PU.1 acts as tumor suppressor for myeloma cells through direct transcriptional repression of IRF4.

We previously reported that PU.1 is downregulated in the majority of myeloma cell lines and primary myeloma cells of certain myeloma patients, and conditional expression of PU.1 in such myeloma cell lines induced cell cycle arrest and apoptosis. We found downregulation of IRF4 protein in the U266 myeloma cell line following induction of PU.1. Previous studies reported that knockdown of IRF4 in myeloma cell lines induces apoptosis, prompting us to further investigate the role of IRF4 downregulation in PU.1-induced cell cycle arrest and apoptosis in myeloma cells. PU.1 induced downregulation of IRF4 at the protein level, cell cycle arrest and apoptosis in six myeloma cell lines. Chromatin immunoprecipitation (ChIP) revealed that PU.1 directly binds to the IRF4 promoter, whereas a reporter assay showed that PU.1 may suppress IRF4 promoter activity. Stable expression of IRF4 in myeloma cells expressing PU.1 partially rescued the cells from apoptosis induced by PU.1. As it was reported that IRF4 directly binds to the IRF7 promoter and downregulates its expression in activated B cell-like subtype of diffuse large B cell lymphoma cells, we performed ChIP assays and found that IRF4 directly binds the IRF7 promoter in myeloma cells. It is known that IRF7 positively upregulates interferon-ß (IFNß) and induces apoptosis in many cell types. Binding of IRF4 to the IRF7 promoter decreased following PU.1 induction, accompanied by downregulation of IRF4 protein expression. Knockdown of IRF7 protected PU.1-expressing myeloma cells from apoptosis. Furthermore, IFNß, which is a downstream target of IRF7, was upregulated in myeloma cells along with IRF7 after PU.1 induction. Finally, we evaluated the mRNA expression levels of PU.1, IRF4 and IRF7 in primary myeloma cells from patients and found that PU.1 and IRF7 were strongly downregulated in contrast to the high expression levels of IRF4. These data strongly suggest that PU.1-induced apoptosis in myeloma cells is associated with IRF4 downregulation and subsequent IRF7 upregulation.

Leukemic survival factor SALL4 contributes to defective DNA damage repair.

SALL4 is aberrantly expressed in human myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). We have generated a SALL4 transgenic (SALL4B Tg) mouse model with pre-leukemic MDS-like symptoms that transform to AML over time. This makes our mouse model applicable for studying human MDS/AML diseases. Characterization of the leukemic initiation population in this model leads to the discovery that Fancl (Fanconi anemia, complementation group L) is downregulated in SALL4B Tg leukemic and pre-leukemic cells. Similar to the reported Fanconi anemia (FA) mouse model, chromosomal instability with radial changes can be detected in pre-leukemic SALL4B Tg bone marrow (BM) cells after DNA damage challenge. Results from additional studies using DNA damage repair reporter assays support a role of SALL4 in inhibiting the homologous recombination pathway. Intriguingly, unlike the FA mouse model, after DNA damage challenge, SALL4B Tg BM cells can survive and generate hematopoietic colonies. We further elucidated that the mechanism by which SALL4 promotes cell survival is through Bcl2 activation. Overall, our studies demonstrate for the first time that SALL4 has a negative impact in DNA damage repair, and support the model of dual functional properties of SALL4 in leukemogenesis through inhibiting DNA damage repair and promoting cell survival.

PU.1 induces apoptosis in myeloma cells through direct transactivation of TRAIL.

We earlier reported that PU.1 was downregulated in myeloma cell lines and myeloma cells in a subset of myeloma patients, and that conditional PU.1 expression in PU.1-negative myeloma cell lines, U266 and KMS12PE, induced growth arrest and apoptosis. To elucidate the molecular mechanisms of the growth arrest and apoptosis, we performed DNA microarray analyses to compare the difference in gene expression before and after PU.1 induction in U266 cells. Among cell cycle-related genes, cyclin A2, cyclin B1, CDK2 and CDK4 were downregulated and p21 was upregulated, although among apoptosis-related genes, tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) was found highly upregulated. When TRAIL was knocked down by small interference RNAs, apoptosis of PU-1-expressing cells was inhibited, suggesting that TRAIL has a critical role in PU.1-induced apoptosis in both U266 and KMS12PE myeloma cells. In both U266 and KMS12PE cells expressing PU.1, PU.1 directly bound to a region 30 bp downstream of the transcription start site of the TRAIL gene. Upregulation of PU.1-induced transactivation of the TRAIL promoter in reporter assays, and disruption of the PU.1-binding site in the TRAIL promoter eliminated this transactivation. Therefore, we conclude that PU.1 is capable of inducing apoptosis in certain myeloma cells by direct transactivation of TRAIL.