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Background Uncontrolled seizures in patients with gliomas have a significant impact on quality of life and morbidity, yet the mechanisms through which these tumors cause seizures remain unknown. Here, we hypothesize that the active metabolite d-2-hydroxyglutarate (d-2-HG) produced by the IDH-mutant enzyme leads to metabolic disruptions in surrounding cortical neurons that consequently promote seizures. Methods We use a complementary study of in vitro neuron-glial cultures and electrographically sorted human cortical tissue from patients with IDH-mutant gliomas to test this hypothesis. We utilize micro-electrode arrays for in vitro electrophysiological studies in combination with pharmacological manipulations and biochemical studies to better elucidate the impact of d-2-HG on cortical metabolism and neuronal spiking activity. Results We demonstrate that d-2-HG leads to increased neuronal spiking activity and promotes a distinct metabolic profile in surrounding neurons, evidenced by distinct metabolomic shifts and increased LDHA expression, as well as upregulation of mTOR signaling. The increases in neuronal activity are induced by mTOR activation and reversed with mTOR inhibition. Conclusion Together, our data suggest that metabolic disruptions in the surrounding cortex due to d-2-HG may be a driving event for epileptogenesis in patients with IDH-mutant gliomas.

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.

BACKGROUND AND HYPOTHESES SCD1, a major enzyme of saturated fatty acids, has been implicated to be important for tumor metabolic reprograming. Our previous study show that a high level of SCD1 mRNA is associated with IDH1mut lower grade gliomas. IDH1mut glioma cells are more sensitive to SFA induced apoptosis. However, the underlying mechanism remains unclear. In this study, we investigate the functions of SCD1 in IDHmut glioma and the potential contribution of SCD1 for cancer therapy of glioma. STUDY DESIGN AND METHODS The genetically engineered IDH wild-type, IDHmut and patient derived glioma cell lines were used to evaluate SCD1 functions. The expression of proteins were checked by Western-blotting assay. SCD1 was silenced by CRISPR or siRNA. The transcriptome change after SCD1 knockdown was profiled by RNA-seq or single cell RNA-seq (scRNA-seq). RESULTS AND CONCLUSIONS SCD1 transient silencing slowed down the cell growth, suggesting that SCD1 may possess an oncogenic property. RNA-seq analysis revealed that SCD1 inhibition decreased the expression of wnt-signaling pathway genes in IDH-1mut cells. scRNA confirmed that CRISPR SCD1 significantly decreased wnt signaling in the patient cell line Ts603. Therefore, we activated wnt pathway using a small chemical compound, BML-2838. Consistent with recent studies, wnt pathway induction led to a dramatically suppression of glioma cells growth. However, SCD1 silencing reversed this inhibitory effect. Further investigation revealed that SCD1 inhibition reduced the nucleus translocation of phosphorylated beta-catenin. Overall, the results suggest that SCD1 is vital for the onset of wnt pathway in glioma cells. High level of SCD1 expression may render the IDHmut glioma cells more sensitive to wnt pathway induced apoptosis. RELEVANCE AND IMPORTANCE In clinical, the 5-years survival rate of glioma remains low. SCD1 have been considered as a target for glioma therapy, recently. Our data provides a new insight on the strategy to target SCD1 in clinical.

The presence of the IDH mutation in gliomas is a major classifier of brain tumor subtypes and has several important implications for cancer growth. Our recent work uncovered that IDH-mutant tumors are susceptible to increased apoptosis via alterations of the sphingolipid pathway due to their excess production of pro-apoptotic ceramides over pro-proliferative sphingosine 1-phosphate (S1P). To that end, we proposed that this rheostat can be modulated to induce cell death in IDHmut tumors by targeting acid ceramidase, a critical sphingolipid enzyme in gliomas. We hypothesize that pharmacological inhibition of acid ceramidase will increase ceramide levels and therefore induce apoptosis in IDHmutgliomas. Using a preliminary drug screen, we have identified a group of haloacetate C2-ceramide derivatives known as SOBRACs that potently inhibit acid ceramidase. We selected five candidate compounds from this family and assessed the effectiveness of each drug in 3 I IDHmut (BT142, TS603, & U251mut) and 3 IDHmut (GSC923, GSC827, U251wt) patient-derived glioma cell lines, as well as non-immortalized normal human astrocytes, using the CCK8 cell viability assay. Our results indicate that the SOBRAC drugs are nearly 10 times more potent in IDH-mutant tumors compared to IDHmut cell lines. Additionally, the SOBRAC drugs are more effective than other known acid ceramidase inhibitors, making them attractive as potential novel therapeutics. To date, azide-SOBRAC is the most potent drug in the family, with EC50 value of 300 nM in BT142 cells (IDHmutmut

Isocitrate dehydrogenase (IDH) enzymes catalyse the oxidative decarboxylation of isocitrate and therefore play key roles in the Krebs cycle and cellular homoeostasis. Major advances in cancer genetics over the past decade have revealed that the genes encoding IDHs are frequently mutated in a variety of human malignancies, including gliomas, acute myeloid leukaemia, cholangiocarcinoma, chondrosarcoma and thyroid carcinoma. A series of seminal studies further elucidated the biological impact of the IDH mutation and uncovered the potential role of IDH mutants in oncogenesis. Notably, the neomorphic activity of the IDH mutants establishes distinctive patterns in cancer metabolism, epigenetic shift and therapy resistance. Novel molecular targeting approaches have been developed to improve the efficacy of therapeutics against IDH-mutated cancers. Here we provide an overview of the latest findings in IDH-mutated human malignancies, with a focus on glioma, discussing unique biological signatures and proceedings in translational research.

Background Little is known about the antineoplastic effect and the mechanism of Stearoyl-CoA desaturase (SCD) inhibitor which catalyzes the biosynthesis of monounsaturated fatty acids (MUFA). Mutant isocitrate dehydrogenase (IDH) catalyzes the NADPH-mediated reduction of α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG) and causes metabolic reprograming of lipid. In this study, to develop a feasible drug for IDH mutant glioma, we have investigated the changes of the lipid distribution and the mechanism of antineoplastic effect of SCD inhibition in IDH mutant glioma. Materials and Methods We prepared genetically engineered glioma cell lines (U251 wild type: U251WT and U251 IDH mutant: U251mut) and patient derived cell lines (TS603 and GSC923). Lipid metabolic analysis was conducted by using Raman imaging spectroscopy and LC-MS, and functional analysis for the role of SCD expression in IDH mutant glioma was investigated by RNA sequence and Western-blotting. Results: In LC-MS analysis of the extracted Endoplasmic Reticulum, MUFAs were distributed significantly higher in IDH mutant than wild type. SCD expression was increased in IDH mutant compared to wild type due to 2HG-mediated upregulation of SCD. Therefore, IDH mutant in which SCD expression level was high indicated high sensitivity to SCD inhibitor, and apoptosis was highly induced in IDH mutant compared to wild type. RNA sequencing was performed in U251mut treated with SCD inhibitor compared to U251mut treated with DMSO, and lipid droplet metabolism-associated RNA expression was significantly changed in U251mut treated with SCD inhibitor. We checked lipid droplet in U251mut with presence or absence of SCD inhibitor, and lipolysis was induced by SCD inhibitor treatment, suggesting that SCD inhibition is associated with the apoptosis in IDH mutant via enhanced lipolysis mechanism. Conclusions 2HG produced in IDH mutant glioma directly induces SCD expression and enhances sensitivity to SCD inhibitor, which suggests that SCD inhibitor is an IDH mutant glioma-specific treatment strategy.

BACKGROUND DNA methylation is an essential component for integrative diagnosis in glioma. Methylation subtype prediction of gliomas is currently done via sample extraction of high-quality of reasonable amount of DNA (~1ug), methylome profiling, followed by probe identification, curation and subsequent analysis via different random forest classifiers. However, the DNA methylation classification is not always available for all the samples. METHODS Raman Spectroscopy performed of the regions of interest using 1mm2 FFPE tissue spots from 45 patient samples with LGm1 to LGm6 methylation subtypes. Spectral information was then used to train a convolutional neural network (CNN) and develop a prediction algorithm. 70 % of dataset - model training while the remaining 30% for validation. Supervised wrapper methods and random forests were used to identify the top 109 most discriminatory Raman frequencies out of 1738. RESULTS We identified the most discriminatory features from these analyses and demonstrated that these frequencies show differential spectral intensities for these frequencies depending upon the glioma subtypes across the larger areas of the tissue. We compared the results of the Ward linkage clustering with the separation induced by the “frequency criterion”, an empirical observation that Raman spectra of tumor spots are characterized by intensities higher than 5000 on some of the frequencies from 1463 to 1473. For each of the 45 samples we ran Ward linkage clustering with a variable number of clusters (from 2 to 7), with the majority cluster corresponding to tumor spots and the others corresponding to (various types of) non-tumor spots. We found that the majority cluster matches very well the tumor spots characterized by the frequency criterion, The average accuracy over all samples was 90:3%, the average precision was 99:6% and the average recall was 90:2%. For most samples, two clusters were sufficient to distinguish between tumor and non-tumor spots with accuracy.

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 render cancer cells dependent on 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 GSH 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, although higher levels of oxidative stress were detected. We observed a compensation from the nutritional intervention revealed as the recovery of cysteine-related metabolites levels in plasma. Our study highlights a time window where cysteine deprivation can be exploited for additional therapeutic strategies.

BACKGROUND IDHwt gliomas exhibit sphingolipid rheostat balance that permits tumors to evade apoptosis by elevating the sphingosine-1-phosphate (S1P)-to-ceramide ratio. Overexpression of sphingosine kinase (SPHK) and consequent accumulation of S1P contribute to progression, chemoresistance, migration, and metastasis in malignant glioblastoma (GBM). We discovered that IDH1mut gliomas present a characteristic sphingolipid rheostat in which pro-apoptotic ceramides and sphingosines are elevated over oncopotent S1P. This characteristic involves inherent silencing of the SPHK2; obliging spheroids to rely on SPHK1 exclusively. We postulated that targeting this unique metabolic vulnerability would abrogate the growth-promoting and anti-apoptotic effects of S1P. METHODS IDH1mut glioma cell lines (TS603, BT142 & NCH1681) and empty vector-induced normal human astrocytes (NHAEV) were cultured and treated with a combination of SPHK1 inhibitor, N,N-dimethylsphingosine and C17-sphingosine to dysregulate sphingolipid rheostat. Biostatic response (i.e., IC50) was measured via spectrophotometric assay. Metabolic and signaling mechanisms were investigated by LC-MS lipidomic and RNA sequencing analysis. Mechanism of apoptosis was determined via western-blotting. RESULTS Following combination treatment, a global increase in ceramides, sphingosines, and their derivatives over S1P was detected in the sphingolipid rheostat. A decline in growth-promoting MAPK signaling enzymes and elevation of enzymes indicative of mitochondria-driven apoptosis occurred. Elevation of TNFα-related regulatory enzymes (NR1H3, MYLIP, INSIG, ABCA1) negatively impacted cholesterol homeostasis along with catalytic enzymes involved in cholesterol (and isoprenoid) biosynthesis. The effective concentration against IDH1mut spheroids was not cytotoxic to NHA spheroids. CONCLUSION The combination treatment potentiated a pro-apoptotic shift in sphingolipidome and revealed a novel mechanism of drug action involving concomitant global attenuation of cholesterol metabolism. While previous studies reported that decreasing cholesterol in gliomas compromises viability and induces apoptosis a link between cholesterol and sphingolipid metabolism remains unknown. Our data demonstrated that targeting sphingolipid rheostat triggers a cascade of pro-apoptotic signaling and metabolic activity that lower the threshold for apoptosis in IDHmut gliomas.

Methylation classification is an essential component for integrative diagnosis in glioma, however, the DNA methylation classification is not always available for all the samples. We hypothesized that Raman spectroscopy might be suitable to predict the glioma methylome, based upon its ability to create a molecular fingerprint of the tumor and would provide biological insights into the discriminatory features. Raman Spectroscopy was used for molecular fingerprinting of the regions of interest using 1mm2 FFPE tissue spots from 45 patient samples with LGm1 to LGm6 methylation subtypes. Spectral information was then used to train a convolutional neural network (CNN), capable of detecting the glioma methylation subtypes. 70 % of the dataset was used for model training while the remaining 30% for validation. We demonstrate that Raman spectroscopy can accurately and rapidly classify gliomas according to their methylation subtype from achieved FFPE samples, as a novel way to obtain classification. For each sample we ran Ward linkage clustering with a variable number of clusters (from 2 to 7), with the majority cluster corresponding to tumor spots and the others corresponding to (various types of) non-tumor spots. The average accuracy over all samples was 90:3%, the average precision was 99:6% and the average recall was 90:2%. We show that Raman spectroscopy together with artificial intelligence can predict the methylome of glioma samples and augment the ability to classify these tumors retrospectively. The non-destructive nature of this method and the ability to be applied on FFPE samples directly, allows the histopathologist to reuse of the same slide for subsequent staining and downstream analyses.

Glioblastoma (GBM) is an aggressive brain malignancy with a dismal prognosis. With emerging evidence to disprove brain-immune privilege, there has been much interest in examining immunotherapy strategies to treat central nervous system (CNS) cancers. Unfortunately, the limited success of clinical studies investigating immunotherapy regimens, has led to questions about the suitability of immunotherapy for these cancers. Inadequate inherent populations of tumor infiltrating lymphocytes (TILs) and limited trafficking of systemic, circulating T cells into the CNS likely contribute to the poor response to immunotherapy. This paucity of TILs is in concert with the finding of epigenetic silencing of genes that promote immune cell movement (chemotaxis) to the tumor. In this study we evaluated the ability of GSK126, a blood-brain barrier (BBB) permeable small molecule inhibitor of EZH2, to reverse GBM immune evasion by epigenetic suppression of T cell chemotaxis. We also evaluated the in vivo efficacy of this drug in combination with anti-PD-1 treatment on tumor growth, survival and T cell infiltration in syngeneic mouse models. GSK126 reversed H3K27me3 in murine and human GBM cell lines. When combined with anti-PD-1 treatment, a significant increase in activated T cell infiltration into the tumor was observed. This resulted in decreased tumor growth and enhanced survival both in sub-cutaneous and intracranial tumors of immunocompetent, syngeneic murine models of GBM. Additionally, a significant increase in CXCR3+ T cells was also seen in the draining lymph nodes, suggesting their readiness to migrate to the tumor. Closer examination of the mechanism of action of GSK126 revealed its ability to promote the expression of IFN-γ driven chemokines CXCL9 and CXCL10 from the tumor cells, that work to traffic T cells without directly affecting T maturation and/or proliferation. The loss of survival benefit either with single agent or combination in immunocompromised SCID mice, suggest that the therapeutic efficacy of GSK126 in GBM is primarily driven by lymphocytes. Taken together, our data suggests that in glioblastoma, epigenetic modulation using GSK126 could improve current immunotherapy strategies by reversing the epigenetic changes that enable immune cell evasion leading to enhanced immune cell trafficking to the tumor.

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&rsquo, 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.

Background Standard therapy for chordoma consists of surgical resection followed by high-dose irradiation. Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase involved in signal transduction, cell cycle progression, cell differentiation, and DNA repair. LB100 is a small-molecule inhibitor of PP2A designed to sensitize cancer cells to DNA damage from irradiation and chemotherapy. A recently completed phase I trial of LB100 in solid tumors demonstrated its safety. Here, we show the therapeutic potential of LB100 in chordoma. Methods Three patient-derived chordoma cell lines were used: U-CH1, JHC7, and UM-Chor1. Cell proliferation was determined with LB100 alone and in combination with irradiation. Cell cycle progression was assessed by flow cytometry. Quantitative γ-H2AX immunofluorescence and immunoblot evaluated the effect of LB100 on radiation-induced DNA damage. Ultrastructural evidence for nuclear damage was investigated using Raman imaging and transmission electron microscopy. A xenograft model was established to determine potential clinical utility of adding LB100 to irradiation. Results PP2A inhibition in concert with irradiation demonstrated in vitro growth inhibition. The combination of LB100 and radiation also induced accumulation at the G2/M phase of the cell cycle, the stage most sensitive to radiation-induced damage. LB100 enhanced radiation-induced DNA double-strand breaks. Animals implanted with chordoma cells and treated with the combination of LB100 and radiation demonstrated tumor growth delay. Conclusions Combining LB100 and radiation enhanced DNA damage-induced cell death and delayed tumor growth in an animal model of chordoma. PP2A inhibition by LB100 treatment may improve the effectiveness of radiation therapy for chordoma.

BACKGROUND Little is known about the mechanisms that render cancer cells dependent on certain nutrients from the microenvironment. Cysteine is a non-essential amino acid, since it can be synthetized from methionine through the transsulfuration pathway; moreover, cysteine is also uptake from the diet as cystine. We have investigated the metabolism of cysteine in glioma cell lines, and how cysteine/cystine-deprivation alters their antioxidant response in addition to the effect of this nutrient restriction to viability and proliferation in vitro and in vivo. METHODS Cysteine metabolism was investigated through LCMS-based 13C-tracing experiments and the expression levels of key enzymes in the transsulfuration pathway were also explored. Finally, a mouse model of IDH1 mutant glioma was subjected to a cysteine/cystine-free diet and tumor metabolism was analyzed by LCMS. RESULTS Herein, we report the dependence of glioma cells on exogenous cysteine/cystine, despite this amino acid being nonessential. Using several 13C-tracers and analysis of cystathionine synthase and cystathioninase levels, we revealed that glioma cells were not able to upregulate the transulfuration pathway cysteine, which allows methionine to be converted to cysteine in cysteine/cystine deprived conditions. We demonstrated that exogenous cysteine/cystine are crucial for glutathione synthesis, and impact growth and viability. Therefore, we explored the nutritional deprivation in a mouse model of glioma. Animals subjected to a cysteine/cystine-free diet survived longer, with concomitant reductions in glutathione and cysteine plasma levels. At the endpoint higher levels of oxidative stress were detected despite the systemic recovery of cysteine-related metabolites in the plasma. CONCLUSION The results presented herein reveal an alternative therapeutic approach combining cysteine/cysteine-deprivation diets and treatments involving ROS production by limiting the ability of glioma cells to quench oxidative stress through dietary interventions. Our study highlights a time window where cysteine deprivation can be exploited for additional therapeutic strategies.

Uncontrolled seizures in patients with low grade gliomas have a significant impact on quality of life and morbidity, yet the mechanisms through which these tumors cause seizures remain unknown. Albeit there are multiple features that contribute to tumor related epileptogenesis, IDH mutations are determined to be an independent factor, although the pathogenesis remains poorly understood. Here, we hypothesize that the active metabolite D-2-hydroxyglutarate (D-2-HG) produced by the IDH-mutant enzyme leads to metabolic disruptions in surrounding cortical neurons that consequently promote seizures. We use a complementary study of in vitro cortical cultures and electrographically sorted human cortical tissue from patients (n=5) with IDH-mutant gliomas to test this hypothesis. We demonstrate that D-2-HG leads to increased neuronal spiking activity (p< 0.0001) and promotes a distinct metabolic profile in surrounding neurons and upregulation of mTOR signaling (p< 0.0001), which is consistent in human epileptic cortex compared to peritumoral nonepileptic cortex. Furthermore, increases in neuronal activity are induced by mTOR activation and reversed with mTOR inhibition. Together, our data suggest that metabolic disruptions and mTOR signaling upregulation in the surrounding cortex due to D-2-HG may be a driving event for epileptogenesis in patients with IDH-mutant low grade gliomas.

BACKGROUND Our study aimed to identify vulnerabilities within sphingolipid metabolism with potential to translate to therapeutics. While the vital role of sphingolipids in maintaining rheostat balance and as secondary messengers for signaling pathways (involving proliferation, invasion, migration, and angiogenesis) has been well-documented, their role has not been widely investigated in gliomas. Therefore, metabolic analysis of sphingolipid pathway for IDH1-R132H (IDH1 mut ) glioma cell lines was conducted in order to elucidate susceptible targets. METHODS Global sphingolipid quantification utilized high-throughput LCMS analysis. Pathway protein expression was measured via Western blots in vitro and derived from patients using The Cancer Genome Atlas analysis. RESULTS We probed the impact of decreasing D-2HG on the sphingolipid metabolism after treating a panel of IDH1 mut glioma cells with IDH1-R132H mut inhibitor, AGI5198. This revealed significant downregulation of N,N-dimethylsphingosine (NDMS), C17-sphingosine, and C18-sphinganine. Coincidentally, sphingosine-1-phosphate (S1P) was significantly upregulated in these gliomas. We conducted rational drug screen which revealed that inhibition of SPHK1 with N,N-dimethylsphingosine in combination with C17-sphingosine triggered biostatic dose-response across IDH1 mut gliomas and low impact on IDH WT glioblastoma (GBM) cells. Western analysis revealed that the IDH1 mut gliomas and IDH WT GBM expressed sphingosine kinase-1 (SPHK1). Data also unveiled a discovery that SPHK2 was highly expressed in the GBM cells while remarkably absent in the glioma cells. CONCLUSION Herein, we provide evidence that certain IDH1 mut gliomas present epigenetic silencing of SPHK2 which creates dependency on SPHK1 for S1P; thus, increasing sensitivity to targeting sphingolipid metabolism, and creating susceptibility to proliferation arrest and subsequent cellular death. S1P production has been reported to be elevated particularly for malignant glioblastomas in prior studies; whereas our research revealed that it is relatively low in IDH mut by comparison with IDH WT tumor cells. These findings suggest targeting the sphingolipid metabolism may present a promising strategy to improve survival for patients diagnosed with IDH1 mut gliomas.

BACKGROUND Increased de novo lipogenesis is a hallmark of cancer metabolism. In this study, we interrogated the role of de novo lipogenesis in IDH1 mutated glioma’s growth and identified the key enzyme, Stearoyl-CoA desaturase 1 (SCD1) that provides this growth advantage. MATERIALS ANDMETHODS We prepared genetically engineered glioma cell lines (U251 wild-type: U251WT and U251 IDHR132H mutant: U251RH) and normal human astrocytes (empty vector induced-NHA: NHAEV and IDHR132H mutant: NHARH). Lipid metabolic analysis was conducted by using LC-MS and Raman imaging microscopy. SCD1 expression was investigated by The Cancer Genome Atlas (TCGA) data analysis and Western-blotting method. Knock-out of SCD1 was conducted by using CRISPR/Cas9 and shRNA. RESULTS Previously, we showed that IDH1 mut glioma cells have increased monounsaturated fatty acids (MUFAs). TCGA data revealed IDH mut glioma shows significantly higher SCD1 mRNA expression than wild-type glioma. Our model systems of IDH1 mut (U251RH, NHARH) showed increased expression of this enzyme compared with their wild-type counterpart. Moreover, addition of D-2HG to U251WT increased SCD1 expression. Herein, we showed that inhibition of SCD1 with CAY10566 decreased relative cell number and sphere forming capacity in a dose-dependent manner. Furthermore, addition of MUFAs were able to rescue the SCD1 inhibitor induced-cell death and sphere forming capacity. Knock out of SCD1 revealed decreased cell proliferation and sphere forming ability. Decreasing lipid content from the media did not alter the growth of these cells, suggesting that glioma cells rely on de novo lipid synthesis rather than scavenging them from the microenvironment. CONCLUSION Overexpression of IDH mutant gene altered lipid composition in U251 cells to enrich MUFA levels and we confirmed that D-2HG caused SCD1 upregulation in U251WT. We demonstrated the glioma cell growth requires SCD1 expression and the results of the present study may provide novel insights into the role of SCD1 in IDH mut gliomas growth.

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.

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 &alpha, 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.

INTRODUCTION: Imipridones selectively target G protein-coupled receptors (GPCRs) that control critical signaling pathways in various cancer cells. Aberrant overexpression of GPCRs has been implicated in tumorigenesis. ONC201, a first generation imipridone that directly antagonizes dopamine receptor D2 (DRD2), continues to be evaluated in clinical trials for advanced cancers. The immediate downstream mechanism(s) of DRD2 inhibition and resulting anti-cancer activity remains an area of active study. METHODS & RESULTS: ONC206, an analog of ONC201, shares the same imipridone core chemical structure and selective antagonism of DRD2, potently inhibits treatment-resistant glioblastoma caused by intra-tumoral heterogeneity with clinically achievable concentrations. In silico analysis of a glioma patient database revealed that alteration of DRD2 mRNA expression was directly connected to global gene expression change. Imaging DRD2 expression by immunofluorescence demonstrated heterogeneous expression of DRD2 in the glioblastoma cells. After DRD2 inhibition, global metabolite profiling in patient-derived glioblastoma stem cells (GSCs) compared with differentiated glioblastoma cells (DGCs) demonstrated globally differential effects in their cellular signaling pathways. Cell viability assay showed that exposure to ONC206 in a dose dependent manner preferentially eliminated GSCs with 50 to 200 nM of IC50 ranges, whereas the IC50 of DGCs ranged from 200 to 1000 nM. In vitro limiting dilution and sphere formation assay showed that ONC206 prevented tumor sphere formation and tumor growth. ONC206 down-regulated protein expression of cancer-related stem cell markers in the GSCs; silencing DRD2 expression confirmed the dependency of DRD2 expression on cancer stem cell niches in glioblastoma. CONCLUSIONS: ONC206 treatment displays the differential effects on glioblastoma cells, more selectively targeting DRD2 in GSCs (at nanomolar concentrations) compared with DGCs in culture and in xenograft models. This suggests that a therapeutic strategy targeting DRD2-expressing GSCs within glioblastoma may be beneficial for overcoming the therapeutic resistance.

Purpose: Glioblastoma (GBM) is highly resistant to treatment, largely due to disease heterogeneity and resistance mechanisms. We sought to investigate a promising drug that can inhibit multiple aspects of cancer cell survival mechanisms and become an effective therapeutic for GBM patients. Experimental Design: To investigate TG02, an agent with known penetration of the blood–brain barrier, we examined the effects as single agent and in combination with temozolomide, a commonly used chemotherapy in GBM. We used human GBM cells and a syngeneic mouse orthotopic GBM model, evaluating survival and the pharmacodynamics of TG02. Mechanistic studies included TG02-induced transcriptional regulation, apoptosis, and RNA sequencing in treated GBM cells as well as the investigation of mitochondrial and glycolytic function assays. Results: We demonstrated that TG02 inhibited cell proliferation, induced cell death, and synergized with temozolomide in GBM cells with different genetic background but not in astrocytes. TG02-induced cytotoxicity was blocked by the overexpression of phosphorylated CDK9, suggesting a CDK9-dependent cell killing. TG02 suppressed transcriptional progression of antiapoptotic proteins and induced apoptosis in GBM cells. We further demonstrated that TG02 caused mitochondrial dysfunction and glycolytic suppression and ultimately ATP depletion in GBM. A prolonged survival was observed in GBM mice receiving combined treatment of TG02 and temozolomide. The TG02-induced decrease of CDK9 phosphorylation was confirmed in the brain tumor tissue. Conclusions: TG02 inhibits multiple survival mechanisms and synergistically decreases energy production with temozolomide, representing a promising therapeutic strategy in GBM, currently under investigation in an ongoing clinical trial. Clin Cancer Res; 24(5); 1124–37. ©2017 AACR.

BACKGROUND: Identification of malignant progression is a major challenge in the field of Neuro-Oncology because of inability of traditional imaging methods to distinguish pseudo-progression from true progression. IDH1-mutant gliomas represent an ideal model system to study the molecular mechanisms associated with malignant progression because they appear initially indolent and non-glycolytic, but eventually a subset progress towards secondary glioblastoma with a Warburg-like phenotype. The mechanisms and molecular features associated with this transformation are poorly understood. METHODS: TS603, NCH1681 and BT142 cell lines harboring IDH1 mutation were cultured in DMEM/F12+ N2 supplement, glutamine, FGF and EGF, then either harvested for metabolomics and methylation analyses or (250,000 cells) were injected into 6-week-old SCID mice brains. MRI was used to monitor tumor size; when tumors reached 100 mm(3), mice were injected in the tail vain with 96 mM 1-(13)C pyruvate which was hyperpolarized using Oxford Hypersense hyperpolarizer and chemical shift images were acquired immediately. Metabolite quantification was done using the Agilent LC/MS 6545 QTOF mass spectrometer. DNA methylation analysis was performed using Ilumina Infinium MethylationEPIC. RESULTS AND CONCLUSIONS: Cell lines that demonstrate aggressive growth both in vitro and in vivo have lost methylation in the promoters of glycolytic enzymes and have increased mRNA and metabolite levels for the glycolysis pathway compared to the non-transformed model. Metabolic (13)C MRI using hyperpolarized(13)C pyruvate confirmed that these aggressive lines have a Warburg-like phenotype (aerobic glycolysis). Moreover, the glycolytic enzyme expression of the aggressive cell line correlated with the subset of patients that are IDH1 mutated and low-G-CIMP in TCGA database. We hypothesize that specific modulation of epigenetic markers is a mechanism of malignant transformation and that monitoring lactate production may be a biomarker of transformation.

BACKGROUND Gliomas with isocitrate dehydrogenase (IDH) mutations in adults evolve from lower-grade gliomas to secondary glioblastomas (GBM), a fatal disease with fast progression. IDH mutation occurs early in tumorigenesis, and persistently contribute to the reprograming of glucose, lipid and amino acid metabolism. This offer a plethora of potential biomarkers of progression. However, because it is extremely difficult to detect the distribution and transfer of metabolites changing in every moment in a single cell, the involvement of metabolites produced by mutant IDH in malignant progression remains understudied. MATERIALS AND METHODS Raman imaging spectroscopy, which can image chemical bonds and concentration of molecules at submicron spatial resolution, enables detection of spatiotemporal changes of metabolomes in live cells. We developed the software called Biomolecular Component Analysis (BCAbox) to deconvolute the recorded raw Raman spectra, leading to detection of unique spectral features of different classes of biomolecules. RESULTS AND CONCLUSIONS We applied Raman imaging spectroscopy to GBM cell lines that were transfected with IDH1 mutant gene. Our results indicated that lipid metabolism has a unique profile in IDH1 mutant gliomas. Subsequent mass spectrometry analysis of extracted organelle revealed the exact classes of lipids altered in the IDH mutant glioma and suggested biomarkers unique to IDH1 mutant. We will report our validation studies of the biomarkers in patient-derived IDH mutant glioma cell lines and patients derived-orthotopic xenograft mouse models with different degrees of aggressiveness and in matched primary versus recurrent gliomas. The results of the present study may provide novel insights into the discovery of metabolic biomarkers for the malignant progression in IDH mutant gliomas.

BACKGROUND: Resistance to therapeutic use of retinoids in glioblastoma (GBM) has been observed for over 20 years; however, the exact mechanism of resistance remains unknown. To better understand retinoic acid resistance in GBM, we studied the turnover mechanism of retinoid receptor proteins in normal neural stem cells and glioma stem-like cells (GSCs). Currently, it is believed that cellular stress induces global sumoylation of proteins in glioma, yet the precise role of sumoylation is not fully understood. METHODS: Protein expression and posttranslational modification (PTM) of the endogenous retinoid receptors were analyzed using Western blots, immunoprecipitations, and siRNA. The discovery of the novel binding partner of retinoid receptors was achieved using immunoprecipitation and mass spectrometry. Promoter luciferase assays were used to measure transcriptional activities. RESULTS AND CONCLUSIONS: Our studies reveal that sumoylation of retinoid receptors occurs in both normal neural stem cells and GSCs; however, protein turnover of the receptor is disrupted in glioma. We show that sumoylation is a PTM required for proteasomal degradation of retinoid receptors. Degradation via the proteasomal pathway is necessary for receptor protein turnover and transcriptional activity. We also identify that the valosin-containing protein (VCP/p97/Cdc48) participates in the PTM of retinoid receptors and impacts the transcriptional activity. The defect in glioma occurs after the sumo modification step and results in the accumulation of high molecular weight forms of the receptors that fail to get degraded. Our findings expand our understanding of the turnover mechanism of nuclear receptors in normal cells. In addition, our findings provide a mechanism for the retinoic acid resistance in glioma cells that involves the disruption of protein turnover and decrease in transcriptional activity. Our studies suggest that the use of combinatory therapies that target retinoid receptors and induce proteasomal degradation of the receptors to ensure protein turnover may be a more effective approach.

Background: AGI-5198 (a specific IDH1mut inhibitor) inhibits the formation of D-2-hydroxyglutarate (D-2HG) typically produced in millimolar quantities by the R132H mutation of isocitrate dehydrogenase I (IDH1). AGI-5198 was found to delay growth, to promote differentiation in cells and to decrease tumor volume in IDH1-mutant (IDH1mut) mice xenographs, and as a result, has been advanced to clinical trials in humans with IDH1mut glioblastomas. However, no metabolic study of this drug has been reported to date. Here, we investigated the metabolic changes induced by AGI-5198 treatment in order to determine if the drug restores the normal metabolic status once D-2HG concentration is decreased. Methods: We treated glioma cells carrying an IDH1 mutation with 10 μM AGI-5198 for 72 h. Metabolic changes were explored using an untargeted metabolomics approach. Cell viability analysis verified that the concentration of drug does not lead to significant cell death. NMR and MS-linked metabolomics together with in vivo cell imaging using RAMAN were used. Principal component analysis was employed to explore the differences in the metabolic profiles comparing treated and untreated glioma cells. Results and Conclusions: We confirmed that AGI5198 treatment reduced D-2HG levels in the cell lines investigated, validating previous reports. Moreover, we observed a decrease in phospholipid levels upon AGI-5198 treatment, which suggests a decreased proliferative potential for these cells; of particular importance for clinical application. Our preliminary analysis also suggests that metabolites involved in one carbon metabolism are altered upon AGI-5198 treatment. This pathway provides the methyl group for the DNA methylation by DNA methylases producing a hypermethylated phenotype. Our findings suggest that combining the AGI-5198 treatment with a DNA methylase inhibitor may improve outcomes in IDH1-mutated gliomas. Citation Format: Victor Ruiz Rodado, Adrian Lita, Tyrone Dowdy, Mark R. Gilbert, Mioara Larion. Profiling the metabolic effects of AGI-5198 treatment on IDH1-mutated gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2508. doi:10.1158/1538-7445.AM2017-2508