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PubMed2003

New mechanisms of AML1 gene alteration in hematological malignancies.

The human AML1 gene (also named CBFA2 or RUNX1), located in the 21q22 chromosomal band, encodes for one of the two subunits forming a heterodimeric transcription factor, the human core binding factor (CBF). AML1 protein contains a highly evolutionary conserved domain of 128 amino acids called runt domain, responsible for both heterodimerization with the beta subunit of CBF and for DNA binding. AML1 is normally expressed in all hematopoietic lineages and acts to regulate the expression of various genes specific to hematopoiesis playing a pivotal role in myeloid differentiation. AML1 is one of the genes most frequently deregulated in leukemia through different mechanisms including translocation, mutation and amplification. Translocations lead to the formation of fusion genes encoding for chimerical proteins such as AML1-ETO which induces leukemogenesis. Recently, new mechanisms of AML1 deregulation by point mutations or amplification have been reported. To our knowledge, 51 patients (among 805 studied) with AML1 point mutations have been described. Forty of them have acute myeloid leukemia (AML) most often M0 AML. In this subtype of AML, the frequency of AML1 mutation is significantly higher; 21.5% of patients mutated (34/158). Mutations have also been found with lower frequency in other FAB subtype AML (6 cases), in myeloproliferative disorders (6 cases), in myelodysplastic syndrome (3 cases) and rarely in acute lymphoblastic leukemia (1 case). AML1 gene amplification has been found essentially in childhood ALL (12 cases) and more rarely in myeloid malignancies (4 cases). Here, we reviewed all these cases of AML1 point mutations and amplification and focused on the mechanisms of AML1 deregulation induced by these alterations.

Acute Disease↗
PubMed2003

The ETO (MTG8) gene family.

Cloning and characterization of the 8;21 chromosomal breakpoint identified AML1 on chromosome 21 and ETO (MTG8) on chromosome 8, and the resultant chimeric gene product, AML-1/ETO. The ETO gene family now includes three human members encoding proteins composed of four evolutionarily conserved domains termed nervy homology regions (NHR) 1-4. ETO associates with N-CoR/Sin3a/HDAC complexes in vivo and acts as a corepressor for the promyelocytic zinc finger protein. Moreover, ETO is nuclear matrix attached at sites coincident with histone deacetylase enzymes and mSin3a. These data suggest that ETO proteins function as transcriptional corepressors. This review focuses on the ETO gene family in terms of expression and function. Specifically, the role of ETO as a co-repressor will be detailed. Additionally, the impact of this recent discovery on treatment of t(8;21)-containing leukemia will be discussed.

Animals↗
PubMed1995

t(8;21) breakpoints are clustered between alternatively spliced exons of MTG8.

1. The (8;21) translocation, which is consistently associated with a subgroup of acute myeloid leukaemia, involves two loci: runt on chromosome 21 and MTG8 on chromosome 8. 2. Breakpoints in runt fall within a single intron that is located immediately downstream of a phylogenetically conserved DNA-binding domain (the 'runt box'). 3. We now show that most breakpoints on chromosome 8 fall within a region between two alternative 5' MTG8 exons. Thus, we predict that chimaeric genes on both the derivative(8) and the derivative(21) chromosomes have the potential to be transcriptionally active.

Acute Disease↗
PubMed2002

Genomic DNA breakpoints in AML1/RUNX1 and ETO cluster with topoisomerase II DNA cleavage and DNase I hypersensitive sites in t(8;21) leukemia.

The translocation t(8;21)(q22;q22) is one of the most frequent chromosome translocations in acute myeloid leukemia (AML). AML1/RUNX1 at 21q22 is involved in t(8;21), t(3;21), and t(16;21) in de novo and therapy-related AML and myelodysplastic syndrome as well as in t(12;21) in childhood B cell acute lymphoblastic leukemia. Although DNA breakpoints in AML1 and ETO (at 8q22) cluster in a few introns, the mechanisms of DNA recombination resulting in t(8;21) are unknown. The correlation of specific chromatin structural elements, i.e., topoisomerase II (topo II) DNA cleavage sites, DNase I hypersensitive sites, and scaffold-associated regions, which have been implicated in chromosome recombination with genomic DNA breakpoints in AML1 and ETO in t(8;21) is unknown. The breakpoints in AML1 and ETO were clustered in the Kasumi 1 cell line and in 31 leukemia patients with t(8;21); all except one had de novo AML. Sequencing of the breakpoint junctions revealed no common DNA motif; however, deletions, duplications, microhomologies, and nontemplate DNA were found. Ten in vivo topo II DNA cleavage sites were mapped in AML1, including three in intron 5 and seven in intron 7a, and two were in intron 1b of ETO. All strong topo II sites colocalized with DNase I hypersensitive sites and thus represent open chromatin regions. These sites correlated with genomic DNA breakpoints in both AML1 and ETO, thus implicating them in the de novo 8;21 translocation.

Adult↗
PubMed2003

Acute myeloid leukemia fusion proteins deregulate genes involved in stem cell maintenance and DNA repair.

Acute myelogenous leukemias (AMLs) are genetically heterogeneous and characterized by chromosomal rearrangements that produce fusion proteins with aberrant transcriptional regulatory activities. Expression of AML fusion proteins in transgenic mice increases the risk of myeloid leukemias, suggesting that they induce a preleukemic state. The underlying molecular and biological mechanisms are, however, unknown. To address this issue, we performed a systematic analysis of fusion protein transcriptional targets. We expressed AML1/ETO, PML/RAR, and PLZF/RAR in U937 hemopoietic precursor cells and measured global gene expression using oligonucleotide chips. We identified 1,555 genes regulated concordantly by at least two fusion proteins that were further validated in patient samples and finally classified according to available functional information. Strikingly, we found that AML fusion proteins induce genes involved in the maintenance of the stem cell phenotype and repress DNA repair genes, mainly of the base excision repair pathway. Functional studies confirmed that ectopic expression of fusion proteins constitutively activates pathways leading to increased stem cell renewal (e.g., the Jagged1/Notch pathway) and provokes accumulation of DNA damage. We propose that expansion of the stem cell compartment and induction of a mutator phenotype are relevant features underlying the leukemic potential of AML-associated fusion proteins.

Calcium-Binding Proteins↗
PubMed1999

Real-time RT-PCR for the detection and quantification of AML1/MTG8 fusion transcripts in t(8;21)-positive AML patients.

AML1/MTG8 was quantified relative to the expression of the GAPDH housekeeping gene by real-time RT-PCR in 22 patients with t(8;21)-positive acute myeloblastic leukaemia (AML) at initial diagnosis and in seven of these patients also during/after chemotherapy and allogeneic bone marrow transplantation. Real-time PCR was able to specifically detect and quantify AML1/MTG8 over a 5 log range. The detection limit for t(8;21)-positive cells was a dilution of 1:105. The AML1/MTG8 expression varied considerably among the 22 AML patients at intial diagnosis with a ratio AML1/MTG8:GAPDH of 0.5135+/-0.536 (range 0.1-2.14, median 0.318). In six patients with t(8;21)-positive AML a marked decline of AML1/MTG8 could be induced by chemotherapy. These patients are in ongoing complete haematological remission (CR) with a constant low-level AML1/MTG8 expression. In another patient a rapid rise of AML1/MTG8 transcripts could be detected in CR after allogeneic bone marrow transplantation and the patient relapsed 10 weeks later. In conclusion, real-time RT-PCR is a suitable approach for the quantification of AML1/MTG8 transcripts in the monitoring of AML patients with t(8;21) during/after chemotherapy and can provide data of prognostic relevance.

Chromosomes, Human, Pair 21↗
PubMed1995

The AML1/ETO fusion protein blocks transactivation of the GM-CSF promoter by AML1B.

The t(8;21) translocation, commonly found in acute myelogenous leukemia (AML), generates a fusion protein containing N-terminal AML1 and C-terminal ETO amino acids. The human AML1 gene encodes several related proteins that specifically bind to the sequence TGT/cGGT, located in the promoter regions of a variety of hematopoietic growth factor genes. To examine the abilities of the AML1B protein (which contains 479 amino acids), a shorter AML1A isoform (which contains amino acids 1-250), and the AML1/ETO fusion protein (which contains AML1A amino acids 1-177) to stimulate transcription from the GM-CSF promoter, we performed co-transfection experiments in T cells using a human GM-CSF promoter-CAT reporter gene plasmid and expression vectors that contain the cDNAs for one of the above proteins. Our data demonstrate that AML1B, but not AML1A or AML1/ETO transactivates the GM-CSF promoter, requiring the TGTGGT sequence contained between base pairs -68 and -53. Furthermore, we show that AML1/ETO, but not AML1A, inhibits the ability of AML1B to stimulate CAT expression. Electrophoretic mobility shift assays demonstrated the specific binding of AML1 proteins to the GM-CSF promoter TGTGGT sequence, which does not require GM-CSF sequences immediately upstream of this binding site. Our data support a role for AML1B as a transcriptional activator and establish that the AML1/ETO fusion protein can act as a dominant negative protein on the human GM-CSF promoter. Although AML1/ETO does not stimulate the transcription of GM-CSF, it may function by inhibiting the normal activity of AML1B in AML cells with the t(8;21) translocation.

Base Sequence↗
PubMed2003

Interactions between the leukaemia-associated ETO homologues of nuclear repressor proteins.

The eight-twenty-one (ETO) homologues, represented by ETO, myeloid transforming gene-related protein 1 (MTGR1) and myeloid transforming gene chromosome 16 (MTG16), are nuclear repressor proteins. ETO is part of the fusion protein acute myeloid leukaemia (AML)1-ETO, resulting from the translocation (8;21). Similarly, MTG16 is disrupted to become part of AML1/MTG16 in t(16;21). The aberrant expression of these chimeras could affect interplay between ETO homologues and contribute to the leukaemogenic process. We investigated possible interactions between the ETO homologues. Ectopic co-expression in COS-cells resulted in heterodimerisation of the various ETO homologues suggesting that they may co-operate. Similarly, the chimeric oncoprotein AML1-ETO interacted with both MTGR1 and MTG16. However, results from cell lines endogenously expressing more than one ETO homologue did not demonstrate co-precipitation. Results from IP-Western and size determination by gel filtration of deletion mutants expressed in COS-cells, indicated an important role of the HHR domain for oligomerisation. A role was also suggested for the Nervy domain in the homologue interactions. Our results suggest that ETO homologues can interact with each other as well as with AML1-ETO, although it is unclear as to what extent these interactions occur in vivo.

Amino Acid Sequence↗
PubMed1999

A novel ubiquitin-specific protease, UBP43, cloned from leukemia fusion protein AML1-ETO-expressing mice, functions in hematopoietic cell differentiation.

Using PCR-coupled subtractive screening-representational difference analysis, we have cloned a novel gene from AML1-ETO knockin mice. This gene is highly expressed in the yolk sac and fetal liver of the knockin mice. Nucleotide sequence analysis indicates that its cDNA contains an 1,107-bp open reading frame encoding a 368-amino-acid polypeptide. Further protein sequence and protein translation analysis shows that it belongs to a family of ubiquitin-specific proteases (UBP), and its molecular mass is 43 kDa. Therefore, we have named this gene UBP43. Like other ubiquitin proteases, the UBP43 protein has deubiquitinating enzyme activity. Protein ubiquitination has been implicated in many important cellular events. In wild-type adult mice, UBP43 is highly expressed in the thymus and in peritoneal macrophages. Among nine different murine hematopoietic cell lines analyzed, UBP43 expression is detectable only in cell lines related to the monocytic lineage. Furthermore, its expression is regulated during cytokine-induced monocytic cell differentiation. We have investigated its function in the hematopoietic myeloid cell line M1. UBP43 was introduced into M1 cells by retroviral gene transfer, and several high-expressing UBP43 clones were obtained for further study. Morphologic and cell surface marker examination of UBP43/M1 cells reveals that overexpression of UBP43 blocks cytokine-induced terminal differentiation of monocytic cells. These data suggest that UBP43 plays an important role in hematopoiesis by modulating either the ubiquitin-dependent proteolytic pathway or the ubiquitination state of another regulatory factor(s) during myeloid cell differentiation.

Amino Acid Sequence↗
PubMed1996

ETO and AML1 phosphoproteins are expressed in CD34+ hematopoietic progenitors: implications for t(8;21) leukemogenesis and monitoring residual disease.

To study acute myelogenous leukemia 1 (AML1) transcription factor, ETO protein, and t(8;21) AML chimeric AML1/ ETO protein in normal hematopoiesis and in leukemia, we raised rabbit antisera to a bacterially expressed polypeptide containing amino acid residues 1 to 220 of ETO and to synthetic peptides extending from residues 528 to 548 of ETO and 32 to 50 of AML1. The latter was selected to have little chance of cross-reactivity with other members of the PEBP2 alpha family. With affinity-purified reagents, we observed immunofluorescent staining for both AML1 and ETO in the nucleus of HEL, K562, and Kasumi-1 leukemic cell lines, the last from a t(8;21) AML. Biochemical analysis confirmed specificity of the antibodies and the nuclear localization of the antigens, the latter being exclusive for AML1 and primary for ETO. Immunoprecipitations of metabolically labeled 32P-proteins from Kasumi-1 cells show that AML1 and ETO are phosphorylated on serine and threonine. Investigations with normal bone marrow reveal AML1 and ETO are coexpressed in megakaryocytes and that each is expressed in a portion of the approximately 10-microns-diameter cells residing there. Using a CD34+ enriched population mobilized to peripheral blood, we found AML1 and, unexpectedly, ETO present in these cells. Because of this, we conclude that the expression of ETO in hematopoietic cells is not by itself leukemogenic. Also, because ETO would not be exclusively expressed as part of chimeric AML1/ETO in leukemic patients, its presence cannot be used to monitor t(8;21) AML residual disease.

Acute Disease↗
PubMed2006

[The use of quantitative assessment of Wilms tumour gene 1 for monitoring of residual disease in acute myeloid leukemia patients].

BACKGROUND: Despite a considerable effort, the majority of acute myeloid leukaemia (AML) patients do not have a suitable specific molecular marker for monitoring minimal residual disease (MRD). The results of some studies suggest the Wilms tumour gene (WT1) as a possible molecular marker of MRD. METHODS AND RESULTS: We measured the expression of WT1 at diagnosis and during treatment of the acute myeloid leukaemia (AML) patients. The expression of WT1 was measured by the quantitative real-time RT-PCR in peripheral leukocytes from 56 AML at diagnosis and 7 patients with AML transformed from myelodysplastic syndromes (MDS). The WT1 expression was significantly elevated (up to 3 orders of magnitude) in peripheral blood samples (PB) of AML patients at diagnosis compared to PB samples of healthy donors (P < 0.0001). The level of WT1 expression depends particularly on FAB AML subtype, with the highest being found in AML patients with subtypes M4, M1, M3 and AML transformed from MDS. Conversely, AML patients with M2 and with the presence of AML1/ET0 at presentation showed a significantly lower expression of the WT1 gene compared to the remaining AML patients at presentation (P = 0,005). Further, sequence samples of 12 AML patients under long-term surveillance were tested for the WT1 expression in parallel with the expression of specific MRD markers--fusion genes: AMLI/ETO, PML/RARalpha and CBFB/MYH11. The levels of WT1 gene expression and the above specific fusion genes significantly correlated. Moreover, 14 patients without the specific MRD marker were tested for the WT1 expression. The results show that haematological relapses were associated with the rise of expression of the specific fusion genes and with the WT1 gene expression. The rise of WT1 expression above the level seen in leucocytes from peripheral blood and/or bone marrow of healthy donors--in four patients under long-term surveillance the "molecular relapse" predicted ongoing haematological relapses as early as 2 months in advance. CONCLUSIONS: Our results, in accordance with some of the previously published ones, show that WT1 expression seems to be a suitable marker of minimal residual disease in AML patients.

Acute Disease↗
PubMed2003

Cryptic chromosomal aberrations leading to an AML1/ETO rearrangement are frequently caused by small insertions.

The translocation t(8;21)(q22;q22), which results in the fusion of the AML1 (RUNX1) and ETO (CBFA2T1) genes, is a recurrent aberration in acute myeloid leukemia (AML), preferentially correlated with FAB M2, and has the highest incidence in childhood AML. Because of the favorable prognosis, the evidence of the t(8;21) or the AML1/ETO fusion gene is mandatory in most of the therapy trials, allowing the stratification of the patients to the correct risk group in terms of treatment. Here we present six out of 59 children with AML who were positive for AML1/ETO by RT-PCR, but showed no evidence of the classical t(8;21)(q22;q22) by conventional cytogenetics. Because of the discrepancies between molecular and cytogenetic analyses, these six patients were further investigated by fluorescence in situ hybridization analysis. Small hidden interstitial insertions resulting in an AML1/ETO rearrangement were detected in five (8.5%) of the 59 patients, whereas the sixth patient showed a cryptic three-way translocation. The insertions could be characterized as ins(21;8) in three patients and ins(8;21) in the remaining two. Additionally, three of the patients showed secondary chromosome aberrations leading to a higher complexity of the karyotype. In conclusion, the combination of more than one standard technique in the analysis of AML1/ETO is useful to reveal the overall frequency of cryptic chromosome rearrangements and permits a better understanding of the mechanisms involved in the generation of this fusion gene.

Adolescent↗
PubMed2004

Insertions generating the 5'RUNX1/3'CBFA2T1 gene in acute myeloid leukemia cases show variable breakpoints.

Translocation t(8;21)(q22;q22) is a common karyotypic abnormality detected in about 15% of acute myeloid leukemia (AML) cases. The rearrangement results in fusion of the RUNX1 (also known as AML1) and CBFA2T1 (also known as ETO) genes, generating a 5'RUNX1/3'CBFA2T1 transcriptionally active fusion gene on derivative chromosome 8, but some cases with ins(21;8) and ins(8;21) have been observed. However, a detailed breakpoint characterization of the insertion events has never been reported. In the present article, we describe six insertion events among 82 (7.3%) AML cases characterized by the RUNX1/CBFA2T1 fusion. Using FISH experiments with appropriate bacterial artificial chromosome (BAC) and P1 artificial chromosome (PAC) probes, we were able to perform a detailed molecular cytogenetic characterization of one case with ins(8;21) and five with ins(21;8). Our analysis revealed that insertions generating the 5'RUNX1/3'CBFA2T1 gene showed variable breakpoints; the size of the inserted elements ranged from 2.4 to 44 Mb.

Core Binding Factor Alpha 2 Subunit↗
PubMed2005

MYND-less splice variants of AML1-MTG8 (RUNX1-CBFA2T1) are expressed in leukemia with t(8;21).

The AML1-MTG8 fusion gene is generated by chromosome translocation t(8;21), which is frequently observed in acute myeloid leukemia. The fusion gene produces a chimeric transcription factor that suppresses the expression of AML1-target genes via the MTG8 part of the chimeric protein, which is thought to be the primary cause of leukemia. The C-terminal region of MTG8 contains the MYND domain, represented by highly conserved zinc-finger-like protein motifs, and is known to interact with corepressor proteins. We found that, instead of the MYND domain, an alternative last exon of MTG8 encoding 27 amino acids in-frame is expressed naturally in human adult testis and in several leukemia cell lines. This type of alternative splicing also occurred in the AML1-MTG8 fusion gene at high levels in leukemia cell lines with t(8;21), as well as in blast cells of leukemia patients with t(8;21). The variant proteins of both MTG8 and AML1-MTG8 reduced transcriptional repressor activity in a mammalian two-hybrid assay. However, mixed expression of these variants with wild-type MTG8 recovered their repressor activity, suggesting that these variants also act as repressors in vivo where wild-type MTG8 and other family members exist in abundance. On the other hand, the MYND-less variants acquired a higher affinity for binding to MTG8 and formed a multimer, whereas the wild-type protein forms a dimer. Thus, expression of the MYND-less variants by the dysregulation of splicing machinery, which stimulates the oligomerization of fusion proteins in leukemia cells, may enhance malignant conversion of hematopoietic cells.

Alternative Splicing↗
PubMed1993

The t(8;21) translocation in acute myeloid leukemia results in production of an AML1-MTG8 fusion transcript.

The t(8;21) translocation is one of the most frequent chromosome abnormalities in acute myeloid leukemia. It has been shown that the t(8;21) breakpoints on chromosome 21 cluster within a single specific intron of the AML1 gene, which is highly homologous to the Drosophila segmentation gene runt. Here we report that this translocation juxtaposes the AML1 gene with a novel gene, named MTG8, on chromosome 8, resulting in the synthesis of an AML1-MTG8 fusion transcript. The fusion protein predicted by the AML1-MTG8 transcript consists of the runt homology region of AML1 and the most part of MTG8, which contains putative zinc finger DNA binding motifs and proline-rich regions constituting a characteristic feature of transcription factors. The MTG8 gene is not expressed in normal hematopoietic cells, whereas AML1 is expressed at high levels. Our results indicate that the production of chimeric AML1-MTG8 protein, probably a chimeric transcription factor, may contribute to myeloid leukemogenesis.

Acute Disease↗
PubMed2003

Gfi-1 attaches to the nuclear matrix, associates with ETO (MTG8) and histone deacetylase proteins, and represses transcription using a TSA-sensitive mechanism.

Gfi-1 and Gfi-1B can repress transcription and play important roles in hematopoietic cell survival and differentiation. Although these proteins are known to bind DNA through a C-terminal zinc-finger domain and may require an N-terminal SNAG domain (SNAIL/Gfi-1) to repress transcription, the mechanism by which Gfi-1 and Gfi-1B act is unknown. A first step towards understanding the mechanism by which these proteins repress transcription is to identify interacting proteins that could contribute to transcriptional repression. ETO (also termed MTG8), was first identified through its involvement in the (8;21) translocation associated with acute myelogenous leukemia. It attaches to the nuclear matrix and associates with histone deacetylases and the co-repressors N-CoR, SMRT, and mSin3A, and may act as a co-repressor for site-specific transcriptions factors. In this report we demonstrate that Gfi-1 interacts with ETO and related proteins both in vitro and in vivo and with histone deacetylase proteins in vivo. We observed that a portion of Gfi-1 and Gfi-1B associated with the nuclear matrix, as is the case with ETO. Moreover, Gfi-1 and ETO co-localize to punctate subnuclear structures. When co-expressed in mammalian cells, Gfi-1 associates with histone deacetylse-1 (HDAC-1), HDAC-2, and HDAC-3. These data identify ETO as a partner for Gfi-1 and Gfi-1B, and suggest that Gfi-1 proteins repress transcription through recruitment of histone deacetylase-containing complexes.

Cells, Cultured↗
PubMed1998

Polymorphism in the 3' untranslated region of MTG8 is associated with obesity in Pima Indian males.

Obesity has a genetic component and predisposes for the development of type 2 diabetes mellitus. One approach to identifying new candidate genes for obesity is to explore potential regulatory factors expressed in fat tissue that may play a role in adipocyte development or metabolic control. Because we found relatively abundant mRNA levels of the putative transcription factor MTG8 in human adipose tissue, a polymorphic microsatellite marker in the 3' untranslated region of this gene was genotyped in 281 Pima Indians, a population with one of the highest reported rates of obesity. We detected a male-specific association with age-adjusted percentage body fat (p = 0.0002), body mass index (p = 0.01), waist circumference (p = 0.008), and thigh circumference (p = 0.02). Comparative analysis of all 13 MTG8 exons in 30 Pimas did not reveal any genetic variants which could explain the association with obesity in males.

Alleles↗