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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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Related in: MedlinePlus

EWS/FLI Off Marker Gene Selection(A) EWS/FLI Western blot analysis with anti-FLI antibody confirms reduction in protein levels with infection of the A673 Ewing sarcoma cell line with retrovirus containing an shRNA construct directed against EWS/FLI (EF-2-RNAi) compared to a control with RNAi directed against EWS/ERG (ERG-RNAi) which is not expressed in A673 cells. Actin is shown as a loading control.(B) Gene expression profiling was performed with shRNA designed against a luciferase control (four replicates) and in duplicate against two different shRNA constructs against EWS/FLI. Using the signal-to-noise ratio, the genes distinguishing luciferase from EWS/FLI directed RNAi were identified and then prioritized for a fold change of at least 2.5 and a baseline level of expression less than 25 in one of the states. These genes were then evaluated in an inducible EWS/FLI rescue system. Exogenous EWS/FLI was induced over 72 h in A673 cells with EWS/FLI knockdown by shRNA, and samples evaluated in duplicate at multiple time points. The EWS/FLI off signature genes are shown in the heat map. Samples are shown in columns and genes in rows. Blue represents poorly expressed genes, and red depicts highly expressed genes.
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pmed-0040122-g001: EWS/FLI Off Marker Gene Selection(A) EWS/FLI Western blot analysis with anti-FLI antibody confirms reduction in protein levels with infection of the A673 Ewing sarcoma cell line with retrovirus containing an shRNA construct directed against EWS/FLI (EF-2-RNAi) compared to a control with RNAi directed against EWS/ERG (ERG-RNAi) which is not expressed in A673 cells. Actin is shown as a loading control.(B) Gene expression profiling was performed with shRNA designed against a luciferase control (four replicates) and in duplicate against two different shRNA constructs against EWS/FLI. Using the signal-to-noise ratio, the genes distinguishing luciferase from EWS/FLI directed RNAi were identified and then prioritized for a fold change of at least 2.5 and a baseline level of expression less than 25 in one of the states. These genes were then evaluated in an inducible EWS/FLI rescue system. Exogenous EWS/FLI was induced over 72 h in A673 cells with EWS/FLI knockdown by shRNA, and samples evaluated in duplicate at multiple time points. The EWS/FLI off signature genes are shown in the heat map. Samples are shown in columns and genes in rows. Blue represents poorly expressed genes, and red depicts highly expressed genes.

Mentions: We first designed a signature for use in the GE-HTS platform. Signature genes were selected based on the following criteria: (i) statistically different expression in the EWS/FLI knockdown versus control A673 cells (at p < 0.05 based on permutation testing); (ii) fold-change over 2.5; (iii) nearly undetectable expression in one of the two conditions; and (iv) reversion of expression with induction of the EWS/FLI rescue construct. We selected 14 genes meeting these criteria (Figure 1). GAPD was included as a control for well-to-well variability in the screen because of its stable expression across the dataset. FLI1 was included to demonstrate EWS/FLI knockdown in positive control wells with EWS/FLI directed shRNA. The final signature thus constituted 16 genes.


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)

EWS/FLI Off Marker Gene Selection(A) EWS/FLI Western blot analysis with anti-FLI antibody confirms reduction in protein levels with infection of the A673 Ewing sarcoma cell line with retrovirus containing an shRNA construct directed against EWS/FLI (EF-2-RNAi) compared to a control with RNAi directed against EWS/ERG (ERG-RNAi) which is not expressed in A673 cells. Actin is shown as a loading control.(B) Gene expression profiling was performed with shRNA designed against a luciferase control (four replicates) and in duplicate against two different shRNA constructs against EWS/FLI. Using the signal-to-noise ratio, the genes distinguishing luciferase from EWS/FLI directed RNAi were identified and then prioritized for a fold change of at least 2.5 and a baseline level of expression less than 25 in one of the states. These genes were then evaluated in an inducible EWS/FLI rescue system. Exogenous EWS/FLI was induced over 72 h in A673 cells with EWS/FLI knockdown by shRNA, and samples evaluated in duplicate at multiple time points. The EWS/FLI off signature genes are shown in the heat map. Samples are shown in columns and genes in rows. Blue represents poorly expressed genes, and red depicts highly expressed genes.
© Copyright Policy
Related In: Results  -  Collection

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

pmed-0040122-g001: EWS/FLI Off Marker Gene Selection(A) EWS/FLI Western blot analysis with anti-FLI antibody confirms reduction in protein levels with infection of the A673 Ewing sarcoma cell line with retrovirus containing an shRNA construct directed against EWS/FLI (EF-2-RNAi) compared to a control with RNAi directed against EWS/ERG (ERG-RNAi) which is not expressed in A673 cells. Actin is shown as a loading control.(B) Gene expression profiling was performed with shRNA designed against a luciferase control (four replicates) and in duplicate against two different shRNA constructs against EWS/FLI. Using the signal-to-noise ratio, the genes distinguishing luciferase from EWS/FLI directed RNAi were identified and then prioritized for a fold change of at least 2.5 and a baseline level of expression less than 25 in one of the states. These genes were then evaluated in an inducible EWS/FLI rescue system. Exogenous EWS/FLI was induced over 72 h in A673 cells with EWS/FLI knockdown by shRNA, and samples evaluated in duplicate at multiple time points. The EWS/FLI off signature genes are shown in the heat map. Samples are shown in columns and genes in rows. Blue represents poorly expressed genes, and red depicts highly expressed genes.
Mentions: We first designed a signature for use in the GE-HTS platform. Signature genes were selected based on the following criteria: (i) statistically different expression in the EWS/FLI knockdown versus control A673 cells (at p < 0.05 based on permutation testing); (ii) fold-change over 2.5; (iii) nearly undetectable expression in one of the two conditions; and (iv) reversion of expression with induction of the EWS/FLI rescue construct. We selected 14 genes meeting these criteria (Figure 1). GAPD was included as a control for well-to-well variability in the screen because of its stable expression across the dataset. FLI1 was included to demonstrate EWS/FLI knockdown in positive control wells with EWS/FLI directed shRNA. The final signature thus constituted 16 genes.

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