Defective Erythropoiesis Research Articles

Eltrombopag Improves Erythroid Differentiation in a Human iPSC Model of Diamond Blackfan Anemia

Diamond Blackfan Anemia (DBA) is a congenital bone marrow (BM) failure syndrome primarily characterized by defective erythropoiesis. In most patients, pathogenic heterozygous mutations have been identified in genes encoding ribosomal proteins (RP). The resulting RP haploinsufficiency was recently shown to delay globin protein translation in erythroid cells, whereas synthesis of heme, the nonprotein iron-containing component of hemoglobin, proceeds normally (Yang et al., Sci Transl Med 2016). Because heme is first synthesized at or just before the proerythroblast stage, free heme is in excess of globin in these cells. High levels of free heme induce proerythroblast cell death, and erythroid differentiation thus halts at the earlier BFU-E/CFU-E progenitor stage. Consistent with these observations, inhibition of heme synthesis with succinylacetone was previously shown to improve erythroid differentiation of DBA marrow cells in vitro. In this study, we investigated whether eltrombopag (Epag), an FDA-approved mimetic of thrombopoietin that promotes trilineage hematopoiesis in subjects with acquired BM failure (Olnes et al., NEJM 2012; Townsley et al., NEJM 2017), could rescue erythropoiesis in DBA. We hypothesized that Epag might inhibit heme synthesis by restricting iron availability due to its robust intracellular iron chelating properties, leading to decrements in iron-induced reactive oxygen species (ROS) and increased proerythroblast survival and maturation. To test this possibility,we first established an induced pluripotent stem cell (iPSC) model of DBA by reprogramming mononuclear cells (MNCs) from a patient with inactivating mutations in RPS19, the most commonly mutated gene in DBA. We also generated a control isogenic iPSC line by CRISPR/Cas9-mediated correction of RPS19 point mutations in the established DBA iPSC line.RPS19 haploinsufficiency was confirmed by Western blot and the expected reduction in 40S/60S ribosomal subunit ratio was detected by polysome profiling of DBA iPSCs. This phenotype normalized in the isogenic iPSCs. Both DBA and isogenic iPSC lines, and iPSCs derived from a healthy donor, were then subjected to hematopoietic differentiation for 21 days using the STEMdiffTMmonolayer-based approach (Ruiz et al., BioRxiv 2019). Hematopoietic cells were harvested between day 19 and 21 of culture when maximum erythroid production is observed in this system. Normal and isogenic iPSCs efficiently gave rise to erythroid cells at various stages of maturation, including CD71+CD45+EPOR+primitive erythroid progenitors (P1), CD71+CD45loEPOR-proerythroblasts (P2), and more mature CD71+CD45-EPOR-erythroblasts (P3) (Figure). In contrast, the majority of erythroid cells detected after differentiation of DBA iPSCs were comprised within P1 with limited maturation to P2 and P3, consistent with a block in differentiation at the early erythroid progenitor stage (Figure). Furthermore, in colony forming unit (CFU) assays, DBA iPSCs generated numbers of myeloid colonies (CFU-G, CFU-M and CFU-GM) comparable to normal and isogenic iPSCs, but erythroid colonies (BFU-E and CFU-E) were undetectable, in keeping with DBA progenitor's inability to differentiate in vitro. Next, DBA iPSCs were differentiated in the presence of Epag 3 µg/mL from day 10 to 21 of culture. Addition of Epag improved late erythroid maturation, as indicated by reduced percentages of early progenitors (P1) and a concomitant increase in more mature P2 and P3 erythroblastic populations (Figure). Investigations are ongoing to confirm Epag-mediated iron restriction and decreased heme synthesis as the primary molecular mechanism underpinning the improved erythroid maturation observed in this study. Overall, our data indicate that directed differentiation of DBA iPSCs recapitulates early erythroid maturation defects in vitro, and erythropoiesis can be rescued in part by addition of Epag during culture. These results suggest that Epag may improve red blood cell production in patients with DBA. Figure Disclosures No relevant conflicts of interest to declare.

Mutation of Murine Rpl5 reveals a New Model for Diamond Blackfan Anemia Characterized By Defective Erythropoiesis

Background Ribosome dysfunction is implicated in several abnormal developmental conditions and diseases in humans. Heterozygous germline ribosomal protein (RP) variants are found in the majority of individuals with Diamond Blackfan anemia (DBA) with RPL5 (uL18) being the second most frequently mutated gene. Patients with RPL5 mutations are for unknown reasons at increased risk of congenital defects when compared to other genotypes. There is currently no available Rpl5 mouse model with anemia and therefore hematopoiesis in this genotype has not been well studied. Methods We characterized a novel mutant mouse with a growth and kinky tail defect, and increased mortality. The mouse was generated in a chromosome 11 balancer ENU mutagenesis screen. Following genetic mapping and whole exome sequencing, we identified a heterozygous intronic Rpl5 missense variant, which segregated with all affected mice. We first performed molecular analyses to validate the impact of this variant on RPL5 and ribosome levels, and then examined the development and physiology of multiple tissues by histology, micro-CT, echocardiogram and measurement of erythroid differentiation to determine the etiology of the observed increased mortality in newborn Rpl5 mutant mice. Results This variant led to decreased Rpl5 mRNA and protein, and consequential polysome defects (decreased 60S and 80S ribosome subunits) consistent with RPL5 haploinsufficiency. All Rpl5+/- neonates exhibited deficient terminal erythroid differentiation. A fraction of the mutants progressed to severe anemia leading to increased mortality while others appeared to undergo spontaneous remission. Surviving adult/aged mice exhibited no anemia although preliminary studies indicate reduced numbers of hematopoietic progenitors. Most newborn mutant mice had large ventricular septal defects (VSD) while surviving adult mice had small VSDs on echocardiogram. Rpl5+/- mice were significantly smaller without craniofacial abnormalities as assessed by micro-CT or histology. Bone and cartilage staining of newborn mice revealed that the kinky tail defect resulted from delayed endochondral ossification. Conclusion This model recapitulates the major developmental and erythroid differentiation defects of DBA in an animal model. We propose that this novel Rpl5 mutant provides an opportunity to study the mechanism of the variable penetrance of anemia and the congenital defects normally encountered in DBA. Disclosures Singh: Emmaus Medical, Inc: Consultancy.

Elucidating the Role of IL6 in Stress Erythropoiesis and in the Development of Anemia Under Inflammatory Conditions

Anemia of inflammation, also known as anemia of chronic disease is the second most common anemia after iron deficiency anemia. The predominant regulators of AI are the cytokine-interleukin-6 (IL6) and the hormone hepcidin (Hamp). IL6 has been implicated in inducing expression of hepcidin. Published data from our lab have shown that lack of IL6 or hepcidin in knockout mouse models (IL6-KO and Hamp-KO) injected with the heat-killed pathogen Brucella abortus(BA) results in recovery from anemia but interestingly the pattern of the recovery was different in IL6-KO and Hamp-KO mice, suggesting that the two proteins contribute independently to AI. Here, we validated the independent role of IL6 and Hamp in AI by generating a double-knockout (DKO) mouse model lacking the expression of both. In the first few days following BA administration, we observed severe reduction in the total number of BM cells in each model followed by a slow recovery in erythroid and multilineage hematopoietic cells. The recovery, initially, was more sustained in the BA-treated-DKO model. In particular, in the first week, BA-treated-DKO mice showed an increased number of erythroblasts in the bone marrow (BM) and spleen as seen in comparison to IL6-KO and Hamp-KO. IL6-KO mice showed an intermediate recovery profile when compared to DKO and Hamp-KO, the last one showing the worst profile in the BM. Interestingly, when the reticulocyte count in the DKO mice was compared to that of IL6-KO and Hamp-KO mice, it showed a biphasic trend, with a significant increase in number during the 2nd week, followed by a significant reduction during the 3rd week. We hypothesized that the initial surge in reticulocyte count in DKO was due to lack of hepcidin, which increases iron availability to erythroid cells, and concurrent lack of IL6, which favors BM erythropoiesis in presence of inflammatory stimuli. However, we also speculated that the excess of iron (as NTBI), which accumulates during the first two weeks, leads to oxidative stress and erythroid cell death in presence of inflammatory cytokines, despite the absence of IL6. We also surmised that, during the second week, a second wave of inflammatory cytokines is triggered by the adaptive response in response to the BA that would explain the negative effect on erythropoiesis after the initial recovery. To assess this hypothesis, we utilized an inflammation panel to analyze the cytokine expression in WT animals treated with PBS or BA at 6 hours, 24 hours and then around ~2 weeks. The cytokine levels were normalized after 24 hours. However, around two weeks, we observed a novel surge of cytokines such as IFN-g and TNFa in the BA treated mice, indicating their role in innate (immediate effect; 6 hours) and adaptive immune response, which activated a second wave of inflammation (around 2 weeks, during the recovery of hematopoiesis in the BM). Interestingly, while we observed oxidative stress and defective erythropoiesis in the bone marrow, this was not seen in the spleen, where increased and extramedullary erythropoiesis sustained some level of RBC production. Since the BA-treated-IL6-KO did not show any major defect in the BM after two weeks, we challenged them with administration of iron dextran. Upon treatment, also the IL6-KO mice treated with both BA and iron dextran shown increased production of reactive oxygen species as well as a defect in bone marrow erythropoiesis, similarly as in DKO or Hamp-KO mice, thereby explaining the plausible reason of reduced erythropoiesis in the bone-marrow. Furthermore, to identify mechanisms leading to oxidative stress, we established an in-vitro culture system where primary murine bone marrow cells were cultured for 18-20 hours in presence of serum isolated after 6hrs from either PBS treated or BA treated C57BL/6 mice. With the help of confocal microscopy, we observed an increase in mitochondrial superoxide in the cells treated with BA serum; interestingly we have also seen a decrease in Ter 119 population in the cells cultured with BA treated serum implicating that the erythroid cells are dying. To further investigate the downstream players related to the death of erythroid progenitors we are currently investigating the role caspase 1 (a major regulator in pyroptosis) and Gata-1. In conclusion, this study is elucidating some of the mechanisms associated with the anemia triggered by inflammation with the potential to identify new targets and treatments. Disclosures Rivella: Disc medicine, Protagonist, LIPC, Meira GTx: Consultancy; Meira GTx, Ionis Pharmaceutical: Membership on an entity's Board of Directors or advisory committees.

ARHGEF12 regulates erythropoiesis and is involved in erythroid regeneration after chemotherapy in acute lymphoblastic leukemia patients.

Hematopoiesis is a finely regulated process in vertebrates under both homeostatic and stress conditions. By whole exome sequencing, we studied the genomics of acute lymphoblastic leukemia (ALL) patients who needed multiple red blood cell (RBC) transfusions after intensive chemotherapy treatment. ARHGEF12, encoding a RhoA guanine nucleotide exchange factor, was found to be associated with chemotherapy-induced anemia by genome-wide association study analyses. A single nucleotide polymorphism (SNP) of ARHGEF12 located in an intron predicted to be a GATA1 binding site, rs10892563, is significantly associated with patients who need RBC transfusion (P=3.469E-03, odds ratio 5.864). A luciferase reporter assay revealed that this SNP impairs GATA1-mediated trans-regulation of ARHGEF12, and quantitative polymerase chain reaction studies confirmed that the homozygotes status is associated with an approximately 61% reduction in ARHGEF12 expression (P=0.0088). Consequently, erythropoiesis was affected at the pro-erythroblast phases. The role of ARHGEF12 and its homologs in erythroid differentiation was confirmed in human K562 cells, mouse 32D cells and primary murine bone marrow cells. We further demonstrated in zebrafish by morpholino-mediated knockdown and CRISPR/Cas9-mediated knockout of arhgef12 that its reduction resulted in erythropoiesis defects. The p38 kinase pathway was affected by the ARHGEF12-RhoA signaling in K562 cells, and consistently, the Arhgef12-RhoA-p38 pathway was also shown to be important for erythroid differentiation in zebrafish as active RhoA or p38 readily rescued the impaired erythropoiesis caused by arhgef12 knockdown. Finally, ARHGEF12-mediated p38 activity also appeared to be involved in phenotypes of patients of the rs10892563 homozygous genotype. Our findings present a novel SNP of ARHGEF12 that may involve ARHGEF12-RhoA-p38 signaling in erythroid regeneration in ALL patients after chemotherapy.

Comparative Proteome-Wide Analysis of Bone Marrow Microenvironment of β-Thalassemia/Hemoglobin E.

β-thalassemia/Hb E is a global health issue, which is characterized by a range of clinical symptoms from a mild and asymptomatic anemia to severe disorders that require transfusions from infancy. Pathological mechanisms of the disease involve the excess of unmatched alpha globin and iron overload, leading to ineffective erythropoiesis and ultimately to the premature death of erythroid precursors in bone marrow (BM) and peripheral organs. However, it is unclear as to how BM microenvironment factors contribute to the defective erythropoiesis in β-thalassemia/Hb E patients. Here, we employed mass spectrometry-based comparative proteomics to analyze BM plasma that was collected from six β-thalassemia/Hb E patients and four healthy donors. We identified that the differentially expressed proteins are enriched in secretory or exosome-associated proteins, many of which have putative functions in the oxidative stress response. Using Western blot assay, we confirmed that atypical lipoprotein, Apolipoprotein D (APOD), belonging to the Lipocalin transporter superfamily, was significantly decreased in BM plasma of the tested pediatric β-thalassemia/Hb E patients. Our results highlight that the disease condition of ineffective erythropoiesis and oxidative stress found in BM microenvironment of β-thalassemia/Hb E patients is associated with the impaired expression of APOD protein.

A Shared Signaling Network That Maintains Erythroid Homeostasis By Activating Stress Erythropoiesis Regulates Inflammation: Implications for the Anemia of Chronic Inflammation

Anemia is a common secondary pathology resulting from inflammatory diseases including cancer or infection. Its exact prevalence is difficult to determine, yet its contributions to the morbidity and mortality of patients and its negative impact on quality of life are clear. Despite the diverse set of factors that can lead to inflammatory anemia, its core pathology of hyperinflammation, iron dysregulation, and lack of red cell production suggests the possibility of a common etiology. Inflammation induces pro-inflammatory cytokines including TNFα, IL-1β and IFNγ that drive myelopoiesis at the expense of steady state bone marrow erythropoiesis. In addition, other cytokines increase the expression of hepcidin, haptoglobin and hemopexin by the liver, leading to the sequestration of iron. While limiting iron can be beneficial in the context of infection, the consequence of this restriction is a further reduction in red cell production in the bone marrow. To compensate for the loss of bone marrow erythropoiesis, inflammation induces stress erythropoiesis in the spleen or liver.Stress erythropoiesis is regulated by different signals which include BMP4 and GDF15 and utilizes stress erythroid progenitors that are distinct from steady state erythroid progenitors. Our work shows that in contrast to steady state erythropoiesis, pro-inflammatory cytokines like TNFα promote the proliferation of stress erythroid progenitors, while anti-inflammatory signals such as PGJ2 and IL-10 promote their differentiation. These studies demonstrate that the expansion and differentiation stages of stress erythropoiesis are coordinated with, and influenced by, signals that initiate and resolve inflammation. In addition, we show that this regulation is reciprocal. Signals that regulate the differentiation of stress erythroid progenitors (GDF15 and BMP4) promote the resolution of inflammation. Mice infected with the model gut pathogen Citrobacter rodentium, exhibit stress erythropoiesis in the spleen, while steady state erythropoiesis in the bone marrow is suppressed until pathogen clearance. We observed that hepcidin expression in the liver increases initially, but then decreases as the expression of erythroferrone by stress erythroid progenitors increased in the spleen, but not the bone marrow. Using mice mutant for GDF15 (GDF15-/-) and for BMP4 signaling (flexed-tail f/f), which exhibit defective stress erythropoiesis, we observed that the expression of hepcidin was dysregulated suggesting that stress erythroid progenitors are responsible for iron regulation at this time. In addition, infection of mutant mice led to increased lethality. During peak infection, we observed morphological differences in the colons of these mice indicative of increased inflammation and systemic infection. These changes were associated with increased expression of pro-inflammatory genes, as well as decreased numbers of FoxP3+ regulatory T-cells (Tregs). Using naïve CD4+ T-cells isolated from uninfected control, f/f or GDF15-/- mice, we observed significantly altered gene expression from mutant T-cells following Treg induction in vitro. However, the addition of BMP4 and GDF15 into these cultures rescued Treg development of mutant naïve T-cells and enhanced Treg development of naïve control T cells. Analysis of the BMP4 and GDF15 signaling pathways in both stress erythroid progenitor differentiation and in Treg development revealed that in both systems these signals converge on the transcription factor HIF1α.Taken together these data support a new model showing that the loss of steady state erythropoiesis due to pro-inflammatory signals is balanced by the activation of stress erythropoiesis by those same factors. Similarly, the differentiation of stress erythroid progenitors appears to regulate iron, and is itself regulated by the same signals that drive the development of Tregs and the expression of anti-inflammatory cytokines during immune resolution. This work supports a novel model where initiation and resolution of inflammatory immune responses are co-regulated with stress erythropoiesis, which allows for a robust immune response while maintaining erythroid homeostasis. Furthermore, this model predicts that alterations to this shared signaling network will underlie the development of chronic inflammatory anemia. DisclosuresNo relevant conflicts of interest to declare.

Mitochondria Clearance Mechanisms Via Interaction with Macrophages in Erythropoiesis

Adult erythropoiesis involves a series of well-coordinated events resulting in the production of mature red blood cells. One of such events is the mitochondria clearance, which is known to occur cell-autonomously via autophagy-dependent mechanisms. Interestingly, we identified a sequential changes in the transcriptional pattern during terminal erythroid differentiation based on the expression of several macroautophagy (e.g. Atg3, Atg5, Atg7 and Atg10) and non-canonical mitophagy (e.g. Pink1, Park2, Bnip3l/Nix, P62 and Ulk1) genes. Hence we hypothesize that the progressive reduction in mitochondria during terminal erythroid differentiation is directed by distinct transcriptionally-regulated programs. Notably, we revealed a gradual reduction of the expression of lysosome related genes (e.g. Lamp1, CD63, and Atp6v) and lysosomal activities from early to late stages of terminal differentiation. On the other hand, P62-Pink1-Parkin mediated ubiquitin proteasome degradation of mitochondria proteins seems to be more prominent during late stage erythropoiesis. Hence our data suggest that mitochondria clearance is predominantly mediated by canonical autophagy during early stages of terminal differentiation, whereas non-canonical mitophagy pathway seem to play a more predominant role to regulate late stages erythroid maturation.Next, we discovered mitochondria transfer activities from erythroblasts to their niche. In the context of erythropoiesis, macrophages are known to interact closely with erythroblasts to provide a specialized niche for erythroid precursors to proliferate, differentiate and enucleate. We showed defective erythropoiesis after macrophage depletion in the bone marrow. Subsequently, we identified a tendency for early erythroblasts to associate with macrophages and isolated those erythroblasts from mito-dendra2 mice with trackable mitochondria to establish a murine primary cell co-culture system. Then we report mitochondria transfer activities in the erythroid niche via different modes including direct uptake, micro-vesicle transfer and tunnelling nanotubes (TNT). Interestingly, interchangeable structures between micro-vesicles and TNTs have been observed, suggesting an interplay between cytoskeleton and membrane lipid molecules in the mitochondria transfer mechanisms. Furthermore, mitochondria transfer activities have also been observed in the co-culture of mito-dendra2 erythroid cells with a macrophage cell line, RAW cells, and are significantly enhanced by the activation of the RAW cells via Tfe3 activation.In summary, our findings may provide insight into the mitochondria clearance machineries that mediates erythroid maturation to fulfil the clinical demand for large scale transfusable blood cell production in vitro. DisclosuresNo relevant conflicts of interest to declare.

Production and Transduction of a Human Recombinant β-Globin Chain into Proerythroid K-562 Cells To Replace Missing Endogenous β-Globin.

Protein replacement therapy (PRT) has been applied to treat severe monogenetic/metabolic disorders characterized by a protein deficiency. In disorders where an intracellular protein is missing, PRT is not easily feasible due to the inability of proteins to cross the cell membrane. Instead, gene therapy has been applied, although still with limited success. β-Thalassemias are severe congenital hemoglobinopathies, characterized by deficiency or reduced production of the adult β-globin chain. The resulting imbalance of α-/β-globin chains of adult hemoglobin (α2β2) leads to precipitation of unpaired α-globin chains and, eventually, to defective erythropoiesis. Since protein transduction domain (PTD) technology has emerged as a promising therapeutic approach, we produced a human recombinant β-globin chain in fusion with the TAT peptide and successfully transduced it into human proerythroid K-562 cells, deficient in mature β-globin chain. Notably, the produced human recombinant β-globin chain without the TAT peptide, used as internal negative control, failed to be transduced into K-562 cells under similar conditions. In silico studies complemented by SDS-PAGE, Western blotting, co-immunoprecipitation and LC-MS/MS analysis indicated that the transduced recombinant fusion TAT-β-globin protein interacts with the endogenous native α-like globins to form hemoglobin α2β2-like tetramers to a limited extent. Our findings provide evidence that recombinant TAT-β-globin is transmissible into proerythroid K-562 cells and can be potentially considered as an alternative protein therapeutic approach for β-thalassemias.

Iron attenuates erythropoietin production by decreasing hypoxia-inducible transcription factor 2α concentrations in renal interstitial fibroblasts

Iron is an essential mineral for oxygen delivery and for a variety of enzymatic activities, but excessive iron results in oxidative cytotoxicity. Because iron is primarily used in red blood cells, defective erythropoiesis caused by loss of the erythroid growth factor erythropoietin (Epo) elevates iron storage levels in serum and tissues. Here, we investigated the effects of iron in a mouse model of Epo-deficiency anemia, in which serum iron concentration was significantly elevated. We found that intraperitoneal injection of iron-dextran caused severe iron deposition in renal interstitial fibroblasts, the site of Epo production. Iron overload induced by either intraperitoneal injection or feeding decreased activity of endogenous Epo gene expression by reducing levels of hypoxia-inducible transcription factor 2α (HIF2α), the major transcriptional activator of the Epo gene. Administration of an iron-deficient diet to the anemic mice reduced serum iron to normal concentration and enhanced the ability of renal Epo production. These results demonstrate that iron overload due to Epo deficiency attenuates endogenous Epo gene expression in the kidneys. Thus, iron suppresses Epo production by reducing HIF2α concentration in renal interstitial fibroblasts.

Management of femoral fractures in adult onset osteopetrosis: A multicentric retrospective study

Background: Osteopetrosis is a relatively rare disease with an incidence of approximately 1 case per 2,00,000 to 5,00,000 population. The clinical features of osteopetrosis includes pathological fractures, severe anemia due to defective erythropoiesis, bone marrow failure, coagulation defects, propensity for developing severe infections. Common fractures seen in these patients include hip and femoral fractures. Management of fractures in these patients is technically difficult and postoperative complications are common. We conducted a retrospective study of 20 patients with osteopetrosis who presented with femoral fractures and were treated in our institute. Materials and Methods: This was a multicentric retrospective study of 20 patients with osteopetrosis who presented with femoral fractures. The patients were included in this study on the basis of predefined inclusion and exclusion criteria. The diagnosis was based upon imaging studies (X-Rays in all the patients and CT in selected cases). The operative procedure post-operative complications, functional outcome and complications during follow up were studied from case papers. The data was tabulated and analyzed using Minitab 17 version software.Results: Out of 20 studied cases there were 16 (80%) males and 4 (20%) females with a M:F ratio of 1: 0.25. The most common affected age group was found to be between 30 – 35 years (35%). Majority of patient sustained fracture following motor vehicular accidents (65%) followed by fall from height (20%) and direct blow or assault (15%). 13 patients were found to be having co-morbidities including anemia, carpal tunnel syndrome and visual disturbances. Open reduction and internal fixation was done in all the cases. Technical difficulties seen during operative procedures included difficult because of obliterated canal, sclerosed medulla and dense swarf. Common complications in these patients included wound infection, hematoma formation and arthritis of hip joint (5%). Repeat surgeries were required in 3 (15%) patients and 1 (5%) patient developed non-union of the fracture ends. Conclusion: Management of femoral fracture in patients with osteopetrosis is challenging for operating orthopedic surgeon. Common problems encountered during open reduction and internal fixation include overheating during drilling, smoke production and increased temperature of both bone and drill bits and production of dense bone swarf. Orthopedic surgeon should be aware of these technical difficulties encountered during operative interventions.

Severely impaired terminal erythroid differentiation as an independent prognostic marker in myelodysplastic syndromes

AbstractAnemia is the defining feature in most patients with myelodysplastic syndromes (MDS), yet defects in erythropoiesis have not been well characterized. We examined freshly obtained bone marrow (BM) samples for stage-specific abnormalities during terminal erythroid differentiation (TED) from 221 samples (MDS, n = 205 from 113 unique patients; normal, n = 16) by measuring the surface expression of glycophorin A, band 3, and integrin α-4. Clinical and biologic associations were sought with presence or absence of TED and the specific stage of erythroid arrest. In 27% of MDS samples (56/205), there was no quantifiable TED documented by surface expression of integrin α-4 and band 3 by terminally differentiating erythroblasts. Absence of quantifiable TED was associated with a significantly worse overall survival (56 vs 103 months, P = .0001) and SRSF2 mutations (7/23, P < .05). In a multivariable Cox proportional hazards regression analysis, absence of TED remained independently significant across International Prognostic Scoring System-Revised (IPSS-R) categories, myeloid/erythroid ratio, and mutations in several genes. In 149/205 MDS samples, the proportion of cells undergoing TED did not follow the expected 1:2:4:8:16 doubling pattern in successive stages. Absence of TED emerged as a powerful independent prognostic marker of poor overall survival across all IPSS-R categories in MDS, and SRSF2 mutations were more frequently associated with absence of TED.

The chromatin remodeling subunit Baf200 promotes normal hematopoiesis and inhibits leukemogenesis

BackgroundAdenosine triphosphate (ATP)-dependent chromatin remodeling SWI/SNF-like BAF and PBAF complexes have been implicated in the regulation of stem cell function and cancers. Several subunits of BAF or PBAF, including BRG1, BAF53a, BAF45a, BAF180, and BAF250a, are known to be involved in hematopoiesis. Baf200, a subunit of PBAF complex, plays a pivotal role in heart morphogenesis and coronary artery angiogenesis. However, little is known on the importance of Baf200 in normal and malignant hematopoiesis.MethodsUtilizing Tie2-Cre-, Vav-iCre-, and Mx1-Cre-mediated Baf200 gene deletion combined with fetal liver/bone marrow transplantation, we investigated the function of Baf200 in fetal and adult hematopoiesis. In addition, a mouse model of MLL-AF9-driven leukemogenesis was used to study the role of Baf200 in malignant hematopoiesis. We also explored the potential mechanism by using RNA-seq, RT-qPCR, cell cycle, and apoptosis assays.ResultsTie2-Cre-mediated loss of Baf200 causes perinatal death due to defective erythropoiesis and impaired hematopoietic stem cell expansion in the fetal liver. Vav-iCre-mediated loss of Baf200 causes only mild anemia and enhanced extramedullary hematopoiesis. Fetal liver hematopoietic stem cells from Tie2-Cre+, Baf200f/f or Vav-iCre+, Baf200f/f embryos and bone marrow hematopoietic stem cells from Vav-iCre+, Baf200f/f mice exhibited impaired long-term reconstitution potential in vivo. A cell-autonomous requirement of Baf200 for hematopoietic stem cell function was confirmed utilizing the interferon-inducible Mx1-Cre mouse strain. Transcriptomes analysis revealed that expression of several erythropoiesis- and hematopoiesis-associated genes were regulated by Baf200. In addition, loss of Baf200 in a mouse model of MLL-AF9-driven leukemogenesis accelerates the tumor burden and shortens the host survival.ConclusionOur current studies uncover critical roles of Baf200 in both normal and malignant hematopoiesis and provide a potential therapeutic target for suppressing the progression of leukemia without interfering with normal hematopoiesis.

NFAT-mediated defects in erythropoiesis cause anemia in Il2-/- mice.

The role of NFAT family transcription factors in erythropoiesis is so far unknown, although their involvement has been suggested previously. We have shown recently that Il2-/- mice develop severe anemia due to defects in KLF1 activity during BM erythropoiesis. Although, KLF1 activity is indispensable for erythropoiesis, the molecular details of Klf1 expression have not yet been elucidated. Here we show that an enhanced NFATc1 activity induced by increased integrin-cAMP signaling plays a critical role in the dysregulation of Klf1 expression and thereby cause anemia in Il2-/- mice. Interestingly, enhanced NFATc1 activity augmented apoptosis of immature erythrocytes in Il2-/- mice. On the other hand, ablation of NFATc1 activity enhanced differentiation of Ter119+ cells in BM. Restoring IL-2 signaling in Il2-/- mice reversed the increase in cAMP-NFAT signaling and facilitated normal erythropoiesis. Altogether, our study identified an NFAT-mediated negative signaling axis, manipulation of which could facilitate erythropoiesis and prevent anemia development.

Isolation and Functional Examination of the Long Non-Coding RNA Redrum.

Here, we report isolation of multiple long non-coding RNAs (lncRNAs) expressed tissue-specifically during murine embryogenesis. One of these, subsequently came to be known as Redrum, is expressed in erythropoietic cells in fetal liver and adult bone marrow. Redrum transcription is also detected during pregnancy in the spleen where extramedullary hematopoiesis takes place. In order to examine the function of Redrum in vivo, we generated a gene-targeted murine model and analyzed its embryonic and adult erythropoiesis. The homozygous mutant embryo showed no apparent deficiency or defect in erythropoiesis. Adult erythropoiesis in bone marrow and in the spleen during pregnancy likewise showed no detectable phenotype as red blood cells matured in normal fashion. The phenotype is in contrast to the reported function of Redrum in vitro, and our observation implies that Redrum plays in vivo an accessory or supplementary role whose loss is compatible with normal erythropoiesis.

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

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. DisclosuresNo relevant conflicts of interest to declare.

NCOR1 modulates erythroid disorders caused by mutations of thyroid hormone receptor \u03b11

Thyroid hormone receptor α (THRA) gene mutations, via dominant negative mode, cause erythroid abnormalities in patients. Using mice expressing a dominant negative TRα1 mutant (TRα1PV; Thra1PV/+ mice), we showed that TRα1PV acted directly to suppress the expression of key erythroid genes, causing erythroid defects. The nuclear receptor corepressor 1 (NCOR1) was reported to mediate the dominant negative effects of mutated TRα1. However, how NCOR1 could regulate TRα1 mutants in erythroid defects in vivo is not known. In the present study, we crossed Thra1PV/+ mice with mice expressing a mutant Ncor1 allele (NCOR1ΔID; Ncor1ΔID mice). TRα1PV mutant cannot bind to NCOR1ΔID. The expression of NCOR1ΔID ameliorated abnormalities in the peripheral blood indices, and corrected the defective differentiation potential of progenitors in the erythroid lineage. The defective terminal erythropoiesis of lineage-negative bone marrow cells of Thra1PV/+ mice was rescued by the expression of NCOR1ΔID. De-repression of key erythroid genes in Thra1PV/+Ncor1ΔID/ΔID mice led to partial rescue of terminal erythroid differentiation. These results indicate that the inability of TRα1PV to recruit NCOR1ΔID to form a repressor complex relieved the deleterious actions of TRα1 mutants in vivo. NCOR1 is a critical novel regulator underpining the pathogenesis of erythroid abnormalities caused by TRα1 mutants.

Defective erythropoiesis caused by mutations of the thyroid hormone receptor α gene.

Patients with mutations of the THRA gene exhibit classical features of hypothyroidism, including erythroid disorders. We previously created a mutant mouse expressing a mutated TRα1 (denoted as PV; Thra1PV/+ mouse) that faithfully reproduces the classical hypothyroidism seen in patients. Using Thra1PV/+ mice, we explored how the TRα1PV mutant acted to cause abnormalities in erythropoiesis. Thra1PV/+ mice exhibited abnormal red blood cell indices similarly as reported for patients. The total bone marrow cells and erythrocytic progenitors were markedly reduced in the bone marrow of Thra1PV/+ mice. In vitro terminal differentiation assays showed a significant reduction of mature erythrocytes in Thra1PV/+ mice. In wild-type mice, the clonogenic potential of progenitors in the erythrocytic lineage was stimulated by thyroid hormone (T3), suggesting that T3 could directly accelerate the differentiation of progenitors to mature erythrocytes. Analysis of gene expression profiles showed that the key regulator of erythropoiesis, the Gata-1 gene, and its regulated genes, such as the Klf1, β-globin, dematin genes, CAII, band3 and eALAS genes, involved in the maturation of erythrocytes, was decreased in the bone marrow cells of Thra1PV/+ mice. We further elucidated that the Gata-1 gene was a T3-directly regulated gene and that TRα1PV could impair erythropoiesis via repression of the Gata-1 gene and its regulated genes. These results provide new insights into how TRα1 mutants acted to cause erythroid abnormalities in patients with mutations of the THRA gene. Importantly, the Thra1PV/+ mouse could serve as a preclinical mouse model to identify novel molecular targets for treatment of erythroid disorders.

SMAD4 has a critical role in erythropoiesis and HSC formation during embryogenesis

Hematopoietic stem cells (HSC) are generated during embryogenesis from specialized vascular beds termed hemogenic endothelium (HE), which includes the aortic endothelium of the aortic-gonado-mesonephros (AGM) region. In the aorta, pre-HSCs are organized in clusters attached to the vascular endothelium. The TGF signaling pathway has a pivotal role in both arterial specification and HSC formation. Previous endothelial SMAD4 loss-of-function studies in mice using a Tie2cre have demonstrated a critical role of this protein in the endothelial to hematopoietic transition (EHT), with notable phenotypes of increased cluster formation and embryonic lethality at E10.5. In order to bypass the early lethality seen in other studies, we used an inducible arterial Cre to delete SMAD4 to evaluate the contribution of the TGF signaling pathway to later EHT events, as well as HSC migration, and colonization of adult hematopoietic organs. We noted embryonic lethality between E13.5-E17.5 in animals with SMAD4 loss of function in arterial endothelial cells. The mutants exhibited decreased HSC numbers, enlarged fetal livers, erythropoiesis defects, and the inability to engraft adult recipients. In addition, we evaluated the deletion of SMAD4 using NG2cre which is expressed within the sub-aortic mesenchyme during EHT. The mutant animals did not exhibit any hematopoietic abnormalities and survived until adult life. Our data suggest that the TGF signaling pathway in arterial endothelial cells is critical for definitive hematopoiesis; however other non-endothelial sources of TGF do not contribute to the endothelial to hematopoietic transition or definitive hematopoiesis.

Abstract 299: RNA editing enzyme ADAR1 accelerates normal hematopoiesis cell cycle by regulation microRNA biogenesis

Abstract Compelling murine studies demonstrate that adenosine-to-inosine (A-to-I) RNA editing mediated by adenosine deaminase associated with RNA1 (ADAR1) is vital for both fetal and adult hematopoiesis. While genetic ablation of ADAR1 leads to murine embryonic lethality due to severe defects in erythropoiesis, conditional deletion in the hematopoietic system impairs maintenance indicative of cell type and context specific roles for ADAR1 in cell fate specification and self-renewal. By regulating mRNA and microRNA (miRNA) stability, ADAR1 exhibit wide-ranging effects on embryonic development and stem cell regulation. We have previously shown that inflammation-responsive ADAR1 plays important roles in both stem cell differentiation and self-renewal in CML (chronic myeloid leukemia) disease progression. Here, we describe a novel function of ADAR1 in cell cycle regulation of normal hematopoietic stem cell and progenitors (HSPC) by regulation of miRNA biogenesis. Our results demonstrated that ADAR1 induces G0 to G1 phase transition in normal cord blood HSPCs, as demonstrated by elevated expression of Ki67, reduced DiR signal, and enhanced in vivo cord blood engraftment. Cell cycle qRT-qPCR microarray of 84 cell cycle transcripts and whole transcriptome RNA-sequencing analysis of KEGG cell cycle pathway indicate that several cell cycle genes are differentially expression upon overexpression of ADAR1 WT or an A-to-I editing deficient ADAR1 mutant (ADAR1E912A). We previously demonstrated that impaired biogenesis of let-7 miRNAs by ADAR1 WT induces enhanced self-renewal in cord blood CD34+ HSPCs. To determine the miRNA targets of ADAR1-mediated RNA editing, we performed miRNome miScript PCR array of 1008 miRNA candidates in cord blood CD34+ HSPCs overexpressing ADAR1 WT or ADAR1E912A. Total of 263 miRNAs were differentially expressed (142 upregulated and 121 downregulated) by comparing ADAR1 WT to the backbone control. Interestingly, ADAR1E912A mutant also exhibit A-to-I editing independent regulation of miRNAs (307 upregulated and 59 downregulated). We found that the expression of miR-26a-5p, a miRNA frequently downregulated in leukemia, is inhibited by ADAR1-mediated RNA editing. ADAR1 directly binds and edits the DROSHA cleavage site of primary miR-26a transcript, thereby prevent miR26a-5p maturation. Moreover, lentiviral expression of mature miR26-5p reverses the effect of ADAR1 WT, including enhanced CDKN1A expression, inhibition of cord blood proliferation in vivo, as well as reduced HSC self-renewal as measured by colony-formation assay. Our finding suggests carefully regulated A-to-I editing by ADAR1 is essential for the maintenance of proper cell proliferation in HSC. For future study, it will be interesting to investigate if the elevated expression of ADAR1 in CML BC LSC contributes to false regulation of cell cycle that leads to the expansion of malignant leukemia stem cells. Citation Format: Qingfei Jiang, Maria Anna Zipeto, Nathan Delos Santos, Sheldon Morris, Catriona Jamieson. RNA editing enzyme ADAR1 accelerates normal hematopoiesis cell cycle by regulation microRNA biogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 299. doi:10.1158/1538-7445.AM2017-299

Splicing factor SF3B1K700E mutant dysregulates erythroid differentiation via aberrant alternative splicing of transcription factor TAL1.

More than 60% of myeloid dysplasia syndrome (MDS) contains mutations in genes encoding for splicing factors such as SF3B1, U2AF, SRSF2 and ZRSR2. Mutations in SF3B1 are associated with 80% cases of refractory anemia with ring sideroblast (RARS), a subtype of MDS. SF3B1K700E is the most frequently mutated site among mutations on SF3B1. Yet the molecular mechanisms on how mutations of splicing factors lead to defective erythropoiesis are not clear. SF3B1K700E mutant binds to an RNA binding protein, RBM15, stronger than the wild type SF3B1 protein in co-immunoprecipitation assays. In addition, K700E mutant alters the RNA splicing of transcription factors TAL1 and GATA1. Via alternative RNA splicing, a novel short TAL1 transcript variant (TAL1s) is generated. Enhanced interaction between SF3B1 and RBM15 promotes the production of full-length TAL1 (TAL1fl) mRNA, while reduction of RBM15 protein level via PRMT1-mediated degradation pathway changes TAL1s/TAL1fl ratio in favor of TAL1s. TAL1s contains the helix-loop-helix DNA binding domain but not the N terminal region upstream of the DNA binding domain. The TAL1s protein loses its interaction with ETO2, which represses early erythropoiesis. In this vein, overexpression of TAL1s stimulates the transcription of β-hemoglobin in human leukemia K562 cells and promotes erythroid differentiation of human cord blood CD34+ cells cultured in erythropoietin-containing medium. Therefore, mutations of SF3B1 may block erythropoiesis via dysregulation of alternative RNA splicing of transcription factor TAL1, and targeting PRMT1 may alleviate the anemic symptoms in MDS patients.