Single-cell transcriptomics and epigenomics unravel the role of monocytes in neuroblastoma bone marrow metastasis.

Metastasis is the major cause of cancer-related deaths. Neuroblastoma (NB), a childhood tumor has been molecularly defined at the primary cancer site, however, the bone marrow (BM) as the metastatic niche of NB is poorly characterized. Here we perform single-cell transcriptomic and epigenomic profiling of BM aspirates from 11 subjects spanning three major NB subtypes and compare these to five age-matched and metastasis-free BM, followed by in-depth single cell analyses of tissue diversity and cell-cell interactions, as well as functional validation. We show that cellular plasticity of NB tumor cells is conserved upon metastasis and tumor cell type composition is NB subtype-dependent. NB cells signal to the BM microenvironment, rewiring via macrophage mgration inhibitory factor and midkine signaling specifically monocytes, which exhibit M1 and M2 features, are marked by activation of pro- and anti-inflammatory programs, and express tumor-promoting factors, reminiscent of tumor-associated macrophages. The interactions and pathways characterized in our study provide the basis for therapeutic approaches that target tumor-to-microenvironment interactions.

High-content drug screening in zebrafish xenografts reveals high efficacy of dual MCL-1/BCL-XL inhibition against Ewing sarcoma.

Ewing sarcoma is a pediatric bone and soft tissue cancer with an urgent need for new therapies to improve disease outcome. To identify effective drugs, phenotypic drug screening has proven to be a powerful method, but achievable throughput in mouse xenografts, the preclinical Ewing sarcoma standard model, is limited. Here, we explored the use of xenografts in zebrafish for high-throughput drug screening to discover new combination therapies for Ewing sarcoma. We subjected xenografts in zebrafish larvae to high-content imaging and subsequent automated tumor size analysis to screen single agents and compound combinations. We identified three drug combinations effective against Ewing sarcoma cells: Irinotecan combined with either an MCL-1 or an BCL-XL inhibitor and in particular dual inhibition of the anti-apoptotic proteins MCL-1 and BCL-XL, which efficiently eradicated tumor cells in zebrafish xenografts. We confirmed enhanced efficacy of dual MCL-1/BCL-XL inhibition compared to single agents in a mouse PDX model. In conclusion, high-content screening of small compounds on Ewing sarcoma zebrafish xenografts identified dual MCL-1/BCL-XL targeting as a specific vulnerability and promising therapeutic strategy for Ewing sarcoma, which warrants further investigation towards clinical application.

LIQUORICE: detection of epigenetic signatures in liquid biopsies based on whole-genome sequencing data.

Summary: Fragmentation patterns of cell-free DNA reflect the chromatin structure of the cells from which these fragments are derived. Nucleosomes protect the DNA from fragmentation, resulting in decreased sequencing coverage in regions of open chromatin. LIQUORICE is a user-friendly software tool that takes aligned whole-genome sequencing data as input and calculates bias-corrected coverage signatures for predefined, application-specific sets of genomic regions. The tool thereby enables a blood-based analysis of cell death in the body, and it provides a minimally invasive assessment of tumor chromatin states and cell-of-origin. With user-defined sets of regions that exhibit tissue-specific or disease-specific open chromatin, LIQUORICE can be applied to a wide range of detection, classification and quantification tasks in the analysis of liquid biopsies. Availability and implementation: LIQUORICE is freely and openly available as a Python package and command-line tool for UNIX-based systems from bioconda. Documentation, examples and usage instructions are provided at http://liquorice.computational-epigenetics.org. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Quantification of Translocation-Specific ctDNA Provides an Integrating Parameter for Early Assessment of Treatment Response and Risk Stratification in Ewing Sarcoma.

PURPOSE: We evaluated the predictive and prognostic value of circulating tumor DNA (ctDNA) in patients with Ewing sarcoma (EWS) treated in the EWING2008 trial. EXPERIMENTAL DESIGN: Plasma samples from 102 patients with EWS enrolled in the EWING2008 trial were obtained before and during induction chemotherapy. Genomic EWSR1 fusion sequence spanning primers and probes were used for highly specific and sensitive quantification of the levels of ctDNA by digital droplet PCR. ctDNA levels were correlated to established clinical risk factors and outcome parameters. RESULTS: Pretreatment ctDNA copy numbers were correlated with event-free and overall survival. The reduction in ctDNA levels below the detection limit was observed in most cases after only two blocks of vincristine, ifosfamide, doxorubicin, and etoposide (VIDE) induction chemotherapy. The persistence of ctDNA after two VIDE blocks was a strong predictor of poor outcomes. ctDNA levels correlated well with most established clinical risk factors; an inverse correlation was found only for the histologic response to induction therapy. ctDNA levels did not provide simple representations of tumor volume, but integrated information from various tumor characteristics represented an independent EWS tumor marker with predictive and prognostic value. CONCLUSIONS: ctDNA copy number in the plasma of patients with EWS is a quantifiable parameter for early risk stratification and can be used as a dynamic noninvasive biomarker for early prediction of treatment response and outcome of patients.

Multimodal analysis of cell-free DNA whole-genome sequencing for pediatric cancers with low mutational burden.

Sequencing of cell-free DNA in the blood of cancer patients (liquid biopsy) provides attractive opportunities for early diagnosis, assessment of treatment response, and minimally invasive disease monitoring. To unlock liquid biopsy analysis for pediatric tumors with few genetic aberrations, we introduce an integrated genetic/epigenetic analysis method and demonstrate its utility on 241 deep whole-genome sequencing profiles of 95 patients with Ewing sarcoma and 31 patients with other pediatric sarcomas. Our method achieves sensitive detection and classification of circulating tumor DNA in peripheral blood independent of any genetic alterations. Moreover, we benchmark different metrics for cell-free DNA fragmentation analysis, and we introduce the LIQUORICE algorithm for detecting circulating tumor DNA based on cancer-specific chromatin signatures. Finally, we combine several fragmentation-based metrics into an integrated machine learning classifier for liquid biopsy analysis that exploits widespread epigenetic deregulation and is tailored to cancers with low mutation rates. Clinical associations highlight the potential value of cfDNA fragmentation patterns as prognostic biomarkers in Ewing sarcoma. In summary, our study provides a comprehensive analysis of circulating tumor DNA beyond recurrent genetic aberrations, and it renders the benefits of liquid biopsy more readily accessible for childhood cancers.

Biomolecular condensation of NUP98 fusion proteins drives leukemogenic gene expression.

NUP98 fusion proteins cause leukemia via unknown molecular mechanisms. All NUP98 fusion proteins share an intrinsically disordered region (IDR) in the NUP98 N terminus, featuring repeats of phenylalanine-glycine (FG), and C-terminal fusion partners often function in gene control. We investigated whether mechanisms of oncogenic transformation by NUP98 fusion proteins are hardwired in their protein interactomes. Affinity purification coupled to mass spectrometry (MS) and confocal imaging of five NUP98 fusion proteins expressed in human leukemia cells revealed that shared interactors were enriched for proteins involved in biomolecular condensation and that they colocalized with NUP98 fusion proteins in nuclear puncta. We developed biotinylated isoxazole-mediated condensome MS (biCon-MS) to show that NUP98 fusion proteins alter the global composition of biomolecular condensates. An artificial FG-repeat-containing fusion protein phenocopied the nuclear localization patterns of NUP98 fusion proteins and their capability to drive oncogenic gene expression programs. Thus, we propose that IDR-containing fusion proteins combine biomolecular condensation with transcriptional control to induce cancer.

Sarcoma treatment in the era of molecular medicine.

Sarcomas are heterogeneous and clinically challenging soft tissue and bone cancers. Although constituting only 1% of all human malignancies, sarcomas represent the second most common type of solid tumors in children and adolescents and comprise an important group of secondary malignancies. More than 100 histological subtypes have been characterized to date, and many more are being discovered due to molecular profiling. Owing to their mostly aggressive biological behavior, relative rarity, and occurrence at virtually every anatomical site, many sarcoma subtypes are in particular difficult-to-treat categories. Current multimodal treatment concepts combine surgery, polychemotherapy (with/without local hyperthermia), irradiation, immunotherapy, and/or targeted therapeutics. Recent scientific advancements have enabled a more precise molecular characterization of sarcoma subtypes and revealed novel therapeutic targets and prognostic/predictive biomarkers. This review aims at providing a comprehensive overview of the latest advances in the molecular biology of sarcomas and their effects on clinical oncology; it is meant for a broad readership ranging from novices to experts in the field of sarcoma.

Retraction Note: Increased survival and cell cycle progression pathways are required for EWS/FLI1-induced malignant transformation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

EWS-FLI1 modulated alternative splicing of ARID1A reveals novel oncogenic function through the BAF complex.

Connections between epigenetic reprogramming and transcription or splicing create novel mechanistic networks that can be targeted with tailored therapies. Multiple subunits of the chromatin remodeling BAF complex, including ARID1A, play a role in oncogenesis, either as tumor suppressors or oncogenes. Recent work demonstrated that EWS-FLI1, the oncogenic driver of Ewing sarcoma (ES), plays a role in chromatin regulation through interactions with the BAF complex. However, the specific BAF subunits that interact with EWS-FLI1 and the precise role of the BAF complex in ES oncogenesis remain unknown. In addition to regulating transcription, EWS-FLI1 also alters the splicing of many mRNA isoforms, but the role of splicing modulation in ES oncogenesis is not well understood. We have identified a direct connection between the EWS-FLI1 protein and ARID1A isoform protein variant ARID1A-L. We demonstrate here that ARID1A-L is critical for ES maintenance and supports oncogenic transformation. We further report a novel feed-forward cycle in which EWS-FLI1 leads to preferential splicing of ARID1A-L, promoting ES growth, and ARID1A-L reciprocally promotes EWS-FLI1 protein stability. Dissecting this interaction may lead to improved cancer-specific drug targeting.

Ewing sarcoma.

Ewing sarcoma is the second most frequent bone tumour of childhood and adolescence that can also arise in soft tissue. Ewing sarcoma is a highly aggressive cancer, with a survival of 70-80% for patients with standard-risk and localized disease and ~30% for those with metastatic disease. Treatment comprises local surgery, radiotherapy and polychemotherapy, which are associated with acute and chronic adverse effects that may compromise quality of life in survivors. Histologically, Ewing sarcomas are composed of small round cells expressing high levels of CD99. Genetically, they are characterized by balanced chromosomal translocations in which a member of the FET gene family is fused with an ETS transcription factor, with the most common fusion being EWSR1-FLI1 (85% of cases). Ewing sarcoma breakpoint region 1 protein (EWSR1)-Friend leukaemia integration 1 transcription factor (FLI1) is a tumour-specific chimeric transcription factor (EWSR1-FLI1) with neomorphic effects that massively rewires the transcriptome. Additionally, EWSR1-FLI1 reprogrammes the epigenome by inducing de novo enhancers at GGAA microsatellites and by altering the state of gene regulatory elements, creating a unique epigenetic signature. Additional mutations at diagnosis are rare and mainly involve STAG2, TP53 and CDKN2A deletions. Emerging studies on the molecular mechanisms of Ewing sarcoma hold promise for improvements in early detection, disease monitoring, lower treatment-related toxicity, overall survival and quality of life.

Correction: Increased survival and cell cycle progression pathways are required for EWS/FLI1-induced malignant transformation.

Since publication of the article, the authors became aware that Figure 6(A,D) contained errors in the bands and loading controls. The newly compiled Figure 6A and 6D is given below.

MIRA: an R package for DNA methylation-based inference of regulatory activity.

Summary: DNA methylation contains information about the regulatory state of the cell. MIRA aggregates genome-scale DNA methylation data into a DNA methylation profile for a given region set with shared biological annotation. Using this profile, MIRA infers and scores the collective regulatory activity for the region set. MIRA facilitates regulatory analysis in situations where classical regulatory assays would be difficult and allows public sources of region sets to be leveraged for novel insight into the regulatory state of DNA methylation datasets. Availability and implementation: http://bioconductor.org/packages/MIRA.

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.

High-throughput RNAi screen in Ewing sarcoma cells identifies leucine rich repeats and WD repeat domain containing 1 (LRWD1) as a regulator of EWS-FLI1 driven cell viability.

A translocation leading to the formation of an oncogenic EWS-ETS fusion protein defines Ewing sarcoma. The most frequent gene fusion, present in 85 percent of Ewing sarcomas, is EWS-FLI1. Here, a high-throughput RNA interference screen was performed to identify genes whose function is critical for EWS-FLI1 driven cell viability. In total, 6781 genes were targeted by siRNA molecules and the screen was performed both in presence and absence of doxycycline-inducible expression of the EWS-FLI1 shRNA in A673/TR/shEF Ewing sarcoma cells. The Leucine rich repeats and WD repeat Domain containing 1 (LRWD1) targeting siRNA pool was the strongest hit reducing cell viability only in EWS-FLI1 expressing Ewing sarcoma cells. LRWD1 had been previously described as a testis specific gene with only limited information on its function. Analysis of LRWD1 mRNA levels in patient samples indicated that high expression associated with poor overall survival in Ewing sarcoma. Gene ontology analysis of LRWD1 co-expressed genes in Ewing tumors revealed association with DNA replication and analysis of differentially expressed genes in LRWD1 depleted Ewing sarcoma cells indicated a role in connective tissue development and cellular morphogenesis. Moreover, EWS-FLI1 repressed genes with repressive H3K27me3 chromatin marks were highly enriched among LRWD1 target genes in A673/TR/shEF Ewing sarcoma cells, suggesting that LRWD1 contributes to EWS-FLI1 driven transcriptional regulation. Taken together, we have identified LRWD1 as a novel regulator of EWS-FLI1 driven cell viability in A673/TR/shEF Ewing sarcoma cells, shown association between high LRWD1 mRNA expression and aggressive disease and identified processes by which LRWD1 may promote oncogenesis in Ewing sarcoma.

Increased survival and cell cycle progression pathways are required for EWS/FLI1-induced malignant transformation.

Ewing sarcoma (ES) is the second most frequent childhood bone cancer driven by the EWS/FLI1 (EF) fusion protein. Genetically defined ES models are needed to understand how EF expression changes bone precursor cell differentiation, how ES arises and through which mechanisms of inhibition it can be targeted. We used mesenchymal Prx1-directed conditional EF expression in mice to study bone development and to establish a reliable sarcoma model. EF expression arrested early chondrocyte and osteoblast differentiation due to changed signaling pathways such as hedgehog, WNT or growth factor signaling. Mesenchymal stem cells (MSCs) expressing EF showed high self-renewal capacity and maintained an undifferentiated state despite high apoptosis. Blocking apoptosis through enforced BCL2 family member expression in MSCs promoted efficient and rapid sarcoma formation when transplanted to immunocompromised mice. Mechanistically, high BCL2 family member and CDK4, but low P53 and INK4A protein expression synergized in Ewing-like sarcoma development. Functionally, knockdown of Mcl1 or Cdk4 or their combined pharmacologic inhibition resulted in growth arrest and apoptosis in both established human ES cell lines and EF-transformed mouse MSCs. Combinatorial targeting of survival and cell cycle progression pathways could counteract this aggressive childhood cancer.

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

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.

Overexpression of HOX genes is prevalent in Ewing sarcoma and is associated with altered epigenetic regulation of developmental transcription programs.

The polycomb proteins BMI-1 and EZH2 are highly overexpressed by Ewing sarcoma (ES), a tumor of stem cell origin that is driven by EWS-ETS fusion oncogenes, most commonly EWS-FLI1. In the current study we analyzed expression of transcription programs that are controlled by polycomb proteins during embryonic development to determine if they are abnormal in ES. Our results show that polycomb target gene expression in ES deviates from normal tissues and stem cells and that, as expected, most targets are relatively repressed. However, we also discovered a paradoxical up regulation of numerous polycomb targets and these were highly enriched for homeobox (HOX) genes. Comparison of HOX profiles between malignant and non-malignant tissues revealed a distinctive HOX profile in ES, which was characterized by overexpression of posterior HOXD genes. In addition, ectopic expression of EWS-FLI1 during stem cell differentiation led to aberrant up regulation of posterior HOXD genes. Mechanistically, this up regulation was associated with altered epigenetic regulation. Specifically, ES and EWS-FLI1+ stem cells displayed a relative loss of polycomb-dependent H3K27me3 and gain of trithorax-dependent H3K4me3 at the promoters of posterior HOXD genes and also at the HOXD11.12 polycomb response element. In addition, a striking correlation was evident between HOXD13 and other genes whose regulation is coordinately regulated during embryonic development by distal enhancer elements. Together, these studies demonstrate that epigenetic regulation of polycomb target genes, in particular HOXD genes, is altered in ES and that these changes are mediated downstream of EWS-FLI1.

Quantitative comparison of genome-wide DNA methylation mapping technologies.

DNA methylation plays a key role in regulating eukaryotic gene expression. Although mitotically heritable and stable over time, patterns of DNA methylation frequently change in response to cell differentiation, disease and environmental influences. Several methods have been developed to map DNA methylation on a genomic scale. Here, we benchmark four of these approaches by analyzing two human embryonic stem cell lines derived from genetically unrelated embryos and a matched pair of colon tumor and adjacent normal colon tissue obtained from the same donor. Our analysis reveals that methylated DNA immunoprecipitation sequencing (MeDIP-seq), methylated DNA capture by affinity purification (MethylCap-seq), reduced representation bisulfite sequencing (RRBS) and the Infinium HumanMethylation27 assay all produce accurate DNA methylation data. However, these methods differ in their ability to detect differentially methylated regions between pairs of samples. We highlight strengths and weaknesses of the four methods and give practical recommendations for the design of epigenomic case-control studies.

Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution.

Bisulfite sequencing measures absolute levels of DNA methylation at single-nucleotide resolution, providing a robust platform for molecular diagnostics. We optimized bisulfite sequencing for genome-scale analysis of clinical samples: here we outline how restriction digestion targets bisulfite sequencing to hotspots of epigenetic regulation and describe a statistical method for assessing significance of altered DNA methylation patterns. Thirty nanograms of DNA was sufficient for genome-scale analysis and our protocol worked well on formalin-fixed, paraffin-embedded samples.

Epigenetic regulation of pluripotency.

Epigenetic regulation refers to the mechanisms that alter gene expression patterns in the absence of changes in the nucleotide sequence of the DNA molecule. The best understood epigenetic marks include posttranslational modifications of the histone tails and DNA methylation. Both play central roles in normal development and in diseases. Pluripotent stem cells have great promise for regenerative medicine and recent efforts have focused on identifying molecular networks that govern pluripotency. This chapter provides an overview of epigenetic regulation in embryonic stem cells. We present a brief introduction into epigenetic mechanisms and focus on their role in pluripotent cells.