MB Tumor Research Articles

G-quadruplexes are a source of vulnerability in BRCA2 deficient granule cell progenitors and medulloblastoma.

Biallelic pathogenic variants in the essential DNA repair gene BRCA2 causes Fanconi anemia, complementation group FA-D1. Patients in this group are highly prone to develop embryonal tumors, most commonly medulloblastoma arising from the cerebellar granule cell progenitors (GCPs). GCPs undergo high proliferation in the postnatal cerebellum under SHH activation, but the type of DNA lesions that require the function of the BRCA2 to prevent tumorigenesis remains unknown. To identify such lesions, we assessed both GCP neurodevelopment and tumor formation using a mouse model with deletion of exons three and four of Brca2 in the central nervous system, coupled with global Trp53 loss. Brca2 Δex3-4 ;Trp53 -/- animals developed SHH subgroup medulloblastomas with complete penetrance. Whole-genome sequencing of the tumors identified structural variants with breakpoints enriched in areas overlapping G-quadruplexes (G4s). Brca2-deficient GCPs exhibited decreased replication speed in the presence of the G4-stabilizer pyridostatin. Pif1 helicase, which resolves G4s during replication, was highly upregulated in tumors, and Pif1 knockout in primary MB tumor cells resulted in increased genome instability upon pyridostatin treatment. These data suggest that G4s may represent sites prone to replication stalling in highly proliferative GCPs and without BRCA2, G4s become a source of genome instability. Tumor cells upregulate G4-resolving helicases to facilitate rapid proliferation through G4s highlighting PIF1 helicase as a potential therapeutic target for treatment of BRCA2-deficient medulloblastomas.

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

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

Abstract 3346: JMJD6 as a novel tumorigenic factor and therapeutic target in group 3 (MYC-driven) medulloblastoma

Abstract Medulloblastoma (MB) is the most common type of childhood brain cancer worldwide. Although current treatment with surgery and extensive chemoradiation has led to increased survival rates, many MB patients still die from the disease. Moreover, surviving patients suffer severe long-term side effects as a consequence of treatment. It is therefore crucial to develop more effective and less toxic therapies. The most aggressive subtype of MB tumors often exhibits amplification or overexpression of the MYC oncogene. Patients with MYC-amplified MB exhibit a high frequency of cerebrospinal tumor dissemination, often experience treatment resistance and have extremely poor prognoses. While the MYC oncogene is established as the oncogenic driver in Group 3 MB, it has remained undruggable. Thus, targeting regulatory components of MYC and the signaling pathways regulated by it is of great potential therapeutic value. Studies have revealed that MB has very few germline mutations in cancer predisposition genes, suggesting that dysregulated epigenetic pathways might be critical in MB pathogenesis. Particularly, dysregulation of epigenetic modifiers, including histone methyltransferases and histone demethylases, is very common in Group 3 MB, compared to other MB subgroups. Therefore, it is important to identify such epigenetic modifiers that may have controls on MYC and its tumorigenic activities and explore these as the epigenetic drug-candidate targets in Group 3 (MYC-driven) MB. In this regard, we found that protein arginine demethylase Jumonji C domain-containing protein 6 (JMJD6), an emerging key epigenetic enzyme in cancers, is a novel regulator of MYC expression in MYC-driven MB. We observed high levels of JMJD6 that not only mirror MYC expression in the most aggressive MB but also correlate with poor outcomes in these patients. Knockdown of JMJD6 decreased MYC expression, cellular proliferation/survival and stemness in MYC-amplified MB cells. Mechanistically, our results revealed that JMJD6 forms complexes with proteins involved in maintaining and regulating the promoter-pause at super-enhancers, suggesting that JMJD6 can regulate MYC at the transcription level. Moreover, our in vivo analyses of JMJD6 inhibition, either with inducible gene knockdown or a pharmacologic small molecule inhibitor, demonstrated anti-MB potential with suppressed MYC expression. Based on this background and preliminary observations, we hypothesize that JMJD6 plays crucial roles in the most aggressive MB by regulating MYC expression and hence MYC-driven tumorigenesis. Accordingly, we hypothesize that targeting the JMJD6-MYC axis by JMJD6 inhibition can serve as a powerful therapeutic strategy for MYC-driven MB. Citation Format: Matthew Kling, Devendra Kumar, Sutapa Ray, Shantaram Joshi, Don Coulter, Nagendra K. Chaturvedi. JMJD6 as a novel tumorigenic factor and therapeutic target in group 3 (MYC-driven) 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 3346.

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

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor, comprised of four distinct molecular subgroups (WNT, SHH, Group 3, and Group 4). A subset of Group 3 MB tumors harbor focal amplifications of the MYC oncogene and are particularly prone to tumor recurrence and leptomeningeal spread which currently remains incurable. Therefore, there is an urgent need for the development of therapeutic modalities that can spare the vulnerable, developing brain while having potent antitumor efficacy against recurrent MB. Through proteomic profiling of cyclophosphamide-resistant patient-derived MYC-driven Group 3 MB cell line SU_MB002, we identified nuclear envelope (NE) proteins to be highly abundant and enriched in Group 3 MB compared to normal tissue. We discovered that LBR, an inner nuclear transmembrane protein, while present in all cells at the NE, is aberrantly presented to the cell surface in MYC-driven Group 3 MB cell lines. Immunohistochemistry analysis of LBR on MB patient tumor samples identified that positive LBR staining correlated with a significantly worse prognosis and was also identified to be enriched in recurrent tissue compared to their matched primary samples. Cell surface expression flow cytometry analysis identified highly abundant cell surface expression in all MYC-driven Group 3 MB cell lines with low to no expression in human neural stem cells and other MB subgroups. Mechanistically, high resolution microscopy of endogenously HALO-tagged LBR reveals LBR cell surface presentation to be linked to ER or ER-like vesicles that are directly trafficked to the cell surface. Transcriptomic analysis of LBR cell surface positive and negative cells identify that cell surface positivity is significantly linked to cell division and mitotic processes (cell cycle process, mitotic cell cycle process, chromosome segregation, cell division, nuclear division). LBR mislocalization stems from mitosis at the time of nuclear envelope dissolution and is enriched in the hyperproliferative, therapy-resistant subpopulation of MB cells following exposure to chemotherapy and radiation in vitro and in vivo and may act as a marker for the brain tumor initiating cells that seed recurrence. Therefore, we demonstrate that LBR is an ideal therapeutic target for patients facing relapsed or treatment-refractory MB and have generated single domain antibodies in an effort to engineer LBR-specific CAR T cells. The development of therapies against NE proteins like LBR may provide patients a novel therapeutic opportunity that may be able to treat therapy-resistant, hyperproliferative MYC-driven Group 3 MB cells. Citation Format: Yujin Suk, Jorge Ibañez-Vega, Iqra Chaudhry, Martin Rossotti, Minomi Subapanditha, Petar Miletic, Stefan Custers, Laura Escudero, Alberto Delaidelli, Takuma Nakashima, Yuxi Xiao, Jason Moffat, Hiromichi Suzuki, Poul Sorensen, Kevin Henry, Chitra Venugopal, Giedre Krenciute, Sheila Singh. Discovery of nuclear envelope mislocalization in MYC-driven group 3 medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5112.

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

Abstract Medulloblastoma is the most common malignant pediatric brain tumor. Patients diagnosed with MYC-amplified group 3 medulloblastoma (G3 MB) have a devastating prognosis with a 5-year survival rate of <50%. Standard of care (SOC) consists of maximal resection surgery followed by chemoradiotherapy, but these extensive treatments leave patients with neurocognitive and developmental deficits. Therefore, the identification of novel targets that can be used alone or in combination with SOC is of utmost importance. Using a batch-normalized bulk RNA-seq dataset (GSE124814) comprised of 1350 MB samples and 291 normal cerebella, we reveal upregulation of Y-box binding protein 1 (YBX1) in MB compared to normal cerebellum. Further analysis of this dataset and others demonstrates G3 tumors that display high MYC levels also have the highest YBX1 expression. Analysis of ChIP-seq data shows strong binding of MYC to the YBX1 promoter in G3 MB, concurrent with H3K27ac and H3K4me3 histone modifications. Using a well-established ex vivo model, we demonstrate increased YBX1 expression as normal cerebellar progenitor cells transition to tumorigenic G3 MB stem-like cells. Recent work in leukemia reveals a reciprocal regulation of MYC by YBX1, however, this mechanism is not well established in G3 MB. We utilized an orthogonal pharmacogenetic approach to inhibit YBX1 function and determine its effects on G3 MB tumor biology. Suppression of YBX1 significantly reduced the proliferation and decreased MYC protein expression of G3 MB cells in vitro. These findings support a reciprocal MYC-YBX1 axis, which could influence tumor growth in vivo. Orthotopic implantation of YBX1 KO G3 MB cells leads to increased animal survival. We next evaluated the therapeutic potential of two recently characterized YBX1 inhibitors which display single agent efficacy in vitro and in vivo. YBX1 inhibitors synergized with SOC in vitro lowering the IC50 for both chemo and radiotherapy, which could reduce adverse effects experienced by patients. In silico molecular docking using PyMOL predicts YBX1 inhibitors interact with amino acids within the cold shock domain of YBX1, likely impairing its ability to bind RNA. We performed formaldehyde RNA immunoprecipitation (fRIP)-sequencing to identify changes in RNA transcript levels following YBX1 pharmacological inhibition. Disruption of YBX1-RNA binding impacts the stability of known oncogenic transcripts which may be required for tumorigenesis. Our findings impart an alluring prospect of targeting YBX1 in combination with standard-of-care, and possibly other compounds to improve patients’ outcomes. Citation Format: Stephen Carney, John Hemenway, Ashmitha Rajendran, Matt Hathaway, Eric Nealy, Christian Bonatto, Dhanir Tailor, Sanjay Malhotra, Myron Evans. Targeting Y box binding protein 1 (YBX1) in MYC-amplified medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 561.

Abstract B003: The OTX2 gene induces tumor growth and triggers leptomeningeal metastasis by regulating the mTOR signaling pathway in medulloblastomas

Abstract B003: The OTX2 gene induces tumor growth and triggers leptomeningeal metastasis by regulating the mTOR signaling pathway in medulloblastomas

CSIG-01. DEFINING THE INVOLVEMENT OF THE PERIVASCULAR NICHE IN BRAIN TUMOR METASTASES

Abstract Medulloblastomas (MB) are the most prevalent malignant pediatric brain tumors, originating in either the cerebellum or the dorsal brainstem. They are classified into four distinct subgroups (WNT, SHH, Group 3, and Group 4), characterized by unique gene expression profiles, metastatic patterns, and recurrence rates. Metastatic progression of MB is frequently associated with poor prognosis, affecting approximately 30% of patients at diagnosis. Despite considerable research on primary tumors, there are currently no approved specific treatments for MB leptomeningeal metastases. Thus, gaining further insights into metastatic MB and developing targeted therapies are imperative. The leptomeningeal niche, where metastatic MB tumor cells reside, comprises a complex network of blood vessels supported by perivascular cells, including pericytes, smooth muscle cells, and fibroblasts. However, the specific adhesion molecules and signaling pathways within the leptomeningeal niche that contribute to tumor cell colonization remain elusive. In this study, we hypothesize that the perivascular niche plays a critical role in the colonization and survival of metastatic cells within the leptomeninges. To investigate this, we employed an innovative sLP-mCherry niche labeling system in conjunction with single-cell RNA sequencing to spatially identify and examine the perivascular cells neighboring metastatic MB tumor cells. Through our analysis, we identified upregulated genes associated with cholesterol export in tumor-associated perivascular cells, suggesting their potential involvement in supporting metastatic cell colonization. To further explore this mechanism, we utilized CRISPR and lentiviral overexpression constructs to modulate cholesterol uptake in MB cell lines, observing subsequent changes in metastatic burden. In summary, our study elucidates the importance of the perivascular niche in metastatic medulloblastomas and provides novel insights into the molecular mechanisms that drive tumor cell colonization within the leptomeninges. These investigations into the lipid transport mechanism in the vascular microenvironment opens doors for the development of innovative treatments targeting MB metastases.

CSIG-26. TARGETING HYPOXIC CANCER CELLS IN MEDULLOBLASTOMA

Abstract The goal of this study is to thoroughly assess the role of hypoxia-induced transcription factors (HIFs) in MB growth and examine HIF as a potential therapeutic target. We first examined patient MB tumor by IHC and identified scattered areas with expression of HIF1a and HIF2a. We then established orthotopic human MB xenografts in mice and found hypoxic areas throughout the tumors using pimonidazole detection and they expressed both HIF1a, and HIF2a. There are only few observations of hypoxic areas within experimental MB. To explore the impact of HIFs on MB growth, we exposed MB cell lines (ONS-76; Shh and D556; Group 3) to 1% hypoxia in cell culture. We observed a decrease in cell growth, an increase in HIF1a and HIF2a protein expression, activation of HIF targets (Glut-1, VEGF), and a hypoxia-response element-driven luciferase reporter assay. To further examine the role of individual HIF isoforms, we employed CRISPR/Cas9 to knockout (KO) the HIF1a and/or HIF2a genes in ONS76 MB cells. The HIF reporter activity was partially canceled with single HIF-KO and completely abrogated with double KO and double KO reduced cell growth, indicating that both HIF-1 and HIF-2 are having roles for activating transcription and cell growth. Treatment of MB cells with 64B, an arylsulfonamide HIF/p300 inhibitor abrogated reporter activity by both HIF-1 and HIF-2 and reduced expression of hypoxia-induced genes. To investigate the potential of targeting HIF in vivo, we injected mice brains with wild-type and HIF1a/2a-KO ONS-76 cells and monitored their growth and survival. Mice injected with double KO HIF cells exhibited prolonged survival compared to those with parental cells (n = 5, p< 0.01). Further experiments to expand this approach with other MB cell line and test the anti-tumor effects of 64B in mice are ongoing. These findings support targeting HIFs as a novel therapeutic strategy in MB treatment.

P04.08.A PATIENT-DERIVED ORGANOIDS FOR MODELING AND CHARACTERIZING PEDIATRIC BRAIN TUMORS

Abstract BACKGROUND Central nervous system (CNS) tumors are the most common solid tumors in the pediatric population and the leading cause of death among childhood cancers. They present common features related to the development and produce symptoms based on their location, rate of tumor growth and age of the child. Tumors of embryonal origin, such as medulloblastoma or primitive neuroectodermal tumor, have a higher incidence in younger patients, whereas older ones tend to present tumors of glial origin. Most of CNS-embryonal tumors are highly malignant and consequently leading to poor prognoses. Despite the understanding of their underlying molecular landscape that has helped in clinical advances to improve life expectancies of the patients, there is still an emerging need to minimize morbidity and manage the long-term therapy effects. MATERIAL AND METHODS Human samples were collected according to protocols approved by the institutional review board with written consent. Patient-Derived Organoids (PDOs) and Patient-Derived Xenograft Organoids (PDXOs) were obtained using scalpels by chopping in small pieces the tumor tissue and were grown in PDOs medium. PDOs and PDXOs were analyzed through DNA methylation, immunostainings, sc-RNA sequencing and orthotopic nude mice engraftment. Therapeutic regimens were adapted according to patients’ treatment protocols depending on the tumor of origin. RESULTS Developing new models to better understand the complexity and heterogeneity of pediatric tumors and to test personalized treatment strategies remains a clinical challenge. We tried to answer this medical need with PDOs and PDXOs established through the direct in vitro culture of different kinds of primary tumors (ependymoma EPN, medulloblastoma MB and low-grade glial tumors) and MB patient derived xenograft (PDX)-derived tumors. We have developed a method that recapitulates mutational profiles, histological features, and cellular heterogeneity of the corresponding parental tumors. We have obtained promising results from the DNA methylation profile analysis, nowadays used for an accurate routine CNS tumors diagnosis. Through this new model, we confirmed the action of clinically used therapeutic regimens, i.e., for EPN with ZFTA-RELA fusion and G3 MB-derived PDOs. PDOs were subjected to the same treatment the patients underwent and responded similarly to the patients. CONCLUSION We found a suitable in vitro culture condition to generate and maintain PDOs from different kinds of pediatric brain tumors. Their reliability has been proved by different analyses, confirming they still resemble the parental tumors. Through this new model, we confirmed the action of clinically used therapeutic regimens, suggesting its potential at the translation level as a reliable tool for testing personalized medicine, drug screening and development of newer and more specific therapeutic strategies.

P04.06.A A PHASE II MONOCENTRIC STUDY OF LIMITED USE OF HIGH DOSE OF CHEMOTHERAPY AND PROTON THERAPY IN PEDIATRIC HIGH RISK MEDULLOBLASTOMA AND OTHER EMBRYONAL CANCERS

Abstract BACKGROUND Despite the multimodal approach, medulloblastoma remains a leading cause of cancer-related death in children. We proposed the preliminary data of an interventional, open-label, phase II study to evaluate the safety and efficacy of standard and high-dose chemotherapy associated with craniospinal proton therapy in patients with metastatic medulloblastoma and other embryonal cancers. MATERIAL AND METHODS A total of 19 patients have been enrolled from the beginning of the study from February 2018 to May 2022 . The median age was 7,5 years (range; 13 males and 6 female). The protocol predicted the first phase of induction with 4 cycles of chemotherapy. After induction patients with favorable histology without evidence of disease were enrolled to proton therapy (with concomitant vinorelbine biweekly). The maintenance phase with Lomustine repeated every 9 weeks and vinorelbine every 3 weeks for overall 12-18 months (PR). Conversely LC/A MB, pineoblastoma or other high risk embryonic tumors were subjected to consolidation phase with high dose of chemotherapy with tiothepa followed by autologous HSC transplant and successive maintenance phase of 6 months. RESULTS At December 2022, the median follow up was 23 months. 18 patients had high risk medulloblastoma and one had a primary leptomeningeal primitive neuroectodermal tumor. After the induction phase 14 patients showed CR, 3 PR, 1PD, 1SD. At subsequent re-evaluations two of the three patients with PR, showed disease control, one CR and the other SD and the patient with PD showed CR. At the end of proton therapy 18 patients had CR. During the reporting period three patient died for progression of the underlying disease. At 15 months from the start of treatment the PFS and OS were 68.8% (95% CI: 34.1% - 87.6%) and 94.7% (95% CI: 68.1% - 99.2%) respectively. The principal expected side effects of chemotherapy were hematology toxicity (fever and neutropenia prevalent after etoposide and carboplatin plus vinorelbine). Among the unexpected side effect was CMV retinopathy with complete blindness and leukoencephalopathy after high dose of thiotepa and proton irradiation. CONCLUSION As the study did not reveal any safety issues, proton therapy combined with intensive chemotherapy, and myeloablative chemotherapy only in selected cases and always before irradiation, proved feasible and successful in treating high-risk medulloblastomas and other embryonal tumors.

MDB-34. “INDUCING IRON IMBALANCE BY TARGETING THE ABCB7/GPX4 AXIS ACCELERATES FERROPTOSIS IN MEDULLOBLASTOMA”

Abstract Medulloblastomas (MB), the most common malignant pediatric brain tumor and a leading cause of childhood mortality, are stratified into four primary subgroups. Deletions within chromosomal locus 17p13.3, which houses multiple tumor suppressor genes including miR-1253, characterize high-risk group 3 tumors. These aggressive tumors also enrich iron transport genes to satisfy their high proliferative need. MiR-1253 targets iron transport by inhibiting the mitochondrial Fe-S transporter, ABCB7. This study elucidated the impact of repressing ABCB7 on cisplatin cytotoxicity in group 3 MB and whether these effects were mediated by ferroptosis. In silico and in vitro analyses revealed specific enrichment of ABCB7 and GPX4, a critical regulator of ferroptosis, in group 3 MB cell lines and tumors. MiR-1253 overexpression (miR-1253OE) resulted in downregulation of both ABCB7 and GPX4, concurrently increasing mitochondrial iron overload, mitochondrial oxidative stress, and lipid peroxidation, leading to cell death and abrogation of medullosphere formation; ABCB7 knockdown (ABCB7KD) recapitulated these effects and abrogated GPX4 expression. Fractionation studies confirmed the inhibitory impact of miR-1253OE and ABCB7KD on GPX4 expression in the cytosol and mitochondria. Seahorse studies revealed that the bulk of ATP generation was occurring in the cytoplasm through glycolysis and not via oxidative phosphorylation, suggesting mitochondrial dysfunction was triggered when ABCB7 was repressed. Cisplatin, a chemotherapeutic agent used in group 3 MB treatment, induces cell death by DNA crosslinking; it also inhibits GPX4 expression. In miR-1253OE and ABCB7KD group 3 cancer cells, cisplatin IC50 was reduced 2-fold. Resultantly, cisplatin treatment augmented oxidative stress and lipid peroxidation, depleted glutathione stores, and culminated in a higher index of ferroptosis. In a mouse model of group 3 tumors, ABCB7KD potentiated cisplatin effects and dramatically prolonged survival. The current study illustrates how targeting iron transport can augment ferroptosis to potentiate cisplatin cytotoxicity in group 3 MB tumors.

MDB-43. GRADIENT ENTROPY-BASED RADIOMIC RISK-SCORE FROM T1-WEIGHTED PRE-TREATMENT MRI SCANS CAN PREDICT SURVIVAL IN PEDIATRIC MEDULLOBLASTOMA

Abstract BACKGROUND Medulloblastoma (MB) is the most frequent malignant brain tumor in children with an inadequate 5-year survival rate of 70-75%. A key determinate in MB treatment (consisting of radiation, chemotherapy) is the accurate risk-stratification of patients into low- and high-risk. The current stratification approaches (Chang’s classification, molecular categorization) are sub-optimal in predicting individual patient outcomes. Reliable risk-assessment approaches can significantly impact treatment management. Our group previously developed a new radiomics feature COLLAGE that measures the orientation of local intensity gradients as an estimate of lesion heterogeneity. We hypothesize that the disorder (i.e., heterogeneity) in orientation of local per-voxel intensity measured by COLLAGE, will tease-out differences in patients with low - versus high-risk MB tumors. METHODS T1-weighted MRI scans of 49 pediatric MB patients were retrospectively collected from Children’s Hospital Los Angeles and Cincinnati Children’s Hospital Medical Center. Registration of the scans was performed via age-specific pediatric atlases, followed by skull stripping and bias correction. Ground truth annotations for tumor sub-compartments; enhancing tumor, edema, and non-enhancing tumor, were generated by two experienced radiologists. Then, 52 Collage features were extracted from each of these sub-compartments, followed by conducting a Lasso model and a cox-proportional hazards model to identify the top features for risk-stratification. A continuous risk score was created for every patient and a risk threshold was obtained from the fitted cox model to stratify patients into low- and high-risk. RESULTS Our risk-score consisted of 13 top COLLAGE features from edema and 14 from non-enhancing tumor sub-compartments, respectively, and demonstrated significant differences between low- and high-risk patients (p = .017 for edema, p =0.0027 for non-enhancing tumor). CONCLUSION Our preliminary analysis suggests that gradient-entropy radiomic risk score maybe able to predict overall survival in pediatric MB tumors. In future, we plan to include additional patients to perform independent multi-site analysis.

MDB-20. IMPROVING THE PERSISTENCE OF CAR T CELLS AGAINST GROUP 3 MEDULLOBLASTOMAS VIA EPIGENETIC REGULATION OF EXHAUSTION

Abstract Medulloblastomas (MB) are the most common malignant pediatric brain tumor with molecular subclass Group 3 (G3) requiring the most intense treatment regimen including surgery, intensive radiation followed by chemotherapy. Despite an overall 40% survival rate, survivors’ quality of life is often impaired due to whole-brain radiation leading to neurocognitive defects. Chimeric antigen receptor (CAR) T cell therapy has emerged clinically as a safe, tumor-targeted immunotherapy with the most success observed against hematological malignancies. Translating CAR T cell therapy to brain tumors has proven challenging due to lack of targetable antigens on the surface of tumor cells and lack of persistence of CAR T cells. We have previously identified that patient samples and PDOX-derived cell lines of G3 MBs consistently over-express B7-H3 (CD267) compared to normal tissue. Herein, we investigated B7-H3 targeted CAR T cell products against G3 MB in vitro and in vivo. Initial results show that B7-H3 CAR T cells are highly effective in clearing human MB tumors in NSG mice, however, 4 out 5 mice experienced late relapses that showed reduced, but still present, B7-H3 antigen expression. We hypothesize that the lack of CAR T cell persistence is ultimately responsible for tumor regrowth. To overcome this limitation, we have identified epigenetic regulators DNMT3A and TET2 that control T cell exhaustion. Using CRISPR/Cas9, we genetically knocked out DNMT3A and TET2 in CAR T cells and investigated the impact on persistence and anti-tumor activity. Our data supports the hypothesis that knock out of DNMT3A and TET2 improves antigen-dependent proliferation and persistence of B7-H3 CAR T cells against G3 MB tumor lines in vitro. DNMT3A KO shows a more sustained effect over TET2. Currently, we are optimizing in vivo experiments to investigate prolonged tumor clearance in both immunocompromised and fully immunocompetent murine models.

MDB-07. MYC AND TGFΒ PROMOTE GROUP 3 MEDULLOBLASTOMA TUMOR RESISTANCE THROUGH DEREGULATION OF KDM2B 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. 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 KDM2B targets culminates in transcriptional changes that lead to aggressive disease. Overall, our studies provide important insight on identifying new therapeutic avenues for patients with MYC and TGFβ driven G3MB.

Metabolism-based targeting of MYC via MPC-SOD2 axis-mediated oxidation promotes cellular differentiation in group 3 medulloblastoma

Group 3 medulloblastoma (G3 MB) carries the worst prognosis of all MB subgroups. MYC oncoprotein is elevated in G3 MB tumors; however, the mechanisms that support MYC abundance remain unclear. Using metabolic and mechanistic profiling, we pinpoint a role for mitochondrial metabolism in regulating MYC. Complex-I inhibition decreases MYC abundance in G3 MB, attenuates the expression of MYC-downstream targets, induces differentiation, and prolongs male animal survival. Mechanistically, complex-I inhibition increases inactivating acetylation of antioxidant enzyme SOD2 at K68 and K122, triggering the accumulation of mitochondrial reactive oxygen species that promotes MYC oxidation and degradation in a mitochondrial pyruvate carrier (MPC)-dependent manner. MPC inhibition blocks the acetylation of SOD2 and oxidation of MYC, restoring MYC abundance and self-renewal capacity in G3 MB cells following complex-I inhibition. Identification of this MPC-SOD2 signaling axis reveals a role for metabolism in regulating MYC protein abundance that has clinical implications for treating G3 MB.

STAT3 Inhibition Attenuates MYC Expression by Modulating Co-Activator Recruitment and Suppresses Medulloblastoma Tumor Growth by Augmenting Cisplatin Efficacy In Vivo

MB is a common childhood malignancy of the central nervous system, with significant morbidity and mortality. Among the four molecular subgroups, MYC-amplified Group 3 MB is the most aggressive type and has the worst prognosis due to therapy resistance. The present study aimed to investigate the role of activated STAT3 in promoting MB pathogenesis and chemoresistance via inducing the cancer hallmark MYC oncogene. Targeting STAT3 function either by inducible genetic knockdown (KD) or with a clinically relevant small molecule inhibitor reduced tumorigenic attributes in MB cells, including survival, proliferation, anti-apoptosis, migration, stemness and expression of MYC and its targets. STAT3 inhibition attenuates MYC expression by affecting recruitment of histone acetyltransferase p300, thereby reducing enrichment of H3K27 acetylation in the MYC promoter. Concomitantly, it also decreases the occupancy of the bromodomain containing protein-4 (BRD4) and phosphoSer2-RNA Pol II (pSer2-RNAPol II) on MYC, resulting in reduced transcription. Importantly, inhibition of STAT3 signaling significantly attenuated MB tumor growth in subcutaneous and intracranial orthotopic xenografts, increased the sensitivity of MB tumors to cisplatin, and improved the survival of mice bearing high-risk MYC-amplified tumors. Together, the results of our study demonstrate that targeting STAT3 may be a promising adjuvant therapy and chemo-sensitizer to augment treatment efficacy, reduce therapy-related toxicity and improve quality of life in high-risk pediatric patients.

Abstract 3569: An animal model of GLI2-amplified medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant brain tumor affecting children. Among the known subtypes of MB, GLI2-amplified SHH-MB associated with P53 mutations has the worst prognosis, and a 5-year survival rate of less than 30%. Moreover, GLI2-amplified MBs are non-responsive to SMO inhibitors, the only targeted treatment option available for SHH-MB. To address this treatment gap, there is an urgent need to identify novel potential therapeutic targets, an effort that requires better understanding of key cellular and molecular mechanisms underlying GLI2-amplified MB. To that end, we generated a mouse model of GLI2-amplified MB and identified GL12 as the critical driver of tumorigenesis, and granule cell progenitors (GCPs) as the cells of tumor origin. We further found that GLI2 specifically drives embryonic, but not neonatal, Math1+ GCPs to form SHH-MB tumors. An scRNA-seq analysis revealed MAPK pathway activation specifically in embryonic Math1+ GCPs. The MAPK pathway was activated in mouse and human GLI2-driven MB tumors, and a MEK/ERK inhibitor significantly inhibited GLI2-driven MB in vivo. Based on these findings, we hypothesize that GLI2-driven MB originates from a specific population of Math1+ GCPs during a discrete temporal interval in cerebellar development, and that targeting MAPK/MEK/ERK pathway components represents a novel approach to treatment of GLI2-amplified MB. Citation Format: Yanxin Pei. An animal model of GLI2-amplified medulloblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3569.

Abstract 4764: The therapeutic potential of lnc-HLX-2-7 in group 3 medulloblastomas in children

Abstract Medulloblastoma (MB) is an aggressive brain tumor that predominantly affects children. Recent high-throughput sequencing studies suggest that the non-coding RNA genome, in particular long non-coding RNAs (lncRNAs), contributes to MB formation and tumor progression. Here we report the identification of a novel lncRNA, lnc-HLX-2-7, as a potential therapeutic target in group 3 MBs. In this study, we report that lnc-HLX-2-7 RNA specifically accumulates in the HLX (host gene of lnc-HLX-2-7) promoter region and activates HLX expression by recruiting multiple factors including enhancer elements. RNA sequencing and chromatin immunoprecipitation revealed that HLX directly binds to the promoters of several tumor-promoting genes, including MYC, and activates their expression. Furthermore, intravenous treatment with antisense oligonucleotides targeting lnc-HLX-2-7 coated with cerium-oxide nanoparticle (CNP-lnc-HLX-2-7) reduced tumor growth (40-50%) in intracranial MB xenograft mouse model (n=10, p<0.01, t-test). We found that the combinatorial therapy of CNP-lnc-HLX-2-7 and cisplatin further inhibits tumor growth and significantly prolongs mouse survival compared to CNP-lnc-HLX-2-7 monotherapy (n=10, p<0.01, t-test) only. We report here the importance of the lnc-HLX-2-7-HLX-MYC axis in regulating group 3 MB progression and provide a strong rationale for using lnc-HLX-2-7 as a specific and potent therapeutic target for the group 3 MBs in children. Citation Format: Keisuke Katsushima, Bongyong Lee, Menglang Yuan, Stacie Stapleton, George Jallo, Sudipta Seal, Charles G. Eberhart, Ranjan J. Perera. The therapeutic potential of lnc-HLX-2-7 in group 3 medulloblastomas in children. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4764.