bluuebunny/crossref_metadata_2025_split huggingface dataset:
MBRS-17. EXAMINING THE ROLE OF LHX9 IN GROUP 3 MEDULLOBLASTOMA
Authors: Injac Sarah , Huang L Frank , Mack Stephen , Braun Frank , Du Yuchen , Kogiso Mari , Lindsay Holly , Parsons D William , Zhao Hong , Lau Ching C , Wong Stephen T C , Li Xiao-nan 
Published: December 2020

Abstract
Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Despite major advances in our understanding of the biology of MB, novel treatments remain urgently needed. Using a chemical-genomics driven drug repositioning strategy, we identified the cardiac glycoside family of compounds as potential treatments for Group 3 MB. We subsequently demonstrated that single-agent treatment with digoxin prolongs survival in a patient-derived xenograft model (PDOX) of Group 3 MB to a degree comparable to radiation therapy, a mainstay in the treatment of MB. Finally, we examined the mechanism of digoxin-mediated cell killing using RNA-seq. This work identified LHX9, a member of the LIM homeobox family of transcription factors, as the gene most significantly down-regulated following treatment (Huang and Injac et al, Sci Trans Medicine, 2018). Homologs of LHX9 play key roles in cerebellar development via spatially and temporally restricted expression and LHX9 has been proposed as a core transcription factor (TF) in the regulatory circuitry of Group 3 tumors. Loss of function of other core TFs has been shown to impact MB growth. The role of LHX9 in MB, however, has not been previously experimentally evaluated. We now report that knockdown of LHX9 in MB-derived cell lines results in marked growth inhibition raising the possibility that loss of LHX9 plays a major role in digoxin-mediated cell killing and that LHX9 represents a key dependency required for the growth of Group 3 MB. Clinical targeting of core TFs would represent a novel approach to targeting this devastating disease.
MBRS-18. TUMOR SUPPRESSOR p53 DEFINES THE THERAPEUTIC RESPONSES IN TREATMENT OF MEDULLOBLASTOMA
Authors: Mohan Avinash L , Amin Anubhav G , Tobias Michael E , Das Mohan K , de Medeiros Raphael S S , Zanon Nelci , Gandhi Chirag D , Epelman Sidnei , Jhanwar-Uniyal Meena 
Published: December 2020

Abstract
Medulloblastoma (MB) is the most common primary pediatric malignant brain tumor. Current molecular analysis classifies MB into 4 groups, classic (WNT), sonic hedgehog (Shh), group 3, and group 4. Furthermore, atypical p53 signaling is associated with disease progression and confers poor prognosis. This study investigated the correlation of mutational status of p53 and iSO17q with disease progression and metastatic potential. In addition, we used small molecule inhibitors of PI3K (Buparlisib; BKM120) and HDAC (LBH-589) on a p53-mutant MB cell line to find novel therapeutic targets. Efficacy of these drugs were assessed using functional assays (cell proliferation, migration, cell cycle and drug resistance). MB tumors (n=53) were evaluated for GLI-1, GAB-1, NPR, KV1, YAP expression and mutant p53 via immunohistochemistry and correlated to patient outcomes. Results demonstrated that: 1) high expression of GAB-1 and YAP were found in the Shh group, while KV1 expression was present in all subtypes; 2) mutant p53 expression was present in various subsets of MB with no apparent correlation with metastasis or disease progression; 3) patients displaying iSO17q (determined by fluorescence in situ hybridization (FISH) technique) exhibited metastatic disease; 4) LBH-589 and BKM120 caused both time and dose-dependent inhibition of MB cell proliferation and migration; 5) combined treatment of BKM120 and LBH-589 had a synergistic effect; 6) MB cells demonstrated drug-resistance to BKM120. In conclusion, these findings underscore use of Buparlisib and LBH-589 in treatment of MB. Further, the role of mutant p53 in disease progression remains elusive, whereas presence of iSO17q defines metastatic potential.
MBRS-19. SYNERGISM OF HDAC AND PARP INHIBITORS IN MYC-DRIVEN GROUP 3 MEDULLOBLASTOMA CELLS
Authors: Vollmer Johanna , Ecker Jonas , Hielscher Thomas , Valinciute Gintvile , Oppermann Sina , Peterziel Heike , Oehme Ina , Witt Olaf , Milde Till 
Published: December 2020

Abstract
Patients with MYC-driven Group 3 medulloblastoma (MB) show particularly poor outcome. It was previously shown that MYC-driven MBs are highly sensitive to class I histone deacetylase inhibition (HDACi). We studied the molecular effects of the class I HDACi entinostat in MYC-driven MB cells to identify potentially synergistic drug combinations, prioritizing drug clinical availability to enable clinical translation. Gene expression profiles of the MYC-amplified group 3 MB cell line HD-MB03 treated with entinostat were analyzed using bioinformatic approaches, identifying 29 altered biomechanisms. Overlay with a translational drug library of n=76 compounds resulted in 44 compounds targeting 9 biomechanisms. Filtering for publications supporting each drug′s role in MYC-driven entities, or functional interaction with HDACs, without publication of this combination in MBs, resulted in 5 compounds (olaparib, idasanutlin, ribociclib, selinexor, vinblastine). Synergism testing identified olaparib as the drug with the strongest synergism. Validation of the combination olaparib and entinostat by p.H2AX and PI staining as well as trypan blue exclusion showed increased double strand breaks (DSBs), increased cell death, loss of viability and cell numbers. Selectivity of MYC-amplified MB cells was shown by comparison to MYC-non amplified cell lines, which showed higher IC50s, and reacted with cell cycle arrest as opposed to cell death to the combination treatment. The role of HDACis in DNA damage repair was confirmed by increased DSBs when entinostat was added to the combination of olaparib with doxorubicin. Our study identified olaparib as a potential combination partner with entinostat for the treatment of MYC-driven Group 3 MB.
MBRS-22. SIGNIFICANCE OF RNF213 IN TUMORGENICITY OF MEDULLOBLASTOMA
Authors: Mineharu Yohei , Oichi Yuki , Kamata Takahiko , Matsui Yasuzumi , Morimoto Takaaki , Tanji Masahiro , Kobayashi Hatasu , Okuda Hiroko , Harada Kouji H , Koizumi Akio , Arakawa Yoshiki , Miyamoto Susumu 
Published: December 2020

Abstract
RNF213 gene, initially identified as a disease-causing gene for moyamoya cerebrovascular disease, has recently been recognized as a tumor regulator. The gene is known to be associated with WNT signaling, lipid metabolism, angiogenesis and genomic instability. The purpose of this study was to investigate the association of RNF213 in tumorgenicity of medulloblastoma. Incidence of medulloblastoma and histopathological findings were compared among ptch1+/-, ptch1+/- rnf213+/-, and ptch1+/- rnf213-/- mice. Knockout of rnf213 in ptch1+/- transgenic mouse model increased the incidence of spontaneous generation of medulloblastoma from 19.8% (ptch1+/-) to 76.5% (rnf213+/- ptch1+/-) at 9 months (p &lt; 0.001). Heterozygous knockout was equivalent to homozygous knockout. Haploinsufficiency of rnf213 seems to be associated with tumorgenicity in medulloblastoma. Molecular mechanism of medulloblastoma generation needs to be further investigated.
MBRS-24. FUNCTIONAL CHARACTERIZATION OF IKBKAP/ELP1 AS A NOVEL SHH MEDULLOBLASTOMA PREDISPOSITION GENE
Authors: Lopez Jesus Garcia , Kutscher Lena , Kojic Marija , Gudenas Brian , Smith Kyle , Hadley Jennifer , Gajjar Amar , Robinson Giles W , Pfister Stefan M , Wainwright Brandon J , Kawauchi Daisuke , Northcott Paul A 
Published: December 2020

Abstract
Medulloblastoma (MB), a common malignant pediatric brain tumor, comprises at least four distinct molecular entities: WNT, SHH, Group 3, and Group 4. SHH-MB is driven by aberrant activation of the Sonic hedgehog (SHH) pathway in granule neuron progenitors (GNPs) and is associated with hereditary cancer predisposition syndromes including Li Fraumeni and Gorlin. We recently identified germline loss of function (LoF) mutations affecting IKBKAP/ELP1, the primary scaffolding subunit of the Elongator complex in a subset of SHH-MB patients. Germline ELP1 mutations account for ~15% of all pediatric SHH-MBs and position ELP1 as the most prevalent hereditary predisposition gene in MB. We genetically engineered Elp1 LoF in mouse GNPs to determine Elp1 function in cerebellar development and SHH-MB. Results of both mechanistic and phenotypic experiments demonstrate that GNPs harboring Elp1 loss exhibit ribosome pausing and protein aggregation, reinforcing the critical role of Elp1 in translational elongation and protein homeostasis. Further, we generated new transgenic mouse models mimicking germline ELP1 LoF mutations observed in SHH-MB patients. Elp1+/- transgenic mice exhibit purkinje cell degeneration and an increased DNA damage response. These mice are currently being evaluated for their capacity to recapitulate ELP1-associated SHH-MB. Additional analyses carried out on SHH-MB patient-derived xenografts showed that ELP1-mutant tumor cells specifically exhibit defects in tRNA biogenesis. Therefore, the function of ELP1 as a translational regulator is severely impaired in ELP1-mutant SHH-MBs. Our ongoing molecular and functional studies will provide important insights into the most common MB predisposition gene and will lay the foundation for future preclinical studies.
MBRS-27. EXOSOMES CARRY DISTINCT miRNAs THAT DRIVE MEDULLOBLASTOMA PROGRESSION
Authors: Jackson Hannah K , Linke Franziska , Kerr Ian , Coyle Beth 
Published: December 2020

Abstract

INTRODUCTION
Extracellular vesicles (EVs) represent an ideal source of functional biomarkers due to their role in intercellular communication and their ability to protect cargo, including RNA, from degradation. The most investigated EV’s are exosomes, nanovesicles secreted by all cell types and able to cross the blood-brain-barrier. Here we characterised the RNA of exosomes isolated from medulloblastoma cell lines, with the aim of investigating exosomal RNA cargo as potential functional biomarkers for medulloblastoma.

METHODS
Exosomes derived from a panel of matched (original tumour and metastasis) medulloblastoma cell lines were isolated and characterised by NanoSight, electron microscopy, western blotting and Nanoscale flow cytometry. Exosomal miRNA and mRNA from our matched cell lines and foetal neuronal stem cells, which were used as a normal control, were analysed by RNA-sequencing technology.

RESULTS
Based on hierarchical clustering, malignant derived exosomes were distinctly separated from normal control exosomes. miRNA profiling revealed several established oncomiRs identified in our malignant derived exosomes compared to control samples. Using interaction pathway analysis, we identified that our malignant exosomes carry numerous miRNAs implicated in migration, proliferation, cellular adhesion and tumour growth. Several previously identified oncomiRs were also identified to be present at higher levels in metastatic exosomes compared to primary and normal, including hsa-miR-455-3p and hsa-miR-92a-3p.

CONCLUSION
This study shows that exosomes from MB cells carry a distinct miRNA cargo which could enhance medulloblastoma progression. The use of circulating exosomes as markers of metastatic disease could be an innovative and powerful non-invasive tool.
MBRS-28. EXOSOMES DRIVE MEDULLOBLASTOMA METASTASIS IN A MMP2 AND EMMPRIN DEPENDENT MANNER
Authors: Jackson Hannah K , Linke Franziska , Kerr Ian , Coyle Beth 
Published: December 2020

Abstract

INTRODUCTION
Recurrent/metastatic medulloblastoma (MB) is a devastating disease with an abysmal prognosis of less than 10% 5-year survival. The secretion of extracellular vesicles (EVs) has emerged as a pivotal mediator for communication in the tumour microenvironment during metastasis. The most investigated EV’s are exosomes, nanovesicles secreted by all cell types and able to cross the blood-brain-barrier. Matrix metalloproteinases (MMPs) are enzymes secreted by tumour cells that can potentiate their dissemination by modification of the extracellular matrix. We hypothesise that exosomal MMP2 and its inducer EMMPRIN could enhance metastasis of MB.

METHODS
Proliferation, invasion and migration assays were used to evaluate the phenotypic behaviour of primary cell lines pre-treated with metastatic tumour cell-derived exosomes. Gelatin zymography and western blotting were performed to confirm MMP2 functional activity in cell lines and exosomes. Nanoscale flow cytometry was used to measure surface exosomal EMMPRIN levels. Exosomal MMP2 and EMMPRIN were modulated at the RNA level.

RESULTS
Number of exosomes is directly related to the migratory behaviour of parental MB cell lines (p&lt;0.01). Notably, functional exosomal MMP2 and EMMPRIN levels also correlate with this. Furthermore, exosomes from metastatic cell lines conferred enhanced migration and invasion on their matched isogenic primary (non-metastatic) cell line pair by ~3.8-fold (p&lt;0.01). Exosomes from metastatic cell lines also conferred increased migration on poorly migratory foetal neuronal stem cells.

CONCLUSION
Together this data suggests that exosomal MMP2 and EMMPRIN may promote medulloblastoma metastasis and supports analysis of exosomal MMP2 and EMMPRIN levels in patient cerebral spinal fluid samples.
MBRS-32. TOPOISOMERASE II β INDUCES NEURONAL, BUT NOT GLIAL, DIFFERENTIATION IN MEDULLOBLASTOMA
Authors: Miyahara Hiroaki , Natsumeda Manabu , Yoshimura Junichi , Fujii Yukihiko , Kakita Akiyoshi , Iwasaki Yasushi , Yoshida Mari 
Published: December 2020

Abstract

BACKGROUND
We previously reported that Gli3, which was a downstream molecule of Sonic Hedgehog signal, induced neuronal and/or glial differentiation in some types of medulloblastoma (desmoplastic/nodular medulloblastoma and medulloblastoma with extensive nodularity), and patients of medulloblastoma with neuronal differentiation showed favorable prognosis, but those with glial differentiation tended to show miserable prognosis (Miyahara H, Neuropathology, 2013). This time, we focused on Topoisomerase II β (Top2β), which was reported to induce neuronal differentiation and inhibit glial differentiation, and examined the expression of Top2β in medulloblastomas with neuronal and glial differentiations.

METHODS
We assessed the expression of Top2β, NeuN, and GFAP using triple fluorescent immunostaining method in medulloblastoma samples with both neuronal and glial differentiations. Furthermore, the expression of Top2β, H3K4me2, and H3K27me3 were also assessed, because Top2βwas positively or negatively regulated by H3K4me2 and H3K27me3, respectively.

RESULTS
Many large nuclei in the nodules, in which differentiated cells were seen, was visualized by Top2β. The Top2β signals were seen in NeuN+ cells but not GFAP+ cells. H3K4me2 signals were visualized in Top2β+ large nuclei, but H3K27me3 and NeuN+ large nuclei were distributed independently.

CONCLUSIONS
These results indicate that Top2β may be a molecule associated with neuronal, but not glial, differentiation of medulloblastoma cells. Drugs targeting histone modification enzymes such as EZH2 inhibitors are possible therapeutic targets as a differentiation-inducing therapy for medulloblastoma.
MBRS-37. RECURRENT ACTIVATING MUTATIONS OF AKT GENES IN WNT-ACTIVATED MEDULLOBLASTOMAS
Authors: Masliah-Planchon Julien , Lechapt-Zalcman Emmanuelle , Aillaud Jean-Baptiste , Ayrault Olivier , Pouponnot Célio , Doz Francois , Delattre Olivier , Bourdeaut Franck 
Published: December 2020

Abstract
Medulloblastoma (MB) can be classified into four distinct molecular subgroups (WNT group, SHH group, group 3, and group 4). Medulloblastoma of the WNT subgroup (WNT-MB) are commonly associated with favorable prognosis. Prospective molecular analysis based on a combination of CGH-array, targeted NGS and Nanostring-based subgrouping on 272 MB was conducted. Our custom targeted NGS panel of 75 genes includes genes recurrently affected in MB together with actionable genes with therapeutic purpose including some involved in the PIK3/AKT signaling pathway. Among the 272 MB analyzed, 26 cases (9.6%) belonged to the WNT subgroup based on CTNNB1 mutations, monosomy of chromosome 6 and Nanostring-based molecular subgrouping. Our targeted NGS revealed three hotspot activating mutations in AKT3 in WNT-MB and only one cases in another MB subgroup (in a group 4 MB; among the 33 cases of confirmed group 4 MB in our cohort). We subsequently performed Sanger sequencing of the hotspot Glu17 codon of AKT1, AKT2, and AKT3 in 42 additional WNT-MB. This analysis revealed six additional activating mutations of AKT genes (four AKT3 and two AKT1 hotspots mutations) in WNT-MB. Altogether, we report 9/68 (13.2%) cases of WNT-MB with AKT genes mutations (two mutations in AKT1 and seven mutations in AKT3). Our molecular analysis revealed AKT hotspot mutations that presumably activate the PIK3/AKT signaling pathway in WNT-MB. Even though WNT-MB is the subgroup of MB with the most favorable prognosis, this result emphasizes a possibility of targeted therapy that need to be further explored in vitro and in vivo.
MBRS-48. IDENTIFICATION OF NOVEL THERAPEUTIC APPROACHES FOR MYC-DRIVEN MEDULLOBLASTOMA
Authors: Taban Kübra , Pauck David , Maue Mara , Marquardt Viktoria , Yu Hua , Ayrault Olivier , Picard Daniel , Bartl Jasmin , Qin Nan , Borkhardt Arndt , Reifenberger Guido , Remke Marc 
Published: December 2020

Abstract
Medulloblastoma (MB) is the most common malignant brain tumor in children and is frequently metastatic at diagnosis. Treatment with surgery, radiation and multi-agent chemotherapy may leave survivors of these brain tumors with long-term deficits as a consequence. One of the four consensus molecular subgroups of MB is the MYC-driven group 3 MB, which is the most malignant type and has a poor prognosis under current therapy. Thus, it is important to discover more effective targeted therapeutic approaches. We conducted a high-throughput drug screening to identify novel compounds showing efficiency in group 3 MB using both clinically established inhibitors (n=196) and clinically-applicable compounds (n=464). More than 20 compounds demonstrated a significantly higher anti-tumoral effect in MYChigh (n=7) compared to MYClow (n=4) MB cell models. Among these compounds, Navitoclax and Clofarabine showed the strongest effect in inducing cell cycle arrest and apoptosis in MYChigh MB models. Furthermore, we show that Navitoclax, an orally bioavailable and blood-brain barrier passing anti-cancer drug, inhibits specifically Bcl-xL proteins. In line, we found a significant correlation between BCL-xL and MYC mRNA levels in 763 primary MB patient samples (Data source: “R2 https://hgserver1.amc.nl”). In addition, Navitoclax and Clofarabine have been tested in cells obtained from MB patient-derived-xenografts, which confirmed their specific efficacy in MYChigh versus MYClow MB. In summary, our approach has identified promising new drugs that significantly reduce cell viability in MYChigh compared to MYClow MB cell models. Our findings point to novel therapeutic vulnerabilities for MB that need to be further validated in vitro and in vivo.
MBRS-57. IDENTIFICATION OF MYC-DEPENDENT THERAPEUTIC VULNERABILITIES FOR TARGETING GROUP 3 MEDULLOBLASTOMA
Authors: Llargués-Sistac Gemma , Lindsey Janet , Swartz Shanel , Selby Matthew , Morcavallo Alaide , Bailey Simon , Chesler Louis , Lord Christopher J , Williamson Daniel , Clifford Steven C 
Published: December 2020

Abstract
Group 3 medulloblastoma (MBGroup3) is a highly aggressive tumour characterised by MYC amplification and elevated expression (17% of MBGroup3). MYC amplification in MBGroup3 confers a dismal prognosis using standard therapies, and there is an urgent unmet need for novel therapeutic approaches. The identification and targeting of MYC’s biological dependencies thus represents a promising strategy to treat MYC-MBGroup3 tumours. Three independent isogenic MYC-regulable MBGroup3 human cell-based models, in which elevated MYC expression can be directly down-regulated by doxycycline-inducible shRNAs, were developed and used initially to establish MYC-dependent growth of each model. Our novel models were then used to investigate MYC-dependent drug sensitivity, by characterising responses to a panel of candidate cancer therapeutics and small molecule inhibitors, including a high-throughput compound screen of &gt;500 established/clinically-relevant small molecule inhibitors. This approach identified several specific, consistently observed, druggable MYC-dependencies (e.g. cell cycle regulators, DNA-damage response controllers, mitotic control machinery) with potential for the development of treatments against MYC-MBGroup3 tumours. PLK1, CHK1 and AURK were identified as prime candidate targets with consistent MYC-dependent response profiles. Subsequent validation of each candidate, by genetic and pharmacological target inhibition, confirmed their MYC-dependent effects, associated with downregulation of MYC and established target-dependent pharmacodynamic biomarkers/pathways. Results were consistent across all of our MBGroup3 models. In summary, our novel models reveal druggable MYC-associated dependencies as a feature of MBGroup3. Our findings support the development of PLK1, CHK1 and AURK inhibition as therapeutic approaches against MYC-dependent MBGroup3. Future work is now essential to validate our findings in vivo, to support the design of future clinical trials.
MBRS-59. SINGLE-CELL WHOLE-GENOME SEQUENCING DISSECTS INTRA-TUMOURAL GENOMIC HETEROGENEITY AND CLONAL EVOLUTION IN CHILDHOOD MEDULLOBLASTOMA
Authors: Danilenko Marina , Zaka Masood , Keeling Claire , Crosier Stephen , Hussain Rafiqul , Schwalbe Edward , Williamson Dan , Coxhead Jonathan , Rand Vikki , Bailey Simon , Clifford Steven 
Published: December 2020

Abstract
Medulloblastomas harbor clinically-significant intra-tumoral heterogeneity for key biomarkers (e.g. MYC/MYCN, β-catenin). Recent studies have characterized transcriptional heterogeneity at the single-cell level, however the underlying genomic copy number and mutational architecture remains to be resolved. We therefore sought to establish the intra-tumoural genomic heterogeneity of medulloblastoma at single-cell resolution. Copy number patterns were dissected by whole-genome sequencing in 1024 single cells isolated from multiple distinct tumour regions within 16 snap-frozen medulloblastomas, representing the major molecular subgroups (WNT, SHH, Group3, Group4) and genotypes (i.e. MYC amplification, TP53 mutation). Common copy number driver and subclonal events were identified, providing clear evidence of copy number evolution in medulloblastoma development. Moreover, subclonal whole-arm and focal copy number alterations covering important genomic loci (e.g. on chr10 of SHH patients) were detected in single tumour cells, yet undetectable at the bulk-tumor level. Spatial copy number heterogeneity was also common, with differences between clonal and subclonal events detected in distinct regions of individual tumours. Mutational analysis of the cells allowed dissection of spatial and clonal heterogeneity patterns for key medulloblastoma mutations (e.g. CTNNB1, TP53, SMARCA4, PTCH1) within our cohort. Integrated copy number and mutational analysis is underway to establish their inter-relationships and relative contributions to clonal evolution during tumourigenesis. In summary, single-cell analysis has enabled the resolution of common mutational and copy number drivers, alongside sub-clonal events and distinct patterns of clonal and spatial evolution, in medulloblastoma development. We anticipate these findings will provide a critical foundation for future improved biomarker selection, and the development of targeted therapies.