bluuebunny/crossref_metadata_2025 huggingface dataset:
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

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

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

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 < 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

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

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

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<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<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

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

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

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

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 >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

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.
MBRS-61. MOLECULAR SUB-GROUPING OF PEDIATRIC MEDULLOBLASTOMA: CORRELATION WITH CLINICAL AND HISTOLOGICAL FEATURES, A SINGLE INSTITUTIONAL STUDY
Authors: Deshpande Gauri , Gurav Mamta , Shetty Omshree , Kadam Vinayak , Chaubey Vishal , Gupta Tejpal , Moiyadi Aliasgar , Chinnaswamy Girish , Epari Sridhar
Published: December 2020

INTRODUCTION

Molecular subgroups of pediatric medulloblastomas are distinctive in infantile and non-infantile age-groups.

METHODS

Real-time quantitative PCR based GEP of customized 12 protein-coding genes was performed on 206 FFPE childhood medulloblastoma samples. FISH for MYC amplification, monosomy 6 and sequencing for CTNNB1 exon 3 mutation were done in relevant cases. H&E and reticulin-stained slides were used for histological subtyping. p53-protein immunoreactivity pattern was noted.

RESULTS

Infantile (n=33) comprised 57.6% SHH-activated (desmoplastic: 73.7%; MBEN: 15.8% and classic: 10.5%), 21.2% group 3 (large cell/anaplastic [LCA]: 28.6% and none were desmoplastic) and 12% group 4. 40% of group 3 patients died of disease and 21% of the SHH-activated (all desmoplastic) had subsequent local recurrence. Non-infantile (n=173) comprised 19.4% WNT-activated, 12.9% SHH-activated (15% classic, 30% desmoplastic, 10% paucinodular), 19.4% group 3 (63.3% classic & 26.7% LCA), 48.4% group 4 (73.3% classic, 5.3% desmoplastic, 10.7% paucinodular & 1.4% LCA), and non-WNT/non-SHH (NWNS), NOS (n=14,9%) and unclassified (n=4,2.6%). None of WNT-activated were desmoplastic/LCA histology. Non-infantile WNT-activated and group 3 MBs showed 90% monosomy 6 & CTNNB1 mutation, and 16.7% MYC-amplification respectively. 17.4% (13% spinal, 4.4% local) WNT-activated, 31% (12.5% local, 18.5% distant [spinal: 12.5%, intracranial:6%]) SHH-activated, 27% (18% both spinal and local, 9% spinal) group 3 and 31.5% (7.4% local, 5.5% intracranial, 11.2% spinal, 7.4% both spinal and local) group 4 showed metastases during follow up.

CONCLUSIONS

SHH-activated and group 3 are the common infantile subgroups but group 4 is not non-existent in infantile age. No desmoplastic (including paucinodular) histological subtype is of WNT- activated and group 3.
MBRS-65. FBXW7 ACTS A TUMOR SUPPRESSOR IN MYC-DRIVEN MEDULLOBLASTOMA BY CONTROLLING A FEED-FORWARD REGULATORY LOOP OF PLK1 AND MYC
Authors: Wang Dong , Pierce Angela , Veo Bethany , Fosmire Susan , Madhavan Krishna , Balakrishnan Ilango , Venkataraman Sujatha , Vibhakar Rajeev
Published: December 2020

Group 3 medulloblastoma (MB) is often accompanied by MYC amplification and has a higher rate of metastatic disease. So, it is critical to have more effective therapies for high MYC expressing sub-groups. Here we report that FBXW7, a substrate recognition component of the SKP1-CUL1-Fbox (SCF) E3 ligase, interacts with and targets c-MYC for polyubiquitination and proteasomal degradation. FBXW7 shows lower expression level in MYC-driven MB compared with other MB subgroups suggesting activity as a tumor suppressor. Genomic deletion or mutation of Fbxw7 has frequently been identified in many human cancers but not in MB. We demonstrate that overexpression of Fbxw7 in MB cells induces apoptosis and suppresses proliferation in vitro and in vivo. Both phospho-deficient (T205A) and phosphomimetic aspartic acid (T205D) mutants deactivate its tumor suppressor function suggesting a conformational change of its protein structure. Mechanistically, PLK1 kinase specifically phosphorylates FBXW7 and promotes its auto-polyubiquitination and proteasomal degradation, counteracting FBXW7-mediated degradation of oncogene substrates, including c-MYC and PLK1. Chip-Seq results show stabilized c-MYC in turn directly activates PLK1 and FBXW7 transcription, constituting a feedforward regulatory loop. Co-immunoprecipitation demonstrates that FBXW7 directly binds to PLK1 and c-MYC, facilitating their protein degradation by promoting the ubiquitination of both proteins. Furthermore, we show that FBXW7 protein can be stabilized by various kinase inhibitors, proposing a mechanism of kinase-targeted agents to treat MYC-driven MB. These results collectively demonstrate how kinase inhibition stabilizes the tumor suppressor FBXW7 in MYC-driven MB, thus revealing an important function of FBXW7 in suppressing MB progression.
MBRS-67. ROLE OF CYCLIN DEPENDENT KINASE-9 IN Myc-ENHANCED MEDULLOBLASTOMA
Authors: Madhavan Krishna , Veo Bethany , Danis Etienne , Balakrishnan Ilango , Pierce Angela , Wang Dong , Abdel-Hafiz Ahmed , Serkova Natalie , Dahl Nathan , Venkataraman Sujatha , Vibhakar Rajeev
Published: December 2020

Myc is highly expressed in group 3 medulloblastoma (Myc-MB) and influences cell growth, proliferation and oncogenesis by directly promoting the activity of RNA polymerases (RNA Pol). Myc driven RNA Pol II activity is mediated by Positive Transcription Elongation Factor b (pTEFb). pTEFb’s catalytic core consists of cyclin dependent kinase-9 (CDK9) and Cyclin T, that phosphorylate and release RNA Pol II into active elongation. CDK9 is over expressed in group 3 MB suggesting that MB may be vulnerable to inhibition of CDK9 (CDK9i). The exact mechanism is not completely known in MB. Genetic depletion of CDK9 suppressed Myc-MB cell clonogenicity in vitro and tumor growth in vivo. CDK9i by two clinically relevant inhibitors, Atuveciclib and AZD4573, suppressed clonogenicity and cell self-renewal of Myc-MB cell lines. CDK9i in Myc-MB cell lines downregulated Myc and RNA Pol II phosphorylation at Ser2 and Ser5, and, upregulated P21. Further, mice with orthotopic xenografts treated with CDK9 inhibitors survived significantly longer than control mice. RNA-Seq-based gene set enrichment analysis showed that CDK9i decreased c-Myc driven transcriptomic programs and enhanced differentiation networks. ChIP-Seq for Pol2 and Myc, demonstrated that the Myc-driven aberrant transcriptional input can be reversed via CDK9i. These findings highlight the role of CDK9 in Myc-driven pathogenesis and that its inhibition is critical to the treatment of Myc-MB.
MODL-02. TARGETING REPLICATION STRESS IN PEDIATRIC BRAIN TUMORS
Authors: Krausert Sonja , Lambo Sander , Mack Norman , Schwalm Benjamin , Pfister Stefan , Kool Marcel
Published: December 2020

Pediatric brain tumors harboring amplifications or high overexpression of MYC-/MYCN are often associated with poor outcome. High MYC(N) expression in these tumors leads to increased transcription, which can be in conflict with DNA replication and subsequently can cause replication stress, R-loops and DNA damage. We hypothesize that high MYC(N) expression makes them vulnerable to DNA damage response inhibitors (DDRi) and even more vulnerable to combinations of DDRi and chemotherapeutics. To test this hypothesis we performed in vitro drug experiments using Group 3 medulloblastoma (MB) and ETMR cell lines. IC50-values were evaluated of topoisomerase inhibitor Irinotecan (SN-38) and Pamiparib (BGB-290), a brain-penetrant PARP-inhibitor, in monotherapy. All cell lines were sensitive for SN-38 and showed IC50-values in the low nM-range but PARP-inhibitors were ineffective. However, a significant decrease in IC50 can be observed when SN-38 and Pamiparib are used in combination. For in vivo treatments, we injected NSG mice with luciferase labelled patient-derived xenograft- (PDX-) cells of various models (MB Group 3, MB SHH, ETMR, RELA EPN), monitored tumor growth via IVIS and randomized the mice into four groups (vehicle, BGB-290, Irinotecan and Irinotecan+Pamiparib) when a predefined threshold of tumor growth was reached. Mice were treated with Irinotecan (or vehicle) once per day i.p. and Pamiparib (or vehicle) twice per day per oral gavage. Treatment with Pamiparib did not show any survival benefit, but mice treated with Irinotecan or the combination showed a clear survival benefit. Treatments are ongoing and more results will be presented at the conference.
MODL-04. MODELING CNS HGNET-BCOR PATHOGENESIS USING NEURAL STEM CELLS
Authors: Nakata Satoshi , Yuan Ming , Raabe Eric , Eberhart Charles
Published: December 2020

Central nervous system high-grade neuroepithelial tumor with BCL6-corepressor alteration (CNS HGNET-BCOR) is a recently identified entity characterized by internal tandem duplication (ITD) of BCOR, a core component of polycomb repressive complex (PRC) 1.1. BCOR-ITD exclusively occurs within an essential binding domain, suggesting aberrant epigenetic activities as a possible mechanism of gliomagenesis; however, the effect of this alteration on the transcriptome and DNA methylation are poorly understood. We have generated new CNS HGNET-BCOR models by lentiviral transduction of the BCOR-ITD into human and murine neural stem cells. In the human model, qRT-PCR and subsequent RNA-seq identified a transient derepression of PRC2-target genes comparing to an isogenic model with overexpression of wildtype-BCOR. A similar effect was found in clinical specimens from previous studies. In the murine-cell model, we confirmed increased clonogenicity in soft-agar assays, and tumors developed in mice flanks. Global DNA methylation levels evaluated by ELISA were significantly lower than those of parent cells, and 177 genes were differentially expressed on RNA-seq analysis comparing to BCOR-overexpressing control cells, including upregulation of known oncogenes. These results suggest that BCOR-ITD and associated alterations in the function of PRC1.1 affect methylation patterns in neural stem cells, driving transcriptional changes and oncogenic transformation into CNS HGNET-BCOR. More detailed analyses, including methylation arrays comparisons with clinical samples and in-silico drug sensitivity testing, are being performed.
MODL-09. FEASIBILITY OF ACUTE SLICE CULTURE-SINGLE CELL SEQUENCING DRUG SCREENING AS A TOOL TO SELECT THERAPY FOR CHILDREN WITH RELAPSED BRAIN TUMORS
Authors: Gampel Bradley , Szalontay Luca , Zhao Wenting , Garvin James , Sethi Chankrit , Stark Eileen , Sims Peter , Canoll Peter , Zacharoulis Stergios
Published: December 2020

Children with relapsed brain tumors are less responsive to treatment. These children often receive therapies without having any robust predictive method of potential benefit. Acute slice culturing(ASC) is a methodology permitting freshly operated tumor to undergo a culturing process preserving the tumor’s micro-environment. With the current study, we investigated the feasibility of obtaining therapeutically meaningful data in a timely manner (3–5 days), performing direct drug testing and single cell sequencing using ASC. Previously, we have combined ex vivo slices of intact, patient-derived Glioblastoma tissue with single-cell RNA-seq for small-scale drug screening and assessment of patient and cell type-specific drug responses. We generated slices from preclinical mouse glioma models and surgical specimens from adult Glioblastoma patients, as well as from children with relapsed Ependymomas, Medulloblastomas, and Gliomas. We demonstrated that these acute slices preserved both the tumor heterogeneity and tumor microenvironment observed in single-cell RNA-seq of cells directly isolated from tumor tissue. Testing drug responses, we then treated tissue slices from the Glioblastoma mouse models and different patients with multiple drugs and combinations. This technique allowed us to identify drug-induced transcriptional responses in specific subpopulations of tumor cells, patient-specific drug sensitivities, and drug effects conserved in both mouse and human tumors. Preliminary data suggests that we can apply this procedure within 5–7 days and provide real-time drug screening/single cell sequencing ASC results to Recurrent/ Progressive pediatric Low-Grade Gliomas, High Grade Gliomas, Ependymomas and Medulloblastomas.
MODL-11. COMPARISON OF HUMAN & MURINE PA/PXA CHARACTERISTICS
Authors: Sommerkamp Alexander C , Sun Pengbo , Wefers Annika K , Ismer Britta , Schramm Kathrin , Wittmann Andrea , Gronych Jan , Korshunov Andrey , von Deimling Andreas , Jäger Natalie , Pfister Stefan M , Jones David T W
Published: December 2020

Pediatric low-grade gliomas (pLGGs) are the most common brain tumors in children. Despite recent advances in the molecular characterization of this heterogeneous set of tumors, the separation of specific tumor types is still not fully established. Pilocytic astrocytoma (PA; WHO grade I) and pleomorphic xanthoastrocytoma (PXA; WHO grade II) are two pLGG types that can be difficult to distinguish based on histology alone. Even though their clinical course is different, they are often grouped as ‘pLGG’ in clinical trials (and therefore treated similarly). Based on a cohort of 89 human pediatric tumor samples, we show that PAs and PXAs have clearly distinct methylation and transcriptome profiles. The difference in gene expression is mainly caused by cell cycle- and development-associated genes, suggesting a key difference in the regulatory circuits involved in tumor growth. In addition to BRAF V600E, we found NTRK fusions and a previously unknown EGFR:BRAF fusion as mutually exclusive driving events in PXAs. Both tumor types show marked signs of immune cell infiltration, but with significant qualitative differences, which might represent therapeutic vulnerabilities. To pave the way for further research on PA and PXA, we developed corresponding mouse models using the virus-based RCAS system, which allows introduction of an oncogenic driver into immunocompetent mice for molecular and preclinical research. The murine tumors do not only histologically resemble their human counterparts but also show a similar growth behavior. Expression analysis revealed that the murine PXAs have a stronger gene signature of proliferation and immune cell infiltration compared to PAs.
MODL-15. THE COMBINATION TREATMENT OF PARP INHIBITOR AND TMZ, OR DAG WILL BE PROMISING TREATMENT IN SF8628
Authors: Ohba Shigeo , Hirose Yuichi
Published: December 2020

Diffuse midline glioma, H3 K27M-mutant (DMG) is a newly defined entity. The prognosis of DMG is poor. Because surgical resection is often incomplete for DMG, radiotherapy and chemotherapy are important. Temozolomide (TMZ) is an alkylating agent that adds a methyl group to DNA (O6-guanine, N7-guanine, and N3-adenine). TMZ-induced cytotoxicity is mainly derived from O6-methylguanine, which is repaired by O6-methylguanine DNA methyltransferase (MGMT). It has been reported that most of DMG lacked MGMT promoter hypermethylation, which is thought to contribute to less effectiveness of TMZ to DMG. The purpose of the study is to explore the way to inhibit the proliferation of DMG. A DMG cell line, SF8628, was used for the experiments. SF8628 had the expression of MGMT and was revealed to be resistant to TMZ. Because N7-methylguanine and N3-methyladenine are repaired via base excision repair, poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitor combined with TMZ was considered to be effective to suppress the proliferation of SF8628. As expected, PARP inhibitor enhanced TMZ-induced cytotoxicity in SF8628. Dianhydrogalactiol (DAG) is a bifunctional DNA-targeting agent forming N7-alkylguanine and inter-strand DNA crosslinks. DAG reduced the clonogenicity of SF8628. Moreover, inhibition of homologous recombination enhanced the DAG-induced cytotoxicity in SF8629. The combination treatment of PARP inhibitor and TMZ, or DAG were revealed to be promising treatments in SF8628.
MODL-17. SHP2 INHIBITORS SHOW ACTIVITY AGAINST NF1-DEFICIENT GLIOMAS AND ENHANCE MAPK PATHWAY INHIBITION IN BRAF-V600E MUTANT GLIOMAS
Authors: Muldoon Daniel , Zhao Guisheng , Batt Carly , Singh Mallika , Nicolaides Theodore
Published: December 2020

INTRODUCTION

Activation of the RAS-MAPK signaling cascade is common in pediatric gliomas. Based on the role of SHP2 in RAS pathway signaling, we hypothesized that NF1-deficient pediatric glioma models would respond to SHP2 inhibitor monotherapy whereas BRAF-V600E gliomas would not. However, we postulated that the latter would exhibit increased sensitivity to a BRAF inhibitor (BRAFi) in combination with SHP2i. Here we demonstrate that the SHP2 inhibitors SHP099 and RMC-4550 (SHP2i) show significant single-agent activity in vitro against NF1-deficient glioma cells and that the combination of RMC-4550 with BRAFi shows increased activity in BRAF-V600E glioma cells relative to the single-agents.

METHODS

Using a panel of NF1 mutant/deficient and BRAF-V600E mutant glioma cell lines we examined effects on cell viability and protein expression levels of total and phosphorylated MEK, ERK, and AKT.

RESULTS

LN229 and U87 NF1-deficient glioma cells are sensitive to SHP2i alone but not A375 cells (melanoma, BRAF-V600E). Additionally, we show that in multiple BRAF-V600E glioma cell lines BRAFi sensitivity increases when combined with a SHP2i. Immunoblots show decreased expression of pERK and pMEK in LN229 cells following SHP2i exposure, while A375 cells maintain MAPK pathway signaling. A sustained decrease in the expression of pERK after 24 hours was observed in BRAF-V600E glioma cells with BRAFi in combination with SHP2i, consistent with relief of feedback inhibition. In vivo studies using orthotopic xenograft models are underway.

CONCLUSION

SHP2i shows preclinical activity in vitro against NF1-deficient pediatric glioma cell lines as a single-agent and against BRAF-V600E gliomas in combination with BRAFi.
MODL-20. A BIOBANK OF ~100 PATIENT-DERIVED MODELS REPRESENTING BIOLOGICAL HETEROGENEITY AND DISTINCT THERAPEUTIC DEPENDENCIES IN PAEDIATRIC HIGH GRADE GLIOMA AND DIPG
Authors: Carvalho Diana , Mackay Alan , Temelso Sara , Izquierdo Elisa , Fernandez Elisabet Potente , Rogers Rebecca , Boult Jessica , Salom Janat Fazal , Simon Natalie , Clarke Matthew , Molinari Valeria , Kessler Ketty , Burford Anna , Bjerke Lynn , Fofana Mariama , Hubank Michael , Pears Jane , Moore Andrew , Carcaboso Angel Montero , Marshall Lynley , Carceller Fernando , Robinson Simon , Hargrave Darren , Vinci Maria , Jones Chris
Published: December 2020

Paediatric high-grade glioma comprise multiple biological and clinical subgroups, the majority of which urgently require novel therapies. Patient-derived models represent useful tools for mechanistic and preclinical investigations based upon their retention of key genetic/epigenetic features and their amenability to high-throughput approaches. We have collected ~100 in vitro models representing multiple subtypes (H3.3/H3.2/H3.1K27M, H3.3G34R/V, BRAF, MYCN_amp, NTRK_fusion, hypermutator, others) established under 2D (laminin) and/or 3D (neurosphere) conditions, credentialed by phenotypic (growth, invasion/migration) and molecular (methylation array, DNA sequencing, RNAseq) comparison to the original tumour sample. These were derived from patients at our local hospitals (n=29), as part of national co-clinical trials (n=19), from international collaborating centres (n=11), or shared directly by research groups worldwide (n=45). These have variously been subjected to pharmacological (approved/experimental drug libraries) and/or genetic screening (whole-genome CRISPR) to identify specific biological dependencies. Many have been established as orthotopic xenografts in vivo (PDX), with detailed pathological and radiological correlations with the clinical disease, and with tumorigenic latencies ranging from 48–435 days. This resource has allowed us to identify genotype-specific synthetic lethalities and responses to targeted inhibitors, including olaparib (PARP) with ATRX, nutlin-3 (MDM2) with PPM1D, AZD1775 (WEE1) with TP53, and CYC065 (CDK9) with MYCN-amplification. Combinatorial screening highlighted synergies in ACVR1-mutant DIPG between novel ALK2 inhibitors and ONC201 (DRD2). Rapid screening allows for feedback of drug sensitivities to treating clinicians at relapse, whilst mechanistic underpinning of these interactions and use of the models to identify specific mediators of resistance will allow for rational future trial design.
MODL-22. DEVELOPING A REAL-TIME PERSONALIZED DRUG TESTING PLATFORM FOR PEDIATRIC CNS CANCERS
Authors: Laternser Sandra , Cosentino Chiara Cianciolo , Przystal Justyna M , Dettwiler Susanne , Rushing Elisabeth Jane , Gerber Nicolas U , Stücklin Ana Guerreiro , Prasad Rachna , Grotzer Michael , Krayenbühl Niklaus , Müller Sabine , Nazarian Javad
Published: December 2020

INTRODUCTION

The relatively small size of biopsied CNS tumors has presented a historical challenge for real-time drug screens. Moreover, in vivo assessment of drug response does not often benefit patients with aggressive gliomas given the relatively long time (>8 months) of tumor engraftment in the classic mouse PDX models. Here, we aimed to develop an innovative real-time in vivo and in vitro drug screening platform capable of analyzing a minimal number (<1E6) of cells obtained at biopsy.

METHODS

Existing primary cells were used to test 6 different culture platforms. The top platform was selected and used to expand tumor cells obtained of DMG biopsy. Tumor cells were validated using the minION sequencing platform. Single and combination drug (n=7) screens were performed. Effective drugs were further evaluated in zebrafish PDX and non-tumor bearing models to assess efficacy and toxicity, respectively.

RESULTS

A total of 8 biopsies were obtained. Successful cell expansion was achieved in 6/8 (75%) and a limited drug screen in 3/6 (50%) of cases. Single and combination drug (n=7) assays identified responder and non-responders to candidate drugs. Systemic toxicity of effective drugs was tested in non-tumor bearing zebrafish. Tumor cells were engrafted in zebrafish providing the opportunity for an in vivo screen. The entire process was completed within 21 days on average.

CONCLUSIONS

A novel platform was developed for rapid in vitro and in vivo drug screens of tumor cells obtained at biopsy. This platform will provide the opportunity to establish personalized therapy for heterogeneous cancers including DMGs.
MODL-24. AN ORGANOTYPIC CHUNK CULTURE TECHNIQUE TO STUDY DISEASE MECHANISM AND DEVELOP TARGETED THERAPEUTICS FOR PEDIATRIC ADAMANTINOMATOUS CRANIOPHARYNGIOMA
Authors: Vijmasi Trinka , Prince Eric , Hengartner Astrid , Staulcup Susan , Griesinger Andrea , Donson Andrew , Gilani Ahmed , Foreman Nicholas , Hankinson Todd
Published: December 2020

BACKGROUND

Advances in the treatment of Adamantinomatous Craniopharyngioma (ACP) face challenges with translation to clinical study due to the absence of robust culture models of the disease. We developed a technique for culturing human ACP tissue in an organotypic chunk culture format that retains the tumor microenvironment for a duration sufficient to evaluate potential targeted therapeutics.

METHODS

Intraoperatively collected tumor tissue from pediatric ACP was cut into volumes of approximately 3 mm3 and rested over a semi-permeable insert placed in the wells of a 6-well plate. Specimens were cultured in (1) Control media, media containing (2) Tocilizumab, (3) Trametinib, and (4) combination of Tocilizumab and Trametinib, for 24 and 96 hours. Specimens were harvested for paraffin embedding, protein and gene expression assays. Supernatants were collected to assay secreted components. Paraffin embedded specimens were sectioned and stained for H&E, Pan-CK, Beta-Catenin, cleaved Caspase-3, Ki-67, and Phospho-ERK.

RESULTS

H&E staining revealed characteristic histologic features of ACP with epithelial cells with palisading nuclei, wet keratin and ghost cells. Tumor sections were markedly positive for epithelial cell markers, Pan-CK and Beta-Catenin. Ki-67 and cleaved Caspase-3 were restricted to a small fraction of cells, indicating low index of proliferation and apoptosis under the culture conditions. The response to drug treatments shall be determined using gene expression assays and evaluation of the secreted components.

CONCLUSION

The organotypic chunk culture technique appears to maintain the viability and integrity of ACP tumors for several days and may serve as an appropriate model for pre-clinical studies to develop targeted therapeutics for pediatric ACP.
MODL-25. REPLICATION REPAIR DEFICIENT MOUSE MODELS PROVIDE INSIGHT ON HYPERMUTANT BRAIN TUMOURS, MECHANISMS OF IMMUNE EVASION, AND COMBINATORIAL IMMUNOTHERAPY
Authors: Galati Melissa , Li Li , Sudhaman Sumedha , Lipman Tatiana , Stengs Lucie , Elshaer Dana , Bridge Taylor , Semenova Dar’ya , Edwards Melissa , Hodel Karl , Forster Victoria J , Nunes Nuno M , Martin Alberto , Bouffet Eric , Pursell Zachary , Hawkins Cynthia , Tabori Uri
Published: December 2020

Replication repair deficiency (RRD) is the leading cause of hypermutant brain tumours in children. RRD is caused by defects in one of four mismatch repair (MMR) genes and mutations in POLE or POLD1. Such tumours are resistant to common therapeutic agents and animal models are needed to study RRD in vivo and test novel therapies like immune checkpoint inhibitors (ICIs). To model RRD brain tumours specifically, we engineered a Pole mutant mouse model harbouring the S459F mutation (PoleS459F). We combined PoleS459F mice with conditional Msh2 knockout (Msh2LoxP) and Nestin-cre mice. All Nestin-cre+Msh2LoxP/LoxPPoleS459F/+ mice rapidly succumbed to posterior fossa brain tumours between 8.6 and 12.4 weeks. Importantly, tumours exhibited hallmark “ultrahypermutation” (~350 mutations/Mb) and the corresponding signatures characteristic of human combined MMR and POLE-proofreading glioblastoma. Interestingly, Nestin-cre+Msh2LoxP/LoxPPoleS459F/S459F mice failed to establish normal cerebella, suggesting such mutational loads may not support normal brain development. Furthermore, OLIG2-cre+Msh2LoxP/LoxPPoleS459F/+ mice failed to develop tumors. Tumors transplanted into syngeneic vs immunocompromised animals egrafted well orthotopically in the mouse hindbrain but significantly less efficiently when engrafted subcutaneously. Furthermore, immunocompromised and subcutaneous tumors revealed striking differences in mutational burden and clonal architecture, suggestive of nonautonomous immunoediting. Finally, anti-PD1 was sufficient to treat subcutaneously engrafted tumors in immunocompetent animals. This first mouse model of immunocompetent, hypermutant brain tumors can be used to uncover unique characteristics of RRD tumour evolution and allow for immune based therapeutic preclinical testing. Experiments to assess combinational ICIs and other therapeutic interventions in orthotopically transplanted tumors will also be presented.