Medulloblastoma Research Articles

NFS-01. EXTREMELY RARE CASE OF CHILDREN WITH NIJMEGEN BREAKAGE SYNDROME AND MEDULLOBLASTOMA – A THERAPEUTIC CHALLENGE

Abstract BACKGROUND Nijmegen Breakage Syndrome (NBS) is an autosomal recessive inherited disorder characterized by DNA repair abnormalities. It manifests with heightened sensitivity to radiotherapy (RT) and predisposes individuals to neoplastic diseases. Antineoplastic treatment in NBS patients is particularly challenging due to concurrent immune disorders, intense toxicities, and contraindications for RT. Hematologic disorders, especially lymphomas, are among the most prevalent malignancies in NBS patients. Solid tumors in NBS are extremely rare, with only two pediatric cases documented in the literature, both succumbing due to severe complications of treatment. METHODS A review and analysis of the medical documentation of a child with NBS and MB treated at the Department of Pediatrics, Hematology, and Oncology, Gdansk, Poland, were conducted. RESULTS A genetically confirmed NBS girl displaying typical phenotypic features presents symptoms at the age of 2 years, such as headaches, reluctance to walk, and drowsiness. Imaging confirmed a tumor in the left cerebellar hemisphere, measuring 40x33x30mm, displaying radiological features consistent with Medulloblastoma (MB). The patient underwent gross total resection of the tumor. The pathology revealed a desmoplastic type of MB with SHH activation, TP53 wild type, and ongoing molecular analysis. Postoperative imaging confirmed clinical remission, and cerebrospinal fluid examination was negative. Considering the substantial toxicities of chemotherapy in NBS patients and the contraindications for RT, the patient received chemotherapy with reduced cytostatic doses. Treatment was complicated by profound and prolonged myelosuppression and infections. Currently, after completing six cycles of chemotherapy, the patient is still in remission. To maintain remission and address NBS, the patient will receive allogeneic bone marrow transplantation from a 9/10 HLA-matched unrelated donor. CONCLUSIONS Managing patients with NBS, complicated by neoplastic diseases, is exceptionally demanding and necessitates an individualized multidisciplinary approach. Modern diagnostic and therapeutic methods enhance the prospects of reducing the toxicity of oncologic treatment, improving quality of life, and extending overall survival.

MDB-09. WNT AND GROUP 4 MEDULLOBLASTOMAS OF EARLY CHILDHOOD TREATED WITH HIGH DOSE CHEMOTHERAPY AND CRANIOSPINAL IRRADIATION SPARING APPROACH

Abstract BACKGROUND WNT and group 4 medulloblastoma (MB) are rare in young children and their outcome when treated with radiation avoidance infant strategies is unknown. METHOD This retrospective international cohort included children less than 7 years of age, with molecularly characterized group 4 or WNT MB, treated with high-dose chemotherapy(HDC) and CSI sparing approach. RESULTS The current cohort includes 29 patients (19M/10F). For the 26 patients with group 4 MB (median age at diagnosis 45.9 months), 16 (61.5%) were M0 and 17 (65.3%) underwent initial gross total resection (GTR). The most common consolidation regimen used was three cycles of high dose carboplatin and thiotepa (65.5%) or a single cycle of high dose of carboplatin, etoposide and thiotepa (31%).Two received adjuvant focal radiotherapy. Sixteen patients (61.5%) relapsed at a median time of 21.4 months from diagnosis (median age of 5.2 years). Relapse was local for 50% of the patients. One patient underwent palliative management and 15 received radiation-based salvage therapy (2 focal, 13 CSI+/- boost).The median dose of CSI was 36Gy (18-36). Five of the 15 died of disease. Overall, at a median follow-up of 37.9 months from diagnosis, 17/26 (65.3%) patients were alive, six had died of disease and two of toxicity. All 3 WNT patients (median age at diagnosis 57.8 months) underwent GTR and two were M0. Two received sequential high dose carboplatin and thiotepa. One patient with initial partial response, progressed 2 months after treatment completion and received salvaged CSI (18Gy). All of them were alive at a median follow up of 88 months from diagnosis. CONCLUSIONS HDC and CSI sparing approaches led to a high rate of relapse for young patient with group 4 (61.5%). However, 2/3 were salvaged with radiation-based therapy given at a median age 5.2 yrs. Additional WNT patients are needed to better describe their management.

MDB-79. THE DEUBIQUITINATING ENZYME USP7 COOPERATES WITH SONIC HEDGEHOG SIGNALING TO PROMOTE MEDULLOBLASTOMA

Abstract BACKGROUND Constitutive activation of the Sonic Hedgehog (SHH) signaling pathway during cerebellar development may lead to medulloblastoma (MB), one of the most common malignant pediatric brain tumor. Clinically, SHH-MB with mutations downstream of SMO or TP53-mutated are a real challenge and alternative targeted therapies are sorely needed for this group of patients. In SHH-MB, the transcription factor Atoh1 is constitutively overexpressed and essential for tumor initiation and progression. Previous work demonstrated that proteasomal-mediated degradation of Atoh1 induces tumor regression in vitro and in vivo, therefore deciphering mechanisms controlling Atoh1 stabilization should provide new avenues for drug development in SHH-MB. METHODS Using a proteomic approach, we identified the deubiquitinating enzyme Usp7 as a positive regulator of Atoh1 in MB cells. We then applied a SILAC-based quantitative ubiquitinomics to investigate the Usp7 target landscape in SHH-MB. We also employed several biochemical approaches to demonstrate the functional interaction between Atoh1 and Usp7 in SHH-MB. In parallel, we validated the impact of Usp7 depletion in vitro and in vivo using relevant SHH-MB models. Last, we investigated large transcriptomic and proteomic datasets to unveil the prognostic value of Usp7 in SHH-MB human tumors. RESULTS We demonstrate that Usp7 binds and accumulates Atoh1 protein by counteracting its ubiquitylation. Post-natal electroporation in vivo directly into the developing cerebella also indicates that overexpression of Usp7 promotes proliferation, whereas in the absence of Atoh1 Usp7 was no longer able to spread cell growth, demonstrating a functional link between the Usp7-Atoh1 axis. In vivo, disruption of Usp7 expression in SHH-MB blocks tumor proliferation. Pharmacological inhibition of Usp7 triggers tumor growth in a syngeneic SHH MB mouse model. Lastly, SHH-MB cells with either TP53 or SMO downstream mutations are sensitive to Usp7 inhibition. CONCLUSIONS Overall, our data pinpoint Usp7 as a promising target against Atoh1, a master regulator of SHH-MB.

OTHR-12. GENERATING A COMPREHENSIVE INTERACTIVE PEDIATRIC BRAIN TUMOR VISUALIZATION PORTAL FROM MOLECULAR AND CLINICAL DATA DERIVED FROM TWO MULTI-INSTITUTIONAL PROTOCOLS (SJMB03, SJYC07) AND NON-PROTOCOL PATIENTS TREATED AT ST JUDE CHILDREN’S RESEARCH HOSPITAL

Abstract BACKGROUND Pediatric central nervous system (CNS) tumors represent a diverse group of neoplasms with heterogeneous molecular and clinical characteristics. The need for well-organized, searchable datasets is paramount to enhance our understanding and improve the treatment approaches for these devastating diseases. To illustrate this, we have organized multifaceted data from patients into an interactive portal equipped with cutting-edge visualization tools. METHODS Clinical and molecular data from the SJMB03 and SJYC07 protocols and select published non-protocol St. Jude patients were collated and harmonized. A data dictionary was constructed to record demographic information, treatment details, histology, methylation classification, mutations, chromosomal aberrations, and outcomes. We implemented five visualization techniques - summary plots, sample view, scatter plots, sample matrix, and survival plots. These techniques are interconnected, enabling interaction and “crosstalk”. RESULTS Data from 710 patients with CNS tumors were incorporated: 386 Medulloblastomas (MB); 88 Atypical teratoid Rhabdoid Tumors (ATRT); 54 Ependymomas (EPN); 49 Pineoblastomas (PB); 28 Embryonal Tumors with Multilayered Rosettes (ETMR), 22 Infant-type hemispheric glioma (IHG), 16 High grade glioma (HGG), 13 Choroid Plexus Tumors (CPT); 11 CNS Sarcoma; 8 CNS tumor with BCOR internal tandem duplication (CNS BCOR); 6 CNS neuroblastoma FOXR2 activated (CNS NB FOXR2); 5 Low grade glioma (LGG); and 24 other entities or unclassified tumors. The portal features enabled the creation of custom cohorts. For example, patients with TP53 mutations (n=32; 15 MB, 7 CPT, 6 HGG, 1 CNS Sarcoma, 1 CNS NB FOXR2, 1 PB, 1 unclassified tumor) or DICER1 mutations (n=10; 7 PB, 2 CNS Sarcoma, and 1 ETMR) could be identified, cross-compared and analyzed adjusting for variables at users’ choice. CONCLUSION The integration of molecular and clinical data from multiple sources into an interactive visualization portal presents a comprehensive resource for research and clinical practice. Future work will focus on expanding the portal to integrate more cases/cohorts.

MDB-57. PROTEOMIC ANALYSIS REVEALS AMBRA1 AS A NEW CRL3REN/KCTD11 SUBSTRATE PROMOTING SONIC HEDGEHOG-DEPENDENT MEDULLOBLASTOMA

Abstract BACKGROUND Sonic Hedgehog (SHH) signaling pathway is fundamental for a proper embryonic development and its uncontrolled activation is responsible for various cancers. One the most relevant SHH-dependent tumors is medulloblastoma (MB), a highly heterogeneous brain malignancy that predominantly occurs in childhood. Understanding the complex networks that govern SHH aberrant activation represents a dramatic challenge for the development of efficient personalized therapies. Here, we identify a new role of the pro-autophagic protein AMBRA1 as interactor of REN/KCTD11 (here REN), the substrate-receptor subunit of the Cullin3-RING ubiquitin ligase complex (CRL3REN) and tumor suppressor lost in ~30% of human SHH-MBs. We show a novel mechanism of action of AMBRA1 in SHH-MB pathogenesis whereby its highly expression, due to loss of REN, drives the activation of GLI1, the final effector of SHH signaling. METHODS Proteomic analysis, co-immunoprecipitation, ubiquitylation and cycloheximide assays have been performed to demonstrate that AMBRA1 is a substrate of ubiquitylation of CRL3REN and to define AMBRA1 mechanism of action in SHH-MB tumorigenesis. Transcriptomic analysis, in vitro and in vivo proliferation assays have been carried out on murine and patient-derived xenograft (PDX) models to examine the effect of AMBRA1 modulation on SHH-MB growth. RESULTS We demonstrate that CRL3REN binds and induces polyubiquitylation and proteasome-mediated degradation of AMBRA1. Interestingly, we found that genetic depletion of AMBRA1 strongly reduces SHH-dependent MB growth both in vitro and in vivo, an effect associated with the decrease in the expression of GLI1 and other SHH target genes. We hypothesize that AMBRA1 could impact on the stability of GLI1 by counteracting the activity of E3 ubiquitin ligases regulating GLI proteins. Remarkably, transcriptomic analysis of SHH PDX upon AMBRA1 knockdown revealed an impairment of SHH activity and an upregulation of cerebellum differentiation. CONCLUSION Our findings identify AMBRA1 as a novel CRL3REN substrate and promising therapeutic target for SHH-dependent cancer.

IMG-18. DIFFERENTIAL DIAGNOSIS OF POSTERIOR FOSSA TUMORS USING RADIOMICS; A FIRST-LINE DIAGNOSTIC STEP REQUIRED FOR DOWNSTREAM ANALYSES

Abstract BACKGROUND Accurate radiographic diagnosis of pediatric brain tumors (PBTs) that originate in the brainstem and posterior fossa, including medulloblastoma (MB), pilocytic astrocytoma (PA), ependymoma (EPN), atypical teratoid/rhabdoid tumor (ATRT), and diffuse intrinsic pontine glioma (DIPG), is crucial for optimizing surgical approaches and enhancing neoadjuvant therapies. Existing research on the radiographic differential diagnosis of posterior fossa tumors has limitations, including small sample sizes, lack of inclusion of certain histologies especially rarer tumors such as ATRTs, and incomplete analysis of the whole tumor, including peritumoral edema. In this study, we aimed to perform a comprehensive analysis using radiomics and machine learning to differentiate among the common posterior fossa and brainstem tumors. METHODS We employed 927 radiomic features extracted from whole tumor regions within treatment-naïve, standard multiparametric MRI sequences (pre-/post-contrast T1-weighted, T2-weighted, FLAIR) of 264 patients (106 MBs, 78 PAs, 28 EPNs, 27 ATRTs, and 25 DIPGs), collected from the Children’s Brain Tumor Network (CBTN). We adopted a one-versus-rest classification strategy, employing Support Vector Machines combined with the Least Absolute Shrinkage and Selection Operator (LASSO) for feature selection, and implementing nested cross-validation for robustness. RESULTS The performances of the classifiers were evaluated using the Area Under the Receiver Operating Characteristic Curve, yielding values of 0.84 for MBs, 0.84 for PAs, 0.70 for EPNs, 0.75 for ATRTs, and 0.71 for DIPGs. CONCLUSIONS Our method effectively differentiates between various tumor types in the posterior fossa and brainstem, paving the path towards the development of comprehensive diagnostic and prognostic AI tools for pre-treatment histological diagnosis of these tumors. These AI tools can lead to more tailored, risk-adjusted treatments for PBTs, reducing morbidities and improving patient outcomes. Based on our promising initial results, we will expand our dataset to include more samples and incorporate rarer tumor types, as well as piloting in different molecular subclassifications.

MDB-92. EFFECT OF REDUCED-DOSE CRANIOSPINAL IRRADIATION AND REDUCED-DOSE ADJUVANT CHEMOTHERAPY ON CHILDREN AND ADOLESCENTS WITH WNT MEDULLOBLASTOMA WITHOUT RESIDUAL OR METASTATIC DISEASE: RESULTS FROM THE SJMB12 CLINICAL TRIAL

Abstract BACKGROUND In response to studies showing medulloblastoma (MB) survival is not only contingent on clinical factors (e.g. metastases, residual disease) but also on molecular factors (e.g. molecular group, gene aberrations), we launched the SJMB12 (NCT01878617) trial to stratify treatment using both clinical and molecular risk factors. Specifically, we sought to evaluate if patients with WNT-group MB, without metastatic (M0) or residual disease (R0), treated with reduced-dose craniospinal irradiation (CSI) and reduced-dose cyclophosphamide-based chemotherapy could maintain a high survival with fewer treatment-related side effects. METHODS Tumors were molecularly stratified via immunohistochemistry and fluorescence in-situ hybridization. Patients with WNT tumors were grouped into 3 strata: W1 (M0, R0, classic histology, monosomy of chromosome 6); W2 (M0, R0, without monosomy 6 or classic histology); W3 [metastatic (M+), residual disease (R+), or MYC/MYCN amplification]. Treatment of W1 consisted of 15-Gy CSI/51-Gy primary site (0.5cm CTV margin) followed by 4 cycles of chemotherapy resulting in cumulative doses of 8 mg/m2 vincristine, 300 mg/m2 cisplatin, 12 gm/m2 cyclophosphamide. Serial audiology, neurocognitive, and endocrine evaluations were conducted over a six-year follow-up period. RESULTS From 2013-2022, 72 patients enrolled onto the W1 stratum: median age was 10.3 years (4.9-22.0); 62.5% female; median time of follow-up was 4.5 years (1.1-10.1). 5-year progression-free survival (PFS) and overall survival were 90.4 ± 5.1% and 98.6 ± 2.1%, respectively. Five patients had PFS events: 4 relapses and 1 accidental death. Relapsed disease was local in 2, distant in 1, and mixed in 1. Time to relapse was > 2 years from enrollment for 3 patients. Severe ototoxicity (SIOP grade ≥ 3) at the end of therapy occurred in 14.4 ± 4.5%. Neurocognitive, endocrine, and detailed molecular data are forthcoming. CONCLUSIONS Patients with low-risk WNT-MB maintained a high PFS (>90%) when treated with reduced-dose CSI and reduced-dose cyclophosphamide-based chemotherapy. Preliminary data suggests fewer treatment-related side effects.

LMIC-16. PRE-IRRADIATION CHEMOTHERAPY AND DELAYED RADIATION THERAPY IN INFANTS AND YOUNG CHILDREN WITH WITH CENTRAL NERVOUS SYSTEM EMBRYONAL TUMOURS

Abstract BACKGROUND Established strategies for infants and young children(IYC) with embryonal tumours of the central nervous system(CNSET) - autologous stem-cell rescue(ASCT) and intraventricular chemotherapy(IVCT) – are not universally available. We report the outcomes of IYC with CNSET treated using the strategy of surgery, pre-irradiation chemotherapy and delayed radiation. METHODS Children <3years diagnosed with CNSET- medulloblastoma(MB), atypical teratoid/rhabdoid tumour(ATRT), embryonal tumour with multilayered rosettes(ETMR), CNS primitive neuroectodermal tumours(PNET)- and pineoblastoma(PB),underwent standard evaluation and staging, maximal safe excision, pre-irradiation chemotherapy(cyclophosphamide, etoposide and carboplatin;CEJ) upto 36months of age and either ris-adapted craniospinal with boost or focal irradiation. We performed a retrospective intention-to-treat outcome analysis. RESULTS Of 117children <3years with CNSET treated January2011- December2022, 72 were treated with this strategy.The median age was 25months (range6-35), 49males,histology medulloblastoma-42(58%), ATRT-9(12.5%), PNET-8(11.1%), ETMR-8(11.1%) and PB-5(6.8%); 23(32%) metastatic.The median chemotherapy cycles were 6(range 1-15). 36 children went on to receive RT; CSI in 33 (20standard-dose,13reduced-dose) and focal in 3. At last follow-up, 27children are in remission, 43 have relapsed/progressed (9 received salvage treatment) and 2 died due to chemotherapy toxicity. The 3-year and 5-year event-free-survival(EFS) are 35% and 3-year and 5-year overall-survival(OS) are 41.8% and 40.2%. The 3-year EFS/OS for MB are 51.1%/ 62.8%,PNET 25%/25%,ATRT 11.1%/11.1%,ETMR 12.5%/12.5% and PB 0%. Only metastasis was prognostic in medulloblastoma (HR 2;95% CI 0.86-4.6 for EFS and HR 1.7;95% CI 0.67-4.5 for OS); age, molecular group and histology were not prognostic. Long-term follow-up (n=17) documented >grade2 late effects in all (endocrine-16, neurocognitive-8, vision-3, hearing -8 and neurological-10). CONCLUSION The use of moderate-intensity pre-irradiation chemotherapy is feasible and effective in infants and young children with CNSETs. Despite inferior outcomes compared to ASCT or IVCT,this strategy could be used in resource-limited settings.

IMMU-20. INNATE IMMUNE EVASION IN MYC-AMPLIFIED MEDULLOBLASTOMA

Abstract BACKGROUND While major advances have been made in improving the quality of life and survival of children with most forms of medulloblastoma (MB), those with MYC-driven tumors (Grp3-MB) still suffer significant morbidity and mortality. Here, we report a cell surface proteome analysis of Group3-MB cell lines after direct MYC inhibition to identify changes in surface proteins and sensitivity to macrophage-mediated phagocytosis. METHODS We had previously demonstrated the preferential activity of HDACi (CI994) in MYC-driven medulloblastoma. CI-994, showed significant cell viability reduction mediated by reduction in the MYC (mRNA and protein), induction of apoptosis in MYC-driven medulloblastoma. In this study, we directly inhibited MYC using MYCi975 and OMO-MYC and carried out a cell surface proteomic analysis on Group3-MB cell lines to reveal CD24 and CD59 as potential immune evasion markers. While CD24 is a potential don’t eat me signal, CD59 is a ubiquitously expressed cell-surface glycosylphosphatidylinositol-anchored protein that acts as an inhibitor to the membrane attack complex and protects cells from CDC. In certain tumors, CD59 expression is enhanced, posing a significant obstacle to treatment by hindering effective monoclonal antibody-induced CDC. We use inhibitors to CD24 and CD59 to confirm their role as innate immune evasion proteins in Group 3MB. RESULTS Meta-analysis of published datasets reveals CD24 and CD59 as strong prognostic indicators in Group3-MB as well as within the Group3g-subtype. Inhibition of MYC reduces surface levels of CD24, increasing macrophage-mediated phagocytosis. The use of a blocking anti-CD24 mAb significantly enhanced macrophage-mediated phagocytosis. Furthermore, a significant reduction in surface expression was observed in CD59. Blocking CD59 mAb significantly enhanced anti-CD47 mAb mediated phagocytosis. Combined treatment of anti-CD24 and anti-CD47 significantly enhanced the survival of Grp3-MB tumor-bearing mice.

MDB-72. BREFELDIN A (BFA) INDUCES CD133 PROTEOLYTIC PROCESSING IN MEDULLOBLASTOMA STEM CELLS (MBSCS)

Abstract BACKGROUND Medulloblastoma (MB) is a malignant brain tumor occurring in children, characterized by elevated resistance to conventional chemotherapy. Medulloblastoma stem cells (MBSCs) represent a fraction of the tumor cell population which has been associated with poor prognosis and refractoriness to conventional therapies. CD133 (Prominin 1, PROM1) is a transmembrane protein serving as a cancer stem cell marker associated with cancer progression. Brefeldin A (BFA) is an antibiotic disrupting the Golgi apparatus, thus triggering Endoplasmic reticulum (ER) stress signaling pathways. BFA cancer-inhibitory properties have been already described in cancer of different tissue origin. However, the effects of BFA on MBCSs have not been investigated yet. Thus, the aim of this study was to characterize the response of MBSCs to BFA and the effects induced by BFA on CD133. METHODS Human group 3 MB (G3MB) cell lines were grown in stem selective medium (B27™) and treated with 0.3 uM BFA for 24h before collection. Apoptosis, clonogenicity and CD133 expression were evaluated. CD133 was immunoprecipitated and the corresponding bands were excised and analyzed by nanoLC-MS/MS. Raw data were processed using PEAKS XPro. RESULTS Our results show that BFA-treated cells induced the overexpression of a CD133 isoform that was missing the N-terminus region. Moreover, we found a downregulation of CD133 activated intracellular signalling together with a reduced exposition on cell membrane. CONCLUSIONS We report here for the first time, a N-terminus proteolytic processing of CD133, potentially associated to the cell trafficking effects induced by BFA. Finally, this study suggests a proof of principle for the design of novel molecules with similar structures and mechanism of action of BFA.

MDB-50. CAR-T CELL-DERIVED EXOSOMES LOADED WITH THERAPEUTIC SIRNA IS A PROMISING TECHNIQUE IN THE TREATMENT OF SUBGROUP 3 MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is a childhood life-threatening tumor that occurs in the cerebellum. This tumor showed resistance against several types of immunotherapies like anti-PD1, cancer vaccines, monoclonal antibodies (mAbs), and even CAR-T cells. Subgroup 3 MB is the worst threatening subtype that poorly responds to treatment. Interestingly, our data showed that the HLX gene a transcription factor that regulates cell growth is highly expressed in G3-MB and associated with poor prognosis. The siRNA-HLX inhibited tumor growth in vitro and in vivo and promisingly showed long survival in the immunocompetent mouse model. We developed CAR-T-Exosomes that can pass brain-blood barriers (BBB) and deliver therapeutic siRNA to the brain tumor. Further analysis revealed that CAR-T-Exosomes contain T cell cytotoxicity materials that can destroy tumor cells and induce anti-tumor immune response. Promisingly, the expression of HLX was significantly inhibited in the tumor cells and the anti-tumor immune response was enhanced. CAR-T-Exosomes have high safety and can induce anti-tumor response without stimulation of immunotoxicity side effects like cytokine release syndrome (CRS). In this study, we are highlighting anti-EPHA2-CAR-Exosomes as a highly specific type of tumor CAR-T-Exos immunotherapy due to a very low expression of EPHA2 in normal cells and overexpression in medulloblastoma, particularly G3-MB. Therefore, we speculate significant inhibition of tumor growth and activation of CTL effector cells in the tumor microenvironment. This study provides a new biological technique for delivering tumor suppressor RNAs and developing specific anti-tumor immune responses. Indeed, the promising safety of anti-EPHA2-CAR-T cell exosomes compared to cell-based immunotherapy.

MDB-13. GERMLINEELP1 DEFICIENCY SENSITIZES CEREBELLAR GRANULE NEURON PROGENITORS TO SHH MEDULLOBLASTOMA

Abstract BACKGROUND Germline loss-of-function (LOF) variants in Elongator complex protein 1 (ELP1) are the most prevalent predisposing genetic events observed in medulloblastoma (MB), accounting for 30% of the Sonic Hedgehog 3 subtype (SHH-3). Molecularly, ELP1-associated SHH-MBs acquire somatic PTCH1 mutations in >80% of cases, and universal loss-of-heterozygosity of the wild-type ELP1 and PTCH1 alleles through loss of chromosome-arm 9q, resulting in their biallelic inactivation. Notably, germline ELP1 LOF occurs mutually exclusive with somatic/germline TP53 mutations in the SHH-3 subtype, suggesting that genetic perturbation of either ELP1 or TP53 may promote similar tumorigenic consequences in cerebellar granule neuron progenitors (GNPs), the accepted cellular origin of SHH-MB. Despite these findings, the molecular, biochemical, and cellular mechanism(s) by which ELP1-deficiency provokes malignant pathogenesis remain unknown. In this study, we sought to close this knowledge gap and functionally determine why children harboring pathogenic ELP1 germline variants are at an increased risk of developing SHH-MB. METHODS Mice were genetically engineered to mimic heterozygous Elp1 LOF (Elp1HET). We studied the effect of Elp1 LOF in GNPs using a combination of molecular profiling, immunophenotyping, and cellular assays. RESULTS GNPs from Elp1HET mice exhibited a spectrum of molecular and biochemical hallmarks of malignant transformation including increased DNA replication stress, DNA damage, accelerated cell cycle progression, and stalled differentiation. Orthotopic transplantation of Elp1HET GNPs engineered to harbor somatic Ptch1 inactivation yielded SHH-MB-like tumors with compromised p53 signaling, providing an explanation for the specificity of ELP1-associated tumors in SHH-3, and their exclusivity with TP53-mutant tumors. Treatment of ELP1-mutant patient-derived xenografts with an FDA-approved MDM2 inhibitor reactivated p53-dependent apoptosis and extended survival. CONCLUSIONS Collectively, our findings functionally substantiate the role of ELP1 deficiency in predisposition to SHH-3 MB and nominate therapeutic strategies to overcome p53 inhibition as a rational treatment option.

MDB-93. A TRANSCRIPTOME-BASED APPROACH TO DRUG REPURPOSING FOR AGGRESSIVE GROUP 3 MEDULLOBLASTOMAS

Abstract BACKGROUND Medulloblastoma (MB) is the most common malignant brain tumor in children, representing 20% of pediatric brain tumors. International consensus has identified molecular subgroups: WNT, SHH, group 3 (G3), and group 4 (G4). Current treatments, guided by histologic tumor grade and stage, do not fully harness these subgroups, resulting in varying prognoses. G3MB particularly faces challenges with rapid recurrence and aggressive disease, necessitating focused intervention. METHODS We conducted differential expression analysis on MB patient datasets (GSE148389, GSE164677) to generate a G3MB gene expression signature. This gene expression signature was analyzed against the LINCS database, yielding candidate antineoplastic compounds. Candidates were filtered for blood-brain barrier permeability and FDA approval. Functional assays evaluated drug effects on cytotoxicity, clonogenicity, wound healing, cell cycle, and apoptosis in G3MB cell lines (HDMB03 and D425). RNA sequencing of treated cells identified differentially regulated pathways to infer mechanism, followed by in vitro validation. Mitochondrial function and membrane potential were measured by Seahorse and TMRM assays, respectively. RESULTS Eighty-one candidates passed filtering. The leading drug class identified was antidepressants. Based on MTT assays, the top four candidates were nortriptyline, simvastatin, fluoxetine, and sertraline. Nortriptyline (NT) emerged as the lead compound, with IC50 values of ~7uM (HDMB03) and ~11uM (D425). Amongst the four drugs, NT induced consistent anti-neoplastic effects on wound healing, colony formation, medullosphere generation, and apoptosis. Mechanistically, NT induced mitochondrial stress, escalated mitochondrial superoxide, and decreased mitochondrial membrane potential. CHRM3, a receptor antagonized by NT and upregulated in G3MB samples was downregulated by NT treatment. Western blotting confirmed the inhibition of CHRM3 downstream effectors. CONCLUSIONS Our pipeline identified cytotoxic compounds against G3MB, including NT, which we hypothesize induces mitochondrial stress via CHRM3 modulation. This approach offers repurposable FDA-approved candidates for high-risk G3MB, potentially reducing current treatment toxicity and improving outcomes.

MDB-20. TOXICITY PROFILE OF TWO CHEMOTHERAPEUTIC REGIMENS ADOPTED FROM COG A9961 AND ACNS0331 FOR STANDARD-RISK MEDULLOBLASTOMA, A COMPARATIVE STUDY

Abstract BACKGROUND Medulloblastoma (MB) is classically treated with surgery, irradiation, and chemotherapy (CTH). Cisplatin-based CTH regimens for standard-risk (SR) MB entail significant toxicities particularly ototoxicity which adversely affect patients’ quality of life. We compared the toxicity profile of two regimens in terms of grade 3 or 4 ototoxicity, hematologic toxicity, hepatotoxicity, nephrotoxicity, and neurotoxicity. METHODS SR-MB patients enrolled at Children Cancer Hospital-Egypt (57357) between January 2016 and December 2019 (cohort A) received adjuvant CTH adopted from COG-A9961 protocol (regimen A; 8 cycles of vincristine, cisplatin, and CCNU; cumulative cisplatin dose 600 mg/m2). Starting from January 2020 (cohort B), patients received CTH adopted from ACNS0331 protocol (9 cycles; 2 cycles of vincristine, cisplatin, CCNU followed by 1 cycle of cyclophosphamide and vincristine, for 3 rounds; cumulative cisplatin dose 450 mg/m2). Toxicity grading was done using Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. RESULTS enrolled patients (n=168; cohort A = 112; 67% and cohort B = 56; 33%). Cohort A had significantly higher rate of grade ≥3 ototoxicity at some point during treatment (27% vs 3.6%, p <0.001). Similarly, cohort A had higher grade ≥3 neurotoxicity (26% vs 12%, p =0.046), anaemia and thrombocytopenia (72% vs 56%, p =0.04). Cumulative rate of leukopenia was very high in both cohorts, 98% of all patients, while the rate of febrile neutropenia was much lower in cohort A (38% vs 67%, p <0.001). No toxic deaths were observed in both cohorts. There was no difference in grade ≥3 nephrotoxicity nor hepatotoxicity between the 2 cohorts. The 2-year OS for cohort A and B were 96.4% and 86.6%, respectively, p=0.11. Similarly, the 2-year EFS were 92.9% and 86.8%, p=0.29. CONCLUSION adjuvant CTH from ACNS0331 protocol showed a better toxicity profile particularly for ototoxicity compared to A9961 CTH regimen, while maintaining comparable survival.

STEM-01. DEVELOPMENT OF A HUMAN-SPECIFIC 3DIN VITRO PAEDIATRIC CEREBELLAR TUMOUR MODEL USING DECELLULARISED BRAIN AUTOPSY TISSUE

Abstract BACKGROUND Medulloblastoma (MB) and Atypical Teratoid/ Rhabdoid Tumours (ATRT) represent paediatric cerebellar tumours with a dismal prognosis. For ATRT in particular, 5-year survival remains at less than 32%. We have developed a 3D in vitro model termed ‘tumoursphere matrix’ which recapitulates in vivo crosstalk between tumour and reactive brain. METHODS AND RESULTS We co-cultured D283 (Group 3-MYC amplified MB) and BT12 (ATRT-MYC) cells with primary human cerebellar astrocytes within a PEGDA hydrogel containing decellularised cerebellar extracellular matrix (ECM) derived from non-disease human autopsy brain. Cerebellar ECM was extensively characterised, showing a significant reduction in cellular material whilst retaining ECM ligands. Reduction of DNA content to 174 ng/mg was corroborated by an absence of nuclei in immunohistochemical staining. Colorimetric assays indicated collagen and glycosaminoglycan retention in decellularised ECM, and Immunofluorescence confirmed retention of fibronectin, laminin and hyaluronic acid. Detection of all ECM ligands was cross-validated by Orbitrap-Secondary Ion Mass Spectrometry. A solubilised ECM/PEGDA hydrogel was used to coat an AggreWell™ plate. Primary human cerebellar astrocytes, D283 and BT12 cells were cultured as spheroids within the ECM/PEGDA hydrogel for 7 days with no significant decrease in cellular viability. Cytokine and Human Matrix Metalloproteinase (MMP) Antibody Arrays showed cytokine and MMP expression in all three cell lines was retained in the presence of ECM material. Furthermore, D283 and BT12 cells were co-cultured with primary astrocytes for 7 days prior to separation using fluorescence activated cell sorting to generate individual cell populations. CONCLUSIONS We have developed a 3D in vitro model which can co-culture cells for over 7 days, whilst recapitulating the in vivo tumour environment. RNA sequencing is currently in progress to identify differentially expressed genes in cerebellar tumour cells upon astrocytic and brain ECM crosstalk. We hypothesise that biochemical signalling underlying this communication will reveal putative therapeutic vulnerabilities.

EPID-04. CAUSES AND LONG-TERM COURSE OF OTOTOXICITY IN PEDIATRIC MEDULLOBLASTOMA AND EPENDYMOMA PATIENTS – CUMULATIVE PLATINUM AND COCHLEAR IRRADIATION DOSE MATTER

Abstract BACKGROUND Ototoxicity is a well-known side effect of treatment with platinum-containing chemotherapy and radiotherapy (RT). Nevertheless, precise dose-effect relationships are still lacking. METHODS Patients from the HIT2000 trial receiving first-line treatment for medulloblastoma (MB) or ependymoma (EPN) who were alive at last follow-up and had at least one audiogram prior to and after administration of chemotherapy with cisplatinum and radiotherapy were included into this study. Treatment details (incl. cochlear irradiation dose [CID]) and audiological outcome were analyzed. The ototoxicity was graded using the HIT scoring. RESULTS Courses of 101 patients were analyzed. Median audiology follow-up was 6 years [range: 1.0–18.1 years]. Ototoxicity grade ≥1 was documented for n=66 patients (65.4%). For n=27 this resulted in treatment modification. Univariable analyses revealed different proportions of ototoxicity (grade ≥1) regarding histology (CMB: 50/65, LCAMB: 3/3, DMB/MBEN: 8/12, EPN: 5/21; p<0.001), tumor location (supratentorial: 3/9, infratentorial: 63/92; p<0.001), metastatic status at diagnosis (M0: 46/77, M+: 20/24; p=0.003), radiation field (CSI: 59/75, local RT: 6/21, no RT: 1/5; p<0.001), CID (continuous variable, p=0.001) and cumulative cisplatinum dose (continuous variable, p<0.001). Certainly, disease specific parameters (histology/staging) were related to treatment intensity (cisplatinum/irradiation dose). Multivariable analyses confirmed the impact of cumulative cisplatinum dose (p=0.01) and CID (p=0.04). ROC-analyses allowed the definition of cut-offs for high risk of ototoxicity grade ≥1 (cisplatinum: 70mg/m2 BSA, CID: 37.5 Gy) and ototoxicity risk estimation, when applying doses above the respective cut-offs (cisplatinum dose increase by 1mg/m2 BSA results in 4% higher risk for ototoxicity grade ≥1; CID dose increase by 1 Gy results in 4.6% higher risk for ototoxicity grade ≥1). CONCLUSIONS Ototoxicity affects a majority of children receiving adjuvant treatment for MB and EPN. To avoid disabilities and negative effect on quality of live, dosage of cisplatinum and radiotherapy should be scrutinized and reduced whenever possible.

MODL-16. CRANIOSPINAL RADIOTHERAPY IN COMBINATION WITH DNA-DAMAGE RESPONSE INHIBITORS IN PATIENT-DERIVED ORTHOTOPIC XENOGRAFT MODELS OF MEDULLOBLASTOMA

Abstract BACKGROUND Despite treatment intensification, survival for high-risk subgroups of medulloblastoma (MB) such as MYC-amplified Group 3 (Gr3-II) and p53-mutant SHH (SHH-3p53mut) have remained dismal with overall survival around 40% and 20% respectively. Our previous studies have shown that the DNA-damage response inhibitors (DDRis) prexasertib and ceralasertib enhance the effects of CSI using medulloblastoma cell line-derived orthotopic mouse models. However, patient-derived orthotopic xenografts (PDOXs) are considered superior models for preclinical testing compared to immortalised cell lines. Here, we developed a CSI protocol for multiple PDOX models of high-risk medulloblastoma and tested the optimal protocol with concurrent DDRis. METHOD For our CSI optimisation studies, MED211FH (Gr3-II) or MED813FH (SHH-3p53mut) tumour-bearing mice were monitored for clinical symptoms of tumour before commencing treatment. Multiple, fractionated CSI dosing schedules using a 5-days-on, 2-days-off treatment cycle were trialled based on our lab’s previously established CSI protocols before a schedule of 10 x 0.5 Gy fractions (5.0 Gy total) proved optimal. This schedule was then used in both PDOX models plus an additional SHH-3p53mut model (MED314mCLFH), either alone or in combination with concurrent prexasertib or ceralasertib. RESULTS MED211FH and MED813FH mice treated with 5.0 Gy CSI alone resulted in significantly improved median survival compared to control mice (p<0.0001 and p<0.005 respectively) While 5.0 Gy CSI plus concurrent prexasertib or ceralasertib showed significantly improved survival when compared to CSI alone for each model used. MED211FH: p<0.01 and p<0.001 respectively, MED813FH: p<0.05 and p<0.01 respectively and MED314mCLFH: p<0.05 for both DDRi combination groups. CONCLUSION Remarkably, concurrent use of prexasertib or ceralasertib with CSI significantly improved survival in fatal Gr3-II and SHH-3p53mut models. With these results, we provide robust preclinical evidence for radiosensitising use of DDRi in paediatric high-risk MB and recommend swift clinical translation.

MDB-53. SALVAGE CRANIOSPINAL IRRADIATION FOR RELAPSED WNT-PATHWAY MEDULLOBLASTOMA PATIENTS TREATED WITH UPFRONT FOCAL CONFORMAL RADIOTHERAPY AND CHEMOTHERAPY AT INITIAL DIAGNOSIS: LONG-TERM AND MATURE OUTCOMES FROM A PROSPECTIVE DE-INTENSIFICATION STUDY

Abstract BACKGROUND Excellent long-term outcomes (5-year overall survival >90%) in WNT-pathway medulloblastoma (MB) patients have prompted systematic attempts at de-intensification of therapy to mitigate treatment-related toxicities. Mature outcomes of a prospective study (FOR-WNT) treating low-risk WNT-MB children with focal conformal radiotherapy (RT) and adjuvant systemic chemotherapy omitting upfront craniospinal irradiation (CSI) are being reported herein. METHODS Seven children with rigorously-defined low-risk WNT-MB were treated with post-operative focal conformal RT to the index tumor-bed (54Gy/30 fractions) followed by 6-cycles of adjuvant systemic chemotherapy after written informed consent/assent. Survival outcomes were computed using Kaplan-Meier method and reported as point estimate with 95% confidence intervals (CI). The trial was terminated prematurely due to unacceptably high risk of neuraxial failures. RESULTS One child succumbed to chemotherapy-related toxicity while 3 of 7 children were detected with leptomeningeal relapse on surveillance neuro-imaging within 2-years of index diagnosis. Full-dose salvage CSI (35Gy/21 fractions) at relapse followed by further chemotherapy led to excellent clinico-radiological response in all 3 children; however, one child developed further local recurrence and leptomeningeal metastases and succumbed to recurrent/progressive disease despite further aggressive salvage therapy. Five of 7 children including the other two treated with salvage CSI and systemic chemotherapy are alive and continue to remain in remission on long-term follow-up. At a median follow up of 61 months, the 5-year Kaplan-Meier estimates of event-free survival, relapse-free survival, and overall survival were 42.9% (95%CI: 18.2-100%), 50.0% (95%CI: 22.5-100%), and 71.4% (44.7-100%) respectively. CONCLUSIONS Omission of upfront CSI in low-risk WNT-MB is associated with unacceptably high-risk of neuraxial failure. Although full-dose salvage CSI achieves excellent response, remissions are not durable resulting in sub-optimal long-term overall survival. Judicious de-escalation with upfront low-dose CSI (18Gy) is currently being tested in a successor study (FOR-WNT 2).

MDB-62. SUBGROUP AND SUBTYPE-SPECIFIC OUTCOMES IN METASTATIC INFANT MEDULLOBLASTOMA

Abstract BACKGROUND Metastatic infant medulloblastoma (MB) is a major challenge given the devastating long-term effects of craniospinal irradiation to the developing brain. However large cohorts of metastatic infant medulloblastoma have not been analysed to date. METHODS A multicentre molecularly informed international cohort of children <6 years old with metastatic medulloblastoma was assembled and analysed. RESULTS Eighty-four patients with a median age of 2.95 (0.66-6.58) years were identified. 48.8% were Group3-MB, 23.8% Group4-MB and 27.4% SHH-MB. 78% of Group3-MB were subtype II (40.4%) and IV (38.1%), 50% of Group 4 were subtype VII, and within SHH, SHHß and SHHγ were 52.2% and 43.5% respectively. 28.5% of all Group3-MB harboured MYC amplification. A multivariable analysis incorporating subgroup, MYC amplification and upfront craniospinal irradiation revealed that survival was superior for SHH patients compared to Group 3 irrespective of MYC status (Group3-veMYCamp: HR 3.05 95%-CI 1.05-8.87 p=0.04, Group3+veMYCamp HR 7.87 95%-CI 2.49-24.87 p=0.00044). Group 4 had a trend to a poor outcome (HR 2.63 95%-CI 0.82-8.39 p=0.1). SHH subjects had a superior outcome with 5-year PFS of 0.657 (95%-CI 0.47-0.92), without a significant difference between SHHß and SHHγ. High-dose chemotherapy regimens portended to a superior outcome for both subtypes of SHH-MB. 5-year PFS for non-radiated SHH-MB was 61% (95%-CI 40.8-91.4), compared to non-radiated Group3-MB (18.5%; 95%-CI 5.4-62.8) and Group4-MB (20%; 95%-CI 3.4-100). SHH-MB patients had more local than metastatic relapses compared to non-SHH-MB (p=0.01). CONCLUSIONS In the largest study dedicated to metastatic infant medulloblastoma assembled to date, outcomes for infants with Group 3- and 4-MB with metastatic disease treated with radiation sparing approaches is dismal despite intensified regimens. MYC amplification portends to a near uniformly fatal prognosis with current treatment irrespective of upfront radiotherapy. Novel radiation sparing approaches are urgently needed for this group. Infants with both metastatic SHHß and SHHγ benefit from intensified therapy.

MDB-15. INHIBITING THE IRON-SULFUR CLUSTER TRANSPORTER ABCB7 CAN POTENTIATE CISPLATIN RESPONSE IN PEDIATRIC GROUP 3 MEDULLOBLASTOMAS BY TRIGGERING FERROPTOSIS

Abstract BACKGROUND Medulloblastoma (MB) is the most common malignant pediatric brain tumor, with group 3 tumors (G3MB) being the most aggressive. Uniquely, their aggressiveness is inversely proportional to ferroptosis activation. Intracellularly, these tumors activate iron-sulfur (Fe-S) cluster export and upregulate the expression of glutathione peroxidase 4 (GPX4), the central regulator of ferroptosis. Our main objective was to investigate whether ferroptosis evasion was a targetable driver of aggressiveness in G3MBs that can be inhibited to potentiate classical chemotherapy. We hypothesized that inhibiting Fe-S cluster export would trigger ferroptosis and potentiate cisplatin cytotoxicity in G3MBs. METHODS We repressed ABCB7 expression (si-/sh-/KO) in classic G3MB cell lines (HDMB03 and D425) and studied its impact on cancer cell growth, ferroptosis activation, mitochondrial function, and cisplatin cytotoxicity. We further investigated the impact of artesunate, an FDA-approved anti-malarial agent capable of inhibiting ABCB7, on cisplatin action and tumor burden. To rescue cells from iron overload and ferroptosis, we used deferoxamine and ferrostatin-1, respectively. RESULTS Silencing ABCB7 resulted in cytoplasmic and mitochondrial iron overload, oxidative stress, and lipid ROS, culminating in ferroptosis and growth retardation. Seahorse studies revealed a switch from oxidative phosphorylation to glycolysis, and TMRM staining revealed loss of mitochondrial membrane potential in ABCB7-silenced cells. Notably, silencing ABCB7 abrogated GPX4 expression and reduced GSH levels. Moreover, ABCB7 silencing reduced the IC50 of cisplatin 2-fold, and ABCB7-silenced cells treated with cisplatin experienced a heightened index of ferroptosis. In vivo, silencing ABCB7 resulted in smaller tumors, dramatically prolonged survival, and potentiated cisplatin action. Artesunate recapitulated ABCB7 silencing by potentiating cisplatin action, reducing tumor burden, and prolonging survival in vivo. CONCLUSION The current study illustrates how targeting mitochondrial Fe-S cluster transport can trigger ferroptosis and potentiate cisplatin in G3MBs. Our study also provides evidence for using an FDA-approved drug with the same mechanism of action to treat G3MB tumors.