Naveed Ishaque

Mutations in the SIX1/2 Pathway and the DROSHA/DGCR8 miRNA Microprocessor Complex Underlie High-Risk Blastemal Type Wilms Tumors

Blastemal histology in chemotherapy-treated pediatric Wilms tumors (nephroblastoma) is associated with adverse prognosis. To uncover the underlying tumor biology and find therapeutic leads for this subgroup, we analyzed 58 blastemal type Wilms tumors by exome and transcriptome sequencing and validated our findings in a large replication cohort. Recurrent mutations included a hotspot mutation (Q177R) in the homeo-domain of SIX1 and SIX2 in tumors with high proliferative potential (18.1% of blastemal cases); mutations in the DROSHA/DGCR8 microprocessor genes (18.2% of blastemal cases); mutations in DICER1 and DIS3L2; and alterations in IGF2, MYCN, and TP53, the latter being strongly associated with dismal outcome. DROSHA and DGCR8 mutations strongly altered miRNA expression patterns in tumors, which was functionally validated in cell lines expressing mutant DROSHA.

The whole-genome landscape of medulloblastoma subtypes

Current therapies for medulloblastoma, a highly malignant childhood brain tumour, impose debilitating effects on the developing child, and highlight the need for molecularly targeted treatments with reduced toxicity. Previous studies have been unable to identify the full spectrum of driver genes and molecular processes that operate in medulloblastoma subgroups. Here we analyse the somatic landscape across 491 sequenced medulloblastoma samples and the molecular heterogeneity among 1,256 epigenetically analysed cases, and identify subgroup-specific driver alterations that include previously undiscovered actionable targets. Driver mutations were confidently assigned to most patients belonging to Group 3 and Group 4 medulloblastoma subgroups, greatly enhancing previous knowledge. New molecular subtypes were differentially enriched for specific driver events, including hotspot in-frame insertions that target KBTBD4 and ‘enhancer hijacking’ events that activate PRDM6. Thus, the application of integrative genomics to an extensive cohort of clinical samples derived from a single childhood cancer entity revealed a series of cancer genes and biologically relevant subtype diversity that represent attractive therapeutic targets for the treatment of patients with medulloblastoma.

Environment-induced epigenetic reprogramming in genomic regulatory elements in smoking mothers and their children

Epigenetic mechanisms have emerged as links between prenatal environmental exposure and disease risk later in life. Here, we studied epigenetic changes associated with maternal smoking at base pair resolution by mapping DNA methylation, histone modifications, and transcription in expectant mothers and their newborn children. We found extensive global differential methylation and carefully evaluated these changes to separate environment associated from genotype‐related DNA methylation changes. Differential methylation is enriched in enhancer elements and targets in particular “commuting” enhancers having multiple, regulatory interactions with distal genes. Longitudinal whole‐genome bisulfite sequencing revealed that DNA methylation changes associated with maternal smoking persist over years of life. Particularly in children prenatal environmental exposure leads to chromatin transitions into a hyperactive state. Combined DNA methylation, histone modification, and gene expression analyses indicate that differential methylation in enhancer regions is more often functionally translated than methylation changes in promoters or non‐regulatory elements. Finally, we show that epigenetic deregulation of a commuting enhancer targeting c‐Jun N‐terminal kinase 2 (JNK2) is linked to impaired lung function in early childhood.

Prenatal maternal stress and wheeze in children: novel insights into epigenetic regulation

Psychological stress during pregnancy increases the risk of childhood wheeze and asthma. However, the transmitting mechanisms remain largely unknown. Since epigenetic alterations have emerged as a link between perturbations in the prenatal environment and an increased disease risk we used whole genome bisulfite sequencing (WGBS) to analyze changes in DNA methylation in mothers and their children related to prenatal psychosocial stress and assessed its role in the development of wheeze in the child. We evaluated genomic regions altered in their methylation level due to maternal stress based of WGBS data of 10 mother-child-pairs. These data were complemented by longitudinal targeted methylation and transcriptional analyses in children from our prospective mother-child cohort LINA for whom maternal stress and wheezing information was available (n = 443). High maternal stress was associated with an increased risk for persistent wheezing in the child until the age of 5. Both mothers and children showed genome-wide alterations in DNA-methylation specifically in enhancer elements. Deregulated neuroendocrine and neurotransmitter receptor interactions were observed in stressed mothers and their children. In children but not in mothers, calcium- and Wnt-signaling required for lung maturation in the prenatal period were epigenetically deregulated and could be linked with wheezing later in children’s life.

Increased vitamin D levels at birth and in early infancy increase offspring allergy risk—evidence for involvement of epigenetic mechanisms

To the Editor: Although a beneficial effect of vitamin D on health is widely accepted, its role in allergy development has been controversial. Both allergy-preventing and allergy-promoting effects have been reported. Thus, a deeper mechanistic understanding of how vitamin D is related to the regulation of immune reactivity and allergic inflammation is required. Vitamin D was shown to modify gene expression through binding of the vitamin D receptor to vitamin D response elements. However, only 26% of the genes identified as regulated by vitamin D have a vitamin D response element in proximity to their transcription start site (TSS), indicating that additional mechanisms are involved in the transcriptional control by vitamin D. As an additional mechanism, epigenetically mediated transcriptional deregulation through vitamin D–induced changes in DNA methylation was suggested. Here, we studied DNA-methylation pattern on a genomewide scale at base-pair resolution in healthy newborn children with high and low vitamin D levels to elucidate the role of vitamin D in epigenetic programming of an allergy-protective or allergypromoting immune reactivity. Within the LINA (Lifestyle and environmental factors and their Influence on Newborns Allergy risk) mother-child cohort, differential DNA methylation was assessed by using whole genome bisulfite sequencing in 6 cord blood samples comparing 3 children with high to 3 children with low

Retrotransposon Alu is enriched in the epichromatin of HL-60 cells

Epichromatin, the surface of chromatin facing the nuclear envelope in an interphase nucleus, reveals a “rim” staining pattern with specific mouse monoclonal antibodies against histone H2A/H2B/DNA and phosphatidylserine epitopes. Employing a modified ChIP-Seq procedure on undifferentiated and differentiated human leukemic (HL-60/S4) cells, >95% of assembled epichromatin regions overlapped with Alu retrotransposons. They also exhibited enrichment of the AluS subfamily and of Alu oligomers. Furthermore, mapping epichromatin regions to the human chromosomes revealed highly similar localization patterns in the various cell states and with the different antibodies. Comparisons with available epigenetic databases suggested that epichromatin is neither “classical” heterochromatin nor highly expressing genes, implying another function at the surface of interphase chromatin. A modified chromatin immunoprecipitation procedure (xxChIP) was developed because the studied antibodies react generally with mononucleosomes and lysed chromatin. A second fixation is necessary to securely attach the antibodies to the epichromatin epitopes of the intact nucleus.

Succession of transiently active tumor‐initiating cell clones in human pancreatic cancer xenografts

Although tumor‐initiating cell (TIC) self‐renewal has been postulated to be essential in progression and metastasis formation of human pancreatic adenocarcinoma (PDAC), clonal dynamics of TICs within PDAC tumors are yet unknown. Here, we show that long‐term progression of PDAC in serial xenotransplantation is driven by a succession of transiently active TICs producing tumor cells in temporally restricted bursts. Clonal tracking of individual, genetically marked TICs revealed that individual tumors are generated by distinct sets of TICs with very little overlap between subsequent xenograft generations. An unexpected functional and phenotypic plasticity of pancreatic TICs in vivo underlies the recruitment of inactive TIC clones in serial xenografts. The observed clonal succession of TIC activity in serial xenotransplantation is in stark contrast to the continuous activity of limited numbers of self‐renewing TICs within a fixed cellular hierarchy observed in other epithelial cancers and emphasizes the need to target TIC activation, rather than a fixed TIC population, in PDAC.

EnrichedHeatmap: an R/Bioconductor package for comprehensive visualization of genomic signal associations

BackgroundHigh-throughput sequencing data are dramatically increasing in volume. Thus, there is urgent need for efficient tools to perform fast and integrative analysis of multiple data types. Enriched heatmap is a specific form of heatmap that visualizes how genomic signals are enriched over specific target regions. It is commonly used and efficient at revealing enrichment patterns especially for high dimensional genomic and epigenomic datasets.ResultsWe present a new R package named EnrichedHeatmap that efficiently visualizes genomic signal enrichment. It provides advanced solutions for normalizing genomic signals within target regions as well as offering highly customizable visualizations. The major advantage of EnrichedHeatmap is the ability to conveniently generate parallel heatmaps as well as complex annotations, which makes it easy to integrate and visualize comprehensive overviews of the patterns and associations within and between complex datasets.ConclusionsEnrichedHeatmap facilitates comprehensive understanding of high dimensional genomic and epigenomic data. The power of EnrichedHeatmap is demonstrated by visualization of the complex associations between DNA methylation, gene expression and various histone modifications.

Abstract 1417: Clonal succession in pancreatic cancer progression is not driven by genetic instability

Our group has recently shown that human pancreatic cancer (PDAC) progression is driven by a succession of transiently active tumor initiating cell (TIC) clones during serial xenotransplantation. Genetic labeling demonstrated that serial PDAC xenograft tumors and even tumors of parallel mice transplanted with cells from the same donor xenografts harbored very little to no overlap of active TIC clones, indicating substantial changes in the proliferative activity of individual TIC predominantly producing progeny without detectable tumor-initiating activity. We now asked whether observed clonal activation and inactivation is caused by acquisition of de novo mutations during evolution of genetic subclones or by functional plasticity of genetically stable TIC clones. Therefore, we monitored somatic non-synonymous mutations in culture and during PDAC progression in genetically marked serial xenografts of two patients. DNA was isolated from xenografts, primary TIC cultures and corresponding normal pancreas or primary tumor tissue. Following paired-end exome sequencing, reads were aligned to a concatenated hs37d5 human and mm10 mouse genome assembly and human specific single nucleotide variants (SNVs) and small insertions/deletions (indels) were identified. We found a total of 45 altered gene coding genomic loci (P1 = 10; P2 = 35) not present in control tissue. Strikingly, most SNVs detected were present in all samples, only very few SNV were acquired during serial transplantation. In P1, 4 novel SNVs not present in the original patient tumor sample were detected within coding regions of TTC13, OR4K15, SSPO and TPGS1. Allele frequencies ranged from 2-27% in serial xenografts. In xenografts of P2 we detected 35 SNVs not present in healthy tissue. Of these, one mutation in the gene C10orf12 aroused after serial transplantation with a maximum altered allele frequency of 17%. None of these mutations is a known cancer driver or was found as recurrent in large scale cancer sequencing approaches. To evaluate whether the clonal TIC dynamics within established tumors are recapitulated in vitro, we analysed individual TIC clone kinetics in serially passaged cultures and in cultures derived from transduced xenografts. Strikingly, the kinetics in vitro were similar to those observed within serially transplanted xenografts. Every culture passage was formed by a distinct set of actively proliferating cell clones without significant overlap between individual serial passages indicating that clonal succession of TIC activity in PDAC is not dependent on the cellular context in tumors in vivo. The remarkable genetic stability of xenografts during serial transplantation strongly indicates that changes in the functional state of PDAC cells and not genetic instability drive clonal succession of TIC activity in PDAC. Citation Format: Karl Roland Ehrenberg, Claudia R. Ball, Felix Oppel, Naveed Ishaque, Taronish D. Dubash, Sebastian M. Dieter, Christopher M. Hoffmann, Ulrich Abel, Moritz Koch, Jens Werner, Frank Bergmann, Manfred Schmidt, Christof von Kalle, Wilko Weichert, Jurgen Weitz, Benedikt Brors, Hanno Glimm. Clonal succession in pancreatic cancer progression is not driven by genetic instability. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1417. doi:10.1158/1538-7445.AM2015-1417