Medulloblastoma Models Research Articles

Development of an orthotopic medulloblastoma zebrafish model for rapid drug testing.

Medulloblastoma (MB) is one of the most common malignant brain tumors in children. Current preclinical in vivo model systems for MB have increased our understanding of molecular mechanisms regulating MB development. However, they may not be suitable for large-scale studies. The aim of this study was to investigate if a zebrafish-based xenograft model can recapitulate MB growth and enable rapid drug testing. Nine different MB cell lines or patient-derived cells were transplanted into blastula-stage zebrafish embryos. Tumor development and migration were then monitored using live imaging. RNA sequencing was performed to investigate transcriptome changes after conditioning cells in neural stem cell-like medium. Furthermore, drug treatments were tested in a 96-well format. We demonstrate here that transplantation of MB cells into the blastula stage of zebrafish embryos leads to orthotopic tumor growth that can be observed within 24 hours after transplantation. Importantly, the homing of transplanted cells to the hindbrain region and the aggressiveness of tumor growth are enhanced by pre-culturing cells in a neural stem cell-like medium. The change in culture conditions rewires the transcriptome towards a more migratory and neuronal phenotype, including the expression of guidance molecules SEMA3A and EFNB1, both of which correlate with lower overall survival in MB patients. Furthermore, we highlight that the orthotopic zebrafish MB model has the potential to be used for rapid drug testing. Blastula-stage zebrafish MB xenografts present an alternative to current MB mouse xenograft models, enabling quick evaluation of tumor cell growth, neurotropism, and drug efficacy.

PP2A activation overcomes leptomeningeal dissemination in group 3 medulloblastoma

Leptomeningeal dissemination (LMD) is the primary cause of treatment failure in children with Group 3 medulloblastoma (MB). Building on our previous work on protein phosphatase 2A (PP2A) activation in MB, here we present pre-clinical and molecular data on the effects of two novel classes of PP2A activators on disease processes of LMD in Group 3 MB. The PP2A activators employed in this study are ATUX-6156 and ATUX-6954 (diarylmethylcycloamine sulfonylureas), and ATUX-1215 and ATUX-5800 (diarylmethyl-4-aminotetrahydropyran-sulfonamides). Treatment with these compounds led to suppression of the endogenous PP2A inhibitor, cancerous inhibitor of PP2A (CIP2A), enhanced phosphatase activity (10-60%), and reduced MB viability, migration, and invasion, prerequisites for MB cells to access the cerebrospinal fluid, affecting the initiation stage of LMD. PP2A activator treatment of MB cells led to apoptosis mediated via caspase 9/PARP signaling due to decreased phosphorylation of Bad, impeding the dispersal stage of LMD. Cell proliferation and LMD-driving cellular traits and molecules pertinent to the third stage, colonization, were also affected. Treatment with ATUX-1215 or ATUX-5800 prevented LMD in an intraventricular murine model of MB, possibly mediated by disruption of the CCL2-CCR2 axis by altered NF-kB phosphorylation via disrupted AKT signaling. The present investigation offers proof-of-principle data for PP2A-based reactivation therapy for Group 3 MB and provides the first indications that PP2A reactivation may challenge the current paradigm in targeting the 3-stage process of MB LMD. Further investigations of PP2A activators are warranted as these compounds may prove beneficial as therapeutics for MB.

A novel indole derivative, 2-{3-[1-(benzylsulfonyl)piperidin-4-yl]-2-methyl-1H-indol-1-yl}-1-(pyrrolidin-1-yl)ethenone, suppresses hedgehog signaling and drug-resistant tumor growth.

The Hedgehog (Hh) signaling pathway plays important roles in various physiological functions. Several malignancies, such as basal cell carcinoma (BCC) and medulloblastoma (MB), have been linked to the aberrant activation of Hh signaling. Although therapeutic drugs have been developed to inhibit Hh pathway-dependent cancer growth, drug resistance remains a major obstacle in cancer treatment. Here, we show that the newly identified, 2-{3-[1-(benzylsulfonyl)-1,2,3,6-tetrahydropyridin-4-yl]-2-methyl-1H-indol-1-yl}-1-(pyrrolidin-1-yl)ethenone analog (LKD1214) exhibits comparable potency to vismodegib in suppressing the Hh pathway activation. LKD1214 represses Smoothened (SMO) activity by blocking its ciliary translocation. Interestingly, we also identified that it has a distinctive binding interface with SMO compared with other SMO-regulating chemicals. Notably, it maintains an inhibitory activity against the SmoD477H mutant, as observed in a patient with vismodegib-resistant BCC. Furthermore, LKD1214 inhibits tumor growth in the mouse model of MB. Collectively, these findings suggest that LKD1214 has the therapeutic potential to overcome drug-resistance in Hh-dependent cancers.

A simple and scalable zebrafish model of Sonic hedgehog medulloblastoma

Medulloblastoma (MB) is the most common malignant brain tumor in children and is stratified into three major subgroups. The Sonic hedgehog (SHH) subgroup represents ∼30% of all MB cases and has significant survival disparity depending upon TP53 status. Here, we describe a zebrafish model of SHH MB using CRISPR to create mutant ptch1, the primary genetic driver of human SHH MB. In these animals, tumors rapidly arise in the cerebellum and resemble human SHH MB by histology and comparative onco-genomics. Similar to human patients, MB tumors with loss of both ptch1 and tp53 have aggressive tumor histology and significantly worse survival outcomes. The simplicity and scalability of the ptch1-crispant MB model makes it highly amenable to CRISPR-based genome-editing screens to identify genes required for SHH MB tumor formation invivo, and here we identify the gene encoding Grk3 kinase as one such target.

Toxoplasma gondii infection supports the infiltration of T cells into brain tumors

Few T cells infiltrate into primary brain tumors, fundamentally hampering the effectiveness of immunotherapy. We hypothesized that Toxoplasma gondii, a microorganism that naturally elicits a Th1 response in the brain, can promote T cell infiltration into brain tumors despite their immune suppressive microenvironment. Using a mouse genetic model for medulloblastoma, we found that T. gondii infection induced the infiltration of activatable T cells into the tumor mass and led to myeloid cell reprogramming toward a T cell-supportive state, without causing severe health issues in mice. The study provides a concrete foundation for future studies to take advantage of the immune modulatory capacity of T. gondii to facilitate brain tumor immunotherapy.

Identification of a prognosis-related phagocytosis regulator gene signature in medulloblastoma

ObjectivesThe aims of this study were to screen for phagocytosis regulator-related genes in tissue samples from children with medulloblastoma (MB) and to construct a prognostic model based on those genes. MethodsDifferentially expressed genes between the MB and control groups were identified using the GSE50161 dataset from the Gene Expression Omnibus database. Prognosis-related phagocytosis regulator genes were selected from the GSE85217 dataset. Intersecting genes of the two datasets (differentially expressed prognosis-related phagocytosis regulator genes) were submitted to unsupervised cluster analysis to identify disease subtypes, after which the association between the subtypes and the immune microenvironment was analyzed. A prognostic risk score model was constructed, and functional, immune-related, and drug sensitivity analyses were performed. ResultsIn total, 23 differentially expressed prognosis-related phagocytosis regulator genes were identified, from which two disease subtypes (clusters 1 and 2) were classified. The prognoses of the patients in cluster 2 were significantly worse than those of the patients in cluster 1. The immune microenvironment differed significantly between the two subtypes. Finally, 10 genes (FAM81A, EZR, NDUFB9, RCOR1, FOXO4, NHLRC2, KIF23, PTPN6, SMAGP, and MED13) were selected to establish the prognostic risk score model. The prognosis in the low-risk group was better than that in the high-risk group. The model genes NDUFB9 and PTPN6 were positively correlated with M2 macrophages. ConclusionTen key phagocytosis regulator genes were screened to construct a prognostic model for MB. These genes may serve as key biomarkers for predicting the prognosis of patients with this type of brain cancer.

Synergy of retinoic acid and BH3 mimetics in MYC(N)-driven embryonal nervous system tumours

BackgroundCertain paediatric nervous system malignancies have dismal prognoses. Retinoic acid (RA) is used in neuroblastoma treatment, and preclinical data indicate potential benefit in selected paediatric brain tumour entities. However, limited single-agent efficacy necessitates combination treatment approaches.MethodsWe performed drug sensitivity profiling of 76 clinically relevant drugs in combination with RA in 16 models (including patient-derived tumouroids) of the most common paediatric nervous system tumours. Drug responses were assessed by viability assays, high-content imaging, and apoptosis assays and RA relevant pathways by RNAseq from treated models and patient samples obtained through the precision oncology programme INFORM (n = 2288). Immunoprecipitation detected BCL-2 family interactions, and zebrafish embryo xenografts were used for in vivo efficacy testing.ResultsGroup 3 medulloblastoma (MBG3) and neuroblastoma models were highly sensitive to RA treatment. RA induced differentiation and regulated apoptotic genes. RNAseq analysis revealed high expression of BCL2L1 in MBG3 and BCL2 in neuroblastomas. Co-treatments with RA and BCL-2/XL inhibitor navitoclax synergistically decreased viability at clinically achievable concentrations. The combination of RA with navitoclax disrupted the binding of BIM to BCL-XL in MBG3 and to BCL-2 in neuroblastoma, inducing apoptosis in vitro and in vivo.ConclusionsRA treatment primes MBG3 and NB cells for apoptosis, triggered by navitoclax cotreatment.

HGG-12. A PHASE IB/II STUDY ON ASIDNA™ IN ASSOCIATION WITH RE-IRRADIATION IN CHILDREN, ADOLESCENTS AND YOUNG ADULTS WITH RELAPSING HIGH-GRADE GLIOMA

Abstract BACKGROUND AsiDNA is a synthetic cholesterol-oligodeoxyribonucleotide conjugate, inducing false DNA damage signaling, which prevents DNA repair in tumor cells. AsiDNA therefore increases the vulnerability of tumor cells to irradiation without increasing toxicity in healthy tissues. Preclinical data showed encouraging results of AsiDNA combined to irradiation in glioma and medulloblastoma models. We aimed to determine the safety and efficacy of AsiDNA, a DNA-repair inhibitor, in children, adolescents and young adults with recurrent High-grade Glioma (HGG) eligible for re-irradiation. METHODS AsiDNA was administered intravenously using a 3 consecutive days loading dose, followed by a weekly dose (350 mg/m2/dose or 600 mg for patients ≥ 50kg) for 11 weeks. Focal radiotherapy, using 1.8 Gy daily fraction, 5 days a week, was administered in combination during the first weeks: 18 Gy for brainstem diffuse midline glioma (DMG) and 36 Gy for supra-tentorial HGG (DMG or non-DMG). The first part of the study aimed to assess the safety and to confirm the recommended phase-II dose based on dose limiting toxicities (DLT) during the first 8 weeks. A second part was planned to assess activity/efficacy of the combination based on the progression-free survival (PFS) rate at 3 months. AsiDNA pharmacodynamics (PD) was studied in peripheral blood mononuclear cells (PMBC). RESULTS Eight (8) patients were enrolled: median age 14 y (6.2-24.5), 5 DMG. No DLT was observed. Median FU was 9.1 months, disease progression was observed in 8 patients; 6 died because of disease progression. Median PFS was 3.9 months. The PD results showed no significant target engagement in PBMCs. CONCLUSIONS Considering the low PFS as well as lack of efficacy on PD markers, the DSMB recommended stopping the study.

MDB-107. MINNELIDE, A PROMISING COMPOUND FOR THE TREATMENT OF HIGH-RISK MEDULLOBLASTOMA

Abstract Despite the overall good outcome of children with medulloblastoma (MB), a subset faces the challenge of highly aggressive metastatic tumors. The poor prognosis for these patients underscores the urgent need for advancing therapeutic approaches. Minnelide, an analog of the natural product triptolide, exhibits potent antitumor activity in preclinical and early clinical settings. Despite its efficacy and tolerable toxicity, Minnelide has not been assessed against MB. Using a bioinformatic dataset integrating cellular drug response and gene expression, we predicted sensitivity of Group 3 (G3) MB, characterized by a poor five-year survival, to triptolide/Minnelide. Subsequent experiments confirmed the ability of both triptolide and Minnelide to reduce the viability of G3 MB cells ex vivo. Transcriptomic analyses identified MYC signaling, a crucial driver of G3 MB, as a downstream target of these drugs. We validated the MYC dependency and demonstrated that triptolide affects both MYC transcription and protein stability. Notably, Minnelide acted in vivo on MYC, leading to reduced tumor growth and metastatic spread, resulting in improved survival in mouse and patient-derived G3 MB models. Furthermore, Minnelide enhanced the efficacy of adjuvant chemotherapy, suggesting its potential repurposing for treating MYC-driven G3 MB patients.

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.

MDB-101. TARGETING CTPS1 AND NUCLEOTIDE BIOSYNTHESIS PATHWAYS AS A NOVEL THERAPEUTIC APPROACH IN MYC-AMPLIFIED MEDULLOBLASTOMA

Abstract Although leukemia is the leading pediatric oncology diagnosis, brain tumors are the leading cause of pediatric cancer-related death. Medulloblastoma (MB) is the most common pediatric brain tumor, representing approximately 20% of all diagnoses. MYC-amplified Group 3 medulloblastoma (G3 MB) tumors are the deadliest, with a < 50% 5-year overall survival rate. High risk patients receive large doses of chemo- and radio- therapy (CT/RT) with most survivors experiencing debilitating neurological and cognitive sequelae making the identification of novel targets of the utmost importance. Utilizing whole-genome CRISPR screening, we identified the enzyme CTPS1 as a strong G3 MB-specific vulnerability. Further analysis revealed dependencies on other enzymes in the de novo pyrimidine synthesis pathway, which is corroborated by recent metabolic studies. Targeted knockdown of CTPS1 restricts sphere formation and reduces proliferation of G3 MB tumor lines. Treatment with a specific CTPS1 inhibitor reduced proliferation but did not induce cell death, suggesting a cytostatic mechanism. Previous work has shown that triggering an imbalance in pyrimidine and purine levels leads to replication stress and activation of the ATR/CHK1 pathway. Both genetic and pharmacologic inhibition of CTPS1 leads to CHK phosphorylation, allowing for cell survival despite lack of dNTPs. Previously, the CHK1 inhibitor, prexasertib, demonstrated robust brain accumulation and targeting of G3 MB in pre-clinical models. Dual inhibition of CHK1 and CTPS1 synergizes to significantly reduce cell viability in vitro and tumor growth in vivo. We further demonstrated robust synergy of CHK1 inhibition with either glutamine antagonists or other inhibitors of the de novo pyrimidine synthesis pathway, prolonging in vivo survival in an orthotopic xenograft G3 MB model. The results of this investigation confirm CTPS1 as a novel target in G3 MB, pinpoint a dependency on de novo pyrimidine rather than purine biosynthesis, and identifies a novel rational combination for treatment in MYC-amplified MB patients.

DIPG-89. ENHANCEMENT OF BLOOD-BRAIN BARRIER PENETRATION AND TUMOR TARGETED DRUG DELIVERY FOR DMG USING A P-SELECTIN TARGETED NANOMEDICINE APPROACH

Abstract The blood-brain barrier (BBB) is one of the greatest barriers for the effective treatment of brain tumors, including H3K27-altered diffuse midline glioma (DMG), a near universally fatal childhood brain cancer. The BBB typically requires drugs to be given at maximally tolerated doses that are limited by systemic toxicities, particularly in settings of combination therapy. We have developed a clinically compatible fucoidan nanoparticle (Fi-NP) that homes to P-selectin on tumor vasculature after low-dose radiation (RT) to breach the BBB through an active caveolin-1-dependent mechanism and deliver several classes of targeted therapies. In non-CNS cancer xenograft models and a transgenic mouse model of SHH-driven medulloblastoma with an intact BBB, this approach improved survival while eliminating on-target systemic toxicities. We have now applied this approach to both brainstem and non-brainstem RCAS-TVA mouse models of DMG with an intact BBB. Specifically, we have found that DMG tumor vasculature expresses P-selectin, and that a single low-dose 2 Gy fraction of ionizing radiation further enhances it in a time-dependent manner up to 24 hours post-treatment. Importantly, we have also found that this P-selectin targeted drug delivery approach facilitates DMG tumor localization of Fi-NP encapsulated EZH2 inhibitor tazemetostat (EPZ-6438) as well as larger macromolecules including the PROTAC BET degrader dBET6, two promising targeted therapies for DMG limited by extremely poor BBB-penetration, while sparing drug delivery to non-tumor healthy brain regions. Work to measure PK, biodistribution, survival benefit, and drug target inhibition is ongoing. Our findings will provide the foundation for this P-selectin targeted Fi-NP approach to be evaluated in clinical trials for children with this lethal cancer.

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-110. A NOVEL TRANSGENIC MOUSE MODEL FOR GLI2-AMPLIFIED MEDULLOBLASTOMA

Abstract BACKGROUND Medulloblastoma (MB) is one of the most common malignant childhood brain tumors. The sonic hedgehog (SHH) subgroup represents 30% of MBs and can arise from granule neuron progenitors (GNPs). SHH-MB is a diverse subgroup of tumors which often exhibit loss-of function mutations in PTCH1 or SUFU, activating mutations in the receptor SMO and amplification of the transcription factor GLI2. Of note, SHH-MBs harboring GLI2-amplification and associated with P53 mutations have extremely poor prognosis with a 5-year survival rate less than 30%. These tumors are non-responsive to SMO inhibitors, which are the only targeted treatment currently available for SHH-MB. Therefore, the development of novel effective therapies for this disease is urgent. Although GLI2 amplification is detected in SHH-MBs, it is not yet known if GLI2 can drive oncogenic activity in this disease. METHOD To elucidate the mechanisms underlying tumorigenesis, our group has developed a novel transgenic model of GLI2-driven MB by expressing a constitutively active form of GLI2 (GLI2A) in Atoh1+ cells in the developing mouse cerebellum to form tumors. To accomplish this, we crossed R26;Gli2A mice with Atoh1-Cre. RESULTS The resulting tumors faithfully recapitulate human GLI2-amplified MB at the cellular and molecular levels. Inactivation of GLI2A resulted in tumor regression, indicating that GLI2 is not only critical for tumorigenesis, but also required for tumor maintenance, suggesting that targeting GLI2 itself or its downstream targets may be effective to inhibit growth of GLI2-driven MB. Interestingly, activation of GLI2A expression in the neonatal cerebellum was not sufficient to drive tumorigenesis, indicating that GLI2-driven MB arises from more primitive cells in the developing cerebellum. CONCLUSION Taken together, our studies demonstrate for the first time the role of GLI2 as an oncogenic driver of MB and introduce a novel mouse model which can be used to develop targeted therapies for this disease.

MODL-06. USING HYDROGEL MODELS TO RECAPITULATE THE TUMOUR-IMMUNE MICROENVIRONMENT OF AGGRESSIVE GROUP 4 MEDULLOBLASTOMA TUMOURS

Abstract BACKGROUND Medulloblastoma is the most common malignant childhood brain-tumour, predominantly affecting children between the ages of one and nine. It is divided into four molecular subgroups amongst which, Group 3 (G3) and Group 4 (G4) subgroups have the worst prognosis. Subgroups possess distinct extracellular-matrix (ECM) and tumour immune microenvironments (TIME) directly influencing therapy-response and metastasis. Current models of medulloblastoma fail to precisely recreate the complex, subgroup-specific TIME and do not mimic intricate immune-tumour heterotypic signalling facilitating immune-evasion. Here we use a HyStem-hydrogel which forms a hyaluronan-rich environment mimicking the natural brain architecture. This overcomes several limitations of current MB models, allowing customisation of subgroup matching of ECM components, adjustable ECM-stiffness and co-culturing of immune cells. METHODS HyStem-hydrogels were co-assembled with ECM components (with/without laminin) and G4 medulloblastoma cell line CHLA-01-MED. Hydrogels were imaged every three-days using an EVOS M7000 system and assessed for cell-viability with PrestoBlue every seven days. Co-culture laminin-supplemented HyStem hydrogels were established with M1-like or M2-like resting/activated macrophages and their growth tracked in the same manner. On day twenty-eight, hydrogels were fixed and stained for markers of cellular proliferation (Ki-67), ECM (laminin, vitronectin) and macrophage markers. Media from hydrogel co-cultures was collected and ELISAs permofmed to assess cytokine production. RESULTS Hydrogels with/without laminin both remained viable and stable for 28 days. Laminin-supplemented hydrogels displayed a mixed laminar-nodular phenotype which mirrored the heterogenous nature of G4 patient-tumours. Co-culture of CHLA-01-MED cells with activated-macrophages led to cytokine production and altered growth patterns. CONCLUSION We show that HyStem hydrogels recapitulate biologically relevant behaviour of aggressive G4-MB tumours in vitro and represent a promising model for G4-tumours. Future research will involve inclusion of additional brain relevant cells/ECM components and comparisons to a fully customisable peptide-amphiphile hydrogel.

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-61. STARVING TUMORS OF POLYAMINES: A NOVEL APPROACH IN TREATING AGGRESSIVE PEDIATRIC MEDULLOBLASTOMA

Abstract BACKGROUND Polyamines, essential intracellular polycations, govern fundamental cell processes and drive oncogenesis. Our research unveiled the dependency of pediatric Diffuse Midline Gliomas (DMG) on the polyamine pathway (Khan et al.,2021). Disruption of this pathway with the synthesis inhibitor DFMO, and the transport inhibitor AMXT1501, potently inhibited DMG tumors, yielding unprecedented survival in-vivo. We hypothesized that dual polyamine pathway targeting could similarly suppress growth in other aggressive pediatric brain tumors. METHODS/RESULTS We analyzed the expression of polyamine pathway regulators in high-risk pediatric brain cancers ZERO precision medicine platform and found elevated expression in medulloblastoma. ODC1, a polyamine synthesis driver, was found to have significant correlation in high-grade gliomas with four key functional tumor states—stemness, cell cycle, hypoxia, and angiogenesis. Medulloblastoma cells were sensitive to treatment with DFMO but showed a compensatory increase in polyamine transporter expression. Conversely, treatment with AMXT1501 increased ODC1 expression. Dual targeting of the polyamine pathway potently and synergistically inhibited cell proliferation and clonogenic potential in Group-3 medulloblastoma cells. Flow cytometric analysis of Annexin V-stained cells showed that treatment with DFMO and AMXT1501 in combination led to induction of apoptosis. Critically, dual polyamine inhibition potently sensitized medulloblastoma cells to the topoisomerase-I inhibitor, SN-38, leading to complete eradication of medulloblastoma cell proliferation and colony formation at nanomolar chemotherapeutic concentrations, with the effect most pronounced in Group-3 medulloblastoma. In triple combination-treated cells, over-representation analysis revealed heightened mitotic DNA damage, macroautophagy, and apoptosis, coupled with diminished neuron/synapse development, glycosylation, and cellular signaling pathways. We are currently testing this treatment strategy in aggressive orthotopic medulloblastoma models. CONCLUSION These findings suggest targeting the polyamine pathway as a promising strategy to sensitize aggressive pediatric medulloblastoma tumors to chemotherapy. DFMO/AMXT1501 is in Phase I evaluation for adults (NCT05500508) and advancing to a Phase I/II trial for children through the CONNECT consortium.

High-throughput neural stem cell-based drug screening identifies S6K1 inhibition as a selective vulnerability in sonic hedgehog-medulloblastoma.

Medulloblastoma (MB) is one of the most common malignant brain tumors in children. Current treatments have increased overall survival but can lead to devastating side effects and late complications in survivors, emphasizing the need for new, improved targeted therapies that specifically eliminate tumor cells while sparing the normally developing brain. Here, we used a sonic hedgehog (SHH)-MB model based on a patient-derived neuroepithelial stem cell system for an unbiased high-throughput screen with a library of 172 compounds with known targets. Compounds were evaluated in both healthy neural stem cells (NSCs) and tumor cells derived from the same patient. Based on the difference of cell viability and drug sensitivity score between normal cells and tumor cells, hit compounds were selected and further validated in vitro and in vivo. We identified PF4708671 (S6K1 inhibitor) as a potential agent that selectively targets SHH-driven MB tumor cells while sparing NSCs and differentiated neurons. Subsequent validation studies confirmed that PF4708671 inhibited the growth of SHH-MB tumor cells both in vitro and in vivo, and that knockdown of S6K1 resulted in reduced tumor formation. Overall, our results suggest that inhibition of S6K1 specifically affects tumor growth, whereas it has less effect on non-tumor cells. Our data also show that the NES cell platform can be used to identify potentially effective new therapies and targets for SHH-MB.

PUMC-MB1 is a novel group 3 medulloblastoma preclinical model, sensitive to PI3K/mTOR dual inhibitor.

Medulloblastoma (MB), a common and heterogeneous posterior fossa tumor in pediatric patients, presents diverse prognostic outcomes. To advance our understanding of MB's intricate biology, the development of novel patient tumor-derived culture MB models with necessary data is still an essential requirement. We continuously passaged PUMC-MB1 in vitro in order to establish a continuous cell line. We examined the in vitro growth using Cell Counting Kit-8 (CCK-8) and in vivo growth with subcutaneous and intracranial xenograft models. The xenografts were investigated histopathologically with Hematoxylin and Eosin (HE) staining and immunohistochemistry (IHC). Concurrently, we explored its molecular features using Whole Genome Sequencing (WGS), targeted sequencing, and RNA sequecing.Guided by bioinformatics analysis, we validated PUMC-MB1's drug sensitivity in vitro and in vivo. PUMC-MB1, derived from a high-risk MB patient, displayed a population doubling time (PDT) of 48.18h and achieved 100% tumor growth in SCID mice within 20 days. HE and Immunohistochemical examination of the original tumor and xenografts confirmed the classification of PUMC-MB1 as a classic MB. Genomic analysis via WGS revealed concurrent MYC and OTX2 amplifications. The RNA-seq data classified it within the Group 3MB subgroup, while according to the WHO classification, it fell under the Non-WNT/Non-SHH MB. Comparative analysis with D283 and D341med identified 4065 differentially expressed genes, with notable enrichment in the PI3K-AKT pathway. Cisplatin, 4-hydroperoxy cyclophosphamide/cyclophosphamide, vincristine, and dactolisib (a selective PI3K/mTOR dual inhibitor) significantly inhibited PUMC-MB1 proliferation in vitro and in vivo. PUMC-MB1, a novel Group 3 (Non-WNT/Non-SHH) MB cell line, is comprehensively characterized for its growth, pathology, and molecular characteristics. Notably, dactolisib demonstrated potent anti-proliferative effects with minimal toxicity, promising a potential therapeutic avenue. PUMC-MB1 could serve as a valuable tool for unraveling MB mechanisms and innovative treatment strategies.

Abstract SY05-03: Circular extrachromosomal DNA promotes tumor heterogeneity and enhancer rewiring

Abstract Circular extrachromosomal DNA (ecDNA) in patient tumors is an important driver of oncogenic gene expression, evolution of drug resistance and poor patient outcomes. Applying computational methods for the detection and reconstruction of ecDNA across a retrospective cohort of 481 medulloblastoma tumors from 465 patients, we identify circular ecDNA in 82 patients (18%). Patients with ecDNA-positive medulloblastoma were more than twice as likely to relapse and three times as likely to die within 5 years of diagnosis. Multimodal sequencing, imaging and CRISPR inhibition experiments in medulloblastoma models reveal intratumoral heterogeneity of ecDNA copy number per cell and frequent ‘enhancer rewiring’ events on ecDNA. This study reveals the frequency and diversity of ecDNA in medulloblastoma, stratified into molecular subgroups, and suggests copy number heterogeneity and enhancer rewiring as oncogenic features of ecDNA. Citation Format: Lukas Chavez. Circular extrachromosomal DNA promotes tumor heterogeneity and enhancer rewiring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY05-03.