Apoptosis Of Erythroid Progenitors Research Articles

Increased BNIP3 activity contributes to mitochondrial dysregulation: A hypothesis on the mechanism of ineffective erythropoiesis in β-thalassemia

Ineffective erythropoiesis (IE) and the premature destruction of erythroblasts play important roles in the pathophysiology of β-thalassemia. Accumulating evidence suggests that the pathology of IE is associated with multiple factors, including apoptosis and reactive oxygen species (ROS) generation. A previous study also indicated that dysregulation of mitochondrial homeostasis and autophagy contributes to IE in the bone marrow of β-thalassemia patients. However, the precise molecular mechanism underlying mitochondria-mediated IE remains poorly understood. Here, we propose that the increased activity of adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) promotes mitochondrial fragmentation, autophagy, and apoptosis of erythroid progenitors in β-thalassemia. The present hypothesis is based on the published literature showing that 1) BNIP3 is involved in mitochondrial dysfunction and autophagy, 2) BNIP3 expression is regulated by p53 and hypoxia-inducible factor-1α, and 3) BNIP3 expression can be attenuated by a number of approaches. Our hypothesis can be verified through both in vitro and in vivo experiments. BNIP3 could be a promising therapeutic target for minimizing the pathophysiology of IE and reducing ROS generation associated with mitochondrial dysfunction in patients with β-thalassemia.

Genetic Iron Overload Aggravates and Pharmacological Iron Restriction Improves MDS Pathophysiology in a Preclinical Mouse Model

Background: Patients with myelodysplastic syndromes (MDS) are prone to develop iron overload as a consequence of ineffective erythropoiesis and chronic transfusion therapy. Although iron overload is a common feature in MDS, it remains unclear whether and how iron excess is detrimental for MDS pathophysiology. Aims: This study aimed at characterizing altered iron homeostasis in a preclinical MDS mouse model, understanding the molecular mechanisms underlying iron toxicity and unraveling the potential therapeutic value of pharmacologic iron restriction in this disease condition. Methods: To this end, we took advantage of complementary approaches and analyzed the effect of iron overload obtained through genetic activation of the iron exporter ferroportin (FPNC326S), and iron restriction achieved through the administration of the FPN inhibitor vamifeport in NUP98-HOXD13 MDS mice, starting at 3 or 5 months of age. Results: At steady-state MDS mice develop an iron overload phenotype, hallmarked by low hepcidin levels, elevated serum iron and transferrin saturation, non-transferrin-bound iron (NTBI) formation and tissue iron deposition. This is associated with anemia and low WBCs in the peripheral blood. FPNC326S MDS mice show an aggravated iron phenotype, with further elevated NTBI and tissue iron accumulation and still inappropriately low hepcidin levels. Vamifeport administration in MDS mice reduced serum iron (194.8 vs 141.8 mg/dl; P<0.05) and NTBI formation (0.62 vs 0 μM; P<0.05) and prevented tissue iron loading. While iron excess in FPNC326S MDS mice did not improve erythropoiesis and hematologic parameters in the peripheral blood, iron restriction by vamifeport significantly ameliorated anemia (Hb 9.5 vs 11.4 g/dl; RBC 5.4 vs 6.9x106 cells/ml; P<0.05) and maturation of bone marrow as well as splenic RBCs in MDS mice. The improved ineffective erythropoiesis was associated with reduced oxidative stress and apoptosis in erythroid progenitors. Importantly, we observed a major role of the iron status in myeloid expansion in MDS. The number of immature myeloid blasts and myeloid progenitors in the bone marrow of MDS mice were aggravated by iron overload in FPNC326S MDS mice and attenuated by iron restriction in vamifeport-treated MDS mice compared to control MDS animals. Myeloid bias, monitored as percentage bone marrow myeloid cells, was exacerbated by iron overload (54.3 vs 43.1% bone marrow mononuclear cells - BMNCs) and alleviated by iron restriction (67.5 vs 74.8% BMNCs; P<0.05). This was associated with increased and reduced inflammatory activation of bone marrow macrophages in FPNC326S MDS and vamifeport-treated MDS mice, respectively. This observation demonstrates a role for NTBI in driving the reported pro-inflammatory activation of MDS monocytes/macrophages (Fuster JJ et al, Science 2017; Jaiswal S et al, NEJM 2017; Cull AH et al, Exp Hematol 2017) and suggests that this iron-driven mechanism contributes to myeloid expansion. Overall, these alterations translated into a faster and delayed transition to AML in iron-overloaded and iron-restricted MDS mice, respectively. Finally, iron overload aggravated and iron restriction alleviated ROS formation, DNA damage and pyroptotic cell death in HSPCs. By reducing the intracellular labile iron pool, vamifeport improved HSPC quality and survival, partially rescuing the stem cell pool size (0.16 vs 0.38% Lin-BMNCs; P<0.05) in MDS mice. The ameliorated HSPC pool, reduced myeloid bias and inflammation, and attenuated ineffective erythropoiesis resulted in improved hematologic parameters for more than 3 months, and a significant survival increment in iron-restricted compared to control MDS mice (>45 days; P=0.037). We further demonstrate that a subgroup of mice can benefit from late iron restriction by vamifeport administration at 5 months of age, resulting in improved anemia and prolonged survival. Conclusion: Our results show for the first time in a preclinical mouse model of MDS that iron excess driven by ineffective erythropoiesis has pathological implication in transfusion-independent MDS. These effects are likely aggravated by transfusions causing additional iron overload. Finally, iron restriction achieved through pharmacologic FPN inhibition by the oral FPN inhibitor vamifeport significantly improves MDS pathophysiology, uncovering the therapeutic potential of early prevention of NTBI formation in MDS.

Malignancies and MDS in Patients with Diamond-Blackfan Anemia (DBA) from the Czech National DBA Registry

Introduction: Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by macrocytic anemia, reticulocytopenia, selective deficiency of erythroid precursors, presence of congenital anomalies and an increased risk of cancer. DBA is caused by germline mutations of genes coding for ribosomal proteins (RP). Interestingly, somatic RP mutations have also been found in several malignant diseases (T-ALL, CLL, Hodgkin lymphoma, myelodysplastic syndrome (MDS) and AML). The only representative overview of malignancies in DBA was published in 2012 based on the data from the North American DBA Registry. The malignancies developed in 3% of patients (MDS and AML in 4 patients and solid tumors in 15 patients). The observed-to-expected ratio for all cancers combined was 5.4 (p&lt;0.05). Median age at diagnosis was 41 years and cumulative incidence of solid tumors/leukemias was approximately 20% at the age of 46 years. Five years later, additional solid tumors were reported, in particular gastrointestinal tumors, and an incidence of MDS was alarming. Our aim was to evaluate cancer incidence within Czech National DBA Registry and characterize underlying molecular pathology. Patients and methods: Czech National DBA Registry currently includes 62 patients. In cooperation with 8 national centers of pediatric and adult hematology, we collected data about patients followed with pre-malignant or malignant condition including a detailed analysis of the course of the disease, treatment and type of developed malignant disorder. In patients with an evolving MDS, a classical cytogenetic analysis, flow cytometry, mutational profile (TruSight Myeloid Sequencing Panel (Illumina) containing 54 genes) and commercial TUNEL assay for detection of apoptotic changes of erythroid cells in the bone marrow were performed. Results: Eight of 62 patients from the Registry (13%) had malignant or pre-malignant condition: four females (6.5%) had solid tumors, 3 males (4.8%) had MDS and one female (1.6%) had multiple myeloma. Age of the onset of these disorders ranged between 25-70 years. Three patients harbored RPL5 mutation, 3 patients RPL11 mutation and 2 patients RPS19 mutation. Cancer incidence was significantly higher within the RPL11 and RPL5 subgroups (p=0.0056, Fisher Exact Test). Two female patients were diagnosed with triple-negative breast carcinoma, both of them during pregnancy. The first patient died despite treatment at the age of 29 years, shortly after delivery of her second child. The second patient is currently undergoing neoadjuvant chemotherapy.One patient developed diffuse large B cell lymphoma (DLBCL), underwent chemotherapy and autologous BMT and is alive in second remission.One patient underwent hemicolectomy for colorectal adenocarcinoma at 53 years of age and is in remission 6 years after the surgery.One patient succumbed to multiple myeloma which evolved from monoclonal gammopathy of unknown significance (MGUS) after 14 years of follow-up.Three male patients developed suspected MDS at the age of 25, 28 and 29 years. Two of them had RPL5 mutation and one harbored RPS19 mutation. None of them had decrease of erythroid cells in the bone marrow, but apoptosis of erythroid progenitors was significantly increased in all cases. All 3 patients had bicytopenia in peripheral blood (anemia, leukopenia) and dysplasia in 2 or 3 hematopoietic lineages in the bone marrow, thus fulfilling criteria for MDS with multilineage dysplasia (MDS-MLD). They had no abnormalities detected by flow cytometry or cytogenetics. The patient with RPS19mut harbored ASXL1 mutation; in patient with RPL5 mut, mutational screening is ongoing. Conclusion: Our results confirmed increased incidence of cancer (13%) in patients with DBA at young age. Our cases of DLBCL and MGUS in DBA are the first published to date. In cases with suspected MDS, cytopenia and dysplastic changes in the bone marrow may either reflect specific features of ribosomopathy, or represent a severe disorder of regulatory mechanisms with propensity to clonal proliferation. International collaboration is required to refine the incidence of malignancies in DBA and to issue consensus guidelines for timely detection of solid tumors, leukemias and MDS. The best approach to DBA patients who developed MDS without harboring any mutation considered prognostically significant in MDS, is another subject of discussion. Support: NV16-32105A Disclosures No relevant conflicts of interest to declare.

Reactivation of Telomerase in Late Generation Tert−/− Mice Results in Reversal of Anemia

AbstractAbstract 1258We previously reported that telomere shortening in a late generation telomerase K/O mouse model results in significantly reduced RBC and erythroblast (CD71+Ter119+) numbers in the peripheral blood and bone marrow, respectively. We also observed significant decreases in erythroid progenitor (Lin-cKit+Sca1-CD34-CD16/CD32-) and HSC (Lin-cKit+Sca1+ CD34-CD150+) numbers in G5 Tert−/− mice. We now report the mechanism underlying the specific defect in the erythroid lineage in these mice. First, there is a higher number of CD34-KLS CD150lo and CD150neg cells in the mutant mice relative to age matched G0 +/− mice. Methyl cellulose colony formation assay showed that CD34-KLS CD150hi cells give rise to a greater number of CFU-GEMM colonies while CD34-KLS CD150lo and CD34-KLS CD150neg cells both had a reduction in the numbers of GEMM colonies, suggesting that the loss of erythroid differentiation is associated with loss of CD150hi expression within the CD34-KLS population. Thus, a significant reduction in CD150hi HSC (p value < 0.001) and an increase in CD150lo (p value < 0.001) and CD150neg (p value < 0.04) HSC in G5 Tert−/− mice underlies the decreased number of erythroblasts in these mice. Secondly, we found that the reduction in telomere length as measured by quantitative PCR is greater in sorted erythroblasts (CD71+Ter119+) than in similarly purified myeloid progenitors (Gr1+Mac1+) in G5 Tert −/− mice. This finding correlates with the presence of increased DNA damage and apoptosis in erythroid progenitors and erythroblasts as compared to myeloid progenitors. In order to establish a cause and effect relationship between telomere shortening and defective erythropoiesis, we reactivated telomerase activity using a tamoxifen-inducible Cre recombinase to excise a Lox-Stop-Lox cassette located in intron 2 of telomerase gene in G5 Tert−/− mice. Reactivation of telomerase activity resulted in increased CD150hi HSC, erythroid progenitors and erythroblasts numbers in the bone marrow of these mice 3 months after tamoxifen treatment. In addition, RBC numbers and Hb levels in the peripheral blood improved in the treated mice, whereas neither improved in control G5 Tert−/− mice treated with sunflower oil. These data show for the first time a direct link between the loss of telomerase function, telomere shortening and a selective defect in erythropoiesis. These results, in conjunction with single-cell “mass cytometry” experiments underway to define the intracellular signaling pathways that are affected in the HSC and erythroid precursors of G5 Tert−/− mice, demonstrate the lineage-specific effects of telomere shortening on erythropoiesis and help to elucidate the relationship between shortened telomeres and the anemia of bone marrow failure syndromes. Disclosures:No relevant conflicts of interest to declare.

Anemia Management in Chronic Kidney Disease: Bursting the Hemoglobin Bubble

In this issue, Palmer and colleagues' meta-analysis confirms earlier findings of harms associated with ESAs in patients with CKD. Remaining questions include whether there is a lower hemoglobin lim...

Recombinant Human Erythropoietin Administration Decreases the Frequency of Excessive Apoptosis in Erythroid Progenitors in the Model Mouse of Anemia Due to Chronic Renal Failure.

Recombinant human erythropoietin (rh-EPO) administration results in a dramatic improvement of anemia due to chronic renal failure (ACRF). We reported that excessive apoptosis in erythroid progenitors was observed in ACRF patients before rh-EPO administration, and that we have established the model mouse of ACRF by administration of adenine-rich diets (ADRD) in ASH meetings 2001 and 2006. In this study, we evaluated the frequency of apoptosis in bone marrow erythroid cells of the model mouse before and after erythropoietin (EPO) administration, to confirm the hypothesis that excessive apoptosis in erythroid progenitors induced anemia via insufficient production of EPO. ADRD was administrated to C57B6 male mice for 8 weeks. Every day subcutaneous administration of EPO started at 6 weeks and continued for 2 weeks. Serum BUN and Crt levels began to elevate from 2 weeks after ADRD administration (Table1). Hb levels began to decrease from 5 weeks after ADRD administration and increased after EPO administration in a dose dependent manner (Table1, Figure1). The frequencies of apoptosis in erythroid progenitors and mature erythroblasts were evaluated by three color flow cytometric analysis using anti-CD34-antibody, anti-EPO-receptor (EPO-R)-antibody and AnnexinV. Erythroid progenitors were identified as CD34(+) and EPO-R(+) cells, and mature erythroblasts identified as CD34(−) and EPO-R(+) cells. Much higher frequency of apoptosis was observed in erythroid progenitors at 6 weeks after ADRD administration (76.4±5.3%, n=4), while, the frequency of apoptosis decreased dramatically after EPO administration in a dose dependent manner (0U/kg/day: 70.1±6.9% at 7 weeks, 73.8±8.7% at 8 weeks, 10U/kg/day: 48.1±5.4%* at 7 weeks, 46.9±4.2%* at 8 weeks, 100U/kg/day: 36.9±2.5%* at 7 weeks, 21.2±6.8%* at 8 weeks, 1000U/kg/day: 18.4±2.9%* at 7 weeks, 15.1±3.5%* at 8 weeks, each n=4, *p<0.01) (Figure2). The frequency of apoptosis was not increased in mature erythroblasts at each week after ADRD administration. Our findings suggested that excessive apoptosis occurred mainly in CD34(+) erythroid progenitors by insufficient production of EPO, and that EPO had the anti-apoptotic effect to erythroid progenitors. Insufficient production of EPO might be one of the important causes of ACRF.Table 1BUN, Crt and Hb levels in C57B6 mice after ADRD administration.0 week2 weeks4 weeks5 weeks6 weeks7 weeks8 weeksBUN (mg/dl)40.1±4.6073.2±14.4150.7±11.6148.8±4.60164.0±8.40161.8±16.4193.0±9.10Crt (mg/dl)0.06±0.040.30±0.030.51±0.070.52±0.090.68±0.090.65±0.040.80±0.17Hb (g/dl)13.6±0.413.3±0.313.2±0.211.1±0.110.2±0.59.6±0.18.3±0.5 [Display omitted] [Display omitted]

Erythroid-Specific Apoptosis Induced by Jun Transcription Factors Employs a Novel Pathway Involving Recruitment of COP1 and Itch Ubiquitin Ligases.

Upregulation of the transcription factor c-Jun has been identified in acute myeloid leukemia, chronic myelogenous leukemia, myelodysplastic syndromes, and in iron deficiency. These conditions all share as a common feature an impairment of erythropoiesis, in some instances manifested by apoptosis of erythroid progenitors. Depending on cellular context, c-Jun may promote either survival or apoptosis, the latter occurring primarily through regulation of target genes such as Bim and FasL. Transcriptional activation by its kinase JNK typically enhances the apoptotic effects of c-Jun. In primary human erythroid progenitors, we have identified a novel Jun-mediated apoptotic pathway that does not depend on transcriptional activation and is associated with retrograde inhibition of JNK signaling. To determine the nature of this pathway, we have employed a system of retroviral transduction of human CD34+ peripheral blood mobilized progenitors, which undergo culture in serum free erythroid medium containing erythroipoietin and stem cell factor. Wild type c-Jun and JunB, as well as c-Jun mutants lacking transactivation and DNA binding functions, all efficiently induced caspase-dependent apoptosis in early erythroid precursors within the CD36-positive GPA-dim compartment. By contrast, transduced cells undergoing megakaryocytic and myeloid differtentiation, as indicated by CD41 and CD13 expression, showed minimal apoptosis regardless of construct. Deletion or replacement of the c-Jun leucine zipper (LZ) dimerization domain completely eliminated the induction of erythroid apoptosis. Introduction of an LZ point mutation E281K, which alters dimerization specificity, also eliminated induction of apoptosis. These results demonstrated the dispensibility of direct target gene regulation but indicated an absolute requirement for specific LZ protein-protein interactions. Because JunD showed relatively inefficient apoptotic induction, we considered a role for recruitment of ubiquitin ligases such as Itch, which selectively binds c-Jun and JunB. Indeed, the c-Jun Y170F mutation, which ablates the PPXY binding site for Itch (and possibly other WW-domain ubiquitin ligases), completely abrogated apoptosis. Surprisingly, the c-Jun mutant VP232/233AA, which eliminates recruitment of the COP1 RING finger ubiquitin ligase, also manifested major defects in apoptosis induction. Biochemical analysis of the transduced cells showed that induction of apoptosis positively correlated with repression of JNK phosphorylation. Wild type c-Jun, JunB, and mutants that retained induction of apoptosis blocked JNK phosphorylation. All mutants tested that were defective in apoptosis induction caused an enhancement of JNK phosphorylation. We therefore postulate that this novel pathway of erythroid apoptosis results from c-Jun nucleation of a protein complex involving an LZ partner and multiple ubquitin ligases. Assembly of this complex then leads to blockade of JNK phosphorylation and induction of apoptosis.

Recent insights into the mechanisms of myeloid leukemogenesis in Down syndrome

AbstractGATA-1 is the founding member of a transcription factor family that regulates growth and maturation of a diverse set of tissues. GATA-1 is expressed primarily in hematopoietic cells and is essential for proper development of erythroid cells, megakaryocytes, eosinophils, and mast cells. Although loss of GATA-1 leads to differentiation arrest and apoptosis of erythroid progenitors, absence of GATA-1 promotes accumulation of immature megakaryocytes. Recently, we and others have reported that mutagenesis of GATA1 is an early event in Down syndrome (DS) leukemogenesis. Acquired mutations in GATA1 were detected in the vast majority of patients with acute megakaryoblastic leukemia (DS-AMKL) and in nearly every patient with transient myeloproliferative disorder (TMD), a “preleukemia” that may be present in as many as 10% of infants with DS. Although the precise pathway by which mutagenesis of GATA1 contributes to leukemia is unknown, these findings confirm that GATA1 plays an important role in both normal and malignant hematopoiesis. Future studies to define the mechanism that results in the high frequency of GATA1 mutations in DS and the role of altered GATA1 in TMD and DS-AMKL will shed light on the multistep pathway in human leukemia and may lead to an increased understanding of why children with DS are markedly predisposed to leukemia.

HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration.

Erythropoietin (Epo) is upregulated by hypoxia and provides protection against apoptosis of erythroid progenitors in bone marrow and brain neurons. Here we show in the adult mouse retina that acute hypoxia dose-dependently stimulates expression of Epo, fibroblast growth factor 2 and vascular endothelial growth factor via hypoxia-inducible factor-1alpha (HIF-1alpha) stabilization. Hypoxic preconditioning protects retinal morphology and function against light-induced apoptosis by interfering with caspase-1 activation, a downstream event in the intracellular death cascade. In contrast, induction of activator protein-1, an early event in the light-stressed retina, is not affected by hypoxia. The Epo receptor required for Epo signaling localizes to photoreceptor cells. The protective effect of hypoxic preconditioning is mimicked by systemically applied Epo that crosses the blood retina barrier and prevents apoptosis even when given therapeutically after light insult. Application of Epo may, through the inhibition of apoptosis, be beneficial for the treatment of different forms of retinal disease.

Apoptosis in erythroid progenitors deprived of erythropoietin occurs during the G1 and S phases of the cell cycle without growth arrest or stabilization of wild-type p53.

Erythropoietin (Epo) inhibits apoptosis in murine proerythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells). We have shown that the apoptotic process in FVA cell populations deprived of Epo is asynchronous as a result of a heterogeneity in Epo dependence among individual cells. Here we investigated whether apoptosis in FVA cells correlated with cell cycle phase or stabilization of p53 tumor suppressor protein. DNA analysis in nonapoptotic FVA cell subpopulations cultured without Epo demonstrated little change in the percentages of cells in G1,S, and G2/M phases over time. Analysis of the apoptotic subpopulation revealed high percentages of cells in G1 and S, with few cells in G2/M at any time. When cells were sorted from G1 and S phases prior to culture without Epo, apoptotic cells appeared at the same rate in both populations, indicating that no prior commitment step had occurred in either G1 or S phase. Steady-state wild-type p53 protein levels were very low in FVA cells compared with control cell lines and did not accumulate in Epo-deprived cultures; however, p53 protein did accumulate when FVA cells were treated with the DNA-damaging agent actinomycin D. These data indicate that erythroblast apoptosis caused by Epo deprivation (i) occurs throughout G1 and S phases and does not require cell cycle arrest, (ii) does not have a commitment event related to cell cycle phase, and (iii) is not associated with conformational changes or stabilization of wild-type p53 protein.

Apoptosis in erythroid progenitors deprived of erythropoietin occurs during the G1 and S phases of the cell cycle without growth arrest or stabilization of wild-type p53.

Erythropoietin (Epo) inhibits apoptosis in murine proerythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells). We have shown that the apoptotic process in FVA cell populations deprived of Epo is asynchronous as a result of a heterogeneity in Epo dependence among individual cells. Here we investigated whether apoptosis in FVA cells correlated with cell cycle phase or stabilization of p53 tumor suppressor protein. DNA analysis in nonapoptotic FVA cell subpopulations cultured without Epo demonstrated little change in the percentages of cells in G1,S, and G2/M phases over time. Analysis of the apoptotic subpopulation revealed high percentages of cells in G1 and S, with few cells in G2/M at any time. When cells were sorted from G1 and S phases prior to culture without Epo, apoptotic cells appeared at the same rate in both populations, indicating that no prior commitment step had occurred in either G1 or S phase. Steady-state wild-type p53 protein levels were very low in FVA cells compared with control cell lines and did not accumulate in Epo-deprived cultures; however, p53 protein did accumulate when FVA cells were treated with the DNA-damaging agent actinomycin D. These data indicate that erythroblast apoptosis caused by Epo deprivation (i) occurs throughout G1 and S phases and does not require cell cycle arrest, (ii) does not have a commitment event related to cell cycle phase, and (iii) is not associated with conformational changes or stabilization of wild-type p53 protein.