Advances in measuring cancer cell metabolism with subcellular resolution.

Characterizing metabolism in cancer is crucial for understanding tumor biology and for developing potential therapies. Although most metabolic investigations analyze averaged metabolite levels from all cell compartments, subcellular metabolomics can provide more detailed insight into the biochemical processes associated with the disease. Methodological limitations have historically prevented the wider application of subcellular metabolomics in cancer research. Recently, however, ways to distinguish and identify metabolic pathways within organelles have been developed, including state-of-the-art methods to monitor metabolism in situ (such as mass spectrometry-based imaging, Raman spectroscopy and fluorescence microscopy), to isolate key organelles via new approaches and to use tailored isotope-tracing strategies. Herein, we examine the advantages and limitations of these developments and look to the future of this field of research.

A Single-Organelle Optical Omics Platform for Cell Science and Biomarker Discovery.

Research in fundamental cell biology and pathology could be revolutionized by developing the capacity for quantitative molecular analysis of subcellular structures. To that end, we introduce the Ramanomics platform, based on confocal Raman microspectrometry coupled to a biomolecular component analysis algorithm, which together enable us to molecularly profile single organelles in a live-cell environment. This emerging omics approach categorizes the entire molecular makeup of a sample into about a dozen of general classes and subclasses of biomolecules and quantifies their amounts in submicrometer volumes. A major contribution of our study is an attempt to bridge Raman spectrometry with big-data analysis in order to identify complex patterns of biomolecules in a single cellular organelle and leverage discovery of disease biomarkers. Our data reveal significant variations in organellar composition between different cell lines. We also demonstrate the merits of Ramanomics for identifying diseased cells by using prostate cancer as an example. We report large-scale molecular transformations in the mitochondria, Golgi apparatus, and endoplasmic reticulum that accompany the development of prostate cancer. Based on these findings, we propose that Ramanomics datasets in distinct organelles constitute signatures of cellular metabolism in healthy and diseased states.

Toward Single-Organelle Lipidomics in Live Cells.

Detailed studies of lipids in biological systems, including their role in cellular structure, metabolism, and disease development, comprise an increasingly prominent discipline called lipidomics. However, the conventional lipidomics tools, such as mass spectrometry, cannot investigate lipidomes until they are extracted, and thus they cannot be used for probing the lipid distribution nor for studying in live cells. Furthermore, conventional techniques rely on the lipid extraction from relatively large samples, which averages the data across the cellular populations and masks essential cell-to-cell variations. Further advancement of the discipline of lipidomics critically depends on the capability of high-resolution lipid profiling in live cells and, potentially, in single organelles. Here we report a micro-Raman assay designed for single-organelle lipidomics. We demonstrate how Raman microscopy can be used to measure the local intracellular biochemical composition and lipidome hallmarks-lipid concentration and unsaturation level, cis/trans isomer ratio, sphingolipids and cholesterol levels in live cells-with a sub-micrometer resolution, which is sufficient for profiling of subcellular structures. These lipidome data were generated by a newly developed biomolecular component analysis software, which provides a shared platform for data analysis among different research groups. We outline a robust, reliable, and user-friendly protocol for quantitative analysis of lipid profiles in subcellular structures. This method expands the capabilities of Raman-based lipidomics toward the analysis of single organelles within either live or fixed cells, thus allowing an unprecedented measure of organellar lipid heterogeneity and opening new quantitative ways to study the phenotypic variability in normal and diseased cells.

Targeting IDH1-Mutated Oligodendroglioma with Acid Ceramidase Inhibitors.

Oligodendroglioma is genetically defined as a tumor harboring isocitrate dehydrogenase 1 or 2 mutations (IDH1 mut /IDH2 mut ) and 1p/19q co-deletions. Previously, we reported that in IDH1 mut gliomas, D-2HG, the product of IDH1 mutant enzyme produces an increase in monounsaturated fatty acid levels that are incorporated into ceramides, tilting the S1P-to-ceramide rheostat toward apoptosis. Herein, we exploited this imbalance to further induce and IDH mut -specific glioma cell death. We report for the first time that the inhibition of acid ceramidase (AC) induces apoptosis and provides a benefit in mice survival in IDH1 mut oligodendroglioma. We demonstrated an IDH1 mut -specific cytotoxicity of SABRAC, an irreversible inhibitor of AC, in patient-derived oligodendroglioma cells. Exploring the mechanism of action of this drug, we found that SABRAC activates both extrinsic and intrinsic apoptosis in an ER stress-independent manner, pointing to a direct action of AC-related ceramides in mitochondria permeability. The activation of apoptosis detected under SABRAC treatment was associated with up to 30-fold increase in some ceramide levels and its derivatives from the salvage pathway. We propose that this novel enzyme, AC, has the potential to increase survival in oligodendroglioma with IDH1 mut and should be considered in the future.

Raman-based machine learning platform reveals unique metabolic differences between IDHmut and IDHwt glioma.

BACKGROUND: Formalin-fixed, paraffin-embedded (FFPE) tissue slides are routinely used in cancer diagnosis, clinical decision-making, and stored in biobanks, but their utilization in Raman spectroscopy-based studies has been limited due to the background coming from embedding media. METHODS: Spontaneous Raman spectroscopy was used for molecular fingerprinting of FFPE tissue from 46 patient samples with known methylation subtypes. Spectra were used to construct tumor/non-tumor, IDH1WT/IDH1mut, and methylation-subtype classifiers. Support vector machine and random forest were used to identify the most discriminatory Raman frequencies. Stimulated Raman spectroscopy was used to validate the frequencies identified. Mass spectrometry of glioma cell lines and TCGA were used to validate the biological findings. RESULTS: Here we develop APOLLO (rAman-based PathOLogy of maLignant glioma) - a computational workflow that predicts different subtypes of glioma from spontaneous Raman spectra of FFPE tissue slides. Our novel APOLLO platform distinguishes tumors from nontumor tissue and identifies novel Raman peaks corresponding to DNA and proteins that are more intense in the tumor. APOLLO differentiates isocitrate dehydrogenase 1 mutant (IDH1mut) from wildtype (IDH1WT) tumors and identifies cholesterol ester levels to be highly abundant in IDHmut glioma. Moreover, APOLLO achieves high discriminative power between finer, clinically relevant glioma methylation subtypes, distinguishing between the CpG island hypermethylated phenotype (G-CIMP)-high and G-CIMP-low molecular phenotypes within the IDH1mut types. CONCLUSIONS: Our results demonstrate the potential of label-free Raman spectroscopy to classify glioma subtypes from FFPE slides and to extract meaningful biological information thus opening the door for future applications on these archived tissues in other cancers.

Targeting the sphingolipid rheostat in IDH1 mut glioma alters cholesterol homeostasis and triggers apoptosis via membrane degradation.

The cross-regulation of metabolism and trafficking is not well understood for the vital sphingolipids and cholesterol constituents of cellular compartments. While reports are starting to surface on how sphingolipids like sphingomyelin (SM) dysregulate cholesterol levels in different cellular compartments (Jiang et al., 2022), limited research is available on the mechanisms driving the relationship between sphingolipids and cholesterol homeostasis, or its biological implications. Previously, we have identified sphingolipid metabolism as a unique vulnerability for IDH1 mut gliomas via a rational drug design. Herein, we show how modulating sphingolipid levels affects cholesterol homeostasis in brain tumors. However, we unexpectedly discovered for the first time that C17 sphingosine and NDMS addition to cancer cells alters cholesterol homeostasis by impacting its cellular synthesis, uptake, and efflux leading to a net decrease in cholesterol levels and inducing apoptosis. Our results reflect a reverse correlation between the levels of sphingosines, NDMS, and unesterified, free cholesterol in the cells. We show that increasing sphingosine and NDMS (a sphingosine analog) levels alter not only the trafficking of cholesterol between membranes but also the efflux and synthesis of cholesterol. We also demonstrate that despite the effort to remove free cholesterol by ABCA1-mediated efflux or by suppressing machinery for the influx (LDLR) and biosynthetic pathway (HMGCR), apoptosis is inevitable for IDH1 mut glioma cells. This is the first study that shows how altering sphingosine levels directly affects cholesterol homeostasis in cancer cells and can be used to manipulate this relationship to induce apoptosis in IDH1 mut gliomas.

Ca11E3C8 (E = Sn, Pb): new complex carbide Zintl phases grown from Ca/Li flux.

New carbide Zintl phases Ca(11)E(3)C(8) (E = Sn, Pb) were grown from reactions of carbon and heavy tetrelides in Ca/Li flux. They form with a new structure type in space group P2(1)/c (a = 13.1877(9)Å, b = 10.6915(7)Å, c = 14.2148(9)Å, ß = 105.649(1)°, and R(1) = 0.019 for the Ca(11)Sn(3)C(8) analog). The structure features isolated E(4-) anions as well as acetylide (C(2)(2-)) and allenylide (C(3)(4-)) anions; the vibrational modes of the carbide anions are observed in the Raman spectrum. The charge-balanced nature of these phases is confirmed by DOS calculations which indicate that the tin analog has a small band gap (E(g) < 0.1 eV) and the lead analog has a pseudogap at the Fermi level. Reactions of these compounds with water produce acetylene and allene.

Metal site-mediated, thermally induced structural changes in Cr6+-silicalite-2 (MEL) molecular sieves.

Cr(6+) ions were incorporated into the lattice sites of phase-pure silicalite-2 made using 3,5-dimethylpiperidinium as a structure-directing agent. The materials exhibited a remarkably well-resolved vibronic emission consisting of a high frequency progression of 987 cm(-1), which was assigned to the fundamental symmetric stretching mode of the (Si-O-)(2)Cr(═O)(2) group dominated by the terminal Cr═O stretch. A low frequency progression at 214 cm(-1), which was assigned to a symmetric O-Cr-O bending mode, was built on each band of the 987 cm(-1) progression. Studies of the vibronic structure of the emission spectrum as a function of temperature and Cr ion concentration reveal an abrupt change in the Franck-Condon factor of the emission at 20 K for samples with very low Cr concentrations (0.03 mol %). The change in the Franck-Condon factor is attributed to a temperature-induced structural change in the coordination sphere of the metal ion. This structural change was found to be accompanied by a concomitant structural change in the lattice structure of the silicalite-2. This structural change, as studied by temperature-dependent X-ray diffraction, did not involve a crystallographic phase change but an abrupt decrease in the unit cell volume, caused specifically by a decrease in the c-axis. This structural change was not observed in pure silicalite-2, indicating that it is not intrinsic to the silicalite lattice. Moreover, no similar structural change was observed at higher Cr loading (1 mol %). This suggests that the presence of the Cr ions and the changes in the coordination geometry they undergo at low temperature induced the observed contraction in the silicalite-2 lattice, in effect acting as a thermal switch that decreases the unit cell volume.

Investigation of the binding of dioxin selective pentapeptides to a polyaniline matrix.

Polyaniline in form of emeraldine salt and emeraldine base was used as a matrix to attach several labeled and non-labeled dioxin selective pentapeptides both directly to the polymer and using glutaraldehyde as a linker. The peptides have been selected as a model to study the binding process due to their smaller size, lower sensitivity to the environment and potential application as solid state extraction reagents for chlorinated toxins. The composition and the properties of the compounds were investigated by means of elemental analysis, XPS, FTIR, UV/vis, and fluorescence spectroscopy. The results have shown that 3.30-7.76% peptides were attached to the emeraldine base both with and without a linker. Glutaraldehyde and the peptides were connected to the matrix via chemical bond resulting in formation of compounds whit similar composition and stability in a broad pH range. The influence of the linker and the peptides on the electronic properties and composition of the polymer have been investigated by principal component analysis.

Probabilistic model checking of cancer metabolism.

Cancer cell metabolism is often deregulated as a result of adaption to meeting energy and biosynthesis demands of rapid growth or direct mutation of key metabolic enzymes. Better understanding of such deregulation can provide new insights on targetable vulnerabilities, but is complicated by the difficulty in probing cell metabolism at different levels of resolution and under different experimental conditions. We construct computational models of glucose and glutamine metabolism with focus on the effect of IDH1/2-mutations in cancer using a combination of experimental metabolic flux data and patient-derived gene expression data. Our models demonstrate the potential of computational exploration to reveal biologic behavior: they show that an exogenously-mutated IDH1 experimental model utilizes glutamine as an alternative carbon source for lactate production under hypoxia, but does not fully-recapitulate the patient phenotype under normoxia. We also demonstrate the utility of using gene expression data as a proxy for relative differences in metabolic activity. We use the approach of probabilistic model checking and the freely-available Probabilistic Symbolic Model Checker to construct and reason about model behavior.

Cysteine is a limiting factor for glioma proliferation and survival.

Nutritional intervention is becoming more prevalent as adjuvant therapy for many cancers in view of the tumor dependence on external sources for some nutrients. However, little is known about the mechanisms that make cancer cells require certain nutrients from the microenvironment. Herein, we report the dependence of glioma cells on exogenous cysteine/cystine, despite this amino acid being nonessential. Using several 13 C-tracers and analysis of cystathionine synthase and cystathioninase levels, we revealed that glioma cells were not able to support glutathione synthesis through the transsulfuration pathway, which allows methionine to be converted to cysteine in cysteine/cystine-deprived conditions. Therefore, we explored the nutritional deprivation in a mouse model of glioma. Animals subjected to a cysteine/cystine-free diet survived longer, although this increase did not attain statistical significance, with concomitant reductions in plasma glutathione and cysteine levels. At the end point, however, tumors displayed the ability to synthesize glutathione, even though higher levels of oxidative stress were detected. We observed a compensation from the nutritional intervention revealed as the recovery of cysteine-related metabolite levels in plasma. Our study highlights a time window where cysteine deprivation can be exploited for additional therapeutic strategies.

Metabolic biomarkers of radiotherapy response in plasma and tissue of an IDH1 mutant astrocytoma mouse model.

Astrocytomas are the most common subtype of brain tumors and no curative treatment exist. Longitudinal assessment of patients, usually via Magnetic Resonance Imaging (MRI), is crucial since tumor progression may occur earlier than clinical progression. MRI usually provides a means for monitoring the disease, but it only informs about the structural changes of the tumor, while molecular changes can occur as a treatment response without any MRI-visible change. Radiotherapy (RT) is routinely performed following surgery as part of the standard of care in astrocytomas, that can also include chemotherapy involving temozolomide. Monitoring the response to RT is a key factor for the management of patients. Herein, we provide plasma and tissue metabolic biomarkers of treatment response in a mouse model of astrocytoma that was subjected to radiotherapy. Plasma metabolic profiles acquired over time by Liquid Chromatography Mass Spectrometry (LC/MS) were subjected to multivariate empirical Bayes time-series analysis (MEBA) and Receiver Operating Characteristic (ROC) assessment including Random Forest as the classification strategy. These analyses revealed a variation of the plasma metabolome in those mice that underwent radiotherapy compared to controls; specifically, fumarate was the best discriminatory feature. Additionally, Nuclear Magnetic Resonance (NMR)-based 13C-tracing experiments were performed at end-point utilizing [U-13C]-Glutamine to investigate its fate in the tumor and contralateral tissues. Irradiated mice displayed lower levels of glycolytic metabolites (e.g. phosphoenolpyruvate) in tumor tissue, and a higher flux of glutamine towards succinate was observed in the radiation cohort. The plasma biomarkers provided herein could be validated in the clinic, thereby improving the assessment of brain tumor patients throughout radiotherapy. Moreover, the metabolic rewiring associated to radiotherapy in tumor tissue could lead to potential metabolic imaging approaches for monitoring treatment using blood draws.

Ligand-passivated Eu:Y2O3 nanocrystals as a phosphor for white light emitting diodes.

Eu(III)-doped Y(2)O(3) nanocrystals are prepared by microwave synthetic methods as spherical 6.4 ± 1.5 nm nanocrystals with a cubic crystal structure. The surface of the nanocrystal is passivated by acetylacetonate (acac) and HDA on the Y exposed facet of the nanocrystal. The presence of acac on the nanocrystal surface gives rise to a strong S(0) → S(1) (π → π*, acac) and acac → Ln(3+) ligand to metal charge transfer (LMCT) transitions at 270 and 370 nm, respectively, in the Eu:Y(2)O(3) nanocrystal. Excitation into the S(0) → S(1) (π → π*) or acac → Ln(3+) LMCT transition leads to the production of white light emission arising from efficient intramolecular energy transfer to the Y(2)O(3) oxygen vacancies and the Eu(III) Judd-Ofelt f-f transitions. The acac passivant is thermally stable below 400 °C, and its presence is evidenced by UV-vis absorption, FT-IR, and NMR measurements. The presence of the low-lying acac levels allows UV LED pumping of the solid phosphor, leading to high quantum efficiency (∼19%) when pumped at 370 nm, high-quality white light color rendering (CIE coordinates 0.33 and 0.35), a high scotopic-to-photopic ratio (S/P = 2.21), and thermal stability. In a LED lighting package luminosities of 100 lm W(-1) were obtained, which are competitive with current commercial lighting technology. The use of the passivant to funnel energy to the lanthanide emitter via a molecular antenna effect represents a new paradigm for designing phosphors for LED-pumped white light.

Synthesis, characterization, and spectroscopic characteristics of chromium(6+) and -(4+) silicalite-2 (ZSM-11) materials.

The systematic incorporation of Cr ions into a phase-pure silicalite-2 lattice was accomplished through hydrothermal synthesis using 3,5-dimethylpiperidinium as a templating agent. The Cr ions, after calcination to remove the template, were in the 6+ oxidation state, with their incorporation into the lattice verified by the systematic expansion of the unit cell as a function of Cr loading. The structures of these materials as revealed by electronic spectroscopy and X-ray absorption near-edge spectroscopy (XANES) were consistent with the dioxo structure typically exhibited by Cr(6+) in an amorphous silica matrix. These materials were highly luminescent, with the emission spectra showing an unusually well-resolved vibronic structure characteristic of an emissive site with little inhomogeneous broadening. The site was reduced under flowing CO to Cr(4+), as characterized by XANES. The reduction of Cr from 6+ to 4+ resulted in unit-cell volumes that are systematically smaller than those observed with Cr(6+), even though the ionic radius of Cr(4+) is larger. This is attributed to the fact that the Cr(6+) site is not a simple metal ion but a significantly larger [CrO(2)](2+) unit, requiring a larger lattice expansion to accommodate it. Through analysis of the XANES preedge and assignment of the ligand-field spectrum of the Cr(4+) ions, it is possible to establish isomorphic substitution into the silicalite lattice.

IDH1 mutations induce organelle defects via dysregulated phospholipids.

Infiltrating gliomas are devastating and incurable tumors. Amongst all gliomas, those harboring a mutation in isocitrate dehydrogenase 1 mutation (IDH1mut) acquire a different tumor biology and clinical manifestation from those that are IDH1WT. Understanding the unique metabolic profile reprogrammed by IDH1 mutation has the potential to identify new molecular targets for glioma therapy. Herein, we uncover increased monounsaturated fatty acids (MUFA) and their phospholipids in endoplasmic reticulum (ER), generated by IDH1 mutation, that are responsible for Golgi and ER dilation. We demonstrate a direct link between the IDH1 mutation and this organelle morphology via D-2HG-induced stearyl-CoA desaturase (SCD) overexpression, the rate-limiting enzyme in MUFA biosynthesis. Inhibition of IDH1 mutation or SCD silencing restores ER and Golgi morphology, while D-2HG and oleic acid induces morphological defects in these organelles. Moreover, addition of oleic acid, which tilts the balance towards elevated levels of MUFA, produces IDH1mut-specific cellular apoptosis. Collectively, these results suggest that IDH1mut-induced SCD overexpression can rearrange the distribution of lipids in the organelles of glioma cells, providing new insight into the link between lipid metabolism and organelle morphology in these cells, with potential and unique therapeutic implications.

Sphingolipid Pathway as a Source of Vulnerability in IDH1mut Glioma.

In addition to providing integrity to cellular structure, the various classes of lipids participate in a multitude of functions including secondary messengers, receptor stimulation, lymphocyte trafficking, inflammation, angiogenesis, cell migration, proliferation, necrosis and apoptosis, thus highlighting the importance of understanding their role in the tumor phenotype. In the context of IDH1mut glioma, investigations focused on metabolic alterations involving lipidomics' present potential to uncover novel vulnerabilities. Herein, a detailed lipidomic analysis of the sphingolipid metabolism was conducted in patient-derived IDH1mut glioma cell lines, as well as model systems, with the of identifying points of metabolic vulnerability. We probed the effect of decreasing D-2HG levels on the sphingolipid pathway, by treating these cell lines with an IDH1mut inhibitor, AGI5198. The results revealed that N,N-dimethylsphingosine (NDMS), sphingosine C17 and sphinganine C18 were significantly downregulated, while sphingosine-1-phosphate (S1P) was significantly upregulated in glioma cultures following suppression of IDH1mut activity. We exploited the pathway using a small-scale, rational drug screen and identified a combination that was lethal to IDHmut cells. Our work revealed that further addition of N,N-dimethylsphingosine in combination with sphingosine C17 triggered a dose-dependent biostatic and apoptotic response in a panel of IDH1mut glioma cell lines specifically, while it had little effect on the IDHWT cells probed here. To our knowledge, this is the first study that shows how altering the sphingolipid pathway in IDH1mut gliomas elucidates susceptibility that can arrest proliferation and initiate subsequent cellular death.

Metabolic Landscape of a Genetically Engineered Mouse Model of IDH1 Mutant Glioma.

Understanding the metabolic reprogramming of aggressive brain tumors has potential applications for therapeutics as well as imaging biomarkers. However, little is known about the nutrient requirements of isocitrate dehydrogenase 1 (IDH1) mutant gliomas. The IDH1 mutation involves the acquisition of a neomorphic enzymatic activity which generates D-2-hydroxyglutarate from α-ketoglutarate. In order to gain insight into the metabolism of these malignant brain tumors, we conducted metabolic profiling of the orthotopic tumor and the contralateral regions for the mouse model of IDH1 mutant glioma; as well as to examine the utilization of glucose and glutamine in supplying major metabolic pathways such as glycolysis and tricarboxylic acid (TCA). We also revealed that the main substrate of 2-hydroxyglutarate is glutamine in this model, and how this re-routing impairs its utilization in the TCA. Our 13C tracing analysis, along with hyperpolarized magnetic resonance experiments, revealed an active glycolytic pathway similar in both regions (tumor and contralateral) of the brain. Therefore, we describe the reprogramming of the central carbon metabolism associated with the IDH1 mutation in a genetically engineered mouse model which reflects the tumor biology encountered in glioma patients.

Metabolic plasticity of IDH1-mutant glioma cell lines is responsible for low sensitivity to glutaminase inhibition.

BACKGROUND: Targeting glutamine metabolism in cancer has become an increasingly vibrant area of research. Mutant IDH1 (IDH1 mut ) gliomas are considered good candidates for targeting this pathway because of the contribution of glutamine to their newly acquired function: synthesis of 2-hydroxyglutarate (2HG). METHODS: We have employed a combination of 13C tracers including glutamine and glucose for investigating the metabolism of patient-derived IDH1 mut glioma cell lines through NMR and LC/MS. Additionally, genetic loss-of-function (in vitro and in vivo) approaches were performed to unravel the adaptability of these cell lines to the inhibition of glutaminase activity. RESULTS: We report the adaptability of IDH1 mut cells' metabolism to the inhibition of glutamine/glutamate pathway. The glutaminase inhibitor CB839 generated a decrease in the production of the downstream metabolites of glutamate, including those involved in the TCA cycle and 2HG. However, this effect on metabolism was not extended to viability; rather, our patient-derived IDH1 mut cell lines display a metabolic plasticity that allows them to overcome glutaminase inhibition. CONCLUSIONS: Major metabolic adaptations involved pathways that can generate glutamate by using alternative substrates from glutamine, such as alanine or aspartate. Indeed, asparagine synthetase was upregulated both in vivo and in vitro revealing a new potential therapeutic target for a combinatory approach with CB839 against IDH1 mut gliomas.

Metabolic reprogramming associated with aggressiveness occurs in the G-CIMP-high molecular subtypes of IDH1mut lower grade gliomas.

BACKGROUND: Early detection of increased aggressiveness of brain tumors is a major challenge in the field of neuro-oncology because of the inability of traditional imaging to uncover it. Isocitrate dehydrogenase (IDH)-mutant gliomas represent an ideal model system to study the molecular mechanisms associated with tumorigenicity because they appear indolent and non-glycolytic initially, but eventually a subset progresses toward secondary glioblastoma with a Warburg-like phenotype. The mechanisms and molecular features associated with this transformation are poorly understood. METHODS: We employed model systems for IDH1 mutant (IDH1mut) gliomas with different growth and proliferation rates in vivo and in vitro. We described the metabolome, transcriptome, and epigenome of these models in order to understand the link between their metabolism and the tumor biology. To verify whether this metabolic reprogramming occurs in the clinic, we analyzed data from The Cancer Genome Atlas. RESULTS: We reveal that the aggressive glioma models have lost DNA methylation in the promoters of glycolytic enzymes, especially lactate dehydrogenase A (LDHA), and have increased mRNA and metabolite levels compared with the indolent model. We find that the acquisition of the high glycolytic phenotype occurs at the glioma cytosine-phosphate-guanine island methylator phenotype (G-CIMP)-high molecular subtype in patients and is associated with the worst outcome. CONCLUSION: We propose very early monitoring of lactate levels as a biomarker of metabolic reprogramming and tumor aggressiveness.

Targeting Glycolysis through Inhibition of Lactate Dehydrogenase Impairs Tumor Growth in Preclinical Models of Ewing Sarcoma.

Altered cellular metabolism, including an increased dependence on aerobic glycolysis, is a hallmark of cancer. Despite the fact that this observation was first made nearly a century ago, effective therapeutic targeting of glycolysis in cancer has remained elusive. One potentially promising approach involves targeting the glycolytic enzyme lactate dehydrogenase (LDH), which is overexpressed and plays a critical role in several cancers. Here, we used a novel class of LDH inhibitors to demonstrate, for the first time, that Ewing sarcoma cells are exquisitely sensitive to inhibition of LDH. EWS-FLI1, the oncogenic driver of Ewing sarcoma, regulated LDH A (LDHA) expression. Genetic depletion of LDHA inhibited proliferation of Ewing sarcoma cells and induced apoptosis, phenocopying pharmacologic inhibition of LDH. LDH inhibitors affected Ewing sarcoma cell viability both in vitro and in vivo by reducing glycolysis. Intravenous administration of LDH inhibitors resulted in the greatest intratumoral drug accumulation, inducing tumor cell death and reducing tumor growth. The major dose-limiting toxicity observed was hemolysis, indicating that a narrow therapeutic window exists for these compounds. Taken together, these data suggest that targeting glycolysis through inhibition of LDH should be further investigated as a potential therapeutic approach for cancers such as Ewing sarcoma that exhibit oncogene-dependent expression of LDH and increased glycolysis. SIGNIFICANCE: LDHA is a pharmacologically tractable EWS-FLI1 transcriptional target that regulates the glycolytic dependence of Ewing sarcoma.