Altered Nucleophosmin Transport in Acute Myeloid Leukaemia with Mutated NPM1: Molecular Basis and Clinical Implications

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 functional 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 nuclear magnetic resonance spectro- that NPM1wt is isolated from cells mainly as oligomers,28 scopy.22 It consists of a well-defined 3-helix right-handed which are usually hexamers.29 The NPM1wt oligomer-forming

Leukemia Altered traffic of nucleophosmin in AML B Falini et al 1733 property resides mainly in its hydrophobic N-terminal core as it lacks the C-terminus nucleolar-binding domain and cannot, structure (Figure 1), which it shares with other nucleoplasmin therefore, bind efficiently to the nucleolus.24 Knowledge of how family members.1 Core amino acids in its two physiological the nucleophosmin functional domains regulate its steady state N-terminal NES motifs (residues 44 and 47; residues 100 and cellular traffic helped elucidate the molecular mechanisms 102) appear to be involved in NPM1 homo- and hetero- underlying aberrant cytoplasmic expression of nucleophosmin dimerization.27 It is still unclear how wild-type nucleophosmin in AML with mutated NPM1 (see below). oligomerization favours nucleolar targeting. The simplest NPM1wt expression in normal and neoplastic human tissues explanation is that a NPM1 oligomer might result in a ‘high was investigated by immunohistochemistry with specific mono- NoLS load’ that reaches the nucleolar targeting threshold. clonal antibodies.7,26,42,43 The protein is found ubiquitously,7,44 Alternatively, oligomerization could confer structural properties and it is usually expressed at higher amounts in the nuclei of on the entire molecular complex that facilitate its targeting to the proliferating, than resting or differentiated cells. For example, nucleolus. we have found that in human bone marrow, early progenitors, It is still unknown whether binding of nucleophosmin to the such as proerythroblasts or promyelocytes, express much more nucleolus occurs through interaction with a protein or a nucleic nuclear nucleophosmin than terminally differentiated cells, such acid, although very recent evidence suggests that other as normoblasts or neutrophils. The usual positivity of NPM1wt nucleolar proteins (that is, rpL23) are required.30 Avrainvilla- in paraffin-embedded samples is ‘nucleus-restricted, diffuse’ mide, a naturally occurring alkaloid with anti-proliferative rather than nucleolar,26,45 reflecting the limits of immunohisto- activity, binds to the NPM1wt C-terminal domain through chemical staining of paraffin sections in detecting subnuclear Cysteine-275.31 Interestingly, Cysteine-275 is located on the structures, such as nucleoli or nuclear bodies.46 In normal same face as Lysine residues 263 and 267.22 Orientated freely human tissues, immunohistochemistry also identifies a small on the C-terminus domain surface, these Lysines appear to play percentage of cells that express nucleophosmin in cytoplasm in an important functional (rather than structural) role in NPM1wt the absence of NPM1 mutations. These represent cells in mitosis nucleolar localization,22 suggesting this protein domain area is as well as post-mitotic and apoptotic elements in which involved in ligand binding. NPM1wt localizes in the cytoplasm because of loss of nuclear Finally, post-translational NPM1wt modifications,32 including membrane (mitosis) or diffusion of the protein from nucleus to phosphorylation,33–35 ubiquitination36 and SUMOylation,37 cytoplasm (apoptosis) (Falini B, unpublished observation). may also contribute to regulating cellular traffic of wild- type NPM1. Nucleophosmin traffic is perturbed in lymphomas and leukaemias carrying NPM1 gene alterations Native NPM1 is a nucleolar protein with ubiquitous tissue The NPM1 gene is translocated or mutated in several expression haematological malignancies,4 and these genetic alterations usually perturb the cellular traffic of nucleophosmin. In this At immunocytochemistry, NPM1wt localizes selectively in the review, we will mainly focus on alterations in nucleophosmin nucleolus,6,7 the most prominent nuclear substructure.20 When traffic in AML with mutated NPM1. observed at electron microscopy, the nucleolus is morpho- logically divided into three main components that reflect the multi-step ribosome biogenesis process: the fibrillar centres Human haematological malignancies carrying (containing DNA sequences encoding ribosomal RNA);38 the translocations involving the NPM1 gene dense fibrillar component (in which nascent pre-rRNA tran- Anaplastic large cell lymphoma (ALCL) expressing the anaplas- scripts enter to undergo a series of early processing steps); and tic lymphoma kinase (ALK) protein is now included as a distinct the granular component, radiating out from the dense fibrillar entity in the 2008 World Health Organization (WHO) classifi- component, in which intermediate or late stages of RNA cation of lymphoid and haemopoietic tissues.47 ALCL cells maturation occur.39 Notably, nucleophosmin expression is harbouring the t(2;5) translocation (about 85% of cases) express restricted to the granular component,20 reflecting its involve- both NPM1–ALK fusion protein48 and NPM1wt (encoded by the ment in later processing and assembly steps and in ribosomal residual NPM1 allele). Because of its ALK moiety, at least a subunit export.40 At first glance, the restricted NPM1wt proportion of the NPM1–ALK protein anchors to ALCL cell nucleolar localization appears surprising in the light of its cytoplasm.46 This accounts for the aberrant cytoplasmic continual shuttling between nucleus and cytoplasm.9,10 How- expression of nucleophosmin that is revealed with antibodies ever, analysis of the dynamics determined by the functional against the NPM1 N-terminus (which is retained in the fusion domains regulating NPM1wt nuclear-cytoplasmic and intra- protein).46,49 In contrast, NPM1wt that can be detected with an nuclear traffic (see above), explains this staining pattern. antibody against NPM1 C-terminus (that is not retained The following scenario can be envisaged. Because of its NLS, in NPM1–ALK), maintains its expected nucleolar expression in NPM1wt migrates from cytoplasm to nucleoplasm and from ALCL with t(2;5),49 suggesting that localization is not perturbed there it can move to the nucleolus because of its strong by the NPM1–ALK fusion product. nucleolar-binding domain. Interaction of Crm1 (exportin 1) with In the rare AML/myelodysplasia carrying the t(3;5)(q25;q35) the two NPM1wt N-terminus NES motifs13,14 (which presum- translocation, leukaemic cells express both NPM1–MLF1 ably occurs both in nucleoplasm and nucleolus) ensures (myelodysplasia/myeloid leukaemia factor 1) fusion protein50 NPM1wt nuclear export. Using a Rev(1.4)-based shuttling assay, and NPM1wt (encoded by the normal NPM1 allele). Like ALCL we found that both NPM1wt NES motifs have low export with t(2;5), AML with t(3;5) shows aberrant nucleophosmin efficiency41 and, therefore, little native protein is exported from expression in cytoplasm as, due to its MLF1 moiety, the NPM1– nucleus to cytoplasm. As NLS-mediated nuclear import of MLF1 fusion protein localizes in leukaemic cell cytoplasm.51 NPM1wt greatly predominates over export,41 most NPM1wt However, unlike ALCL with t(2;5), NPM1wt is also dislocated resides within the nucleolus (Figure 2). In contrast, the rare into cytoplasm of AML cells carrying the t(3;5)51 through a NPM1.2 (or B23.2) truncated isoform localizes to nucleoplasm,8 mechanism that still needs to be deciphered.

Leukemia Altered trafﬁc of nucleophosmin in AML B Falini et al 1734

Figure 2 (Top, left) Mechanism of nucleo-cytoplasmic shuttling of wild-type NPM1 (NPM1wt). The nuclear import of the protein (arrow) greatly predominates over the nuclear export (dotted arrow). Thus, NPM1wt mainly resides in the nucleolus. (Top, right) Mechanism of aberrant cytoplasmic expression of nucleophosmin (both mutated and wild-type proteins) in AML with mutated NPM1 gene. Nuclear export (thick arrows) overcomes nuclear import (thin arrow). Purple squares indicate tryptophans 288 and 290; black squares indicate mutated tryptophans; turquoise rectangle indicates the nuclear localization signal (NLS); the red circles indicate the nuclear export signal (NES) motifs (two in the wild-type protein and three in the leukaemic NPM1 mutant). (Bottom, a) Confocal 3D-reconstruction of NIH-3T3 cells expressing enhanced green fluorescent protein (eGFP)-NPM1wt fusion protein. The nucleus (red) was cut electronically to analyse the localization of NPM1wt protein. NPM1wt (green) localizes selectively in the nucleoli. (Bottom, b) Confocal 3D-reconstruction of NIH-3T3 cells expressing enhanced green fluorescent protein (eGFP)-NPM1 mutant A fusion protein. The NPM1 mutant (green) is present only in the cytoplasm; nucleus is counterstained in red with propidium iodide. (Bottom, c) Expression of enhanced green fluorescent protein (eGFP)-NPM1 mutant A fusion protein in NIH-3T3 cells after incubation with the CRM1 inhibitor leptomycin B. The nucleus (counterstained in red with propidium iodide) was cut electronically to analyse the localization of the NPM1 mutant. Leptomycin B re-localizes the NPM1 mutant (green) from cytoplasm to nucleoplasm. Images a–c represent confocal 3D- reconstruction, Imaris software, Bitplane, Zurich, CH, Switzerland.

The NPM1–RARa fusion protein was detected in extremely AML with mutated NPM1 (NPMc þ AML) rare cases of acute promyelocytic leukaemia carrying the t(5;17) Having observed that in ALCL with t(2;5) the presence of the translocation.52 In two patients investigated by immunocyto- NPM1–ALK fusion protein was associated with ectopic expres- chemistry,53,54 nucleophosmin showed diffuse nuclear positivity sion of nucleophosmin in tumour cell cytoplasm,49 we decided that clearly differed from the expected nucleolar expression of to adopt immunohistochemical detection of cytoplasmic NPM1wt. nucleophosmin, as a simple, rapid screening test for putative The transforming role of ALK, MLF1 and RAR-a fusion protein NPM1 gene alterations in a wide range of human malignancies. moieties in the above malignancies is well established.46 This approach led, in 2005, to the discovery that about 35% Nevertheless, the view that the NPM1 moiety of the chimeric of adult AML had aberrant nucleophosmin expression in protein only provides a dimerization substrate for the C-terminal leukaemic cell cytoplasm.26 onco-protein has recently been challenged by in vivo evidence We initially named such AML cases NPMc þ (cytoplasmic that NPM1 is an haploinsufﬁcient tumour suppressor gene.55 positive) to distinguish them from AML with nucleus-restricted Therefore, its heterozygous loss could also contribute to the NPM expression26 (named NPMcÀ). As NPMc þ AML was pathogenesis of the disease.11,55 closely associated with normal karyotype and other distinctive

Leukemia Altered traffic of nucleophosmin in AML B Falini et al 1735 features,26 an unidentified genetic lesion seemed a strong NPM dislocation into cytoplasm: a critical event for possibility as underlying cause of nucleophosmin dislocation leukaemogenesis? into cytoplasm. As known NPM1 fusion gene/transcripts were not found in NPMc þ AML leukaemic cells,26 we sequenced the Several observations clearly point to nucleophosmin dislocation entire coding sequence of NPM1 and found heterozygous into cytoplasm as the most distinguishing functional conse- insertions at exon-12 of the gene causing a reading frame-shift quence of NPM1 mutations in AML. They include the following. that resulted in a slightly longer protein with a different C-terminal amino-acidic sequence.26 Whole genomic sequencing is emerging as a powerful tool for Nucleophosmin dislocation into cytoplasm is specific solving complex problems, such as identification of underlying 56 for AML genetic lesions in AML with normal karyotype. On the other Aberrant nucleophosmin expression in cytoplasm was detected hand, the story of how NPM1 mutations were discovered in by immunohistochemistry in about 35% of adult AML.26 In AML teaches that sometimes solutions to complex problems contrast, NPM1 is not mutated nor aberrantly expressed in may derive from a simple observation at the microscope using cytoplasm in any other human haemopoietic or extra-haemo- inexpensive techniques, like immunohistochemistry. The crucial poietic neoplasm.26,44,59 Moreover, ectopic nucleophosmin step was taken when immunoscreening for nucleophosmin expression is usually observed in the entire leukaemic popula- expression was extended to tumours other than ALCL with t(2;5). tion. One exception is NPM1-mutated AML with M5b morphol- However, had we not, several years previously, chosen to ogy, in which cytoplasmic NPM1 is seen mainly in monoblasts generate monoclonal antibodies directed against fixative-resis- 26 7 but not in the more mature monocytic forms. This expression tant epitopes of the NPM1 molecule, aberrant nucleophosmin pattern is in keeping with experimental in vitro observations of dislocation in AML cell cytoplasm might have been difficult to NPM1 downregulation during maturation.60 Finally, in AML, recognize, as this staining pattern is optimally detected only in 26,45 aberrant NPM1 expression in cytoplasm was associated with a fixed, paraffin-embedded sections. Another factor also distinct gene expression profile61 and microRNA signature.62 played a role in the discovery. Even though NPM1 is a nuclear-cytoplasmic shuttling protein,9 the very small fraction that is physiologically present in cytoplasm is fortunately below Generation of a NES motif through duplication appears the immunohistochemistry detection threshold. Therefore, unique to the NPM1 mutation immunohistochemical detection of cytoplasmic nucleophosmin About 80% of AML with mutated NPM1 bear the so-called could be exploited for specific recognition of an NPM1 mutation mutation A, that is, a duplication of a TCTG tetranucleotide at in AML cells. positions 956–959 of the NPM1 gene.58 A genome-wide computational analysis designed to evaluate whether duplication of 3–6 nucleotide combinations could lead to generation of a known NES motif in any human gene showed that duplication- Molecular mechanism of cytoplasmic accumulation of linked generation of a NES motif was unique to the NPM1 nucleophosmin in AML with mutated NPM1 mutation,44 an AML-specific genetic event. Aberrant cytoplasmic expression of nucleophosmin is the immunohistochemical hallmark45 of AML with mutated NPM1. All NPM1 mutations, despite molecular heterogeneity Nucleophosmin cytoplasmic expression is a stable (about 50 mutation variants identified so far), result in a shift of event in AML with mutated NPM1 the reading frame leading to common changes at the very end of Many observations indicate that NPM1 mutations are 63–65 the NPM1 protein C-terminus25 that are responsible for stable. Accordingly, in patients with NPM1-mutated AML cytoplasmic mislocalization of the NPM1 leukaemic mutants. who were investigated by immunohistochemistry during the Mutation-related changes causing this effect, shown in Figure 1, course of the disease, cytoplasmic nucleophosmin was con- are (i) generation of a new NES motif,57 which reinforces the sistently detected at relapse not only in the bone marrow but Crm1-dependent nuclear export of NPM1 protein and (ii) loss of also in extramedullary sites. Persistence of NPM1 expression in tryptophan residues 288 and 290 (or residue 290 alone),58 leukaemic cell cytoplasm over a period of many years was 66,67 which cause the 3-helix structure of NPM1 C-terminal domain demonstrated in patients at late relapse and in a xenotrans- to unfold,22 thus preventing or decreasing NPM1 binding to the plant model of AML with mutated NPM1 in immunodeficient 65 nucleolus. mice. Moreover, the OCI-AML3 cell line, first established Both alterations are critical for perturbing traffic of NPM1 about 20 years ago, stably harbours NPM1 mutation A and 68 mutants in AML cells25 and an hypothetical scheme of how this displays aberrant nucleophosmin expression in cytoplasm. It occurs is shown in Figure 2. As NPM1 leukaemic mutants retain should be noted that loss of NPM1 mutation at relapse has been 69,70 their NLS, they move efficiently from cytoplasm to nucleoplasm. rarely observed in NPM1-mutated AML. Discrepancies with In the nucleus, however, the capability of NPM1 mutants to bind other reports may be due to technical reasons or one might nucleolus is hindered either completely (when both tryptophans speculate that these cases represent treatment-related or even 288 and 290 are mutated) or partially (when only tryptophan second de-novo AMLs rather than relapses from previous 71 290 is altered).41 Consequently, a significant fraction of NPM1 leukaemia. Extensive investigation by FISH and other mole- mutant protein roams in nucleoplasm in which it is more prone cular techniques are warranted to clarify the issue. to bind to Crm1 through the additional C-terminus NES motif introduced by the mutation41 (Figure 1). Under these circumstances, the mutation-driven NES-Crm1-mediated nucle- NPM1 leukaemic mutants are born to be exported ar export is more efficient than import into the nucleolus Two major observations suggest NPM1 mutations act to and, therefore, the NPM1 mutants accumulate in cytoplasm maximize mutant export into cytoplasm underscoring this event (Figure 2). as critical for leukaemogenesis.

Leukemia Altered traffic of nucleophosmin in AML B Falini et al 1736 All NPM1 mutations result in dislocation of the encoded NPM1 leukaemic mutants dislocate diverse protein partners proteins into cytoplasm. Although NPM1 mutations in AML into cytoplasm usually occur at exon-12 of the gene,26 they may very occasionally involve exon-972 or exon-11.73,74 Yet, these very How mutated NPM1 contributes to AML development is still rare NPM1 mutants localize aberrantly in leukaemic cell unclear. One interesting hypothesis is that the NPM1 mutants cytoplasm73 through the same mechanism described in AML exert their oncogenic action by binding and dislocating into carrying typical exon-12 NPM1 mutations, that is, through the leukaemic cell cytoplasm NPM1wt (encoded by the normal concerted action of tryptophan(s) changes and a new NES motif allele)25,58 and other protein partners, including tumour at the C-terminus.25 In particular, mutations involving exon-11 suppressor p19Arf,78,79 thereby interfering with their functions. generate a truncated NPM1 protein (274 instead of 294 amino Proteins whose subcellular distribution and function are altered acids) with consequent loss of tryptophans at positions 288 and by NPM1 mutants are discussed below. 290,73,75 which are critical for nucleolar binding.24 Deletion of the two C-terminus tryptophans has the same detrimental effect on the nucleolus-binding capability of the truncated protein as Delocalization of NPM1wt their replacement in AML carrying typical exon-12 NPM1 In vitro transfection studies25,78 and immunohistochemical mutations.26 Even though the reduced nucleolus-binding cap- observations in samples from AML patients25 clearly demon- ability increases mutant affinity for Crm1/Exportin,25 it is not strated that NPM1 mutants recruit NPM1wt from nucleoli and sufficient to cause aberrant mutant accumulation in cytoplasm. delocalize it into nucleoplasm and cytoplasm. In the same way As with exon-12 NPM1 mutations, the additional force as NPM1wt with NPM1 fusion proteins,4,46,80 NPM1 mutants contributing to nuclear export and ectopic cytoplasmic accu- form heterodimers with NPM1wt25,58 through the N-terminal mulation of the leukaemic mutant is a new NES motif at the dimerization domain, which is conserved in all NPM1 same C-terminus end of NPM1.73,75 Interestingly, the only mutants.4,58 Core amino acids in the two physiological amino- NPM1 mutation that was found to involve the splicing donor site terminal NES motifs seem to play a major role in mediating of NPM1 exon 972 also caused deletion of C-terminus NPM1 homo- and hetero-dimerization, by dictating in a ‘dose- tryptophans and creation of a new NES motif. These findings dependent tug of war’ fashion the subcellular distribution of in AML also explain why B23.2, the physiologically truncated wild-type and mutated NPM1 proteins.27 In fact, transfected NPM1 isoform, localizes in nucleoplasm.8 In fact, even though cells showed that, when NPM1wt and mutant proteins were B23.2 lacks tryptophans 288 and 290, there is no new NES motif expressed at equimolar doses, a fraction of NPM1wt was at its C-terminus to drive accumulation of the protein in consistently retained in the nucleoli (as is most often observed in cytoplasm. These observations clearly show that all NPM1 AML samples). On the other hand, in the presence of excess of mutations inevitably cause export of the leukaemic mutants into mutant NPM1, all NPM1wt protein was pulled out of the cytoplasm. nucleoli into nucleoplasm and cytoplasm,27 as seen in some patients with NPM1-mutated AML. On the contrary, excess doses of NPM1wt relocated NPM1 leukaemic mutant from In rare NPM1 leukaemic mutants with residual nucleo- cytoplasm to the nucleoli of transfected cells,27 a pattern never lar targeting ability, stronger NES are selected.In observed in NPM1-mutated AML patients in whom the mutant NPM1-mutated AML, 495% mutants lack both tryptophans was always cytoplasmic restricted.81 This evidence points at a 288 and 290, whereas rare mutants retain tryptophan 288 and dose-dependent relationship between wild-type and mutated show residual nucleolar targeting ability.58 Despite this differ- NPM1 proteins, and supports the view that NPM1 leukaemic ence, all mutants aberrantly accumulate in leukaemic cell mutants are ‘born to be exported’.41 cytoplasm, implying that their nuclear export occurs even when tryptophan 288 is in place. As NES sequences may differ in export efficiency of their cargo proteins,76,77 we hypothesized p19Arf protein delocalization that these rare NPM1 mutants could have stronger NES motifs The tumour suppressor p14ARF, a crucial positive regulator of to ensure their efficient cytoplasmic accumulation even in the the p53 response to oncogenic stimuli, closely interacts with presence of a residual force driving the proteins to the nucleolus. NPM1wt.82 Most studies used murine cell lines to study Indeed, when both tryptophans were mutated, we found NPM1 nucleophosmin interaction with ARF, thereby focusing on its mutants always bore the most common NES motif (that is, L-xxx- murine ortholog p19Arf. P19Arf co-localizes with NPM1wt V-xx-V-x-L).41,58 In contrast, when tryptophan 288 was retained, protein in the nucleolus in which it forms high molecular NPM1 mutants consistently carried variant NES motifs (that is, complexes.83 As p19Arf has a stable structure only when bound Valine at the second position was replaced with Leucine, to NPM1wt,84 the NPM1wt–p19Arf interaction appears not only Phenyl-alanine, Cysteine or Methionine).41,58 Interestingly, we to protect p19Arf from the rapid proteasome-mediated degrada- demonstrated that these variant NES motifs were endowed with tion85,86 (which many misfolded proteins are subjected to87), stronger export efficiency than L-xxx-V-xx-V-x-L motif,41 which but also to dictate its nucleolar location.84 As p19Arf stability counteracted the force of tryptophan 288 driving mutants to the and subcellular distribution are essential for maintenance of its nucleolus.24 basal levels and functional activity,88 cytoplasmic NPM1 mutant The above findings strongly suggest that NPM1 mutants are may contribute to AML development by inactivating p19Arf. In ‘born to be exported’,41 and that aberrant nucleophosmin fact, in NIH-3T3 fibroblasts that were engineered to produce a accumulation in AML cell cytoplasm may play, through a yet zinc-inducible p19Arf protein, NPM1 mutant A delocalized unknown mechanism, a critical role in leukaemogenesis. As NPM1wt and p19Arf from nucleoli to cytoplasm,78 which very low levels of NPM1wt are transiently present in cytoplasm reduced p53-dependent (Mdm2 and p21cip1 induction) and under physiological conditions9 (due to shuttling activity), the p53-independent (sumoylation of NPM) p19Arf activities.78 amount of NPM1 mutant that accumulates in leukaemic cell When complexed with the NPM1 mutant, p19Arf stability was cytoplasm and/or the highly increased shuttling rate of the greatly compromised and the p53-dependent cell-cycle arrest at mutated protein may be crucial for transforming activity. the G1/S boundary was weaker.79

Leukemia Altered traffic of nucleophosmin in AML B Falini et al 1737 HEXIM1 protein delocalization another valuable diagnostic approach, as detection of aberrant Hexamethylene bis-acetamide inducible protein 1 (HEXIM1) nucleophosmin expression in leukaemic cell cytoplasm is fully inhibits the positive transcription elongation factor b (P-TEFb), predictive of NPM1 mutations.45 which is a key RNA polymerase II (Pol II) transcriptional Optimal detection of nucleophosmin using anti-NPM anti- regulator. Gurumurthy et al89 reported that NPM1wt binds to bodies is easily achieved in paraffin-embedded samples, while HEXIM1 in vitro and in vivo, and functions as a negative fresh material (smears or cytospins) is not suitable for this HEXIM1 regulator. In transfected cells, mutated NPM1 asso- purpose.26 For routine immunohistochemical diagnosis of ciated with, and sequestered, HEXIM1 in cytoplasm, resulting in NPMc þ AML, the most reliable reagents are monoclonal higher transcription of RNA pol II target genes, among which antibodies directed against fixative-resistant epitopes that were some positive regulators of cell-cycle progression such as are shared by wild-type and mutated NPM1 proteins7,26,45 cyclin D1 and anti-apoptotic proteins such as Mcl-1.89 (Figure 3). When stained with these reagents, AML cells harbouring an NPM1 mutation appear positive in the nucleus (which contains a fraction of NPM1wt) and in cytoplasm that NF-kB delocalization contains mutated NPM1 and NPM1wt (recruited by the mutant Activated nuclear factor kappaB may confer resistance to through heterodimer formation)45 (Figures 3 and 4). Although 90 91 chemotherapy in AML patients. Cilloni et al showed that polyclonal antibodies recognizing only mutant NPM1 pro- mutated NPM1 bound to NF-kappaB and sequestered it in tein45,68 are very useful for investigating lineage involvement81 cytoplasm, leading to its inactivation. The authors hypothesized and for diagnosing AML with mutated NPM1 by western that this mechanism underlies the high sensitivity of AML with blotting,97 they are of limited value in immunohistochemical 91 mutated NPM1 to chemotherapy. detection of cytoplasmic nucleophosmin on paraffin sections81 because antigenic epitopes are frequently denaturated by Delocalization of proteins involved in control of c-Myc fixation/decalcification procedures. and p15 and p21 In most NPMc þ AML samples, immunostaining results are striking and easy to interpret. Problems may occasionally be In MEF transfected cells, Bonetti et al92 found that the NPM1 encountered in the following circumstances: (i) weak nucleo- mutant stabilized cMyc by dislocating to cytoplasm and phosmin positivity in leukaemic cell cytoplasm; (ii) cytoplasmic degrading the F-box protein Fbw7g, a component of the E3 expression of nucleophosmin in only a percentage of leukaemic ligase complex, which is involved in c-Myc ubiquitination cells, as usually occurs in AML cases with M5b morphology26 and proteasome degradation.93 Thus, NPM1 mutant-mediated and (iii) determining nucleophosmin positivity in small-sized enhanced proliferative activity (by increasing c-Myc protein blasts with limited cytoplasmic rim. Corollary evidence is levels) may be a potential player in leukaemogenesis.94 obtained by staining parallel paraffin sections with a mono- Wanzel et al30 found that the NPM1 leukaemic mutant clonal antibody against a different shuttling nucleolar protein interacted with the Myc-associated zinc-finger protein Miz1, such as C23/nucleolin.9 Nucleus-restricted localization of this dislocating it into cytoplasm. When complexed with the NPM1 protein in the above situations confirms the diagnosis of mutant in cytoplasm, Miz1 no longer exerted its physiological NPMc þ AML26 (Figure 3). Finally, non-specific nucleophosmin action of negatively regulating cell proliferation by inducing expression in cytoplasm (in the absence of NPM1 mutation) may expression of cell-cycle inhibitors p15 and p21. occur in AML cells in association with apoptosis and extensive The role of NPM1 mutant-induced protein mislocalizations in bone marrow necrosis. These cases are easily recognized leukaemogenesis, however, still remains unclear. In fact, information on the altered subcellular distribution of NPM1 partners (ARF, HEXIM1, Fbw7g and Miz1) was derived only AML with AML with from the study of transfected non-haemopoietic cells, and no mutated NPM1 germline NPM1 immunohistochemical evidence was provided that these proteins were really expressed aberrantly in the cytoplasm of NPM wt NPMc þ AML cells from patients. Strong in vitro and in vivo N N evidence of a transforming role of mutated NPM1 in haematopoietic cells is indeed still lacking. The only positive cell NPM mut transformation assay was achieved in mouse embryonic fibroblasts by combining NPM1 mutant with the adenoviral protein E1A, as expression of mutated NPM1 alone only conferred a senescent phenotype in this cell model.95 C23/nucleolin N N Diagnostic and clinical implications of aberrant cytoplasmic expression of nucleophosmin in AML with mutated NPM1 Figure 3 Mouse monoclonal antibody against NPM1 (clone 376, Ectopic expression of nucleophosmin in leukaemic cell cyto- Dakocytomation, Glostrup, Denmark) recognizes a fixative-resistant plasm has not only a biological impact but also diagnostic and epitope at the N-terminus of both wild-type and mutated NPM1 clinical implications. proteins. Mouse monoclonal antibody anti-C23/nucleolin (sc-8031, Santa Cruz Biotechnology, Santa Cruz, CA, USA) is raised against full- length C23 of human origin and is suitable for staining fixed paraffin- Cytoplasmic expression of nucleophosmin as embedded samples. Subcellular distribution patterns of nucleophosmin and nucleolin/C23 in AML are indicated in red. AML with immunohistochemical marker for AML mutated NPM1 is characterized by nuclear plus cytoplasmic expres- Many haematological centres identify AML patients with sion of NPM1 and nucleus-restricted positivity for nucleolin/C23. AML mutated NPM1 by directly searching for NPM1 mutations by with germline NPM1 gene is characterized by nuclear-restricted means of molecular techniques.96 Immunohistochemistry is positivity for NPM1 and nucleolin/C23.

Leukemia Altered trafﬁc of nucleophosmin in AML B Falini et al 1738

Figure 4 Aberrant cytoplasmic expression of nucleophosmin in AML and myeloid sarcoma with mutated NPM1.(a) AML with mutated NPM1 (bone marrow biopsy, paraffin section). Marked marrow infiltration by myeloid blasts. The arrow indicates a dysplastic megakaryocyte (hematoxylin–eosin, Â 800). (b) Myeloid blasts and a dysplastic megakaryocyte (arrow) show aberrant cytoplasmic expression of nucleophosmin (in addition to the expected nuclear positivity); this staining pattern indicates that both myeloid blasts and the megakaryocyte belong to the same leukaemic clone (bone marrow paraffin section, hematoxylin counterstaining, Â 800); (c) Myeloid sarcoma with mutated NPM1 of lymph node. Marked infiltration by myeloid blasts (asterisk). LF indicates a residual lymphoid follicle (paraffin section, hematoxylin–eosin, Â 400). (d) Myeloid blasts infiltrate the interfollicular area of the lymph node (asterisk) and show nuclear plus aberrant cytoplasmic expression of nucleophosmin; cells of the residual lymphoid follicle (LF) show the expected nucleus-restricted positivity of wild-type NPM1 (paraffin section, hematoxylin counterstaining, Â 400). (b and d) Immunostaining with monoclonal antibody against NPM1 (clone 376) using the immuno-alkaline phosphatase anti-alkaline phosphatase (APAAP) technique.

because immunostainings show that C23/nucleolin is also and immunohistochemical studies can be applied in parallel, to aberrantly dislocated into cytoplasm in a non-specific manner. complement each other (Figure 5). Immunohistochemical detection of cytoplasmic nucleophos- It should be emphasized that immunohistochemistry is not min in AML has several diagnostic applications. It is a first- suitable for monitoring minimal residual disease in NPM1- choice assay for recognizing NPM1-mutated AML in cases of mutated AML because in normal bone marrow a few cells dry-tap or in extramedullary sites,66,98 and for detecting (mitoses, post-mitotic and apoptotic cells) may show cyto- haemopoietic lineage involvement81 (Figure 4). As cytoplasmic plasmic positivity for NPM1wt (in the absence of NPM1 nucleophosmin is fully predictive of NPM1 mutations,45 mutation). Thus, quantitative PCR analysis is the first-choice immunohistochemistry could be used as a first-line screening assay for detecting minimal amounts of NPM1 mutants.101,102 to rationalize cytogenetic and molecular studies in AML. This procedure is ideally carried out on bone marrow aspirates. A potential algorythm is shown in Figure 5. As immunohisto- In conclusion, availability of several techniques for detecting chemistry is a cheap, easy, and readily available technique, NPM1 mutations offers a great advantage as it may facilitate applying it to detect cytoplasmic nucleophosmin should application of the WHO classification. Investigators can choose facilitate implementation of the 2008 WHO classification of an appropriate technique (or a combination of techniques) on myeloid neoplasms, which now incorporates AML with mutated the basis of their experience, equipment, preferred diagnostic NPM1 (synonym: NPMc þ AML) as a new provisional entity.99 procedure (for example, bone marrow biopsy and/or aspirate) Trephine biopsies are not, however, performed in all and type of analysis (initial mutational screening, monitoring of countries and cases of acute leukaemia. Under these circum- minimal residual disease, etc.). stances, PCR-based screening for NPM1 mutations in peripheral or bone marrow blast cells from AML patients is the first-choice approach (Figure 5). A method for rapid detection of cytoplas- Impact of mutated cytoplasmic nucleophosmin on mic nucleophosmin using intracellular flow cytometry has clinical management of AML recently been proposed100 and may emerge in the future as a The flexible diagnostic approach depicted in Figure 5 has valuable alternative procedure for the initial screening of AML important prognostic and clinical implications. AML with samples. Finally, for well-equipped laboratories, both molecular mutated NPM1 usually carries normal karyotype and, according

Leukemia Altered trafﬁc of nucleophosmin in AML B Falini et al 1739 AML SAMPLE

Analysis of NPM1 Immunohistochemistry mutations in all AML of NPM1 in all AML

Anti-NPM1

PB cells or BM aspirate (DNA, RNA) Bone marrow biopsy

NPM1 nuclear NPM1 cytoplasmic (NPMc-) (NPMc+)

AML with wild-type AML with mutated NPM1 NPM1

Establish type of NPM1 mutation* + - All other FLT3-ITD FLT3-ITD Minimal Residual FLT3-ITD+ FLT3-ITD- Disease (MRD) studies monitoring Bad Good Minimal Residual Bad Good Disease (MRD) prognosis prognosis prognosis prognosis monitoring All other studies

Figure 5 Possible approaches for diagnosis of AML with mutated NPM1: mutation analysis by molecular techniques (left side), immunohistochemical detection of cytoplasmic NPM1 (right side) or a combination of the two methods (bi-directional arrows). The approach on the left side implies that mutational analysis is applied to all AML patients at ﬁrst diagnosis. The immunohistochemical approach (right side) can be used as ﬁrst screening to restrict mutation analysis of NPM1 gene to cases showing aberrant cytoplasmic expression of nucleophosmin. Evaluation of the FLT3 status is performed in all AML with mutated NPM1 as it allows to identify patients with the NPM1-mutated/FLT3-ITD negative genotype who have a good prognosis. PB indicates peripheral blood; BM indicates bone marrow.

to several studies, is associated with good outcome when FLT3- In patients 460 years old, the favourable impact of NPM1 ITD is absent.103–108 About 15% of NPM1-mutated AML mutations may be less important.112 A recent report113 harbour chromosomal aberrations (other than the recurrent suggested that elderly patients may benefit from the addition ones), which are likely to represent secondary events. In a recent of all-trans retinoic acid (ATRA) (see below). study designed to establish the prognostic value of these additional genetic abnormalities, we found that prognosis in AML with mutated NPM1 is not influenced by karyotype.109 Perturbed cellular traffic of nucleophosmin in AML as Given its favourable prognosis, it is recommended that therapeutic target NPM1-mutated/FLT3-ITD negative AML be treated by conven- The process of developing drugs targeting altered nucleo- tional chemotherapy regimens with or without autologous stem cytoplasmic shuttling in cancer cells is hindered by several cell transplantation. Schlenk et al108 recently reported that obstacles, which include (i) specificity (that is, targeting neoplastic NPM1-mutated AML patients without FLT3-ITD did not appear but not normal cells) and (ii) technical difficulties in creating small to benefit from allogeneic stem cell transplantation in first molecules that are able to interfere with protein–protein inter- complete remission. actions.16 The role of allogeneic transplantation in AML patients with One example of specificity-related problems are inhibitors of mutated NPM1 and a concomitant FLT3-ITD remains contro- karyopherin-B family members, such as leptomycin B, a natural versial.107 As the mutant NPM1 might be recognized by product that binds covalently and irreversibly inhibits Crm1, the immune cells,110 it would be interesting to explore whether it export receptor for NES-containing proteins.114,115 Another elicits a specific GvL effect. It should be added that detection of problem with Crm1 inhibitors is how to re-direct the NPM1 minimal residual disease may emerge in the future as an mutant straight to the nucleolus, as one or both C-terminus important assay for assessing quality of therapy response and for tryptophans are missing. Indeed, incubation of transfected or guiding therapy.102,111 NPMc þ AML cells with leptomycin B causes relocalization of

Leukemia Altered traffic of nucleophosmin in AML B Falini et al 1740 the mutant only in the nucleoplasm (Figure 2, bottom, c). A third Finally, it could be feasible to screen a library of pharmaco- obstacle to the use of leptomycin B in therapy is its toxicity, logical agents on the OCI-AML3 cell line68 or primary NPMc þ which is hardly surprising as Crm1 is critical for nuclear export AML cells from patients, in a search for drug sensitivity that is of most proteins and RNAs in normal and neoplastic cells. specifically associated with the NPM1 mutation. Schlenk Indeed, phase I clinical trials with leptomycin B were associated et al113 retrospectively analysed the prognostic impact of with several adverse side effects, particularly profound anorexia NPM1, CEBPA, FLT3 and MLL gene mutations on the effects and malaise.116 Generation of less toxic Crm1 inhibitors with of ATRA treatment in AML patients 460 years old (excluding improved therapeutic windows could overcome these pro- APL) who were enrolled in the HD98B randomized trial. blems.117 Notably, ATRA, given as adjunct to intensive induction therapy A more rational approach involves developing small mole- (idarubicin, cytarabine and etoposide), significantly improved cules to re-direct wrongly located proteins, like mutated NPM1, relapse-free and overall survival only in patients bearing the to the proper cellular compartment (that is, the nucleolus). NPM1 mutation without concomitant FLT3-ITD. On the other Designing small molecules that specifically interfere with NPM1 hand, the NPM1-mutated/FLT3-ITD negative genotype did not mutant traffic will require better knowledge of the 3D structure emerge as a predictive marker for response to ATRA in a large of different nucleophosmin domains.118 Theoretically, in the series of non-APL patients from the UK MRC AML12 trial.124 future, libraries of small interfering molecules might be screened Different ATRA administration schedules and/or inclusion of in a high-throughput format16 to search for compounds that, in etoposide in the regimen may account for discrepancies. an appropriate revealing assay, change the subcellular localiza- Etoposide in combination with ATRA seems to exert a beneficial tion of mutated NPM1. synergistic effect in NPM1-mutated/FLT3-ITD negative AML125 An alternative therapeutic intervention in NPMc þ AML is to possibly because this leukaemia subtype frequently associates interfere with the localization/function of the residual NPM1wt with myelomonocytic/monocytic differentiation which is be- encoded by the normal allele.119 The rationale for this approach lieved to confer particular sensitivity to etoposide. Prospective is that NPM1 mutations in AML are always heterozygous,58 clinical trials are ongoing to clarify the issue. The molecular suggesting that a certain amount of NPM1wt may be necessary mechanism through which ATRA exerts its effects in AML with for leukaemic cell survival. Indeed, experimental evidence mutated NPM1 remains elusive. Interestingly, ATRA induced demonstrated that complete deletion of the NPM1 gene in apoptosis (rather than differentiation) of NPMc þ AML cells, and knock-out mice led to death during embryogenesis.3 Therefore, this effect was associated with specific downregulation of the we predict that small molecules blocking residual NPM1wt NPM1 mutant.126 activity in NPMc þ AML cells (for example, disassembling nucleolar structure and dislocating nucleophosmin in the nucleoplasm) may enhance the propensity of these cells to die Conflict of interest or be killed by concomitant administration of chemotherapeutic drugs. As NPM1-mutated AML cells contain much less nucleolar B Falini applied for a patent on the clinical use of NPM1 NPM1wt than normal haemopoietic cells, tuning the dose of the mutants. small molecules designed to interfere with NPM1wt could also become a strategy to target leukaemic cells more selectively Acknowledgements than normal cells. Another potential strategy is to interfere with NPM1 mutant- This work was supported by grants from Associazione Italiana induced changes in traffic of various proteins. As an example, Ricerca Cancro (AIRC) and Fondazione Cassa di Risparmio di NPM1 mutants dislocate HEXIM1 into cytoplasm and the Perugia, project code 2007.0099.020. We thank Roberta Pacini consequent increase in P-TEFb-dependent transcription was and Manola Carini for performing the immunohistochemical advocated as a potential target for P-TEFb inhibitors, such as studies and Dr Geraldine Boyd for helping editing the manuscript. flavopiridol and CYC202.120 The current list of molecules that are mislocalized by the NPM1 mutants25,30,78,79,89,91,92 may be only the ‘tip of the iceberg’ and the use of mass spectrometry and immunohistochemistry is expected to identify other target References proteins and possibly help defining the functional significance of their dislocation. Better understanding of the structural bases of 1 Frehlick LJ, Eirin-Lopez JM, Ausio J. 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