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).     

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.     

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

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

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.     

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.     

AML1-EVI1转基因斑马鱼模型的建立及对造血转录因子的调控

目的:通过建立表达急性髓系白血病(AML)1-同向性病毒整合位点(EVI)1融合基因斑马鱼模型,研究AML1-EVI1融合基因对造血转录因子的影响。方法:构建携带绿色荧光蛋白的AML1-EVI1质粒,显微注射到斑马鱼胚胎单细胞期,建立生殖系稳定转染的转基因模型,常规养殖到F2代。以F2代转基因斑马鱼作为实验对象,运用反转录(RT)-PCR验证AML1-EVI1融合基因的表达,外周血涂片、肾细胞涂片检测血细胞形态学改变,全胚胎原位杂交及荧光实时定量PCR(RFQ-PCR)检测融合基因对造血转录因子[GATA结合蛋白1(GATA1)、髓过氧化物酶(MPO)]的影响。结果:在性成熟146条嵌合表达的F0代斑马鱼中鉴定得到3条(2.1%)转基因阳性斑马鱼,其中雄鱼2条,雌鱼1条。外周血涂片及肾细胞涂片显示转基因斑马鱼出现造血细胞分化障碍,幼稚细胞增多,全胚胎原位杂交及RFQ-PCR均显示,与野生型相比,转基因型斑马鱼造血转录因子MPO基因表达量无论造血细胞发育早期还是晚期都明显下降,转录因子GATA1则在发育早期上调,晚期呈现明显下调。结论:AML1-EVI1融合基因通过调节转录因子抑制髓系分化,红系发育,促进原始细胞增殖。     

Involvement of the AML1 gene in the t(3;21) in therapy-related leukemia and in chronic myeloid leukemia in blast crisis

A nonrandom translocation between chromosomes 3 and 21, t(3;21)(q26.2;q22) has been detected in patients with a myelodysplastic syndrome or acute myeloid leukemia after treatment (t-MDS/t-AML) for a primary malignant disease and in chronic myelogenous leukemia in blast crisis (CML-BC). In these patients, the breakpoint on chromosome 21 is at band 21q22. This band is also involved in the t(8;21)(q22;q22) detected in 40% of the patients with acute myeloid leukemia subtype M2 (AML-M2) de novo who have an abnormal karyotype. In the t(8;21), the AML1 gene is the site of the breakpoint on chromosome 21. The AML1 gene is transcribed from telomere to centromere, and in the t(8;21) the 5' part of AML1 is fused to the ETO gene on chromosome 8 to produce the chimeric AML1/ETO on the der(8) chromosome. We found that AML1 is also rearranged in two t-AML patients and in one CML-BC patient with the t(3;21), but the breakpoints are approximately 40 to 60 kb downstream to those of AML-M2 patients. This region contains at least one additional exon of AML1, as determined by using an AML1 cDNA as a probe in Southern blot analysis. The t(3;21) breakpoints for the remaining patients could not be determined because, by fluorescence in situ hybridization analysis, the breaks are outside of the region covered by the available probes.     

AML1/MTG8 oncogene suppression by small interfering RNAs supports myeloid differentiation of t(8;21)-positive leukemic cells

The translocation t(8;21) yields the leukemic fusion gene AML1/MTG8 and is associated with 10%-15% of all de novo cases of acute myeloid leukemia. We demonstrate the efficient and specific suppression of AML1/MTG8 by small interfering RNAs (siRNAs) in the human leukemic cell lines Kasumi-1 and SKNO-1. siRNAs targeted against the fusion site of the AML1/MTG8 mRNA reduce the levels of AML1/MTG8 without affecting the amount of wild-type AML1. These data argue against a transitive RNA interference mechanism potentially induced by siRNAs in such leukemic cells. Depletion of AML1/MTG8 correlates with an increased susceptibility of both Kasumi-1 and SKNO-1 cells to tumor growth factor beta(1) (TGF beta(1))/vitamin D(3)-induced differentiation, leading to increased expression of CD11b, macrophage colony-stimulating factor (M-CSF) receptor, and C/EBP alpha (CAAT/enhancer binding protein). Moreover, siRNA-mediated AML1/MTG8 suppression results in changes in cell shape and, in combination with TGF beta(1)/vitamin D(3), severely reduces clonogenicity of Kasumi-1 cells. These results suggest an important role for AML1/MTG8 in preventing differentiation, thereby propagating leukemic blast cells. Therefore, siRNAs are promising tools for a functional analysis of AML1/MTG8 and may be used in a molecularly defined therapeutic approach for t(8;21)-positive leukemia.