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Pyrrole-imidazole polyamide targeted to the REL/ELK1 overlapping sequences in the EVI1 minimal promoter

ABSTRACT: Pyrrole-imidazole polyamides (PIPs) have been shown to inhibit gene expression by interrupting the DNA-protein interface. Human Ectopic viral integration site 1 (EVI1) is an oncogenic transcription factor which plays a key role in many aggressive forms of cancer. We have developed a novel pyrrole–imidazole polyamide, PIP1 targeting the REL/ELK1 binding site in the EVI1 minimal promoter that can significantly repress the expression of EVI1 in MDA-MB-231 cells. Whole-transcriptome analysis revealed that a fraction of EVI1-driven genes were modulated by PIP1. Global expression changes in MDA-MB-231 cells were evaluated after treating the cells with PIP1 and DMSO for 48 hours. The vehicle DMSO is used as a negative control. Each condition is performed in technical replicates.

ORGANISM(S): Homo sapiens

SUBMITTER: Syed Junetha

PROVIDER: S-ECPF-GEOD-59502 | biostudies-other |

REPOSITORIES: biostudies-other

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Pyrrole-imidazole polyamide targeted to the REL/ELK1 overlapping sequences in the EVI1 minimal promoter

Project description:Pyrrole-imidazole polyamides (PIPs) have been shown to inhibit gene expression by interrupting the DNA-protein interface. Human Ectopic viral integration site 1 (EVI1) is an oncogenic transcription factor which plays a key role in many aggressive forms of cancer. We have developed a novel pyrroleM-bM-^@M-^Simidazole polyamide, PIP1 targeting the REL/ELK1 binding site in the EVI1 minimal promoter that can significantly repress the expression of EVI1 in MDA-MB-231 cells. Whole-transcriptome analysis revealed that a fraction of EVI1-driven genes were modulated by PIP1. Global expression changes in MDA-MB-231 cells were evaluated after treating the cells with PIP1 and DMSO for 48 hours. The vehicle DMSO is used as a negative control. Each condition is performed in technical replicates.

2014-07-28 | E-GEOD-59502 | biostudies-arrayexpress

Pyrrole-imidazole polyamide targeted to the REL/ELK1 overlapping sequences in the EVI1 minimal promoter

Project description:Pyrrole-imidazole polyamides (PIPs) have been shown to inhibit gene expression by interrupting the DNA-protein interface. Human Ectopic viral integration site 1 (EVI1) is an oncogenic transcription factor which plays a key role in many aggressive forms of cancer. We have developed a novel pyrrole–imidazole polyamide, PIP1 targeting the REL/ELK1 binding site in the EVI1 minimal promoter that can significantly repress the expression of EVI1 in MDA-MB-231 cells. Whole-transcriptome analysis revealed that a fraction of EVI1-driven genes were modulated by PIP1.

2014-07-28 | GSE59502 | GEO

Targeting a DNA binding motif of the EVI1 protein by a pyrrole-imidazole polyamide.

Project description: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.

| S-EPMC3619939 | biostudies-literature

Antitumor activity of a pyrrole-imidazole polyamide

Project description:We report that a DNA minor groove binding hairpin pyrrole-imidazole (Py-Im) polyamide interferes with RNA polymerase II (RNAP2) activity in cell culture. Genome-wide mapping of RNAP2 binding shows reduction of occupancy preferentially at transcription start sites (TSS), while occupancy at enhancer sites are is unchanged. Genome-wide mapping of RNA polymerase II in LNCaP cells treated with DHT and DHT with Py-Im polyamide.

2013-01-31 | E-GEOD-43253 | biostudies-arrayexpress

Antitumor activity of a pyrrole-imidazole polyamide.

Project description:Many cancer therapeutics target DNA and exert cytotoxicity through the induction of DNA damage and inhibition of transcription. We report that a DNA minor groove binding hairpin pyrrole-imidazole (Py-Im) polyamide interferes with RNA polymerase II (RNAP2) activity in cell culture. Polyamide treatment activates p53 signaling in LNCaP prostate cancer cells without detectable DNA damage. Genome-wide mapping of RNAP2 binding shows reduction of occupancy, preferentially at transcription start sites, but occupancy at enhancer sites is unchanged. Polyamide treatment results in a time- and dose-dependent depletion of the RNAP2 large subunit RPB1 that is preventable with proteasome inhibition. This polyamide demonstrates antitumor activity in a prostate tumor xenograft model with limited host toxicity.

| S-EPMC3562772 | biostudies-literature

Suppression of GPR56 expression by pyrrole-imidazole polyamide represents a novel therapeutic drug for AML with high EVI1 expression.

Project description:G protein-coupled receptor 56 (GPR56) is highly expressed in acute myeloid leukemia (AML) cells with high EVI1 expression (EVI1high AML). Because GPR56 is a transcriptional target of EVI1 and silencing of GPR56 expression induces apoptosis, we developed a novel drug to suppress GPR56 expression in EVI1high AML cells. For this purpose, we generated pyrrole-imidazole (PI) polyamides specific to GPR56 (PIP/56-1 or PIP/56-2) as nuclease-resistant novel compounds that interfere with the binding of EVI1 to the GPR56 promoter in a sequence-specific manner. Treatment of EVI1high AML cell lines (UCSD/AML1 and Kasumi-3) with PIP/56-1 or PIP/56-2 effectively suppressed GPR56 expression by inhibiting binding of EVI1 to its promoter, leading to suppression of cell growth with increased rates of apoptosis. Moreover, intravenous administration of PIP/56-1 into immunodeficient Balb/c-RJ mice subcutaneously transplanted with UCSD/AML1 cells significantly inhibited tumor growth and extended survival. Furthermore, organ infiltration by leukemia cells in immunodeficient Balb/c-RJ mice, which were intravenously transplanted using UCSD/AML1 cells, was successfully inhibited by PIP/56-1 treatment with no apparent effects on murine hematopoietic cells. In addition, PIP treatment did not inhibit colony formation of human CD34+ progenitor cells. Thus, PI polyamide targeting of GPR56 using our compound is promising, useful, and safe for the treatment of EVI1high AML.

| S-EPMC6137133 | biostudies-literature

Antitumor activity of a pyrrole-imidazole polyamide

2013-01-31 | GSE43253 | GEO

Tandem trimer pyrrole-imidazole polyamide probes targeting 18 base pairs in human telomere sequences.

Project description:The binding of molecules to specific DNA sequences is important for imaging genome DNA and for studying gene expression. Increasing the number of base pairs targeted by these molecules would provide greater specificity. N-Methylpyrrole-N-methylimidazole (Py-Im) polyamides are one type of such molecules and can bind to the minor groove of DNA in a sequence-specific manner without causing denaturation of DNA. Our recent work has demonstrated that tandem hairpin Py-Im polyamides conjugated with a fluorescent dye can be synthesized easily and can serve as new probes for studying human telomeres under mild conditions. Herein, to improve their selectivities to telomeres by targeting longer sequences, we designed and synthesized a fluorescent tandem trimer Py-Im polyamide probe, comprising three hairpins and two connecting regions (hinges). The new motif bound to 18 bp dsDNA in human telomeric repeats (TTAGGG) n , the longest sequence for specific binding reported for Py-Im polyamides. We compared the binding affinities and the abilities to discriminate mismatch, the UV-visible absorption and fluorescence spectra, and telomere staining in human cells between the tandem trimer and a previously developed tandem hairpin. We found that the tandem trimer Py-Im polyamide probe has higher ability to recognize telomeric repeats and stains telomeres in chemically fixed cells with lower background signal.

| S-EPMC5645774 | biostudies-literature

Characterization and solubilization of pyrrole-imidazole polyamide aggregates.

Project description:To optimize the biological activity of pyrrole-imidazole polyamide DNA-binding molecules, we characterized the aggregation propensity of these compounds through dynamic light scattering and fractional solubility analysis. Nearly all studied polyamides were found to form measurable particles 50-500 nm in size under biologically relevant conditions, while HPLC-based analyses revealed solubility trends in both core sequences and peripheral substituents that did not correlate with overall ionic charge. The solubility of both hairpin and cyclic polyamides was increased upon addition of carbohydrate solubilizing agents, in particular, 2-hydroxypropyl-?-cyclodextrin (Hp?CD). In mice, the use of Hp?CD allowed for improved injection conditions and subsequent investigations of the availability of polyamides in mouse plasma to human cells. The results of these studies will influence the further design of Py-Im polyamides and facilitate their study in animal models.

| S-EPMC3375050 | biostudies-literature

Inhibition of DNA Methylation at the <i>MLH1</i> Promoter Region Using Pyrrole-Imidazole Polyamide.

Project description:Aberrant DNA methylation causes major epigenetic changes and has been implicated in cancer following the inactivation of tumor suppressor genes by hypermethylation of promoter CpG islands. Although methylated DNA regions can be randomly demethylated by 5-azacytidine and 5-aza-2'-deoxycytidine, site-specific inhibition of DNA methylation, for example, in the promoter region of a specific gene, has yet to be technically achieved. Hairpin pyrrole (Py)-imidazole (Im) polyamides are small molecules that can be designed to recognize and bind to particular DNA sequences. In this study, we synthesized the hairpin polyamide MLH1_-16 (Py-Im-β-Im-Im-Py-γ-Im-Py-β-Im-Py-Py) to target a CpG site 16 bp upstream of the transcription start site of the human MLH1 gene. MLH1 is known to be frequently silenced by promoter hypermethylation, causing microsatellite instability and a hypermutation phenotype in cancer. We show that MLH1_-16 binds to the target site and that CpG methylation around the binding site is selectively inhibited in vitro. MLH1_non, which does not have a recognition site in the MLH1 promoter, neither binds to the sequence nor inhibits DNA methylation in the region. When MLH1_-16 was used to treat RKO human colorectal cancer cells in a remethylating system involving the MLH1 promoter under hypoxic conditions (1% O2), methylation of the MLH1 promoter was inhibited in the region surrounding the compound binding site. Silencing of the MLH1 expression was also inhibited. Promoter methylation and silencing of MLH1 were not inhibited when MLH1_non was added. These results indicate that Py-Im polyamides can act as sequence-specific antagonists of CpG methylation in living cells.

| S-EPMC6044701 | biostudies-literature

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