New Strategies in Ewing Sarcoma: Lost in Translation?

Published OnlineFirst April 22, 2014; DOI: 10.1158/1078-0432.CCR-13-0633

Clinical Cancer CCR New Strategies Research

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); 1–7. Ó2014 AACR.

Background other fusions have been described (10, 11). This fusion gives Ewing sarcoma is the second most common pediatric origin to a chimeric transcription factor that is responsible malignant bone tumor, and it can also present as a soft- for the Ewing sarcoma oncogenic program. This aberrant tissue malignancy. Approximately 225 new cases are diag- factor modifies the transcription machinery, activating or nosed each year in the United States in patients between the quite often repressing transcription of target genes (12, 13). ages of 1 and 20 years (1). The presence of macrometastatic Although the cell of origin has been much debated, growing disease continues to be the single most significant predictor evidence suggests that Ewing sarcoma could possibly orig- of poor clinical outcome (2): Patients with localized disease inate in mesenchymal stem cells (14). treated with multimodality therapy can achieve a 5-year Despite a growing body of knowledge about Ewing event-free survival rate of 70% (3, 4), whereas the 5-year sarcoma biology, the successful application of basic discov- overall survival rate of patients who present with overt bone eries to the clinic has remained elusive. Preclinical results or bone marrow metastatic disease at diagnosis is less than have not always been predictive of clinical trial outcomes, 20% (1, 5, 6). Traditional therapeutic approaches include highlighting the need for better models with the capability local control of the primary lesion that involves surgery to identify targetable drivers of disease. Improved preclin- and/or radiation therapy, and treatment of disseminated ical models and application of innovative clinical trial disease with multiagent cytotoxic chemotherapy. designs are needed, including the incorporation of combi- These approaches have led to significant improvements natorial therapy in early-phase therapeutic development. in outcomes over the past decades, particularly in patients However, several recent contributions hold promise for the with localized disease (7). However, novel therapeutic future and are discussed below (Table 1). approaches are clearly needed, not only to increase survival in patients with relapsed or metastatic disease (2), but also On the Horizon to continue to improve survival of patients with localized Targeting the EWS–Fli1 fusion protein disease as well as to decrease the acute and chronic toxicities Efforts to suppress the oncogenicity of the fusion protein associated with current cytotoxic drugs. can be described in two fronts: inhibition of the transcrip- The molecular hallmark of Ewing sarcoma is the trans- tion factor activity and inhibition of selected downstream location between EWS, an FET family protein, and an ETS target genes (Fig. 1). The discovery of the EWSR1–Fli1 (EF) transcription factor (8). The FET family includes nuclear fusion protein was reported in 1992 (8). This fusion protein proteins such as FUS, EWS, and TAF15, which are involved generated by the Ewing-specific t(11;22) translocation func- in abnormal rearrangements with transcription factors (9). tions as an aberrant transcription factor that leads to an In 85% of cases, the t(11;22)(q24;q12) translocation altered transcriptional profile of both upregulated and between the EWSR1 and FLI1 is detected. However, many downregulated transcripts (12, 13). Preclinical models support the potential of the EF fusion protein as a therapeutic target in Ewing sarcoma. Synthetic RNAi targeting of the in vivo in vitro Authors' Affiliation: Pediatric Oncology Branch, National Cancer Institute, fusion led to inhibition of tumor growth and . NIH, Bethesda, Maryland In these models, RNAi knockdown was associated with Corresponding Author: Fernanda I. Arnaldez, National Cancer Institute, decreased expression of the EF fusion protein and down- 10 Center Drive, Bldg 10CRC Room 13816, Bethesda, MD 20892. Phone: regulation of EF transcriptional targets such as c-Myc (15). 301-451-7014; Fax: 301-451-7010; E-mail: [email protected] Inhibition or knockdown of EWS–Fli1 in vitro leads to doi: 10.1158/1078-0432.CCR-13-0633 decreased cell viability in vitro (16). Concurrent adminis- Ó2014 American Association for Cancer Research. tration of rapamycin and antisense oligonucleotides

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

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Insulin-like growth factors Immune clearance IGF1R antibodies IGF1R inhibitors Receptor

TKIs Signaling Lexatumumab NK cells CAR Vorinostat

Figure 1. Current therapeutic RNA interference EWS-FlI1 strategies in Ewing sarcoma. YK-4-279 These include growth factor receptor blockade, intracellular signal inhibition, epigenetic modulation, immune clearance PARP inhibitors enhancement, and manipulation of Mithramycin the EWS–Fli1 transcriptional signature.

Target gene expression/repression

Ewing sarcoma cell growth and survival

complete response (CR)/partial response (PR) rate of 10% that the most rational combinatorial therapies are used on (36). Similarly, the single-agent activities of cixutumumab the properly selected patients. (IMC-A12) and ﬁgitumumab, different human antibodies targeting the same receptor, were reported as 10% and 14%, PARP inhibitors respectively, in patients with refractory Ewing sarcoma (37, PARP inhibitors are an area of growing interest for the 38). A major limitation of these studies was the inability to Ewing sarcoma community. In 2012, a marked sensitivity of identify strategies to select patients most likely to respond to Ewing sarcoma to olaparib, a PARP inhibitor (AZD2281), this therapeutic intervention. was observed in a high-throughput screen of 639 cancer cell Even in responding patients, the majority of responses are lines aimed to detect drug sensitivity patterns as a function short lived; thus, as in other targeted therapies, acquired of genomic features (42). In a different study, sensitivity of resistance to IGF1R blockade has also emerged as a major Ewing sarcoma cells to olaparib was documented both in problem in targeting the IGF1R in Ewing sarcoma, and it is vitro and in tumor xenografts (43). Ewing sarcoma cells were very likely that combination strategies will be needed in the more sensitive to PARP inhibition than prostate cancer cells future to optimize the likelihood of sustained responses. harboring the TMPRSS2-ERG translocation. Remarkably, There is some emerging evidence of mTOR and ERK acti- the combination of temozolamide and olaparib was syn- vation in patients who develop resistance to these therapies ergistic in abrogating progression of Ewing sarcoma xeno- (39). A recent trial combining cixutumumab with temsir- grafts (43). An effect of the EWS–FLI1 fusion transcript in oliumus reported that 35% patients had SD longer than 5 the DNA damage response was suggested more than a months or CR/PR (40). In addition, activation of MAPK and decade ago (44). Interestingly, a high expression of PARP insulin receptor (41) could be involved in resistance to in Ewing sarcoma cells has been reported (45). However, IGF1R inhibition. the exact role of PARP in Ewing sarcoma biology continues Given that the activity of these IGF1R-blocking agents has to be an area of active research. been established, it is imperative that we identify predictors The enthusiasm about these results resulted in a phase II of response as well as mechanisms of acquired resistance so clinical trial of olaparib in recurrent/metastatic Ewing

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sarcoma following failure of prior chemotherapy. Unfortu- cancer immunotherapy, particularly the positive results seen nately, no CR/PR was seen with 4 of 12 patients achieving after PD-1 blockade in solid tumors (58, 59), have renewed SD at a maximum of 18.4 weeks with a median time to enthusiasm about therapeutic manipulation of the immune progression of 5.7 weeks. Further accrual to this trial was system with the aim of tumor eradication. discontinued (46). Unfortunately, molecular diagnosis was A trial of consolidative immunotherapy for high-risk not a requisite for enrollment, and it is hard to speculate pediatric sarcomas, including Ewing sarcoma, using autol- about the biologic reasons for these results, which could be ogous T cells and dendritic cells pulsed with peptides related not only to lack of the FET–ETS translocation but derived from tumor-specific translocation, was performed also to general lack of predictiveness of current preclinical at the NCI. This approach proved to be feasible and led to models as well as pharmacologic factors. However, it is 31% 5-year overall survival (60). quite possible that other PARP inhibitors, or, combination TRAIL is a member of the TNF superfamily with antitu- therapies, such as with temozolomide, might have a more moral activity secreted primarily by natural killer (NK) cells. auspicious outcome. Ewing sarcoma cells express the TRAIL death receptors, and have been shown to be sensitive to TRAIL-induced caspase- Epigenetic therapies 8–mediated apoptosis in vitro. Tumor progression using Polycomb repressor genes. One of the known down- xenografts and transgene TRAIL expression showed associ- stream targets of EWS–FLi1 is EZH2, which is the catalytic ation of ligand expression with delayed tumor progression subunit of the polycomb repressor gene 2 associated with (61). In a recent phase I trial evaluating lexatumumab, a "stemness" features in tumor cells (47). Expression of EWS– fully human agonistic antibody against TRAIL receptor 2 in FLi1 leads to EZH2 upregulation in mesenchymal stem cells which 4 patients with Ewing sarcoma were enrolled, the (48) and expression of both EZH2 and BIM1 in human agent was well tolerated but no CRs or PRs were observed neural crest cells, although BIM1 is not a direct transcrip- (62). Interestingly, there is potential for synergistic combi- tional target of EWS–FLI1 (49). These findings suggest a nation of immune-based therapies and HDAC inhibitors. rationale for the exploration of the use of the new EZH2 Ewing sarcoma cells treated with vorinostat had increased inhibitors in this tumor (50, 51). sensitivity to TRAIL-induced apoptosis via increased acti- Histone deacetylases. It has been shown that EWS–FLi1 vation of caspase 8 (63). has a transcriptional repressive function (13). One of the Preclinical studies have demonstrated sensitivity of downstream targets of the fusion protein that is required for Ewing sarcoma cells to expanded NK cells in vitro and in oncogenic transformation is NKX2.2 (52). This gene vivo (64). This is congruent with the previous findings that encodes for a transcription factor with both activating and NK cells are able to recognize and destroy Ewing sarcoma repressing domains. NKX2.2 is thought to exert its transcrip- cells by signaling through NKG2D and DNAM-1 receptors tional repression via TLE (transducin-like enhancers of (65). Clinical trials exploring the feasibility of NK-based split)–associated recruitment of histone deacetylases therapy with and without stem cell transplantation in (HDAC). TLE proteins are the homologues of Groucho in patients with high-risk sarcomas, including Ewing sarcoma, humans. They are a family of proteins that act as transcrip- are ongoing (clinicaltrials.gov: NCT01287104 and tional modulators. Expression of individual TLE genes cor- NCT01875601). relates with immature epithelial cells that are progressing Once again, HDAC inhibition has been linked with toward their terminally differentiated state, suggesting a role increased expression of NKG2D ligands in Ewing sarcoma during epithelial differentiation (53). TLE proteins are cells that increased sensitivity to NK cell–mediated cytolysis expressed in Ewing sarcoma and are believed to exert their (66). Ligand upregulation has also been linked to DNA repressive function via recruitment of HDACs. This is a damage—for instance using radiation—(67), all suggesting possible mechanism that could explain preclinical activity that optimal combination or sequential therapies may of HDAC inhibitors in these tumors (54). In vitro HDAC enhance this therapeutic approach. inhibition using vorinostat in the Ewing sarcoma A673 cells Finally, chimeric antigen receptor (CAR)–based therapy led to growth inhibition by abrogation of the transcription- is currently being developed for Ewing sarcoma. Modified T al-repressivefunction (54), which is consistentwith findings cells have shown promising results in hematologic malig- from prior studies in which Ewing sarcoma xenografts nancies (68). Surface receptors expressed in Ewing sarcoma, showed sensitivity to HDAC inhibition (55). Moreover, such as the ganglioside antigen GD2, are being actively combination of 5-aza-20-deoxycytidine, an inhibitor of explored as potential targets for Ewing sarcoma CAR therapy DNA methylation, and an HDAC inhibitor in vitro showed (69, 70). reactivation of tumor suppressor genes and decreased clo- In summary, several recent discoveries are opening a new nogenicity in vitro in Ewing sarcoma cell lines (56). Although era of clinical investigation in Ewing sarcoma. Inhibition of initial clinical trials of this approach have not shown the aberrant fusion protein, PARP inhibition, epigenetic responses (57), this avenue has not been fully explored yet. manipulation, IGF1R blockade, and immune therapies all hold the promise of achieving improved outcomes in Immunotherapy patients with Ewing sarcoma. Immunotherapyshouldbeconsideredasavalidapproach However, despite a sizable number of scientific discov- to Ewing sarcoma therapy. The recent developments in eries in Ewing sarcoma biology, these findings have not led

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to a significant improvement in outcomes, particularly in Another issue that may be impeding progress in this rare, patients presenting with metastatic disease. A recent Chil- highly aggressive sarcoma is the paucity of preclinical and dren’s Oncology Group trial demonstrated benefit of inter- clinical models testing rational combinations of signaling val-compressed chemotherapy in patients with localized pathway–targeted agents. With the limited number of disease (7), but as was the case for previous improvements patients available for phase II testing, we may be better in the treatment of Ewing sarcoma, this approach had no served by early testing of combination-targeted agents based impact on the outcome of patients with advanced meta- upon scientific rationale, rather than insisting upon single- static disease. Although the reasons for this remain elusive, agent activity in the phase II setting. some assessment of possible reasons for a disconnect Finally, inadequate correlative studies early on in the between these advances and clinical advances seems development of agents for Ewing sarcoma have markedly warranted. hindered progress. Although tissue acquisition in pediatric The lack of reproducibility of preclinical data, particularly oncology continues to be an area of heated debate, one in oncology, has emerged as a growing concern. Reasons for could argue the dubious ethics of enrolling patients in a trial this could be multifactorial: technical difficulties, lack of in which key biologic questions will remain unanswered. adequate models or even well-meaning experimental bias, In summary, although much biologic insight has been and error. However, it has been argued that the increasing gained in understanding Ewing sarcoma, we must work pressure in the current "publish or perish" culture, difficul- harder to ensure these gains are translated to the clinic. ties in publishing negative results, and scarcity of funding Combination of different approaches in a rational and might have an impact in variable experimental results. creative manner continues to be a challenge for the future. One result of this well-documented phenomenon could To overcome this hurdle, it will be necessary to foster be lack of preclinical rigor required before observations collaboration between very distinct investigative app- are tested in the clinic. For example, many published articles roaches and to appeal to innovative clinical trial designs. using xenograft models demonstrate that a novel inter- If the recent and exciting biologic discoveries can be trans- vention leads to a slowing of tumor growth, but very few lated into effective therapies able to optimize outcomes publications demonstrate actual tumor shrinkage. while minimizing toxicities, we will be able to convey Although tumor shrinkage per se may not predict clinical renewed optimism to patients affected by this deadly tumor efficacy, clearly better preclinical predictors are necessary. In who are in desperate need of new therapeutic approaches. a previous report comparing activity of novel cytotoxic agents in xenografts with activity in phase II studies, the Disclosure of Potential Conflicts of Interest best predictor of clinical activity was when a compound had No potential conflicts of interest were disclosed. activity in at least 33% of multiple histologies in multiple xenograft models (71). As noted above, these data were Authors' Contributions Conception and design: F.I. Arnaldez, L.J. Helman generated using cytotoxic agents, and currently we simply Development of methodology: F.I. Arnaldez do not know what best predicts for clinical activity with Acquisition of data (provided animals, acquired and managed patients, newer "targeted" agents other than a specific activating provided facilities, etc.): L.J. Helman Analysis and interpretation of data (e.g., statistical analysis, biosta- mutation in a kinase predicting for response to a specific tistics, computational analysis): L.J. Helman kinase inhibitor. Unfortunately, no such activating kinase Writing, review, and/or revision of the manuscript: F.I. Arnaldez, mutations have been found in Ewing sarcomas, reinforcing L.J. Helman the point that better predictors for clinical activity are Received August 8, 2013; revised April 4, 2014; accepted April 13, 2014; necessary for this tumor. published OnlineFirst April 22, 2014.

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www.aacrjournals.org Clin Cancer Res; 20(12) June 15, 2014 OF7

New Strategies in Ewing Sarcoma: Lost in Translation?

Fernanda I. Arnaldez and Lee J. Helman

Clin Cancer Res Published OnlineFirst April 22, 2014.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-13-0633

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