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Signature-based small molecule screening identifies cytosine arabinoside as an EWS/FLI modulator in Ewing sarcoma.

Stegmaier K, Wong JS, Ross KN, Chow KT, Peck D, Wright RD, Lessnick SL, Kung AL, Golub TR - PLoS Med. (2007)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Oncology, Dana-Farber Cancer Institute and Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

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.

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In vivo Efficacy of ARA-C in a Mouse Model of Ewing Sarcoma(A) A673-Luciferase-positive xenografts were established for 2–3 wk in NCr nude mice, with tumor burden monitored by bioluminescence imaging. Mice with logarithmically growing tumors were treated with intraperitoneal injection of ARA-C at 50 mg/kg daily for four doses or vehicle control. In both a pilot experiment A: and a larger study with cohorts of nine mice in each group B: control mice continued to have logarithmic tumor growth, necessitating sacrifice within 1 wk of starting treatments. By comparison, ARA-C-treated mice had stable disease or tumor regression with seven of nine animals showing regression.(B) Arrows indicate days of drug dosing, and the asterisk indicates p < 0.05 by Student's t-test (two-tailed assuming samples with equal variance). Error bars show the standard deviation across nine replicates. Bioluminescence was measured as photonic flux through standardized regions of interest (photons per second per region of interest), which did not include reflected light along the left flank (e.g., right animal in part [A] at day 17 of ARA-C treatment).
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pmed-0040122-g006: In vivo Efficacy of ARA-C in a Mouse Model of Ewing Sarcoma(A) A673-Luciferase-positive xenografts were established for 2–3 wk in NCr nude mice, with tumor burden monitored by bioluminescence imaging. Mice with logarithmically growing tumors were treated with intraperitoneal injection of ARA-C at 50 mg/kg daily for four doses or vehicle control. In both a pilot experiment A: and a larger study with cohorts of nine mice in each group B: control mice continued to have logarithmic tumor growth, necessitating sacrifice within 1 wk of starting treatments. By comparison, ARA-C-treated mice had stable disease or tumor regression with seven of nine animals showing regression.(B) Arrows indicate days of drug dosing, and the asterisk indicates p < 0.05 by Student's t-test (two-tailed assuming samples with equal variance). Error bars show the standard deviation across nine replicates. Bioluminescence was measured as photonic flux through standardized regions of interest (photons per second per region of interest), which did not include reflected light along the left flank (e.g., right animal in part [A] at day 17 of ARA-C treatment).

Mentions: A xenograft model of Ewing sarcoma was used to evaluate the effects of ARA-C on in vivo tumor growth. A673 cells were engineered to express firefly luciferase, and tumor burden was monitored by in vivo bioluminescence imaging as previously described [14]. After inoculation of cells in the flanks of nude mice, tumors were allowed to establish for 2–3 wk. Mice with logarithmically growing tumors were divided into cohorts and treated with either systemic ARA-C at 50 mg/kg daily for four doses or vehicle control. Whereas vehicle-treated animals had tumors that continued to grow logarithmically, ARA-C-treated animals had significantly reduced tumor growth or tumor regression (Figure 6A and 6B). There was no significant effect on body weight in ARA-C treated animals, suggesting no significant systemic toxicity at this dose (unpublished data).


Signature-based small molecule screening identifies cytosine arabinoside as an EWS/FLI modulator in Ewing sarcoma.

Stegmaier K, Wong JS, Ross KN, Chow KT, Peck D, Wright RD, Lessnick SL, Kung AL, Golub TR - PLoS Med. (2007)

In vivo Efficacy of ARA-C in a Mouse Model of Ewing Sarcoma(A) A673-Luciferase-positive xenografts were established for 2–3 wk in NCr nude mice, with tumor burden monitored by bioluminescence imaging. Mice with logarithmically growing tumors were treated with intraperitoneal injection of ARA-C at 50 mg/kg daily for four doses or vehicle control. In both a pilot experiment A: and a larger study with cohorts of nine mice in each group B: control mice continued to have logarithmic tumor growth, necessitating sacrifice within 1 wk of starting treatments. By comparison, ARA-C-treated mice had stable disease or tumor regression with seven of nine animals showing regression.(B) Arrows indicate days of drug dosing, and the asterisk indicates p < 0.05 by Student's t-test (two-tailed assuming samples with equal variance). Error bars show the standard deviation across nine replicates. Bioluminescence was measured as photonic flux through standardized regions of interest (photons per second per region of interest), which did not include reflected light along the left flank (e.g., right animal in part [A] at day 17 of ARA-C treatment).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1851624&req=5

pmed-0040122-g006: In vivo Efficacy of ARA-C in a Mouse Model of Ewing Sarcoma(A) A673-Luciferase-positive xenografts were established for 2–3 wk in NCr nude mice, with tumor burden monitored by bioluminescence imaging. Mice with logarithmically growing tumors were treated with intraperitoneal injection of ARA-C at 50 mg/kg daily for four doses or vehicle control. In both a pilot experiment A: and a larger study with cohorts of nine mice in each group B: control mice continued to have logarithmic tumor growth, necessitating sacrifice within 1 wk of starting treatments. By comparison, ARA-C-treated mice had stable disease or tumor regression with seven of nine animals showing regression.(B) Arrows indicate days of drug dosing, and the asterisk indicates p < 0.05 by Student's t-test (two-tailed assuming samples with equal variance). Error bars show the standard deviation across nine replicates. Bioluminescence was measured as photonic flux through standardized regions of interest (photons per second per region of interest), which did not include reflected light along the left flank (e.g., right animal in part [A] at day 17 of ARA-C treatment).
Mentions: A xenograft model of Ewing sarcoma was used to evaluate the effects of ARA-C on in vivo tumor growth. A673 cells were engineered to express firefly luciferase, and tumor burden was monitored by in vivo bioluminescence imaging as previously described [14]. After inoculation of cells in the flanks of nude mice, tumors were allowed to establish for 2–3 wk. Mice with logarithmically growing tumors were divided into cohorts and treated with either systemic ARA-C at 50 mg/kg daily for four doses or vehicle control. Whereas vehicle-treated animals had tumors that continued to grow logarithmically, ARA-C-treated animals had significantly reduced tumor growth or tumor regression (Figure 6A and 6B). There was no significant effect on body weight in ARA-C treated animals, suggesting no significant systemic toxicity at this dose (unpublished data).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Oncology, Dana-Farber Cancer Institute and Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus