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 detected in myelodysplasia and T-cell lymphoblastic leukemia (for 9,10 Recently, the sequencing of 200 acute myeloid leukemia (AML) example, DNMT3A) or solid tumors (for example, IDH1/2 and 11 exomes or genomes1 revealed that they carry hundreds of gene NRAS). Fourth, NPM1 mutations are mutually exclusive with other mutations. The majority of these are likely to represent ‘passenger’ recurrent genetic abnormalities that define distinct AML entities in mutations and only about 20 are regarded as ‘drivers’ on the basis the World Health Organization (WHO) classification of lympho- 12 that they occur in at least 2% of AML patients.1 Interestingly, the hemopoietic tumors. Finally, NPM1-mutated AMLs show 13,14 AML genome appears less complex than that of other adult cancer distinctive mRNA and miRNA expression profiles, regardless types. Specifically, the median mutation frequency in AML is 0.28 of the presence or absence of karyotypic abnormalities.15 per megabase (Mb), whereas other tumors show an average of Deregulated genes in these signatures include several homeobox over 1 mutation per Mb.2 (HOX), CD34, miR-10a, miR-10b and let-7 family members. Overall, The most commonly mutated genes in AML include nucleo- these molecular effects of NPM1 mutations deeply influence phosmin (NPM1), FLT3 (each in about 30% of cases) and DNMT3A hematopoietic development and maintenance of stem/progenitor (in ~ 20% of cases).1 Mutations of other genes (for example, cell properties. IDH1/2, NRAS) occur at a frequency ⩽ 10%.1 Mutated genes in AML NPM1 mutations also associate with distinctive clinicopatholo- are organized into functional categories including transcription- gical features,4,5 including female sex, higher white blood cell factor fusions, tumor suppressors, DNA-methylation-related genes, count with increased blast percentage (especially in association activated signaling genes, chromatin modifiers, myeloid transcrip- with FLT3-ITD), frequent M4/M5 morphology, absent or low CD34 tion factors, cohesion complex genes, spliceosome complex genes expression, strong positivity for CD33, good response to induction and NPM1, which occupies its own category.1 therapy and favorable prognosis (mostly in the absence of FLT3- We discovered NPM1 mutations in AML in 20053 and ITD).16 For these reasons, NPM1-mutated AML has been included subsequently found that they associate with unique biological as a new provisional entity in the 2008 WHO classification.17 and clinical features.4 Several lines of evidence point to NPM1 In spite of these advances, the pathogenic role of NPM1 mutations as a driving event defining a distinct AML entity.5 First, mutations in AML remains incompletely understood. In vitro they are highly recurrent in AML (about one-third of cases) and analysis of cells transfected with mutant NPM1, as well as studies represent the most common genetic alteration underlying AML of NPM1-mutated human AML cell lines18 and primary blasts from with normal cytogenetics, accounting for 50–60% of patients.3,4 patients, contributed to the clarification of mechanisms respon- Second, NPM1 mutations remain stable over the course of sible for the aberrant accumulation of nucleophosmin in the disease4,6 and are usually detected at relapse, even many years cytoplasm of leukemic cells19,20 and AML development.21 How- after the initial diagnosis of AML.7 Third, NPM1 mutations and ever, to gain insight into the early transformation events leading aberrant cytoplasmic expression of nucleophosmin are specific for to the generation of leukemic stem cells, there is a need for AML.2,3,8 In contrast, other common AML-associated mutations are prospective in vivo models. 1Institute 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 Altogether, these findings strongly suggest that the DNMT3A models (xenotransplant, transgenic and knock-in) that are mutations preceded NPM1 mutations during leukemogenesis. currently available to understand NPM1-driven 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 XENOTRANSPLANTATION MODELS OF NPM1-MUTATED AML cells gave rise mostly to multilineage (lymphoid and myeloid) Xenografting of immunocompromised mice with human primary engraftments devoid of NPM1 mutations.26 Moreover, the leukemic cells represents a valuable tool for studying the biology of xenograft models highlighted a competitive growth advantage AML in vivo.22 Severe combined immunodeficiency (SCID) mice that of the preleukemic DNMT3A-mutated HSCs over non-mutated were subcutaneously injected with NPM1-mutated AML blasts from normal HSCs.26 These findings support a model of leukemogenesis apatient6 served as the first in vivo model for the disease. The wherein an ancestral DNMT3A-mutated HSC generates an engraftment represented the expansion of true leukemia-initiating expanded pool of HSCs and downstream multilineage progeni- cells, as the engraftment potential and genetic characteristics were tors, but not leukemia. Overt AML develops when NPM1 mutations retained in secondary and tertiary recipients through many passages are subsequently acquired, probably within a DNMT3A mutant for 48years.6 Moreover, the characteristic aberrant expression of HSC or possibly a granulocyte monocyte or multilymphoid nucleophosmin in the cytoplasm of leukemic cells3 was maintained progenitor, which is transformed by the mutation.26 This over time. These findings fully mimic those observed in patients observation is of potential clinical relevance because the experiencing late relapses of NPM1-mutated AML.7 persistence of chemoresistant preleukemic cells at remission Xenotransplantation studies have shown that AML is a stem-cell represents a reservoir from which a relapse may potentially arise disease, with individual leukemia cases containing a variable through acquisition of de novo mutations.26 population of CD34-positive leukemia-initiating cells (LICs).22 NPM1-mutated AML is frequently CD34-negative3 and in only GENETICALLY ENGINEERED MOUSE MODELS OF MUTANT NPM1 about 10% of cases it expresses CD34 at low intensity.23 Two studies in NPM1-mutated AML demonstrated the presence of After the description of xenotransplant models, efforts focused on NPM1 mutations in the rare fraction of CD34-positive cells, the development of transgenic and knock-in models of NPM1- providing evidence that they belong to the leukemic clone.23,24 mutated AML using several strategies (Figures 2 and 3). This NPM1-mutated gene and/or protein was also confirmed in a approach allows the generation of large cohorts of inbred mice subpopulation of CD34-positive cells with the immunophenotype with a constant genotype and a reproducible phenotype, thus of leukemic stem cells (that is, CD38-/CD123+/CD33+/CD90 − ).23 facilitating detailed functional studies. It is noteworthy that NPM1- Interestingly, NPM1-mutated/CD34-positive blasts could be mutated alleles were sometimes referred to as NPMc+ or NPM1c to successfully transplanted into immunocompromised NOD/SCID indicate the aberrant cytoplasmic localization of