Rapid mobilisation of HSC using a small molecule to modulate stem cell interactions with their niche

B Cao1,2, Z Zhang1, J Grassinger3, B Williams1,2, CK Heazlewood1,2, QI Churches1, SA James4, S Li1,2, T Papayannopoulou5 and SK Nilsson1,2

  1. Biomedical Manufacturing, CSIRO, Melbourne, Victoria 3169, Australia
  2. ARMI, Monash University, Clayton, Victoria 3800, Australia
  3. University Hospital Regensburg, Department of Internal Medicine III, Regensburg, Germany
  4. Australian Synchrotron, Clayton, Victoria 3168, Australia
  5. Department of Medicine/Hematology, University of Washington, Seattle, Washington 98195-7710, USA

Currently mobilised peripheral blood (PB) is one of the most common sources of haematopoietic stem cells (HSC) for transplant. This routinely involves the administration of daily doses of granulocyte-colony stimulating factor (G-CSF), but inherent disadvantages of the use of this cytokine have driven efforts to identify faster and more effective agents. Recently this has involved trialling the use of single doses of small molecules. For example, the CXCR4 antagonist AMD3100 (Plerixafor/Mozobil) rapidly mobilises HSC, but 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) which targets both α9β1 and α4β1 integrins expressed by HSC and dislodges them the bone marrow microenvironment. A single dose of BOP rapidly mobilised long-term multi-lineage reconstituting HSC, either alone or synergistically when used in combination with AMD3100. Impressively, HSC in PB mobilised with the combination of BOP+AMD3100 effectively out competed equivalent numbers of HSC in equivalent volumes of PB mobilised with G-CSF. The significantly greater augmentation of HSC in PB using BOP+AMD3100 was recapitulated in humanised NODSCIDIL2Rγ-/- mice, demonstrating its applicability in the clinical context. α4β1 is ubiquitously expressed by all haematopoietic cells but the expression of α9β1 is restricted to HSC/progenitors. Consistent with these results, we demonstrated inhibiting α4β1 alone results in mobilisation of WBC and progenitor cells but inhibiting α9β1 is critical for effective HSC mobilisation. 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 marrow. Together, these results identify and validate targeting α9β1 integrin for stem cell mobilisation and highlights the combination of the small molecule integrin antagonist BOP with AMD3100 as an alternative, effective, rapid and transient mobiliser of long-term reconstituting HSC with promising clinical applications.