Assessment of patient-derived tumour xenografts (PDXs) as a discovery tool for cancer epigenomics

Background: The use of tumour xenografts is a well-established research tool in cancer genomics but has not yet been comprehensively evaluated for cancer epigenomics. Methods: In this study, we assessed the suitability of patient-derived tumour xenografts (PDXs) for methylome analysis using Infinium 450 K Beadchips and MeDIP-seq. Results: Controlled for confounding host (mouse) sequences, comparison of primary PDXs and matching patient tumours in a rare (osteosarcoma) and common (colon) cancer revealed that an average 2.7% of the assayed CpG sites undergo major (Δβ ≥ 0.51) methylation changes in a cancer-specific manner as a result of the xenografting procedure. No significant subsequent methylation changes were observed after a second round of xenografting between primary and secondary PDXs. Based on computational simulation using publically available methylation data, we additionally show that future studies comparing two groups of PDXs should use 15 or more samples in each group to minimise the impact of xenografting-associated changes in methylation on comparison results. Conclusions: Our results from rare and common cancers indicate that PDXs are a suitable discovery tool for cancer epigenomics and we provide guidance on how to overcome the observed limitations

Assessment of RainDrop BS-seq as a method for large-scale, targeted bisulfite sequencing.

We present a systematic assessment of RainDrop BS-seq, a novel method for large-scale, targeted bisulfite sequencing using microdroplet-based PCR amplification coupled with next-generation sequencing. We compared DNA methylation levels at 498 target loci (1001 PCR amplicons) in human whole blood, osteosarcoma cells and an archived tumor tissue sample. We assessed the ability of RainDrop BS-seq to accurately measure DNA methylation over a range of DNA quantities (from 10 to 1500 ng), both with and without whole-genome amplification (WGA) following bisulfite conversion. DNA methylation profiles generated using at least 100 ng correlated well (median R = 0.92) with those generated on Illumina Infinium HumanMethylation450 BeadChips, currently the platform of choice for epigenome-wide association studies (EWAS). WGA allowed for testing of samples with a starting DNA amount of 10 and 50 ng, although a reduced correlation was observed (median R = 0.79). We conclude that RainDrop BS-seq is suitable for measuring DNA methylation levels using nanogram quantities of DNA, and can be used to study candidate epigenetic biomarker loci in an accurate and high-throughput manner, paving the way for its application to routine clinical diagnostics

Meta-analysis of IDH-mutant cancers identifies EBF1 as an interaction partner for TET2

Isocitrate dehydrogenase (IDH) genes 1 and 2 are frequently mutated in acute myeloid leukaemia (AML), low-grade glioma, cholangiocarcinoma (CC) and chondrosarcoma (CS). For AML, low-grade glioma and CC, mutant IDH status is associated with a DNA hypermethylation phenotype, implicating altered epigenome dynamics in the aetiology of these cancers. Here we show that the IDH variants in CS are also associated with a hypermethylation phenotype and display increased production of the oncometabolite 2-hydroxyglutarate, supporting the role of mutant IDH-produced 2-hydroxyglutarate as an inhibitor of TET-mediated DNA demethylation. Meta-analysis of the acute myeloid leukaemia, low-grade glioma, cholangiocarcinoma and CS methylation data identifies cancer-specific effectors within the retinoic acid receptor activation pathway among the hypermethylated targets. By analysing sequence motifs surrounding hypermethylated sites across the four cancer types, and using chromatin immunoprecipitation and western blotting, we identify the transcription factor EBF1 (early B-cell factor 1) as an interaction partner for TET2, suggesting a sequence-specific mechanism for regulating DNA methylation

A C19MC-LIN28A-MYCN Oncogenic Circuit Driven by Hijacked Super-enhancers Is a Distinct Therapeutic Vulnerability in ETMRs: A Lethal Brain Tumor

© 2019 Elsevier Inc. Embryonal tumors with multilayered rosettes (ETMRs) are highly lethal infant brain cancers with characteristic amplification of Chr19q13.41 miRNA cluster (C19MC) and enrichment of pluripotency factor LIN28A. Here we investigated C19MC oncogenic mechanisms and discovered a C19MC-LIN28A-MYCN circuit fueled by multiple complex regulatory loops including an MYCN core transcriptional network and super-enhancers resulting from long-range MYCN DNA interactions and C19MC gene fusions. Our data show that this powerful oncogenic circuit, which entraps an early neural lineage network, is potently abrogated by bromodomain inhibitor JQ1, leading to ETMR cell death. Sin-Chan et al. uncover a C19MC-LIN28A-MYCN super-enhancer-dependent oncogenic circuit in embryonal tumors with multilayered rosettes (ETMRs). The circuit entraps an early neural lineage network to sustain embryonic epigenetic programming and is vulnerable to bromodomain inhibition, which promotes ETMR cell death

Genome-wide analysis of differential transcriptional and epigenetic variability across human immune cell types

Abstract Background A healthy immune system requires immune cells that adapt rapidly to environmental challenges. This phenotypic plasticity can be mediated by transcriptional and epigenetic variability. Results We apply a novel analytical approach to measure and compare transcriptional and epigenetic variability genome-wide across CD14+CD16− monocytes, CD66b+CD16+ neutrophils, and CD4+CD45RA+ naïve T cells from the same 125 healthy individuals. We discover substantially increased variability in neutrophils compared to monocytes and T cells. In neutrophils, genes with hypervariable expression are found to be implicated in key immune pathways and are associated with cellular properties and environmental exposure. We also observe increased sex-specific gene expression differences in neutrophils. Neutrophil-specific DNA methylation hypervariable sites are enriched at dynamic chromatin regions and active enhancers. Conclusions Our data highlight the importance of transcriptional and epigenetic variability for the key role of neutrophils as the first responders to inflammatory stimuli. We provide a resource to enable further functional studies into the plasticity of immune cells, which can be accessed from: http://blueprint-dev.bioinfo.cnio.es/WP10/hypervariability

SMuRF: a novel tool to identify regulatory elements enriched for somatic point mutations

Abstract Background Single Nucleotide Variants (SNVs), including somatic point mutations and Single Nucleotide Polymorphisms (SNPs), in noncoding cis-regulatory elements (CREs) can affect gene regulation and lead to disease development. Several approaches have been developed to identify highly mutated regions, but these do not take into account the specific genomic context, and thus likelihood of mutation, of CREs. Results Here, we present SMuRF (Significantly Mutated Region Finder), a user-friendly command-line tool to identify these significantly mutated regions from user-defined genomic intervals and SNVs. We demonstrate this using publicly available datasets in which SMuRF identifies 72 significantly mutated CREs in liver cancer, including known mutated gene promoters as well as previously unreported regions. Conclusions SMuRF is a helpful tool to allow the simple identification of significantly mutated regulatory elements. It is open-source and freely available on GitHub (https://github.com/LupienLab/SMURF)

Comparative methylome analysis identifies new tumour subtypes and biomarkers for transformation of nephrogenic rests into Wilms tumour

Background Wilms tumours (WTs) are characterised by several hallmarks that suggest epimutations such as aberrant DNA methylation are involved in tumour progression: loss of imprinting at 11p15, lack of recurrent mutations and formation of nephrogenic rests (NRs), which are lesions of retained undifferentiated embryonic tissue that can give rise to WTs. Methods To identify such epimutations, we performed a comprehensive methylome analysis on 20 matched trios of micro-dissected WTs, NRs and surrounding normal kidneys (NKs) using Illumina Infinium HumanMethylation450 Bead Chips and functionally validated findings using RNA sequencing. Results Comparison of NRs with NK revealed prominent tissue biomarkers: 629 differentially methylated regions, of which 55% were hypermethylated and enriched for domains that are bivalent in embryonic stem cells and for genes expressed during development (P = 2.49 × 10-5). Comparison of WTs with NRs revealed two WT subgroups; group-2 WTs and NRs were epigenetically indistinguishable whereas group-1 WTs showed an increase in methylation variability, hypomethylation of renal development genes, hypermethylation and relative loss of expression of cell adhesion genes and known and potential new WT tumour suppressor genes (CASP8, H19, MIR195, RB1 and TSPAN32) and was strongly associated with bilateral disease (P = 0.032). Comparison of WTs and NRs to embryonic kidney highlighted the significance of polycomb target methylation in Wilms tumourigenesis. Conclusions Methylation levels vary during cancer evolution. We have described biomarkers related to WT evolution from its precursor NRs which may be useful to differentiate between these tissues for patients with bilateral disease