RAB14 Regulates Human Erythropoiesis and Megakaryopoiesis

We recently reported that RAB GTPase 14 (RAB14) knockdown (KD) increased the frequency and total numbers of erythroid cells generated in vitro in response to erythropoietin (EPO) from either the TF1 human leukemia erythropoietic model cell line or from primary human CD34+ hematopoietic stem-progenitor cells (HSPCs). RAB14 overexpression (OE) had the opposite effect. Thus, RAB14 functions as an endogenous inhibitor of human erythropoiesis (Kim et al., Br. J. Haematol., 2015). In contrast to the greater cell numbers generated in the presence of EPO, RAB14 KD TF1 cells grown in standard culture media containing granulocyte-macrophage colony-stimulating factor (GM-CSF; as the only cytokine) generated fewer total cells, compared to empty vector-transduced control TF1 cells. Furthermore, RAB14 KD TF1 cells cultured in GM-CSF media generated greater numbers of erythroid (CD34-/CD71+/CD235a+) cells, as compared to control TF1 cells, suggesting that RAB14 KD stimulated erythropoiesis even in the absence of EPO. Cells generated from RAB14 KD TF1 cells had higher GATA1 and lower GATA2 transcription factor expression, as compared to controls, demonstrating the cells had undergone the "GATA1/2 switch," a hallmark of erythropoiesis. Consistent with higher GATA1 levels, RAB14 KD TF1 cells generated cells with higher levels of b- and g-hemoglobins. Similarly, RAB14 KD in primary human CD34+ HSPCs generated greater numbers of erythroid cells, with or without exogenous EPO. RAB14 KD CD34+ HSPCs cultured in GM-CSF media generated fewer monocytic/granulocytic (CD13+/CD33+) cells, as compared to control CD34+ HSPCs. Interestingly, RAB14 OE CD34+ HSPCs cultured in thrombopoietin (TPO)-containing media generated higher numbers of megakaryocytic (CD34-/CD41a+/CD42b+) cells, as compared to control CD34+ HSPCs. In summary, (1) RAB14 KD in TF1 cells or primary human CD34+ HSPCs increased erythropoiesis in the presence or absence of EPO, but reduced myeloid cell differentiation, probably via the GATA1/2 switch; and (2) RAB14 OE in CD34+ HSPCs increased megakaryopoiesis in the presence of TPO. Thus, RAB14 normally serves as an endogenous hematopoietic decision-maker, physiologically inhibiting erythropoiesis and stimulating megakaryopoiesis (and possibly, to a lesser extent, mono-granulopoiesis). Disclosures No relevant conflicts of interest to declare.

Download Full-text

Two Distinct Mechanisms Contribute to Granulomonocytic Hyperplasia in Chronic Myelomonocytic Leukemias (CMML)

Blood ◽

10.1182/blood.v120.21.309.309 ◽

2012 ◽

Vol 120 (21) ◽

pp. 309-309

Author(s):

Raphael Itzykson ◽

Olivier Kosmider ◽

Aline Renneville ◽

Margot Morabito ◽

Dorothee Buet ◽

...

Keyword(s):

Conflicts Of Interest ◽

Cytokine Signaling ◽

Single Mutation ◽

Monocyte Count ◽

Kras Mutations ◽

Hematopoietic Stem ◽

Gm Csf ◽

Cd34 Cells ◽

Recurrent Mutations

Abstract Abstract 309 Background: The granulomonocytic (GM) hyperplasia of CMML has been attributed to GM-CSF hypersensitivity triggered by mutations in the CBL/RAS pathway according to the prevailing model in juvenile myelomonocytic leukemias (Kotecha Cancer Cell 2008). Recurrent mutations affecting epigenetic (eg TET2 and ASXL1) and splicing (eg SRSF2) machineries, or cytokine signaling (N/KRAS, CBL, JAK2) are present in most CMML cases, but none is specific of CMML. In 224 CMML patients (pts), we found TET2 (58%), SRSF2 (47%) and ASXL1 (38%) to be the most frequently mutated genes; only 66 (35%) cases had mutations in cytokine signaling genes (CBL, N/KRAS, JAK2, FLT3, KIT) (abstract submitted). We analyzed the differentiation of CD34+populations from genetically annotated CMML pts to address the mechanisms of GM hyperplasia in CMML. Methods: CD34+ populations (hematopoietic stem cells [HSC]; multipotent [MPP]; common myeloid [CMP] and granulomonocytic progenitors [GMP] defined by the CD34/CD38/CD90/CD123/CD45RA panel; Majeti Cell Stem Cell 2007) from 28 genetically annotated CMML and TET2 mutated MPN (n=8) or MDS (n=5) cases were cloned and genotyped for each mutation identified in mature CD14+ cells, and differentiated in vitro. Results: Early clonal dominance, with at least one mutation in > 75% of HSC/MPP clones, was found in all cases. In 18/19 pts with ≥2 mutations, a linear succession of mutations was found, with signaling mutations often following TET2 or ASXL1 mutations. Contrasting with the dominance of first events in HSC/MPP, second events reached clonal dominance in GMP, suggesting that they provide a selective advantage during the early steps of myeloid differentiation. We next analyzed the clonogenicity of peripheral blood (PB) CD34+ cells in the presence of GM-CSF (10 ng/mL) in 20 CMML cases and 4 controls. GM-CSF hypersensitivity (clonogenicity > mean+2SD of controls) was found in 7 (35%) cases. A mutation in a signaling gene was found in 6/7 pts (1 homozygous JAK2, 1 homozygous CBL, 4 heterozygous N/KRAS mutations), compared to 3/13 in pts without GM-CSF hypersensitivity (2 JAK2, 1 CBL, all heterozygous; P=.02) Median WBC was 29.2 and 11.4 G/L in pts with and without GM-CSF hypersensitivity, respectively (P=.08). The proportion of GMP in bone marrow (BM) CD34+cells was not significantly different in 33 CMML pts compared to 15 age-matched controls. Clonogenicity of GMP was similar in CMML and controls, except for a trend toward increased clonogenicity in pts with mutations in signaling genes. In contrast, the proportion of MPP and CMP was higher in CMML than in controls (P<.01 and P <.05, resp.). In erythromyeloid conditions (SCF, IL-3, G-CSF & EPO), both CMP and to a lesser extent MPP had an increased ability to form GM colonies at the expense of erythroid colonies (P <.001 and P<.01, resp.). Compared to healthy CMP, CMML CMP had and increased ability to mature into GMP in short-term culture, and increased PU.1 mRNA expression (P<.05), without significant changes in the levels of GATA1, CEBPA and CEBPB. Finally, in 16 pts, the proportion of GM colonies differentiating from CMP at the expense of erythroid colonies was inversely correlated to patient hemoglobin level (P=.002). Thus, premature GM differentiation of CMP, and to a lesser extent MPP, appears as the dominant mechanism of GM hyperplasia in CMML, whereas GM-CSF hypersensitivity and GMP expansion contribute only in the minority of patients with mutations in signaling genes. We next explored a possible link between early clonal dominance of TET2 mutations and premature GM differentiation. In TET2 mutated MPN (n=8) or MDS (n=5), the PB monocyte count was significantly correlated to the size of the TET2-mutated clone in the CD34+/CD38− (P=.006) rather than in the CD34+/CD38+ population (P=.08). Finally, functional invalidation by shRNA of TET2 in CD34+/CD38− followed by culture in the presence of SCF, IL-3, G-CSF & EPO caused a GM expansion that was not observed in CD34+/CD38+ cells. Similar analyses are underway for ASXL1. Conclusion: Our results suggest that early clonal dominance of mutations affecting the epigenetic machinery leading to premature GM differentiation of multipotent progenitors, rather than GM-CSF hypersensitivity, is the main mechanism of GM hyperplasia in CMML. This suggests a model whereby a single mutation can lead to different phenotypes, depending on the stage of differentiation at which the mutation has gained clonal dominance. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Angptl-4 Is Upregulated Under Inflammatory Conditions In The BM Of Mice, Expands Myeloid Progenitors and Accelerates Reconstitution Of Platelets After Myelosuppressive Therapy

Blood ◽

10.1182/blood.v122.21.3677.3677 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3677-3677

Author(s):

Anne Schumacher ◽

Till Braunschweig ◽

Bernd Denecke ◽

Tim H. Brümmendorf ◽

Patrick Ziegler

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Myeloid Cell ◽

Relative Increase ◽

T Test ◽

Gm Csf ◽

Emergency Situations ◽

Inflammatory Conditions

Abstract The concerted action of hematopoiesis supporting cytokines such as G-CSF, GM-CSF or IL-6 regulates hematopoiesis during steady state and emergency situations. Respective knockout mice show defects both in production and function of myelopoietic effector cells. However, alternative pathways are likely to exist as mice with single or combined deficiencies for G-CSF, GM-CSF, and IL-6 or G-CSF and GM-CSF are still able to mount reactive neutrophilia responses during inflammatory conditions. In order to identify pathways for inflammation induced enhancement of hematopoiesis as well as to find new cytokines, which enhance myeloid cell regeneration, we analyzed the bone marrow (BM) of lipopolysaccharide (LPS) and vehicle injected wild type (WT) mice (single IP- injection) by gene expression microarray. Focusing on the identification of genes encoding for secreted or membrane proteins, we found 83 candidates to be up- and 14 to be downregulated after LPS treatment. Among known candiates, we found angiopoietin-like 4 (Angptl-4) as a predominantly upregulated gene in the BM of LPS-treated WT-mice. Upregulation was confirmed by RT-PCR as well as by Elisa in the BM of LPS treated mice and bone marrow stromal cells (BMSC) were identified as candidate producer cells. Functionally, we found recombinant Angptl-4 to stimulate the proliferation of myeloid colony-forming units (CFU) in vitro. In mice, repeated injections of Angptl-4 increased BM progenitor cell frequency and this was paralleled by a relative increase in phenotypically defined granulocyte-macrophage progenitors (GMPs). Furthermore, in vivo treatment with Angptl-4 resulted in elevated platelet counts both in untreated animals and after myelosuppressive therapy. After lethal irradiation and transplantation of syngeneic BM cells repetitive injections of recombinant Angptl-4 for 5 consecutive days resulted in an accelerated reconstitution of platelets starting at day 8 after transplantation. The 50% pre-treatment platelet count was reached on day 14 in Angptl-4-treated animals as compared to day 21 for transplanted controls receiving no Angptl-4 (n=8; p=0.03, student´s T test). In contrast, transplantation of BM cells from Angptl-4 pre-treated donor mice had no effect on the recovery of platelets in this setting. The frequency of CD41lowCD61+ immature megakaryocytes was significantly increased in the BM of Angptl-4 injected as compared to control mice (27% vs 19% of total megakaryocytes; p= 0.008, student´s T test). Furthermore, bone marrow cytology revealed local accumulation of megakaryocytes carrying dysplastic features in Angptl-4 injected mice. In summary, our data suggest that Angptl-4 plays a complementary role on hematopoiesis during emergency situations like sepsis. The use of Angptl-4 in the setting of autologous stem cell transplantation could represent a potential approach to accelerate the reconstitution of megakaryopoiesis. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The Growth Response of Erythrocyte Production to Increased Dense Condition

Blood ◽

10.1182/blood.v118.21.4812.4812 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4812-4812

Author(s):

Hye sook Choi ◽

Eun-mi Lee ◽

Brijesh Siddaveerappa Ajjappala ◽

Ji Young Huh ◽

Hwi-Joong Yoon ◽

...

Keyword(s):

Conflicts Of Interest ◽

Cell Communication ◽

Surface Characteristics ◽

Cell Interaction ◽

Maturation Stage ◽

Erythroid Cells ◽

Human Cd34 ◽

Cd34 Cells ◽

Final Maturation

Abstract Abstract 4812 Erythroid precursors proliferate, differentiate, and enucleate in the bone marrow niches called erythroblastic islands in contact with macrophages. However, the cell-to-cell communication between erythroblasts which are undergoing terminal maturation leading to enucleation is not well known. Moreover, the cell-to-cell interaction at a terminal erythroblast maturation stage was hardly detected in conventional suspension culture systems due to cell surface characteristics of electrostatic repulsion. To elucidate the communication between late maturing erythroblasts, erythroid cells, which were derived from human CD34+ cells, were cultured at conventional or supra-optimal densities. Surprisingly, the final yields of red blood cells (RBCs) were significantly increased in supra-optimal culture density, even in the absence of feeder stromal cells and serum or plasma. Also observed were increases in the rates of cell survival, expansion, and enucleation. Contact between cells observed in these supra-optimal cultures was associated with increases in adhesion-related signaling proteins (150 kDa and 123 kDa), the latter of which was previously unknown to be involved in erythropoiesis. The cultured erythroid cells showed erythropoiesis specific markers such as CD71, GPA, GATA-1 and b-globin. Therefore, we firstly elucidated that the erythroid cells which were in the final maturation step can communicate each other and affect their survival and maturation including enucleation without the help of macrophages. Also, the increased attachments between erythroid cells evoked adhesion-related signals. We anticipate that the culture of these cells at supra-optimal density may enhance in vitro RBC production for human transfusion. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

In vitro expansion of fetal liver hematopoietic stem cells

Scientific Reports ◽

10.1038/s41598-021-91272-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Rashmi Bhardwaj ◽

Lalit Kumar ◽

Deepika Chhabra ◽

N. K. Mehra ◽

Atul sharma ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Mononuclear Cells ◽

Culture Media ◽

Fetal Liver ◽

Antigen Expression ◽

Proliferative Potential ◽

Hematopoietic Stem ◽

Gm Csf

AbstractFetal liver hematopoietic stem and progenitor cells (HSPCs) have been considered appropriate for the management of aplastic anemia owing to their proliferative potential. Bone marrow recovery was possible in some cases; the engraftment potential of these cells, however was unsatisfactory, possibly due to the availability of a smaller number of these cells from a single fetus. The present study explores how we can expand fetal liver hematopoietic stem cells under in vitro conditions. We isolated mononuclear cells from fetal liver and hematopoietic stem cells were identified and analyzed by cell surface marker CD34. CD34+ fetal liver HSPCs cells were separated by magnetic cell sorting positive selection method. HSPCs (CD34+) were cultured by using 5 cytokines, stem cell factor (SCF), granulocyte macrophages-colony stimulating factor (GM-CSF), interleukin-6 (IL-6), Fms-related tyrosine kinase 3 (FLT-3) and erythropoietin (EPO), in 4 different combinations along with supplements, in serum-free culture media for 21 days. Cell viability continued to be greater than 90% throughout 21 days of culture. The cells expanded best in a combination of media, supplements and 5 cytokines, namely SCF, FLT-3, IL6, EPO and GM-CSF to yield a large number of total (CD34+ & CD34-) cells. Even though the total number of nucleated cells increased in culture significantly, levels of CD34 antigen expression declined steadily over this period.

Download Full-text

GATA1 Is Overexpressed in Patients with Essential Thrombocythemia and It Is Normalized By Anagrelide Treatment

Blood ◽

10.1182/blood.v124.21.5574.5574 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5574-5574 ◽

Cited By ~ 1

Author(s):

Lilia Brown ◽

Ciaren Graham ◽

Yvette Martyn ◽

Ciro Rinaldi

Keyword(s):

Transcription Factor ◽

Conflicts Of Interest ◽

Ectopic Expression ◽

Therapy Response ◽

Myeloid Cell ◽

Hematopoietic Stem ◽

Thrombotic Risk ◽

Mutational Status ◽

Monitoring Therapy

Abstract GATA1 is the founding member of the GATA transcription factor family and is essential for cell maturation and differentiation within the erythroid and megakaryocytic lineages. GATA1 is a pleotropic transcription factor, whose expression is essential in these lineages; its function depends upon its ability to bind to both DNA and protein partners. Disturbance of either of these functions causes severe hematopoietic dysfunction and can result in blood disorders, such as thrombocytopenia, anaemia and even leukaemia. Ectopic expression of GATA1 in the murine myeloid cell line M1 induces c-Mpl appearance and megakaryocyte (MK) differentiation. The same phenomenon has been showed to occur in hematopoietic stem cells; in that forced increased expression of GATA1 blocked self-renewal and induced the exclusive generation of MegE lineages. Several studies, have suggested a connection between GATA1 and myeloproliferative neoplasia (MPN). We previously reported that high GATA1 transcript levels are found in the bone marrow of patients with ET and PV, independent of JAK2V617F or CALR mutation status. However, over expression of GATA1 is not seen in other MPNs. Anagrelide (ANA) has been proven to be an effective drug in reducing platelet count and thrombotic risk in management of ET and PMF patients. However, the mechanism by which this drug induces this effect is still unclear. Recently Erusalimsky and colleges have reported that ANA results in down-modulation of GATA1 and its co-factor FOG1 in MK during in vitro differentiation. In this study, we analysed the expression of GATA1 in peripheral blood (PB) samples from 38 patients with ET and compared the levels of expression before and after treatment with common cytoreductive agents such as hydroxyurea (HU) and ANA. We confirmed the data obtained in BM, with a significant up-regulation of GATA1 in ET compared to controls. When we measured the expression of GATA1 before and during treatment with ANA, there was a significant reduction of the GATA1 expression at 3 and 6 months of therapy, concomitant with a reduction in platelets (PLT) counts. Interestingly, this was not equally observed in patients treated with HU, where despite reduction in PLT counts, the GATA1 levels rose. Furthermore, when we analysed patients on combination treatment ANA + HU, GATA1 expression reduced only when patients were taking ANA. When ANA treatment was stopped and the patient continued only on HU, GATA1 levels rose again. This data suggests a direct effect of ANA on GATA1 expression. Although, no direct correlation between GATA1 expression and CALR mutations was found. However, a significant up regulation of CALR mRNA was observed independently from the CALR mutational status. GATA1 may represent a generic molecular marker in ET and a possible additional diagnostic criteria in thrombocytosis. GATA1 overexpression is independent from JAK2 mutations, and responds specifically to ANA therapy suggesting a role in monitoring therapy response. Disclosures No relevant conflicts of interest to declare.

Download Full-text

The Epigenetic Regulators CBP and p300 Facilitate Leukemogenesis and Represent Therapeutic Targets In Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v122.21.3732.3732 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3732-3732

Author(s):

George Giotopoulos ◽

Wai-In Chan ◽

David Ruau ◽

Paolo Gallipoli ◽

Alexis Fowler ◽

...

Keyword(s):

Conflicts Of Interest ◽

Therapeutic Targets ◽

Selective Advantage ◽

Human Leukemia ◽

Hematopoietic Stem ◽

Molecule Inhibitor ◽

Stem And Progenitor Cells ◽

Clonogenic Potential

Abstract Although molecularly and clinically heterogeneous, AML is characterised by aberrant transcription and abnormal epigenetic regulation. The transcriptional co-activators CBP and p300 have well characterised roles in hematopoiesis and hematopoietic stem cell (HSC) function. These are mutated in AML and also bind a number of AML-associated oncogenes. However, despite this, their roles in the induction and maintenance of AML are poorly understood. To address this question, we have combined genetic and pharmacological inhibition of CBP and p300 in AML and normal hematopoiesis. Using a murine model where Cbp was conditionally deleted from murine hematopoietic stem and progenitor cells (HSPC), either prior to, or following expression of a number of AML-associated oncogenes, we assessed the role of Cbp in the induction and maintenance of AML. We demonstrated that although not an absolute requirement, Cbp confers a selective advantage for robust immortalisation in vitro, and that Cbp is also an important requirement for the generation and maintenance of AML in vivo. Furthermore, redundancy between Cbp and p300 in myeloid transformation was demonstrated, as p300 knockdown further decreased proliferation in Cbp-/- AML cells. We next validated CBP/p300 as potential therapeutic targets, using a pharmacological strategy. Using a selective small molecule inhibitor (C646) of the lysine acteyltranferase (KAT) activity of CBP/p300, we demonstrated a significant decrease in growth and clonogenic potential across multiple AML subtypes in vitro. This was mediated through induction of apoptosis and cell cycle arrest. Importantly, no alteration in the growth of normal murine and human hematopoietic progenitors was detected at similar doses. We further demonstrated that inhibition of CBP/p300 KAT activity in human leukemia cells alters a transcriptional programme associated with genomic integrity, linking transcriptional changes to the cellular phenotype. Finally, we demonstrated the efficacy of the HAT inhibitors to decrease clonogenic growth across a panel of primary AML patient samples, representing multiple genetic subtypes. Taken together, these data suggest that CBP/p300 are involved in leukemogenesis across multiple subtypes in AML and that targeting these proteins may be possible with an acceptable side-effect profile. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

ER71/ETV2 Is Essential for Hematopoietic Regeneration after Injury

Blood ◽

10.1182/blood.v126.23.2359.2359 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2359-2359

Author(s):

Canxin Xu ◽

Kyunghee Choi ◽

Tae-Jin Lee ◽

Nagisa Sakurai

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Myeloid Cell ◽

Conditional Knockout ◽

Cell Recovery ◽

Drug Induced ◽

Hematopoietic Stem ◽

Anticancer Chemotherapy ◽

Hematopoietic Reconstitution

Abstract Hematopoietic stem and progenitor cell (HSPC) expansion and regeneration is requisite for effective hematopoietic transplantation and recovery from anticancer chemotherapy drug induced bone marrow injury. In chemotherapy-mediated hematopoietic injury model, we found that key developmental genes that are critical for HSPC generation were upregulated in HSPCs when treated with 5-FU. ETS transcription factor Er71/ETV2 is essential for the development of endothelial and blood cells during mammalian embryogenesis. Here, we generated different tissue specific Er71 conditional knockout (CKO) mice as well as transgenic mice that coexpress Er71, GATA2 and Scl (EGS) in a doxycycline inducible manner to investigate the Er71 role in adult hematopoiesis. While Vav-Cre; Er71 CKO and Tie2-Cre; Er71 CKO mice did not display any hematopoietic defects in homeostatic conditions, Vav-Cre; Er71 CKO bone marrow was significantly compromised in hematopoietic reconstitution potential compared to controls. In addition, Vav-Cre; Er71 CKO and Tie2-Cre; Er71 CKO but not VECadherin-Cre; Er71 CKO mice showed reduced HSPC proliferation and deficient HSPC and myeloid cell recovery after 5-FU injury. On the other hand, temporal Er71, GATA2 and Scl (EGS) coexpression promoted HSPC proliferation, hematopoietic reconstitution potential and regeneration after 5-FU injury. These results demonstrate that hematopoietic Er71 is critical for HSPC expansion and regeneration. Future studies warrant Er71 application for in vitro hematopoietic stem cell expansion and bone marrow regeneration in response to hematopoietic injury. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Absence of the Hemochromatosis Gene Hfe Confers Protection Under Conditions of Stress Erythropoiesis.

Blood ◽

10.1182/blood.v114.22.4044.4044 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4044-4044

Author(s):

Pedro Ramos ◽

Ella Guy ◽

Nan Chen ◽

Sara Gardenghi ◽

Robert W. Grady ◽

...

Keyword(s):

Steady State ◽

Iron Overload ◽

Iron Uptake ◽

Conflicts Of Interest ◽

Type I ◽

Erythroid Cells ◽

Hematopoietic Stem ◽

Stress Erythropoiesis ◽

In Erythroid Cells

Abstract Abstract 4044 Poster Board III-979 Hereditary hemochromatosis type-I (HH) is a disease associated mainly with the C282Y-HFE mutation and characterized by iron overload. HFE was shown to participate in the regulation of hepcidin and, therefore, in iron absorption. Additionally, in vitro studies have shown that Hfe controls cellular iron uptake by interfering with the binding of holo-transferrin to transferrin receptor-1 (TfR1), decreasing internalization of the complex. TfR1 is highly expressed in erythroid cells, being essential for iron uptake during early stages of erythroid maturation. Additionally, some studies have reported altered erythropoietic values in HH patients. Therefore, we hypothesize that Hfe might play a role in early steps of erythropoiesis. To test this hypothesis, we have tried to discriminate between the contribution of iron overload and a potential intrinsic role for this protein in erythroid cells. Complete blood counts, flow cytometry profiles and organ iron contents were determined in Hfe-KO and wt mice at 2, 5 and 12 months. Lentiviral vectors were used to overexpress Hfe in the liver of Hfe-KO animals. Compared to wt animals, Hfe-KO mice had increased hemoglobins, MCHs, MCVs and higher proportions of immature erythroid cells in the bone marrow (BM) and spleen (p≤0.05). Older Hfe-KO animals also showed a decrease in RBC counts. When erythropoiesis was challenged by either phlebotomy or phenylhydrazine, we observed that Hfe-KO mice were able to recover faster from anemia (p≤0.05). In order to confirm that the results observed were not exclusively due to iron overload, we attempted to eliminate excess iron by two different strategies: 1) re-establishing expression of Hfe in the liver of Hfe-KO mice; and 2) transplantation of Hfe-KO BM into lethally irradiated wt recipients. To achieve our first goal, a lentiviral vector carrying Hfe driven by a liver specific promoter (THW) was injected into the liver of 3-day-old Hfe-KO pups. This approach was sufficient to significantly increase hepcidin levels and to decrease the liver, spleen and serum iron content in Hfe-KO mice compared to animals harboring a control vector. No differences in hematological parameters relative to controls were seen in Hfe-KO animals expressing Hfe specifically in the liver. Regarding our second goal, we transplanted Hfe-KO or wt hematopoietic stem cells (HSCs) into wt recipients, designated Hfe→wt and wt→wt, respectively. At steady state we observed that Hfe→wt animals had decreased RBC counts, slightly increased MCHs (less dramatic than seen in Hfe-KO mice at steady state) and an increase of immature erythroid cells in the spleen when compared to wt→wt mice. Other parameters were unchanged. Recovery from induced anemia was faster in Hfe→wt than wt→wt mice suggesting that lack of Hfe in the BM is protective under conditions of stress erythropoiesis even in the absence of iron overload. To compare the maturation of erythroid cells while minimizing potential differences in the microenvironment, animals were phlebotomized and erythroid cells at an early stage of differentiation were isolated from both Hfe-KO and wt animals. These cells were cultured in vitro for 48 hours in presence of the erythropoietin. We detected expression of Hfe in the wt cells. We also found that the proliferation of Hfe-KO cells was 25% greater than that of wt cells (p≤0.01). This result was confirmed by mixing the same number of cultured cells from the two genotypes, after labeling them with different dyes. We observed that the percentage of Hfe-KO cells was consistently higher than that of wt cells. From these results, we can conclude that while iron overload undoubtedly contributes to increased erythropoiesis as seen in the Hfe-KO mice, reduced expression of Hfe in erythroid cells might have a beneficial role under conditions of stress erythropoiesis. Expression of Hfe may control iron uptake in erythroid progenitors so as to avoid excessive iron intake and associated toxicity. However, in conditions of acute anemia, lack of Hfe might be protective leading to faster recovery. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Reduced Expression of DOCK4 Leads to Increased Protein Phosphorylation and Migration of Hematopoietic Stem/Progenitor Cells

Blood ◽

10.1182/blood-2018-99-114320 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2563-2563

Author(s):

Sriram Sundaravel ◽

Wen Liang-Kuo ◽

Hui Liu ◽

Jong Jeong ◽

Gaurav Choudhary ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Mammalian Cells ◽

Conflicts Of Interest ◽

Percent Level ◽

Fold Increase ◽

Immunoblot Analysis ◽

Signaling Networks ◽

Hematopoietic Stem ◽

Human Cd34

Abstract Dedicator of cytokinesis 4 (DOCK4) is a large, multi-domain signaling protein involved in modulating signaling networks in mammalian cells. Recent studies have highlighted its role across multiple tissue types in maintaining homeostasis. Deletion and/or mutations in the DOCK4 gene lead to malignant conditions and has been attributed to poor long-term prognosis. Within the hematopoietic compartment reduced expression of DOCK4 leads to dysplasia in the erythroid lineage (Sundaravel et al, PNAS, 2015). However, its functions during early stages of hematopoietic cell development have not been defined. Identification of signaling networks regulated by DOCK4 within the hematopoietic compartment will provide insights into developing strategies in overcoming functional deficits that arise due to reduced expression of DOCK4. In this study, using primary human hematopoietic stem/early progenitors (HSPCs), we identified the major downstream signaling networks regulated by DOCK4 and functional consequences of reduced expression of DOCK4. Global phospho-proteomic analysis was performed on primary human CD34+/CD90+ hematopoietic cells expressing normal levels or reduced levels of DOCK4. A similar set of experiments were also performed on cells after they have committed to the erythroid lineage. These initial studies, using 1D and 2D phospho immunoblot analysis, we showed that cells expressing reduced levels of DOCK4 exhibit increased global tyrosine phosphorylation compared to cells expressing normal levels of DOCK4 regardless of whether cells exposed to hematopoietic cytokines. Further analysis of CD34+/CD90+ cells using liquid chromatography coupled mass spectrometry (LC/MS/MS) enabled us to precisely identify multiple proteins that were differentially phosphorylated. Among the proteins that were hyper-phosphorylated, phosphatases, PTPN6 (SHP1), INPP5D (SHIP1) and LYN kinase exhibited the greatest increase in their levels of tyrosine phosphorylation. These results were further validated by immunoblot analysis using site-specific phospho-antibodies against each of the three proteins. Co-immunoprecipitation experiments using HSPCs showed that DOCK4 interacts directly with INPP5D but not with PTPN6 or LYN suggesting that INPP5D is part of the DOCK4 protein complex but not PTPN6 or LYN. We then abrogated DOCK4-INPP5D interaction by knockdown of DOCK4, which led to increased localization of phospho-INPP5D (Y1021) from the cytoplasmic compartment to the cell membrane in CD34+/CD90+ HSPCs. To determine the functional pathways impacted by reduced expression of DOCK4, we performed in-silico DAVID/GO pathway analysis using our phospho proteomic data and found that cell migration function as one of the highly enriched pathways affected, when DOCK4 levels were reduced in HSPCs. We confirmed these findings by examining the migratory abilities of HSPCs expressing low levels of DOCK4 by an in vitro transwell assay, which showed approximately 2-fold increase in HSPC migration in the cell population where DOCK4 was expressed at fifty percent level. In addition, these cells also exhibited increased cell spreading and filopodia formation. Finally, pharmacologic inhibition of enzyme activities/phosphorylation of INPP5D, LYN or PTPN6 decreased the migration of HSPCs. Based on these results; we conclude that DOCK4 is a critical signaling intermediate, which acts within the hematopoietic stem/early progenitor compartment to regulate primary human HSPCs migration by modulating downstream kinases and phosphatases. Therefore using pharmacological inhibitors of INPP5D, LYN or PTPN6 may provide an avenue to restore proper migratory patterns of HSPCs within the hematopoietic niche and potentially reverse differentiation block as observed in dysplasia. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Monocyte-Derived Macrophages Are Impaired in Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v128.22.5522.5522 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5522-5522

Author(s):

Yu Han ◽

Huaquan Wang ◽

Zonghong Shao

Keyword(s):

Myelodysplastic Syndrome ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Regulatory Protein ◽

Control Group ◽

Hematopoietic Stem ◽

Gm Csf ◽

Increased Risk ◽

Macrophage Colony

Abstract The myelodysplastic syndrome (MDS) comprises a group of clonal hematopoietic stem cell diseases characterized by cytopenia, dysplasia in one or more of the major myeloid lineages, ineffective hematopoiesis, and increased risk of development of acute myeloid leukemia (AML). Macrophages are innate immune cells that ingest and degrade abnormal cells, debris, and foreign material and orchestrate inflammatory processes. Tumor-associated macrophages (TAMs) play an important role in the pathophysiology of human malignancies. They support growth of cancer cells by promoting angiogenesis, inhibiting tumor cell apoptosis and anti-tumor immune reactions. In this study, we analyzed the role of macrophages from MDS patients in vitro. Macrophages were induced from peripheral blood of patients with MDS via granulocyte macrophage colony-stimulating factor (GM-CSF). Compared with that in the normal control group, the number of monocytes increased in MDS patients. However, the monocytes showed impaired ability to induce macrophages and the number of macrophages induced from MDS samples was lower. Further, we demonstrated that the ex vivo phagocytic function of macrophages from MDS patients was impaired and levels of reorganization receptors CD206 and signal regulatory protein alpha (SIRPα) were lower. Levels of inducible nitric oxide synthase(iNOS) secreted by macrophages in MDS were increased. In conclusion, monocyte-derived macrophages are impaired in myelodysplastic syndromes. Disclosures No relevant conflicts of interest to declare.

Download Full-text