LMO2 Research Articles

Nuclear localization of lymphocyte-specific protein tyrosine kinase (Lck) and its role in regulating LIM domain only 2 (Lmo2) gene

LIM domain only protein 2 (Lmo2) is a transcription factor that plays a critical role in the development of T-acute lymphoblastic leukemia (T-ALL). A previous report established a link between Lmo2 expression and the nuclear presence of oncogenic Janus kinase 2 (JAK2), a non-receptor protein tyrosine kinase. The oncogenic JAK2 kinase phosphorylates histone H3 on Tyr 41 that leads to the relief of Lmo2 promoter repression and subsequent gene expression. Similar to JAK2, constitutive activation of lymphocyte-specific protein tyrosine kinase (Lck) has been implicated in lymphoid malignancies. However, it is not known whether oncogenic Lck regulates Lmo2 expression through a similar mechanism. We show here that Lmo2 expression is significantly elevated in T cell leukemia LSTRA overexpressing active Lck kinase and in HEK 293 cells expressing oncogenic Y505FLck kinase. Nuclear localization of active Lck kinase was confirmed in both Lck-transformed cells by subcellular fractionation and immunofluorescence microscopy. More importantly, in contrast to oncogenic JAK2, oncogenic Lck kinase does not result in significant increase in histone H3 phosphorylation on Tyr 41. Instead, chromatin immunoprecipitation experiment shows that oncogenic Y505FLck kinase binds to the Lmo2 promoter in vivo. This result raises the possibility that oncogenic Lck may activate Lmo2 promoter through direct interaction.

High expression of LMO2 in Hodgkin, Burkitt and germinal center diffuse large B cell lymphomas

Background and aimThe LMO2 gene encodes a transcription factor that regulates key events in erythropoiesis, angiogenesis, and embryogenesis and is highly expressed at the most immature stages of lymphopoiesis. Its implication in Hodgkin lymphoma (HL), Burkitt lymphoma (BL) and diffuse large B cell lymphoma (DLBCL) is limited in the literature. Material and methodsAn immunohistochemical study was performed on 68 lymphoma specimens showing different types including Hodgkin lymphoma (23), Burkitt lymphoma (10) and diffuse large B cell lymphoma (35). Also, seven specimens of the reactive nodal tissue were included as control. A monoclonal anti-human antibody has been used to detect LMO2. ResultsLMO2 was detected in all cases of HL (100%), in nine cases of BL (90%) and in all cases of DLBCL of germinal center (GC) subtype 20/35 (57.1%) but is completely negative in non-germinal center (NGC) DLBCL. In normal control of reactive nodes, LMO2 was expressed in germinal center area but not expressed in other areas including mantle, marginal, or T cell zones. In DLBCL; there was no statistically significant relation between LMO2 positive cases and the studied clinicopathological parameters including patient’s age, sex and tumor site, stage and histological subtype. On the other hand, it was statistically significant regarding immunophenotyping of GC versus NGC. ConclusionsLMO2 expression is a special feature of GC DLBCL which can be used as a diagnostic marker and therapeutic target. Further studies regarding its prognostic role in patients are recommended.

Abstract 16786: Er71 Transcriptionally Activates Lmo2 and Regulates Endothelial Development

During early embryonic development, mesodermal progenitor cells give rise to multiple lineages including the cardiovascular, endothelial and hematopoietic lineages. Studies suggest that the extraembryonic yolk sac is a progenitor source for endothelial and hematopoietic lineages during embryogenesis. We have previously shown that Ets related protein 71(ER71) regulates the development of endothelial and hematopoietic lineages in the early yolk sac and embryo proper utilizing an ER71 gene knock out mouse model, an ER71 promoter driven EYFP-expressing transgenic mouse model and an doxycycline-inducible ER71-overexpressing ES/EB model. To decipher the ER71 mediated mechanism for lineage specification, we evaluated candidate downstream targets of ER71 using qPCR, transcriptome analysis, ChIP and transcriptional assays in the ER71 global gene knockout mouse and doxycycline inducible ER71 overexpressing ES/EB models. Using qPCR, we observed an absence of hematopoietic and endothelial restricted genes in the ER71 mutant yolk sacs and embryo proper compared to their wildtype littermates. We further observed a significant downregulation of Lmo2 in the mutant embryo compared to the wildtype control. Moreover, forced overexpression of ER71 using an inducible ES/EB system revealed that Lmo2 was significantly upregulated within 12 hours following ER71 induction. Analysis of approximately 100kb of the Lmo2 gene revealed multiple potential Ets binding motifs and evaluation of each of these sites using ChIP assays focused our efforts on the 1 kb region in the first intron of the Lmo2 gene. Using an array of biochemical techniques including EMSA, transcriptional assays and mutagenesis our data collectively support the hypothesis that Lmo2 is a direct downstream target of ER71. In summary, the present studies define an ER71-Lmo2 transcriptional network that governs endothelial specification and differentiation during cardiovascular development.

AlkIs a Transcriptional Target of LMO4 and ERα That Promotes Cocaine Sensitization and Reward

Previously, we showed that the mouse LIM-domain only 4 (Lmo4) gene, which encodes a protein containing two zinc-finger LIM domains that interact with various DNA-binding transcription factors, attenuates behavioral sensitivity to repeated cocaine administration. Here we show that transcription of anaplastic lymphoma kinase (Alk) is repressed by LMO4 in the striatum and that Alk promotes the development of cocaine sensitization and conditioned place preference, a measure of cocaine reward. Since LMO4 is known to interact with estrogen receptor α (ERα) at the promoters of target genes, we investigated whether Alk expression might be controlled by a similar mechanism. We found that LMO4 and ERα are associated with the Alk promoter by chromatin immunoprecipitation and that Alk is an estrogen-responsive gene in the striatum. Moreover, we show that ERα knock-out mice exhibit enhanced cocaine sensitization and conditioned place preference and an increase in Alk expression in the nucleus accumbens. These data define a novel regulatory network involved in behavioral responses to cocaine. Interestingly, sex differences in several behavioral responses to cocaine in humans and rodents have been described, and estrogen is thought to mediate some of these differences. Our data suggest that estrogen regulation of Alk may be one mechanism responsible for sexually dimorphic responses to cocaine.

STAT3, Constitutively Activated In ABC-Like DLBCL, Regulates Expression of the Prognostic Factor Cyclin D2

AbstractAbstract 705As the most common subtype of non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL) remains clinically heterogeneous. Genome-scale expression profiles have defined two distinct molecular subtypes of DLBCL: Germinal Center B-Cell like (GCB-like) and Activated B-Cell like (ABC-like), with separate postulated tumor cells of origin and different pathogenesis, e.g. activation of NF-kB and Signal Transducer and Activator of Transcription 3 (STAT3) in the ABC-like DLBCLs. These subtypes are also characterized by distinct clinical outcomes. Since global gene expression is not practical for routine prognosis determination, we established an IPI independent 6-gene model based on the expression of BCL6, LMO2, FN1, BCL2, SCYA3 and cyclin D2 (CCND2) genes, which was capable of predicting patients' survival. While some of these genes may merely reflect different postulated tumor cells of origin, others may represent distinct pathogenetic mechanisms. Herein we demonstrate that CCND2, whose expression correlates with shorter survival, is positively regulated by the STAT3 signaling pathway, activated in the ABC-like DLBCL. Immunohistochemical staining of phosphorylated STAT3 (pSTAT3) demonstrated strong positive pSTAT3 staining (greater than 30% of nuclei) in 85 of 204 (41.7%) primary DLBCLs. Hierarchical clustering analysis of 143 DLBCL specimens stained for GCB and ABC markers demonstrated that expression of the pSTAT3 protein correlated with the expression patterns of non-GCB markers MUM1/IRF4 and BCL2 but not with GCB-specific markers HGAL, BCL6, and CD10. However, pSTAT3 expression was not different between the GCB-like and non-GCB-like DLBCLs defined by Han's model (Hans et al, Blood 2004). Examination of pSTAT3 levels in GCB-like DLBCL cell lines (OCI-LY7, OCI-LY19, and Sud-HL6) and the ABC-like cell lines (OCI-LY3 and OCI-LY10) by Western blotting demonstrated its expression in OCI-LY3 and OCI-LY10 but not in other cell lines. pSTAT3 expression in these cells was confirmed by phospho-flow cytometry. Interestingly, pSTAT3 expressing cell lines also expressed high levels of Cyclin D2, while it was not detected by Western blotting in the DLBCL cell lines not expressing pSTAT3. Analysis of the CCND2 promoter showed a single potential STAT3 binding site. Chromatin immunoprecipitation (ChIP) assay confirmed specific binding of STAT3 to this site in the CCND2 promoter. BCL6 is reported to negatively regulate expression of CCND2 and to competitively bind to STAT3 binding sites. Consequently, using quantitative ChIP assay we measured BCL6 and STAT3 binding to the CCND2 promoter in Sud-HL6, Oci-LY7 and Oci-LY19 GCB-like and in Oci-LY3 and Oci-LY10 ABC-like DLBCL cell lines. Levels of the bound STAT3 and BCL6 proteins were significantly higher in the Oci-LY3 and Oci-LY10 cell lines compared to their GCB counterparts. Treatment with BCL6 peptide inhibitor (BPI) slightly increased CCND2 mRNA expression in both GCB-like and ABC-like DLBCL, confirming the inhibitory effect of BCL6 on the CCND2 expression. To examine the effect of pSTAT3 on Cyclin D2 in the physiological context, we have knocked-down expression of STAT3 and pSTAT3 by specific siRNA in the OCI-LY3 cell line. A 37% decrease in the total levels of STAT3 led to a 52% decrease in pSTAT3 and a 55% reduction in the Cyclin D2 protein levels and was associated with a statistically significant decrease in cell proliferation as measured by the MTT assay. Overall our results demonstrate that a constitutively active STAT3 signaling pathway contributes to the Cyclin D2 expression and proliferation of the ABC-like DLBCLs. These findings elucidate the mechanism regulating the expression of prognostic factor Cyclin D2 and link it to the STAT3 signaling pathway constitutively activated in ABC-like DLBCL. Disclosures:Patricelli:ActivX Biosciences: Employment. Nomanbhoy:ActivX Biosciences: Employment.

Loss of p19Arf Increases the Self-Renewal Capacity of Immature Thymocytes That Over-Express LMO2

AbstractAbstract 4198LMO2 is a lim-only domain transcription factor that mediates protein-protein interactions in a multimeric complex containing LDB1, E47, Tal1 and either GATA1 or GATA2. It was discovered as a result of chromosomal translocations in T-cell leukemia; where it becomes abnormally regulated by T-cell receptor elements and is involved in approximately 9% of cases. LMO2 transgenic mice develop T-cell leukemia with a latency of between 10–12 months indicating that other co-operating genetic events are required. In recent X-SCID gene therapy trials 4 patients developed T-cell leukemia due to vector–induced activation of LMO2 gene expression. Two out of the 4 patients had loss of the CDKN2A locus with another having an activating insertion in BMI1, a known repressor of the CDKN2A locus. The CDKN2A locus encodes both p19Arf and p16INK4A, and p19Arf suppression is known to increase self-renewal in a variety of different stem cell systems. Because LMO2 has been shown to promote thymocyte self-renewal (Curtis et al, Science 2010); we investigated whether Arf loss could collaborate with LMO2 in regulating self-renewal of thymocyte progenitors. We utilized OP9-DL1 stromal cells that express the Notch ligand; Delta-ligand 1 which enables differentiation of immature bone marrow cells and thymocytes into mature T-cells. When cultured on OP9-DL1 stromal cells LMO2+Arf+/+ and LMO2+Arf-/- populations had a block in progression to mature T-cells at the DN2 stage of thymocyte differentiation. These DN2 thymocytes were sorted and replated onto fresh OP9-DL1 stromal cells and analysis 7 days later showed that both populations had 99% DN2 stage thymocytes, indicating that LMO2 was inducing a complete block in thymocyte differentiation. LMO2+ Arf-/- DN2 thymocytes had 3 fold more thymocytes than the Arf+/+ population 11 days after the thymocytes had been sorted. This indicates that Arf loss may be increasing the self-renewal of the DN2 population. We next transplanted either 2×105 LMO2+ Arf-/- or 2×105 LMO2 Arf+/+ DN2 thymocytes together with 2×105 WT bone marrow cells into 2 groups of 10 WT lethally irradiated syngenic recipients. After only three weeks LMO2+ Arf-/-(n=7) cells were detected in the thymi of recipient mice. Marked cells came to represent about 18% of total thymocytes between weeks 6 to 15 post-transplant, these LMO2+ Arf-/- thymocytes were able to differentiate into mature T cell lineages in the thymus. By week 18 the LMO2+ Arf-/- cells dramatically increased to 97% of total thymocytes indicating that they maybe a preleukemic pool. In contrast, no thymic repopulation with LMO2 Arf+/+ (N=7) cells was noted. Serial transplant experiments were performed by isolating mCherry+, LMO2+ thymocytes 12 weeks post transplant. Then 2×105 LMO2+ thymocytes were transplanted into secondary Rag2-/-γc-/- recipients. After 6 weeks the thymi of 2 secondary mice were analyzed and were 100% mCherry+, LMO2+ and contained mature T-cells. These LMO2+ Arf-/- thymocytes had the capacity to be serially transplanted demonstrating that Arf loss was contributing to the self-renewal potential of these LMO2+DN2 thymocytes. Comparing our data to the published result for LMO2+Arf+/+, a 100 fold less thymocytes were transplanted in our assay potentially explaining the lack of engraftment with our LMO2+Arf+/+ cells. Furthermore we observed activation of the Arf locus when these thymocytes were transplanted in vivo, indicating that somehow these LMO2+thymocytes were inducing Arf activation at later times after transplant. p19Arf expression in thymocytes has been reported to induce p53 dependent apoptosis (Miyazaki et al, Immunity 2008). This supports the concept that Arf loss contributes to the self-renewal potential of thymocytes in and above the context of LMO2 over-expression. The combination of an increase in self-renewal induced by LMO2 and loss of the gatekeeper function of self-renewal through p19Arf loss leads to acceleration in the pathogenesis of LMO2 induced T-cell malignancies. Disclosures:No relevant conflicts of interest to declare.

Identification of DeltaEF1 as a novel target that is negatively regulated by LMO2 in T‐cell leukemia

The lmo2 gene is a specific oncogene in T-cell leukemia, for its ectopic expression causes both increased pro-T-cell proliferation and differentiation arrest, leading to the onset of leukemia. Notably, DeltaEF1 (also known as ZEB1), a member of zinc finger-homeodomain family transcription factor, also exhibits crucial function in promoting T-cell differentiation. In this study, we found that DeltaEF1 was positively regulated by T-lineage-specific transcriptional regulator GATA3, while ectopically expressed LMO2 targeted to DeltaEF1 promoter by interaction with GATA3 and inhibited DeltaEF1 expression in transcriptional level. Moreover, LMO2 interacted with the N-terminal zinc finger domain of DeltaEF1 protein and inhibited its positive transcriptional regulatory function by this interaction. Taken together, our findings revealed that ectopically expressed LMO2 impaired the function of DeltaEF1 in both transcriptional and protein levels and identified DeltaEF1 as a novel pathogenic target of LMO2 in T-cell leukemia.

A DNA-binding mutant of TAL1 cooperates with LMO2 to cause T cell leukemia in mice

The most common translocation in childhood T-cell acute lymphoblastic leukemia (T-ALL) involves the LMO2 locus, resulting in ectopic expression of the LMO2 gene in human thymocytes. The LMO2 gene was also activated in patients with X-linked Severe Combined Immune Deficiency treated with gene therapy because of retroviral insertion in the LMO2 locus. The LMO2 insertions predisposed these children to T-ALL, yet how LMO2 contributes to T cell transformation remains unclear. The LIM (Lin 11, Isl-1, Mec-3) domain containing LMO2 protein regulates erythropoiesis as part of a large transcriptional complex consisting of LMO2, TAL1, E47, GATA1 and LDB1 that recognizes bipartite E-box-GATA1 sites on target genes. Similarly, a TAL1/E47/LMO2/LDB1 complex is observed in human T-ALL and Tal1 and Lmo2 expression in mice results in disease acceleration. To address the mechanism(s) of Tal1/Lmo2 synergy in leukemia, we generated Lmo2 transgenic mice and mated them with mice that express wild-type Tal1 or a DNA-binding mutant of TAL1. Tal1/Lmo2 and MutTAL1/Lmo2 bitransgenic mice exhibit perturbations in thymocyte development due to reduced E47/HEB transcriptional activity and develop leukemia with identical kinetics. These data demonstrate that the DNA-binding activity of Tal1 is not required to cooperate with Lmo2 to cause leukemia in mice and suggest that Lmo2 may cooperate with Tal1 to interfere with E47/HEB function(s).

Aberrant induction of LMO2 by the E2A-HLF chimeric transcription factor and its implication in leukemogenesis of B-precursor ALL with t(17;19)

AbstractLMO2, 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.

Gene Coexpression Analysis in Single Cells Indicates Lymphomyeloid Copriming in Short-Term Hematopoietic Stem Cells and Multipotent Progenitors

Progressive restriction to a differentiation pathway results from both activation and silencing of particular gene expression programs. To identify the coexpression and the expression levels of regulatory genes during hematopoietic stem cell (HSC) differentiation toward the T cell branch, we applied a new single-cell RT-PCR technique to analyze the simultaneous expression of 13 genes in 9 functionally purified populations from the bone marrow and the thymus. We report in this paper that Lin(-)Sca1(+)ckit(+) HSCs display, at the single-cell level, a homogeneous and high transcriptional activity as do early thymic progenitors. Moreover, the coexpression of lymphoid and myeloid genes is an early event detected in approximately 30% of short-term HSC and most multipotent progenitors, suggesting novel sources for the generation of early thymic progenitors, common lymphoid progenitors (CLPs), and common myeloid progenitors. Loss of multipotency in Lin(-)Sca1(+)ckit(+) cells directed to the lymphoid branch is characterized by Lmo2 and Gata2 gene expression downregulation. Indeed, highest levels of Gata2 expression are detected only in long-term and short-term HSC populations. Complete shutdown of Pu1 gene expression in all triple-negative (TN)3 stage thymic pre-T cells is indicative of total T cell commitment. Interestingly, this is also observed in 30% of TN2 cells and 25% of CLP in the bone marrow, suggesting a possible initiation of T cell engagement in TN2 and CLP. Also, our strategy highlights similar gene patterns among HSCs and intrathymic progenitors, proposing, therefore, that identical activation signals are maintained until further maturation and generation of CD4 and CD8 coreceptors bearing thymocytes.

Homo-binding character of LMO2 isoforms and their both synergic and antagonistic functions in regulating hematopoietic-related target genes

BackgroundThe human lmo2 gene plays important roles in hematopoiesis and is associated with acute T lymphocyte leukemia. The gene encodes two protein isoforms, a longer form LMO2-L and a shorter form LMO2-S. Both isoforms function as bridge molecules to assemble their partners together to regulate their target genes. A typical LMO2 binding site consists of two elements, a GATA site and an E-box, with an interval of 9~12 bp.MethodsIn this study, the combination of MBP pulldown assay and mammalian two hybrid assay were used to confirm the homo-binding character of LMO2-L/-S isoforms. Luciferase reporter assay and Real-time PCR assay were used to detect expression levels and relative promoter activities of LMO2-L/-S isoforms. Co-transfection and Luciferase reporter assay were used to reveal the detailed regulatory pattern of LMO2-L/-S isoforms on their targets.ResultsHerein we report the homo-interaction character of LMO2-L and LMO2-S and their major difference in manner of regulating their target genes. Our results showed that LMO2-L and LMO2-S could only bind to themselves but not each other. It was also demonstrated that LMO2-L could either positively or negatively regulate the transcription of its different target genes, depending on the arrangement and strand location of the two elements GATA site and E-box, LMO2-S, however, performed constitutively transcriptional inhibiting function on all target genes.ConclusionThese results suggest that LMO2 isoforms have independent functions while there is no interaction between each other and they could play synergetic or antagonistic roles precisely in regulating their different genes involved in normal and aberrant hematopoiesis.

Expression of the leukemia oncogene Lmo2 is controlled by an array of tissue-specific elements dispersed over 100 kb and bound by Tal1/Lmo2, Ets, and Gata factors

AbstractThe Lmo2 gene encodes a transcriptional cofactor critical for the development of hematopoietic stem cells. Ectopic LMO2 expression causes leukemia in T-cell acute lymphoblastic leukemia (T-ALL) patients and severe combined immunodeficiency patients undergoing retroviral gene therapy. Tightly controlled Lmo2 expression is therefore essential, yet no comprehensive analysis of Lmo2 regulation has been published so far. By comparative genomics, we identified 17 highly conserved noncoding elements, 9 of which revealed specific acetylation marks in chromatin-immunoprecipitation and microarray (ChIP-chip) assays performed across 250 kb of the Lmo2 locus in 11 cell types covering different stages of hematopoietic differentiation. All candidate regulatory regions were tested in transgenic mice. An extended LMO2 proximal promoter fragment displayed strong endothelial activity, while the distal promoter showed weak forebrain activity. Eight of the 15 distal candidate elements functioned as enhancers, which together recapitulated the full expression pattern of Lmo2, directing expression to endothelium, hematopoietic cells, tail, and forebrain. Interestingly, distinct combinations of specific distal regulatory elements were required to extend endothelial activity of the LMO2 promoter to yolk sac or fetal liver hematopoietic cells. Finally, Sfpi1/Pu.1, Fli1, Gata2, Tal1/Scl, and Lmo2 were shown to bind to and transactivate Lmo2 hematopoietic enhancers, thus identifying key upstream regulators and positioning Lmo2 within hematopoietic regulatory networks.

Acute Myeloid Leukemia in Mice Associated with Retrovirally-Mediated Overexpression of HOXB4

Numerous studies have established that retrovirally-mediated overexpression of the homeobox gene HOXB4 in murine bone marrow cells (BMCs) enhances expansion of hematopoietic stem cells (HSCs) in vivo and in vitro without causing hematopoietic abnormalities or impairing the ability of HSCs to give rise to differentiated progeny. More recently, however, retrovirally-mediated overexpression of HOXB4 in BMCs of large animals, such as dogs and macaques, has been implicated in leukemogenesis, but there are no prior reports of such a phenomenon occurring in mice transplanted with HOXB4-transduced BMCs. We report here two independent occurrences of acute myeloid leukemia (AML) in mice that received transplants of BMCs transduced with a HOXB4-overexpressing retrovirus. 5-FU treated BMCs were transduced with a MSCV-HOXB4-IRES-GFP retrovirus and transplanted into lethally irradiated mice. Two of the 54 mice (#1869 & #2050), transplanted with HOXB4-transduced BMCs, developed AML at 4 and 9.5 months, respectively, following transplantation. None of the 90 control mice, transplanted with BMCs transduced with the parent MSCV-IRES-GFP retrovirus, developed leukemia. Leukemia 1869 (L1) manifested marked leukocytosis, thrombocytopenia and mild anemia. Results of morphological and immunophenotyping analyses of bone marrow were consistent with AML. Leukemia 2050 (L2) also showed marked leukocytosis and significant anemia, but a near normal platelet count. Morphological analysis revealed a myelomonocytic phenotype, while immunophenotying showed the presence of multi-lineage cell surface markers, including myeloid (CD11b, Gr-1), erythroid (Ter119), and B lymphoid (B220) markers. A small percentage of cells that expressed the stem cell markers c-kit and Sca1 were identified in both leukemias. Both leukemias could be passaged by serial transplantation of leukemic bone marrow or spleen cells into wild type recipient mice. Analysis of retroviral integration sites identified ten separate integration sites in L1 and two in L2. This correlated with 6 fold higher levels of HOXB4 mRNA in L1 BMCs compared to L2 BMCs, as assessed by northern blot analysis that showed undetectable levels of HOXB4 mRNA in RNA of control BMCs. Some of the integration sites occurred in introns of genes known to be involved in leukemogenesis and resulted in aberrant expression of these genes. In L1, one of the retroviral integration sites occured in intron 1 of the N-ras gene and was associated with markedly increased levels of its mRNA in RNA of leukemic BMCs (15–20 fold higher levels compared to that in control BMCs). Another integration site in L1 was in intron 2 of the Lmo2 gene and was associated with a mild (~2 fold) increase in its mRNA level in leukemic cells. In L2, one of the retroviral integration sites was in intron 1 of the Prdm16 gene and was associated with markedly increased levels of its mRNA in leukemic cells. The second retroviral integration site in L2 was located ~50 kb upstream of the Notch1 gene and there was ~2 fold increase in its mRNA in BMCs of leukemic mice. We speculate that overexpression of HOXB4 acts in a cooperative or combinatorial fashion with overexpression of proto-oncogenes at the retroviral integration sites to induce AML in these mice. To test this hypothesis, we are testing various culture conditions to allow viable growth of the leukemic cells ex vivo and assess the effects of knockdown of HOXB4 mRNA accumulation on growth and differentiation of the cells in vitro, as well as on their ability to transmit leukemia in vivo. We are also characterizing the nature of the cells in the total leukemic cell population that are associated with in vivo leukemia-initiating activity. This is the first report of AML in mice associated with retrovirally-mediated overexpression of HOXB4.

Robust Generation of Hemangioblastic Progenitors from Human Embryonic Stem Cells

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

An experimental system for the evaluation of retroviral vector design to diminish the risk for proto-oncogene activation

Pathogenic activation of the LMO2 proto-oncogene by an oncoretroviral vector insertion in a clinical trial for X-linked severe combined immunodeficiency (X-SCID) has prompted safety concerns. We used an adeno-associated virus vector to achieve targeted insertion of a gamma-retroviral long terminal repeat (LTR) driving a GFP expression cassette with flanking loxP sites in a human T-cell line at the precise location of vector integration in one of the patients with X-SCID. The LTR-GFP cassette was inserted into the first intron of the LMO2 gene, resulting in strong activation of LMO2. Cre-mediated cassette exchange was used to replace the original LTR-GFP cassette with one flanked by insulator elements leading to a several fold reduction in LMO2 expression. The LTR-GFP cassette was also replaced with a globin gene regulatory cassette that failed to activate the LMO2 gene in lymphoid cells. A gamma-retroviral vector with 2 intact LTRs resulted in activation of the LMO2 gene when inserted into the first intron, but a self-inactivating lentiviral vector with an internal cellular promoter and flanking insulator elements did not activate the LMO2 gene. Thus, this system is useful for comparing the safety profiles of vector cassettes with various regulatory elements for their potential for proto-oncogene activation.

Downregulation of microRNA-142 by proto-oncogene LMO2 and its co-factors

Downregulation of microRNA-142 by proto-oncogene LMO2 and its co-factors

The Lim-only protein LMO2 acts as a positive regulator of erythroid differentiation

LMO2, a member of the LIM-only protein family, is essential for the regulation of hematopoietic stem cells and formation of erythroid cells. It is found in a transcriptional complex comprising LMO2, TAL1, E47, GATA-1, and LDB1 which regulates erythroid genes. While TAL1 has been shown to induce erythroid differentiation, LMO2 appears to suppress fetal erythropoiesis. In addition to LMO2, the closely related LMO4 gene is expressed in hematopoietic cells, but has unknown functions. Here we demonstrate that LMO2 and LMO4 are expressed at the same level in erythroid colonies from mouse bone marrow, implying a function in erythroid differentiation. However, while LMO2 induced erythroid differentiation, LMO4 had no such effect. Interestingly, both LMO2 and TAL1 were able to partially suppress myeloid differentiation, implying that they activate erythroid differentiation in uncommitted bone marrow progenitors. Both LMO2 and LMO4 interacted strongly to LDB1, which was required for their localization to the nucleus.

Activation of the LMO2 gene in human lymphoid cells by targeted insertion of a single retroviral LTR expression cassette into the first intron

Activation of the LMO2 gene in human lymphoid cells by targeted insertion of a single retroviral LTR expression cassette into the first intron

The Effect of a Chromatin Insulator (5′cHS4) on Enhanced Expression of the LMO2 Proto-Oncogene by an Oncoretroviral Long Terminal Repeat.

Activation of the LMO2 proto-oncogene by the insertional mutagenic effect of an oncoretroviral vector contributed to leukemia in two patients otherwise successfully treated for Severe Combined Immunodeficiency (SCID) in a gene therapy trial (Science 302: 415–419, 2003). Various facets of vector redesign have been proposed to improve the safety profile of retroviral gene therapy. We recreated the insertional mutation that occurred in one of the SCID patients by introducing an expression cassette into the first intron of the LMO2 gene in a human T-cell leukemia line (Jurkat) using recombinant adeno-associated viral (rAAV) vector mediated gene targeting. The rAAV targeting vector was constructed to have 1.3 kb of LMO2 intron sequences from both upstream and downstream of the desired insertion site flanking an expression cassette consisting of GFP coding sequences under the control of a single LTR. The expression cassette was in a reverse orientation to the LMO2 gene and flanked by LoxP sites (floxed). Jurkat cells were transduced with the rAAV targeting vector, GFP expressing cells were selected by FACS and single cell clones were recovered. Four clones showed the predicted bands on PCR and Southern blot analysis in addition to wildtype bands indicating successful targeting into one of the LMO2 alleles. As measured by real-time quantitative RT-PCR (qRT-PCR), expression of LMO2 mRNA was at a very low level in untransduced Jurkat cells but was upregulated in all 4 clones ranging from an 1100 to 3000-fold increase over baseline. LMO2 protein was highly expressed in all 4 clones, as shown by Western blot analysis, but was not detected in control Jurkat cells. Cells containing the floxed LTR-GFP cassette in the LMO2 first intron were transduced with a rAAV Cre expression cassette. Twenty-four hours later, the cells were transfected with a plasmid containing a floxed LTR-DsRed expression cassette and GFP−, DsRed+ cells were identified. Four clones were documented to have undergone Cre-mediated cassette exchange by Southern blot and PCR analysis. The LMO2 gene remained highly expressed in these clones. To explore the effect of an insulator element on proto-oncogene activation, a 1.2 kb fragment containing the 5′ DNase Hypersensitive Site 4 (5′cHS4) from the chicken β-globin locus was placed on each side of the retroviral LTR-GFP expression cassette. LoxP sites were located outside of the 5′ and 3′ insulator fragments. DsRed expressing Jurkat cells were transduced with the rAAV-Cre vector and then transfected with the plasmid containing the LoxP-5′cHS4-GFP-LTR-5′cHS4-LoxP cassette. GFP positive clones were isolated and nine were shown to have undergone cassette exchange by PCR and Southern blot analysis. Six clones were studied by qRT-PCR and/or Western blot analysis and all expressed LMO2 gene at much higher levels than the control Jurkat cells although expression was decreased 2- to 10-fold compared to clones containing the LTR-GFP or LTR-DsRed expression cassette. Our results indicated that the 5′cHS4 insulator could reduce LMO2 expression, but was not sufficient to prevent gene activation by the retroviral LTR suggesting that other safety measures should be considered when designing vectors. rAAV-mediated gene targeting and Cre-mediated cassette exchange provide a useful strategy for evaluating the potential safety profile of various retroviral expression cassettes in planning future gene therapy trials.

Hematopoietic Stem and Progenitor Cell Biology in the Zebrafish.

Over the past decade, the development of forward genetic approaches in the zebrafish system has provided unprecedented power in understanding the molecular basis of vertebrate blood development. Establishment of cellular and hematological approaches to better understand the biology of resulting blood mutants, however, has lagged behind these efforts. We have recently developed the means to identify zebrafish hematopoietic stem cells (HSCs), transgenic lines to mark hematopoietic precursors and their progeny, and the assays to test these populations functionally. Like other vertebrates, zebrafish demonstrate differential waves of hematopoiesis during embryogenesis. These waves can be visualized directly by fluorescent transgenesis in living embryos. The earliest blood-forming cells in the zebrafish embryo express the scl and lmo2 genes. By directing expression of GFP to early blood precursors using the lmo2 promoter, we have isolated early hematopoietic cells by flow cytometry and tested them functionally by transplantation. Transplantation of lmo2::GFP+ cells isolated from embryos at 14 hours post-fertilization (hpf) resulted in only transient reconstitution of erythrocytes, suggesting that the earliest identifiable blood-forming cells are committed to the erythroid lineage. Later in embryogenesis, lmo2:GFP+ GATA-1:dsRED+ cells are found in the posterior blood island (PBI) from approximately 30–60 hpf. Molecular and functional characterization of these cells suggests that they possess limited myeloid and erythroid, but not lymphoid differentiation potentials. This suggests that committed progenitors with definitive hematopoietic potential arise in the embryo before HSCs can be identified. Additional studies have suggested that the first multipotent HSCs are born later in the zebrafish aorta/gonad/mesonephros (AGM) region. We have visualized putative HSCs in the AGM by their expression of the lmo2 and cd41 transgenes. Using confocal timelapse imaging in living embryos, lmo2::GFP+ cells have been observed to emigrate from the AGM region into circulation. Transplantation studies are underway to test putative HSC populations for repopulation activity. Taken together, our findings suggest that at least three independent waves of blood cell precursors are formed during zebrafish embryogenesis.