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Review in translational hematology
Acute myeloid leukemia carrying cytoplasmic/mutated nucleophosmin
(NPMc⫹ AML): biologic and clinical features
Brunangelo Falini,1 Ildo Nicoletti,2 Massimo F. Martelli,1 and Cristina Mecucci1
1Institutes
of Hematology and 2Internal Medicine, University of Perugia, Perugia, Italy
The nucleophosmin (NPM1) gene encodes for a multifunctional nucleocytoplasmic shuttling protein that is localized
mainly in the nucleolus. NPM1 mutations
occur in 50% to 60% of adult acute myeloid leukemia with normal karyotype
(AML-NK) and generate NPM mutants that
localize aberrantly in the leukemic-cell
cytoplasm, hence the term NPM-cytoplasmic positive (NPMcⴙ AML). Cytoplasmic
NPM accumulation is caused by the concerted action of 2 alterations at mutant
C-terminus, that is, changes of tryptophan(s) 288 and 290 (or only 290) and
creation of an additional nuclear export
signal (NES) motif. NPMcⴙ AML shows
increased frequency in adults and females, wide morphologic spectrum, multilineage involvement, high frequency of
FLT3-ITD, CD34 negativity, and a distinct
gene-expression profile. Analysis of mutated NPM has important clinical and
pathologic applications. Immunohistochemical detection of cytoplasmic NPM
predicts NPM1 mutations and helps rationalize cytogenetic/molecular studies in
AML. NPM1 mutations in absence of FLT3ITD identify a prognostically favorable
subgroup in the heterogeneous AML-NK
category. Due to their frequency and stability, NPM1 mutations may become a
new tool for monitoring minimal residual
disease in AML-NK. Future studies should
focus on clarifying how NPM mutants
promote leukemia, integrating NPMcⴙ
AML in the upcoming World Health Organization leukemia classification, and eventually developing specific antileukemic
drugs. (Blood. 2007;109:874-885)
© 2007 by The American Society of Hematology
Introduction
The NPM1 gene, mapping to chromosome 5q35 in humans,
contains 12 exons19 (Figure 1A). It encodes for 3 alternatively
spliced nucleophosmin isoforms: B23.1 (np_002511), B23.2 (np_
954654), and B23.3 (np_001032827).
B23.1, the prevalent isoform,20 is a 294–amino acid protein
of about 37 kDa, which shares a conserved N-terminal region
with the other nucleophosmin isoforms and has multiple functional domains.21,22 The N-terminus portion contains a hydrophobic region,21,22 regulating self-oligomerization23 and NPM chaperone activity toward proteins, nucleic acids, and histones.24,25
In resting and proliferating cells, more than 95% of NPM is
present as an oligomer26; both the nonpolar N-terminus region21
and multimeric state of NPM26 appear to be crucial for proper
assembly of maturing ribosomes in the nucleolus. The middle
portion of NPM contains 2 acidic stretches that are critical for
histone binding25; the segment between the acidic stretches
exerts ribonuclease activity.21 The C-terminal domain, with its
basic regions involved in nucleic acid binding and ribonuclease
activity,21,22 is followed by an aromatic short stretch encompassing tryptophans 288 and 290, which are critical for NPM binding
to nucleolus.27 B23.1 is also equipped with a bipartite nuclear
localization signal (NLS),21,22 leucine-rich nuclear export signal
(NES) motifs,28,29 and several phosphorylation sites regulating
its association with centrosomes30 and subnuclear compartments.31,32 In immunocytochemical analysis, B23.1 shows restricted nucleolar localization.20
B23.2, a truncated isoform, lacks the last 35 C-terminal
amino acids of B23.1 (containing the nucleolar-binding domain)
and is found in tissues at very low levels. In immunocytochemical analysis, B23.2 is located in nucleoplasm.33 Little information is available on the B23.3 isoform, which consists of 259
amino acids.
Submitted July 10, 2006; accepted September 11, 2006. Prepublished
online as Blood First Edition Paper, October 2, 2006; DOI 10.1182/blood-
2006-07-012252.
© 2007 by The American Society of Hematology
Acute myeloid leukemia (AML) is clinically, cytogenetically, and
molecularly heterogeneous.1 About 30% of cases carry recurrent chromosomal abnormalities that identify leukemia entities with distinct
clinical and prognostic features,2 and 10% to 15% of AMLs have
nonrandom chromosomal abnormalities. In cytogenetic analysis, 40%
to 50% of AMLs show normal karyotype (AML-NK) and, biologically
and clinically, are the most poorly understood group.1,3,4 Attempts to
stratify AML-NK using gene microarrays5,6 succeeded in associating
gene-expression patterns with differences in response to treatment, but
no specific genetic subgroups emerged. Molecular analyses ofAML-NK
identified several mutations that target genes encoding for transcription
factors (AML1 and CEBPA: 2%-3% and 15%-20% of cases, respectively),7-10 receptor tyrosine kinases (FLT3 and KIT: 25%-30% and 1%,
respectively),11-13 and the RAS genes (10%),14 as well as a partial tandem
duplication of MLL gene (MLL-PTD: 5%-10%).15-17
We discovered that nucleophosmin (NPM1) gene mutations
target 50% to 60% of adult AML-NK.18 This is the most common
genetic lesion described to date in adult de novo AML (about one
third of patients).18 Since NPM1 mutations dislocate nucleophosmin into cytoplasm, we named this subgroup NPM-cytoplasmic
positive (NPMc⫹) AML,18 and here we review its biologic and
clinical features.
Nucleophosmin (NPM1) gene and protein
Structure of gene and protein
874
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BLOOD, 1 FEBRUARY 2007 䡠 VOLUME 109, NUMBER 3
Figure 1. The NPM1 gene encodes for a protein involved in multiple functions.
(A) The NPM1 gene contains 12 exons. NPM1 is translated from exons 1 to 9 and 11
to 12. The portion encoding isoform B23.2 contains exons 1 to 10. In the protein, the
N-terminus is characterized by a nonpolar domain responsible for oligomerization
and heterodimerization. Functional nuclear export signal (NES) motifs and a
metal-binding (MB) domain are present in this region. The central portion of the
protein contains 2 acidic stretches (Ac) that are important for binding to histones, and
a bipartite nuclear localization signal (NLS); this region confers ribonuclease activity.
The C-terminus of the protein shows ribonuclease activity and contains basic regions
involved in nucleic-acid binding. The latter are followed by an aromatic stretch, unique
to NPM isoform 1, which contains 2 tryptophan residues (288 and 290), which are
required for nucleolar localization of the protein (NoLS). (B) NPM is a nucleolar
phosphoprotein that shuttles between the nucleus and cytoplasm. Shuttling plays a
fundamental role in ribosome biogenesis, since NPM transports preribosomal
particles. In cytoplasm, NPM binds to the unduplicated centrosome and regulates its
duplication during cell division. Furthermore, NPM interacts with p53 and its
regulatory molecules (ARF, Hdm2/Mdm2) influencing the ARF-Hdm2/Mdm2-p53
oncosuppressive pathway.
Expression and functions of NPM protein
Nucleophosmin is a highly conserved phosphoprotein that is
ubiquitously expressed in tissues.34 It is one of the most abundant
of the approximately 700 proteins identified to date in the nucleolus
by proteomics.35,36 Although the bulk of NPM resides in the
granular region of the nucleolus,36,37 it shuttles continuously
between nucleus and cytoplasm,38,39 as proven by interspecies
heterokaryon assays. NPM nucleocytoplasmic traffic is strictly
regulated, since important NPM functions, including transport of
ribosome components to the cytoplasm and control of centrosome
duplication, are closely related to its ability to actively mobilize
into distinct subcellular compartments.28,29 Here, we briefly discuss
some of the major functions of NPM that have been recognized so
far (Figure 1B).
NPM plays a key role in ribosome biogenesis providing the
necessary export signals and chaperoning capabilities that are
required to transport components of the ribosome from nucleus to
cytoplasm. A major role of NPM is to mediate, through a
Crm1-dependent mechanism, nuclear export of the ribosomal
protein L5/5S rRNA subunit complex.29 Other elements that
implicate NPM in the processing and/or assembly of ribosomes are
its nucleocytoplasmic shuttling properties and intrinsic RNAse
activity,40 and the ability to bind nucleic acids,41 to process
pre-RNA molecules,42 and to act as a chaperone,24 thus preventing
BIOLOGIC AND CLINICAL FEATURES OF NPMc⫹ AML
875
protein aggregation in the nucleolus during ribosome assembly.23
Because it can directly access maturing ribosomes and potentially
regulate the protein translational machinery,29 NPM seems to play a
central role in balancing protein synthesis, and cell growth and
proliferation. This view concurs with evidence that closely correlates NPM overexpression with cell proliferation43; for example, in
bone marrow, NPM positivity progressively decreases as cells
mature from proerythroblasts to normoblasts and from promyelocytes to neutrophils (B.F., personal observation, January 2005).
Moreover, NPM overexpression promotes survival and recovery of
hematopoietic stem cells under stress conditions44; high NPM
levels in malignant cells promote aberrant cell growth by enhancing ribosome machinery. Conversely, inhibition of NPM shuttling
or NPM loss block protein translation and produce cell-cycle arrest.29
NPM also maintains genomic stability,45,46 controlling DNA
repair47 and centrosome duplication48 during mitosis. NPM associates with the unduplicated centrosome in resting cells and dissociates from it after CDK2-cyclinE–mediated phosphorylation on
threonine 199,49 thus enabling proper chromosome duplication.
NPM reassociates with centrosomes at the mitotic spindle during
mitosis as a result of phosphorylation on serine 4 by PLK150 and
NEK2A.51 NPM seems to protect from centrosome hyperamplification as its inactivation causes unrestricted centrosome duplication
and genomic instability,45,52 with increased risk of cellular
transformation.
Finally, NPM interacts with the oncosuppressors p53 and
p19Arf and their partners (ie, Hdm2/Mdm2), thus controlling cell
proliferation and apoptosis.53
(1) Interaction with p53. NPM regulates p53 levels and
activity. A functional link exists between nucleolar integrity, NPM,
and p53 stability,54,55 since stimuli inducing cellular stress (eg, UV
radiation or drugs interfering with rRNA processing) lead to loss of
nucleolar integrity, relocation of NPM from nucleolus to nucleoplasm, and p53 activation.55,56 Nucleoplasmic NPM increases p53
stability by binding to, and inhibiting, Hdm2/Mdm257 (a p53
E3-ubiquitin ligase), although “nucleolar stress” can also activate
p53 in an Hdm2/Mdm2-independent fashion.58,59 Thus, under
stress conditions, the nucleolus functions as a “stress-sensor” with
NPM playing a key role in potentiating p53-dependent cell-cycle
arrest.57 NPM-p53 cross talk may also involve proteins that are
downstream targets of p53 (eg, GADD45␣).60
(2) Interaction with ARF. In cultured unstressed cells, most
NPM and ARF colocalize in the nucleolus, in high-molecularweight complexes containing other nucleolar proteins.61 The NPMARF interaction helps stabilize both proteins, since ARF influences
NPM polyubiquitination62 and NPM protects ARF from degradation.46,63 The functional significance of NPM and ARF interactions
is still in debate.64 There is evidence that ARF targets NPM into
nucleoli for degradation,62 that the ARF-NPM complex sequesters
Hdm2/Mdm2 into the nucleolus thereby activating p53 in the
nucleoplasm,65 that ARF impedes NPM shuttling, thereby producing cell-cycle arrest in a p53-independent fashion,66 and that NPM
sequesters ARF into the nucleolus, impairs ARF-Hdm2/Mdm2
association, and inhibits ARF’s p53-dependent activation.67 Despite these uncertainties, NPM and ARF activities appear closely
coordinated and regulate cell proliferation and apoptosis65 through
precise subnuclear compartmentalization.68
In response to cellular stress, NPM and ARF are redistributed to
the nucleoplasm.68 Competition between Hdm2/Mdm2 and NPM
for ARF binding promotes formation of NPM-Hdm2/Mdm2 and
ARF-Hdm2/Mdm2 binary complexes and strongly activates the
p53 pathway.69 Thus, through its nucleolar association with NPM,
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FALINI et al
ARF may directly access ribosome function to inhibit cell growth,
and via its nucleoplasmic interaction with Hdm2/Mdm2 and
ARF-BP1,70 it may regulate the p53 cell-cycle pathway.
Discovery of NPM1 mutations in AML
NPM1 is translocated to several partner genes in various hematopoietic malignancies,71 including CD30⫹ anaplastic large-cell lymphoma with t(2;5),72 the rare myelodysplasia/AML with t(3;5),73
and exceptional cases of promyelocytic leukemia with t(5;17).74
Translocations create chimeric genes encoding for fusion proteins
(NPM-ALK,72 NPM-MLF1,73 and NPM-RAR␣74), which form
heterodimers with wild-type NPM (NPMwt) and promote cellular
transformation. Since proper NPM function is closely dependent on
its levels in different cell compartments, we wondered whether
NPM fusion proteins have a different subcellular distribution to
NPMwt and whether this finding could be exploited for diagnostic
purposes. In the late 1980s, we generated monoclonal antibodies
against fixative-resistant epitopes of NPM protein34 to investigate
NPM subcellular expression in anaplastic large-cell lymphoma
carrying the NPM-ALK fusion protein compared with normal
tissues. We found that, because of NPM-ALK chimeric protein, this
lymphoma was associated with NPM expression in cytoplasm75
instead of in the nucleolus as in normal tissues.34 Reasoning that
detection of aberrant cytoplasmic NPM might provide a simple
way of immunoscreening routine bioptic samples for potential
NPM1 gene alterations, we extended our immunohistochemical
investigations to a range of human tumors, including leukemias. In
1999, we observed our first AML cases with cytoplasmic NPM but
no known NPM fusion proteins, and named them NPMc⫹ AML to
distinguish them from NPMc⫺ AML, which showed the expected
nucleus-restricted NPM expression. Subsequently, in 591 AML
patients enrolled in the GIMEMA/AML12 EORTC trial, we
observed cytoplasmic NPM is mutually exclusive of major chromosomal abnormalities, closely associates with normal karyotype, and
has distinctive biologic and clinical features.18 These findings led
us to hypothesize that an as-yet-unidentified genetic lesion of
NPM1 gene underlayed NPMc⫹ AML, and indeed gene sequencing
revealed mutations at exon 12.18
Characteristics of NPM1 mutations
NPM1 mutations are reliably identified by various molecular
biology techniques,76,77 which also allow simultaneous detection of
NPM1 and FLT3-ITD or CEBPA mutations78,79 (all prognostically
relevant in AML-NK10,80-83).
Immunohistochemical detection of cytoplasmic NPM in AML
is predictive of NPM1 mutations.84 The main advantages of
immunohistochemistry are simplicity, low cost, and ability to
correlate cytoplasmic NPM with morphology and topographic
distribution of leukemic cells. Moreover, immunohistochemistry is
the first-choice technique for detecting NPM1 mutations in cases of
dry-tap or small biopsies (eg, skin involvement in myeloid
sarcoma85), and also helps rationalize cytogenetic/molecular studies in AML.84
The choice of technique for detecting NPM1 mutations at
diagnosis may be based on the investigator’s experience, costs,
available equipment, application of bone marrow trephine for AML
diagnosis, among other factors. Immunohistochemistry is not
suitable for monitoring minimal residual disease in NPMc⫹ AML.
BLOOD, 1 FEBRUARY 2007 䡠 VOLUME 109, NUMBER 3
Specificity of NPM1 mutations
NPM1 mutations are AML specific since other human neoplasms
consistently show nucleus-restricted NPM expression.18 They have
been sporadically detected in chronic myeloproliferative disorders
(5/200 cases investigated; 2.5%).86,87 All 5 cases were chronic
myelomonocytic leukemias and 4 quickly progressed to overt AML
in less than 1 year, suggesting they represented M4 or M5 AMLs
with marked monocytic differentiation (often targeted by NPM1
mutations18,80). NPM1 mutations were found in only 2 (5.2%) of 38
myelodysplastic patients.88 However, caution is needed when defining a
case as “NPM1-mutated myelodysplastic syndrome” since NPMc⫹
AML frequently shows multilineage involvement and dysplastic
features.89 NPM1 mutations closely associate with de novo AML,
as AML secondary to myeloproliferative disorders/myelodysplasia
and therapy-related AML rarely show cytoplasmic NPM.18
Types, frequency, and stability of NPM1 mutations
A polymorphism of nucleotide T deletion at position 1146 in the
NPM1 3⬘-untranslated region has been reported in 60% to 70% of
AMLs and in healthy volunteers.81,90 NPM1 mutations are characteristically heterozygous and retain a wild-type allele. In children
with AML, their incidence ranges from 2.1% in Taiwan90 to
6.5% in Western countries,91,92 accounting for 9% to 26.9% of
all childhood AML-NK.90-92 In about 3000 adult AML patients,
the frequency of NPM1 mutations ranged between 25% and
35%,80-83,90,93,94 accounting for 45.7% to 63.8% of adult AMLNK.80-83,90,93,94 This suggests the molecular pathogenesis of
AML-NK is different in adults and children.
Except for 2 cases involving the splicing donor site of NPM1
exon 995 and exon 11,96 mutations are restricted to exon
1218,80-83,93,94 (Figure 2). The most common NPM1 mutation, which
we named mutation A,18 is a duplication of a TCTG tetranucleotide
at position 956 to 959 of the reference sequence (GenBank
accession number NM_002520) and accounts for 75% to 80% of
cases. Mutations B and D are observed in about 10% and 5% of
NPMc⫹ AML; other mutations are very rare (Figure 2).
Alterations of the NPM1 gene appear more stable than those of
FLT3 gene.93 Loss of NPM1 mutations at relapse is rare and may be
due either to emergence of a different leukemic clone or inability to
detect mutations because few leukemic blasts are infiltrating the
marrow.90 In some cases, loss of NPM1 mutations was associated
with change from normal to abnormal karyotype.90,93 AML patients
with a germ-line NPM1 gene at diagnosis do not appear to acquire
NPM1 mutations during the course of disease, suggesting that
mutations play a scarce role in disease progression.90
Relationship between NPM1 mutations, karyotype,
and other mutations
Cytoplasmic NPM closely associates with normal karyotype (Figure 3A) and is mutually exclusive of recurrent genetic abnormalities,18 as confirmed by NPM1 mutational analysis of thousands of
AML patients.80-83,91,93,94 Chromosomal abnormalities observed in
a minority of NPMc⫹ AMLs (14%)18 are probably secondary
because (1) they are similar, in type and frequency, to secondary
chromosomal changes occurring in AML with recurrent genetic
abnormalities97; (2) cells with an abnormal karyotype represent
subclones within the leukemic population with normal karyotype18;
and (3) these abnormalities occasionally occur at relapse in patients
with AML-NK at diagnosis (B.F., unpublished observations, September 2005).
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BLOOD, 1 FEBRUARY 2007 䡠 VOLUME 109, NUMBER 3
BIOLOGIC AND CLINICAL FEATURES OF NPMc⫹ AML
877
Figure 2. List of exon 12 NPM1 mutations so far identified in AML patients. Red coloring indicates nucleotides inserted by mutations; green, leucine-rich NES motif; violet,
tryptophan (W) residues; L, leucine; C, cysteine; V, valine; M, methionine; and F, phenyl-alanine. Mutations A to F refer to those we originally identified.18 Mutations E to H refer
to those we identified in childhood AML.91 Mutations J to Q refer to those subsequently identified in Falini et al.84 Mutations Gm to Qm refer to those we identified in the protocol
99 of the German AML cooperative group.80 Mutations 1, 3, 4, 6, 7, 12, 13, 10, and 14 are according to Dohner et al.81 Mutation I* is according to Verhaak et al.82 *NM_002520.
†Mutations G, I, H, and J are according to Suzuki et al.93
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BLOOD, 1 FEBRUARY 2007 䡠 VOLUME 109, NUMBER 3
Figure 3. NPM1 mutations closely associate with AML-NK and FLT3-ITD. (A) Pie
chart showing cytogenetic alterations in adult AML and frequency of NPM1 gene
mutations in AML with normal karyotype (AML-NK). (B) Pie chart showing relationship
between NPM1 mutations and FLT3-ITD in AML-NK.
Several studies80-83,93 confirmed our original observation that
NPMc⫹ AML is preferentially targeted by FLT3-ITD18 (Figure 3B).
NPM1 and FLT3-TKD correlated in some studies,80,81,93 but not in
others.18,82 Thiede et al83 found NPM1 mutations are primary
events that precede acquisition of FLT3-ITD or other mutations.
MLL-PTD rarely coincides with cytoplasmic/mutated NPM.18
Some authors80,81 reported no difference in frequency of CEBPA
mutations in NPM1-mutated versus NPM1-unmutated AML-NK,
while Chou et al90 found a negative correlation between NPM1 and
CEBPA mutations. KIT and NRAS mutations were not significantly
associated with NPM1 mutations.80,81 KRAS mutations were reported in 5 of 8 AML with NPM1 mutations.82 Unsurprisingly,
TP53 mutations rarely occur in NPMc⫹ AML,93 as they mostly
associate with karyotypic abnormalities,98,99 while NPM1 mutations are typical of AML-NK.
Nucleocytoplasmic traffic of wild-type NPM
Although NPMwt is a nucleocytoplasmic shuttling protein,38 its
expression is restricted to the nucleolus.34,37 Explanation comes
from analysis of NPM functional domains (Figure 4 top). The NLS
signal drives NPM from cytoplasm to nucleoplasm, where it is
translocated to the nucleolus through its nucleolar-binding domain,
particularly tryptophans 288 and 290.27 Nuclear export of NPM
(and of proteins in general) is mediated by the evolution-conserved
Crm1 (also called exportin 1),100,101 the export receptor of proteins
containing leucine-rich NES motifs with the generally accepted
loose consensus L-x(2,3)-(LIVFM)-x(2,3)-L-x-(LI),102 a core of
closely spaced leucines or other large hydrophobic amino acids.
Two such highly conserved NES motifs have been identified in the
NPMwt protein: one with the sequence I-xx-P-xx-L-x-L within
residues 94 to 10228 and another at the N-terminus within amino
Figure 4. NPM, a shuttling protein, is mainly localized in the nucleolus. (Top)
Mechanism of nucleocytoplasmic traffic of NPM wild-type (NPMwt). Green boxes
indicate NPMwt protein; red circles, tryptophan residues; NES motif, nuclear export
signal motif; and NLS, nuclear localization signal. The nuclear import of NPM (arrows)
greatly predominates over the nuclear export (dotted arrow). Thus, NPM is mainly
localized in the nucleolus. (Middle) Confocal analysis of NIH-3T3 cells expressing
enhanced green fluorescent protein (eGFP)–NPMwt fusion protein. The bulk of
NPMwt (green) colocalizes in nucleoli with C23/nucleolin (red), as revealed using a
specific antibody. Images were collected with a Zeiss LSM 510 confocal microscope
(Zeiss, Jena, Germany) using a Plan Apochromat 100⫻/1.4 NA oil objective and the
LSM 5 software (Zeiss) for image acquisition. An Alexa 543-conjugated secondary
antibody (Molecular Probes, Eugene, OR) was used for C23/nucleolin staining;
nuclei were stained with To-Pro3 (Molecular Probes). Three-dimensional reconstruction of the confocal images and electronic cuts of nucleus and nucleoli to analyze the
subcellular distribution of NPMwt (green) and C23 (red) proteins were performed with
Imaris software (Bitplane, Zurich, Switzerland).
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BLOOD, 1 FEBRUARY 2007 䡠 VOLUME 109, NUMBER 3
acids 42 to 61, with leucines 42 and 44 as critical nuclear export
residues.29 However, despite the NES motifs, NPM remains
localized in nucleoli (Figure 4 middle and bottom panels). Thus, in
physiologic conditions, nuclear import of NPMwt greatly predominates over export (Figure 4 top panel), since NPMwt’s NES motifs
exhibit the general property of NES motifs (ie, weak interaction
with Crm1).103,104 Knowledge of how NPMwt nucleocytoplasmic
traffic is regulated at the steady state is fundamental to understanding the molecular mechanisms underlying aberrant cytoplasmic
NPM expression in AML.18
Altered nucleocytoplasmic traffic of
nucleophosmin in NPMcⴙ AML
In NPMc⫹ AML, aberrant cytoplasmic NPM localization, which is
easily detected by immunohistochemistry (Figure 5), is caused by 2
mutation-related alterations at the C-terminus of NPM leukemic
mutants105: (1) generation of an additional leucine-rich NES
motif,106 reinforcing Crm1-dependent nuclear export of mutated
NPM proteins105; and (2) loss of tryptophan residues 288 and 290
(or residue 290 alone)18, which determine NPM nucleolar localization.27 Both alterations are crucial for perturbing NPM mutant
traffic105 (Figure 6).
BIOLOGIC AND CLINICAL FEATURES OF NPMc⫹ AML
879
Role of the additional C-terminus NES motif
Specific exportin-1/Crm1-inhibitors107 (leptomycin-B and ratjadones) block cytoplasmic NPM accumulation, demonstrating that
export of NPM mutants is NES dependent105 (Figure 6A top
panels). How can we reconcile this finding with the observation
that NPMwt, despite the presence of physiologic NES motifs,28,29 is
restricted to nucleolus? Comparative analysis of artificial and
leukemic NPM mutants provides the answer. Artificial NPMwt
mutants lacking tryptophans 288 and 290 delocalize from nucleolus to nucleoplasm but do not accumulate into cytoplasm,27,28 due
to the weak activity of the physiologic NES motifs; a similar
picture is observed with the C-terminus truncated B23.2 isoform.33
In contrast, NPM leukemic mutants, which also lack C-terminus
tryptophan(s), accumulate in the cytoplasm, since the new Cterminus NES motif acts as the additional force causing their
overexport from nucleus to cytoplasm.105
Role of C-terminal tryptophans
The C-terminus NES motif is, however, insufficient in itself, for
nuclear export of NPM mutants105 because reinserting residues 288
and 290 (which are both critical for nucleolar binding27) relocates
NPM mutants to nucleoli (Figure 6A bottom panels).
Figure 5. Cytoplasmic NPM and multilineage involvement in NPMcⴙ AML. (Top, left) Myeloid sarcoma (skin, paraffin section). Nuclear plus aberrant cytoplasmic
expression of NPM is seen in leukemic blasts. Normal residual vessel endothelial cells show the expected nucleus-restricted NPM expression (arrow). Immunostaining with
monoclonal antibody 376 that recognizes both wild-type and mutated NPM (alkaline phosphatase anti–alkaline phosphatase [APAAP] technique; hematoxylin counterstaining;
image was collected using an Olympus BX51 microscope and a UPlanApo 40⫻0.85 NA objective). (Top, right) NPMc⫹ AML (bone marrow biopsy, paraffin section).
Cytoplasmic-restricted expression of mutant NPM protein in leukemic cells (arrow). A normal residual megakaryocyte is negative (double arrows). Immunostaining with a
polyclonal antibody (Sil-A) that recognizes mutated but not wild-type NPM (APAAP technique; hematoxylin counterstaining; image was collected using an Olympus BX51
microscope and a UPlan FL 100⫻/1.3 NA oil objective). (Bottom, left) NPMc⫹ AML with multilineage involvement (bone marrow biopsy, image was collected using an Olympus
BX51 microscope and a UplanApo 40⫻/0.85 NA objective). Double APAAP/immunoperoxidase staining for NPM (blue) and glycophorin (brown). Aberrant cytoplasmic
expression of NPM is seen in proerythroblasts (arrow), megakaryocytes (double arrows), and myeloid blasts surrounding a blood vessel (asterisk). (Bottom, right) Higher
magnification (image was collected using an Olympus BX51 microscope and a UPlan FL 100⫻/1.3 NA oil objective) from a different field of the same case. Proerythroblasts are
strongly NPMc⫹ (blue) and weakly positive or negative for glycophorin (brown) (long arrow), while more mature erythroid precursors show nucleus-restricted NPM(blue)/strong
surface glycophorin (brown) (short arrow). Double arrows indicate a NPMc⫹/glycophorin-negative megakaryocyte. A myeloid blast is positive for cytoplasmic NPM (blue) and
negative for surface glycophorin (arrowhead).
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BLOOD, 1 FEBRUARY 2007 䡠 VOLUME 109, NUMBER 3
ated with variants of this NES motif (ie, replacement of valine at
the second position with leucine, phenyl-alanine, cysteine, or
methionine).105 Therefore, to ensure NPM mutants are dislocated
into cytoplasm, variant NES motifs likely need to be more efficient
than L-xxx-V-xx-V-x-L, so as to counterbalance the action of
tryptophan 288, which drives mutants to the nucleolus.27 That NES
motifs may differ in their activity, influencing the ability of a given
protein to be exported from the nucleus more or less efficiently,
is a known phenomenon,108,109 which may also apply to NPM
leukemic mutants.
A hypothetical scheme of NPM nucleocytoplasmic traffic in
NPMc⫹ AML is shown in Figure 6B. NPM mutants retain the NLS
and can, therefore, enter the nucleus. Their capability to bind
nucleoli is hindered completely (when both tryptophans are
mutated) or partially (when only tryptophan 290 is altered). The
additional C-terminus NES motif (L-xxx-V-xx-V-x-L if both
tryptophans are mutated, or a variant NES motif if tryptophan 288
is retained) probably renders the mutants more available for
binding to Crm1 in nucleoplasm. Under these circumstances, the
NES-Crm1–mediated nuclear export of NPM mutants is more
efficient than nuclear import and they accumulate in cytoplasm.
There is now evidence that NPM mutants recruit NPMwt from
nucleoli and delocalize it into nucleoplasm and cytoplasm,105,110
perhaps altering its functions. Recruitment occurs through the
N-terminal dimerization domain that is conserved in all NPM
mutants, allowing them to form heterodimers with NPMwt, as
occurs between NPMwt and NPM fusion proteins.71,111,112 NPM
also interacts with ARF and delocalizes it into cytoplasm.110,113
Role of NPM mutants in leukemogenesis
Figure 6. Mechanism of perturbed NPM nucleocytoplasmic traffic in NPMcⴙ
AML. (A) Cytoplasmic accumulation of NPM is NES-dependent. OCI-AML3 cell line
carrying the NPM1 mutation A shows cytoplasmic (and nucleolar) positivity for NPM
(green) (top, left). NPM is relocated into the nucleus after exposure to the Crm1
inhibitor leptomycin B (top, right). Cytoplasmic accumulation of NPM is tryptophan
dependent. In NIH-3T3 cells, NPM mutant A is relocated from cytoplasm (green,
bottom, left) into nucleoli (green, bottom right), after the 2 tryptophans (W) are
reinserted at positions 288 and 299. Images were collected with a Zeiss LSM 510
confocal microscope using a Plan Apochromat 100⫻/1.4 NA oil objective and LSM 5
software for image acquisition. Nuclei were stained with propidium iodide (Molecular
Probes). Three-dimensional reconstruction of the confocal images and electronic
cuts of cells were performed with Imaris software. (B) Hypothetical mechanism of
altered nucleocytoplasmic traffic of NPM mutant A and NPMwt. Green boxes indicate
NPMwt protein; blue boxes, mutated NPM protein; red circles, tryptophan residues;
black circles, mutated tryptophans; magenta boxes, NES motif; and NLS, nuclear
localization signal.
More than 95% of NPM leukemic mutants lack tryptophans 288
and 290; the others retain tryptophan 288. Despite this difference,
all mutants are aberrantly dislocated in the cytoplasm, implying
that their nuclear export occurs even when tryptophan 288 is
present. The explanation comes from analysis of the relationship
between mutated tryptophans and types of NES motif at the
C-terminus of leukemic mutants.105 Mutations of both tryptophans
are usually associated with the most common NES motif (ie,
L-xxx-V-xx-V-x-L); retention of tryptophan 288 is always associ-
NPM1 belongs to a new category of genes that function both as
oncogenes and tumor-suppressor genes,114 depending on gene
dosage, expression levels, interacting partners, and compartmentalization. NPM1 appears implicated in promoting cell growth, as its
expression increases in response to mitogenic stimuli and abovenormal amounts are detected in highly proliferating and malignant
cells. On the other hand, since NPM contributes to growthsuppressing pathways through its interaction with ARF, loss of
NPM expression or function can contribute to tumorigenesis.
At present, no experimental models exist for NPMc⫹ AML. In
NPM knock-out mice, NPM is involved in control of primitive
hematopoiesis (especially erythropoiesis), and NPM is haploinsufficient for the control of proper centrosome duplication, resulting in
genomic instability and development of a hematologic syndrome
reminiscent of human myelodysplasia.45 In NPMc⫹ AML, since
one NPM allele is mutated and NPM mutants dislocate the NPMwt
encoded by the uninvolved allele into cytoplasm, one might argue
that blasts show a form of NPM haploinsufficiency. However,
NPMc⫹ AML is characterized by normal karyotype as NPM
mutants are still effective in binding to, and controlling, centrosome duplication. Furthermore, NPM1 mutations are extremely
rare in human myelodysplasia, being characteristically associated
with de novo AML.
Therefore, other mechanisms, probably related to inherent NPM
mutant properties, may operate in humans to promote leukemic
transformation. Cytoplasmic NPM may contribute to AML development by inactivating ARF, a critical antioncogene.115 In NIH3T3 fibroblasts engineered to produce a zinc-inducible ARF
protein, human leukemic NPM mutant A delocalizes NPMwt and
ARF from nucleoli to cytoplasm,110 resulting in reduced p53-
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BLOOD, 1 FEBRUARY 2007 䡠 VOLUME 109, NUMBER 3
dependent (Hdm2/Mdm2 and p21cip1 induction) and p53independent (ARF-induced sumoylation of NPM and Hdm2/
Mdm2) ARF activities.110 When complexed with NPM mutant,
ARF stability was greatly compromised and the p53-dependent
cell-cycle arrest at the G1/S boundary was appreciably lower.113
ARF is only one of the potential targets of NPM mutants, and new
molecules are expected to be found that could interact with NPM
mutants and contribute to leukemogenesis. A hypothetical scheme
of the molecular events underlying NPMc⫹ AML is shown in
Figure 7.
Pathologic and clinical features
of NPMcⴙ AML
NPMc⫹ AML shows a wide morphologic spectrum.18 However,
NPM1 mutations are more frequent in M4 and M5 FAB categories,18,80,81 and in AML with prominent nuclear invaginations
BIOLOGIC AND CLINICAL FEATURES OF NPMc⫹ AML
881
(“cuplike” nuclei).116 More than 95% of NPMc⫹ AMLs are
CD34⫺.18 Involvement of several cell lineages (myeloid, monocytic, erythroid, and megakaryocytic but not lymphoid)89 (Figure
5) is another distinctive, apparently intrinsic feature of NPMc⫹
AML that is independent of additional genetic alterations, such as
FLT3-ITD. High frequency of multilineage involvement is somewhat surprising since NPMc⫹ AML is usually a de novo leukemia,18 and multilineage involvement is mainly associated with
secondary leukemias.117
The blast count is higher in NPM-mutated than NPM–germ-line
AML-NK,81,83 and rises with concomitant FLT3-ITD83 and LDH
serum levels.81 NPM1 mutations correlate with extramedullary
involvement, mainly gingival hyperplasia and lymphadenopathy,81
possibly because this extramedullary dissemination pattern is
mostly seen with M4 and M5 AML,2 which are frequently targeted
by NPM1 mutations.80,81
Platelet counts are higher in NPM-mutated than NPM–germline AML-NK.81,83 Of interest, bone marrow biopsies from NPMc⫹
AML (especially M4) frequently show increased number of
megakaryocytes carrying mutated NPM and exhibiting dysplastic
features.89 These findings suggest that megakaryocytes with NPM1
mutations retain a certain capacity for thrombocytic differentiation.
NPM1 mutations are more frequent in female patients,80,81,83
strangely, as AML incidence is higher in males.83 This appears to be
specific for NPM1 mutations, as a similar association is not seen for
FLT3-ITD,83 which is also common in AML-NK. To date, only the
5q syndrome has been identified as a prevalent genetic alteration in
females.118 Therefore, sex-specific differences might underlie the
mechanism of NPMc⫹ AML development.
Clinical impact of NPM1 mutations
Analysis of mutated/cytoplasmic NPM has important clinical
applications.
Response to therapy and prognostic value of NPM1 mutations
Figure 7. Hypothetical mechanism leading to NPMcⴙ AML.
After induction therapy, AML-NK carrying mutated/cytoplasmic
NPM shows a higher complete remission rate than AML-NK
without NPM1 mutations.18,80,83,93 Dohner et al81 found NPM1
mutation status is not an independent predictor for responsiveness
to chemotherapy, because the best complete remission rate was
observed only in the NPM1-mutated/FLT3-ITD–negative group,
while the lowest response rate occurred in AML patients carrying
both mutations.
In 4 large European studies80-83 on more than 1000 AML-NKs,
NPM1 mutations in the absence of FLT3-ITD identified a subgroup
of patients with favorable prognosis. Notably, younger AML
patients81 carrying NPM1 mutations without concomitant FLT3ITD showed approximately 60% probability of survival at 5 years,
that is, similar to long-term outcomes in AML carrying corebinding factor (CBF)119,120 or AML-NK with CEBPA mutations.121
In a donor versus no-donor comparison, the favorable prognostic
group of NPM-mutated/FLT3-ITD–negative patients did not benefit from allogeneic stem cell transplantation.81
Thus, 2 “dueling” mutations122 act in AML-NK, because NPM1
and FLT3-ITD are, respectively, associated with good and bad
prognosis. In patients with both mutations, FLT3-ITD–induced
antiapoptotic and proproliferative pathways, especially via STAT5,
might dominate the leukemic phenotype,123 explaining why 2 other
groups,93,94 which did not stratify patients according to NPM1 and
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882
FALINI et al
FLT3-ITD mutations, could not document differences in survival in
NPM1-mutated cases. These studies emphasize the value of
comprehensive molecular genetic screening in AML-NK, so as to
improve risk stratification.
It is unclear why NPM1-mutated/FLT3-ITD–negative patients
respond reasonably well to therapy. Nucleoplasmic and cytoplasmic recruitment of NPMwt mediated by the NPM mutants might
interfere with its functions. NPMwt protects hematopoietic cells
from p53-induced apoptosis in conditions of cellular stress124; the
level of genotoxic stress appears to regulate this effect.56 It is
tempting to speculate that NPM mutants fail to protect cells and
render them more susceptible to chemotherapy-induced high-level
genotoxic stress. Of interest, daunorubicin-induced dislocation of
NPMwt into nucleoplasm is associated with increased apoptosis.125
Monitoring of minimal residual disease
Monitoring of minimal residual disease in AML-NK is hampered
by the lack of reliable molecular markers. FLT3-ITD can be used
for this purpose,11 but is detected in only about 30% of AMLNK.11,12 Since NPM1 mutations appear more stable than FLT3-ITD
over the course of disease90,93 and occur in 50% to 60% of
AML-NK,18,80-83 they may become a new tool for monitoring
minimal residual disease. Preliminary results using a highly-sensitive
real-time quantitative polymerase chain reaction (PCR) assay126 are
encouraging but need to be validated in large clinical trials.
Classification of leukemias
Identification of NPMc⫹ AML impacts on the current World Health
Organization (WHO) classification of AML,2 which encompasses 4
major categories: (1) AML with recurrent genetic abnormalities;
(2) AML with multilineage dysplasia; (3) therapy-related AML;
and (4) AML “not otherwise characterized.” The latter bin category
(currently subclassified according to slightly modified FAB criteria) accounts for 60% to 70% of de novo adult AML, including
AML-NK and, therefore, NPMc⫹ AML. Our observation that the
wide morphologic spectrum of NPMc⫹ AML derives from various
combinations, at diverse ratios, of NPMc⫹ cells of different
lineages (all belonging to the leukemic clone)89 question the value
of FAB criteria for subclassifying “AML not otherwise characterized.” The frequent finding of multilineage dysplasia in NPMc⫹
AML89 also raises doubts as to whether dysplasia is a reproducible
criterion for assigning a case to WHO category 3. When updating
WHO classification, we propose the term “AML not otherwise
characterized” should be restricted to AML for which no genetic
lesions have as yet been identified, and NPMc⫹ AML be provisionally considered as a separate entity, because of its high frequency
and distinctive clinical and biologic features. Correct definition of
the heterogeneous WHO AML categories 3 and 4 and their
prognostic significance will require a more genetically oriented
approach, including systematic chromosome banding analyses and
screening for the prognostically relevant mutations (FLT3, CEBPA,
and NPM1).
Future perspectives
In spite of the remarkable progress achieved in biologic, diagnostic, and clinical characterization of NPM1 mutations since their first
identification in early 2005,18 several important issues still remain
to be explored. Although multilineage involvement points to
derivation from a common myeloid or an earlier progenitor without
BLOOD, 1 FEBRUARY 2007 䡠 VOLUME 109, NUMBER 3
the ability to differentiate into lymphoid lineages,89 the exact cell of
origin of NPMc⫹ AML remains unknown. How does the NPMc⫹
AML cell of origin correlate with the recently revised road map for
adult blood lineage development?127 In NPMc⫹ AML, how can we
reconcile consistent CD34 negativity with up-regulation of HOX
genes involved in stem-cell maintenance?82,128 What are the
characteristics of leukemic stem cells in NPMc⫹ AML? These
issues need to be addressed by analyzing flow-sorted progenitor
populations and their engraftment potential in mice.
How mutated NPM contributes to leukemogenesis still remains
elusive. What we know at present could well be only part of the
picture since NPM is an extremely eclectic molecule that is
involved in multiple functions; others could well be identified in
the future. Analysis of rare mutations occurring in NPM1 exons
other than 1295,96 and the close relationship between tryptophan
mutations and type of NES motif at NPM mutants C-terminus105
suggest the main aim of mutations is to place NPM in cytoplasm.
One might infer that aberrant NPM localization in cytoplasm is, in
some way, essential for transforming normal hematopoietic cells
into leukemic blasts. Since very low levels of NPMwt (due to
shuttling activity) are physiologically present in cytoplasm, the
amount of NPM mutant that accumulates in cytoplasm may also be
crucial for transforming activity. Thus, the goal of NPM1 mutations
appears to be to export mutants into cytoplasm at the maximum
efficient dose. Interference of the mutants with key functions of
NPMwt and oncosuppressor ARF through their recruitment into
cytoplasm may play a role in transforming activity, but one cannot
discount that mutant NPM may interfere with the functions of
other, as yet unidentified, proteins in the nucleus or cytoplasm.
Mass spectrometry analysis may help characterize complexes
formed by NPM mutants and these putative partners. The role of
mutated NPM in leukemogenesis needs to be addressed in experimental models, which, due to close association of FLT3-ITD and
NPM1 mutations in clinical practice,18,80-83 will also need to
investigate whether FLT3-ITD plays a cooperative role (Figure 7).
The NPM1 gene is an appealing target for therapy. Retargeting
NPM from cytoplasm to its physiologic site (eg, nucleolus) is
challenging because the NES motif and mutated tryptophan(s) at
the NPM mutant C-terminus constitute 2 obvious obstacles.105
Analysis of differences in the 3-dimensional structure of the
C-terminus of NPM mutants and NPMwt may lead to designing
small molecules that can interfere with the abnormal NPM mutant
traffic. However, efforts to pharmacologically target NPM might be
hindered by the need to tread a very fine line in regulating its
function so as to prevent the detrimental consequences of underfunctioning or overfunctioning. In fact, according to expression levels
and gene dosage, NPM1 functions both as oncogene and tumorsuppressor gene114 and partial functional loss and aberrant overexpression both lead, through separate mechanisms, to abnormal cell growth.
Several clinical issues yet need to be addressed. Immunohistochemistry (detection of cytoplasmic NPM) should be integrated
with cytogenetics/fluorescence in situ hybridization (FISH)/
molecular studies, to achieve maximum rationalization of biologic
investigations in AML.84 As NPM1 mutations without FLT3-ITD
identify a good prognostic subgroup, mutational studies should, in
the future, be used to stratify risk in the heterogeneous category of
AML-NK.18,80-83,129 Linking NPM1 mutations to good prognosis
raises other clinically important questions. Does the prognostic
value of mutated NPM1 apply to AML patients older than 60 years
who are recognized as a poor prognostic category? Should adult
NPM1-mutated/FLT3-ITD–negative AML-NK patients be exempted from allogeneic stem cell transplantation in first-line
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BLOOD, 1 FEBRUARY 2007 䡠 VOLUME 109, NUMBER 3
therapy,130 as is current practice for AMLs carrying t(8;21) or
Inv(16)? Can we exploit FLT3 inhibitors to reverse NPM1-mutated/
FLT3-ITD–positive patients into the more prognostically favorable
category of NPM1 mutated/FLT3-ITD negative? Which molecular
mechanism underlies the sensitivity of NPMc⫹ AML to chemotherapy? Does NPMc⫹ AML show selective sensitivity to any
known chemotherapeutic agents? Can we exploit quantitative
detection of NPM1-mutated copies126 to predict patients with
long-term survival? Future prospective clinical trials and availability of a human cell line (OCI-AML3) carrying cytoplasmic/
mutated NPM131 for drug testing should help provide answers.
Acknowledgments
This work was supported by the Associazione Italiana per la
Ricerca sul Cancro (AIRC).
We are indebted to Niccolò Bolli, Maria Paola Martelli,
Arcangelo Liso, Roberto Rosati, Paolo Gorello, Barbara Bigerna,
Alessandra Pucciarini, Roberta, Pacini, Alessia Tabarrini, and
Roberta Mannucci for generating some of the data described in the
paper. We would also like to thank Claudia Tibidò for her excellent
BIOLOGIC AND CLINICAL FEATURES OF NPMc⫹ AML
883
secretarial assistance and Dr Geraldine Anne Boyd for her help in
editing this paper.
Authorship
Contribution: B.F. had the original idea, which, inspired by his
immunohistochemical studies on ALK-positive anaplastic largecell lymphoma, led to the discovery of NPM cytoplasmic/mutated
acute myeloid leukemia (NPMc⫹ AML); B.F. is also the head of the
project on NPM in acute myeloid leukemia; I.N. carried out the
confocal microscopy studies; M.F.M. analyzed clinical data of
NPMc⫹ AML patients; C.M. was involved in the study of NPM1
mutations. All authors contributed to writing the paper.
Conflict-of-interest disclosure: B.F. and C.M. applied for a
patent on clinical use of NPM mutants and have a financial interest
in the patent.
Correspondence: Brunangelo Falini, Institute of Hematology,
Policlinico, Monteluce, 06122 Perugia, Italy; e-mail:
[email protected]; Cristina Mecucci, Institute of Hematology,
Policlinico, Monteluce, 06122 Perugia, Italy; e-mail:
[email protected].
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2007 109: 874-885
doi:10.1182/blood-2006-07-012252 originally published
online September 28, 2006
Acute myeloid leukemia carrying cytoplasmic/mutated nucleophosmin
(NPMc + AML): biologic and clinical features
Brunangelo Falini, Ildo Nicoletti, Massimo F. Martelli and Cristina Mecucci
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