Therapeutic targeting and rapid mobilization of endosteal HSC using a small molecule integrin antagonist

Abstract

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. Here, we show targeting α9β1/α4β1 integrins with a single dose of a small molecule antagonist (BOP (N-(benzenesulfonyl)-L-prolyl-L-O-(1-pyrrolidinylcarbonyl)tyrosine)) rapidly mobilizes long-term multi-lineage reconstituting HSC. Synergistic engraftment augmentation is observed when BOP is co-administered with AMD3100. Impressively, HSC in equal volumes of peripheral blood (PB) mobilized with this combination effectively out-competes PB mobilized with G-CSF. The enhanced mobilization observed using BOP and AMD3100 is recapitulated in a humanized NODSCIDIL2Rγ−/− model, demonstrated by a significant increase in PB CD34+ cells. 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 α9β1/α4β1 within the endosteal niche. These results support using dual α9β1/α4β1 inhibitors as effective, rapid and transient mobilization agents with promising clinical applications.

Niche Extracellular Matrix Components and Their Influence on HSC

“…The dominant form of OPN in human and murine BM is thrombin-cleaved OPN (tcOPN), to which integrins a 4 b 1 and a 9 b 1 expressed on HSC can bind [Grassinger et al, 2009]. TcOPN has been demonstrated to be critical in the attraction, retention, and negative regulation of HSC proliferation and differentiation within the endosteal region [Nilsson et al, 2005;Stier et al, 2005;Grassinger et al, 2009] and recently we demonstrated that integrins a 4 b 1 and a 9 b 1 on HSC within the endosteal BM region are endogenously activated; and are therefore in a more ligand receptive state than their central BM counterpart [Cao et al, 2016a]. Moreover, as previously mentioned, tcOPN is produced locally within the BM, with megakaryocytes releasing all the required factors to cleave full length OPN [Storan et al, 2015].…”

“…While calcium is the most recognized metal ion in the BM niche, magnesium and manganese ions have also been identified to be elevated in the endosteal region [Cao et al, ], which along with calcium, are known modulators of integrin activity [Pepinsky et al, ]. In particular, the greater calcium, magnesium and manganese content in the endosteal region has been shown to differentially prime/activate α 4 β 1 and α 9 β 1 integrins that are expressed by endosteal HSC [Cao et al, ] as demonstrated by binding of a divalent‐cation dependent α 4 β 1 /α 9 β 1 integrin probe [Cao et al, ].…”

“…While calcium is the most recognized metal ion in the BM niche, magnesium and manganese ions have also been identified to be elevated in the endosteal region [Cao et al, ], which along with calcium, are known modulators of integrin activity [Pepinsky et al, ]. In particular, the greater calcium, magnesium and manganese content in the endosteal region has been shown to differentially prime/activate α 4 β 1 and α 9 β 1 integrins that are expressed by endosteal HSC [Cao et al, ] as demonstrated by binding of a divalent‐cation dependent α 4 β 1 /α 9 β 1 integrin probe [Cao et al, ]. Although the physiological role for differential integrin activity on BM HSC has not been determined, it is possible that the greater homing ability of endosteal HSC, compared to central HSC which are phenotypically identical for the eight markers used to isolate them, is due in part to the enhanced integrin activity of HSC in this region [Grassinger et al, ].…”

Cytokine‐induced hematopoietic stem and progenitor cell mobilization: unraveling interactions between stem cells and their niche

“…The small molecule N-(benzenesulfonyl)-l-prolyl-l-O-(1-pyrrolidinylcarbonyl)tyrosine (BOP), which is a dual ␣ 9 ␤ 1 /␣ 4 ␤ 1 integrin antagonist, mobilizes multilineage reconstituting HSPCs after a single dose in mice. 133 Administration of a single dose of BOP in combination with a single dose of AMD3100 mobilizes similar numbers of HSPCs as is observed after 4 days of G-CSF. However, in comparison with G-CSF, the combination of BOP and AMD3100 results in significantly enhanced short-and longterm engraftment in mice, indicating that this combination may be a rapid and effective alternative to G-CSF.…”

In Vivo Hematopoietic Stem Cell Transduction

“…Furthermore, BIO5192 can be combined with G-CSF alone or with AMD31000 to increase levels of mobilized HSCs. BOP (N-(benzenesulfonyl)-L-prolyl-L-O-(1-pyrrolidinylcarbonyl)tyrosine), a small molecule targeting α9β1/α4β1 integrins also rapidly mobilizes long-term multi-lineage reconstituting HSC 10 . Synergistic engraftment augmentation is observed when BOP is co-administered with AMD3100.…”