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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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ARA-C Abrogates Anchorage-Independent Growth, a Hallmark of Oncogenic Transformation(A) ARA-C at 30 nM attenuates anchorage-independent growth as assessed by a soft agar assay and abrogates it at 60 nM as does shRNA against the EWS/FLI translocation.(B) ARA-C at 30 and 60 nM dosing decreases mean cumulative population doubling time by only 18% and 32%, respectively, at day 15. Error bars show the standard deviation across three replicates.
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pmed-0040122-g005: ARA-C Abrogates Anchorage-Independent Growth, a Hallmark of Oncogenic Transformation(A) ARA-C at 30 nM attenuates anchorage-independent growth as assessed by a soft agar assay and abrogates it at 60 nM as does shRNA against the EWS/FLI translocation.(B) ARA-C at 30 and 60 nM dosing decreases mean cumulative population doubling time by only 18% and 32%, respectively, at day 15. Error bars show the standard deviation across three replicates.

Mentions: It had previously been reported that EWS/FLI RNAi knockdown in A673 cells abrogates anchorage-independent growth, one of the hallmarks of oncogenic transformation [14]. We sought to determine whether ARA-C would phenocopy this effect in A673 cells. We treated A673 cells with ARA-C at doses below the 3 d EC50 (30 and 60 nM) and evaluated colony formation in soft agar. At 30-nM ARA-C, cells were limited in their ability to form colonies. A total of 60 nM ARA-C nearly completely abrogated colony formation (Figure 5A). It is possible that loss of colony formation is merely a result of ARA-C inhibition of A673 cell viability rather than independent inhibition of transformation. In order to address this issue, we simultaneously performed a 3T10 serial passage growth assay to evaluate effects of these conditions on A673 doubling time (Figure 5B). A total of 15 d of treatment with ARA-C at 30 and 60 nM decreased mean population doubling time by 18% and 32%, respectively, compared to parental controls. Thus, while we see only modest effects of low dose ARA-C on cell growth, we see dramatic effects on transformation.


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)

ARA-C Abrogates Anchorage-Independent Growth, a Hallmark of Oncogenic Transformation(A) ARA-C at 30 nM attenuates anchorage-independent growth as assessed by a soft agar assay and abrogates it at 60 nM as does shRNA against the EWS/FLI translocation.(B) ARA-C at 30 and 60 nM dosing decreases mean cumulative population doubling time by only 18% and 32%, respectively, at day 15. Error bars show the standard deviation across three replicates.
© Copyright Policy
Related In: Results  -  Collection

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

pmed-0040122-g005: ARA-C Abrogates Anchorage-Independent Growth, a Hallmark of Oncogenic Transformation(A) ARA-C at 30 nM attenuates anchorage-independent growth as assessed by a soft agar assay and abrogates it at 60 nM as does shRNA against the EWS/FLI translocation.(B) ARA-C at 30 and 60 nM dosing decreases mean cumulative population doubling time by only 18% and 32%, respectively, at day 15. Error bars show the standard deviation across three replicates.
Mentions: It had previously been reported that EWS/FLI RNAi knockdown in A673 cells abrogates anchorage-independent growth, one of the hallmarks of oncogenic transformation [14]. We sought to determine whether ARA-C would phenocopy this effect in A673 cells. We treated A673 cells with ARA-C at doses below the 3 d EC50 (30 and 60 nM) and evaluated colony formation in soft agar. At 30-nM ARA-C, cells were limited in their ability to form colonies. A total of 60 nM ARA-C nearly completely abrogated colony formation (Figure 5A). It is possible that loss of colony formation is merely a result of ARA-C inhibition of A673 cell viability rather than independent inhibition of transformation. In order to address this issue, we simultaneously performed a 3T10 serial passage growth assay to evaluate effects of these conditions on A673 doubling time (Figure 5B). A total of 15 d of treatment with ARA-C at 30 and 60 nM decreased mean population doubling time by 18% and 32%, respectively, compared to parental controls. Thus, while we see only modest effects of low dose ARA-C on cell growth, we see dramatic effects on transformation.

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