Glioblastoma Stem-like Cells Research Articles

TRIM25 promotes glioblastoma cell growth and invasion via regulation of the PRMT1/c-MYC pathway by targeting the splicing factor NONO

BackgroundUbiquitination plays an important role in proliferating and invasive characteristic of glioblastoma (GBM), similar to many other cancers. Tripartite motif 25 (TRIM25) is a member of the TRIM family of proteins, which are involved in tumorigenesis through substrate ubiquitination.MethodsDifference in TRIM25 expression levels between nonneoplastic brain tissue samples and primary glioma samples was demonstrated using publicly available glioblastoma database, immunohistochemistry, and western blotting. TRIM25 knockdown GBM cell lines (LN229 and U251) and patient derived GBM stem-like cells (GSCs) GBM#021 were used to investigate the function of TRIM25 in vivo and in vitro. Co-immunoprecipitation (Co-IP) and mass spectrometry analysis were performed to identify NONO as a protein that interacts with TRIM25. The molecular mechanisms underlying the promotion of GBM development by TRIM25 through NONO were investigated by RNA-seq and validated by qRT-PCR and western blotting.ResultsWe observed upregulation of TRIM25 in GBM, correlating with enhanced glioblastoma cell growth and invasion, both in vitro and in vivo. Subsequently, we screened a panel of proteins interacting with TRIM25; mass spectrometry and co-immunoprecipitation revealed that NONO was a potential substrate of TRIM25. TRIM25 knockdown reduced the K63-linked ubiquitination of NONO, thereby suppressing the splicing function of NONO. Dysfunctional NONO resulted in the retention of the second intron in the pre-mRNA of PRMT1, inhibiting the activation of the PRMT1/c-MYC pathway.ConclusionsOur study demonstrates that TRIM25 promotes glioblastoma cell growth and invasion by regulating the PRMT1/c-MYC pathway through mediation of the splicing factor NONO. Targeting the E3 ligase activity of TRIM25 or the complex interactions between TRIM25 and NONO may prove beneficial in the treatment of GBM.

Proteomic, Metabolomic, and Fatty Acid Profiling of Small Extracellular Vesicles from Glioblastoma Stem-Like Cells and Their Role in Tumor Heterogeneity.

Glioblastoma is a deadly brain tumor for which there is no cure. The presence of glioblastoma stem-like cells (GSCs) contributes to the heterogeneous nature of the disease and makes developing effective therapies challenging. Glioblastoma cells have been shown to influence their environment by releasing biological nanostructures known as extracellular vesicles (EVs). Here, we investigated the role of GSC-derived nanosized EVs (<200 nm) in glioblastoma heterogeneity, plasticity, and aggressiveness, with a particular focus on their protein, metabolite, and fatty acid content. We showed that conditioned medium and small extracellular vesicles (sEVs) derived from cells of one glioblastoma subtype induced transcriptomic and proteomic changes in cells of another subtype. We found that GSC-derived sEVs are enriched in proteins playing a role in the transmembrane transport of amino acids, carboxylic acids, and organic acids, growth factor binding, and metabolites associated with amino acid, carboxylic acid, and sugar metabolism. This suggests a dual role of GSC-derived sEVs in supplying neighboring GSCs with valuable metabolites and proteins responsible for their transport. Moreover, GSC-derived sEVs were enriched in saturated fatty acids, while their respective cells were high in unsaturated fatty acids, supporting that the loading of biological cargos into sEVs is a highly regulated process and that GSC-derived sEVs could be sources of saturated fatty acids for the maintenance of glioblastoma cell metabolism. Interestingly, sEVs isolated from GSCs of the proneural and mesenchymal subtypes are enriched in specific sets of proteins, metabolites, and fatty acids, suggesting a molecular collaboration between transcriptionally different glioblastoma cells. In summary, this study revealed the complexity of GSC-derived sEVs and unveiled their potential contribution to tumor heterogeneity and critical cellular processes commonly deregulated in glioblastoma.

The paracaspase MALT1 controls cholesterol homeostasis in glioblastoma stem-like cells through lysosome proteome shaping

Glioblastoma stem-like cells (GSCs) compose a tumor-initiating and -propagating population remarkably vulnerable to variation in the stability and integrity of the lysosomal compartment. Previous work has shown that the expression and activity of the paracaspase MALT1 control GSC viability via lysosome abundance. However, the underlying mechanisms remain elusive. By combining RNA sequencing (RNA-seq) with proteome-wide label-free quantification, we now report that MALT1 repression in patient-derived GSCs alters the homeostasis of cholesterol, which accumulates in late endosomes (LEs)-lysosomes. This failure in cholesterol supply culminates in cell death and autophagy defects, which can be partially reverted by providing exogenous membrane-permeable cholesterol to GSCs. From a molecular standpoint, a targeted lysosome proteome analysis unraveled that Niemann-Pick type C (NPC) lysosomal cholesterol transporters are diluted when MALT1 is impaired. Accordingly, we found that NPC1/2 inhibition and silencing partially mirror MALT1 loss-of-function phenotypes. This supports the notion that GSC fitness relies on lysosomal cholesterol homeostasis.

Development of an intraventricular adeno-associated virus-based labeling strategy for glioblastoma cells nested in the subventricular zone.

Glioblastoma (GBM) is a dreadful brain tumor, with a particular relationship to the adult subventricular zone (SVZ) that has been described as relevant to disease initiation, progression, and recurrence. We propose a novel strategy for the detection and tracking of xenografted GBM cells that are located in the SVZ, based on an intracerebroventricular (icv) recombinant adeno-associated virus (AAV)-mediated color conversion method. We used different patient-derived GBM stem-like cells (GSCs), which we transduced first with a retroviral vector (LRLG) that included a lox-dsRed-STOP-lox cassette, upstream of the eGFP gene, then with rAAVs expressing the Cre-recombinase. Red and green fluorescence is analyzed in vitro and in vivo using flow cytometry and fluorescence microscopy. After comparing the efficiency of diverse rAAV serotypes, we confirmed that the in vitro transduction of GSC-LRLG with rAAV-Cre induced a switch from red to green fluorescence. In parallel, we verified that rAAV transduction was confined to the walls of the lateral ventricles. We, therefore, applied this conversion approach in 2 patient-derived orthotopic GSC xenograft models and showed that the icv injection of an rAAV-DJ-Cre after GSC-LRLG tumor implantation triggered the conversion of red GSCs to green, in the periventricular region. Green GSCs were also found at distant places, including the migratory tract and the tumor core. This study not only sheds light on the putative outcome of SVZ-nested GBM cells but also shows that icv injection of rAAV vectors allows to transduce and potentially modulate gene expression in hard-to-reach GBM cells of the periventricular area.

Glioblastoma Stem-like Cell Detection using Advanced MR Imaging: results from the STEMRI trial

Glioblastoma Stem-like Cell Detection using Advanced MR Imaging: results from the STEMRI trial

Nuclear Glycoprotein A Repetitions Predominant (GARP) Is a Common Trait of Glioblastoma Stem-like Cells and Correlates with Poor Survival in Glioblastoma Patients.

Glioblastoma (GB) is notoriously resistant to therapy. GB genesis and progression are driven by glioblastoma stem-like cells (GSCs). One goal for improving treatment efficacy and patient outcomes is targeting GSCs. Currently, there are no universal markers for GSCs. Glycoprotein A repetitions predominant (GARP), an anti-inflammatory protein expressed by activated regulatory T cells, was identified as a possible marker for GSCs. This study evaluated GARP for the detection of human GSCs utilizing a multidimensional experimental design that replicated several features of GB: (1) intratumoral heterogeneity, (2) cellular hierarchy (GSCs with varied degrees of self-renewal and differentiation), and (3) longitudinal GSC evolution during GB recurrence (GSCs from patient-matched newly diagnosed and recurrent GB). Our results indicate that GARP is expressed by GSCs across various cellular states and disease stages. GSCs with an increased GARP expression had reduced self-renewal but no alterations in proliferative capacity or differentiation commitment. Rather, GARP correlated inversely with the expression of GFAP and PDGFR-α, markers of astrocyte or oligodendrocyte differentiation. GARP had an abnormal nuclear localization (GARPNU+) in GSCs and was negatively associated with patient survival. The uniformity of GARP/GARPNU+ expression across different types of GSCs suggests a potential use of GARP as a marker to identify GSCs.

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.

TMIC-26. TAMS AND HYPOXIA ENHANCE GLIOBLASTOMA STEM-LIKE CELL ENRICHMENT AND MAY IMPACT RADIORESISTANCE

Abstract The dismal survival rates of glioblastoma (GBM) patients are due, at least in part, to therapeutic resistance of GBM stem-like cells (GSCs) and their ability to repopulate recurrent tumors. Our goal was to determine if tumor-associated macrophages (TAMs) or hypoxia enhance the enrichment of GSCs in a manner that could impact radioresistance. First, we established GBM-brain cortical organoids (GBM-BCOs) to model human GBM. We co-cultured GFP-labeled GBM neurospheres with BCOs for 14 days and observed a tumor take rate of 100%. Next, we evaluated whether hypoxia enhanced TAM migration into GBM-BCOs by incorporating THP-1 cells into GFP-GBM-BCOs and incubating them for 24 h under normoxic or hypoxic conditions. We found that hypoxia significantly increased THP-1 cell influx into GBM-BCOs. Further, we investigated whether TAMs influenced the enrichment of GSCs in GBM neurospheres and in GBM-BCOs. We found that co-culture of THP-1 cells with either GBM neurospheres or with GBM-BCOs enhanced the CD133+ population after 72 h hypoxia, correlating with our IF studies of human GBM samples that show increased CD133+ and IBA1+ cells in hypoxic peri-necrotic zones compared to non-necrotic regions. Next, we established Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI), an advanced methodology for monitoring cell cycle phases for investigating the role of hypoxia and radiation on GBM. Flow cytometry analysis of patient-derived GBM cells revealed that the fraction of G1 cells decreased and the fraction of G2/M cells increased 24 h after 3 Gy radiation compared to non-irradiated cells in normoxia. In contrast, GBM cells exposed to hypoxia do not show such cell cycle shifts after radiation and have an enriched population of cells in G0 in both radiated and non-irradiated cells. Our results indicate that hypoxia and TAMs play critical roles in GSC enrichment and may impact radioresistance.

DNAR-13. TARGETING THE DNA DAMAGE RESPONSE IN GLIOBLASTOMA PROPAGATING CELLS: FROM MOLECULAR MECHANISMS TO PRECISION MEDICINE

Abstract Glioblastoma (GBM) is the most aggressive and lethal primary tumor of the central nervous system (CNS). Despite surgical resection and a combination of radiotherapy and alkylation chemotherapy, the median survival for patients with newly diagnosed GBM is only ~ 14 months. Among the many challenges confronting clinicians are subpopulations of tumor-propagating cells named GBM Stem-like Cells (GSCs), which have been proposed to drive treatment resistance and foster tumor relapse. At the mechanistic level, GSCs have been shown to display constitutive activation of the DNA damage response (DDR) due to chronic replication stress (RS), which enhances their ability to repair the DNA lesions induced by chemoradiation. Thus, developing new therapeutic strategies against glioblastoma is of paramount importance. Cell cycle regulation and DNA damage response are two crucial molecular networks altered in most cancer types. In this context, we have previously identified a DNA repair and cell cycle gene expression signature that results in the classification of GBM tumors in two major groups, G1 and G3, which differ in the expression of survival and proliferation pathways. Using bioinformatics analysis of the GLASS consortium database, we have created an exhaustive oncopanel of the molecular alterations defining the G1 and G3 groups. Furthermore, using G1- and G3-patient-derived GSC cell lines as experimental models, we have identified crucial differences in the mechanisms orchestrating the cell cycle, DDR, and treatment resistance between G1 and G3 cells. Finally, we have also identified group-specific therapeutic strategies combining repositioned FDA-approved drugs with the inhibition of crucial DDR factors. Our long-term goals are i) to understand the molecular basis behind the differential response to DNA damage and replication stress developed by G1 and G3 cells and ii) to propose group-specific, DDR-based combination therapies that prevent treatment resistance and promote long-lasting treatment.

CNSC-28. CELLULAR AND FUNCTIONAL REMODELING OF HUMAN CEREBRAL ORGANOIDS BY GLIOBLASTOMA STEM-LIKE CELLS

Abstract The burgeoning field of cancer neuroscience focuses on deciphering the role of normal neural cells in brain tumor growth. Until recently, representation of normal human neural cells in brain tumor model systems was lacking. By utilizing human induced-pluripotent stem cells derived cerebral organoids (COs) co-cultured with glioblastoma stem-like cells (RFP-luciferase tagged MDA-GSCs, n = 3 representing each glioblastoma molecular sub-type), we investigated the effects of GSCs on CO-derived neural cells and their activity. COs and CO+MDA-GSCs were subjected to single-cell RNA-sequencing (scRNA-seq). As per typical micro-anatomy of COs, neural stem/progenitor cells are interiorly distributed whereas differentiated neurons are positioned on the surfaces, enabling measurement of neuronal action potential on surfaces of COs by micro-electrode arrays (MEA). The scRNA-seq data from CO+MDA-GSC samples were subset into CO-derived (luciferase-ve) and GSCs-derived (luciferase+ve). Compared with COs, co-cultured samples had significantly higher percentage of non-telencephalon neural progenitor cells (ntNPCs) (CO_vs._CO+MDA-GSC(luciferase-ve): 15.6% vs. 47.2%-63.4%) while the percentages of cortical NPC (cNPCs) and intermediate progenitors (IPs) was lower (CO_vs._CO+MDA-GSC(luciferase-ve): 39.2% vs. 12.0%-20.5%). These alterations in NPC/IPs abundance were accompanied by decreased proportions of cortical excitatory neurons (CO_vs._CO+MDA-GSC(Luciferase-ve): 33.2% vs. 7.9%-23.4%). Among luciferase+ve MDA-GSCs-derived cells in co-culture samples, mesenchymal MDA-GSCs had higher abundance of ntNPCs versus cNPCs (30.4% versus 63.6%) whereas, among classical and proneural MDA-GSCs ntNPCs were 11.45-22.8 times higher. Interestingly, 14.9% of proneural MDA-GSCs derived cells displayed inhibitory neuron signatures. After 3 weeks, the mean firing rate measured by MEA was significantly lower in co-cultures compared to COs (CO_vs._CO+MDA-GSC: 0.052 Hz vs. 0.0081 Hz p-value 0.000219), suggesting functional remodeling. Our results show that cellular remodeling of normal cells resulted in progenitor-like cell-state whereas that of MDA-GSCs varied across molecular sub-type. These results suggest interactions between normal and tumor cells can potentially be leveraged to manage brain tumor growth and its impact on normal brain.

CSIG-17. NICOTINAMIDE N-METHYLTRANSFERASE (NNMT) AS A POTENTIAL THERAPEUTIC TARGET FOR EGFRVIII-DRIVEN GLIOBLASTOMA

Abstract Nicotinamide N-methyltransferase (NNMT) is an enzymatic catalyst involved in converting methyl groups from S-adenosyl-L-methionine (SAM) to nicotinamide (NAM), generating S-adenosine hyperblastoids (SAH) and 1-methylnicotinamide (MNA). Increased NNMT expression is frequently observed in various cancers and is associated with tumor progression, metastasis, unfavorable clinical outcomes, and drug resistance. Notably, NNMT stands out as one of the most up-regulated metabolism-related genes in glioblastoma, although the underlying mechanism for its overexpression remains elusive. Glioblastoma commonly exhibits genetic lesions in the epidermal growth factor receptor (EGFR) gene, including amplification or mutation, with EGFRvIII being the predominant mutant contributing to glioblastoma cell proliferation, invasion, therapeutic resistance, metastasis, and stemness. In this study, we demonstrate that activation of wild-type EGFR by EGF or the constitutively active mutant EGFRvIII promotes NNMT expression and enzyme activity in established glioblastoma cell lines and glioblastoma stem-like cells via the p38 mitogen-activated protein kinase pathway. Additionally, we identify STAT1 as a downstream transcription factor essential for EGFR/EGFRvIII-induced NNMT upregulation. RNA interference-mediated knockdown of NNMT in glioblastoma cells significantly inhibits EGFRvIII-promoted invasion and proliferation in vitro. Targeting NNMT effectively suppresses EGFRvIII-driven glioblastoma tumorigenicity and prolongs the survival of tumor-bearing mice. Clinically, NNMT is highly expressed and positively correlated with EGFR expression in human glioblastoma tumors. Collectively, these findings establish NNMT as a previously unrecognized downstream target gene of EGFR/EGFRvIII expression and highlight its potential as a therapeutic target for inhibiting EGFRvIII-driven glioblastoma.

CCRG-03. KAT5 REGULATES DEVELOPMENTAL TRANSITIONS AND CELLULAR HETEROGENEITY IN AN IN VIVO MODEL OF GLIOBLASTOMA

Abstract In solid tumors, G0-like states are likely critical for maintaining developmental hierarchies and cellular heterogeneity and promoting tumor growth/recurrence, yet little is known about tumor G0 states or regulation of their ingress/egress. To discover G0-like states and their regulators for glioblastoma (GBM), we previously performed a genome-wide CRISPR-Cas9 screen in patient-derived GBM stem-like cells (GSCs) for genes that trap cells in G0 when inhibited. We identify the protein acetyltransferase KAT5 as a key regulator of GBM G0 ingress/egress. Here, we show in an in vivo GSC-derived orthotopic xenograft model that KAT5 regulates transcriptional, epigenetic, and proliferative heterogeneity impacting transitions into G0-like states. We show that KAT5 activity suppresses the emergence of non-dividing subpopulations with oligodendrocyte progenitor and radial glial cell characteristics. With regard to chromatin regulation, we show that loss of KAT5 activity alters sites of mixed epigenetic valency (which have both activating and repressive marks in bulk tumor populations) to promote emergence of OPC/RG G0-like populations in tumors. Our data reveal KAT5-associated transcriptional and epigenetic changes in tumors, which indicate that KAT5 activity biases cell state distribution away from OPC and RG-like subpopulations. In addition, loss of KAT5 resulted in a less invasive phenotype and prolonged survival of mice harboring orthotopic xenografts.

The Semaphorin 3A-Neuropilin-1 Pathway Promotes Clonogenic Growth of Glioblastoma via Activation of TGFβ Signaling.

Glioblastoma (GBM) is the most lethal brain cancer with a dismal prognosis. Stem-like GBM cells (GSCs) are a major driver of GBM propagation and recurrence, thus understanding the molecular mechanisms that promote GSCs may lead to effective therapeutic approaches. Through in vitro clonogenic growth-based assays, we determined mitogenic activities of the ligand molecules that are implicated in neural development. We have identified that Semaphorin 3A (Sema3A), originally known as an axon guidance molecule in the central nervous system, promotes clonogenic growth of GBM cells but not normal neural progenitor cells (NPCs). Mechanistically, Sema3A binds to its receptor Neuropilin-1 (NRP1) and facilitates an interaction between NRP1 and TGF receptor 1 (TGFR1), which in turn leads to activation of canonical TGF signaling in both GSCs and NPCs. TGF signaling enhances self-renewal and survival of GBM tumors through induction of key stem cell factors, but it evokes cytostatic responses in NPCs. Blockage of the Sema3A-NRP1 axis via shRNA-mediated knockdown of Sema3A or NRP1 impeded clonogenic growth and TGF pathway activity in GSCs and inhibited tumor growth in vivo. Taken together, these findings suggest that the Sema3A-NRP1-TGFR1 signaling axis is a critical regulator of GSC propagation and a potential therapeutic target for GBM.

Dauricine attenuates Oct4/sonic hedgehog co-activated stemness and induces reactive oxygen species-mediated mitochondrial apoptosis via AKT/β-catenin signaling in human neuroblastoma and glioblastoma stem-like cells.

Neuroblastoma and glioblastoma are primary malignant tumors of the nervous system, with frequent relapse and limited clinical therapeutic drugs. The failure of their treatment is due to the tumor cells exhibiting cancer stem-like cells (CSLCs) properties. Octamer binding transcription factor 4 (Oct4) is involved in mediating CSLCs, our previous work found that Oct4-driven reprogramming of astrocytes into induced neural stem cells was potentiated with continuous sonic hedgehog (Shh) stimulation. In this study, we aimed to study the importance of Oct4 and Shh combination in the stemness properties induction of neuroblastoma and glioblastoma cells, and evaluate the anti-stemness effect of dauricine (DAU), a natural product of bis-benzylisoquinoline alkaloid. The effect of Oct4 and Shh co-activation on cancer stemness was evaluated by tumor spheres formation model and flow cytometry analysis. Then the effects of DAU on SH-SY5Y and T98-G cells were assessed by the MTT, colony formation, and tumor spheres formation model. DAU acts on Oct4 were verified using the Western blotting, MTT, and so on. Mechanistic studies were explored by siRNA transfection assay, Western blotting, and flow cytometry analysis. We identified that Shh effectively improved Oct4-mediated generation of stemness in SH-SY5Y and T98-G cells, and Oct4 and Shh co-activation promoted cell growth, the resistance of apoptosis. In addition, DAU, a natural product, was found to be able to attenuate Oct4/Shh co-activated stemness and induce cell cycle arrest and apoptosis via blocking AKT/β-catenin signaling in neuroblastoma and glioblastoma, which contributed to the neuroblastoma and glioblastoma cells growth inhibition by DAU. In summary, our results indicated that the treatment of DAU may be served as a potential therapeutic method in neuroblastoma and glioblastoma.

Wnt/β‑catenin signaling is a novel therapeutic target for tumor suppressor CYLD‑silenced glioblastoma cells.

Tumor suppressor cylindromatosis (CYLD) dysfunction by its downregulation is significantly associated with poor prognosis in patients with glioblastoma (GBM), the most aggressive and malignant type of glioma. However, no effective treatment is currently available for patients with CYLD‑downregulated GBM. The aim of the present study was to identify the crucial cell signaling pathways and novel therapeutic targets for CYLD downregulation in GBM cells. CYLD knockdown in GBM cells induced GBM malignant characteristics, such as proliferation, metastasis, and GBM stem‑like cell (GSC) formation. Comprehensive proteomic analysis and RNA sequencing data from the tissues of patients with GBM revealed that Wnt/β‑catenin signaling was significantly activated by CYLD knockdown in patients with GBM. Furthermore, a Wnt/β‑catenin signaling inhibitor suppressed all CYLD knockdown‑induced malignant characteristics of GBM. Taken together, the results of the present study revealed that Wnt/β‑catenin signaling is responsible for CYLD silencing‑induced GBM malignancy; therefore, targeting Wnt/β‑catenin may be effective for the treatment of CYLD‑negative patients with GBM with poor prognosis.

Defining super-enhancers by highly ranked histone H4 multi-acetylation levels identifies transcription factors associated with glioblastoma stem-like properties

BackgroundSuper-enhancers (SEs), which activate genes involved in cell-type specificity, have mainly been defined as genomic regions with top-ranked enrichment(s) of histone H3 with acetylated K27 (H3K27ac) and/or transcription coactivator(s) including a bromodomain and extra-terminal domain (BET) family protein, BRD4. However, BRD4 preferentially binds to multi-acetylated histone H4, typically with acetylated K5 and K8 (H4K5acK8ac), leading us to hypothesize that SEs should be defined by high H4K5acK8ac enrichment at least as well as by that of H3K27ac.ResultsHere, we conducted genome-wide profiling of H4K5acK8ac and H3K27ac, BRD4 binding, and the transcriptome by using a BET inhibitor, JQ1, in three human glial cell lines. When SEs were defined as having the top ranks for H4K5acK8ac or H3K27ac signal, 43% of H4K5acK8ac-ranked SEs were distinct from H3K27ac-ranked SEs in a glioblastoma stem-like cell (GSC) line. CRISPR-Cas9–mediated deletion of the H4K5acK8ac-preferred SEs associated with MYCN and NFIC decreased the stem-like properties in GSCs.ConclusionsCollectively, our data highlights H4K5acK8ac’s utility for identifying genes regulating cell-type specificity.

P02.08.B ROLE OF THE PROTEIN NETWORK OF THE PARACASPASE MALT1 IN SELF-RENEWAL PROPERTIES OF GLIOBLASTOMA CELLS

Abstract BACKGROUND Glioblastoma is the most common and most aggressive brain tumor in adults. Despite standard treatment combining surgery, radiotherapy and chemotherapy, relapse is inevitable, making this cancer a major public health issue. Within this tumor reside cells with a stem-like character, called GSCs for Glioblastoma Stem-like Cells, which maintain, initiate, and propagate the tumor, making them a potential target to hinder glioblastoma. These cells constitute a reservoir of heterogeneous cells capable of differentiating into neural lineage cell types. In this context, lysosomal homeostasis plays a key role in the maintenance of the GSC reservoir. Furthermore, previous studies demonstrated that the paracaspase MALT1 is a major regulator of lysosomal homeostasis: MALT1 is required for GSC viability as well as for the maintenance of stemness by modulating the abundance of lysosomal proteins. This current study addresses the issue of the balance between self-renewal and viability of GSCs. In addition, this project aims to identify and characterize an actor of self-renewal mechanisms that does not impact the survival-death balance of primary cells from patients. MATERIAL AND METHODS An initial analysis combining DepMap and String Network databases allowed us to select potential relays of the MALT1 activity in GSCs. A functional screening was then set up and performed in GSCs derived from patients to identify among 20 candidates those regulating cell expansion without impacting viability. RESULTS The strategy of functional screening allowed the selection of 14 genes with a global viability effect, while only 2 genes were affecting self-renewal while sparing viability in vitro. We next pursued with one of the identified targets, from the IKK family. The involvement of this serine/threonine protein kinase was then confirmed in multiple patient cells and in CRISPR-mediated knock-out. Future experiments will decipher the involvement of this kinase in the self-renewal properties of GSCs. CONCLUSION This study highlights a putative candidate specifically operating on the self-renewal of glioblastoma cells, that does not affect the life-death balance. Future investigations will permit to characterize and understand the underlying mechanisms.

Stationary-to-migratory transition in glioblastoma stem-like cells driven by a fatty acid-binding protein 7-RXRα neurogenic pathway.

Glioblastoma (GBM) stem-like cells (GSCs) are crucial drivers of treatment resistance and tumor recurrence. While the concept of "migrating" cancer stem cells was proposed a decade ago, the roles and underlying mechanisms of the heterogeneous populations of GSCs remain poorly defined. Cell migration using GBM cell lines and patient-derived GSCs was examined using Transwell inserts and the scratch assay. Single-cell RNA sequencing data analysis were used to map GSC drivers to specific GBM cell populations. Xenografted mice were used to model the role of brain-type fatty acid-binding protein 7 (FABP7) in GBM infiltration and expansion. The mechanism by which FABP7 and its fatty acid ligands promote GSC migration was examined by gel shift and luciferase gene reporter assays. A subpopulation of FABP7-expressing migratory GSCs was identified, with FABP7 upregulating SOX2, a key modulator for GBM stemness and plasticity, and ZEB1, a prominent factor in GBM epithelial-mesenchymal transition and invasiveness. Our data indicate that GSC migration is driven by nuclear FABP7 through activation of RXRα, a nuclear receptor activated by polyunsaturated fatty acids (PUFAs). Infiltrative progression in GBM is driven by migratory GSCs through activation of a PUFA-FABP7-RXRα neurogenic pathway.

Advancing glioblastoma imaging: Exploring the potential of organic fluorophore-based red emissive carbon dots

Over time, the interest in developing stable photosensitizers (PS) which both absorb and emit light in the red region (650 and 950 nm) has gained noticeable interest. Recently, carbon dots (CDs) have become the material of focus to act as a PS due to their high extinction coefficient, low cytotoxicity, and both high photo and thermal stability. In this work, a Federal and Drug Association (FDA) approved Near Infra-Red (NIR) organic fluorophore used for photo-imaging, indocyanine green (ICG), has been explored as a precursor to develop water-soluble red emissive CDs which possess red emission at 697 nm. Furthermore, our material was found to yield favorable red-imaging capabilities of glioblastoma stem-like cells (GSCs) meanwhile boasting low toxicity. Additionally with post modifications, our CDs have been found to have selectivity towards tumors over healthy tissue as well as crossing the blood-brain barrier (BBB) in zebrafish models.