Published OnlineFirst April 22, 2014; DOI: 10.1158/1078-0432.

CCR-13-0633

Clinical
Cancer
CCR New Strategies Research

New Strategies in Ewing Sarcoma: Lost in Translation?

Fernanda I. Arnaldez and Lee J. Helman

Abstract
Ewing sarcoma is the second most common pediatric malignant bone tumor. Aggressive multimodality
therapy has led to an improvement in outcomes, particularly in patients with localized disease. However,
therapy-related toxicities are not trivial, and the prognosis for patients with relapsed and/or metastatic
disease continues to be poor. In this article, we outline some of the promising therapies that have the
potential to change the Ewing sarcoma therapeutic paradigm in the not-too-distant future: insulin-like
growth factor receptor inhibitors, targeting of the fusion protein, epigenetic manipulation, PARP inhibitors,
and immunotherapy. Clin Cancer Res; 20(12); 3050–6. !2014 AACR.

Background
Ewing sarcoma is the second most common pediatric
malignant bone tumor, and it can also present as a soft-
tissue malignancy. Approximately 225 new cases are diag-
nosed each year in the United States in patients between the
ages of 1 and 20 years (1). The presence of macrometastatic
disease continues to be the single most significant predictor
of poor clinical outcome (2): Patients with localized disease
treated with multimodality therapy can achieve a 5-year
event-free survival rate of 70% (3, 4), whereas the 5-year
overall survival rate of patients who present with overt bone
or bone marrow metastatic disease at diagnosis is less than
20% (1, 5, 6). Traditional therapeutic approaches include
local control of the primary lesion that involves surgery
and/or radiation therapy, and treatment of disseminated
disease with multiagent cytotoxic chemotherapy.
These approaches have led to significant improvements
in outcomes over the past decades, particularly in patients
with localized disease (7). However, novel therapeutic
approaches are clearly needed, not only to increase survival
in patients with relapsed or metastatic disease (2), but also
to continue to improve survival of patients with localized
disease as well as to decrease the acute and chronic toxicities
associated with current cytotoxic drugs.
The molecular hallmark of Ewing sarcoma is the trans-
location between EWS, an FET family protein, and an ETS
transcription factor (8). The FET family includes nuclear
proteins such as FUS, EWS, and TAF15, which are involved
in abnormal rearrangements with transcription factors (9).
In 85% of cases, the t(11;22)(q24;q12) translocation
between the EWSR1 and FLI1 is detected. However, many
Authors' Affiliation: Pediatric Oncology Branch, National Cancer Institute,
NIH, Bethesda, Maryland
Corresponding Author: Fernanda I. Arnaldez, National Cancer Institute,
10 Center Drive, Building 10CRC, Room 13816, Bethesda, MD 20892.
Phone: 301-451-7014; Fax: 301-451-7010; E-mail: arnaldezf@mail.nih.gov
doi: 10.1158/1078-0432.CCR-13-0633
!2014 American Association for Cancer Research.
other fusions have been described (10, 11). This fusion gives
origin to a chimeric transcription factor that is responsible
for the Ewing sarcoma oncogenic program. This aberrant
factor modifies the transcription machinery, activating or
quite often repressing transcription of target genes (12, 13).
Although the cell of origin has been much debated, growing
evidence suggests that Ewing sarcoma could possibly orig-
inate in mesenchymal stem cells (14).
Despite a growing body of knowledge about Ewing
sarcoma biology, the successful application of basic discov-
eries to the clinic has remained elusive. Preclinical results
have not always been predictive of clinical trial outcomes,
highlighting the need for better models with the capability
to identify targetable drivers of disease. Improved preclin-
ical models and application of innovative clinical trial
designs are needed, including the incorporation of combi-
natorial therapy in early-phase therapeutic development.
However, several recent contributions hold promise for the
future and are discussed below (Table 1).
On the Horizon
Targeting the EWS–Fli1 fusion protein
Efforts to suppress the oncogenicity of the fusion protein
can be described in two fronts: inhibition of the transcrip-
tion factor activity and inhibition of selected downstream
target genes (Fig. 1). The discovery of the EWSR1–Fli1 (EF)
fusion protein was reported in 1992 (8). This fusion protein
generated by the Ewing-specific t(11;22) translocation func-
tions as an aberrant transcription factor that leads to an
altered transcriptional profile of both upregulated and
downregulated transcripts (12, 13). Preclinical models sup-
port the potential of the EF fusion protein as a therapeutic
target in Ewing sarcoma. Synthetic RNAi targeting of the
fusion led to inhibition of tumor growth in vivo and in vitro.
In these models, RNAi knockdown was associated with
decreased expression of the EF fusion protein and down-
regulation of EF transcriptional targets such as c-Myc (15).
Inhibition or knockdown of EWS–Fli1 in vitro leads to
decreased cell viability in vitro (16). Concurrent adminis-
tration of rapamycin and antisense oligonucleotides

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New Strategies in Ewing Sarcoma

Table 1. Novel therapeutic approaches in Ewing sarcoma

Outcome
for phase
Strategy Agent Development II studies Refs.
Inhibition of EF RNAi Preclinical (14, 15, 17, 19)
YK-4-279
Inhibition of EF transcriptional Cytarabine Clinical (phase II) Negative (22)
signature Trabectedine Clinical (phase II) Negative (23, 24)
Mithramycin Clinical (phase I/II) Ongoing (26)
Midostaurin Preclinical (25)
Inhibition of tumor growth signaling Insulin growth factor receptor I blockade Clinical (phase II) Positive (34)
pathways (antibodies, kinase inhibitors)
Disruption of DNA repair PARP inhibitors Clinical (phase II) Negative (45)
Epigenetic deregulation HDAC inhibitors (vorinostat) Clinical (phase I) (53, 56)
Immune-based therapies Lexatumumab Clinical (phase I) (60)
NK cell–based therapies Clinical (phase I) (65, 66)
CAR Preclinical (67, 68)

Abbreviations: CAR, chimeric antigen receptor; HDAC, histone deacetylase.

showed delayed tumor growth in murine xenografts (17).
Although these results are encouraging, in vivo delivery of
RNAi has proved to be challenging. Using a synthetic
polymeric nanoparticle as a carrier, an RNAi directed
toward the EWS–Fli1 fusion is being studied in murine
xenografts. The strategy used to target tumor cells is to
couple the siRNA-carrying nanoparticles with a monoclo-
nal antibody against CD99, an antigen widely expressed in
the surface of Ewing sarcoma cells. Efficacy measures of this
creative approach will be of interest in the near future (18).
Thus, it has become clear that the EF fusion transcription
factor is necessary for tumor growth, and targeting the
mutant transcription factor itself or critical downstream
targets of this protein are attractive therapeutic strategies.
However, direct inhibition of transcription factors has
proved to be difficult. The fusion protein has been shown
to interact with RNA helicase A (RHA) using phage display
and chromatin immunoprecipitation techniques (19). Fol-
low-up studies showed that RHA stimulated the activity
of promoters regulated by EWS–Fli1. Using Plasmon reso-
nance screening, YK-4-279, an inhibitor of the EWS–Fli1–
RHA interaction has been identified. This compound was
associated with decreased tumor growth in orthotopic
xenografts, but has not progressed to clinical development
yet (20, 21).
The transcriptional signature of the EF fusion has been
described (12), and as noted above, another way to target
the EF-mutant transcription factor is to develop approaches
to alter the downstream targets. Creative approaches have
been used to screen compounds that are able to negatively
regulate the expression of EWS–FLi1 target genes. A func-
tional screen of a library of 1,040 compounds was per-
formed in search for an EWS–FLi1 "off’" signature, and
identified cytarabine as a negative modulator of transcrip-
tional activity (22). Unfortunately, cytarabine did not show
benefit in a phase II clinical trial and was associated with
significant hematologic toxicity (23). In a similar fashion,
although preclinical work showed that trabectedin could
interfere with the transcriptional activity of EWS–Fli1 (24),
only 1 patient with Ewing sarcoma achieved stable disease
(SD) in a recent phase II trial (25).
More recently, 1,280 compounds were functionally
screened for suppression of EWS–Fli1 activity. Midostaurin,
a multikinase inhibitor, was shown to modulate the expres-
sion of EWS–Fli1 target genes, and it was associated with
decreased tumor growth in xenografts (26). Although mid-
ostaurin is undergoing clinical evaluation for the treatment
of hematologic malignancies, no clinical data in solid
tumors are available at this time. Finally, a library of
50,000 compounds was functionally screened using a
reporter of EWS transcriptional activity (27), and mithra-
mycin, an antineoplastic antibiotic, was identified as the
leading hit. Interestingly, reports of Ewing sarcoma re-
sponses to this drug had been reported decades ago (28).
Mithramycin treatment led to decreased tumor growth in
xenograft models, as well as inhibition of the EWS–Fli1
signature. A phase I/II clinical trial evaluating the activity of
mithramycin in patients with Ewing sarcoma is ongoing.
Insulin-like growth factor receptor blockade
The relevance of insulin-like growth factor receptor
(IGF1R) signaling in Ewing sarcoma has been established.
The fusion protein positively regulates the expression of
IGF1R (29, 30), which has been shown to be necessary for
EWS–FLi1-mediated transformation of fibroblasts (31).
Ewing sarcoma cells are sensitive to IGF1R inhibition
in vitro and in vivo (32–35). A phase II trial of R1507, a
fully human IGF1R-blocking antibody, showed an overall

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Arnaldez and Helman
Insulin-like growth factors
IGF1R antibodies
IGF1R inhibitors Receptor
TKIs Signaling
Vorinostat
RNA interference
YK-4-279 EWS-FlI1
PARP inhibitors
Mithramycin
Target gene expression/repression
Ewing sarcoma cell growth and survival
© 2014 American Association for Cancer Research
complete response (CR)/partial response (PR) rate of 10%
(36). Similarly, the single-agent activities of cixutumumab
(IMC-A12) and figitumumab, different human antibodies
targeting the same receptor, were reported as 10% and 14%,
respectively, in patients with refractory Ewing sarcoma (37,
38). A major limitation of these studies was the inability to
identify strategies to select patients most likely to respond to
this therapeutic intervention.
Even in responding patients, the majority of responses are
short lived; thus, as in other targeted therapies, acquired
resistance to IGF1R blockade has also emerged as a major
problem in targeting the IGF1R in Ewing sarcoma, and it is
very likely that combination strategies will be needed in the
future to optimize the likelihood of sustained responses.
There is some emerging evidence of mTOR and ERK acti-
vation in patients who develop resistance to these therapies
(39). A recent trial combining cixutumumab with temsir-
oliumus reported that 35% patients had SD longer than 5
months or CR/PR (40). In addition, activation of MAPK and
insulin receptor (41) could be involved in resistance to
IGF1R inhibition.
Given that the activity of these IGF1R-blocking agents has
been established, it is imperative that we identify predictors
of response as well as mechanisms of acquired resistance so
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Immune clearance
Lexatumumab
NK cells
CAR
Figure 1. Current therapeutic
strategies in Ewing sarcoma.
These include growth factor
receptor blockade, intracellular
signal inhibition, epigenetic
modulation, immune clearance
enhancement, and manipulation of
the EWS–Fli1 transcriptional
signature.
that the most rational combinatorial therapies are used on
the properly selected patients.
PARP inhibitors
PARP inhibitors are an area of growing interest for the
Ewing sarcoma community. In 2012, a marked sensitivity of
Ewing sarcoma to olaparib, a PARP inhibitor (AZD2281),
was observed in a high-throughput screen of 639 cancer cell
lines aimed to detect drug sensitivity patterns as a function
of genomic features (42). In a different study, sensitivity of
Ewing sarcoma cells to olaparib was documented both in
vitro and in tumor xenografts (43). Ewing sarcoma cells were
more sensitive to PARP inhibition than prostate cancer cells
harboring the TMPRSS2-ERG translocation. Remarkably,
the combination of temozolamide and olaparib was syn-
ergistic in abrogating progression of Ewing sarcoma xeno-
grafts (43). An effect of the EWS–FLI1 fusion transcript in
the DNA damage response was suggested more than a
decade ago (44). Interestingly, a high expression of PARP
in Ewing sarcoma cells has been reported (45). However,
the exact role of PARP in Ewing sarcoma biology continues
to be an area of active research.
The enthusiasm about these results resulted in a phase II
clinical trial of olaparib in recurrent/metastatic Ewing
Clinical Cancer Research
 Published OnlineFirst April 22, 2014; DOI: 10.1158/1078-0432.CCR-13-0633
sarcoma following failure of prior chemotherapy. Unfortu-
nately, no CR/PR was seen with 4 of 12 patients achieving
SD at a maximum of 18.4 weeks with a median time to
progression of 5.7 weeks. Further accrual to this trial was
discontinued (46). Unfortunately, molecular diagnosis was
not a requisite for enrollment, and it is hard to speculate
about the biologic reasons for these results, which could be
related not only to lack of the FET–ETS translocation but
also to general lack of predictiveness of current preclinical
models as well as pharmacologic factors. However, it is
quite possible that other PARP inhibitors, or, combination
therapies, such as with temozolomide, might have a more
auspicious outcome.
Epigenetic therapies
Polycomb repressor genes. One of the known down-
stream targets of EWS–FLi1 is EZH2, which is the catalytic
subunit of the polycomb repressor gene 2 associated with
"stemness" features in tumor cells (47). Expression of EWS–
FLi1 leads to EZH2 upregulation in mesenchymal stem cells
(48) and expression of both EZH2 and BIM1 in human
neural crest cells, although BIM1 is not a direct transcrip-
tional target of EWS–FLI1 (49). These findings suggest a
rationale for the exploration of the use of the new EZH2
inhibitors in this tumor (50, 51).
Histone deacetylases. It has been shown that EWS–FLi1
has a transcriptional repressive function (13). One of the
downstream targets of the fusion protein that is required for
oncogenic transformation is NKX2.2 (52). This gene
encodes for a transcription factor with both activating and
repressing domains. NKX2.2 is thought to exert its transcrip-
tional repression via TLE (transducin-like enhancers of
split)–associated recruitment of histone deacetylases
(HDAC). TLE proteins are the homologues of Groucho in
humans. They are a family of proteins that act as transcrip-
tional modulators. Expression of individual TLE genes cor-
relates with immature epithelial cells that are progressing
toward their terminally differentiated state, suggesting a role
during epithelial differentiation (53). TLE proteins are
expressed in Ewing sarcoma and are believed to exert their
repressive function via recruitment of HDACs. This is a
possible mechanism that could explain preclinical activity
of HDAC inhibitors in these tumors (54). In vitro HDAC
inhibition using vorinostat in the Ewing sarcoma A673 cells
led to growth inhibition by abrogation of the transcription-
al-repressive function (54), which is consistent with findings
from prior studies in which Ewing sarcoma xenografts
showed sensitivity to HDAC inhibition (55). Moreover,
combination of 5-aza-20 -deoxycytidine, an inhibitor of
DNA methylation, and an HDAC inhibitor in vitro showed
reactivation of tumor suppressor genes and decreased clo-
nogenicity in vitro in Ewing sarcoma cell lines (56). Although
initial clinical trials of this approach have not shown
responses (57), this avenue has not been fully explored yet.
Immunotherapy
Immunotherapy should be considered as a valid approach
to Ewing sarcoma therapy. The recent developments in
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New Strategies in Ewing Sarcoma
cancer immunotherapy, particularly the positive results seen
after PD-1 blockade in solid tumors (58, 59), have renewed
enthusiasm about therapeutic manipulation of the immune
system with the aim of tumor eradication.
A trial of consolidative immunotherapy for high-risk
pediatric sarcomas, including Ewing sarcoma, using autol-
ogous T cells and dendritic cells pulsed with peptides
derived from tumor-specific translocation, was performed
at the NCI. This approach proved to be feasible and led to
31% 5-year overall survival (60).
TRAIL is a member of the TNF superfamily with antitu-
moral activity secreted primarily by natural killer (NK) cells.
Ewing sarcoma cells express the TRAIL death receptors, and
have been shown to be sensitive to TRAIL-induced caspase-
8–mediated apoptosis in vitro. Tumor progression using
xenografts and transgene TRAIL expression showed associ-
ation of ligand expression with delayed tumor progression
(61). In a recent phase I trial evaluating lexatumumab, a
fully human agonistic antibody against TRAIL receptor 2 in
which 4 patients with Ewing sarcoma were enrolled, the
agent was well tolerated but no CRs or PRs were observed
(62). Interestingly, there is potential for synergistic combi-
nation of immune-based therapies and HDAC inhibitors.
Ewing sarcoma cells treated with vorinostat had increased
sensitivity to TRAIL-induced apoptosis via increased acti-
vation of caspase 8 (63).
Preclinical studies have demonstrated sensitivity of
Ewing sarcoma cells to expanded NK cells in vitro and
in vivo (64). This is congruent with the previous find-
ings that NK cells are able to recognize and destroy
Ewing sarcoma cells by signaling through NKG2D and
DNAM-1 receptors (65). Clinical trials exploring the
feasibility of NK-based therapy with and without stem
cell transplantation in patients with high-risk sarcomas,
including Ewing sarcoma, are ongoing (clinicaltrials.gov:
NCT01287104 and NCT01875601).
Once again, HDAC inhibition has been linked with
increased expression of NKG2D ligands in Ewing sarcoma
cells that increased sensitivity to NK cell–mediated cytolysis
(66). Ligand upregulation has also been linked to DNA
damage—for instance using radiation—(67), all suggesting
that optimal combination or sequential therapies may
enhance this therapeutic approach.
Finally, chimeric antigen receptor (CAR)–based therapy
is currently being developed for Ewing sarcoma. Modified T
cells have shown promising results in hematologic malig-
nancies (68). Surface receptors expressed in Ewing sarcoma,
such as the ganglioside antigen GD2, are being actively
explored as potential targets for Ewing sarcoma CAR therapy
(69, 70).
In summary, several recent discoveries are opening a new
era of clinical investigation in Ewing sarcoma. Inhibition of
the aberrant fusion protein, PARP inhibition, epigenetic
manipulation, IGF1R blockade, and immune therapies all
hold the promise of achieving improved outcomes in
patients with Ewing sarcoma.
However, despite a sizable number of scientific discov-
eries in Ewing sarcoma biology, these findings have not led
Clin Cancer Res; 20(12) June 15, 2014 3053
 Published OnlineFirst April 22, 2014; DOI: 10.1158/1078-0432.CCR-13-0633
Arnaldez and Helman
to a significant improvement in outcomes, particularly in
patients presenting with metastatic disease. A recent Chil-
dren’s Oncology Group trial demonstrated benefit of inter-
val-compressed chemotherapy in patients with localized
disease (7), but as was the case for previous improvements
in the treatment of Ewing sarcoma, this approach had no
impact on the outcome of patients with advanced meta-
static disease. Although the reasons for this remain elusive,
some assessment of possible reasons for a disconnect
between these advances and clinical advances seems
warranted.
The lack of reproducibility of preclinical data, particularly
in oncology, has emerged as a growing concern. Reasons for
this could be multifactorial: technical difficulties, lack of
adequate models or even well-meaning experimental bias,
and error. However, it has been argued that the increasing
pressure in the current "publish or perish" culture, difficul-
ties in publishing negative results, and scarcity of funding
might have an impact in variable experimental results.
One result of this well-documented phenomenon
could be lack of preclinical rigor required before observa-
tions are tested in the clinic. For example, many pub-
lished articles using xenograft models demonstrate that
a novel intervention leads to a slowing of tumor growth,
but very few publications demonstrate actual tumor shrink-
age. Although tumor shrinkage per se may not predict clini-
cal efficacy, clearly better preclinical predictors are necessary.
In a previous report comparing activity of novel cytotoxic
agents in xenografts with activity in phase II studies, the best
predictor of clinical activity was when a compound had
activity in at least 33% of multiple histologies in multiple
xenograft models (71). As noted above, these data were
generated using cytotoxic agents, and currently we simply
do not know what best predicts for clinical activity with
newer "targeted" agents other than a specific activating muta-
tion in a kinase predicting for response to a specific kinase
inhibitor. Unfortunately, no such activating kinase muta-
tions have been found in Ewing sarcomas, reinforcing the
point that better predictors for clinical activity are necessary
for this tumor.
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Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: F.I. Arnaldez, L.J. Helman
Development of methodology: F.I. Arnaldez
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): L.J. Helman
Analysis and interpretation of data (e.g., statistical analysis, biosta-
tistics, computational analysis): L.J. Helman
Writing, review, and/or revision of the manuscript: F.I. Arnaldez,
L.J. Helman
Received August 8, 2013; revised April 4, 2014; accepted April 13, 2014;
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 Published OnlineFirst April 22, 2014; DOI: 10.1158/1078-0432.CCR-13-0633

New Strategies in Ewing Sarcoma: Lost in Translation?

Fernanda I. Arnaldez and Lee J. Helman

Clin Cancer Res 2014;20:3050-3056. Published OnlineFirst April 22, 2014.

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