Kortine Kleinheinz

Decoding the regulatory landscape of medulloblastoma using DNA methylation sequencing

Epigenetic alterations, that is, disruption of DNA methylation and chromatin architecture, are now acknowledged as a universal feature of tumorigenesis. Medulloblastoma, a clinically challenging, malignant childhood brain tumour, is no exception. Despite much progress from recent genomics studies, with recurrent changes identified in each of the four distinct tumour subgroups (WNT-pathway-activated, SHH-pathway-activated, and the less-well-characterized Group 3 and Group 4), many cases still lack an obvious genetic driver. Here we present whole-genome bisulphite-sequencing data from thirty-four human and five murine tumours plus eight human and three murine normal controls, augmented with matched whole-genome, RNA and chromatin immunoprecipitation sequencing data. This comprehensive data set allowed us to decipher several features underlying the interplay between the genome, epigenome and transcriptome, and its effects on medulloblastoma pathophysiology. Most notable were highly prevalent regions of hypomethylation correlating with increased gene expression, extending tens of kilobases downstream of transcription start sites. Focal regions of low methylation linked to transcription-factor-binding sites shed light on differential transcriptional networks between subgroups, whereas increased methylation due to re-normalization of repressed chromatin in DNA methylation valleys was positively correlated with gene expression. Large, partially methylated domains affecting up to one-third of the genome showed increased mutation rates and gene silencing in a subgroup-specific fashion. Epigenetic alterations also affected novel medulloblastoma candidate genes (for example, LIN28B), resulting in alternative promoter usage and/or differential messenger RNA/microRNA expression. Analysis of mouse medulloblastoma and precursor-cell methylation demonstrated a somatic origin for many alterations. Our data provide insights into the epigenetic regulation of transcription and genome organization in medulloblastoma pathogenesis, which are probably also of importance in a wider developmental and disease context.

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.

Applying the ResFinder and VirulenceFinder web-services for easy identification of acquired antibiotic resistance and E. coli virulence genes in bacteriophage and prophage nucleotide sequences

Extensive research is currently being conducted on the use of bacteriophages for applications in human medicine, agriculture and food manufacturing. However, phages are important vehicles of horisontal gene transfer and play a significant role in bacterial evolution. As a result, concern has been raised that this increased use and dissemination of phages could result in spread of deleterious genes, e.g., antibiotic resistance and virulence genes. Meanwhile, in the wake of the genomic era, several tools have been developed for characterization of bacterial genomes. Here we describe how two of these tools, ResFinder and VirulenceFinder, can be used to identify acquired antibiotic resistance and virulence genes in phage genomes of interest. The general applicability of the tools is demonstrated on data sets of 1,642 phage genomes and 1,442 predicted prophages.

HostPhinder: A Phage Host Prediction Tool.

The current dramatic increase of antibiotic resistant bacteria has revitalised the interest in bacteriophages as alternative antibacterial treatment. Meanwhile, the development of bioinformatics methods for analysing genomic data places high-throughput approaches for phage characterization within reach. Here, we present HostPhinder, a tool aimed at predicting the bacterial host of phages by examining the phage genome sequence. Using a reference database of 2196 phages with known hosts, HostPhinder predicts the host species of a query phage as the host of the most genomically similar reference phages. As a measure of genomic similarity the number of co-occurring k-mers (DNA sequences of length k) is used. Using an independent evaluation set, HostPhinder was able to correctly predict host genus and species for 81% and 74% of the phages respectively, giving predictions for more phages than BLAST and significantly outperforming BLAST on phages for which both had predictions. HostPhinder predictions on phage draft genomes from the INTESTI phage cocktail corresponded well with the advertised targets of the cocktail. Our study indicates that for most phages genomic similarity correlates well with related bacterial hosts. HostPhinder is available as an interactive web service [1] and as a stand alone download from the Docker registry [2].

Screening drug effects in patient-derived cancer cells links organoid responses to genome alterations

Cancer drug screening in patient‐derived cells holds great promise for personalized oncology and drug discovery but lacks standardization. Whether cells are cultured as conventional monolayer or advanced, matrix‐dependent organoid cultures influences drug effects and thereby drug selection and clinical success. To precisely compare drug profiles in differently cultured primary cells, we developed DeathPro, an automated microscopy‐based assay to resolve drug‐induced cell death and proliferation inhibition. Using DeathPro, we screened cells from ovarian cancer patients in monolayer or organoid culture with clinically relevant drugs. Drug‐induced growth arrest and efficacy of cytostatic drugs differed between the two culture systems. Interestingly, drug effects in organoids were more diverse and had lower therapeutic potential. Genomic analysis revealed novel links between drug sensitivity and DNA repair deficiency in organoids that were undetectable in monolayers. Thus, our results highlight the dependency of cytostatic drugs and pharmacogenomic associations on culture systems, and guide culture selection for drug tests.

Alterations of microRNA and microRNA-regulated messenger RNA expression in germinal center B-cell lymphomas determined by integrative sequencing analysis

MicroRNA are well-established players in post-transcriptional gene regulation. However, information on the effects of microRNA deregulation mainly relies on bioinformatic prediction of potential targets, whereas proof of the direct physical microRNA/target messenger RNA interaction is mostly lacking. Within the International Cancer Genome Consortium Project “Determining Molecular Mechanisms in Malignant Lymphoma by Sequencing”, we performed miRnome sequencing from 16 Burkitt lymphomas, 19 diffuse large B-cell lymphomas, and 21 follicular lymphomas. Twenty-two miRNA separated Burkitt lymphomas from diffuse large B-cell lymphomas/follicular lymphomas, of which 13 have shown regulation by MYC. Moreover, we found expression of three hitherto unreported microRNA. Additionally, we detected recurrent mutations of hsa-miR-142 in diffuse large B-cell lymphomas and follicular lymphomas, and editing of the hsa-miR-376 cluster, providing evidence for microRNA editing in lymphomagenesis. To interrogate the direct physical interactions of microRNA with messenger RNA, we performed Argonaute-2 photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation experiments. MicroRNA directly targeted 208 messsenger RNA in the Burkitt lymphomas and 328 messenger RNA in the non-Burkitt lymphoma models. This integrative analysis discovered several regulatory pathways of relevance in lymphomagenesis including Ras, PI3K-Akt and MAPK signaling pathways, also recurrently deregulated in lymphomas by mutations. Our dataset reveals that messenger RNA deregulation through microRNA is a highly relevant mechanism in lymphomagenesis.

Integrative genomic and transcriptomic analysis of leiomyosarcoma

Leiomyosarcoma (LMS) is an aggressive mesenchymal malignancy with few therapeutic options. The mechanisms underlying LMS development, including clinically actionable genetic vulnerabilities, are largely unknown. Here we show, using whole-exome and transcriptome sequencing, that LMS tumors are characterized by substantial mutational heterogeneity, near-universal inactivation of TP53 and RB1, widespread DNA copy number alterations including chromothripsis, and frequent whole-genome duplication. Furthermore, we detect alternative telomere lengthening in 78% of cases and identify recurrent alterations in telomere maintenance genes such as ATRX, RBL2, and SP100, providing insight into the genetic basis of this mechanism. Finally, most tumors display hallmarks of “BRCAness”, including alterations in homologous recombination DNA repair genes, multiple structural rearrangements, and enrichment of specific mutational signatures, and cultured LMS cells are sensitive towards olaparib and cisplatin. This comprehensive study of LMS genomics has uncovered key biological features that may inform future experimental research and enable the design of novel therapies.The molecular genetic landscape of leiomyosarcoma (LMS) is largely unknown. Here, the authors identify frequent DNA copy number alterations, whole-genome duplication, TP53 and RB1 inactivation, alternative telomere lengthening, and genomic imprints of defective DNA repair via homologous recombination as a potential therapeutic target in LMS patients.

The evolutionary history of 2,658 cancers

Cancer develops through a process of somatic evolution. Here, we use whole-genome sequencing of 2,778 tumour samples from 2,658 donors to reconstruct the life history, evolution of mutational processes, and driver mutation sequences of 39 cancer types. The early phases of oncogenesis are driven by point mutations in a small set of driver genes, often including biallelic inactivation of tumour suppressors. Early oncogenesis is also characterised by specific copy number gains, such as trisomy 7 in glioblastoma or isochromosome 17q in medulloblastoma. By contrast, increased genomic instability, a nearly four-fold diversification of driver genes, and an acceleration of point mutation processes are features of later stages. Copy-number alterations often occur in mitotic crises leading to simultaneous gains of multiple chromosomal segments. Timing analysis suggests that driver mutations often precede diagnosis by many years, and in some cases decades, providing a window of opportunity for early cancer detection.

Genetic subclone architecture of tumor clone-initiating cells in colorectal cancer

A hierarchically organized cell compartment drives colorectal cancer (CRC) progression. Genetic barcoding allows monitoring of the clonal output of tumorigenic cells without prospective isolation. In this study, we asked whether tumor clone-initiating cells (TcICs) were genetically heterogeneous and whether differences in self-renewal and activation reflected differential kinetics among individual subclones or functional hierarchies within subclones. Monitoring genomic subclone kinetics in three patient tumors and corresponding serial xenografts and spheroids by high-coverage whole-genome sequencing, clustering of genetic aberrations, subclone combinatorics, and mutational signature analysis revealed at least two to four genetic subclones per sample. Long-term growth in serial xenografts and spheroids was driven by multiple genomic subclones with profoundly differing growth dynamics and hence different quantitative contributions over time. Strikingly, genetic barcoding demonstrated stable functional heterogeneity of CRC TcICs during serial xenografting despite near-complete changes in genomic subclone contribution. This demonstrates that functional heterogeneity is, at least frequently, present within genomic subclones and independent of mutational subclone differences.

Meningiomas induced by low-dose radiation carry structural variants of NF2 and a distinct mutational signature

Felix Sahm1,2 · Umut H. Toprak3 · Daniel Hübschmann3,4,5 · Kortine Kleinheinz3 · Ivo Buchhalter3 · Martin Sill6 · Damian Stichel2 · Matthias Schick7 · Melanie Bewerunge‐Hudler7 · Daniel Schrimpf1,2 · Gelareh Zadeh8,9 · Ken Aldape8,10 · Christel Herold‐Mende11 · Katja Beck12 · Ori Staszewski13 · Marco Prinz13,14 · Carmit Ben Harosh15 · Roland Eils3,4,12 · Dominik Sturm5,16 · David T. W. Jones16 · Stefan M. Pfister5,16 · Werner Paulus17 · Zvi Ram15,18 · Matthias Schlesner3 · Rachel Grossman15,18 · Andreas von Deimling1,2