bluuebunny/crossref_metadata_2025_split huggingface dataset:
STAT5-Regulated miRNA193b Controls Hematopoietic Stem and Progenitor Cell Expansion By Fine Tuning Cytokine Signaling
Authors: Haetscher Nadine , Feuermann Yonatan D , Wingert Susanne , Rehage Maike , Weiser Christian , Thalheimer Frederic B. , Bohnenberger Hanibal , Schroeder Timm , Serve Hubert , Oellerich Thomas , Hennighausen Lothar , Rieger Michael A. 
Published: October 2019

Abstract
Hematopoietic stem cells (HSCs) regenerate the blood system life-long. Their number must be strictly controlled to avoid either exhaustion or excessive expansion leading to leukemia. Not only proteins but also regulatory RNAs are necessary to maintain HSCs. Hematopoiesis requires the right composition of microRNAs (miRNA), small, non-coding RNAs with the ability to simultaneously target multiple mRNAs for the orchestration of complex gene expression networks.
We identified miR193b to be induced in long-term repopulating (LT)-HSCs by the self-renewal promoting cytokine thrombopoietin via STAT5 signaling. To understand the physiological function of miR193b expression in HSCs, we generated a miR193b knock-out mouse model. These mice showed a selective expansion of the pool of functional LT-HSCs over time that were fully capable of multilineage blood reconstitution. MiR193b-deficient LT-HSCs were less quiescent than wildtype LT-HSCs in homeostasis, and were therefore more susceptible for repeated 5-Fluorouracil-induced stress. Further, they showed a higher expansion in vitro, not by changing the cell cycle length or death rate, but by slowing differentiation. Conversely, ectopic miR193b expression in LT-HSCs restricted their expansion and they were unable to repopulate the blood of lethally irradiated recipients. Long-term videomicroscopy-based tracking of LT-HSCs and their progeny revealed an increased cell death and slowed cell cycle upon miR193b expression. MiR193b-deficient hematopoietic stem and progenitor cells (HSPCs) showed a higher basal level of cytokine receptor signaling (pSTAT5 and pAKT), and an extended signal after cytokine stimulation, determined by phosphoflow cytometry at single cell resolution and in vitro PamGene kinase activity assays. Differential next generation RNA-sequencing of HPSCs with and without miR193b confirmed the regulation of signaling-related gene clusters and revealed potential mRNA targets. We identified the receptor tyrosine kinase ckit as a direct target of the miR193b, suggesting that miR193 modulates cytokine receptor expression and thereby signaling in HSCs.
Here we show a regulatory circuit that prevents excessive HSC self-renewal by upregulation of miR193b upon self-renewal promoting thrombopoietin – Mpl - STAT5 signaling, that in turn restricts cytokine signaling by targeting cytokine receptor expression. Thereby, we shed light into the essential function of miR193b as a modulator of HSC self-renewal.

Disclosures
No relevant conflicts of interest to declare.
Toll like Receptor 2 Signaling Regulates Hematopoietic Stem Cell Function and Promotes G-CSF Mediated HSC Mobilization
Authors: Herman Angela , Romine Molly , Monlish Darlene , Schuettpelz Laura G. 
Published: October 2019

Abstract
Toll like receptors (TLRs) are a family of pattern recognition receptors that play a central role in pathogen recognition and shaping the innate immune response. While most of the studies of the role of TLRs have focused on mature immune cell populations, recent reports suggest that TLR signaling may regulate the immune response from the level of the hematopoietic stem cell (HSC). In this study, we sought to further elucidate the effects of systemic TLR ligand exposure on HSCs and determine the cell-intrinsic versus extrinsic effects of such exposure. We specifically focused on TLR2 signaling, as although TLR2 is expressed on HSCs, it’s role in their regulation is not clear. Furthermore, enhanced TLR2 signaling is associated with myelodysplastic syndrome (Wei et al, Leukemia 2013), suggesting that aberrant signaling through this receptor may have clinically significant effects on HSC function.
To elucidate the role of TLR2 signaling in regulating HSCs, we used mice with genetic loss of TLR2, as well as a synthetic agonist of TLR2 (PAM3CSK4) to determine the effects of TLR2 signaling loss or gain, respectively, on HSC cycling, mobilization and function. While TLR2 expression is not required for normal HSC function, treatment of wild-type mice with PAM3CSK4 leads to expansion of HSCs in the bone marrow and spleen, increased HSC cycling, and loss of HSC function in competitive bone marrow transplantation experiments. As TLR2 is expressed on a variety of stromal and hematopoietic cell types, we used bone marrow chimeras (Tlr2-/- + Tlr2+/+ marrow transplanted into Tlr2+/+ recipients) to determine if the effects of PAM3CSK4 treatment are cell intrinsic or extrinsic. The data suggests that HSC cycling and expansion in the marrow and spleen upon PAM3CSK4 treatment are extrinsic (occurring in both transplanted HSC populations), and are associated with increased serum levels of G-CSF. Indeed, inhibition of G-CSF using either a neutralizing antibody or mice lacking the G-CSF receptor (Csf3r-/-) leads to even further enhanced HSC bone marrow expansion upon G-CSF treatment but significantly reduced numbers of spleen HSCs compared to similarly treated wild-type mice. This suggests mobilization in response to TLR2 signaling is an indirect, G-CSF-mediated process. Ongoing studies are aimed at determining the contribution of G-CSF to the PAM3CSK4- induced loss of HSC function, and determining the source (stromal vs hematopoietic) of G-CSF production upon PAM3CSK4 exposure. Collectively, this data suggest that TLR2 signaling affects HSCs in a largely extrinsic fashion, with G-CSF playing a major role in regulating the effects of TLR2 ligand exposure on HSCs.

Disclosures
No relevant conflicts of interest to declare.
Molecular Regulation of Adult Hematopoiesis By GATA-2
Authors: Li Haiyan , Jin Jin , Sun shao-Cong , Watowich Stephanie S. 
Published: October 2019

Abstract
GATA-2 is a zinc finger-containing transcriptional regulator that plays important roles in embryonic and adult hematopoiesis. Mutations in human GATA2 are associated with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), as well as immunodeficiency disorders that present with a profound loss of monocytes, dendritic cells and other myeloid lineage populations. Recent work reveals crucial roles for GATA-2 in definitive hematopoietic stem/progenitor cell activity, vascular integrity and lymphatic development. However, the molecular mechanisms by which GATA-2 controls adult hematopoiesis via hematopoietic-cell autonomous functions are largely unknown. To address this question, we generated a tamoxifen-inducible Gata2-deficient mouse strain by breeding Gata2flox/flox mice with Cre-ER transgenic animals. Following tamoxifen treatment, Cre-ER Gata2flox/flox mice show a rapid and profound loss of circulating neutrophils, monocytes and lymphocytes, concomitant with development of anemia. These results are consistent with the requirement for GATA-2 in hematopoietic stem/progenitor cells, and may also reflect GATA-2 function in endothelial cells within the vascular niche. To explore hematopoietic-specific GATA-2 activity, we generated bone marrow chimeric mice with hematopoietic-restricted Gata2-deficiency by transplanting Cre-ER Gata2flox/flox hematopoietic cells into wild type recipients. Cre-ER Gata2flox/flox bone marrow chimeras show rapid development of cytopenias upon tamoxifen exposure, suggesting a cell autonomous role for GATA-2 in maintaining adult hematopoiesis. Strikingly, hematopoietic progenitor cells rapidly lose c-Kit expression upon inducible Gata2 deletion. Chromatin immunoprecipitation and reporter assays suggest GATA-2 cooperates with C/EBPa in regulating kit transcription. Our study suggests conditional deletion of Gata2 restricted to the hematopoietic compartment provides a model for bone marrow failure associated with MDS and mutant GATA2 human immunodeficiencies that may enable further insight into the molecular network by which GATA-2 mediates definitive hematopoiesis.
Supported by grants from NIH (AI098099) and the MD Anderson Center for Cancer Epigenetics.(SSW) and the MD Anderson Center for Cancer and Inflammation (HSL).

Disclosures
No relevant conflicts of interest to declare.
HoxA3 Controls Notch Pathway to Repress Blood Development
Authors: Sanghez Valentina , Kee Dustin , Beuder Steven , Rux Danielle , Osawa Mitsujiro , Markson Joe , Elowitz Michael , Kyba Michael , Iacovino Michelina 
Published: October 2019

Abstract
Hematopoietic stem cells (HSC) are generated from a specialized subset of endothelial cells, hemogenic endothelium. Previous studies performed by our group showed that HoxA3 restrains the cell at the hemogenic endothelium stage, inhibiting further differentiation toward blood by direct repression of Runx1. Building on our previous work, we show here that overexpression of HoxA3 affects the Notch pathway. Upon HoxA3 upregulation in endothelial cells, Jag1 is induced, Mfng (Manic) and Lfng (Lunatic) fringes are downregulated, and there is a trend towards Notch target gene repression. These data suggest that in the presence of HoxA3, endothelial cells are blocked from receiving Notch signal through ligand cis-inhibition with resulting blood inhibition. In order to test this hypothesis, we evaluated the effect of activation or inhibition of the Notch pathway during blood development. We show here that the number of blood progenitors originating from the hemogenic endothelium is decreased when the Notch pathway is inhibited. Conversely, induction of the pathway by upregulation of NICD (Notch1 Intra Cellular Domain) promotes an increase in the number of blood progenitors originating from hemogenic endothelium. Furthermore, inhibition of the pathway when HoxA3 is upregulated has little or no effect in blood while induction of the pathway in HoxA3 presence in part promotes blood development. Taken together, these results demonstrate that Notch pathway is both sufficient and essential for blood development. Specifically HoxA3 inhibits Notch signal reception in two ways: 1) HoxA3 increases Jag1 ligand expression that acts in cis-inhibition; 2) represses Manic and Lunatic fringes both necessary to increase the affinity of Notch receptors for the Delta ligands. When this blockage is bypassed by NICD upregulation, blood is formed, demonstrating that HoxA3-dependent Notch inhibition results in blood suppression.

Disclosures
No relevant conflicts of interest to declare.
The Endocannabinoid/Endovanilloid N-Arachidonoyl Dopamine Suppresses the Plasma Membrane Translocation and Neoplastic Transforming Activity of NRAS and KRAS4A
Authors: Wu Min , Jiao Bo , Ren Ruibao 
Published: October 2019

Abstract
The RAS family includes three RAS genes, which encode four highly homologous proteins: H-, N-, and KRAS4A and 4B, the latter two being alternatively spliced forms differing only at the carboxyl terminus. RAS oncogenic mutations, mostly KRAS and NRAS, are common in human cancers. Since the enzymatic activity of RAS is used to turn itself off and its GTP binding affinity is very high, RAS proteins have been difficult to target. Identification of alternative means to block the RAS oncogenic signaling is critical for developing therapies against RAS-driven cancer. We took a chemical biology approach to discover novel therapeutic strategies for RAS-related cancer. Since the biological activity of RAS proteins relies upon lipid modifications and RAS regulates lipid metabolisms in cancer cells, we used a RAS specific cell viability assay to screen a bioactive lipid library for potential therapeutics against RAS-driven cancer. We found that endocannabinoid/endovanilloid N-arachidonoyl dopamine (NADA) could effectively kill RAS-transformed cells. The structure-activity analysis showed that the anti-RAS activity of NADA relied on both the dopamine and arachidonoyl moieties in the same molecule and was independent of its ability to engage cannabinoid or vanilloid receptors. Further analysis showed that NADA was more active in killing cells transformed by oncogenic NRAS or KRAS4A than that of KRAS4B. Consistently, NADA was more active in inhibiting the phosphorylation of ERK in NRAS-transformed cells than that in KRAS4B-transformed cells. Interestingly, NADA blocked the plasma membrane translocation of NRAS and KRAS4A, but not that of KRAS4B. Significant effect of NADA on palmitoylated NRAS levels was not detected, suggesting that NADA inhibits NRAS and KRAS4A plasma membrane translocation through a novel mechanism. These results demonstrate that NADA possesses a novel property of inhibiting the plasma membrane translocation and neoplastic transforming activity of NRAS and KRAS4A. Since NRAS mutations are common in hematological malignancies as well as in solid tumors such as melanoma and colorectal carcinoma, and KRAS4A is required for carcinogen-induced lung cancer, the new findings would help to develop novel therapies for a broad range of human cancers.

Disclosures
No relevant conflicts of interest to declare.
Platelet-Derived Growth Factor Has a Potent Effect on MK Proliferation, F-Actin Reorganization and Proplatelet Formation Via PDGFR and p-Akt or p-ERK1/2 Pathways
Authors: Yang Mo , Cheng Yuan-shan , Li Su-yi , Meng Fan-yi , Lian Qi-zhou 
Published: October 2019

Abstract
Platelet-derived growth factor (PDGF) may intimate the existence of an autocrine and/or paracrine loop constructed by megakaryocytes (MKs) and their granular constituents in the regulation of megakaryopoiesis and thrombopoiesis. Our study demonstrated that PDGF has a promoting effect on thrombopoiesis in an irradiated-mice model (Ye et al, Haematologica, 2010). PDGF may enhance megakaryopoiesis and platelet production via PDGFR and Erk/Akt pathways. PDGF receptors were identified on platelets and megakaryocytic cells (MKs) by ELISA, flow cytometry and immunocytochemical staining. MK proliferation and platelet formation were studied by CFU-MK and pro-platelet assay. Both PDGFR subtype were identified on platelets and MKs; Levels of PDGF beta-receptors exceeded that of alpha-receptors. PDGF-BB significantly stimulated MKs proliferation; this mitogenic effect was partially neutralized by antibodies directed against the PDGF beta-receptors. Western blot also determind that these receptors mediate PDGF-BB-inducible expression of c-fos. In the MKs apoptotic model, PDGF had a similar anti-apoptotic effect as TPO on MKs. We demonstrated that PDGF activated the Akt signaling pathway, while addition of imatinib mesylate reduced p-Akt expression, suggesting that the PDGF-initiated protective effect is likely to be mediated via PDGF receptors with subsequent activation of the Akt pathway. PDGF also promoted proplatelet formation in MKs and this effect was reduced by PDGFR antibodies. In PDGF-BB (50ng/ml) and TPO (50 ng/ml) groups had more proplatelet bearing MKs; in 200 MKs, proplatelet bearing MKs in PDGF-BB treatment group was (15 % ± 3.2), and in TPO group was (18 % ± 2.5), which were significantly more than the control group (7 % ± 2.6) (n=5). Whether PDGF has effects on cytoskeleton reorganization of MKs, and whether the effect can be reduced by Erk1/2 inhibitor PD98059, MKs were staining with F-actin specific binder rhodamine-phalloidin. We observed that polymerized actin level is lower in control group refer to PDGF-BB group and distributed throughout the cytoplasm with occasionally few aggregation. In contrast, polymerization actin level was higher in PDGF-BB group. Adding PD98059, the fluorescence intensity was reduced (n=5). Our data demonstrated that treated with PDGF-BB for 30 minutes Erk1/2 was activated in MKs. This study suggests that PDGF has a potent effect on megakarpoiesis and platelet formation. This effect is likely mediated via PDGF beta-receptors with subsequent activation of p-ERK1/2 or p-Akt, which leads to MKs proliferation, F-actin reorganization and proplatelet formation.

Disclosures
No relevant conflicts of interest to declare.
Identification of Secreted Factors with a Role in Hematopoietic Stem and Progenitor Cell Engraftment in the Developing Zebrafish
Authors: Blaser Bradley Wayne , Li Brian , Tamplin Owen J. , Binder Vera , Prober David , Schier Alexander , Vidal Marc , Zon Leonard I. 
Published: October 2019

Abstract
The hematopoietic microenvironment regulates the behavior of hematopoietic stem and progenitor cells (HSPC) throughout vertebrate development. We sought to identify secreted factors that may play a role in HSPC engraftment in the caudal hematopoietic territory (CHT), an endothelial cell-rich vascular plexus that serves as the primary site of hematopoiesis in the developing zebrafish from 3-6 days post fertilization (dpf). We hypothesized that such factors would be highly expressed in endothelial cells relative to hematopoietic stem cells (HSCs). To identify these factors, endothelial cells and HSCs were purified from 3 dpf Flk1:mcherry; Runx1:GFP double transgenic zebrafish embryos and gene expression profiling was performed by microarray analysis. Gene set enrichment analysis of these data showed that zebrafish chemokines, cytokines, TGF-β, TNF, Notch and non-canonical WNT family members were enriched in the endothelial cell fraction with a nominal P ≤ 0.2. Genes from the leading edge of these gene sets were then used as candidates for gain-of-function testing. Coding sequences from candidate genes were cloned downstream of the zebrafish HSP70l promoter and microinjected into wildtype zebrafish embryos at the single-cell stage. Gene expression was induced in F0 transgenic animals by heat shock at 36 and 48 hours post fertilization. HSPC numbers were assayed by performing whole-mount in situ hybridization to identify runx1- and cmyb-expressing cells at 3 dpf. WNT5A was found to enhance HSPC numbers in this assay (P = 0.00046). We conclude non-canonical WNT family members, in particular WNT5A, regulate HSPC engraftment in the developing zebrafish.

Disclosures
Tamplin: Boston Children's Hospital: Patents & Royalties. Zon:FATE Therapeutics, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Stemgent: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
Downregulation of Dicer1 Promotes Cellular Senescence and Decreases the Differentiation and Stem Cell-Supporting Capacities of MSCs in Patients with Myelodysplastic Syndrome
Authors: Zhao Youshan , Wu Dong , Fei Chengming , Guo Juan , Gu Shucheng , Xu Feng , Zhang Zheng , Chang Chunkang , Li Xiao 
Published: October 2019

Abstract
Myelodysplastic syndrome (MDS) is characterized by decreased blood counts caused by ineffective hematopoiesis. Although the insufficient hematopoietic support capabilities of mesenchymal stromal cells (MSCs) have been reported in patients with MDS, the underlying mechanisms remain elusive. In this study, we found that MSCs from patients with MDS displayed a significant increase in senescence, as evidenced by their decreased proliferative capacity, flattened morphology and increased expression of SA-β-gal and p21.Senescent MDS-MSCs exhibited decreased osteoblastic and adipogenic differentiation potential and decreased stem cell support capacity. Next, we found that Dicer1 expression was downregulated in MSCs from patients with lower-risk (LR)-MDS and higher-risk (HR)-MDS. Gene knockdown of Dicer1 induced senescence in the MSCs. The differentiation of MSCs into osteoblasts and adipocytes was significantly inhibited by Dicer1 knockdown (Dicer1-KD). Moreover, Dicer1-KD MSCs exhibited less vascular endothelial growth factor (VEGF) and granulocyte colony-stimulating factor (G-CSF) expression than normal MSCs. Long-term culture assays showed that layers of MSCs with the Dicer1 gene knocked down are inefficient at supporting hematopoiesis. Further, we observed that the key senescence mediators, p21,but not p16, was elevated in Dicer1-KD MSCs. We also identified reduced expression in the miR-17 family (miR-17-5p, miR-20a/b, miR-106a/b and miR-93) as a potential factor responsible for increased p21 expression in Dicer1-KD MSCs. A verification experiment showed that miR-93 and miR-20a expression levels were significantly reduced in MSCs from patients with MDS. Taken together, our study shows that MSCs from MDS patients are prone to senescence and that Dicer1downregulation promotes cellular senescence and decreases the differentiation and stem cell-supporting capacities of MSCs in MDS patients. Dicer1 downregulation seems to contribute to the insufficient hematopoietic support capacities of MSCs in patients with MDS.

Disclosures
No relevant conflicts of interest to declare.
Pharmacokinetics and Tolerability of Etoposide in Newly Diagnosed Lymphoma Patients with Hepatic Impairment
Authors: Lai Catherine , Cole Diane , Lucas Nicole , Steinberg Seth M. , Widemann Brigitte C , Dunleavy Kieron , Wilson Wyndham H 
Published: October 2019

Abstract
Background: The addition of etoposide to chemotherapy regimens (e.g.- CHOEP and EPOCH) may be beneficial for the treatment of aggressive lymphomas. Previous studies have shown decreased clearance of etoposide in hepatic impairment leading to a dose reduction or removal of drug. Total clearance of etoposide does not change significantly in patients with elevated bilirubin. However, free (or unbound) etoposide levels are a more accurate measurement of drug clearance (Stewart el al. Changes in the Clearance of Total and Unbound Etoposide in Patients with Liver Dysfunction. J Clin Oncol. 1990 Nov;8(11):1874-9.). No studies have analyzed etoposide pharmacokinetics and clinical toxicity in lymphoma patients with hepatic impairment.
Methods: Patients with newly diagnosed aggressive lymphoma received 1 to 8 cycles of DA-EPOCH +/-R at the NCI. Of 56 pts studied, 48 had normal and 8 had elevated bilirubin (median 3.4mg/dL, range 1.4-22.5mg/dL). All patients received full dose etoposide at 200mg/m2 over 96-hours on cycle 1. Blood samples were collected at 0, 22 and 96 hours after infusion began.
Results: There was no significant difference between free etoposide clearance (Cl) and the free etoposide concentration (Css) in patients with elevated versus normal bilirubin (see table below). In all patients, irrespective of bilirubin, there was no significant correlation between free etoposide Cl, free etoposide Css and creatinine clearance (CrCl) or age. Neutropenia and thrombocytopenia was higher in patients with elevated bilirubin. Complete response (CR) was achieved in 75% (6/8) versus 85% (41/48) and PR in 0% (0/8) vs 4% (2/48) of patients with elevated vs normal bilirubin.
Conclusions: Etoposide pharmacokinetics were not altered in patients with abnormal versus normal hepatic function. Although there was an increase in hematologic toxicities, there was no difference in Css or Cl of etoposide. Importantly, there were no significant differences in febrile neutropenia or grade 3/4 toxicities. The toxicity difference is likely attributed to other drugs. Response rates were similar between the 2 groups. Our results do not support the empiric dose reduction of etoposide in patients with aggressive lymphomas receiving potentially curative treatment.
Table Results Elevated Bilirubin Normal Bilirubin p-value Median age (yrs) 39 (17-59) 54 (20-75) 0.15 IPI (int hi/hi risk) 75% (6/8) 23% (11/48) 0.0070 ANC nadir < 500/mm3 (C1) 100% (8/8) 54% (26/48) 0.017 PLT nadir < 25x103/mm3 (C1) 38% (3/8) 4% (2/48) 0.017 Febrile Neutropenia (C1) 13% (1/8) 13% (6/48) 1.00 G3/4 GI and neuro tox (C1) 38% (3/8) 13% (6/48) 0.11 Median free etoposide clearance on C1 (ml/min/m2) 724 (438-1413) 663 (199-1148) 0.40 Median free etoposide concentration on C1 (μM) 0.082 (0.044-0.135) 0.089 (0.052-0.298) 0.44

Disclosures
No relevant conflicts of interest to declare.