EPCO-47. DISSECTING MYC-DRIVEN EPIGENETIC MECHANISMS THAT PROMOTE GROUP 3 MEDULLOBLASTOMA UTILIZING HUMANIZED IPSC MODELS

Medulloblastoma (MB) is one of the most prevalent malignant brain tumors in children, with tremendous cognitive and neuroendocrine disability among survivors. Group 3 MB (G3MB) has poor overall survival at <50%, high frequencies of metastases, and no targeted therapies. Amplification of MYC and activation of TGFβ signaling occur frequently in G3MB. Many tumors have no reported mutations, suggesting epigenetic drivers. We here describe novel humanized models for G3MB from human induced pluripotent stem cells (hiPSC). By transducing hiPSC-derived neuroepithelial stem cells (NESC), we determined that: 1) both MYC and TGFβ effectors drove tumors in vivo; 2) MYC/TGFβR1 in combination led to more aggressive tumors and resistance to clinical inhibitors of TGFβ, and 3) NESC-derived tumors clustered with human G3MB. To decipher mechanisms, we integrated RNA-sequencing with CUT&RUN (for MYC genomic localization and post-translational modification of histones). MYC-bound neural developmental genes were repressed in MYC/TGFβR1 co-driven lines. Gene signatures associated with the Polycomb Repressive Complex (PRC) demarcated with H3K27me3; the histone mark directly regulated by PRC. We identified JARID1B, a MYC binding partner and H3K4me3 demethylase, as a regulator of repressed neural genes. Primary G3MB also showed increased levels of H3K27me3 concurrent with higher expression of JARID1B. Knockdown of JARID1B in human G3MB cell lines reduced growth, supporting potential as a therapeutic target. We conclude that a MYC-TGFβ-JARID1B axis represses target genes to drive G3MB and present new humanized models for G3MB to understand epigenetic dysregulation in G3MB.

KDM6A/B Demethylases in Gut Homeostasis

Medulloblastoma (MB) is the most common malignant pediatric posterior fossa tumor. Recent genetic, epigenetic, and transcriptomic analyses have classified MB into three subgroups, Wingless Type (WNT), Sonic Hedgehog (SHH), and non-WNT/non-SHH (originally termed Group 3 and Group 4), with discrete patient profiles and prognoses. WNT is the least common subgroup with the best prognosis, characterized by nuclear β-catenin expression, mutations in Catenin beta-1 (CTNNB1), and chromosome 6 monosomy. SHH tumors contain mutations and alterations in GLI1, GLI2, SUFU, and PTCH1 genes, which constitutively activate the SHH pathway. Originally, the presence of TP53 gene alterations and/or MYC amplifications was considered the most reliable prognostic factor. However, recent molecular analyses have subdivided SHH MB into several subtypes with distinct characteristics such as age, TP53 mutation, MYC amplification, presence of metastases, TERT promoter alterations, PTEN loss, and other chromosomal alterations as well as SHH pathway-related gene mutations. The third non-WNT/non-SHH MB (Group3/4) subgroup is genetically highly heterogeneous and displays several molecular patterns, including MYC and OTX2 amplification, GFI1B activation, KBTBD4 mutation, GFI1 rearrangement, PRDM6 enhancer hijacking, KDM6A mutation, LCA histology, chromosome 10 loss, isochromosome 17q, SNCAIP duplication, and CDK6 amplification. However, based on molecular profiling and methylation patterns, additional non-WNT/non-SHH MB subtypes have been described. Recent WHO (2021) guidelines stratified MB into four molecular subgroups with four and eight further subgroups for SHH and non-WNT/non-SHH MB, respectively. In this review, we discuss advancements in genetics, epigenetics, and transcriptomics for better characterization, prognostication, and treatment of MB using precision medicine.

Microcephaly-associated mutations disrupt microtubule-dependent WDR62 translocation from the Golgi complex to the mitotic spindle poles, impair mitotic progression, and alter neurogenic trajectories in patient induced Pluripotent Stem Cell-derived 2D and 3D models of human neurodevelopment.

Medulloblastoma (MB), the most common embryonal tumour of the Central Nervous System, occurs in the cerebellum. Treatment regimens involve surgery, craniospinal radiotherapy, and chemotherapy. The greatest mortality is associated with disseminated disease, almost exclusively found in the leptomeningeal space. Unfortunately, knowledge about the aetiology of MB spread is limited and the need for kinder and efficacious therapy remains an unmet goal. Of the four molecular classified MB groups, Group3 (Gr3) MB presents with a high frequency of metastasis at diagnosis, with the worst overall survival. Gr3 MB tumours are dominated by primitive progenitor-like cells and cMYC deregulation; often, p53 deficiency is observed at relapse. To dissect the biology of primary and metastatic Gr3 MB, we have developed a new germline genetically engineered mouse model (GEMM), harbouring cMYC amplification in a Tamoxifen-inducible p53 functional background (Trp53ERTAM strain). A novel LSL-cMYC-CopGFP-Luciferase transgene was integrated into the Rosa-26 locus of the mouse genome. Transgenic mice were crossed with a strain expressing Cre recombinase under the Blbp promoter targeting embryonic neural progenitors, and subsequently bred to Trp53ERTAM mice. As result, the cMYC overexpression was sufficient to generate tumours. Tumour penetrance was observed in all the expected tumour bearing genotypes, with increased aggressiveness in a non-functional p53 background. Bioluminescence imaging demonstrated tumour onset in the brain and dissemination along the spinal cord. CopGFP positive tumour cells were isolated from primary and metastatic tumours. Pathological interrogation confirmed that tumours present large cell/anaplastic (LCA) histology. Analysis of preliminary transcriptional profiling data proved that tumours cluster with human Gr3 MB. Ongoing methylation profiling and multi-omics approaches will inform on the tumour cells of origin and clonal divergence of primary tumour versus metastasis. In conclusion, we have successfully developed a novel immunocompetent mouse model of metastatic Gr3 MB with which we can investigate therapeutic vulnerabilities of MB.

&lt;div&gt;Abstract&lt;p&gt;Medulloblastoma (MB) is the most common malignant pediatric brain tumor. MYC-driven MBs, commonly found in the group 3 MB, are aggressive and metastatic with the worst prognosis. Modeling MYC-driven MB is the foundation of therapeutic development. Here, we applied a synthetic mRNA-driven strategy to generate neuronal precursors from human-induced pluripotent stem cells (iPSCs). These neuronal precursors were transformed by the &lt;i&gt;MYC&lt;/i&gt; oncogene combined with &lt;i&gt;p53&lt;/i&gt; loss of function to establish an MYC-driven MB model recapitulating the histologic and transcriptomic hallmarks of group 3 MB. We further show that the marine compound Frondoside A (FA) effectively inhibits this MYC-driven MB model without affecting isogenic neuronal precursors with undetectable MYC expression. Consistent results from a panel of MB models support that MYC levels are positively correlated with FA's antitumor potency. Next, we show that FA suppresses MYC expression and its downstream gene targets in MB cells, suggesting a potential mechanism underlying FA's inhibitory effects on MYC-driven cancers. In orthotopic xenografts of MYC-driven MB, intratumoral FA administration potently induces cytotoxicity in tumor xenografts, significantly extends the survival of tumor-bearing animals, and enhances the recruitment of microglia/macrophages and cytotoxic T lymphocytes to tumors. Moreover, we show that MYC levels also predict FA potency in glioblastoma and non–small cell lung cancer cells. Taken together, this study provides an efficient human iPSC-based strategy for personalizable cancer modeling, widely applicable to mechanistic studies (e.g., genetic predisposition to cancer) and drug discovery. Our preclinical results justify the clinical translation of FA in treating MYC-driven MB and other human cancers.&lt;/p&gt;&lt;/div&gt;

Group 4 medulloblastoma (MB) is the most common medulloblastoma subgroup and shows high incidence of metastasis and late-onset relapse. Group 4 MBs lack a unifying oncogenic driver and treatment targets, despite extensive genomic characterization. Group 4 MBs are characterized by recurrent genetic alterations in chromatin modifiers, amplification of stemness genes, and putative enhancer "hijacking" events. A substantial fraction of Group 4 MBs are characterized by enhancer hijacking through tandem duplication of SNCAIP, resulting in high expression of PRDM6, a putative transcriptional repressor and histone methyltransferase. Some PRDM6-overexpressing MBs show additional mutations in chromatin regulators and high MYCN expression. We set out to elucidate the impact and oncogenic potential of sustained PRDM6 expression in early neural stem cell populations and patient-derived medulloblastoma cells. We find that PRDM6 expression in human iPSC-derived neuroepithelial stem cells (NESCs) results in tumor growth in mice, albeit at low penetrance. Moreover, we find that PRDM6 expression in MYCN-overexpressing NESCs does not further alter tumor aggressiveness or survival in vivo. Notably, PRDM6 overexpression in the patient-derived Group 3 MYC-amplified D283-Med cell line causes significantly increased aggressiveness of tumor growth and shorter survival in mice. At the cellular level, PRDM6 localizes to the nucleus, suggesting a role in gene expression regulation. Consistent with this notion, ATAC-seq and RNA-seq analysis of PRDM6-NESCs and PRDM6-D283-Med cells reveals major changes in chromatin accessibility and gene expression, including upregulation of integrin family members and genes related to focal adhesion and extracellular matrix-receptor interaction pathways. We conclude that PRDM6 promotes tumor growth in NESCs and may play a role in tumor maintenance through interactions with the extracellular matrix and tumor microenvironment.

Medulloblastoma (MB) is the most common malignant brain tumor in childhood and is stratified into four molecular groups ‒ Wingless and Int-1 (WNT), Sonic hedgehog (SHH), Group 3 and Group 4. Group 3 MB patients exhibit the poorest prognosis, with a 5-year overall survival of <60%, followed by Group 4 MB patients. Apart from MYC amplification in a subset of Group 3 MBs, the molecular pathomechanisms driving aggressiveness of these tumors remain incompletely characterized. The gene encoding the mTOR substrate and mRNA translation inhibitor eukaryotic translation initiation factor 4E-binding protein 1 (EIF4EBP1) represents a possible MYC target gene whose corresponding protein, 4EBP1, was shown to be more active in Group 3 versus Group 4 MBs. However, the prognostic role of 4EBP1 in MB and the mechanisms supporting 4EBP1 overexpression in Group 3 MB are still elusive. We analyzed EIF4EBP1 mRNA expression in publicly available data sets and found an upregulation in MB as compared to non-neoblastic brain. EIF4EBP1 mRNA expression levels were higher in Group 3 compared to Group 4 MBs. EIF4EBP1 mRNA expression was correlated with MYC expression, most prominently in Group 3 MBs. Survival analyses highlighted that high EIF4EBP1 mRNA expression was associated with reduced overall and event-free survival across all MB patients and in Group 3/Group 4 MB patients. Immunohistochemical evaluation of 4EBP1 protein expression in MB tissues confirmed that high levels of 4EBP1 are associated with poor outcome. Functional analyses revealed that MYC directly regulates EIF4EBP1 promoter activity, providing a mechanism for increased EIF4EBP1 mRNA levels in Group 3 MBs. Finally, we observed that 4EBP1 may support colony formation of in vitro cultured MB cells. Our data highlight that transcriptional upregulation of EIF4EBP1 by MYC promotes in vitro tumorigenicity of MB cells and associates with shorter survival of MB patients.

H3K27 trimethylation, catalyzed by the Polycomb Repressive Complex 2 (PRC2), is mainly associated with transcriptional repression of target genes. PRC2 activity is counterbalanced by UTX and JMJD3, belonging to the family of Jmj-C-containing H3K27me3 demethylases. UTX acts in concert with members of the Mixed Lineage Leukemia (MLL) family of H3K4 methyltransferases to sustain gene transcription. In vivo studies have assigned to UTX important roles in hematopoiesis, particularly in immune cell differentiation. We showed that Utx is expressed throughout B cell development. To study the role of UTX in B lymphopoiesis, we crossed Utx conditional knock-out mice to the Mb1cre/+ strain, to inactivate the demethylase throughout B cell development, starting from pro-B cells. Our results suggest that UTX influences B cell physiology in multiple ways, controlling mature B cell subset differentiation and survival, B cell distribution among lymphoid organs, Ig isotype switching and antigen-driven germinal center (GC) B cell clonal selection. As a result of these defects, B cell-specific Utx deficiency causes a mild humoral immunodeficiency, affecting primarily intestinal IgA-linked immunity. Importantly, these defects resemble those described in patients affected by the Kabuki Syndrome, a genetic disorder caused by mutations in either UTX or in the MLL methyltransferase KMT2D. Chromatin immunoprecipitation studies coupled to sequencing performed on Utx mutant resting and GC B cells started to unveil the identity of the transcriptional and epigenetic networks under the influence of UTX. These studies could improve our understanding of how deregulated UTX participates to the pathogenesis of immunological disorders, including B cell malignancies.

NF2-related schwannomatosis is a tumor predisposition syndrome caused by mutations in the NF2 gene and associated with spinal ependymomas (SP-EPN). SP-EPNs are suspected to originate from aberrations in the radial glia (RG) cell lineage. These tumors are only effectively treated through high-risk surgical resection, emphasizing the critical need for identification of targets for medical therapy. Yet, the role of NF2-dependent disruption in RG cell development is poorly understood. We hypothesize that this mutation may be halting normal RG cell differentiation, resulting in a RG-like stem cell that is driving SP-EPN formation. An NF2-knockout was generated in neuroepithelial stem (NES) cells using CRISPR/Cas9. Knockouts were validated using Western blot and Sanger sequencing. In vitro differentiation was induced with removal of growth factors. NF2-knockout phenotypes were assessed with real-time polymerase chain reaction and compared with wildtype NES cells. Preliminary data has shown that NF2-knockout cells express similar levels of early pan-neural and neural stem cell genes compared to wildtype after CRISPR editing. The NF2-knockout cells retain this stem cell-like gene expression following attempted differentiation, whereas wildtype cells take on a primarily neural phenotype. The knockout cells also form what appears to be pre-neoplastic spheres when allowed to differentiate. NF2-knockout NES cells fail to differentiate normally compared to wildtype NES cells. They retain early RG-like markers, including SOX2, BLBP, and GFAP. Furthermore, formation of spheres when growth factors were removed hints at NF2 loss being relevant to formation of pre-neoplastic growths. Future work will include investigating downstream effects of NF2 loss in this model.

Simple SummaryGroup 3 medulloblastoma (MB) is often accompanied by MYC amplification and has a poor prognosis. FBXW7, a critical tumor suppressor in many types of cancer, regulates the proteasome-mediated degradation of oncoproteins including MYC. However, the role of FBXW7 in the tumorigenesis of group 3 MB has not been well studied. In this study, we show that FBXW7 is downregulated in group 3 MB patient samples, and FBXW7 stabilization is crucial for inhibiting c-MYC. We identified a FBXW7-MYC-PLK1 regulatory loop in MYC-driven MB, which provides a mechanism of using protein kinase inhibitors for translation in the future.Polo-like kinase 1 (PLK1) is highly expressed in group 3 medulloblastoma (MB), and it has been preclinically validated as a cancer therapeutic target in medulloblastoma. Here, we demonstrate that PLK1 inhibition with PCM-075 or BI6727 significantly reduces the growth of MB cells and causes a decrease of c-MYC mRNA and protein levels. We show that MYC activates PLK1 transcription, while the inhibition of PLK1 suppresses MB tumor development and causes a decrease in c-MYC protein level by suppressing FBXW7 auto poly-ubiquitination. FBXW7 physically interacts with PLK1 and c-MYC, facilitating their protein degradation by promoting ubiquitination. These results demonstrate a PLK1-FBXW7-MYC regulatory loop in MYC-driven medulloblastoma. Moreover, FBXW7 is significantly downregulated in group 3 patient samples. The overexpression of FBXW7 induced apoptosis and suppressed proliferation in vitro and in vivo, while constitutive phosphorylation mutation attenuated its tumor suppressor function. Altogether, these findings demonstrated that PLK1 inhibition stabilizes FBXW7 in MYC-driven MB, thus revealing an important function of FBXW7 in suppressing medulloblastoma progression.

MYC-driven Group 3 (G3) medulloblastoma (MB) is the most aggressive of four molecular subgroups classified by transcriptome, genomic landscape and clinical outcomes. Mouse models that recapitulate human G3 MB all rely on retroviral vector-induced Myc expression driven by viral regulatory elements (Retro-Myc tumors). We used nuclease-deficient CRISPR/dCas9-based gene activation with combinatorial single guide RNAs (sgRNAs) to enforce transcription of endogenous Myc in Trp53-null neurospheres that were orthotopically transplanted into the brains of naïve animals. Three combined sgRNAs linked to dCas9-VP160 induced cellular Myc expression and large cell anaplastic MBs (CRISPR-Myc tumors) which recapitulated the molecular characteristics of mouse and human G3 MBs. The BET inhibitor JQ1 suppressed MYC expression in a human G3 MB cell line (HD-MB03) and CRISPR-Myc, but not in Retro-Myc MBs. This G3 MB mouse model in which Myc expression is regulated by its own promoter will facilitate pre-clinical studies with drugs that regulate Myc transcription.

INTRODUCTION Molecular classification of medulloblastomas(MB) is prognostically and therapeutically relevant and helps in better risk-stratification. Translation of this subgrouping to routine practice still remains a challenge. The most pathologist-accessible techniques for molecular subgrouping include immunohistochemistry(IHC), fluorescent in-situ hybridization(FISH) and Nanostring assay. Objectives:(1)Molecular subgrouping of MBs by IHC and FISH, and Nanostring Assay (2)To compare their efficacy against sequencing and DNA methylation, and cost for applicability in resource-constrained centers METHODS Ninety-five cases of MB with adequate tissue were included. Molecular subgrouping was performed by IHC for β-catenin, GAB1, YAP1, and p53; FISH for MYC amplification, and sequencing for CTNNB1, and by Nanostring Assay on the same set of MBs. Further, a subset of cases were subjected to 850k DNA methylation array. RESULTS IHC+FISH classified MBs into 15.8% WNT, 16.8% SHH, and 67.4% non-WNT/non-SHH subgroups; with MYC amplification identified in 20.3% cases of non-WNT/non-SHH. A single showed diffuse strong p53 positivity among the SHH subgroup. Nanostring successfully classified 91.5% MBs into 25.3% WNT, 17.2% SHH, 23% Group3 and 34.5% Group4. However, Nanostring assay failure was seen in eight cases, all of which were &gt;8-years-old formalin-fixed paraffin-embedded tissue blocks. Concordant subgroup assignment was noted in 88.5% cases, while subgroup switching was seen in 11.5% cases. Both methods showed prognostic correlation. Among the 5 discrepant cases, which switched to WNT subgroup by Nanostring, only 2 were found to have CTNNB1 mutation. Methylation profiling performed on discordant cases revealed 1 out of 4 extra WNT identified by Nanostring to be WNT, others aligned with IHC subgroups; extra SHH by Nanostring turned out to be SHH by methylation. CONCLUSIONS Both IHC supplemented by FISH and Nanostring are robust methods for molecular subgrouping, albeit with few disadvantages. IHC cannot differentiate between Groups 3 and 4, while Nanostring cannot classify older-archived tumors, and is not available at most centres. WNT subgroup with the best prognosis is best detected by IHC or IHC followed by sequencing for confirmation. Nanostring Assay is better suitable to separate Group 3, the worst prognostic group from Group 4. Thus, both the methods complement each other and can be used in concert for high confidence allotment of molecular subgroups in clinical practice. The cost of IHC plus Nanostring will almost be the same as IHC plus FISH. We recommend a cost-efficient algorithmic approach using histopathological subtype and IHC as the first step followed by Nanostring or FISH, wherever suitable.

Medulloblastoma (MB) is the most common solid malignant pediatric brain neoplasm, with Group 3 (G3) MB representing the most aggressive subgroup. MYC amplification is an independent poor prognostic factor in G3 MB, however, therapeutic targeting of the MYC pathway remains limited and alternative therapies for G3 MB are urgently needed. Here we show that an RNA-binding protein, Musashi-1 (MSI1) is an essential mediator of G3 MB in both MYC-overexpressing mouse models and patient-derived xenografts. Unbiased integrative multi-omics analysis of MSI1 function in human G3 MB suggests a paradigm shift beyond traditional gene-based profiling of oncogenes. Here we identify MSI1 as an oncogene in G3 MB driving stem cell self-renewal through stabilization of HIPK1 mRNA, a downstream context-specific therapeutic target for drug discovery.

SummaryNeural crest cells (NCCs) contribute to several tissues during embryonic development. NCC formation depends on activation of tightly regulated molecular programs at the neural plate border (NPB) region, which initiate NCC specification and epithelial-to-mesenchymal transition (EMT). Although several approaches to investigate NCCs have been devised, these early events of NCC formation remain largely unknown in humans, and currently available cellular models have not investigated EMT. Here, we report that the E6 neural induction protocol converts human induced pluripotent stem cells into NPB-like cells (NBCs), from which NCCs can be efficiently derived. NBC-to-NCC induction recapitulates gene expression dynamics associated with NCC specification and EMT, including downregulation of NPB factors and upregulation of NCC specifiers, coupled with other EMT-associated cell-state changes, such as cadherin modulation and activation of TWIST1 and other EMT inducers. This strategy will be useful in future basic or translational research focusing on these early steps of NCC formation.