Glioma Stem-like Cells Research Articles

P12.06.A PERICYTE-LIKE CELLS EXPRESSING FIBROBLAST ACTIVATION PROTEIN ARE MAIN PRODUCERS OF FIBRILLAR EXTRACELLULAR MATRIX IN GLIOBLASTOMA

Abstract BACKGROUND Compared to the normal brain, glioblastomas (GBMs) contain higher amounts of extracellular matrix (ECM) composed of fibrillar proteins such as collagen I and fibronectin. These proteins are associated with glioma cell invasion and poor clinical outcome of GBM patients, but their origin remains unclear. The highest levels of fibrillar ECM can be found in perivascular niches, which are, among others, resided by fibroblast activation protein (FAP) expressing pericyte-like cells. The aim of this study was to determine the role of FAP+ pericyte-like cells in the production of fibrillar ECM proteins and their importance in glioma cell biology. MATERIAL AND METHODS FAP+ pericyte-like cells, glioma stem-like cells (GSCs) and primary endothelial cells were isolated from human GBMs. M2 macrophages were differentiated from monocytes isolated from healthy donors´ buffy coats. The expression of FAP, collagen I and fibronectin in GBM tissues and different cell types (FAP+ pericyte-like cells, glioma cell lines, GSCs, M2 macrophages) was analyzed by immunohistochemistry and ELISA. FAP+ pericyte-like cells and glioma cells were used to prepare extracellular matrices, which were analyzed using mass spectrometry. Solubilized ECM was used in a modified transwell migration assay. RESULTS Compared to the non-tumorous brain, high levels of collagen I and fibronectin were present in the vicinity of FAP+ pericyte-like cells in GBMs. The expression of collagen I and fibronectin was higher in GBMs with higher FAP expression. In vitro, FAP+ pericyte-like cells produced the largest amount of collagen I and fibronectin compared to other cell types and secreted these proteins extracellularly, resulting in production of 3D extracellular matrices rich in collagen I and fibronectin. Mass spectrometry revealed that the ECM produced by FAP+ pericyte-like cells strongly differs in composition compared to glioma cell ECM. The ECM produced by FAP+ pericyte-like cells enhanced migration of glioma stem-like cells. CONCLUSION FAP+ pericyte-like cells are the main producers of fibrillar ECM proteins such as collagen I and fibronectin in GBM tissue. By this means, their presence within the GBM microenvironment affect the migration of glioma stem-like cells. Funding: Supported by the National institute for cancer research (LX22NPO5102), Charles University Grant Agency (GAUK 1084120) and Centre for Tumour Ecology (CZ.02.1.01/0.0/0.0/16_019/0000785). EATRIS-CZ (LM2023053), Cooperatio Program, research area ,,Oncology and Haematology", and Ministry of Health of the Czech Republic (NV19 03 00501).

P15.03.B MOLECULARLY TARGETED PROTEASE-ACTIVATED PROBES FOR THE VISUALIZATION OF GLIOBLASTOMA: A COMPARISON WITH 5-ALA

Abstract BACKGROUND Surgery is the main treatment option for glioblastoma. However, due to its highly infiltrative growth, it is difficult to distinguish the tumor from normal brain tissue. The aim of this work was to evaluate the ability of recently developed molecularly targeted near-infrared (NIR) protease-activated probes to be specifically activated in brain tumor tissue and to compare the most promising candidate with the FDA approved 5-aminolevulinic acid (5-ALA). MATERIAL AND METHODS The cysteine cathepsin-cleavable probes 6QC-ICG and 6QC-Cy5 (single-substrate), the cysteine cathepsin and caspase 3-cleavable dual, AND-GATE substrate probes AG2-FNIR and AG2-Cy5 (double-substrate) were administered intravenously to mice with orthotopic tumors. Activation of the probes was further evaluated in glioma cell lines, patient-derived glioma stem-like cells, M2-polarized macrophages in vitro, and biopsy material from patients with glioblastoma ex vivo. Tumor to normal brain tissue fluorescence ratio (TNR) was quantified in tissue sections using preclinical and clinical visualization platforms. Fluorescence in tissue homogenates and cell suspensions was quantified by spectrofluorimetry. RESULTS 6QC-ICG was activated in glioma cells and macrophages in vitro and the resulting fluorophore accumulated intracellularly. In experimental glioblastomas, single- and double-substrate probes visualized tumor tissue with a minimum signal generated in healthy brain. TNR was highest for 6QC-ICG and AG2-FNIR. Nevertheless, the signal intensity was higher for 6QC-ICG. Using xenograft and syngeneic mouse models, we confirmed the ability of 6QC-ICG to specifically visualize glioma tissue based on image analysis and spectrofluorimetric measurement. Furthermore, we confirmed that 6QC-ICG was activated in human glioblastoma tissue using ex vivo biopsy material and could be visualized on a clinical visualization platform. Finally, a comparison with 5-ALA in animals co-administered with both compounds revealed a higher TNR for 6QC-ICG in experimental glioblastomas. CONCLUSION 6QC-ICG is activated by glioma cells and tumor-associated macrophages leading to a high contrast between tumor and non-tumorous brain tissue that is superior to that of the current standard 5-ALA. In addition to their well-defined mechanism of action, the protease-activated probes using NIR fluorophores such as ICG offer the advantage of low absorption and scattering of the NIR light and lower tissue autofluorescence. Our results thus suggest the potential of 6QC-ICG to become a targeted agent for the intraoperative detection of glioblastoma. Supported by National Institute for Cancer Research (LX22NPO5102), Centre for Tumour Ecology (CZ.02.1.01/0.0/0.0/16_019/0000785), EATRIS-CZ (LM2023053), Cooperatio Program-,,Oncology and Haematology", Ministry of Health of the Czech Republic (NV190300501).

Ex vivo drug sensitivity screening predicts response to temozolomide in glioblastoma patients and identifies candidate biomarkers

BackgroundPatient-derived glioma stem-like cells (GSCs) have become the gold-standard in neuro-oncological research; however, it remains to be established whether loss of in situ microenvironment affects the clinically-predictive value of this model. We implemented a GSC monolayer system to investigate in situ-in vitro molecular correspondence and the relationship between in vitro and patient response to temozolomide (TMZ).MethodsDNA/RNA-sequencing was performed on 56 glioblastoma tissues and 19 derived GSC cultures. Sensitivity to TMZ was screened across 66 GSC cultures. Viability readouts were related to clinical parameters of corresponding patients and whole-transcriptome data.ResultsTumour DNA and RNA sequences revealed strong similarity to corresponding GSCs despite loss of neuronal and immune interactions. In vitro TMZ screening yielded three response categories which significantly correlated with patient survival, therewith providing more specific prediction than the binary MGMT marker. Transcriptome analysis identified 121 genes related to TMZ sensitivity of which 21were validated in external datasets.ConclusionGSCs retain patient-unique hallmark gene expressions despite loss of their natural environment. Drug screening using GSCs predicted patient response to TMZ more specifically than MGMT status, while transcriptome analysis identified potential biomarkers for this response. GSC drug screening therefore provides a tool to improve drug development and precision medicine for glioblastoma.

SDPS-30 ANALYSIS OF THE BRAIN TUMOR MATRISOME REVEALS CANCER ASSOCIATED FIBROBLASTS PREDICT POOR IMMUNOTHERAPY RESPONSE IN GLIOBLASTOMA PATIENTS

Abstract Excessive deposition of extracellular matrix (ECM) is a hallmark of solid tumors; however, it remains poorly understood which cellular and molecular components contribute to the formation of ECM stroma in central nervous system (CNS) tumors. Here, we undertook a pan-CNS analysis of retrospective gene expression datasets to characterize inter- and intra-tumoral heterogeneity of ECM remodeling signatures. We found that glioblastoma can be divided into two ECM-based subtypes that are influenced by the presence of cells resembling cancer associated fibroblasts (CAFs) and are conserved across a number of CNS lesions. In glioblastoma, CAF-like cells are correlated with unfavorable response to immune checkpoint blockade and poor patient survival across a subset of CNS tumors. We show that CAF-like cells reside in the perivascular niche, activate chemoattractant signaling pathways to recruit tumor-associated macrophages, and promote glioma stem-like cell phenotype. Our analysis provides insights into mechanisms underlying immunotherapy resistance in glioblastoma and suggests that targeting CAF-like cells may provide an effective approach to improve patient survival in a subset of CNS tumors.

Long non-coding RNA RP5-821D11.7 promotes proliferation, migration, and epithelial-mesenchymal transition in glioma and glioma stem-like cells

Long noncoding RNA (lncRNA) has been recently revealed as a main regulatory molecule, implicating many cellular functions. Studies showed that lncRNA is abnormally expressed and involved in the progression and tumorigenesis of glioma. The present study identified a novel lncRNA associated with glioma, glioma stem-like cells (GSCs) and then revealed their potential functions. During the screening of lncRNAs, we found lncRNA RP5-821D11.7 (lncRNA-RP5) overexpress in GSCs compared to glioma cells. Lentivirus-mediated shRNA for lncRNA-RP5 was constructed and transfected into glioma cells. Transfected stable glioma cells were transplanted into nude mice and tumor growth was determined. Knockdown of lncRNA-RP5 significantly inhibits proliferation, migration and reduces epithelial-mesenchymal transition (EMT) by activating the Wnt/β-catenin pathway. Additionally, the results showed that lncRNA RP5 knockdown enhances cell apoptosis through endoplasmic reticulum stress. Therefore, this study may provide a better understanding and demonstrates that lncRNA-RP5 may be a potential therapeutic target in glioma.

DIPG-28. IDENTIFICATION AND TARGETING OF METABOLIC VULNERABILITIES IN COMBINATION WITH PDGFRA-INHIBITION IN DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline gliomas (DMG) are driven by self-renewing, stem-like glioma cells stalled in an oligodendrocyte precursor cell (OPC)-like state that are characterized by high expression of PDGFRA. This lineage overexpression of PDGFRA, together with commonly found genomic amplifications and mutations in PDGFRA, has been shown to cooperate with H3-K27M mutations in tumor development. Therefore, targeting PDGFRA has long been proposed as a potential treatment strategy for DMG. Our previous work revealed that Avapritinib, a small molecule inhibitor of PDGFRA, is highly effective in DMG cells in vitro and in vivo. Following these preclinical findings, nine DMG patients were treated with Avapritinib for the first time. While 50% of all treated patients experienced a strong clinical response from Avapritinib, tumor progression and treatment escape were eventually observed in most patients. Here, we investigated which mechanisms DMG cells use to escape Avapritinib treatment and explored how these resistance mechanisms can be therapeutically exploited. Transcriptional analysis of a DMG cell line previously identified as Avapritinib-resistant revealed an upregulation of genes associated with fatty acid metabolism and oxidative phosphorylation (OXPHOS) following treatment with Avapritinib. Functional assays confirmed elevated OXPHOS in Avapritinib-resistant cells with significant (i) increase in mitochondrial energy transduction, (ii) increase in palmitate- (a product of fatty acid metabolism) driven oxygen consumption rate and (iii) greater incorporation of palmitate-derived carbons into the tricarboxylic acid cycle. These metabolic changes could be genetically reproduced in the same cell line after CRISPR-Cas9-mediated knockout of PDGFRA. To determine which therapies could target the dependency of Avapritinib-resistant cells on OXPHOS and fatty acid metabolism, we performed a metabolic drug screening and identified two metabolic drugs to have synergistic cytotoxic effects with Avapritinib. Together, we revealed distinct metabolic changes in Avapritinib-resistant DMG cells and showed that targeting these metabolic vulnerabilities might further increase the clinical benefit of Avapritinib.

Oncolytic herpes simplex viruses for the treatment of glioma and targeting glioblastoma stem-like cells.

Glioblastoma (GBM) is one of the most lethal cancers, having a poor prognosis and a median survival of only about 15 months with standard treatment (surgery, radiation, and chemotherapy), which has not been significantly extended in decades. GBM demonstrates remarkable cellular heterogeneity, with glioblastoma stem-like cells (GSCs) at the apex. GSCs are a subpopulation of GBM cells that possess the ability to self-renew, differentiate, initiate tumor formation, and manipulate the tumor microenvironment (TME). GSCs are no longer considered a static population of cells with specific markers but are quite flexible phenotypically and in driving tumor heterogeneity and therapeutic resistance. In light of these features, they are a critical target for successful GBM therapy. Oncolytic viruses, in particular oncolytic herpes simplex viruses (oHSVs), have many attributes for therapy and are promising agents to target GSCs. oHSVs are genetically-engineered to selectively replicate in and kill cancer cells, including GSCs, but not normal cells. Moreover, oHSV can induce anti-tumor immune responses and synergize with other therapies, such as chemotherapy, DNA repair inhibitors, and immune checkpoint inhibitors, to potentiate treatment effects and reduce GSC populations that are partly responsible for chemo- and radio-resistance. Herein, we present an overview of GSCs, activity of different oHSVs, clinical trial results, and combination strategies to enhance efficacy, including therapeutic arming of oHSV. Throughout, the therapeutic focus will be on GSCs and studies specifically targeting these cells. Recent clinical trials and approval of oHSV G47Δ in Japan for patients with recurrent glioma demonstrate the efficacy and promise of oHSV therapy.

Insights into Gene Regulation under Temozolomide-Promoted Cellular Dormancy and Its Connection to Stemness in Human Glioblastoma.

The aggressive features of glioblastoma (GBM) are associated with dormancy. Our previous transcriptome analysis revealed that several genes were regulated during temozolomide (TMZ)-promoted dormancy in GBM. Focusing on genes involved in cancer progression, Chemokine (C-C motif) Receptor-Like (CCRL)1, Schlafen (SLFN)13, Sloan-Kettering Institute (SKI), Cdk5 and Abl Enzyme Substrate (Cables)1, and Dachsous Cadherin-Related (DCHS)1 were selected for further validation. All showed clear expression and individual regulatory patterns under TMZ-promoted dormancy in human GBM cell lines, patient-derived primary cultures, glioma stem-like cells (GSCs), and human GBM ex vivo samples. All genes exhibited complex co-staining patterns with different stemness markers and with each other, as examined by immunofluorescence staining and underscored by correlation analyses. Neurosphere formation assays revealed higher numbers of spheres during TMZ treatment, and gene set enrichment analysis of transcriptome data revealed significant regulation of several GO terms, including stemness-associated ones, indicating an association between stemness and dormancy with the involvement of SKI. Consistently, inhibition of SKI during TMZ treatment resulted in higher cytotoxicity, proliferation inhibition, and lower neurosphere formation capacity compared to TMZ alone. Overall, our study suggests the involvement of CCRL1, SLFN13, SKI, Cables1, and DCHS1 in TMZ-promoted dormancy and demonstrates their link to stemness, with SKI being particularly important.

Uncovering transcriptomic landscape alterations of CAN-2409 in in vitro and in vivo glioma models.

CAN-2409 is a locally delivered oncolytic therapy, which results in vaccination against the injected tumor. CAN-2409 consists of a non-replicating adenovirus armed with the Herpes virus thymidine kinase, which metabolizes ganciclovir into a phosphorylated nucleotide that is incorporated into the tumor cell's genome, thereby inflicting immunogenic cancer cell death. While CAN-2409's immunological impact has been well characterized, its effects on the tumor cells transcriptome remains unknown. We compared the transcriptomic landscape after treatment of glioblastoma models with CAN-2409 in vitro and in vivo to assess how the interplay with the tumor microenvironment influences CAN-2409-mediated transcriptome alterations. We performed RNA-Seq with CAN-2409 treated patient-derived glioma stem-like cells and tumors of C57/BL6 mice and compared KEGG pathway usage and differential gene expression focusing on immune cell and cytokine profiles. T-cell -killing assays were performed to assess candidate effectors. PCA analysis showed distinct clustering of control and CAN-2409 samples under both conditions. KEGG pathway analysis revealed significant enrichment for p53 signaling and cell cycle pathway, with similar dynamics for key regulators of both pathways in vitro and in vivo, including MYC, CCNB1, PLK1 and CDC20. Selected alterations (PLK1 and CCNB1) were validated at the protein level. Cytokine expression analysis revealed upregulation of pro-inflammatory IL12a under both conditions; immune cell gene profiling showed reduction of myeloid associated genes. T-cell-killing assays showed increased killing in the presence of IL-12. CAN-2409 significantly alters the transcriptome both in vitro and in vivo. Comparison of pathway enrichment revealed mutual and differential utilization of pathways under both conditions, suggesting a modulating influence on the cell cycle in tumor cells, and of the tumor microenvironment on the transcriptome in vivo. IL-12 synthesis likely depends on interactions with the tumor microenvironment, and it facilitates CAN-2409 cell killing. This dataset provides potential to understand resistance mechanisms and identify potential biomarkers for future studies.

The Antiepileptic Drug Oxcarbazepine Inhibits the Growth of Patient-Derived Isocitrate Dehydrogenase Mutant Glioma Stem-like Cells.

Patients diagnosed with isocitrate dehydrogenase mutant (IDHmut) gliomas suffer frequently from seizures. Although the clinical course is less aggressive than that of its IDH wildtype counterpart, recent discoveries have shown that epileptic activity can promote tumor proliferation. However, it is not known if antiepileptic drugs confer additional value by inhibiting tumor growth. In this study, the antineoplastic properties of 20 FDA-approved antiepileptic drugs (AEDs) were tested in six patient-derived IDHmut glioma stem-like cells (GSCs). Cell proliferation was assessed using the CellTiterGlo-3D assay. Two of the screened drugs (oxcarbazepine and perampanel) demonstrated an antiproliferative effect. A subsequent eight-point dose-response curve proved the dose-dependent growth inhibition for both drugs, but only oxcarbazepine reached an IC50 value below 100 µM in 5/6 GSCs (mean 44.7 µM; range 17.4-98.0 µM), approximating the possible cmax for oxcarbazepine in patient serums. Furthermore, the treated GSC spheroids were 82% smaller (mean volume 1.6 nL vs. 8.7 nL; p = 0.01 (live/deadTM fluorescence staining)), and the apoptotic events increased by more than 50% (caspase-3/7 activity; p = 0.006). Taken together, this drug screen of a large series of antiepileptic drugs identified oxcarbazepine as a potent proapoptotic drug in IDHmut GSCs, which combines antiepileptic and antineoplastic properties to treat this seizure-prone patient population.

Sema3C signaling is an alternative activator of the canonical WNT pathway in glioblastoma

The Wnt pathway is frequently dysregulated in many cancers, underscoring it as a therapeutic target. Wnt inhibitors have uniformly failed in clinical trials. Here, we report a mechanism of WNT pathway activation through the Semaphorin 3 C neurodevelopmental program in glioma stem-like cells. Sema3C directs β-catenin nuclear accumulation in a Rac1-dependent process, leading to transactivation of Wnt target genes. Sema3C-driven Wnt signaling occurred despite suppression of Wnt ligand secretion, suggesting that Sema3C drives canonical Wnt signaling independent of Wnt ligand binding. In a mouse model of glioblastoma, combined depletion of Sema3C and β-catenin partner TCF1 extended animal survival more than single target inhibition alone. In human glioblastoma, Sema3C expression and Wnt pathway activation were highly concordant. Since Sema3C is frequently overexpressed in glioblastoma, Sema3C signaling may be a significant mechanism of resistance to upstream Wnt pathway inhibitors. Dual targeting of Sema3C and Wnt pathways may achieve clinically significant Wnt pathway inhibition.

Upregulation of the ZNF148/PTX3 axis promotes malignant transformation of dendritic cells in glioma stem-like cells microenvironment.

The recent development of dendritic cell (DC)-based immunotherapy has resulted in advances in glioblastoma multiforme (GBM) treatment. However, the cell fate of DCs in the GBM microenvironment, especially in microenvironments in which glioma stem cell (GSCs)-mediated remodeling has resulted in highly immunosuppressive conditions, has not yet been fully investigated. Observed the interaction between GSCs and primary cultured DCs in a dual-color tracing model, monoclonal and continuously passaged highly proliferative DCs, and named transformed DCs (t-DCs). The expression of DC-specific surface markers was analyzed using RT-PCR, chromosome karyotype, and flow cytometry. The expression of long pentraxin 3 (PTX3) and its transcription factor zinc finger protein 148 (ZNF148) in t-DCs was detected using qRT-PCR and western blot. CCK8 and transwell assays were conducted to assess the effect of ZNF148 and PTX3 on the proliferation, migration, and invasion of t-DCs. Bioinformatics analysis, dual-luciferase reporter assay, and chromatin immunoprecipitation (ChIP)-qPCR assay were used to explore the relation between ZNF148 and PTX3. Transformed DCs (t-DCs) still expressed DC-specific surface markers, namely, CD80 and CD11c, and immune-related costimulatory molecules, namely, CD80, CD86, CD40, and ICAM-1. However, the expression levels of these molecules in t-DCs decreased moderately compared to those in naive DCs. Stable overexpression of PTX3 further promoted the proliferation and migration of t-DCs in vitro, decreased the expression of costimulatory molecules, and increased the tumorigenicity of t-DCs in vivo. The transcription factor zinc finger protein 148 (ZNF148) was directly bound to the PTX3 promoter region and enhanced PTX3 expression. Downregulation of ZNF148 significantly decreased PTX3 expression and reduced the proliferation and migration of t-DCs. Overexpression of ZNF148 significantly increased PTX3 expression and promoted the proliferation and migration of t-DCs, achieving the same biological effects as PTX3 overexpression in t-DCs. Simultaneously, the downregulation of ZNF148 partially reversed the effect of PTX3 overexpression in t-DCs. The ZNF148/PTX3 axis played an important role in regulating the malignant transformation of DCs after cross-talk with GSCs, and this axis may serve as a new target for sensitizing GBM to DC-based immunotherapy.

OHSV-P10 reduces glioma stem cell enrichment after oncolytic HSV therapy

Longstanding evidence implicate glioma stem-like cells as the main drivers contributing toward glioblastoma (GBM) therapy resistance and tumor recurrence. Although oncolytic herpes simplex virus (oHSV) viral therapy is a promising biological therapy recently approved for melanoma (in the United States and Europe) and GBM (in Japan); however, the impact of this therapy on GBM stem-like cells (GSCs) is understudied. Here we show that post-oHSV virotherapy activated AKT signaling results in an enrichment of GSC signatures in glioma, which mimics the enrichment in GSC observed after radiation treatment.We also uncovered that a second-generation oncolyticvirusarmed with PTEN-L (oHSV-P10) decreases this by moderating IL6/JAK/STAT3 signaling. This ability was retained in the presence of radiation treatment and oHSV-P10-sensitized intracranial GBM to radiotherapy. Collectively, our findings uncover potential mechanisms to overcome GSC-mediated radiation resistance via oHSV-P10.

Oncogenic long noncoding RNA LINC02283 enhances PDGF receptor A-mediated signaling and drives glioblastoma tumorigenesis.

Long noncoding RNAs (lncRNAs) regulate the etiology of complex diseases and cancers, including glioblastoma (GBM). However, lncRNA-based therapies are limited because the mechanisms of action of many lncRNAs with their binding partners are not completely understood. We used transcriptomic and genomic data to analyze correlations between LINC02283 and PDGFRA (platelet-derived growth factor receptor A). The biological functions of the novel lncRNA were assessed in vivo using patient-derived glioma stem-like cells (GSCs), and orthotopic GBM xenografts. Immunoblotting, qRT-PCR, RNA pull down, crosslinked RNA immunoprecipitation, fluorescence in situ hybridization, and antisense oligo-mediated knockdown were performed to explore the regulation of LINC02283 on PDGFRA signaling. Expression of LINC02283 in clinical samples was assessed using pathologically diagnosed GBM patient samples. We identified a novel oncogenic lncRNA, LINC02283, that is highly expressed in the PDGFRA mutation-driven cohort of glioma patients and associated with worse prognosis. LINC02283 gene co-amplifies with the PDGFRA locus and shows high correlation with PDGFRA expression. Deprivation of LINC02283 in GSCs with PDGFRA amplification mutation, attenuated tumorigenicity and enhanced survival in orthotopic GBM xenograft models, while overexpression of LINC02283 in GSCs with wild-type PDGFRA, enhances PDGFRA signaling, and decreases survival. Further, LINC02283 interacts with PDGFRA to enhance its signaling and that of its downstream targets AKT and ERK, thus promoting oncogenesis in GBM. Our results provide strong evidence of LINC02283 as a regulator of PDGFRA oncogenic activity and GBM malignancy and support the potential of lncRNAs as possible therapeutic targets.

Xihuang Pill-destabilized CD133/EGFR/Akt/mTOR cascade reduces stemness enrichment of glioblastoma via the down-regulation of SOX2

BackgroundOur previous study found that XHP could induce GBM cells to undergo apoptosis. A lot of evidence suggests that glioma stem-like cells (GSCs) are key factors that contribute to disease progression and poor prognosis of glioblastoma multiforme (GBM). Traditional Chinese medicine has been applied in clinical practice as a complementary and alternative therapy for glioma. PurposeTo evaluate the effect and the potential molecular mechanism of Xihuang pill (XHP) on GSCs. MethodsUPLC-QTOF-MS analysis was used for constituent analysis of XHP. Using network pharmacology and bioinformatics methods, a molecular network targeting GSCs by the active ingredients in XHP was constructed. Cell viability, self-renewal ability, apoptosis, and GSC markers were detected by CCK-8 assay, tumor sphere formation assay and flow cytometry, respectively. The interrelationship between GSC markers (CD133 and SOX2) and key proteins of the EGFR/Akt/mTOR signaling pathway was evaluated using GEPIA and verified by western blot. A GBM cell line stably overexpressing Akt was constructed using lentivirus to evaluate the role of Akt signaling in the regulation of glioma stemness. The effect of XHP on glioma growth was analyzed by a subcutaneously transplanted glioma cell model in nude mice, hematoxylin-eosin staining was used to examine pathological changes, TUNEL staining was used to detect apoptosis in tumor tissues, and the expression of GSC markers in tumor tissues was identified by western blot and immunofluorescence. ResultsBioinformatics analysis showed that 55 matched targets were related to XHP targets and glioma stem cell targets. In addition to causing apoptosis, XHP could diminish the number of GBM 3D spheroids, the proportion of CD133-positive cells and the expression level of GSC markers (CD133 and SOX2) in vitro. Furthermore, XHP could attenuate the expression of CD133, EGFR, p-Akt, p-mTOR and SOX2 in GBM spheres. Overexpression of Akt significantly increased the expression level of SOX2, which was prohibited in the presence of XHP. XHP reduced GSC markers including CD133 and SOX2, and impeded the development of glioma growth in xenograft mouse models in vivo. ConclusionWe demonstrate for the first time that XHP down-regulates stemness, restrains self-renewal and induces apoptosis in GSCs and impedes glioma growth by down-regulating SOX2 through destabilizing the CD133/EGFR/Akt/mTOR cascade.

Pharmacogenomic profiling reveals molecular features of chemotherapy resistance in IDH wild-type primary glioblastoma

BackgroundAlthough temozolomide (TMZ) has been used as a standard adjuvant chemotherapeutic agent for primary glioblastoma (GBM), treating isocitrate dehydrogenase wild-type (IDH-wt) cases remains challenging due to intrinsic and acquired drug resistance. Therefore, elucidation of the molecular mechanisms of TMZ resistance is critical for its precision application.MethodsWe stratified 69 primary IDH-wt GBM patients into TMZ-resistant (n = 29) and sensitive (n = 40) groups, using TMZ screening of the corresponding patient-derived glioma stem-like cells (GSCs). Genomic and transcriptomic features were then examined to identify TMZ-associated molecular alterations. Subsequently, we developed a machine learning (ML) model to predict TMZ response from combined signatures. Moreover, TMZ response in multisector samples (52 tumor sectors from 18 cases) was evaluated to validate findings and investigate the impact of intra-tumoral heterogeneity on TMZ efficacy.ResultsIn vitro TMZ sensitivity of patient-derived GSCs classified patients into groups with different survival outcomes (P = 1.12e−4 for progression-free survival (PFS) and 3.63e−4 for overall survival (OS)). Moreover, we found that elevated gene expression of EGR4, PAPPA, LRRC3, and ANXA3 was associated to intrinsic TMZ resistance. In addition, other features such as 5-aminolevulinic acid negative, mesenchymal/proneural expression subtypes, and hypermutation phenomena were prone to promote TMZ resistance. In contrast, concurrent copy-number-alteration in PTEN, EGFR, and CDKN2A/B was more frequent in TMZ-sensitive samples (Fisher’s exact P = 0.0102), subsequently consolidated by multi-sector sequencing analyses. Integrating all features, we trained a ML tool to segregate TMZ-resistant and sensitive groups. Notably, our method segregated IDH-wt GBM patients from The Cancer Genome Atlas (TCGA) into two groups with divergent survival outcomes (P = 4.58e−4 for PFS and 3.66e−4 for OS). Furthermore, we showed a highly heterogeneous TMZ-response pattern within each GBM patient using in vitro TMZ screening and genomic characterization of multisector GSCs. Lastly, the prediction model that evaluates the TMZ efficacy for primary IDH-wt GBMs was developed into a webserver for public usage (http://www.wang-lab-hkust.com:3838/TMZEP).ConclusionsWe identified molecular characteristics associated to TMZ sensitivity, and illustrate the potential clinical value of a ML model trained from pharmacogenomic profiling of patient-derived GSC against IDH-wt GBMs.

Feedforward loop between IMP1 and YAP/TAZ promotes tumorigenesis and malignant progression in glioblastoma.

YAP/TAZ have been identified as master regulators in malignant phenotypes of glioblastoma (GBM); however, YAP/TAZ transcriptional disruptor in GBM treatment remains ineffective. Whether post-transcriptional dysregulation of YAP/TAZ improves GBM outcome is currently unknown. Here, we report that insulin-like growth factor 2 (IGF2) mRNA-binding protein 1 (IGF2BP1 or IMP1) is upregulated in mesenchymal GBM compared with proneural GBM and correlates with worse patient outcome. Overexpression of IMP1 in proneural glioma stem-like cells (GSCs) promotes while IMP1 knockdown in mesenchymal GSCs attenuates tumorigenesis and mesenchymal signatures. IMP1 binds to and stabilizes m6A-YAP mRNA, leading to activation of YAP/TAZ signaling, depending on its m6A recognition and binding domain. On the other hand, TAZ functions as enhancer for IMP1 expression. Collectively, our data reveal a feedforward loop between IMP1 and YAP/TAZ maintaining GBM/GSC tumorigenesis and malignant progression and a promising molecular target in GBM.

Selective and brain-penetrant lanosterol synthase inhibitors target glioma stem-like cells by inducing 24(S),25-epoxycholesterol production

Glioblastoma (GBM) is an aggressive adult brain cancer with few treatment options due in part to the challenges of identifying brain-penetrant drugs. Here, we investigated the mechanism of MM0299, a tetracyclic dicarboximide with anti-glioblastoma activity. MM0299 inhibits lanosterol synthase (LSS) and diverts sterol flux away from cholesterol into a "shunt" pathway that culminates in 24(S),25-epoxycholesterol (EPC). EPC synthesis following MM0299 treatment is both necessary and sufficient to block the growth of mouse and human glioma stem-like cells by depleting cellular cholesterol. MM0299 exhibits superior selectivity for LSS over other sterol biosynthetic enzymes. Critical for its application in the brain, we report an MM0299 derivative that is orally bioavailable, brain-penetrant, and induces the production of EPC in orthotopic GBM tumors but not normal mouse brain. These studies have implications for the development of an LSS inhibitor to treat GBM or other neurologic indications.

Neuronal differentiation drives the antitumor activity of mitogen-activated protein kinase kinase (MEK) inhibition in glioblastoma.

Epidermal growth factor receptor (EGFR) amplification is found in nearly 40%-50% of glioblastoma cases. Several EGFR inhibitors have been tested in glioblastoma but have failed to demonstrate long-term therapeutic benefit, presumably because of acquired resistance. Targeting EGFR downstream signaling with mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) inhibitors would be a more effective approach to glioblastoma treatment. We tested the therapeutic potential of MEK1/2 inhibitors in glioblastoma using 3D cultures of glioma stem-like cells (GSCs) and mouse models of glioblastoma. Several MEK inhibitors were screened in an unbiased high-throughput platform using GSCs. Cell death was evaluated using flow cytometry and Western blotting (WB) analysis. RNA-seq, real-time quantitative polymerase chain reaction, immunofluorescence, and WB analysis were used to identify and validate neuronal differentiation. Unbiased screening of multiple MEK inhibitors in GSCs showed antiproliferative and apoptotic cell death in sensitive cell lines. An RNA-seq analysis of cells treated with trametinib, a potent MEK inhibitor, revealed upregulation of neurogenesis and neuronal differentiation genes, such as achaete-scute homolog 1 (ASCL1), delta-like 3 (DLL3), and neurogenic differentiation 4 (NeuroD4). We validated the neuronal differentiation phenotypes in vitro and in vivo using selected differentiation markers (β-III-tubulin, ASCL1, DLL3, and NeuroD4). Oral treatment with trametinib in an orthotopic GSC xenograft model significantly improved animal survival, with 25%-30% of mice being long-term survivors. Our findings demonstrated that MEK1/2 inhibition promotes neuronal differentiation in glioblastoma, a potential additional mechanism of action of MEK1/2 inhibitors. Thus, MEK inhibitors could be efficacious in glioblastoma patients with activated EGFR/MAPK signaling.

CHRDL1 Regulates Stemness in Glioma Stem-like Cells

Glioblastoma (GBM) still presents as one of the most aggressive tumours in the brain, which despite enormous research efforts, remains incurable today. As many theories evolve around the persistent recurrence of this malignancy, the assumption of a small population of cells with a stem-like phenotype remains a key driver of its infiltrative nature. In this article, we research Chordin-like 1 (CHRDL1), a secreted protein, as a potential key regulator of the glioma stem-like cell (GSC) phenotype. It has been shown that CHRDL1 antagonizes the function of bone morphogenic protein 4 (BMP4), which induces GSC differentiation and, hence, reduces tumorigenicity. We, therefore, employed two previously described GSCs spheroid cultures and depleted them of CHRDL1 using the stable transduction of a CHRDL1-targeting shRNA. We show with in vitro cell-based assays (MTT, limiting dilution, and sphere formation assays), Western blots, irradiation procedures, and quantitative real-time PCR that the depletion of the secreted BMP4 antagonist CHRDL1 prominently decreases functional and molecular stemness traits resulting in enhanced radiation sensitivity. As a result, we postulate CHRDL1 as an enforcer of stemness in GSCs and find additional evidence that high CHRDL1 expression might also serve as a marker protein to determine BMP4 susceptibility.