Transcriptional Programs Define Intratumoral Heterogeneity of Ewing Sarcoma at Single-Cell Resolution

Sakina Zaïdi,Olivier Saulnier,Aziz Fouché,Valentina Boeva,Sylvain Baulande,Virginie Raynal,Didier Surdez,Olivier Mirabeau,Franck Tirode,Ulykbek Kairov,Jean-Philippe Vert,Olivier Delattre,Sandrine Grossetête,Svetlana Gribkova,Thomas G.P Grünewald,Marie-Ming Aynaud,Simon Durand,Emmanuel Barillot,Mylène Bohec ,Isabelle Janoueix‐Lerosey ,Nadège Gruel ,Andreï Zinovyev

doi:10.1016/j.celrep.2020.01.049

Abstract

EWSR1-FLI1, the chimeric oncogene specific for Ewing sarcoma (EwS), induces a cascade of signaling events leading to cell transformation. However, it remains elusive how genetically homogeneous EwS cells can drive the heterogeneity of transcriptional programs. Here, we combine independent component analysis of single-cell RNA sequencing data from diverse cell types and model systems with time-resolved mapping of EWSR1-FLI1 binding sites and of open chromatin regions to characterize dynamic cellular processes associated with EWSR1-FLI1 activity. We thus define an exquisitely specific and direct enhancer-driven EWSR1-FLI1 program. In EwS tumors, cell proliferation and strong oxidative phosphorylation metabolism are associated with a well-defined range of EWSR1-FLI1 activity. In contrast, a subpopulation of cells from below and above the intermediary EWSR1-FLI1 activity is characterized by increased hypoxia. Overall, our study reveals sources of intratumoral heterogeneity within EwS tumors.

Highlights

Ewing sarcoma (EwS) is a highly aggressive pediatric bone cancer that is defined by a pathognomonic recurrent somatic mutation: a fusion between the EWSR1 gene and an ETS family member, most frequently the FLI1 gene (Delattre et al, 1992; Gru€newald et al, 2018)
Experimental Design for Collecting EwS Single-Cell RNA Sequencing Time-Resolved Profiles In order to explore the dynamics of individual cell transcriptomes under EWSR1-FLI1 expression, we used the previously developed A673/TR/shEF inducible cellular model derived from the A673 EwS cell line, where the expression of EWSR1FLI1 can be modulated through doxycycline-controlled short hairpin RNA (Carrillo et al, 2007)
We tested in vivo the impact of EWSR1-FLI1 on gene expression

Summary

Introduction

Ewing sarcoma (EwS) is a highly aggressive pediatric bone cancer that is defined by a pathognomonic recurrent somatic mutation: a fusion between the EWSR1 gene and an ETS family member, most frequently the FLI1 gene (Delattre et al, 1992; Gru€newald et al, 2018). This leads to the expression of EWSR1-FLI1, an aberrant and potent chimeric transcription factor. EWSR1-FLI1 can act both as a transcriptional activator and as a repressor, depending on the sequences of DNA binding sites and on the presence of additional co-factors (Bilke et al, 2013; Riggi et al, 2014). Through binding to these sites, EWSR1-FLI1 has been reported to act directly or indirectly on many key cellular processes including the cell cycle, apoptosis, angiogenesis, metabolism, and cell migration (Gru€newald et al, 2018)

Methods

Results

Discussion

Conclusion