Insights into the biology of mobilized hematopoietic stem/progenitor cells through innovative treatment schedules of the CXCR4 antagonist AMD3100

Abstract The exopeptidase CD26 (also known as DPPIV/dipeptidylpeptidase IV) cleaves dipeptides from the N-terminus of proteins that contain the required X-Pro or X-Ala motif. We have previously reported that inhibition or loss of CD26 activity results in a deficiency in normal granulocyte-colony stimulating factor (G-CSF) induced mobilization, suggesting that CD26 is a necessary component of mobilization (Christopherson, et al Blood 2003 and Christopherson, et al Exp Hematol 2003). The chemokine CXCL12 (SDF-1, stromal cell derived factor-1) contains the appropriate recognition sequence for CD26 induced cleavage. This combined with the importance of CXCL12 in the trafficking of hematopoietic stem and progenitor cells (HSC/HPC) suggests CXCL12 as a likely functional target of CD26 during G-CSF induced mobilization. For this reason we therefore decided to investigate whether genetically engineered mice lacking CD26 (CD26−/−) could be mobilized utilizing the CXCR4 antagonist, AMD3100. To evaluate this, ten week old C57BL/6 and CD26−/− mice (also on a C57BL/6 background) received a single subcutaneous injection of AMD3100 (1mg/1kg). One hour following injection the mice were euthanized by CO2 inhalation. Peripheral blood was then obtained by heart stick with a 1.2 ml syringe containing EDTA as an anticoagulant. A complete blood count was taken for each peripheral blood sample. Following red blood cell lysis, cells were plated for myeloid colony formation in a standard 1% methylcellulose colony assay containing the appropriate cytokines. Following 7 days of incubation at 5% O2, 5% CO2 and 37°C plates were scored for colony-forming units-granulocyte macrophage (CFU-GM), burst-forming units-erythroid (BFU-E), and colony-forming units-granulocyte, erythroid, macrophage, and megakaryocytic (CFU-GEMM). Data is presented as the number of colonies per femur for the bone marrow and as the number of colonies per ml of whole blood for the peripheral blood. AMD3100 treatment resulted in an increase in white blood cell (WBC) counts from 5.05±0.48 × 106/ml in untreated mice to 10.21±0.88×106/ml in treated mice (p≤0.01). An increase in WBC counts was also observed during AMD3100 treatment in CD26−/− mice from 7.77±1.28×106/ml in untreated mice to 16.7 ±2.11 × 106/ml in treated mice (p<0.01). AMD3100 treatment resulted in an increase in circulating myeloid progenitors in the peripheral blood of C57BL/6 and CD26−/− mice as compared to untreated C57BL/6 and CD26−/− mice respectively (p≤0.01). Specifically, a 2.38, 3.75, 12.33 fold increase in CFU-GM, BFU-E, and CFU-GEMM were observed in the peripheral blood of C57BL/6 mice after treatment. A 2.63, 5.48, 14.29 fold increase in CFU-GM, BFU-E, and CFU-GEMM were observed in the peripheral blood of CD26−/− mice after treatment. Existing pre-clinical and clinical data suggest that the CXCR4 antagonist, AMD3100, rapidly mobilizes hematopoietic progenitor cells from the bone marrow into the periphery. The results presented here provide pre-clinical evidence that disruption of the interaction between the CXCR4 chemokine receptor and CXCL12, via sub-cutaneous injection of AMD3100, mobilizes significant numbers of myeloid progenitors in mice, even in the absence of CD26. These results support the notion that CD26 is downstream of G-SCF treatment. Additionally, these results support the potential use of AMD3100 to treat patients that may have an altered ability to respond to G-CSF treatment as a result of a reduction or loss in CD26 activity. Future studies are warranted to evaluate potential variations in CD26 levels or activity in the general population, in differing patient populations, and during different treatment regimens.

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Mobilization Studies in Complement-Deficient Mice Reveal That AMD3100 Mobilization Depends On Complement Cascade Activation and Suggest Involvement of “Membrane Attack Complex - MAC” in Egress of Hematopoietic Stem/Progenitor Cells.

Blood ◽

10.1182/blood.v114.22.367.367 ◽

2009 ◽

Vol 114 (22) ◽

pp. 367-367

Author(s):

Marcin Wysoczynski ◽

HakMo Lee ◽

Rui Liu ◽

Wan Wu ◽

Janina Ratajczak, ◽

...

Keyword(s):

Progenitor Cells ◽

Conflicts Of Interest ◽

Membrane Attack Complex ◽

Classical Pathway ◽

Compensatory Mechanism ◽

Complement Cascade ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Deficient Mice

Abstract Abstract 367 We reported that complement cascade (CC) becomes activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPCs) by immunoglobulin (Ig)-dependent pathway and/or by alternative Ig-independent pathway as seen during G-CSF- or Zymosan mobilization, respectively. As a result, several potent bioactive CC anaphylatoxins (C3 and C5 cleavage fragments) are released that regulate egress of HSPCs (Blood 2003;101,3784; Blood 2004;103,2071; Blood 2005;105,40, Leukemia 2009; in press.). This explains why: i) NOD/SCID and RAG-/- animals that do not activate the Ig-dependent CC classical pathway; ii) C2fB-/- and C3-/- mice that do not activate the classical and alternative CC pathways; and iii) C5-/- mice that do not activate the distal pathway of CC are all poor G-CSF- and/or Zymosan mobilizers. In this study, we evaluated the role of CC in mobilization induced by CXCR4 antagonist AMD3100. We noticed that all CC activation-deficient mice mentioned above, except C5-/- mice, mobilize normally in response to AMD3100 administration. Accordingly, the number of mobilized CD34- SKL cells, leucocytes, and CFU-GM clonogeneic progenitors in mutant mice was similar to wt littermates. More important we observed that AMD3100 mobilization of HSPCs was preceded by a massive egress of leucocytes from BM and that AMD3100 was able to stimulate in these cells i) phosphorylation of MAPKp42/44 and ii) secretion of MMP-9. At the same time, ELISA data to detect CC activation revealed that serum levels of CC cleavage fragments, which were low in the initial phase of AMD3100 mobilization during granulocyte egress, become elevated later during HSPC egress. Thus, our data show that despite a fact that G-CSF and AMD3100 mobilize HSPCs by involving different mechanisms, activation of CC is a common phenomenon occurring during mobilization induced by both compounds. This further supports a pivotal role of CC activation in the egress of HSPCs from BM; however, both compounds activate CC differently. While G-CSF administration initiates CC activation at its proximal C1q-C3 level, AMD3100 induces CC activation at the distal C5 level, pointing to a crucial role of C5 cleavage in executing mobilization. To support this, all mice employed in our studies that display defects in activation of proximal stages of CC (NOD/SCID, RAG, C2fB-/-, and C3-/-) are normal AMD3100 mobilizers. However, C5 is cleavage required for mobilization occurs in the plasma of these animals latter on - directly by proteases released from AMD3100-stimulated granulocytes that egress from the BM as a first wave of mobilized cells. This compensatory mechanism cannot occur from obvious reasons in C5-/- mice. We conclude that AMD3100-directed mobilization similarly as G-CSF-induced one depends on activation of CC; however, AMD3100 in contrast to G-CSF activates CC at distal stages – directly by proteases released from mobilized/activated granulocytes. Cleavage of C5 and release of C5a and desArgC5a create a sinusoid-permissive environment in BM for HSPCs egress. This suggests involvement of both C5 cleavage fragments as well as a potential role of downstream elements of CC activation - membrane attack complex - MAC (C5b-C9) in stem cell mobilization. Therefore, some poor AMD3100 patient responders could possess a defect in activation of the distal steps of CC. Disclosures: No relevant conflicts of interest to declare.

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ALL Cells Mobilized by AMD3100 Are More Proliferative, and Remain In the Circulation Longer Than Normal HSC, Enhancing the Action of Vincristine

Blood ◽

10.1182/blood.v116.21.2137.2137 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2137-2137 ◽

Cited By ~ 1

Author(s):

Linda J. Bendall ◽

Robert Welschinger ◽

Florian Liedtke ◽

Carole Ford ◽

Aileen Dela Pena ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Peripheral Blood ◽

Bone Marrow Microenvironment ◽

Hematopoietic Progenitors ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Cell Mobilization ◽

Cycle Dependent

Abstract Abstract 2137 The chemokine CXCL12, and its receptor CXCR4, play an essential role in homing and engraftment of normal hematopoietic cells in the bone marrow, with the CXCR4 antagonist AMD3100 inducing the rapid mobilization of hematopoietic stem and progenitor cells into the blood in mice and humans. We have previously demonstrated that AMD3100 similarly induces the mobilization of acute lymphoblastic leukemia (ALL) cells into the peripheral blood. The bone marrow microenvironment is thought to provide a protective niche for ALL cells, contributing to chemo-resistance. As a result, compounds that disrupt leukemic cell interactions with the bone marrow microenvironment are of interest as chemo-sensitizing agents. However, the mobilization of normal hematopoietic stem and progenitor cells may also increase bone marrow toxicity. To better evaluate how such mobilizing agents affect normal hematopoietic progenitors and ALL cells, the temporal response of ALL cells to the CXCR4 antagonist AMD3100 was compared to that of normal hematopoietic progenitor cells using a NOD/SCID xenograft model of ALL and BALB/c mice respectively. ALL cells from all 7 pre-B ALL xenografts were mobilized into the peripheral blood by AMD3100. Mobilization was apparent 1 hour and maximal 3 hours after drug administration, similar to that observed for normal hematopoietic progenitors. However, ALL cells remained in the circulation for longer than normal hematopoietic progenitors. The number of ALL cells in the circulation remained significantly elevated in 6 of 7 xenografts examined, 6 hours post AMD3100 administration, a time point by which circulating normal hematopoietic progenitor levels had returned to baseline. No correlation between the expression of the chemokine receptor CXCR4 or the adhesion molecules VLA-4, VLA-5 or CD44, and the extent or duration of ALL cell mobilization was detected. In contrast, the overall motility of the ALL cells in chemotaxis assays was predictive of the extent of ALL cell mobilization. This was not due to CXCL12-specific chemotaxis because the association was lost when correction for background motility was undertaken. In addition, AMD3100 increased the proportion of actively cells ALL cells in the peripheral blood. This did not appear to be due to selective mobilization of cycling cells but reflected the more proliferative nature of bone marrow as compared to peripheral blood ALL cells. This is in contrast to the selective mobilization of quiescent normal hematopoietic stem and progenitor cells by AMD3100. Consistent with these findings, the addition of AMD3100 to the cell cycle dependent drug vincristine, increased the efficacy of this agent in NOD/SCID mice engrafted with ALL. Overall, this suggests that ALL cells will be more sensitive to effects of agents that disrupt interactions with the bone marrow microenvironment than normal progenitors, and that combining agents that disrupt ALL retention in the bone marrow may increase the therapeutic effect of cell cycle dependent chemotherapeutic agents. Disclosures: Bendall: Genzyme: Honoraria.

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The novel CXCR4 antagonist POL5551 mobilizes hematopoietic stem and progenitor cells with greater efficiency than Plerixafor

Leukemia ◽

10.1038/leu.2013.266 ◽

2013 ◽

Vol 27 (12) ◽

pp. 2322-2331 ◽

Cited By ~ 37

Author(s):

D Karpova ◽

K Dauber ◽

G Spohn ◽

D Chudziak ◽

E Wiercinska ◽

...

Keyword(s):

Progenitor Cells ◽

The Novel ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

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CXCR4 antagonist (BL-8040) to enhance antitumor effects by increasing tumor infiltration of antigen-specific effector T-cells.

Journal of Clinical Oncology ◽

10.1200/jco.2018.36.5_suppl.73 ◽

2018 ◽

Vol 36 (5_suppl) ◽

pp. 73-73 ◽

Cited By ~ 4

Author(s):

Pankaj Gaur ◽

Vivek Verma ◽

Seema Gupta ◽

Ella Sorani ◽

Abi Vainstein Haras ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

Tumor Growth ◽

Progenitor Cells ◽

Specific Antigen ◽

Mouse Tumor ◽

Cxcr4 Antagonist ◽

Antitumor Effects ◽

Hematopoietic Stem ◽

Effector Cells

73 Background: C-X-C chemokine receptor type 4 (CXCR4) helps to retain the hematopoietic stem cells (HSC) in the bone marrow (BM). CXCR4 binds to its ligand CXCL12/SDF1 which is constitutively expressed in the BM thereby inhibiting the mobilization of CXCR4 expressing immune progenitor cells. Moreover, increased numbers of effector cells in the tumor microenvironment (TME) are directly correlated to enhanced immunotherapeutic efficacy. Therefore, we hypothesized that CXCR4 antagonism will result in movement of immune progenitor cells from BM to periphery leading to increased availability of T-cells in the periphery and better infiltration of effector cells into the TME. Methods: In TC-1 mouse tumor-model, we tested the effects of CXCR4 antagonist (BL-8040; 4 doses; 24 h apart; 20 mg/kg) on the tumor growth and mice survival in the presence of tumor-specific antigen priming using E7 peptide vaccine (3 doses, one week apart). Anti-tumor immune responses were determined in the tumor tissues obtained 3-4 days after the second vaccination using flow cytometry. Results: We found that BL-8040 given with specific antigenic stimulation results in significantly enhanced anti-tumor immune response, leading to a decrease in tumor growth (p≤0.001 at day 21) and prolonged mice survival. At day 35 after tumor implantation, 80% of mice survived in the BL-8040 + vaccine treatment group compared to 0% survival following BL-8040 or vaccine treatments. Interestingly, we also found that BL-8040 leads to a significant increase in the numbers of antigen-specific CD8+ T-cells in the TME. We also found that starting BL-8040 prior to or together with the priming of the mice did not affect the outcome, suggesting that the scheduling of BL-8040 does not affect the therapeutic outcomes. Conclusions: These results suggest that BL-8040 treatment enhances anti-tumor immune response by potentially increasing the immune progenitor cells in the periphery leading to a better immune response. These results also suggest that BL-8040, a CXCR4 antagonist, is a promising immune-modulatory agent with potent anti-tumor effects.

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Oncostatin M Is a Novel Niche Factor That Restrains Hematopoietic Stem Cell Mobilization in Response to G-CSF and CXCR4 Antagonist Plerixafor

Blood ◽

10.1182/blood-2019-127431 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4469-4469

Author(s):

Kavita Bisht ◽

Crystal McGirr ◽

Kylie A Alexander ◽

Whitney Fleming ◽

Natalie Sims ◽

...

Keyword(s):

Progenitor Cells ◽

Stromal Cells ◽

Receptor Gene ◽

Fold Increase ◽

Oncostatin M ◽

Distribution Analysis ◽

Mesenchymal Progenitor Cells ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem

Granulocyte colony-stimulating factor (G-CSF) and the CXCR4 chemokine receptor antagonist Plerixafor are used to mobilize hematopoietic stem and progenitor cells (HSPC). However, in the autologous setting, prior chemotherapy and radiotherapy impair HSPC mobilizing response to G-CSF and Plerixafor thereby precluding autologous HSC transplantation. We now demonstrate in mice that oncostatin M (OSM) production in the bone marrow (BM) is dramatically increased in response to G-CSF and that OSM counteracts the mobilizing effect of G-CSF and Plerixafor indirectly through the BM stroma. Indeed, by immunohistochemistry, a 4-day G-CSF treatment strongly increased OSM protein expression in cell clusters containing F4/80+ macrophages in the endosteal and central region of the mobilized BM. OSM concentration was 7-fold higher in BM fluids of G-CSF mobilized mice compared to control mice as measured by ELISA. To investigate OSM effects on HSPC mobilization, C57BL/6 mice (WT) and mice lacking the OSM receptor gene (OSMR-/-) were treated with 125 μg/kg human G-CSF bidaily for 2-6 days and/or Plerixafor (10 mg/kg) one hour before harvest. Absence of OSMR increased HSPC mobilization with a 4-fold increase in colony forming cells (CFC), phenotypic Lin-Kit+Sca1+ HSPC (LKS+ cells) and LKS+Flt3-CD48-CD150+ HSC into the blood at days 2 and 4 of G-CSF treatment and increased accumulation of HSPC and HSC in the spleen of OSMR-/- mice at day 6 of G-CSF treatment. In a competitive repopulation assay with serial dilutions of mobilized blood, deletion of OSMR gene enhanced ≈4-fold mobilization of functional reconstituting HSC in response to G-CSF as shown by Poisson's distribution analysis. HSPC mobilization in response to Plerixafor was also significantly enhanced with a doubling of CFC, HSPC and HSC into the blood of OSMR-/- mice compared to WT. OSMR gene deletion also significantly enhanced HSPC mobilization (3-fold) in response to G-CSF plus Plerixafor combination, the most effective mobilization regimen currently approved. To further demonstrate that endogenous OSM restrains HSPC mobilization in response to G-CSF, we produced a recombinant mouse OSM-trap, a dimeric protein chimera made of the extracellular domain of mouse OSMR and mouse gp130 fused with a mutant Fc fragment of mouse IgG2a. Co-administration of this OSM-trap together with G-CSF also increased HSPC and HSC mobilization into the blood in response to G-CSF compared to mice treated with G-CSF and control trap construct. This negative effect of endogenous OSM on HSPC mobilization was indirectly mediated by BM stromal cells. qRT-PCR on BM myeloid and stromal cells sorted from steady-state or mobilized mice showed that Osm mRNA is mostly expressed by CD11b+F4/80+CD169+VCAM1+ macrophages and CD11b+F4/80-Ly6G+ neutrophils. In sharp contrast, Osmr mRNA was undetectable in any sorted BM leukocyte population, but abundantly expressed by CD45-Lin-CD31+Sca1+CD105+ endothelial and CD45-Lin-CD31-CD51+PDGFRα+/-Sca-1+/- mesenchymal progenitor cells. OSMR protein was expressed by vascular beds in the BM as detected by IHC. Confirming the indirect effect of OSM, we found that recombinant OSM had no effect on HSPC chemotaxis in response to CXCL12, or HSPC adhesion to immobilized VCAM-1 or selectins in vitro. However, HSPC freshly isolated from the BM of OSMR-/- mice had enhanced chemotaxis in response to CXCL12 compared to HSPC isolated from WT mice. In conclusion, G-CSF increases OSM expression in the BM, and this appears to restrain HSPC mobilization in response to G-CSF via OSMR-expressing endothelial and mesenchymal cells that decreases HSPC responsiveness to CXCL12 gradient. Disclosures Winkler: GlycoMimetics: Patents & Royalties. Levesque:GlycoMimetics: Equity Ownership.

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Subacute AMD3100 Treatment Is Not Efficient in Neonatal Hypoxic-Ischemic Rats

Stroke ◽

10.1161/strokeaha.120.033768 ◽

2021 ◽

Author(s):

Daiane Aparecida Spiess ◽

Raquel Maria Pereira Campos ◽

Luciana Conde ◽

Nadine Didwischus ◽

Johannes Boltze ◽

...

Keyword(s):

Progenitor Cells ◽

Brain Atrophy ◽

Water Maze ◽

Motor Coordination ◽

Separate Analysis ◽

Therapeutic Effects ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Cell Therapies ◽

Combining Data

Background and Purpose: Despite the advances in treating neonatal hypoxic-ischemic encephalopathy (HIE) with induced hypothermia, the rates of severe disability are still high among survivors. Preclinical studies have indicated that cell therapies with hematopoietic stem/progenitor cells could improve neurological outcomes in HIE. In this study, we investigated whether the administration of AMD3100, a CXCR4 antagonist that mobilizes hematopoietic stem/progenitor cells into the circulation, has therapeutic effects in HIE. Methods: P10 Wistar rats of both sexes were subjected to right common carotid artery occlusion or sham procedure, and then were exposed to hypoxia for 120 minutes. Two subcutaneous injections of AMD3100 or vehicle were given on the third and fourth day after HIE. We first assessed the interindividual variability in brain atrophy after experimental HIE and vehicle treatment in a small cohort of rats. Based on this exploratory analysis, we designed and conducted an experiment to test the efficacy of AMD3100. Brain atrophy on day 21 after HIE was defined as the primary end point. Secondary efficacy end points were cognitive (T-water maze) and motor function (rotarod) on days 17 and 18 after HIE, respectively. Results: AMD3100 did not decrease the brain atrophy in animals of either sex. Cognitive impairments were not observed in the T-water maze, but male hypoxic-ischemic animals exhibited motor coordination deficits on the rotarod, which were not improved by AMD3100. A separate analysis combining data from animals of both sexes also revealed no evidence of the effectiveness of AMD3100 treatment. Conclusions: These results indicate that the subacute treatment with AMD3100 does not improve structural and functional outcomes in a rat HIE model.

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Pan-histone deacetylase inhibitor panobinostat depletes CXCR4 levels and signaling and exerts synergistic antimyeloid activity in combination with CXCR4 antagonists

Blood ◽

10.1182/blood-2010-05-284414 ◽

2010 ◽

Vol 116 (24) ◽

pp. 5306-5315 ◽

Cited By ~ 34

Author(s):

Aditya Mandawat ◽

Warren Fiskus ◽

Kathleen M. Buckley ◽

Kelly Robbins ◽

Rekha Rao ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Histone Deacetylase ◽

Histone Deacetylase Inhibitor ◽

Cxcr4 Antagonist ◽

Lethal Effects ◽

Hematopoietic Stem ◽

Deacetylase Inhibitor ◽

Cxcr4 Antagonists

Abstract Stromal cell derived factor-1 (SDF-1 or CXCL12) and its receptor CXCR4 are involved in the directional homing to the bone marrow niches and in peripheral mobilization of normal and transformed hematopoietic stem and myeloid progenitor cells. Elevated CXCR4 expression confers poor prognosis, whereas inhibition of CXCR4 signaling overcomes stroma-mediated chemoresistance in acute myeloid leukemia (AML). Here, we demonstrate that treatment with the pan-histone deacetylase inhibitor panobinostat (PS) depleted the mRNA and protein levels of CXCR4 in the cultured and primary AML cells. PS-induced acetylation of the heat shock protein (hsp) 90 reduced the chaperone association between CXCR4 and hsp90, directing CXCR4 to degradation by the 20S proteasome. PS treatment also depleted G protein–coupled receptor kinase 3, as well as attenuated the phosphorylation of AKT and ERK1/2 in AML cells, which was not affected by cotreatment with CXCL12. Compared with each agent alone, cotreatment with PS and CXCR4 antagonist AMD3100 or FC-131 synergistically induced apoptosis of cultured and primary AML cells. PS and FC-131 exerted more lethal effects on primary AML versus normal CD34+ bone marrow progenitor cells. These findings support the rationale to test the in vivo efficacy of PS in enhancing the lethal effects of CXCR4 antagonists against AML cells.

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Expansion and Maintenance of Hematopoietic Stem and Progenitor Cells in Course of Long-Term Inhibition of CXCR4/CXCL12 Signaling

Blood ◽

10.1182/blood.v128.22.2648.2648 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2648-2648 ◽

Cited By ~ 1

Author(s):

Darja Karpova ◽

Julie Ritchey ◽

Matthew Holt ◽

Darlene Monlish ◽

Laura G. Schuettpelz ◽

...

Keyword(s):

Cell Cycle ◽

Steady State ◽

Continuous Infusion ◽

Progenitor Cells ◽

Small Molecule ◽

Critical Role ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Abstract During the past two decades peripheral blood stem cells have become the favored graft source for HSCT with 80 % of allogeneic and almost 100 % of autologous HSCT performed with mobilized blood. The critical role of the interaction between the chemokine receptor CXCR4 and its chief ligand CXCL12 for retention and migration of hematopoietic stem and progenitor cells (HSPC) has been well established. Interference with CXCR4/CXCL12 signalling iscurrentlybeing exploited as a strategy to mobilize HSPC indirectly with the most clinically relevant mobilizing agent to date, G-CSF as well as directly with the bicyclam CXCR4 antagonist Plerixafor (AMD3100).In this study, qualitative and quantitative effects of long-term pharmacologic inhibition of CXCR4/CXCL12 axis within the HSPC compartment were investigated in healthy C57BL/6 mice using the non-peptidic small molecule CXCR4 antagonists Plerixafor and ALT1188 along with the Protein-EpitopeMimeticsInhibitor POL5551. Up to 12-14 fold higher mobilization efficiency was achieved by applying the antagonists via two weeks of continuous infusion (up to 8-10x104 CFU-C and LSK/ml) as compared to bolus treatment (4-6x103 CFU-C and LSK/ml) or 5-day course of G-CSF (3-6x103 CFU-C/ml).Despite dramatic increase in numbers of circulating HSPC, the BM HSPC pool dis not decrease; in fact it expanded up to 2-4-fold compared to steady state reservoir (sham-operated control mice) as measured by immunophenotypical (LSK SLAM) and functional (e.g. serial competitive transplantation) properties of the cells. Thus, in contrast to genetically CXCR4 ablatedHSPC, the reversible long-term blockade of the receptor did not diminish the long-term repopulating capacity of HSPC. Cell cycle analysis showed a 2-3-fold increase in cycling activity of BM HSPC: only 10-20% of LSK and 30-40 % of LSK SLAM cells were found to be quiescent (in G0 phase of the cell cycle) after two weeks of CXCR4 antagonist infusion versus 50-60 % of LSK and 70 % of LSK SLAM found in G0 under homeostatic conditions. This increased proliferation was very similar to the one induced transiently at day 3 G-CSF treatmentand would conceivably explain the sustained mobilization without concomitant depletion of the BM HSPC pool. Profiling of differentially treated BM HSC (LSK SLAM) via microarray analysis did not reveal substantial effects of CXCR4 inhibitor infusion on the expression signature. Ofnote, major cytological changes typically associated with G-CSF induced mobilization, e.g. depletion of bone lining osteoblast lineage cells and macrophages, were not detected in continuous infusion of POL5551 exposed BM suggesting limitedeffects within the BM niche compartment. Moreover analysis of the BM HSPC after different washout periods at the end of continuous infusion treatment revealed a rapid (within 1-3 days after discontinuation of infusion) reestablishment of steady state HSPC numbers in the BM.Our data suggest that prolonged pharmacologic blockade of the CXCR4/CXCL12 axis using multiple small molecule inhibitorsrepresents an approach thatreleasesHSPCwith efficiency superiorto any other knownmobilization strategybut also may serve as an effective method induce cell cycling and thus expand BM HSPCs. Figure Competitive transplantation of POL5551 treated andcontrol BM (n=5 recipients per group, mean±SEM) Figure. Competitive transplantation of POL5551 treated andcontrol BM (n=5 recipients per group, mean±SEM) Disclosures Levesque: GlycoMimetics: Equity Ownership.

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Novel Mechanistic Insight Into Mobilization of Hematopoietic Stem/Progenitor Cells (HSPCs): Complement Cascade and Membrane Attack Complex Activated in Bone Marrow Sinusoids During Mobilization Release From Erythrocytes Sphingosine-1 Phosphate – An Underappreciated Chemoattractant Executing Egress of HSPCs.

Blood ◽

10.1182/blood.v114.22.31.31 ◽

2009 ◽

Vol 114 (22) ◽

pp. 31-31 ◽

Cited By ~ 1

Author(s):

HakMo Lee ◽

Marcin Wysoczynski ◽

Wan Wu ◽

Rui Liu ◽

Magdalena Kucia ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Conflicts Of Interest ◽

Membrane Attack Complex ◽

Heat Inactivation ◽

Cationic Peptides ◽

Complement Cascade ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Sphingosine 1 Phosphate

Abstract Abstract 31 We reported that complement cascade (CC) is activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPCs) and that CC clevage fragments direct egress of HSPCs from BM into peripheral blood (PB) (Blood 2003;101,3784; Blood 2004;103,2071; Blood 2005;105,40). We also reported that C5 cleavage fragments play a crucial role in the mobilization process by: i) inducing proteolytic activity in the BM environment; ii) directing BM egress of granulocytes that “pave a road” for HSPCs; and iii) inducing secretion of cationic peptides from activated granulocytes that prime HSPC egress (Leukemia 2009; in press). In this study, we sought to determine which major chemottractant is present in PB that is responsible for egress of HSPCs and whether activation of CC plays some role in its level/expression. We noticed that plasma derived from normal and mobilized PB strongly chemoattracts murine and human HSPCs. This chemotactic effect was not dependent on plasma SDF-1 levels because: i) it occurs unaffectedly in the presence of CXCR4 antagonist AMD3100; ii) it was still robust to heat-inactivated sera; and iii) ELISA studies revealed negligible concentrations of SDF-1, which did not correlate with good or poor mobilizer status. However, to our surprise, we noticed that plasma isolated from G-CSF-mobilized mice and patients contains traces of free hemoglobin, which suggests some level of hemolysis occurs in mobilized PB. As such, we performed chemotactic assays in the presence of different concentrations of lysed erythrocytes and noticed that such diluted lysates are potent chemoattractants for HSPCs. The chemotactic activities of these lysates were resistant to heat inactivation similarly as patient sera. Based on this, we focused on S1P, a thermo-resistant lipid that, as reported, chemoattracts HSPCs and whose major reservoirs are erythrocytes (FASEB J 2007:21;1202). In fact we found by ELISA that S1P level increases during mobilization in PB and that SP1 is the most potent chemoattractant for BM-residing HSPCs, much stronger than SDF-1 - if both compounds are compared in physiologically relevant concentrations. Furthermore, activation of S1P receptors on BM-derived HSPCs augmented responsiveness to SDF-1 gradient up to 50%. However, these chemotactic effects of S1P were not visible for previously mobilized PB or umbilical cord blood HSPCs, which we explain by a fact that these mobilized cells are already desensitized to S1P gradient. Therefore, we propose the following scenario. First, a mobilizing agent (e.g., G-CSF) induces activation of CC in BM that subsequently contributes to the release of protelolytic enzymes from granulocytes that perturb SDF-1-CXCR4/VLA-4-VCAM1 interactions and stimulate egress of activated granulocytes from BM that “pave a road” for egress of HSPCs. Simultaneously, the final product of CC activation (C5b-C9), the membrane attack complex (MAC), induces in BM sinusoids the release of S1P from erythrocytes. S1P accumulating in BM sinusoids and cationic peptides released from activated granulocytes, but not changes in plasma SDF-1 levels, are crucial executors of HSPCs egress from BM into PB. Thus, our results provide novel evidence that CC activation/membrane attack complex (MAC)-induced elevated plasma S1P level is essential for egress/mobilization of HSPCs. Disclosures: No relevant conflicts of interest to declare.

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