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Glioblastoma is characterized by a pronounced resistance to therapy with dismal prognosis. Transcriptomics classify glioblastoma into proneural (PN), mesenchymal (MES) and classical (CL) subtypes that show differential resistance to targeted therapies. The aim of this study was to provide a viable approach for identifying combination therapies in glioblastoma subtypes. Proteomics and phosphoproteomics were performed on dasatinib inhibited glioblastoma stem cells (GSCs) and complemented by an shRNA loss-of-function screen to identify genes whose knockdown sensitizes GSCs to dasatinib. Proteomics and screen data were computationally integrated with transcriptomic data using the SamNet 2.0 algorithm for network flow learning to reveal potential combination therapies in PN GSCs. In vitro viability assays and tumor spheroid models were used to verify the synergy of identified therapy. Further in vitro and TCGA RNA-Seq data analyses were utilized to provide a mechanistic explanation of these effects. Integration of data revealed the cell cycle protein WEE1 as a potential combination therapy target for PN GSCs. Validation experiments showed a robust synergistic effect through combination of dasatinib and the WEE1 inhibitor, MK-1775, in PN GSCs. Combined inhibition using dasatinib and MK-1775 propagated DNA damage in PN GCSs, with GCSs showing a differential subtype-driven pattern of expression of cell cycle genes in TCGA RNA-Seq data. The integration of proteomics, loss-of-function screens and transcriptomics confirmed WEE1 as a target for combination with dasatinib against PN GSCs. Utilizing this integrative approach could be of interest for studying resistance mechanisms and revealing combination therapy targets in further tumor entities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Glioblastoma (GBM), an aggressive brain malignancy with a cellular hierarchy dominated by GBM stem cells (GSCs), evades antitumor immunity through mechanisms that remain incompletely understood. Like most cancers, GBMs undergo metabolic reprogramming toward glycolysis to generate lactate. Here, we show that lactate production by patient-derived GSCs and microglia/macrophages induces tumor cell epigenetic reprogramming through histone lactylation, an activating modification that leads to immunosuppressive transcriptional programs and suppression of phagocytosis via transcriptional upregulation of CD47, a "don't eat me" signal, in GBM cells. Leveraging these findings, pharmacologic targeting of lactate production augments efficacy of anti-CD47 therapy. Mechanistically, lactylated histone interacts with the heterochromatin component chromobox protein homolog 3 (CBX3). Although CBX3 does not possess direct lactyltransferase activity, CBX3 binds histone acetyltransferase (HAT) EP300 to induce increased EP300 substrate specificity toward lactyl-CoA and a transcriptional shift toward an immunosuppressive cytokine profile. Targeting CBX3 inhibits tumor growth by both tumor cell-intrinsic mechanisms and increased tumor cell phagocytosis. Collectively, these results suggest that lactate mediates metabolism-induced epigenetic reprogramming in GBM that contributes to CD47-dependent immune evasion, which can be leveraged to augment efficacy of immuno-oncology therapies.

Aims: The incidence of recurrent gliomas is high, exerting low survival rates and poor prognoses. Transcription factor AP-2α has been reported to regulate the progression of primary glioblastoma (GBM). However, the function of AP-2α in recurrent gliomas is largely unclear., Methods: The expression of AP-2α and O6-methylguanine DNA-methyltransferase (MGMT) was detected in recurrent glioma tissues and cell lines by Western blots, the regulation mechanisms between AP-2α/MGMT promoter and RA/AP-2α promoter were studied by luciferase reporter assays, EMSA, and chIP assays. The effects of AP-2α and TMZ/RA treatment on cell viability in vitro and in vivo were investigated by MTT assays, γH 2 AX staining, comet assays and intracranial injection., Key Findings: AP-2α expression negatively correlates with the expression of MGMT in glioma samples. AP-2α could directly bind with the promoter of the MGMT gene, suppresses transcriptional levels of MGMT and downregulate MGMT expression in TMZ-resistant U87MG-R and T98G cells, but TMZ treatment decreases AP-2α expression and increases MGMT expression. The extended TMZ treatment and increased TMZ concentrations reversed these effects. Moreover, AP-2α overexpression combines with TMZ to decrease cell viability, concurrently with improved DNA damage marker γH 2 AX. Furthermore, retinoic acid (RA) activates RAR/RXR heterodimers, which bind to RA-responsive elements (RAREs) of the AP-2α promoter, and activates AP-2α expression in recurrent glioma cells. Finally, in intracranial relapsed glioma mouse model, both RA and TMZ could retard tumor development and prolong the mouse survival., Significance: AP-2α activation by gene overexpression or RA treatment reveals the suppressive effects on glioma relapse, providing a novel therapeutic strategy against malignant refractory gliomas., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Tumor-associated microglia and macrophages (TAM) make up the largest immune cell population in the glioblastoma (GBM) tumor microenvironment. Given the heterogeneity and plasticity of TAMs in the GBM tumor microenvironment, understanding the context-dependent cancer cell-TAM symbiotic interaction is crucial for understanding GBM biology and developing effective therapies. In a recent issue of Cell, Kloosterman and colleagues identified a subpopulation of glycoprotein nonmetastatic melanoma protein Bhigh lipid-laden microglia and macrophages (LLM) in GBM. Mesenchymal-like GBM cells help generate the LLM phenotype. Reciprocally, LLMs are epigenetically rewired to recycle myelin and transfer the lipid from myelin to cancer cells, fueling mesenchymal-like GBM progression in a liver X receptor/ABCA1-dependent manner. Together, leveraging LLMs opens new therapeutic possibilities for rewiring the metabolism-mediated tumor-TAM interaction during GBM progression., (©2024 American Association for Cancer Research.)

Galectin-9 (Gal-9) is a multifaceted regulator of various pathophysiologic processes that exerts positive or negative effects in a context-dependent manner. In this study, we elucidated the distinctive functional properties of Gal-9 on myeloid cells within the brain tumor microenvironment (TME). Gal-9-expressing cells were abundant at the hypoxic tumor edge in the tumor-bearing ipsilateral hemisphere compared with the contralateral hemisphere in an intracranial mouse brain tumor model. Gal-9 was highly expressed in microglia and macrophages in tumor-infiltrating cells. In primary glia, both the expression and secretion of Gal-9 were influenced by tumors. Analysis of a human glioblastoma bulk RNA sequencing dataset and a single-cell RNA sequencing dataset from a murine glioma model revealed a correlation between Gal-9 expression and glial cell activation. Notably, the Gal-9high microglial subset was functionally distinct from the Gal-9neg/low subset in the brain TME. Gal-9high microglia exhibited properties of inflammatory activation and higher rates of cell death, whereas Gal-9neg/low microglia displayed a superior phagocytic ability against brain tumor cells. Blockade of Gal-9 suppressed tumor growth and altered the activity of glial and T cells in a mouse glioma model. Additionally, glial Gal-9 expression was regulated by hypoxia-inducible factor-2α in the hypoxic brain TME. Myeloid-specific hypoxia-inducible factor-2α deficiency led to attenuated tumor progression. Together, these findings reveal that Gal-9 on myeloid cells is an immunoregulator and putative therapeutic target in brain tumors. Significance: Galectin-9 serves as an immune checkpoint molecule that modulates the functional properties of microglia in the brain tumor microenvironment and could potentially be targeted to effectively treat brain tumors., (©2024 American Association for Cancer Research.)

Prognosis of glioblastoma patients is still poor despite multimodal therapy. The highly brain-infiltrating growth in concert with a pronounced therapy resistance particularly of mesenchymal glioblastoma stem-like cells (GSCs) has been proposed to contribute to therapy failure. Recently, we have shown that a mesenchymal-to-proneural mRNA signature of patient derived GSC-enriched (pGSC) cultures associates with in vitro radioresistance and gel invasion. Importantly, this pGSC mRNA signature is prognostic for patients' tumor recurrence pattern and overall survival. Two mesenchymal markers of the mRNA signature encode for IK Ca and BK Ca Ca 2+ -activated K + channels. Therefore, we analyzed here the effect of IK Ca - and BK Ca -targeting concomitant to (fractionated) irradiation on radioresistance and glioblastoma spreading in pGSC cultures and in pGSC-derived orthotopic xenograft glioma mouse models. To this end, in vitro gel invasion, clonogenic survival, in vitro and in vivo residual DNA double strand breaks (DSBs), tumor growth, and brain invasion were assessed in the dependence on tumor irradiation and K + channel targeting. As a result, the IK Ca - and BK Ca -blocker TRAM-34 and paxilline, respectively, increased number of residual DSBs and (numerically) decreased clonogenic survival in some but not in all IK Ca - and BK Ca -expressing pGSC cultures, respectively. In addition, BK Ca - but not IK Ca -blockade slowed-down gel invasion in vitro. Moreover, systemic administration of TRAM-34 or paxilline concomitant to fractionated tumor irradiation increased in the xenograft model(s) residual number of DSBs and attenuated glioblastoma brain invasion and (numerically) tumor growth. We conclude, that K Ca -blockade concomitant to fractionated radiotherapy might be a promising new strategy in glioblastoma therapy., (© 2024 The Author(s). International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Glioblastoma is an aggressive brain cancer with a poor prognosis. The O6-methylguanine-DNA methyltransferase (MGMT) gene methylation status is crucial for treatment stratification, yet economic constraints often limit access. This study aims to develop an artificial intelligence (AI) framework for predicting MGMT methylation. Diagnostic magnetic resonance (MR) images in public repositories were used for training. The algorithm created was validated in data from a single institution. All images were segmented according to widely used guidelines for radiotherapy planning and combined with clinical evaluations from neuroradiology experts. Radiomic features and clinical impressions were extracted, tabulated, and used for modeling. Feature selection methods were used to identify relevant phenotypes. A total of 100 patients were used for training and 46 for validation. A total of 343 features were extracted. Eight feature selection methods produced seven independent predictive frameworks. The top-performing ML model was a model post-Least Absolute Shrinkage and Selection Operator (LASSO) feature selection reaching accuracy (ACC) of 0.82, an area under the curve (AUC) of 0.81, a recall of 0.75, and a precision of 0.75. This study demonstrates that integrating clinical and radiotherapy-derived AI-driven phenotypes can predict MGMT methylation. The framework addresses constraints that limit molecular diagnosis access., Competing Interests: Declarations Competing interests The authors declare no competing interests. Institutional review board (IRB) approval number This study was conducted in accordance with the principles of the Declaration of Helsinki and was submitted and approved by the Brazilian National Health Council through the Brazil platform (identifier 63591922.6.0000.5461). As the study involved a retrospective analysis of hospital database records, a waiver for obtaining Informed Consent was requested and approved by the Research Ethics Committee of the Sírio-Libanês Hospital (ref. 5461), which also granted approval for both the research and the waiver of the informed consent form., (© 2024. The Author(s).)

Background: The interaction between mesenchymal stem cells (MSC) and Glioblastoma (GBM), although potentially of the highest importance, is ill-understood. This is due, in part, to the lack of relevant experimental models. The similarity between the in vitro situations and the in vivo situation can be improved by 3D co-culture as it reproduces key cell-cell interactions between the tumor microenvironment (TME) and cancer cells., Methods: MSC Can acquired characteristics of cancer associated fibroblasts (CAF) by being cultured with conditioned medium from GBM cultures and thus are called MSC CAF . We co Cultured MSC CAF with patient derived GBM in a scaffold 3D bioprinted model. We studied the response to current GBM therapy (e.g. Temozolomide + /Radiation) on the co cultures by bulk transcriptomic (RNA Seq) and epigenetic (ATAC Seq) analyses RESULTS: The transcriptomic modifications induced by standard GBM treatment in bioprinted scaffolds of mono- or co-cultures of GBM ± MSC can be analyzed. We found that mitochondrial encoded OXPHOS genes are overexpressed under these conditions and are modified by both co-culture and treatment (chemotherapy ± radiation). We have identified two new markers of MSC/GBM interactions, one epigenetically regulated (i.e. TREM-1) associated with an increased overall survival in GBM patients and another implicated in post-transcriptional regulation (i.e. the long non-coding RNA, miR3681HG), which is associated with a reduced overall survival in GBM patients., Competing Interests: Declarations Ethics approval and consent to participate The bio-collection used for this analysis is the Glioblastoma collection belonging to the approved project, “pediatric research program of the University Hospital of Nantes, France (Ref. MESR DC-2014–2206)”, having obtained an institutional approval from committee patients protection board, “CPP Ouest IV, approval number “Dossier 06/15”, date of approval 08/04/2015. The patients or their guardians/legally authorized representatives/next of kin provided written informed consent for participation in the use of samples. After informed consent, tumor samples classified as GBM, based on the World Health Organization criteria, were obtained from patients undergoing surgical intervention at the Department of Neurosurgery at “Centre Hospitalier Universitaire de Nantes” and the “Tumorothèque IRCNA”. Declaration of use of AI The authors declare that artificial intelligence is not used in this study. Competing Interests The authors declare that they have no competing of interest., (© 2024. The Author(s).)

Purpose: Extent of resection, MGMT promoter methylation status, age, functional level, and residual tumor volume are established prognostic factors for overall survival in glioblastoma patients. Preoperative tumor volume has also been investigated, but the results have been inconclusive. We hypothesized that the surface area and the shape were more representative of the tumor's infiltrative capacities, and thus, the purpose of this study was to assess the prognostic value of tumor size and shape in patients with glioblastoma., Methods: In total, 271 patients with primary, unifocal glioblastoma were included from two centers in Norway and Sweden, respectively. All tumors were automatically segmented on preoperative MRI scans and manually validated. Tumor volume was used as a measurement of size, whereas sphericity index and area-to-volume ratio defined the shape complexity of the tumor. Contact surface area of the tumor was considered a measurement of both size and shape. Multivariable Cox proportional hazards models were used to assess the prognostic value of the respective tumor measurements, with previously established prognostic factors as covariates., Results: There were no associations between preoperative tumor volume and overall survival. Contact surface area (HR = 1.013, p = 0.002) and sphericity index (HR = 2.223, p = 0.001) were both significant independent prognostic factors for survival in the multivariable Cox models. Contact surface area was also associated with MGMT promoter methylation (p = 0.039) and extent of resection (p = 0.017)., Conclusion: Tumor shape complexity appears to be an independent prognostic factor in glioblastoma patients and may also be associated with MGMT promoter methylation status and extent of surgical resection., Competing Interests: Declarations Ethical approval The project was approved by the Regional Committee for Medical and Health Research Ethics (REK) in Norway (REK-reference 2019/510) and the Swedish Ethical Review Authority (Dnr: 702 − 18). The study was conducted in accordance with the Declaration of Helsinki, and all patients provided either written informed consent (Norway) or passive consent (Sweden). Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Glioblastoma (GBM) is the most malignant brain cancer and one of the leading causes of cancer-related death globally. So, identifying potential molecular signatures and associated drug molecules are crucial for diagnosis and therapies of GBM. This study suggested GBM-causing ten key genes (ASPM, CCNB2, CDK1, AURKA, TOP2A, CHEK1, CDCA8, SMC4, MCM10, and RAD51AP1) from nine transcriptomics datasets by combining supervised and unsupervised learning results. Differential expression patterns of key genes (KGs) between GBM and control samples were verified by different independent databases. Gene regulatory network (GRN) detected some important transcriptional and post-transcriptional regulators for KGs. The KGs-set enrichment analysis unveiled some crucial GBM-causing molecular functions, biological processes, cellular components, and pathways. The DNA methylation analysis detected some hypo-methylated CpG sites that might stimulate the GBM development. From the immune infiltration analysis, we found that almost all KGs are associated with different immune cell infiltration levels. Finally, we recommended KGs-guided four repurposable drug molecules (Fluoxetine, Vatalanib, TGX221 and RO3306) against GBM through molecular docking, drug likeness, ADMET analyses and molecular dynamics simulation studies. Thus, the discoveries of this study could serve as valuable resources for wet-lab experiments in order to take a proper treatment plan against GBM., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Adult glioblastoma (GBM) is a highly aggressive primary brain tumor, accounting for nearly half of all malignant brain tumors, with a median survival rate of only 8 months. Treatment for GBM is largely ineffective due to the highly invasive nature and complex tumor composition of this malignancy. MicroRNAs (miRNA) are short, non-coding RNAs that regulate gene expression by binding to messenger RNAs (mRNA). While specific miRNA have been associated with GBM, their precise roles in tumor development and progression remain unclear. In this study, the analysis of miRNA expression data from 743 adult GBM cases and 59 normal brain samples identified 94 downregulated miRNA and 115 upregulated miRNA. Many of these miRNA were previously linked to GBM pathology, confirming the robustness of our approach, while we also identified novel miRNA that may act as potential regulators in GBM. By integrating miRNA predictions with gene expression data, we were able to associate downregulated miRNA with tumor microenvironment factors, including extracellular matrix remodeling and signaling pathways involved in tumor initiation, while upregulated miRNA were found to be associated with essential neuronal processes. This analysis highlights the significance of miRNA in GBM and serves as a foundation for further investigation., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Nervous system cancers exhibit diverse transcriptional cell states influenced by normal development, injury response, and growth. However, the understanding of these states' regulation and pharmacological relevance remains limited. Here we present "single-cell regulatory-driven clustering" (scregclust), a method that reconstructs cellular regulatory programs from extensive collections of single-cell RNA sequencing (scRNA-seq) data from both tumors and developing tissues. The algorithm efficiently divides target genes into modules, predicting key transcription factors and kinases with minimal computational time. Applying this method to adult and childhood brain cancers, we identify critical regulators and suggest interventions that could improve temozolomide treatment in glioblastoma. Additionally, our integrative analysis reveals a meta-module regulated by SPI1 and IRF8 linked to an immune-mediated mesenchymal-like state. Finally, scregclust's flexibility is demonstrated across 15 tumor types, uncovering both pan-cancer and specific regulators. The algorithm is provided as an easy-to-use R package that facilitates the exploration of regulatory programs underlying cell plasticity., (© 2024. The Author(s).)

Purpose: To develop a novel approach for increasing radiosensitivity in glioblastoma (GBM) by using targeted nanoparticles to deliver siRNA aimed at silencing the EGFR and RELA/P65 genes, which are implicated in radioresistance., Patients and Methods: We engineered biodegradable, tumor-targeted, self-assembled, and stimuli-responsive peptide nanoparticles for efficient siRNA delivery. We evaluated the nanoparticles' ability to induce gene silencing and enhance DNA damage under radiation in vitro and in vivo. The nanoparticles were designed to exhibit pH-responsive endosomal escape and αvβ3 integrin targeting, allowing for preferential accumulation at tumor sites and traversal of the blood-brain tumor barrier., Results: The application of these nanoparticles resulted in significant gene silencing, increased apoptosis, and decreased cell viability. The treatment impaired DNA repair mechanisms, thereby enhancing radiosensitivity in GBM cells. In a GBM mouse model, the combination of nanoparticle treatment with radiotherapy notably prolonged survival without apparent toxicity., Conclusion: Our findings suggest that nanoparticle-mediated dual gene silencing can effectively overcome GBM radioresistance. This strategy has the potential to improve clinical outcomes in GBM treatment, proposing a promising therapeutic avenue for this challenging malignancy., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Cen et al.)

Glioma associated macrophages/microglia (GAMs) play an important role in glioblastoma (GBM) progression, due to their massive recruitment to the tumor site and polarization to a tumor promoting phenotype. GAMs secrete a variety of cytokines, which facilitate tumor cell growth and invasion, and prevent other immune cells from mounting an immune response against the tumor. Here, we demonstrate that zinc finger and BTB containing domain 18 (ZBTB18), a transcriptional repressor with tumor suppressive function in glioblastoma, impairs the production of key cytokines, which function as chemoattractant for GAMs. Consistently, we observe a reduced migration of GAMs when ZBTB18 is expressed by glioblastoma cells, both in cell culture and in vivo experiments. Moreover, RNA sequencing analysis shows that the presence of ZBTB18 in glioblastoma cells alters the commitment of conditioned microglia, suggesting the loss of the immune-suppressive phenotype and the acquisition of pro-inflammatory features. Thus, therapeutic approaches to increase ZBTB18 expression in GBM cells could represent an effective adjuvant to immune therapy in GBM., (© 2024. The Author(s).)

A hallmark of glioma cells, particularly glioblastoma multiforme (GBM) cells, is their resistance to apoptosis. Accumulating evidences has demonstrated that CARD16, a caspase recruitment domain (CARD) only protein, enhances both anti-apoptotic and tumorigenic properties. Nevertheless, there is a limited understanding of the expression and functional role of CARD16 in glioma. This study seeks to investigate, through in silico analysis and clinical specimens, the role of CARD16 as a potential tumor promoter in glioma. Functional assays and molecular studies revealed that CARD16 promotes tumorigenesis and suppresses apoptosis in glioma cells. Moreover, knockdown of CARD16 enhances the expression of the FOXO1/TRAIL axis in GBM cells. Additionally, FOXO1 downregulation in CARD16 knockdown GBM cells restores proliferation and reduces apoptosis. Further investigation demonstrated that elevated P21 expression inhibits CDK2-mediated FOXO1 phosphorylation and ubiquitination in CARD16-knockdown GBM cells. Collectively, these findings suggest that CARD16 is a tumor-promoting molecular in glioma via downregulating FOXO1/TRAIL axis, and suppressing TRAIL-induced apoptosis. The CARD16 gene presents significant potential for prognostic prediction and advances in innovative apoptotic therapeutics., (© 2024. The Author(s).)

Glioblastoma is one of the most common and aggressive primary brain tumors. The aberration of metabolism is the important character of GBM cells and is tightly related to the malignancy of GBM. We mainly verified the regulatory effects of KHDRBS1, SNORD51 and ZBED6 on pentose phosphate pathway and malignant biological behavior in glioblastoma cells, such as proliferation, migration and invasion. KHDRBS1 and SNORD51 were upregulated in GBM tissues and cells. But ZBED6 had opposite tendency in GBM tissues and cells. KHDRBS1 may improve the stability of SNORD51 by binding to SNORD51, thus elevating the expression of SNORD51. More importantly, SNORD51 can competitively bind to WDR33 with 3'UTR of ZBED6 pre-mRNA which can inhibit the 3' end processing of ZBED6 pre-mRNA, thereby inhibiting the expression of ZBED6 mRNA. ZBED6 inhibited the transcription of G6PD by binding to the promoter region of G6PD. Therefore, the KHDRBS1/SNORD51/ZBED6 pathway performs an important part in regulating the pentose phosphate pathway to influence malignant biological behavior of GBM cells, providing new insights and potential targets for the treatment of GBM., (© 2024. The Author(s).)

Background: Resistance acquired after radiotherapy is directly related to the failure of various cancer treatments, including GBM. Because the mechanism for overcoming radioresistance has not yet been clearly identified, the development of diagnostic and therapeutic markers to treat radioresistance is necessary. Since increased expression of stemness- and EMT-related markers are reported to be closely correlated with radioresistance, research is underway to develop new drugs targeting these factors., Methods: To develop an anticancer drug that overcomes radioresistance, a library of drugs already approved by the FDA was used. After treating radioresistant GBM cells with each drug, the expression of stemness- and EMT-related markers was confirmed by qRT-PCR, and as a result, Atomoxetine (ATX) was selected. It was confirmed that radioresistance-induced cell migratory, invasive, sphere formation abilities, and tumor growth using a xenograft mouse model were suppressed upon ATX treatment. Using a miRNA prediction tool, we discovered miR-520d-5p, which targets Notch2 and Hey1, key factors in radioresistance, and discovered circATIC targeting this miRNA, revealing its relationship with ATX. We demonstrated the expression regulation mechanism and signaling mechanism between circATIC, miR-520d-5p, Notch2, and Hey1 factors using a luciferase reporter assay. In addition, the results at the cellular level were clinically verified by confirming the correlation between radiation, miR-520d-5p, and circATIC using patient plasma by qRT-PCR., Results: ATX showed potential as a treatment for radioresistance by suppressing the malignant phenotype by regulating the circATIC/miR-520d-5p/Notch2-Hey1 signaling mechanism in vitro and in vivo using radioresistant GBM cells., Conclusions: This study revealed that ATX suppresses radioresistance through the circATIC/miR-520d-5p/Notch2-Hey1 signaling pathway. These results showed the potential of ATX as a new drug that can overcome radioresistance, a major challenge in cancer treatment, and the signaling factors identified in this mechanism suggest the possibility of use as potential targets for the diagnosis and treatment of radioresistance., (© 2024. The Author(s).)

Tumors reprogram their metabolism to generate complex neoplastic ecosystems. Here, we demonstrate that glioblastoma (GBM) stem cells (GSCs) display elevated activity of the malate-aspartate shuttle (MAS) and expression of malate dehydrogenase 2 (MDH2). Genetic and pharmacologic targeting of MDH2 attenuated GSC proliferation, self-renewal, and in vivo tumor growth, partially rescued by aspartate. Targeting MDH2 induced accumulation of alpha-ketoglutarate (αKG), a critical co-factor for dioxygenases, including the N6-methyladenosine (m6A) RNA demethylase AlkB homolog 5, RNA demethylase (ALKBH5). Forced expression of MDH2 increased m6A levels and inhibited ALKBH5 activity, both rescued by αKG supplementation. Reciprocally, targeting MDH2 reduced global m6A levels with platelet-derived growth factor receptor-β (PDGFRβ) as a regulated transcript. Pharmacological inhibition of MDH2 in GSCs augmented efficacy of dasatinib, an orally bioavailable multi-kinase inhibitor, including PDGFRβ. Collectively, stem-like tumor cells reprogram their metabolism to induce changes in their epitranscriptomes and reveal possible therapeutic paradigms., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Brain tumors, particularly glioblastoma (GBM), are devastating and challenging to treat, with a low 5-year survival rate of only 6.6%. Mouse models are established to understand tumorigenesis and develop new therapeutic strategies. Large-scale genomic studies have facilitated the identification of genetic alterations driving human brain tumor development and progression. Genetically engineered mouse models (GEMMs) with clinically relevant genetic alterations are widely used to investigate tumor origin. Additionally, syngeneic implantation models, utilizing cell lines derived from GEMMs or other sources, are popular for their consistent and relatively short latency period, addressing various brain cancer research questions. In recent years, the success of immunotherapy in specific cancer types has led to a surge in cancer immunology-related research which specifically necessitates the utilization of immunocompetent mouse models. In this review, we provide a comprehensive summary of GEMMs and syngeneic mouse models for adult brain tumors, emphasizing key features such as model origin, genetic alteration background, oncogenic mechanisms, and immune-related characteristics. Our review serves as a valuable resource for the brain tumor research community, aiding in the selection of appropriate models to study cancer immunology., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Glioblastoma, the most frequent primary malignant brain tumour in adults, is characterised by profound yet dynamic hypoxia and nutrient depletion. To sustain survival and proliferation, tumour cells are compelled to acquire metabolic plasticity with the induction of adaptive metabolic programs. Here, we interrogated the pathways necessary to enable processing of nutrients other than glucose. We employed genetic approaches (stable/inducible overexpression, CRISPR/Cas9 knockout), pharmacological interventions with a novel inhibitor of AMP-activated protein kinase (AMPK) in glioblastoma cell culture systems and a proteomic approach to investigate mechanisms of metabolic plasticity. Moreover, a spatially resolved multiomic analysis was employed to correlate the gene expression pattern of PGC-1α with the local metabolic and genetic architecture in human glioblastoma tissue sections. A switch from glucose to alternative nutrients triggered an activation of AMPK, which in turn activated PGC-1α-dependent adaptive programs promoting mitochondrial metabolism. This sensor-effector mechanism was essential for metabolic plasticity with both functional AMPK and PGC-1α necessary for survival and growth of cells under nonglucose nutrient sources. In human glioblastoma tissue specimens, PGC-1α-expression correlated with nonhypoxic tumour niches defining a specific metabolic compartment. Our findings reveal a cell-intrinsic nutrient sensing and switching mechanism. The exposure to alternative fuels triggers a starvation signal that subsequently is passed on via AMPK and PGC-1α to induce adaptive programs necessary for broader spectrum nutrient metabolism. The integration of spatially resolved transcriptomic data confirms the relevance of PGC-1α especially in nonhypoxic tumour regions. Thus, the AMPK-PGC-1α axis is a candidate for therapeutic inhibition in glioblastoma. KEY POINTS/HIGHLIGHTS: AMPK activation induces PGC-1α expression in glioblastoma during nutrient scarcity. PGC-1α enables metabolic plasticity by facilitating metabolism of alternative nutrients in glioblastoma. PGC-1α expression is inversely correlated with hypoxic tumour regions in human glioblastomas., (© 2024 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Glioblastoma (GBM) is categorized by the World Health Organization (WHO) as a grade 4 glioma and is a uniformly fatal tumor of the central nervous system. With the discovery of specific gene anomalies, GBM classification has been modified several times to provide better diagnostic and prognostic accuracy. Survival outcomes remain dismal despite the current therapeutic modalities, which include a combination of surgical resection, adjuvant chemotherapy, and radiotherapies, providing brief control of tumor progression. GBM remains aggressive and reoccurs primarily due to the presence of a unique population of untreatable glioblastoma stem cells (GSC). The presence of high mutation rates and a dysregulated transcriptional landscape increase GSC resistance to conventional chemotherapy and radiation therapy, contributing to poor outcomes seen in GBM patients. Accordingly, GSCs have emerged as targets of interest in new GBM treatment paradigms. Consequently, it is important to understand their distinct properties, such as GSC interactions with the hypoxic microenvironment, enhancing their growth. The epigenomic regulators and fundamental molecular components of the signaling pathways represent potential targets for GBM therapies. In this review, we aimed to describe the evolution of GBM classification and highlight the current therapeutic modalities, including gene and immunotherapies, and mammalian target of rapamycin (mTOR) inhibitors to target GBM. Furthermore, we explored the molecular pathway of GSCs and the ongoing investigation of circulating tumor cells (CTC), along with precision therapeutics, which aim to provide novel discoveries and effective treatments for GBM with improved survival., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Glioblastoma multiforme (GBM) is the most prevalent and aggressive brain tumor found in adult humans with a poor prognosis and average survival of 14-15 months. In order to have a comprehensive understanding of proteome and identify novel therapeutic targets, this study focused mainly on the differentially abundant proteins (DAPs) of Ras V12 -induced GBM. Ras V12 is a constitutively active Ras mutant form essential for tumor progression by continuously activating signaling pathways leading to uncontrolled tumor growth. This study used a transgenic Drosophila model with Ras V12 overexpression using the repo-GAL4 driver line, specifically in glial cells, to study GBM. The high-resolution mass spectrometry (HRMS)-based proteomic analysis of the GBM larval central nervous system identified three novel DAPs specific to mitochondria. These DAPs, probable maleylacetoacetate isomerase 2 (Q9VHD2), bifunctional methylene tetrahydrofolate dehydrogenase (Q04448), and glutamine synthetase1 (P20477), identified through HRMS were further validated by qRT-PCR. The protein-protein interaction analysis revealed interactions between Ras V12 and DAPs, with functional links to mitochondrial dynamics regulators such as Drp1, Marf, Parkin, and HtrA2. Notably, altered expressions of Q9VHD2, P20477, and Q04448 were observed during GBM progression, which offers new insights into the involvement of mitochondrial dynamic regulators in Ras V12 -induced GBM pathophysiology.

Chemokines and their receptors form a complex interaction network, crucial for precise leukocyte positioning and trafficking. In cancer, they promote malignant cell proliferation and survival but are also critical for immune cell infiltration in the tumor microenvironment. Glioblastoma (GBM) is the most common and lethal brain tumor, characterized by an immunosuppressive TME, with restricted immune cell infiltration. A better understanding of chemokine-receptor interactions is therefore essential for improving tumor immunogenicity. In this study, we assessed the expression of all human chemokines in adult-type diffuse gliomas, with particular focus on GBM, based on patient-derived samples. Publicly available bulk RNA sequencing datasets allowed us to identify the chemokines most abundantly expressed in GBM, with regard to disease severity and across different tumor subregions. To gain insight into the chemokines-receptor network at the single cell resolution, we explored GBmap, a curated resource integrating multiple scRNAseq datasets from different published studies. Our study constitutes the first patient-based handbook highlighting the relevant chemokine-receptor crosstalks, which are of significant interest in the perspective of a therapeutic modulation of the TME in GBM., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Glioblastoma (GBM) is a fatal adult brain tumor with an extremely poor prognosis. GBM poses significant challenges for targeted therapies due to its intra- and inter-tumoral heterogeneity, a highly immunosuppressive microenvironment, diffuse infiltration into normal brain parenchyma, protection by the blood-brain barrier and acquisition of therapeutic resistance. Recent studies have implicated epigenetic modifiers as key players driving tumorigenesis, resistance, and progression of GBM. While the vast majority of GBM research on epigenetic modifiers thus far has focused predominantly on elucidating the functional roles and targeting of DNA methyltransferases and histone deacetylases, emerging evidence indicates that histone acetyltransferases (HATs) also play a key role in mediating plasticity and therapeutic resistance in GBM. Here, we will provide an overview of HATs, their dual roles and functions in cancer as both tumor suppressors and oncogenes and focus specifically on their implications in GBM resistance. We also discuss the technical challenges in developing selective HAT inhibitors and highlight their promise as potential anti-cancer therapeutics for treating intractable cancers such as GBM., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Glioma is a kind of brain tumor that develops in the central nervous system and is classified based on its histology and molecular genetic features. The lifespan of patients does not exceed 22 months. One of the motives for the low effectiveness of glioma treatment is its radioresistance and chemoresistance. Noncoding RNAs (ncRNAs) are a diverse set of transcripts that do not undergo translation to become proteins in glioma. The ncRNAs have been identified as significant regulators of several biological processes in different cell types and tissues, and their abnormal function has been linked to glioma. They are known to impact important occurrences, including carcinogenesis, progression, and enhanced treatment resistance in glioma cells. The ncRNAs control cell proliferation, migration, epithelial-to-mesenchymal transition (EMT), invasion, and drug resistance in glioma cells. The main focus of this study is to inspect the involvement of ncRNAs in the drug resistance of glioma., Competing Interests: Declaration of Competing Interest I declare that there is no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier GmbH.)

In vitro and ex-vivo target identification strategies often fail to predict in vivo efficacy, particularly for glioblastoma (GBM), a highly heterogenous tumor rich in resistant cancer stem cells (GSCs). An in vivo screening tool can improve prediction of therapeutic efficacy by considering the complex tumor microenvironment and the dynamic plasticity of GSCs driving therapy resistance and recurrence. This study proposes lipid nanoparticles (LNPs) as an efficient in vivo CRISPR-Cas9 gene editing tool for target validation in mesenchymal GSCs. LNPs co-delivering mRNA (mCas9) and single-guide RNA (sgRNA) were successfully formulated and optimized facilitating both in vitro and in vivo gene editing. In vitro, LNPs achieved up to 67 % reduction in green fluorescent protein (GFP) expression, used as a model target, outperforming a commercial transfection reagent. Intratumoral administration of LNPs in GSCs resulted in ∼80 % GFP gene knock-out and a 2-fold reduction in GFP signal by day 14. This study showcases the applicability of CRISPR-Cas9 LNPs as a potential in vivo screening tool in GSCs, currently lacking effective treatment. By replacing GFP with a pool of potential targets, the proposed platform presents an exciting prospect for therapeutic target validation in orthotopic GSCs, bridging the gap between preclinical and clinical research., (Copyright © 2024. Published by Elsevier B.V.)

Phenotypic plasticity is a cause of glioblastoma therapy failure. We previously showed that suppressing the oligodendrocyte-lineage regulator SOX10 promotes glioblastoma progression. Here, we analyze SOX10-mediated phenotypic plasticity and exploit it for glioblastoma therapy design. We show that low SOX10 expression is linked to neural stem-cell (NSC)-like glioblastoma cell states and is a consequence of temozolomide treatment in animal and cell line models. Single-cell transcriptome profiling of Sox10-KD tumors indicates that Sox10 suppression is sufficient to induce tumor progression to an aggressive NSC/developmental-like phenotype, including a quiescent NSC-like cell population. The quiescent NSC state is induced by temozolomide and Sox10-KD and reduced by Notch pathway inhibition in cell line models. Combination treatment using Notch and HDAC/PI3K inhibitors extends the survival of mice carrying Sox10-KD tumors, validating our experimental therapy approach. In summary, SOX10 suppression mediates glioblastoma progression through NSC/developmental cell-state transition, including the induction of a targetable quiescent NSC state. This work provides a rationale for the design of tumor therapies based on single-cell phenotypic plasticity analysis., (© 2024. The Author(s).)

Background: Glioblastoma (GBM) poses a significant medical challenge due to its aggressive nature and poor prognosis. Mitochondrial unfolded protein response (UPRmt) and the heat shock factor 1 (HSF1) pathway play crucial roles in GBM pathogenesis. Post-translational modifications, such as SUMOylation, regulate the mechanism of action of HSF1 and may influence the progression of GBM. Understanding the interplay between SUMOylation-modified HSF1 and GBM pathophysiology is essential for developing targeted therapies., Methods: We conducted a comprehensive investigation using cellular, molecular, and in vivo techniques. Cell culture experiments involved establishing stable cell lines, protein extraction, Western blotting, co-immunoprecipitation, and immunofluorescence analysis. Mass spectrometry was utilized for protein interaction studies. Computational modeling techniques were employed for protein structure analysis. Plasmid construction and lentiviral transfection facilitated the manipulation of HSF1 SUMOylation. In vivo studies employed xenograft models for tumor growth assessment., Results: Our research findings indicate that HSF1 primarily undergoes SUMOylation at the lysine residue K298, enhancing its nuclear translocation, stability, and downstream heat shock protein expression, while having no effect on its trimer conformation. SUMOylated HSF1 promoted the UPRmt pathway, leading to increased GBM cell proliferation, migration, invasion, and reduced apoptosis. In vivo studies have confirmed that SUMOylation of HSF1 enhances its oncogenic effect in promoting tumor growth in GBM xenograft models., Conclusion: This study elucidates the significance of SUMOylation modification of HSF1 in driving GBM progression. Targeting SUMOylated HSF1 may offer a novel therapeutic approach for GBM treatment. Further investigation into the specific molecular mechanisms influenced by SUMOylated HSF1 is warranted for the development of effective targeted therapies to improve outcomes for GBM patients., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Cellular senescence is a natural barrier of the transition from premalignant cells to invasive cancer. Pharmacological induction of senescence has been proposed as a possible anticancer strategy. In this study, we found that deoxyarbutin inhibited the growth of glioblastoma (GBM) cells by inducing cellular senescence, independent of tyrosinase expression. Instead, deoxyarbutin induced mitochondrial oxidative stress and damage. These aberrant mitochondria were key to the p53-dependent senescence of GBM cells. Facilitating autophagy or mitigating mitochondrial oxidative stress both suppressed p53 expression and alleviated cellular senescence induced by deoxyarbutin. Thus, our study reveals that deoxyarbutin induces mitochondrial oxidative stress to trigger the p53-dependent senescence of GBM cells. Importantly, deoxyarbutin treatment resulted in accumulation of p53, induction of cellular senescence, and inhibition of tumor growth in a subcutaneous tumor model of mouse. In conclusion, our study reveals that deoxyarbutin has therapeutic potential for GBM by inducing mitochondrial oxidative stress for p53-dependent senescence of GBM cells., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

l-2-Hydroxyglutarate (l-2-HG) has been regarded as a tumor metabolite, and it plays a crucial role in adaptation of tumor cells to hypoxic conditions. However, the role of l-2-HG in tumor radioresistance and the underlying mechanism have not yet been revealed. Here, we found that l-2-HG exhibited to have radioresistance effect on U87 human glioblastoma cells, which could reduce DNA damage and apoptosis caused by irradiation, promote cell proliferation and migration, and impair G2/M phase arrest. Mechanistically, l-2-HG upregulated the protein level of hypoxia-inducible factor-1α (HIF-1α) and the expression levels of HIF-1α downstream target genes. The knockdown of l-2-hydroxyglutarate dehydrogenase (L2HGDH) gene promoted the tumor growth and proliferation of U87 cells in nude mice by increasing HIF-1α expression level in vivo. In addition, the low expression level of L2HGDH gene was correlated with the short survival of patients with glioma or kidney cancer. In conclusion, our study revealed the role and mechanism of l-2-HG in tumor radioresistance and may provide a new perspective for overcoming tumor radioresistance and broaden our comprehension of the role of metabolites in tumor microenvironment., (© 2024 Wiley Periodicals LLC.)

Glioblastoma (GBM) is the highest grade of glioma for which no effective therapy is currently available. Despite extensive research in diagnosis and therapy, there has been no significant improvement in GBM outcomes, with a median overall survival continuing at a dismal 15-18 months. In recent times, glioblastoma stem cells (GSCs) have been identified as crucial drivers of treatment resistance and tumor recurrence, and GBM therapies targeting GSCs are expected to improve patient outcomes. We used a multistep in silico screening strategy to identify repurposed candidate drugs against selected therapeutic molecular targets in GBM with potential to concomitantly target GSCs. Common differentially expressed genes (DEGs) were identified through analysis of multiple GBM and GSC datasets from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA). For identification of target genes, we selected the genes with most significant effect on overall patient survival. The relative mRNA and protein expression of the selected genes in TCGA control versus GBM samples was also validated and their cancer dependency scores were assessed. Drugs targeting these genes and their corresponding proteins were identified from LINCS database using Connectivity Map (CMap) portal and by in silico molecular docking against each individual target using FDA-approved drug library from the DrugBank database, respectively. The molecules thus obtained were further evaluated for their ability to cross blood brain barrier (BBB) and their likelihood of resulting in drug resistance by acting as p-glycoprotein (p-Gp) substrates. The growth inhibitory effect of these final shortlisted compounds was examined on a panel of GBM cell lines and compared with temozolomide through the drug sensitivity EC50 values and AUC from the PRISM Repurposing Secondary Screen, and the IC50 values were obtained from GDSC portal. We identified RPA3, PSMA2, PSMC2, BLVRA, and HUS1 as molecular targets in GBM including GSCs with significant impact on patient survival. Our results show GSK-2126458/omipalisib, linifanib, drospirenone, eltrombopag, nilotinib, and PD198306 as candidate drugs which can be further evaluated for their anti-tumor potential against GBM. Through this work, we identified repurposed candidate therapeutics against GBM utilizing a GSC inclusive targeting approach, which demonstrated high in vitro efficacy and can prospectively evade drug resistance. These drugs have the potential to be developed as individual or combination therapy to improve GBM outcomes., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Gliomas are primary brain tumors and are among the most malignant types. Adult-type diffuse gliomas can be classified based on their histological and molecular signatures as IDH-wildtype glioblastoma, IDH-mutant astrocytoma, and IDH-mutant and 1p/19q-codeleted oligodendroglioma. Recent studies have shown that each subtype of glioma has its own specific distribution pattern. However, the mechanisms underlying the specific distributions of glioma subtypes are not entirely clear despite partial explanations such as cell origin. To investigate the impact of multi-scale brain attributes on glioma distribution, we constructed cumulative frequency maps for diffuse glioma subtypes based on T1w structural images and evaluated the spatial correlation between tumor frequency and diverse brain attributes, including postmortem gene expression, functional connectivity metrics, cerebral perfusion, glucose metabolism, and neurotransmitter signaling. Regression models were constructed to evaluate the contribution of these factors to the anatomic distribution of different glioma subtypes. Our findings revealed that the three different subtypes of gliomas had distinct distribution patterns, showing spatial preferences toward different brain environmental attributes. Glioblastomas were especially likely to occur in regions enriched with synapse-related pathways and diverse neurotransmitter receptors. Astrocytomas and oligodendrogliomas preferentially occurred in areas enriched with genes associated with neutrophil-mediated immune responses. The functional network characteristics and neurotransmitter distribution also contributed to oligodendroglioma distribution. Our results suggest that different brain transcriptomic, neurotransmitter, and connectomic attributes are the factors that determine the specific distributions of glioma subtypes. These findings highlight the importance of bridging diverse scales of biological organization when studying neurological dysfunction., (© 2024 UICC.)

Purpose: This phase 1/2 study aimed to evaluate the safety and preliminary efficacy of combining disulfiram and copper (DSF/Cu) with radiation therapy (RT) and temozolomide (TMZ) in patients with newly diagnosed glioblastoma (GBM)., Methods and Materials: Patients received standard RT and TMZ with DSF (250-375 mg/d) and Cu, followed by adjuvant TMZ plus DSF (500 mg/d) and Cu. Pharmacokinetic analyses determined drug concentrations in plasma and tumors using high-performance liquid chromatography-mass spectrometry., Results: Thirty-three patients, with a median follow-up of 26.0 months, were treated, including 12 IDH-mutant, 9 NF1-mutant, 3 BRAF-mutant, and 9 other IDH-wild-type cases. In the phase 1 arm, 18 patients were treated; dose-limiting toxicity probabilities were 10% (95% CI, 3%-29%) at 250 mg/d and 21% (95% CI, 7%-42%) at 375 mg/d. The phase 2 arm treated 15 additional patients at 250 mg/d. No significant difference in overall survival or progression-free survival was noted between IDH- and NF1-mutant cohorts compared with institutional counterparts treated without DSF/Cu. However, extended remission occurred in 3 BRAF-mutant patients. Diethyl-dithiocarbamate-copper, the proposed active metabolite of DSF/Cu, was detected in plasma but not in tumors., Conclusions: The maximum tolerated dose of DSF with RT and TMZ is 375 mg/d. DSF/Cu showed limited clinical efficacy for most patients. However, promising efficacy was observed in BRAF-mutant GBM, warranting further investigation., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Proteomics has been little used for the identification of novel prognostic and/or therapeutic markers in isocitrate dehydrogenase (IDH)-wildtype glioblastoma (GB). In this study, we analyzed 50 tumor and 30 serum samples from short- and long-term survivors of IDH-wildtype GB (STS and LTS, respectively) by data-independent acquisition mass spectrometry (DIA-MS)-based proteomics, with the aim of identifying such markers. DIA-MS identified 5422 and 826 normalized proteins in tumor and serum samples, respectively, with only three tumor proteins and 26 serum proteins displaying significant differential expression between the STS and LTS groups. These dysregulated proteins were principally associated with the detoxification of reactive oxygen species (ROS). In particular, GB patients in the STS group had high serum levels of malate dehydrogenase 1 (MDH1) and ribonuclease inhibitor 1 (RNH1) and low tumor levels of fatty acid-binding protein 7 (FABP7), which may have enabled them to maintain low ROS levels, counteracting the effects of the first-line treatment with radiotherapy plus concomitant and adjuvant temozolomide. A blood score built on the levels of MDH1 and RNH1 expression was found to be an independent prognostic factor for survival based on the serum proteome data for a cohort of 96 IDH-wildtype GB patients. This study highlights the utility of circulating MDH1 and RNH1 biomarkers for determining the prognosis of patients with IDH-wildtype GB. Furthermore, the pathways driven by these biomarkers, and the tumor FABP7 pathway, may constitute promising therapeutic targets for blocking ROS detoxification to overcome resistance to chemoradiotherapy in potential GB STS., (© 2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Purpose: Glioblastoma (GBM), characterized by astrocytic tumorigenesis, remains one of the most prognostically challenging tumor types. Targeting entire GBM microenvironment using novel therapeutic factors is currently desired investigation approach. In this study, we focused on progranulin (PGRN), a regulator of diverse cellular functions. Recent studies implicated PGRN in the poor prognostics of GBM patients. However, the specific role of PGRN in the GBM microenvironment remains elusive., Methods: We utilized public databases of GBM patient and previous single-cell RNA sequence to examine association between PGRN expression and patient survival/grade, and expression levels of PGRN in each cell constituting the tumor microenvironment. To clarify the role of PGRN in Tumor-associated macrophage (TAM), we examined cell proliferation and expression of some proteins in murine GBM cells when cell supernatants derived from TAM of PGRN knockout (Grn -/- ) or wild type mice were treated with murine GBM cells., Results: Our results reveal significant PGRN expression in macrophages within the GBM environment, suggesting an association between increased PGRN expression in macrophages and tumor malignancy. TAM induction led to PGRN expression enhancement. Treatment with Grn -/- mouse -derived bone marrow-derived macrophage (BMDM) supernatant resulted in diminished GBM cell proliferation and cell cycle- and mesenchymal GBM subtype-associated reduced protein expression. Furthermore, the Grn -/- mouse-derived BMDM supernatant treatment reduced the phosphorylated STAT3 expression in GBM cells, while the expression of IL-6 and IL-10, known STAT3 pathway activators, diminished in Grn -/- mouse-derived BMDMs., Conclusion: Our results suggest that macrophage-derived PGRN is pivotal for fostering malignant transformations within the tumor microenvironment., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Glyphosate, the world's most widely used herbicide, has a low toxicity rating despite substantial evidence of adverse health effects. Furthermore, glyphosate-based formulations (GBFs) contain several other chemicals, some of which are known to be harmful. Additionally, chronic, and acute exposure to GBFs among rural workers may lead to health impairments, such as neurodegenerative diseases and cancer. P53 is known as a tumor suppressor protein, acting as a key regulator of the cellular response to stress and DNA damage. Therefore, mutations in the TP53 gene, which encodes p53, are common genetic alterations found in various types of cancer. Therefore, this study aimed to evaluate the cytotoxicity and genotoxicity of GBF in two glioblastoma cell lines: U87MG (TP53-proficient) and U251MG (TP53-mutant). Additionally, the study aimed to identify the main proteins involved in the response to GBF exposure using Systems Biology in a network containing p53 and another network without p53. The MTT assay was used to study the toxicity of GBF in the cell lines, the clonogenic assay was used to investigate cell survival, and the Comet Assay was used for genotoxicity evaluation. For data analysis, bioinformatics tools such as String 12.0 and Stitch 5.0 were applied, serving as a basis for designing binary networks in the Cytoscape 3.10.1 program. From the in vitro test analyses, it was observed a decrease in cell viability at doses starting from 10 ppm. Comet Assay at concentrations of 10 ppm and 30 ppm for the U251MG and U87MG cell lines, respectively observed DNA damage. The network generated with systems biology showed that the presence of p53 is important for the regulation of biological processes involved in genetic stability and neurotoxicity, processes that did not appear in the TP53-mutant network., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Fernanda Rabaioli da Silva reports equipment, drugs, or supplies was provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (Brazil). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Purpose: To propose a novel recursive partitioning analysis (RPA) classification model in patients with IDH-wildtype glioblastomas that incorporates the recently expanded conception of the extent of resection (EOR) in terms of both supramaximal and total resections., Experimental Design: This multicenter cohort study included a developmental cohort of 622 patients with IDH-wildtype glioblastomas from a single institution (Severance Hospital) and validation cohorts of 536 patients from three institutions (Seoul National University Hospital, Asan Medical Center, and Heidelberg University Hospital). All patients completed standard treatment including concurrent chemoradiotherapy and underwent testing to determine their IDH mutation and MGMTp methylation status. EORs were categorized into either supramaximal, total, or non-total resections. A novel RPA model was then developed and compared with a previous Radiation Therapy Oncology Group (RTOG) RPA model., Results: In the developmental cohort, the RPA model included age, MGMTp methylation status, Karnofsky performance status, and EOR. Younger patients with MGMTp methylation and supramaximal resections showed a more favorable prognosis [class I: median overall survival (OS) 57.3 months], whereas low-performing patients with non-total resections and without MGMTp methylation showed the worst prognosis (class IV: median OS 14.3 months). The prognostic significance of the RPA was subsequently confirmed in the validation cohorts, which revealed a greater separation between prognostic classes for all cohorts compared with the previous RTOG RPA model., Conclusions: The proposed RPA model highlights the impact of supramaximal versus total resections and incorporates clinical and molecular factors into survival stratification. The RPA model may improve the accuracy of assessing prognostic groups. See related commentary by Karschnia et al., p. 4811., (©2024 American Association for Cancer Research.)

ALDH1A3 is a marker for mesenchymal glioblastomas characterized by a greater degree of aggressiveness compared to other major subtypes. ADH1A3 has been implicated in the regulation of stemness and radioresistance mediated by glioblastoma stem cells. Mechanisms by which ALDH1A3 promotes malignant progression of glioblastoma remain elusive posing a challenge for rationalization of ALDH1A3 targeting in glioblastoma, and it is also unclear how ALDH1A3 regulates glioblastoma cells stemness. Usage of different models with diverse genetic backgrounds and often unknown degree of stemness is one possible reason for discrepant views on the role of ALDH1A3 in glioblastoma stem cells. This study clarifies ALDH1A3 impacts on glioblastoma stem cells by modelling ALDH1A3 expression in an otherwise invariable genetic background with consideration of the impacts of inherent plasticity and proliferative changes associated with transitions between cell states. Our main finding is that ALDH1A3 exerts cell-state dependent impact on proliferation of glioblastoma stem cells. We provide evidence that ALDH1A3 augments radiation-induced inhibition of self-renewal and promotes the proliferation of differentiated GSC progenies. Congruent effects ALDH1A3 and radiation on self-renewal and proliferation provides a framework for promoting glioblastoma growth under radiation treatment.

HOX transcript antisense RNA (HOTAIR) is upregulated in glioblastoma (GBM) and associated with temozolomide (TMZ) resistance. However, the mechanisms underlying HOTAIR-mediated TMZ resistance remains poorly understood. HOTAIR expression in glioma-related public datasets and drug response estimation were analyzed using bioinformatics. These findings were verified by overexpressing HOTAIR in TMZ-sensitive U251 cells and/or silencing HOTAIR in resistant U251 cells (U251R). The cytotoxic effects were evaluated using cell viability assay and flow cytometry analysis of cell cycle and apoptosis. In this study, we found that HOTAIR was upregulated in TMZ-resistant GBM cell lines and patients with high HOTAIR expression responded poorly to TMZ therapy. HOTAIR knockdown restored TMZ sensitivity in U251R cells, while HOTAIR overexpression conferred TMZ resistance in U251 cells. Wnt/β-catenin signaling was enriched in patients with high HOTAIR expression; consistently, HOTAIR positively regulated β-catenin expression in U251 cells. Moreover, HOTAIR-mediated TMZ resistance was associated with increased MGMT protein level, which resulted from the HOTAIR/miR-214-3p/β-catenin network. Besides, GBM with high HOTAIR expression exhibited sensitivity to methotrexate. Methotrexate enhanced TMZ sensitivity in U251R cells, accompanied by reduced expression of HOTAIR and β-catenin. Thus, we conlcude that HOTAIR is a risk factor for TMZ resistance and methotrexate may represent a potential therapeutic drug for patients with high HOTAIR expression level., (© 2024. The Author(s).)

Objective: To investigate the association between disulfidptosis-related genes (DFRGs) and patient prognosis, while concurrently identifying potential therapeutic targets in glioblastoma (GBM)., Methods: We retrieved RNA sequencing data and clinical characteristics of GBM patients from the TCGA database. We found there was a total of 6 distinct clusters in GBM, which was identified by the t-SNE and UMAP dimension reduction analysis. Prognostically significant genes in GBM were identified using the limma package, coupled with univariate Cox regression analysis. Machine learning algorithms were then applied to identify central genes. The CIBERSORT algorithm was utilized to assess the immunological landscape across different GBM subtypes. In vitro and in vivo experiments were conducted to investigate the role of SPAG4 in regulating the proliferation, invasion of GBM, and its effects within the immune microenvironment., Results: 23 genes, termed DFRGs, were successfully identified, demonstrating substantial potential for establishing a prognostic model for GBM. Single cell analysis revealed a significant correlation between DFRGs and the progression of GBM. Utilizing individual risk scores derived from this model enabled the stratification of patients into two distinct risk groups, revealing significant variations in immune infiltration patterns and responses to immunotherapy. Utilizing the random survival forests algorithm, SPAG4 was identified as the gene with the highest prognostic significance within our model. In vitro studies have elucidated SPAG4's significant role in GBM pathogenesis, potentially through the regulation of fatty acid metabolism pathways. Our in vivo investigations using a subcutaneous xenograft model have confirmed SPAG4's influence on tumor growth and its capacity to modulate the immune microenvironment. Advanced research hints that SPAG4 might achieve immune evasion by increasing CD47 expression, consequently reducing phagocytosis., Conclusions: These findings highlight SPAG4 as a potential GBM therapeutic target and emphasize the complexity of the immune microenvironment in GBM progression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Chen, Zeng, Chen, Ding, Lin, Ye, Wu, Yang and Yang.)

Glioblastoma multiforme is one of the most common primary tumors of the central nervous system, with a very poor prognosis. Cancer cells have been observed to upregulate pH regulators, such as monocarboxylate transporters (MCTs), with an increase in MCT4 expression being observed in several malignancies. MCT4/ recombinant cluster of differentiation 147 (CD147) transporter complex was reported to stimulate vascular endothelial growth factor (VEGF) via the phosphatidylinositol 3 kinase (PI3K) /protein kinase B (Akt) pathway, which has been proven to mediate glioblastoma invasion and migration. The present study aimed to clarify the role of the MCT4/CD147 transporter complex in glioblastoma cell proliferation, migration, and invasion. In this work, lentiviral vectors were used to overexpress MCT4/CD147 and small interfering RNA (siRNA) was used to silence MCT4/CD147 in the human glioma cell lines U87 and U251, respectively. The effects on cell proliferation, migration and invasiveness, as well as the protein expression levels of MCT4 and CD147, extracellular lactate content and Akt activation were assessed by MTT, wound-healing and invasion assays, western blotting and colorimetric method, respectively. The analysis results suggested that cell proliferation, migration, invasion, and Akt activation were decreased by siRNA in all cell lines, but were increased by lentivirus-mediated MCT4 overexpression. These findings suggest that inhibiting the activity and expression of the MCT4/CD147 transporter complex via metabolic-targeting drugs, particularly in cells with a high rate of glycolysis, should be explored as a novel strategy for glioblastoma treatment., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Gao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Glioblastoma is the commonest primary malignant brain tumor, with a very poor prognosis and short overall survival. It is characterized by its high intra- and intertumoral heterogeneity, in terms of both the level of single-nucleotide variants, copy number alterations, and aneuploidy. Therefore, routine diagnosis can be challenging in some cases. We present a complicated case of glioblastoma, which was characterized with five cytogenomic methods: interphase fluorescence in situ hybridization, multiplex ligation-dependent probe amplification, comparative genomic hybridization array and single-nucleotide polymorphism, targeted gene panel, and whole-genome sequencing. These cytogenomic methods revealed classical findings associated with glioblastoma, such as a lack of IDH and TERT mutations, gain of chromosome 7, and loss of chromosome 10. At least three pathological clones were identified, including one with whole-genome duplication, and one with loss of 1p and suspected loss of 19q. Deletion and mutation of the TP53 gene were detected with numerous breakends on 17p and 20q. Based on these findings, we recommend a combined approach to the diagnosis of glioblastoma involving the detection of copy number alterations, mutations, and aneuploidy. The choice of the best combination of methods is based on cost, time required, staff expertise, and laboratory equipment. This integrated strategy could contribute directly to tangible improvements in the diagnosis, prognosis, and prediction of the therapeutic responses of patients with brain tumors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Aghova, Lhotska, Lizcova, Svobodova, Hodanova, Janeckova, Vucinic, Gregor, Konecna, Kramar, Soukup, Netuka and Zemanova.)

We presented a method to find potential cancer attractors using single-cell RNA sequencing (scRNA-seq) data. We tested our method in a Glioblastoma Multiforme (GBM) dataset, an aggressive brain tumor presenting high heterogeneity. Using the cancer attractor concept, we argued that the GBM's underlying dynamics could partially explain the observed heterogeneity, with the dataset covering a representative region around the attractor. Exploratory data analysis revealed promising GBM's cellular clusters within a 3-dimensional marker space. We approximated the clusters' centroid as stable states and each cluster covariance matrix as defining confidence regions. To investigate the presence of attractors inside the confidence regions, we constructed a GBM gene regulatory network, defined a model for the dynamics, and prepared a framework for parameter estimation. An exploration of hyperparameter space allowed us to sample time series intending to simulate myriad variations of the tumor microenvironment. We obtained different densities of stable states across gene expression space and parameters displaying multistability across different clusters. Although we used our methodological approach in studying GBM, we would like to highlight its generality to other types of cancer. Therefore, this report contributes to an advance in the simulation of cancer dynamics and opens avenues to investigate potential therapeutic targets., (© 2024. The Author(s).)

Background: Tumorigenicity represents a critical challenge in stem cell-based therapies requiring rigorous monitoring. Conventional approaches for tumorigenicity evaluation are based on animal models and have numerous limitations. Brain organoids, which recapitulate the structural and functional complexity of the human brain, have been widely used in neuroscience research. However, the capacity of brain organoids for tumorigenicity evaluation needs to be further elucidated., Methods: A cerebral organoid model produced from human pluripotent stem cells (hPSCs) was employed. Meanwhile, to enhance the detection sensitivity for potential tumorigenic cells, we created a glioblastoma-like organoid (GBM organoid) model from TP53 -/- /PTEN -/- hPSCs to provide a tumor microenvironment for injected cells. Midbrain dopamine (mDA) cells from human embryonic stem cells were utilized as a cell therapy product. mDA cells, hPSCs, mDA cells spiked with hPSCs, and immature mDA cells were then injected into the brain organoids and NOD SCID mice. The injected cells within the brain organoids were characterized, and compared with those injected in vivo to evaluate the capability of the brain organoids for tumorigenicity evaluation. Single-cell RNA sequencing was performed to identify the differential gene expression between the cerebral organoids and the GBM organoids., Results: Both cerebral organoids and GBM organoids supported maturation of the injected mDA cells. The hPSCs and immature mDA cells injected in the GBM organoids showed a significantly higher proliferative capacity than those injected in the cerebral organoids and in NOD SCID mice. Furthermore, the spiked hPSCs were detectable in both the cerebral organoids and the GBM organoids. Notably, the GBM organoids demonstrated a superior capacity to enhance proliferation and pluripotency of spiked hPSCs compared to the cerebral organoids and the mouse model. Kyoto Encyclopedia of Genes and Genomes analysis revealed upregulation of tumor-related metabolic pathways and cytokines in the GBM organoids, suggesting that these factors underlie the high detection sensitivity for tumorigenicity evaluation., Conclusions: Our findings suggest that brain organoids could represent a novel and effective platform for evaluating the tumorigenic risk in stem cell-based therapies. Notably, the GBM organoids offer a superior platform that could complement or potentially replace traditional animal-based models for tumorigenicity evaluation., (© 2024. The Author(s).)

Background: NF1 inactivation is associated with sensitivity to MEK inhibitor targeted therapy in low-grade and some high-grade gliomas. NF1 loss may also be a harbinger of exploitable vulnerabilities in IDH-wildtype glioblastoma (GBM). Accurate and consistent detection of NF1 loss, however, is fraught given the large gene size, challenges with complete coverage and variant calling upon sequencing, and mechanisms of mRNA and protein regulation that result in early degradation in the absence of genomic alterations. Here, we seek to perform a composite analysis for NF1 loss accounting for genomic alterations and protein expression via immunohistochemistry. We also characterize the landscape of NF1 alterations in GBM., Methods: We assembled a single-institution, retrospective cohort of 542 IDH-wildtype GBM with somatic next generation sequencing to investigate the frequency and nature of detected NF1 alterations. We selected 69 GBMs from which to build a tissue microarray (TMA) of 44 NF1-wildtype and 25 NF1-mutant cases. We performed NF1 immunohistochemistry using two different NF1 antibodies (NFC, Sigma-Aldrich; and iNF-07E, iNFixion Bioscience) and correlated results with clinical, genomic, and other immunohistochemical features., Results: In our retrospective cohort, we identified 88 IDH-wildtype GBM with NF1 alterations (16%). NF1 alterations were mutually exclusive with EGFR and MDM2 alterations (p-adj < 0.001, 0.05, respectively), but co-occurred with PIK3R1 alterations (Log 2 (OR) = - 1.6, p-adj = 0.03). Of the 63 scorable sporadic GBMs in the TMA, 14 harbored NF1 inactivating alterations and of those, 12 (86%) demonstrated minimal NF1 immunoreactivity by NFC antibody, compared to 8 (57%) by iNF-07E antibody. Among the 42 scorable NF1-wildtype GBM in the TMA, NF1 immunostaining was minimal in 18 (43%) by NFC antibody compared to 4 (10%) by iNF-07E antibody, potentially reflecting false positives or differential protein regulation. Minimal immunoreactivity by NFC antibody was associated with decreased median overall survival (8.5 vs. 16.4 months, p = 0.011). Cox proportional hazards model correcting for prognostic variables in this subset revealed HR 3.23 (95% CI 1.29-8.06, p = 0.01) associated with decreased NF1 expression by IHC., Conclusion: NF1 immunostaining may serve as a sensitive surrogate marker of NF1 genomic inactivation and a valuable extension to next-generation sequencing for defining NF1 status. Minimal NF1 immunoreactivity is a poor prognostic marker, even in IDH-wildtype glioblastoma without apparent NF1 genomic alterations, but the underlying molecular mechanism requires further investigation., (© 2024. The Author(s).)

Glioblastoma (GB) is an aggressive malignant central nervous system tumor that is currently incurable. One of the main pitfalls of GB treatment is resistance to the chemotherapeutic standard of care, temozolomide (TMZ). The role of aldehyde dehydrogenases (ALDHs) in the glioma stem cell (GSC) subpopulation has been related to chemoresistance. ALDHs take part in processes such as cell proliferation, differentiation, invasiveness or metastasis and have been studied as pharmacological targets in cancer treatment. In the present work, three novel α,β-acetylenic amino thiolester compounds, with demonstrated efficacy as ALDH inhibitors, were tested in vitro on a panel of six human GB cell lines and one murine GB cell line. Firstly, the expression of the ALDH1A isoforms was assessed, and then inhibitors were tested for their cytotoxicity and their ability to inhibit cellular ALDH activity. Drug combination assays with TMZ were performed, as well as an assessment of the cell death mechanism and generation of ROS. A knockout of several ALDH genes was carried out in one of the human GB cell lines, allowing us to discuss their role in cell proliferation, migration capacity and resistance to treatment. Our results strongly suggest that ALDH inhibitors could be an interesting approach in the treatment of GB, with EC50 values in the order of micromolar, decreasing ALDH activity in GB cell lines to 40-50%.

Inhibitory receptors are critical for regulating immune cell function. In cancer, these receptors are often over-expressed on the cell surface of T and NK cells, leading to reduced anti-tumor activity. Here, through the analysis of 11 commonly studied checkpoint and inhibitory receptors, we discern that only HAVCR2 (TIM3) and ENTPD1 (CD39) display significantly greater gene expression in glioblastoma compared to normal brain and lower grade glioma. Cell surface TIM-3, but not ENTPD1, was also elevated on activated CD4 + and CD8 + T cells, as well as on NK cells from glioblastoma patients compared to healthy donor T and NK cells. A subsequent analysis of molecules known to co-ordinate TIM-3 function and regulation was performed, which revealed that BAT3 expression was significantly reduced in CD4 + and CD8 + T cells, as well as NK cells from glioblastoma patients compared to counterparts from healthy donors. These pro-inhibitory changes are also correlated with reduced levels of the activation marker CD69 and the pro-inflammatory cytokine IFNγ in CD4 + and CD8 + T cells, as well as NK cells from glioblastoma patients. Collectively, these data reveal that glioblastoma-mediated CD4 + and CD8 + T cell and NK cell suppression is due, at least in part, to dysregulated TIM-3 and BAT3 expression and the associated downstream immunoregulatory and dysfunctional effects.

Background: Certain cancers present challenges for treatment because they are resistant to immune checkpoint blockade (ICB), attributed to low tumor mutational burden and the absence of T cell-inflamed features. Among these, glioblastoma (GBM) is notoriously resistant to ICB. To overcome this resistance, the identification of T cells with heightened stemness marked by T-cell factor 1 (TCF1) expression has gained attention. Several studies have explored ways to preserve stem-like T cells and prevent terminal exhaustion. In this study, we investigate a target that triggers stem-like properties in CD8 T cells to enhance the response to ICB in a murine GBM model., Methods: Using Fcgr2b -/- mice and a murine GL261 GBM model, we confirmed the efficacy of anti-programmed cell death protein-1 (PD-1) immunotherapy, observing improved survival. Analysis of immune cells using fluorescence-activated cell sorting and single-cell RNA sequencing delineated distinct subsets of tumor-infiltrating CD8 T cells in Fcgr2b -/- mice. The crucial role of the stem-like feature in the response to anti-PD-1 treatment for reinvigorating CD8 T cells was analyzed. Adoptive transfer of OT-I cells into OVA-expressing GL261 models and CD8 T cell depletion in Fcgr2b -/- mice confirmed the significance of Fcgr2b -/- CD8 T cells in enhancing the antitumor response. Last, S1P 1 inhibitor treatment confirmed that the main source of tumor antigen-specific Fcgr2b -/- CD8 T cells is the tumor-draining lymph nodes (TdLNs)., Results: In a murine GBM model, anti-PD-1 monotherapy and single-Fc fragment of IgG receptor IIb (FcγRIIB) deletion exhibit limited efficacy. However, their combination substantially improves survival by enhancing cytotoxicity and proliferative capacity in tumor-infiltrating Fcgr2b -/- CD8 T cells. The improved response to anti-PD-1 treatment is associated with the tumor-specific memory T cells (Ttsms) exhibiting high stemness characteristics within the tumor microenvironment (TME). Ttsms in the TdLN thrives in a protective environment, maintaining stem-like characteristics and serving as a secure source for tumor infiltration. This underscores the significance of FcγRIIB ablation in triggering Ttsms and enhancing ICB therapy against GBM., Conclusions: Deletion of FcγRIIB on CD8 T cells leads to the generation of a Ttsms, which is localized in TdLN and protected from the immunosuppressive TME. Incorporating these highly stemness-equipped Ttsms enhances the response to anti-PD-1 therapy in immune-suppressed brain tumors., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Glioblastoma (GBM) is the most aggressive form of glioma, characterized by high mortality and poor prognosis. Dysregulation of microRNAs (miRNAs) plays a critical role in the progression and metastasis of GBM. This study aimed to investigate the role and molecular mechanism of miR-124-3p in GBM. Levels of miR-124-3p, EPHA2, and ALKBH5 were measured using quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, migration, invasion, and stemness were assessed using the Cell Counting Kit-8 (CCK-8), colony formation, Transwell, and sphere formation assays, respectively. Bioinformatics prediction, dual-luciferase reporter assays, and RNA pull-down experiments were employed to validate the target of miR-124-3p. RNA binding protein immunoprecipitation (RIP) and methylated RNA immunoprecipitation (Me-RIP) were utilized to evaluate the regulation of miR-124-3p maturation by ALKBH5. The results indicated that overexpression of miR-124-3p inhibited the proliferation, migration, invasion, and stemness of GBM cells. EPHA2 was identified as a direct downstream target of miR-124-3p, and its overexpression reversed the inhibitory effects of miR-124-3p on cellular functions. Furthermore, miR-124-3p targeted EPHA2 to inactivate the Wnt/β-catenin pathway. Additionally, ALKBH5 negatively regulated miR-124-3p by impeding its processing. In conclusion, knockdown of ALKBH5 promoted the processing of pri-miR-124-3p, increasing mature miR-124-3p levels, which inhibited the malignant behaviors of GBM cells by targeting EPHA2. These findings highlight the importance of the ALKBH5/miR-124-3p/EPHA2 axis in GBM., (© 2024. The Author(s) under exclusive licence to Japan Human Cell Society.)

Glioblastoma accounts for nearly half of all primary malignant brain tumors in adults, and despite an aggressive standard of care, including excisional surgery and adjuvant chemoradiation, recurrence remains universal, with an overall median survival of 14.6 months. Recent work has revealed the importance of passenger mutations as critical mediators of metabolic adaptation in cancer progression. In our previous work, we identified a role for the epigenetic modifier ID-1 in temozolomide resistance in glioblastoma. Here, we show that ID-1-mediated glioblastoma tumourigenesis is accompanied by upregulation of one-carbon (1-C) mediated de novo purine synthesis. ID-1 knockout results in a significant reduction in the expression of 1-C metabolism and purine synthesis enzymes. Analysis of glioblastoma surgical specimens at initial presentation and recurrence reveals that 1-C purine synthesis metabolic enzymes are enriched in recurrent glioblastoma and that their expression correlates with a shorter time to tumor recurrence. Further, we show that the 1-C metabolic phenotype underlies proliferative capacity and temozolomide resistance in glioblastoma cells. Supplementation with exogenous purines restores proliferation in ID-1-deficient cells, while inhibition of purine synthesis with AICAR sensitizes temozolomide-resistant glioblastoma cells to temozolomide chemotherapy. Our data suggest that the metabolic phenotype observed in treatment-resistant glioma cells is a potential therapeutic target in glioblastoma., (© 2024. The Author(s).)