in 2005, before the recognition of the involvement of IDH1/2-mutations in gliomagenesis. The E478 xenograft line has been grown successfully for over 35 passages now, is genetically stable and produces elevated levels of the oncometabolite D-2HG.
It is still not completely understood how IDH1 mutations in gliomas contribute to tumorigenesis and at the same time are correlated to good prognosis as compared to gliomas with wtIDH [21, 47]. Likely, the effects are multifactorial. Elevated D-2HG levels have been proposed to result in a block of differentiation via epigenetic alterations , including the induction of a hypermethylated DNA phenotype . Hypermethylation of genes encoding DNA repair enzymes such as MGMT is a predictor for the response to alkylating chemotherapy . However prognosis of patients with IDH1 mutated glioma is not confined to those receiving chemotherapy, suggesting that other factors may also be important. Models such as the E478 xenograft may contribute to elucidate the underlying mechanisms of IDH mutations with respect to gliomagenesis and prognostic relevance. Our data indicate that some answers may be found in the field of tumor metabolism as we observed mitochondrial hyperactivity in E478 xenografts. It remains unfortunate that only a limited number of endogenous IDH1-R132H mutant glioma models is currently available [38, 40] making it difficult to confirm our findings in other models.
We found that NADP+-dependent isocitrate conversion was not decreased in E478 xenografts as compared to IDH wt glioma, indicating that cells compensate for loss of IDH1 activity by increasing mitochondrial IDH2 activity by inducing mitochondrial biosynthesis. This is in agreement with our finding that α-KG levels are not diminished in E478 xenografts as compared to IDH wt tumors. These data suggest that D-2HG, rather than a shortage of α-KG, is involved in the specific features of IDH1-mutated glioma and is in line with D-2HG-mediated competition of α-KG dependent enzymes. Yet, our measurements do not allow discrimination between mitochondrial and cytosolic α-KG levels, and a role for a specific depletion of the cytosolic pool of α-KG in tumor biology cannot be excluded. For instance, fatty acid synthesis is an essential condition for tumor growth, and requires cytosolic α-KG and acetyl-CoA as precursors . Depletion of cytosolic α-KG may compromise lipid biosynthesis and might require import of mitochondrial α-KG into the cytosol. An attractive hypothesis is that such processes require a lot of energy for cell survival at the expense of extensive tumor cell proliferation. This may be a possible explanation for the better prognosis of patients with IDH-mutated gliomas.
An alternative manner to deal with decreased cytosolic α-KG, apart from increasing IDH2 activity, may be increased import of glutamine via specific importers in glioma cells. Glutaminase converts glutamine to glutamate which is subsequently converted to α-KG by glutamate dehydrogenase . This metabolic adaptation may be in line with the increased dependency of IDH1-mutant gliomas on exogenous glutamine, the low levels of glutamate in IDH1-mutant gliomas  and the sensitivity of these tumors to glutaminase inhibitors . The relative contribution of these systems in α-KG homeostasis remains to be determined.
It is still enigmatic why mutations in IDH genes are only found in the NADP+-dependent enzymes. It is tempting to speculate that this is related to a specific aspect of NADP+ metabolism. During conversion of α-KG to D-2HG by IDH1-R132H, NADPH is oxidized to NADP+ and is not available for generation of reduced glutathione and other detoxifying systems . As reduced glutathione is essential for scavenging reactive oxygen species (ROS), this may result in increased oxidative stress in IDH1-mutant glioma cells, a phenomenon that will be further augmented by increased mitochondrial density. Indeed, glutathione levels appear to be lower in IDH1-mutated tumors . How IDH1-mutant tumors cope with this stress is currently under investigation in our laboratory.
Based on our findings we propose that tumor cells that carry the IDH1 mutation undergo a metabolic switch involving increased mitochondrial activity leading to impaired proliferation and a relatively good prognosis. Cells acquiring IDH1 mutations will become tumorigenic based on the oncogenic activity of D-2HG, possibly in combination with acquired mutations in TP53 [34, 54]. Acquiring IDH1 mutations comes at a price however, as it will pose the cell with an excessive need for exogenous sources of α-KG for sustained membrane synthesis. The increased requirement for α-KG results in an increased dependency on exogenous glutamine. Interestingly, glutamine is produced at high levels by glial cells and is under normal conditions used by neurons as a precursor for the neurotransmitter glutamate. It would be interesting to investigate whether the dependency on glutamine or glutamate as external carbon source has a causal relation to the diffuse infiltrative growth in neuroglial tissue that is characteristic of diffuse gliomas. Indeed, such a dependency suggests that tumor cells would benefit from the nearby presence of non-neoplastic glial cells or neurons.
This model thus provides novel handles for metabolic targeting of low grade gliomas. Especially inhibition of glutaminolysis may be an effective way to interfere with glioma metabolism, although the effects of such an intervention on neuronal glutamate function will require careful evaluation. Combination approaches to further increase redox stress and/or glycolysis may further arrest tumor cells.
The E478 xenograft line represents a stable tumor model with the endogenous IDH1-R132H mutation. We show that this model has high mitochondrial activity, produces high levels of D-2HG and maintains near-physiological levels of α-KG. We propose that this model will be of high value for investigating novel therapies for the large group of gliomas that carry IDH1-R132H mutations.
Athymic female BALB/c nu/nu mice (18–25 gram, age 6–8 weeks) were kept under specified pathogen-free conditions and received food and water ad libitum. The local Animal Experimental Committee of the Radboud University Nijmegen Medical Centre approved all experiments. Glioma cell suspensions, directly obtained from surgically resected tumor tissue of a patient with an anaplastic oligodendroglioma, which was later shown to carry the IDH1-R132H mutation, were injected intracranially as described previously . Animals were closely monitored and sacrificed when evident signs of tumor burden (especially weight loss >15% in two days, severe neurological abnormalities) were observed. The xenograft line has been maintained by direct intracerebral passaging of tumor cell suspensions, generated from E478-bearing mouse brains as described .
Cryopreservation of xenograft brains
To test whether E478 cancer cells retain their tumorigenic potential upon cryopreservation, cell suspensions of tumor-bearing brains were generated in PBS and washed twice followed by suspension in DMEM (containing 0.45% w/v glucose; PAA Laboratories, Pasching, Austria) with 5% DMSO (Merck, Nottingham, UK). Cell suspensions were frozen at −1°C/min in Nalgene Mr. Frosty containers (ThermoScientific, Landsmeer, The Netherlands) at −80°C and subsequently stored in liquid nitrogen for at least a month. For re-injection, cells were rapidly thawed at 37°C, washed twice and suspended in PBS, followed by intracranial injection in mice (n = 5) as described above. When animals developed tumor-related symptoms, a tumor of one of the mice was transplanted into new animals (n = 5).
Array comparative genomic hybridization (aCGH)
aCGH was carried out with DNA of the originating patient tumor and several passages of the derived xenografts. DNA was digested using the restriction enzymes RsaI and AluI, followed by labeling using the Bio Prime CGH Genomic Labeling Kit (Invitrogen, Carlsbad, CA) and Cy3 and Cy5 dyes (GE Healthcare, Buckinghamshire, UK), according to standard protocols for Agilent CGH. Commercially available female DNA pooled from multiple donors (Promega Cat:G1521) was used as reference. Labeled DNA was competitively hybridized to SurePrint G3 Human 2×400k CGH microarrays (G4448A, Agilent Technologies, Amstelveen, The Netherlands) following standard protocols. The slides were scanned at 3 μm resolution using the Agilent High-Resolution Microarray scanner and the image data were extracted with Feature Extraction software (Agilent Technologies). Feature extraction files were imported into Genomic Workbench 7.0 for visualization and analysis. Briefly, after diploid centralization and GC correction, aberrations were called using the ADM2 algorithm with a threshold setting of 20, centralization on with a threshold of 25 and an aberration filter min Probes = 5 and minAvgAbsLogRatio = 0.25 for amplifications and deletions.
Animals with tumor-related symptoms were sacrificed by cervical dislocation and brains were removed for fixation in buffered formalin and subsequent paraffin embedding. Parts were also snap frozen in liquid nitrogen and stored for protein analysis and DNA/RNA isolation, D-2HG measurements and metabolic mapping.
H&E and immunohistochemical stainings were performed as described previously  including the use of antibodies directed against IDH1-R132H (clone H09, Dianova, Hamburg, Germany), Ki67 (for proliferation index assessment, clone Sp6, Neomarkers, Fremont, CA), cleaved caspase 3A (for detection of apoptotic cells, clone C92-605, BD Pharmingen, Franklin Lakes, NJ), CD34 (for endothelial cell staining, clone MEC14.7, Hycult Biotech, Uden, The Netherlands), GLUT-1 (Neomarkers), mouse IgG (Vector, Burlingame, CA), MCT-1 and MCT-4 (clones C-20 and H-90 respectively, Santa Cruz, CA). Primary antibody incubations were performed using 4 μm-thick sections of formalin-fixed paraffin-embedded tumor samples. Appropriate biotinylated secondary antibodies were used for detection using the ABC-method (Vector Laboratories). Specific signals were visualized by staining with 3-amino-9-ethyl-carbazole (Scytek Laboratories, West-Logan, Ut). All sections were counterstained with haematoxylin and mounted in Imsol Mount medium (Klinipath B.V., Duiven, The Netherlands). For all stainings, control incubations were carried out by omitting the primary antibody.
Activities of NADPH- and NADH-producing dehydrogenases were visualized using metabolic mapping [53, 56] using 10 μm thick unfixed cryostat sections of mouse brains infiltrated with E478, E434 or E98 glioblastoma xenografts . The wt IDH1 status of E434 and E98 xenograft lines (oligodendroglioma and glioblastoma, respectively) was previously confirmed. Control incubations to establish the specificity of the enzyme reactions, were performed in the absence of relevant substrates [53, 56].
Tissue samples of approximately 1–2 mm3 were fixed in 2% glutaraldehyde in cacodylate buffer (100 mM) for 4 hours, rinsed in cacodylate buffer and post-fixed for 1 hour in a solution of 1% osmium tetroxide containing 1% potassium hexacyanoferrat. Semithin (1 μm) sections and ultrathin (70 nm) sections were cut on an ultramicrotome (Leica EM UC6). Semithin sections were stained with toluidin blue for light microscopical previewing and ultrathin sections were collected on 200 mesh copper grids and contrasted with uranyl acetate and lead citrate. All sections were examined and images generated on a JEM1200 transmission electron microscope (Jeol, The Netherlands).
D-2HG measurements by isotope dilution LC-MS
D-2HG levels in serum and tissue extracts were measured using stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS). D-2HG for the preparation of calibration standards was purchased from Sigma Aldrich. Samples of 100 μl were mixed with 50 μl of 13C5-2-HG stable isotope solution (Chiralex, Nijmegen, The Netherlands; 10 μM in deionized water) before passing it through a Microcon YM-30 filter (Millipore) by centrifugation (14,000xg; 30 min). After acidification of the filtrate with 10 μl 4% formic acid in deionized water, 5 μl was injected into a Luna PFP column (2.1 mm*100 mm*3 μm, Phenomenex). The mobile phase consisted of methanol and water containing 0.3% formic acid. 2-HG was separated from its isomers 3-hydroxyglutarate and 2-hydroxy-2-methylsuccinate using a water-to-methanol gradient at 250 μl/min. The column was connected to an electrospray tandem mass spectrometer (Quattro LC, Micromass) operated in negative mode (capillary voltage 3 kV, cone voltage 20V) with an argon filled collision cell (0.18 Pa, 9eV). The tandem mass spectrometer was set to monitor the water loss of both D-2HG and 13C5-2-HG recording the mass transitions of m/z 147 to 129 and m/z 152 to 134, respectively. The temperature settings for the source and ion block were 400°C and 100°C respectively. Nitrogen was used as drying and nebulizer gas set at flow rates of respectively 650 L/h and 100 L/h.
In situ metabolite quantification by LESA-nano ESI-FTICR
In a separate set of experiments, we quantified D-2HG and α-KG levels via LESA (Liquid Extraction Surface Analysis, Nanomate, Advion) coupled to ESI-FTICR (Fourier transform ion cyclotron resonance, Solarix 7T, Bruker Daltonics, Bremen) and Quantinetix Software (ImaBiotech, France) allowing very sensitive detection and quantification of metabolites in small tissue plugs taken from 10 μm thick cryostat brain sections (Cryostat HM560, Microme). The Nanomate system was used in LESA mode with a 400 μm diameter surface extraction. Sample plates were cooled down to 12°C during analyses. Spray parameters were set as follows: Voltage to apply 1.30 kV and gas pressure 0.40 psi. Extraction solvent consisted of 65:15:20 MeOH:IPA:Water + 5 mM ammonium acetate using LCMS quality solvents. 0.6 μL extraction solvent was used to extract analytes from the surface and was injected then into the nano-electrospray source. Tissue suppression was calculated using Quantinetix to address matrix effect and to normalize signals of 2-HG and α-KG. A dilution range of pure metabolites mixed with brain tissue was applied next to the samples for absolute quantification and data for each LESA spot were analyzed using Quantinetix™ (ImaBiotech, France) taking 10 mDa mass tolerance around theoretical m/z of 2-HG and α-KG. Data were normalized against a standard included in the extraction solvent (m/z 141.019). Mass spectrometry was performed using the negative mode with the nano-electrospray source and CASI mode (isolation of m/z 150 +/− 50 Da in the quadrupole) in the mass range 71–160 Da. Each acquisition was a result of 80 accumulated spectra.
This work was supported by a grant from the Hersenstichting (grant 2010(1)-01) and by the CORE grant ESCAPE (C10/BM/784322) from the FNR in Luxembourg. ACN was supported by a RUNMC grant. We thank Jeroen Mooren for excellent technical assistance with xenograft transplantations and Nathalie Nicot from the Genomics Research Unit of CRP-Santé for expert technical assistance in genomics analysis.
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