Brain Tumor-initiating Cells Research Articles

CS-30 * PROTEIN KINASE CK2 IS IMPORTANT FOR THE FUNCTION OF GLIOBLASTOMA BRAIN TUMOR INITIATING CELLS

Casein kinase 2 (CK2) is an ubiquitously expressed serine/threonine kinase composed of two catalytic subunits (α and/or α') and two regulatory (β) subunits. The expression and kinase activity of protein kinase CK2 are elevated in many different cancers, including glioblastoma (GBM). Brain tumor initiating cells (BTICs) are a subset of cells that are tumorigenic and are considered to promote the resistance of GBM to current therapies. Previously our lab has reported that CK2 activity promotes prosurvival signaling in GBM; however, the role of CK2 in BTICs has not been examined. In this study, we found that the expression of CK2α was increased in CD133+ BTICs compared to CD133− cells from GBM xenografts. We found that GBM cells are sensitive to cell death caused by CX-4945, an ATP-competitive inhibitor of CK2. Inhibition of CK2 also reduced the frequency of CD133+ BTICs over the course of 7 days, indicating a role for CK2 in BTIC persistence and survival. Additionally, using an in vitro limiting dilution assay, we found that CX-4945 reduced neurosphere formation in bulk cells from GBM xenografts. On the other hand, inhibition of CK2 had no effect on the proliferation or neurosphere formation of normal murine neural precursor cells. We are currently investigating the mechanism of cell death and reduced neurosphere formation and have found a reduction in the constitutive activation of signal transduction pathways dysregulated in GBM including NF-kB and MAPK pathways. Due to the integration of CK2 in multiple signaling pathways important for BTIC survival, protein kinase CK2 is a promising target in GBM.

ET-46 * ONCOLYTIC VIRAL THERAPY FOR MALIGNANT GLIOMAS USING MYXOMA VIRUS DELETED FOR ANTI-APOPTOTIC M11L GENE

Brain Tumour Initiating Cells (BTICs) are stem-like cells hypothesized to mediate recurrence in high-grade gliomas. Myxoma virus (MyxV) is a promising oncolytic virus, which is highly effective in conventional long term resistant glioma cell lines and less effective in BTICs. We hypothesized that one possible factor limiting efficacy in BTICs is that cell death following infection with MyxV is inhibited by virally encoded anti-apoptotic proteins, such as the Bcl-2 structural homologue, M011L. To test this we evaluated and compared the efficacy of wtMYXV versus the viral construct MyxV-M011L-KO (in which the anti-apoptotic protein M11L has been deleted) in BTICs. We found that WT-MyxV does not induce significant level of apoptosis in infected BTICs, but that MyxV-M011L-KO induces dramatically more apoptosisas shown by caspase activation, PARP cleavage, and Cytochrome C release from the mitochondria M11L from the WT-MyxV localized to the mitochondrial membrane and prevented the association of Bax with the mitochondrial membrane. Finally, silencing of Bax using specific siRNAs significantly blocked the induction of apoptosis and cell death that occurs after infection with mutant MyxV-M011L-KO virus. Therefore MyxV-M011L-KO, which is has the anti-apoptotic virally derived gene M11L, dramatically improves the oncolytic efficacy in BTICs and this is dependent on the presence of the pro-apoptotic host protein, Bax. This is the first demonstration, that the MyxV mutant, genetically modified to promote apoptosis in tumor initiating cells, is significantly more efficacious than the wildtype virus. Strategies, such as this one, that promotes apoptosis in tumor initiating cells might be particularly effective.

SC-33 * POLYCOMB GROUP PROTEIN Bmi1 CONTROLS NEURAL STEM AND PROGENITOR CELL MAINTENANCE AND GLIOBLASTOMA TUMOUR DEVELOPMENT

Bmi1, a polycomb group protein is required for self-renewal of neural stem and progenitor cells (NSPCs). Elevated Bmi1 expression is observed in most brain tumours, including glioblastoma (GBM). GBM, one of the most aggressive and heterogeneous tumours is known to possess a subpopulation of brain tumour initiating cells (BTICs) that have acquired stem cell properties and are exclusively capable of driving tumour formation in a human-mouse xenograft model. These observations raise the question of whether over-expressing Bmi1 is sufficient to promote self-renewal or tumorigenesis by NSPCs and whether loss of Bmi1 in GBM BTICs affects the progression of brain tumours. Using lentiviral constructs, we studied the gain of Bmi1 in human NSPCs and observed increased self-renewal and proliferative capacity of these cells. Subsequently, OEBmi1 NSPCs were injected in NOD-SCID mouse brains to assess their tumorigenic ability. Furthermore, knockdown of Bmi1 was performed in human GBM BTICs, which resulted in inhibition of clonogenic potential in vitro as well as decreased tumour cell mass in vivo. Lastly, to determine the comprehensive Bmi1 transcriptional network in human NSPCs and GBM BTICs, we applied ChIP-seq strategy to discover if Bmi1 binds to the promoter region of genes critical for neural lineage commitment and if these genes are involved in brain tumour formation. Together with RNA-seq data from human NSPCs and GBM BTICs sorted for stem cell marker CD133, this data will generate a comprehensive genome-wide Bmi1 binding profile and identify critical downstream Bmi1 targets & signaling pathways important for brain tumour maintenance.

ET-35 * PACRITINIB: A NOVEL JAK/STAT INHIBITOR WITH TRANSLATIONAL RELEVANCE FOR GBM

Glioblastoma multiforme (GBM) is characterized by an aggressive clinical course, therapeutic resistance, and striking molecular heterogeneity. GBM-derived brain tumor initiating cells (BTICs) closely model this molecular heterogeneity and may have a key role in tumor recurrence and therapeutic resistance. Recent evidence, from our lab and others, indicates that the Janus kinase (JAK)2/signal transducer and activator of transcription (STAT)3 signaling pathway is an important mediator of tumor cell survival, growth, and invasion in a large group of GBMs. Thus, inhibition of the JAK/STAT3 pathway may hold great promise as a therapeutic strategy for GBM. However, to date, drugs targeting activated STAT3 in clinically relevant models have not been successfully transitioned into clinical studies. Here we investigated the efficacy of a novel JAK/STAT inhibitor, Pacritinib, in BTIC lines cultured from GBM patients. Pacritinib is currently in a multicenter, randomized, Phase 3 trial comparing its efficacy and safety with that of best available therapy in patients with primary myelofibrosis. In GBM BTICs, Pacritinib administration resulted in on-target JAK2/STAT3 inhibition at 1-2 µΜ that dramatically reduced cell survival in a large number of lines, regardless of endogenous MGMT promoter methylation or EGFR, PTEN, and TP53 mutational status. Pacritinib was also found to cross the blood-brain barrier in NOD-SCID mice, by liquid chromatography-mass spectrophometry, with no toxicity observed on repeated administration of up to 200mg/kg per day by oral gavage. Previous studies on different JAK/STAT3 inhibitors, by others and us, demonstrated toxicity at doses required to shut-off STAT3 signaling and achieve tumour cell death in vivo. We are currently investigating the actions of Pacritinib in orthotopic BTIC xenograft animal survival studies. Given its previously established human safety profile and demonstrated activity in the clinically relevant BTIC model, Pacritinib may hold considerable promise for clinical translation in GBM.

CS-14 * NEUROTROPHIN RECEPTORS TrkB AND TrkC ARE REQUIRED FOR SURVIVAL OF BRAIN TUMOR INITIATING CELLS

Neurotrophins (NGF, BDNF and NT3) and their receptors (TrkA, TrkB, TrkC and p75NTR) are frequently expressed in malignant gliomas, yet their functions are largely unknown. Previously, we have shown that p75NTR is required for glioma invasion and proliferation. However, the role of other Trk receptors has not been examined. In this study, we investigated the importance of neurotrophin receptors TrkB and TrkC in survival of brain tumor initiating cells (BTICs). We used human malignant glioma tissues and also tumor-initiating cells isolated from fresh human malignant gliomas and examined the expression of neurotrophin receptors and studied their role in survival/growth these cells in vitro, we have also examined the mechanism underlie. Here, we show that human malignant glioma tissues and tumor-initiating cells express the neurotrophin receptors TrkB and TrkC, not TrkA and they also express neurotrophins NGF, BDNF and NT3. Specific activation of TrkB and TrkC receptors by ligands BDNF and NT3 stimulates Trk phosphorylation, activates Erk and Akt and enhances tumor-initiating cell viability and growth. Conversely, TrkB and TrkC knockdown or pharmacologic inhibition using inhibitors of Trk, Erk and Akt, decreases neurotrophin-dependent Trk phosphorylation, Erk and Akt activation and BTIC survival. Importantly, attenuation of BTIC growth by EGFR inhibitors could be overcome by activation of neurotrophin signaling and further neurotrophin signaling is required for maintains long-term BTIC growth in the absence of EGF and FGF, and this effect is attenuated with Trk, Erk and Akt inhibitors. In vivo experiments are under progress. Our results highlight a novel role for neurotrophin signaling in malignant glioma and suggest that Trks could be a target for combinatorial treatment.

CS-04 * STAT3 INVOLVEMENT IN AN EMT-LIKE PROCESS IN GLIOBLASTOMA BRAIN TUMOR INITIATING CELLS

Glioblastoma Multiforme (GBM) is the most aggressive subtype of brain tumour with a median survival of 15 months. Currently, GBM is managed by a combination of maximal safe resection followed by radiation and chemotherapy. However, GBM invariably recurs, highlighting the need to better delineate the basis of recurrent disease and develop novel more effective and targeted therapies. The Signal Transducer and Activator of Transcription 3 (STAT3) is abnormally active in GBM. A growing body of evidence supports the hypothesis that STAT3 is a major oncogenic molecular hub in patient-derived Brain Tumor Initiating Cells (BTICs) leading to stemness, proliferation, invasion and therapeutic resistance. Recently, there has been an increasing awareness that Epithelial to Mesenchymal Transition (EMT) is an essential process promoting the generation of highly tumorigenic cells with stem-like characteristics. The mesenchymal GBM profile, characterized by significantly shortened progression-free and overall survival, suggests that an EMT-like process has clinical relevance in GBM. Moreover, recent studies support a possible role for STAT3 in an EMT-like process in GBM. Here we show in that SLUG (encoded by SNAI2) appears to be expressed at higher levels than other EMT regulators in patient samples and matching BTICs. High SLUG protein level is observed in a large number of BTIC lines and positively correlates with activated STAT3. Moreover, SLUG is significantly decreased after pharmacological inhibition of STAT3 signalling using a well-characterized STAT3 inhibitor (Stattic) and a novel clinically relevant JAK2 inhibitor. Furthermore, IL-6 treatment or overexpression of a constitutively active form of STAT3 promote SLUG expression. SLUG and the subsequent EMT-like process may play an important role in the aggressive mesenchymal phenotype of GBM and the archetypal characteristics of BTICs: self-renewal, invasion, tumorigenicity and therapeutic resistance. We propose that STAT3-dependent EMT-like process and the key regulator SLUG are interesting novel therapeutic targets in GBM.

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.

Abstract 3153: Slit-2 directs the migration of primary cultured human GBM tumor initiating cells

Abstract Glioblastoma multiforme (GBM), the most aggressive and proliferative primary brain tumor, has an extremely high recurrence incidence. This is mainly due to cancer cell invasion of brain parenchyma beyond surgical boundaries. Brain tumor initiating cells (BTICs) are stem-like cells with properties of self-renewal and multipotency. BTICs are able to migrate to initiate tumor formation away from the original tumor and are highly resistant to chemo and radiotherapy, suggesting that they are responsible for tumor recurrence. Understanding the mechanisms affecting the migration of BTICs may shed light on mechanisms for preventing GBM recurrence. Cell migration and proliferation events present during brain development are recapitulated in the fundamental hallmarks of cancer. In rodents, Slit-Robo signaling functions as a chemorepulsive ligand-receptor system involved in guiding the migration of newly generated cells from the subventricular zone to other regions of the central nervous system. In neural cancers, Slit2 affects the migration of glioma and medulloblastoma cell lines. We hypothesized that Slit proteins have a chemorepellant effect on primary-cultured human GBM-derived BTICs, which promotes tumor dispersal. Primary BTIC cultures were established from GBM intraoperative samples and maintained in neurosphere culture media containing EGF and bFGF, heparin and LIF, which maintains the presence of stem cells. Expression of Slit and Robo was then evaluated at the mRNA and protein level. Our results revealed that Slit2 induces a chemorepulsive effect on Robo-expressing BTICs, as evaluated by transwell migration and chemotaxis assays (p&amp;lt;0.05). Slit2 also induces a significant increase in cell speed (p&amp;lt;0.05). Moreover, we did not see any effects of Slit2 on cell proliferation or viability. We further observed that Slit2 stimulation of BTICs induces a decrease in AKT activity and an increase in MYPT1/MLC2; proteins involved in actin polymerization. In summary, we demonstrated that Robo1 is expressed in different primary human GBM BTICs. As result, Slit2 acts on the migration of these cells in a chemorepellant manner, while increasing speed. The mechanisms responsible for these observations involve actin polymerization and Akt signaling. While further research is required, the effects of Slit-2 proteins on the migration behavior of human GBM BTIC cells may be related to the invasive nature of these tumors. Citation Format: Hugo Guerrero-Cazares, Vivian Capilla-Gonzalez, Emily A. Lavell, Alejandro Ruiz-Valls, Linda Chen, Gabrielle Drummond, Sural Ranamukhaarachchi, Alfredo Quinones-Hinojosa. Slit-2 directs the migration of primary cultured human GBM tumor initiating cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3153. doi:10.1158/1538-7445.AM2014-3153

Abstract 5513: Targeting brain tumor initiating cells in atypical teratoid/rhabdoid tumors: Aldehyde dehydrogenase inhibition with disulfiram

Abstract Atypical teratoid/rhabdoid tumors (AT/RT) are one of the most malignant pediatric brain tumors with a dismal prognosis. Cells with high aldehyde dehydrogenase (ALDH) activity from brain tumors have a number of characteristics that are expected of brain tumor initiating cells (BTICs). This study aimed to evaluate the therapeutic potential of ALDH inhibition using disulfiram (DSF) against BTICs from AT/RT. Primary cultured BTICs from AT/RT were stained with Aldefluor and isolated by fluorescence activated cell sorting. Therapeutic effect of DSF against BTICs from AT/RT was confirmed using in vitro and in vivo studies. AT/RT showed high expression of ALDH. DSF significantly inhibited ALDH enzyme activity of AT/RT cells. DSF decreased self-renewal ability, cell viability, proliferation potential and induced apoptosis and cell cycle arrest on ALDH+ AT/RT cells. DSF showed more potent cytotoxic effect to ALDH+ AT/RT cells compared to standard anti-cancer agents including ifosfamide, carboplatin and etoposide. Importantly, DSF reduced metabolite (metabolism?) of ALDH+ AT/RT cells by increasing NAD+ ratio and regulating SIRT1, NF-κB, Lin28A/B and miRNA let-7g. Notably, treatment with DSF did not have considerable effect on the normal neural stem cells and fibroblasts. Animals in the DSF-treated group demonstrated a significant survival benefit (105 days of median survival in the DSF-treated group versus 91 days in the control group, p = 0.0219). Our study demonstrated the therapeutic potential of DSF against BTICs from AT/RT and suggested the possibility of ALDH inhibition for clinical application. Note: This abstract was not presented at the meeting. Citation Format: Seung Ah Choi, Jung Won Choi, Kyu-Chang Wang, Ji Hoon Phi, Ji Yeoun Lee, Yong Hwy Kim, Young-Hoon Kim, Sung-Hye Park, Seung-Ki Kim. Targeting brain tumor initiating cells in atypical teratoid/rhabdoid tumors: Aldehyde dehydrogenase inhibition with disulfiram. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5513. doi:10.1158/1538-7445.AM2014-5513

Response-predictive gene expression profiling of glioma progenitor cells in vitro.

BackgroundHigh-grade gliomas are amongst the most deadly human tumors. Treatment results are disappointing. Still, in several trials around 20% of patients respond to therapy. To date, diagnostic strategies to identify patients that will profit from a specific therapy do not exist.MethodsIn this study, we used serum-free short-term treated in vitro cell cultures to predict treatment response in vitro. This approach allowed us (a) to enrich specimens for brain tumor initiating cells and (b) to confront cells with a therapeutic agent before expression profiling.ResultsAs a proof of principle we analyzed gene expression in 18 short-term serum-free cultures of high-grade gliomas enhanced for brain tumor initiating cells (BTIC) before and after in vitro treatment with the tyrosine kinase inhibitor Sunitinib. Profiles from treated progenitor cells allowed to predict therapy-induced impairment of proliferation in vitro.ConclusionFor the tyrosine kinase inhibitor Sunitinib used in this dataset, the approach revealed additional predictive information in comparison to the evaluation of classical signaling analysis.

Abstract LB-207: mTORC2/Akt signaling is modulated by noncanonical mitochondrial Notch1/PINK1 interaction in myc-amplified medulloblastoma tumorigenesis

Abstract Medulloblastoma is known to be the most malignant pediatric brain tumor. The armamentarium of targeted therapies to currently treat medulloblastoma and similar pediatric central nervous system malignancies is extremely limited, often necessitating the need to combat such tumors with modified regimens of therapeutic options designed originally to target adult neoplasms. Given such limited therapies, a budding focus on the role of mitochondrial dysregulation in the tumorigenesis of such pathologies merits consideration. Mitochondria are known to play fundamental roles in multiple processes conserved across eukaryotic species. Aside from their key role in energy production through oxidative phosphorylation, the organelles also serve as the sites of essential metabolic pathways, redox regulation, calcium homeostasis, apoptosis, and cell fate determination and differentiation. Recently, we documented the role of noncanonical Notch signaling and mitochondrial involvement in adult glioblastoma brain tumor-initiating cells (Lee KS et al., Genes and Development, 2013). Although the canonical Notch pathway and is generally well-characterized, involving the ligand-induced cleavage of Notch for transcriptional regulation, only more recently has credible evidence surfaced documenting the role of a second noncanonical pathway, where Notch can function independently of ligand and transcription through a mechanism that remains to be fully elucidated. The regulatory self-renewal versus differentiation choice of Drosophila and mammalian human neural stem cells requires Notch signaling, and in our work, we found noncanonical Notch pathway interaction with PTEN-induced putative kinase 1 (PINK1) to influence mitochondrial function, activating mTORC2/Akt signaling. siRNA-induced knockdown preferentially impaired the maintenance of Drosophila and human glioblastoma cancer stem cell-like tumor-forming cells to a far greater degree than normal stem cell counterparts. Further experiments have elucidated similar findings of increased Notch1/PINK1 mitochondrial interaction and mTORC2/Akt activity in patient-derived Group 3 (myc-amplified) medulloblastoma primary lines to levels greater than normal or glioblastoma samples. Additionally these medulloblastoma samples appear to be more susceptible to siRNA-induced knockdown of PINK1 than their counterparts. In vivo experiments are ongoing to address the role of mitochondrial Notch1/Pink1 interaction in tumor initiation and its targeting by small molecule inhibitors. Such results underscore the importance of mitochondria in both normal and cancer stem cell biology, a highly conserved mechanism across species, with exciting implications for the treatment of pediatric central nervous system malignancies. Citation Format: Abdullah H. Feroze, Kyu-Sun Lee, Sharareh Gholamin, Zhihao Wu, Irving Weissman, Bingwei Lu, Siddhartha S. Mitra, Samuel Cheshier. mTORC2/Akt signaling is modulated by noncanonical mitochondrial Notch1/PINK1 interaction in myc-amplified medulloblastoma tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-207. doi:10.1158/1538-7445.AM2014-LB-207

Casein kinase 2α regulates glioblastoma brain tumor-initiating cell growth through the β-catenin pathway.

Glioblastoma (GBM) is the most common and fatal primary brain tumor in humans and it is essential that new and better therapies are developed to treat this disease. Previous research suggests that casein kinase 2 (CK2), may be a promising therapeutic target for GBMs. CK2 has enhanced expression or activity in numerous cancers, including GBM and it has been demonstrated that inhibitors of CK2 regressed tumor growth in GBM xenograft mouse models. Our studies demonstrate that the CK2 subunit, CK2α, is overexpressed in and plays an important role in regulating brain tumor initiating cells (BTIC) in GBM. Initial studies showed that two GBM cell lines (U87-MG and U138) transduced with CK2α had enhanced proliferation and anchorage-independent growth. Inhibition of CK2α using siRNA or small molecule inhibitors (TBBz, CX-4945) reduced cell growth and decreased tumor size and increased the survival rate in GBM xenograft mouse models. We also verified that inhibition of CK2α decreased the activity of a well-known GBM initiating cell regulator, β-catenin. Loss of CK2α decreased two β-catenin-regulated genes that are involved in GBM initiating cell growth, OCT4 and NANOG. To determine the importance of CK2α in GBM stem cell maintenance, we reduced CK2α activity in primary GBM samples and tumor spheres derived from GBM patients. We discovered that loss of CK2α activity reduced the sphere forming capacity of BTIC and decreased numerous GBM stem cell markers including CD133, CD90, CD49f, and A2B5. Our study suggests that CK2α is involved in GBM tumorigenesis by maintaining BTIC through the regulation of β-catenin.

P17.39 * TURN OLD INTO NEW: METFORMIN AS ANTINEOPLASTIC AGENT IN GLIOBLASTOMA

Metformin is one of the most widely prescribed oral antidiabetic drugs. In addition to its known hypoglycemic effects there is current evidence, that metformin may also reduce the risk and mortality of cancer in diabetic patients. The antineoplastic role of metformin has been extensively studied in breast-, prostate-, pancreatic-, lung- and colon cancer, however, the effect of metformin on glioblastomas, one of the most devastating forms of human tumors, has not been thoroughly investigated. We examined the effects of metformin on a variety of brain tumor initiating cells (BTIC) from fresh tumor resections of primary glioblastomas using proliferation assays (cell count, BRDU-assay) and migration assays (scratch and spheroid migration assay), and investigated differentiation of cells by means of immunocytochemistry. Tumor cell metabolism was investigated by measuring extracellular lactate levels, mitochondrial respiration by high resolution respirometry and determination of cellular oxygen consumption under cell culture conditions using the PreSens-assay. Important signalling molecules, such as AMPK, Akt and mTOR were investigated by means of Western Blot analysis. Functional assays were compared in BTIC and differentiated tumor cells from the same patients. In BTIC, we observed a markedly impaired proliferation and migration of cells after treatment with metformin and inhibition of complex I of the respiratory chain. In addition, we observed increased extracellular lactate levels and activation of the AMPK/mTOR signalling cascade. Metformin-treated BTIC showed reduced expression of stem cell markers. Differentiated tumor cells were distinctly less responsive to treatment with metformin. We hypothesize that differential sensitivity of brain tumor initiating cells and differentiated tumor cells to metformin is due to different use of and dependence on oxidative energy production and impaired glycolytic and glutaminolytic rescue pathways in BTIC. Further investigation of metformin as a possible antineoplastic agent in glioblastoma is warranted, especially in the light of a multitude of promising ongoing clinical studies on metformin and other tumor types.

P03.02 * IN SITU ORGANOTYPIC SLICE CULTURE MIGRATION MODEL TO DISSECT INFILTRATIVE FROM STATIONARY HUMAN BRAIN TUMOR INITIATING CELLS

Glioblastomas (GBM) are highly malignant brain tumors, hallmarked by infiltrating growth of tumor cells. Brain Tumor Initiating Cells (BTIC) may initiate GBM pathogenesis according to recent publications. In particular the subventricular zone and hippocampus are areas where BTICs may arise and migrate to distinct areas to initiate the tumor bulk via differentiation into the glial linage. As a valuable tool an in situ model of organotypic brain slice cultures is utilized to visualize and analyse migration and invasion in a setting that simulates normal brain tissue conditions. This method allows the inoculation of tagged BTIC in the hippocampal region of fresh brain slice cultures from newborn rats (P8-12), and to monitor tumor cell invasion for up to three weeks. During this process the original cell population divides into different subpopulations: a stationary that proliferates at the implantation site, and an infiltrating one. Stationary and infiltrating cells differ in morphology and behaviour. In order to reveal why an initial cell population separates into subpopulations, a method was developed to microdissect single cells from the slices after an inoculation time of up to three weeks. cDNA libraries were generated for subsequent microarray analysis to compare the subgroups regarding gene expression profiles of genes associated with e.g. proliferation, invasion, cell-cell contacts, and stemness. Besides the different phenotypic appearance of the subpopulations, the genetic pattern of cells from the subpopulations determined by microarray analysis should unveil genes driving tumor invasion. These newly identified targets will be further characterized and regulated to manipulate the behaviour of BTICs.

P01.11 * ATF4 AS A MEDIATOR OF RESISTANCE TO TARGETED THERAPY IN HIGH-GRADE GLIOMAS

Small molecule inhibitors have been investigated in a large set of clinical trials in malignant gliomas. Despite promising preclinical studies, results of pilot trials have been generally disappointing. A deeper understanding of the complex biology of malignant glioma cells, and their adaptation to targeted agents is therefore critical for further therapy development. We analyzed gene expression in 18 short-term serum-free cultures of high-grade gliomas enhanced for brain tumor initiating cells (BTIC) before and after in vitro treatment with the tyrosine kinase inhibitor Sunitinib. Geneset enrichment analysis (GSEA) revealed that some of the regulated patterns are associated to the amino acid metabolism. In gene ontology annotations, additional evidence was raised that ER-stress response signaling which is connected to the amino acid metabolism is induced by Sunitinib. Based on the observation of gene network analysis (STRING) we hypothesize that the central mediator of the integrated stress response ATF4 (activating transcription factor 4) plays an important role in the regulation of the Sunitinib induced expression profiles and probably in the adaptation to treatment conditions. First, we confirmed a concentration dependent induction of ATF4 protein expression in Sunitinib treated BTICs. For further evaluation, we analyzed the expression of ATF4 in paraffin embedded tissue blocks from high grade glioma patients treated with Sunitinib by immunohistochemistry. ATF4 significantly correlated with shorter overall survival from the beginning of Sunitinib treatment. Interestingly, expression data from high grade gliomas retrieved from a public platform (REMBRANDT) revealed that transcription of the ATF4-induced TRB3 pseudokinase is negatively correlated with overall survival independent of treatment. In summary our data suggest that ATF4 expression may be predictive for response to Sunitinib and might also be involved in general resistance mechanisms in high grade gliomas. Further studies are planned to elucidate the induction of apoptosis and autophagy in dependence of ATF4 expression and its contribution to therapy resistance.

BRAIN TUMOUR INITIATING CELLS AND TARGETING STAT3 ONCOGENIC SIGNALLING IN GBM

BACKGROUND: Glioblastoma (GBM) is a devastating disease and the most lethal of adult brain tumours. Despite standard surgery, radiation and chemotherapy, the median survival is 15 months, thought to be due, in part, to recurrence from a small reservoir of brain tumour initiating cells. Our laboratory discovered adult neural stem cells, now found to be present in the brains of all adult mammals, through the development of the clonal neurosphere assay. This assay has contributed to the identification of adult human brain tumour initiating cells (BTICs), which may represent a reservoir that leads to GBM recurrence and death. Building upon the identification of growth factors and cytokines that converge on the cytoplasmic signal transducer and activator of transcription 3 (STAT3) to maintain the adult neural stem cell undifferentiated state, and the fact that STAT3 is abnormally active in GBM and may be one of the causes of tumour growth and therapeutic resistance, targeting the janus kinase (JAK)/STAT3 signalling pathway has become a major research focus for our laboratory. Here, we report the GBM translational potential of R333, a JAK/SYK inhibitor in development for other indications by Rigel Pharmaceuticals. METHODS: We have developed a cellular resource of more than 100 BTIC lines derived from GBM patients. Collaborative studies with academic and industry partners has resulted in the identification of promising therapeutic candidates that target JAK/STAT3 signalling in GBM. These compounds are tested for their ability to attenuate growth of BTICs in cell culture, on target activity in vitro and in vivo, and for their impact on the the survival of immunocompromised mice that have received BTIC xenografts. We employed this approach to examine the therapeutic potential of R333. RESULTS: In a collection of diverse patient-derived BTIC lines, R333 demonstrated potent, on-target JAK/STAT3 inhibition at low micromolar doses that effectively decreased BTIC viability and clonogenic potential, without affecting human fetal astrocytes. Importantly, R333 could be co-administered with temozolomide without reducing therapeutic response. Using phosphoproteomics, we confirmed the primary target of R333 in BTSCs as activated STAT3. In a BTIC orthotopic xenograft model, systemic treatment with R348, the pro-drug form of R333, resulted in BBB penetration and significant increased overall median survival without major side effects. CONCLUSIONS: These data suggest that R333 (R348) is a potent, BBB-permeable JAK/STAT3 inhibitor, worthy of further clinical evaluation for GBM treatment. These findings support our overall hypothesis that targeting the STAT3 signalling pathway may represent an effective therapeutic approach to managing GBM. SECONDARY CATEGORY: Tumor Biology.

SOX2 IDENTIFIES THE TREATMENT-REFRACTORY STEM CELL POPULATION IN GROUP 2 MEDULLOBLASTOMA

BACKGROUND: Brain tumors represent the leading cause of childhood cancer mortality, of which medulloblastoma (MB) is the most frequent malignant pediatric brain tumor. Recent studies have demonstrated the presence of several MB molecular subgroups, each distinct in terms of prognosis and predicted therapeutic response. Although, group 2 MBs characterized by activation of the sonic hedgehog (Shh) signaling pathway have been of particular therapeutic interest, treatment-resistance has posed a significant challenge. Given the identification of brain tumor-initiating cells (BTICs), which have the ability to initiate and maintain tumor growth while promoting resistance to radio- and chemotherapy, we investigated the mechanistic profile of treatment-resistant Shh-dependent MB BTICs. METHODS: Through investigating a differential stem cell gene expression profile of 325 primary human MBs, followed by a subsequent series of step-wise knockdown, overexpression, chromatin immunoprecipitation (ChIP), and in vitro self-renewal analyses we assessed the mechanistic role of Sox2 as a novel downstream target of Shh effector proteins, Gli1 and Gli2. We further determined the clinical utility of this mechanism by treating our MB BTICs with conventional radio- and chemotherapy as per the Children's Oncology Group (COG) protocol for MB patients in order to assess the validity of Sox2 as a marker of Shh-dependent treatment-refractory MB BTICs. RESULTS: Activation and inhibition of the Shh pathway and Sox2 expression using small molecule Shh agonists and antagonists, respectively, were observed only in distinct subsets of tumor cells (CD15+ MB BTICs). ChIP experiments further demonstrated the presence of a differential Shh signaling mechanism within distinct cell populations of the bulk tumor mass as Gli proteins showed Sox2 promoter binding only in CD15+ MB BTICs. The functional relevance of this cell-specific signaling mechanism was assessed through an in vitro and in vivo increase in treatment-resistant Sox2+ MB BTICs following conventional and combinatorial radio- and chemotherapy with Shh pathway inhibitors. CONCLUSIONS: Our work demonstrates for the first time that Sox2 expression in MB is regulated by the Shh signaling pathway and Sox2+ MB BTICs represent the treatment-resistant clone in Group 2 MBs, suggesting a need for combinatorial therapy as tumors evolve over the course of treatment. Aside from implicating developmental genes and pathways in oncogenesis, we have also demonstrated the importance of studying cancer at a cellular level, as distinct differences in rare subsets of tumor cells may not otherwise be appreciated with genomic profiling of the bulk tumor mass. SECONDARY CATEGORY: Neuropathology & Tumor Biomarkers.

Brain metastasis-initiating cells: survival of the fittest.

Brain metastases (BMs) are the most common brain tumor in adults, developing in about 10% of adult cancer patients. It is not the incidence of BM that is alarming, but the poor patient prognosis. Even with aggressive treatments, median patient survival is only months. Despite the high rate of BM-associated mortality, very little research is conducted in this area. Lack of research and staggeringly low patient survival is indicative that a novel approach to BMs and their treatment is needed. The ability of a small subset of primary tumor cells to produce macrometastases is reminiscent of brain tumor-initiating cells (BTICs) or cancer stem cells (CSCs) hypothesized to form primary brain tumors. BTICs are considered stem cell-like due to their self-renewal and differentiation properties. Similar to the subset of cells forming metastases, BTICs are most often a rare subpopulation. Based on the functional definition of a TIC, cells capable of forming a BM could be considered to be brain metastasis-initiating cells (BMICs). These putative BMICs would not only have the ability to initiate tumor growth in a secondary niche, but also the machinery to escape the primary tumor, migrate through the circulation, and invade the neural niche.

Effect of inhibition of α-secretase activity on cleavage of p75 neurotrophin receptor (p75NTR) and proliferation of brain tumor initiating cells (BTICs) and malignant gliomas (MGs).

2086 Background: MGs are highly invasive, proliferative, and are resistant to treatment. The identification of Glioma Stem Cells (GSCs a.k.a. BTICs) suggests MGs may arise from these cells. By usin...