Mouse models of NPM1-mutated acute myeloid leukemia - Nature

Leukemia (2015) 29, 269–278 © 2015 Macmillan Publishers Limited All rights reserved 0887-6924/15 www.nature.com/leu

CONCISE REVIEW

Mouse models of NPM1-mutated acute myeloid leukemia: biological and clinical implications P Sportoletti1, E Varasano1, R Rossi1, A Mupo2, E Tiacci1, G Vassiliou2, MP Martelli1 and B Falini1 Acute myeloid leukemia (AML) carrying nucleophosmin (NPM1) mutations displays distinct biological and clinical features that led to its inclusion as a provisional disease entity in the 2008 World Health Organization (WHO) classification of myeloid neoplasms. Studies of the molecular mechanisms underlying the pathogenesis of NPM1-mutated AML have benefited greatly from several mouse models of this leukemia developed over the past few years. Immunocompromised mice xenografted with NPM1-mutated AML served as the first valuable tool for defining the biology of the disease in vivo. Subsequently, genetically engineered mouse models of the NPM1 mutation, including transgenic and knock-in alleles, allowed the generation of mice with a constant genotype and a reproducible phenotype. These models have been critical for investigating the nature of the molecular effects of these mutations, defining the function of leukemic stem cells in NPM1-mutated AML, identifying chemoresistant preleukemic hemopoietic stem cells and unraveling the key molecular events that cooperate with NPM1 mutations to induce AML in vivo. Moreover, they can serve as a platform for the discovery and validation of new antileukemic drugs in vivo. Advances derived from the analysis of these mouse models promise to greatly accelerate the development of new molecularly targeted therapies for patients with NPM1-mutated AML. Leukemia (2015) 29, 269–278; doi:10.1038/leu.2014.257

INTRODUCTION Recently, the sequencing of 200 acute myeloid leukemia (AML) exomes or genomes1 revealed that they carry hundreds of gene mutations. The majority of these are likely to represent ‘passenger’ mutations and only about 20 are regarded as ‘drivers’ on the basis that they occur in at least 2% of AML patients.1 Interestingly, the AML genome appears less complex than that of other adult cancer types. Specifically, the median mutation frequency in AML is 0.28 per megabase (Mb), whereas other tumors show an average of over 1 mutation per Mb.2 The most commonly mutated genes in AML include nucleophosmin (NPM1), FLT3 (each in about 30% of cases) and DNMT3A (in ~ 20% of cases).1 Mutations of other genes (for example, IDH1/2, NRAS) occur at a frequency ⩽ 10%.1 Mutated genes in AML are organized into functional categories including transcriptionfactor fusions, tumor suppressors, DNA-methylation-related genes, activated signaling genes, chromatin modifiers, myeloid transcription factors, cohesion complex genes, spliceosome complex genes and NPM1, which occupies its own category.1 We discovered NPM1 mutations in AML in 20053 and subsequently found that they associate with unique biological and clinical features.4 Several lines of evidence point to NPM1 mutations as a driving event defining a distinct AML entity.5 First, they are highly recurrent in AML (about one-third of cases) and represent the most common genetic alteration underlying AML with normal cytogenetics, accounting for 50–60% of patients.3,4 Second, NPM1 mutations remain stable over the course of disease4,6 and are usually detected at relapse, even many years after the initial diagnosis of AML.7 Third, NPM1 mutations and aberrant cytoplasmic expression of nucleophosmin are specific for AML.2,3,8 In contrast, other common AML-associated mutations are

detected in myelodysplasia and T-cell lymphoblastic leukemia (for example, DNMT3A)9,10 or solid tumors (for example, IDH1/2 and NRAS).11 Fourth, NPM1 mutations are mutually exclusive with other recurrent genetic abnormalities that define distinct AML entities in the World Health Organization (WHO) classification of lymphohemopoietic tumors.12 Finally, NPM1-mutated AMLs show distinctive mRNA and miRNA expression profiles,13,14 regardless of the presence or absence of karyotypic abnormalities.15 Deregulated genes in these signatures include several homeobox (HOX), CD34, miR-10a, miR-10b and let-7 family members. Overall, these molecular effects of NPM1 mutations deeply influence hematopoietic development and maintenance of stem/progenitor cell properties. NPM1 mutations also associate with distinctive clinicopathological features,4,5 including female sex, higher white blood cell count with increased blast percentage (especially in association with FLT3-ITD), frequent M4/M5 morphology, absent or low CD34 expression, strong positivity for CD33, good response to induction therapy and favorable prognosis (mostly in the absence of FLT3ITD).16 For these reasons, NPM1-mutated AML has been included as a new provisional entity in the 2008 WHO classification.17 In spite of these advances, the pathogenic role of NPM1 mutations in AML remains incompletely understood. In vitro analysis of cells transfected with mutant NPM1, as well as studies of NPM1-mutated human AML cell lines18 and primary blasts from patients, contributed to the clarification of mechanisms responsible for the aberrant accumulation of nucleophosmin in the cytoplasm of leukemic cells19,20 and AML development.21 However, to gain insight into the early transformation events leading to the generation of leukemic stem cells, there is a need for prospective in vivo models.

1 Institute of Hematology, University of Perugia, Perugia, Italy and 2The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK. Correspondence: Dr P Sportoletti or Professor B Falini, Institute of Hematology, University of Perugia, Ospedale S. Maria della Misericordia, S. Andrea delle Fratte, 06132 Perugia, Italy. E-mail: [email protected] or [email protected] Received 24 July 2014; revised 25 August 2014; accepted 26 August 2014; accepted article preview online 2 September 2014; advance online publication, 23 September 2014

Mouse models of NPM1-mutated AML P Sportoletti et al

270 In this review, we provide a synopsis of three types of mouse models (xenotransplant, transgenic and knock-in) that are currently available to understand NPM1-driven leukemogenesis. XENOTRANSPLANTATION MODELS OF NPM1-MUTATED AML Xenografting of immunocompromised mice with human primary leukemic cells represents a valuable tool for studying the biology of AML in vivo.22 Severe combined immunodeficiency (SCID) mice that were subcutaneously injected with NPM1-mutated AML blasts from a patient6 served as the first in vivo model for the disease. The engraftment represented the expansion of true leukemia-initiating cells, as the engraftment potential and genetic characteristics were retained in secondary and tertiary recipients through many passages for 48 years.6 Moreover, the characteristic aberrant expression of nucleophosmin in the cytoplasm of leukemic cells3 was maintained over time. These findings fully mimic those observed in patients experiencing late relapses of NPM1-mutated AML.7 Xenotransplantation studies have shown that AML is a stem-cell disease, with individual leukemia cases containing a variable population of CD34-positive leukemia-initiating cells (LICs).22 NPM1-mutated AML is frequently CD34-negative3 and in only about 10% of cases it expresses CD34 at low intensity.23 Two studies in NPM1-mutated AML demonstrated the presence of NPM1 mutations in the rare fraction of CD34-positive cells, providing evidence that they belong to the leukemic clone.23,24 NPM1-mutated gene and/or protein was also confirmed in a subpopulation of CD34-positive cells with the immunophenotype of leukemic stem cells (that is, CD38-/CD123+/CD33+/CD90 − ).23 Interestingly, NPM1-mutated/CD34-positive blasts could be successfully transplanted into immunocompromised NOD/SCID IL2 receptor gamma chain knockout (NSG) mice to generate an AML that recapitulated the features of the original patient's disease, including the loss of CD34 expression in the leukemic population bulk (Figure 1). In contrast, engraftment in NSG mice transplanted with NPM1-mutated/CD34-negative leukemic cells was less consistent and LIC activity was not always present, suggesting that this fraction was relatively depleted in LICs. The existence of an ancestral population of preleukemic hemopoietic stem cells (HSCs) capable of differentiation has been postulated in AML.25 The analysis of nonleukemic populations of HSCs and progenitors, as well as of mature B and T cells sorted from NPM1-mutated/DNMT3A-mutated AML patients at diagnosis and remission, revealed that, unlike AML blasts, they usually contain the DNMT3A but not the NPM1 mutations.26 Similarly, CorcesZimmerman et al.27 using a genomic and functional analysis of de novo AML and patient-matched HSCs demonstrated that mutations in NPM1 or in genes involved in activated signaling were significantly absent in preleukemic cells, whereas mutations in genes involved in processes such as DNA methylation, histone modification and chromatin looping were significantly enriched in preleukemic cells.

Altogether, these findings strongly suggest that the DNMT3A mutations preceded NPM1 mutations during leukemogenesis. Notably, the DNMT3A-mutated preleukemic populations in NPM1-mutated AML appear to be functionally competent. In fact, in xenograft repopulation assays, DNMT3A-mutated preleukemic cells gave rise mostly to multilineage (lymphoid and myeloid) engraftments devoid of NPM1 mutations.26 Moreover, the xenograft models highlighted a competitive growth advantage of the preleukemic DNMT3A-mutated HSCs over non-mutated normal HSCs.26 These findings support a model of leukemogenesis wherein an ancestral DNMT3A-mutated HSC generates an expanded pool of HSCs and downstream multilineage progenitors, but not leukemia. Overt AML develops when NPM1 mutations are subsequently acquired, probably within a DNMT3A mutant HSC or possibly a granulocyte monocyte or multilymphoid progenitor, which is transformed by the mutation.26 This observation is of potential clinical relevance because the persistence of chemoresistant preleukemic cells at remission represents a reservoir from which a relapse may potentially arise through acquisition of de novo mutations.26 GENETICALLY ENGINEERED MOUSE MODELS OF MUTANT NPM1 After the description of xenotransplant models, efforts focused on the development of transgenic and knock-in models of NPM1mutated AML using several strategies (Figures 2 and 3). This approach allows the generation of large cohorts of inbred mice with a constant genotype and a reproducible phenotype, thus facilitating detailed functional studies. It is noteworthy that NPM1mutated alleles were sometimes referred to as NPMc+ or NPM1c to indicate the aberrant cytoplasmic localization of the mutant protein, as first reported by Falini et al.3 (resulting from the disruption of a C-terminal nucleolar localization signal and generation of a de novo nuclear export signal). Because NPM1 mutations are AML specific,2,8 some mouse models have been engineered to express NPMc+ specifically in either the myeloid progenitor or HSC compartments using specific Cre transgenic lines (Figure 3). Examples of such models are discussed below. Transgenic model of NPM1-mutated gene (NPMc+) Cheng et al.28 developed a transgenic mouse expressing the NPM1-mutated gene under the control of the human MRP8 promoter (Figures 2 and 3a). This promoter drives the expression of NPMc+ in common myeloid progenitors, as well as in mature granulocytes and monocytes.28 The transgenic NPMc+ mice developed a late-onset myeloproliferative disease with splenomegaly and increased number of Gr-1/Mac-1+ve mature myeloid cells in the bone marrow and spleen.28 None of these transgenic mice developed acute leukemia. A similar expansion of myeloid cells was obtained in zebrafish embryos overexpressing mutant NPMc+ ubiquitously.29 In this

Figure 1. A xenograft model of human NPM1-mutated AML: CD34+ cells from NPM1-mutated AML generate CD34-negative NPMc+ AML in immunocompromised NOD/SCID IL2 receptor gamma chain knockout (NSG) mice. (a) Human NPM1-mutated AML xenotransplant model. (b) Flow cytometric analysis showing CD34 antigen expression in the CD34+ cell fraction (carrying NPM1 mutation) isolated from a patient with NPM1-mutated AML and injected in NSG mice (left). Flow cytometric analysis of mouse bone marrow (8 weeks after the injection) shows engraftment of NPM1-mutated human myeloid (CD33+/CD117+) cells that are mainly CD34 − (3% CD34+ cells). (c) Femoral head paraffin section showing extensive bone marrow infiltration by human leukemic cells with aberrant cytoplasmic expression of nucleophosmin (NPM1), characteristic of NPM1-mutated AML. In comparison, a nonleukemic cell showing nuclear NPM1 staining is indicated (arrow). Immunostaining was performed with an anti-NPM1 mouse monoclonal antibody (clone 376) (Alkaline Phosphatase/Anti-alkaline Phosphatase (APAAP) method; hematoxylin counterstain). (d) Mouse bone marrow cells express mutant NPM on western blot analysis with a specific anti-mutant NPM1 rabbit polyclonal antibody (anti-NPMmut), as is also seen with the unprocessed original patient sample (total) and the CD34+ cell fraction (CD34+). Lysate from the OCI/AML3 human cell line harboring NPM1 mutation is used as positive control. (e and f) Tibial paraffin sections showing preferential leukemic infiltration of bone marrow endosteal regions at low (e) and high (f) magnification. Immunostaining was performed with a specific anti-human CD45 monoclonal antibody (APAAP; hematoxylin counterstaining). Images were collected using an Olympus B61 microscope (Olympus Italia, Segrate, Italy) a UPlanApo 40 × /0.85 (c and f) and UPlanApo 10 × /0.40 (e); Camedia 4040, Dp_soft Version 3.2 (Olympus Italia); and Adobe Photoshop 7.0 (Adobe Systems, Mountain View, CA, USA). Leukemia (2015) 269 – 278

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272 Transgenic

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Mupo et al. 2013 Mallardo et al. 2013 Rau et al. 2013

Figure 2. Summary of NPM1 mutant mouse models and their phenotypic characteristics. The first genetically engineered mouse model of NPM1 mutation was a transgenic mouse expressing the mutated gene under the control of the MRP8 hematopoietic promoter. These mice developed myeloproliferation with low frequency and late onset. Two different ‘nonconventional’ conditional knock-in mouse models targeted the human NPM1 mutation A into a highly recombinogenic mouse genomic locus in embryonic stem cells. Conditional expression of the mutant into the hematopoietic compartment was obtained using Mx1-Cre mice. Mice developed hematopoietic defects including megakaryocytic expansion, myeloid proliferation and a late-onset leukemia. A conventional knock-in strategy has been used in two different models in order to induce the expression of either a human or a mouse NPMc+ in BM cells. The human NPM1 mutant led to late leukemia development, whereas the mouse mutant induced myeloproliferation with low penetrance. Three different groups have generated compound mutant mice carrying the NPM1 mutation and Flt3-ITD expression in the hematopoietic compartment. All these models developed a fully penetrant leukemic phenotype rapidly, although with different latencies. AML, Acute Myeloid Leukemia; LICs, Leukemia-Initiating Cells; Mk, Megakaryocytes; MPD, Myeloproliferative Disease; NPMc+, NPM1 mutation; NSG, NOD/SCID IL2 receptor gamma chain knockout.

setting, embryos injected with the human NPM1-mutated mRNA demonstrated abnormal cytoplasmic localization of the mutated protein, similar to human NPM1-mutated AML cells. The expression of the NPM1 mutant perturbed primitive myelopoiesis inducing a marked proliferation of early pu.1-expressing myeloid cells. In addition, embryos showed increased numbers of hematopoietic stem cells (c-myb+/cd41+), but follow-up for AML development was not possible owing to the transient expression of the NPM1 mutant.29 These initial in vivo observations indicated that the NPM1 mutant was sufficient to induce a myeloproliferative disorder, but was unable to generate AML recapitulating the phenotypic features observed in humans. The absence of a leukemic phenotype may have been due to a number of reasons. First, NPM1-mutated AML has a characteristic stem cell-like gene expression signature Leukemia (2015) 269 – 278

characterized by the upregulation of HOX genes,13 pointing to the possible need to specifically target the HSC compartment. Second, NPM1 mutations in AML patients are consistently heterozygous4 and result in a mutant to wild-type NPM1 expression ratio that is instrumental to induce cytoplasmic delocalization of both NPM1 forms.20 In contrast, the degree of cytoplasmic mislocalization of the NPM1 forms and any effects of NPM1 heterozygosity was lower in the NPMc+ transgenic model. This is likely owing to the fact that the expression levels of the NPM1 mutant were too low compared with endogenous expression, as both Npm1 wild-type alleles were preserved. This view is supported by in vitro transfection experiments showing that an excess of the wild-type NPM1 protein relocated the NPM1 mutant from the cytoplasm to the nucleoli dampening the mutant's gain-of-function activity and impairing its transforming abilities.30 Besides NPM1 © 2015 Macmillan Publishers Limited


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Figure 3. Schematic representation of the targeting strategies for generating NPM1-mutated mouse models. (a) Transgenic mouse model. The transgene construct was generated by cloning the NPM1 mutation A cDNA (hNPMc+) under the control of the human MRP8 promoter (hMRP8 promoter). The construct was flag-tagged at the N terminus (FLAG) of the NPM1 mutation. The NPMc+ protein was expressed in common myeloid progenitors and mature granulocytes and monocytes. (b) Sportoletti et al.33 and Mallardo et al.34 models: the human cDNA of the Type A mutation of NPM1 (hNPMc+) was inserted into the Rosa26 and Hprt genetic loci, respectively. A stop cassette (STOP) flanked by loxP sites (triangles) was inserted between a strong CAG promoter (CAG pr.) and the hNPMc+ cassette, thus allowing the conditional expression of the NPM mutant protein upon Cre-mediated recombination. NPM1 mutant mice were crossed with Mx1-Cre mice, and excision of the STOP-Neo cassette was induced by polyinosinic-polycytidylic acid (pIpC) treatment in vivo. (c) Vassiliou et al.38 model: a constitutive knock-in allele was engineered to have NPM1 TCTG duplication in exon 11 (asterisk) of the mouse Npm1 gene. The humanized mutant exon 11 was directly used to replace the wild-type mouse sequence (mouse exon 11 is homologous to human exon 12). Two loxP sites (triangle) were flanking the wild-type mouse exon 11 and a Purodelta-TK cassette (puΔTK). The NPM1 mutant was expressed under the control of the endogenous mouse Npm1 regulatory elements. The conditional allele allowed the expression of a mouse NPM1 protein that was converted into a humanized mutant NPM1 upon Cre recombination. NPM1 conditional knock-in mice were crossed with Mx1-Cre mice, and the expression of the mutant in the hematopoietic compartment was induced by pIpC treatment in vivo. (d) Chou et al.39 model: the conditional knock-in allele was generated by introducing a mutated NPM1 exon 11 with a floxed Neo cassette (Neo) in intron 10 of the mouse NPM1 gene. This mouse-mutated exon 11 was obtained by the insertion of a TCTG after nucleotide c.857 of murine Npm1 coding sequence. This pattern mimics human NPM1 mutation without any ‘humanized’ sequence. The loxP-neoloxP cassette was excised in mouse embryonic stem cells before their implantation into foster mothers. The mouse Npm1 mutant was constitutively expressed under the control of the endogenous mouse Npm1 regulatory elements.

cytoplasmic dislocation effects, reduced levels of wild-type NPM1 owing to the loss of one NPM1 functional allele may also contribute to leukemogenesis. Indeed, NPM1 haploinsufficiency led to myeloid-specific defects ex vivo31 and Npm1 heterozygous mice developed both myeloid and lymphoid leukemias.32 Third, the association between NPM1 and other frequent mutations, such as FLT3-ITD, DNMT3A and IDH11,5 in human AML strongly suggests that additional genetic lesions are required to induce a leukemic phenotype in vivo. © 2015 Macmillan Publishers Limited

Knock-in models expressing NPMc cDNA from permissive loci A number of permissive genomic loci are routinely used for knocking-in gene cDNAs by homologous recombination, so that mRNAs are then expressed under the control of the endogenous regulatory elements of these loci. Mice harboring the NPM1 mutation A (representing 80% of all NPM1 mutations)4 have been generated using this approach to circumvent some limitations of conventional transgenics, such as low expression and susceptibility to epigenetic silencing after random genomic Leukemia (2015) 269 – 278


274 integration into susceptible sites. Two mouse models have been developed through insertion of the human NPM1 mutation A cDNA into the Rosa26 and Hprt loci, respectively33,34 (Figures 2 and 3b). In addition, the targeting vector was designed to contain a strong and ubiquitous pCAG promoter along with a stop cassette flanked by loxP sites between the promoter and the cDNA (Figure 3b), thus allowing the conditional expression of the NPM1 mutant protein upon Cre‐mediated recombination. These mouse models expressed the mutant NPM1 gene in addition to the two normal copies of the endogenous mouse Npm1 gene. Nevertheless, the mutant NPM1 expression levels were comparable to the levels of NPM1 wild-type protein encoded by the endogenous loci at least in homozygous transgenic mice.33 Upon Cre induction, the NPM1 mutant expression significantly perturbed adult hematopoiesis in both conditional models. As described by Sportoletti et al.,33 NPMc+ induced myeloproliferation in a fraction of mice and a fully penetrant block of megakaryocytic development. Although none of these mice developed leukemia after a long follow-up, the model mirrored some features of the human NPM1-mutated AML, such as megakaryocytic expansion35,36 (Figure 4) and deregulation of specific miRNAs in bone marrow.14 In particular, expanded CD41+ megakaryocytes of NPMc+ mice overexpressed miR-10a, miR-10b and miR-20a that are also deregulated in NPM1-mutated AML patients.14 Interestingly, these miRNAs are known to control megakaryocytic lineage development and platelet function, as their downregulation allows expression of target genes involved in megakaryocytic differentiation.37 In the model developed by Mallardo et al.,34 the mutant NPM1 induced AML after a very long latency and in a minority of mice. In particular, 30% of animals developed leukemia and about 50% of leukemias expressed both myeloid and B-lymphoid markers. In contrast, human acute lymphoblastic leukemias never carry NPM1 mutations.3 Conventional knock-in models of NPM1 mutation This strategy has been used to introduce the mutation into the endogenous NPM1 locus, thus keeping its expression under the control of the endogenous promoter and genocopying what happens in patients' leukemic cells. This replacement reduces the normal Npm1 gene to heterozygosity such that the relative expression levels of the normal and mutated genes are, in principle, similar and comparable with the situation in human AML (NPM1 mutations are consistently heterozygous in AML patients). Two NPM1-mutated knock-in mice have been developed including a conditional model that allows the induction of NPMc+ expression in HSCs at controlled time points after birth and a constitutive model expressing the NPM1 mutant in all tissues (Figure 2; Figures 3c and d). Vassiliou et al.38 (Figure 3c), directly used the human NPM1 type A mutation nucleotide sequence to replace the wild-type mouse sequence. The goal was to have the same consequences at the protein level as seen in human AML. Activation of the humanized Npm1 knock-in allele in mouse HSCs caused Hox gene overexpression (a characteristic feature of NPM1-mutated AML13,14), enhanced self-renewal and expanded myelopoiesis. One-third of the mice developed late-onset leukemia, demonstrating that the NPM1 mutation is an AML-driving lesion. Furthermore, to accelerate leukemogenesis, these mice were subjected to hemopoieticspecific insertional mutagenesis with the Sleeping Beauty transposon, and in this context 480% of mice developed AML. More recently, Chou et al.39 developed a constitutive knock-in mouse model by introducing the same TCTG duplication seen in human type A mutations, but without ‘humanizing’ the surrounding sequence leading to a mutant protein sequence predicted to have a weaker nuclear export signal and not seen in human AML (Figure 3d). As described in other NPM1-mutated models, a percentage of mice developed a myeloproliferative disease with extramedullary hematopoiesis. In addition, mice showed a Leukemia (2015) 269 – 278

perturbation in the niche function with a decline of cobblestone area formation and a defective CXCR4/CXCL12 pathway that was similar to NPM1-mutated AML patients.39 COMPOUND MUTANT MICE RELEVANT TO NPM1-MUTATED AML Although data gained from knock-in mouse models have added new insights into the pathogenesis of NPM1-mutated AML, they have also demonstrated that sustained NPM1 mutant expression in the HSC compartment is not sufficient for leukemia onset. Additional stochastic mutations that some of the animals acquire during their life span are likely needed to drive a full leukemia phenotype. This view is also supported by in vitro co-transfection studies showing that cooperative genetic events are required for NPM1 mutation leukemogenesis.21 Moreover, it is consistent with the experimental observation that, as with NPM1, other AML oncogenes (for example, RUNX1-RUNX1T1 or CBF-MYH11) are not sufficient to cause leukemia alone in mice, but require cooperating events to do so.40,41 Indeed, some animal studies support the classic two-hit model of leukemogenesis42 in which the cooperation between two classes of genetic alterations is necessary for leukemic transformation: for example, a class I mutation, which activates signal transduction pathways (for example, FLT3-ITD) and confers a proliferative advantage, and a class II mutation, which affects transcription (for example, RUNX1/RUNX1T1)40 and causes a differentiation block. Findings from whole-genome sequencing studies point to an even more complex configuration in many AMLs that might involve several mutations, serving complementary roles including those affecting chromatin landscaping and epigenetic pathways. On the basis of such observations, an alternative ‘slot machine’ model has been recently proposed,43 in which the late steps would be, to some extent, constrained by the initial ones (clonal dominance, cooperations/exclusions). A number of clinical and experimental lines of evidence point to a particular complementarity in AML development between NPM1 and FLT3-ITD mutations, which are present in 30–40% of NPM1-mutated cases.44 Moreover, in conditional Npm1c+/ Sleeping Beauty transposon compound mutant mice, recurrent activating integrations were identified in Flt3 and the related gene Csf2 (encoding GM-CSF).38 To better understand whether FLT3-ITD and NPM1 mutations can significantly cooperate to induce leukemia in vivo, three different groups have generated compound mutant mice carrying both these mutations in the hematopoietic compartment34,45,46 (Figure 2). Notably, the combination of Flt3/ITD and NPMc+ always resulted in the development of AML with features that closely recapitulated those observed in patients, including monocytic differentiation and very high white blood cell counts. Moreover, spontaneous loss of heterozygosity (LOH) of the wild-type Flt3 allele occurred with a high frequency, and the extent of LOH closely correlated with the level of leukocytosis. Notably, FLT3-LOH is a well-described feature of NPM1-mutated/FLT3-ITD+ AML patients and is associated with a poor outcome.47 The rapidity of leukemia onset depended on the NPM1 mutant mouse line that had been crossed with Flt3-ITD mice and reflected different NPM1 mutant expression levels. In transgenic NPMc+/Flt3-ITD mice described by Rau et al.,46 leukemia occurred with a rather long latency probably because of the low expression of NPMc+ and the high levels of wild-type NPM1 (two endogenous alleles). By comparison, in the model developed by Mallardo et al.,34 NPM1 mutant expression was significantly increased and the NPMc+/Flt3-ITD mice died of leukemia between 35 and 161 days (median 72 days). Mupo et al.45 described an even shorter latency to disease onset using the humanized NPM1-mutated model, in which all double-mutant mice developed leukemia in o2 months without the need for formal Cre induction (Figure 5a), but driven instead by low-level © 2015 Macmillan Publishers Limited


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Figure 4. NPM1 conditional mutant mice show a megakaryocytic compartment expansion mimicking that observed in human NPM1-mutated AML patients. (a) The NPM1 mutation induces an expansion of the immature megakaryocytes in mice. Representative colonies ( ×10 magnification) from (ii) NPM1-mutated compared with (i) wild-type control mice showing an increase in the CFU-MK potential of bone marrow (BM) cells from NPM1-mutated mice. Flow cytometric analysis of BM cells from representative conditional NPM1-mutated (lower dot plot) and wild-type control (upper dot plot) mice demonstrates an increase in the percentage of Lin–Kit1Sca-1–CD150+CD41+ megakaryocytic progenitor populations. (b) Megakaryocyte expansion in BM trephines of NPM1-mutated AML patients. Representative BM sections from a nonleukemic control were stained with (i) hematoxylin and eosin and (ii) a mouse monoclonal antibody against the linker for activation of T-cell (LAT) protein, which is an excellent marker for megakaryocytes in BM biopsies. (iii-iv) BM trephine sections from an NPM1-mutated AML patient with an increased number of megakaryocytes (40 × magnification), as assessed by (iii) hematoxylin and eosin and (iv) immunostaining for human LAT.

‘leaky’ expression of Cre from the Mx1-Cre allele and by subsequent acquisition of LOH for Flt3-ITD (Figure 5b). This is not surprising because in this setting both tumor suppressor effects of NPM1 haploinsufficency and oncogenic properties of the mutated NPMc+ protein operate to drive leukemogenesis. The rapid onset of leukemia in all compound mutant mice suggests that coexpression of NPM1 and FLT3‐ITD mutations may be enough to initiate and promote leukemogenesis. However, it cannot be excluded that additional mutations are acquired very rapidly in a population of murine cells prone to leukemic transformation. © 2015 Macmillan Publishers Limited

An in vivo insertional mutagenesis study conducted by Vassiliou et al.38 showed that NPM1 mutations could also cooperate with activated Ras signaling to cause AML in NPM1 knock-in mice. Indeed, compound Npmc+/Nras-G12D mutated mice develop AML with 80% penetrance and a 3-month median survival.48 Interestingly, exome analysis of these leukemias revealed the accumulation of additional mutations, including an Idh1R132Q (synonymous to human R132H) substitution that is known to co-occur with NPM1 and NRAS-G12D in human AML. Leukemia (2015) 269 – 278


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Flt3-ITD-LOH/Npmc+ cell

Figure 5. Molecular synergy and clonal evolution in Npmc+ /Flt3-ITD-driven murine AML. (a) Npmc+ and Flt3-ITD single-mutant mice have a significantly reduced survival compared with wild-type mice owing to an excess of myeloid malignancies (*Po 0.01). All double-mutant mice develop very early-onset universal AML, in keeping with a strong synergy between the two mutations. (b) Clonal evolution of AML in doublemutant mice: the process starts in prenatal or early postnatal life with activation by ‘leaky’ Mx1-Cre expression of the Npmc+ conditional allele to generate a small number of double-mutant HSCs. Double-mutant cells grow rapidly to dominate hemopoiesis with an evidence of AML by the time of weaning (3–4 weeks), while a small number of these cells acquire LOH for Flt3-ITD. LOH gives the latter cell an additional advantage and they outgrow double-mutant cells without LOH by the time mice develop full-blown AML.

NPM1-TARGETED THERAPY AND MOUSE MODELS NPM1 mutations are a particularly attractive therapeutic target as they are common, behave as founder genetic lesions and lead to AML development through mechanisms that appear to differ from those of other mutations.5 Clinically, NPM1 mutations are associated with a favorable prognosis. However, about 40% of NPM1-mutated/FLT3-ITD-negative and the majority of NPM1mutated/FLT3-ITD-positive AML patients succumb to their disease.16 Thus, there is clearly a need for new therapeutic strategies against NPM1-mutated AML. Wild-type NPM1 is a nucleolar protein that shuttles between the nucleus and the cytoplasm20 and exerts multiple functions.49 Characteristically, all NPM1 mutations cause similar changes at the C-terminal portion of the wild-type protein, disrupting a nucleolar localization and creating a new nuclear export motif in its place.19 These alterations perturb the nucleo-cytoplasmic traffic of nucleophosmin, leading to its aberrant accumulation in the cytoplasm of AML cells, in what appears to be a critical leukemogenic event.19,20 However, we found that a small amount of residual wild-type NPM1 is always detectable in the nucleolus of NPM1-mutated leukemic cells, strongly suggesting that it may be required for their survival.19 This is in keeping with the observation that NPM1 mutations in AML are consistently heterozygous4,20 and that the complete knockdown of Npm1 alleles is embryonic lethal in mice.50 These data suggest that AML cells harboring NPM1 mutations could be potentially targeted using at least two different strategies that make use of the aberrant localization of the mutant protein (reviewed by Falini et al.51). One approach is to focus on relocation of the cytoplasmic NPM1 mutant to the nucleus using Leptomycin-B19 or newer Crm1 inhibitors with improved therapeutic windows.52 These compounds redirect the Leukemia (2015) 269 – 278

mutant to the nucleoplasm but unfortunately not in the nucleolus (because of the lack of one or both C-terminal tryptophans at positions 288 and 290).19 An alternative therapeutic strategy derives from the assumption that the nucleolus in NPM1-mutated AML cells is more vulnerable than in other cells,51 because it contains a lower amount of wildtype NPM1 (because of both haploinsufficiency and cytoplasmic dislocation).19 This establishes the rationale for using compounds that are capable of disrupting nucleolar integrity51,53 by dislocating the residual nucleolar wild-type NPM1 to the nucleoplasm. Because NPM1 is a hub protein that in its oligomeric form is essential for maintaining nucleolus homeostasis,54 its dislocation (and possibly that of other nucleolar components) to the nucleoplasm55 can trigger apoptotic signals.56 We predict that in NPM1-mutated AML a number of compounds affecting ribosome biogenesis57 or inhibiting the oligomerization of NPM153 may exert antileukemic activity. Recent studies indicate that wild-type NPM1 binds to the nucleolus by interacting with G-quadruplexes of ribosomal DNA, suggesting also the potential of using selective ligands of the G-quadruplexes that compete with NPM1 binding,58 to disrupt the structure of the nucleolus. Preclinical testing of compounds against NPM1-mutated AML cells, both in vitro and in animal models, will be critical for their rapid translation into clinical use. The OCI–AML318 and the IMSM259 human cell lines, which recapitulate the features of NPM1mutated AML, are useful tools for in vitro studies. Genetically engineered mouse models of NPM1 mutation and compound mutants may further contribute to high-throughput drug screening in vivo. For example, they could be used for the generation of immortalized cell lines harboring selected genetic lesions and be used in drug sensitivity screens. Mouse models could be also useful in designing clinical trials. As an example, Schlenk et al.60 found that all-trans retinoic acid (ATRA) significantly improved © 2015 Macmillan Publishers Limited


277 survival only in AML patients harboring the NPM1 mutation in the absence of FLT3-ITD. However, these findings were not confirmed in another study from the UK MRC AML12 trial.61 Different ATRA administration schedules and/or inclusion of etoposide in the regimen have been claimed to account for these conflicting results. The availability of NPM mutant mouse models could help clarify this controversial issue. A strong association has been found between NPM1 mutations and those affecting the FLT3, DNMT3A, IDH1/2, RAS and cohesincomplex genes in AML with normal cytogenetics.1 This suggests that, in order to eradicate the leukemic clone, drugs targeting more than one mutation could be combined, hence the importance of having compound mutant mouse models as a platform for testing drug combinations. As an example, therapy of NPM1-mutated AML with a high burden of FLT3-ITD is a medical need because this genotype is usually associated with a poor prognosis. The double-mutated (NPMc+/FLT3-ITD) mouse models34,45,46 represent a valuable preclinical tool for testing drugs targeting the residual nucleolar wild-type NPM1 (see above) in combination with second-generation FLT3-ITD inhibitors (for example, quizartinib)62 or with bromodomain and extra-terminal (BET) inhibitors.63 Generation of mouse models mimicking clinically relevant examples of cooperative mutations other than NPMc+/FLT3-ITD, for example, NPM1, DNMT3A and IDH1/2 mutations in various combinations, would be very useful for evaluating multitargeted therapeutic approaches—for instance, the use of compounds targeting NPM1 together with epigenetic drugs such as 5-azacytidine (that have already shown activity in the preemptive therapy of NPM1-mutated AML64) or the recently developed inhibitors of IDH165 and IDH266 mutants. Finally, xenotransplant models may serve as a tool for assessing the capability of drugs to kill LSCs. Recent microarray analyses of enriched CD34+ LSCs from NPM1-mutated AML patients revealed overexpression of genes involved in T-cell immunogenicity, such as CD96 and IL12RB1 in NPM1-mutated as compared with NPM1 wild-type cases.67 Because CD96 is not significantly expressed in normal HSCs, specific anti-CD96 antibodies may have the potential to eradicate NPM1-mutated CD34+ leukemic stem cells. CONCLUSIONS Current mouse models of NPM1-mutated AML have been important for (i) demonstrating that the NPM1 mutation alone displays a low leukemogenic activity in vivo but can lead to leukemia after a long latency required to acquire collaborating mutations; (ii) showing that the combination of mutant NPM1 with Flt3-ITD can lead to the rapid development of AML in mice, often displaying LOH for Flt3-ITD and mimicking the situation in AML patients; (iii) identifying a functionally competent population of preleukemic hemopoietic stem cells; and for (iv) improving our understanding of the molecular mechanisms underlying deregulated growth induced by NPM1 mutants. They also represent robust in vivo platforms for the evaluation of novel therapies alone or in combination with established drugs. Information gained from these models can help the development of new rational treatments for NPM1-mutated AML patients. CONFLICT OF INTEREST B. Falini applied for a patent on the clinical use of NPM1 mutants. The remaining authors declare no conflict of interest.

ACKNOWLEDGEMENTS This work was supported by the Associazione Italiana Ricerca Cancro (AIRC) (IG 2013 n.14595), the Associazione Umbra contro le Leucemie e i Linfomi (AULL) and Italian Minister of Health, Project ‘Ricerca Finalizzata 2008’ (Grant n. RF-UMB-2008-1198396).


GV is funded by a Welcome Trust Senior Fellowship in Clinical Science. AM is funded by a Kay Kendall Leukaemia Fund project grant.

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