Glioblastoma Stem-like Cell Lines Research Articles

DDDR-08. DISCOVERY OF ER STRESS-INDUCING COMPOUNDS FOR GBM: A PROMISING APPROACH TOWARDS THERAPEUTIC DEVELOPMENT

Abstract The unfolded protein response (UPR) is a pro-survival mechanism triggered by cellular stresses encountered by tumor cells, such as hypoxia, acidosis, reactive oxygen species and even radiation and chemotherapy. In response to increased unfolded or mis-folded proteins in the endoplasmic reticulum (ER), the UPR initiates specific cell signaling pathways aiming to alleviate ER stress and restore ER proteostasis which include increasing the expression of chaperones to aid in protein folding, enhancing protein degradation and reducing global protein synthesis. However, if homeostasis cannot be restored, the UPR switches from survival to cell death. The UPR is a relatively novel therapeutic target for glioblastoma (GBM), and specifically GBM stem-like cells (GSCs) which maintain tumor heterogeneity, drive GBM tumor growth and therapy resistance. Here we tested a series of curcumin derivatives against three GSC lines and identified a trimethoxy bis-chalcone which promotes robust GSC death with an average IC50 of just below 300 nM, a 100-fold increase in activity compared to curcumin. Furthermore, this bis-chalcone was non-toxic to normal human mesenchymal stem cells. Western blot analysis indicated that the bis-chalcone induced robust activation of UPR pro-death protein C/EBP Homologous Protein (CHOP) while simultaneously decreasing the expression of pro-survival chaperone GRP78/Bip. Lastly our bis-chalcone induced caspase 7 expression and PARP cleavage indicating apoptosis. Most ER stress inducing agents promote both the survival, upregulation of Grp78, and cell death arms of the UPR. Here we identified a compound that only promotes UPR cell death signaling and is highly toxic to radiation and temozolomide resistant GSCs while demonstrating no toxicity to non-tumor cells. This is a promising lead compound for the development of UPR targeted GSC/GBM therapeutics.

MODL-49. NEW MURINE GLIOBLASTOMA STEM-LIKE CELL MODELS

Abstract BACKGROUND There is a dearth of mouse glioblastoma (GBM) preclinical models in immunocompetent mice for the development of immunotherapeutic agents. In this study, we characterized five murine GBM stem-like cell (GSC) models derived from lentivirus induced tumors in transgenic mice that are driven by the activation of the Nf1-Ras MAP kinase signalling pathway due to HRasV12 or NF1 shRNA, and inactivation of Tp53. METHODS Murine GSC lines, 005, NF53, RIG, C1, and C3, were cultured as spheres in serum-free stem cell media. Whole exome sequencing (WES) was conducted to quantify single nucleotide polymorphisms (SNPs) using the C57BL/6J genome as reference. Stem cell properties were characterized by in vitro assays and tumorigenicity was examined by intracerebral implantation into C57BL/6 mice. Brain tissues were processed for histological analysis, such as immunohistochemistry, or for FACS to examine markers of stemness and tumor-infiltrating immune cells. RESULTS WES identified a large variation in coding sequence SNPs, likely due to the mixed backgrounds of the parental mice, with 005 having the highest number of SNPs, and C1 and C3 the lowest. All GSC lines were capable of clonogenic sphere formation, indicative of self-renewal, and showed varying levels of cell surface CD133. In vivo, all the GSCs efficiently established tumors that were histologically consistent with malignant glioma with strong CD44 expression. Median survival varied, ranging from 27 days (005) to 82 days (C1), upon 20,000-cell implantation. CD68+ macrophages were consistently dominant in the tumor microenvironment, while C1 and C3 exhibited fewer CD4+ and CD8+ T cells. CONCLUSIONS Despite the similar molecular strategy used to generate these GBMs, we observed a range of tumor phenotypes and immune microenvironmental profiles. These models can serve as valuable tools for studying GBM biology and exploring immunotherapeutic strategies. Keywords: Glioblastoma, glioblastoma stem-like cell (GSC), genetically engineered mouse models, Tumor microenvironment.

Genome-wide profiling of patient-derived glioblastoma stem-like cells reveals recurrent genetic and transcriptomic signatures associated with brain tumors

PurposePatient-derived cancer cell lines can be very useful to investigate genetic as well as epigenetic mechanisms of transformation and to test new drugs. In this multi-centric study, we performed genomic and transcriptomic characterization of a large set of patient-derived glioblastoma (GBM) stem-like cells (GSCs).Methods94 (80 I surgery/14 II surgery) and 53 (42 I surgery/11 II surgery) GSCs lines underwent whole exome and trascriptome analysis, respectively.ResultsExome sequencing revealed TP53 as the main mutated gene (41/94 samples, 44%), followed by PTEN (33/94, 35%), RB1 (16/94, 17%) and NF1 (15/94, 16%), among other genes associated to brain tumors. One GSC sample bearing a BRAF p.V600E mutation showed sensitivity in vitro to a BRAF inhibitor. Gene Ontology and Reactome analysis uncovered several biological processes mostly associated to gliogenesis and glial cell differentiation, S − adenosylmethionine metabolic process, mismatch repair and methylation. Comparison of I and II surgery samples disclosed a similar distribution of mutated genes, with an overrepresentation of mutations in mismatch repair, cell cycle, p53 and methylation pathways in I surgery samples, and of mutations in receptor tyrosine kinase and MAPK signaling pathways in II surgery samples. Unsupervised hierarchical clustering of RNA-seq data produced 3 clusters characterized by distinctive sets of up-regulated genes and signaling pathways.ConclusionThe availability of a large set of fully molecularly characterized GCSs represents a valuable public resource to support the advancement of precision oncology for the treatment of GBM.

Abstract 2413: Glioblastoma stem-like cells are resistant to the negative effects of increased aneuploidy on in vitro survival and radiosensitivity

Abstract Glioblastomas (GBM) have long been classified as radioresistant tumors. Glioblastoma stem-like cells (GSCs) are a portion of GBM’s heterogenous cell population and are generally considered to be crucial drivers of tumorigenesis and radioresistance. As for most tumor cells, GSCs display a moderate level of chromosomal instability and aneuploidy. However, whether aneuploidy promotes or inhibits GSC survival and its effects on radiosensitivity remain unclear. In this study we compared the radiation response of GSC lines (NSC11 and NSC20) and the standard glioma cell line U251 after an increase in aneuploidy. This increase was induced by treatment with CFI-402257, a selective inhibitor of the mitotic kinase TTK, which plays a key role in spindle-assembly checkpoint (SAC) regulation. After an initial drug exposure of 24h, the drug was removed and cells were allowed to grow for 5 days. In both GSC lines and U251, as demonstrated by interphase chromosome FISH, this protocol resulted in approximately 150%, 50%, and 125% increases in aneuploidy of chromosomes 2, 7 and 10 respectively. After these increases in aneuploidy, the clonogenicity (in vitro survival) of U251 cells was reduced by 80%, whereas it was reduced by 50% and 30% in NSC11 and NSC20 respectively. Moreover, the increase in aneuploidy significantly enhanced the radiosensitivity of U251 cells, whereas it had no effect on the radiosensitivity of the GSCs. These changes could not be attributed to residual inhibition of TTK or deficiencies in the SAC: at 5 days after CFI inhibition no changes in nocodazole-induced SAC arrest in mitosis were detected. Additionally, after aneuploid induction no changes in the expression of the stem cell marker CD133 were detected in the GSC lines, indicating no change in differentiation status. To begin to investigate the mechanism behind these differences in radiosensitivity, γH2AX foci dispersal, a surrogate marker for DNA double strand break (DSB) repair was evaluated. After the increase in aneuploidy, cells were irradiated (2Gy) and collected 1-24h after for analysis of γH2AX. In U251 cells, the increase in aneuploidy resulted in slower foci dispersal, indicative of a decrease in DSB repair; whereas in GSCs the increased aneuploidy had no effect on foci dispersal as compared to control. Mitotic catastrophe, the primary mechanism of solid tumor cell death after irradiation, was also evaluated. The increase in aneuploidy significantly increased the frequency of radiation-induced mitotic catastrophe in U251 cells, but no changes in either GSC line were detected. Overall, these results suggest that GSCs have an enhanced ability to tolerate the negative consequences of aneuploidy on survival as well as on radiosensitivity. Such aneuploid tolerance may provide a mechanism through which GBMs exploit karyotype diversity to survive under harsh environmental conditions and after treatment. Citation Format: Ian C. Sutton, Charlotte M. Degorre, Komal Rawal, Philip J. Tofilon. Glioblastoma stem-like cells are resistant to the negative effects of increased aneuploidy on in vitro survival and radiosensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2413.

Effectiveness of Flattening-Filter-Free versus Flattened Beams in V79 and Glioblastoma Patient-Derived Stem-like Cells

Literature data on the administration of conventional high-dose beams with (FF) or without flattening filters (FFF) show conflicting results on biological effects at the cellular level. To contribute to this field, we irradiated V79 Chinese hamster lung fibroblasts and two patient-derived glioblastoma stem-like cell lines (GSCs-named #1 and #83) using a clinical 10 MV accelerator with FF (at 4 Gy/min) and FFF (at two dose rates 4 and 24 Gy/min). Cell killing and DNA damage induction, determined using the γ-H2AX assay, and gene expression were studied. No significant differences in the early survival of V79 cells were observed as a function of dose rates and FF or FFF beams, while a trend of reduction in late survival was observed at the highest dose rate with the FFF beam. GSCs showed similar survival levels as a function of dose rates, both delivered in the FFF regimen. The amount of DNA damage measured for both dose rates after 2 h was much higher in line #1 than in line #83, with statistically significant differences between the two dose rates only in line #83. The gene expression analysis of the two GSC lines indicates gene signatures mimicking the prognosis of glioblastoma (GBM) patients derived from a public database. Overall, the results support the current use of FFF and highlight the possibility of identifying patients with candidate gene signatures that could benefit from irradiation with FFF beams at a high dose rate.

MEOX2 Regulates the Growth and Survival of Glioblastoma Stem Cells by Modulating Genes of the Glycolytic Pathway and Response to Hypoxia.

Simple SummaryGlioblastoma is the most common incurable primary brain tumor in adults, typically leading to death within 15 months of diagnosis. Although there is an ongoing debate in the scientific community about the precise cellular origin of this tumor, glioblastoma stem cells (GSCs), which are able to self-renew, yield a full tumor mass, and determine chemo- and radio-resistance, are recognized to have a pivotal role. Our research aims to understand the role of the mesenchyme homeobox 2 (MEOX2) transcription factor in GSCs where it is strongly and specifically expressed. We have found that MEOX2 is indeed important for the survival of these cells. In fact, when we reduce its expression in two different GSC lines, they undergo a massive death accompanied by the inhibition of key genes of the glycolytic metabolism, the main source of energy for these cells. Our results reveal a novel function for MEOX2 in glioblastoma and suggest a mechanism through which GSCs may survive even in unfavorable conditions.The most widely accepted hypothesis for the development of glioblastoma suggests that glioblastoma stem-like cells (GSCs) are crucially involved in tumor initiation and recurrence as well as in the occurrence of chemo- and radio-resistance. Mesenchyme homeobox 2 (MEOX2) is a transcription factor overexpressed in glioblastoma, whose expression is negatively correlated with patient survival. Starting from our observation that MEOX2 expression is strongly enhanced in six GSC lines, we performed shRNA-mediated knock-down experiments in two different GSC lines and found that MEOX2 depletion resulted in the inhibition of cell growth and sphere-forming ability and an increase in apoptotic cell death. By a deep transcriptome analysis, we identified a core group of genes modulated in response to MEOX2 knock-down. Among these genes, the repressed ones are largely enriched in genes involved in the hypoxic response and glycolytic pathway, two strictly related pathways that contribute to the resistance of high-grade gliomas to therapies. An in silico study of the regulatory regions of genes differentially expressed by MEOX2 knock-down revealed that they mainly consisted of GC-rich regions enriched for Sp1 and Klf4 binding motifs, two main regulators of metabolism in glioblastoma. Our results show, for the first time, the involvement of MEOX2 in the regulation of genes of GSC metabolism, which is essential for the survival and growth of these cells.

Screening the Significant Hub Genes by Comparing Tumor Cells, Normoxic and Hypoxic Glioblastoma Stem-like Cell Lines Using Co-Expression Analysis in Glioblastoma.

Glioblastoma multiforme (GBM) is categorized by rapid malignant cellular growth in the central nervous system (CNS) tumors. It is one of the most prevailing primary brain tumors, particularly in human male adults. Even though the combination therapy comprises surgery, chemotherapy, and adjuvant therapies, the survival rate is on average 14.6 months. Glioma stem cells (GSCs) have key roles in tumorigenesis, progression, and counteracting chemotherapy and radiotherapy. In our study, firstly, the gene expression dataset GSE45117 was retrieved and differentially expressed genes (DEGs) were spotted. The co-expression network analysis was employed on DEGs to find the significant modules. The most significant module resulting from co-expression analysis was the turquoise module. The turquoise module related to the tumor cells, hypoxia, normoxic treatments of glioblastoma tumor (GBT), and GSCs were screened. Sixty-one common genes in the turquoise module were selected generated through the co-expression analysis and protein–protein interaction (PPI) network. Moreover, the GO and KEGG pathway enrichment results were studied. Twenty common hub genes were screened by the NetworkAnalyst web instrument constructed on the PPI network through the STRING database. After survival analysis via the Kaplan–Meier (KM) plotter from The Cancer Genome Atlas (TCGA) database, we identified the five most significant hub genes strongly related to the progression of GBM. We further observed these five most significant hub genes also up-regulated in another GBM gene expression dataset. The protein–protein interaction (PPI) network of the turquoise module genes was constructed and a KEGG pathway enrichments study of the turquoise module genes was performed. The VEGF signaling pathway was emphasized because of the strong link with GBM. A gene–disease association network was further constructed to demonstrate the information of the progression of GBM and other related brain neoplasms. All hub genes assessed through this study would be potential markers for the prognosis and diagnosis of GBM.

A Phase I Study of Autologous Dendritic Cell Vaccine Pulsed with Allogeneic Stem-like Cell Line Lysate in Patients with Newly Diagnosed or Recurrent Glioblastoma

Abstract Purpose: Glioblastoma (GBM) is a heterogeneous malignancy with multiple subpopulations of cancer cells present within any tumor. We present the results of a phase I clinical trial using an autologous dendritic cell (DC) vaccine pulsed with lysate derived from a GBM stem-like cell line. Patients and Methods: Patients with newly diagnosed and recurrent GBM were enrolled as separate cohorts. Eligibility criteria included a qualifying surgical resection or minimal tumor size, ≤ 4-mg dexamethasone daily dose, and Karnofsky score ≥70. Vaccine treatment consisted of two phases: an induction phase with vaccine given weekly for 4 weeks, and a maintenance phase with vaccines administered every 8 weeks until depletion of supply or disease progression. Patients with newly diagnosed GBM also received standard-of-care radiation and temozolomide. The primary objective for this open-label, single-institution trial was to assess the safety and tolerability of the autologous DC vaccine. Results: For the 11 patients with newly diagnosed GBM, median progression-free survival (PFS) was 8.75 months, and median overall survival was 20.36 months. For the 25 patients with recurrent GBM, median PFS was 3.23 months, 6-month PFS was 24%, and median survival was 11.97 months. A subset of patients developed a cytotoxic T-cell response as determined by an IFNγ ELISpot assay. Conclusions: In this trial, treatment of newly diagnosed and recurrent GBM with autologous DC vaccine pulsed with lysate derived from an allogeneic stem-like cell line was safe and well tolerated. Clinical outcomes add to the body of evidence suggesting that immunotherapy plays a role in the treatment of GBM.

Elesclomol-induced increase of mitochondrial reactive oxygen species impairs glioblastoma stem-like cell survival and tumor growth

BackgroundGlioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults, characterized by a poor prognosis mainly due to recurrence and therapeutic resistance. It has been widely demonstrated that glioblastoma stem-like cells (GSCs), a subpopulation of tumor cells endowed with stem-like properties is responsible for tumor maintenance and progression. Moreover, it has been demonstrated that GSCs contribute to GBM-associated neovascularization processes, through different mechanisms including the transdifferentiation into GSC-derived endothelial cells (GdECs).MethodsIn order to identify druggable cancer-related pathways in GBM, we assessed the effect of a selection of 349 compounds on both GSCs and GdECs and we selected elesclomol (STA-4783) as the most effective agent in inducing cell death on both GSC and GdEC lines tested.ResultsElesclomol has been already described to be a potent oxidative stress inducer. In depth investigation of the molecular mechanisms underlying GSC and GdEC response to elesclomol, confirmed that this compound induces a strong increase in mitochondrial reactive oxygen species (ROS) in both GSCs and GdECs ultimately leading to a non-apoptotic copper-dependent cell death. Moreover, combined in vitro treatment with elesclomol and the alkylating agent temozolomide (TMZ) enhanced the cytotoxicity compared to TMZ alone. Finally, we used our experimental model of mouse brain xenografts to test the combination of elesclomol and TMZ and confirmed their efficacy in vivo.ConclusionsOur results support further evaluation of therapeutics targeting oxidative stress such as elesclomol with the aim of satisfying the high unmet medical need in the management of GBM.

Identification of Novel Cancer Stem Cell Markers in Glioblastoma by Comparing Tumor Cells with Stem-cell-like Cell Lines

Aim. The aim of this study is to identify differential gene expression for glioblastoma tumor cells, normoxic and hypoxic glioblastoma stem-like cell lines. Finding the upregulated and downregulated gene and pathway interactions. Analysis to find the differential expression genes and pathway interactions. Materials and methods. The gene expression profiling data from the microarray dataset GSE45117 from the Gene Expression Omnibus (GEO) database, as well as differentially expressed genes (DEGs) between the 2 categories, are used in this analysis. 4 Samples of Glioblastoma tumors were considered as group 1 and 4 samples of normoxic and Hypoxic glioblastoma stem-like cell lines were considered as group 2 in the GEO2R web tool that has been used to screen them. Results. The gene-gene interactions among the DEGs and the GGI network with 37 nodes and 13 edges. The stem-cell-like cell lines showed lower expression of endothelin-related genes such as EDN3 and EDNRA along with dysregulation of enzymes such as PDK1, PGK1 which points to dysregulation of cellular respiratory pathways. This effect in consensus with under expression of cell attachment genes such as COL2A1, COL5A2, COL15A1 denotes a strong shift toward metastasis. Conclusion. Thus, a computational pipeline for identifying the significant genes and pathways involved in the glioblastoma tumors and glioblastoma stem-like cell lines. This study provides a path towards discovering potential leads for the treatment of glioblastoma and aids in comprehending the underlying novel molecular mechanisms.

In Vitro Methods for the Study of Glioblastoma Stem-Like Cell Radiosensitivity.

Ionizing radiation is a critical component of glioblastoma (GBM) therapy. Recent data have implicated glioblastoma stem-like cells (GSCs) as determinants of GBM development, maintenance, and treatment response. Understanding the response of GSCs to radiation should thus provide insight into the development of improved GBM treatment strategies. Towards this end, in vitro techniques for the analysis of GSC radiosensitivity are an essential starting point. One such method, the clonogenic survival assay has been adapted to assessing the intrinsic radiosensitivity of GSCs and is described here. As an alternative method, the limiting dilution assay is presented for defining the radiosensitivity of GSC lines that do not form colonies or only grow as neurospheres. In addition to these cellular strategies, we describe γH2AX foci analysis, which provides a surrogate marker for radiosensitivity at the molecular level. Taken together, the in vitro methods presented here provide tools for defining intrinsic radiosensitivity of GSCs and for testing agents that may enhance GBM radioresponse.

Abstract 1929: A confirmatory study to probe the reproducibility of cancer cell lines as lab tools to identify molecular subtype-specific therapies and therapeutic targets against glioblastoma stem-like cells

Abstract Objective: Evidence that some high-impact biomedical results cannot be repeated has stimulated interest in development of standardization and normalization tools. Cancer cell lines are the workhorse of preclinical cancer research and their ability as test-matrix to prove reproducible results has been challenged. Stratifying tumors according to their DNA-methylation and transcriptome has revolutionized neuropathology and raises hopes to develop personalized management options for brain cancer patients. Here, we probe the reproducibility of glioblastoma stem-like cell lines (GSCs) to function as a study tool for the identification of tumor-subtype-specific therapeutic targets and therapies. Methods: A collection of phenotypically diverse GSCs was assembled. In vitro aging was enforced by controlled passaging of n=30 passages and comparative analysis of aging markers before and at the end of the experiment. High-resolution (at least n=5 different concentrations of the substances), medium-throughput (231 clinical-relevant drugs) pharmacology was performed using automation-assistance. Therapy response is presented by quantification of IC50 and AUC in terms of inhibition of cellular growth. Quantification of gene expression was performed using whole transcriptome RNA sequencing and global DNA-methylation was characterized using EPIC BeadChip. In silico prediction of drug response was performed by correlating the transcriptome with gene expression/drug-response data retrieved from publically-available sources. Results: Classification of cell models into transcriptional tumor subtypes was feasible but models show limited difference in DNA-methylation. Reproducibly of drug-response was limited with significant alterations observed in every 8th of the tested compounds. Our comparative results provide a large and technical most accurate alignment of in silico drug screen of GSCs to its physical acquired counterpart. Conclusions: GSCs are a practical study tool to perform throughput pharmacology testing in molecular subtype-specific manner. Unequivocal association of cell models into established molecular subtypes as identified in clinical specimens is partly feasible. Meanwhile our project is ongoing, we hypothesize the generation of a multi OMICS/chemo responsome compendium is an valuable approach to increase the robustness of cell line-guided studies aiming to discover therapeutic targets and therapeutics. Citation Format: Ann-Christin Nickel, Daniel Picard, Gonzalo Torga, Hans-Jakob Steiger, Daniel Hänggi, Maria Stella Carro, Marc Remke, Ulf Dietrich Kahlert. A confirmatory study to probe the reproducibility of cancer cell lines as lab tools to identify molecular subtype-specific therapies and therapeutic targets against glioblastoma stem-like cells [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 1929.

Mir-370-3p Impairs Glioblastoma Stem-Like Cell Malignancy Regulating a Complex Interplay between HMGA2/HIF1A and the Oncogenic Long Non-Coding RNA (lncRNA) NEAT1

Glioblastoma (GBM) is the most aggressive and prevalent form of a human brain tumor in adults. Several data have demonstrated the implication of microRNAs (miRNAs) in tumorigenicity of GBM stem-like cells (GSCs). The regulatory functions of miRNAs in GSCs have emerged as potential therapeutic candidates for glioma treatment. The current study aimed at investigating the function of miR-370-3p in glioma progression, as aberrant expression of miR-370-3p, is involved in various human cancers, including glioma. Analyzing our collection of GBM samples and patient-derived GSC lines, we found the expression of miR-370-3p significantly downregulated compared to normal brain tissues and normal neural stem cells. Restoration of miR-370-3p expression in GSCs significantly decreased proliferation, migration, and clonogenic abilities of GSCs, in vitro, and tumor growth in vivo. Gene expression analysis performed on miR-370-3p transduced GSCs, identified several transcripts involved in Epithelial to Mesenchymal Transition (EMT), and Hypoxia signaling pathways. Among the genes downregulated by the restored expression of miR-370-3p, we found the EMT-inducer high-mobility group AT-hook 2 (HMGA2), the master transcriptional regulator of the adaptive response to hypoxia, Hypoxia-inducible factor (HIF)1A, and the long non-coding RNAs (lncRNAs) Nuclear Enriched Abundant Transcript (NEAT)1. NEAT1 acts as an oncogene in a series of human cancers including gliomas, where it is regulated by the Epidermal Growth Factor Receptor (EGFR) pathways, and contributes to tumor growth and invasion. Noteworthy, the expression levels of miR-370-3p and NEAT1 were inversely related in both GBM tumor specimens and GSCs, and a dual-luciferase reporter assay proved the direct binding between miR-370-3p and the lncRNAs NEAT1. Our results identify a critical role of miR-370-3p in the regulation of GBM development, indicating that miR-370-3p acts as a tumor-suppressor factor inhibiting glioma cell growth, migration and invasion by targeting the lncRNAs NEAT1, HMGA2, and HIF1A, thus, providing a potential candidate for GBM patient treatment.

The TRIM26-SOX2 Ubiquitin Signaling Axis Regulates the Maintenance and Tumorigenicity of Glioblastoma Stem-like Cells

Abstract INTRODUCTION Glioblastomas harbor inter and intratumoral genetic diversity, posing a challenge for targeted therapies. A major question is whether shared mechanisms might control the malignant phenotypes of genetically diverse glioblastoma cells. We reasoned that ubiquitin-dependent regulation of pluripotency-related transcription factor SOX2, which is indispensable for the maintenance of tumorigenic glioblastoma stem-like cells (GSC), may represent one such mechanism. TRIM26, an E3-ubiquitin ligase with immune-related functions, is highly expressed in glioblastoma tumors compared to normal brain. Immunoprecipitation followed by mass spectrometry suggested TRIM26 interacts with SOX2. We hypothesized that TRIM26 plays an essential role in GSCs by regulating SOX2 function. METHODS Direct protein-protein interactions were assessed by in vitro binding assays. In Vitro ubiquitination assays were performed. Lentiviral TRIM26 overexpression and knockdown were used to test the role of TRIM26 in regulating SOX2 stability, activity, and ubiquitination. The functional relevance of TRIM26 in GSCs was assessed by in Vitro self-renewal and in Vivo tumorigenicity assays. RESULTS We found that TRIM26 directly interacts with SOX2 via the C-terminal PRY-SPRY domain. Unexpectedly, TRIM26 overexpression resulted in decreased SOX2 polyubiquitination in cells. In line with this observation, TRIM26 knockdown in GSCs decreased SOX2 protein stability without changing SOX2 mRNA levels. Functionally, TRIM26 knockdown reduced SOX2 transcriptional activity, self-renewal, and in Vivo tumorigenicity in multiple genetically divergent GSC lines. Mechanistically, we discovered TRIM26 stabilizes SOX2 protein by competitively reducing the interaction of SOX2 with WWP2, a bonafide SOX2 E3 ligase in GSCs. Accordingly; WWP2 depletion in the setting of TRIM26 knockdown in GSCs rescued SOX2 protein levels, self-renewal, and tumorigenicity. CONCLUSION Together, these results suggest that TRIM26 maintains GSCs by protecting SOX2 from WWP2-mediated ubiquitination and subsequent proteasomal degradation. These findings raise the intriguing possibility that modulating ubiquitin-dependent regulation of SOX2 in genetically heterogeneous GSCs may represent a unifying therapeutic strategy.

Genetically distinct glioma stem-like cell xenografts established from paired glioblastoma samples harvested before and after molecularly targeted therapy

Intratumoural heterogeneity underlies tumour escape from molecularly targeted therapy in glioblastoma. A cell-based model preserving the evolving molecular profiles of a tumour during treatment is key to understanding the recurrence mechanisms and development of strategies to overcome resistance. In this study, we established a matched pair of glioblastoma stem-like cell (GSC) cultures from patient glioblastoma samples before and after epidermal growth factor receptor (EGFR)-targeted therapy. A patient with recurrent glioblastoma (MGG70R) harboring focal, high-level EGFR amplification received the irreversible EGFR tyrosine kinase inhibitor dacomitinib. The tumour that subsequently recurred (MGG70RR) showed diploid EGFR, suggesting inhibitor-mediated elimination of EGFR-amplified tumour cells and propagation of EGFR non-amplified cell subpopulations. The MGG70R-GSC line established from MGG70R formed xenografts retaining EGFR amplification and EGFR overexpression, while MGG70RR-GSC established from MGG70RR generated tumours that lacked EGFR amplification and EGFR overexpression. MGG70R-GSC-derived intracranial xenografts were more proliferative than MGG70RR-GSC xenografts, which had upregulated mesenchymal markers, mirroring the pathological observation in the corresponding patient tumours. In vitro MGG70R-GSC was more sensitive to EGFR inhibitors than MGG70RR-GSC. Thus, these molecularly distinct GSC lines recapitulated the subpopulation alteration that occurred during glioblastoma evasion of targeted therapy, and offer a valuable model facilitating therapeutic development for recurrent glioblastoma.

SWIM tool application to expression data of glioblastoma stem-like cell lines, corresponding primary tumors and conventional glioma cell lines

BackgroundIt is well-known that glioblastoma contains self-renewing, stem-like subpopulation with the ability to sustain tumor growth. These cells – called cancer stem-like cells – share certain phenotypic characteristics with untransformed stem cells and are resistant to many conventional cancer therapies, which might explain the limitations in curing human malignancies. Thus, the identification of genes controlling the differentiation of these stem-like cells is becoming a successful therapeutic strategy, owing to the promise of novel targets for treating malignancies.MethodsRecently, we developed SWIM, a software able to unveil a small pool of genes – called switch genes – critically associated with drastic changes in cell phenotype. Here, we applied SWIM to the expression profiling of glioblastoma stem-like cells and conventional glioma cell lines, in order to identify switch genes related to stem-like phenotype.ResultsSWIM identifies 171 switch genes that are all down-regulated in glioblastoma stem-like cells. This list encompasses genes like CAV1, COL5A1, COL6A3, FLNB, HMMR, ITGA3, ITGA5, MET, SDC1, THBS1, and VEGFC, involved in “ECM-receptor interaction“ and “focal adhesion” pathways. The inhibition of switch genes highly correlates with the activation of genes related to neural development and differentiation, such as the 4-core OLIG2, POU3F2, SALL2, SOX2, whose induction has been shown to be sufficient to reprogram differentiated glioblastoma into stem-like cells. Among switch genes, the transcription factor FOSL1 appears as the brightest star since: it is down-regulated in stem-like cells; it highly negatively correlates with the 4-core genes that are all up-regulated in stem-like cells; the promoter regions of the 4-core genes harbor a consensus binding motif for FOSL1.ConclusionsWe suggest that the inhibition of switch genes in stem-like cells could induce the deregulation of cell communication pathways, contributing to neoplastic progression and tumor invasiveness. Conversely, their activation could restore the physiological equilibrium between cell adhesion and migration, hampering the progression of cancer. Moreover, we posit FOSL1 as promising candidate to orchestrate the differentiation of cancer stem-like cells by repressing the 4-core genes’ expression, which severely halts cancer growth and might affect the therapeutic outcome. We suggest FOSL1 as novel putative therapeutic and prognostic biomarker, worthy of further investigation.

Abstract 4444: The XPO1 inhibitor Selinexor attenuates global translation and enhances the radiosensitivity of glioblastoma cells grown in vitro and in vivo

Abstract Radiation therapy remains a primary treatment modality for glioblastoma (GBM). To identify novel targets for GBM radiosensitization, the radiation-induced translatome was defined for a series of glioblastoma stem-like cells (GSCs). Analysis of the radiation-induced translatome of GSCs identified an interacting network in which Exportin 1 (XPO1) serves as a major hub protein. XPO1 transports a variety of proteins and RNAs from the nucleus to the cytoplasm via the nuclear pore complex and is dysregulated in multiple types of cancers, including GBM. To determine whether XPO1 provides a target for GBM radiosensitization, we defined the effects of the clinically relevant XPO1-inhibitor Selinexor (KPT-330) on the radiosensitivity of GSC lines. Using clonogenic survival analysis, Selinexor was shown to enhance the in vitro radiosensitivity of GSCs but not normal fibroblast cell lines. Because XPO1 mediates the nuclear export of ribosomal subunits, we tested the effect of Selinexor on gene translation using polysomes profiles to calculate translational efficiency and O-propargyl-puromycin (OPP) incorporation to measure de novo protein synthesis. Selinexor significantly reduced translational efficiency in GSCs beginning at 1-6h after treatment, which was accompanied by a decrease in protein synthesis. Moreover, Selinexor exposure increased in 18S and 5S rRNA accumulation in the nucleus with corresponding decreases in the cytoplasm suggesting that the inhibition of rRNA export plays a role in the Selinexor-mediated decrease in protein synthesis. In contrast to the tumor cells, Selinexor had no effect on protein synthesis in normal fibroblast cell lines and astrocytes. To determine the effect of XPO1 inhibition on GBM cells in vivo, mice bearing orthotopic xenografts initiated from a GSC line (NSC11) were treated with Selinexor (20 mg/kg) and tumors were collected over 48 hours. Polysome profiles generated from these orthotopic xenografts indicated that Selinexor treatment resulted in a decrease in translational efficiency with the greatest effect at 24 hours after treatment. These results suggest that consistent with in vitro data, Selinexor inhibits global translation in GBM cells grown under orthotopic, in vivo conditions. To determine the effect of XPO1 inhibition on radioresponse in vivo, mice bearing orthotopic xenografts were treated with Selinexor in combination with fractionated radiation. Whereas Selinexor alone had no effect on survival, the XPO1 inhibitor enhanced radiation-induced prolongation of animal survival with an apparent dose enhancement factor of 2. These results suggest that Selinexor delivered in combination with radiotherapy may improve the effectiveness of GBM treatment. Citation Format: Amy Wahba, John W. O'Neill, Barbara H. Rath, Kevin Camphausen, Philip J. Tofilon. The XPO1 inhibitor Selinexor attenuates global translation and enhances the radiosensitivity of glioblastoma cells grown in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4444.

Computational identification of specific genes for glioblastoma stem-like cells identity

Glioblastoma, the most malignant brain cancer, contains self-renewing, stem-like cells that sustain tumor growth and therapeutic resistance. Identifying genes promoting stem-like cell differentiation might unveil targets for novel treatments. To detect them, here we apply SWIM – a software able to unveil genes (named switch genes) involved in drastic changes of cell phenotype – to public datasets of gene expression profiles from human glioblastoma cells. By analyzing matched pairs of stem-like and differentiated glioblastoma cells, SWIM identified 336 switch genes, potentially involved in the transition from stem-like to differentiated state. A subset of them was significantly related to focal adhesion and extracellular matrix and strongly down-regulated in stem-like cells, suggesting that they may promote differentiation and restrain tumor growth. Their expression in differentiated cells strongly correlated with the down-regulation of transcription factors like OLIG2, POU3F2, SALL2, SOX2, capable of reprogramming differentiated glioblastoma cells into stem-like cells. These findings were corroborated by the analysis of expression profiles from glioblastoma stem-like cell lines, the corresponding primary tumors, and conventional glioma cell lines. Switch genes represent a distinguishing feature of stem-like cells and we are persuaded that they may reveal novel potential therapeutic targets worthy of further investigation.

Meta-Analysis and Experimental Validation Identified FREM2 and SPRY1 as New Glioblastoma Marker Candidates.

Glioblastoma (GB) is the most aggressive brain malignancy. Although some potential glioblastoma biomarkers have already been identified, there is a lack of cell membrane-bound biomarkers capable of distinguishing brain tissue from glioblastoma and/or glioblastoma stem cells (GSC), which are responsible for the rapid post-operative tumor reoccurrence. In order to find new GB/GSC marker candidates that would be cell surface proteins (CSP), we have performed meta-analysis of genome-scale mRNA expression data from three data repositories (GEO, ArrayExpress and GLIOMASdb). The search yielded ten appropriate datasets, and three (GSE4290/GDS1962, GSE23806/GDS3885, and GLIOMASdb) were used for selection of new GB/GSC marker candidates, while the other seven (GSE4412/GDS1975, GSE4412/GDS1976, E-GEOD-52009, E-GEOD-68848, E-GEOD-16011, E-GEOD-4536, and E-GEOD-74571) were used for bioinformatic validation. The selection identified four new CSP-encoding candidate genes—CD276, FREM2, SPRY1, and SLC47A1—and the bioinformatic validation confirmed these findings. A review of the literature revealed that CD276 is not a novel candidate, while SLC47A1 had lower validation test scores than the other new candidates and was therefore not considered for experimental validation. This validation revealed that the expression of FREM2—but not SPRY1—is higher in glioblastoma cell lines when compared to non-malignant astrocytes. In addition, FREM2 gene and protein expression levels are higher in GB stem-like cell lines than in conventional glioblastoma cell lines. FREM2 is thus proposed as a novel GB biomarker and a putative biomarker of glioblastoma stem cells. Both FREM2 and SPRY1 are expressed on the surface of the GB cells, while SPRY1 alone was found overexpressed in the cytosol of non-malignant astrocytes.

Yeast display biopanning identifies human antibodies targeting glioblastoma stem-like cells

Glioblastoma stem-like cells (GSC) are hypothesized to evade current therapies and cause tumor recurrence, contributing to poor patient survival. Existing cell surface markers for GSC are developed from embryonic or neural stem cell systems; however, currently available GSC markers are suboptimal in sensitivity and specificity. We hypothesized that the GSC cell surface proteome could be mined with a yeast display antibody library to reveal novel immunophenotypes. We isolated an extensive collection of antibodies that were differentially selective for GSC. A single domain antibody VH-9.7 showed selectivity for five distinct patient-derived GSC lines and visualized orthotopic GBM xenografts in vivo after conjugation with a near-infrared dye. These findings demonstrate a previously unexplored high-throughput strategy for GSC-selective antibody discovery, to aid in GSC isolation, diagnostic imaging, and therapeutic targeting.