F-MuLV acceleration of myelomonocytic tumorigenesis in SV40 large T antigen transgenic mice is accompanied by retroviral insertion at Fli1 and a novel locus, Fim4.

We describe here the development of a murine system for the identification of genes involved in myelomonocytic neoplasms. Transgenic C57BL/6J mice expressing SV40 early region under a myelomonocytic promoter develop histiocytic sarcomas with a latency of 167 days. We used retroviral proviral tagging to accelerate tumorigenesis and to uncover genetic changes that contribute to tumor development. Infection of transgenic mice with Friend murine leukemia virus (F-MuLV) shortened the latency of morbidity to 103 days (P< 0.001); this was associated with clonal proviral integrations in tumor DNA. As expected for F-MuLV, proviral insertions occurred at Fli1 in both transgenic and nontransgenic tumors. Four insertions were found at a novel locus, termed Fim4, on chromosome 6. This region is syntenic to human 7q32, a region that is commonly deleted in human myelodysplastic syndrome and acute myeloid leukemia. A murine BAC containing Fim4 was sequenced and analyzed, and while there was significant human-mouse homology in the area of the insertions, no candidate gene has been identified. Thus we have established a system to identify genes involved in myelomonocytic tumors, and have used it to identify Fim4, a new common site of proviral insertion. Study of this locus may provide insight into genes involved in AML-associated 7q32 deletions in humans.

Retroviral insertional activation of the EVI1 oncogene does not prevent G-CSF-induced maturation of the murine pluripotent myeloid cell line 32Dcl3.

Evi1 is a myeloid-specific protooncogene that encodes 145 kDa and 88 kDa proteins via alternative splicing. Overexpression of the gene via retroviral insertion in murine tumors or chromosomal rearrangement in human tumors is associated with myeloid leukemias and myelodysplasias; however, the mechanism by which such overexpression leads to transformation is not clear. It has been postulated that overexpression of evi1 acts to block normal myelopoiesis. In attempts to assess the effect of overexpression of evi1 on myelopoiesis, we chose to utilize the IL-3-dependent murine 32Dcl3 cell line, which has been shown to differentiate in culture in response to G-CSF. Previous experiments with this cell line, which we have confirmed, showed that overexpression of evi1, mediated by retroviral vector transfer, caused a block to G-CSF-induced cell survival and differentiation. We report here that the naive 32Dcl3 cell line contains a rearrangement of the evi1 locus and constitutively overexpresses evi1 mRNA and protein; this expression is downregulated only slightly during G-CSF-induced myeloid maturation. The steady state levels, molecular weight and DNA binding characteristics of the EVI1 protein in these cells is comparable to that seen in NFS 58, a myeloid leukemia cell line with retroviral insertion at evi1. The observed ability of the murine 32Dcl3 cells to fully differentiate in the presence of G-CSF while evi1 continues to be expressed indicates that, at the levels expressed in naive 32Dcl3, evi1 does not block G-CSF-induced survival and differentiation. Thus, retroviral insertions at evi1 may have been selected for in 32Dcl3 cells due to effects other than that on G-CSF-induced cell survival.