Exploring the bone marrow micro environment in thalassemia patients: potential therapeutic alternatives.

Genetic mutations in the ß-globin gene lead to a decrease or removal of the ß-globin chain, causing the build-up of unstable alpha-hemoglobin. This condition is referred to as beta-thalassemia (BT). The present treatment strategies primarily target the correction of defective erythropoiesis, with a particular emphasis on gene therapy and hematopoietic stem cell transplantation. However, the presence of inefficient erythropoiesis in BT bone marrow (BM) is likely to disturb the previously functioning BM microenvironment. This includes accumulation of various macromolecules, damage to hematopoietic function, destruction of bone cell production and damage to osteoblast(OBs), and so on. In addition, the changes of BT BM microenvironment may have a certain correlation with the occurrence of hematological malignancies. Correction of the microenvironment can be achieved through treatments such as iron chelation, antioxidants, hypoglycemia, and biologics. Hence, This review describes damage in the BT BM microenvironment and some potential remedies.

Therapeutic Relevance of Inducing Autophagy in ß-Thalassemia.

The ß-thalassemias are inherited genetic disorders affecting the hematopoietic system. In ß-thalassemias, more than 350 mutations of the adult ß-globin gene cause the low or absent production of adult hemoglobin (HbA). A clinical parameter affecting the physiology of erythroid cells is the excess of free α-globin. Possible experimental strategies for a reduction in excess free α-globin chains in ß-thalassemia are CRISPR-Cas9-based genome editing of the ß-globin gene, forcing "de novo" HbA production and fetal hemoglobin (HbF) induction. In addition, a reduction in excess free α-globin chains in ß-thalassemia can be achieved by induction of the autophagic process. This process is regulated by the Unc-51-like kinase 1 (Ulk1) gene. The interplay with the PI3K/Akt/TOR pathway, with the activity of the α-globin stabilizing protein (AHSP) and the involvement of microRNAs in autophagy and Ulk1 gene expression, is presented and discussed in the context of identifying novel biomarkers and potential therapeutic targets for ß-thalassemia.

Updates on clinical and laboratory aspects of hereditary dyserythropoietic anemias.

Hereditary dyserythropoietic anemias, or congenital dyserythropoietic anemias (CDAs), are rare disorders disrupting normal erythroid lineage development, resulting in ineffective erythropoiesis and monolinear cytopenia. CDAs include three main types (I, II, III), transcription-factor-related forms, and syndromic forms. The widespread use of next-generation sequencing in the last decade has unveiled novel causative genes and unexpected genotype-phenotype correlations. The discovery of the genetic defects underlying the CDAs not only facilitates accurate diagnosis but also enhances understanding of CDA pathophysiology. Notable advancements include identifying a hepatic-specific role of the SEC23B loss-of-function in iron metabolism dysregulation in CDA II, deepening CDIN1 dysfunction during erythroid differentiation, and uncovering a recessive CDA III form associated with RACGAP1 variants. Current treatments primarily rely on supportive measures tailored to disease severity and clinical features. Comparative studies with pyruvate kinase deficiency have illuminated new therapeutic avenues by elucidating iron dyshomeostasis and dyserythropoiesis mechanisms. We herein discuss recent progress in diagnostic methodologies, novel gene discoveries, and enhanced comprehension of CDA pathogenesis and molecular genetics.

Nrf2 Plays a Key Role in Erythropoiesis during Aging.

Aging is characterized by increased oxidation and reduced efficiency of cytoprotective mechanisms. Nuclear factor erythroid-2-related factor (Nrf2) is a key transcription factor, controlling the expression of multiple antioxidant proteins. Here, we show that Nrf2-/- mice displayed an age-dependent anemia, due to the combined contributions of reduced red cell lifespan and ineffective erythropoiesis, suggesting a role of Nrf2 in erythroid biology during aging. Mechanistically, we found that the expression of antioxidants during aging is mediated by activation of Nrf2 function by peroxiredoxin-2. The absence of Nrf2 resulted in persistent oxidation and overactivation of adaptive systems such as the unfolded protein response (UPR) system and autophagy in Nrf2-/- mouse erythroblasts. As Nrf2 is involved in the expression of autophagy-related proteins such as autophagy-related protein (Atg) 4-5 and p62, we found impairment of late phase of autophagy in Nrf2-/- mouse erythroblasts. The overactivation of the UPR system and impaired autophagy drove apoptosis of Nrf2-/- mouse erythroblasts via caspase-3 activation. As a proof of concept for the role of oxidation, we treated Nrf2-/- mice with astaxanthin, an antioxidant, in the form of poly (lactic-co-glycolic acid) (PLGA)-loaded nanoparticles (ATS-NPs) to improve its bioavailability. ATS-NPs ameliorated the age-dependent anemia and decreased ineffective erythropoiesis in Nrf2-/- mice. In summary, we propose that Nrf2 plays a key role in limiting age-related oxidation, ensuring erythroid maturation and growth during aging.

The interactions between ineffective erythropoiesis and ferroptosis in ß-thalassemia.

In Guangxi, Hainan, and Fujian Province in southern China, ß-thalassemia is a frequent monogenic hereditary disorder that is primarily defined by hemolytic anemia brought on by inefficient erythropoiesis. It has been found that ineffective erythropoiesis in ß-thalassemia is closely associated with a high accumulation of Reactive oxygen species, a product of oxidative stress, in erythroid cells. During recent years, ferroptosis is an iron-dependent lipid peroxidation that involves abnormalities in lipid and iron metabolism as well as reactive oxygen species homeostasis. It is a recently identified kind of programmed cell death. ß-thalassemia patients experience increased iron release from reticuloendothelial cells and intestinal absorption of iron, ultimately resulting in iron overload. Additionally, the secretion of Hepcidin is inhibited in these patients. What counts is both ineffective erythropoiesis and ferroptosis in ß-thalassemia are intricately linked to the iron metabolism and Reactive oxygen species homeostasis. Consequently, to shed further light on the pathophysiology of ß-thalassemia and propose fresh ideas for its therapy, this paper reviews ferroptosis, ineffective erythropoiesis, and the way they interact.

Luspatercept for the treatment of congenital sideroblastic anemia: Two case reports.

Congenital sideroblastic anemia (CSA) is a group of disorders caused by different genetic mutations that result in low iron utilization and ineffective erythropoiesis. Current treatments are limited, and some patients do not respond to vitamin B6 therapy. Luspatercept is a novel erythropoietic maturation agent approved for adult ß-thalassemia and Myelodysplastic syndromes with ring sideroblasts (MDS-RS) associated with ineffective erythropoiesis. Here we report 2 patients with CSA due to mutations in ALAS2 and SLC25A38 genes who became unresponsive after a period of treatment with vitamin B6 and iron chelators but achieved transfusion independence and a markedly reduced spleen after combination with luspatercept.

Iron chelation improves ineffective erythropoiesis and iron overload in myelodysplastic syndrome mice.

Myelodysplastic syndrome (MDS) is a heterogeneous group of bone marrow stem cell disorders characterized by ineffective hematopoiesis and cytopenias, most commonly anemia. Red cell transfusion therapy for anemia in MDS results in iron overload, correlating with reduced overall survival. Whether the treatment of iron overload benefits MDS patients remains controversial. We evaluate underlying iron-related pathophysiology and the effect of iron chelation using deferiprone on erythropoiesis in NUP98-HOXD13 transgenic mice, a highly penetrant well-established MDS mouse model. Our results characterize an iron overload phenotype with aberrant erythropoiesis in these mice which was reversed by deferiprone-treatment. Serum erythropoietin levels decreased while erythroblast erythropoietin receptor expression increased in deferiprone-treated MDS mice. We demonstrate, for the first time, normalized expression of the iron chaperones Pcbp1 and Ncoa4 and increased ferritin stores in late-stage erythroblasts from deferiprone-treated MDS mice, evidence of aberrant iron trafficking in MDS erythroblasts. Importantly, erythroblast ferritin is increased in response to deferiprone, correlating with decreased erythroblast ROS. Finally, we confirmed increased expression of genes involved in iron uptake, sensing, and trafficking in stem and progenitor cells from MDS patients. Taken together, our findings provide evidence that erythroblast-specific iron metabolism is a novel potential therapeutic target to reverse ineffective erythropoiesis in MDS.

Up-regulation of microRNA 101-3p during erythropoiesis in ß-thalassemia/HbE.

Ineffective erythropoiesis is the main cause of anemia in ß-thalassemia. The crucial hallmark of ineffective erythropoiesis is the high proliferation of erythroblast. microRNA (miR/miRNA) involves several biological processes, including cell proliferation and erythropoiesis. miR-101 was widely studied and associated with proliferation in several types of cancer. However, the miR-101-3p has not been studied in ß-thalassemia/HbE. Therefore, this study aims to investigate the expression of miR-101-3p during erythropoiesis in ß-thalassemia/HbE. The results showed that miR-101-3p was upregulated in the erythroblast of ß-thalassemia/HbE patients on day 7, indicating that miR-101-3p may be involved with high proliferation in ß-thalassemia/HbE. Therefore, the mRNA targets of miR-101-3p including Rac1, SUB1, TET2, and TRIM44 were investigated to determine the mechanisms involved with high proliferation of ß-thalassemia/HbE erythroblasts. Rac1 expression was significantly reduced at day 11 in severe ß-thalassemia/HbE compared to normal controls and mild ß-thalassemia/HbE. SUB1 gene expression was significantly lower in severe ß-thalassemia/HbE compared to normal controls at day 9 of culture. For TET2 and TRIM44 expression, a significant difference was not observed among normal and ß-thalassemia/HbE. However, the high expression of miR-101-3p at day 7 and these target genes was not correlated, suggesting that this miRNA may regulate ineffective erythropoiesis in ß-thalassemia/HbE via other target genes.

New Cases and Mutations in SEC23B Gene Causing Congenital Dyserythropoietic Anemia Type II.

Congenital dyserythropoietic anemia type II (CDA II) is an inherited autosomal recessive blood disorder which belongs to the wide group of ineffective erythropoiesis conditions. It is characterized by mild to severe normocytic anemia, jaundice, and splenomegaly owing to the hemolytic component. This often leads to liver iron overload and gallstones. CDA II is caused by biallelic mutations in the SEC23B gene. In this study, we report 9 new CDA II cases and identify 16 pathogenic variants, 6 of which are novel. The newly reported variants in SEC23B include three missenses (p.Thr445Arg, p.Tyr579Cys, and p.Arg701His), one frameshift (p.Asp693GlyfsTer2), and two splicing variants (c.1512-2A>G, and the complex intronic variant c.1512-3delinsTT linked to c.1512-16_1512-7delACTCTGGAAT in the same allele). Computational analyses of the missense variants indicated a loss of key residue interactions within the beta sheet and the helical and gelsolin domains, respectively. Analysis of SEC23B protein levels done in patient-derived lymphoblastoid cell lines (LCLs) showed a significant decrease in SEC23B protein expression, in the absence of SEC23A compensation. Reduced SEC23B mRNA expression was only detected in two probands carrying nonsense and frameshift variants; the remaining patients showed either higher gene expression levels or no expression changes at all. The skipping of exons 13 and 14 in the newly reported complex variant c.1512-3delinsTT/c.1512-16_1512-7delACTCTGGAAT results in a shorter protein isoform, as assessed by RT-PCR followed by Sanger sequencing. In this work, we summarize a comprehensive spectrum of SEC23B variants, describe nine new CDA II cases accounting for six previously unreported variants, and discuss innovative therapeutic approaches for CDA II.

Profile of Luspatercept in the Treatment of Anemia in Adults with Non-Transfusion-Dependent ß-Thalassemia (NTDT): Design, Development and Potential Place in Therapy.

Over the past decade, evidence has been mounting on the detrimental clinical sequelae of untreated anemia in patients with non-transfusion-dependent ß-thalassemia (NTDT). There are no pharmacologic agents that are specifically approved for the management of anemia in NTDT, and available options such as splenectomy, transfusion therapy, and hydroxyurea each come with their own shortcomings, especially for long-term use. Luspatercept is an erythroid maturation agent that has been evaluated in a Phase 2, randomized trial and showed a significant benefit in raising hemoglobin level by at least 1 g/dL in adults with NTDT and a baseline hemoglobin level ≤10 g/dL. These data led to luspatercept's approval by the European Commission for the treatment of anemia in adults with NTDT and presents the first evidence-based approach for a novel agent that is able to ameliorate anemia in this patient population.

Treating Thalassemia Patients with Luspatercept: An Expert Opinion Based on Current Evidence.

Luspatercept has recently been approved for the treatment of beta-thalassemia and its use in clinical practice has been increasing. As it is the first erythroid maturation drug available for this diagnosis, the expertise about its use is still limited. To address this point, and to promote awareness and guide the clinical use of luspatercept in beta-thalassemia, this paper was developed as a consensus by experts from the Italian Society of Thalassemia and Hemoglobinopathies (SITE). After a brief presentation of the core features of luspatercept, a comprehensive set of questions is addressed, covering relevant aspects for the practical management of this new therapeutic option.

A deep dive into future therapies for microcytic anemias and clinical considerations.

INTRODUCTION: Microcytic anemias (MA) have frequent or rare etiologies. New discoveries in understanding and treatment of microcytic anemias need to be reviewed. AREAS COVERED: Microcytic anemias with a focus on the most frequent causes and on monogenic diseases that are relevant for understanding biocellular mechanisms of MA. All treatments except gene therapy, with a focus on recent advances. PubMed search with references selected by expert opinion. EXPERT OPINION: As the genetic and cellular backgrounds of dyserythropoiesis will continue to be clarified, collaboration with bioengineering of treatments acting specifically at the protein domain level will continue to provide new therapies in hematology as well as oncology and neurology.

Single-cell profiling of ineffective erythropoiesis in a mouse model of ß-thalassaemia intermedia.

Beta-thalassaemia is an inherited haemoglobin disorder characterised by ineffective erythropoiesis (IE). The detailed pathogenesis of IE remains unclear. In this study, we used single-cell RNA sequencing (scRNA-seq) to examine IE in Th3/+ ß-thalassaemic mice. The results showed that the erythroid group was remarkably expanded, and genes involved in biological processes such as iron metabolism, haeme synthesis, protein folding, and response to heat were significantly upregulated from erythroid progenitors to reticulocytes in ß-thalassaemic mice. In particular, we identified a unique cell population close to reticulocytes, named ThReticulocytes, characterised by a high level of heat shock protein 70 (Hsp70) expression and dysregulation of iron metabolism and haeme synthesis signalling. Treatment of ß-thalassaemic mice with the haeme oxygenase inhibitor tin-mesoporphyrin effectively improved the iron disorder and IE, and the ThReticulocyte population and Hsp70 expression were significantly suppressed. This study revealed in detail the progression of IE at the single-cell level and possibly provided clues to find therapeutic targets in thalassaemia.

Pathogenic Mechanisms in Thalassemia I: Ineffective Erythropoiesis and Hypercoagulability.

Erythropoiesis is the physiological process that results in the production of red blood cells (RBCs). In conditions of pathologically altered erythropoiesis or ineffective erythropoiesis, as in the case of ß-thalassemia, the reduced ability of erythrocytes to differentiate, survive and deliver oxygen stimulates a state of stress that leads to the ineffective production of RBCs. We herein describe the main features of erythropoiesis and its regulation in addition to the mechanisms behind ineffective erythropoiesis development in ß-thalassemia. Finally, we review the pathophysiology of hypercoagulability and vascular disease development in ß-thalassemia and the currently available prevention and treatment modalities.

Pathogenic Mechanisms in Thalassemia II: Iron Overload.

Iron overload remains a lethal complication of ß-thalassemia and other anemias caused by ineffective erythropoiesis. This review discusses the pathogenetic mechanisms of iron overload in thalassemia, at organismal, cellular, and molecular levels.

Clinical Complications and Their Management.

The diversity of disease-related complications among patients with ß-thalassemia is complicated by the wide spectrum of genotypes and clinical risk factors. The authors herein present the different complications seen in patients with ß-thalassemia, the pathophysiology underlying these complications and their management.

TMPRSS6 as a Therapeutic Target for Disorders of Erythropoiesis and Iron Homeostasis.

TMPRSS6 is a serine protease highly expressed in the liver. Its role in iron regulation was first reported in 2008 when mutations in TMPRSS6 were shown to be the cause of iron-refractory iron deficiency anemia (IRIDA) in humans and in mouse models. TMPRSS6 functions as a negative regulator of the expression of the systemic iron-regulatory hormone hepcidin. Over the last decade and a half, growing understanding of TMPRSS6 biology and mechanism of action has enabled development of new therapeutic approaches for patients with diseases of erythropoiesis and iron homeostasis.ClinicalTrials.gov identifier NCT03165864.

Erythropoiesis and Malaria, a Multifaceted Interplay.

One of the major pathophysiologies of malaria is the development of anemia. Although hemolysis and splenic clearance are well described as causes of malarial anemia, abnormal erythropoiesis has been observed in malaria patients and may contribute significantly to anemia. The interaction between inadequate erythropoiesis and Plasmodium parasite infection, which partly occurs in the bone marrow, has been poorly investigated to date. However, recent findings may provide new insights. This review outlines clinical and experimental studies describing different aspects of ineffective erythropoiesis and dyserythropoiesis observed in malaria patients and in animal or in vitro models. We also highlight the various human and parasite factors leading to erythropoiesis disorders and discuss the impact that Plasmodium parasites may have on the suppression of erythropoiesis.