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Targeting a DNA Binding Motif of the EVI1 Protein by a Pyrrole−Imidazole Polyamide

Zhang, Yi and Sicot, Géraldine and Cui, Xiaohui and Vogel, Marion and Wuertzer, Charles A. and Lezon-Geyda, Kimberly and Wheeler, John and Harki, Daniel A. and Muzikar, Katy A. and Stolper, Daniel A. and Dervan, Peter B. and Perkins, Archibald S. (2011) Targeting a DNA Binding Motif of the EVI1 Protein by a Pyrrole−Imidazole Polyamide. Biochemistry, 50 (48). pp. 10431-10441. ISSN 0006-2960. PMCID PMC3619939. http://resolver.caltech.edu/CaltechAUTHORS:20120105-071520702

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Abstract

The zinc finger protein EVI1 is causally associated with acute myeloid leukemogenesis, and inhibition of its function with a small molecule therapeutic may provide effective therapy for EVI1-expressing leukemias. In this paper we describe the development of a pyrrole–imidazole polyamide to specifically block EVI1 binding to DNA. We first identify essential domains for leukemogenesis through structure–function studies on both EVI1 and the t(3;21)(q26;q22)-derived RUNX1-MDS1-EVI1 (RME) protein, which revealed that DNA binding to the cognate motif GACAAGATA via the first of two zinc finger domains (ZF1, encompassing fingers 1–7) is essential transforming activity. To inhibit DNA binding via ZF1, we synthesized a pyrrole–imidazole polyamide 1, designed to bind to a subsite within the GACAAGATA motif and thereby block EVI1 binding. DNase I footprinting and electromobility shift assays revealed a specific and high affinity interaction between polyamide 1 and the GACAAGATA motif. In an in vivo CAT reporter assay using NIH-3T3-derived cell line with a chromosome-embedded tet-inducible EVI1-VP16 as well as an EVI1-responsive reporter, polyamide 1 completely blocked EVI1-responsive reporter activity. Growth of a leukemic cell line bearing overexpressed EVI1 was also inhibited by treatment with polyamide 1, while a control cell line lacking EVI1 was not. Finally, colony formation by RME was attenuated by polyamide 1 in a serial replating assay. These studies provide evidence that a cell permeable small molecule may effectively block the activity of a leukemogenic transcription factor and provide a valuable tool to dissect critical functions of EVI1 in leukemogenesis.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/bi200962uDOIArticle
http://pubs.acs.org/doi/abs/10.1021/bi200962uPublisherArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3619939/PubMed CentralArticle
ORCID:
AuthorORCID
Stolper, Daniel A.0000-0003-3299-3177
Dervan, Peter B.0000-0001-8852-7306
Additional Information:© 2011 American Chemical Society. Received: June 23, 2011. Revised: September 14, 2011. Publication Date (Web): November 1, 2011. We thank Giuseppina Nucifora for providing the pBS-AML1-MDS1-EVI1 plasmid. Support for this work comes from NIH [R01 CA-112188 (A.S.P.), R01 GM51747 (P.B.D.)], the AA-MDS Foundation, and the Dobranski Foundation. D.A.H. thanks the California Tobacco-Related Disease Research Program (16FT-0055) for a postdoctoral fellowship. The National Science Foundation Chemistry Research Instrumentation and Facilities Program (CHE-0541745) is acknowledged for providing the UPLC-MS instrument.
Funders:
Funding AgencyGrant Number
NIHR01 CA-112188
NIHR01 GM51747
AA-MDS FoundationUNSPECIFIED
Dobranski Foundation UNSPECIFIED
California Tobacco-Related Disease Research Program16FT-0055
NSFCHE-0541745
PubMed Central ID:PMC3619939
Record Number:CaltechAUTHORS:20120105-071520702
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20120105-071520702
Official Citation:Targeting a DNA Binding Motif of the EVI1 Protein by a Pyrrole–Imidazole Polyamide Yi Zhang, Géraldine Sicot, Xiaohui Cui, Marion Vogel, Charles A. Wuertzer, Kimberly Lezon-Geyda, John Wheeler, Daniel A. Harki, Katy A. Muzikar, Daniel A. Stolper, Peter B. Dervan, and Archibald S. Perkins Biochemistry 2011 50 (48), 10431-10441
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:28660
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:05 Jan 2012 15:37
Last Modified:19 Nov 2016 00:54

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