Chimeric EWSR1-FLI1 regulates the Ewing sarcoma susceptibility gene EGR2 via a GGAA microsatellite

Abstract

Deciphering the ways in which somatic mutations and germline susceptibility variants cooperate to promote cancer is challenging. Ewing sarcoma is characterized by fusions between EWSR1 and members of the ETS gene family, usually EWSR1-FLI1, leading to the generation of oncogenic transcription factors that bind DNA at GGAA motifs1–3. A recent genome-wide association study4 identified susceptibility variants near EGR2. Here we found that EGR2 knockdown inhibited proliferation, clonogenicity and spheroidal growth in vitro and induced regression of Ewing sarcoma xenografts. Targeted germline deep sequencing of the EGR2 locus in affected subjects and controls revealed 291 Ewing-associated SNPs. At rs79965208, the A risk allele connected adjacent GGAA repeats by converting an interspaced GGAT motif into a GGAA motif, thereby increasing the number of consecutive GGAA motifs and thus the EWSR1-FLI1–dependent enhancer activity of this sequence, with epigenetic characteristics of an active regulatory element. EWSR1-FLI1 preferentially bound to the A risk allele, which increased global and allele-specific EGR2 expression. Collectively, our findings establish cooperation between a dominant oncogene and a susceptibility variant that regulates a major driver of Ewing sarcomagenesis.

Widespread Chromatin Accessibility at Repetitive Elements Links Stem Cells with Human Cancer

“…The absence of accessible chromatin at repetitive elements in differentiated cell types may restrict EWSR1-FLI1 targeting offering an explanation of why this oncogene fails to broadly transform cells (Owen and Lessnick, 2006). Simple repeats, when bound by EWSR1-FLI1, gain enhancer activity to regulate the transcription of multiple genes known to be important for Ewing Sarcoma (Gangwal et al, 2008; Grunewald et al, 2015; Kinsey et al, 2006; Luo et al, 2009; Patel et al, 2012; Smith et al, 2006). Further, germline variation in repetitive element composition has recently been associated with disease risk (Beck et al, 2012; Grunewald et al, 2015).…”

“…Simple repeats, when bound by EWSR1-FLI1, gain enhancer activity to regulate the transcription of multiple genes known to be important for Ewing Sarcoma (Gangwal et al, 2008; Grunewald et al, 2015; Kinsey et al, 2006; Luo et al, 2009; Patel et al, 2012; Smith et al, 2006). Further, germline variation in repetitive element composition has recently been associated with disease risk (Beck et al, 2012; Grunewald et al, 2015). The differences in location and composition of these repetitive regions relative to critical genes across species may partially explain the challenge in generating an animal model that faithfully recapitulates features of Ewing sarcoma (Lin et al, 2008).…”

“…At these sites EWSR1-FLI1 is necessary to maintain a fully accessible chromatin landscape marked by enhancer associated histone modifications (Patel et al, 2012; Riggi et al, 2014). Many of the genes implicated in tumor development and regulated by EWSR1-FLI1 are located proximally to these microsatellite repeats (Grunewald et al, 2015; Kinsey et al, 2006; Luo et al, 2009). Despite its chromatin remodeling activity, EWSR1-FLI1 only demonstrates cancer-like targeting in Ewing sarcoma cells.…”

Abundant contribution of short tandem repeats to gene expression variation in humans

“…Several candidate-gene studies in human indeed reported that STR variations modulate gene expression 19,33–37 and alternative splicing 23,38,39 . In one example, a recent study found that that underlying mechanism behind a GWAS signal for Ewing Sarcoma is a sequence variant in an AAGG repeat that increases the binding of the EWSR1-FLI1 oncoprotein resulting in EGF2 overexpression 40 . Despite these accumulating lines of evidence, there has been no systematic evaluation of the contribution of STRs to gene expression in humans.…”

Molecular genetics of Ewing sarcoma, model systems and finding novel (immuno-) therapeutic targets

“…A recent GWAS identified 3 EWS-associated single nucleotide polymorphisms (SNPs), near EGR2, BMF and TARDBP genes [34] . At the locus near EGR2, the causative SNP links two GGAA repeat stretches [35] . In addition to classic ETS transcription factor binding sites, EWSR1-FLI1 preferentially binds to such stretches of GGAA repeats, where its activity increases with the increase of amount of repeats [36,37] .…”