bluuebunny/crossref_metadata_2025 huggingface dataset:
TAMI-63. GLIOBLASTOMA ASSOCIATED MESENCHYMAL STEM LIKE CELLS (G-MSC) WHICH INFILTRATE TUMORS IN HUMAN AND MOUSE ARE STRONGLY ASSOCIATED WITH IMMUNOSUPPRESSION
Authors: Dharma Sanam , Figel Sheila , Barone Tara , Ciesielski Michael , Fenstermaker Robert
Published: December 2020

Glioblastoma (GBM) is the most aggressive form of brain cancer with an overall survival (OS) less than 16 months. Glioblastoma associated mesenchymal stem like cells (G-MSC) were identified in human patient samples, and higher presence of these cells in patient samples co-relates with lower overall survival. Our lab and others have also identified these cells in orthotopic GL261 glioblastoma murine models. We hypothesize that infiltration of G-MSC plays a key role in creating the highly immunosuppressive environment seen in GBMs. In order to investigate this, we utilized data from the TCGA database to stratify patients into two groups, showing higher or lower expression of the G-MSC markers (CD73, CD90, CD105). Patients expressing higher levels of G-MSC markers showed higher expression of CD4+ cells, as compared to patients with lower G-MSC marker expression. Further, patients with higher G-MSC markers showed high levels of PTGS2, the gene encoding cyclooxygenase 2 (COX2), a known tumor growth promoting and immunosuppressive molecule. We further investigated CD4+ T cell infiltration using GL261 orthotopic implants and observed that CD4+ T cells positively corelated with levels of G-MSC in these tumors. Our studies indicate that levels of G-MSC co-relate with immune cell infiltration and the expression of immunosuppressive factors such as COX2, underlining the importance of these cells in immunosuppression-driven resistance to therapy. These studies will be later utilized to develop rationale combination therapies to improve the overall survival in GBM.
TMOD-04. INHIBITION OF MELANOMA BRAIN METASTASIS BY TARGETING miR146a
Authors: Wang Jiwei , Lunavat Taral , Zhou Wenjing , Feng Zichao , Hoang Tuyen , Thorsen Frits
Published: December 2020

INTRODUCTION

It is well known that cancer patients with brain metastasis (BM) have a poor prognosis and current treatments are not effective. It has been shown that BM development is caused by molecular interactions between the tumor cells and the normal brain, constituting the so-called brain metastatic niche. In this respect, miRNAs delivered by exosomes released by the primary tumor cells seem to be an important factor in BM niche establishment yet the mechanisms behind this process is poorly understood.

AIM

The aim of this project was to determine miRNAs released by exosomes from melanomas that may be important in establishing the BM niche, and to validate these findings in vitro and in vivo.

METHODS

We performed a miRNA profiling on exosomes collected from 3 human melanoma BM cell lines and from human astrocytes and melanocytes, to determine the most important differently expressed miRNAs. Functional in vitro validation was performed by cell growth and migration assays, cytokine arrays, qPCR and Western blots. Functional in vivo studies were performed after miR-knockdown in BM cell lines, in an experimental animal/tumor model.

RESULTS AND DISCUSSION

The miRNA profiling showed that miR-146a was by far the most differentially upregulated miRNA in exosomes from melanoma BM cell lines, compared to exosomes from normal cells. Mir-146a mimics activated human astrocytes, shown by increased proliferation and migration, elevated expression of GFAP in vitro and in mouse brain tumor samples, and increased cytokine production. These effects were reversed by adding anti-miR-146a to the experiments. In animal studies, knockdown of miR-146 in melanoma BM cells injected intracardially into nod/scid mice resulted in a significant reduction in BM burden, compared to wild type cells, and increased animal survival.

CONCLUSIONS

Inhibition of miR-146a reduces activation of the BM niche, leading to inhibition of BM development.
TMOD-07. HUMAN DIFFUSE MIDLINE GLIOMA AVATARS AS A PLATFORM TO SEARCH FOR NOVEL THERAPEUTIC TARGETS
Authors: Koga Tomoyuki , Miki Shunichiro , Skinner Kasey R , Venneti Sriram , Malicki Denise , Wechsler-Reya Robert , Gruener Robert , Huang R Stephanie , Chen Clark , Miller C Ryan , Furnari Frank
Published: December 2020

Diffuse midline glioma is the leading cause of brain tumor death among the pediatric population. Drugs that show notable promise in preclinical models inevitably fail to demonstrate efficacy in clinical trials, likely due to the inadequacy of preclinical models. We have recently proposed glioblastoma models derived from human induced pluripotent stem cells (hiPSCs) genetically engineered with different combinations of glioblastoma-associated genetic alterations as a platform to search for therapeutic targets. These glioblastoma avatars authentically recapitulated the different pathobiology of glioblastoma subtypes, depending on what genetic alterations to be introduced. To investigate the biology and to develop novel therapeutics for diffuse midline glioma with H3K27M mutation, we have established a novel model by introducing H3.3 K27M mutation together with one of the most common concurrent genetic alterations, TP53 R248Q mutation, into hiPSCs through CRISPR/Cas9 genome engineering. Orthotopic engraftment of the neural progenitor cells derived from these edited hiPSCs formed diffusely invasive brainstem tumors with histological features of the diffuse midline glioma. These tumor avatars presented a global reduction in H3K27me3 accompanied by the expression of H3K27M. Transcriptome analyses of these models revealed that these avatars with H3K27M cluster apart from the pediatric glioma samples without this particular mutation, and that they present signatures of oligodendroglial progenitor differentiation as discovered in patient samples with this mutation. Using these models faithfully recapitulating histology and pathobiology of the patient tumors, we have performed drug screening and confirmed that their sensitivity to known drugs, including an EZH2 inhibitor and histone deacetylase inhibitors. On these faithful human avatars of diffuse midline glioma with H3K27M, we have applied bioinformatics algorithms of drug sensitivity prediction aiming at developing novel therapeutics for this devastating pediatric glioma.
TMOD-08. PRIMARY AND RECURRENT GLIOMA PATIENT-DERIVED ORTHOTOPIC XENOGRAFTS (PDOX) REPRESENT RELEVANT PATIENT AVATARS FOR PRECISION MEDICINE
Authors: Golebiewska Anna , Hau Ann-Christin , Oudin Anais , Stieber Daniel , Yabo Yahaya A , Kwon Yong-Jun , Klink Barbara , Skupin Alexander , Hertel Frank , Bjerkvig Rolf , Niclou Simone P
Published: December 2020

Patient-derived cancer models are essential tools for studying tumor biology and for preclinical interventions. Although numerous clinical cancer trials are being conducted, many fail due to inappropriate selection of compounds at the preclinical stage. Therefore, better preclinical models are crucial for predicting successful clinical impact. Orthotropic patient-derived xenograft (PDOX) models are of particular importance for brain cancers, as they allow to better recapitulate the brain tumor environment and the blood brain barrier. We created a large collection of PDOXs from primary and recurrent gliomas with and without mutations in IDH1. PDOX models were based on 3D organoids, derived from mechanically minced patient material. Organoids were implanted in the brain of immunodeficient mice and further propagated by serial intracranial transplantations. High grade glioma PDOX models, starting with viable patient-derived organoids, have generally a high tumor take rate, a reproducible phenotype and tumor development time. PDOXs retain histopathological, genetic, epigenetic and transcriptomic features of patient tumors with no mouse-specific clonal evolution. Longitudinal PDOX models confirmed limited evolution of gliomas upon treatment observed in patient tumors. PDOX-derived standardized tumor organoid cultures enabled assessment of drug responses, which were validated in mice. PDOXs showed clinically relevant responses to Temozolomide and to targeted treatments such as EGFR and CDK4/6 inhibitors in (epi)genetically defined groups, according to MGMT promoter and EGFR/CDK status respectively. These data indicate that glioma PDOXs represent clinically relevant avatars for personalized treatment. The use of these models should lead to a more realistic evaluation of the efficacy of novel drugs, thereby increasing the success of clinical studies.
TMOD-10. EFFECT OF BRAIN TUMORIGENESIS ON CEREBRAL CORTICAL FUNCTIONAL CONNECTIVITY IN THE MOUSE
Authors: Cramer Samuel , Popa Laurentiu , Haley Samuel , Dhawan Sanjay , Carter Russell , Ning Jianfang , Aronson Justin , Kodandaramaiah Suhasa , Ebner Timothy , Chen Clark
Published: December 2020

INTRODUCTION

Neuro-cognitive decline is near universal in glioblastoma patients and negatively impacts the quality of life for afflicted patients. Yet, there is little information on longitudinal effects of brain tumor growth on cerebral cortical function and network connectivity.

OBJECTIVE

To address this knowledge gap, we examined in vivo Ca2+ imaging in a transgenic murine glioblastoma model.

METHODS

Mesoscopic Ca2+ imaging was performed after implant of GL261 glioblastoma cells into a transgenic mice strain (Thy1-GCaMP6f) that expresses the fast calcium indicator GCaMP6f in Layer II/III and Layer V pyramidal neurons. Independent component analysis (ICA), correlation matrix and graph theory approaches were used to assess changes in network connectivity.

RESULTS

ICA defined canonical cerebral network consisting of nodal convergence and connectivity between nodes. The overall network structure remained unaltered after tumor implant. A decrease in the strength of connectivity was observed immediately following tumor implant. This temporary suppression was followed by progressive, global increase in the strength of nodal connectivity (p < 0.0001). By two weeks post-tumor implant, 50% of the nodes exhibited increased connectivity compared to baseline. Progressive activation of select nodes was also observed in the weeks following tumor implant (p < 0.01). In aggregate, these results suggest that activation of select network nodes as well as enhanced connectivity as means to compensate for the deleterious effects of glioblastoma growth.

CONCLUSIONS

Our results indicate that focal brain tumor growth induces a reorganization of both local and remote cortical activity. The finding bears pertinence to the pathogenesis of neuro-cognitive decline and tumor-associated epilepsy.
TMOD-18. TARGETING THE PI3K/AKT PATHWAY IN MYCN AMPLIFIED HIGH GRADE GLIOMAS
Authors: Halligan Katharine , Stanton Ann-Catherine , Halbert Matthew , Golbourn Brian , Mack Stephen , Pollack Ian , Agnihotri Sameer
Published: December 2020

Pediatric glioblastoma (pGBM) are incurable brain tumors with overall poor prognosis and response to treatments due to molecular and epigenetic heterogeneity. In particular, the MYCN subtype of pGBM are a highly aggressive form of GBM with a dismal median survival of only 14 months. Furthermore, this subtype is enriched with loss of the tumor suppressor genes TP53 and PTEN, leading to aberrantly active PI3K-AKT signaling pathway and DNA-checkpoint abnormalities. Here, we report the generation of a novel syngeneic mouse model that recapitulates the features of the MYCN subtype of pGBM. We isolated Sox2-Cre neural stem cells from C57BL/6 mice and transduced inverted retroviral-cassettes of the murine Mycn oncogene simultaneously with shRNA targeting tumor suppressor genes p53 and Pten. Retroviral-cassettes are flanked by tandem LoxP sites arranged so that Cre recombinase expression inverts the cassettes in frame allowing for MYCN protein expression and loss of the P53/PTEN proteins. Transgene activation is accompanied with selectable cell surface markers and fluorescent tags enabling for fluorescent activated cell sorting (FACS) of the desired cell populations. Neural stem cells with MYCN protein expression and concurrent silencing of P53 and PTEN protein (NPP cells) result in significantly increased proliferation and activation of PI3K-AKT pathway as compared to control neural stem cells and have. Injection of NPP cells into the forebrain of immune competent C57BL/6 mice result in the formation of invasive high-grade gliomas with a lethal phenotype at ~50 days post injection. Using several next generation brain penetrant small molecule inhibitors of the PI3K-AKT pathway, we show inhibition of tumorigenesis in vitro. Moreover, we have identified several novel mechanisms of PI3KAKT treatment resistance and are currently identifying therapies that may overcome this resistance through RNA seq analysis. In summary, well defined genetic drivers of GBM can lead to informed mouse model generation to test promising therapies.
TMOD-20. A SWINE MODEL OF GLIOBLASTOMA INDUCED BY SOMATIC GENE MODIFICATION
Authors: Tschida Barbara , Duerre Dylan , Taisto Mandy , Watson Adrienne
Published: December 2020

Glioblastoma (GBM) is the most common and malignant primary brain tumor. Novel therapeutic development for GBM is needed since the standard of care universally fails to cure patients and the five-year survival rate remains below 10%. GBM therapeutic development is hampered by the lack of relevant large animal models for preclinical studies. To mitigate this problem, we are developing a model of GBM in outbred, immune-proficient swine which have comparable brain size and anatomy to humans. We developed methods for introducing genome engineering tools to minipig brain cells in vivo by direct injection of gene delivery reagents to the lateral ventricle. Using this technique, we have delivered a combination of expression vectors for oncogenes and targeted nucleases to disrupt tumor suppressor genes commonly altered in human GBM to alter six major human GBM-associated signaling pathways in a cohort of minipigs (Ras, Pi3k, p53, Rb/E2F, Pdgf, and the alternative lengthening of telomeres (ALT) pathways). These minipigs are being monitored for tumorigenesis using a secreted reporter, detectable through a simple luminescence-based blood test. Resulting tumors will be examined molecularly to detect the pathway-associated alterations in tumor tissue and determine the resemblance to human GBM. We hypothesize that this somatic cell gene-modification platform we have developed in the minipig will facilitate the efficient production of brain tumors that histologically and genetically resemble human GBM. It will allow the production of tumors that are genetically heterogeneous, of specified molecular subclasses, containing therapeutic targets of interest, and in the context of genetic backgrounds of interest. This minipig model of GBM will be applied towards preclinical therapeutic studies, imaging studies using human clinical grade equipment, and surgical technique development, to improve clinical trial success rates and patient outcomes. Funding for this study is provided by the National Institutes of Health through SBIR grant # 1R43CA235837-01A1.
TMOD-22. AKALUC BIOLUMINESCENCE OFFERS SUPERIOR SENSITIVITY TO TRACK IN VIVO GBM EXPANSION
Authors: Bozec Dominique , Sattiraju Anirudh , Bouras Alexandros , Raj Joe Gerald Jesu , Rivera Daniel , Huang Yong , Alves Chrystian Junqueira , Tejero Rut , Zou Hongyan , Hadjipanayis Constantinos , Friedel Roland H
Published: December 2020

Longitudinal tracking of tumor growth using non-invasive bioluminescence imaging (BLI) is a key approach for in vivo cancer studies, but the current method of firefly luciferase (Fluc) BLI has quantitative limitations, as it is only suited for detection of tumors of considerable sizes at advanced stage, typically in the order of >105 cells. Recently, Akaluciferase (Akaluc) has been developed as an alternative BLI system that offers higher signal strength and better light penetration of tissue due to its red-shifted emission. Here, we established Akaluc BLI as a new sensitive method for in vivo tracking of glioblastoma (GBM) expansion in intracranial transplant models. In multiple GBM cell lines, including the frequently used U87MG and GL261, as well as patient-derived glioma stem cells (GSC), we demonstrate that Akaluc-expressing GBM cells produced more than 50-times brighter BLI signals in vitro and up to 100-fold higher signal intensities in vivo over Fluc-expressing counterparts. The higher sensitivity of Akaluc BLI permits early in vivo detection of intracranial GBM transplants starting as early as 4 hours after implantation and with as little as 5,000 transplanted GSC. We also reveal a prolonged engraftment period in intracranial GSC transplants before wide dissemination into host brain parenchyma. Akaluc BLI is also advantageous for longitudinal monitoring of therapeutic effects of chemoradiation for GBM and detection of early phase of tumor relapse. Thus, Akaluc BLI offers an important addition to the tool box for cancer research. SIGNIFICANCE: The high sensitivity of Akaluc bioluminescence is a significant improvement for the non-invasive tracking of tumors in preclinical cancer studies, including detection of small incipient tumors and micro-metastasis.
TMOD-23. A NOVEL BRAF V600E LOW-GRADE GLIOMA MOUSE MODEL HIGHLIGHTS EXOMIC AND TUMOR IMMUNE ALTERATIONS AND DIFFERING THERAPEUTIC RESPONSES IN LOW- AND HIGH-GRADE GLIOMAS
Authors: Marie Barrette Anne , Meyer Lasse , Hirata Yoko , Grossauer Stefan , Lu Edbert , Tamaki Whitney , Petritsch Claudia , Wang Wei
Published: December 2020

Pediatric low-grade glioma (pLGG), the most common brain cancer in children, is difficult to treat especially at recurrence. The BRAF V600E mutation is the second most common mutation in pLGG, and in a high-risk group for progression is associated with deletion of the tumor suppressor CDKN2A. A better understanding of the factors contributing to progression, in particular the role of the immune infiltrate is needed, but studies have been hindered by the lack of low-grade glioma mouse models. We utilized transgenic mice with a cre-activatable (CA) allele of BRAF V600E to generate endogenous models for low-grade gliomas. We found that BRAF V600E expression cooperates with hemizygous CDKN2A deletion to induce low-grade gliomas, with tumors forming at a greater latency than by homozygous deletion. Cell line derivatives from low-grade lesions continue to grow slower upon orthotopic injection than those we previously derived from high-grade tumors. Murine LGG can progress to higher grade tumors within the mouse lifespan and we observe exomic changes and alterations in the tumor immune infiltrate associated with progression, the details of which will be discussed at the meeting. High-grade cells’ phenotypic changes within in vivo passage are accompanied by exomic changes. The high-grade glioma immune infiltrate is altered by dual MAPK pathway inhibition with dabrafenib and trametinib. Adding dual immune checkpoint inhibition by anti-PD-L1 and anti-CTLA-4 antibodies significantly extends survival of dabrafenib-trametinib dual treatment. Human BRAF V600E mutant tumors reportedly have a higher tumor immune infiltrate than that of BRAF wildtype gliomas, consistent with our murine RESULTS: Here we present a novel model for BRAF V600E mutant gliomas in mice that has a frequent rate of progression, similar to human BRAF V600E mutant gliomas, and an active immune infiltrate in high grade tumors which makes them susceptible to the immunostimulatory effects of dual checkpoint inhibition.
TMOD-24. GENERATION AND CHARACTERIZATION OF A NOVEL TRANSGENIC IDO REPORTER MOUSE FOR IDO POSTTRANSLATIONAL MODIFICATION ANALYSIS IN SITU
Authors: Bell April , Zhai Lijie , Ladomersky Erik , Lauing Kristen , Bollu Lakshmi , Wainwright Derek
Published: December 2020

Glioblastoma (GBM) is the most common and aggressive primary central nervous system tumor in adults with a median survival of 14.6 months. GBM is a potently immunosuppressive cancer due in-part to the prolific expression of immunosuppressive indoleamine 2,3 dioxygenase 1 (IDO). Tumor cell IDO facilitates the intratumoral accumulation of regulatory T cells (Tregs; CD4+CD25+FoxP3+). Although immunosuppressive IDO activity is canonically characterized by the conversion of tryptophan into kynurenine, we have utilized transgenic and syngeneic mouse models and mutant glioma lines to demonstrate that tumor cell IDO increases Treg accumulation independent of tryptophan metabolism. Here, we address the gap in our understanding of IDO signaling activity in vivo. Subcutaneously-engrafted human GBM expressing human IDO-GFP cDNA was isolated from immunodeficient humanized NSG-SGM3 mice. The tumor was immunoprecipitated for the GFP tag using GFP-TRAP followed by mass spectrometry which revealed a novel methylation site on a lysine residue at amino acid 373 in the IDO C-terminus region. Western blot analysis of IDO protein also revealed the presence of tyrosine phosphorylation. Additionally, we recently created a new transgenic IDO reporter mouse model whereby endogenous IDO is fused to GFP via a T2A linker (IDO→GFP). This model allows for the isolation of IDO+ cells in real-time and without causing cell death, thereby creating the opportunity for downstream molecular analysis of in situ-isolated GFP+ cells. Collectively, our work suggests that IDO non-enzyme activity may involve the post-translational modifications we recently identified. As IDO activity may differ between in vitro and in vivo modeling systems, we will use the new IDO→GFP reporter mouse model for an improved mechanistic understanding of how immunosuppressive IDO facilitates Treg accumulation in vivo.
TMOD-27. EFFICACY OF SOLUBLE PANOBINOSTAT (MTX110) IN PRECLINICAL MODELS OF ADULT GLIOBLASTOMA
Authors: Ashley David , Keir Stephen T , Roskoski Martin A , Davies Rhian , Williams Ben , Damment Stephen J P , Conlon Kelly
Published: December 2020

INTRODUCTION

Panobinostat is a histone deacetylase (HDAC) inhibitor with antineoplastic and anti-angiogenic effects in glioma. HDAC inhibitors have demonstrated therapeutic efficacy preclinically via multiple mechanisms, including the induction of cell-cycle arrest, differentiation, senescence, apoptosis, mitotic cell death, autophagy, generation of reactive oxygen species, inhibition of angiogenesis and metastasis, and improvement in tumor immunity. One of the biggest challenges in treating glioblastoma is achieving effective local drug concentrations, and trials using systemic administration of therapeutics have failed in GBM despite compelling pre clinical evidence. MTX110 (Midatech Pharma plc) is a water-soluble form of panobinostat currently in clinical development for DIPG and medulloblastoma using direct tumour delivery. We explored the therapeutic potential of MTX110 in GBM in our preclinical in vivo adult brain tumor models.

METHODS

Tumor-bearing mice were stratified to either the vehicle control or treatment group based on median tumor volume and were treated with Panobinostat/MTX110 at a dose of 15mg/kg IP, twice weekly for 5 consecutive weeks. The response to treatment was assessed by delay in tumor growth in days, expressed as a T (Treated) - C (Control) value. Statistical analysis was performed using SAS (a statistical analysis program) and the Wilcoxon rank order test.

RESULTS

MTX110 produced non-significant growth delays of 10.76 and 2.01 days (p Value ≥ 0.072 NS) in IDH wild type xenograft lines. However, in an IDH1 mutated line, MTX110 produced a significant growth delay of 23.35 days (p Value ≤ 0.018). Further work is ongoing to look at the therapeutic potential of MTX110 in combination with radiation in both IDH wild type and IDH mutated xenograft lines.
TMOD-30. IDENTIFYING NEW DRIVERS OF GROUP 3 MEDULLOBLASTOMA
Authors: Beigi Masihi Meher , Lee Catherine , Garancher Alexandra , Furnari Grace , Wechsler-Reya Robert
Published: December 2020

Medulloblastoma (MB) is the most common malignant childhood brain tumor. MB can be divided into four major subgroups – WNT, Sonic hedgehog (SHH), Group 3 (G3-MB), and Group 4 (G4-MB) – that exhibit distinct genetic alterations, gene expression profiles, and clinical outcomes. Patients with G3-MB have the worst prognosis, and a deeper understanding of this form of the disease is critical for development of new therapies. Most G3-MBs express high levels of the MYC oncogene, suggesting that MYC plays an important role in tumorigenesis. However, MYC overexpression is not sufficient to drive tumor formation. To identify genes that cooperate with MYC to promote development of G3-MB, we performed an in vivo mutagenesis screen using mice expressing the Sleeping Beauty (SB) transposon. Cerebellar stem cells isolated from transposon/transposase-expressing transgenic mice were infected with viruses encoding Myc, and these cells were transplanted into the cerebellum of adult hosts. Tumors that arose were subjected to DNA and RNA sequencing to identify candidate genes, and these genes were subjected to functional analysis to determine whether they could cooperate with Myc to drive G3-MB. These studies identified the transcription factor Ras-responsive element binding protein 1 (Rreb1) as a potent Myc-cooperating gene. Tumors driven by Myc and Rreb1 (MR tumors) resemble G3-MB at a histological and molecular level. Moreover, RREB1 is overexpressed in human G3-MB, and knockdown of RREB1 expression impairs growth of G3-MB cell lines and patient-derived xenografts. Ongoing studies are aimed at identifying the molecular mechanisms by which Rreb1 contributes to tumor growth. Our studies demonstrate an important role for RREB1 in G3-MB, and provide a new model that can be used to identify therapeutic targets and develop more effective and less toxic therapies for this devastating pediatric brain tumor.
TMOD-31. NOVEL GENETIC CHARACTERIZATION OF MURINE MEDULLOBLASTOMA CELL LINES
Authors: Rumler Sarah , Maienschein-Cline Mark , Noronha Melline Fontes , Song Fei , Lock Andrew , Loeb Jeffrey , Nicholas M Kelly
Published: December 2020

Pre-clinical research in medulloblastoma (MB) is conducted either with human-derived cell lines or tumors arising in genetically engineered mice (GEM). Murine MB cell lines are not readily available. To begin in vitro immuno-oncology MB research, we identified murine MB cell lines from research laboratories to determine their fidelity to human cell lines and GEM models. Using PubMed, we identified murine MB cell lines of potential interest. Seven cell lines form two independent laboratories were identified. We characterized the cell lines’ behavior in culture and performed next generation sequencing (NGS) on four of them (two from each group) to determine the extent to which these cell lines resemble both human and GEM MB models. DNA and RNA were extracted from MB cell lines, quality and quantity assessed. Whole-exome sequencing was performed and post-capture libraries sequenced. The cell lines identified were derived from either FVB/N TP53 null mice carrying the N-MYC oncogene (GTML3 and GTML5) or C57Bl6/J TP53 null heterozygous ptch (-/+) mice (MM1 and MM3). GTML cells grow in suspension and form clusters. MM cells are adherent, growing in monolayers. Whole exome sequencing of the 4 lines revealed similarities between both GEM and human MB cell lines, demonstrating 25% of sixteen genes known to be affected in both GEM tumors and human MB cell lines. Furthermore, the four cell Iines shared five deleterious mutations previously not described in MB, suggesting they might play a role in MB tumorigenesis: Pde4d, Whrln, Olr156, RIKEN cDNA D630045J12, and Olfr159. These are the first data to describe these murine MB cell lines and demonstrates 1) they resemble both GEM tumors and human MB cell lines at the genome level, and 2) they offer the potential to identify heretofore unrecognized mechanisms in MB tumorigenesis.
TMOD-32. MUTATIONAL ANALYSIS OF THE C11orf95 DOMAIN AND SINGLE CELL RNASEQ PROFILE OF A MOUSE MODEL OF SUPRATENTORIAL EPENDYMOMA
Authors: Troung Kevin , He James , Randazzo Ericka , Dunnack Jesse , LoTurco Joseph
Published: December 2020

We used a recently developed mouse model to better understand the cellular and molecular determinants of tumors driven by the oncogenic fusion protein C11orf95-RELA. Our approach makes use of in utero electroporation and a binary transposase system to introduce human C11orf95-RELA sequence, wild type and mutant forms, into neural progenitors. We used single cell RNA-seq to profile the cellular constituents within the resulting tumors in mice. We find that approximately 70% of the cells in the tumors do not express the oncogene C11orf95-RELA and these non-oncogene expressing cells are a combination of different non-tumor cell cell-types, including significant numbers of T-cells, and macrophages. The C11orf95-RELA expressing tumor cells have a unique transcriptomic profile that includes both astrocytic and neural progenitor marker genes, and is distinct from glioblastoma transcriptomic profiles. Since C11orf95-RELA is believed to function through a combination of both activation of NF-κB response genes by constitutive activation of RELA, and genes not activated by NF-κB, we assessed the expression of NF-κB response genes across the populations of cells in the tumor. Interestingly, the genes that best defined the oncogene expressing tumor cell population were not enriched for NF-κB response genes relative. We hypothesized that the C110rf95 domain may function to bring RELA transcriptional activation to inappropriate non-NF-κB targets, and we therefore performed a point mutation analysis of the C110rf95 domain. We found that mutations in either of the cysteines or histidines that make up a possible zinc finger domain in C11orf95 eliminate the ability of the fusion to induce tumors. In cell lines, these loss-of-function point mutants still trafficked to nuclei, and activated NF-κB pathways. We are currently using RNAseq and CRISPR loss-of function to identify genes downstream of C11orf95-RELA that are required for tumorigenesis.
TMOD-36. THE DEVELOPMENT OF A NOVEL MOUSE MODEL TO STUDY THE ROLE OF HISTONE MUTATIONS AND MODIFICATIONS IN PEDIATRIC HIGH-GRADE GLIOMA
Authors: Grigore Florina , Day Charles , Hanson Nicholas , Langfald Alyssa , Sarkaria Jann , Daniels David , Hinchcliffe Edward , Robinson James
Published: December 2020

Pediatric glioblastoma and diffuse intrinsic pontine glioma are high-grade gliomas of children (pHGG) with a median overall survival of under 15 months and among the most lethal cancers. Mutations in histone H3.3 and H3.1 occur as an early event in pHGG. H3.3G34R/V-mutations occur in pHGG of cerebral hemispheres, and H3.3K27M mutations occur in midline pHGGs. Post-translational histone modifications (PTMs) serve to regulate gene expression by relaxing or compacting chromatin and by recruiting proteins, with subsequent silencing or activating effects. H3.3 Serine 31 (S31) shows reduced phosphorylation during mitosis in H3.3G34R/V and H3.3K27M mutant cell. Phosphorylation at S31 is restored in wildtype H3.3K27 CRISPR revertants. Serine to alanine (A) mutant H3.3 S31A are nonphosphorylatable in vitro. To study the influence of histone mutations and the role of altered PTM and including the loss of methylation and phosphorylation on tumorigenesis, we have developed an innovative model based on the RCAS/N-TVA mouse model. In this system, the expression of an oncogenic driver is linked to mutant histone expression using a self-cleaving peptide, and tumors develop following viral delivery to neural stem cells in newborn mice. This approach is necessary, as otherwise, clonal selection could prevent tumors from forming with mutations detrimental to growth. To establish the model, N-TVA mice were injected with RCAS H3.3K27M-P2A-PDGFB, RCAS H3.3G34R-P2A-PDGFB, or H3.3WT-P2A-PDGFB. The mean survival of mice injected with H3.3K27M and H3.3S31A was 81 and 68 days, respectively, and 100% of S31A mice developed HGG. In contrast, H3.3WT caused only low-grade tumors in 46% of the mice, and all mice survived until 100 days. In ongoing experiments with H3.3G34R, 23% of mice succumb to tumors by 80 days. These results provide mechanistic insights into the early establishment of pHGGs and established a new mouse model to study the role of histone mutation and PTMs in tumor development.
TMOD-37. IN VIVO COMPRESSION AND IMAGING FOR CAUSAL STUDIES OF MECHANICAL FORCES IN THE BRAIN
Authors: Datta Meenal , Nia Hadi , Seano Giorgio , Roberge Sylvie , Huang Peigen , Munn Lance , Jain Rakesh
Published: December 2020

Clinicians have long observed the effects of abnormal mechanical forces – edema (fluid pressure) in particular – in brain tumors and the surrounding normal brain tissue. However, it was not previously possible to dissect the direct effects of solid stress (i.e., “mass effect”), a mechanopathology resulting from solid components of the tumor tissue, on the brain from the biological and physiological adverse effects exerted by cancer cells. We recently developed for the first time an in vivo compression device that allows for causal and mechanistic studies that delineate the solid mechanical forces of a tumor growing in the brain from its biological effects. The brain poses a unique anatomical consideration of abnormal mechanical forces due to its physical confinement by the skull. We adapted standard transparent cranial windows normally used for intravital imaging studies in mice to include a tunable screw for controlled and acute or chronic compression and decompression in the brain. This compressive cranial window allows for longitudinal imaging of the surrounding brain tissue (cortex or cerebellum) over time (weeks or months) as the screw is lowered further into the brain tissue to recapitulate tumor growth-induced solid stress. Using this device, we have demonstrated that solid stress is causally linked to vascular and neurological dysfunction in the brain. We have also been able to utilize this preclinical system to screen for effective therapeutic interventions to reduce solid stress-induced neuronal death and improve neurological function. Beyond cancer, this technique can be used to study a variety of diseases or disorders that present with abnormal solid masses in the brain, including cysts and benign growths. Thus, mechanistic studies enabled by the compressive cranial window can elucidate the role of mechanics in brain tumor progression, and reveal novel targets for treatment.
ATRT-04. INHERITED RHABDOID PREDISPOSITION SYNDROME: A CASE OF CHOROID PLEXUS CARCINOMA AND ATYPICAL TERATOID RHABDOID TUMOR IN SIBLINGS
Authors: Judd Alexis , Wright Erin , Rush Sarah
Published: December 2020

Choroid plexus carcinoma (CPC) and Atypical teratoid/rhabdoid tumor (ATRT) are aggressive, malignant brain cancers most commonly arising in children less than 3 years of age. These tumors often have genetic alterations in the tumor suppressor gene SMARCB1/INI1. Rhabdoid predisposition syndrome (RTPS) categorizes patients with germline mutations in SMARCB1 or SMARCA4, leading to a markedly increased risk of developing rhabdoid tumors. Both CPC and ATRT have been demonstrated in patients with these rhabdoid predisposition syndromes. In general, these tumors tend to have a poor prognosis. However, with the presence of a SMARCB1 mutation they may have improved overall survival. We present two interesting cases of siblings with maternally inherited SMARCB1 mutations: one a 21-month-old male who presented with an ATRT and another a 10 month old female who presented with a CPC. The ATRT was treated as per the Children’s Oncology Group study ACNS0333 with high dose chemotherapy and stem cell rescue as well as cranial radiation. The CPC was treated as per CPT-SIOP 2009 with etoposide, cyclophosphamide and vincristine. Unlike other patients with these aggressive tumors, both of these patients are alive without evidence of disease recurrence 8 and 7 years post therapy, respectively. Additional genomic testing on both tumors is currently pending in order to potentially identify other mutations that may impact survival. These cases further illustrate the similar profile of two very different tumors with improved overall survival that may be secondary to mutations in SMARCB1 in RTPS.
ATRT-09. IDENTIFICATION OF HUB GENES IN ATYPICAL TERATOID/RHABDOID TUMORS BY MULTIPLE-MICROARRAY ANALYSIS
Authors: Liu Wei , Chai Yi , Wang Junhua , Zhang Yuqi
Published: December 2020

BACKGROUND

Atypical teratoid/rhabdoid tumors (ATRT) are rare, highly malignant neoplasms arising in infants and young children. However, the biological basis of ATRTs remains poorly understood. In the present study, we employed integrated bioinformatics to investigate the hub genes and potential molecular mechanism in ATRT.

METHODS

Three microarray datasets, GSE35943, GSE6635 and GSE86574, were downloaded from Gene Expression Omnibus (GEO) which contained a total of 79 samples including 32 normal brain tissue samples and 47 ATRT samples. The RobustRankAggreg method was employed to integrate the results of these gene expression datasets to obtain differentially expressed genes (DEGs). The GO function and KEGG pathway enrichment analysis were conducted at the Enrichr database. The hub genes were screened according to the degree using Cytoscape software. Finally, transcription factor (TF) of hub genes were obtained by the NetworkAnalyst algorithm.

RESULTS

A total of 297 DEGs, consisting of 94 downregulated DEGs and 103 upregulated DEGs were identified. Functional enrichment analysis revealed that these genes were associated with cell cycle, p53 signaling pathway and DNA replication. Protein-protein interaction (PPI) network analysis revealed that CDK1, CCNA2, BUB1B, CDC20, KIF11, KIF20A, KIF2C, NCAPG, NDC80, NUSAP1, PBK, RRM2, TPX2, TOP2A and TTK were hub genes and these genes could be regulated by MYC, SOX2 and KDM5B according to the results of TF analysis.

CONCLUSIONS

Our study will improve the understanding of the molecular mechanisms and provide novel therapeutic targets for ATRT.
ATRT-10. ATYPICAL TERATOID/RHABDOID TUMOR OF THE PINEAL REGION IN A PEDIATRIC PATIENT
Authors: Almeida Carlos , Mançano Bruna Minniti , Matsushita Marcus , Previdelli Stephanie , Lamim Marina Lopes , Faustino Fabio Costa , Souza Fernanda Magalhaes , Lourenço Lucas Dias
Published: December 2020

BACKGROUND

Atypical teratoid/rhabdoid tumor (ATRT) is a malignant neoplasm of the central nervous system and corresponds to 1.5% of all intracranial tumors. Mainly affects children under three years of age and shows aggressive behavior (most pediatric patients succumb to their disease within a year after the initial diagnosis, despite the treatment performed). Its place of occurrence in children is preferably in the posterior fossa, and it is rare to appear in other regions. There are only seven patients with ATRT reported on literature; all of them are adults. We present the case of a pediatric patient with a tumor in the pineal region diagnosed as ATRT. CASE REPORT: Three-year-old female patient admitted with occipital headache, vomiting, and seizure. Magnetic resonance imaging (MRI) showed obstructive hydrocephalus secondary to a solid-cystic lesion located at the pineal region that was 3.0 x 3.0 x 3.5 cm in size. Spine MRI did not reveal leptomeningeal spreading. We performed an occipital transtentorial approach to achieve the best safe resection possible, and a ventriculoperitoneal shunt. Histological examination revealed ATRT. The patient received adjuvant treatment with radiotherapy and chemotherapy according to the “Head Start” protocol. One year after the surgery, MRI did not identify any remaining lesion.

CONCLUSION

ATRT is an aggressive and rare neoplasm whose clinical picture depends on the location of the tumor; however, it must be considered in the differential diagnosis of tumors of the pineal region in the pediatric population.
ATRT-11. PREVALENCE OF GERMLINE VARIANTS IN SMARCB1 INCLUDING SOMATIC MOSAICISM IN AT/RT AND OTHER RHABDOID TUMORS
Authors: Shirai Ryota , Osumi Tomoo , Terashima Keita , Kiyotani Chikako , Uchiyama Meri , Tsujimoto Shinichi , Yoshida Masanori , Yoshida Kaoru , Uchiyama Toru , Tomizawa Daisuke , Shioda Yoko , Sekiguchi Masahiro , Watanabe Kenichiro , Keino Dai , Ueno-Yokohata Hitomi , Ohki Kentaro , Takita Junko , Ito Shuichi , Deguchi Takao , Kiyokawa Nobutaka , Ogiwara Hideki , Hishiki Tomoro , Ogawa Seishi , Okita Hajime , Matsumoto Kimikazu , Yoshioka Takako , Kato Motohiro
Published: December 2020

BACKGROUND

Genetic hallmark of atypical teratoid/rhabdoid tumor (AT/RT) is loss-of-function variants or deletions in SMARCB1 gene on 22q11.2 chromosome, which is common to extracranial malignant rhabdoid tumors (MRT). Previous studies demonstrated that approximately one-thirds of AT/RT and extracranial MRT patients harbored germline SMARCB1 variants as the rhabdoid tumor predisposing syndrome. We studied herein intensive analysis of the SMARCB1 gene in AT/RT and extracranial MRT patients focusing on prevalence of germline genetic variants. PROCEDURE: In total, 16 patients were included. Both tumor-derived DNA and germline DNA were obtained from all patients. First, screening for SMARCB1 alterations in the tumor specimens was done by direct sequencing, ddPCR and SNP array analysis. Then, analysis of germline DNA samples focusing on the genomic abnormalities detected in the paired tumors in each case was performed.

RESULTS

In eight of 16 cases (50%), genomic alterations observed in the tumor-derived DNA were also detected in the germline DNA. It is worth noting that three patients had germline mosaicism. Two of three patients had mosaic deletion, including SMARCB1 region, and the average copy number of the deleted region in the SMARCB1 gene in the germline was 1.60 and 1.76. For another patient, the fraction of SMARCB1 variants in normal cells was as low as 1.7%.

CONCLUSIONS

Approximately half the MRT cases in this study had SMARCB1 germline alterations. Considering the presence of low-frequency mosaicisms which conventional methods might overlook, inherited germline variants in predisposition genes are more important than previously assumed for the pathogenesis of pediatric cancers.
ATRT-25. INTEGRATED QUANTITATIVE SWATH-MS PROTEOMICS ANALYSIS OF ATRTs UNCOVERS NEW THERAPEUTIC OPPORTUNITIES
Authors: Finetti Martina Anna , Hacking James , Lishman-Walker Emma , Synowsky Silvia A , Crosier Stephen , Martin Emmma , Burns Jess , Shirran Sally L , Bailey Simon , Clifford Steven C , Huang Paul , Williamson Daniel
Published: December 2020

The consequences of SMARCB1 loss in Atypical Teratoid Rhabdoid Tumors (ATRTs) have been extensively characterized at the epigenetic/transcriptomic level. In this study we detail the functional effect of SMARCB1 mutation on the MRT proteome, its relationship with RNA deregulation or lack thereof. We performed unlabeled discovery proteomics using MS-SWATH on MRT cells in which SMARCB1 was forcibly re-expressed (5 cell lines, +/-SMARCB1); analyzing changes in protein abundance within 3 fractions (total, membrane, nuclear). We generated a custom spectral library, covering >8,000 proteins, for analysis of the ATRT proteome using a pH fractionated pool of each cellular subfraction. This SMARCB1-dependent ATRT spectral library constitutes a powerful tool for profiling proteins of potentially therapeutic relevance in both model systems and primary ATRT samples. We show that whilst gene expression and protein abundance are significantly related there are many instances whereby expression changes do not reliably predict protein abundances. Several hundred proteins show significantly increased abundance (p<0.01) with no concomitant change by RNA-seq. SMARCB1 mutation is able to invoke critical changes in post-transcriptional/translational regulation as well as sub-cellular localization. By integration with whole-genome CRISPR/cas9 screening we describe functionally essential SMARCB1 dependent pathway/membrane biomarkers, evident at the protein but not the RNA level. We describe several which are druggable and suggest certain therapeutic modalities e.g. specific combinations of RTKs, RNA-binding proteins/splicing factors (SpliceosomeA, U4:U5:U6 tri-snRNP complexes). Our analysis links, for the first time in ATRT, genome-wide transcriptomic and proteome aberrations and reveals broad mechanisms underlying the effect of SMARCB1 mutation.
DIPG-01. REIRRADIATION PRACTICES FOR DIFFUSE INTRINSIC PONTINE GLIOMA
Authors: Cacciotti Chantel , Liu Kevin , Haas-Kogan Daphne , Warren Katherine
Published: December 2020

INTRODUCTION

Diffuse intrinsic pontine gliomas (DIPG) are a leading cause of brain tumor deaths in children. Current standard of care includes focal radiation therapy (RT). Despite clinical improvement in the majority of patients, the effect is temporary and median survival is less than one year. The use of reirradiation and possible benefit has been reported in progressive DIPG, yet standardized approaches are lacking. We conducted an internet-based survey to assess physicians’ practices in pediatric DIPG.

METHODS

A 14-question REDCap survey regarding re-irradiation practices was emailed to 396 physicians identified through an International Pediatric Neuro-Oncology and Radiation-Oncology database.

RESULTS

Response rate was 35% overall (radiation-oncologists, 28%; pediatric oncologists, 57%). Two participants were excluded (did not treat DIPG). Participants included radiation-oncologists (62%), pediatric oncologists (7%), and pediatric neuro-oncologists (29%). Most physicians (62%) treated 1–5 DIPG patients per year, with 10% treating >10/year. Reirradiation was considered a treatment option in 88%. Progressive disease or worsening clinical status were the most common reasons to consider reirradiation. The majority (84%) considered reirradiation a minimum of 6 months following initial RT. Doses varied, with median total dose 24Gy (range 12–60); 2Gy/fraction (range 1–9). Concurrent use of systemic agents with reirradiation was considered in 46%, mainly with targeted agents (37%), biologics (34%), or immunotherapy (25%). One-time reirradiation was the most common practice (71%). Interestingly, 9% of respondents would not consider reirradiation.

CONCLUSION

Although, the vast majority of physicians agree with re-irradiation as a treatment option for DIPG the total doses varied, and further clinical trials are needed.
DIPG-10. OPTIMAL HDAC INHIBITION IN DIFFUSE INTRINSIC PONTINE GLIOMA
Authors: Vitanza Nicholas , Biery Matt , Myers Carrie , Ferguson Eric , Park Giulia , Noll Alyssa , Pakiam Fiona , Girard Emily , Morris Shelli , Cole Bonnie , Brusniak Mi-Youn , Mhyre Andrew , Olson James
Published: December 2020

As the majority of diffuse intrinsic pontine glioma (DIPG) have H3K27M mutations, epigenetic-targeting agents have been studied, though evaluations have been limited by their model systems, untranslatable drug concentrations, and/or evasive mechanisms of action. To develop a more translational model, we used biopsy samples from newly diagnosed DIPG patients to create treatment-naïve in vitro and in vivo models (molecular aberrations in parentheses), including PBT-09FH (H3FA3, PI3KCA), PBT-22FH (H3F3A, TP53), PBT-24FH (PMS2), and PBT-27FH (HIST1H3B, TP53, NTRK2). Models demonstrated radiation-resistance similar to the patient from whom the culture was generated, supporting the models’ relevance (e.g. cell viability after 8 Gy was 36%, 81%, 71%, and 61% in PBT-09FH, -22FH, -24FH, and -27FH, respectively, compared to 7% in the medulloblastoma model MED-411FH). We evaluated cell viability and apoptosis following treatment with a panel of HDAC inhibitors, identifying the low nanomolar IC50 of quisinostat (~50 nM) and romidepsin (~5 nM). While RNA expression changes induced by 100 nM panobinostat and quisinostat included shared overexpression of the top 20/25 genes (e.g. FSTL5, ITIH5) and shared downregulation of the top 22/25 (e.g. GPR37L1, HEPACAM), only 9/25 were downregulated by panobinostat, quisinostat, and romidepsin (e.g. C21orf62, IFIT2), identifying these as potential vulnerabilities or biomarkers of lethal HDAC inhibition. Mass-spectrometry (LC-MS) demonstrated panobinostat as the greatest acetylator of cortactin, potentially related to thrombocytopenia. While PBT-09 flank models demonstrated quisinostat’s on-target acetylation and efficacy, orthotopic xenograft models did not, supporting our model’s intact blood-brain barrier and emphasizing the need for CNS penetrant versions of potentially efficacious agents.