Andreas Schönegger

Somatic Mutations of Calreticulin in Myeloproliferative Neoplasms

BACKGROUND Approximately 50 to 60% of patients with essential thrombocythemia or primary myelofibrosis carry a mutation in the Janus kinase 2 gene (JAK2), and an additional 5 to 10% have activating mutations in the thrombopoietin receptor gene (MPL). So far, no specific molecular marker has been identified in the remaining 30 to 45% of patients. METHODS We performed whole-exome sequencing to identify somatically acquired mutations in six patients who had primary myelofibrosis without mutations in JAK2 or MPL. Resequencing of CALR, encoding calreticulin, was then performed in cohorts of patients with myeloid neoplasms. RESULTS Somatic insertions or deletions in exon 9 of CALR were detected in all patients who underwent whole-exome sequencing. Resequencing in 1107 samples from patients with myeloproliferative neoplasms showed that CALR mutations were absent in polycythemia vera. In essential thrombocythemia and primary myelofibrosis, CALR mutations and JAK2 and MPL mutations were mutually exclusive. Among patients with essential thrombocythemia or primary myelofibrosis with nonmutated JAK2 or MPL, CALR mutations were detected in 67% of those with essential thrombocythemia and 88% of those with primary myelofibrosis. A total of 36 types of insertions or deletions were identified that all cause a frameshift to the same alternative reading frame and generate a novel C-terminal peptide in the mutant calreticulin. Overexpression of the most frequent CALR deletion caused cytokine-independent growth in vitro owing to the activation of signal transducer and activator of transcription 5 (STAT5) by means of an unknown mechanism. Patients with mutated CALR had a lower risk of thrombosis and longer overall survival than patients with mutated JAK2. CONCLUSIONS Most patients with essential thrombocythemia or primary myelofibrosis that was not associated with a JAK2 or MPL alteration carried a somatic mutation in CALR. The clinical course in these patients was more indolent than that in patients with the JAK2 V617F mutation. (Funded by the MPN Research Foundation and Associazione Italiana per la Ricerca sul Cancro.).

Single-Cell DNA Methylome Sequencing and Bioinformatic Inference of Epigenomic Cell-State Dynamics

Summary Methods for single-cell genome and transcriptome sequencing have contributed to our understanding of cellular heterogeneity, whereas methods for single-cell epigenomics are much less established. Here, we describe a whole-genome bisulfite sequencing (WGBS) assay that enables DNA methylation mapping in very small cell populations (μWGBS) and single cells (scWGBS). Our assay is optimized for profiling many samples at low coverage, and we describe a bioinformatic method that analyzes collections of single-cell methylomes to infer cell-state dynamics. Using these technological advances, we studied epigenomic cell-state dynamics in three in vitro models of cellular differentiation and pluripotency, where we observed characteristic patterns of epigenome remodeling and cell-to-cell heterogeneity. The described method enables single-cell analysis of DNA methylation in a broad range of biological systems, including embryonic development, stem cell differentiation, and cancer. It can also be used to establish composite methylomes that account for cell-to-cell heterogeneity in complex tissue samples.

Epigenome mapping reveals distinct modes of gene regulation and widespread enhancer reprogramming by the oncogenic fusion protein EWS-FLI1.

Summary Transcription factor fusion proteins can transform cells by inducing global changes of the transcriptome, often creating a state of oncogene addiction. Here, we investigate the role of epigenetic mechanisms in this process, focusing on Ewing sarcoma cells that are dependent on the EWS-FLI1 fusion protein. We established reference epigenome maps comprising DNA methylation, seven histone marks, open chromatin states, and RNA levels, and we analyzed the epigenome dynamics upon downregulation of the driving oncogene. Reduced EWS-FLI1 expression led to widespread epigenetic changes in promoters, enhancers, and super-enhancers, and we identified histone H3K27 acetylation as the most strongly affected mark. Clustering of epigenetic promoter signatures defined classes of EWS-FLI1-regulated genes that responded differently to low-dose treatment with histone deacetylase inhibitors. Furthermore, we observed strong and opposing enrichment patterns for E2F and AP-1 among EWS-FLI1-correlated and anticorrelated genes. Our data describe extensive genome-wide rewiring of epigenetic cell states driven by an oncogenic fusion protein.

KRAS and CREBBP mutations: a relapse-linked malicious liaison in childhood high hyperdiploid acute lymphoblastic leukemia

High hyperdiploidy defines the largest genetic entity of childhood acute lymphoblastic leukemia (ALL). Despite its relatively low recurrence risk, this subgroup generates a high proportion of relapses. The cause and origin of these relapses remains obscure. We therefore explored the mutational landscape in high hyperdiploid (HD) ALL with whole-exome (n=19) and subsequent targeted deep sequencing of 60 genes in 100 relapsing and 51 non-relapsing cases. We identified multiple clones at diagnosis that were primarily defined by a variety of mutations in receptor tyrosine kinase (RTK)/Ras pathway and chromatin-modifying genes. The relapse clones consisted of reappearing as well as new mutations, and overall contained more mutations. Although RTK/Ras pathway mutations were similarly frequent between diagnosis and relapse, both intergenic and intragenic heterogeneity was essentially lost at relapse. CREBBP mutations, however, increased from initially 18–30% at relapse, then commonly co-occurred with KRAS mutations (P<0.001) and these relapses appeared primarily early (P=0.012). Our results confirm the exceptional susceptibility of HD ALL to RTK/Ras pathway and CREBBP mutations, but, more importantly, suggest that mutant KRAS and CREBBP might cooperate and equip cells with the necessary capacity to evolve into a relapse-generating clone.

DNA methylation heterogeneity defines a disease spectrum in Ewing sarcoma

Developmental tumors in children and young adults carry few genetic alterations, yet they have diverse clinical presentation. Focusing on Ewing sarcoma, we sought to establish the prevalence and characteristics of epigenetic heterogeneity in genetically homogeneous cancers. We performed genome-scale DNA methylation sequencing for a large cohort of Ewing sarcoma tumors and analyzed epigenetic heterogeneity on three levels: between cancers, between tumors, and within tumors. We observed consistent DNA hypomethylation at enhancers regulated by the disease-defining EWS-FLI1 fusion protein, thus establishing epigenomic enhancer reprogramming as a ubiquitous and characteristic feature of Ewing sarcoma. DNA methylation differences between tumors identified a continuous disease spectrum underlying Ewing sarcoma, which reflected the strength of an EWS-FLI1 regulatory signature and a continuum between mesenchymal and stem cell signatures. There was substantial epigenetic heterogeneity within tumors, particularly in patients with metastatic disease. In summary, our study provides a comprehensive assessment of epigenetic heterogeneity in Ewing sarcoma and thereby highlights the importance of considering nongenetic aspects of tumor heterogeneity in the context of cancer biology and personalized medicine.

Complex Patterns of Chromosome 11 Aberrations in Myeloid Malignancies Target CBL, MLL, DDB1 and LMO2

Exome sequencing of primary tumors identifies complex somatic mutation patterns. Assignment of relevance of individual somatic mutations is difficult and poses the next challenge for interpretation of next generation sequencing data. Here we present an approach how exome sequencing in combination with SNP microarray data may identify targets of chromosomal aberrations in myeloid malignancies. The rationale of this approach is that hotspots of chromosomal aberrations might also harbor point mutations in the target genes of deletions, gains or uniparental disomies (UPDs). Chromosome 11 is a frequent target of lesions in myeloid malignancies. Therefore, we studied chromosome 11 in a total of 813 samples from 773 individual patients with different myeloid malignancies by SNP microarrays and complemented the data with exome sequencing in selected cases exhibiting chromosome 11 defects. We found gains, losses and UPDs of chromosome 11 in 52 of the 813 samples (6.4%). Chromosome 11q UPDs frequently associated with mutations of CBL. In one patient the 11qUPD amplified somatic mutations in both CBL and the DNA repair gene DDB1. A duplication within MLL exon 3 was detected in another patient with 11qUPD. We identified several common deleted regions (CDR) on chromosome 11. One of the CDRs associated with de novo acute myeloid leukemia (P=0.013). One patient with a deletion at the LMO2 locus harbored an additional point mutation on the other allele indicating that LMO2 might be a tumor suppressor frequently targeted by 11p deletions. Our chromosome-centered analysis indicates that chromosome 11 contains a number of tumor suppressor genes and that the role of this chromosome in myeloid malignancies is more complex than previously recognized.

Abstract PR13: DNA methylation mapping and computational modeling in a large Ewing sarcoma cohort identifies principles of tumor heterogeneity and their impact on clinical phenotypes

Ewing sarcoma is an excellent model for studying the role of epigenetic deregulation and tumor heterogeneity, given its low mutation rates and the well-defined oncogenic driver. We have recently shown that the fusion oncogene EWS-FLI1 induces widespread epigenetic rewiring in proximal and distal enhancers (Tomazou et al. Cell Reports 2015). In the current study, we validate the clinical relevance of our results in a large cohort of primary tumors, and we explore the prevalence, characteristics, and clinical impact of epigenetic tumor heterogeneity in Ewing sarcoma. We used reduced representation bisulfite sequencing (RRBS) to generate genome-wide profiles of DNA methylation in 141 Ewing sarcoma primary tumors, 17 Ewing sarcoma cell lines, and 32 primary mesenchymal stem cell (MSC) samples. Deep sequencing resulted in DNA methylation measurements for an average of 3.5 million unique CpGs per sample with excellent data quality (>98% bisulfite conversion efficiency). In addition, for three primary tumors we generated comprehensive reference epigenome maps using whole genome bisulfite sequencing (WGBS) and ChIP-seq for seven histone marks (H3K4me3, H3K4me1, H3K27me3, H3K27ac, H3K56ac, H3K36me3, and H3K9me3). We show that DNA methylation data can be used to infer enhancer activity differences among tumors, allowing us to exploit our large primary tumor dataset to systematically compare the regulation of EWS-FLI1 correlated and anticorrelated enhancers. We also identified Ewing-specific DNA methylation patterns. For example, Ewing sarcoma samples consistently show higher DNA methylation than MSCs at AP-1 binding sites, but lower DNA methylation at EWS-FLI1 binding sites. To explore epigenetic heterogeneity within individual tumors, we developed a bioinformatic algorithm that quantifies DNA methylation disorder. Using individual reads containing multiple DNA methylation measurements from single cells, we assign scores at single-nucleotide resolution. This method uses a probabilistic model to account for overall methylation rate and expected disorder levels. By evaluating the likelihood of the data in a model that assumes that the DNA methylation status of a CpG is independent of the methylation status of a nearby CpG, we identify extremely heterogeneous as well as highly epigenetically conserved genomic elements. These different region types show distinct patterns of enrichment for regulatory modes and transcription factor binding. We also compared the observed DNA methylation disorder in 141 Ewing tumors to those observed in 17 Ewing sarcoma cell lines, 32 primary MSC samples, and several hundred additional tumor and normal samples that are unrelated to Ewing sarcoma. This analysis identified Ewing-specific patterns of DNA methylation heterogeneity and stratifies patients based on epigenetic heterogeneity. Our dataset constitutes the largest available resource of genome-scale DNA methylation maps in a solid pediatric tumor. It strongly confirms the relevance of enhancer reprogramming and tumor heterogeneity in Ewing sarcoma, and it constitutes a starting point to develop DNA methylation biomarkers for prognosis and patient stratification. This study is supported by the Austrian National Bank (OeNB project #15714) and the Kapsch group (https://www.kapsch.net/). This abstract is also presented as Poster A24. Citation Format: Nathan C. Sheffield, Franck Tirode, Sandrine Grossetete-Lalami, Paul Datlinger, Andreas Schonegger, Johanna Hadler, Diana Walder, Ingeborg M. Ambros, Ana Teresa Amaral, Enrique de Alava, Katharina Schallmoser, Dirk Strunk, Beate Rinner, Bernadette Liegl-Atzwanger, Berthold Huppertz, Andreas Leithner, Uta Dirksen, Peter Ambros, Olivier Delattre, Heinrich Kovar, Christoph Bock, Eleni M. Tomazou. DNA methylation mapping and computational modeling in a large Ewing sarcoma cohort identifies principles of tumor heterogeneity and their impact on clinical phenotypes. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR13.

Abstract A60: An epigenetic map of Ewing sarcoma revealed by comprehensive epigenome sequencing

Epigenetic marks, including DNA methylation, histone modifications and long non-coding RNAs, are frequently deregulated in pediatric cancers. Even in pediatric cancers that are driven by well-defined genetic events, changes in the epigenome are prevalent, suggesting that epigenetic defects constitute an emerging hallmark of pediatric cancers and a promising new target for drug development. To define the epigenetic basis of Ewing sarcoma, we performed comprehensive epigenome sequencing in a cellular model that provides precise temporal control of the EWS/FLI1 fusion gene, which is the main cause and driving event in Ewing sarcoma. Specifically, we established reference epigenome maps in the A673 cell line in the presence and absence of the EWS/FLI1 oncogene. All mapping experiments were performed in accordance with the standards of the International Human Epigenome Consortium, thus adhering to stringent requirements for quality control, sequencing depth, and reproducibility in biological replicates. The epigenome maps we established include comprehensive DNA methylation maps obtained by whole genome bisulfite sequencing (WGBS), genome-wide maps of eight histone modifications (H3K4me3, H3K4me1, H3K27me3, H3K9me3, H3K27ac, H3K36me, H3K56ac, H4K16ac) obtained by ChIP-seq and deep transcript annotations, and expression maps obtained by RNA-seq. The epigenome datasets were further complemented by ChIP-seq maps of EWS/FLI1-binding throughout the genome. Integrative bioinformatic analysis of our dataset enables testing of the following hypothesis: i. EWS/FLI1 induces changes in the global distribution of specific epigenetic marks ii. EWS/FLI1 introduces de novo enhancers that rewire the transcriptional network in Ewing sarcoma iii. EWS/FLI1 binding of super-enhancers provides a mechanism for oncogene addiction iv. EWS/FLI1 regulates a novel class of long non-coding RNAs that are expected to play a role in EWS/FLI1-induced tumorgenesis In summary, the comprehensive nature of our dataset (10 different epigenetic marks, including DNA methylation, histone modifications, and long non-coding RNAs, mapped genome-wide in 4 different cell populations) constitutes a unique resource for the Ewing sarcoma community, and it provides a foundation that allowed us to conduct a systematic bioinformatic dissection of the interplay between EWS/FLI1 and the epigenome. These results shed light on the functional relevance of the epigenetic deregulation in Ewing sarcoma cells, and have the potential to provide a rational basis for testing the therapeutic utility of epigenetic drugs in preclinical models. This study is supported by the 7th framework program of the European commission, grant 259348 (“ASSET”) and a generous donation by the Kapsch group for next generation sequencing. EMT is a recipient of a Lise Meitner Fellowship of the Austrian Science Fund (FWF, M1448-B13). Citation Format: Eleni Marina Tomazou, Nathan Sheffield, Michael Schuster, Andreas Schonegger, Maximilian Kauer, Christoph Bock, Heinrich Kovar. An epigenetic map of Ewing sarcoma revealed by comprehensive epigenome sequencing. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A60.