EVI1 Gene Research Articles

Arsenic Trioxide Targets the MDS1 and EVI1- Containing Oncoproteins for Degradation by Proteasome Dependent and Independent Mechanisms.

The EVI1 gene was first identified as a common site of retroviral integration in murine myeloid tumors and expresses a zinc finger-containing transcriptional regulator. It also encodes for MDS1/EVI1, a longer, alternatively spliced form of EVI1. In blood development EVI1 is normally expressed in hematopoietic stem cells (HSCs) but not in committed cells. However, aberrant expression of EVI1 or MDS1/EVI1 has been frequently found in myelodysplastic syndrome (MDS), and in cases of chronic myeloid leukemia during blast phase (CML-BP) and acute myelogenous leukemia (AML). In addition, the MDS1/EVI1 gene fuses to the AML1 (Runx1) gene as a consequence of the t(3;21)(q26;q22) translocation found in patients with CML-BP and therapy related MDS and AML, producing the AML1/MDS1/EVI1 (AME) or AML1/MDS1 fusion genes. We have previously shown that expression of the AME fusion protein in mouse bone marrow cells by retroviral transduction impaired hematopoiesis and eventually induced an AML. EVI1 was also reported to induce MDS in mice. These results indicate that the EVI1 and AME oncoproteins play causal roles in t(3;21)- and EVI1-positive hematological malignancies. Therapies targeting oncoproteins that play causal roles in tumorigenesis have been shown to be effective in treating cancers. Arsenic trioxide (ATO) has been found as an effective treatment for patients suffering from acute promyelocytic leukemia (APL) that harbor the t(15;17) translocation. The success of ATO in treating APL patients has prompted many to consider expanding the drug's use to target other translocations and/or mutations found in hematological malignancies. In this study we found that ATO could target the EVI1, MDS1/EVI1, AML1/MDS1 and AME oncoproteins for degradation. We further found that ATO targeted EVI1 for degradation through the ubiquitin-proteasome pathway. Interestingly, ATO induced degradation of MDS1-containing oncoproteins via a proteasome-independent mechanism. Consistent with the downregulation of AME, the ATO treatment induced differentiation and apoptosis in AME leukemic cells in vitro and reduced splenomegally in mice transplanted with AME leukemic cells. Our results suggest that ATO could be used as a part of targeted therapy for AME-, AML1/MDS1-, MDS1/EVI1- and EVI1-positive hematological malignancies. We are currently studying the mechanism of increased ubiquitination of EVI1 by ATO, as well as the proteasome-independent mechanism by which ATO downregulates MDS1-containing proteins. Such research will help further the identification of new therapeutic targets and the development of new therapeutic strategies for human cancer.

Regulation of the expression of the oncogene EVI1 through the use of alternative mRNA 5′-ends

The EVI1 gene plays important roles in development and leukemogenesis. Recently, human EVI1 has been shown to give rise to at least six different mRNA variants with alternative 5′-ends, only some of which are conserved in mice. In order to gain a basic understanding of the regulation and potential biological importance of these alternative transcripts, we confirmed their expression by Northern blot, and, using real time quantitative RT-PCR, compared their abundance and stability under different conditions. The general expression patterns of the EVI1 5′-end variants in a panel of 20 human tissues were similar, but particularly high or low levels of some of them were noted in certain tissues. Pronounced differences in the expression of the 5′-end variants were noted in response to all- trans retinoic acid: in a human teratocarcinoma cell line, only the EVI1 transcript variants containing alternative exons 1a and 1b were upregulated in response to this agent. This induction required transcriptional activity of RNA polymerase, but was also associated with a substantial increase in the stability of these mRNA variants.

Oligomerization of Evi-1 regulated by the PR domain contributes to recruitment of corepressor CtBP

Evi-1 is a transcription factor that is implicated in leukemic transformation of hematopoietic cells. Two distinct alternative forms, Evi-1a and Evi-1c, are generated from the EVI-1 gene. Whereas Evi-1a is widely recognized as an oncoprotein, a role for Evi-1c, which has an additional PR domain in the amino-terminus of Evi-1a, in leukemogenesis, has not been elucidated thus far. Aberrant oligomerization of transcription factors has recently emerged as a prevalent mechanism for activating their oncogenic potential in hematopoietic malignancies. Here, to study the mechanisms that underlie Evi-1-mediated oncogenesis, we investigated formation of oligomeric complexes by the Evi-1 proteins. We show that Evi-1a forms homo-oligomers, whereas Evi-1c exclusively exists as a monomer in mammalian cells. Remarkably, Evi-1c has lost the ability to interact with CtBP, a transcriptional corepressor that associates with Evi-1a. As a consequence, the ability of Evi-1c to repress transforming growth factor-beta (TGF-beta) signaling is significantly abrogated. These results identify a novel function of a PR domain to regulate oligomerization of transcription factors and suggest that homo-oligomerization may play a critical role in corepressor recruitment by the Evi-1 proteins. In addition, we found that the chimeric oncoprotein acute myelocytic leukemia (AML)1-Evi-1, generated in t(3;21) leukemia, also forms homo-oligomers and hetero-oligomers with Evi-1a, while it did not interact with Evi-1c. Consistent with the results, repression of TGF-beta by AML1-Evi-1 was significantly enhanced by Evi-1a, whereas it was hardly affected by the presence of Evi-1c. These results suggest that oligomerization may contribute to the oncogenic potential of Evi-1-containing proteins.

Evi-1 expression in Xenopus

The Evi-1 gene was first identified as a site for viral integration in murine myeloid leukemia. Evi-1 is a zinc finger transcription factor that has been implicated in the development of myeloid neoplasia. In humans, disruption of the Evi-1 locus, by chromosomal rearrangements, is associated with myeloid leukemia and myelodyplastic syndromes. Here, we report the cloning and developmental pattern of expression of Xenopus Evi-1. xEvi-1 is expressed during oogenesis and during embryonic development. In situ hydridization reveals that xEvi-1 has a dynamic expression profile during early embryonic development. Expression of Evi-1 is detected by in situ hybridization in the pronephric tissue, the brain and in neural crest derivatives of the head and neck.

AML-M6 Associated with Diabetes Insipidus: Potential Role of Hypophyseal Dysfunction, Monosomy-7 and Impairment of TGF-β1 Signaling in Erythroleukemogenesis.

AML with erythroid differentiation (M6), is usually caused by leukaemic transformation of MDS or following drug therapy. AML is rarely associated with diabetes insipidus(DI). Numerous explanations for the link between DI and M6 have been proposed, the most common being leukaemic infiltration of the pituitary gland. Two cases of M6 with MRI evidence of pituitary damage are presented, in one an over expression of EVI-1 and in both a low circulating level of TGF-ß1 was observed. Interestingly, following chemotherapy the DI resolved.#1, a 27 year old man with an 8 month history of DI was diagnosed of M6 and monosomy 7. Brain-MRI showed an empty sella. Serological investigations prior to chemotherapy showed; normal GnRH and TRH-TSH stimulation tests, elevated FSH, elevated erythropoietin, low Vitamin D3, normal calcemia and parathormone and low TGF- ß 1. Partial morphological remission and resolution of DI were observed after AML chemotherapy. An over expression of EVI-1 locus was found on RT-PCR. The patient had further AML therapy, followed by a sibling BMT. He died 3 months later from interstitial pneumonitis.#2, a 51 years old woman admitted with anaemia and a one-year history of polyuria and polydipsia. She was diagnosed with M6 with complex karyotype, a diagnosis of DI was made post serological investigations. Brain MRI showed smooth alterations of the hypophyseal peduncle. Investigations post admission revealed: normal GnRH and TRH-TSH stimulation tests, elevated erythropoietin, low Vitamin D3, normal calcemia and parathormone and low TGF-ß1. Partial morphological remission and resolution of DI was observed following standard AML chemotherapy. Despite further chemotherapy and an autologous BMT with pulses of interleukin 2, progressive disease was observed. The patient died of sepsis after BMT.To our knowledge, this is the first documented case of AML-M6 linked with DI and monosomy-7. We have shown for the first time a significant diminished TGF-ß1 serum levels. Furthermore, the higher EVI-1 gene expression level in patient 1 may indicate a selective and additional block of TGF-ß1 signal transduction pathway (STP). Kurokawa et al. have shown that EVI-1gene product can bind and inhibit Smad-3, a mediator of STP. These findings suggest an interesting association in these leukemic patients. Monosomy 7 may be a more general cytogenetic abnormality associated with AML and DI. Also, Monosomy 7 may play an important role in the pathobiology of M6 associated with DI. Haemopoietic stem cells (HSC's) with such marker could be committed into different sub lineages, as shown in mixed lineage leukemia's (Rovigatti et al.). Interestingly, human myeloid precursor cells display CD34+ on their surface, as receptor for TGF-ß1 and both described cases were CD-34+. The significant lower TGF-ß1 serum levels could explain why HSC's in this compartment stop cycling and are allowed to shift to and to be committed towards erythroid blasts. High levels of erythropoietin in these patients are consistent with this shift. EVI-1 gene over expression in patient #1 could also inhibit TGF-ß1 STP, favouring shifting into the erythroid compartment.In conclusion, we suggest an interesting association between TGF-ß1 STP/hypophyseal damage and AML-M6 + DI with recurrent abnormal karyotypic patterns. Further studies are warranted to dissect the underlying molecular mechanism.

Oligomerization of Evi-1 Contributes to Recruitment of Transcriptional Corepressor CtBP and Repression of TGF-β Signaling.

Aberrant oligomerization of transcription factors has recently emerged as a prevalent mechanism for activating their oncogenic potential in hematopoietic malignancies. Furthermore, for some chimeric transcription factors generated in leukemia-associated chromosomal translocations including PML-RARα and AML1-ETO, it has also been proposed that aberrant recruitment of corepressor complexes caused by oligomerization is critical for the development of leukemia. Among leukemia-associated transcription factors is Evi-1, which is frequently activated in cases with myeloid malignancies. Evi-1a and Evi-1c, with the latter also called MDS1-Evi-1, are two alternative forms derived from the Evi-1 gene. Evi-1a is a Zn finger transcription factor which recruits CtBP as a transcriptional corepressor and possesses the ability to repress TGF-β signaling. Amino-terminal to the Evi-1a, Evi-1c harbors an additional sequence similar to the PR domain, which was originally identified in PRDI-BF1 and RIZ1. In contrast to frequent upregulation of Evi-1a in myeloid malignancies, expression of Evi-1c widely varies depending on cases. Thus, although several lines of evidence suggest that Evi-1a is involved in leukemic transformation of hematopoietic cells, a role for Evi-1c in leukemogenesis has remained elusive. To elucidate the mechanism that underlies leukemogenesis by Evi-1, we investigated the ability of Evi-1 proteins to form oligomeric complexes. We found that Evi-1a forms a homo-oligomer in mammalian cells, whereas Evi-1c exclusively exists as a monomer. Remarkably, Evi-1c has lost the ability to interact with CtBP and failed to efficiently repress TGF-β signaling. We previously reported that AML1-Evi-1, a chimeric transcription factor generated in t(3;21) leukemia, interacts with CtBP as Evi-1a does. In the current study, we also found that AML1-Evi-1 forms a homo-oligomer, suggesting a close relationship between oligomer formation and CtBP binding. Taken together, these data indicate that homo-oligomerization may contribute to the oncogenic potential of the Evi-1 proteins by regulating the interaction with CtBP. These results also identify a novel function of PR domain to dictate oligomerization of transcription factors.

Molecular Epidemiological Study in Pediatric Acute Myeloid Leukemia.

It is hypothesized that AML arises from two cooperative types of mutations: type I mutations mainly induce proliferation and type II mutations involved in the maturation arrest. AML is a rare disease in children and few molecular data are available on pediatric AML. We therefore studied N-RAS, K-RAS, FLT3-ITD, FLT3 , C-KIT mutations (type I), and CEBPA mutations (type II) as well as FLT3, EVI-1 and WT1 gene expression in 77 de novo AML.Patients and methods: All the patients (aged 1 month-17 years, median age: 6.9 years, male/female ratio 1.26) were treated for de novo AML between 1995 and 2003 in two French institutions and prospectively enrolled in LAM91, LAM01 and APLs French collaborative protocols. According to the FAB classification the repartition was: M0:6.5%, M1: 5.2%, M2: 22%, M3: 13%, M4 :14.3%, M5 :30%, M7: 6.5% and unclassified :2.5%. Cytogenetics features according to the MRC classification were favorable, intermediate or poor in 25% (t(8;21) n=5; t(15,17) n=8, inv(16) n=5), 65% (normal n=20 and 11q23 abnormalities n=15) and 10% (−7, n=4) respectively. With a median follow-up of 26 months (range 2–98 months), Complete Remission was obtained in 92% (71/77) of patients, OS was 71% and EFS 61%. CEBPA, N-RAS, K-RAS, C-KIT and FLT3 mutations detection was performed by direct sequencing. FLT3, EVI-1 and WT1 transcripts were quantified by RQ-PCR.Results: (1) Frequency of N-RAS and K-RAS mutations were 11% (8/75) and 16% (12/75) respectively. RAS-mutated patients belonged to favorable (30%), intermediate (60%) and poor (10%) cytogenetic subgroups. In univariate analysis only N-RAS mutations is associated with adverse outcome (OS 37% vs 79%, p<0.05). (2) CEBPA mutations were found in 8% (6/75), mostly belonged to the intermediate cytogenetic risk subgroup (66%). (3) C-KIT mutations were observed in 4% (4/75) always associated with fusion CBFb/MYH11 transcript and excellent outcome. (4) FLT3-ITD and FLT3 Asp835 mutations were obtained in 12% (9/74) and 4% (3/74) of patients respectively. Cytogenetic subgroups were favorable (33%) and intermediate (67%). (5) At diagnosis FLT3 overexpression was detected in 34% (24/70) and 11q23 abnormalities were associated in 7/24 patients. (6) EVI-1 overexpression was found in 21% (16/76), belonged to intermediate (85%) and poor (15%) cytogenetic subgroups with a significant frequency in monosomy 7 (p<0.025). The EVI-1 expression was specifically expressed (p<0.001) in M5 and M7-FAB subtypes. (7) WT1 overexpression was detected in 81% (62/76).Conclusion: In total, 48% of de novo AML in children had a mutation in N-RAS, K-RAS, FLT3-ITD, FLT3 Asp835, C-KIT or CEBPA with a high frequency of RAS mutations (27%) compared to adult AML and a significantly bad survival. Additional gene expression quantification of EVI-1, FLT3 and WT1 allows MDR detection in 95% of patients.

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.

14. Absence of T-Shaped Structure and Deletions of B and C Hairpins Have Minimal Effects on Essential Functions of AAV Inverted Terminal Repeats

Recent reports on severe adverse effects induced by retroviral integration into the LMO2 proto-oncogene in humans and the demonstration of preferential AAV serotype 2 (rAAV2) vector integration into genes in our previous study with a limited number of integration sites in mice1, have raised a possible concern about rAAV-mediated insertional mutagenesis. In order to further address this issue, we have expanded our previous study and performed a high-throughput analysis of rAAV2 integration sites isolated from in vivo selected hereditary tyrosinemia type I (HTI) mouse hepatocytes transduced with a human fumaryl acetoacetate hydrolase (FAH)-expressing rAAV2 shuttle vector, AAV-EF1α-hFAH.AOS. Briefly, total liver DNA was isolated from 4 HTI mice that underwent a 7-month in vivo selection of transplanted hepatocytes isolated from the donor HTI mice having received 3.0×1011 vector genomes of AAV-EF1α-hFAH.AOS via the portal vein and undergone an 8-week in vivo selection. The whole proviral rAAV2 vector genome and flanking genomic DNA sequences were isolated as a plasmid from the liver DNA by a plasmid rescue technique as previously described1. All four mice had a different donor HTI mouse, therefore, any integrations found in different mice should be considered as independent integration events. To date, we have characterized 307 independent integration events and found: 1) 191 of 307 (62%) of integrations occurred in genes; 2) there was a strong bias toward integrating into host genomes around transcription start sites, as has been observed in murine leukemia virus integrations (approximately a quarter of the total rAAV2 integrations occurred within ±1 kb from transcription start sites); 3) rAAV integration involved large genomic deletions of over 1 kb in 15% of the cases, suggesting that such large deletions may not be rare events associated with rAAV2 integration. The most striking finding was that among the 307 independent integration events, at least 4 integrations from 3 different mice (1.5 % of total integrations and 2.1 % of the genes targeted by rAAV) were found within a 30-kb region in the mouse ubiquitin C gene, and at least 4 integrations from 4 different mice fell on another sequence stretch of approximately 2.5 kb. Our preliminary analysis by restriction enzyme mapping and sequencing over a thousand base pairs has indicated that the hot spot of 2.5 kb in length likely resides in a proto-oncogene, the Evi-1 gene. It should be noted that murine leukemia found in a retroviral gene marking study was induced by insertional mutagenesis at this locus. Although the oncogenic potential of this gene in the liver is not known, overexpression of this gene can transform not only hematopoietic cells but also other cell types. Thus, the preliminary results in our high-throughput rAAV2 integration site analysis further emphasize the importance of pursuing the study on the elucidation of the mechanisms of rAAV vector integration and its consequences in the host.

A novel EVI1 gene family, MEL1, lacking a PR domain (MEL1S) is expressed mainly in t(1;3)(p36;q21)-positive AML and blocks G-CSF–induced myeloid differentiation

AbstractWe have identified a novel gene MEL1 (MDS1/EVI1-like gene 1) encoding a zinc finger protein near the breakpoint of t(1; 3)(p36;q21)-positive human acute myeloid leukemia (AML) cells. Here, we studied the structure, expression pattern, and function of MEL1 in leukemia cells. In this study, we have identified 3 transcription start sites, 1 in exon 1 and 2 in exon 2, and 2 kinds of translation products, 170 kDa (MEL1) and 150 kDa (MEL1S). Notably, the 150-kDa band of MEL1S was detected mainly in the t(1;3)(p36;q21)-positive AML cells. By immunoblot analysis and proteolytic mapping, it is suggested that the 150-kDa band of MEL1S in the leukemia cells is translated from the internal initiation codon ATG597 in exon 4 and is mostly lacking the amino-terminal PR domain of MEL1. By the cyclic amplification and selection of targets (CASTing) method for identifying consensus sequences, it was shown that the consensus sequences of MEL1 were included in 2 different consensus sequences for DNA-binding domain 1 and 2 (D1-CONS and D2-CONS) of EVI1. In reporter gene assays, MEL1S activated transcription via binding to D2-CONS; however, the fusion of MEL1 or MEL1S to GAL4 DNA-binding domain (DBD) made them GAL4 binding site–dependent transcriptional repressors. Moreover, overexpression of MEL1S blocked granulocytic differentiation induced by granulocyte colony-stimulating factor (G-CSF) in interleukin-3 (IL-3)–dependent murine myeloid L-G3 cells, while MEL1 could not block the differentiation. Thus, it is likely that overexpression of the zinc finger protein lacking the PR domain (EVI1 and MEL1S) in the leukemia cells is one of the causative factors in the pathogenesis of myeloid leukemia.

Detection of NPM/ MLF1 fusion in t(3;5)-positive acute myeloid leukemia and myelodysplasia

Balanced translocations are rare in myelodysplasia (MDS) and acute myeloid leukemia (AML) with multilineage dysplasia; however, the t(3;5)(q25;q35) and insertion variant occur in a subset of patients. To evaluate the possible genes involved in this translocation, we studied 6 cases with a t(3;5) by fluorescence in situ hybridization with probes directed against the nucleophosmin ( NPM), EVI1, and Ribophorin genes, as well as a newly developed myeloid leukemia factor 1 ( MLF1) BAC clone. The histologic spectrum of the cases was variable, ranging from refractory cytopenia with multilineage dysplasia to AML with multilineage dysplasia in the World Health Organization classification. An NPM/ MLF1 fusion was identified in 5 of 6 cases, whereas the EVI1 and Ribophorin genes were not involved in any of the cases. The NPM/ MLF1-positive cases were predominantly young adult males (median age, 33 years) who responded well to hematopoietic stem cell transplantation. These findings suggest that an NPM/ MLF1 fusion is the primary molecular abnormality in t(3;5) MDS and AML with multilineage dysplasia, and also that cases with NPM/ MLF1 may be clinically distinct from other MDS-associated disease.

EVI1 Is Expressed in Megakaryocyte Cell Lineage and Enforced Expression of EVI1 in UT-7/GM Cells Induces Megakaryocyte Differentiation

3q21q26 syndrome, an acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) with chromosomal translocations or inversions between the bands 3q21 and 3q26, is frequently associated with dysmegakaryocytopoiesis and increased platelet counts at the initial diagnosis. Since the EVI1 gene at 3q26 is transcriptionally activated in 3q21q26 syndrome, we assessed the role of EVI1 gene expression in the abnormal megakaryocytic differentiation in 3q21q26 syndrome. RT-PCR analysis of various types of hematopoietic cells revealed that the EVI1 gene is expressed specifically in CD34+ cells, megakaryocytes, and platelets. UT-7 is a human immature megakaryoblastic leukemia cell line with dependence for the growth on granulocyte–macrophage colony-stimulating factor (GM-CSF) (designated at UT-7/GM) and with a differentiation capacity to erythroid (UT-7/EPO) and megakaryocytic lineages (UT-7/TPO) by erythropoietin (EPO) and thrombopoietin (TPO), respectively. Among three UT-7 sublines, UT-7/GM, UT-7/EPO, and UT-7/TPO, expression of the EVI1 gene was detected at low levels in UT-7/GM and UT-7/EPO cells, but was detected at a higher level in UT-7/TPO cells. When UT-7/GM cells were cultured with TPO, the level of EVI1 expression was increased, along with increased numbers of polynuclear megakaryocytes and expression of the platelet factor 4 (PF-4) gene. Furthermore, forced expression of the EVI1 gene in UT-7/GM cells changed their morphology to polynuclear megakaryocytes, stopped their growth, and induced cell death within a month. These data indicate that expression of the EVI1 gene is involved in progression of megakaryocytic differentiation and, thus, the dysmegakaryocytopoiesis in 3q21q26 syndrome could be partly due to an enhanced differentiation capacity of leukemia cells and/or megakaryocytes by constitutive expression of the EVI1 gene.

Cytogenetic and Molecular Genetic Evolution of Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is genetically characterized by the presence of the reciprocal translocation t(9;22)(q34;q11), resulting in a BCR/ABL gene fusion on the derivative chromosome 22 called the Philadelphia (Ph) chromosome. In 2–10% of the cases, this chimeric gene is generated by variant rearrangements, involving 9q34, 22q11, and one or several other genomic regions. All chromosomes have been described as participating in these variants, but there is a marked breakpoint clustering to chromosome bands 1p36, 3p21, 5q13, 6p21, 9q22, 11q13, 12p13, 17p13, 17q21, 17q25, 19q13, 21q22, 22q12, and 22q13. Despite their genetically complex nature, available data indicate that variant rearrangements do not confer any specific phenotypic or prognostic impact as compared to CML with a standard Ph chromosome. In most instances, the t(9;22), or a variant thereof, is the sole chromosomal anomaly during the chronic phase (CP) of the disease, whereas additional genetic changes are demonstrable in 60–80% of cases in blast crisis (BC). The secondary chromosomal aberrations are clearly nonrandom, with the most common chromosomal abnormalities being +8 (34% of cases with additional changes), +Ph (30%), i(17q) (20%), +19 (13%), –Y (8% of males), +21 (7%), +17 (5%), and monosomy 7 (5%). We suggest that all these aberrations, occurring in >5% of CML with secondary changes, should be denoted major route abnormalities. Chromosome segments often involved in structural rearrangements include 1q, 3q21, 3q26, 7p, 9p, 11q23, 12p13, 13q11–14, 17p11, 17q10, 21q22, and 22q10. No clear-cut differences as regards type and prevalence of additional aberrations seem to exist between CML with standard t(9;22) and CML with variants, except for slightly lower frequencies of the most common changes in the latter group. The temporal order of the secondary changes varies, but the preferred pathway appears to start with i(17q), followed by +8 and +Ph, and then +19. Molecular genetic abnormalities preceding, or occurring during, BC include overexpression of the BCR/ABL transcript, upregulation of the EVI1 gene, increased telomerase activity, and mutations of the tumor suppressor genes RB1, TP53, and CDKN2A. The cytogenetic evolution patterns vary significantly in relation to treatment given during CP. For example, +8 is more common after busulfan than hydroxyurea therapy, and the secondary changes seen after interferon-alpha treatment or bone marrow transplantation are often unusual, seemingly random, and occasionally transient. Apart from the strong phenotypic impact of addition of acute myeloid leukemia/myelodysplasia-associated translocations and inversions, such as inv(3)(q21q26), t(3;21)(q26;q22), and t(15;17)(q22;q12–21), in CML BC, only a few significant differences between myeloid and lymphoid BC are discerned, with i(17q) and TP53 mutations being more common in myeloid BC and monosomy 7, hypodiploidy, and CDKN2A deletions being more frequent in lymphoid BC. The prognostic significance of the secondary genetic changes is not uniform, although abnormalities involving chromosome 17, e.g., i(17q), have repeatedly been shown to be ominous. However, the clinical impact of additional cytogenetic and molecular genetic aberrations is most likely modified by the treatment modalities used.

Rearrangements of Chromosome Band 3q21 in Myeloid Leukemia

Chromosome rearrangements affecting band 3q21, namely, the inv(3)(q21q26), the t(3;3)(q21;q26), and the t(1;3)(p36;q21), are associated with a particularly poor prognosis in myeloid leukemia or myelodysplasia. Originally, inv(3) and t(3;3) breakpoints have been reported to cluster in a region (breakpoint cluster region, BCR) of approximately 30 kb, which is located centromeric and downstream of the ribophorin I (RPN-I) gene. More recently, we established a PAC contig that includes the 3q21 BCR, and used these PAC clones to map breakpoints in patient samples by both metaphase and interphase fluorescence in situ hybridization (FISH) analysis. A significant proportion of inv(3) and t(3;3) breakpoints was located at sometimes considerable distances centromeric of the originally described BCR, in a region recently also implicated in t(1;3) rearrangements. These breakpoints may thus define a second, centromeric BCR (BCR-C), or extend the original 3q21 BCR to a size of approximately 100 kb. Activation of the EVI-1 gene in 3q26 by regulatory sequences of the housekeeping gene RPN-I has been suggested as a leukemogenic mechanism in patients with inv(3) and t(3;3). However, despite a number of characteristics that make EVI-1 an attractive candidate oncogene, its biological properties fail to fully explain the phenotype of leukemias carrying 3q rearrangements. Several additional candidate genes have been identified in or near the 3q21 breakpoint region, but their possible contribution to the characteristics of leukemias with 3q21 rearrangements remains to be explored.

Donor lymphocyte infusion followed by interferon-α plus low dose cyclosporine A for modulation of donor CD3 cells activity with monitoring of minimal residual disease and cellular chimerism in a patient with first hematologic relapse of chronic myelogenous leukemia after allogeneic bone marrow transplantation

A 15-year-old girl with Ph-positive chronic myelogenous leukemia in first chronic phase received bone marrow from her human leukocyte antigen matched brother. Twenty three months after bone marrow transplantation hematological relapse occured which was treated with two infusions of donor lymphocytes (DLI) (0.5×10 8 CD3/kg b.w./infusion). To enforce the graft-versus-leukemia effect (GvL), the first DLI was followed by administration of interferon-α (INF-α) 6×10 6 U/day for 30 days, whereas, after the second infusion INF-α was given at the same dose until hematological remission was achieved (80 doses). Minimal residual disease (MRD) was detected by conventional cytogenetics (Ph chromosome), fluorescence in situ hybridization (FISH) cytogenetics (BCR/ABL translocation) and reverse transcriptase-polymerase chain reaction (RT-PCR) Ecotropic virus integration site-1 (EVI-1 gene expression), whereas cellular chimerism was monitored by assessment of microsatellite markers PCR and Y-chromosomal DNA content FISH. When hematological remission was achieved the pancytopenia was observed and the cytogenetic and molecular investigations revealed only partial remission and mixed chimerism, however, with predominance of donor origin hematopoiesis. To diminish the myelosupressive effect of donor CD3 cells without switching-off the GvL effect, a low dose of cyclosporine A was given. Further observation revealed significant improvement of hematopoiesis with parallel gradual decline of MRD and increase of donor hematopoiesis up to complete chimerism. Graft-versus-host disease was not observed at any stage of the treatment.

A novel gene, MEL1, mapped to 1p36.3 is highly homologous to the MDS1/EVI1 gene and is transcriptionally activated in t(1;3)(p36;q21)-positive leukemia cells

AbstractThe reciprocal translocation t(1;3)(p36;q21) occurs in a subset of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), which is frequently characterized by trilineage dysplasia, in particular dysmegakaryocytopoiesis, and poor prognosis. Previously, the breakpoint cluster region (BCR) at 3q21 was identified within a 60-kilobase (kb) region centromeric to the BCR of 3q21q26 syndrome and that at 1p36.3 within a 90-kb region. In this study, genes were searched near the breakpoints at 1p36.3, and a novel gene was isolated that encoded a zinc finger protein with a PR domain, which is highly homologous to theMDS1/EVI1 gene. The novel gene, designated asMEL1(MDS1/EVI1-like gene 1), with 1257 amino acid residues is 64% similar in nucleotide and 63% similar in amino acid sequences to MDS1/EVI1 with the same domain structure. The MEL1 gene is expressed in leukemia cells with t(1;3) but not in other cell lines or bone marrow, spleen, and fetal liver, suggesting that MEL1 is specifically in the t(1;3)(p36;q21)-positive MDS/AML. On the basis of the positional relationship between the EVI1 and MEL1 genes in each translocation, it was suggested that both genes are transcriptionally activated by the translocation of the 3q21 region with the Ribophorin I gene. Because of the transcriptional activation of the EVI1 family genes in both t(1;3)(p36;q21)-positive MDS/AML and 3q21q26 syndrome, it is suggested that they share a common molecular mechanism for the leukemogenic transformation of the cells.

A novel gene, MEL1, mapped to 1p36.3 is highly homologous to the MDS1/EVI1 gene and is transcriptionally activated in t(1;3)(p36;q21)-positive leukemia cells

The reciprocal translocation t(1;3)(p36;q21) occurs in a subset of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), which is frequently characterized by trilineage dysplasia, in particular dysmegakaryocytopoiesis, and poor prognosis. Previously, the breakpoint cluster region (BCR) at 3q21 was identified within a 60-kilobase (kb) region centromeric to the BCR of 3q21q26 syndrome and that at 1p36.3 within a 90-kb region. In this study, genes were searched near the breakpoints at 1p36.3, and a novel gene was isolated that encoded a zinc finger protein with a PR domain, which is highly homologous to theMDS1/EVI1 gene. The novel gene, designated asMEL1(MDS1/EVI1-like gene 1), with 1257 amino acid residues is 64% similar in nucleotide and 63% similar in amino acid sequences to MDS1/EVI1 with the same domain structure. The MEL1 gene is expressed in leukemia cells with t(1;3) but not in other cell lines or bone marrow, spleen, and fetal liver, suggesting that MEL1 is specifically in the t(1;3)(p36;q21)-positive MDS/AML. On the basis of the positional relationship between the EVI1 and MEL1 genes in each translocation, it was suggested that both genes are transcriptionally activated by the translocation of the 3q21 region with the Ribophorin I gene. Because of the transcriptional activation of the EVI1 family genes in both t(1;3)(p36;q21)-positive MDS/AML and 3q21q26 syndrome, it is suggested that they share a common molecular mechanism for the leukemogenic transformation of the cells.

Identification of a zinc finger protein whose subcellular distribution is regulated by serum and nerve growth factor.

A subclass of zinc finger proteins containing a unique protein motif called the positive regulatory (PR) domain has been described. The members include the PRDI-BF1/Blimp-1 protein, the Caenorhabditis elegans egl-43 and EVI1 gene products, and the retinoblastoma interacting protein RIZ. Here we describe a member of this family, SC-1, that exhibits several distinctive features. First, SC-1 interacts with the p75 neurotrophin receptor and is redistributed from the cytoplasm to the nucleus after nerve growth factor (NGF) treatment of transfected COS cells. The translocation of SC-1 to the nucleus was specific for p75, as NGF binding to the TrkA receptor did not lead to nuclear localization of SC-1. Thus, SC-1 provides a downstream transducer for the effects of NGF through the p75 neurotrophin receptor. Under normal growth conditions, SC-1 was found predominantly in the cytoplasm. On serum-starvation, SC-1 also translocated into the nucleus. A direct correlation between nuclear expression of SC-1 with the loss of BrdUrd incorporation was observed. These results imply that SC-1 may be involved in events associated with growth arrest.

Activation of EVI1 transcripts with chromosomal translocation joining the TCRVbeta locus and the EVI1 gene in human acute undifferentiated leukemia cell line (Kasumi-3) with a complex translocation of der(3)t(3;7;8).

A cell line (Kasumi-3) established from acute myeloid leukemia (AML-M0) had unique phenotypes of undifferentiated leukemia cells with expression of both T cell and myeloid antigens. Kasumi-3 cells with t(3;7)(q26;q22) highly expressed a 6 kb transcript of EVI1, which is located on chromosome 3q26. Therefore, we further characterized the chromosomal breakpoint by pulsed-field gel electrophoresis near EVI1. We identified and isolated the chromosomal breakpoint at approximately 80 kb upstream from the 5' end of EVI1. Sequence analysis of the breakpoint revealed that the whole Vbeta region from T cell receptor beta (TCRbeta) at 7q35 was translocated to the upstream of EVI1. A 1.0 kb TCRbeta transcript was expressed in the Kasumi-3 cells, suggesting that TCRbeta rearrangement occurred as Dbeta-Jbeta joining events. Fluorescence in situ hybridization analysis revealed that the inverted chromosome 7q22-q35 segment between TCRbeta and the region proximal to the erythropoietin gene at 7q22 was translocated to the region distal to EVI1 in der(3). Since the telomeric region of chromosome 8 q was also translocated to the inverted chromosome 7q22-q35 segment in der(3), the chromosomal abnormalities of der(3) were defined as being der(3)t(3;7;8)(3pter-3q26::7q35-7q22::8q22 -8qter). It is suggested that a translocated enhancer element in the TCRbeta locus and/or loss of a negative regulatory element near EVI1 might function to enhance the EVI1 expression. Therefore, the enhanced EVI1 expression may contribute to the development of a subset of undifferentiated leukemia.

Evi-1 ZF1 DNA binding activity and a second distinct transcriptional repressor region are both required for optimal transformation of Rat1 fibroblasts.

The Evi-1 gene encodes a zinc finger transcriptional repressor protein that normally plays a role in development and is frequently activated in myeloid leukaemias. Evi-1 has two distinct DNA binding domains, ZF1 and ZF2, and a defined repressor domain but the function of the remainder of the molecule is unknown. The ZF2 and repressor domains have been shown to be required for transformation and we show here that ZF1 is also required. An alternative splice variant of Evi-1, designated delta324, encodes a protein which lacks a portion of the ZF1 DNA binding domain and the intervening amino acids 239-514 (designated IR) located between ZF1 and the repressor domain. We show that delta324 can neither bind ZF1, repress transcription through this site nor transform Rat1 fibroblasts. Reconstitution studies demonstrate that the defect in delta324 is partially complemented by recreating the ZF1 DNA binding activity. However, full function also requires the IR region which has transcriptional repressor activity. This study shows therefore, that ZF1, ZF2 and repressor domains and the IR region all contribute to the transformation efficiency of the Evi-1 protein.