Aggressive Primary Brain Cancer Research Articles

Editorial: fate mapping of human glioblastoma reveals an invariant stem cell hierarchy

Glioblastoma (GB) is the most aggressive primary brain cancer of adulthood, associated with an average 5-year survival rate of 2% (1). A small population of tumor-initiating cells, termed cancer stem cells (CSCs), are the putative origin of GB, accounting for tumor development, progression and recurrence (2).

A Review on Tumor-Treating Fields (TTFields): Clinical Implications Inferred From Computational Modeling.

Tumor-treating fields (TTFields) are a cancer treatment modality that uses alternating electric fields of intermediate frequency (∼100-500kHz) and low intensity (1-3 V/cm) to disrupt cell division. TTFields are delivered by transducer arrays placed on the skin close to the tumor and act regionally and noninvasively to inhibit tumor growth. TTFields therapy is U.S. Food and Drug Administration approved for the treatment of glioblastoma multiforme, the most common and aggressive primary human brain cancer. Clinical trials testing the safety and efficacy of TTFields for other solid tumor types are underway. The objective of this paper is to review computational approaches used to characterize TTFields. The review covers studies of the macroscopic spatial distribution of TTFields generated in the human head, and of the microscopic field distribution in tumor cells. In addition, preclinical and clinical findings related to TTFields and principles of its operation are summarized. Particular emphasis is put on outlining the potential clinical value inferred from computational modeling.

DNM3, p65 and p53 from exosomes represent potential clinical diagnosis markers for glioblastoma multiforme.

Background:Glioblastoma multiforme (GBM) is the most aggressive and deadly primary brain cancer that arises from astrocytes and classified as grade IV. Recently, exosomes have been reported as an essential mediator in diverse cancer carcinogenesis and metastasis. However, their role in GBM is still unclear. In this study, we aimed to investigate whether blood exosomes can be potential clinical diagnostic markers for GBM.Methods:We used a xenograft orthotopic mouse model to detect the differentially expressed genes in the brain and blood exosomes of original/recurrent GBM.Results:We found that recurrent GBM had stronger growth capacity and lethality than original GBM in the mouse model. A gene microarray of original tumors and blood exosomes from GBM orthotopic xenografts results showed that DNM3, p65 and CD117 expressions increased, whereas PTEN and p53 expressions decreased in both original tumors and blood exosomes. In the recurrent GBM tumor model, DNM3 and p65 showed increased expressions, whereas ST14 and p53 showed decreased expressions in tumor and blood exosomes of the recurrent GBM mouse model.Conclusion:In summary, we found that DNM3, p65 and p53 had a similar trend in brain and blood exosomes both for original and recurrent GBM, and could serve as potential clinical diagnostic markers for GBM.

Impact of mesenchymal stem cells\u2019 secretome on glioblastoma pathophysiology

BackgroundGlioblastoma (GBM) is a highly aggressive primary brain cancer, for which curative therapies are not available. An emerging therapeutic approach suggested to have potential to target malignant gliomas has been based on the use of multipotent mesenchymal stem cells (MSCs), either unmodified or engineered to deliver anticancer therapeutic agents, as these cells present an intrinsic capacity to migrate towards malignant tumors. Nevertheless, it is still controversial whether this innate tropism of MSCs towards the tumor area is associated with cancer promotion or suppression. Considering that one of the major mechanisms by which MSCs interact with and modulate tumor cells is via secreted factors, we studied how the secretome of MSCs modulates critical hallmark features of GBM cells.MethodsThe effect of conditioned media (CM) from human umbilical cord perivascular cells (HUCPVCs, a MSC population present in the Wharton’s jelly of the umbilical cord) on GBM cell viability, migration, proliferation and sensitivity to temozolomide treatment of U251 and SNB-19 GBM cells was evaluated. The in vivo chicken chorioallantoic membrane (CAM) assay was used to evaluate the effect of HUCPVCs CM on tumor growth and angiogenesis. The secretome of HUCPVCs was characterized by proteomic analyses.ResultsWe found that both tested GBM cell lines exposed to HUCPVCs CM presented significantly higher cellular viability, proliferation and migration. In contrast, resistance of GBM cells to temozolomide chemotherapy was not significantly affected by HUCPVCs CM. In the in vivo CAM assay, CM from HUCPVCs promoted U251 and SNB-19 tumor cells growth. Proteomic analysis to characterize the secretome of HUCPVCs identified several proteins involved in promotion of cell survival, proliferation and migration, revealing novel putative molecular mediators for the effects observed in GBM cells exposed to HUCPVCs CM.ConclusionsThese findings provide novel insights to better understand the interplay between GBM cells and MSCs, raising awareness to potential safety issues regarding the use of MSCs as stem-cell based therapies for GBM.

Memantine-derived drugs as potential antitumor agents for the treatment of glioblastoma

Glioblastoma is one of the most aggressive malignant primary brain cancer in adults. To date, surgery, radiotherapy and current pharmacological treatments are not sufficient to manage this pathology that has a high mortality rate (median survival 12–15months).Recently, anticancer multi-targeted compounds have attracted much attention with the aim to obtain new drugs able to hit different biological targets that are involved in the onset and progression of the disease.Here, we report the synthesis of novel memantine-derived drugs (MP1–10) and their potential antitumor activities in human U87MG glioblastoma cell line. MP1–10 were synthetized joining memantine, which is a NMDA antagonist, to different histone deacetylase inhibitors to obtain one molecule with improved therapeutic efficacy.Biological results indicated that MP1 and MP2 possessed more potent anti-proliferative effects on U87MG cells than MP3–10 in a dose-dependent manner. MP1 and MP2 induced significant cell death by apoptosis characterized by apoptotic morphological changes in Hoechst staining. Both drugs also exhibited non-genotoxic and only mild cytotoxic effects in human whole blood cells. However, only MP1, showing good chemico-physical properties (solubility, LogP) and enzymatic stabilities in gastric and intestinal fluids, can be considered a suitable candidate for in vivo pharmacokinetic studies.

Abstract CT054: Phase II study of dianhydrogalactitol in patients with MGMT-unmethylated bevacizumab-naive recurrent glioblastoma

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain cancer. Current standard of care includes surgery, radiation and treatment with temozolomide (TMZ), however nearly all tumors recur and the prognosis for recurrent GBM is dismal. Most GBM tumors have unmethylated promoter status for O6-methylguanine-DNA-methyltransferase (MGMT); a validated biomarker for TMZ-resistance. Second-line treatment with anti-angiogenic agent bevacizumab has not improved overall survival (OS) and 5-year survival is less than 3%. Dianhydrogalactitol (VAL-083) is a bi-functional alkylating agent targeting N7-Guanine and inducing interstrand cross-links, DNA double-strand breaks and cell-death in GBM cell-lines and GBM cancer stem cells. VAL-083’s cytotoxicity is independent of MGMT status and VAL-083 overcomes TMZ-resistance in vitro. Our recent phase I/II clinical trial in recurrent GBM patients failing both TMZ and bevacizumab, suggested VAL-083 offers clinically meaningful survival benefits for patients with recurrent GBM and pinpointing a new dosing regimen (40 mg/m2/d on days 1,2,3 of a 21-day cycle). A pivotal Phase 3 study in recurrent GBM after failing both TMZ and bevacizumab is being planned. If successful, this study will serve as the basis for a New Drug Application (NDA) submission for VAL-083. In addition, a single-arm Phase 2 study to confirm the tolerability of the new dosing regimen in combination with radiotherapy and to explore the activity of VAL-083 in newly diagnosed MGMT-unmethylated GBM patients whose tumors are known to express high MGMT levels is proceeding. In the present Phase 2 clinical trial, the main goal is to assess the overall survival (OS) in MGMT-unmethylated, recurrent, bevacizumab-naïve GBM. RATIONALE: The vast majority of GBM patients experience recurrent/progressive disease within a year from initial diagnosis and median survival after recurrence is 3-9 months. Chemotherapy regimens for these patients are lacking and there is a significant unmet medical need. Given VAL-083’s novel alkylating mechanism, promising clinical benefit, and favorable safety profile, a trial studying VAL-083 in MGMT-unmethylated recurrent GBM is warranted. METHOD: Open label, single-arm, biomarker-driven Phase 2 clinical trial in MGMT-unmethylated adult GBM patients at first recurrence/progression, prior to bevacizumab. 48 patients will be enrolled to determine if treatment with VAL-083 will improve OS at 9-months compared to historical control with lomustine. The patients will receive VAL-083 40mg/m2/day on days 1,2,3 of a 21-day cycle. Patients will be followed until death or for at least 9 months from enrollment, whichever occurs earlier. Survival will be compared to recently published EORTC26101 for recurrent MGMT-unmethylated GBM patients treated with lomustine. Secondary outcome measures include progression-free survival and overall response rate. Clinicaltrials.gov identifier: NCT02717962. Citation Format: Barbara J. O'Brien, Jeffrey A. Bacha, Dennis M. Brown, Anne Steino, Richard Schwartz, Sarath Kanekal, Lorena Lopez, Marta Penas-Prado. Phase II study of dianhydrogalactitol in patients with MGMT-unmethylated bevacizumab-naive recurrent glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT054. doi:10.1158/1538-7445.AM2017-CT054

Expression of metabolic enzymes as predictors of prognosis in patients with aggressive primary brain cancers.

e13529 Background: Aggressive primary brain cancers like glioblastoma multiforme (GBM) are metabolically active tumors. We hypothesis that abnormalexpression of metabolic enzymes may be useful in predicting survival. Methods: 60 patients (45 with GBM, 11 with anaplastic gliomas and 4 with gliosarcoma) were evaluated using Immunohistochemistry (IHC) to measure the relative expression of metabolic enzymes known to be predictors of prognosis including hexokinase-II (HK2) and mutated isocitrate dehydrogenase-1 (IDH1-R132H) as well as several ketolytic enzymes including cytosolic type 2 (R)-beta-hydroxybutyrate dehydrogenase (BDH2)..The tumors were graded based on the percentage of immunoreactive tumor cells: Positive (≥20%), Negative ( < 20%). Survival was calculated using Kaplan Meier (KM), and an age adjusted hazard ratio (aHR) for death was obtained using cox proportional analysis for enzyme expression. Significance p ≤0.05. Results: Demographics for 60 patients; 19 females and 41 males, median age 62 years, and median survival for all the patients 5.9 months. Univariate analysis showed positive HK2 and negative BDH2 expression had decreased survival with (HR of 1.8 p = 0.06 and 2.6 p = 0.01), respectively. In a multivariate analysis after adjusting to age, histology and other enzyme expression, positive HK2 and negative BDH2 expression were statistically associated with decreased survival (aHR of 11.3 p = 0.001 and 32.6, p = 0.0004), respectively. The group with favorable prognosis (negative HK2 and positive BDH2 expression) (n = 36) had a median survival of 9.7 months (95% CI 4.2 -13.5) whereas the group with non-favorable prognosis (neither a negative HK2 nor a positive BDH, n = 24) had a median survival of 3.1 months (95% CI 1.8 – 5.8). Because only 3 patients had GBM that were positive for the IDH1-R132H mutant protein, IDH1-R132H was not used as a statistically valid predictor in this study. Conclusions: Our current result confirms the poor prognosis associated with positive expression of HK2, and suggests that negative IHC expression of BDH2 may be a new indicator of poor prognosis for patients with aggressive primary brain cancers.

Clinical trials of VAL-083 in patients with chemo-resistant glioblastoma.

TPS2082 Background: Glioblastoma (GBM) is the most common and aggressive primary brain cancer. Current standard of care includes surgery, radiation and treatment with temozolomide (TMZ), however nearly all tumors recur and the prognosis for recurrent GBM is dismal. Most GBM tumors have unmethylated promoter status for O6-methylguanine-DNA-methyltransferase (MGMT); a validated biomarker for TMZ-resistance. Second-line treatment with anti-angiogenic agent bevacizumab has not improved overall survival (OS) and 5-year survival is less than 3%. Dianhydrogalactitol (VAL-083) is a bi-functional alkylating agent targeting N7-Guanine and inducing interstrand DNA cross-links, double-strand breaks and cell death in GBM cell lines and GBM cancer stem cells, independent of MGMT status in vitro. VAL-083 readily crosses the blood-brain barrier and accumulates in brain tumor tissue. Our recent phase I/II clinical trial in recurrent GBM patients failing both TMZ and bevacizumab, suggested that VAL-083 offers clinically meaningful survival benefits for patients with recurrent GBM and pinpointed a new dosing regimen (40 mg/m2/d on days 1,2,3 of a 21-day cycle) which was well-tolerated and was selected for study in subsequent GBM trials. Methods: These trials include i) an ongoing single-arm, biomarker driven, Phase 2 study to determine if VAL-083 treatment of MGMT-unmethylated adult GBM patients at first recurrence/progression, prior to bevacizumab improves overall survival at 9 months, compared to historical control with lomustine (clinicaltrials.gov identifier: NCT02717962). ii) A pivotal Phase 3 study in recurrent GBM after failing both TMZ and bevacizumab. The control arm will consist of a limited number of salvage chemotherapies currently used in bevacizumab-failed GBM. If successful, this study will serve as the basis for a New Drug Application (NDA) submission for VAL-083. iii) A single arm, biomarker driven, Phase 2 study to confirm the tolerability and efficacy of VAL-083 in combination with radiotherapy in newly diagnosed MGMT-unmethylated GBM patients whose tumors are known to express high MGMT levels. The results of these studies may support a new treatment paradigm in chemotherapeutic regimens for the treatment of GBM. Clinical trial information: NCT02717962.

IL13RA2 targeted alpha particle therapy against glioblastomas.

Glioblastoma (GBM) is the most aggressive primary malignant brain cancer that invariably results in a dismal prognosis. Chemotherapy and radiotherapy have not been completely effective as standard treatment options for patients due to recurrent disease. We and others have therefore developed molecular strategies to specifically target interleukin 13 receptor alpha 2 (IL13RA2), a GBM restricted receptor expressed abundantly on over 75% of GBM patients. In this work, we evaluated the potential of Pep-1L, a novel IL13RA2 targeted peptide, as a platform to deliver targeted lethal therapies to GBM. To demonstrate GBM-specificity, we radiolabeled Pep-1L with Copper-64 and performed in vitro cell binding studies, which demonstrated specific binding that was blocked by unlabeled Pep-1L. Furthermore, we demonstrated real-time GBM localization of [64Cu]Pep-1L to orthotopic GBMs using small animal PET imaging. Based on these targeting data, we performed an initial in vivo safety and therapeutic study using Pep-1L conjugated to Actinium-225, an alpha particle emitter that has been shown to potently and irreversibly kill targeted cells. We infused [225Ac]Pep-1L into orthotopic GBMs using convection-enhanced delivery and found no significant adverse events at injected doses. Furthermore, our initial data also demonstrated significantly greater overall, median and mean survival in treated mice when compared to those in control groups (p < 0.05). GBM tissue extracted from mice treated with [225Ac]Pep-1L showed double stranded DNA breaks, lower Ki67 expression and greater propidium iodide internalization, indicating anti-GBM therapeutic effects of [225Ac]Pep-1L. Based on our results, Pep-1L warrants further investigation as a potential targeted platform to deliver anti-cancer agents.

Secreted reporter proteins, a valuable complementary tool for non-invasive preclinical monitoring of brain tumour growth

Malignant glioma and glioblastoma multiforme (GBM) are the most common and aggressive primary brain cancers with a very low dismal mean survival after initial diagnosis (1), emphasizing the need for novel treatment options. Several novel therapeutics have been identified for the treatment of malignant brain tumours, but despite strong preclinical data, very few successes have been achieved in clinical trials (2). The dichotomy between potential successful preclinical therapeutic agents and the ineffectiveness of the drug in patients underscores the need for better predictive preclinical models. The ideal model is a highly reproducible tumour model that mimics the key histopathological, genetic and imaging characteristics encountered in human GBMs. The most relevant models are isogenic orthotopic models, in which the complex micro-environmental interaction between glioma cells, the central nervous system and the immune system are present during tumour growth and treatment (3).

MPTH-16. EXPRESSION OF METABOLIC ENZYMES AS PREDICTORS OF PROGNOSIS IN PATIENTS WITH AGGRESSIVE PRIMARY BRAIN CANCERS

Aggressive primary brain cancers like glioblastoma multiforme (GBM) are metabolically active tumors. We hypothesis that abnormal expression of metabolic enzymes may reflect the tumors increased metabolism and be useful in predicting survival. 60 patients (45 with GBM, 11 with anaplastic gliomas and 4 with gliosarcoma) were evaluated using Immunohistochemistry (IHC) to measure the relative concentrations of the glycolytic enzyme hexokinase-II (HK2) a known predictor of poor prognosis and several ketolytic enzymes including cytosolic type 2(R)-hydroxybutyrate dehydrogenase (BDH-2). The immunoreactivities were graded based on the percentage of positive cells: Positive (≥20%), Negative (< 20%). Survival was calculated using Kaplan Meier (KM) and an age adjusted hazard ratio (aHR) for death was obtained using Cox proportional analysis for enzyme expression. Significance p ≤ 0.05. Demographics for 60 patients; 19 female and 41 males, median age 62 years, and median survival for all the patients 5.9 months. Univariate analysis showed positive HK2 and negative BDH-2 expression had decreased survival with HR of 1.8 (p=0.06) and 2.6 (p=0.01) respectively. In a multivariate analysis after adjusting to age, histology and other enzyme expression, positive HK2 and negative BDH-2 expression were statistically associated with decreased survival (aHR of 11.3 p= 0.001 and 32.6, p=0.0004), respectively. The group with favorable prognosis (negative HK2 or positive BDH-2 expression) (n=36) had a median survival of 9.7 months (95% CI 4.2 -13.5) whereas the group with non-favorable prognosis (n=24) had a median survival of 3.1months (95% CI 1.8 – 5.8). IHC helps to confirm the poor prognosis associated with positive HK-2 and suggests that negative IHC expression of BDH-2 may be a new indicator of poor prognosis for patients with aggressive primary brain cancers.

Abstract 3861: Ruxolitinib inhibits STAT-3 activation in glioblastoma

Abstract Glioblastoma (GBM), a primary Grade IV astrocytoma, is the most aggressive primary brain cancer in humans. Despite combined radiotherapy and chemotherapy, the median survival is 14 months. We and others have validated that levels of phosphorylated JAK1/2 and STAT-3 are increased in GBM tissues. When stimulated with IL-6 family members, JAK1/2 recruits and phosphorylates STAT-3. Phosphorylated STAT-3 dimerizes and translocates to the nucleus where it regulates genes involved in proliferation, angiogenesis, anti-apoptotic activity and immunosuppression. Elevated JAK/STAT activation is generated through an increase in IL-6 family cytokines as well as a decrease in JAK/STAT-3 negative regulators. To therapeutically inhibit the JAK/STAT-3 pathway in GBM we employed ruxolitinib, an FDA approved JAK1/JAK2 inhibitor. Human GBM cell lines U87-MG and U251-MG, as well as patient derived GBM xenograft lines JX6 and JX12, were treated with ruxolitinib in doses ranging from 0.01 to 10 μM. Ruxolitinib was observed to inhibit STAT-3 activation in a dose-dependent manner. Pre-treatment of U87-MG and U251-MG cells with ruxolitinib inhibited stimulus-induced STAT-3 activation and downstream gene expression in a dose-dependent manner. Future studies will examine ruxolitinib effects on migration, invasion, apoptosis and proliferation in vitro. Collectively, these data indicate the potential for ruxolitinib therapy for GBM patients. Citation Format: Samuel Fehling, Braden McFarland, Etty Benveniste. Ruxolitinib inhibits STAT-3 activation in glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3861.

Molecular Predictors of Long-Term Survival in Glioblastoma Multiforme Patients

Glioblastoma multiforme (GBM) is the most common and aggressive adult primary brain cancer, with <10% of patients surviving for more than 3 years. Demographic and clinical factors (e.g. age) and individual molecular biomarkers have been associated with prolonged survival in GBM patients. However, comprehensive systems-level analyses of molecular profiles associated with long-term survival (LTS) in GBM patients are still lacking. We present an integrative study of molecular data and clinical variables in these long-term survivors (LTSs, patients surviving >3 years) to identify biomarkers associated with prolonged survival, and to assess the possible similarity of molecular characteristics between LGG and LTS GBM. We analyzed the relationship between multivariable molecular data and LTS in GBM patients from the Cancer Genome Atlas (TCGA), including germline and somatic point mutation, gene expression, DNA methylation, copy number variation (CNV) and microRNA (miRNA) expression using logistic regression models. The molecular relationship between GBM LTS and LGG tumors was examined through cluster analysis. We identified 13, 94, 43, 29, and 1 significant predictors of LTS using Lasso logistic regression from the somatic point mutation, gene expression, DNA methylation, CNV, and miRNA expression data sets, respectively. Individually, DNA methylation provided the best prediction performance (AUC = 0.84). Combining multiple classes of molecular data into joint regression models did not improve prediction accuracy, but did identify additional genes that were not significantly predictive in individual models. PCA and clustering analyses showed that GBM LTS typically had gene expression profiles similar to non-LTS GBM. Furthermore, cluster analysis did not identify a close affinity between LTS GBM and LGG, nor did we find a significant association between LTS and secondary GBM. The absence of unique LTS profiles and the lack of similarity between LTS GBM and LGG, indicates that there are multiple genetic and epigenetic pathways to LTS in GBM patients.

Inactivation of the ATMIN/ATM pathway protects against glioblastoma formation.

Glioblastoma multiforme (GBM) is the most aggressive human primary brain cancer. Using a Trp53-deficient mouse model of GBM, we show that genetic inactivation of the Atm cofactor Atmin, which is dispensable for embryonic and adult neural development, strongly suppresses GBM formation. Mechanistically, expression of several GBM-associated genes, including Pdgfra, was normalized by Atmin deletion in the Trp53-null background. Pharmacological ATM inhibition also reduced Pdgfra expression, and reduced the proliferation of Trp53-deficient primary glioma cells from murine and human tumors, while normal neural stem cells were unaffected. Analysis of GBM datasets showed that PDGFRA expression is also significantly increased in human TP53-mutant compared with TP53-wild-type tumors. Moreover, combined treatment with ATM and PDGFRA inhibitors efficiently killed TP53-mutant primary human GBM cells, but not untransformed neural stem cells. These results reveal a new requirement for ATMIN-dependent ATM signaling in TP53-deficient GBM, indicating a pro-tumorigenic role for ATM in the context of these tumors.

EPID-26EVALUATION OF METABOLIC ENZYME EXPRESSION AS PREDICTORS OF SURVIVAL IN AGGRESSIVE GLIOMAS

INTRODUCTION: Aggressive primary brain cancers like glioblastoma multiforme (GBM) are metabolically active tumors with a poor prognosis. We hypothesize that expression of metabolic enzymes may be reflective of the tumors increased metabolism and might be useful in predicting survival. METHODS: Immunohistochemistry (IHC) reactions using formalin fixed paraffin embedded sections from brains, were performed with antibodies raised against the glycolytic enzyme Hexokinase-II (HK2) and several ketolytic enzymes [Succinyl CoA: 3-oxoacid CoA transferase 1 (OXCT1) cytosolic type 2 (R)-hydroxybutyrate dehydrogenase (BDH2) and Acetyl-CoA acetyltransferase (ACAT1)] on 23 patients, 18 with GBM and 5 with anaplastic gliomas. The immunoreactivities were graded by comparing the IHC staining observed in normal brain cells present in the periphery of the tissue with tumor cells using a semi-quantitative scale based on the percentage of positive cells: Positive (≥20%), Negative (< 20%). The survival of patients was evaluated using Kaplan Meier and an age adjusted hazard ratio (aHR) for death was obtained using cox proportional analysis for enzyme expression. RESULTS: Out of 23 patients 6 were female and 17 males, median age was 55 years with the median survival for all the patients of 11.3 months. Univariate analysis for survival using HK2 showed increased survival with decreased IHC expression with a aHR of 0.3 p = 0.02. Patients with increased expression of the ketolytic enzymes had increased survival: BDH2 with an aHR of 7.6 p = 0.008; ACAT1 aHR 0.2, p = 0.02; OXCT1 aH1.6, p = 0.08. In a multivariate analysis HK2 was the only enzyme that was statistically significant; median survival in 17 negative patients was 13.5 months and 3.8 months in 6 positive patients, aHR 0.3, p = 0.02. CONCLUSION: IHC analysis of metabolic enzymes may help to predict survival in patients with aggressive primary brain cancers, but this requires confirmation using a larger number of subjects.

TM-01 * IDENTIFICATION OF GLUCOSE-6-PHOSPHATASE ALPHA AS A KEY METABOLIC REGULATOR FOR GLIOBLASTOMA CELL INVASION

Glioblastoma (GBM) remains the most aggressive primary brain cancer in adults. Similar to other cancers, GBM cells undergo metabolic reprogramming to promote proliferation and survival. Glycolytic inhibition is widely used to target such reprogramming. However, the stability of glycolytic inhibition in GBM remains unclear. In this study, we found that the expression of glucose-6-phosphatase-α (G6PC) was elevated in GBM when compared to normal brain ) (p < 0.001). Human-derived brain tumor initiating cells (BTICs) utilize this enzyme to counteract glycolytic inhibition induced by 2-Deoxy-D-glucose (2DG) and sustain malignant progression. Down-regulation of G6PC renders the majority of these cells unable to survive glycolytic inhibition, and promotes glycogen accumulation through the activation of glycogen synthase (GYS1) and inhibition of glycogen phosphorylase (PYGL). Moreover, BTICs that survive G6PC knockdown are less aggressive (reduced migration, invasion, proliferation, and increased astrocytic differentiation) (p < 0.001). Interestingly, after performing immunohistochemistry staining for G6PC in brain sections derived from animals implanted with G6PC knockdown cells for a survival study, we found a complete rescue of its expression in the tumor cells of these animals. These results explain the absence of survival differences observed between G6PC knockdown and wild type mice. Furthermore, these findings also suggest a very important role for G6PC in cell survival, since only the cells that carried G6PC where able to survive and invade in our animal model. Collectively, our findings establish G6PC as a key enzyme with pro-malignant functional consequences that has not been previously reported in GBM and identify it as a potential therapeutic target.

Glucose-6-phosphatase is a key metabolic regulator of glioblastoma invasion.

Glioblastoma (GBM) remains the most aggressive primary brain cancer in adults. Similar to other cancers, GBM cells undergo metabolic reprogramming to promote proliferation and survival. Glycolytic inhibition is widely used to target such reprogramming. However, the stability of glycolytic inhibition in GBM remains unclear especially in a hypoxic tumor microenvironment. In this study, it was determined that glucose-6-phosphatase (G6PC/G6Pase) expression is elevated in GBM when compared with normal brain. Human-derived brain tumor-initiating cells (BTIC) use this enzyme to counteract glycolytic inhibition induced by 2-deoxy-d-glucose (2DG) and sustain malignant progression. Downregulation of G6PC renders the majority of these cells unable to survive glycolytic inhibition, and promotes glycogen accumulation through the activation of glycogen synthase (GYS1) and inhibition of glycogen phosphorylase (PYGL). Moreover, BTICs that survive G6PC knockdown are less aggressive (reduced migration, invasion, proliferation, and increased astrocytic differentiation). Collectively, these findings establish G6PC as a key enzyme with promalignant functional consequences that has not been previously reported in GBM and identify it as a potential therapeutic target. This study is the first to demonstrate a functional relationship between the critical gluconeogenic and glycogenolytic enzyme G6PC with the metabolic adaptations during GBM invasion.

NT-31 * KETOGENIC DIET USED AS TREATEMENT FOR PRIMARY AGGRESSIVE BRAIN CANCERS: EXPERIENCE IN HUMANS

BACKGROUND: A ketogenic diet (KD) has been proposed as an alternative therapy for treatment of malignant gliomas. Unlike normal brain, malignant brain tumors may not be able to efficiently metabolize ketones. The ketogenic diet exploits this metabolic difference as a possible therapeutic approach. This report reviews the past reported experiences in humans using a ketogenic diet to treat aggressive primary brain cancers and includes our experience treating 2 patients with an energy restricted ketogenic diet (ERKD). METHODS: Patients in this report include 5 detailed case reports, 2 new patients treated with an ERKD protocol and 19 recently reported patients from Germany. RESULTS: Patients were treated using different protocols. Two were monitored with twice daily measurements of blood glucose and ketones and daily weights while the remaining patients' ketones and glucose and weight measurements were intermittent and not at prescribed intervals. Prolonged remissions ranging from more than 5 years to 4 months were reported in detailed case reports. Only one of these patients was treated using the ketogenic diet as monotherapy. The best responses reported in more recent patient series were stable disease for approximately 6 weeks. Two patients with progressive disease after 12 weeks of diet therapy had tissue expression of at least one of 2 mitochondrial ketolytic enzymes succinyl CoA: 3-oxoacid CoA transferase (OXCT-1), β-3-hydroxybutyrate dehydrogenase 1 (BDH1). The 19 patients from Germany treated with a ketogenic diet demonstrated that the diet was safe with no major side effects. CONCLUSION: 1. Treatment with a ketogenic diet may be effective in controlling some patients with primary brain cancers, but additional protocol studies are needed; 2. Ketosis can be induce using customary foods and; 3. A ketogenic diet is safe without major side effects. TRIAL REGISTRATION: ClinicalTrials.gov# {type:clinical-trial,attrs:{text:NCT01535911,term_id:NCT01535911}}NCT01535911

Propentofylline Targets TROY, a Novel Microglial Signaling Pathway

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain cancer, with a median survival of less than 2 years after diagnosis with current available therapies. The tumor microenvironment serves a critical role in tumor invasion and progression, with microglia as a critical player. Our laboratory has previously demonstrated that propentofylline, an atypical methylxanthine with central nervous system glial modulating and anti-inflammatory actions, significantly decreases tumor growth in a GBM rodent model by preferentially targeting microglia. In the present study, we used the CNS-1 rat glioma model to elucidate the mechanisms of propentofylline. Here we demonstrate that propentofylline targets TROY, a novel signaling molecule up-regulated in infiltrating microglia, and not macrophages, in response to CNS-1 cells. We identify Pyk2, Rac1 and pJNK as the downstream signaling molecules of TROY through western blot analysis and siRNA transfection. We demonstrate that inhibition of TROY expression in microglia by siRNA transfection significantly inhibits microglial migration towards CNS-1 cells similar to 10 µM propentofylline treatment. These results identify TROY as a novel molecule expressed in microglia, involved in their migration and targeted by propentofylline. Furthermore, these results describe a signaling molecule that is differentially expressed between microglia and macrophages in the tumor microenvironment.

Abstract 3450: Functional characterization of a DNA damage response network in glioblastoma associated with long-term patient survival

Abstract Glioblastoma multiforme (GBM) is the most aggressive primary brain cancer in humans with a median survival of less than 14 months. However, a small minority of GBM patients have a median survival of over three years. In an effort to identify genetic and epigenetic events that might explain this relative long-term survival, an integrative genomic approach was utilized. We show that in contrast to most GBM patients, relative long-term survivors have tumors that lack a biological network that responds to DNA damage. This network is comprised of pro-inflammatory genes and genes normally regulated through interferon signaling. By overlaying the network with phenotype information, key genes and gene-gene interactions that strongly associate with patient survival were identified to facilitate the discovery of important genes and genetic dependencies in the network. One such gene is Interferon-Stimulated Gene 15 (ISG15), which is part of the interferon subnetwork and encodes for an ubiquitin-like protein. Targeted disruption of ISG15 in various GBM cell lines unmasked a DNA damage response as characterized by delayed S-phase entry and G2/M accumulation, increased γH2AX, and senescence. Various genes that displayed a predicted interaction with ISG15 were tested for evidence for a biological interaction. Functional validation confirmed that these predicted interactions with ISG15 were mirrored by genetic and regulatory interactions that resulted in rescue of the effects of ISG15 knockdown. We also investigated genes that control the pro-inflammatory subnetwork. Targeted disruption of one such gene, MYD88, resulted in decreased invasion and increased sensitivity to DNA damage. In summary, we have identified a biological network that responds to and regulates DNA damage. The absence of network genes results in susceptibility to DNA damage, senescence, and decreased invasion. GBM patients with tumors lacking the network are rare long-term survivors of an otherwise uniformly and rapidly fatal tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3450. doi:1538-7445.AM2012-3450