A Knock-In Npm1 Mutation in Mice Results in Myeloproliferation and Implies a Perturbation in Hematopoietic Microenvironment

et al. (2012) A Knock-In Npm1 Mutation in Mice Results in Myeloproliferation and Implies a Perturbation in Hematopoietic Microenvironment. PLoS ONE 7(11): e49769. doi:10.1371/journal.pone.0049769 A Knock-In Npm1 Mutation in Mice Results in Myeloproliferation and Implies a Perturbation in Hematopoietic Microenvironment Shiu-Huey Chou 0 1 Bor-Sheng Ko 0 1 Ji-Shain Chiou 0 1 Yueh-Chwen Hsu 0 1 Mong-Hsun Tsai 0 1 Yu-Chiao Chiu 0 1 I-Shing Yu 0 1 Shu-Wha Lin 0 1 Hsin-An Hou 0 1 Yi-Yi Kuo 0 1 Hsiu-Mei Lin 0 1 Ming-Fang Wu 0 1 Wen-Chien Chou 0 1 Hwei-Fang Tien 0 1 Kevin D. Bunting, Emory University, United States of America 0 1 Department of Life Science, Fu-Jen University , Taipei, Taiwan , 2 Division of Hematology, Department of Internal Medicine, National Taiwan University Hospital , Taipei, Taiwan , 3 Institute of Cellular and Systemic Medicine, National Health Research Institute, MiaoLi County, Taiwan, 4 Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine , Taipei, Taiwan , 5 Institute of Biotechnology, National Taiwan University , Taipei, Taiwan , 6 Graduate Institute of Biomedical Electronics and Bioinformatics, National Taiwan University , Taipei, Taiwan, 7 Transgenic Mouse Models Core , National Taiwan University College of Medicine , Taipei, Taiwan , 8 Department of Laboratory Medicine, National Taiwan University Hospital , Taipei, Taiwan , 9 Animal Medicine Center, National Taiwan University College of Medicine , Taipei , Taiwan 1 A Mouse Model of Npm1 Mutation Somatic Nucleophosmin (NPM1) mutation frequently occurs in acute myeloid leukemia (AML), but its role in leukemogenesis remains unclear. This study reports the first ''conventional'' knock-in mouse model of Npm1 mutation, which was achieved by inserting TCTG after nucleotide c.857 (c.854_857dupTCTG) to mimic human mutation without any ''humanized'' sequence. The resultant mutant peptide differed slightly different from that in humans but exhibited cytoplasmic pulling force. Homozygous (Npm1c+/c+) mice showed embryonic lethality before day E8.5, wheras heterozygous (Npm1wt/c+) mice appeared healthy at birth and were fertile. Approximately 36% of Npm1wt/c+ mice developed myeloproliferative disease (MPD) with extramedullary hematopoiesis. Those Npm1wt/c+ mice that did not develop MPD nevertheless gradually developed monocytosis and showed increased numbers of marrow myeloid precursors. This second group of Npm1wt/c+ mice also showed compromised cobblestone area formation, suggesting pathology in the hematopoietic niche. Microarray experiments and bioinformatic analysis on mice myeloid precursor cells and 227 human samples revealed the expression of CXCR4/CXCL12-related genes was significantly suppressed in mutant cells from both mice and humans. Thus, our mouse model demonstrated that Npm1 mutation can result in MPD, but is insufficient for leukemogenesis. Perturbation of hematopoietic niche in mutant hematopoietic stem cells (implied by underrepresentation of CXCR4/CXCL12-related genes) may be important in the pathogenesis of NPM1 mutations. - Funding: This work was supported by grants NSC 98-2314-B-002-033-MY3, NSC 98-2314-B-030-003-MY3, NSC 100-2325-B-002-032, NSC 100-2325-B-002-033, and NSC 101-2325-B-002-029 from National Science Council (Taiwan), NHRI-EX97-9731BI from National Health Research Institute, and DOH101-TD-C-111-001 from Department of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. NPM1 mutation in acute myeloid leukemia (AML) was first identified by the aberrant cytoplasmic localization of NPM1 protein, which is normally located in the nucleoli of non-mitotic cells [1]. The mutation consists of a tetra-nucleotide insertion near the C terminal end of the coding sequence of NPM1. The most frequent form of mutation is duplication of TCTG (type A, c.860_863dupTCTG), which results in alteration of the peptide sequence from DLWQWRKSL* to DLCL AVEEVSLRK* [1,2]. The insertion is near the stop codon of the gene, but nonetheless disrupts 2 tryptophan residues that are critical for the localization of wild-type NPM1 protein in nucleoli [3]. Moreover, the mutant peptide contains a nuclear export signal (NES), causing the mutant NPM1 protein to shift into cytoplasm [4]. Some less frequent mutant forms involve insertion of 4 nucleotides between coding sequence position 863 and 864 or further 39 ends; invariably, these mutations disrupt at least 1 tryptophan residue [2]. In vitro study has shown that an increased number of disrupted tryptophan residues is associated with incremental traction power of mutant NPM1 protein toward the cytoplasm [5]. The NPM1 protein is ubiquitously expressed in mammalian cells. The protein shuttles between nuclei and cytoplasm, with pleiotropic functions including ribosomal biogenesis [6,7], centrosome duplication [8], and regulation of p53 and ARF proteins [9,10]. Constitutional knock-out of Npm1 in mice results in embryonic lethality [11]. Heterozygous Npm1 knock-out produced phenotypes mimicking human myelodysplastic syndrome or malignancies of myeloid or lymphoid cells [12]. These findings suggest a haploinsufficient tumor suppressor role of Npm1 in vivo. However, the phenotypes of Npm1 haploinsufficiency of differ from those seen in AML patients with NPM1 mutation. Lymphoid malignancies and cytogenetic changes are associated with the phenotypes of Npm1 haploinsufficiency, whereas these 2 phenomena are rarely seen in human AML patients with NPM1 mutation [12]. This finding, together with the fairly fixed pattern of mutation in the NPM1 gene, suggests that the mutation is likely a gain-of-function one rather than simple haploinsufficiency. Although the clinical and biological features of AML patients with NPM1 mutations have been well characterized [1,1318], the pathophysiological roles of NPM1 mutation remain to be defined. In mice bearing transgenic human NPM1 mutation, a number of animals developed myeloproliferation [19], indicating a pathological role of mutant NPM1 protein in myeloid disorders. Similarly, zebrafish bearing enforced human NPM1 mutant expression showed an expansion of hematopoietic cells [20]. Furthermore, a mouse model of knock-in of humanized NPM1 mutation (which resulted in a peptide sequence identical to the human mutation) showed expansion of the myeloid pool but shrinkage of B cells. In addition, AML phenotypes were evident in a percentage of aging mice [21]. All these animal models suggest that mutant NPM1 causes hematopoietic expansion, but is insufficeint for leukemogenesis. The pathogenetic role of NPM1 mutation in AML awaits further clarification. This study was the firstto document a mouse Npm1 mutant knock-in model. In a previous study using a humanized NPM1 mutant knock-in model (...truncated)