Human astrocytomas and oligodendrogliomas are defined by mutations of the metabolic enzymes isocitrate dehydrogenase (IDH) 1 or 2, resulting in the production of the abnormal metabolite D-2 hydroxyglutarate. Here, we studied the effect of mutant IDH on cell proliferation and apoptosis in a glioma mouse model. Tumors were generated by inactivating Pten and p53 in forebrain progenitors and compared with tumors additionally expressing the Idh1 R132H mutation. Idh-mutant cells proliferated less in vitro and mice with Idh-mutant tumors survived significantly longer compared with Idh-wildtype mice. Comparison of miRNA and RNA expression profiles of Idh-wildtype and Idh-mutant cells and tumors revealed miR-183 was significantly upregulated in IDH-mutant cells. Idh-mutant cells were more sensitive to endoplasmic reticulum (ER) stress, resulting in increased apoptosis and thus reduced cell proliferation and survival. This was mediated by the interaction of miR-183 with the 5′ untranslated region of semaphorin 3E, downregulating its function as an apoptosis suppressor. In conclusion, we show that mutant Idh1 delays tumorigenesis and sensitizes tumor cells to ER stress and apoptosis. This may open opportunities for drug treatments targeting the miR-183–semaphorin axis.

Significance:

The pathologic metabolite 2-hydroxyglutarate, generated by IDH-mutant astrocytomas, sensitizes tumor cells to ER stress and delays tumorigenesis.

The grading of gliomas, previously largely based on histologic criteria, has over the last years been complemented with diagnostic or prognostic biomarkers (1). The discovery of “neomorphic” mutations in the isocitrate dehydrogenase (IDH) genes 1 or 2 (2) has provided a diagnostic and prognostic biomarker of oligodendrogliomas and astrocytomas. This delineates them from IDH-wildtype gliomas, which carry a poorer prognosis (3, 4). IDH-wildtype and IDH-mutant glioblastomas are histologically similar but molecularly distinct, and differ not only by their IDH mutation status. Therefore, a direct comparison and evaluation of the effects of mutant IDH is possible only in experimental settings. Cells expressing mutant IDH1/2 catalyze α-ketoglutarate into the D-enantiomer of 2-hydroxyglutarate (D2HG), which causes the DNA hypermethylator phenotype (5). Developmental expression of mutant Idh (R132H) in the mouse brain causes early postnatal lethal brain hemorrhages, possibly due to stabilized hypoxic-inducible hypoxia-inducible factor (HIF)-1α, (6), impaired collagen maturation, and basement membrane structure and function. This lethal phenotype precluded the study of neoplastic effects of mutant Idh. A study of inducible Nestin-CreER(T2)–mediated expression of Idh1 R132H in adult neural progenitors of the subventricular zone (SVZ) circumvented embryonic lethality. These mice showed an enlarged SVZ with increased numbers of quiescent neural stem cells and their progeny but did not form brain tumors. Instead they developed hydrocephalus, a sign of brain atrophy (7). Instead, the growth-delaying effects of high intracellular levels of R(–)-2-hydroxyglutarate (2-HG; ref. 8), appear contradictory to the tumor-promoting role of IDH1 R132H. The combination of mutant Idh1, Atrx and Pten in an experimental model formed gliomas, but has not addressed the role of Idh1 R132H itself in neoplastic neural progenitor formation (9). A role of mutant Idh1 in suppressing immune infiltrating cells in experimental gliomas has been shown in an RCAS/tva model (10).

We hypothesize that mutant IDH and the production of 2-HG induces a growth delay (8) by triggering cellular pathways that reduce proliferation or render tumor cells prone to cell death. This hypothesis was tested in mouse and cell models to selectively investigate the role of mutant Idh1 in the context of an established intrinsic brain tumor model. The Idh-mutant glioma-initiating cells and tumors corresponded well to human IDH-mutant astrocytomas and glioblastomas, producing high levels of 2-HG, and showing a proneural expression profile. We chose this approach to compare the transcriptome of Idh-wildtype and Idh-mutant tumors, and we have identified gene sets enriched for endoplasmic reticulum (ER) stress response and the miRNA family 96-182-183, controlling semaphorins and mediating ER stress response.

Mice

Mouse studies were performed under approval and license granted by the UK Home Office [Animals (Scientific Procedures) Act 1986], project license number PA79953C0, and conformed to UCL institutional and Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (http://www.nc3rs.org.uk/arrive-guidelines). All mice were kept at the Biological Service Facility, UCL. The following published mouse strains were used: p53loxP/loxP (11); PtenloxP/loxP (12); and GLAST-cre ER(T2) (13). All mice used in this study have a reporter gene lacZloxP/loxP in the ROSA26 locus [Gt(ROSA)26Sortm1Sor]. Mice were genotyped as described previously (14).

The Idh1 PM/Flex mouse (B6N-Idh1tm1(R132H)Avd/N) containing a conditional point mutation (R132H) in Idh1 exon 3 was generated in the Mouse Clinical Institute (MCI, Illkirch, France) using Flex transgenesis technology. The procedure of the conditional knock-in of the Idh1 R132H (395-396 GA>AT) into the murine Idh1 locus under the endogenous promoter is shown in Supplementary Fig. S1. Primers for Idh1 genotyping can be found in Supplementary Table S1.

Retrovirus production and injection

The PDGFB-Ires-Cre retroviral construct was kindly provided by Prof. Peter Canoll (Columbia University Medical Center, New York, NY) and the use of this retrovirus to generate gliomas have been described previously (15). Retrovirus was transfected into Platinum E cells using Fugene (Promega) according to the manufacturer's protocol. The Platinum E cells were kindly provided by Prof. Verdon Taylor (University of Basel, Basel, Switzerland). Supernatant containing retrovirus was concentrated using Retro-X concentrator (ClonTech, PT5063-2). Just before injection, polybrene (final concentration 8 μg/mL) was supplemented to retrovirus solution to facility infection rates. One microliter of retrovirus solution was injected to the subventricular zone of neonatal mice (age P0–P1).

Tunicamycin administration in vivo and imaging

Tunicamycin injection method has been described in detail previously (16). Briefly, tunicamycin (New England Biolabs NEB, #12819S) was first dissolved in DMSO to make a 10 μg/μL stock solution, and then diluted to 0.3 μg/μL with 150 mmol/L glucose for injection. Each mouse received 3 μg/g body weight (10 μL/g) tunicamycin twice by subcutaneous injection. Two such doses were administered with an interval of 2 hours. Mice were sacrificed 24 hours after the second tunicamycin injection. For in vivo comparison of ER stress–induced apoptosis, Annexin-vivo 750 (Perkin Elmer NEV11053) fluorescent imaging agent was administered intravenously 2 hours prior to imaging on an IVIS III preclinical imaging system. Automatic unmixing as per manufacturer's guidelines was performed to separate Annexin-vivo 750 signal from background autofluorescence.

RNA and miRNA sequencing

RNA sequencing was performed at UCL Genomics (UCL Institute of Child Health, London, United Kingdom). The library preparation, sequencing, and data analysis were described previously (17). Gene-set enrichment analysis was performed using datasets downloaded from the Broad Institute (http://software.broadinstitute.org/gsea/msigdb/index.jsp) as described previously (18). miRNA sequencing was performed by Exiqon (17). miRNA sequencing libraries were prepared from 500 ng total RNA using NEBNEXT library generation kit (New England Biolabs) according to manufacturer's protocols. Adaptors were ligated to each individual RNA sample, allowing amplification of RNAs with specific primers using RT-PCR for 15 cycles. After amplification, libraries were purified QiaQuick (Qiagen) columns and cDNA with approximate size of 142 nt (120 nt plus ∼22 nt miRNAs) were selected on a LabChip XT (Perkin Elmer). Samples were sequenced on the Illumina NextSeq 500 system (Illumina). Sequencing data were mapped to mouse annotated miRNA (miRBase 20), and normalized using trimmed mean of M-value method (19). Differential expression analysis was performed on R using EdgeR package (Bioconductor). RNA and miRNA sequencing data are available in Gene Expression Omnibus under GSE119741 and GSE119740, respectively.

Cell culture

Freshly prepared murine neural stem cells or brain tumor–initiating cells (BTIC) derived from in vitro recombined neural stem cells were cultured in DMEM/F12 medium, supplemented with B27 (2%), penicillin/streptomycin (1%), EGF (20 ng/mL), and FGF (20 ng/mL). Cell proliferation assay was performed in an IncuCyte interval image capture chamber (Essen Bioscience). Short oligonucleotides including siRNA, miRNA mimics/inhibitors, and negative controls (Supplementary Table S1) were transfected into cells with TransIT-X2 reagent (Mirus), and plasmids were transfected with Lipofectamine (Invitrogen) according to the manufacturer's instructions. No established or externally acquired cell lines were used.

D2HG quantification

The cellular level of D2HG was assessed using an enzymatic assay described previously (20). Briefly, cells were lysed in NP40 lysis buffer, which contains 0.1% NP40 (Calbiochem, 492016), 135 mmol/L NaCl, and 45 mmol/L Tris-HCl (pH 8.0). Protein amount used for normalization was determined using bicinchoninic acid assay (Thermo Fisher Scientific, 23225). Commercial D2HG (Sigma, #H8378) with a range of concentrations (0.5, 1, 2.5, 5, 7.5, 10, 25, 50, and 100 μmol/L) was set as standards. Samples and standards were deproteinized using a deproteinization kit (Biovision, K808-200) according to manufacturer's protocol. The deproteinized samples and standards in triplicate were incubated with assay solution, which contains 100 mmol/L of pH 8.0 HEPES (Applichem, H0887), 100 μmol/L NAD+ (Applichem, #A1124), 5 μmol/L resazurin (Applichem, #A2830), 0.01 U/mL diaphorase (MP Biomedicals, #150843), and 1 μg/mL D-2-hydroxyglutarate dehydrogenase (HGDH, kindly provided by Prof. A. von Deimling), at room temperature for 30 minutes in black 96-well plate (BRANDTech, #781608). The fluorescent intensity (FI) was detected using FLUOstar microplate reader (BMG Labtech) with excitation at 540 nm and emission at 610 nm. The standard curve was created using 4-parameter fit method in Omega Data Analysis software to determine the D2HG concentration in each sample.

Caspase-3/7 activity assay

Cells were lysed (#7018; Cell Signaling Technology) and, 5–10 μg of the lysate was mixed with 150 μL protease assay buffer containing 20 mmol/L HEPES (pH 7.5), 10% glycerol, and 2 mmol/L DTT, and 20 μmol/L fluorogenic substrate Ac-DEVD-AMC (#556449, BD Pharmingen), followed by a 2-hour incubation at 37°C in a black 96-well plate. Fluorescence intensity was determined (excitation, 360 nm; emission, 460 nm) with a FLUOstar Omega plate reader (BMG labtech).

PCR and qRT-RCR

Primers used for PCR or qRT-PCR were listed in Supplementary Table S1. The use of Xbp1 splicing primers were reported previously (21). For qRT-PCR, cDNA was synthesized using RevertAid RT kit (Thermo Fisher Scientific Inc.), and SYBR Green was used as reporter. GAPDH levels were used as normalization and fold changes were calculated using the 2−ΔΔCt method on DataAssist 3.1 software (Thermo Fisher Scientific). PCR analysis of the Idh and wild-type alleles on brain tumors was performed on genomic DNA extracted from different tumors, microdissected from paraffin sections as described previously (14).

Immunoblots

Cell lysate for immunoblots was collected with RIPA buffer (Thermo Fisher Scientific, 89900) containing protease/phosphatase inhibitor (Cell Signaling Technology, 5872). The protein concentration was assessed using bicinchoninic acid assay (Thermo Fisher Scientific, 23225). Equal amount of protein from each sample was separated using 10% SDS-PAGE, and transferred onto polyvinylidene difluoride membrane (GE Healthcare, 10600023). Membranes were blocked with 5% nonfat milk for 1 hour at room temperature, and incubated with primary antibody overnight at 4°C, followed by secondary antibody conjugated with HRP incubation at room temperature for 1 hour, and signal was detected with ECL Prime Western Blotting Detection Kit (GE Healthcare, RPN2232). Antibody information is listed in Supplementary Table S1.

Luciferase assay

The 3′UTR of Sema3E (174 bp) containing wild-type or mutant miR-183-5p binding site were synthesized commercially (GeneArt, Invitrogen). The 3′UTR fragment was inserted to pMIRREPORT luciferase vector between HindIII and SpeI restriction sites. Luciferase signal was measured 48 hours posttransfection using Dual-Light kit (Applied Biosystems).

IHC staining

Brains were fixed in 10% formalin, embedded in paraffin, cut into 3-μm sections and processed for hematoxylin and eosin staining. Staining was carried out using a Ventana Discovery automated staining instruments (ROCHE Ventana Medical Systems) following the manufacturer's guidelines, using horseradish peroxidase–conjugated streptavidin complex and diaminobenzidine as a chromogen. Antibodies are listed in Supplementary Table S1.

Image analysis

Histologic slides were digitized on LEICA SCN400 scanner (LEICA), or Hamamatsu S360 (Hamamatsu) at ×40 magnification, and images were captured from the LEICIA Slidepath slide management software. Digital image analysis was performed on Definiens Developer 2.4 as published previously (17, 22) or on the open source software QuPath (23) Version 0.2m2.

Human tissue resources

The use of human tissue samples was licensed by the National Research Ethics Service (NRES), University College London Hospitals NRES license for using human tissue samples: Project ref 08/0077 (S. Brandner). The storage of human tissue is licensed by the Human Tissue Authority, UK, License #12054 (to S. Brandner). Glioma tissue blocks and associated clinical and molecular information were from the archives of the National Hospital for Neurology and Neurosurgery (NHNN).

Expression of Idh1 R132H increases intracellular D2HG levels and inhibits cell proliferation

To study the pathologic function of mutant IDH1 R132 in tumor initiation and propagation, we generated a knock-in mouse model of Idh1LoxP(R132H) (Fig. 1A; Supplementary Fig. S1). Expression of Cre replaces exon 3 of the Idh1 gene with the mutant exon 3 (Fig. 1B) and the resulting Idh1 R132H can be detected with the mutation-specific antibody H09 (Fig. 1C; ref. 24). Idh1LoxP(R132H) knock-in mice were crossed with wild-type, or p53LoxP/LoxP, or PtenLoxP/LoxP; p53LoxP/LoxP mice, resulting in Idh1LoxP(R132H)/+, or Idh1LoxP(R132H)/+; p53LoxP/LoxP, or Idh1LoxP(R132H)/+; PtenLoxP/LoxP; p53LoxP/LoxP mice, and controls, respectively (in short “p53,” “Pten/p53,” and “Idh/p53,”Idh/Pten/p53”). All mice were kept in a ROSA26lox/lox reporter [Gt(ROSA)26Sortm1Sor] background (25) to identify recombined cells. Mutant IDH1 catalyzes α-ketoglutarate into D2HG, which accumulates in Idh-mutant tumors (7, 20, 26), and in our model (Fig. 1D), which replicates the metabolic changes of human IDH-mutant tumors.

To establish the effect of Idh1 R132H on naïve neural stem cells, we derived neural stem/progenitor cells from the murine SVZ and induced Idh1 R132 expression with adeno-cre as described previously (Fig. 1C; ref. 14). Unexpectedly, nonrecombined, β-galactosidase negative (Idh-wildtype) cells or spheres grew faster than the Idh-mutant moiety over multiple passages (Fig. 1E). Repeat transfection with adeno-cre “restored” a population of β-galactosidase–positive, Idh-mutant cells, indicating that Idh-wildtype cells had a growth advantage over Idh-mutant cells (Fig. 1F). The growth reduction of Idh-mutant cells was overcompensated by the additional homozygous deletion of p53, giving Idh1/p53 cells a growth advantage over the wild-type cells (Fig. 1G). All Idh1-mutant cultures had consistently a reduced proliferation compared with wild-type counterparts (Idh1 vs. wt, Idh1/p53 vs. p53, and Idh1/Pten/p53 vs. Pten/p53; Fig. 1G). A caspase-3/7 activity assay (Fig. 1H) shows 1.5-fold increase (P < 0.05) of apoptosis in Idh-mutant cell cultures. The formation of colonies, a measure of self-renewal, was significantly reduced (P < 0.01) in Idh/Pten/p53 cultures compared with Pten/p53 controls (Fig. 1I). In conclusion, the introduction of the Idh1 R132H into naïve neural stem cells reduces cell proliferation and increases apoptosis in vitro.

Idh1 R132H is not sufficient for tumorigenesis, delays tumor growth in vivo, and is associated with increased apoptosis

We assessed the effect of Idh1 R132H on stem cell growth and tumorigenesis in vivo by crossing Idh1LoxP(R132H) mice with GLAST-cre ER(T2) (13) and ROSA26lox/lox reporter mice. Tamoxifen was administered systemically (Fig. 2A) and led to Cre expression in the SVZ stem cell population (Fig. 2B; Supplementary Fig. S2; ref. 27). Five weeks after Cre activation, the proliferation in the SVZ was determined by Ki67 labeling. There was overall a smaller number of recombined cells in the SVZ and the subcallosal zone of GLAST-cre ER(T2); Idh1LoxP(R132H) mice compared with controls, but proportionally there was still a significant reduction of Ki-67–labeled nuclei in IDH-mutant SVZ-derived cells (Fig. 2C). Long-term observation of tamoxifen-induced GLAST-cre ER(T2); ROSA26lox/lox; Idh1LoxP(R132H) mice or of adeno-cre–injected ROSA26lox/lox; Idh1LoxP(R132H) mice showed populations of recombined SVZ stem/progenitor cells but no tumors (Fig. 2C; Supplementary Fig. S3).

To confirm the tumor growth-delaying effects of mutant Idh1, we generated Idh/p53, and Idh/Pten/p53 mice. Idh1LoxP(R132H) mice and wild-type mice served as controls. Intraventricular injection of a retrovirus expressing PDGF-IRES-Cre into new-born Idh-wildtype or Idh-mutant mice induced glioblastomas (15) with >90% incidence (Fig. 3A; Supplementary Table S1) within 30–50 days. Tumors of Idh-mutant mice contained high levels of D2HG, consistent with findings in human IDH-mutant astrocytomas (Fig. 3B). While controls with an Idh mutation alone or no mutation developed tumors with long latencies (Fig. 3C), p53 mice (n = 16) developed tumors already at 30 days (SEM ± 0.93 day). The additional expression of mutant Idh1 (n = 13) delayed tumor formation to 35 days (SEM ± 1.16 days; P < 0.0001; Fig. 3D; Supplementary Table S1). Further deletion of both Pten alleles led to an acceleration of tumorigenesis in both, Pten/p53 and Idh/Pten/p53 mice and again, the Idh-wildtype group (Pten/p53, n = 28), survived shorter (29 days, SEM ± 0.66 day) than the Idh-mutant group (Idh/Pten/p53, n = 24, 34 days, SEM ± 1.27 days; Fig. 3E). Idh-mutant and wild-type tumors showed no difference in neural and glial marker expression (Fig. 3A; Supplementary Fig. S4A and S4B), or tumor microenvironment (microglia, T cells, macrophages; Fig. 3F–H). Neonatal ROSA26lox/lox reporter mice injected with PDGF-IRES-Cre retrovirus (wt or Idh) developed small infiltrative gliomas after more than 150 days (wt, n = 6, Idh, n = 10; Fig. 3C; Supplementary Fig. S4C), as previously shown (28), again confirming growth-delaying role of mutant Idh1. The Idh mutation in these tumors was confirmed on DNA (Supplementary Fig. S4D and protein level; Fig. 3A; Supplementary Fig. S4A–S4C). In conclusion, we show here that mutant Idh1 delays tumorigenesis. Adeno-cre–induced tumors (14) in Pten/p53 and Idh/Pten/p53 mice again were morphologically indistinguishable (Supplementary Fig. S4A) but occurred with lower incidence (Idh/Pten/p53, 3/46 and Pten/p53 group, 6/57). In keeping with the lower incidence in IDH-mutant adeno Cre-induced tumors, Idh1/Pten/p53 or Pten/p53 cells grafted into the striatum of NOD-SCID also showed a difference, with lower tumor incidence of Idh1/Pten/p53 tumors (P = 0.047; Supplementary Fig. S4E).

Idh1 R132H–mutant BTICs are sensitive to endoplasmic reticulum stress

To understand how mutant Idh1 reduces cell growth and promotes apoptosis, we performed RNA sequencing on Idh1/Pten/p53 and Pten/p53 BTICs. Gene set enrichment analysis showed significant enrichment of ER response and ER stress–induced apoptosis pathways in Idh1/Pten/p53 cells (Fig. 4A). Treatment of Idh1-mutant cells with ER stress inducers tunicamycin (16) or thapsigargin (29) increased cell death in Idh1/Pten/p53 BTIC (Fig. 4B). To explore whether this ER stress sensitization was mediated by D2HG, we treated Idh-mutant cells with AGI-5198, an inhibitor of the catalytic pocket of Idh1 R132 (30), and conversely, Idh1 wild-type cells with D2HG, and then exposed them to tunicamycin and thapsigargin. Idh1/Pten/p53 BTIC showed 4-fold increase of apoptosis after tunicamycin treatment, compared with 1.5-fold in Pten/p53 BTICs. Inhibition with AGI-5198 prevented ER stress–induced apoptosis in Idh1/Pten/p53 BTICs (Fig. 4C), and in turn, treatment of Pten/p53 BTICs with 10 mg/mL (D)-2-Hydroxyglutaric acid disodium salt significantly sensitized Pten/p53 BTIC to ER stress (Fig. 4C) while treatment with AGI-5198 or D2HG alone, without tunicamycin or thapsigargin showed no effect (Fig. 4D).

Next, we analyzed the activation of ER stress–triggered unfolded protein response (UPR) pathways. The UPR is mediated by three stress receptors, (i) inositol-requiring transmembrane kinase/endoribonuclease 1 (IRE1), (ii) protein kinase RNA (PKR)-like ER kinase (PERK); and (iii) activating transcription factor-6 (ATF6; ref. 31). ATF4 and CHOP/Ddit3 are activated downstream of PERK. Without tunicamycin or thapsigargin treatment, Idh1/Pten/p53 and Pten/p53 BTICs expressed similar levels of the downstream targets of PERK (Atf4, Chop/Ddit3), of Atf6 and its target Grp94 (Hsp90b1) and of the target of X-box-binding protein 1 (XBP1), Grp78 (Hspa5 or Bip; Fig. 4E and F), although Chop protein is undetectable at this condition. Tunicamycin or thapsigargin treatment upregulated UPR genes in Idh1/Pten/p53, but not in Pten/p53 BTICs; Fig. 4E and F). Splicing of XBP1 mRNA is a direct indicator of IRE1 pathway activation (32–34) and indeed Xbp1 splicing during ER stress is increased in tunicamycin and thapsigargin treated Idh1/Pten/p53 BTICs (Fig. 4G and H). This effect was also seen in vivo in tunicamycin-treated tumor-bearing mice (16). We then assessed the alteration of protein levels of UPR genes during tunicamycin and thapsigargin treatment for 8 hours, in Idh1/Pten/p53 and Pten/p53 BTICs. In both BTIC genotypes, Chop (and phosphorylated eIf2α and PERK) gradually accumulated, but at a higher rate in Idh1/Pten/p53 BTIC (Fig. 4I). Protein levels of Grp78/Bip instead remained constant in both genotypes even though Grp78 transcripts were differentially expressed (Supplementary Fig. S5). In vivo treatment of tumor-bearing mice with tunicamycin showed nearly absent caspase-3–labeled apoptotic nuclei in the DMSO-treated Pten/p53 group (n = 7) and rare positive nuclei in the tunicamycin-treated group (n = 4) with no significant difference between the two groups (Fig. 4J). Instead, Idh1/Pten/p53 tumors had a higher (P < 0.05) baseline caspase-3/7 activity than Pten/p53 tumors and a significant increase of apoptosis in tunicamycin-treated Idh1/Pten/p53 tumors (P < 0.05), that is, tunicamycin sensitizes Idh-mutant tumors to apoptosis in vivo (Fig. 4J; Supplementary Fig. S5C). Expression analysis of transcripts of the ER stress pathway shows upregulation of Chop transcripts in Idh1/Pten/p53 tumors compared with Pten/p53 controls (Fig. 4K). Overall, we show that the Idh1 R132H mutation sensitizes BTICs to ER stress in vitro and in vivo.

miR-183 is most significantly differentially expressed between Idh-mutant and Idh-wildtype BTIC

To identify the mechanism of Idh1 R132H-mediated ER stress sensitization, we compared miRNA expression profiles of Idh1/Pten/p53 and Pten/p53 BTIC. A functional connection between ER-induced UPR (ERUPR) signaling and miRNA expression has revealed mechanisms of protein homeostasis regulation, and miRNA biogenesis is regulated by the ERUPR (35). We found 31 significantly differentially expressed miRNAs (DE-miR; Fig. 5A, P < 0.05). Validation of the top 14 DE-miRs qRT-PCR identified miR-183-5p as most significantly differentially regulated (Fig. 5B). miR-183 is part of a miRNA cluster that comprises miR-182 and miR-96 (36). qRT-PCR confirmed significant upregulation of all 3 miRNAs in Idh1/Pten/p53 cells (Fig. 5B). This was the starting point for the analysis of these miRNAs in the involvement in regulating apoptotic pathways (37).

Regulation of ER stress response by mutant Idh1 is mediated by miR-183 but not miR-182 and miR-96

To establish the roles of miR-183, miR-182, and miR-96 in modulating proliferation and apoptosis in Idh1/Pten/p53 and Pten/p53 BTICs, we transfected them with mimics and antagomirs. Cell confluence and growth rates were determined by live-cell imaging (17). Pten/p53 BTICs transfected with miR-183 mimics (Fig. 5C) and antagomiR-183–treated Idh1/Pten/p53 BTIC (Fig. 5D) showed a significant growth increase compared with controls (P < 0.0001), while transfection with miR-96 or miR-182 mimics (Fig. 5B and C) did not significantly change their growth rate. To investigate how miR-183 sensitizes cells to tunicamycin, we transfected Pten/p53 cells, in which miR-183 baseline levels are low, with miR-183 mimic (Supplementary Fig. S6). Tunicamycin treatment of Pten/p53-miR-183mimic cells significantly increases caspase-3/7 activity (Fig. 5E). Correspondingly, transfecting Idh/Pten/p53 cells (high miR-183 base levels) with miR-183 antagomir significantly reduces tunicamycin-induced cell death (P < 0.001), that is, antagonizing miR-183 desensitizes cells to tunicamycin-induced ER stress, and in this context against Idh R132H-mediated effects (Fig. 5F). Instead, miR-182 and miR-96 had no significant effect on cell proliferation in vitro (Fig. 5C and D) and did not modulate tunicamycin-induced ER stress in Idh1/Pten/p53 or Idh1/Pten/p53 BTIC. In conclusion, we show here that miR-183-mediates Idh1 mutation–associated ER stress sensitization in BTICs. Baseline expression levels of miR-183 are higher in IDH-mutant tumors (Fig. 5G). We have confirmed its pathobiological role in vivo by inserting miR-183 or miR-183 antagomir expression cassette into a PDGF-IRES-cre retroviral vector. Tumors were induced with PDGF-IRES-cre-miR-183ant in Idh/Pten/p53 mice (n = 3; Fig. 5H) and in Pten/p53 mice (n = 5) with PDGF-IRES-cre-miR-183mimic (Pten/p53-miR-183mimic; Fig. 5I). Pten/p53- miR-183mimic mice (n = 5; controls n = 28) showed a significant delay in tumorigenesis, with an increased survival of 29 days (P = 0.001), while Idh/Pten/p53-miR-183ant mice (n = 3; controls n = 24) showed a significant acceleration of tumor formation with a reduction by 9 days (P < 0.0001). In conclusion, miR-183 mediates ER stress response in IDH-mutant BTIC in vitro and in IDH-mutant experimental gliomas in vivo.

miR-183 downregulates semaphorin 3E, an axonal guidance, and apoptosis suppressor protein

To identify the target binding sites of miR-183 with relevance to apoptosis, we performed a TargetScan and identified among 23 differentially regulated genes, semaphorin 3E (Sema3E; Fig. 6A). Sema3E regulates tumor cell survival by suppressing an apoptotic pathway (38). The interaction of miR-183 with Sema3E was confirmed by expression of miR-183 mimic in Pten/p53 BTIC, resulting in a highly significant (P <0.001) downregulation of Sema3E transcripts. Conversely, expression of miR-183 inhibitor in Idh1/Pten/p53 cells, significantly upregulated Sema3E transcripts (Fig. 6B). A luciferase reporter assay of miR-183 binding (Fig. 6C) to the 5′ untranslated region (UTR) of Sema3E containing wild-type or mutated miR-183-5p–binding site showed a reduction of the luciferase signal upon miR-183 binding to the Sema3E UTR, but not to the mutated control Sema3E UTR (Fig. 6D), that is, there is a functional interaction of miR-183 with the Sema3E promoter. In keeping, knockdown of Sema3E with two different shRNAs (Fig. 6E) reduces proliferation of Pten/p53 cells (Fig. 6F), sensitizes them to tunicamycin-induced ER stress (Fig. 6G) and induces apoptosis. This interaction is further confirmed in Idh/Pten/p53 BTIC (miR-183high), which show a downregulation of Sema3E (Fig. 6H). In conclusion, we show here the role of miR-183 in targeting Sema3E and reducing its expression, thus antagonizing the apoptosis-suppressing effect, rendering cells more sensitive to ER stress.

miR-183 and semaphorin 3E expression inversely correlate in brain tumors in vivo

To demonstrate the inverse correlation of Sema3E and miR-183 expression, we analyzed transcripts of miR-183 and found a statistically nonsignificant trend of increased miR-183 expression in Idh1/Pten/p53 tumors compared with Pten/p53 controls, and inversely, a nonsignificant trend of reduced Sema3E expression in Idh1/Pten/p53 tumors. Immunostaining for Sema3E protein on murine brain tumors showed patchy expression in tumor cells and widespread expression in selected neuronal populations. Overall, however, there was no significant difference between Idh-mutant and IDH-wildtype tumors.

Semaphorin 3E is downregulated in IDH-mutant gliomas

To translate the experimental findings to the biology of human gliomas, we analyzed miR-183 and Sema3E transcripts in a selection of human gliomas. IDH-mutant astrocytomas and oligodendrogliomas were compared with IDH-wildtype astrocytic gliomas with molecular signature of glioblastoma (4), and to IDH-wildtype GBM (1). The tumors used in this study were previously described and molecularly characterized in detail (17). First, we determined transcript levels of miR-183 in IDH-mutant (n = 13; 5 oligodendrogliomas and 8 astrocytomas) and IDH-wildtype gliomas (n = 10; 4 IDH-wildtype astrocytomas with molecular profile corresponding to GBM and 6 GBM) and found a significantly higher miR-183 expression in IDH-mutant gliomas (Fig. 7A), in keeping with the mouse model where Idh-mutant tumors have higher miR-183 transcript levels (P < 0.05; Fig. 5G). miR-183 downregulates Sema3E through binding to its 3′UTR (Fig. 6C). In keeping, Sema3E transcript levels are low in all IDH-mutant tumors (Fig. 7B and C), and significantly higher in IDH-wildtype gliomas (P < 0.05), consistent with the prediction from the in vitro experiments (Fig. 5). Importantly, higher Sema3E expression levels are associated with shorter survival in patients from our own cohort (Fig. 7D) as well as The Cancer Genome Atlas (TCGA) cohorts (Fig. 7E and F). The longer survival of patients with lower Sem3E expression is explained by an overrepresentation of IDH-mutant tumors in this cohort, consistent with our data in Fig. 7B and C). In conclusion, we show that IDH-mutant gliomas express high levels of miR-183, which downregulates its target Sema3E, and Sema3E expression inversely correlates with survival.

In this study, we show a novel role of mutant Idh1 (R132H) in sensitizing glioma cells to ER stress through upregulation of miR-183 and suppression of its target SemaE3, leading to apoptosis. We induced Idh1 R132H expression and loss of Pten and p53 in the SVZ (14) of newborns. This model (i) allows the tumor to develop in an environment similar to the SVZ of young human adults, that is, during a certain developmental stage of the cell of origin (“window of opportunity”); (ii) combines Idh1 R132H and p53 mutations that are commonly found in astrocytomas; (iii) overexpresses PDGFR, a feature of IDH-mutant astrocytomas (39), and (iv) rapidly forms tumors to allow the study of large cohorts and the effects of inhibitory or stimulating signals. The effects of mutant Idh1 were selectively studied by comparing Idh-mutant with wild-type cells, that is, Idh1/p53 with p53-mutant tumors and Idh1/Pten/p53 with Pten/p53-mutant tumors. Corresponding to IDH-mutant human tumors (5), Idh R132H-mutant murine cells and tumors produce the metabolite D2HG (Fig. 1D). The Idh R132H mutation delays self-renewal and proliferation of neural stem/progenitor cells in vitro (Fig. 1E) and in vivo. Interestingly, this is in discrepancy with previous reports where the presence of mutant Idh1 R132H in stem/progenitor cells led to an expansion of the progenitor pool (7). It is possible that our study more accurately tracked the fate of recombined, Idh1 R132H cells with a mutation-specific antibody, while the previous study examined only indirect evidence of recombination (7). A key finding in human IDH-mutant gliomas is the relatively slow growth compared with IDH-wildtype “counterparts”, although it should be acknowledged that these “counterparts” are biologically distinct entities that have only morphologic features in common (IDH-mutant and IDH-wildtype GBM, and correspondingly IDH-mutant and IDH-wildtype astrocytomas). Thus, a direct comparison of the pathobiology of IDH-mutant and IDH-wildtype gliomas made it difficult to dissect the specific role of mutant IDH. Therefore, our model was designed to identify biological effects related to IDH1 R132H comparing Idh1/p53 with p53-mutant tumors and Idh1/Pten/p53 with Pten/p53-mutant tumors. Corresponding to the human counterparts, our experimental IDH-mutant and wild-type gliomas are morphologically indistinguishable (Fig. 3A), and the IDH-mutant gliomas produce D2HG (Fig. 3B; refs. 5, 40). Importantly, while it is not possible to directly demonstrate a growth-inhibiting effect of mutant IDH in human tumors (due to the lack of a biologically equivalent, direct comparator), we can demonstrate that Idh1 (R132H) significantly delays tumorigenesis (Fig. 3C). Our experimental paradigm differs from previous studies (9), combining some, but not all genetic lesions of IDH-mutant astrocytomas. The study by Philip and colleagues (9) has a lot in common with our model, such as the expression of mutant IDH1 targeting of Pten, but not p53. Instead, their model also targets Atrx, which is a diagnostic mutation in IDH-mutant astrocytomas (41), and it required additional CDKN2A/B deletion to elicit tumors (42, 43).

Direct comparison of IDH-mutant with wild-type tumors (in an otherwise identical genetic background) revealed that mutant IDH sensitizes BTICs to ER stress (Fig. 4). This led to the identification of DE-miRs. miRNAs control many biological processes, such as cell cycle, apoptosis, stem cell differentiation, and immune responses (44), and they also play a key role in the differentiation and maintenance of tissue identity (45). We found that miR-183, a member of a family comprising miR-183, miR-182, and miR-96 was differentially regulated. This miRNA cluster is highly conserved and miR-183 has varied effects during development, in mental health and cancer (36), utilizing multiple mechanisms governing development, energy and metabolism, and immune signaling (36). A mouse model where the miRNA-183/96/182 cluster was inactivated showed retina degeneration but no additional phenotype (46). In lung cancer, overexpression of miR-183 inhibits growth, consistent with our findings (47), and overexpression was also shown in medulloblastoma (48). In malignant gliomas, an effect of miR-183 on the expression of IDH2 and HIF1α has been described (49), but no mechanistic context has been given.

We have identified Sema3E as a direct target of miR-183. Semaphorins are a large family of conserved, secreted, and membrane-associated proteins that possess a semaphorin domain and a PSI domain (found in plexins, semaphorins, and integrins) in the N-terminal extracellular portion (50). The semaphorin guidance molecules and their receptors, the plexins, are often inappropriately expressed in cancers. Sema3E has recently been identified to regulate tumor cell survival in breast cancer by suppressing an apoptotic pathway (38) by suppressing an apoptotic pathway triggered by the Plexin D1 dependence receptor. In analogy with our findings where higher Sema3E levels are associated with the more aggressive IDH-wildtype glioma (Fig. 7B and C), it has been shown that Sema3E level correlates with metastatic progression of human breast cancers (38). Silencing of Sema3E induces apoptosis in the 4T1 mouse mammary breast cancer, consistent with our findings that IDH-mutant, Sema3Elow cells are more sensitive to apoptosis, in particular under ER stress (Fig. 6E–G). Lower Sema3E expression is associated with better survival (Fig. 7), as Sema3Elow tumors are strongly associated with the IDH mutation that correlates with better clinical outcome. Our findings suggest that drugs inhibiting the enzymatic effect of IDH1 R132H (e.g., Ag inhibitors) may counteract the growth-delaying, apoptosis-promoting effect of D2HG (Fig. 3C and D), thus potentially reducing the efficacy of treatments that aim to induce cytotoxic stress and cell death.

No potential conflicts of interest were disclosed.

Conception and design: Y. Zhang, N. Li, S. Brandner

Development of methodology: Y. Zhang, S. Pusch, J. Innes, K. Sidlauskas, M. Ellis, A. von Deimling, N. Li, S. Brandner

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Zhang, S. Pusch, J. Innes, K. Sidlauskas, J. Lau, T. El-Hassan, N. Aley, A. Richard-Loendt, J. de Boer, A. von Deimling, N. Li

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y. Zhang, J. Innes, K. Sidlauskas, M. Ellis, L. Wang, A. von Deimling, N. Li

Writing, review, and/or revision of the manuscript: Y. Zhang, S. Pusch, M. Ellis, A. von Deimling, N. Li, S. Brandner

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Innes, J. Lau, N. Aley, F. Launchbury, A. Richard-Loendt, A. von Deimling, N. Li

Study supervision: J. de Boer, N. Li, S. Brandner

Other (carried out experiments involving in vivo imaging of brain tumors requested as revisions): J. Innes

The PDGFB-Ires-Cre retroviral construct was kindly provided by Prof. Peter Canoll (Columbia University Medical Center, New York, NY). Platinum E cells were kindly provided by Prof. Verdon Taylor (University of Basel, Basel, Switzerland). We thank G. Graham, C. Fitzhugh, R. Labesse-Garbal, and other staff of the MRC Prion Unit Biological Services facility for animal observation and care. This work was supported, in part, by a grant from the Brain Tumour Charity UK (to N. Li and S. Brandner; BTC, 8/128) and by a Centre grant from BTC (8/197). N. Li and J. Innes were also supported by a Centre of Excellence grant to Queen May University London from Brain Tumour Research UK. S. Brandner is supported by the Department of Health's NIHR Biomedical Research Centre's funding scheme and BRC399/NS/RB/101410. The tissue resource was supported by the CRUK Accelerator Ed-UCL Grant C416/A23615. Y. Zhang was supported by a PhD Overseas research scholarship (UCL-ORS) and UCLH Trustees. M. Ellis was funded by a Cancer Research UK Accelerator grant Cl 15121 A 20256.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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