Although aldehyde dehydrogenase (ALDH) activity has become a surrogate of hematopoietic stem and progenitor cells (HSPCs), its function during hematopoiesis was unclear. Here, we examined its role in zebrafish hematopoiesis based on pharmacological inhibition and morpholino (MO) knockdown. Zebrafish embryos were treated with diethylaminobenzaldehyde (DEAB, 1 μmol/l) between 0- and 48 hour-post-fertilization (hpf). MOs targeting aldhs were injected between 1 and 4-cell stage. The effects on hematopoiesis were evaluated at different stages. DEAB treatment between 0 and 18 hpf increased gene expression associated with HSPC (scl, lmo2), erythropoiesis (gata1, α- and β-eHb) and myelopoiesis (spi1) as well as gfp(+) cells in dissociated Tg(gata1:gfp) embryos. The effects were ameliorated by all-trans retinoic acid (1 nmol/l). Definitive hematopoiesis and the erythromyeloid precursors were unaffected. In all, 14 out of 15 zebrafish aldhs were detectable by reverse transcription PCR in 18 hpf embryos, of which only aldh1a2 and aldh16a1 were expressed in sites pertinent to hematopoiesis. Molecular targeting by MOs was demonstrated for 15 aldhs, but none of them, even in combined aldh1a2 and aldh1a3 knockdown, recapitulated the hematopoietic expansion in DEAB-treated embryos. In conclusion, DEAB expands HSPC population during primitive hematopoiesis through inhibition of aldh and retinoic acid synthesis. The specific aldh isoform(s) remains to be determined.

BACKGROUND

Human embryonic stem cells (hESCs) are a potentially inexhaustible source of cells for replacement therapy. However, successful preclinical and clinical progress requires efficient and controlled differentiation towards the specific differentiated cell fate.

METHODS

We previously developed a strategy to generate blast cells (BCs) from hESCs that were capable of differentiating into vascular structures as well as into all hematopoietic cell lineages. Although the BCs were shown to repair damaged vasculature in multiple animal models, the large-scale generation of cells under these conditions was challenging. Here we report a simpler and more efficient method for robust generation of hemangioblastic progenitors.

RESULTS

In addition to eliminating several expensive factors that are unnecessary, we demonstrate that bone morphogenetic protein (BMP)-4 and VEGF are necessary and sufficient to induce hemangioblastic commitment and development from hESCs during early stages of differentiation. BMP-4 and VEGF significantly upregulate T-brachyury, KDR, CD31 and Lmo2 gene expression, while dramatically downregulating Oct-4 expression. The addition of basic FGF during growth and expansion was found to further enhance BC development, consistently generating approximately 1 x 10(8) BCs from one six well plate of hESCs.

CONCLUSIONS

This new method represents a significantly improved system for generating hemangioblasts from hESCs, and although simplified, results in an eightfold increase in cell yield.

GATA-binding protein 2 (GATA2) and LIM domain only 2 (Lmo2) form common transcription complexes during hematopoietic differentiation. Here we show that these two transcription factors also play a key role in endothelial cells (EC) and lymphatic EC (LEC) function. Primary EC and tumor-associated blood vessels expressed GATA2 and Lmo2. VEGF-induced sprouting angiogenesis in both differentiating embryonic stem cells (embryoid bodies) and primary EC increased GATA2 and Lmo2 levels. Conversely, silencing of GATA2 and Lmo2 expression in primary EC inhibited VEGF-induced angiogenic activity, including EC migration and sprouting in vitro, two key steps of angiogenesis in vivo. This inhibition of EC function was associated with downregulated expression of neuropilin-2 (NRP2), a co-receptor of VEGFRs for VEGF, at the protein, mRNA and promoter levels. NRP2 overexpression partially rescued the impaired angiogenic sprouting in the GATA2/Lmo2 knockdown EC, confirming that GATA2 and Lmo2 mediated EC function, at least in part, by directly regulating NRP2 gene expression. Furthermore, it was found that primary LEC expressed GATA2 and Lmo2 as well. Silencing of GATA2 and Lmo2 expression in LEC inhibited VEGF-induced LEC sprouting, also in a NRP2-dependent manner. In conclusion, our results demonstrate that GATA2 and Lmo2 cooperatively regulate VEGF-induced angiogenesis and lymphangiogenesis via NRP2.

Nodal marginal zone lymphoma (NMZL) represents a rare and heterogeneous group that lacks markers specific for the diagnosis. We evaluated morphologic and immunoarchitectural features of 51 NMZLs, and the following immunostains were performed: CD20, CD21, CD23, CD5, CD3, CD43, CD10, Ki-67, BCL1, BCL2, BCL6, HGAL, and LMO2. Four immunoarchitectural patterns were evident: diffuse (38 [75%]), well-formed nodular/follicular (5 [10%]), interfollicular (7 [14%]), and perifollicular (1 [2%]). Additional features included a monocytoid component (36 [71%]), admixed large cells (20 [39%]), plasma cells (24 [47%]), compartmentalizing stromal sclerosis (13 [25%]), and prominent blood vessel sclerosis (10 [20%]). CD21 highlighted disrupted follicular dendritic cell meshwork in 35 (71%) of 49 cases, and CD43 coexpression was present in 10 (24%) of 42 cases. A panel of germinal center-associated markers was helpful in eliminating cases of diffuse follicle center lymphoma. Our results highlight the histologic and immunoarchitectural spectrum of NMZL and the usefulness of immunohistochemical analysis for CD43, CD23, CD21, BCL6, HGAL, and LMO2 in the diagnosis of NMZL.

Cre/loxP system is a powerful tool to manipulate the genome. Transgenic animals expressing Cre recombinase in specific tissues or cells have been widely used for conditional gene targeting, lineage tracing, and other genetic analyses. In zebrafish, the transgenic line with stable expression of Cre in specific tissues and cell subtypes has not been generated and its functional activity remains to be defined. Here we report the establishment of a stable transgenic fish Tg(zlmo2:Cre), which specifically expresses Cre in the primitive hematopoietic progenitors and vascular endothelial cells, under the control of lmo2 promoter. Our result shows that the Cre expression pattern recapitulates the endogenous lmo2 expression pattern during embryogenesis. Crossing of the Tg(zlmo2:Cre) line with another established transgenic reporter line Tg(zlmo2:loxP-DsRed-loxP-EGFP), induces a robust recombination activity in hematopoietic progenitors and vascular endothelial cells. Thus, the Tg(zlmo2:Cre) transgenic line provides an invaluable tool to dissect genetic pathways in hematopoietic development and diseases.

Several identified genes play key roles in the specification of the blood-forming system, from commitment of mesoderm to differentiation of hemopoietic and endothelial cells. We have thoroughly analyzed the expression dynamics of some of these genes during yolk sac erythropoiesis in the chick embryo. The study includes transcription factors which are known to participate in multimeric complexes: GATA-1, -2, SCL/tal-1 and Lmo2 (whose avian orthologue we have cloned), VEGF-R2, a critical regulator of hemopoietic and endothelial commitment, and hemoglobin used as a marker of the last step in erythroid differentiation. Several findings were unexpected. (1) Two distinct patterns were revealed for GATA-2, first: low expression, ubiquitous in all mesodermal cells, as soon as cells ingress through the primitive streak; secondly: high, blood island-specific expression. (2) VEGF-R2 is coexpressed with GATA-2 at the level of the primitive streak. (3) SCL and Lmo2 expression is restricted to presumptive hemangioblasts. (4) The up-regulation of GATA-2 in newly formed blood islands is shortly followed by GATA-1 expression. (5) Lmo2 is up-regulated in blood island angioblasts thus appearing as one of the earliest markers for endothelial cell commitment. VEGF-R2 is down-regulated in hemopoietic cells prior to GATA-2, SCL/tal-1, Lmo2 and GATA-1 in erythroblasts.

The oncogenic fusion protein E2A-HLF is a chimeric transcription factor that arises from the t(17;19) translocation in childhood B-cell acute lymphoblastic leukemias (B-precursor ALL) and is associated with very poor outcome. We show that retroviral-mediated expression of E2A-HLF alone is sufficient to immortalize primary lymphoid progenitors. We identify Lmo2 and Bcl-2 as direct target genes downstream of E2A-HLF. We use real-time PCR analysis to show that LMO2 and BCL-2 expression is preferentially upregulated both in biopsy material from t(17;19) B-precursor ALL patients and lymphoid cell lines derived from t(17;19) leukemias. Co-expression of Lmo2 and Bcl-2 was sufficient to immortalize lymphoid progenitor cells resulting in a similar phenotype to that induced by E2A-HLF alone. Both shRNA-mediated knockdown of Lmo2 expression and pharmacological inhibition of BCL-2 function in E2A-HLF immortalized cells severely compromised their viability. These data suggest that both Lmo2 and Bcl-2 are required for the action of E2A-HLF in leukemogenesis.

Gene transfer vectors derived from gamma-retroviruses or lentiviruses are currently used for the gene therapy of genetic or acquired diseases. Retroviral vectors display a non-random integration pattern in the human genome, targeting either regulatory regions (gamma-retroviruses) or the transcribed portion of expressed genes (lentiviruses), and have the potential to deregulate gene expression at the transcriptional or post-transcriptional level. A recently developed alternative vector system derives from the avian sarcoma-leukosis alpha-retrovirus (ASLV) and shows favorable safety features compared to both gamma-retroviral and lentiviral vectors in preclinical models. We performed a high-throughput analysis of the integration pattern of self-inactivating (SIN) alpha-retroviral vectors in human CD34+ hematopoietic stem/progenitor cells (HSPCs) and compared it to previously reported gamma-retroviral and lentiviral vectors integration profiles obtained in the same experimental setting. Compared to gamma-retroviral and lentiviral vectors, the SIN-ASLV vector maintains a preference for open chromatin regions, but shows no bias for transcriptional regulatory elements or transcription units, as defined by genomic annotations and epigenetic markers (H3K4me1 and H3K4me3 histone modifications). Importantly, SIN-ASLV integrations do not cluster in hot spots and target potentially dangerous genomic loci, such as the EVI2A/B, RUNX1 and LMO2 proto-oncogenes at a virtually random frequency. These characteristics predict a safer profile for ASLV-derived vectors for clinical applications.

Ets transcription factors play important roles during the development and maintenance of the haematopoietic system. One such factor, Elf-1 (E74-like factor 1) controls the expression of multiple essential haematopoietic regulators including Scl/Tal1, Lmo2 and PU.1. However, to integrate Elf-1 into the wider regulatory hierarchies controlling haematopoietic development and differentiation, regulatory elements as well as upstream regulators of Elf-1 need to be identified. Here, we have used locus-wide comparative genomic analysis coupled with chromatin immunoprecipitation (ChIP-chip) assays which resulted in the identification of five distinct regulatory regions directing expression of Elf-1. Further, ChIP-chip assays followed by functional validation demonstrated that the key haematopoietic transcription factor PU.1 is a major upstream regulator of Elf-1. Finally, overexpression studies in a well-characterized erythroid differentiation assay from primary murine fetal liver cells demonstrated that Elf-1 downregulation is necessary for terminal erythroid differentiation. Given the known activation of PU.1 by Elf-1 and our newly identified reciprocal activation of Elf-1 by PU.1, identification of an inhibitory role for Elf-1 has significant implications for our understanding of how PU.1 controls myeloid-erythroid differentiation. Our findings therefore not only represent the first report of Elf-1 regulation but also enhance our understanding of the wider regulatory networks that control haematopoiesis.

Little is known about the genes that control the embryonic erythroid program. Laser capture microdissection was used to isolate primitive erythroid precursors and epithelial cells from frozen sections of the embryonic day 9.5 yolk sac. The RNA samples were amplified and labeled for hybridization to Affymetrix GeneChip Mouse Genome 430A 2.0 arrays. Ninety-one genes are expressed significantly higher in erythroid than in epithelial cells. Ingenuity pathway analysis indicates that many of these erythroid-enriched genes cluster in highly significant biological networks. One of these networks contains RBTN2/LMO2, SCL/TAL1, and EKLF/KLF1, three of the very few genes required for primitive erythropoiesis. Quantitative real-time polymerase chain reaction was used to verify that platelet factor 4, reelin, thrombospondin-1, and muscleblind-like 1 mRNA is erythroid-enriched. These genes have established roles in development or differentiation in other systems, and are, therefore, good candidates for regulating primitive erythropoiesis. These results provide a catalog of genes expressed during primitive erythropoiesis.

LMO2, a critical transcription regulator of hematopoiesis, is involved in human T-cell leukemia. The binding site of proline and acidic amino acid-rich protein (PAR) transcription factors in the promoter of the LMO2 gene plays a central role in hematopoietic-specific expression. E2A-HLF fusion derived from t(17;19) in B-precursor acute lymphoblastic leukemia (ALL) has the transactivation domain of E2A and the basic region/leucine zipper domain of HLF, which is a PAR transcription factor, raising the possibility that E2A-HLF aberrantly induces LMO2 expression. We here demonstrate that cell lines and a primary sample of t(17;19)-ALL expressed LMO2 at significantly higher levels than other B-precursor ALLs did. Transfection of E2A-HLF into a non-t(17;19) B-precursor ALL cell line induced LMO2 gene expression that was dependent on the DNA-binding and transactivation activities of E2A-HLF. The PAR site in the LMO2 gene promoter was critical for E2A-HLF-induced LMO2 expression. Gene silencing of LMO2 in a t(17;19)-ALL cell line by short hairpin RNA induced apoptotic cell death. These observations indicated that E2A-HLF promotes cell survival of t(17;19)-ALL cells by aberrantly up-regulating LMO2 expression. LMO2 could be a target for a new therapeutic modality for extremely chemo-resistant t(17;19)-ALL.

SCL, Lmo2 and GATA factors form common transcription complexes during hematopoietic differentiation. The overlapping expression of SCL with GATA-2 and GATA-3 in the developing brain indicated that these factors might collaborate also in the course of neural tissue differentiation. The expression pattern of Lmo2 in the developing CNS, however, is not well understood. Here, we show that neural cells in the early embryonic chick mid- and hindbrain express SCL and GATA-2, while Lmo2 is expressed only in vascular elements. The lack of Lmo2 transcripts in neural cells demonstrated that SCL and GATA-2 cannot form common complexes with Lmo2 in the developing brain. In the course of neural tissue genesis, GATA-2 mRNA appeared prior to the SCL transcript. While GATA-2 expression decreased with maturation, SCL expression persisted at a high level also in post-neurogenic periods. The temporal pattern of SCL and GATA-2/3 expression was investigated also in vitro, in the course of induced neurogenesis by NE-4C neural stem cells. While GATA-2 expression increased from the very beginning of differentiation, SCL expression appeared only in more differentiated cells expressing proneural genes. GATA-3 expression, on the other hand, was detected only in advanced stages of the neuronal maturation, which were characterised by the activation of the Math2 neuronal gene. Similarly to the hematopoietic differentiation, GATA-2 expression precedes the activation of both SCL and GATA-3, and may play roles in the activation of the SCL gene in neuronal development. In contrast to hematopoietic differentiation, however, our results failed to demonstrate co-assembling of GATA factors or SCL with Lmo2. While overlapping expression of GATA-2/3 and SCL was detected, Lmo2 activation could not be demonstrated in neural cells in the investigated period of neuronal development.

BACKGROUND

T-cell acute lymphoblastic leukemia (T-ALL) is caused by a defect in T-cell maturation to the mature T cell. T-ALL is a poor prognostic hematopoietic malignancy. In order to establish a successful therapeutic treatment plan, it is essential to understand the biology of T-cell development and molecules that contribute to this process. This study uses Jurkat T cells, as a well-established model for in vitro study of T-ALL to investigate the role of the microRNA (miRNA), miR-146a, on gene expressions involved in T-cell differentiation.

METHODS

The permanent over-expression of miR-146a was established using a lentivector that expressed GFP hsa-mir-146a miRNA. We used quantitative real-time polymerase chain reaction and flow cytometry for T-cell differentiation to monitor induction of the differentiation process by assessing changes in expression of some distinct transcription factors and cell surface markers.

RESULTS

Ectopic expression of miR-146a resulted in significant up-regulation of PU.1, c-Fos, CCAAT/enhancer-binding protein alpha (C/EBPα) and GATA3, and slight up-regulation of Foxp3 and Runx1. There was a significant, moderate down-regulation in the expressions of Notch1, LIM-domain only (Lmo2), son of sevenless 1 (SOS1), Ikaros, and signal transducer and activator of transcription 3 (STAT3).

CONCLUSIONS

Our results indicated that ectopic expression of miR-146a could not independently induce differentiation in lymphoblastic cells. However, the expression of multiple genes involved in T-cell differentiation and T-cell CD markers were found to be affected. These results have suggested a potential tumor suppressive, immunomodulatory and cell activator role for miR146-a.

Somatic mutations within noncoding genomic regions that aberrantly activate oncogenes have remained poorly characterized. Here we describe recurrent activating intronic mutations of LMO2, a prominent oncogene in T-cell acute lymphoblastic leukemia (T-ALL). Heterozygous mutations were identified in PF-382 and DU.528 T-ALL cell lines in addition to 3.7% of pediatric (6 of 160) and 5.5% of adult (9 of 163) T-ALL patient samples. The majority of indels harbor putative de novo MYB, ETS1, or RUNX1 consensus binding sites. Analysis of 5'-capped RNA transcripts in mutant cell lines identified the usage of an intermediate promoter site, with consequential monoallelic LMO2 overexpression. CRISPR/Cas9-mediated disruption of the mutant allele in PF-382 cells markedly downregulated LMO2 expression, establishing clear causality between the mutation and oncogene dysregulation. Furthermore, the spectrum of CRISPR/Cas9-derived mutations provides important insights into the interconnected contributions of functional transcription factor binding. Finally, these mutations occur in the same intron as retroviral integration sites in gene therapy-induced T-ALL, suggesting that such events occur at preferential sites in the noncoding genome.

Follicular lymphoma (FL) can exhibit variant histologic patterns that can lead to confusion with other B-cell lymphomas and reactive conditions. Diagnostic markers such as CD10 and BCL2 may be difficult to interpret in variant FL patterns, and are often diminished or absent in the interfollicular and diffuse components. We evaluated 2 recently characterized germinal center B-cell markers, human germinal center associated lymphoma (HGAL), and LIM-only transcription factor 2 (LMO2), in 127 FL patient biopsies (94 nodal, 33 extranodal), and correlated the findings with histologic pattern, cellular composition, grade, and additional immunostains (CD20, CD3, CD21, CD10, BCL2, and BCL6). Architectural patterns included predominantly follicular (75%) and follicular and diffuse components (25%); 10 cases showed marginal zone differentiation and 3 were floral variants. Eighty-nine cases were low grade (38 grade 1; 51 grade 2) and 38 were grade 3 (29 grade 3A and 9 grade 3B). HGAL had the highest overall sensitivity of detecting FL and was superior in detecting the interfollicular and diffuse components compared with BCL2, LMO2, CD10, and BCL6. All 28 cases that lacked CD10, expressed HGAL, and the majority also expressed LMO2. Our results show that HGAL and LMO2 are sensitive markers for FL diagnosis. The addition of HGAL and LMO2 to the immunohistologic panel is beneficial in the work-up of nodal and extranodal B-cell lymphomas and the efficacy of HGAL in detecting the follicular, interfollicular and diffuse components of FL is of particular value in the setting of variant immunoarchitectural patterns.

The first wave of erythropoiesis in amniotic animals generates all primitive erythrocytes and takes place exclusively in yolk sac mesoderm. It is less clear, however, to what extent and for how long the yolk sac contributes to the second wave of erythropoiesis which gives rise to definitive erythrocytes for later embryonic and adult use. Here, we examine the initiation, duration, and site of definitive erythrocyte formation in chicken yolk sac. We show that the earliest definitive erythrocytes are generated in yolk sac venous vessels surrounding major arteries at embryonic day (E) 4-4.5, and that mature definitive erythrocytes enter circulating at E4.5-E5. This takes place at a time when yolk sac vasculature remodels extensively to generate paired arterial/venous vessels. The yolk sac remains the predominant site for definitive erythropoiesis from E5 to E10, and continues to generate definitive erythrocytes at least until E15. Similar to primitive erythropoiesis, definitive erythropoiesis in the yolk sac is accompanied by the expression of transcriptional regulators gata1, scl, and lmo2. Furthermore, our data suggest that one main source of definitive erythropoietic cells is the pre-existing vascular endothelial cells. It remains unclear whether yolk sac derived hematopoietic progenitors that do not undergo erythropoiesis in the yolk sac may take up intraembryonic niches and contribute to erythropoietic stem cell population after hatching.

The ectopic expression of LMO1 or LMO2 in T cell acute leukaemias resulting from chromosomal translocations t(11;14)(p15;qll) or t(11;14)(p13;q11) respectively in a causal factor in tumorigenesis. LMO1 has been found as a heterodimer with a 46 Kd protein in a T cell line derived from a childhood T-acute leukaemia. This 46 Kd protein is the LIM-binding protein LDB1/NLI. The latter is a phosphoprotein and binds to LMO1 in its phosphorylated state and essentially all the LMO1 and LDB1 protein in the T cell line is part of the complex. Therefore, the LMO1-LDB1 interaction is likely to be involved in tumorigenesis after LMO1 is ectopically expressed following chromosomal translocation in T cells prior to development of acute leukaemias.

Transcription factors are key regulators of both normal and malignant hematopoiesis. Chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-Seq) has become the method of choice to interrogate the genome-wide effect of transcription factors. We have collected and integrated 142 publicly available ChIP-Seq datasets for both normal and leukemic murine blood cell types. In addition, we introduce the new bioinformatic tool Gene Set Control Analysis (GSCA). GSCA predicts likely upstream regulators for lists of genes based on statistical significance of binding event enrichment within the gene loci of a user-supplied gene set. We show that GSCA analysis of lineage-restricted gene sets reveals expected and previously unrecognized candidate upstream regulators. Moreover, application of GSCA to leukemic gene sets allowed us to predict the reactivation of blood stem cell control mechanisms as a likely contributor to LMO2 driven leukemia. It also allowed us to clarify the recent debate on the role of Myc in leukemia stem cell transcriptional programs. As a result, GSCA provides a valuable new addition to analyzing gene sets of interest, complementary to Gene Ontology and Gene Set Enrichment analyses. To facilitate access to the wider research community, we have implemented GSCA as a freely accessible web tool (http://bioinformatics.cscr.cam.ac.uk/GSCA/GSCA.html).

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.

Deletion of caudal/cdx genes alters hox gene expression and causes defects in posterior tissues and hematopoiesis. Yet, the defects in hox gene expression only partially explain these phenotypes. To gain deeper insight into Cdx4 function, we performed chromatin immunoprecipitation sequencing (ChIP-seq) combined with gene-expression profiling in zebrafish, and identified the transcription factor spalt-like 4 (sall4) as a Cdx4 target. ChIP-seq revealed that Sall4 bound to its own gene locus and the cdx4 locus. Expression profiling showed that Cdx4 and Sall4 coregulate genes that initiate hematopoiesis, such as hox, scl, and lmo2. Combined cdx4/sall4 gene knockdown impaired erythropoiesis, and overexpression of the Cdx4 and Sall4 target genes scl and lmo2 together rescued the erythroid program. These findings suggest that auto- and cross-regulation of Cdx4 and Sall4 establish a stable molecular circuit in the mesoderm that facilitates the activation of the blood-specific program as development proceeds.

Diffuse large B-cell lymphoma (DLBCL) includes two prognostically important subtypes, the germinal center B-cell (GCB) and the non-GCB types. The aim of this study was to evaluate immunohistochemical approaches for predicting the survival of patients with DLBCL following autologous hematopoietic stem cell transplantation (AHSCT). We identified 62 patients with DLBCL who either had an initial complete remission (17 patients) or received salvage chemotherapy for relapsed or refractory disease (45 patients), followed by AHSCT. Tissue microarrays were immunostained with monoclonal antibodies against GCET1, CD10, BCL6, MUM1, FOXP1 and LMO2. Using the Hans algorithm, we classified 50% of the cases as GCB type, whereas the Choi algorithm classified 58% as GCB type and LMO2 was positive in 69%. However, no significant differences were found in the 5-year overall or event-free survivals using any of these approaches. In conclusion, cell of origin fails to predict survival of DLBCL patients treated with AHSCT.

Two general features have emerged about genes that are activated after chromosomal translocations in acute forms of cancer. The protein products of these genes are transcription regulators and are involved in developmental processes, and it seems that the subversion of these normal functions accounts for their role in tumorigenesis. The features of the LMO family of genes, which encode LIM-domain proteins involved in T-cell acute leukemia through chromosomal translocations, typify these abnormal functions in tumorigenesis. For example, the LMO2 protein is involved in the formation of multimeric DNA-binding complexes, which may vary in composition at different stages of hematopoiesis and function to control differentiation of specific lineages. In T cells, enforced expression of Lmo2 causes aberrant protein complex formation that primarily seems to hinder the T-cell differentiation program. These observations underscore the conclusion that protein-protein interaction (in this case, through the LIM domain) is a key determinant in tumorigenesis. Furthermore, the study of chromosomal translocations as naturally occurring mutations has been informative about mechanisms in hematopoiesis as well as in tumor etiology.

Several cases of T-cell leukemia caused by gammaretroviral insertional mutagenesis in children treated for x-linked severe combined immunodeficiency (SCID) by transplantation of autologous gene-modified stem cells were reported. In a comparative analysis, we recently showed that mature T cells, on the contrary, are highly resistant to transformation by gammaretroviral gene transfer. In the present study, we observed immortalization of a single T-cell clone in vitro after gammaretroviral transduction of the T-cell protooncogene LMO2. This clone was CD4/CD8 double-negative, but expressed a single rearranged T-cell receptor. The clone was able to overgrow nonmanipulated competitor T-cell populations in vitro, but no tumor formation was observed after transplantation into Rag-1 deficient recipients. The retroviral integration site (RIS) was found to be near the IL2RA and IL15RA genes. As a consequence, both receptors were constitutively upregulated on the RNA and protein level and the immortalized cell clone was highly IL-2 dependent. Ectopic expression of both, the IL2RA chain and LMO2, induced long-term growth in cultured primary T cells. This study demonstrates that insertional mutagenesis can contribute to immortalization of mature T cells, although this is a rare event. Furthermore, the results show that signaling of the IL-2 receptor and the protooncogene LMO2 can act synergistically in maligniant transformation of mature T lymphocytes.

Transcriptional corepressor ETO2 is a component of a protein complex containing master regulators of hematopoiesis, including GATA-1, SCL/TAL1, LMO2, and LDB1. To elucidate the role of ETO2 during erythroid differentiation, including the effects of ETO2 on GATA-1 targets, we performed gene expression profiling using K562 cells overexpressed with ETO2. The analysis demonstrated that 667 and 598 genes were upregulated and downregulated (more than twofold), respectively, in ETO2-overexpressing cells. ETO2-repressed genes included those encoding prototypical erythroid proteins. To test what percentages of ETO2-repressed genes could be direct target genes of GATA-1 in K562 cells, we merged the microarray results with ChIP-seq profile (n = 5,749), demonstrating that 23.1% of ETO2-repressed genes contained significant GATA-1 in their loci. However, there was no significant enrichment of PU.1 target genes among ETO2-repressed genes. Gene ontology analysis among ETO2-repressed genes revealed significant enrichment of genes related to "oxygen transporter," corresponding to globin genes. Quantitative chromatin immunoprecipitation and ETO2 knockdown analyses confirmed that ETO2 directly regulates globin genes in K562 cells. Next, we evaluated the role of ETO2 in human primary erythroblasts, derived from cord blood CD34-positive cells. In an ex vivo model of erythroid differentiation from CD34-positive cells, ETO2 protein level peaked at day 2-4 and almost diminished at the later stage of differentiation. Furthermore, short hairpin RNA-mediated knockdown and retroviral vector-mediated overexpression of ETO2 in primary erythroblasts suggested that ETO2 significantly represses HBB, HBA, and ALAS2 expression. In summary, ETO2 regulates GATA-1 target genes critical for erythroid differentiation, and the decrease of ETO2 levels during erythroid differentiation would contribute to the activation of these targets.