Malignant Pediatric Brain Tumor Research Articles

Multiomic profiling of medulloblastoma reveals subtype-specific targetable alterations at the proteome and N-glycan level

Medulloblastomas (MBs) are malignant pediatric brain tumors that are molecularly and clinically heterogenous. The application of omics technologies—mainly studying nucleic acids—has significantly improved MB classification and stratification, but treatment options are still unsatisfactory. The proteome and their N-glycans hold the potential to discover clinically relevant phenotypes and targetable pathways. We compile a harmonized proteome dataset of 167 MBs and integrate findings with DNA methylome, transcriptome and N-glycome data. We show six proteome MB subtypes, that can be assigned to two main molecular programs: transcription/translation (pSHHt, pWNT and pG3myc), and synapses/immunological processes (pSHHs, pG3 and pG4). Multiomic analysis reveals different conservation levels of proteome features across MB subtypes at the DNA methylome level. Aggressive pGroup3myc MBs and favorable pWNT MBs are most similar in cluster hierarchies concerning overall proteome patterns but show different protein abundances of the vincristine resistance-associated multiprotein complex TriC/CCT and of N-glycan turnover-associated factors. The N-glycome reflects proteome subtypes and complex-bisecting N-glycans characterize pGroup3myc tumors. Our results shed light on targetable alterations in MB and set a foundation for potential immunotherapies targeting glycan structures.

LMIC-24. ASSESSING THE TREATMENT OUTCOMES OF PEDIATRIC PATIENTS WITH MEDULLOBLASTOMA UNDER A U.S.-MEXICO CROSS-BORDER NEURO-ONCOLOGY PROGRAM

Abstract BACKGROUND Treating pediatric brain tumors in low-income countries is associated with worse outcomes, likely due to requiring a highly trained interdisciplinary team with ample resources. The Cross-Border Neuro-Oncology Program (CBNP) between Rady Children’s Hospital, San Diego (RCHSD), and Hospital General, Tijuana (HGT), was established to provide comprehensive care for children with brain tumors diagnosed at HGT. Given the most common malignant pediatric brain tumor is medulloblastoma, assessing treatment outcomes for this tumor is essential to establish this program’s validity. METHODS Pediatric patients with medulloblastoma were recruited from 2012-2021 as part of CBNP and risk-stratified into one of two treatment protocols (low - (K) versus high-risk (Y)). Patients underwent surgical resection and chemotherapy, which included cisplatin, etoposide, vincristine, and cyclophosphamide; patients with higher risk additionally received radiation therapy and higher-dose chemotherapy. Clinicopathologic profiles, extent of resection, methylation status and protocol type were analyzed and compared to known prognostic values for pediatric medulloblastoma. RESULTS Seventeen patients with medulloblastoma (age at diagnosis=6.8±5.0 years) were treated as part of CBNP during the study period. Patients were admitted to HGT, underwent resection at RCHSD, and returned to HGT for collaborative management. The mean symptom duration preceding diagnosis was 72.1±99.2 days. Gross total resection was achieved in 53% of cases and 5-year overall survival was 53% (9/17). Eight (47%) patients had residual post-treatment cerebellar signs. Survival data correlated with protocol type and methylation status will be presented. CONCLUSION Our cohort demonstrated a 5-year overall survival of 53%, moderately lower than the reported average 5-year overall survival of ~70% for pediatric medulloblastoma, with the majority of our cohort comprising of higher risk subtype and pathology. This indicates that this twinning model is a valid archetype for treating pediatric patients with brain tumors in low-income countries.

MDB-29. SALL4 IS A CRL3REN/KCTD11 SUBSTRATE THAT DRIVES SONIC HEDGEHOG-DEPENDENT MEDULLOBLASTOMA

Abstract BACKGROUND Medulloblastoma (MB) is the most common malignant pediatric brain tumor arising from alterations in cerebellum development. The Sonic Hedgehog variant (SHH MB) is the best genetically characterized, however the molecular mechanisms responsible for its pathogenesis are not fully understood and therapeutic benefits are still limited. Mutations in key components of the SHH pathway and cytogenetic alterations (such as chromosome 17p deletion) lay the pathogenetic foundation for SHH MB subgroup. RENKCTD11 (here REN) is a tumor suppressor and substrate-receptor subunit of the Cullin3 Ring E3 ubiquitin ligase (CRL3) complex mapping on chromosome 17p and frequently deleted in SHH MB. We wished to expand the knowledge of CRL3REN activity to unveil molecular circuitries whose deregulation can contribute to SHH MB onset and to uncover novel therapeutic targets. METHODS Affinity purification coupled to mass spectrometry was performed to identify new REN interactors. Co-immunoprecipitation and ubiquitylation assays validated proteomic data. The proliferation of primary murine SHH MB cells and Patient Derived Xenografts (PDXs) models was evaluated in vitro by using the IncuCyte imaging system. Heterotopic and orthotopic allograft in vivo models of SHH MB were established in immunodeficient mice. RESULTS Proteomic analysis revealed that the pro-oncogenic stemness Spalt-like transcriptional factor 4 (SALL4) interacts with REN. We found that SALL4 is re-expressed in mouse SHH MB models and its high levels correlate with worse overall survival in SHH MB patients. We demonstrate that CRL3REN induces polyubiquitylation and degradation of wild type SALL4, but not of a SALL4 mutant lacking zinc finger cluster 1 domain (ΔZFC1). Interestingly, SALL4 binds GLI1 and cooperates with HDAC1 to potentiate GLI1 deacetylation and transcriptional activity. Notably, inhibition of SALL4 suppress SHH MB growth both in murine and PDX models. CONCLUSIONS Our findings identify SALL4 as a CRL3REN substrate and a promising therapeutic target in SHH-dependent cancers.

QOL-04.IN VIVO MODELLING RECAPITULATES RADIOTHERAPY DELIVERY AND LATE-EFFECT PROFILE FOR CHILDHOOD MEDULLOBLASTOMA

Abstract BACKGROUND Medulloblastoma (MB) is the most common malignant paediatric brain tumour, with 5-year survival rates >70%. Survivors frequently suffer a wide-range of late-effects due to their tumour and its treatment. Cranial radiotherapy (CRT) to the whole-brain, with posterior fossa boost (PFB), underpins treatment for non-infants, however, radiotherapeutic insult to normal brain tissue has deleterious consequences to neurocognitive and physical functioning, and causes accelerated ageing/frailty. Unfortunately, approaches to ameliorate radiotherapy-induced late-effects are lacking and a paucity of appropriate model systems hinders their development. METHODS We have developed a clinically-relevant in vivo model system that recapitulates the radiotherapy dose, delivery and late-effect profile of childhood medulloblastoma, at an equivalent developmental stage. Consistent with human regimens, age-equivalent (postnatal days 28-37) C57Bl/6J mice received targeted, CT image-guided, CRT (human-equivalent 38 Gy, n=12) or CRT with PFB (human-equivalent 49 Gy, n=12), via the small animal radiation research platform (SARRP) and were longitudinally assessed for over a year. Late-effects were compared to a sham-irradiated group (n=12). RESULTS CRT was well tolerated, independent of PFB receipt, and no mice suffered severe acute toxicity. Mice were significantly more frail following irradiation (frailty index; p=0.0002) and had reduced physical functioning; time to fall from a rotating rod and grip strength were significantly lower (rotarod; p=0.026 and grip strength; p=0.006, respectively). Neurocognitive deficits were consistent with those observed in childhood MB survivors; irradiated mice displayed significantly worse working memory (Y-maze; p=0.009) and exhibited spatial memory deficits (Barnes maze; p=0.029). Receipt of PFB did not induce a more severe late-effect profile. CONCLUSION We conclude that our in vivo model of childhood MB radiotherapy recapitulates the late-effect profile of MB survivors. Our clinically-relevant model will facilitate both the elucidation of novel/target mechanisms underpinning MB late-effects and the development of novel interventions for their amelioration.

MDB-104. TARGETING Y BOX BINDING PROTEIN (YBX1) IN PEDIATRIC MEDULLOBLASTOMA

Abstract Medulloblastoma is the most common malignant pediatric brain tumor, with some patients having abysmal survival despite an intense therapeutic regimen. A significant barrier to effective outcomes is cell-intrinsic resistance to chemo- and radio- therapy. Using a batch-normalized bulk RNA-seq dataset (GSE124814) comprised of 1350 MB samples and 291 normal cerebella, we reveal upregulation of Y-box binding protein 1 (YBX1) in MB compared to normal cerebellum. Further analysis of this dataset and others demonstrates a significant correlation between MYC and YBX1 expression in G3 tumors. Proteomics analysis also reveals higher YBX1 in G3 tumors characterized by hyperstability of MYC. Using a well-established ex vivo model, we demonstrate increased YBX1 expression as normal cerebellar progenitor cells transition to tumorigenic G3 MB stem-like cells. Deletion of YBX1 in these progenitor cells limits tumorigenesis suggesting a requirement for YBX1 in tumor initiation. We utilized an orthogonal pharmacogenetic approach to inhibit YBX1 and determine its effects on G3 MB tumor biology. Genetic depletion of YBX1 significantly reduced cellular proliferation and antagonized MYC expression in vitro. Orthotopic implantation of these YBX1KO G3 MB cells leads to increased tumor latency and animal survival. Transcriptomic analysis reveals a de-repression of neuronal target genes as well as suppression of MYC-activated genes. Evaluation of SU056, a YBX1 targeting small molecule, revealed both single agent efficacy as well as synergism with chemo- and radio- therapy. Further analysis revealed efficacy in SHH MB, even in tumors with mutant p53, suggesting cross-subgroup use for SU056. In silico docking identified binding of SU056 in the mRNA recognition domain of YBX1. Disruption of YBX1-RNA binding impacts the stability of known oncogenic transcripts, important for tumorigenesis and progression. Our findings corroborate previous identification of YBX1 as a chemoresistance factor and validate pre-clinical targeting of YBX1 to improve patient outcomes.

MDB-68. DISCOVERING DRUG SENSITIVITIES IN MEDULLOBLASTOMA USING PATIENT- AND PDX-DERIVED TUMOROIDS

Abstract BACKGROUND Medulloblastoma (MB) is one of the most common malignant pediatric brain tumor types and is subdivided into four major types: WNT, SHH, Group 3 and Group 4. Treatment, including resection, cranio-spinal radiotherapy, and chemotherapy, often leads to undesirable long-term side effects. MB prognosis depends on type, subtype, and genetic alterations, reflecting the high level of heterogeneity observed in these tumors and indicating that specific patient populations with particularly poor prognosis require alternative treatment approaches. Therefore, preclinical models are essential for recapitulating MB heterogeneity and developing subtype-specific treatments. METHODS Long-term in vitro MB tumoroid models were developed from patient and PDX tumors and molecularly characterized using DNA methylation and transcriptomic analyses. High-throughput single drug screening (225 compounds) and combination screening were performed to identify (subtype-specific) drug sensitivities. RESULTS Fifteen tumoroid lines were established from TP53-mutated SHH (n=6), Group 3 (G3) MYC-amplified (n=4) and non-amplified (n=5) patient (n=6) and PDX (n=9) tumors. DNA methylation analyses confirmed the maintenance of molecular identity in these tumoroids after long-term culture (>5 passages). Transcriptome analyses reveal a more proliferative, progenitor-like phenotype in these tumoroids compared to patient/PDX tumors. Hight-throughput drug screens identified both common and type-specific drug sensitivities within MB. Notably, drugs targeting CDK7, CDK12, CHECK1, BCL2L, and HDAC demonstrated efficacy in both G3 and TP53-mutated SHH-MB, while TOP1/2 and MDM2 inhibitors were specific for G3-MB. Currently, these drugs are being further investigated to select the most effective synergistic drug combinations for in vivo validation in PDX models. CONCLUSIONS Thus far, we have established already fifteen MB tumoroid models representing SHH-MB and G3-MB and identified several drug targets, including cell cycle kinases and HDACs. Tumoroids closely resemble patient tumors, based on methylome and transcriptome, enhancing their relevance for drug discovery applications in a high-throughput fashion and for prioritizing new treatment strategies for MB patients in clinical trials.

Central pathological review and molecular classification of medulloblastoma in countries with limited resources: A commitment to equitable access.

10062 Background: Medulloblastoma, the most common malignant pediatric brain tumor, exhibits molecular heterogeneity influencing prognosis and treatment decisions. This study focuses on feasibility the pathological review and molecular classification with cost-effective methods. Methods: From December 2018 to November 2023, we analysed samples of patients referred to our laboratory. Data were collected on patient demographics (age, gender, origin), pathological review and specific medulloblastoma subtypes identified. The study was performed without costs for parents or institutions. Results: During the study period, 146 patients were included. The predominant age groups being 0 to 4 years (20.1%) and 5 to 9 years (36.1%) The mean age was 10.2 years. 55.6% of patients were female and 43.8% were male. Ninety-three patients were from Brazil and 7% from Latin America. Of the 117 samples that could be analyzed, 43% was classified as group 4, 37% were sonic hedgehog (SHH) group, 10% were group 3 and 4% were WNT group. Five percent of the samples had inconclusive results, with two patients had diagnoses other than medulloblastoma. Seventy percent of samples had a turnaround time for molecular classification results around 30 days. Conclusions: This study highlights the importance of central review pathology and molecular classification in medulloblastoma management. Our results emphasize the feasibility of centralized pathology review and molecular classification in countries with limited resources and provide equity for all patients. Based on this experience, we have expanded for other brain and pediatric tumors. Our effort contributes to ensuring that every patient benefits from the advancements in personalized medicine and this represents a real step to offer the same chances of cure for everyone.

Drivers Underlying Metastasis and Relapse in Medulloblastoma and Targeting Strategies.

Medulloblastomas comprise a molecularly diverse set of malignant pediatric brain tumors in which patients are stratified according to different prognostic risk groups that span from very good to very poor. Metastasis at diagnosis is most often a marker of poor prognosis and the relapse incidence is higher in these children. Medulloblastoma relapse is almost always fatal and recurring cells have, apart from resistance to standard of care, acquired genetic and epigenetic changes that correlate with an increased dormancy state, cell state reprogramming and immune escape. Here, we review means to carefully study metastasis and relapse in preclinical models, in light of recently described molecular subgroups. We will exemplify how therapy resistance develops at the cellular level, in a specific niche or from therapy-induced secondary mutations. We further describe underlying molecular mechanisms on how tumors acquire the ability to promote leptomeningeal dissemination and discuss how they can establish therapy-resistant cell clones. Finally, we describe some of the ongoing clinical trials of high-risk medulloblastoma and suggest or discuss more individualized treatments that could be of benefit to specific subgroups.

270 Inducing Neural Maturation and Increasing Survival in SHH Medulloblastoma Using EZH2 Inhibition in a Mouse Model

INTRODUCTION: Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Genetically, MB can be divided into four subgroups of which the SHH subtype histologically shows nodular architecture. Within this nodular architecture, there are islands of mature cells, with more abundant neuropil and a low proliferation rate, scattered among sheets of primitive cells. METHODS: We performed laser capture microdissection followed by whole transcriptome analysis, spatial transcriptomics using Digital Spatial Profiling, whole genome DNA methylation and ChIP-Seq analysis of mature and primitive areas from 8 medulloblastomas. We developed a genetically-engineered mouse model of SHH MB showing spontaneous maturation and lack of maturation with a conditional EZH2 genetic ablation or EZH2 overactivation respectively. Finally, we developed a fucoidan-based nanoparticle drug delivery across the blood brain barrier (BBB) for targeted molecular inhibition. RESULTS: Using whole transcriptome and DNA methylation analysis, we identified ∼120 differentially expressed genes between primitive and mature regions with enrichment for genes regulated by H3K4me3 and H3K27me3. ChIP-Seq analysis showed striking differences in H3K27me3 enrichment between primitive and mature medulloblastoma cells including at the EZH2 locus. Medulloblastoma specific EZH2 genetic ablation resulted in diffuse tumor cell differentiation and prolonged survival in mice (n = 10 per group, log-rank p = 0.01). Conversely, conditional EZH2 (Y641F) activation prevented medulloblastoma differentiation. A fucoidan-based nanoparticle successfully delivered the EZH2 inhibitor (EPZ-6438) across the murine BBB to achieve significant extension of mouse survival (median 70 days compared to 21 days in control mice; *p = 0.01, Mantel-Cox). CONCLUSIONS: Spontaneous maturation of medulloblastoma cells can be induced by inhibition of EZH2. Fucoidan-based nanoparticle delivery systems allows tumor specific targeted delivery across the BBB extending survival in mice.

Roles and interactions of tumor microenvironment components in medulloblastoma with implications for novel therapeutics.

Medulloblastomas, the most common malignant pediatric brain tumors, can be classified into the wingless, sonic hedgehog (SHH), group 3, and group 4 subgroups. Among them, the SHH subgroup with the TP53 mutation and group 3 generally present with the worst patient outcomes due to their high rates of recurrence and metastasis. A novel and effective treatment for refractory medulloblastomas is urgently needed. To date, the tumor microenvironment (TME) has been shown to influence tumor growth, recurrence, and metastasis through immunosuppression, angiogenesis, and chronic inflammation. Treatments targeting TME components have emerged as promising approaches to the treatment of solid tumors. In this review, we summarize progress in research on medulloblastoma microenvironment components and their interactions. We also discuss challenges and future research directions for TME-targeting medulloblastoma therapy.

Abstract 1261: In vitro medulloblastoma leptomeningeal metastasis models reveal adhesion signaling as a therapeutic vulnerability

Abstract Introduction: How tumor cells colonize an inherently distinct environment and survive the pressures of therapeutic treatment during metastasis is incompletely understood. In patients with medulloblastoma, the most common malignant pediatric brain tumor, mortality is almost exclusively dictated by metastasis to the leptomeninges, the inner covering of the brain and spinal cord. We aim to mechanistically understand the molecular drivers of leptomeningeal colonization, including how leptomeningeal adhesion mediates therapy resistance. Experimental approach: Progress in leptomeningeal metastasis research has been hindered by the scarcity of matched primary and metastatic tissue from patients given the lack of clinical benefit to obtaining metastatic tissue. Furthermore, end points from pre-clinical human xenograft mouse models are often driven by primary cerebellar tumors, not leptomeningeal dissemination. These barriers underscore the necessity for novel in vitro leptomeningeal metastasis models that allow for rigorous experimental analysis. These models must simultaneously recapitulate tumor cell-host and tumor cell-environment interactions driving colonization to produce translatable findings. Accordingly, our lab has developed two novel in vitro leptomeningeal metastasis models that allow for cell-based experimental analysis: a primary meningeal cell-medulloblastoma organotypic co-culture and an acellular leptomeningeal matrix mimetic monoculture. Results: Proteomic analysis identified that leptomeningeal cells promote medulloblastoma chemotactic honing to the leptomeninges through CXCL12-CXCR4 signaling. Additionally, loss of CXCL12/CXCR4 signaling impaired medulloblastoma adhesion to the leptomeninges. CXCL12/CXCR4 are known to mediate adhesion of stem cells in stromal niches via activation of integrin receptor ITGA4/ITGB1 (VLA4) binding to stromal VCAM1. Indeed, the VLA4-VCAM1 axis is required for medulloblastoma leptomeningeal adhesion and requires activation by CXCL12/CXCR4 signaling. From a translational perspective, we found that leptomeningeal adhesion promotes therapy resistance and that inhibition of adhesion signaling sensitized adherent medulloblastoma cells to therapy. Conclusions: These findings provide proof-of-concept that appropriate in vitro models of metastasis can be developed and utilized to define the biology driving adhesion and chemotherapy resistance in medulloblastoma metastasis. Furthermore, we show that medulloblastoma leptomeningeal adhesion is mediated by a CXCL12/CXCR4-VLA4/VCAM1 axis. Finally, we demonstrate the therapeutic potential of targeting this axis in sensitizing medulloblastoma metastases to current therapies. Citation Format: Leyre Jimenez Garcia, Amy C. Gross, Ryan D. Roberts, James B. Reinecke. In vitro medulloblastoma leptomeningeal metastasis models reveal adhesion signaling as a therapeutic vulnerability [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 1261.

Abstract 5112: Discovery of nuclear envelope mislocalization in MYC-driven group 3 medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor, comprised of four distinct molecular subgroups (WNT, SHH, Group 3, and Group 4). A subset of Group 3 MB tumors harbor focal amplifications of the MYC oncogene and are particularly prone to tumor recurrence and leptomeningeal spread which currently remains incurable. Therefore, there is an urgent need for the development of therapeutic modalities that can spare the vulnerable, developing brain while having potent antitumor efficacy against recurrent MB. Through proteomic profiling of cyclophosphamide-resistant patient-derived MYC-driven Group 3 MB cell line SU_MB002, we identified nuclear envelope (NE) proteins to be highly abundant and enriched in Group 3 MB compared to normal tissue. We discovered that LBR, an inner nuclear transmembrane protein, while present in all cells at the NE, is aberrantly presented to the cell surface in MYC-driven Group 3 MB cell lines. Immunohistochemistry analysis of LBR on MB patient tumor samples identified that positive LBR staining correlated with a significantly worse prognosis and was also identified to be enriched in recurrent tissue compared to their matched primary samples. Cell surface expression flow cytometry analysis identified highly abundant cell surface expression in all MYC-driven Group 3 MB cell lines with low to no expression in human neural stem cells and other MB subgroups. Mechanistically, high resolution microscopy of endogenously HALO-tagged LBR reveals LBR cell surface presentation to be linked to ER or ER-like vesicles that are directly trafficked to the cell surface. Transcriptomic analysis of LBR cell surface positive and negative cells identify that cell surface positivity is significantly linked to cell division and mitotic processes (cell cycle process, mitotic cell cycle process, chromosome segregation, cell division, nuclear division). LBR mislocalization stems from mitosis at the time of nuclear envelope dissolution and is enriched in the hyperproliferative, therapy-resistant subpopulation of MB cells following exposure to chemotherapy and radiation in vitro and in vivo and may act as a marker for the brain tumor initiating cells that seed recurrence. Therefore, we demonstrate that LBR is an ideal therapeutic target for patients facing relapsed or treatment-refractory MB and have generated single domain antibodies in an effort to engineer LBR-specific CAR T cells. The development of therapies against NE proteins like LBR may provide patients a novel therapeutic opportunity that may be able to treat therapy-resistant, hyperproliferative MYC-driven Group 3 MB cells. Citation Format: Yujin Suk, Jorge Ibañez-Vega, Iqra Chaudhry, Martin Rossotti, Minomi Subapanditha, Petar Miletic, Stefan Custers, Laura Escudero, Alberto Delaidelli, Takuma Nakashima, Yuxi Xiao, Jason Moffat, Hiromichi Suzuki, Poul Sorensen, Kevin Henry, Chitra Venugopal, Giedre Krenciute, Sheila Singh. Discovery of nuclear envelope mislocalization in MYC-driven group 3 medulloblastoma [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 5112.

Abstract 5111: Rational synergistic radiosensitization strategies for group 3 medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor. MB is comprised of 4 groups, termed Wnt, Shh, Group 3 and Group 4, each characterized by distinct molecular and clinical profiles. MYC-amplified Group 3 MB is associated imminent fatal metastatic relapse following standard treatment approaches, which currently include surgery, craniospinal irradiation and chemotherapy, highlighting an urgent need for identification of therapeutic vulnerabilities for these tumours. As such, using 4 novel patient-derived models of Group 3 MB that faithfully recapitulate the molecular and histological tumor characteristics, we characterised response to radiation of Group 3 MB in vitro to show that these tumors do not possess intrinsic radiation resistance. Furthermore, cumulative radiation experiments do not increase bulk radiation resistance in MB cells, highlighting the contribution of dynamic cell states to radiation response. In contrast, exposure to radiation leads to changes in cell clonogenic capacity as measured by limited dilution assays. Furthermore, RNA-seq data uncovered altered transcriptomic programs in response to cumulative radiation treatment. In particular, we observe differential regulation of genes encoding potassium ion channels and their regulators as well as genes encoding ECM and cell adhesion proteins. As part of our goal to evaluate the current multimodal treatment strategy of MB, we also show that a set of standard chemotherapeutic agents (etoposide, thiotepa and lomustine) currently used in the clinic do not produce a potent response at optimal doses in vitro, potentially accounting for abysmal survival outcomes seen in chemotherapy treated MB patients. As such, we conducted a screen of 144 FDA approved cancer drugs and identified therapeutics that selectively target Group 3 MB cells in contrast to normal tissue controls (foetal neural stem cells) in the low nanomolar dose range. In particular, topoisomerase inhibitor topotecan produces a potent response specific to MYC-amplified Group 3 MB, and sensitizes 2 patient-derived MB models to gamma irradiation. Altogether, our data characterizes Group 3 MB molecular and cellular responses to current treatment modalities and provides rationale for new clinical strategies to sensitize patients to radiation that will potentially allow to reduce the required radiation doses to treat these aggressive tumors, reduce the incidence of radiation treatment-related morbidity as well as improve cure rates. Citation Format: Julija Povilaikaite, Kaitlin Stitz, Nikhita Austin, Vijay Ramaswamy. Rational synergistic radiosensitization strategies for group 3 medulloblastoma [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 5111.

Abstract 561: Targeting Y box binding protein 1 (YBX1) in MYC-amplified medulloblastoma

Abstract Medulloblastoma is the most common malignant pediatric brain tumor. Patients diagnosed with MYC-amplified group 3 medulloblastoma (G3 MB) have a devastating prognosis with a 5-year survival rate of <50%. Standard of care (SOC) consists of maximal resection surgery followed by chemoradiotherapy, but these extensive treatments leave patients with neurocognitive and developmental deficits. Therefore, the identification of novel targets that can be used alone or in combination with SOC is of utmost importance. Using a batch-normalized bulk RNA-seq dataset (GSE124814) comprised of 1350 MB samples and 291 normal cerebella, we reveal upregulation of Y-box binding protein 1 (YBX1) in MB compared to normal cerebellum. Further analysis of this dataset and others demonstrates G3 tumors that display high MYC levels also have the highest YBX1 expression. Analysis of ChIP-seq data shows strong binding of MYC to the YBX1 promoter in G3 MB, concurrent with H3K27ac and H3K4me3 histone modifications. Using a well-established ex vivo model, we demonstrate increased YBX1 expression as normal cerebellar progenitor cells transition to tumorigenic G3 MB stem-like cells. Recent work in leukemia reveals a reciprocal regulation of MYC by YBX1, however, this mechanism is not well established in G3 MB. We utilized an orthogonal pharmacogenetic approach to inhibit YBX1 function and determine its effects on G3 MB tumor biology. Suppression of YBX1 significantly reduced the proliferation and decreased MYC protein expression of G3 MB cells in vitro. These findings support a reciprocal MYC-YBX1 axis, which could influence tumor growth in vivo. Orthotopic implantation of YBX1 KO G3 MB cells leads to increased animal survival. We next evaluated the therapeutic potential of two recently characterized YBX1 inhibitors which display single agent efficacy in vitro and in vivo. YBX1 inhibitors synergized with SOC in vitro lowering the IC50 for both chemo and radiotherapy, which could reduce adverse effects experienced by patients. In silico molecular docking using PyMOL predicts YBX1 inhibitors interact with amino acids within the cold shock domain of YBX1, likely impairing its ability to bind RNA. We performed formaldehyde RNA immunoprecipitation (fRIP)-sequencing to identify changes in RNA transcript levels following YBX1 pharmacological inhibition. Disruption of YBX1-RNA binding impacts the stability of known oncogenic transcripts which may be required for tumorigenesis. Our findings impart an alluring prospect of targeting YBX1 in combination with standard-of-care, and possibly other compounds to improve patients’ outcomes. Citation Format: Stephen Carney, John Hemenway, Ashmitha Rajendran, Matt Hathaway, Eric Nealy, Christian Bonatto, Dhanir Tailor, Sanjay Malhotra, Myron Evans. Targeting Y box binding protein 1 (YBX1) in MYC-amplified medulloblastoma [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 561.

Abstract 5470: ONC206 inhibits tumour growth and is a potential novel therapeutic strategy for refractory medulloblastoma

Abstract Medulloblastoma is the most common malignant pediatric brain tumor with a for certain molecular subgroups very low overall survival and therefore an urgent need for novel treatment approaches. Dordaviprone (ONC201) and its chemical derivative with nanomolar potency ONC206 are inhibitors of the dopamine receptor 2 (DRD2) and more importantly induce apoptosis of cancer cells by activation of the mitochondrial caseinolytic protease P (ClpP). ONC201 has shown objective responses in patients with diffuse midline gliomas and is currently being evaluated in the Phase 3 ACTION study. ONC206 is currently in Phase I clinical trials for pediatric and adult patients with primary brain tumors. In this study, we evaluated the preclinical therapeutic effects of ONC206 in medulloblastoma in vitro and in vivo and investigated the molecular mode of action for this imipridone. We found evidence for high expression of ClpP at both the RNA and protein level in medulloblastoma tumours, compared to very low expression in normal brain and normal cerebellum. In addition, we saw a pronounced reduction in cell viability of human Group 3 and Group 4 and murine medulloblastoma cells treated with ONC206 with extremely low IC-50s (range: 14nM - 2μM).After treatment with ONC206, we observed a significant downregulation of mitochondrial electron transport chain complexes I and III and mitochondrial hyperpolarization, as well as an induction of the ATF4 pathway, thereby implying that ONC206 induces an integrated stress response and mitochondrial damage. In order to test the efficacy of ONC206 in vivo, we used murine models of SHH-driven and Group 3 medulloblastoma as well as Group 3 patient-derived xenografts (PDXs). ONC206 led to a significant prolongation of survival in both murine models, with the SHH mice demonstrating a remarkable survival benefit. Mice from both Group 3 PDXs also responded to ONC206, with 25% of ONC206 treated animals exhibiting long-term tumor-free survival. Our results highlight ONC206 as a novel attractive therapeutic option for patients with relapsed medulloblastoma and importantly pave the way for a clinical trial testing the efficacy of ONC206 in the treatment of these patients. Citation Format: Theophilos Tzaridis, Jingbo Liu, Varun Prabhu, Robert Wechsler-Reya, Tobey MacDonald. ONC206 inhibits tumour growth and is a potential novel therapeutic strategy for refractory medulloblastoma [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 5470.

Abstract 6505: Targeting PINK1 reduces medulloblastoma progression in animal models

Abstract Medulloblastoma is a brain cancer that mainly arises during infancy and childhood. Medulloblastoma is the most common pediatric malignant brain tumor worldwide. It comprises of approximately 20% of all childhood brain tumors and 63% of intracranial embryonic tumors. One of the major effects of proliferating cancer cells is altered mitochondrial metabolism, as well as aberrant metabolism. Medulloblastoma patients suffer severe complications due to intensive and long-term treatments. Thus, we aim to improve applications against medulloblastoma by targeting the PINK1 gene. We hypothesize that PINK1 inactivation slows down medulloblastoma progression. PINK1 is a mitochondrial serine/threonine protein kinase that protects cells by controlling mitochondria quality in response to cellular stress. PINK1 recruits parkin to target damaged mitochondria for degradation through autophagy. In this study, PINK1 has been knocked-out using gene editing technology (CRISPR/Cas9) in two commercial medulloblastoma cell lines (DAOY, and D283 cell lines). We validated PINK1 editing using RT-PCR and Western blot. Then, cell viability was studied in comparison to the control sample. The control sample contained scramble fragments with empty CRISPR/Cas9. We studied apoptosis and performed cell cycle analysis using flow cytometry. Next, orthotopic medulloblastoma mouse model was generated which showed differential survivals comparing the PINK1 knockout group and control. The results showed a low expression of PINK1 within the PINK1 knockout group. In addition, the Western blot showed reductions in PINK1 expression in the knockout group. The knockout group showed more apoptosis (cell death) 40% more than the control. The mouse model showed increased survival of the PINK1 knockout group compared to the control with a p-value of 0.0288 (log-rank). Our analysis shows that survival differences approximately extend human life by one year. We conclude that inhibiting the PINK1 gene in medulloblastoma decreases cell metabolic activities. PINK1 Knockout in medulloblastoma enhances the apoptosis pathways. Engrafted medulloblastoma mouse model treated with PINK1 inhibitor showed superior survival than the control. These results qualify PINK1 to be used as potential therapeutic target for medulloblastoma. The Future work needs additional preclinical studies to investigate the proper dose of PINK1 inhibitor and measurement of toxicity index. Citation Format: Reema N. Alsubaiee, Amani Alghamdi, Bahauddeen M. Alrfaei. Targeting PINK1 reduces medulloblastoma progression in animal 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 6505.

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

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

Development of a high-throughput screening platform to identify new therapeutic agents for Medulloblastoma Group 3

Pediatric brain tumors (PBTs) represent about 25 % of all pediatric cancers and are the most common solid tumors in children and adolescents. Medulloblastoma (MB) is the most frequently occurring malignant PBT, accounting for almost 10 % of all pediatric cancer deaths. MB Group 3 (MB G3) accounts for 25–30 % of all MB cases and has the worst outcome, particularly when associated with MYC amplification. However, no targeted treatments for this group have been developed so far.Here we describe a unique high throughput screening (HTS) platform specifically designed to identify new therapies for MB G3. The platform incorporates optimized and validated 2D and 3D efficacy and toxicity models, that account for tumor heterogenicity, limited efficacy and unacceptable toxicity from the very early stage of drug discovery. The platform has been validated by conducting a pilot HTS campaign with a 1280 lead-like compound library. Results showed 8 active compounds, targeting MB reported targets and several are currently approved or in clinical trials for pediatric patients with PBTs, including MB. Moreover, hits were combined to avoid tumor resistance, identifying 3 synergistic pairs, one of which is currently under clinical study for recurrent MB and other PBTs.

Elucidating cellular response to treatment with viral immunotherapies in pediatric high-grade glioma and medulloblastoma

HSV G207, a double-stranded, DNA virus, and the polio:rhinovirus chimera, PVSRIPO, a single positive-strand RNA virus, are viral immunotherapies being used to treat pediatric malignant brain tumors in clinical trials. The purpose of this work is to elucidate general response patterns and putative biomarkers of response. Multiple pediatric high-grade glioma and medulloblastoma cell lines were treated with various multiplicities of infection of G207 or PVSRIPO. There was a significant inverse correlation between expression of one HSV cellular receptor, CD111, and the lethal dose of 50% of cells (LD50) of cells treated with G207 (r = -0.985, P<0.001) but no correlation between PVSRIPO cellular receptor expression (CD155) and LD50. RNA sequencing of control cells and cells treated for 8 and 24 h revealed that there were few shared differentially expressed (DE) genes between cells treated with PVSRIPO and G207: GCLM, LANCL2, and RBM3 were enriched whilst ADAMTS1 and VEGFA were depleted. Likewise, there were few shared DE genes enriched between medulloblastoma and high-grade glioma cell lines treated with G207: GPSM2, CHECK2, SEPTIN2, EIF4G2, GCLM, GDAP1, LANCL2, and PWP1. Treatment with G207 and PVSRIPO appear to cause disparate gene enrichment and depletion suggesting disparate molecular mechanisms in malignant pediatric brain tumors.

SALL4 is a CRL3REN/KCTD11 substrate that drives Sonic Hedgehog-dependent medulloblastoma

The Sonic Hedgehog (SHH) pathway is crucial regulator of embryonic development and stemness. Its alteration leads to medulloblastoma (MB), the most common malignant pediatric brain tumor. The SHH-MB subgroup is the best genetically characterized, however the molecular mechanisms responsible for its pathogenesis are not fully understood and therapeutic benefits are still limited. Here, we show that the pro-oncogenic stemness regulator Spalt-like transcriptional factor 4 (SALL4) is re-expressed in mouse SHH-MB models, and its high levels correlate with worse overall survival in SHH-MB patients. Proteomic analysis revealed that SALL4 interacts with REN/KCTD11 (here REN), a substrate receptor subunit of the Cullin3-RING ubiquitin ligase complex (CRL3REN) and a tumor suppressor lost in ~30% of human SHH-MBs. We demonstrate that CRL3REN induces polyubiquitylation and degradation of wild type SALL4, but not of a SALL4 mutant lacking zinc finger cluster 1 domain (ΔZFC1). Interestingly, SALL4 binds GLI1 and cooperates with HDAC1 to potentiate GLI1 deacetylation and transcriptional activity. Notably, inhibition of SALL4 suppresses SHH-MB growth both in murine and patient-derived xenograft models. Our findings identify SALL4 as a CRL3REN substrate and a promising therapeutic target in SHH-dependent cancers.