Pediatric Brain Cancer Research Articles

MDB-46. ARE RAB40 SMALL GTPASES DRIVERS OF MEDULLOBLASTOMA PATHOGENESIS?

Abstract BACKGROUND Medulloblastoma is the most common solid paediatric neoplasm and is among the leading causes of cancer-related deaths in children. Patients associated with high-risk molecular subgroups convey poor outcomes due to frequent relapse and metastasis, with two thirds of survivors experiencing life-changing developmental deficits. Poor patient outcomes and significant co-morbidities highlight an urgent need for specific, molecularly targeted therapies. The RAB40 subfamily of atypical small GTPases regulate cytoskeletal structure and cell adhesion via their ubiquitin ligase activity. Although previous studies have linked the RAB40 subfamily with cell invasiveness, their role in paediatric brain cancer remains undefined. METHODS Patient methylation and survival data from Cavalli et. al (2017) was analysed via the R2 Genomics Analysis and Visualisation Platform. RAB40 gene and protein expression were determined in eight in vitro models of medulloblastoma via qRT-PCR and western blotting. Cells were transfected with EGFP-Cas9 mRNA and RAB40-targeted CRISPR sgRNA and seeded singly using fluorescence-activated cell sorting. RAB40 knockout clones were identified via western blot and Sanger sequencing. RESULTS Patient data demonstrated that higher RAB40 gene expression correlates with worse outcomes in group 3 and 4 medulloblastoma. RAB40 genes were also significantly more highly expressed in high-risk groups 3 and 4 compared to low-risk WNT group medulloblastoma. RAB40 mRNA was enriched in cell lines derived from high-risk group 3 and 4 medulloblastoma relative to SHH controls. RAB40 knockout models were subsequently generated in HD-MB03, an in vitro model of metastatic Group 3 medulloblastoma using CRISPR/Cas9. CONCLUSIONS Our data supports the hypothesis that RAB40 small GTPases are enriched in high-risk medulloblastoma subgroups, and that their expression correlates with worse patient outcomes. We have generated knockout models which will be used to characterise the impact of RAB40 GTPases on cell growth, adhesion, invasiveness, and chemotherapeutic tolerance.

MDB-59. INTEGRATIVE PROTEO-GENOMICS TO DECIPHER MOLECULAR MECHANISMS UNDERLYING SUBGROUP-SPECIFIC HETEROGENEITY IN MEDULLOBLASTOMA

Abstract BACKGROUND Medulloblastoma (MB), an aggressive pediatric brain cancer, poses significant treatment challenges due to its complex disease biology, comprising a collection of discrete molecular subtypes that require different treatment. Although the genomics, transcriptomics, and epigenetic landscapes of biologically distinct MB subgroups are well-annotated and have contributed to enhanced diagnostics and risk stratification, the intersection of these molecular layers with the MB proteome remains poorly defined METHODS To overcome this knowledge gap, we sought to create a deep proteomics and phospho-proteomics cohort from a large MB clinical trial cohort (SJMB12), comprising 166 primary patient tumors, complemented with matched DNA methylation, whole-exome sequencing (WES), and RNA sequencing (RNA-seq) datasets. Proteomics data were interrogated using unsupervised computational techniques and evaluated for pathway enrichment and kinase activity. To complement these bulk tumor datasets, we investigated the tumor microenvironment focusing on immune and glial cell populations, using single-nuclei RNA and ATAC sequencing (snRNA-seq and snATAC-seq) for validation RESULTS Distinct proteomics subgroups were identified, correlating with established MB clinical subgroups and unveiling previously unrecognized proteomics subgroups. The integration of proteomics with mutational data revealed downstream effects of genetic alterations not evident in the transcriptome, implicating novel molecular mechanisms contributing to disease pathogenesis. Protein-protein interaction networks discovered novel signaling hubs and pathways, highlighting the power of proteomics to detect subgroup-specific drivers of disease that may represent attractive therapeutic vulnerabilities CONCLUSIONS Our multi-omics approach applied to a large series of primary MB patient tumors revealed novel mechanisms underlying MB subgroup biology that were previously hidden at the genetic, epigenetic, and transcriptional level. These findings offer a comprehensive, systems biology view of MB pathogenesis, fostering provocative insights that serve as a foundation for future discovery and the development of more effective personalized treatment strategies.

LGG-02. ELUCIDATING THE ROLE OF FGFR1 ALTERATIONS IN PEDIATRIC GLIOMAS

Abstract BACKGROUND Pediatric brain cancers are the leading cause of cancer-related deaths in children. We (and others) have recently found that close to 10% of all pediatric gliomas, encompassing low-grade gliomas (LGGs) and high-grade gliomas (HGGs), harbor recurrent driver alterations in FGFR proteins, most frequently FGFR1, the gene encoding Fibroblast Growth Factor Receptor 1. In pediatric gliomas, FGFR1 alterations present as either structural variants (SVs), including kinase duplications and fusion proteins, or kinase-activating single nucleotide variants (SNVs). Recurrent FGFR1 alterations represent a promising therapeutic target for precision medicine approaches, however a challenge has been the lack of clinical trials for FGFR inhibitors. A major goal of this project is to identify preclinical FGFR inhibitor candidates for FGFR1-altered pediatric gliomas. METHODS We have generated isogenic mouse and human neural stem cell (NSC) models driven by FGFR1 and BRAF alterations commonly seen in pediatric gliomas to test the oncogenic properties and therapeutic capacities of these alterations. RESULTS We found that NSC lines grow independent of growth factors and form tumors in mice. RNA-sequencing of these lines revealed the FGFR1-altered lines are enriched in neuronal gene programs. The FGFR1-altered models are exquisitely sensitive to the four FDA-approved panFGFR inhibitors Infigratinib, Erdafitinib, Pemigatinib, and Futibatinib. However, the FGFR1 SNV + PTPN11 co-occurring SNV line is the least sensitive to FGFR inhibition, which suggests combination therapies may be needed. Combination drug studies with FGFR inhibition in our FGFR1-altered lines reveal the best synergy with mTOR inhibition. Finally, we present examples of case studies of patients with FGFR1-altered tumors treated with targeted inhibitors. CONCLUSIONS Taken together, our studies have established models that have generated key insights into the biology of FGFR1-altered gliomas and how to best target them therapeutically.

MDB-108. MRI-GUIDED FOCUSED ULTRASOUND BLOOD BRAIN/TUMOR BARRIER DISRUPTION FOR AUGMENTATION OF ANTIBODY DELIVERY TO HIGH-RISK MEDULLOBLASTOMAS

Abstract BACKGROUND Medulloblastoma is the most common pediatric brain cancer, and among its four molecular subgroups, group 3 (G3MB) harbors the worst prognosis (5-year survival rate <50%). Effective treatment of G3MB remains difficult due to disease complexity, its challenging niche within the immunologically- and pharmacologically-privileged central nervous system, and significant risk of long-term developmental deficits due to treatment toxicities. MRI-guided focused ultrasound (MRgFUS) holds the potential to revolutionize care as a non-invasive, non-ionizing approach for spatially precise intervention in G3MBs. Given that the utility of FUS in MBs remains unknown, we implemented FUS-mediated blood brain/tumor barrier disruption (BBB/BTB-D) in a high-fidelity model of G3MB and evaluated its impact on localized antibody delivery – with an eye toward future applications in immuno-oncology. METHODS An orthotopic syngeneic model was established via stereotactic implantation of G3MB cells (Gfi1/c-MYC driven) into the cerebellum. G3MB growth kinetics were validated via serial MRI. Multiple protocols for naive cerebellum or G3MB BBB/BTB-D were established with a 1.15 MHz FUS system under 9.4T MRI guidance. Across both settings, mice were sonicated with concomitant intravenous administration of microbubbles and fluorescently-labeled IgG. Safety was confirmed by acoustic emissions monitoring, susceptibility-weighted imaging, histology, and acute activity monitoring. Antibody delivery was evaluated via ex vivo epifluorescence imaging. RESULTS Post-contrast T1w-MRI depicted clear evidence of localized BBB/BTB-D in naive and G3MB mice. No damage was noted by imaging or histopathologic analysis, and mice displayed normal cardiorespiratory and motor functions following FUS. FUS conferred significantly increased epifluorescence signal in targeted cerebellum (1.3x) and G3MBs (1.5x) relative to controls, indicating improved antibody access. CONCLUSIONS Our findings support the safety and feasibility of FUS BBB/BTB-D in naive and G3MB-bearing cerebellum. We demonstrate that FUS markedly improves antibody penetrance within G3MBs. Ongoing studies are evaluating FUS in combination with novel immunotherapeutic antibodies for G3MB therapy.

MDB-78. ISOCITRATE DEHYDROGENASE 1 INHIBITION PRIMES GROUP-3 MEDULLOBLASTOMAS FOR CUPROPTOSIS

Abstract BACKGROUND MYC-driven Group-3 medulloblastomas (MB) are deadly and malignant pediatric brain cancers and we sought to define actionable metabolic dependencies in these tumors. METHODS To identify uniquely upregulated genes in Group-3 MB, we performed transcriptomic analysis on two previously published medulloblastoma RNA-seq datasets. To elucidate the relationship between c-MYC/IDH1/DLAT and assess impact on tumor metabolism, we performed metabolic and transcriptional profiling of Group-3 MB cell lines that were either untreated or were subjected to shRNA-mediated knockdown of DLAT or treatment with IDH1 inhibitor. We also treated Group-3 MB cell lines containing varying levels of DLAT expression with copper ionophore elesclomol and assessed its ability to induce toxicity. Finally, we established in vivo models of Group-3 MB via orthotopic implantation to assess the effect of DLAT knockdown, IDH1 inhibition, and cuproptosis induction on tumor growth and survival outcomes. RESULTS We identified upregulation of dihydrolipoyl transacetylase (DLAT), the E2-subunit of pyruvate dehydrogenase complex (PDC) in a subset of Group-3 MB. DLAT was induced by c-MYC and targeting DLAT lowered TCA-cycle metabolism and glutathione synthesis in Group-3 MB cells. We also noted upregulation of isocitrate dehydrogenase 1 (IDH1) in Group-3 MB. Remarkably, genetic and pharmacologic suppression of IDH1 epigenetically reduced c-MYC and downstream DLAT levels. DLAT is a central regulator of cuproptosis, a copper-dependent cell death mechanism induced by the copper ionophore elesclomol. DLAT expression in Group-3 MB cells correlated with increased sensitivity to cuproptosis. Elesclomol was CNS-penetrant and suppressed tumor growth in vivo in Group-3 MB animal models. CONCLUSIONS Our data uncover an IDH1/c-MYC dependent vulnerability that regulates DLAT levels and can be targeted to kill Group-3 MB by cuproptosis.

DIPG-58. TARGETING DISORDERED DNA METHYLATION IN DIPG TO CONSTRAIN VARIABILITY AND INDUCE IMMUNE SIGNALING

Abstract BACKGROUND Diffuse Intrinsic Pontine Glioma (DIPG) is a universally fatal pediatric brain cancer characterized by the histone H3 K27M mutation. The known downstream consequences of this mutation include loss of repressive chromatin marks, global DNA hypomethylation, and altered gene expression. METHODS We sought to investigate the role of epigenetic variability as a basis of DIPG cellular heterogeneity and plasticity by using Whole Genome Bisulfite Sequencing (WGBS). Existing methods of analysis of WGBS are limited in that they average/smooth methylation data and fail to capture intrinsic variability. We performed (WGBS) on 23 primary patient samples of DIPG and applied an analysis that captures the variability of DNA methylation reads to characterize methylation stochasticity. We then sought to modulate the epigenome pharmacologically using a DNA methyltransferase inhibitor and assessed changes through RNA-Seq and immunopeptidomics. RESULTS We find that DIPG has a marked increase in methylation stochasticity (quantified as methylation entropy). Additionally, while DIPG is globally hypomethylated, it retains DNA methylation at specific regulatory regions, such as bivalent promoters, and key genes, such as FOXG1, CXCR4, and KLF4. We sought to reverse these changes using the DNA methyltransferase inhibitor, decitabine, in 4 DIPG neurosphere cell lines. Treatment with decitabine reversed the entropy seen in DIPG and induced innate immune signaling and interferon signaling in DIPG cells. Using RNA-seq, we found that decitabine treatment dramatically alters gene expression, including activation of endogenous retroviral elements, activation of interferon signaling, de-repression of hypermethylated targets, and expression of putative neoantigens, such as PRAME and DAZL. Immunopeptidomics profiling of MHC Class I bound canonical and non-canonical peptides of DIPG cell lines after decitabine treatment also revealed decitabine-induced differences. CONCLUSIONS Taken together, our study finds that the methylome of DIPG is highly disordered but is also highly responsive to pharmacologic modulation by hypomethylating agents. Furthermore, hypomethylating drugs can increase the immunogenicity of DIPG, thus offering the potential for future combination with immunotherapy.

Laminin-associated integrins mediate Diffuse Intrinsic Pontine Glioma infiltration and therapy response within a neural assembloid model

Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive and fatal pediatric brain cancer. One pre-requisite for tumor cells to infiltrate is adhesion to extracellular matrix (ECM) components. However, it remains largely unknown which ECM proteins are critical in enabling DIPG adhesion and migration and which integrin receptors mediate these processes. Here, we identify laminin as a key ECM protein that supports robust DIPG cell adhesion and migration. To study DIPG infiltration, we developed a DIPG-neural assembloid model, which is composed of a DIPG spheroid fused to a human induced pluripotent stem cell-derived neural organoid. Using this assembloid model, we demonstrate that knockdown of laminin-associated integrins significantly impedes DIPG infiltration. Moreover, laminin-associated integrin knockdown improves DIPG response to radiation and HDAC inhibitor treatment within the DIPG-neural assembloids. These findings reveal the critical role of laminin-associated integrins in mediating DIPG progression and drug response. The results also provide evidence that disrupting integrin receptors may offer a novel therapeutic strategy to enhance DIPG treatment outcomes. Finally, these results establish DIPG-neural assembloid models as a powerful tool to study DIPG disease progression and enable drug discovery.

Management of patients with diffuse intrinsic pontine glioma in Australia and New Zealand: Australian and New Zealand Children's Haematology/Oncology Group position statement.

The main mission of the Australian and New Zealand Children's Haematology and Oncology Group (ANZCHOG) is to develop and facilitate local access to the world's leading evidence-based clinical trials for all paediatric cancers, including brain tumours, as soon as practically possible. Diffuse intrinsic pontine gliomas (DIPGs) - a subset of a larger group of tumours now termed diffuse midline glioma, H3K27-altered (DMG) - are paediatric brain cancers with less than 10% survival at two years. In the absence of any proven curative therapies, significant recent advancements have been made in pre-clinical and clinical research, leading many to seek integration of novel therapies early into standard practice. Despite these innovative therapeutic approaches, DIPG remains an incurable disease for which novel surgical, imaging, diagnostic, radiation and systemic therapy approaches are needed. All patients with DIPG should be discussed in multidisciplinary neuro-oncology meetings (including pathologists, neuroradiologists, radiation oncologists, neurosurgeons, medical oncologists) at diagnosis and at relapse or progression. Radiation therapy to the involved field remains the local and international standard of care treatment. Proton therapy does not yield a superior survival outcome compared with photon therapy and patients should undergo radiation therapy with the available modality (photon or proton) at their treatment centre. Patients may receive concurrent chemotherapy or radiation-sensitising agents as part of a clinical trial. Biopsy should be offered to facilitate consideration of experimental therapies and eligibility for clinical trial participation. After radiation therapy, each patient should be managed individually with either observation or considered for enrolment on a clinical trial, if eligible, after full discussion with the family. Re-irradiation can be considered for progressive disease. Every child diagnosed with DIPG should be offered enrolment on a clinical trial where available. Access to investigational drugs without biological rationale outside the clinical trial setting is not supported. In case of potentially actionable target identification with molecular profiling and absence of a suitable clinical trial, rational targeted therapies can be considered through compassionate access programs.

Identifying and Targeting Immunotherapeutic Resistance in Pediatric Brain Cancer

Identifying and Targeting Immunotherapeutic Resistance in Pediatric Brain Cancer

EDA2R reflects the acute brain response to cranial irradiation in liquid biopsies.

Cranial radiotherapy is standard of care for high-grade brain tumors and metastases; however, it induces debilitating neurocognitive impairments in cancer survivors, especially children. As the numbers of pediatric brain cancer survivors continue improving, the numbers of individuals developing life-long neurocognitive sequalae are consequently expected to rise. Yet, there are no established biomarkers estimating the degree of the irradiation-induced brain injury at completion of radiotherapy to predict the severity of the expected neurocognitive complications. We aimed to identify sensitive biomarkers associated with brain response to irradiation that can be measured in easily accessible clinical materials, such as liquid biopsies. Juvenile mice were subjected to cranial irradiation with 0.5, 1, 2, 4, and 8 Gy. Cerebrospinal fluid (CSF), plasma, and brains were collected at acute, subacute, and subchronic phases after irradiation, and processed for proteomic screens, and molecular and histological analyses. We found that the levels of ectodysplasin A2 receptor (EDA2R), member of tumor necrosis factor receptor superfamily, increased significantly in the CSF after cranial irradiation, even at lower irradiation doses. The levels of EDA2R were increased globally in the brain acutely after irradiation and decreased over time. EDA2R was predominantly expressed by neurons, and the temporal dynamics of EDA2R in the brain was reflected in the plasma samples. We propose EDA2R as a promising potential biomarker reflecting irradiation-induced brain injury in liquid biopsies. The levels of EDA2R upon completion of radiotherapy may aid in predicting the severity of IR-induced neurocognitive sequalae at a very early stage after treatment.

Abstract 145: Unraveling SMARCB1 depletion in hiPSC-derived neural crest cells: Implications for ATRT cell of origin

Abstract Atypical teratoid rhabdoid tumors (ATRTs) are highly aggressive pediatric brain cancers that lack standardized treatment regimens and are associated with poor prognoses. Despite being driven by biallelic inactivation of a single gene, SMARCB1, ATRTs exhibit three epigenetic subgroups (SHH, TYR, and MYC), each with different clinical outcomes. Like many pediatric brain tumors, ATRTs share the fundamental underlying mechanism of stalled differentiation, suggesting that it may arise from neural progenitor cells that fail to terminally differentiate. However, unlike other brain tumors, ATRTs share molecular similarities with malignancies found outside of the CNS, especially in the kidney and soft tissues. This unique feature implies a putative cell of origin that is not restricted to the CNS: neural crest cells, a transient embryonic cell population that emerges from the neuroectoderm but then migrates throughout the embryo during development. To study SMARCB1 loss in a genetically defined neural progenitor cellular context with isogenic controls, the Furnari lab engineered a doxycycline inducible SMARCB1 knockdown system into human induced pluripotent stem cells (hiPSCs). After simultaneous SMARCB1 knockdown with directed neural differentiation, the resulting SMARCB1-depleted neural progenitor cells exhibited transcriptomic profiles that were similar to ATRTs, particularly to the SHH subgroup. Building upon these findings, the engineered hiPSCs were differentiated into neural crest cells using a modified version of a published protocol. Via RT-qPCR, these hiPSC-derived neural crest cells were confirmed to express typical neural crest marker genes and lose expression of SMARCB1 in the presence of doxycycline. Remarkably, ATP assays demonstrated that neural crest cells that were differentiated without SMARCB1 acquired enhanced proliferation in comparison to those differentiated with SMARCB1 intact. Further work aims to tease apart this proliferative phenotype in the neural crest cellular context via RNAseq, evaluate the impact of cell identity upon subgroup status, and to determine whether SMARCB1 depletion post-differentiation also enhances proliferation. Together, these findings feature the utility of hiPSC-derived models for investigating mechanisms underlying ATRTs to hopefully identify novel subgroup-specific targets. Citation Format: Clark Wang, Frank Furnari. Unraveling SMARCB1 depletion in hiPSC-derived neural crest cells: Implications for ATRT cell of origin [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 145.

Abstract 5109: Novel acylfulvene LP184 shows single agent and combinatorial efficacy with PARP inhibitors in atypical teratoid rhabdoid tumors

Abstract Atypical Teratoid Rhabdoid Tumor (AT/RT) is an aggressive form of pediatric brain cancer that spreads quickly and leads to survival rates of less than 40%. Current treatments are insufficient at reducing this poor prognosis. A hallmark feature of ATRT is a SMARCB1 mutation. Illudins are a class of cytotoxic compounds with activity against pediatric solid tumors, including AT/RT. LP-184 is a novel synthetic illudin with improved brain penetration and activity against adult high-grade glioma orthotopic xenografts. We tested LP-184 against a battery of AT/RT cell lines including BT37, CHLA06, CHLA05, and CHLA266. LP-184 decreases viability at nanomolar concentrations with IC50s ranging from 9.91nM - 23.92nM. LP-184 decreased proliferation as measured by immunofluorescence and confirmed by western blots (BT37 p=0.0001, CHLA06 =0.0001, CHLA05=0.033). LP-184 also induced apoptosis as demonstrated in immunofluorescent images and western blots (BT37 p=0.0001, CHLA06 =0.0001, CHLA05=0.0015, CHLA266 p=0.002). Furthermore, LP-184 shows strong synergy with the PARP inhibitor Rucaparib, suggesting that inhibition of DNA repair potentiates LP-184 activity. LP-184 reduces viable cell count by as much as 50% and combination with Rucaparib further decreases viability by 75% (BT37=0.0001, CHLA06=0.0002, CHLA05=0.0077). The dual therapy also demonstrates cytostatic and cytotoxic effects as confirmed by immunofluorescence and western blots (BT37 BrdU = 0.0004, BT37 CC3 = 0.0002, CHLA06 BrdU = 0.0033, CHLA06 CC3 = 0.0117 compared to control). Further studies in vivo are being conducted to test single agent and combination therapies of LP-184 and Rucaparib in ATRT, as our data indicates that the two could provide a compelling treatment option for patients with this poor prognosis brain tumor. Citation Format: Abigail Tindall, Hyuk J. Kwon, Istabraq Musa, Tyler Findlay, Su Chan Lee, Charles Eberhart, Eric Raabe. Novel acylfulvene LP184 shows single agent and combinatorial efficacy with PARP inhibitors in atypical teratoid rhabdoid tumors [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 5109.

Abstract 5474: Biorepository yields potential for personalized medicine approach in pediatric brain cancer patients

Abstract Childhood brain cancer, with a 12% mortality rate, stands as the most lethal cancer among children. Out of the annual 4,000 diagnoses, 30% involve low-grade gliomas, with a troubling 10% progressing to lethal high-grade gliomas within 2 years. While surgery can often cure the remaining 90%, 50% necessitate adjuvant therapies like chemotherapy and molecular targeting, with radiation sparingly used. Alarmingly, 66% of survivors grapple with long-term health, mental, and cognitive issues due to treatment toxicity, emphasizing the urgent need for innovative biological therapies in pediatric glioma treatment.Our proposed strategy involves the acute analysis of freshly discarded tissue from brain cancer craniotomies, departing from the use of FFPE-preserved tissue, which may exhibit suboptimal quality. All patients provided informed consent, and by prospectively preserving viable tissue, we enable its utilization in ten or more subsequent analyses. These encompass cell-culture-based assays, viability assays, bulk, single cell, and single nucleus RNA sequencing, DNA sequencing, single-cell proteome analyses, flow cytometry, and immunofluorescence.Our findings highlight a direct correlation between surgical sample size and analysis feasibility. Bulk RNA sequencing proves valuable in acutely isolated surgical tissue and cryostored or flash-frozen tissue for identifying novel therapeutic targets in individual patients. Viable cells retrieved from cryopreserved tissue of the same patient allow ex vivo functional validation of therapeutic targets, eliminating the need for establishing patient-derived cell lines and xenografts, particularly challenging for low-grade gliomas. These findings establish the feasibility of a personalized bedside-to-bench approach, contingent on an adequate supply of surgical material.Expanding this approach to encompass all childhood brain cancer types, we plan to investigate using our comprehensive biorepository, housing viable tissue and single-cell suspensions from 122 childhood brain cancer patients. Among these, 51 cases yielded tissue for single-cell RNA-seq and viable cryoprotection. The most common tumor histology identified was low-grade glioma (32), primarily pilocytic astrocytoma (19), followed by embryonal (12) and ependymal tumors (4).In summary, we developed a standard operating procedure for analyzing fresh and cryopreserved surgical discards, identifying novel targets in childhood brain cancer tissue, and functionally validating them in patient-matched samples. This personalized medicine approach holds promise in circumventing the tedious development of patient-derived models. Citation Format: Anna Pronina, Emon Nasajpour, Caitlynn Tran, Troy Schouten, Eden Gibson, Jeffrey Nirschl, Laura Marie Prolo, Kelly Mahaney, Hannes Vogel, Claudia Katharina Petritsch. Biorepository yields potential for personalized medicine approach in pediatric brain cancer patients [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 5474.

Abstract 3173: Impact of hypoxia on the integrated stress response activated by imipridones ONC201 and ONC206 in pediatric diffuse midline glioma cells

Abstract Diffuse midline gliomas (DMGs) are highly aggressive, high-grade gliomas which typically arise in children and young adults. With currently approved treatments the median survival from time of diagnosis for pediatric DMG is 8-10 months with only 10% of children living to 2 years post diagnosis. Despite being only 15% of the cases it makes up 40% of the pediatric brain cancer deaths making it the leading cause of death for all pediatric glioma cases. Two recent clinical studies, NCT03416530 and NCT03134131, have shown the clinical efficacy of Dordaviprone (ONC201/TIC10) for the treatment of DMG; increasing median survival to 22 months in patients following radiation treatment prior to recurrence. Imipridone ONC206, a chemical derivative of ONC201, is under clinical development for treatment of pediatric and adult patients with primary brain tumors (NCT04732065 and NCT04541082). To further improve treatment, we must continue to study how the tumor microenvironment impacts the efficacy of imipridones. One crucial aspect of all brain cancers is hypoxia, so the IC50s of the SU-DIGP-25, SU-DIPG-XIII and SU-DIPGIV pediatric DMG cell lines treated with ONC201 or ONC206 were measured using the CellTiter-Glo assay under conditions of hypoxia and normoxia. Imipridones mediate apoptosis through the upregulation of the TRAIL death receptor DR5 and the activation of the integrated stress response (ISR), so western blots were used to show changes in the ISR protein expression in ONC201 treated DMG cells at multiple different degrees of hypoxia. To understand how hypoxia inducible factors (HIFs) play a role in resistance and susceptibility to ONC201 or ONC206, HIF-1a, HIF-2a, and HIF-3a were knocked down showing altered ISR protein expression after treating with the imipridones under hypoxic and normoxic conditions. Citation Format: Tyler J. Roady, Nolan Stubbs, Josephine Chen, Yutong Xia, Ashley Sanchez Sevilla Uruchurtu, Xiaobing Tian, Lanlan Zhou, Wafik S. El-Deiry. Impact of hypoxia on the integrated stress response activated by imipridones ONC201 and ONC206 in pediatric diffuse midline glioma cells [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 3173.

Abstract 1719: Targeting H3.3 serine 28 phosphorylation as a novel therapeutic strategy for diffuse midline glioma

Abstract Background: Diffuse midline glioma (DMG) is a highly aggressive pediatric brain cancer with a poor prognosis [1]. Mechanistically, DMG harbors a lysine-to-methionine substitution at position 27 of histone H3.3 (H3.3K27M), which leads to global hypomethylation and a dramatically altered epigenetic landscape [2, 3]. Aim: This study aims to explore the H3.3K27M mutation from a novel perspective by investigating its effects on H3.3 ubiquitination, with the goal of identifying effective, novel therapeutic targets. Methods: Mass spectrometry was used to identify potential E3 ligases for H3.3, while immunoprecipitation to verify this interaction. Cell viability and survival were determined by MTS and clonogenic survival assays, respectively. Nucleosome fractionation was performed to detect the distribution of wild-type (WT) or K27M mutant H3.3 histones. Results: Mass spectrometry identified UBR7, an E3 ligase, as exhibiting a stronger binding affinity with WT H3.3 than its K27M mutant counterpart. Further nucleosome fractionation assay revealed that K27M histones are more likely deposited in nucleosomes than WT H3.3. Further analysis demonstrated that a non-phosphorylation mimic mutation (serine to alanine) at amino acid 28 (S28A) restored the binding affinity of K27M mutant H3.3 with UBR7. Importantly, this mutation disrupted H3.3 phosphorylation and significantly diminished cell survival, whereas the constitutively activated phosphorylation mutation (S to glutamine (E)) on S28 (S28E) mimics K27M mutation in promoting cell proliferation and survival. Conclusion: These findings suggest that S28ph impairs UBR7-mediated K27M ubiquitination and promotes DMG cell growth, suggesting that targeting S28ph is an effective therapeutic strategy. However, the underlying mechanisms by which K27M interplays with S28ph in promoting tumorigenesis require further investigation. Citation Format: Yuqian Yan. Targeting H3.3 serine 28 phosphorylation as a novel therapeutic strategy for diffuse midline 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 1719.

Evaluation of sonobiopsy feasibility and safety in a mouse model of diffuse intrinsic pontine glioma

Diffuse intrinsic pontine glioma (DIPG) is a leading cause of pediatric brain cancer mortality, emphasizing the need for precise molecular diagnosis to advance therapeutic development. The tumor's location in the brainstem poses challenges for invasive biopsy, prompting the exploration of liquid biopsies. However, both blood-based and cerebrospinal fluid (CSF)-based liquid biopsies exhibit limited sensitivity in diagnosing DIPG. Sonobiopsy, an innovative technique utilizing focused ultrasound and microbubbles, aims to enhance the sensitivity of liquid biopsies. This study assessed the feasibility and safety of sonobiopsy in a DIPG mouse model. The model was established by intracranial injection of enhanced green-fluorescent-protein (eGFP) transduced DIPG tumor cells. Mice were divided into sonobiopsy and conventional liquid biopsy groups. Sonobiopsy, employing MRI-guided focused ultrasound and microbubbles, demonstrated increases in plasma and CSF eGFP DNA, RNA, and protein concentrations compared to conventional liquid biopsy. The enrichment effect varied based on the biomarker type. No observed hemorrhage or tissue damage attributed to sonication suggests the safety of this approach. These results affirm the feasibility and safety of sonobiopsy in enriching DIPG-specific biomarkers in both plasma and CSF, suggesting sonobiopsy as a promising noninvasive molecular diagnostic tool for DIPG.

Deep learning segmentation of organs-at-risk with integration into clinical workflow for pediatric brain radiotherapy.

Radiation therapy (RT) of pediatric brain cancer is known to be associated with long-term neurocognitive deficits. Although target and organs-at-risk (OARs) are contoured as part of treatment planning, other structures linked to cognitive functions are often not included. This paper introduces a novel automatic segmentation tool specifically designed for the unique challenges posed by pediatric patients undergoing brain RT, as well as its seamless integration into the existing clinical workflow. Images of 47 pediatric brain cancer patients aged 1 to 20 years old and 33 two-year-old healthy infants were used to train a vision transformer, UNesT, for the segmentation of five brain OARs. The trained model was then incorporated to clinical workflow via DICOM connections between a treatment planning system (TPS) and a server hosting the trained model such that scans are sent from TPS to the server, automatically segmented, and sent back to TPS for treatment planning. The proposed automatic segmentation framework achieved a median dice similarity coefficient of 0.928 (frontal white matter), 0.908 (corpus callosum), 0.933 (hippocampi), 0.819 (temporal lobes), and 0.960 (brainstem) with a mean±SD run time of 1.8±0.67s over 20 test cases. The pediatric brain segmentation tool showed promising performance on five OARs linked to neurocognitive functions and can easily be extended for additional structures. The proposed integration to the clinic enables easy access to the tool from clinical platforms and minimizes disruption to existing workflow while maximizing its benefits.

An Update on Emergent Nano-Therapeutic Strategies against Pediatric Brain Tumors.

Pediatric brain tumors are the major cause of pediatric cancer mortality. They comprise a diverse group of tumors with different developmental origins, genetic profiles, therapeutic options, and outcomes. Despite many technological advancements, the treatment of pediatric brain cancers has remained a challenge. Treatment options for pediatric brain cancers have been ineffective due to non-specificity, inability to cross the blood-brain barrier, and causing off-target side effects. In recent years, nanotechnological advancements in the medical field have proven to be effective in curing challenging cancers like brain tumors. Moreover, nanoparticles have emerged successfully, particularly in carrying larger payloads, as well as their stability, safety, and efficacy monitoring. In the present review, we will emphasize pediatric brain cancers, barriers to treating these cancers, and novel treatment options.

A Benzarone Derivative Inhibits EYA to Suppress Tumor Growth in SHH Medulloblastoma.

Development of a benzarone derivative that inhibits EYA1 and impedes the growth of SHH medulloblastoma provides an avenue for improving treatment of this malignant pediatric brain cancer.

Lipidomic-Based Approach to 10 s Classification of Major Pediatric Brain Cancer Types with Picosecond Infrared Laser Mass Spectrometry.

Picosecond infrared laser mass spectrometry (PIRL-MS) is shown, through a retrospective patient tissue study, to differentiate medulloblastoma cancers from pilocytic astrocytoma and two molecular subtypes of ependymoma (PF-EPN-A, ST-EPN-RELA) using laser-extracted lipids profiled with PIRL-MS in 10 s of sampling and analysis time. The average sensitivity and specificity values for this classification, taking genomic profiling data as standard, were 96.41 and 99.54%, and this classification used many molecular features resolvable in 10 s PIRL-MS spectra. Data analysis and liquid chromatography coupled with tandem high-resolution mass spectrometry (LC-MS/MS) further allowed us to reduce the molecular feature list to only 18 metabolic lipid markers most strongly involved in this classification. The identified 'metabolite array' was comprised of a variety of phosphatidic and fatty acids, ceramides, and phosphatidylcholine/ethanolamine and could mediate the above-mentioned classification with average sensitivity and specificity values of 94.39 and 98.78%, respectively, at a 95% confidence in prediction probability threshold. Therefore, a rapid and accurate pathology classification of select pediatric brain cancer types from 10 s PIRL-MS analysis using known metabolic biomarkers can now be available to the neurosurgeon. Based on retrospective mining of 'survival' versus 'extent-of-resection' data, we further identified pediatric cancer types that may benefit from actionable 10 s PIRL-MS pathology feedback. In such cases, aggressiveness of the surgical resection can be optimized in a manner that is expected to benefit the patient's overall or progression-free survival. PIRL-MS is a promising tool to drive such personalized decision-making in the operating theater.