Retroviral Integration Sites Research Articles

Efficient and stable MGMT-mediated selection of long-term repopulating stem cells in nonhuman primates.

HSC transplantation using genetically modified autologous cells is a promising therapeutic strategy for various genetic diseases, cancer, and HIV. However, for many of these conditions, the current efficiency of gene transfer to HSCs is not sufficient for clinical use. The ability to increase the percentage of gene-modified cells following transplantation is critical to overcoming this obstacle. In vivo selection with mutant methylguanine methyltransferase (MGMTP140K) has been proposed to overcome low gene transfer efficiency to HSCs. Previous studies have shown efficient in vivo selection in mice and dogs but only transient selection in primates. Here, we report efficient and stable MGMTP140K-mediated multilineage selection in both macaque and baboon nonhuman primate models. Treatment consisting of both O6-benzylguanine (O6BG) and N,N'-bis(2-chloroethyl)-N-nitroso-urea (BCNU) stably increased the percentage of transgene-expressing cells from a range of initial levels of engrafted genetically modified cells, with the longest follow-up after drug treatment occurring over 2.2 years. Drug treatment was well tolerated, and selection occurred in myeloid, lymphoid, and erythroid cells as well as platelets. Retrovirus integration site analysis before and after drug treatments confirmed the presence of multiple clones. These nonhuman primate studies closely model a clinical setting and should have broad applications for HSC gene therapy targeting human diseases of malignant, genetic, and infectious nature, including HIV.

In vivo expansion of MDR1‐transduced cells accompanied by a post‐transplantation chemotherapy regimen with mitomycin C and methotrexate

A recurrent breast cancer patient received high-dose chemotherapy, a transplant of multidrug resistance 1 (MDR1)-transduced cells and four different protocols of post-transplantation chemotherapy. We report the analysis of MDR1-transduced cells in this patient. MDR1 transgene levels in the peripheral blood mononuclear cells of the patient were evaluated by polymerase chain reaction (PCR). Retroviral integration sites of the MDR1-transduced cells were identified by linear amplification-mediated (LAM)-PCR. Twelve days after transplantation, approximately 1% of the peripheral blood mononuclear cells were MDR1 transgene-positive. The transgene levels decreased quickly, and were at low levels until day 504. A remarkable increase in MDR1 transgene-positive cells was observed on day 532, during combination chemotherapy with mitomycin C and methotrexate. Using LAM-PCR, 31 MDR1-transduced clones were identified, and eight of these were long-life clones that survived for more than 500 days. Among the 31 clones, ten had a retroviral integration site near genes listed in the Retroviral Tagged Cancer Gene (RTCG) Database. Two long-life clones, N-30 and N-31, had retroviral integration sites within the MDS1-EVI1 locus. Another two long-life clones had integration sites close to PRDM16 or CUEDC1. These results suggest that MDR1-transduced cells were enriched in vivo by an MDR1 substrate, mitomycin C. The possible activation of EVI1 or other RTCGs by retroviral insertion may have affected the survival and persistence of a proportion of the transduced cells.

Dynamics of gene-modified progenitor cells analyzed by tracking retroviral integration sites in a human SCID-X1 gene therapy trial

AbstractX-linked severe-combined immunodeficiency (SCID-X1) has been treated by therapeutic gene transfer using gammaretroviral vectors, but insertional activation of proto-oncogenes contributed to leukemia in some patients. Here we report a longitudinal study of gene-corrected progenitor cell populations from 8 patients using 454 pyrosequencing to map vector integration sites, and extensive resampling to allow quantification of clonal abundance. The number of transduced cells infused into patients initially predicted the subsequent diversity of circulating cells. A capture-recapture analysis was used to estimate the size of the gene-corrected cell pool, revealing that less than 1/100th of the infused cells had long-term repopulating activity. Integration sites were clustered even at early time points, often near genes involved in growth control, and several patients harbored expanded cell clones with vectors integrated near the cancer-implicated genes CCND2 and HMGA2, but remain healthy. Integration site tracking also documented that chemotherapy for adverse events resulted in successful control. The longitudinal analysis emphasizes that key features of transduced cell populations—including diversity, integration site clustering, and expansion of some clones—were established early after transplantation. The approaches to sequencing and bioinformatics analysis reported here should be widely useful in assessing the outcome of gene therapy trials.

346. High-Definition Mapping of Retroviral Integration Sites Defines the Fate of Allogeneic T Cells after Donor Lymphocyte Infusion

346. High-Definition Mapping of Retroviral Integration Sites Defines the Fate of Allogeneic T Cells after Donor Lymphocyte Infusion

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.

A retroviral vector common integration site between leupaxin and zinc finger protein 91 ( ZFP91) observed in baboon hematopoietic repopulating cells

Retroviral vector proviruses can lead to aberrant expression of nearby genes in hematopoietic repopulating cells, leading to an over-representation of clones with dysregulated genes that affect hematopoiesis. Common integration sites (CISs) identified using the vector provirus as a molecular tag can be used to identify these genes. Here we characterized a retroviral CIS observed at high frequency in baboon hematopoietic repopulating cells that has not been described previously. Gammaretroviral vector integration sites in baboon repopulating cells identified by polymerase chain reaction amplification were localized to the human genome to identify a CIS. The presence of each clone was tracked over time using allele-specific polymerase chain reaction. In three different animals that received gammaretrovirally transduced CD34-enriched bone marrow cells, vector proviruses were identified at three distinct sites within a window of 664 base pairs between leupaxin and zinc finger protein 91 (ZFP91). All three integrants of the CIS occurred within a CpG island between leupaxin and zinc finger protein 91 (ZFP91). We describe a novel CIS between leupaxin and ZFP91 in hematopoietic repopulating cells. Our data suggest that leupaxin and/or ZFP91 may play a role in hematopoietic repopulating cells.

Proviruses Selected for High and Stable Expression of Transduced Genes Accumulate in Broadly Transcribed Genome Areas

Retroviruses and retrovirus-derived vectors integrate nonrandomly into the genomes of host cells with specific preferences for transcribed genes, gene-rich regions, and CpG islands. However, the genomic features that influence the transcriptional activities of integrated retroviruses or retroviral vectors are poorly understood. We report here the cloning and characterization of avian sarcoma virus integration sites from chicken tumors. Growing progressively, dependent on high and stable expression of the transduced v-src oncogene, these tumors represent clonal expansions of cells bearing transcriptionally active replication-defective proviruses. Therefore, integration sites in our study distinguished genomic loci favorable for the expression of integrated retroviruses and gene transfer vectors. Analysis of integration sites from avian sarcoma virus-induced tumors showed strikingly nonrandom distribution, with proviruses found prevalently within or close to transcription units, particularly in genes broadly expressed in multiple tissues but not in tissue-specifically expressed genes. We infer that proviruses integrated in these genomic areas efficiently avoid transcriptional silencing and remain active for a long time during the growth of tumors. Defining the differences between unselected retroviral integration sites and sites selected for long-terminal-repeat-driven gene expression is relevant for retrovirus-mediated gene transfer and has ramifications for gene therapy.

Retroviral Integration Site Selection

The stable insertion of a copy of their genome into the host cell genome is an essential step of the life cycle of retroviruses. The site of viral DNA integration, mediated by the viral-encoded integrase enzyme, has important consequences for both the virus and the host cell. The analysis of retroviral integration site distribution was facilitated by the availability of the human genome sequence, revealing the non-random feature of integration site selection and identifying different favored and disfavored genomic locations for individual retroviruses. This review will summarize the current knowledge about retroviral differences in their integration site preferences as well as the mechanisms involved in this process.

Angiotensin II Receptor-Associated Protein (AGTRAP) Synergizes with Mpl Signaling to Promote Survival and to Increase Proliferation Rate of Hematopoietic Cells.

Abstract 3606 Poster Board III-542 The Mpl proto-oncogene plays a basic role in megakaryocytic development and platelet production as well as performs substantial function in hematopoietic stem cell (HSC) homeostasis and self-renewal. Several, mutations in Mpl have been shown to be associated with the myeloproliferative disorders. In an attempt to identify cellular factors that could cooperate with Mpl signaling leading to increases in cell survival and proliferation, we performed retroviral insertional mutagenesis using MSCV based vector coding for a drug dependent, dimerizable, fusion protein which contains the cytoplasmic domain of Mpl. Such vector construct can interrupt gene structure through insertion, and has an intact long terminal repeats which can activate adjacent genes. We used that vector construct to transduce the human leukemia cell line K562. After transduction, the endogenous transforming BCR-ABL kinase was blocked using Imatinib, and cells dependent on Mpl signaling were selected by the addition of dimerizing drug, AP20187. In the absence of Mpl signaling the cells underwent erythroid differentiation and died. Cells that acquired proliferative advantage and were dependent on Mpl function were expected to point to mutations synergistic with Mpl signaling. Cloning of retroviral integration sites (RIS) from the selected populations would allow to identify overrepresented clones. Indeed, overrepresented RIS clones were found in several independent transductions. Such overrepresented clones were not observed in an independent experiment where RIS were cloned from cells transduced with the same vector and sorted but not selected on Imatinib and AP20187. RIS from the overrepresented clones pointed to candidate Mpl cooperating genes. AGTRAP a member of renin-angiotensin system (RAS) was identified among the candidate genes. Multiple lines of evidence implicate RAS in hematopoietic cell proliferation and differentiation. RAS components, including renin, angiotensin-converting enzyme(ACE), angiotensin II, angiotensin type 1 and type 2 receptors (AT1 andAT2), and tetrapeptide N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP an inhibitory hematopoietic peptide) have been isolated in the bone marrow as well as in circulating hematopoietic cells. Angiotensin II was found to interact with the AT1 receptor to stimulate erythroid differentiation and increase hematopoietic progenitor cell proliferation. Moreover, stimulation of AT1/AT2 receptors was found to exert an stimulatory/inhibitory action on JAK-STAT pathway, which is directly linked to physiologic functions of erythropoietin, thrombopoietin and other cytokines. Finally, ACE was implicated in enhancing the recruitment of primitive stem cells into the S-phase by degrading AcSDKP. We have tested the AGTRAP function in hematopoietic cells. For that purpose we overexpressed AGTRAP protein in the K562 cells and in K562 cells driven by Mpl signaling. AGTRAP overexpression stimulated the growth of plain K562 cells decreasing the cells division time from 16 to 12 hours. Importantly, it synergized with Mpl signaling to promote the proliferation rate of Mpl dependent K562 cells (expressing the dimerizable construct of Mpl activated with AP20187 while the transforming Bcr-Abl protein was inactivated with Imatinib) decreasing the cells division time from 13 to 9.6 hours. The survival of Mpl dependent K562 cells over-expressing AGTRAP was still dependent on Mpl signaling and on Jak2 function as tested by withdrawal of AG20187 and by treatment with Jak2 inhibitor AG490. Overexpression of AGTRAP protein over-sensitized the UT7/TPO cells to treatment with recombinant human megakaryocyte growth and development factor (rhMGDF), increasing growth rate of the cells at low rhMGDF concentrations. The cells proliferation rate was increased by 22.5% and 39.3% at 1/100 and 1/1000 of optimal concentrations of rhMGDF respectively. There was no growth advantage from AGTRAP expression at optimal concentration of rhMGDF, however in the presence of AGTRAP the UT7/TPO cells were able to proliferate in the absence of rhMGDF altogether. Surprisingly, the overexpression of AGTRAP negatively regulated the growth of HEL cells, known to carry a homozygous transforming mutation of Jak2V617F. In conclusion our data implicate the RAS member AGTRAP in hematopoietic cell proliferation and survival and point to its synergistic effect with the proliferation promoting function of Mpl. Disclosures: No relevant conflicts of interest to declare.

A Mouse Model of Evi1-Related Leukemia.

Abstract 1958Poster Board I-981 Background:Evi1 gene is located on chromosome 3q26 and aberrantly expressed in acute myeloid leukemia (AML) patients with or without 3q26 abnormalities, and inappropriate expression of Evi1 associates with poor prognosis. Evi-1 is originally identified in a common integration site of murine leukemia retrovirus and enhanced expression of Evi1 by retrovirus integration is thought to be responsible for leukemogenesis in mouse models. However, retroviral expression of marker genes such as GFP has not induced leukemia even if some clones possessing integration at Evi1 site have been identified. These data indicate that Evi1 requires cooperative factors to induce progressive leukemia, whereas overexpression of Evi1 is enough to lead to clonal expansion of hematopoietic cells. Therefore, identifying genes collaborating with Evi1 is one of the key issues of understanding Evi1-related leukemogenesis. Recently, we demonstrated that a point mutation of the transcription factor AML1 (AML1-D171N) can induce myelodysplastic syndrome (MDS) that progresses to AML in association with overexpression of Evi1 through a mouse bone marrow transplantation model. In that work, we analyzed mice transplanted with BM cells transduced Evi1 alone as control and surprisingly confirmed that all of the mice developed leukemia within 6-11 months after the transplant. In this report, we will describe interesting findings in the novel mouse model of Evi1-induced leukemia. Result: C57BL6/Ly-5.1 murine BM cells infected with retroviruses harboring Evi1 were transplanted into irradiated syngeneic Ly-5.2 mice. The mice looked fine until 5 months, but GFP-positive-Evi1 expressing cells were gradually increased in the peripheral blood (PB), and then the mice died at 6-11 months after the transplantation. The mice showed dysplastic features in myeloid and erythroid cells, increase of blasts in the PB and the BM, hepatosplenomegaly, slight anemia, and some of the mice showed severe leukocytosis. The mice were thought to die of multiple organ failure due to invasion of leukemic cells not due to anemia. The phenotype is different from that of the mouse BMT model expressing Evi1 by retrovirus reported by another group, in which the mice died about 10 months with severe peripheral cytopenia and finally the disease did not progress to AML. Therefore, we considered that Evi1 might have collaborated with unknown genes near retrovirus integration sites in our case and analyzed integration sites by the bubble PCR method. Interestingly, frequent integration at 3' side of C/EBPb gene was found in six mice out of eight mice transplanted with Evi1-transduced BM cells. The integrations were located at 62.5-86.7kb downstream of C/EBPb gene. Next, we examined the expression level of C/EBPb, Tmem189, and Ptpn1, all of which are located near the integration site, and confirmed that C/EBPb showed elevated expression although neither Tmem189 nor Ptpn1 did. We also identified Bcas1, Rps6ka1, and Rapgef4 genes at the retroviral integration site in the other two mice without integration near C/EBPb. Discussion: C/EBPb, also known as NF-IL6, is a transcription factor that specifically binds to an IL1-responsive element in the IL-6 gene and has a role in regulation not only for the IL-6 gene but also for several cytokine genes such as TNF, IL-8, and G-CSF. The hematopoietic progenitor cells of C/EBPb-deficient mice have been reported to respond imperfectly to GM-CSF and G-CSF. Furthermore, C/EBPb is a downstream target of the Ras-Raf pathway. The locus of C/EBPb gene has been reported as a common integration site in the Retrovirus Tagged Cancer Gene Database (RTCGD), which is a database of retroviral insertional mutagenesis in mouse tumors. AKxD mice, Cdkn2a-KO mice, NUP98/HOXD13 transgenic mice, and MYC/Runx2 transgenic mice were reported to develop myeloid or lymphoid leukemia by retroviral insertion into 3' side of C/EBPb gene. In this study, we identified frequent integration at 3' side of C/EBPb gene in Evi1-transduced leukemic cells, whereas we have not identified this locus in AML1-mutants-transduced leukemic cells. Based on these findings and our results, C/EBPb is supposed to be a candidate gene to collaborate with Evi1 in leukemogenesis. Conclusion: We identified involvement of C/EBPb in Evi1-induced leukemogenesis. The novel mouse model that we generated in this study could help understanding the molecular basis of Evi1-related leukemia. Disclosures:No relevant conflicts of interest to declare.

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.

Genotoxic Potential of Lineage-specific Lentivirus Vectors Carrying the β-Globin Locus Control Region

Insertional mutagenesis by long terminal repeat (LTR) enhancers in gamma-retrovirus-based vectors (GVs) in clinical trials has prompted deeper investigations into vector genotoxicity. Experimentally, self-inactivating (SIN) lentivirus vectors (LVs) and GV containing internal promoters/enhancers show reduced genotoxicity, although strong ubiquitously-active enhancers dysregulate genes independent of vector type/design. Herein, we explored the genotoxicity of beta-globin (BG) locus control region (LCR), a strong long-range lineage-specific-enhancer, with/without insulator (Ins) elements in LV using primary hematopoietic progenitors to generate in vitro immortalization (IVIM) assay mutants. LCR-containing LV had approximately 200-fold lower transforming potential, compared to the conventional GV. The LCR perturbed expression of few genes in a 300 kilobase (kb) proviral vicinity but no upregulation of genes associated with cancer, including an erythroid-specific transcription factor occurred. A further twofold reduction in transforming activity was observed with insulated LCR-containing LV. Our data indicate that toxicology studies of LCR-containing LV in mice will likely not yield any insertional oncogenesis with the numbers of animals that can be practically studied.

Long-term polyclonal and multilineage engraftment of methylguanine methyltransferase P140K gene-modified dog hematopoietic cells in primary and secondary recipients

AbstractOverexpression of methylguanine methyltransferase P140K (MGMTP140K) has been successfully used for in vivo selection and chemoprotection in mouse and large animal studies, and has promise for autologous and allogeneic gene therapy. We examined the long-term safety of MGMTP140K selection in a clinically relevant dog model. Based on the association of provirus integration and proto-oncogene activation leading to leukemia in the X-linked immunodeficiency trial, we focused our analysis on the distribution of retrovirus integration sites (RIS) relative to proto-oncogene transcription start sites (TSS). We analyzed RIS near proto-oncogene TSS before (n = 157) and after (n = 129) chemotherapy in dogs that received MGMTP140K gene-modified cells and identified no overall increase of RIS near proto-oncogene TSS after chemotherapy. We also wanted to determine whether in vivo selected cells retained fundamental characteristics of hematopoietic stem cells. To that end, we performed secondary transplantation of MGMTP140K gene-modified cells after in vivo selection in dog leukocyte antigen (DLA)–matched dogs. Gene-modified cells achieved multilineage repopulation, and we identified the same gene-modified clone in both dogs more than 800 and 900 days after transplantation. These data suggest that MGMTP140K selection is well tolerated and should allow clinically for selection of gene-corrected cells in genetic or infectious diseases or chemoprotection for treatment of malignancy.

Pathogenetic significance of ecotropic viral integration site‐1 in hematological malignancies

The ecotropic viral integration site-1 (Evi-1) gene was first identified as a common locus of retroviral integration in murine leukemia models. In humans, EVI-1 is located on chromosome 3q26, and rearrangements on chromosome 3q26 often activate EVI-1 expression in hematological malignancies. Overexpression of EVI-1 also occurs with high frequency in leukemia patients without 3q26 abnormalities, and importantly, high EVI-1 expression is an independent negative prognostic indicator irrespective of the presence of 3q26 rearrangements. Recent gene targeting studies in mice revealed that Evi-1 is preferentially expressed in hematopoietic stem cells and plays an essential role in proliferation and maintenance of hematopoietic stem cells. In addition, intense attention has been focused on the EVI-1 gene complex as retrovirus integration sites because transcription-activating integrations into the EVI-1 locus confer survival and self-renewing ability to hematopoietic cells. The experimental results using animal models suggest that activation of Evi-1 in hematopoietic cells leads to clonal expansion or dysplastic hematopoiesis, whereas onset of full-blown leukemia requires cooperative genetic events. EVI-1 possesses diverse functions as an oncoprotein, including suppression of transforming growth factor-beta-mediated growth inhibition, upregulation of GATA2, inhibition of the Jun kinase pathway, and stimulation of cell growth via activator protein-1. In this article, we summarize current knowledge regarding the biochemical properties and biological functions of EVI-1 in normal and malignant hematopoiesis, with specific focus on its pathogenetic significance in hematological malignancies.

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.

Long-Term Vector Integration Site Analysis Following Retroviral Mediated Gene Transfer to Hematopoietic Stem Cells for the Treatment of HIV Infection

We previously reported the efficacy of nonmyeloablative allogeneic transplantation in 2 HIV positive recipients, one of whom received retrovirus transduced hematopoietic stem cells to confer resistance to HIV. Here we report an assessment of retroviral integration sites (RISs) recovered out to 3 years post-transplantation. We identified 213 unique RISs from the patient's peripheral blood samples by linear amplification-mediated PCR (LAM-PCR). While vector integration patterns were similar to that previously reported, only 3.76% of RISs were common among early (up to 3 months) and late samples (beyond 1 year). Additionally, common integration sites were enriched among late samples (14.9% vs. 36.8%, respectively). Three RISs were found near or within known oncogenes, but 2 were limited to early timepoints. Interestingly, an integration site near the MDS1 gene was detected in long-term follow-up samples; however, the overall contribution of MDS1 integrated clone remained stably low during follow-up.

Long-Term Vector Integration Site Analysis Following Retroviral Mediated Gene Transfer to Hematopoietic Stem Cells for the Treatment of HIV Infection.

We previously reported the efficacy of nonmyeloablative allogeneic transplantation in 2 HIV positive recipients, one of whom received retrovirus transduced hematopoietic stem cells to confer resistance to HIV (Blood . 2002; 99:698–701). Half of the donor cells were genetically modified with a Moloney murine leukemia virus (MoMLV) based HIV resistance vector containing a transdominant negative mutant Rev (TdRev) (2.58×10e8 cells) or a control vector MoMLV based vector encoding GP91phox (4.04×10e8 cells). Here we report an assessment of retroviral integration sites recovered out to 3 years post-transplantation.We identified 213 unique retroviral integration sites (RISs) from the patient's peripheral blood samples myeloid and lymphoid cells from 1 to 36 months after reinfusion of genetically modified CD34+ cells by linear amplification-mediated PCR (LAM-PCR). While overall vector integration patterns were similar to that previously reported, only 3.75% of RISs were common among early (up to 3 months) and late samples (beyond 1 year). This low percentage of overlap offers further evidence that the early phase of hematopoiesis after transplantation derives primarily from short-term repopulating cells. Additionally, we identified 14 common integration sites (CISs). Interestingly, common integration sites were enriched among late samples; 14.9% of early RISs were CISs vs. 36.8% late.A total of 3 RISs were found near or within known oncogenes, but 2 (Integrin alpha 9 [ITGA9] and ADP-ribosylation factor-like 11 [ARL11]) were limited to early time points. An integration site near the MDS1 gene was detected in a late follow-up sample by LAM-PCR. We confirmed the integration site near the MDS1 gene by PCR with integration site-specific primers amplifying the region between the 3'-LTR of the provirus and the MDS1 locus. The MDS1 integration was not detected in early, but became detectable at all time points from 6 months to 3 years post transplant from both lymphoid and myeloid populations. Q-PCR using an integration specific Taqman probe was utilized to assess the level of clonal contribution to hematopoiesis from the clone containing the MDS1 RIS. The overall contribution of the MDS1 integrated clone remained stable during followup. Given an overall gene marking level of 0.001-0.01% with an MDS1 marking level estimated at 0.00001% in the follow up samples, the frequency of the MDS1 integrated clone is predicted to be 1/1000 marked LT-HSCs. We infused an estimated 1324 transduced LT-HSCs based upon cell dose, transduction efficiency and an estimated LT-HSC frequency of 5 per 10e3 CD34+ cells. The single integration in MDS1 in the context of non-LT-HSC limited hematopoiesis may thus account for the stability observed over time.In summary, the pattern of contribution by genetically modified cells is distinct between the early and late phase post transplantation and emphasizes the importance of long-term studies to assess the risk of integrating vectors. Additionally, the enrichment for CISs in the late phase supports the concept that integrations in the LT-HSCs favors genes that may be involved in “stemness”. Furthermore, integrations in or near putative oncogenes are likely insufficient alone as a cause of oncogenesis. Finally, LT-HSC dose may be an important determinant of the risk of integrating vectors in the context of HSC gene transfer.

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.