A Mouse Model of Evi1-Related Leukemia.

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

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Evi-2, a common integration site involved in murine myeloid leukemogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4658-4666.1990 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4658-4666

Author(s):

A M Buchberg ◽

H G Bedigian ◽

N A Jenkins ◽

N G Copeland

Keyword(s):

Leucine Zipper ◽

Transmembrane Domain ◽

Integration Site ◽

Transmembrane Protein ◽

Structural Features ◽

Viral Integration ◽

Integration Sites ◽

Leukemia Viruses ◽

Common Integration Site

BXH-2 mice have the highest incidence of spontaneous retrovirally induced myeloid leukemia of any known inbred strain and, as such, represent a valuable model system for identifying cellular proto-oncogenes involved in myeloid disease. Chronic murine leukemia viruses often induce disease by insertional activation or mutation of cellular proto-oncogenes. These loci are identified as common viral integration sites in tumor DNAs. Here we report on the characterization of a novel common viral integration site in BXH-2 myeloid leukemias, designated Evi-2. Within the cluster of viral integration sites that define Evi-2, we identified a gene that has the potential for encoding a novel protein of 223 amino acids. This putative proto-oncogene possesses all of the structural features of a transmembrane protein. Within the transmembrane domain is a "leucine zipper," suggesting that Evi-2 is involved in either homopolymer or heteropolymer formation, which may play an important role in the normal functioning of Evi-2. Interestingly, the human homolog of Evi-2 has recently been shown to be tightly linked to the von Recklinghausen neurofibromatosis locus, suggesting a role for Evi-2 in human disease as well.

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Sox4 and CREB Cooperate in Myeloid Transformation,

Blood ◽

10.1182/blood.v118.21.3452.3452 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3452-3452

Author(s):

Salemiz Sandoval ◽

Michelle Cho ◽

Christina Kraus ◽

Er-Chieh Cho ◽

Linda Wolff ◽

...

Keyword(s):

Bone Marrow ◽

Transgenic Mice ◽

Progenitor Cells ◽

Genomic Dna ◽

Bone Marrow Cells ◽

Mouse Bone Marrow ◽

Wild Type ◽

Bone Marrow Progenitor Cells ◽

Bone Marrow Progenitor ◽

Integration Sites

Abstract Abstract 3452 The cAMP Response Element Binding Protein, CREB, is a transcription factor that is critical for cell proliferation and survival in neuronal and hematopoietic cells. We previously reported that CREB is overexpressed in leukemic blasts from patients with AML. CREB overexpression is also associated with an increased risk of relapse and decreased event-free survival in AML patients. We generated transgenic mice in which CREB is expressed under the control of the myeloid specific promoter, hMRP8. In vitro, bone marrow progenitors from CREB transgenic mice have increased proliferative potential and replating ability and these mice develop myeloproliferative disease, but not AML. Therefore, CREB is not sufficient to fully transform hematopoietic cells. To identify genes that cooperate with CREB, we performed retroviral insertional mutagenesis with CREB or wildtype C57/Blk6 mice. Newborn mice were infected with the MOL4070LTR retrovirus. Leukemia was observed in both CREB transgenic and wild type mice, however, latency for disease was significantly shortened in CREB transgenic mice at 9 months vs. 14 months for wild type mice (p<0.001). Infected mice developed hepatosplenomegaly (weighing up to 10-fold more than spleens from uninfected wild type mice). The leukemic phenotype by FACs analysis showed that 75% (24/32 mice analyzed) had AML with blast cells expressing Gr-1/Mac-1. Therefore, wild type MOL4070LTR infected mice had decreased penetrance for myeloid disease (45% or 5/11 mice analyzed). To identify potential cooperating oncogenes, genomic DNA was subjected to long-mediated PCR analysis to amplify genomic sequences upstream of the virus. Viral integration sites were identified by Southern blot analysis with genomic DNA from spleens from mice with leukemia. These sequences were mapped to the mouse genome to identify the chromosomal location. Blast analysis of retroviral integration sites (RIS) was performed on the publicly available Ensembl database and compared to known common integration sites on the Retroviral Tagged Cancer Gene Database (RTCGD http://rtcgd.ncifcrf.gov/). Sequencing of integration sites identified previously known oncogenes, such as Evi1/Mds, Evi5/Gfi1, Myb/Ahi, and Ras. Common integration sites identified in multiple mice include, sox4, Evi5/Gfi1, Myb/Ahi, Cbfa2t3. The highest incidence of integration in the mutagenesis screen was in the sox4 gene. Sox4 is a transcription factor that regulates embryonic development and cell fate. Transduction of CREB transgenic mouse bone marrow progenitor cells with a sox4 retrovirus increased survival and self-renewal by 2-fold (p<0.01) in serial replating and methylcellulose colony assays compared to wild type bone marrow progenitors. CREB overexpressing bone marrow progenitor cells stained with Brdu had a 10% increase in cycling cells compared to wild type bone marrow progenitor cells transduced with sox4 retrovirus (p<0.0001). Consistent with this observation is that sox4 retroviral transduction of CREB transgenic bone marrow cells led to increased CREB and cyclin D expression (p<0.01 and p<0.001, respectively). CREB transgenic mouse bone marrow transduced with sox4 retrovirus and stained with Annexin/7AAD also showed decreased apoptosis when cultured in low serum and absence of growth factor (p<0.05). Furthermore, the expression of the sox4 gene was increased in bone marrow cells from AML patients that also overexpressed CREB. Our results indicate that sox4 and CREB cooperate and contribute to transformation of hematopoietic progenitor cells. Disclosures: No relevant conflicts of interest to declare.

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Retrovirus insertion site analysis of LGL leukemia patient genomes

10.1101/535997 ◽

2019 ◽

Author(s):

Weiling Li ◽

Lei Yang ◽

Robert S. Harris ◽

Lin Lin ◽

Thomas L. Olson ◽

...

Keyword(s):

Integration Site ◽

Insertion Site ◽

Endogenous Retrovirus ◽

European Ancestry ◽

Human Endogenous Retrovirus ◽

Reference Database ◽

Leukemia Patient ◽

Integration Sites ◽

Retrovirus Integration ◽

Lgl Leukemia

AbstractBackgroundLarge granular lymphocyte (LGL) leukemia is an uncommon cancer characterized by a sustained clonal proliferation of LGL cells. Antibodies reactive to retroviruses have been documented in the serum of patients with LGL leukemia. Culture or molecular approaches have to date not been successful in identifying a retrovirus.MethodsBecause a retrovirus must integrate into the genome of an infected cell, we focused our efforts on detecting a novel retrovirus integration site in the clonally expanded LGL cells. We present a new computational tool that uses long-insert mate pair sequence data to search the genome of LGL leukemia cells for retrovirus integration sites. We also utilize recently published methods to interrogate the status of polymorphic human endogenous retrovirus type K (HERV-K) provirus in patient genomes.ResultsWhile our analysis did not reveal any new retrovirus insertions in LGL genomes from LGL leukemia patients, we did identify four HERV-K provirus integration sites that are polymorphic in the human population and absent from the human reference genome, hg19. To determine if the prevalence of these or other polymorphic proviral HERV-Ks differed between LGL leukemia patients and the general population, we applied a recently developed approach that reports all sites in the human genome occupied by a proviral HERV-K. Using the 1000 genomes project (KGP) data as a reference database for HERV-K proviral prevalence at each polymorphic site, we show that there are significant differences in the number of polymorphic HERV-Ks in the genomes of LGL leukemia patients of European origin compared to individuals with European ancestry in the KGP data.ConclusionsOur study confirms that the integration of a new infectious or endogenous retrovirus does not cause the clonal expansion of LGL cells in LGL leukemia, although we do not rule out that these cells could be responding to retroviral antigens produced in other cell types. However, it is of interest that the burden of polymorphic proviral HERV-K is elevated in LGL leukemia patient genomes. Our research emphasizes the merits of comprehensive genomic assessment of HERV-K in cancer samples and suggests that further analyses to determine contributions of HERV-K to LGL leukemia are warranted.

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The Role of microRNA-155 in Mouse Models of MLL -AML

Blood ◽

10.1182/blood.v126.23.2446.2446 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2446-2446

Author(s):

Anna Staffas ◽

Edith Schneider ◽

Linda Fogelstrand ◽

Linda Röhner ◽

Michael Heuser ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Therapeutic Target ◽

Conflicts Of Interest ◽

Micro Rna ◽

Blood Analysis ◽

Leukemic Cells ◽

Mouse Bone Marrow ◽

Fusion Genes

Abstract Background: Genetic rearrangements that fuse the mixed lineage leukemia (MLL) gene, now termed KMT2A, to one of a variety of partners are seen in 5% - 20% of acute myeloid leukemia (AML). MLL -fusions are especially common in childhood AML and many of them are associated with poor prognosis. The MLL -fusions perturb transcription through different mechanisms and they are often associated with high expression of the transcription factors HOXA9 and MEIS1. Based on a micro-RNA screen in an AML mouse model mimicking the step-wise development of aggressive AML we have found that concurrent Hoxa9 and Meis1 overexpression is associated with upregulation of micro-RNA-155 (miR-155). Expression of miR-155 was also found to be higher in bone marrow samples from patients with MLL- AML compared with bone marrow from healthy donors (p <0.001), as were the expression of HOXA9 and MEIS1 (p <0.05). In lymphomas, miR-155 plays a pivotal role as an oncogene. It is frequently upregulated in samples from lymphoma patients and a mouse model of lymphoma showed a certain degree of miR-155-addiction which could be targeted by miR-155 inhibitors. Despite the differences in the pathobiology of AML and lymphoma, the upregulation of miR-155 in AML with high HOXA9 and MEIS1 expression may indicate miR-155 as a relevant therapeutic target also in MLL -AML. Methods: To test the importance of miR-155 and its potency as a drug target in MLL -AML we used a miR-155 knock-out mouse model (miR-155-/-) (Thai et al, Science, 2007). MLL -fusion genes of varying leukemic potential; MLL-AF5 (KMT2A-AFF4), MLL-ENL (KMT2A-MLLT1), MLL-AF9 (KMT2A-MLLT3) were retrovirally expressed in miR-155-/- mouse bone marrow (mbm) cells and in wild-type mbm cells (miR-155+/+). Results: In concordance with the previous findings in human AML patient samples, miR-155+/+ cells expressing MLL-AF5, MLL-ENL, or MLL-AF9 showed upregulation of miR-155 (p < 0.05). Also, Hoxa9 and Meis1 transcripts were increased (p<0.05). Interestingly, the magnitude of upregulation of both miR-155 and Meis1 correlated with the degree of aggressiveness based on disease latency and survival observed in these leukemia models with highest upregulation in MLL-ENL and MLL-AF9 and lowest in MLL-AF5 (p<0.05). Expression of the MLL-fusion genes in miR-155-/- mbm cells resulted in similar induction of Hoxa9 and Meis1 expression as in miR-155+/+ mbm cells, indicating that miR-155 is downstream of the Hoxa9/Meis1 axis. To determine the leukemic potential in vivo, we transplanted recipient mice with miR-155+/+ mbm cells and miR-155-/- mbm cells expressing MLL-ENL or MLL-AF9. Engraftment of leukemic cells, based on peripheral blood analysis, did not differ between mice transplanted with miR-155+/+ mbm cells and miR-155-/- mbm cells expressing MLL-fusions. Also, disease development induced by MLL-AF9 and MLL-ENL (4-8 weeks and 10-32 weeks, respectively) was similar in mice transplanted with miR-155-/- mbm cells and mice transplanted with miR-155+/+ mbm cells. In accordance with the in vivo results, functional studies in vitro showed that the proliferative capacity and colony forming ability of MLL -fusion expressing cells were similar in miR-155+/+ mbm cells and miR-155-/- mbm cells, indicating that miR-155 is not essential for MLL-ENL- or MLL-AF9-induced leukemic transformation. Conclusions: In summary, miR-155 is upregulated in MLL-AML in both mice and man, seemingly through an MLL>HOXA9/MEIS1>miR-155 axis. Since absence of miR-155 does not alter the leukemic potential induced by MLL-AF9 or MLL-ENL, miR-155 may contribute to, but is not pivotal for MLL leukemogenesis. We therefore conclude that miR-155 is not a therapeutic target in MLL- AML. Disclosures No relevant conflicts of interest to declare.

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A novel β-myosin heavy chain gene mutation, p.Met531Arg, identified in isolated left ventricular non-compaction in humans, results in left ventricular hypertrophy that progresses to dilation in a mouse model

Clinical Science ◽

10.1042/cs20070179 ◽

2008 ◽

Vol 114 (6) ◽

pp. 431-440 ◽

Cited By ~ 15

Author(s):

Tomoya Kaneda ◽

Chie Naruse ◽

Atsuhiro Kawashima ◽

Noboru Fujino ◽

Toru Oshima ◽

...

Keyword(s):

Mouse Model ◽

Transgenic Mice ◽

Dilated Cardiomyopathy ◽

Myosin Heavy Chain ◽

Heavy Chain ◽

Ventricular Hypertrophy ◽

Left Ventricular ◽

Chain Gene ◽

The Novel ◽

Sarcomeric Protein

Mutations in the βMHC (β-myosin heavy chain), a sarcomeric protein are responsible for hypertrophic and dilated cardiomyopathy. However, the mechanisms whereby distinct mutations in the βMHC gene cause two kinds of cardiomyopathy are still unclear. In the present study we report a novel βMHC mutation found in a patient with isolated LVNC [LV (left ventricular) non-compaction] and the phenotype of a mouse mutant model carrying the same mutation. To find the mutation responsible, we searched for genomic mutations in 99 unrelated probands with dilated cardiomyopathy and five probands with isolated LVNC, and identified a p.Met531Arg mutation in βMHC in a 13-year-old girl with isolated LVNC. Next, we generated six lines of transgenic mice carrying a p.Met532Arg mutant αMHC gene, which was identical with the p.Met531Arg mutation in the human βMHC. Among these, two lines with strong expression of the mutant αMHC gene were chosen for further studies. Although they did not exhibit the features characteristic of LVNC, approx. 50% and 70% of transgenic mice in each line displayed LVH (LV hypertrophy) by 2–3 months of age. Furthermore, LVD (LV dilation) developed in approx. 25% of transgenic mice by 18 months of age, demonstrating biphasic changes in LV wall thickness. The present study supports the idea that common mechanisms may be involved in LVH and LVD. The novel mouse model generated can provide important information for the understanding of the pathological processes and aetiology of cardiac dilation in humans.

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CVnCoV and CV2CoV protect human ACE2 transgenic mice from ancestral B BavPat1 and emerging B.1.351 SARS-CoV-2

Nature Communications ◽

10.1038/s41467-021-24339-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Donata Hoffmann ◽

Björn Corleis ◽

Susanne Rauch ◽

Nicole Roth ◽

Janine Mühe ◽

...

Keyword(s):

Mouse Model ◽

Transgenic Mice ◽

Neutralizing Antibody ◽

Transgenic Mouse Model ◽

Host Immunity ◽

The Novel ◽

Viral Strain ◽

Fast Development ◽

Antibody Titers ◽

Ancestral Strain

AbstractThe ongoing SARS-CoV-2 pandemic necessitates the fast development of vaccines. Recently, viral mutants termed variants of concern (VOC) which may escape host immunity have emerged. The efficacy of spike encoding mRNA vaccines (CVnCoV and CV2CoV) against the ancestral strain and the VOC B.1.351 was tested in a K18-hACE2 transgenic mouse model. Naive mice and mice immunized with a formalin-inactivated SARS-CoV-2 preparation were used as controls. mRNA-immunized mice develop elevated SARS-CoV-2 RBD-specific antibody and neutralization titers which are readily detectable, but significantly reduced against VOC B.1.351. The mRNA vaccines fully protect from disease and mortality caused by either viral strain. SARS-CoV-2 remains undetected in swabs, lung, or brain in these groups. Despite lower neutralizing antibody titers compared to the ancestral strain BavPat1, CVnCoV and CV2CoV show complete disease protection against the novel VOC B.1.351 in our studies.

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Sox4 cooperates with CREB in myeloid transformation

Blood ◽

10.1182/blood-2011-05-357418 ◽

2012 ◽

Vol 120 (1) ◽

pp. 155-165 ◽

Cited By ~ 28

Author(s):

Salemiz Sandoval ◽

Christina Kraus ◽

Er-Chieh Cho ◽

Michelle Cho ◽

Juraj Bies ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Transcription Factor ◽

Transgenic Mice ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Mouse Bone Marrow ◽

Mouse Bone Marrow Cells ◽

Acute Myeloid ◽

Marrow Cells

Abstract The cAMP response element-binding protein (CREB) is a nuclear transcription factor that is critical for normal and neoplastic hematopoiesis. Previous studies have demonstrated that CREB is a proto-oncogene whose overexpression promotes cellular proliferation in hematopoietic cells. Transgenic mice that overexpress CREB in myeloid cells develop a myeloproliferative disease with splenomegaly and aberrant myelopoiesis. However, CREB overexpressing mice do not spontaneously develop acute myeloid leukemia. In this study, we used retroviral insertional mutagenesis to identify genes that accelerate leukemia in CREB transgenic mice. Our mutagenesis screen identified several integration sites, including oncogenes Gfi1, Myb, and Ras. The Sox4 transcription factor was identified by our screen as a gene that cooperates with CREB in myeloid leukemogenesis. We show that the transduction of CREB transgenic mouse bone marrow cells with a Sox4 retrovirus increases survival and self-renewal of cells in vitro. Furthermore, leukemic blasts from the majority of acute myeloid leukemia patients have higher CREB, phosphorylated CREB, and Sox 4 protein expression. Sox4 transduction of mouse bone marrow cells results in increased expression of CREB target genes. We also demonstrate that CREB is a direct target of Sox4 by chromatin immunoprecipitation assays. These results indicate that Sox4 and CREB cooperate and contribute to increased proliferation of hematopoietic progenitor cells.

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A Transgenic Mouse Model To Study JAK2-V617F In Vivo .

Blood ◽

10.1182/blood.v108.11.665.665 ◽

2006 ◽

Vol 108 (11) ◽

pp. 665-665 ◽

Cited By ~ 2

Author(s):

Ralph Tiedt ◽

Hui Hao-Shen ◽

Radek C. Skoda

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Transgenic Mice ◽

Jak2 V617f ◽

Genetic Alterations ◽

Mouse Bone Marrow ◽

Upstream Sequence ◽

Wild Type ◽

Copy Numbers ◽

V617f Mutation

Abstract It is generally assumed that the JAK2-V617F mutation is the cause of disease in a large proportion of patients with myeloproliferative disorders. However, recent evidence suggests that additional genetic alterations might be collaborating with JAK2-V617F. Most importantly, screenings of hematopoietic cells from JAK2-V617F positive patients using quantitative PCR-based methods have revealed a strong variability in the fraction of cells that carry the mutation and in some patients a striking discrepancy with the extent of clonal hematopoiesis. On the other hand, retroviral overexpression of JAK2-V617F in mouse bone marrow transplantation models causes phenotypes that resemble human polycythemia vera, the MPD entity in which the incidence of JAK2-V617F is highest. In the mouse transplantation models described to date, retroviral vectors were used that bear the risk of insertional mutagenesis and in most cases lead to a strong overexpression of JAK2-V617F. These limitations might hamper an accurate reproduction of the human disease. To express the mutant JAK2 at physiological levels in a mouse model, we generated JAK2-V617F transgenic mice. The transgene was constructed based on a human BAC containing JAK2 exons 1–12 and approximately 100 kb of upstream sequence. A cDNA fragment covering exons 13–25 of JAK2, which includes the V617F mutation, was appended by homologous recombination in bacteria. We also generated an identical construct with the JAK2 wild type sequence as a control. Pronuclear microinjection of the control construct yielded 8 viable and fertile founders with transgene copy numbers ranging from 1 to 18. We also obtained 3 mice that were positive for the JAK2-V617F transgene. One of these mice died 2 days after birth, a second mouse died at the age of 5 weeks. This mouse had 3 integrated copies of the transgene and interestingly an approximately 10-fold enlarged spleen. Unfortunately, we were unable to obtain additional data from this mouse. The third mouse with one integrated copy of the transgene was viable and gave rise to a transgenic line (VF1). The VF1 line expresses the transgene at approximately 25% of the endogenous wild-type Jak2 level. We analyzed blood counts at the age of 5–6 weeks and observed a small but significant elevation of platelet numbers (approximately 1.2-fold), which was reproduced when transplanting JAK2-VF1 bone marrow into wild type recipients. No changes in hematocrit or white blood cell count were detected. The low yield of JAK2-V617F transgenic mice together with the early death of two of the founders suggested that the transgene could be lethal when expression exceeds a certain level. To overcome this potential problem and to allow for inducible expression of the JAK2-V617F transgene, we generated a second construct in which exons 13–25 of the original transgene were inverted to abolish expression of catalytically active Jak2 in this configuration. Antiparallel loxP sites were placed at the borders of this inverted fragment to allow Cre-mediated repair of the transgene. These loxP sites contain previously characterized mutations to prevent continuous recombination. We have obtained 4 founders of this Cre-activated JAK2-V617F transgene with copy numbers between 1 and 9. After crossing to Mx-Cre mice and inducing Cre we observed repair of the transgene at the DNA level. Furthermore, mRNA of the recombined JAK2-V617F allele was detectable in peripheral blood. The effects of the JAK2-V617F transgene induction on the hematopoietic system are currently being studied in detail and will be presented.

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Evi-2, a common integration site involved in murine myeloid leukemogenesis.

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4658 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4658-4666 ◽

Cited By ~ 67

Author(s):

A M Buchberg ◽

H G Bedigian ◽

N A Jenkins ◽

N G Copeland

Keyword(s):

Leucine Zipper ◽

Transmembrane Domain ◽

Integration Site ◽

Transmembrane Protein ◽

Structural Features ◽

Viral Integration ◽

Integration Sites ◽

Leukemia Viruses ◽

Common Integration Site

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

Blood ◽

10.1182/blood.v112.11.4484.4484 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4484-4484

Author(s):

Hui Zhang ◽

Wendy Chen ◽

Svetlana Rogulina ◽

Han M. Lee ◽

Bernard G. Forget

Keyword(s):

Bone Marrow ◽

Myeloid Leukemia ◽

Integration Site ◽

Fold Increase ◽

Leukemic Cells ◽

Retroviral Integration ◽

Intron 1 ◽

Integration Sites

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

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