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1
Kubo, Yoshinao, Hideki Hayashi, Toshifumi Matsuyama, Hironori Sato, and Naoki Yamamoto. "Retrovirus Entry by Endocytosis and Cathepsin Proteases." Advances in Virology 2012 (2012): 1–14. http://dx.doi.org/10.1155/2012/640894.
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Retroviruses include infectious agents inducing severe diseases in humans and animals. In addition, retroviruses are widely used as tools to transfer genes of interest to target cells. Understanding the entry mechanism of retroviruses contributes to developments of novel therapeutic approaches against retrovirus-induced diseases and efficient exploitation of retroviral vectors. Entry of enveloped viruses into host cell cytoplasm is achieved by fusion between the viral envelope and host cell membranes at either the cell surface or intracellular vesicles. Many animal retroviruses enter host cells through endosomes and require endosome acidification. Ecotropic murine leukemia virus entry requires cathepsin proteases activated by the endosome acidification. CD4-dependent human immunodeficiency virus (HIV) infection is thought to occur via endosomes, but endosome acidification is not necessary for the entry whereas entry of CD4-independent HIVs, which are thought to be prototypes of CD4-dependent viruses, is low pH dependent. There are several controversial results on the retroviral entry pathways. Because endocytosis and endosome acidification are complicatedly controlled by cellular mechanisms, the retrovirus entry pathways may be different in different cell lines.
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Andersen, Klaus Bahl. "Characterization of retrovirus-induced SC-1 cell fusion." Virus Research 37, no. 3 (August 1995): 177–98. http://dx.doi.org/10.1016/0168-1702(95)00032-l.
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Andersen, K. B., and H. Skov. "Retrovirus-induced Cell Fusion Is Enhanced by Protease Treatment." Journal of General Virology 70, no. 7 (July 1, 1989): 1921–27. http://dx.doi.org/10.1099/0022-1317-70-7-1921.
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Ellis, T. M., G. E. Wilcox, and W. F. Robinson. "Characteristics of cell fusion induced by a caprine retrovirus." Archives of Virology 86, no. 3-4 (September 1985): 263–73. http://dx.doi.org/10.1007/bf01309830.
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Rein, Alan. "Murine Leukemia Viruses: Objects and Organisms." Advances in Virology 2011 (2011): 1–14. http://dx.doi.org/10.1155/2011/403419.
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Murine leukemia viruses (MLVs) are among the simplest retroviruses. Prototypical gammaretroviruses encode only the three polyproteins that will be used in the assembly of progeny virus particles. These are the Gag polyprotein, which is the structural protein of a retrovirus particle, the Pol protein, comprising the three retroviral enzymes—protease, which catalyzes the maturation of the particle, reverse transcriptase, which copies the viral RNA into DNA upon infection of a new host cell, and integrase, which inserts the DNA into the chromosomal DNA of the host cell, and the Env polyprotein, which induces the fusion of the viral membrane with that of the new host cell, initiating infection. In general, a productive MLV infection has no obvious effect upon host cells. Although gammaretroviral structure and replication follow the same broad outlines as those of other retroviruses, we point out a number of significant differences between different retroviral genera.
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Zavorotinskaya, Tatiana, and Lorraine M. Albritton. "Suppression of a Fusion Defect by Second Site Mutations in the Ecotropic Murine Leukemia Virus Surface Protein." Journal of Virology 73, no. 6 (June 1, 1999): 5034–42. http://dx.doi.org/10.1128/jvi.73.6.5034-5042.1999.
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ABSTRACT Entry of ecotropic murine leukemia virus initiates when the envelope surface protein recognizes and binds to the virus receptor on host cells. The envelope transmembrane protein then mediates fusion of viral and host cell membranes and penetration into the cytoplasm. Using a genetic selection, we isolated an infectious retrovirus variant containing three changes in the surface protein—histidine 8 to arginine, glutamine 227 to arginine, and aspartate 243 to tyrosine. Single replacement of histidine 8 with arginine (H8R) resulted in almost complete loss of infectivity, even though the mutant envelope proteins were stable and efficiently incorporated into virions. Virions carrying H8R envelope were proficient at binding cells expressing receptor but failed to induce cell-cell fusion of XC cells, indicating that the histidine at position 8 plays an essential role in fusion during penetration of the host cell membrane. Thus, there is at least one domain in SU that is involved in fusion; the fusion functions do not reside exclusively in TM. In contrast, envelope with all three changes induced cell-cell fusion of XC cells and produced virions that were 10,000-fold more infectious than those containing only the H8R substitution, indicating that changes at positions 227 and 243 can suppress a fusion defect caused by loss of histidine 8 function. Moreover, the other two changes acted synergistically, indicating that both compensate for the loss of the same essential function of histidine 8. The ability of these changes to suppress this fusion defect might provide a means for overcoming postbinding defects found in targeted retroviral vectors for use in human gene therapy.
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Kinzler, Eric R., and Teresa Compton. "Characterization of Human Cytomegalovirus Glycoprotein-Induced Cell-Cell Fusion." Journal of Virology 79, no. 12 (June 15, 2005): 7827–37. http://dx.doi.org/10.1128/jvi.79.12.7827-7837.2005.
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ABSTRACT Human cytomegalovirus (CMV) infection is dependent on the functions of structural glycoproteins at multiple stages of the viral life cycle. These proteins mediate the initial attachment and fusion events that occur between the viral envelope and a host cell membrane, as well as virion-independent cell-cell spread of the infection. Here we have utilized a cell-based fusion assay to identify the fusogenic glycoproteins of CMV. To deliver the glycoprotein genes to various cell lines, we constructed recombinant retroviruses encoding gB, gH, gL, and gO. Cells expressing individual CMV glycoproteins did not form multinucleated syncytia. Conversely, cells expressing gH/gL showed pronounced syncytium formation, although expression of gH or gL alone had no effect. Anti-gH neutralizing antibodies prevented syncytium formation. Coexpression of gB and/or gO with gH/gL did not yield detectably increased numbers of syncytia. For verification, these results were recapitulated in several cell lines. Additionally, we found that fusion was cell line dependent, as nonimmortalized fibroblast strains did not fuse under any conditions. Thus, the CMV gH/gL complex has inherent fusogenic activity that can be measured in certain cell lines; however, fusion in fibroblast strains may involve a more complex mechanism involving additional viral and/or cellular factors.
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Tomasson, Michael H., Ifor R. Williams, Shaoguang Li, Jeffrey Kutok, Danielle Cain, Silke Gillessen, Glenn Dranoff, Richard A. Van Etten, and D. Gary Gilliland. "Induction of myeloproliferative disease in mice by tyrosine kinase fusion oncogenes does not require granulocyte-macrophage colony-stimulating factor or interleukin-3." Blood 97, no. 5 (March 1, 2001): 1435–41. http://dx.doi.org/10.1182/blood.v97.5.1435.
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Tyrosine kinase fusion oncogenes that occur as a result of chromosomal translocations have been shown to activate proliferative and antiapoptotic pathways in leukemic cells, but the importance of autocrine and paracrine expression of hematopoietic cytokines in leukemia pathogenesis is not understood. Evidence that leukemic transformation may be, at least in part, cytokine dependent includes data from primary human leukemia cells, cell culture experiments, and murine models of leukemia. This report demonstrates that interleukin (IL)-3 plasma levels are elevated in myeloproliferative disease (MPD) caused by the TEL/tyrosine kinase fusions TEL/platelet-derived growth factor beta receptor (PDGFβR), TEL/Janus kinase 2 (JAK2), and TEL/neurotrophin-3 receptor (TRKC). Plasma granulocyte-macrophage colony-stimulating factor (GM-CSF) levels were elevated by TEL/PDGFβR and TEL/JAK2. However, all of the fusions tested efficiently induced MPD in mice genetically deficient for both GM-CSF and IL-3, demonstrating that these cytokines are not necessary for the development of disease in this model system. Furthermore, in experiments using normal marrow transduced with TEL/PDGFβR retrovirus mixed with marrow transduced with an enhanced green fluorescent protein (EGFP) retrovirus, the MPD induced in these mice demonstrated minimal stimulation of normal myelopoiesis by the TEL/PDGFβR-expressing cells. In contrast, recipients of mixed GM-CSF–transduced and EGFP-transduced marrow exhibited significant paracrine expansion of EGFP-expressing cells. Collectively, these data demonstrate that, although cytokine levels are elevated in murine bone marrow transplant models of leukemia using tyrosine kinase fusion oncogenes, GM-CSF and IL-3 are not required for myeloproliferation by any of the oncogenes tested.
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Sugimoto, Jun, Sehee Choi, Megan A. Sheridan, Iemasa Koh, Yoshiki Kudo, and Danny J. Schust. "Could the Human Endogenous Retrovirus-Derived Syncytialization Inhibitor, Suppressyn, Limit Heterotypic Cell Fusion Events in the Decidua?" International Journal of Molecular Sciences 22, no. 19 (September 23, 2021): 10259. http://dx.doi.org/10.3390/ijms221910259.
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Proper placental development relies on tightly regulated trophoblast differentiation and interaction with maternal cells. Human endogenous retroviruses (HERVs) play an integral role in modulating cell fusion events in the trophoblast cells of the developing placenta. Syncytin-1 (ERVW-1) and its receptor, solute-linked carrier family A member 5 (SLC1A5/ASCT2), promote fusion of cytotrophoblast (CTB) cells to generate the multi-nucleated syncytiotrophoblast (STB) layer which is in direct contact with maternal blood. Another HERV-derived protein known as Suppressyn (ERVH48-1/SUPYN) is implicated in anti-fusogenic events as it shares the common receptor with ERVW-1. Here, we explore primary tissue and publicly available datasets to determine the distribution of ERVW-1, ERVH48-1 and SLC1A5 expression at the maternal-fetal interface. While SLC1A5 is broadly expressed in placental and decidual cell types, ERVW-1 and ERVH48-1 are confined to trophoblast cell types. ERVH48-1 displays higher expression levels in CTB and extravillous trophoblast, than in STB, while ERVW-1 is generally highest in STB. We have demonstrated through gene targeting studies that suppressyn has the ability to prevent ERVW-1-induced fusion events in co-culture models of trophoblast cell/maternal endometrial cell interactions. These findings suggest that differential HERV expression is vital to control fusion and anti-fusogenic events in the placenta and consequently, any imbalance or dysregulation in HERV expression may contribute to adverse pregnancy outcomes.
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Caudell, David, Rachel M. Pierce, David P. Harper, Rachel L. Novak, Christopher Slape, Linda Wolff, and Peter D. Aplan. "Identification of Genes That Collaborate with CALM-AF10 to Induce Leukemia by Retroviral Insertional Mutagenesis and Candidate Gene Sequencing." Blood 112, no. 11 (November 16, 2008): 3787. http://dx.doi.org/10.1182/blood.v112.11.3787.3787.
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Abstract The t(10;11) translocation results in a CALM-AF10 fusion gene in a subset of patients with ALL or AML. Expression of a CALM-AF10 fusion gene in transgenic mice is leukemogenic, however, the prolonged latency period, and incomplete penetrance suggests that additional events are necessary to complement CALM-AF10 mediated leukemic transformation. We used retroviral insertional mutagenesis (RIM) to identify complementary genetic events that might collaborate with CALM-AF10 during leukemic transformation. Newborn CALM-AF10 mice were infected with a modified replication competent retrovirus (Mol4070LTR); by 7 months of age, 90% of the transgenic mice developed acute leukemia. Acute leukemia developed more rapidly in CALM-AF10 infected mice than either wild-type mice infected with retrovirus (p=0.004) or CALM-AF10 mice not infected with retrovirus (p=0.037). Ligation-mediated PCR of DNA isolated from leukemic spleens identified potential collaborating genes near the retroviral insertion sites. 262 unique integrations were identified in 40 CALM-AF10 mice. Of these, 55 integrations occurred at 20 common insertion sites, including Zeb2, Mn1, Evi1, Nf1, Ift57, Mpl, Kras, Vav1, and Gata1. Of the candidate genes identified, Zeb2 and Nf1 are of particular interest. Zeb2 encodes the transcriptional co-repressor Smad-interacting protein 1, which is a member of the TGF beta signaling pathway. Previous studies using RIM identified Zeb2 insertions only sporadically, never more than once in a single study, and in association with B-cell, T-cell, and myeloid leukemia. In this study retroviral integrations near Zeb2 were identified either by PCR or Southern blot analysis in 26% (11/42) of the CALM-AF10 mice. RIM studies often generate oligoclonal leukemias, with two or more independent leukemias arising in the same mouse. Southern blot analysis demonstrated that the Zeb2 integration was present in the dominant leukemic clone in most of these mice, and the Zeb2 transcript was overexpressed compared to wild-type spleen and bone marrow. Most leukemias that arise in CALM-AF10 mice without retroviral insertions are myeloid (17/20). However, almost all (10/11) of the CALM-AF10 mice with Zeb2 insertions developed B-lineage ALL suggesting that expression of Zeb2 strongly influences the phenotype of CALM-AF10 leukemias. The tumor suppressor gene Nf1 (Neurofibromatosis type 1) functions as a GTPase activating protein that regulates the activity of Ras proteins involved in cellular proliferation, and acts as a tumor suppressor gene in immature myeloid cells. Retroviral integrations into the Nf1 locus were identified by PCR or Southern blot in 26% (11/42) of the mice in this study. In at least two cases, Southern blot showed loss of the germline allele. The high frequency of Nf1 integrations identified in the RIM assay suggested that Ras pathway activation complemented the CALM-AF10 transgene. Interestingly, 4 other retroviral integrations were identified near Ras genes; 2 near Kras (one with a Kras activating mutation), and 2 near Rras2. Given the high frequency of Nf1 or Ras insertions in the RIM study, we sequenced Nras and Kras genes from CALM-AF10 mice not infected with retrovirus. Thus far, we have identified 3 activating mutations in 15 mice, demonstrating that spontaneous Ras mutations develop as complementary events. We have shown that retroviral infection accelerates the onset of acute leukemia, and identified genes that potentially collaborate with the CALM-AF10 fusion gene in the leukemic transformation process. Furthermore, by sequencing candidate genes, we have demonstrated that pathways (ie, Ras) identified by RIM can also be activated by spontaneous mutations and potentially collaborate with CALM-AF10 to induce leukemia.
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Katsumoto, Takuo, and Issay Kitabayashi. "MOZ Is Critical for MOZ/MLL-Fusion-Induced HoxA9/Meis1 Expression and Leukemia Development,." Blood 118, no. 21 (November 18, 2011): 3467. http://dx.doi.org/10.1182/blood.v118.21.3467.3467.
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Abstract Abstract 3467 Monocytic leukemia Zinc finger (MOZ), a Myst-type histone acetyltransferase, is involved in chromosome translocations associated with FAB M4/M5 subtypes of acute myeloid leukemia (AML). We have shown that MOZ is critical for hematopoietic cell development and self-renewal of hematopoietic stem cells (HSCs). Recent reports suggest that hematopoietic and leukemic stem cells shares molecular basis for their self-renewal. To clarify the roles of endogenous MOZ in malignant hematopoiesis, we tested MOZ-deficient cells for leukemogenesis by leukemia-associated fusion genes such as MOZ-TIF2, MLL-AF10 and PML-RARa. Hematopoietic stem/progenitor cells (HSPCs), prepared with MOZ+/− or MOZ−/− fetal livers, were stimulated with SCF, IL-3, IL-6 and Osteopontin M for 1 day and infected with retrovirus encoding each fusion genes. Five days after infection, the infected cells were transplanted into recipient mice or cultured in methylcellulose medium with IL-3, SCF and GM-CSF. MOZ+/− HSPCs expressing MOZ-TIF2 or MLL-AF10 fusion gene formed colonies repeatedly and the recipient mice developed AML 2–3 month after transplantation. However, the MOZ−/− HSPCs expressing the MOZ/MLL-fusions showed neither colony-forming activity nor AML-inducing acyivity. On the other hand, when PML-RARa was introduced, both MOZ+/− and MOZ−/− cells showed continuous colony-forming activity. Expression of HoxA9 and Meis1 were high in MOZ+/− cells expressing MOZ/MLL-fusions, but they were very low in MOZ−/− cells expressing MOZ/MLL fusions. We have previously reported that expression of HoxA9 and Meis1 was significantly decreased in HSPCs of MOZ−/− fetal liver. When MOZ−/− HSPCs were stimulated in a condition without IL-6 prior to infection of MOZ-TIF2, the MOZ−/− cells acquired ability to generate colonies serially. In this condition, high HoxA9 expression was induced in the MOZ−/− cells. However, Meis1 expression was low and AML was not induced in recipient mice. These results indicate that endogenous MOZ plays critical roles in MOZ/MLL-fusion-induced AML and in HoxA9 and Meis1 gene expression. Disclosures: No relevant conflicts of interest to declare.
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Côté, Marceline, Yi-Min Zheng, Lorraine M. Albritton, and Shan-Lu Liu. "Fusogenicity of Jaagsiekte Sheep Retrovirus Envelope Protein Is Dependent on Low pH and Is Enhanced by Cytoplasmic Tail Truncations." Journal of Virology 82, no. 5 (December 19, 2007): 2543–54. http://dx.doi.org/10.1128/jvi.01852-07.
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ABSTRACT Jaagsiekte sheep retrovirus (JSRV) envelope (Env) is an active oncogene responsible for neoplastic transformation in animals and cultured cells. In this study, we used syncytium induction and fluorescence-based cell fusion assays to investigate JSRV Env fusion and its modulation by the cytoplasmic tail (CT). We found that JSRV Env induced syncytia in cells overexpressing the receptor for JSRV and that a low pH was required for this process to occur. Fusion kinetics studies revealed that cell-cell fusion by JSRV Env at neutral pH was poor, taking up to a day, in sharp contrast to fusion at low pH, which peaked within 2 min following a low-pH trigger. Deletion of the C-terminal 7 or 16 amino acids of the JSRV Env CT had no or little effect on fusion, yet additional truncation toward the membrane-spanning domain, resulting in mutants retaining as little as 1 amino acid of the CT, led to progressively increased syncytium formation at neutral pH that was further enhanced by low-pH treatment. Notably, the severely truncated mutants showed elevated levels of surface subunits in culture medium, suggesting that the CT truncations resulted in conformational changes in the ectodomain of Env that impaired surface subunit associations. Taken together, this study reveals for the first time that the fusion activity of the JSRV Env protein is dependent on a low pH and is modulated by the CT, whose truncation overcomes, at least partially, the low-pH requirement for fusion and enhances Env fusion activity and kinetics.
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Earp, Laurie J., Sue E. Delos, Robert C. Netter, Paul Bates, and Judith M. White. "The Avian Retrovirus Avian Sarcoma/Leukosis Virus Subtype A Reaches the Lipid Mixing Stage of Fusion at Neutral pH." Journal of Virology 77, no. 5 (March 1, 2003): 3058–66. http://dx.doi.org/10.1128/jvi.77.5.3058-3066.2003.
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ABSTRACT We previously showed that the envelope glycoprotein (EnvA) of avian sarcoma/leukosis virus subtype A (ASLV-A) binds to liposomes at neutral pH following incubation with its receptor, Tva, at ≥22°C. We also provided evidence that ASLV-C fuses with cells at neutral pH. These findings suggested that receptor binding at neutral pH and ≥22°C is sufficient to activate Env for fusion. A recent study suggested that two steps are necessary to activate avian retroviral Envs: receptor binding at neutral pH, followed by exposure to low pH (W. Mothes et al., Cell 103:679-689, 2000). Therefore, we evaluated the requirements for intact ASLV-A particles to bind to target bilayers and fuse with cells. We found that ASLV-A particles bind stably to liposomes in a receptor- and temperature-dependent manner at neutral pH. Using ASLV-A particles biosynthetically labeled with pyrene, we found that ASLV-A mixes its lipid envelope with cells within 5 to 10 min at 37°C. Lipid mixing was neither inhibited nor enhanced by incubation at low pH. Lipid mixing of ASLV-A was inhibited by a peptide designed to prevent six-helix bundle formation in EnvA; the same peptide inhibits virus infection and EnvA-mediated cell-cell fusion (at both neutral and low pHs). Bafilomycin and dominant-negative dynamin inhibited lipid mixing of Sindbis virus (which requires low pH for fusion), but not of ASLV-A, with host cells. Finally, we found that, although EnvA-induced cell-cell fusion is enhanced at low pH, a mutant EnvA that is severely compromised in its ability to support infection still induced massive syncytia at low pH. Our results indicate that receptor binding at neutral pH is sufficient to activate EnvA, such that ASLV-A particles bind hydrophobically to and merge their membranes with target cells. Possible roles for low pH at subsequent stages of viral entry are discussed.
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Green, Kathy, Li Wang, Randolph Noelle, and William Green. "MDSC suppression of B cell responses in murine retrovirus-induced immunodeficiency: a role for VISTA (IRC4P.603)." Journal of Immunology 194, no. 1_Supplement (May 1, 2015): 57.20. http://dx.doi.org/10.4049/jimmunol.194.supp.57.20.
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Abstract Inhibition by myeloid derived suppressor cells (MDSC) of T cell responses is well established in tumor microenvironments. We demonstrated (Green et al., 2013) induction of monocytic MDSCs during infection of B6 mice by LP-BM5 retrovirus, which causes profound immunodeficiency. These MDSCs suppressed not only T, but also B cell, responsiveness ex vivo. Whereas MDSC inhibition of stimulated T cell proliferation and IFN-gamma production was ~100%, iNOS/NO dependent, MDSC suppression of B-cell responses was only ~50% dependent on iNOS/NO - as shown by using iNOS inhibitors and iNOS k.o. mice as a source of MDSCs. We then discovered an additional mechanism(s) in MDSC inhibition of (only) B cell responsiveness that involved VISTA, a new negative checkpoint regulator. Using anti-VISTA blocking mAb, or LP-BM5 infected VISTA-/- MDSCs, MDSC inhibition of B cell responses was dependent on MDSC-expressed VISTA — again accounting for ~50% of total MDSC suppression. Combining the use of reagents to block both iNOS/NO and VISTA lead to an additive, if not synergistic, abrogation of MDSC suppression of B cell responsiveness. Consistent with a direct role of VISTA, in the absence of MDSCs, a VISTA-Ig fusion protein also partially inhibited B cell responsiveness. These results were compatible with a role for MDSC in LP-BM5-induced immunodeficiency and highlight involvement of multiple and unique suppressive pathways in the under-studied area of MDSC suppression of B cell responses.
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Côté, Marceline, Yi-Min Zheng, and Shan-Lu Liu. "Receptor Binding and Low pH Coactivate Oncogenic Retrovirus Envelope-Mediated Fusion." Journal of Virology 83, no. 22 (September 2, 2009): 11447–55. http://dx.doi.org/10.1128/jvi.00748-09.
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ABSTRACT Fusion of enveloped viruses with host cells is triggered by either receptor binding or low pH but rarely requires both except for avian sarcoma leukosis virus (ASLV). We recently reported that membrane fusion mediated by an oncogenic Jaagsiekte sheep retrovirus (JSRV) envelope (Env) requires an acidic pH, yet receptor overexpression is required for this process to occur. Here we show that a soluble form of the JSRV receptor, sHyal2, promoted JSRV Env-mediated fusion at a low pH in normally fusion-negative cells and that this effect was blocked by a synthetic peptide analogous to the C-terminal heptad repeat of JSRV Env. In contrast to the receptor of ASLV, sHyal2 induced pronounced shedding of the JSRV surface subunit, as well as unstable conformational rearrangement of its transmembrane (TM) subunit, yet full activation of JSRV Env fusogenicity, associated with strong TM oligomerization, required both sHyal2 and low pH. Consistently, sHyal2 enabled transduction of nonpermissive cells by JSRV Env pseudovirions, with low efficiency, but substantially blocked viral entry into permissive cells at both binding and postbinding steps, indicating that sHyal2 prematurely activates JSRV Env-mediated fusion. Altogether, our study supports a model that receptor priming promotes fusion activation of JSRV Env at a low pH, and that the underlying mechanism is likely to be different from that of ASLV. Thus, JSRV may provide a useful alternate model for the better understanding of virus fusion and cell entry.
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Talarico, D., M. M. Ittmann, R. Bronson, and C. Basilico. "A retrovirus carrying the K-fgf oncogene induces diffuse meningeal tumors and soft-tissue fibrosarcomas." Molecular and Cellular Biology 13, no. 4 (April 1993): 1998–2010. http://dx.doi.org/10.1128/mcb.13.4.1998-2010.1993.
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The K-fgf/hst oncogene encodes a growth factor of the fibroblast growth factor (FGF) family and transforms cells through an autocrine mechanism which requires extracellular activation of its receptor(s). To identify the cell and tissue targets of K-fgf oncogenic potential in vivo, we constructed a recombinant retrovirus carrying the human K-fgf cDNA and injected it, together with helper Moloney murine leukemia virus, into immunocompetent as well as nude mice. The original construct was highly transforming in tissue culture but produced no detectable pathologies in vivo with the exception of a single fibrosarcoma which arose after a long latency. The virus produced by this tumor appears to have undergone a complex series of recombination events involving the helper Moloney murine leukemia virus. It encodes an Env/K-FGF fusion protein whose expression is under the control of a hybrid long terminal repeat. This virus (designated MFS, for meningeal fibrosarcoma) induces tumors in mice with high frequency and short latency. These neoplasms consist of aggressive fibrosarcomas of soft tissue as well as diffuse meningeal tumors originating from the dura mater that surround the whole central nervous system and cause severe hydrocephalus. The Env/K-FGF fusion protein expressed by the MFS virus has retained all of the biological properties of native K-FGF, including secretion, mitogenic activity, heparin binding, and neutralization by anti-K-FGF antibodies. These and other results indicate that the tumors induced by the MFS virus result from the oncogenic potential of K-FGF.
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Goodings, Charnise, and Utpal P. Dave. "Enforced E47 Expression Has Differential Effects on Lmo2-Induced T-Cell Leukemia." Blood 118, no. 21 (November 18, 2011): 4637. http://dx.doi.org/10.1182/blood.v118.21.4637.4637.
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Abstract Abstract 4637 Lmo2 is one of the most commonly deregulated oncogenes in human T-cell acute lymphoblastic leukemia (T-ALL). In mouse models, Lmo2 overexpression causes a differentiation block before the onset of T-ALL at a developmental stage that is similar to the block seen in E47 knockout mice. Furthermore, Lmo2 and E47 are part of an oligomeric protein complex in hematopoietic stem and progenitor cells. Since E47 knockout mice also develop T-ALL, it has been hypothesized that Lmo2 may induce T-ALL by redirecting E47 activity away from its normal target genes. We noted downregulation of many E2A targets in Lmo2-induced T-ALL. To directly test whether E47 is required in Lmo2-induced T-ALLs, we transduced four stable T-ALL lines established from Lmo2 transgenic mice with retrovirus expressing E47 fused with estrogen receptor. All 4 lines tolerated stable high- level protein expression of E47-ER with no change in from their baseline growth rates. The E47-ER fusion protein allowed forced dimerization upon treatment with 4-hydroxytamoxifen. Tamoxifen treatment increased expression CD4 and other described E2A targets in all 4 T-ALL lines; but two lines underwent G0/G1 cell cycle arrest. Our data suggest that E47 deficiency is not a universal feature of Lmo2- induced T-ALL and E47 forced expression has differential effects on T-ALL. Disclosures: No relevant conflicts of interest to declare.
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Zavorotinskaya, Tatiana, Zhaohui Qian, John Franks, and Lorraine M. Albritton. "A Point Mutation in the Binding Subunit of a Retroviral Envelope Protein Arrests Virus Entry at Hemifusion." Journal of Virology 78, no. 1 (January 1, 2004): 473–81. http://dx.doi.org/10.1128/jvi.78.1.473-481.2004.
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ABSTRACT The transmembrane subunits of viral envelope proteins are thought to perform all of the functions required for membrane fusion during entry of enveloped viruses. However, changes in a conserved SPHQ motif near the N terminus of the receptor binding subunit of a murine leukemia virus (MLV) envelope protein block infection and induction of cell-cell fusion but not receptor binding. Here we report evidence that a histidine-to-arginine change at position 8 (H8R) in the SPHQ motif of Moloney MLV blocks infection by arresting virus-cell fusion at the hemifusion state. In cell-cell fusion assays, H8R envelope protein induced mixing of membrane outer leaflet lipids but did not lead to content mixing, a finding indicative of fusion pore formation. Kinetic studies of virus-cell fusion showed that lipid mixing of H8R virus membranes begins much later than for wild-type virus. The length of the delay in lipid mixing decreased upon addition of two second-site changes that increase H8R virus infection to 100-fold less than the wild-type virus. Finally, chlorpromazine, dibucaine, and trifluoperazine, agents that induce pores in an arrested hemifusion state, rescued infection by H8R virus to within 2.5-fold of the level of wild-type virus infection and cell-cell fusion to half that mediated by wild-type envelope protein. We interpret these results to indicate that fusion progressed to the hemifusion intermediate but fusion pore formation was inhibited. These results establish that membrane fusion of Moloney MLV occurs via a hemifusion intermediate. We also interpret these findings as evidence that histidine 8 is a key switch-point residue between the receptor-induced conformation changes that expose fusion peptide and those that lead to six-helix bundle formation.
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Gavrilescu, L. Cristina, Nicholas C. P. Cross, and Richard A. Van Etten. "Distinct Leukemogenic Activity and Imatinib Responsiveness of a BCR-PFGFRα Fusion Tyrosine Kinase." Blood 108, no. 11 (November 16, 2006): 3634. http://dx.doi.org/10.1182/blood.v108.11.3634.3634.
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Abstract Chromosomal rearrangements of the platelet-derived growth factor receptor alpha (PDGFRA) gene on 4q12 have been described in a subset of patients with idiopathic hypereosinophilic syndrome/chronic eosinophilic leukemia (IHES/CEL). The most common, found in 12–17% of CEL patients, is the FIP1L1-PDGFRA fusion, resulting from a cytogenetically invisible deletion on 4q12 (Cools et al., NEJM2003; 348:1201; Griffin et al., PNAS2003; 100:7830). FIP1L1-PDGFRA+ CEL patients have an excellent response to low-dose imatinib therapy, often accompanied by complete molecular remission (Pardanani et al., Leuk. Res.2006; 30:965). Additional fusion partners of PDGFRA include KIF5B and CDK5RAP2 in CEL, and BCR in atypical chronic myeloid leukemia with eosinophilia and t(4;22)(q12;q11) (Baxter et al., Hum. Mol. Genet.2002; 11:1391). However, the oncogenic activity and imatinib responsiveness of these other activated PDGFRA alleles are unknown. In this study, we assessed the imatinib sensitivity of BCR-PDGFRα and FIP1L1-PDGFRα in hematopoietic cell lines, and compared their leukemogenic activity in a mouse retroviral bone marrow transduction/transplantation model. Like FIP1L1-PDGFRα, BCR-PDGFRα transformed Ba/F3 cells to become independent of IL-3 for survival and growth. In the absence of IL-3, FIP1L1-PDGFRα-expressing Ba/F3 cells were 30-fold more sensitive to imatinib than BCR-PDGFRα (IC50 = 3 nM vs. 90 nM) for inhibition of proliferation and induction of apoptosis. A FIP1L1-PDGFRα N659D mutant (Cools et al., Cancer Cell2003; 3:459) was relatively resistant to imatinib (IC50 = 200 nM), while the corresponding BCR-PDGFRα N659D mutant displayed increased imatinib resistance (IC50 > 2 μM). Inhibition of cellular proliferation correlated with decreased tyrosyl phosphorylation of the respective fusion kinase and of the downstream substrate PLCγ1. In leukemogenesis experiments, recipients of bone marrow transduced with retrovirus expressing either FIP1L1-PDGFRα or BCR-PDGFRα developed fatal myeloproliferative disease, with similar median and overall survival. This was accompanied by significant eosinophilia, relative to the disease induced by BCR-ABL in this model system (Li et al., Blood2001; 97:1442). Treatment of recipients of FIP1L1-PDGFRA- or BCR-PDGFRA-transduced marrow with imatinib (125 mg/kg/day by oral gavage for 20 days) suppressed leukocytosis and prolonged their survival. These results suggest that distinct signaling pathways activated by leukemogenic PDGFRα fusions in hematopoietic progenitors induce eosinophila in vivo. However, different fusion partners of PDGFRα can significantly influence the sensitivity of the fusion kinase to imatinib treatment.
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Hernandez, Lorraine D., Reuben J. Peters, Sue E. Delos, John A. T. Young, David A. Agard, and Judith M. White. "Activation of a Retroviral Membrane Fusion Protein: Soluble Receptor-induced Liposome Binding of the ALSV Envelope Glycoprotein." Journal of Cell Biology 139, no. 6 (December 15, 1997): 1455–64. http://dx.doi.org/10.1083/jcb.139.6.1455.
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It is not known how membrane fusion proteins that function at neutral pH, for example the human immunodeficiency virus envelope (Env) glycoprotein and intracellular fusion machines, are activated for target bilayer binding. We have addressed this question using a soluble oligomeric form of an avian retroviral Env glycoprotein (API) and soluble forms of its receptor. Binding of soluble receptor to API induces API to bind to liposomes composed of phosphatidylcholine and cholesterol at neutral pH. Liposome binding only occurs at fusion permissive temperatures (T > 20°C), is complete between 2 to 5 min at 37°C, and is stable to high salt, carbonate, and urea. Liposome binding is mediated by the ectodomain of the transmembrane subunit of API, and a mutant with a Val to Glu substitution in the Env fusion peptide (located in the ectodomain of the transmembrane subunit) shows significantly reduced liposome binding. Moreover, under conditions of equivalent binding to API, a mutant receptor that does not support infection (Zingler, K., and J.A.T. Young. 1996. J. Virol. 70:7510–7516) does not induce significant liposome binding. Our results indicate that a highly specific interaction between an avian retroviral Env and its receptor activates the retroviral glycoprotein for target bilayer binding at neutral pH in much the same way as low pH activates the influenza hemagglutinin. Our findings are discussed in terms of the mechanisms of viral and cellular fusion proteins that function at neutral pH.
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Hein, Sibyll, Vladimir Prassolov, Yuanming Zhang, Dmitry Ivanov, Jürgen Löhler, Susan R. Ross, and Carol Stocking. "Sodium-Dependent myo-Inositol Transporter 1 Is a Cellular Receptor for Mus cervicolor M813 Murine Leukemia Virus." Journal of Virology 77, no. 10 (May 15, 2003): 5926–32. http://dx.doi.org/10.1128/jvi.77.10.5926-5932.2003.
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ABSTRACT Retrovirus infection is initiated by binding of the surface (SU) portion of the viral envelope glycoprotein (Env) to specific receptors on cells. This binding triggers conformational changes in the transmembrane portion of Env, leading to membrane fusion and cell entry, and is thus a major determinant of retrovirus tissue and species tropism. The M813 murine leukemia virus (MuLV) is a highly fusogenic gammaretrovirus, isolated from Mus cervicolor, whose host range is limited to mouse cells. To delineate the molecular mechanisms of its restricted host range and its high fusogenic potential, we initiated studies to characterize the cell surface protein that mediates M813 infection. Screening of the T31 mouse-hamster radiation hybrid panel for M813 infectivity localized the receptor gene to the distal end of mouse chromosome 16. Expression of one of the likely candidate genes (slc5a3) within this region in human cells conferred susceptibility to both M813 infection and M813-induced fusogenicity. slc5a3 encodes sodium myo-inositol transporter 1 (SMIT1), thus adding another sodium-dependent transporter to the growing list of proteins used by MuLVs for cell entry. Characterization of SMIT1 orthologues in different species identified several amino acid variations within two extracellular loops that may restrict susceptibility to M813 infection.
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Lu, Chi-Wei, and Monica J. Roth. "Role of the Mutation Q252R in Activating Membrane Fusion in the Murine Leukemia Virus Surface Envelope Protein." Journal of Virology 77, no. 20 (October 15, 2003): 10841–49. http://dx.doi.org/10.1128/jvi.77.20.10841-10849.2003.
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ABSTRACT Entry of retroviruses into host cells requires the fusion between the viral and cellular membranes. It is unclear how receptor binding induces conformational changes within the surface envelope protein (SU) that activate the fusion machinery residing in the transmembrane envelope protein (TM). In this report, we have isolated a point mutation, Q252R, within the proline-rich region of the 4070A murine leukemia virus SU that altered the virus-cell binding characteristics and induced cell-cell fusion. Q252R displays a SU shedding-sensitive phenotype. Cell-cell fusion is receptor dependent and is observed only in the presence of MuLV Gag-Pol. Both cellular binding and fusion by Q252R are greatly enhanced in conjunction of G100R, a mutation within the SU variable region A which increases viral binding through an independent mechanism. Deletion of a conserved histidine (His36) at the SU N terminus abolished cell-cell fusion by G100R/Q252R Env without compromising virus-cell binding. Although G100R/Q252R virus has no detectable titer, replacement of the N-terminal nine 4070A SU amino acids with the equivalent ecotropic MuLV sequence restored viral infectivity. These studies provide insights into the functional cooperation between multiple elements of SU required to signal receptor binding and activate the fusion machinery.
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Gurevich, Rhonna M., Peter D. Aplan, and R. Keith Humphries. "Generation of a Pre-Leukemic, Transplantable Cell Line from the AML-Associated NUP98-TOP1 Fusion Gene as a New Model To Test Potential Collaborating Genes." Blood 104, no. 11 (November 16, 2004): 1969. http://dx.doi.org/10.1182/blood.v104.11.1969.1969.
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Abstract Over 15 distinct chromosomal translocations have been documented in human leukemias that involve rearrangement of the nucleoporin gene, NUP98. While most NUP98 fusion partners are HOX transcription factors, the remaining 7 have no obvious common unifying function or unique role in hematopoiesis. The translocation t(11;20) identified the novel NUP98 fusion partner Topoisomerase I (TOP1), a catalytic enzyme known for its role in relaxing supercoiled DNA. We recently demonstrated that over-expression of NUP98-TOP1 induces a lethal, transplantable AML in a murine bone marrow (BM) transplantation model (Gurevich et al, Blood 2004). To further explore the mechanistic process of leukemic transformation we sought to establish BM cell lines that retain the preleukemic-inducing properties of NUP98-TOP1. Murine BM was transduced with GFP-linked NUP98-TOP1 retrovirus. Following 4 weeks of culture, there was a striking out-growth of GFP+ cells (expansion of 1% to >80% GFP+ cells) and generation of poly-clonal cell lines (herein called NT 12.1 and NT 12.2) exhibiting features of primitive cells (blast-like morphology, lin−/lo, increased levels of c-Kit, Sca-1 and CD34). NT cell lines had limited differentiation ability when plated in GM-CSF, G-CSF or M-CSF but in methylcellulose colony assays generated large granulo-monocytic colonies at high frequency (1 in 12) when plated in IL-3, IL-6, SCF and EPO. Strikingly, NT cell lines demonstrated significant levels of short-term in vivo repopulating ability when transplanted into lethally irradiated recipients. Transplant doses of 104-106 cells yielded >50% engraftment at 1 month post transplant (tx) (54±13% GFP+ WBC; n=12) which diminished to levels of <20% by 2 months. The frequency of cells with repopulating potential was estimated at 1 in 1609 following limit dilution assay which yielded repopulation of 3 of 4 recipients 1 month post-tx (range; 5–46%). While the level of engraftment diminished in NT 12.1 mice, two NT 12.2 mice developed fatal MPD/AML after a latency of 103 and 166 days. Thus these NUP98-TOP1 cell lines demonstrate a differentiation block in vitro, high level short term repopulating capacity and the potential to give rise to AML with a long latency that is consistent with a preleukemic phenotype. These lines now provide a novel platform to test candidate genes for their ability to co-operate with NUP98-TOP1 to induce leukemia. As an initial candidate, we tested the collaborating potential of Meis1, a HOX co-factor known to co-operate with several NUP98-HOX fusions to induce a rapid leukemia and may thus represent a common collaborating gene in NUP98 associated leukemias. When a representative cell line (NT12.2) was infected with a YFP-linked Meis1 retrovirus and transplanted into mice, the mice contained only GFP+/YFP+ circulating WBC by 1 months post-tx. However, despite co-expression of both genes in all transplanted mice, there was no apparent acceleration of induction of AML with 5 of 6 recipients succumbing to leukemia by 9 months post-tx, (median survival 164 days; range 101–193). This long latency argues that Meis1 does not collaborate with NUP98-TOP1 to induce leukemia and that NUP98-TOP1 has distinct mechanisms of leukemogenesis compared to NUP98-HOX fusions. These novel pre-leukemic, transplantable cell lines generated by NUP98-TOP1 will facilitate further large scale screens for potential cooperative genes and delineation of the mechanistic basis of NUP98-TOP1 induced transformation.
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Muroi, Yoshikage, Toshihiro Sakurai, Akira Hanashi, Kentaro Kubota, Kentaro Nagaoka, and Kazuhiko Imakawa. "CD9 regulates transcription factor GCM1 and ERVWE1 expression through the cAMP/protein kinase A signaling pathway." REPRODUCTION 138, no. 6 (December 2009): 945–51. http://dx.doi.org/10.1530/rep-09-0082.
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ERVWE1 (SYNCYTIN-1), a membrane protein originating from the envelope gene of human endogenous retrovirus-W (HERV-W), mediates the fusion of mononucleated cytotrophoblasts into multinucleated syncytiotrophoblast. Though ERVWE1 has been characterized since its discovery, regulatory mechanisms associated with ERVWE1 expression have not been firmly established. We hypothesized that membrane protein CD9, involved in cell–cell fusion of fertilization and myogenesis, could be involved in the regulation ofERVWE1gene expression. In this study, regulatory mechanisms of ERVWE1 expression were studied using human choriocarcinoma BeWo cells. Forskolin is an activator of adenylate cyclase, which increased CD9 and ERVWE1 expression. The increase in CD9 expression was inhibited by a protein kinase A (PKA) inhibitor, Rp-cAMPS. These results indicate that CD9 expression is regulated by the cAMP/PKA signaling pathway. Overexpression ofCD9increased expression levels of ERVWE1 as well as GCM1 (hGCMa), which is a transcription factor known to activateERVWE1gene transcription. However, high ERVWE1 expression induced byCD9overexpression did not result in the increase in chorionic gonadotropin, beta polypeptide production. Moreover,CD9-induced increase inERVWE1andGCM1expressions were inhibited by Rp-cAMPS. These results suggest that CD9 increases GCM1 expression via the cAMP/PKA signaling pathway, resulting in the increase in ERVWE1 expression.
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Zhang, Qi-Yao, Ping Liu, Mengmeng Pan, and Sai-Juan Chen. "Human NUP98/Iqcg Fusion Gene Induced An Acute Myelomonocytic Leukemia In Mice." Blood 122, no. 21 (November 15, 2013): 3725. http://dx.doi.org/10.1182/blood.v122.21.3725.3725.
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Abstract NUP98 gene encoding a nucleoporin located at 11p15 has been reported to be fused with about 30 different partner genes by chromosomal translocations in hematological malignances. NUP98/IQCG was cloned in a patient of Myeloid/ T-lymphoid bi-phenotypic leukemia with 47, XX, t(3;11)(q29q13;p15)der(3)(q29), +21 karyotype. However, NUP98/IQCG’s ability for leukemogenesis has not been identified yet.In this study, we established a retrovirus-mediated murine bone marrow transduction and transplantation (BMT) model of NUP98/IQCG to investigate its oncogenicity. In our model, half of the NUP98/IQCG mice developed a penetrable and transplantable acute myelomonocytic leukemia ,which was similar to the phenotype of patient with t(3;11). It suggested that NUP98/IQCG could induce the disease development. To investigate how the fusion gene promoted leukemogenesis, we transduced NUP98/IQCG into primary bone marrow cells, and found that NUP98/IQCG-expressing cells retained the ability to generate colonies in serial replating, while seldom control cells did, which indicated the increasing self-renewal ability caused by NUP98/IQCG. Meanwhile, when induced by Macrophage-Colony Stimulating Factor, NUP98/IQCG -expression bone marrow cells showed enhanced proliferation in vitro. Further molecular mechanism studies revealed that NUP98/IQCG could be involved in both NFκB and CREB pathways during leukemia development. In summary, we showed that NUP98/IQCG promoted leukemogenesis in BMT mouse model through increasing the bone marrow cells’ proliferation and self-renewal capacity, which explained the fusion gene’s oncogenicity in patient with t(3;11). Our mouse model will be a powerful tool both to investigate the leukemogenic mechanism of NUP98-related fusion gene, and to find the drugs for treating the disease. Disclosures: No relevant conflicts of interest to declare.
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Yang, Zhongfa, Cong Peng, Yaoyu Chen, Junling Wang, Xuejun Zhu, Karen Drumea, Shaoguang Li, and Alan G. Rosmarin. "Without GABP Transcription Factor, BCR-ABL Cannot Transform HSCs to Leukemic Stem Cells Nor Induce Chronic Myelogenous Leukemia in Mice." Blood 118, no. 21 (November 18, 2011): 965. http://dx.doi.org/10.1182/blood.v118.21.965.965.
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Abstract Abstract 965 Chronic Myelogenous Leukemia (CML) is driven by the fusion oncogene, BCR-ABL, which transforms normal hematopoietic stem cells (HSCs) to leukemic stem cells (LSCs). Tyrosine kinase inhibitors, such as imatinib mesylate, control the massive expansion of leukemic cells in most patients with CML, but cannot eradicate CML LSCs. Several genetic pathways have been shown to be critical for the growth and survival of CML LSCs, including signaling molecules, tumor suppressors, and metabolic regulators. However, the role of transcription factors in functional regulation of LSCs in CML has not been widely studied. GA Binding Protein (GABP) is an ets transcription factor that is required for entry of fibroblasts into the cell cycle, and expression of Gabpa (the DNA-binding component of the complex), alone, was sufficient to induce quiescent, serum-starved cells to enter the cell cycle. Thus, Gabp is both necessary and sufficient for cell cycle entry. Conditional deletion of Gabpa in mouse bone marrow decreased hematopoietic progenitor cells more than 100-fold, but hematopoietic stem cells (HSCs) were relatively preserved. Gabpα null HSCs exhibited significant cell cycle arrest. We sought to determine if the cell cycle arrest caused by Gabpa loss could impair development of CML cells in a mouse model. We used retroviral infection of bone marrow from 5-FU-treated mice (to enrich for stem and progenitor cells) to generate a rapidly fatal CML-like syndrome in mice. Bone marrow from mice with loxP-flanked (floxed) Gabpa and wild type control mice was infected with a retrovirus that co-expresses BCR-ABL, Cre recombinase, and green fluorescent protein (GFP). As expected, transplantation into recipient mice of control mouse bone marrow infected with BCR-ABL-Cre-GFP retrovirus caused a rapidly fatal myeloproliferative neoplasm, with a median survival of approximately three weeks; mice died with massive infiltration of GFP+ myeloid cells in peripheral blood cell, spleen, bone marrow, and other organs. In floxed Gabpa bone marrow, the retrovirus deleted floxed Gabpa in cells that express the BCR-ABL fusion oncogene, and these cells were identifiable based on GFP expression. Transplantation of floxed Gabpa bone marrow infected with BCR-ABL-Cre-GFP retrovirus failed to induce CML during six months of observation. Importantly, GFP+ peripheral blood granulocytes were observed for at least 6 months after transplantation; these CD11b+, Gr1+ cells continued to express BCR-ABL and were shown to be Gabpa null. These results indicate that the lack of Gabpa severely impaired the function of LSCs. In addition, secondary transplantation of bone marrow from these mice again demonstrated the presence of BCR-ABL-expressing peripheral blood myeloid cells. We conclude that Gabp transcription factor is required for the transformation of HSCs to LSCs by BCR-ABL. Furthermore, the persistence of BCR-ABL-expressing myeloid cells without the development of leukemia provides a unique model that permits analysis of the biological properties of BCR-ABL in vivo. The continued generation of BCR-ABL-expressing cells without CML development is unprecedented, and represents a unique model of leukemia tumor suppression. Disclosures: No relevant conflicts of interest to declare.
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Iwasaki, Masayuki, Takeshi Kuwata, Yukari Yamazaki, Nancy A. Jenkins, Neal G. Copeland, Motomi Osato, Yoshiaki Ito, Evert Kroon, Guy Sauvageau, and Takuro Nakamura. "Identification of cooperative genes for NUP98-HOXA9 in myeloid leukemogenesis using a mouse model." Blood 105, no. 2 (January 15, 2005): 784–93. http://dx.doi.org/10.1182/blood-2004-04-1508.
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Abstract The chromosomal translocation t(7; 11)(p15;p15), observed in human myeloid leukemia, results in a NUP98 and HOXA9 gene fusion. We generated a transgenic mouse line that specifically expressed the chimeric NUP98-HOXA9 gene in the myeloid lineage. While only 20% of the transgenic mice progressed to leukemia after a latency period, myeloid progenitor cells from nonleukemic transgenic mice still exhibited increased proliferative potential. This suggested that the NUP98-HOXA9 fusion induced a preleukemic phase, and other factors were required for complete leukemogenesis. NUP98-HOXA9 expression promoted the onset of retrovirus-induced BXH2 myeloid leukemia. This phenomenon was used to identify cooperative disease genes as common integration sites (CISs). Meis1, a known HOX cofactor, was identified as a CIS with a higher integration frequency in transgenic than in wild-type BXH2 mice. By the same means we identified further 4 candidate cooperative genes, Dnalc4, Fcgr2b, Fcrl, and Con1. These genes cooperated with NUP98-HOXA9 in transforming NIH 3T3 cells. The system described here is a powerful tool to identify cooperative oncogenes and will assist in the clarification of the multistep process of carcinogenesis.
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Crescenzi, M., D. H. Crouch, and F. Tatò. "Transformation by myc prevents fusion but not biochemical differentiation of C2C12 myoblasts: mechanisms of phenotypic correction in mixed culture with normal cells." Journal of Cell Biology 125, no. 5 (June 1, 1994): 1137–45. http://dx.doi.org/10.1083/jcb.125.5.1137.
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To study the effects of myc oncogene on muscle differentiation, we infected the murine skeletal muscle cell line C2C12 with retroviral vectors encoding various forms of avian c- or v-myc oncogene. myc expression induced cell transformation but, unlike many other oncogenes, prevented neither biochemical differentiation, nor commitment (irreversible withdrawal from the cell cycle). Yet, myotube formation by fusion of differentiated cells was strongly inhibited. Comparison of uninfected C2C12 myotubes with differentiated myc-expressing C2C12 did not reveal consistent differences in the expression of several muscle regulatory or structural genes. The present results lead us to conclude that transformation by myc is compatible with differentiation in C2C12 cells. myc expression induced cell death under growth restricting conditions. Differentiated cells escaped cell death despite continuing expression of myc, suggesting that the muscle differentiation programme interferes with the mechanism of myc-induced cell death. Cocultivation of v-myc-transformed C2C12 cells with normal fibroblasts or myoblasts restored fusion competence and revealed two distinguishable mechanisms that lead to correction of the fusion defect.
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Daenke, S., and S. Booth. "HTLV-1-induced cell fusion is limited at two distinct steps in the fusion pathway after receptor binding." Journal of Cell Science 113, no. 1 (January 1, 2000): 37–44. http://dx.doi.org/10.1242/jcs.113.1.37.
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Human T-cell leukemia virus type 1 (HTLV-1) is notable among retroviruses for its poor ability to infect permissive cells, particularly as cell free virus. The virus is most efficiently transmitted between individuals by infected cells, where it is presumed that intracellular particles and viral RNA are transferred to target cells following fusion. Although the mandatory first step for HTLV-1 fusion is the binding of envelope SU (gp46) to the receptor, the events which follow this interaction and lead to fusion and infection have not been well characterized. To investigate these events, we studied two HTLV-1 chronically infected cell lines with different abilities to fuse with K562 target cells. Although not inherently fusion incompetent, the HTLV-1 envelope protein on MT2 cells was poorly able to undergo a change in membrane hydrophobicity required for fusion with the target cell membrane after binding to the receptor. High level expression of a fusion-competent HTLV-1 envelope protein on MT2 cells had little effect on improving this suggesting that the defect was encoded by the parent cell. Visible syncytia were seen after incubation of these cells with K562 target cells but complete fusion as measured by transfer of cellular contents into the recipient cell was not observed. In C91-PL cells, binding of SU to the receptor resulted in a sustained hydrophobic change of envelope accompanied by a cytopathic effect in mixed cell cultures and complete fusion. However, in C91-PL cells, overexpression of envelope protein blocked the transfer of cell contents after receptor engagement and initiation of cytopathic membrane changes, indicating that post binding fusion events were blocked. These data suggest that HTLV-1 fusion is a multistep process which is susceptible to inhibition at two seperate stages of the fusion pathway post receptor binding. This, and the inefficient infection by cell-free virions, may explain the poor infectivity of HTLV-1 in vivo and suggests strategies for preventative therapy.
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Ceccaldi, Pierre-Emmanuel, Frédéric Delebecque, Marie-Christine Prevost, Arnaud Moris, Jean-Pierre Abastado, Antoine Gessain, Olivier Schwartz, and Simona Ozden. "DC-SIGN Facilitates Fusion of Dendritic Cells with Human T-Cell Leukemia Virus Type 1-Infected Cells." Journal of Virology 80, no. 10 (May 15, 2006): 4771–80. http://dx.doi.org/10.1128/jvi.80.10.4771-4780.2006.
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ABSTRACT Interactions between the oncogenic retrovirus human T-cell leukemia virus type 1 (HTLV-1) and dendritic cells (DCs) are poorly characterized. We show here that monocyte-derived DCs form syncytia and are infected upon coculture with HTLV-1-infected lymphocytes. We examined the role of DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), a C-type lectin expressed in DCs, in HTLV-1-induced syncytium formation. DC-SIGN is known to bind with high affinity to various viral envelope glycoproteins, including human immunodeficiency virus (HIV) and hepatitis C virus, as well as to the cellular receptors ICAM-2 and ICAM-3. After cocultivating DCs and HTLV-1-infected cells, we found that anti-DC-SIGN monoclonal antibodies (MAbs) were able to decrease the number and size of HTLV-1-induced syncytia. Moreover, expression of the lectin in epithelial-cell lines dramatically enhanced the ability to fuse with HTLV-1-positive cells. Interestingly, in contrast to the envelope (Env) glycoproteins of HIV and other viruses, that of HTLV-1 does not bind directly to DC-SIGN. The facilitating role of the lectin in HTLV-1 syncytium formation is mediated by its interaction with ICAM-2 and ICAM-3, as demonstrated by use of MAbs directed against these adhesion molecules. Altogether, our results indicate that DC-SIGN facilitates HTLV-1 infection and fusion of DCs through an ICAM-dependent mechanism.
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Krivtsov, Andrei V., Matthew C. Stubbs, Renee Wright, Zhaohui Feng, Andrew L. Kung, and Scott A. Armstrong. "MLL-AF9 Initiates Leukemia from a Single Committed Hematopoietic Progenitor." Blood 108, no. 11 (November 16, 2006): 2534. http://dx.doi.org/10.1182/blood.v108.11.2534.2534.
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Abstract Recent experiments have demonstrated that MLL-translocation associated fusion proteins can transform either hematopoietic stem cells (HSC) or granulocyte macrophage progenitors (GMP) into leukemia stem cells. However, it may be that leukemogenic process differs when HSC are the cell of origin as compared to myeloid progenitors. We transduced either HSC or GMP with a retrovirus expressing MLL-AF9 and GFP followed by single cell sorting of transduced cells. Approximately 80% of singly sorted MLL-AF9 transduced GMP (MLL-AF9-GMP) and about 25% of MLL-AF9 transduced HSC (MLL-AF9-HSC) could be serially re-plated over 9 passages. Upon transplantation into syngeneic mice, 83% (n=12) of MLL-AF9-HSC single cell derived clones induced AML with a median latency 70 days. Approximately 30% (n=20) of MLL-AF9-GMP single cell derived clones induced AML, with median latency 112 days. When MLL-AF9-GMP single cell derived clones were co-infected with an empty retrovirus (to provide additional oncogenic events as a result of retroviral integration) before transplantation into recipient mice, 93% of the transplanted mice (n=15) developed AML with mean latency 65 days, similar to leukemia initiated from HSC. This suggests that either single GMP or HSC can be transformed into leukemia initiating cells. However, extra mutations appear to be required to induce leukemia from committed progenitors. Consistent with this hypothesis, southern blot analysis performed on leukemias initiated from 5,000 and 15,000 MLL-AF9 transduced HSC or GMP demonstrated polyclonal AML arising from HSC compared to oligoclonal AML arising from GMP. Next, we used bioluminescent imaging to follow disease kinetics. When 15,000 MLL-AF9 transduced HSC were injected into recipient mice, the disease accumulated in a linear fashion over 42 days. However, when 15,000 MLL-AF9 transduced GMP were injected the disease developed more slowly over 75 days. Immunophenotypic analysis of the resultant leukemias demonstrated that the HSC-derived and GMP-derived leukemias were quite similar, with a GMP-like population containing LSC in both cases. Globally, the two cell types were also very similar with their gene expression profile being more similar to GMP than any other progenitor or stem cell population. However, we found that in addition to the previously reported 363-gene “self-renewal associated signature” LSC derived from HSC also possessed high-level expression of genes such as Flt3, Mcl1, and Notch-1. Preliminary analysis also suggests that gene expression differences between HSC and GMP-derived leukemia stem cells may have prognostic significance in human AML. These data suggest that AML derived from different cells of origin, while globally quite similar, require a different number of genetic events, and have gene expression differences that may influence drug response.
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Nilsson, Tina, Ahmed Waraky, Anders Östlund, Laleh Arabanian, Julia Asp, Linda Fogelstrand, and Lars Palmqvist. "Recreation of t(7;12)(q36;p13) in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9 Generates a Useful Model for Studying Acute Myeloid Leukemia." Blood 134, Supplement_1 (November 13, 2019): 2736. http://dx.doi.org/10.1182/blood-2019-123004.
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Introduction Acute myeloid leukemia (AML) is the result of aberrant hematopoietic processes, such as enhanced proliferation, blocked differentiation, and dysregulated apoptosis of hematopoietic stem and progenitor cells, and frequently these changes are initiated by chromosomal translocations in leukemia. Efficient methods for modelling leukemia and to recreate leukemia-associated genetic aberrations, such as chromosome translocations, are therefore crucial for investigating how leukemia is initiated. Today, most such models are murine and usually based on introduction of fusion gene transcripts of interest under the control of a constitutive active promoter using lenti- or retroviral transduction rather than the chromosomal translocation itself. The aim of the current project was to create a human cellular model of a chromosomal translocation that is typically found in AML in children under 24 months of age, the translocation t(7;12)(q36;p13). This translocation has been associated with poor prognosis, and leads to a gene fusion MNX1-ETV6 but also aberrant MNX1 gene expression. Its mechanism for leukemia initiation is so far unknown, mainly due to lack of a suitable experimental model. Material and methods CRISPR/Cas9 was used to reconstruct the genetics of the t(7;12)(q36;p13) rearrangement in human induced pluripotent stem cells (iPSC) while maintaining the genomic architecture and regulatory elements. Ribonucleoprotein (RNP) complex was delivered by lipofection (Nucleofection, Amaxa 4D system) into undifferentiated iPSC (ChiPSC 22, Cellartis). An ATTO550 tag on tracrRNA/RNP complex was used to sort out positive cells by flow cytometry and then seeded as single-cells in 96-well plates. Genomic DNA from the single-cell derived iPSC clones were screened by PCR for the presence of the translocation and positive clones were verified with a FISH probe specific for t(7;12)(q36;p13) (Double Fusion Break Apart probe, Metasystem). RT-qPCR was used to detect and quantify the expression of MNX1-ETV6 fusion and MNX1 transcripts. Differentiation potential was tested with the Trilineage Differentiation and Hematopoietic Kits (STEMdiff, STEMCELL Technologies). Results Using CRISPR/Cas9, we could successfully generate iPSC with the t(7;12)(q36;p13) translocation. The translocation was confirmed using conventional karyotyping and FISH and the mRNA expression of the fusion was confirmed with RT-qPCR. No additional chromosomal aberrations were seen. The t(7;12)(q36;p13) iPSC showed similar growth and differentiation properties as the parental iPSC. They showed propensity to differentiate into all three germ layers, confirming their pluripotent stem cell properties. The potential for differentiation into hematopoietic progenitor cells was shown by expression of CD34+, CD43+ and CD45+. In AML with t(7;12)(q36;p13), MNX1 mRNA expression is increased and this may play a role for leukemia development. In the t(7;12)(q36;p13) iPSC, RT-qPCR indeed showed increased expression of MNX1 expression compared with iPSC without the translocation. This increase of MNX1 was not seen in murine adult bone marrow or fetal liver cells transduced with retrovirus expressing the MNX1-ETV6 fusion. Further characterization of the t(7;12)(q36;p13) iPSC, e.g. whole exome and transcriptome sequencing and engraftment potential in immunocompromised mice (NSG-SGM), is ongoing. Conclusion In summary, we have using CRISPR/Cas9 successfully created a t(7;12)(q36;p13) iPSC line with potential to differentiate into hematopoietic progenitor cells and with gene expression pattern similar to what is seen in human AML samples with the t(7;12)(q36;p13). The introduction of the MNX1-ETV6 fusion in its correct genomic context could recapitulate local gene regulation, making it superior to models based on lenti- or retroviral introduction of fusion genes transcripts. In conclusion, this created cell line will be a valuable tool to study the mechanisms behind t(7;12)(q36;p13) AML, a severe form of AML associated with poor prognosis. Disclosures No relevant conflicts of interest to declare.
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Watanabe-Okochi, Naoko, Jiro Kitaura, Hironori Harada, Ryoichi Ono, Hideaki Nakajima, Tetsuya Nosaka, Toshiya Inaba, and Toshio Kitamura. "A BMT Model Mice for Myelodysplastic Syndromes (MDS) and Transformation to AML." Blood 108, no. 11 (November 16, 2006): 2633. http://dx.doi.org/10.1182/blood.v108.11.2633.2633.
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Abstract To understand myelodysplastic syndromes (MDS) at the molecular level, we used a mouse bone marrow transplant (BMT) model. Several leukemogenic fusion proteins were demonstrated to induce leukemia in mouse BMT models. However, analysis of molecular basis of MDS has been hampered by the lack of mouse MDS models. Mutations of a transcription factor AML-1/Runx-1 were identified in 15–40% of MDS-RAEB and MDS/AML and 5–10% of de novo AML. To test transforming abilities of the mutant AML-1, we transduced AML-1 with different mutations (#5 and #27) into mouse bone marrow cells using our highly efficient retrovirus-mediated gene transfer, transplanted the transduced cells to irradiated mice, and found that most mice developed leukemia within 4–13 months after the transplant (table1 and figure1). The leukemic mice exhibited marked hepato-splenomegaly, and morphological abnormalities of myeloid and erythroid lineages were frequently observed. Some mice developed to leukemia after a long latency with MDS-like symptoms, either with pancytopenia or leukocytosis with anemia, thus mimicking the human disease. To clarify the differences between the leukemic mice with early onsets and those that developed leukemia after a long period of MDS-like symptoms in the mutant AML-1-induced mouse diseases, the integration sites of the transduced retroviruses were investigated using a polymerase chain reaction (PCR)-based technique bubble PCR. Our preliminary results suggested that the integration sites of the retroviruses harboring the mutant AML-1 contribute to the early onset of leukemia. In conclusion, we have developed a useful mouse in vivo model of MDS/AML that should help understand the molecular basis of MDS and progression of MDS to leukemia. penetrance and latency mutation number position form penetrance latency #5 D171N point mutation in RHD 69.6 % 4–13 months #27 S291fsX300 truncation type 100 % 4–9 months Figure Figure
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Matte, Catherine C., James Cormier, Britt E. Anderson, Ioanna Athanasiadis, Jinli Liu, Stephen G. Emerson, Warren Pear, and Warren D. Shlomchik. "Graft-versus-leukemia in a retrovirally induced murine CML model: mechanisms of T-cell killing." Blood 103, no. 11 (June 1, 2004): 4353–61. http://dx.doi.org/10.1182/blood-2003-10-3735.
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Abstract The graft-versus-leukemia (GVL) effect, mediated by donor T cells, has revolutionized the treatment of leukemia. However, effective GVL remains difficult to separate from graft-versus-host disease (GVHD), and many neoplasms are GVL resistant. Murine studies aimed at solving these problems have been limited by the use of leukemia cell lines with limited homology to human leukemias and by the absence of loss-of-function leukemia variants. To address these concerns, we developed a GVL model against murine chronic-phase chronic myelogenous leukemia (mCP-CML) induced with retrovirus expressing the bcr-abl fusion cDNA, the defining genetic abnormality of chronic-phase CML (CP-CML). By generating mCP-CML in gene-deficient mice, we have studied GVL T-cell effector mechanisms. mCP-CML expression of Fas or tumor necrosis factor (TNF) receptors is not required for CD8-mediated GVL. Strikingly, maximal CD4-mediated GVL requires cognate interactions between CD4 cells and mCP-CML cells as major histocompatibility complex-negative (MHC II-/-) mCP-CML is relatively GVL resistant. Nevertheless, a minority of CD4 recipients cleared MHC II-/- mCP-CML; thus, CD4 cells can also kill indirectly. CD4 GVL did not require target Fas expression. These results suggest that CPCML's GVL sensitivity may in part be explained by the minimal requirements for T-cell killing, and GVL-resistance may be related to MHC II expression. (Blood. 2004;103:4353-4361)
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Roger, R., C. Issaad, M. Pallardy, MC Leglise, AG Turhan, J. Bertoglio, and J. Breard. "BCR-ABL does not prevent apoptotic death induced by human natural killer or lymphokine-activated killer cells." Blood 87, no. 3 (February 1, 1996): 1113–22. http://dx.doi.org/10.1182/blood.v87.3.1113.bloodjournal8731113.
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The erythromyeloid cell line, K562, the most sensitive target in human natural killer (NK) cell mediated cytotoxicity, is derived from a chronic myeloid leukemia (CML) patient and expresses the characteristic reciprocal translocation t(9;22). The resulting BCR-ABL fusion protein has been shown to mediate the unusual resistance of K562, and other BCR- ABL expressing lines, to apoptosis induced by a variety of agents (irradiation, UV light, cytotoxic drugs). Here we show that human NK and lymphokine-activated killer (LAK) cells, when tested at low effector to target ratio, can readily induce apoptotic death in K562 cells. This was accompanied with classical DNA oligonucleosomal fragmentation, an unexpected finding given the reported lack of such fragmentation when apoptosis is induced in K562 by chemical agents, after downregulation of BCR-ABL. Apoptosis was assessed by several means: morphological studies, 125I-DNA versus 51Cr release, DNA agarose gel electrophoresis, and results were always concordant, with a delayed kinetics for DNA oligonucleosomal fragmentation. Similar data were obtained with a pluripotent human hematopoietic cell line, UT-7, infected with a defective amphotropic p210 BCR-ABL retrovirus. The BCR- ABL expressing subclone UT-7/9, while being no longer sensitive to cytotoxic drugs or to tumor necrosis factor, a lytic mediator to which UT-7 cells are sensitive, underwent apoptotic death when exposed to LAK effector cells to the same degree as the parental UT-7 line. With these targets, DNA oligonucleosomal fragmentation occurred concomitantly with isotope release. Results obtained with several inhibitors of exocytosis strongly suggest that cytotoxic granules mediate NK and LAK cell- induced apoptotic death. In conclusion, NK and LAK cell-induced apoptotic signals, unlike those activated by chemotherapeutic agents, are unaffected by the antiapoptotic action of BCR-ABL. This unique property may support the observed curative effect of allogeneic bone marrow transplantation in CML.
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Huleihel, M., M. Goldsborough, J. Cleveland, M. Gunnell, T. Bonner, and U. R. Rapp. "Characterization of murine A-raf, a new oncogene related to the v-raf oncogene." Molecular and Cellular Biology 6, no. 7 (July 1986): 2655–62. http://dx.doi.org/10.1128/mcb.6.7.2655-2662.1986.
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A 1.6-kilobase cDNA (A-raf) has been isolated from a murine spleen cDNA library which encodes part of a protein related to the raf oncogene. Its amino acid sequence has 85% homology to raf in a central portion of 100 amino acids. In contrast to raf, A-raf shows a highly restricted tissue distribution of expression, with highest levels observed in epididymis, followed by intestine. When incorporated into a retrovirus, the resulting gag-A-raf fusion gene causes transformation in vitro and induces tumors in newborn mice. Thus, A-raf represents a new proto-oncogene. Transformation by A-raf is independent of ras gene function, as is the case for raf and mos but not other oncogenes.
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Sørensen, Annette Balle, Anders H. Lund, Steen Ethelberg, Neal G. Copeland, Nancy A. Jenkins, and Finn Skou Pedersen. "Sint1, a Common Integration Site in SL3-3-Induced T-Cell Lymphomas, Harbors a Putative Proto-Oncogene with Homology to the Septin Gene Family." Journal of Virology 74, no. 5 (March 1, 2000): 2161–68. http://dx.doi.org/10.1128/jvi.74.5.2161-2168.2000.
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ABSTRACT The murine retrovirus SL3-3 is a potent inducer of T-cell lymphomas when inoculated into susceptible newborn mice. Previously, DNAs from twenty SL3-3-induced tumors were screened by PCR for provirus integration sites. Two out of 20 tumors demonstrated clonal provirus insertion into a common region. This region has now been isolated and characterized. The region, named SL3-3 integration site 1 (Sint1), maps to the distal end of mouse chromosome 11, corresponding to human chromosome 17q25, and may be identical to a mouse mammary tumor virus integration site in a T-cell lymphoma,Pad3. Two overlapping genomic λ clones spanning about 35 kb were isolated and used as a starting point for a search for genes in the neighborhood of the virus integration sites. A genomic fragment was used as a hybridization probe to isolate a 3-kb cDNA clone, the expression of which was upregulated in one of two tumors harboring a provirus in Sint1. The cDNA clone is predicted to encode a protein which shows 97.0% identity to a human septin-like protein encoded by a gene which has been found as a fusion partner gene of MLL in an acute myeloid leukemia with a t(11;17)(q23;q25). Together these findings raise the possibility that a proto-oncogene belonging to the septin family, and located about 15 kb upstream of the provirus integration sites, is involved in murine leukemia virus-induced T-cell lymphomagenesis.
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Matsuzawa, Ayumi, Jiyoung Lee, So Nakagawa, Johbu Itoh, Mahoko Takahashi Ueda, Satomi Mitsuhashi, Yuta Kochi, Tomoko Kaneko-Ishino, and Fumitoshi Ishino. "HERV-Derived Ervpb1 Is Conserved in Simiiformes, Exhibiting Expression in Hematopoietic Cell Lineages Including Macrophages." International Journal of Molecular Sciences 22, no. 9 (April 26, 2021): 4504. http://dx.doi.org/10.3390/ijms22094504.
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(1) Background: The ERVPb1 gene in humans is derived from an envelope (Env) gene of a human endogenous retrovirus group, HERV-P(b). The ERVPb1 gene reportedly has a conserved open reading frame (ORF) in Old World monkeys. Although its forced expression led to cell-fusion in an ex vivo cell culture system, like other Env-derived genes such as syncytin-1 and -2, its mRNA expression is not placenta-specific, but almost ubiquitous, albeit being quite low in human tissues and organs, implying a distinct role for ERVPb1. (2) Methods: To elucidate the cell lineage(s) in which the ERVPb1 protein is translated in human development, we developed a novel, highly sensitive system for detecting HERV-derived proteins/peptides expressed in the tissue differentiation process of human induced pluripotent stem cells (iPSCs). (3) Results: We first determined that ERVPb1 is also conserved in New World monkeys. Then, we showed that the ERVPb1 protein is translated from a uniquely spliced ERVPb1 transcript in hematopoietic cell lineages, including a subset of macrophages, and further showed that its mRNA expression is upregulated by lipopolysaccharide (LPS) stimulation in primary human monocytes. (4) Conclusions: ERVPb1 is unique to Simiiformes and actually translated in hematopoietic cell lineages, including a subset of macrophages.
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Norton, P. A., and J. M. Coffin. "Bacterial beta-galactosidase as a marker of Rous sarcoma virus gene expression and replication." Molecular and Cellular Biology 5, no. 2 (February 1985): 281–90. http://dx.doi.org/10.1128/mcb.5.2.281-290.1985.
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We have developed a convenient and sensitive assay of eucaryotic gene expression which uses the Escherichia coli lacZ gene product, beta-galactosidase, as a nonselectable marker. This system has been applied to the analysis of Rous sarcoma virus replication and gene expression. Avian cells were transfected with plasmids encoding in-frame gene fusions of the N-terminal portion of the gag gene to a 'lacZ gene, which requires both transcriptional and translational initiation signals; these were supplied by the virus long terminal repeat and leader region. Readily detectable quantities of beta-galactosidase were synthesized in transfected cells; it was demonstrated that the levels of enzyme activity induced in such cultures increased linearly with the input DNA concentration and also correlated with mRNA levels. By using a Rous sarcoma virus-derived vector containing the src gene and a related virus as a helper, it was shown that lac sequences were compatible with all phases of the virus life cycle. gag-lacZ fusion proteins were immunoprecipitable from cultures which stably expressed lacZ as well as src. Virus rescued from stably transfected cultures resulted in continued lac and src expression in recipient cells. One particular construction was efficiently transmitted as virus, although it lacked sequences thought to be important for encapsidation of RNA into virions. The data presented here demonstrate the use of lacZ as a marker of retrovirus gene expression and replication.
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Lei, Tei Chi, and David W. Scott. "Blockade of Inhibitor Formation by B-Cell Delivered Tolerance to Immunodominant A2 and C2 Domains." Blood 104, no. 11 (November 16, 2004): 3187. http://dx.doi.org/10.1182/blood.v104.11.3187.3187.
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Abstract A major impediment in the treatment of hemophilia is the formation of inhibitory antibodies, which occurs in approximately 25–30% of Hemophilia A patients treated with therapeutic Factor VIII (fVIII). We have focused on the development of a gene therapy protocol for tolerance induction, with an emphasis on the elimination of inhibitor production. Our lab has demonstrated that LPS-stimulated B-cell blasts, transfected with a retrovirus encoding an IgG-peptide fusion protein, such as fVIII domains, are tolerogenic in both normal and primed recipients. Last year, we reported (http://www.abstracts-on-line.com/abstracts/hemphiladelphia03; Scott and Lei 2003) that specific tolerance to the immunodominant epitopes in the C2 domain of fVIII (a major target of inhibitors) could be induced by our protocol. However, the immune response to full length fVIII was only modestly affected. Most inhibitory antibodies are reactive with conformational epitopes on the exposed surfaces of the A2, as well as the C2, domain of fVIII. Therefore, in this study, we inserted residues S2173-Y2332 of the C2 domain and S373-R740 of the A2 domain onto the IgG heavy chain backbone, respectively, to induce tolerance in hemophilic mice. Specific tolerance to each domain was induced by this protocol. Importantly, a combination of A2-IgG and C2-IgG expressing B cells induced tolerance to the full length fVIII molecule, a result which supports the dominance of these domains in the immune response to fVIII. Tolerance was manifested in terms of ELISA, T-cell proliferation and especially Bethesda Unit titers (95% reduction). Similar results were obtained even when treatment was initiated after priming injections of fVIII. In conclusion, this protocol offers great promise for prevention and potential reversal of this serious complication of fVIII replacement therapy. (Supported by HL61883 and a Laboratory Grant from the National Hemophilia Foundation).
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Talarico, D., M. M. Ittmann, R. Bronson, and C. Basilico. "A retrovirus carrying the K-fgf oncogene induces diffuse meningeal tumors and soft-tissue fibrosarcomas." Molecular and Cellular Biology 13, no. 4 (April 1993): 1998–2010. http://dx.doi.org/10.1128/mcb.13.4.1998.
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The K-fgf/hst oncogene encodes a growth factor of the fibroblast growth factor (FGF) family and transforms cells through an autocrine mechanism which requires extracellular activation of its receptor(s). To identify the cell and tissue targets of K-fgf oncogenic potential in vivo, we constructed a recombinant retrovirus carrying the human K-fgf cDNA and injected it, together with helper Moloney murine leukemia virus, into immunocompetent as well as nude mice. The original construct was highly transforming in tissue culture but produced no detectable pathologies in vivo with the exception of a single fibrosarcoma which arose after a long latency. The virus produced by this tumor appears to have undergone a complex series of recombination events involving the helper Moloney murine leukemia virus. It encodes an Env/K-FGF fusion protein whose expression is under the control of a hybrid long terminal repeat. This virus (designated MFS, for meningeal fibrosarcoma) induces tumors in mice with high frequency and short latency. These neoplasms consist of aggressive fibrosarcomas of soft tissue as well as diffuse meningeal tumors originating from the dura mater that surround the whole central nervous system and cause severe hydrocephalus. The Env/K-FGF fusion protein expressed by the MFS virus has retained all of the biological properties of native K-FGF, including secretion, mitogenic activity, heparin binding, and neutralization by anti-K-FGF antibodies. These and other results indicate that the tumors induced by the MFS virus result from the oncogenic potential of K-FGF.
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Palmqvist, Lars, Nicolas Pineault, Patricia Rosten, and Keith R. Humphries. "Redundant Leukemogenicity of NUP98-HOX Fusion Genes in Primary Murine Bone Marrow Cells Correlates with Gene Expression Changes Consistent with Common Key Target Genes." Blood 104, no. 11 (November 16, 2004): 1134. http://dx.doi.org/10.1182/blood.v104.11.1134.1134.
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Abstract Several Abd-B HOX genes have been found in translocations with the nucleoporin gene NUP98 in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). We have previously tested both known and engineered NUP98-HOX fusions in the murine bone marrow transplantation model (N. Pineault et al., MCB24:1907, 2004). Strikingly, an engineered NUP98-HOXA10 (NA10) fusion, not observed in patients, and the AML-associated fusion gene NUP98-HOXD13 (ND13) have a virtually indistinguishable ability to transform myeloid progenitor cells and to induce leukemia in collaboration with MEIS1. Importantly, their transforming ability is lost when the DNA-binding homeodomain is mutated. This functional overlap provides a potentially powerful strategy to identify key genes/pathways mediating HOX-induced leukemias by looking for overlapping gene expression changes induced by different NUP98-HOX fusion genes. 5-FU bone marrow cells were transduced with retroviral vectors encoding for the leukemogenic ND13 or NA10 fusion genes or a non-leukemogenic ND13 gene with a N51S homeodomain mutation or the empty MIG vector. RNA was extracted from transduced GFP+ Sca1+ Lin- cells and linear RNA amplification was performed before the analysis on the Affymetrix GeneChip MOE430. Three independent experiments were conducted and analyzed. Correlation analysis showed a high degree of similarity between ND13 and NA10 in their overall gene expression profiles, compared to the N51S mutant or the MIG control. Validation with real-time quantitative RT-PCR on non-amplified RNA revealed good agreement between the gene array and the PCR, with a tendency for bigger fold-changes with the PCR method. Close to 500 genes were found differentially expressed (changed ≥2-fold vs. MIG ctrl and t-test p-value <0.05) and some 100 of these were changed by both ND13 and NA10 but not by the N51S homeodomain mutant. These genes are strong candidates as direct and/or immediate downstream targets involved in leukemic transformation. Remarkably, among these were genes previously identified as a NUP98 fusion partner in human leukemia (DEAD-box protein, Ddx10), or part of the same family of genes found in NUP98-fusions (Ddx4 and the paired mesoderm homeobox gene, Pmx2). This suggests a possible molecular link in leukemogenicity between HOX- and non-HOX-NUP98 fusions. Other interesting genes that were induced by ND13 and NA10, but not by the N51S homeodomain mutant, were genes previously implicated in leukemia (e.g. Flt3, Evi1) as well as Hox-related genes, such as the Hox cofactor Pbx3 and several Hox A cluster members. Furthermore, approximately one third were ESTs or genes with unknown function. In conclusion, our results document similar changes in gene expression induced by functionally redundant but different NUP98-HOX fusions and should facilitate the identification of common target genes involved in leukemic transformation.
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Sakurai, Toshihiro, So Nakagawa, Hanako Bai, Rulan Bai, Kazuya Kusama, Atsushi Ideta, Yoshito Aoyagi, et al. "Novel endogenous retrovirus-derived transcript expressed in the bovine placenta is regulated by WNT signaling." Biochemical Journal 474, no. 20 (October 10, 2017): 3499–512. http://dx.doi.org/10.1042/bcj20170531.
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Endogenous retroviruses (ERVs) are involved in placentation; perhaps, the most well-known ERVs are the syncytins, actively transcribed env genes involved in cell–cell fusion and possible morphological variations. However, ERVs other than syncytins that play an important role in placental development have not been well characterized. To identify ERV genes expressed during the onset of placentation in the bovine species, we characterized the expression profiles of bovine conceptus transcripts during the peri-attachment period using RNA-seq analysis, and confirming some candidates through real-time PCR. Using in silico and PCR analyses, we identified a novel ERV proviral sequence derived from a gag region, designated bovine endogenous retroviruses (BERV)-K3, containing Gag_p10 and Gag_p24, zinc finger domain. Initial expression of this ERV in bovine conceptuses was on day 20 (day 0 = day of estrus), soon after conceptus attachment to the endometrial epithelium, and its high placental expression was maintained up to the middle of pregnancy. The BERV-K3 transcript was also found in the uterine luminal and glandular epithelia, liver, kidney, intestine, and skin. BERV-K3 is located on chromosome 7 and integrated within LOC100848658, from which noncoding RNA could be transcribed. Furthermore, the expression of endogenous BERV-K3 in bovine trophoblast cell lines was induced by a WNT agonist, a signaling system common to genes expressed in placentas. These data support the argument that during the evolutionary process, mammals incorporated not only similar ERV sequences, but also ERVs unique to individual species. BERV-K3 is in the latter case, likely providing functions unique to ruminant gestation.
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Imam, Sabrina, Sarah Talley, Rachel S. Nelson, Adarsh Dharan, Christopher O'Connor, Thomas J. Hope, and Edward M. Campbell. "TRIM5α Degradation via Autophagy Is Not Required for Retroviral Restriction." Journal of Virology 90, no. 7 (January 13, 2016): 3400–3410. http://dx.doi.org/10.1128/jvi.03033-15.
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ABSTRACTTRIM5α is an interferon-inducible retroviral restriction factor that prevents infection by inducing the abortive disassembly of capsid cores recognized by its C-terminal PRY/SPRY domain. The mechanism by which TRIM5α mediates the disassembly of viral cores is poorly understood. Previous studies demonstrated that proteasome inhibitors abrogate the ability of TRIM5α to induce premature core disassembly and prevent reverse transcription; however, viral infection is still inhibited, indicating that the proteasome is partially involved in the restriction process. Alternatively, we and others have observed that TRIM5α associates with proteins involved in autophagic degradation pathways, and one recent study found that autophagic degradation is required for the restriction of retroviruses by TRIM5α. Here, we show that TRIM5α is basally degraded via autophagy in the absence of restriction-sensitive virus. We observe that the autophagy markers LC3b and lysosome-associated membrane protein 2A (LAMP2A) localize to a subset of TRIM5α cytoplasmic bodies, and inhibition of lysosomal degradation with bafilomycin A1 increases this association. To test the requirement for macroautophagy in restriction, we examined the ability of TRIM5α to restrict retroviral infection in cells depleted of the autophagic mediators ATG5, Beclin1, and p62. In all cases, restriction of retroviruses by human TRIM5α, rhesus macaque TRIM5α, and owl monkey TRIM-Cyp remained potent in cells depleted of these autophagic effectors by small interfering RNA (siRNA) knockdown or clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome editing. Collectively, these results are consistent with observations that the turnover of TRIM5α proteins is sensitive to autophagy inhibition; however, the data presented here do not support observations that the inhibition of autophagy abrogates retroviral restriction by TRIM5 proteins.IMPORTANCERestriction factors are a class of proteins that inhibit viral replication. Following fusion of a retrovirus with a host cell membrane, the retroviral capsid is released into the cytoplasm of the target cell. TRIM5α inhibits retroviral infection by promoting the abortive disassembly of incoming retroviral capsid cores; as a result, the retroviral genome is unable to traffic to the nucleus, and the viral life cycle is extinguished. In the process of restriction, TRIM5α itself is degraded by the proteasome. However, in the present study, we have shown that in the absence of a restriction-sensitive virus, TRIM5α is degraded by both proteasomal and autophagic degradation pathways. Notably, we observed that restriction of retroviruses by TRIM5α does not require autophagic machinery. These data indicate that the effector functions of TRIM5α can be separated from its degradation and may have further implications for understanding the mechanisms of other TRIM family members.
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Green, Kathy A., Randolph J. Noelle, William R. Green, and Li Wang. "Checkpoint Regulator VISTA plays a role in Suppression of B-Cell Responsiveness by Monocytic Myeloid Derived Suppressor Cells from LP-BM5 retrovirus-infected Mice." Journal of Immunology 196, no. 1_Supplement (May 1, 2016): 195.14. http://dx.doi.org/10.4049/jimmunol.196.supp.195.14.
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Abstract MDSC inhibition of tumor directed T cell responses is well described. We have reported an increase of monocytic MDSC (M-MDSC) during infection of B6 mice by LP-BM5 immunodeficiency causing retrovirus. These M-MDSCs suppressed T, and B cell responsiveness ex vivo. M-MDSC inhibition of stimulated T-cell proliferation and IFN-gamma production was ~100% iNOS/NO dependent; whereas suppression of B-cells was only ~50% dependent on iNOS/NO. An additional mechanism(s) for M-MDSC inhibition of B cell responsiveness involved V-domain Ig Suppressor of T cell Activation (VISTA), negative checkpoint regulator. Using anti-VISTA blocking mAb, ~50% of total MDSC suppression of B-cell responses was dependent on MDSC-expressed VISTA. The combination of iNOS/NO inhibitors and VISTA lead to additive, if not synergistic, blockade of M-MDSC suppression of B cell responsiveness. Regarding the LP-BM5-infection dependency of M-MDSCs, spleens from uninfected mice yielded ~3-fold fewer enriched M-MDSCs; and on a per-cell basis, the M-MDSCs from uninfected mice were also substantially less suppressive (4.3 fold); than those from infected mice. Consistent with a direct role of VISTA, in the absence of M-MDSCs, a VISTA-Ig fusion protein also partially inhibited B-cell responsiveness. Initial experiments show differential VISTA expression for the LP-BM5 M-MDSC CD11b+hiLy6C+hi subpopulations we have recently defined (O’Connor et al, 2015, Virology). Thus, LP-BM5 infected, when compared to analogous subsets from uninfected mice, contain substantially more VISTA+ cells. These results suggest an unique role for M-MDSCs in LP-BM5 induced immunodeficiency, and highlight multiple suppressive pathways in the area of MDSC suppression of B cell responses.
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Ehrenfeld, Sophia, Khalid Shoumariyeh, Teresa Poggio, Robin Khan, Desiree Melanie Redhaber, Stefanie Kreutmair, Cathrin Klingeberg, Anna Lena Illert, Justus Duyster, and Cornelius Miething. "Towards Improved Models of NPM-ALK Induced T-Cell Lymphoma." Blood 132, Supplement 1 (November 29, 2018): 1342. http://dx.doi.org/10.1182/blood-2018-99-115872.
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Abstract Time- and tissue-specific expression of transgenes is essential for the accurate representation of human disease in in vivo models. To improve flexibility but also fidelity of ALK+ ALCL models, we developed new approaches enabling lineage specific expression of NPM-ALK cDNAs, as well as a novel system for CRISPR/Cas induced Npm-Alk recombination. First, we have designed a new system for lineage-restricted expression of transgenes based on a retroviral vector incorporating a translational stop-cassette flanked by loxP recombination sites. Conditional transgene expression in chimeric mice is rapidly achieved through retroviral infection and subsequent transplantation of hematopoietic stem cells (HSC) derived from transgenic mice expressing Cre-recombinase from a lineage specific promoter. To validate the model, we directed expression of NPM-ALK, the fusion oncogene present in anaplastic large cell lymphoma (ALCL), to T-cells by infecting hematopoietic stem cells from Lck-Cre transgenic mice with a retroviral construct containing the Npm-Alk cDNA preceded by a translational stop cassette. The approach efficiently induced T-cell lymphomas within 12-16 weeks closely resembling the human disease including the expression of the ALCL hallmark antigen CD30. Since NPM-ALK overexpressed from a cDNA acts as a very strong oncogene transforming a range of cell types, we were interested to develop a more physiologic model based on chromosomal recombination, enabling NPM-ALK expression from the endogenous Npm promoter. To achieve this, we have designed guide RNAs (gRNAs) directed to either the intron between exon 4 and 5 for Npm1 on mouse chr. 11, or the intron between exons 19 and 20 for Alk on mouse chr. 17., enabling targeted translocation between the two chromosomes. For further analysis, the IL-3 dependent murine pro-B cell line Ba/F3 stably expressing the Cas9 recombinase was transduced with the respective gRNAs and subsequently grown in the absence of IL-3 to allow positive selection of cells transformed by productive t(11;17) NA recombination. A PCR reaction on genomic DNA using primers covering the translocation breakpoint resulted in a product and Sanger sequencing of the amplicon confirmed the intended recombination at the targeted genomic positions. The translocation was also detectable by fluorescence in-situ hybridization (FISH), and Western blot analysis demonstrated expression of a highly phosphorylated Npm-Alk fusion protein. To further probe for oncogene dependency, we treated Npm-Alk translocated cells and control cells with a specific Alk inhibitor, resulting in rapid cell depletion of the Npm-Alk translocated cells, but not controls. The described Cre/loxP-based system represents a versatile tool for the rapid functional analysis of gene function in a defined lineage or in a developmental stage in vivo, and faithfully recapitulates many features of ALCL. Furthermore, using Crispr/Cas to induce targeted double-strand breaks, we have been able to generate specific Npm-Alk translocations in murine cells, paving the way for novel models which may help to further define the initial pathogenetic event underlying lymphomagenesis in ALCL. Disclosures No relevant conflicts of interest to declare.
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Maeda, Yosuke, Hiromi Terasawa, Yuetsu Tanaka, Chisho Mitsuura, Kaori Nakashima, Keisuke Yusa, and Shinji Harada. "Separate Cellular Localizations of Human T-Lymphotropic Virus 1 (HTLV-1) Env and Glucose Transporter Type 1 (GLUT1) Are Required for HTLV-1 Env-Mediated Fusion and Infection." Journal of Virology 89, no. 1 (October 22, 2014): 502–11. http://dx.doi.org/10.1128/jvi.02686-14.
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ABSTRACTInteraction of the envelope glycoprotein (Env) of human T-lymphotropic virus 1 (HTLV-1) with the glucose transporter type 1 (GLUT1) expressed in target cells is essential for viral entry. This study found that the expression level of GLUT1 in virus-producing 293T cells was inversely correlated with HTLV-1 Env-mediated fusion activity and infectivity. Chimeric studies between GLUT1 and GLUT3 indicated that the extracellular loop 6 (ECL6) of GLUT1 is important for the inhibition of cell-cell fusion mediated by Env. When GLUT1 was translocated into the plasma membrane from intracellular storage sites by bafilomycin A1 (BFLA1) treatment in 293T cells, HTLV-1 Env-mediated cell fusion and infection also were inhibited without the overexpression of GLUT1, indicating that the localization of GLUT1 in intracellular compartments rather than in the plasma membrane is crucial for the fusion activity of HTLV-1 Env. Immunoprecipitation and laser scanning confocal microscopic analyses indicated that under normal conditions, HTLV-1 Env and GLUT1 do not colocalize or interact. BFLA1 treatment induced this colocalization and interaction, indicating that GLUT1 normally accumulates in intracellular compartments separate from that of Env. Western blot analyses of FLAG-tagged HTLV-1 Env in virus-producing cells and the incorporation of HTLV-1 Env in virus-like particles (VLPs) indicate that the processing of Env is inhibited by either overexpression of GLUT1 or BFLA1 treatment in virus-producing 293T cells. This inhibition probably is due to the interaction of the Env with GLUT1 in intracellular compartments. Taken together, separate intracellular localizations of GLUT1 and HTLV-1 Env are required for the fusion activity and infectivity of HTLV-1 Env.IMPORTANCEThe deltaretrovirus HTLV-1 is a causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although HTLV-1 is a complex retrovirus that has accessory genes, no HTLV-1 gene product has yet been shown to regulate its receptor GLUT1 in virus-producing cells. In this study, we found that a large amount of GLUT1 or translocation of GLUT1 to the plasma membrane from intracellular compartments in virus-producing cells enhances the colocalization and interaction of GLUT1 with HTLV-1 Env, leading to the inhibition of cell fusion activity and infectivity. The results of our study suggest that GLUT1 normally accumulates in separate intracellular compartments from Env, which is indeed required for the proper processing of Env.
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Gong, Shenglan, Mengqiao Guo, Gusheng Tang, Jianmin Yang, and Huiying Qiu. "Overexpression of TEL-MN1 Fusion Enhances Resistance of HL-60 Cells to Idarubicin." Chemotherapy 63, no. 6 (2018): 308–14. http://dx.doi.org/10.1159/000495073.
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Background: The translocation t(12; 22) (p13;q12) is a recurrent but infrequent chromosome abnormality in human myeloid malignancies. To date, the role of TEL-MN1 fusion in leukemogenic process and drug resistance is still largely unknown. Methods: In the present study, the TEL-MN1 fusion was transfected into HL-60 cells to upregulate TEL-MN1 expression via a retroviral vector. MTT assay was employed to examine cell viability and flow cytometry was performed to evaluate cell apoptosis. Idarubicin was used to treat HL-60 cells for estimating the effect of TEL-MN1 fusion on the chemotherapy resistance. Results: The results showed that overexpression of TEL-MN1 in HL-60 cells could promote cell proliferation, suggesting that TEL-MN1 may be involved in the leukemogenesis process. HL-60 cells treated with idarubicin showed a weakened cell viability, whereas TEL-MN1 overexpression attenuated the idarubicin-induced inhibition of cell viability and acceleration of cell apoptosis of HL-60 cells. Conclusion: Taken together, our results indicated that TEL-MN1 fusion is an oncogene involved in the leukemogenesis process and TEL-MN1 overexpression enhanced resistance of HL-60 cells to idarubicin, which may provide a useful tool for studying the mechanism of leukemogenesis and drug resistance.
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Krivtsov, Andrei V., Amit U. Sinha, Matthew C. Stubbs, Andrew Kung, and Scott Armstrong. "Cell of Origin Influences Leukemia Stem Cell Phenotype." Blood 114, no. 22 (November 20, 2009): 3459. http://dx.doi.org/10.1182/blood.v114.22.3459.3459.
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Abstract Abstract 3459 Poster Board III-347 MLL-fusion proteins can transform either hematopoietic stem cells (HSC) or granulocyte macrophage progenitors (GMP) into leukemia stem cells (LSC). However, the leukemogenic process in HSC may differ from that in GMP. We transduced HSC and GMP with MLL-AF9 or control retroviruses. Single-cell sorted MLL-AF9 expressing HSC or GMP could be serially replated for over 9 passages. Upon transplantation into syngeneic mice, 86.3% (n=22) of HSC:MLL-AF9 single cell derived clones (SCC) induced AML with a median latency of 61 days, while 33.3% of GMP:MLL-AF9 SCC induced AMLs with median latency of 100 days. Immunophenotype analysis of the resultant leukemias demonstrated that long-term repopulating HSC (LT-HSC) and GMP-derived leukemias were quite similar, with a GMP-like (LGMP) population enriched in LSC in both cases. Gene expression analysis demonstrated that globally the LGMP isolated from HSC derived AMLs (AML:HSC) and GMP derived AMLs (AML:GMP) were similar to each other but possessed specific genetic programs reminiscent of the cell of origin (HSC or GMP). For example Evi1, Jun, and Fos oncogenes were highly expressed in HSC and AML:HSC, but expressed at low level in GMP or AML:GMP. The genetic program that distinguished LGMP:HSC from LGMP:GMP was found to be enriched in hematopoietic stem cells compared to more differentiated myeloid progenitors and correlate with genetic programs in and human MLL-rearranged AML associated with a poor clinical outcome in two independent MLL-rearranged AML data sets. In order to directly assess differences in treatment response for leukemias derived from different cells of origin, we treated leukemic mice with a chemotherapeutic agent often used to treatment human AMLs. Treatment of leukemic mice with Etoposide reduced the spleen weights in mice transplanted with AML:HSC to a lesser extent (28%) than in mice transplanted with AML:GMP (88%). Altogether, these data indicate that cell of origin of AML can influence the genetic program of fully developed leukemia, and thus could account for some of the heterogeneity in human leukemias and perhaps outcome. Disclosures No relevant conflicts of interest to declare.
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Berthiaume, Sara, James A. Kennedy, Anne Bergeron, Mathieu Belanger, John E. Dick, and Frederic Barabe. "Comparing Human Fetal Liver, Cord Blood and Adult Bone Marrow Stem/Progenitor Hematopoietic Cells As Cells of Origin of Human MLL Leukemias." Blood 118, no. 21 (November 18, 2011): 2956. http://dx.doi.org/10.1182/blood.v118.21.2956.2956.
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Abstract Abstract 2956 Acute leukemias in newborns, children, adults and elderly share the same name, but their genetic, biology and prognosis are significantly different. These differences may be explained by many factors including the nature of the recurrent genetic abnormalities, tolerance to chemotherapy, co-morbidities, etc. One noteworthy potential difference is the cell of origin in which the initial genetic event occurs, which can be a fetal liver, cord blood or adult bone marrow stem/progenitor cell. Genetically engineered mouse models of leukemias and gene expression studies on human acute lymphoblastic leukemias (ALL) suggest that leukemias developing in utero (i.e. in an early ontogenic cell of origin) are biologically distinct from child and adult leukemias. However, direct comparison of human hematopoietic stem/progenitor cells at different stages of ontogeny has not been tested experimentally. Over 70% of infant acute leukemias and 5–10% of adult leukemias exhibit rearrangements of the mixed-lineage leukemia gene (MLL). Therefore, MLL translocations are highly relevant oncogenes to investigate the leukemic potential of the different cells of origin. The MLL-ENL (MLL-MLLT1) fusion gene, from the t(11;19) (q23;p13.3), is one of the most frequent MLL fusion and can occur in infants and adults. It is found in both acute myeloid leukemias (AML) and ALL (both B-cell and T-cell ALL) and we have previously shown that it generates human B-ALL in immunodeficient mice when introduced into lineage depleted human cord blood cells using a retrovirus (Barabé et al, Science 2007). The same experimental design was used to study the cells of origin; CD34+ cells from human fetal liver (FL), cord blood (CB) and adult bone marrow (BM) were infected with either a retrovirus encoding MLL-ENL and an enhanced green fluorescent protein (EGFP) marker gene, or a control retrovirus encoding EGFP only, then injected into sub-lethally irradiated immunodeficient mice. After transduction with MLL-ENL, FL cells generated leukemias in 8/10 mice, CB cells in 6/6 mice and adult BM cells in only 2/7 mice, although four times more cells were transduced and injected. Adult BM cells were very difficult to infect with retroviruses and were clearly not as potent to reconstitute a human graft in immunodeficient mice in comparison to CB cells. All generated leukemias were B-ALL, regardless of the cell of origin. The leukemic phenotype (CD19+, HLA-DR+, CD20-, IgM-, IgG-, CD34 variable) was similar in all three cell types. Mice injected with MLL-ENL-transduced FL and adult BM cells that were not leukemic had a normal human graft but no EGFP cells. The frequency of leukemia-initiating cells (L-IC) was evaluated at 0.2–1% by limiting dilution in secondary mice and no significant differences were observed between the three cell types. Leukemic cells were harvested from the BM of primary mice and cultured in vitro under conditions supportive of both B-lymphoid and myeloid cells. All leukemias were able to grow in vitro and at least one culture derived from each cell type (FL, CB and adult BM) switched to a myeloid phenotype (monocytic morphology with CD33+ and loss of CD19), demonstrating that leukemias derived from all 3 cell types still have myeloid potential. Thus, our results suggest that MLL-ENL can generate leukemia in FL, CB and adult BM CD34+ cells. The lower penetrance of leukemia with adult BM cells is probably due to the difficulty to infect these cells and their limited reconstitution ability. MLL-ENL induces a leukemic program in all three cell types with similar clinical features, cell surface phenotype, frequency of L-IC and conserved bi-lineage potential (B and myeloid). Gene expression profiling and clonality studies are currently performed on leukemias generated from the 3 different cell types. So far, no significant differences between FL, CB and adult BM as cells of origin of MLL leukemias have been identified, suggesting that the nature of the oncogenic hit is probably more important than the ontogenic stage of the cell of origin. Disclosures: No relevant conflicts of interest to declare.