Pediatric High-grade Gliomas Research Articles

HGG-48. THE BLOOD-BASED TELOMERIC DNA C-CIRCLE ASSAY CAN DETECT ALTERNATIVE LENGTHENING OF TELOMERES (ALT) IN PEDIATRIC HIGH-GRADE GLIOMAS

Abstract BACKGROUND High-grade gliomas (HGG) comprise ~10% of pediatric brain tumors; 40% of pediatric HGG use the alternative lengthening of telomeres (ALT) mechanism to maintain replicative immortality. ALT cancers share a unique biology providing potential therapeutic targets. Extrachromosomal telomere DNA repeats, termed C-circles, can be detected by a unique PCR assay, providing a sensitive and specific biomarker for ALT cancers. C-circles have been detected in ALT tumors and serum of ALT patients. We have adapted the C-circle assay (CCA) to provide a sensitive and specific assay with cfDNA in patient plasma. Here we determined if cfDNA in plasma can be used to detect patients with ALT-positive HGG. METHODS Frozen plasma samples were collected in Streck tubes at time of surgery prior to resection from patients with HGG. DNA was extracted and quantified. Isothermal rolling C-circle amplification and real-time telomere PCR was performed followed by analysis. The C-circle positive neuroblastoma cell line, CHLA-90, and C-circle negative neuroblastoma cell line, CHLA-20, served as the positive and negative controls respectively. CCA values in plasma of 2 and above indicate positivity for C-circles. RESULTS Plasma samples were analyzed by CCA from three pediatric patients whose brain tumors were previously biopsied and identified as ATRX mutation positive. ATRX mutation indicates ALT however, not all ALT cancers have ATRX mutations. All plasma samples were above the 2.0 cutoff at 3.37, 3.13, and 4.59 and thus classified as C-circle positive. CONCLUSIONS This preliminary cohort shows that the CCA has potential for identifying ALT pediatric brain tumors using cfDNA from plasma. As novel therapies effective against ALT HGG are developed, the plasma CCA may allow accelerated initiation of neoadjuvant therapy to shrink tumor prior to surgical resection and lessen or prevent the need for tumor biopsy. Expansion of the initial test cohort is underway. Supported by NCI UO1 CA63988.

The Landscape of Pediatric High-Grade Gliomas: The Virtues and Pitfalls of Pre-Clinical Models.

Pediatric high-grade gliomas (pHGG) are malignant and usually fatal central nervous system (CNS) WHO Grade 4 tumors. The majority of pHGG consist of diffuse midline gliomas (DMG), H3.3 or H3.1 K27 altered, or diffuse hemispheric gliomas (DHG) (H3.3 G34-mutant). Due to diffuse tumor infiltration of eloquent brain areas, especially for DMG, surgery has often been limited and chemotherapy has not been effective, leaving fractionated radiation to the involved field as the current standard of care. pHGG has only been classified as molecularly distinct from adult HGG since 2012 through Next-Generation sequencing approaches, which have shown pHGG to be epigenetically regulated and specific tumor sub-types to be representative of dysregulated differentiating cells. To translate discovery research into novel therapies, improved pre-clinical models that more adequately represent the tumor biology of pHGG are required. This review will summarize the molecular characteristics of different pHGG sub-types, with a specific focus on histone K27M mutations and the dysregulated gene expression profiles arising from these mutations. Current and emerging pre-clinical models for pHGG will be discussed, including commonly used patient-derived cell lines and in vivo modeling techniques, encompassing patient-derived xenograft murine models and genetically engineered mouse models (GEMMs). Lastly, emerging techniques to model CNS tumors within a human brain environment using brain organoids through co-culture will be explored. As models that more reliably represent pHGG continue to be developed, targetable biological and genetic vulnerabilities in the disease will be more rapidly identified, leading to better treatments and improved clinical outcomes.

Capmatinib is an effective treatment for MET-fusion driven pediatric high-grade glioma and synergizes with radiotherapy

BackgroundPediatric-type diffuse high-grade glioma (pHGG) is the most frequent malignant brain tumor in children and can be subclassified into multiple entities. Fusion genes activating the MET receptor tyrosine kinase often occur in infant-type hemispheric glioma (IHG) but also in other pHGG and are associated with devastating morbidity and mortality.MethodsTo identify new treatment options, we established and characterized two novel orthotopic mouse models harboring distinct MET fusions. These included an immunocompetent, murine allograft model and patient-derived orthotopic xenografts (PDOX) from a MET-fusion IHG patient who failed conventional therapy and targeted therapy with cabozantinib. With these models, we analyzed the efficacy and pharmacokinetic properties of three MET inhibitors, capmatinib, crizotinib and cabozantinib, alone or combined with radiotherapy.ResultsCapmatinib showed superior brain pharmacokinetic properties and greater in vitro and in vivo efficacy than cabozantinib or crizotinib in both models. The PDOX models recapitulated the poor efficacy of cabozantinib experienced by the patient. In contrast, capmatinib extended survival and induced long-term progression-free survival when combined with radiotherapy in two complementary mouse models. Capmatinib treatment increased radiation-induced DNA double-strand breaks and delayed their repair.ConclusionsWe comprehensively investigated the combination of MET inhibition and radiotherapy as a novel treatment option for MET-driven pHGG. Our seminal preclinical data package includes pharmacokinetic characterization, recapitulation of clinical outcomes, coinciding results from multiple complementing in vivo studies, and insights into molecular mechanism underlying increased efficacy. Taken together, we demonstrate the groundbreaking efficacy of capmatinib and radiation as a highly promising concept for future clinical trials.

CD57 defines a novel cancer stem cell that drive invasion of diffuse pediatric-type high grade gliomas.

Diffuse invasion remains a primary cause of treatment failure in pediatric high-grade glioma (pHGG). Identifying cellular driver(s) of pHGG invasion is needed for anti-invasion therapies. Ten highly invasive patient-derived orthotopic xenograft (PDOX) models of pHGG were subjected to isolation of matching pairs of invasive (HGGINV) and tumor core (HGGTC) cells. pHGGINV cells were intrinsically more invasive than their matching pHGGTC cells. CSC profiling revealed co-positivity of CD133 and CD57 and identified CD57+CD133- cells as the most abundant CSCs in the invasive front. In addition to discovering a new order of self-renewal capacities, i.e., CD57+CD133- > CD57+CD133+ > CD57-CD133+ > CD57-CD133- cells, we showed that CSC hierarchy was impacted by their spatial locations, and the highest self-renewal capacities were found in CD57+CD133- cells in the HGGINV front (HGGINV/CD57+CD133- cells) mediated by NANOG and SHH over-expression. Direct implantation of CD57+ (CD57+/CD133- and CD57+/CD133+) cells into mouse brains reconstituted diffusely invasion, while depleting CD57+ cells (i.e., CD57-CD133+) abrogated pHGG invasion. We revealed significantly increased invasive capacities in HGGINV cells, confirmed CD57 as a novel glioma stem cell marker, identified CD57+CD133- and CD57+CD133+ cells as a new cellular driver of pHGG invasion and suggested a new dual-mode hierarchy of HGG stem cells.

Rapid P-TEFb-dependent transcriptional reorganization underpins the glioma adaptive response to radiotherapy

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. P-TEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock, but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma, we show that rapid genome-wide reorganization of active chromatin facilitates P-TEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of P-TEFb disrupts this chromatin reorganization and blunts transcriptional induction, abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent, synergistic therapeutic potential agnostic of glioma subtype, leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for P-TEFb underpinning the early adaptive response to radiotherapy, opening avenues for combinatorial treatment in these lethal malignancies.

Fractionated radiation therapy alters energy metabolism and induces cellular quiescence exit in patient-derived orthotopic xenograft models of high-grade glioma

Radiation is one of the standard therapies for pediatric high-grade glioma (pHGG), of which the prognosis remains poor. To gain an in-depth understanding of biological consequences beyond the classic DNA damage, we treated 9 patient-derived orthotopic xenograft (PDOX) models, including one with DNA mismatch repair (MMR) deficiency, with fractionated radiations (2 Gy/day x 5 days). Extension of survival time was noted in 5 PDOX models (P < 0.05) accompanied by γH2AX positivity in >95 % tumor cells in tumor core and >85 % in the invasive foci as well as ∼30 % apoptotic and mitotic catastrophic cell death. The model with DNA MMR (IC-1406HGG) was the most responsive to radiation with a reduction of Ki-67(+) cells. Altered metabolism, including mitochondria number elevation, COX IV activation and reactive oxygen species accumulation, were detected together with the enrichment of CD133+ tumor cells. The latter was caused by the entry of quiescent G0 cells into cell cycle and the activation of self-renewal (SOX2 and BMI1) and epithelial mesenchymal transition (fibronectin) genes. These novel insights about the cellular and molecular mechanisms of fractionated radiation in vivo should support the development of new radio-sensitizing therapies.

Immunohistochemical Approach to Mismatch Repair Deficiency in Pediatric High-Grade Glioma.

Knowledge of the molecular pathways of pediatric high-grade gliomas is increasing. Gliomas with mismatch repair deficiency do not currently comprise a distinct group, but data on this topic have been accumulating in recent publications. Immunohistochemistry can effectively determine mismatch repair status, indirectly suggesting the microsatellite instability of the tumor. This study aimed to determine the number of mismatch repair-deficient pediatric high-grade gliomas in a tertiary institution and assess the relationship between the survival and mismatch repair status of the patients. It also aimed to assess the potential for further clinical studies including immunotherapy. Of 24 patients with high-grade gliomas, 3 deceased patients were mismatch repair-deficient. Mismatch repair deficiency was significantly associated with shorter survival ( P =0.004). Immunotherapy trials need to progress, and patients with mismatch repair-deficient pediatric high-grade gliomas are the most suitable candidates for such studies.

Advances in pediatric gliomas: from molecular characterization to personalized treatments.

Pediatric gliomas, consisting of both pediatric low-grade (pLGG) and high-grade gliomas (pHGG), are the most frequently occurring brain tumors in children. Over the last decade, several milestone advancements in treatments have been achieved as a result of stronger understanding of the molecular biology behind these tumors. This review provides an overview of pLGG and pHGG highlighting their clinical presentation, molecular characteristics, and latest advancements in therapeutic treatments. Conclusion: The increasing understanding of the molecular biology characterizing pediatric low and high grade gliomas has revolutionized treatment options for these patients, especially in pLGG. The implementation of next generation sequencing techniques for these tumors is crucial in obtaining less toxic and more efficacious treatments. What is Known: • Pediatric Gliomas are the most common brain tumour in children. They are responsible for significant morbidity and mortality in this population. What is New: • Over the last two decades, there has been a significant increase in our global understanding of the molecular background of pediatric low and high grade gliomas. • The implementation of next generation sequencing techniques for these tumors is crucial in obtaining less toxic and more efficacious treatments, with the ultimate goal of improving both the survival and the quality of life of these patients.

133 BMAL1 is Upregulated in Pediatric High-Grade Gliomas

INTRODUCTION: Pediatric high-grade gliomas (pHGG) are highly invasive brain tumors with poor survival outcomes, and novel therapeutic approaches are urgently needed. Recently, a retrospective study found timing of Temozolomide administration affects survival in adult patients with glioblastoma (Damato et al. 2021). There has, however, been limited exploration into circadian gene expression in pediatric gliomas. METHODS: We analyzed expression of BMAL1, a key circadian gene, in pediatric gliomas using bioinformatic approaches, Western blotting, and immunocytochemistry (ICC). We first compared BMAL1 expression in pLGGs and pHGGs using published publicly available data from cBioPortal for Cancer Genomics. Immunoblotting was utilized to determine BMAL1 protein expression levels in patient specimens of pHGG, pLGG and cortex from epilepsy surgery. Immunocytochemistry on synchronized primary tumor lines was used to show localization of BMAL1 protein. RESULTS: Using data from cBioPortal, we found significantly higher expression of BMAL1 in pHGGs compared to pLGGs (0.349 + 1.05 vs 0.0545 + 0.666, p &lt; 0.001). Western blotting showed increased expression of BMAL1 in HGG tumor specimens compared to LGG and cortical specimens. Immunostaining demonstrated cellular localization of BMAL1. CONCLUSIONS: In this study, we found that the circadian gene BMAL1 is expressed in pHGGs, and that expression is higher in pHGGs compared to pLGGs and cortex. Next steps will be to understand how timing of Temozolomide in relationship to the circadian cycle affects tumor cell survival.

I smell something weird - Ictal olfactory hallucinations in patients with primary brain tumors

IntroductionIctal olfactory hallucinations (the experience of a smell due to a focal seizure in the absence of an environmental stimulus for the sensation) are rare. They often appear in a context of a brain tumor located in the orbitofrontal or mesotemporal region. However, their accurate prevalence, etiology and anatomical origin remains unclear, as few studies focused on this type of seizures specifically.ObjectivesTo evaluate the clinical, neurophysiological and imaging characteristics of patients with brain tumors and olfactory seizures.MethodsWe present a 3-year retrospective patient record study carried out at the Portuguese Institute of Oncology in Lisbon. Clinical records of 572 patients admitted due to a primary Central Nervous System (CNS) tumor, for their first neuro-oncology appointment, between July 2020 and July 2023, were reviewed.Results8 patients with olfactory seizures were identified. Five were men. The mean age was 57.75 (ages between 15 and 70 years old). In seven patients, olfactory seizures constituted the initial clinical presentation of the tumor. In two patients, focal olfactory seizures had progression to bilateral tonic clonic. Most seizures were perceived as unpleasant (smells of metal, ammonia, “hot blood”, “dead bodies” were described). Tumors involved the temporal lobe in all patients, the insula in two of them and, for the majority, the lesion was right-sided. Six patients were diagnosed with Glioblastoma IDH wildtype (Grade 4, WHO), one patient with Oligodendroglioma, IDH-mutated and 1p/19q-codeleted (Grade 2, WHO) and the pediatric patient with a diffuse pediatric type high-grade glioma, H3 and IDH wildtype. The average follow-up time was 6.8 months, two patients died.ConclusionsThis is the first retrospective study carried out in Portugal that documents the prevalence of olfactory seizures in patients with primary CNS tumors. Given the scarce literary evidence, we consider that olfactory seizures may be more frequent than documented, particularly in the presentation of brain tumors. As so, active semiological investigation may contribute to an earlier diagnosis.Disclosure of InterestNone Declared

The oncolytic adenovirus Delta-24-RGD in combination with ONC201 induces a potent antitumor response in pediatric high-grade and diffuse midline glioma models.

Pediatric high-grade gliomas (pHGGs), including diffuse midline gliomas (DMGs), are aggressive pediatric tumors with one of the poorest prognoses. Delta-24-RGD and ONC201 have shown promising efficacy as single agents for these tumors. However, the combination of both agents has not been evaluated. The production of functional viruses was assessed by immunoblotting and replication assays. The antitumor effect was evaluated in a panel of human and murine pHGG and DMG cell lines. RNAseq, the seahorse stress test, mitochondrial DNA content, and γH2A.X immunofluorescence were used to perform mechanistic studies. Mouse models of both diseases were used to assess the efficacy of the combination in vivo. The tumor immune microenvironment was evaluated using flow cytometry, RNAseq, and multiplexed immunofluorescence staining. The Delta-24-RGD/ONC201 combination did not affect the virus replication capability in human pHGG and DMG models in vitro. Cytotoxicity analysis showed that the combination treatment was either synergistic or additive. Mechanistically, the combination treatment increased nuclear DNA damage and maintained the metabolic perturbation and mitochondrial damage caused by each agent alone. Delta-24-RGD/ONC201 cotreatment extended the overall survival of mice implanted with human and murine pHGG and DMG cells, independent of H3 mutation status and location. Finally, combination treatment in murine DMG models revealed a reshaping of the tumor microenvironment to a proinflammatory phenotype. The Delta-24-RGD/ONC201 combination improved the efficacy compared to each agent alone in in vitro and in vivo models by potentiating nuclear DNA damage and in turn improving the antitumor (immune) response to each agent alone.

Abstract 5105: Aberrant CLK1 splicing is a key dependency factor in pediatric high-grade gliomas

Abstract Pediatric brain cancer is the leading cause of disease-related mortality in children. With a paucity of therapeutically targetable alterations, there is a pressing need to identify effective strategies for many of these tumors. In this study, we systematically examine aberrant alternative splicing in pediatric brain tumors from the Open Pediatric Brain Tumor Atlas, and additional tumors from the Children's Brain Tumor Network and the Pacific Pediatric Neuro-Oncology Consortium (n = 1831). Notably, high-grade gliomas (HGGs), including those with and without H3 histone mutations, were enriched in the upper quartile of all tumors with high splicing burden (SB). HGGs demonstrated the greatest SB heterogeneity of all tumor types and we identified a total of 19,229 aberrant splicing events in 8,577 genes in midline tumors compared to non-tumor brainstem controls (ΔPSI &amp;gt;= |.20|, p &amp;lt; 0.05). We next integrated protein annotations from the UniProt Knowledgebase to assess and prioritize differential alternative splicing events affecting protein function. This approach led to the discovery of 3,754 splice variants in 5,036 genes resulting in the gain or ablation of functional sites. The protein kinase CDC-like kinase 1 (CLK1), a recognized master splicing factor and cell-cycle modulator, exhibited aberrant splicing of exon 4, whereby inclusion promoted the gain of two known phosphorylation sites. Additionally, we observed a strong correlation between CLK1 splicing, its own RNA expression and proteomic activity of the Serine/Arginine-rich (SR) family of splicing factors. The distribution of exon 4 splicing was significantly different (p = 0.019) between tumors with low vs high splicing burden; tumors with high splicing burden had increased exon 4 inclusion. Further, we performed experimental modulation of CLK1 exon 4 aberrant splicing in the brain tumor cell line KNS42 and demonstrated that forced exon 4 skipping leads to reduced CLK1 protein abundance and significantly reduces cell proliferation (p &amp;lt; 0.01 vs. control). Following bulk RNA-Seq analysis of these cell lines, we identified that forced CLK1 exon 4 skipping resulted in 4,518 dysregulated genes enriched for known cancer pathways such as mitotic spindle, E2F targets, and G2M checkpoint. Our data suggest a role for CLK1 in driving transcriptional dysregulation in pediatric high-grade gliomas and uncover a potential therapeutic vulnerability. Citation Format: Ammar S. Naqvi, Priyanka Seghal, Ryan Corbett, Komal Rathi, Brian Ennis, Bo Zhang, Miguel Brown, Daniel Miller, Adam Kraya, Joseph Dybas, Katharina Hayer, Shehbeel Arif, Zhuangzhuang Geng, Antonia Chroni, Aditya Lahiri, Karina Conkrite, Madison Hollawell, Sharon Diskin, Peter Madsen, Jessica B. Foster, Mateusz P. Koptyra, Andrei Thomas-Tikhonenko, Adam Resnick, Phillip J. Storm, Jo Lynne Rokita. Aberrant CLK1 splicing is a key dependency factor in pediatric high-grade gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5105.

Abstract 5110: Exploring the effects of Indirubins in pediatric diffuse high grade glioma models

Abstract Diffuse midline glioma (DMG) is an invasive pediatric brain tumor which grows in the pons of the brainstem. The current course of treatment is focal radiation however, DMG continues to have a very poor prognosis with a median survival of less than 10 months. A major barrier for chemotherapeutic treatment of DMG is the blood brain barrier (BBB) which tightly controls access of molecules to the brain. This limits the efficacy of drugs to treat brain cancer and other diseases of the central nervous system. Previous studies from the Lawler lab and others have shown that the indirubin derivative, 6-bromoindirubin-3′-acetoxime(BIO-acetoxime/BIA), has anti-invasive properties and can enhance survival in glioblastoma xenograft models. Here, we investigated the effects of BIA on pediatric glioma models using a range of relevant assays. In an in vitro collagen migration assay, BIA was observed to significantly slow cell migration at a concentration of 1µM in DIPG 36 cells, over 72 hours. Furthermore, we have also shown that BIA modulates the tumor vasculature, which could limit tumor growth, and improves drug delivery to tumors by targeting tight junctions in tumor associated endothelium. The effect on BBB drug penetration was assessed in a multicellular 3D in vitro BBB model and effects of BIA on endothelial barrier functions was determined using trans-endothelial resistance assays (TEER). BIA treatment increased dextran uptake into 3D BBB models, and also lowered endothelial cell permeability in vitro via a reduction in the expression of tight junction proteins as shown by staining and a decrease in TEER values. In order to assess potential synergistic interactions, a drug screen was performed on a panel of distinct pediatric glioma cell lines (DIPG 36 and KNS42). This identified several FDA approved drugs that combine well with BIA. From this screen, BIA was shown to synergize with DNA damaging chemotherapy to enhance toxicity and in combination with cisplatin promote DNA damage in pediatric glioma cell lines. Other drugs identified in the screen are currently under investigation and details will be reported. Thus, our hypothesis is that BIA may provide an effective approach for further development as a DMG therapeutic through targeting invasion and enhancing drug potency and delivery via modulation of endothelial cell tight junctions. Interrogating the mechanisms involved and developing drug formulations informed by the drug screen for in vivo studies to determine the translational potential of this approach is our focus for future studies. Citation Format: Jasmine S. Clark, Jorge Jimenez-Macias, Philippa Vaughn-Beaucaire, Shengliang Zhang, Wafik El-Deiry, Rishi Lulla, Sean Lawler. Exploring the effects of Indirubins in pediatric diffuse high grade glioma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5110.

Abstract 3955: A proteogenomic study of high-grade glioma among adolescents and young adults

Abstract To unravel the molecular mechanisms underlying high-grade glioma (HGG) in adolescent and young adult (AYA) patients, we conducted a comprehensive proteogenomic analysis for 34 AYA (age 15-40) and 59 pediatric (age 0-15) HGG cases. Our approach involved whole genome sequencing, methylation profiling, RNA sequencing, and a suite of mass spectrometry-based proteomic experiments, including global proteomic, phosphoproteomic, and glycoproteomic profiling. The proteomics study successfully identified and quantified approximately 11,000 proteins, 33,000 phosphosites, and 3000 glycopeptides with a 50% missing filtering threshold. To identify the unique characteristics of AYA HGG in contrast to both pediatric and adult HGG, we further integrated a proteogenomic dataset of 99 adult GBM tumors previously published by CPTAC and collaborators (PMID: 33577785). Our study unveiled a collection of mutations, copy number variations, epigenetic modifications and gene fusions that exhibited different frequencies across tumors from pediatric, AYA, and adult patients. Moreover, the influence of these genetic variations on RNA/protein activities also varied across different age groups. Clustering analysis using age-dependent molecular profiles revealed striking differences in RNA/protein/phosphosite activities between patients aged 15-26 and 26-40. Additionally, significantly better overall survival (OS) was observed in the 26-40 age group compared to all other age groups, highlighting distinct biological characteristics within the AYA category that differentiate adolescents from young adults.By leveraging proteogenomic datasets from normal brain tissues (PMID: 30518843), we identified genes, proteins, and pathways with differential age-dependent molecular profiles between tumor and normal brain tissues. Notably, this analysis highlighted significant alterations in proteins from the oxidative phosphorylation, tricarboxylic acid (TCA) cycle, and myelin sheath pathways in tumors compared to normal tissues, some of which were found to be sex-specific.To search for prognostic markers, we introduced a novel approach called Trans-Population Survival Analysis through Interpolation of Age-Dependent Tumor Molecular Profiles. Applying this method on the kinase activity scores derived from the phosphoproteomic data, we identified 150 kinases whose activities were significantly associated with OS among AYA patients. Additionally, employing a causal network analysis tool, we pinpointed six kinases causally linked to OS, suggesting their potential as treatment targets. This study not only characterizes the molecular landscape of AYA HGG but also explores the disease trajectory across the lifespan. Our findings shed light on the intricate interplay of various factors influencing glioma etiology and patient outcomes, paving the way for a deeper understanding of HGG in the AYA population. Citation Format: Nicole Tignor, Mateusz P. Koptyra, Shrabanti Chowdhury, Weiping Ma, Jo Lynne Rokita, Marina Gritsenko, Xiaoyu Song, Giacomo B. Marino, Eden Z. Deng, Francesca Petralia, Azra Krek, Dmitry Rykunov, Felipe da Veiga Leprevost, Noshad Hosseini, Komal S. Rathi, Yingwei Hu, Simona Migliozzi, Tomer Yaron, Weijia Fu, Bo Zhang, Yuankun Zhu, Miguel A. Brown, Jeffrey R. Whiteaker, Clinical Proteomics Tumor Analysis Consortium (CPTAC), Children’s Brain Tumor Network (CBTN), Mehdi Mesri, Ana I. Robles, Karin Rodland, Lewis C. Cantley, Antonio Iavarone, Kenneth Aldape, Marcin Cieślik, Alexey I. Nesvizhskii, Joseph E. Ippolito, Joshua B. Rubin, Amanda G. Paulovich, Hui Zhang, Avi Ma'ayan, Tao Liu, Phillip B. Storm, Adam C. Resnick, Brian R. Rood, Pei Wang. A proteogenomic study of high-grade glioma among adolescents and young adults [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3955.

Abstract 2541: The landscape of circular extrachromosomal amplification in pediatric cancers

Abstract Circular extrachromosomal DNA (ecDNA, also known as double minutes, dm) has emerged as a driver of oncogenic copy number amplification, intratumoral heterogeneity, tumor evolution and therapy resistance. We recently screened the whole genomes of 481 medulloblatomas for ecDNA and concluded that medulloblastoma tumors harboring ecDNA are more than twice as likely to progress within 5 years of diagnosis. To evaluate the frequency and clincical impact of ecDNA across the spectrum of childhood cancers, we performed a retrospective analysis of 1671 tumors from 1482 patients across 47 pediatric tumor types from two large cloud pediatric cancer genomics resources, the Children’s Brain Tumor Network and St. Jude Cloud. ecDNA was detected in 15 pediatric tumor types, including ETMR (100%, n=4), osteosarcoma (47%, n=57), rhabdomyosarcoma (40%, n=35), neuroblastoma (30%, n=106), high-grade glioma (20%, n=157), and retinoblastoma (19%, n=32). Patients with tumors containing ecDNA had significantly poorer 5-year overall survival. In sequential samples from the same tumor at diagnosis and relapse, we observe evidence of ecDNA evolution including sequence instability and generation of new high-copy ecDNA amplification not detected at initial diagnosis. These results comprise the most detailed survey to date of the landscape of extrachromosomal amplifications across childhood cancers. Citation Format: Sunita Sridhar, Owen S. Chapman, Shanqing Wang, Aditi Dutta, Jill P. Mesirov, Lukas Chavez. The landscape of circular extrachromosomal amplification in pediatric cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2541.

Abstract 5113: Fractionated radiation therapy alters energy metabolism and induces cellular quiescence exit in patient-derived orthotopic xenograft models of high-grade glioma

Abstract Radiation is one of the standard therapies for pediatric high-grade glioma (pHGG), of which the prognosis remains poor. To gain an in-depth understanding of the biological activities beyond the classic DNA damage, we treated 6 patient-derived orthotopic xenograft (PDOX) models, including one with DNA mismatch repair (MMR) deficiency, with fractionated radiations (2 Gy/day x 5 days). Extension of survival time was noted in 4/6 PDOX models (P&amp;lt;0.05) and widespread γH2AX positivity in &amp;gt;95% tumor cells in tumor core and &amp;gt;85% in the invasive foci, accompanied by ~30% apoptotic and mitotic catastrophic cell death. The model with DNA MMR (IC-1406HGG) was the most responsive to radiation with a reduction of Ki-67(+) cells. Altered metabolism, including mitochondria number elevation, COX IV activation and reactive oxygen species buildup, were detected together with the accumulation of CD133+ tumor cells. The latter was caused by the entry of quiescent G0 cells into cell cycle and the activation of self-renewal (SOX2 and BMI1) and epithelial mesenchymal transition (fibronectin) genes. These novel insights about the cellular and molecular mechanisms of fractionated radiation in vivo should support the development of new radio-sensitizing therapies. Citation Format: Zi-Lu Huang, Zhi-Gang Liu, Qi Lin, Ya-Lan Tao, Xinzhuoyun Li, Patricia Baxter, Jack MF Su, Adekunle M. Adesina, Chris Man, Murali Chintagumpala, Wan Yee Teo, Yu-Chen Du, Yun-Fei Xia, Xiao-Nan Li. Fractionated radiation therapy alters energy metabolism and induces cellular quiescence exit in patient-derived orthotopic xenograft models of high-grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5113.

Abstract 5108: Immunomodulatory and anti-tumor effect of radiation and induced telomere damage to treat pediatric high-grade gliomas

Abstract Pediatric high-grade gliomas (pHGGs), including diffuse intrinsic pontine glioma (DIPG) are among the most treatment refractory cancers in children. Despite multimodal therapy, prognosis is dismal with survival of &amp;lt;1 year for DIPG and a 5-year overall survival &amp;lt;10% for other pHGGs. Radiotherapy remains the only standard of care for DIPG that extends the survival by few months. In recent years, immunotherapy is emerging as a potential alternative treatment option. However, several factors have hindered the use of immunotherapy for pHGGs including “cold” immune environment with lack of immune cells infiltration, low tumor mutational burden, and lack of neo-antigen expression that prevents tumor recognition by the immune system. Therefore, novel therapies that activate the immune system while evading the tumor immunosuppression are highly needed. Here, we aim to demonstrate the immunomodulatory anti-tumor effects of THIO (6-thio-2’deoxyguanosine), a nucleoside analog and a telomerase-dependent telomere targeting agent, in DIPG mouse models. Our preclinical in vitro and in vivo data have demonstrated the efficacy of THIO in inducing telomeric damage, G2/M cell cycle arrest, and apoptosis in high-risk medulloblastoma and DIPG. Importantly, we have demonstrated that THIO crosses the blood-brain barrier and specifically targets tumor cells in an orthotopic mouse model of DIPG. THIO was shown to induce anti-tumor immunity through micronuclei formation leading to cGAS-STING pathway mediated activation of the innate and adaptive immune response in colon, lung, and hepatocellular carcinoma tumor models. Our preliminary data demonstrate the THIO-dependent induction of micronuclei formation co-localizing with cGAS and leading to STAT1 activation in DIPG cell lines. We have also shown that THIO synergistically sensitizes DIPG cells to ionizing radiation (IR), significantly decreasing cell proliferation. We are currently testing the combination of THIO+IR in an orthotopic mouse model for DIPG. We will present results on the potential of THIO and IR treatments to stimulate anti-tumor immunity through the activation of STING pathway in a syngeneic mouse model of DIPG. These preclinical studies will support the potential use of THIO and IR to treat children with high-risk pediatric brain tumors. Citation Format: Umaru Banlanjo, Shiva Senthil Kumar, Sergei Gryaznov, Rachid Drissi. Immunomodulatory and anti-tumor effect of radiation and induced telomere damage to treat pediatric high-grade gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5108.

Myeloid cells as potential targets for immunotherapy in pediatric gliomas.

Pediatric high-grade glioma (pHGG) including pediatric glioblastoma (pGBM) are highly aggressive pediatric central nervous system (CNS) malignancies. pGBM comprises approximately 3% of all pediatric CNS malignancies and has a 5-year survival rate of approximately 20%. Surgical resection and chemoradiation are often the standard of care for pGBM and pHGG, however, even with these interventions, survival for children diagnosed with pGBM and pHGG remains poor. Due to shortcomings associated with the standard of care, many efforts have been made to create novel immunotherapeutic approaches targeted to these malignancies. These efforts include the use of vaccines, cell-based therapies, and immune-checkpoint inhibitors. However, it is believed that in many pediatric glioma patients an immunosuppressive tumor microenvironment (TME) possess barriers that limit the efficacy of immune-based therapies. One of these barriers includes the presence of immunosuppressive myeloid cells. In this review we will discuss the various types of myeloid cells present in the glioma TME, including macrophages and microglia, myeloid-derived suppressor cells, and dendritic cells, as well as the specific mechanisms these cells can employ to enable immunosuppression. Finally, we will highlight therapeutic strategies targeted to these cells that are aimed at impeding myeloid-cell derived immunosuppression.

Therapeutically targeting the unique disease landscape of pediatric high-grade gliomas.

Pediatric high-grade gliomas (pHGG) are a rare yet devastating malignancy of the central nervous system's glial support cells, affecting children, adolescents, and young adults. Tumors of the central nervous system account for the leading cause of pediatric mortality of which high-grade gliomas present a significantly grim prognosis. While the past few decades have seen many pediatric cancers experiencing significant improvements in overall survival, the prospect of survival for patients diagnosed with pHGGs has conversely remained unchanged. This can be attributed in part to tumor heterogeneity and the existence of the blood-brain barrier. Advances in discovery research have substantiated the existence of unique subgroups of pHGGs displaying alternate responses to different therapeutics and varying degrees of overall survival. This highlights a necessity to approach discovery research and clinical management of the disease in an alternative subtype-dependent manner. This review covers traditional approaches to the therapeutic management of pHGGs, limitations of such methods and emerging alternatives. Novel mutations which predominate the pHGG landscape are highlighted and the therapeutic potential of targeting them in a subtype specific manner discussed. Collectively, this provides an insight into issues in need of transformative progress which arise during the management of pHGGs.

Abstract B033: Histone H3.3 K27M and G34R/V glioma driver mutations inhibit mitotic phosphorylation of H3.3 S31, driving tumor formation by inducing chromosomal instability

Abstract Diffuse gliomas are pediatric high-grade gliomas that frequently harbor heterozygous histone H3.3 K27M, G34R, or G34V mutations. These mutations alter histone methylations, although the precise mechanism(s) of gliomagenesis remains uncertain. H3.3 contains a unique Serine at position 31 that is phosphorylated in prophase and dephosphorylated in anaphase during normal cell division. Previously we have reported that chromosome missegregation triggers H3.3 S31 phosphorylation along chromosome arms in ana/telophase leading to p53-induced cell cycle arrest. Here we show, in vitro and in vivo, that H3.3 K27M, G34R, and G34V suppress S31 phosphorylation by its mitotic kinase, Chk1. In diploid cells, overexpression of H3.3 K27M, G34R, G34V, or non-phosphorylatable S31A significantly increases the frequency of chromosome missegregation, while H3.3 K36M did not. This missegregation phenotype could be rescued by overexpression of K27M/S31E or G34R/S31E double mutants. Next, we used CRISPR/Cas9 gene editing to revert K27M or G34V patient-derived DMG cells to WT or replace the mutant allele with S31A. Reversion to WT increased metaphase S31 phosphorylation and reduced chromosome missegregations, but inserting an S31A mutation caused more mitotic defects. Following a mitotic error, newly formed aneuploid cells overexpressing K27M, G34R, or G34V suppress p53 levels and continue to divide, while WT overexpressing cells always arrest. To test the effects of diminished S31 phosphorylation on gliomagenesis, we utilized a novel mouse model of glioma. Expression of PDGFß linked to H3.3K27M, H3.3G34R, and H3.3S31A in neural stem cells in vivo promoted the development of high-grade gliomas in under 100 days, whereas PDFß-H3.3WT did not. Notably, the S31A tumors had histologically normal levels of K27me3 and K36me3. This work shows that a single glioma-associated point mutation in H3.3 can generate chromosomal instability, and which is a major contributing factor to H3.3 K27M and G34-altered glioma formation. Citation Format: Charles A. Day, Florina Grigore, Alyssa Langfald, Faruck Hakkim, David Daniels, Kevin Vaughan, James Robinson, Edward Hinchcliffe. Histone H3.3 K27M and G34R/V glioma driver mutations inhibit mitotic phosphorylation of H3.3 S31, driving tumor formation by inducing chromosomal instability [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 B033.