Retroviral integration at the Epi1 locus cooperates with Nf1 gene loss in the progression to acute myeloid leukemia

Abstract

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.

FIG. 1

The Epi1 locus. (A) Southern blot analysis showing rearrangements of the Epi1 locus in two independent tumors. Normal DNA (N) and tumor DNAs were digested with BglII and hybridized with probe C from the Epi1 locus. (B) Map of the Epi1 region relative to the Myb gene. The large line represents the region 3′ of the Myb gene. The small hash marks represent BamHI sites in the region, the large box is exon 15 of the Myb gene, the large arrow shows the direction of transcription of Myb, and the small boxes above the line are the probes isolated from the region. Probe C is the Ahi1 probe (18). (C) Location and orientation of 13 proviruses integrated in Epi1 relative to the Myb gene. The small arrows above the line show the location and orientation of the viral integrations in the Epi1 locus.

FIG. 2

Epi1 maps in the proximal region of mouse chromosome 10. Epi1 was placed on mouse chromosome 10 by interspecific backcross analysis. The segregation patterns of Epi1 and flanking genes in 128 backcross animals that were typed for all loci are shown at the top of the figure. For individual pairs of loci, more than 128 animals were typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J × M. spretus)F₁ parent. The black boxes represent the presence of a C57BL/6J allele, and white boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 10 linkage map showing the location of Epi1 in relation to linked genes is shown at the bottom of the figure. Recombination distances between loci in centimorgans are shown to the left of the chromosome, and the positions of loci in human chromosomes, where known, are shown to the right. References for the human map positions of loci cited in this study can be obtained from the Genome Data Base, a computerized database of human linkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore, Md.).

FIG. 3

Northern blot analysis of Myb expression in Epi1 and non-Epi1 tumors. RNA derived from four tumors containing somatic viral insertion at Epi1 (+) and four tumors without this viral insertion (−) were analyzed by Northern blotting. The top panel shows the hybridization with a Myb cDNA probe, and the bottom panel shows the same blot stripped and rehybridized with a control probe for Gapd.

FIG. 4

Leukemia morphology. (A) Bone marrow histology showing immature cells and numerous pseudo-Gaucher histiocytes. Magnification, ×250. (B) Spleen histology showing expanded red pulp with leukemic cells accompanied by large megakaryocytes and few small dark-staining erythroid precursors. Residual white pulp is visible at lower right. Magnification, ×100. (C) Liver histology showing massive infiltration of leukemic cells. Magnification, ×100. H&E stain was used for panels A to C. (D) Cytology (Wright's Giemsa stain) of leukemic cells prepared from lymph node. Magnification, ×500.

FIG. 5

Flow cytometric analysis of a representative leukemia. Data in panel CD45/SSC are ungated; for those in the remaining panels, the gate was set at R1.