bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract

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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract

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

Abstract
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

Abstract

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 (&gt;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 (&lt;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

Abstract

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.