Detection of t(3 ; 21) (q26 ; q22) with AML1/EVI1 mRNA during progression of myelodysplastic syndrome to acute myeloid leukemia]

A 74-year-old woman had myelodysplastic syndrome (MDS) in 1986. In June 1994, she suffered exacerbation of pancytopenia with no chromosomal abnormalities, but AML1/EVI1 chimeric mRNA was detected by RT-PCR. Two months later, an increase in bone marrow blasts (5%) was noted, and chromosomal analysis detected t(3 ; 21) (q26 ; 22), del(7) (q22), del(11) (q23). In 1995, the marrow blasts increased to 30% and the patient died of disease progression. The AML1/EVI1 gene has been shown to cause blast crisis in chronic myelogenous leukemia. This case suggested that the AML1/EVI1 gene may be involved in the progression of MDS together with del(7) (q22) and del(11) (q23).     

Expression of EVI1 in myelodysplastic syndromes and other hematologic malignancies without 3q26 translocations

The EVI1 gene encodes a zinc-finger, DNA-binding protein originally described as the transforming gene associated with a common ecotropic viral insertion site in myeloid leukemias. Previous studies demonstrated EVI1 expression in human leukemias in cases with 3q26 translocations, but not in normal blood or bone marrow. These studies also suggested an association between EVI1 expression and chromosome 7 deletion (del). Because of this association, we examined expression of EVI1 using RNA polymerase chain reaction (PCR) in patients with myelodysplastic syndromes (MDS) and acute leukemia with and without 3q26 translocations. EVI1 RNA was expressed in 29% of 34 (95% confidence interval, 20% to 50%) patients with the MDS subtypes refractory anemia (RA), refractory anemia with excess blasts (RAEB), or refractory anemia with excess blasts in transformation (RAEB-T). The vast majority of these cases occurred in patients with RAEB and RAEB-T. EVI1 expression was not detected in patients with chronic myelomonocytic leukemia (CMML), normal bone marrow or cord blood, or a variety of other hematologic malignancies. EVI1 RNA was detected in three of 18 patients with acute myelogenous leukemia (AML) and in two of four patients with acute promyelocytic leukemia (APL). Karyotypes showed that only one AML patient had karyotype 3q26 abnormalities, indicating that EVI1 expression is associated with cases that do not have structural abnormalities involving chromosome 3q26. These studies document for the first time the abnormal expression of EVI1 RNA by patients with MDS, and suggest an important role for EVI1 in the pathogenesis or progression of some myeloid malignancies.     

Dysplastic definitive hematopoiesis in AML1/EVI1 knock-in embryos

The AML1/EVI1 chimeric gene is created by the t(3;21)(q26;q22) chromosomal translocation seen in patients with leukemic transformation of myelodysplastic syndrome or blastic crisis of chronic myelogenous leukemia. We knocked-in the AML1/EVI1 chimeric gene into mouse Aml1 genomic locus to explore its effect in developmental hematopoiesis in vivo. AML1/EVI1/+ embryo showed defective hematopoiesis in the fetal liver and died around embryonic day 13.5 (E13.5) as a result of hemorrhage in the central nervous system. The peripheral blood had yolk-sac-derived nucleated erythroblasts but lacked erythrocytes of the definitive origin. Although E12.5 fetal liver contained progenitors for macrophage only, E13.5 fetal liver contained multilineage progenitors capable of differentiating into dysplastic myelocyte and megakaryocyte. No erythroid progenitor was detected in E12.5 or E13.5 fetal liver. Hematopoietic progenitors from E13.5 AML1/EVI1/+ fetal liver were highly capable of self-renewal compared with those from wild-type liver. Maintained expression of PU.1 gene and decreased expression of LMO2 and SCL genes may explain the aberrant hematopoiesis in AML1/EVI1/+ fetal liver.     

An activated receptor tyrosine kinase,TEL/PDGFbetaR,cooperates with AML1/ETO to induce acute myeloid leukemia in mice

The t(8;21)(q22;q22) translocation, occurring in 40% of patients with acute myeloid leukemia (AML) of the FAB-M2 subtype (AML with maturation), results in expression of the RUNX1-CBF2T1 [AML1-ETO (AE)] fusion oncogene. AML/ETO may contribute to leukemogenesis by interacting with nuclear corepressor complexes that include histone deacetylases, which mediate the repression of target genes. However, expression of AE is not sufficient to transform primary hematopoietic cells or cause disease in animals, suggesting that additional mutations are required. Activating mutations in receptor tyrosine kinases (RTK) are present in at least 30% of patients with AML. To test the hypothesis that activating RTK mutations cooperate with AE to cause leukemia, we transplanted retrovirally transduced murine bone marrow coexpressing TEL-PDGFRB and AE into lethally irradiated syngeneic mice. These mice (19/19, 100%) developed AML resembling M2-AML that was transplantable in secondary recipients. In contrast, control mice coexpressing with TEL-PDGFRB and a DNA-binding-mutant of AE developed a nontransplantable myeloproliferative disease identical to that induced by TEL-PDGFRB alone. We used this unique model of AML to test the efficacy of pharmacological inhibition of histone deacetylase activity by using trichostatin A and suberoylanilide hydroxamic acid alone or in combination with the tyrosine kinase inhibitor, imatinib mesylate. We found that although imatinib prolonged the survival of treated mice, histone deacetylase inhibitors provided no additional survival benefit. These data demonstrate that an activated RTK can cooperate with AE to cause AML in mice, and that this system can be used to evaluate novel therapeutic strategies.     

Acute myelogenous leukemia with the t(3;12)(q26;p13) translocation: case report and review of the literature

Translocations involving the EVI1/MDS1 gene at 3q26 and the TEL gene at 12p13 are comparatively common in acute leukemia, but a translocation between the two genes has been reported only in a handful of cases. We report an additional case of acute myelogenous leukemia (AML) preceded by a myelodysplastic syndrome (MDS) with the translocation t(3;12)(q26;p13) in a 36-year-old woman. The translocation was present early in the disease and long before the MDS progressed to AML 3 years after diagnosis. At the time of progression to AML, an additional chromosomal abnormality, a monosomy 22, was discovered. The patient was treated with the protocol MAQ, which comprised mitoxantrone, aracytine, and quinine, as her blasts expressed the p-glycoprotein, but she failed to obtain remission. A second treatment with the same protocol resulted in only minimal response. The patient was treated again with high-dose Ara-C and idarubicine in an attempt to achieve a response before allogeneic unrelated transplantation, but she did not respond to the treatment and died shortly thereafter. A review of the literature revealed 12 other cases of the t(3;12)(q26;p13) translocation. Characteristics of those cases are reviewed in this article.     

EVI1 is critical for the pathogenesis of a subset of MLL-AF9-rearranged AMLs

The proto-oncogene EVI1 (ecotropic viral integration site-1), located on chromosome band 3q26, is aberrantly expressed in human acute myeloid leukemia (AML) with 3q26 rearrangements. In the current study, we showed, in a large AML cohort carrying 11q23 translocations, that ～ 43% of all mixed lineage leukemia (MLL)-rearranged leukemias are EVI1(pos). High EVI1 expression occurs in AMLs expressing the MLL-AF6, -AF9, -AF10, -ENL, or -ELL fusion genes. In addition, we present evidence that EVI1(pos) MLL-rearranged AMLs differ molecularly, morphologically, and immunophenotypically from EVI1(neg) MLL-rearranged leukemias. In mouse bone marrow cells transduced with MLL-AF9, we show that MLL-AF9 fusion protein maintains Evi1 expression on transformation of Evi1(pos) HSCs. MLL-AF9 does not activate Evi1 expression in MLL-AF9-transformed granulocyte macrophage progenitors (GMPs) that were initially Evi1(neg). Moreover, shRNA-mediated knockdown of Evi1 in an Evi1(pos) MLL-AF9 mouse model inhibits leukemia growth both in vitro and in vivo, suggesting that Evi1 provides a growth-promoting signal. Using the Evi1(pos) MLL-AF9 mouse leukemia model, we demonstrate increased sensitivity to chemotherapeutic agents on reduction of Evi1 expression. We conclude that EVI1 is a critical player in tumor growth in a subset of MLL-rearranged AMLs.     

The ETO domain is necessary for the developmental abnormalities associated with AML1-ETO expression in the hematopoietic stem cell compartment in vivo

Translocation of the ETO gene on human chromosome 8 with the AML1 gene on chromosome 21 (AML1-ETO) is a recurrent cytogenetic abnormality associated with approximately 12% of acute myelogenous leukemia (AML) cases. To understand the contribution of the t(8;21) to AML, we transduced purified hematopoietic stem cells (HSC) with a retroviral vector that coexpressed AML1-ETO or just the AML1 portion (AML1d) of the translocation along with a green fluorescent protein reporter gene. Animals reconstituted with AML1-ETO-expressing cells exhibited many of the hematopoietic developmental abnormalities seen in the bone marrow of human patients with the t(8;21), although the animals did not develop acute leukemia. We noted a gradual increase in primitive myeloblasts that accounted for approximately 10% of bone marrow by 10 months posttransplant. Consistent with this observation was a 50-fold increase in myeloid colony-forming cells in vitro. In addition, accumulation of late stage metamyelocytes was observed in bone marrow along with an increase in immature eosinophil myelocytes that showed abnormal basophilic granulation. There was also a gradual increase in both the frequency and absolute number of AML1-ETO-expressing HSC so that by 10 months posttransplant, there were 29-fold greater HSC numbers than in transplant-matched control mice. These phenotypes were not observed in animals reconstituted with cells expressing only the DNA-binding domain of AML1, suggesting that the ETO domain is necessary to establish the developmental abnormalities associated with AML1-ETO expression in HSC.     

AML1-ETO rapidly induces acute myeloblastic leukemia in cooperation with the Wilms tumor gene, WT1

AML1-ETO, a chimeric gene frequently detected in acute myelogenous leukemia (AML), inhibits the differentiation of myeloid progenitors by suppressing genes associated with myeloid differentiation and increases the replating ability of clonogenic myeloid progenitors. However, AML1-ETO alone cannot induce AML and thus additional genetic events are required for the onset of AML. The Wilms tumor gene (WT1), which has been identified as the gene responsible for Wilms tumor, is expressed at high levels in almost all human leukemias. In this study, we have generated transgenic mice (WT1-Tg) that overexpress WT1 in hematopoietic cells to investigate the effects of WT1 on AML1-ETO-associated leukemogenesis. AML1-ETO-transduced bone marrow (BM) cells from WT1-Tg mice exhibited inhibition of myeloid differentiation at more immature stages and higher in vitro colony-forming ability compared with AML1-ETO-transduced BM cells from wild-type mice. Most importantly, all of the mice that received a transplant of AML1-ETO-transduced BM cells from the WT1-Tg mice rapidly developed AML. These results demonstrate that AML1-ETO may exert its leukemogenic function in cooperation with the expression of WT1.