Up to 5% allogeneic healthy donors and up to 40–60% of chemotherapy-treated patients in autologous setting, fail to reach minimal threshold of 2×106 blood CD34+cells/kg in response to G-CSF, precluding transplantation. Plerixafor, a small inhibitor of the chemokine receptor CXCR4, used for 4 days in combination with G-CSF enables this minimal threshold to be reached in up to 60% patients who previously failed to mobilise in response to G-CSF alone. However, the remaining 40% of patients who failed to mobilise in response to G-CSF alone, still fail to mobilize adequately with G-CSF + Plerixafor. In an attempt to further boost HSC mobilization in response to combinations of G-CSF and Plerixafor, we have investigated the role of the hypoxia-sensing pathway in HSC mobilization.
HIF-1α (Hypoxia-inducible factor-1α) controls HSC proliferation and self-renewal in poorly perfused hypoxic bone marrow (BM) niches where very quiescent HSC with highest self-renewal potential reside. When O2 concentration is above 2% in the cell microenvironment, HIF-1α protein is rapidly hydroxylated on Pro residues by prolyl hydroxylases PHD1-3. This recruits the E3 ubiquitin ligase VHL, which targets HIF-1α to rapid proteasomal degradation. When O2concentration is below 2% (hypoxia), PHDs are inactive; HIF-1α protein is stabilized, associates with its β subunit ARNT, translocates to the nucleus and activates of transcription and hypoxia-responsive genes.
In this study, we have investigated the effect of pharmacological stabilization of HIF-1α protein on HSC mobilization in mice using the HIF-PHD inhibitor FG-4497. We report that FG-4497 treatment stabilizes HIF-1α protein in mouse BM. We find that FG-4497 synergizes with G-CSF and Plerixafor to enhance HSC mobilization. C57/Bl6 mice were in 4 treatment groups: (G) 250μg/kg/day G-CSF alone for 2 days; (GF) G-CSF for 2 days + 20mg/kg/day FG-4497 for 3 days; (GP) G-CSF for 2 days together with16mg/kg Plerixafor 1 hour prior harvest; (GPF) G-CSF together with Plerixafor and FG-4497 with same dosing as above. Mobilization of colony-forming cells (CFC), phenotypic Lin-CD41-Sca1+Kit+CD48-CD150+ HSC, and functional HSC in long-term competitive transplantation assays were measured. Mice in the GF group (G-CSF + FG-4497) mobilized CFC to the blood 4-fold and phenotypic HSC 3-fold more than mice mobilized with G-CSF alone (p<0.005), whereas FG-4497 alone had a poor mobilizing effect. This demonstrates synergy between G-CSF and PHD inhibition. Expectedly, Plerixafor enhanced mobilization of CFC 10-fold and phenotypic HSC 2-fold in response to G-CSF (p<0.005). Most interestingly, addition of FG-4497 boosted 4-fold mobilization of CFC and phenotypic HSC in response to G-CSF+Plerixafor (p<0.005). This was confirmed in competitive repopulation assays following transplantation of 20μL mobilized blood in competition with 2×105BM cells from congenic donors. CD45.2/CD45.1 chimerism showed that combination of G-CSF+Plerixafor+FG-4497 mobilized competitive repopulating HSC 6-fold more than G-CSF+Plerixafor (p<0.005), the best mobilizing cocktail used in the clinic currently.
To determine which cell types drive HSC mobilization in a HIF-1α-dependant manner, we crossed HIF1αflox/floxmice with osxCre (HIF-1α gene deletion in osteoprogenitors), LysMCre (deletion in myeloid cells), or with SclCreER mice (tamoxifen-induced deletion in HSC). While studies in LysMCre and SclCreER mice are ongoing, we find that deletion of HIF-1α gene in osteoprogenitors (osxCre mice) decreased 2.5-fold the number of CFU/mL blood following 2 and 3 days treatment with G-CSF. This suggests that HIF-1α in osteoprogenitors and their osteoblastic progenies is necessary for optimal mobilization in response to G-CSF.
In conclusion, our data highlight the importance of HIF-1α in HSC mobilization and provide a novel therapeutic strategy for increasing HSC mobilization above levels obtained with combinations of G-CSF and Plerixafor. Thus PHD inhibitors could be useful agents in patients who still fail to mobilize in response to G-CSF and plerixafor.
Walkinshaw: Fibrogen Inc.: Employment, Equity Ownership.