Poster Board II-417
Hematopoietic stem cells (HSCs) and progenitor cells (HPCs) reside in the bone marrow and give rise to blood-forming cells. Mobilization of these cells from the bone marrow is clinically important as these cells can be harvested from the peripheral blood, and used in transplantation. G-CSF (granulocyte colony stimulating factor) is used clinically and is considered the ‘gold standard' for human progenitor cell mobilization. However, there is broad variability in mobilization between G-CSF-treated patients, with some heavily-treated patients exhibiting very poor mobilization. Therefore, novel agents to mobilize HSCs and HPCs are urgently required. The G protein-coupled chemokine receptor, CXCR4, and its ligand, stromal derived factor-1a (SDF-1a), regulate a diverse array of cellular processes, including hematopoiesis, stem cell homing to the bone marrow, and survival of HSCs and HPCs. SDF-1a secreted from stromal cells in the bone marrow acts on CXCR4-expressing HSCs and HPCs to maintain these cells within the bone marrow microenviroment. Therefore, disruption of the interaction between SDF-1a and CXCR4 is a potential strategy for mobilization of these cells to the peripheral blood.
In this study, we employed cell-penetrating lipopeptide ‘pepducins' which interact with the intracellular loops of CXCR4 to specifically antagonize SDF-1a-mediated CXCR4 activity. We characterized a number of pepducins that are targeted to different intracellular loop regions of CXCR4. We demonstrated that two pepducins based on the first intracellular (i1) loop, PZ-218 and PZ-305, significantly inhibited SDF-1a-mediated calcium flux and chemotaxis of human neutrophils. A pepducin targeted to the third intracellular loop (i3), PZ-210, also inhibited calcium flux and migration of neutrophils, and caused a conformational change in CXCR4 that inhibited binding of the 12G5 CXCR4 antibody. Conversely, no such conformational change was elicited by the i1 pepducins PZ-218 and PZ-305, suggesting that different pepducins have distinct modes of action on CXCR4 signaling.
To examine the efficacy of these pepducins in mobilization of stem cells, colony forming unit (CFU) assays and transplantation studies were performed. We determined that, when used in combination with G-CSF, the i1 and i3-loop pepducins mobilized up to three-fold more progenitor cells from the bone marrow than G-CSF treatment alone. Furthermore, lethally irradiated mice were rescued with peripheral blood from pepducin-treated donor mice, and cells mobilized by pepducin treatment had long-term repopulating ability in donor mice. These data demonstrate the potential therapeutic application of cell-penetrating pepducins in CXCR4 antagonism for the purpose of stem cell mobilization and modulation of the effects of SDF-1a on hematopoietic cells.
O'Callaghan:Ascent Therapeutics Inc: Research Funding. Covic:Ascent Therapeutics Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Kuliopulos:Ascent Therapeutics Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.
Asterisk with author names denotes non-ASH members.