Background The presence of tumor-specific mutations in the cancer genome represents a potential opportunity for pharmacologic intervention to therapeutic benefit. Unfortunately, many classes of oncoproteins (e.g., transcription factors) are not amenable to conventional small-molecule screening. Despite the identification of tumor-specific somatic mutations, most cancer therapy still utilizes nonspecific, cytotoxic drugs. One illustrative example is the treatment of Ewing sarcoma. Although the EWS/FLI oncoprotein, present in the vast majority of Ewing tumors, was characterized over ten years ago, it has never been exploited as a target of therapy. Previously, this target has been intractable to modulation with traditional small-molecule library screening approaches. Here we describe a gene expression–based approach to identify compounds that induce a signature of EWS/FLI attenuation. We hypothesize that screening small-molecule libraries highly enriched for FDA-approved drugs will provide a more rapid path to clinical application. Methods and Findings A gene expression signature for the EWS/FLI off state was determined with microarray expression profiling of Ewing sarcoma cell lines with EWS/FLI-directed RNA interference. A small-molecule library enriched for FDA-approved drugs was screened with a high-throughput, ligation-mediated amplification assay with a fluorescent, bead-based detection. Screening identified cytosine arabinoside (ARA-C) as a modulator of EWS/FLI. ARA-C reduced EWS/FLI protein abundance and accordingly diminished cell viability and transformation and abrogated tumor growth in a xenograft model. Given the poor outcomes of many patients with Ewing sarcoma and the well-established ARA-C safety profile, clinical trials testing ARA-C are warranted. Conclusions We demonstrate that a gene expression–based approach to small-molecule library screening can identify, for rapid clinical testing, candidate drugs that modulate previously intractable targets. Furthermore, this is a generic approach that can, in principle, be applied to the identification of modulators of any tumor-associated oncoprotein in the rare pediatric malignancies, but also in the more common adult cancers.
References
[1]
Shapiro DN, Sublett JE, Li B, Downing JR, Naeve CW (1993) Fusion of PAX3 to a member of the forkhead family of transcription factors in human alveolar rhabdomyosarcoma. Cancer Res 53: 5108–5112.
[2]
Delattre O, Zucman J, Plougastel B, Desmaze C, Melot T, et al. (1992) Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature 359: 162–165.
[3]
Knezevich SR, McFadden DE, Tao W, Lim JF, Sorensen PH (1998) A novel ETV6-NTRK3 gene fusion in congenital fibrosarcoma. Nat Genet 18: 184–187.
[4]
Delattre O, Zucman J, Melot T, Garau XS, Zucker JM, et al. (1994) The Ewing family of tumors—A subgroup of small-round-cell tumors defined by specific chimeric transcripts. N Engl J Med 331: 294–299.
[5]
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.
[6]
May WA, Gishizky ML, Lessnick SL, Lunsford LB, Lewis BC, et al. (1993) 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 90: 5752–5756.
[7]
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.
[8]
Tanaka K, Iwakuma T, Harimaya K, Sato H, Iwamoto Y (1997) EWS-Fli1 antisense oligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma and primitive neuroectodermal tumor cells. J Clin Invest 99: 239–247.
[9]
Toretsky JA, Connell Y, Neckers L, Bhat NK (1997) Inhibition of EWS-FLI-1 fusion protein with antisense oligodeoxynucleotides. J Neurooncol 31: 9–16.
[10]
Kovar H, Aryee DN, Jug G, Henockl C, Schemper M, et al. (1996) EWS/FLI-1 antagonists induce growth inhibition of Ewing tumor cells in vitro. Cell Growth Differ 7: 429–437.
[11]
Ouchida M, Ohno T, Fujimura Y, Rao VN, Reddy ES (1995) Loss of tumorigenicity of Ewing's sarcoma cells expressing antisense RNA to EWS-fusion transcripts. Oncogene 11: 1049–1054.
[12]
Prieur A, Tirode F, Cohen P, Delattre O (2004) EWS/FLI-1 silencing and gene profiling of Ewing cells reveal downstream oncogenic pathways and a crucial role for repression of insulin-like growth factor binding protein 3. Mol Cell Biol 24: 7275–7283.
[13]
Matsunobu T, Tanaka K, Nakamura T, Nakatani F, Sakimura R, et al. (2006) The possible role of EWS-Fli1 in evasion of senescence in Ewing family tumors. Cancer Res 66: 803–811.
[14]
Smith R, Owen LA, Trem DJ, Wong JS, Whangbo JS, et al. (2006) Expression profiling of EWS/FLI identifies NKX2.2 as a critical target gene in Ewing's sarcoma. Cancer Cell 9: 405–416.
[15]
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.
[16]
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.
[17]
Stegmaier K, Ross KN, Colavito SA, O'Malley S, Stockwell BR, et al. (2004) Gene expression-based high-throughput screening (GE-HTS) and application to leukemia differentiation. Nat Genet 36: 257–263.
[18]
Lessnick SL, Dacwag CS, Golub TR (2002) The Ewing's sarcoma oncoprotein EWS/FLI induces a p53-dependent growth arrest in primary human fibroblasts. Cancer Cell 1: 393–401.
[19]
Kinsey M, Smith R, Lessnick SL (2006) NR0B1 Is required for the oncogenic phenotype mediated by EWS/FLI in Ewing's sarcoma. Mol Cancer Res 4: 851–859.
[20]
Tweddle DA, Malcolm AJ, Bown N, Pearson AD, Lunec J (2001) Evidence for the development of p53 mutations after cytotoxic therapy in a neuroblastoma cell line. Cancer Res 61: 8–13.
[21]
Walker DR, Bond JP, Tarone RE, Harris CC, Makalowski W, et al. (1999) Evolutionary conservation and somatic mutation hotspot maps of p53: Correlation with p53 protein structural and functional features. Oncogene 18: 211–218.
[22]
Golub TR, Slonim DK, Tamayo P, Huard C, Gaasenbeek M, et al. (1999) Molecular classification of cancer: Class discovery and class prediction by gene expression monitoring. Science 286: 531–537.
[23]
Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, et al. (2005) Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 102: 15545–15550.
[24]
Nilsson M, Barbany G, Antson DO, Gertow K, Landegren U (2000) Enhanced detection and distinction of RNA by enzymatic probe ligation. Nat Biotechnol 18: 791–793.
[25]
Landegren U, Kaiser R, Sanders J, Hood L (1988) A ligase-mediated gene detection technique. Science 241: 1077–1080.
[26]
Peck D, Crawford ED, Ross KN, Stegmaier K, Golub TR, et al. (2006) A method for high-throughput gene expression signature analysis. Genome Biol 7: R61.
[27]
Mateo-Lozano S, Tirado OM, Notario V (2003) Rapamycin induces the fusion-type independent downregulation of the EWS/FLI-1 proteins and inhibits Ewing's sarcoma cell proliferation. Oncogene 22: 9282–9287.
[28]
Mateo-Lozano S, Gokhale PC, Soldatenkov VA, Dritschilo A, Tirado OM, et al. (2006) Combined transcriptional and translational targeting of EWS/FLI-1 in Ewing's sarcoma. Clin Cancer Res 12: 6781–6790.
[29]
Hofbauer S, Hamilton G, Theyer G, Wollmann K, Gabor F (1993) Insulin-like growth factor-I-dependent growth and in vitro chemosensitivity of Ewing's sarcoma and peripheral primitive neuroectodermal tumour cell lines. Eur J Cancer 29A: 241–245.
[30]
Johnson SA (2000) Clinical pharmacokinetics of nucleoside analogues: Focus on haematological malignancies. Clin Pharmacokinet 39: 5–26.
[31]
Tomizawa D, Tabuchi K, Kinoshita A, Hanada R, Kigasawa H, et al. (2006) Repetitive cycles of high-dose cytarabine are effective for childhood acute myeloid leukemia: Long-term outcome of the children with AML treated on two consecutive trials of Tokyo children's cancer study group. Pediatr Blood Cancer. E-pub 28 June 2006.
[32]
Kern W, Estey EH (2006) High-dose cytosine arabinoside in the treatment of acute myeloid leukemia: Review of three randomized trials. Cancer 107: 116–124.
[33]
Estlin EJ, Yule SM, Lowis SP (2001) Consolidation therapy for childhood acute lymphoblastic leukaemia: Clinical and cellular pharmacology of cytosine arabinoside, epipodophyllotoxins and cyclophosphamide. Cancer Treat Rev 27: 339–350.
[34]
Tomlins SA, Rhodes DR, Perner S, Dhanasekaran SM, Mehra R, et al. (2005) Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science 310: 644–648.
[35]
Tomlins SA, Mehra R, Rhodes DR, Smith LR, Roulston D, et al. (2006) TMPRSS2:ETV4 gene fusions define a third molecular subtype of prostate cancer. Cancer Res 66: 3396–3400.
