Integrin α5β1 Plays a Critical Role in Resistance to Temozolomide by Interfering with the p53 Pathway in High-Grade Glioma

High-grade glioma is one of the most lethal human cancers characterized by extensive tumor heterogeneity. In order to identify cellular and molecular mechanisms that drive tumor heterogeneity of this lethal disease, we performed single-cell RNA sequencing analysis of one high-grade glioma. Accordingly, we analyzed the individual cellular components in the ecosystem of this tumor. We found that tumor-associated macrophages are predominant in the immune microenvironment. Furthermore, we identified five distinct subpopulations of tumor cells, including one cycling, two OPC/NPC-like and two MES-like cell subpopulations. Moreover, we revealed the evolutionary transition from the cycling to OPC/NPC-like and MES-like cells by trajectory analysis. Importantly, we found that SPP1/CD44 interaction plays a critical role in macrophage-mediated activation of MES-like cells by exploring the cell-cell communication among all cellular components in the tumor ecosystem. Finally, we showed that high expression levels of both SPP1 and CD44 correlate with an increased infiltration of macrophages and poor prognosis of glioma patients. Taken together, this study provided a single-cell atlas of one high-grade glioma and revealed a critical role of macrophage-mediated SPP1/CD44 signaling in glioma progression, indicating that the SPP1/CD44 axis is a potential target for glioma treatment.

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EPCO-20. PEDIATRIC HIGH-GRADE GLIOMA EXHIBITS DISTINCT 3D GENOME STRUCTURE THAT IMPACTS TRANSCRIPTION REGULATION

Neuro-Oncology ◽

10.1093/neuonc/noab196.019 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi6-vi6

Author(s):

Tina Huang ◽

Juan Wang ◽

Ye Hu ◽

Andrea Piunti ◽

Elizabeth Bartom ◽

...

Keyword(s):

Transcription Regulation ◽

Cell Lines ◽

Regulatory Networks ◽

Critical Role ◽

Genome Structure ◽

High Grade Glioma ◽

High Grade ◽

Sequencing Data ◽

Structural Variations ◽

The Impact

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.

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Epigenomic landscape and 3D genome structure in pediatric high-grade glioma

Science Advances ◽

10.1126/sciadv.abg4126 ◽

2021 ◽

Vol 7 (23) ◽

pp. eabg4126

Author(s):

Juan Wang ◽

Tina Yi-Ting Huang ◽

Ye Hou ◽

Elizabeth Bartom ◽

Xinyan Lu ◽

...

Keyword(s):

Regulatory Networks ◽

Critical Role ◽

Genome Structure ◽

Three Dimensional ◽

High Grade Glioma ◽

Diffuse Intrinsic Pontine Glioma ◽

Chromatin Accessibility ◽

High Grade ◽

Structural Variations ◽

Type Specimens

Pediatric high-grade gliomas (pHGGs), including glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG), are morbid brain tumors. Even with treatment survival is poor, making pHGG the number one cause of cancer death in children. Up to 80% of DIPGs harbor a somatic missense mutation in genes encoding histone H3. To investigate whether H3K27M is associated with distinct chromatin structure that alters transcription regulation, we generated the first high-resolution Hi-C maps of pHGG cell lines and tumor tissue. By integrating transcriptome (RNA-seq), enhancer landscape (ChIP-seq), genome structure (Hi-C), and chromatin accessibility (ATAC-seq) datasets from H3K27M and wild-type specimens, we identified tumor-specific enhancers and regulatory networks for known oncogenes. We identified genomic structural variations that lead to potential enhancer hijacking and gene coamplification, including A2M, JAG2, and FLRT1. Together, our results imply three-dimensional genome alterations may play a critical role in the pHGG epigenetic landscape and contribute to tumorigenesis.

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HGG-01. 3D GENOME STRUCTURE IMPACTS GENE EXPRESSION IN PEDIATRIC HIGH-GRADE GLIOMA

Neuro-Oncology ◽

10.1093/neuonc/noab090.067 ◽

2021 ◽

Vol 23 (Supplement_1) ◽

pp. i17-i17

Author(s):

Tina Huang ◽

Juan Wang ◽

Ye Hou ◽

Andrea Piunti ◽

Elizabeth Bartom ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Regulatory Networks ◽

Critical Role ◽

Genome Structure ◽

High Grade Glioma ◽

High Grade ◽

Sequencing Data ◽

Structural Variations ◽

The Impact

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. 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, including CRISPR knock-down of target enhancer regions, is therefore underway.

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Integrin α5β1 and p53 convergent pathways in the control of anti-apoptotic proteins PEA-15 and survivin in high-grade glioma

Cell Death and Differentiation ◽

10.1038/cdd.2015.131 ◽

2015 ◽

Vol 23 (4) ◽

pp. 640-653 ◽

Cited By ~ 32

Author(s):

G Renner ◽

H Janouskova ◽

F Noulet ◽

V Koenig ◽

E Guerin ◽

...

Keyword(s):

High Grade Glioma ◽

High Grade ◽

Integrin Α5β1 ◽

Apoptotic Proteins

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Integrin α5β1 regulates both survival and migration of high-grade glioma cells through divergent AKT-driven pathways

European Journal of Cancer ◽

10.1016/s0959-8049(16)61622-2 ◽

2016 ◽

Vol 61 ◽

pp. S175-S176 ◽

Cited By ~ 1

Author(s):

G. Renner ◽

M.C. Mercier ◽

F. Noulet ◽

M. Dontenwill

Keyword(s):

Glioma Cells ◽

High Grade Glioma ◽

High Grade ◽

Integrin Α5β1 ◽

And Migration

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OMRT-6. Optimizing MDM2 inhibition for the treatment of high-grade glioma

Neuro-Oncology Advances ◽

10.1093/noajnl/vdab070.031 ◽

2021 ◽

Vol 3 (Supplement_2) ◽

pp. ii8-ii8

Author(s):

Veronica Rendo ◽

Leslie Lupien ◽

Nicholas Khuu ◽

Kristine Pelton ◽

Sophie Lu ◽

...

Keyword(s):

Cell Cycle ◽

Drug Response ◽

High Grade Glioma ◽

High Grade ◽

Wild Type ◽

P53 Pathway ◽

In Vivo Models ◽

Mesenchymal Transition ◽

Transcriptional Changes ◽

Wild Type P53

Abstract Over 80% of high-grade gliomas have alterations in members of the p53 pathway, a central regulator of cell cycle progression and apoptosis that becomes activated in response to cellular stress and DNA damage. For tumors that retain wild-type p53, pathway deregulation frequently occurs through the amplification of negative regulators of p53, including the E3 ubiquitin ligase MDM2. The p53/MDM2 interaction axis has served as basis for the development of several classes of MDM2 inhibitors, with AMG232 being the most potent molecule currently undergoing clinical evaluation. As the effects of MDM2 inhibition (MDM2i) remain poorly understood in high-grade glioma, we performed genomic and transcriptomic analyses in patient-derived models to better characterize sensitive tumors and identify putative biomarkers of drug response. Treatment with AMG232 impaired the growth of cell lines with wild-type p53 status, particularly in tumors with additional amplification of MDM4 or PPM1D activating mutations. Treatment with AMG232 upregulated both cell cycle arrest and apoptotic cellular responses, as measured by annexin V/PI staining and immunoblotting. Interestingly, the dynamics of these two downstream p53 signaling axis were dependent on treatment duration across models. In addition to p53 pathway activation and apoptotic induction, RNA-sequencing revealed MDM2i to be associated with the activation of oncogenic MAPK and KRAS signaling as well as epithelial to mesenchymal transition markers. In most solid tumors, resistance to MDM2i is mainly mediated by acquisition of p53 inactivating mutations. We hypothesized that resistance mechanisms in glioma may be partially driven by transcriptional changes, as these tumors consist of subpopulations with diverse cell differentiation states. By chronic AMG232 treatment, we have developed in vitro and in vivo models of acquired MDM2i resistance that are not mediated by p53 inactivation. Ongoing work is focused on characterizing the transcriptional profile of these cells to identify transcriptional changes leading to decreased drug response.

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