RUNX1 mutations promote leukemogenesis of myeloid malignancies in ASXL1-mutated leukemia

Abstract Background Additional sex combs-like 1 (ASXL1) mutations have been described in all forms of myeloid neoplasms including chronic myelomonocytic leukemia (CMML) and associated with inferior outcomes, yet the molecular pathogenesis of ASXL1 mutations (ASXL1-MT) remains poorly understood. Transformation of CMML to secondary AML (sAML) is one of the leading causes of death in CMML patients. Previously, we observed that transcription factor RUNX1 mutations (RUNX1-MT) coexisted with ASXL1-MT in CMML and at myeloid blast phase of chronic myeloid leukemia. The contribution of RUNX1 mutations in the pathogenesis of myeloid transformation in ASXL1-mutated leukemia, however, remains unclear. Methods To evaluate the leukemogenic role of RUNX1-MT in ASXL1-mutated cells, we co-expressed RUNX1-MT (R135T) and ASXL1-MT (R693X) in different cell lines and performed immunoblot, co-immunoprecipitation, gene expression microarray, quantitative RT-PCR, cell proliferation, differentiation, and clonogenic assays for in vitro functional analyses. The in vivo effect was investigated using the C57BL/6 mouse bone marrow transplantation (BMT) model. Results Co-expression of two mutant genes increased myeloid stem cells in animal model, suggesting that cooperation of RUNX1 and ASXL1 mutations played a critical role in leukemia transformation. The expression of RUNX1 mutant in ASXL1-mutated myeloid cells augmented proliferation, blocked differentiation, and increased self-renewal activity. At 9 months post-BMT, mice harboring combined RUNX1 and ASXL1 mutations developed disease characterized by marked splenomegaly, hepatomegaly, and leukocytosis with a shorter latency. Mice transduced with both ASXL1 and RUNX1 mutations enhanced inhibitor of DNA binding 1 (ID1) expression in the spleen, liver, and bone marrow cells. Bone marrow samples from CMML showed that ID1 overexpressed in coexisted mutations of RUNX1 and ASXL1 compared to normal control and either RUNX1-MT or ASXL1-MT samples. Moreover, the RUNX1 mutant protein was more stable than WT and increased HIF1-α and its target ID1 gene expression in ASXL1 mutant cells. Conclusion The present study demonstrated the biological and functional evidence for the critical role of RUNX1-MT in ASXL1-mutated leukemia in the pathogenesis of myeloid malignancies.

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Abstract 454: Myeloid CD98hc Deficiency Reduces Atherosclerotic Plaque Development via Impaired Proliferation of Macrophages

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.454 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Sara McCurdy ◽

William A Boisvert

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

Atherosclerotic Plaque ◽

Bone Marrow Cells ◽

Transmembrane Protein ◽

Mouse Bone Marrow ◽

Plaque Development ◽

Primary Mouse

Macrophage accumulation is a key process affecting all stages of atherosclerosis. Whether these cells accumulate in plaque solely by recruitment of monocytes from circulation or by proliferation within the plaque is an important question that has garnered much interest in recent years. Originally identified as a lymphocyte activation marker, CD98hc (SLC3A2) is a transmembrane protein involved in cell proliferation and survival via integrin signaling and MAP kinase activation. We hypothesized that CD98hc deficiency in myeloid cells would have a protective effect on atherosclerosis development and plaque composition by limiting macrophage proliferation. For the studies described, we utilized mice with myeloid-specific deletion of the CD98hc ( CD98hc fl/fl LysMCre + ) to determine the effects of CD98hc deficiency on macrophage function in the context of atherosclerosis . We performed in vitro assays to investigate the role of CD98hc in the proliferation and survival of primary mouse bone marrow derived macrophages. Although we found no differences in the number of bone marrow cells isolated from control or CD98hc -/- animals, after differentiation with MCS-F for 7 days, the number of macrophages obtained from CD98hc -/- mice was approximately 80% lower (7.2 ± 2.2 x 10 6 vs. 42.4 ± 4.6 x 10 6 per mouse) compared to control mice. Proliferation assays in vitro using EdU revealed approximately 50% (15.4 ± 2.5% vs. 7.5±1.8%) reduced cell proliferation in CD98hc -/- macrophages compared to control cells that could not be rescued with the addition M-CSF. In a 6-week atherosclerosis study using Ldlr -/- CD98hc fl/fl LysMCre + mice, Oil-Red O staining of whole aortae as well as aortic sinus sections showed that atherosclerotic plaque development was reduced compared to Ldlr -/- CD98hc fl/fl LysMCre - control mice. Additionally, immunohistochemical staining of atherosclerotic tissues revealed a reduction in macrophage abundance and proliferation within the plaque of Ldlr -/- CD98hc fl/fl LysMCre + mice compared to control mice. These findings support an important role of CD98hc in macrophage proliferation within the plaque environment, and provide a novel target for reducing atherosclerosis.

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CREB Plays a Critical Role in the Regulation of Normal and Malignant Hematopoiesis.

Blood ◽

10.1182/blood.v110.11.1224.1224 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1224-1224

Author(s):

Jerry C. Cheng ◽

Dejah Judelson ◽

Kentaro Kinjo ◽

Jenny Chang ◽

Elliot Landaw ◽

...

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

Progenitor Cells ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Leukemia Cells ◽

Mouse Bone Marrow ◽

T315i Mutation

Abstract The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, memory, and glucose metabolism. We previously demonstrated that CREB overexpression is associated with an increased risk of relapse in a small cohort of adult acute myeloid leukemia (AML) patients. Transgenic mice that overexpress CREB in myeloid cells develop myeloproliferative/myelodysplastic syndrome after one year. Bone marrow cells from these mice have increased self-renewal and proliferation. To study the expression of CREB in normal hematopoiesis, we performed quantitative real-time PCR in both mouse and human hematopoietic stem cells (HSCs). CREB expression was highest in the lineage negative population and was expressed in mouse HSCs, common myeloid progenitors, granulocyte/monocyte progenitors, megakaryocyte/erythroid progenitors, and in human CD34+38- cells. To understand the requirement of CREB in normal HSCs and myeloid leukemia cells, we inhibited CREB expression using RNA interference in vitro and in vivo. Bone marrow progenitor cells infected with CREB shRNA lentivirus demonstrated a 5-fold decrease in CFU-GM but increased Gr-1/Mac-1+ cells compared to vector control infected cells (p<0.05). There were fewer terminally differentiated Mac-1+ cells in the CREB shRNA transduced cells (30%) compared to vector control (50%), suggesting that CREB is critical for both myeloid cell proliferation and differentiation. CREB downregulation also resulted in increased apoptosis of mouse bone marrow progenitor cells. Given our in vitro results, we transplanted sublethally irradiated mice with mouse bone marrow cells transduced with CREB or scrambled shRNA. At 5 weeks post-transplant, we observed increased Gr-1+/Mac-1+ cells in mice infused with CREB shRNA transduced bone marrow compared to controls. After 12 weeks post-transplant, there was no difference in hematopoietic reconstitution or in the percentage of cells expressing Gr-1+, Mac-1+, Gr-1/Mac-1+, B22-+, CD3+, Ter119+, or HSCs markers, suggesting that CREB is not required for HSC engraftment. To study the effects of CREB knockdown in myeloid leukemia cells, K562 and TF-1 cells were infected with CREB shRNA lentivirus, sorted for GFP expression, and analyzed for CREB expression and proliferation. Within 72 hours, cells transduced with CREB shRNA demonstrated decreased proliferation and survival with increased apoptosis. In cell cycle experiments, we observed increased numbers of cells in G1 and G2/M with CREB downregulation. Expression of cyclins A1 and D, which are known target genes of CREB, was statistically significantly decreased in TF-1 and K562 cells transduced with CREB shRNA lentivirus compared to controls. To study the in vivo effects of CREB knockdown on leukemic progression, we injected SCID mice with Ba/F3 cells expressing bcr-abl or bcr-abl with the T315I mutation and the luciferase reporter gene. Cells were transduced with either CREB or scrambled shRNA. Disease progression was monitored using bioluminescence imaging. The median survival of mice injected with CREB shRNA transduced Ba/F3 bcr-abl or bcr-abl with the T315I mutation was increased with CREB downregulation compared to controls (p<0.05). Our results demonstrate that CREB is a critical regulator of normal and neoplastic hematopoiesis both in vitro and in vivo.

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G-CSF Mediated Decrease in Bone-Marrow SDF-1 Level Is Neutrophil Dependent but CD26 Independent

Blood ◽

10.1182/blood.v112.11.3469.3469 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3469-3469

Author(s):

Pratibha Singh ◽

Seiji Fukuda ◽

Janardhan Sampath ◽

Louis M. Pelus

Keyword(s):

Bone Marrow ◽

Magnetic Beads ◽

Critical Role ◽

Alternative Mechanism ◽

Wild Type ◽

Hematopoietic Stem ◽

Osteoblast Activity

Abstract Interaction of CXCR4 expressed on hematopoietic stem and progenitor cells (HSPC) with bone-marrow stromal SDF-1 is believed to play a central role in retention or mobilization of HSPC. Recently, a mobilization regimen of G-CSF was shown to decrease osteoblast number resulting in reduced levels of bone-marrow SDF-1, however the detailed mechanism leading to this reduction is currently unknown. It is unlikely that G-CSF directly regulates osteoblast SDF-1 production since osteoblasts do not express G-CSF receptor. Proteolytic cleavage of SDF-1 by peptidase CD26 in the bone-marrow may be an alternative mechanism responsible for reduction of SDF-1 level. Although CD26 can cleave SDF-1 in vitro, direct evidence of SDF-1 cleavage by CD26 in vivo during G-CSF induced HSPC mobilization has not been demonstrated. We previously demonstrated that neutrophils are required for G-CSF induced HSPC mobilization and that CD26 expression on neutrophils, rather than HSPC, is critical for mobilization. To more fully understand the role of CD26 in altering SDF-1 protein/activity during G-CSF induced HSPC mobilization, we quantitated bone-marrow SDF-1 levels in CD26−/− and wild-type CD26+/+ mice by ELISA during G-CSF administration. A standard 4 day G-CSF mobilization regimen (100 μg/kg bid, sc × 4 days) decreased bone-marrow total SDF-1 from 4.55±0.3 to 0.52±0.06 ng/femur in wild-type CD26+/+ mice (8.7-fold) and from 4.51±0.3 to 0.53±0.05 ng/femur (8.5-fold) in CD26−/− mice. However, despite an equivalent decrease in SDF-1, total CFU mobilization and the absolute number of mobilized SKL cells were decreased (3.1 and 2.0 fold lower, respectively) in CD26−/− mice compared to wild-type CD26+/+ controls. These results suggest that the decrease in total SDF-1 level in marrow seen following G-CSF treatment is independent of CD26. Cytological examination of bone-marrow smears showed that the reduction in SDF-1 levels in bone-marrow of both wild-type CD26+/+ and CD26−/− mice following G-CSF administration correlated with an increase in total absolute bone-marrow neutrophil cell number, suggesting a role for neutrophils in modulation of SDF-1 protein. To determine if neutrophils affect osteoblast SDF-1 production, bone marrow Gr-1+ neutrophils from wild-type CD26+/+ and CD26−/− mice were purified using anti-Ly6G magnetic beads and co-cultured with MC3T3-E1 preosteoblasts in vitro. Gr-1+ neutrophils from both wild-type and CD26−/− mice decreased pre-osteoblast SDF-1 production by similar amounts (15.4-fold vs 14.8-fold respectively), while Gr-1 neg cells from both wild-type CD26+/+ or CD26−/− were without effect on SDF-1 levels. Similarly, Gr-1+ neutrophils from both wild-type and CD26−/− mice decreased SDF-1 produced by MC3T3-E1-derived osteoblasts from 1.85±0.3 to 0.52±0.06 ng/ml (3.5 fold) and 0.56±0.07 ng/ml (3.3 fold) respectively, with Gr-1neg cells having no effect. Gr-1+ neutrophils either from wild-type or CD26−/− mice, but not Gr-1neg cells, significantly induced apoptosis of MC3T3-E1 cells as measured by Annexin-V staining (70.5%±10.2 vs 71.2%±12.5 for wild-type CD26+/+ and CD26−/− neutrophils respectively) and significantly inhibited osteoblast activity (20-fold vs 20.6-fold for CD26+/+ and CD26−/− neutrophils respectively) as measured by osteocalcin expression. Furthermore, irrespective of G-CSF treatment, an inverse correlation between absolute neutrophil number and SDF-1 protein levels was observed, suggesting that G-CSF induces neutrophil expansion but does not directly affect SDF-1 production. Collectively, these results provide additional support for the critical role of neutrophils in G-CSF induced mobilization and strongly suggested that neutrophils directly regulate bone-marrow SDF-1 levels independent of CD26 activity.

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Genotoxicity Assessment of Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)

International Journal of Toxicology ◽

10.1080/10915810500366922 ◽

2005 ◽

Vol 24 (6) ◽

pp. 427-434 ◽

Cited By ~ 9

Author(s):

Gunda Reddy ◽

Gregory L. Erexson ◽

Maria A. Cifone ◽

Michael A. Major ◽

Glenn J. Leach

Keyword(s):

Bone Marrow ◽

World War Ii ◽

Environmental Protection Agency ◽

Bone Marrow Cells ◽

Metabolic Activation ◽

Maximum Tolerated Dose ◽

Mouse Bone Marrow ◽

Mouse Lymphoma

Hexahydro-1,3,5-trinitro-1,3,5-triazine, a polynitramine compound, commonly known as RDX, has been used as an explosive in military munitions formulations since World War II. There is considerable data available regarding the toxicity and carcinogenicity of RDX. It has been classified as a possible carcinogen (U.S. Environmental Protection Agency, Integrated Risk Information System, 2005, www.epa.gov/IRIS/subst/0313.htm ). In order to better understand its gentoxic potential, the authors conducted the in vitro mouse lymphoma forward mutation and the in vivo mouse bone marrow micronucleus assays. Pure RDX (99.99%) at concentrations ranging from 3.93 to 500 μg/ml showed no cytotoxicity and no mutagenicity in forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, with and without metabolic activation. This finding was also confirmed by repeat assays under identical conditions. In addition, RDX did not induce micronuclei in mouse bone marrow cells when tested to the maximum tolerated dose of 250 mg/kg in male mice. These results show that RDX was not mutagenic in these in vitro and in vivo mammalian systems.

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ASXL2 Is Recurrently Mutated in t(8;21) AML and Regulates Hematopoietic Development

Blood ◽

10.1182/blood.v128.22.1051.1051 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1051-1051

Author(s):

Vikas Madan ◽

Lin Han ◽

Norimichi Hattori ◽

Anand Mayakonda ◽

Qiao-Yang Sun ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Research Funding ◽

Hematopoietic Differentiation ◽

Wild Type ◽

Flow Cytometric ◽

Deficient Mice

Abstract Chromosomal translocation t(8;21) (q22;q22) leading to generation of oncogenic RUNX1-RUNX1T1 fusion is a cytogenetic abnormality observed in about 10% of acute myelogenous leukemia (AML). Studies in animal models and recent next generation sequencing approaches have suggested cooperativity of secondary genetic lesions with t(8;21) in inducing leukemogenesis. In this study, we used targeted and whole exome sequencing of 93 cases (including 30 with matched relapse samples) to profile the mutational landscape of t(8;21) AML at initial diagnosis and post-therapy relapse. We identified recurrent mutations of KIT, TET2, MGA, FLT3, NRAS, DHX15, ASXL1 and KMT2Dgenes in this subtype of AML. In addition, high frequency of truncating alterations in ASXL2 gene (19%) also occurred in our cohort. ASXL2 is a member of mammalian ASXL family involved in epigenetic regulation through recruitment of polycomb or trithorax complexes. Unlike its closely related homolog ASXL1, which is mutated in several hematological malignancies including AML, MDS, MPN and others; mutations of ASXL2 occur specifically in t(8;21) AML. We observed that lentiviral shRNA-mediated silencing of ASXL2 impaired in vitro differentiation of t(8;21) AML cell line, Kasumi-1, and enhanced its colony forming ability. Gene expression analysis uncovered dysregulated expression of several key hematopoiesis genes such as IKZF2, JAG1, TAL1 and ARID5B in ASXL2 knockdown Kasumi-1 cells. Further, to investigate implications of loss of ASXL2 in vivo, we examined hematopoiesis in Asxl2 deficient mice. We observed an age-dependent increase in white blood cell count in the peripheral blood of Asxl2 KO mice. Myeloid progenitors from Asxl2 deficient mice possessed higher re-plating ability and displayed altered differentiation potential in vitro. Flow cytometric analysis of >1 year old mice revealed increased proportion of Lin-Sca1+Kit+ (LSK) cells in the bone marrow of Asxl2 deficient mice, while the overall bone marrow cellularity was significantly reduced. In vivo 5-bromo-2'-deoxyuridine incorporation assay showed increased cycling of LSK cells in mice lacking Asxl2. Asxl2 deficiency also led to perturbed maturation of myeloid and erythroid precursors in the bone marrow, which resulted in altered proportions of mature myeloid populations in spleen and peripheral blood. Further, splenomegaly was observed in old ASXL2 KO mice and histological and flow cytometric examination of ASXL2 deficient spleens demonstrated increased extramedullary hematopoiesis and myeloproliferation compared with the wild-type controls. Surprisingly, loss of ASXL2 also led to impaired T cell development as indicated by severe block in maturation of CD4-CD8- double negative (DN) population in mice >1 year old. These findings established a critical role of Asxl2 in maintaining steady state hematopoiesis. To gain mechanistic insights into its role during hematopoietic differentiation, we investigated changes in histone marks and gene expression affected by loss of Asxl2. Whole transcriptome sequencing of LSK population revealed dysregulated expression of key myeloid-specific genes including Mpo, Ltf, Ngp Ctsg, Camp and Csf1rin cells lacking Asxl2 compared to wild-type control. Asxl2 deficiency also caused changes in histone modifications, specifically H3K27 trimethylation levels were decreased and H2AK119 ubiquitination levels were increased in Asxl2 KO bone marrow cells. Global changes in histone marks in control and Asxl2 deficient mice are being investigated using ChIP-Sequencing. Finally, to examine cooperativity between the loss of Asxl2 and RUNX1-RUNX1T1 in leukemogenesis, KO and wild-type fetal liver cells were transduced with retrovirus expressing AML1-ETO 9a oncogene and transplanted into irradiated recipient mice, the results of this ongoing study will be discussed. Overall, our sequencing studies have identified ASXL2 as a gene frequently altered in t(8;21) AML. Functional studies in mouse model reveal that loss of ASXL2 causes defects in hematopoietic differentiation and leads to myeloproliferation, suggesting an essential role of ASXL2 in normal and malignant hematopoiesis. *LH and NH contributed equally Disclosures Ogawa: Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

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A comparative study of the effects of genistein, estradiol and raloxifene on the murine skeletal system.

Acta Biochimica Polonica ◽

10.18388/abp.2009_2458 ◽

2009 ◽

Vol 56 (2) ◽

Cited By ~ 15

Author(s):

Leszek Sliwiński ◽

Joanna Folwarczna ◽

Barbara Nowińska ◽

Urszula Cegieła ◽

Maria Pytlik ◽

...

Keyword(s):

Mechanical Properties ◽

Bone Marrow ◽

Mrna Expression ◽

Bone Marrow Cells ◽

Bone Mineralization ◽

Mouse Bone Marrow ◽

Skeletal System ◽

Mouse Bone Marrow Cells

Genistein, a major phytoestrogen of soy, is considered a potential drug for prevention and treatment of postmenopausal osteoporosis. The aim of the present study was to compare the effects of genistein, estradiol and raloxifene on the skeletal system in vivo and in vitro. Genistein (5 mg/kg), estradiol (0.1 mg/kg) or raloxifene hydrochloride (5 mg/kg) were administered daily by a stomach tube to mature ovariectomized Wistar rats for 4 weeks. Bone mass, mineral and calcium content, macrometric parameters and mechanical properties were examined. Also the effects of genistein, estradiol and raloxifene (10(-9)-10(-7) M) on the formation of osteoclasts from neonatal mouse bone marrow cells and the activity of osteoblasts isolated from neonatal mouse calvariae were compared. In vivo, estrogen deficiency resulted in the impairment of bone mineralization and bone mechanical properties. Raloxifene but not estradiol or genistein improved bone mineralization. Estradiol fully normalized the bone mechanical properties, whereas genistein augmented the deleterious effect of estrogen-deficiency on bone strength. In vitro, genistein, estradiol and raloxifene inhibited osteoclast formation from mouse bone marrow cells, decreasing the ratio of RANKL mRNA to osteoprotegerin mRNA expression in osteoblasts. Genistein, but not estradiol or raloxifene, decreased the ratio of alkaline phosphatase mRNA to ectonucleotide pyrophosphatase phosphodiesterase 1 mRNA expression in osteoblasts. This difference may explain the lack of genistein effect on bone mineralization observed in ovariectomized rats in the in vivo study. Concluding, our experiments demonstrated profound differences between the activities of genistein, estradiol and raloxifene towards the osseous tissue in experimental conditions.

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Frequent in Vivo redundancy of C/EBPβ and C/EBPε during Myeloid Development

Blood ◽

10.1182/blood.v112.11.2440.2440 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2440-2440

Author(s):

Nils Heinrich Thoennissen ◽

Tadayuki Akagi ◽

Sam Abbassi ◽

Daniel Nowak ◽

Ann George ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Transcription Factors ◽

Bone Marrow Cells ◽

Double Knockout ◽

Hematopoietic Stem ◽

Gm Csf ◽

Marrow Cells

Abstract CCAAT/enhancer binding protein (C/EBP) transcription factors are involved in a variety of cellular responses including proliferation and differentiation. Although C/EBPβ and C/EBPε are believed to be most important for macrophage and granulocyte activity, respectively, experiments by others and ourselves suggest a possible overlap in their function in myelopoiesis. In order to explore further this potential redundancy, we assessed the in vivo and in vitro function of both transcription factors by generating a double knockout (KO) germline murine model (C/EBPβ/ε−/−/−/−) and compared their hematopoiesis to those of single deficient (C/EBPβ−/−, C/EBPε−/−) and wild-type (WT) mice. Gene expression analysis of bone marrow cells showed expression of C/EBPβ in C/EBPε−/− and WT mice, and vice versa. The weight of the double-KO mice was significantly less as measured at 4 weeks of age (11.5 ± 0.9 g) compared to WT (13.4 ± 0.6 g), C/EBPβ−/− (14.5 ± 1.4 g), and C/EBPε−/− mice (15.4 ± 2.3 g) (p < 0.05). The double-KO mice were prone to infections of the eyes, lungs, liver, and peritoneum. In contrast, C/EBPβ−/−, C/EBPε−/− and WT mice demonstrated no signs of infection. Microscopic imaging of peripheral blood showed metamyelocytes and myelocytes in the double-KO mice. FACS analysis found that the fraction of bone marrow cells which were Lin(−) (no expression of B220, CD3, Gr1, Ter119, and Mac1) were modestly elevated in double-KO and C/EBPβ−/− mice (8.42 % and 8.1 %, respectively) compared to C/EBPε−/− (4.24 %) and WT (3.93 %) mice. A subanalysis highlighted an elevated level of B220(−)/Gr1(−) bone marrow cells in the double-KO mice (54 %) compared to the levels in the C/EBPβ−/− (31 %), C/EBPε−/− (33 %) and WT (21.5 %) mice. Moreover, the proportion of hematopoietic stem cells in the bone marrow were significantly increased in the hematopoietic stem cell compartment [Sca1(+)/c-Kit(+)] in the double-KO mice (20.8 %) compared to the C/EBPβ−/− (6.9 %), C/EBPε−/− (5.9 %) and WT (6.9 %) mice. When given a cytotoxic stress (5-FU) to kill cycling hematopoietic progenitor cells, the mean neutrophil count at their nadir (day 4) was 0.14 × 109 cells/L in the double-KO mice compared to 0.71 × 109 cells/L in the WT mice (p < 0.001); both reached normal values again on day 10. Taken together, these results indicated a relatively higher percentage of immature hematopoietic cells in the double-KO mice compared to the WT mice. Nevertheless, clonogenic assays in methylcellulose using bone marrow cells of the double-KO showed a significant decreased number of myeloid colonies. For example, in the presence of G-CSF, GM-CSF, and SCF, a mean of 83 ± 10 hematopoietic colonies formed in the double-KO mice compared to 135 ± 6 in C/EBPβ−/−, 159 ± 12 in C/EBPε−/− and 165 ± 2 in WT mice (p < 0.001, double-KO vs. WT). Similar clonogenic results occurred when bone marrow cells were stimulated with either G-CSF, GM-CSF or SCF/G-CSF alone. Although our in vitro experiments suggested that double-KO mice had a decreased clonogenic response to G-CSF, their bone marrow cells had normal levels of phosphorylated STAT3 protein when stimulated with G-CSF. Hence, the G-CSFR and its secondary signaling pathway seemed to be intact. In further experiments, downstream targets of the C/EBP transcription factors were examined. Bone marrow macrophages activated with LPS and IFNγ from both double-KO and C/EBPβ−/− mice had decreased gene expression of IL6, IL12p35, TNFα, and G-CSF compared to the levels detected in macrophages of C/EBPε−/− and WT. Interestingly, expression levels of cathelicidin antimicrobial peptide (CAMP) were similarly robust in the macrophages from C/EBPβ−/−, C/EBPε−/−, and WT mice. In sharp contrast, CAMP expression was undetectable in the activated macrophages of the double-KO mice. In conclusion, the phenotype of the double-KO mice was often distinct from the C/EBPβ−/− and C/EBPε−/− mice suggesting a redundancy of activity of both transcription factors in myeloid hematopoiesis.

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In Vitro expanded STAT1−/− Regulatory T Cells Prevent Acute Graft Versus Host Disease (GVHD) and Promote Donor Cell Engraftment In Allogeneic Fully MHC-Mismatched Bone Marrow Transplant

Blood ◽

10.1182/blood.v116.21.732.732 ◽

2010 ◽

Vol 116 (21) ◽

pp. 732-732

Author(s):

Huihui Ma ◽

Caisheng Lu ◽

Judy Ziegler ◽

Suzanne Lentzsch ◽

Markus Y Mapara

Keyword(s):

Bone Marrow ◽

T Cells ◽

Bone Marrow Cells ◽

Donor Cell ◽

Treg Cells ◽

Cell Engraftment ◽

Lethal Irradiation

Abstract Abstract 732 Treg cells have been recognized as critical regulators of the immune response and shown to prevent the development of GVHD. However, little is known about of the role of STAT1 signaling in Treg cells during the development of GVHD. In this study, we tried to investigate how STAT1 signaling controls donor Treg development and function in the setting of GVHD. For this purpose we studied the role of STAT1 in natural and inducible Treg (nTreg and iTreg, respectively). To better understand the influence of STAT1-deficiency on the proliferation of nTreg cells, purified splenic STAT1−/− or STAT1+/+ CD4+CD25+ cells were labeled with Carboxyfluorescein succinimidyl ester (CFSE) and cultured on anti-CD3 coated plates in the presence of anti-CD28 and IL-2 for 3 days and analyzed for proliferation and viability. After 72h of in vitro culture 50% of the STAT1+/+ starting population were no longer viable compared to only 10% of STAT1−/− cells. Furthermore, we noted a significantly increased expansion of STAT1-deficient CD4+CD25+Foxp3+ Treg cells compared to STAT1+/+ Treg cells (p<0.001). In line with these findings, STAT1-deficiency resulted in a significantly higher proportion of CFSElo cells indicating vigorous proliferation (85% Foxp3+CFSElo in STAT1−/− compared to only 65% Foxp3+CFSElo in STAT1+/+ Treg cells. Furthermore, at the end of the culture 30% of the STAT1+/+ CD4+CD25+ population were Foxp3-negative compared to only 10% of the STAT1−/− cells. We next determined the impact of STAT1 on the generation of iTreg cells in vitro. For this purpose CD4+CD25− cells from STAT1−/− or STAT1+/+ mice were cultured for 3 days on anti-CD3 coated plates in the presence of anti-CD28 antibodies, hTGF-β, mIL-2, anti-IFN-γ and anti-IL-4 for 3 days. Compared to STAT1+/+, we observed significantly enhanced generation of iTregs from STAT1−/− splenocytes (19.9%±3.0% vs. 10.6%±1.3%, p=0.008). We then performed studies to assess the in vivo generation of iTreg. For that purpose BALB/c mice were reconstituted with T Cell Depleted (TCD) 129.STAT1+/+Bone Marrow Cells (BMC) following lethal irradiation and recipients were co-injected with CD4+CD25− cells purified from either 129.STAT1+/+ or 129.STAT1−/− splenocytes. We again noted a significantly higher proportion of CD4+CD25+ Foxp3+ cells in recipients of CD4+CD25−STAT1−/− cells compared to recipients of STAT1+/+ T cells indicating a significantly increased conversion of CD4+CD25- cells into Treg cells. To confirm the in vitro results we tested the functional ability of in vitro expanded (using anti-CD3, anti-CD28, IL-2 and TGF-β) STAT1+/+ or STAT1−/− Treg cells to block induction of GVHD. GVHD was induced in BALB/c mice following lethal irradiation (800rad) and fully MHC-mismatched BMT using 129.STAT1+/+ bone marrow cells plus 129.STAT+/+ conventional T cells (Tcon). Animals were co-injected with expanded Treg cells from either 129.STAT1+/+ or 129.STAT1−/− donors at a ratio of 1:1 or 1:4 (Treg:Tcon). STAT1−/− or STAT1+/+ Treg cells were equipotent in completely preventing GVHD mortality. However, compared to recipients of STAT1+/+ Treg recipients of STAT1−/− Treg showed reduced signs of GVHD morbidity as determined by a significantly improved weight development. Furthermore, recipients of STAT1−/− Treg showed significantly increased donor cell engraftment compared to recipients of STAT1+/+Treg (donor CD4+ [87% vs. 60%, p=0.03], CD8+[99% vs. 96%, p=0.04], Mac1+[96% vs. 77%, p=0.02] and B220+[100% vs. 96%, p=0.007]) cells in the recipient spleen. These observations clearly demonstrate that STAT1 is a critical regulator of Treg cell development and expansion and that targeting STAT1 in CD4+ T cells may facilitate in vitro and in vivo generation/expansion of Treg cells for therapeutic use in GVHD while also promoting donor cell engraftment. Disclosures: Lentzsch: Celgene Corp: Research Funding. Mapara:Resolvyx: Research Funding; Gentium: stocks.

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Cellular and Molecular Targets of MLL-AF9 in a Novel Conditional Mouse Model

Blood ◽

10.1182/blood.v120.21.1280.1280 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1280-1280

Author(s):

Vaia Stavropoulou ◽

Susanne Kaspar ◽

Laurent Brault ◽

Sabine Juge ◽

Stefano Morettini ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Mouse Model ◽

Prognostic Markers ◽

Bone Marrow Cells ◽

Median Latency ◽

Hematopoietic Stem ◽

Marrow Cells

Abstract Abstract 1280 Previous studies have shown that the expression of several leukemia-associated mixed lineage leukemia (MLL) fusion genes transformed human and mouse bone marrow cells in vitro and in vivo. In order to dissect the molecular and cellular targets of the MLL-AF9 fusion, we generated a novel inducible doxycycline (DOX)-regulated transgenic mouse model. Conditional ex vivo activation of MLL-AF9 induced aberrant self-renewal and impaired differentiation of long-term or short-term hematopoietic stem (LT-HSC and ST-HSC), common myeloid progenitor (CMP) and granulocyte-macrophage progenitor (GMP) cells in a fully reversible manner. Direct activation of the fusion in vivo or after transplantation of transgenic bone marrow cells into irradiated hosts induced an aggressive and transplantable disease after a median latency of 80days characterized as acute myelo-monocytic leukemia closely mimicking the human disease. Fusion gene expression and leukemia induction was DOX dosage dependent and reversible upon DOX removal. Activation of MLL-AF9 in isolated LT-HSC or GMP cells in vitro or in vivo resulted in the accumulation of immature blast-like cells with similar immunophenotypes. However, MLL-AF9-expressing stem and progenitor cells displayed distinct properties such as colony formation, differentiation and resistance to chemotherapeutic drugs. Turning-off the fusion resulted in multi-lineage differentiation of LT-HSC-derived cells, whereas GMP-derived cells were limited to mature macrophages and granulocytes suggesting partial maintenance of their original identity. In line with these in vitro observations, lower cell numbers of transplanted LT-HSCs induced a more aggressive leukemia with a significantly shorter latency as compared to ST-HSC, CMP or GMPs. Immunophenotypically 15% of the LT-HSC derived leukemias displayed a CMP–like phenotype and had a median latency of 37d (“early”) whereas the rest of the cases displayed a GMP-like phenotype with a median latency of 73d (“late”). In contrast, only GMP-like phenotypes and longer latencies were observed upon transplanting ST-HSCs (75d), CMPs (72d) or GMPs (100d). Transplantation of blasts from “early” LT-HSC- and GMP-derived leukemias into secondary recipients induced the disease after similar latency, however, cytarabine (Ara-C) treatment significantly delayed only the disease induced by GMP- but not by LT-HSC-derived blasts. Gene expression profiling in immortalized pre-leukemic cells revealed down-regulation of over 300 genes, including several well-known MLL targets such as Meis1, HoxA5, HoxA9 and HoxA10 upon reducing the levels of MLL-AF9 expression. Likewise, we observed a global decrease in histone H3 lysine 79 dimethylation consistent with a Dot1l function in MLL-AF9 driven leukemia. LT-HSC-derived (“early”) blasts displayed distinct genetic signatures with > 400 genes highly and > 1300 genes lowly expressed (p001 fc1.5), clearly separating them from the GMP-derived blasts. Evi-1 and Erg, two prognostic markers in patient-derived gene signatures, stood out among these genes. The aggressive “early” LT-derived murine leukemias showed high Evi-1 and Erg expression levels (Evi-1 high, Erg high) as compared to the “late” LT-derived (Evi-1 low, Erg high) or the GMP-derived leukemias (Evi-1 low, Erg low). These observations suggest that the previously reported poor prognosis associated with elevated EVI-1 and/or ERG expression might directly reflect the cell of origin of the disease. We are currently exploiting our highly informative MLL-AF9 disease model to evaluate the functional relevance of novel origin-dependent MLL-AF9 target genes and to identify novel prognostic markers and therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

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ROLE OF VASOPRESSIN IN THE MITOTIC RESPONSE OF RAT BONE MARROW CELLS TO HAEMORRHAGE

Journal of Endocrinology ◽

10.1677/joe.0.0720005 ◽

1977 ◽

Vol 72 (1) ◽

pp. 5-16 ◽

Cited By ~ 35

Author(s):

N. H. HUNT ◽

A. D. PERRIS ◽

P. A. SANDFORD

Keyword(s):

Bone Marrow ◽

Mitotic Activity ◽

Bone Marrow Cells ◽

Cell Numbers ◽

Severe Haemorrhage ◽

Mitogenic Action ◽

Stimulate Bone Marrow

SUMMARY Two days after a severe haemorrhage plasma calcium concentrations and bone marrow mitotic activity in rats were significantly increased and so remained for a further 5–6 days until the haematocrit had returned to normal. The first 48 h after bleeding were characterized by hypocalcaemia. During this phase two significant peaks in mitotic activity were observed at 4 and 18 h after haemorrhage. The mitotic surge 4 h after bleeding was still present in adrenalectomized and parathyroidectomized animals but in rats which were either hypophysectomized or had congenital diabetes insipidus this mitotic response was absent. Vasopressin was shown to stimulate bone marrow mitotic activity both in vivo and in vitro whereas angiotensin, aldosterone and erythropoietin had no rapid, direct mitogenic action on these cells. This novel hypophysial–bone marrow system suggests that vasopressin may assist in post-haemorrhagic recovery in blood cell numbers in the circulation.

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