Patient-derived Glioblastoma Multiforme Cells Research Articles

A facile and scalable in production non-viral gene engineered mesenchymal stem cells for effective suppression of temozolomide-resistant (TMZR) glioblastoma growth

BackgroundMesenchymal stem cells (MSCs) serve as an attractive vehicle for cell-directed enzyme prodrug therapy (CDEPT) due to their unique tumour-nesting ability. Such approach holds high therapeutic potential for treating solid tumours including glioblastoma multiforme (GBM), a devastating disease with limited effective treatment options. Currently, it is a common practice in research and clinical manufacturing to use viruses to deliver therapeutic genes into MSCs. However, this is limited by the inherent issues of safety, high cost and demanding manufacturing processes. The aim of this study is to identify a facile, scalable in production and highly efficient non-viral method to transiently engineer MSCs for prolonged and exceptionally high expression of a fused transgene: yeast cytosine deaminase::uracil phosphoribosyl-transferase::green fluorescent protein (CD::UPRT::GFP).MethodsMSCs were transfected with linear polyethylenimine using a cpg-free plasmid encoding the transgene in the presence of a combination of fusogenic lipids and β tubulin deacetylase inhibitor (Enhancer). Process scalability was evaluated in various planar vessels and microcarrier-based bioreactor. The transfection efficiency was determined with flow cytometry, and the therapeutic efficacy of CD::UPRT::GFP expressing MSCs was evaluated in cocultures with temozolomide (TMZ)-sensitive or TMZ-resistant human glioblastoma cell lines. In the presence of 5-fluorocytosine (5FC), the 5-fluorouracil-mediated cytotoxicity was determined by performing colometric MTS assay. In vivo antitumor effects were examined by local injection into subcutaneous TMZ-resistant tumors implanted in the athymic nude mice.ResultsAt > 90% transfection efficiency, the phenotype, differentiation potential and tumour tropism of MSCs were unaltered. High reproducibility was observed in all scales of transfection. The therapeutically modified MSCs displayed strong cytotoxicity towards both TMZ-sensitive and TMZ-resistant U251-MG and U87-MG cell lines only in the presence of 5FC. The effectiveness of this approach was further validated with other well-characterized and clinically annotated patient-derived GBM cells. Additionally, a long-term suppression (> 30 days) of the growth of a subcutaneous TMZ-resistant U-251MG tumour was demonstrated.ConclusionsCollectively, this highly efficient non-viral workflow could potentially enable the scalable translation of therapeutically engineered MSC for the treatment of TMZ-resistant GBM and other applications beyond the scope of this study.

Abstract 4488: Immunomodulatory activity of epigenetic drugs: Laying the ground for new combined immunotherapeutic strategies for glioblastoma

Abstract Rationale: Glioblastoma multiforme (GBM) is the most common and deadliest primary brain tumour in adults, with a very dismal prognosis and limited effective therapies. Immunotherapy may play a role in treating GBM, though the initial clinical results are still poor, and novel combined treatment(s) could possibly improve its efficacy. Manipulating the epigenome has been considered a therapeutic opportunity in GBM, since epigenetic modifications regulate glioma proliferation, metastasis, invasion, and prognosis. In addition, epigenetic alterations may negatively regulate the cross-talk between GBM and immune cells in GBM microenvironment (TME), thus limiting the efficacy of immunotherapy. We previously demonstrated the immunomodulatory potential of epigenetic drugs in cancer cells, and translated this notion in the clinic to improve efficacy of immunotherapy of melanoma patients (Di Giacomo AM, CCR 2019). Accordingly, this study aimed to evaluate the immunomodulatory potential of epigenetic drugs in GBM, and to provide the scientific rationale for new combinatorial therapeutic approaches in GBM patients. Methods: Patient-derived GBM (pGBM) cell lines (n=10) were established from surgically-removed GBM lesions and treated with the DNA hypomethylating agent guadecitabine (1 µM); commercially available human GBM (cGBM) cell lines (n=3) were treated with guadecitabine (1 µM) and/or with the EZH2-inhibitor GSK126 (10 µM). Expression of HLA class I and ICAM-1 by GBM cells was investigated by flow-cytometry; cancer testis antigens (CTA, i.e., NY-ESO, MAGE-A1 and MAGE-A3) and the epithelial-mesenchymal transition (EMT)-regulating cadherins (i.e., CDH1 and CDH2) expression was analyzed by quantitative Real-Time PCR. Results: A positive modulation of immune profile was observed in all investigated guadecitabine-treated GBM cells. Specifically, in untreated vs guadecitabine-treated cells, MFI mean values ± SD of HLA class I were 402±326 vs 442±303 in pGBM cells, and 277±127 vs 381±138 in cGBM cells; MFI mean values ± SD of ICAM-1 were 53±72 vs 92±116 in pGBM cells, and 5±6 vs 26±17 in cGBM cells. Though a slight increment was induced by GSK126 for HLA class I (312±169) and ICAM-1 (9±11) expression in cGBM, an additive effect was detected with GSK126+guadecitabine (405±261 for HLA class I and 35±22 for ICAM-1) in cGBM cells. Guadecitabine induced NY-ESO-1, MAGE-A1, and MAGE-A3 expression in 9/10, 6/10, and 5/8 pGBM cells, respectively and in all cGBM cells. Levels of CTA expression were increased in cGBM by GSK126+guadecitabine vs guadecitabine with mean Fold Change (FC) of 1.5, 1.6 and 1.6 for NY-ESO-1, MAGE-A1 and MAGE-A3 expression. Finally, CDH1 expression was induced in 1/3 and in 2/3 cGBM cells by guadecitabine and guadecitabine+GSK126, respectively; CDH2-positive expression was upregulated by guadecitabine (mean FC=2.45) but not by guadecitabine+GSK126. Conclusions: Immunomodulatory properties of epigenetic drugs in GBM should be exploited to improve the efficacy of GBM immunotherapy. Citation Format: Maria Fortunata Lofiego, Francesca Piazzini, Carolina Fazio, Ornella Cutaia, Carla Chiarucci, Sara Cannito, Elisabetta Gambale, Monica Valente, Anna Maria Di Giacomo, Sandra Coral, Clelia Miracco, Giuseppe Oliveri, Michele Maio, Alessia Covre. Immunomodulatory activity of epigenetic drugs: Laying the ground for new combined immunotherapeutic strategies for glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4488.

Abstract 2998: Linear confined space migration induces treatment resistance and gene expression changes in glioblastoma multiforme

Abstract Background: Glioblastoma Multiforme (GBM), an aggressive brain cancer, is known for its treatment resistant recurrence and lethality. The diffuse migration and invasion of GBM cells commonly occurs along brain white matter tracts in linear confined spaces between axons and has significant clinical implications as these cells survive initial therapies forming the main source of recurrent tumors resistant to therapies. In this context, this project aims to analyze the treatment resistance mechanism associated with linear confined space migrating cells (LCSM) to devise a better treatment strategy for GBM. Methods: We used a combination of microfluidic technology (5 µM wide channels in PDMS block) emulating linear white matter tracts in brain and standard molecular analysis (confocal microscopy, high content imaging, western blot, and flow cytometry) to investigate LCSM cells compared to Standard Monolayer Culture cells (SMCs) for the mechanism of drug resistance. Both established and patient derived GBM cell lines were used in the study. Results: LCSM cells were more resistant to chemo-drugs (Temozolomide and Doxorubicin) than SMCs as observed in cell viability assays. Enhanced drug efflux was observed in LCSM cells compared to SMC cells indicating the activation of drug efflux pumps. Protein expression analysis indicated that ABC drug efflux transporters (ABCB1, ABCC1, ABCG2) were activated or enhanced at varying levels in different LCSM cells compared to SMCs. Pharmacological inhibition of the prominently expressed ABCG2 protein using KO143 resulted in enhanced cell death by chemo drugs, suggesting that chemo resistance in LCSM, at least in part, is conferred by ABCG2 or other efflux transporters. In addition, CD133 (a marker of treatment resistant stem cell population in GBM) and AQP4 (Aquaporin 4; pro-migratory marker in GBM) were also enhanced in LCSM compared to SMC suggesting their possible roles in treatment resistance. Conclusion: Preliminary studies reveal that the enhanced expression ABC transporters, prominently ABCG2, might be responsible for enhanced drug efflux and low therapeutic sensitivity in GBM- LCSM cells. The over expression of AQP4 and CD133, and differential expression of other genes suggest that more complex mechanisms might be involved, which are currently under investigation. Citation Format: Anish Babu, Patrick McKernan, Xue Cai, James Battiste. Linear confined space migration induces treatment resistance and gene expression changes in glioblastoma multiforme [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2998.

Temozolomide in Combination With NF-κB Inhibitor Significantly Disrupts the Glioblastoma Multiforme Spheroid Formation.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor, accounting for 50% of all cases. GBM patients have a five-year survival rate of merely 5.6% and a median overall survival of 14.6 months with the “Stupp” regimen, 20.9 months with tumor treatment fields (TTF, OptuneR) in patients who participated in clinical trials, and 11 months for all GBM patients prior to TTF use. Objective: Our group recently developed a brain cancer chip which generates tumor spheroids, and provides large-scale assessments on the response of tumor cells to various concentrations and combinations of drugs. This platform could optimize the use of tumor samples derived from GBM patients to provide valuable insight on the tumor growth and responses to drug therapies. To minimize any sample loss in vitro, we improved our brain cancer chip system by adding an additional laminar flow distribution layer, which reduces sample loss during cell seeding and prevents spheroids from escaping from the microwells. Methods: In this study, we cultured 3D spheroids from GBM cell lines and patient-derived GBM cells in vitro, and investigated the effect of the combination of Temozolomide and nuclear factor-κB inhibitor on tumor growth. Results: Our study revealed that these drugs have synergistic effects in inhibiting spheroid formation when used in combination. Conclusions: These results suggest that the brain cancer chip enables large-scale, inexpensive and sample-effective drug screening to 3D cancer tumors in vitro, and could be applied to related tissue engineering drug screening studies.

BET and Aurora Kinase A inhibitors synergize against MYCN-positive human glioblastoma cells

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults. Patients usually undergo surgery followed by aggressive radio- and chemotherapy with the alkylating agent temozolomide (TMZ). Still, median survival is only 12–15 months after diagnosis. Many human cancers including GBMs demonstrate addiction to MYC transcription factor signaling and can become susceptible to inhibition of MYC downstream genes. JQ1 is an effective inhibitor of BET Bromodomains, a class of epigenetic readers regulating expression of downstream MYC targets. Here, we show that BET inhibition decreases viability of patient-derived GBM cell lines. We propose a distinct expression signature of MYCN-elevated GBM cells that correlates with significant sensitivity to BET inhibition. In tumors showing JQ1 sensitivity, we found enrichment of pathways regulating cell cycle, DNA damage response and repair. As DNA repair leads to acquired chemoresistance to TMZ, JQ1 treatment in combination with TMZ synergistically inhibited proliferation of MYCN-elevated cells. Bioinformatic analyses further showed that the expression of MYCN correlates with Aurora Kinase A levels and Aurora Kinase inhibitors indeed showed synergistic efficacy in combination with BET inhibition. Collectively, our data suggest that BET inhibitors could potentiate the efficacy of either TMZ or Aurora Kinase inhibitors in GBM treatment.

CBMT-01. ALANINE FUELS ENERGY METABOLISM OF GLIOBLASTOMA CELLS

Abstract Despite available aggressive treatment options, glioblastoma multiforme (GBM) has a median survival of 16 months indicating an urgent unmet need to develop new therapeutic strategies. Earlier, we have demonstrated that both glucose and acetate are the two major nutrients leading to synthesis of approximately ~60% acetyl-CoA in the brain tumors. Therefore, there must be other nutrients that may contribute to bioenergetic needs of highly proliferative GBM cells. Alanine is one of the non-essential amino acids (NEAA) that is constantly produced in cancer cells through Warburg glycolysis and its role in cancer metabolism is not well understood. Recently, it has been shown that alanine produced by pancreatic stellar cells contributed to the energy metabolism in pancreatic cancer cells. However, it is not known whether GBM can use alanine as an energy source. Here, we test whether GBM cells have the ability to metabolize alanine as a fuel to meet its increased energy requirements. Patient-derived GBM cells were cultured with 2.0 mM [3-13C]alanine for the final 24 hours, harvested in 50% methanol, snap-frozen in liquid N2, freeze-thaw cycle 3 times and lysates were stored at -80 °C. Derivatized material from the frozen lysates were used for GC-MS analysis to determine carbon mass isotopomer distribution (MID) of various glycolytic and TCA cycle intermediates. Our results indicated that [3-13C]alanine entered the GBM cells and produced [3-13C]lactate via pyruvate. Also, alanine-derived [3-13C]pyruvate led to the generation of [2-13C]acetyl-CoA, which entered TCA cycle and produced M+1 13C isotopomers of citrate, glutamate, malate and aspartate. MID showed the following 13C enrichment (M+1) values: citrate, 6.9 % ± 0.3%; glutamate, 4.4% ± 0.3%; malate, 2.1% ± 0.6%; 2.1% ± 0.5%. This preliminary data shows that GBM cells are capable of utilizing alanine to generate energy and produce precursors for biomolecular synthesis.

CBMT-33. VISUALIZING THE METABOLISM OF GLIOBLASTOMA PATIENT-DERIVED ORTHOTOPIC XENOGRAFTS BY MASS SPECTROMETRY IMAGING

Abstract INTRODUCTION. Glioblastoma multiforme (GBM) has a dismal prognosis, which has been attributed to extensive inter- and intra-tumoural heterogeneity. To investigate the metabolic heterogeneity of GBM, we employed desorption electrospray ionization (DESI) imaging on snap frozen samples of GBM patient-derived orthotopic xenografts (PDOX) in rat brains, following infusion of [U-13C]glucose. METHODS. Athymic nude rats (n=3) were infused with [U-13C]glucose (0.4 mg/g body weight bolus, 0.012 mg/g/min at 300 µL/h infusion [1]) for 40, 80 and 120 min, followed by cardiac puncture to measure blood [U-13C]glucose levels using 13C NMR spectroscopy. A second set of rats (n=2) were implanted intracranially with patient-derived GBM cells. The presence of tumour was confirmed by T2-weighted MRI. One animal was infused with [U-13C]glucose for 2 h, while another with [12C]glucose. The brains were snap frozen in liquid nitrogen followed by analysis with DESI imaging in negative ion mode (35 µm resolution). RESULTS. After a 2 h infusion, 43% of the circulating glucose was 13C labelled. Unsupervised spatial clustering of the MSI data resulted in segmentation of normal brain from tumour. Levels of [12C and 13C]glucose were high in the peri-tumoural region and low within the tumour. [12C and 13C]lactate showed the opposite distribution, indicating that the rate of glucose utilization within the tumour exceeded the rate of delivery. The peri-necrotic and peri-tumoural areas had higher levels of glutamate compared to the rest of the tumour. CONCLUSIONS. The results demonstrate that the PDOX is metabolically heterogeneous and differs from surrounding brain. While the tumour was predominately glycolytic, the presence of glutamate in peri-necrotic and peri-tumoral regions indicates the presence of increased TCA cycle activity. Comparison of MSI data with histological staining of brain sections should yield additional information about the relationship between the tumour and its microenvironment.

The STAT3/Slug Axis Enhances Radiation-Induced Tumor Invasion and Cancer Stem-like Properties in Radioresistant Glioblastoma.

Glioblastoma multiforme (GBM) requires radiotherapy (RT) as a part of definitive management strategy. RT is highly effective, destroying cancer cells that may exist around the surgical tumor bed. However, GBM still has a poor prognosis and a high local recurrence rate after RT. Accumulating research indicates that GBM contains cancer stem-like cells (CSCs), which are radioresistant and result in therapeutic failure. Additionally, GBM cells can aggressively invade normal brain tissue, inducing therapeutic failure. Using clinical observations, we evaluated the effect of radiation on tumor control. We also explored the biomolecular pathways that connect radioresistance and CSC- and epithelial-mesenchymal transition (EMT)-associated phenotypes in patient-derived GBM cells. Transwell and microarray assay demonstrated that radioresistant GBM cells (GBM-R2I2) exhibit increased invasion and self-renewal abilities compared with parental GBM cells. Finally, to identify potential mechanisms underlying these observations, we used a PCR array to search for molecular markers of cell motility. Signal transducer and activator of transcription 3 (STAT3) directly bound to the Slug promoter in a chromatin immunoprecipitation assay. Reduced STAT3 decreased Slug expression and suppressed cell invasion in GBM-R2I2 cells while increasing Slug reversed these effects. In addition, STAT3 knockdown significantly inhibited CSC properties, synergistically increased the radiotherapeutic effect, and effectively increased the survival rate in vivo. We deciphered a new pathway of GBM radioresistance, invasion, and recurrence via the STAT3/Slug axis that could be a new target of GBM therapy.

EPH Profiling of BTIC Populations in Glioblastoma Multiforme Using CyTOF.

The ability to elucidate the phenotype of brain tumor initiating cell (BTIC) in the context of bulk tumor in glioblastoma multiforme (GBM) provides significant therapeutic benefits for therapeutic evaluation. For the identification of such an elusive and rare subpopulation of cells, a single cell analysis technology with deep profiling capabilities known as Mass Cytometry (CyTOF) can prove to be highly useful. CyTOF circumvents the spectral overlap limitations of traditional flow cytometry by replacing fluorophores with metal isotope tags, allowing the accurate detection of significantly more parameters at the same time. In this chapter, we demonstrate that synthetic antibodies can be conjugated with metal isotope tags for CyTOF analysis, resulting in the development of a highly tailored, custom multi-parameter panel. This toolset was used to stain patient-derived GBM cells, which was analyzed via CyTOF. Analysis software viSNE and SPADE were applied to study the co-expression patterns of the Eph Receptor (EphR) family and several putative BTIC markers in GBM, resulting in the identification of a distinct group of cells consistent with a BTIC subpopulation. This approach can be readily adapted to the detection of cancer stem-like cells in other cancer types.

BMP9 counteracts the tumorigenic and pro-angiogenic potential of glioblastoma.

Glioblastoma multiforme (GBM) is a highly vascularized and aggressive brain tumor, with a strong ability to disseminate and invade the surrounding parenchyma. In addition, a subpopulation of GBM stem cells has been reported to possess the ability to transdifferentiate into tumor-derived endothelial cells (TDECs), supporting the resistance to anti-angiogenic treatments of newly formed blood vessels. Bone Morphogenetic Protein 9 (BMP9) is critically involved in the processes of cancer cell differentiation, invasion and metastasis, representing a potential tool in order to impair the intrinsic GBM aggressiveness. Here we demonstrate that BMP9 is able to trigger the activation of SMADs in patient-derived GBM cells, and to strongly inhibit proliferation and invasion by reducing the activation of PI3K/AKT/MAPK and RhoA/Cofilin pathways, respectively. Intriguingly, BMP9 treatment is sufficient to induce a strong differentiation of GBM stem-like cells and to significantly counteract the already reported process of GBM cell transdifferentiation into TDECs not only in in vitro mimicked TDEC models, but also in vivo in orthotopic xenografts in mice. Additionally, we describe a strong BMP9-mediated inhibition of the whole angiogenic process engaged during GBM tumor formation. Based on these results, we believe that BMP9, by acting at multiple levels against GBM cell aggressiveness, can be considered a promising candidate, to be further developed, for the future therapeutic management of GBM.

Deciphering molecular mechanisms of arginine deiminase-based therapy – Comparative response analysis in paired human primary and recurrent glioblastomas

Arginine auxotrophy constitutes the Achilles' heel for several tumors, among them glioblastoma multiforme (GBM). Hence, arginine-depleting enzymes such as arginine deiminase (ADI) from Streptococcus pyogenes are promising for treatment of primary and maybe even refractory GBM. Based on our previous study in which ADI-susceptibility was shown on a panel of patient-derived GBM cell lines, we here aimed at deciphering underlying molecular mechanisms of ADI-mediated growth inhibition. We found that ADI (35 mU/mL) initially induces a cellular stress-response that is characterized by upregulation of genes primarily belonging to the heat-shock protein family. In addition to autophagocytosis, we show for the first time that senescence constitutes another cellular response mechanism upon ADI-treatment and that this bacterial enzyme is able to act as radiosensitizer (¼ cases). Long-term treatment schedules revealed no resistance development, with treated cells showing morphological signs of cell stress. Next, several combination strategies were employed to optimize ADI-based treatment. Simultaneous and sequential S. pyogenes ADI-based combinations included substances acting at different molecular pathways (curcumin, resveratrol, quinacrine, and sorafenib, 2 × 72 h treatment). Adding drugs to GBM cell lines (n = 4, including a matched pair of primary and recurrent GBM in one case) accelerated and potentiated ADI-mediated cytotoxicity. Autophagy was identified as the main cause of tumor growth inhibition. Of note, residual cells again showed classical signs of senescence in most combinations.Our results suggest an alternative treatment regimen for this fatal cancer type which circumvents many of the traditional barriers. Using the metabolic defect in GBM thus warrants further (pre-) clinical evaluation.

Abstract 4067: BPM31510, a clinical stage metabolic modulator demonstrates therapeutic efficacy in an in vivo C6 rat glioma model and synergizes with temozolomide

Abstract BPM31510, a parenteral nanodispersion of ubidecarenone that is in clinical testing for solid tumors, effectuates an anti-cancer effect in highly metabolic cancers by eliciting an anti-Warburg effect. Intraperitoneal (i.p.) administration of BPM31510 reverses paraplegia in a rat CNS leukemia model demonstrating the bioavailability of the drug to the central nervous system. The study describes data demonstrating the therapeutic utility of BPM31510 in glioblastoma multiforme (GBM). BPM31510 decreased cell proliferation rates in patient derived GBM cell lines compared to temozolamide (TMZ), pretreatment with BPM31510 followed by TMZ challenge was associated with significant synergy in reducing cell proliferation compared to monotherapy. In a C6 glioma allograft rat model treatment with BPM31510 alone (n=30/group) (i.p., 50mg/kg, b.i.d) increased overall survival compared to untreated control group. BPM31510 in combination with procarbazine (oral, 60mg/kg; d 8, 21) and vincristine (IV, 1.4mg/kg; d 8, 29) significantly improved overall survival compared to BPM31510 alone or chemotherapy. Furthermore, long term survival was achieved in four of twenty rats with C6 gliomas receiving BPM31510 doses between 10 and 50 mg/kg compared with none treated with either vehicle or irradiation (P < 0.02, Fisher exact test). Interestingly, the long term survival persisted for over two months after all treatment was discontinued; MR imaging confirmed the presence of static masses in these long term survivors. Collectively, the data demonstrate that BPM31510 has a robust effect in a preclinical model of GBM. Intravenous BPM31510 is currently in Phase 1/2 clinical trial for TMZ+BEV refractory GBM patients. Citation Format: Stephane Gesta, Semma Nagpal, Tulin Dadali, Milton Merchant, Taichang Jan, Anne R. Diers, Michael Kiebish, Joaquin Jiminez, Vivek K. Vishnudas, Niven R. Narain, Rangaprasad Sarangarajan, Lawrence Recht. BPM31510, a clinical stage metabolic modulator demonstrates therapeutic efficacy in an in vivo C6 rat glioma model and synergizes with temozolomide [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 4067. doi:10.1158/1538-7445.AM2017-4067

High-Dose Compound Heat Map for 3D-Cultured Glioblastoma Multiforme Cells in a Micropillar and Microwell Chip Platform.

Glioblastoma multiforme (GBM) is recognized as the most common and lethal form of central nervous system cancer. To cure GBM patients, many target-specific chemotherapeutic agents have been developing. However, 2D monolayer cell-based toxicity and efficacy tests did not efficiently screen agents due to the pool reflection of in vivo microenvironments (cell-to-cell and cell-to-extracellular matrix interaction). In this study, we used a 3D cell-based, high-throughput screening method reflecting the microenvironments using a micropillar and microwell chip platform to draw a high-dose heat map of the cytotoxicity and efficacy of 70 compounds, with two DMSO controls. Moreover, the high-dose heat map model compared the responses of four 3D-cultured patient-derived GBM cells and astrocytes to high dosages of compounds with respect to efficacy and cytotoxicity, respectively, to discern the most efficacious drug for GBM. Among the 70 compounds tested, cediranib (a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases) exhibited the lowest cytotoxicity to astrocytes and high efficacy to GBM cells in a high-dose heat map model.

Disruption of KIF3A in patient-derived glioblastoma cells: effects on ciliogenesis, hedgehog sensitivity, and tumorigenesis.

KIF3A, a component of the kinesin-2 motor, is necessary for the progression of diverse tumor types. This is partly due to its role in regulating ciliogenesis and cell responsiveness to sonic hedgehog (SHH). Notably, primary cilia have been detected in human glioblastoma multiforme (GBM) tumor biopsies and derived cell lines. Here, we asked whether disrupting KIF3A in GBM cells affected ciliogenesis, in vitro growth and responsiveness to SHH, or tumorigenic behavior in vivo. We used a lentiviral vector to create three patient-derived GBM cell lines expressing a dominant negative, motorless form of Kif3a (dnKif3a). In all unmodified lines, we found that most GBM cells were capable of producing ciliated progeny and that dnKif3a expression in these cells ablated ciliogenesis. Interestingly, unmodified and dnKif3a-expressing cell lines displayed differential sensitivities and pathway activation to SHH and variable tumor-associated survival following mouse xenografts. In one cell line, SHH-induced cell proliferation was prevented in vitro by either expressing dnKif3a or inhibiting SMO signaling using cyclopamine, and the survival times of mice implanted with dnKif3a-expressing cells were increased. In a second line, expression of dnKif3a increased the cells' baseline proliferation while, surprisingly, sensitizing them to SHH-induced cell death. The survival times of mice implanted with these dnKif3a-expressing cells were decreased. Finally, expression of dnKif3a in a third cell line had no effect on cell proliferation, SHH sensitivity, or mouse survival times. These findings indicate that KIF3A is essential for GBM cell ciliogenesis, but its role in modulating GBM cell behavior is highly variable.

Abstract B66: Identification of genetic and non-genetic dependencies in glioblastoma cell lines targeted by small molecules

Abstract Introduction. Glioblastoma multiforme (GBM) is the most common and malignant brain tumor. This single histopathological entity includes a devastating collection of heterogeneous high-grade tumors showing a broad spectrum of cellular morphologies and molecular diversity. These tumors display a uniform and very short survival time. Until now and in spite of improvements achieved in neurosurgery tools and molecular biology understanding, there is still no effective treatment. For this purpose we pursue a systematic analysis of genetic and non-genetic features that correlate with chemical sensitivity in order to identify molecular dependencies. Experimental procedures. We profiled 430 small chemical compounds at 16 different duplicated concentrations in 78 GBM cell lines belonging to two different models and collections: Patient Derived GBM Cell Lines (PDGCL) and Long Term GBM Cell Lines (LTGCL) from gCCLE collection. All cell lines were deeply characterized at genotype and phenotype level. As non-genomic determinants we considered cell line model, growth rate, passages, origin, behavior, stem cell markers (OLIG2, SOX2, NESTIN), differentiation markers (GFAP, O4, TUJ1), stem cell clonality markers (CD44, CD133, CD15) and overall sensitivity displayed by all cell lines across the drug panel. As genomic determinants we take into account significant copy number variations (CNV) and driver mutations (Whole Exome Sequencing). Mutations and CNV were integrated to determine oncogene activation and tumor suppressor gene inactivation of genes involved in three major signaling pathways (p53, RB and TRK). At transcriptomic level we considered the expression of 20.647 genes and transcription patters depicted according molecular fingerprints described in high-grade gliomas (Proneural, Mesenchymal, Proliferative), and primary GBMs (Proneural, Neural, Classical, Mesenchymal). At the end 28.503 features, were surveyed against 381 filtered small chemical profiles, rendering a total 10.859.643 interactions. Summary. Three major types of chemical sensitivities associated with molecular determinants were identified: 1) Dependencies related with genotype; sensitivities specifically driven by genetic alterations (TP53 mutations and CDKN1A overexpression to MDM2 inhibitor). 2) Dependencies related with phenotype; sensitivities specifically ruled by gene expression changes associated with Mesenchymal features (VEGFR, MET, PDGFRA inhibitors), or un-specifically driven by epigenetic changes (HDCA inhibitors). 3) Dependencies dominated by enzymatic activities (NAMPT inhibitors) indirectly associated with genetic alterations or gene expression changes. Statement. Obtained results explained chemical sensitivities targeting specific genotype and molecular phenotype features (differentiation state and chromatin status determined by gene expression) represented in two models of cell lines: PDGCL and LTGCL. Both states of cellular phenotype might co-exist at GBM tumor in different proportions. Our analysis provided molecular insights to drive targeted therapies in the new era of precision medicine. Citation Format: Ruben Ferrer-Luna, Shakti H. Ramkissoon, Lori A. Ramkissoon, Rebecca Lamothe, Jaime H. Cheah, Steven E. Schumacher, Sam Haidar, Paul A. Clemons, Alykhan Shamji, Keith L. Ligon, Rameen Beroukhim. Identification of genetic and non-genetic dependencies in glioblastoma cell lines targeted by small molecules. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B66.

Abstract 1369: Inhibition of Notch- and EGFR signaling reduces cell viability and angiogenesis in glioblastoma multiforme

Abstract The high-grade glioma, Glioblastoma Multiforme (GBM), is the most prevalent and aggressive brain tumour in adults with a median survival of fifteen months. Thus new therapeutic targets are urgently needed. GBM is characterized by extensive vascularisation why targeting angiogenesis has promising potential in improving the treatment of GBM patients. Aberrant EGFR signaling is another characteristic of GBM, and increased EGFR downstream signaling is believed to correlate with poor prognosis. However, little is known of the involvement of EGFR signaling in GBM angiogenesis. Notch signaling is known for its role in angiogenesis during foetal development and in other cancers, but the importance of Notch signaling in GBM angiogenesis is yet to be elucidated. Recently, it has been shown, that crosstalk between Notch- and EGFR signaling occurs in cancer cells, and aberrant EGFR signaling may therefore play a functional role in GBM angiogenesis through crosstalk with Notch signaling. The aim of this study was to investigate the effect of combined Notch- and EGFR inhibition on GBM cell viability and on ability of GBM cells to induce angiogenesis in endothelial cells. In vitro assays of GBM angiogenesis were established using two patient derived GBM cell cultures, one expressing the mutated EGFR, the EGFRvIII, and one expressing wild type EGFR. The functional relevance of EGFR- and Notch signaling in GBM cells was tested using the tyrosine kinase inhibitor Iressa for EGFR inhibition, and the gamma-secretase inhibitor DAPT for Notch inhibition. We show that combined inhibition of EGFR- and Notch signaling caused additive inhibition of cell viability of both cell cultures in vitro. Abrogated EGFR/Notch signaling in GBM cells further reduced angiogenesis in endothelial cells exposed to conditioned media from treated GBM cells. Moreover, when GBM cells were treated with the inhibitors, this inhibited phosphorylation and activation of the two survival signalling pathways ERK and Akt, and abrogated secretion and expression of the most prominent angiogenic factor - VEGF. The presented results indicate that EGFR- and Notch signaling play a functional role in GBM angiogenesis and that combined treatment with drugs targeting both the EGFR- and Notch pathway could prove beneficial over single drug therapy in targeting both the tumorigenic and angiogenic potential of GBM cells. Citation Format: Mikkel Staberg, Signe Regner Michaelsen, Louise Stobbe Olsen, Mette Kjølhede Nedergaard, Mette Villingshøj, Hans Skovgaard Poulsen. Inhibition of Notch- and EGFR signaling reduces cell viability and angiogenesis in glioblastoma multiforme. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1369. doi:10.1158/1538-7445.AM2015-1369

Abstract LB-218: Protein arginine methyltransferase inhibition of malignant gliomas leads to restored chemokine expression and enhanced immune effector function

Abstract Patients with Glioblastoma Multiforme (GBM) face a poor prognosis despite multimodal therapy, thus, there is an unmet need for discovery of novel therapeutic targets and approaches. The immune-privileged nature of the central nervous system and down-modulation of cytokines and chemokines in GBM tumors led us to explore epigenetic approaches to restore expression of immune-relevant genes. PRMT5 is a type II arginine methyltransferase that catalyzes symmetric dimethylation of arginine residues on histone proteins leading to transcriptional repression. Our previous work showed PRMT5 overexpression correlates with poor clinical outcome of GBM patients and that PRMT5 silencing inhibited tumor growth in vitro and in vivo. Microarray transcriptional studies following PRMT5 depletion identified potential PRMT5 target genes interferon-inducible protein 10 (CXCL10) and interferon-inducible T cell α chemoattractant (CXCL11). Increased secretion of CXCL10 and CXCL11 has been shown to facilitate homing of innate and adaptive immune-effector populations that promote anti-GBM activity in vivo. Thus we further investigated the significance of restored CXCL10 and CXLC11 with PRMT5 inhibition in GBM. CXCL10 protein expression was found to be significantly lower in GBM tumors (N = 15) by immunochemistry staining, compared with grade I gliomas (N = 7) (p = 0.0001). PRMT5 knockdown led to increased mRNA and protein expression of CXCL10 and CXCL11. Chromatin immunoprecipitation (ChIP) experiments identified CXCL10 and CXCL11 promoters to be directly targeted by PRMT5 repressive complexes. Previous work has shown that PRMT5 associates with other co-repressor molecules including HDAC2, DNA methyltransferase 3a (DNMT3a) and methyl binding domain protein 2 (MBD2). ChIP studies confirmed that PRMT5, HDAC2, DNMT3a and MBD2 proteins were all recruited to the promoters of CXCL10 and CXCL11. Interestingly, when two patient-derived GBM cell lines were transfected with siRNA targeting PRMT5, recruitment of all co-repressor proteins, PRMT5, HDAC2, DNMT3a, and MBD2 was lost on CXCL10 and CXCL11 promoters. These findings suggest that PRMT5 is a master transcriptional repressor that plays a central role in coordinating and assembling co-repressor chromatin remodeling complexes on PRMT5 target gene promoters. PRMT5 silencing resulted in secretion of biologically relevant levels of CXCL10 and CXCL11 in culture medium that promoted immune cell recruitment in transwell migration assays toward target glioma cells. Our data suggests that in addition to restoring regulatory and tumor suppressor gene expression that promotes direct GBM cell death, PRMT5 silencing may enhance immune-mediated anti-tumor activity. These findings further justify the development experimental therapeutic strategies targeting PRMT5 to modulate activity of immune-relevant genes. Citation Format: Fengting Yan, Yeshavanth Banasavadi-Siddegowda, John T. Patton, Mark Lustberg, Xin Wu, Balveen Kaur, Rober A. Baiocchi. Protein arginine methyltransferase inhibition of malignant gliomas leads to restored chemokine expression and enhanced immune effector function. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-218. doi:10.1158/1538-7445.AM2015-LB-218

Dedifferentiation of patient-derived glioblastoma multiforme cell lines results in a cancer stem cell-like state with mitogen-independent growth.

Emerging evidence shows that glioblastoma multiforme (GBM) originates from cancer stem cells (CSCs). Characterization of CSC-specific signalling pathways would help identify new therapeutic targets and perhaps lead to the development of more efficient therapies selectively targeting CSCs. Here; we successfully dedifferentiated two patient-derived GBM cell lines into CSC-like cells (induced glioma stem cells, iGSCs) through expression of Oct4, Sox2 and Nanog transcription factors. Transformed cells exhibited significant suppression of epidermal growth factor receptor and its downstream pathways. Compared with parental GBM cells, iGSCs formed large neurospheres even in the absence of exogenous mitogens; they exhibited significant sensitivity to salinomycin and chemoresistance to temozolomide. Further characterization of iGSCs revealed induction of NOTCH1 and Wnt/β-catenin signalling and expression of CD133, CD44 and ALDH1A1. Our results indicate that iGSCs may help us understand CSC physiology and lead to development of potential therapeutic interventions aimed at differentiating tumour cells to render them more sensitive to chemotherapy or other standard agents.

Glioblastoma specific antigens, GD2 and CD90, are not involved in cancer stemness.

Glioblastoma multiforme (GBM) is the most malignant World Health Organization grade IV brain tumor. GBM patients have a poor prognosis because of its resistance to standard therapies, such as chemotherapy and radiation. Since stem-like cells have been associated with the treatment resistance of GBM, novel therapies targeting the cancer stem cell (CSC) population is critically required. However, GBM CSCs share molecular and functional characteristics with normal neural stem cells (NSCs). To elucidate differential therapeutic targets of GBM CSCs, we compared surface markers of GBM CSCs with adult human NSCs and found that GD2 and CD90 were specifically overexpressed in GBM CSCs. We further tested whether the GBM CSC specific markers are associated with the cancer stemness using primarily cultured patient-derived GBM cells. However, results consistently indicated that GBM cells with or without GD2 and CD90 had similar in vitro sphere formation capacity, a functional characteristics of CSCs. Therefore, GD2 and CD90, GBM specific surface markers, might not be used as specific therapeutic targets for GBM CSCs, although they could have other clinical utilities.

Genetic modification of neurons to express bevacizumab for local anti-angiogenesis treatment of glioblastoma.

The median survival of glioblastoma multiforme (GBM) approximately 1 yr. Following surgical removal, systemic therapies are limited by the blood-brain barrier. To circumvent this, we developed a method to modify neurons with the genetic sequence for therapeutic monoclonal antibodies using adeno-associated virus (AAV) gene transfer vectors, directing persistent, local expression in the tumor milieu. The human U87MG GBM cell line or patient-derived early passage GBM cells were administered to the striatum of NOD/SCID immunodeficient mice.AAVrh.10BevMab, an AAVrh.10-based vector coding for bevacizumab (Avastin®), an anti-human vascular endothelial growth factor (VEGF) monoclonal antibody, was delivered to the area of the GBM xenograft. Localized expression of bevacizumab was demonstrated by quantitative PCR, ELISA and Western. Immunohistochemistry showed the bevacizumab was expressed in neurons. Concurrent administration of AAVrh.10BevMab with the U87MG tumor reduced tumor blood vessel density, and tumor volume and increased survival. Administration of AAVrh.10BevMab 1 wk after U87MG xenograft reduced growth and increased survival. Studies with patient-derived early passage GBM primary cells showed a reduction in primary tumor burden with an increased survival. This data supports the strategy of AAV-mediated CNS gene therapy to treat GBM, overcoming the blood-brain barrier through local, persistent delivery of an anti-angiogenesis monoclonal antibody.