Medulloblastoma Tumors Research Articles

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.

MODL-10. COMPUTATIONAL DRUG SENSITIVITY PREDICTS TAILORED THERAPY FOR MEDULLOBLASTOMA SUBGROUPS

Abstract BACKGROUND Medulloblastoma is the most common malignant pediatric brain tumor. Tumors are typically characterized as SHH α- δ, WNT α-β, Group 3α- γ, or Group 4 α- γ. Current standard-of-care includes surgery, radiation, and chemotherapy; however, treatment response and prognosis vary widely between subgroups. Despite extensive characterization of medulloblastoma tumors into four molecular subgroups, few subgroup specific therapies have advanced to the clinic and a universal approach is unlikely to be effective due to the considerable intertumor heterogeneity of medulloblastoma. Hence, there is an urgent demand for innovative therapeutic strategies. METHODS To address heterogeneity between patients, we have developed a novel platform called DrugSeq, which predicts drug sensitivities in patients and is able to stratify drug response based on subgroup. We first calculated disease signatures for each patient by normalizing patient gene expression to median gene expression across the Cavalli dataset. Using previously developed transcriptional consensus signatures (TCSs), which contain genes that are consistently dysregulated following drug treatment, we then calculated the discordance between each patient disease signature and drug TCS. In doing so, we were able to identify drugs to target specific molecular subtypes based on Cavalli molecular classifications. RESULTS As a proof of concept we identified vismodegib and curcumin as selective for SHH-MB. Furthermore, we correlated predicted drug response with target protein expression. For example, we found that predicted sensitivity to vismodegib correlates closely with GLI1 protein expression. CONCLUSIONS Collectively, we show that DrugSeq may identify novel therapies and facilitate patient stratification in clinical trials, leading to more successful targeted medulloblastoma therapies.

QOL-39. FATIGUE AS A RISK FACTOR FOR REDUCED SOCIAL CONNECTEDNESS IN PEDIATRIC BRAIN TUMOR SURVIVORS

Abstract BACKGROUND Pediatric brain tumor survivors (PBTS) experience reduced social connectedness, including fewer friendships, less social acceptance, and greater isolation. Identifying modifiable factors contributing to social issues would yield treatment opportunities. Fatigue is common among PBTS and may relate to social challenges. This study evaluated fatigue among PBTS and its associations with various indices of social connectedness. METHODS Survivors (N = 34) of three types of brain tumors—medulloblastoma (N = 11), pilocytic astrocytoma (N = 15), and craniopharyngioma (N = 8)—completed self-reported measures of fatigue and social connectedness. Laboratory studies assessed thyroid function and growth hormone status. Caregivers completed measures of social functioning. Descriptive statistics summarized fatigue and endorsement of fatigue items. ANOVAs, t-tests, and Pearson correlations evaluated fatigue according to various medical factors, and Pearson correlations evaluated associations between self-reported fatigue and indices of social connectedness. RESULTS Over 20% of survivors reported elevated fatigue, with 53% and 35% indicating fatigue interfering with attention and socialization, respectively. We did not detect associations between self-reported fatigue and diagnosis, current age or age at diagnosis, time since end of treatment, or history of chemotherapy or cranial radiation therapy. We did not detect an association between BMI Z-score and collected hormones. Mean T scores on the self-reported peer relationships measure were below the reference mean (M = 45.8, SD = 11.9), with 22% of survivors endorsing concerns. Higher self-reported fatigue was significantly correlated with poorer self-reported peer relationships (r = -.62, p < 0.001) and more parent-reported social impairments (r =.61, p < 0.001). CONCLUSIONS Fatigue is common among PBTS and may interfere with peer relationships. Clinicians should screen for fatigue as a risk factor for reduced social connectedness. Further research is needed to identify predictors of fatigue and social difficulties and determine temporal associations between fatigue and social connectedness.

REST-dependent downregulation of von Hippel-Lindau tumor suppressor promotes autophagy in SHH-medulloblastoma

The RE1 silencing transcription factor (REST) is a driver of sonic hedgehog (SHH) medulloblastoma genesis. Our previous studies showed that REST enhances cell proliferation, metastasis and vascular growth and blocks neuronal differentiation to drive progression of SHH medulloblastoma tumors. Here, we demonstrate that REST promotes autophagy, a pathway that is found to be significantly enriched in human medulloblastoma tumors relative to normal cerebella. In SHH medulloblastoma tumor xenografts, REST elevation is strongly correlated with increased expression of the hypoxia-inducible factor 1-alpha (HIF1α)—a positive regulator of autophagy, and with reduced expression of the von Hippel-Lindau (VHL) tumor suppressor protein – a component of an E3 ligase complex that ubiquitinates HIF1α. Human SHH-medulloblastoma tumors with higher REST expression exhibit nuclear localization of HIF1α, in contrast to its cytoplasmic localization in low-REST tumors. In vitro, REST knockdown promotes an increase in VHL levels and a decrease in cytoplasmic HIF1α protein levels, and autophagy flux. In contrast, REST elevation causes a decline in VHL levels, as well as its interaction with HIF1α, resulting in a reduction in HIF1α ubiquitination and an increase in autophagy flux. These data suggest that REST elevation promotes autophagy in SHH medulloblastoma cells by modulating HIF1α ubiquitination and stability in a VHL-dependent manner. Thus, our study is one of the first to connect VHL to REST-dependent control of autophagy in a subset of medulloblastomas.

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.

The OTX2 Gene Induces Tumor Growth and Triggers Leptomeningeal Metastasis by Regulating the mTORC2 Signaling Pathway in Group 3 Medulloblastomas.

Medulloblastoma (MB) encompasses diverse subgroups, and leptomeningeal disease/metastasis (LMD) plays a substantial role in associated fatalities. Despite extensive exploration of canonical genes in MB, the molecular mechanisms underlying LMD and the involvement of the orthodenticle homeobox 2 (OTX2) gene, a key driver in aggressive MB Group 3, remain insufficiently understood. Recognizing OTX2's pivotal role, we investigated its potential as a catalyst for aggressive cellular behaviors, including migration, invasion, and metastasis. OTX2 overexpression heightened cell growth, motility, and polarization in Group 3 MB cells. Orthotopic implantation of OTX2-overexpressing cells in mice led to reduced median survival, accompanied by the development of spinal cord and brain metastases. Mechanistically, OTX2 acted as a transcriptional activator of the Mechanistic Target of Rapamycin (mTOR) gene's promoter and the mTORC2 signaling pathway, correlating with upregulated downstream genes that orchestrate cell motility and migration. Knockdown of mTOR mRNA mitigated OTX2-mediated enhancements in cell motility and polarization. Analysis of human MB tumor samples (N = 952) revealed a positive correlation between OTX2 and mTOR mRNA expression, emphasizing the clinical significance of OTX2's role in the mTORC2 pathway. Our results reveal that OTX2 governs the mTORC2 signaling pathway, instigating LMD in Group 3 MBs and offering insights into potential therapeutic avenues through mTORC2 inhibition.

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.

Accuracy of diffusion-weighted imaging in differentiation between medulloblastoma and ependymoma tumors

Accuracy of diffusion-weighted imaging in differentiation between medulloblastoma and ependymoma tumors

Targeting Group 3 Medulloblastoma by the Anti-PRUNE-1 and Anti-LSD1/KDM1A Epigenetic Molecules.

Medulloblastoma (MB) is a highly malignant childhood brain tumor. Group 3 MB (Gr3 MB) is considered to have the most metastatic potential, and tailored therapies for Gr3 MB are currently lacking. Gr3 MB is driven by PRUNE-1 amplification or overexpression. In this paper, we found that PRUNE-1 was transcriptionally regulated by lysine demethylase LSD1/KDM1A. This study aimed to investigate the therapeutic potential of inhibiting both PRUNE-1 and LSD1/KDM1A with the selective inhibitors AA7.1 and SP-2577, respectively. We found that the pharmacological inhibition had a substantial efficacy on targeting the metastatic axis driven by PRUNE-1 (PRUNE-1-OTX2-TGFβ-PTEN) in Gr3 MB. Using RNA seq transcriptomic feature data in Gr3 MB primary cells, we provide evidence that the combination of AA7.1 and SP-2577 positively affects neuronal commitment, confirmed by glial fibrillary acidic protein (GFAP)-positive differentiation and the inhibition of the cytotoxic components of the tumor microenvironment and the epithelial-mesenchymal transition (EMT) by the down-regulation of N-Cadherin protein expression. We also identified an impairing action on the mitochondrial metabolism and, consequently, oxidative phosphorylation, thus depriving tumors cells of an important source of energy. Furthermore, by overlapping the genomic mutational signatures through WES sequence analyses with RNA seq transcriptomic feature data, we propose in this paper that the combination of these two small molecules can be used in a second-line treatment in advanced therapeutics against Gr3 MB. Our study demonstrates that the usage of PRUNE-1 and LSD1/KDM1A inhibitors in combination represents a novel therapeutic approach for these highly aggressive metastatic MB tumors.

GD2-Targeting CAR T-cell Therapy for Patients with GD2+ Medulloblastoma.

Medulloblastoma (MB), the most common childhood malignant brain tumor, has a poor prognosis in about 30% of patients. The current standard of care, which includes surgery, radiation, and chemotherapy, is often responsible for cognitive, neurologic, and endocrine side effects. We investigated whether chimeric antigen receptor (CAR) T cells directed toward the disialoganglioside GD2 can represent a potentially more effective treatment with reduced long-term side effects. GD2 expression was evaluated on primary tumor biopsies of MB children by flow cytometry. GD2 expression in MB cells was also evaluated in response to an EZH2 inhibitor (tazemetostat). In in vitro and in vivo models, GD2+ MB cells were targeted by a CAR-GD2.CD28.4-1BBζ (CAR.GD2)-T construct, including the suicide gene inducible caspase-9. GD2 was expressed in 82.68% of MB tumors. The SHH and G3-G4 subtypes expressed the highest levels of GD2, whereas the WNT subtype expressed the lowest. In in vitro coculture assays, CAR.GD2 T cells were able to kill GD2+ MB cells. Pretreatment with tazemetostat upregulated GD2 expression, sensitizing GD2dimMB cells to CAR.GD2 T cells cytotoxic activity. In orthotopic mouse models of MB, intravenously injected CAR.GD2 T cells significantly controlled tumor growth, prolonging the overall survival of treated mice. Moreover, the dimerizing drug AP1903 was able to cross the murine blood-brain barrier and to eliminate both blood-circulating and tumor-infiltrating CAR.GD2 T cells. Our experimental data indicate the potential efficacy of CAR.GD2 T-cell therapy. A phase I/II clinical trial is ongoing in our center (NCT05298995) to evaluate the safety and therapeutic efficacy of CAR.GD2 therapy in high-risk MB patients.

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 5107: 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 is the most common pediatric brain tumor, representing approximately 20% of all diagnoses. Of all four defined subgroups, 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 from the Pediatric Cancer Dependency Map, we identified the enzyme CTPS1 as a strong G3 MB-specific vulnerability. Recent studies corroborate our findings and suggest a dependence on the de novo pyrimidine biosynthesis pathway in MB. Targeted knockdown of CTPS1 restricts sphere formation and reduces proliferation of G3 MB tumor lines. We confirmed this genetic approach using a specific CTPS1 inhibitor, which also reduced proliferation but did not induce cell death, suggesting a cytostatic mechanism. Strikingly, we demonstrate that G3 MB is only dependent upon de novo pyrimidine synthesis and not de novo purine synthesis, despite regulation of both pathways by oncogenic MYC signaling. 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. Citation Format: Matthew Hathaway, Hawa Jagana, Michael Meechan, John Hemenway, Kate E. Gadek, Aya Miyaki, Isabella Terrones, Barbara Slusher, Siobhan Pattwell, Nicholas Vitanza, Myron K. Evans. Targeting CTPS1 and nucleotide biosynthesis pathways as a novel therapeutic approach 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 5107.

Abstract 7458: Inhibition of brain cancer progression via resolution of inflammation

Abstract While inflammation is a hallmark of cancer, the mechanisms that disrupt its resolution in cancer are not well understood. A paradigm shift is emerging in our understanding that the resolution of inflammation is an active process controlled by specialized pro-resolving mediators (SPMs), including protectins and resolvins, which are endogenous lipid autacoids present in multiple tissues including the brain and cerebrospinal fluid. SPMs counter-regulate pro-inflammatory cytokine production and stimulate phagocytosis of cellular debris (“pro-resolution”) without being immunosuppressive. We hypothesized treatment with pro-resolving agonists such as SPMs presents a novel debris-clearing and resolution-enhancing therapeutic approach to complement cytotoxic brain cancer therapies. Here, we demonstrate the failure of resolution of inflammation via dysregulated SPMs may be a critical risk factor for aggressive brain cancer growth. Moreover, chemotherapy and targeted therapy further disrupt inflammation resolution via a tumor cell death (“debris”)-induced cytokine storm by macrophages. The SPM receptors GPR37 (protectin D1), GPR18/DRV2 (resolvin (Rv) D2), GPR32 (RvD1), and ChemR23/ERV (RvE1) are expressed in tumor-infiltrating macrophages and endothelial cells in the brain tumor microenvironment. Plasma levels of SPMs were significantly reduced in the medulloblastoma-bearing mice, suggesting that brain cancer progression is associated with a failure of endogenous resolution of inflammation. To determine whether failed resolution in brain cancer can be rescued by lipid mediator agonists, we systemically administered SPMs to subcutaneous and orthotopic glioblastoma and medulloblastoma murine brain tumor models. Significantly, RvD1 and RvD2 each inhibited the growth of human glioblastoma tumors by 15-fold at nanogram concentrations without toxicity. Likewise, RvD2 inhibited the growth of human medulloblastoma tumors by 3-fold. Protectins, PDX and PD1n-3, each inhibited growth of human medulloblastoma in mice by approximately 5-fold. Systemic administration of PDX or RvD2 prolonged survival of mice injected with orthotopic human D556 medulloblastoma and also glioblastoma models. Flow cytometry analysis of orthotopic glioblastoma tumors revealed increased immune infiltration into the brain tumor microenvironment in the PCTR1-treatment group when compared to the tumor-bearing non-treated group. At nanogram per day doses, protectins and resolvins suppressed orthotopic debris-stimulated brain tumor growth by activating macrophage and microglial phagocytosis of tumor cell debris and counter-regulating pro-inflammatory cytokines, including TNF-α, CCL2, CXCL1, and CCL4. Thus, stimulating the resolution of inflammation via the clearance of cellular debris with pro-resolving agonists represents a novel therapeutic approach to brain cancer, including glioblastoma and medulloblastoma. Citation Format: Jacqueline Capuano, Kimberly Vasquez, Katherine Quinlivan, Rachel Bayer, Sarina Virani, Michael Gillespie, Mark Kieran, Charles Serhan, Diane Bielenberg, Dipak Panigrahy. Inhibition of brain cancer progression via resolution of inflammation [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 7458.

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

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

Abstract 2271: Conservation and faithful representation of circular extrachromosomal DNA in orthotopic patient-derived medulloblastoma xenografts

Abstract Background: Extrachromosomal DNA (ecDNA) is a driver of tumor heterogeneity and is associated with poor survival in many different adult and childhood cancer types. Patient-derived xenograft (PDX) models facilitate preclinical drug testing in an in vivo environment that enables analysis of tumor growth and survival and molecular tumor profiling. These models, combined with single-cell profiling, can be used to study intratumoral heterogeneity under therapeutic pressure and the emergence of drug resistance. Here we investigate the abundance and conservation of ecDNA in PDX models with the aim of assessing the faithful representation of ecDNA compared to the tumors from which they are derived. Methods: Whole-genome sequencing (WGS) was applied to 27 medulloblastoma (MB) PDX models and their tumors of origin using Amplicon Architect, a tool used to reconstruct focally amplified regions of DNA to provide insights into the landscape of cancer genomes. The frequency of ecDNA in these models was also compared to that of ecDNA in the MB patient population. Optical genome mapping enabled accurate and reliable ecDNA sequence reconstructions. Multiome single-cell RNA and ATAC-sequencing of a PDX tumor enabled analysis of ecDNA conservation and intratumoral heterogeneity compared to similar data previously obtained from the primary tumor. Results: Comparative analyses revealed that ecDNA is largely conserved in PDX models, emphasizing their relevance as faithful models of the primary tumors. In addition, we find that ecDNA is significantly more frequent in MB PDX models (16 out of 27 analyzed patient-derived tumors) as compared to their frequency in the overall patient population (~18% of all medulloblastoma tumors contain ecDNA), suggesting ecDNA as a driver for successful establishment of MB PDX models. Single-cell analysis revealed that ecDNA is omnipresent in almost all PDX tumor cells, while its presence was limited to only ~9% of tumor cells in the primary tumor. Conclusion: Analysis of ecDNA in medulloblastoma primary tumors and in PDX models revealed that ecDNA is preserved in PDX models. This is an important characteristic that renders patient-derived mouse xenografts as faithful models of their primary tumors. These results emphasize the relevance of PDX models for preclinical in vivo studies aimed at analyzing the role of ecDNA under therapeutic pressure and for analyzing clonal selection along with molecular evolution of ecDNA during the development of therapeutic resistance. Citation Format: Rishaan Kenkre, Owen Chapman, Jens Luebeck, Vineet Bafna, Robert Wechsler-Reya, Jill Mesirov, Lukas Chavez. Conservation and faithful representation of circular extrachromosomal DNA in orthotopic patient-derived medulloblastoma xenografts [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 2271.

Abstract 2864: Transcriptional repressor REST controls autophagy in Sonic Hedgehog medulloblastoma through the VHL-HIF1α axis

Abstract Introduction: Medulloblastoma (MB) is the most common malignant brain tumor in children. It is categorized into four molecular subgroups, each with unique genetic and epigenetic alterations, molecular signatures, and differences in clinical features and treatment responses. Targeted therapy for MB has failed in the clinic, prompting new studies of mechanisms involved in MB tumorigenesis and progression. Experimental Procedure: The transcriptomic information of the MB patients was obtained from the publicly available RNAseq datasets and was analyzed for differential expression and clustering analysis. Patient-derived orthotopic xenografts of medulloblastoma tumors with high- and low-REST expression were histologically and biochemically studied for autophagy markers. Furthermore, the SHH-MB cell lines were studied to get a mechanistic understanding of the REST-driven autophagy in this MB subgroup. Summary: Our data showed that autophagy, a process responsible for cellular recycling, and genes associated with this pathway exhibit distinctive expression patterns in human MB subgroups. Specifically, we demonstrate that autophagy is induced in one of the MB subgroups (Sonic Hedgehog-SHH) in response to elevated expression of the RE1-silencing transcription factor (REST), a transcriptional repressor and a canonical regulator of neuronal differentiation genes. REST is also a driver of SHH-MB metastasis. Pharmacological inhibition of autophagy decreases MB cell growth, suggesting it is a pro-survival pathway in REST-driven SHH MBs. In mechanistic studies, we found REST-dependent autophagy induction to involve upregulation of the hypoxia-inducible factor 1-alpha (HIF1α) due to REST-mediated silencing of the von Hippel-Lindau (VHL) gene. The VHL gene product promotes HIF1α ubiquitination and proteasomal degradation, and its loss in SHH-MBs with elevated REST expression drives HIF1α stabilization and nuclear localization to activate target autophagy-related genes. Conclusion: Our work is the first to link VHL to MB pathology. VHL loss of function in cancers has been mainly attributed to mutational events. Here, we report a novel mechanism of VHL loss, namely its epigenetic silencing by REST. Our findings provide the foundation for future pre-clinical investigations to examine the feasibility of autophagy blockade as a therapeutic strategy for REST-driven SHH MBs. Citation Format: Ashutosh Singh, Donghang Cheng, Jyothishmathi Swaminathan, Yan Zheng, Nancy Gordon, Vidya Gopalakrishnan. Transcriptional repressor REST controls autophagy in Sonic Hedgehog medulloblastoma through the VHL-HIF1α axis [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 2864.

Abstract 2848: MYC and TGFβ promote group 3 medulloblastoma tumor resistance through deregulation of Polycomb targets

Abstract Medulloblastoma (MB) is one of the most prevalent malignant brain tumors in children, with tremendous cognitive and neuroendocrine disability among survivors. Group 3 (G3) MBs have poor overall survival at <50%, few recurrent mutations, higher frequency of metastasis, and no targeted therapies. Amplification of MYC and activation of TGFβ signaling are frequent in G3MB. Remarkably, some MB tumors have no reported mutations, suggesting roles for epigenetic mechanisms in driving disease. We hypothesize that the TGFβ pathway and MYC contribute to the intrinsic resistance of G3MB through deregulation of key genes and pathways. We previously established humanized models for SHHMB by introducing MYCN or PTCH1 deletions into neuroepithelial stem cells (NESC) derived from normal human induced pluripotent stem cells (hIPSCs). In this study, we transduced NESCs with TGFb effectors activated in G3MB alone and/or in combination with MYC, prioritizing combinations observed in patients. Excitingly, both MYC and TGFβ effectors drove tumor formation in vivo with the combination of TGFβ effectors with MYC leading to more aggressive tumors. Moreover, they clustered with human G3/G4MB samples, indicating they phenocopy the relevant tumor of interest. We next found that NESCs expressing MYC with either TGFβR1 or TGFβ1 showed resistance to clinical TGFβR1 inhibitors, compared to cells driven by either TGFβR1 or TGFβ1 alone. To decipher mechanisms of resistance, we integrated CUT&RUN to probe for MYC genomic localization and relevant histone PTMs with RNA-seq analysis and discovered a subset of genes upregulated in MYC and TGFb-driven lines that are targets of the histone demethylase KDM2B. Loss of function mutations in KDM2B occur in G3MB patients, indicating that KDM2B deregulated genes are critical in G3MB. We postulate that epigenetic remodeling via MYC and recruitment of other MYC-interacting cofactors to PRC targets culminates in transcriptional changes that lead to aggressive disease. Work from others suggests that PRC-mediated chromatin regulation is critical in many adult and pediatric brain tumors. Overall, our studies provide important insight on identifying new therapeutic avenues for patients with MYC and TGFβ driven G3MB. Citation Format: Zulekha A. Qadeer, Samanta Westelman, Mackenzie O. Johnson, Elise Hou, Shane Grele, Kyle Smith, Liam Hendrikse, Linyu Wang, Sarah Husain, Mary Clare Beytagh, Christin Schmidt, Miller Huang, Michael D. Taylor, Paul Northcott, William A. Weiss. MYC and TGFβ promote group 3 medulloblastoma tumor resistance through deregulation of Polycomb targets [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 2848.

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.

PTCH1-mutant human cerebellar organoids exhibit altered neural development and recapitulate early medulloblastoma tumorigenesis.

Patched 1 (PTCH1) is the primary receptor for the sonic hedgehog (SHH) ligand and negatively regulates SHH signalling, an essential pathway in human embryogenesis. Loss-of-function mutations in PTCH1 are associated with altered neuronal development and the malignant brain tumour medulloblastoma. As a result of differences between murine and human development, molecular and cellular perturbations that arise from human PTCH1 mutations remain poorly understood. Here, we used cerebellar organoids differentiated from human induced pluripotent stem cells combined with CRISPR/Cas9 gene editing to investigate the earliest molecular and cellular consequences of PTCH1 mutations on human cerebellar development. Our findings demonstrate that developmental mechanisms in cerebellar organoids reflect in vivo processes of regionalisation and SHH signalling, and offer new insights into early pathophysiological events of medulloblastoma tumorigenesis without the use of animal models.