A Mouse Model of Evi1-Related Leukemia.

Evi1 gene is located on chromosome 3q26 and aberrantly expressed in acute myeloid leukemia (AML) patients with or without 3q26 abnormalities, and inappropriate expression of Evi1 associates with poor prognosis. Evi-1 is originally identified in a common integration site of murine leukemia retrovirus and enhanced expression of Evi1 by retrovirus integration is thought to be responsible for leukemogenesis in mouse models. However, retroviral expression of marker genes such as GFP has not induced leukemia even if some clones possessing integration at Evi1 site have been identified. These data indicate that Evi1 requires cooperative factors to induce progressive leukemia, whereas overexpression of Evi1 is enough to lead to clonal expansion of hematopoietic cells. Therefore, identifying genes collaborating with Evi1 is one of the key issues of understanding Evi1-related leukemogenesis. Recently, we demonstrated that a point mutation of the transcription factor AML1 (AML1-D171N) can induce myelodysplastic syndrome (MDS) that progresses to AML in association with overexpression of Evi1 through a mouse bone marrow transplantation model. In that work, we analyzed mice transplanted with BM cells transduced Evi1 alone as control and surprisingly confirmed that all of the mice developed leukemia within 6-11 months after the transplant. In this report, we will describe interesting findings in the novel mouse model of Evi1-induced leukemia. Result: C57BL6/Ly-5.1 murine BM cells infected with retroviruses harboring Evi1 were transplanted into irradiated syngeneic Ly-5.2 mice. The mice looked fine until 5 months, but GFP-positive-Evi1 expressing cells were gradually increased in the peripheral blood (PB), and then the mice died at 6-11 months after the transplantation. The mice showed dysplastic features in myeloid and erythroid cells, increase of blasts in the PB and the BM, hepatosplenomegaly, slight anemia, and some of the mice showed severe leukocytosis. The mice were thought to die of multiple organ failure due to invasion of leukemic cells not due to anemia. The phenotype is different from that of the mouse BMT model expressing Evi1 by retrovirus reported by another group, in which the mice died about 10 months with severe peripheral cytopenia and finally the disease did not progress to AML. Therefore, we considered that Evi1 might have collaborated with unknown genes near retrovirus integration sites in our case and analyzed integration sites by the bubble PCR method. Interestingly, frequent integration at 3' side of C/EBPb gene was found in six mice out of eight mice transplanted with Evi1-transduced BM cells. The integrations were located at 62.5-86.7kb downstream of C/EBPb gene. Next, we examined the expression level of C/EBPb, Tmem189, and Ptpn1, all of which are located near the integration site, and confirmed that C/EBPb showed elevated expression although neither Tmem189 nor Ptpn1 did. We also identified Bcas1, Rps6ka1, and Rapgef4 genes at the retroviral integration site in the other two mice without integration near C/EBPb. Discussion: C/EBPb, also known as NF-IL6, is a transcription factor that specifically binds to an IL1-responsive element in the IL-6 gene and has a role in regulation not only for the IL-6 gene but also for several cytokine genes such as TNF, IL-8, and G-CSF. The hematopoietic progenitor cells of C/EBPb-deficient mice have been reported to respond imperfectly to GM-CSF and G-CSF. Furthermore, C/EBPb is a downstream target of the Ras-Raf pathway. The locus of C/EBPb gene has been reported as a common integration site in the Retrovirus Tagged Cancer Gene Database (RTCGD), which is a database of retroviral insertional mutagenesis in mouse tumors. AKxD mice, Cdkn2a-KO mice, NUP98/HOXD13 transgenic mice, and MYC/Runx2 transgenic mice were reported to develop myeloid or lymphoid leukemia by retroviral insertion into 3' side of C/EBPb gene. In this study, we identified frequent integration at 3' side of C/EBPb gene in Evi1-transduced leukemic cells, whereas we have not identified this locus in AML1-mutants-transduced leukemic cells. Based on these findings and our results, C/EBPb is supposed to be a candidate gene to collaborate with Evi1 in leukemogenesis. Conclusion: We identified involvement of C/EBPb in Evi1-induced leukemogenesis. The novel mouse model that we generated in this study could help understanding the molecular basis of Evi1-related leukemia.

No relevant conflicts of interest to declare.