Neuro-Oncology

BackgroundGlioblastoma (GBM) is the most aggressive primary brain tumor in adults. Radioresistance, partly mediated by glioma stem-like cells, represents a major clinical challenge which could be overcome by the identification of the modulators of radioresistance. Existing CRISPR screens in human GBM models have largely used two-dimensional cultures with short-term viability readouts, failing to capture the long-term clonogenic behaviour underlying tumour recurrence after radiotherapy. MethodWe developed ClonoScreen3D-CRISPRi, combining CRISPRi-mediated gene knockdown with three-dimensional clonogenic survival assays. Two GBM cell lines (G7 and GBML20), differing in MGMT promoter methylation status, were engineered to express the KRAB-dCas9 editor. Nine candidate radiosensitivity modifiers, selected through transcriptomic analysis, pharmacological studies, and literature review, were examined in both lines. Target validation was performed using full radiation dose-response assays and a pharmacological inhibitor. ResultsThe majority of candidate genes significantly altered survival fraction following irradiation in both cell lines. Knockdown of NFKB2, RELB, and CDK9 produced the most potent radiosensitization, with sensitizer enhancement ratios of 1.39-1.70 in validation studies -- exceeding those of established radiosensitizers including PARP and ATM inhibitors. Notably, knockdown of these genes induced no significant cytotoxicity in the absence of radiation. Pharmacological validation using an IKK inhibitor confirmed these findings, implicating non-canonical NF-{kappa}B signalling and CDK9-dependent transcriptional elongation as critical adaptive mechanisms in GBM radioresistance. ConclusionsClonoScreen3D-CRISPRi is a scalable, physiologically relevant platform for identifying genetic modifiers of radioresistance. The non-canonical NF-{kappa}B pathway and CDK9 represent promising radiosensitizing targets, and larger screens could enable systematic prioritisation of candidates for clinical translation. Key PointsO_LIClonoScreen3D-CRISPRi combines gene knockdown with 3D clonogenic survival assays C_LIO_LIWe identified NFKB2, RELB, and CDK9 as modifiers of radioresistance in two GBM cell lines C_LIO_LIValidation experiments show ClonoScreen3D-CRISPRi reliably identifies radiosensitizers in GBM C_LI Importance of the studyGlioblastoma (GBM) remains one of the most lethal human cancers, with radioresistance representing a central barrier to improved patient outcomes. While CRISPR-based screens have begun to illuminate genetic drivers of GBM biology, prior approaches using human models have largely relied on two-dimensional culture systems and short-term viability readouts that inadequately model the disease. This study introduces ClonoScreen3D-CRISPRi, a novel platform that integrates CRISPRi-mediated gene knockdown with three-dimensional clonogenic survival assays in patient-derived GBM cells -- more faithfully recapitulating the long-term clonogenic potential that underlies post-radiotherapy recurrence. Using this platform, we identified NFKB2, RELB, and CDK9 as potent genetic modifiers of radioresistance, with sensitizer enhancement ratios exceeding those of established clinical radiosensitizers such as PARP and ATM inhibitors. Pharmacological validation of the non-canonical NF-{kappa}B pathway demonstrates direct translational relevance, providing a rationale for targeting this axis in combination with radiotherapy to improve GBM treatment.

BackgroundTelomerase reactivation, a hallmark of many cancers, is associated with expression of its catalytic subunit, hTERT. However, the prognostic significance of telomere maintenance mechanisms in pediatric medulloblastoma remains poorly defined. MethodsIn this multi-institutional retrospective study of telomerase expression and hTERT regulation in newly diagnosed children with medulloblastoma, hTERT and MYC expression were assessed by qRT-PCR, normalized to non-neoplastic brain control samples. hTERT promoter methylation was analyzed using quantitative pyrosequencing and Illumina 450k methylation array. Cox proportional-hazard regression analyses evaluated the association of hTERT expression with progression-free survival (PFS) or overall survival (OS). Spearman correlation and Kruskal-Wallis tests correlated hTERT promoter methylation and expression and assessed variations among medulloblastoma subgroups, respectively. ResultsAmong 74 patients with available hTERT expression and outcome data, higher expression was associated with worse OS (HR=1.22, 95% CI: 1.01-1.47, p=0.036) and PFS (HR=1.17, 95% CI: 1.00-1.37, p=0.051) after adjusting for subgroup. Similar results were obtained when adjusting for metastatic status. Group 3 patients had the highest hTERT expression (p=0.001). Pyrosequencing data were available for 61 patients and 450k methylation array data for 292 patients. hTERT promoter was differentially methylated across subgroups with WNT followed by group 3 demonstrating the highest methylation on 450k (p<0.0001), findings that were confirmed by pyrosequencing. hTERT promoter methylation positively correlated with hTERT expression (Spearman correlation=0.42, p=0.02 by 450k and 0.34, p=0.007 by pyrosequencing). No significant correlation was observed between hTERT and MYC expression. ConclusionElevated hTERT expression is associated with worse PFS and OS in medulloblastoma across subgroups, supporting telomerase inhibition as a potential therapeutic strategy.

BackgroundGroup 3 (MYC-driven) medulloblastoma (MB) is a highly aggressive brain tumor with poor-prognosis and limited treatment options. We previously identified protein-arginine methyltransferase-5 (PRMT5) as a promising target in Group 3 MB with its control on MYC protein stability. In this follow up study, we further mechanistically investigated PRMT5 control on MYC transcription and targeted it pharmacologically for therapeutic proof-of-concept. MethodsUsing pharmacogenetic inhibition approaches against PRMT5 in MYC-amplified (Group 3) MB cell line and neurosphere models in vitro and in vivo, we investigated molecular mechanism(s) and anti-cancer efficacy of PRMT5 inhibition. ResultsOur experiments demonstrated that PRMT5 epigenetically regulates MYC transcription in MYC-amplified MB cells by binding to the proximal-promoter region of the MYC gene and contributing to the enriched symmetric-dimethylation of histone H4R3 in the same region. We further showed that PRMT5 is recruited to the MYC promoter by its interaction with BRD4, the major BET-protein responsible for MYC transcription. PRMT5 inhibition caused the suppression of MYC-induced transcriptional programs and target genes, with widespread disruption of splicing across the transcriptome, particularly affecting metabolism-related gene products. Pharmacologic inhibition of PRMT5 using a panel of selective small-molecule inhibitors demonstrates suppression of cell growth/survival in a MYC-dependent manner in MB cells. Moreover, our in vivo analyses of PRMT5 inhibition, in mice treated with one of the potent pharmacologic inhibitors, particularly a lipid-decorated form of it, demonstrated reduced cerebellar tumor growth with suppressed MYC expression and prolonged survival of mice with MYC-amplified MB xenografts. ConclusionsOur findings establish a functional link between PRMT5 and MYC-mediated transcriptional regulation, suggesting a promising therapeutic approach targeting the PRMT5-MYC axis for MYC-driven MB. Key PointsO_LIPRMT5 acts as an epigenetic regulator of MYC transcription, RNA splicing and associated energy metabolism in MYC-driven MB. C_LIO_LIPRMT5 inhibition selectively suppresses cell growth/survival in MYC-driven MB. C_LIO_LIPRMT5 inhibition reduces tumor burden and prolongs survival in a MYC-driven MB mouse model. C_LI Importance of the StudyGroup 3 medulloblastoma is a highly aggressive pediatric brain tumor marked by MYC amplification, malignant clinical behavior, and poor survival outcomes despite intensive multimodal therapy. Because MYC remains largely undruggable, there is an urgent need for effective and less toxic treatment options for affected children. This study identifies protein arginine methyltransferase 5 (PRMT5) as a key epigenetic regulator of MYC transcription and MYC-dependent oncogenic programs in Group 3 MB. We show that PRMT5 is recruited to the MYC promoter via BRD4, sustains MYC-driven transcription and RNA splicing networks associated with metabolism, and supports MB tumor growth. Importantly, pharmacologic inhibition of PRMT5 using a selective brain-penetrant inhibitor suppresses MYC expression, reduces cerebellar tumor burden, and prolongs survival in MYC-amplified MB models. These findings provide a strong translational rationale for PRMT5 inhibition as a targeted therapeutic strategy for high-risk MB, with the potential to improve outcomes while reducing treatment-related toxicity.

BackgroundThe Netrin-1 dependence receptor pathway plays critical roles in neural development, but its expression landscape and prognostic significance in glioblastoma (GBM) remain poorly characterized. MethodsSingle-cell RNA-seq data from 148,019 cells across 34 tumors (Neftel et al., 2019) were analyzed to map Netrin-1 pathway gene expression across GBM cellular states. Differential gene expression and pathway enrichment analyses were performed on NEO1-defined subpopulations. Bulk RNA-seq survival analysis was conducted across three independent GBM cohorts TCGA (n=106), CGGA mRNAseq_325 (n=137), and CGGA mRNAseq_693 (n=237), totaling 480 patients. Primary analysis used continuous Cox regression (per-SD hazard ratios); meta-analysis employed fixed-effects inverse-variance weighting. ResultsIn GBM single-cell data, Netrin-1 pathway genes showed state-specific enrichment --NEO1, DCC, NTN1, and RGMB were predominantly expressed in oligodendrocyte-precursor (OPC) and neural-progenitor (NPC) states. Cells positive for NEO1 were enriched for neural differentiation programs (nervous system development, p=9.6x10-; Axon Guidance, p=2.8x10-), whereas NEO1-negative cells were dominated by ribosomal/translational and immune activation programs. In the 3-cohort survival meta-analysis, NTN1 (Netrin-1 ligand) emerged as the sole gene reaching meta-analytic significance as a risk factor (Meta HR=1.163 per SD, 95% CI 1.056-1.281, p=0.0021, I{superscript 2}=0%, 3/3 cohorts concordant), while DCC and RGMB showed directionally consistent protective trends (DCC: Meta HR=0.938, 95% CI 0.858-1.025, p=0.156; RGMB: Meta HR=0.979, 95% CI 0.881-1.087, p=0.686; both 3/3 cohorts concordant). NEO1 itself did not independently predict survival (Meta HR=1.008, 95% CI 0.885-1.147, p=0.910). After Bonferroni correction for 10 genes tested (threshold p<0.005), only NTN1 met strict significance. In exploratory sex-stratified analysis of a single cohort (CGGA 693, n=237), NEO1 and NTN1 exhibited female-specific risk enhancement (NEO1: HR=1.417, p=0.014; NTN1: HR=1.249, p=0.019), with minimal effects in males. UNC5B showed context-dependent risk in MGMT-unmethylated tumors (HR=1.331, p=0.037). These sex-dimorphic findings require independent validation. ConclusionsThe Netrin-1 pathway exhibits divergent prognostic trends in GBM, with NTN1 as a risk factor and DCC trending toward protection--consistent with the dependence receptor model. These findings, which should be interpreted as hypothesis-generating, nominate NTN1 as a candidate therapeutic target and highlight the potential importance of sex-stratified evaluation in future Netrin-1-directed trials. Independent replication in larger cohorts is warranted.

Oligodendroglioma is a primary central nervous system tumor classified by the presence of isocitrate dehydrogenase (IDH) mutations and codeletion of 1p/19q. Here we describe the generation of an IDH-mutant 1p/19q-codeleted oligodendroglioma mouse model using in utero electroporation. We identified IDH1R132H, PIK3CAE545K, CicKO, Fubp1KO and Cdkn2aKO as the optimal combination (termed OligoCdkn2a) to drive fully penetrant tumors that histologically resemble human grade II/III IDH-mutant, 1p/19q-codeleted oligodendroglioma. Replacing Cdkn2a with Trp53 loss in this mouse model shifted tumor histology towards high grade astrocytoma. OligoCdkn2a tumors displayed metabolic and transcriptional changes associated with IDH and CIC mutations, and single cell sequencing identified a bias towards oligodendrocyte differentiation compared to an IDH wild-type glioblastoma mouse model. OligoCdkn2a tumors represent the first mouse model system to recapitulate the genetic, histological and transcriptional features of human IDH-mutant 1p/19q-codeleted oligodendrogliomas, offering a platform to further dissect tumor biology and test new therapeutic strategies.

Background/ObjectivesIncreasing evidence indicates that gliomas co-opt mechanisms of excitatory synaptic transmission and plasticity to support tumor progression, yet these processes remain poorly characterized in lower-grade gliomas (LGGs). Here, we investigated whether genes associated with excitatory synaptic function are linked to patient prognosis in LGG. MethodsA curated panel of 36 synaptic genes was analyzed in LGG using RNA-sequencing and clinical data from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) datasets. Correlations among gene expression levels were analyzed using the Evergene platform. ResultsAmong the genes investigated, DLG2, DLG3, and DLG4, which encode the postsynaptic scaffolding proteins PSD-93, SAP-102, and PSD-95, respectively, showed strong associations with patient overall survival (OS). Higher expression of each gene was consistently associated with longer OS across both datasets. Expression of DLG2-DLG4 was higher in oligodendroglioma and IDH-mutant, 1p/19q co-deleted tumors, and lower in astrocytoma and IDH-wild-type tumors. Furthermore, expression of all three genes positively correlated with a broad gene signature related to excitatory synaptic transmission and synaptic plasticity, including multiple components of glutamatergic signaling and postsynaptic organization. ConclusionsThese findings suggest that elevated expression of DLG2-DLG4 is associated with a transcriptional program resembling differentiated neuronal-like features and favorable clinical outcome in LGG. Simple SummaryLower-grade gliomas are brain tumors with highly variable outcomes, and better markers are needed to predict how patients will fare. Recent research suggests that these tumors may use mechanisms normally involved in communication between brain cells, but this is not well understood in these cancer types. In this study, we analyzed large patient datasets to examine genes related to synaptic function. We found that higher expression of three genes involved in synaptic membrane organization, DLG2, DLG3, and DLG4 was consistently associated with longer patient survival. These genes were also linked to a broader pattern of gene expression suggestive of neural transmission and plasticity. Our findings suggest that some lower-grade gliomas may adopt characteristics of normal brain cells that are associated with less aggressive behavior. This work may help guide future research on prognostic markers and improve understanding of brain tumor biology.

Gliomas comprise a heterogeneous group of central nervous system tumors in which gene fusions (GFs) are significant oncogenic drivers and emerging diagnostic and therapeutic biomarkers. In cancer diagnosis, GF detection largely relies on targeted short-read sequencing fusion panels, such as the Childrens Hospital of Philadelphia (CHOP) Fusion Panel (FUSIP). While these panels are effective for detecting recurrent, well-characterized GFs, they are limited to predefined gene sets and cannot identify full-length transcripts. Here, we analyzed 49 high-and low-grade gliomas previously classified as fusion-negative by FUSIP using an untargeted whole-transcriptome RNA sequencing approach with Oxford Nanopore Technologies (ONT) long-read sequencing. This enabled transcriptome-wide fusion discovery of additional known and potentially novel oncogenic GFs beyond panel constraints. Long-read sequencing further allowed direct resolution of full-length fusion transcripts and their associated isoform structures. By integrating GF detection with isoform-level transcript analysis, we identified fusion-associated transcript isoforms with alternative splicing patterns that aligned near reported GF breakpoints, including ZNF254::GNAS and PTPRK::NOX3, which have not been reported in literature or existing fusion databases. To assess functional relevance, candidate GFs were evaluated using the Drosophila melanogaster model, with ventral nerve cord (VNC) morphology serving as a quantitative in vivo readout of fusion-induced disruption of glial regulation. VNC enlargement or elongation reflects abnormal glial growth or defects in brain tissue organization. Of the 15 candidate GFs subjected to experimental functional testing, 8 induced significant VNC abnormalities relative to wild-type controls, indicating fusion-specific disruption and oncogenic potential. Notably, CLDND1::WRN and DUSP22::APOE produced the most pronounced VNC phenotypes. Together, these findings demonstrate that untargeted transcriptome-wide GF discovery, coupled with long-read isoform-level analysis and in vivo functional validation, enables the identification and prioritization of potentially novel and clinically relevant GFs that are missed by standard targeted short-read fusion panels in glioma.

Background and ObjectivesIntramedullary spinal cord tumors (IMSCTs) are rare, and the extent of surgical resection may influence overall survival (OS). Gross total resection (GTR) may offer superior outcomes compared to subtotal resection (STR) or biopsy. Our study seeks to quantify the benefits of resection extent on OS in patients with spinal gliomas (SGs). MethodsA systematic review was conducted using the following databases: Scopus, Embase, and PubMed. Studies reporting OS in patients who underwent GTR, STR, or biopsy for low- or high-grade SG. We used a random-effects model to calculate pooled hazard ratios (HRs) and 95% confidence intervals (CIs); this was performed separately for low-grade (WHO grade I-II) and high-grade (III-IV) SGs. Subgroup analysis was performed for radiotherapy. I2 statistic and Cochrans Q tests evaluated study heterogeneity, Eggers and funnel plot asymmetry tests assessed publication bias, and Risk Of Bias In Non-randomized Studies of Exposure (ROBINS-E) evaluated individual study bias. ResultsIn a pooled analysis of 5 studies, GTR was not associated with improvement in OS compared to STR or biopsy in high grade SGs (HR=0.48, 95% CI: 0.19 -1.26). However, low-grade SGs revealed significant benefit in overall survival with GTR (HR=0.27, 95% CI: 0.15-0.46). Patients treated with radiotherapy were associated with worse outcomes following GTR in low-grade SGs (HR=1.48, 95% CI: 1.30-1.69) but no survival differences in high-grade SGs (HR=1.21, 95% CI: 0.52-2.83). ROBINS-E determined only 1 study with high risk of bias. ConclusionGTR for intramedullary spinal gliomas may not confer a significant benefit in overall survival for high-grade lesions but may provide benefit in lower grades. Radiotherapy confers a worse survival in lower-grade tumors, potentially due to their infiltrative nature. Future studies should stratify outcomes based on tumor biology, as well as follow functional outcomes overtime.

Diffuse intrinsic pontine glioma (DIPG) is a devastating pediatric brain tumor with limited treatment options. Emerging evidence indicates infiltration of tumor-associated macrophages (TAMs) within tumor sites, accompanied by an immunosuppressive tumor microenvironment (TME). However, the mechanisms underlying macrophage recruitment and communication between TAMs and other cellular compartments within brain tumors remain poorly understood. Bulk RNA sequencing of 26 DIPG autopsy specimens with matched normal brain tissue, single-cell RNA sequencing data from eight DIPG patients integrated with public pediatric high-grade glioma (pHGG) datasets, and in vitro transwell and flow cytometry assays collectively indicated that DIPG tumors actively recruit monocytes through chemokine-mediated mechanisms. The chemokine expression of tumor cells is driven by a mesenchymal-like (MES-like) lineage state rather than histone mutations, as evidenced by significant correlation between MES-like lineage scores and chemokine expression scores across 46 pHGG cell lines. CellChat analysis identified APP-CD74 signaling as a prominent tumor cell-TAM interaction pathway, supported by immunofluorescence validation. Notably, APP expression was significantly reduced in DIPG tumor tissues compared with normal brain tissue at both the RNA and protein levels. Recombinant APP stimulation of THP-1-derived macrophages induced a robust proinflammatory response, including upregulation of M1-like markers, enrichment of interferon-related pathways, and elevated secretion of inflammatory cytokines. Collectively, these findings indicate that APP suppression in tumors attenuates the antitumor activity of TAMs and promotes an immunosuppressive microenvironment. Furthermore, protein modeling and docking analyses identified the APP-CD74 binding interface, providing a structural basis for therapeutic targeting.

Importance: Glioblastoma (GBM) cells integrate into neuronal circuits, and preclinical work implicates multiple neurotransmitter (NT) networks as key drivers of invasion and treatment resistance. Whether the integration of GBM within NT-defined large-scale brain networks conveys prognostic information for overall survival (OS) is unknown. Objective: To determine whether NT-specific network involvement of GBM is associated with OS in patients with newly diagnosed Isocitrate dehydrogenase (IDH)-wildtype(wt) GBM. Design, Setting, and Participants: In this observational multicenter cohort study, we analyzed two independent cohorts of adults with histopathologically confirmed IDH-wt GBM. Cohort 1 included 153 patients treated at the University Medical Center Hamburg-Eppendorf, Germany (2012-2024), and cohort 2 comprised 264 patients from the University of Pennsylvania Health System, USA (2006-2018). Preoperative contrast-enhanced MRI was used to derive individual tumor masks, which were spatially mapped onto normative NT-informed structural connectomes spanning 19 receptor and transporter systems. Exposures: Preoperative contrast-enhancing GBM lesions, quantified as patient-specific involvement scores (0-1) within each NT-defined brain network. Statistics: We used partial least-squares regression for variable selection and multivariable Cox proportional-hazards models alongside regularized logistic regression with out-of-sample prediction, adjusted for age, methylguanine methyltransferase (MGMT) promoter methylation, and extent of resection, to test associations between NT-specific GBM network involvement and OS. Results: Across 417 patients in two cohorts, greater GBM involvement within cholinergic networks, defined by normative vesicular acetylcholine transporter (VAChT)-weighted as well as dopaminergic D2 receptor involvement, was consistently associated with reduced OS, independent of age, MGMT status, and resection extent. Further, cholinergic network involvement showed the strongest contribution to the prediction models. Other NT networks did not show reproducible prognostic effects across cohorts. Tumor-intrinsic hypomethylation of acetylcholine receptor-associated regions correlated with imaging-based cholinergic network involvement and mirrored its prognostic relevance. Conclusion and Relevance: Tumor integration into neurotransmitter-specific brain networks is an independent predictor of poorer survival in GBM. By combining routine clinical MRI with normative NT-informed connectome data, this approach delineates a novel systems-level marker of tumor aggressiveness and supports cholinergic inhibition as a putative therapeutic target in GBM.

BackgroundFLASH-RT defines a promising treatment modality against medulloblastoma, as it minimizes treatment-related complications. To support its clinical translation, we dissected the cellular and molecular determinants of the FLASH response in the tumor-microenvironment (TME) and healthy hippocampus using an orthotopic human medulloblastoma mouse model treated with a hypo-fractionated FLASH regimen. MethodsFive cohorts of 4 weeks-old UW228-MB-bearing female nude mice (n=57) were irradiated, or sham-irradiated using 3x10 Gy (BED=60), delivered 48h apart at 0.1 Gy/s (CONV) or 5.5x106 Gy/s (FLASH) using an electron beam (eRT6). Digital spatial profiling (DSP) was performed 24h after radiotherapy in one cohort, while the four other cohorts were followed for long-term tumor response, cognition, and neuroinflammation. ResultsBoth CONV and FLASH-RT induced a complete and long-lasting anti-tumor response in 100% of animals associated with cognitive decline. However, more mice maintained a very good discrimination score after FLASH exposure (38%) than CONV (7%). DSP revealed a sustained microglial activation in the cerebellar tumor micro-environment, where FLASH enhanced expression of genes with phagocytic and proteolytic activity. In the tumor free hippocampus, FLASH exposure induced a preferential neuron/astrocyte transcriptional crosstalk, which manifested over protracted times to minimize neuroinflammation and cognitive complications. ConclusionThe study shows the tumor-ablative efficacy of hypo-fractionated FLASH-RT in a human medulloblastoma mouse model. It is associated with qualitatively distinct transcriptional signatures prone to tumor and debris clearance mediated by microglial cells of the TME. Moreover, in the hippocampus, FLASH mitigates radiation-induced neurotoxicity by enhancing genes involved in synaptic plasticity, attenuating neuroinflammation, and preserving metabolic function. Key PointsO_LIComplete response of medulloblastoma and reduction of neurotoxicity with hypo-fractionated FLASH regimen. C_LIO_LIClearance-prone phagocytic and proteolytic activity in the microglia of the TME. C_LIO_LINeuron/astrocyte transcriptional crosstalk in the hippocampus. C_LI Importance of the studyThis study constitutes a milestone for the future implementation of FLASH-RT in the treatment of children with brain cancer. It shows that FLASH does not protect medulloblastoma and on the contrary can be ablative when delivered in 3 fractions of 10 Gy. FLASH promotes a metabolically active, phagocytosis-prone phenotype in microglial cells consistent with immune activation and tumor surveillance, in contrast to the proliferative and immunosuppressive signaling programs induced by CONV. It also shows how FLASH may differentially shape long-term brain function in patients with brain tumors by modifying the transcriptional program of hippocampal subregions known to be critical for memory encoding, pattern separation, and consolidation. In summary, this study supports the idea that FLASH has the potential to shift treatment paradigms and change the dismal therapeutic outcome in patients with brain cancer.

Glioblastoma (GBM) is the most prevalent primary brain cancer, with poor prognosis and limited therapeutic options available. The genetic and cellular heterogeneity characteristic of GBM contributes to poor response rates. Activating mutations of the epidermal growth factor receptor (EGFR) gene are among the most frequent alterations in GBM, occurring in roughly half of cases. Despite the prevalence of EGFR mutations, EGFR inhibition has shown limited success in GBM. The transcription factor C/EBP{beta} is a master regulator of the mesenchymal transformation in GBM, an aggressive state characterized by increased invasiveness and resistance to chemotherapy. Lucicebtide is a C/EBP{beta} antagonist peptide with demonstrated single agent activity in patients with recurrent GBM that is currently being evaluated in a clinical trial in combination with radiation and temozolomide in patients with newly-diagnosed GBM (NCT04478279), with emerging data supporting clinical activity in that setting. Here we show that in the TCGA-GBM dataset, patients with EGFR mutations display significant enrichment of a high C/EBP{beta} activity signature. Functionally, genetic inactivation of EGFR by CRISPR results in synthetic lethality in the presence of lucicebtide in GBM cell lines, and synergistic in vitro cytotoxicity and suppression of C/EBP{beta} target gene expression was observed in combination experiments with lucicebtide and EGFR inhibitors. Finally, enhanced anti-tumor activity was demonstrated in vivo in the combination setting, as combined subpharmacologic dose levels of lucicebtide and the EGFR inhibitor osimertinib potently suppressed GBM xenograft growth. These data identify EGFR and C/EBP{beta} dependencies in GBM and support lucicebtide combination with EGFR inhibitors as a potential therapeutic option for a sizable fraction of GBM patients.

BackgroundGlioblastoma (GB) is a highly aggressive brain tumor with a median survival of approximately 14 months, primarily due to its ability to infiltrate healthy brain tissue both as single cells and in collectives. A deeper understanding of GB cell motility, both individual and collective, is crucial for developing patient-specific therapies. We aimed to characterize migration in patient-derived GB cells using advanced modeling to identify stratification markers and therapeutic vulnerabilities. MethodsWe developed Single-Cell Behavior Live Imaging (ScBLI), an approach integrating live imaging with computational analysis, applied to 30 GB primary cell cultures. Trajectories and morphological features were tracked and analyzed. Diffusion Entropy Analysis (DEA) was applied to classify trajectories based on the Delta Scaling parameter ({delta} scaling). We evaluated functional responses correlating all findings with clinical outcomes and transcriptomic profiles. ResultsWe analyzed 4,279 cell trajectories. Based on {delta} scaling (range 0.28-0.837), we defined three distinct motility groups: Low (L, {delta} scaling [&le;]0.5), Medium (M, 0.5 < {delta} scaling [&le;] 0.7), and High (H, {delta} scaling >0.7). Functional assays demonstrated that Group H cells are more performant in both positive and negative chemotaxis. Clinically, the three groups showed a clear linear progression with patient survival: High {delta} scaling correlated with the shortest survival (poorer prognosis), while Low {delta} correlated with the longest survival, suggesting that structured motility drives invasiveness. Integrative multi-omic analysis, encompassing both exome and transcriptome profiling, demonstrated that these groups are defined by distinct molecular landscapes rather than poor behavioral traits. Moreover, exome data revealed that Group H is significantly enriched in PTEN alterations (75% vs. 8% in Group L), with PTEN gain-of-function (GoF) mutations exclusively restricted to this group (100% vs 0% in Group L). Notably, within our extended cohort (n=51) currently characterized by whole-exome sequencing, we observed that specific PTEN GoF mutations were associated with a significantly shorter survival compared to PTEN wild-type cases (median OS 6.4 vs 16.6 months; p=0.02), which typically harbor the canonical loss of chromosome 10q. A similar clinical trend was observed when comparing directly GoF carriers to patients with truncating (Ter) alterations (median OS 6.4 vs 14.3 months; p=0.09). Conversely, no survival difference was found between truncating (Ter) mutations and wild-type cases. ConclusionOur findings demonstrate for the first time that migratory efficiency, quantified through DEA, represents a powerful predictor of glioblastoma aggressiveness. Tumor cells adopting highly efficient exploration strategies are strongly associated with poor clinical outcomes and are characterized by distinct molecular signatures, notably PTEN gain-of-function alterations. Statement of significanceOur multi-scale computational framework elucidates emergent behavioral phenotypes as pivotal drivers of glioblastoma progression. By demonstrating a correlation between enhanced migratory efficiency, PTEN gain-of-function, and significantly reduced overall survival, we establish a foundational paradigm for deciphering the emergent complexity governing tumor invasiveness.

Background Platinum refractory paediatric germ cell tumours (GCTs) carry a poor prognosis, with five year survival below 30% and no validated molecular stratification tool. The biological mechanisms underlying platinum resistance in this population remain poorly defined, limiting the development of targeted therapeutic strategies and early warning biomarkers. Methods We performed integrated plasma multi-omics profiling in 105 pediatric GCT patients (54 refractory and 51 treatment naive) using data-independent acquisition proteomics, untargeted metabolomics, and exploratory lipidomics. Candidate biomarkers were validated using ELISA and spatial multiplex immunofluorescence. Predictive models were constructed using logistic regression and evaluated by ROC analysis, calibration, and decision-curve analysis. Results Multiomics integration has revealed the coordinated dysregulation of sphingolipid metabolism, extracellular matrix remodeling, and immune checkpoint signaling in refractory diseases. Lipidomic analysis demonstrated a significant depletion of sphingolipid associated species, including lysophosphatidylserine, lysophosphatidylethanolamine, and phosphatidylserine. Proteomic profiling identified the upregulation of LAG3 and HTRA1, which was validated by ELISA. Multiplex immunofluorescence demonstrated the spatial enrichment of exhausted CD8 + LAG3 T cells adjacent to CK-PAN tumor cells in refractory tumors. A plasma biomarker panel integrating LAG3, HTRA1, and AFP showed improved discrimination of refractory disease (AUC = 0.821) compared with AFP alone. Conclusions Our study identified a sphingolipid HTRA1 LAG3 immune evasion axis as a defining molecular feature of refractory pediatric germ cell tumors and proposed a clinically applicable plasma biomarker panel for early risk stratification.

Background: Extent of resection remains central to meningioma management, yet Simpson grading is subjective and may not reflect measurable postoperative residual disease. We compared surgeon-reported Simpson grade, report-derived radiological grading, and residual tumour volumetry across a multicentre cohort. Methods: We performed a retrospective study across two tertiary neurosciences centres comprising four hospitals, including patients undergoing primary cranial meningioma resection from 2006 to 2025. Postoperative magnetic resonance imaging (MRI) reports were harmonised using weakly supervised natural language processing based on term frequency-inverse document frequency (TF-IDF) and a linear support vector machine classifier. Residual tumour volume was segmented from contrast-enhanced postoperative MRI and log-transformed. Concordance between Simpson and radiological gross-total/subtotal resection classification was assessed using absolute agreement and prevalence-adjusted bias-adjusted kappa (PABAK). Cox models assessed recurrence-free survival, with bootstrap validation and anatomical and scan-timing sensitivity analyses. Results: Among 912 patients, recurrence or residual progression occurred in 281. Surgical-radiological agreement was substantial but imperfect (absolute agreement 74%; PABAK 0.61), with lower agreement in skull-base and parafalcine-parasagittal tumours. In adjusted models, recurrence hazard increased with Simpson grade (hazard ratio 1.54, 95% confidence interval 1.37-1.72), radiological grade (1.92, 1.68-2.20), and log-transformed residual volume (1.20, 1.16-1.24; all p<0.0005). Optimism corrected concordance increased from Simpson grade to radiological grade and log-volumetry (0.692, 0.733, and 0.748), with this ranking preserved across sensitivity analyses. Conclusions: Imaging-based postoperative residual disease measures outperformed Simpson grade. TF-IDF-assisted report-derived grading provides a scalable bridge to volumetry, while quantitative residual volume offers the strongest prognostic representation.

BackgroundGangliogliomas (GGs) are low-grade glioneuronal tumors that frequently present with drug-resistant epilepsy. Although their indolent course contrasts with their high epileptogenic potential, the oncogenic mechanisms sustaining neuronal precursor-like populations within the tumor microenvironment remain poorly defined. MethodsWe performed spatial transcriptomic profiling on eight histologically confirmed GGs and matched healthy cortex to map the cellular and molecular architecture of the tumor microenvironment. Integrated analysis with weighted gene correlation network analysis (WGCNA) defined recurrent oncogenic programs and spatially resolved tumor-stroma interactions. ResultsEight conserved gene modules emerged, encompassing physiological cortical, reactive glial, and oncopathological programs. The latter captured extracellular matrix (ECM) remodeling, vascular-immune signaling, and persistence of immature, proliferative neuronal-like states. Spatial modeling revealed that these oncopathological programs form structured niches at the tumor-brain interface, where radial glia-derived neuronal-like tumor cells coexist with immune and stromal elements engaged in ECM turnover and cytokine signaling. ConclusionsGanglioglioma represents a hybrid glioneuronal neoplasm in which developmental neuronal programs are co-opted by tumor-associated stromal and immune cues. This convergence establishes a permissive oncogenic niche that sustains precursor-like tumor cells and provides a mechanistic basis for both the tumors benign growth and its intrinsic epileptogenicity. Key PointsO_LISpatial transcriptomics identifies reproducible transcriptional programs that define the ganglioglioma microenvironment. C_LIO_LITumor-associated regions show transcriptional programs consistent with immature neuronal states together with ECM remodelling and immune activity. C_LIO_LISingle-cell reference data indicate that immature neuronal programs in ganglioglioma resemble radial glia-derived developmental states. C_LI Importance of the StudyGanglioglioma is a low-grade glioneuronal tumor that combines benign growth with pronounced epileptogenicity, yet the molecular basis of this dual behavior remains poorly understood. Through spatial transcriptomics integrated with single-cell analysis, we reveal that ganglioglioma architecture is defined by two interacting transcriptional axes: a residual glioneuronal network and a tumoral niche enriched for extracellular-matrix, vascular, and immune programs. Within these niches, immature neuronal-like tumor cells persist in a developmentally arrested state maintained by ECM-immune signaling. This spatially organized interplay between physiological and pathological programs explains both the low oncologic aggressiveness and high excitability of these lesions. Our findings provide molecular signatures that may refine diagnostic classification within the LEAT spectrum, delineate epileptogenic zones, and identify candidate pathways for therapeutic modulation of the ganglioglioma microenvironment.

Atypical teratoid rhabdoid tumor (ATRT) is a malignant brain tumor of children that has an overall survival of less than 40 percent even with aggressive therapy. We identified upregulation of the mitogen activated protein (MAP) kinase pathway in ATRT. The novel, brain-penetrant MEK inhibitor mirdametinib inhibited the growth of ATRT cell lines in culture at nanomolar concentrations. Mirdametinib suppressed proliferation as measured by BrdU incorporation and induced apoptosis as measured by cPARP and Annexin V staining. Monotherapy with mirdametinib extended the life of mice bearing orthotopic xenografts. Combination therapy with the brain-penetrant cyclin dependent kinase 4/6 inhibitor abemaciclib further suppressed growth and BrdU incorporation in ATRT cell lines representing all molecular subgroups. Mirdametinib and abemaciclib combined to extend survival of mice bearing orthotopic ATRT xenografts. In conclusion, mirdametinib has single agent activity against ATRT and combines with abemaciclib to decrease proliferation and extend survival in orthotopic xenograft models of ATRT.

BackgroundConvection-enhanced delivery of topotecan enables sustained local chemotherapy for recurrent glioblastoma and was associated with reduced tumor proliferation in our previous phase 1B clinical trial. That trial incorporated a paired pre- and post-treatment biopsy design - rare in glioblastoma clinical research - enabling tissue-anchored assessment of drug effect without reliance on radiographic or survival endpoints, which are notoriously difficult to interpret in this disease. However, the cellular and molecular consequences of local chemotherapy within the treated tumor microenvironment remain incompletely defined. MethodsWe integrated paired, MRI-localized pre- and post-treatment biopsies from a first-in-human CED-topotecan trial (n=5), leveraging the paired biopsy architecture, in which each patient serves as their own control and post-treatment specimens are spatially annotated relative to the MRI-defined infusion zone, to generate tissue-based evidence of drug effect without requiring large patient numbers. These biopsies were integrated with complementary experimental models, including a time-resolved syngeneic murine glioma CED model, acute patient-derived glioblastoma slice cultures, and in vitro human microglial and glioma systems. Clinical biopsies were analyzed by bulk RNA-seq, cell-type deconvolution, and multiplex immunofluorescence. Murine tumors were analyzed by survival, immunofluorescence, and single-cell RNA-seq; patient-derived slice cultures were profiled by single-cell RNA-seq. ResultsIn paired human biopsies, CED-topotecan induced spatially restricted transcriptional remodeling within the infusion zone, characterized by suppression of proliferative tumor programs and enrichment of inflammatory, interferon, hypoxia, and mesenchymal signatures. Cell-type deconvolution and immunofluorescence linked this response to myeloid remodeling, including enrichment of monocyte-derived tumor-associated macrophage states, increased MARCO-positive myeloid populations, and pH2AX-positive genotoxic stress within Iba1-positive myeloid cells. In the murine CED model, topotecan prolonged survival and reduced tumor cellularity, while also inducing inflammatory and DNA-damage programs in tumor-associated macrophages that evolved by 7-days toward hypoxia, angiogenesis, TGF-{beta} signaling, and mesenchymal/tissue-remodeling programs. Human slice culture and in vitro microglial systems confirmed stress-coupled inflammatory and DNA-damage responses in human myeloid cells. ConclusionsLocal topotecan delivery produces spatially structured tumor cytotoxicity together with a genotoxic, stress-coupled inflammatory myeloid response that evolves toward mesenchymal macrophage remodeling. By integrating paired clinical biopsies with time-resolved and mechanistic experimental models, this study provides a framework for understanding how local chemotherapy reshapes the glioblastoma microenvironment and for future studies evaluating dose, schedule, treatment duration, and combination strategies. These findings demonstrate that paired, spatially annotated tissue sampling from small, precisely characterized clinical cohorts can yield mechanistic insight that conventional radiographic and survival endpoints cannot provide, and support tissue-based response assessment as the appropriate paradigm for evaluating novel locoregional therapies in glioblastoma.

IntroductionCraniopharyngioma (CP) comprises two distinct histological subtypes, adamantinomatous craniopharyngioma (ACP) and papillary craniopharyngioma (PCP), which are often challenging to distinguish preoperatively. Approximately 95% of PCP harbor the BRAF V600E mutation, whereas ACP lacks this alteration, making PCP uniquely sensitive to BRAF and MEK inhibition. However, in the absence of a reliable preoperative classification strategy, targeted therapy has been limited to recurrent disease or to cases with histological confirmation. This study aims to describe and prospectively evaluate a pragmatic preoperative classification strategy and short-course neoadjuvant BRAF and MEK inhibition followed by surgery in newly diagnosed, preoperatively classified PCP. Methods and analysisThis is a prospective, single-arm, open-label study. Patients with newly diagnosed craniopharyngioma will be screened using an integrated preoperative strategy combining imaging-based prediction and selective cerebrospinal fluid (CSF) cell-free DNA testing for BRAF V600E in indeterminate cases. Twelve participants preoperatively predicted as PCP and BRAF V600E positive will receive dabrafenib 150 mg twice daily plus trametinib 2 mg once daily for up to three 28-day cycles, followed by transnasal endoscopic surgery. Assessments are scheduled at days 7, 14, 28, 56, and 84 until surgery. The primary endpoint is objective response rate, assessed by contrast-enhanced MRI using RANO 2.0 criteria. Secondary outcomes include progression-free survival, local disease control, endocrine outcomes of the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-thyroid axes, visual and cognitive outcomes, postoperative diabetes insipidus, surgical complexity, and concordance between the preoperative classification strategy and postoperative pathology and BRAF V600E status. Exploratory analyses will evaluate treatment-related changes in tumor vascularity, tissue characteristics, and post-treatment molecular alterations in tumor tissue. Ethics and disseminationThis protocol has been approved by the Ethics Committee of Huashan Hospital, Fudan University (KY2024-028). Written informed consent will be obtained from all participants. Results will be disseminated through peer-reviewed publications and scientific conferences. Trial registration numberChiCTR2400081636 STRENGTHS AND LIMITATIONS OF THIS STUDYO_ST_ABSStrengthC_ST_ABS[tpltrtarr] This study proposes an integrated, clinically applicable preoperative strategy that combines imaging-based prediction with selective cerebrospinal fluid cell-free DNA analysis to identify papillary craniopharyngioma (PCP) prior to surgery. [tpltrtarr]It prospectively evaluates short-course neoadjuvant BRAF and MEK inhibition in newly diagnosed PCP, addressing a clinically relevant gap in current management. [tpltrtarr]Standardized, multidimensional assessments are performed across the neoadjuvant, perioperative, and early postoperative periods, capturing radiographic, surgical, endocrine, visual, and cognitive outcomes. Limitation[tpltrtarr] The single-arm, open-label design without a surgical control group limits direct comparison with upfront surgery. [tpltrtarr]Despite the integrated prediction strategy, preoperative misclassification cannot be excluded entirely.

Meningiomas are the most common primary central nervous system tumors in adults, posing a significant burden to society. Although a large percentage of lower-grade meningiomas are curable by surgery or radiation alone, high-grade and a subset of low-grade meningiomas demonstrate recurrences and complications from treatment. Systemic therapies for meningioma remain ineffective, and no targeted treatments are approved. Despite the central role of YAP1/TAZ-TEAD signaling in NF2-deficient/mutant tumors, no studies have systematically examined TEAD inhibition across molecularly defined meningioma subtypes or investigated mechanisms of resistance in this disease. We have recently shown that YAP1/TAZ signaling is an oncogenic driver of meningioma. Here, using established and patient-derived meningioma cell lines, we demonstrate that genetic ablation of YAP1/TAZ suppresses growth in both NF2 mutant and NF2 wild type cell lines, establishing YAP1/TAZ-TEAD signaling as a shared oncogenic dependency. Pharmacologic TEAD inhibition suppressed growth of benign NF2 mutant and a subset of higher-grade NF2 mutant meningiomas, whereas NF2 wild type meningiomas were generally more resistant. RNA-Seq and Western Blot analysis identified compensatory activation of MEK-ERK, mTOR-S6, and FAK signaling in resistant lines exhibit. Importantly, co-targeting these pathways was able to overcome resistance to TEADi and was superior to MEK/mTOR/FAK inhibition alone. These studies provide a compelling proof-of-concept that TEADi represents a novel therapeutic vulnerability in meningioma and reveal adaptive signaling responses that can be therapeutically exploited.