Type: Oral
Session: 113. Hemoglobinopathies, Excluding Thalassemia—New Genetic Approaches to Sickle Cell Disease: New Insights Into Sickle Cell Disease Pathophysiology
Hematology Disease Topics & Pathways:
Anemias, sickle cell disease, Diseases, Hemoglobinopathies
In the present study, using both in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. We explored the mechanistic basis of the ineffective erythropoiesis in SCD using biochemical, cellular and imaging techniques.
In vitro erythroid differentiation using CD34+ cells isolated from SCD patients and from healthy donors was performed. A 2-phase erythroid differentiation protocol was used and cultures were performed at two different oxygen conditions, i.e. normoxia and partial hypoxia (5% O2). We found that hypoxia induces cell death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). This inference was supported by flow cytometry data showing higher percentages of dead cells within the non-F-cell population as compared to the F-cell population for SCD cells. Moreover, SCD dead cells showed higher levels of chaperon protein HSP70 in the cytoplasm than live cells, while no difference was detected between both subpopulations for control cells, suggesting that cell death of SCD erythroblasts was probably due to HSP70 cytoplasmic sequestration. This was supported by western-blot experiments showing less HSP70 in the nucleus of SCD erythroblasts under hypoxia, associated with decreased levels of GATA-1. At the molecular level, HSP70 was co-immunoprecipitated with HbS under hypoxia indicating that both proteins were in the same complex and suggesting interaction between HSP70 and HbS polymers in the cyotplasm. Importantly, we confirm these results in vivo by showing that in bone marrow of SCD patients (n = 5) cell loss occurs during terminal erythroid differentiation, with a significant drop in the cell count between the polychromatic and the orthochromatic stages (Figure 1).
In order to specifically address the role of HbF in cell survival, we used a CRISPR-Cas9 approach to mimic the effect of hereditary persistence of fetal hemoglobin (HPFH). CD34+ cells were transfected either with a gRNA targeting the LRF binding site (-197) or a gRNA targeting an unrelated locus (AAVS1) (Weber, Frati, et al. 2020). As expected, the disruption of the LRF binding site resulted in HbF induction as shown by higher %F-cells compared to AAVS1 control. These higher levels of F-cells resulted in decreased apoptosis, under both normoxic and hypoxic conditions, clearly demonstrating the positive and selective effect of HbF on SCD cell survival (Figure 2).
In summary, our study shows that HbF has a dual beneficial effect in SCD by conferring a preferential survival of F-cells in the circulation and by decreasing ineffective erythropoiesis. These findings thus bring new insights into the role of HbF in modulating clinical severity of anemia in SCD by both regulating red cell production and red cell destruction.
Disclosures: No relevant conflicts of interest to declare.
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