title: Targeting a DNA binding motif of the EVI1 protein by a pyrrole-imidazole polyamide.
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.
authors: Department of Pathology and Lab Medicine, University of Rochester Medical Center, Rochester, New York 14642, United States.
authors: Yi
authors: Y
authors: Zhang
authors: Géraldine
authors: G
authors: Sicot
authors: Xiaohui
authors: X
authors: Cui
authors: Marion
authors: M
authors: Vogel
authors: Charles A
authors: CA
authors: Wuertzer
authors: Kimberly
authors: K
authors: Lezon-Geyda
authors: John
authors: J
authors: Wheeler
authors: Daniel A
authors: DA
authors: Harki
authors: Katy A
authors: KA
authors: Muzikar
authors: Daniel A
authors: DA
authors: Stolper
authors: Peter B
authors: PB
authors: Dervan
authors: Archibald S
authors: AS
authors: Perkins
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D016503:Drug Delivery Systems / Q000379:methods; D006131:Growth Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D007093:Imidazoles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D000074008:MDS1 and EVI1 Complex Locus Protein; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D022423:Myeloid Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D009757:Nylons / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding / Q000235:genetics; D011519:Proto-Oncogenes / Q000235:genetics; D011758:Pyrroles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D012190:Retroviridae / Q000235:genetics; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D006131:Growth Inhibitors; D007093:Imidazoles; D000074008:MDS1 and EVI1 Complex Locus Protein; C063575:MECOM protein, human; D009757:Nylons; D011758:Pyrroles; D014157:Transcription Factors