EVI1 Blocks Apoptosis in DA-1 Myeloid Leukemia Cells Via Enhanced Transcription of the Prosurvival Gene Bcl2a1 (A1)

Abstract

Retroviral insertion at the Evi1 locus causes high level of expression of the gene in myeloid neoplasms in mice and in nonmalignant expansions of myeloid cells in humans and monkeys. In these settings, it is suggested that EVI1 confers a survival advantage on myeloid cells. Here, we investigate the survival phenotype in DA-1 cells, a leukemic cell line with provirally activated Evi1. We report that short hairpin-mediated suppression of Evi1 in DA-1 cells induces apoptosis via the intrinsic/mitochondrial pathway: DNA fragmentation and histone release are both induced, as is reduction in mitochondrial membrane potential. In addition, procasapses 3 and 9, but not caspase 8 or Bid, are cleaved following Evi1 knockdown; phosphoAKT remains unchanged. Furthermore, mRNA expression profiling following Evi1 suppression show transcriptional changes in several apoptotic regulators, including a 3.5-fold decrease in Bcl2a1 (bfl/A1), a prosurvival member of the Bcl-2 family. To assess whether EVI1 regulates Bcl2a1 expression in a cell type other than DA-1 cells, we transduced primary murine Lin-/Sca-1+/c-Kit+ cells with EVI1 via retroviral vector, and then assessed the expression of Bcl2a1. This revealed that EVI1 induced a more than five-fold increase in Bcl2a1 mRNA expression, as measured by quantitative PCR. Furthermore, transduction of primary murine bone marrow cells with retroviruses bearing either Bcl2a1 or Evi1 resulted in a significant decrease in spontaneous apoptosis, as assessed by the activity of caspases 3 and 7. To further test if Bcl2a1 is necessary for leukemic transformation by Evi1, we assessed the ability of Evi1 to confer serial replating ability on primary bone marrow cells from Bcl2a1−/− mice. Bone marrow was harvested from Bcl2a1−/− and C57BL6 mice and transduced with retrovirus containing either no gene or Evi1. While in C57BL6 mice, Evi1 induced a significant increase in colonies, bone marrow from Bcl2a1−/− mice were resistant to transformation by Evi1. To show that this was due to the lack of Bcl2a1, the gene was added back via retrovirus. While Bcl2a1 by itself did not induce significant number of colonies over vector, when introduced into Bcl2a1−/− cells together with Evi1, there was a significant increase in colony formation. These data indicate that transformation of bone marrow cells by Evi1 depends on the presence of the Bcl2a1 gene. We further show EVI1 can transcriptionally upregulate a BCL2A1::luc reporter that harbors 1.37 kb of the human BCL2A1 upstream sequence. Our analysis of the Bcl2a1 promoter indicates that the effect of EVI1 is likely indirect. Based on our findings, we propose that EVI1 acts to block apoptosis in DA-1 cells by transcriptionally activating Bcl2a1.