Neuro-Oncology

BackgroundMost genomic studies of adult-type diffuse gliomas have focused on predominantly European ancestry populations, limiting the generalizability of molecular classifications and precision medicine approaches. We assembled a multi-institutional glioma cohort of diverse patients to investigate how germline ancestry, molecular subtypes, and mutational processes shape tumor biology and clinical outcomes. MethodsWe analyzed 1,102 adults with WHO 2021-classified diffuse gliomas (IDH-mutant, 1p/19q-codeleted oligodendroglioma; IDH-mutant astrocytoma; IDH-wildtype glioma) from seven U.S. institutions. Whole-exome sequencing (WES) of FFPE tumors identified somatic alterations and COSMIC SBS v3.2 mutational signatures. Genetic ancestry was estimated from WES using 1000 Genomes reference populations. Overall survival was assessed using Kaplan-Meier and multivariable models. ResultsThe cohort included 66.9% European (EUR), 21.1% Admixed American/Hispanic (AMR), 10.3% Admixed African (AFR), and 1.6% Asian (AS) ancestry. Survival followed expected molecular hierarchy (median overall survival: oligodendroglioma 15.7 years, astrocytoma 10.6 years, IDH-wildtype glioma 1.9 years). Within oligodendroglioma, AMR patients showed improved survival versus EUR (HR 0.67, 95% CI 0.48-0.94; p=0.011), with similar trends across subtypes. Somatic profiling confirmed canonical subtype-defining alterations and revealed higher ATRX alterations in AFR and AMR IDH-wildtype tumors compared with EUR. ATRX alterations were associated with improved survival only in AFR (p=0.003). Mutational signature analysis identified subtype-specific signatures, including therapy-associated signatures. Chemotherapy-related signatures were more frequent in EUR and AMR than in AFR. ConclusionsThis ancestrally diverse glioma cohort confirms established molecular classifications and identifies ancestry-associated differences in survival, somatic alterations, and mutational processes, indicating the critical need for broad representation to inform precision neuro-oncology. Key PointsO_LIA multi-institutional glioma cohort validates subtype and survival patterns across ancestries. C_LIO_LITherapy-associated mutational signatures differ by ancestry, suggesting distinct treatment-related mutational processes. C_LIO_LIAdmixed American patients show improved survival, particularly in oligodendroglioma. C_LI Importance of the StudyMost genomic studies of adult-type diffuse glioma have focused on populations of predominantly European ancestry which limits the ability to examine variation in tumor biology and clinical outcomes across populations. In this study, we assembled one of the largest ancestrally diverse cohorts of molecularly characterized adult diffuse gliomas, integrating germline ancestry inference with tumor whole-exome sequencing and mutational signature analysis. We confirm that established molecular classifications and survival hierarchies remain robust across ancestry groups. However, we also identified ancestry-associated differences in survival within specific tumor subtypes, higher ATRX alteration frequencies in African American and admixed American patients with IDH-wildtype tumors, and variation in therapy-associated mutational signatures across ancestry groups. These findings highlight the importance of incorporating population differences into genomic studies of glioma and provide a resource for future multi-ancestry investigations of glioma risk, tumor evolution, and treatment response, ultimately supporting more inclusive precision neuro-oncology.

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.

Medulloblastoma and ependymoma are common pediatric central nervous system tumors with significant molecular and clinical heterogeneity. We collected bulk RNA sequencing data from 888 medulloblastoma and 370 ependymoma tumors to establish a comprehensive reference landscape. Following rigorous batch effect correction, normalization, and dimensionality reduction, we constructed a unified landscape to explore gene expression, signaling pathways, RNA fusions, and copy number variations. Our analysis revealed distinct clustering patterns, including two primary ependymoma compartments, EPN-E1 and EPN-E2, each with specific RNA fusions and molecular signatures. In medulloblastoma, we observed precise stratification of Group 3/4 tumors by subtype and in SHH tumors by patient age. This landscape serves as a vital resource for identifying biomarkers, refining diagnoses, and enables the mapping of new patients bulk RNA-seq data onto the reference framework to predict biology and outcome from nearest neighbor analysis facilitate accurate disease subtype identification. The landscape is accessible via Oncoscape, an interactive platform, empowering global exploration and application. One Sentence SummaryA landscape built using only Transcriptomic analysis for medulloblastoma and ependymoma reveals novel insights about subtype-specific biology.

BackgroundDiffuse midline glioma (DMG) is a lethal pediatric brain tumor driven by the H3K27M oncohistone, which disrupts epigenetic regulation and promotes tumor proliferation. While prior studies show that H3K27M is essential for tumor initiation, its role in established tumors, tumor microenvironment (TME) regulation, and therapeutic response remain unclear. MethodsHere, we developed inducible and reversible H3.3K27M and H3.1K27M cell and mouse models to study oncohistone-dependent effects on tumor growth, recurrence, and the immune/stromal microenvironment. We generated a tetracycline-inducible PiggyBac-based oncohistone expression cassette in patient- and murine-derived models and validated inducible and reversible H3K27M expression. ResultsRe-expression of H3K27M in knockout cells induced morphological changes and suppressed astrocytic markers. Chromatin accessibility profiling revealed distinct states between ON, OFF, and OFF-ON groups, including PD1-mediated immunosuppressive mechanisms associated with H3K27M expression. Single-cell RNA sequencing demonstrated that the oncohistone reshapes the TME. H3K27M expression promotes tumor-neuron interactions, enhances neuronal excitability, excitatory/inhibitory imbalance, and synaptic connectivity that supports tumor proliferation. These effects are associated with increased glutamatergic signaling and enhanced tumor-neuron coupling through glutamate transport and receptor pathways, including EAAT1 (SLC1A3) and AMPARs (GRIA3). Conversely, H3K27M inhibition reduces neuronal excitation, disrupts tumor-associated signaling, and partially restores neuron-neuron and neuron-immune communications. These findings identify H3K27M as a key driver of excitatory neuron-to-tumor coupling and immunosuppression in DMG. ConclusionsOverall, our findings demonstrate that H3K27M extensively reshapes TME in DMG and support direct oncohistone targeting as a potential therapeutic strategy, including potential CRISPR-based or small-molecule approaches for patients with H3K27M-mutant DMG. Key PointsO_LIWe developed inducible and reversible H3K27M DMG models to investigate the role of H3K27M in the tumor microenvironment. C_LIO_LIH3K27M promotes tumor-neuron communication, while its inhibition disrupts these interactions, supporting H3K27M-targeted therapies for DMG. C_LI Importance of StudyDiffuse midline glioma (DMG) remains one of the deadliest pediatric brain tumors, with limited effective treatment options and poor patient survival. Although the H3K27M oncohistone is recognized as a key driver of tumor initiation, its role in maintaining tumor progression and shaping the tumor microenvironment is unclear. In this study, we developed inducible and reversible H3.3K27M and H3.1K27M murine and patient-derived DMG cell- and mouse-models that enabled precise control of the oncohistone expression. Using these models, we demonstrate that H3K27M actively promotes tumor-neuron interactions, neuronal excitability, and glutamatergic signaling pathways that support tumor growth. Importantly, inhibition of H3K27M disrupted these tumor-associated signaling networks and partially restored neuron-immune communication within the tumor microenvironment. Together, these findings demonstrate that H3K27M extensively reshapes the tumor microenvironment in these Diffuse Midline Gliomas and provides strong rationale for directly targeting the oncohistone as a therapeutic strategy for patients with H3K27M-mutant DMG. Lay SummaryDiffuse Midline Glioma (DMG) is a devastating childhood brain cancer. Despite decades of research, radiation remains the primary treatment and provides only temporary benefit. Most DMGs carry a mutation called H3K27M, which is an attractive target for new treatments such as directly inhibiting or removing this mutation using gene-editing. However, it remains unclear whether inhibiting H3K27M alone will be sufficient to stop the growth of established tumors. In this study, we developed human and mouse models that allow H3K27M to be turned on and off. We found that H3K27M helps tumors communicate with surrounding cells, particularly neurons. Inhibiting H3K27M disrupted tumor-promoting interactions and partially restored normal communication, supporting direct H3K27M-targeted therapies as a promising strategy for children with DMG.

BackgroundMedulloblastoma is the most common malignant pediatric brain tumor, and has an urgent need for novel treatment approaches. Dordaviprone (ONC201) and its chemical derivative with nanomolar potency, ONC206, induce apoptosis of cancer cells by activation of the mitochondrial caseinolytic protease P (ClpP). ONC206 is currently in Phase I clinical trials for pediatric patients with primary brain tumors. MethodsIn this study, we evaluated the preclinical therapeutic effects of ONC206 in medulloblastoma and investigated its mechanism of action. ResultsWe found evidence for high expression of ClpP at both the RNA and protein level in medulloblastoma tumors, compared to very low expression in normal brain tissue. In addition, we saw a pronounced reduction in cell viability of human Group 3 and Group 4 and murine SHH-driven and Group 3 medulloblastoma cells treated with ONC206 with low IC-50s. After treatment with ONC206, we observed an induction of integrated stress response and mitochondrial damage. To test the efficacy of ONC206 in vivo, we used murine models of SHH-driven and Group 3 medulloblastoma as well as Group 3 and Group 4 patient-derived xenografts (PDXs). ONC206 led to a significant prolongation of survival in both murine models, with the SHH mice demonstrating survival extension from 70 to 140 days. PDX-bearing mice also responded to ONC206, which led to a significant survival benefit. ConclusionOur results highlight ONC206 as a novel therapeutic option for patients with high-risk medulloblastoma and provide strong rationale for testing the efficacy of ONC206 in the treatment of these patients. Key points (2-3)ONC206 potently kills medulloblastoma cells by inducing integrated stress response and mitochondrial damage. ONC206 prolongs survival of medulloblastoma-bearing mice in both murine and patient-derived xenograft models. Importance of studyThere is an unmet need for better therapies for high-risk medulloblastoma patients. ONC201 has shown promising responses and recently received FDA approval for diffuse midline glioma. ONC206 is a chemical derivative with higher potency and better brain penetrance. In this study, we analyzed the therapeutic potential of ONC206 for high-risk medulloblastoma and found that the drug effectively killed mouse and human medulloblastoma cells with high nanomolar potency. We also saw that ONC206 very significantly prolonged survival of medulloblastoma-bearing mice, both in genetically engineered mouse models and patient-derived xenografts. Our study provides a strong rationale for testing the efficacy of ONC206 in the treatment of patients with medulloblastoma and has set the stage for a clinical trial with this agent in pediatric patients with recurrent malignant brain tumors, including medulloblastoma (NCT04732065).

In order to better understand the relationship between normal and neoplastic brain, we combined five publicly available large-scale datasets, correcting for batch effects and applying Uniform Manifold Approximation and Projection (UMAP) to RNA-seq data. We assembled a reference Brain-UMAP including 702 adult gliomas, 802 pediatric tumors and 1409 healthy normal brain samples, which can be utilized to investigate the wealth of information obtained from combining several publicly available datasets to study a single organ site. Normal brain regions and tumor types create distinct clusters and because the landscape is generated by RNA seq, comparative gene expression profiles and gene ontology patterns are readily evident. To our knowledge, this is the first meta-analysis that allows for comparison of gene expression and pathways of interest across adult gliomas, pediatric brain tumors, and normal brain regions. We provide access to this resource via the open source, interactive online tool Oncoscape, where the scientific community can readily visualize clinical metadata, gene expression patterns, gene fusions, mutations, and copy number patterns for individual genes and pathway over this reference landscape.

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.

The cerebellar rhombic lip generates cerebellar progenitors and neurons that ultimately differentiate to comprise over half of all neurons in the adult human brain. Standard clustering approaches often fragment or miss rhombic lip progenitor populations entirely due to their transient nature, small size, and rapid state transitions, leaving fundamental questions unanswered about normal cerebellar development and how such processes may be hijacked in pediatric brain cancer. Medulloblastoma, the most common malignant pediatric brain tumor, affects approximately 500 children annually in the United States with overall survival rates varying dramatically by subgroup. Sonic hedgehog (SHH) medulloblastoma, comprising 25-30% of cases, arises from rhombic lip-derived granule neuron precursors (GNP) within the external granule layer (EGL) and has particularly poor outcomes in several subtypes (5-year survival [~]41%). Using our topic modeling framework on over one million fetal cerebellar nuclei, we identify proliferative rhombic lip and EGL states that bifurcate into distinct glial and neuronal lineages through intermediate progenitors and capture a portion of the developmental spectrum form outer EGL (oEGL) proliferation through inner EGL (iEGL) differentiation. These developmental signatures (topics) persist in medulloblastoma, validating GNP origins of SHH tumors and revealing age-specific molecular programs that correspond to distinct stages of EGL development within SHH subtypes. Our transferable framework enables systematic comparison of developmental and disease states across technologies without data integration, solving a fundamental challenge as genomic atlases expand.

Gliomas are complex and heterogeneous primary brain tumors with a high degree of therapeutic resistance, particularly glioblastomas, which carry a poor prognosis. Cellular plasticity and stem cell-like features, or "stemness," are increasingly recognized as key contributors to tumor progression and treatment resistance. In this study, we introduce two machine-learning-based prediction models designed to assess stemness in glioma samples using bulk gene expression data. One model captures fetal astrocyte characteristics (ASTsi), while the other identifies glioma stem cell traits (GSCsi). ASTsi was notably correlated with poor prognosis in IDHmut gliomas, whereas GSCsi was more indicative of stemness in IDHwt subtypes. Longitudinal data analysis showed that IDHwt and grade IV gliomas exhibit shifts in stemness indices upon recurrence, suggesting a phenotypic change linked to therapy resistance. Additionally, single-cell transcriptomic analysis confirmed that GSCsi can detect stem-like cell subsets in IDHwt gliomas. This approach enhances our understanding of glioma heterogeneity and reveals potential therapeutic targets.

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.

BackgroundMutations of the isocitrate dehydrogenase (IDH) gene occur in over 80% of low-grade gliomas and secondary glioblastomas. Despite considerable efforts, endogenous in vitro IDH-mutated glioma models remain scarce. Availability of these models is key for the development of new therapeutic interventions. MethodsCell cultures were established from fresh tumor material and expanded in serum-free culture media. D-2-Hydroxyglutarate levels were determined by mass-spectrometry. Genomic and transcriptomic profiling were carried out on the Illumina Novaseq platform, methylation profiling was performed with the Infinium MethylationEpic BeadChip array. Mitochondrial respiration was measured with the Seahorse XF24 Analyzer. Drug screens were performed with an NIH FDA-approved anti-cancer drug set and two IDH-mutant specific inhibitors. ResultsA set of twelve patient-derived IDHmt cell cultures was established. We confirmed high concordance in driver mutations, copy number and methylation profiles between the tumors and derived cultures. Homozygous deletion of CDKN2A/B was observed in all cultures. IDH-mutant cultures had lower mitochondrial reserve capacity. IDH-mutant specific inhibitors did not affect cell viability or global gene expression. Screening of 107 FDA-approved anti-cancer drugs identified nine compounds with potent activity against IDHmt gliomas, including three compounds with favorable pharmacokinetic characteristics for CNS penetration: teniposide, omacetaxine mepesuccinate, and marizomib. ConclusionsOur twelve IDH-mutant cell cultures show high similarity to the parental tissues and offer a unique tool to study the biology and drug sensitivities of high-grade IDHmt gliomas in vitro. Our drug screening studies reveal lack of sensitivity to IDHmt inhibitors, but sensitivity to a set of nine available anti-cancer agents. Key pointsO_LIIDHmt glioma cultures closely resemble their parental tumors C_LIO_LIMicroscopic monitoring of early passages and colony isolation increases IDH1mt culture success C_LIO_LIDrug screening identified nine candidate repurposed drugs for IDHmt glioma C_LI Importance of the studyIDH-mutations are highly prevalent in low grade and secondary high-grade gliomas. Despite this high frequency however, very few in vitro models have been reported for IDH-mutated gliomas. In this manuscript we describe and characterize in detail twelve primary cultures from IDH-mutant astrocytomas. We show that these cultures retain most of the genetic, epigenetic and metabolic features of their respective parental tumors. Because of these similarities, these independent model systems will not only help understand the molecular defects driven by the mutation, but are also vital to identify means to target these tumors. Screening of 107 FDA-approved anti-cancer agents on these cultures identified a set of highly effective agents that may offer candidates for either systemic or assisted delivery treatment of this tumor subtype.

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.

In contrast to most other tumors, the anatomical extent of brain tumors is not objectified and quantified through staging. Staging systems are built on the understanding of the anatomical sequence of tumor progression and its relation to histopathological dedifferentiation and survival. While major advances in the understanding of primary brain tumors at a histological, cellular and molecular level have been achieved in recent decades, our understanding at a macroscopic anatomical level is limited. The aim of this study was to describe the anatomical phenotype of the most frequent brain tumor entities based on topographic probability and growth behavior analysis. The association of anatomical tumor features with survival probability was assessed and a prototypical staging system for WHO grade II-IV glioma was proposed based on the hypothesized anatomical sequence of tumor progression. The analysis is based on data from a consecutive cohort of 1000 patients with first diagnosis of a primary or secondary brain tumor. On preoperative MRI, the relative tumor density (RTD) of different topographic, phylogenetic and ontogenetic parcellation units was derived through normalization of the relative tumor prevalence to the relative volume of the respective structure. While primary central nervous system lymphoma (PCNSL) showed a high RTD along white matter tracts, the RTD in metastases was highest along terminal arterial flow areas. Neuroepithelial tumors (NT) demonstrated a high and homogeneous RTD along all sectors of the ventriculo-cortical axis, avoiding adjacent units, consistent with a transpallial behavior within phylo-ontogenetic radial units. Additionally, the topographic probability in NT correlated with morphogenetic processes of convergence and divergence of radial units during phylo- and ontogenesis. The anatomical tumor growth behavior was analyzed by comparing pre- and postoperative MRI, showing that a ventriculofugal growth dominates in NT. With progressive histopathological dedifferentiation of NT, a gradual deviation from this neuroepithelial anatomical behavior was found. By comparing survival probability, we identified prognostically critical steps in the anatomical behavior of NT. Based on a hypothesized sequence of anatomical tumor progression, we developed a three-level prototypical staging system for WHO grade II-IV glioma. This staging system proved to be accurate across histological, molecular, radiomorphological and clinical strata based on Kaplan Meier curves and multivariable survival analysis. Similar to staging systems for other tumors, a staging system such as this one may have the potential to inform stage-adapted treatment decisions.

Somatic mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 occur at high frequency in several tumour types. Even though these mutations are confined to distinct hotspots, we show that gliomas are the only tumour type with an exceptionally high percentage of IDH1R132H mutations. This high prevalence is important as IDH1R132H is presumed to be relatively poor at producing D-2-hydroxyglutarate (D-2HG) whereas high concentrations of this oncometabolite are required to inhibit TET2 DNA demethylating enzymes. Indeed, patients harbouring IDH1R132H mutated tumours have lower levels of genome-wide DNA-methylation, and an associated increased gene expression, compared to tumours with other IDH1/2 mutations ("non-R132H mutations"). This reduced methylation is seen in multiple tumour types and thus appears independent of site of origin. For 1p/19q non-codeleted glioma patients, we show that this difference is clinically relevant: in samples of the randomised phase III CATNON trial, patients harbouring non-R132H mutated tumours have better outcome (HR 0.41, 95% CI [0.24, 0.71], p=0.0013). Non-R132H mutated tumours also had a significantly lower proportion of tumours assigned to prognostically poor DNA-methylation classes (p<0.001). IDH mutation-type was independent in a multivariable model containing known clinical and molecular prognostic factors. To confirm these observations, we validated the prognostic effect of IDH mutation type on a large independent dataset. The observation that non-R132H mutated 1p/19q non-codeleted gliomas have a more favourable prognosis than their IDH1R132H mutated counterpart is clinically relevant and should be taken into account for patient prognostication. Single sentence summaryAstrocytoma patients with tumours harbouring IDH mutations other than p.R132H have increased DNA methylation levels and longer survival

BackgroundGlioblastoma (GB) is a highly aggressive and treatment-resistant brain cancer with poor prognosis. Surgical resection followed by radiotherapy (RT) with the chemotherapeutic, temozolomide (TMZ), is the standard GB treatment; yet recurrence often occurs. GB is organized hierarchically with a small population of radiation-resistant glioma-initiating cells (GICs) that self-renew and drive tumor growth. Importantly, RT can induce a subset of cells from non-tumor-initiating into glioma-initiating cells (iGICs). Both GICs and iGICs contribute to tumor recurrence and therapy resistance. Thus, without effective elimination of non-tumorigenic GB and prevention or targeting of GICs, a cure is unlikely. The objective of this study is to identify small molecules that block RT-induced phenotypic conversion to occur. MethodWe conducted a high-throughput screen of NCIs Cancer Therapy Evaluation Program (CTEP) compounds with evidence for crossing the blood-brain-barrier. To identify "stemness" or reprogramming of cells, we transduced GB cell lines representing each TCGA subtype to express a fluorescent reporter for proteasomal activity that distinguishes non-tumor-initiating cells from GICs. We tested CTEP agents at 10 different concentrations in combination with radiation. ResultsOur results identified selumetinib as a candidate compound that effectively prevents radiation-induced phenotype conversion. Furthermore, in combination with radiation, selumetinib decreased stem cell maintenance in GICs with differential effects on viability in non-tumorigenic cells. ConclusionTaken together, these findings suggest that repurposing FDA-approved compounds alongside current therapies may effectively target the cellular and molecular heterogeneity of GB--and because these agents are already clinically approved, this approach can be rapidly implemented in the clinic.

Medulloblastoma (MB) is the most common malignant primary pediatric brain tumor. Current therapies are ineffective for targeting proliferation, leptomeningeal migration, and metastasis of MB cancer cells to visceral organs and therefore, novel treatments are needed. The small GTPase, RAC1, has emerged as an important regulator of actin cytoskeletal dynamics, proliferation, and migration in several cancers. However, it has not been characterized in MB and no clinical drug candidates have been described for RAC1 in MB. Here we demonstrate that RAC1 levels are higher in MB tissue relative to normal cerebellum. Further, RAC1 depletion significantly reduces proliferation and migration of Shh-MB cells in vitro. Mechanistically, RAC1 controls the mRNA and protein levels of the main transcription factors in the Shh pathway, GLI1 and GLI2. RAC1 binds to the GLI1 promoter highlighting a novel role in transcriptional regulation in Shh-dependent cancers. We demonstrate that the RAC1 inhibitor, GYS32661, is brain penetrant, and reduces MB growth and increases mouse survival in an orthotopic model of Shh-MB. Importantly, GYS32661 is a non-toxic clinical candidate, suggesting that it may be a novel potential drug for the treatment of either the pediatric or adult forms of MB. Collectively, our studies identify RAC1 as a druggable target in Shh-dependent MB. Graphical abstractRAC1 controls Shh-MB progression by binding to the GLI1 promoter. Graphical abstract demonstrating RAC1 localizes to the nucleus and binds to the GLI1 promoter and plays an important role in its transcription. RAC1 genetic or GYS32661 mediated inhibition leads to RAC1 dissociation from the GLI1 promoter and transcriptional repression of Shh-MB biomarkers GLI2, DNMT1 and UHRF1. This eventually causes inhibition of Shh-MB tumor cell proliferation and migration, which leads to a decrease in Shh-MB development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/655563v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@e4def9org.highwire.dtl.DTLVardef@1eab826org.highwire.dtl.DTLVardef@dfe68borg.highwire.dtl.DTLVardef@198efb9_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundThe current WHO classification integrates histological and molecular features of brain tumors. The aim of this study was to identify generalizable topological patterns with the potential to add an anatomical dimension to the classification of brain tumors. MethodsWe applied non-negative matrix factorization as an unsupervised pattern discovery strategy to the fine-grained topographic tumor profiles of 936 patients with primary and secondary brain tumors. From the anatomical features alone, this machine learning algorithm enabled the extraction of latent topological tumor patterns, termed meta-topologies. The optimal parts-based representation was automatically determined in 10,000 split-half iterations. We further characterized each meta-topologys unique histopathologic profile and survival probability, thus linking important biological and clinical information to the underlying anatomical patterns ResultsIn primary brain tumors, six meta-topologies were extracted, each detailing a transpallial pattern with distinct parenchymal and ventricular compositions. We identified one infratentorial, one allopallial, three neopallial (parieto-occipital, frontal, temporal) and one unisegmental meta-topology. Each meta-topology mapped to distinct histopathologic and molecular profiles. The unisegmental meta-topology showed the strongest anatomical-clinical link demonstrating a survival advantage in histologically identical tumors. Brain metastases separated to an infra- and supratentorial meta-topology with anatomical patterns highlighting their affinity to the cortico-subcortical boundary of arterial watershed areas. ConclusionsUsing a novel data-driven approach, we identified generalizable topological patterns in both primary and secondary brain tumors Differences in the histopathologic profiles and prognosis of these anatomical tumor classes provide insights into the heterogeneity of tumor biology and might add to personalized clinical decision making.

AbstractO_ST_ABSBackgroundC_ST_ABSGlioblastoma (GB) is the most aggressive primary brain tumor, characterized by therapy resistance, attributed to a multitude of epi-genetic changes resulting in phenotypic plasticity with altered cell states. To uncover druggable epigenetic vulnerabilities, we disturbed GB-derived spheres and observed coordinated repression of the aberrantly activated hemopoietic stem-like cell signature, dominated by HOXA genes. This signature has been associated with poor prognosis and resistance to therapy in GB. Here we investigate biological vulnerabilities associated with the deregulated epigenetic landscape in high-HOX GB. MethodsGB-derived spheres (GS) were treated with an inhibitor of Bromodomain and extra-terminal motif proteins (BETi) (JQ1) or transduced with inducible constructs to genetically modulate HOXA10 expression (shRNA for knockdown, ectopic HOXA10). Functional effects were evaluated through proliferation, neurosphere formation, and senescence assays. Epigenomic profiling incorporated RNA-seq, ChIP-seq, ATAC-seq, promoter capture MicroC, and DNA methylation. ResultsBETi-mediated rapid, coordinated downregulation of the HOX-signature, suggested direct transcriptional regulation. Knockdown of HOXA10 alone yielded similar effects, decreasing expression of HOXA genes, reducing proliferation, self-renewal capacity, and triggering senescence. Conversely, ectopic HOXA10 expression was ineffective in reactivating the HOXA cluster, or reverse BETi-mediated biological effects. Integrative epigenomic analysis of high-HOX-GS revealed concerted activation of the HOXA region, with broad domains of H3K27ac/H3K4me3 associated with super-enhancer activity, open chromatin (ATAC) and focal DNA hypomethylation. Architectural changes included altered CTCF interactions and increased promoter-anchored looping. ConclusionThese results position the HOX-signature as a potential therapeutic target and offer a mechanistic rationale for disrupting BET-dependent transcriptional regulation in high-HOX GB. Key pointsO_LIEpigenetic activation of stem cell-related high-HOX signature in GB is associated with a super-enhancer encompassing the HOXA locus. C_LIO_LITargeting this vulnerability by BETi or HOXA10 knockdown results in concerted repression and loss of stemness features. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/727851v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@5bf4b9org.highwire.dtl.DTLVardef@11fa405org.highwire.dtl.DTLVardef@497a2eorg.highwire.dtl.DTLVardef@1f47084_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Chiesi, D. (2026) https://BioRender.com/eknk0ez Importance of studyGlioblastoma (GB) are the most aggressive brain tumors in adults that are difficult to treat, due to their high plasticity resulting invariably to resistance to therapies. Here we report on the identification of epigenetic vulnerabilities that may be leveraged in combination therapies. Disturbing GB-derived stem-like cells with epigenetic drugs, we uncovered that a HOXA gene dominated hematopoietic stem cell-related signature, previously associated with aggressiveness and treatment resistance, can be repressed in a coordinated manner, resulting in loss of stem cell features. Analysis of the underlying epigenetic landscape revealed that the HOXA region was activated in high-HOX glioblastoma through the formation of a super-enhancer. This feature presents a particular vulnerability that may be leveraged by BETi as strategy of a combination therapy.

While CDKN2A loss is classically associated with cell cycle deregulation through the p16-Cdk4-Rb axis, our findings suggest an additional layer of metabolic vulnerability arising from altered NAD homeostasis in CDKN2A-deleted glioblastoma, revealing a previously unrecognized metabolic-genetic interface for rationally revisiting NAD+ targeting strategies, moving beyond the broad inhibition approaches.

BackgroundTo achieve replicative immortality, most cancers develop a telomere maintenance mechanism, such as reactivation of telomerase or alternative lengthening of telomeres (ALT). There are limited data on the prevalence and clinical significance of ALT in pediatric brain tumors, and ALT-directed therapy is not available. MethodsWe performed C-circle analysis (CCA) on 579 pediatric brain tumors that had corresponding tumor/normal whole genome sequencing through the Open Pediatric Brain Tumor Atlas (OpenPBTA). We detected ALT in 6.9% (n=40/579) of these tumors and completed additional validation by ultrabright telomeric foci in situ on a subset of these tumors. We used CCA to validate TelomereHunter for computational prediction of ALT status and focus subsequent analyses on pediatric high-grade glioma (pHGG) Finally, we examined whether ALT is associated with recurrent somatic or germline alterations. ResultsALT is common in pHGG (n=24/63, 38.1%), but occurs infrequently in other pediatric brain tumors (<3%). Somatic ATRX mutations occur in 50% of ALT+ pHGG and in 30% of ALT-pHGG. Rare pathogenic germline variants in mismatch repair (MMR) genes are significantly associated with an increased occurrence of ALT. Conclusions: We demonstrate that ATRX is mutated in only a subset of ALT+ pHGG, suggesting other mechanisms of ATRX loss of function or alterations in other genes may be associated with the development of ALT in these patients. We show that germline variants in MMR are associated with development of ALT in patients with pHGG. Key PointsATRX alterations are frequent, but not required, for an ALT phenotype in pHGGs pHGG patients with germline mismatch repair variants have higher rate of ALT + tumors TelomereHunter is validated to predict ALT in pHGGs Importance of the StudyWe performed orthogonal molecular and computational analyses to detect the presence of alternative lengthening of telomeres in a highly characterized cohort of pediatric brain tumors. We demonstrate that many pHGG utilize ALT without a mutation in ATRX, suggesting either loss of function of ATRX via an alternative mechanism or an alternate means of development of ALT. We show that germline variants in MMR genes are significantly associated with ALT in pHGG. Our work adds to the biological understanding of the development of ALT and provides an approach to stratify patients who may benefit from future ALT-directed therapies in this patient population.