Early Noninvasive Metabolic Biomarkers of Mutant IDH Inhibition in Glioma.
ABSTRACT: Approximately 80% of low-grade glioma (LGGs) harbor mutant isocitrate dehydrogenase 1/2 (IDH1/2) driver mutations leading to accumulation of the oncometabolite 2-hydroxyglutarate (2-HG). Thus, inhibition of mutant IDH is considered a potential therapeutic target. Several mutant IDH inhibitors are currently in clinical trials, including AG-881 and BAY-1436032. However, to date, early detection of response remains a challenge. In this study we used high resolution 1H magnetic resonance spectroscopy (1H-MRS) to identify early noninvasive MR (Magnetic Resonance)-detectable metabolic biomarkers of response to mutant IDH inhibition. In vivo 1H-MRS was performed on mice orthotopically-implanted with either genetically engineered (U87IDHmut) or patient-derived (BT257 and SF10417) mutant IDH1 cells. Treatment with either AG-881 or BAY-1436032 induced a significant reduction in 2-HG. Moreover, both inhibitors led to a significant early and sustained increase in glutamate and the sum of glutamate and glutamine (GLX) in all three models. A transient early increase in N-acetylaspartate (NAA) was also observed. Importantly, all models demonstrated enhanced animal survival following both treatments and the metabolic alterations were observed prior to any detectable differences in tumor volume between control and treated tumors. Our study therefore identifies potential translatable early metabolic biomarkers of drug delivery, mutant IDH inhibition and glioma response to treatment with emerging clinically relevant therapies.
Project description:B7H3 (also known as CD276) is a co-stimulator checkpoint protein of the cell surface B7 superfamily. Recently, the function beyond immune regulation of B7H3 has been widely studied. However, the expression preference and the regulation mechanism underlying B7H3 in different subtypes of gliomas is rarely understood. We show here that B7H3 expression is significantly decreased in IDH-mutated gliomas and in cultured IDH1-R132H glioma cells. Accumulation of 2-HG leads to a remarkable downregulation of B7H3 protein and the activity of IDH1-R132H mutant is responsible for B7H3 reduction in glioma cells. Inhibition of autophagy by inhibitors like leupeptin, chloroquine (CQ), and Bafilomycin A1 (Baf-A1) blocks the degradation of B7H3 in glioma cells. In the meantime, the autophagy flux is more active with higher LC3B-II and lower p62 in IDH1-R132H glioma cells than in IDH1-WT cells. Furthermore, sequence alignment analysis reveals potential LC3-interacting region (LIR) motifs "F-V-S/N-I/V" in B7H3. Moreover, B7H3 interacts with p62 and CQ treatment significantly enhances this interaction. Additionally, we find that <i>B7H3</i> is positively correlated with <i>VEGFA</i> and <i>MMP2</i> by bioinformatics analysis in gliomas. B7H3 and VEGFA are decreased in IDH-mutated gliomas and further reduced in 2-HG<sup>high</sup> gliomas compared to 2-HG<sup>low</sup> glioma sections by IHC staining. Our study demonstrates that B7H3 is preferentially overexpressed in IDH wild-type gliomas and could serve as a potential theranostic target for the precise treatment of glioma patients with wild-type IDH.
Project description:The oncogenes that are expressed in gliomas reprogram particular pathways of glucose, amino acids, and fatty acid metabolism. Mutations in isocitrate dehydrogenase genes (IDH1/2) in diffuse gliomas are associated with abnormally high levels of 2-hydroxyglutarate (2-HG) levels. The aim of this study was to determine whether metabolic reprogramming associated with IDH mutant gliomas leads to additional ¹H MRS-detectable differences between IDH1 and IDH2 mutations, and to identify metabolites correlated with 2-HG. A total of 21 glioma patients (age= 37 ± 11, 13 males) were recruited for magnetic resonance spectroscopy (MRS) using semi-localization by adiabatic selective refocusing pulse sequence at an ultra-high-field (7T). For 20 patients, the tumor mutation subtype was confirmed by immunohistochemistry and DNA sequencing. LCModel analysis was applied for metabolite quantification. A two-sample t-test was used for metabolite comparisons between IDH1 (<i>n</i> = 15) and IDH2 (<i>n</i> = 5) mutant gliomas. The Pearson correlation coefficients between 2-HG and associated metabolites were calculated. A Bonferroni correction was applied for multiple comparison. IDH2 mutant gliomas have a higher level of 2-HG/tCho (total choline=phosphocholine+glycerylphosphorylcholine) (2.48 ± 1.01vs.0.72 ± 0.38, <i>P<sub>c</sub></i> < 0.001) and myo-Inositol/tCho (2.70 ± 0.90 vs. 1.46 ± 0.51, <i>P<sub>c</sub></i> = 0.011) compared to IDH1 mutation gliomas. Associated metabolites, myo-Inositol and glucose+taurine were correlated with 2-HG levels. These results show the improved characterization of the metabolic pathways in IDH1 and IDH2 gliomas for precision medicine.
Project description:BACKGROUND:BAY1436032 is a fluorine-containing inhibitor of the R132X-mutant isocitrate dehydrogenase (mIDH1). It inhibits the mIDH1-mediated production of 2-hydroxyglutarate (2-HG) in glioma cells. We investigated brain penetration of BAY1436032 and its effects using 1H/19F-Magnetic Resonance Spectroscopy (MRS). METHODS:19F-Nuclear Magnetic Resonance (NMR) Spectroscopy was conducted on serum samples from patients treated with BAY1436032 (NCT02746081 trial) in order to analyze 19F spectroscopic signal patterns and concentration-time dynamics of protein-bound inhibitor to facilitate their identification in vivo MRS experiments. Hereafter, 30 mice were implanted with three glioma cell lines (LNT-229, LNT-229 IDH1-R132H, GL261). Mice bearing the IDH-mutated glioma cells received 5 days of treatment with BAY1436032 between baseline and follow-up 1H/19F-MRS scan. All other animals underwent a single scan after BAY1436032 administration. Mouse brains were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). RESULTS:Evaluation of 1H-MRS data showed a decrease in 2-HG/total creatinine (tCr) ratios from the baseline to post-treatment scans in the mIDH1 murine model. Whole brain concentration of BAY1436032, as determined by 19F-MRS, was similar to total brain tissue concentration determined by Liquid Chromatography with tandem mass spectrometry (LC-MS/MS), with a signal loss due to protein binding. Intratumoral drug concentration, as determined by LC-MS/MS, was not statistically different in models with or without R132X-mutant IDH1 expression. CONCLUSIONS:Non-invasive monitoring of mIDH1 inhibition by BAY1436032 in mIDH1 gliomas is feasible.
Project description:<h4>Background</h4>Recurrent mutations in the isocitrate dehydrogenase 1 (IDH1) and IDH2 genes, which are frequent in gliomas, result in marked accumulation of the metabolic by-product 2-hydroxyglutarate (2-HG) within tumors. In other malignancies, such as acute myeloid leukemia, presence of IDH mutation is associated with elevated 2-HG levels in serum or urine compartments. Circulating 2-HG in patients with glial malignancies has not been thoroughly investigated.<h4>Methods</h4>In this study, we analyzed 2-HG levels in the serum and urine of a large set of patients with IDH-mutant and IDH-wild-type glioma, and the cerebrospinal fluid (CSF) from a subset of this cohort.<h4>Results</h4>We found that 2-HG was elevated in the urine of patients with IDH-mutant versus IDH-wild-type glioma, although no significant differences in 2-HG levels were observed in the serum or the small set of CSF samples obtained. Among patients with IDH-mutant glioma, 2-HG levels did not differ based on the histopathologic grade, genetic subtype (TP53 mutant or 1p/19q codeleted), presence of a canonical (IDH1 R132H) or noncanonical (any other IDH variant) mutation, or treatment type.<h4>Conclusion</h4>Our finding suggests that urinary 2-HG is increased among patients with IDH-mutant gliomas, and may represent a future surrogate, noninvasive biomarker to aid in diagnosis, prognosis, and management.<h4>Implications for practice</h4>Patients with glioma who harbor mutations in isocitrate dehydrogenase genes showed selective elevation of the oncometabolite 2-hydroxyglutarate in the urine. Similar elevations were not identified in the serum or cerebrospinal fluid. 2-Hydroxyglutarate may serve as a useful, noninvasive biomarker to stratify patients newly diagnosed with glioma with regard to prognosis and management.
Project description:B7H3 (also known as CD276) is a co-stimulator checkpoint protein of the cell surface B7 superfamily. Recently, the function beyond immune regulation of B7H3 has been widely studied. However, the expression preference and the regulation mechanism underlying B7H3 in different subtypes of gliomas is rarely understood. We show here that B7H3 expression is significantly decreased in IDH-mutated gliomas and in cultured IDH1-R132H glioma cells. Accumulation of 2-HG leads to a remarkable downregulation of B7H3 protein and the activity of IDH1-R132H mutant is responsible for B7H3 reduction in glioma cells. Inhibition of autophagy by inhibitors like leupeptin, chloroquine (CQ), and Bafilomycin A1 (Baf-A1) blocks the degradation of B7H3 in glioma cells. In the meantime, the autophagy flux is more active with higher LC3B-II and lower p62 in IDH1-R132H glioma cells than in IDH1-WT cells. Furthermore, sequence alignment analysis reveals potential LC3-interacting region (LIR) motifs 'F-V-S/N-I/V' in B7H3. Moreover, B7H3 interacts with p62 and CQ treatment significantly enhances this interaction. Additionally, we find that <i>B7H3</i> is positively correlated with <i>VEGFA</i> and <i>MMP2</i> by bioinformatics analysis in gliomas. B7H3 and VEGFA are decreased in IDH-mutated gliomas and further reduced in 2-HG<sup>high</sup> gliomas compared to 2-HG<sup>low</sup> glioma sections by IHC staining. Our study demonstrates that B7H3 is preferentially overexpressed in IDH wild-type gliomas and could serve as a potential theranostic target for the precise treatment of glioma patients with wild-type IDH.
Project description:Mutation in the IDH1 or IDH2 genes occurs frequently in gliomas and other human malignancies. In intermediate grade gliomas, IDH1 mutation is found in over 70% of tumors. These mutations impart the mutant IDH enzyme with a neomorphic activity - the ability to synthesize 2-hydroxyglutarate (2-HG). This ability leads to a reprogramming of chromatin state, a block in differentiation, and the establishment of the glioma hypermethylator phenotype (G-CIMP). It has been hypothesized but not proven that the extensive DNA methylation that occurs in G-CIMP tumors helps maintain and "lock in" glioma cancer cells in a dedifferentiated state. Here, we tested this hypothesis by treating patient derived IDH1 mutant glioma initiating cells (GIC) with non-cytotoxic, epigenetically targeted doses of the DNMT inhibitor decitabine. Global methylome analysis of treated IDH1 mutant GICs showed that DAC treatment resulted in reversal of DNA methylation marks induced by IDH and the re-expression of genes associated with differentiation. Accordingly, treatment of IDH1 mutant glioma cells resulted in a dramatic loss of stem-like properties and efficient adoption of markers of differentiation, effects not seen in decitabine treated IDH wild-type GICs. Induction of differentiation was much more efficient than that seen following treatment with a specific inhibitor of mutant IDH enzyme (Agios). Decitabine also decreased replicative potential and tumor growth in vivo. Reexpression of polycomb regulated genes accompanied these DAC-induced phenotypes. In total, our data indicates that targeting the pathologic DNA methylation in IDH mutant cells can reverse mutant IDH induced hypermethylation and block in differentiation and promote tumor control. These findings have substantial impact for exploring new treatment strategies for patients with IDH mutant gliomas.
Project description:Gliomas with Isocitrate dehydrogenase 1 (IDH1) mutation have alterations in several enzyme activities, resulting in various metabolic changes. The aim of this study was to determine a mechanism for the better prognosis of gliomas with IDH mutation by performing metabolomic analysis. To understand the metabolic state of human gliomas, we analyzed clinical samples obtained from surgical resection of glioma patients (grades II-IV) with or without the IDH1 mutation, and compared the results with U87 glioblastoma cells overexpressing IDH1 or IDH1<sup>R132H</sup>. In clinical samples of gliomas with IDH1 mutation, levels of D-2-hydroxyglutarate (D-2HG) were increased significantly compared with gliomas without IDH mutation. Gliomas with IDH mutation also showed decreased intermediates in the tricarboxylic acid cycle and pathways involved in the production of energy, amino acids, and nucleic acids. The marked difference in the metabolic profile in IDH mutant clinical glioma samples compared with that of mutant IDH expressing cells includes a decrease in ?-oxidation due to acyl-carnitine and carnitine deficiencies. These metabolic changes may explain the lower cell division rate observed in IDH mutant gliomas and may provide a better prognosis in IDH mutant gliomas.
Project description:Mutations in the isocitrate dehydrogenase genes (IDH1/2) occur often in diffuse gliomas, where they are associated with abnormal accumulation of the oncometabolite 2-hydroxyglutarate (2-HG). Monitoring 2-HG levels could provide prognostic information in this disease, but detection strategies that are noninvasive and sufficiently quantitative have yet to be developed. In this study, we address this need by presenting a proton magnetic resonance spectroscopy ((1)H-MRS) acquisition scheme that uses an ultrahigh magnetic field (? 7T) capable of noninvasively detecting 2-HG with quantitative measurements sufficient to differentiate mutant cytosolic IDH1 and mitochondrial IDH2 in human brain tumors. Untargeted metabolomics analysis of in vivo (1)H-MRS spectra discriminated between IDH-mutant tumors and healthy tissue, and separated IDH1 from IDH2 mutations. High-quality spectra enabled the quantification of neurochemical profiles consisting of at least eight metabolites, including 2-HG, glutamate, lactate, and glutathione in both tumor and healthy tissue voxels. Notably, IDH2 mutation produced more 2-HG than IDH1 mutation, consistent with previous findings in cell culture. By offering enhanced sensitivity and specificity, this scheme can quantitatively detect 2-HG and associated metabolites that may accumulate during tumor progression, with implications to better monitor patient responses to therapy.
Project description:<i>Isocitrate dehydrogenase</i> (<i>IDH)-1</i> mutation is an important prognostic factor and a potential therapeutic target in glioma. Immunohistological and molecular diagnosis of <i>IDH</i> mutation status is invasive. To avoid tumor biopsy, dedicated spectroscopic techniques have been proposed to detect D-2-hydroxyglutarate (2-HG), the main metabolite of <i>IDH</i>, directly in vivo. However, these methods are technically challenging and not broadly available. Therefore, we explored the use of machine learning for the non-invasive, inexpensive and fast diagnosis of <i>IDH</i> status in standard <sup>1</sup>H-magnetic resonance spectroscopy (<sup>1</sup>H-MRS). To this end, 30 of 34 consecutive patients with known or suspected glioma WHO grade II-IV were subjected to metabolic positron emission tomography (PET) imaging with O-(2-<sup>18</sup>F-fluoroethyl)-L-tyrosine (<sup>18</sup>F-FET) for optimized voxel placement in <sup>1</sup>H-MRS. Routine <sup>1</sup>H-magnetic resonance (<sup>1</sup>H-MR) spectra of tumor and contralateral healthy brain regions were acquired on a 3 Tesla magnetic resonance (3T-MR) scanner, prior to surgical tumor resection and molecular analysis of <i>IDH</i> status. Since 2-HG spectral signals were too overlapped for reliable discrimination of <i>IDH</i> mutated (<i>IDHmut</i>) and <i>IDH</i> wild-type (<i>IDHwt</i>) glioma, we used a nested cross-validation approach, whereby we trained a linear support vector machine (SVM) on the complete spectral information of the <sup>1</sup>H-MRS data to predict <i>IDH</i> status. Using this approach, we predicted <i>IDH</i> status with an accuracy of 88.2%, a sensitivity of 95.5% (95% CI, 77.2-99.9%) and a specificity of 75.0% (95% CI, 42.9-94.5%), respectively. The area under the curve (AUC) amounted to 0.83. Subsequent ex vivo <sup>1</sup>H-nuclear magnetic resonance (<sup>1</sup>H-NMR) measurements performed on metabolite extracts of resected tumor material (eight specimens) revealed myo-inositol (M-ins) and glycine (Gly) to be the major discriminators of <i>IDH</i> status. We conclude that our approach allows a reliable, non-invasive, fast and cost-effective prediction of <i>IDH</i> status in a standard clinical setting.