CD200 Limits Monopoiesis and Monocyte Recruitment in Atherosclerosis

Rationale: Inflammation is a basic component of the pathogenesis of atherosclerosis. CD200 is an immune checkpoint known to control macrophage activation. CD200 recently emerged in the Framingham Heart Study and 2 other cohorts as being potentially relevant in CVD. The role of this pathway in CVD is unknown. Objective: We sought to examine the role of CD200 in atherosclerosis. Methods and Results: Using hypercholesterolemic ApoE-/- mice, we demonstrate that whole-body CD200 deficiency augments atherosclerotic lesion formation and vulnerability. Administration of a CD200-Fusion protein reduces neointima formation. Our data show that the CD200-CD200R pathway restrains activation of CD200R+ lesional macrophages, their production of CCR2 ligands, and monocyte recruitment in vitro and in vivo in an air pouch model. Loss of CD200 leads to an excessive accumulation of classical Ly6Chi monocytes and CCR2+ macrophages within the atherosclerotic aorta, as assessed by mass cytometry. Moreover, we uncover a previously uncharacterised effect of the CD200/CD200R pathway in limiting dysregulated monopoiesis and Ly6Chi monocytosis in hypercholesterolemic mice. Bone marrow chimera experiments demonstrate that the CD200-CD20R pathway enables two complementary and tissue-dependent strategies to limit atherogenesis: CD200 expression by bone-marrow derived cells limits systemic monocytosis, while CD200 expression by non-haematopoietic cells, e.g. endothelial cells, prevents local plaque growth. We show that CD200R signalling controls monopoiesis and macrophage activation through inhibiting phosphorylation of STAT1. Finally, CD200R expression on classical monocytes in peripheral blood of patients with coronary artery disease (CAD) is associated with a lower burden of CAD and a more favourable Virtual Histology plaque profile. Conclusions: The CD200 checkpoint is a key limiting factor for monopoiesis, monocyte-macrophage activation and recruitment in atherosclerosis with conserved features in human and mouse. It thus offers a novel potential therapeutic pathway to treat CVD.

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Low Intensity Shockwave Treatment Modulates Macrophage Functions Beneficial to Healing Chronic Wounds

International Journal of Molecular Sciences ◽

10.3390/ijms22157844 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7844

Author(s):

Jason S. Holsapple ◽

Ben Cooper ◽

Susan H. Berry ◽

Aleksandra Staniszewska ◽

Bruce M. Dickson ◽

...

Keyword(s):

Macrophage Activation ◽

Molecular Mechanisms ◽

Chronic Wounds ◽

Immunohistochemical Analysis ◽

Therapeutic Effects ◽

Gene Expressions ◽

Extracorporeal Shock Wave

Extracorporeal Shock Wave Therapy (ESWT) is used clinically in various disorders including chronic wounds for its pro-angiogenic, proliferative, and anti-inflammatory effects. However, the underlying cellular and molecular mechanisms driving therapeutic effects are not well characterized. Macrophages play a key role in all aspects of healing and their dysfunction results in failure to resolve chronic wounds. We investigated the role of ESWT on macrophage activity in chronic wound punch biopsies from patients with non-healing venous ulcers prior to, and two weeks post-ESWT, and in macrophage cultures treated with clinical shockwave intensities (150–500 impulses, 5 Hz, 0.1 mJ/mm2). Using wound area measurements and histological/immunohistochemical analysis of wound biopsies, we show ESWT enhanced healing of chronic ulcers associated with improved wound angiogenesis (CD31 staining), significantly decreased CD68-positive macrophages per biopsy area and generally increased macrophage activation. Shockwave treatment of macrophages in culture significantly boosted uptake of apoptotic cells, healing-associated cytokine and growth factor gene expressions and modulated macrophage morphology suggestive of macrophage activation, all of which contribute to wound resolution. Macrophage ERK activity was enhanced, suggesting one mechanotransduction pathway driving events. Collectively, these in vitro and in vivo findings reveal shockwaves as important regulators of macrophage functions linked with wound healing. This immunomodulation represents an underappreciated role of clinically applied shockwaves, which could be exploited for other macrophage-mediated disorders.

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The Use of Erythropoietin in the Treatment of Post-bone Marrow Transplatation Anemia

The International Journal of Artificial Organs ◽

10.1177/039139889301605s02 ◽

1993 ◽

Vol 16 (5_suppl) ◽

pp. 8-12 ◽

Cited By ~ 1

Author(s):

A.M. Vannucchi ◽

A. Bosi ◽

A. Grossi ◽

S. Guidi ◽

R. Saccardi ◽

...

Keyword(s):

Bone Marrow ◽

Clinical Trials ◽

Bone Marrow Transplantation ◽

Blood Transfusion ◽

In Vitro Models ◽

Immunosuppressive Drug ◽

Number Of Patients

The issue of the role of erythropoietin (Epo) in the erythroid reconstitution after bone marrow transplantation (BMT) has been addressed in several recent studies. A defective Epo production in response to anemia has been shown to occur in patients undergoing allogeneic BMT unlike in most of those subjected to an autologous rescue. The factors involved in the inadeguate Epo production in BMT are discussed, with particular attention to the role of the immunosuppressive drug cyclosporin-A, which has been shown to inhibit Epo production in both in vivo and in vitro models. The observation of defective Epo production eventually led to the development of clinical trials of recombinant human Epo (rhEpo) administration in BMT patients; the aims of these studies were to stimulate erythroid engraftment, hence reducing blood transfusion exposure. Although the number of patients studied up to now is relatively small, a benefit from rhEpo administration in terms of accelerated erythroid engraftment seems very likely, and it may also be associated with decreased transfusional needs in most treated patients. However, further studies are needed to better define indications, dosages and schedules of rhEpo in BMT patients.

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Increases in circulating megakaryocyte growth-promoting activity in the plasma of rats following whole body irradiation

Blood ◽

10.1182/blood.v63.5.1060.1060 ◽

1984 ◽

Vol 63 (5) ◽

pp. 1060-1066 ◽

Cited By ~ 15

Author(s):

M Miura ◽

CW Jackson ◽

SA Lyles

Keyword(s):

Bone Marrow ◽

Plasma Concentration ◽

Bone Marrow Cells ◽

Single Cells ◽

Rat Plasma ◽

Whole Body ◽

Growth Promoting ◽

Marrow Cells

Abstract To gain insight into the regulation of megakaryocyte precursors in vivo, we assayed (in vitro) megakaryocyte growth-promoting activity (Meg-GPA) in plasma of rats in which both marrow hypoplasia and thrombocytopenia had been induced by irradiation. Rats received whole body irradiation of 834 rad from a 137Cs source. Plasma was collected at intervals of hours to days, up through day 21 postirradiation, and was tested, at a concentration of 30%, for Meg-GPA on bone marrow cells cultured in 1.1% methylcellulose with 5 X 10(-5) M 2-mercaptoethanol. With normal rat plasma, no megakaryocyte colonies (defined as greater than or equal to 4 megakaryocytes) were seen and only a few single megakaryocytes and clusters (defined as 2 or 3 megakaryocytes) were formed. Two peaks of plasma Meg-GPA were observed after irradiation. The first appeared at 12 hr, before any decrease in marrow megakaryocyte concentration or platelet count. The second occurred on days 10–14 after irradiation, after the nadir in megakaryocyte concentration and while platelet counts were at their lowest levels. A dose-response study of plasma concentration and megakaryocyte growth, using plasma collected 11 days postirradiation, demonstrated that patterns of megakaryocyte growth were related to plasma concentration; formation of single megakaryocytes was optimal over a range of 20%-30% plasma concentration, while cluster and colony formation were optimal at a plasma concentration of 30%. All forms of megakaryocyte growth were decreased with 40% plasma. There was a linear relationship between the number of bone marrow cells plated and growth of single cells, clusters, and colonies using a concentration of 30% plasma collected 11 days after irradiation. We conclude that irradiation causes time- related increases in circulating megakaryocyte growth-promoting activity. We suggest that the irradiated rat is a good model for studying the relationships between Meg-GPA and megakaryocyte and platelet concentration in vivo.

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Activation of heme oxygenase-1 by Ginkgo biloba extract differentially modulates endothelial and smooth muscle-like progenitor cells for vascular repair

Scientific Reports ◽

10.1038/s41598-019-53818-7 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Tao-Cheng Wu ◽

Jia-Shiong Chen ◽

Chao-Hung Wang ◽

Po-Hsun Huang ◽

Feng-Yen Lin ◽

...

Keyword(s):

Bone Marrow ◽

Smooth Muscle ◽

Progenitor Cells ◽

Heme Oxygenase ◽

Neointimal Hyperplasia ◽

Vascular Repair ◽

Vascular Progenitor Cells

AbstractVascular progenitors such as endothelial progenitor cells (EPCs) and smooth muscle-like progenitor cells (SMPCs) may play different roles in vascular repair. Ginkgo biloba extract (GBE) is an exogenous activator of heme oxygenase (HO)-1, which has been suggested to improve vascular repair; however, the detailed mechanisms have yet to be elucidated. This study aimed to investigate whether GBE can modulate different vascular progenitor cells by activating HO-1 for vascular repair. A bone marrow transplantation mouse model was used to evaluate the in vivo effects of GBE treatment on wire-injury induced neointimal hyperplasia, which is representative of impaired vascular repair. On day 14 of GBE treatment, the mice were subjected to wire injury of the femoral artery to identify vascular reendothelialization. Compared to the mice without treatment, neointimal hyperplasia was reduced in the mice that received GBE treatment for 28 days in a dose-dependent manner. Furthermore, GBE treatment increased bone marrow-derived EPCs, accelerated endothelial recovery, and reduced the number of SMPCs attached to vascular injury sites. The effects of GBE treatment on neointimal hyperplasia could be abolished by co-treatment with zinc protoporphyrin IX, an HO-1 inhibitor, suggesting the in vivo role of HO-1. In this in vitro study, treatment with GBE activated human early and late EPCs and suppressed SMPC migration. These effects were abolished by HO-1 siRNA and an HO-1 inhibitor. Furthermore, GBE induced the expression of HO-1 by activating PI3K/Akt/eNOS signaling in human late EPCs and via p38 pathways in SMPCs, suggesting that GBE can induce HO-1 in vitro through different molecular mechanisms in different vascular progenitor cells. Accordingly, GBE could activate early and late EPCs, suppress the migration of SMPCs, and improve in vivo vascular repair after mechanical injury by activating HO-1, suggesting the potential role of pharmacological HO-1 activators, such as GBE, for vascular protection in atherosclerotic diseases.

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Role of Il-2, Il-7, and IL-15 in T Cell Mediated Graft-vs-Leukemia Against Human Acute Lymphoblastic Leukemia in a NOD/scid Chimeric Mouse Model.

Blood ◽

10.1182/blood.v106.11.5256.5256 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5256-5256

Author(s):

Doug Cipkala ◽

Kelly McQuown ◽

Lindsay Hendey ◽

Michael Boyer

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Mouse Model ◽

Scid Mouse ◽

In Vitro Cytotoxicity ◽

Scid Mouse Model

Abstract The use of cytotoxic T-lymphocytes (CTL) has been attempted experimentally with various tumors to achieve disease control. Factors that may influence GVT include CTL cytotoxicity, ability to home to disease sites, and survival of T cells in the host. The objective of our study is to evaluate the GVL effects of human alloreactive CTL against ALL in a chimeric NOD/scid mouse model. CTL were generated from random blood donor PBMCs stimulated with the 697 human ALL cell line and supplemented with IL-2, -7, or -15. CTL were analyzed for in vitro cytotoxicity against 697 cells, phenotype, and in vitro migration on day 14. NOD/scid mice were injected with 107 697 ALL cells followed by 5x106 CTL. Mice were sacrificed seven days following CTL injection and residual leukemia was measured in the bone marrow and spleen via flow cytometry. The ratios of CD8/CD4 positive T cells at the time of injection were 46/21% for IL-2, 52/31% for IL-7, and 45/14% for IL-15 cultured CTL (n=13). Control mice not receiving CTL had a baseline leukemia burden of 2.01% and 0.15% in the bone marrow and spleen, respectively (n=15). Mice treated with IL-15 cultured CTL had a reduction in tumor burden to 0.2% (n=13, p=0.01) and 0.05% (n=13, p=0.01) in bone marrow and spleen, respectively. Those treated with IL-2 or IL-7 cultured CTL showed no significant difference in leukemia burden in either the bone marrow (IL-2 1.28%, Il-7 5.97%) or spleen (IL-2 0.4%, IL-7 0.33%). No residual CTL could be identified in the bone marrow or spleen at the time of sacrifice in any CTL group. CTL grown in each cytokine resulted in similar in vitro cytotoxicity at an effector:target ratio of 10:1 (IL-2 41.3%, IL-7 37.7%, IL-15 45.3%, n=12–15, p>0.05 for all groups) and had statistically similar intracellular perforin and granzyme-B expression. In vitro CTL migration to a human mesenchymal stem cell line was greatest with IL-15 CTL (30.5%, n=4), followed by IL-7 CTL (18.9%, n=4), and least in IL-2 CTL (17.9%, n=4), though the differences were not significant. In vitro CTL migration was analyzed to an SDF-1α gradient as CXCR4/SDF-1α interactions are necessary for hematopoietic progenitor cell homing to the bone marrow. IL-15 cultured CTL showed the highest migration (48.8%, n=8) as compared to IL-2 (21.7%, n=6, p=0.048) or IL-7 CTL (35.9%, n=8, p>0.05). However, surface expression of CXCR4 measured by flow cytometry was significantly higher in IL-7 CTL (89.4%, n=9) compared to IL-2 CTL (52.2%, n=9, p<0.001) and IL-15 CTL (65.4%, n=10, p=0.002). Experiments are currently underway to further evaluate the role of CXCR4/SDF-1α in GVL. Preliminary in vivo experiments do not suggest any significant differences in CTL engraftment when evaluated at 24 hours post injection. Expression of the anti-apoptotic bcl-2 protein was greatest on IL-7 (MFI=5295, n=13) and IL-15 (MFI=4865, n=14) when compared to IL-2 CTL (MFI=3530, n=13, p=0.02 vs. IL-7, p=0.05 vs. IL-15), suggesting an increased in vivo survival ability. We hypothesize that IL-15 cultured CTL have greater GVL effects due to either higher in vivo survival, greater bone marrow homing efficiency, or both. Future experiments are planned to evaluate in vivo administration of IL-2 to enhance CTL survival in the host. In conclusion, IL-15 cultured CTL had significantly greater in vivo GVL effects compared to IL-2 and IL-7 CTL in the NOD/scid mouse model. This model can be utilized to evaluate the mechanism of T cell mediated GVL against ALL and potentially other human malignancies.

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FIP1L1/PDGFRa Synergizes with SCF/c-Kit Signaling To Induce Systemic Mastocytosis in a Chronic Eosinophilic Leukemia Murine Model

Blood ◽

10.1182/blood.v110.11.1540.1540 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1540-1540

Author(s):

Yoshiyuki Yamada ◽

Jose A. Cancelas ◽

Eric B. Brandt ◽

Abel Sanchez-Aguilera ◽

Melissa McBride ◽

...

Keyword(s):

Bone Marrow ◽

Small Intestine ◽

Murine Model ◽

Fusion Gene ◽

Systemic Mastocytosis ◽

Wild Type ◽

Chronic Eosinophilic Leukemia

Abstract Systemic mastocytosis (SM) associated with chronic eosinophilic leukemia (CEL)/hypereosinophilic syndrome (HES) is a result of expression of the Fip1-like1 (FIP1L1)/platelet-derived growth factor receptor alpha (PDGFRa) (F/P) fusion gene. We have previously described a murine CEL/HES model (CEL-like mice) induced by F/P fusion gene transduction and T-cell overexpression of IL-5 (Yamada Y et al., Blood 2006). We have now validated a preclinical murine model of F/P-induced SM/CEL and analyzed the pathogenesis of SM in this model. F/P+ mast cells (MC, defined as EGFP+/c-kit+/FceRI+) were significantly increased in the small intestine, bone marrow (BM) and spleen of CEL-like mice compared to wild-type mice (Table). CEL-like mice also developed cutaneous MC infiltration. In addition, mMCP-1 serum levels, which correlate well with MC expansion and activation in vivo, were significantly higher in CEL-like mice than in wild-type mice (64,000 ± 23,800 and 38 ± 41.4 pg/ml, respectively). F/P induces increased expansion of BM-derived MC in vitro (∼2,000-fold) and F/P+ BM-derived MC survive longer than wild-type MC in cytokine-deprived medium (28.0 ± 2.3% vs. 8.7 ± 3.1% 7AAD−/Annexin V− cells after 48 hours). This correlated with increased Akt phosphorylation in the F/P+ MC. Since c-kit mutations are the most frequent cause of SM, we analyzed the possible synergistic role of SCF and F/P signaling. F/P and SCF/c-kit signaling indeed synergize in the development of BM-derived MC (16-fold greater expansion than in the absence of SCF) and F/P+ BM-derived MC showed a 3.7-fold greater migratory response to SCF than wild-type BM-derived MC. In order to determine the role of SCF/c-kit signaling in F/P+ MC development, activation and tissue infiltration in vivo,these responses were evaluated in mice that were treated with a blocking anti-c-kit blocking antibody, ACK-2, or an isotype-matched control antibody. ACK-2 treatment suppressed intestinal MC infiltration and elevated plasma levels of mMCP-1 induced by F/P expression by 95 ± 6.0% and 98 ± 0.76%, respectively, whereas MC and plasma mMCP-1 were completely undetectable in wild-type mice treated with ACK2. This suggests that SCF/c-kit interactions may synergize with F/P to induce SM. In summary, mice with CEL-like disease also develop SM. F/P-induced SM is a result of increased in vivo MC proliferation, survival, activation and tissue infiltration. SCF/c-kit signaling synergizes with F/P in vivo and in vitro to promote mast cell development, activation and survival. EGFP+/c-kit+/FcεRI+ cell frequency in tissues of control and CEL-like mice (%) Control mice CEL-like mice Small intestine 1.0±0.95 47±21.4* Bone marrow 0.2±0.14 3±1.9* Spleen 0.05±0.01 3±0.8*

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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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Direct Activation of STAT5 by TEL-Lyn Fusion Protein Promotes Induction of Myeloproliferative Neoplasms with Myelofibrosis

Blood ◽

10.1182/blood.v116.21.4114.4114 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4114-4114

Author(s):

Yusuke Takeda ◽

Chiaki Nakaseko ◽

Hiroaki Tanaka ◽

Masahiro Takeuchi ◽

Makiko Yui ◽

...

Keyword(s):

Bone Marrow ◽

Myeloproliferative Neoplasms ◽

Jak2 V617f ◽

Signaling Molecules ◽

Janus Kinase ◽

Idiopathic Myelofibrosis ◽

Lyn Kinase

Abstract Abstract 4114 Background Myeloproliferative neoplasms (MPN), a group of hematopoietic stem cell (HSC) disorders, are often accompanied by myelofibrosis. The V617F somatic mutation in the Janus kinase 2 (JAK2) gene has recently been found in the majority of patients with polycythemia vera (PV) and more than half of patients with essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). The expression of JAK2 V617F causes a PV-like disease with myelofibrosis in a murine bone marrow (BM) transplant model. In addition, a gain-of-function c-MPL W515 mutation was described in nearly 10% of patients with JAK2 V617F-negative IMF. However, the mechanism responsible for MPD and the formation of myelofibrosis in patients without JAK2 or c-MPL mutations is still unclear. We previously identified the fusion of the TEL gene to the Lyn gene (TEL-Lyn) in idiopathic myelofibrosis with ins(12;8)(p13;q11q21). The introduction of TEL-Lyn into HSCs resulted in fatal MPN with massive myelofibrosis in mice, implicating the rearranged Lyn kinase in the pathogenesis of MPN with myelofibrosis. However, the signaling molecules directly downstream from and activated by TEL-Lyn remain unknown. Design and Methods We examined the signaling pathways activated by TEL-Lyn by Western blotting, immunoprecipitation, and in vitro kinase assay using a TEL-Lyn kinase-dead mutant as a control. We further characterized the functional properties of Stat5-deficient HSCs transduced with TEL-Lyn by colony-forming assay and bone marrow transplantation to evaluate the role of STAT5 in TEL-Lyn-induced MPN. Results TEL-Lyn was demonstrated to be constitutively active as a kinase through autophosphorylation. In TEL-Lyn-expressing cells, STAT5, STAT3, and Akt were constitutively activated. Among these signaling molecules, STAT5 was activated most prominently and this occurred without the activation of Jak2, the major kinase for STAT5. TEL-Lyn was co-immunoprecipitated with STAT5, and STAT5 was phosphorylated when incubated with TEL-Lyn, but not with TEL-Lyn kinase-dead mutant. These results indicate that TEL-Lyn interacts with STAT5 and directly activates STAT5 both in vitro and in vivo. Of note, the capacity of TEL-Lyn to support the formation of hematopoietic colonies under cytokine-free conditions in vitro and to induce MPN with myelofibrosis in vivo was profoundly attenuated in a Stat5-null background. Conclusions In this study, we clearly showed that TEL-Lyn directly activates STAT5 and the capacity of TEL-Lyn to induce MPN with myelofibrosis was profoundly attenuated in the absence of STAT5. Our findings of TEL-Lyn in this study support the role of the Src family kinases in the regulation of STAT pathways and implicate active Lyn in the alternative pathway for STAT activation in pathological cytokine signaling. Our mouse model of MPD with myelofibrosis would be beneficial for the analysis of therapeutic approaches for myelofibrosis. Disclosures: No relevant conflicts of interest to declare.

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MicroRNAs 15a and 16 regulate tumor proliferation in multiple myeloma

Blood ◽

10.1182/blood-2009-01-198408 ◽

2009 ◽

Vol 113 (26) ◽

pp. 6669-6680 ◽

Cited By ~ 224

Author(s):

Aldo M. Roccaro ◽

Antonio Sacco ◽

Brian Thompson ◽

Xavier Leleu ◽

Abdel Kareem Azab ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Expression Profiling ◽

Map Kinases ◽

Qrt Pcr ◽

Ribosomal Protein S6 ◽

Genomic Studies

Abstract Detailed genomic studies have shown that cytogenetic abnormalities contribute to multiple myeloma (MM) pathogenesis and disease progression. Nevertheless, little is known about the characteristics of MM at the epigenetic level and specifically how microRNAs regulate MM progression in the context of the bone marrow milieu. Therefore, we performed microRNA expression profiling of bone marrow derived CD138+ MM cells versus their normal cellular counterparts and validated data by qRT-PCR. We identified a MM-specific microRNA signature characterized by down-expression of microRNA-15a/-16 and overexpression of microRNA-222/-221/-382/-181a/-181b (P < .01). We investigated the functional role of microRNA-15a and -16 and showed that they regulate proliferation and growth of MM cells in vitro and in vivo by inhibiting AKT serine/threonine-protein-kinase (AKT3), ribosomal-protein-S6, MAP-kinases, and NF-κB-activator MAP3KIP3. Moreover, miRNA-15a and -16 exerted their anti-MM activity even in the context of the bone marrow milieu in vitro and in vivo. These data indicate that microRNAs play a pivotal role in the biology of MM and represent important targets for novel therapies in MM.

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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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