TEL-AML1 Expression Research Articles

Expression differences in TEL-AML1 fusion gene in leukemia glucocorticoid-sensitive and -resistant cell lines.

We investigated the expression differences of the TEL-AML1 fusion gene in a leukemia glucocorticoid (GC)-sensitive cell line (CEM) and a GC-resistant cell line (Jurkat). Changes in TEL-AML1 expression before and after GC exposure were analyzed. Expression of GC-sensitive and GC-resistant leukemia cells following initial diagnosis and during treatment was simulated. Leukemia cells were divided into a GC-unexposed or a GC-exposed group. A methyl thiazolyl tetrazolium assay was used to detect cell proliferation inhibition, flow cytometry was used to observe cell apoptosis, reverse transcription-polymerase chain reaction was used to detect the mRNA expression of TEL-AML1 before and after exposure, and western blotting was used to analyze protein levels of TEL-AML1 before and after exposure. Inhibitory concentrations of 50% of cells in the Jurkat and CEM cells at 24 h were 382 and 9 mM, respectively, and at 48 h they were 216 and 2 mM. The proliferation inhibition effect of dexamethasone sodium phosphate on Jurkat cells was much lower than that on CEM cells. Jurkat cells showed obvious apoptosis after exposure to 100 mM dexamethasone sodium phosphate for 48 h. In the exposed group, Jurkat cells showed higher TEL-AML1 expression than did CEM cells (P < 0.05). In the unexposed group, TEL-AML1 gene expression in Jurkat cells was not affected by GC exposure (P > 0.05), while the CEM cells presented significant differences before and after exposure (P < 0.05). Sustained high expression of TEL-AML1 participated in and maintained the occurrence of GC resistance. Inhibition of TEL-AML1 may provide a new therapeutic approach to reverse GC resistance.

The impact of TEL-AML1 (ETV6-RUNX1) expression in precursor B cells and implications for leukaemia using three different genome-wide screening methods

The reciprocal translocation t(12;21)(p13;q22), the most common structural genomic alteration in B-cell precursor acute lymphoblastic leukaemia in children, results in a chimeric transcription factor TEL-AML1 (ETV6-RUNX1). We identified directly and indirectly regulated target genes utilizing an inducible TEL-AML1 system derived from the murine pro B-cell line BA/F3 and a monoclonal antibody directed against TEL-AML1. By integration of promoter binding identified with chromatin immunoprecipitation (ChIP)-on-chip, gene expression and protein output through microarray technology and stable labelling of amino acids in cell culture, we identified 217 directly and 118 indirectly regulated targets of the TEL-AML1 fusion protein. Directly, but not indirectly, regulated promoters were enriched in AML1-binding sites. The majority of promoter regions were specific for the fusion protein and not bound by native AML1 or TEL. Comparison with gene expression profiles from TEL-AML1-positive patients identified 56 concordantly misregulated genes with negative effects on proliferation and cellular transport mechanisms and positive effects on cellular migration, and stress responses including immunological responses. In summary, this work for the first time gives a comprehensive insight into how TEL-AML1 expression may directly and indirectly contribute to alter cells to become prone for leukemic transformation.

Initially disadvantaged, TEL-AML1 cells expand and initiate leukemia in response to irradiation and cooperating mutations

Although TEL-AML1 is present in one-fourth of childhood precursor B-cell acute lymphoblastic leukemia (pre-B ALL), the expression of the ETV6-RUNX1 gene encoding this fusion protein has been a poor initiator of leukemia in multiple mouse models.1, 2, 3, 4, 5, 6, 7, 8 To gain insight into why this common lesion does not readily initiate disease, we took advantage of a novel mouse model, in which TEL-AML1 expression was linked to a fluorescence marker and could be induced in a subset of hematopoietic cells during embryonic development. The laboratory of Corrinne Lobe had generated a reporter mouse in which enhanced green fluorescent protein (EGFP) was expressed following Cre-mediated excision (designated herein as ZEG mice), and subsequently generated an analogous mouse, in which a human TEL-AML1 encoding complementary DNA and an internal ribosomal entry site were placed upstream of EGFP (designated herein as TA1 mice). We crossed the inducible ZEG and TA1 mice with mice expressing Cre under the control of the Tie2(Tek) promoter, which is expressed in endothelial cells as well as during early hematopoietic development. These crosses resulted in mice carrying either the Tie2-Cre and inducible ZEG transgenes (Cre/ZEG mice) or the Tie2-Cre and inducible TA1 transgenes (Cre/TA1 mice). Mice carrying the inducible TA1 transgene but lacking Cre were also generated (Cre-/TA1+ control mice).

TEL-AML1 Affects the Regulation of Cytoskeleton and Causes Alteration In Cellular Adhesive and Migratory Properties In An In Vitro Model of Pre-Leukemia.

AbstractAbstract 3624 Introduction:The t(12;21) chromosome translocation generating TEL-AML1 chimeric fusion gene is a frequent initiating event in childhood leukaemia. Its impact is to generate a clone of covert, clinically silent pre-leukemic B cell progenitors. The leukemia arises only following second, post-natal hit/genetic events occurring years later. Moreover, relapse of leukemia is frequently arising from the pre-leukemic clone. Aimof our study is to investigate how TEL-AML1 expression can sustain this covert condition for many years. In a recent paper we described that the fusion gene rendered the B precursors resistant to the inhibitory activity of TGFbeta. Here we want to inquire into other factors that can explain the positive selection of the pre-leukemic clones over the normal counterpart. In particular, given the importance of the interaction with the microenvironment for survival signals for normal and leukemic stem cells, we question if the fusion gene causes changes in cellular adhesive and migratory properties. Methods:the study was performed by using two different models: i) a TEL-AML1 inducible expression system on the murine pro-B Ba/F3 cell line and ii) murine primary B lymphocytes (pre-BI cells) isolated from fetal liver, stably transduced with the pMIGR1-TEL-AML1-IRES-GFP construct. Gene expression assays were performed by using TaqMan (Applied Biosystems) and PCR Array technologies (SABioscences). Results:The expression of TEL-AML1 in Ba/F3 cell line causes over-expression of genes regulators of the cytoskeleton, specifically involved in cellular movement and in the regulation of actin dynamics. This gene expression alteration results in changes in the cellular morphology and phenotype: the cells acquire long extensions and several molecules involved in cell adhesion and migration are disregulated. Moreover, the TEL-AML1 positive cells present an increased ability to adhere to the ICAM1 substrate, but they also show a significant defect in the chemotactic response to CXCL12 in transwell migration assays in vitro, although the expression and the recycling of CXCR4 receptor are unaffected. This inability is not due to defects to migrate in general, as spontaneous motility is enhanced, but it is associated with a defect in CXCR4 signaling. In particular, CXCL12 calcium flux and ERK phosphorylation were inhibited. Those results have been confirmed in murine PreBI primary cells. Conclusions:in our murine models, TEL-AML1 affects the cytoscheleton regulation and causes alteration in cellular adhesive and migratory properties. We are now investigating how these alterations can give advantages to the pre-leukemic cells in the pathogenesis of TEL-AML1–expressing leukemia. Disclosures:No relevant conflicts of interest to declare.

ETV6-RUNX1 (TEL-AML1) Deregulates the Erythropoietin Receptor (EPOR) and Promotes Cell Survival.

AbstractAbstract 3641ETV6-RUNX1 (TEL-AML1) fusion is usually an initiating and pre-natal event in childhood acute lymphoblastic leukaemia (ALL). Transformation results in the generation of a persistent pre-leukemic clone, which post-natally converts to ALL following the acquisition of necessary secondary genetic alterations. The erythropoietin receptor gene (EPOR) is consistently highly expressed ectopically in TEL-AML1+ ALL but the presence of a functional receptor on the cell surface and its role, if any, in leukemogenesis driven by TEL-AML1 remains to be confirmed. Similarly, there is much debate as to the role played by the cytokine erythropoietin (EPO) and signaling through EPOR in relation to non-erythroid cell survival. Here, we show by using biotinylated erythropoietin (EPO) and flow analysis that the pre-B ALL TEL-AML1+ cell line (REH) appeared to have higher levels of EPO receptor than other non-TEL-AML1 cell lines. Furthermore, when we “blind screened” CD19+ peripheral blood or bone marrow cells from 10 patients with pre-B ALL, we identified 5 patients that showed high expression of ligand-binding EPO receptor on the cell surface, 4 of which were subsequently shown to be TEL-AML1+. We show that the inducible expression of TEL-AML1 in lymphoid BaF3 cells or its constitutive expression in either a murine transgenic model or in normal human cord blood cells is also sufficient to increase expression of EPOR. In order to further assess the direct regulation of EPOR by TEL-AML1 we next performed EMSA and ChIP experiments to confirm occupancy of AML1 consensus binding sites within the EPOR promoter and luciferase reporter assays to confirm up-regulation of EPOR promoter activity in the presence of TEL-AML1. Given the proposed pro-survival properties of EPO on non-erythroid cells, we next asked if the observed increase in expression of the EPOR in cells expressing TEL-AML1 could correlate with an increased cell survival in the presence of EPO. Cell survival experiments including growth curves, Annexin V staining and analysis of anti-apoptotic gene markers revealed that IL3-dependent cells expressing TEL-AML1 showed a prolonged survival in the presence of EPO alone and the consequent activation of anti-apoptotic pathways. The subsequent removal of TEL-AML1 expression in these cells resulted in cell death even in the presence of EPO, further suggesting that this effect is a consequence solely of fusion gene expression. Moreover, the observed survival in the presence of TEL-AML1 and EPO was enhanced by the co-addition of stem cell factor (SCF). Interestingly, SCF associated intracellular tyrosines have been shown to play an essential role in signaling through EPOR-SCFR interactions. In the presence of EPO, signaling via EPOR was confirmed by phosophorylation of JAK2, STAT5 and AKT and the subsequent up-regulation of BCL-XL. Similarly, EPOR functionality was demonstrated in TEL-AML1 positive patient cells by analysis of JAK2 pathway signaling after EPO induction. We propose that such activation lead to the increase in cell survival also observed in patient cells in the presence of EPO - again suggesting a role for enhanced cell survival through the EPO-EPOR axis. In our final model of pre-leukemia, CD34+ cord blood cells were transduced in vitro with a lentivirus capable of expressing both TEL-AML1 and GFP and ‘primed’ for pre-B lineage commitment. GFP (TEL-AML1) positive cord blood cells also showed increased levels of cell surface EPO receptor when compared to empty vector controls. Collectively, these data support the contention that TEL-AML1 directly activates ectopic expression of a functional EPO receptor and provides cell survival signals that may contribute critically to persistence of the pre-malignant clone in patients. Disclosures:No relevant conflicts of interest to declare.

Folylpolyglutamate Synthetase Gene Transcription is Regulated by a Multiprotein Complex that Binds the TEL-AML1 Fusion in Acute Lymphoblastic Leukemia

Acute Lymphoblastic Leukemia (ALL) non-random fusions influence clinical outcome and alter the accumulation of MTX-PGs in vivo. Analysis of primary ALL samples uncovered subtype-specific patterns of folate gene expression. Using an FPGS-luciferase reporter gene assay, we determined that E2A-PBX1 and TEL-AML1 expression decreased FPGS transcription. ChIP assays uncovered HDAC1,AML1, mSin3A, E2F, and Rb interactions with the FPGS promoter region. We demonstrate that FPGS expression is epigenetically regulated through binding of selected ALL fusions to a multiprotein complex, which also controls the cell cycle dependence of FPGS expression. This study provides insights into the pharmacogenomics of MTX in ALL subtypes.

Abstract 2395: Prediction of HLA-DRB1-restricted CD4+ T cell epitopes in the TEL-AML1 fusion oncoprotein of childhood ALL: population coverage, and design of heteroclitic peptides

Abstract In-frame fusion of two transcription factor genes, TEL (ETV6) on chromosome 12p and AML1 (RUNX1) on chromosome 21q, is thought to be an initiating event in ∼25% of childhood acute lymphoblastic leukemias (ALL). TEL-AML1 fusion leads to the expression of a chimeric oncoprotein which interferes with the function of the normal AML1 protein, leading to arrested B cell development, and enhanced B cell progenitor self-renewal. In vitro studies showed that a TEL-AML1 junctional peptide induced HLA-DP-restricted CD4+ T cell responses. Although expression of TEL-AML1 by pro-leukaemic cells makes it an attractive immunotherapeutic target, information on the number, position, and population distribution of HLA-DR-restricted CD4+ T cell epitopes in TEL-AML1 has not been published. Here, we used the artificial neural network algorithm, NetMHCIIpan (Nielsen et al, PLoS Computational Biology, 2008, 4(7), e1000107) to predict DR-restricted T cell epitopes in TEL-AML1. The 797 residue fusion oncoprotein was screened using NetMHCIIpan to identify 9-mer DR-binding core peptides. 15-mer peptide sequences were submitted in FASTA format to the NetMHCIIpan server (http://www.cbs.dtu.dk/services/NetMHCIIpan/) to facilitate discovery of peptides with strong binding affinity (IC50:&amp;lt;50 nM). Consensus T cell epitopes predicted to provide maximum coverage across common DR alleles in 4 ethnic groups were identified. Overlapping 15-mer junctional sequences were screened to identify a 9-mer DR-binding core sequence, and P1,4,6, and 9 substituted with all 20 amino acids to identify junctional sequences with enhanced (heteroclitic) binding affinity. NetMHCIIpan predicted 45 TEL-AML1 epitopes binding to 22 common DR alleles in 4 ethnic groups. Fourteen epitopes are in TEL and 31 in AML1, with 5 epitopes binding to 5-8 DR alleles, and 1 binding to 10 alleles. Three TEL peptides achieve the greatest population coverage across all ethnic groups, one peptide (266IQLMPSPIM274) giving maximal coverage (25.5-50.1%). Weak DRB1*0101-restricted binding of a junctional peptide (332IGRIA/ECIL340) (415 nM) was strongly increased (52 nM) by substituting serine for glutamic acid at the P6 position. A junctional peptide (332FGRMA/SCIV343) with substitutions at P1, P4 and P9 had enhanced DRB1*0101 affinity (13 nM). These predictions suggest wide population coverage of a small number of strongly binding TEL-AML1 epitopes. The binding affinity of a junctional peptide is enhanced by substituting key anchor residues. Functional studies are now required to distinguish CD4+ T cell-activating TEL-AML1 epitopes inducing anti-leukemic, autoimmune, and regulatory T cell responses. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2395.

TEL-AML1 Corrupts Hematopoietic Stem Cells to Persist in the Bone Marrow and Initiate Leukemia

The initial steps in the pathogenesis of acute leukemia remain incompletely understood. The TEL-AML1 gene fusion, the hallmark translocation in Childhood Acute Lymphoblastic Leukemia and the first hit, occurs years before the clinical disease, most often in utero. We have generated mice in which TEL-AML1 expression is driven from the endogenous promoter and can be targeted to specific populations. TEL-AML1 renders mice prone to malignancy after chemical mutagenesis when expressed in hematopoietic stem cells (HSCs), but not in early lymphoid progenitors. We reveal that TEL-AML1 markedly increases the number of HSCs and predominantly maintains them in the quiescent (G(0)) stage of the cell cycle. TEL-AML1(+) HSCs retain self-renewal properties and contribute to hematopoiesis, but fail to out-compete normal HSCs. Our work shows that stem cells are susceptible to subversion by weak oncogenes that can subtly alter their molecular program to provide a latent reservoir for the accumulation of further mutations.

The TEL-AML1 leukemia fusion gene dysregulates the TGF-β pathway in early B lineage progenitor cells

Chromosome translocation to generate the TEL-AML1 (also known as ETV6-RUNX1) chimeric fusion gene is a frequent and early or initiating event in childhood acute lymphoblastic leukemia (ALL). Our starting hypothesis was that the TEL-AML1 protein generates and maintains preleukemic clones and that conversion to overt disease requires secondary genetic changes, possibly in the context of abnormal immune responses. Here, we show that a murine B cell progenitor cell line expressing inducible TEL-AML1 proliferates at a slower rate than parent cells but is more resistant to further inhibition of proliferation by TGF-beta. This facilitates the competitive expansion of TEL-AML1-expressing cells in the presence of TGF-beta. Further analysis indicated that TEL-AML1 binds to a principal TGF-beta signaling target, Smad3, and compromises its ability to activate target promoters. In mice expressing a TEL-AML1 transgene, early, pre-pro-B cells were increased in number and also showed reduced sensitivity to TGF-beta-mediated inhibition of proliferation. Moreover, expression of TEL-AML1 in human cord blood progenitor cells led to the expansion of a candidate preleukemic stem cell population that had an early B lineage phenotype (CD34+CD38-CD19+) and a marked growth advantage in the presence of TGF-beta. Collectively, these data suggest a plausible mechanism by which dysregulated immune responses to infection might promote the malignant evolution of TEL-AML1-expressing preleukemic clones.

TEL-AML1 Dysregulates the TGFβ Pathway: A Basis for Pre-Leukaemic Stem Cell Selection.

TEL-AML1 fusion is the most frequent, defined genetic abnormality in paediatric cancer and is usually an early or initiating and pre-natal event in childhood acute lymphoblastic leukaemia (ALL). Transformation results in the generation of a persistent pre-leukaemic clone, which converts to frank ALL post-natally (at 1-15years) following the acquisition of secondary genetic alterations. The mechanism by which the transcriptional dysregulation imposed by TEL-AML1 impacts on the pre-leukaemic phenotype and disease natural history is unknown. Using regulated expression of TEL-AML1, we show that, in murine progenitor cells, this protein blocks the TGFβ response pathway including the critical regulator of cell cycle inhibition, p27. This inhibition facilitates the selective expansion of otherwise more slowly expanding TEL-AML1 progenitor cells. The mechanism of action of TEL-AML1 appears to operate downstream of SMAD2/3 phosphorylation (by TGFβ signalling) and involves binding of a transcriptional repressor complex to TGFβ target genes. In transgenic Eμ TEL-AML1 mice, we show that early pro-B cells (but not pre-B cells) are selectively expanded and appear less sensitive to the inhibitory effects of TGFβ than equivalent wild-type cells. Finally, we show that expression of TEL-AML1 in human cord blood progenitor cells leads to the expansion of a candidate pre-leukaemic stem cell population with an early B lineage phenotype (CD34+/CD38-/CD19+) that has a selective growth advantage in the presence of TGFβ.

High Hyperdiploidy and t(12; 21) (p13; q22) Translocation Is Not a so Rare Association: A Report of 4 Cases in the Experience of Armand Trousseau Hospital (Paris).

INTRODUCTION. Since the discovery of the cryptic t(12; 21) translocation, many secondary genetic abnormalities have been described in association with TEL-AML1 fusion gene. Extensive studies of karyotypes in a few series of TEL-AML1 positive leukaemia revealed a heterogeneous pattern of chromosomal abnormalities. Numerical and structural abnormalities are often present together. The modal chromosome number does not exceed 49 so that high hyperdiploidy and TEL-AML1 fusion are so far considered mutually exclusives. Here we reported that such association is found in a small group of patients and that relapse may still occur in those patients despite the good prognostic impact commonly attributed to each lesion.PATIENTS and METHODS. Between April 1994 and April 2006, 105 children were consecutively diagnosed with TEL-AML1 positive B-ALL. TEL-AML1 expression was detected by RT-PCR in Bone Marrow (BM) diagnostic samples and the t(12;21) explored by FISH with LSI TEL-AML1 dual-color probe (Vysis) in cases with low level of fusion gene expression or lacking molecular study. Conventional cytogenetics with G and R banding was performed on BM cells after overnight and 24 hours cultureRESULTS and DISCUSSION. We detected numerical and/or structural abnormalities in 82/105 (78%) of the karyotypes. Aneuploidy alone was found in 4/105(3.8%): two cases had an extra chromosome 21, one an extra chromosome 10 and one lost 1 chromosome X. Structural abnormalities alone were present in 18/105 (17.1%) and up to 58 /105 (55.2%) presented both. The most frequent structural change was observed on chromosome 12p13 (50% of all structural abnormalities) whereas the most frequent chromosome gain was +21 (14%) and +10 (6.6%). Interestingly, in four cases (3.8%) we detected high hyperdiploidy with classical supplementary chromosomes; in two of these cases a partial trisomy of chromosome 1 was also present. Karyotypes are presented:Pt1: 52,XX,+10,+16,+18,+21,+21,+22 [22]/46, XX [4]Pt 2: 54,XY,+X,+4,+ 6?[del(6q)],+9,+14,+15,+18,+21,+21,-22 [17]/46,XY [3]Pt 3: 55,XX,+X,dup(1)(q21q31),+4,+6,+10,+14,+17,+18,+21,+21 [1]/56,idem+19 [4]/56,idem,+14 [5] / 46,XX [10]Pt 4: 7,XX,+der(X)(t(X;1)(q26;q12),+4,+5,+6,+8,+10,+14,+17,+18,+21,+21 [17]/46,XX [3]Pt1 present CNS relapse at 24 months follow up without involvement of BM which tested negative for TEL-AML1 expression. Patients 2,3,4 are in continuous complete remission at respectively 6, 6 and 4 years follow up. Since t(12;21) translocation and high hyperdiploidy seem to be primary events in leukaemia our observation raises the question if the two lesions coexist in a same cell or are independent events in different clones. In two cases (Pt 3,4), FISH analysis indicated the presence of TEL-AML1 gene fusion in 7% and in 5% of nuclei respectively. The distribution and the number of the signals on AML1 locus let think that t(12;21) occurred independently from hyperdiploidy. Microarrays studies have revealed distinct gene expression signatures associated to TEL-AML1 fusion and hyperdiploidy over 50 chromosomes, our observation points out the existence of cases which could be difficult to assign to one or the other of these classes.

Childhood acute lymphocytic leukemia and perspectives on risk assessment of early-life stage exposures

Recognition that children are a potentially susceptible subpopulation has led to the development of child-specific sensitivity factors. Establishing reliable sensitivity factors in support of risk assessment of early-life stage exposures can be aided by evaluating studies that enhance our understanding both of the biological basis of disease processes and the potential role of environmental exposures in disease etiology. For these reasons, we evaluated childhood acute lymphocytic leukemia (ALL) studies from the point of view of mechanism and etiology. ALL is the most common form of childhood cancer proposed to result from a prenatal primary event and a postnatal second event. This multi-stage model is supported by the observation that chromosomal translocations/fusion genes (e.g., TEL-AML1) involved in producing ALL are detected at birth (prenatal event), and a postnatal event (e.g., TEL deletion) is required for disease manifestation. It appears that a proportion of ALL cases are the result of environmental exposures, in which case preconceptional, prenatal, and postnatal stages are likely to be critical exposure windows. To this end, we recognized postnatal infection-related risk factors as potential candidates associated with the ALL second event. Additionally, we discuss use of ALL-associated fusion genes and genetic polymorphisms, together or separately, as indicators of ALL susceptibility and increased risk. The possibility of using fusion genes alone as biomarkers of response is also discussed because they can serve as predictors of key events in the development of a mode of action (a sequence of key events, starting with interaction of an agent with a cell, ultimately resulting in cancer formation) for particular environmental exposures. Furthermore, we discuss use of an initiated animal model for ALL, namely transgenic mice with TEL-AML1 expression, for exploring mechanisms by which different classes of environmental exposures could be involved in inducing the postnatal step in ALL formation.

TEL-AML1 Transgenic Zebrafish Model of Acute Lymphoblastic Leukemia (ALL).

TEL-AML1 (ETV6-RUNX1) fusion is the product of the t(12;21) chromosomal translocation, the most common chromosomal rearrangement in childhood cancer. The translocation fuses two highly conserved transcription factors, TEL and AML1 that have essential roles in hematopoiesis. Genetic studies of identical twins with concordant leukemia, the detection of leukemia-specific fusion genes in neonatal blood spots, and the existence of multiple leukemic subclones at ALL diagnosis point to prenatal origin of the fusion and long latency before leukemia development. Additionally, a study in growth factor-dependent cell lines and transgenic mice, and several mouse transplant models suggest that TEL-AML1 is insufficient by itself for leukemic transformation. To determine whether TEL-AML1 has selective transforming impact on a particular stem cell lineage, we established stable zebrafish transgenic lines expressing the TEL-AML1 fusion protein both constitutively, and within the lymphoid progenitors. In these lines, either the zebrafish β-actin (ZBA) promoter or the xenopus elongation factor-1 (XEF1) promoter drives TEL-AML1 expression constitutively, while the zebrafish RAG-2 promoter (RAG2) selectively drives TEL-AML1 expression in committed lymphocyte precursors, but not in earlier multilineage hematopoietic precursors. TEL-AML1 expression, alone or fused to EGFP, was detected at 24-hour post fertilization (hpf) with fluorescent microscopy or RT-PCR and confirmed with western blot analysis. In-Situ Hybridization and confocal microscopy revealed that transgenic zebrafish maintained TEL-AML1 expression throughout adulthood. The expression of TEL-AML1 was associated with accumulation of immature hematopoietic progenitor cells, mostly in the kidney and spleen of several transgenic zebrafish lines, within a few weeks of development, indicating an expansion of the progenitor cell population. To date, a small number (5 out of 391 transgenic fish; 1.3%) of founders and progeny of zebrafish transgenic for ZBA- or XEF1-TEL-AML1 fusion developed an infiltrating lymphoid neoplasm, most likely acute lymphoblastic leukemia with a latency of 8–12 month. The lymphoblasts were negative for myeloperoxidase and PAS staining, with abundant expression of both EGFP in lymphoblasts from EGFP-TEL-AML1 line, and pre-B-cell leukemia transcription factor-1 (PBX1; also called cALLa) indicating that Leukemias originated in a TEL-AML1 expressing cell(s), and mimic the human CD10-positive precursor-B cell ALL. None of 350 wild-type, 125 control EGFP fish or 353 RAG2-TEL-AML1 transgenic zebrafish had any leukemia or hematopoietic changes. Therefore, the expression of TEL-AML1 fusion in our ZBA- and XEF1-TEL-AML1 transgenic lines generates a premalignant state, which appears to require additional genetic events for acquisition of the leukemic phenotype. The fact that none of the RAG2 zebrafish expressing TEL-AML1 in committed lymphoid progenitors developed leukemia or progenitor expansion indicates that the leukemic stem cell associated with TEL-AML1 leukemia is an immature progenitor earlier than the committed lymphoid progenitors where RAG2 expression is detected. The TEL-AML1 transgenic zebrafish lines provide a basis for large scale and targeted mutagenesis screens aimed at identifying mutations and cooperating events that are required for TEL-AML1 induced leukemias.

TEL/AML1 and E2A/PBX1 Tranlsocations Lead to Altered Folylpoly-γ-Glutamate Synthetase (FPGS) Expression in ALL.

Expression of the human folylpoly-γ-glutamate synthetase (FPGS) gene is controlled by tissue/lineage specific and proliferation-dependent mechanisms. Levels of FPGS mRNA, protein, and enzyme activity are 2–3 fold higher in B-precursor (Bp) ALL cell lines and primary cells when compared to T-lineage ALL. These differences correlate with intracellular accumulation of long chain methotrexate (MTX) polyglutamates (PG), and more important with clinical sensitivity to MTX. However, significant heterogeneity of FPGS expression exists within hematopoietic cells of the same lineage, suggesting additional factors influence FPGS expression in Bp- and T-ALL. cDNA microarray data was analyzed for expression of folate-related genes (DHFR, FPGS, RFC, MTHFR, and γ-GGH) in six prognostic leukemia subtypes (T-ALL, hyperdiploid (> 50 chromosomes), BCR-ABL, E2A-PBX1, TEL-AML1, and MLL) (Yeoh et al, Cancer Cell, 2002). It was found that only the FPGS gene exhibited significant heterogeneity of expression compared the others analyzed. Therefore, we investigated the effect of distinct non-random genomic translocations on FPGS gene expression to determine whether gene fusions may be responsible for the heterogeneity of FPGS expression in ALL cells. Using quantitative fluorescence real-time RT-PCR, we first detected reduced levels of FPGS mRNA expression in the RCH-ACV (Bp-ALL, t(1:19)/E2A-PBX1) and REH (Bp-ALL, t(12:21)/TEL-AML1) human leukemia cell lines expressing non-random chromosomal gene fusions when compared to control (NALM6). To determine if expression of fusion proteins encoded by the E2A-PBX1 and TEL-AML1 translocations lead to decrease FPGS promoter activity and mRNA expression, we used a FPGS-luciferase reporter gene assay. The CHO FPGS null mutant cell line AUXB1, was triple transfected with constructs expressing luciferase (pFPGS-luc), β-galactosidase (pCMVβ), and E2A-PBX1 (pCMVKJ7) or TEL-AML1 (pTEL-AML1) and assayed for luciferase and β-galactosidase activity. Expression of E2A-PBX1 and TEL-AML1, determined by quantitative RT-PCR and Western blot, resulted in 45% and 30% decrease in the level of normalized FPGS-luciferase activity, respectively. These data indicate that FPGS gene transcription is decreased by both translocations, either by direct binding to regulatory elements or indirectly via downstream pathways influenced by E2A-PBX1 and TEL-AML1. Therefore, to confirm these findings and to identify genes or pathways in Bp-ALL regulated by E2A-PBX1 and TEL-AML1 gene fusions, we constructed NALM6 stable cell lines expressing E2A-PBX1 or TEL-AML1 using vectors pCI-neo/E2A-PBX1 and pCI-neo/TEL-AML1. In both NALM6 stable cell lines the level of FPGS mRNA expression was decreased by more than 50% compared to wild type (NALM6). Total RNA was then extracted from NALM6, NALM6/E2A-PBX1, and NALM6/TEL-AML1 cells and analyzed by cDNA microarray. Gene expression profile analysis from existing databases and our NALM6/E2A-PBX1 and NALM6/TEL-AML1 stable cell lines identified genes differentially expressed compared to NALM6 control. These included transcription factors (CSDA, AKAP12, ARL2, PBX1, TEL), cell cycle regulators (CDK2P1), signal transduction (PIK3C3), proliferation and cell migration (PTPRK), zinc finger binding protein (PHF2), among others. This study is the first to demonstrate the causal relationship between the presence of E2A-PBX1 and TEL-AML1 gene fusions and altered FPGS expression and may explain the differential sensitivity to MTX exhibited by these childhood ALL phenotypes.

Expression Levels of TEL, AML1, and the Fusion Products TEL-AML1 and AML1-TEL versus Drug Sensitivity and Clinical Outcome in t(12;21)-Positive Pediatric Acute Lymphoblastic Leukemia

t(12;21)(p13; q22), present in approximately 25% of pediatric precursor B-ALL, is highly sensitivity to L-asparaginase and the prognosis depends on the intensity of the treatment protocol. This study analyzes the relationship between the mRNA expression of the genes and fusion products involved in t(12;21), in vitro sensitivity to prednisolone, vincristine, and L-asparaginase, and long-term clinical outcome in t(12;21)+ acute lymphoblastic leukemia (ALL) patients. Long-term clinical outcome in 45 t(12;21)+ ALL patients was related to mRNA expression of TEL, AML1, TEL-AML1, and AML1-TEL, determined by real-time quantitative PCR, and the in vitro sensitivity to prednisolone, vincristine, and L-asparaginase, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. A significant approximately 3.5-fold lower TEL expression in t(12;21)+ compared with t(12;21)- ALL samples (P = 0.006) and normal controls (P = 0.004) was found. Expression of AML1 did not differ between t(12;21)+ and t(12;21)- ALL. However, AML1 expression in the leukemic cells was 2-fold higher compared with normal controls (P = 0.02). The TEL-AML1 fusion product was expressed in all t(12;21)+ cases, whereas the reciprocal fusion product AML1-TEL was expressed in only 76%. High expression levels of TEL-AML1 [hazard ratio (HR), 1.3; 95% confidence interval (95% CI), 1.10-1.57; P = 0.003], AML1-TEL (HR, 4.9; 95% CI, 1.99-12.40; P = 0.001) and AML1 (HR, 1.1; 95% CI, 1.03-1.22; P = 0.006) were associated with a poor long-term clinical outcome within t(12;21)+ ALL. Cellular drug resistance towards prednisolone, vincristine, and L-asparaginase could not explain this predictive value. Multivariate analysis including age and WBC showed that only high AML1-TEL expression is an independent poor prognostic factor in t(12;21)+ childhood ALL. High AML1-TEL expression is an independent poor prognostic factor in t(12;21)+ childhood ALL.

Modeling first-hit functions of the t(12;21) TEL-AML1 translocation in mice.

The t(12;21) translocation, which generates the TEL-AML1 (ETV6-RUNX1) fusion gene, is the most common structural chromosome change in childhood cancer and is exclusively associated with the common B cell precursor subset of acute lymphoblastic leukemia (ALL). Evidence suggests that the translocation usually occurs in utero during fetal hemopoiesis and most probably constitutes an initiating or first-hit mutation that is necessary but insufficient for the development of overt, clinical leukemia. The mechanism by which TEL-AML1 contributes to this early stage of leukemogenesis is unknown. To address this question we have analyzed hemopoiesis in mice syngeneically transplanted with TEL-AML1-transduced bone marrow stem cells. TEL-AML1 expression was associated with an accumulation/expansion of primitive c-kit-positive multipotent progenitors and a modest increase in myeloid colony-forming cells. TEL-AML1 expression was, however, permissive for myeloid differentiation. Analysis of B lymphopoiesis revealed an increase in early, pro-B cells but a differentiation deficit beyond that stage, resulting in reduced B cell production in the marrow. TEL-AML1-positive B cell progenitors exhibited reduced expression of the surrogate light-chain component lambda5 and the IL-7 receptor, both of which may contribute to impedance of differentiation in vivo and account for their reduced in vitro clonogenicity in IL-7. A selective differentiation deficit of B lineage progenitors (i) is consistent with the phenotype of TEL-AML1-associated leukemia in children and (ii) provides a potential mechanism for the protracted preleukemic state that often precedes ALL. These results provide mechanistic insight into the role of the t(12;21) translocation in the initiation of common B cell precursor ALL.

Relapse of TEL-AML1--positive acute lymphoblastic leukemia in childhood: a matched-pair analysis.

The aim of this study was to investigate whether, in relapsed childhood acute lymphoblastic leukemia (ALL), the frequent genetic feature of TEL-AML1 fusion resulting from the cryptic chromosomal translocation t(12;21)(p13;q22) is an independent risk factor. A matched-pair analysis was performed within a homogeneous group of children with first relapse of BCR-ABL-negative B-cell precursor (BPC) ALL treated according to relapse trials ALL-Rezidiv (REZ) of the Berlin-Frankfurt-Münster Study Group. A total of 249 patients were eligible for this study: 53 (21%) were positive for TEL-AML1, and 196 (79%) were negative. Positive patients were matched for established most-significant prognostic determinants at relapse, time point, and site of relapse, as well as age and peripheral blast cell count at relapse. Fifty pairs matching the aforementioned criteria could be determined. The probabilities with SE of event-free survival and survival at 5 years for matched TEL-AML1 positives and negatives are 0.63 +/- 0.10 versus 0.38 +/- 0.10 (P =.09) and 0.82 +/- 0.09 versus 0.42 +/- 0.19 (P =.10), respectively. These results were confirmed by multivariate analysis, revealing an independent prognostic significance of time point and site of relapse (both P <.001) but not of TEL-AML1 expression (P =.09). TEL-AML1 expression does not constitute an independent risk factor in relapsed childhood BCP-ALL after matching for relevant prognostic parameters. It undoubtedly characterizes genetically an ALL entity associated with established favorable prognostic parameters. High-risk therapeutic procedures such as allogeneic SCT should be considered restrictively.

TEL-AML1 Fusion Transcript in Relapsed Childhood Acute Lymphoblastic Leukemia

AbstractThe cryptic translocation t(12;21)(p13;q22) has been recently recognized as the most common genetic rearrangement in B-lineage childhood acute lymphoblastic leukemia (ALL). The resulting fusion transcript, termed TEL-AML1, has been associated with an excellent prognosis at initial ALL diagnosis. Hence, we postulated that the incidence of TEL-AML1 fusion should be lower in patients with ALL relapse. To address this assumption and to investigate the prognostic significance of TEL-AML1 expression in relapsed childhood ALL, bone marrow samples of 146 children were analyzed by reverse-transcriptase (RT)-polymerase chain reaction (PCR). All children were treated according to Berlin-Frankfurt-Münster (BFM) ALL relapse trial protocols (ALL-REZ BFM 90-96). Their clinical features and outcome were compared with those of 262 patients who could not be tested due to lack of bone marrow samples. Thirty-two of 146 children with relapsed ALL were TEL-AML1–positive. Four of the negative patients had T-lineage and nine Philadelphia chromosome (Ph1)-positive leukemia. Thus, the incidence ofTEL-AML1 in relapsed Ph1-negative, B-cell precursor ALL is 32 of 133 (24%). The 32 TEL-AML1–positive and 101 negative patients differed significantly with respect to duration of last remission (42.5 v 27 months; P = .0001) and age at initial diagnosis (53.5 v 74 months;P = .0269). At a median follow-up time of 21.5 months, children positive for TEL-AML1 had a significantly (P = .0011) higher probability of event-free survival (EFS; 0.79 v 0.33). The predominant majority of patients had been treated for initial ALL according to German multicenter BFM (108 of 133) or Cooperative ALL study group (CoALL) (19 of 133) frontline protocols. The comparison of tested and not-tested (N = 262) patients showed no significant difference.TEL-AML1 positivity predicted a favorable short-term outcome; long-term results are unknown. Screening for TEL-AML1 should become routine at relapse diagnosis and might be used for therapy stratification in future trials.

TEL-AML1 Fusion Transcript in Relapsed Childhood Acute Lymphoblastic Leukemia

The cryptic translocation t(12;21)(p13;q22) has been recently recognized as the most common genetic rearrangement in B-lineage childhood acute lymphoblastic leukemia (ALL). The resulting fusion transcript, termed TEL-AML1, has been associated with an excellent prognosis at initial ALL diagnosis. Hence, we postulated that the incidence of TEL-AML1 fusion should be lower in patients with ALL relapse. To address this assumption and to investigate the prognostic significance of TEL-AML1 expression in relapsed childhood ALL, bone marrow samples of 146 children were analyzed by reverse-transcriptase (RT)-polymerase chain reaction (PCR). All children were treated according to Berlin-Frankfurt-Münster (BFM) ALL relapse trial protocols (ALL-REZ BFM 90-96). Their clinical features and outcome were compared with those of 262 patients who could not be tested due to lack of bone marrow samples. Thirty-two of 146 children with relapsed ALL were TEL-AML1–positive. Four of the negative patients had T-lineage and nine Philadelphia chromosome (Ph1)-positive leukemia. Thus, the incidence ofTEL-AML1 in relapsed Ph1-negative, B-cell precursor ALL is 32 of 133 (24%). The 32 TEL-AML1–positive and 101 negative patients differed significantly with respect to duration of last remission (42.5 v 27 months; P = .0001) and age at initial diagnosis (53.5 v 74 months;P = .0269). At a median follow-up time of 21.5 months, children positive for TEL-AML1 had a significantly (P = .0011) higher probability of event-free survival (EFS; 0.79 v 0.33). The predominant majority of patients had been treated for initial ALL according to German multicenter BFM (108 of 133) or Cooperative ALL study group (CoALL) (19 of 133) frontline protocols. The comparison of tested and not-tested (N = 262) patients showed no significant difference.TEL-AML1 positivity predicted a favorable short-term outcome; long-term results are unknown. Screening for TEL-AML1 should become routine at relapse diagnosis and might be used for therapy stratification in future trials.

The majority of myeloid-antigen-positive (My+) childhood B-cell precursor acute lymphoblastic leukaemias express TEL-AML1 fusion transcripts.

The t(12:21) translocation fuses the TEL and AML1 genes and has been found in up to 28% of paediatric B-cell precursor acute lymphoblastic leukaemias (BCP-ALL). The AML1 gene is a transcription factor which regulates expression of several myeloid differentiation associated genes. A molecular analysis of TEL-AML1, E2A-PBX1, MLL-AF4, BCR-ABL expression and an immunophenotypic study of CD13/CD33 myeloid antigen expression have been performed prospectively on tumour cells from 96 paediatric BCP-ALL patients. Percentages of CD13 or CD33 expressing leukaemic cells were found to be higher in TEL-AML1 positive cases (n = 22) than in TEL-AML1 negative (n = 74) cases (P<0.001). In 22/96 cases (23%) >10% of neoplastic cells were found to express at least one of the two markers. In 14 of these cases (63%), TEL-AML1 expression was detected, whereas t(4;11), t(11;19) and t(9;22) translocations were found by molecular methods in only three cases (14%). In four cases (18%) no molecular marker was found. These data show that TEL-AML1 expression is significantly associated with myeloid antigen expression by leukaemic cells and suggests that the prognostic significance of myeloid antigen expression in paediatric ALLs should be re-evaluated in the light of molecular cytogenetic markers.