Oxygen-Independent Stabilization of the Oxygen-Labile Transcription Factor HIF-1α with Dimethyloxalyl Glycine or FG-4497 Increases Hematopoietic Stem Cell Quiescence In Vivo and Mobilization in Response to G-CSF

Abstract 2334

The endosteal region of the bone marrow (BM) is hypoxic in steady-state and most quiescent hematopoietic stem cells (HSC) reside in hypoxic, poorly perfused niches. Mobilizing doses of G-CSF renders most of the BM space hypoxic. Most cellular effects of hypoxia are mediated by O₂-labile hypoxia-inducible transcription factors (HIF). At O₂ concentration above 2%, HIF-α is rapidly hydroxylated on Pro residues by the prolyl hydroxylases PHD1, PHD2, and PHD3. HIF-α prolyl hydroxylation recruits the E3 ubiquitin ligase VHL, which targets HIF-α to the proteasome. In hypoxia (O₂ < 2%), HIF-α proteins are stable, associate with the β subunit ARNT, and translocate to the nucleus to activate transcription. It has emerged that HIF-1α regulates HSC proliferation and is critical to maintain long-term HSC self-renewal in vivo.

In this study, we investigated the effect of pharmacological stabilization of HIF-1α protein on HSC cycling and mobilization in mice using two different HIF prolyl hydroxylase (PHD) inhibitors, dimethyloxalyl glycine (DMOG) and FG-4497. We first assessed whether DMOG and FG-4497 stabilized HIF-1α protein in BM leukocytes in vivo by western-blot. Following a single injection of 400mg/kg DMOG, HIF-1α protein was stabilized for up to 6 hrs in BM leukocytes. With 20mg/kg FG-4497, HIF-1α protein persisted over 12 hours. HIF-1α protein was below detection in the BM from saline injected animals.

C57BL/6 mice were injected daily with 400mg/kg DMOG to measure effect on HSC cycling and BrdU incorporation by flow cytometry. Using Hoecht33342 and FITC-conjugated anti-Ki67 antibody, a 18 day DMOG treatment increased the proportion of Lin-negative Kit+ Sca1+ CD48- (L-K+S+48-) HSC in phase G0 from 61±11% to 84±6% (p<0.001, 5 mice/group). Conversely, DMOG decreased the proportion of HSC in phase G1 from 31±5% to 14±5% (p<0.001). Similar enhancement of quiescence was observed in less primitive cells such as L-K+S+48+ cells and L-K+S- myeloid progenitors. In mice given BrdU in their drinking water for the last 3 days of the experiment, a 18 day DMOG treatment reduced 3-fold the proportion of L-K+S+48- HSC that incorporated BrdU and halved the proportion of BrdU+ L-K+S+48+ progenitors. Shorter DMOG treatments (6 days or 12 days) did not alter hematopoietic stem and progenitor cell (HSPC) cycling or BrdU incorporation. In contrast, mice treated with 20mg/kg/day FG-4497 had significantly increased proportion of HSPC in G0 phase after only 6 days of treatment, likely due to the more lasting effect of FG-4497 on HIF-1α stabilization. Proportion of HSPC that incorporated BrdU was also significantly decreased. As HIF-PHD inhibitors also increase erythropoietin (EPO) expression by stabilizing HIF in the kidney, we injected a parallel cohort of mice with EPO daily for 18 days. Despite a very strong increase in red cells and hemoglobin in the blood, EPO had no effect of HSPC cycling and BrdU incorporation. Therefore the effects of HIF-PHD inhibitors on HSPC cycling are not an indirect effect of increased EPO.

Since HIF-PHD inhibitors slow HSC cycling in vivo, we tested whether they could protect HSC from sublethal irradiation. Mice were treated with 400mg/kg DMOG or saline for 22 days and then irradiated with 9.0Gy. Both cohorts were leukopenic betweens days 7 and 14 post-irradiation but DMOG treated mice had significantly higher blood leukocytes at days 22 and 30, and higher platelet numbers day 22 and all subsequent time-points suggesting that DMOG enhances HSC survival with higher blood recovery.

Finally mice were injected with DMOG for 4 days and pegylated rhuG-CSF 3 and 1.5 days before harvest. DMOG doubled mobilization in response to G-CSF with 1,620±530 CFC / mL blood in response G-CSF alone, and 3,250±830 CFC / mL in the G-CSF + DMOG group (p<0.05). Without G-CSF, circulating CFC were less than 10 / mL blood. Similarly, FG-4497 pre-treatment increased the number of CFC mobilized into the spleen 4-fold compared to G-CSF alone (p<0.01).

In conclusion, these data highlight the importance of the hypoxia pathway and HIF in the regulation of HPSC cycling and trafficking in vivo. Furthermore HIF-PHD inhibitors may provide therapeutic opportunities to protect HSC from deleterious effects of irradiation as well as increasing mobilization efficiency for transplantation.