Abstract
Mobilized peripheral blood (PB) has largely replaced bone marrow (BM) as the source of hematopoietic stem cells (HSC) for transplantation. Today, the administration of daily doses of granulocyte-colony stimulating factor (G-CSF) is routinely used for clinical mobilization, but its inherent disadvantages has driven efforts to identify faster and more effective agents based on single doses of small molecules. While the CXCR4 antagonist AMD3100 (Plerixafor/Mozobil) rapidly mobilizes HSC, it is only clinically effective when used in combination with G-CSF. Here, we have identified a novel small molecule antagonist (BOP; N-(Benzene-sulfonyl)-L-prolyl-L-O-(1-Pyrrolidinylcarbonyl)tyrosine) that targets both alpha9beta1 and alpha4beta1 integrins expressed by BM HSC. A single dose of BOP rapidly mobilized long-term multi-lineage reconstituting HSC, either alone or synergistically when used in combination with AMD3100. Impressively, HSC in PB mobilized using the BOP+AMD3100 combination effectively outcompeted equivalent numbers of HSC detected in equivalent volumes of PB mobilized with G-CSF. The significantly greater augmentation of HSC in PB using BOP+AMD3100 was recapitulated in a humanized NODSCIDIL2Rγ−/− model, demonstrating its applicability in the clinical context. Alpha4beta1 is ubiquitously expressed on all hematopoietic cells but the expression of alpha9beta1 is restricted to HSC/progenitors. Consistent with these results, we demonstrated inhibiting alpha4beta1 alone results in mobilization of WBC and progenitor cells but inhibiting alpha9beta1 is critical for effective HSC mobilization. Interestingly, BOP was also found to preferentially bind human and murine HSC and progenitors through endogenously primed/activated integrins within the endosteal niche. Differential targeting of endosteal HSC/progenitors was shown to be due to greater concentrations of integrin-activating metal ions (calcium, magnesium and manganese), with a metal ion concentration gradient emanating from the endosteal BM demonstrated. Together, these results identify and validate the integrin alpha9beta1 as a novel target for stem cell mobilization and highlights the combination of the small molecule integrin antagonist BOP with AMD3100 as an alternative, effective, rapid and transient mobilizer of long-term reconstituting HSC with promising clinical applications.
Highlights
The inherent disadvantages of using granulocyte colony-stimulating factor (G-CSF) for hematopoietic stem cell (HSC) mobilization have driven efforts to identify alternate strategies based on single doses of small molecules
Significant advances have been made to address the shortcomings of G-CSF such as the development of CXCR4 antagonists including the Food and Drug Administration (FDA)-approved AMD3100 and experimental agents such as ALT-1188 and TG-0054
HSC are known to express a large number of integrins including a4b1 and a9b1, which have critical roles in HSC regulation in BM16
Summary
The inherent disadvantages of using granulocyte colony-stimulating factor (G-CSF) for hematopoietic stem cell (HSC) mobilization have driven efforts to identify alternate strategies based on single doses of small molecules. Using a related fluorescent analogue of BOP (R-BC154), we show that this class of antagonists preferentially bind human and mouse HSC and progenitors via endogenously primed/activated a9b1/a4b1 within the endosteal niche These results support using dual a9b1/a4b1 inhibitors as effective, rapid and transient mobilization agents with promising clinical applications. G-CSF-based mobilization requires multiple doses over a number of days, is known to alter the function of the HSC niche as well as bone formation[3], can cause bone pain and spleen enlargement and on rare occasions G-CSF results in splenic rupture, myocardial infarction or cerebral ischaemia (reviewed in refs 4,5) These inherent disadvantages of G-CSF have driven efforts to identify alternate mobilization strategies based on the use of single doses of small molecules[6,7]. The therapeutic targeting of endosteal HSC for mobilization should provide better transplant outcomes
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