Vacuolin-1 Promotes Enucleation of Cultured Human and Murine Erythroblasts

Abstract 2321

Enucleation of erythroblasts is the final step of differentiation before reticulocytes enter circulation in mammals. In culture, enucleation efficiencies vary widely, between 0 and 90%: improving this process is a necessary step for large-scale production of reticulocytes for transfusion. Recently, we demonstrated that the formation, movement and coalescence of vesicles and vacuoles play a key role in enucleation. Moreover, we showed that vacuolin-1, a small molecule that induces the formation and fusion of vacuoles, increases the enucleation of primary erythroblasts. Separately, we recently discovered that the anti-apoptotic and chromosomal passenger protein survivin plays a novel role in enucleating erythroblasts. Survivin interacts with clathrin and EPS-15, proteins that play important roles in vesicle trafficking, and together these proteins contribute to enucleation. In order to further understand the specific role played by survivin in vesicle trafficking and enucleation, we treated fetal liver Ter119- cells derived from survivin fl/fl mice with TAT-Cre to excise the survivin gene and assess the consequences of survivin loss on enucleation. We observed a significant reduction in the extent of enucleation upon deletion of survivin. Of note, survivin depleted cells harbored fewer numbers of vacuoles compared to control cells, and the vacuoles that were present were smaller than normal. Survivin deleted cells also lacked autophagolysosomes, which are formed by fusion of late endosomes/lysosomes to autophagosomes. Since vacuolin-1 is known to induce fusion of endosomes and lysosomes, we hypothesized that vacuolin-1 would rescue this enucleation defect observed in survivin KO erythroblasts. Indeed, vacuolin-1 reversed the inhibitory effect of survivin KO on enucleation. Surprisingly, we also observed that vacuolin-1 promoted the proliferation of human erythroblasts at the CFU-E stage, leading to a 4-fold increase in the percentage of glycophorin-positive cells on day 12. Thus, we conclude that vacuolin-1 acts at multiple stages of differentiation to improve the production of erythrocytes in vitro. Finally, to gain insights into the mechanism of action of the “hit” vacuolin-1, and to identify more potent small molecules that enhance enucleation in vitro, we designed and studied a further small set of vacuolin-1 structural analogs (N=12). Four vacuolin-1 analogs increase enucleation of erythroblasts with greater potency than that of vacuolin-1, providing us with initial insights into the small molecule structure-activity relationships that might govern this process. We are currently assaying the activity of these next generation compounds in erythroblast cultures as a means to improve production of red blood cells for human use.