Juvenile myelomonocytic leukemia (JMML) is a disease that occurs in young children and is associated with a high mortality rate. In most patients, JMML has a progressive course leading to death by virtue of infection, bleeding, or progression to acute myeloid leukemia (AML). As it is known that children with neurofibromatosis type 1 syndrome have a markedly increased risk of developing JMML, we have previously developed a mouse model of JMML through reconstitution of lethally irradiated mice with hematopoietic stem cells homozygous for a loss-of-function mutation in the Nf1 gene (D. L. Largaespada, C. I. Brannan, N. A. Jenkins, and N. G. Copeland, Nat. Genet. 12:137-143, 1996). In the course of these experiments, we found that all these genetically identical reconstituted mice developed a JMML-like disorder, but only a subset went on to develop more acute disease. This result strongly suggests that additional genetic lesions are responsible for disease progression to AML. Here, we describe the production of a unique tumor panel, created using the BXH-2 genetic background, for identification of these additional genetic lesions. Using this tumor panel, we have identified a locus, Epi1, which maps 30 to 40 kb downstream of the Myb gene and appears to be the most common site of somatic viral integration in BXH-2 mice. Our findings suggest that proviral integrations at Epi1 cooperate with loss of Nf1 to cause AML.Juvenile myelomonocytic leukemia (JMML) is a disease characterized by a young age of onset, a tendency to affect boys, prominent enlargement of the liver and spleen, leukocytosis, and the absence of the Philadelphia chromosome. JMML has a poor prognosis, with either progression to acute myeloid leukemia (AML) or death from bleeding or infection (36). It has been estimated that at least 10% of children with JMML also have neurofibromatosis type 1 (NF1) syndrome, an autosomal dominant disorder found in 1/3500 individuals (1,7,14,32). However, the actual frequency of children with NF1 and JMML is likely higher than 10% as the peak incidence of childhood leukemia occurs at an age when NF1 often goes undiagnosed (13, 34). In fact, one study found that 15% of JMML patients had mutations in the NF1 gene even though there was no previous clinical diagnosis of NF1 (33), suggesting that approximately 25% of JMML cases are associated with NF1.While RAS gene point mutations are commonly found in JMML patients without NF1, they are not found in JMML patients with NF1 (20), providing genetic evidence that NF1 and RAS are involved in the same pathway. This idea is supported by the fact that neurofibromin, the protein product of NF1, contains a region that has extensive homology with the catalytic domain of GTPase activating proteins that are known to accelerate the intrinsic GTPase activity of Ras, thereby negatively regulating Ras GTP levels (15). This suggests that inactivating mutations in NF1 are equivalent to activating mutations in RAS. Consistent with the hypothesis, analysis of bone marrow taken from children with ...