Signature-Based Small Molecule Screening Identifies Cytosine Arabinoside as an EWS/FLI Modulator in Ewing Sarcoma

Autor: Todd R. Golub, David Peck, Stephen L. Lessnick, Andrew L. Kung, Kwan T. Chow, Jenny S. Wong, Kenneth N. Ross, Renee D. Wright, Kimberly Stegmaier
Rok vydání: 2007
Předmět:
Oncogene Proteins
Fusion
Pediatrics and Child Health
lcsh:Medicine
Biochemistry
Mice
Drug Delivery Systems
0302 clinical medicine
RNA interference
Gene duplication
Fluorometry
Regulation of gene expression
Genetics
0303 health sciences
Molecular Structure
Cytarabine
General Medicine
Microspheres
3. Good health
Gene Expression Regulation
Neoplastic
Oncology
030220 oncology & carcinogenesis
RNA Interference
Sarcoma
Research Article
medicine.drug
Antimetabolites
Antineoplastic
Bone Neoplasms
Sarcoma
Ewing
Pharmacology and Toxicology
Biology
03 medical and health sciences
Cell Line
Tumor
Drugs and Adverse Drug Reactions
medicine
Animals
Humans
Chemotherapy
Gene
Transcription factor
Fluorescent Dyes
030304 developmental biology
Proto-Oncogene Protein c-fli-1
Gene Expression Profiling
lcsh:R
Gene Amplification
Genetics and Genomics
medicine.disease
Xenograft Model Antitumor Assays
Molecular Weight
Gene expression profiling
Cancer research
Drug Screening Assays
Antitumor
RNA-Binding Protein EWS
Transcription Factors
Zdroj: PLoS Medicine, Vol 4, Iss 4, p e122 (2007)
PLoS Medicine
ISSN: 1549-1676
DOI: 10.1371/journal.pmed.0040122
Popis: 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.
Todd Golub and colleagues show that a gene expression-based screen of small-molecule libraries can identify candidate drugs that modulate cancer-associated oncoproteins.
Editors' Summary Background. Cancer occurs when cells accumulate genetic changes (mutations) that allow them to divide uncontrollably and to travel throughout the body (metastasize). Chemotherapy, a mainstay of cancer treatments, works by killing rapidly dividing cells. Because some normal tissues also contain dividing cells and are therefore sensitive to chemotherapy drugs, it is hard to treat cancer without causing serious side effects. In recent years, however, researchers have identified some of the mutations that drive the growth of cancer cells. This raises the possibility of designing drugs that kill only cancer cells by specifically targeting “oncoproteins” (the abnormal proteins generated by mutations that transform normal cells into cancer cells). Some “targeted” drugs have already reached the clinic, but unfortunately medicinal chemists do not know how to inhibit the function of many classes of oncoproteins with the small organic molecules that make the best medicines. One oncoprotein in this category is EWS/FLI. This contains part of a protein called EWS fused to part of a transcription factor (a protein that controls cell behavior by telling the cell which proteins to make) called FLI. About 80% of patients with Ewing sarcoma (the second commonest childhood cancer of bone and soft tissue) have the mutation responsible for EWS/FLI expression. Localized Ewing sarcoma can be treated with nontargeted chemotherapy (often in combination with surgery and radiotherapy), but treatment for recurrent or metastatic disease remains very poor. Why Was This Study Done? Researchers have known for years that EWS/FLI expression drives the development of Ewing sarcoma by activating the expression of target genes needed for tumor formation. However, EWS/FLI has never been exploited as a target for therapy of this cancer—mainly because traditional approaches used to screen libraries of small molecules do not identify compounds that modulate the activity of transcription factors. In this study, the researchers have used a new gene expression–based, high-throughput screening (GE-HTS) approach to identify compounds that modulate the activity of EWS/FLI. What Did the Researchers Do and Find? The researchers used a molecular biology technique called microarray expression profiling to define a 14-gene expression signature that differentiates between Ewing sarcoma cells in which the EWS/FLI fusion protein is active and those in which it is inactive. They then used this signature to screen a library of about 1,000 chemicals (many already approved for other clinical uses) in a “ligation-mediated amplification assay.” For this, the researchers grew Ewing sarcoma cells with the test chemicals, extracted RNA from the cells, and generated a DNA copy of the RNA. They then added two short pieces of DNA (probes) specific for each signature gene to the samples. In samples that expressed a given signature gene, both probes bound and were then ligated (joined together) and amplified. Because one of each probe pair also contained a unique “capture sequence,” the signature genes expressed in each sample were finally identified by adding colored fluorescent beads, each linked to DNA complementary to a different capture sequence. The most active modulator of EWS/FLI activity identified by this GE-HTS approach was cytosine arabinoside (ARA-C). At levels achievable in people, this compound reduced the abundance of EWS/FLI protein in and the viability and cancer-like behavior of Ewing sarcoma cells growing in test tubes. ARA-C treatment also slowed the growth of Ewing sarcoma cells transplanted into mice. What Do These Findings Mean? These findings identify ARA-C, which is already used to treat children with some forms of leukemia, as a potent modulator of EWS/FLI activity. More laboratory experiments are needed to discover how ARA-C changes the behavior of Ewing sarcoma cells. Nevertheless, given the poor outcomes currently seen in many patients with Ewing sarcoma and the historical reluctance to test new drugs in children, these findings strongly support the initiation of clinical trials of ARA-C in children with Ewing sarcoma. These results also show that the GE-HTS approach is a powerful way to identify candidate drugs able to modulate the activity of some of the oncoproteins (including transcription factors and other previously intractable targets) that drive cancer development. Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040122. Cancerquest from Emory University, provides information on cancer biology (also includes information in Spanish, Chinese and Russian) The MedlinePlus encyclopedia has pages on Ewing sarcoma Information for patients and health professionals on Ewing sarcoma is available from the US National Cancer Institute Cancerbackup offers information for patients and their parents on Ewing sarcoma Wikipedia has pages on DNA microarrays and expression profiling (note that Wikipedia is a free online encyclopedia that anyone can edit)
Databáze: OpenAIRE
Externí odkaz: