Article

FG-4497, a pharmacological stabilizer of HIF-1alpha protein, synergistically enhances hematopoietic stem cells (HSC) mobilization in response to G-CSF and plerixafor

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Abstract

216 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. Disclosures Walkinshaw: Fibrogen Inc.: Employment, Equity Ownership.

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... For instance, granulocyte colony-stimulating factor (G-CSF) mobilizes HSCs from the bone marrow into the circulation by inhibiting macrophages that regulate HSC niches, causing the down-regulation of CXCL12 and VCAM-1 (88,90,91), two key proteins expressed by mesenchymal progenitor cells and endothelial cells to retain HSCs within the bone marrow . G-CSF also causes a reduction of oxygen availability in the bone marrow, which contributes to HSC mobilization (97,98). These discoveries have enabled the development of drugs targeting these niche interactions (also called nichotherapies) such as CXCR4 inhibitors (99)(100)(101) and prolylhydoxylase inhibitors (98) to further boost HSC mobilization for transplantation, particularly in patients undergoing autologous transplantations as prior successive rounds of radiotherapy and chemotherapy compromise subsequent HSC mobilization and transplantation (102). ...
... G-CSF also causes a reduction of oxygen availability in the bone marrow, which contributes to HSC mobilization (97,98). These discoveries have enabled the development of drugs targeting these niche interactions (also called nichotherapies) such as CXCR4 inhibitors (99)(100)(101) and prolylhydoxylase inhibitors (98) to further boost HSC mobilization for transplantation, particularly in patients undergoing autologous transplantations as prior successive rounds of radiotherapy and chemotherapy compromise subsequent HSC mobilization and transplantation (102). Furthermore, it has recently emerged that leukaemia initiating cells, which represent a small subset of leukemia cells that self-renew and clonally reinitiate the leukemia (103,104), hijack HSC niches in the leukemic bone marrow where they are sheltered and protected from the cytotoxic effects of chemotherapy (105,106). ...
Chapter
The dynamics of the hematopoietic system in steady state requires precise tuning to maintain the pool of deeply dormant hematopoietic stem cells (HSCs), while allowing the more proliferative HSCs to actively regenerate hematopoietic tissue. This chapter reviews some recent advances in dissecting the interactions between mesenchymal stem cells (MSCs) and HSCs firstly in mice and then in humans. More refined flow cytometry and 3D confocal fluorescent microscopy analyses of Nes&;#x02010;green fluorescent protein (GFP) transgenic mice revealed GFP positivity did not define a single population of bone marrow MSCs. Despite the complexity of HSC niches in the mouse bone marrow and the fact that exquisitely sensitive genetic studies with conditional gene induction or ablation in vivo in mice cannot be repeated in humans, progress has been made to better characterize genuine clonal multipotent MSCs and their HSC&;#x02010;supportive role in human bone marrow.
... (ii) Other novel agents currently evaluated in preclinical studies are FG-4497 that stabilizes HIF-1 through inhibition of its hydroxylation [106], Gro (CXCL2), a chemokine whose exact mechanism is unclear [107], and S1P agonists [52]. ...
Article
Full-text available
Following chemotherapy and/or the administration of growth factors, such as granulocyte-colony stimulated factor (G-CSF), hematopoietic stem cells (HSC) mobilize from bone marrow to peripheral blood. This review aims to systematically present the structure of the HSC "niche" and elucidate the mechanisms of their mobilization. However, this field is constantly evolving and new pathways and molecules have been shown to contribute to the mobilization process. Understanding the importance and the possible primary pathophysiologic role of each pathway is rather difficult, since they share various overlapping components. The primary initiating event for the mobilization of HSC is chemotherapy-induced endogenous G-CSF production or exogenous G-CSF administration. G-CSF induces proliferation and expansion of the myelomonocytic series, which leads to proteolytic enzyme activation. These enzymes result in disruption of various receptor-ligand bonds, which leads to the disanchorage of HSC from the bone marrow stroma. In everyday clinical practice, CXC chemokine receptor-4 (CXCR4) antagonists are now being used as mobilization agents in order to improve HSC collection. Furthermore, based on the proposed mechanisms of HSC mobilization, novel mobilizing agents have been developed and are currently evaluated in preclinical and clinical studies.
Chapter
Hematopoietic stem cells egress and traffic to the bone marrow throughout the normal human life span. This egress process is essential during inflammation and is exploited for peripheral blood stem cell mobilization. This chapter reviews the bone marrow microenvironment and its role in stem cell mobilization. It reviews critical components of the bone marrow microenvironment and data on stem cell trafficking to the niche and mobilization from the niche. It highlights studies that describe the niche and normal stem cell homeostasis and the biology of stem cell mobilization. Finally, stem cell mobilizing agents are described and their mechanism is reviewed.
Article
Full-text available
Hypoxia-inducible factors (HIFs) are oxygen-sensitive transcription factors regulated by oxygen-dependent prolyl hydroxylase domain (PHD) enzymes and are key to cell adaptation to low oxygen. The hematopoietic stem cell (HSC) niche in the bone marrow is highly heterogeneous in terms of microvasculature and thus oxygen concentration. The importance of hypoxia and HIFs in the hematopoietic environment is becoming increasingly recognized. Many small compounds that inhibit PHDs have been developed, enabling HIFs to be pharmacologically stabilized in an oxygen-independent manner. The use of PHD inhibitors for therapeutic intervention in hematopoiesis is being increasingly investigated. PHD inhibitors are well established to increase erythropoietin production to correct anemia in hemodialysis patients. Pharmacological stabilization of HIF-1α protein with PHD inhibitors is also emerging as an important regulator of HSC proliferation and self-renewal. Administration of PHD inhibitors increases quiescence and decreases proliferation of HSCs in the bone marrow in vivo, thereby protecting them from high doses of irradiation and accelerating hematological recovery. Recent findings also show that stabilization of HIF-1α increases mobilization of HSCs in response to granulocyte colony-stimulating factor and plerixafor, suggesting that PHD inhibitors could be useful agents to increase mobilization success in patients requiring transplantation. These findings highlight the importance of the hypoxia-sensing pathway and HIFs in clinical hematology.
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