Enhancing Hematopoietic Stem and Progenitor Cell Mobilization with 27-Hydroxycholesterol and Cyp7b1 Inhibitors

Adult hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow, sustaining blood production. HSPC transplantation is used to treat hematologic malignancies and disorders. Current HSPC collection methods primarily use granulocyte colony-stimulating factor (G-CSF, filgrastim) to mobilize HSPCs from the bone marrow into the bloodstream for collection via apheresis. For donors and patients who do not sufficiently respond to G-CSF alone, AMD3100 (plerixafor), a CXCR4 antagonist, can be used in combination with G-CSF to enhance HSPC mobilization. However, some donors and patients do not respond efficiently to these HSPC mobilizing agents, leading to multiple apheresis sessions or HSPC transplantation failure. Additionally, G-CSF can trigger fatal sickle-cell crisis in patients with sickle-cell disease (SCD), and AMD3100 alone is not as effective as G-CSF for mobilizing HSPCs. The need for alternative HSPC mobilizing agents is increasing, especially with the emergence of gene therapies to treat SCD via autologous HSPC transplantation.

In our previous study, we observed increased HSPC mobilization during pregnancy, dependent on the levels of 27-hydroxycholesterol (27HC), an oxygenated derivative of cholesterol and an endogenous selective estrogen receptor modulator (Oguro et al., J Clin Invest. 2017). Treatment of mice with 27HC in combination with G-CSF significantly increased the number of mobilized HSPCs compared to G-CSF alone.

In this study, we investigated the effect of elevating 27HC levels on AMD3100-induced HSPC mobilization. Using colony-forming unit (CFU) assay with peripheral blood from mice, we assessed the number of CFUs that gave rise to granulocyte, macrophage, erythroblast, and megakaryocyte lineages (CFU-gmEM) to estimate the number of immature HSPCs with multi-lineage differentiation capacity. Four-day subcutaneous 27HC injections significantly enhanced HSPC mobilization in mice treated with AMD3100 compared to AMD3100 alone. 27HC injections also enhanced HSPC mobilization by G-CSF and AMD3100 combination treatment.

Given that 27HC is the most abundant oxysterol in human plasma, achieving sufficient 27HC increase through injections can be challenging in clinical settings. Cyp7b1 catalyzes the metabolism of 27HC into a downstream metabolite via its oxysterol 7α-hydroxylase enzyme activity, and Cyp7b1-deficient mice exhibit elevated 27HC levels in tissues and plasma. Interestingly, Cyp7b1 is highly expressed by HSCs compared to hematopoietic progenitors and differentiated cells in the bone marrow. The antifungal medication clotrimazole is known to inhibit Cyp7b1 function, leading us to hypothesize that pharmacological inhibition of Cyp7b1 increases intracellular 27HC levels, enhancing HSPC mobilization. In similar mobilization experiments, mice treated with AMD3100, with or without G-CSF, had a greater number of mobilized HSPCs when intraperitoneally treated with clotrimazole for four days. To examine the effect of clotrimazole on human HSPC mobilization, we used humanized mice transplanted with human cord blood-derived HSPCs. Four months post-transplantation, treatment with clotrimazole and AMD3100 significantly enhanced human HSPC mobilization compared to AMD3100 alone. These data indicate that clotrimazole enhances HSPC mobilization induced by clinically used HSPC mobilizing agents.

To determine if clotrimazole also enhances AMD3100-induced HSPC mobilization in a SCD mouse model, we conducted similar experiments on Townes SCD model mice. The combination of clotrimazole and AMD3100 significantly increased HSPC mobilization in SCD mice as quantified by the number of CFU-gmEM. We also tested other Cyp7b1-binding azoles that are FDA approved for systemic treatment (Yantsevich et al., FEBS J. 2014), as clotrimazole is not FDA approved for systemic use in clinics. In turn, we found that four-day intravenous treatment of mice with voriconazole, an antifungal medication used for treatment post-HSC transplant in clinics, similarly enhanced AMD3100-induced HSPC mobilization.

Our findings indicate that Cyp7b1-inhibiting azoles clinically used for antifungal treatment can enhance HSPC mobilization, offering a new strategy for developing novel HSPC mobilization options for donors and patients, especially those with SCD undergoing gene therapies.

Kanaujiya:Alexion Pharmaceuticals: Current Employment.