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• W2090932363 abstract "Cytoplasmic nucleophosmin (NPMc+) mutations and FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations are two of the most common known molecular alterations in acute myeloid leukemia (AML); they frequently occur together, suggesting cooperative leukemogenesis. To explore the specific relationship between NPMc+ and FLT3/ITD in vivo, we crossed Flt3/ITD knock-in mice with transgenic NPMc+ mice. Mice with both mutations develop a transplantable leukemia of either myeloid or lymphoid lineage, definitively demonstrating cooperation between Flt3/ITD and NPMc+. In mice with myeloid leukemia, functionally significant loss of heterozygosity of the wild-type Flt3 allele is common, similar to what is observed in human FLT3/ITD+ AML, providing further in vivo evidence of the importance of loss of wild-type FLT3 in leukemic initiation and progression. Additionally, in vitro clonogenic assays reveal that the combination of Flt3/ITD and NPMc+ mutations causes a profound monocytic expansion, in excess of that seen with either mutation alone consistent with the predominance of myelomonocytic phenotype in human FLT3/ITD+/NPMc+ AML. This in vivo model of Flt3/ITD+/NPMc+ leukemia closely recapitulates human disease and will therefore serve as a tool for the investigation of the biology of this common disease entity. Cytoplasmic nucleophosmin (NPMc+) mutations and FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations are two of the most common known molecular alterations in acute myeloid leukemia (AML); they frequently occur together, suggesting cooperative leukemogenesis. To explore the specific relationship between NPMc+ and FLT3/ITD in vivo, we crossed Flt3/ITD knock-in mice with transgenic NPMc+ mice. Mice with both mutations develop a transplantable leukemia of either myeloid or lymphoid lineage, definitively demonstrating cooperation between Flt3/ITD and NPMc+. In mice with myeloid leukemia, functionally significant loss of heterozygosity of the wild-type Flt3 allele is common, similar to what is observed in human FLT3/ITD+ AML, providing further in vivo evidence of the importance of loss of wild-type FLT3 in leukemic initiation and progression. Additionally, in vitro clonogenic assays reveal that the combination of Flt3/ITD and NPMc+ mutations causes a profound monocytic expansion, in excess of that seen with either mutation alone consistent with the predominance of myelomonocytic phenotype in human FLT3/ITD+/NPMc+ AML. This in vivo model of Flt3/ITD+/NPMc+ leukemia closely recapitulates human disease and will therefore serve as a tool for the investigation of the biology of this common disease entity. Patients with acute myeloid leukemia (AML) represent a large and heterogeneous group. Much work has been undertaken to identify unique molecular genetic alterations in AML that are clinically and functionally significant, work greatly facilitated recently by next generation sequencing technology. Internal tandem duplication (ITD) mutations of the FMS-like tyrosine kinase 3 (FLT3) gene and mutations of the nucleophosmin gene (NPM1) are two of the most frequently identified mutations [1Patel J.P. Gonen M. Figueroa M.E. et al.Prognostic relevance of integrated genetic profiling in acute myeloid leukemia.N Engl J Med. 2012; 366: 1079-1089Crossref PubMed Scopus (1419) Google Scholar, 2The Cancer Genome Atlas Research NetworkGenomic and Epigenomic Landscapes of Adult De Novo Acute Myeloid Leukemia.N Engl J Med. 2013; 368: 2059-2074Crossref PubMed Scopus (3189) Google Scholar].NPM1 encodes nucleophosmin (NPM), which is a ubiquitously expressed nucleocytoplasmic shuttling phosphoprotein. Although the bulk of NPM resides in the nucleolus, it constantly exchanges between the nucleus and cytoplasm [3Cordell J.L. Pulford K.A. Bigerna B. et al.Detection of normal and chimeric nucleophosmin in human cells.Blood. 1999; 93: 632-642Crossref PubMed Google Scholar]. NPM plays a key role in several cellular functions, including ribosome biogenesis and maintenance of genomic stability via regulation of centrosome duplication and control of DNA repair [4Falini B. Nicoletti I. Martelli M.F. Mecucci C. Acute myeloid leukemia carrying cytoplasmic/mutated nucleophosmin (NPMc+ AML): biologic and clinical features.Blood. 2007; 109: 874-885Crossref PubMed Scopus (426) Google Scholar]. NPM also interacts with the oncosuppressors p53 and ARF and their partners, thus controlling cell proliferation and apoptosis [4Falini B. Nicoletti I. Martelli M.F. Mecucci C. Acute myeloid leukemia carrying cytoplasmic/mutated nucleophosmin (NPMc+ AML): biologic and clinical features.Blood. 2007; 109: 874-885Crossref PubMed Scopus (426) Google Scholar]. Acquired mutations in exon 12 of NPM1 were first reported in 2005 and are found frequently in patients with AML, particularly in those with a normal karyotype (NK-AML) [5Falini B. Mecucci C. Tiacci E. et al.Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1403) Google Scholar]. These mutations are characteristically heterozygous with the mutated allele encoding a protein that aberrantly localizes to the cytoplasm, thus the designation NPM-cytoplasmic positive (NPMc+) AML. NPM1 exon 12 mutations target 30%–35% of all adult AML and up to 50%–60% of adult NK-AML [5Falini B. Mecucci C. Tiacci E. et al.Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1403) Google Scholar]. In childhood AML, the prevalence is significantly less, with approximately 8% of all AML and approximately 20% of NK-AML [6Brown P. McIntyre E. Rau R. et al.The incidence and clinical significance of nucleophosmin mutations in childhood AML.Blood. 2007; 110: 979-985Crossref PubMed Scopus (160) Google Scholar, 7Hollink I.H. Zwaan C.M. Zimmermann M. et al.Favorable prognostic impact of NPM1 gene mutations in childhood acute myeloid leukemia, with emphasis on cytogenetically normal AML.Leukemia. 2009; 23: 262-270Crossref PubMed Scopus (128) Google Scholar]. In most studies, NPMc+ mutation is associated with improved prognosis, with a significantly higher CR rate [5Falini B. Mecucci C. Tiacci E. et al.Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1403) Google Scholar, 8Gale R.E. Green C. Allen C. et al.The impact of FLT3 internal tandem duplication mutant level, number, size, and interaction with NPM1 mutations in a large cohort of young adult patients with acute myeloid leukemia.Blood. 2008; 111: 2776-2784Crossref PubMed Scopus (563) Google Scholar, 9Schnittger S. Schoch C. Kern W. et al.Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype.Blood. 2005; 106: 3733-3739Crossref PubMed Scopus (588) Google Scholar, 10Thiede C. Koch S. Creutzig E. et al.Prevalence and prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid leukemia (AML).Blood. 2006; 107: 4011-4020Crossref PubMed Scopus (577) Google Scholar] and, in many studies, longer OS and EFS [7Hollink I.H. Zwaan C.M. Zimmermann M. et al.Favorable prognostic impact of NPM1 gene mutations in childhood acute myeloid leukemia, with emphasis on cytogenetically normal AML.Leukemia. 2009; 23: 262-270Crossref PubMed Scopus (128) Google Scholar, 8Gale R.E. Green C. Allen C. et al.The impact of FLT3 internal tandem duplication mutant level, number, size, and interaction with NPM1 mutations in a large cohort of young adult patients with acute myeloid leukemia.Blood. 2008; 111: 2776-2784Crossref PubMed Scopus (563) Google Scholar, 9Schnittger S. Schoch C. Kern W. et al.Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype.Blood. 2005; 106: 3733-3739Crossref PubMed Scopus (588) Google Scholar, 10Thiede C. Koch S. Creutzig E. et al.Prevalence and prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid leukemia (AML).Blood. 2006; 107: 4011-4020Crossref PubMed Scopus (577) Google Scholar]. Given its distinctive biologic and clinical features and its clear clinical relevance, NPMc+ AML is included as a provisional entity in the 2008 World Health Organization classification of myeloid malignancies [11Vardiman J.W. Thiele J. Arber D.A. et al.The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes.Blood. 2009; 114: 937-951Crossref PubMed Scopus (3404) Google Scholar].FLT3 is a receptor tyrosine kinase that, together with its ligand FL, plays important roles in the proliferation, survival, and differentiation of hematopoietic stem and progenitor cells [12Small D. Levenstein M. Kim E. et al.STK-1, the human homolog of Flk-2/Flt-3, is selectively expressed in CD34+ human bone marrow cells and is involved in the proliferation of early progenitor/stem cells.Proc Natl Acad Sci U S A. 1994; 91: 459-463Crossref PubMed Scopus (373) Google Scholar, 13Rosnet O. Schiff C. Pebusque M.J. et al.Human FLT3/FLK2 gene: cDNA cloning and expression in hematopoietic cells.Blood. 1993; 82: 1110-1119Crossref PubMed Google Scholar]. Upon binding FL the receptor dimerizes, activating its tyrosine kinase domain resulting in autophosphorylation [14Levis M. Small D. FLT3: ITDoes matter in leukemia.Leukemia. 2003; 17: 1738-1752Crossref PubMed Scopus (372) Google Scholar]. Several important signaling proteins such as Ras-GAP, PLC-b, PI3-kinase, STAT5, PIM1, and MAP kinase have been linked to FLT3 activation [14Levis M. Small D. FLT3: ITDoes matter in leukemia.Leukemia. 2003; 17: 1738-1752Crossref PubMed Scopus (372) Google Scholar, 15Kim K.T. Baird K. Ahn J.Y. et al.Pim-1 is up-regulated by constitutively activated FLT3 and plays a role in FLT3-mediated cell survival.Blood. 2005; 105: 1759-1767Crossref PubMed Scopus (192) Google Scholar]. Mutations in FLT3 have been reported in approximately 20%–35% of patients with AML [14Levis M. Small D. FLT3: ITDoes matter in leukemia.Leukemia. 2003; 17: 1738-1752Crossref PubMed Scopus (372) Google Scholar, 16Nakao M. Yokota S. Iwai T. et al.Internal tandem duplication of the flt3 gene found in acute myeloid leukemia.Leukemia. 1996; 10: 1911-1918PubMed Google Scholar, 17Meshinchi S. Woods W.G. Stirewalt D.L. et al.Prevalence and prognostic significance of Flt3 internal tandem duplication in pediatric acute myeloid leukemia.Blood. 2001; 97: 89-94Crossref PubMed Scopus (423) Google Scholar, 18Stirewalt D.L. Radich J.P. The role of FLT3 in haematopoietic malignancies.Nat Rev Cancer. 2003; 3: 650-665Crossref PubMed Scopus (691) Google Scholar, 19Gilliland D.G. Griffin J.D. The roles of FLT3 in hematopoiesis and leukemia.Blood. 2002; 100: 1532-1542Crossref PubMed Scopus (1165) Google Scholar]. These mutations are either ITD mutations, most commonly occurring in the juxtamembrane domain, or point mutations in the kinase domain, which result in the constitutive dimerization and activation of FLT3 independent of FL. FLT3/ITD mutations confer a poor prognosis in studies of pediatric and adult AML [14Levis M. Small D. FLT3: ITDoes matter in leukemia.Leukemia. 2003; 17: 1738-1752Crossref PubMed Scopus (372) Google Scholar, 19Gilliland D.G. Griffin J.D. The roles of FLT3 in hematopoiesis and leukemia.Blood. 2002; 100: 1532-1542Crossref PubMed Scopus (1165) Google Scholar, 20Meshinchi S. Alonzo T.A. Stirewalt D.L. et al.Clinical implications of FLT3 mutations in pediatric AML.Blood. 2006; 108: 3654-3661Crossref PubMed Scopus (300) Google Scholar].Importantly, NPMc+ mutations and FLT3/ITD mutations coexist frequently in AML. FLT3/ITD mutations are approximately twofold more frequent in NPMc+ leukemia compared with leukemia lacking the NPM1 mutation [5Falini B. Mecucci C. Tiacci E. et al.Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1403) Google Scholar, 6Brown P. McIntyre E. Rau R. et al.The incidence and clinical significance of nucleophosmin mutations in childhood AML.Blood. 2007; 110: 979-985Crossref PubMed Scopus (160) Google Scholar, 8Gale R.E. Green C. Allen C. et al.The impact of FLT3 internal tandem duplication mutant level, number, size, and interaction with NPM1 mutations in a large cohort of young adult patients with acute myeloid leukemia.Blood. 2008; 111: 2776-2784Crossref PubMed Scopus (563) Google Scholar]. Given the frequency with which these two mutations coexist in AML, we hypothesized that they cooperate to cause leukemia. To investigate the relationship between NPMc+ and FLT3/ITD mutations specifically, we crossed mice with Flt3/ITD constitutively knocked-in with NPMc+ transgenic mice. Flt3/ITD knock-in mice develop a fatal myeloproliferative neoplasm with a relatively long latency, but do not develop leukemia [21Lee B.H. Williams I.R. Anastasiadou E. et al.FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model.Oncogene. 2005; 24: 7882-7892Crossref PubMed Scopus (57) Google Scholar, 22Li L. Piloto O. Nguyen H.B. et al.Knock-in of an internal tandem duplication mutation into murine FLT3 confers myeloproliferative disease in a mouse model.Blood. 2008; 111: 3849-3858Crossref PubMed Scopus (113) Google Scholar]. NPMc+ transgenic mice develop a nonfatal myeloproliferation and do not develop overt leukemia [23Cheng K. Sportoletti P. Ito K. et al.The cytoplasmic NPM mutant induces myeloproliferation in a transgenic mouse model.Blood. 2010; 115: 3341-3345Crossref PubMed Scopus (77) Google Scholar]. Indeed, combination of Flt3/ITD and NPMc+ resulted in the development of leukemia in mice, providing an in vivo model of Flt3/ITD+/NPMc+ leukemia, which closely recapitulates human disease, thus making an in-depth investigation of disease biology possible.MethodsMiceMice with an 18bp-ITD mutation knocked into the juxtamembrane domain of the murine Flt3 gene (FLT3wt/ITD) and transgenic mice expressing Flag-tagged human NPMc+ mutant A driven by human MRP8 promoter (hMRP8-NPMc+) were generated as reported previously [22Li L. Piloto O. Nguyen H.B. et al.Knock-in of an internal tandem duplication mutation into murine FLT3 confers myeloproliferative disease in a mouse model.Blood. 2008; 111: 3849-3858Crossref PubMed Scopus (113) Google Scholar, 23Cheng K. Sportoletti P. Ito K. et al.The cytoplasmic NPM mutant induces myeloproliferation in a transgenic mouse model.Blood. 2010; 115: 3341-3345Crossref PubMed Scopus (77) Google Scholar]. Mice were categorized as wild-type (WT), positive for the NPMc+ mutation alone (NPMc+), positive for the Flt3/ITD mutation alone (ITD), or positive for both mutations (ITD/NPMc+) based on PCR of germline DNA using the primers mITD-5F + mITD-3R, NPM874F + MRP8R (sequences in Supplementary Table E1, online only, available at www.exphem.org).For transplantation experiments, CD45.1+ mice received 700 cGy of gamma irradiation. Next, 1 × 106 whole bone marrow cells isolated from leukemic ITD/NPMc+ CD45.2 mice were injected via retroorbital injection. Engraftment was evaluated by flow cytometric determination of the percentage of CD45.2+ cells in the peripheral blood. All animal experiments were reviewed and approved by the Johns Hopkins University Institutional Animal Care and Use Committee.Flow cytometric analysisFlow cytometric analysis of murine bone marrow (BM) cells, splenocytes, and cells of other infiltrated organs was performed with the following monoclonal antibodies: lineage mixture (Caltag Laboratories–Invitrogen, Carlsbad, CA, USA), Ly-6A/E (Sca-1; Invitrogen), CD135, CD117 (c-Kit), CD41, Ter119, CD11b, Gr1, CD24, CD43, CD19, CD45R/B220, CD4, CD3, and CD8a (BD Pharmingen, San Jose, CA, USA). BM cells were obtained, washed, fixed, and permeabilized for intracellular flow cytometry. Alexa Fluor 488 conjugated anti-phosphoSTAT5 (pY694; BD biosciences) was used to detect phosphorylated STAT5. Analysis was performed using FlowJo software (TreeStar, Ashland, OR, USA).Reverse-transcriptase PCRRNA was isolated from murine tissues and was reverse transcribed and amplified using the following primers (primer sequences found in Supplementary Table E1, online only, available at www.exphem.org). For Flag-tagged NPMc+: exon 1F + NPM500R; for HPRT: HPRT1 + HPRT2. For Flt3: mFLT3-RT-5F + mFLT3-RT-3R.In vitro clonogenic assayLineage-negative murine BM cells from 2-month-old mice were placed in liquid culture with RPMI 1640 plus 20% fetal bovine serum, murine stem cell factor (SCF; 50 ng/mL), IL-3 (10 ng/mL), and IL-6 (10 ng/mL) or plated in Methocult M3434 medium supplemented with recombinant murine SCF (50 ng/mL), IL-3 (10 ng/mL), IL-6 (10 ng/mL), GM-CSF (10 ng/mL), and EPO (3 U/mL). Cells in liquid culture were counted daily and on day 7 were analyzed with flow cytometry for lineage commitment. On methylcellulose, all colonies were scored 9–11 days after plating. Cells were then isolated from the plates and analyzed with flow cytometry for cKit, Mac1, Gr1, and Ter119. Experiments were performed in triplicate.In vitro cytotoxicity assayMurine BM cells were cultured in RPMI 1640 (Gibco–Invitrogen, Carlsbad, CA, USA) plus 10% heat inactivated fetal bovine serum (FBS), 1% L-glutamine, 50 ng/mL SCF, 10 ng/mL IL-3, and 10 ng/mL IL-6 (PeproTech, Rocky Hill, NJ, USA). Cells were treated with increasing concentrations of lestaurtinib (Cephalon, Malvern, PA, USA) for 48 hours. WST-1 reagent (Roche Diagnostics, Manheim, Germany) was added to the culture medium (1:10 dilution), and absorbance was measured at 450nm using a Bio-Rad model 680 microplate reader (BioRad). Assays were performed in triplicate.StatisticsKaplan-Meier survival curve was generated and analyzed using Graph Pad Prism version 4.0 software (GraphPad, San Diego, CA, USA). Log-rank test was performed to compare the generated survival curves. Analysis of variance followed by pairwise t tests were used where appropriate (GraphPad).ResultsFlt3/ITD and NPMc+ mutations cooperate to cause acute leukemiaMice with both Flt3/ITD and NPMc+ mutations (ITD/NPMc+) die between 6 and 18 months, with a median survival of 420 days (Fig. 1A). Their survival is significantly shorter than the survival of WT mice and NPMc+ mice (p < 0.0001). Mice with Flt3/ITD mutation alone (ITD) succumb to a fatal myeloproliferative neoplasm (MPN) between 6 and 20 months [22Li L. Piloto O. Nguyen H.B. et al.Knock-in of an internal tandem duplication mutation into murine FLT3 confers myeloproliferative disease in a mouse model.Blood. 2008; 111: 3849-3858Crossref PubMed Scopus (113) Google Scholar] and in our cohort had a median survival of 550 days, which was longer than ITD/NPMc+ mice, though this difference did not reach statistical significance. ITD/NPMc+ mice become moribund prior to death. Moribund mice were sacrificed (N = 40) and found to have splenomegaly (mean spleen weight, 0.741 ± 0.06 g vs. 0.13 ± 0.005 g for WT mice, 0.2 ± 0.04 g in NPMc+ mice, and 0.37 ± 0.05 g in ITD mice), leukocytosis (mean white blood cell count, 124.5 ± 22.5 103/μL vs. 10.7 ± 1.3 103/μL in WT mice, 11.1 ± 3.4 103/μL in NPMc+ mice, and 30.4 ± 8 103/μL in ITD mice), anemia (mean red blood cell count, 5.3 ± 0.4 109/μL vs. 8.5 ± 0.2 109/μL in WT mice, 8.2 ± 0.4 109/μL in NPMc+ mice, and 6.3 ± 0.5 109/mL in ITD mice), and thrombocytopenia (mean platelet count, 352.3 ± 23 103/μL vs. 735 ± 37.1 103/μL in WT mice, 795.5 ± 42 103/μL in NPMc+ mice, and 629.3 ± 72.2 103/μL in ITD mice; Fig. 1B–D). All these parameters were statistically significant compared with WT littermate controls (N = 19), ill ITD mice (N = 12), and littermate control NPMc+ mice (N = 8) except red blood cell count, which trended toward more severe in the ITD/NPMc+ mice but did not reach statistical significance compared with ill ITD mice. On gross examination, there was pathologic enlargement of multiple organs, including spleens and livers of all ill ITD/NPMc+ mice and kidneys and thymuses of a subset of ill ITD/NPMc+ mice (Fig. 2A–D). On histologic examination, large monotonous cells were seen infiltrating the BM and infiltrating and disrupting the normal architecture of the livers of leukemic mice with both mutations (Fig. 2E). Additionally, the malignant cells infiltrating the BM are found in high numbers circulating in the peripheral blood of ill ITD/NPMc+ mice (Supplementary Figure E1, online only, available at www.exphem.org). Furthermore, the spleens of all ill ITD/NPMc+ mice and the kidneys and meninges of a subset of ill ITD/NPMc+ mice were infiltrated with large monotonous cells on hematoxylin and eosin (H&E) staining, indicating a highly aggressive, infiltrative disease (Supplementary Figure E1, online only, available at www.exphem.org).Figure 2The leukemia that develops in ITD/NPMc+ mice is aggressive, infiltrating multiple organs. Gross evaluation of representative leukemic ITD/NPMc+ mice, revealing involvement of the (A) spleen, (B) liver, (C) kidneys, and in some cases the (D) thymus. (E) H&E stains of BM (i–v) and liver (vi–x) of representative mice of each genotype. Scale bars are as follows: i–v, 10 μm; vi–x, 100 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mice harboring both NPMc+ and FLT3/ITD mutations develop transplantable myeloid and lymphoid leukemiasTo characterize the disease of ill ITD/NPMc+ mice, flow cytometry was performed on BM cells, splenocytes, and cells of other infiltrated tissue. Wild-type littermate controls were sacrificed and analyzed at the same time as ill ITD/NPMc+ mice (Fig. 3A, row 1). A number of ill mice with Flt3/ITD alone or littermate control mice with NPMc+ alone were also analyzed in the same fashion (Fig. 3A, rows 2 and 3, respectively). As anticipated, there was a high frequency of myeloid leukemia in ITD/NPMc+ mice. Most commonly, ITD/NPMc+ mice developed myeloid leukemia with maturation, characterized by high cKit positivity, and Mac1+/Gr1+ myeloblasts (Fig. 3A, row 4) [24Kogan S.C. Ward J.M. Anver M.R. et al.Bethesda proposals for classification of nonlymphoid hematopoietic neoplasms in mice.Blood. 2002; 100: 238-245Crossref PubMed Scopus (340) Google Scholar]. We also observed a number of myeloid leukemias without maturation, characterized by high cKit positivity, but myeloblasts lacking cell surface markers indicative of maturation (Fig. 3A, row 5) [24Kogan S.C. Ward J.M. Anver M.R. et al.Bethesda proposals for classification of nonlymphoid hematopoietic neoplasms in mice.Blood. 2002; 100: 238-245Crossref PubMed Scopus (340) Google Scholar]. In addition, we observed a number of T cell acute lymphoblastic leukemias (ALLs) characterized by cKit−, CD3+/CD4+/CD8+ lymphoblasts (Fig. 3A, row 6) or less commonly CD3+/CD4–/CD8+ lymphoblasts (Supplementary Figure E2, online only, available at www.exphem.org). A number of ITD/NPMc+ mice also developed a mixed lineage T/myeloid disease with high cKit positivity and both Mac1+/Gr1+ myeloblasts and CD3+/CD4+/CD8+ lymphoblasts (Fig. 3A, rows 7). All the leukemic mice also had a paucity of normal maturing erythrocytes as demonstrated by decreased Ter119+ cells in the BM and maturing B lymphocytes indicated by decreased B220+/CD19+ cell in the BM (Fig. 3, columns 3 and 4, respectively). The relative frequency with which each of the leukemic subtypes occurred in the ITD/NPMc+ mice is summarized in Figure 3B with greater than 50% of ill mice having a myeloid leukemia.Figure 3ITD/NPMc+ mice develop myeloid and lymphoid leukemias. (A) Flow cytometry plots demonstrating the characteristic phenotype of WT mice (row 1), ITD mice with myeloproliferative neoplasm (row 2), NPMc+ mice with myeloproliferation (row 3), and each of the four most common types of acute leukemia diagnosed in ITD/NPMc+ mice (rows 4–7). All the leukemic mice also have a paucity of normal maturing erythrocytes, as demonstrated by decreased Ter119+ cells in the BM and maturing B lymphocytes indicated by decreased B220+/CD19+ cell in the BM. (B) Disease distribution of the 40 fully characterized ITD/NPMc+ mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In transplantation experiments, sublethally irradiated syngeneic mice injected with 1 × 106 whole BM cells from leukemic mice with either myeloid or T lineage disease engrafted by 4 weeks after transplant and develop disease with the same phenotypic characteristics as the leukemic donor mouse (Fig. 4A–D). The kinetics of the transplanted leukemias were variable, with transplanted T cell ALL causing more rapid decline than transplanted AML (Fig. 4D, E).Figure 4Both T cell and myeloid leukemias are transplantable. (A) Experimental design of transplantation experiment. (B) Phenotype of the donor mouse with T cell ALL (row 1). Engraftment (as determined by percent CD45.2 positivity) and phenotype of a representative recipient (row 2). (C) Phenotype of the leukemic donor mouse with AML without maturation (row 1). Engraftment and phenotype of a representative recipient (row 2). (D) Summary of transplantation experiments. (E) Kaplan-Meier survival curves demonstrating disease kinetics engrafted mice of the three disease phenotypes; 2 of 4 mice that engrafted with AML with maturation had not succumbed to illness when the experiment was terminated at 32 weeks, and were therefore censored.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Expression of both Flt3/ITD and the flag-tagged NPMc+ transgene mRNA in the BM and infiltrated nonhematopoietic tissue of leukemic mice was demonstrated by reverse-transcriptase (RT) PCR (Supplementary Figure E3, online only, available at www.exphem.org). Given the likely importance of NPMc+ localization to the cytoplasm in its role in leukemogenesis, we confirmed mutant transgenic NPMc+ protein localization to the cytoplasm by immunofluorescence and immunohistochemistry (Supplementary Figure E3, online only, available at www.exphem.org). To demonstrate functional effects of NPMc+ mutation, we examined expression of Hox cluster genes known to be overexpressed in human NPMc+ AML and other NPMc+ animal models [4Falini B. Nicoletti I. Martelli M.F. Mecucci C. Acute myeloid leukemia carrying cytoplasmic/mutated nucleophosmin (NPMc+ AML): biologic and clinical features.Blood. 2007; 109: 874-885Crossref PubMed Scopus (426) Google Scholar, 25Mallardo M. Caronno A. Pruneri G. et al.NPMc+ and FLT3_ITD mutations cooperate in inducing acute leukaemia in a novel mouse model.Leukemia. 2013; 27: 2245-2251Crossref Scopus (35) Google Scholar, 26Vassiliou G.S. Cooper J.L. Rad R. et al.Mutant nucleophosmin and cooperating pathways drive leukemia initiation and progression in mice.Nat Genet. 2011; 43: 470-475Crossref PubMed Scopus (159) Google Scholar]. Using quantitative RT-PCR, we found increased expression of both HoxA9 and HoxA10 in whole BM cells from 2-month-old NPMc+ and ITD/NPMc+ mice (Supplementary Figure E3, online only, available at www.exphem.org). We also found increased expression of HoxA9 in mice with Flt3/ITD mutation alone, as has been reported previously [27Greenblatt S. Li L. Slape C. et al.Knock-in of a FLT3/ITD mutation cooperates with a NUP98-HOXD13 fusion to generate acute myeloid leukemia in a mouse model.Blood. 2012; 119: 2883-2894Crossref PubMed Scopus (52) Google Scholar]. However, the ITD/NPMc+ mice had higher levels of HoxA9 expression than did mice with either mutation alone, suggesting an additive effect. HoxA10 expression was increased only in mice with NPMc+ mutations, and to the same degree both in the presence and absence of concomitant Flt3/ITD mutation, suggesting that this increased expression was due solely to the effects of the NPMc+ mutation.The occurrence of T lineage leukemia was an unexpected finding because NPMc+ mutations are not associated with T cell ALL, and activating FLT3 mutations rarely occur in T cell ALL [5Falini B. Mecucci C. Tiacci E. et al.Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype.N Engl J Med. 2005; 352: 254-266Crossref PubMed Scopus (1403) Google Scholar, 28Paietta E. Ferrando A.A. Neuberg D. et al.Activating FLT3 mutations in CD117/KIT(+) T-cell acute lymphoblastic leukemias.Blood. 2004; 104: 558-560Crossref PubMed Scopus (83) Google Scholar]. Furthermore, the hMRP8 promoter that drives expression of NPMc+ is expected to be expressed only in myeloid lineage Mac1+/Gr1+ cells. We hypothesized that there may be a degree of infidelity of the hMRP8 promoter allowing for NPMc+ expression in the T cells of mice with the hMRP8-NPMc+ transgene. We therefore isolated T cells from the spleens of 2-month-old mice of each genotype. Using RT-PCR, NPMc+ expression was demonstrated in T cells of mice with the NPMc+ transgene (Supplementary Figure E4, online only, available at www.exphem.org). Furthermore, similar T cell disease has been documented in other mouse models utilizing the hMPR8 promoter [29Jaiswal S. Traver D. Miyamoto T. Akashi K. Lagasse E. Weissman I.L. Expression of BCR/ABL and BCL-2 in myeloid progenitors leads to myeloid leukemias.Proc Natl Acad Sci U S A. 2003; 100: 10002-10007Crossref PubMed Scopus (146) Google Scholar].Loss of heterozygosity of the wild-type Flt3 allele occurs with a high frequency in myeloid leukemia and is functionally significantIn human AML, loss of the WT" @default.
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