Common Site Of Retroviral Integration Research Articles

Cooperation between SS18-SSX1 and miR-214 in Synovial Sarcoma Development and Progression.

SS18-SSX fusion proteins play a central role in synovial sarcoma development, although, the genetic network and mechanisms of synovial sarcomagenesis remain unknown. We established a new ex vivo synovial sarcoma mouse model through retroviral-mediated gene transfer of SS18-SSX1 into mouse embryonic mesenchymal cells followed by subcutaneous transplantation into nude mice. This approach successfully induced subcutaneous tumors in 100% recipients, showing invasive proliferation of short spindle tumor cells with occasional biphasic appearance. Cytokeratin expression was observed in epithelial components in tumors and expression of TLE1 and BCL2 was also shown. Gene expression profiling indicated SWI/SNF pathway modulation by SS18-SSX1 introduction into mesenchymal cells and Tle1 and Atf2 upregulation in tumors. These findings indicate that the model exhibits phenotypes typical of human synovial sarcoma. Retroviral tagging of the tumor identified 15 common retroviral integration sites within the Dnm3 locus as the most frequent in 30 mouse synovial sarcomas. miR-199a2 and miR-214 upregulation within the Dnm3 locus was observed. SS18-SSX1 and miR-214 cointroduction accelerated sarcoma onset, indicating that miR-214 is a cooperative oncomiR in synovial sarcomagenesis. miR-214 functions in a cell non-autonomous manner, promoting cytokine gene expression (e.g., Cxcl15/IL8). Our results emphasize the role of miR-214 in tumor development and disease progression.

Ecotropic Viral Integration Site 5 (EVI5) variants are associated with multiple sclerosis in Iranian population

BackgroundMultiple sclerosis (MS) is a multifactorial disorder with immunological basis. Numerous genetic and environmental factors contribute in its pathogenesis. Several genetic loci have been shown to be associated with MS risk. Among genes whose participation in MS has been evaluated is Ecotropic Viral Integration Site 5 (EVI5). EVI5 is a common site of retroviral integration with a possible role in T-cell lymphomagenesis. MethodsIn the current study, we aimed to confirm association of the single nucleotide polymorphisms (SNPs) within EVI5 gene with MS in 410 relapsing-remitting MS patients and 410 controls from Iranian population. The rs6680578, rs10537781 and rs11810217 genotypes were defined by amplification-refractory mutation system (ARMS)-PCR method. Results: The allele and genotype frequencies of rs6680578 and rs11810217 were not significantly different between cases and controls. However, in the rs10735781 the GG genotype was significantly associated with MS risk in recessive (P = 0.03, OR (95%CI) = 1.84 (1.05–3.19)) and co-dominant models (P = 0.02, OR 95%CI) = 1.90 (1.08–3.35)). In addition, T G T haplotype (rs6680578, rs10735781 and rs11810217 respectively) was associated with MS risk while T C C, A G T and A C T had a protective effect against MS. ConclusionThe results of the current study provide further evidences for participation of EVI5 in MS pathogenesis.

Multiple Roles for the Zebrafish Homologue of the Murine Evi1 Gene during Primitive Myelopoiesis and HSC Development

The Evi1 locus was originally identified as a common site of retroviral integration in murine myeloid tumors. Several reports associate Evi1 expression with aggressiveness in myeloid leukemia. Since developmental pathways often reactivate in cancer, we hypothesized that Evi1 also plays critical roles during developmental hematopoiesis. Here, we employ the zebrafish model to study how evi1 modulates early blood development. We find that indeed zebrafish evi1 co-localizes with the hematopoietic markers scl, gata1, pu.1 and gata2 in the posterior lateral mesoderm and the rostral blood islands, indicating involvement in primitive hematopoiesis. Knockdown of evi1 via three independent Morpholino Oligonucleotides impairs embryonic myelopoiesis as shown by reduced pu.1, mpo and l-plastin staining, while not affecting hemangioblast formation and primitive erythropoiesis. Additionally, we observe reduced levels of cd41 expression upon evi1 knockdown, indicating that megakaryopoiesis is also impaired.Notably, at later time-points, evi1 is also expressed in the AGM region and evi1 morphants show strong reduction of runx1/c-myb expression in this region, demonstrating an additional role in hematopoietic stem cell (HSC) formation. Consistently, evi1 morphants lack ikaros+ lymphocyte precursor cells and rag1+ T-lymphocytes, and less circulating globin+, lyz+ and cd41+ cells are detected in transgenic fish analyzed by flow cytometry at 5 days post fertilization. To dissect the mechanisms by which evi1 regulates HSC formation, we furthermore analyzed genes specifically expressed in the dorsal aorta, where HSCs emerge from the hemogenic endothelium. We hypothesize that evi1 is indeed regulating HSC specification from hemogenic endothelial cells, since detailed analyses show defective aortic expression of efnb2a and dlc. How this effect is mediated is currently being investigated. Furthermore, TUNEL, anti-activated Caspase-3, anti-phospho Histone H3 and BrdU assays show both increased apoptosis and decreased proliferation in the AGM region of evi1 morphants as compared to control injected fish, suggesting important additional effects of evi1 on HSC biology beyond the specification step. Similar effects are seen in the rostral blood islands and are possibly responsible for the observed reduction of primitive myeloid progenitors.We also examined potential downstream targets of evi1. Previous reports in adult murine hematopoietic cells suggest that Evi1 affects HSC proliferation through regulation of Gata2. Indeed, gata2 co-injection enhances hematopoietic cell viability also in zebrafish embryos, and co-injection of gata2 mRNA is able to fully rescue both primitive myelopoiesis and HSC formation. We are currently investigating whether gata2 expression can compensate evi1 requirement during HSC specification or, alternatively, might rescue HSC numbers by amplifying residual HSCs that escape evi1 inhibition.Taken together, our data demonstrate that evi1 plays multiple roles during hematopoietic development, critically regulating the biology of primitive myeloid progenitors and pre-formed HSCs, as well as HSC specification from the hemogenic endothelium. Currently, we analyze the molecular mechanisms that mediate evi1 effects during these different regulatory steps and use the CRISPR/Cas9 system to generate evi1 mutant fish for further validation of our findings. DisclosuresNo relevant conflicts of interest to declare.

Cover Picture: Eur. J. Immunol. 9'14

Our cover features an electron micrograph of two mouse plasmacytoma cells expressing endogenous mouse leukemia virus (MLV, between the cells). MLV buds from the cell membrane into the culture supernatant, and its genome is reverse‐transcribed and reintegrated into the cellular genome, contributing to tumorigenesis by activating proto‐oncogenes or destroying tumor suppressor genes. The image is taken from Knittel et al., in which the authors show that mineral oil‐induced plasmacytomas produce large amounts of endogenous retroelements, including MLV. The authors identified up to 10 insertions per plasmacytoma, mostly in genes with common retroviral integration sites. These findings suggest that endogenous retroelements might be partially responsible for the formation of plasmacytomas in mice.

Insertional hypermutation in mineral oil-induced plasmacytomas.

Unless stimulated by a chronic inflammatory agent, such as mineral oil, plasma cell tumors are rare in young BALB/c mice. This raises the questions: What do inflammatory tissues provide to promote mutagenesis? And what is the nature of mutagenesis? We determined that mineral oil-induced plasmacytomas produce large amounts of endogenous retroelements--ecotropic and polytropic murine leukemia virus and intracisternal A particles. Therefore, plasmacytoma formation might occur, in part, by de novo insertion of these retroelements, induced or helped by the inflammation. We recovered up to ten de novo insertions in a single plasmacytoma, mostly in genes with common retroviral integration sites. Additional integrations accompany tumor evolution from a solid tumor through several generations in cell culture. The high frequency of de novo integrations into cancer genes suggests that endogenous retroelements are coresponsible for plasmacytoma formation and progression in BALB/c mice.

The DN2 Myeloid-T (DN2mt) Progenitor is a Target Cell for Leukemic Transformation by the TLX1 Oncogene.

Inappropriate activation of the TLX1 (T-cell leukemia homeobox 1) gene by chromosomal translocation is a recurrent event in human T-cell Acute Lymphoblastic Leukemia (T-ALL). Ectopic expression of TLX1 in murine bone marrow progenitor cells using a conventional retroviral vector efficiently yields immortalized cell lines and induces T-ALL-like tumors in mice after long latency. To eliminate a potential contribution of retroviral insertional mutagenesis to TLX1 immortalizing and transforming function, we incorporated the TLX1 gene into an insulated self-inactivating retroviral vector. Retrovirally transduced TLX1-expressing murine bone marrow progenitor cells had a growth/survival advantage and readily gave rise to immortalized cell lines. Extensive characterization of 15 newly established cell lines failed to reveal a common retroviral integration site. This comprehensive analysis greatly extends our previous study involving a limited number of cell lines, providing additional support for the view that constitutive TLX1 expression is sufficient to initiate the series of events culminating in hematopoietic progenitor cell immortalization. When TLX1-immortalized cells were co-cultured on OP9-DL1 monolayers under conditions permissive for T-cell differentiation, a latent T-lineage potential was revealed. However, the cells were unable to transit the DN2 myeloid-T (DN2mt)-DN2 T-lineage determined (DN2t) commitment step. The differentiation block coincided with failure to upregulate the zinc finger transcription factor gene Bcl11b, the human ortholog of which was shown to be a direct transcriptional target of TLX1 downregulated in the TLX1+ T-ALL cell line ALL-SIL. Other studies have described the ability of TLX1 to promote bypass of mitotic checkpoint arrest, leading to aneuploidy. We likewise found that diploid TLX1-expressing DN2mt cells treated with the mitotic inhibitor paclitaxel bypassed the mitotic checkpoint and displayed chromosomal instability. This was associated with elevated expression of TLX1 transcriptional targets involved in DNA replication and mitosis, including Ccna2 (cyclin A2), Ccnb1 (cyclin B1), Ccnb2 (cyclin B2) and Top2a (topoisomerase IIα). Notably, enforced expression of BCL11B in ALL-SIL T-ALL cells conferred resistance to the topoisomerase IIα poison etoposide. Taken together with previous findings, the data reinforce a mechanism of TLX1 oncogenic activity linked to chromosomal instability resulting from dysregulated expression of target genes involved in mitotic processes. We speculate that repression of BCL11B expression may provide part of the explanation for the observation that aneuploid DNA content in TLX1+ leukemic T cells does not necessarily portend an unfavorable prognosis. This TLX1 hematopoietic progenitor cell immortalization/T-cell differentiation assay should help further our understanding of the mechanisms of TLX1-mediated evolution to malignancy and has the potential to be a useful predictor of disease response to novel therapeutic agents in TLX1+ T-ALL.

A Dual Role of Evi-1 During Developmental Hematopoiesis

AbstractAbstract 765The Evi-1 locus was originally identified as a common site of retroviral integration in murine myeloid tumors. Several reports associate Evi-1 expression with aggressiveness in myeloid leukemia. More recent studies suggest involvement of Evi-1 in developmental hematopoiesis. Here, we analyzed zebrafish embryo and human induced pluripotent stem (iPS) cells to understand how Evi-1 modulates early blood development. We found that the zebrafish homologue evi-1 co-localizes with the hematopoietic markers scl, gata1 and gata2 during embryogenesis, indicating a role in primitive hematopoiesis. To analyse the functional role of evi-1, we performed in vivo loss-of-function studies by injecting control and inhibitory morpholino oligonucleotides (MO) suppressing evi-1 pre-mRNA splicing in zebrafish zygotes. To control for off-target effects, three separate MO were designed and injected. N = 100 zebrafish embryos were analyzed per experiment in each group. In situ hybridization analyses performed in evi-1 morphants versus control fish revealed strongly impaired formation of myeloid embryonic cells as shown by reduced pu.1, mpo and l-plastin expression and suppressed numbers of DsRed positive lyz-expressing cells in Tg(lyz:DsRed) fish. Interestingly, scl expression was not affected, suggesting that evi-1 acts at later stages, regulating specification and/or survival/proliferation of pu.1 expressing progenitor cells. Consistent with this notion, no changes were observed in primitive erythroid progenitor cells as monitored by gata1 and hbae3 expression and flow cytometry analyses of GFP-positive globin-expressing cells in Tg(globin:eGFP) fish. Furthermore, runx1+/cmyb+ cells were strongly reduced, suggesting a separate role of evi-1 in definitive hematopoiesis. Supporting this hypothesis, evi-1 morphants displayed profound suppression of ikzf1+ lymphoid progenitors and rag1+ T-lymphocytes, which was not due to impaired thymus development, since foxn1 expression remained unaltered in evi-1 morphants. Previous reports in adult murine hematopoietic cells suggest that Evi-1 affects HSC proliferation through regulation of Gata2. Indeed, we could show that co-injection of gata2 mRNA was able to rescue the impaired myeloid as well as the HSC phenotype in evi-1 morphants. However, data generated by TUNEL assays indicate that evi-1 also confers apoptosis resistance to developing AGM-HSC. Whether this effect is also mediated by gata2 is currently being explored. We further investigated the effect of EVI-1 during differentiation of hematopoietic cells from human iPS cells. Confirming our results collected in zebrafish embryo, inhibition of EVI-1 strongly reduced the numbers of CD34+ and CD45+ blood progenitor cells and the generation of myeloid colonies in colony forming assays, while leaving SCL expression unaltered. Taken together, our data suggest that Evi-1 plays conserved roles in zebrafish and human hematopoietic development, regulating embryonic myelopoiesis and HSC formation while leaving primitive erythropoiesis unaltered. The precise molecular basis of evi-1 mediated effects on these separate phases of developmental hematopoiesis is subject of ongoing investigations. Disclosures:No relevant conflicts of interest to declare.

Evi-1 Regulates Myelopoiesis and Hematopoietic Stem Cell Development in Zebrafish and Human Pluripotent Stem Cells

Abstract 1281The Evi-1 locus was originally identified as a common site of retroviral integration in murine myeloid tumors. Over the last years, Evi-1 evolved as one of the most potent oncogenes associated with human and murine myeloid leukemia. More recent studies in knockout mice suggest also involvement of Evi-1 in the regulation of developmental hematopoiesis, but the role of Evi-1 in this context is poorly understood. Here, we analyzed zebrafish embryo and human pluripotent stem cells (PSC) to understand how Evi-1 modulates early hematopoietic development.We examined the hematopoietic development in zebrafish embryo by in situ hybridization (ISH) for hematopoietic markers. The zebrafish homologue evi-1 was shown to be expressed in co-localization with scl in the posterior blood islands, indicating a role during early blood development. We also performed loss-of-function studies were by injecting morpholino oligonucleotides (MO) in zebrafish zygotes to inhibit evi-1 pre-mRNA splicing. Inhibition of evi-1 was confirmed in MO-injected versus control embryos. N=100 zebrafish embryos were analyzed per experiment in each group. To control for off-target effects, two separate MO were designed and injected. MO mediated evi-1 knockdown severely reduced numbers of circulating blood cells and induced hemorrhages. ISH performed in evi-1 morphants versus control fish revealed strongly impaired formation of myeloid embryonic cells (measured by pu.1 expression), while no changes were observed in primitive erythroid progenitor cells (monitored by gata1 expression) or overall in blood and endothelial precursors in the posterior lateral plate mesoderm (as monitored by scl expression). Moreover, analyses at 36 hours and 5 days post fertilization showed strong reduction of runx1+/cmyb+ cells and rag1+ lymphoid cells, indicating a role of evi-1 in developing hematopoietic stem cells (HSC).Previous reports in adult murine hematopoietic cells suggest that Evi-1 affects hematopoietic stem cell proliferation through regulation of Gata2. To test whether Gata2 is a putative downstream regulator of Evi-1 in our system, we performed a rescue experiment and co-injected gata2 mRNA in evi-1 MO treated fish. Indeed, ectopic gata2 rescued the impaired myeloid phenotype, as shown by re-occurrence of mpo, l-plastin as well as pu.1 expressing cells.To assess whether these molecular interactions are conserved during human developmental hematopoiesis, we surveyed in vitro differentiating human pluripotent stem cells (PSC) genetically modified to suppress EVI-1. EVI-1 expression was detected during differentiation of human PSC in embryoid bodies, especially around day 9 when hematopoietic progenitors start to emerge in this system. Treatment with EVI-1 shRNA strongly reduced the generation of myeloid colonies from human PSC in vitro as well as the numbers of emerging CD34+ and CD45+ cells. Molecularly, EVI-1 suppression inhibited the expression of PU.1 and GATA2 during the course of development, while leaving SCL and GATA1 expression unaltered.Taken together, our data suggest that, in both fish and human, Evi-1 regulates embryonic myelopoiesis through interactions with Gata2 and independently of Gata1 and embryonic erythropoiesis. Moreover, Evi-1 appears crucial for HSC development. Currently ongoing experiments in our laboratory focus on the further elucidation of the molecular mechanisms underlying the Evi-1 effects during developmental hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Acetylation of Lysine 564 Adjacent to the C-terminal Binding Protein-binding Motif in EVI1 Is Crucial for Transcriptional Activation of GATA2

Ecotropic viral integration site 1 (EVI1) is an important transcription factor for leukemogenesis. EVI1 is a member of a group of transcription factors with C-terminal binding protein (CtBP)-binding motifs that act as transcriptional co-repressors; however, we recently found that EVI1 directly activates GATA2 transcription, which is an important gene for the maintenance of hematopoietic stem cells. We show here that EVI1-activated GATA2 transcripts derive from exon 1S of GATA2, which is specifically activated in neural and hematopoietic cells. EVI1 was acetylated by the histone acetyltransferase p300/CBP association factor (P/CAF) in myeloid leukemia cells and hematopoietic progenitor cells. Acetylation at Lys(564), which is adjacent to the CtBP-binding consensus sequence of EVI1, was found to be important for transcriptional activation of GATA2. Mutation of Lys(564) to alanine (K564A) markedly reduced the ability of EVI1 to bind DNA and activate transcription of GATA2. Furthermore, we confirmed that Lys(564) in EVI1 was specifically acetylated in leukemia and primary hematopoietic cells by using an antibody directed against an acetylated Lys(564) EVI1 peptide. Moreover, co-transfection of P/CAF with EVI1 overcame the suppressive effect of the CtBP co-repressor and resulted in GATA2 transcriptional activation; nonetheless, CtBP2 was still included in the protein complex with EVI1 and P/CAF on the EVI1-binding site in the GATA2 promoter region. Thus, acetylation of EVI1 at Lys(564) by P/CAF enhances the DNA binding capacity of EVI1 and thereby contributes to the activation of GATA2.

The Zebrafish Homologue of the Murine Ecotropic Viral Integration Site-1 (. Evi-1) gene Regulates Zebrafish Embryonic Blood Development.

Abstract 1461Poster Board I-484The ecotropic viral integration site-1 (Evi-1) locus was originally identified as a common site of retroviral integration in murine myeloid tumors and was later shown to be one of the most potent oncogenenes associated with murine and human myeloid leukemia. More recent data suggest involvement of Evi-1 in embryonic hematopoiesis (Goyama et al, Cell Stem Cell 2008; Yuasa et al, EMBO J, 2005), yet the precise role and molecular regulation of Evi-1 during blood development remains poorly understood. The zebrafish model offers powerful tools for genetic and embryonic studies. Here, we study zebrafish embryonic development and human pluripotent stem cells to understand how evi-1 modulates early hematopoietic development.Loss-of-function studies were performed in vivo by injecting Morpholino oligonucleotides in zebrafish zygotes to inhibit evi-1 pre-mRNA splicing. To control for off-target effects, two separate morpholinos were designed and injected. N=100 zebrafish were analysed pro experiment in each group. Inhibition of evi-1 was confirmed by quantitative PCR comparison in morpholino-injected and control embryos. Hematopoietic development was followed in both morphants and wild-type embryos by simple microscopy and in situ hybridizations using known hematopoeitic markers in order to investigate the developmental time-point in which evi-1 regulates blood development. evi-1 morpholino injected zebrafisch embryo showed severely reduced numbers of circulating blood cells, consistent with the phenotype observed in Evi-1−/− mice. Additionally, hemorrhages were observed, suggesting concomittant defects of the endothelial lineage in evi-1 deficient fish. In situ hybridization analysis on 11-12 somite stage embryos revealed strong reduction of myeloid embryonic hematopoiesis (measured by pu.1 expression in the anterior lateral plate mesoderm), while no change was observed in primitive erythroid progenitor cells (monitored by gata1 expression) or overall in blood and endothelial precursors in the posterior lateral plate mesoderm (as monitored by scl expression). Taken together, our studies demonstrate a strong impact of evi-1 on zebrafish blood development, confirming the results from Evi-1−/− mice. As gata1 expression and therefore erythroid precursor cells in the posterior blood islands are unaffected in evi-1 morphants, our results support the hypothesis that the reduction of primitive yolk-sac erythrocytes in mutant mice was caused from hemorrhages from pericardial effusions. Since erythroid and myeloid cells derive from a common precursor, but gata1 expression was unaffected in knock-down embryos, we anticipate that evi-1 plays a specific role in the myeloid lineage, as shown by abolished pu.1 expression in the anterior LPM. evi-1 therefore probably affects differentiation, survival or proliferation of myeloid cells.Previous reports in adult hematopoietic cells show that evi-1 can interact with both gata1 and pu.1. However, our data suggest that this is not the case during embryonic development, since gata1 expression remained unaltered in morpholino-injected embryos. Furthermore, data in mice suggest that Evi-1 may modulate embryonic hematopoiesis by affecting hematopoietic stem cell proliferation through regulation of Gata2. Currently ongoing experiments in our laboratories focus on characterization of genetic interactions between evi-1, gata2 and pu.1 during zebrafish blood development. Amongst other, gata2 and respectively pu.1 mRNA are co-injected in evi-1 morphants to analyse whether they can rescue the blood phenotype. Moreover, selected findings in zebrafish embryonic development will be verified in the human using using in vitro differentiating human induced pluripotent stem (iPS) cells. First expression data generated by real-time PCR analysis showed differential expression of EVI-1 in embryoid bodies generated from human iPS cells, confirming our hypothesis that EVI-1 has specific effects during human blood development. DisclosuresNo relevant conflicts of interest to declare.

Analysis of BCL11A gene Variants in Hematological Malignancies.

Abstract 2956Poster Board II-932 Background.The B-cell leukemia 11A gene (BCL11A/Evi9/CTIP1) is essential for normal lymphoid development and genetic association studies have shown its potential regulator effect in blood related phenotypes. BCL11A encodes a Krüppel-like zinc-finger protein and functions as a transcriptional repressor through its interaction with several proteins including BCL6. The corresponding mouse gene is a common site of retroviral integration in myeloid leukemia, and may function as a leukemia oncogene. It is down-regulated during hematopoietic cell differentiation and abnormalities involving this gene have been detected in a variety of B-cell malignancies in humans.We genotyped SNP rs11886868 in the BCL11A gene, which has been previously associated with HbF production, in patients with hematological malignancies from Sardinia to investigate a possible contribution of this gene in determining genetic susceptibility to onco-hematological diseases. Patients and Methods.We screened a total of 325 patients with hematological malignancies for rs11886868 SNP at the BCL11A locus using the TaqMan allelic discrimination assay: 51 B-cell Non Hodgkin's lymphoma (NHL), 27 Hodgkin's disease (HD), 42 Chronic Lymphocytic Leukemia (CLL), 52 Multiple Myeloma, 35 Cutaneous T-cell Lymphomas (CTCL), 11 Acute Lymphoblastic Leukemia (ALL), 19 Myelodysplastic Syndromes (MDS), 31 Acute Non Lymphoblastic Leukemia (ANLL), 36 Philadelphia negative Myeloproliferative Disorders (MPD), 21 Chronic Myelogenous Leukemia. Fifty–four DNAs from healthy individuals were used as population controls. Both patients and controls originated from central Sardinia. The frequencies comparisons between controls and cases were performed using chi-square test and Odds Ratio (OR) analysis with Cornfield 95% confidence intervals (CI). Results.Allele frequencies for BCL11A rs11886868 were 22% for the “C” allele and 78% for the “T” allele. No statistically significant difference was observed between cases and controls. All genotypes were in Hardy-Weinberg equilibrium for both patients and controls groups. The genotype frequencies were 65% (T/T), 26% (C/T) and 9% (C/C) in controls and 53% (T/T), 40.5% (C/T), and 6.5% (C/C) in hematological malignancies. When compared with the genotype frequencies reported for Caucasian and healthy controls from Sardinia no statistically significant difference was observed (p=0.4). However, the C/T genotype was more frequent in cases than controls (41% vs 26%) conferring an increased risk for hematological malignancies with an estimated OR=1,9 (95%CI 1.08-3.6; p=0.03). In detail, statistically significant differences in genotype distribution were observed in CTCL (p< 0.0001), MPD (p=0.0006), NHL (p=0.008), HD (p=0.002) and ALL patients (p=0.02). The C/C genotype was not observed in CTCL and HD patients, while heterozygousity conferred an increased risk of 4.2 (2.3-7.7; p value <0.0001) and 2.6 (1.6-4.7; p value <0.002), respectively. The C/T genotype was also overrepresented in MPD with an estimated OR of 3.2 (1.7-5.8; p value= 0.0001) and NHL with OR of 2.7 (1.5-4.9; p value <0.001). Stratification for clinical and biological parameters showed that among CLLs, the C/C genotype was present in 4/27 (15%) of the CD38-negative patients and in none of the CD38-positive subgroup. By contrast, the homozygousity for the ancestral “T” allele was not observed in Mantle Cell and Marginal Zone Lymphomas. Conclusions.We found genetic association of BCL11A gene in several blood disorders with the strongest association for Cutaneous T-cell Lymphomas and Myeloproliferative disorders suggesting a possible role of BCL11A in both lymphoid and myeloid lineages. Specific BCL11A genotypes have been associated with different BCL11A expression levels that influence HbF production. We speculate that BCL11A sequence variants may influence expression of different isoforms that may have effect on cell pathways involved in oncogenetic events as well as in globin gene regulation.This work was supported by Associazione Italiana contro le Leucemie e Linfomi (AIL) Disclosures:No relevant conflicts of interest to declare.

EVI1 Is Activated During the Generation of Induced Pluripotent Stem (iPS) Cells.

Abstract 5048EVI1 gene was first identified as a common site of retroviral integration in murine leukemia models. This gene is part of a complex genomic locus, MDS1-EVI1, that has been described as a target for retroviral integration that may lead to the emergence of a non-malignant dominant hematopoietic stem cell (HSC) clone in mice, in primates, and in humans. These studies suggested that one of the genes encoded by this locus could affect the self-renewal potential of HSC. Recent studies in mice revealed that indeed EVI1 plays an essential role in cell proliferation and it also enhances the self-renewal ability of HSC. The intense attention focused on the MDS1-EVI1 locus as retrovirus integration site prompted us to investigate whether EVI1 might have a role in somatic cell re-programming generated with retroviruses. Recent developments in stem cell research have enabled the re-programming of somatic cells to a pluripotent state using exogenous factors. Induced pluripotent stem (iPS) cells have the potential to differentiate into any cells types and that might be used in the future for clinical therapy. In order to elucidate the molecular events allowing the conversion of adult somatic into pluripotent stem cell, we evaluated EVI1 expression during this process. We found that EVI1 is activated in the early stages of re-programming and then it is silenced once the cells has been fully re-programmed. EVI1 seems to facilitate the initiation of cell re-programming by up-regulating a subset of genes previously described as potent stimulators of stem cells expansion. DisclosuresNo relevant conflicts of interest to declare.

The Role of Zinc Finger Protein 521/Early Hematopoietic Zinc Finger Protein in Erythroid Cell Differentiation

ZNF521 (zinc finger protein 521) is a transcription factor with an N-terminal transcriptional repressor motif and 30 zinc finger domains. Although a high expression level of ZNF521 in human CD34+ progenitors and hematopoietic malignancies has been demonstrated, the functional role of ZNF521 in hematopoietic cell differentiation has not been clarified. In this study, we analyzed the role of ZNF521 in erythroid cell differentiation using the short hairpin RNA (shRNA)-mediated gene silencing method. Down-regulation of ZNF521 mediated by transient expression of shRNA for ZNF521 resulted in increased synthesis of hemoglobin in K562 and HEL cell lines as compared with control cells. K562-derived clones in which ZNF521 was constitutively silenced by shRNA also showed marked synthesis of hemoglobin and an increased expression level of glycophorin A. Since GATA-1 is the key regulator of erythroid differentiation, the effect of ZNF521 on transcription activity of GATA-1 was analyzed using a luciferase assay. GATA-1 activity was markedly inhibited by ZNF521 in a dose-dependent manner. Deletion analysis of ZNF521 showed that the repressive effect requires an N-terminal repression motif. Furthermore, the direct interaction of ZNF521 with GATA-1 was demonstrated. These results indicate that ZNF521 modulates erythroid cell differentiation through direct binding with GATA-1.

MicroRNAs and lymphomas

MicroRNAs (miRNAs) are small non-coding RNAs which regulate the expression of target mRNAs through base pairing to partially (in mammals) or perfectly (in plants) complementary sites, mainly, but not exclusively, in the 3#-untranslated region (3#-UTR) of their targets [1]. Should a perfect complementarity occur, the mRNA is cleaved and degraded, whereas a translational silencing is the main miRNA regulatory mechanism in the case of imperfect base pairing [1]. MiRNAs have important regulatory functions, including cell cycle, apoptosis and differentiation [2]. Systematic miRNA expression profiling has revealed widespread aberrant miRNA expression in cancer with respect to the normal tissues, including lymphomas [3–6]. Earlier studies have shown that miR-155 is up-regulated in pediatric Burkitt’s lymphoma [4], diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma (PMBL) and Hodgkin’s lymphoma [5–6] (Table 1). This miRNA is encoded by the final portion of the BIC (B-cell integration cluster) gene, which was originally identified as a common retroviral integration site in avian leukosis virusinduced B-cell lymphomas [7]. Our group demonstrated that mice overexpressing miR-155 in B lymphocytes develop polyclonal pre-leukemic pre-B-cell proliferation followed by full-blown B-cell malignancy [8]. More recently two knock-out mice models have demonstrated a critical role of miR-155 in immunity by showing that BIC/miR-155 have defective dendritic cell functions, impaired cytokine secretion and TH cells intrisically biased toward TH2 differentiation [9–10]. Moreover miR-155 could represent the connection between inflammation, immunity and cancer since its expression can be induced by mediators of the flogosis and is involved in the response to endotoxic shock [11]. He et al. [12] reported that the miR-17 92 polycistron, which is located in 13q31–32, a region commonly amplified in B-cell lymphoma12, was up-regulated in 65% of the B-cell lymphoma patients. The authors overexpressed the miR-17 92 cluster in HPCs from mice that carry the MYC transgene and reported that the miR-17 92 cluster acted, together with MYC expression, to accelerate tumor development [12]. A different group recently generated mice with high miR-17 92 expression in lymphocytes and found that these mice developed a lymphoproliferative disease and autoimmunity and died prematurely (Table 1) [13]. The transgenic lymphocytes exhibited enhanced proliferation and survival due to the downregulation of PTEN and the pro-apoptotic Bim (which controls B-lymphocyte apoptosis) by the miR-17 92 cluster [13]. Insights about the regulation of this cluster in lymphoma was provided by O’Donnell et al. [14]. Using an inducible system of MYC expression and Chip assays, the authors showed that MYC binds and activates the expression of the miR-17 92 cluster at chromosome 13q31. Furthermore, they indicated that E2F transcription factor 1 (E2F1) is down-regulated by two miRNAs of the cluster, miR-17–5p and miR-20a. E2F1 is a direct target of MYC that promotes cell-cycle progression [15]. Thus, MYC simultaneously activates E2F1 and limits its translation through a miRNA-based mechanism, enabling a tightly controlled proliferative signal [14].

Generation of Pluripotent Stem Cells from Adult Mouse Liver and Stomach Cells

Induced pluripotent stem (iPS) cells have been generated from mouse and human fibroblasts by the retroviral transduction of four transcription factors. However, the cell origins and molecular mechanisms of iPS cell induction remain elusive. This report describes the generation of iPS cells from adult mouse hepatocytes and gastric epithelial cells. These iPS cell clones appear to be equivalent to embryonic stem cells in gene expression and are competent to generate germline chimeras. Genetic lineage tracings show that liver-derived iPS cells are derived from albumin-expressing cells. No common retroviral integration sites are found among multiple clones. These data suggest that iPS cells are generated by direct reprogramming of lineage-committed somatic cells and that retroviral integration into specific sites is not required.

Trib1 and Evi1 cooperate with Hoxa and Meis1 in myeloid leukemogenesis

AbstractCooperative activation of Meis1 and Hoxa9 perturbs myeloid differentiation and eventually leads myeloid progenitors to leukemia, yet it remains to be clarified what kinds of subsequent molecular processes are required for development of overt leukemia. To understand the molecular pathway in Hoxa9/Meis1-induced leukemogenesis, retroviral insertional mutagenesis was applied using retrovirus-mediated gene transfer. The mice that received Hoxa9/Meis1-transduced bone marrow cells developed acute myeloid leukemia (AML), and Trib1, Evi1, Ahi1, Rarα, Pitpnb, and AK039950 were identified as candidate cooperative genes located near common retroviral integration sites. Trib1 and Evi1 were up-regulated due to retroviral insertions, and coexpression of these genes significantly accelerated the onset of Hoxa9/Meis1-induced AML, suggesting that Trib1 and Evi1 are the key collaborators. Furthermore, Trib1 by itself is a novel myeloid oncogene, enhancing phosphorylation of ERK, resulting in inhibition of apoptosis. These results demonstrate the importance of specific oncogene interaction in myeloid leukemogenesis.

Arsenic Trioxide Targets the MDS1 and EVI1- Containing Oncoproteins for Degradation by Proteasome Dependent and Independent Mechanisms.

The EVI1 gene was first identified as a common site of retroviral integration in murine myeloid tumors and expresses a zinc finger-containing transcriptional regulator. It also encodes for MDS1/EVI1, a longer, alternatively spliced form of EVI1. In blood development EVI1 is normally expressed in hematopoietic stem cells (HSCs) but not in committed cells. However, aberrant expression of EVI1 or MDS1/EVI1 has been frequently found in myelodysplastic syndrome (MDS), and in cases of chronic myeloid leukemia during blast phase (CML-BP) and acute myelogenous leukemia (AML). In addition, the MDS1/EVI1 gene fuses to the AML1 (Runx1) gene as a consequence of the t(3;21)(q26;q22) translocation found in patients with CML-BP and therapy related MDS and AML, producing the AML1/MDS1/EVI1 (AME) or AML1/MDS1 fusion genes. We have previously shown that expression of the AME fusion protein in mouse bone marrow cells by retroviral transduction impaired hematopoiesis and eventually induced an AML. EVI1 was also reported to induce MDS in mice. These results indicate that the EVI1 and AME oncoproteins play causal roles in t(3;21)- and EVI1-positive hematological malignancies. Therapies targeting oncoproteins that play causal roles in tumorigenesis have been shown to be effective in treating cancers. Arsenic trioxide (ATO) has been found as an effective treatment for patients suffering from acute promyelocytic leukemia (APL) that harbor the t(15;17) translocation. The success of ATO in treating APL patients has prompted many to consider expanding the drug's use to target other translocations and/or mutations found in hematological malignancies. In this study we found that ATO could target the EVI1, MDS1/EVI1, AML1/MDS1 and AME oncoproteins for degradation. We further found that ATO targeted EVI1 for degradation through the ubiquitin-proteasome pathway. Interestingly, ATO induced degradation of MDS1-containing oncoproteins via a proteasome-independent mechanism. Consistent with the downregulation of AME, the ATO treatment induced differentiation and apoptosis in AME leukemic cells in vitro and reduced splenomegally in mice transplanted with AME leukemic cells. Our results suggest that ATO could be used as a part of targeted therapy for AME-, AML1/MDS1-, MDS1/EVI1- and EVI1-positive hematological malignancies. We are currently studying the mechanism of increased ubiquitination of EVI1 by ATO, as well as the proteasome-independent mechanism by which ATO downregulates MDS1-containing proteins. Such research will help further the identification of new therapeutic targets and the development of new therapeutic strategies for human cancer.

A Genome-Wide Retroviral Insertional Mutagenesis Screen for Genes Cooperating with Truncated, Oncogenic GATA1s.

Somatic mutations in the hematopoietic transcription factor GATA1 are found in megakaryoblasts of Down Syndrome (DS) patients with transient myeloproliferative disorder (TMD, or transient leukemia or TL) and the related acute megakaryoblastic leukemia (DS-AMKL, or DS- AML M7). These mutations lead to production of a GATA1 variant (GATA1s) lacking its N-terminal domain. Mice carrying GATA1s mutation have normal adult hematopoiesis. However, during embryonic/fetal development, we have identified a transient population of abnormal yolk sac/fetal liver megakaryocytic progenitors in mutant mice. We proposed that these progenitor cells are the target for transformation in DS-AMKL/TMD. GATA1s mice (either during development or as adults) do not develop myeloproliferative disorder or leukemia. To recapitulate human DS TMD in mice, we bred GATA1s mice to mouse DS models (Ts65Dn and Ts1Cje) and generated GATA1s/DS double mutants. The phenotype of GATA1s/DS mice is not different from that of GATA1s mice, suggesting that these mouse DS models (representing ~166 and 112 trisomic genes on human chromosome 21, respectively, including Runx1, Ets2, and Erg) do not accurately recapitulate the effects of trisomy in DS. To search for genes that cooperate with GATA1s in an unbiased fashion, we established a genome-wide retroviral insertional mutagenesis screen. GATA1s mutant fetal liver progenitors proliferate in culture in the presence of thrombopoietin (Tpo) for about 4–5 weeks. We infected mutant fetal progenitors with MSCV retrovirus and selected in vitro in the presence of Tpo for immortalized cell lines. Retroviral integration sites in these cell lines were determined by Splinkerette PCR, and confirmed by genomic PCR. Genes that were affected by retroviral integration were confirmed by real-time PCR for their elevated expression or knock-down. From the genetic screen performed thus far, we identified two common retroviral integration sites, Evi1 and Prdm16 (PR domain containing 16). Interestingly, Evi1 is also overexpressed in M7 leukemias, though its expression in non-DS M7 leukemia is higher than in DS M7 leukemia. By retroviral overexpression, we have confirmed that ectopic expression of Evi1 in GATA1s mutant fetal progenitors further enhanced proliferation. Currently we are testing the in vivo leukemogenic abilities of these cell lines by transplantation. By this approach, we will identify genes that cooperate with GATA1s in cellular transformation and, thereby, gain insights into the mechanism of leukemogenesis in DS-AMKL/TMD.

Hematopoietic cells and osteoblasts are derived from a common marrow progenitor after bone marrow transplantation.

Bone and bone marrow are closely aligned physiologic compartments, suggesting that these tissues may represent a single functional unit with a common bone marrow progenitor that gives rise to both osteoblasts and hematopoietic cells. Although reports of multilineage engraftment by a single marrow-derived stem cell support this idea, more recent evidence has challenged claims of stem cell transdifferentiation and therefore the existence of a multipotent hematopoietic/osteogenic progenitor cell. Using a repopulation assay in mice, we show here that gene-marked, transplantable marrow cells from the plastic-nonadherent population can generate both functional osteoblasts/osteocytes and hematopoietic cells. Fluorescent in situ hybridization for the X and Y chromosomes and karyotype analysis of cultured osteoblasts confirmed the donor origin of these cells and excluded their generation by a fusion process. Molecular analysis demonstrated a common retroviral integration site in clonogenic hematopoietic cells and osteoprogenitors from each of seven animals studied, establishing a shared clonal origin for these ostensibly independent cell types. Our findings indicate that the bone marrow contains a primitive cell able to generate both the hematopoietic and osteocytic lineages. Its isolation and characterization may suggest novel treatments for genetic bone diseases and bone injuries.

Evi3, a common retroviral integration site in murine B-cell lymphoma, encodes an EBFAZ-related Krüppel-like zinc finger protein

Retroviral insertional mutagenesis in inbred mouse strains provides a powerful method for cancer gene discovery. Here, we show that a common retroviral integration site (RIS) in AKXD B-cell lymphomas, termed Evi3, encodes a novel zinc finger protein with 30 Krüppel-like zinc finger repeats. Most integrations at Evi3 are located upstream of the first translated exon and result in 3' long-terminal repeat (LTR)-driven overexpression of Evi3. Evi3 is highly related to the early B-cell factor-associated zinc finger gene (Ebfaz), and all 30 zinc fingers found in EVI3 are conserved in EBFAZ. EBFAZ binds to and negatively regulates early B-cell factor (EBF) (also known as olfactory-1, OLF1), a basic helix-loop-helix (bHLH) transcription factor required for B-lineage commitment and the development of the olfactory epithelium. EBFAZ also binds to SMA- and MAD-related protein-1 (SMAD1) and SMAD4 in response to bone morphogenetic protein-2 (BMP2) signaling, which in turn activates the homeobox regulator of Xenopus mesoderm and neural development Xvent-2. Surprisingly, while Ebfaz and Evi3 are coexpressed in many tissues, and both proteins are nuclear, we could not detect Ebfaz expression in B cells by reverse transcriptase-polymerase chain reaction (RT-PCR), whereas Evi3 expression could be detected at all stages of B-cell development. Our results suggest that EVI3, like EBFAZ, is a multifunctional protein that participates in many signaling pathways via its multiple zinc fingers. Furthermore, our results suggest that EVI3, not EBFAZ, is the member of this protein family that interacts with and regulates EBF in B cells.