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, Archibald S. Perkins

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26 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)10431-10441
Number of pages11
JournalBiochemistry
Volume50
Issue number48
DOIs
StatePublished - Dec 6 2011

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    Zhang, Y., Sicot, G., Cui, X., Vogel, M., Wuertzer, C. A., Lezon-Geyda, K., Wheeler, J., Harki, D. A., Muzikar, K. A., Stolper, D. A., Dervan, P. B., & Perkins, A. S. (2011). Targeting a DNA binding motif of the EVI1 protein by a pyrrole-imidazole polyamide. Biochemistry, 50(48), 10431-10441. https://doi.org/10.1021/bi200962u