bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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 (&gt;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

Abstract
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

Abstract
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

Abstract

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&gt;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&gt;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&lt;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&lt;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

Abstract
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

Abstract
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

Abstract
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

Abstract
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 &lt;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

Abstract
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

Abstract
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

Abstract
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.