XPO1 (Exportin-1) Is a Major Regulator of Human Erythroid Differentiation. Potential Clinical Applications to Decrease Ineffective Erythropoiesis of Beta-Thalassemia

Background
We and others have shown that normal human erythroid cell maturation requires a transient activation of caspase-3 at late stages of maturation (Zermati et al, J Exp Med 2001). We further documented that, in human erythroblasts, the chaperone HSP70 is constitutively expressed and, at late stages of maturation, translocates into the nucleus and protects GATA-1, the master transcriptional factor critical for erythropoiesis, from caspase-3 cleavage (Ribeil et al, Nature 2007). During the maturation of human β-TM erythroblasts, HSP70 is sequestrated by excess of α-globin chains in the cytoplasm and as a consequence, GATA-1 is no longer protected from caspase-3 cleavage resulting in end-stage maturation arrest and apoptosis (Arlet et al, Nature 2013). Understanding the molecular mechanisms that regulate the localization of HSP70 during erythroid differentiation may help to find new therapeutic targets to reduce ineffective erythropoiesis in beta-thalassemia.
Methods
CD34 positive cells from normal and thalassemic peripheral blood were cultured in IMDM/BIT media in the presence of SCF, IL3, IL6 for seven days and subsequently cultured for additional 7 to 9 days in media containing SCF, IL3 and Epo. Erythroblasts differentiation, HSP70 localization were analysed by FACS, AMNIS stream, confocal microscopy and western blot analysis. RNAseq and proteomic analysis of highly purified erythroid cells at all distinct stages of differentiation were used to assess expression levels of various exportins. Duolink and Octet analyses were used to assess protein proximity and affinity of interactions, respectively.
Results
During erythroid differentiation, Hikeshi, the cognate nuclear importin of HSP70, is constitutively expressed and enables HSP70 nucleus entry as assessed by siRNA experiments. However, its expression was not regulated during erythroid differentiation. In contrast, exportin expression analysis showed marked differences in expression levels of XPO1 and XPO7 during erythroid differentiation. XPO1 expression being reduced at the time of c-kit down-regulation and caspase 3 activation while there was a marked increase in XPO7 expression at the late stages of terminal erythroid differentiation. XPO1 interacted in vivo (Duolink analysis) and in vitro with HSP70 (Octet analysis). Likewise, the previously described HSP70 S400A mutant (in the Leucine-rich Nuclear Export Sequence), which is constitutively located in the nucleus interacted with XPO1 with lower affinity compared to HSP70 WT. Stem Cell Factor (SCF) starvation and Pi3k inhibition led to decreased in vivo HSP70/XPO1 interactions. However, neither phosphorylation of HSP70 nor XPO1 were detected by Nanopro and proteomic analysis, and XPO1 expression was not regulated by Pi3K pathway. Expression of RanGTP Activating Protein (RanGAP), a protein critical for XPO1/cargo interaction, was down-regulated at the moment of caspase 3 activation during erythroid maturation, which may explain the decrease in HSP70/XPO-1 interactions. Inhibitors of XPO1 (leptomycin B and KPT 251) were able to induce HSP70 nuclear localization at early stages of differentiation (proE).
In erythroid progenitors from β-TM patients, treatment with the Selective Inhibitor of Nuclear Export compound KPT-251 rescued nuclear HSP70 localization and GATA1 expression, and resulted in improved of erythroid terminal differentiation, without cytotoxicity, of thalassemic erythroid progenitors.
Conclusion
XPO1 is a major regulator of erythropoiesis through the regulation of HSP70 nuclear localization and is a potential new target to decrease ineffective erythropoiesis of thalassemia. Specific XPO-1 inhibitors currently in clinical development are being tested for potential therapy in thalassemic erythroid progenitors.