Oncogenic fusion proteins & epigenome: partners in crime?

Significance: Cancer is one of the leading causes of death among children in the developed world. Advances in treatment have raised survival rates to 80% for many paediatric cancers, but certain rare tumours including Ewing sarcoma continue to be associated with poor survival. The discovery of epigenetic aberrations in paediatric cancers (such as changes in DNA methylation and histone modification patterns) suggests a promising route toward new therapies. Epigenetic aberrations can be reversed by an emerging class of epigenetic drugs, which are already used for cancer treatment in the elderly. The prospect that epigenetic events may broadly contribute to the development of paediatric tumours opens up exciting opportunities for epigenetic cancer therapy in children. Goal: The project will study epigenetic aberrations with relevance to Ewing sarcoma, a rare paediatric tumour of the bone that lacks satisfactory treatment strategies. Ewing sarcoma is driven by the oncogenic fusion protein EWS/FLI1, thus providing a powerful model for dissecting the interplay of fusion proteins and epigenetic regulation on a genome-wide scale. Innovation: An in vivo mouse transplantation assay will be used to investigate the regulatory networks coordinated by EWS/FLI1. This approach provides a major advance over prior research, which has been restricted to in vitro cancer cell lines. The in vivo assay will provide insights into the chain of regulatory events that lead to carcinogenesis, and it will establish an assay for pre-clinical testing of epigenetic drugs. Approach: Aim 1. Development of a flexible and tractable assay for EWS/FLI1-induced transformation Mesenchymal stem cells (MSCs) are considered the tissue of origin of Ewing sarcoma. An in vivo transplantation assay will be established based on EWS/FLI1-expressing MSCs, which induces transformation and carcinogenesis. This model will be complemented by in vitro assays using both mouse and human MSCs. Aim 2. Identification of epigenetic states and events associated with EWS/FLI1-induced transformation Epigenome mapping of EWS/FLI1-induced tumours will be combined with gene expression and EWS/FLI-binding analysis. Integrated bioinformatic analysis of the resulting dataset will identify candidate epigenetic events relevant for carcinogenesis. Aim 3. Functional characterisation of epigenetic events contributing to EWS/FLI1-induced transformation Potentially causal epigenetic events will be functionally characterized by knock-down and over-expression of epigenetic regulators in vitro. The clinical relevance of these events will be confirmed by epigenetic analysis of human tumour samples and by epigenetic drug treatment of the in vivo mouse model. Alignment with Funding Strategy: The proposed project addresses all three aims of the Lise Meitner program: (i). it enables a scientist trained in world-class labs to pursue a research project at one of Austrias leading cancer research institutes, while exploiting local expertise in genomic and mouse research through collaborations; (ii). it introduces a new scientific field to Austria; and (iii). it establishes a formal collaboration between an Austrian institute and a leading research institute abroad.

Pediatric cancers have a low rate of somatic mutations compared to most adult cancers, suggesting that mechanisms other than the gradual accumulation of genetic alterations contribute to their etiology. I proposed to study epigenetic aberrations that could be relevant to Ewing sarcoma, a rare pediatric tumor of the bone lacking satisfactory treatment strategies (overall 60% long term survival). The term epigenetics refers to mechanisms of gene regulation that are not directly encoded in the DNA sequence. Epigenetic marks, such as DNA methylation and histone modifications, control the packaging of the DNA in the nucleus and regulate gene expression in a way that is faithfully propagated over multiple cell divisions. The oncogenic fusion protein EWS-FLI1 is the master regulator of Ewing sarcoma and can be used as a model system for the identification of key epigenetic events crucial for tumor progression. I have established the first comprehensive reference epigenome of a pediatric solid cancer driven by an oncogenic fusion protein. I found that EWS-FLI1 dynamically interacts with the epigenome and identified the chromatin states that are regulated by EWS-FLI1. In summary, the data generated contribute to our understanding of synergy between genetic and epigenetic mechanisms in a fusion protein driven cancer, and they underline the potential value of epigenetic drugs for Ewing sarcoma and other pediatric solid cancers.