Isocitrate Dehydrogenase 1 R132H Research Articles

TROP-2, 5hmC, and IDH1 Expression in Anaplastic Thyroid Carcinoma.

Anaplastic thyroid carcinoma (ATC), a highly aggressive malignancy, has no effective treatment to date. Trophoblast cell-surface antigen 2 (TROP-2), a transmembrane glycoprotein, has been suggested to be a promising novel target for sacituzumab govitecan, an antibody-drug conjugate. 5-Hydroxymethylcytosine (5hmC) has a role in tumor suppression and promoting modification. Additionally, isocitrate dehydrogenase 1 (IDH1) mutations are strongly associated with increased overall survival in gliomas and worse prognosis in leukemias. This study attempts to evaluate the immunoexpression of TROP-2, 5hmC, and IDH1 in ATCs and to determine their potential impact in targeted therapy. Twenty-four ATCs were retrieved, with 9 cases that occurred de novo and 15 cases derived from either papillary thyroid carcinoma (PTC) or follicular thyroid carcinoma (FTC). Sections were immunostained with TROP-2, 5hmC, and IDH1 antibodies, and evaluated using the QuPath program. The t tests were performed using SPSS software. TROP-2 was detected in 12 ATCs with 9 cases demonstrating a high expression and in all PTC components, and absent in all FTC components of secondary ATCs. 5hmC expression was moderately reduced in PTC and FTC components and markedly reduced in ATC. The entire cohort showed a total absence of IDH1. Increased TROP-2 immunoexpression in some ATCs supports that these patients may potentially benefit from an antibody-drug conjugate therapy targeting TROP-2. Markedly reduced 5hmC expression suggests that 5hmC may be used as potential therapeutic targets for ATC. The total lack of IDH1 R132H mutation by immunostain indicates that it has no prognostic and therapeutic value in ATC.

Prognostic significance of L-type amino acid transporter-1 (LAT-1) expression in human astrocytic gliomas

BackgroundGliomas are the most prevalent type of primary brain tumors. Clarifying the correlation between glioma markers and disease progression provide a basis for adjuvant treatments. We examined Isocitrate dehydrogenase 1 (IDH1)expression, Ki-67, programmed death-ligand 1 (PD-L1) and L-type amino acid transporter 1 (LAT-1) with respect to overall survival in glioma patients. MethodsImmunohistochemistry (IHC) results from 25 glioma samples were analyzed semi-quantitatively. By WHO criteria, eleven specimens were diagnosed as grade II, 7 as grade III, and 7 as grade IV. Grade II was classified as lower grade glioma (LGG); grade III-IV were classified as high grade glioma (HGG). ResultsIn this study,immuno-characterizationrevealed HGG was associated with highKi-67, PD-L1,and LAT-1 expression (p < 0.05), but not with IDH1 R132H mutant expression (p = 0.6217). Both LAT-1 and PD-L1 were associated with Ki-67 expression (p < 0.05). LAT-1 was found to be correlated with PD-L1 expression (r = 0.525,p = 0.007). Upon, multivariate analysis, LAT-1 expression in human astrocytic gliomas was meaningful as an independent prognosis factor (HR = 0.004 (0.00004–0.41)). ConclusionLAT-1 and PD-L1 expression by HGG contribute to the disease progression. LAT-1 expression in astrocytic gliomas serves an independent prognosis factor. Therefore, LAT-1 and PD-L1 examination is potentially valuable diagnostic markers for adjuvant therapies.

Discovery of a Novel Chemical Scaffold Against Mutant Isocitrate Dehydrogenase 1 (IDH1).

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are frequently found in various cancer types. IDH1 mutants produce 2-hydroxyglutarate (2-HG), an oncometabolite, from alpha-ketoglutarate (α-KG). This 2-HG plays a key role in tumorigenesis via inhibition of α-KG dependent enzymes. For this reason, IDH1 mutant could be an ideal target for the treatment of cancer. To find a new IDH1 inhibitor, 8,364 compounds were obtained from Korea Chemical Bank. Using high-throughput screening (HTS) of a chemical library, we unveiled a compound that could inhibit the IDH1 mutant. According to the enzyme assay, our compound (KRC-09) effectively inhibited the activity of IDH1 R132H mutant. In addition, KRC-09 decreased the concentration of intracellular 2-HG in the U-87 MG cell line harboring IDH1 R132H. In this article, we present a novel chemical scaffold that suppresses the activity of an IDH1 mutant.

IDH1 R132H gene mutation reduces cell proliferation and sensitizes recurrent Glioblastoma to hydrogen peroxide

Glioblastoma (GBM) recurrence rate is 90% resulting in 15 months median survival only. Isocitrate dehydrogenase 1 (IDH1) mutations in gliomas significantly improved patient’s prognosis. Therefore, understanding common IDH1 mutation, IDH1 R132H in recurrent GBM is necessary to improve poor survival rate. IDH1 R132H recurrent GBM was developed to investigate cell proliferation rate and sensitivity towards oxidative stress induced by hydrogen peroxide. The cell death mechanism induced by hydrogen peroxide were further investigated. Malaysian recurrent GBM cell line was authenticated via Short Tandem Repeat and screened for IDH1 gene via PCR. IDH1 R132H gene expression in GBM was confirmed via real-time PCR and western blot. The effect of IDH1 R132H mutation on cell proliferation rate and cytotoxicity using hydrogen peroxide were determined using MTT assay. The angiogenesis, apoptotic genes and cell cycle induced by hydrogen peroxide in IDH1 wild-type GBM were determined via real-time PCR and flow-cytometry. Malaysian GBM cell line is unique and harbors IDH1 wild-type gene. IDH1 R132H gene mutation significantly reduced the growth rate and sensitized the GBM cells to hydrogen peroxide at 72 hours (p

MP16-09 THE ANDROGEN RECEPTOR REPRESSES PROSTATE CANCER INVASION INDUCED BY ONCOMETABOLITE R-2-HYDROXYGLUTARATE THROUGH CIRC51217/MIR-646/TGFβ1/P-SMAD2/3 SIGNALING

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I (MP16)1 Apr 2020MP16-09 THE ANDROGEN RECEPTOR REPRESSES PROSTATE CANCER INVASION INDUCED BY ONCOMETABOLITE R-2-HYDROXYGLUTARATE THROUGH CIRC51217/MIR-646/TGFβ1/P-SMAD2/3 SIGNALING Hua Xu* and Ding-wei Ye Shanghai Hua Xu*Hua Xu* More articles by this author and Ding-wei Ye ShanghaiDing-wei Ye Shanghai More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000841.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: IDH1 (Isocitrate dehydrogenase 1) mutation occurring at codon 132 (R132) in prostate cancer (PCa) is considered as a classifier for a subgroup PCa with accumulation of oncometabolite R-2HG (R-2-hydroxyglutarate). Besides its association with early onset of PCa, little is known about the biological effect of IDH1 mutation and R-2HG in PCa. In this study, we focus on the biological effect of IDH1 R132H mutation, which is the most frequent hotspot mutation, and oncometabolite R-2HG in PCa cell invasion. METHODS: A retrospective cohort of 1012 PCa patients in Fudan University Shanghai Cancer Center were included for next-generation sequencing. IDH1 R132H mutation was discovered as a hot spot mutation for further research. The in vitro study was designed to explore the biological function of IDH1 R132H mutation as well as R-2HG in PCa. Transwell assay was applied to test the cell invasiveness of PC3 and C4-2 PCa cell lines. CircRNA and microRNA candidates were screened based on database and manipulation of their expression was used test their function in the signaling pathway. RESULTS: Based on the next-generation sequencing data from a retrospective cohort of 1012 PCa patients, IDH1 R132H was found to be on the most frequent mutations. We discovered 9 PCa patients (0.9%) carried IDH1 R132H mutation. In the in vitro study, we found that over-expressing mutant IDH1 R132H as well as R-2HG promotes cell invasion in PCa with negative or low expression of androgen receptor (AR). Mechanism dissection revealed that R-2HG can induce circ51217 generation through increasing N6-methyladenosine (m6A) level of TGFβ1 (Transforming growth factor-β 1) mRNA, and in turn sponge miR-646 to further increase TGFβ1 protein expression and TGFβ1/p-Smad2/3 signaling. AR can suppress R-2HG effect at multiple levels, namely repressing TGFβ1 transcription and inhibiting circ51217generation via regulating ADAR2 (Double-stranded RNA-specific editase 1) expression. Preclinical study with in vivo xenograft mouse model also shows that PCa with IDH1 R132H mutation is more invasive in experimental metastasis. CONCLUSIONS: This study demonstrates that IDH1 R132H mutation as well as oncometabolite R-2HG increases cell invasiveness of PCa with negative or low expression of AR through circ51217/miR-646/TGFβ1/p-Smad2/3 signaling. AR can negate R-2HG effect via suppressing transcription of TGFβ1 and generation of circ51217. Source of Funding: National Natural Science Foundation of China (No. 81472377 and 81802570) © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e217-e217 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hua Xu* More articles by this author Ding-wei Ye Shanghai More articles by this author Expand All Advertisement PDF downloadLoading ...

Androgen receptor reverses the oncometabolite R-2-hydroxyglutarate-induced prostate cancer cell invasion via suppressing the circRNA-51217/miRNA-646/TGFβ1/p-Smad2/3 signaling.

IDH1 (Isocitrate dehydrogenase 1) mutation occurring at codon 132 (R132) in prostate cancer (PCa) is considered as a classifier for a subgroup of PCas with accumulation of oncometabolite R-2HG (R-2-hydroxyglutarate). Here we found that adding R-2HG or the mutant IDH1 R132H could promote PCa cell invasion in androgen receptor (AR)-negative PC3 cells or suppressing the AR in AR-positive C4-2cells. Mechanism dissection revealed that R-2HG could increase circRNA-51217 expression to sponge miRNA-646, which might then lead to increase TGFβ1 expression and thus induce TGFβ1/p-Smad2/3 signaling to increase PCa cell invasion. AR can suppress this R-2HG/circRNA-51217/miRNA-646/TGFβ1/p-Smad2/3 signaling-increased PCa cell invasion via repressing TGFβ1 transcription and inhibiting circRNA-51217 expression through regulating ADAR2 expression. Preclinical studies with an in vivo xenograft mouse model also revealed that PCa cells with the IDH1 R132H mutation have more invasive metastasis. This study demonstrates that IDH1 R132H mutation with increased oncometabolite R-2HG in PCa cells may play important roles to increase PCa cell invasion.

Clinico-neuropathological features of isocitrate dehydrogenase 2 gene mutations in lower-grade gliomas.

Background:Mutations in the isocitrate dehydrogenase 1 (IDH1) and IDH2 genes are important for both the integrated diagnosis and the prognosis of diffuse gliomas. The p.R132H mutation of IDH1 is the most frequently observed IDH mutation, while IDH2 mutations were relatively rarely studied. The aim of the study was to determine the pathological and genetic characteristics of lower-grade gliomas that carry IDH2 mutations.Methods:Data from 238 adult patients with lower-grade gliomas were retrospectively analyzed. The status of IDH1/2 gene mutations, telomerase reverse transcriptase (TERT) promoter mutations, O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation, 1p/19q co-deletion and the expressions of IDH1 R132H, alpha-thalassemia X-linked mental retardation, and p53 were evaluated. Progression-free survival (PFS) and overall survival (OS) were calculated via Kaplan-Meier estimation using the log-rank test.Results:Totally, 71% (169/238) of patients were positive for IDH mutations, including 12 patients harboring mutations in IDH2. Among the 12 patients with IDH2 mutations, ten patients harbored the R172K mutation, one patient harbored the R172S mutation and one harbored the R172W mutation. Of these, 11 tumors occurred in the frontal lobe and showed morphology typical of oligodendroglioma. The proportion of grade II tumors was higher than that of grade III tumors in IDH2 mutant-gliomas. IDH2 mutations were frequently associated with TERT promoter mutations, 1p/19q co-deletion and MGMT promoter methylation. IDH2 mutations were associated with better outcomes compared with IDH wild-type gliomas (P < 0.05). However, the PFS and OS did not differ from that of IDH1 mutant patients (P = 0.95 and P = 0.60, respectively).Conclusions:IDH2 mutations are more frequent in oligodendrogliomas and associated with a better prognosis. IDH2 mutations may segregate in distinct clinico-pathological and genetic subtypes of gliomas, and therefore may merit routine investigation.

EXTH-45. THERAPEUTIC EFFICACY OF MUTANT ISOCITRATE DEHYDROGENASE 1 (IDH1) INHIBITOR SYC-435 WITH STANDARD THERAPY IN PATIENT-DERIVED IDH1 MUTANT GLIOMA XENOGRAFT MOUSE MODELS

Abstract Mutation in isocitrate dehydrogenase 1 (IDH1) occurs in &gt;70% of WHO grade II/III astrocytomas, oligodendrogliomas and secondary glioblastoma. The mutant enzyme catalyzes the reduction of α-ketoglutaric acid to D-2-hydroxyglutaric acid, leading to cancer initiation. In this study, we examined therapeutic efficacy of SYC-435 (1-hydroxypyridin-2-one), a newly developed mutant IDH1inhibitor, in IDH1 mutant gliomas. IDH1 R132H mutation (homozygous) was detected in BT142 anaplastic oligoastrocytoma (AOA) and R132C mutation (mutant allele frequency 39–50%) in V0914AOA by pyrosequencing. Suppression of cell growth by SYC-435 was observed with more sensitive of mutant over wild-type IDH1. Patient-derived orthotopic xenograft mouse models of BT142AOA and V0914AOA were treated with vehicle, SYC-435 (15 mg/kg/day x 28 days), temozolomide (50 mg/kg/day x 5 days)/fractionated radiation (2 Gy/day x 5 days) (standard therapy), and SYC-435/standard therapy starting 2 weeks after intracranial tumor implantation. Log rank analysis showed SYC-435 alone did not alter survival times. Although standard therapy significantly prolonged survival times in both models (P&lt; 0.0005), SYC-435/standard therapy further extended survival times (P&lt; 0.05) in V0914AOA and exhibited similar trend in BT142AOA. Elevation of 2-HG/α-KG ratio and methylation of H3K4/H3K9 in V0914AOA tumor compared to wild-type model was detected at the end of treatments. SYC-435 with/without standard therapy tended to reduce 2-HG/α-KG ratio and dramatically reduced methylation of H3K4/H3K9. RNA-seq analysis showed sirtuin signaling pathway, mitochondrial dysfunction and oxidative phosphorylation pathways with mitochondrial DNA (mtDNA) encoded molecules were highly affected by all treatments. However, mtDNA regulation did not correlate to survival benefits. In conclusion, SYC-435 possesses anti-tumor effects that are more sensitive in IDH1 mutant gliomas and generated strong synergistic activities with standard therapies for survival benefits with reduced methylation of H3K4/H3K9. Treatments significantly affected mtDNA but significance to survival benefits remains to be elucidated. Our data support the clinical testing of SYC-435 in patients with IDH1 mutant glioma.

EXTH-20. HYPERPOLARIZED [2-13C] PYRUVATE TO [5-13C] GLUTAMATE AS BIOMARKERS OF IDH1 MUTANT GLIOMA RESPONSE TO TEMOZOLOMIDE THERAPY

Abstract Low-grade gliomas, driven by mutations in the cytosolic isocitrate dehydrogenase 1 (IDH1) gene, are less aggressive than primary glioblastoma, but nonetheless always recur and ultimately lead to patient death. The treatment of IDH1 mutant patients with Temozolomide (TMZ) improves survival, but there remains a need for complementary imaging methods to assess response to therapy at an early time point. The goal of this study was, therefore, to determine the value of magnetic resonance spectroscopy (MRS)-based metabolic imaging biomarkers for detection of response to treatment. To this end we investigated NHA and U87 cells expressing IDH1 R132H mutant gene (NHAIDHmut and U87IDHmut) and first used 1H MRS combined with chemometrics to examined the metabolic alterations that occurred following treatment with the IC50 value of TMZ. We observed a significant increase in 2-hydroxyglutarate (2-HG), glutamate, and glutamine, and metabolic pathway analysis showed tricarboxylic acid (TCA) cycle and pyruvate metabolism to be significantly altered pathways following TMZ treatment compared to DMSO control. To confirm changes in TCA cycle flux and to assess the metabolic pathways contributing to the increase in 2-HG and glutamate/glutamine, cells were then labelled with [1-13C] glucose and [3-13C] glutamine. Our data indicated that both glucose flux via the TCA to glutamate and 2HG, and the contribution of glutamine to glutamate and 2HG were increased following TMZ treatment. Finally, we used hyperpolarized 13C-MRS to dynamically probe the metabolism of hyperpolarized [2-13C] pyruvate and its conversion to hyperpolarized [5-13C] glutamate via the TCA cycle. Consistent with our previous findings, we observed that hyperpolarized [5-13C] glutamate synthesis was significantly higher in TMZ-treated cells compared to controls. Collectively, our findings identify 1H MRS-detectable elevation of 2-HG and glutamate/glutamine as well as hyperpolarized 13C-MRS-detectable [5-13C] glutamate production from [2-13C] pyruvate as potentially translatable metabolic biomarkers of response to TMZ therapy in mutant IDH1 glioma.

OS12.1 Editing of IDH1 R132H mutation in human induced pluripotent stem cells to investigate tumor genesis in glioma

Abstract BACKGROUND Low grade gliomas and secondary glioblastomas are characterised by a hot-spot mutation in the Isocitrate dehydrogenase 1 (IDH1). The mutation causes a new catalytic function of the enzyme resulting in the production of 2-Hydroxyglutarat (2-HG), which is known as an oncometabolite. To this day, the pathomechanism by which IDH1 mutation promotes tumorigenesis is not completely understood and the tumour cell of origin for glioma is not known. Moreover, reliable cell models reflecting the patient ́s situation are not available. Thus, we aim at creating human induced pluripotent stem cells (hiPSC) carrying the IDH1 R132H mutation. We will utilize this cell model to investigate how the mutation influences stem cell properties and cell differentiation in neuronal progenitor cells. MATERIAL AND METHODS For designing the cell model, we applied the novel CRISPR/Cas9 based genome editing tool Base Editor 3 and 4. We transiently transfected the cells with two plasmids containing the Cas9 enzyme and sgRNA respectively. To determine editing efficiency, cells were investigated using the T7 Endonuclease 1 assay and Next Generation Sequencing. Single cell clones were picked to analyse allele status using allele-specific PCR with subsequent Sanger Sequencing. RESULTS Using fluorescence-activated cell sorting we measured a transfection efficiency in iPSCs for one plasmid of 33.8% ± 5.92%. Out of 96 picked clones one clone showed the IDH1 mutation. Thus, the overall editing efficiency was about 1%. We confirmed expression of mutated IDH1 R132H by Western Blot. Measurement of the tricarboxylic acid cycle metabolites using liquid chromatography tandem mass spectrometry (LC-MS/MS) showed a forty times increased concentration of 2-HG in IDH-mutated compared to the wildtype iPSCs, proving that the mutated enzyme is functional. To investigate effects of the IDH1 R132H on cell differentiation, we generated cerebral organoids from our iPSC-models. The IDH1 R132H mutation did not inhibit the cell differentiation in maturation of cerebral organoids. However, we observed differences in the growth between mutated and wildtype brain organoids. We additionally characterized our cell models using genome wide methylation arrays and RNA-Seq; these results will also be presented. CONCLUSION We successfully established a heterozygous IDH1 R132H mutated hiPSC cell line, which represents a promising cell model for future studies of early glioma genesis.

Analysis of risk factors influencing the progression-free survival in WHO II grade gliomas

Objective To investigate the risk factors that influence the progression-free survival (PFS) in WHO Ⅱ-grade gliomas in adults. Methods Clinical data obtained from 72 patients with WHO Ⅱ grade gliomas undergoing surgical resection between January 2008 and December 2015 at Department of Neurosurgery, Sanbo Brain Hospital, Capital Medical University were analyzed. Immunohistochemistry staining method was employed to detect mutation of isocitrate dehydrogenase 1(IDH1) R132H, α thalassemia/mental retardation syndrome X-linked(ATRX) and the expression of regulator of telomere elongation helicase 1(RTEL1). Survival analysis was estimated by Kaplan-Meier analysis. Cox regression modelling was used for univariate analysis and multivariate analysis. Results The results of immunohistochemical staining in 72 patients with gliomas revealed 39 (54.2%) cases of IDH1 R132H mutant positive, 33 (45.8%) cases of negative; 56 (77.8%) cases of ATRX missing mutant negative, 16 (22.2%) cases of positive; 29 (40.3%) cases of RTEL1 positive and 43 (59.7%) cases of negative. The results of survival analysis showed that the cumulative PFS rate of IDH1 R132H negative patients was lower than that of IDH1 R132H positive(P 0.05). The cumulative PFS rate of RTEL1 positive patients was lower than that of RTEL1 negative patients (P 40 years (OR=3.618, 95% CI: 1.688-7.753, P=0.001), partial resection (OR=0.204, 95% CI: 0.064-0.656, P=0.008), expression of IDH1 R132H negative (OR=2.867, 95% CI: 1.129-7.282, P=0.027) and RTEL1 positive (OR=0.354, 95% CI: 0.151-0.827, P=0.016) were independent factors influencing PFS of glioma patients, and could be independently used for prognostic judgment of glioma patients. Conclusion Age (>40 years), degree of tumor resection (partial resection), expression of IDH1 R132H (negative) and RTEL1 expression (positive) are independent risk factors affecting the PFS of WHO Ⅱ-grade gliomas. Key words: Glioma; Progression-free survival; Isocitrate dehydrogenase 1; α thalassemia/mental retardation syndrome X-linked; Telomere elongation helicase 1

Inhibitor potency varies widely among tumor-relevant human isocitrate dehydrogenase 1 mutants.

Mutations in isocitrate dehydrogenase 1 (IDH1) drive most low-grade gliomas and secondary glioblastomas and many chondrosarcomas and acute myeloid leukemia cases. Most tumor-relevant IDH1 mutations are deficient in the normal oxidization of isocitrate to α-ketoglutarate (αKG), but gain the neomorphic activity of reducing αKG to D-2-hydroxyglutarate (D2HG), which drives tumorigenesis. We found previously that IDH1 mutants exhibit one of two reactivities: deficient αKG and moderate D2HG production (including commonly observed R132H and R132C) or moderate αKG and high D2HG production (R132Q). Here, we identify a third type of reactivity, deficient αKG and high D2HG production (R132L). We show that R132Q IDH1 has unique structural features and distinct reactivities towards mutant IDH1 inhibitors. Biochemical and cell-based assays demonstrate that while most tumor-relevant mutations were effectively inhibited by mutant IDH1 inhibitors, R132Q IDH1 had up to a 16 300-fold increase in IC50 versus R132H IDH1. Only compounds that inhibited wild-type (WT) IDH1 were effective against R132Q. This suggests that patients with a R132Q mutation may have a poor response to mutant IDH1 therapies. Molecular dynamics simulations revealed that near the NADP+/NADPH-binding site in R132Q IDH1, a pair of α-helices switches between conformations that are more wild-type-like or more mutant-like, highlighting mechanisms for preserved WT activity. Dihedral angle changes in the dimer interface and buried surface area charges highlight possible mechanisms for loss of inhibitor affinity against R132Q. This work provides a platform for predicting a patient's therapeutic response and identifies a potential resistance mutation that may arise upon treatment with mutant IDH inhibitors.

Isocitrate Dehydrogenase 1 Expression in Canine Gliomas

Mutation of the isocitrate dehydrogenase 1 (IDH1) gene at codon 132 has been identified in approximately 70% of low-grade (II and III) human gliomas and secondary glioblastomas, with the IDH1 R132H point mutation representing 92.7% of these mutations. In people, the presence of an IDH1 gene mutation is associated with a better prognosis (both progression-free survival time and overall survival time)and a better response to therapy, including chemotherapy and radiation therapy. Furthermore, IDH1 mutations are included in diagnostic panels to improve diagnosis and molecular classification. Canine gliomas resemble their human counterpart both morphologically and immunohistochemically, therefore they are likely to share similar genetic abnormalities. The IDH1 gene is also comparable between man and dogs. If the IDH1 R132H point mutation is demonstrated in canine gliomas, the prognostic significance of this mutation in people may be transferable to the dog. The objective of this study was to investigate canine gliomas for the IDH1 R132H point mutation using immunohistochemistry. Thirty-one formalin-fixed and paraffin wax-embedded canine gliomas were examined for both IDH1 R132H expression and pan-IDH1 (IDH1 wild-type and point mutated IDH1). Glial tumour specimens were recorded to be either positive or negative for expression. Pan-IDH1 expression was identified in all 31 tumours (100%), while the IDH1 R132H point mutation was identified in none of the tumours (0%). Therefore, the IDH1 R132H point mutation was not identified in this population of canine gliomas and may not be a suitable biomarker or treatment target in canine gliomas. Further investigation is required to determine if other point mutations occur in the IDH1 gene of canine gliomas.

Inhibition of Mutated Isocitrate Dehydrogenase 1 in Cancer.

R132H mutation of isocitrate dehydrogenase 1 (IDH1) is found in ~75% of low-grade gliomas and secondary glioblastomas as well as in several other types of cancer. More chemotypes of inhibitors of IDH1(R132H) are therefore needed. The study aimed to develop a new class of IDH1(R132H) inhibitors as potent antitumor agents. A biochemical assay was developed to find inhibitors of IDH1(R132H) mutant enzyme. Chemical synthesis and structure-activity relationship studies were used to find compounds with improved potency. Antitumor activities of selected compounds were evaluated. A series of aromatic sulfonamide compounds was found to be novel, potent inhibitors of IDH1(R132H) with Ki values as low as 0.6 µM. Structure-activity relationships of these compounds are discussed. Enzyme kinetics studies showed that one compound is a competitive inhibitor against the substrate α-KG and a non-competitive inhibitor against the cofactor NADPH. Several inhibitors were found to have no activity against wild-type IDH1, showing a high selectivity. Two potent inhibitors exhibited strong activity against proliferation of BT142 glioma cells with IDH1 R132H mutation, while these compounds did not significantly affect the growth of glioma cells without IDH1 mutation. This novel series of IDH1(R132H) inhibitors have potential to be further developed for the treatment of glioma with IDH1 mutation.

Abstract 531: Heterozygous IDH1 R132H/WT created by “single base editing” inhibits human astroglial cell growth by downregulating YAP

Abstract Mutations in the isocitrate dehydrogenase 1 (IDH1) gene have been identified in a number of cancer types, including brain cancer. The Cancer Genome Atlas project has revealed that IDH1 mutations occur in 70-80% of grade II and grade III gliomas. Until recently, most of the functional studies of IDH1 mutations in cellular models have been conducted in overexpression systems with the IDH1 wild type background. In this study, we employed a modified CRISPR/Cas9 genome editing technique called “single base editing”, and efficiently introduced heterozygous IDH1 R132H mutation in human astroglial cells (~20%). Compared with astroglial cells with IDH1 wild type, monoallelic IDH1 R132H mutation (IDH1R132H/WT) inhibited cell proliferation and promoted cell migration. Global gene expression analysis identified molecular targets and pathways altered by IDH1R132H/WT, including cell proliferation, extracellular matrix, and cell migration, consistent with our phenotype characterization. Global DNA methylation profiling showed hypermethylation as well as hypomethylation induced by IDH1R132H/WT. Using our mutated IDH1 models generated by genome editing, we identified novel molecular targets of IDH1R132H/WT, namely Yes-associated protein (YAP) and its downstream signaling pathway Notch, to mediate the cell growth-inhibiting effect of IDH1R132H/WT. Our findings are in line with the clinical observations that brain tumor patients with mutated IDH1 have a better prognosis. In summary, the “single base editing” strategy has successfully created heterozygous IDH1 R132H mutation that recapitulate the naturally occurring IDH1 mutant. Our isogenic cellular systems that differ in a single nucleotide in one allele of the IDH1 gene provide a valuable model for novel discoveries of IDH1R132H/WT-driven biological events. Citation Format: Shuang Wei, Jie Wang, Olutobi Oyinlade, Ding Ma Ma, Shuyan Wang, Shuli Xia. Heterozygous IDH1R132H/WT created by “single base editing” inhibits human astroglial cell growth by downregulating YAP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 531.

Combination of isocitrate dehydrogenase 1 (IDH1) mutation and podoplanin expression in brain tumors identifies patients at high or low risk of venous thromboembolism

Essentials Risk stratification for venous thromboembolism (VTE) in patients with brain tumors is challenging.Patients with IDH1 wildtype and high podoplanin expression have a 6‐month VTE risk of 18.2%.Patients with IDH1 mutation and no podoplanin expression have a 6‐month VTE risk of 0%. IDH1 mutation and podoplanin overexpression in primary brain tumors appear to be exclusive. SummaryBackgroundVenous thromboembolism (VTE) is a frequent complication in primary brain tumor patients. Independent studies revealed that podoplanin expression in brain tumors is associated with increased VTE risk, whereas the isocitrate dehydrogenase 1 (IDH1) mutation is associated with very low VTE risk.ObjectivesTo investigate the interrelation between intratumoral podoplanin expression and IDH1 mutation, and their mutual impact on VTE development.Patients/MethodsIn a prospective cohort study, intratumoral IDH1 R132H mutation and podoplanin were determined in brain tumor specimens (mainly glioma) by immunohistochemistry. The primary endpoint of the study was symptomatic VTE during a 2‐year follow‐up.ResultsAll brain tumors that expressed podoplanin to a medium‐high extent showed also an IDH1 wild‐type status. A score based on IDH1 status and podoplanin expression levels allowed prediction of the risk of VTE. Patients with wild‐type IDH1 brain tumors and high podoplanin expression had a significantly increased VTE risk compared with those with mutant IDH1 tumors and no podoplanin expression (6‐month risk 18.2% vs. 0%).Conclusions IDH1 mutation and podoplanin overexpression seem to be exclusive. Although brain tumor patients with IDH1 mutation are at very low risk of VTE, the risk of VTE in patients with IDH1 wild‐type tumors is strongly linked to podoplanin expression levels.

Heterozygous IDH1R132H/WT created by "single base editing" inhibits human astroglial cell growth by downregulating YAP.

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene have been identified in a number of cancer types, including brain cancer. The Cancer Genome Atlas project has revealed that IDH1 mutations occur in 70–80% of grade II and grade III gliomas. Until recently, most of the functional studies of IDH1 mutations in cellular models have been conducted in overexpression systems with the IDH1 wild type background. In this study, we employed a modified CRISPR/Cas9 genome editing technique called “single base editing”, and efficiently introduced heterozygous IDH1 R132H mutation (IDH1R132H/WT) in human astroglial cells. Global DNA methylation profiling revealed hypermethylation as well as hypomethylation induced by IDH1R132H/WT. Global gene expression analysis identified molecular targets and pathways altered by IDH1R132H/WT, including cell proliferation, extracellular matrix, and cell migration. Our phenotype analysis indicated that compared with IDH1 wild type cells, IDH1R132H/WT promoted cell migration by upregulating integrin β4 (ITGB4), and significantly inhibited cell proliferation. Using our mutated IDH1 models generated by “single base editing”, we identified novel molecular targets of IDH1R132H/WT, namely Yes-associated protein (YAP) and its downstream signaling pathway Notch, to mediate the cell growth-inhibiting effect of IDH1R132H/WT. In summary, the “single base editing” strategy has successfully created heterozygous IDH1 R132H mutation that recapitulates the naturally occurring IDH1 mutation. Our isogenic cellular systems that differ in a single nucleotide in one allele of the IDH1 gene provide a valuable model for novel discoveries of IDH1R132H/WT-driven biological events.

Oncogenic MicroRNA-20a is downregulated by the HIF-1α/c-MYC pathway in IDH1 R132H-mutant glioma

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene have been identified as one of the earliest events in gliomagenesis, occurring in over 70% of low grade gliomas and are present in the vast majority of secondary glioblastoma (GBM) that develop from these low-grade lesions. The aim of this study was to investigate whether the IDH1 R132H mutation influences the expression of oncogenic miR-20a and shed light on the underlying molecular mechanisms. The findings of the current study demonstrate presence of the IDH1 R132H mutation in primary human glioblastoma cell lines with upregulated HIF-1α expression, downregulating c-MYC activity and resulting in a consequential decrease in miR-20a, which is responsible for cell proliferation and resistance to standard temozolomide treatment. Elucidating the mechanism of oncogenic miR-20a activity introduces its role among well-established signaling pathways (i.e. HIF/c-MYC) and may be a meaningful prognostic biomarker or target for novel therapies among patients with IDH1-mutant glioma.

Oncogenic IDH1 Mutations Promote Enhanced Proline Synthesis through PYCR1 to Support the Maintenance of Mitochondrial Redox Homeostasis

SummarySince the discovery of mutations in isocitrate dehydrogenase 1 (IDH1) in gliomas and other tumors, significant efforts have been made to gain a deeper understanding of the consequences of this oncogenic mutation. One aspect of the neomorphic function of the IDH1 R132H enzyme that has received less attention is the perturbation of cellular redox homeostasis. Here, we describe a biosynthetic pathway exhibited by cells expressing mutant IDH1. By virtue of a change in cellular redox homeostasis, IDH1-mutated cells synthesize excess glutamine-derived proline through enhanced activity of pyrroline 5-carboxylate reductase 1 (PYCR1), coupled to NADH oxidation. Enhanced proline biosynthesis partially uncouples the electron transport chain from tricarboxylic acid (TCA) cycle activity through the maintenance of a lower NADH/NAD+ ratio and subsequent reduction in oxygen consumption. Thus, we have uncovered a mechanism by which tumor cell survival may be promoted in conditions associated with perturbed redox homeostasis, as occurs in IDH1-mutated glioma.

Differential expression of the TWEAK receptor Fn14 in IDH1 wild-type and mutant gliomas.

The TNF receptor superfamily member Fn14 is overexpressed by many solid tumor types, including glioblastoma (GBM), the most common and lethal form of adult brain cancer. GBM is notable for a highly infiltrative growth pattern and several groups have reported that high Fn14 expression levels can increase tumor cell invasiveness. We reported previously that the mesenchymal and proneural GBM transcriptomic subtypes expressed the highest and lowest levels of Fn14 mRNA, respectively. Given the recent histopathological re-classification of human gliomas by the World Health Organization based on isocitrate dehydrogenase 1 (IDH1) gene mutation status, we extended this work by comparing Fn14 gene expression in IDH1 wild-type (WT) and mutant (R132H) gliomas and in cell lines engineered to overexpress the IDH1 R132H enzyme. We found that both low-grade and high-grade (i.e., GBM) IDH1 R132H gliomas exhibit low Fn14 mRNA and protein levels compared to IDH1 WT gliomas. Forced overexpression of the IDH1 R132H protein in glioma cells reduced Fn14 expression, while treatment of IDH1 R132H-overexpressing cells with the IDH1 R132H inhibitor AGI-5198 or the DNA demethylating agent 5-aza-2'-deoxycytidine increased Fn14 expression. These results support a role for Fn14 in the more aggressive and invasive phenotype associated with IDH1 WT tumors and indicate that the low levels of Fn14 gene expression noted in IDH1 R132H mutant gliomas may be due to epigenetic regulation via changes in DNA methylation.