Isocitrate Dehydrogenase 1 R132H Research Articles

MODL-46. RECAPITULATING CELLULAR HETEROGENEITY AND TREATMENT-SPECIFIC EFFECTS THROUGH SINGLE-CELL ANALYSIS OF PATIENT-DERIVED TUMOR SLICE CULTURE MODELS

Abstract INTRODUCTION Malignant brain tumors are almost universally fatal despite standard-of-care (SOC) therapy with radiation (XRT) and temozolomide (TMZ). Low Grade Glioma (LGG) is a subclass of malignant glioma that can harbor an R132H mutation in Isocitrate Dehydrogenase 1 (IDH1). Extensive clinical trials have explored the clinical utility of IDH1 R132H mutation small molecule inhibitors such as ivosidenib and vorasidenib; however, the direct intracellular and intercellular effects of IDH1 R132H inhibition relative to SOC remain largely unknown. Objective This study examines these issues by developing a novel patient-specific tumor slice culture model to study the effects of both SOC and IDH1 R132H inhibition on IDH1 mutant glioma. Method En-bloc tumor samples from a patient with IDH mutant LGG undergoing surgery were sectioned into 400 um thick slices using a vibratome and cultured on porous microwell inserts for up to 14 days. Tumor slices were then either left untreated or treated with SOC (XRT/TMZ) or ivosidenib and harvested at various time points. Samples were hashed, and single-cell RNA sequencing was performed and analyzed. Result: Patient-derived tumor slice cultures were able to grow and were observed to contain heterogeneous cell populations, including tumor cells, astrocytes, neurons, oligodendrocytes, and microglia. When slice cultures were treated with SOC and ivosidenib treatment, separate tumor subpopulations were shown to be sensitive to either treatment. Furthermore, scRNA analysis revealed unique subpopulations that were insensitive to either treatment. We further demonstrate that slice cultures treated with Ivosidenib enriched for gene signatures related to protein translation and antigen processing. Intriguingly, we find that tumor cultures treated with ivosidenib abrogated the Notch signaling pathways between tumor-tumor and tumor-immune clusters. CONCLUSION Herein, we demonstrate that patient-derived tumor slice cultures are a viable strategy for understanding drug effects and tumor sensitivity to different therapies.

An intermediate phenotype in IDH related enchondromatosis spectrum

Mosaic variants of IDH1 (isocitrate dehydrogenase-1) R132 and IDH2 (isocitrate dehydrogenase-2) R172 loci were detected in most of the bone cysts of Ollier and Maffucci series and in the blood and tissue samples of metaphyseal enchondromatosis with D-2-hydroxyglutaric aciduria (MC-HGA) patients. We aimed to report an intermediate phenotype comparing with the reported cases. The proband was a 9-year-old boy with widespread metaphyseal enchondromatosis involving metaphyses of long tubular bones, iliac bones and tubular bones of both hands and feet and sparing spine and flat and short bones. He underwent quad whole exome sequencing (index-both parents-healthy sibling). Sanger sequencing was performed for confirmation and segregation purposes. Heterozygous IDH1 R132H (c.395G > A) variant was detected in his blood via whole exome sequencing and Sanger analysis in mosaic state, 22% of the reads and Sanger signal. He had no D-2-hydroxyglutaric aciduria in urinary organic acid analysis. Our case is unique with the presence of IDH1 R132H variant in blood with metaphyseal enchondromatosis without D-2-hydroxyglutaric aciduria. It was a transitional phenotype. With his phenotype, we expand the IDH1/IDH2 related enchondromatosis phenotypes.

Targeting IDH1-Mutated Pre-Leukemic Hematopoietic Stem Cells in Myeloid Disease, Including CCUS and AML

Introduction: Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 have been reported in clonal hematopoiesis and are recurrent, early events in the development of acute myeloid leukemia (AML). A subsequent stepwise acquisition of additional genetic aberrations leads to the development of frank leukemia. At the time of AML diagnosis, rare pre-leukemic hematopoietic stem cells (pHSC) can be detected containing some, but not all, of the mutations in the corresponding leukemia, although these pHSCs are phenotypically indistinguishable from residual wildtype hematopoietic stem cells (HSC). The frequency of pHSCs at the time of diagnosis is known to correlate to overall survival in AML patients (Corces et al, Nat Genet 2016), indicating the potential relevance of therapeutically targeting these cells to prevent relapse. Methods: To model these rare pre-leukemic cells, we introduced IDH1 R132H and IDH2 R140Q mutants or their wildtype counterparts into human CD34+ hematopoietic stem and progenitor cells (HSPC) using CRISPR/Cas9 and AAV6-mediated homology directed repair (HDR). Successfully edited cells were then sorted by FACS based on GFP expression for subsequent analyses including 2HG quantification, colony formation, differentiation capacity, RNA sequencing, and metabolic assays. Additionally, primary pHSCs from diagnostic bone marrow aspirates from AML patients were also isolated by FACS and subjected to treatment, colony formation assays, and genotyping by ddPCR. Results: Human CD34+ HSPCs engineered to express either IDH1 R132H or IDH2 R140Q produced 2HG and showed a striking reduction in colony formation and differentiation capacity, recapitulating the cytopenic phenotype observed in IDH-mutated clonal cytopenia of undetermined significance (CCUS). RNA-seq of these cells revealed large transcriptomic changes shared between IDH1 R132H and IDH2 R140Q mutations, but gene set enrichment analyses revealed the specific and significant downregulation of oxidative phosphorylation-related genes in R132H-mutated cells. These R132H cells were further confirmed to have lower basal metabolic activity coupled to a higher mitochondrial dependency as determined by SCENITH (Argüello et al, Cell Metab 2021). These metabolic alterations suggested that it might be possible to selectively therapeutically target R132H mutated cells. To evaluate this, we treated HSPCs engineered to express GFP, IDH1 wildtype, or IDH1 R132H, with the known mutant-specific inhibitor ivosidenib, the OXPHOS-inhibitor IACS-010759, or DMSO as a control. Strikingly, ivosidenib restored the defect in colony formation conferred by R132H and increased the total number of colonies whereas IACS-010759 effectively inhibited R132H colony formation (Figure 1A). To further investigate this vulnerability, we sorted pHSCs from diagnostic bone marrow samples from primary IDH1 and IDH2-mutated AML samples according to the phenotype CD45dimCD3-CD19-CD20-CD34+CD38-CD99-TIM3-. These cells were subjected to treatment with DMSO, ivosidenib, enasidenib, or IACS-010759 and subsequently plated for colony formation. Because the sorted cell population contained a mixture of mutant pHSCs and residual wildtype HSCs, all colonies were picked and genotyped for IDH mutations using ddPCR, revealing that only IDH1 wildtype colonies grew out after IACS-010759 treatment whereas all other conditions resulted in a mixture of wildtype and mutant colonies (Figure 1B). Conclusions: The OXPHOS-inhibitor IACS-010759 can eradicate IDH1-mutated pHSCs whereas ivosidenib cannot. This effect is specific for IDH1 mutations and the same effect is not seen in IDH2-mutated pHSCs. These results have potential clinical implications when considering therapeutic options in AML as well as IDH1-mutated pre-leukemic conditions such as CCUS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cooperative Inhibitory Effect of IDH1 Mutation and ASXL1 Mutation on Normal Myeloid Differentiation

Somatic mutations in the additional sex comb-like 1, ASXL1, have been described in various types of myeloid malignancies, including myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia (AML). In addition, ASXL1 mutations are frequently found in healthy population, which predispose them to not only myeloid malignancies, but also cardiovascular diseases. This condition is known as clonal hematopoiesis with intermediate potential (CHIP), although the full etiology of this preleukemic condition is not elucidated. As for AML, patients with ASXL1 mutations are known to have very poor prognosis compared to non-mutated patients. Although previous studies demonstrated that mutant ASXL1 promoted myeloid leukemogenesis via both loss-of-function and gain-of-function manners, mechanisms how ASXL1 mutations induce myeloid transformation is not fully elucidated. We analyzed publicly available datasets of AML patients, and found that isocitrate dehydrogenase 1 (IDH1) is co-mutated in 13% of AML with ASXL1. As the mechanism by which ASXL1 and IDH1 mutations cooperate to promote AML is not understood, we generated a model of AML with ASXL1 and IDH1 mutations using 32D-cl3 cells and discovered that mutant IDH1 could block myeloid differentiation only when mutant ASXL1 was present. At first, in our experiments, we retrovirally overexpressed mutant (G646WfsX12) or normal form of ASXL1 in 32D-cl3, which is a commonly used IL-3 dependent cell-line of murine myeloid precursor cell. We found that Gr.1 expression levels were decreased in these cells compared to 32D-cl3 with mutant ASXL1 (11.1% vs 26.5%, respectively; p=0.014), and that the proportion of cells in which Gr1 expression was induced by treatment with G-CSF was significantly lower (29.1% vs 54.8%, respectively; p=0.003). Next, we generated a tetracycline-inducible mutant (R132H) form of IDH1 lentiviral vector, and transfected them to these 32D-cl3 cells. We found that tetracycline-inducible mutant IDH1 inhibited G-CSF induced Gr.1 expression in the presence of mutant ASXL1 (7.1% with doxycycline vs 29.9% without doxycycline, respectively; p<0.01), while in the absence of mutant ASXL1, forced expression of mutant IDH1 could not block G-CSF induced Gr.1 expression (59.6% with doxycycline vs 59.2% without doxycycline, respectively; p=0.035). In accordance to these analyses of surface markers, we found that reduction of expression levels of CEBPβ induced by tetracycline-inducible ectopic expression of mutant IDH1 was only observed in 32D-cl3 cells with mutant ASXL1. Furthermore, we found that growth rate of 32D-cl3 cells with mutant ASXL1 and mutant IDH1 was significantly higher than those with mutant ASXL1 alone. These findings suggested that mutant IDH1 can exert its inhibitory effect of normal myeloid differentiation only when mutant ASXL1 is present. Herein, we hypothesized that mutant ASXL1 and mutant IDH1 had unknown cooperative mechanisms to suppress normal differentiation and promote leukemogenesis, and performed transcriptome analysis of the 32D-cl3 cells treated with G-CSF. Tendency toward decreased expression of genes associated with myeloid differentiation in the presence of IDH1 was only observed in cells with mutant ASXL1. Further, it was revealed that specifically dysregulated genes such as HDAC6 or KMT2A in leukemic stem cells were enriched in 32D-cl3 cells with mutant ASXL1, which suggested that mutant IDH1 could induce leukemogenesis only in the presence of ASXL1. In our study, we found that the presence of IDH1 R132H mutation in 32D-cl3 cells had an inhibitory effect on normal myeloid differentiation only in the presence of ASXL1 mutation. Transcriptome analysis was performed to explore genes dysregulated by cooperation of mutant ASXL1 and mutant IDH1. We demonstrated that there might be cooperative mechanisms between ASXL1 and IDH1 to induce differentiation and promote leukemogenesis. Our study would not only provide a new role for IDH1 mutation in ASXL1 mutated AML, but also identify a new therapeutic target for ASXL1-mutatated AML.

High EZH2 Protein Expression Is a Poor Prognostic Predictor in IDH1 R132H-Negative Gliomas.

Accumulating data indicates that enhancer of zeste homology 2 (EZH2) and isocitrate dehydrogenase 1 (IDH1) are implicated in promoting tumourigenesis in a myriad of malignancies including gliomas. We aimed to determine the immunoexpression of EZH2 in gliomas and its correlation with clinicopathological variables. The prognostic value of the combined immunoexpression of EZH2 and IDH1 was further explored in a retrospective analysis involving 56 patients with histologically confirmed gliomas in Universiti Kebangsaan Malaysia Medical Centre from 2010 to 2016. The patients were then followed up for a period of five years. EZH2 and IDH1 R132H immunoexpressions were performed and analysed on respective tissue blocks. Five-year progression-free survival (PFS) and overall survival (OS) were estimated by Kaplan–Meier analysis. Univariate and multivariate Cox proportional hazard regression models were performed to evaluate the value of EZH2 as an independent factor for the prediction of PFS and OS. High EZH2 immunoexpression was demonstrated in 27 (48.2%) gliomas. High EZH2 expression was significantly correlated with older age (p = 0.003), higher tumour grade (p < 0.001), negative IDH1 R132H immunoexpression (p = 0.039), a poor 5-year PFS (mean = 9.7 months, p < 0.001) and 5-year OS (mean = 28.2 months, p = 0.007). In IDH1 R132H-negative gliomas, there was a trend toward shorter 5-year PFS (mean = 8.0 months, p = 0.001) and 5-year OS (mean = 28.7 months, p = 0.06) in gliomas demonstrating high EZH2 expression compared with those with low EZH2 expression. High EZH2 immunoexpression is an unfavourable independent prognostic predictor of poor survival in gliomas. EZH2 analysis might therefore be of clinical value for risk stratification, especially in patients with IDH1 R132H-negative gliomas.

Low expression of isocitrate dehydrogenase 1 (IDH1) R132H is associated with advanced pathological features in laryngeal squamous cell carcinoma.

Recent developments in genomic sequencing have led to the identification of somatic mutations in isocitrate dehydrogenase 1 (IDH1) in various malignancies. IDH1 R132H is the most common mutation of IDH1, which affects codon 132 and results in the conversion of amino acid residue arginine (R) to histidine (H). This study is designed to evaluate the association between the expression of IDH1 R132H and clinicopathological characteristics in laryngeal squamous cell carcinoma (LSCC). The expression pattern and clinical significance of IDH1 R132H were investigated in tissue microarrays (TMAs) of 50 LSCC tumors as well as adjacent normal tissues using immunohistochemistry. Then the exons of the 12 tumor samples with negative/weak positive staining were sequenced by applying polymerase chain reaction(PCR). The results demonstrated that the cytoplasmic expression of IDH1 R132H was downregulated in tumor cells compared to adjacent normal tissues. A statistically significant association was found between a low level of cytoplasmic expression of IDH1 R132H protein and an increase in histological grade (p < 0.001), perineural invasion (p = 0.019), and lymph node involvement (p < 0.001). The exon4 sequencing results showed that only one sample was positive for IDH1 R132H mutation. IDH1 R132H expression was observed in 39 (78.0%) LSCC samples. These findings indicate that low cytoplasmic expression of IDH1 R132H may have clinical significance in LSCC patients and is associated with more aggressive tumor behavior and progression of the disease, which can help improve potential treatment in patients with LSCC. Further investigations are needed to understand the biological function of IDH1 R132H and larger sample size to confirm our findings.

P12.06.A Relationship between ascorbate and DNA methylation markers in clinical glioma tumours

Abstract Background Members of the 2-oxoglutarate-dependent dioxygenase (OGDD) enzyme family play an important role in gliomas as they regulate epigenetic modifications and response to hypoxia. The OGDDs require 2-OG and O2 as substrates, and ferrous iron and ascorbate as cofactors. Both hypoxia and aberrant DNA methylation are prognostic indicators for gliomas. The ten-eleven translocase (TET) DNA demethylases are OGDDs that convert 5-methyl cytosine (5mC) to 5-hydroxymethylcytosine (5hmC), with 5hmC levels related to better prognosis. Despite this, there is limited data on the OGDD enzymes and their substrates/cofactors in glioma tissues. Our previous study showed an association between ascorbate content and markers of the hypoxic response in glioblastoma tissue. Here we determine whether there is an association between ascorbate and DNA methylation in glioma. In addition, we assess whether methylation of the methylguanine-DNA methyltransferase (DNA repair enzyme MGMT) promoter is associated with ascorbate content. Materials and methods Frozen clinical glioma samples from 37 patients (n=11 WHO grade I-III, n=26 glioblastoma) were obtained from the Cancer Society Tissue Bank (Ethics approval H19/163). Isocitrate dehydrogenase 1 (IDH1) mutation status was determined by sequencing. Samples were processed on dry ice in liquid nitrogen and analysed for ascorbate (high-performance liquid chromatography), global DNA methylation (mass spectrometry) and MGMT promoter analyses (methylation specific PCR). Results Many grade I-III tumours were IDH1 R132H mutant (6/11), and most glioblastomas were not (2/26). Glioblastoma had significantly lower ascorbate content than grade I-III tumours (p=0.026). Glioblastoma also had lower global 5hmC levels (p=0.0013). IDH1 R132H tumours tended to have a lower ascorbate content (p=0.09). Ascorbate and 5hmC levels were directly correlated (Spearman r= 0.466, p=0.004). However, cytosine and 5mC showed no association with grade or ascorbate. MGMT promoter methylation status was not associated with global methylation or ascorbate content (p=0.97, p=0.96, respectively). Conclusion Our data suggests that ascorbate supports TET activity in clinical glioma. It also appears that site-specific (promoter) methylation was not affected by ascorbate availability. These findings may have clinical implications, as higher 5hmC levels are associated with improved outcome, whilst continued MGMT suppression suggests chemotherapy responsiveness. However, evidence that raising tumour ascorbate leads to increased 5hmC levels, or an associated improvement in survival, requires intervention trials.

Resistance to the isocitrate dehydrogenase 1 mutant inhibitor ivosidenib can be overcome by alternative dimer-interface binding inhibitors

Ivosidenib, an inhibitor of isocitrate dehydrogenase 1 (IDH1) R132C and R132H variants, is approved for the treatment of acute myeloid leukaemia (AML). Resistance to ivosidenib due to a second site mutation of IDH1 R132C, leading to IDH1 R132C/S280F, has emerged. We describe biochemical, crystallographic, and cellular studies on the IDH1 R132C/S280F and R132H/S280F variants that inform on the mechanism of second-site resistance, which involves both modulation of inhibitor binding at the IDH1 dimer-interface and alteration of kinetic properties, which enable more efficient 2-HG production relative to IDH1 R132C and IDH1 R132H. Importantly, the biochemical and cellular results demonstrate that it should be possible to overcome S280F mediated resistance in AML patients by using alternative inhibitors, including some presently in phase 2 clinical trials.

Clinical application of a highly sensitive digital PCR assay to detect a small fraction of IDH1 R132H-mutant alleles in diffuse gliomas.

The current World Health Organization classification of diffuse astrocytic and oligodendroglial tumors requires the examination of isocitrate dehydrogenase 1 (IDH1) or IDH2 mutations. Conventional analysis tools, including Sanger DNA sequencing or pyrosequencing, fail in detecting these variants of low frequency owing to their limited sensitivity. Digital polymerase chain reaction (dPCR) is a recently developed, highly sensitive, and precise quantitative rare variant assay. This study aimed to establish a robust limit of quantitation of the dPCR assay to detect a small fraction of IDH1 R132H mutation. The dPCR assays with serially diluted IDH1 R132H constructs detected 0.05% or more of mutant IDH1 R132H in samples containing mutant DNA. The measured target/total value of the experiments was proportional to the dilution factors and was almost equal to the actual frequencies of the mutant alleles. Based on the average target/total values, together with a twofold standard deviation of the normal DNA, a limit of quantitation of 0.25% was set to secure a safe margin to judge the mutation status of the IDH1 R132H dPCR assay. In clinical settings, detecting IDH1 R132H using dPCR assays can validate ambiguous immunohistochemistry results even when conventional DNA sequencing cannot detect the mutation and assure diagnostic quality.

A high-throughput screening assay for mutant isocitrate dehydrogenase 1 using acoustic droplet ejection mass spectrometry.

Acoustic droplet ejection mass spectrometry (ADE-MS) has recently emerged as a promising label-free, MS-based readout method for high throughput screening (HTS) campaigns in early pharmaceutical drug discovery, since it enables high-speed analysis directly from 384- or 1536-well plates. In this manuscript we describe our characterization of an ADE-MS based high sample content enzymatic assay for mutant isocitrate dehydrogenase 1 (IDH1) R132H with a strong focus on assay development. IDH1 R132H has become a very attractive therapeutic target in the field of antitumor drug discovery, and several pharmaceutical companies have attempted to develop novel small molecule inhibitors against mutant IDH1. With the development of an mIDH1 ADE-MS based HTS assay and a detailed comparison of this new readout technique to the commonly used fluorescence intensity mIDH1 assay, we demonstrated good correlation of both methods and were able to identify new potent inhibitors of mIDH1.

Minimally Invasive Detection of IDH1 Mutation With Cell-Free Circulating Tumor DNA and D-2-Hydroxyglutarate, D/L-2-Hydroxyglutarate Ratio in Gliomas.

Isocitrate dehydrogenase-1 (IDH1) mutation is accepted as one of the earliest events in tumorigenesis in gliomas. This mutation causes preferential accumulation of D- relative to L-enantiomer of 2-hydroxyglutarate (2-HG). Minimally invasive techniques to detect IDH1 mutation may prove useful for clinical practice. We adopted 2 different diagnostic approaches to detect IDH1 mutation status in glioma patients: Evaluation of D- and L-2-HG levels in cerebrospinal fluid (CSF), urine, and plasma, and identification of IDH1 mutation using cell-free circulating tumor DNA (ctDNA) in CSF and plasma. Forty-nine glioma patients in different stages were included. Levels of D- and L-2-HG were determined using liquid chromatography-tandem mass spectrometry; IDH1 R132H mutation was determined by digital-PCR. D-2-HG levels and D/L-2-HG ratio (rDL) in CSF and rDL in plasma were significantly higher in the mutant group than in the wild-type group (p = 0.029, 0.032, 0.001, respectively). The IDH1 mutation detection rates in CSF- and plasma-ctDNA were 63.2% and 25.0%, respectively. These data indicate that D-2-HG values in CSF and rDL in plasma and CSF can be considered as significant contributors to the identification of IDH1 mutation status. In addition, detection of IDH1 mutation in CSF-ctDNA from glioma patients provides a basis for future use of ctDNA for minimally invasive clinical assessment of gliomas.

Effects of the IDH1 R132H Mutation on the Energy Metabolism: A Comparison between Tissue and Corresponding Primary Glioma Cell Cultures.

The R132H mutation in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) is the most important prognostic factor for the survival of glioma patients. Subsequent studies led to the discovery of a panel of enzymes mainly involved in glutamate anaplerosis and aerobic glycolysis that change in abundance as a result of the IDH1 mutation. To further study these changes, appropriate glioma models are required that accurately mimic in vivo metabolism. To investigate how metabolism is affected by in vitro cell culture, we here compared surgically obtained snap-frozen glioma tissues with their corresponding primary glioma cell culture models with a previously developed targeted mass spectrometry proteomic assay. We determined the relative abundance of a panel of metabolic enzymes. Results confirmed increased glutamate use and decreased aerobic glycolysis in resected IDH1 R132H glioma tissue samples. However, these metabolic profiles were not reflected in the paired glioma primary cell cultures. We suggest that culture conditions and tumor microenvironment play a crucial role in maintaining the in vivo metabolic situation in cell culture models. For this reason, new models that more closely resemble the in vivo microenvironment, such as three-dimensional cell co-cultures or organotypic multicellular spheroid models, need to be developed and investigated.

IDH1/2 Mutations in Patients With Diffuse Gliomas: A Single Centre Retrospective Massively Parallel Sequencing Analysis.

Patients below 55 years were genetically studied because the prevalence of isocitrate dehydrogenase 1 (IDH1) decreases in older patients and on grounds of cost-effectiveness, as suggested by the World Health Organization (WHO) in 2016. The aim of our study was to use novel massively parallel sequencing (MPS) approaches to examine rare variants of IDH1/2 in Czech diffuse astrocytic and oligodendroglial tumors (gliomas) patients below 55 years of age who had been immunohistochemically (IHC) diagnosed as IDH1 R132H negative. The IHC IDH1 status (wild type or mutant) of 275 tissue samples was analyzed using antibodies against the IDH1 R132H protein. Sixty-three samples of 55 years old patients with IHC IDH1 WT status were genotyped using two different MPS technologies to detect rare IDH1 and IDH2 variants. The tiered IHC (60 positive) and molecular (10 positive) approach thus revealed that 70 of the 275 samples (25%) bore IDH1/IDH2 mutations. The combined molecular and IHC approach thus revealed that 70 of the 275 samples (25%) considered in the study bore IDH1/IDH2 mutations. IHC detection of the IDH1 R132H variant should be routinely complemented with MPS to detect rare IDH1/2 variants in glioma patients below 55 years of age with negative IHC result of IDH R132H variant.

YAP1-mediated regulation of mitochondrial dynamics in IDH1 mutant gliomas.

Mutation of the isocitrate dehydrogenase 1 (IDH1) gene leads to the production of oncometabolite D-2-hydroxyglutarate (2-HG) from α-ketoglutarate and is associated with better prognosis in glioma. As Yes-associated protein 1 (YAP1) is an important regulator of tumor progression, its role in glioma expressing IDH1 with an R132H mutation was investigated. Diminished nuclear levels of YAP1 in IDH1 mutant glioma tissues and cell lines were accompanied by decreased levels of mitochondrial transcription factor A (TFAM). Luciferase reporter assays and chromatin immunoprecipitation were used to investigate the functionality of the TEAD2-binding site on the TFAM promoter in mediating its YAP1-dependent expression. YAP1-dependent mitochondrial fragmentation and ROS generation were accompanied by decreased telomerase reverse transcriptase (TERT) levels and increased mitochondrial TERT localization in IDH1 R132H cells. Treatment with the Src kinase inhibitor bosutinib, which prevents extranuclear shuttling of TERT, further elevated ROS in IDH1 R132H cells and triggered apoptosis. Importantly, bosutinib treatment also increased ROS levels and induced apoptosis in IDH1 wild-type cells when YAP1 was concurrently depleted. These findings highlight the involvement of YAP1 in coupling mitochondrial dysfunction with mitochondrial shuttling of TERT to constitute an essential non-canonical function of YAP1 in the regulation of redox homeostasis. This article has an associated First Person interview with the first author of the paper.

TMOD-10. THE ROLE OF IDH1 R132H MUTATION IN GLIOMA – AN INVESTIGATION BY GENOME-EDITING IN HUMAN INDUCED PLURIPOTENT STEM CELLS

Abstract BACKGROUND Hot-spot mutations in the Isocitrate dehydrogenase 1 (IDH1) cause a new catalytic function resulting in the production of 2-HG, a hallmark in the development of low-grade glioma. The tumorigenic mechanism of this mutation as well as the cell of origin are not known and there is a lack of suitable disease models. Thus, we aim to create a model mimicking glioma development by introducing the IDH1 R132H into human induced pluripotent stem cells (hiPSC) and investigate the influence on stem cell properties and cell differentiation in neuronal progenitor cells. MATERIAL AND METHODS We use CRISPR/Cas9 based genome editing to induce the IDH1 R132H mutation into healthy-control-derived hiPSCs. Successful introduction of the mutation was confirmed on DNA, RNA and protein level. The hiPSCs are then differentiated into cerebral organoids and characterized using transcriptome sequencing and methylation arrays. RESULTS We successfully introduced the IDH1 R132H mutation into hiPSCs and confirmed expression of the mutated protein by Western Blot. Metabolite measurement 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 hiPSCs, proving that the mutated enzyme is functional. To investigate effects of IDH1 R132H on cell differentiation, we generated cerebral organoids from our iPSC-models. The IDH1 R132H mutation did not inhibit cell differentiation or maturation of cerebral organoids but led to a downregulation of splicosome, proteasome and DNA repair enzymes as well as an upregulation of ECM components. CONCLUSION AND OUTLOOK hiPSCs with R132H mutation pose a promising model for investigations on early glioma development. We are currently step-wise including TP53 and ATRX loss of function mutations in our hiPSC models to recapitulating tumor development in vivo.

Synthesis of [1-13 C-5-12 C]-alpha-ketoglutarate enables noninvasive detection of 2-hydroxyglutarate.

Isocitrate dehydrogenase 1 (IDH1) mutations that generate the oncometabolite 2-hydroxyglutarate (2-HG) from α-ketoglutarate (α-KG) have been identified in many types of tumors and are an important prognostic factor in gliomas. 2-HG production can be determined by hyperpolarized carbon-13 magnetic resonance spectroscopy (HP-13 C-MRS) using [1-13 C]-α-KG as a probe, but peak contamination from naturally occurring [5-13 C]-α-KG overlaps with the [1-13 C]-2-HG peak. Via a newly developed oxidative-Stetter reaction, [1-13 C-5-12 C]-α-KG was synthesized. α-KG metabolism was measured via HP-13 C-MRS using [1-13 C-5-12 C]-α-KG as a probe. [1-13 C-5-12 C]-α-KG was synthesized in high yields, and successfully eliminated the signal from C5 of α-KG in the HP-13 C-MRS spectra. In HCT116 IDH1 R132H cells, [1-13 C-5-12 C]-α-KG allowed for unimpeded detection of [1-13 C]-2-HG. 12 C-enrichment represents a novel method to circumvent spectral overlap, and [1-13 C-5-12 C]-α-KG shows promise as a probe to study IDH1 mutant tumors and α-KG metabolism.

Efficacy of 5-Azacitidine on IDH1/2 Acute Myeloid Leukemia/Myelodysplastic Syndromes and Correlation with 2-Hydroxyglutarate Production

Background. Isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) mutations are found in 10 and 15% of acute myeloid leukemia (AML) patients, respectively. IDH1/2 mutants' proteins produce high 2-hydroxyglutarate (2HG) levels that inhibit α-ketoglutarate-dependent dioxygenases including histone and DNA demethylases, as TET2 proteins. Furthermore, serum 2HG can act as a biomarker predictive of the presence of IDH1 and IDH2 mutations at diagnosis (cut-off value >2µM) but also correlate with response after intensive chemotherapy. 5-azacitidine (AZA) is a DNA methyltransferase inhibitor that demonstrated significant clinical benefit not only in high-risk MDS but also in AML. Hypomethylating agents yield higher response rate in the context of CpG island hypermethylation phenotype induced by TET2 mutations in myelodysplastic syndrome (MDS). Thus, we evaluate AZA response in the context of IDH1/2 mutations and the effect of AZA on serum 2HG production.Patients and methods. The present study retrospectively focused on IDH1/2 mutant AML/MDS patients treated in our center with at least 1 cycle of AZA. IDH1/2 mutational status was evaluated in all patients with Sanger or Next-Generation sequencing before therapy. All patients received subcutaneous AZA 75mg/m2/d for 7 days every 28 days. Response to therapy was evaluated according to modified 2003 IWG criteria's for AML. Hematological improvement (HI) was evaluated according to IWG 2006 criteria's for MDS. Overall survival (OS) was censored in case of IDH1/2 inhibitor therapy after AZA. Serum samples were analyzed for total 2HG by reverse-phase liquid chromatography coupled to mass spectrometry.Results. Thirty-six patients (34 AML, 2 MDS) were retrospectively analyzed. Initial characteristics of the patients are shown on Table 1. Median age was 74.3 years (range, 48.4-86). Thirteen patients (36%) had IDH1 mutations (R132C: 10, R132H: 2, R132G: 1) and 22 patients (61%) had IDH2 mutations (R140Q: 19, R172K: 3). One patient had both IDH1 R132H and IDH2 R140Q mutations. Twenty-nine patients had intermediate cytogenetic (including normal karyotype in 20 patients) and 6/36 unfavorable. IDH1/2 mutations were associated with SRSF2 mutations in 13 patients, DNMT3A in 11 patients, NPM1 mutation in 9 patients, ASXL1 in 5 patients and FLT3 -ITD in 5 patients. Ten patients (28%) were treated frontline with AZA while the others received AZA as second line (50%, including 6 patients with primary refractory AML to intensive chemotherapy) or subsequent lines (22%). Five patients received IDH1/2 inhibitors (AG221: 3, AG120: 2) prior to AZA treatment. Patients received a median of 4.5 cycles of AZA (range, 1-23). Four patients (11%) achieved complete remission (CR) and 7 (19%) obtained HI with persistent excess of blasts (overall response rate: 30%). Median OS was 9.5 months (FU range, 0.2-41.6) with a trend toward shorter OS in IDH1 mutant AML patient (6.4 vs 10.1; p≤0.0764) (Figure 1). No difference was observed between patients in 1st or subsequent treatment lines (p≤0.7265).Median 2HG concentration (available in 25/36 patients) before the onset of AZA treatment was 9.7µM. Seventeen patients had available serum during follow-up (median: 2 measures/patient). When 2HG values in responding and non-responding patients were compared at different time-points, 2HG values remained elevated in responders (median 2HG: 6.8µM; range 0.99-13.5) without significant difference compared to baseline or relapse values (Figure 2). All responders to AZA ultimately relapsed or progressed during follow-up. IDH1 or IDH2 mutations persisted at relapse along with elevated 2HG levels (median 2HG: 4.5µM).Discussion. In the present series, IDH1/2 mutations were not associated with an increased sensitivity to AZA, as opposed to TET2 mutations. AZA may decrease production of 2HG in responders compared to diagnosis values. However, unlike intensive chemotherapy, AZA is unable to normalize 2HG values in responding patients. This suggests that 2HG cannot be used as a surrogate marker of AZA response and AZA efficacy might not be related to inhibition of 2HG production in IDH1/2 AML/MDS patients. Thus, combinations of AZA with IDH1/2 mutant proteins inhibitors might lead to an additive or synergistic antitumor effect. [Display omitted] DisclosuresRenneville:CELGENE: Research Funding. Ribrag:Roche: Honoraria, Other: travel, accommodation, expenses; BMS: Consultancy, Honoraria; MSD: Consultancy, Honoraria; Infinity: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Nanostring: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria; Pharmamar: Consultancy; Esai: Honoraria, Research Funding; ArgenX: Research Funding; Servier: Consultancy, Honoraria. de Botton:Celgene Corporatation: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Servier: Honoraria; Agios Pharmaceuticals, Inc.: Honoraria, Research Funding.

IDH1 gene mutation activates Smad signaling molecules to regulate the expression levels of cell cycle and biological rhythm genes in human glioma U87‑MG cells.

Isocitrate dehydrogenase1 (IDH1) mutation is the most important genetic change in glioma. The most common IDH1 mutation results in the amino acid substitution of arginine 132 (Arg/R132), which is located at the active site of the enzyme. IDH1 Arg132His (R132H) mutation can reduce the proliferative rate of glioma cells. Numerous diseases follow circadian rhythms, and there is growing evidence that circadian disruption may be a risk factor for cancer in humans. Dysregulation of the circadian clock serves an important role in the development of malignant tumors, including glioma. Brain-Muscle Arnt-Like protein 1 (BMAL1) and Circadian Locomotor Output Cycles Kaput (CLOCK) are the main biological rhythm genes. The present study aimed to further study whether there is an association between IDH1 R132H mutation and biological rhythm in glioma, and whether this affects the occurrence of glioma. The Cancer Genome Atlas (TCGA) database was used to detect the expression levels of the biological rhythm genes BMAL1 and CLOCK in various types of tumor. Additionally, U87-MG cells were infected with wild-type and mutant IDH1 lentiviruses. Colony formation experiments were used to detect cell proliferation in each group, cell cycle distribution was detected by flow cytometry and western blotting was used to detect the expression levels of wild-type and mutant IDH1, cyclins, biological rhythm genes and Smad signaling pathway-associated genes in U87-MG cells. TCGA database results suggested that BMAL1 and CLOCK were abnormally expressed in glioma. Cells were successfully infected with wild-type and mutant IDH1 lentiviruses. Colony formation assay revealed decreased cell proliferation in the IDH1 R132H mutant group. The cell cycle distribution detected by flow cytometry indicated that IDH1 gene mutation increased the G1 phase ratio and decreased the S phase ratio in U87-MG cells. The western blotting results demonstrated that IDH1 R132H mutation decreased the expression levels of the S phase-associated proteins Cyclin A and CDK2, and increased the expression levels of the G1 phase-associated proteins Cyclin D3 and CDK4, but did not significantly change the expression levels of the G2/M phase-associated protein Cyclin B1. The expression levels of the positive and negative rhythm regulation genes BMAL1, CLOCK, period (PER s (PER1, 2 and 3) and cryptochrom (CRY)s (CRY1 and 2) were significantly decreased, those of the Smad signaling pathway-associated genes Smad2, Smad3 and Smad2-3 were decreased, and those of phosphorylated (p)-Smad2, p-Smad3 and Smad4 were increased. Therefore, the present results suggested that the IDH1 R132H mutation may alter the cell cycle and biological rhythm genes in U87-MG cells through the TGF-β/Smad signaling pathway.

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.