Altered Nucleophosmin Transport in Acute Myeloid Leukaemia with Mutated NPM1: Molecular Basis and Clinical Implications
Total Page：16
File Type：pdf, Size：1020Kb
Leukemia (2009) 23, 1731–1743 & 2009 Macmillan Publishers Limited All rights reserved 0887-6924/09 $32.00 www.nature.com/leu REVIEW Altered nucleophosmin transport in acute myeloid leukaemia with mutated NPM1: molecular basis and clinical implications B Falini1, N Bolli1,5, A Liso2, MP Martelli1, R Mannucci3, S Pileri4 and I Nicoletti3 1The Institute of Haematology, University of Perugia, IBiT Foundation, Fondazione IRCCS Biotecnologie nel Trapianto, Perugia, Italy; 2Institute of Haematology, University of Foggia, Foggia, Italy; 3Institute of Internal Medicine, University of Perugia, Perugia, Italy and 4Unit of Haematopathology, Policlinico S. Orsola, University of Bologna, Bologna, Italy Nucleophosmin (NPM1) is a highly conserved nucleo-cytoplas- n. NM_001037738) with a distinct C-terminus; it accounts for mic shuttling protein that shows a restricted nucleolar localiza- minimal nucleophosmin content in tissues. NPM1 (B23.1) and tion. Mutations of NPM1 gene leading to aberrant cytoplasmic NPM1.2 (B23.2) have different subcellular distribution patterns:5 dislocation of nucleophosmin (NPMc þ ) occurs in about one 6,7 third of acute myeloid leukaemia (AML) patients that exhibit NPM1 protein is localized only in the nucleolus and NPM1.2 8 distinctive biological and clinical features. We discuss the mainly in the nucleoplasm. A third variant (accession number latest advances in the molecular basis of nucleophosmin traffic NM_199185) lacks an alternate in-frame exon compared with under physiological conditions, describe the molecular ab- variant 1, resulting in a shorter protein whose functions and normalities underlying altered transport of nucleophosmin in expression pattern are unknown. NPM1-mutated AML and present evidences supporting the view that cytoplasmic nucleophosmin is a critical event for leukae- Experiments on NPM1/B23 migration in interspecies (chick- mogenesis. We then outline how a highly specific immunohis- mouse) heterokaryons and studies on nuclear accumulation of tochemical assay can be exploited to diagnose NPM1-mutated cytoplasmically microinjected anti-nucleophosmin antibodies AML and myeloid sarcoma in paraffin-embedded samples by provided conclusive evidence that NPM1 (B23.1), despite its looking at aberrant nucleophosmin accumulation in cytoplasm nucleolar localization,6,7 shuttles constantly back and forth of leukaemic cells. This procedure is also suitable for detection between nucleus and cytoplasm.9 As NPM1 gene mutations in of haemopoietic multilineage involvement in bone marrow trephines. Moreover, use of immunohistochemistry as surro- acute myeloid leukaemia (AML) always result in traffic altera- gate for molecular analysis can serve as first-line screening in tions of the most common isoform 1, we will refer to it AML and should facilitate implementation of the 2008 World constantly as NPM1 in this review. Health Organization classification of myeloid neoplasms that now incorporates AML with mutated NPM1 (synonym: NPMc þ AML) as a new provisional entity. Finally, we discuss the future therapeutic perspectives aimed at reversing the altered nucleo- Functional domains regulating cellular traffic of wild-type phosmin transport in AML with mutated NPM1. NPM1 Leukemia (2009) 23, 1731–1743; doi:10.1038/leu.2009.124; published online 11 June 2009 Nuclear-cytoplasmic shuttling of wild-type NPM1 (NPM1wt)9,10 Keywords: nucleophosmin; NPM1; mutations; myeloid leukaemia; is critical for most of its functions, including regulation of cell transport; monoclonal antibodies ribosome biogenesis and control of centrosome duplication.11 NPM1wt shuttles across cytoplasm and nucleoplasm as well as between nucleoplasm and nucleolus. Distinct NPM1wt func- tional domains regulate this complex shuttling activity. Nucleophosmin is a nuclear-cytoplasmic shuttling protein NPM1wt transport between cytoplasmic and nuclear Nucleophosmin (NPM1), also named B23 or Numatrin, is an compartments ubiquitously expressed protein belonging to the nucleoplasmin This process requires energy and closely depends on two family of nuclear chaperones.1 It is encoded by the NPM1 gene 2 NPM1wt functional domains (Figure 1). A bipartite nuclear that, in humans, maps to chromosome 5q35. NPM1 is essential localization signal (NLS),12 which is located between the two for embryonic development3 and is frequently translocated or 4 acidic regions in the nucleophosmin mid-region, drives mutated in haematological malignancies. NPM1wt from cytoplasm to nucleoplasm. Moreover, NPM1wt Three nucleophosmin isoforms are generated through alter- contains two leucine-rich nuclear export signal (NES) motifs13,14 native splicing. NPM1 (or B23.1), the dominant isoform 5 with the generally accepted loose consensus L-x(2,3)-(LIVFM)- (accession n. NM_002520), is a 294-amino acid phospho- x(2,3)-L-x-(LI) (that is, a core of closely spaced leucines or other protein of about 37 kDa that is expressed in all tissues. NPM1.2 large hydrophobic amino acids). One is found within residues 0 (or B23.2) uses an alternate 3 -terminal exon compared with 94–102 with the sequence I-xx-P-xx-L-x-L13 and the other at the variant 1, resulting in a 259-amino acid protein (accession N-terminus within amino acids 42 to 49 (sequence L-x-L-xx-V-x-L), with leucines 42 and 44 being critical nuclear export residues.14 Correspondence: Professor B Falini, Institute of Haematology, These NES motifs mediate NPM1wt nuclear export by interact- University of Perugia, Perugia, Italy. ing with the evolutionary conserved export receptor Crm116 E-mail: [email protected] (also called exportin 1) (Figure 1). Fluorescence recovery after 5Current address: Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. photobleaching (FRAP) showed Crm1 travels unimpeded within Received 12 March 2009; revised 6 May 2009; accepted 13 May the nuclear compartment and interacts with NES-containing 2009; published online 11 June 2009 proteins both in nucleoplasm and nucleolus.17 Altered traffic of nucleophosmin in AML B Falini et al 1732 Structure of NPM1 C-terminus DIMERIZATION W288 Wild-type NPM1 NUCLEAR NUCLEOLAR DOMAIN LOCALIZATION LOCALIZATION SIGNAL SIGNAL W290 NES NES CRM1 DIMERIZATION W288 Mutated NPM1 NUCLEAR NUCLEOLAR DOMAIN LOCALIZATION LOCALIZATION SIGNAL SIGNAL W290 NES NES NES CRM1 Figure 1 Traffic of wild-type NPM1 is mainly dictated by three functional motifs: two leucine-rich nuclear export signal (NES) motifs located in the N-terminal portion of the protein (dimerization domain); a bipartite nuclear localization signal (NLS) motif (residues 152–157 and 190–197, respectively); and an aromatic region at the C-terminus of the protein containing the nucleolar localization signal with tryptophans at positions 288 and 290. The 3-helix structure of C-terminus with the two tryptophans 288 and 290 (arrows) embedded in the hydrophobic core is shown on the top (by courtesy of Prof. Alan Warren and Dr Mark Bycroft, University of Cambridge, UK). The mutated NPM1 protein in AML differs from wild- type NPM1 because of mutations of both tryptophans or tryptophan 290 only and the presence of a new NES motif at the C-terminus of the protein. CRM1 (Exportin 1) binds specifically to the wild-type and mutated NPM1 proteins through the NES motifs. NPM1wt traffic between nucleoplasm and nucleolus bundle22 that appears similar to the archeal ribosomal protein Intranuclear trafficking of NPM1wt (and other nuclear proteins) S17e23 (Figure 1). Maintenance of C-terminus tertiary conforma- is mostly driven by diffusion, a rapid, non-directional process tion closely depends on tryptophans 288 and 290 that form part that usually occurs through energy-independent mechanisms.18 of the folded domain’s hydrophobic core (Figure 1), although FRAP analysis revealed that, in a continuous exchange with phenylalanines 268 and 273 may also play a role.22 Stabiliza- nucleoplasm,18,19 nucleophosmin (like other typical nucleolar tion of the C-terminus 3-helix structure by the two tryptophans proteins) rapidly associates with, and dissociates from the is, in turn, critical for NPM1wt binding to the nucleolus.22 nucleolar component, which is in keeping with lack of Accordingly, earlier findings that artificially induced mutations nucleolus membrane. Indeed, the structure of the nucleolus is of tryptophans 288 and 290 in NPM1wt prevented its binding to thought to be determined by protein on-rate versus off-rate the nucleolus24,25 are now understood to be the result of loss of ratio.18 According to this model, nucleophosmin contributes to C-terminal domain folding.22 C-terminus tryptophan mutations build-up the nucleolar compartment in which it is one of the that consistently occur in natural NPM1 mutants in AML patients most abundant components20 among the approximately 700 prevent or reduce mutant binding to the nucleolus in the same proteins identified by proteomics.21 In fact, flux of nucleophosmin manner,25,26 thus contributing to their aberrant accumulation in at the nucleolus–nucleoplasm interface is definitively unba- leukaemic cell cytoplasm (see below). lanced towards the nucleolus. This is, in turn, the consequence The ability of NPM1wt protein to form homodimers also helps of the very efficient nucleolar-binding domain (NoLS) that drive it to the nucleolus. Indeed, artificial NPM1 mutants that NPM1wt contains at its C-terminus. contain the nucleolar-binding domain but are unable to form The structure of nucleophosmin C-terminal domain has oligomers cannot enter the nucleolus.27 Thus, it is not surprising recently been revealed by