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ASSET: Analysing and Striking the Sensitivities of Embryonal Tumours

Final Report Summary - ASSET (ASSET: Analysing and Striking the Sensitivities of Embryonal Tumours)

Executive Summary:
Executive Summary

ASSET applied a systems level approach to study embryonal tumours (ET), which include neuroblastoma (NB), medulloblastoma (MB) and Ewing sarcoma family tumours (ESFT). These tumours arise in babies and children, and are devastating for the patients and their families. Combining state-of-the-art genomics, transcriptomics, proteomics and mathematical modelling, ASSET aimed to (i) deconvolute the plethora of molecular pathogenetic cancer aetiologies to the common core principles; (ii) develop better patient stratification; and (iii) devise new drug targets, drugs, and drug combinations that will open new therapy options for ETs.

ASSET was highly successful and not only delivered on its objectives but also developed new research directions, e.g. the adoption of zebrafish as an animal model, which is rapidly becoming a new gold standard animal model for neuroblastoma. Another major success was the integration between basic, translational and clinical research through computational modelling. This aspect of the project can serve as a blueprint for implementing systems medicine approaches that leverage breakthroughs in basic research to accelerate translational research and clinical applications.

ASSET scientific discovery highlights
• New molecular mechanistic insights into the pathogenesis of ETs arising from the integrated application of omics technologies and computational modelling
• New methods to stratify patients based on advanced interpretation of omics results
• New drug targets identified through systems approaches and data integration
• New drug combinations identified
• New computational resources developed
• Identifying the role of miRNAs in ETs
• Development of new computational biology resources
o A comprehensive resource for data collection and analysis: the ASSET Data Warehouse
o Development of data analysis pipelines for mutations
o New tools for data integration and contextualisationIntegration of genomic and transcriptomics data for enhanced patient stratification
o Development of statistical tools for analysing timecourse series of gene expression data
o Developing new statistical tools for analysing drug cooperation
o Developing reusable code for conducting Markov chain Monte Carlo (MCMC) inference

ASSET impact highlights
ASSET has generated a number of scientific innovations that will have lasting impact on science and the scientific community. The most important achievements in this respect are briefly listed below:
• Proof of concept for systems medicine approaches
• Systems guided drug design and deployment
• New software tools for the analysis and interpretation of omics data
• New tools for data integration and data comparison
• Nucleating a community of researchers and clinicians interested in systems medicine approaches
• Broad and effective dissemination of results

Project Context and Objectives:
Project aims and objectives

ASSET applied a systems level approach to study embryonal tumours (ET), which include the entities neuroblastoma (NB), medulloblastoma (MB) and Ewing sarcoma family tumours (ESFT). These tumours arise in babies and children, and are devastating for the patients and their families. ETs pose significant clinical challenges in terms of disease stratification for prognosis and treatment as well as in the paucity of drugs available for treatment. ETs seem to share common aberrations in core signalling networks with “modulator” pathways determining disease-specific manifestations. Combining state-of-the-art genomics, transcriptomics, proteomics and mathematical modelling, ASSET has analyses ETs with the aims (i) to deconvolute the plethora of molecular pathogenetic cancer aetiologies to the common core principles; (ii) to develop better patient stratification and to (iii) devise new drug targets, drugs, and drug combinations that will open new therapy options for ETs.

Towards these aims the ASSET objectives were
using a systems biology-driven discovery and validation engine for
• the combined analysis of genomic mutations, transcriptome, miRNA expression and dynamic proteome changes in ET model cell lines
• mathematical modelling to elucidate molecular pathogenetic networks and their emergent properties
• systematic perturbations to probe and refine these networks
• implementation of a virtuous cycle of model making and validation in relevant biological model systems (cell culture models and preclinical mouse models) and clinical samples
• using a systems medicine approach in order to identify new drug targets, drugs and drug combinations based on a molecular mechanistic understanding of pathogenic network aberrations

The ASSET concept and workflow - see attached diagram.

ASSET outcomes and achievements

ASSET was highly successful and not only delivered on its objectives but also developed new research directions, e.g. the adoption of zebrafish as an animal model, which is rapidly becoming a new gold standard animal model for neuroblastoma. Another major success was the integration between basic, translational and clinical research through computational modelling. This aspect of the project can serve as a blueprint for implementing systems medicine approaches that leverage breakthroughs in basic research to accelerate translational research and clinical applications.

ASSET research highlights
Ewing Sarcoma
• Analysis of aberrant signalling in Ewing sarcoma leading to the discovery of new targets
• New functional insights into EWS-FLI1, the main driver oncogene of Ewing Sarcoma
• A central role for the histone deacetylase SIRT1 in Ewing Sarcoma
• Post-transcriptional gene regulation exerted by EWS-FLI1 dependent microRNAs
• Role of miRNAs in Ewing sarcoma
• Small molecules and new targets for Embryonal Tumours
• New drug combinations for treating Embryonal Tumours
• Insights from mathematical models of EWS-FLI1 signalling
• Single cell transcriptomics
• Whole genome sequencing of Ewing sarcomas

Neuroblastoma
• Targeting MYCN signalling through a network based approach
• Mathematical models as informative biomarkers in neuroblastoma (NB)
• Insights from whole genome sequencing in neuroblastoma
• Identifying vulnerable nodes in neuroblastoma by synthetic siRNA screens
• Modelling and predicting chemotherapy survival of neuroblastoma cells
• New roles for MYCN in neuroblastoma (NB)
• New roles for ALK in neuroblastoma (NB)
• Proteomic analysis of TrkA signaling

Medulloblastoma (MB)
• The role of miRNAs in MB
• Synthetic lethal siRNA screens in MB
• Synthetic lethal kinome screens in MB

Crosscutting Findings
• The role of miRNAs in ETs
• Crosstalk between receptor tyrosine kinases (RTKs) and nuclear receptors for steroid hormones

Technology Advances
• Systematic and elective high throughput perturbation screens to identify vulnerabilities in Embryonal Tumour (ET) cells
• New targets arising from the screens
• Investigating the dynamic behaviour of protein signalling networks by reverse protein microarrays

Development of new computational biology resources
• A comprehensive resource for data collection and analysis: the ASSET Data Warehouse ASSETmart
• Development of data analysis pipelines for mutations
• New tools for data integration and contextualisation
o Cell-type specific models to simulate the different cell lines and predict the effect of drugs
o Tools to perform analyses and comparison of the mutational profiles of the cell lines and primary samples within the context of large international cancer patient datasets
o Tools for comparing gene expression profiles from cell lines and primary patient samples
• Integration of genomic and transcriptomics data for enhanced patient stratification
• Development of statistical tools for analysing timecourse series of gene expression data
• Developing new statistical tools for analysing drug cooperation
• Developing reusable code for conducting Markov chain Monte Carlo (MCMC) inference

ASSET impact highlights
ASSET has generated a number of scientific innovations that will have lasting impact on science and the scientific community. The most important achievements in this respect are briefly listed below:
• Mathematical models as informative biomarkers in neuroblastoma
• New biomarkers for high risk neuroblastoma
• Systems guided drug design and deployment
• New software tools for the analysis and interpretation of omics data
o Atlas of Cancer Signalling Network (ACSN) database
o NaviCell Web Service
o A new version of BiNoM Cytoscape plugin
o NBPrognostics, a comprehensive catalogue of prognostic down- und up-regulated genes in NB
o SwitchFinder is a novel statistical method developed for the analysis of time-series data.
o Lora is a package for Markov chain Monte Carlo (MCMC) inference
• New therapeutic targets
• Circadian regulation of RTK signalling as a new type of therapeutic intervention
• Development of new technologies for analysing pathogenetic mechanisms in ETs
• Establishment of ASSETmart, a data warehouse and analysis platform for ETs for use by the scientific community
• New tools for data integration and data comparison
• Proof of concept for systems medicine approaches
• Nucleating a community of researchers and clinicians interested in systems medicine approaches
• Broad and effective dissemination of results

Project Results:
Advanced Insights into in the Molecular Pathology of Embryonal Tumours (ETs)

Ewing Sarcoma
The driving force of Ewing sarcoma pathogenesis is the chimeric gene regulatory protein EWS-FLI1. It combines the DNA binding domain of an ETS transcription factor with a portion of a nuclear protein, EWS, with functions in general transcription, RNA splicing, and DNA recombination. EWS-FLI1 perturbs gene expression on a genome-wide level leading to unlimited proliferation and blocked differentiation. In this project we applied a systems approach to identify hubs in the gene regulatory network of EWS-FLI1 and unravel mechanisms of expression dysregulation. To this end, a Ewing sarcoma model cell line (A673) with switchable EWS-FLI1 expression was fully characterized on the genomic, epigenomic, transcriptional and proteomic level in EWS-FLI1 on and off states. We obtained a genome-wide map of EWS-FLI1 binding 1 and described the first Ewing sarcoma specific epigenome 2 and microRNA regulome (Schwentner et al., submitted). Integrating the high throughput data sets, patterns of EWS-FLI1 driven gene activation and repression were obtained, among them modifiers of therapeutic response 3.

Analysis of aberrant signalling in Ewing sarcoma leading to the discovery of new targets
Ewing sarcoma is hallmarked by the expression of a EWS-FLI1 fusion oncogene. Careful analysis of time series data of Ewing sarcoma cell line conditionally inhibited for EWS-FLI1 expression, led to the identification of new promising targets for new therapeutic approaches in Ewing sarcoma. One of such was Protein Kinase C Beta (PRKCβ) as a gene strongly activated by EWS-FLI1. Comparison with other paediatric or bone cancers showed that PRKCβ is highly and specifically overexpressed in Ewing sarcomas. Its transcriptional activation is directly regulated by the EWS-FLI1 oncogene. This protein is responsible for the phosphorylation of histone H3T6, allowing global maintenance of H3K4 trimethylation on a variety of gene promoters. In the long term, PRKCβ RNA interference induces apoptosis in vitro. In xenograft mice models, complete impairment of tumour engraftment and even tumour regression were observed upon PRKCβ inhibition, highlighting PRKCβ as a most valuable therapeutic target for new therapeutic approaches in Ewing sarcoma.

New functional insights into EWS-FLI1, the main driver oncogene of Ewing Sarcoma
We found widespread EWS-FLI1 dependent reprogramming of the Ewing sarcoma epigenome resulting in super-activation of proliferation associated promoters, repression of widely active and differentiation associated enhancers, and de novo generation of Ewing sarcoma specific super-enhancers. The most dynamic EWS-FLI1 dependent epigenetic modification was histone H3K27 acetylation 2. We could also demonstrate that genome-wide methylation profiling allows prediction of enhancer activity and established inter- and intra-tumour variability along two dimensions, associated with the relative strength of a mesenchymal versus stem cell signature and with the strength of the EWS-FLI1 regulatory signature (Sheffield N et al., submitted). Consistent with the importance of epigenomic reprogramming in Ewing sarcoma, epigenetic drugs, predominantly inhibitors of histone deacetylases (HDACs) and drugs targeting apoptosis inhibitors were among the agents that scored most highly when interrogating a library of >3000 compounds for EWS-FLI1 dependent toxicity (Tsafou K et al., in prep). Finally, by a high-throughput druggable siRNA screen, we identified a reader of the repressive histone H3K27 tri-methylation mark, the leucine rich repeats and WD repeat domain containing 1 (LRWD1) protein as the most sensitive target discriminating viability of EWS-FLI1 high and low Ewing sarcoma cells (Iljin K et al., submitted). Among epigenetic readers, the polycomb proteins BMI-1 and EZH2 are highly overexpressed in Ewing sarcoma. Polycomb target gene expression in Ewing sarcoma deviates from normal tissues and stem cells and most targets are relatively repressed. However, our studies contributed to the discovery of a EWS-FLI1 dependent paradoxical up-regulation of numerous polycomb targets highly enriched for posterior homeobox group D (HOXD) genes due to a relative loss of polycomb-dependent histone H3K27 tri-methylation and gain of histone H3K4 tri-methylation at the promoters of posterior HOXD genes 4.

Central to the aberrant activation of proliferation genes by EWS-FLI1 we described a transcriptional module in which direct EWS-FLI1 binding to the promoter leads to the exchange of a repressive by an activating E2F transcription factor in a feed-forward loop. We reported an evolutionary conserved architecture of the E2F/ETS transcriptional module that we demonstrated to be aberrantly activated not only in EWS-FLI1 expressing Ewing sarcoma but also in ERG rearranged prostate cancer 1. Time-resolved measurements of gene expression were used to mathematically model this module and confirm a direct functional interaction between EWS-FLI1 and E2F3, which we had predicted by genome-binding studies of these transcription factors and mutational analysis of their binding sites 5. EWS-FLI1 had previously been reported to potentially associate with serum response factor (SRF) to drive the expression of growth factor induced gene sets. In healthy tissue, in response to signals delivered by RAS or RHO pathways, SRF either complexes with the ETS transcription factors ELK1 or SAP1, or with myocardin related transcription factors 1 or 2 (MRTF1/2) depending on the growth factor signal and the sequence context of SRF response elements. Integration of RNAseq and transcription factor binding studies by chromatin immune precipitation sequencing (ChIP-seq) revealed a significant overlap between EWS-FLI1 and MRTF2 binding in Ewing sarcoma cell lines. By single and combined knockdown of these transcription factors we obtained evidence that EWS-FLI1 hijacks MRTF2 target genes independent of SRF leading to growth factor independent aberrant activation (Katschnig A et al., in prep.). Motif analysis of genes bound by MRTF2 suggested that this mechanism affected predominantly target genes of the developmental JAP/TAZ signalling pathway. A further EWS-FLI1 dependent transcriptional module involves FOXO1 as a central player regulated by EWS-FLI1. We observed direct transcriptional suppression of FOXO1 expression as well as indirect regulation of FOXO1 subcellular localization via the direct EWS-FLI1/E2F regulated kinases CDK2 and AKT. We found FOXO1 to be responsible for a repressive sub-signature of the chimeric oncoprotein. We demonstrated that FOXO1 re-activation by selenic acid reduces Ewing tumour cell growth in vitro and in a xenograft mouse model 6.

A central role for the histone deacetylase SIRT1 in Ewing Sarcoma
In contrast to most adult cancers, the tumour suppressor TP53 is rarely mutated in Ewing sarcoma. Yet, basal TP53 expression and activity is indirectly modulated by EWS-FLI1 through