Pediatric High-grade Gliomas Research Articles

DYSREGULATION OF M6A RNA METHYLATION, R-LOOPS AND THE DNA DAMAGE RESPONSE IN PAEDIATRIC HIGH-GRADE GLIOMAS

Abstract AIMS Paediatric high-grade gliomas (pHGG) are virtually incurable malignant brain tumours. The regulation of R- loops is necessary for ensuring genomic stability. The formation of R-loops at DNA damage sites promotes DNA repair via m6A methylation of the RNA strand. We aimed to investigate whether m6A methylation, R-loop homeostasis and the DNA damage response is dysregulated in pHGG and whether inhibitors, such as ATM inhibitors, could be potential therapeutics for pHGG. METHOD To assess the global levels of m6A, R-loops, yH2AX, total ATM and phosphorylated ATM in paediatric HGG cell lines compared to control human astrocytes, immunocytochemistry was initially performed. Immunocyto- chemistry was also performed to assess localisation between m6A and R-loops, R-loops and yH2AX, R-loops and total/phosphorylated ATM, m6A and total/phosphorylated ATM, and total/phosphorylated ATM and yH2AX in pHGG cell lines and human astrocytes. We are also exploring two ATM inhibitors, AZD1390 and AZD0156, and are currently investigating their effects on m6A methylation, R-loops and the DNA damage response in pHGG. As a molecular mechanism involving the tumour suppressor ARID1A was recently discovered that is crucial for maintaining genomic stability, we are also exploring the role of this protein in pHGG. We are also investigating the potential involvement of m6A readers in the pathogenesis of pHGG. RESULTS Immunocytochemistry revealed that dysregulation of global m6A, R-loop, yH2AX and ATM occurs in pHGG cell lines as higher levels of expression was generally observed compared to human astrocytes. Moreover, m6A, R-loops, yH2AX and ATM appear to colocalise as high Pearson’s correlation coefficient values were obtained. Experiments exploring ATM inhibitors, ARID1A and m6A readers in pHGG are currently in progress, however the results will be presented. CONCLUSION It appears that dysregulation of m6A methylation, R-loop homeostasis and the DNA damage response occurs in pHGG and that molecular mechanisms involving m6A methylation and R-loops could be impaired.

IDENTIFYING YB1 AND NPM1 AS PART OF THE MUTANT HISTONE 3 PROTEIN INTERACTOME IN H3 MUTATED PHGG

Abstract AIMS Paediatric high-grade gliomas (pHGG) are the leading cause of childhood cancer deaths, accounting for over 40% of deaths in the UK. pHGG tumours harbour histone H3 mutations, defining 50% of pHGG cases and the major mutations are H3.3K27M and H3.3G34R/V. Our previous proteomic research on H3 intercatomes, identified unique and common interacting binding partners of H3-G34R and H3-G34V mutant proteins relative to wild type H3 protein. Among these binding proteins we have focused on YB-1 and Nucleophosmin 1 (NPM1). YB-1, is multifunctional transcription factor and a well- characterised oncogenic protein. NPM1 is a molecular chaperone and frequently overexpressed in cancers. Our aim is to analyse the role of YB-1 and NPM1 in H3 mutated pHGG, in order to identify targeted therapeutic options for these difficult to treat tumours. METHOD H3 mutated glioma, H3 wild type pHGG and control cell lines were cultured and then subjected to comparative immunofluorescence microscopy, and western blot analyses to investigate YB-1 and NPM1 expression. RESULTS Results showed statistically significant altered protein expression and localisation of YB-1 and NPM1 in H3 mutated pHGG in comparison to wild type and control cells. YB-1 expression was increased in H3 mutated cells and NPM1 showed increased cytoplasmic localization in H3 mutated cells in comparison to H3 WT glioma and control cells. CONCLUSION Results of the study support that YB-1 and NPM1 are important interacting proteins in H3G34-mutated pHGGs and can be explored as potential therapeutic targets in H3 mutated pHGG.

H3K27me3 Loss in Central Nervous System Tumors: Diagnostic, Prognostic, and Therapeutic Implications.

Central nervous system (CNS) tumors represent a formidable clinical challenge due to their molecular complexity and varied prognostic outcomes. This review delves into the pivotal role of the epigenetic marker H3K27me3 in the development and treatment of CNS tumors. H3K27me3, specifically the trimethylation of lysine 27 on the histone H3 protein, plays a crucial role in regulating gene expression and maintaining chromatin architecture (e.g., in X-chromosome inactivation). Notably, a reduction in H3K27me3 levels, frequently tied to mutations in the H3 gene family such as H3F3A and HIST1H3B, is evident in diverse brain tumor variants, including the diffuse midline glioma characterized by the H3K27M mutation and certain pediatric high-grade gliomas. The loss of H3K27me3 has been linked to more aggressive behavior in meningiomas, with the trimethylation loss associated with significantly shorter recurrence-free survival (RFS) among grade 2 meningiomas, albeit not within grade 1 tumors. Pediatric posterior fossa ependymomas characterized by a lowered H3K27me3 and DNA hypomethylation exhibit poor prognosis, underscoring the prognostic significance of these epigenetic alterations in CNS tumors. Comprehending the role of H3K27me3 in CNS tumors is vital for advancing diagnostic tools and therapeutic interventions, with the goal of enhancing patient outcomes and quality of life. This review underscores the importance of ongoing investigations into H3K27me to refine and optimize management strategies for CNS tumors, paving the way for improved personalized medicine practices in oncology.

Microglia and monocyte-derived macrophages drive progression of pediatric high-grade gliomas and are transcriptionally shaped by histone mutations

Pediatric high-grade gliomas (pHGGs), including hemispheric pHGGs and diffuse midline gliomas (DMGs), harbor mutually exclusive tumor location-specific histone mutations. Using immunocompetent de novo mouse models of pHGGs, we demonstrated that myeloid cells were the predominant infiltrating non-neoplastic cell population. Single-cell RNA sequencing (scRNA-seq), flow cytometry, and immunohistochemistry illustrated the presence of heterogeneous myeloid cell populations shaped by histone mutations and tumor location. Disease-associated myeloid (DAM) cell phenotypes demonstrating immune permissive characteristics were identified in murine and human pHGG samples. H3.3K27M DMGs, the most aggressive DMG, demonstrated enrichment of DAMs. Genetic ablation of chemokines Ccl8 and Ccl12 resulted in a reduction of DAMs and an increase in lymphocyte infiltration, leading to increased survival of tumor-bearing mice. Pharmacologic inhibition of chemokine receptors CCR1 and CCR5 resulted in extended survival and decreased myeloid cell infiltration. This work establishes the tumor-promoting role of myeloid cells in DMG and the potential therapeutic opportunities for targeting them.

Characterization of aberrant splicing in pediatric central nervous system tumors reveals CLK1 as a candidate oncogenic dependency.

Pediatric brain cancer is the leading cause of disease-related mortality in children, and many aggressive tumors still lack effective treatment strategies. We characterized aberrant alternative splicing across pediatric brain tumors, identifying pediatric high-grade gliomas (HGGs) among the most heterogeneous. Annotating these events with UniProt, we identified 11,940 splice events in 5,368 genes leading to potential protein function changes. We discovered CDC-like kinase 1 (CLK1) is aberrantly spliced to include exon 4, resulting in a gain of two phosphorylation sites and subsequent activation. Inhibition of CLK1 with Cirtuvivint significantly decreased both cell viability and proliferation in the pediatric HGG KNS-42 cell line. Morpholino-mediated depletion of CLK1 exon 4 splicing reduced RNA expression, protein abundance, and cell viability with concurrent differential expression of 78 cancer genes and differential splicing at functional sites in 193 cancer genes. Our findings highlight a dependency of pediatric HGGs on CLK1 and represent a promising therapeutic strategy.

Pediatric metastatic extracranial high-grade glioma: A case report and literature review

Abstract We report a case of a 10-year-old male with a right frontal diffuse pediatric-type high-grade glioma (HGG), H3-wild-type (WT), and IDH-WT, diagnosed at the age of 9 years, who underwent gross total resection, 60 Gy focal proton radiation in 30 fractions to the resection cavity with concurrent temozolomide followed by maintenance chemotherapy with temozolomide and lomustine. One month after completion of maintenance chemotherapy, he developed subcutaneous swelling in the right temporal region and was treated with antibiotics for presumed lymphadenitis. Two months later, he developed a recurrent painless right parietal soft tissue mass that failed to respond to antibiotic therapy. This prompted evaluation by MRI which revealed new enhancing masses in the cerebellum and extracranial soft tissue mass in the right temporal region. He underwent gross total resection of both masses. Pathologic analysis confirmed both masses as recurrent HGG. Molecular markers, however, differed between the 2 sites of recurrence. He proceeded to complete hypofractionated proton therapy at sites of recurrence. Three months later, he was found to have tumor dissemination into the spine and brain for which he received proton therapy to the whole spine and brain. Due to the presence of CDK4 amplification at diagnosis and both sites of tumor recurrence, he then received palliative treatment with the CDK4/6 inhibitor, abemaciclib, for the final 5 months of his life. Since extracranial HGG is a rare presentation, with few cases reported in the pediatric population, we report this case and review previously published literature.

112 (PB100): The novel ATR inhibitor elimusertib is efficacious as monotherapy and in combination with radiation in pdox models of pediatric high-grade glioma

112 (PB100): The novel ATR inhibitor elimusertib is efficacious as monotherapy and in combination with radiation in pdox models of pediatric high-grade glioma

Emerging and Biological Concepts in Pediatric High-Grade Gliomas.

Primary central nervous system tumors are the most frequent solid tumors in children, accounting for over 40% of all childhood brain tumor deaths, specifically high-grade gliomas. Compared with pediatric low-grade gliomas (pLGGs), pediatric high-grade gliomas (pHGGs) have an abysmal survival rate. The WHO CNS classification identifies four subtypes of pHGGs, including Grade 4 Diffuse midline glioma H3K27-altered, Grade 4 Diffuse hemispheric gliomas H3-G34-mutant, Grade 4 pediatric-type high-grade glioma H3-wildtype and IDH-wildtype, and infant-type hemispheric gliomas. In recent years, we have seen promising advancements in treatment strategies for pediatric high-grade gliomas, including immunotherapy, CAR-T cell therapy, and vaccine approaches, which are currently undergoing clinical trials. These therapies are underscored by the integration of molecular features that further stratify HGG subtypes. Herein, we will discuss the molecular features of pediatric high-grade gliomas and the evolving landscape for treating these challenging tumors.

Pediatric Hemispheric High-Grade Gliomas and H3.3-G34 Mutation: A Review of the Literature on Biological Features and New Therapeutic Strategies.

Pediatric high-grade glioma (pHGG) encompasses a wide range of gliomas with different genomic, epigenomic, and transcriptomic features. Almost 50% of pHGGs present a mutation in genes coding for histone 3, including the subtype harboring the H3.3-G34 mutation. In this context, histone mutations are frequently associated with mutations in TP53 and ATRX, along with PDGFRA and NOTCH2NL amplifications. Moreover, the H3.3-G34 histone mutation induces epigenetic changes in immune-related genes and exerts modulatory functions on the microenvironment. Also, the functionality of the blood-brain barrier (BBB) has an impact on treatment response. The prognosis remains poor with conventional treatments, thus eliciting the investigation of additional and alternative therapies. Promising molecular targets include PDGFRA amplification, BRAF mutation, EGFR amplification, NF1 loss, and IDH mutation. Considering that pHGGs harboring the H3.3-G34R mutation appear to be more susceptible to immunotherapies (ITs), different options have been recently explored, including immune checkpoint inhibitors, antibody mediated IT, and Car-T cells. This review aims to summarize the knowledge concerning cancer biology and cancer-immune cell interaction in this set of pediatric gliomas, with a focus on possible therapeutic options.

Journey through tumorverse: Creating models to decode PXA mysteries

Pleomorphic xanthoastrocytoma (PXA) is a rare pediatric low-grade glioma (pLGG), of which 60%-80% exhibit the BRAF V600E mutation, that enhances the aggressiveness and progression to an eventual pediatric high-grade glioma (pHGG). Despite the aggressiveness of this mutational status, the mechanisms underlying the progression of BRAF V600E tumors remain poorly understood, primarily due to limited insights into their invivo growth dynamics. In this issue,Rajappa and colleagues leverage a novel immunocompetent RCAS-BRAF V600E murine glioma model to profile the immunological dynamics taking place in BRAF V600E pLGG.

Molecular and Pathological Features of Paediatric High-Grade Gliomas.

Paediatric high-grade gliomas are among the most common malignancies found in children. Despite morphological similarities to their adult counterparts, there are profound biological and molecular differences. Furthermore, and thanks to molecular biology, the diagnostic pathology of paediatric high-grade gliomas has experimented a dramatic shift towards molecular classification, with important prognostic implications, as is appropriately reflected in both the current WHO Classification of Tumours of the Central Nervous System and the WHO Classification of Paediatric Tumours. Emphasis is placed on histone 3, IDH1, and IDH2 alterations, and on Receptor of Tyrosine Kinase fusions. In this review we present the current diagnostic categories from the diagnostic pathology perspective including molecular features.

AB001. Development of tumour-targeted therapy for the treatment of adult and paediatric high-grade gliomas.

Brain cancer patients, especially those suffering from high-grade gliomas (HGGs) face a bleak future with very dismal long-term disease-free survival outcomes due to the limited treatment options currently available. Therefore, there is an unmet need for new therapeutic intervention that extends patients' progress-free survival and improves their quality of life. A significant hurdle is the inability of current chemotherapy agents to cross the blood-brain barrier (BBB). BBB acts as a protective shield that filters the blood to ensure nothing harmful makes it to the brain. This protection is usually good, but it becomes a problem if you want to deliver therapeutic cancer drugs through it. This barrier blocks 98% of drugs from entering the brain. Even the ones that cross BBB are unevenly distributed in the normal brain and tumour tissue, resulting in mediocre treatment and severe side effects. We are developing drug delivery systems that can cross the BBB and facilitate the specific accumulation of drugs in the tumour tissue. This will significantly improve the efficacy of anticancer drugs in treating various brain cancers and reduce systemic toxicity. Our group has explored and developed BBB crossing and tumour targeting near infra-red dyes, which can be covalently attached to Food and Drug Administration (FDA)-approved chemotherapy agents (drug-dye conjugates), thereby delivering it to the tumour tissue. We synthesized such drug-dye conjugates to target various aberrant pathways in HGG and tested these conjugates against patient-derived HGG cell lines. One such conjugate was tested on a mouse model of glioblastoma, an aggressive form of HGG, and shown to cross the BBB and specifically accumulate in tumour tissue, bringing forth tumour burden reduction. The results obtained from this work serve as proof of principle that enables tumour-specific drug delivery to treat HGG. This work also paves the way for treating other brain cancers and central nervous system (CNS) disorders like Parkinson's and Alzheimer's disease, for which no adequate therapy exists.

AB005. Development of tumour specific therapy for treatment of diffuse intrinsic pontine glioma (DIPG).

Diffuse intrinsic pontine glioma (DIPG), is an aggressive form of paediatric high-grade glioma (pHGG) that affects children below the age of 10 months. The survival period for a child suffering from DIPG has not changed in decades (approximately 10 months). This pattern is similar for most pHGG; even though the survival period is more extended, tumour recurrence and death are almost inevitable. This is primarily due to the presence of the blood-brain barrier (BBB), which blocks the entry of most therapeutics into the brain, and also due to tumour heterogeneity associated with central nervous system (CNS) tumours that blunt the efficacy of targeted therapy. The development of a meaningful cure for paediatric brain cancer hinges on discovering chemotherapy agents that (I) can cross the BBB; (II) accumulates explicitly in tumour tissues; and (III) can block pathways leading to the escape of cancer stem cells, promoting recurrence. This project aims to develop therapeutics that can cross the BBB, a significant hindrance to delivering medicines across the brain, and specifically target cancer cells without affecting normal brain cells. We will accomplish this by attaching novel dyes possessing tumour specificity to various classes of chemotherapy agents. The compounds will be tested on patient-derived paediatric brain cancer cell lines and the most potent compounds will be progressed to an animal model of DIPG. Several drug-dye conjugates were designed and synthesized to target various aberrant pathways involved in disease initiation and progression of DIPG. These were tested first in patient-derived DIPG cell lines. Several of these drug-dye conjugates showed potent antiproliferative effect in various DIPG cell lines. One of these conjugates is currently undergoing maximum tolerated dose study in an animal model of DIPG. The present work details an effort to develop BBB crossing tumour specific therapeutic agents for the treatment of DIPG. The work has resulted in several promising drug-dye conjugates showing antiproliferative activity in various patient-derived DIPG cell lines, enabling the progression of such conjugates into animal models of DIPG. Such studies will inform the utility of such drug-dye conjugates for application in difficult to treat pHGGs such as DIPG.

Co-culturing Glutamatergic Neurons and Pediatric High-Grade Glioma Cells

Co-culturing Glutamatergic Neurons and Pediatric High-Grade Glioma Cells

Co-culturing Glutamatergic Neurons and Pediatric High-Grade Glioma Cells

Co-culturing Glutamatergic Neurons and Pediatric High-Grade Glioma Cells

Utility of OLIG2 immunostaining in pediatric brain tumors with embryonal morphology.

This study evaluates the diagnostic utility of OLIG2 immunohistochemistry for distinguishing between pediatric high-grade gliomas (pHGG) and embryonal tumors (ETs) of the CNS. Utilizing a retrospective pediatric cohort (1990-2021) of 56 CNS tumors, classified initially as primitive neuroectodermal tumors or CNS ET, we reclassified the cases based on WHO CNS5 criteria after comprehensive review and additional molecular testing that included next-generation sequencing and DNA methylation profiling. Our results indicate that OLIG2 immunopositivity was negative or minimal in a significant subset of pHGG cases (6 out of 11). At the same time, it showed diffuse expression in all cases of CNS neuroblastomas with FOXR2 activation (5/5), demonstrating its limited specificity in differentiating between pHGG and ET. Variable OLIG2 expression in other ETs, ATRT, and ETMR suggests the broader diagnostic implications of the marker. Furthermore, incidental findings of OLIG2 positivity in cases traditionally expected to be negative, such as medulloblastoma and ependymoma, introduce an additional layer of complexity. Together, these findings highlight the challenges of relying solely on OLIG2 immunostaining for accurate tumor classification in pediatric CNS neoplasms and underscore the importance of an integrated diagnostic approach.

Somatic CpG hypermutation is associated with mismatch repair deficiency in cancer

Somatic hypermutation in cancer has gained momentum with the increased use of tumour mutation burden as a biomarker for immune checkpoint inhibitors. Spontaneous deamination of 5-methylcytosine to thymine at CpG dinucleotides is one of the most ubiquitous endogenous mutational processes in normal and cancer cells. Here, we performed a systematic investigation of somatic CpG hypermutation at a pan-cancer level. We studied 30,191 cancer patients and 103 cancer types and developed an algorithm to identify somatic CpG hypermutation. Across cancer types, we observed the highest prevalence in paediatric leukaemia (3.5%), paediatric high-grade glioma (1.7%), and colorectal cancer (1%). We discovered germline variants and somatic mutations in the mismatch repair complex MutSα (MSH2-MSH6) as genetic drivers of somatic CpG hypermutation in cancer, which frequently converged on CpG sites and TP53 driver mutations. We further observe an association between somatic CpG hypermutation and response to immune checkpoint inhibitors. Overall, our study identified novel cancer types that display somatic CpG hypermutation, strong association with MutSα-deficiency, and potential utility in cancer immunotherapy.

Canadian Consensus for Treatment of BRAF V600E Mutated Pediatric and AYA Gliomas.

Background: The treatment of BRAF V600E gliomas with BRAF inhibitors (BRAFis) and MEK inhibitors (MEKis) has been increasingly integrated into clinical practice for pediatric low-grade gliomas (PLGGs) and pediatric high-grade gliomas (HGGs). However, some questions remain unanswered, such as the best time to start targeted therapy, duration of treatment, and discontinuation of therapy. Given that no clinical trial has been able to address these critical questions, we developed a Canadian Consensus statement for the treatment of BRAF V600E mutated pediatric as well as adolescent and young adult (AYA) gliomas. Methods: Canadian neuro-oncologists were invited to participate in the development of this consensus. The consensus was discussed during monthly web-based national meetings, and the algorithms were revised until a consensus was achieved. Results: A total of 26 participants were involved in the development of the algorithms. Two treatment algorithms are proposed, one for the initiation of treatment and one for the discontinuation of treatment. We suggest that most patients with BRAF V600E gliomas should be treated with BRAFis ± MEKis upfront. Discontinuation of treatment can be considered in certain circumstances, and we suggest a slow wean. Conclusions: Based on expert consensus in Canada, we developed algorithms for treatment initiation of children and AYA with BRAF V600E gliomas as well as a discontinuation algorithm.

H3K27-Altered Diffuse Midline Glioma of the Brainstem: From Molecular Mechanisms to Targeted Interventions.

Pediatric high-grade gliomas are a devastating subset of brain tumors, characterized by their aggressive pathophysiology and limited treatment options. Among them, H3 K27-altered diffuse midline gliomas (DMG) of the brainstem stand out due to their distinct molecular features and dismal prognosis. Recent advances in molecular profiling techniques have unveiled the critical role of H3 K27 alterations, particularly a lysine-to-methionine mutation on position 27 (K27M) of the histone H3 tail, in the pathogenesis of DMG. These mutations result in epigenetic dysregulation, which leads to altered chromatin structure and gene expression patterns in DMG tumor cells, ultimately contributing to the aggressive phenotype of DMG. The exploration of targeted therapeutic avenues for DMG has gained momentum in recent years. Therapies, including epigenetic modifiers, kinase inhibitors, and immunotherapies, are under active investigation; these approaches aim to disrupt aberrant signaling cascades and overcome the various mechanisms of therapeutic resistance in DMG. Challenges, including blood-brain barrier penetration and DMG tumor heterogeneity, require innovative approaches to improve drug delivery and personalized treatment strategies. This review aims to provide a comprehensive overview of the evolving understanding of DMG, focusing on the intricate molecular mechanisms driving tumorigenesis/tumor progression and the current landscape of emerging targeted interventions.

MODL-15. CHARACTERIZATION OF SPONTANEOUS CANINE HIGH-GRADE GLIOMA MODELS AS CLINICALLY-RELEVANT SURROGATES OF PEDIATRIC HIGH-GRADE GLIOMA

Abstract BACKGROUND High-grade glioma (HGG) occurring spontaneously in canines shows histological and genetic resemblance to pediatric high-grade glioma (PHGG). Both canine HGG and PHGG are treated with combinations of surgical resection, radiation therapy, and/or chemotherapy, but outcomes for both groups are poor. Studying canine HGG both serves companion dogs and provides an orthogonal preclinical model of PHGG. METHODS Canine HGG lines J3T, SDT3G, and G06A were characterized and compared with H3K27M-mutant PHGG lines DIPG7, DIPG13, and BT245 using neurosphere dilution assay and drug screening with all FDA-approved anti-cancer agents. J3T and SDT3G were orthotopically implanted into mice and monitored using MRI and bioluminescence. RESULTS Like PHGG, the canine lines J3T and SDT3G were highly sensitive to proteasome and XPO1 inhibition; compared to PHGG, the canine lines were most sensitive to protein translation inhibitors and DNA intercalators. Canine HGG lines were not as sensitive to other targeted inhibitors effective in PHGG. Compared to PHGG cell lines, canine cell lines had greater variance in self-renewal capability. J3T and SDT3G were also injected into the cortex of immunodeficient mice. Bioluminescent signal confirmed J3T engraftment one week post-surgery with rapid tumor growth, while SDT3G presented initial engraftment by MRI two weeks post-surgery but did not exhibit sustained growth. DISCUSSION Canine HGG and PHGG showed similar sensitivity to proteasome and XPO1 inhibition; they differed in self-renewal capability and susceptibility to other anti-cancer agents. This could be because canine target proteins often differ from their human counterparts and thus may respond differently to targeted medications designed for humans. Overall, canine HGG provides a novel, primary, immunocompetent surrogate that resembles PHGG and may provide a step for clinical investigation between xenograft models and PHGG trials and/or opportunities for co-clinical trials.