CXC Chemokine Receptor 4 Activation Research Articles

In vitro effects and mathematical modelling of CTCE-9908 (a chemokine receptor 4 antagonist) on melanoma cell survival.

CTCE-9908, a CXC chemokine receptor 4 (CXCR4) antagonist, prevents CXCR4 phosphorylation and inhibits the interaction with chemokine ligand 12 (CXCL12) and downstream signalling pathways associated with metastasis. This study evaluated the in vitro effects of CTCE-9908 on B16 F10 melanoma cells with the use of mathematical modelling. Crystal violet staining was used to construct a mathematical model of CTCE-9908 B16 F10 (melanoma) and RAW 264.7 (non-cancerous macrophage) cell lines on cell viability to predict the half-maximal inhibitory concentration (IC50). Morphological changes were assessed using transmission electron microscopy. Flow cytometry was used to assess changes in cell cycle distribution, apoptosis via caspase-3, cell survival via extracellular signal-regulated kinase1/2 activation, CXCR4 activation and CXCL12 expression. Mathematical modelling predicted IC50 values from 0 to 100 h. At IC50, similar cytotoxicity between the two cell lines and ultrastructural morphological changes indicative of cell death were observed. At a concentration 10 times lower than IC50, CTCE-9908 induced inhibition of cell survival (p = 0.0133) in B16 F10 cells but did not affect caspase-3 or cell cycle distribution in either cell line. This study predicts CTCE-9908 IC50 values at various time points using mathematical modelling, revealing cytotoxicity in melanoma and non-cancerous cells. CTCE-9908 significantly inhibited melanoma cell survival at a concentration 10 times lower than the IC50 in B16 F10 cells but not RAW 264.7 cells. However, CTCE-9908 did not affect CXCR4 phosphorylation, apoptosis,\ or cell cycle distribution in either cell line.

Simultaneous activation of CXC chemokine receptor 4 and histamine receptor H1 enhances calcium signaling and cancer cell migration

C-X-C chemokine receptor 4 (CXCR4) is widely overexpressed in various types of cancer and is involved in several cancer phenotypes including tumor growth, survival, and metastasis. The roles of histamine and histamine receptor H1 (HRH1) in cancer pathogenesis remain controversial. Here, we show that HRH1 is widely expressed in various cancer cell lines and cancer tissues and that coexpression of CXCR4 and HRH1 is associated with poor prognosis in breast cancer. Using bimolecular fluorescence complementation and bioluminescence resonance energy transfer donor saturation assays, we demonstrate that CXCR4 and HRH1 can assemble into a heteromeric complex. Simultaneous activation of CXCR4 and HRH1 synergistically increases calcium flux in MDA-MB-231 cells that endogenously express CXCR4 and HRH1 but not in cells deficient in CXCR4 or HRH1. Costimulation of CXCR4 and HRH1 also significantly enhances CXCL12-induced MDA-MB-231 cell migration, while histamine alone does not induce cell migration. Synergistic effects on calcium flux and cell migration are inhibited by the Gαi inhibitor pertussis toxin and the Gαq inhibitor YM254890, suggesting that the Gαi and Gαq pathways are involved in the synergy. Enhanced calcium signaling and cell migration are also observed in NCI-H23 and HeLa cells, which coexpress CXCR4 and HRH1. Taken together, our findings demonstrate an interplay between CXCR4 and HRH1, and suggest the possibility of the CXCR4-HRH1 heteromer as a potential therapeutic target for anticancer therapy.

C-X-C chemokine receptor type 7 antibody enhances neural plasticity after ischemic stroke.

Stromal cell-derived factor-1 and its receptor C-X-C chemokine receptor 4 (CXCR4) have been shown to regulate neural regeneration after stroke. However, whether stromal cell-derived factor-1 receptor CXCR7, which is widely distributed in the developing and adult central nervous system, participates in neural regeneration remains poorly understood. In this study, we established rat models of focal cerebral ischemia by injecting endothelin-1 into the cerebral cortex and striatum. Starting on day 7 after injury, CXCR7-neutralizing antibody was injected into the lateral ventricle using a micro drug delivery system for 6 consecutive days. Our results showed that CXCR7-neutralizing antibody increased the total length and number of sprouting corticospinal tract fibers in rats with cerebral ischemia, increased the expression of vesicular glutamate transporter 1 and growth-related protein 43, markers of the denervated spinal cord synapses, and promoted the differentiation and maturation of oligodendrocyte progenitor cells in the striatum. In addition, CXCR7 antibody increased the expression of CXCR4 in the striatum, increased the protein expression of RAS and ERK1/2 associated with the RAS/ERK signaling pathway, and improved rat motor function. These findings suggest that CXCR7 improved neural functional recovery after ischemic stroke by promoting axonal regeneration, synaptogenesis, and myelin regeneration, which may be achieved by activation of CXCR4 and the RAS/ERK1/2 signaling pathway.

Fermented Ginger Extract in Natural Deep Eutectic Solvent Enhances Cytotoxicity by Inhibiting NF-κB Mediated CXC Chemokine Receptor 4 Expression in Oxaliplatin-Resistant Human Colorectal Cancer Cells.

Ginger extracts have been shown to have health-promoting pharmacological activity and beneficial effects, including antioxidant and anticancer properties. The extraction of ginger by natural deep eutectic solvents (NaDES) has been shown to enhance bioactivity, but the cytotoxicity of NaDES extracts needs to be further determined. Signaling through the CXC chemokine receptor 4 (CXCR4) expressed on colorectal cancer (CRC) cells has a pivotal role in tumor cell chemosensitivity. Oxaliplatin is a third-generation platinum compound used as an effective chemotherapeutic drug for CRC treatment. However, whether ginger extract and oxaliplatin could induce a synergistic cytotoxic effect in oxaliplatin-resistant CRC cells through modulating CXCR4 expression is not known. In this study, oxaliplatin-resistant HCT-116 (HCT-116/R) cells were generated first. Ginger was extracted using the NaDES mixture betaine/lactate/water (1:2:2.5). Lactobacillus reuteri fermentation of NaDES-ginger extract increased the total polyphenol content (12.42 mg gallic acid/g in non-fermented NaDES-ginger extract and 23.66 mg gallic acid/g in fermented NaDES-ginger extract). It also increased the antioxidant activity by about 20–30% compared to non-fermented NaDES-ginger extract. In addition, it achieved low cytotoxicity to normal colonic mucosal cells and enhanced the anticancer effect on HCT-116/R cells. On the other hand, the inhibition of NF-κB activation by fermented NaDES-ginger extract significantly decreased the CXCR4 expression (p < 0.05) in HCT-116/R cells. The inactivation of NF-κB by pharmacological inhibitor pyrrolidine dithiocarbamate further enhanced the fermented NaDES-ginger extract-reduced CXCR4 expression levels (p < 0.05). Moreover, fermented NaDES-ginger extract could synergistically increase the cytotoxicity of oxaliplatin by inhibiting CXCR4 expression and inactivating NF-κB, resulting in HCT-116/R cell death. These findings demonstrate that fermented NaDES-ginger extract reduces the NF-kB-mediated activation of CXCR4 and enhances oxaliplatin-induced cytotoxicity in oxaliplatin-resistant CRC cells.

Association of Cytoplasmic CXCR4 With Loss of Epithelial Marker and Activation of ERK1/2 and AKT Signaling Pathways in Non–Small-Cell Lung Cancer

ObjectivesCompelling evidence demonstrates that CXC-chemokine receptor 4 (CXCR4) is involved in tumor invasion, angiogenesis, metastasis, and resistance to chemotherapy in addition to being one of the coreceptors for T-tropic human immunodeficiency virus entry into T cells. However, it remains controversial as to how to identify functionally activated CXCR4 in tumor biopsies, which would assist in determining which patients may benefit from potential CXCR4-targeted therapy. Materials and MethodsImmunohistochemistry (IHC) staining on archival tissues of patients with non–small-cell lung cancer (NSCLC) was used to detect a panel of biomarkers, including phospho-ERK1/2, phospho-AKT, and E-cadherin, which are relevant to downstream signaling of CXCR4 and epithelial to mesenchymal transition (EMT). We also examined whether subcellular localization of CXCR4 could help define possible activation of CXCR4. ResultsA total of 94 primary tumor tissue samples from patients with NSCLC were included. Sixty-six patients had both cytomembranous and nuclear staining of CXCR4, 22 had solely nuclear staining, 5 had solely cytomembranous staining, and 1 had negative staining. Cytoplasmic location of CXCR4 with or without nuclear location was associated with loss of the epithelial marker E-cadherin (P = .0015) and activation of ERK1/2 (P = .0121) and AKT (P = .0024), suggesting EMT in these tumors; whereas tumors with only nuclear location of CXCR4 were more indolent and preserved an epithelial phenotype. ConclusionsOur study suggests that different subcellular localization of CXCR4 may be associated with different activation states; cytoplasmic CXCR4 seems to correlate with biomarker changes associated with EMT in NSCLC.

EZH2-mediated loss of miR-622 determines CXCR4 activation in hepatocellular carcinoma.

The CXC chemokine receptor 4 (CXCR4) exerts a variety of functions at different steps of hepatocellular carcinoma (HCC) progression. The molecular mechanisms and therapeutic value of CXCR4 in the development of HCC remain undefined. Here we show that aberrant CXCR4 overexpression is associated with poor prognosis and aggressive characteristics of HCC. Suppression of CXCR4 activity via CXCR4 knockdown, AMD3100 or neutralizing antibody administration inhibits hepatoma cell tumorigenesis in vitro and in vivo. CXCR4 overexpression displays the opposite effects. Using Mir library screening we identify miR-622 as a regulator of CXCR4. Further studies show that miR-622 directly target the 3′ untranslated region of CXCR4 and is transcriptionally repressed by EZH2-induced H3K27 trimethylation and promoter methylation. EZH2/miR-622 promotes tumorigenesis through CXCR4. EZH2-mediated loss of miR-622 is found to correlate with CXCR4 overexpression and unfavourable prognosis in HCC patients. This study establishes EZH2/miR-622/CXCR4 as a potential adverse prognostic factor and therapeutic target for HCC patients.

CXCR4 Antagonists: A Screening Strategy for Identification of Functionally Selective Ligands

The CXC chemokine receptor 4 (CXCR4) is a widely expressed G protein–coupled receptor implicated in several diseases. In cancer, an increased number of surface CXCR4 receptors, in parallel with aberrant signaling, have been reported to influence several aspects of malignancy progression. CXCR4 activation by the specific ligand C-X-C motif chemokine 12 (CXCL12) induces several intracellular signaling pathways that have been selectively related to malignancy depending on the tissue or cell type. We developed a panel of CXCR4 screening assays investigating Gαi-mediated cyclic adenosine monophosphate modulation, β-arrestin recruitment, and receptor internalization. All of the assays were set up in recombinant cells and were used to test four reported CXCR4 antagonists. Consequently, a set of hit compounds, deriving from a screening campaign of a 30,000-small-molecule internal library, was profiled with the different assays. We identified several compounds showing a pathway-selective activity: antagonists on a Gαi-dependent pathway; antagonists on both the β-arrestin and Gαi-dependent pathways, some of which induce receptor internalization; and compounds with an antagonist behavior in all of the readouts. The identified biased antagonists induce different functional states on CXCR4 and preferentially affect specific downstream responses from the activated receptor, thus providing an improved therapeutic profile for correction of CXCR4 abnormal signaling.

The Disparate Twins: A Comparative Study of CXCR4 and CXCR7 in SDF-1α–Induced Gene Expression, Invasion and Chemosensitivity of Colon Cancer

In colorectal cancer, increased expression of the CXC chemokine receptor 4 (CXCR4) has been shown to provoke metastatic disease due to the interaction with its ligand stromal cell-derived factor-1 (SDF-1). Recently, a second SDF-1 receptor, CXCR7, was found to enhance tumor growth in solid tumors. Albeit signaling cascades via SDF-1/CXCR4 have been intensively studied, the significance of the SDF-1/CXCR7-induced intracellular communication triggering malignancy is still only marginally understood. In tumor tissue of 52 patients with colorectal cancer, we observed that expression of CXCR7 and CXCR4 increased with tumor stage and tumor size. Asking whether activation of CXCR4 or CXCR7 might result in a similar expression pattern, we performed microarray expression analyses using lentivirally CXCR4- and/or CXCR7-overexpressing SW480 colon cancer cell lines with and without stimulation by SDF-1α. Gene regulation via SDF-1α/CXCR4 and SDF-1α/CXCR7 was completely different and partly antidromic. Differentially regulated genes were assigned by gene ontology to migration, proliferation, and lipid metabolic processes. Expressions of AKR1C3, AXL, C5, IGFBP7, IL24, RRAS, and TNNC1 were confirmed by quantitative real-time PCR. Using the in silico gene set enrichment analysis, we showed that expressions of miR-217 and miR-218 were increased in CXCR4 and reduced in CXCR7 cells after stimulation with SDF-1α. Functionally, exposure to SDF-1α increased invasiveness of CXCR4 and CXCR7 cells, AXL knockdown hampered invasion. Compared with controls, CXCR4 cells showed increased sensitivity against 5-FU, whereas CXCR7 cells were more chemoresistant. These opposing results for CXCR4- or CXCR7-overexpressing colon carcinoma cells demand an unexpected attention in the clinical application of chemokine receptor antagonists such as plerixafor.

Anti-invasive effects of celastrol in hypoxia-induced fibroblast-like synoviocyte through suppressing of HIF-1α/CXCR4 signaling pathway

Rheumatoid arthritis (RA) joints are in a hypoxic condition. Hypoxia-induced migration and invasion of fibroblast-like synoviocytes (FLSs) are considered to play a critical role in the pathogenesis of RA. Among the key genes upregulated by hypoxia-inducible factor-1α (HIF-1α), CXC chemokine receptor 4 (CXCR4) plays an important role in FLS migration and invasion. Our previous studies have shown that celastrol exerts anti-arthritic effects by inhibiting FLS migration and invasion under normoxic conditions. However, the effect and molecular mechanisms underlying the effect of celastrol on hypoxia-induced FLS migration and invasion are poorly understood. In the present study, we assessed the effect of celastrol on hypoxia-induced FLS migration and invasion. Results showed that celastrol suppressed hypoxia-induced FLS migration and invasion. In addition, we also found that celastrol inhibited hypoxia-induced CXCR4 expression at both the mRNA and the protein levels in RA-FLSs. Meanwhile, it is revealed that celastrol inhibited the transcriptional activity of CXCR4 under hypoxic conditions by suppressing the binding activity of HIF-1α in the CXCR4 promoter, and blocked hypoxia-induced accumulation of nuclear HIF-1α. Furthermore, treatment with HIF-1α inhibitor reduced the hypoxia-induced expression and transcriptional activity of CXCR4. In conclusion, our results indicate that celastrol inhibits hypoxia-induced migration and invasion via suppression of HIF-1α mediated CXCR4 expression in FLSs under hypoxic conditions. These results provide a strong rationale for further testing and validation of the use of celastrol as a new alternative for using in the treatment of RA.

Whole Genome Sequencing Identifies Recurring Somatic Mutations in the C-Terminal Domain of CXCR4, Including a Gain of Function Mutation in Waldenstrom's Macroglobinemia.

Abstract 2715 Introduction:Waldenstrom's Macroglobulinemia (WM) is an indolent non-Hodgkin's lymphoma characterized by the accumulation of IgM secreting lymphoplasmacytic cells (LPC) in the bone marrow. Using paired normal/WM lymphoplasmacytic cell paired tissues and whole genome sequencing (WGS), we identified somatic mutations in the CXC chemokine receptor 4 (CXCR4) gene which were present in 16/55 (29%) WM patients. CXCR4 is a G-protein-coupled receptor, together with its ligand, the stromal cell- derived factor-1(CXCL12/SDF-1), play an important role in leukocyte and lymphocyte hematopoiesis and trafficking. Upon SDF-1 stimulation, CXCR4 is phosphorylated and interacts with b-arrestins, which then trigger extracellular signal-regulated kinase (ERK) MAPKs and chemotaxis. CXCR4 signaling is then terminated through receptor internalization which is mediated via phosphorylation of its C-terminal tail. Methods:Sanger sequencing was used to validate WGS results. To clarify the functional significance of one of the most common somatic mutation identified (C1013G), cloning by PCR was undertaken from CD19+ bone marrow cells from a WM patient with the C1013G CXCR4 (C1013G-CXCR4) mutation. Wild type (WT) and C1013G-CXCR4 cDNAs were subcloned into plenti-IRES-GFP vector, and transduced using an optimized lentiviral based strategy for WM cells into BCWM.1 WM cells. Five days after transduction, GFP positive cells were sorted and used for functional studies. Surface expression of CXCR4 was determined by FACS using PE-conjugated anti-CXCR4 monoclonal antibody 12G5. CXCR4 internalization was studied by comparing CXCR4 surface expression before and after SDF-1 stimulation. Chemotaxis studies were performed using a transwell assay. The expression of phosphorylated ERK1/2 and total ERK2 was determined by western blot. Results:Validated somatic mutations in CXCR4 were all present in the C-terminal domain and included premature stop codons (C1013A; C1013G), and frameshift mutations. Importantly, the identified mutations are similar to those reported in the germline of patients with WHIM (Warts, Hypogammaglobulinemia, Infections and Myelokathexis) Syndrome, a dominant autosomal genetic disorder caused by tonic CXCR4 activation by impairment of CXCR4 internalization, and stimulation of G-protein-dependent responses, and chemotaxis. Consistent with such a role, SDF-1a stimulation showed decreased internalization of CXCR4 in C1013G-CXCR4 versus WT CXCR4 transduced BCWM.1 WM cells. SDF-1a stimulated ERK1/2 phosphorylation was also more robust C1013G-CXCR4 versus WT CXCR4 expressing cells. Lastly, C1013G-CXCR4 transduced cells displayed stronger migratory response toward SDF-1a versus WT CXCR4 expressing BCWM.1 cells. Conclusions:C-terminal domain somatic mutations are common in WM and overlap with germline mutation identified in WHIM syndrome. Moreover, the most common of these mutations (C1013G) confers gain of function including decreased CXCR4 internalization, more robust ERK ½ phosphorylation, and chemotaxis. The findings provide new insights into the pathogenesis of WM, and a framework for the study of CXCR4 inhibitors for WM therapy. Disclosures:Treon:Onyx: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees.

ChemInform Abstract: Drug Discovery Research Targeting the CXC Chemokine Receptor 4 (CXCR4)

AbstractReview: 203 refs.

Effects of specific DNMT gene depletion on cancer cell transformation and breast cancer cell invasion; toward selective DNMT inhibitors

A hallmark of cancer is aberrant DNA methylation, consisting of global hypomethylation and regional hypermethylation of tumor suppressor genes. DNA methyltransferase inhibitors have been recognized as promising candidate anticancer drugs. Drug development has focused on DNA methylation inhibitors with the goal of activating tumor suppressor genes silenced by DNA methylation. 5-azacytidine (5-AC; Vidaza), a global DNA methyltransferase inhibitor, was Food and Drug Administration approved to treat myelodysplastic syndromes and is clinically tested for solid tumors. In this paper, it was demonstrated that 5'-aza-2'-deoxycytidine (5-azaCdR) activated both silenced tumor suppressor genes and pro-metastatic genes by demethylation, raising the concern that it would promote metastasis. 5-AzaCdR treatment increased the invasiveness of non-invasive breast cancer cell lines MCF-7 cells and ZR-75-1 and dramatically induced pro-metastatic genes; Urokinase plasminogen activator (uPA), matrix metalloproteinase 2 (MMP2), metastasis-associated gene (H-MTS1; S100A4) and C-X-C chemokine receptor 4 (CXCR4). The hypothesis that the blocking of cellular transformation activity of DNA methyltransferase inhibitor could be separated from the pro-metastatic activity was tested using short interfering RNA (siRNA) targeted to the different DNA methyltransferase (DNMT) genes. Although depletion of DNMT1 had the strongest effect on colony growth suppression in cellular transformation assays, it did not result in demethylation and activation of uPA, S100A4, MMP2 and CXCR4 in MCF-7 cells. Depletion of DNMT1 did not induce cellular invasion in MCF-7 and ZR-75-1 non-invasive breast cancer cell lines. These data have implications on the design of new DNA methyltransferase inhibitor and on the proper utilization of current inhibitors.

Red wine consumption improves in vitro migration of endothelial progenitor cells in young, healthy individuals

Endothelial progenitor cells (EPCs) contribute to the maintenance of vascular endothelial function. The moderate consumption of red wine provides cardiovascular protection. We investigated the underlying molecular mechanism of EPC migration in young, healthy individuals who drank red wine. Fourteen healthy volunteers consumed 250 mL red wine daily for 21 consecutive days. Vascular endothelial function, plasma stromal cell-derived factor 1alpha (SDF1alpha) concentrations, and the number, migration, and nitric oxide production of EPCs were determined before and after the daily consumption of red wine. EPCs were glucose stressed to study the effect of red wine on EPC migration, proliferation, and senescence and to study the expressions of CXC chemokine receptor 4 (CXCR4) and members of the Pi3K/Akt/eNOS (phosphatidylinositol 3-kinase/protein kinase B/endothelial nitric oxide synthase) signaling pathway by Western blotting. Daily red wine consumption for 21 consecutive days significantly enhanced vascular endothelial function. Although plasma SDF1alpha concentrations were unchanged, EPC count and migration were significantly increased after this 21-d consumption period. Red wine increased the migration, proliferation, CXCR4 expression, and activity of the Pi3K/Akt/eNOS signaling pathway and decreased the extent of apoptosis in glucose-stressed EPCs. The results of the present study indicate that red wine exerts its effect through the up-regulation of CXCR4 expression and activation of the SDF1alpha/CXCR4/Pi3K/Akt/eNOS signaling pathway, which results in increased EPC migration and proliferation and decreased extent of apoptosis. Our findings suggest that these effects could be linked to the mechanism of cardiovascular protection that is associated with the regular consumption of red wine.

Tonic Activation of CXC Chemokine Receptor 4 in Immature Granule Cells Supports Neurogenesis in the Adult Dentate Gyrus

Stromal-cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) play a well-established role during embryonic development of dentate gyrus granule cells. However, little is known about the regulation and function of CXCR4 in the postnatal dentate gyrus. Here, we identify a striking mismatch between intense CXCR4 mRNA and limited CXCR4 protein expression in adult rat subgranular layer (SGL) neurons. We demonstrate that CXCR4 protein expression in SGL neurons is progressively lost during postnatal day 15 (P15) to P21. This loss of CXCR4 protein expression was paralleled by a reduction in the number of SDF-1-responsive SGL neurons and a massive upregulation of SDF-1 mRNA in granule cells. Intraventricular infusion of the CXCR4-antagonist AMD3100 dramatically increased CXCR4 protein expression in SGL neurons, suggesting that CXCR4 is tonically activated and downregulated by endogenous SDF-1. Infusion of AMD3100 also facilitated detection of CXCR4 protein in bromodeoxyuridine-, nestin-, and doublecortin-labeled cells and showed that the vast majority of adult-born granule cells transiently expressed CXCR4. Chronic AMD3100 administration impaired formation of new granule cells as well as neurogenesis-dependent long-term recognition of novel objects. Therefore, our findings suggest that tonic activation of CXCR4 in newly formed granule cells by endogenous SDF-1 is essential for neurogenesis-dependent long-term memory in the adult hippocampus.

Cloning and characterizing mutated human stromal cell-derived factor-1 (SDF-1): C-terminal α-helix of SDF-1α plays a critical role in CXCR4 activation and signaling, but not in CXCR4 binding affinity

A novel C-terminal alpha-helix-defective mutant of human stromal cell-derived factor-1 (SDF-1), hSDF-154, was designed and produced in order to develop an optimal CXC chemokine receptor 4 (CXCR4) antagonist. Human native SDF-1 and alpha-helix defective SDF-1 (hSDF-154) were cloned from human bone marrow stromal cells by reverse transcription polymerase chain reaction, inserted into vector pET-30a(+), and transformed into Escherichia coli strain BL21(DE3). The recombinant hSDF-154 was purified and refolded under optimized conditions and its functional characteristics were compared with the native form of SDF-1. Functional evaluation includes migration of Jurkat and MOLT4 cells assessed by chemotaxis assay, intracellular calcium influx in these cells measured by flow cytometry, extracellular signal-regulated kinase (ERK) phosphorylation analyzed by Western blot assay, receptor binding affinity examined by sequential concentrations of unlabeled SDF-1alpha, hSDF-154 competition with (125)I- SDF-1alpha, and internalization of CXCR4 on the cell surface detected by flow cytometry. hSDF-154 had significantly decreased chemotaxic ability, such as cell migration, as compared to the native hSDF-1. hSDF-154 failed to trigger CXCR4 to induce transient calcium influx and ERK phosphorylation. However, both hSDF-154 and the native hSDF-1 have similar binding affinity to CXCR4 and a similar ability to induce CXCR4 internalization. These results indicate that hSDF-154, which has a defective C-terminal alpha-helix, a normal N-terminus, and a normal central beta-strand scaffold structure, retains normal binding affinity to CXCR4 and normal induction of CXCR4 internalization, but fails to activate CXCR4-mediated cellular signaling and chemotaxis. Therefore, the C-terminal alpha-helix of hSDF-1 plays a critical role for CXCR4 stimulation. The hSDF-154, which efficiently binds to and induces internalization of CXCR4 without activating CXCR4-related intracellular signaling and cell migration, may serve as an optimal CXCR4 antagonist.

Highlights from: The 46th Annual Meeting of the American Society of Hematology; San Diego, CA, December 2004

The CXC chemokine receptor 4 (CXCR4) is a widely expressed G protein–coupled receptor implicated in several diseases. In cancer, an increased number of surface CXCR4 receptors, in parallel with aberrant signaling, have been reported to influence several aspects of malignancy progression. CXCR4 activation by the specific ligand C-X-C motif chemokine 12 (CXCL12) induces several intracellular signaling pathways that have been selectively related to malignancy depending on the tissue or cell type. We developed a panel of CXCR4 screening assays investigating Gαi-mediated cyclic adenosine monophosphate modulation, β-arrestin recruitment, and receptor internalization. All of the assays were set up in recombinant cells and were used to test four reported CXCR4 antagonists. Consequently, a set of hit compounds, deriving from a screening campaign of a 30,000-small-molecule internal library, was profiled with the different assays. We identified several compounds showing a pathway-selective activity: antagonists on a Gαi-dependent pathway; antagonists on both the β-arrestin and Gαi-dependent pathways, some of which induce receptor internalization; and compounds with an antagonist behavior in all of the readouts. The identified biased antagonists induce different functional states on CXCR4 and preferentially affect specific downstream responses from the activated receptor, thus providing an improved therapeutic profile for correction of CXCR4 abnormal signaling.

GRK6 deficiency is associated with enhanced CXCR4-mediated neutrophil chemotaxis in vitro and impaired responsiveness to G-CSF in vivo.

The stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) signaling pathway is thought to play an important role in the induction of neutrophil mobilization from the bone marrow in response to granulocyte-colony stimulating factor (G-CSF) treatment. CXCR4 belongs to the family of G protein-coupled receptors. Multiple members of this receptor family are desensitized by agonist-induced G protein-coupled receptor kinase (GRK)-mediated phosphorylation. Here, we demonstrate that in vitro SDF-1-induced chemotaxis of bone marrow-derived neutrophils from GRK6-deficient mice is significantly enhanced and that desensitization of the calcium response to SDF-1 is impaired in GRK6-/- neutrophils. CXCR4 activation by SDF-1 provides a key retention signal for hematopoietic cells in the bone marrow. It is interesting that we observed that in the absence of GRK6, the G-CSF-induced increase in circulating neutrophils is profoundly impaired. Three days after injection of pegylated-G-CSF, significantly lower numbers of circulating neutrophils were observed in GRK6-/- as compared with wild-type (WT) mice. In addition, early/acute neutrophil mobilization in response to G-CSF (3 h after treatment) was also impaired in GRK6-/- mice. However, blood neutrophil levels in untreated GRK6-/- and WT mice were not different. Moreover, the percentage of neutrophils in the bone marrow after G-CSF treatment was increased to the same extent in WT and GRK6-/- mice, indicating that neutrophil production is normal in the absence of GRK6. However, the increased chemotactic sensitivity of GRK6-/- neutrophils to SDF-1 was retained after G-CSF treatment. In view of these data, we suggest that the impaired G-CSF-induced neutrophil mobilization in the absence of GRK6 may be a result of enhanced CXCR4-mediated retention of PMN in the bone marrow.