Epigenetic modifications such as carbon 5 methylation of the cytosine base in a CpG dinucleotide context are involved in the onset and progression of human diseases. A comprehensive understanding of ...the role of genome-wide DNA methylation patterns, the methylome, requires quantitative determination of the methylation states of all CpG sites in a genome. So far, analyses of the complete methylome by whole-genome bisulfite sequencing (WGBS) are rare because of the required large DNA quantities, substantial bioinformatic resources and high sequencing costs. Here we describe a detailed protocol for tagmentation-based WGBS (T-WGBS) and demonstrate its reliability in comparison with conventional WGBS. In T-WGBS, a hyperactive Tn5 transposase fragments the DNA and appends sequencing adapters in a single step. T-WGBS requires not more than 20 ng of input DNA; hence, the protocol allows the comprehensive methylome analysis of limited amounts of DNA isolated from precious biological specimens. The T-WGBS library preparation takes 2 d.
Glioblastoma frequently exhibits therapy-associated subtype transitions to mesenchymal phenotypes with adverse prognosis. Here, we perform multi-omic profiling of 60 glioblastoma primary tumours and ...use orthogonal analysis of chromatin and RNA-derived gene regulatory networks to identify 38 subtype master regulators, whose cell population-specific activities we further map in published single-cell RNA sequencing data. These analyses identify the oligodendrocyte precursor marker and chromatin modifier SOX10 as a master regulator in RTK I-subtype tumours. In vitro functional studies demonstrate that SOX10 loss causes a subtype switch analogous to the proneural-mesenchymal transition observed in patients at the transcriptomic, epigenetic and phenotypic levels. SOX10 repression in an in vivo syngeneic graft glioblastoma mouse model results in increased tumour invasion, immune cell infiltration and significantly reduced survival, reminiscent of progressive human glioblastoma. These results identify SOX10 as a bona fide master regulator of the RTK I subtype, with both tumour cell-intrinsic and microenvironmental effects.
Abstract
Background
Common diseases manifest differentially between patients, but the genetic origin of this variation remains unclear. To explore possible involvement of gene ...transcriptional-variation, we produce a DNA methylation-oriented, driver-gene-wide dataset of regulatory elements in human glioblastomas and study their effect on inter-patient gene expression variation.
Results
In 175 of 177 analyzed gene regulatory domains, transcriptional enhancers and silencers are intermixed. Under experimental conditions, DNA methylation induces enhancers to alter their enhancing effects or convert into silencers, while silencers are affected inversely. High-resolution mapping of the association between DNA methylation and gene expression in intact genomes reveals methylation-related regulatory units (average size = 915.1 base-pairs). Upon increased methylation of these units, their target-genes either increased or decreased in expression. Gene-enhancing and silencing units constitute
cis
-regulatory networks of genes. Mathematical modeling of the networks highlights indicative methylation sites, which signified the effect of key regulatory units, and add up to make the overall transcriptional effect of the network. Methylation variation in these sites effectively describe inter-patient expression variation and, compared with DNA sequence-alterations, appears as a major contributor of gene-expression variation among glioblastoma patients.
Conclusions
We describe complex
cis
-regulatory networks, which determine gene expression by summing the effects of positive and negative transcriptional inputs. In these networks, DNA methylation induces both enhancing and silencing effects, depending on the context. The revealed mechanism sheds light on the regulatory role of DNA methylation, explains inter-individual gene-expression variation, and opens the way for monitoring the driving forces behind deferential courses of cancer and other diseases.
We studied how intratumoral genetic heterogeneity shapes tumor growth and therapy response for isocitrate dehydrogenase (IDH)-wild-type glioblastoma, a rapidly regrowing tumor. We inferred the ...evolutionary trajectories of matched pairs of primary and relapsed tumors based on deep whole-genome-sequencing data. This analysis suggests both a distant origin of de novo glioblastoma, up to 7 years before diagnosis, and a common path of early tumorigenesis, with one or more of chromosome 7 gain, 9p loss, or 10 loss, at tumor initiation. TERT promoter mutations often occurred later as a prerequisite for rapid growth. In contrast to this common early path, relapsed tumors acquired no stereotypical pattern of mutations and typically regrew from oligoclonal origins, suggesting sparse selective pressure by therapeutic measures.
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•We inferred evolutionary trajectories of pairs of primary/relapsed glioblastomas•Chromosome 7 gain, 9p loss, or 10 loss commonly occurred at tumor initiation•TERT promoter mutations often occurred later as a prerequisite for rapid growth•Relapsed tumors typically regrew from oligoclonal origins
By analyzing 21 paired primary and locally relapsed IDH-wild-type glioblastomas (GBM), Körber et al. show that most GBM initiate by gains and losses of specific chromosomes; TERT promoter mutations often occur later as a prerequisite for rapid growth, and relapsed GBM acquire few stereotypical mutations.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Cerebral gliomas of World Health Organization (WHO) grade II and III represent a major challenge in terms of histological classification and clinical management. Here, we asked whether large-scale ...genomic and transcriptomic profiling improves the definition of prognostically distinct entities. We performed microarray-based genome- and transcriptome-wide analyses of primary tumor samples from a prospective German Glioma Network cohort of 137 patients with cerebral gliomas, including 61 WHO grade II and 76 WHO grade III tumors. Integrative bioinformatic analyses were employed to define molecular subgroups, which were then related to histology, molecular biomarkers, including isocitrate dehydrogenase 1 or 2 (
IDH1/2
) mutation, 1p/19q co-deletion and telomerase reverse transcriptase (
TERT
) promoter mutations, and patient outcome. Genomic profiling identified five distinct glioma groups, including three
IDH1/2
mutant and two
IDH1/2
wild-type groups. Expression profiling revealed evidence for eight transcriptionally different groups (five
IDH1/2
mutant, three
IDH1/2
wild type), which were only partially linked to the genomic groups. Correlation of DNA-based molecular stratification with clinical outcome allowed to define three major prognostic groups with characteristic genomic aberrations. The best prognosis was found in patients with
IDH1/2
mutant and 1p/19q co-deleted tumors. Patients with
IDH1/2
wild-type gliomas and glioblastoma-like genomic alterations, including gain on chromosome arm 7q (+7q), loss on chromosome arm 10q (−10q),
TERT
promoter mutation and oncogene amplification, displayed the worst outcome. Intermediate survival was seen in patients with
IDH1/2
mutant, but 1p/19q intact, mostly astrocytic gliomas, and in patients with
IDH1/2
wild-type gliomas lacking the +7q/−10q genotype and
TERT
promoter mutation. This molecular subgrouping stratified patients into prognostically distinct groups better than histological classification. Addition of gene expression data to this genomic classifier did not further improve prognostic stratification. In summary, DNA-based molecular profiling of WHO grade II and III gliomas distinguishes biologically distinct tumor groups and provides prognostically relevant information beyond histological classification as well as
IDH1/2
mutation and 1p/19q co-deletion status.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
During the ongoing COVID-19 pandemic, PCR testing and antigen tests have proven critical for helping to stem the spread of its causative agent, SARS-CoV-2. However, these methods suffer from either ...general applicability and/or sensitivity. Moreover, the emergence of variant strains creates the need for flexibility to correctly and efficiently diagnose the presence of substrains. To address these needs we developed the diagnostic test ADESSO (Accurate Detection of Evolving SARS-CoV-2 through SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) Optimization) which employs Cas13 to diagnose patients in 1 h without sophisticated equipment. Using an extensive panel of clinical samples, we demonstrate that ADESSO correctly identifies infected individuals at a sensitivity and specificity comparable to RT-qPCR on extracted RNA and higher than antigen tests for unextracted samples. Altogether, ADESSO is a fast, sensitive and cheap method that can be applied in a point of care setting to diagnose COVID-19 and can be quickly adjusted to detect new variants.
Purpose: The CD133 antigen has been identified as a putative stem cell marker in normal and malignant brain tissues. In gliomas, it
is used to enrich a subpopulation of highly tumorigenic cancer ...cells. According to the cancer stem cell hypothesis, CD133-positive
cells determine long-term tumor growth and, therefore, are suspected to influence clinical outcome. To date, a correlation
between CD133 expression in primary tumor tissues and patients' prognosis has not been reported.
Experimental Design: To address this question, we analyzed the expression of the CD133 stem cell antigen in a series of 95 gliomas of various
grade and histology by immunohistochemistry on cryostat sections. Staining data were correlated with patient outcome.
Results: By multivariate survival analysis, we found that both the proportion of CD133-positive cells and their topological organization
in clusters were significant ( P < 0.001) prognostic factors for adverse progression-free survival and overall survival independent of tumor grade, extent
of resection, or patient age. Furthermore, proportion of CD133-positive cells was an independent risk factor for tumor regrowth
and time to malignant progression in WHO grade 2 and 3 tumors.
Conclusions: These findings constitute the first conclusive evidence that CD133 stem cell antigen expression correlates with patient survival
in gliomas, lending support to the current cancer stem cell hypothesis.
To identify new tumor‐suppressor gene candidates relevant for human hepatocarcinogenesis, we performed genome‐wide methylation profiling and vertical integration with array‐based comparative genomic ...hybridization (aCGH), as well as expression data from a cohort of well‐characterized human hepatocellular carcinomas (HCCs). Bisulfite‐converted DNAs from 63 HCCs and 10 healthy control livers were analyzed for the methylation status of more than 14,000 genes. After defining the differentially methylated genes in HCCs, we integrated their DNA copy‐number alterations as determined by aCGH data and correlated them with gene expression to identify genes potentially silenced by promoter hypermethylation. Aberrant methylation of candidates was further confirmed by pyrosequencing, and methylation dependency of silencing was determined by 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) treatment. Methylation profiling revealed 2,226 CpG sites that showed methylation differences between healthy control livers and HCCs. Of these, 537 CpG sites were hypermethylated in the tumor DNA, whereas 1,689 sites showed promoter hypomethylation. The hypermethylated set was enriched for genes known to be inactivated by the polycomb repressive complex 2, whereas the group of hypomethylated genes was enriched for imprinted genes. We identified three genes matching all of our selection criteria for a tumor‐suppressor gene (period homolog 3 PER3, insulin‐like growth‐factor–binding protein, acid labile subunit IGFALS, and protein Z). PER3 was down‐regulated in human HCCs, compared to peritumorous and healthy liver tissues. 5‐aza‐dC treatment restored PER3 expression in HCC cell lines, indicating that promoter hypermethylation was indeed responsible for gene silencing. Additionally, functional analysis supported a tumor‐suppressive function for PER3 and IGFALS in vitro. Conclusion: The present study illustrates that vertical integration of methylation data with high‐resolution genomic and transcriptomic data facilitates the identification of new tumor‐suppressor gene candidates in human HCC. (HEPATOLOGY 2012;56:1817–1827)
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Quinolinic acid is a product of tryptophan degradation and may serve as a precursor for NAD(+), an important enzymatic cofactor for enzymes such as the DNA repair protein PARP. Pathologic ...accumulation of quinolinic acid has been found in neurodegenerative disorders including Alzheimer and Huntington disease, where it is thought to be toxic for neurons by activating the N-methyl-D-aspartate (NMDA) receptor and inducing excitotoxicity. Although many tumors including gliomas constitutively catabolize tryptophan, it is unclear whether quinolinic acid is produced in gliomas and whether it is involved in tumor progression. Here, we show that quinolinic acid accumulated in human gliomas and was associated with a malignant phenotype. Quinolinic acid was produced by microglial cells, as expression of the quinolinic acid-producing enzyme 3-hydroxyanthranilate oxygenase (3-HAO) was confined to microglia in glioma tissue. Human malignant glioma cells, but not nonneoplastic astrocytes, expressed quinolinic acid phosphoribosyltransferase (QPRT) to use quinolinic acid for NAD(+) synthesis and prevent apoptosis when de novo NAD(+) synthesis was blocked. Oxidative stress, temozolomide, and irradiation induced QPRT in glioma cells. QPRT expression increased with malignancy. In recurrent glioblastomas after radiochemotherapy, QPRT expression was associated with a poor prognosis in two independent datasets. Our data indicate that neoplastic transformation in astrocytes is associated with a QPRT-mediated switch in NAD(+) metabolism by exploiting microglia-derived quinolinic acid as an alternative source of replenishing intracellular NAD(+) pools. The elevated levels of QPRT expression increase resistance to oxidative stress induced by radiochemotherapy, conferring a poorer prognosis. These findings have implications for therapeutic approaches inducing intracellular NAD(+) depletion, such as alkylating agents or direct NAD(+) synthesis inhibitors, and identify QPRT as a potential therapeutic target in malignant gliomas.
Cancer stem cells (CSCs) have been suggested as potential therapeutic targets for treating malignant tumors, but the in vivo supporting evidence is still missing. Using a GFP reporter driven by the ...promoter of the nuclear receptor tailless (Tlx), we demonstrate that Tlx+ cells in primary brain tumors are mostly quiescent. Lineage tracing demonstrates that single Tlx+ cells can self-renew and generate Tlx− tumor cells in primary tumors, suggesting that they are brain tumor stem cells (BTSCs). After introducing a BTSC-specific knock-out of the Tlx gene in primary mouse tumors, we observed a loss of self-renewal of BTSCs and prolongation of animal survival, accompanied by induction of essential signaling pathways mediating cell-cycle arrest, cell death, and neural differentiation. Our study demonstrates the feasibility of targeting glioblastomas and indicates the suitability of BTSCs as therapeutic targets, thereby supporting the CSC hypothesis.
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•A mouse brain tumor model for tumor stem cell-specific gene targeting is presented•Tlx is only expressed in brain tumor stem cells of mouse primary gliomas in vivo•BTSC-specific knock-out of Tlx leads to the loss of BTSCs and prolonged survival•Loss of BTSCs leads to cell death, cell-cycle arrest, and differentiation
Zhu et al. demonstrate that cells expressing the neural stem cell marker Tlx in glioblastoma are largely quiescent, but can self-renew to generate Tlx− tumors. Inducibly targeting TLx in Nestin-expressing brain tumor cells results in cell death, cell-cycle arrest, and neural differentiation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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