Bone Marrow Myeloid Progenitor Cells Research Articles

The ratio of bone marrow myeloid progenitor cell proportion to mature lymphocytes proportion can effectively differentiate aplastic anemia and hypoplastic myelodysplastic syndrome and evaluate the quality of bone marrow aspirates.

Aplastic anemia (AA) and hypoplastic myelodysplastic syndrome (MDS-h) are bone marrow failure disease and difficult to distinguish merely by morphological analysis. In this study, we investigated the value of flow cytometry (FCM) in the differential diagnosis of AA and MDS-h. We included 822 patients (626 control, 69 AA, 22 MDS-h and 105 dilution patients) from January 2017 to December 2022 for a retrospective study. Bone marrow myeloid progenitor (MP) cell and mature lymphocytes proportions were analyzed by FCM. The ratio of MP cell proportion and mature lymphocytes proportion, MPLR, was calculated. Data were compared by Kruskal-Wallis test. Differential diagnostic efficacy was evaluated by receiver operating characteristic (ROC) curve. Cutoff value was determined by the maximum Youden index. Bone marrow MP cell proportion and MPLR of MDS-h patients were higher than AA patients. Mature lymphocytes proportion of MDS-h patients was lower than AA patients. Area under ROC curve (AUC of ROC) of MP cell proportion, MPLR and mature lymphocytes proportion to distinguish AA from MDS-h were 0.992, 0.988, and 0.850, respectively. Moreover, MPLR of dilution patients was higher than AA patients but lower than MDS-h patients. The AUC of ROC curves of MPLR to distinguish MDS-h and AA from dilution were 0.854 and 0.871, respectively. Bone marrow MP cell proportion and MPLR can effectively discriminate AA from MDS-h with similar differential efficacy, which is higher than mature lymphocytes proportion. Moreover, MPLR can evaluate the quality of bone marrow aspirates, which would interfere with the differential diagnosis.

Novel Small Molecule, UTS-1401, as a Radioprotector for Total-Body Irradiation.

We report on a new radioprotector, UTS-1401, a small molecule that was synthesized (by one of us, JS) and evaluated here for its radioprotective effect against total-body irradiation (TBI). Female and male NIH Swiss mice were subjected to TBI at doses of 6.5, 7.5 and 8.5 Gy either with or without a 24 h pretreatment of UTS-1401 given ip and observed for 30 days. Survival rates were significantly increased when mice were treated with UTS-1401 compared to those not treated. The radioprotective effect of UTS-1401 was drug-dose dependent for male mice exposed to 8.5 Gy TBI with 150 mg/kg of UTS-1401 as the optimal dose. The radioprotective effect of UTS-1401 on female mice exposed to 8.5 Gy TBI was observed at 50, 100, and 150 mg/kg, with no dose response relationship noted. Female mice were more radioresistant than male mice with LD50/30 values of 7.8 Gy vs. 6.8 Gy, respectively. Weight changes after UTS-1401 alone showed a significant body weight increase at 150 mg/kg. Both the ip and iv route for UTS-1401 were similarly effective for male mice exposed to 8 Gy TBI. Further analysis using an endogenous spleen colony assay demonstrated that pretreatment of UTS-1401 for up to 72h prior to TBI protected both spleen weight and hematopoietic stem cells with a treated/untreated ratio between 2.0 and 3.2 for the latter for times between 0.5 h and 72 h. A separate invivo study showed that pretreatment of UTS-1401 protected bone marrow CFU-GM for mice exposed to TBI. In summary, UTS-1401 is a promising small-molecule radioprotective agent as demonstrated by whole animal, hematopoietic stem cell and bone marrow myeloid progenitor cell survival.

Tristetraprolin regulates the skeletal phenotype and osteoclastogenic potential through monocytic myeloid-derived suppressor cells.

Tristetraprolin (TTP; also known as NUP475, GOS24, or TIS11), encoded by Zfp36, is an RNA-binding protein that regulates target gene expression by promoting mRNA decay and preventing translation. Although previous studies have indicated that TTP deficiency is associated with systemic inflammation and a catabolic-like skeletal phenotype, the mechanistic underpinnings remain unclear. Here, using both TTP-deficient (TTPKO) and myeloid-specific TTPKO (cTTPKO) mice, we reveal that global absence or loss of TTP in the myeloid compartment results in a reduced bone microarchitecture, whereas gain-of-function TTP knock-in (TTPKI) mice exhibit no significant loss of bone microarchitecture. Flow cytometry analysis revealed a significant immunosuppressive immune cell phenotype with increased monocytic myeloid-derived suppressor cells (M-MDSCs) in TTPKO and cTTPKO mice, whereas no significant changes were observed in TTPKI mice. Single-cell transcriptomic analyses of bone marrow myeloid progenitor cell populations indicated a dramatic increase in early MDSC marker genes for both cTTPKO and TTPKO bone marrow populations. Consistent with these phenotypic and transcriptomic data, invitro osteoclastogenesis analysis of bone marrow M-MDSCs from cTTPKO and TTPKO displayed enhanced osteoclast differentiation and functional capacity. Focused transcriptomic analyses of differentiated M-MDSCs showed increased osteoclast-specific transcription factors and cell fusion gene expression. Finally, functional data showed that M-MDSCs from TTP loss-of-function mice were capable of osteoclastogenesis and bone resorption in a context-dependent manner. Collectively, these findings indicate that TTP plays a central role in regulating osteoclastogenesis through multiple mechanisms, including induction of M-MDSCs that appear to regulate skeletal phenotype.

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology.

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and adaptive immune responses. In vitro study of macrophages can be undertaken by ex vivo culture from the peritoneum or through differentiation of myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). A common approach to macrophage differentiation from precursors involves the use of conditioned media from L929 cells (LCM). This media is easy to self-produce but suffers from batch variability, and its constituents are undefined. Similarly, Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture of undefined molecules that may vary between batches. These methods are not adequate for the study of nitric oxide biology and redox mechanisms as they both contain substantial amounts of small molecules that either interfere with redox mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), required for the production of NO from inducible nitric oxide synthase (iNOS). In this report, we present an optimized protocol allowing for control of the NO-redox environment by reducing the levels of exogenous biopterin while maintaining conditions suitable for cell growth and differentiation. Tight control of culture media composition helps ensure experimental reproducibility and facilitates accurate interpretation of results. In this protocol, BMDMs were obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs was performed with media containing either (i) conditioned LCM, or (ii) recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and NO-deficient culture conditions - thus allowing for the reproducible study of NO-redox biology and immunometabolism in vitro.

Isolation and <em>In vitro</em> Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and adaptive immune responses. In vitro study of macrophages can be undertaken by ex vivo culture from the peritoneum or through differentiation of myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). A common approach to macrophage differentiation from precursors involves the use of conditioned media from L929 cells (LCM). This media is easy to self-produce but suffers from batch variability, and its constituents are undefined. Similarly, Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture of undefined molecules that may vary between batches. These methods are not adequate for the study of nitric oxide biology and redox mechanisms as they both contain substantial amounts of small molecules that either interfere with redox mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), required for the production of NO from inducible nitric oxide synthase (iNOS). In this report, we present an optimized protocol allowing for control of the NO-redox environment by reducing the levels of exogenous biopterin while maintaining conditions suitable for cell growth and differentiation. Tight control of culture media composition helps ensure experimental reproducibility and facilitates accurate interpretation of results. In this protocol, BMDMs were obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs was performed with media containing either (i) conditioned LCM, or (ii) recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and NO-deficient culture conditions - thus allowing for the reproducible study of NO-redox biology and immunometabolism in vitro.

Abrogated GADD45b-Mediated Integrity Control of Hematopoietic Stem Cells upon ER Stress and DNA Damage Triggers Lymphopoiesis over Granulopoiesis in Congenital Neutropenia

In patients with severe congenital neutropenia (CN) with maturation arrest of bone marrow granulopoiesis, hematopoietic and myeloid progenitor cells exhibit elevated endoplasmic reticulum (ER) stress and unfolded protein response (UPR) due to inherited mutations in ELANE (neutrophil elastase, NE) or HAX1 (HCLS1-associated protein X-1). UPR and ER stress may induce genetic instability in hematopoietic stem and progenitor cells (HSPCs). Indeed, we recently reported severe changes in cell cycle control and DNA repair pathways (e.g. CHEK1 and BRCA1) in HSPCs of CN patients, as compared to G-CSF treated healthy individuals. We further studied the molecular mechanism of the hyper-sensitivity of CN HSPC to stress conditions. We found that expression of Growth Arrest and DNA-Damage-inducible, beta (GADD45b), which functions as stress sensor regulating cell cycle, survival and apoptosis in response to ER stress or DNA damage, was markedly reduced in myeloid progenitors of CN patients, as compared to G-CSF-treated healthy individuals. GADD45b is essential in stress-induced murine hematopoiesis and myeloid differentiation is severely compromised in gadd45b knockout mice (Gupta S. et al., 2006). Less is known about the functions of GADD45b in the DNA damage response and myeloid differentiation of human HSPCs. To study GADD45b functions in human hematopoiesis, we generated human induced pluripotent stem cells (iPSC) of healthy individuals haplo-insufficient for GADD45b using CRISPR/Cas9-mediated knockout of GADD45b . Human iPSCs were transfected with GADD45b -specific CRISPR/Cas9-gRNA RNP and gene-edited clones were selected for further study. We could not detect any iPSCs clones with frameshift homozygote or compound heterozygote mutations in exon 1 of GADD45b leading to the complete knockout of GADD45b . Most probably GADD45b-deficient iPSCs are lethal. GADD45b+/- iPSCs also presented a profound phenotype. While these cells retained pluripotency, we detected elevated γH2AX levels in GADD45b+/- iPSCs upon UV light exposure, as compared to GADD45b-WT cells. In line with up-regulated γH2AX levels, we measured strongly induced DNA damage, as quantified by Lord-Q DNA damage assay. We further analyzed myeloid differentiation of GADD45b+/- iPSCs using embryoid body (EB)-based method and found that GADD45b+/- iPSCs cells completely lost the capacity to differentiate into hematopoietic cells. We were able to make EBs of GADD45b+/- cells, these EBs survived and showed no signs of degradation or cell death, but they produced no floating (hematopoietic) cells. To study the effects of GADD45b inhibition in hematopoietic cells we transfected bone marrow CD34+ cells with GADD45b -specific CRISPR/Cas9-gRNA RNP. Similar to iPSCs, we detected markedly elevated UV-induced DNA damage and diminished G-CSF-mediated myeloid differentiation of GADD45b-edited CD34+cells in colony-forming units (CFU) assay. These data strongly support the essential role of GADD45b in hematopoietic and myeloid differentiation. Diminished GADD45b expression in CN HSPCs may increase susceptibility to stress and may contribute to malignant transformation.In order to prevent the appearance and clonal outgrowth of stressed HSPCs with inherited disease-causing mutations in combination with acquired oncogenic mutations, intrinsic DNA damage must be tightly controlled. Failures in the control mechanisms may affect the fate of HSPCs as for instance altered proliferation and wrongly biased differentiation. Therefore, we further evaluated the composition of the HSPC pool in GADD45b-deficient bone marrow cells of CN patients (CN- ELANE n=5, CN- HAX1 n=5), in comparison to healthy individuals treated (n=5) or not (n =7) with G-CSF using multicolor FACS panel of J. Dick. Treatment of healthy individuals with G-CSF induces increase of common myeloid progenitors (CMP, 7-fold) and decrease of multi-lymphoid progenitors (MLP, 10-fold). At the same time, the hematopoietic differentiation in CN patients is substantially shifted from CMP (14-fold decrease) to MLP (36-fold increase) lineage with dramatically reduced numbers of GMPs (3-fold) and increased numbers of B/NK progenitors (5-fold). These data suggest that deregulated integrity control of HSPC pool due to elevated UPR and ER stress and reduced GADD45b expression in hematopoietic cells of CN patients may change composition of HSPC pool and G-CSF response. DisclosuresNo relevant conflicts of interest to declare.

Reprogramming of bone marrow myeloid progenitor cells in patients with severe coronary artery disease.

Atherosclerosis is the major cause of cardiovascular disease (CVD). Monocyte-derived macrophages are the most abundant immune cells in atherosclerotic plaques. In patients with atherosclerotic CVD, leukocytes have a hyperinflammatory phenotype. We hypothesize that immune cell reprogramming in these patients occurs at the level of myeloid progenitors. We included 13 patients with coronary artery disease due to severe atherosclerosis and 13 subjects without atherosclerosis in an exploratory study. Cytokine production capacity after ex vivo stimulation of peripheral blood mononuclear cells (MNCs) and bone marrow MNCs was higher in patients with atherosclerosis. In BM-MNCs this was associated with increased glycolysis and oxidative phosphorylation. The BM composition was skewed towards myelopoiesis and transcriptome analysis of HSC/GMP cell populations revealed enrichment of neutrophil- and monocyte-related pathways. These results show that in patients with atherosclerosis, activation of innate immune cells occurs at the level of myeloid progenitors, which adds exciting opportunities for novel treatment strategies.

Langerhans cell histiocytosis of the rib

Abstract Langerhans cell histiocytosis (LCH) is a rare systemic disease characterized by the proliferation and accumulation of Langerhans cells derived from bone marrow myeloid progenitor cells. The disease can affect a single organ or multi-organ system. The main sites involved in LCH include bones, lungs, central nervous system, liver, thymus, skin, and lymph nodes, causing focal or systemic effects. We report a rare case of LCH showing a single osteolytic lesion on the posterolateral side of the tenth rib of a 2-year-old boy who presented with upper back pain.

Isolation and culture of murine bone marrow-derived macrophages for nitric oxide and redox biology

Macrophages are mononuclear phagocytes derived from haematopoietic progenitors that are widely distributed throughout the body. These cells participate in both innate and adaptive immune responses and lie central to the processes of inflammation, development, and homeostasis. Macrophage physiology varies depending on the environment in which they reside and they exhibit rapid functional adaption in response to external stimuli. To study macrophages in vitro, cells are typically cultured ex vivo from the peritoneum or alveoli, or differentiated from myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). BMDMs represent an efficient and cost-effective means of studying macrophage biology. However, the inherent sensitivity of macrophages to biochemical stimuli (such as cytokines, metabolic intermediates, and RNS/ROS) makes it imperative to control experimental conditions rigorously. Therefore, the aim of this study was to establish an optimised and standardised method for the isolation and culture of BMDMs. We used classically activated macrophages isolated from WT and nitric oxide (NO)-deficient mice to develop a standardised culture method, whereby the constituents of the culture media are defined. We then methodically compared our standardised protocol to the most commonly used method of BMDM culture to establish an optimal protocol for the study of nitric oxide (NO)-redox biology and immunometabolism in vitro.

CD205-positive, Sepharose-induced peritoneal exudate cells: a new resource for DC research in the chicken.

Dendritic cells (DC) are important antigen-presenting cells and are among the least characterized immune cells in the chicken. In order to obtain chicken DC, current protocols require isolation of bone marrow myeloid progenitor cells and induction of DC differentiation with supplemental cytokines or negative selection of splenic cell preparations. Chicken peritoneal exudate cells (PEC) have traditionally been a source of various immune cells for ex vivo studies, primarily to investigate heterophils and macrophages. In this study, we observe the presence of CD205+ PEC populations, a marker of DC, as an additional resource to isolate and study chicken primary DCs. A panel of monoclonal antibodies was developed against the chicken CD205 DC marker and used to isolate CD205+ DC from the PEC population using magnetic bead cell sorting. This study reports the development of new anti-CD205 monoclonal antibodies as a reagent for chicken DC research, as well as PEC as a potential source of CD205+ DC for ex vivo studies in the chicken.

Apigenin Inhibits UVB-Induced Skin Carcinogenesis: The Role of Thrombospondin-1 as an Anti-Inflammatory Factor

We have previously demonstrated that apigenin promotes the expression of antiangiogenic protein thrombospondin-1 (TSP1) via a mechanism driven by mRNA-binding protein HuR. Here, we generated a novel mouse model with whole-body THBS-1 gene knockout on SKH-1 genetic background, which allows studies of UVB-induced acute skin damage and carcinogenesis and tests TSP1 involvement in apigenin's anticancer effects. Apigenin significantly inhibited UVB-induced carcinogenesis in the wild-type (WT) animals but not in TSP1 KO (TKO) mice, suggesting that TSP1 is a critical component of apigenin's chemopreventive function in UVB-induced skin cancer. Importantly, TKO mice presented with the elevated cutaneous inflammation at baseline, which was manifested by increased inflammatory infiltrates (neutrophils and macrophages) and elevated levels of the two key inflammatory cytokines, IL-6 and IL-12. In agreement, maintaining normal TSP1 expression in the UVB-irradiated skin of WT mice using topical apigenin application caused a marked decrease of circulating inflammatory cytokines. Finally, TKO mice showed an altered population dynamics of the bone marrow myeloid progenitor cells (CD11b+), with dramatic expansion of the population of neutrophil progenitors (Ly6ClowLy6Ghigh) compared to the WT control. Our results indicate that the cutaneous tumor suppressor TSP1 is a critical mediator of the in vivo anticancer effect of apigenin in skin, specifically of its anti-inflammatory action.

Abstract 1882: Preclinical evaluation of the tyrosine kinase inhibitor ARQ 531 in AML

Abstract Acute Myeloid Leukemia (AML) is a rapidly progressing hematopoietic malignancy arising from bone marrow myeloid progenitor cells. Treatment with cytotoxic chemotherapy has not changed for over four decades resulting in poor survival. The dismal prognosis could be attributed to the heterogeneity of this disease, where multiple genetically aberrant clones exist within the same patient. Mutations in the FMS-like tyrosine kinase (FLT3) occurs in 30% of AML patients, typically as an internal tandem duplication (ITD) resulting in a constitutively active FLT3 survival pathway. This has prompted the generation of selective FLT3 inhibitors such as Quizartinib and Gilteritinib which are currently being pursued in clinical trials. Still, acquired resistance to these selective FLT3 inhibitors due to the acquisition of tyrosine kinase domain mutations (TKD) can occur. This suggests that the use of an agent with a broader kinome inhibition profile (such as the recently granted FDA approved Midostaurin) could achieve more durable clinical benefit. ARQ 531 is a novel potent BTK inhibitor currently being investigated in a Phase 1 trial in patients with relapsed/refractory hematological malignancies (ClinicalTrials.gov Identifier: NCT03162536). We have found that ARQ 531 also has inhibitory activity against members of the Src family of kinases (SFK; including downstream target SYK) as well as FLT3. SYK directly binds to and trans-activates FLT3 which is essential for FLT3-ITD tumorigenicity, suggesting that ARQ 531 has therapeutic potential in AML. Therefore we have investigated the in vitro and in vivo efficacy of ARQ 531 in AML. Our preliminary studies demonstrate cytotoxicity for ARQ 531 in patient-derived primary AML cells harboring FLT3 wild type and FLT3-ITD, as well as multiple AML cell lines. Importantly, ARQ 531 is effective in a MOLM-13 tyrosine kinase inhibitor (TKI) resistant cell line harboring a FLT3-ITD-TKD-D835Y mutation. Furthermore, we show that ARQ 531 can reduce the level of phosphorylated FLT3, but unlike selective FLT3 inhibitors, it can also inhibit Src family phosphorylation and SYK phosphorylation. Additionally, ARQ 531 exhibited an anti-clonogenic effect on primary patient blasts using Methocult colony forming unit assay. Finally, to investigate the in vivo effect of ARQ 531, we used an aggressive AML MOLM-13 disseminated xenograft mouse model. NSG mice were randomized one-week post engraftment to either vehicle or daily oral gavage of 50 mg/kg ARQ 531. The estimated median survival for the ARQ 531 group was 23 days compared to 21 days for the vehicle group (p = 0.002) suggesting in vivo efficacy for ARQ 531 in AML. Collectively, we provide for the first time promising preclinical efficacy for ARQ 531 in AML supporting further mechanistic investigation of this agent, and potentially, expansion of the ongoing clinical studies to include AML patients. E. H. and J.C. B. contributed equally as co-senior authors to this work Citation Format: Ola A. Elgamal, Bridget Carmichael, Amy Lehman, Shelley J. Orwick, Minh Tran, Virginia M. Goettl, Shaneice Mitchell, Rosa Lapalombella, Jae Yoon Jeon, Sharyn D. Baker, Sudharshan Eathiraj, Brian Schwartz, Erin Hertlein, John C. Byrd. Preclinical evaluation of the tyrosine kinase inhibitor ARQ 531 in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1882.

Cell-surface phosphatidylserine regulates osteoclast precursor fusion

Bone-resorbing multinucleated osteoclasts that play a central role in the maintenance and repair of our bones are formed from bone marrow myeloid progenitor cells by a complex differentiation process that culminates in fusion of mononuclear osteoclast precursors. In this study, we uncoupled the cell fusion step from both pre-fusion stages of osteoclastogenic differentiation and the post-fusion expansion of the nascent fusion connections. We accumulated ready-to-fuse cells in the presence of the fusion inhibitor lysophosphatidylcholine and then removed the inhibitor to study synchronized cell fusion. We found that osteoclast fusion required the dendrocyte-expressed seven transmembrane protein (DC-STAMP)-dependent non-apoptotic exposure of phosphatidylserine at the surface of fusion-committed cells. Fusion also depended on extracellular annexins, phosphatidylserine-binding proteins, which, along with annexin-binding protein S100A4, regulated fusogenic activity of syncytin 1. Thus, in contrast to fusion processes mediated by a single protein, such as epithelial cell fusion in Caenorhabditis elegans, the cell fusion step in osteoclastogenesis is controlled by phosphatidylserine-regulated activity of several proteins.

The Interferon Consensus Sequence Binding Protein (Icsbp/Irf8) Is Required for Termination of Emergency Granulopoiesis

Emergency granulopoiesis occurs in response to infectious or inflammatory challenge and is a component of the innate immune response. Some molecular events involved in initiating emergency granulopoiesis are known, but termination of this process is less well defined. In this study, we found that the interferon consensus sequence binding protein (Icsbp/Irf8) was required to terminate emergency granulopoiesis. Icsbp is an interferon regulatory transcription factor with leukemia suppressor activity. Expression of Icsbp is decreased in chronic myeloid leukemia, and Icsbp(-/-) mice exhibit progressive granulocytosis with evolution to blast crisis, similar to the course of human chronic myeloid leukemia. In this study, we found aberrantly sustained granulocyte production in Icsbp(-/-) mice after stimulation of an emergency granulopoiesis response. Icsbp represses transcription of the genes encoding Fas-associated phosphatase 1 (Fap1) and growth arrest-specific 2 (Gas2) and activates genes encoding Fanconi C and F. After stimulation of emergency granulopoiesis, we found increased and sustained expression of Fap1 and Gas2 in bone marrow myeloid progenitor cells from Icsbp(-/-) mice in comparison with the wild type. This was associated with resistance to Fas-induced apoptosis and increased β-catenin activity in these cells. We also found that repeated episodes of emergency granulopoiesis accelerated progression to acute myeloid leukemia in Icsbp(-/-) mice. This was associated with impaired Fanconi C and F expression and increased sensitivity to DNA damage in bone marrow myeloid progenitors. Our results suggest that impaired Icsbp expression enhances leukemogenesis by deregulating processes that normally limit granulocyte expansion during the innate immune response.

SH2B3/LNK Loss of Function Promotes Atherosclerosis and Thrombosis

Human genome-wide association studies (GWAS) have revealed many novel genetic loci that are associated with coronary heart disease (CHD) but do not involve traditional risk factors. However, the relevant genes and mechanisms are largely unknown. One such locus resides in SH2B3/LNK, which is expressed in hematopoietic cells and suppresses thrombopoietin (TPO) signaling via its receptor (MPL). The common risk single nucleotide polymorphism (SNP) of SH2B3/LNK is associated with CHD, increased platelet and myeloid cell counts in peripheral blood and JAK2V617F positive myeloproliferative neoplasms.Analysis of human cord blood revealed that samples with TT risk SNP (R262W) in SH2B3/LNK was associated with expansion of hematopoietic stem cells (HSCs), increased MPL signaling in HSCs, increased megakaryopoiesis, and a paradoxical increased expression of LNK mRNA, indicating reduced LNK function but induction of LNK transcription downstream of increased MPL signaling. Since humans are hypercholesterolemic relative to mice, to assess the role of reduced LNK function in athero-thrombosis, we transplanted WT or Lnk-/- bone marrow (BM) cells into irradiated WT or low-density lipoprotein receptor knock-out (Ldlr-/-) recipient mice and fed the recipients with chow and western type diet (WTD), respectively. Although Lnk-/- BM recipients showed similarly increased platelet counts on both diets, we found that platelet activation and aggregation, formation of platelet/leukocyte aggregates, atherosclerosis and arterial thrombosis were markedly increased by the combination of hyperlipidemia and hematopoietic LNK deficiency. These diet-genotype interactions mirrored an increase in mean platelet volume, protease-activated receptor-4 (PAR4) agonist-induced P-selectin exposure on platelets and platelet protein kinase C (PKC) activity, suggesting increased platelet granule secretion. We also observed an increased number of bone marrow myeloid progenitor cells expressing elevated levels of the common-beta subunit (CBS) of the interleukin-3/granulocyte-macrophage colony-stimulating factor (IL-3/GM-CSF) receptor, which promotes leukocytosis and the formation of platelet/leukocyte aggregates in the bloodstream. The increased P-selectin exposure, platelet aggregation and PKC activity were largely reversed by high-density lipoprotein (HDL) or cyclodextrin, reagents that remove cholesterol from platelets. Consistently, cholesterol loading of platelets from the chow-fed mice led to increased P-selectin exposure and PKC activity upon PAR4 agonist stimulation but the increase was more pronounced in Lnk-/- platelets, indicating a cholesterol-genotype interaction. LNK deficiency also resulted in defective MPL-mediated TPO internalization in platelets, leading to increased plasma TPO levels in mice on both diets. Elevated TPO levels and enhanced MPL signaling likely acted together to increase the pool of HSCs and megakaryopoiesis, leading to marked thrombocytosis.These studies suggest that SH2B3/LNK loss of function variants lead to HSC expansion, overproduction of platelets, and in the setting of hypercholesterolemia, markedly increased myeloid cells, platelet activation and formation of platelet/leukocyte aggregates, leading to accelerated atherosclerosis and arterial thrombosis. Our studies point to the importance of treating dyslipidemia in the setting of platelet and myeloid cell overproduction, as observed in subjects with SH2B3/LNK risk alleles. DisclosuresLevine:Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy. Tall:Amgen: Consultancy; Arisaph: Consultancy; CSL: Consultancy.

Homozygous Mutations in PHRF1 and PPARG in a Patient with Congenital Neutropenia and Profound Monocytosis: Understanding the Pathway Beyond Monocyte Versus Granulocyte Cell-Fate Choice

Myelopoiesis is a strictly regulated process of monocytes and granulocytes production, originating from common myeloid progenitors. Studies on patients with inherited severe congenital neutropenia (CN) could help to better elucidate myelopoiesis regulation. In CN, maturation arrest of granulocytic precursors at the promyelocytic stage leads to a shift towards monopoiesis and an ineffective granulopoiesis. Hereby we report on a particular CN patient with a typical "maturation arrest" of granulopoiesis at the stage of promyelocytes, very low levels of neutrophils but extremely high levels of monocytes in the peripheral blood and bone marrow. Upon G-CSF treatment (5μg/kg/day) this patient produces high levels of monocytes (up to 24.8x103/μL, more than 50 % of total leukocytes) and only up to 1x103/μl neutrophils. Sanger sequencing of DNA from this patient revealed no mutations in CN-related genes such as ELANE, HAX1 and G6PC3. Therefore we performed whole genome sequencing (Complete Genomics. Inc, Mountain View, CA) of DNA from blood of this patient and his mother to discover causative gene mutations. We identified a homozygous deletion in PHRF1 (PHD and Ring Finger Domain-Containing Protein 1) (p.R1015-G1019, NP_065952.2; rs144630030) and a homozygous missense mutation in PPARG (Peroxisome proliferator-activated receptor gamma) (p.P12A, NP_0569553.2; rs1801282). Both mutations are heterozygote in the patient's mother. Population frequency for heterozygote allele of these two variations was reported to be 13% and 7%, respectively, but no homozygote variants were reported till date. PHRF1 functions as an essential component of the TGF-ß tumour suppressor pathway by triggering degradation of the homeodomain repressor factor TGIF (TG-Interacting Factor) and a consequent retinoic acid signalling activation in haematopoiesis and monopoiesis. PPARG interacts with Retinoid X Receptors (RXR) and controls the expansion of macrophages. In order to evaluate the functional role of the detected mutations on disturbed G-CSF-triggered myelopoiesis in reference CN patient, CD33+ bone marrow myeloid progenitor cells of two healthy controls and this patient were treated with G-CSF in vitro and mRNA expression profiles were analysed in an Affymetrix Microarray platform, followed by Ingenuity Pathway Analysis (IPA). We found, that 'TREM1 signalling' was among the top three pathways with most significant differences (p<9x10-7) between this CN patient and healthy individuals. 'Granulocyte adhesion and diapedesis' and 'LXR/RXR pathway' were the next two significantly affected gene sets (p<8x10-6 and p<1.3x10-5, respectively). TREM1 (Triggering Receptor Expressed on Myeloid cells 1) is a chemokine receptor that is expressed by neutrophils and monocytes, however the ligand that activates this receptor is yet unknown. TREM-1 is involved in neutrophil apoptosis and is known to positively regulate monopoiesis by activation of M-CSF synthesis. Intriguingly, M-CSF was 4.2-fold upregulated in myeloid cells of patient, in comparison to healthy individuals. Other known components of TREM 1 signalling were also among the top 10 differentially expressed genes identified by IPA: HSD11B1 (+20 fold), ATP1B2 (+14 fold) and THBS1 (-10 fold). Functional connections between PPARG and TREM1 is known. PPARG mutation could lead to TREM1 signalling activation that consequently lead to M-CSF over-expression (+4.2 fold). In addition to the activation of TREM1 signalling, deletion in the PHFR1 gene could be the causative effect of marked upregulation of ALDH1A2 (+17 fold), which also could lead to an increase in M-CSF levels and in a retinoid acid signalling activation ultimately leading to increased monocyte production. Together, PPARG and PHRF1 mutations could hyper-activate the secretion of M-CSF by myeloid progenitors leading to a strong shift towards monopoiesis upon G-CSF treatment. Disclosures No relevant conflicts of interest to declare.

Stop-Codon Mutation In The Extracellular Part Of G-CSFR Leading To G-CSF Unresponsiveness Due To Defects In Erk1/2 Phopsphorylation In a Patient With Severe Congenital Neutropenia

We identified a 10 year old female severe congenital neutropnia patient who did not respond to the treatment with G-CSF (Filgrastim) up to 100 µg/kg/day, s.c. By search of possible reasons of G-CSF unresponsiveness, we detected by deep sequencing of the CSF3R (SOLID 5500xl, 3125 reads) a novel mutation within the extracellular part of CSF3R. The mutation was also confirmed by convential Sanger sequencing. It is a heterozygous stop-codon mutation C1641T (NM_000760), p.W547* (NP_000751) which is localized proximal to the cell membrane in the fifth fibronectin type III domain of the CSF3R. This is a first report describing a stop-codon mutation in the extracellular part of CSF3R in severe congenital neutropenia. FACS analysis revealed severely diminished surface expression levels of G-CSFR on CD33+ bone marrow myeloid progenitor cells of this patient as compared to G-CSFR levels on CD33+ cells of healthy individuals. In line with reduced expression of G-CSFR, we found disturbed G-CSFR-triggered intracellular signaling pathways activated by G-CSFR. MAPK/Erk1/2 activation by phosphorylation is known to be crucial for G-CSF- induced myeloid differentiation and STAT5 signaling is important for proliferation and survival of myeloid cells upon stimulation with G-CSF. We demonstrated abrogated phosphorylation of ERK1/2 and of STAT5 in bone marrow CD33+ myeloid cells of CN patient treated with G-CSF in vitro for 2, 5, 10 and 15 minutes, comparing with healthy donor. Since this patient has no known inherited CN-associated mutations (e.g. ELANE, HAX1, G6PC3), it may represent a new subgroup of CN patients characterized by non-responsiveness to G-CSF due to lack of binding of G-CSF to its receptor. Since the majority of investigations on CSF3R mutations are targeting the cytoplasmic part of the receptor only, we suggest that CN patients who do not respond to G-CSF treatment should be tested also for mutations within the extracellular part of the CSF3R. Disclosures:No relevant conflicts of interest to declare.

Pegylation of High-Density Lipoprotein Decreases Plasma Clearance and Enhances Antiatherogenic Activity

Infusions of apolipoprotein AI (apoAI), mimetic peptides, or high-density lipoprotein (HDL) remain a promising approach for the treatment of atherosclerotic coronary disease. However, rapid clearance leads to a requirement for repeated administration of large amounts of material and limits effective plasma concentrations. Because pegylation of purified proteins is commonly used as a method to increase their half-life in the circulation, we determined whether pegylation of apoAI or HDL would increase its plasma half-life and in turn its antiatherogenic potential. Initial pegylation attempts using lipid-poor apoAI showed a marked tendency to form multi-pegylated (PEG) species with reduced ability to promote cholesterol efflux from macrophage foam cells. However, pegylation of human holo-HDL or reconstituted phospholipid/apoAI particles (rHDL) led to selective N-terminal monopegylation of apoAI with full preservation of cholesterol efflux activity. The plasma clearance of PEG-rHDL was estimated after injection into hypercholesterolemic Apoe-/- mice; the half-life of pegylated PEG-apoAI after injection of PEG-rHDL was increased ≈7-fold compared with apoAI in nonpegylated rHDL. In comparison with nonpegylated rHDL, infusion of PEG-rHDL (40 mg/kg) into hypercholesterolemic Apoe-/- mice led to more pronounced suppression of bone marrow myeloid progenitor cell proliferation and monocytosis, as well as reduced atherosclerosis and a stable plaque phenotype. We describe a novel method for effective monopegylation of apoAI in HDL particles, in which lipid binding seems to protect against pegylation of key functional residues. Pegylation of apoAI in rHDL markedly increases its plasma half-life and enhances antiatherogenic properties in vivo.

New G-CSF-Dependent Signaling Pathway and Its Role In Patients with Severe Congenital Neutropenia and Acute Myeloid Leukemia

AbstractAbstract 385We demonstrated that stimulation of hematopoietic CD34+ cells with granulocyte-colony stimulating factor (G-CSF) leads to phosphorylation of hematopoietic cell-specific Lyn substrate 1 (HCLS1) protein and HCLS1 interaction with G-CSF receptor-associated tyrosine kinases Lyn and Syk. Activated HCLS1 binds to lymphoid-enhancer factor 1 (LEF-1) protein, inducing LEF-1-dependent autoregulation of the LEF-1 gene promoter. Transcription factor LEF-1 is a downstream effector of Wnt signaling and a master regulator of myeloid differentiation. In patients with severe congenital neutropenia (CN), inherited mutations in the HCLS1-associated protein × 1 (HAX1) lead to profound defects in the G-CSF-triggered expression and phosphorylation of HCLS1 protein and subsequently, to reduced expression of LEF-1 and abrogated granulopoiesis. Using immunoprecipitation assay, we identified endogenous HAX1 protein in the complex with LEF-1 and HCLS1 proteins. HAX1 is known as a mitochondrial protein, however using cell fractionation experiments of different myeloid cell lines (NB4, HL60, THP-1) and of primary CD34+ cells we identified endogenous HAX1 protein in different intracellular compartments. Thus, HAX1 protein was expressed in mitochondria, cytoplasm, nucleus, cytosol and in membranous fraction. Expression levels of HAX1 protein varied between different cell lines and between maturation stage of the cells. In vitro inhibition of HCLS1 or HAX1 by shRNA severely disrupted granulocytic differentiation of CD34+ cells, due to a lack of LEF-1 and C/EBPa protein expression and subsequently abolished activation of LEF-1 and C/EBPa gene promoters. Intriguingly, LEF-1 in turn induced HCLS1 mRNA and protein expression by direct binding to the HCLS1 gene promoter and inhibition of LEF-1 in CD34+ cells leads to severe downregulation of HCLS1 protein levels. These findings demonstrated a reciprocal feed-back regulation between LEF-1 and HCLS1 proteins. Moreover, HCLS1-/- mice are neutropenic due to a lack of LEF-1 protein expression and defective in vitro G-CSF-triggered F-actin rearrangement in bone marrow myeloid progenitor cells, as compared to WT mice. At the same time, in bone marrow biopsies of 88.46 % of tested patients (46 out of 52 patients) with acute myeloid leukemia (AML), we found significantly elevated levels of HCLS1 protein. Inhibition of HCLS1 in the AML cell lines and primary blasts of AML patients resulted in reduced proliferation and increased apoptosis. Taken together, we described a novel G-CSFR downstream signaling pathway, with dose-dependent effects on myelopoiesis: diminished expression of HCLS1 and LEF-1 resulted in a “maturation arrest” of granulopoiesis and severe neutropenia, but hyperactivation of one of these proteins led to a hyperproliferation of myeloid progenitors and AML. Disclosures:No relevant conflicts of interest to declare.

Bone marrow lymphoid and myeloid progenitor cells are suppressed in 7,12-dimethylbenz(a)anthracene (DMBA) treated mice

In this study we used colony forming unit (CFU) assays to demonstrate rapid suppression (within 6 h) of lymphoid (CFU-preB) and myeloid (CFU-GM) progenitor cells in DMBA-treated mice. The duration of these changes were consistent with the blood levels of DMBA and its metabolites that were achieved by either IP or oral DMBA administration. CFU-GM and CFU-preB activities returned to control levels by 2 and 7 days after oral DMBA exposure, respectively, but remained suppressed through 7 days after IP DMBA administration. The continued presence of low levels of DMBA in the bloodstream following IP administration was associated with sustained suppression of CFU-preB, total bone marrow lymphoid cells and peripheral blood lymphocytes. The changes noted above were not observed in Cyp1b1 null mice, demonstrating the need for local DMBA metabolism in the bone marrow by Cyp1b1 to impair bone marrow CFU-preB and CFU-GM. Furthermore, these data provide evidence that myeloid-lineage cells are restored more quickly than lymphoid-lineage cells after DMBA exposure.