Interactions of the natural product (+)-avrainvillamide with nucleophosmin and exportin-1 Mediate the cellular localization of nucleophosmin and its AML-associated mutants

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 of the disease, and result in aberrant, cytoplasmic localization of the mutant protein. We have previously shown that the electrophilic antiproliferative natural product (+)-avrainvillamide (1) binds to proteins, including nucleophosmin, by S-alkylation of cysteine residues. Here, 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 exportin-1 (Crm1). Immunofluorescence and mass spectrometric experiments employing a series of different NPM1 constructs suggest that a specific interaction between avrainvillamide and Cys275 of certain NPM1 mutants mediates the relocalization of these proteins to the nucleolus. Avrainvillamide treatment is also shown to inhibit nuclear export of Crm1 cargo proteins, including AML-associated NPM1 mutants. We also observe that avrainvillamide treatment displaces Thr199-phosphorylated NPM1 from duplicated centrosomes, leads to an accumulation of supernumerary centrosomes, and inhibits dephosphorylation of Thr199-phosphorylated NPM1 by protein phosphatase 1. Avrainvillamide is the first small molecule reported to relocalize specific cytoplasmic AML-associated NPM1 mutants to the nucleolus, providing an important demonstration of principle that small molecule induction of a wild-type NPM1 localization phenotype is feasible in certain human cancer cells.

Figure 1

Primary structures of three NPM1 proteins and chemical structures of avrainvillamide and leptomycin B. (A) C-terminal sequences of wild-type NPM1 and two AML-NPMc+ variants. The NoLS is shown in bold; the C-terminal NES introduced in the mutants is underlined. (B) Structures of (+)-avrainvillamide and leptomycin B. Red asterisks indicate sites of cysteine addition.

Figure 2

Avrainvillamide binds the C-terminal domain of NPM1 and AML-associated NPM1 variants. (A) ESI-TOF mass spectrum of wild-type NPM1 C-terminal peptide prior to (black trace) and 2 h after the addition of 3 mol equiv of avrainvillamide (blue trace). (B) Fraction of NPM1 C-terminal peptides bound by avrainvillamide vs time. (C) Relative rates of formation of the NPM1 C-terminal peptide– avrainvillamide complexes. Results are the mean ± standard deviation of three independent experiments.

Figure 3

Avrainvillamide influences the localization of NPM1 and an NPMc+ variant in AML cells. (A) OCI-AML2 and (B) OCI-AML3 cells treated with DMSO (vehicle control) or avrainvillamide as indicated for 48 h. Colors: green, pan-NPM1; red, nucleolin (nucleolar marker); blue, DNA (nuclear marker). Scale bars = 10 μm.

Figure 4

Cys²⁷⁵ of NPM1 Mutant A is required for the nucleolar relocalization of NPM1 by avrainvillamide. (A) HCT-116 cells transfected with eGFP-NPM plasmids and treated with DMSO (vehicle control) or avrainvillamide (1 μM, 24 h). Colors: green, eGFP; red, nucleolin; blue, DNA. Scale bar = 10 μm. (B) Subcellular localization of the eGFP-NPM Mutant A-derived constructs following treatment with DMSO or avrainvillamide. For each dosing condition, a minimum of 150 cells was counted in each of three separate experiments. Error bars indicate mean ± standard deviation.

Figure 5

Avrainvillamide inhibits Crm1-mediated nuclear export. (A) HCT-116 cells treated with DMSO (vehicle control), avrainvillamide (1 μM, 4 h), or leptomycin B (100 nM, 4 h). Colors: green, RanBP1; blue DNA (nuclear marker). Scale bar = 10 μm. (B) Percentage of cells with RanBP1 observed in the cytoplasm or nucleus. Results are the mean ± standard deviation of three separate experiments; a minimum of 150 cells was counted per experiment.

Figure 6

Avrainvillamide interferes with proper centrosome duplication. (A) Mitotic and (B) interphase HCT-116 cells treated with DMSO (vehicle control) or avrainvillamide (1 μM, time points as indicated). Twenty-four-hour images taken at 1.6× magnification relative to the 7 h images. Colors: green, NPM1pThr199; red, γ-tubulin; blue, DNA. Scale bar = 5 μm.

Figure 7

Avrainvillamide inhibits the dephosphorylation of NPM1 by PP1β. (A) Inhibition of NPM1pTh199 dephosphorylation in an in vitro PP1β activity assay. CA = calyculin A, 10 nM, LMB = leptomycin B, 10 μM. Total NPM1 shown as a loading control. (B) Inhibition of PP1β activity in a standard activity assay with 4-nitrophenyl phosphate as the substrate.