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

Husam Qanash,Jizhong Zou,Sara Young-Baird,Yongqin Li,Kaari Linask,Andre Larochelle,Richard H Smith,Keyvan Keyvanfar,John S Choy,Waleed Hakami

doi:10.3390/cells10040734

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.

Highlights

Diamond Blackfan Anemia (DBA) is a dominantly inherited bone marrow failure syndrome primarily characterized by defective erythropoiesis
To investigate whether EPAG can improve erythroid maturation in DBA, we first established an induced pluripotent stem cell (iPSC) disease model by Sendai vector-mediated overexpression of Oct4, Sox2, Klf4 and c-Myc in Mononuclear cells (MNCs) collected from three patients, each with a distinct point mutation within the RPS19 gene (Table 1)
We established genetically defined isogenic sibling iPSC clones to ensure that phenotypes observed in DBA iPSCs are driven by disease-specific mutations and do not result from genetic or epigenetic alterations introduced during reprogramming

Summary

Introduction

Diamond Blackfan Anemia (DBA) is a dominantly inherited bone marrow failure syndrome primarily characterized by defective erythropoiesis. In more than half of patients with DBA, heterozygous mutations have been identified in genes encoding ribosomal proteins (RP), resulting in RP haploinsufficiency and abnormal ribosome biogenesis or function [1,2]. RPS19 is the most commonly mutated gene in DBA, and monoallelic silencing of RPS19 results in aberrant assembly of the small 40S ribosomal subunits. In select subjects with DBA, hematopoietic stem and progenitor cell (HSPC) transplantation can offer a potential cure. Corticosteroids and long-term transfusion support remain the mainstay of treatment. Steroid toxicity precludes long-term use and acquired transfusional hemochromatosis may have serious clinical sequelae. Novel drug candidates with therapeutic potential in DBA, including the translation enhancer

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