Kinuko Mitani

AML-1 is required for megakaryocytic maturation and lymphocytic differentiation, but not for maintenance of hematopoietic stem cells in adult hematopoiesis

Embryonic development of multilineage hematopoiesis requires the precisely regulated expression of lineage-specific transcription factors, including AML-1 (encoded by Runx1; also known as CBFA-2 or PEBP-2αB). In vitro studies and findings in human diseases, including leukemias, myelodysplastic syndromes and familial platelet disorder with predisposition to acute myeloid leukemia (AML), suggest that AML-1 has a pivotal role in adult hematopoiesis. However, this role has not been fully uncovered in vivo because of the embryonic lethality of Runx1 knockout in mice. Here we assess the requirement of AML-1/Runx1 in adult hematopoiesis using an inducible gene-targeting method. In the absence of AML-1, hematopoietic progenitors were fully maintained with normal myeloid cell development. However, AML-1-deficient bone marrow showed inhibition of megakaryocytic maturation, increased hematopoietic progenitor cells and defective T- and B-lymphocyte development. AML-1 is thus required for maturation of megakaryocytes and differentiation of T and B cells, but not for maintenance of hematopoietic stem cells (HSCs) in adult hematopoiesis.

The oncoprotein Evi-1 represses TGF-|[beta]| signalling by inhibiting Smad3

Evi-1 encodes a zinc-finger protein that may be involved in leukaemic transformation of haematopoietic cells. Evi-1 has two zinc-finger domains, one with seven repeats of a zinc-finger motif and one with three repeats, and it has characteristics of a transcriptional regulator,. Although Evi-1 is thought to be able to promote growth and to block differentiation in some cell types, its biological functions are poorly understood. Here we study the mechanisms that underlie oncogenesis induced by Evi-1 by investigating whether Evi-1 perturbs signalling through transforming growth factor-β (TGF-β), one of the most studied growth-regulatory factors, which inhibits proliferation of a wide range of cell types. We show that Evi-1 represses TGF-β signalling and antagonizes the growth-inhibitory effects of TGF-β. Two separate regions of Evi-1 are responsible for this repression; one of these regions is the first zinc-finger domain. Through this domain, Evi-1 interacts with Smad3, an intracellular mediator of TGF-β signalling, thereby suppressing the transcriptional activity of Smad3. These results define a new function of Evi-1 as a repressor of signalling through TGF-β.

Generation of the AML1-EVI-1 fusion gene in the t(3;21)(q26;q22) causes blastic crisis in chronic myelocytic leukemia.

The t(3;21)(q26;q22) translocation, which is one of the consistent chromosomal abnormalities found in blastic crisis of chronic myelocytic leukemia (CML), is thought to play an important role in the leukemic progression of CML to an acute blastic crisis phase. The AML1 gene, which is located at the translocation breakpoint of the t(8;21)(q22;q22) translocation found in acute myelocytic leukemia, was also rearranged by the t(3;21)(q26;q22) translocation. Screening of a cDNA library of the t(3;21)‐carrying leukemic cell line cells (SKH1) resulted in the isolation of two potentially complete AML1‐EVI‐1 chimeric cDNAs of 6 kb. Two species of AML1‐EVI‐1 fusion transcripts of 8.2 and 7.0 kb were detected in SKH1 cells. These cells expressed the 180 kDa AML1‐EVI‐1 fusion protein containing an N‐terminal half of AML1 including a runt homology domain which is fused to the entire zinc finger EVI‐1 protein. The AML1‐EVI‐1 fusion transcript was consistent in all three cases of the t(3;21)‐carrying leukemia examined by RNA‐based PCR. These findings strongly suggest that the t(3;21) translocation results in the formation of a new class of chimeric transcription factor which could contribute to the leukemic progression of CML through interference with cell growth and differentiation.

An acute myeloid leukemia gene, AML1, regulates hemopoietic myeloid cell differentiation and transcriptional activation antagonistically by two alternative spliced forms.

The AML1 gene on chromosome 21 is disrupted in the (8;21)(q22;q22) and (3;21)(q26;q22) translocations associated with myelogenous leukemias and encodes a DNA binding protein. From the AML1 gene, two representative forms of proteins, AML1a and AML1b, are produced by alternative splicing. Both forms have a DNA binding domain but, unlike AML1b, AML1a lacks a putative transcriptional activation domain. Here we demonstrate that overexpressed AML1a totally suppresses granulocytic differentiation and stimulates cell proliferation in 32Dcl3 murine myeloid cells treated with granulocyte colony‐stimulating factor. These effects of AML1a were canceled by the concomitant overexpression of AML1b. Such biological phenomena could be explained by our observations that (i) AML1a, which on its own has no effects as a transcriptional regulator, dominantly suppresses transcriptional activation by AML1b, and (ii) AML1a exhibits the higher affinity for DNA binding compared with AML1b. These antagonistic actions could be important in leukemogenesis and/or myeloid cell differentiation because more than half of myelogenous leukemia patients showed an increase in the relative amounts of AML1a.

The extracellular signal-regulated kinase pathway phosphorylates AML1, an acute myeloid leukemia gene product, and potentially regulates its transactivation ability.

AML1 (also called PEBP2alphaB, CBFA2, or CBFalpha2) is one of the most frequently disrupted genes in chromosome abnormalities seen in human leukemias. It has been reported that AML1 plays several pivotal roles in myeloid hematopoietic differentiation and other biological phenomena, probably through the transcriptional regulation of various relevant genes. Here, we investigated the mechanism of regulation of AML1 functions through signal transduction pathways. The results showed that AML1 is phosphorylated in vivo on two serine residues within the proline-, serine-, and threonine-rich region, with dependence on the activation of extracellular signal-regulated kinase (ERK) and with interleukin-3 stimulation in a hematopoietic cell line. These in vivo phosphorylation sites of AML1 were phosphorylated directly in vitro by ERK. Although differences between wild-type AML1 and phosphorylation site mutants in DNA-binding affinity were not observed, we have shown that ERK-dependent phosphorylation potentiates the transactivation ability of AML1. Furthermore the phosphorylation site mutations reduced the transforming capacity of AML1 in fibroblast cells. These data indicate that AML1 functions are potentially regulated by ERK, which is activated by cytokine and growth factor stimuli. This study provides some important clues for clarifying unidentified facets of the regulatory mechanism of AML1 function.

Acetylation of GATA‐3 affects T‐cell survival and homing to secondary lymphoid organs

Acetylation of a transcription factor has recently been shown to play a significant role in gene regulation. Here we show that GATA‐3 is acetylated in T cells and that a mutation introduced into amino acids 305–307 (KRR‐GATA3) creates local hypoacetylation in GATA‐3. Remarkably, KRR‐GATA3 possesses the most potent suppressive effect when compared with other mutants that are disrupted in putative acetylation targets. Expressing this mutant in peripheral T cells results in defective T‐cell homing to systemic lymphnodes, and prolonged T‐cell survival after activation. These findings have significant implications in that the acetylation state of GATA‐3 affects its physiological function in the immune system and, more importantly, provides evidence for the novel role of GATA‐3 in T‐cell survival and homing to secondary lymphoid organs.

Dual functions of the AML1/Evi-1 chimeric protein in the mechanism of leukemogenesis in t(3;21) leukemias.

The chromosomal translocation t(3;21)(q26;q22), which is found in blastic crisis in chronic myelogenous leukemias and myelodysplastic syndrome-derived leukemias, produces AML1/Evi-1 chimeric transcription factor and is thought to play important roles in acute leukemic transformation of hemopoietic stem cells. We report here the functional analyses of AML1/Evi-1. It was revealed that AML1/Evi-1 itself does not alter the transactivation level through mouse polyomavirus enhancer-binding protein 2 (PEBP2; PEA2) sites (binding site of AML1) but dominantly suppresses the transactivation by intact AML1, which is assumed to be a stimulator of myeloid cell differentiation. DNA-binding competition is a putative mechanism of such dominant negative effects of AML1/Evi-1 because it binds to PEBP2 sites with higher affinity than AML1 does. Furthermore, AML1/Evi-1 stimulated c-fos promoter transactivation and increased AP-1 activity, as Evi-1 (which is not normally expressed in hemopoietic cells) did. Experiments using deletion mutants of AML1/Evi-1 showed that these two functions are mutually independent because the dominant negative effects on intact AML1 and the stimulation of AP-1 activity are dependent on the runt domain (DNA-binding domain of AML1) and the zinc finger domain near the C terminus, respectively. Furthermore, we showed that AML1/Evi-1 blocks granulocytic differentiation, otherwise induced by granulocyte colony-stimulating factor, of 32Dcl3 myeloid cells. It was also suggested that both AML1-derived and Evi-1-derived portions of the fusion protein play crucial roles in this differentiation block. We conclude that the leukemic cell transformation in t(3;21) leukemias is probably caused by these dual functions of AML1/Evi-1 chimeric protein.

The evi-1 oncoprotein inhibits c-Jun N-terminal kinase and prevents stress-induced cell death

Evi‐1 encodes a nuclear protein involved in leukemic transformation of hematopoietic cells. Evi‐1 possesses two sets of zinc finger motifs separated into two domains, and its characteristics as a transcriptional regulator have been described. Here we show that Evi‐1 acts as an inhibitor of c‐Jun N‐terminal kinase (JNK), a class of mitogen‐activated protein kinases implicated in stress responses of cells. Evi‐1 physically interacts with JNK, although it does not affect its phosphorylation. This interaction is required for inhibition of JNK. Evi‐1 protects cells from stress‐induced cell death with dependence on the ability to inhibit JNK. These results reveal a novel function of Evi‐1, which provides evidence for inhibition of JNK by a nuclear oncogene product. Evi‐1 blocks cell death by selectively inhibiting JNK, thereby contributing to oncogenic transformation of cells.

Characterization of stage progression in chronic myeloid leukemia by DNA microarray with purified hematopoietic stem cells.

Chronic myeloid leukemia (CML) is characterized by the clonal expansion of hematopoietic stem cells (HSCs). Without effective treatment, individuals in the indolent, chronic phase (CP) of CML undergo blast crisis (BC), the prognosis for which is poor. It is therefore important to clarify the mechanism underlying stage progression in CML. DNA microarray is a versatile tool for such a purpose. However, simple comparison of bone marrow mononuclear cells from individuals at different disease stages is likely to result in the identification of pseudo-positive genes whose change in expression only reflects the different proportions of leukemic blasts in bone marrow. We have therefore compared with DNA microarray the expression profiles of 3456 genes in the purified HSC-like fractions that had been isolated from 13 CML patients and healthy volunteers. Interestingly, expression of the gene for PIASy, a potential inhibitor of STAT (signal transducer and activator of transcription) proteins, was down-regulated in association with stage progression in CML. Furthermore, forced expression of PIASy has induced apoptosis in a CML cell line. These data suggest that microarray analysis with background-matched samples is an efficient approach to identify molecular events underlying the stage progression in CML.

Antisense repression of proto-oncogene c-Cbl enhances activation of the JAK-STAT pathway but not the ras pathway in epidermal growth factor receptor signaling.

Many growth factors including epidermal growth factor (EGF) induce tyrosine phosphorylation of the c-Cbl proto-oncogene product, whose function, however, remains unclear. Recently, Sli-1, a Caenorhabditis elegans homologue of c-Cbl, was found to be a negative regulator of let-23-mediated vulval induction pathway, suggesting that c-Cbl may negatively regulate EGF receptor (EGFR)-mediated signaling. In this study, by an antisense RNA approach, we examined the effects of expression level of c-Cbl on EGFR signaling and showed that overexpression of c-Cbl reduces and antisense repression of c-Cbl enhances autophosphorylation of EGF receptors and activation of the JAK-STAT pathway. However, in contrast to the Sli-1 protein, the expressed amount of c-Cbl does not affect activation of the Ras pathway, suggesting that the EGFR-mediated signaling pathways are differently regulated by c-Cbl among nematodes and mammals.