Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Mutant nucleophosmin and cooperating pathways drive leukemia initiation and progression in mice


Acute myeloid leukemia (AML) is a molecularly diverse malignancy with a poor prognosis whose largest subgroup is characterized by somatic mutations in NPM1, which encodes nucleophosmin1. These mutations, termed NPM1c, result in cytoplasmic dislocation of nucleophosmin1 and are associated with distinctive transcriptional signatures2, yet their role in leukemogenesis remains obscure. Here we report that activation of a humanized Npm1c knock-in allele in mouse hemopoietic stem cells causes Hox gene overexpression, enhanced self renewal and expanded myelopoiesis. One third of mice developed delayed-onset AML, suggesting a requirement for cooperating mutations. We identified such mutations using a Sleeping Beauty3,4 transposon, which caused rapid-onset AML in 80% of mice with Npm1c, associated with mutually exclusive integrations in Csf2, Flt3 or Rasgrp1 in 55 of 70 leukemias. We also identified recurrent integrations in known and newly discovered leukemia genes including Nf1, Bach2, Dleu2 and Nup98. Our results provide new pathogenetic insights and identify possible therapeutic targets in NPM1c+ AML.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

Figure 1: Conditional mouse model of type A NPM1c mutation.
Figure 2: Hematopoietic changes and incidence of AML in Npm1cA/+ mice.
Figure 3: Npm1cA and the GrOnc transposon synergize to cause AML.
Figure 4: Common integration sites in transposon-derived leukemias.
Figure 5: A model for Npm1cA/+-driven leukemogenesis.

Accession codes




  1. Falini, B. et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N. Engl. J. Med. 352, 254–266 (2005).

    Article  CAS  PubMed  Google Scholar 

  2. Alcalay, M. et al. Acute myeloid leukemia bearing cytoplasmic nucleophosmin (NPMc+ AML) shows a distinct gene expression profile characterized by up-regulation of genes involved in stem-cell maintenance. Blood 106, 899–902 (2005).

    Article  CAS  PubMed  Google Scholar 

  3. Dupuy, A.J., Akagi, K., Largaespada, D.A., Copeland, N.G. & Jenkins, N.A. Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature 436, 221–226 (2005).

    Article  CAS  PubMed  Google Scholar 

  4. Collier, L.S., Carlson, C.M., Ravimohan, S., Dupuy, A.J. & Largaespada, D.A. Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse. Nature 436, 272–276 (2005).

    Article  CAS  PubMed  Google Scholar 

  5. Okuwaki, M. The structure and functions of NPM1/Nucleophsmin/B23, a multifunctional nucleolar acidic protein. J. Biochem. 143, 441–448 (2008).

    Article  CAS  PubMed  Google Scholar 

  6. Grisendi, S., Mecucci, C., Falini, B. & Pandolfi, P.P. Nucleophosmin and cancer. Nat. Rev. Cancer 6, 493–505 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. Hingorani, K., Szebeni, A. & Olson, M.O. Mapping the functional domains of nucleolar protein B23. J. Biol. Chem. 275, 24451–24457 (2000).

    Article  CAS  PubMed  Google Scholar 

  8. Falini, B. et al. Altered nucleophosmin transport in acute myeloid leukaemia with mutated NPM1: molecular basis and clinical implications. Leukemia 23, 1731–1743 (2009).

    Article  CAS  PubMed  Google Scholar 

  9. Falini, B. et al. NPM1 mutations and cytoplasmic nucleophosmin are mutually exclusive of recurrent genetic abnormalities: a comparative analysis of 2562 patients with acute myeloid leukemia. Haematologica 93, 439–442 (2008).

    Article  CAS  PubMed  Google Scholar 

  10. Rocquain, J. et al. Combined mutations of ASXL1, CBL, FLT3, IDH1, IDH2, JAK2, KRAS, NPM1, NRAS, RUNX1, TET2 and WT1 genes in myelodysplastic syndromes and acute myeloid leukemias. BMC Cancer 10, 401 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Cheng, K. et al. The cytoplasmic NPM mutant induces myeloproliferation in a transgenic mouse model. Blood 115, 3341–3345 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Sportoletti, P. et al. Npm1 is a haploinsufficient suppressor of myeloid and lymphoid malignancies in the mouse. Blood 111, 3859–3862 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Lavau, C., Szilvassy, S.J., Slany, R. & Cleary, M.L. Immortalization and leukemic transformation of a myelomonocytic precursor by retrovirally transduced HRX-ENL. EMBO J. 16, 4226–4237 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Kogan, S.C. et al. Bethesda proposals for classification of nonlymphoid hematopoietic neoplasms in mice. Blood 100, 238–245 (2002).

    Article  CAS  PubMed  Google Scholar 

  15. Smith, G.S., Walford, R.L. & Mickey, M.R. Lifespan and incidence of cancer and other diseases in selected long-lived inbred mice and their F1 hybrids. J. Natl. Cancer Inst. 50, 1195–1213 (1973).

    Article  CAS  PubMed  Google Scholar 

  16. Rad, R. et al. PiggyBac transposon mutagenesis: a tool for cancer gene discovery in mice. Science 330, 1104–1107 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Voisin, V., Barat, C., Hoang, T. & Rassart, E. Novel insights into the pathogenesis of the Graffi murine leukemia retrovirus. J. Virol. 80, 4026–4037 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. de Ridder, J., Uren, A., Kool, J., Reinders, M. & Wessels, L. Detecting statistically significant common insertion sites in retroviral insertional mutagenesis screens. PLOS Comput. Biol. 2, e166 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Dührsen, U., Stahl, J. & Gough, N.M. In vivo transformation of factor-dependent hemopoietic cells: role of intracisternal A-particle transposition for growth factor gene activation. EMBO J. 9, 1087–1096 (1990).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Rogers, S.Y., Bradbury, D., Kozlowski, R. & Russell, N.H. Evidence for internal autocrine regulation of growth in acute myeloblastic leukemia cells. Exp. Hematol. 22, 593–598 (1994).

    CAS  PubMed  Google Scholar 

  21. Young, D.C. & Griffin, J.D. Autocrine secretion of GM-CSF in acute myeloblastic leukemia. Blood 68, 1178–1181 (1986).

    CAS  PubMed  Google Scholar 

  22. Takeda, A., Sarma, N.J., Abdul-Nabi, A.M. & Yaseen, N.R. Inhibition of CRM1-mediated nuclear export of transcription factors by leukemogenic NUP98 fusion proteins. J. Biol. Chem. 285, 16248–16257 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kim, R. et al. Genome-based identification of cancer genes by proviral tagging in mouse retrovirus-induced T-cell lymphomas. J. Virol. 77, 2056–2062 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Lauchle, J.O. et al. Response and resistance to MEK inhibition in leukaemias initiated by hyperactive Ras. Nature 461, 411–414 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Nakamura, T., Largaespada, D.A., Shaughnessy, J.D. Jr., Jenkins, N.A. & Copeland, N.G. Cooperative activation of Hoxa and Pbx1-related genes in murine myeloid leukemias. Nat. Genet. 12, 149–153 (1996).

    Article  CAS  PubMed  Google Scholar 

  26. Ayton, P.M. & Cleary, M.L. Transformation of myeloid progenitors by MLL oncoproteins is dependent on Hoxa7 and Hoxa9. Genes Dev. 17, 2298–2307 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lawrence, H.J. et al. Mice bearing a targeted interruption of the homeobox gene HOXA9 have defects in myeloid, erythroid, and lymphoid hematopoiesis. Blood 89, 1922–1930 (1997).

    CAS  PubMed  Google Scholar 

  28. Verhaak, R.G. et al. Mutations in nucleophosmin (NPM1) in acute myeloid leukemia (AML): association with other gene abnormalities and previously established gene expression signatures and their favorable prognostic significance. Blood 106, 3747–3754 (2005).

    Article  CAS  PubMed  Google Scholar 

  29. Ley, T.J. et al. DNMT3A mutations in acute myeloid leukemia. N. Engl. J. Med. 363, 2424–2433 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Liu, P., Jenkins, N.A. & Copeland, N.G. A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res. 13, 476–484 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Chen, Y.T. & Bradley, A. A new positive/negative selectable marker, puDeltatk, for use in embryonic stem cells. Genesis 28, 31–35 (2000).

    Article  CAS  PubMed  Google Scholar 

  32. Kühn, R., Schwenk, F., Aguet, M. & Rajewsky, K. Inducible gene targeting in mice. Science 269, 1427–1429 (1995).

    Article  PubMed  Google Scholar 

  33. Li, J. et al. JAK2 V617F impairs hematopoietic stem cell function in a conditional knock-in mouse model of JAK2 V617F-positive essential thrombocythemia. Blood 116, 1528–1538 (2010).

    Article  CAS  PubMed  Google Scholar 

  34. Yang, Y.H. et al. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 30, e15 (2002).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Smyth, G.K. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, Article3 (2004).

    Article  PubMed  Google Scholar 

  36. Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc., B 57, 289–300 (1995).

    Google Scholar 

  37. Uren, A.G. et al. A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites. Nat. Protoc. 4, 789–798 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Akagi, K., Suzuki, T., Stephens, R.M., Jenkins, N.A. & Copeland, N.G. RTCGD: retroviral tagged cancer gene database. Nucleic Acids Res. 32, D523–D527 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Collier, L.S. et al. Whole-body sleeping beauty mutagenesis can cause penetrant leukemia/lymphoma and rare high-grade glioma without associated embryonic lethality. Cancer Res. 69, 8429–8437 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Uren, A.G. et al. Large-scale mutagenesis in p19(ARF)- and p53-deficient mice identifies cancer genes and their collaborative networks. Cell 133, 727–741 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references


We acknowledge the use of the Research Support Facility at the Wellcome Trust Sanger Institute, the Department of Pathology Tissue Bank and the Cambridge National Institute of Health Research (NIHR) Biomedical Research Centre, University of Cambridge. We thank F. Law and J. Gadiot for assistance in generating the Npm1flox-CA and Rosaflox-SB mice; F. Foyer and B. Graham for help with fluorescent microscopy; B. Ling, W. Cheng, R. Macintyre and P. Chan for help with flow cytometry; R. Bautista for help with gene expression images; C. Hale and A. Nyzhnyk for help with ELISAs; B. Huntly, D. Adams, J. Cadinanos, H. Prosser, N. Conte, K. Yusa and Q. Liang for helpful discussions during the project; and P. Campbell and A. Green for critical reading of the manuscript. This work was supported by a Clinician Scientist Fellowship from Cancer Research UK (G.S.V.).

Author information

Authors and Affiliations



G.S.V. and A.B. designed the study. G.S.V. generated Npm1flox-cA mice, GrOnc mice and GFP-NPM1 constructs, managed mouse colonies, designed and validated polyclonal anti-Npm1c sera and carried out protein blots. J.L.C. and G.S.V. performed mouse genotyping, tumor processing and banking and K562 transfections. G.S.V., J.L.C., R.R. and L.R. performed mouse necropsies. G.S.V., J.L.C. and J.L. performed hemopoietic analyses. G.S.V. and C.G. performed quantitative PCR. G.S.V., P.E. and R.A. performed gene expression analysis studies. S.R., G.S.V. and R.R. performed mapping and analysis of transposon integration sites. G.S.V. and R.B. performed fluorescence in situ hybridization. W.W. and P.L. generated the Stella-Cre mice. A.U. generated the Rosaflox-SB mice. P.W. and M.A. performed histological analyses. A.B. supervised the study. All authors contributed to the writing of the manuscript.

Corresponding authors

Correspondence to George S Vassiliou or Allan Bradley.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11 and Supplementary Tables 1–9. (PDF 1031 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Vassiliou, G., Cooper, J., Rad, R. et al. Mutant nucleophosmin and cooperating pathways drive leukemia initiation and progression in mice. Nat Genet 43, 470–475 (2011).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer