Yutaka Okuno

Acute myeloid leukemia induced by graded reduction of a lineage-specific transcription factor, PU.1

Transcription factors are believed to have a dominant role in acute myeloid leukemia (AML). This idea is supported by analysis of gene-knockout mice, which uncovered crucial roles of several transcription factors in normal hematopoiesis, and of individuals with leukemia, in whom transcription factors are frequently downregulated or mutated. However, analysis of knockout animals has not shown a direct link between abrogated transcription factors and the pathogenesis of AML. Sfpi1, encoding the lineage-specific transcription factor PU.1, is indispensable for normal myeloid and lymphoid development. We found that mice carrying hypomorphic Sfpi1 alleles that reduce PU.1 expression to 20% of normal levels, unlike mice carrying homo- or heterozygous deletions of Sfpi1, developed AML. Unlike complete or 50% loss, 80% loss of PU.1 induced a precancerous state characterized by accumulation of an abnormal precursor pool retaining responsiveness to G-CSF with disruption of M- and GM-CSF pathways. Malignant transformation was associated with a high frequency of clonal chromosomal changes. Retroviral restoration of PU.1 expression rescued myeloid differentiation of mutant progenitors and AML blasts. These results suggest that tightly graded reduction, rather than complete loss, of a lineage-indispensable transcription factor can induce AML.

PU.1 is a major downstream target of AML1 (RUNX1) in adult mouse hematopoiesis

Both PU.1 (also called SFPI1), an Ets-family transcription factor, and AML1 (also called RUNX1), a DNA-binding subunit of the CBF transcription factor family, are crucial for the generation of all hematopoietic lineages, and both act as tumor suppressors in leukemia. An upstream regulatory element (URE) of PU.1 has both enhancer and repressor activity and tightly regulates PU.1 expression. Here we show that AML1 binds to functionally important sites within the PU.1 upstream regulatory element and regulates PU.1 expression at both embryonic and adult stages of development. Analysis of mice carrying conditional AML1 knockout alleles and knock-in mice carrying mutations in all three AML1 sites of the URE proximal region demonstrated that AML1 regulates PU.1 both positively and negatively in a lineage dependent manner. Dysregulation of PU.1 expression contributed to each of the phenotypes observed in these mice, and restoration of proper PU.1 expression rescued or partially rescued each phenotype. Thus, our data demonstrate that PU.1 is a major downstream target gene of AML1.

Potential Autoregulation of Transcription Factor PU.1 by an Upstream Regulatory Element

ABSTRACT Regulation of the hematopoietic transcription factor PU.1 (Spi-1) plays a critical role in the development of white cells, and abnormal expression of PU.1 can lead to leukemia. We previously reported that the PU.1 promoter cannot induce expression of a reporter gene in vivo, and cell-type-specific expression of PU.1 in stable lines was conferred by a 3.4-kb DNA fragment including a DNase I hypersensitive site located 14 kb upstream of the transcription start site. Here we demonstrate that this kb −14 site confers lineage-specific reporter gene expression in vivo. This kb −14 upstream regulatory element contains two 300-bp regions which are highly conserved in five mammalian species. In Friend virus-induced erythroleukemia, the spleen focus-forming virus integrates into the PU.1 locus between these two conserved regions. DNA binding experiments demonstrated that PU.1 itself and Elf-1 bind to a highly conserved site within the proximal homologous region in vivo. A mutation of this site abolishing binding of PU.1 and Elf-1 led to a marked decrease in the ability of this upstream element to direct activity of reporter gene in myelomonocytic cell lines. These data suggest that a potential positive autoregulatory loop mediated through an upstream regulatory element is essential for proper PU.1 gene expression.

Differential regulation of the human and murine CD34 genes in hematopoietic stem cells.

Human CD34 (hCD34)-positive cells are used currently as a source for hematopoietic transplantation in humans. However, in steady-state murine hematopoiesis, hematopoietic stem cells (HSCs) with long-term reconstitution activity are found almost exclusively in the murine CD34 (mCD34)-negative to low fraction. To evaluate the possible differences in hCD34 and mCD34 gene expression in hematopoiesis, we made transgenic mouse strains with human genomic P1 artificial chromosome clones spanning the entire hCD34 genomic locus. In all transgenic mouse strains, a vast majority of phenotypic and functional HSC populations including mCD34−/lo express the hCD34 transgene. These data strongly support the notion that hCD34+ human bone marrow cells contain long-term HSCs that can maintain hematopoiesis throughout life.

Activation of the endoplasmic reticulum stress pathway is associated with survival of myeloma cells.

The endoplasmic reticulum (ER) is an organelle in which proteins are modified. When unfolded proteins accumulate in the ER under various stresses, ER stress (ERS) pathways, including the induction of chaperones, are activated to protect the cell. However, when ERS is excessive, the cell undergoes apoptosis. This study investigated ERS in multiple myeloma cells (MMCs) because they contain a well-developed ER due to M-protein production. The myeloma cell line 12-PE underwent apoptosis via caspase-3 after treatment with thapsigargin (thap), an ERS inducer, while another cell line, U266, did not. To understand the mechanism regulating this heterogeneity, the induction of chaperones by thap was analysed. Chaperones were up-regulated in U266 cells but down-regulated in 12-PE cells, suggesting that chaperones contribute to cell survival under ERS. Analysis of XBP-1, a transcriptional inducer of chaperones, in freshly isolated MMCs from 22 myeloma cases revealed 10 cases with active XBP-1, who also showed significantly poorer survival (p < 0.05), suggesting that chaperone expression protects MMCs from apoptosis, thereby allowing tumor cell expansion. These results suggest that MMCs are subjected to ERS under certain circumstances and that chaperones are induced to protect the cells against such ERS. Inhibition of chaperones could be a new target for myeloma therapy.

Down-regulation of PU.1 by methylation of distal regulatory elements and the promoter is required for myeloma cell growth.

The transcription factor PU.1 is essential for myeloid and B-cell development. Down-regulation of PU.1 by disruption of its 14-kb 5 upstream regulatory element induced acute myeloid leukemia, T-cell lymphoma, and chronic lymphocytic leukemia-like disease in murine models. In the present study, we found that PU.1 was down-regulated in the majority of human myeloma cell lines and a subset of freshly isolated myeloma cells, in contrast to relatively high expression of PU.1 in normal plasma cells. Patients in this low PU.1 expression subset may have a poor prognosis. In human myeloma cell lines, the 17-kb 5 upstream enhancer and the promoter region of the PU.1 gene were highly methylated, and this is consistent with disappearance of DNase I-hypersensitive sites in these regions. To elucidate the significance of down-regulation of PU.1, we generated stable myeloma cell lines with an inducible PU.1 expression system. Exogenous expression of PU.1 in PU.1 null myeloma cell lines, U266 and KMS12PE, induced complete growth arrest and cell death. Up-regulation of PU.1 by 5-aza-2-deoxycytidine also induced growth arrest of KMS12PE and KHM11 myeloma cells. These data suggest that down-regulation of PU.1 is an essential step for the survival of a subset of myeloma cells and that up-regulation of PU.1 by demethylation agents or other types of agents may represent a new therapeutic strategy for treatment of multiple myeloma patients.

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 30u2009bp 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.