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holding chromosome pairs together during cell division, and in the context of cancer also affects genome or-
[77]
ganization and transcriptional regulation . STAG2 is functionally redundant with STAG1, and depletion of
STAG1 resulted in lethality in EWS cells with mutated STAG2, but not in cells with wildtype STAG2, making
[78]
it an interesting target for therapeutics .
Epigenetics
Despite the quite homogenous genetic make-up of EWS, disease course and presentation varies significantly
between patients. This may be attributed to a heterogeneity in the epigenetic profile of EWS tumours. A re-
cent study characterized epigenetic heterogeneity of EWS compared to other tumour types, between differ-
[79]
ent EWS cases, and within individual tumours . Based on genome-wide methylation profiles, EWS can be
separated from other cancer types and normal tissue. Between patients, no epigenetically defines subtypes
are found: samples fall along a mid-to-high range of variation for methylation. Intra-tumour heterogeneity
was is also high and varying.

The epigenetic profile of EWS has been directly linked to the activity of EWSR1-FLI1 [24,80] . EWSR1-FLI1 pref-
erentially binds to sites with GGAA repeats, and classical ETS binding sites. At GGAA repeat sites, EWSR1-
FLI1 acts as an enhancer and causes opening of the chromatin, while at ETS sites it displaces ETS transcrip-
tion factors, deactivating their enhancer activity.

Efforts are being made to determine epigenetic changes specific to EWS, and the consequences they may
have on disease development and progression [81-83] . The methylation of NPTX2 or PHF11 has been linked to
[82]
a less favourable prognosis . Another study showed that hypermethylation of PTRF leads to suppression of
[83]
this tumour suppressor gene, and that reintroduction could lead to p53 activation and subsequent apoptosis .

MODEL SYSTEMS
The relatively simple genetic make-up of EWS makes it an appealing tumour for which to develop a
transgenic animal model. However, despite many attempts made, no research group as of yet has been
[33]
successful . Introduction of the EWS oncogene EWSR1-FLI1 in mice has led to embryonic lethality, or
development of non-EWS-resembling cancer [84,85] . For a comprehensive overview of the efforts made by
six independent laboratories to generate a murine model for EWS, we refer to the 2016 publication by
[33]
Minas et al. . In addition to murine models, development of zebrafish transgenic models for EWS has also
been attempted. The transient expression of EWSR1-FLI1 in zebrafish embryos led to mitotic defect in the
developing embryo . A stable transgenic zebrafish line with expression of EWSR1-FLI1 developed solid
[86]
-/-
tumours with histologic features of small round blue cell tumours in a TP53 background, but the model
simultaneously gave rise to malignant peripheral nerve sheet tumours. Another zebrafish model showed that
loss of one or both wildtype alleles of the zebrafish EWSR1 orthologue ewsa led to an increase in tumour
-/-
formation in a TP53 background, suggesting that EWSR1 deficiency may also contribute to the malignancy
[87]
of EWSR1-translocation driven tumours . Given the lack of transgenic animal models, the EWS research
community looks for different models in which to test potential novel therapeutics for the disease.

In vitro models
In order to test novel therapeutics, classic 2D culture is widely used. Though rapid and convenient, the con-
tribution of the tumour microenvironment cannot be assessed in these assays, while this could alter drug
response significantly. Cells undergo biomechanical stimulation from shear stress due to blood flow, or
mechanical forces from surrounding muscle contractility or body impact, and alter their behaviour in re-
sponse [88,89] . EWS cells are no different, and several models have been reported taking this into account [90-92] .
Shear stress was applied to EWS cells by growing them on 3D scaffolds within a flow perfusion bioreac-
tor. This resulted in an increased production of IGF1, and a shear-stress dependant alteration in response
to IGF1-R inhibitors . Continued development of this model involves the addition of mesenchymal stem
[90]