Giuseppina Nucifora

EVI1 induces myelodysplastic syndrome in mice

Myelodysplasia is a hematological disease in which genomic abnormalities accumulate in a hematopoietic stem cell leading to severe pancytopenia, multilineage differentiation impairment, and bone marrow (BM) apoptosis. Mortality in the disease results from pancytopenia or transformation to acute myeloid leukemia. There are frequent cytogenetic abnormalities, including deletions of chromosomes 5, 7, or both. Recurring chromosomal translocations in myelodysplasia are rare, but the most frequent are the t(3;3)(q21;q26) and the inv(3)(q21q26), which lead to the inappropriate activation of the EVI1 gene located at 3q26. To better understand the role of EVI1 in this disease, we have generated a murine model of EVI1-positive myelodysplasia by BM infection and transplantation. We find that EVI1 induces a fatal disease of several stages that is characterized by severe pancytopenia. The disease does not progress to acute myeloid leukemia. Comparison of in vitro and in vivo results suggests that EVI1 acts at two levels. The immediate effects of EVI1 are hyperproliferation of BM cells and downregulation of EpoR and c-Mpl, which are important for terminal erythroid differentiation and platelet formation. These defects are not fatal, and the mice survive for about 10 months with compensated hematopoiesis. Over this time, compensation fails, and the mice succumb to fatal peripheral cytopenia.

Bacterial resistance ATPases: primary pumps for exporting toxic cations and anions

Bacterial plasmid resistance systems that maintain low intracellular levels of toxic heavy metals by pumping the substrates out as rapidly as they accumulate sometimes work at the biochemical level as efflux ATPases. The two systems responsible for arsenic and cadmium resistance have recently been sequenced. Comparison of the deduced amino acid sequences with those of better characterized ATPases has revealed certain structural and sequence similarities.

The EVI1 gene in myeloid leukemia

Leukemia is an acquired genetic disease caused by the accumulation of chromosomal abnormalities which modify either the biochemical property or the level of expression of proteins. Frequent genetic abnormalities identified in human leukemia are chromosomal rearrangements such as chromosomal translocations and inversions. Chromosome band 3q26 is the site of the breakpoint of recurring translocations and inversions observed in patients with myeloid leukemias. Two genes located at 3q26 have been implicated in development or progression of myeloid leukemia. They are MDS1 and EVI1. MDS1, first identified as part of a fusion transcript resulting from the t(3;21)(q26;q22), encodes a small protein of unknown function. EVI1 encodes a zinc finger protein inappropriately overexpressed by chromosomal rearrangements (in man) or by retroviral insertion (in the mouse). Both genes are rearranged by the t(3;21)(q26;q22) and by the t(3;12)(p13;q22). As a result of the translocation, they are expressed as fusion genes either with AML1 or with TEL. EVI1 and MDS1 are unusual in that they can either encode separate proteins, or they can be expressed as one protein which we named MDS1/EVI1. EVI1 and MDS1/EVI1 have opposite functions as transcription factors. In this report, we review the current information on the two genes, and on their involvement in myeloid leukemia.

MDS1/EVI1 enhances TGF-β1 signaling and strengthens its growth-inhibitory effect, but the leukemia-associated fusion protein AML1/MDS1/EVI1, product of the t(3;21), abrogates growth-inhibition in response to TGF-β1

MDS1/EVI1, located on chromosome 3 band q26, encodes a zinc-finger DNA-binding transcription activator not detected in normal hematopoietic cells but expressed in several normal tissues. MDS1/EVI1 is inappropriately activated in myeloid leukemias following chromosomal rearrangements involving band 3q26. The rearrangements lead either to gene truncation, and to expression of the transcription repressor EVI1, as seen in the t(3;3)(q21;q26) and inv(3)(q21q26), or to gene fusion, as seen in the t(3;21)(q26;q22) which results in the fusion protein AML1/MDS1/EVI1. This fusion protein contains the DNA-binding domain of the transcription factor AML1 fused in-frame to the entire MDS1/EVI1 with the exclusion of its first 12 amino acids. In this report, we have analyzed the response of the hematopoietic precursor cell line 32Dcl3, expressing either the normal protein MDS1/EVI1 or the fusion protein AML1/MDS1/EVI1, to factors that control cell differentiation or cell replication. The 32Dcl3 cells are IL-3-dependent for growth and they differentiate into granulocytes when exposed to G-CSF. They are growth-inhibited by TGF-β1. We show that whereas the expression of MDS1/EVI1 has no effect on granulocytic differentiation induced by G-CSF, expression of AML1/MDS1/EVI1 blocks differentiation resulting in cell death. This effect is similar to that previously described by others for 32Dcl3 cells that express transgenic Evi1. Furthermore, we show that whereas the expression of the fusion protein AML1/MDS1/EVI1 completely abrogates the growth-inhibitory effect of TGF-β1 and allows 32Dcl3 cells to proliferate, expression of the normal protein MDS1/EVI1 has the opposite effect, and it strengthens the response of cells to the growth-inhibitory effect of TGF-β1. By using the yeast two-hybrid system, we also show that EVI1 (contained in its entirety in MDS1/EVI1 and AML1/MDS1/EVI1) physically interacts with SMAD3, which is an intracellular mediator of TGF-β1 signaling. Finally, we have correlated the response of the cells to G-CSF or TGF-β1 with the ability of the normal and fusion proteins to activate or repress promoters which they can directly regulate by binding to the promoter site. We propose that mutations of MDS1/EVI1 either by gene truncation resulting in the transcription repressor EVI1 or by gene fusion to AML1 lead to an altered cellular response to growth and differentiation factors that could result in leukemic transformation. The different response of myeloid cells ectopically expressing the normal or the fusion protein to G-CSF and TGF-β1 could depend on the different transactivation properties of these proteins resulting in divergent expression of downstream genes regulated by the two proteins.

Small ubiquitin-like modifier conjugation regulates nuclear export of TEL, a putative tumor suppressor

Posttranslational modification by small ubiquitin-like modifier (SUMO) conjugation regulates the subnuclear localization of several proteins; however, SUMO modification has not been directly linked to nuclear export. The ETS (E-Twenty-Six) family member TEL (ETV6) is a transcriptional repressor that can inhibit Ras-dependent colony growth in soft agar and induce cellular aggregation of Ras-transformed cells. TEL is frequently disrupted by chromosomal translocations such as the t(12;21), which is associated with nearly one-fourth of pediatric B cell acute lymphoblastic leukemia. In the vast majority of t(12;21)-containing cases, the second allele of TEL is deleted, suggesting that inactivation of TEL contributes to the disease. Although TEL functions in the nucleus as a DNA-binding transcriptional repressor, it has also been detected in the cytoplasm. Here we demonstrate that TEL is actively exported from the nucleus in a leptomycin B-sensitive manner. TEL is posttranslationally modified by sumoylation at lysine 99 within a highly conserved domain (the “pointed” domain). Mutation of the sumo-acceptor lysine or mutations within the pointed domain that affect sumoylation impair nuclear export of TEL. Mutation of lysine 99 also results in an increase in TEL transcriptional repression, presumably because of decreased nuclear export. We propose that the ability of TEL to repress transcription and suppress growth is regulated by sumoylation and nuclear export.

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.

AML1 gene over-expression in childhood acute lymphoblastic leukemia

The present study was conducted on a series of 41 Egyptian children with newly diagnosed acute lymphoblastic leukemia (ALL) to investigate TEL and AML1 abnormalities. The TEL-AML1 fusion was observed in six patients both by RT-PCR and FISH analyses, with a frequency of 22.2% among the B-lineage group, whereas TEL deletion was seen by FISH analysis in seven patients (17.1%). By FISH analysis, nine patients (22%) showed evidence of extra AML1 copies. In five of these patients the extra copies were due to non-constitutional trisomy 21, whereas in the remaining four cases they were due to tandem AML1 copies on der(21), as evidenced by metaphase FISH. Unexpectedly however, enhanced AML1 expression levels were seen by real-time quantitative RT-PCR in 18 out of the 41 ALL patients (43.9%). This high level of AML1 expression could be an important factor contributing to the pathogenesis and progression of childhood ALL. One key mechanism for over-expression seems to be the extra copies of AML1, but other mechanisms may involve an alteration of the activity of the AML1 promoter. Here, we also report two novel findings. The first is an intragenic deletion of TEL exon 7 in a case of T cell ALL. This deletion creates a frame-shift and results in a truncated protein lacking the C-terminus that includes the ETS domain. This shorter TEL is presumably unable to bind DNA. The second finding is a rearrangement of AML1 in a case of T cell ALL due to t(4;21)(q31;q22). This is the first reported chromosomal translocation where AML1is rearranged in childhood T cell ALL.

Human Menkes X-chromosome disease and the staphylococcal cadmium-resistance ATPase: a remarkable similarity in protein sequences.

A search with the proposed amino acid translation product from the new ‘candidate gene’ for human Menkes disease against protein sequence libraries showed a remarkable similarity to that for the cadmium efflux ATPase from Staphylococcus aureus resistance plasmids. The Menkes sequence appears closer to the CadA Cd2+ sequence than to P‐type ATPases from animal sources. Menkes syndrome is an X‐chromosome invariably fatal disease that results from abberant copper metabolism. The gene that is defective in Menkes patients, i.e. the Menkes candidate gene, encodes a P‐type ATPase, whose properties satisfactorily explain the phenotype of the disease. P‐type ATPases are all cation pumps, either for uptake (e.g. the bacterial Kdp K+ ATPase), for efflux (e.g. the muscle sarcoplasmic reticulum Ca2+ ATPase), or for cation exchange (e.g. the animal cell Na+/K+ ATPase). These enzymes have a conserved aspartate residue that is transiently phosphorylated from ATP during the transport cycle, hence the name ‘P‐type’ ATPase. The Menkes sequence shares with the staphylococcal CadA ATPase those regions common to all P‐type ATPases and also an N‐terminal dithiol region that was proposed to be a ‘metal‐binding motif’. There are one or two copies of this motif in the available CadA sequences and six copies in the Menkes sequence.

Forced expression of the leukemia-associated gene EVI1 in ES cells: a model for myeloid leukemia with 3q26 rearrangements.

Chromosome band 3q26 is the locus of two genes, MDS1/EVI1 and EVI1. The proteins encoded by these genes are nuclear factors each containing two separate DNA-binding zinc finger domains. The proteins are identical, aside from the N-terminal extension of MDS1/EVI1, which is missing in EVI1. However, they have opposite functions as transcription factors. In contrast to MDS1/EVI1, EVI1 is often activated inappropriately by chromosomal rearrangements at 3q26 leading to inappropriate expression of the protein in hematopoietic cells and to myeloid leukemias, which are often characterized by abnormal megakaryopoiesis. We previously showed that the two proteins affect replication and differentiation of progenitor hematopoietic cell lines in opposite ways: whereas EVI1 inhibits the response of 32Dc13 cells to G-CSF and TGFβ1, MDS1/EVI1 has no effect on the G-CSF-induced differentiation of the 32Dc13 cells, and it enhances the growth-inhibitory effect of TGFβ1. In the present study, we analyzed the endogenous expression of the two genes during in vitro hematopoietic differentiation of murine embryonic stem (ES) cells and evaluated the effects of their forced expression on the ability of ES cells to produce differentiated hematopoietic colonies. We found that the expression of the two genes is independently and tightly controlled during differentiation. In addition, the forced expression of EVI1 led to a much higher rate of cell growth before and during differentiation, whereas the expression of MDS1/EVI1 repressed cell growth and strongly reduced the number of differentiated hematopoietic colonies. Finally, our study also found that the forced expression of EVI1 resulted in the differentiation of abnormally high numbers of megakaryocytic colonies, thus providing one of the first experimental models showing a clear correlation between inappropriate expression of EVI1 and abnormalities in megakaryopoiesis.

The role of EVI1 in normal and leukemic cells

One of the genes associated with both murine and human myeloid leukemia is EVI1 (ecotropic viral integration 1 site). EVI1 was first identified as a common locus of retroviral integration in myeloid tumors found in AKXD mice. The exact mechanism by which EVI1 induces leukemogenesis is not clear. Studies of the function of EVI1 in the bone marrow and in cell lines have shown that the inappropriate expression of EVI1 prohibits terminal differentiation of the bone marrow progenitor cells in granulocytes and erythroid cells, but strongly favors hematopoietic differentiation along the megakaryocytic lineage. We summarize recent data showing that EVI1 is a complex transcription factor with multiple functions, and this complexity is further demonstrated by the ability of EVI1 to interact with coactivators and corepressors and to abrogate cellular response to cytokines.