Mechanism of Altered Nucleo-Cytoplasmic Traffic of Nucleophosmin in Acute Myelogenous Leukemia Carrying Exon-12 NPM Mutations (NPMc+ AML).

We recently found that about one-third of adult AML (60% of all AML with normal karyotype) display aberrant cytoplasmic expression of nucleophosmin (NPM) which is due to mutations occurring at the exon-12 of NPM gene (Falini B, et al., Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype..N Engl J Med2005; 352: 254–266). Hereby, we clarify the molecular mechanism underlying cytoplasmic NPM accumulation that yet remained to be elucidated. AML-associated mutated NPM alleles encode abnormal NPM proteins (25 mutants so far identified) which have acquired at the C-terminus a nuclear export signal-(NES) motif and lost at least one of tryptophan residues 288 and 290 which determine nucleolar localization. Both alterations are crucial for mutant NPM export from nucleus to cytoplasm. In fact, the cytoplasmic NPM accumulation is blocked by leptomycin-B and ratjadones which are specific inhibitors of exportin-1/CRM1, and by re-insertion of tryptophan residues 288 and 290, which respectively relocate NPM mutants in the nucleoplasm and nucleoli. Thus, for cytoplasmic accumulation of NPM to occur, the NES motif and tryptophan mutations must act in concert. Possibly, when NPM mutans enter the nucleus by virtue of their nuclear localization signals (NLS), their capability to bind nucleoli must be hindered at least partially to become a CRM-1 target. Specific antibodies anti-NPM mutant proteins showed that the mutants localized exclusively in the cytoplasm and recruited in that site the wild-type NPM protein which is physiologically located in the nucleoli. These findings suggest that the NPM mutants may interfere with the functions of wild-type NPM and possibly contribute to leukemogenesis. Immunostaining of 393 AML cases using anti-NPM monoclonal antibodies predicted the presence of NPM exon-12 mutations in all 191 NPM-cytoplasmic positive cases. This finding is consistent with the fact that, despite genomic heterogeneity, all NPM mutants contain a NES motif which, in the presence of altered tryptophans, promotes their rapid export from the nucleus to the cytoplasm. The immunohistochemical test is diagnostically relevant since it can be used as simple first-step procedure in molecular-genetic characterization of AML and as a surrogate for mutational analysis in selected cases. These findings are also clinically relevant since cytoplasmic NPM/NPM mutations are predictors of good response to induction therapy and favourable prognosis in AML with a normal karyotype.