Identification of an Inhibitor of the EWS-FLI1 Oncogenic Transcription Factor by High-Throughput Screening
Background Chromosomal translocations generating oncogenic transcription factors are the hallmark of a variety of tumors, including many sarcomas. Ewing sarcoma family of tumors (ESFTs) are characterized by the t(11;22)(q24;q12) translocation that generates the Ewing sarcoma breakpoint region 1 and...
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| creator | Grohar, Patrick J. Woldemichael, Girma M. Griffin, Laurie B. Mendoza, Arnulfo Chen, Qing-Rong Yeung, Choh Currier, Duane G. Davis, Sean Khanna, Chand Khan, Javed McMahon, James B. Helman, Lee J. |
| description | Background
Chromosomal translocations generating oncogenic transcription factors are the hallmark of a variety of tumors, including many sarcomas. Ewing sarcoma family of tumors (ESFTs) are characterized by the t(11;22)(q24;q12) translocation that generates the Ewing sarcoma breakpoint region 1 and Friend leukemia virus integration 1 (EWS-FLI1) fusion transcription factor responsible for the highly malignant phenotype of this tumor. Although continued expression of EWS-FLI1 is believed to be critical for ESFT cell survival, a clinically effective small-molecule inhibitor remains elusive likely because EWS-FLI1 is a transcription factor and therefore widely felt to be "undruggable."
Methods
We developed a high-throughput screen to evaluate more than 50 000 compounds for inhibition of EWS-FLI1 activity in TC32 ESFT cells. We used a TC32 cell-based luciferase reporter screen using the EWS-FLI1 downstream target NR0B1 promoter and a gene signature secondary screen to sort and prioritize the compounds. We characterized the lead compound, mithramycin, based on its ability to inhibit EWS-FLI1 activity in vitro using microarray expression profiling, quantitative reverse transcription-polymerase chain reaction, and immunoblot analysis, and in vivo using immunohistochemistry. We studied the impact of this inhibition on cell viability in vitro and on tumor growth in ESFT xenograft models in vivo (n = 15-20 mice per group). All statistical tests were two-sided.
Results
Mithramycin inhibited expression of EWS-FLI1 downstream targets at the mRNA and protein levels and decreased the growth of ESFT cells at half maximal inhibitory concentrations between 10 (95% confidence interval [CI] = 8 to 13 nM) and 15 nM (95% CI = 13 to 19 nM). Mithramycin suppressed the growth of two different ESFT xenograft tumors and prolonged the survival of ESFT xenograft-bearing mice by causing a decrease in mean tumor volume. For example, in the TC32 xenograft model, on day 15 of treatment, the mean tumor volume for the mithramycin-treated mice was approximately 3% of the tumor volume observed in the control mice (mithramycin vs control: 69 vs 2388 mm3, difference = 2319 mm3, 95% CI = 1766 to 2872 mm3, P < .001).
Conclusion
Mithramycin inhibits EWS-FLI1 activity and demonstrates ESFT antitumor activity both in vitro and in vivo. |
| doi_str_mv | 10.1093/jnci/djr156 |
| format | Article |
| fullrecord | <record><control><sourceid>proquest_pubme</sourceid><recordid>TN_cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_3119649</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><oup_id>10.1093/jnci/djr156</oup_id><sourcerecordid>907168146</sourcerecordid><originalsourceid>FETCH-LOGICAL-c566t-baa497c7a76f3a6b3b0afba6ec887a2712641aa02950a92fae0994a1b90fd2c3</originalsourceid><addsrcrecordid>eNp9kc1r3DAQxUVoaTZJT7kXU2hyKE70YcurSyGEbLKwkEMWehRjrWTLeCVXsgv576vNbtKPQ3URQr95M28eQucEXxEs2HXnlL3edIGU_AjNSMFxTgku36EZxrTK5_OqOEYnMXY4HUGLD-iYEl4yQdkMdcuNdqM1VsFovcu8ycBlS9fa2o4-7N5jq7O770_5YrUk2aNTvtHOqmwdwEUV7PBStwC1w-vn7ME2bb5ug5-adpjG7EkFnQpcc4beG-ij_ni4T9F6cbe-fchXj_fL25tVrkrOx7wGKESlKqi4YcBrVmMwNXCtkhOgFaG8IACYihKDoAY0FqIAUgtsNlSxU_RtLztM9VZvVLIXoJdDsFsIz9KDlX__ONvKxv-UjBDBC5EELg8Cwf-YdBzl1kal-x6c9lOUAleEz9OeE_n5H7LzU3DJnExLFwynHBL0dQ-p4GMM2ryNQrDcBSh3Acp9gIn-9Of0b-xrYgn4cgAgKuhNSkHZ-JsrGK34C3ex5_w0_LfjL-hQsvM</addsrcrecordid><sourcetype>Open Access Repository</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>874930210</pqid></control><display><type>article</type><title>Identification of an Inhibitor of the EWS-FLI1 Oncogenic Transcription Factor by High-Throughput Screening</title><source>Oxford University Press Journals All Titles (1996-Current)</source><source>MEDLINE</source><source>Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals</source><source>Alma/SFX Local Collection</source><creator>Grohar, Patrick J. ; Woldemichael, Girma M. ; Griffin, Laurie B. ; Mendoza, Arnulfo ; Chen, Qing-Rong ; Yeung, Choh ; Currier, Duane G. ; Davis, Sean ; Khanna, Chand ; Khan, Javed ; McMahon, James B. ; Helman, Lee J.</creator><creatorcontrib>Grohar, Patrick J. ; Woldemichael, Girma M. ; Griffin, Laurie B. ; Mendoza, Arnulfo ; Chen, Qing-Rong ; Yeung, Choh ; Currier, Duane G. ; Davis, Sean ; Khanna, Chand ; Khan, Javed ; McMahon, James B. ; Helman, Lee J.</creatorcontrib><description>Background
Chromosomal translocations generating oncogenic transcription factors are the hallmark of a variety of tumors, including many sarcomas. Ewing sarcoma family of tumors (ESFTs) are characterized by the t(11;22)(q24;q12) translocation that generates the Ewing sarcoma breakpoint region 1 and Friend leukemia virus integration 1 (EWS-FLI1) fusion transcription factor responsible for the highly malignant phenotype of this tumor. Although continued expression of EWS-FLI1 is believed to be critical for ESFT cell survival, a clinically effective small-molecule inhibitor remains elusive likely because EWS-FLI1 is a transcription factor and therefore widely felt to be "undruggable."
Methods
We developed a high-throughput screen to evaluate more than 50 000 compounds for inhibition of EWS-FLI1 activity in TC32 ESFT cells. We used a TC32 cell-based luciferase reporter screen using the EWS-FLI1 downstream target NR0B1 promoter and a gene signature secondary screen to sort and prioritize the compounds. We characterized the lead compound, mithramycin, based on its ability to inhibit EWS-FLI1 activity in vitro using microarray expression profiling, quantitative reverse transcription-polymerase chain reaction, and immunoblot analysis, and in vivo using immunohistochemistry. We studied the impact of this inhibition on cell viability in vitro and on tumor growth in ESFT xenograft models in vivo (n = 15-20 mice per group). All statistical tests were two-sided.
Results
Mithramycin inhibited expression of EWS-FLI1 downstream targets at the mRNA and protein levels and decreased the growth of ESFT cells at half maximal inhibitory concentrations between 10 (95% confidence interval [CI] = 8 to 13 nM) and 15 nM (95% CI = 13 to 19 nM). Mithramycin suppressed the growth of two different ESFT xenograft tumors and prolonged the survival of ESFT xenograft-bearing mice by causing a decrease in mean tumor volume. For example, in the TC32 xenograft model, on day 15 of treatment, the mean tumor volume for the mithramycin-treated mice was approximately 3% of the tumor volume observed in the control mice (mithramycin vs control: 69 vs 2388 mm3, difference = 2319 mm3, 95% CI = 1766 to 2872 mm3, P < .001).
Conclusion
Mithramycin inhibits EWS-FLI1 activity and demonstrates ESFT antitumor activity both in vitro and in vivo.</description><identifier>ISSN: 0027-8874</identifier><identifier>EISSN: 1460-2105</identifier><identifier>DOI: 10.1093/jnci/djr156</identifier><identifier>PMID: 21653923</identifier><identifier>CODEN: JNCIEQ</identifier><language>eng</language><publisher>Cary, NC: Oxford University Press</publisher><subject>Animals ; Antineoplastic Agents - pharmacology ; Biological and medical sciences ; Bones ; Cell Line, Tumor ; Cell Proliferation - drug effects ; Cell Survival - drug effects ; Cell Survival - genetics ; DNA Damage - drug effects ; Dose-Response Relationship, Drug ; Gene expression ; Gene Expression Regulation, Neoplastic - drug effects ; Genotype & phenotype ; High-Throughput Screening Assays - methods ; Humans ; Immunoblotting ; Immunohistochemistry ; Inhibitor drugs ; Medical sciences ; Medical screening ; Mice ; Microscopy, Confocal ; Oncogene Proteins, Fusion - drug effects ; Oncogene Proteins, Fusion - genetics ; Plicamycin - pharmacology ; Protein Array Analysis ; Proto-Oncogene Protein c-fli-1 - drug effects ; Proto-Oncogene Protein c-fli-1 - genetics ; Reverse Transcriptase Polymerase Chain Reaction ; RNA-Binding Protein EWS - drug effects ; RNA-Binding Protein EWS - genetics ; Sarcoma, Ewing - drug therapy ; Sarcoma, Ewing - genetics ; Transcription Factors - genetics ; Transcription, Genetic - drug effects ; Translocation, Genetic - drug effects ; Transplantation, Heterologous ; Tumors</subject><ispartof>JNCI : Journal of the National Cancer Institute, 2011-06, Vol.103 (12), p.962-978</ispartof><rights>Published by Oxford University Press 2011. 2011</rights><rights>2015 INIST-CNRS</rights><rights>Copyright Oxford Publishing Limited(England) Jun 22, 2011</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c566t-baa497c7a76f3a6b3b0afba6ec887a2712641aa02950a92fae0994a1b90fd2c3</citedby><cites>FETCH-LOGICAL-c566t-baa497c7a76f3a6b3b0afba6ec887a2712641aa02950a92fae0994a1b90fd2c3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,314,776,780,881,1578,27901,27902</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=24327623$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21653923$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Grohar, Patrick J.</creatorcontrib><creatorcontrib>Woldemichael, Girma M.</creatorcontrib><creatorcontrib>Griffin, Laurie B.</creatorcontrib><creatorcontrib>Mendoza, Arnulfo</creatorcontrib><creatorcontrib>Chen, Qing-Rong</creatorcontrib><creatorcontrib>Yeung, Choh</creatorcontrib><creatorcontrib>Currier, Duane G.</creatorcontrib><creatorcontrib>Davis, Sean</creatorcontrib><creatorcontrib>Khanna, Chand</creatorcontrib><creatorcontrib>Khan, Javed</creatorcontrib><creatorcontrib>McMahon, James B.</creatorcontrib><creatorcontrib>Helman, Lee J.</creatorcontrib><title>Identification of an Inhibitor of the EWS-FLI1 Oncogenic Transcription Factor by High-Throughput Screening</title><title>JNCI : Journal of the National Cancer Institute</title><addtitle>J Natl Cancer Inst</addtitle><description>Background
Chromosomal translocations generating oncogenic transcription factors are the hallmark of a variety of tumors, including many sarcomas. Ewing sarcoma family of tumors (ESFTs) are characterized by the t(11;22)(q24;q12) translocation that generates the Ewing sarcoma breakpoint region 1 and Friend leukemia virus integration 1 (EWS-FLI1) fusion transcription factor responsible for the highly malignant phenotype of this tumor. Although continued expression of EWS-FLI1 is believed to be critical for ESFT cell survival, a clinically effective small-molecule inhibitor remains elusive likely because EWS-FLI1 is a transcription factor and therefore widely felt to be "undruggable."
Methods
We developed a high-throughput screen to evaluate more than 50 000 compounds for inhibition of EWS-FLI1 activity in TC32 ESFT cells. We used a TC32 cell-based luciferase reporter screen using the EWS-FLI1 downstream target NR0B1 promoter and a gene signature secondary screen to sort and prioritize the compounds. We characterized the lead compound, mithramycin, based on its ability to inhibit EWS-FLI1 activity in vitro using microarray expression profiling, quantitative reverse transcription-polymerase chain reaction, and immunoblot analysis, and in vivo using immunohistochemistry. We studied the impact of this inhibition on cell viability in vitro and on tumor growth in ESFT xenograft models in vivo (n = 15-20 mice per group). All statistical tests were two-sided.
Results
Mithramycin inhibited expression of EWS-FLI1 downstream targets at the mRNA and protein levels and decreased the growth of ESFT cells at half maximal inhibitory concentrations between 10 (95% confidence interval [CI] = 8 to 13 nM) and 15 nM (95% CI = 13 to 19 nM). Mithramycin suppressed the growth of two different ESFT xenograft tumors and prolonged the survival of ESFT xenograft-bearing mice by causing a decrease in mean tumor volume. For example, in the TC32 xenograft model, on day 15 of treatment, the mean tumor volume for the mithramycin-treated mice was approximately 3% of the tumor volume observed in the control mice (mithramycin vs control: 69 vs 2388 mm3, difference = 2319 mm3, 95% CI = 1766 to 2872 mm3, P < .001).
Conclusion
Mithramycin inhibits EWS-FLI1 activity and demonstrates ESFT antitumor activity both in vitro and in vivo.</description><subject>Animals</subject><subject>Antineoplastic Agents - pharmacology</subject><subject>Biological and medical sciences</subject><subject>Bones</subject><subject>Cell Line, Tumor</subject><subject>Cell Proliferation - drug effects</subject><subject>Cell Survival - drug effects</subject><subject>Cell Survival - genetics</subject><subject>DNA Damage - drug effects</subject><subject>Dose-Response Relationship, Drug</subject><subject>Gene expression</subject><subject>Gene Expression Regulation, Neoplastic - drug effects</subject><subject>Genotype & phenotype</subject><subject>High-Throughput Screening Assays - methods</subject><subject>Humans</subject><subject>Immunoblotting</subject><subject>Immunohistochemistry</subject><subject>Inhibitor drugs</subject><subject>Medical sciences</subject><subject>Medical screening</subject><subject>Mice</subject><subject>Microscopy, Confocal</subject><subject>Oncogene Proteins, Fusion - drug effects</subject><subject>Oncogene Proteins, Fusion - genetics</subject><subject>Plicamycin - pharmacology</subject><subject>Protein Array Analysis</subject><subject>Proto-Oncogene Protein c-fli-1 - drug effects</subject><subject>Proto-Oncogene Protein c-fli-1 - genetics</subject><subject>Reverse Transcriptase Polymerase Chain Reaction</subject><subject>RNA-Binding Protein EWS - drug effects</subject><subject>RNA-Binding Protein EWS - genetics</subject><subject>Sarcoma, Ewing - drug therapy</subject><subject>Sarcoma, Ewing - genetics</subject><subject>Transcription Factors - genetics</subject><subject>Transcription, Genetic - drug effects</subject><subject>Translocation, Genetic - drug effects</subject><subject>Transplantation, Heterologous</subject><subject>Tumors</subject><issn>0027-8874</issn><issn>1460-2105</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kc1r3DAQxUVoaTZJT7kXU2hyKE70YcurSyGEbLKwkEMWehRjrWTLeCVXsgv576vNbtKPQ3URQr95M28eQucEXxEs2HXnlL3edIGU_AjNSMFxTgku36EZxrTK5_OqOEYnMXY4HUGLD-iYEl4yQdkMdcuNdqM1VsFovcu8ycBlS9fa2o4-7N5jq7O770_5YrUk2aNTvtHOqmwdwEUV7PBStwC1w-vn7ME2bb5ug5-adpjG7EkFnQpcc4beG-ij_ni4T9F6cbe-fchXj_fL25tVrkrOx7wGKESlKqi4YcBrVmMwNXCtkhOgFaG8IACYihKDoAY0FqIAUgtsNlSxU_RtLztM9VZvVLIXoJdDsFsIz9KDlX__ONvKxv-UjBDBC5EELg8Cwf-YdBzl1kal-x6c9lOUAleEz9OeE_n5H7LzU3DJnExLFwynHBL0dQ-p4GMM2ryNQrDcBSh3Acp9gIn-9Of0b-xrYgn4cgAgKuhNSkHZ-JsrGK34C3ex5_w0_LfjL-hQsvM</recordid><startdate>20110622</startdate><enddate>20110622</enddate><creator>Grohar, Patrick J.</creator><creator>Woldemichael, Girma M.</creator><creator>Griffin, Laurie B.</creator><creator>Mendoza, Arnulfo</creator><creator>Chen, Qing-Rong</creator><creator>Yeung, Choh</creator><creator>Currier, Duane G.</creator><creator>Davis, Sean</creator><creator>Khanna, Chand</creator><creator>Khan, Javed</creator><creator>McMahon, James B.</creator><creator>Helman, Lee J.</creator><general>Oxford University Press</general><general>Oxford Publishing Limited (England)</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7TO</scope><scope>7U7</scope><scope>7U9</scope><scope>C1K</scope><scope>H94</scope><scope>K9.</scope><scope>NAPCQ</scope><scope>7TM</scope><scope>5PM</scope></search><sort><creationdate>20110622</creationdate><title>Identification of an Inhibitor of the EWS-FLI1 Oncogenic Transcription Factor by High-Throughput Screening</title><author>Grohar, Patrick J. ; Woldemichael, Girma M. ; Griffin, Laurie B. ; Mendoza, Arnulfo ; Chen, Qing-Rong ; Yeung, Choh ; Currier, Duane G. ; Davis, Sean ; Khanna, Chand ; Khan, Javed ; McMahon, James B. ; Helman, Lee J.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c566t-baa497c7a76f3a6b3b0afba6ec887a2712641aa02950a92fae0994a1b90fd2c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Animals</topic><topic>Antineoplastic Agents - pharmacology</topic><topic>Biological and medical sciences</topic><topic>Bones</topic><topic>Cell Line, Tumor</topic><topic>Cell Proliferation - drug effects</topic><topic>Cell Survival - drug effects</topic><topic>Cell Survival - genetics</topic><topic>DNA Damage - drug effects</topic><topic>Dose-Response Relationship, Drug</topic><topic>Gene expression</topic><topic>Gene Expression Regulation, Neoplastic - drug effects</topic><topic>Genotype & phenotype</topic><topic>High-Throughput Screening Assays - methods</topic><topic>Humans</topic><topic>Immunoblotting</topic><topic>Immunohistochemistry</topic><topic>Inhibitor drugs</topic><topic>Medical sciences</topic><topic>Medical screening</topic><topic>Mice</topic><topic>Microscopy, Confocal</topic><topic>Oncogene Proteins, Fusion - drug effects</topic><topic>Oncogene Proteins, Fusion - genetics</topic><topic>Plicamycin - pharmacology</topic><topic>Protein Array Analysis</topic><topic>Proto-Oncogene Protein c-fli-1 - drug effects</topic><topic>Proto-Oncogene Protein c-fli-1 - genetics</topic><topic>Reverse Transcriptase Polymerase Chain Reaction</topic><topic>RNA-Binding Protein EWS - drug effects</topic><topic>RNA-Binding Protein EWS - genetics</topic><topic>Sarcoma, Ewing - drug therapy</topic><topic>Sarcoma, Ewing - genetics</topic><topic>Transcription Factors - genetics</topic><topic>Transcription, Genetic - drug effects</topic><topic>Translocation, Genetic - drug effects</topic><topic>Transplantation, Heterologous</topic><topic>Tumors</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Grohar, Patrick J.</creatorcontrib><creatorcontrib>Woldemichael, Girma M.</creatorcontrib><creatorcontrib>Griffin, Laurie B.</creatorcontrib><creatorcontrib>Mendoza, Arnulfo</creatorcontrib><creatorcontrib>Chen, Qing-Rong</creatorcontrib><creatorcontrib>Yeung, Choh</creatorcontrib><creatorcontrib>Currier, Duane G.</creatorcontrib><creatorcontrib>Davis, Sean</creatorcontrib><creatorcontrib>Khanna, Chand</creatorcontrib><creatorcontrib>Khan, Javed</creatorcontrib><creatorcontrib>McMahon, James B.</creatorcontrib><creatorcontrib>Helman, Lee J.</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Oncogenes and Growth Factors Abstracts</collection><collection>Toxicology Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Environmental Sciences and Pollution Management</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Nursing & Allied Health Premium</collection><collection>Nucleic Acids Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>JNCI : Journal of the National Cancer Institute</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Grohar, Patrick J.</au><au>Woldemichael, Girma M.</au><au>Griffin, Laurie B.</au><au>Mendoza, Arnulfo</au><au>Chen, Qing-Rong</au><au>Yeung, Choh</au><au>Currier, Duane G.</au><au>Davis, Sean</au><au>Khanna, Chand</au><au>Khan, Javed</au><au>McMahon, James B.</au><au>Helman, Lee J.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Identification of an Inhibitor of the EWS-FLI1 Oncogenic Transcription Factor by High-Throughput Screening</atitle><jtitle>JNCI : Journal of the National Cancer Institute</jtitle><addtitle>J Natl Cancer Inst</addtitle><date>2011-06-22</date><risdate>2011</risdate><volume>103</volume><issue>12</issue><spage>962</spage><epage>978</epage><pages>962-978</pages><issn>0027-8874</issn><eissn>1460-2105</eissn><coden>JNCIEQ</coden><abstract>Background
Chromosomal translocations generating oncogenic transcription factors are the hallmark of a variety of tumors, including many sarcomas. Ewing sarcoma family of tumors (ESFTs) are characterized by the t(11;22)(q24;q12) translocation that generates the Ewing sarcoma breakpoint region 1 and Friend leukemia virus integration 1 (EWS-FLI1) fusion transcription factor responsible for the highly malignant phenotype of this tumor. Although continued expression of EWS-FLI1 is believed to be critical for ESFT cell survival, a clinically effective small-molecule inhibitor remains elusive likely because EWS-FLI1 is a transcription factor and therefore widely felt to be "undruggable."
Methods
We developed a high-throughput screen to evaluate more than 50 000 compounds for inhibition of EWS-FLI1 activity in TC32 ESFT cells. We used a TC32 cell-based luciferase reporter screen using the EWS-FLI1 downstream target NR0B1 promoter and a gene signature secondary screen to sort and prioritize the compounds. We characterized the lead compound, mithramycin, based on its ability to inhibit EWS-FLI1 activity in vitro using microarray expression profiling, quantitative reverse transcription-polymerase chain reaction, and immunoblot analysis, and in vivo using immunohistochemistry. We studied the impact of this inhibition on cell viability in vitro and on tumor growth in ESFT xenograft models in vivo (n = 15-20 mice per group). All statistical tests were two-sided.
Results
Mithramycin inhibited expression of EWS-FLI1 downstream targets at the mRNA and protein levels and decreased the growth of ESFT cells at half maximal inhibitory concentrations between 10 (95% confidence interval [CI] = 8 to 13 nM) and 15 nM (95% CI = 13 to 19 nM). Mithramycin suppressed the growth of two different ESFT xenograft tumors and prolonged the survival of ESFT xenograft-bearing mice by causing a decrease in mean tumor volume. For example, in the TC32 xenograft model, on day 15 of treatment, the mean tumor volume for the mithramycin-treated mice was approximately 3% of the tumor volume observed in the control mice (mithramycin vs control: 69 vs 2388 mm3, difference = 2319 mm3, 95% CI = 1766 to 2872 mm3, P < .001).
Conclusion
Mithramycin inhibits EWS-FLI1 activity and demonstrates ESFT antitumor activity both in vitro and in vivo.</abstract><cop>Cary, NC</cop><pub>Oxford University Press</pub><pmid>21653923</pmid><doi>10.1093/jnci/djr156</doi><tpages>17</tpages><oa>free_for_read</oa></addata></record> |
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| source | Oxford University Press Journals All Titles (1996-Current); MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
| subjects | Animals Antineoplastic Agents - pharmacology Biological and medical sciences Bones Cell Line, Tumor Cell Proliferation - drug effects Cell Survival - drug effects Cell Survival - genetics DNA Damage - drug effects Dose-Response Relationship, Drug Gene expression Gene Expression Regulation, Neoplastic - drug effects Genotype & phenotype High-Throughput Screening Assays - methods Humans Immunoblotting Immunohistochemistry Inhibitor drugs Medical sciences Medical screening Mice Microscopy, Confocal Oncogene Proteins, Fusion - drug effects Oncogene Proteins, Fusion - genetics Plicamycin - pharmacology Protein Array Analysis Proto-Oncogene Protein c-fli-1 - drug effects Proto-Oncogene Protein c-fli-1 - genetics Reverse Transcriptase Polymerase Chain Reaction RNA-Binding Protein EWS - drug effects RNA-Binding Protein EWS - genetics Sarcoma, Ewing - drug therapy Sarcoma, Ewing - genetics Transcription Factors - genetics Transcription, Genetic - drug effects Translocation, Genetic - drug effects Transplantation, Heterologous Tumors |
| title | Identification of an Inhibitor of the EWS-FLI1 Oncogenic Transcription Factor by High-Throughput Screening |
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