Cytoplasmic localization of NPM in myeloid leukemias is dictated by gain-of-function mutations that create a functional nuclear export signal

Abstract

Nucleophosmin (NPM) is a nucleus-cytoplasmic shuttling protein that is implicated in centrosome duplication, cell cycle progression and stress response. At the steady state, NPM localizes mainly in the nucleolus, where it forms a complex with different cellular proteins. One-third of acute myeloid leukemias (AML) are characterized by aberrant cytoplasmic localization of NPM, due to mutations within its last coding exon (exon 12) that cause a frameshift and the formation of novel C-termini. We report here our investigations on the molecular basis for the aberrant localization of mutated NPM. Alignment of the C-terminus of the various NPM mutants revealed the obligatory presence of four amino-acid residues that match a CRM1-dependent nuclear export signal (NES). Single alanine-substitutions at these sites provoked nuclear re-localization, while fusion of the mutated C-terminus to a heterologous nuclear protein induced CRM1-dependent cytoplasmic localization. Molecular characterization of one exceptional AML carrying cytoplasmic NPM and germ line exon 12 revealed a somatic mutation in the splicing donor site of exon 9 that caused the formation of a functional NES. It appears, therefore, that AMLs are frequently characterized by gain-of-function mutations of NPM that create functional NES, suggesting that alterations of nuclear export might represent a general mechanism of leukemogenesis and a novel target for therapeutic intervention.

A dose-dependent tug of war involving the NPM1 leukaemic mutant, nucleophosmin, and ARF

“…The above findings further support the concept that NPM1 mutants are 'born to be exported', 26 as it is suggested by the findings that (i) rare truncated mutants generated by mutations at exon 9 or 11 also acquire a new C-terminus NES and localize in cytoplasm; [30][31][32][33] (ii) NPM1 leukaemic mutants retaining tryptophan 288 harbour a stronger C-terminus NES motif than mutants lacking both tryptophans 288 and 290, to ensure the most efficient cytoplasmic export; 26 and (iii) the capability of NPM1 mutants to dislocate to the cytoplasm is stable over time. 34,35 Thus, all the evidences to date point to natural selection of a mutational event on the basis of its ability to promote cytoplasmic dislocation of the resulting protein, pointing to this event as critical for leukaemogenesis.…”

Role of cysteine 288 in nucleophosmin cytoplasmic mutations: sensitization to toxicity induced by arsenic trioxide and bortezomib

“…[43][44][45][46] Several studies have provided evidence that a new leucine-rich NES created by the NPMc þ mutation promotes CRM1-dependent nuclear export of NPMc þ , thus resulting in accumulation of NPMc þ in the cytoplasm. [5][6][7]47,48 In the current study, we demonstrate that the redox-sensitive C288 generated by the NPMc þ mutation has an important role in regulating the cytoplasmic accumulation of NPMc þ . Mutation of C288 to alanine enables NPMc þ to remain in the nucleolus, suggesting that this cysteine residue may be sufficient to cause cytoplasmic localization independent of the loss of W288 and the acquisition of additional leucine-rich nuclear export motifs resulting from the C-terminal extension.…”

Clinical outcome of de novo acute myeloid leukaemia patients with normal cytogenetics is affected by molecular genetic alterations: a concise review

“…In a subsequent study, NPM1 mutations were found in 53% of CN-AML patients and were associated with a higher CR rate and longer EFS compared with non-mutated cases . Each of at least 40 different NPM1 mutations described to date causes not only loss of tryptophan residues 288 and 290 (or only 290) that are critical for nucleolar protein localisation, but also acquisition of an additional nuclear export signal motif at the C-terminus of nucleophosmin Mariano et al, 2006), that lead to abnormal localisation of nucleophosmin in the cytoplasm.…”