The Stapled Peptide PM2 Stabilizes p53 Levels and Radiosensitizes Wild-Type p53 Cancer Cells.

The tumor suppressor p53 is a key mediator of cellular stress and DNA damage response cascades and is activated after exposure to ionizing radiation. Amplifying wild-type p53 expression by targeting negative regulators such as MDM2 in combination with external beam radiotherapy (EBRT) may result in increased therapeutic effects. The novel stapled peptide PM2 prevents MDM2 from suppressing wild-type p53, and is thus a promising agent for therapeutic combination with EBRT. Effects of PM2 and potential PM2-induced radiosensitivity were assessed in a panel of cancer cell lines using 2D cell viability assays. Western Blot and flow cytometric analyses were used to investigate the mechanisms behind the observed effects in samples treated with PM2 and EBRT. Finally, PM2-treatment combined with EBRT was evaluated in an in vitro 3D spheroid model. PM2-therapy decreased cell viability in wild-type p53, HPV-negative cell lines. Western Blotting and flow cytometry confirmed upregulation of p53, as well as initiation of p53-mediated apoptosis measured by increased cleaved caspase-3 and Noxa activity. Furthermore, 3D in vitro tumor spheroid experiments confirmed the superior effects of the combination, as the only treatment regime resulting in growth inhibition and complete spheroid disintegration. We conclude that PM2 induces antitumorigenic effects in wt p53 HPV-negative cancer cells and potentiates the effects of EBRT, ultimately resulting in tumor eradication in a 3D spheroid model. This strategy shows great potential as a new wt p53 specific tumor-targeting compound, and the combination of PM2 and EBRT could be a promising strategy to increase therapeutic effects and decrease adverse effects from radiotherapy.

Identification and Characterization of an eIF4e DNA Aptamer That Inhibits Proliferation With High Throughput Sequencing.

Development of DNA aptamer screens that are both simple and informative can increase the success rate of DNA aptamer selection and induce greater adoption. High eIF4e levels contribute to malignancies, thus eIF4e presents itself as a valuable target for DNA aptamer-based inhibition screen. Here, we demonstrate a method for the rapid selection of looped DNA aptamers against eIF4e by combining negative selection and purification in a single step, followed by characterization with high throughput sequencing. The resulting aptamers show functional binding to eIF4e and inhibit translation initiation in biochemical assays. When transfected into cells, eIF4e aptamers cause a dramatic loss of cell proliferation in tumor cells as seen with eIF4e knockdown with antisense oligonucleotides, shRNAs, and siRNAs, hinting at therapeutic possibilities. With the large data set provided by high throughput sequencing, we demonstrate that selection happens in waves and that sequencing data can be used to infer aptamer structure. Lastly, we show that ligation of looped aptamers can enhance their functional effects. These results demonstrate a rapid protocol to screen and optimize aptamers against macromolecules of interest.

New insights into p53 based therapy.

A common step in human cancer is the inactivation of the p53 tumor suppressor pathway. This occurs either by mutations in the coding region of the p53 gene itself, or equally commonly, by inactivation of pathways that are required for p53 to exert its cellular function. Dramatic new results from animal models and the widespread availability of p53 activating small molecules are yielding important new insights into the therapeutic and toxic effects of p53 and how these can be exploited for improving therapy of cancer and other diseases.

The electrostatic surface of MDM2 modulates the specificity of its interaction with phosphorylated and unphosphorylated p53 peptides.

Florescence anisotropy measurements using FAM-labelled p53 peptides showed that the binding of the peptides to MDM2 was dependant upon the phosphorylation of p53 at Thr18 and that this binding was modulated by the electrostatic properties of MDM2. In agreement with computational predictions, the binding to phosphorylated p53 peptide, in comparison to the unphosphorylated p53 peptide, was enhanced upon mutation of 3 key residues on the MDM2 surface.