EWS/FLI and its Targets in Ewing’s Sarcoma:
A Progress Report and Future Directions


Ewing’s sarcoma is a highly aggressive and enigmatic tumor of children and young adults.1 While this tumor most commonly arises in and around bones, it does not appear to be of bony origin. Indeed, the type of cell that transforms into Ewing’s sarcoma is not currently known. This makes it difficult to study the process of Ewing’s sarcoma formation.

Figure 1

Figure 1: EWS/FLI schematic.

Tumors are generally thought to arise because of mutations in normal genes. In the case of Ewing’s sarcoma, the most important mutation is called a chromosomal translocation.2,3 Chromosomes are large segments of linearly-arranged DNA that contain many genes. Humans typically have 23 pairs of chromosomes. In Ewing’s sarcoma tumor cells, chromosomes 11 and 22 have traded portions of their DNA, creating two abnormal chromosomes.2,3 At the exact portion where the two chromosomes have traded genetic material, two different genes, EWS (or more properly, EWSR1) and the other called FLI, have become fused together in an abnormal way.4 This creates a mutant protein called EWS/FLI (Figure 1).4 Rarely, Ewing’s sarcoma tumors will have different chromosomal translocations that generate different, but related, mutant proteins. EWS/FLI (or one of the related proteins) is found in nearly every case of Ewing’s sarcoma.5 From here on out I will refer to EWS/FLI exclusively, but recognize that the themes discussed are conserved (or presumed to be conserved) for the other related fusions as well. EWS/FLI is only found in the tumor cells of the patient, not in the normal cells. These data suggest that EWS/FLI is the critical mutation required for the development of Ewing’s sarcoma.

The diagnosis of Ewing’s sarcoma can be problematic. When surgeons or interventional radiologists remove a small piece of the tumor, called a biopsy, the tumor sample is given to a pathologist to evaluate for diagnosis. This involves a variety of procedures, including looking at the tumor under the microscope, and staining the tumor for proteins that are usually found in Ewing’s sarcoma. Unfortunately, neither of these approaches are perfectly specific, and diagnostic dilemmas still occur (i.e., there are cases in which a diagnosis can not be made). A complementary approach is to look for the chromosomal translocation (or its products) that is usually associated with Ewing’s sarcoma. While this approach tends to be more specific, it requires that the tumor material be of high quality, and processed in a rigorous way. Unfortunately, not all tumor biopsies are of adequate quality or processed appropriately. New approaches that are more sensitive, and more forgiving, would aid in the diagnosis of Ewing’s sarcoma.

One of the projects that we have focused on, using a grant awarded by the Liddy Shriver Sarcoma Initiative, is to develop an improved method to detect the products of Ewing’s sarcoma-associated chromosomal translocations. We have devised an approach that combines a series of new technologies to detect EWS/FLI, or any of the related mutants found in Ewing’s sarcoma. We hope that this technique will be more sensitive, and will thus allow for the detection of the fusion products in samples that are of low quality, or not processed adequately for more "typical" approaches. At this point, the technology is still quite early, but we have some promising results that suggest that the approach will be feasible.

In addition to improving the diagnosis of Ewing’s sarcoma, our lab is focused on identifying new therapeutic approaches for this disease. We believe that understanding the details of how EWS/FLI causes Ewing’s sarcoma will allow us to find new ways to treat this disease. A second project that we have been pursuing, with ongoing funding from the Liddy Shriver Sarcoma Initiative, is analyzing the EWS/FLI target gene NR0B1.

Figure 2

Figure 2: The "knock-down/rescue" approach.

EWS/FLI is thought to function as a transcription factor.4,6,7 Transcription factors bind directly to DNA, and alter the expression of nearby genes. In order to study EWS/FLI in its "normal" cellular context, we developed a system that allowed us to analyze the function of EWS/FLI in Ewing’s sarcoma cells themselves, rather than heterologous cell types (Figure 2).8 We used RNA-interference (RNAi) to "knock-down" (diminish) the amount of EWS/FLI in patient-derived Ewing’s sarcoma cell lines.9 Following the RNAi treatment, we have the ability to reintroduce EWS/FLI expression using a version that is immune to the RNAi effect. We then used high-density microarrays to identify many of the RNA transcripts whose expression levels change when EWS/FLI is knocked down in these cells.9,10

One of the genes we found whose expression was upregulated by EWS/FLI was NR0B1.10,11 Importantly, in recent work (also supported by the Liddy Shriver Sarcoma Initiative), we also found that NR0B1 was directly regulated by EWS/FLI (in other words, EWS/FLI bound directly to the NR0B1 promoter) via a GGAA-containing microsatellite in its promoter.12 This is interesting and unique, as microsatellites are simple repetitive elements that are located throughout the human genome, and are often considered "junk" DNA with no real function. The identification of a GGAA-microsatellite as an EWS/FLI response element demonstrates an important role for these sequences in Ewing’s sarcoma development. This work was recently published in the Proceedings of the National Academy of Sciences.12

The normal function of the NR0B1 protein is not entirely clear. It is important for adrenal gland development and for the development and function of the hypothalamic-pituitary-adrenal-gonadal axis. Since its first identification in 1994,13 a series of functions have been ascribed to the protein, including direct DNA binding and transcriptional repression,13,14 inhibition of other transcription factors such as the retinoic acid receptor,13 SF-115, LRH-116 and the estrogen receptor,17 interaction with corepressors such as NCoR18 and Alien,19 and binding to RNA and directing its shuttling between the nucleus and cytoplasm.20 Loss of NR0B1 expression or function results in adrenal hypoplasia congenita, in which the fetal adrenal gland fails to undergo differentiation into the adult gland.13 Duplication of the NR0B1 gene (with resulting increased expression) results in a sex-reversal phenotype.13

Using our newly-developed Ewing’s sarcoma-based model system, we verified that knock-down of EWS/FLI resulted in a decreased expression of NR0B1.10 We then found that knock-down of NR0B1 expression resulted in a nearly complete loss of oncogenic transformation.10 This effect was specific to NR0B1 loss, and wasn’t an "off-target" effect of the RNAi, because oncogenic transformation was restored by re-expression of NR0B1, using a version that was immune to the RNAi construct.10 Finally, using a series of over thirty different tumor cell lines, we showed that NR0B1 expression was specific for Ewing’s sarcoma.10 These data suggest that NR0B1 may be a new therapeutic target for Ewing’s sarcoma.

Unfortunately, a drug targeting NR0B1 does not currently exist, nor do we have an understanding of the molecular mechanisms that NR0B1 uses to mediate oncogenic transformation in Ewing’s sarcoma. Our lab is now focused on understanding the function of NR0B1 in Ewing’s sarcoma. We recognize that various potential functional domains have been identified in NR0B1, such as a putative ligand binding domain, transcriptional repression domains, and LXXLL protein binding motifs. We are in the process of determining which of these domains are required for NR0B1 to participate in oncogenic transformation. Furthermore, we have identified a number of potential protein binding partners of NR0B1 in Ewing’s sarcoma cells. We hope that by defining key domains and protein partners of NR0B1, we will develop a better understanding of NR0B1 function in Ewing’s sarcoma. This may then allow us to identify new approaches that might functionally inhibit NR0B1, and serve as a new therapeutic direction for Ewing’s sarcoma.

Summary and Conclusions

Ewing’s sarcoma remains an enigmatic tumor. Through a combination of better diagnostic and prognostic approaches and new therapeutic agents, we believe that great opportunities exist for improving the care of patients with this disease. While much work is left to be done, the foundation is firmly placed, and can now be used to build the crucial molecular understanding that is required to cure this devastating illness.

By Stephen L. Lessnick, MD, PhD
Associate Professor of Pediatrics
Adjunct Assistant Professor of Oncological Sciences,
Investigator, Center for Children at Huntsman Cancer Institute
University of Utah School of Medicine
Salt Lake City, UT 84112


1. Rodriguez-Galindo C, Spunt SL, Pappo AS. Treatment of Ewing sarcoma family of tumors: current status and outlook for the future. Med Pediatr Oncol 2003; 40:276-87.

2. Turc-Carel C, Philip I, Berger MP, Philip T, Lenoir GM. Chromosome study of Ewing's sarcoma (ES) cell lines. Consistency of a reciprocal translocation t(11;22)(q24;q12). Cancer genet. cytogenet. 1984; 12:1-19.

3. Whang-Peng J, Triche TJ, Knutsen T, et al. Chromosome translocation in peripheral neuroepithelioma. New England Journal of Medicine 1984; 311:584-585.

4. Delattre O, Zucman J, Plougastel B, et al. Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature 1992; 359:162-5.

5. Barr FG, Womer RB. Molecular diagnosis of ewing family tumors: too many fusions... ? J Mol Diagn 2007; 9:437-40.

6. May WA, Gishizky ML, Lessnick SL, et al. Ewing sarcoma 11;22 translocation produces a chimeric transcription factor that requires the DNA-binding domain encoded by FLI1 for transformation. Proc Natl Acad Sci U S A 1993; 90:5752-6.

7. May WA, Lessnick SL, Braun BS, et al. The Ewing's sarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptional activator and is a more powerful transforming gene than FLI-1. Mol Cell Biol 1993; 13:7393-8.

8. Owen LA, Lessnick SL. Identification of Target Genes in Their Native Cellular Context: An Analysis of EWS/FLI in Ewing's Sarcoma. Cell Cycle 2006; 5:2049-53.

9. Smith R, Owen LA, Trem DJ, et al. Expression profiling of EWS/FLI identifies NKX2.2 as a critical target gene in Ewing's sarcoma. Cancer Cell 2006; 9:405-16.

10. Kinsey M, Smith R, Lessnick SL. NR0B1 Is Required for the Oncogenic Phenotype Mediated by EWS/FLI in Ewing's Sarcoma. Mol Cancer Res 2006; 4:851-9.

11. Hancock JD, Lessnick SL. A transcriptional profiling meta-analysis reveals a core EWS-FLI gene expression signature. Cell Cycle 2008; 7:250-6.

12. Gangwal K, Sankar S, Hollenhorst PC, et al. Microsatellites as EWS/FLI response elements in Ewing's sarcoma. Proc Natl Acad Sci U S A 2008; 105:10149-54.

13. Zanaria E, Muscatelli F, Bardoni B, et al. An unusual member of the nuclear hormone receptor superfamily responsible for X-linked adrenal hypoplasia congenita. Nature 1994; 372:635-41.

14. Zazopoulos E, Lalli E, Stocco DM, Sassone-Corsi P. DNA binding and transcriptional repression by DAX-1 blocks steroidogenesis. Nature 1997; 390:311-5.

15. Ito M, Yu R, Jameson JL. DAX-1 inhibits SF-1-mediated transactivation via a carboxy-terminal domain that is deleted in adrenal hypoplasia congenita. Mol Cell Biol 1997; 17:1476-83

16. Suzuki T, Kasahara M, Yoshioka H, Umesono K, Morohashi K. LXXLL motifs in Dax-1 have target specificity for the orphan nuclear receptors Ad4BP/SF-1 and LRH-1. Endocr Res 2002; 28:537.

17. Zhang H, Thomsen JS, Johansson L, Gustafsson JA, Treuter E. DAX-1 functions as an LXXLL-containing corepressor for activated estrogen receptors. J Biol Chem 2000; 275:39855-9.

18. Crawford PA, Dorn C, Sadovsky Y, Milbrandt J. Nuclear receptor DAX-1 recruits nuclear receptor corepressor N-CoR to steroidogenic factor 1. Mol Cell Biol 1998; 18:2949-56.

19. Altincicek B, Tenbaum SP, Dressel U, Thormeyer D, Renkawitz R, Baniahmad A. Interaction of the corepressor Alien with DAX-1 is abrogated by mutations of DAX-1 involved in adrenal hypoplasia congenita. J Biol Chem 2000; 275:7662-7.

20. Lalli E, Ohe K, Hindelang C, Sassone-Corsi P. Orphan receptor DAX-1 is a shuttling RNA binding protein associated with polyribosomes via mRNA. Mol Cell Biol 2000; 20:4910-21.

Grant Funding

The Liddy Shriver Sarcoma Initiative announced the funding of this $100,000 grant in August 2008. The grant was for a new study by Dr. Stephen Lessnick entitled, "Analysis of NR0B1 in Ewing’s sarcoma." The study was made possible, in part, by a generous gift from the Arlo and Susan Ellison family and by a generous gift from Truus van der Spek, in loving memory of her son Paul Onvlee.

This study led to the publication of an article in Oncogene and an article in the Journal of Clinical Investigation. The study's findings were covered in the following press release by Georgetown University:

New hope for deadly childhood bone cancer

Surprising discovery made by studying so-called 'junk DNA'

SALT LAKE CITY, Aug. 31, 2009 — Researchers at Huntsman Cancer Institute (HCI) at the University of Utah have shed new light on Ewing's sarcoma, an often deadly bone cancer that typically afflicts children and young adults. Their research shows that patients with poor outcomes have tumors with high levels of a protein known as GSTM4, which may suppress the effects of chemotherapy. The research is published online today in the journal Oncogene.

"Doctors and researchers have long known that certain Ewing's sarcoma patients respond to chemotherapy, but others don't even though they have the same form of cancer," says HCI Investigator Stephen Lessnick, M.D., Ph.D. "Our research shows that GSTM4 is found in high levels among those patients where chemotherapy doesn't seem to work. It's found in low levels in patients where chemotherapy is having a more positive effect."

The research could lead to drugs that can suppress GSTM4 in certain patients. It also could lead to a screening test that could reveal which therapies will be most effective for patients. "GSTM4 doesn't seem to suppress the benefits of all chemotherapy drugs, just certain ones. A GSTM4-based test could help to identify the best therapy for each individual patient," Lessnick says.

Ewing's sarcoma is the second most common bone cancer in children and adolescents. The five-year survival rate is considered poor at about 30 percent if the cancer has spread by the time it is diagnosed, and there is an even poorer prognosis for patients who have suffered a relapse.

For this study, researchers focused on an abnormal protein known as EWS-FLI, which is found in most Ewing's sarcoma tumors. What they discovered is that EWS-FLI causes increased amounts of the GSTM4 gene – and the protein it produces – to be expressed in tumors, a previously unknown effect that led them to make the connection between poor outcomes and high levels of GSTM4. The discovery was made by focusing on repetitive DNA sequences called microsatellites. Microsatellites are sometimes referred to as "junk DNA" because they are not thought to have a normal role in the genome. By examining how EWS-FLI interacts with certain microsatellites, Lessnick and his team were able to identify GSTM4.

Lessnick says the next step in research is to focus on testing and treatments that may lead to better survival rates in patients. "Personalized medicine is the next frontier in the battle against cancer," he says. "We now know all cancers are not the same. By focusing on how these proteins are expressed in individual tumors, we may soon be able to offer the treatment that will work best for each patient, and that could lead to higher cure rates," he says.

Liddy Shriver Sarcoma InitiativeLessnick is director of HCI's Center for Children's Cancer Research, and is a Jon and Karen Huntsman Presidential Professor in Cancer Research. This research was supported by funds from the Terri Anna Perine Sarcoma Fund, the Liddy Shriver Sarcoma Initiative, the Sunbeam Foundation, the Huntsman Cancer Foundation, and Alex's Lemonade Stand Foundation.

Huntsman Cancer Institute (HCI) at the University of Utah marks its 10th anniversary in 2009. HCI was founded by Jon M. Huntsman to fulfill his dream of finding a cure for cancer through genetic research. In the last 10 years, HCI has grown to become one of America's major cancer research centers. HCI is part of the University of Utah healthcare system and is ranked consistently by U.S. News & World Report as one of the top cancer hospitals in the country.

  • Figure 1. EWS/FLI schematic.
    Wild-type (normal) EWS is encoded on chromosome 22, and contains an "amino-terminal domain" and a series of domains in the carboxy terminus that are likely RNA binding regions. Wild-type FLI is encoded on chromosome 11, and contains a weak transcriptional activation domain (TAD) in its amino terminus, and a DNA binding domain in its carboxy terminus. The t(11;22)(q24;q12) translocation encodes the fusion EWS/FLI protein, that contains the amino terminal domain of EWS, fused in-frame, to the carboxy terminus of FLI. In this setting, the amino terminus of EWS functions as a strong transcriptional activation domain, while FLI contributes its DNA binding domain to the fusion.
  • Figure 2. The "knock-down/rescue" approach.
    Patient-derived Ewing’s sarcoma cell lines are grown in the laboratory. These normally express EWS/FLI. Treatment with RNA interference (RNAi) results in a "knock-down" of EWS/FLI mRNA and protein levels. EWS/FLI can be re-expressed using a version that is resistant to the RNAi effect. Thus, the function of EWS/FLI can be studied in the cells in which it is normally found: Ewing’s sarcoma tumor cells.

Funding Dr. Lessnick's Research

December, 2006: The Liddy Shriver Sarcoma Initiative announced the funding of two research projects at the Huntsman Cancer Institute at the University of Utah. The two grants, totaling $50,000, are being made in memory of Liddy Shriver, Brian Morden, Krystle Smith, Shane Duffy, Conor O'Sullivan, Paul Onvlee, and Allen Strehlow and to honor those currently fighting this disease.

These grants were made possible because a number of people worked very hard in obtaining donations to sponsor much needed sarcoma research. Most of the donations were contributed in conjunction with the Team Sarcoma 2006 Initiative held earlier this year. We are extremely grateful to all of those involved in raising these funds, in particular, supporters of the Liddy Shriver Sarcoma Initiative, supporters of the Brian Morden Foundation, three Irish families — Patricia and Chris Smith, Joan Duffy, and William and Catherine Walsh, and three members of the Adult Bone Cancer Survivors support group — Mary Sorens, Rachel Baumgartner and Ashley Frost. We are also grateful to the family and friends of those who lost a loved one to Ewing’s sarcoma whose donations are helping sponsor these grants, in particular Maribeth Allen and Val Strehlow and Truus van der Spek. There is, as you can plainly see, strength in numbers.

Both of the studies will be directed by Stephen Lessnick, M.D., Ph.D. at the Huntsman Cancer Institute. The ultimate goal of the first study, "New Approaches for EWS/ETS Detection in Ewing’s Sarcoma" is to improve physicians’ abilities to provide an accurate diagnosis of Ewing’s sarcoma to patients, to provide physicians’ with molecular data which may be relevant to prognosis, and to provide a new non-invasive assay for the measurement of treatment response. The goal of the second study, "Analysis of NR0B1 in Ewing’s sarcoma" is to help to characterize the molecular mechanisms involved in Ewing’s sarcoma development. Additionally, by fully understanding these mechanisms, Dr. Lessnick and his team hope to identify new therapeutic approaches for patients with this devastating disease.

New Approaches for EWS/ETS Detection in Ewing’s Sarcoma

Ewing’s sarcomas are highly associated with recurrent chromosomal translocations. These translocations encode EWS/ETS fusion proteins. Detection of EWS/ETS fusions is a clinically-important adjunct in the diagnosis of patients with Ewing’s sarcoma. Unfortunately, the current technology for EWS/ETS detection is limited in sensitivity, and in some cases, specificity. A new methodology with improved characteristics could be used to assist in accurately diagnosing patients with Ewing’s sarcoma. Indeed, a highly sensitive and specific assay may be able to detect Ewing’s sarcoma cells in the blood and bone marrow of patients, thereby providing a new biomarker for Ewing’s sarcoma. This could result in diagnosing patients with Ewing’s sarcoma from a blood sample, in measuring their response to treatment, and in providing prognostic information to patients. Dr. Lessnick proposes to adapt a newly reported methodology, LMF, towards the detection of EWS/ETS fusion transcripts. He and his team will first develop the system to detect any of the various EWS/ETS fusion transcripts that have been reported. Next, they will assess the sensitivity and specificity of LMF on laboratory-based samples, and compare these results to the current gold standard, RT-PCR. Next, they will extend their analysis to determine if the methodology can be used to detect Ewing’s sarcoma cells in experimentally derived blood samples. Finally, they will assess this methodology to an animal model of Ewing’s sarcoma to mimic a likely clinical scenario. If they are able to successfully develop this assay, and if it is more sensitive and specific than the standard RT-PCR assay, they plan to further develop this into a clinical assay through ongoing collaborations with experts in the field of molecular testing for solid tumors. The ultimate goal of Dr. Lessnick and his team is to use this assay to improve physicians’ abilities to provide an accurate diagnosis to patients, to provide them with molecular data which may be relevant to prognosis, and to provide a new non-invasive assay for the measurement of treatment response.

Analysis of NR0B1 in Ewing’s sarcoma

Ewing’s sarcoma is a highly aggressive bone-associated tumor that is primarily diagnosed in adolescents and young adults. Most cases of Ewing’s sarcoma express a unique fusion oncoprotein, EWS/FLI. EWS/FLI functions as a transcription factor to dysregulate genes involved in cancer development. Because the cell of origin of Ewing’s sarcoma is unknown, prior work has studied the EWS/FLI fusion in heterologous cell types with uncertain relevance to the human disease. Dr. Lessnick and his colleagues recently developed a system to circumvent this difficulty. By "knocking-down" endogenous EWS/FLI expression in patient-derived Ewing’s sarcoma cell lines, using retroviral-mediated RNAi, they have been able to define the full complement of EWS/FLI gene targets in Ewing’s sarcoma. They have identified two of these as being critically important for tumorigenesis mediated by the fusion protein. One of these, NR0B1, is an orphan nuclear receptor whose only prior known role is in adrenal and sexual differentiation. Thus, they defined a new role for this factor. Dr. Lessnick now proposes to characterize the structure-function relationships present in NR0B1. He and his team will use a "knock-down/rescue" system to remove endogenous NR0B1 expression, and replace it with mutant forms of the protein. In this way they will be able to determine which domains are important for the function of NR0B1 in tumor formation. These will serve as important preliminary data as we move forward to characterize the molecular mechanisms involved in Ewing’s sarcoma development. Additionally, by fully understanding these mechanisms, they hope to identify new therapeutic approaches for patients with this devastating disease.