Search Results (26)

Isocitrate dehydrogenase (IDH) catalyzes the conversion of NAD(P)⁺ and isocitrate to NAD(P)H and α-ketoglutarate (αKG). The cytoplasmic enzyme IDH1 is important for producing NADPH for biosynthesis and for protecting against oxidative stress. IDH1 mutants, such as R132H found in glioblastomas and other types of human cancers, have a neomorphic activity that uses NADPH to reduce αKG to 2-hydroxyglutarate (2HG). 2HG interferes with the activity of important enzymes such as histone demethylases and TET demethylases. We hypothesized that Caenorhabditis elegans could be a good model system for studying oncogenic properties of mutant IDH1. To test this, we purified C. elegans cytoplasmic IDH-1 and two mutants, G98N and R133H, which correspond to human IDH1 mutants G97N and R132H, respectively. We found that the wild-type IDH-1 had similar kinetic properties to human IDH1, and it could produce small amounts of 2HG. We also found that the R133H mutant had a lower K_M for αKG than human R132H in steady-state enzyme kinetic experiments, and it produced almost exclusively 2HG in the presence of NADPH and αKG. The G98N mutant had a higher k_cat in the forward direction than the comparable human G97N mutant, and the G98N mutant produced a smaller amount of 2HG compared to the R133H mutant. These results suggest that C. elegans strains with IDH-1 mutations could be a good model system for studying the effects of 2HG in eukaryotic organisms. Full article

Background: Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are enzymes that catalyze the oxidative decarboxylation of isocitrate to alpha-ketoglutarate (α-KG), which is essential for many metabolic processes, including some steps in DNA repair. In tumors, notably in gliomas, IDH1 and IDH2 are frequently mutated. The mutation found in different cancers is functionally active, causing, instead of α-KG, the formation of 2-hydroxyglutarate (2-HG), which inhibits α-KG-dependent enzymes. Gliomas harboring mutated IDH1/2 show a better prognosis than IDH1 wild-type (wt) tumors of the same grade, which might result from the inhibition of DNA repair functions. A DNA repair enzyme dependent on α-KG is alkB homolog 2 (ALKBH2), which removes several lesions from DNA. These findings prompted us to investigate the response of glioma cells to artesunate (ART), a plant ingredient with genotoxic and anticancer activity currently used in several trials. Materials and Methods: We used isogenic glioblastoma cell lines that express IDH1 wild-type or, based on a TET-inducible system, the IDH1 mutant (mt) protein, and treated them with increasing doses of artesunate. We also treated glioblastoma cells with 2-HG, generated ALKBH2 knockout cells, and checked their sensitivity to the cytotoxic effects of artesunate. Results: We show that the cell-killing effect of ART is enhanced if the IDH1 mutant (R132H) is expressed in glioblastoma cells. Further, we show that 2-HG imitates the effect of IDH1mt as 2-HG ameliorates the cytotoxicity of ART. Finally, we demonstrate that the knockout of ALKBH2 causes the sensitization of glioblastoma cells to ART. Conclusions: The data indicate that ALKBH2 protects against the anticancer effect of ART, and the mutation of IDH1/2 commonly occurring in low-grade gliomas sensitizes to ART via an ALKBH2-dependent mechanism. The data support the use of ART in the therapy of IDH1/2-mutated cancers both in combination with chemotherapy and adjuvant treatment. Full article

Mutations in IDH1 and TP53 have a significant impact on glioma prognosis and progression; however, their roles in tumor cell invasion in terms of interactions with particular components of the extracellular matrix (ECM) are still unclear. Using gene editing protocol based on CRISPR-Cas 9 with cytidine deaminase, we introduced point mutations into U87MG glioblastoma cells to establish modified cell lines with heterozygous IDH1 R132H, homozygous TP53 R248Q and heterozygous IDH1 R132H, homozygous TP53 R248Q genotypes. A comparative study of cell migration on major ECM components was carried out by high-content microscopy. IDH1 R132H mutation introduced to U87MG glioblastoma cells was shown to decrease the migration speed on Matrigel and collagen IV substrates compared to the wild-type. This data were supported by cell adhesion quantification via the lateral shift assay performed by atomic force microscopy (AFM). TP53 R248Q mutation increased cell adhesion to various substrates and significantly promoted cell migration on hyaluronic acid and chondroitin sulfate but did not change the migration rates on laminin and collagens IV and I. A double-mutant genotype produced by consequently introducing IDH1 R132H and TP53 R248Q to parental glioblastoma cells was characterized by the highest migration among all the cell lines, with particularly faster motility on chondroitin sulfate. These findings underscore the complex interactions between glioma cells, with the most important driver mutations and specific ECM components regulating cancer cell migration, offering valuable insights for potential therapeutic targets in glioma treatment. Full article

Glioblastoma (GBM) is the most common primary brain malignancy in the U.S. with a 5-year overall survival < 5% despite an aggressive standard of care. Laser interstitial thermal therapy (LITT) is a surgical approach to treating GBM that has gained traction, providing a safe option for reducing intracranial tumor burden. LITT is believed to potentially modulate GBM immune responses; however, the biochemical mechanisms underlying the modulation of immune checkpoints in GBM cells have been poorly characterized. The present study aimed to preliminarily evaluate the effects of thermal therapy and radiation on PD-L1 modulation in vitro, as a function of IDH mutational status. U87 cells and their IDH-mutant counterpart (U87^R132H), which was generated using a crispr-cas9 knock-in approach, were utilized for this preliminary evaluation. Cell heating was achieved by harvesting with trypsin centrifugation where the cell pellets were treated on a heat block for the associated time and temperature. Following thermal therapy, cells were resuspended and irradiated using a 37-Cesium irradiator at 0.6 Gy min⁻¹. Immediately following treatment, cells were either plated as single cells to allow colonies to form, and stained with Coomassie blue to be counted approximately 10–14 days later or harvested for Western blot analysis. Cell lysates were analyzed for PD-L1 expression with respect to various iron metabolic parameters (mortalin (HSPA9), transferrin receptor, and ferritin heavy chain) using a Western blotting approach. In both U87 and U87^R132H cell lines, thermal therapy showed a temperature-dependent cell-killing effect, but U87^R132H cells appeared more sensitive to thermal treatment when treated at 43 °C for 10 min. Moreover, thermal therapy had minimal effects on cell responses to 2 Gy irradiation. Treatment with thermal therapy downregulated PD-L1 expression in U87^R132H cells, which was associated with increased expression of the mitochondrial iron metabolic enzyme, HSPA9. Thermal therapy reversed the radiation-induced overexpression of PD-L1, transferrin receptor, and ferritin heavy chain in U87^R132H cells. No effects were observed in wild-type U87 cells. Moreover, Ga(NO₃)₃ depleted mitochondrial iron content which, in turn, significantly enhanced the sensitivity of U87^R132H cells to thermal therapy and 2 Gy irradiation and caused a significant increase in PD-L1 expression. These results suggest that thermal therapy alone can modulate the immune checkpoint PD-L1. This effect was more pronounced when thermal therapy was combined with radiation. Mechanistically, mitochondrial iron trafficking through HSPA9 may coordinate the regulation of PD-L1 in the context of thermal therapy and ionizing radiation, which can be targeted with gallium-based therapy. These novel, preliminary findings warrant further mechanistic investigations in pre-clinical models of LITT. Full article

Ollier disease (OD), acute myeloid leukemia (AML), and brain glioma (BG) are three apparently completely different neoplasms in terms of histopathology, clinic, natural history, and management, but they can affect the same patient. This study aimed to identify the common molecular pathways involved in the pathogenesis of all three diseases and discuss their current and potential role as therapeutic targets. A detailed and comprehensive systematic literature review according to PRISMA guidelines on OD patients harboring BG and/or AML was made. In addition, the unique case of a patient affected by all three considered diseases has been added to our case series. Demographic, pathological, treatment, and outcome data were analyzed and discussed, mainly focusing on the molecular findings. Twenty-eight studies reported thirty-three patients affected by OD and BG, and only one study reported one patient with OD and AML, while only our patient harbored all three pathologies. The IDH R132H mutation was the only genetic alteration shared by all three pathologies and was simultaneously detected in enchondromas and brain glioma in 100% (3/3) of OD patients with BG and also in the neoplastic blood cells of the single patient hosting all three diseases. The IDH1-R132H gene mutation is the etiopathogenetic common denominator among three apparently different tumors coexisting in the same patient. The adoption of mutant-specific IDH1 inhibitor molecules could represent a potential panacea for these conditions in the era of targeted therapies. Further studies with larger clinical series are needed to confirm our results and hypothesis. Full article

How hematopoietic stem and progenitor cell (HSPC) fate decisions are affected by genetic alterations acquired during AML leukemogenesis is poorly understood and mainly explored in animal models. Here, we study isocitrate dehydrogenase (IDH) gene mutations in the human model of HSPC and discuss the available literature on this topic. IDH1/2 mutations occur in ~20% of AML cases, are recognized among the mutations earliest acquired during leukemogenesis, and are targets of specific inhibitors (ivosidenib and enasidenib, respectively). In order to investigate the direct effects of these mutations on HSPCs, we expressed IDH1-R132H or IDH2-R140Q mutants into human CD34+ healthy donor cells via lentiviral transduction and analyzed the colony-forming unit (CFU) ability. CFU ability was dramatically compromised with a complete trilineage block of differentiation. Strikingly, the block was reversed by specific inhibitors, confirming that it was a specific effect induced by the mutants. In line with this observation, the CD34+ leukemic precursors isolated from a patient with IDH2-mutated AML at baseline and during enasidenib treatment showed progressive and marked improvements in their fitness over time, in terms of CFU ability and propensity to differentiate. They attained clonal trilinear reconstitution of hematopoiesis and complete hematological remission. Full article

Isocitrate Dehydrogenase-1 (IDH1) is commonly mutated in lower-grade diffuse gliomas. The IDH1R132H mutation is an important diagnostic tool for tumor diagnosis and prognosis; however, its role in glioma development, and its impact on response to therapy, is not fully understood. We developed a murine model of proneural IDH1R132H-mutated glioma that shows elevated production of 2-hydroxyglutarate (2-HG) and increased trimethylation of lysine residue K27 on histone H3 (H3K27me3) compared to IDH1 wild-type tumors. We found that using Tazemetostat to inhibit the methyltransferase for H3K27, Enhancer of Zeste 2 (EZH2), reduced H3K27me3 levels and increased acetylation on H3K27. We also found that, although the histone deacetylase inhibitor (HDACi) Panobinostat was less cytotoxic in IDH1R132H-mutated cells (either isolated from murine glioma or oligodendrocyte progenitor cells infected in vitro with a retrovirus expressing IDH1R132H) compared to IDH1-wild-type cells, combination treatment with Tazemetostat is synergistic in both mutant and wild-type models. These findings indicate a novel therapeutic strategy for IDH1-mutated gliomas that targets the specific epigenetic alteration in these tumors. Full article

Intracranial chondroid tumors are a heterogeneous group of neoplasms characterized by the presence of a cartilage matrix. These tumors exhibit overlapping clinical and histological features. Mutations in IDH1/2 genes serve as important diagnostic markers of tumor type, particularly chondrosarcoma. To improve the accuracy of IDH1/2 diagnostics, we compared three methods: biochip assay, real-time PCR with DNA melting analysis using TaqMan probes and sequencing (qPCR-DMA-Sanger), and immunohistochemistry (IHC). Tumor samples from 96 patients were investigated. The IDH1 mutations were detected in 34/64 (53%) chondrosarcomas; IHC detected 27/56 (48.2%) mutations, the qPCR-DMA-Sanger method 27/59 (46%) mutations, and the biochip assay revealed 29/60 (48.3%) mutations. The detection of IDH1 mutations in chordoma (2/15) and osteosarcoma (2/7) suggested the need for a revised diagnosis. In benign tumors, IDH1 mutations were present in chondroma (4/6), but absent in chondromyxoid fibroma (0/4). The most frequent IDH1 mutations were R132C (60%), R132L, and R132G (13.5% each), R132H (8%), and R132S (5%). The concordance between the biochip assay and IHC was 90%, between IHC and PCR-DMA-Sanger 83%, and between biochip assay and qPCR-DMA-Sanger was 98%, respectively. No IDH2 mutations were found. The use of independent diagnostic methods may improve the detection of IDH-mutant specimens in chondroid tumors. Full article

While the central nervous system (CNS) tumor classification has increasingly incorporated molecular parameters, there is a paucity of literature reporting molecular alterations found in intraventricular glioblastoma (IVGBM), which are rare. We present a case series of nine IVGBMs, including molecular alterations found in standardized next-generation sequencing (NGS). We queried the clinical charts, operative notes, pathology reports, and radiographic images of nine patients with histologically confirmed IVGBM treated at our institution (1995–2021). Routine NGS was performed on resected tumor tissue of two patients. In this retrospective case series of nine patients (22% female, median (range) age: 64.3 (36–85) years), the most common tumor locations were the atrium of the right lateral ventricle (33%) and the septum pellucidum (33%). Five patients had preoperative hydrocephalus, which was managed with intraoperative external ventricular drains in three patients and ventriculoperitoneal shunts in one patient. Hydrocephalus was managed with subtotal resection of a fourth ventricular IVGBM in one patient. The most common surgical approach was transcortical intraventricular (56%). Gross total resection was achieved in two patients, subtotal resection was achieved in six patients, and one patient received a biopsy only. Immunohistochemistry for IDH1 R132H mutant protein was performed in four cases and was negative in all four. Genetic alterations common in glioblastoma, IDH-wildtype, were seen in two cases with available NGS data, including EGFR gene amplification, TERT promoter mutation, PTEN mutation, trisomy of chromosome 7, and monosomy of chromosome 10. Following surgical resection, four patients received adjuvant chemoradiation. Median survival among our cohort was 4.7 months (IQR: 0.9–5.8 months). Management of IVGBM is particularly challenging due to their anatomical location, presentation with obstructive hydrocephalus, and fast growth, necessitating prompt intervention. Additional studies are needed to better understand the genetic landscape of IVGBM compared to parenchymal glioblastoma and may further elucidate the unique pathophysiology of these rare tumors. Full article

Histological identification of dispersed glioma cells in small biopsies can be challenging, especially in tumours lacking the IDH1 R132H mutation or alterations in TP53. We postulated that immunohistochemical detection of proteins expressed preferentially in gliomas (EGFR, MEOX2, CD34) or during embryonal development (SOX11, INSM1) can be used to distinguish reactive gliosis from glioma. Tissue microarrays of 46 reactive glioses, 81 glioblastomas, 34 IDH1-mutant diffuse gliomas, and 23 gliomas of other types were analysed. Glial neoplasms were significantly more often (p < 0.001, χ²) positive for EGFR (34.1% vs. 0%), MEOX2 (49.3% vs. 2.3%), SOX11 (70.5% vs. 20.4%), and INSM1 (65.4% vs. 2.3%). In 94.3% (66/70) of the glioblastomas, the expression of at least two markers was observed, while no reactive gliosis showed coexpression of any of the proteins. Compared to IDH1-mutant tumours, glioblastomas showed significantly higher expression of EGFR, MEOX2, and CD34 and significantly lower positivity for SOX11. Non-diffuse gliomas were only rarely positive for any of the five markers tested. Our results indicate that immunohistochemical detection of EGFR, MEOX2, SOX11, and INSM1 can be useful for detection of glioblastoma cells in limited histological samples, especially when used in combination. Full article

Mutations in homodimeric isocitrate dehydrogenase (IDH) enzymes at specific arginine residues result in the abnormal activity to overproduce D-2 hydroxyglutarate (D-2HG), which is often projected as solid oncometabolite in cancers and other disorders. As a result, depicting the potential inhibitor for D-2HG formation in mutant IDH enzymes is a challenging task in cancer research. The mutation in the cytosolic IDH1 enzyme at R132H, especially, may be associated with higher frequency of all types of cancers. So, the present work specifically focuses on the design and screening of allosteric site binders to the cytosolic mutant IDH1 enzyme. The 62 reported drug molecules were screened along with biological activity to identify the small molecular inhibitors using computer-aided drug design strategies. The designed molecules proposed in this work show better binding affinity, biological activity, bioavailability, and potency toward the inhibition of D-2HG formation compare to the reported drugs in the in silico approach. Full article

Atomic force microscopy (AFM) recently burst into biomedicine, providing morphological and functional characteristics of cancer cells and their microenvironment responsible for tumor invasion and progression, although the novelty of this assay needs to coordinate the malignant profiles of patients’ specimens to diagnostically valuable criteria. Applying high-resolution semi-contact AFM mapping on an extended number of cells, we analyzed the nanomechanical properties of glioma early-passage cell cultures with a different IDH1 R132H mutation status. Each cell culture was additionally clustered on CD44+/− cells to find possible nanomechanical signatures that differentiate cell phenotypes varying in proliferative activity and the characteristic surface marker. IDH1 R132H mutant cells compared to IDH1 wild-type ones (IDH1wt) characterized by two-fold increased stiffness and 1.5-fold elasticity modulus. CD44+/IDH1wt cells were two-fold more rigid and much stiffer than CD44-/IDH1wt ones. In contrast to IDH1 wild-type cells, CD44+/IDH1 R132H and CD44-/IDH1 R132H did not exhibit nanomechanical signatures providing statistically valuable differentiation of these subpopulations. The median stiffness depends on glioma cell types and decreases according to the following manner: IDH1 R132H mt (4.7 mN/m), CD44+/IDH1wt (3.7 mN/m), CD44-/IDH1wt (2.5 mN/m). This indicates that the quantitative nanomechanical mapping would be a promising assay for the quick cell population analysis suitable for detailed diagnostics and personalized treatment of glioma forms. Full article

The current diagnosis of diffuse glioma involves isocitrate dehydrogenase (IDH) mutation testing. Most IDH mutant gliomas carry a G-to-A mutation at IDH1 position 395, resulting in the R132H mutant. R132H immunohistochemistry (IHC), therefore, is used to screen for the IDH1 mutation. In this study, the performance of MRQ-67, a recently generated IDH1 R132H antibody, was characterized in comparison with H09, a frequently used clone. Selective binding was demonstrated by an enzyme-linked immunosorbent assay for MRQ-67 to the R132H mutant, with an affinity higher than that for H09. By Western and dot immunoassays, MRQ-67 was found to bind specifically to the IDH1 R1322H, with a higher capacity than H09. IHC testing with MRQ-67 demonstrated a positive signal in most diffuse astrocytomas (16/22), oligodendrogliomas (9/15), and secondary glioblastomas tested (3/3), but not in primary glioblastomas (0/24). While both clones demonstrated a positive signal with similar patterns and equivalent intensities, H09 exhibited a background stain more frequently. DNA sequencing on 18 samples showed the R132H mutation in all IHC positive cases (5/5), but not in negative cases (0/13). These results demonstrate that MRQ-67 is a high-affinity antibody suitable for specific detection of the IDH1 R132H mutant by IHC and with less background as compared with H09. Full article

The IDH1^R132H mutation in glioma results in the neoenzymatic function of IDH1, leading to the production of the oncometabolite 2-hydroxyglutarate (2-HG), alterations in energy metabolism and changes in the cellular redox household. Although shifts in the redox ratio NADPH/NADP⁺ were described, the consequences for the NAD⁺ synthesis pathways and potential therapeutic interventions were largely unexplored. Here, we describe the effects of heterozygous IDH1^R132H on the redox system in a CRISPR/Cas edited glioblastoma model and compare them with IDH1 wild-type (IDH1^wt) cells. Besides an increase in 2-HG and decrease in NADPH, we observed an increase in NAD⁺ in IDH1^R132H glioblastoma cells. RT-qPCR analysis revealed the upregulation of the expression of the NAD⁺ synthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). Knockdown of NAMPT resulted in significantly reduced viability in IDH1^R132H glioblastoma cells. Given this dependence of IDH1^R132H cells on NAMPT expression, we explored the effects of the NAMPT inhibitors FK866, GMX1778 and GNE-617. Surprisingly, these agents were equally cytotoxic to IDH1^R132H and IDH1^wt cells. Altogether, our results indicate that targeting the NAD⁺ synthesis pathway is a promising therapeutic strategy in IDH mutant gliomas; however, the agent should be carefully considered since three small-molecule inhibitors of NAMPT tested in this study were not suitable for this purpose. Full article

Characterization of hypoxia and tissue acidosis could advance the understanding of glioma biology and improve patient management. In this study, we evaluated the ability of a pH- and oxygen-sensitive magnetic resonance imaging (MRI) technique to differentiate glioma genotypes, including isocitrate dehydrogenase (IDH) mutation, 1p/19q co-deletion, and epidermal growth factor receptor (EGFR) amplification, and investigated its prognostic value. A total of 159 adult glioma patients were scanned with pH- and oxygen-sensitive MRI at 3T. We quantified the pH-sensitive measure of magnetization transfer ratio asymmetry (MTR_asym) and oxygen-sensitive measure of R₂’ within the tumor region-of-interest. IDH mutant gliomas showed significantly lower MTR_asym × R₂’ (p < 0.001), which differentiated IDH mutation status with sensitivity and specificity of 90.0% and 71.9%. Within IDH mutants, 1p/19q codeletion was associated with lower tumor acidity (p < 0.0001, sensitivity 76.9%, specificity 91.3%), while IDH wild-type, EGFR-amplified gliomas were more hypoxic (R₂’ p = 0.024, sensitivity 66.7%, specificity 76.9%). Both R₂’ and MTR_asym × R₂’ were significantly associated with patient overall survival (R₂’: p = 0.045; MTR_asym × R₂’: p = 0.002) and progression-free survival (R₂’: p = 0.010; MTR_asym × R₂’: p < 0.0001), independent of patient age, treatment status, and IDH status. The pH- and oxygen-sensitive MRI is a clinically feasible and potentially valuable imaging technique for distinguishing glioma subtypes and providing additional prognostic value to clinical practice. Full article