Abstract
Anaemia is a major global health problem arising from diverse causes and for which improved therapeutic strategies are needed. Erythroid cells can undergo apoptotic cell death and loss of pro-survival BCL-XL is known to trigger apoptosis during late-stage erythroid development. However, the mechanism by which loss or pharmacological blockade of BCL-XL leads to erythroid cell apoptosis remains unclear. Here we sought to identify the precise stage of erythropoiesis that depends on BCL-XL. We also tested whether deficiency of BIM or PUMA, the two main pro-apoptotic antagonists of BCL-XL, could prevent reticulocyte death and anaemia caused by BCL-XL loss. Using an in vivo mouse model of tamoxifen-inducible Bclx gene deletion and in vitro assays with a BCL-XL-selective inhibitor, we interrogated each stage of erythrocyte differentiation for BCL-XL dependency. This revealed that reticulocytes, but not orthochromatic erythroblasts, require BCL-XL for their survival. Surprisingly, concurrent loss of BIM or PUMA had no significant impact on the development of anemia following acute BCL-XL deletion in vivo. However, analysis of mixed bone marrow chimaeric mice revealed that loss of PUMA, but not loss of BIM, partially alleviated impaired erythropoiesis caused by BCL-XL deficiency. Insight into how the network of pro-survival and pro-apoptotic proteins works will assist the development of strategies to mitigate the effects of abnormal cell death during erythropoiesis and prevent anaemia in patients treated with BCL-XL-specific BH3-mimetic drugs.
Highlights
Anaemia affects over 1.5 billion people world-wide[1] and is a major cause of morbidity that requires improved therapeutic interventions
Aberrant apoptosis of red blood cells and erythroid progenitors is a common feature of chronic anaemias and is likely to contribute to their severity
Given that BIM is essential for the aberrant apoptosis of erythroid progenitors in embryonic mice caused by the absence of BCL-XL,[25] we investigated the role of this pro-apoptotic BH3-only protein in adult erythropoiesis
Summary
Bone marrow cells were cultured in semi-solid agar in the presence of SCF+IL-3+EPO for 7 days. Following Bclx gene deletion we observed significantly decreased percentages of mature red blood cells, but not reticulocytes in the bone marrow with a corresponding increase in the percentages of the more immature precursors (Figure 2b) This phenomenon was mirrored in the spleen of the tamoxifen-treated Bclxfl/fl; RosaCreERT2Ki/+ mice, where normally only few immature erythroid progenitor cells are found. (b) n = 5–6, unpaired Students t-test, adjusted for multiple testing (Holm–Sidak); *Po0.002 analysis gated on donor, non-transfused cells (GFP−) in the spleen and bone marrow revealed elevated proportions of immature erythroid progenitors with a decrease in the proportions of reticulocytes and mature red blood cells in the tamoxifen-treated Bclxfl/fl;RosaCreERT2Ki/+ mice (Supplementary Figure 4).
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