Podocalyxin Negatively Regulates Stress Myelopoiesis, Directly Binds Rap-1A and Modulates Its G-CSF-Induced Activity

Abstract Podocalyxin (PodxL) is a CD34 family member previously identified to mark hematopoietic stem cells (HSCs) and other progenitor cells. Previously, we discovered PodxL as a potent erythropoietin (EPO) response gene and demonstrated to promote egression of immature reticulocytes from bone marrow into circulation. PodxL is upregulated in several cancers, including myeloid and lymphoid leukemia. Herein, we aim to define the functional role of PodxL in hematopoiesis - specifically myelopoiesis - by employing conditional PodxL knock out (KO) mouse models. Hematopoietic-specific deletion was achieved using Cre mice with a Vav1 driver and myeloid-specific deletion was achieved with Lyzm2 - Cre driver. We confirmed the deletion of exons 3-7 at the gene, transcript and protein levels using PCR, RT-qPCR and western blotting, respectively. Peripheral blood analysis revealed no difference in blood cell lineages for either KO mouse strain. At steady state, colony forming unit-granulocyte/macrophage (CFU-GM) assay also showed no difference between the KO strains and wild type. In order to examine the functional role of PodxL during stress myelopoiesis, PodxL-/- ; Vav1-Cre mice were treated with 5-Fluorouracil (5FU), a chemotherapeutic agent induces myeloablation. Notably, during rebound of neutrophils, the PodxL-/- ; Vav1-Cre mice showed a sharp increase in neutrophil counts at day 12.5, which at later time points reverted to normal levels comparable to wild type mice. Previously, our in silico analyses combined with outcomes from truncated EpoR knock-in alleles had revealed that PodxL is a potential STAT5 transcriptional target. Here, we tested if G-CSF induces PodxL expression in hematopoietic progenitors. In vivo, G-CSF significantly induced PodxL expression four fold. We then tested the role of PodxL in G-CSF induced neutrophil formation in vivo. Both KO strains (Podxl-/-;Vav1-Cre and Podxl-/-;Lyzm2-Cre) and wild type were treated with G-CSF (125ug/kg/day) for 5 days. Peripheral blood analysis revealed increased neutrophil and monocyte levels in the PodxL-/-;Vav1-Cremice. In order to then determine a possible role of PodxL at the progenitor level, CFU-GM assays were performed. PodxL-/- ; lyzm2-Cre mice had increased colony forming capabilities but there was no difference in PodxL-/-;Vav1-Cre mice compared to wild type. Our results imply that PodxL is playing a negative regulatory role in stress myelopoiesis. Interestingly, the deletion of PodxL in hematopoietic progenitors (Vav1-Cre) resulted in enhanced migration of neutrophils, whereas deletion of PodxL in myeloid compartment (Lyzm2-Cre) resulted in decreased neutrophil migration. This may be in part due to a compensatory effect by CD34 in the hematopoietic compartment. To dissect the molecular mechanism of PodxL during stress myelopoiesis, upon in vivo G-CSF treatment, bone marrow derived hematopoietic progenitors were isolated and PodxL protein was immunoprecipitated. LC-MS/MS proteomic analysis was performed to identify the interacting partners with PodxL. Rap-1A, a small GTPase and member of the RAS family, was among the top interacting proteins. Rap-1A has been shown to promote adhesion and migration of myeloid cells. The association of PodxL with Rap-1A was further confirmed in hematopoietic progenitors by immunoprecipitation and western blotting. To determine if the interaction of PodxL directly regulates Rap-1A activity, a GTP-Rap-1A activity assay was performed in response to G-CSF, GM-CSF and IL-3. Rap-1A activity was significantly elevated in hematopoietic progenitors upon G-CSF treatment in PodxL-/-:Vav1-Cre mice compared to wild type, followed by IL3; however, GM-CSF did not affect Rap-1A activity. In conclusion, our results indicate an important functional role for PodxL in stress myelopoiesis, a function likely mediated via Rap-1A. A complete understanding of the PodxL/Rap-1A axis may reveal important molecular insights into G-CSF-induced mobilization of neutrophils and provide mechanistic understanding into the pathological role of PodxL in aggressive cancers, including leukemia, which in turn may facilitate identification of novel therapeutic targets in PodxL associated cancers. Disclosures No relevant conflicts of interest to declare.

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Dissection of the Role of Connexin-43 in Hematopoietic Progenitors and Stromal Cells Unveils Distinct Hematopoietic Intrinsic and Extrinsic Regulatory Functions.

Blood ◽

10.1182/blood.v110.11.607.607 ◽

2007 ◽

Vol 110 (11) ◽

pp. 607-607

Author(s):

Lina Li ◽

Bhuvana Murali ◽

Dealma N. Worsham ◽

Susan K. Dunn ◽

Jose A. Cancelas

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Connexin 43 ◽

Chimeric Mice ◽

Wild Type ◽

Hematopoietic Progenitors ◽

Hematopoietic Progenitor ◽

Cx43 Expression

Abstract Bone marrow (BM) stromal cells seem to be crucial in the establishment of the hematopoietic niches in bone marrow. BM stromal cells can communicate through gap junctions, which consist of narrow channels between contacting cells and are composed by connexins. Connexin 43 (Cx43) is expressed by BM stromal cells and upon adhesion to stroma, by hematopoietic stem cells and progenitors (HSC/P). Cx43 has been shown to be essential in controlling osteoblast and fibroblast function. We have previously reported that Cx43 is critical for the interaction between stroma and HSC/P in CAFC assays (Cancelas J.A. et al., Blood 2000) and in adult hematopoiesis after 5-fluorouracil (5-FU) administration in Mx1-Cre-Tg;Cx43KO mice (Presley C, et al., Cell Comm. Adh., 2005). We have also previously shown that after 5-FU administration, Cx43 is predominantly expressed in the endosteum and the deficiency of Cx43 in stroma of Collagen I (ColI)-Cre;Cx43KO and chimeric mice impairs their hematopoiesis by impairing the homing of wild-type (WT) hematopoietic progenitors and after 5-FU administration, the hematopoietic progenitor cycling inducing a ∼30% expansion of the long-term stem cell compartment in BM (Li L et al., ASH 2006). Interestingly, stromal Cx43-deficient mice contain around twice as many CFU-F as wild-type (WT) mice. Now, we have further investigated the role of stromal Cx43 expression in the regulation of hematopoietic progenitor adhesion to stroma, trans-stromal migration and mobilization. Cx43-deficient stromal cells display complete absence of intercellular communication as assayed by calcein dye transfer which can be reverted by retroviral transduction of Cx43. Trans-stromal migration of hematopoietic progenitors through Cx43-deficient irradiated stroma is impaired (7.8% vs 13.8% in WT stroma, p=0.015) but primary adhesion to Cx43-deficient irradiated stroma and in vivo mobilization response to G-CSF in ColI-Cre;Cx43KO mice were similar to WT controls, suggesting that stromal Cx43 plays a role in the regulation of the post-adhesion migration of HSC/P. On the other hand, Cx43-deficient HSC/P from Vav1-Cre;Cx43KO primary and chimeric mice show severe impairment of blood cell formation during the recovery phase after 5-FU administration (day +14) compared to wild-type controls (ANC: 0.23±0.12 vs 1.40±1.25 x 109 neutrophils/L; Platelet count: 135±91 vs 572±205 x 109 platelets/L; p < 0.05). Cx43 deficiency in hematopoietic progenitors did not significantly impair their homing ability in wild-type mice. Taken together, these studies indicate that Cx43 expression plays distinct roles in the regulation of hematopoietic intrinsic and extrinsic mechanisms. While Cx43 expression in stroma seems to be crucial in the regulation of the stromal progenitor and HSC pool content as well as HSC/P trans-stromal migration and homing, deficiency of Cx43 in either hematopoietic cells or stromal cells independently induce a significant impairment in the post-chemotherapy blood formation in vivo, suggesting that, under stress, hematopoietic regeneration depends on complete Cx43 channels communicating HSC/P and stromal cells.

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Eosinophil-specific deletion of IκBα in mice reveals a critical role of NF-κB–induced Bcl-xL for inhibition of apoptosis

Blood ◽

10.1182/blood-2014-10-607788 ◽

2015 ◽

Vol 125 (25) ◽

pp. 3896-3904 ◽

Cited By ~ 28

Author(s):

Christian Schwartz ◽

Ralf Willebrand ◽

Silke Huber ◽

Rudolf A. Rupec ◽

Davina Wu ◽

...

Keyword(s):

Helminth Infection ◽

Critical Role ◽

Gm Csf ◽

Eosinophil Survival ◽

Key Points ◽

Specific Deletion ◽

Specific Inhibitors

Key Points IL-3, IL-5, and GM-CSF promote eosinophil survival by NF-κB–induced upregulation of Bcl-xL, which can be blocked by specific inhibitors. Specific and constitutive deletion of the inhibitor of NF-κB (IκBα) in eosinophils in vivo reduced apoptosis during helminth infection.

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Contribution of R domain phosphoserines to the function of CFTR studied in Fischer rat thyroid epithelia

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2000.279.5.l835 ◽

2000 ◽

Vol 279 (5) ◽

pp. L835-L841 ◽

Cited By ~ 14

Author(s):

Olafur Baldursson ◽

Herbert A. Berger ◽

Michael J. Welsh

Keyword(s):

Functional Role ◽

Transmembrane Conductance Regulator ◽

Regulatory Domain ◽

Wild Type ◽

Transmembrane Conductance ◽

Dependent Protein ◽

Rat Thyroid ◽

Cystic Fibrosis Transmembrane

The regulatory domain of cystic fibrosis transmembrane conductance regulator (CFTR) regulates channel activity when several serines are phosphorylated by cAMP-dependent protein kinase. To further define the functional role of individual phosphoserines, we studied CFTR containing previously studied and new serine to alanine mutations. We expressed these constructs in Fischer rat thyroid epithelia and measured transepithelial Cl− current. Mutation of four in vivo phosphorylation sites, Ser660, Ser737, Ser795, and Ser813 (S-Quad-A), substantially decreased cAMP-stimulated current, suggesting that these four sites account for most of the phosphorylation-dependent response. Mutation of either Ser660 or Ser813 alone significantly decreased current, indicating that these residues play a key role in phosphorylation-dependent stimulation. However, neither Ser660 nor Ser813 alone increased current to wild-type levels; both residues were required. Changing Ser737 to alanine increased current above wild-type levels, suggesting that phosphorylation of Ser737 may inhibit current in wild-type CFTR. These data help define the functional role of regulatory domain phosphoserines and suggest interactions between individual phosphoserines.

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Requirement of CREB in Normal and Malignant Hematopoiesis.

Blood ◽

10.1182/blood.v108.11.1168.1168 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1168-1168

Author(s):

Jerry C. Cheng ◽

Deepa Shankar ◽

Stanley F. Nelson ◽

Kathleen M. Sakamoto

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

Western Blot ◽

Blot Analysis ◽

Hematopoietic Cells ◽

Wild Type ◽

Qrt Pcr ◽

T315i Mutation

Abstract CREB is a nuclear transcription factor that plays an important role in regulating cellular proliferation, memory, and glucose homeostasis. We previously demonstrated that CREB is overexpressed in bone marrow cells from a subset of patients with acute leukemia at diagnosis. Furthermore, CREB overexpression is associated with an increased risk of relapse and decreased event-free survival in adult AML patients. Transgenic mice that overexpress CREB in myeloid cells developed myeloproliferative/myelodysplastic syndrome after one year. To further understand the role of CREB in leukemogenesis and in normal hematopoiesis, we employed RNA interference methods to inhibit CREB expression. To achieve sustained, CREB-specific gene knockdown in leukemia and normal hematopoietic cells, a lentiviral-based small hairpin (shRNA) approach was taken. Three CREB specific shRNAs were generated and tested for efficiency of gene knockdown in 293T cells. Knockdown efficiency approached 90 percent by Western blot analysis compared to vector alone and luciferase controls. Human myeloid leukemia cell lines, K562, TF1, and MV411, were then infected with CREB shRNA lentivirus, sorted for GFP expression, and analyzed using quantitative real time (qRT)-PCR, Western blot analysis, and growth and viability assays. Lentiviral CREB-shRNA achieved between 50 to 90 percent knockdown of CREB compared to control shRNAs at the protein and mRNA levels. To control for non-specific effects, we performed qRT-PCR analysis of the interferon response gene, OAS1, which was not upregulated in cells transduced with CREB shRNA constructs. Within 72 hours, cells transduced with CREB shRNA had decreased proliferation and survival. Similar results were obtained with murine leukemia cells (NFS60 and BA/F3 bcr-abl).To study the role of CREB in normal hematopoiesis, both primary murine and human hematopoietic cells were transduced with our shRNA constructs, and methylcellulose-based colony assays were performed. Primary hematopoietic cells infected with CREB shRNA lentivirus demonstrated a 5-fold decrease in colony number compared to control virus-infected cells (p<0.05). Bone marrow colonies consisted of myeloid progenitor cells that were mostly Mac-1+ by FACs analysis. Interestingly, there were fewer differentiated cells in the CREB shRNA transduced cells compared to vector control or wild type cells, suggesting that CREB is critical for both myeloid cell proliferation and differentiation. To study the in vivo effects of CREB knockdown on leukemia progression, we studied mice injected with BA/F3 cells that express both bcr/abl with the T315I mutation and a luciferase reporter gene. BA/F3 cells expressing the T315I mutation have a 2-fold increase in CREB overexpression compared to wild-type cells. Disease progression was monitored using bioluminescence imaging with luciferin. CREB knockdown was 90 percent after transduction and prior to injection into SCID mice. We observed improved survival of mice injected with CREB shRNA transduced BA/F3 bcr-abl (T315I) compared to vector control cells. To understand the mechanism of growth suppression resulting from CREB downregulation, we performed microarray analysis with RNA from CREB shRNA transduced K562 and TF1 cells. Several genes were downregulated using a Human Affymetrix chip. Most notable was Beclin1, a tumor suppressor gene often deleted in prostate and breast cancer that has been implicated in autophagy. Our results demonstrate that CREB is required for normal and leukemic cell proliferation both in vitro and in vivo.

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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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Bone Marrow Stroma-Derived Prolactin Is Involved in Basal and Platelet-Activating Factor–Stimulated In Vitro Erythropoiesis

Blood ◽

10.1182/blood.v90.1.21.21_21_27 ◽

1997 ◽

Vol 90 (1) ◽

pp. 21-27 ◽

Cited By ~ 1

Author(s):

Graziella Bellone ◽

Paola Astarita ◽

Elisa Artusio ◽

Stefania Silvestri ◽

Katia Mareschi ◽

...

Keyword(s):

Bone Marrow ◽

Cdna Probe ◽

Platelet Activating Factor ◽

Pcr Amplification ◽

Erythroid Differentiation ◽

Hematopoietic Progenitors ◽

Gm Csf ◽

Colony Efficiency

Cooperation between in vitro exogenous prolactin (PRL), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3) at an early step of in vitro erythroid differentiation has been shown in a previous study. To gain more insight into the role of PRL in in vivo hematopoiesis, we have now addressed the involvement of endogenous PRL in the growth of hematopoietic progenitors in a bone marrow (BM) stroma environment. The possible modulation of local PRL production by the inflammatory mediator platelet-activating factor (PAF), which is known to be produced by BM cells and to regulate pituitary PRL release, has also been evaluated. Development of burst-forming unit-erythroid (BFU-E) colonies from CD34+ hematopoietic progenitors cultured on a BM stroma cells (BMSC) layer was slightly, but significantly, reduced in the presence of an antihuman PRL antibody. Pretreatment of BMSC with PAF increased the BFU-E colony efficiency of cocultured CD34+ cells, and this effect was completely abrogated by the antiserum. PAF-modulated release of PRL by BMSC was confirmed by an enzyme-linked-immunospot (Elispot) technique. In addition, immunoprecipitation and Western blotting experiments showed two immunoreactive products in the BMSC culture medium. These corresponded to the nonglycosylated (23 kD) and glycosylated (25.5 kD) forms of pituitary PRL that are also expressed by the B-lymphoblastoid cell line IM9-P3. Specific increase of the nonglycosylated form and decrease of the glycosylated form was observed after PAF treatment. Polymerase chain reaction (PCR) amplification of reverse transcribed RNA using PRL-specific primers showed the presence of PRL message in BMSC and IM9-P3 cells. In situ hybridization experiments with a rat PRL cDNA probe cross-reacting with human PRL mRNA confirmed its presence in a small fraction of unstimulated BMSC and in the majority of PAF-stimulated BMSC. The enhancing effect of PAF on PRL-mediated colony formation, PRL release, and mRNA activation was counteracted by pretreating BMSC with the PAF-receptor (R) antagonist WEB 2170. Lastly, responsiveness of BMSC to PAF was substantiated by the presence of the PAF-R mRNA on these cells.

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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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MBNL1 As a New Therapeutic Target in MLL-Fusion Gene Leukemia

Blood ◽

10.1182/blood.v126.23.462.462 ◽

2015 ◽

Vol 126 (23) ◽

pp. 462-462 ◽

Cited By ~ 2

Author(s):

Svetlana S Itskovich ◽

Jason Clark ◽

James C. Mulloy ◽

Matthew D Disney ◽

Ashish R Kumar

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Peripheral Blood ◽

Fusion Gene ◽

Leukemia Cell ◽

Leukemia Cells ◽

Transformed Cells ◽

Wild Type

Abstract Translocations of the Mixed Lineage Leukemia (MLL) gene located on chromosome 11 are commonly found in infants with AML or ALL and in secondary leukemia at all ages. A majority of patients with these translocations have a poor prognosis. Gene expression profiling studies demonstrate that one of the most consistently overexpressed genes in these leukemias (compared to all other leukemias) is muscleblind-like 1 (MBNL1). Further, MBNL1 was also identified as a direct transcriptional target of MLL-fusion proteins. An RNA-binding protein, MBNL1 is known to be a key factor in the pathophysiology of Myotonic Dystrophy Type I (DM), where sequestration of MBNL1 leads to splicing defects in muscle and neuronal cells. However, the role of MBNL1 in hematopoiesis and leukemogenesis is unknown. To determine the role of MBNL1 in normal hematopoiesis we studied MBNL1-/- mice. Compared to littermate controls, MBNL1-/- mice showed no differences in peripheral blood counts or bone marrow cellularity. When challenged with 5-FU, both MBNL1-/- and wild type mice displayed similar kinetics of peripheral blood cytopenia and recovery. Next we examined the role of MBNL1 in hematopoietic stem cell function using a competitive transplantation assay. Lethally irradiated mice were transplanted with a 1:1 mix of CD45.1 and CD45.2 bone marrow, with the latter being wild-type or MBNL1-/-. Flow cytometry analysis of peripheral blood at 4 weeks post-transplant showed donor chimerism being 53±4.14% in recipients of wild type marrow and 25±5.41 % in the MBNL1-/- recipients. Successive analyses every 4 weeks showed the chimerism to be stable over the next 16 weeks. To determine the role of MBNL1 in leukemia, we transformed MBNL1-/- or wild type bone marrow cells with various oncogenes delivered via retroviral transduction and compared them in methylcellulose colony replating assays. Absence of MBNL1 significantly reduced colony formation in MLL-AF9 and E2A-HLF transformed cells by 59.5% (± 27.1) and 50.7% (± 23) respectively, compared to controls. To assess the role of MBNL1 in leukemia in vivo, we transplanted MLL-AF9-transformed wild type or MBNL1-/- cells into irradiated mice. All recipients injected with wild-type MLL-AF9-transformed cells succumbed to leukemia with a median time of 106 days. In contrast, the majority of recipients of MBNL1-/- cells survived leukemia-free for at least 140 days post-transplantation (p=0.0017, log rank test). We next assessed the role of MBNL1 in human leukemia cells. Lentiviral-shRNA knockdown of MBNL1 in leukemia cell lines (MV4;11, THP-1) significantly inhibited cell growth, both in liquid culture and methylcellulose colony forming assays. To determine the requirement of MBNL1 for leukemia propagation in vivo, we used cord blood-derived leukemia cells bearing the MLL-AF9 fusion gene and mutant NRAS (MA9NRAS). MA9NRAS cells transduced with MBNL1-specific or control (non-targeting, NT) shRNA were transplanted into immunodeficient mice. Six weeks after transplant, bone marrow aspirates showed persistence of lentiviral-transduced cells in 85% of the NT-group. On the other hand, MBNL1-shRNA transduced cells were not detected in any of the recipient mice. These results suggest that MBNL1 is essential for leukemia cell propagation in vivo. Finally, we tested therapeutic targeting of MBNL1 in MLL-fusion gene leukemia. A lead inhibitor that prevents binding of MBNL1 to its targets was recently identified. Treatment of MA9NRAS cells with the inhibitor for 48 hours led to significant apoptosis whereas normal cord blood CD34+ cells were relatively less sensitive. Blockade of MBNL1 in leukemia cells either by shRNA-knockdown or by the inhibitor showed identical changes in splicing patterns of known MBNL1 target genes. Collectively, our data suggest that MBNL1 is required for the initiation and propagation of MLL-fusion gene leukemia while it appears relatively dispensable for normal hematopoiesis. Further, we have identified a promising lead inhibitor that could be developed for novel treatments for therapy-resistant leukemias. Disclosures No relevant conflicts of interest to declare.

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Activation of JAK/STAT Signaling in Megakaryocytes Is Necessary and Sufficient for Myeloproliferation In Vivo

Blood ◽

10.1182/blood.v128.22.949.949 ◽

2016 ◽

Vol 128 (22) ◽

pp. 949-949

Author(s):

Q. Jeremy Wen ◽

Brittany Woods ◽

Qiong Yang ◽

Chiu Sophia ◽

Gu Lillu ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Diphtheria Toxin ◽

Bone Marrow Cells ◽

Erythroid Cell ◽

Spleen Weight ◽

Wild Type ◽

Myeloid Progenitor

Abstract Aberrant megakaryopoiesis is a hallmark of the myeloproliferative neoplasms (MPN). It is has been long known that abnormal megakaryocytes secrete elevated levels of cytokines such as TGFβ, resulting in pathologies including bone marrow fibrosis. Two recent studies showed that megakaryocytes regulate the quiescence of HSCs, raising the possibility that megakaryocytes may promote the MPNs by influencing the biology of non-malignant HSCs. However, the mechanism by which megakaryocytes regulate the initiation and progression of MPNs is largely unknown. To study the role of megakaryocytes in the MPNs, we analyzed the phenotype of PF4-Cre/Jak2V617F mice in which Jak2 is expressed in the megakaryocyte lineage from the endogenous locus, in contrast to previous studies, which used transgenic models. Selective activation of Jak2V617F was confirmed by allele-specific qPCR. CD41+ cells were positive for mutant Jak2, whereas sorted stem/progenitor cells and erythroid cells were Jak2 wild-type. Furthermore, flow cytometry showed that Stat5 activation was present in megakaryocytes, but not in erythroid or myeloid cells. Activation of JAK-STAT signaling caused an expansion of megakaryocytes in the bone marrow and spleen and a modest increase in the platelet count. Surprisingly, PF4-Cre/Jak2V617F mice also displayed a robust expansion of TER119(low)/CD71(high) and TER119(high)/CD71(high) red cells in the spleen, increased hematocrit and splenomegaly. Histological examination of the spleen revealed expansion of the erythroid lineage coupled with disrupted splenic architecture and fibrosis. This PV-like phenotype was fully penetrant and comparable to that of Vav-Cre/Jak2V617F mice, which express mutant Jak2 in all hematopoietic lineages. Profiling of hematopoietic progenitors by flow cytometry demonstrated that myeloid progenitor populations were expanded and skewed toward the erythroid-megakaryocyte lineage with a significant increase in Pre Meg-E, Pre CFU-E and MKPs in the PF4Cre/Jak2V617F mice. In addition, LSK cells were increased in both the bone marrow and spleen. Cytokine profiling of the plasma revealed increased levels of several cytokines, including Il-6, which is known to be upregulated in human JAK2 mutant PV megakaryocytes. Significant increases in Cxcl1, Cxcl2, and Ccl11 were also detected. Real-time qPCR analysis confirmed increased expression of these cytokines/chemokines in Jak2V617F-mutant CD41+ cells. Furthermore, IL6 treatment increased EPO-dependent colony formation of wild type LSKs and MEPs, and also enhanced expression of the erythroid cell markers CD71 and Ter119. To further explore the role of megakaryocytes in the MPNs, we used a strategy in which expression of the diphtheria toxin receptor (DTR) sensitizes cells to diphtheria toxin (DT). We transduced c-Kit+ cells from PF4-Cre/iDTR+/- mice with MPLW515L and transplanted the cells to irradiated mice. As expected, both iDTR+/- and PF4-Cre/iDTR+/- mice developed a PMF-like phenotype, including leukocytosis, thrombocytosis, splenomegaly and myelofibrosis (Fig 1). Treatment of these animals with DT caused significant reductions in megakaryocytes in the bone marrow and spleen as well as a decrease in the platelet count of PF4-Cre/iDTR+/- mice. Of note, DT also significantly reduced the white count and spleen weight, while restoring splenic architecture. PF4Cre/iDTR+/- mice also showed significant reduction of c-Kit+ myeloid progenitor cells. Therefore, depletion of megakaryocytes significantly attenuated the disease phenotype of MPLW515L induced MPN in vivo. Together, these two model systems reveal that JAK2 activation in megakaryocytes is sufficient and necessary for MPNs and support the development of megakaryocyte differentiation therapy in the disease. Moreover our data resonate with studies in MPN patients in which a JAK2V617F low allele burden in the setting of full-blown, clinical MPN. figure 1 Depletion of megakaryocytes attenuated the MPN phenotype induced by MPLW515L. c-Kit+ bone marrow cells of IDTR+/- mice with or without PF4Cre were transduced with retroviruses carrying MPLW515L. Injection of diphtheria toxin (DT) was initiated on day 28 post-transplant. Depletion of megakaryocytes by DT reduced platelet and white count (A, B), decreased spleen weight (C) and reduced megakaryocyte and erythroid cell infiltration in the spleen (D). *, p<0.05, **, p<0.01. figure 1. Depletion of megakaryocytes attenuated the MPN phenotype induced by MPLW515L. c-Kit+ bone marrow cells of IDTR+/- mice with or without PF4Cre were transduced with retroviruses carrying MPLW515L. Injection of diphtheria toxin (DT) was initiated on day 28 post-transplant. Depletion of megakaryocytes by DT reduced platelet and white count (A, B), decreased spleen weight (C) and reduced megakaryocyte and erythroid cell infiltration in the spleen (D). *, p<0.05, **, p<0.01. Disclosures Levine: Novartis: Consultancy; Qiagen: Membership on an entity's Board of Directors or advisory committees.

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