Medulloblastoma Cells Research Articles

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.

Functional expression of the proton sensors ASIC1a, TMEM206, and OGR1 together with BKCa channels is associated with cell volume changes and cell death under strongly acidic conditions in DAOY medulloblastoma cells

Fast growing solid tumors are frequently surrounded by an acidic microenvironment. Tumor cells employ a variety of mechanisms to survive and proliferate under these harsh conditions. In that regard, acid-sensitive membrane receptors constitute a particularly interesting target, since they can affect cellular functions through ion flow and second messenger cascades. Our knowledge of these processes remains sparse, however, especially regarding medulloblastoma, the most common pediatric CNS malignancy. In this study, using RT-qPCR, whole-cell patch clamp, and Ca2+-imaging, we uncovered several ion channels and a G protein-coupled receptor, which were regulated directly or indirectly by low extracellular pH in DAOY and UW228 medulloblastoma cells. Acidification directly activated acid-sensing ion channel 1a (ASIC1a), the proton-activated Cl− channel (PAC, ASOR, or TMEM206), and the proton-activated G protein-coupled receptor OGR1. The resulting Ca2+ signal secondarily activated the large conductance calcium-activated potassium channel (BKCa). Our analyses uncover a complex relationship of these transmembrane proteins in DAOY cells that resulted in cell volume changes and induced cell death under strongly acidic conditions. Collectively, our results suggest that these ion channels in concert with OGR1 may shape the growth and evolution of medulloblastoma cells in their acidic microenvironment.

392 Targeting One-Carbon Metabolism in Brain Cancer

OBJECTIVES/GOALS: Glioblastoma (GBM) is the most malignant brain tumor in adults and remains incurable with an average survival of 15 months after diagnosis. There is great need for treatment options without side effects that are devastating to the quality of life for patients. GBM tumors can circumvent cellular damage by upregulating antioxidant production. METHODS/STUDY POPULATION: Highly aggressive tumors tend to exhibit increased oxidative metabolism, and thus rely on a mechanism to eliminate reactive oxygen species (ROS) in order for cells to evade autophagy and cell death. We propose that recurrent GBM cells achieve this is by promoting methionine metabolism, upregulating glutathione production and preventing ROS accumulation. We investigated the expression of AHCY and MAT2A, two key enzymes in the methionine pathway, at the gene and protein level in both GBM and non-GBM tissues. We probed for markers of cell death following pharmacological inhibition and siRNA knockdown, and performed metabolite-mediated rescue experiments. Finally, we evaluated changes in cellular respiration using the Seahorse XFe96 real-time mitochondrial stress test following inhibitor treatment. RESULTS/ANTICIPATED RESULTS: The selective AHCY inhibitor markedly reduced cell viability in different cancer cell types, but significantly reduced cell viability in recurrent GBM cells compared to newly diagnosed GBM (p=.009; MD: -0.828, 95% CI -1.350 to -0.306) and normal astrocytes (p=.073; MD: -0.609, 95% CI -1.305 to 0.085). AHCY and MAT2a protein expression appeared to be higher in GBM cells compared to normal astrocytes, medulloblastoma cells and other cancer cell lines. Genetic knockdown of AHCY and MAT2A demonstrated reduced cell viability, increased Caspase, SOD2, LC3-II and Transferrin receptor expression. Acute treatment with the AHCY inhibitor induced cell death, markedly reduced oxygen consumption rate and ablated spare respiratory capacity in recurrent GBM cells compared to newly diagnosed GBM cells. DISCUSSION/SIGNIFICANCE: Oxidative damage was induced following interference with key methionine pathway enzymes by pharmacological inhibition, while a similar concentration of drug largely preserved normal astrocyte viability. These results point to a novel targetable mechanism of disease progression and expand the realm of treatment options for recurrent GBM.

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

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

Protective Effects of Curcumin Against Medulloblastoma: A Review

Medulloblastoma (MB) is the most common childhood tumor with a poor prognosis. Primary approaches for treating MB comprise surgical resection along with radiotherapy and chemotherapy. However, these methods have not created a promising outlook for subjects with this neuroepithelial tumor due to their low efficiency. On the other hand, these therapeutic strategies are associated with many side effects. So, there is an unmet need to find an alternative way to overcome MB. Currently, there is a significant focus on natural compound-based therapies, particularly curcumin obtained from Curcuma longa, for ameliorating different disorders like cancer. Extensive research has also been conducted to provide evidence supporting the beneficial effects of curcumin in the treatment of MB. This polyphenolic compound can exert its suppressive effects on the proliferation and growth of MB cells by affecting several molecular pathways and agents, such as suppressing Wnt/β-catenin, NF-κB, and SHH signaling pathways, triggering apoptosis-related genetic effectors (eg, Bax, Bcl-2, PARP, caspase-3, and caspase-9), potentiating tubulin acetylation, and decreasing HDAC4 function. Hence, in this literature review, we aimed to debate documents pertaining to MB therapy with curcumin and other formulations in vitro and in vivo with a mechanistic insight.

Identification and validation of a metabolism-related gene signature for predicting the prognosis of paediatric medulloblastoma

Medulloblastoma (MB) is a malignant brain tumour that is highly common in children and has a tendency to spread to the brain and spinal cord. MB is thought to be a metabolically driven brain tumour. Understanding tumour cell metabolic patterns and characteristics can provide a promising foundation for understanding MB pathogenesis and developing treatments. Here, by analysing RNA-seq data of MB samples from the Gene Expression Omnibus (GEO) database, 12 differentially expressed metabolic-related genes (DE-MRGs) were chosen for the construction of a predictive risk score model for MB. This model demonstrated outstanding accuracy in predicting the outcomes of MB patients and served as a standalone predictor. An evaluation of functional enrichment revealed that the risk score showed enrichment in pathways related to cancer promotion and the immune response. In addition, a high risk score was an independent poor prognostic factor for MB in patients with different ages, sexes, metastasis stages and subgroups (SHH and Group 4). Consistently, the metabolic enzyme ornithine decarboxylase (ODC1) was upregulated in MB patients with poor survival time. Inhibition of ODC1 in primary and metastatic MB cell lines decreased cell proliferation, migration and invasion but increased immune infiltration. This study could aid in identifying metabolic targets for MB as well as optimizing risk stratification systems and individual treatment plans for MB patients via the use of a metabolism-related gene prognostic risk score signature.

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 5114: Deciphering the implications of THOC7 in medulloblastoma: Unraveling its role in tumor growth and progression

Abstract Background: Medulloblastoma (MB) is the most common malignant brain cancer in children. Despite the progress made in treating MB, the 5-year survival rate for high-risk tumors remains poor and risk of recurrence within 2 years of treatment is still high. Unfortunately, patients who do survive have reduced quality of life because of the highly toxic side effects of radiation and chemotherapy. Those facts underline the importance of identifying new drivers of MB and understanding the mechanism by which those drivers may promote MB. Using CRISPR-based dependency analysis, we discovered THO Complex Subunit 7 Homolog (THOC7) as one of the highly differentially expressed genes that may uniquely provide survival advantage to MB cells. THOC7 is a component of the THO subcomplex of the TREX complex, which is known to couple mRNA transcription, processing and nuclear export associated with spliced mRNA. Methods: To understand the role of THOC7 in MB, we silenced the expression of THOC7 using siRNAs in multiple MB cell lines. The knockdown efficiency was confirmed by western blot and qPCR. The effect of THOC7 silencing on the viability of MB cells was assessed by short-term viability and long-term colony formation assays. The effect of THOC7 silencing on MB cell proliferation was measured using an IncuCyte. To assess whether THOC7 may affect MB progression, we performed Transwell migration assay. To understand the mechanism by which THOC7 may play a role in medulloblastomogenesis, we measured the effect of THOC7 silencing on the cell cycle progression and apoptosis. As presence of cancer stem cells is linked to relapse and makes MB refractory to radiation therapy, we asked whether THOC7 may support MB stem cell proliferation using neurosphere assay. Results: Our analysis revealed that MB is highly addicted to THOC7. Importantly, higher expression of THOC7 is strongly correlated with lower overall survival of MB patients. We showed that depletion of THOC7 inhibited the short- and long-term viability as well as proliferation of MB cells. In addition, silencing of THOC7 inhibited migration/invasion of MB cells. THOC7 depletion led to cell cycle arrest and increased apoptosis of MB cells. Furthermore, our neurosphere assay demonstrated that silencing of THOC7 blocked the MB stem cell proliferation. Conclusion: Our results suggest that THOC7 may be a novel driver of MB growth and progression. Inhibition of MB stem cell proliferation in THOC7 depleted cells indicates that THOC7 inhibition may sensitize refractory MB to radiation therapy. Experiments are currently underway to understand the mechanism by which THOC7 may support MB growth and progression. In conclusion, our results showing no effect of THOC7 silencing on normal cell viability suggest that approaches aimed at silencing THOC7 may be a safe and viable approach for treating MB. Citation Format: Shahad M. Abdulsahib, Santosh Timilsina, Saif Nirzhor, Daisy Medina, Prabhakar V. Pitta, Panneerdoss Subbarayalu, Manjeet K. Rao. Deciphering the implications of THOC7 in medulloblastoma: Unraveling its role in tumor growth and progression [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 5114.

Abstract 152: Exploring the impact of heat shock protein 90 on the antitumor efficacy and stemness in medulloblastoma cells

Abstract Background: Medulloblastoma (MB) is the most common children’s malignant brain tumor. Although MB can be treated with conventional chemotherapies, chemoresistance has been frequently developed and thereby hampers their therapeutic effectiveness. The preliminary reports of our research showed that some heat shock proteins have higher levels in MB cells. Among these heat shock proteins, HSP70 and HSP90 expressions are particularly high in MB CD133+ cancer stem cells (CSCs), which arouses our attention and interest. Numerous studies demonstrated that heat shock proteins are prominent in some chemotherapy-resistant cancer cells and CSCs. As known HSP90 and its client proteins are required for the maintenance of self-renewal/stemness and regulation of cell cycle, synergistic killing of cancer cells by simultaneous inhibition of HSP90 expression and activity may be a considerable good therapeutic strategy for treating MB. The purpose of this study is to verify the regulatory function of HSP90 in chemoresistance and stemness of MB. Materials and Methods: The levels of HSP90, its client proteins (P13K/pAKT, Erk, NFκB, and c-Myc), and stemness makers were detected using immunoblotting and immunoprecipitation after the treatment of HSP90 inhibitor (17-AAG) or knockdown of HSP90 by siRNA in MB and MB CSCs. The antitumor effects of HSPs inhibitors or knockdown of HSPs on these cells were evaluated by MTT assay, Annexin V/PI analysis and cell cycle analysis. Results: Our findings revealed elevated expression of HSP90 and its target client proteins in MB cells compared to normal astrocytes. Silencing or inhibiting HSP90 expression induced cell cycle arrest in MB cells. Notably, CD133+ MB CSCs exhibited higher HSP90 levels than parental MB cells. Inhibition of HSP90 led to reduced sphere formation and expression of stemness markers in CD133+ MB CSCs. Treatment with 17-AAG demonstrated antitumor activity in CD133+ MB CSCs, and a synergistic effect was observed when combining conventional chemotherapeutic agents with 17-AAG in MB cells Conclusion: These results illustrated that inhibition of HSP90 could eliminate CSCs, and thereby overcome resistance to chemotherapeutic agents in MB. Chemotherapies in combination with HSP90 blockade may be an emerging MB therapy in the future. Citation Format: Shu-Mei Chen, Hsieng-Yun Huang, Chien-Kai Wang, Yi-Yuan Yang, Sy-Jye Leu, Ying-Ying Li. Exploring the impact of heat shock protein 90 on the antitumor efficacy and stemness in medulloblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 152.

Abstract 5929: HuR-targeted therapy promotes the adaptive immune response in medulloblastoma through the cGAS-Sting pathway

Abstract HuR is an RNA binding protein overexpressed in multiple human cancers and is involved in tumorigenesis, invasion, and metastasis. Genetic and pharmacologic inhibition of HuR demonstrated antitumor activity establishing HuR as a molecular target for cancer therapy. However, little is known about HuR’s role in medulloblastoma (MB). In the present study, we investigated the effect of CMLD2, a small molecule inhibitor of HuR, on MB cell growth and on the adaptive immune response by assessing the cyclic GMP-AMP synthase (cGAS) - stimulator of interferon genes (STING) pathway. The cytoplasmic double-stranded DNA (dsDNA) sensing cGAS-STING signaling pathway plays pivotal role in host defense against cancers. Activation of the cGAS-STING signaling cascade induces the expression of pro-inflammatory cytokines and type I interferons promoting anti-tumor immunity. Human MB Daoy cells were treated with DMSO (control) or CMLD2 (20uM and 30uM). At 48 and 72 h after treatment, cells were collected and cell viability was determined using Trypan blue assay. A time and dose-dependent cytotoxicity was observed with 22.5% and 32.5% reduction in cell number with 20uM treatment (p<0.01) and 38.3% and 50.2% reduction with 30uM treatment (p<0.001) at 48 h and 72 h respectively compared to DMSO-treated control. Western blot analysis showed CMLD2 treatment inhibited HuR and markedly increased the expression of cGAS-STING pathway proteins (p-STING Ser366, p-TBK1 Ser172, p-IRF3Ser396 and pNF-KBSer536) at the two drug concentrations tested albeit greater increase in cGAS-STING protein expression was observed with 20uM treatment. Analysis for chemokines (CXCL10 and CCL5) and cytokine (IL-6) associated with the cGAS-STING pathway in cell culture supernatant collected from 20uM CMLD2 treatment showed significant increase in CXCL10 (1.96 ± 0.12 and 2.12 ± 0.06 fold increase at 48 h and 72 h; p<0.01) and CCL5 (1.21 ± 0.09 and 1.86 ± 0.05 fold increase at 48 h and 72 h; p=ns at 48 h; p<0.01 at 72 h) over DMSO-treated control. IL-6 expression significantly increased at 72h (1.98± 0.24 fold, p<0.001) but not at 48h after 20uM CMLD2 treatment compared to DMSO treated control. Our study results demonstrate CMLD2 mediated HuR targeting induces MB cell cytotoxicity and concurrently activates the cGAS-STING pathway. Testing of CMLD2 treatment in additional MB cell models and studying the impact of the cGAS-STING activation on immune cells (T cells, NK cells and macrophages) will aid in developing novel HuR-targeted treatment strategies in MB. Funding: The study was supported in part by a grant received from the National Cancer Institute of the National Institutes of Health (R01 CA 282735-01) and funds from the Jim and Christy Everest Endowed Chair in Cancer Developmental Therapeutics. Citation Format: Meghna Mehta, Rajeswari Raguraman, Anupama Munshi, Rajagopal Ramesh. HuR-targeted therapy promotes the adaptive immune response in medulloblastoma through the cGAS-Sting pathway [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 5929.

Abstract 4599: A novel small molecule inhibitor targeting MYC oncogenic signaling as an enhancer of HDAC inhibitors for the treatment of high-risk medulloblastoma

Abstract Background: Medulloblastoma (MB) is the most prevalent malignant childhood brain tumour. Although prognosis has improved over time, 95% of children with high-risk or relapsed MB eventually succumb to the disease. A key driver gene in high-risk MB is the c-MYC oncogene, for which there are currently no approved inhibitors. Histone deacetylases (HDACs) are transcriptional repressors that are dysregulated in many cancers, making them an attractive therapeutic target. Although several HDAC inhibitors have been FDA-approved for cancer treatment, they have been associated with high toxicity and limited efficacy as single agents. Therefore, simultaneously targeting c-MYC and HDACs may result in synergistic effects that could overcome drug resistance in high-risk MB. Methods and Results: We previously reported a novel pyrido-benzimidazole analogue, SE486-11, which enhanced the therapeutic effect of HDAC inhibitors in MYCN-driven cancers. We recently developed analogues (UNSW-SC compounds) with more potent activity (IC50: 0.017 to 3.70 µM). Here, we showed that these compounds significantly reduced cell viability and induced apoptosis in MB cells. MYC status was a key determinant of sensitivity of UNSW-SC compounds, with more than 3-fold higher cytotoxicity in high MYC compared to low MYC MB cells. The lead compound, UNSW-SC-22, was shown to reduce MYC protein expression and was found to be highly synergistic in enhancing the efficacy of HDAC inhibitors in MB cells. Most importantly, we demonstrated that UNSW-SC-22 was able to freely cross the blood brain barrier, reaching a concentration of 10.8 μM, with a half-life of 1.22 hours. UNSW-SC-22 directly bound to c-MYC and MYCN proteins at low micromolar equilibrium dissociation constant (KD) values which was demonstrated by surface plasmon resonance assay. In a MYCN-driven MB mouse model, we found that UNSW-SC-22 as a single agent at 90 mg/kg decreased tumor growth and prolonged survival. Through RNA-sequencing, we identified 512 differentially expressed genes after in vitro treatment with UNSW-SC-22. Importantly, GSEA analysis revealed MYC targets and cell cycle pathways as highly downregulated pathways, and upregulation in the p53 pathway. Several genes, such as MELK and USP1 were validated as down-stream targets of UNSW-SC-22. As UNSW-SC-22 is a first-in-class anticancer drug, which we are currently testing extensively in other MYC-driven MB mice models. Conclusion: Collectively, our data strongly suggest that c-Myc and MYCN are the molecular targets of UNSW-SC compounds, and these compounds have strong potential to serve as specific targeted therapy to treat subgroup 3 and 4 of MB patients with c-Myc and MYCN overexpression and amplification. Citation Format: Sin Wi Ng, Satyanarayana Gadde, Qian Wang, Belamy B. Cheung, Glenn M. Marshall. A novel small molecule inhibitor targeting MYC oncogenic signaling as an enhancer of HDAC inhibitors for the treatment of high-risk 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 4599.

Abstract 3156: Unveiling the functional role of HDGFRP3 in pediatric medulloblastoma: Implications for growth, progression, and radioresistance

Abstract Introduction: Medulloblastoma (MB) is the most common malignant brain cancer in children. Although long-term survival has improved in recent years, only a modest percentage of patients survive high-risk disease. The quality of life for those who survive is substantially reduced due to the high toxicity associated with the radiation and chemotherapy. Therefore, identification of safe and potent therapeutic approaches that can sensitize the therapy response in MB without inducing toxicity will lead to more efficient treatment of MB patients. We recently discovered that targeting HDGFRP3, a protein highly expressed in MB, and can block MB growth and improve the efficacy of radiation therapy. HDGFRP3 is a multifaceted protein that plays important roles in diverse cellular processes. Our investigation sheds light on the functional role of HDGFRP3 in MB, focusing on its involvement in growth, progression, and radioresistance where we report HDGFRP3 as a novel oncogenic driver of these processes. Methods: Human MB cell lines (DAOY, D556, D425, and HDMB03) were purchased from the American Type Culture Collection and cultured in standard medium. MB cancer cells were transfected with siRNA specific to HDGFRP3. These knockdown cells were analyzed for cell viability, migration, invasion, colony formation, stemness, cell cycle, apoptosis assays for functional role assessment. Real-time qPCR and western blotting were performed to validate the downstream targets of HDGFRP3 in MB models. Results: Our findings demonstrate that HDGFRP3 functions as an oncogene in MB, as its overexpression promotes cancer growth, while its depletion results in decreased tumor growth in MB models. Furthermore, we provide evidence that HDGFRP3 knockdown results in decreased migratory potential of MB cells as well as a reduction in stemness. Interestingly, HDGFRP3 depletion improved the efficacy of standard of care radiation in multiple MB models. At present, in vivo studies are validate the oncogenic role of HDGFRP3 in preclinical animal models of MB. Conclusions: In conclusion, our data suggests HDGFRP3 facilitates MB growth, progression, stemness as well as resistance to radiation therapy. Our comprehensive investigation into HDGFRP3's functional role in MB presented here provides valuable insights into its potential as a therapeutic target and opens avenues for further research into novel treatment strategies for the pediatric brain tumors. Citation Format: Saif S. Nirzhor, Prabhakar Pitta Venkata, Shahad Abdulsahib, Panneerdoss Subbarayalu, Santosh Timilsina, Manjeet K. Rao. Unveiling the functional role of HDGFRP3 in pediatric medulloblastoma: Implications for growth, progression, and radioresistance [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 3156.

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

Abstract Introduction: How tumor cells colonize an inherently distinct environment and survive the pressures of therapeutic treatment during metastasis is incompletely understood. In patients with medulloblastoma, the most common malignant pediatric brain tumor, mortality is almost exclusively dictated by metastasis to the leptomeninges, the inner covering of the brain and spinal cord. We aim to mechanistically understand the molecular drivers of leptomeningeal colonization, including how leptomeningeal adhesion mediates therapy resistance. Experimental approach: Progress in leptomeningeal metastasis research has been hindered by the scarcity of matched primary and metastatic tissue from patients given the lack of clinical benefit to obtaining metastatic tissue. Furthermore, end points from pre-clinical human xenograft mouse models are often driven by primary cerebellar tumors, not leptomeningeal dissemination. These barriers underscore the necessity for novel in vitro leptomeningeal metastasis models that allow for rigorous experimental analysis. These models must simultaneously recapitulate tumor cell-host and tumor cell-environment interactions driving colonization to produce translatable findings. Accordingly, our lab has developed two novel in vitro leptomeningeal metastasis models that allow for cell-based experimental analysis: a primary meningeal cell-medulloblastoma organotypic co-culture and an acellular leptomeningeal matrix mimetic monoculture. Results: Proteomic analysis identified that leptomeningeal cells promote medulloblastoma chemotactic honing to the leptomeninges through CXCL12-CXCR4 signaling. Additionally, loss of CXCL12/CXCR4 signaling impaired medulloblastoma adhesion to the leptomeninges. CXCL12/CXCR4 are known to mediate adhesion of stem cells in stromal niches via activation of integrin receptor ITGA4/ITGB1 (VLA4) binding to stromal VCAM1. Indeed, the VLA4-VCAM1 axis is required for medulloblastoma leptomeningeal adhesion and requires activation by CXCL12/CXCR4 signaling. From a translational perspective, we found that leptomeningeal adhesion promotes therapy resistance and that inhibition of adhesion signaling sensitized adherent medulloblastoma cells to therapy. Conclusions: These findings provide proof-of-concept that appropriate in vitro models of metastasis can be developed and utilized to define the biology driving adhesion and chemotherapy resistance in medulloblastoma metastasis. Furthermore, we show that medulloblastoma leptomeningeal adhesion is mediated by a CXCL12/CXCR4-VLA4/VCAM1 axis. Finally, we demonstrate the therapeutic potential of targeting this axis in sensitizing medulloblastoma metastases to current therapies. Citation Format: Leyre Jimenez Garcia, Amy C. Gross, Ryan D. Roberts, James B. Reinecke. In vitro medulloblastoma leptomeningeal metastasis models reveal adhesion signaling as a therapeutic vulnerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1261.

Abstract 6505: Targeting PINK1 reduces medulloblastoma progression in animal models

Abstract Medulloblastoma is a brain cancer that mainly arises during infancy and childhood. Medulloblastoma is the most common pediatric malignant brain tumor worldwide. It comprises of approximately 20% of all childhood brain tumors and 63% of intracranial embryonic tumors. One of the major effects of proliferating cancer cells is altered mitochondrial metabolism, as well as aberrant metabolism. Medulloblastoma patients suffer severe complications due to intensive and long-term treatments. Thus, we aim to improve applications against medulloblastoma by targeting the PINK1 gene. We hypothesize that PINK1 inactivation slows down medulloblastoma progression. PINK1 is a mitochondrial serine/threonine protein kinase that protects cells by controlling mitochondria quality in response to cellular stress. PINK1 recruits parkin to target damaged mitochondria for degradation through autophagy. In this study, PINK1 has been knocked-out using gene editing technology (CRISPR/Cas9) in two commercial medulloblastoma cell lines (DAOY, and D283 cell lines). We validated PINK1 editing using RT-PCR and Western blot. Then, cell viability was studied in comparison to the control sample. The control sample contained scramble fragments with empty CRISPR/Cas9. We studied apoptosis and performed cell cycle analysis using flow cytometry. Next, orthotopic medulloblastoma mouse model was generated which showed differential survivals comparing the PINK1 knockout group and control. The results showed a low expression of PINK1 within the PINK1 knockout group. In addition, the Western blot showed reductions in PINK1 expression in the knockout group. The knockout group showed more apoptosis (cell death) 40% more than the control. The mouse model showed increased survival of the PINK1 knockout group compared to the control with a p-value of 0.0288 (log-rank). Our analysis shows that survival differences approximately extend human life by one year. We conclude that inhibiting the PINK1 gene in medulloblastoma decreases cell metabolic activities. PINK1 Knockout in medulloblastoma enhances the apoptosis pathways. Engrafted medulloblastoma mouse model treated with PINK1 inhibitor showed superior survival than the control. These results qualify PINK1 to be used as potential therapeutic target for medulloblastoma. The Future work needs additional preclinical studies to investigate the proper dose of PINK1 inhibitor and measurement of toxicity index. Citation Format: Reema N. Alsubaiee, Amani Alghamdi, Bahauddeen M. Alrfaei. Targeting PINK1 reduces medulloblastoma progression in animal models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6505.

Abstract 1672: PKC-ι and 14-3-3 are crucial regulators of c-Raf and the Erk pathway in human medulloblastoma

Abstract Medulloblastoma is the second most common childhood cancer, behind leukemia, and the most common pediatric brain tumor, comprising 20% of all childhood brain cancers. Current treatment options are limited to radiation therapy, surgical resection, and chemotherapy, which are extremely invasive. This underscores the need for more targeted and effective therapeutic treatments to improve patient outcomes. The following study will focus on exploring the PKC-ι specific inhibitor 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) and its effects on medulloblastoma cell lines Daoy and ONS-76. Our in-vitro treatments showed that PKC-ι functions an oncogenic protein that is necessary for Daoy and ONS-76 cell proliferation and that ICA-1S was found to be cytostatic to both Daoy and ONS-76 cells. The inhibition of PKC-ι in both cell lines resulted in a decreased phosphorylation of PKC-ι as well as downregulation of 14-3-3 and crucial components of the c-Raf/Erk pathway including total and phosphorylated levels of c-Raf, Mek1/2, and Erk1/2. This comes as a result of ICA-1S blocking the association of PKC-ι with 14-3-3 while PKC-ι and c-Raf were not found to directly associate with each other, indicating indirect modulation of c-Raf by PKC-ι. Additionally, PKC-ι inhibition decreased its association with Stat3. Expression levels of Stat1, Stat3, c-Fos, and c-Myc transcription factors were significantly downregulated, which is likely the result of reduced transcriptional activation due to upstream inhibition of PKC-ι and Erk signaling. Treatment with ICA-1S was also shown to substantially decrease cellular invasion of Daoy and ONS-76 cells and induce apoptosis. Taken together, these data suggest that PKC-ι likely plays a critical role in the regulation of the c-Raf/Erk pathway, which controls the proliferation and invasion of human medulloblastoma. Citation Format: Luke Lajmi, Wishrawana S. Ratnayake, Joseph Palathinkal, Sloan Breedy, Aaron Todman, Mildred Acevedo-Duncan. PKC-ι and 14-3-3 are crucial regulators of c-Raf and the Erk pathway in human 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 1672.

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.

MiR-124-3p and miR-194-5p regulation of the PI3K/AKT pathway via ROR2 in medulloblastoma progression

Medulloblastoma (MB), a prevalent pediatric central nervous system tumor, is influenced by microRNAs (miRNAs) that impact tumor initiation and progression. However, the specific involvement of miRNAs in MB tumorigenesis remains unclear. Using single-cell RNA sequencing, we identified ROR2 expression in normal human fetal cerebellum. Subsequent analyses, including immunofluorescence, quantitative real-time PCR (qRT-PCR), and Western blot, assessed ROR2 expression in MB tissues and cell lines. We investigated miR-124-3p and miR-194-5p and their regulatory role in ROR2 expression through the dual-luciferase reporter, qRT-PCR, and western blot assays. Mechanistic insights were gained through functional assays exploring the impact of miR-124-3p, miR-194-5p, and ROR2 on MB growth in vitro and in vivo. We observed significantly reduced miR-124-3p and miR-194-5p expression and elevated ROR2 expression in MB tissues and cell lines. High ROR2 expression inversely correlated with overall survival in WNT and SHH subgroups of MB patients. Functionally, overexpressing miR-124-3p and miR-194-5p and inhibiting ROR2 suppressed in vitro malignant transformation and in vivo tumorigenicity. Mechanistically, miR-124-3p and miR-194-5p synergistically regulated the ROR2/PI3K/Akt pathway, influencing MB progression. Our findings indicate that miR-124-3p and miR-194-5p function as tumor suppressors, inhibiting MB progression via the ROR2/PI3K/Akt axis, suggesting a key mechanism and therapeutic targets for MB patients.

Revealing the Ferroptotic Phenotype of Medulloblastoma.

The interaction of iron and oxygen is an integral part of the development of life on Earth. Nonetheless, this unique chemistry continues to fascinate and puzzle, leading to new biological ventures. In 2012, a Columbia University group recognized this interaction as a central event leading to a new type of regulated cell death named "ferroptosis." The major feature of ferroptosis is the accumulation of lipid hydroperoxides due to (1) dysfunctional antioxidant defense and/or (2) overwhelming oxidative stress, which most frequently coincides with increased content of free labile iron in the cell. This is normally prevented by the canonical anti-ferroptotic axis comprising the cystine transporter xCT, glutathione (GSH), and GSH peroxidase 4 (GPx4). Since ferroptosis is not a programmed type of cell death, it does not involve signaling pathways characteristic of apoptosis. The most common way to prove this type of cell death is by using lipophilic antioxidants (vitamin E, ferrostatin-1, etc.) to prevent it. These molecules can approach and detoxify oxidative damage in the plasma membrane. Another important aspect in revealing the ferroptotic phenotype is detecting the preceding accumulation of lipid hydroperoxides, for which the specific dye BODIPY C11 is used. The present manuscript will show how ferroptosis can be induced in wild-type medulloblastoma cells by using different inducers: erastin, RSL3, and iron-donor. Similarly, the xCT-KO cells that grow in the presence of NAC, and which undergo ferroptosis once NAC is removed, will be used. The characteristic "bubbling" phenotype is visible under the light microscope within 12-16 h from the moment of ferroptosis triggering. Furthermore, BODIPY C11 staining followed by FACS analysis to show the accumulation of lipid hydroperoxides and consequent cell death using the PI staining method will be used. To prove the ferroptotic nature of cell death, ferrostatin-1 will be used as a specific ferroptosis-preventing agent.

Abstract B044: Elongation control of mRNA translation drives Group 3 medulloblastoma adaptation to nutrient deprivation

Abstract Background: Group 3 affiliation and MYC genetic amplification are associated with poor life expectancy and substantial morbidity in children suffering from medulloblastoma (MB). The high metabolic demand induced by MYC-driven transformation sensitizes MYC-overexpressing MB to cell death under conditions of nutrient deprivation (ND). Additionally, MYC-driven transformation is known to promote mitochondrial oxidative phosphorylation (OXPHOS). We previously reported that eukaryotic Elongation Factor Kinase 2 (eEF2K), the master regulator of mRNA translation elongation, promotes survival of MYC-overexpressing tumors under ND. Interestingly, eEF2K is overexpressed in MYC-driven MB and our preliminary proteomics data highlight large-scale alterations in OXPHOS components affecting eEF2K deficient MB cells. We therefore hypothesized that eEF2K activity is required for the selective translation of mRNAs needed for efficient OXPHOS, and for the progression of MYC-driven MB. METHODS: Multiplexed enhanced Protein Dynamic Mass Spectrometry performed in eEF2K knockdown MYC-overexpressing D425 MB cells to identify mRNAs selectively translated upon eEF2K activation. Time course experiments under ND were conducted in eEF2K knockout (KO) D425 cells to assess the presence of electron transport chain (ETC) complexes I-IV in their native state (via BN-PAGE), as well as transcript expression of individual ETC complex components (by qPCR). The effects of eEF2K inactivation on oxygen consumption, metabolic fluxes and mitochondrial membrane potential were studied with Seahorse technology and JC1/TMRE staining. The viability of eEF2K KO D425 cells was assessed by Incucyte system. Finally, MB orthotopic xenograft mouse models were used to confirm in vitro observations. RESULTS: Multiple (9 out of 10 detected) components of the mitochondrial OXPHOS pathway are selectively translated upon eEF2K activation. Inactivation of eEF2K by genetic KO leads to the disassembly of ETC complexes I-IV without affecting mRNA levels of their respective components. Consistently, eEF2K KO MB cells display decreased mitochondrial membrane potential and ~20% increased proton leak through the mitochondrial membrane. In addition, eEF2K inactivation results in increased D425 cell death under ND and doubles survival of MB bearing mice fed with calorie restricted diets (p<0.05). CONCLUSION: Control of mRNA translation elongation by eEF2K is critical for mitochondrial ETC complex assembly and efficient OXPHOS in MYC-overexpressing MB, likely representing an adaptive response by which MYC-driven MB cells cope with acute metabolic stress. Future therapeutic studies will aim to combine eEF2K inhibition with caloric restriction mimetic drugs as eEF2K activity appears critical under metabolic stress conditions. Citation Format: Alberto Delaidelli, Fares Burwag, Jessica Oliveira de Santis, Betty Yao, Haifeng Zhang, Yue Zhou Huang, Gian Luca Negri, Christopher Hughes, Gabriel Leprivier, Poul Sorensen. Elongation control of mRNA translation drives Group 3 medulloblastoma adaptation to nutrient deprivation [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 B044.