bluuebunny/crossref_metadata_2025 huggingface dataset:
BIMG-05. TO BE OR NOT TO BE GLYCOLYTIC: DEUTERATED GLUCOSE-BASED ASSESSMENT OF THE WARBURG EFFECT ALLOWS NON-INVASIVE IMAGING OF TUMOR BURDEN AND TREATMENT RESPONSE IN MUTANT IDH GLIOMAS IN VIVO
Authors: Taglang Celine , Batsios Georgios , Tran Meryssa , Gillepsie Anne Marie , Luchman Hema Artee , Pieper Russell O , Ronen Sabrina M , Viswanath Pavithra
Published: March 2021

The Warburg effect, characterized by elevated glucose uptake and flux to lactate, is a metabolic hallmark of cancer. Recent studies have identified deuterium 2H-magnetic resonance spectroscopy (MRS) using 6,6’-2H-glucose as a novel method of imaging the Warburg effect in high-grade primary glioblastomas (GBMs). However, its utility for imaging low-grade gliomas has not been tested. The goal of this study was to determine whether 6,6’-2H-glucose can be used for imaging tumor burden and treatment response in mutant isocitrate dehydrogenase (IDHmut) low-grade gliomas in vivo. We examined mice bearing orthotopic tumors of the patient-derived BT257 astrocytoma model. 1H-MRS, providing a readout of steady-state metabolite levels, confirmed the presence of 2-hydroxyglutarate, the product of IDHmut, in BT257 tumor tissue but not normal brain. Previous studies comparing IDHmut gliomas with GBMs suggest that IDHmut gliomas undergo lactate dehydrogenase silencing, potentially leading to a non-glycolytic phenotype. Nevertheless, our results indicated that, compared to normal brain, glucose uptake and concomitant flux to lactate were significantly higher in BT257 tumor tissue. Importantly, 6,6’-2H-glucose metabolism to lactate was observed in BT257 tumor-bearing mice, but not tumor-free mice. Furthermore, imaging studies confirmed spatial localization of lactate production to the tumor vs. contralateral normal brain. We then examined the ability of 6,6’-2H-glucose to assess treatment response. Poly-(adenosine 5′-diphosphate-ribose) polymerase inhibitors (PARPi) inhibit IDHmut glioma growth and are in clinical trials for IDHmut glioma patients. Treatment with the PARPi niraparib reduced 6,6’-2H-glucose flux to lactate in BT257 tumor-bearing mice. Importantly, this reduction was observed at early time-points when no difference in tumor volume could be detected using anatomical imaging, pointing to the ability of 6,6’-2H-glucose to assess pseudoprogression. Collectively, our results suggest that IDHmut gliomas display a glycolytic phenotype amenable to non-invasive 2H-MRS-based imaging of tumor burden and treatment response.
BIMG-15. LACTATE AND GLUTATHIONE LEVELS DETECTED WITH PROTON MR SPECTROSCOPY ARE ASSOCIATED WITH POOR SURVIVAL IN IDH WILD TYPE TERTP MUTANT DIFFUSE GLIOMAS
Authors: Sacli-Bilmez Banu , Danyeli Ayça Ersen , Yakicier Cengiz , Pamir M Necmettin , Özduman Koray , Dincer Alp , Ozturk-Isik Esin
Published: March 2021

Telomerase reverse transcriptase promoter (TERTp) mutations in the absence of IDH mutations (TERTp-only) are recently proposed as a characteristic of glioblastoma regardless of the morphological grade. This study aims to analyze metabolic profiles of TERTp-only gliomas using proton magnetic resonance spectroscopy (1H-MRS) and evaluate the effect of metabolite concentrations on progression-free survival (PFS) and overall survival (OS). The patient cohort consisted of 56 TERTp-only gliomas (35M/21F, mean age: 58±10.37 years, 44 glioblastomas (GBM), and 12 lower-grade gliomas with TERTp-mutation but no IDH-mutations). All patients underwent preoperative diagnostic 1H-MRS using a 3T MR scanner with a 32-channel head coil. 1H-MRS was obtained from a manually placed region of interest with no necrosis, edema, and hemorrhage, using a Point Resolved Spectroscopy (PRESS) sequence (TR/TE=2000/30 ms, 1024 points). LCModel spectral fitting program was used for quantification of MR spectroscopic peak concentrations of 17 metabolites. The patients were divided into two groups using median values of the metabolite intensities, and Kaplan-Meier survival analysis followed by a log-rank test was used to determine the effects of metabolite concentrations on OS and PFS. Median PFS and OS of TERTp-only gliomas were 11 and 17 months, respectively. TERTp-only LGG patients had longer OS and PFS than TERTp-only GBM patients (p=0.022 for OS and p=0.018 for PFS). Significantly shorter OS and PFS were identified in TERTp-only gliomas, who had higher GSH/tCr (p=0.011 for OS and p=0.004 for PFS) and higher Lac/tCr (p=0.014 for OS and p=0.012 for PFS). Lactate is a marker of necrosis and a sign of malignancy. On the other hand, both tCr and GSH were lower, which resulted in higher GSH/tCr, in TERTp-only GBM. The results of this study indicate that high Lac/tCr and GSH/tCr might be indicators of poor prognosis in TERTp-only gliomas. This study has been supported by TUBITAK 1003 grant 216S432.
BIMG-16. TRACKING TTFIELDS-INDUCED ALTERATIONS IN GLIOBLASTOMA METABOLISM WITH [18F]DASA-23, A NON-INVASIVE PROBE OF PYRUVATE KINASE M2 (PKM2)
Authors: Patel Chirag , Beinat Corinne , Xie Yuanyang , Chang Edwin , Gambhir Sanjiv
Published: March 2021

Despite the anti-proliferative and survival benefits from tumor treating fields (TTFields) in human glioblastoma (hGBM), little is known about the effects of this form of alternating electric fields therapy on the aberrant glycolysis of hGBM. [18F]FDG is the most common radiotracer in cancer metabolic imaging, but its utility in hGBM is impaired due to high glucose uptake in normal brain tissue. With TTFields, radiochemistry, Western blot, and immunofluorescence microscopy, we identified pyruvate kinase M2 (PKM2) as a biomarker of hGBM response to therapeutic TTFields. We used [18F]DASA-23, a novel radiotracer that measures PKM2 expression and which has been shown to be safe in humans, to detect a shift away from hGBM aberrant glycolysis in response to TTFields. Compared to unexposed hGBM, [18F]DASA-23 uptake was reduced in hGBM exposed to TTFields (53%, P< 0.05) or temozolomide chemotherapy (33%, P > 0.05) for 3 d. A 6-d TTFields exposure resulted in a 31% reduction (P = 0.043) in 60-min uptake of [18F]DASA-23. [18F]DASA-23 was retained after a 10 but not 30-min wash-out period. Compared to [18F]FDG, [18F]DASA-23 demonstrated a 4- to 9-fold greater uptake, implying an improved tumor-to-background ratio. Furthermore, compared to no-TTFields exposure, a 6-d TTFields exposure caused a 35% reduction in [18F]DASA-23 30-min uptake compared to only an 8% reduction in [18F]FDG 30-min uptake. Quantitative Western blot analysis and qualitative immunofluorescence for PKM2 confirmed the TTFields-induced reduction in PKM2 expression. This is the first study to demonstrate that TTFields impairs hGBM aberrant glycolytic metabolism through reduced PKM2 expression, which can be non-invasively detected by the [18F]DASA-23 radiotracer.
BIMG-18. ELEVATED CYSTATHIONINE IN MEDULLOBLASTOMA DEMONSTRATES TUMOR-SPECIFIC METHIONINE METABOLISM
Authors: Tikunov Andrey , Rosen Elias , Babcock Ben , Weir Seth , Parnham Stuart , Macdonald Jeffrey
Published: March 2021

We investigated tumor-specific metabolism medulloblastoma using a non-biased MS-imaging screen and identified a pattern of methionine flux that may present a therapeutic opportunity. We studied brain tumors that form in mice genetically engineered to develop Sonic Hedgehog (SHH)-driven medulloblastoma. We subjected sagittal sections including brain and medulloblastoma to MS-imaging, generating concentration maps for hundreds of metabolites MW 100–400. We then confirmed results by analyzing tumor, brain and blood by LC-MS/MS, high-resolution NMR and 2D NMR-TOCSY, and used immunohistochemistry to determine the cellular localization of implicated enzymes. MS imaging, accomplished by matrix-assisted laser desorption electrospray ionization (MALDESI), detected cystathionine at an order of magnitude higher concentration in medulloblastomas compared to adjacent brain. No other metabolite showed such a strong, tumor-specific localization. LC-MS/MS and NMR methods confirmed cystathionine elevation. As cystathionine is the product of homocysteine and serine, catalyzed by cystathionine beta-synthase (CBS), we investigated CBS expression by IHC. Consistent with prior studies, we found that only astrocytes expressed CBS, both in the normal brain and within the tumors. ScRNA-seq confirmed Cbs only in astrocytes, and showed tumor cells express methionine-metabolizing enzymes Mat2a, Dnmt1, Ancy and Mtr. Together, these findings show that tumor cells generate and export homocysteine, which astrocytes convert to cystathionine. Tumor cystathionine generation responded to changes in methionine- cycle metabolites. In vivo, systemic administration of homocysteine increased tumor cystathionine which decreased in response to systemic folate, the methyl donor for homocysteine methyltransferase. Cystathionine itself was inert in tumors as tumor cells cultured in up to 8 mM cystathionine showed no change in cell cycle progression. Our studies show that medulloblastomas utilize methionine and generate homocysteine, but avoid folate-dependent homocysteine-methionine recycling by exporting homocysteine for detoxification by local astrocytes. This model suggests that treatments that impose methionine scarcity, folate scarcity or CBS inhibition may produce anti-tumor effects in medulloblastoma.
BIMG-21. DEUTERIUM METABOLIC IMAGING (DMI), A NEW, MRI-BASED TECHNIQUE FOR MAPPING BRAIN TUMOR METABOLISM IN VIVO
Authors: Corbin Zachary , Fulbright Robert , Rothman Douglas , de Graaf Robin , De Feyter Henk
Published: March 2021

Deuterium Metabolic Imaging (DMI) combines 3D deuterium (2H) magnetic resonance spectroscopic imaging (MRSI) with administration of a 2H-labeled substrate to map uptake and metabolism of the substrate. DMI has been implemented on a 4 Tesla clinical research MRI scanner, and on an 11.7 Tesla preclinical MRI scanner, and has been used with 2H-labeled glucose, acetate and choline. DMI data are presented as color maps of concentration of the 2H-labeled substrate and its metabolites, overlaid on anatomical MR images. In rat and mouse models of glioblastoma, DMI data acquired at 5 to 8 uL resolution following intravenous 2H-glucose infusion clearly showed the Warburg effect in the tumor lesions. The Warburg effect is indicated by the ratio of 2H-labeled lactate/glutamate+glutamine (Glx). High levels of 2H-labeled lactate and low levels of 2H-labeled Glx are the result of a high rate of glycolysis and low rate of oxidative glucose metabolism. Because DMI detects both glucose and its downstream metabolism, the technique does not suffer from low image contrast with normal brain, as is the case with FDG-PET that detects glucose uptake only. For clinical research studies patients orally consumed 0.75g/kg of 2H-glucose dissolved in water. The observations made in the animal models were confirmed in several patients with recurrent GBM, showing hotspots in the lac/Glx maps (8 mL resolution), coinciding with the area of the tumor lesion. In patients with meningioma, no Warburg effect was detected using DMI. Furthermore, DMI data acquired in a patient with GBM one week after finishing 30 days of radiation therapy, also showed no high levels of 2H-labeled lactate in the lesion. These data indicate that the presence of the Warburg effect could correlate with tumor grade and/or aggressiveness, and that DMI of glucose metabolism could potentially be a biomarker of therapy effect.
DDRE-02. SMOOTHENED-ACTIVATING LIPIDS DRIVE RESISTANCE TO CDK4/6 INHIBITION IN HEDGEHOG-ASSOCIATED MEDULLOBLASTOMA
Authors: Daggubati Vikas , Hochstelter Jordan , Bommireddy Ani , Choudhury Abrar , Krup Alexis , Kaur Pervinder , Tong Pakteema , Li Amy , Xu Libin , Reiter Jeremy , Raleigh David
Published: March 2021

BACKGROUND

Medulloblastoma is an aggressive pediatric brain tumor that is associated with misactivation of the Hedgehog (HH) pathway. Our lab has shown that CDK6, a critical activator of the cell cycle, is a direct transcriptional target of oncogenic HH signaling, and that inhibiting CDK6 blocks the growth of HH-associated medulloblastoma in mice. A clinical trial exploring the efficacy of CDK6 inhibition in medulloblastoma patients is underway, but prior attempts to target the HH pathway in medulloblastoma have been encumbered by resistance to molecular monotherapy. Thus, we sought to identify mechanisms of resistance to CDK6 inhibition in HH-associated medulloblastoma.

METHODS

We performed orthogonal CRISPR and CRISPR interference screens in HH-associated medulloblastoma cells treated with pharmacologic inhibitors of CDK6 in vitro, and RNA-sequencing of HH-associated medulloblastomas with genetic deletion of CDK6 in vivo. Mechanistic and functional validation of resistance pathways was performed using CRISPR interference, immunoblotting, immunofluorescence, genetics, and pharmacology. Lipid quantification was carried out by ultra-high performance liquid chromatography-tandem mass spectrometry.

RESULTS

Our results reveal that decreased ribosomal protein expression underlies resistance to CDK6 inhibition in HH-associated medulloblastoma, leading to endoplasmic reticular (ER) stress and activation of the unfolded protein response (UPR). We show that ER stress and the UPR increase the activity of enzymes producing Smoothened-activating sterol lipids that sustain oncogenic HH signaling in medulloblastoma despite CDK6 inhibition. These discoveries suggest that combination molecular therapy against CDK6 and HSD11ß2, an enzyme producing Smoothened-activating lipids, may be an effective treatment for HH-associated medulloblastoma. In support of this hypothesis, we demonstrate that concurrent genetic deletion or pharmacological inhibition of CDK6 and HSD11ß2 additively blocks the growth of multiple models of HH-associated medulloblastoma in mice.

CONCLUSIONS

Smoothened-activating lipid biosynthesis underlies resistance to CDK6 inhibition in HH-associated medulloblastoma, revealing a novel combination therapy to treat the most common malignant brain tumor in children.
DDRE-04. THE COMBINED TREATMENT OF L-ASPARAGINASE AND 6-DIAZO-5-OXO-L-NORLEUCINE INHIBIT THE PROLIFERATION OF TEMOZOLOMIDE-SENSITIVE OR RESISTANT GLIOBLASTOMA CELLS
Authors: Ohba Shigeo , Hirose Yuichi
Published: March 2021

Glioblastoma is one of the aggressive brain tumors with a 5-year survival rate of < 10%. The standard treatment is maximal safe resection, followed by radiation therapy and temozolomide (TMZ). Clinically, the resistance to TMZ is a big problem. Cancer cells have been revealed to show different metabolism from normal cells. The object of this study is to evaluate whether cancer metabolism, especially asparagine, could be a new target of treatment in primary and recurrent glioblastoma. Glioblastoma cells (U251 and U87) were treated with L-asparaginase and/or 6-diazo-5-oxo-L-norleucine (DON). L-asparaginase converts asparagine into aspartate and depletes asparagine. DON is a glutamine analog that inhibits several glutamine-utilizing enzymes, including asparagine synthetase. L-asparaginase or DON suppressed the proliferation of U251, and U87 cells in a dose-dependent manner. Combined treatment with these drugs had a synergistic antiproliferative effect in these cell lines. The effect was counteracted by exogenous asparagine. The combined treatment induced greater apoptosis and autophagy than did single-drug treatment. Several clones of TMZ-resistant U251 were obtained after long treatment of TMZ to U251. The expression of MSH6, one of the mismatch repair proteins, was suppressed in these resistant clones. The synergistic effect of L-asparaginase and DON was detected in these U251-derived TMZ-resistant clones. These results suggest that the combination of L-asparaginase and DON could be a new therapeutic option for patients with primary and recurrent glioblastoma.
DDRE-07. FATTY ACID SYNTHESIS IS REQUIRED FOR BREAST CANCER BRAIN METASTASIS
Authors: Ferraro Gino , Ali Ahmed , Luengo Alba , Kodack David , Deik Amy , Abbott Keene , Bezwada Divya , Blanc Landry , Prideaux Brendan , Jin Xin , Possada Jessica , Chen Jiang , Chin Christopher , Amoozgar Zohreh , Ferreira Raphael , Chen Ivy , Naxerova Kamila , Ng Christopher , Westermark Anna , Duquette Mark , Roberge Sylvie , Lyssiotis Costas , Duda Dan , Golub Todd , Cantley Lewis , Asara John , Davidson Shawn , Fukumura Dai , Dartois Véronique , Clish Clary , Heiden Matthew Vander , Jain Rakesh
Published: March 2021

Brain metastases are refractory to therapies that otherwise control systemic disease in patients with human epidermal growth factor receptor 2 (HER2+) breast cancer, and the unique brain microenvironment contributes to this therapy resistance. Nutrient availability can vary across tissues, therefore metabolic adaptations required for breast cancer growth in the brain microenvironment may also introduce liabilities that can be exploited for therapy. Here, we assessed how metabolism differs between breast tumors growing in the brain versus extracranial sites and found that fatty acid synthesis is elevated in breast tumors growing in the brain. We determine that this phenotype is an adaptation to decreased lipid availability in the brain relative to other tissues, which results in a site-specific dependency on fatty acid synthesis for breast tumors growing at this site. Genetic or pharmacological inhibition of fatty acid synthase (FASN) reduces HER2+ breast tumor growth in the brain, demonstrating that differences in nutrient availability across metastatic sites can result in targetable metabolic dependencies.
DDRE-10. METABOLIC TARGETING OF HUMAN GLIOBLASTOMA USING 5-AMINOLEVULINIC ACID (ALA)-MEDIATED SONODYNAMIC THERAPY: A FIRST-IN-HUMAN STUDY
Authors: Marcus Stuart , Sanai Nader
Published: March 2021

Heme biosynthesis is altered in glioblastoma (GBM). Systemic dosing with ALA, the first committed molecule in the heme pathway, results in accumulation of the fluorescent intermediate, protoporphyrin IX (PpIX) only within tumor tissue (Gleolan label, 2019). PpIX is a photosensitizer that is effective in photodynamic therapy (PDT); in recurrent GBM patients, the safety and feasibility of ALA PDT has been demonstrated (Johansson A, et al. Lasers Surg Med 2013;45:225), although the practicality of this strategy in clinical care remains uncertain. Importantly, preclinical models of GBM show that PpIX is also a sonosensitizer and, in combination with transcranial MRI-guided focused ultrasound (MRgFUS), leads to non-ablative cytotoxic effects in vivo (Jeong EJ et al, Ultrasound in Medicine and Biology 2013:38;2143, Suehiro S et al, J Neurosurg 2018: 1377, Wu et al Nature Sci Reports 2019: 9;10465). The Ivy Brain Tumor Center is conducting a first-in-human study of 5-ALA sonodynamic therapy (SDT) for recurrent GBM (NCT 04559685). In this Phase 0/1 clinical trial, nontherapeutic, single-treatment SDT is administered prior to planned tumor resection. A Dose-Escalation Arm varies the power/energy of the MRgFUS while using a fixed time-interval from exposure to surgery. A subsequent Time-Escalation Arm varies the interval between MRgFUS and surgical resection, but fixes the power/energy of the delivered ultrasound. In both Arms, patient tumor tissue is assessed for sonodynamic and pharmacodynamic effects. In each patient, half of the tumor volume is not targeted with SDT and serves as an internal control. This first-in-human study will demonstrate the safety and feasibility of ALA sonodynamic therapy in GBM and may provide the first-ever biological evidence of sonosensitization in a brain tumor patient. If successful, this Phase 0 trial will introduce a new, metabolically-driven, GBM treatment modality that may be applicable to any brain tumor that selectively accumulates PpIX after ALA administration.
DDRE-17. A PHASE I CLINICAL TRIAL OF DOSE-ESCALATED METABOLIC THERAPY COMBINED WITH CONCOMITANT RADIATION THERAPY IN HIGH-GRADE GLIOMA
Authors: Porper Keren , Shpatz Yael , Plotkin Luba , Pechthold Ronit Goldman , Talianski Alisa , Hemi Rina , Mardor Yael , Jan Elisheva , Genssin Hili , Symon Zvi , Yair Anikster , Zach Leor , Lawrence Yaacov R
Published: March 2021

BACKGROUND

Animal brain-tumor models have demonstrated a synergistic interaction between radiation therapy and a ketogenic diet (KD). Metformin has in-vitro anti-cancer activity, through AMPK activation and mTOR inhibition. We sought to assess the feasibility of combined radiation, KD and metformin in adults with high grade gliomas.

METHODS

A prospective single-institution phase I clinical trial of combined metabolic therapy and radiotherapy. Radiotherapy was either 60Gy over six weeks or 35Gy over two weeks for newly diagnosed and recurrent gliomas, respectively. The dietary intervention consisted of a KD supplemented with medium chain triglycerides (MCT). There were three cohorts 1) dietary intervention alone, 2) low-dose metformin combined with dietary intervention and 3) high-dose metformin combined with dietary intervention. Clinicaltrials.gov NCT02149459.

RESULTS

A total of 13 patients were accrued, median age 61 years, of whom 6 had newly diagnosed and 7 with recurrent disease. All completed radiation therapy; 5 patients stopped the metabolic intervention early. Metformin 850mg three-times daily was poorly tolerated. There were no grade 4 / 5 adverse events, and only one grade 3 event (nausea). The median level of ketones during the intervention was 0.5 mM. Ketone levels were associated with dietary factors (high fat, low carbohydrates, MCT intake), use of metformin and low insulin levels. Median progression free survival was 10 months for newly diagnosed disease and 4 months for recurrent disease.

CONCLUSIONS

The intervention was fairly well tolerated, however only moderate ketones levels were obtained. Metformin use and dietary intake were associated with higher serum ketone levels. The recommended phase II dose is the 8 weeks of a low-carbohydrate diet combined with 850mg metformin twice daily.
DDRE-18. THE MITOCHONDRIAL PYRUVATE CARRIER—A METABOLIC TARGET OF MALIGNANT GLIOMA
Authors: Tiburcio Patricia , Murnyak Balazs , Coffman Kimberly , Huang Eric
Published: March 2021

Metabolic reprogramming has been recognized as crucial to the survival and proliferation of cancer cells through the reduction of glucose oxidation (the Warburg effect) and diversion of pyruvate and glycolytic metabolites to fuel anabolic processes. The mitochondrial pyruvate carrier (MPC) protein complex, consisting of MPC1 and MPC2, has been identified as essential for pyruvate transport into mitochondria. In most human cancers, MPC1 is frequently deleted or downregulated and, therefore, the MPC activity is low. Restoration of MPC levels increases pyruvate oxidation and markedly inhibits tumor growth. Despite being tumor suppressive in general, the role of MPC in malignant glioma seems complex. We reported previously that unlike MPC1 in IDH-mutant glioma, MPC2 expression correlated with worsened survival. Interestingly, MPC2 homo-oligomers have been identified recently as an efficient autonomous pyruvate transporter, which can be inhibited by an insulin sensitizer rosiglitazone but not by the MPC heterotypic oligomer inhibitor UK-5099. In this study, we report that glioma cells show low MPC1 expression but much higher MPC2 expression. Analysis of glioma patient data revealed that the mean MPC2 expression increased in a grade-dependent fashion whereas the mean MPC1 expression remained essentially low, thereby resulting in an increased MPC2/MPC1 ratio in association with glioma progression. Importantly, we show that malignant glioma cells are extremely sensitive to the mitochondrion-specific, PPARγ-sparing insulin sensitizer mitoglitazone but not UK-5099 whereas MPC-proficient glial cells are highly responsive to the latter, indicating MPC2 as an alternative pyruvate transporter in glioma. Furthermore, the addition of glutamate fully rescues the growth arrest of Mpc1-/- murine glial cells, suggesting the involvement of cerebral cortex-specific microenvironment in glioma growth. We are employing newly developed RCAS/tva Mpc1fl/fl mouse models to test therapeutic targeting of Mpc2 in spontaneously developed glioma.
DDRE-25. INVESTIGATING MITOCHONDRIAL SLC25A TRANSPORTERS INVOLVED IN SUPPORTING BRAIN TUMOUR METABOLISM AND SURVIVAL UNDER HYPOXIC CONDITIONS
Authors: Eales Katherine , Finch Alina , Wykes Victoria , Watts Colin , Tennant Daniel
Published: March 2021

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
Authors: De Martino Mara , Daviaud Camille , Vanpouille-Box Claire
Published: March 2021

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 IFN-beta 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-34. TARGETING RESISTANCE IN MEDULLOBLASTOMA
Authors: Chang Simone , Meiman Evan , Telang Sucheta
Published: March 2021

Medulloblastoma is the most commonly diagnosed pediatric brain tumor. Although therapeutic advances have improved survival from this cancer, they result in devastating sequelae and, additionally, have proven inadequate in metastatic disease and recurrence where survival remains <5%. Effective therapies are urgently needed to improve outcomes in medulloblastoma. Medulloblastoma development is driven by dysregulation of normal cerebellar proliferation. Mutations in the sonic hedgehog (Shh) pathway are found in ~30% of these tumors and responsible for their aggressive growth. The poor outcomes in Shh-driven medulloblastoma have prompted the evaluation of Shh-targeting agents in their treatment – with limited success likely attributable in part to the upregulation of alternate survival pathways (e.g. Ras/MAPK and HIF-1α). These alternate mechanisms stimulate glycolysis, in part by increasing the activity of the 6-phosphofructo-2-kinase/fructose-2,6 bisphosphatases (PFKFB1-4) to produce fructose-2,6-bisphosphate (F26BP), a potent activator of the rate-limiting glycolytic enzyme, 6-phosphofructo-1-kinase. In recent studies, we have determined that the PFKFB4 enzyme is highly expressed in patient-derived Shh medulloblastomas. We have found that hypoxia, through HIF-1α, strongly induced PFKFB4 expression in Shh-driven medulloblastoma cells and that silencing PFKFB4 suppressed F26BP, glycolysis and proliferation in normoxia and, more markedly, in hypoxia, indicating that PFKFB4 may be required for growth under hypoxia. We found that simultaneously silencing PFKFB4 and Shh pathway effectors significantly reduced cell survival and that co-targeting PFKFB4 (with a novel inhibitor) and Shh effectors synergistically decreased cell viability. In order to simulate Shh antagonist resistance, we have now subjected Shh medulloblastoma cells to prolonged Shh inhibitor exposure and found that these cells exhibit increased proliferation, glycolysis and PFKFB4. Studies are underway to delineate their metabolic alterations. Taken together, our data indicate that targeting PFKFB4 may be a valid therapeutic option in aggressive, treatment-resistant medulloblastoma and strongly support the further examination of PFKFB4 inhibitors in these tumors.
ETMM-05. LACTIC ACID FACILITATES GLIOBLASTOMA GROWTH THROUGH MODULATION OF THE EPIGENOME
Authors: Torrini Consuelo , Nguyen Trang , Shu Chang , Mela Angeliki , Humala Nelson , Mahajan Aayushi , Karpel-Massler Georg , Bruce Jeffrey , Canoll Peter , Siegelin Markus
Published: March 2021

Glioblastoma (GBM) is the most common primary malignant brain tumor with an unfavorable prognosis. While GBMs utilize glucose, there are other carbon sources at their disposal. Lactate accumulates to a significant amount in the infiltrative margin of GBMs. In the current study, we demonstrated that lactate rescued patient-derived xenograft (PDX) GBM cells from nutrient deprivation mediated cell death and inhibition of growth. Transcriptome analysis, ATAC-seq and CHIP-seq. showed that lactic acid exposure entertained a signature of cell cycle progression and oxidative phosphorylation (OXPHOS) /tricarboxylic acid (TCA)-cycle. LC/MS analysis demonstrated that U-13C-Lactate elicited substantial labeling of TCA-cycle metabolites, acetyl-CoA and histone protein acetyl-residues in PDX derived GBM cells. Given that acetyl-CoA is pivotal for histone acetylation we observed a dose-dependent elevation of histone marks (e.g. H3K27ac), which was rescued by genetic and pharmacological inhibition of lactic acid-uptake, ATP-citrate lyase, p300 histone-acetyl-transferase and OXPHOS, resulting in reversal of lactate mediated protection from cell death. CHIP-seq. analysis demonstrated that lactic acid facilitated enhanced binding of H3K27ac to gene promoters and cis-regulatory elements. Consistently, ATAC-seq. analysis highlighted enhanced accessibility of the chromatin by lactic acid. In a combined tracer experiment (U-13C-glucose and 3-C13-lactate), we made the fundamental observation that lactic acid carbons were predominantly labeling the TCA cycle metabolites over glucose, implying a critical role of lactic acid in GBMs. Finally, pharmacological blockage of the TCA-cycle, using a clinically validated drug, extended overall survival in an orthotopic PDX model in mice without induction of toxicity, implying a critical role of lactic acid in GBMs and establishing lactic acid metabolism as a novel drug target for GBM.
ETMM-07. HYPOXIC REGULATION OF METABOLIC AND STRUCTURAL GENES IN T98 GLIOBLASTOMA MULTIFORME CELLS BY RNA SEQUENCING
Authors: White Brian E , Liu Edward , Hakonarson Hakon , Buono Russell J
Published: March 2021

Glioblastoma multiforme (GBM) is the most common primary brain cancer and carries a very poor prognosis. The GBM tumor microenvironment is characterized by regions of profound hypoxia, which are associated with a variety of alterations in gene expression that confer survival, proliferation, and resistance to therapy. Multiple mechanisms have been implicated in hypoxia-associated GBM behavior including upregulation of pathways involved in angiogenesis, immunosuppression, and glucose metabolism. Our study aimed to identify changes in gene expression induced by hypoxia among T98G cells via total RNA sequencing. Human T98 GBM cell lines were cultured in a humidified incubator at 37° C and 5% CO2 and were grown in normoxia (21% O2) or hypoxia (95% N2, 5% C02) for 72 hours. Total RNA was harvested, and global gene expression was evaluated via total RNA sequencing. Standard bioinformatics analysis was performed to identify changes in expression associated with hypoxia. Hypoxia in T98 cells led to significant upregulation of genes implicated in canonical glycolysis, focal adhesion, extracellular matrix reorganization, and endoplasmic reticulum-associated protein processing. We document 690 genes and 11 associated KEGG pathways that demonstrated significant enrichment (p ≤0.01 with Bonferroni, Benjamini, and False Discovery Rate corrections) induced by hypoxia. Notably, upregulation of the IRE1-mediated unfolded protein response was observed. DrugBank database analysis identified four molecules targeting genes upregulated in hypoxic T98G cells: tenecteplase (p = 0.013, 5 gene targets), succinic acid (p = 0.02, 7 targets), artenimol (p = 0.013, 13 targets), and copper (p = 0.0015, 22 targets). We document 733 genes and 6 associated KEGG pathways significantly downregulated (p ≤0.01) in hypoxia, including genes associated with DNA replication and repair, mitotic processes, and spliceosome function. Total RNA sequencing showed hypoxic upregulation of genes involved in various pathways associated with neoplastic GBM behavior and identified multiple candidate molecules which may hold therapeutic potential.
ETMM-09 TARGETING GLIOBLASTOMA MULTIFORME METABOLISM AT THE INVASIVE TUMOR FRONT
Authors: Garcia Joseph H , Jain Saket , Akins Erin A , Beniwal Angad S , Wolfe Kayla J , Cha Jungwha , Kumar Sanjay , Aghi Manish K
Published: March 2021

Glioblastoma (GBM) is a primary malignant brain tumor with a median survival under two years. The poor prognosis GBM caries is largely due to cellular invasion, which enables escape from resection and drives inevitable recurrence. Numerous factors have been proposed as the primary driving forces behind GBM’s ability to invade adjacent tissues rapidly, including alterations in its cellular metabolism. Though studies have investigated links between GBM’s metabolic profile and its invasive capabilities, these studies have had two notable limitations. First, while infiltrating GBM cells utilize adaptive cellular machinery to overcome stressors in their microenvironment, the cells at the invasive tumor front have rarely been sampled in previous studies, which have primarily used banked tissue taken from the readily accessible tumor core. Second, studies of invasion have primarily used two-dimensional (2D) culture systems, which fail to capture the dimensionality, mechanics, and heterogeneity of GBM invasion. To address these limitations, our team developed two complementary approaches: acquisition of site-directed biopsies from patient GBMs to define regional heterogeneity in invasiveness, and engineering of three dimensional (3D) platforms to study invasion in culture. Through utilization of these platforms, and by taking advantage of the system-wide, unbiased screens of metabolite profile and gene expression available, our team looked to accomplish the goal of identifying targetable metabolic factors which drive cellular invasion in GBM. Pilot RNA-Sequencing data revealed 87 of the top 250 (35%) genes preferentially expressed in the tumor invasive edge, and 30 of the top 250 (12%) genes preferentially expressed in the tumor core were involved in cellular metabolism. KEGG pathways analysis demonstrated enrichment of glycolytic, pentose phosphate, and response to amino acid starvation pathways at the tumor invasive edge. These preliminary studies demonstrate a distinct metabolic phenotype in invasive GBM cells which will be further explored with system wide screens.
FSMP-01. ID1 MEDIATES ONE-CARBON MEDIATED PURINE SYNTHESIS IN GLIOBLASTOMA
Authors: Ghannad-Zadeh Kimia , Wu Megan , Wilson Taylor , Flick Robert , Das Sunit
Published: March 2021

Inhibitor of DNA-binding-1 (ID1) is a transcriptional regulatory protein involved in maintenance of self-renewal and inhibition of differentiation, and acts as a key regulator of tumorigenesis in glioblastoma. Studies suggest that de novo purine synthesis is essential for the maintenance of rapid proliferation rates in glioma initiating cells. We hypothesise that ID1 plays a role in reprogramming one-carbon mediated de novo purine synthesis, thereby metabolically contributing to the tumorigenic advantage seen in ID1-high glioblastoma cells. The effect of ID1 regulation on metabolic reprogramming of glioblastoma was studied using ID1-knockout U251 glioblastoma cell lines. Protein expression analysis and liquid chromatography mass-spectrometry were respectively used to assess expression and concentration of metabolic enzymes and intermediates of one-carbon and de novo purine synthesis pathways. CD44 expression was analyzed as a marker of cancer stem cells. The expression of DHFR and MTHFD2 was significantly decreased after ID1 knockout. Furthermore, PAICS expression, and overall concentration of IMP, AMP, GMP, and ATP were reduced after ID1 knockout. ID1 expression in glioblastoma tumor xenografts was associated with positive expression of one-carbon metabolism and purine synthesis enzymes, while ID1-/- cells within the same xenograft had significantly reduced expression of these enzymes. The expression of CD44 was reduced after ID1 knockout. This data suggests that ID1 mediates an increase in one-carbon mediated de novo purine synthesis, thereby regulating metabolic reprogramming in glioblastoma cells. The correlation between CD44 and ID1 expression provides further support that ID1 maintains a less differentiated phenotype in a subset of glioblastoma cells, and metabolic reprogramming is one of the mechanisms through which this phenotype, and the capacity for self-renewal are maintained. Further elucidation of the mechanisms through which ID1 mediates metabolic reprograming of glioblastoma cells can lead to developing effective combination therapies coupling chemotherapeutic strategies with targeting of metabolic programs used by cancer initiating cells.
FSMP-05. KETOGENIC DIETS FOR HIGH-GRADE GLIOMA
Authors: Immidisetti Amanda , Boison Detlev
Published: March 2021

BACKGROUND

Given therapeutic challenges posed by high-grade glioma (HGG), multiple concomitant therapies, including metabolic adjuncts to standard of care, are warranted. Tumor cells are almost exclusively energy-dependent on glucose. Preclinical data supports the use of ketogenic diets (KDs) in this population to deplete the tumor microenvironment of glucose, thereby exerting anti-tumor effects while the surrounding parenchymal tissue utilizes ketones.

OBJECTIVE

The aim of this study was to conduct an up-to-date systematic review of the clinical use of ketogenic diets (KD) in the setting of high-grade glioma treatment and compare study designs, outcomes, and challenges in the translation of these methods from bench to bedside.

METHODS

We conducted comprehensive searches of both the national clinical trials database (clinicaltrials.gov) and pubmed.gov. Trials were included in our review if they were conducted in a patient population with high-grade glioma (either early or refractory) and at least one study arm included the use of a KD.

RESULTS

The clinicaltrials.gov search yielded 12 studies of which 11 met inclusion criteria. Five of these trials reported results. The PubMed search yielded 2 additional studies. Seven clinical trials with reported results on a total of 69 patients were considered.

CONCLUSIONS

The use of KD has proven to be safe and tolerable in early trials, however, further studies are warranted to examine efficacy. Challenges to feasibility include low patient enrollment and compliance, as dietary changes were reported to negatively affect quality of life. Additionally, variability between animal and plant-based KDs, duration of KD regimen, carbohydrate: fat ratio, underlying genetic factors that affect the induction of ketosis, and use of steroid therapy in this patient population may all contribute to inconsistent clinical data when compared to preclinical studies. Future larger scale clinical trials and prospective studies are needed to clarify the role of KDs in the treatment of HGG.
FSMP-06. IN VIVO MONITORING OF LDHA EXPRESSION IN GLIOBLASTOMA USING QUANTITATIVE EXCHANGED-LABEL TURNOVER 1H MRS TECHNIQUE
Authors: Bagga Puneet , Rich Laurie , Haris Mohammad , Wilson Neil , Schnall Mitch , Detre John , Patay Zoltan , Gajjar Amar , Akers Walter , Steinberg Jeffrey , Krenciute Giedre , Baker Suzanne , Reddy Ravinder
Published: March 2021

Most cancers, including glioblastomas (GBMs), rely extensively on glycolysis to support growth, proliferation, and survival. A hallmark of this elevated glycolysis is overexpression of Lactate dehydrogenase-A (LDHA) protein leading to increased uptake of glucose and overproduction of lactate. Various clinical trials using LDHA as a target for diagnosis and treatment have yielded encouraging results. However, in vivo monitoring of LDHA expression has been challenging due to either requirement of administration of radioactive substrates or specialized hardware. In this presentation, we will demonstrate a new method-quantitative exchanged-label turnover MRS (QELT, or simply qMRS)-that increases the sensitivity of magnetic resonance-based metabolic mapping without the requirement for specialized hardware. qMRS relies on the administration of deuterated (2H-labeled) substrates to track the production of downstream metabolites. Since 2H is invisible on 1H MRS, replacement of 1H with 2H due to metabolic turnover leads to an overall reduction in 1H MRS signal for the corresponding metabolites. We applied our qMRS technique to monitor the rate of lactate production in a preclinical GBM model. Infusion of [6,6’-2H2]glucose led to downstream deuterium labeling of lactate, thereby resulting in a reduction in the 1.33 ppm lactate-CH3 peak on 1H MRS over time. The subtraction of post-administration 1H MR spectra from the pre-infusion spectra aided in the determination of the kinetics of the lactate turnover. We believe that the detection and quantification of lactate production kinetics may provide crucial information regarding tumor LDHA expression non-invasively in GBMs without requiring biopsies. Hence, qMRS is expected to open up new opportunities to probe LDHA expression differences in a variety of gliomas, including GBMs and astrocytomas. This method takes advantage of the universal availability and ease of implementation of 1H MRS on all clinical and preclinical magnetic resonance scanners.
FSMP-08. TARGETING PYRIMIDINE SYNTHESIS ACCENTUATES MOLECULAR THERAPY RESPONSE IN GLIOBLASTOMA STEM CELLS
Authors: Yang Kailin , Wang Xiuxing , Wu Qiulian , Kim Leo , Morton Andrew , Gimple Ryan , Prager Briana , Tao Weiwei , Qiu Zhixin , Zhao Linjie , Agnihotri Sameer , Mischel Paul , Mack Stephen , Bao Shideng , Rich Jeremy
Published: March 2021

Glioblastoma stem cells (GSCs) reprogram glucose metabolism by hijacking high-affinity glucose uptake to survive in a nutritionally dynamic microenvironment. Here, we trace metabolic aberrations in GSCs to link core genetic mutations in glioblastoma to dependency on de novo pyrimidine synthesis. Targeting the pyrimidine synthetic rate-limiting step enzyme carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, dihydroorotase (CAD) or the critical downstream enzyme dihydroorotate dehydrogenase (DHODH) inhibited GSC survival, self-renewal, and in vivo tumor initiation through the depletion of the pyrimidine nucleotide supply in rodent models. Mutations in EGFR or PTEN generated distinct CAD phosphorylation patterns to activate carbon influx through pyrimidine synthesis. Simultaneous abrogation of tumor-specific driver mutations and DHODH activity with clinically approved inhibitors demonstrated sustained inhibition of metabolic activity of pyrimidine synthesis and GSC tumorigenic capacity in vitro. Higher expression of pyrimidine synthesis genes portends poor prognosis of patients with glioblastoma. Collectively, our results demonstrate a therapeutic approach of precision medicine through targeting the nexus between driver mutations and metabolic reprogramming in cancer stem cells.