An epigenetic approach for Ewing sarcoma patients

Abstract

[eng] CHAPTER 1:

Ewing sarcoma (EwS) is the most common malignant neoplasm in bone and soft tissues among adolescents and young adults in Spain, characterized by a chromosomal translocation between an ETS factor and a member of the FET family of genes. In the 85% of cases, it gives rise to EWSR1-FLI1 fusion protein, which acts as an aberrant transcription factor affecting gene expression (Delattre et al., 1992; Sankar et al., 2013). Although EWSR1-FLI1 is essential for tumorigenesis, it is not solely sufficient for malignant transformation, highlighting the importance of cell specificity and developmental epigenome in its activity (Crompton et al., 2014). EWSR1-FLI1 is capable to induce chromatin opening and create de novo enhancers that interact with its target gene promoters, creating specific enhancer and super-enhancer signatures that are dependent on the oncogene expression ( (Riggi et al., 2014; Tomazou et al., 2015). Enhancers are cis- regulatory elements that drive cell-type-specific gene expression, activating transcription of their target genes at great distances in the genome (Calo & Wysocka, 2013). Although traditionally repressive, RING1B collaborates with transcription activation at super-enhancers in cancers such as melanoma and breast cancer. Results from our lab demonstrate that RING1B co-localizes with EWSR1-FLI1 at active enhancers, regulating genes critical for EwS tumorigenesis like NKX2-2, SOX-2, and IGF-1 (Sánchez-Molina et al., 2020). Despite the important role of RING1B in the activation of oncogenic enhancers, specific inhibitors have been only described for its repressive E3 ubiquitin ligase activity. Thus, we hypothesize that inhibition of AURKB may represent a novel and specific targeted therapy for RING1B modulation, whose activating role at enhancers we have already demonstrated as necessary for EwS tumorigenesis. As single-agent targeted therapies are rarely curative, and AZD-1152 showed toxicity in previous clinical trials, we propose that combining AURKB inhibitors with drugs already proven effective in Ewing sarcoma, such as enhancer disruptors, could enable the use of lower, more effective doses while minimizing side effects. To elaborate on this hypothesis, we will address three aims:

• Aim 1: To describe genome wide the distribution of AURKB and its colocalization with RING1B and EWSR1-FLI1 at active enhancers.

• Aim 2: To elucidate the mechanisms behind the regulation of RING1B and EWSR1-FLI1 at enhancers by AURKB.

• Aim 3: To evaluate the synergistic combination of AZD-1152 and different enhancer disruptors in vitro and in vivo.

CHAPTER 2:

The GEIS-21 conducted in our institution confirmed in a multivariate analysis the importance of age (≥18 years cutoff) as an independent predictor of worse outcome, superior to the detection of metastasis (Mora et al., 2017). In the epigenetic context of EwS, characterizing chromatin modifications is crucial to further elucidate the tumorigenesis process of the disease. Using as putative cell of origin mesenchymal stem cells isolated from bone marrow of different age donors (grouped below or above 18 years) we aim to understand how the epigenomic background affects EwS development. We hypothesize that the epigenetic changes produced in mesenchymal stem cells coming from different age donors (adult or pediatric) will be different enough to help us understand the pathogenetic mechanisms underlying age (18 years as cut-off) as prognostic biomarker. However, there is no reported comparison between chromatin states generated by the oncogene in different types of mesenchymal cells. Furthermore, age has not been correlated with the epigenetic background to explain its prognostic significance. • Aim 1: To stablish different hMSC models from younger (pediatric) or older (adult) than 18 years olds including healthy donors (hpMSC-HD or hMSC-HD) and EwS patients (hpMSC-P or hMSC-P). • Aim 2: Using ChIP-seq technology, analyze chromatin marks for active enhancers in samples generated from different bone marrow derived hMSC (HD or P).