On the Biological Activity of the Natural Product (+)-Avrainvillamide

Abstract

Nucleophosmin (NPM1) is a multifunctional phosphoprotein localized predominantly within the nucleoli of eukaryotic cells. Mutations within its C-terminal domain are frequently observed in patients with acute myeloid leukemia (AML), are thought to play a key role in the initiation and progression of the disease, and result in aberrant, cytoplasmic localization of the mutant protein. It has previously been demonstrated that the electrophilic antiproliferative natural product (+)-avrainvillamide binds to proteins, including nucleophosmin, by ¬S-alkylation of cysteine residues. In this thesis we report that the biological activity of avrainvillamide is mediated by NPM1 and the nuclear export receptor exprtin-1 (Crm1). Using mass spectrometry, we demonstrate that the antiproliferative activity of a series of avrainvillamide analogs correlates with their ability to bind C-terminal NPM1 truncation constructs; it is also observed that the interaction between avrainvillamide and the C-terminal domain of NPM1 is fully reversible under our experimental conditions. We report that avrainvillamide restores nucleolar localization of certain AML-associated mutant forms of NPM1 and provide evidence that this relocalization is mediated by interactions of avrainvillamide with mutant NPM1 and Crm1. Immunofluorescence and mass spectrometric experiments employing a series of different NPM1 constructs suggest that a specific interaction between avrainvillamide and cysteine-275 of certain NPM1 mutants mediates the relocalization of these proteins to the nucleolus. Avrainvillamide is further shown to inhibit nuclear export of Crm1 cargo proteins, including AML-associated NPM1 mutants; this marks the first evidence that avrainvillamide directly influences the biology of a cellular target other than NPM1. We also observe that avrainvillamide treatment displaces thr199-phosphorylated NPM1 from duplicated centrosomes, leads to an accumulation of supernumerary centrosomes, and causes mitotic defects in vitro. Finally, we show that avrainvillamide treatment increases levels of thr199-phosphorylated NPM1 by inhibiting the action of protein phosphatase 1 beta on phosphorylated NPM1, thereby indirectly displacing NPM1 from nucleoli and destabilizing nucleolar and nuclear structure.