M P Martelli

Altered nucleophosmin transport in acute myeloid leukaemia with mutated NPM1: molecular basis and clinical implications

Nucleophosmin (NPM1) is a highly conserved nucleo-cytoplasmic shuttling protein that shows a restricted nucleolar localization. Mutations of NPM1 gene leading to aberrant cytoplasmic dislocation of nucleophosmin (NPMc+) occurs in about one third of acute myeloid leukaemia (AML) patients that exhibit distinctive biological and clinical features. We discuss the latest advances in the molecular basis of nucleophosmin traffic under physiological conditions, describe the molecular abnormalities underlying altered transport of nucleophosmin in NPM1-mutated AML and present evidences supporting the view that cytoplasmic nucleophosmin is a critical event for leukaemogenesis. We then outline how a highly specific immunohistochemical assay can be exploited to diagnose NPM1-mutated AML and myeloid sarcoma in paraffin-embedded samples by looking at aberrant nucleophosmin accumulation in cytoplasm of leukaemic cells. This procedure is also suitable for detection of haemopoietic multilineage involvement in bone marrow trephines. Moreover, use of immunohistochemistry as surrogate for molecular analysis can serve as first-line screening in AML and should facilitate implementation of the 2008 World Health Organization classification of myeloid neoplasms that now incorporates AML with mutated NPM1 (synonym: NPMc+ AML) as a new provisional entity. Finally, we discuss the future therapeutic perspectives aimed at reversing the altered nucleophosmin transport in AML with mutated NPM1.

Cell line OCI/AML3 bears exon-12 NPM gene mutation-A and cytoplasmic expression of nucleophosmin

We recently identified a new acute myeloid leukemia (AML) subtype characterized by mutations at exon-12 of the nucleophosmin (NPM) gene and aberrant cytoplasmic expression of NPM protein (NPMc+). NPMc+ AML accounts for about 35% of adult AML and it is associated with normal karyotype, wide morphological spectrum, CD34-negativity, high frequency of FLT3-ITD mutations and good response to induction therapy. In an attempt to identify a human cell line to serve as a model for the in vitro study of NPMc+ AML, we screened 79 myeloid cell lines for mutations at exon-12 of NPM. One of these cell lines, OCI/AML3, showed a TCTG duplication at exon-12 of NPM. This mutation corresponds to the type A, the NPM mutation most frequently observed in primary NPMc+ AML. OCI/AML3 cells also displayed typical phenotypic features of NPMc+ AML, that is, expression of macrophage markers and lack of CD34, and the immunocytochemical hallmark of this leukemia subtype, that is, the aberrant cytoplasmic expression of NPM. The OCI/AML3 cell line easily engrafts in NOD/SCID mice and maintains in the animals the typical features of NPMc+ AML, such as the NPM cytoplasmic expression. For all these reasons, the OCI/AML3 cell line represents a remarkable tool for biomolecular studies of NPMc+ AML.

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

In acute myeloid leukaemia (AML), nucleophosmin-1 (NPM1) mutations create a nuclear export signal (NES) motif and disrupt tryptophans at NPM1 C-terminus, leading to nucleophosmin accumulation in leukaemic cell cytoplasm. We investigated how nucleophosmin NES motifs (two physiological and one created by the mutation) regulate traffic and interaction of mutated NPM1, NPM1wt and p14ARF. Nucleophosmin export into cytoplasm was maximum when the protein contained all three NES motifs, as naturally occurs in NPM1-mutated AML. The two physiological NES motifs mediated NPM1 homo/heterodimerization, influencing subcellular distribution of NPM1wt, mutated NPM1 and p14ARF in a ‘dose-dependent tug of war’ fashion. In transfected cells, excess doses of mutant NPM1 relocated completely NPM1wt (and p14ARF) from the nucleoli to the cytoplasm. This distribution pattern was also observed in a proportion of NPM1-mutated AML patients. In transfected cells, excess of NPM1wt (and p14ARF) relocated NPM1 mutant from the cytoplasm to the nucleoli. Notably, this distribution pattern was not observed in AML patients where the mutant was consistently cytoplasmic restricted. These findings reinforce the concept that NPM1 mutants are naturally selected for most efficient cytoplasmic export, pointing to this event as critical for leukaemogenesis. Moreover, they provide a rationale basis for designing small molecules acting at the interface between mutated NPM1 and other interacting proteins.

Aberrant subcellular expression of nucleophosmin and NPM-MLF1 fusion protein in acute myeloid leukaemia carrying t(3;5): a comparison with NPMc+ AML.

Aberrant subcellular expression of nucleophosmin and NPM-MLF1 fusion protein in acute myeloid leukaemia carrying t(3;5): A comparison with NPMc+ AML

Aberrant cytoplasmic expression of C-terminal-truncated NPM leukaemic mutant is dictated by tryptophans loss and a new NES motif.

Aberrant cytoplasmic expression of C-terminal-truncated NPM leukaemic mutant is dictated by tryptophans loss and a new NES motif

A western blot assay for detecting mutant nucleophosmin (NPM1) proteins in acute myeloid leukaemia

A western blot assay for detecting mutant nucleophosmin (NPM1) proteins in acute myeloid leukaemia

The NPM1 wild-type OCI-AML2 and the NPM1 -mutated OCI-AML3 cell lines carry DNMT3A mutations

The NPM1 wild-type OCI-AML2 and the NPM1 -mutated OCI-AML3 cell lines carry DNMT3A mutations

NPM1 -mutated acute myeloid leukaemia occurring in JAK2-V617F + primary myelofibrosis: de-novo origin?

NPM1 -mutated acute myeloid leukaemia occurring in JAK2-V617F + primary myelofibrosis: de-novo origin?

Reply to Pitiot et al.

We thank Pitiot et al.1 for clarifying that nucleolar positivity in their patients leukaemic cells could be due to the wild-type NPM protein. We fully agree with this interpretation, which is also in keeping with the results of previous immunohistochemical studies.2

Erratum: Aberrant subcellular expression of nucleophosmin and NPM-MLF1 fusion protein in acute myeloid leukaemia carrying t(3;5): A comparison with NPMc + AML (Leukemia (2006) vol. 20 (368-371) 10.1038/sj.leu.2404068)

B Falini1, B Bigerna1, A Pucciarini1, E Tiacci1,8, C Mecucci1, SW Morris2, N Bolli1, R Rosati1, S Hanissian2, Z Ma2, Y Sun2, E Colombo3, DA Arber4, R Pacini1, R La Starza1, BV Galletti1, A Liso5, MP Martelli1, D Diverio6, P-G Pelicci3, FL Coco7 and MF Martelli1 Correction to: Leukemia (2006) 20, 368–371.