bluuebunny/crossref_metadata_2025 huggingface dataset:
DIPG-11. DISSECTING THE DIFFUSE MIDLINE GLIOMA TUMOR MICROENVIRONMENT COMMUNICATIONS USING MULTI-OMICS APPROACHES
Authors: Collot Raphael , Moreno Cristian Ruiz , Wezenaar Amber , Zomer Anoek , Johnson Hannah , Stunnenberg Henk , Rios Anne
Published: June 2023

Diffuse midline glioma (DMG) represents a highly aggressive pediatric brain tumor with no chance of survival. Treatment options are urgently needed, and efforts have been put towards developing T-cell immunotherapy. However, this should consider DMG’s unique tumor microenvironment (TME), both in terms of anatomical location and developmental stage. To gain knowledge on this unique tumor niche, we implemented an immuno-competent, syngeneic mouse model based on in-utero electroporation (IUE) of the key driver mutations to induce tumor formation at an embryonic stage. We characterized DMG’s spatial and molecular landscape by combining snRNAseq, spatial transcriptomics, and state-of-the-art imaging. We observed a high intratumoral heterogeneity with spatially restricted cell populations. Importantly, their molecular profiles correlate with patient data, illustrating the clinical relevance of the IUE model. Analysis of cell-cell communications in the TME revealed novel signaling between DMG and immunosuppressive myeloid cells that could impact T-cell response. Using an advanced patient-derived DMG organoid co-culture model with macrophages and engineered T-cells, we are currently investigating the role of this pathway on T-cells mediated killing of DMG organoids. Thus, this project fills a critical need to delineate and overcome the immune-suppressive environment in DMG and potentiate T cell targeting.
DIPG-13. NEW IMMUNOCOMPETENT GENETICALLY-ENGINEERED MOUSE MODELS OF PEDIATRIC-TYPE DIFFUSE HIGH-GRADE GLIOMAS REVEAL MECHANISMS OF TUMOR INITIATION AND PROGRESSION
Authors: Tian Ruxiao , Wu Annette , Laugesen Eric , Mak Tak , Fortin Jerome
Published: June 2023

Pediatric-type diffuse high-grade gliomas (pHGGs) form a group of aggressive brain tumors with very poor prognosis. Recurrent driver mutations in TP53 and histone H3-encoding genes, most commonly H3F3A, are observed in a large proportion of these tumors. H3-K27M substitutions are particularly prevalent in midline gliomas, and H3-G34R mutations occur in supratentorial tumors. To better understand the molecular mechanisms driving pHGG emergence and progression, we set out to develop immunocompetent mouse models recapitulating the oncogenic lesions observed in human tumors. Given that oligodendrocyte progenitor cells (OPCs) may be the cells of origin of some pHGGs, we investigated the effect of mutation induction in the OLIG2-expressing cell lineage, using the Olig2Cre driver. Interestingly, deletion of the P53-encoding gene, Trp53, in Trp53flox/flox;Olig2Cre/+ mice led to the development of diffuse gliomas in nearly all animals. Latency to humane endpoint was relatively long (>1 year), and tumors developed throughout the brain with a mild preference for infratentorial (e.g. brainstem) localization. The gliomas diffusely infiltrated the brain parenchyma, and were heterogeneously immuno-reactive for OPC, astrocytic, and neural progenitor cell (NPC) markers. Based on RNA sequencing, the tumors segregated in two groups: one enriched in expression of OPC genes, and another with a prominent NPC-like signature. Whole exome sequencing revealed frequent Ccnd1 and Kras/Hras copy number gains in advanced tumors. To model additional oncogenic lesions, we generated knock-in mice in which the H3f3aK27M or the H3f3aG34R mutations can be induced in any cell type with Cre recombinase, and extinguished with Flp recombinase. These were combined with conditional Trp53 loss-of-function alleles in various cell lineages, and their effect on tumor initiation and progression was monitored. Collectively, our studies provide new reagents for accurate disease modeling of pHGG in mice, opening opportunities to better understand tumor initiation and progression at high resolution, and for pre-clinical therapeutic testing.
DIPG-14. PROTEOGENOMICS PROFILING REVEALS ENRICHED NON-HISTONE PROTEIN METHYLTRANSFERASES AS NEW THERAPEUTIC TARGETS IN DIPG
Authors: Anguraj Vadivel Arun Kumaran , Pajovic Sanja , Patel Palak , Siddaway Robert , Zhu Sabrina , Phillips Lauren , Hawkins Cynthia
Published: June 2023

Diffuse intrinsic pontine glioma (DIPG) is a devastating brain tumour arising in the brainstems of children. Despite advances in genomics and treatments, the survival rate remains zero, with a median of less than a year, making it the leading cause of death among children with brain tumours. A mutation in histone H3 protein (H3K27M) has been identified as a genetic initiation event and affects global K27 trimethylation on histone H3 proteins and DNA methylation in DIPG. The epigenetic changes caused by the H3K27M mutation suggest the existence of an H3K27M-specific transcriptome and proteome. To deepen our knowledge of DIPG, we conducted a proteogenomic analysis of DIPG tissues by using high-resolution mass spectrometry for comprehensive proteome profiling, including total proteome, phosphoproteome, and methylproteome. Integrating proteomics data with DNA methylation and transcriptomics (bulk RNAseq and single-cell RNAseq) provided new insights into DIPG tumorigenesis. Our multi-omics analyses reveal enriched translation machinery, negative regulation of apoptosis process, and non-histone protein methyltransferase proteins, suggesting their previously unknown roles in DIPG cell growth and survival. Furthermore, our findings indicate that DIPG tissues have lower global mean protein phosphorylation and higher global mean protein methylation compared to normal brains, implying that DIPG may use methyl-signaling rather than phospho-signaling for tumour growth. The translation elongation proteins EEF1A1 and EEF1A2 are the most highly methylated proteins in DIPG. Methylation modifications such as K55me2, K79me3, and K165me2 of EEF1A1 are significantly higher in DIPG tissues compared to normal brains. These enriched methylpeptides are substrates of the non-histone methyltransferases METTL13 and METTL21B, which are enriched in the multi-omics analysis. Knocking down these methyltransferases in DIPG cells significantly decreases their target protein methylation, reduces global protein synthesis, and inhibits cell growth in vitro. Thus, proteogenomic analysis of DIPG reveals tumour-enriched non-histone methyltransferases, METTL13 and METTL21B as new therapeutic targets.
DIPG-21. LINKING DETERMINATION OF CELL FATE IN THE DEVELOPING CENTRAL NERVOUS SYSTEM TO DIFFUSE MIDLINE GLIOMA
Authors: Furst Liam , Roussel Enola , Leung Ryan , George Ankita , Firestein Ron , Faux Maree , Eisenstat David
Published: June 2023

Diffuse midline glioma, K27-altered (DMG) is a treatment resistant, uniformly fatal cancer that arises predominantly in children. Next-generation sequencing has revealed that the majority of patients with DMG harbor K27M mutations in canonical histone variants H3.1 and H3.3, leading to global epigenetic dysregulation and an oligodendrocyte precursor cell (OPC) transcriptional profile. DLX2 is a homeobox transcription factor that directly represses OPC cell fate and promotes a GABAergic interneuron cell fate during neurogenesis. We identified DLX2 occupancy of early (Olig2, Nkx2.2) and late (Myt1, Plp1) genes required in OPC differentiation in vivo. Co-expression of DLX2 to target sequences reduced reporter gene expression in vitro. The Dlx1/Dlx2 double knockout (DKO) mouse showed increases in Olig2, Nkx2.2, and Plp-1 expression in vivo. Transient over-expression of a Dlx2-GFP construct into murine DMG cells resulted in significant increased expression of Gad1/2 isoforms and decreased Olig2 and Nkx2.2, global restoration of H3K27me3, as well as a reduction in migration, invasion, and colony formation in vitro. An RNAseq database containing 45 human DMG cell lines showed an inverse correlation between DLX2 expression and OLIG1/2. Cell fate changes in DLX2high/OLIG1/2low cell lines were evaluated by knockout of DLX2 using CRISPR/Cas9. Concurrently, OLIG1/2high/DLX2low expressing cell lines underwent stable transfection of an DLX2-mCherry overexpression construct. Successful alteration of DLX2 expression was confirmed by qPCR and immunoblot in knockout and overexpressing human DMG cell lines. Analysis of histone H3 marks (K27me3, K27ac), K27M, and OPC signature genes will be presented along with comparison of altered OPC genes from the Dlx1/Dlx2 DKO mouse as determined by RNAseq. The manipulation of DLX2 expression in human DMG cells both in vitro and in vivo provides a model system for assessing the contribution of a homeobox transcription factor required for neurogenesis to cell fate regulation and effects on tumorigenicity in DMG.
DIPG-26. CLINICAL AND GENOMIC ANALYSIS OF H3K27M-DMG LONG-TERM SURVIVORS REVEALS ENRICHMENT IN MAPK PATHWAY ALTERATIONS
Authors: Roberts Holly , Picca Alberto , Sanson Marc , Schüller Ulrich , Picart Thiébaud , Ducray François , Green Adam , Nakano Yoshiko , Sturm Dominik , Abdullaev Zied , Aldape Kenneth , Waszak Sebastian , Venneti Sriram , Pratt Drew , Koschmann Carl
Published: June 2023

INTRODUCTION

H3K27M-mutant diffuse midline gliomas (H3K27M-DMGs) share a generally poor prognosis with a median overall survival (OS) of 9–12 months. However, patients with histone-mutant central nervous system (CNS) tumors and long-term survival (LTS) have been reported, in some instances beyond three years. We aim to determine characteristics of patients with H3K27M-mutant CNS tumors associated with LTS (OS>36 months).

METHODS

We performed a multi-site and literature case review of patients with confirmed H3K27M-DMG (including H3.1, H3.3, or by IHC) and LTS (OS>36 months) with extraction of demographic, diagnostic, molecular, therapeutic, and outcome data. We obtained a final cohort of 72 patients (median age 14 years, IQR 7-31 years). A control cohort of 453 patients with confirmed H3K27M-DMG and OS<18 months was created through literature review. Between the cohorts, demographic and clinical differences were compared for statistical significance (unpaired t-test and chi-squared test), excluding unknown variables, and frequent genetic alterations were compared.

RESULTS

There was no association with H3.3 versus H3.1 when comparing LTS (15% H3.1) and control (13.6% H3.1) cohorts. Alterations in MAPK pathway genes [NF1 (n=10), FGFR1 (n=10), and BRAF (n=9)] were more frequent in the LTS cohort, with H3.3/BRAFV600E co-mutation being reported in 8/72 (11%) LTS patients and only 5/453 (1.1%) control patients. The LTS group had a greater proportion of patients with non-infiltrative (17.5%) and low-grade (26.6%) histology tumors than the control group (1.7%, p<0.001 and 11.4%, p=0.001, respectively). DNA methylation analysis and RNA sequencing are currently being performed for 29 LTS patients to determine molecular sub-groups within H3K27M-DMG with improved prognosis.

CONCLUSIONS

Our early findings in this ongoing study suggest that both histological and molecular characteristics of H3K27M-DMG influence survival. We identify enrichment of alterations in MAPK pathway-related genes in patients with LTS. These findings hold promise for further defining and classifying H3K27M-DMG to improve prognostication.
DIPG-30. HIF2 OVEREXPRESSION DICTATES AGGRESSIVENESS ADDITIONALLY TO TP53 MUTATIONS IN H3.3K27M PEDIATRIC HIGH-GRADE GLIOMAS THROUGH RAS-MAPK AND WNT/BCATENIN PATHWAYS
Authors: Fuchs Quentin , Legras Stephanie , Lhermitte Benoit , Vauchelles Romain , Cosset Erika , Gorka Lucas , Outilaft Hassiba , Namer Izzie Jacques , Guerin Eric , Mura Carole , Burckel Hélène , Noel Georges , Hibaoui Youssef , Feki Anis , Chammas Agathe , Wolf Thibaut , Coca Andres Hugo , Foppolo Sophie , Dontenwill Monique , Entz-Werlé Natacha
Published: June 2023

Diffuse midline (DMG) and brainstem (DIPG) high-grade gliomas (HGG) bearing H3.3K27M driver mutation are aggressive and uncurable brain tumors. We evidenced by the past HIF-2a and its transcriptional gene, EPAS1, as a marker of resistance when targeting mTor/HIF-1a pathway. To understand HIF-2α potential role in chronically active hypoxic environment in DIPG/DMG, we investigate its status in a cohort of pediatric HGGs comprising notably H3.3K27M mutant tumors and in their paired in vitro derived models. Using concomitantly the low-input chromatin immunoprecipitation sequencing method CUT&RUN, RNAsequencing and metabolomics, we profiled the landscape of promoters and pathways regulated by HIF-2a in HGGs, where H3.3K27M and TP53 mutations are associated. Elucidation of HIF-2a downstream genes reveal statistically significant pathways linked to DIPGs (e.g., mTor and canonical Wnt pathways) and to DMGs (glutamatergic synapse and non-canoncial Wnt signaling). Finally, HIF-2a was evidenced as a new therapeutic target opening the path for its use concomitantly to irradiation.
DIPG-31. HIF-1A AND HIF-2A COMBINATORIAL TARGETING OR HOW TO REVERSE THEIR BALANCED ONCOGENIC IMPACT
Authors: Fuchs Quentin , Vauchelles Romain , Cosset Erika , Gorka Lucas , Lhermitte Benoit , Guerin Eric , Hubsch Clémence , Foppolo Sophie , Hibaoui Youssef , Feki Anis , Wolf Thibaut , Dontenwill Monique , ENTZ-WERLE Natacha
Published: June 2023

HIF-1a and HIF-2a are master regulators operating during tumor hypoxia. Few data are available on their role in pediatric high-grade gliomas (pHGGs), in which recent insights on metabolism and immunotolerance induction through hypoxia-driven mechanisms might opening the path to study further this process and their molecular actors. Our study investigated and confirmed the balanced dependency of both transcription factors in pHGGs at protein levels, but not in their transcriptional reprogramming. We also examined how HIF-1a and HIF-2a are operating during acute and chronic hypoxia in a spatiotemporal cell setting. HIF-1a was clearly correlated within pHGGs samples and models to acute and moderate hypoxia, whereas HIF-2a was associated to H3.3K27M mutant DIPG and chronic hypoxia. The impact of targeting both transcription factors in pHGG-derived models was on cell proliferative and invasive mechanisms, especially when studying this drug testing in engineered neural tissues. We might position those new targets as an innovative way to counterpart their hyperactivation in tumor core hypoxia and to propose them as an innovative approach interfering with cell-to-cell adhesion and synaptic interactions with normal brain cells.
DIPG-35. IDENTIFYING DRIVER-SPECIFIC VULNERABILITIES IN PAEDIATRIC HIGH GRADE GLIOMA SUBTYPES
Authors: De Cola Antonella , McNicholas Michael , Foss Amelia , Lloyd Cameron , Hébert Steven , Faury Damien , Andrade Augusto Faria , Jabado Nada , Kleinman Claudia , Pathania Manav
Published: June 2023

Paediatric high-grade gliomas (pHGGs) are incurable malignant brain tumours and a leading cause of cancer-related death in children. The majority of pHGGs carry lysine-to-methionine (K27M) or glycine-to-arginine/valine (G34R/V) mutations in histone variants H3.1 or H3.3. Moreover, histone mutations associate with different anatomical locations and co-segregating mutations defining distinct tumour subtypes within pHGG. However whether these co-occuring mutations can act as drivers to modify tumour phenotypes and drug sensitivities is currently unknown. In order to functionally evaluate the role of partner alterations and to identify new therapeutic targets, we developed in vivo tumour models of pHGG subtypes using in utero electroporation (IUE) in combination with piggyBac transposon and CRISPR technology. We also established ex vivo glioma stem cell (GSCs) lines from mouse models with different co-segregating mutations, able to engraft in syngeneic immune-competent mice. Then, we performed transcriptome analysis and drug screening identifying selective pharmacological vulnerabilities. Our approach represents a preclinical platform to evaluate subtype-specific precision therapies identifying new pathways involved in brain tumour initiation, progression and maintenance.
DIPG-36. H3K27M HISTONE MUTANT GLIOMAS ARE SENSITIVE TO METHIONINE LOSS AND MAT2A INHIBITION THROUGH A NOVEL FEEDBACK MECHANISM BETWEEN AMD1 AND METTL16
Authors: Chilukuri Akanksha , Halbert Matthew , Golbourn Brian , Varadharajan Srinidhi , Krug Brian , Mbah Nneka , Casillo Stephanie , Gatesman Taylor , Cruz Andrea , Zapotocky Michael , Hu Baoli , Kohanbash Gary , Bertrand Kelsey , Kleinman Claudia , Lyssiotis Costas , Jabado Nada , Pollack Ian , Mack Stephen , Agnihotri Sameer
Published: June 2023

Diffuse Midline gliomas (DMGs) are grade IV tumors by the World Health Organization. They are inoperable and resistant to chemo/radiotherapies resulting in a median survival of 8-11 months and a 5-year survival of <2%. DMG is an epigenetic disease characterized by mutations on histone H3.3 K27M resulting in global transcriptional reprogramming. This disease lacks appropriate models to predict disease biology and response to treatment. Therefore, we developed a novel syngeneic H3K27M mouse model using clinically relevant co-alterations in Olig2+ neural progenitor cells (NPCs). Using an unbiased systems biology approach, we identified a reliance of H3K27M but not isogenic controls to the amino acid methionine, and the enzymes methionine adenosyltransferase 2A (MAT2A), and adenosylmethionine decarboxylase 1 (AMD1). MAT2A is a master regulator of methionine metabolism that converts methionine into the universal methyl donor S-adenosylmethionine (SAM) which is later converted into decarboxylated SAM (dcSAM) by AMD1 for polyamine metabolism. We postulated that targeting methionine regulator MAT2A through genetic/pharmacological abrogation would selectively alter DMG viability by disrupting the methylome. We discovered a novel mechanism demonstrating H3K27M cells are sensitive to MAT2A loss independent of methylthioadenosine phosphorylase (MTAP) deletions but rather through AMD1 overexpression. The current paradigm shows that MAT2A protein expression is inversely correlated with cellular SAM concentrations as sensed by splicing complex and m6A reader methyltransferase-like protein 16 (METTL16). To investigate the molecular mechanism by which H3K27M represses MAT2A, we postulated that dcSAM, the resultant metabolite of AMD1, promote(s) high turnover of METTL16–MAT2A transcript interactions like SAM, thereby diminishing MAT2A transcript and protein expression. We found that exogenous dcSAM promoted MAT2A intron retention and lower mature transcript levels. Our findings demonstrate that H3K27M leads to increased AMD1 protein expression resulting in diminished MAT2A expression. Combinatorial treatments inhibiting MAT2A and AMD1 may presents exploitable therapeutic vulnerabilities in these gliomas.
DIPG-41. EXPLORING BRAIN PENETRANT PEPTIDE-DRUG CONJUGATES IN DIFFUSE INTRINSIC PONTINE GLIOMA MODELS
Authors: Clark Jasmine , McKinnon Samuel , JImenez-Macias Jorge_Luis , Miller Edward , Loas Andrei , Pentelute Brad , Lulla Rishi , Lawler Sean
Published: June 2023

The blood brain barrier (BBB) tightly controls access of molecules to the brain, limiting the efficacy of therapeutics for diseases of the central nervous system. We recently developed a novel approach for drug delivery across the BBB using a perfluoroaryl macrocyclic peptide-drug conjugate, M13-Pt(IV). This approach showed accumulation of platinum (Pt) within the brain, and improved survival in murine xenograft models using adult patient-derived glioblastoma cells. We also found that uptake of M13-Pt(IV) into tumor tissue is further enhanced by 6-bromo-indirubin acetoxime (BIA), a small molecule with anti-invasive properties. Because diffuse intrinsic pontine glioma (DIPG) is an invasive brain tumor protected from systemic drugs by an intact BBB, we hypothesized that this approach may be of particular benefit in this tumor type. Therefore our goal here was to investigate peptide-mediated drug delivery across the BBB in DIPG, using M13-Pt(IV) as a tool compound. When tested across a panel of four distinct DIPG lines in vitro the M13-Pt(IV) conjugate showed tumor cell killing effects superior to Pt(IV). BIA significantly slowed DIPG cell migration and lowered endothelial cell permeability in vitro via reduced expression of tight junction proteins as shown by immunostaining and decrease in trans-endothelial resistance. Bi-weekly dose regimens of M13-Pt(IV) are well-tolerated in nude mice at a dose of 15mg/kg, 3-fold more than the reported tolerable cisplatin dose at 5 mg/kg. Moreover, in vivo BIA also enhanced drug M13-Pt(IV) uptake into adult glioblastoma tumors. In vivo uptake and efficacy studies in DIPG tumor-bearing mice are underway. Overall, our data using a prototype peptide-drug conjugate support the further development of BBB-crossing peptide-drug conjugates for DIPG. Identification of the optimal therapeutic combinations for clinical application is our focus for future studies.
DIPG-43. CLINICAL AND MOLECULAR CHARACTERISTIC OF A NEW SUBTYPE OF DMG, H3K27-ALTERED WITH MAPK-ACTIVATING CO-DRIVER MUTATIONS
Authors: Auffret Lucie , Ajlil Yassine , Kergrohen Thomas , Tauziède-Espariat Arnault , Puiseux Chloé , Riffaud Laurent , Picca Alberto , Touat Medhi , Sanson Marc , Beccaria Kevin , Blauwblomme Thomas , Dangouloff-Ros Volodia , Boddaert Nathalie , Varlet Pacale , Debily Marie-Anne , Grill Jacques , Castel David
Published: June 2023

Diffuse midline gliomas (DMG) represent a big challenge in neuro-oncology. These tumors occur more frequently in children and are presently incurable. They are characterized by a K27M substitution in H3.1 or H3.3 histone tail or the overexpression of EZHIP (EZH Inhibitory Protein). These three alterations induce a global loss of trimethylation in H3K27 with a specific epigenic and transcriptomic remodeling. The additional oncogenic events and the clinical behavior are also distinct according to the driver event. Based on these differences, the H3K27-altered DMG is now classified in 4 subtypes by the latest edition of the WHO Classification of CNS tumors. Even with this new subclassification, the H3.3K27M subgroup still appears heterogenous. Recent publications reported that rare patients presenting a co-occurrence of H3.3K27M with BRAF or FGFR1 alterations tend to have a better prognosis.

To better study the role of these co-driver alterations that activate the mitogen activated protein kinase (MAPK) signaling, we assembled a large pediatric and adult cohort of H3K27-altered DMG comprising 25 new DMG patients mutated in FGFR1 or BRAFV600E and 37 previous cases from the literature. We performed a comprehensive histological, radiological, genomic, transcriptomic and DNA methylome analysis on this extended cohort. Interestingly, the results show clear differences with other DMG subtypes, including: specific DNA methylation profile, senescence signature, better overall survival (median around 3 years), older age at diagnosis, specific histological and radiological presentations with calcifications or more circumscribed tumors. Additionally, in specific cases, we show that the MAPK-activating mutation occurred subsequently to the histone H3K27M mutation.

In conclusion, DMG, H3K27-altered harboring MAPK activating mutations represent a new subtype of DMG also frequent in adults, and deserve further attention with respect to specific therapeutic challenges.
DIPG-44. DIFFUSE MIDLINE GLIOMA WITH EXTRACRANIAL METASTASIS
Authors: Larsen Alexandra Giantini , Rivera Maricruz , Pisapia David , Sait Sameer Farouk , Karajannis Matthias , Greenfield Jeffrey , Souweidane Mark
Published: June 2023

Diffuse midline gliomas (DMGs) are malignant infiltrative gliomas enriched in the pediatric population and characterized by loss of the H3 K27me3 epigenetic marker, most frequently via mutation of the H3F3A gene. Few cases of extracranial DMG metastasis with redemonstrated H3F3A p.K28M (K27M) mutation in metastatic tissue are reported in the literature. Here, we report two such patients, both females (ages 12 and 15), with DMG and extracranial metastasis who died 5 years after initial diagnosis. The first patient with thalamic DMG underwent biopsy, radiation, chemotherapy, and subsequent decompression of spinal metastasis. Initial tumor biopsy demonstrated mutation in BRAF p.V600E, H3F3A K27M, and TERT promoter. The epidural spinal metastasis had mutations in BRAF p.V600E, H3F3A, TERT, and TP53. The second patient with brainstem DMG, diagnosed by radiographic appearance, underwent conventional external beam radiation therapy, chemotherapy including panobinostat and direct delivery of radioimmunotherapy, intra-arterial delivery of bevacizumab, and subsequent decompression of spinal metastasis. The histology and molecular profile for the spinal metastasis was consistent with DMG H3 K27M-mutant, with mutations in BRAF p.G464V, PTPN11 p.R498L, PDGFRA, and KIT. Bone marrow biopsy demonstrated atypical extrinsic cells with strong and diffuse immunolabeling for OLIG2. Autopsy revealed solid organ and osseous metastases. Both patients significantly exceeded the median life expectancy for DMG, raising the possibility that prolonged overall survival permitted progression to a rarely observed disseminated state of disease. There was absence of TP53/p53 modulating pathway mutation seen in classic DMG in the thalamic biopsy of the first patient, as well as the metastatic disease for the second patient, which may contribute to the prolonged survival observed. Molecular analysis of metastatic disease is important, as clinically and prognostically relevant alterations that vary from the primary site of disease may be detected, which shed light on clonal evolution patterns and further our understanding of disease biology.
DIPG-53. LSD1 DEGRADATION SYNERGIZES WITH HDAC INHIBITION IN PEDIATRIC H3K27M DIFFUSE MIDLINE GLIOMA
Authors: Haase Rebecca , Groves Andrew , Mire Hafsa , Qi Jun , Filbin Mariella
Published: June 2023

H3K27M-mutated diffuse midline glioma (H3K27M DMG) is an aggressive, lethal pediatric brain tumor which resists conventional cancer treatments. To identify new therapeutic targets, our lab executed a focused CRISPR negative selection screen in patient-derived H3K27M cell lines after treatment with the histone deacetylase (HDAC) inhibitor panobinostat, and discovered a strong co-dependence with the histone demethylase LSD1. We further explored the therapeutic potential of this synergistic interaction with a screen of HDAC- and LSD1- targeting drugs to identify synergistic combinations, which show penetration of the blood brain barrier suitable for clinical translation. Interestingly, conventional catalytic LSD1 inhibitors did not seem to synergize with HDAC inhibitors tested on cell viability, while a recently described CoREST/LSD1 degrader phenocopied the effects seen in our CRISPR screen. We hypothesized that the CoREST/LSD1 degrader induces synergy with HDAC inhibitors through elimination of a scaffolding function of LSD1. To prove this, we knocked out LSD1 using CRISPR/Cas9 in H3K27M cells and subsequently treated with a panel of HDAC inhibitors. These showed a significant sensitization compared to controls cells. Further, we confirm the high specificity of the CoREST/LSD1 degrader by performing streptavidin bead pull down with a newly synthesized biotin-conjugated probe. This revealed that it interacts with the entire CoREST complex (members include LSD1, RCOR1, HDAC1/2). In conclusion, we show that degrading CoREST/LSD1 in combination with HDAC inhibition is a synergistic strategy in H3K27M DMG. For future translational studies, we will assess biomarkers upon treatment and analyse compound potency in vivo.
EPEN-03. ZFTA-RELA LOCALIZATION DYNAMICS REGULATE TRANSCRIPTIONAL CONTROL
Authors: Arabzade Amir , Laboe Nic , Ippagunta Siri , Emanus Eric , Kardian Alisha , Mack Stephen
Published: June 2023

ZFTA gene fusions are the most frequent driver events in supratentorial ependymoma (EPN). ZFTA-fusion proteins act as oncogenic transcription factors (TFs) to aberrantly activate oncogenic transcriptional programs. Our data indicates that ZFTA fusion proteins form distinct punctate structures with heterogeneous patterns of liquid-phase-like characteristics. These condensates are closely associated with transcription as seen by nascent RNA FISH and recruitment of various members of the transcription machinery, such as BRD4, MED1, and RNA POLII. Our genomic footprinting data indicates that many key transcription factors, such as Sox9, share similar genomic occupancy patterns with ZFTA fusions suggesting that these transcription factors are potentially recruited to these condensates, establishing transcriptional hubs. Our mutagenesis studies reveal that the ZFTA zinc finger domain is indispensable for condensate formation, and may bridge ZFTA fusion proteins to DNA at unique genomic loci. Condensate-forming deficient mutants fail to establish the same transcriptional program and are unable to drive tumor formation. This mechanistic data has led to our efforts to establish drug-chemical screen approaches against ZFTA-RELA localization/dynamics to identify novel therapeutic targets.
EPEN-06. CHARACTERISING THE DEVELOPMENTAL ORIGINS AND TRAJECTORIES OF ZFTA-RELA FUSION EPENDYMOMA IN A NOVEL MOUSE MODEL
Authors: Bonner Sigourney , King Claire , Rarhmann Katherine Wickham , Cooper Elizabeth , Katyal Gunjan , Gilbertson Richard
Published: June 2023

Ependymomas driven by the ZFTA-RELA fusion account for >70% of all supratentorial ependymomas. These tumours are now recognised in the WHO classification of CNS tumours and have been associated with a poor prognosis. Seven ZFTA-RELA fusion variants have been described: around two thirds of cases are fusion 1. No spontaneous genetically modified mouse models (GEMMS) have been described and current models require invasive intracranial injection (of transduced cells or RCAS-TVA system). Here we describe the first spontaneous GEMM of ZFTA-RELA fusion-driven ependymoma. Nestin-Flx-STOP-Flx-ZFTA-RELA (Fusion 1) open reading frames were targeted together with luciferase to the Rosa-26 locus. Breeding these mice with Nestin-CreERT2 or Blbp-Cre lines that drive recombination in neural progenitor cells resulted in forebrain tumours that could be tracked with bioluminescence imaging from P20. Tumours displayed NF-kB and L1CAM expression and ZFTA-RELA protein was detected using a novel in-house antibody. Tumours display expression of a known ZFTA-RELA fusion ependymoma transcriptomic signature. ZFTA-RELA tumours can be grown as neurospheres and passaged as allografts in nude mice and respond to irradiation. We provide the first spontaneous GEMM of this important group of ependymomas. Here we present our histologic and transcriptomic developmental timecourse of these tumours, their comparison to the human disease and further understanding of the lineage origins and potential novel targets.
EPEN-08. COMBINED PDGFR INHIBITOR AXITINIB AND RETINOID TRETINOIN DECREASES PROLIFERATION AND HIGH-RISK NEOPLASTIC SUBPOPULATION PROPORTIONS PFA EPENDYMOMA
Authors: Donson Andrew , Grimaldo Enrique , Riemondy Kent , Griesinger Andrea , Amani Vladimir , Brunt Breauna , Pierce Angela , Foreman Nicholas
Published: June 2023

Ependymoma (EPN) is fatal in over 50% of children and has not seen any therapeutic improvements in over 30 years. Based on data from single-cell RNA sequencing (scRNAseq) and large-scale screening of FDA-approved oncology compounds in posterior fossa group A (PFA) EPN, we hypothesized that combination of PDGFR inhibitors and retinoids therapy will selectively deplete aggressive mesenchymal and undifferentiated EPN subpopulations resulting in therapeutic benefit. In vitro screening of a panel of three receptor PDGFR inhibitors (PDGFRIs) and nine retinoid analogues in high-risk chromosome 1q+/6q- PFA cell lines MAF-811 and MAF-928 was performed to identify those drugs with (i) anti-proliferative effect (tritiated thymidine incorporation and live-cell imaging), (ii) depletion of high-risk associated neoplastic subpopulations (qRT-PCR using subpopulation specific markers and scRNAseq), and (iii) synergistic effects. PDGFRIs axitinib and pazopanib and retinoids tazarotene, adapalene and tretinoin showed superior potency versus other analogues. qRT-PCR showed that tazarotene and axitinib demonstrated beneficial subpopulation perturbation. Combination of the top two PDGFRIs (axitinib and pazopanib) and retinoids (tazarotene and tretinoin) demonstrated that axitinib combined with tretinoin provided the strongest synergistic anti-proliferative effect and depletion of high-risk-associated undifferentiated subpopulations. More definitive scRNAseq subpopulation analysis revealed a reduction of mesenchymal cells in MAF-811 in response to combined axitinib/tretinoin treatments. In both MAF-811 and MAF-928 there was a net increase in the proportion of differentiated subpopulations, which are associated with a more favorable clinical outcome, in response to combined axitinib/tretinoin, collectively supporting our original hypothesis. These results demonstrate that combined RTKi axitinib and retinoid tretinoin treatment has a synergistic anti-proliferative effect and reduction of high-risk subpopulation proportions. The effect of combined axitinib and tretinoin on survival and subpopulation perturbation is now being explored in vivo.
EPEN-09. UTILITY OF RADIATION THERAPY IN SPINAL CORD MYXOPAPILLARY EPENDYMOMAS: A REVIEW OF THE LITERATURE
Authors: Jackson Kasey , Kleinknecht Sarah , Packer Roger , Hwang Eugene
Published: June 2023

INTRODUCTION

Myxopapillary ependymomas (MPEs) are slow-growing tumors that comprise about 25% of spinal ependymomas. Although historically designated as grade 1, the recent WHO classification has upgraded MPEs to grade 2 given their potential for recurrence and dissemination. While the National Comprehensive Cancer Network guidelines recommend avoiding radiation therapy (RT) unless a gross-total resection (GTR) is not achieved, several reports have suggested a potential benefit of adjuvant RT in improving relapse-free survival in children after GTR. This literature review was conducted to compare available data to determine the utility of upfront irradiation in these patients.

METHODS

A comprehensive literature review from 1984-2021 was performed on PubMed using keywords: “pediatric myxopapillary ependymoma” and “radiation.” One case was censored for unknown radiation status.

RESULTS

17 analyses from 1984-2021 were reviewed, yielding 140 pediatric cases of MPE, of whom 59% (n=83) received a GTR. Of those that received a GTR, average age was 13.7 yrs (range, 6-21), and 66% were male. The most common location was lumbosacral (25%), followed by thoracolumbar (19%). 22.8% (n=19) of patients received upfront focal RT. Relapse occurred in eight (42%) patients who received upfront RT, and 30 (47%) of those who did not, with no clear age or gender discrimination in those that relapsed. Distant recurrence occurred in 75% of those who received RT, and 68% of those that did not. The duration of event-free survival will be included in the final presentation.

CONCLUSIONS

While some individual studies have suggested a survival advantage in pediatric MPE after focal RT, in this meta-analysis, both relapse rate and location seemed similar regardless of receipt of RT. From this available evidence, the efficacy of upfront RT has not been demonstrated.
EPEN-12. COMPARING CELLULAR HIERARCHIES AND SPATIAL ARCHITECTURE ACROSS MOLECULAR SUBGROUPS OF SUPRATENTORIAL EPENDYMOMAS
Authors: Jeong Daeun , Maass Kendra K , Ghasemi David R , Englinger Bernhard , Jiang Li , Nascimento Andrezza , Hack Olivia A , Shaw McKenzie L , Pfister Stefan M , Kool Marcel , Gojo Johannes , Pajtler * Kristian W , Filbin * Mariella G
Published: June 2023

Ependymomas (EPN) are among the most common and fatal pediatric brain tumors with currently no targeted therapies available. Molecular studies including genome-wide DNA methylation profiling have shown that supratentorial ependymoma is subdivided into subgroups characterized by distinct fusion proteins: ZFTA-RELA fusion-positive (ZFTA-RELA), YAP1 fusion-positive (ST-YAP1), and four alternative ZFTA fusion-positive (ZFTA-Cluster 1, ZFTA-Cluster 2, ZFTA-Cluster 3, and ZFTA-Cluster 4). ZFTA-RELA tumors have been identified as the most common and aggressive molecular group with high intra-tumoral heterogeneity. However, limited sample size and interpatient variability in previous studies highlight a critical need for comprehensive profiling of supratentorial ependymomas across a larger patient cohort. In particular, how ZFTA-RELA compares to other subgroups of supratentorial ependymoma is unknown. Here, we collected and profiled 44 supratentorial patient tumors encompassing ZFTA-RELA (n=21), ZFTA-Cluster 1 (n=6), ZFTA-Cluster 2 (n=4), ZFTA-Cluster 3 (n=7), ZFTA-Cluster 4 (n=2), and ST-YAP1 (n=4). We profiled all tumors by single cell/single nucleus RNA-sequencing, and further characterized ZFTA-RELA by single cell spatial transcriptomics. Within each patient’s tumor we find multiple normal and aberrant differentiation trajectories from neural progenitor-like cells towards glial-like, mesenchymal-like and ependymal-like cells. Using sequencing-based lineage tracing, we experimentally validate these computationally inferred cellular hierarchies in vitro. Notably, compared to ZFTA-RELA ependymomas we observed predominantly higher proportion of early neural progenitor-like cells in ZFTA-Cluster 2 and ZFTA-Cluster 3, while in ST-YAP1 greater representation of mature ependymal-like cells. These findings highlight differences in developmental and aberrant differentiation trajectories driven by combination of oncogenic fusions and cell of origin in supratentorial ependymoma subgroups. In addition, neural progenitor-like cells most highly express ZFTA-fusions and are spatially distinct from other cell states, highlighting oncogenic fusions driving tumorigenesis organized in spatial niches. Together, this study reveals previously unknown intra- and intertumoral heterogeneity across subgroups of supratentorial ependymoma, as well as distinct cellular and spatial architecture.
METB-05. NON-INVASIVE DIAGNOSIS OF BRAINSTEM GLIOMAS IN PEDIATRIC, ADOLESCENT, AND YOUNG ADULT PATIENTS THROUGH CEREBROSPINAL FLUID CELL-FREE DNA SEQUENCING
Authors: Hill Katherine , Giantini-Larsen Alexandra , Hickman Richard A , Holle Bridget , Alano Tina , Doe-Tetteh Seyram , Bouvier Nancy , Li Shanita , Stockfisch Emily , Huereca Claudia , DiNapoli Sara , Hertz Charli Ann , Lee Alex , Szalontay Luca , Li Bryan Kincheon , Riviere-Cazaux Cecile , Burns Terry C , Haque Sofia , Bale Tejus , Benhamida Jamal , Dogan Snjezana , Vanderbilt Chad , Borsu Laetitia , Ross Dara , Chang Jason , Zampieri Caroline Barbosa , Donzelli Maria , Dunkel Ira J , Kramer Kim , Sait Sameer Farouk , Khakoo Yasmin , Gilheeney Stephen , Souweidane Mark , Greenfield Jeffery , Berger Michael , Arcila Maria , Ladanyi Marc , Mellinghoff Ingo , Karajannis Matthias , Miller Alexandra
Published: June 2023

Intrinsic brainstem tumors arising in pediatric, adolescent, and young adult patients comprise a spectrum of entities, predominantly diffuse midline gliomas (DMG) and IDH mutant astrocytomas. Accurate molecular diagnosis is essential for prognostication and optimal therapy. Therapeutic considerations include inclusion (in IDH mutant) or exclusion (in H3K27M) of adjuvant Temozolomide post radiotherapy and use of molecular targeted therapy (IDH inhibitors). Surgical biopsy, however, is associated with increased risk of permanent neurological deficits and may yield insufficient or non-diagnostic tissue. We hypothesized that minimally-invasive “liquid biopsy” of cerebrospinal fluid (CSF) could represent a superior diagnostic modality in this patient population, employing molecular analysis of cell free DNA (cfDNA). We analyzed 44 CSF samples from 38 unique patients for recurrent driver mutations in brainstem gliomas, including H3K27M, IDH1, and IDH2. MSK-IMPACT, a NY state-authorized hybridization capture-based next-generation panel DNA sequencing assay, was used for analysis. Samples without a detectable driver mutation by MSK-IMPACT testing were further subjected to gene targeted testing by droplet digital PCR. In all, 10/44 (22.7%) samples had mutations detected by MSK-IMPACT using standard calling criteria and 13 of the remaining 34 samples (38.2%) had supporting evidence of a mutation based on manual review. Further testing by ddPCR was performed on a subset of cases (based on DNA availability) which confirmed 7 of the previous low-level mutations and uncovered 4 additional mutations. Overall, 27/44 (61.4%) of cases had detectable evidence of a mutation, and of the 23 patients without a known tissue diagnosis, a driver in the CSF was identified for 47.8% of patients (11/23). Minimally-invasive analysis of CSF cfDNA in patients with intrinsic brainstem tumors has a high diagnostic yield and may obviate the need for tissue biopsy in a majority of patients. Testing with high sensitivity assays is valuable to maximize the rate of detection.
METB-09. GERMLINE PATHOGENIC VARIANTS IN 838 PEDIATRIC BRAIN TUMOR PATIENTS
Authors: McQuaid Shelly W , Kaufman Rebecca , Corbett Ryan J , Vaksman Zalman , Bornhorst Miriam , Brown Miguel A , Guo Yiran S , Zhu Yuankun , Heath Allison P , Storm Phillip B , Resnick Adam C , Waanders Angela J , Cole Kristina A , Rokita Jo Lynne , Macfarland Suzanne , Diskin Sharon J
Published: June 2023

The genetic contribution of rare pathogenic germline variation in cancer patients without a family history remains unclear. We sought to characterize the prevalence, spectrum, and clinical significance of pathogenic germline variation in cancer predisposition genes (CPGs) in pediatric brain tumor patients. Paired tumor and normal whole genome or exome sequencing was performed for 838 patients in the Pediatric Brain Tumor Atlas (PBTA). Rare variants in 196 CPGs were annotated as pathogenic (P) or likely pathogenic (LP) in an automated manner consistent with American College of Medical Genetics criteria and reviewed by an interdisciplinary panel. To assess enrichment, the frequency of CPG P-LP variants in cases was compared to the gnomAD v3.1 cancer-free control cohort (n=74,023). Second hit, mutational signature, and gene set enrichment analyses (GSEA) were performed on matched tumor sequencing to characterize differences in patients with and without germline P-LP variants in DNA repair genes. We observed 178 germline CPG P-LP variants in 163 PBTA patients (19.5%). CPG P-LP variants were most prevalent among Neurofibroma plexiform patients (64.3%), and NF1 and TSC2 harbored the most significant P-LP variant burden compared to controls (OR=69.5, p=2.4E-23, CI=34.6-137 and OR=357, p=1.1E-14, CI=71-3669, respectively). DNA repair gene P-LP variants were enriched in cases compared to controls (OR=4.4, p=8.3E-30). Patients harboring mismatch repair (MMR) gene P-LP variants exhibited significantly higher tumor-associated MMR-deficiency mutational signature exposures (p<0.05) and DNA repair GSEA scores relative to other patients (p=0.04). Second hit SNVs in tumors were identified in four patients with P-LP germline variants in NF1 (n=2), APC (n=1), and SUFU (n=1). Germline P-LP variants are enriched in PBTA patients, and DNA repair variants are predictive of repair-deficiency signatures in tumor samples. Our work emphasizes the need for germline sequencing to inform patient care.
RARE-03. GENOME-WIDE ASSOCIATION STUDY OF INTRACRANIAL GERM CELL TUMORS: A COMMON DELETION AT BAK1 ATTENUATES THE ENHANCER ACTIVITY AND CONFERS RISK FOR THE BRAIN TUMORS IN CHILDREN ADOLESCENTS AND YOUNG ADULTS
Authors: Terashima Keita , Sonehara Kyuto , Kimura Yui , Nakano Yoshiko , Ozawa Tatsuya , Takahashi Meiko , Suzuki Kenji , Fujii Takashi , Matsuhita Yuko , Tomiyama Arata , Kishikawa Toshihiro , Yamamoto Kenichi , Naito Tatsuhiko , Suzuki Tomonori , Yamaguchi Shigeru , Miwa Tomoru , Sasaki Hikaru , Kitagawa Masashi , Ohe Naoyuki , Fukai Junya , Ogiwara Hideki , Kawamura Atsufumi , Miyawaki Satoru , Matsuda Fumihiko , Kiyokawa Nobutaka , Ichimura Koichi , Nishikawa Ryo , Okada Yukinori
Published: June 2023

Intracranial germ cell tumors (IGCTs) are rare brain tumors that mainly occur in children, adolescents and young adults with a particularly high incidence in East Asian populations. The biological basis of these tumors is still largely unknown. We conduct a genome-wide association study (GWAS) of 133 patients with IGCTs and 762 controls of Japanese ancestry by using Infinium Asian Screening Array and other molecular biology methods. A common 4-bp deletion polymorphism in an enhancer adjacent to BAK1 is significantly associated with the disease risk (rs3831846; P = 2.4 x 10-9, odds ratio = 2.46 [95% CI: 1.83-3.31], minor allele frequency = 0.43). Rs3831846 is in strong linkage disequilibrium with a testicular GCTs susceptibility variant rs210138. Expression quantitative trait locus (eQTL) analysis using the GTEx dataset reveals that the risk allele of rs3831846 has a down-regulating effect on BAK1 expression in a wide range of tissues. Further in-vitro reporter assays reveal rs3831846 to be a functional variant attenuating the enhancer activity. BAK1 is a pro-apoptotic member of the BCL-2 family, thus our results suggested that the risk allele may contribute to IGCTs predisposition through down-regulating BAK1 expression. Risk alleles of testicular GCTs derived from the European GWAS show significant positive correlations in the effect sizes with the Japanese IGCTs GWAS (P = 1.3 x 10-4, Spearman’s ρ = 0.48). Of the 57 loci, 11 exhibit significant association with IGCTs and these loci were implicated in a broad range of biological pathways, including KIT/KITLG signaling, apoptosis regulation, and telomerase activity. The risk allele frequency of rs3831846 is higher in east Asia than Europe (0.49 vs. 0.20), which may provide a partial explanation for the ethnic difference in incidence. Nevertheless, our results suggest the shared genetic susceptibility of GCTs beyond ethnicity and primary sites.
RARE-06. DISSECTING CELLULAR DIFFERENTIATION HETEROGENEITY IN CHOROID PLEXUS CARCINOMA PATHOGENESIS
Authors: Faltings Lukas , Mian Noreen , Siluveru Anjali , Modi Siddhi , Virga James , Cao Ping , Zhao Haotian
Published: June 2023

Choroid plexus (CP) neoplasms are rare, predominantly pediatric, brain tumors ranging from benign CP papilloma to aggressive CP carcinoma (CPC) associated with poor survival. Survivors often experience devastating side effects from current treatment strategies. There is an urgent need for safer, more effective therapies for CPC. A subset of human CPC exhibits abnormal NOTCH activity, whereas mutations of tumor suppressor gene TP53 are detected in >50% of CPC and are associated with increased genetic tumor instability and worse prognosis. CPC is one hallmark of Li-Fraumeni syndrome (LFS), a rare genetic disorder associated with germline TP53 mutations. Human CP tumors also frequently display aberrant expression of transcriptional regulators of roof plate/CP differentiation, including LIM homeodomain transcription factors LMX1A, and SRY-Box Transcription Factor 2 (SOX2). To investigate the role of transcriptional regulation in CP tumorigenesis, animal models were developed driven by molecular defects commonly found in CP tumors in humans. Accordingly, sustained NOTCH and Sonic Hedgehog (SHH) activation or combined deletion of Rb1 and Trp53 tumor suppressors led to malignant CP tumors with characteristics of CPC in humans. NOTCH-driven CP tumors arose from monociliated roof plate progenitors and exhibited increased levels of Lmx1a, Lmx1b, and Sox2. In contrast, their expression was significantly reduced, while OTX2 levels were markedly increased in Rb1/Trp53-deficient CPC. Consistently, single-cell transcriptomics data show a proliferative Otx2+ tumor bulk and a small Lmx1a+/Sox2+ population in Rb1/Trp53-deficient CPC. Importantly, deletion of Sox2 in Lmx1a+ progenitors blocked NOTCH-driven CP tumor and reduced Lmx1a/b expression. Thus, LMX1A and SOX2 expression marks tumor-initiating cells, while OTX2 labels differentiated tumor cells in CPC. Together, our studies revealed intra- and inter-tumoral heterogeneity in CPC driven by distinct oncogenic signals. Knowledge of the molecular and cellular basis of heterogeneity in CP tumor development may facilitate the development of innovative diagnostic and therapeutic strategies in CPC.