CXCR4 Agonist Research Articles

Deubiquitination of CXCR4 by USP14 Is Critical for Both CXCL12-induced CXCR4 Degradation and Chemotaxis but Not ERK Activation

The chemokine receptor CXCR4 plays important roles in the immune and nervous systems. Abnormal expression of CXCR4 contributes to cancer and inflammatory and neurodegenerative disorders. Although ligand-dependent CXCR4 ubiquitination is known to accelerate CXCR4 degradation, little is known about counter mechanisms for receptor deubiquitination. CXCL12, a CXCR4 agonist, induces a time-dependent association of USP14 with CXCR4, or its C terminus, that is not mimicked by USP2A, USP4, or USP7, other members of the deubiquitination catalytic family. Co-localization of CXCR4 and USP14 also is time-dependent following CXCL12 stimulation. The physical interaction of CXCR4 and USP14 is paralleled by USP14-catalyzed deubiquitination of the receptor; knockdown of endogenous USP14 by RNA interference (RNAi) blocks CXCR4 deubiquitination, whereas overexpression of USP14 promotes CXCR4 deubiquitination. We also observed that ubiquitination of CXCR4 facilitated receptor degradation, whereas overexpression of USP14 or RNAi-induced knockdown of USP14 blocked CXCL12-mediated CXCR4 degradation. Most interestingly, CXCR4-mediated chemotactic cell migration was blocked by either overexpression or RNAi-mediated knockdown of USP14, implying that a CXCR4-ubiquitin cycle on the receptor, rather than a particular ubiquitinated state of the receptor, is critical for the ligand gradient sensing and directed motility required for chemokine-mediated chemotaxis. Our observation that a mutant of CXCR4, HA-3K/R CXCR4, which cannot be ubiquitinated and does not mediate a chemotactic response to CXCL12, indicates the importance of this covalent modification not only in marking receptors for degradation but also for permitting CXCR4-mediated signaling. Finally, the indistinguishable activation of ERK by wild typeor 3K/R-CXCR4 suggests that chemotaxis in response to CXCL12 may be independent of the ERK cascade.

The Combination of AMD3100 Plus GROβ Rapidly Mobilizes Hematopoietic Stem Cells with Enhanced Homing, Adhesion and Survival Properties

Mobilized peripheral blood stem cells (PBSC) are the primary cell source for hematopoietic transplantation. In mice, combined single administration of AMD3100, a bicyclam antagonist of the chemokine receptor CXCR4 with GROβ, a CXCR2 agonist, rapidly mobilizes hematopoietic stem and progenitor cells (HSPC) within 15 minutes of administration that is equal or greater in magnitude to a multiday regimen of G-CSF and demonstrates significant synergy compared to either compound used alone. In non-competitive transplant models, AMD3100 plus GROβ mobilized 6.2±0.3 CRU/105 PBSC compared to 1.8±0.2, 1.9±0.2 and 2.0±0.3 CRU, respectively, for G-CSF, AMD3100 or GROβ used alone. Furthermore, AMD3100 plus GROβ mobilized long-term repopulating cells that show superior hematopoietic engrafting capacity in lethally irradiated mice. Analysis of chimerism at 6 months post competitive transplant of mobilized PBSC compared to normal bone marrow at a ratio of 1:1, showed 15±2%, 10±2% and 28±2% donor chimerism for G-CSF, AMD3100 and GROβ mobilized PBSC, respectively, whereas the equivalent number of AMD3100 plus GROβ mobilized PBSC produced 55±6% donor chimerism. In order to define the mechanism(s) responsible for increased engraftment of AMD3100 plus GROβ mobilized PBSC, we performed extensive phenotypic and functional analysis of combination AMD3100 plus GROβ mobilized HSPC and compared them to HSPC mobilized by G-CSF, AMD3100 or GROβ used alone. Flow cytometry analysis of highly enriched HSC using SLAM family receptor markers demonstrated that the AMD3100 plus GROβ mobilized PBSC population contained 1.8±0.1, 3.2±0.15 and 1.6±0.18 fold more CD150+ CD48− SKL cells, respectively, than the PBSC products mobilized by G-CSF, AMD3100 or GROβ used alone. In addition, a higher proportion of AMD3100 plus GROβ mobilized CD150+ CD48− SKL cells expressed higher levels of the adhesion receptors CD11a, CD49e and CD49d. Cell cycle analysis demonstrated that AMD3100 plus GROβ mobilized PBSC contained more cells (92±4 %) in G0/G1 phase of the cell cycle than PBSC mobilized by G-CSF (82±6%), AMD3100 (76±4%) and GROβ (85±2%) alone. In homing studies using CFSE labeled mobilized mononuclear cells injected into lethally irradiated mice, ~2-fold more SKL cells mobilized by AMD3100 plus GROβ homed to bone-marrow compared to SKL cells mobilized by G-CSF, AMD3100 or GROβ used alone. In addition, analysis of apoptosis in homed CFSE+ SKL cells indicated that 2.92±0.4% of homed SKL cells mobilized by AMD3100 plus GROβ were Annexin-Vpositive vs. 6.4±1.5%, 10.4±2.6% and 4.5±1.2%, respectively, for homed SKL cells mobilized by G-CSF, AMD3100 or GROβ alone, suggesting that mobilization by AMD3100 and GROβ in combination results in enhanced survival of mobilized HSC. Our results suggest that the enhanced engraftment observed upon transplantation of PBSC mobilized by combination treatment with AMD3100 plus GROβ in results from mobilization of a greater number of more primitive quiescent HSC that have enhanced intrinsic homing, adhesion and survival properties.

The CXCR4 Antagonist AMD3100 Stimulates the Proliferation of Myeloma Cells.

AMD3100, an antagonist of the chemokine receptor CXCR4, is about to be used clinically for the peripheral mobilization of hematopoietic stem cells, especially in patients with lymphoma and multiple myeloma. However, AMD3100 has been shown to activate a G protein coupled with CXCR4 and thus acts as a partial CXCR4 agonist in vitro. Although stromal cell-derived factor-1 (SDF-1) alone has minimal or negligible effects on the growth and survival of myeloma cells in vitro, many reports are consistent with the SDF-1/CXCR4 axis being involved in the progression of myeloma. Therefore, it is necessary to address the question of whether AMD3100 functions as a partial agonist for CXCR4 in myeloma cells, before it is released for wide clinical application. In this study, we explored whether AMD3100 affects the proliferation and survival of myeloma cells in vitro. As demonstrated previously, AMD3100 markedly inhibited the SDF-1 induced chemotaxis of myeloma cells, including three cell lines (RPMI8226, U266, and ARH77 cells), and CD138+ primary human myeloma cells. AMD3100 also induced internalization of CXCR4. SDF-1 alone did not stimulate the proliferation of these myeloma cells, nor did it rescue the cells from apoptosis induced by serum deprivation. By contrast, AMD3100 at 10−5M enhanced the proliferation of all three myeloma cell lines in serum-free condition by up to 2-folds in 3-day cultures, which was abrogated by pretreating the cells with pertussis toxin (PTX). This phenomenon was also observed with CD138+ primary human myeloma cells. In addition, AMD3100 enhanced the proliferation of U266 and ARH77 cells induced by interleukin-6 (IL-6). AMD3100 partially inhibited the apoptosis induced by serum deprivation, and the anti-apoptotic effect of AMD3100 was further enhanced in the presence of IL-6. AMD3100 on its own induced the phosphorylation of Akt and ERK1/2 but not Stat3 and p38/MAPK in RPMI8226 and U266 cells. The phosphorylation was also inhibited by pretreating the cells with PTX. The signal blocking agents wortmannin, PD98056, SB203580, and rapamycin did not affect the AMD3100-induced proliferation of RPMI8226 cells. LY294002 at concentrations not inhibiting spontaneous proliferation (5 μM or less) did not affect AMD3100-induced proliferation of the cells. AG490 inhibited spontaneous proliferation and AMD3100-induced proliferation of the cells in a dose-dependent manner. Accordingly, the signal-blocking effects were unclear. However, these results show that AMD3100 partially overcame the strong growth inhibition of myeloma cells induced by AG490. Our results indicate that AMD3100 can induce the phosphorylation of signaling molecules and stimulate the proliferation of myeloma cells, through signaling via G-protein pathway.

Abstract 5547: CXCR4 Signaling Exacerbates Vein Graft Neointimal Hyperplasia

Vein graft neointimal hyperplasia can cause vein graft failure directly or, by promoting accelerated atherosclerosis, indirectly. Because most vein graft neointimal cells originate from outside the vein graft, and because CXCR4, the SDF-1α receptor, contributes significantly to recruitment of vascular progenitor cells, we tested the hypothesis that CXCR4 activity exacerbates vein graft neointimal hyperplasia. We performed carotid artery interposition vein grafting in congenic WT and cxcr4 −/+ mice, using the inferior vena cava of WT and cxcr4 −/+ mice as vein grafts. We created 3 surgically chimeric groups, designated by vein graft donor/vein graft recipient: WT/WT, cxcr4 −/+ /WT, and cxcr4 −/+ / cxcr4 −/+ . Immunofluorescence for SDF-1, the CXCR4 agonist, was evident in 2-wk-old vein grafts (with expanding neointimas), but not in 6-wk-old vein grafts (in which neointimal hyperplasia had reached steady state). In 6-wk-old WT/WT, cxcr4 −/+ /WT, and cxcr4 −/+ / cxcr4 −/+ vein grafts, respectively, neointimal areas were 0.12 ± 0.01, 0.118 ± 0.008, and 0.066 ± 0.008 mm 2 , while medial areas were 0.20 ± 0.02, 0.26 ± 0.01, and 0.32 ± 0.02 mm 2 . Thus, relative to WT/WT grafts, neointimal area was reduced only in cxcr4 −/+ / cxcr4 −/+ vein grafts, by 47% ( p <0.01), while medial area was increased in cxcr4 −/+ /WT and cxcr4 −/+ / cxcr4 −/+ vein grafts, by 26% and 56%, respectively ( p <0.05). However, vein graft re-endothelialization was equivalent in all 3 vein graft groups (judged by immunostaining for Tie2). SMC-like cells comprised 100% of neointimal cells, but < 50% of medial cells. In cxcr4 −/+ / cxcr4 −/+ vein grafts, however, the prevalence of medial SMC actin-positive cells was 45±20% greater than in WT/WT grafts (p<0.04), while the prevalence of collagen I-positive cells was 40±5% less (p<0.01), as assessed by quantitative immunofluorescence. We conclude that CXCR4 contributes to vein graft neointimal hyperplasia through endothelium-independent mechanisms that alter homing of graft-extrinsic cells to the vein graft. Moreover, while CXCR4 activity augments neointimal hyperplasia, it reduces the medial hypertrophy that is believed to be desirable for reducing vein graft wall stress. These results suggest that CXCR4 antagonist therapy may alleviate vein graft disease.

Abstract 1962: CXCR4-mediated Bone Marrow Stem Cell Homing and Niche Maintenance Is Modulated by the Functional Status of c-kit

Objective: The bone marrow (BM) “niche”, where the stem cells are dynamically maintained, is an attractive target for cell-based cardiovascular repair. Both CXCR4 antagonist and c-kit blocking antibody can rapidly mobilize BM stem cells; however, the functional interaction between CXCR4 and c-kit remains unclear. We hypothesize that CXCR4 crosstalk with c-kit to maintain BM stem cell niche. Methods and Results: We designed an in vitro adhesion assay to mimic BM niche. Mouse total BM cells were harvested and applied onto VCAM1-coated plates for 15 min, and followed by addition of CXCR4 agonist SDF-1 or antagonist AMD3100. The adhesion of BM cells to VCAM1 resulted in marked c-kit phosphorylation. Interestingly, AMD3100 significantly decreased while SDF-1 augmented the c-kit phosphorylation, suggesting that CXCR4 may regulate the functional status of c-kit. To determine the role of c-kit in CXCR4-regulated stem cell niche maintenance, we developed an in vivo homing assay. The c-kit mutant (c-kit W/W-V ) and WT mice (n=5) were first treated with AMD3100 to disrupt CXCR4-mediated niche, and 2 h later, transplanted with eGFP-transgenic BM cells. After 3 h, BM cells were isolated and analyzed by FACS. The percentages of donor-derived eGFP+, eGFP+Lin+, and eGFP+Lin− cells were similar between the two groups. However, the donor-derived primitive stem cells, including eGFP+Lin-Sca1+ and eGFP+Lin-ckit+ cells, were significantly fewer in c-kit W/W-V mice than in WT mice (c-kit W/W-V vs. WT: 94.1% vs. 96.0%, p<0.05, and 33.82% vs. 55.27%, p<0.01, respectively), indicating that c-kit-deficient BM niche specifically affects primitive stem cell homing and maintenance. Importantly, although donor-derived CXCR4-expressing total (eGFP+CXCR4+) cells were similar in both groups, CXCR4-expressing stem cells, including eGFP+Lin-CXCR4+, eGFP+Lin-Sca1+CXCR4+, and eGFP+Lin-ckit+CXCR4+ cells, were much fewer in c-kit W/W-V than in WT mice (c-kit W/W-V vs. WT: 19.7% vs. 30.6%, p<0.01, 20.3% vs. 29.1%, P<0.05, and 6.7% vs. 17.9%, p<0.001, respectively), suggesting c-kit deficiency reduces the homing and maintenance of stem cells that express CXCR4. Conclusion: CXCR4-mediated BM stem cell homing and niche maintenance is modulated by the functional status of c-kit. This research has received full or partial funding support from the American Heart Association, AHA National Center.

Technique for Generating Three-Dimensional Alignments of Multiple Ligands from One-Dimensional Alignments

We describe and demonstrate a method for the simultaneous, fully flexible alignment of multiple molecules with a common biological activity. The key aspect of the algorithm is that the alignment problem is first solved in a lower dimensional space, in this case using the one-dimensional representations of the molecules. The three-dimensional alignment is then guided by constraints derived from the one-dimensional alignment. We demonstrate using 10 hERG channel blockers, with a total of 72 rotatable bonds, that the one-dimensional alignment is able to effectively isolate key conserved pharmacophoric features and that these conserved features can effectively guide the three-dimensional alignment. Further using 10 estrogen receptor agonists and 5 estrogen receptor antagonists with publicly available cocrystal structures we show that the method is able to produce superpositions comparable to those derived from crystal structures. Finally, we demonstrate, using examples from peptidic CXCR3 agonists, that the method is able to generate reasonable binding hypotheses.

Transcriptome of Hypoxic Immature Dendritic Cells: Modulation of Chemokine/Receptor Expression

Hypoxia is a condition of low oxygen tension occurring in inflammatory tissues. Dendritic cells (DC) are professional antigen-presenting cells whose differentiation, migration, and activities are intrinsically linked to the microenvironment. DCs will home and migrate through pathologic tissues before reaching their final destination in the lymph node. We studied the differentiation of human monocytes into immature DCs (iDCs) in a hypoxic microenvironment. We generated iDC in vitro under normoxic (iDCs) or hypoxic (Hi-DCs) conditions and examined the hypoxia-responsive element in the promoter, gene expression, and biochemical KEGG pathways. Hi-DCs had an interesting phenotype represented by up-regulation of genes associated with cell movement/migration. In addition, the Hi-DC cytokine/receptor pathway showed a dichotomy between down-regulated chemokines and up-regulated chemokine receptor mRNA expression. We showed that CCR3, CX3CR1, and CCR2 are hypoxia-inducible genes and that CCL18, CCL23, CCL26, CCL24, and CCL14 are inhibited by hypoxia. A strong chemotactic response to CCR2 and CXCR4 agonists distinguished Hi-DCs from iDCs at a functional level. The hypoxic microenvironment promotes the differentiation of Hi-DCs, which differs from iDCs for gene expression profile and function. The most prominent characteristic of Hi-DCs is the expression of a mobility/migratory rather than inflammatory phenotype. We speculate that Hi-DCs have the tendency to leave the hypoxic tissue and follow the chemokine gradient toward normoxic areas where they can mature and contribute to the inflammatory process.

Analysis of the pharmacokinetic/pharmacodynamic relationship of a small molecule CXCR3 antagonist, NBI‐74330, using a murine CXCR3 internalization assay

Pharmacokinetic/pharmacodynamic (PK/PD) models are necessary to relate the degree of drug exposure in vivo to target blockade and pharmacological efficacy. This manuscript describes a murine agonist-induced CXCR3 receptor internalization assay and demonstrates its utility for PK/PD analyses. Activated murine DO11.10 cells were incubated with agonist in the presence or absence of a CXCR3 antagonist and changes in surface CXCR3 expression were detected by flow cytometry. For PK/PD analysis, mice were dosed with a small molecule CXCR3 antagonist, NBI-74330, (100 mg kg(-1)) orally or subcutaneously and plasma samples taken at specified timepoints for the CXCR3 internalization assay. Surface CXCR3 expression was specifically decreased in response to CXCL9, CXCL10 and CXCL11. CXCL11 was the most potent CXCR3 agonist in buffer (pA50=9.23+/-0.26) and the pA50 for CXCL11 was unaltered in murine plasma (pA50=9.17+/-0.15). The affinity of a small molecule CXCR3 antagonist, NBI-74330, was obtained in the absence or presence of plasma (buffer pA2 value: 7.84+/-0.14; plasma pKB) value 6.36+/-0.01). Administration of NBI-74330 to mice resulted in the formation of an N-oxide metabolite, also an antagonist of CXCR3. Both antagonists were detectable up to 7 h post oral dose and 24 h post subcutaneous dose. Measurement of CXCR3 internalization demonstrated significant antagonism of this response ex vivo, 24 h following subcutaneous administration of NBI-74330. The CXCR3 receptor internalization assay provides a robust method for determining agonist potency orders, antagonist affinity estimates and PK/PD analyses, which discriminate between dosing regimens for the CXCR3 antagonist NBI-74330.

Allosteric Theory: Taking Therapeutic Advantage of the Malleable Nature of GPCRs

The description of the allosteric modification of receptors to affect changes in their function requires a model that considers the effects of the modulator on both agonist affinity and efficacy. A model is presented which describes changes in affinity in terms of the constant α (ratio of affinity in the presence vs the absence of modulator) and also the constant ξ (ratio of intrinsic efficacy of the agonist in the presence vs absence of modulator). This allows independent effects of both affinity and efficacy and allows the modeling of any change in the dose-response curve to an agonist after treatment with modulator. Examples are given where this type of model can predict effects of modulators that reduce efficacy but actually increase affinity of agonist (i.e. ifenprodil) and also of modulators that block the action of some agonists (the CXCR4 agonist SDF-1α by the antagonist AMD3100) but not others for the same receptor (SDF-1α peptide fragments RSVM and ASLW). ‘All models are wrong…but some are useful…’anonymous environmental scientist

Allosteric Transinhibition by Specific Antagonists in CCR2/CXCR4 Heterodimers

Chemokine receptors are presently used as targets for candidate drugs in the frame of inflammatory diseases and human immunodeficiency virus infection. They were shown to dimerize, but the functional relevance of dimerization in terms of drug action remains poorly understood. We reported previously the existence of negative binding cooperativity between the subunits of CCR2/CCR5 heterodimers. In the present study, we extend these observations to heterodimers formed by CCR2 and CXCR4, which are more distantly related. We also show that specific antagonists of one receptor inhibit the binding of chemokines to the other receptor as a consequence of their heterodimerization, both in recombinant cell lines and primary leukocytes. This resulted in a significant functional cross-inhibition in terms of calcium mobilization and chemotaxis. These data demonstrate that chemokine receptor antagonists regulate allosterically the functional properties of receptors on which they do not bind directly, with important implications on the effects of these potential therapeutic agents.

The SDF-1–CXCR4 signaling pathway: a molecular hub modulating neo-angiogenesis

Pro-angiogenic bone marrow (BM) cells include subsets of hematopoietic cells that provide vascular support and endothelial progenitor cells (EPCs), which under certain permissive conditions could differentiate into functional vascular cells. Recent evidence demonstrates that the chemokine stromal-cell derived factor-1 (SDF-1, also known as CXCL12) has a major role in the recruitment and retention of CXCR4(+) BM cells to the neo-angiogenic niches supporting revascularization of ischemic tissue and tumor growth. However, the precise mechanism by which activation of CXCR4 modulates neo-angiogenesis is not clear. SDF-1 not only promotes revascularization by engaging with CXCR4 expressed on the vascular cells but also supports mobilization of pro-angiogenic CXCR4(+)VEGFR1(+) hematopoietic cells, thereby accelerating revascularization of ischemic organs. Here, we attempt to define the multiple functions of the SDF-1-CXCR4 signaling pathway in the regulation of neo-vascularization during acute ischemia and tumor growth. In particular, we introduce the concept that, by modulating plasma SDF-1 levels, the CXCR4 antagonist AMD3100 acutely promotes, while chronic AMD3100 treatment inhibits, mobilization of pro-angiogenic cells. We will also discuss strategies to modulate the mobilization of essential subsets of BM cells that participate in neo-angiogenesis, setting up the stage for enhancing revascularization or targeting tumor vessels by exploiting CXCR4 agonists and antagonists, respectively.

Inhibition of Gαi2 Activation by Gαi3 in CXCR3-mediated Signaling

G protein-coupled receptors (GPCRs) convey extracellular stimulation into dynamic intracellular action, leading to the regulation of cell migration and differentiation. T lymphocytes express G alpha(i2) and G alpha(i3), two members of the G alpha(i/o) protein family, but whether these two G alpha(i) proteins have distinguishable roles guiding T cell migration remains largely unknown because of a lack of member-specific inhibitors. This study details distinct G alpha(i2) and G alpha(i3) effects on chemokine receptor CXCR3-mediated signaling. Our data showed that G alpha(i2) was indispensable for T cell responses to three CXCR3 ligands, CXCL9, CXCL10, and CXCL11, as the lack of G alpha(i2) abolished CXCR3-stimulated migration and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) incorporation. In sharp contrast, T cells isolated from G alpha(i3) knock-out mice displayed a significant increase in both GTPgammaS incorporation and migration as compared with wild type T cells when stimulated with CXCR3 agonists. The increased GTPgammaS incorporation was blocked by G alpha(i3) protein in a dose-dependent manner. G alpha(i3)-mediated blockade of G alpha(i2) activation did not result from G alpha(i3) activation, but instead resulted from competition or steric hindrance of G alpha(i2) interaction with the CXCR3 receptor via the N terminus of the second intracellular loop. A mutation in this domain abrogated not only G alpha(i2) activation induced by a CXCR3 agonist but also the interaction of G alpha(i3) to the CXCR3 receptor. These findings reveal for the first time an interplay of G alpha(i) proteins in transmitting G protein-coupled receptor signals. This interplay has heretofore been masked by the use of pertussis toxin, a broad inhibitor of the G alpha(i/o) protein family.

The Many Facets of SDF-1α, CXCR4 Agonists and Antagonists on Hematopoietic Progenitor Cells

Stromal cell-derived factor-1alpha (SDF-1α) has pleiotropic effects on hematopoietic progenitor cells (HPCs). We have monitored podia formation, migration, proliferation, and cell-cell adhesion of human HPC under the influence of SDF-1α, a peptide agonist of CXCR4 (CTCE-0214), a peptide antagonist (CTCE-9908), and a nonpeptide antagonist (AMD3100). Whereas SDF-1α induced migration of CD34+ cells in a dose-dependent manner, CTCE-0214, CTCE-9908, and AMD3100 did not induce chemotaxis in this concentration range albeit the peptides CTCE-0214 and CTCE-9908 increased podia formation. Cell-cell adhesion of HPC to human mesenchymal stromal cells was impaired by the addition of SDF-1α, CTCE-0214, and AMD3100. Proliferation was not affected by SDF-1α or its analogs. Surface antigen detection of CXCR4 was reduced upon treatment with SDF-1α or AMD3100 and it was enhanced by CTCE-9908. Despite the fact that all these molecules target the same CXCR4 receptor, CXCR4 agonists and antagonists have selective effects on different functions of the natural molecule.

WHIM syndrome myelokathexis reproduced in the NOD/SCID mouse xenotransplant model engrafted with healthy human stem cells transduced with C-terminus–truncated CXCR4

AbstractWHIM(warts, hypogammaglobulinemia, recurrent bacterial infection, and myelokathexis) syndrome is a rare immunodeficiency caused in many cases by autosomal dominant C-terminal truncation mutations in the chemokine receptor CXCR4. A prominent and unexplained feature of WHIM is myelokathexis (hypercellularity with apoptosis of mature myeloid cells in bone marrow and neutropenia). We transduced healthy human CD34+ peripheral blood–mobilized stem cells (PBSCs) with retrovirus vector encoding wild-type (wt) CXCR4 or WHIM-type mutated CXCR4 and studied these cells ex vivo in culture and after engraftment in a nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse xenograft model. Neither wt CXCR4 nor mutated CXCR4 transgene expression itself enhanced apoptosis of neutrophils arising in transduced PBSC cultures even with stimulation by a CXCR4 agonist, stromal cell–derived factor-1 (SDF-1 [CXCL12]). Excess wt CXCR4 expression by transduced human PBSCs enhanced marrow engraftment, but did not affect bone marrow (BM) apoptosis or the release of transduced leukocytes into PB. However, mutated CXCR4 transgene expression further enhanced BM engraftment, but was associated with a significant increase in apoptosis of transduced cells in BM and reduced release of transduced leukocytes into PB. We conclude that increased apoptosis of mature myeloid cells in WHIM is secondary to a failure of marrow release and progression to normal myeloid cell senescence, and not a direct effect of activation of mutated CXCR4.

CCR5 and CXCR4 antagonist and agonist binding sites

Several small molecule antagonists of the HIV co-receptors CCR5 and CXCR4 are being developed as HIV entry inhibitors, but side effects have been observed in clinical trials that are likely due to agonist activity and/or cross-reactivity with closely related receptors. In order to develop high resolution maps of the binding sites for these antagonists that can be exploited to improve their activity and specificity, we are synthesizing derivatives of CCR5 and CXCR4 antagonists which contain photo-crosslinking groups at a variety positions in the molecules and that retain high affinity and activity against the receptors. Derivatives of two CCR5 antagonists and one CXCR4 antagonist have been crosslinked to their receptors on cells, and the interaction sites are being mapped by mass spectrometry. Techniques for purification, crosslinking, CNBr and/or trypsin digestion, and LC/MS/MS mass spectrometry of highly hydrophobic peptides initially developed using the rhodopsin GPCR system have been applied with some success to CCR5 and CXCR4, although these represent particular challenges due to the relative paucity of Met residue cleavage sites compared to rhodopsin and resistance to tryptic digestion. Nevertheless, peptides covering most of the extracellular and transmembrane regions of CCR5 that could interact with an antagonist can be identified, and affinity purification methods are being developed to isolate CXCR4 peptides crosslinked to derivatives of the antagonist T-140 that should facilitate the identification and localization of the crosslinking sites within the proteins.

Suprasynergistic Peripheral Blood Stem Cell Mobilization in Normal and Fanconi Anemia Knockout Mice by the Combination of G-CSF Plus the CXCR4 Antagonist AMD3100 and the CXCR2 Agonist GRO β

In mice, the CXCR4 antagonist AMD3100 and the CXCR2 agonist GROβ rapidly mobilizes short and long term repopulating hematopoietic stem and progenitor cells (HSPC). Synergy in mobilization is observed using GRO plus G-CSF or AMD plus G-CSF, and we have recently shown synergy in rapid mobilization using AMD plus GROβ. In general, a common feature of mobilization is that only a relatively small percentage of HSPC egress from marrow. We therefore evaluated whether added benefit in HSPC mobilization could be attained by using all three mobilizers in combination. Although this alters the paradigm of rapid mobilization, it addresses shortcomings of poor mobilization response, requirements for multiple aphereses and the need for large numbers of HSPC in transplant and gene therapy applications. We mobilized BALB/c mice with AMD (5 mg/kg SC, 60 min), GROβ (2.5 mg/kg SC, 15 min), G-CSF (100 ug/kg/day, bid, SC x 4 days) or the G-CSF regimen followed by GROβ, AMD or GRO+AMD administered on day 5 and harvest of peripheral blood 15 (GRO; GRO+AMD) or 60 (AMD) min later. Significant CFU-GM/mL blood were mobilized by G-CSF (4362±996), GRO (2562±396) and AMD3100 (991±121) used alone as expected. Single administration of GRO or AMD to mice mobilized by G-CSF and harvest of blood 15 (GRO) and 60 (AMD) min later, resulted in synergistic mobilization of (12,246±2751) and (12,379±953) CFU-GM, respectively. Rapid mobilization by simultaneous injection of GRO+AMD was similar in magnitude (10,709±1041) at 15 min post administration to mobilization by GRO or AMD in combination with a multiday G-CSF regimen. Administration of the combination of GRO+AMD to mice mobilized by G-CSF resulted in suprasynergistic mobilization of 32,510±3569 CFU-GM/mL after 15 min, representing ~5% of total marrow CFU-GM, with no adverse effects. Fanconi Anemia patients mobilize poorly to G-CSF. FancC −/− mice present a phenotype similar to FancC patients and mobilize poorly to G-CSF, which can be improved by the addition of AMD. We evaluated mobilization by GRO, AMD and G-CSF alone and in combination in +/+ C57Bl and FancC −/− mice using the regimens described above. Mobilization by G-CSF was 45% lower in FancC −/− mice (858±21) compared to +/+ controls (1451±80) and AMD+G-CSF synergistically mobilized CFU-GM more effectively in FancC −/− mice (5078±597) than controls (2981±267). Similarly, CFU-GM mobilization by GRO was lower in FancC −/− mice and GRO+G-CSF synergistically mobilized CFU-GM more effectively in FancC −/− mice. The combination of GRO+AMD mobilized CFU-GM within 15 min that was similar in magnitude to mobilization by AMD+G-CSF in wild type (2077±541 vs 2511±176) as well as FancC −/− mice (4924±577 vs 5078±1597). Mobilization by addition of the rapid acting combination of GRO+AMD to mice mobilized by G-CSF was suprasynergistic reaching 44,669±2974 and 41,068±5630 CFU-GM/mL blood in wild type and −/− mice, respectively. In preliminary studies, transduction of mobilized blood cells with FancC and transplant in FancC −/−mice demonstrated durable engraftment. These studies identify highly effective, rapid GRO+AMD mobilization regimens for standalone application in normal donors and combination regimens for potential application in patients who respond poorly to G-CSF or when large quantities of HSPC are required, for example in gene therapy applications.

The CXCR4 Antagonist AMD3100 and the CXCR2 Agonist GROβ Synergistically Mobilize Hematopoetic Stem Cells (HSC) with Short and Long Term Repopulating Activity.

The CXCR4 antagonist AMD3100 mobilizes short and long term repopulating HSC peaking at 60 minutes post SC administration in mice. The CXCR2 agonist GROβ (CXCL2Δ4) similarly mobilizes HSC peaking at 15 minutes post SC administration, which is polymorphonuclear neutrophil (PMN) and active matrix metalloproteinase-9 (MMP-9) dependent. Based upon preliminary findings that CXCR4 signaling has a negative effect on MMP-9 release, we evaluated mobilization by the combination of AMD3100 plus GROβ. Initial sequential administration produced only additive mobilization in mice, however, simultaneous SC administration resulted in dramatic synergy in mobilization of CFU-GM, reaching >71 fold at 15 minutes post administration (8695±648/ml blood) compared to 6 fold by AMD alone (699±166) and 20 fold by GRO alone (2505±929) at 60 and 15 minutes, respectively. In addition, combination mobilization remained synergistically elevated for >2.5 hrs. Combination AMD plus GRO mobilization was significantly higher than a multiday regimen of G-CSF (100 ug/kg/day × 4 days)(2404±333 CFU-GM/ml blood). Extensive dose ranging and timing analysis confirm a requirement for early/simultaneous administration of both compounds for maximal mobilization response. Spearman's Rank analysis showed a direct association between synergistic mobilization and synergistically elevated plasma proMMP-9 levels (r=0.84; P<0.001), suggesting that CXCR4 antagonism upregulates signaling through CXCR2 resulting in enhanced release of proMMP-9. Synergistic mobilization and elevaton of plasma MMP-9 by AMD plus GROβ, like GROβ, demonstrated inter-strain variability, being highest in DBA and lowest in C57Bl mice, while no strain variability was observed for AMD alone. Synergistic mobilization was absent in mice depleted of PMN with anti-GR-1 monoclonal antibody, in MMP-9 knockouot mice, and in normal mice treated with a monoclonal antibody that blocks MMP-9 activation, consistent with a requirement for synergistically enhanced PMN derived MMP-9 in response to AMD plus GROβ. A partial MMP-9 requirement was observed for mobilization induced by AMD alone. In limiting dilution transplant studies, peripheral blood mononuclear cells (PBMC) mobilized by GROβ or AMD alone resulted in 80% and 60% survival of lethally irradiated mice at 180 days, respectively. The combination of GRO plus AMD mobilized significantly more LTRC with 100% survival observed using 2×106 cells and 50% survival at 2×104 cells, whereas PBMC mobilized by GRO or AMD alone provided no survival at 2×104 cells. In limiting dilution primary competitive congenic mouse transplant studies, both GRO and AMD alone mobilized competitive repopulating units (CRU) with the combination mobilizing significantly more CRU than expected, using nonparamatric ANOVA analysis. In addition, AMD plus GROβ mobilized significantly more CRU (2–4 fold) than G-CSF. A competitive advantage for PBSC mobilized by AMD plus GRO was more obvious in secondary non competitive transplants, where marrow cells from mice transplanted with PBSC mobilized by AMD plus GRO showed 89–99% engraftment in secondary recipients over a diluton range of 1:1 to 4:1 mobilized PBMC to congenic bone marrow. In summary, these studies suport a significant role of chemokines and chemokine receptors in HSC mobiliztion and identify a novel highly effective mobilizing regimen for rapid mobilization of HSC with enhanced repopulating activity.

HIV-1 coreceptors CCR5 and CXCR4 both mediate neuronal cell death but CCR5 paradoxically can also contribute to protection

The chemokine receptors CCR5 and CXCR4 serve, in addition to CD4, as coreceptors for human immunodeficiency virus-1 (HIV-1), and infection with HIV-1 can cause dementia. In brain-derived cells, HIV-1 envelope glycoprotein gp120 initiates a signaling cascade that involves p38 mitogen-activated protein kinase and leads to neuronal cell death. Using mixed neuronal/glial cultures from rats and mice genetically deficient in one or both HIV coreceptors, we show here that CCR5, CXCR4 or both can mediate HIV/gp120 neurotoxicity depending on the viral strain. Paradoxically, we also found evidence for a CCR5-mediated neuroprotective pathway. We identify protein kinase Akt/PKB as an essential component of this pathway, which can be triggered by the CCR5 agonists macrophage inflammatory protein-1beta and regulated-and-normal-T-cell-expressed-and-secreted. Moreover, these CCR5 ligands prevent neuronal cell death induced by stromal cell-derived factor-1, a CXCR4 agonist. Both neurons and glia coexpress CXCR4 and CCR5. Ca2+ imaging experiments demonstrate that engagement of CCR5 prevents CXCR4-triggered increases in intracellular free Ca2+. This finding suggests that CCR5 ligands can protect neurons at least, in part, by modulating CXCR4-mediated toxicity through heterologous desensitization.

Characterization of the molecular pharmacology of AMD3100: A specific antagonist of the G-protein coupled chemokine receptor, CXCR4

The chemokine receptor CXCR4 is widely expressed on different cell types, is involved in leukocyte chemotaxis, and is a co-receptor for HIV. AMD3100 has been shown to be a CXCR4 receptor antagonist, and to block HIV infection of T-tropic, X4-using, virus in vitro and in vivo. AMD3100 is an effective mobilizer of hematopoietic stem cells and is being investigated in clinical trials in multiple myeloma and non-Hodgkins lymphoma patients. Using the CCRF–CEM T-cell line that constitutively expresses CXCR4 we confirmed that AMD3100 was an antagonist of SDF-1/CXCL12 ligand binding (IC 50 = 651 ± 37 nM). We have also shown that AMD3100 inhibits SDF-1 mediated GTP-binding (IC 50 = 27 ± 2.2 nM), SDF-1 mediated calcium flux (IC 50 = 572 ± 190 nM), and SDF-1 stimulated chemotaxis (IC 50 = 51 ± 17 nM). AMD3100 did not inhibit calcium flux against cells expressing CXCR3, CCR1, CCR2b, CCR4, CCR5 or CCR7 when stimulated with their cognate ligands, nor did it inhibit receptor binding of LTB 4. AMD3100 did not, on its own, induce a calcium flux in the CCRF–CEM cells, which express multiple GPCRs including CXCR4, CCR4 and CCR7. Furthermore, AMD3100 neither stimulated GTP-binding, an assay for GPCR activation, in CEM cell membranes; nor chemotaxis of CCRF–CEM cells. These data therefore demonstrate that AMD3100 is a specific antagonist of CXCR4, is not cross-reactive with other chemokine receptors, and is not an agonist of CXCR4.

Structural determinants of unexpected agonist activity in a retro-peptide analogue of the SDF-1α N-terminus

We have synthesised two retro-peptide analogues of the stromal cell derived growth factor 1 (SDF-1α) segment known to be critical for CXCR4 receptor binding, corresponding to the sequences HSEFFRCPCRFFESH and HSEFFRGGGRFFESH. We have assayed the ability of these peptides to activate extracellular signal-regulated kinase 1/2 phosphorylation in cells over expressing the SDF-1α receptor, finding that the first variant was able to serve as an agonist of CXCR4, whereas the second one was inactive. Finally, by comparing representative solution structures of the two peptides, we have found that the biological response of HSEFFRCPCRFFESH may be ascribed to a β-β-type turn motif centred on Phe 4–Phe 5.