PU.1-Induced IRF4 Down-Regulation and Subsequent IRF7 up-Regulation in Myeloma Cells

PU.1 is an Ets family transcription factor, which is necessary for differentiation of both myeloid and lymphoid lineages. It was previously reported that conditional knockout of the upstream regulatory element (URE) located in 14 kb 5' of the PU.1 gene resulted in down-regulation of PU.1 expression in granulocytes and B lymphocytes by 80% compared to that of wild type and induced acute myeloid leukemia and CLL-like diseases in mice. Since the URE contains a suppressor region for PU.1 expression in T cells, such mice express PU.1 in T cells and develop T cell lymphoma. Thus, the failure of proper expression of PU.1 in certain differentiation stages in certain cell lineages appears to result in hematological malignancies. We previously reported that PU.1 is down-regulated in various myeloma cell lines. In addition, PU.1 is expressed in normal plasma cells and PU.1 is down-regulated in myeloma cells of certain myeloma patients, who appear to have poor prognosis. In those myeloma cell lines, the promoter and URE of the PU.1 gene are highly methylated. A demethylation agent, 5-aza-2'-deoxycytidine, induced PU.1 up-regulation, growth arrest, and apoptosis in myeloma cell lines, KMS12PE and KHM11. In addition, conditionally expressed PU.1 induced cell growth arrest and apoptosis in PU.1-low-negative myeloma cell lines, U266 and KMS12PE, suggesting that PU.1 is a tumor suppressor for myeloma cells. To elucidate the mechanisms of the cell growth arrest and apoptosis in myeloma cells induced by PU.1, we performed DNA microarray analysis to compare gene expression levels before and after PU.1 expression. Among cell-cycle related genes, p21^WAF1/CIP1 was found up-regulated in U266 cells, while among apoptosis related genes, TRAIL was highly up-regulated in both U266 and KMS12PE cell lines. With further investigation, we concluded that PU.1 directly transactivated the TRAIL gene in myeloma cells, leading to apoptosis.

Based on the DNA microarray data generated, we found that IRF4 is downregulated in U266 myeloma cells after PU.1 induction. It has been reported that knockdown of IRF4 induces apoptosis in myeloma cell lines. Therefore, we examined whether IRF4 was down-regulated in three myeloma cell lines, U266, KMS12PE, and KHM11 following PU.1 induction. Conditional expression of PU.1 by tet-off system induced IRF4 down-regulation in U266and KMS12PE cells. With lentiviral transduction method, ectopic expression of PU.1 also induced IRF4 down-regulation, cell-cycle arrest, and apoptosis in KHM11 cells. To investigate the role of IRF4 in PU.1-expressing U266 cells, we stably expressed IRF4, partially rescuing U266 cells from apoptosis. IRF4 is known to directly bind to the IRF7 promoter and down-regulate IRF7 expression in activated B cell-like (ABC) subtype of diffuse large B-cell lymphoma cells. Therefore, we examined whether IRF4 bound to the IRF7 promoter in KMS12PEand U266cells using chromatin immunoprecipitation assays. We found that IRF4 directly bound to the IRF7 promoter in both myeloma cell lines. When we overexpressed PU.1, IRF4 levels were decreased and the IRF4 binding to the IRF7 promoter was significantly reduced in those cell lines. Moreover, knockdown of IRF7 significantly rescued PU.1-expressing U266cells from apoptosis. These data strongly suggest that PU.1-induced apoptosis is associated with IRF4 down-regulation and subsequent IRF7 up-regulation in myeloma cells. Since IRF4 is essential transcription factor for myeloma cell survival, up-regulation of PU.1 by demethylation agents, including 5-aza-2'-deoxycytidine may serve as a promising therapeutic modality of multiple myeloma by inducing down-regulation of IRF4.