Mutations of the HIPK2 gene in acute myeloid leukemia and myelodysplastic syndrome impair AML1- and p53-mediated transcription.

The AML1 transcription factor complex is the most frequent target of leukemia-associated chromosomal translocations. Homeodomain-interacting protein kinase 2 (HIPK2) is a part of the AML1 complex and activates AML1-mediated transcription. However, chromosomal translocations and mutations of HIPK2 have not been reported. In the current study, we screened mutations of the HIPK2 gene in 50 cases of acute myeloid leukemia (AML) and in 80 cases of myelodysplastic syndrome (MDS). Results indicated there were two missense mutations (R868W and N958I) in the speckle-retention signal (SRS) domain of HIPK2. Subcellular localization analyses indicated that the two mutants were largely localized to nuclear regions with conical or ring shapes, and were somewhat diffused in the nucleus, in contrast to the wild type, which were mainly localized in nuclear speckles. The mutations impaired the overlapping localization of AML1 and HIPK2. The mutants showed decreased activities and a dominant-negative function over wild-type protein in AML1- and p53-dependent transcription. These findings suggest that dysfunction of HIPK2 may play a role in the pathogenesis of leukemia.

Dual inhibition of EZH1/2 breaks the quiescence of leukemia stem cells in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive and lethal blood cancer originating from rare populations of leukemia stem cells (LSCs). AML relapse after conventional chemotherapy is caused by a remaining population of drug-resistant LSCs. Selective targeting of the chemoresistant population is a promising strategy for preventing and treating AML relapse. Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27 to maintain the stemness of LSCs. Here, we show that quiescent LSCs expressed the highest levels of enhancer of zeste (EZH) 1 and EZH2, the PRC2 catalytic subunits, in the AML hierarchy, and that dual inactivation of EZH1/2 eradicated quiescent LSCs to cure AML. Genetic deletion of Ezh1/2 in a mouse AML model induced cell cycle progression of quiescent LSCs and differentiation to LSCs, eventually eradicating AML LSCs. Quiescent LSCs showed PRC2-mediated suppression of Cyclin D, and Cyclin D-overexpressing AML was more sensitive to chemotherapy. We have developed a novel EZH1/2 dual inhibitor with potent inhibitory activity against both EZH1/2. In AML mouse models and patient-derived xenograft models, the inhibitor reduced the number of LSCs, impaired leukemia progression, and prolonged survival. Taken together, these results show that dual inhibition of EZH1/2 is an effective strategy for eliminating AML LSCs.

The retinoblastoma gene product RB stimulates Sp1-mediated transcription by liberating Sp1 from a negative regulator.

Studies have demonstrated that the retinoblastoma susceptibility gene product, RB, can either positively or negatively regulate expression of several genes through cis-acting elements in a cell-type-dependent manner. The nucleotide sequence of the retinoblastoma control element (RCE) motif, GCCACC or CCACCC, and the Sp1 consensus binding sequence, CCGCCC, can confer equal responsiveness to RB. Here, we report that RB activates transcription of the c-jun gene through the Sp1-binding site within the c-jun promoter. Preincubation of crude nuclear extracts with monoclonal antibodies to RB results in reduction of Sp1 complexes in a mobility shift assay, while addition of recombinant RB in mobility shift assay mixtures with CCL64 cell extracts leads to an enhancement of DNA-binding activity of SP1. These results suggest that RB is directly or indirectly involved in Sp1-DNA binding activity. A mechanism by which RB regulates transactivation is indicated by our detection of a heat-labile and protease-sensitive Sp1 negative regulator(s) (Sp1-I) that specifically inhibits Sp1 binding to a c-jun Sp1 site. This inhibition is reversed by addition of recombinant RB proteins, suggesting that RB stimulates Sp1-mediated transactivation by liberating Sp1 from Sp1-I. Additional evidence for Sp1-I involvement in Sp1-mediated transactivation was demonstrated by cotransfection of RB, GAL4-Sp1, and a GAL4-responsive template into CV-1 cells. Finally, we have identified Sp1-I, a approximately 20-kDa protein(s) that inhibits the Sp1 complexes from binding to DNA and that is also an RB-associated protein. These findings provide evidence for a functional link between two distinct classes of oncoproteins, RB and c-Jun, that are involved in the control of cell growth, and also define a novel mechanism for the regulation of c-jun expression.

The AML1-MTG8 leukemic fusion protein forms a complex with a novel member of the MTG8(ETO/CDR) family, MTGR1.

The AML1-CBFbeta transcription factor complex is essential for the definitive hematopoiesis of all lineages and is the most frequent target of chromosomal rearrangements in human leukemia. In the t(8;21) translocation associated with acute myeloid leukemia (AML), the AML1(CBFA2/PEBP2alphaB) gene is juxtaposed to the MTG8(ETO/CDR) gene. We show here that the resultant AML1-MTG8 gene product specifically and strongly interacts with an 85-kDa phosphoprotein. Molecular cloning of cDNA indicated that the AML1-MTG8-binding protein (MTGR1) is highly related to MTG8 and similar to Drosophila Nervy. Comparison of amino acid sequences among MTGR1, MTG8, and Nervy revealed four evolutionarily conserved regions (NHR1 to NHR4). Ectopic expression of AML1-MTG8 in L-G murine myeloid progenitor cells inhibits differentiation to mature neutrophils and induces cell proliferation in response to granulocyte colony-stimulating factor (G-CSF). Analysis with C-terminal deletion mutants of AML1-MTG8 indicated that the region of 51 residues (488 to 538), which contains NHR2, is essential for the induction of G-CSF-dependent cell proliferation. Immunoprecipitation analysis indicates that this region is required for AML1-MTG8 to form a stable complex with MTGR1. Overexpression of MTGR1 stimulates AML1-MTG8 to induce G-CSF-dependent proliferation of L-G cells and to interfere with AML1-dependent transcription. These results suggest that AML1-MTG8 could function as a complex with MTGR1 and that the complex might be important in promoting leukemogenesis.

Physical association of the patient-specific GATA1 mutants with RUNX1 in acute megakaryoblastic leukemia accompanying Down syndrome.

Mutations of the GATA1 gene on chromosome X have been found in almost all cases of transient myeloproliferative disorder and acute megakaryoblastic leukemia (AMKL) accompanying Down syndrome (DS). Although most GATA1 mutations lead to the expression of GATA1s lacking the N-terminal activation domain, we recently found two novel GATA1 proteins with defects in another N-terminal region. It has been suggested that loss of the N-terminal portion of GATA1 might interfere with physiological interactions with the critical megakaryocytic transcription factor RUNX1, and this would imply that GATA1s is not able to interact properly with RUNX1. However, the interaction domain of GATA1 remains controversial. In this study, we show that GATA1 binds to RUNX1 through its zinc-finger domains, and that the C-finger is indispensable for synergy with RUNX1. All of the patient-specific GATA1 mutants interacted efficiently with RUNX1 and retained their ability to act synergistically with RUNX1 on the megakaryocytic GP1balpha promoter, whereas the levels of transcriptional activities were diverse among the mutants. Thus, our data indicate that physical interaction and synergy between GATA1 and RUNX1 are retained in DS-AMKL, although it is still possible that increased RUNX1 activity plays a role in the development of leukemia in DS.

MLL is essential for NUP98-HOXA9-induced leukemia.

Rearrangements involving the NUP98 gene resulting in fusions to several partner genes occur in acute myeloid leukemia and myelodysplastic syndromes. This study demonstrates that the second FG repeat domain of the NUP98 moiety of the NUP98-HOXA9 fusion protein is important for its cell immortalization and leukemogenesis activities. We demonstrate that NUP98-HOXA9 interacts with mixed lineage leukemia (MLL) via this FG repeat domain and that, in the absence of MLL, NUP98-HOXA9-induced cell immortalization and leukemogenesis are severely inhibited. Molecular analyses indicate that MLL is important for the recruitment of NUP98-HOXA9 to the HOXA locus and for NUP98-HOXA9-induced HOXA gene expression. Our data indicate that MLL is crucial for NUP98-HOXA9 leukemia initiation.

Induction by adenovirus-5 E1A of the differentiation phenotype of F9 teratocarcinoma cells involves a conserved region (CR1) of E1A.

The effects of the E1A protein of adenovirus-5 on the differentiation program of F9 teratocarcinoma cells were examined by the stable introduction of plasmids that expressed wild-type or mutated forms of E1A. Constitutive expression of plasmids for most of the mutant E1As induced loss of expression of the cell-surface antigen SSEA-1 and the enhanced expression of genes specific for the differentiated phenotype of F9 cells, such as genes for laminin B1, tissue-type plasminogen activator (tPA) and type IV collagen, as well as the altered cell morphology that is associated with the differentiated state. However, such changes were not observed in the case of genes for mutant proteins from which a conserved region (CR1) of E1A had been deleted. Furthermore, no significant induction of expression of the c-jun gene or transactivation of the c-jun-CAT reporter gene were observed when the sequence that encodes CR1 of E1A had been deleted. A palindromic sequence element (DRE) of the c-jun promoter was essential for the E1A-mediated up-regulation of the c-jun gene. These results imply that CR1 is required for activation of the c-jun gene and that it is implicated in the growth arrest, expression of parietal endoderm-specific functions and the orderly differentiation of F9 cells.

Fusion of MOZ and p300 histone acetyltransferases in acute monocytic leukemia with a t(8;22)(p11;q13) chromosome translocation.

Histone acetyltransferase p300 functions as a transcriptional co-activator which interacts with a number of transcription factors. Monocytic leukemia zinc finger protein (MOZ) has histone acetyltransferase activity. We report the fusion of the MOZ gene to the p300 gene in acute myeloid leukemia with translocation t(8;22)(p11;q13). FISH and Southern blot analyses showed the rearrangement of the MOZ and p300 genes. We determined the genomic structure of the p300 and the MOZ genes and the breakpoints of the translocation. Analysis of fusion transcripts indicated that the zinc finger and acetyltransferase domains of MOZ are fused to a largely intact p300. These results suggest that MOZ-p300, which has two acetyltransferase domains, could be involved in leukemogenesis through aberrant regulation of histone acetylation.

Hes1 suppresses acute myeloid leukemia development through FLT3 repression.

In leukemogenesis, Notch signaling can be up and downregulated in a context-dependent manner. The transcription factor hairy and enhancer of split-1 (Hes1) is well-characterized as a downstream target of Notch signaling. Hes1 encodes a basic helix-loop-helix-type protein, and represses target gene expression. Here, we report that deletion of the Hes1 gene in mice promotes acute myeloid leukemia (AML) development induced by the MLL-AF9 fusion protein. We then found that Hes1 directly bound to the promoter region of the FMS-like tyrosine kinase 3 (FLT3) gene and downregulated the promoter activity. FLT3 was consequently upregulated in MLL-AF9-expressing immortalized and leukemia cells with a Hes1- or RBPJ-null background. MLL-AF9-expressing Hes1-null AML cells showed enhanced proliferation and ERK phosphorylation following FLT3 ligand stimulation. FLT3 inhibition efficiently abrogated proliferation of MLL-AF9-induced Hes1-null AML cells. Furthermore, an agonistic anti-Notch2 antibody induced apoptosis of MLL-AF9-induced AML cells in a Hes1-wild type but not a Hes1-null background. We also accessed two independent databases containing messenger RNA (mRNA) expression profiles and found that the expression level of FLT3 mRNA was negatively correlated with those of HES1 in patient AML samples. These observations demonstrate that Hes1 mediates tumor suppressive roles of Notch signaling in AML development, probably by downregulating FLT3 expression.

Structure and expression pattern of a human MTG8/ETO family gene, MTGR1.

AML1-MTG8 fusion protein, which is produced from the rearranged gene formed between AML1 and MTG8 in myeloid leukemia with t(8;21) chromosomal translocation, plays an important role in the pathogenesis of leukemia. We previously showed that ectopically expressed AML1-MTG8 fusion protein is associated with an MTG8-like protein in the mouse myeloid precursor cell line L-G, and this association seemed to be required for AML1-MTG8 to stimulate proliferation. As a candidate cDNA for this MTG8-like protein, a 6.4 kb MTGR1 cDNA encoding human MTGR1b protein of 604 amino acids was isolated. Since this cDNA was shorter than the main mRNA (about 7.5 kb), the 5'-end of the MTGR1 cDNA was extended using Marathon Ready cDNA. When the newly obtained 5'-sequence was combined with the previous cDNA, the resultant MTGR1 cDNA (6995 bp), including exon 3 that the previous cDNA lacked, could encode MTGR1a protein of 575 amino acids. Transcripts of the MTGR1 gene were expressed ubiquitously in the human tissues and cell lines examined. PCR analyses of the cDNAs from human tissues showed the presence of various splicing variants with regard to the 5'-region including exons 1, 2 and 3. The MTGR1 gene consists of 14 exons and spans about 68 kb. The genomic structure of MTGR1 is highly similar to those of other MTG 8-family genes, MTG8 and MTG16. MTG16 was recently cloned from the translocation breakpoint of myeloid malignancies with t(16;21) chromosomal translocation.

Rapid method for construction of yeast artificial chromosome human DNA libraries involving the trapping of cells in agarose films.

A simple method for the molecular cloning of fragments of more than one hundred kilobase pairs of exogenous DNA, by the encapsulation of cells in agarose beads, was reported previously for the construction of a human genomic DNA library in a yeast artificial chromosome (YAC) vector (in situ YAC construction) [1]. The efficiency of this procedure is impaired by the step in which agarose beads that contain human DNA fragments are melted before transformation. The incomplete solubility of the ligated human DNA fragment-YAC vector often results in lower than desirable frequencies of transformation. To overcome this problem we have developed a new improved method that involves use of an agarose film. The technical manipulations involved in the construction of clones of very large segments of human DNA are discussed.

A novel pathway for retinoic acid-induced differentiation of F9 cells that is distinct from receptor-mediated trans-activation.

Retinoic acid (RA) has striking effects on vertebrate development and induces differentiation of several lines of cells including embryonal carcinoma F9 cells. It is generally accepted that the actions of RA are mediated by nuclear receptors for RA. However, we now provide evidence that F9 cells can differentiate in response to RA without trans-activation by nuclear receptors. Irreversible differentiation of F9 cells was induced by 18 h of exposure to RA with subsequent incubation in the absence of RA. This induction of differentiation was not blocked after inhibition of protein synthesis and mRNA synthesis during the 18-h treatment with RA, but the endogenous RA receptors failed to activate transcription from their target genes that contain the receptor-binding sequences. During the commitment to RA-induced differentiation, at least five sets of four phosphorylated proteins underwent changes in the absence of protein synthesis de novo. These results suggest that there is a novel pathway for the action of RA that is independent of nuclear receptor-mediated trans-activation.

Phosphorylation of PML is essential for activation of C/EBP epsilon and PU.1 to accelerate granulocytic differentiation.

Promyelocytic leukemia (PML) is a nuclear protein that functions as a regulator of transcription, cell proliferation, apoptosis and myeloid cell differentiation. PML is subjected to post-translational modifications such as sumoylation and phosphorylation. However, the physiological significance of these modifications, especially for myeloid cell differentiation, remains unclear. In this report, we found that four serine residues in the PML C-terminal region are highly phosphorylated in a myeloid cell line. Wild-type PML accelerated G-CSF-induced granulocytic differentiation, but a phosphorylation-deficient PML mutant failed. PML interacted with C/EBP epsilon, a transcription factor essential for granulopoiesis, activated C/EBP epsilon-mediated transcription in concert with p300 and accelerated C/EBP epsilon-induced granulocytic differentiation. Phosphorylation of PML was required for stimulating C/EBP epsilon-dependent transcription and accelerating C/EBP epsilon-induced granulocytic differentiation. We also found that PML phosphorylation was required for stimulation of PU.1-dependent transcription and acceleration of PU.1-induced granulocytic differentiation. These results suggest that phosphorylation plays essential roles in the regulation of PML to accelerate granulocytic differentiation through multiple pathways.

Interaction and functional cooperation of the leukemia-associated factors AML1 and p300 in myeloid cell differentiation.

The AML1 transcription factor and the transcriptional coactivators p300 and CBP are the targets of chromosome translocations associated with acute myeloid leukemia and myelodysplastic syndrome. In the t(8;21) translocation, the AML1 (CBFA2/PEBP2alphaB) gene becomes fused to the MTG8 (ETO) gene. We previously found that the terminal differentiation step leading to mature neutrophils in response to granulocyte colony-stimulating factor (G-CSF) was inhibited by the ectopic expression of the AML1-MTG8 fusion protein in L-G murine myeloid progenitor cells. We show here that overexpression of normal AML1 proteins reverses this inhibition and restores the competence to differentiate. Immunoprecipitation analysis shows that p300 and CREB-binding protein (CBP) interact with AML1. The C-terminal region of AML1 is responsible for the induction of cell differentiation and for the interaction with p300. Overexpression of p300 stimulates AML1-dependent transcription and the induction of cell differentiation. These results suggest that p300 plays critical roles in AML1-dependent transcription during the differentiation of myeloid cells. Thus, AML1 and its associated factors p300 and CBFbeta, all of which are targets of chromosomal rearrangements in human leukemia, function cooperatively in the differentiation of myeloid cells.

E1A dependent up-regulation of c-jun/AP-1 activity.

E1A, the early region 1A transcription unit of human adenovirus, exhibits multiple functions that regulate the expression of some cellular genes and promote cell growth and division. We found that E1A stimulated c-jun gene expression at least fifty-fold in rat 3Y1 cells in a serum-independent manner, concomitantly with E1A down-regulation of jun B expression. The E1A-dependent induction of c-jun transcription resulted in increase amount of cJun/AP1. This induction was mediated by the enhancement of the binding activity of the transcription factor cJun/AP1 to an AP1 binding site in the c-jun promoter. Additionally, this induction can be repressed by introducing junB into the cells. Taken collectively, these results suggest that the differential expression of two closely related proteins greatly expands their cellular regulation. Induction of c-jun expression by E1A as well as c-jun autoregulation may amplify the action of E1A during adenovirus infection. Therefore, some of the biological effects of E1A may include mediating the constitutive activation of c-jun, which is important in transcriptional regulation and oncogenic transformation.

Analysis of genes under the downstream control of the t(8;21) fusion protein AML1-MTG8: overexpression of the TIS11b (ERF-1, cMG1) gene induces myeloid cell proliferation in response to G-CSF.

The AML1-MTG8 fusion transcription factor generated by t(8;21) translocation is thought to dysregulate genes that are crucial for normal differentiation and proliferation of hematopoietic progenitors to cause acute myelogenous leukemia (AML). Although AML1-MTG8 has been shown to repress the transcription of AML1 targets, none of the known targets of AML1 are probably responsible for AML1-MTG8-mediated leukemogenesis. In this study, 24 genes under the downstream control of AML1-MTG8 were isolated by using a differential display technique. Analysis with deletion mutants of AML1-MTG8 demonstrated that the regulation of the majority of these genes requires the region of 51 residues (488-538) containing the Nervy homology region 2 (NHR2), through which AML1-MTG8 interacts with MTGR1. Among the 24 genes identified, 10 were considered to be genes under the control of AML1, because their expression was altered by AML1b or AML1a or both. However, the other 14 genes were not affected by either AML1b or AML1a, suggesting the possibility that AML1-MTG8 regulates a number of specific target genes that are not normally regulated by AML1. Furthermore, an up-regulated gene, TIS11b (ERF-1, cMG1), was highly expressed in t(8;21) leukemic cells, and the overexpression of TIS11b induced myeloid cell proliferation in response to granulocyte colony-stimulating factor. These results suggest that the high-level expression of TIS11b contributes to AML1-MTG8-mediated leukemogenesis. (Blood. 2000;96:655-663)

AML1-MTG8 leukemic protein induces the expression of granulocyte colony-stimulating factor (G-CSF) receptor through the up-regulation of CCAAT/enhancer binding protein epsilon.

The t(8;21) translocation is one of the most frequent chromosomal abnormalities associated with acute myeloid leukemia (AML). In this translocation, the AML1 (CBFA2/PEBP2aB) gene is disrupted and fused to the MTG8 (ETO) gene. The ectopic expression of the resulting AML1-MTG8 fusion gene product in L-G and 32Dcl3 murine myeloid precursor cells stimulates cell proliferation without inducing morphologic terminal differentiation into mature granulocytes in response to granulocyte-colony stimulating factor (G-CSF). This study found that the ectopic expression of AML1-MTG8 elevates the expression of the G-CSF receptor (G-CSFR). Analysis of the promoter region of the G-CSFR gene revealed that up-regulation of G-CSFR expression by AML1-MTG8 does not depend on the AML1-binding sequence, but on the C/EBP (CCAAT/enhancer binding protein) binding site. The results suggest that the overproduction of G-CSFR is at least partly mediated by C/EBPepsilon, whose expression is activated by AML1-MTG8. The ectopic expression of G-CSFR in L-G cells induced cell proliferation in response to G-CSF, but did not inhibit cell differentiation into mature neutrophils. Overexpression of C/EBPepsilon in L-G cells also stimulated G-CSF-dependent cell proliferation. High expression levels of G-CSFR were also found in the leukemic cells of AML patients with t(8;21). Therefore, G-CSF-dependent cell proliferation of myeloid precursor cells may be implicated in leukemogenesis.

Two cis-regulatory elements that mediate different signaling pathways for serum-dependent activation of the junB gene.

Transcription of the junB gene is rapidly and transiently induced by a variety of extracellular signals. We report here that expression directed by a junB promoter/chloramphenicol acetyltransferase reporter construct (junB/CAT) is induced by fetal bovine serum, 12-O-tetradecanoylphorbol-13-acetate (TPA), epidermal growth factor (EGF), platelet-derived growth factor, and fibroblast growth factor in mouse fibroblast 3T6 cells. Deletion analysis of the promoter region of the junB gene indicates that there are at least two cis-regulatory elements that confer the capacity for serum-dependent induction. These two serum response elements (SRE1 and SRE2) are mapped between nucleotides -1451 and -1425 and between nucleotides -3100 and -2500, respectively, relative to the site of initiation of transcription. SRE1, the nucleotide sequence of which resembles that of the serum response element of the c-fos gene, is activated by TPA, platelet-derived growth factor, and fibroblast growth factor, but these growth-stimulating factors do not induce SRE2-mediated transcription. Pretreatment of the cells with phorbol dibutyrate, which reduces the level of protein kinase C activity in cells, almost completely abolishes the activation of SRE1 by TPA. Pretreatment with phorbol dibutyrate also reduces (but does not eliminate) the serum-dependent activation of SRE1. By contrast, the induction of SRE2 by serum is not affected by this pretreatment. Herbimycin A, an inhibitor of protein kinases, inhibits the activity of SRE2, but not that of SRE1. These results suggest that transcription of the junB gene can be induced by at least two distinct signaling pathways, which are mediated by SRE1 and SRE2, respectively. In addition, EGF induces expression of junB/CAT as strongly as does serum, but neither SRE1 nor SRE2 is sufficient for responsiveness to EGF.

The conserved domain CR2 of Epstein-Barr virus nuclear antigen leader protein is responsible not only for nuclear matrix association but also for nuclear localization.

There is a growing body of evidence for the importance of the nuclear matrix in various nuclear events including gene expression and DNA replication. Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is a nuclear matrix-associated protein that has been suggested to play an important role in EBV-induced transformation. To define the biological significance of the association of EBNA-LP with the nuclear matrix, we mapped the domain of EBNA-LP responsible for nuclear matrix association and investigated the functions of the EBNA-LP mutant mutagenized by substitution of alanines for the cluster of arginine residues in the mapped region. The results of the present study were as follows. (i) Transiently expressed EBNA-LP in COS-7 or BOSC23 cells was associated with the nuclear matrix, similarly to that in EBV-infected B cells. (ii) Mutational analysis of EBNA-LP revealed that a 10-amino acid segment of EBNA-LP is critical for nuclear matrix association of the protein. Interestingly, the identified region overlapped with the region CR2 of EBNA-LP conserved among a subset of primate gammaherpesviruses. The identified segment is referred to as EBNA-LP NMTS (nuclear matrix targeting signal). (iii) The EBNA-LP mutant with the arginine to alanine substitutions in NMTS was no longer localized not only to the nuclear matrix but also to the nucleus. (iv) The EBNA-LP mutant lacked its ability to coactivate EBNA-2-dependent transactivation. These results indicated that EBNA-LP needs to be localized in the nucleus and/or associated with the nuclear matrix through CR2 to elicit its function such as the coactivation of the EBNA-2-dependent transcriptional activation.