TAL1 Expression Research Articles

Core Transcriptional Regulatory Circuit Controlled by the TAL1 Complex in Human T Cell Acute Lymphoblastic Leukemia

The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human Tcell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. We show that TAL1 forms a positive interconnected autoregulatory loop with GATA3 and RUNX1 and that the TAL1 complex directly activates the MYB oncogene, forming a positive feed-forward regulatory loop that reinforces and stabilizes the TAL1-regulated oncogenic program. One of the critical downstream targets in this circuitry is the TRIB2 gene, which is oppositely regulated by TAL1 and E2A/HEB and is essential for the survival of T-ALL cells.

Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated red blood cells

Background In many countries, the available supply of transfusable red blood cells (RBCs) is often inadequate. This problem has stimulated considerable research around the world into the development of a robust method for in vitro production of transfusable RBCs. Some possible methods are under active investigation such as the production of RBCs from hematopoietic stem/progenitor cells and from embryonic stem (ES) or induced Pluripotent Stem (iPS) cells. Although it may be feasible to produce RBCs from their immediate precursor cells, it is not easy to obtain sufficient numbers of these precursor cells. One solution to the latter problem may be to establish immortalized erythroid progenitor cell lines able to produce transfusable RBCs in vitro; these immortalized cells would form a very valuable resource. We previously developed a robust method for generating immortalized erythroid progenitor cell lines following the induction of hematopoietic differentiation of mouse ES cells, and successfully established several immortalized erythroid progenitor cell lines that we designated Mouse ES cell Derived‐Erythroid Progenitor (MEDEP) cell lines. Although the precise characteristics of these cell lines varied, each of them could differentiate in vitro into more mature erythroid cells, including enucleated RBCs. Transplantation of MEDEP cells into mice suffering from acute anemia resulted in transient proliferation of the cells, which subsequently differentiated into functional RBCs and significantly ameliorated the acute anemia.Aims Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated RBCs.Methods We sought to establish immortalized erythroid progenitor cell lines following the induction of hematopoietic differentiation of human ES cells and iPS cells using a similar method as for establishment of MEDEP cell lines (Hiroyama et al., PLoS ONE 3: e1544, 2008).Results Using the original method developed in mice, we failed to establish immortalized human erythroid progenitor cell lines. This may have been due to the low efficiency of inducing erythroid cells from ES and iPS cells. It was previously reported that enforced expression of the transcription factor TAL1, which plays essential roles in early hematopoiesis and differentiation of erythroid cells and megakaryocytes, improved the efficiency of induction of hematopoietic cells from ES cells of the common marmoset. We therefore enforced expression of TAL1 in human iPS cells and found that this resulted in a significant improvement in the rate of production of hematopoietic cells, in particular of erythroid cells. However, our attempts to establish immortalized human erythroid progenitor cell lines from iPS cells expressing Tal1 were unsuccessful. Recently, it was reported that enforced expression of HPV16‐E6/E7 in CD36‐positive erythroid cells aided the establishment of immortalized human erythroid progenitor cell lines. We decided to adopt this strategy with some modifications and succeeded in establishing immortalized human erythroid progenitor cell lines able to produce enucleated RBCs.Summary/Conclusions We have successfully established immortalized Human Umbilical Cord Blood Derived‐Erythroid Progenitor (HUDEP) cell lines and Human iPS cell Derived‐Erythroid Progenitor (HiDEP) cell lines that are able to produce enucleated RBCs.

Characterization of non-oxidative transaldolase and transketolase enzymes in the pentose phosphate pathway with regard to xylose utilization by recombinant Saccharomyces cerevisiae

The activity of transaldolase and transketolase, key enzymes in the non-oxidative pentose phosphate pathway, is rate-limiting for xylose utilization in recombinant Saccharomyces cerevisiae. Overexpression of TAL1 and TKL1, the major transaldolase and transketolase genes, increases the flux from the pentose phosphate pathway into the glycolytic pathway. However, the functional roles of NQM1 and TKL2, the secondary transaldolase and transketolase genes, especially in xylose utilization, remain unclear. This study focused on characterization of NQM1 and TKL2, together with TAL1 and TKL1, regarding their roles in xylose utilization and fermentation. Knockout or overexpression of these four genes on the phenotype of xylose-utilizing S. cerevisiae strains was also examined. Transcriptional analysis indicated that the expression of TAL1, NQM1, and TKL1 was up-regulated in the presence of xylose. A significant decrease in both growth on xylose and xylose-fermenting ability in tal1Δ and tkl1Δ mutants confirmed that TAL1 and TKL1 are essential for xylose assimilation and fermentation. Gene disruption analysis using a tkl1Δ mutant revealed that TKL1 is also required for utilization of glucose. Growth on xylose and xylose-fermenting ability were slightly influenced by deletion of NQM1 or TKL2 when xylose was used as the sole carbon source. Moreover, the rate of xylose consumption and ethanol production was slightly impaired in TKL1- and TKL2-overexpressing strains. NQM1 and TKL2 may thus play a physiological role via an effect on the non-oxidative pentose phosphate pathway in the xylose metabolic pathway, although their roles in xylose utilization and fermentation are less important than those of TAL1 and TKL1.

Upregulation of T-Cell-Specific Transcription Factor Expression in Pediatric T-Cell Acute Lymphoblastic Leukemia (T-ALL)

Objective: T-cell acute lymphoblastic leukemia (T-ALL) is associated with recurrent chromosomal aberrations andabnormal ectopic gene expression during T-cell development. In order to gain insight into the pathogenesis of T-ALLthis study aimed to measure the level of expression of 7 T-cell oncogenes (LMO2, LYL1, TAL1, TLX1, TLX3, BMI1, andCALM-AF10) in pediatric T-ALL patientsMaterial and Methods: LMO2, LYL1, TLX1, TLX3, BMI1, TAL1, and CALM-AF10 expression was measured usingquantitative real-time PCR in 43 pediatric T-ALL patients.Results: A high level of expression of LMO2, LYL1, TAL1, and BMI1 genes was observed in a large group of T-ALL.Several gene expression signatures indicative of leukemic arrest at specific stages of normal thymocyte development(LYL1 and LMO2) were highly expressed during the cortical and mature stages of T-cell development. Furthermore,upregulated TAL1 and BMI1 expression was observed in all phenotypic subgroups. In all, 6 of the patients had TLX1and TLX3 proto-oncogene expression, which does not occur in normal cells, and none of the patients had CALM-AF10fusion gene transcription. Expression of LYL1 alone and LMO2-LYL1 co-expression were associated with mediastinalinvolvement; however, high-level oncogene expression was not predictive of outcome in the present pediatric T-ALLpatient group, but there was a trend towards a poor prognostic impact of TAL1 and/or LMO2 and/or LYL1 protooncogeneexpression.Conclusion: Poor prognostic impact of TAL1 and/or LMO2 and/or LYL1 proto-oncogene expression indicate the needfor extensive study on oncogenic rearrangement and immunophenotypic markers in T-ALL, and their relationship totreatment outcome.Conflict of interest:None declared.

Efficient Induction of CD34+ Progenitor Cells From Human Bone Marrow Mesenchymal Stem Cell-Derived Induced Pluripotent Stem Cells with Defined Chemicals

Abstract 2347Induced pluripotent stem cells (iPSCs) hold great potential in clinical application with indefinite passages, pluripotency of forming three germ layers and immunological compatibility with patients. Human bone marrow mesenchymal stem cell-derived iPSCs (hBMMSC-iPSCs) may have more virtues than other types of cell–derived iPSCs do in diseases treatment for their wide application in clinic for many years. Efficient differentiation of induced pluripotent stem cells (iPSCs) to a specialized cell type is a crucial step for iPSC clinical application. CD34+ progenitor cells derived from hBMMSC-iPSCs by treatment with defined chemicals have not been reported. Here, base on the efficient generation of hBMMSC-iPSCs with cDNA encoding four transcription factors OCT4, SOX2, KLF4 and c-MYC, and the compounds of p53 siRNA, VPA and Vc, we proposed that CD34+ progenitor cells, retaining hematopoietic and endothelial cell potential, could be efficiently obtained from hBMMSC-iPSCs by treatments with chemicals exerting function on mesoderm and hematopoietic cells. The differentiated hBMMSC-iPSCs treated with the chemicals comprised nearly 20% proportion of CD34+ progenitor cells, about 20-fold increasing compared to the spontaneous differentiation hBMMSC-iPSCs and higher than that from human skin fibroblast-derived iPSCs (hFib-iPSCs) (nearly 15%) and human embryonic stem cell line H1 (nearly 17%) by flow cytometry analysis. Those cells retained the hematopoietic and endothelial cell potential as CD34+ progenitor cells derived from umbilical cord blood. After coculture with the first chemical cocktails including BMP4 and SCF for 5 days, a higher proportion of the mesoderm cells were induced from hBMMSC-iPSCs than that from the untreated groups (P<0.05) by analysis the expression of Brachyury and GATA2 with RT-PCR and immunochemistry analysis. The efficient mesoderm cell activity was then directed to CD34+ progenitor cells with the following chemicals as SCF and IL-3 for another 7–9 days by analysis of cellular phenotype and the expression of the hematopoietic transcription factors TAL-1 and GATA-2. Sorted by CD34 magnetic separation and exposed to semisolid media with the hematopoietic cytokines including EPO and G-CSF, CD34+ progenitor cells formed various hematopoietic colonies as BFU-E, CFU-E, CFU-G, CFU-GM, and CFU-GEMM. CFUs derived from hBMMSC-iPSC groups treated with the chemicals outnumbered those from hFib-iPSCs, the spontaneous differentiation hBMMSC-iPSCs and H1 groups. Hematopoietic cell lineages as erythroid, granulocyte, and megakaryocyte were identified with Wright-Giemsa staining. During the hematopoietic commitment of hBMMSC-iPSCs, the pluripotent gene Oct4 continued to be down-regulated and completely vanished at day 14 after the induction. However, the expression of the hematologic transcription factor GATA-2 experienced a dynamic process with up-regulation at the initial induction stage and then gradual down-regulation after CD34+ progenitor cell commitment to CFU forming. Endothelial cell potential of CD34+ progenitor cells was also verified by demonstration of the vascular tube-like structure formation and the expression of endothelial specific markers CD31 and VE-CADHERIN. The efficient induction of CD34+ progenitor cells from hBMMSC-iPSCs with defined chemicals, avoiding contamination from indentified factors with using mouse feeder lays or other poorly defined induction components, gives an opportunity for the generation of unlimited HLA matched transplantable cells, provides cell models for study the mechanism of haemopoiesis and related diseases, and therefore promotes iPSC clinical application. Disclosures:No relevant conflicts of interest to declare.

Sequence Specific Single Stranded DNA Binding Protein 2 (SSBP2) Is a Regulator of Hematopoietic Stem Cell Activity

Abstract 1384Interstitial deletions of chromosome 5q are common, non-random anomalies in myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). While most deletions are large, spanning 5q13–33, a subset of patients deletes only the 5q11–14 region. We isolated SSBP2, a candidate gene from a critical region of loss at 5q14. SSBP2 expression is undetectable in AML cell lines, primary AML and MDS samples with chromosome 5q deletions. Decreased Ssbp2 expression due to viral integration has been reported in a case of primate AML resulting from HOXB4 retroviral transduction1. We have established SSBP2, to be a member of Lim Domain Binding Protein (LDB1)-Lim Only 2(LMO2)- TAL1/LYL1containing transcriptional complexes. SSBP2-LDB1, interactions promote gene expression by protecting LDB1 from proteosomal degradation2,3. LDB1-LMO2-bHLH complexes are also critical regulators of hematopoietic stem cells (HSC), a target of transformation in MDS and AML. The transcriptional cofactor LDB1 is required for hematopoiesis as part of an autoregulatory loop, which maintains the expression of TAL1, LMO2, E2A and GATA14.Since Ssbp2 is a candidate gene from 5q and an in vivo regulator of LDB1 stability3, we sought to determine its role in hematopoietic stem cell homeostasis. In normal marrow, Ssbp2 expression is highest in Lin−, Sca1+, c-Kit+ (LSK) cells and decreases during lineage commitment. Ssbp2−/− mice are apparently normal upto six months of age although multiple hematopoitic tissues exhibit modest hypoplasia. However, hematopoietic recovery from 5-Fluoro uracil treatment is considerably delayed in these mice. Furthermore, competitive repopulation assays reveal significantly reduced short and long-term engraftment with Ssbp2−/− bone marrow. Enforced expression of Ssbp2 rescues the competitive repopulating activity. Increased expression of E2A and its’ transcriptional target CDKN1A in Ssbp2−/− LSK cells appears to underlie the impaired stem cell activity. Our results identify SSBP2 to be a novel regulator of HSC function by maintaining the LDB1 mediated regulatory loop. Furthermore, these findings suggest defective stem cell homeostasis in the absence of SSBP2, when combined with mitogenic signals might contribute to the evolution of MDS/AML in a subset of patients. Disclosures:No relevant conflicts of interest to declare.

Core Transcriptional Regulatory Circuit Controlled by the TAL1 Complex in T-Cell Acute Lymphoblastic Leukemia,

Abstract 3453Aberrant expression of TAL1 is one of the most frequent abnormalities in T-cell acute lymphoblastic leukemia (T-ALL), yet little is known about the transcriptional network controlled by this oncogenic transcription factor, posing a major obstacle to understanding T-ALL pathogenesis. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, GATA3 and RUNX1 in T-ALL cells. We determined direct transcriptional targets of TAL1 and its regulatory partners by ChIP-seq analysis, and found that TAL1 binds to the majority of HEB- and E2A-enriched regions and that these commonly bound regions are frequently overlapping with the GATA3- and RUNX1-enriched regions. We found that TAL1 forms an interconnected auto-regulatory loop with its partners, which likely contribute to the sustained upregulation of its direct target genes. TAL1 core regulatory circuit activates genes involved in T-cell development and hematopoesis. Microarray gene expression analysis revealed that TAL1 and GATA3 predominantly act as positive regulators of the expression of their direct target genes in T-ALL. Importantly, we found the MYB oncogenic transcription factor is directly activated by the TAL1 complex and positively regulates many of the same target genes, thus forming a feed-forward positive regulatory loop that further promotes the TAL1-regulated oncogenic program. Moreover, we found that a specific subset of TAL1 target genes are oppositely regulated by TAL1 and its obligate partner proteins E2A and HEB, uncovering a leukemogenic pattern that previously emerged from studies in murine models. These findings underscore the importance of TAL1 as a critical regulator of an aberrant gene expression program in T-ALL, and indicate how these networks maintain the malignant state in thymocytes. Disclosures:No relevant conflicts of interest to declare.

Infantile Hemangioma: A Tumor Involving Hemogenic Endothelium with Erythropoetic Potential

AbstractAbstract 4305 BackgroundInfantile hemangioma (IH) is a tumor of the microvasculature predominantly composed of proliferating endothelial cells, but also contains progenitor cells of mesenchymal and hematopoietic lineages. We have previously shown that IH has a primitive mesodermal origin. Here we investigated expression of hematopoietic associated proteins (Tal-1, GATA-2, CD133 & CD34) as well as the erythropoietin receptor (EPOR) and the embryonically-associated hemoglobin zeta chain (HBZ) by both the cells of the endothelium and the cells emanating from proliferating IH explants. Additionally we investigated if the cells emanating from the explants generate erythrocytes in vitro. PurposeTo investigate the expression of hematopoietic associated proteins and the primitive erythropoietic capacity of proliferating IH. MethodsProliferating IH biopsies from 5 patients were (i) processed for IHC staining for Tal-1, GATA-2, CD133, CD34, HBZ and EPOR; (ii) used to determine the relative expression of Tal-1, GATA-2, CD133, CD34, HBZ & EPOR transcripts by qRT-PCR; (ii) cultured as explants using our in vitro model and differentiated the cells down the erythropoietic lineage. ResultsIHC showed that the cells that line the endothelium forming the capillaries of proliferating IH expressed Tal-1, GATA-2, CD133, CD34, HBZ and EPOR. qRT-PCR confirmed the IHC data as abundant expression mRNA transcripts for the same proteins was detected in both the tissue and the cells emanating from the cultured explants. Following 5 months in culture morphological biconcave cells were visible, and were confirmed to be erythrocytes by immunoreactivity to the erythrocyte-specific cell surface marker glycophorin A. ConclusionThe mRNA and protein expression of hematopoietic associated proteins in proliferating IH supports our previous studies showing this tumor expressing markers of primitive mesoderm and confirms a hemogenic endothelium phenotype. The expression of the primitive erythropoiesis-associated proteins HBZ and EPOR by proliferating IH and its functional ability of proliferating IH explants to form erythrocytes in vitro confirms the erythropoietic potential. Proliferating IH thus represents an extra-medullary site of tumor-associated erythropoiesis. Disclosures:No relevant conflicts of interest to declare.

Targets of the Tal1 Transcription Factor in Erythrocytes: E2 UBIQUITIN CONJUGASE REGULATION BY Tal1

The Tal1 transcription factor is essential for the development of the hematopoietic system and plays a role during definitive erythropoiesis in the adult. Despite the importance of Tal1 in erythropoiesis, only a small number of erythroid differentiation target genes are known. A chromatin precipitation and cloning approach was established to uncover novel Tal1 target genes in erythropoiesis. The BirA tag/BirA ligase biotinylation system in combination with streptavidin chromatin precipitation (Strep-CP) was used to co-precipitate genomic DNA bound to Tal1. Tal1 was found to bind in the vicinity of 31 genes including the E2-ubiquitin conjugase UBE2H gene. Binding of Tal1 to UBE2H was confirmed by chromatin immunoprecipitation. UBE2H expression is increased during erythroid differentiation of hCD34(+) cells. Tal1 expression activated UBE2H expression, whereas Tal1 knock-down reduced UBE2H expression and ubiquitin transfer activity. This study identifies parts of the ubiquitinylation machinery as a cellular target downstream of the transcription factor Tal1 and provides novel insights into Tal1-regulated erythropoiesis.

Defective Erythropoiesis in Transgenic Mice Expressing Dominant-Negative Upstream Stimulatory Factor

Transcription factor USF is a ubiquitously expressed member of the helix-loop-helix family of proteins. It binds with high affinity to E-box elements and, through interaction with coactivators, aids in the formation of transcription complexes. Previous work demonstrated that USF regulates genes during erythroid differentiation, including HoxB4 and beta-globin. Here, we show that the erythroid cell-specific expression of a dominant-negative mutant of USF, A-USF, in transgenic mice reduces the expression of all beta-type globin genes and leads to the diminished association of RNA polymerase II with locus control region element HS2 and with the beta-globin gene promoter. We further show that the expression of A-USF reduces the expression of several key erythroid cell-specific transcription factors, including EKLF and Tal-1. We provide evidence demonstrating that USF interacts with known regulatory DNA elements in the EKLF and Tal-1 gene loci in erythroid cells. Furthermore, A-USF-expressing transgenic mice exhibit a defect in the formation of CD71(+) progenitor and Ter-119(+) mature erythroid cells. In summary, the data demonstrate that USF regulates globin gene expression indirectly by enhancing the expression of erythroid transcription factors and directly by mediating the recruitment of transcription complexes to the globin gene locus.

Gene Specificity of Suppression of Transgene-Mediated Insertional Transcriptional Activation by the Chicken HS4 Insulator

Insertional mutagenesis has emerged as a major obstacle for gene therapy based on vectors that integrate randomly in the genome. Reducing the genotoxicity of genomic viral integration can, in first approximation, be equated with reducing the risk of oncogene activation, at least in the case of therapeutic payloads that have no known oncogenic potential, such as the globin genes. An attractive solution to the problem of oncogene activation is the inclusion of insulators/enhancer-blockers in the viral vectors. In this study we have used Recombinase-Mediated Cassette Exchange to characterize the effect of integration of globin therapeutic cassettes in the presence or absence of the chicken HS4 and three other putative insulators inserted near Stil, Tal1 and MAP17, three well-known cellular proto-oncogenes in the SCL/Tal1 locus. We show that insertion of a Locus Control Region-driven globin therapeutic globin transgene had a dramatic activating effect on Tal1 and Map17, the two closest genes, a minor effect on Stil, and no effect on Cyp4x1, a non-expressed gene. Of the four element tested, cHS4 was the only one that was able to suppress this transgene-mediated insertional transcriptional activation. cHS4 had a strong suppressive effect on the activation expression of Map17 but has little or no effect on expression of Tal1. The suppressive activity of cHS4 is therefore promoter specific. Importantly, the observed suppressive effect of cHS4 on Map17 activation did not depend on its intercalation between the LCR and the Map 17 promoter. Rather, presence of one or two copies of cHS4 anywhere within the transgene was sufficient to almost completely block the activation of Map17. Therefore, at this complex locus, suppression of transgene-mediated insertional transcriptional activation by cHS4 could not be adequately explained by models that predict that cHS4 can only suppress expression through an enhancer-blocking activity that requires intercalation between an enhancer and a promoter. This has important implications for our theoretical understanding of the possible effects of the insertion of cHS4 on gene therapy vectors. We also show that cHS4 decreased the level of expression of the globin transgene. Therefore, the benefits of partially preventing insertional gene activation are in part negated by the lower expression level of the transgene. A cost/benefit analysis of the utility of incorporation of insulators in gene therapy vectors will require further studies in which the effects of insulators on both the therapeutic gene and the flanking genes are determined at a large number of integration sites. Identification of insulators with minimal promoter specificity would also be of great value.

Multicellular sprouting during vasculogenesis

Vasculogenesis, the de novo assembly of vessels from endothelial precursors, is a fundamental process common to both embryonic development and certain pathophysiologies. We studied vasculogenic sprouting, the multicellular patterning mechanisms of vasculogenesis, with time‐lapse microscopy in avian embryos, and in cultures of endothelial cells invading 3D collagen I gels. In quail embryos cell‐autonomous endothelial cell motility and tissue movements were distinguished based on a new method utilizing digitally filtered displacements of immuno‐labeled extracellular matrix components fibrillin and fibronectin. Recently developed TIE1‐GFP transgenic quail lines enabled us to investigate the motility of endothelial cells inside the forming vessels. We correlate the degree of in situ cell‐autonomous motility with the level of TAL1 expression, the morphology of the surrounding vessel network and the structure of the extracellular matrix environment.

NKX3.1 Is a Direct TAL1 Target Gene That Mediates the TAL1 Dependent Proliferation of Human T-Cell Acute Lymphoblastic Leukemia (T-ALL)

The TAL1/SCL gene encodes a bHLH (basic Helix-Loop-helix) protein that acts as a master gene in hematopoiesis. The TAL1/SCL gene is also the most frequently activated gene in human T-ALL but the oncogenic transcriptional programs, downstream of TAL1 in human T-ALL, are not well characterized. Using RNA interference to knockdown TAL1 expression, we show that TAL1 regulates both cell proliferation and death of human T-ALL cells. To determine the TAL1 target genes in human T-ALL, we combine TAL1 knockdown and gene expression profiling and show that TAL1 activates and repress a common subset of genes in cell lines. This subset includes known TAL1 target genes but also the NKX3.1 gene that is a homeobox gene, specifically expressed in the prostate epithelium during prostate development and in adulthood. NKX3.1 gene inactivation is one of the earliest events that occur in prostate cancer initiation, defining NKX3.1 as a major tumor suppressor gene of this cancer. TAL1 expression is associated with NKX3.1 activation in human T-ALL cell lines and NKX3.1 is expressed in TAL1 expressing human T-ALL blasts. TAL1 and GATA-3 are specifically bound in vivo to the [−870/−570] region of the human NKX3.1 gene promoter, and ex vivo, TAL1 can either directly binds an E-box [position −738] or be recruited by GATA-3 on a GATA binding site [position −697]. Finally, functional analyses of the NKX3.1 promoter indicate that these binding sites mediate the transcriptional activity of this promoter in T-cell lines. Sequences analysis of the human and mouse NKX3.1 promoters show that the regulatory sequences involved in the TAL1 activation of the human NKX3.1 gene are not conserved in the mouse gene, indicating why the NKX3.1 gene is not expressed in mouse models of TAL1 mediated leukemogenesis. NKX3.1 knockdown shows that NKX3.1 is necessary for the proliferation of TAL1 expressing T-ALL cell lines and NKX3.1 overexpression can complement the proliferation defects associated with TAL1 knockdown in T-ALL cell lines. Microarray analyses show that TAL1 and NKX3.1 regulate a common subset of genes in T-ALL that includes numerous genes encoding proteins known to be involved in T-cell proliferation and/or signaling. Finally, using a new culture system that enables proliferation of primary human leukemic cells, we show that the NKX3.1 gene is specifically activated in human TAL1 expressing T-ALL together with the defined potential TAL1 and/or NKX3.1 target genes. These results characterize NKX3.1 as the first gene directly activated by TAL1 and involved in the TAL1 dependent proliferation of human T-cell Acute Lymphoblastic Leukemia.

Erythropoietin Receptor Expression: A Role for the Tal1/SCL and GATA-1 Complex

Tal1/SCL is required for hematopoietic stem cell development and is essential for normal erythropoiesis. Although Tal1-DNA binding is not required for hematopoietic development, Tal1 participates in a complex with other binding partners GATA-1, LMO2, Ldb1 and possibly Sp1. During erythropoiesis, Tal1 can activate expression via direct association with E-box-GATA DNA sequence motifs for lineage specific genes such as Protein 4.2. In mice, loss of Tal1 in adult erythropoiesis also affects TER119 expression and BFU-E growth resulting in anemia. We found that knock down of Tal1 expression in primary erythropoietin (EPO) stimulated hematopoietic progenitor cells in culture inhibited EPO receptor (EPOR) expression and erythroid differentiation, consistent with the anemia observed in mice with targeted deletion of adult Tal1 expression. In contrast, overexpression of Tal1 in erythroid cells increased both EPOR expression and erythroid differentiation. In fact, overexpression of EPOR was sufficient to increase differentiation in erythroid progenitor cell cultures. Increased EPOR expression by Tal1 was mediated by 3 conserved E-boxes in the 5′ UTR. In reporter gene assays in K562 erythroid cells, EPOR promoter activity was lost with the mutation of these E-boxes, which are located 75 bp downstream of the required GATA-1 binding motif in the human EPOR proximal promoter. Tal1/E2A dimer binding to the E-box region was demonstrated by gel mobility shift assay. Tal1 transactivated EPOR mRNA and chromatin immunoprecipitation (ChIP) analysis confirmed that Tal1 bound directly to the E-box region in intact erythroid cells. We previously showed that GATA-1 also transactivates EPOR gene expression and is required for high level of EPOR transcription activity. In ChIP assays, an antibody to Tal1 also pulled down chromatin containing the GATA-1 binding site as well as the E-box region and conversely, antibodies to GATA-1 pulled down chromatin corresponding to the GATA-1 binding site and the E-box region. These data show that the complex containing Tal1 and GATA-1 bound to the E-box region and to the GATA-1 site in the EPOR proximal promoter and that the complex has greater occupancy at the GATA-1 site in the proximal promoter. High-level Tal1 expression increased chromatin containing Tal1 or GATA-1 associated with the GATA-1 binding site as well as the down stream E-box region, with a greater proportionate increase in the E-box region. Hence, in addition to the GATA-1 binding site, the downstream E-box region is necessary for high-level EPOR expression. These data suggest that transactivation of the EPOR promoter via these sites is mediated by a Tal1 and GATA-1 containing complex and that during erythropoiesis EPO induction of both Tal1 and GATA-1 regulates the high expression of its own receptor. We hypothesize that the high binding of GATA-1 and Tal1 at the GATA site opens the upstream chromatin structure. The over-expression of Tal1 promotes the open status and facilitates the spreading of open chromatin along the chromatin to the downstream E-box region. Subsequent E-box binding by Tal1 and its interaction with GATA-1 maintains the open chromatin and activates transcription.

Novel Role of the TAL1/SCL Transcription Factor in Murine Monocytopoiesis.

The basic helix-loop-helix (bHLH) transcription factor TAL1/SCL plays a critical role in hematopoiesis and vascular remodeling. A mouse Tal1 cDNA was first cloned from a bone marrow (BM) macrophage cDNA library, and we and others observed expression ofTal1 protein by BM mononuclear cells. To characterize Tal1 expression during monocyte/macrophage differentiation, we isolated common myeloid precursors (CMPs) from BM of 3-5 week old C57BL/6J mice and induced them to terminally differentiate according to a published method (Genes & Dev. , 16:1721, 2002). Using real-time PCR analysis,Tal1 mRNA was expressed in a biphasic pattern from CMP to post-mitotic macrophage, including lipopolysaccharide- and interferon-ã-activated macrophages. To elucidate Tal1'sfunctions in murine monocytopoiesis we deleted the Tal1 gene in murine BM monocytes and monocytic precursors in culture. To that end, C57BL/6 mice with loxP sequences flanking the third coding exon of Tal1 were bred with C57BL/6 mice with a lacZ gene replacing Tal1 coding exons 1, 2, and 3. Tal1fl/fl/lacZ progeny were identified by PCR genotyping, and BM mononuclear cells were cultured with mouse interleukin-3 and macrophage colony-stimulating factor (M-CSF). To render the cells Tal1-null, Cre coding sequences were introduced with the MSCV-GFP retroviral vector and GFP-positive cells were then sorted and cultured with M-CSF alone. Real-time PCR analysis showed near-total abolition of Tal1 mRNA expression in Cre-transduced relative to vector-transduced cells. Gene expression analysis for other transcripts showed an approximately 4-foldreduction in Gata2 expression over the same culture period but no difference in Aml1,PU.1, Csfr1, Msr1 (mouse scavenger receptor), Cd68, or Il6ra. Biologically, the most significant effect of Tal1 knockout was on cell number, which increased by 80% in control cells but not at all in Tal1-null cells. Transduction of wild-type BM monocytes with MSCV-GFP-Cre (or the parental MSCV-GFP) vector had no effect on cell proliferation, precluding any nonspecific or toxic effect of Cre (or retroviral infection) in this cell type. Dye dilution analysis of virus-transduced cells with the fluorescent membrane-intercalating dye PKH26 revealed a delay and absolute reduction in proliferation of Tal1-null compared to control cells. In contrast, little or no difference was noted in annexin V staining ofTal1-null compared to heterozygous knockout (knock-in) cells, indicating a lack of effect on apoptosis. Finally, serial analysis of CD31 and Ly6c expression in differentiating Tal1hemizygous and nullizygous BM monocytes showed that loss of Tal1 caused a slight acceleration in terminal monocyte-macrophage differentiation. In summary, these studies confirm our earlier finding that the Tal1 gene is expressed in differentiating mouse BMmonocytes. In addition, they reveal a novel function of this bHLH transcription factor in proliferation of murine monocyte/macrophage precursors. Finally, they place Tal1upstream of Gata2 in cells of this lineage.

575 POSTER Targeting metastatic tumor cell functions by inhibition of new blood vessel formation with the selective VEGF signal inhibitor, cediranib (RECENTIN™, AZD2171) or Src signaling interference using the small molecule Src inhibitor, AZD0530

The mechanisms controlling blood vessel formation during early embryonal development have only partly been elucidated. Shb is an adaptor protein previously implicated in the angiogenic response to vascular endothelial growth factor (VEGF). To elucidate a possible role of Shb in embryonic vascular development, wild-type and SH2 domain mutated (R522K) Shb were overexpressed in murine embryonic stem (ES) cells. Embryoid bodies (EBs) differentiating from Shb-overexpressing ES cells in vitro were stained for CD31 or VEGFR-2 to visualize the formation of vascular structures. We found that Shb promotes the outgrowth of blood vessels in EBs both in the absence and presence of growth factors. This response may be the consequence of an increased number of VEGFR-2 positive cells at an early stage of EB development, a finding corroborated by both immunostaining and real-time RT-PCR. In addition, Shb overexpression upregulated the expression of PDGFR-β, CD31, CD41 and Tal1. Cells co-expressing VEGFR-2 and PDGFR-β were commonly observed when Shb was overexpressed and inhibition of PDGF-BB signaling reduced the amount of VEGFR-2 mRNA under these conditions. EBs expressing the Shb R522K-mutant did not form vascular structures. Microarray analysis of VEGFR-2/CD31 positive cells after 6 days of differentiation revealed numerous changes of expression of genes relating to an endothelial/hematopoietic phenotype in response to Shb overexpression. The findings suggest that Shb may play a crucial role during early ES cell differentiation to vascular structures by transducing VEGFR-2 and PDGFR-β signals.

Impairment of Granulo-Monocytic Development of Human Common Myeloid Progenitors but Not of Granulo-Monocytic Progenitors by Decreasing Stem Cell Leukemia/T-Cell Acute Leukemia 1 Expression

We recently showed that Stem Cell Leukemia/T-cell Acute Leukemia 1 (SCL/TAL1) regulates hematopoiesis from hematopoietic stem cells to committed myeloid progenitors compartment. However, in this heterogeneous compartment, the precise role of TAL1, that is largely debated, remains to be clearly defined, notably at the common myeloid progenitor (CMP) and granulo-monocytic progenitor (GMP) levels. Using small hairpin (sh)RNA lentiviral constructs, we decreased TAL1 expression in sorted human CMP and GMP subpopulations that were then assayed for erythroid and granulo-monocytic (GM) differentiation. Decreased TAL1 expression in CMP resulted in rare erythroid colonies, in a 2-3 fold reduction of GM colony number in clonogenic assays and in a 3.6-5.6 decreased production of CD14(+)CD15(+) GM cells in liquid culture. Moreover, analysis of transcript profile of gene involved in GM differentiation showed that GM cells expressing shRNA-TAL1 construct displayed decreased levels of g-csfr, c/ebpalpha, and mpo and high levels of gata-2 transcripts, indicating a blocking of GM differentiation. In contrast, GM differentiation of GMP remained unaffected when TAL1 transcript levels were decreased. These data definitively delineate the human myeloid progenitors that are regulated by TAL1. Disclosure of potential conflicts of interest is found at the end of this article.

Ubiquitination and Degradation of Tal1/SCL Are Induced by Notch Signaling and Depend on Skp2 and CHIP

Notch signaling controls diverse eukaryotic differentiation processes in multiple cell types, thus demanding versatile tools with which Notch triggers downstream events. Ubiquitin-mediated proteolysis has previously been shown to be one such tool with which Notch regulates the turnover of the basic helix-loophelix transcription factor, E47. Here, we show that Notch signaling also accelerated the degradation of Tal1/SCL (T cell acute leukemia 1/stem cell leukemia) protein, a basic helix-loop-helix protein involved in the development of hematopoietic, vascular, and neuronal tissues. Notch-induced Tal1/SCL degradation was mediated by ubiquitination and proteasomes. The sequence responsible for Tal1 degradation was localized to a region in the C terminus of Tal1, which is evolutionarily conserved, thus suggesting a functional significance. Analogous to the situation for E47, Notch-induced Tal1/SCL degradation not only required Skp2, a substrate-binding subunit of SCF ubiquitin ligase complexes, but also relied on CHIP, a chaperone-binding protein with a ubiquitin ligase activity. In contrast to the fact that the N-terminal tetratricopeptide region (TPR) domain of CHIP is necessary and sufficient for E47 ubiquitination and degradation, CHIP promoted Tal1 degradation with both chaperone binding and ubiquitin ligase activities, which are mediated by its TPR domain and U box, respectively. Although the TPR domain was not involved in Tal1/SCL binding, it was required for enhancing its degradation. Likewise, the ubiquitin ligase activity of CHIP was dispensable for Tal1/SCL binding but essential for degradation. These findings provide both novel mechanistic insights into the operation of cullin-based ubiquitin ligase complexes and potential means by which Notch and Tal1/SCL regulate eukaryotic development.

Prognostic Significance of Molecular-Cytogenetic Abnormalities in Pediatric T-ALL Is Not Explained by Immunophenotypic Differences.

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is characterized by recurrent chromosomal rearrangements that may have prognostic significance. T-ALL cases with specific aberrations may arrest at specific T-cell development stages. Two classification systems were developed to classify T-ALL into developmental subgroups, i.e. the European Group for the Immunological Characterization of Leukemias (EGIL) and the T-cell receptor (TCR) classification system. In this study, we investigated the relationship between molecular-cytogenetic abnormalities and T-cell development stage. We investigated whether arrest at specific T-cell stages explains the prognostic significance of molecular-cytogenetic abnormalities. To this aim, we extensively studied 72 pediatric T-ALL cases by FISH and RQ-PCR for the presence of genetic abnormalities and expression of transcription factors, by PCR and sequencing for NOTCH1 mutations and by flow-cytometry to determine the T-cell receptor status as well as CD marker expression. The median clinical follow-up was 5 years. HOX11 rearranged cases were CD1 positive consistent with a cortical stage, but as 4 out of 5 cases lacked cytoplasmatic-beta expression, developmental arrest may precede beta-selection. HOX11L2 was especially confined to immature and pre-AB developmental stages, but 3 out of 17 HOX11L2 mature cases were restricted to the γδ-lineage. TAL1 rearrangements were restricted to the αβ-lineage with most cases being TCRαβ positive. NOTCH1 mutations were present in all molecular-cytogenetic subgroups without restriction to a specific developmental stage. The CALM-AF10 translocation as detected in 3 T-ALL patients was significantly associated with early relapse. TAL1 or HOX11L2 rearrangements were associated with trends to good and poor outcome, respectively. Cases with high TAL1 expression levels also demonstrated a trend towards good outcome, whereas cases with the lowest TAL1 levels had a poor outcome and were mostly HOX11L2 or CALM-AF10 positive. NOTCH1 mutations did not predict for poor outcome. Classification into T-cell developmental subgroups did not predict for outcome. In conclusion, the present study shows that differences in outcome for various molecular-cytogenetic subgroups cannot be attributed to differences in T-cell maturation stage.

Prognostic significance of molecular-cytogenetic abnormalities in pediatric T-ALL is not explained by immunophenotypic differences

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is characterized by chromosomal rearrangements possibly enforcing arrest at specific development stages. We studied the relationship between molecular-cytogenetic abnormalities and T-cell development stage to investigate whether arrest at specific stages can explain the prognostic significance of specific abnormalities. We extensively studied 72 pediatric T-ALL cases for genetic abnormalities and expression of transcription factors, NOTCH1 mutations and expression of specific CD markers. HOX11 cases were CD1 positive consistent with a cortical stage, but as 4/5 cases lacked cytoplasmatic-beta expression, developmental arrest may precede beta-selection. HOX11L2 was especially confined to immature and pre-AB developmental stages, but 3/17 HOX11L2 mature cases were restricted to the gammadelta-lineage. TAL1 rearrangements were restricted to the alphabeta-lineage with most cases being TCR-alphabeta positive. NOTCH1 mutations were present in all molecular-cytogenetic subgroups without restriction to a specific developmental stage. CALM-AF10 was associated with early relapse. TAL1 or HOX11L2 rearrangements were associated with trends to good and poor outcomes, respectively. Also cases with high vs low TAL1 expression levels demonstrated a trend toward good outcome. Most cases with lower TAL1 levels were HOX11L2 or CALM-AF10 positive. NOTCH1 mutations did not predict for outcome. Classification into T-cell developmental subgroups was not predictive for outcome.