Glioma Stem-like Cells Research Articles

Expression profiling of single cells and patient cohorts identifies multiple immunosuppressive pathways and an altered NK cell phenotype in glioblastoma.

SummaryGlioblastoma (GBM) is an aggressive cancer with a very poor prognosis. Generally viewed as weakly immunogenic, GBM responds poorly to current immunotherapies. To understand this problem more clearly we used a combination of natural killer (NK) cell functional assays together with gene and protein expression profiling to define the NK cell response to GBM and explore immunosuppression in the GBM microenvironment. In addition, we used transcriptome data from patient cohorts to classify GBM according to immunological profiles. We show that glioma stem‐like cells, a source of post‐treatment tumour recurrence, express multiple immunomodulatory cell surface molecules and are targeted in preference to normal neural progenitor cells by natural killer (NK) cells ex vivo. In contrast, GBM‐infiltrating NK cells express reduced levels of activation receptors within the tumour microenvironment, with hallmarks of transforming growth factor (TGF)‐β‐mediated inhibition. This NK cell inhibition is accompanied by expression of multiple immune checkpoint molecules on T cells. Single‐cell transcriptomics demonstrated that both tumour and haematopoietic‐derived cells in GBM express multiple, diverse mediators of immune evasion. Despite this, immunome analysis across a patient cohort identifies a spectrum of immunological activity in GBM, with active immunity marked by co‐expression of immune effector molecules and feedback inhibitory mechanisms. Our data show that GBM is recognized by the immune system but that anti‐tumour immunity is restrained by multiple immunosuppressive pathways, some of which operate in the healthy brain. The presence of immune activity in a subset of patients suggests that these patients will more probably benefit from combination immunotherapies directed against multiple immunosuppressive pathways.

A ketogenic diet attenuates proliferation and stemness of glioma stem‑like cells by altering metabolism resulting in increased ROS production.

Abnormal metabolism serves a critical role in the development and progression of different types of malignancies including glioblastoma (GBM), and may therefore serve as a promising target for treatment of cancer. Preclinical studies have indicated that a ketogenic diet (KD) may exhibit beneficial effects in patients with GBM; however, the underlying mechanisms remain incompletely understood. The aim of the present study was to evaluate the effects of a KD on glioma stem‑like cells (GSCs), by culturing patient‑derived primary GSCs as well as a GSC cell line in glucose‑restricted, β‑hydroxybutyrate‑containing medium (BHB‑Glow) which was used to mimic clinical KD treatment. GSCs cultured in BHB‑Glow medium exhibited reduced proliferation and increased apoptosis compared with cells grown in the control medium. Furthermore, decreased expression of stem cell markers, diminished self‑renewal invitro, and reduced tumorigenic capacity invivo, providing evidence that the stemness of GSCs was compromised. Mechanistically, culturing in BHB‑Glow medium reduced glucose uptake and inhibited glycolysis in GSCs. Furthermore, culturing in the BHB‑Glow medium resulted in morphological and functional disturbances to the mitochondria of GSCs. These metabolic changes may have reduced ATP production, promoted lactic acid accumulation, and thus, increased the production of reactive oxygen species (ROS) in GSCs. The expression levels and activation of mammalian target of rapamycin, hypoxia‑inducible factor 1 and B‑cell lymphoma 2 were decreased, consistent with the reduced proliferation of GSCs in BHB‑Glow medium. ROS scavenging reversed the inhibitory effects of a KD on GSCs. Taken together, the results demonstrate that treatment with KD inhibited proliferation of GSCs, increased apoptosis and attenuated the stemness in GSCs by increasing ROS production.

The Phenotypes of Proliferating Glioblastoma Cells Reside on a Single Axis of Variation.

Although tumor-propagating cells can be derived from glioblastomas (GBM) of the proneural and mesenchymal subtypes, a glioma stem-like cell (GSC) of the classic subtype has not been identified. It is unclear whether mesenchymal GSCs (mGSC) and/or proneural GSCs (pGSC) alone are sufficient to generate the heterogeneity observed in GBM. We performed single-cell/single-nucleus RNA sequencing of 28 gliomas, and single-cell ATAC sequencing for 8 cases. We found that GBM GSCs reside on a single axis of variation, ranging from proneural to mesenchymal. In silico lineage tracing using both transcriptomics and genetics supports mGSCs as the progenitors of pGSCs. Dual inhibition of pGSC-enriched and mGSC-enriched growth and survival pathways provides a more complete treatment than combinations targeting one GSC phenotype alone. This study sheds light on a long-standing debate regarding lineage relationships among GSCs and presents a paradigm by which personalized combination therapies can be derived from single-cell RNA signatures, to overcome intratumor heterogeneity. SIGNIFICANCE: Tumor-propagating cells can be derived from mesenchymal and proneural glioblastomas. However, a stem cell of the classic subtype has yet to be demonstrated. We show that classic-subtype gliomas are comprised of proneural and mesenchymal cells. This study sheds light on a long-standing debate regarding lineage relationships between glioma cell types.See related commentary by Fine, p. 1650.This article is highlighted in the In This Issue feature, p. 1631.

Abstract A054: Extracellular vesicles modulate glioblastoma intratumor heterogeneity and treatment resistance

Abstract Glioblastoma (GBM) is the most common primary malignant brain tumor and despite optimal treatment, long-term survival remains uncommon. GBM can be roughly divided into three different molecular subtypes, each varying in aggressiveness and treatment resistance. Interestingly, recent evidence shows plasticity between these subtypes in which the proneural (PN) glioma stem-like cells undergo transition into the more aggressive mesenchymal (MES) subtype leading to therapeutic resistance. Extracellular vesicles (EVs) are membranous structures secreted by nearly every cell and have been shown to play a key role in GBM progression by acting as multifunctional signaling complexes. Here we show that EVs derived from MES cells educate PN cells to increase stemness, cell proliferation, tumor aggressiveness, and therapeutic resistance by modulating mesenchymal transition through NF-κB activation. Our findings could potentially help to explore new treatment strategies for GBM and indicate that EVs may also play a role in the mesenchymal transition of different tumor types. Citation Format: Markus W Schweiger, Elie Tabet, Mao Li, Tian Tian, Max Zinter, Renata Fleming, Litia A Carvalho, Michael J Saad, Bakhos A Tannous. Extracellular vesicles modulate glioblastoma intratumor heterogeneity and treatment resistance [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A054. doi:10.1158/1535-7163.TARG-19-A054

IDH1-R132H mutation radiosensitizes U87MG glioma cells via epigenetic downregulation of TIGAR.

Isocitrate dehydrogenase 1 (IDH1) is the most frequently mutated gene in World Health Organization grade II–III and secondary glioma. The majority of IDH1 mutation cases involve the substitution from arginine to histidine at codon 132 (IDH1-R132H). Although the oncogenic role of IDH1-R132H has been confirmed, patients with IDH1-R132H brain tumors exhibit a better response to radiotherapy compared with those with wild-type (WT) IDH1. In the present study, the potential mechanism of radiosensitization mediated by IDH1-R132H was investigated by overexpressing IDH1-R132H in U87MG glioma cells. The results demonstrated decreased clonogenic capacity of IDH1-R132H-expressing cells, as well as delayed repair of DNA double-strand breaks compared with IDH1-WT. Data from The Cancer Genome Atlas were analyzed, which demonstrated that the expression of TP53-induced glycolysis and apoptosis regulator (TIGAR) was lower in patients with glioma harboring IDH1 mutations compared with that in patients with IDH1-WT. TIGAR-knockdown increases the radiosensitivity of glioma cells; in U87MG cells, IDH1-R132H suppressed TIGAR expression. Chromatin immunoprecipitation assays revealed increased levels of repressive H3K9me3 markers at the TIGAR promoter in IDH1-R132H compared with IDH1-WT. These data indicated that IDH1-R132H may overcome radioresistance in glioma cells through epigenetic suppression of TIGAR expression. However, these favorable effects were not observed in U87MG glioma stem-like cells. The results of the present study provide an improved understanding of the functionality of IDH1 mutations in glioma cells, which may improve the therapeutic efficacy of radiotherapy.

CBMT-24. CHARACTERIZATION OF PRIMARY CILIUM IN RECURRENT GLIOBLASTOMA: IMPLICATIONS FOR NEW THERAPEUTIC TARGETS

Abstract INTRODUCTION Primary cilium is a highly conserved, dynamic cellular organelle which plays several roles in embryonic development, intracellular signaling, and cell cycle. Structural alterations of primary cilium have been described in human gliomas including glioblastoma (GBM), however, its actual role in pathogenesis and treatment resistance of these tumors is largely unknown. METHODS We investigated cilium morphology and expression of cilium-related genes in human glioma of various WHO grade and in couples of patient-derived glioma stem-like cells (GSCs) that were established from the very same GBM at first diagnosis and at recurrence. Immunohistochemistry with anti-Arl13b antibody was used to assess cilium morphology. The expression levels of genes involved in ciliary disassembly complex (CDC) were analyzed by quantitative real-time PCR, using neural progenitor cells (NPCs) as control. Lastly, we assessed 3 GSC cultures that were treated with a drug inhibiting cilia disassembly (CCB-Cil). RESULTS Anaplastic oligodendroglioma and proneural GBM showed the highest percentage of ciliated cells. In GBM, we found the highest percentage of fragmented cilia. GSCs derived from newly diagnosed GBMs displayed lower percentages of ciliated cells than those derived from recurrent GBMs (20% vs 70%). Morphological analysis indicated that GSCs from recurrent GBM show cilia with extremely various morphology compared with GSCs from newly diagnosed GBM and NPCs. Gene analysis showed reduced expression of CDC-related genes in GSCs from newly diagnosed GBM with respect to those from recurrent GBMs. CCB-Cil treatment determined a global reduction of CDC-related genes, increased expression of differentiation markers (GFAP), and reduction of stemness markers (SOX2). CONCLUSIONS The increased percentage of ciliated cells in GSCs from recurrent GBM may be related to a compensatory response of CDC and to an accelerated ciliary turnover. Blocking cilia disassembly reduces stemness features and induces differentiation in GSCs, suggesting that this approach could represent a promising strategy for targeting GBM.

GENE-14. BMP4 INDUCES TO ASYMMETRIC CELL DIVISION IN HUMAN GLIOMA STEM-LIKE CELLS

Abstract Glioblastoma (GBM) is one of the human malignant tumors with a high recurrence rate and the poorest prognosis. Therefore, a new treatment strategy is required. It has also come to be thought that cancer is a heterogeneous, which composes of cancer stem cells (CSCs), which have self-renewal ability, multipotency and treatment resistance. Although various strategies targeting glioma stem-like cells (GSCs) have been studied, a median survival remains to be less than 2 years. In this study, we focused on strategies targeting GSCs through induction of differentiation using BMP4. We examined the expression of CD133, a cancer stem cell marker, under BMP4-treatment in GSCs using flowcytometry analysis, western blotting and qPCR. We also examined immunofluorescent staining of GSCs to study cell division. The treatment of BMP4 caused downregulation of CD133 expression in GSCs. In addition, BMP4-treatment induced to asymmetric cell division in GSCs. Tumor sphere assay showed that BMP4 suppresses self-renewal ability. These findings may provide a new perspective how BMP4 reduces the tumorigenicity of GSCs.

IMMU-35. COMPLEMENT SYSTEM AND GLIOBLASTOMA: AN INTRICATE RELATIONSHIP

Abstract The complement system is a vital part of the innate immune system which plays a critical role in immune surveillance and inflammatory processes. Malignant and host cells express various complement inhibitory proteins on their surface to protect against complement mediated cytotoxicity. Imbalanced complement activation triggers inflammation and alters the tumor microenvironment. Complement activation has been shown to induce proliferation and migration of breast, ovarian and lung cancer cells. At this time, the expression and functional role of the complement cascade in glioblastoma (GBM) remain elusive. Here, we investigated the role of complement proteins and their receptor expression in human primary glioma stem-like cells (GSCs) and human GBM tissues. Western blot data demonstrated a high level of expression of central complement components and their receptors in GSCs. RT-PCR data further confirmed the high expression of complement genes which was similar or higher to normal human astrocytes (HA), a cell with high baseline expression levels of complement genes. Flow cytometry analysis revealed that almost 95% of GSCs expressed the anaphylatoxin complement receptors C3aR and C5aR on their cell surfaces which was consistent with our immunohistochemistry analysis of freshly resected GBM tissues. Furthermore, anaphylatoxin C5a exposure increased the proliferation of a subgroup of GSCs while reduced in another subset. Interestingly, C5a exposure was found to increase the expression of various pro-inflammatory markers in GSCs with reduced proliferation. Fluorescence-activated cell sorting (FACS) analysis of freshly isolated human GBM tissue revealed predominant expression of C5aR on cancer cells (CD11b-/CD45- cells) rather than on immune cells. RT-PCR analysis also demonstrated high expression levels of complement genes with concomitant decrease in complement inhibitory genes in human GBM tissue. Evaluation of the differential role of the complement system in GSCs along with their role in in vivo glioma models is ongoing.

ANGPTL4 Induces TMZ Resistance of Glioblastoma by Promoting Cancer Stemness Enrichment via the EGFR/AKT/4E-BP1 Cascade.

Glioblastoma (GBM) is the most aggressive type of brain tumor, with strong invasiveness and a high tolerance to chemotherapy. Despite the current standard treatment combining temozolomide (TMZ) and radiotherapy, glioblastoma can be incurable due to drug resistance. The existence of glioma stem-like cells (GSCs) is considered the major reason for drug resistance. However, the mechanism of GSC enrichment remains unclear. Herein, we found that the expression and secretion of angiopoietin-like 4 protein (ANGPTL4) were clearly increased in GSCs. The overexpression of ANGPTL4 induced GSC enrichment that was characterized by polycomb complex protein BMI-1 and SRY (sex determining region Y)-box 2 (SOX2) expression, resulting in TMZ resistance in GBM. Furthermore, epidermal growth factor receptor (EGFR) phosphorylation induced 4E-BP1 phosphorylation that was required for ANGPTL4-induced GSC enrichment. In particular, ANGPTL4 induced 4E-BP1 phosphorylation by activating phosphoinositide 3-kinase (PI3K)/AKT and extracellular signal–regulated kinase (ERK) cascades for inducing stemness. To elucidate the mechanism contributing to ANGPTL4 upregulation in GSCs, chromatin immunoprecipitation coupled with sequencing (ChIP-Seq) revealed that specificity protein 4 (Sp4) was associated with the promoter region, −979 to −606, and the luciferase reporter assay revealed that Sp4 positively regulated activity of the ANGPTL4 promoter. Moreover, both ANGPTL4 and Sp4 were highly expressed in GBM and resulted in a poor prognosis. Taken together, Sp4-mediated ANGPTL4 upregulation induces GSC enrichment through the EGFR/AKT/4E-BP1 cascade.

STEM-25. HDAC1 IS ESSENTIAL FOR GLIOMA STEM CELL SURVIVAL

Abstract OLIG2 is a central nervous system-specific transcription factor that is expressed in almost all diffuse gliomas. It is also one of the key core transcription factors that can reprogram differentiated glioma cells to highly tumorigenic glioma stem-like cells (GSCs). We have previously shown that expression of OLIG2 is critical for glioma growth both in a genetically relevant mouse model as well as in patient-derived xenograft models. Our work suggests that a small molecule inhibitor of OLIG2 could serve as a highly targeted therapy for high-grade glioma; however, transcription factors are generally very difficult to target because their interactions with DNA and co-regulatory proteins involve large and complex surface area contacts. Our laboratory has shown that OLIG2 functions are regulated through interactions with distinct co-regulator proteins in normal neural stem cells. However, there are currently no reports on interactors that promote the proto-oncogenic functions of OLIG2 in malignant glioma. In this study, we employed two independent proteomics screens identify tumor-specific, druggable OLIG2 co-regulators as possible surrogate targets to suppress OLIG2 function in glioma. These screens led to the identification of a novel OLIG2 partner protein: Histone Deacetylase 1 (HDAC1). We confirmed that this interaction occurs in both murine and human glioma models. Although HDACs are ubiquitously expressed and are known to be functionally redundant, we show that ablation of HDAC1 alone significantly decreases the stemness and proliferation capacity of patient-derived GSCs in a p53-dependent manner, while having a minimal impact on normal human neural stem cells and astrocytes. Furthermore, we demonstrate that knockdown of HDAC1, in combination with ionizing radiation treatment, significantly alters the growth pattern of intracranial tumors in vivo. We demonstrate that HDAC1 function is critical for GSC growth and provide a strong rationale for targeting the OLIG2-HDAC1 interaction in malignant glioma.

TMIC-63. ENDOCAN BRIDGING THE GAP: TUMOR ENDOTHELIAL CELL SECRETOME AND THE VASCULATURE-PROXIMAL TUMOR EDGE

Abstract BACKGROUND Glioblastoma (GBM) is a highly invasive, angiogenic and therapeutically resistant primary brain tumor. It is impossible to completely resect the tumor, which subsequently leaves behind tumor “edge” that can act as seeds for recurrence. We recently reported the leading “edge” and tumor core have the presence of two distinct types of glioma stem-like cells (GSCs) that resemble proneural (PN) and mesenchymal (MES) subtypes, respectively. GSCs are enriched in perivascular niches, and vascular endothelial (VE) cells are known to provide paracrine factors, radioresistance properties to GSCs. Therapeutically, targeting GBM using an anti-angiogenic anti-VEGF inhibitor, Bevacizumab, did not aid in overall survival and instead resulted in aggressive MES phenotype. Hence, it is imperative to study this nexus in greater detail to develop novel targeted therapies for VE and GBM cells. The purpose of this study was to understand the molecular mechanisms emanating from VE cells that cause the invading “edge” to recur and determine if targeting these molecular cascades can provide therapeutic benefit. RESULTS Using unbiased approach to identify paracrine factors from GBM patient derived VE cells, we identified endocan, a vascular endothelial cell secreted molecule, as a pro-tumorigenic factor essential for neovascularization, radioresistance, and stemness of GBM edge tumor cells. Utilizing patient-derived and mouse glioblastoma sphere models and the corresponding intracranial tumor models, we demonstrate that endocan maintains the “edge”-located glioma stem cell signature and protects glioblastoma cells from radiation-induced differentiation and apoptosis. Mechanistically, we show that endocan is a novel ligand that interacts with PDGFRa and induce its phosphorylation, leading to phosphorylation of the downstream targets PI3K and MAPK in GBM. CONCLUSIONS Collectively, these findings demonstrate a novel signaling axis of endocan-PDGFRa in GBM that is activated by tumor endothelial cell and plays an essential role in glioma stem cells maintenance and radioresistance in tumor “edge”.

DDIS-32. MEK INHIBITORS INDUCES NEURONAL DIFFERENTIATION IN EGFR AMPLIFIED GLIOMA STEM LIKE CELLS

Abstract The median survival for patients with glioblastoma (GBM) is 12–15 months highlighting the need for better therapeutic strategies for this deadly disease. Genomic and epigenomic sequencing analysis at the single cell level have identified multiple genomic aberrations as potential targets for therapeutic intervention in GBM. EGFR and PDGFR amplification are evident in nearly 40% and 12% of human GBM, respectively. Although the first and second-generation EGFR tyrosine kinase small molecule inhibitors failed to show long term therapeutic benefit in GBM patients, multiple factors such as incorrect patient selection, acquired resistance, and drug-target heterogeneity may all lead to clinical failure of targeted therapies. Although the multilevel genomic characterization of gliomas are increasing, the clinical translation of these findings is beginning to unravel. In this study, we attempted to correlate the genomic variations using an unbiased high throughput drug screen using primary glioma stem-like cell (GSCs) as our model system. An unbiased high-throughput screen utilizing our GSC models identified that glioblastoma cells harboring focal EGFR amplification are sensitive to mitogen-activated protein kinase (MEK) inhibitors. MEK inhibition induced apoptosis in EGFR amplified cells at low concentration. RNA sequence analysis of cells treated with MEK inhibitors revealed upregulation of genes related to neuronal differentiation and down regulation of MEK target genes in MEK sensitive glioma stem cells. Additionally, RNA sequencing of GSCs with acquired MEK inhibitor resistance demonstrated an upregulation of oncogenic transcription factor ETS Variant Gene 1 (ETV1) as a mediator of resistance. Overall our data suggest that the MEK inhibition in combination with ETV inhibitors could be a potential therapeutic target for a subset of GBM patients.

EXTH-70. ENHANCEMENT OF ANTITUMOR ACTIVITY BY USING 5-ALA–MEDIATED SONODYNAMIC THERAPY TO INDUCE APOPTOSIS AND NECROSIS IN A MOUSE GLIOMA MODEL

Abstract OBJECTIVE High invasiveness of malignant gliomas frequently causes local tumor recurrence. To control such recurrence, novel therapies targeted toward infiltrating glioma cells are required. Here, we examined cytotoxic effects of sonodynamic therapy (SDT) combined with a sonosensitizer, 5-aminolevulinic acid (5-ALA), on malignant gliomas both in vitro and in vivo. METHODS In vitro cytotoxicity of 5-ALA-SDT was evaluated in U87 and U251 glioma cells and in U251Oct-3/4 glioma stemlike cells. Treatment-related apoptosis was analyzed using flow cytometry. Intracellular reactive oxygen species (ROS) were measured and the role of ROS in treatment-related cytotoxicity was examined. Effects of 5-ALA-SDT with high-intensity focused ultrasound (HIFU) on tumor growth, survival of glioma-transplanted mice, and histological features of the mouse brains were investigated. RESULTS The 5-ALA-SDT inhibited cell growth and changed cell morphology. Flow cytometric analysis indicated that 5-ALA-SDT induced apoptotic cell death. The 5-ALA-SDT generated higher ROS than in the control group, and inhibition of ROS generation completely eliminated the cytotoxic effects of 5-ALA-SDT. In the in vivo study, 5-ALA-SDT with HIFU greatly prolonged survival of the tumor-bearing mice compared with that of the control group (p < 0.05). Histologically, 5-ALA-SDT produced mainly necrosis of the tumor tissue in the focus area and induced apoptosis of the tumor cells in the perifocus area around the target of the HIFU-irradiated field. Normal brain tissues around the ultrasonic irradiation field of HIFU remained intact. CONCLUSIONS The 5-ALA-SDT was cytotoxic toward malignant gliomas. Generation of ROS by the SDT was thought to promote apoptosis of glioma cells. The 5-ALA-SDT with HIFU induced tumor necrosis in the focus area and apoptosis in the perifocus area of the HIFU-irradiated field. These results suggest that 5-ALA-SDT with HIFU may present a less invasive and tumor-specific therapy, not only for a tumor mass but also for infiltrating tumor cells in malignant gliomas.

CBMT-22. REVERSING THE WARBURG EFFECT BY TARGETING NICOTINAMIDE PHOSPHORIBOSYLTRANSFERASE (NAMPT), THE RATE-LIMITING ENZYME OF NAD+ SALVAGE PATHWAY INCREASES, CHEMOSENSITIVITY TO TEMOZOLOMIDE IN GLIOMA CELLS

Abstract BACKGROUND Glioma cells metabolize glucose primarily through incomplete, non-oxidative glycolysis even in the presence of oxygen (the Warburg effect) which provides a constant supply of substrate for proliferation and generates an acidic environment that favors invasion and immunosuppression. NAMPT, the rate-limiting enzyme of the NAD+ salvage pathway, regulates key metabolic processes preferentially used in glioma energy metabolism. Inhibition of NAMPT could have global impact on glioma cell metabolism and could be a promising strategy for glioma therapy. This study examined the effect of NAMPT inhibition using KPT-9274 and FK866 (NAMPTi) on glycolysis and glioma cell metabolism. METHODS Metabolic changes related to NAMPT inhibition were examined in glioma cell lines and patient derived glioma stem-like cells (GSC). Mass spectrometry was used for metabolic profiles and the generated data was annotated by comparison against an extensive metabolite library to identify relevant metabolites. The Agilent Seahorse assay was utilized to determine treatment-related changes in glycolysis. RESULTS NAMPT inhibition caused NAD, NADH, NADP, NADPH, and ATP depletion in glioma cells. Global metabolomics results showed an accumulation of GMP, IMP, AMP and myo-inositol indicating reprogramming of glioma cell metabolism for de novo purine biosynthesis. Additionally, an accumulation of glucose- and fructose-1,6 bisphosphate in NAMPTi treated cells indicated dysfunctional glycolysis. This was confirmed by the Agilent glycolysis stress test by which NAMPTi-treated glioma cells showed a reduction in glycolysis, glycolytic capacity and reserves. NAMPTi treated cells showed downregulation of LDHA due to NAD depletion. Further, downregulation of LDHA enhanced the chemosensitivity of glioma cells to temozolomide. CONCLUSIONS NAMPTi cause profound disruption of glycolysis, reduced glycolytic capacity and glycolytic reserve in gliomas and GSCs irrespective of O[6]-methylguanine-DNA methyltransferase promoter methylation or IDH1 status. Targeting NAMPT to disrupt the Warburg effect is a novel strategy for developing effective therapies against glioma to overcome heterogeneity.

GENE-22. GENOME-WIDE METHYLATION PROFILING OF GLIOBLASTOMA EXTRACELLULAR VESICLE DNA ALLOWS TUMOR CLASSIFICATION

Abstract BACKGROUND Genome-wide methylation profiling has recently been developed into a tool that allows subtype tumor classification in central nervous system (CNS) tumors. Extracellular vesicles (EVs) are released by CNS tumor cells and contain high molecular weight tumor DNA, rendering EVs a potential biomarker source to identify tumor subgroups, stratify patients and monitor therapy by liquid biopsy. We investigated whether the DNA in glioma-derived EVs reflects genome-wide tumor methylation profiles and allows tumor subtype classification. METHODS DNA was isolated from EVs secreted by cultured glioma stem-like cells (GSC) as well as from the cells of origin and from the original tumor samples (n=3 patients). EVs were classified by nanoparticle analysis (NTA), immunoblotting, imaging flow cytometry (IFCM), multiplex EV assays and electron microscopy. Genome-wide DNA methylation profiling was performed using an 850k Illumina EPIC array and results were classified according to the DKFZ brain tumor classifier. RESULTS The size range of GSC-derived EVs was 120–150 nm, as measured by NTA. The majority of secreted EVs exhibited high expression of common EV markers (i.e. CD9, CD63 and CD81), as characterized by IFCM and multiplex EV assays. Genome-wide methylation profiling of GSC-derived EVs correctly identified the methylation class of the original tumor, including information on the IDH mutation status and subclass classification (RTK1, RTK2). In addition, copy number alterations and the MGMT metyhlation status matched the pattern of the parental GSCs and original tumor samples. CONCLUSION EV DNA faithfully reflects the tumor methylation class as well as the MGMT methylation status and copy number variations present in the parental cells and the original tumor. Methylation profiling of circulating tumor EV DNA could become a useful tool to detect and classify CNS tumors.

Retinoid receptor turnover mediated by sumoylation, ubiquitination and the valosin-containing protein is disrupted in glioblastoma

Resistance to therapeutic use of retinoids in glioma has been observed for over 20 years; however, the exact mechanism of resistance remains unknown. To understand retinoic acid resistance in glioma, we studied the turnover mechanism of retinoid receptor proteins in neural stem cells and glioma stem-like cells. Here, we show that in normal neural stem cells, proteasomal degradation of retinoid receptors involves sumoylation, ubiquitination and recognition by the valosin-containing protein (VCP/p97/Cdc48). We find that Sumo1 modification has a dual role to stabilize the retinoid receptor from unwanted degradation and signal additional modification via ubiquitination. Subsequently, the modified receptor binds to the VCP chaperone and both proteins are degraded by the proteasome. Additionally, we reveal that all trans retinoic acid (ATRA) induces VCP expression, creating a positive feedback loop that enhances degradation. In contrast, the pathway is impaired in the glioma stem-like cells resulting in the accumulation of sumoylated and high molecular weight forms of retinoid receptors that lack transcriptional activity and fail to be recognized by the proteasome. Moreover, modified receptor accumulation occurs before ATRA treatment; therefore, the transcritptional defect in glioma is due to a block in the proteasomal degradation pathway that occurs after the sumo modification step.

Iguratimod encapsulated PLGA-NPs improves therapeutic outcome in glioma, glioma stem-like cells and temozolomide resistant glioma cells

Glioma is the most common neoplasm of the central nervous system, with the highest mortality rate. The present study was designed to examine the therapeutic effect of Iguratimod (IGU) encapsulated-poly (lactic-co-glycolic acid) PLGA nanoparticles (IGU-PLGA-NPs), which showed inhibition of glioma cells proliferation both in vitro and in vivo. IGU encapsulated in PLGA nanoparticles with an average size of 100-200 nm was prepared using modified double-emulsion (W1/O/W2) method. Cell Counting Kit-8 (CCK-8) analysis of Glioma cancer cells and glioma stem-like cells (GSCs) demonstrated significant inhibition of their growth treated with IGU-PLGA-NPs. IGU-PLGA-NPs inhibit migration in glioma cells as well as tumor sphere formation in GSCs. Treatment with IGU-PLGA-NPs showed a significant decrease in tumor growth through the apoptotic pathway in mice model without any visible organ toxicity and it can successfully cross the blood brain barrier (BBB). Most Importantly, IGU-PLGA-NPs significantly depleted growth of U251 Temozolomide-resistant (U251TMZ-R) cells.

SRSF3-Regulated RNA Alternative Splicing Promotes Glioblastoma Tumorigenicity by Affecting Multiple Cellular Processes.

Misregulated alternative RNA splicing (AS) contributes to the tumorigenesis and progression of human cancers, including glioblastoma (GBM). Here, we showed that a major splicing factor, serine and arginine rich splicing factor 3 (SRSF3), was frequently upregulated in clinical glioma specimens and that elevated SRSF3 was associated with tumor progression and a poor prognosis for patients with glioma. In patient-derived glioma stem-like cells (GSC), SRSF3 expression promoted cell proliferation, self-renewal, and tumorigenesis. Transcriptomic profiling identified more than 1,000 SRSF3-affected AS events, with a preference for exon skipping in genes involved with cell mitosis. Motif analysis identified the sequence of CA(G/C/A)CC(C/A) as a potential exonic splicing enhancer for these SRSF3-regulated exons. To evaluate the biological impact of SRSF3-affected AS events, four candidates were selected whose AS correlated with SRSF3 expression in glioma tissues, and their splicing pattern was modified using a CRISPR/Cas9 approach. Two functionally validated AS candidates were further investigated for the mechanisms underlying their isoform-specific functions. Specifically, following knockout of SRSF3, transcription factor ETS variant 1 (ETV1) gene showed exon skipping at exon 7, while nudE neurodevelopment protein 1 (NDE1) gene showed replacement of terminal exon 9 with a mutually exclusive exon 9'. SRSF3-regulated AS of these two genes markedly increased their oncogenic activity in GSCs. Taken together, our data demonstrate that SRSF3 is a key regulator of AS in GBM and that understanding mechanisms of misregulated AS could provide critical insights for developing effective therapeutic strategies against GBMs. SIGNIFICANCE: SRSF3 is a significant regulator of glioma-associated alternative splicing, implicating SRSF3 as an oncogenic factor that contributes to the tumor biology of GBM.

TGF-β-activated lncRNA LINC00115 is a critical regulator of glioma stem-like cell tumorigenicity.

Long non-coding RNAs (lncRNAs) are critical regulators in cancer. However, the involvement of lncRNAs in TGF-β-regulated tumorigenicity is still unclear. Here, we identify TGF-β-activated lncRNA LINC00115 as a critical regulator of glioma stem-like cell (GSC) self-renewal and tumorigenicity. LINC00115 is upregulated by TGF-β, acts as a miRNA sponge, and upregulates ZEB1 by competitively binding of miR-200s, thereby enhancing ZEB1 signaling and GSC self-renewal. LINC00115 also promotes ZNF596 transcription by preventing binding of miR-200s to the 5'-UTR of ZNF596, resulting in augmented ZNF596/EZH2/STAT3 signaling and GBM tumor growth. Inhibition of EZH2 by genetic approaches or a small molecular inhibitor markedly suppresses LINC00115-driven GSC self-renewal and tumorigenicity. Moreover, LINC00115 is highly expressed in GBM, and LINC00115 expression or correlated co-expression with ZEB1 or ZNF596 is prognostic for clinical GBM survival. Our work defines a critical role of LINC00115 in GSC self-renewal and tumorigenicity, and suggests LINC00115 as a potential target for GBM treatment.

Epigenetic downregulation of STAT6 increases HIF-1\u03b1 expression via mTOR/S6K/S6, leading to enhanced hypoxic viability of glioma cells

Multifunctional signal transducer and activator of transcription (STAT) proteins play important roles in cancer. Here, we have shown that STAT6 is epigenetically silenced in some cases of malignant glioblastoma, which facilitates cancer cell survival in a hypoxic microenvironment. This downregulation results from hypermethylation of CpG islands within the STAT6 promoter by DNA methyltransferases. STAT6 interacts with Rheb under hypoxia and inhibits mTOR/S6K/S6 signaling, in turn, inducing increased HIF-1α translation. STAT6 silencing and consequent tumor-promoting effects are additionally observed in glioma stem-like cells (GSC). Despite recent advances in cancer treatment, survival rates have shown little improvement. This is particularly true in the case of glioma, where multimodal treatment and precision medicine is needed. Our study supports the application of epigenetic restoration of STAT6 with the aid of DNA methyltransferase inhibitors, such as 5-aza-2-deoxycytidine, for treatment of STAT6-silenced gliomas.