Absence or Blockage of E-Selectin-Mediated Cell Adhesion Delays Hematopoietic Stem Cell (HSC) Turn-Over and Enhances Chemoresistance.

Abstract 564

The behaviour of a hematopoietic stem cell (HSC) is regulated by its immediate micro-environment or niche. We have identified a novel function for the adhesion molecule E-selectin which is constitutively expressed on bone marrow (BM) vasculature. Using mice knocked-out for E- (E^-/-) or P-selectin (P^-/-) genes, we investigated whether selectin absence alters HSC behaviour in vivo. We found HSC cycling in the absence of E-selectin to be significantly delayed 2.5-fold in BrdU incorporation assays compared to either P^-/- or WT (mice were administered BrdU for 3d then BrdU incorporation in BM Lineage^-KIT⁺Sca1⁺(LKS⁺)CD34^- or LKS⁺CD48^-CD150⁺cells measured). To confirm these findings, LKS⁺ cells were stained with rhodamine123, a vital dye retained by metabolically active cells but not quiescent HSC. More LKS⁺ cells from E^-/- mice were rhodamine dull (34±2%) than WT (23±1%; p=0.037) confirming that a greater proportion of HSC from E^-/- mice are quiescent. We then determined whether administration of E-selectin antagonists alone could similarly delay HSC turnover. Mice were administered the glycomimetic E-selectin antagonist GMI-1070, for set periods of time before harvest. We found HSC turnover to be significantly delayed following GMI-1070 administration (1.4 fold less BrdU incorporation, p=0.011) with a concomitant 1.4-fold increase in the number of Rho123 dull LSK+ quiescent HSC per femur (p=0.020). Non-cycling, quiescent HSC are known to be more resistant to chemotherapy and irradiation. Indeed 7 days following 5-FU administration, we found that E^-/- mice had faster BM HSC recovery / less HSC damage compared to WT mice, both by phenotype analysis and in a competitive long-term reconstituting assay. Following 5-FU administration the number of reconstituting units/femur in WT mice decreased 5.1-fold but only decreased 2.3-fold in similarly treated E^-/- mice. Interestingly, when mice were pre-treated with GMI-1070 before 5-FU, there was significantly enhanced blood neutrophil recovery compared to mice administered 5-FU alone (blood neutrophils were 710±205 ×10³/mL with GMI-1070, compared to 234±141 ×10³/mL without, at day 9 post-5-FU, p=0.0001). Similarly when mice were severely irradiated and test bleeds performed weekly, a more rapid haematopoietic recovery was observed in E^-/- compared to WT mice. In summary, we have identified a novel function for the adhesion molecule E-selectin. HSC turnover is dramatically reduced in E^-/- mice an effect that can be replicated by transient administration of E-selectin antagonist mimetics. Furthermore blood leukocyte and HSC numbers recover faster following cytotoxic or irradiation injury in the absence or blockage of E-selectin-mediated cell adhesion. Thus E-selectin may well be a crucial component of the proliferative HSC niche regulating HSC turnover. Blockage of E-selectin adhesive interaction by GMI-1070, a novel E-selectin antagonist that has completed phase I clinical trails, may represent a promising treatment for the protection of HSC during chemotherapy.