Encoding Isocitrate Dehydrogenase Research Articles

Acute and prolonged effects of Bacillus amyloliquefaciens GF424-derived SOD on antioxidant defense in healthy individuals challenged with intense aerobic exercise

Reactive oxygen species (ROS) play a vital role in cellular functions but can lead to oxidative stress and contribute to degenerative diseases when produced in excess. Maintaining redox balance is essential and can be achieved through innate defense mechanisms or external antioxidants. Superoxide dismutase (SOD) is a key enzyme that mitigates intracellular oxidative stress by converting harmful free radicals into hydrogen peroxide, which is subsequently neutralized by catalase and glutathione peroxidase. Previous studies have demonstrated the antioxidant capabilities of SOD derived from Bacillus amyloquefaciens GF424 (BA-SOD) in murine models exposed to either irradiation or SOD1 gene deletion. In this study, a randomized clinical trial was conducted to evaluate the antioxidative benefits of BA-SOD in healthy individuals undergoing acute aerobic exercise (AAE). Eighty participants were randomly assigned to receive either BA-SOD or a placebo for 8 weeks. Antioxidant enzyme activities and glutathione levels were measured before, immediately after, and 30 min post-exercise. A single dose of BA-SOD significantly reduced ROS levels induced by AAE, primarily by enhancing SOD activity in erythrocytes and activating glutathione peroxidase. Continuous BA-SOD administration was associated with a sustained increase in catalase activity and elevated levels of reduced glutathione (GSH). Transcriptomic and metabolomic analyses revealed that a single BA-SOD dose facilitated GSH oxidation, as evidenced by decreased levels of serine, glutamine, and glycine, and increased pyroglutamate levels. Additionally, repeated dosing led to increased expression of genes encoding isocitrate dehydrogenase and malic enzyme, which are involved in NADPH synthesis, as well as nicotinamide phosphoribosyl transferase and NAD kinase, which are essential for NADP availability–critical for converting oxidized glutathione (GSSG) back to GSH. These molecular insights align with clinical observations, suggesting that both acute and long-term BA-SOD supplementation may effectively enhance antioxidant defenses and maintain redox balance under oxidative stress conditions.

Abstract 556: The Idylla™ IDH1-2 Mutation Assay Kit: A tool for mutation detection in IDH1 and IDH2 genes

Abstract Background: Mutations in the genes encoding isocitrate dehydrogenase (IDH1 and IDH2) are frequently detected in various cancers, including acute myeloid leukemia (~20%) and glioma (up to ~80%). They commonly involve a single amino acid substitution at codon R132 in IDH1, and R140 or R172 in IDH2 and are important for diagnosis and prognosis as well as therapeutic decisions. With advancements in molecular diagnostics and precision therapy, IDH inhibitors such as ivosidenib, vorasidenib and enasidenib are now mainstay in management of patients with a susceptible mutation. Methods: The Idylla™ IDH1-2 Mutation Assay Kit (RUO) is a fully automated qPCR assay for the qualitative detection of 15 common mutations in IDH1 (R132C/H/G/S/L) and IDH2 (R140Q/L/G/W, R172K/M/G/W/S) at codon level. The assay consists of a ready-for-use Idylla™ DNA Cartridge and an assay specific Idylla™ Vial. It runs on the Idylla™ Platform and covers the entire process from sample to result including specimen processing, real-time PCR amplification, detection, data analysis, and result reporting, within 100 minutes.Five clinical sites in US and Europe participated in an early access program to evaluate performance of the assay and establish a final classification algorithm by testing a total of 241 specimens, including FFPE, human whole blood and bone marrow samples, and extracted DNA from these sample types. Reference results were obtained using a range of established methods with different mutations in scope, such as Sanger sequencing, immunohistochemistry, NGS and qPCR. Results: Of 241 Cartridges, one invalid result was obtained, while 13 results were flagged as having low DNA input. Compared to available reference methods, overall concordance was 98.3% (235/239), 100% (140/140) and 99.6% (230/231) for IDH1 R132, IDH2 R140 and R172, respectively (see table). Conclusion: The Idylla™ IDH1-2 Mutation Assay Kit demonstrates a high concordance with established reference methods across a range of different specimen types. Reference method(s) PPA (%) NPA (%) OPA (%) MUT WT Total Idylla™ IDH1-2 Mutation Assay Kit IDH1_R132x 111 3 114 99.1 97.6 98.3 WT 1 124 125 Total 112 127 239 IDH2_R140x 10 0 10 100.0 100.0 100.0 WT 0 130 130 Total 10 130 140 IDH2_R172x 25 0 25 96.2 100.0 99.6 WT 1 205 206 Total 26 205 231 Total 148 462 610 Citation Format: Bram De Craene, Katrien Trappeniers, Ana Marcelino, Lee Robertson, Phillippe Taniere, Jo Van Dorpe, Malaïka Van der Linden, Maria Arcila, Khedoudja Nafa, Jochen K. Lennerz, Richard Craig Carson, Richard D. Ledding, Sofie Metsu. The Idylla™ IDH1-2 Mutation Assay Kit: A tool for mutation detection in IDH1 and IDH2 genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 556.

Real-World Treatment Patterns and Clinical Outcomes in Newly Diagnosed Acute Myeloid Leukemia with and without mIDH1 Treated with Intensive Chemotherapy from an International Real-World Database (REAL-IDH)

Introduction Intensive chemotherapy (IC) remains the main backbone therapy in fit, newly diagnosed, acute myeloid leukemia (AML) patients. Clinical trials assessing the efficacy of IC under-represent select subgroups of patients, such as those with certain comorbidities, unique characteristics, or rare genomic abnormalities. Real-world data provides additional insights into treatment patterns and clinical outcomes in patients with AML receiving first-line IC. REAL-IDH is an international network that generates evidence to improve real-world understanding of AML, including patients with a mutation in the gene encoding isocitrate dehydrogenase 1 (m IDH1). This abstract presents data from the REAL-IDH study, specifically on treatment patterns and outcomes in newly diagnosed AML patients who were treated with IC, including those with or without m IDH1 disease. Methods This retrospective, longitudinal, observational study was conducted using databases from the USA (Cancer Outcomes Tracking and Analysis [COTA]), UK (Cancer Analysis System [CAS]), and France (DATAML registry). Data were collected on adult patients with newly diagnosed AML between 2013-2020 (USA and UK) and 2017-2021 (France).Similar inclusion/exclusion criteria were applied across countries. Here we report treatment patterns, efficacy and safety outcomes, including complete (CR) and partial response (PR), CR with incomplete count recovery (CRi), minimal residual disease (MRD), morphological leukemia-free state (MLFS), time to treatment response (TTR), objective response rate (ORR; defined as rate of CR, CRi, PR and MLFS in the total treated population), mortality rate, and overall survival (OS) in patients receiving first-line IC. Results 13,013 patients with AML were included in the REAL-IDH study, with 7406 eligible patients receiving IC as first-line treatment (USA: n=1145; France: n=800; UK: n=5461). In the IC population, of those tested for the IDH mutation, m IDH1 was detected in 70 (6.1%), 84 (10.5%), and 15 (9.3%) patients in the USA, France, and UK, respectively. The most frequent first-line IC, in all three databases, was cytarabine + anthracycline (7+3 regimen) in the total populations and in the m IDH1 and wild-type subgroups. In France, lomustine was added to 7+3 in patients >60 years. The most frequent second-line therapy in the whole IC population was salvage chemotherapy with high-dose or intermediate-dose cytarabine or a combination of either of these with anthracycline (daunorubicin, idarubicin, or amsacrine) in France and decitabine or the combination of cytarabine, etoposide and mitoxantrone in the USA. Azacitidine/venetoclax combination was used in second and third line in France and USA and it was the most frequent third-line therapy (35.8%) in France. Patients received IC in combination with targeted therapies (mainly midostaurin) in 18% of the IC population in France and 12.4% of the IC population in the USA. Rates of CR, MLFS, TTR and ORR were similar in the USA and France ( Table 1). Mortality rates were similar across all three countries ( Table 1). Adverse events (AE) were reported in 43.7% of patients (500; N=1145) in the USA and 76.3% (610; N=800) in France with a reported infection in 13% (149; N=1145; CTCAE coded infection AE) and 43.1% (345; N=800; fungal/bacterial at induction or during treatment; septic shock/pneumopathy at induction or during first 3 months of treatment leading to admission to an intensive care unit; or infection leading to death) of patients in the USA and France, respectively. Conclusion This large-scale population-based study provides insight into treatment patterns and clinical outcomes in patients with AML who received first-line IC in the real-world setting, including patients with or without m IDH1 disease. Intensive treatment combinations have not significantly changed over the past decade with the exception of the addition of targeted therapies. Despite the lack of a common standard IC regimen and protocol across countries, and different healthcare systems, mortality rates were similar with IC across each country. In the REAL-IDH study we observed that treatment patterns in m IDH1 or wild type patients were similar. More specific data is needed regarding the sequencing of treatment regimens and overall outcomes in m IDH1 AML patients.

Flavopiridol Suppresses Cell Proliferation and Migration and Induces Apoptotic Cell Death by Inhibiting Oncogenic FOXM1 Signaling in IDH Wild-Type and IDH-Mutant GBM Cells.

Glioblastoma multiforme (GBM) remains one of the most challenging solid cancers to treat due to its highly aggressive and drug-resistant nature. Flavopiridol is synthetic flavone that was recently approved by the FDA for the treatment of acute myeloid leukemia. Flavopiridol exhibits antiproliferative activity in several solid cancer cells and currently evaluated in clinical trials in several solid and hematological cancers. In this study, we investigated the molecular mechanisms underlying antiproliferative effects of flavopiridol in GBM cell lines with wild-type and mutant encoding isocitrate dehydrogenase 1 (IDH1). We found that flavopiridol inhibits proliferation, colony formation, and migration and induces apoptosis in IDH1 wild-type and IDH-mutant cells through inhibition of FOXM1 oncogenic signaling. Furthermore, flavopiridol treatment also inhibits of NF-KB, mediators unfolded protein response (UPR), including, GRP78, PERK and IRE1α, and DNA repair enzyme PARP, which have been shown to be potential therapeutic targets by downregulating FOXM1 in GBM cells. Our findings suggest for the first time that flavopiridol suppresses proliferation, survival, and migration and induces apoptosis in IDH1 wild-type and IDH1-mutant GBM cells by targeting FOXM1 oncogenic signaling which also regulates NF-KB, PARP, and UPR response in GBM cells. Flavopiridol may be a potential novel therapeutic strategy in the treatment of patients IDH1 wild-type and IDH1-mutant GBM.

Improved Glutamic Acid Production Capacity of Corynebacterium glutamicum by the ARTP Mutagenesis Method

Glutamic acid is an important amino acid that is used widely in the fields of food, medicine, and agriculture. One of the methods of glutamic acid production is direct microbial fermentation, so the genetic stability and glutamic-acid-producing capacity of the producing strain are the keys to improving glutamic acid concentration. Experiments were carried out using Corynebacterium glutamicum GL−6 as the parental strain, with two iterations of mutagenesis by atmospheric and room temperature plasma (ARTP) and screening with agar plates tolerant to high sugar and malonic acid, and the best strains with stable phenotypes were verified by fermentation in 20 L tanks. The results show that the optimal mutagenesis time of ARTP was 140 s, with lethality and positive mutation rates of 93.0% and 15.6%, respectively. The concentrations of the high-sugar and malonic acid agar plates were 240 g/L and 35 g/L, respectively. A mutant strain, P−45, with improved glutamic acid production capacity and genetic stability, was obtained through two rounds of iterative mutagenesis screening. The concentration of this strain in the Erlenmeyer flasks was 17.7 g/L, which was 18.8% higher than that of the parental strain, GL−6, and could be inherited stably for 10 generations. In the glutamic acid synthesis pathway, the upregulation of the gene encoding citrate synthase (cs), gene encoding isocitrate dehydrogenase (icdh), and gene encoding glutamate dehydrogenase (gdh), and the downregulation of the gene encoding oxoglutarate dehydrogenase complex (odhc) increased the carbon flows of the TCA cycle and its branch metabolic flow to glutamic acid synthesis. P−45 showed a glutamic acid concentration of 147.0 g/L under fed-batch fermentation conditions in 20 L tanks, which was 81.5% higher than the starting strain, GL−6. This study provides a new technical solution for improving microbial metabolites and genetic stability.

Targeting Cell Adhesion Molecules in IDH Mutant Myeloid Malignances

Neomorphic mutations in the genes encoding isocitrate dehydrogenase (IDH) are common in human cancers including myeloid malignancies. The oncometabolite (R)-2-hydroxyglutarate (R-2-HG) produced by mutant IDH promotes leukemia development by competitively inhibiting α-ketoglutarate (α-KG)-dependent dioxygenases, causing epigenetic dysregulation of histone and DNA methylation in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). FDA-approved allosteric inhibitors (ivosidenib and enasidenib targeting mutant IDH1 and IDH2 respectively) are used for treating newly diagnosed and/or relapsed/refractory AML patients with IDH mutations. However, the low response rate and primary resistance to IDHmut inhibitors are major clinical challenges. Moreover, acquired resistance to IDH inhibition can occur through multiple mechanisms, including R-2-HG-restoring second-site IDH mutations and the selection of sub-clonal mutations in RUNX1/CEBPA or RAS-RTK pathway. These findings underscore the importance of developing new and more effective approaches for the treatment of IDH-mutant MDS and AML patients. To identify mutant IDH-mediated alterations and actionable dependencies, we generated isogenic TF-1 myeloid leukemia cell lines containing endogenous, hotspot IDH mutations (IDH1R132H, IDH2R140Q, IDH2R140W, and IDH2R172K) by CRISPR/Cas9-mediated base-editing. By comparative transcriptomic (RNA-seq) and epigenomic (ATAC-seq and ChIP-seq) profiling of base-edited versus parental isogenic leukemia cells, we identified cell adhesion molecules, especially CD44, as the top upregulated genes in IDH-mutant leukemia cells. CD44 expression is significantly upregulated in IDH-mutant AML patient samples relative to normal CD34+ hematopoietic stem/progenitor cells (HSPCs) or other AML samples without IDH mutations. Increased CD44 expression is also associated with overall poorer survival and therapy resistance in independent AML cohorts. Importantly, we found that ectopic CD44 expression promotes cytokine-independent cell growth of TF-1 myeloid leukemia cells, resembling the effect observed upon expression of mutant IDH. Moreover, CD44 depletion led to decreased R-2-HG levels in base-edited IDH-mutant TF-1 leukemia cells, and impaired the propagation of IDH-mutant leukemia cells in vitro and in xenotransplanted recipient mice. These studies establish direct evidence that IDH mutations cause aberrant activation of cell adhesion molecules such as CD44, and CD44 is required for the propagation of IDH-mutant myeloid leukemia cells. Mechanistically, we found that the aberrantly activated CD44 is first cleaved by matrix metalloproteinases MMP2 and MMP9 for proteolytic release of its ectodomain from membrane, followed by γ-secretase-mediated intramembranous cleavage to produce the CD44 intracellular domain (CD44ICD). CD44ICD subsequently activates the pentose phosphate pathway (PPP) for the production of NADPH, a critical cofactor for mutant IDH enzymes to convert α-KG to R-2-HG. Hence, mutant IDH-mediated activation of CD44 functions to rewire intracellular metabolism to facilitate IDHmut-catalyzed R-2-HG production, supporting a model for mutant IDH-induced feed-forward oncogenic programs. CD44 also activates AKT/ERK signaling pathway to promote the proliferation and cytokine-independent growth of IDH-mutant leukemia cells. More importantly, primary and resistant IDH-mutant leukemia cells are sensitive to combination regimens consisting of IDHmut inhibitors and humanized monoclonal antibody specific for CD44 (RG7536) or MMP2/9 inhibitors (SB-3CT) invitro and invivo. Taken together, our findings uncover the cell adhesion molecule-mediated metabolic signaling as a new targetable dependency of IDH-mutant leukemia cells, with therapeutic implications for more effective treatment of primary and resistant IDH-mutant myeloid malignancies.

Isocitrate dehydrogenase of Bacillus cereus is involved in biofilm formation.

Isocitrate dehydrogenase (IDH), a key enzyme in the TCA cycle, participates in the formation of biofilms in Staphylococcus aureus, but it remains to be clarified whether it is involved in the formation of Bacillus cereus biofilms. In this study, we scanned the genome of B. cereus 0-9 and found a gene encoding isocitrate dehydrogenase (FRY47_22620) named icdH. The IcdH protein was expressed and purified. The enzyme activity assay showed that the protein had IDH activity dependent on NADP+, indicating that this gene encoded an IDH. The ΔicdH mutant and its complemented strains were obtained by a homologous recombination strategy, and crystal violet data and CLSM were measured. The results showed that the biofilm yield of the mutant ΔicdH decreased, and the biofilm morphology also changed, while the growth of ΔicdH was not affected. The extracellular pH and citric acid content results showed that the ΔicdH mutant exhibited citric acid accumulation and acidification of the extracellular matrix. In addition, the addition of excess Fe3+ restored the biofilm formation of the ΔicdH mutant. It is speculated that IDH in B. cereus may regulate biofilm formation by modulating intracellular redox homeostasis. In addition, we found that the icdH deletion of B. cereus 0-9 could result in a reduced sporulation rate, which was significantly different from sporulation in B. subtilis caused by interruption of the stage I sporulation process due to icdH loss. All the above results provide us with new insights for further research on IDH.

IDH Mutations in Glioma: Double-Edged Sword in Clinical Applications?

Discovery of point mutations in the genes encoding isocitrate dehydrogenases (IDH) in gliomas about a decade ago has challenged our view of the role of metabolism in tumor progression and provided a new stratification strategy for malignant gliomas. IDH enzymes catalyze the conversion of isocitrate to alpha-ketoglutarate (α-KG), an intermediate in the citric acid cycle. Specific mutations in the genes encoding IDHs cause neomorphic enzymatic activity that produces D-2-hydroxyglutarate (2-HG) and result in the inhibition of α-KG-dependent enzymes such as histone and DNA demethylases. Thus, chromatin structure and gene expression profiles in IDH-mutant gliomas appear to be different from those in IDH-wildtype gliomas. IDH mutations are highly common in lower grade gliomas (LGG) and secondary glioblastomas, and they are among the earliest genetic events driving tumorigenesis. Therefore, inhibition of mutant IDH enzymes in LGGs is widely accepted as an attractive therapeutic strategy. On the other hand, the metabolic consequences derived from IDH mutations lead to selective vulnerabilities within tumor cells, making them more sensitive to several therapeutic interventions. Therefore, instead of shutting down mutant IDH enzymes, exploiting the selective vulnerabilities caused by them might be another attractive and promising strategy. Here, we review therapeutic options and summarize current preclinical and clinical studies on IDH-mutant gliomas.

Maffucci Syndrome, Calcium Homeostasis, and Endocrine Challenges in Management

Maffucci syndrome is a rare disorder characterized by enchondromatosis and hemangiomata. It can occur due to sporadic, de novo, mosaic pathogenic variants in the gene encoding isocitrate dehydrogenase 1 (IDH1) or isocitrate dehydrogenase 2 (IDH2). IDH1 variants are associated with endocrine manifestations, such as pituitary adenomas. However, literature is limited in describing other clinical features and available treatments in severe phenotypes. We report a pediatric patient with uniquely complex and severe Maffucci syndrome. Case: A 5-year-old boy was evaluated by pediatric endocrinology for chronic hypercalcemia as part of a multidisciplinary evaluation of his severe Maffucci syndrome. Past medical history included prematurity, restrictive lung disease, developmental delay, seizures, 2-OH glutaric aciduria, angiomas, and bicytopenia. Physical exam revealed angiomas, scoliosis, and severe bony deformities throughout the entire skeleton. During admission, laboratory assays revealed normal parathyroid hormone, phosphorus, 1,25-OH2D, and C-telopeptide; elevated serum calcium and PTH-related peptide; and low 25-OHD, alkaline phosphatase, and osteocalcin. Low-dose ACTH stimulation test yielded a peak cortisol level of 16.8. A 24-hour urine study confirmed hypercalciuria. Renal ultrasound demonstrated nephrocalcinosis. Skeletal survey revealed diffuse and chondromatous changes of nearly every bone. Whole-exome sequencing detected a presumed, mosaic de novo IDH1 variant. DEXA scan revealed total body BMD z-score of -3.8. Discussion: Hypercalcemia in Maffucci syndrome is a rare phenomenon. The most likely etiology was due to the severe and chronic bony breakdown from the underlying progressive enchondromatosis. Subsequently, the body attempted to adapt to these chronic processes with abnormal mineral homeostasis, as seen in his laboratory assays. Chronic primary hyperparathyroidism was not likely, as his PTH, phosphorus, and 1,25 OH2D levels were not congruent with that diagnosis. Familial hypocalciuric hypercalcemia was not likely, as his urine calcium clearance ratio was >0.01. Finally, his slightly elevated PTHrP level was not due to PTHrP-mediated hypercalcemia of malignancy, as his bone marrow biopsy was negative. The options for short-term hypercalcemia management had their own inherent risks and were not suitable for long-term management. Although there is a lack of pediatric data to guide therapy in Maffucci syndrome, decision was made to proceed with bisphosphonate infusion given the benefits in the setting of his nephrocalcinosis, chronic hypercalcemia, and results of his DEXA scan. Given the rarity of Maffucci syndrome, few characteristics are well-described in the pediatric population. A multidisciplinary approach is necessary to review the severity of the disease and to determine the best treatment approach based on this information.

Relationship of Molecular Genetic Tumor Marker IDH with Oxidative Status in Gliomas.

The presence of mutations in the gene encoding isocitrate dehydrogenase (IDH1), parameters of pro- and antioxidant activity in glioma tumor tissue, and their correlation were studied using bioinformatic analysis using STRING, IntAct, BioGrid, UniProt, SwissProt databases. The rate of detection of IDH1 gene mutations varied in different degree of anaplasia, being maximum in grade III gliomas. The intensity of free radical oxidation and activities of superoxide dismutase and catalase in the tumor tissue were significantly higher than in the control. The dependence of these parameters on the degree of anaplasia was shown. Mutations in IDH1 are associated with a decrease in antioxidant activity in the tissues of gliomas, which is critically important for the initial diagnosis, the choice of treatment options, and prediction of patient survival.

An integrative analysis of genome-wide 5-hydroxymethylcytosines in circulating cell-free DNA detects noninvasive diagnostic markers for gliomas.

BackgroundGliomas, especially the high-grade glioblastomas (GBM), are highly aggressive tumors in the central nervous system (CNS) with dismal clinical outcomes. Effective biomarkers, which are not currently available, may improve clinical outcomes through early detection. We sought to develop a noninvasive diagnostic approach for gliomas based on 5-hydroxymethylcytosines (5hmC) in circulating cell-free DNA (cfDNA).MethodsWe obtained genome-wide 5hmC profiles using the 5hmC-Seal technique in cfDNA samples from 111 prospectively enrolled patients with gliomas and 111 age-, gender-matched healthy individuals, which were split into a training set and a validation set. Integrated models comprised 5hmC levels summarized for gene bodies, long noncoding RNAs (lncRNAs), cis-regulatory elements, and repetitive elements were developed using the elastic net regularization under a case–control design.ResultsThe integrated 5hmC-based models differentiated healthy individuals from gliomas (area under the curve [AUC] = 84%; 95% confidence interval [CI], 74–93%), GBM patients (AUC = 84%; 95% CI, 74–94%), WHO II-III glioma patients (AUC = 86%; 95% CI, 76–96%), regardless of IDH1 (encoding isocitrate dehydrogenase) mutation status or other glioma-related pathological features such as TERT, TP53 in the validation set. Furthermore, the 5hmC biomarkers in cfDNA showed the potential as an independent indicator from IDH1 mutation status and worked in synergy with IDH1 mutation to distinguish GBM from WHO II-III gliomas. Exploration of the 5hmC biomarkers for gliomas revealed relevance to glioma biology.ConclusionsThe 5hmC-Seal in cfDNA offers the promise as a noninvasive approach for effective detection of gliomas in a screening program.

Metabolic engineering of an acid-tolerant yeast strain Pichia kudriavzevii for itaconic acid production

Itaconic acid (IA), or 2-methylenesuccinic acid, has a broad spectrum of applications in the biopolymer industry owing to the presence of one vinyl bond and two acid groups in the structure. Its polymerization can follow a similar mechanism as acrylic acid but additional functionality can be incorporated into the extra beta acid group. Currently, the bio-based production of IA in industry relies on the fermentation of the filamentous fungus Aspergillus terreus. However, the difficulties associated with the fermentation undertaken by filamentous fungi together with the pathogenic potential of A. terreus pose a serious challenge for industrial-scale production. In recent years, there has been increasing interest in developing alternative production hosts for fermentation processes that are more homogenous in the production of organic acids.Pichia kudriavzevii is a non-conventional yeast with high acid tolerance to organic acids at low pH, which is a highly desirable trait by easing downstream processing. We introduced cis-aconitic acid decarboxylase gene (cad) from A. terreus (designated At_cad) into this yeast and established the initial titer of IA at 135 ​± ​5 ​mg/L. Subsequent overexpression of a native mitochondrial tricarboxylate transporter (herein designated Pk_mttA) presumably delivered cis-aconitate efficiently to the cytosol and doubled the IA production. By introducing the newly invented CRISPR-Cas9 system into P. kudriavzevii, we successfully knocked out both copies of the gene encoding isocitrate dehydrogenase (ICD), aiming to increase the availability of cis-aconitate. The resulting P. kudriavzevii strain, devoid of ICD and overexpressing Pk_mttA and At_cad on its genome produced IA at 505 ​± ​17.7 ​mg/L in shake flasks, and 1232 ​± ​64 ​mg/L in fed-batch fermentation. Because the usage of an acid-tolerant species does not require pH adjustment during fermentation, this work demonstrates the great potential of engineering P. kudriavzevii as an industrial chassis for the production of organic acid.

Blockade of Glutathione Metabolism in IDH1-Mutated Glioma.

Mutations in genes encoding isocitrate dehydrogenases (IDH) 1 and 2 are common cancer-related genetic abnormalities. Malignancies with mutated IDHs exhibit similar pathogenesis, metabolic pattern, and resistance signature. However, an effective therapy against IDH1-mutated solid tumor remains unavailable. In this study, we showed that acquisition of IDH1 mutation results in the disruption of NADP+/NADPH balance and an increased demand for glutathione (GSH) metabolism. Moreover, the nuclear factor erythroid 2-related factor 2 (Nrf2) plays a key protective role in IDH1-mutated cells by prompting GSH synthesis and reactive oxygen species scavenging. Pharmacologic inhibition of the Nrf2/GSH pathway via brusatol administration exhibited a potent tumor suppressive effect on IDH1-mutated cancer in vitro and in vivo Our findings highlight a possible therapeutic strategy that could be valuable for IDH1-mutated cancer treatment.

Conventional chondrosarcoma with focal clear cell change: a clinicopathological and molecular analysis.

AimsClear cell chondrosarcomas are known to occasionally contain areas of low‐grade conventional chondrosarcoma; however, the opposite phenomenon has not yet been described. We identified five cases of conventional chondrosarcoma alongside clear cell chondrosarcoma. Here, we report on their clinicopathological and molecular characteristics, and investigate whether these hybrid lesions should be considered to be a collision tumour, conventional chondrosarcoma with clear cell change, or clear cell chondrosarcoma with extensive areas of conventional chondrosarcoma, as this has clinical implications.Methods and resultsClinicohistopathological features were characterised, immunohistochemistry was performed for H3 histone family member 3B (H3F3B), histone H3 trimethylated on lysine 27 (H3K27me3), and p53, and genetic alterations of IDH1 (encoding isocitrate dehydrogenase 1), IDH2 (encoding isocitrate dehydrogenase 2), TP53 and H3F3B were evaluated. All five chondrosarcomas consisted predominantly of areas with conventional chondrosarcoma. Different grades were found [grade I (n = 1), grade II (n = 2), and grade III (n = 2)]. Up to 20% of the tumour consisted of classic features of clear cell chondrosarcoma. Gradual merging between both components was observed. Molecular analysis of conventional chondrosarcoma components revealed an IDH1 c.395G>T, p.(Arg132Leu) mutation in two cases, and an IDH1 c.394C>T, p.(Arg132Cys) mutation in one case, with identical IDH mutations in the clear cell chondrosarcoma counterpart (100%). Two cases were IDH wild‐type. In all cases, none of the components harboured H3F3B mutations. High‐grade tumours had an aggressive course, as three patients died of the disease.ConclusionOn the basis of clinicopathological characterisation and genetic alterations, it is suggested that these lesions should be considered to be conventional chondrosarcoma, with clear cell change. Pathologists should be aware of their existence to avoid confusion with clear cell chondrosarcoma, dedifferentiated chondrosarcoma, or chondroblastic osteosarcoma.

Biochemical characterization of human D-2-hydroxyglutarate dehydrogenase and two disease related variants reveals the molecular cause of D-2-hydroxyglutaric aciduria

D-2-hydroxyglutaric aciduria is a neurometabolic disorder, characterized by the accumulation of D-2-hydroxyglutarate (D-2HG) in human mitochondria. Increased levels of D-2HG are detected in humans exhibiting point mutations in the genes encoding isocitrate dehydrogenase, citrate carrier, the electron transferring flavoprotein (ETF) and its downstream electron acceptor ETF-ubiquinone oxidoreductase or D-2-hydroxyglutarate dehydrogenase (hD2HGDH). However, while the pathogenicity of several amino acid replacements in the former four proteins has been studied extensively, not much is known about the effect of certain point mutations on the biochemical properties of hD2HGDH.Therefore, we recombinantly produced wild type hD2HGDH as well as two recently identified disease-related variants (hD2HGDH-I147S and -V444A) and performed their detailed biochemical characterization. We could show that hD2HGDH is a FAD dependent protein, which is able to catalyze the oxidation of D-2HG and D-lactate to α-ketoglutarate and pyruvate, respectively. The two variants were obtained as apo-proteins and were thus catalytically inactive. The addition of FAD failed to restore enzymatic activity of the variants, indicating that the cofactor binding site is compromised by the single amino acid replacements. Further analyses revealed that both variants form aggregates that are apparently unable to bind the FAD cofactor.Since, D-2-hydroxyglutaric aciduria may also result from a loss of function of either the ETF or its downstream electron acceptor ETF-ubiquinone oxidoreductase, ETF may serve as the cognate electron acceptor of reduced hD2HGDH. Here, we show that hD2HGDH directly reduces recombinant human ETF, thus establishing a metabolic link between the oxidation of D-2-hydroxyglutarate and the mitochondrial electron transport chain.

Rapid diagnosis of IDH1-mutated gliomas by 2-HG detection with gas chromatography mass spectrometry

The metabolic genes encoding isocitrate dehydrogenase (IDH1, 2) are frequently mutated in gliomas. Mutation of IDH defines a distinct subtype of glioma and predicts therapeutic response. IDH mutation has a remarkable neomorphic activity of converting α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG), which is now commonly referred to as an oncometabolite and biomarker for gliomas. PCR-sequencing (n = 220), immunohistochemistry staining (IHC, n = 220), and gas chromatography mass spectrometry (GC-MS, n = 87) were applied to identify IDH mutation in gliomas, and the sensitivity and specificity of these strategies were compared. PCR-sequencing and IHC staining are reliable for retrospective assessment of IDH1 mutation in gliomas, but both methods usually take 1–2 days, which hinders their application for rapid diagnosis. GC-MS-based methods can detect 2-HG qualitatively and quantitatively, offering information on the IDH1 mutation status in gliomas with the sensitivity and specificity being 100%. Further optimization of the GC-MS based methodology (so called as the mini-column method) enabled us to determine 2-HG within 40 min in glioma samples without complex or time-consuming preparation. Most importantly, the ratio of 2-HG/glutamic acid was shown to be a reliable parameter for determination of mutation status. The mini-column method enables rapid identification of 2-HG, providing a promising strategy for intraoperative diagnosis of IDH1-mutated gliomas in the future.

Study of Isocitrate Dehydrogenase 1and 2 Mutations in Adult Egyptian Patients with Denovo Acute Myeloblastic Leukemia, Their Relation to Clinical Characteristics, FLT3/ITD and Nucleophosmin 1 Mutations and Impact on Treatment Outcome

Background: Acute myeloid leukaemia (AML) is a malignancy that is heterogeneous in nature characterized by genetic abnormalities some of which are established in the diagnosis and prognosis of the disease. An additional role for alterations in epigenetic mechanisms has been also highlighted in the pathogenesis of the disease. This may have a role in determining the disease outcome, impact the treatment decision and provide options for targeted therapies especially in patients who lack genetic aberrations. One of the modes of epigenetic dysregulation is mutation in genes encoding isocitrate dehydrogenase 1 and 2 that has been observed in AML with a higher incidence in patients with normal karyotype (NK).
 Aim of the work: The aim of this work was to study the frequency of IDH1 (R132) and IDH2 (R140Q, R172K) mutations in adult Egyptian patients with de novo acute myeloblastic leukemia (AML), their relation with clinical characteristics, other molecular markers (the internal tandem duplication (ITD)) mutation of FLT3 gene and NPM1 gene mutation) and impact on treatment outcome.
 Methods: Peripheral blood samples from 50 adult patients with denovo acute myeloid leukemia, admitted to the haematology unit at Alexandria Main University Hospital from February 2015 to February 2017, were used. The polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP) was used for detection of IDH1 codon R132 and IDH2 codons (R140, R172) mutations on genomic DNA. PCR was used for detection of FLT3-ITD mutation on genomic DNA. PCR was used for detection of NPM1 mutation on RNA.
 Results: IDH 1and 2 mutations occurred in 30% of newly diagnosed AML patients and 47.6% of NK patients. Both mutations did not co-occur except in one case. IDH positive patients were significantly older than IDH negative patients (p=0.003). There was no statistically significant correlation between any of the clinical parameters and the IDH mutations. FAB-M2 was the most common FAB subtype among IDH positive patients. No correlation between IDH mutations and NPM1 or FLT3 could be demonstrated. IDH positive patients had significantly lower CR rates after induction chemotherapy than IDH negative patients (p=0.021).
 Conclusion: IDH1, 2 mutations are recurring genetic alterations in AML with a higher incidence in patients with normal karyotype and they may have an unfavorable impact on clinical outcome in adult AML patients.

Treatment Patterns and Healthcare Resource Utilization in Patients with Relapsed/Refractory Acute Myeloid Leukemia and a Subset with IDH1-Mutation: A United States Medical Chart Review Study

▪Introduction: In the United States (US), acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. Patients with relapsed or refractory AML (R/R) have a particularly poor prognosis, and more effective and less toxic treatments are urgently needed. The gene encoding isocitrate dehydrogenase 1 (IDH1) is mutated in 6-10% of AML patients. Drugs that target the altered enzyme have shown significant clinical activity in the R/R setting, with the first such agent, ivosidenib, recently receiving FDA approval. We conducted a retrospective chart review to evaluate current treatments and healthcare resource use in an R/R AML population, with a sub-analysis focusing on the population with IDH1 mutations (mIDH1). Findings from this study will help to better understand the future impact of targeted IDH1 inhibitors in this difficult-to-treat patient population.Methods:US-based hematologists and oncologists were recruited from existing physician panels to extract information from eligible patients' medical charts. Each physician extracted information, including patient demographics, medical history, AML treatment, and AML-related healthcare resource use. Eligible patients must have been initially diagnosed with R/R AML and tested for mIDH1 between January 2012 and June 2017. Patients were excluded if they had ever been enrolled in a clinical trial for an IDH1 inhibitor. The index date was defined as the date of initial R/R diagnosis and information was collected through end of follow-up, defined as the end date of care, date of death, or date of data collection, whichever occurred first. Patient characteristics, treatments, and AML-related healthcare resource use were described. mIDH1 patients were oversampled to achieve the targeted sample size of 300 patient charts, evenly split between mIDH1-positive and IDH1 wild type (wt) patients. As a result, findings reported for the overall patient population were reweighted to reflect an 8.0% prevalence of mIDH1.Results:Data were collected from 182 oncologists/hematologists, providing chart information for 304 patients (154 mIDH1-positivepatients and 150 mIDH1-negativepatients). Median follow-up time for all patients was 11.1 months. After reweighting, the mean age for the overall patient population was 57.7 years old and most patients were first diagnosed with de novoAML (92.8%), as opposed to secondary AML (7.2%). The population had a higher percentage of relapsed patients than primary refractory patients (73.7% versus 26.3%) and a higher percentage of patients treated in a community-based setting than an academic-based setting (64.2% versus 35.8%).After the initial diagnosis of R/R AML, 90.9% of all patients received some type of active treatment, with intensive chemotherapy being the most common initial treatment (65.0%), followed by less-intensive chemotherapy regimens (19.3%), and hypomethylating agents (14.9%). 44.8% and 11.0% of patients also received a second- or third-line treatment regimen, respectively. 10.4% of patients received a stem cell transplant during the follow-up period.There was significant health resource utilization in this population. 82.0% of patients had at least one inpatient admission during the follow-up period, with the overall population averaging 0.28 admissions per-patient-per-month. The most common reason for AML-related inpatient admissions was chemotherapy administration. 66.4% of patients were hospitalized for treatment administration, 31.8% for infections, and 27.1% for febrile neutropenia. In addition, 39.6% of patients had at least one emergency room (ER) visit and 14.2% of patients had at least one intensive care unit (ICU) admission. The treatment and healthcare resource use burden was similarly high for the subset of mIDH1-positive patients. No significant differences were found between the mIDH1-positive and IDH1 wtpatients.Conclusions:Using retrospective medical chart data, this study highlights current treatment and healthcare resource use for adult patients with R/R AML. There is a high burden associated with R/R AML due to the health issues associated with rapidly progressive disease and the toxicity of available treatments, which require inpatient admissions. As more targeted drugs become available for patients in the R/R setting, such as ivosidenib, it will be of interest to re-evaluate the burden and clinical outcomes in the R/R AML population. DisclosuresGriffin:RXi Pharmaceuticals: Consultancy; Analysis Group: Consultancy; Myeloproliferative Neoplasia Foundation: Other: Grant ; Lilly Pharmaceuticals: Other: Grant; Sun Pharmaceuticals: Consultancy; Novartis Pharma: Other: Grant, Patents & Royalties: Royalties ; Astellas Pharma: Consultancy. Storm:Agios Pharmaceuticals: Employment. Wilhelm:Agios Pharmaceuticals: Employment. Boscoe:Agios Pharmaceuticals: Employment. Macaulay:Analysis Group, Inc.: Employment. Zhou:Celgene Corporation: Research Funding; Analysis Group, Inc.: Employment. Faust:Analysis Group, Inc.: Employment. Cheung:Analysis Group, Inc.: Employment.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Maffucci Syndrome with Clival Enchondroma in Nasopharynx: A Case Report.

Maffucci syndrome is an extremely rare sporadic disease, characterized by multiple enchondromas and associated with multiple hemangiomas and/or lymphangiomas. First case was reported in 1881, and fewer than 200 case have been reported to date. Potential sarcomatous malignant transformation have been noticed in previous cases. This report describes the case of 21years-old male complain of right sided nasal obstruction for 10months, and right sided aural fullness. Examination revealed multiple asymptomatic nodules in both hands since childhood, that have not been investigated and a large nasopharyngeal mass. Computed tomography, showed a 36mm in 30mm diameter lesion extending from the inferior aspect of the clivus and basisphenoid into the nasopharynx, which did not seems to be highly vascular with angiogram. Incidental radiological findings of multiple bubbly lytic bony lesions. Endoscopic sinus surgery and clival lesion excision was performed. Pathology confirmed diagnosis of clival enchondroma with clinical and radiological features consistent with Maffucci syndrome. Clear etiology have not been established yet. Mutations in gene encoding parathyroid hormone receptor 1 found in 10% of enchondromatosis. Mutations in gene encoding isocitrate dehydrogenase 1 and 2 (IDH 1&2) occur in some enchondromas and spindle cell hemangiomas. Maffucci syndrome usually presents with asymmetrical distribution of multiple enchondromas. Malignant transformation is the most concerning potential sequel. Many studies have shown sarcomatous degeneration of enchondromas to chondrosarcomas in average of 25% of cases. Patient counselling and education are crucial in the management. Surgical excision usually for symptomatic patients and suspicious lesions. Nevertheless, all patient of Maffucci syndrome will require a long term follow up and surveillance for the lifelong risk of malignant transformation. Maffucci syndrome diagnosis based on clinical presentation, radiological and histopathological findings. Surgical excision offered for symptomatic patients and for suspicious lesions. Lifelong risk of sarcomatous malignant transformation necessitate a long term surveillance.