Eltrombopag Improves Erythroid Differentiation in a Human Induced Pluripotent Stem Cell Model of Diamond Blackfan Anemia

Abstract

Diamond Blackfan Anemia (DBA) is a congenital macrocytic anemia associated with ribosomal protein haploinsufficiency. Ribosomal dysfunction delays globin synthesis, resulting in excess toxic free heme in erythroid progenitors, early differentiation arrest, and pure red cell aplasia. In this study, DBA induced pluripotent stem cell (iPSC) lines were generated from blood mononuclear cells of DBA patients with inactivating mutations in RPS19 and subjected to hematopoietic differentiation to model disease phenotypes. In vitro differentiated hematopoietic cells were used to investigate whether eltrombopag, an FDA-approved mimetic of thrombopoietin with robust intracellular iron chelating properties, could rescue erythropoiesis in DBA by restricting the labile iron pool (LIP) derived from excessive free heme. DBA iPSCs exhibited RPS19 haploinsufficiency, reduction in the 40S/60S ribosomal subunit ratio and early erythroid differentiation arrest in the absence of eltrombopag, compared to control isogenic iPSCs established by CRISPR/Cas9-mediated correction of the RPS19 point mutation. Notably, differentiation of DBA iPSCs in the presence of eltrombopag markedly improved erythroid maturation. Consistent with a molecular mechanism based on intracellular iron chelation, we observed that deferasirox, a clinically licensed iron chelator able to permeate into cells, also enhanced erythropoiesis in our DBA iPSC model. In contrast, erythroid maturation did not improve substantially in DBA iPSC differentiation cultures supplemented with deferoxamine, a clinically available iron chelator that poorly accesses LIP within cellular compartments. These findings identify eltrombopag as a promising new therapeutic to improve anemia in DBA.

Induced Pluripotent Stem Cells as a Tool for Modeling Hematologic Disorders and as a Potential Source for Cell-Based Therapies

“…Another group also used a DBA-iPSC model and identified a new therapy, namely, eltrombopag (EPAG), a Food and Drug Administration (FDA)-approved synthetic small-molecule mimetic of TPO. EPAG partially rescued erythropoiesis by mediating intracellular iron restriction in DBA-iPSC-derived erythroid cells, which was in common with the effects of deferasirox (DFX), a clinically licensed iron chelator, on DBA-iPSCs [156].…”

Human cellular model systems of β-thalassemia enable in-depth analysis of disease phenotype

“…The copyright holder for this this version posted September 2, 2022. ; https://doi.org/10.1101/2022.09.01.506225 doi: bioRxiv preprint keeping, catalytic activity of erythroid specific ALAS2, the first enzyme in the heme synthesis pathway, can be modulated by small molecules binding a hotspot around its autoinhibitory Ct-extension 52 . Alternatively, use of succinylacetone, an inhibitor of the second step of heme synthesis, and the iron chelator Eltrombopag have shown promise in alleviating disease phenotype in Diamond Blackfan Anemia (DBA) erythroid cells in vitro 53,54 , further supporting the potential benefits of an iron reduction approach to treating β-thalassemia.…”

Long‐term eltrombopag for bone marrow failure depletes iron

“…This disconnect remains a barrier to conclusively demonstrating a causative relationship between iron chelation and hematopoietic responses. Data from prospective trials are limited, dosing and schedule of chelation varies, and DFO, historically the most prevalent chelator, enters cells much less efficiently than EPAG 23,24 …”