4 research outputs found
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Epitranscriptomic and Epigenetic Engineering as Novel Therapeutic Approaches in Glioma
Glioma is the most common form of primary brain cancer and suffers from a paucity of efficacious therapies. Decades of research have successfully characterized glioma subtypes based on genetic, epigenetic, and molecular signatures that act as important prognostic indicators. Here, we present two novel approaches to designing therapeutics that convert malignant glioma and glioblastoma subtypes into more treatment-amenable subtypes. We first describe how the IDH1mut → D-2-HG ⊣ FTO axis establishes a unique epitranscriptomic profile that we term G-RAMP, which results in reduced tumor cell proliferation in both patient tumor samples and patient-derived gliomaspheres via inhibition of the anti-apoptotic regulator ATF5. G-RAMP was characterized using MeRIP-Seq unbiased screening of N6-methyladenosine (m6A) enrichment sites in IDH1mut gliomas, and small molecule inhibitors of FTO were employed to recapitulate IDH1mut growth phenotypes in more malignant IDH1wt lines. Our second approach utilized dCas9-DNMT3a epigenetic editing platforms to induce high-density methylation of the MGMT promoter and exon 1 region. MGMT methylation is a positive prognostic indicator in gliomas that predicts patient tumor responses to the standard-of-care antineoplastic agent temozolomide. We demonstrate that unmethylated MGMT gliomas can be converted to exhibit MGMT methylated profiles, which results in decreased expression of MGMT and enhanced sensitivity to temozolomide. The utilization of epitranscriptomic and epigenetic engineering approaches thus represent two novel means of effecting subtype conversions in glioma that demonstrate promising potential as neoadjuvant therapies
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dCas9/CRISPR-based methylation of O-6-methylguanine-DNA methyltransferase enhances chemosensitivity to temozolomide in malignant glioma
BackgroundMalignant glioma carries a poor prognosis despite current therapeutic modalities. Standard of care therapy consists of surgical resection, fractionated radiotherapy concurrently administered with temozolomide (TMZ), a DNA-alkylating chemotherapeutic agent, followed by adjuvant TMZ. O-6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme, removes alkylated lesions from tumor DNA, thereby promoting chemoresistance. MGMT promoter methylation status predicts responsiveness to TMZ; patients harboring unmethylated MGMT (~60% of glioblastoma) have a poorer prognosis with limited treatment benefits from TMZ.MethodsVia lentiviral-mediated delivery into LN18 glioma cells, we employed deactivated Cas9-CRISPR technology to target the MGMT promoter and enhancer regions for methylation, as mediated by the catalytic domain of the methylation enzyme DNMT3A. Methylation patterns were examined at a clonal level in regions containing Differentially Methylation Regions (DMR1, DMR2) and the Methylation Specific PCR (MSP) region used for clinical assessment of MGMT methylation status. Correlative studies of genomic and transcriptomic effects of dCas9/CRISPR-based methylation were performed via Illumina 850K methylation array platform and bulk RNA-Seq analysis.ResultsWe used the dCas9/DNMT3A catalytic domain to achieve targeted MGMT methylation at specific CpG clusters in the vicinity of promoter, enhancer, DMRs and MSP regions. Consequently, we observed MGMT downregulation and enhanced glioma chemosensitivity in survival assays in vitro, with minimal off-target effects.ConclusiondCas9/CRISPR is a viable method of epigenetic editing, using the DNMT3A catalytic domain. This study provides initial proof-of-principle for CRISPR technology applications in malignant glioma, laying groundwork for subsequent translational studies, with implications for future epigenetic editing-based clinical applications
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Targeted next-generation sequencing of 565 neuro-oncology patients at UCLA: A single institution experience
BackgroundTargeted next-generation sequencing (NGS) is frequently obtained at the University of California, Los Angeles (UCLA) for clinical characterization of CNS tumors. In this study, we describe the diagnostic reliability of the Foundation Medicine (FM) targeted NGS platform and its ability to explore and identify tumor characteristics of prognostic significance in gliomas.MethodsNeuro-oncology patients seen at UCLA who have received FM testing between August 2012 and March 2019 were included in this study, and all mutations from FM test reports were recorded. Initial tumor diagnoses and diagnostic markers found via standard clinical methods were obtained from pathology reports. With overall and progression-free survival data, elastic net regularized Cox regression and Cox proportional hazards models were used to determine whether any mutations of unknown significance detected by FM could predict patient outcome in glioblastoma (GBM).ResultsSix hundred and three samples tested by FM from 565 distinct patients were identified. Concordance of diagnostic markers was high between standard clinical testing methods and FM. Oligodendroglial markers detected via FM were highly correlated with 1p19q codeletion in IDH mutated gliomas. FM testing of multiple tumor samples from the same patient demonstrated temporal and spatial mutational heterogeneity. Mutations in BCORL1, ERBB4, and PALB2, which are mutations of unknown significance in GBM, were shown to be statistically significant in predicting patient outcome.ConclusionsIn our large cohort, we found that targeted NGS can both reliably and efficiently detect important diagnostic markers in CNS tumors
The conservation status of the world’s reptiles
Effective and targeted conservation action requires detailed information about species, their distribution, systematics and ecology as well as the distribution of threat processes which affect them. Knowledge of reptilian diversity remains surprisingly disparate, and innovative means of gaining rapid insight into the status of reptiles are needed in order to highlight urgent conservation cases and inform environmental policy with appropriate biodiversity information in a timely manner. We present the first ever global analysis of extinction risk in reptiles, based on a random representative sample of 1500 species (16% of all currently known species). To our knowledge, our results provide the first analysis of the global conservation status and distribution patterns of reptiles and the threats affecting them, highlighting conservation priorities and knowledge gaps which need to be addressed urgently to ensure the continued survival of the world’s reptiles. Nearly one in five reptilian species are threatened with extinction, with another one in five species classed as Data Deficient. The proportion of threatened reptile species is highest in freshwater environments, tropical regions and on oceanic islands, while data deficiency was highest in tropical areas, such as Central Africa and Southeast Asia, and among fossorial reptiles. Our results emphasise the need for research attention to be focussed on tropical areas which are experiencing the most dramatic rates of habitat loss, on fossorial reptiles for which there is a chronic lack of data, and on certain taxa such as snakes for which extinction risk may currently be underestimated due to lack of population information. Conservation actions specifically need to mitigate the effects of human-induced habitat loss and harvesting, which are the predominant threats to reptiles