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DDRE-25. INVESTIGATING MITOCHONDRIAL SLC25A TRANSPORTERS INVOLVED IN SUPPORTING BRAIN TUMOUR METABOLISM AND SURVIVAL UNDER HYPOXIC CONDITIONS

Abstract Advancements in prevention, detection and treatment over the last 40 years have significantly transformed cancer healthcare however there are a few cancers, such as brain tumours, which are consistently lagging behind. The most common adult brain tumour is glioma; a highly aggressive cancer that invades deep into the surrounding brain consequently making treatment challenging. The severe hypoxic nature of glioma adds further complications to therapeutic efficacy as hypoxia limits efficient drug delivery as well as increasing treatment resistance. Therapies that therefore target both the hypoxic tumour microenvironment and metabolic pathways that sustain growth have significant potential to improve patient prognosis. It is well known that cancer cells demonstrate an abnormal metabolism, resulting in an altered requirement for amino acids to aid uncontrolled proliferation. Furthermore, tumour metabolism can also be influenced by this hostile hypoxic microenvironment, leading to a more malignant phenotype. We are therefore interested in a family of mitochondrial transporters, SLC25A, which translocate numerous solutes across the mitochondrial membrane and are crucial for many metabolic reactions. TCGA analysis has shown that many of these amino acid carriers are upregulated in glioma. Remarkably however, around 23 of the 53 mammalian SLC25A members lack defined substrate selectivity and so we are interested in identifying which transporters are particularly important in the metabolic adaptation to hypoxia. Using CRISPR and siRNA technologies we have identified transporters that are functionally required to maintain cell proliferation of glioma cell lines and patient tumour cells. Furthermore, using stable isotope-enriched nutrients, we have identified novel means by which glioma cell metabolism can be perturbed by inhibition of these transporters. Characterising which SLC25A transporters are important for hypoxic tumour metabolism could therefore expose a way to exploit these hypoxic areas subsequently making them more vulnerable to treatment and thus impacting patient survival.

The brain environment is low in amino acids, including serine and glycine, both of which are important for tumor growth as they are precursors of proteins and nucleotide bases. How tumor cells overcome these conditions to proliferate and survive in the brain is incompletely understood. Here, we show that 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the first and rate-limiting step of glucose-derived serine synthesis, enables brain metastasis in multiple human types and in preclinical models. Genetic suppression and small molecule inhibition of PHGDH attenuated brain metastasis, but not extra cranial tumors, and improved the overall survival of mice bearing brain metastasis. These results demonstrate that the tumor nutrient microenvironment determines tumor cell sensitivity to loss of serine synthesis pathway activity and raise the possibility that serine synthesis inhibitors may be useful in the treatment of brain metastases.

DDRE-23. A COMPREHENSIVE CHARACTERIZATION OF THE GBM LIPIDOME REVEALS A MOLECULARLY-DEFINED SUB-GROUP WITH HEIGHTENED SENSITIVITY TO LIPID PEROXIDATION INDUCED CELL DEATH
Danielle Morrow, Jenna Minami, Nicholas Bayley, Kevin Williams, Steven Bensinger, Robert Prins, Linda Liau, Timothy Cloughesy, David Nathanson; University of California Los Angeles, Los Angeles, CA, USA Cancers, including the universally lethal glioblastoma (GBM), have reprogrammed lipid metabolism to fuel tumor growth and promote survival. However, the full extent to which lipid content is altered across molecularly heterogeneous patient tumors has yet to be fully elucidated. Additionally, the molecular alterations responsible for aberrant lipid metabolism, and the potential for identifying new therapeutic opportunities are not fully understood. To systematically investigate the GBM lipidome, we performed integrated transcriptomic, genomic and shotgun lipidomic analysis of an extensive library of molecularly diverse patient-derived GBM tumors across tumor microenvironments both in vivo (n=23) and in vitro (n=30). Using this comprehensive approach, we discovered two GBM sub-groups defined by their combined molecular and lipidomic profile. Triacylglycerides (TAGs) enriched in polyunsaturated fatty acids (PUFAs) were among the most significantly altered lipids between the two groups of GBM tumors. TAGs are the main components of lipid droplets, which have been shown to sequester PUFAs away from membrane phospholipids where their sensitivity to peroxidation leads to cell death. The GBM subgroup with a depletion of PUFA TAGs showed heightened sensitivity to lipid peroxidation both under basal conditions and in response to pro-oxidant compounds in vitro. Our findings suggest a novel association between specific molecular signatures of GBM, lipid metabolism and lipid peroxidation-induced cell death. This relationship may present a new therapeutic opportunity to target reprogrammed lipid metabolism in a molecularly-defined subset of GBMs.
Intratumoral genomic heterogeneity in glioblastoma (GBM) is a barrier to overcoming radiation (RT) resistance. To discover genotype-independent mediators of RT resistance, we correlated RT resistance with the concentration of approximately 700 metabolites across 23 GBM cell lines. Purine metabolites, especially those containing the base guanine, were most correlated with RT resistance. Similarly, increased abundance of tumor purines was associated with decreased survival in GBM patients treated with RT. This relationship is causal. Purine supplementation protected RT-sensitive GBMs from RT and promoted the repair of RT-induced double strand DNA breaks (DSBs). In vitro and in vivo stable isotope tracing confirmed that GBM cell lines and orthotopic patient-derived xenografts primarily generated purines through the de novo synthetic pathway. RT treatment further increased de novo purine synthesis in GBM through signaling via the DNA damage response. Inhibition of de novo GTP synthesis with mycophenolic acid (MPA) sensitized multiple GBM cell lines and neurospheres to RT by slowing the repair of RT-induced DSBs. MPA-induced radiosensitization was GTPdependent as it was rescued by nucleoside supplementation. Modulating pyrimidine metabolism affected neither RT resistance nor DSB repair, suggesting these GTP-specific effects are due to active signaling rather than its ability to act as a physical substrate for DNA repair and candidate signaling molecules have been identified. These results were recapitulated in vivo with mycophenolate mofetil (MMF), the orally bioavailable FDA-approved prodrug of MPA. MMF potentiated RT efficacy, reduced tumor guanylates and slowed the repair of RT-induced DSBs across multiple models. Because de novo purine synthesis is activated by many of the oncogenic alterations that drive GBM, its inhibition is a promising genotype-independent strategy to overcome GBM RT resistance. We have now begun a clinical trial to determine whether combining MMF and RT is safe and potentially efficacious in patients with GBM. Advancements in prevention, detection and treatment over the last 40 years have significantly transformed cancer healthcare however there are a few cancers, such as brain tumours, which are consistently lagging behind. The most common adult brain tumour is glioma; a highly aggressive cancer that invades deep into the surrounding brain consequently making treatment challenging. The severe hypoxic nature of glioma adds further complications to therapeutic efficacy as hypoxia limits efficient drug delivery as well as increasing treatment resistance. Therapies that therefore target both the hypoxic tumour microenvironment and metabolic pathways that sustain growth have significant potential to improve patient prognosis. It is well known that cancer cells demonstrate an abnormal metabolism, resulting in an altered requirement for amino acids to aid uncontrolled proliferation. Furthermore, tumour metabolism can also be influenced by this hostile hypoxic microenvironment, leading to a more malignant phenotype. We are therefore interested in a family of mitochondrial transporters, SLC25A, which translocate numerous solutes across the mitochondrial membrane and are crucial for many metabolic reactions. TCGA analysis has shown that many of these amino acid carriers are upregulated in glioma. Remarkably however, around 23 of the 53 mammalian SLC25A members lack defined substrate selectivity and so we are interested in identifying which transporters are particularly important in the metabolic adaptation to hypoxia. Using CRISPR and siRNA technologies we have identified transporters that are functionally required to maintain cell proliferation of glioma cell lines and patient tumour cells. Furthermore, using stable isotope-enriched nutrients, we have identified novel means by which glioma cell metabolism can be perturbed by inhibition of these transporters. Characterising which SLC25A transporters are important for hypoxic tumour metabolism could therefore expose a way to exploit these hypoxic areas subsequently making them more vulnerable to treatment and thus impacting patient survival.

DDRE-26. THE IMMUNO-METABOLIC ENZYME FASN PREVENTS CANCER-CELL INTRINSIC TYPE I INTERFERON RESPONSES IN GLIOBLASTOMA
Mara De Martino 1 , Camille Daviaud 1 , Claire Vanpouille-Box 1,2 ; 1 Department of Radiation Oncology, Weill Cornell Medicine, New York, NY, USA, 2 Sandra and Edward Meyer Cancer Center, New York, NY, USA Glioblastoma (GBM) is a devastating primary brain cancer with a median survival of 11-15 months. Radiation therapy (RT), the standard of care for GBM, can generate type I interferon responses (IFN-I) to jumpstart antitumor immunity. However, these effects are sometimes mitigated by inhibitory mechanisms that are exacerbated by RT. RT can modify GBM metabolism to promote de novo lipogenesis via the fatty acid synthase (FASN). Because FASN was found to impair IFN-I in antiviral immunity, we hypothesize that FASN is preventing RT-induced IFN-I responses to promote GBM survival and evade immune recognition. We first defined RT-induced metabolic changes in the GL261 murine GBM model. We observed an increase in mitochondrial respiration, glycolysis and in lipid metabolism-related pathways in 10 gray (Gy) irradiated GL261 cells. Additionally, we found upregulation of FASN by western blot and lipid accumulation by BODIPY staining in 10 Gy-GL261 cells. RT-induced lipid accumulation was reverted when GL261 cells were incubated with a FASN inhibitor. Next, to ask whether FASN was impairing IFN-I, GL261 cells were engineered to express an inducible shRNA silencing FASN (GL261shFASN) or its non-silencing control (GL261shNS). As expected, irradiation of GL261shNS cells enhanced the secretion of IFNbeta and CXCL10. This effect was more pronounced when FASN was abrogated in GL261 independently from the presence of RT. Finally, GL261shNS and GL261shFASN cells were orthotopically implanted in mice and IFN-I signaling was blocked by anti-IFN-I receptor antibody (a-IFNAR). Mice bearing GL261shFASN tumors presented a median survival of 51 days vs. 35 days for GL261shNS tumors, a significant prolongation of mice survival that was completely abrogated with a-IFNAR treatment. Overall, our findings suggest that FASN-mediated lipogenesis prevents RT-induced cancer cell intrinsic IFN-I to promote GBM survival. Consequently, it is possible that FASN acts as an immuno-metabolic checkpoint capable to regulate the immune system upon metabolic cues generated by RT.

DDRE-27. IDH MUTATED GLIOMAS PROMOTE EPILEPTOGENESIS VIA D-2-HYDROXYGLUTARATE DEPENDENT MTOR HYPERACTIVATION
Armin Mortazavi 1 , Islam Fayed 1,2 , Muzna Bachani 1 , Tyrone Dowdy 1 , Joseph Steiner 3 , Dragan Maric 1 , Chun Zhang Yang 1 , Mioara Larion 1 , Alexander Ksendzovsky 4 , Kareem Zaghloul 1 ; 1 National Institute of Health, Bethesda, MD, USA, 2 Medstar Georgetown University Hospital, Washington, DC, USA, 3 National Institute of Health, Bethesda, MD, USA, USA, 4 University of Maryland School of Medicine, Baltimore, MD, USA INTRODUCTION: Epileptic seizures in patients with low-grade, isocitrate dehydrogenase (IDH) mutated gliomas reach 90%, a major source of morbidity for these patients. 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. We demonstrate IDH-mutated tumors promote epileptogenesis through D-2hydroxyglutarate (D-2-HG) dependent mTOR hyperactivation and metabolic reprogramming. METHODS: Human epileptic and nonepileptic cortex were identified via subdural electrodes in patients with IDH-mutated gliomas (n=5). An in vitro rat cortical neuronal model on microelectrode arrays were utilized to investigate the role of D-2-HG on neuronal excitability. mTOR and lysine demethylase (KDM) modulators were applied to elucidate the epileptogenic mechanism. Tetrodotoxin was utilized to evaluate the contribution of neuronal activity to mTOR signaling and metabolism. mTOR signaling was evaluated through western blot analysis and multiplex immunofluorescence. Metabolic function were analyzed via Seahorse assays and metabolomic analysis. RESULTS: D-2-HG increased normalized bursting rate in the neuronal cultures (p<0.0001). Inhibition of mTOR with rapamycin corrected bursting levels to control levels. Furthermore, D-2-HG induced mTOR hyperactivation, independent of bursting activity, which correlated with upregulation of mTOR signaling in human epileptic tissue. KDM inhibition resulted in mTOR hyperactivation and neuronal hyperexcitability, which we demonstrated with D-2-HG, succinate, and PFI-90, a small molecule KDM inhibitor. Epileptic cortex and D-2-HG-treated neurons, have distinct metabolisms independent of neuronal activity compared to peritumoral nonepileptic cortex and control, respectively. CONCLUSION: We demonstrate IDHmutated gliomas promote epileptogenesis through a D-2-HG dependent mTOR hyperactivation via KDM inhibition, a putative mechanism and potential therapeutic targets. Furthermore, we argue mTOR hyperactivation results in metabolic reprogramming, independent of neuronal firing, which may contribute to epileptogenesis, a heretofore unrecognized aspect of pathologic mTOR signaling in neurological diseases.

DDRE-28. MECHANISTIC AND THERAPEUTIC LINKS BETWEEN PURINE BIOSYNTHESIS AND DNA DAMAGE IN GLIOBLASTOMA
Andrew Scott, Weihua Zhou, Kari Wilder-Romans, Jiane Feng, Zhe Wu, Anthony Andren, Li Zhang, Peter Sajjakulnukit, Maureen Kachman, Yoshie Umemura, Melanie Schmitt, Nathan Qi, Theodore Lawrence, Costas Lyssiotis, Daniel Wahl; University of Michigan, Ann Arbor, MI, USA Glioblastoma (GBM) is the most common and aggressive adult brain cancer. Radiation therapy (RT) is a critical treatment modality, and development of RT resistance is the predominant cause of recurrence and mortality in GBM patients. Using cell line models as well as patient-derived xenografts and neurospheres in orthotopic brain tumor models, we have identified increased rates and dependence upon de novo purine biosynthesis as a hallmark of GBM RT resistance. More recently, we have discovered that radiation treatment acutely stimulates flux through de novo purine synthesis in cell line and neurosphere models of GBM. This RT-induced increase in de novo purine synthesis is dependent on signaling through the DNA damage response and thus appears to be an adaptive mechanism to supply purines to repair radiation-induced DNA damage. To determine whether this regulatory mechanism also exists in vivo, we have used advanced metabolomic and metabolic tracing techniques with 13 C-labeled glucose and 15 N-labeled glutamine in mice bearing RT-resistant GBM patient-derived orthotopic brain tumors. We found that that orthotopic GBM PDXs had elevated activity of de novo purine synthesis that increased further after RT, while normal cortex had little activity even after RT. These observations have therapeutic relevance, as targeting this metabolic pathway with the FDA-approved purine biosynthesis inhibitor mycophenolate mofetil (MMF) overcomes GBM radiation resistance in mouse models in vivo. The lack of de novo purine synthesis in normal cortex suggests that targeting this pathway may be tumor specific. Collectively our data suggest that de novo synthesis of purines mediates RT resistance in GBM and that treatment of brain tumors with MMF in combination with RT may be a promising therapeutic strategy in patients.