Medulloblastoma Samples Research Articles

TMIC-04. SPATIAL SEQUENCING OF MEDULLOBLASTOMA REVEALS BIOLOGICAL NICHES CORRELATED WITH HIGH-RISK FEATURES AND RELAPSE

Abstract Medulloblastoma (MB) defines a heterogeneous set of neuroepithelial cancers of the posterior fossa. The MB tumor microenvironment (MB-TME) influences tumor progression and therapy response and has emerged as a growing target for novel therapeutic approaches. Prior single-cell analysis has characterized the MB-TME composition but does not reveal the spatial orientation of components. To address this gap, we performed 10X Visium spatial sequencing on sixteen pediatric MB samples obtained at surgical resection with samples representing the four molecular subtypes (WNT, N=1; SHH, N=6; Group 3, N=2; Group 4, N=5) and two matching samples at diagnosis and relapse. Spatial voxel clustering yielded clusters corresponding to malignant, immune, and stromal components of the TME, including tumor-associated astrocytes (TAAs), macrophages (TAMs), and vascular endothelium. Ten distinct clusters emerged representing malignant cell states, defined primarily by programs of cell cycle progression and neuronal differentiation. Notably, these MB cell clusters were differentially abundant between the MB subgroups, highlighting heterogeneity between disease subtypes. Moreover, examining the patterns of cellular spatial distribution in high-risk (HR) patients revealed dense regions of quiescent MB progenitors, while standard-risk patients showed greater heterogeneity within their TME. Proximity-dependent intercellular signaling further highlighted increased signaling by TAAs and vascular endothelium with decreased heat shock response signaling in HR patients. Comparing samples from relapse to diagnosis, TAMs, TAAs, and vascular endothelium were found to constitute a greater proportion of the TME. Subsequent niche-dependent gene expression and pathway analysis for TAAs localized near tumor vasculature in relapsed samples revealed increased expression of metastasis-associated pathways. Collectively, the results of our study enable new insight into the heterogeneity of the MB-TME, with close spatial association of quiescent MB progenitors correlating with high-risk clinical features. Quiescent MB progenitors have been documented to confer resistance through cellular dormancy, and identifying its molecular drivers may reveal potential targets for anticancer therapy.

EPCO-01. FROM HIERARCHY TO GEOGRAPHY: MULTI-CLONES DRIVE THE CELLULAR DIVERSITY AND EVOLUTION OF HUMAN MEDULLOBLASTOMA

Abstract We’ve curated a comprehensive multi-omics database of medulloblastoma (MB) serving as a resource to understand spatiotemporal organization and guide therapeutic strategies. Herein, our analysis encompasses single-nucleus transcriptome (n = 51), chromatin accessibility (n = 46), and spatial transcriptomic profiles (n = 31) from human MB samples and patient-derived xenograft lines spanning four subgroups, augmented by bulk RNA (n = 322), whole-genome short-sequencing (n = 279), and bisulfite sequencing (n = 300) from matched samples. We delineate the malignant cellular hierarchy, identifying MB cancer stem-like cells (MB-CSCs), cycling cells, and more differentiated populations. Gene signatures of different cell subpopulations strongly correlate to clinical outcomes, while spatial character of patient-derived materials emphasizes the geographically heterogeneous nature of MB. Examination of somatic copy number variants (CNVs) and transcriptional signatures at single-nucleus resolution reveals geographically defined subclones harboring distinct CNVs, suggesting a competitive agglomeration of co-existing multi-clones for MB. Alongside the trajectorial transition, we observe distinct chromosomal alterations in apical MB-CSCs compared to more differentiated cells, indicative of extensive subclone-primed spatiotemporal evolution. Further analysis of reagent-gene interactions of subclonal lineages contributes to predicting the druggable candidates for treatment optimization. Integration of spatial architecture data highlights the recapitulation of subclone-determined niches through colocation with identified malignant lineages and their own inflammatory or fibrotic adaptions. Taken together, this comprehensive database, comprising five or six kinds of datatypes on the same MB cases, facilitates combinatorial analyses across different genomic modalities, offering genetic and geographic insights into therapeutic advancements.

VISTA Emerges as a Promising Target against Immune Evasion Mechanisms in Medulloblastoma.

Relapsed medulloblastoma (MB) poses a significant therapeutic challenge due to its highly immunosuppressive tumor microenvironment. Immune checkpoint inhibitors (ICIs) have struggled to mitigate this challenge, largely due to low T-cell infiltration and minimal PD-L1 expression. Identifying the mechanisms driving low T-cell infiltration is crucial for developing more effective immunotherapies. We utilize a syngeneic mouse model to investigate the tumor immune microenvironment of MB and compare our findings to transcriptomic and proteomic data from human MB. Flow cytometry reveals a notable presence of CD45hi/CD11bhi macrophage-like and CD45int/CD11bint microglia-like tumor-associated macrophages (TAMs), alongside regulatory T-cells (Tregs), expressing high levels of the inhibitory checkpoint molecule VISTA. Compared to sham control mice, the CD45hi/CD11bhi compartment significantly expands in tumor-bearing mice and exhibits a myeloid-specific signature composed of VISTA, CD80, PD-L1, CTLA-4, MHCII, CD40, and CD68. These findings are corroborated by proteomic and transcriptomic analyses of human MB samples. Immunohistochemistry highlights an abundance of VISTA-expressing myeloid cells clustering at the tumor-cerebellar border, while T-cells are scarce and express FOXP3. Additionally, tumor cells exhibit immunosuppressive properties, inhibiting CD4 T-cell proliferation in vitro. Identification of VISTA's binding partner, VSIG8, on tumor cells, and its correlation with increased VISTA expression in human transcriptomic analyses suggests a potential therapeutic target. This study underscores the multifaceted mechanisms of immune evasion in MB and highlights the therapeutic potential of targeting the VISTA-VSIG axis to enhance anti-tumor responses.

Medulloblastoma Spatial Transcriptomics Reveals Tumor Microenvironment Heterogeneity with High-Density Progenitor Cell Regions Correlating with High-Risk Disease.

The tumor microenvironment (TME) of medulloblastoma (MB) influences progression and therapy response, presenting a promising target for therapeutic advances. Prior single-cell analyses have characterized the cellular components of the TME but lack spatial context. To address this, we performed spatial transcriptomic sequencing on sixteen pediatric MB samples obtained at diagnosis, including two matched diagnosis-relapse pairs. Our analyses revealed inter- and intra-tumoral heterogeneity within the TME, comprised of tumor-associated astrocytes (TAAs), macrophages (TAMs), stromal components, and distinct subpopulations of MB cells at different stages of neuronal differentiation and cell cycle progression. We identified dense regions of quiescent progenitor-like MB cells enriched in patients with high-risk (HR) features and an increase in TAAs, TAMs, and dysregulated vascular endothelium following relapse. Our study presents novel insights into the spatial architecture and cellular landscape of the medulloblastoma TME, highlighting spatial patterns linked to HR features and relapse, which may serve as potential therapeutic targets.

BIOM-11. TUMOUR SUPPRESSIVE ROLES OF MIR-206 AND MIR-383 VIACORO1C ANDSV2B IN AGGRESSIVE PAEDIATRIC AND ADULT BRAIN MALIGNANCIES

Abstract Medulloblastoma (MB), the most prevalent brain malignancy in children, poses challenges in paediatric oncology due to its aggressiveness and potential for metastasis. Tailored treatment strategies are essential due to the impact of treatment-related toxicities and long-term side effects on children’s developing brains. This study aimed to identify therapeutic targets for paediatric MB and explore common biosignatures with glioblastoma multiforme (GBM), the most aggressive adult brain tumour. Total RNA and microRNAs (miRNAs) were extracted from eleven paediatric brain tissues (normal brain tissues, n=2; MB tumour tissue, n=8; pilocytic astrocytoma, n=1). High-throughput small-RNA sequencing (sRNA-seq) and transcriptomics were performed to identify the most deregulated miRNAs in paediatric MB tissue samples. Selected miRNAs, gene targets and encoded proteins were evaluated in vitro and ex vivo via real-time polymerase chain reaction (RT-qPCR), western blotting and immunohistochemistry (IHC). The sRNA-seq demonstrated that miR-383 and miR-206 were highly down regulated in MB samples suggesting their tumour suppressor properties. Bioinformatics analysis using miRSystem and TargetScan identified SV2B and CORO1C as their direct targets, respectively. Transcriptomics showed significant upregulation of SV2B and CORO1C in MB tissue samples. RT-qPCR analysis confirmed overexpression of SV2B and CORO1C within GBM cell lines and IHC depicted significant overexpression of SV2B and CORO1C proteins in paediatric MB and GBM patient cohorts (n=28) compared to their normal brain counterparts (n=32). Elevated CORO1C expression was also detected in adult MB (n=23) and GBM (n=73) tissue samples, when compared to normal brain tissue. Western blotting further verified the significant overexpression of CORO1C and SV2B within GBM cells, suggesting their role as potential oncoproteins. Collectively, our preliminary study proposes the clinical significance of miR-206 and miR-383 when overexpressed in the therapy of aggressive paediatric and adult brain malignancies.

MDB-12. NANOPORE SEQUENCING AS A CUTTING-EDGE TECHNOLOGY FOR MEDULLOBLASTOMA CLASSIFICATION

Abstract INTRODUCTION Medulloblastoma (MB) is one of the most prevalent embryonal malignant brain tumors, divided into four distinct molecular subgroups (WNT, SHH, group 3, and group 4) with its individual prognosis. A more extensive classification of MB has recently been provided, identifying numerous subtypes, some with poor prognosis, leading to a significant number of patients succumbing to the disease while many survivors experience long-term side effects, highlighting the need for efficient subgrouping methods. Distinct genome-wide DNA methylation profiles characterize each MB subgroup, and the advent of Nanopore DNA sequencing has positioned itself as a reliable technique for comprehensive copy number and methylation profiling. MATERIAL AND METHODS We evaluated the ability of Nanopore sequencing to provide clinically relevant methylation and copy number profiles of MB, on a discovery cohort of 45 frozen MB samples, benchmarked against the gold standard EPIC array, and subsequently validated on a cohort of 116 MB samples. RESULTS The majority of MB in both the discovery cohort (43/45; 95.6%) and the validation cohort (110/116; 94.8%) were accurately subgrouped by Nanopore sequencing. Flongle flow cells for 18 MB allowed for a more rapid and cost-effective analysis, with 94.4% (17/18) being correctly classified. Additionally, Nanopore sequencing allowed us to correctly subtype 24/30 (80.0%) within MB groups. DISCUSSION We provided proof of concept that Nanopore sequencing can subgroup MB using DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples and cell-free DNA obtained from cerebrospinal fluid. This first large-scale study establishes the proof of concept that this modern and innovative technology is well-suited for MB classification. We confirm the use of Nanopore sequencing on circulating DNA and present an initial exploration of its application on DNA extracted from FFPE samples. Its ability to deliver quick and cost-effective results firmly establishes Nanopore sequencing as a game-changer in the field of MB classification.

MDB-22. DECIPHERING MEDULLOBLASTOMA’S MOSAIC: UNVEILING SUBGROUP CLASSIFICATION THROUGH METHYLATION PROFILING

Abstract BACKGROUND Medulloblastoma represents the most prevalent malignant brain tumor in children, posing significant treatment challenges despite advances in multimodal therapies. The heterogeneity of medulloblastomas necessitates precise classification to improve therapeutic outcomes. This study explores the utility of genome-wide DNA methylation profiling in categorizing medulloblastomas into molecular subgroups, aiming to refine diagnostic and treatment strategies. METHODS This investigation involved the analysis of medulloblastoma samples from pediatric patients at the King Faisal Hospital and Research Centre. We employed the Illumina Infinium 850K EPIC assay to assess the CpG methylation status in six medulloblastoma cases. The methylation data were then compared with immunohistochemical findings to validate the methylation-based subgroup classification. RESULTS The Infinium 850K EPIC assay successfully classified all six medulloblastoma cases into the recognized subgroups: WNT, SHH, Group 3, and Group 4. This classification was further supported by immunohistochemical analysis, demonstrating the assay’s robustness in accurately identifying the molecular subtypes. Notably, the assay also distinguished a control case within the cohort, illustrating its potential for widespread clinical application. The study highlights the feasibility of using formalin-fixed, paraffin-embedded (FFPE) tissues, which offers a practical advantage by eliminating the need for fresh tissue samples. CONCLUSIONS Genome-wide methylation profiling emerges as a superior method for the classification of pediatric medulloblastomas, facilitating the identification of molecular subgroups with distinct prognostic and therapeutic implications. The ability to utilize FFPE tissues broadens the applicability of this approach, particularly in settings where fresh samples are not readily available. This advancement holds significant promise for enhancing the clinical management of medulloblastoma in pediatric populations.

MDB-36. 22-HDOHE, A PREDICTIVE MARKER IN CSF OF PATIENTS WITH RECURRENT MEDULLOBLASTOMA TREATED WITH MEMMAT

Abstract BACKGROUND With the antiangiogenic metronomic MEMMAT combinatorial regimen, we could achieve sustained responses in a quarter of the patients with previously irradiated recurrent medulloblastoma. There are currently no molecular biomarkers predicting response to MEMMAT treatment for recurrent medulloblastoma. Lipid mediators, including classical arachidonic acid-derived eicosanoids and docosanoids, are locally acting bioactive signaling lipids that regulate a diverse set of homeostatic and inflammatory processes. Before initiation of MEMMAT treatment for relapsed medulloblastoma, we examined the bioactive lipid mediator profile in the cerebrospinal fluid (CSF) of patients and compared it with samples from patients with different neurological disorders, but without cancer. METHODS We analyzed CSF samples from 23 patients with relapsed MBL before initiation of MEMMAT treatment and from 12 controls. Lipid mediators were enriched via solid phase extraction and analyzed using liquid chromatography coupled to a high resolution orbitrap Exploris 480 mass spectrometer. RESULTS Mass spectrometry-based untargeted oxylipin analysis led to the identification of 98 lipid mediators. Several molecules were downregulated in medulloblastoma samples versus control, such as lipoxin A4 and 14(15)-DiHETE. Non-responders had lower levels of these molecules than responders. Glycodeoxycholic acid and deoxycholic acid were up-regulated in medulloblastoma patients, again with higher levels in non-responders. Remarkably, the docosahexaenoic acid (DHA)-derived 22-HDoHE was specifically identified in the CSF samples of medulloblastoma patients but not in control samples. CONCLUSION An oxylipin pattern in CSF might serve as a predictive biomarker signature for response in patients with recurrent medulloblastoma treated with the antiangiogenic metronomic MEMMAT regimen. Our findings advocate for the prospective assessment of CSF-derived lipid markers in future trials for recurrent medulloblastoma.

MDB-98. HISTOPATHOLOGY IS A ROBUST TOOL TO DIFFERENTIATE BETWEEN SHH AND WNT MEDULLOBLASTOMA VERSUS NON-SHH/NON-WNT MEDULLOBLASTOMA: COMPARATIVE ANALYSIS OF HISTOPATHOLOGY, CHROMOSOMAL MICROARRAY, NANOSTRING BASED 22-GENE ASSAY, AND DNA METHYLATION FOR MEDULLOBLASTOMA SUBGROUP ASSIGNMENT ON “HEAD START” 4 CLINICAL TRIAL

Abstract BACKGROUND “Head Start-4” (HS-4) is a prospective, randomized clinic trial that tailors treatment based on medulloblastoma molecular subgroups (WNT, SHH, Group 3, and Group 4) and response to induction chemotherapy, and compares the efficacy of one versus three (tandem) cycles of myeloablative therapy. Here we compare different methodologies used to distinguish between WNT/SHH medulloblastoma (low-risk arm) and non-SHH/non-WNT medulloblastoma (high-risk arm) during the course of the trial. METHODS When HS-4 trial began enrolling patients in 2015, in the absence of a CAP-CLIA certified test for methylation and gene expression profile, we utilized histopathology/immunochemistry (HP/IHC) and chromosomal microarray (CMA) via OncoScanTM (Thermo Fisher) to classify medulloblastoma samples into either WNT, SHH, or non-WNT/non-SHH (Subgroups 3 and 4) at the time of diagnosis. Retrospectively, we performed both NanoString based 22-gene assay and DNA methylation profiling on all patient samples. RESULTS We have HP/IHC, CMA, NanoString and methylation profiling for 54 infants and young children with medulloblastoma enrolled on HS-4. While indeterminate result occurred with CMA in three cases and NanoString in two cases, HP/IHC successfully assigned samples to SHH/WNT and non-SHH/non-WNT arms of the study in all 54 cases. We have HP/IHC, CMA and DNA methylation profiling for additional 33 patients. While pathology/IHC was indeterminate in two cases (one WNT and one group 3 MB), remaining cases accurately categorized medulloblastoma methylation subtype as WNT/SHH and non-WNT/non-SHH. CONCLUSION Due to the long turnaround time, waiting for medulloblastoma molecular subtype confirmation by DNA methylation array may delay treatment initiation. HS-4 data displays robust prediction of WNT/SHH versus non-WNT/non-SHH molecular subtypes by HP/IHC in comparison to DNA methylation profiling and provides a platform to initiate treatment based on HP/IHC while awaiting molecular information. In addition, these data support relying on histopathology for medulloblastoma subtypes in resource poor countries where methylation profiling is not readily available.

Identification and validation of a metabolism-related gene signature for predicting the prognosis of paediatric medulloblastoma

Medulloblastoma (MB) is a malignant brain tumour that is highly common in children and has a tendency to spread to the brain and spinal cord. MB is thought to be a metabolically driven brain tumour. Understanding tumour cell metabolic patterns and characteristics can provide a promising foundation for understanding MB pathogenesis and developing treatments. Here, by analysing RNA-seq data of MB samples from the Gene Expression Omnibus (GEO) database, 12 differentially expressed metabolic-related genes (DE-MRGs) were chosen for the construction of a predictive risk score model for MB. This model demonstrated outstanding accuracy in predicting the outcomes of MB patients and served as a standalone predictor. An evaluation of functional enrichment revealed that the risk score showed enrichment in pathways related to cancer promotion and the immune response. In addition, a high risk score was an independent poor prognostic factor for MB in patients with different ages, sexes, metastasis stages and subgroups (SHH and Group 4). Consistently, the metabolic enzyme ornithine decarboxylase (ODC1) was upregulated in MB patients with poor survival time. Inhibition of ODC1 in primary and metastatic MB cell lines decreased cell proliferation, migration and invasion but increased immune infiltration. This study could aid in identifying metabolic targets for MB as well as optimizing risk stratification systems and individual treatment plans for MB patients via the use of a metabolism-related gene prognostic risk score signature.

Single-cell transcriptomic sequencing identifies subcutaneous patient-derived xenograft recapitulated medulloblastoma.

Medulloblastoma (MB) is one of the most common malignant brain tumors that mainly affect children. Various approaches have been used to model MB to facilitate investigating tumorigenesis. This study aims to compare the recapitulation of MB between subcutaneous patient-derived xenograft (sPDX), intracranial patient-derived xenograft (iPDX), and genetically engineered mouse models (GEMM) at the single-cell level. We obtained primary human sonic hedgehog (SHH) and group 3 (G3) MB samples from six patients. For each patient specimen, we developed two sPDX and iPDX models, respectively. Three Patch+/- GEMM models were also included for sequencing. Single-cell RNA sequencing was performed to compare gene expression profiles, cellular composition, and functional pathway enrichment. Bulk RNA-seq deconvolution was performed to compare cellular composition across models and human samples. Our results showed that the sPDX tumor model demonstrated the highest correlation to the overall transcriptomic profiles of primary human tumors at the single-cell level within the SHH and G3 subgroups, followed by the GEMM model and iPDX. The GEMM tumor model was able to recapitulate all subpopulations of tumor microenvironment (TME) cells that can be clustered in human SHH tumors, including a higher proportion of tumor-associated astrocytes and immune cells, and an additional cluster of vascular endothelia when compared to human SHH tumors. This study was the first to compare experimental models for MB at the single-cell level, providing value insights into model selection for different research purposes. sPDX and iPDX are suitable for drug testing and personalized therapy screenings, whereas GEMM models are valuable for investigating the interaction between tumor and TME cells.

Abstract B047: Inhibition of the polyamine pathway: a novel therapeutic approach to treat aggressive pediatric medulloblastoma

Abstract Medulloblastoma is the most common pediatric malignant brain tumor. Polyamines, critical intracellular polycations governing key cell processes such as DNA replication, translation, and cell proliferation, are frequently upregulated in cancer. We previously showed that the polyamine pathway is critical to the growth and survival of pediatric diffuse midline gliomas and MYC amplified neuroblastoma. Given the activity of polyamine inhibition in DMG, and given the critical role of MYC in medulloblastoma, we sought to investigate the potential of polyamine pathway inhibition as a therapeutic approach for medulloblastoma. We compared the expression of regulators of the polyamine pathway in a cohort of pediatric medulloblastoma tumors from patients enrolled in the Zero Childhood Cancer (ZERO) Personalized Medicine Program and found significant over-expression compared with normal fetal brain samples. Analysis of Cavalli dataset of 763 medulloblastoma samples, found that high expression of polyamine biosynthetic genes predicted poor overall survival, and low expression of catabolic flux genes significantly correlated with better outcomes. We next treated medulloblastoma cells treated with a polyamine synthesis inhibitor, DFMO, and found that led to a compensatory increase in polyamine transporter protein expression. Conversely, treatment with the polyamine transport inhibitor AMXT 1501 led to compensatory increase in ODC1, the key polyamine synthetic enzyme. Dual targeting of the polyamine pathway, (DFMO and AMXT 1501) showed a potent and synergistic inhibition of cell proliferation and reduced the clonogenic potential of medulloblastoma cells in soft agar clonogenic assays. Flow cytometric analysis of Annexin V-stained cells showed that treatment with DFMO and AMXT 1501 in combination led to induction of apoptosis. Furthermore, we found that dual polyamine inhibition potently sensitized medulloblastoma cells, particularly Group 3, to the chemotherapeutic agent SN-38, leading to complete eradication of medulloblastoma cells in both cytotoxicity assays and clonogenic assays at nanomolar chemotherapeutic concentrations. We are currently validating this treatment strategy in aggressive orthotopic medulloblastoma models. Our findings suggest that inhibiting both polyamine synthesis and uptake holds promise for sensitizing highly aggressive pediatric medulloblastoma tumors to chemotherapy. Currently, AMXT 1501 combined with DFMO is undergoing evaluation in a Phase I clinical trial for adults (NCT05500508). Building on these promising results, we are developing a Phase I/II clinical trial for children with aggressive brain tumors through the international CONNECT consortium. Citation Format: Aaminah Khan, Anjana Gopalakrishnan, Jie Liu, Mark R. Burns, Maria Tsoli, David S. Ziegler. Inhibition of the polyamine pathway: a novel therapeutic approach to treat aggressive pediatric medulloblastoma [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B047.

Development and validation of a 23-gene expression signature for molecular subtyping of medulloblastoma in a long-term Chinese cohort.

Medulloblastoma is the most common childhood malignant brain tumor andis a leading cause of cancer-related death in children. Recent transcriptional studies have shown that medulloblastomas comprise at least four molecular subgroups, each with distinct demographics, genetics, and clinical outcomes. Medulloblastoma subtyping has become critical for subgroup-specific therapies. The use of gene expression assays to determine the molecular subgroup of clinical specimens is a long-awaited application of molecular biology for this pediatric cancer. In the current study, we established a medulloblastoma transcriptome database of 460 samples retrieved from three published datasets (GSE21140, GSE37382, and GSE37418). With this database, we identified a 23-gene signature that is significantly associated with the medulloblastoma subgroups and achieved a classification accuracy of 95.2%. The 23-gene signature was further validated in a long-term cohort of 142 Chinese medulloblastoma patients. The 23-gene signature classified 21 patients as WNT (15%), 41 as SHH (29%), 16 as Group 3 (11%), and 64 as Group 4 (45%). For patients of WNT, SHH, Group 3, and Group 4, 5-year overall-survival rate reached 80%, 62%, 27%, and 47%, respectively (p < 0.0001), meanwhile 5-year progression-free survival reached 80%, 52%, 27%, and 45%, respectively (p < 0.0001). Besides, SHH/TP53-mutant tumors were associated with worse prognosis compared with SHH/TP53 wild-type tumors and other subgroups. We demonstrated that subgroup assignments by the 23-gene signature and Northcott's NanoString assay were highly comparable with a concordance rate of 96.4%. In conclusion, we present a novel gene signature that is capable of accurately and reliably assigning FFPE medulloblastoma samples to their molecular subgroup, which may serve as an auxiliary tool for medulloblastoma subtyping in the clinic. Future incorporation of this gene signature into prospective clinical trials is warranted to further evaluate its clinical.

FANCD2 deficiency sensitizes SHH medulloblastoma to radiotherapy via ferroptosis.

Radiotherapy is one of the standard therapeutic regimens for medulloblastoma (MB). Tumor cells utilize DNA damage repair (DDR) mechanisms to survive and develop resistance during radiotherapy. It has been found that targeting DDR sensitizes tumor cells to radiotherapy in several types of cancer, but whether and how DDR pathways are involved in the MB radiotherapy response remain to be determined. Single-cell RNA sequencing was carried out on 38 MB tissues, followed by expression enrichment assays. Fanconi anemia group D2 gene (FANCD2) expression was evaluated in MB samples and public MB databases. The function of FANCD2 in MB cells was examined using cell counting assays (CCK-8), clone formation, lactate dehydrogenase activity, and in mouse orthotopic models. The FANCD2-related signaling pathway was investigated using assays of peroxidation, a malondialdehyde assay, a reduced glutathione assay, and using FerroOrange to assess intracellular iron ions (Fe2+ ). Here, we report that FANCD2 was highly expressed in the malignant sonic hedgehog (SHH) MB subtype (SHH-MB). FANCD2 played an oncogenic role and predicted worse prognosis in SHH-MB patients. Moreover, FANCD2 knockdown markedly suppressed viability, mobility, and growth of SHH-MB cells and sensitized SHH-MB cells to irradiation. Mechanistically, FANCD2 deficiency led to an accumulation of Fe2+ due to increased divalent metal transporter 1 expression and impaired glutathione peroxidase 4 activity, which further activated ferroptosis and reduced proliferation of SHH-MB cells. Using an orthotopic mouse model, we observed that radiotherapy combined with silencing FANCD2 significantly inhibited the growth of SHH-MB cell-derived tumors in vivo. Our study revealed FANCD2 as a potential therapeutic target in SHH-MB and silencing FANCD2 could sensitize SHH-MB cells to radiotherapy via inducing ferroptosis. © 2024 The Pathological Society of Great Britain and Ireland.

Prognostic significance of molecular subgroups in survival outcome for children with medulloblastoma in Malaysia.

Advancements in genomic profiling led to the discovery of four major molecular subgroups in medulloblastoma (MB), which have now been incorporated into the World Health Organization classification of central nervous system tumors. The current study aimed to determine the prognostic significance of the MB molecular subgroups among children in Malaysia. We assembled MB samples from children <18 years between January 2003 and June 2017 from four pediatric oncology centers in Malaysia. MB was sub-grouped using 850k DNA methylation testing at German Cancer Research Centre, Heidelberg, Germany. Fifty samples from patients diagnosed and treated as MB were identified. Two (4%) of the 50 patients' tumor DNA samples were insufficient for analysis. Of the remaining 48 patients, 41 (85%) samples were confirmed as MB, while for 7 (15%) patients, DNA methylation classification results were discrepant with the histopathological diagnosis of MB, with various other diagnoses. Of the 41 MB patients, 15 patients were stratified as standard-risk (SR), 16 patients as high-risk (HR), and ten as infants (age <3 years old). Molecular subgrouping of the whole cohort revealed four (14%) WNT, 11 (27%) SHH, 10 (24%) Group 3, and 16 (39%) Group 4. Treatment abandonment rates for older children and infants were 22.5% and 10%, respectively. After censoring treatment abandonment, for SR patients, the 5-year event-free survival (EFS) and overall survival (OS) were 43.1% ± 14.7% and 46.9 ± 15.6%, respectively, while in HR, 5-year EFS and OS were both 63.6% ± 14.5%. Infants had a 5-year EFS and OS of 55.6% ± 16.6% and 66.7% ± 15.7%, respectively. WNT tumors had the best 5y-OS, followed by Group 3, Group 4, and SHH in children ≥3 years old. In younger children, SHH MB patients showed favorable outcomes. The study highlights the importance of DNA methylation profiling for diagnostic accuracy. Most infants had SHH MB, and their EFS and OS were comparable to those reported in high-income countries. Due to the relatively small cohort and the high treatment abandonment rate, definite conclusions cannot be made regarding the prognostic significance of molecular subgroups of MB. Implementing this high-technology investigation would assist pathologists in improving the diagnosis and provide molecular subgrouping of MB, permitting subgroup-specific therapies.

MBMethPred: a computational framework for the accurate classification of childhood medulloblastoma subgroups using data integration and AI-based approaches.

Childhood medulloblastoma is a malignant form of brain tumor that is widely classified into four subgroups based on molecular and genetic characteristics. Accurate classification of these subgroups is crucial for appropriate treatment, monitoring plans, and targeted therapies. However, misclassification between groups 3 and 4 is common. To address this issue, an AI-based R package called MBMethPred was developed based on DNA methylation and gene expression profiles of 763 medulloblastoma samples to classify subgroups using machine learning and neural network models. The developed prediction models achieved a classification accuracy of over 96% for subgroup classification by using 399 CpGs as prediction biomarkers. We also assessed the prognostic relevance of prediction biomarkers using survival analysis. Furthermore, we identified subgroup-specific drivers of medulloblastoma using functional enrichment analysis, Shapley values, and gene network analysis. In particular, the genes involved in the nervous system development process have the potential to separate medulloblastoma subgroups with 99% accuracy. Notably, our analysis identified 16 genes that were specifically significant for subgroup classification, including EP300, CXCR4, WNT4, ZIC4, MEIS1, SLC8A1, NFASC, ASCL2, KIF5C, SYNGAP1, SEMA4F, ROR1, DPYSL4, ARTN, RTN4RL1, and TLX2. Our findings contribute to enhanced survival outcomes for patients with medulloblastoma. Continued research and validation efforts are needed to further refine and expand the utility of our approach in other cancer types, advancing personalized medicine in pediatric oncology.

Smurf1 and Smurf2 mediated polyubiquitination and degradation of RNF220 suppresses Shh-group medulloblastoma

Sonic hedgehog (Shh)-group medulloblastoma (MB) (Shh-MB) encompasses a clinically and molecularly distinct group of cancers originating from the developing nervous system with aberrant high Shh signaling as a causative driver. We recently reported that RNF220 is required for sustained high Shh signaling during Shh-MB progression; however, how high RNF220 expression is achieved in Shh-MB is still unclear. In this study, we found that the ubiquitin E3 ligases Smurf1 and Smurf2 interact with RNF220, and target it for polyubiquitination and degradation. In MB cells, knockdown or overexpression of Smurf1 or Smurf2 promotes or inhibits cell proliferation, colony formation and xenograft growth, respectively, by controlling RNF220 protein levels, and thus modulating Shh signaling. Furthermore, in clinical human MB samples, the protein levels of Smurf1 or Smurf2 were negatively correlated with those of RNF220 or GAB1, a Shh-MB marker. Overall, this study highlights the importance of the Smurf1- and Smurf2-RNF220 axes during the pathogenesis of Shh-MB and provides new therapeutic targets for Shh-MB treatment.

EYA2 tyrosine phosphatase inhibition reduces MYC and prevents medulloblastoma progression.

Medulloblastoma is the most common pediatric brain malignancy. Patients with the Group 3 subtype of medulloblastoma (MB) often exhibit MYC amplification and/or overexpression and have the poorest prognosis. While Group 3 MB is known to be highly dependent on MYC, direct targeting of MYC remains elusive. Patient gene expression data were used to identify highly expressed EYA2 in Group 3 MB samples, assess the correlation between EYA2 and MYC, and examine patient survival. Genetic and pharmacological studies were performed on EYA2 in Group 3 derived MB cell models to assess MYC regulation and viability in vitro and in vivo. EYA2 is more highly expressed in Group 3 MB than other MB subgroups and is essential for Group 3 MB growth in vitro and in vivo. EYA2 regulates MYC expression and protein stability in Group 3 MB, resulting in global alterations of MYC transcription. Inhibition of EYA2 tyrosine phosphatase activity, using a novel small molecule inhibitor (NCGC00249987, or 9987), significantly decreases Group 3 MB MYC expression in both flank and intracranial growth in vivo. Human MB RNA-seq data show that EYA2 and MYC are significantly positively correlated, high EYA2 expression is significantly associated with a MYC transcriptional signature, and patients with high EYA2 and MYC expression have worse prognoses than those that do not express both genes at high levels. Our data demonstrate that EYA2 is a critical regulator of MYC in Group 3 MB and suggest a novel therapeutic avenue to target this highly lethal disease.

Low Expression of the NRP1 Gene Is Associated with Shorter Overall Survival in Patients with Sonic Hedgehog and Group 3 Medulloblastoma.

Medulloblastoma (MB) is the most common type of malignant pediatric brain tumor. Neuropilin-1 (NRP1), encoded by the NRP1 gene, is a transmembrane glycoprotein overexpressed in several types of cancer. Previous studies indicate that NRP1 inhibition displays antitumor effects in MB models and higher NRP1 levels are associated with poorer prognosis in MB patients. Here, we used a large MB tumor dataset to examine NRP1 gene expression in different molecular subgroups and subtypes of MB. We found overall widespread NRP1 expression across MB samples. Tumors in the sonic hedgehog (SHH) subgroup showed significantly higher NRP1 transcript levels in comparison with Group 3 and Group 4 tumors, with SHH samples belonging to the α, β, Δ, and γ subtypes. When all MB subgroups were combined, lower NRP1 expression was associated with significantly shorter patient overall survival (OS). Further analysis showed that low NRP1 was related to poorer OS, specifically in MB subgroups SHH and Group 3 MB. Our findings indicate that patients with SHH and Group 3 tumors that show lower expression of NRP1 in MB have a worse prognosis, which highlights the need for subgroup-specific investigation of the NRP1 role in MB.

Musashi-1 regulates cell cycle and confers resistance to cisplatin treatment in Group 3/4 medulloblastomas cells.

Groups (Grp) 3 and 4 are aggressive molecular subgroups of medulloblastoma (MB), with high rates of leptomeningeal dissemination. To date, there is still a paucity of biomarkers for these subtypes of MBs. In this study, we investigated the clinical significance and biological functions of Musashi-1 (MSI1) in Grp3 and Grp4-MBs. First, we assessed the expression profile of MSI1 in 59 primary MB samples (15-WNT, 18-SHH, 9-Grp3, and 17-Grp4 subgroups) by qRT-PCR. MSI1 mRNA expression levels were also validated in an additional public dataset of MBs (GSE85217). The ROC curve was used to validate the diagnostic standards of MSI1 expression. Next, the potential correlated cell-cycle genes were measured by RNA-Seq. Cell cycle, cell viability, and apoptosis were evaluated in a Grp3/Grp4 MB cell line after knockdown of MSI1 and cisplatin treatment. We identified an overexpression of MSI1 with a high accuracy to discriminate Grp3/Grp4-MBs from non-Grp3/Grp4-MBs. We identified that MSI1 knockdown not only triggered transcriptional changes in the cell-cycle pathway, but also affected G2/M phase in vitro, supporting the role of knockdown of MSI1 in cell-cycle arrest. Finally, MSI1 knockdown decreased cell viability and sensitized D283-Med cells to cisplatin treatment by enhancing cell apoptosis. Based on these findings, we suggest that MSI1 modulates cell-cycle progression and may play a role as biomarker for Grp3/Grp4-MBs. In addition, MSI1 knockdown combined with cisplatin may offer a potential strategy to be further explored in Grp3/Grp4-MBs.