O-Fucose and Fringe-modified NOTCH1 extracellular domain fragments as decoys to release niche-lodged hematopoietic progenitor cells

Abstract

Abstract Successful hematopoietic progenitor cell (HPC) transplant therapy is improved by mobilizing HPCs from the bone marrow niche in donors. Notch receptor–ligand interactions are known to retain HPCs in the bone marrow, and neutralizing antibodies against Notch ligands, Jagged-1 or Delta-like ligand (DLL4), or NOTCH2 receptor potentiates HPC mobilization. Notch–ligand interactions are dependent on posttranslational modification of Notch receptors with O-fucose and are modulated by Fringe-mediated extension of O-fucose moieties. We previously reported that O-fucosylglycans on Notch are required for Notch receptor–ligand engagement controlling hematopoietic stem cell quiescence and retention in the marrow niche. Here, we generated recombinant fragments of NOTCH1 or NOTCH2 extracellular domain carrying the core ligand-binding regions (EGF11–13) either as unmodified forms or as O-fucosylglycan-modified forms. We found that the addition of O-fucose monosaccharide or the Fringe-extended forms of O-fucose to EGF11–13 showed substantial increases in binding to DLL4. Furthermore, the O-fucose and Fringe-extended NOTCH1 EGF11–13 protein displayed much stronger binding to DLL4 than the NOTCH2 counterpart. When assessed in an in vitro 3D osteoblastic niche model, we showed that the Fringe-extended NOTCH1 EGF11–13 fragment effectively released lodged HPC cells with a higher potency than the NOTCH2 blocking antibody. We concluded that O-fucose and Fringe-modified NOTCH1 EGF11–13 protein can be utilized as effective decoys for stem cell niche localized ligands to potentiate HPC egress and improve HPC collection for hematopoietic cell therapy.