RAS mutations are common in myeloid malignancies; however, it is not known whether oncogenic RAS can initiate leukemia. We show that expressing mutant K-Ras G12D protein from the endogenous murine locus rapidly induces a fatal myeloproliferative disorder with 100% penetrance characterized by tissue infiltration, hypersensitivity to growth factors, and hyperproliferation. Hematopoietic cells from diseased mice demonstrated increased levels of Ras-GTP, but effector kinases were not constitutively phosphorylated and responded normally to growth factors. Oncogenic RAS is sufficient to initiate myeloid leukemogenesis in mice, and this provides an in vivo system for biologic and preclinical studies.

The NF1 tumor suppressor gene encodes a guanosine triphosphotase (GTPase)-activating protein that negatively regulates Ras signaling and is inactivated in a subset of juvenile myelomonocytic leukemias (JMMLs). Adoptive transfer of fetal liver cells from Nf1 mutant mice models JMML; however, this system has important limitations as a platform for performing biologic and preclinical studies. We have exploited the interferon-inducible Mx1-Cre transgene to ablate a conditional mutant Nf1 allele in hematopoietic cells. Somatic inactivation of Nf1 induces a myeloproliferative disorder with 100% penetrance that is associated with a subacute clinical course, tissue infiltration by myeloid cells, hypersensitivity to granulocyte-macrophage colony stimulating factor, hyperproliferation, and resistance to apoptosis. These Mx1-Cre, Nf1 flox/flox mice establish a tractable experimental model for testing therapeutics and for identifying mutations that cooperate with hyperactive Ras in myeloid leukemogenesis. IntroductionJuvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disease (MPD) characterized by monocytosis, thrombocytopenia, splenomegaly, and malignant infiltration of the skin, lymph nodes, lungs, liver, and other organs (reviewed in Emanuel et al 1 and Arico et al 2 ). The clinical course is relentless, and bone marrow transplantation is the only treatment that cures more than 10% of patients. Selective hypersensitivity of granulocyte-macrophage colony-forming unit (CFU-GM) progenitors to granulocyte-macrophage colony-stimulating factor (GM-CSF) is an in vitro hallmark of JMML. 3,4 The incidence of JMML is increased more than 200-fold in children with neurofibromatosis type 1 (NF1) 5,6 ; this observation provided a starting point for elucidating the molecular basis of aberrant myeloid growth in this disorder. The NF1 gene encodes neurofibromin, a guanosine triphosphotase (GTPase)-activating protein (GAP) that negatively regulates p21 ras (Ras) output by accelerating GTP hydrolysis (reviewed in Boguski and McCormick,7 Bernards, 8 and Donovan et al 9 ). Analysis of JMML cells from children with NF1 revealed homozygous NF1 inactivation because of somatic loss of the normal allele, which is associated with hyperactive Ras. [10][11][12][13] Two groups used homologous recombination in embryonic stem cells to disrupt Nf1, the murine homolog of NF1. 14,15 Approximately 10% of heterozygous (Nf1 ϩ/Ϫ ) mutant mice spontaneously develop a MPD that resembles JMML during the second year of life. 14 Homozygous mutant (Nf1 Ϫ/Ϫ ) embryos fail around embryonic day 13 (E13) with cardiovascular defects 14,15 ; however, CFU-GM colonies derived from mutant fetal livers show hypersensitive growth in response to GM-CSF that is similar to human JMML cells. 11,16 Importantly, adoptive transfer of Nf1 Ϫ/Ϫ fetal liver cells consistently induces a JMML-like MPD in irradiated recipient mice. 16 Nf1 inactivation leads to deregulated growth in multiple hematopoietic compartments and confers a durable proliferative advantage i...

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