Understanding the role of metabolome in Diamond-Blackfan Anemia erythropoiesis

Diamond-Blackfan anemia (DBA) is a ultra-rare inherited bone marrow failure syndrome characterized by severe hyporegenerative macrocytic anemia, congenital abnormalities, and an increased risk of malignancies. The disorder is mainly caused by heterozygous mutations in genes encoding ribosomal proteins, leading to defective ribosome biogenesis and impaired protein synthesis. This reduces proliferation, increases apoptosis, and promote defective differentiation of DBA erythroid progenitors. Despite significant advances in understanding the molecular basis of DBA, the mechanisms linking ribosomal defects to abnormal erythropoiesis remain poorly known, and therapeutic options are still limited to supportive treatments or hematopoietic stem cell transplantation. The aim of this study was to Identification of new therapeutic option(s) for DBA pathologic erythropoiesis. To address these questions, two complementary in vitro models of DBA were developed and characterized. The first model consisted of the erythroid progenitor cell line HUDEP-1 in which the ribosomal protein gene RPS26 was silenced using siRNA-mediated nucleofection. The second model involved primary hematopoietic progenitor cells isolated from peripheral blood or bone marrow of healthy donors and DBA patients. CD34⁺ progenitor cells were cultured under erythroid-promoting conditions to analyze erythroid commitment and differentiation. Flow cytometry was used to identify erythroid progenitor populations, including BFU-E, transitional progenitors, and CFU-E, based on the expression of specific surface markers. Additional analyses were performed to assess cell proliferation, apoptosis, and cell cycle progression. RPS26-silenced HUDEP-1 cells displayed reduced cell viability, increased apoptosis, and impaired erythroid differentiation compared to control cells, confirming the validity of this model in recapitulating key features of DBA erythropoiesis. In parallel, cultures of primary CD34⁺ progenitors derived from DBA patients showed decreased proliferation, reduced cell viability, and altered distribution of erythroid progenitor populations, characterized by an accumulation of early progenitors and impaired transition toward more mature stages. Based on these data, we carried out a metabolomic analysis of healthy and DBA progenitors. We found abnormalities of metabolic pathways associated with cellular energy production and redox balance, indicating a novel and key role of metabolic dysregulation in pathophysiology of DBA. Treatment with the pyruvate kinase activator Mitapivat was evaluated in erythroid cultures derived from DBA patient progenitors. Pharmacological activation of pyruvate kinase improved erythroid cell proliferation and survival and promoted a more efficient progression of erythroid differentiation in DBA patients carrying RPs mutations compared with untreated cultures. In conclusion, our study provides new insights into the interplay between ribosomal dysfunction, metabolic regulation, and erythroid differentiation in DBA. The results support the potential therapeutic relevance of metabolic reprogramming DBA erythroid progenitors by mitapivat as pyruvate kinases.