scholarly journals RGL2 Deficiency Impairs Human Erythropoiesis By Altering Terminal Erythroid Differentiation and Apoptosis

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 8-8
Author(s):  
Harry Lesmana ◽  
Georgios E Christakopoulos ◽  
Mary Risinger ◽  
Haripriya Sakthivel ◽  
Omar Niss ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Signaling Pathways ◽  
Ex Vivo ◽  
Bone Marrow Failure ◽  
Flow Cytometric Analysis ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Guanine Nucleotide ◽  
Splicing Variant ◽  
Terminal Erythroid Differentiation

Abstract Several members of the Ras-GTPases have been shown to play major roles as molecular switches in various signaling pathways in hematopoietic cells. The roles of Ras/MAPKs/ERK signaling pathways in EPO-induced erythroid differentiation have been explored in the past on animal models. Recently, the Ras/RalGEFs/Ral pathway has been a focus of research in oncology given its roles in tumorigenesis. However, its role in normal human erythropoiesis is poorly understood. The seven RalGEF family members are guanine nucleotide exchange factors with unique as well as overlapping roles. They bridge activated Ras to activation of RalA and RalB while they have also been shown to promote Akt activation. Recently, we identified and characterized a homozygous splicing variant (c.1580+4T>A) in RGL2 (Ral Guanine Nucleotide Dissociation Stimulator-Like 2) gene, one of the RalGEFs, in a 16 month-old female patient with a recessive form of familial syndromic pancytopenia, more severe in the erythroid lineage with transfusion-dependent anemia, hepatosplenomegaly, liver fibrosis, and cardiac dysfunction. We verified by RT-PCR that this splicing variant was associated by a decrease in the RGL2 expression levels by 70-96% in the white blood cells and reticulocytes of the patient. We hypothesized that decreased RGL2 expression leads to defective human erythropoiesis by altering terminal erythroid differentiation, erythroblast cell cycle progression and/or apoptosis. To test our hypothesis, we transduced human bone marrow derived CD34+ cells (donated by healthy volunteers under an IRB-approved research protocol in our institution) with lentivirus encoding shRNA targeting RGL2 mRNA and then cultured them in a previously validated 3-phase culture system that recapitulates erythropoiesis ex vivo. A scrambled shRNA sequence was used as control. The efficiency of the shRNAs used to knock-down (KD) RGL2 expression was initially validated by qRT-PCR after transduction of K562 cells. Our study demonstrated that knockdown of RGL2 expression resulted in decreased erythroid cell growth in ex vivo erythropoiesis accompanied by increased apoptosis. We further showed that knockdown of RGL2 expression also led to delayed terminal erythroid differentiation by flow cytometric analysis using glycophorin A (GPA), band 3, and α4-integrin as markers of maturing erythroblasts. This finding was corroborated by morphology studies using Wright staining in cytospins from RGL2-KD cultures and control. Together, our studies indicate an essential role for RGL2 and the Ras/RGL2/Ral pathway in human erythropoiesis, affecting both terminal erythroid differentiation and apoptosis. Our findings not only provide insights into regulation of normal erythropoiesis but also indicate that RGL2 may be a novel candidate gene associated with inherited bone marrow failure syndromes. Disclosures No relevant conflicts of interest to declare.

scholarly journals Identification of RPS14 as the 5q-Syndrome Gene by RNA Interference Screen.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1-1 ◽  
Author(s):  
Benjamin L. Ebert ◽  
Jennifer Pretz ◽  
Jocelyn Bosco ◽  
Cindy Y. Chang ◽  
Pablo Tamayo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Rna Interference ◽  
Ribosomal Protein ◽  
Translational Control ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Ribosomal Subunit ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
The Common

Abstract We report here that haploinsufficiency of the ribosomal protein encoding RPS14 gene causes the characteristic hematologic phenotype that defines the 5q- syndrome. The 5q- syndrome, a subtype of myelodysplastic syndrome (MDS), was first described in 1974 as a distinct clinical entity associated with deletions of the long arm of chromosome 5. Since that time, the common deleted region has been narrowed in search of the causal gene within the region. However, no biallelic inactivation events have been identified for any of the 41 genes within the critical region, suggesting that a haploinsufficiency mechanism may explain the disease phenotype. We addressed this question through a functional genomic approach, utilizing systematic RNA interference (RNAi) to interrogate the function of each gene in the common deleted region. We introduced 3–5 unique, lentivirally expressed short hairpin RNAs (shRNAs) targeting each of the 41 genes in the region into normal CD34+ human bone marrow hematopoietic progenitor cells and assayed the effects of each shRNA on hematopoietic differentiation. We found that knock-down of a single gene, RPS14, recapitulated the phenotype of the 5q- syndrome: namely, a block in erythroid differentiation (leading to erythroid cell apoptosis) with relative preservation of megakaryocyte differentiation as measured by FACS analysis. Importantly, forced expression of an RPS14 cDNA in primary bone marrow cells from patients with the 5q- syndrome rescued the phenotype, yet had no effect on cells from MDS patients without 5q deletions. In addition, we found that RPS14 haploinsufficiency caused a block in the processing of pre-ribosomal RNA and in the formation of the 40S ribosomal subunit. This ribosomal processing defect is highly analogous to the functional defect seen in Diamond Blackfan Anemia (also characterized by an erythroid differentiation defect and predisposition to AML), thereby establishing an unexpected link between the molecular pathophysiology of acquired 5q- syndrome and congenital bone marrow failure syndromes. These results indicate that the 5q- syndrome is caused by a defect in ribosomal protein function, highlighting the importance of translational control in hematologic malignancy. The results further suggest that RNAi screening is an effective strategy for identifying causal haploinsufficiency disease genes.

scholarly journals RASA3 Deficiency Contributes to Anemia By Multiple Mechanisms

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 920-920
Author(s):  
Elena C. Brindley ◽  
Emily Hartman ◽  
Julien Papoin ◽  
Brian Dulmovits ◽  
Steven L. Ciciotte ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Cell Cycle Progression ◽  
Bone Marrow Failure ◽  
Erythroid Differentiation ◽  
Ras Signaling ◽  
Cycle Progression ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Terminal Erythroid Differentiation

Abstract RASA3, a Ras GTPase activating protein, is critical to vertebrate erythropoiesis and megakaryopoiesis. The autosomal recessive mouse model scat (severe combined anemia and thrombocytopenia) carries a G125V mutation in Rasa3 that leads to profound bone marrow failure with characteristics of aplastic anemia. The phenotype is cyclic, and mice alternate between periods of crisis and remission. Our previous studies demonstrated that this mutation in Rasa3 causes defects in several aspects of erythropoiesis, including a significant delay of erythroid differentiation at the polychromatophilic stage, decreased hemoglobinization, defects in cell cycle progression past the G1 checkpoint, and increased reactive oxygen species (ROS) during terminal erythroid differentiation as well as in scat peripheral blood reticulocytes and red blood cells. We previously reported that the mislocalization of mutated RASA3 to the cytosol of reticulocytes and mature red cells plays a role in the erythropoietic defect in scat, and the observed cell cycle arrest and increased ROS likely also contribute to this unique disease phenotype. Our current efforts are focused on further elucidation of the mechanism and specific disruptions in Ras signaling that lead to anemia, membrane fragmentation, and the cyclic phenotype in scat. Interestingly, we report here that apoptosis is not increased during scat crisis, and that mitochondria, a potential source of ROS, are normally eliminated at the reticulocyte stage. The dramatic nature of remission, with complete normalization of all hematologic parameters, led us to hypothesize that a secreted factor may be mediating the cyclic phenotype of scat. Differences in the cytokine profile of the serum of scat mice compared to wild type suggest that, indeed, one or several secreted factor(s) may be influencing the occurrence of bone marrow failure. Levels of galectin-1, a known mediator of cell-cell interactions and intracellular signaling in the hematopoietic niche, are consistently decreased in scat serum according to a multispot anti-cytokine antibody array (23,326.5 ± 21,439.7 integrated density in scat vs. 31,019.6± 20,110.7 in controls, p<0.05).Studies exploring the influence of the galectin family on erythropoiesis and Ras signaling in the context of scat are underway. Strengthening the notion that RASA3 has a critical conserved role in vertebrate terminal erythropoiesis, the characteristics of bone marrow failure seen in scat have been reproduced in human CD34+ cells using siRNA and shRNA knockdowns of Rasa3 . Similar to the changes seen in scat, cells with decreased RASA3 demonstrated delayed terminal erythroid differentiation and defective hemoglobinization. Finally, analysis of Ras expression and functional pull-down studies in human CD34+ cells revealed that, while K-Ras is the major active isoform expressed during terminal erythroid differentiation, H-Ras is also active during human erythropoiesis. Future studies with CD34+ Rasa3 knockdown cells will explore the influence of RASA3 on human K- and H-Ras signaling in erythropoiesis. Taken together, our studies further characterize the vital role of RASA3 in hemoglobinization, cell cycle progression, and cell survival during terminal erythroid differentiation, as well as identify novel targets for investigation of unknown mechanisms (e.g., dysregulated cytokine secretion) of bone marrow failure syndromes. Disclosures No relevant conflicts of interest to declare.

scholarly journals Morphologic Assessment of Terminal Erythroid Differentiation Predicts Outcomes in Myelodysplastic Syndromes

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3011-3011
Author(s):  
Maria Julia Montoro ◽  
Ana Perez ◽  
Silvia Saumell ◽  
Maida Navarrete ◽  
Gael Roue ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Research Funding ◽  
Erythroid Differentiation ◽  
Response Monitoring ◽  
Erythroid Cell ◽  
Categorical Variables ◽  
Differential Count ◽  
Advisory Committees ◽  
Terminal Erythroid Differentiation

Introduction Terminal erythroid differentiation (TED) follows a doubling pattern of maturation in each erythroid stage from proerythroblast to orthochromatic erythroblast. Recently, it has been shown by flow cytometry that erythroid differentiation is profoundly abnormal across all MDS subtypes and that the absence of quantifiable cells undergoing TED by well-defined cell surface markers is strongly associated with inferior overall survival (OS). Analysis of TED is not easy to perform and, notably, TED in MDS has not been systematically assessed by morphology, the gold standard analysis for MDS diagnosis. Against this background, we studied the pattern of TED maturation by conventional microscopy and compared clinical and prognostic characteristics between MDS patients with normal and abnormal TED. Methods Cytological analysis of erythroid cells was assessed in bone marrow smears in 500-cell differential count from patients diagnosed with MDS from 2011 to 2018 at our institution. TED cell subsets were categorized in proerythroblast, basophilic, polychromatic and orthochromatic erythroblasts. Patients were categorized as TED-negative when total TED was <15% of total cells in bone marrow and/or did not follow the expected doubling pattern of maturation. Erythroid stages were calculated based on the total erythroid cell population. Chi-square test was used for categorical variables, t-test for continuous variables and a Kaplan-Meier for survival analysis. Results A total of 247 patients diagnosed with MDS were included, whom main characteristics are detailed in table 1. Twenty percent of MDS patients were considered TED-abnormal, 8 (16%) because insufficient TED and 41 (84%) because abnormal TED maturation pattern. From the clinical standpoint, TED-negative patients exhibit lower hemoglobin and platelet counts, higher blasts, and worse IPSS-R (Table 1). No differences in the subtype of MDS (WHO 2016 classification) were observed between abnormal or normal TED. Median OS of the series was 28.6 months (CI95% 8-43.4). In TED-negative MDS, median OS was 24.1 months (CI95%: 7.3-50.7), whereas it was of 46 months (15.4-82.5) for normal TED (HR 0.53; p=0.001) (Figure 1). Risk of transformation to acute myeloid leukemia at 4 years was of 11% (CI95% 6-20) and 26% (CI95% 14-46) in the TED-negative and TED-positive patients, respectively (HR 0.27; p=0.001) (Figure 2). Conclusions Morphological analysys revealed that 20% of of MDS patients do not follow the expected pattern of maturation in successive erythroid stages. Importantly, the presence of an abnormal TED arises as a reliable prognostic marker of poor outcome in MDS patients. With the advent of the new erythroid maturation agents in the field of the MDS, analysis of TED maturation might become of paramount importance for treatment selection and response monitoring. Disclosures Salamero: Novartis: Honoraria; Celgene: Honoraria; Pfizer: Honoraria; Daichii Sankyo: Honoraria. Bosch:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AstraZeneca: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd/Genentech, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Acerta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding. Valcarcel:MSD: Consultancy, Honoraria, Speakers Bureau; Astellas: Consultancy, Honoraria, Speakers Bureau; JAZZ: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau.

Impairment of human terminal erythroid differentiation by histone deacetylase 5 deficiency

Blood ◽  
2021 ◽  
Author(s):  
Yaomei Wang ◽  
Wei Li ◽  
Vince Schulz ◽  
Huizhi Zhao ◽  
Xiaoli Qu ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
Molecular Mechanisms ◽  
Chromatin Condensation ◽  
Erythroid Differentiation ◽  
Chromatin Accessibility ◽  
Erythroid Cell ◽  
Global Changes ◽  
Human Cd34 ◽  
Wide Range ◽  
Terminal Erythroid Differentiation

Histone deacetylases (HDACs) are a group of enzymes catalyzing the removal of acetyl groups from histone and non-histone proteins. HDACs have been shown to play diverse functions in a wide range of biological processes. However, their roles in mammalian erythropoiesis remain to be fully defined. We show here that of the eleven classic HDAC family members, six of them (HDAC 1,2,3 and HDAC 5,6,7) are expressed in human erythroid cells with HDAC5 most significantly up regulated during terminal erythroid differentiation. Knockdown of HDAC5 by either shRNA or siRNA in human CD34+ cells followed by erythroid cell culture led to increased apoptosis, decreased chromatin condensation, and impaired enucleation of erythroblasts. Biochemical analyses revealed that HDAC5 deficiency resulted in activation of p53 in association with increased acetylation of p53. Furthermore, while acetylation of histone 4 (H4) is decreased during normal terminal erythroid differentiation, HDAC5 deficiency led to increased acetylation of H4 (K12) in late stage erythroblasts. This increased acetylation was accompanied by decreased chromatin condensation, implying a role for H4 (K12) deacetylation in chromatin condensation. ATAC-seq and RNA-seq analyses revealed that HDAC5 knockdown leads to increased chromatin accessibility genome wide and global changes in gene expression. Moreover, pharmacological inhibition of HDAC5 by the inhibitor LMK235 also led to increased H4 acetylation, impaired chromatin condensation and enucleation. Taken together, our findings have uncovered previously unrecognized roles and molecular mechanisms of action for HDAC5 in human erythropoiesis. These results may provide insights into understanding the anemia associated with HDAC inhibitor treatment.

scholarly journals Decreased PGC1β expression results in disrupted human erythroid differentiation, impaired hemoglobinization and cell cycle exit

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Taha Sen ◽  
Jun Chen ◽  
Sofie Singbrant
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Mitochondrial Function ◽  
Iron Homeostasis ◽  
Erythroid Differentiation ◽  
Refractory Anemia ◽  
Cell Cycle Exit ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Terminal Erythroid Differentiation

AbstractProduction of red blood cells relies on proper mitochondrial function, both for their increased energy demands during differentiation and for proper heme and iron homeostasis. Mutations in genes regulating mitochondrial function have been reported in patients with anemia, yet their pathophysiological role often remains unclear. PGC1β is a critical coactivator of mitochondrial biogenesis, with increased expression during terminal erythroid differentiation. The role of PGC1β has however mainly been studied in skeletal muscle, adipose and hepatic tissues, and its function in erythropoiesis remains largely unknown. Here we show that perturbed PGC1β expression in human hematopoietic stem/progenitor cells from both bone marrow and cord blood results in impaired formation of early erythroid progenitors and delayed terminal erythroid differentiation in vitro, with accumulations of polychromatic erythroblasts, similar to MDS-related refractory anemia. Reduced levels of PGC1β resulted in deregulated expression of iron, heme and globin related genes in polychromatic erythroblasts, and reduced hemoglobin content in the more mature bone marrow derived reticulocytes. Furthermore, PGC1β knock-down resulted in disturbed cell cycle exit with accumulation of erythroblasts in S-phase and enhanced expression of G1-S regulating genes, with smaller reticulocytes as a result. Taken together, we demonstrate that PGC1β is directly involved in production of hemoglobin and regulation of G1-S transition and is ultimately required for proper terminal erythroid differentiation.

Characteristics of De Novo Acute Leukemia Patients with Glycosylphosphatidylinositol (GPI) Protein-Negative Population of Leukemic Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4462-4462
Author(s):  
Hideyoshi Noji ◽  
Tsutomu Shichishima ◽  
Masatoshi Okamoto ◽  
Kazuhiko Ikeda ◽  
Akiko Nakamura ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Leukemia ◽  
De Novo ◽  
Bone Marrow Failure ◽  
Flow Cytometric Analysis ◽  
Leukemic Cells ◽  
Remission Induction ◽  
Color Flow ◽  
Flow Cytometric ◽  
Number Of Patients

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is considered to be an acquired stem cell disorder affecting all hematopoietic lineages, which lack GPI-anchored membrane proteins, such as CD59, because of abnormalities in the phosphatidylinositol glycan-class A (PIG-A) gene. Also, PNH is one disorder of bone marrow failure syndromes, including aplastic anemia and myelodysplastic syndrome, which are considered as pre-leukemic states. In this study, to know some characteristics of patients with de novo acute leukemia, we investigated expression of CD59 in leukemic cells from 25 patients (female: male=8: 17; mean age ± standard deviation, 57.8 ± 19.5 years) with de novo acute leukemia by single-color flow cytometric analysis. In addition, the PIG-A gene from CD59− leukemic cells sorted by FACS Vantage in 3 patients with acute leukemia was examined by sequence analysis. All the patients had no past history of PNH. Based on the French-American-British criteria, the diagnosis and subtypes of acute leukemia were determined. The number of patients with subtypes M1, M2, M3, M4, M5, and M7 was 1, 14, 2, 4, 2, and 2, respectively. Two of the patients were classified into acute myeloid leukemia with trilineage myelodysplasia from morphological findings in bone marrow. Chromosomal analyses presented abnormal karyotypes in 14 of 25 patients. Flow cytometric analyses showed that leukemic cells from 16 of 25 patients (64%) had negative populations of CD59 expression and the proportion of the populations was 63.3 ± 25.7%, suggesting the possibility that CD59− leukemic cells from patients with de novo acute leukemia might be derived from PNH clones. In fact, the PIG-A gene analyses showed that monoclonal or oligoclonal PIG-A mutations in coding region were found in leukemic cells from 3 patients with CD59− leukemic cells and all of the clones with the PIG-A mutations were minor. Then, various clinical parameters, including rate of complete remission for remission-induction chemotherapy, peripheral blood, bone marrow blood, and laboratory findings, and results of chromosomal analyses were statistically compared between 2 groups of patients with (n=16) and without (n=9) CD59− leukemic cells. The reticulocyte counts (10.5 ± 13.0 x 104/μl) and proportions of bone marrow erythroblasts (17.5 ± 13.9%) in patients with only CD59+ leukemic cells were significantly higher than those (2.5 ± 1.7 x 104/μl, p&lt;0.05; and 5.6 ± 6.2%, p&lt;0.01, respectively) in patients with CD59− leukemic cells. The proportions of bone marrow blasts (69.3 ± 21.1%) in patients with CD59− leukemic cells were significantly higher than those (45.5 ± 19.3%, p&lt;0.02) in patients with only CD59+ leukemic cells. In conclusion, our findings indicate that leukemic cells derived from PNH clones may be common in de novo acute leukemia patients, suggesting that bone marrow failure may have already occurred in localized bone marrow even in de novo acute leukemia.

RAP-536 Promotes Terminal Erythroid Differentiation and Reduces Anemia in a Murine Model of Myelodysplastic Syndromes

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3796-3796 ◽  
Author(s):  
Rajasekhar NVS Suragani ◽  
Robert Li ◽  
Dianne Sako ◽  
Asya Grinberg ◽  
R. Scott Pearsall ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Erythroid Differentiation ◽  
Severe Anemia ◽  
Ineffective Erythropoiesis ◽  
Employment Equity ◽  
Erythroid Hyperplasia ◽  
Erythroid Precursors ◽  
Equity Ownership ◽  
Terminal Erythroid Differentiation

Abstract Abstract 3796 Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell disorders characterized by peripheral blood cytopenias such as anemia, neutropenia or thrombocytopenia. Ineffective erythropoiesis due to increased proliferation and abortive maturation of precursors leads to severe anemia, the most common cytopenia observed in MDS syndromes. Despite elevated erythropoietin (EPO) and erythroid hyperplasia, MDS patients are often given recombinant EPO therapy to stimulate erythropoiesis. However, only a small proportion of patients respond to EPO therapy. Frequent blood transfusions as supportive care result in iron overloading and recently iron overloading is also linked to enhanced progression to AML. Therefore, alternative therapies are necessary to treat anemia in MDS patients. Signaling by members of the TGFβ superfamily are known regulators of erythropoiesis. We developed ACE-536, a ligand trap consisting of a modified activin receptor Type IIB extracellular domain linked to a human Fc domain. In vitro assays revealed that ACE-536 inhibits smad 2/3 ligands of the signaling pathway but not smad 1/5/8 ligands. Dose dependent studies using ACE-536 in mice, rats and monkeys revealed that ACE-536 treatment resulted in increased red blood parameters but did not affect other cell types. These data suggests that ACE-536 inhibits smad 2/3 phosphorylation modulating the expression of downstream genes involved in erythroid development pathway. BFU-E and CFU-E colony formation assays from bone marrow and spleen in mice following ACE-536 treatment revealed that ACE-536 did not affect the proliferation stages of erythropoiesis. In mice, terminal erythroid differentiation analysis by flow cytometry at 72hrs following RAP-536 (10mg/kg) treatment demonstrated decreased basophilic and increased ortho- and poly-chromatophilic erythroblasts and reticulocytes compared to VEH treatment. Cell cycle analysis of bone marrow and splenic erythroblasts counterstained with BrdU and 7-AAD after RAP-536 (10mg/kg, for 24 hours) or VEH treatment to EPO pre-treated (1500 units/kg, for 40 hours) mice (N=5/group) revealed that EPO+RAP-536 treatment resulted in significant decrease in S-phase and increase in G1/G2-phases of cell cycle compared to EPO+VEH treatment. In addition, EPO+RAP-536 treatment resulted in a greater increase in RBC parameters than either of the treatments alone. Together, these results demonstrate that ACE-536 increases red blood cell formation by promoting maturation of late stage erythroblasts. We then investigated the effect of ACE-536 on anemia in NUP98-HOXD13 (NHD13) transgenic murine model of MDS. NHD13 mice develop anemia, neutropenia and lymphopenia, with normal or hyper cellular bone marrow. A Majority of the mice die by 14 months due to severe pancytopenia or progression to acute myeloid leukemia. In this study, mice were divided into three groups based on age. Early (∼4 months old), mid (∼8 months old) and late stage (∼10 months) groups were randomized and dosed with either RAP-536 at 10 mg/kg or VEH twice per week for 6–8 weeks. NHD13 mice in each group had severe anemia characterized by reduced RBC, Hemoglobin and HCT and compared to wild-type littermates prior to treatment. Treatment of RAP-536 for 6–8 weeks significantly increased RBC parameters and reversed anemia at all stages. Peripheral blood smear analysis revealed no indication of increased leukemic progression due to RAP-536 treatment. Cell differential and flow cytometric evaluation of erythroid precursors from bone marrow demonstrated decreased erythroid precursors and hyperplasia after RAP-536 treatment compared to vehicle treated control. Our data demonstrate that RAP-536 can increase hematology parameters by enhancing maturation of terminally differentiated red blood cells. We have shown RAP-536 corrects ineffective erythropoiesis, decreases erythroid hyperplasia and normalizes myeloid: erythroid ratios without enhanced progression to AML in a murine MDS model. Therefore ACE-536 may represent a novel treatment for anemia associated with MDS, particularly in patients that are refractory to EPO therapy. ACE-536 has completed Phase I clinical trials in healthy human volunteers and Phase II study in MDS patients is planned. Disclosures: Suragani: Acceleron Pharma Inc: Employment, Equity Ownership. Li:Acceleron Pharma Inc: Employment, Equity Ownership. Sako:Acceleron Pharma Inc: Employment, Equity Ownership. Grinberg:Acceleron Pharma Inc: Employment, Equity Ownership. Pearsall:Acceleron Pharma Inc: Employment, Equity Ownership. Kumar:Acceleron Pharma Inc: Employment, Equity Ownership.

scholarly journals Acute Myeloid Leukemia with Basophilic Differentiation Transformed from Myelodysplastic Syndrome

2017 ◽  
Vol 2017 ◽  
pp. 1-6 ◽  
Author(s):  
Yasuhiro Tanaka ◽  
Atsushi Tanaka ◽  
Akiko Hashimoto ◽  
Kumiko Hayashi ◽  
Isaku Shinzato
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myelodysplastic Syndrome ◽  
Myeloid Leukemia ◽  
Bone Marrow Failure ◽  
Immunosuppressive Treatment ◽  
Flow Cytometric Analysis ◽  
Chromosomal Analysis ◽  
Monosomy 7 ◽  
Acute Myeloid

Myelodysplastic syndrome (MDS) terminally transforms to acute myeloid leukemia (AML) or bone marrow failure syndrome, but acute myeloid leukemia with basophilic differentiation has been rarely reported. An 81-year-old man was referred to our department for further examination of intermittent fever and normocytic anemia during immunosuppressive treatment. Chromosomal analysis showed additional abnormalities involving chromosome 7. He was diagnosed as having MDS. At the time of diagnosis, basophils had not proliferated in the bone marrow. However, his anemia and thrombocytopenia rapidly worsened with the appearance of peripheral basophilia three months later. He was diagnosed as having AML with basophilic differentiation transformed from MDS. At that time, monosomy 7 was detected by chromosomal analysis. We found that basophils can be confirmed on the basis of the positivity for CD203c and CD294 by flow cytometric analysis. We also found by cytogenetic analysis that basophils were derived from myeloblasts. He refused any chemotherapy and became transfusion-dependent. He died nine months after the transformation. We should keep in mind that MDS could transform to AML with basophilic differentiation when peripheral basophilia in addition to myeloblasts develops in patients with MDS.

scholarly journals TLR7 ligation augments haematopoiesis in Rps14 (uS11) deficiency via paradoxical suppression of inflammatory signalling and enhanced differentiation

2020 ◽  
Author(s):  
Oscar A Peña ◽  
Alexandra Lubin ◽  
Jasmine Rowell ◽  
Catherine Hockings ◽  
Youngrock Jung ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Failure ◽  
Erythroid Differentiation ◽  
Gene Signature ◽  
Erythroid Progenitors ◽  
Pathway Activation ◽  
Age Dependent ◽  
Small Molecule Screen ◽  
Inflammatory Signalling

AbstractMyelodysplastic syndrome (MDS) is a haematological malignancy characterised by blood cytopenias and predisposition to acute myeloid leukaemia (AML). Therapies for MDS are lacking, particularly those that impact the early stages of disease. We developed a model of MDS using zebrafish using knockout of Rps14, the primary mediator of the anaemia associated with del (5q) MDS. These mutant animals display dose- and age-dependent abnormalities in haematopoiesis, culminating in bone marrow failure with dysplastic features. We utilized rps14 knockdown to undertake an in vivo small molecule screen to identify compounds that ameliorate the MDS phenotype, identifying imiquimod, an agonist of TLR7 and TLR8. Imiquimod alleviates anaemia by promoting haematopoietic stem and progenitor cell expansion and erythroid differentiation, the mechanism of which is dependent on TLR7 ligation. TLR7 activation in this setting paradoxically promoted an anti-inflammatory gene signature suggesting crosstalk between pro-inflammatory pathways endogenous to Rps14 loss and TLR7 pathway activation. Finally, we show that in highly purified human bone marrow samples from anaemic patients, imiquimod leads to an increase in erythroid output from myelo-erythroid progenitors and common myeloid progenitors. Our findings have both specific implications for the development of targeted therapeutics for del (5q) MDS and wider significance identifying a potential role for TLR7 ligation in modifying anaemia.

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