VRK3 depletion in Pontine Diffuse Midline Glioma (DMG)-K27 altered cells
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PROVIDER: EGAS00001007047 | EGA |
REPOSITORIES: EGA
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Project description:In order to investigate possible mechanisms underlying the phenotype of cell fitness decline observed following decrease of VRK3 in pontine DMG-K27 altered cells 7, we examined differences in global gene expression. RNA-seq was performed 44h and 60h post-transduction with two distinct shRNAs targeting VRK3 to be able to evaluate the early impact of VRK3 knock-down (KD) in four independent in vitro models of DMG
| EGAD00001010097 | EGA
Project description:We previously identified VRK3 as a specific vulnerability in DMG-H3K27M cells in a synthetic lethality screen targeting the whole kinome. The aim of the present study was to elucidate the mechanisms by which VRK3 depletion impact DMG-H3K27M cell fitness. Gene expression studies after VRK3 knockdown emphasized the inhibition of genes involved in G1/S transition of the cell cycle resulting in growth arrest in G1. Additionally, a massive modulation of genes involved in chromosome segregation was observed, concomitantly with a reduction in the level of phosphorylation of serine 10 and serine 28 of histone H3 supporting the regulation of chromatin condensation during cell division. This last effect could be partly due to a concomitant decrease of the chromatin kinase VRK1 in DMG following VRK3 knockdown. Furthermore, a metabolic switch specific to VRK3 function was observed towards increased oxidative phosphorylation without change in mitochondria content, that we hypothesized would represent a cell rescue mechanism. This study further explored the vulnerability of DMG-H3K27M cells to VRK3 depletion suggesting potential therapeutic combinations, e.g. with the mitochondrial ClpP protease activator ONC201.
| S-EPMC10599138 | biostudies-literature
Project description:Diffuse midline glioma with two components: classical glial and ependymal.
| S-EPMC10711257 | biostudies-literature
Project description:Abstract Effective treatments are urgently needed for the incurable paediatric brainstem tumour Diffuse Midline Glioma (DMG). Most DMGs contain Histone H3 mutations (H3K27M), which produce extensive epigenetic dysregulation by inhibiting EZH2-mediated trimethylation of H3K27 (H3K27me3). Global depletion of the repressive H3K27me3 modification results in aberrantly open chromatin and is central to DMG tumorigenesis. Thus, targeting the epigenome is a promising avenue of treatment for DMG. We found that targeting the histone chaperone complex Facilitates Chromatin Transcription (FACT) with the curaxin drug CBL0137 to have potent pre-clinical efficacy against DMG, leading to a paediatric Phase I/II clinical trial for CBL0137 which includes a DMG/DIPG cohort (NCT04870944). In this project we aim to elucidate CBL0137’s molecular mechanism in DMG. FACT is critical for maintaining chromatin homeostasis during DNA replication, transcription, and repair. We therefore hypothesised that FACT maintains the aberrant epigenetic landscape resulting from H3K27M. Consistently, we found CBL0137 to be more cytotoxic against H3K27M-mutant, compared to H3-WT or isogenic DMG cells lacking the mutation. Furthermore, FACT directly interacts with H3K27M, and FACT inhibition increases both EZH2 catalytic activity and global H3K27me3 levels. We are now using ChIP-seq to discover the genome-wide distribution of FACT and H3K27M, and will interrogate the consequence of CBL0137 treatment on the epigenome and transcriptome. Preliminary results suggest that FACT is located at certain genes co-occupied by H3K27M, the active histone mark H3K27ac, and the BET protein BRD4. This implies that FACT is involved in maintaining open chromatin and perhaps transcription at these regions. Combining CBL0137 with other epigenetic drugs, such as BET inhibitors, could therefore represent a rational therapeutic opportunity for DMG. This work will ultimately inform mechanism-based targeted therapies to combine with CBL0137 to improve its efficacy and uncover valuable new insights into DMG epigenetics and pathobiology.
| S-EPMC9165134 | biostudies-literature
Project description:Diffuse intrinsic pontine glioma (DIPG) is a uniformly lethal brainstem tumor of childhood, driven by mutations in histone H3 at lysine 27 (K27M) leading to altered epigenetic regulation. Epigenomic analyses of DIPG have shown global loss of repressive chromatin marks accompanied by DNA hypomethylation. However, studies providing a static view of the epigenome do not adequately capture the regulatory underpinnings of DIPG intratumoral heterogeneity and phenotypic plasticity. To address this, we performed whole-genome bisulfite sequencing (WGBS) on 23 primary patient DIPG specimens, 4 patient-derived DIPG neurosphere cell lines, and 5 normal controls and applied a novel framework for analysis of DNA methylation variability, permitting the derivation of comprehensive genome-wide DNA methylation potential energy landscapes that capture intrinsic epigenetic variation. We show that DIPG exhibits a markedly disordered epigenome, with increasingly stochastic DNA methylation localizing to key regulatory elements and genes. We demonstrate marked epigenetic instability at genes regulating pluripotency and developmental identity, potentially enabling cells to sample a diverse range of transcriptional programs and differentiation states. We then evaluated the responsiveness of the DIPG epigenetic landscape to pharmacologic modulation. Treatment with the DNA hypomethylating agent decitabine produced profound genome-wide demethylation and reduced DNA methylation stochasticity at active enhancers, bivalent promoters, and promoters of key developmental genes. Decitabine treatment elicited changes in gene expression, including upregulation of immune signaling, and sensitized DIPG cells to the effects of histone deacetylase inhibition. This study provides a resource for dissecting the epigenetic instability that underlies DIPG heterogeneity and plasticity, suggesting the application of epigenetic therapies to constrain the range of epigenetic states available to DIPG cells.
2024-02-25 | GSE224500 | GEO
Project description:BackgroundDiffuse midline gliomas (DMG) are highly invasive brain tumors with rare survival beyond two years past diagnosis and limited understanding of the mechanism behind tumor invasion. Previous reports demonstrate upregulation of the protein ID1 with H3K27M and ACVR1 mutations in DMG, but this has not been confirmed in human tumors or therapeutically targeted.MethodsWhole exome, RNA, and ChIP-sequencing was performed on the ID1 locus in DMG tissue. Scratch-assay migration and transwell invasion assays of cultured cells were performed following shRNA-mediated ID1-knockdown. In vitro and in vivo genetic and pharmacologic [cannabidiol (CBD)] inhibition of ID1 on DMG tumor growth was assessed. Patient-reported CBD dosing information was collected.ResultsIncreased ID1 expression in human DMG and in utero electroporation (IUE) murine tumors is associated with H3K27M mutation and brainstem location. ChIP-sequencing indicates ID1 regulatory regions are epigenetically active in human H3K27M-DMG tumors and prenatal pontine cells. Higher ID1-expressing astrocyte-like DMG cells share a transcriptional program with oligo/astrocyte-precursor cells (OAPCs) from the developing human brain and demonstrate upregulation of the migration regulatory protein SPARCL1. Genetic and pharmacologic (CBD) suppression of ID1 decreases tumor cell invasion/migration and tumor growth in H3.3/H3.1K27M PPK-IUE and human DIPGXIIIP* in vivo models of pHGG. The effect of CBD on cell proliferation appears to be non-ID1 mediated. Finally, we collected patient-reported CBD treatment data, finding that a clinical trial to standardize dosing may be beneficial.ConclusionsH3K27M-mediated re-activation of ID1 in DMG results in a SPARCL1+ migratory transcriptional program that is therapeutically targetable with CBD.
| S-EPMC9825316 | biostudies-literature
Project description:Diffuse midline gliomas (DMG) are aggressive tumors with a poor prognosis. In this study, a technique called t-SNE analysis was used to cluster tumors based on their methylation profiles. DMG subtype with a co-occurrence of H3.3K27M and BRAF or FGFR1 mutations have been identified. This subtype has a more favorable prognosis, with a median overall survival of 3 years.
2024-07-30 | E-MTAB-13471 | biostudies-arrayexpress
Project description:Conjugation of ubiquitin (Ub) to numerous substrate proteins regulates virtually all cellular processes. Eight distinct ubiquitin polymer linkages specifying different functional outcomes are generated in cells. However, the roles of some atypical poly-ubiquitin topologies, in particular linkages via lysine 27 (K27), remain poorly understood due to a lack of tools for their specific detection and manipulation. Here, we adapted a cell-based ubiquitin replacement strategy to enable selective and conditional abrogation of K27-linked ubiquitylation, revealing that this ubiquitin linkage type is essential for proliferation of human cells. We demonstrate that K27-linked ubiquitylation is predominantly a nuclear modification whose ablation deregulates nuclear ubiquitylation dynamics and impairs cell cycle progression in an epistatic manner with inactivation of the ATPase p97/VCP. Moreover, we show that a p97-proteasome pathway model substrate (Ub(G76V)-GFP) is directly modified by K27-linked ubiquitylation, and that disabling the formation of K27-linked ubiquitin signals or blocking their decoding via overexpression of the K27 linkage-specific binder UCHL3 impedes Ub(G76V)-GFP turnover at the level of p97 function. Our findings suggest a critical role of K27-linked ubiquitylation in supporting cell fitness by facilitating p97-dependent processing of ubiquitylated nuclear proteins.
2022-04-14 | PXD023579 | Pride
Project description:BackgroundCross-sectional tumor measures are traditional clinical trial endpoints; however volumetric measures may better assess tumor growth. We determined the correlation and compared the prognostic impact of cross-sectional and volumetric measures of progressive disease (PD) among patients with DIPG.MethodsImaging and clinical data were abstracted from the International DIPG Registry. Tumor volume and cross-sectional product (CP) were measured with mint Lesion™ software using manual contouring. Correlation between CP and volume (segmented and mathematical [ellipsoid] model) thresholds of PD were assessed by linear regression. Landmark analyses determined differences in survival (via log-rank) between patients classified as PD versus non-PD by CP and volumetric measurements at 1, 3, 5, 7, and 9 months postradiotherapy (RT). Hazard ratios (HR) for survival after these time points were calculated by Cox regression.ResultsA total of 312 MRIs (46 patients) were analyzed. Comparing change from the previous smallest measure, CP increase of 25% (PD) correlated with a segmented volume increase of 30% (R2 = 0.710), rather than 40% (spherical model extrapolation). CP-determined PD predicted survival at 1 month post-RT (HR = 2.77), but not other time points. Segmented volumetric-determined PD (40% threshold) predicted survival at all imaging timepoints (HRs = 2.57, 2.62, 3.35, 2.71, 16.29), and 30% volumetric PD threshold predicted survival at 1, 3, 5, and 9 month timepoints (HRs = 2.57, 2.62, 4.65, 5.54). Compared to ellipsoid volume, segmented volume demonstrated superior survival associations.ConclusionsSegmented volumetric assessments of PD correlated better with survival than CP or ellipsoid volume at most time points. Semiautomated tumor volume likely represents a more accurate, prognostically-relevant measure of disease burden in DIPG.
| S-EPMC9435485 | biostudies-literature
Project description:We used microarray to determine the changes in gene expression profile after KD of Jmjd2b and Jmjd2c compared to Anti-GFP KD from mES cells Mouse ES cells were infected with AntiGFP, Jmjd2b and Jmjd2c shRNAs lentivirus, puromycin selected, passage them 2-3 times, collected for RNA isolation and gene expression
2013-12-01 | E-GEOD-43059 | biostudies-arrayexpress