bluuebunny/crossref_metadata_2025_split huggingface dataset:
EPCO-16. DISRUPTING DOT1L EPIGENETIC ACTIVITY REPROGRAMS GLIOBLASTOMA STEM CELLS TOWARDS A DOPAMINERGIC NEURONAL-LIKE STATE
Authors: Assaf Samir , Bozek Danielle , Luchman H Artee , Weiss Samuel 
Published: November 2023

Abstract
Glioblastoma is the most common and aggressive primary adult brain tumor, remaining incurable despite current therapeutic strategies. Through a genome-wide essentiality screen, we previously identified disruptor of telomeric silencing-1-like (DOT1L) as one of the most important epigenetic regulators in glioblastoma stem cells (GSCs). Chemogenic inhibition of DOT1L epigenetic activity was found to increase the expression of neuronal markers while decreasing the expression of cancer stem cell markers at the transcriptional and protein level, suggesting GSC differentiation towards a neuronal lineage. Thus, we sought to investigate the mechanism of DOT1L in GSCs and whether disruption of DOT1L epigenetic activity promotes functional integration of GSCs into neural circuits. Here, we profiled the overall transcriptional and chromatin accessibility landscape of GSCs following DOT1L inhibition. Interestingly, we identified that DOT1L inhibition results in increased chromatin accessibility at key gene loci involved in neural differentiation, synaptic transmission, in addition to dopamine synthesis, packaging, and release, which correlated with upregulated gene expression. Furthermore, these gene loci exhibited alterations of the DOT1L epigenetic mark, H3K79me2. Transcriptional motif analysis of GSCs following DOT1L inhibition revealed dysregulated accessibility of transcription factor motifs known to be involved in regulating cellular response to neuronal signals. Cell viability assessment of GSCs treated with a DOT1L inhibitor and a panel of neurotransmitters further confirmed that DOT1L inhibition sensitizes GSCs to neuronal signals. These data suggest that disrupting the epigenetic activity of DOT1L reprograms the epigenetic landscape of GSCs to promote a functional, dopaminergic, neuronal-like state that exhibits enhanced responsiveness to neuronal signals. Future experiments aim to investigate the impact of disrupted DOT1L epigenetic activity on GSC-neuronal bidirectional communication and the mechanisms by which this influences the progression and aggressiveness of glioblastoma.
EPCO-17. 3D GENOME STRUCTURE REGULATES TRANSCRIPTION IN PEDIATRIC HIGH-GRADE GLIOMA
Authors: Huang Tina , Wang Juan , Hu Ye , Piunti Andrea , Bartom Elizabeth , Yue Feng , Saratsis Amanda 
Published: November 2023

Abstract

INTRODUCTION
Pediatric high-grade gliomas (pHGGs), including glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG), are highly morbid brain tumors. Up to 80% of DIPGs harbor a somatic missense mutation in genes encoding Histone H3. To investigate whether the H3K27M mutant protein is associated with distinct chromatin structure affecting transcription regulation, we generated the first high-resolution Hi-C and ATAC-Seq maps of pHGG cell lines, and integrated these with tissue and cell genomic data.

METHODS
We generated sequencing data from patient-derived cell lines (DIPG n=6, GBM n=3, normal n=2) and frozen tissue specimens (DIPG n=1, normal brainstem n=1). Analyses included cell line RNA-Seq, ChIP-Seq (H3K27ac, H3K27me3, H3K27M) and genome-wide chromatin conformation capture (Hi-C), as well as tissue ATAC-Seq. Publicly available pediatric glioma tissue ChIP-Seq data was integrated with cell data. CRISPR knock-down of target enhancer regions was performed.

RESULTS
We identified tumor-specific enhancers and regulatory networks for known oncogenes in DIPG and GBM. In DIPG, FOX, SOX, STAT and SMAD families were among top H3K27Ac enriched motifs. Significant differences in Topologically Associating Domains (TADs) and DNA looping were observed at OLIG2 and MYCN in H3K27M mutant DIPG, relative to wild-type GBM and normal cells. Pharmacologic treatment targeting H3K27Ac (BET and Bromodomain inhibition) altered these 3D structures. Functional analysis of differentially enriched enhancers in DIPG implicated SOX2, SUZ12, and TRIM24 as top activated upstream regulators. Distinct genomic structural variations leading to enhancer hijacking and gene co-amplification were identified at A2M, JAG2, and FLRT1.

CONCLUSION
We show genome structural variations enhancer-promoter interactions that impact gene expression in pHGG in the presence and absence of the H3K27M mutation. Our results imply that tridimensional genome alterations may play a critical role in the pHGG epigenetic landscape and thereby contribute to pediatric gliomagenesis. Further studies examining the impact of the alterations is therefore underway.
EPCO-19. TUMOR-WIDE NEOANTIGEN-SPECIFIC T-CELLS INFILTRATING MUTANT IDH1 LOW-GRADE GLIOMAS AND PERSISTING IN PERIPHERAL BLOOD ALLOW FOR PERSONALIZED TCR-BASED IMMUNOTHERAPIES
Authors: Kwok Darwin , Zhang Michael , Wang Cliff , Stevers Nicholas , Borrman Tyler , Pan Zheng , Yuen Benjamin , Peng Songming , Nguyen Diana , Martin Michael , Hong Chibo , Hilz Stephanie , Phillips Joanna , Shai Anny , Bush Nancy Ann Oberheim , Hervey-Jumper Shawn , McDermott Michael , Mandl Stefanie , Okada Hideho , Costello Joseph 
Published: November 2023

Abstract

BACKGROUND
The low mutational burden and immunologically “cold” microenvironment of mutant IDH1 low-grade gliomas (LGG) are considerable challenges in immunotherapy for these tumor types. However, we hypothesize that LGG-targeting T-cells may exist at low frequency and with limited regional infiltration within the tumor. Through multi-region tumor sampling coupled with high-throughput T-cell receptor (TCR) profiling, we identified tumor-wide neoantigens and corresponding neoantigen-specific T-cells regionally infiltrating the tumor and persisting in peripheral blood.

METHODS
Maximally-distanced anatomical sampling of at least 10 distinct tumor regions was performed at the initial resection for three WHO Grade II diffuse astrocytoma patients for exome-based prediction of clonally and subclonally expressed neoantigens, RNAseq analysis of regional immune cell composition, and TCR beta deep sequencing. We used these predictions to generate a barcoded library of patient-specific peptide-HLA multimers loaded with predicted neoepitopes. With this library, neoantigen-specific CD8+ T-cells were captured and isolated from patients’ peripheral blood. Single-cell TCR sequencing allowed us to identify the neoantigen-reactive TCR clonotypes which were transduced subsequently into Jurkat76 cell lines for functional validation.

RESULTS
We screened patient-derived peripheral blood drawn two years after initial resection in 3 mutant IDH1 LGG patients and detected a total of 20 TCR clonotypes recognizing neoepitopes derived from truncal, tumor-wide mutations in CNTNAP1 (n=8), TP53 (n=3), and MRPL46 (n=2) as well as subclonal mutations in PRMT5 (n=1) and ZDHHC5 (n=6). Jurkat76 cells transduced with the mutant-PRMT5-specific TCR demonstrated dose-dependent neoantigen-specific immune responses when co-cultured with mutant-PRMT5 pulsed-antigen presenting cells expressing HLA-A*0201.

CONCLUSION
Our study demonstrates the existence and persistence of neoantigen-targeting T-cells within the blood and tumor of mutant IDH1 LGG patients. We identified a TCR clonotype that successfully recognizes and induces an immune response against mutant-PRMT5. These findings suggest a feasible methodology to develop personalized T-cell-based immunotherapies for patients with mutant IDH1 LGGs.
EPCO-24. DNA METHYLATION-BASED SUBCLASS PREDICTS THE EFFECT OF THE EXTENT OF RESECTION ON SURVIVAL IN IDH-WILDTYPE GLIOBLASTOMA
Authors: Dankmeijer Luuk , Westerink Lucas , Wesseling Pieter , Hamer Philip de Witt , Kouwenhoven Mathilde , Kooi Evert-Jan , Verburg Niels 
Published: November 2023

Abstract

BACKGROUND
DNA methylation-based classification of diffuse glioma is increasingly used in daily practice. Yet, little is known regarding the prognostic and predictive value of DNA methylation-based subclasses of IDH-wildtype glioblastoma. A recent study found a predictive value for the effect of extent or resection (EOR) on survival for DNA methylation-based subclasses using the v11.4 classifier. We set out to validate these findings in an independent cohort and to test if these results are similar using a more recent version (v12.5) of the classifier.

METHODS
This concerns a single-center retrospective cohort study of all patients with a glioblastoma, IDH-wildtype of whom DNA methylation was obtained. Patient and tumor characteristics, pre-operative Karnofsky performance status and EOR (complete, subtotal, partial, biopsy) were retrieved. DNA methylation-based classification and MGMT-promoter status were obtained using version 11.4 and 12.5 of the Heidelberg classifier. Progression-free survival (PFS) and overall survival (OS) were collected and validated using the national brain tumor registry.

RESULTS
Of 149 patients, the subtype was Mesenchymal in 57 (38%), RTK1 in 41 (28%), and RTK2 in 51 patients (34%). PFS and OS did not differ between the three subclasses, also not after pooling RTK1 and RTK2 into one Classical subclass. Complete or subtotal resection resulted in a longer OS than partial resection or biopsy in the entire cohort. This was similar for the pooled Classical subclass. In the Mesenchymal subclass, however, there was no survival advantage for complete or subtotal resection over partial resection or biopsy. These results were similar with the 11.4 and 12.5 classifier version.

CONCLUSION
In IDH-wildtype glioblastoma of the Mesenchymal subclass complete or subtotal resection does not result in a survival benefit. Our findings evoke the need for pre- or intraoperative assessment of the DNA-methylation based subclass of IDH-wildtype glioblastoma.
EPCO-26. INTRATUMORAL HETEROGENEITY OF GBM IDENTIFIED AND CHARACTERIZED BY A MULTISAMPLING APPROACH AND METHYLATION PROFILING
Authors: Singh Omkar , Abdullaev Zied , Matsumoto Yuji , Brem Steven , O'Rourke Donald M , Davatzikos Christos , Aldape Kenneth , Nasrallah MacLean P 
Published: November 2023

Abstract
Intratumoral heterogeneity in glioblastoma is known and is considered a key feature of GBM’s clinical aggressiveness. To more fully characterize this phenomenon, we conducted methylation profiling on the EPIC array for 79 regions from 20 glioblastomas. Using the Heidelberg classifier, each of the 20 tumors had at least 1 region with a match to a GBM subtype with &gt;0.84 score. Among these, 6 tumors (30%) showed homogeneous-high score matches to the same GBM subclass, while the remaining 14 tumors (70%) showed heterogeneity in GBM subclass, with either lower scores, or disparate matches to different glioma subtypes. Comparing these subclass-homogeneous versus subclass-heterogeneous groups of tumors using methylCIBERSORT deconvolution, the subclass-heterogeneous cases showed a significantly higher proportions of CD8+ T cells and microglia, and significantly lower proportions of CD4+ T cells, B cells and monocytes. While low tumor purity can be a cause of lower confidence scores in the methylation classifier, we did not find that the subclass-heterogeneous tumors had lower tumor purity compared to subclass-homogeneous cases, suggesting that this heterogeneity was due to factors other than tumor purity. Within the subclass-heterogeneous tumors we identified the common intratumorally most variable probes and examined their characteristics. Compared to the overall distribution of probes on the array, these intratumoral-variable probes were significantly more likely to be intergenic and located on enhancers. Conversely, they were significantly less likely to be on gene promoters and CpG islands. Gene ontology analysis showed that genes associated with these probes were enriched for pathways involved in DNA repair. Overall, our multisampling methylation profiling analysis identifies specific biologic correlates of intratumoral heterogeneity that is not simply due to tumor purity. Methylation changes associated with intratumoral heterogeneity converge on specific genomic patterns that warrant further study.
EPCO-28. MULTIPLATFORM MOLECULAR AND FUNCTIONAL GENOMIC ANALYSES OF NF1 MUTANT GLIOBLASTOMA IDENTIFY DISTINCT CO-MUTATION PATTERNS AND DRUGGABLE DEPENDENCIES UNDERLYING SELUMETINIB RESPONSE
Authors: Mirchia Kanish , Payne Emily , Pan Sixuan , Al-Adli Nadeem , Young Jacob , Chen William , Pak Joanna , Seo Kyounghee , Ozawa Tomoko , Reddy Alyssa T , Perry Arie , Liu John , Berger Mitchel , Raleigh David , Vasudevan Harish 
Published: November 2023

Abstract
The tumor suppressor NF1 is recurrently mutated in sporadic IDH-wildtype glioblastoma and activates Ras/Raf/MEK signaling, motivating the use of targeted MEK inhibition. Here, we combine multiplatform molecular analysis of NF1 mutant glioblastoma human resection specimens with functional genomic CRISPRi screens to the MEK inhibitor selumetinib. METHODS: We identified 123 consecutive patients treated at the University of California San Francisco between 2016-2022 with NF1 mutant, IDH-wildtype glioblastoma. DNA methylation data was processed using the minfi package, and hierarchical clustering, principal component analysis (PCA), and survival analysis was performed in R. Human NF1 mutant GBM43 and mouse NRAS mutant SB28 GBM cells expressing CRISPRi machinery were used for genome wide selumetinib-resistance screens. Immunoblotting was performed to validate CRISPRi screen hits and selumetinib responses in cell lines. RESULTS: DNA sequencing of human glioblastomas identified 12 recurrent alterations in greater than 10% of cases including cell cycle regulators (CDKN2A/B, TP53, RB1), PI3K signaling (PTEN, PIK3R1, PIK3CA), epigenetic regulators (ATRX, SETD2), and Ras signaling (EGFR, PDGFRA, PTPN11). Methylation data (n=55 tumors) identified three groups marked by differences in MGMT methylation (p=0.04, chi-square test) and CDKN2A/B loss (p=0.01, chi-square test). CRISPRi screens in GBM43 and SB28 cells identified driver phenotypes in cell cycle regulators (TP53, CDKN2A, CDK4), PI3K signaling (PTEN, MTOR), and Ras signaling (RASA2, PTPN11) consistent with co-occurring mutations in the human NF1 mutant glioblastoma cohort. In contrast, mediators of selumetinib response converged on regulators of Ras/Raf/MEK signaling including the Ras effector SHOC2. Biochemical analysis validated SHOC2 repression is sufficient for selumetinib sensitization. CONCLUSIONS: Using a combination of human tumor genomic analysis, functional genetic screens, and biochemical validation, we identify three epigenetic groups of NF1 mutant glioblastoma with distinct co-mutation patterns and validate compensatory Ras/Raf/MEK activation as a conserved mechanism of selumetinib resistance, suggesting combination molecular therapy as a rational therapeutic combination.
EPCO-30. UNCOVERING MECHANISMS OF RESISTANCE IN PEDIATRIC HIGH-GRADE GLIOMA (PHGG)
Authors: Mukherji Devarshi , Haase Santiago , Welch Joshua , Lowenstein Pedro , Castro Maria 
Published: November 2023

Abstract
Pediatric high-grade gliomas (pHGGs) are currently the leading cause of cancer-related deaths in children, remaining incurable in most cases. A molecular subgroup of pHGGs harbor G34R mutations in the histone H3.3, although the mechanisms of the H3.3-G34R/V are not well established. In most of the patients, the tumors resist radiation and relapse after standard of care (SOC). We aim to study the molecular mechanisms mediating resistance in these tumors. Knowing why certain tumors resist treatment allows us to implement targeted therapies. We performed single cell RNA sequencing on 3 non-treated (NT) glioma bearing mice, 3 mice resistant to treatment (R), and 3 mice susceptible to treatment (S). We isolated the tumor tissue (including tumor microenvironment) and processed the cells for single cell RNA sequencing. Using R, the single-cell RNA sequencing data was converted to Seurat datasets for analysis and subsequently combined and clustered using UMAP. We used the ScType and SingleR software tools to annotate cell types. After cell annotation, Gene Set Enrichment Analysis (GSEA) was performed between R and S tumors for each cell cluster as well as NT vs Treated. GSEA showed that susceptible tumor cells showed increased immune responses when compared to resistant cells. Additionally, resistant cells showed increased neuronal phenotypes as well as suppression of the ERK 1/2 cascade pathway in comparison to susceptible cells. There were also numerous differences in immune cell subpopulations, which may help uncover mechanisms of resistance. For example, certain immune cells show signs of increased epigenetic modifications in resistant vs. susceptible glioma cells. Our results indicate that the resistance of pHGG to treatment is associated with the establishment of immunosuppressive signals from tumor cells, and the suppression of tumor growth mediated by downregulation of the MAPK/ERK cascade.
EPCO-33. H3K4 METHYLATION IS A PREREQUISITE OF CELL FATE CHANGE DURING IDH-MUTANT GLIOMAGENESIS
Authors: Koch Marilin , Deo Minh , Turcan Sevin , Hoetker Michael , Herold-Mende Christel , Wick Wolfgang 
Published: November 2023

Abstract
RATIONALE: Low grade gliomas (LGGs) are defined by isocitrate dehydrogenase (IDH)-mutations that mediate broad epigenetic changes. However, additional determinants of low-grade gliomagenesis remain unknown, including factors necessary for the activation of LGG gene programs. Here, we aim at identifying epigenetic prerequisites of IDH-dependent gliomagenesis by modulating key chromatin modifiers in a conditionally IDHR132H-expressing immortalized human astrocyte (IHA) model system. METHODS: Using chemical compounds, we targeted key histone modifications and DNA methylation in an inducibly IDHR132H expressing IHA cell line. Flow cytometry for the LGG-associated marker L1CAM served as a primary read-out. Compound candidates were further assessed with RNA-Seq and mass spectrometry. Phenotypic evaluation included sphere formation, clonogenicity, cell viability and invasion assays. Relevant findings were additionally assessed in established LGG cell lines TS603, NCH612 and NCH1681. RESULTS: L1CAM was significantly altered by DOT1L inhibitor EPZ00477, MLL1 inhibitor MM-102, and EZH2 inhibitor GSK-126. MM-102 treatment was associated with broad transcriptional downregulation. Intersecting data from MM-102 and GSK126 treated cells, we identified potential bivalent genes directly affected by inhibitor treatment that were enriched for Wnt signaling and developmental transcription factors implicated in gliomagenesis. Overlapping transcriptional and proteomics data, MM-102-treated cells exhibited a signature of predominantly downregulated genes associated with invasion/migration, extracellular matrix (ECM) and cholesterol metabolism. Fittingly, cells showed impaired invasion, clonal proliferation and sphere forming capacity. The negative effect on sphere formation tracked inversely with aggressiveness of established LGG cell lines. CONCLUSION: Interfering with histone H3K4 methylation through MLL1 inhibition is associated with broad downregulation of gene sets related to cell identity and ECM/migration, reflected by disruption of clonal proliferation in IDHR132H astrocytes and LGG cell lines. This points to deposition of H3K4 methylation as a prerequisite of cell fate change in LGG development. Persisting effects on established LGG cell lines warrant its investigation as a potential therapeutic target.
EPCO-35. IDENTIFICATION OF DIVERSE IMMUNE PHENOTYPES IN NF-1 GLIOMA BY SINGLE NUCLEI GENE EXPRESSION ANALYSIS
Authors: Garofano Luciano , Oh Young Taek , Najac Ryan D , Ceccarelli Michele , Sanson Marc , Lasorella Anna , Iavarone Antonio 
Published: November 2023

Abstract
Neurofibromatosis type 1 (NF-1) is the most common cancer predisposition syndrome in which 15-20% of affected individuals develop glioma. Large scale DNA and RNA bulk profiling showed the high molecular complexity of NF-1 glioma with the tumor cellular ecosystem constituted by multiple malignant clones and heterogenous immune microenvironment. However, the composition and function of infiltrating cells was mostly hidden in the bulk tumor mass, and the extended granularity of NF-1 glioma immune microenvironment remained still unexplored. Here, to address this challenge, we collected glioma samples from 53 NF-1 patients including 27 high-grade and 26 low-grade tumors, and we analyzed their gene expression at the highest resolution through single nuclei RNA sequencing. A total of 288,372 single cells have been classified by integrating multiple computational approaches (including genomic copy number inference, gene signature enrichment, pathway-based deconvolution and integrative non-negative matrix factorization). The non-tumor cell compartment (124,082 cells, 43%) has been dissected for the deep characterization of each cell type that populate the microenvironment of NF-1 glioma. We unraveled the complexity of the immune infiltration and identified different subpopulations exhibiting specific immune functions within myeloid and lymphoid components. Different glioma niches have been revealed by comparing the relative composition of the immune microenvironment across the tumors. Recruitment and activation of cytotoxic CD8+ T cells and natural killers by an active crosstalk with dendritic and pro-inflammatory myeloid cells defined an immune-supportive phenotype that can mediate a potential anti-tumor response in low-grade NF-1 glioma. Regulatory T cell infiltration and effector T cell exhaustion, instead, induced immune suppression in another tumor niche. Conversely, the complete absence of lymphocytes characterized a large set of cold tumors, mostly high-grade glioma. The elucidation of the complex immune interplay in the different tumor niches provides novel insights for the application of immunotherapies in NF-1 glioma patients.
EPCO-42. A SYSTEMS SCALE MODEL FOR PERSONALIZED PRECISION ONCOLOGY APPLICATIONS TO GBM
Authors: Turkarslan Serdar , He Yu , Hothi Parvinder , Murie Carl , Nicolas Abran , Kannan Kavya , Park James H , Pan Min , Patel Anoop , Cobbs Charles , Baliga Nitin S 
Published: November 2023

Abstract
Glioblastoma (GBM) is the most aggressive and lethal type of brain tumor, and its treatment remains challenging due to the complexity of the underlying molecular mechanisms. Here, we present gbmMINER, a causal and mechanistic transcriptional regulatory network model for GBM that integrates multi-omics and clinical data with a new network quantization approach using the MINER algorithm. gbmMINER accurately grouped 9,728 genes into 3,797 regulons and identified 179 transcriptional programs that stratify GBM into 23 disease states, associated with distinct clinical outcomes. We demonstrate that the activity profiles of regulons and programs in the quantized network model accurately predict overall survival, identifying patients with significantly longer OS of up to 42 months. Through extensive characterization using independent cohort datasets and genome-wide CRISPR screening data, we demonstrate the accuracy of gbmMINER in delineating causal and mechanistic insights into well-known and novel prognostic somatic mutations in GBM. Finally, through high-throughput drug screening, we demonstrate that the activity of regulons and programs accurately predicts the sensitivity of patient-derived glioma stem-like cells (PD-GSCs) to anticancer drugs. In theory, gbmMINER could be implemented to better stratify patients based on prognosis and potentially identify precision therapeutic strategies based on network activity.