Novel Mechanisms of Thrombopoietin Generation: The Essential Role of Kupffer Cells

Abstract Thrombopoietin (TPO) is the physiological regulator of hemopoietic stem cell niche and megakaryocyte differentiation, and therefore platelet production. Prevailing theory posits that TPO is constitutively expressed by hepatocytes, and levels are fine-tuned through platelet and megakaryocyte internalization/clearance via the c-Mpl receptor. Our lab has previously shown that platelet glycoprotein (GP) Ibα is indispensable for platelet-mediated TPO generation (Blood 2018), and recent reports have demonstrated that Kupffer cells, the tissue resident macrophages of the liver, contribute to the clearance of desialylated platelets. However, whether Kupffer cells may contribute to TPO generation has never been explored. To determine the possible role of Kupffer cells in TPO production, clodronate liposome was intravenously administered to deplete Kupffer cells in wild-type mice. Wild-type, Kupffer cell depleted mice showed a TPO decrease of 43.6% (±16%) 2 days post depletion, with only a gradual insignificant increase in TPO levels to day 6. Interestingly, TPO levels could not be significantly increased in wild-type Kupffer cell depleted mice even when transfused 2x10 8 wild-type or desialylated platelets, or 50mU neuraminidase. Kupffer cell depletion in IL4Rα/GPIbα-transgenic mice, which lack platelet-mediated TPO generation, showed a TPO decrease of 22.5% (±5%) from baseline 2 days post depletion, with only a gradual increase in levels to day 6, suggesting that Kupffer cells are required for constitutive in addition to platelet-mediated TPO production. As our lab has previously shown that platelet GPIbα drives platelet-mediated TPO generation, and that Kupffer cells now required, WT and GPIbα -/- platelets were co-cultured with Kupffer cells to assess interaction. Desialylated WT platelets interacted significantly more with Kupffer cells as analyzed by flow cytometry than GPIbα -/- platelets. Interestingly, desialylation of GPIbα -/- platelets did not increase binding to Kupffer cells, consolidating that desialylated GPIbα is required for Kupffer cell interaction, and subsequent TPO generation. This data demonstrates the novel and unexpected finding that Kupffer cells are required for both platelet-mediated and baseline hepatocellular TPO generation. Elucidation of the role of Kupffer cells in this crucial mechanism will provide a better understanding of why thrombocytopenias may occur in pathological states, as well as contribute to the development of TPO mimetic therapies. Disclosures No relevant conflicts of interest to declare.

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Negative Effects of GM-CSF Signaling In t(8;21)-Induced Leukemia

Blood ◽

10.1182/blood.v116.21.3163.3163 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3163-3163

Author(s):

Shinobu Matsuura ◽

Ming Yan ◽

Eun-Young Ahn ◽

Miao-Chia Lo ◽

David Dangoor ◽

...

Keyword(s):

Myeloid Leukemia ◽

Sex Chromosome ◽

De Novo ◽

Conflicts Of Interest ◽

Hematopoietic Cells ◽

Receptor Activation ◽

Pseudoautosomal Region ◽

Wild Type ◽

Gm Csf

Abstract Abstract 3163 The t(8;21)(q22;q22) translocation is one of the most common chromosomal translocations in de novo acute myeloid leukemia (AML). The 8;21 translocation is often associated with additional cytogenetic abnormalities. The loss of the sex chromosome (LOS) is by far the most frequent abnormality found in association with the t(8;21) leukemia, accounting for 32–59% of patients, in contrast to other types of AML in which the LOS occurs in less than 5% of patients. To evaluate the role of sex chromosome deletion in t(8;21)-related leukemogenesis, hematopoietic cells from a mouse line with only one sex chromosome were used in retrovirus-mediated t(8;21) (AML1-ETO) expression and transplantation assays. The absence of leukemia in those animals suggested that a gene present in the pseudoautosomal region of sex chromosomes in humans but not in mice may be the target gene in LOS. The granulocyte-macrophage colony-stimulating factor receptor α (GM-CSFRα) gene is one such gene and is also known to be involved in myeloid cell survival, proliferation and differentiation. The GM-CSFRα gene is specifically down-regulated in AML patients with t(8;21), but not in other common translocations (Valk PJM et al, NEJM, 2004). The GM-CSFR complex is composed of α and βc subunits that assemble into a complex for receptor activation and signaling. To investigate the role of GM-CSFR signaling in t(8;21)-mediated leukemogenesis, GM-CSFR common β subunit knockout (GM-CSFRβc-/-) mice were used in our studies as a model for deficient GM-CSFR signaling. Transduction of AML1-ETO in hematopoietic cells from GM-CSFRβc-/- resulted in myeloid leukemia of a median survival time of 225 days, high percentage of blasts in peripheral blood and bone marrow, anemia, thrombocytopenia, hepatomegaly and splenomegaly. Comparison of wild-type and GM-CSFRβc-/- cells in the same transplantation resulted in development of AML1-ETO-induced leukemia at higher penetrance in GM-CSFRβc-/- cells (28.5% vs 100%). Moreover, the latency of leukemia was shorter in GM-CSFRβc-/- cells than in wild-type cells after transduction of AML1-ETO9a. Analysis of the hematopoietic compartment of healthy GM-CSFRβc-/- mice detected no significant abnormalities in the immature hematopoietic compartment (LSK, CMP, GMP, MEP), suggesting that AML1-ETO expression is required for leukemia to occur. In vitro, expression of AML1-ETO alone is sufficient for the immortalization of normal hematopoietic cells, as demonstrated by serial replating capacity of cells in methylcellulose colony assay. Addition of mGM-CSF to the basic cytokine cocktail (mIL-3, hIL-6, mSCF, hEPO) did not significantly affect number, type, size, and cell composition of colony cells. In contrast, the addition of mGM-CSF eliminated the replating capacity of AML1-ETO expressing cells, although they survived longer than control vector-infected cells. The results suggest that activation of GM-CSF signaling can specifically abrogate the self-renewal ability of potential leukemic stem cells in the early immortalization phase. These results support a possible tumor suppressor role of GM-CSF in leukemogeneis by AML1-ETO and may provide clues to understand how AML1-ETO corrupts normal GM-CSF signals to its own advantage for leukemogenic transformation. Disclosures: No relevant conflicts of interest to declare.

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Neutrophils Clearance by Endothelial Cells Regulates Homeostasis and Coagulation.

Blood ◽

10.1182/blood.v116.21.3782.3782 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3782-3782

Author(s):

Wen Li ◽

Shuchuan Liu ◽

Yueyue Fu ◽

Jinxiao Hou ◽

Xiushuai Dong ◽

...

Keyword(s):

Endothelial Cells ◽

Kupffer Cells ◽

Milk Fat ◽

Conflicts Of Interest ◽

Umbilical Vein ◽

Large Fraction ◽

Background Level ◽

Procoagulant Activity ◽

Phagocytic Cells

Abstract Abstract 3782 Neutrophils, also called polymorphonuclear leukocytes (PMN's) have a half-life of only 6 hours in the blood. Inflammation can further shorten the circulating life-time. A large fraction of the bone marrow capacity is committed to ongoing production of these short-lived cells but the manner of their clearance from the circulation is less well understood. We have previously demonstrated that PMN's are cleared by liver macrophages. However, the details of PMN adhesion-induced PMN clearance in the liver are unknown. The aim of this study is to evaluate a pathway of PMNs clearance by endothelial cells, which are not ordinarily considered phagocytes. Lactadherin is a glycoprotein of milk fat globules and is also secreted by stimulated macrophages. Lactadherin binds phosphatidylserine on apoptotic cells via tandem lectin-homology domains with homology to factor VIII and binds αvβ3 and αvβ5 integrins on phagocytic cells via an RGD sequence in an epithelial growth factor domain. Lactadherin aids in engulfment of senescent lymphocytes by splenic macrophages and mediates an anti-inflammatory response. We utilized lactadherin as a probe to detect phosphatidylserine exposure on aging PMN's and evaluated the lactadherin-dependent engulfment of these PMN's by endothelial cells. Cultured human PMNs from healthy donors, with 95% purity, were 40% and 96% PS-exposure positive at 9 and 24 h, respectively. They displayed a parallel increase in procoagulant supporting, activity related to the PS exposure. Coculture of the aging PMNs and human umbilical vein endothelial cells resulted in phagocytosis of the PMN's, observed by confocal microscopy and electron microscopy. Exogenous lactadherin increased phagocytosis by 3–5 fold during 120 minutes of observation. An anti-lactadherin RGD antibody and an anti-lactadherin C2 domain antibody inhibited phagocytosis to approx 1/2 the background level suggesting that lactadherin secreted by PMN's or neutrophils contributes to the base level of phagocytosis. Clearance of the senescent neutrophils by endothelial cells decreased procoagulant activity >70% and blockade of neutrophil PS with lactadherin reduced procoagulant activity by > 90% indicating the potential role of neutrophil uptake in limiting prothrombotic activity. In a rat model of neutrophil homeostasis we injected low dose lipopolysaccharide (LPS) and gadolinium chloride intravenously to increase the number circulating PMN's and block clearance by Kupffer cells. This allowed observation of PMN adhesion and sequestration in the liver. The number of PMNs peaked at 9 h and decreased to the normal range at 24 h after blockade of Kupffer cells. Blocking the endothelial P-selectin significantly delayed PMN's removal in the liver. Injection of lactadherin promoted the PMNs accumlation and removal. The current results suggest that ECs contribute to maintaining the homeostasis of PMNs in the circulation and a possible role of lactadherin in the EC-mediated clearance. Our results also indicate that lactadherin-mediated clearance may limit procoagulant or prothrombotic activity of senescent PMN's. Disclosures: No relevant conflicts of interest to declare.

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Desialylated Platelets Are Cleared From the Circulation by the Hepatic Asialoglycoprotein Receptor,

Blood ◽

10.1182/blood.v118.21.3257.3257 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3257-3257

Author(s):

Renata Grozovsky ◽

Silvia Giannini ◽

Karin M. Hoffmeister

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Fold Increase ◽

Asialoglycoprotein Receptor ◽

Regulatory Mechanisms ◽

Wild Type ◽

Cell Counts ◽

Blood Cell Counts ◽

Terminal Galactose

Abstract Abstract 3257 The regulatory mechanisms of platelet homeostasis remain elusive. We investigated here the role of hepatic asialoglycoprotein receptor (a.k.a. Ashwell-Morell receptor) in platelet clearance. Mice lacking the hepatic asialoglycoprotein receptor Asgpr2 subunit had increased platelet survivals (T1/2 = 49.5±2h) when compared to wild type (WT, T1/2 = 31±4h) mice. Consequently, Asgpr2−/− mice had platelet counts increased by ∼20%, compared to WT, with increased terminal galactose exposure, as demonstrated using the galactose specific lectin RCA1. Bone marrow and spleen megakaryocyte numbers were reduced by ∼15% and ∼20% in Asgpr2−/− mice, compared to WT mice. Sialidase (NA, Clostidium perfringens, 50mU/mice) maximally desialylated circulating platelets when injected intravenously, as evidenced by increased RCA1 binding. Sialidase injection resulted in a ∼60% depletion of circulating platelets after 24h in Asgpr2−/− mice, compared to >90% in WT mice, indicating that desialylated platelets were partially removed by Asgpr1/2. In contrast to platelets, red blood cell counts were unaffected by sialidase treatment. Sialidase injection for 72h resulted in a 2.3-fold and 1.2-fold increase in megakaryocyte numbers in the spleen and bone marrow of WT mice, respectively, but not in Asgpr2−/− mice. In contrast to sialidase treatment, injections of rabbit anti-mouse platelet serum (RAMPS) depleted >95% of circulating platelets and increased by 70% bone marrow, but not spleen MK numbers in both WT and Asgpr2−/− mice. The data shows that removal of desialylated, i.e, senescent, platelets by the hepatic Ashwell-Morell receptor differs to that of antibody-mediated platelet clearance. Disclosures: No relevant conflicts of interest to declare.

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Non-Canonical NF-Kb Signaling Regulates Hematopoietic Stem Cell Self-Renewal and Microenvironment Interactions

Blood ◽

10.1182/blood.v118.21.859.859 ◽

2011 ◽

Vol 118 (21) ◽

pp. 859-859 ◽

Cited By ~ 1

Author(s):

Chen Zhao ◽

Yan Xiu ◽

John M Ashton ◽

Lianping Xing ◽

Yoshikazu Morita ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Wild Type ◽

Double Knockout ◽

Self Renewal ◽

Hematopoietic Stem ◽

Physiological Processes ◽

Marrow Cells

Abstract Abstract 859 RelB and NF-kB2 are the main effectors of NF-kB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-kB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-kB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

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The Clinical Role of Micrornas (miRs) in Cytogenetically Normal (CN) Acute Myeloid Leukemia (AML): miR-155 Upregulation Independently Identifies High-Risk Patients (Pts)

Blood ◽

10.1182/blood.v120.21.1387.1387 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1387-1387 ◽

Cited By ~ 1

Author(s):

Guido Marcucci ◽

Kati Maharry ◽

Klaus H. Metzeler ◽

Stefano Volinia ◽

Yue-Zhong Wu ◽

...

Keyword(s):

Clinical Outcome ◽

Conflicts Of Interest ◽

Disease Free Survival ◽

Gene Expression Signature ◽

White Blood Count ◽

Categorical Variables ◽

Prognostic Impact ◽

Continuous Variables ◽

Wild Type

Abstract Abstract 1387 miR-155 is upregulated in aggressive subtypes of solid tumors and leukemia. In AML, higher miR-155 expression is associated with FLT3-ITD. However, whether miR-155 upregulation impacts on clinical outcome independently from FLT3-ITD and other prognosticators is unknown. We evaluated the prognostic impact of miR-155 in 363 CN-AML pts (153 age <60 y; 210 age ≥60 y) that were treated with cytarabine-daunorubicin-based regimens and had a median follow-up of 7.9 y (range, 2.3–12.9). miR-155 levels were measured in pretreatment marrow or blood by the NanoString nCounter assay quantifying expression of the encoding gene MIR155HG; other molecular markers were assessed centrally. High miR-155 expressers (miR-155) had higher WBC (P<.001) and were more often FLT3-ITD-positive (pos; P<.001), RUNX1-mutated (mut; <.001), WT1-mut (P=.03), ↑ ERG (P=.02) and ↑ BAALC (P=.002), and less often CEBPA-mut (P=.003), IDH2-mut (P=.004) and FLT3-TKD-pos (P=.08) than low expressers (↓ miR-155). ↑ miR-155 had lower CR rates (P<.001), shorter DFS (P=.001) and OS (P<.001), than ↓ miR-155. In multivariable analyses (MVA; Table), ↑ miR-155 was associated with lower CR rates (P=.007) and shorter OS (P<.001). Among younger pts, ↑ miR-155 had lower CR rates (P=.03) and shorter DFS (P<.001) and OS (P<.001) than ↓ miR-155. In MVA (Table), ↑ miR-155 status remained associated with worse CR rate (P=.06), shorter DFS (P=.003) and OS (P=.01). Among older pts, ↑ miR-155 had lower CR rates (P=.008) and shorter OS (P<.001); in MVA (Table), ↑ miR-155 remained associated with worse CR (P=.03) and shorter OS (P=.05). In the European LeukemiaNet classification, younger ↑ miR-155 in the Favorable (Fav) Genetic Group (GG; CEBPA-mut and/or NPM1-mut without FLT3-ITD) had lower CR rates (P=.03) and shorter DFS (P=.04) and OS (P=.02) than ↓ miR-155. In the younger Intermediate-I (Int-I) GG pts (with wild-type CEBPA, NPM1-mut with FLT3-ITD, or wild-type NPM1), miR-155 expression did not impact independently on outcome. In older pts, ↑ miR-155 had a shorter OS both in the Fav (P=.06) and Int-I GGs (P=.05) than ↓ miR-155. To gain biologic insights, we derived an Affymetrix gene-expression signature that comprised 196 mRNAs significantly correlated with miR-155 expression. Consistent with previous mechanistic studies, Gene Ontology analysis revealed that the ↑ miR-155-associated signature was enriched for genes involved in anti-apoptotic, proliferative and inflammatory activities (FDR<0.05). ↑ miR-155 was not significantly correlated with that of any other microRNAs (miRs) thereby supporting the unique role of miR-155 among the miRs in AML. In summary, miR-155 expression is independently associated with clinical outcome in CN-AML and may allow for better evaluation of molecular risk, especially in pts lacking FLT3-ITD, like those in the ELN Fav GG. Moreover, given its role in deregulation of fundamental mechanisms of cell homeostasis and the emergence of miR inhibitors, miR-155 may become a novel therapeutic target. Table. MVA in pts with primary CN-AML Group CR DFS OS OR P HR P HR P All pts miR-155 expression not significantly associated with DFS a miR-155, ↑ v ↓ 0.46 .007 1.62 <.001 NPM1, mut v wt 2.42 .005 BAALC, ↑ v ↓ 0.37 .002 2.16 <.001 WBC, each 50 units 0.65 <.001 Age group, older v younger 0.43 .003 2.38 <.001 FLT3-ITD, pos v neg 1.78 <.001 Race, white v nonwhite 1.62 .03 Pts age < 60 y miR-155, ↑ v ↓ 0.39 .06 2.13 .003 1.84 .01 RUNX1, mut v wt 0.21 .01 Age, each 10 y increase 0.45 .004 WBC, each 50 units 1.49 <.001 FLT3-ITD, pos v neg 2.82 <.001 1.82 .01 FLT3-TKD, pos v neg 3.27 <.001 BAALC, ↑ v ↓ 2.66 <.001 2.33 <.001 Race, white v nonwhite 2.81 .02 CEBPA, mut v wt 0.47 .02 WT1, mut v wt 2.25 .005 Pts age ≥ 60 y miR-155 expression not significantly associated with DFS miR-155, ↑ v ↓ 0.46 .03 1.36 .05 NPM1, mut v wt 2.45 .03 BAALC, ↑ v ↓ 0.32 .004 2.18 <.001 WBC, each 50 units 0.65 .005 Age, each 10 y increase 0.48 .02 FLT3-ITD, pos v neg 1.56 .006 ↑, high expression; ↓, low expression; CR, complete remission; DFS, disease-free survival; HR, hazard ratio; mut, mutated; neg, negative; OR, odds ratio; OS, overall survival; pos, positive; WBC, white blood count; wt, wild-type. Odds ratios > (<) 1.0 mean higher (lower) CR rate and HRs > (<) 1.0 mean higher (lower) risk of relapse or death (DFS) or death (OS) for the higher values of continuous variables and the 1st category listed for categorical variables. Disclosures: No relevant conflicts of interest to declare.

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Platelets Protect From Lipopolysaccharide-Induced Lethal Endotoxemia by Inhibiting Macrophage-Dependent Inflammation Via the Cyclooxygenase 1 (COX1) Signaling Pathway

Blood ◽

10.1182/blood.v120.21.93.93 ◽

2012 ◽

Vol 120 (21) ◽

pp. 93-93

Author(s):

Binggang Xiang ◽

Guoying Zhang ◽

Xiang-An Li ◽

Andrew J. Morris ◽

Alan Daugherty ◽

...

Keyword(s):

Septic Shock ◽

Platelet Transfusion ◽

Conflicts Of Interest ◽

Plasma Concentrations ◽

Platelet Inhibition ◽

Severe Thrombocytopenia ◽

Wild Type ◽

Activated Platelets ◽

Platelet Depletion

Abstract Abstract 93 Sepsis is a tremendous burden for health-care systems. Patients with sepsis often have low platelet counts, and septic patients with severe thrombocytopenia have a poor prognosis and higher mortality. However, the role of platelets in the pathogenesis of sepsis has not been well elucidated. We investigated the role of platelets in septic shock using a mouse model of lipopolysaccharide (LPS)-induced endotoxemia. Depletion of platelets by intraperitoneal injection of a rat anti-mouse GPIb monoclonal antibody increased mortality and aggravated organ failure in endotoxemic mice as evident by increases in plasma aminotransferase (ALT), aspartate aminotransferase (AST), Lactate dehydrogenase (LDH), and Creatine kinase (CK) concentrations, while transfusion of platelets reduced mortality. Increases in mortality rate in thrombocytopenic mice by LPS challenge was not due to inflammatory hemorrhage, because there was no significantly hemorrhage observed in brains and lungs from mice pre-treated with either control IgG or the anti-GPIba antibody and blood RBC and Hb concentrations between IgG pre-treated mice and the anti-GPIba antibody pre-treated mice were similar. TNF-a, which is produced mainly by macrophages in vivo, plays critical roles in the development of disseminated intravascular coagulation, acute respiratory distress syndrome and shock in sepsis. Our data indicate that plasma concentrations of proinflammatory cytokines, TNF-a and IL-6, were markedly increased by platelet depletion and decreased by platelet transfusion in the mice challenged with LPS. Effects of platelet depletion on TNF-a production were eliminated in the mice that macrophages were pre-depleted. Furthermore, LPS- or thrombin-activated platelets or releasates from activated platelets inhibited TNF-a and IL-6 production in macrophages in vitro. Inhibition of TNF-a and IL-6 production in macrophages by activated platelets was prevented by pre-incubation of platelets with a COX1 inhibitor aspirin. Moreover, platelets from wild type mice but not COX1 deficient mice inhibited LPS-induced TNF-a and IL-6 production in macrophages. Transfusion of COX1 deficient platelets failed to protect against endotoxemia. Washed platelets from wild-type mice or platelet releasates from thrombin-activated wild-type mice inhibited LPS-induced TNF-a and IL-6 production in macrophages lacking TXA2 receptor, TP, suggesting that a metabolite other than TXA2 is responsible for platelet inhibition of macrophage function. We found that stimulation of platelets with thrombin or LPS induced PGE2 production and pre-incubation of macrophages with an antagonist of PGE2 receptor EP4 reversed platelet inhibition on TNF-a and IL-6 production in macrophages. Our results indicate that platelets protect against septic shock by inhibiting macrophage-dependent inflammatory response via the COX1/PGE2/EP4 dependent pathway. Thus, these findings demonstrate a previously unappreciated role for platelets in septic shock and suggest that platelet transfusion may be effective in treating septic patients. Disclosures: No relevant conflicts of interest to declare.

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Gtpase Immunity-Associated Protein Family Member 4 Contributes to the Enhanced Expansion of HSPCs in RUNX1 Deficient Mice

Blood ◽

10.1182/blood.v124.21.4344.4344 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4344-4344

Author(s):

Amanda Scholl ◽

Kentson Lam ◽

Alex Muselman ◽

Tingdong Tang ◽

Shinobu Matsuura ◽

...

Keyword(s):

Family Member ◽

Conflicts Of Interest ◽

Protein Family ◽

Dominant Negative ◽

Negative Regulator ◽

Wild Type ◽

Double Knockout ◽

Hematopoietic Stem ◽

Myeloid Progenitor

Abstract RUNX1 is the transcription factor described as the master regulator of hematopoiesis. Due to its central role during blood development, numerous RUNX1 mutations have been reported in hematologic abnormalities. Mice null for Runx1 die during embryogenesis, lacking definitive HSCs. Conditional Runx1Δ/Δ mice are viable, but exhibit a variety of blood abnormalities. The most salient defect in these Runx1Δ/Δ mice is expansion of the hematopoietic stem and progenitor cell (HSPC) population, measured as an increase in number of lineage negative, Sca1 positive, cKit positive (LSK) cells. A shortened form of RUNX1 (RUNX1SF) lacking the C-terminal and part of the N-terminal domain (41-214) acts as a dominant negative regulator of RUNX1 and hence also models RUNX1 loss-of-function. A differential gene expression analysis of HSPCs derived from Runx1Δ/Δ compared to wild type mice uncovered GTPase immunity-associated protein family member 4 (GIMAP4) as one of the genes most highly upregulated. Previous studies have focused almost exclusively on the role of GIMAP4 as a pro-apoptotic protein during T-cell development. This study illuminates a novel non-apoptotic role of GIMAP4 in a formerly unstudied HSPC context. Runx1Δ/Δ mice were crossed with Gimap4-/- mice to generate a double knockout (dKO) mouse line. These dKO mice exhibited attenuated HSPC proliferation in comparison to Runx1Δ/Δ mice, suggesting that GIMAP4 functions in this HSPC expansion phenotype. BMT experiments using lethally irradiated C57 mice and RUNX1SF transduced wild type versus Gimap4-/-bone marrow confirmed this result. GIMAP4 also worked independently and coordinately with RUNX1 to influence individual progenitor populations. Common lymphoid progenitors (CLP) were affected only by GIMAP4. Gimap4-/- mice exhibited an expansion of the CLP population, consistent with its pro-apoptotic role in lymphoid populations. Conversely, both RUNX1 and GIMAP4 coordinately exerted an effect on myeloid progenitor populations. Runx1Δ/Δ mice harbored expanded granulocyte-macrophage progenitor (GMP) and common myeloid progenitor (CMP) populations. This expansion was not observed when GIMAP4 was also ablated. This suggests a pro-proliferative role of GIMAP4 specifically in myeloid populations. These opposing roles of GIMAP4 in lymphoid versus myeloid cells suggest a more contextual, cell-specific role of this GTPase protein. Ultimately, this study provides insight into how RUNX1 and GIMAP4 may coordinate to maintain HSPC homeostasis. Disclosures No relevant conflicts of interest to declare.

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Targeting on the Stem Cell Niche Can Protect Hematopoietic Stem Cell from Chemotherapy and G-CSF

Blood ◽

10.1182/blood.v124.21.5790.5790 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5790-5790

Author(s):

Sidan Li ◽

Qiongli Zhai ◽

Dehui Zou ◽

Changhong Li ◽

Lugui Qiu

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Mouse Models ◽

Cell Function ◽

Conflicts Of Interest ◽

Hematopoietic Stem ◽

Transplant Patients ◽

Cell Engraftment ◽

Cell Niche

Abstract The majority of hematopoietic stem/progenitor cells (HSPCs) reside in the bone marrow surrounded by specialized bone-shielded environment. The specialized microenvironment or niche not only provides a favorable habitat for HSPC maintenance and development but also governs stem cell function. Here we investigated the potential role of bone remodeling osteoblasts and osteoclasts in homeostasis and stress-induced mobilization of hematopoietic progenitors, then further tested the hypothesis that targeting the niche might improve stem cell–based therapies using six mouse models to mimic the multiple rounds of chemotherapy followed by autologous hematopoietic stem cells (HSCs) transplantation in a clinical setting. Herein, we show that multiple rounds treatment of cytotoxic drugs influence niche. Serum osteocalcin level declined obviously (22.19 ± 1.08 ng/mL, before treatment vs 16.08 ± 2.12 ng/mL, steady state, P=0.01) in autologous HSPCs transplant patients. In mouse models, the number of CD45- Ter119- OPN+ osteoblast was significantly reduced (untreated, 3993 ± 129 cells/femur; CTLs, 1937 ±196 cells/femur; Gs, 1055 ± 43 cells/femur; P<0.01). Pharmacologic use of parathyroid hormone (PTH) or receptor activator of nuclear factor kappa-B ligand (RANKL) increases the number of HSC mobilized into the peripheral blood for stem cell harvests and protects stem cells from repeated exposure to cytotoxic chemotherapy. Ttreatment with granulocyte colony stimulating factor (G-CSF) plus PTH led to relative preservation of the HSC pool (G vs PTH, P<0.01; CTL vs PTH, P<0.05). Recipient mice transplanted with circulation HSPCs of P+R and P+R+G groups also showed more robust myeloid and lymphatic cell engraftment than did HSCs from either CTL or G group. These data provide evidence that targeting the HSPC niche may improve the efficacy of HSPC mobilization. Disclosures No relevant conflicts of interest to declare.

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Lin28b-Let-7-PRC1 Axis Guides Developmental Maturation of the Hematopoietic System

Blood ◽

10.1182/blood-2021-146004 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 21-21

Author(s):

Mayuri Tanaka-Yano ◽

Dahai Wang ◽

Eleanor Meader ◽

Melissa A. Kinney ◽

Vivian Morris ◽

...

Keyword(s):

Young Adult ◽

Developmental Stage ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Polycomb Group ◽

Wild Type ◽

Hematopoietic Stem ◽

Protein Levels ◽

Developmental Maturation

Abstract Polycomb group (PcG) proteins are a well-studied group of chromatin modifiers belonging to one of two distinct multi-protein complexes: Polycomb repressive complex 1 (PRC1) and PRC2. With definitive hematopoiesis, PRCs contribute to many aspects of fetal and adult blood formation. However, it is largely unknown how many of the age-specific effects of PRCs in hematopoiesis are regulated. Here, we show that the definitive hematopoietic stem and progenitor cell (HSPC) compartment is remodeled from the fetus to the neonate and into young adulthood coordinated with changes in mature blood cell output. This process is in part dependent on the PRC1 component Cbx2, which is regulated by the heterochronic Lin28b/let-7 axis. First, we quantified various population of definitive hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) using midgestation fetal liver (FL, embryonic day 14.5 (E14.5)), newborn bone marrow (BM, postnatal day 0-1), or young adult (postnatal age 6 to 8 weeks) BM. The lymphoid biased multipotent progenitor 4 (MPP4, ~0.9-fold) declined as the mice matured and aged. We also found erythroid-biased MPP2 diminished (~0.7-fold) while myeloid-biased MPP3 increased (~1.7-fold) with maturation. Using isolated long-term (LT) HSCs from these three stages, we found that E14.5 FL (~8.0-fold) and neonatal LT-HSC (~4.0-fold) showed more rapid B-cell reconstitution compared to young adult LT-HSCs upon transplantation. We found that many of these effects were regulated by Lin28b/let-7. Next, we aimed to determine the downstream mediators of Lin28/let-7's effect on HSPCs maturation. By interrogating gene regulatory subnetworks differentially active across mouse HSPC maturation and mining these subnetworks for predicted let-7 target transcripts, we found Cbx2 enriched in E14.5 FL (P=0.003) and adult HSPCs ectopically expressing LIN28B relative to wild-type adult HSPCs. In cell-based assays, we confirmed that let-7 microRNAs directly regulated CBX2 protein levels. Thus, the Lin28b/let-7 axis governs CBX2 protein levels, leading us to hypothesize that this axis exerts its wide-ranging effects on hematopoietic maturation by regulating PRC1 by controlling Cbx2 levels. As CBX2's developmental stage-specific functions have not been investigated, we generated Cbx2-/-embryos and investigated definitive FL hematopoiesis. We observed skewing of myeloerythorid progenitors to an adult-like myeloid-predominant distribution in Cbx2-/- embryos (P=0.0002), and B-cells in Cbx2-/- neonatal spleens were diminished (P=0.04). We further examined this effect using transplanted Cbx2-/- MPP4 from E14.5 FL which resulted in a decreased donor derived B-lymphoid output compared to wild-type littermates (~0.7-fold). To understand the functional role of Cbx2/PRC1 in juvenile hematopoiesis, we next investigated the role of Cbx2 in maintaining histone H2A monoubiquitinylation (H2AK119Ub) - the histone modification placed by PRC1 - in FL HSPCs. In Cbx2-/- FL HSPCs, the global distribution of H2AK119Ub localization did not change, but several specific H2AK119Ub peaks were altered. We observed differential H2AK119Ub abundance associated with a candidate enhancer within the Erg gene, suggestive of control of Erg expression by Lin28b/let-7/Cbx2. We confirmed that this enhancer activated transcription from a minimal promoter (~8-fold). Erg expression was increased in perinatal spleens of Cbx2-/- mice compared to Cbx2+/+ littermates (~4-fold). Moreover, we found that Cbx2 could repress ERG expression as well as other master HSPC transcription factors. Overall, our findings show that the Lin28b/let-7-axis controls developmental stage-specific hematopoietic output through PRC1-mediated chromatin remodeling. These findings demonstrate a key mechanism by which HSPCs alter their properties during developmental maturation with relevance to age-skewed blood disorders. Disclosures No relevant conflicts of interest to declare.

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C-CBL Is a Critical Negative Regulator of Megakaryopoesis

Blood ◽

10.1182/blood.v124.21.1581.1581 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1581-1581

Author(s):

Sebastian J. Saur ◽

Melanie Märklin ◽

Alexandra Poljak ◽

Manuela Ganser ◽

David E. James ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Negative Regulator ◽

Cytokine Signaling ◽

Wild Type ◽

Platelet Factor ◽

Physiological Level ◽

Hematopoietic Stem ◽

Primary Mouse

Abstract Megakaryopoiesis is controlled by a variety of hematopoietic growth factors in order to maintain a physiological level of circulating platelets. Thrombopoietin (TPO) is the main regulator of megakaryopoiesis modulating megakaryocyte differentiation, promoting endomitosis and proplatelet formation and as such supports the self-renewal and survival of hematopoietic stem cells. To allow proper proliferation and differentiation of different hematopoetic lineages, TPO signal transduction must be tightly regulated. Several mechanisms negatively modulating hematopoiesis and differentiation of the megakaryocytic lineage have previously been identified. Among those are suppressors cytokine signaling, protein phosphatases as well as a multitude of negative regulatory signaling pathways. However, one of the most effective mechanisms to permanently disable activated signaling proteins is by targeted degradation via lysosomes or proteasomes. In this study, we investigated the mechanisms that regulate TPO-mediated MPL degradation in primary mouse cells. Previous studies have identified CBL as an E3 ligase responsible for the ubiquitination of MPL in cell lines. In order to determine the potential role of c-CBL in murine thrombopoiesis, we used Cre/loxP technology to specifically delete c-CBL in the megakaryocytic lineage. Mice expressing two floxed c-CBL alleles were crossed to mice expressing Cre recombinase under the control of the platelet factor 4 (PF4) promoter. This yielded progeny with the desired genotype of c-CBLfl/fl PF4-Cre (CBL ko) after two generations of breeding. The desired cohort exhibited a quantitative absence of c-CBL in megakaryocytes and platelets as assessed by western blotting compared with wild type C57/BL6 mice. The expression of CBL in other hematopoietic cells such as B cells, T cells, neutrophils, monocytes and dendritic cells remained unaffected in this conditional ko strain. The experimental cohort showed significantly higher numbers of megakaryocytes in the bone marrow and of platelets in the peripheral blood as compared to wild type mice (1.2 mio vs. 1.8 mio cells/µl, p<0.0001). In addition, the platelets from the mutant mouse strain were of significantly smaller size (43 vs. 38 fL, p=0.0022). To evaluate the role of c-CBL in mature megakaryocytes, total bone marrow was collected from 12 wk old CBL ko mice and grown in TPO-containing culture medium for 72 h. Megakaryocytes derived from the bone marrow of wild type mice served as controls. Mature megakaryocytes were eventually isolated on a BSA-density gradient. Subsequent Western Blot analysis revealed a significant reduction of MPL ubiquitination in the CBL ko mice as compared to wild type mice, thereby identifying c-CBL as a critical negative regulator of megakaryopoesis. Taken together, we have successfully ablated c-CBL specifically from the megakaryocyte lineage and could demonstrate that this has profound effects on platelet counts and platelet size. In addition, we were able to show that c-CBL ablation leads to reduced ubiquitination of MPL and a consecutively longer half life of this protein culminating in substantially increased megakaryopoiesis in the c-CBL ko cohort. In summary, these data enhance our understanding of the regulation of TPO signaling and the physiological role of CBL in the megakaryocytic lineage. Disclosures No relevant conflicts of interest to declare.

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