bluuebunny/crossref_metadata_2025 huggingface dataset:
SURG-22. PATTERN OF RECURRENCE ALONG EXTENT OF RESECTION IN PATIENTS WITH GLIOBLASTOMA, IDH-WILDTYPE: A RETROSPECTIVE STUDY
Authors: Yoo Jihwan , Kang Seok-Gu , Yoon Seon-Jin , Kim Kyung Hwan , Jung Inho , Kim Woohyun , Yoon Hong In , Kim Se Hoon , Moon Ju Hyung , Kim Eui Hyun , Chang Jong Hee
Published: December 2020

OBJECTIVE

The concept for maximal resection of glioblastoma(GBM) is changing to resect not only contrast-enhanced (CE) tumor but non-contrast-enhanced (NCE) tumor. Although maximal resection is associated with longer survival, the pattern of recurrence in glioblastoma, isocitrate dehydrogenase (IDH)-wildtype patient is poorly understood.

METHODS

This retrospective, single-center study included 358 eligible patients through November 1, 2005 to December 31, 2018. Those patients were grouped along extent of resection (subtotal resection (STR, n=123), gross-total resection (GTR, n=194), and supratotal resection (SupTR, n=41)). The pattern of recurrence was classified based on the range of radiation treatment, into infield gross target volume (GTV), infield clinical target volume (CTV), marginal, outfield and CSF seeding.

RESULTS

Tumor recurrence was observed for each group as follows: STR group, 80.5%, GTR group, 75.3%, and SupTR group, 63.4%. Statistical differences in pattern of recurrence among each group were observed in infield GTV (STR group:75.8%, GTR group: 71.9%, and SupTR group: 42.3%, p=0.003) and Outfield (STR group:11.0%, GTR group: 21.2%, SupTR group: 34.6%, p=0.012) recurrence. The survival for SupTR group was significantly longer than that for the STR and GTR in terms of both overall survival (OS) and progression free survival (PFS) (OS, STR: 15.7m, GTR: 21.6m, SupTR: 44.7m, PFS, STR: 10.4m, GTR: 14.3m, SupTR: 35.5m)

CONCLUSIONS

As the extent of resection increased, local recurrence decreased and distant recurrence increased. During treatment of glioblastoma, expectation
SURG-27. TREATING HYDROCEPHALUS IN DIFFUSE MIDLINE GLIOMAS WITH AN H3 K27M MUTATION
Authors: Coombs L , LaRocca R , Hata Jessica , Nickols H , Spalding A , Mutchnick Ian , Moriarity Thomas , Gump William , Sun D
Published: December 2020

BACKGROUND

Diffuse midline gliomas (DMG) are a subset of malignant gliomas that share a characteristic Histone H3K27M mutation. These tumors are centrally located and may cause hydrocephalus on initial presentation. DMG lack characteristic imaging that distinguish from other primary brain tumors in the midline. We conducted this retrospective chart review of 43 consecutive patients presenting with midline tumors to determine: how many had a DMG; whether DMG patients with hydrocephalus were candidates for resection; and what the outcomes of endoscopic third ventriculostomy (ETV) versus ventriculoperitoneal shunt (VPS) placement were, as compared to wild type (WT) tumors.

METHODS

We conducted an IRB approved retrospective chart review of patients presenting with midline tumors from 9/2016-3/2020 to determine H3K27M mutation status, hydrocephalus, and neurosurgery intervention.

RESULTS

The median age of all midline tumor patients was 19.1 years (range 1.1-80.1). 26% (11/43) of midline tumors presented with H3K27M mutation, with a higher rate of hydrocephalus compared to patients without mutation [7/11 (65%) for DMG vs. 6/32 (19%) for WT, p< 0.05]. Of the seven H3K27M patients presenting with hydrocephalus, none were candidates for resection, 5 underwent ETV, and 2 underwent VPS placement as initial management. 4 out of these 5 ETVs failed within an average of 24 days (6-42 days). 2 patients then underwent VP shunt placement; the other 2 underwent secondary ETV but both failed and required VP shunting as well. All 6 WT tumor patients had one procedure (1 ETV, 5 resection) to relieve hydrocephalus, and no patients had recurrent hydrocephalus.

CONCLUSIONS

Both pediatric and adult patients may present with DMG associated with a higher rate of unresectable tumors and hydrocephalus on presentation. Furthermore, these data suggest that neuroendoscopic third ventriculostomy and septum pellucidum fenestration for the management of obstructive hydrocephalus in patients with DMG may be less robust than shunting.
SURG-39. 5-AMINOLEVULINIC ACID FLUORESCENCE (5-ALA) GUIDED RESECTION OF GLIOBLASTOMA, LESSON LEARNED FROM 26 CASES
Authors: Cho Jinmo
Published: December 2020

INTRODUCTION

5-ALA is known as useful tool for high grade glioma resection and the accumulation extent of 5-ALA is known as far beyond gadolinium enhancement. Extent of resection is key factor for favorable outcome and long-term survival for high grade glioma patients and 5-ALA might increase extent of resection. We present our experience of 5-ALA guided glioma surgeries.

MATERIALS AND METHODS

Total 26 patients were performed 5-ALA guided surgery. They ingested 20mg/kg, four hours before craniotomy. We tried to perform supra-total resection rather than gross total resection according to the tumor consistency and if the tumor located relatively non-eloquent area, we tried to perform lobectomy rather than lesionectomy. After tumor resection, we inspect the tumor bed under 5-ALA fluorescence, and we confirmed the complete loss of fluorescence on the tumor resected bed. We check the MRI within 48 hour after operation and assess the extent of resection.

RESULTS

Among the 26 patients, 22 patients were confirmed glioblastoma and 3 anaplastic astrocytoma and 1 anaplastic oligoastrocytoma. We confirmed all enhancing lesion was completely removed, however, 4 patients show residual non-enhancing lesion in post-operative MRI. Two patients suffered temporary hemiparesis and 2 patients show permanent visual field defect.

CONCLUSION

5-ALA is useful tool for glioma surgery. Resection extent could be increased, however, non-enhancing lesion in the high grade gliomas, might be missed under 5-ALA guidance.
TAMI-01. NEOADJUVANT PD-1 ANTIBODY BLOCKADE REMODELS THE IMMUNE MICROENVIRONMENT OF METASTATIC BRAIN TUMORS
Authors: Sun Lu , Kienzler Jenny , Lee Alexander , Chow Frances , Chavez Carolina , Yong William , Everson Richard , Kim Won , Hugo Willy , Prins Robert
Published: December 2020

Brain metastases (BM) commonly arise in patients with melanoma, lung, and breast cancer. Currently, there are limited options for GBM and BM patients who have failed the first-line standard treatment, underscoring the importance of developing new therapeutic strategies. Last year, we and other groups evaluated the neoadjuvant timing of anti-PD-1 checkpoint blockade therapy in recurrent GBM (rGBM) patients, which resulted in a modest survival benefit. In light of the known effectiveness of anti-PD-1 as a systemic therapy to control melanoma and non-small cell lung cancer BM, we set out to study the anti-tumor immune response of BM patients to anti-PD-1 in the neoadjuvant setting. We posited that neoadjuvant anti-PD-1 in patients with BM would result in a stronger antitumoral immune response, which could be quantified at the single cell level. To test this, we made use of contemporary single cell techniques, including multiplex immunofluorescence, time-of-flight mass cytometry (CyTOF) and single-cell RNA sequencing (scRNAseq), to characterize the intratumoral immune cell populations and their transcriptomic profiles. We found that neoadjuvant anti-PD-1 significantly increased the number of tumor infiltrating T lymphocytes in BM compared to rGBM (2.5 fold in BM, p= 0.02 vs. 1.4 fold in rGBM, p= 0.19). Multiplex immunofluorescence analysis of T cells in BM samples revealed a change from T cell exclusion to a diffusely infiltrating phenotype after anti-PD-1 treatment. Importantly, BM showed a higher fraction of effector/cytotoxic T cells compared to rGBM (7.3% vs. 0.9% of lymphoid cells, p= 0.005) and anti-PD-1 further enhanced this population. In the myeloid compartment of BM, neoadjuvant anti-PD-1 increased the frequency of HLA-DR+CD206- M1-like macrophages, implicating a pro-inflammatory microenvironment. In summary, our study delineated the immune cell subtypes altered by neoadjuvant anti-PD-1 and offers insights into new combination therapies that can help understand the clinical efficacy of immunotherapy for BM and GBM patients.
TAMI-02. ALTERATIONS IN C-X3-C MOTIF CHEMOKINE RECEPTOR 1 (CX3CR1) EXPRESSION INFLUENCE MICROGLIAL AND MACROPHAGE RESPONSE IN DIFFERENT STEPS OF CEREBRAL METASTASIS FORMATION
Authors: Zhang Wenlong , von Mücke-Heim Iven-Alex , Mulazzani Matthias , Karschnia Philipp , Straube Andreas , von Baumgarten Louisa
Published: December 2020

Metastasis to the brain is a frequent complication in lung cancer and is still associated with a dismal prognosis. Current treatment strategies not only target tumor cells but also focus on cells of the tumor microenvironment like tumor associated microglia/macrophages (TAMs). The interactions between tumor cells and TAMs during different steps of cerebral metastasis formation of lung cancer brain metastasis are poorly characterized. Moreover, the role of CX3CR1 in this process remains unclear. We established a syngeneic cerebral metastasis mouse model by combining a chronic cranial window and two-photon laser scanning microscopy (TPLSM), which allows the tracking of single fluorescent metastasizing tumor cells and the tumor microenvironment on a cellular resolution in vivoover time for a period of weeks. Transgenic CX3CR1 proficient and deficient mice (CX3CRGFP/wt and CX3CR1GFP/GFP) were injected with red fluorescent Lewis lung carcinoma cells. During different steps of metastasis formation (extravasation, formation of micro- and macrometastasis) the density and cell body volume of TAMs, their interaction with tumor cells and possible influence on the fate of single metastatic tumor cells were investigated using serial TPLSM. We found that during metastasis formation TAM density was significantly lower in CX3CR1 deficient mice. However, activation as assessed by TAM morphology did not differ in the absence of CX3CR1. Strikingly, CX3CR1 deficiency was associated with a significant increase of tumor cells successfully extravasating the cerebral vasculature. However, subsequent steps (mirco- and macrometastasis formation) were observed less frequent in CX3CR1 deficient mice. In summary, our results highlight a complex role of CX3CR1 for TAMs during cerebral metastasis formation, indicating anti-tumorous properties of CX3CR1 at early steps and possible pro-tumorous effects at later stages (micro- and macrometastasis formation).
TAMI-06. PRECLINICAL MODELS REVEAL BRAIN-MICROENVIRONMENT SPECIFIC METABOLIC DEPENDENCIES IN GLIOBLASTOMA
Authors: Minami Jenna , Bayley Nicholas , Tse Christopher , Zhu Henan , Morrow Danielle , Yong William , Liau Linda , Cloughesy Timothy , Graeber Thomas , Nathanson David
Published: December 2020

Metabolic reprogramming is a hallmark of cancer, and malignant cells must acquire metabolic adaptations to fuel neoplastic progression. Mutations or changes in metabolic gene expression can impose nutrient dependencies in tumors, and even in the absence of metabolic defects, cancer cells can become auxotrophic for particular nutrients or metabolic byproducts generated by other cells in the tumor microenvironment (TME). Conventional cell lines do not recapitulate the metabolic heterogeneity of glioblastoma (GBM), while primary cultured cells do not account for the influences of the microenvironment and the blood brain barrier on tumor biology. Additionally, these systems are under strong selective pressure divergent from that in vivo, leading to reduced heterogeneity between cultured tumor cells. Here, we describe a biobank of direct-from-patient derived orthotopic xenografts (GliomaPDOX) and gliomaspheres that reveal a subset of gliomas that, while able to form in vivo, cannot survive in vitro. RNA sequencing of tumors that can form both in vivo and in vitro (termed “TME-Indifferent”) compared to that of tumors that can only form in vivo (termed “TME-Dependent”) revealed transcriptional changes associated with altered nutrient availability, emphasizing the unique metabolic programs impacted by the tumor microenvironment. Furthermore, TME-dependent tumors lack metabolic signatures associated with nutrient biosynthesis, thus indicating a potential dependency of these tumors on scavenging specific nutrients from the extracellular milieu. Collectively, these data emphasize the metabolic heterogeneity within GBM, and reveal a subset of gliomas that lack metabolic plasticity, indicating a potential brain-microenvironment specific metabolic dependency that can be targeted for therapy.
TAMI-13. IMPORTANCE OF CD38 INHIBITION IN PRE-RECURRENT GLIOBLASTOMA
Authors: Hirte Renee , Olson Ian E , Rahman Masum , Mansour Moustafa , Casabella Amanda Munoz , Burns Terry C
Published: December 2020

Glioblastoma multiforme (GBM) is the most aggressive CNS neoplasm with a mere 15 month progression-free survival following current standards-of-care. Furthermore, conventional therapies have been effective, in part, by inducing a senescent-like phenotype in at least a proportion of glioma, as well as within the tumor-adjacent, healthy brain cells. Through an N-glyco-capture large-scale screening method, we were able to identify 25 genes exclusively overexpressed on glioma cell surfaces. Of those 25 targets, CD38 was an attractive target due to pre-existing FDA approved therapeutics. Recent studies have shown, however, that senescent cells, such as those induced via chemo- and radiotherapies, secrete a pro-inflammatory array of cytokines, known collectively as senescence associated secretory phenotype (SASP), that are capable of increasing CD38 expression in monocytes. CD38 functions as an ectoenzyme to convert extracellular NAD+ to nicotinamide (required for glioma cell salvage pathway NAD+ synthesis) and ADPR/cADPR (a cellular proliferation signal). To examine the effects of radiation on human glioma tissue, we performed reverse cyclase assays (RCA) on paired primary and recurrent human glioma tissue samples and found an increase in CD38 activity in post-irradiated tissues (recurrent glioma) compared to pre-irradiated (primary glioma). Through proliferation assays, we also found an increase in glioma cell growth following treatment with cADPR compared to untreated. Our results demonstrate that CD38 activity is tumorgenic, and furthermore that conventional chemo- and radiotherapies increase this CD38 activity. This indicates that treating CD38 with previously FDA approved therapeutics may provide hope for increasing progression free survival in our GBM patient population, and moreover emphasize the importance of leveraging novel large scale screening methods for identifying additional opportunities for treatment.
TAMI-15. THE EFFECT OF INTERSTITIAL FLUID FLOW AND ASTROCYTES / MICROGLIA ON INVASION, PROLIFERATION AND STEMNESS OF PATIENT-DERIVED GLIOMA STEM CELLS
Authors: Atay Naciye , Yuan Jessica , Cornelison Chase , Munson Jennifer
Published: December 2020

SIGNIFICANCE

Glioblastoma is a highly infiltrative, malignant, and deadly glioma that can be classified into subtypes based on molecular classification. Treatment resistant glioma stem cells (GSCs) depend on the tumor microenvironment (TME) to drive recurrence. Cellular composition and interstitial fluid flow (IFF) are significant aspects of the TME. IFF and astrocyte and microglia (A+M) presence have independently been shown to mediate invasion. This study’s goal is to expand our knowledge of IFF and A+M effects on invasion to proliferation and stemness.

METHODS

Seven patient-derived GSC lines were tested in an in vitro 3D model, which consists of GSCs ± A+M resuspended in 0.2% hyaluronan / 0.12% rat tail collagen I gel. The gel was applied to an 8um pore 96-well transwell system. Flow and static conditions were modeled with and without a pressure head above the gel, respectively. Cells beyond the transwell membrane after 18 hrs of incubation were considered invaded. Stemness and proliferation were determined via flow cytometry for CD71 and Ki67, respectively.

RESULTS/CONCLUSIONS

The three mesenchymal GSC lines tested exhibited the largest IFF fold increases in stemness, proliferation, and invasion with averages of 23.9, 19.1, and 2.1, respectively. CD44+ cell populations, highest in mesenchymal cells, had a strong correlation with proliferation (R=0.8439) and stemness (R=0.7829) under flow. Furthermore, depending on the cell line/subtype, the addition of A+M either amplified, reduced, reversed, mitigated, or kept constant the effect of IFF on invasion and proliferation. Incorporating A+M never amplified the effect of IFF on stemness. Adding A+M had a strong effect on the IFF fold change of at least one parameter in six of the cell lines. This is the first presentation showing that IFF, patient-specific, and context-specific factors contribute to both increased proliferation, and maintenance of stem-like phenotypes in glioma.
TAMI-16. BIOMATERIAL MATRICES TO STUDY GLIOBLASTOMA INVASION
Authors: Sohrabi Alireza , Guivatchian Elnaz , Xiao Weikun , Liang Jesse , Ehsanipour Arshia , Condro Michael , Kornblum Harley , Seidlits Stephanie
Published: December 2020

INTRODUCTION

Glioblastoma (GBM) is a highly infiltrative and lethal brain cancer. Previous studies have suggested that GBM tumors are stiffer than healthy brain tissues. We posit that local changes in the mechanical microenvironment near the tumor/tissue interface promote migration of GBM cells away from primary tumors. These studies seek to improve our understanding of how mechanical changes to the microenvironment of GBM tumors and surrounding brain tissue drive GBM progression using an in vitro, 3D model incorporating patient-derived primary GBM cells and a brain matrix-mimetic scaffold in which mechanical modulus can be varied.

METHODS

GBM xenografts were explanted and vibratome sectioning of fresh tissues for AFM. Hydrogels were fabricated by crosslinking thiolated hyaluronic acid (HA-SH), 8-arm polyethylene glycol (PEG)-norbornene and 4-arm PEG-thiol in the presence of photoinitiator UV light. Mechanical properties were measured using both rheology and AFM. Patient-derived GBM cells were maintained in 3D culture and migration monitored.

RESULTS AND DISCUSSION

AFM measurements of xenograft and brain tissue suggest that modulus is highest near the tumor center, decreases near the tumor border and is lowest in surrounding brain tissue. Hydrogels were tuned to match in vivo mechanics by altering total thiol content while matrix composition and effective pore size remained constant. GBM cells invaded readily in soft hydrogels, but were not able to migrate in stiff hydrogels. When cultured in platforms including defined regions in which moduli were varied, GBM cells seeded in stiffer regions readily migrated towards softer regions, while cells seeded into a softer matrix did not migrate to stiffer regions. Mechanical properties of the microenvironment appear to have marked effects on tumor cell migration and may drive GBM migration into surrounding tissues. In the long-term, we expect these studies to inform development of new therapeutics targeting GBM tumor invasion through improved understanding of underlying mechanisms.
TAMI-19. METABOLIC INTERACTIONS BETWEEN TUMOR CELLS AND THE IMMUNE SYSTEM IN GBM: A POTENTIAL ACHILLES HEEL OF GBM FOR NOVEL THERAPEUTICS
Authors: Tian Guimei , Yang Changlin , Andrews Michael , Karachi Aida , Dajac Mariana , Doshi Devshri , Mendez-Gomez Hector , Moore Ginger , Flores-Toro Joseph , Rahman Maryam , Huang Jianping , Sayour Elias , Harrison Jeffrey , Mitchell Duane , Deleyrolle Loic
Published: December 2020

INTRODUCTION

Glioblastoma (GBM) contains cell populations with distinct metabolic requirements, with fast-cycling cells harnessing aerobic glycolysis, and treatment-resistant slow-cycling cells (SCCs) preferentially engaging lipid metabolism. How the different tumor cells interact with immune cells and how this metabolic heterogeneity shapes the immune landscape in GBM has yet to be understood.

OBJECTIVES

The objectives are to unravel the various molecular signals and metabolic link that underlie the interaction of SCCs with the GBM microenvironment, in particular with the suppressive immune compartment, and to effectively target these interactions for better therapeutics.

METHODS

Multiple murine glioma cell lines were used to establish metabolic heterogeneity and communications, while various genetic and pharmacological approaches were applied to assess the effect of disrupting the metabolic interplay between SCCs and the immune system.

RESULTS

We determined that SCCs exhibit distinct metabolic dependencies, involving preferential lipid metabolism supported by enhanced fatty acid uptake. We also found that tumor progression is regulated by the interaction of SCCs with the immune system and established that SCCs recruit immune suppressive M2-like macrophages to the tumor microenvironment, which in turn work against tumor immune rejection by inhibiting T cell anti-tumor activity. The immune microenvironment shaped by SCCs is marked by specific metabolic features enhancing lipid exchange capacities that are exploited by SCCs to support their survival and functions. Importantly, disrupting lipid metabolic exchange sensitized tumors to chemotherapy.

CONCLUSION

Our results reveal that metabolic interactions between SCCs and tumor-associated macrophages within the GBM microenvironment play a critical role in the development of drug and immune resistant tumors. This study delineates these metabolic communications and assesses the potential therapeutic effect of disrupting these interactions to treat GBM. The insights generated from this project uncover fundamental principles of the emerging connections between the tumor microenvironment, cell metabolism, anti-tumor immunity, and associated therapeutic vulnerabilities.
TAMI-20. POLY(ADP-RIBOSE) GLYCOHYDROLASE INHIBITION SEQUESTERS NAD+ TO POTENTIATE THE METABOLIC LETHALITY OF ALKYLATING CHEMOTHERAPY IN IDH MUTANT TUMOR CELLS
Authors: Nagashima Hiroaki , Lee Christine , Tateishi Kensuke , Higuchi Fumi , Subramanian Megha , Rafferty Seamus , Melamed Lisa , Miller Julie , Wakimoto Hiroaki , Cahill Daniel
Published: December 2020

Mutations in IDH1 or IDH2 characterize the majority of diffuse infiltrative gliomas of younger adulthood. DNA alkylator chemotherapy has proven to be an effective treatment for IDH mutant glioma, yet recurrences remain frequent and improved treatments are needed. Upon exposure of cancer cells to DNA alkylating agents, poly(ADP-ribose) polymerases (PARPs) catalyze the transient oligomerization of nicotinamide adenine dinucleotide (NAD+) into poly-ADP-ribose (PAR) chain-assemblies, which stimulate the DNA damage response. Here, we find that, in IDH mutant glioma cells, alkylator-induced cytotoxicity is dramatically augmented by pharmacologic inhibition or genetic knockout of the PAR breakdown enzyme poly(ADP-ribose) glycohydrolase (PARG). Mechanistically, we show this augmentation is driven by a metabolic state pairing amplified alkylator-induced DNA damage with catastrophic depletion of NAD+. IDH mutant cancer cells have low basal levels of NAD+, rendering them vulnerable to prolonged NAD+ depletion. Either of two clinically-utilized monofunctional alkylators, procarbazine (PCZ) or temozolomide (TMZ), when exposed to multiple IDH mutant lines, activated PARP conversion of cellular NAD+ monomers into polymerized PAR. Subsequent PAR breakdown and recycling of NAD+ monomers was then halted by concomitant PARG inactivation, amplifying PARylation-mediated DNA damage signaling and critically depleting free NAD+, resulting in profound cytotoxicity. This effect was partially reversed by NAD+ rescue supplementation, confirming the key contribution of metabolic stress. Combined alkylator treatment and PARG inhibition in vivo maximized both NAD+ depletion and cytotoxicity in an endogenous IDH mutant cancer model. Thus, alkyating DNA damaging agents expose a metabolic liability in IDH mutant cancers, and subsequent targeted blockade of PAR breakdown and NAD+ metabolite recovery can exploit this vulnerability to provide genotype-specific therapeutic benefit.
TAMI-29. MULTIFACTORIAL UPREGULATION OF ID1 DRIVES DIPG INVASIVENESS AND IS THERAPEUTICALLY TARGETABLE
Authors: Nand Yadav Viveka , Harris Micah K , Stallard Stefanie , Woo Rinette , Siddaway Robert , Qin Tingting , Cummings Jessica , Mullan Brendan , Miklja Zachary , Siada Ruby , Ravindran Ramya , Cao Xuhong , Pasternak Amy , Castro Maria G , Lowenstein Pedro , Mody Rajen , Chinnaiyan Arul , Hawkins Cynthia , Desprez Pierre , McAllister Sean , Venneti Sriram , Koschmann Carl
Published: December 2020

Diffuse intrinsic pontine gliomas (DIPGs) are lethal brain tumors with no effective therapies other than radiation. Inhibitor of DNA binding (ID) proteins, key regulators of lineage commitment during embryogenesis, are implicated in tumorigenesis in multiple human cancers. Prior work showed that recurrent H3F3A and ACVR1 mutations increase ID1 expression in cultured astrocytes. However, this has not been validated in human DIPG. The regulation and targetability of ID1 in DIPG has not been explored either. Exome and transcriptome sequencing analysis of multi-focal DIPG tumors and normal brain tissue from autopsy (n=52) revealed that ID1 expression is significantly elevated in DIPG tissues. Higher ID1 expression correlates with reduced survival in DIPG patients and increased regional invasion in multi-focal autopsy samples. Analyses of developing mouse brain RNA/ChiP-Seq data revealed high ID1 expression and H3K27ac promoter binding in prenatal hind brain compared to all other prenatal and postnatal brain regions. ChIP-qPCR for H3K27ac and H3K27me3 revealed that ID1 gene regulatory regions are epigenetically poised for upregulation in DIPG tissues compared to normal brain, regardless of H3/ACVR1 mutational status. These data support that the developing pons is regionally poised for ID1 activation. Genetic (shRNA) ID1 knockdown in primary human H3.3K27M-DIPG cells (DIPG007) resulted in significantly reduced invasion and migration in vitro. Additionally, DIPG-ID1-KO cells showed improved sensitivity to radiation therapy. Phospho-kinase array analysis of DIPG cells revealed that Akt and WNK1 activity were significantly downregulated upon ID1 knockdown, which was previously shown in lung tumors. Treatment of DIPG007 cells with cannabidiol (CBD) reduced ID1 expression levels and viability/proliferation of DIPG cells in vitro. ID1 knockdown and CBD treatment studies in vivo are ongoing. In summary, our findings indicate that multifactorial (genetic and regional) epigenetic upregulation of ID1 drives DIPG invasiveness and targeting ID1 using CBD may be a potential strategy for the treatment of DIPGs.
TAMI-33. AURKA INHIBITION REPROGRAMS METABOLISM AND IS SYNTHETICALLY LETHAL WITH FATTY ACID OXIDATION INHIBITION IN GLIOBLASTOMA
Authors: Nguyen Trang , Shang Enyuan , Shu Chang , Mela Angeliki , Humala Nelson , Mahajan Aayushi , Quinzii Catarina , Westhoff Mike-Andrew , Karpel-Massler Georg , Bruce Jeffrey , Canoll Peter , Siegelin Markus
Published: December 2020

Aurora kinase A (AURKA) has emerged as a viable drug target for glioblastoma (GBM), the most common malignant primary brain tumor in adults with a life expectancy of 12-15 months. However, resistance to therapy remains a critical issue, which partially may be driven by reprogramming of metabolism. By integration of transcriptome, chromatin immunoprecipitation with sequencing (CHIP-seq.), assay for transposase-accessible chromatin with sequencing (ATAC-seq.), proteomic and metabolite screening followed by carbon tracing (U-13C-Glucose, U-13C-Glutamine and U-13C-Palmitic acid) and extracellular flux analysis we provided evidence that genetic (shRNA and CRISPR/Cas9) and pharmacological (Alisertib) AURKA inhibition elicited substantial metabolic reprogramming mediated in part by inhibition of MYC targets and concomitant activation of PPARA (e.g. PGC1A) signaling. While glycolysis was suppressed by AURKA inhibition, we noted a compensatory increase in oxygen consumption rate (OCR) fueled by fatty acid oxidation (FAO). Whereas interference with AURKA elicited a suppression of c-Myc, we detected an upregulation of PGC1A, a master regulator of oxidative metabolism, upon AURKA inhibition. Silencing of PGC1A reversed the increase in OCR and sensitized GBM cells to AURKA inhibition mediated reduction in cellular viability. CHIP experiments confirmed binding of c-Myc to the promoter region of PGC1A, which is abrogated by AURKA inhibition and in turn unleashed PGC1A expression. ATAC-seq. confirmed higher accessibility of the MYC binding region within the PGC1A promoter. Forced expression of c-Myc blocked AURKA inhibition mediated increase of PGC1A, suggesting that c-Myc acted as a repressor. To interfere with this oxidative metabolic reprogramming, we combined AURKA inhibitors with blockers of FAO (etomoxir), which elicited substantial synergistic growth inhibition and extension of overall survival in orthotopic patient derived xenografts of GBM in mice without induction of toxicity in normal tissue. Taken together, these data support that simultaneous targeting of oxidative metabolism and AURKA inhibition might be a potential novel therapy against GBM.
TAMI-34. SEX-SPECIFIC METABOLIC ADAPTATIONS TO THE KETOGENIC DIET IN A MOUSE MODEL OF GLIOBLASTOMA
Authors: Zhang Shanshan , Sponagel Jasmin , Rubin Joshua , Ippolito Joseph
Published: December 2020

Glioblastoma (GBM) is a lethal brain tumor that has a higher incidence and mortality in males compared to females. GBM tumors consume glucose and glutamine as the major fuels for growth and strategies have been developed that disrupt these nutrient utilization pathways. Both pre-clinical and clinical reports have identified the ketogenic diet (KD) as an approach which consists of high fat and low carbohydrates. It reduces circulating glucose and increases ketones resulting in restricted tumor growth. Because of published studies showing that carbohydrate utilization is higher in healthy males, we hypothesized that the KD would have greater inhibition of male GBM growth. We compared sex differences in response to the KD using an established GBM mouse model (Nf1-/-DNp53), which has previously yielded important insights into sex differences in GBM. Flank tumors were implanted and tumor size measurements were performed. Unexpectedly, the KD had no effect on male GBM tumor growth, but female tumors grew significantly slower in mice that were on the KD compared to control diet. To understand how male GBM tumors adapt their metabolism to the KD, we measured serial blood glucose and ketone levels. We discovered expected physiologic responses to the KD with decreased serum glucose and increased serum ketones, suggesting that males adapt to the KD through changes in cell-intrinsic metabolism. Using FDG-PET imaging to investigate glucose utilization, we found that FDG uptake was increased in both tumor and normal brain of male but not female mice that were on the KD compared to control diet. Taken together, our results show that the KD has a greater impact on female GBM tumor growth and that sex differences in metabolic adaptations such as increased glucose transport may underlie the male-specific resistance to the KD. This study has the potential to develop sex-specific metabolic treatment approaches for GBM.
TAMI-38. CYSTEINE-PROMOTING COMPOUNDS INDUCE MITOCHONDRIAL TOXICITY IN GLIOBLASTOMA THROUGH ALTERED PYRUVATE AND SERINE METABOLISM
Authors: Noch Evan , Palma Laura , Yim Isaiah , Binder Bhavneet , Benedetti Elisa , Krumsiek Jan , Elemento Olivier , Cantley Lewis
Published: December 2020

Glioblastoma (GBM) remains a poorly treatable disease with high mortality. Tumor metabolism in GBM is a critical mechanism responsible for accelerated growth because of upregulation of glucose, amino acid, and fatty acid utilization. However, little is known about the metabolic alterations that are specific to GBM and that are targetable with FDA-approved compounds. To investigate tumor metabolism signatures unique to GBM, we interrogated the TCGA and a cancer metabolite database for alterations in glucose and amino acid signatures in GBM relative to other human cancers and relative to low-grade glioma. From these analyses, we found that GBM exhibits the highest levels of cysteine and methionine pathway gene expression of 32 human cancers and that GBM exhibits high levels of cysteine-related metabolites compared to low-grade gliomas. To study the role of cysteine in GBM pathogenesis, we treated patient-derived GBM cells with a variety of FDA-approved cyst(e)ine-promoting compounds in vitro, including N-acetylcysteine (NAC) and the cephalosporin antibiotic, Ceftriaxone (CTX), which induces cystine import through System Xc transporter upregulation. Cysteine-promoting compounds, including NAC and CTX, inhibit growth of GBM cells, which is exacerbated by glucose deprivation. This growth inhibition is associated with reduced mitochondrial metabolism, manifest by reduction in ATP, NADPH/NADP+ ratio, mitochondrial membrane potential, and oxygen consumption rate. Metabolic tracing experiments with 13C6-glucose demonstrate that L-serine is rapidly depleted in GBM cells upon treatment with NAC and CTX, and exogenous serine rescues NAC- and CTX-mediated cell growth inhibition. In addition, these compounds reduce GBM mitochondrial pyruvate transport. We show that cysteine-promoting compounds reduce cell growth and induce mitochondrial toxicity in GBM, which may be due to rapid serine depletion and reduced mitochondrial pyruvate transport. This metabolic phenotype is exacerbated by glucose deprivation. This pathway is targetable with FDA-approved cysteine-promoting compounds and could synergize with glucose-lowering treatments, including the ketogenic diet, for GBM.
TAMI-43. IMPACT OF SEX AND RADIATION ON IRON TRAFFICKING IN BONE MARROW DERIVED MACROPHAGES
Authors: Shenoy Ganesh , Snyder Amanda , Slagle-Webb Becky , Marshall Savannah , Schell Todd , Chroneos Zissis , Davalos Dimitrios , Berens Michael , Lathia Justin , Barnholtz-Sloan Jill S , Rubin Joshua , Connor James
Published: December 2020

The tumor microenvironment in glioblastoma provides cancer cells with favorable conditions to proliferate and invade surrounding tissues. Macrophages comprise a large portion of the glioblastoma tumor microenvironment (TME) both in terms of volume and function. These cells have been reported to influence tumor progression by modulating immune responses, remodeling extracellular matrix, and providing nutrients to cancer cells among numerous other functions. Radiation therapy forms one of the pillars of glioblastoma management along with surgical resection and chemotherapy. Here we investigated the effects of radiation on macrophage iron metabolism. Using mouse bone-marrow-derived macrophages (BMDMs) we performed in-vitro 59Fe radiotracer assays to study how radiation exposure modified iron trafficking in these cells. We found that low dose radiation at 0.25, 0.5, or 2 Gy from a 60Co source stimulated iron release from the BMDMs with maximal release occurring at 0.5 Gy. Moreover, we observed that iron release was dependent on the amount of serum present in culture media with cells cultured in 20% fetal bovine serum (FBS) showing reduced iron release profiles compared to those cultured in 10% or 1% FBS. Since glioblastoma patients exhibit sexually dimorphic survival outcomes, we investigated whether these radiation-induced responses occurred in a sexually dimorphic pattern. At radiation doses of 0.25 Gy we observed that male macrophages tended to release more iron than female macrophages despite no differences in iron uptake between the sexes – raising the question as to whether differential iron trafficking in response to treatment contributes to the poorer survival outcomes observed in males. Our data suggest that delineating how supporting cells such as macrophages respond to glioblastoma treatment regimens may provide insights into addressing mechanisms of treatment resistance and further our understanding of the sexual dimorphism observed in patient outcomes.
TAMI-45. PHENOGENOMIC CHARACTERIZATION OF IMMUNOMODULATORY PURINERGIC SIGNALING IN GLIOBLASTOMA
Authors: Coy Shannon , Rashid Rumana , Stopka Sylwia , Lin Jia-Ren , Euskirchen Philipp , Hwang Jaeho , Khadka Prasidda , Bandopadhayay Pratiti , Wen Patrick , Sorger Peter , Agar Nathalie Y R , Ligon Keith , Touat Mehdi , Santagata Sandro
Published: December 2020

INTRODUCTION

Purinergic signaling plays critical roles in the regulation of tumor growth and anti-tumor immunity via autocrine/paracrine binding of metabolites to receptors on neoplastic and non-neoplastic populations. Extracellular purine concentrations are mediated by the ectonucleotidase enzymes CD39 and CD73, which catabolize ATP to adenosine. Within tumors such as glioblastoma, neoplastic, immune, and stromal cells expressing these enzymes may co-localize to generate immunosuppressive adenosine-rich environments. However, the composition, architecture, and phenotypic properties of these tumor ecosystems and their relationship to tumor genotype are poorly characterized.

METHODS

We quantified CD73 expression by immunohistochemistry in a cohort of CNS tumors [meningiomas(n=222), gliomas(n=244), ependymomas(n=44), medulloblastomas(n=24), and craniopharyngiomas(n=38)]. We used publicly-available single-cell RNA-seq data and 36-marker multiplexed tissue imaging (t-CyCIF) of 139 clinically and genomically annotated glioblastoma resections to characterize CD39 and CD73-expressing populations, define the immune architecture and tumor cell-states at single cell resolution, and identify markers of clinical outcome. We used mass spectrometry imaging (MALDI-MSI) to generate spatially-resolved quantification of purine metabolite levels in glioblastoma resections (n=10).

RESULTS

CD73 exhibited strong expression in a subset of gliomas and meningiomas but was typically not expressed in ependymomas or medulloblastomas. CD73 expression correlated with poor progression-free-survival in IDH-wildtype glioblastoma (p=0.04). scRNA-seq and t-CyCIF in glioblastoma showed CD73 expression in tumor cells, and CD39 expression in macrophages and endothelial cells. MALDI-MSI showed significantly greater adenosine concentrations (3.5-fold;p=0.04) in glioblastomas with high CD73 expression. scRNA-seq showed direct correlations between stem-like mRNA expression, proliferation, and CD73 expression in DIPG. CD73 expression significantly correlated with EGFR amplification, interferon signaling, and PD-L1 expression in glioblastoma.

CONCLUSIONS

Phenogenomic analysis of purinergic immunomodulatory signaling revealed significant interplay between CD73 activity and genotype, adenosine concentration, differentiation-state, clinical outcome, and possible interaction between CD39-positive macrophages and CD73-positive neoplastic cells. Anti-CD73 therapy may provide therapeutic benefits in glioblastoma by blunting immunosuppressive and oncogenic adenosine signaling.
TAMI-46. TNFα SECRETED BY GLIOMA ASSOCIATED MACROPHAGES PROMOTES ENDOTHELIAL ACTIVATION AND RESISTANCE AGAINST ANTI-ANGIOGENIC THERAPY
Authors: Wei Qingxia , Singh Olivia , Ekinci Can , Gill Jaspreet , Li Mira , Yasin Mamatjan , Karimi Shirin , Bunda Severa , Mansouri Sheila , Aldape Kenneth , Zadeh Gelareh
Published: December 2020

Glioblastoma (GBM) remains a universal fatal disease and improving survival remains a challenge. One of the most prominent features of GBM is hyper-vascularization, characterized by abnormally dilated, distorted, and leaky vessels. Bone marrow (BM)-derived macrophages are recognized to be an important host cell population that are actively recruited to the tumor and referred to as glioma-associated macrophages (GAMs). GAMs are found to associate closely with blood vessels, and thought to provide a critical role in tumor neo-vascularization. However, the mechanisms by which GAMs regulate and promote endothelial cells (ECs) in the process of tumor vascularization and response to anti-angiogenic therapy (AATx) is not understood. To understand how GBM, GAMs and EC interacts, we designed and developed a novel GBMs-GAMs-EC co-culture system as well as a unique in vivo model in which the BM of NOD/SCID mice were reconstituted with red fluorescent protein (RFP)-BM cells to create a chimeric mouse and then using intracranial injection of GFP-U87 cells to create a GBM xenograft. Our study is the first to demonstrate that glioma cells-secreted IL8 and CCL2 which stimulate GAMs to secrete TNFα and promote EC activation. TNFα inhibition led to normalization of the tumor vasculature and significant improvement in survival in glioma mice model. More importantly, we validate these findings using clinical data showing that TNFα is a predictor of overall survival and response to AATx in GBM patients. We further demonstrated that increased macrophage recruitment and upregulation of TNFα in GAMs activating EC in response to bevacizumab treatment is one of the critical molecular mechanisms underlying the failure of AATx in GBM. Collectively, these results provide compelling evidence to further explore the clinical impact of inhibiting TNFα concurrent with AATx.
TAMI-57. MEDULLOBLASTOMA UTILIZE GABA TRANSAMINASE TO SURVIVE IN THE CEREBROSPINAL FLUID MICROENVIRONMENT AND PROMOTE LEPTOMENINGEAL DISSEMINATION
Authors: Martirosian Vahan , Deshpande Krutika , Zhou Hao , Shen Keyue , Stepanosyan Vazgen , Remsik Jan , Boire Adrienne , Isakov Danielle , De Feyter Henk , Hurth Kyle , Nakamura Brooke , Shao Ling , Northcott Paul , Wiemels Joe , Li Shaobo , Neman Josh
Published: December 2020

Medulloblastoma (MB) is a malignant pediatric brain tumor. Studies have shown heterogeneous cells amongst the tumor bulk which mirror normal neural cells in various neurodevelopmental stages. To discern exploited mechanisms promoting MB leptomeningeal disease, we drew conclusions from developmental neurobiology. In normal differentiation, the metabolic phenotype in proliferating neural progenitor cells evolves from a glycolysis-dependent to an oxidative phosphorylation-reliant energetic profile in quiescent differentiated neurons. Cancer cells mirror this evolution, which also grants them the capability to utilize alternative nutrients in the microenvironment as an energy source. Considering metastatic cells are typically in a dormant state and primarily utilize oxidative phosphorylation, we hypothesized metastatic MB cells emulate a quiescent neuron-like cellular profile to survive in the cerebrospinal fluid and form leptomeningeal metastases. To examine this, we query the expression of GABA catabolic enzyme GABA transaminase (ABAT) in MB. GABA is found in the cerebellar and leptomeningeal microenvironments, and is utilized by metastatic cancer cells in the CNS as an energy source. We correlate an increase in ABAT expression with neurodevelopment and show heterogeneous expression of this protein in primary MB tumors. MB cells with increased expression of ABAT were slower-dividing, expressed a genetic and metabolic phenotype reminiscent of quiescent neuron-like cells, and had increased capability to metabolize GABA. Conversely, lower expression of ABAT was associated with an increased proliferation rate and correlated with a progenitor-like cellular profile. Transplantation of MB cells into the leptomeningeal compartment decreased proliferative capacity and enhanced ABAT expression. Xenograft models showed MB cells with ABAT knockdown had increased growth in the cerebellar microenvironment. Conversely, MB cells with ABAT overexpression transplanted into the cerebrospinal fluid formed leptomeningeal metastases whereas ABAT knockdown cells could not. These results suggest ABAT expression in MB cells can be modulated by the tumor microenvironment and is required to form leptomeningeal metastases.
TAMI-58. A NOVEL ROLE FOR REST IN THE CONTROL OF THE MEDULLOBLASTOMA MICROENVIRONMENT
Authors: Shaik Shavali , Maegawa Shinji , Sharma Ajay , Haltom Amanda , Dobson Tara , Yang Yanwen , Schadler Keri , Gopalakrishnan Vidya
Published: December 2020

The REI Silensing Transcription Factor (REST) is a transcriptional repressor and a canonical regulator of neurogenesis. Its expression is elevated in human sonic hedgehog (SHH) subgroup of medulloblastomas (MBs), where functional studies shown its elevated expression to promote proliferation and block neuronal specification. A role for REST in the control of the MB tumor microenvironment (TME) has not been described previously. Here, we demonstrate that REST also controls the MB-TME, and specifically vascular remodeling. Using our unique RESTTG mouse model, we show that conditional expression of human REST transgene in cerebellar granule neuron progenitors (CGNPs), the cell of origin of a subset of SHH MBs, promoted increased vascular growth. In the context of constitutive activation of SHH signaling, a key driver of SHH-MB development, REST elevation drove tumor progression by altering the tumor vasculature. These findings were validated in mouse orthotopic models of human MB cell lines and through analyses of publicly available transcriptomic database of human MB samples. A strong positive correlation between REST and that of endothelial genes CD31/VEGFR1/ETS1 was seen in samples from patients with SHH-MBs subtypes that are associated with the worst prognosis. Proteomic analyses identified increased secretion of a number of pro-angiogenic factors in the context of upregulated REST expression in MB cells. Unexpectedly, in vitro and in vivo studies showed that MB cells expressed these endothelial markers and co-localized with endothelial cells suggesting that REST elevation may have altered the fate of cells that were destined to become neurons. Finally, ETS1 knockdown in MB cell lines not only downregulated VEGFR1 levels in these cells, and blocked tube formation in vitro, but also caused a reduction in tumor cell co-localization with endothelial cells. Collectively, these data suggest that REST elevation remodels MB vasculature through cell-intrinsic and cell-extrinsic mechanisms.
TAMI-60. EXPLORING METABOLIC DIFFERENCES IN VARYING GRADES OF MENINGIOMAS THROUGH NONTARGETED MASS SPECTROMETRY
Authors: Rabow Zach , Morningstar Taryn , Lechpammer Mirna , Fiehn Oliver
Published: December 2020

Emerging molecular data demonstrates the importance of genomic and epigenetic factors in pathogenesis of meningioma. Understanding the metabolic landscape using mass spectrometry is needed to overcome significant uncertainty in predicting tumor behavior and the risk of recurrence. Current technologies lack sensitivity and selectivity, which hinders the discovery of potential novel diagnostic and prognostic features. Here, we present a nontargeted metabolomics approach applied to meningiomas for biomarker discovery and identification of potential therapeutic targets. Fresh frozen tissue from 36 patients (57% women, 43% men; mean age: 48) who underwent surgical resection for newly diagnosed meningiomas and 9 patient samples with non-neoplastic dura were used for case/control comparison. Additionally, 16 patient-derived meningioma cell lines (Grade I-III), 2 immortalized human cell lines (IOMM-Lee and Ch157MN) and immortalized arachnoid cells were used to further explore metabolic changes in meningiomas. Metabolites were extracted from tissue and cells for GC-TOF (primary metabolism), RPLC ESI (±) (lipidomics) and HILIC ESI (±) (biogenic amines) coupled to high-resolution mass spectrometry for analysis. Metabolites were identified using authentic standards, retention time, and MS2 fragmentation. Over one thousand known metabolites were identified and annotated as well as over 300 unknown metabolites. Metabolites were grouped into one of fifteen classes based on chemical ontology and function. Bis(monoacylglycero)phosphates were over 2-fold increase in atypical (Grade II) meningiomas versus Grade I, indicating lysosomal activation. One carbon metabolism pathway showed significant upregulation in neoplastic tissue vs. tumor involved dura, as well as neoplastic cells compared to arachnoid cells, indicating folate-dependent pathways as a potential therapeutic target. Using novel combined untargeted metabolomics, we found multiple classes of metabolites that were either enriched or suppressed in neoplastic tissue and cells compared to non-neoplastic cells. Further studies are warranted for a better understanding of possible oncogenic signaling pathways and to detect potential biomarkers useful for diagnosis and treatment.