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Mechanisms of EWS/FLI1-GLI1 Activation in Ewing Sarcoma

By William A. May, MD

Background

Transcriptional deregulation by EWS/FLI1 is a key phenomenon is Ewing Sarcoma development. One undeniable testament to the multifunctional importance of the EWS/FLI1 class of oncoproteins is the extremely low mutation frequency demonstrated in Ewing tumors.^1-3 Oncogenic functions which might be provided via mutation in other tumor systems are provided by the direct and indirect actions of this class of chimeric transcription factors. In this context, a deeper understanding of the mechanisms of the EWS/FLI1 class of oncogenic transcription factors is an important area of investigation for Ewing tumors.

Figure 1: EWS/FLI1 Target Classes — Report Figure 1: Hedgehog-GLI in Human Cancers

Many important targets of EWS/FLI1 transcriptional deregulation have been identified and studied. In the original proposal, we outline the evidence for the role of the Hedgehog-GLI pathway (HH-GLI) in the biology of Ewing tumors, consistent with the role of this pathway in diverse cancers (Figure 1). We also detail the finding of a surprising degree of overlap between the transcriptional targets of EWS/FLI1 and of GLI1 in Ewing cells, a finding which has been corroborated by colleagues in the study of Ewing tumors. With this and other evidence detailed in the proposal, we formulated a hypothesis that these two oncoproteins act in concert at at least some of these many jointly regulated loci to collaborate to make the Ewing transcriptome the uniquely malignant entity that afflicts our patients.

This research built on these findings by using genome wide studies to corroborate the findings, then using the knowledge gained to facilitate therapeutic development.

Results

Aim 1: To use ChIP-Sequencing to characterize EWS/FLI1 and GLI1 binding at a genome wide level

Effort was expended optimizing conditions for ChIP including the antibody used and the conditions for chromatin fragmentation in the A673 cell line. Experiments using known targeted loci including NR0B1 for EWS/FLI1 and Patched1 for GLI1 confirmed the suitability of conditions.

Having optimized conditions, we undertook large scale ChIP. Recovered DNA fragments were processed for library construction and subsequent high throughput sequencing. This sequencing confirmed that we had attained a sufficient number of unique reads for valid data interpretation. Preliminary analysis of this initial data set indicates a statistically significant association of loci bound by EWS/FLI1 and those bound by GLI1. A second independent experiment was similarly processed and is currently undergoing sequencing.Our hope is that this data set will confirm these findings.

Further analysis is underway to establish the binding sites for each protein and for correspondence between jointly bound loci and our transcript data for jointly regulated loci. Further work to confirm our preliminary data demonstrating physical association of EWS/FLI1 and GLI1 in Ewing cells has been difficult to reproduce in other Ewing lines. So, the cooperation between EWS/FLI1 and GLI1 may not depend on direct physical association much as has been recently observed in other EWS/FLI1 transcriptional interactions.

Aim 2: To develop a reporter system to identify compounds which interfere with the interaction of EWS/FLI1 and GLI1

Efforts to conduct the proposed experiments have been hampered by high background reporter activation in several lines. We determined that a reworking of the reporter system would await full analysis of our ChIP data to guide and inform our redesign efforts.

However, we have generated experimental evidence that binding of endogenous GLI2 is the cause of this high background activity. Further work, conducted in part with funding via this grant, indicates that GLI2 is expressed abundantly in Ewing cell lines and tumors. Since GLI2 transactivation is the major factor in upregulation of GLI1 in physiologic and many oncologic systems, we hypothesized that GLI2 played a role in GLI1 regulation in Ewing cells. Experiments using GLI2 overexpression and shRNA directed to GLI2 demonstrate that GLI2 is an important factor GLI1 regulation in Ewing cells. Further evidence demonstrates that experimental targeting of GLI2 in Ewing cells is, in several aspects, more effective at diminishing oncogenic phenotype than targeting GLI1 alone. These results make sense since GLI2 is further upstream in the HH-GLI pathway than GLI1 (Figure 2). Therefore, GLI2 knockdown encompasses the effects of both GLI2 and GLI1 ablation. Despite this linkage, numerous experiments have failed to demonstrate that EWS/FLI1 transcriptionally regulates GLI2. In recent experiments, we have been able to show that GLI2 nuclear localization, an important checkpoint in GLI2 regulation,⁴ is enhanced by a mechanism dependent on EWS/FLI1 expression. GLI2 upregulation has been found to play a role in other tumor systems and plays an important role in mediating one mechanism of resistance to HH-GLI inhibition clinically by the Smoothened inhibitor Vismodegib.⁵ We believe that EWS/FLI1 regulates GLI2 via this presumably indirect mechanism and that this mechanism is an important therapeutic choke point in Ewing tumors.

So while it is unfortunate that the enhanced amount of GLI2 in the nuclei of Ewing cells raises reporter background, our elucidation of the mechanism for this phenomenon has advanced our understanding of the process of HH-GLI activation in Ewing tumors.

Conclusions

We think that the combination of these findings will, when soon published, further the evidence of HH-GLI pathway activation being an important mediator of EWS/FLI1 in Ewing tumors. They establish that Ewing tumors are among the class of malignancies which should benefit from ongoing efforts to target with this pathway therapeutically via mechanisms not dependent on Smoothened inhibition. With all that is coming to light with regard to both genetic and epigenetic deregulation of gene expression in cancer, it is an exciting time for all in the field.

I and those in my lab want to thank the Bruce Shriver for his many years of admirable and much needed efforts to further the study of Ewing tumors and other sarcomas. Efforts such as his have helped raise the profile of this often overlooked class of malignancies.

By William A. May, MD
Professor of Pediatrics at the David Geffen School of Medicine, University of California Los Angeles
Attending Physician at Mattel Children’s Hospital

References

1. Brohl, A. S. et al. The genomic landscape of the Ewing Sarcoma family of tumors reveals recurrent STAG2 mutation. PLoS genetics 10, e1004475, doi:10.1371/journal.pgen.1004475 (2014).

2. Tirode, F. et al. Genomic landscape of Ewing sarcoma defines an aggressive subtype with co-association of STAG2 and TP53 mutations. Cancer discovery 4, 1342-1353, doi:10.1158/2159-8290.CD-14-0622 (2014).

3. Crompton, B. D. et al. The genomic landscape of pediatric Ewing sarcoma. Cancer discovery 4, 1326-1341, doi:10.1158/2159-8290.CD-13-1037 (2014).

4. Kim, J., Kato, M. & Beachy, P. A. Gli2 trafficking links Hedgehog-dependent activation of Smoothened in the primary cilium to transcriptional activation in the nucleus. Proc Natl Acad Sci U S A 106, 21666-21671, doi:0912180106 [pii]
10.1073/pnas.0912180106 (2009).

5. Sharpe, H. J. et al. Genomic analysis of smoothened inhibitor resistance in basal cell carcinoma. Cancer cell 27, 327-341, doi:10.1016/j.ccell.2015.02.001 (2015).

Mechanisms of EWS/FLI1-GLI1 Activation in Ewing Sarcoma

An ESUN Experimental Plan
By William A. May, MD

Background

Ewing Sarcoma is one of the most common bone and soft tissue tumors of childhood, adolescence, and young adulthood. Its successful treatment requires aggressive multimodal therapy, even in cases of apparently localized disease.¹ While 65-70% of patients with localized disease can be cured with aggressive therapy, the dismal prognosis² of those with metastatic and recurrent disease means that close to half of those diagnosed with this cancer will die of it. The aggressive nature of therapy means that the incidence of secondary malignancies can be alarmingly high³ and that those who otherwise survive can experience a very high rate of life-long debility.⁴ While recent intensifications in therapy have resulted in a modest improvement for patients with localized disease,⁵ nothing in the last two decades has changed the poor prognosis for metastatic or recurrent patients.^6,7 New treatment approaches using targeted therapeutics are needed but the fundamental understanding which forms the basis of such strategies has lagged.

EWS/ETS oncogenic fusion proteins (see sidebar) are uniquely found in virtually all Ewing tumors. These chimera are the only well established driver mutation in this cancer. The EWS/FLI1 chimera is found in approximately 85% of Ewing tumors and is thought to function by aberrantly regulating the expression of target genes which drive tumor phenotype.⁸ To date, many targets have been identified, some direct and some indirect.⁹ Regrettably, none has yet led to a proven prognostic or therapeutic application. Given the fact that ETS transcription factors characteristically depend on interaction with other transcription factors for specificity,¹⁰ it has long been speculated¹¹ that alteration in these interactions by the substitution of amino-terminal EWS may be important to this altered biology. Some evidence for such interactions has emerged over the years.^12-14

GLI1 is a downstream target whose expression is upregulated EWS/FLI1 in Ewing tumors. It has been shown to be important to Ewing tumor biology.^15-18 GLI1 is a critical component of the Hedgehog-GLI pathway, a pathway of known importance in many cancers.^19,20 We have preliminary evidence that EWS/FLI1 and GLI1 target sets in Ewing tumor cell lines show a surprising amount of overlap. Similar evidence for the importance to GLI1 in the Ewing transcriptome has recently been published by others.²¹ We have further evidence that EWS/FLI1 interacts with GLI1 at select promoters in Ewing cells to produce synergistic deregulation of target genes. We believe the study of this interaction can enhance our understanding of a critical process in Ewing tumor development.

Purpose of Investigation

We believe that cooperative interaction between EWS/FLI1 and GLI1 is a powerful determinant of Ewing tumor biology (see Figure 1). Since all cooperative targets are, by definition, direct targets of two professional oncogenes, there is a higher likelihood they will be drivers of tumor formation with therapeutic potential rather than passengers in the process of tumor propagation. Beyond this, the key oncogenic driver in Ewing Sarcoma is EWS/FLI1 transcriptional deregulation. Our data demonstrates that a direct interaction between EWS/FLI1 and GLI1 is a major element of this deregulation. We therefore propose two aims, one to provide needed additional detail to our understanding of this mechanism and one to translationally exploit our present state of knowledge.

Aim 1: To use ChIP-Sequencing to characterize EWS/FLI1 and GLI1 binding at a genome wide level

To accomplish this, we propose to use Chromatin Immunoprecipitation combined with high throughput sequencing (ChIP-Seq).

To assure consistency in this assay, we will express versions of EWS/FLI1 and GLI1 which have been tagged with the same FLAG epitope (see Figure 2). This approach has proved useful in deciphering gene regulation by GLI1 in developmental systems.^22,23 Sonicated chromatin will be incubated with antibody to FLAG. As a control for background binding which can be found with FLAG antisera, we will include a control FLAG IP of Ewing cells transfected with empty vector only. Input chromatin will be used as a negative control. Immunoprecipitation will be performed using magnetic beads. After washing, the chromatin will be eluted and the resulting DNA will be purified and subjected to next generation sequencing (see Figure 2).

Sequence data will be assembled to the reference genome. Genomic regions enriched by ChIP will be determined in software and will be statistically analyzed. The regions enriched will be mapped using and classified based on their location relative to known genes. This will yield a set of jointly bound loci which would then be correlated with our extensive microarray expression data. The result will be a set of loci directly bound by both EWS/FLI1 and GLI1 and which are regulated by both.

Once identified, top ranking targets will be further analyzed to confirm that they are regulated as predicted. As before we will use Ewing cell lines with shRNA targeting EWS/FLI1, GLI1, or of a non-targeting control. Transcript level expression from microarray will be confirmed by via real time quantitative PCR (qPCR). Protein expression changes will be confirmed by western blot. With this confirmation, future studies will focus on loci which demonstrate joint binding of regulatory regions and confirmed joint regulation of expression. The validity of the epitope tagged CHIP-Seq data will be confirmed by CHIP experiments using native antibody with physiologic expression levels.

The analysis of sequence adjacent to transcription factor DNA binding peaks has been used to predict transcription factor interactions by analyzing polymorphic binding site sequence variants for other transcription factors.²⁴ As part of this analysis, we will also analyze regions enriched by ChIP for the non-random association of binding motifs of other classes of transcription factor. These other motifs will represent other DNA binding transcription factors which may potentially bind in the same region as EWS/FLI1 and GLI1. These other classes of transcription factor may cooperate in transcriptional deregulation along with EWS/FLI1 and GLI1. These candidate cooperative transcription factors will be further analyzed in future studies since such binding partners could alter EWS/FLI1 and GLI1 protein stability as well as their transcriptional activity. As such, these partner proteins could potentially be therapeutic targets in themselves.

Aim 2: To develop a reporter system to identify compounds which interfere with the interaction of EWS/FLI1 and GLI1

Another way to exploit the biologic importance of this interaction is to develop systems which, once validated, can be used to perform mass screening for compounds which interfere with this interaction. In Ewing cells, both EWS/FLI1 and GLI1 have been shown to be important to the oncogenic phenotype individually. Since reporter based biologic screens have been critical in the identification of novel therapeutic compounds directed at specific pathways,^25,26 a system for the identification of compounds interfering with the concerted action of this protein pair would be a new and potentially useful approach to Ewing therapeutics.

We have a developed reporter system incorporating only minimal EWS/FLI1 and GLI1 binding sequences confirmed by gene specific ChIP in our preliminary studies. These minimal sequences have been concatenated into a luciferase reporter construct and demonstrate good activity (see Figure 3). We will take our optimized construct into Ewing cells. To minimize experimental variability, we will develop a reporter line in which this construct has stably integrated. Clones expressing high levels of the reporter at baseline will be chosen for further screening. We will then screen clones for their responsiveness to EWS/FLI1 and GLI1 individually using shRNA directed to each protein. Those clones demonstrating a combination of high native activity and high dependence on the expression of EWS/FLI1 and GLI1 will be used for future work.

To demonstrate the workability of this system, we will test our system with compounds which are known to interfere with the function of either EWS/FLI1 or GL11. To interfere with EWS/FLI1, we will use compounds previously shown to inhibit EWS/FLI1 activity²⁷ or expression.²⁸ To interfere with GLI1, we will use compounds such as GANT61, HPI1, and HPI4, which have all been shown to inhibit GLI1 downstream of Smoothened.²⁹ These known compounds will be applied to our reporter line as a test of its utility. The IC50 for each of these compounds in inhibiting reporter expression will be determined. The reporter line would be assessed for effects on cell proliferation and possible apoptosis, either of which may complicate the interpretation of data. Once validated, this system will be used to screen large libraries of compounds.

Impact Statement

Twenty years have passed since early findings identified the major function of EWS/ETS proteins.^30,31 Since that time, many biologic studies of Ewing tumors have concentrated on the identification of EWS/ETS transcriptional targets, both direct and indirect, seeking to identify some key target which would allow us to alter the deadly behavior of these tumor. Frustratingly, this “key target” approach has not yet resulted in clinically useful prognostic or therapeutic advances. One alternative to this “key target approach” is to focus on the essential mechanisms by which EWS/ETS chimera act, as such mechanisms will apply to many targets. The work we propose will detail the novel mechanism we have uncovered, one which applies to a broad class of EWS/ETS deregulated genes. We will also develop a system by which mass compound screening for interference with this mechanism could be subsequently undertaken. By focusing on one of the fundamental mechanisms of EWS/ETS biology, we hope to more effectively attack these deadly tumors.

Glossary of Terms

Chromatin Immunoprecipitation (ChIP): A powerful technique for studying what transcription factors do in living cells. Normally, transcription factors bind DNA near genes to regulate their expression. In ChIP, cells are treated to permanently attach these transcription factors to the DNA they bind. We can then use specific antibodies to grab onto specific proteins and pull them out solution. The DNA that gets dragged along in this process can be purified and sequenced. The sequences which are recovered most often tell us where transcription factor binds.

Downstream Target: A gene whose expression is altered by the action of transcription factor is often called a downstream target. In this perspective, the transcription factor is the causal event and the change in expression of the targeted gene is the effect.

Epitope: A small portion of a protein that is recognized by an antibody. Sometimes the naturally occurring epitopes of a protein are too weak to permit a good experiment. So, to increase our ability to detect or isolate these proteins, we genetically engineer a really strong epitope onto the protein and express the protein artificially. Epitopes are often just a handful of amino acids, so they can leave normal protein function intact.

Fusion Protein: A type of oncogenic mutation. In most Ewing Sarcomas, a piece of Chromosome 11 becomes attached to Chromosome 22. This cuts in half the EWS gene on chromosome 22 and the FLI1 gene on Chromosome 11. The result is a fusion of the two proteins producing a protein which has the front half of EWS fused to the back half of FLI1. These fusions are sometimes called chimera, a name derived from mythological creatures fusing two different animals.

Oncogene: Normal cellular genes that cause cancer to form. They cause cancer because they are altered in some way. Sometimes they are proteins that have mutated to have altered function. This mutation may be a change in as little as a single amino acid (out of potentially hundreds), or a deletion of part of the protein, or a fusion of the protein with another protein. Some normal proteins can also become oncogenes simply by being expressed in the wrong tissue, at the wrong time, or to the wrong degree.

Transcription Factors: A broad class of cellular proteins whose function is to turn on or turn off the expression of other cellular proteins. They typically do so by binding DNA in the region of a gene. This binding either enhances or inhibits that gene from being written as an RNA copy (transcribed). This is an important step in controlling how a cell behaves. Many cancer causing genes function by altering the expression of other genes.

Transcriptome: At any given time, there are thousands and thousands of genes being expressed in a cell. All genes start on the path to expression by having the DNA of the gene transcribed into an RNA copy. This RNA copy is called a transcript. The collection of these many thousands of transcripts is called the transcriptome. Transcripts carry information from the DNA in the nucleus of the cell out to the cytoplasm where it can be made into protein, which are the molecules that mostly carry out the work of the cell.

By William A. May, MD
Associate Professor of Pediatrics at the Keck School of Medicine, University of Southern California
Attending Physician on the Bone and Soft Tissue Tumor Service at Children’s Hospital in Los Angeles

References

1. Rodriguez-Galindo C, Spunt SL, Pappo AS (2003) Treatment of Ewing sarcoma family of tumors: current status and outlook for the future. Med Pediatr Oncol 40: 276-287.

2. Miser JS, Krailo MD, Tarbell NJ, Link MP, Fryer CJ, et al. (2004) Treatment of metastatic Ewing's sarcoma or primitive neuroectodermal tumor of bone: evaluation of combination ifosfamide and etoposide--a Children's Cancer Group and Pediatric Oncology Group study. J Clin Oncol 22: 2873-2876.

3. Bhatia S, Krailo MD, Chen Z, Burden L, Askin FB, et al. (2007) Therapy-related myelodysplasia and acute myeloid leukemia after Ewing sarcoma and primitive neuroectodermal tumor of bone: A report from the Children's Oncology Group. Blood 109: 46-51.

4. Oeffinger KC, Mertens AC, Sklar CA, Kawashima T, Hudson MM, et al. (2006) Chronic health conditions in adult survivors of childhood cancer. N Engl J Med 355: 1572-1582.

5. Womer RB, West DC, Krailo MD, Dickman PS, Pawel BR, et al. (2012) Randomized controlled trial of interval-compressed chemotherapy for the treatment of localized Ewing sarcoma: a report from the Children's Oncology Group. J Clin Oncol 30: 4148-4154.

6. Grier HE, Krailo MD, Tarbell NJ, Link MP, Fryer CJ, et al. (2003) Addition of ifosfamide and etoposide to standard chemotherapy for Ewing's sarcoma and primitive neuroectodermal tumor of bone. N Engl J Med 348: 694-701.

7. Leavey PJ, Mascarenhas L, Marina N, Chen Z, Krailo M, et al. (2008) Prognostic factors for patients with Ewing sarcoma (EWS) at first recurrence following multi-modality therapy: A report from the Children's Oncology Group. Pediatr Blood Cancer 51: 334-338.

8. Arvand A, Denny CT (2001) Biology of EWS/ETS fusions in Ewing's family tumors. Oncogene 20: 5747-5754.

9. Janknecht R (2005) EWS-ETS oncoproteins: the linchpins of Ewing tumors. Gene 363: 1-14.

10. Verger A, Duterque-Coquillaud M (2002) When Ets transcription factors meet their partners. Bioessays 24: 362-370.

11. Lessnick SL, Braun BS, Denny CT, May WA (1995) Multiple domains mediate transformation by the Ewing's sarcoma EWS/FLI-1 fusion gene. Oncogene 10: 423-431.

12. Kinsey M, Smith R, Iyer AK, McCabe ER, Lessnick SL (2009) EWS/FLI and its downstream target NR0B1 interact directly to modulate transcription and oncogenesis in Ewing's sarcoma. Cancer Res 69: 9047-9055.

13. Toretsky JA, Erkizan V, Levenson A, Abaan OD, Parvin JD, et al. (2006) Oncoprotein EWS-FLI1 activity is enhanced by RNA helicase A. Cancer Res 66: 5574-5581.

14. Erkizan HV, Uversky VN, Toretsky JA (2010) Oncogenic partnerships: EWS-FLI1 protein interactions initiate key pathways of Ewing's sarcoma. Clin Cancer Res 16: 4077-4083.

15. Zwerner JP, Joo J, Warner KL, Christensen L, Hu-Lieskovan S, et al. (2008) The EWS/FLI1 oncogenic transcription factor deregulates GLI1. Oncogene 27: 3282-3291.

16. Joo J, Christensen L, Warner K, States L, Kang HG, et al. (2009) GLI1 is a central mediator of EWS/FLI1 signaling in Ewing tumors. PLoS ONE 4: e7608.

17. Beauchamp EM, Ringer L, Bulut G, Sajwan KP, Hall MD, et al. (2011) Arsenic trioxide inhibits human cancer cell growth and tumor development in mice by blocking Hedgehog/GLI pathway. J Clin Invest 121: 148-160.

18. Beauchamp E, Bulut G, Abaan O, Chen K, Merchant A, et al. (2009) GLI1 is a direct transcriptional target of EWS-FLI1 oncoprotein. J Biol Chem 284: 9074-9082.

19. Ruiz i Altaba A, Sanchez P, Dahmane N (2002) Gli and hedgehog in cancer: tumours, embryos and stem cells. Nat Rev Cancer 2: 361-372.

20. Zhao C, Chen A, Jamieson CH, Fereshteh M, Abrahamsson A, et al. (2009) Hedgehog signalling is essential for maintenance of cancer stem cells in myeloid leukaemia. Nature 458: 776-779.

21. Sankar S, Tanner JM, Bell R, Chaturvedi A, Randall RL, et al. (2013) A Novel Role for Keratin 17 in Coordinating Oncogenic Transformation and Cellular Adhesion in Ewing sarcoma. Mol Cell Biol.

22. Peterson KA, Nishi Y, Ma W, Vedenko A, Shokri L, et al. (2012) Neural-specific Sox2 input and differential Gli-binding affinity provide context and positional information in Shh-directed neural patterning. Genes Dev 26: 2802-2816.

23. Vokes SA, Ji H, McCuine S, Tenzen T, Giles S, et al. (2007) Genomic characterization of Gli-activator targets in sonic hedgehog-mediated neural patterning. Development 134: 1977-1989.

24. Karczewski KJ, Tatonetti NP, Landt SG, Yang X, Slifer T, et al. (2011) Cooperative transcription factor associations discovered using regulatory variation. Proc Natl Acad Sci U S A 108: 13353-13358.

25. Lauth M, Bergstrom A, Shimokawa T, Toftgard R (2007) Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists. Proc Natl Acad Sci U S A 104: 8455-8460.

26. Kim J, Lee JJ, Gardner D, Beachy PA (2010) Arsenic antagonizes the Hedgehog pathway by preventing ciliary accumulation and reducing stability of the Gli2 transcriptional effector. Proc Natl Acad Sci U S A 107: 13432-13437.

27. Erkizan HV, Kong Y, Merchant M, Schlottmann S, Barber-Rotenberg JS, et al. (2009) A small molecule blocking oncogenic protein EWS-FLI1 interaction with RNA helicase A inhibits growth of Ewing's sarcoma. Nat Med 15: 750-756.

28. Stegmaier K, Wong JS, Ross KN, Chow KT, Peck D, et al. (2007) Signature-based small molecule screening identifies cytosine arabinoside as an EWS/FLI modulator in Ewing sarcoma. PLoS Med 4: e122.

29. Firestone AJ, Chen JK (2011) Small-Molecule Inhibitors of the Hedgehog Pathway. In: Xie J, editor. Hedgehog Signaling Activation in Human Cancer and its Clinical Implications. New York: Springer pp. 187-212.

30. May WA, Gishizky ML, Lessnick SL, Lunsford LB, Lewis BC, et al. (1993) Ewing sarcoma 11;22 translocation produces a chimeric factor that requires the DNA-binding domain encoded by FLI1 for transformation. Proc Natl Acad Sci U S A 90: 5752-5756.

31. May WA, Lessnick SL, Braun BS, Klemsz M, Lewis BC, et al. (1993) 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 13: 7393-7398.

32. Dijkgraaf GJ, Alicke B, Weinmann L, Januario T, West K, et al. (2011) Small molecule inhibition of GDC-0449 refractory smoothened mutants and downstream mechanisms of drug resistance. Cancer Res 71: 435-444.

Grant Funds Research on GLI1 in Ewing's Sarcoma

An ESUN Announcement

The Liddy Shriver Sarcoma Initiative is pleased to partner with the Brian Morden Foundation in the funding of a $50,000 grant for Ewing's sarcoma research at the Children's Hospital Los Angeles. In the study, William A. May, MD will work with the well-known EWS/FLI1 mutation in a new way by targeting one of its helper proteins, GLI1.

EWS/FLI1 is the name for the most common form of mutant gene in Ewing's sarcoma. It is a type of protein that functions by altering the expression of many other genes in Ewing's cells. This puts it in a class of proteins called transcription factors that control expression by altering the transcription of genes. Transcription factors cannot act in isolation; they usually recruit other proteins to help them.

Scientists have not yet created or discovered a drug that will stop EWS/FLI1 from functioning. Dr. May believes that targeting GLI1 might be almost as effective as targeting EWS/FLI1 itself. He writes:

Data from my lab has shown that a majority of targets transcriptionally deregulated by EWS/FLI1 are co-regulated by GLI1. This demonstrates a central importance for Hedgehog-GLI (HH-GLI) in Ewing tumors. We now have evidence that the reason for this tremendous target overlap involves a novel mechanism in which GLI1 interacts with EWS/FLI1 to synergistically upregulate jointly-targeted promoters. Since it adds key details to the only proven driver mutation in this disease, this synergism represents a novel aspect of EWS/FLI1 and GLI1 biology that can have tremendous translational implications for Ewing tumors.

GLI1 is the most critical member of an important cancer pathway known as Hedgehog. Hedgehog is a key pathway in quite a large number of cancers, and GLI1 is an indispensable part of the Hedgehog pathway. As a result, there is a lot of interest in discovering drugs to interfere with GLI1. The work funded by this grant will establish a framework so that drugs targeting GLI1 can be used in Ewing's sarcoma patients at the earliest possible stages of development.

Dr. May's Research

William A. May, MD Cancer is a devastating problem for kids and for families. I appreciated this early in my medical career, and I went into Pediatric Oncology to try to make a difference. But, as a practicing oncologist, there are enormous frustrations when what we do fails to help the patient. I believe we fail because, despite all we have learned, we are all still profoundly ignorant about the causes of cancer. So I have dedicated myself to fighting cancer in two ways: by doing the very best I can for my patients with the knowledge we presently possess and by fighting to learn what I can about the causes of cancer so that we can better treat future patients.

Working with the Initiative

By Dr. William A. May

In most cases, you submit a grant for review and the reviewers tell you they liked it or they did not. Perhaps the reason they disliked it is something addressable and simple. However, the only way of recovering from that failure is to resubmit the grant in the next funding cycle. This takes a long time and slows progress.

With the Liddy Shriver Sarcoma Initiative, I was given the chance to immediately address the shortcomings of the original proposal. The result is that the work is funded sooner and can move ahead with less delay. Overall, conventional peer review is like a decree. The Liddy Shriver process is more like a conversation.

I hope that the publication of my experimental plan and research results in ESUN makes the process of cancer investigation more accessible to people. I hope this leads to a better understanding of what is done in this country with research dollars and what results from it. If it can do that, then perhaps people will see the value of what researchers do. If this tremendous value is better appreciated, perhaps the funding of research into so many serious diseases can be insulated from the budgetary and political struggles which have so hindered research in the US in recent years.

Funding

This grant is co-funded by the Brian Morden Foundation and the Liddy Shriver Sarcoma Initiative. It is also made possible by generous donations to the Initiative from the families and friends of Mike Homan, Christi Campbell and Michael McMahan, all of whom lost their lives to Ewing's sarcoma.

Targets of EWS/FLI1 have long been classed as direct or indirect. We will investigate a new class of targets which are cooperatively regulated by EWS/FLI1 and its target GLI1.
Epitope tagged versions of EWS/FLI1 and GLI1 will be expressed in Ewing cells and cross-linked to DNA. Following immunoprecipitation, recovered DNA fragments will be sequence and mapped to the human genome. Peak areas of enrichment will be identified.
An 8x concatemer containing minimal promoter sequences shown to bind EWS/FLI1 and GLI1 in Ewing cells was cloned upstream of a minimal luciferase promoter. This construct was transfected into A673 cells and luciferase activity was measured 48-72 hours later. The concatemer exhibits good signal, exceeding the full length promoter from which it was derived.
Extracellular Sonic Hedgehog (SHH) binds Patched (PTCH) in the cell membrane. SHH+PTCH then releases Smoothened (SMO) from inhibition. This allows inactive GLI2 to enter to the nucleus, where its active form (GLI2-a) increases expression of GLI1 and other targets.