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Diffuse midline gliomas (DMGs) bearing driver mutations of histone 3 lysine 27 (H3K27M) are incurable brain tumors with unique epigenomes. Here, we generated a syngeneic H3K27M mouse model to study the amino acid metabolic dependencies of these tumors. H3K27M mutant cells were highly dependent on methionine. Interrogating the methionine cycle dependency through a short-interfering RNA screen identified the enzyme methionine adenosyltransferase 2A (MAT2A) as a critical vulnerability in these tumors. This vulnerability was not mediated through the canonical mechanism of MTAP deletion; instead, DMG cells have lower levels of MAT2A protein, which is mediated by negative feedback induced by the metabolite decarboxylated S-adenosyl methionine. Depletion of residual MAT2A induces global depletion of H3K36me3, a chromatin mark of transcriptional elongation perturbing oncogenic and developmental transcriptional programs. Moreover, methionine-restricted diets extended survival in multiple models of DMG in vivo. Collectively, our results suggest that MAT2A presents an exploitable therapeutic vulnerability in H3K27M gliomas.

First-line cancer therapies such as alkylating agents and radiation have limited survival benefits for Glioblastoma (GBM) patients. Current research strongly supports the notion that inhibition of aberrant tumor metabolism holds promise as a therapeutic strategy when used in combination with radiation and chemotherapy. Hexokinase 2 (HK2) has been shown to be a key driver of altered metabolism in GBM, and presents an attractive therapeutic target. To date, no study has fully assessed the therapeutic value of targeting HK2 as a mechanism to sensitize cells to standard therapy, namely in the form of radiation and temozolomide (TMZ). Using cell lines and primary cultures of GBM, we showed that inducible knockdown of HK2 altered tumor metabolism, which could not be recapitulated by HK1 or HK3 loss. HK2 loss diminished both in vivo tumor vasculature as well as growth within orthotopic intracranial xenograft models of GBMs, and the survival benefit was additive with radiation and TMZ. Radio-sensitization following inhibition of HK2 was mediated by increased DNA damage, and could be rescued through constitutive activation of ERK signaling. This study supports HK2 as a potentially effective therapeutic target in GBM.

Purpose: Hexokinase II (HK2) protein expression is elevated in glioblastoma (GBM), and we have shown that HK2 could serve as an effective therapeutic target for GBM. Here, we interrogated compounds that target HK2 effectively and restrict tumor growth in cell lines, patient-derived glioma stem cells (GSCs), and mouse models of GBM. Experimental Design: We performed a screen using a set of 15 drugs that were predicted to inhibit the HK2-associated gene signature. We next determined the EC50 of the compounds by treating glioma cell lines and GSCs. Selected compounds showing significant impact in vitro were used to treat mice and examine their effect on survival and tumor characteristics. The effect of compounds on the metabolic activity in glioma cells was also assessed in vitro. Results: This screen identified the azole class of antifungals as inhibitors of tumor metabolism. Among the compounds tested, ketoconazole and posaconazole displayed the greatest inhibitory effect on GBM both in vitro and in vivo. Treatment of mice bearing GBM with ketoconazole and posaconazole increased their survival, reduced tumor cell proliferation, and decreased tumor metabolism. In addition, treatment with azoles resulted in increased proportion of apoptotic cells. Conclusions: Overall, we provide evidence that azoles exert their effect by targeting genes and pathways regulated by HK2. These findings shed light on the action of azoles in GBM. Combined with existing literature and preclinical results, these data support the value of repurposing azoles in GBM clinical trials.

Gliomas are a heterogeneous group of tumors that show variable proliferative potential, invasiveness, aggressiveness, histological grading, and clinical behavior. In this review, we focus on glioblastoma multiforme (GBM), a grade IV glioma, which is the most common and malignant of primary adult brain tumors. Research over the past several decades has revealed the existence of extensive cellular, molecular, genetic, epigenetic, and metabolic heterogeneity among tumors of the same grade and even within individual tumors. Evaluation of different tumor types has shown that tumors with advanced grade and clinical aggressiveness also display enhanced molecular, cellular, and microenvironmental heterogeneity. From a therapeutic standpoint, this heterogeneity is a major clinical hurdle for devising effective therapeutic strategies for patients and challenges personalized medicine. In this review, we will highlight key aspects of GBM heterogeneity, directing special attention to regional heterogeneity, hypoxia, genomic heterogeneity, tumor-specific metabolic reprogramming, neovascularization or angiogenesis, and stromal immune cells. We will further discuss the clinical implications of GBM heterogeneity in the context of therapy.

Glioblastomas (GBM) are one of the most malignant adult primary brain tumors. Through decades of research using various model systems and GBM patients, we have gained considerable insights into the mechanisms regulating GBM pathogenesis, but have mostly failed to significantly improve clinical outcome. For the most part GBM heterogeneity is responsible for this lack of progress. Here, we have discussed sources of cellular and microenvironmental heterogeneity in GBMs and their potential regulation through microRNA mediated mechanisms. We have focused on the role of individual microRNAs (miRNA) through their specific targets and miRNA mediated RNA-RNA interaction networks with the potential to influence various aspects of GBM heterogeneity including tumor neo-vascularization. We believe a better understanding of such mechanisms for regulation of GBM pathogenesis will be instrumental for future therapeutic options.

BACKGROUND: Hexokinase I and 2 (HK1/HK2) catalyze the first committed step in glucose metabolism, ensuring a sustained glucose concentration gradient. Glioblastomas (GBMs) over-express HK2, and we have previously shown that its loss sensitizes GBM cells to treatment. In this study, we have conducted a systematic small-drug screen to identify potential HK2 inhibitors. METHODS: Pathway analysis was conducted using Gene Set Enrichment Analysis on differentially expressed genes in control and HK2 siRNA-treated samples. The top 200 up- and down-regulated genes were used to query the Connectivity Map database for potential inhibitors. 15 candidate drugs were identified and their EC(50) was determined in glioma cell lines, glioma stem cells (GSCs), and normal human astrocytes. Dynamic metabolic flux analysis with (13)C-glucose labeling followed by liquid chromatography-mass spectrometry (LC-MS) was used to assess effect of candidate drugs on tumor cell glycolytic intermediates. Xenograft mice bearing glioma stem cells or U87 cells were treated with vehicle, ketoconazole, or posaconazle (25mg/kg). Mice were sacrificed when moribund and immunohistochemistry was used to assess proliferation (Ki67) and apoptosis (TUNEL assay). Mouse brain tissue drug concentration was determined using HPLC-MS/MS. RESULTS: HK2 knockdown affected glycolysis and angiogenesis. The EC(50) of ketoconazole and posaconazole was within clinically achievable doses and below the concentration needed to affect normal human astrocytes or stem cells (

INTRODUCTION: GBMs are characterized by elevated glycolysis, however to date effective therapies targeting tumor glycolysis have not been identified. Also, the contribution of metabolic pathways other than glycolysis, such as glutamine and fatty acid oxidation, that are key in providing carbon to TCA cycle, have not been explored in GBMs. Aim: In this study we aim to decipher the adaptive mechanisms brought about by the inhibition of tumor glycolysis in GBMs. We have investigated the metabolic changes occurring in GBM cells after the knockdown of key glycolytic enzyme, hexokinase2 (HK2). METHODS AND RESULTS: We have generated doxycyclin-inducible HK2 knockdown GBM cells by transfecting shRNA in U87 and three Glioma Stem Cell lines with different baseline molecular characteristics We confirm specific knockdown of HK2, with no change in HK1 or HK3, using western blot. HK2 knockdown reduced cell number and proliferative capacity, in addition to decrease in lactate production and O2 consumption. HK2 loss significantly increased NADP:NADPH ratio. Metabolic profiling by LC/MS targeted metabolomics was conducted after exposing GBM cells to C13-labeled glucose media. LC/MS metabolomics demonstrate that decrease in HK2 significantly reduced the levels of metabolites belonging to glycolysis and Pentose Phosphate Pathway. These two pathways are known to be associated with NADPH and biomass production for rapidly proliferating tumor cells. In contrast, metabolites in TCA cycle increased in response to HK2 inhibition. CONCLUSION: Our data strongly suggests that GBM cells activate alternate metabolic pathways in response to inhibition of HK2 dependent glycolysis. These results have important clinical implications in designing combinatorial targeted therapy for blocking tumor metabolism in GBMs. Our ongoing work is focused on establishing the metabolites involved in glutamine catabolism and fatty acid oxidation, both in-vitro and in-vivo GBM models.

METB-08. INHIBITION OF HEXOKINASE 2 USING TUMOR GLYCOLYSIS INHIBITORS IDENTIFIED THROUGH A DRUG SCREEN INHIBITS GLIOBLASTOMA GROWTH IN VITRO AND IN VIVO Gelareh Zadeh1,2, Kenneth Aldape1, Mira Li1, Sameer Agnihotri1, Kelly Burrell1, Alenoush Vartanian1, Amir Alamsahebpour1, and Shahrzad Jalali1; MacFeeters Hamilton Center for Neuro-Oncology, Toronto, ON, Canada; University Health Network, Toronto, ON, Canada Current research in cancer has demonstrated that normal and cancer cells use glucose differently. Normal cells will use glycolysis and oxidative phosphorylation to generate ATP, whereas proliferating tumor cells have additional demands including biomass generationand harbor glycolysis rates up to 200 times higher than normal cells. To generate biomass, proliferating tumor cells preferentially use the Warburg effect or aerobic glycolysis and in the process consume less oxygen and produce large amounts of lactate. Our ongoing work has demonstrated that hexokinase 2 (HK2) but not HK1 or HK3 is a critical mediatorof the Warburg effect in glioblastomas (GB). Currently, no direct inhibitor of HK2 exists. By using a systems biology approach and a rationale drug screen we identified several azole antifungal agents as inhibitors of tumor metabolism that reduce proliferation, lactate production, glucose uptake in GB cells but not in primary normal human astrocytes or normal neural stem cells. Dynamic metabolic flux analysis with 13C-labeling experiments followed by liquid chromatography-mass spectrometry (LC-MS) demonstrated that GB cell lines and GB glioma stem cell cultures treated with several azoles reduced some pro-anabolic glycolytic intermediates. Loss of HK2inGBcells dampened theeffectof several azoles suggesting that themechanism of action is mediated in part through HK2. Furthermore, we tested several azole compounds known to cross the blood brain barrier in vivo. Clinically achievable doses of azoles as single agents increased survival in several orthotopic xenograft GB mouse models. Current studies are underway to determine the oncogenic molecular pathways inhibited by these drug compounds and whether azole treatment with the current standard of care (temozolomide and radiation) has a synergistic effect in vivo. In summary, the azole class of antifungals may represent a new way of targeting tumor metabolism in tumors dependent on aerobic glycolysis. Neuro-Oncology 17:v135–v137, 2015. doi:10.1093/neuonc/nov221.8 Published by Oxford University Press on behalf of the Society for Neuro-Oncology 2015.

The role of cancer stem cells in tumor formation and tumor heterogeneity is currently one of the most researched topics in cancer biology, and microRNAs likely have functional relevance in regulation of critical genes and parameters implicated in glioma stem cell (GSC) behavior and differentiation. To address this, we investigated global role of microRNA in GSCs, focusing on DICER, which regulates double-stranded RNA processing for microRNA biogenesis. Analysis of data from the Cancer Genome Atlas (TCGA) database suggests that high Dicer expression level is correlated with better prognosis of GBM patients. Gene signatures correlated with DICER expression levels suggest DICER/miRNA mediated mechanisms potentially regulate a multitude of cellular pathways in GBMs. Immunohistochemistry analysis of GBM tissue microarray reveals that of 54 tumor samples, 26 (48%) of GBM patients have low or undetectable levels of DICER1 protein, indicating frequent inactivation in high grade glioma. To characterize this functionally, we utilized various in vitro approaches, including exposure to hypoxia, to characterize the GSC properties after knockdown of DICER (GSCs did not have significant variation in endogenous DICER levels). Using three different GSC lines, we found in all cases with Dicer knockdown resulted in increased proliferation of three independent GSC lines with concomitant decrease in levels of stem cell markers (Sox2, Bmi1 etc.), a phenotype observed even upon exposure to hypoxia, a state that is known to preserve and promote the stem-like cell phenotype. Results from self-renewal assays as well as expression profiling show that GSCs with depleted levels of Dicer lose stem cell characteristics and acquire a progenitor-cell like state. Our results highlight the role of DICER as potential regulators of GSC stem-like versus progenitor-like state.

Rapidly proliferating tumour cells preferentially use aerobic glycolysis over oxidative phosphorylation (OXPHOS) to support growth and survive unfavorable microenvironment conditions. This metabolic reprogramming is referred to as the “Warburg effect” and offers a novel way to target cancer cells including glioblastoma (GBM), the most common malignant brain tumor. Here we demonstrate that Hexokinase 2 (HK2) but not HK1 or HK3 is a critical mediator of metabolic reprograming in GBMs and its inhibition is a potential therapeutic strategy for sensitization of GBM tumors to radiation (RAD) and temozolomide (TMZ). In GBM xenografts, conditional HK2 loss sensitizes tumors to concomitant RAD/TMZ and results in a significant survival benefit. Loss of HK2 resulted in increased necrosis, hypoxia, inflammatory infiltration and reduced vascularization. Currently, no direct inhibitor of HK2 exists so we explored whether a system biology approach to identify gene networks associated with HK2 could lead to the identification of HK2 inhibitors. Using HK2 knockdown in established GBM cell lines and primary cultures we established gene signatures and networks associated with HK2 expression. Loss of HK2 led to attenuation of several pro GBM signaling pathways affecting tumour cell invasion, glucose metabolism and proliferation. Using a small drug screen targeting potential HK2 regulated gene expression networks we identified the azole class of antifungals as inhibitors of tumour metabolism by reducing proliferation, lactate production, glucose uptake in GBM cells but not primary normal human astrocytes or normal neural stem cells. Interestingly, several azole compounds were more potent at killing GBM cells in hypoxic conditions. Azoles in combination with further HK2 knockdown, RT or in combination with other metabolic therapeutics including pyruvate kinase M2 (PKM) activators led to synergistic tumour cell death. In summary, the azole class of antifungals may represent a new way of targeting tumour metabolism in tumours dependent on aerobic glycolysis.