Abstract

The ecotropic viral integration site-1 (Evi-1) gene was first identified as a common locus of retroviral integration in murine leukemia. In humans, Evi-1 is located on chromosome 3q26, and rearrangements on chrmosome 3q26 often activate Evi-1 expression in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Patients with these karyotypes are characterized by the elevated platelet count and lack of response to antileukemic therapy. Elevated Evi-1 expression occurs with high frequency in AML patients without 3q26 abnormalities, and is also associated with unfavorable outcomes. Thus, Evi-1 is one of the key factors that predict poor survival in leukemia patients. Evi-1 is a member of the SET/PR domain family of transcription factors and it contains a total of 10 zinc finger motifs organized in two discrete domains, comprising 7 and 3 repeats respectively, which have distinct DNA binding specificities. Recently, we generated Evi-1-mutant mice and showed that Evi-1 plays an essential role in proliferation of both hematopoietic stem cells (HSCs) and transformed leukemic cells. Furthermore, we identified candidate target genes of Evi-1 using gene expression profiling analysis in HSCs combined with the gene expression data of AML samples. These genes include Gata1, Gata2, Angpt1, Mpl, Jag2, Pbx-1, Setbp1 and Itga2b. In this study, we first analyzed relative gene expression of these candidate genes in control- or Evi-1-transduced hematopoietic stem/progenitors (c-Kit+ cells). Among these candidate genes, Evi-1 up-regulates transcription of Pbx-1, Mpl, Setbp1 and Itga2b. Next, we cloned 5 ′ up-stream genomic regions of these four genes into the pGL-4 luciferase reporter vector, and found that Evi-1 increased luciferase activity of Pbx-1 reporter construct in COS7 cells. Deletion of reporter constructs revealed that Evi-1 binds to -0.5kb upstream of the transcription start site of Pbx-1. We then examined the transcription activity of a series of Evi-1 mutants and found that both the first and second zinc finger domains are required for the Pbx-1 up-regulation. Furthermore, chromatin immunoprecipitation (ChIP) analysis revealed that Evi-1 directly binds to the promoter region of Pbx-1. We next evaluated a role for Pbx-1 in Evi-1-induced myeloid transformation. Bone marrow progenitors transduced with Evi-1 showed sustained colony formation in the serial replating assay. After establishment of sustained clonogenic activity following more than three rounds of replating in methylcellulose medium, the cells were transduced with control or Pbx-1-shRNA. Interestingly, reduction of Pbx-1 levels through RNAi-mediated knockdown significantly inhibited Evi-1-induced transformation. In contrast, knockdown of Pbx-1 did not impair bone marrow transformation by transcription factor E2A-hepatic leukemia factor (E2A-HLF), suggesting that Pbx-1 is specifically, as opposed to generally, required for maintenance of transformation mediated by Evi-1. Taken together, these results indicate that Pbx-1 is one of the direct target genes of Evi- 1 in hematopoietic cells, and aberrant Pbx-1 expression is responsible, at least in part, for the oncogenic activity of Evi-1. Because Pbx-1 is known as a critical regulator of hematopoietic stem cells and leukemia development, the Evi-1-Pbx-1 pathway may be a key modulator of stem cell activity in normal and malignant hematopoiesis.

Disclosures: No relevant conflicts of interest to declare.

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