Masayuki Towatari

Tandem-duplicated Flt3 constitutively activates STAT5 and MAP kinase and introduces autonomous cell growth in IL-3-dependent cell lines

We have recently identified an internal tandem duplication of the human Flt3 gene in approximately 20% of acute myeloid leukemia (AML) cases. In the present study, the wild-type and the mutant Flt3 genes were transfected into two IL-3-dependent cell lines, 32D and BA/F3 cells. Mutant Flt3-transfected cells exhibited autonomous growth while wild-type Flt3-transfected cells with the continuous stimulation of Flt3 ligand exhibited a minimal proliferation. Cells expressing mutant Flt3 showed constitutive activation of STAT5 and MAP kinase. In contrast, Flt3 ligand stimulation caused rapid activation of MAP kinase but not STAT5 in cells expressing wild-type Flt3. Finally, we found constitutive activation of MAP kinase and STAT5 in all clinical samples of AML patients with mutant Flt3. Our study shows the significance of internal tandem duplication of Flt3 receptors for leukemia cell expansion.

Internal tandem duplication of the FLT3 gene is a novel modality of elongation mutation which causes constitutive activation of the product.

An internal tandem duplication (ITD) of the FLT3 gene is found in nearly 20% of acute myeloid leukemia (AML) and 5% of myelodysplastic syndrome cases. Our serial studies on 51 samples with the FLT3 gene mutation indicated that the ITD was frequently (47/51) clustered in the tyrosine-rich stretch from codon 589 to 599 and rarely (3/51) in its downstream region, both of which are located within the juxtamembrane (JM) domain. One remaining sample had an insertion into the JM domain of nucleotides of unknown origin. To eludicate the biological relevance of the ITD or the insertion, we expressed various types of mutant FLT3 in Cos 7 cells. All mutant FLT3 studied were ligand-independently dimerized and their tyrosine residues were phosphorylated. The Y589 of FLT3 was essential for the phosphorylation in the wild FLT3, but a Y589F conversion did not affect the phosphorylation status of the mutant FLT3. These findings suggest that the elongation of the JM domain rather than increase of tyrosine residues causes gain-of-function of FLT3. Thus, ITD is a novel modality of somatic mutation which activates its product. Since the DNA corresponding to codon 593 to 602 potentially forms a palindromic intermediate, we propose that a DNA-replication error might be associated with generating the ITD of the FLT3 gene.

Histone deacetylase inhibitors are the potent inducer/enhancer of differentiation in acute myeloid leukemia : a new approach to anti-leukemia therapy

We investigated the effect of the histone deacetylase inhibitors (HDIs), trichostatin A and trapoxin A on leukemia cells and cell lines from the viewpoint of differentiation induction. TSA induced differentiation in erythroid cell lines by itself, whereas it synergistically enhanced the differentiation that was directed by all-trans retinoic acid (ATRA) or vitamin D3 in U937, HL60 and NB4 cells. The combined treatment of HDI with ATRA induced differentiation in ATRA-resistant HL60 and NB4 cells. The transcriptional expression during the treatment with HDI was examined in HL60, U937 and MEG-O1. Cell cycle-regulator genes (p21waf1 and p16INK4A) were upregulated or constantly expressed, erythroid-specific genes (GATA-1, β-globin) were silent or downregulated, and housekeeping genes (β-actin and GAPDH) were constantly expressed. Twelve of 35 (34%) clinical samples from AML patients ranging from M0 to M7 also displayed both phenotypical and morphological changes by the treatment with TSA alone. HDIs are thus the potent inducer or enhancer of differentiation in acute myeloid leukemia and regulate transcription in an ordered manner.

Constitutive activation of mitogen-activated protein kinase pathway in acute leukemia cells

Mitogen-activated protein (MAP) kinase appears to be one of the key regulators of cell proliferation and differentiation. Very little, however, has been revealed as to how MAP kinase is involved in leukemogenesis. We have studied the activation of the MAP kinase pathway in 100 human primary leukemia cells including 73 acute myelogenous leukemias (AMLs). Forty acute leukemia samples (40% of the total), including 37 AML samples (51% of AML), showed activation of MAP kinase as revealed by the mobility shift of the phosphorylated form of the protein and by in vitro kinase assay. This activation was correlated with MAP kinase kinase activity in these cells. In contrast, none of 14 chronic myelogenous leukemia samples showed the activation of MAP kinase. These results suggest that the MAP kinase pathway is constitutively activated in a subset of primary acute leukemias, and thus indicate the possible role of the constitutively activated MAP kinase in leukemogenesis.

c-Myb acetylation at the carboxyl-terminal conserved domain by transcriptional co-activator p300

Transcription factor c-Myb plays important roles in cell survival and differentiation in immature hematopoietic cells. Here we demonstrate that c-Myb is acetylated at the carboxyl-terminal conserved domain by histone acetyltransferase p300 both in vitro and in vivo. The acetylation sites in vivo have been located at the lysine residues of the conserved domain (K471, K480, K485) by the use of the mutant Myb (Myb-KAmut), in which all three lysine residues are substituted into alanine. Electrophoretic mobility shift assay reveals that Myb-KAmut shows higher DNA binding activity than wild type c-Myb and that acetylation of c-Myb in vitro by p300 causes dramatic increase in DNA binding activity. Accordingly, transactivation activity of both mim-1 and CD34 promoters by Myb-KAmut is higher than that driven by wild type c-Myb. Furthermore, the bromodomain of p300, in addition to the histone acetyltransferase (HAT) domain, is required for effective acetylation of c-Myb, and hGCN5 is revealed to be a factor acetyltransferase for c-Myb in vitro. We present a new manner of post-translational modification of the c-Myb protein and the potential significance of the acetylation in c-Myb.

In vivo treatment of mutant FLT3-transformed murine leukemia with a tyrosine kinase inhibitor

Somatic mutation of the FLT3 gene, in which the juxtamembrane domain has an internal tandem duplication, is found in 20% of human acute myeloid leukemias and causes constitutive tyrosine phosphorylation of the products. In this study, we observed that the transfection of mutant FLT3 gene into an IL3-dependent murine cell line, 32D, abrogated the IL3-dependency. Subcutaneous injection of the transformed 32D cells caused leukemia in addition to subcutaneous tumors in C3H/HeJ mice. To develop a FLT3-targeted therapy, we examined tyrosine kinase inhibitors for in vitro growth suppression of the transformed 32D cells. A tyrosine kinase inhibitor, herbimycin A, remarkably inhibited the growth of the transformed 32D cells at 0.1 μM, at which concentration it was ineffective in parental 32D cells. Herbimycin A suppressed the constitutive tyrosine phosphorylation of the mutant FLT3 but not the phosphorylation of the ligand-stimulated wild-type FLT3. In mice transplanted with the transformed 32D cells, the administration of herbimycin A prolonged the latency of disease or completely prevented leukemia, depending on the number of cells inoculated and schedule of drug administration. These results suggest that mutant FLT3 is a promising target for tyrosine kinase inhibitors in the treatment of leukemia.

Altered interaction of HDAC5 with GATA-1 during MEL cell differentiation

The transcription factor GATA-1 plays a significant role in erythroid differentiation and association with CBP stimulates its activity by acetylation. It is possible that histone deacetylases (HDACs) repress the activity of GATA-1. In the present study, we investigated whether class I and class II HDACs interact with GATA-1 to regulate its function and indeed, GATA-1 is directly associated with HDAC3, HDAC4 and HDAC5. The expression profiling and our previous observation that GATA-2 interacts with members of the HDAC family prompted us to investigate further the biological relevance of the interaction between GATA-1 and HDAC5. Coexpression of HDAC5 suppressed the transcriptional potential of GATA-1. Our results demonstrated that GATA-1 and HDAC5 colocalized to the nucleus of murine erythroleukemia (MEL) cells. Furthermore, a portion of HDAC5 moved to the cytoplasm concomitant with MEL cell erythroid differentiation, which was induced by treatment with N,N′-hexamethylenebisacetamide. These observations support the suggestion that control of the HDAC5 nucleocytoplasmic distribution might be associated with MEL cell differentiation, possibly through regulated GATA-1 transactivation.

Differential constitutive activation between STAT‐related proteins and MAP kinase in primary acute myelogenous leukaemia

Many cytokines and growth factors stimulate multiple signal transduction pathways essential for proliferation in human acute leukaemia cells, including a mitogen‐activated protein (MAP) kinase pathway and a Janus kinase (JAK)‐STAT (signal transducers and activators of transcription) pathway. We have previously shown constitutive activation of MAP kinase in approximately 50% of acute myelogenous leukaemia (AML) samples. Recently, STAT proteins have been reported to be constitutively activated in 10–20% of AML cases. STAT3 and STAT5 are the main STAT proteins activated in haemopoietic progenitors in response to cytokines such as IL‐3, GM‐CSF, erythropoietin and thrombopoietin. Although the possibility of STAT1 protein as a substrate for MAP kinase at a serine residue has been suggested, the cross‐talk between STATs and MAP kinase pathways in vivo, especially in leukaemia cells, remains unknown.

Functional regulation of GATA-2 by acetylation

The transcription factor GATA‐2 is expressed in hematopoietic stem and progenitor cells and is functionally implicated in their survival and proliferation. In the present study, we show that GATA‐2 exists as an acetylated protein in immature precursor cells, KG1. GATA‐2 was acetylated in vitro by p300 and GCN5. We have identified multiple acetylation sites by p300 on GATA‐2, which include sites outside the zinc finger domain. We confirmed that GATA‐2 acetylation occurred in transiently transfected 293T cells at sites similar to those induced by p300 in vitro. We have successfully shown that acetylation of GATA‐2 in vitro increased its DNA‐binding activity. In addition, GATA‐2 displayed a transcriptional synergism with p300 that was impaired by mutation of each acetylation site. More importantly, each mutation in the acetylation sites of GATA‐2 abolished its growth inhibitory effect on an interleukin‐3‐dependent progenitor, 32D. We conclude that acetylation provides multiple control points for the regulation of GATA‐2 function.

In vivo effects of a histone deacetylase inhibitor, FK228, on human acute promyelocytic leukemia in NOD / Shi-scid/scid mice.

Histone acetylation and deacetylation are closely linked to transcriptional activation and repression, respectively. In acute promyelocytic leukemia (APL), histone deacetylase inhibitors (HDACIs) have a synergistic effect with all‐iraws retinoic acid (ATRA) in vitro to induce differentiation. Here we report in vitro and in vivo effects of a HDACI, FK228 (formerly FR901228 or depsipeptide), on the human APL cell line NB4. FK228 had a strong and irreversible cytotoxicity compared with another HDACI, trichostatin A. In vivo administration of ATRA or FK228 alone partly inhibited the growth of established tumors of NB4 subcutaneously transplanted in NOD/Shi‐scid/scid mice, and the combination was synergistically effective. Histopathological examination revealed that the combination induced apoptosis and differentiation as well as histone acetylation. Intravenous injection of NB4 in NOD/Shi‐scid/scid mice followed by combination treatment significantly prevented leukemia death, whereas single administration did not. These findings suggest that FK228 is a promising agent to enhance ATRA‐sensitivity in the treatment of APL.