Molecular determinants of antagonist interactions with chemokine receptors CCR2 and CCR5.

By driving monocyte chemotaxis, the chemokine receptor CCR2 shapes inflammatory responses and the formation of tumor microenvironments. This makes it a promising target in inflammation and immuno-oncology; however, despite extensive efforts, there are no FDA-approved CCR2-targeting therapeutics. Cited challenges include the redundancy of the chemokine system, suboptimal properties of compound candidates, and species differences that confound the translation of results from animals to humans. Structure-based drug design can rationalize and accelerate the discovery and optimization of CCR2 antagonists to address these challenges. The prerequisites for such efforts include an atomic-level understanding of the molecular determinants of action of existing antagonists. In this study, using molecular docking and artificial-intelligence-powered compound library screening, we uncover the structural principles of small molecule antagonism and selectivity towards CCR2 and its sister receptor CCR5. CCR2 orthosteric inhibitors are shown to universally occupy an inactive-state-specific tunnel between receptor helices 1 and 7; we also discover an unexpected role for an extra-helical groove accessible through this tunnel, suggesting its potential as a new targetable interface for CCR2 and CCR5 modulation. By contrast, only shape complementarity and limited helix 8 hydrogen bonding govern the binding of various chemotypes of allosteric antagonists. CCR2 residues S1012.63 and V2446.36 are implicated as determinants of CCR2/CCR5 and human/mouse orthosteric and allosteric antagonist selectivity, respectively, and the role of S1012.63 is corroborated through experimental gain-of-function mutagenesis. We establish a critical role of induced fit in antagonist recognition, reveal strong chemotype selectivity of existing structures, and demonstrate the high predictive potential of a new deep-learning-based compound scoring function. Finally, this study expands the available CCR2 structural landscape with computationally generated chemotype-specific models well-suited for structure-based antagonist design.

Chemokine CXCL4 interactions with extracellular matrix proteoglycans mediate widespread immune cell recruitment independent of chemokine receptors.

Leukocyte recruitment from the vasculature into tissues is a crucial component of the immune system but is also key to inflammatory disease. Chemokines are central to this process but have yet to be therapeutically targeted during inflammation due to a lack of mechanistic understanding. Specifically, CXCL4 (Platelet Factor 4, PF4) has no established receptor that explains its function. Here, we use biophysical, in vitro, and in vivo techniques to determine the mechanism underlying CXCL4-mediated leukocyte recruitment. We demonstrate that CXCL4 binds to glycosaminoglycan (GAG) sugars on proteoglycans within the endothelial extracellular matrix, resulting in increased adhesion of leukocytes to the vasculature, increased vascular permeability, and non-specific recruitment of a range of leukocytes. Furthermore, GAG sulfation confers selectivity onto chemokine localization. These findings present mechanistic insights into chemokine biology and provide future therapeutic targets.

The dual-function chemokine receptor CCR2 drives migration and chemokine scavenging through distinct mechanisms.

C-C chemokine receptor 2 (CCR2) is a dual-function receptor. Similar to other G protein-coupled chemokine receptors, it promotes monocyte infiltration into tissues in response to the chemokine CCL2, and, like atypical chemokine receptors (ACKRs), it scavenges chemokine from the extracellular environment. CCR2 therefore mediates CCL2-dependent signaling as a G protein-coupled receptor (GPCR) and also limits CCL2 signaling as a scavenger receptor. We investigated the mechanisms underlying CCR2 scavenging, including the involvement of intracellular proteins typically associated with GPCR signaling and internalization. Using CRISPR knockout cell lines, we showed that CCR2 scavenged by constitutively internalizing to remove CCL2 from the extracellular space and recycling back to the cell surface for further rounds of ligand sequestration. This process occurred independently of G proteins, GPCR kinases (GRKs), ß-arrestins, and clathrin, which is distinct from other "professional" chemokine scavenger receptors that couple to GRKs, ß-arrestins, or both. These findings set the stage for understanding the molecular regulators that determine CCR2 scavenging and may have implications for drug development targeting this therapeutically important receptor.

CCR2-Mediated Uptake of Constitutively Produced CCL2: A Mechanism for Regulating Chemokine Levels in the Blood.

C-C chemokine receptor 2 (CCR2) is a key driver of monocyte/macrophage trafficking to sites of inflammation and has long been considered a target for intervention in autoimmune disease. However, systemic administration of CCR2 antagonists is associated with marked increases in CCL2, a CCR2 ligand, in the blood. This heretofore unexplained phenomenon complicates interpretation of in vivo responses to CCR2 antagonism. We report that CCL2 elevation after pharmacological CCR2 blockade is due to interruption in a balance between CCL2 secretion by a variety of cells and its uptake by constitutive internalization and recycling of CCR2. We observed this phenomenon in response to structurally diverse CCR2 antagonists in wild-type mice, and also found substantially higher CCL2 plasma levels in mice lacking the CCR2 gene. Our findings suggest that CCL2 is cleared from blood in a CCR2-dependent but G protein (Gαi, Gαs or Gαq/11)-independent manner. This constitutive internalization is rapid: on a given monocyte, the entire cell surface CCR2 population is turned over in <30 minutes. We also found that constitutive receptor internalization/recycling and ligand uptake are not universal across monocyte-expressed chemokine receptors. For example, CXCR4 does not internalize constitutively. In summary, we describe a mechanism that explains the numerous preclinical and clinical reports of increased CCL2 plasma levels following in vivo administration of CCR2 antagonists. These findings suggest that constitutive CCL2 secretion by monocytes and other cell types is counteracted by constant uptake and internalization by CCR2-expressing cells. The effectiveness of CCR2 antagonists in disease settings may be dependent upon this critical equilibrium.

Heparan Sulfate Microarray Reveals That Heparan Sulfate-Protein Binding Exhibits Different Ligand Requirements.

Heparan sulfates (HS) are linear sulfated polysaccharides that modulate a wide range of physiological and disease-processes. Variations in HS epimerization and sulfation provide enormous structural diversity, which is believed to underpin protein binding and regulatory properties. The ligand requirements of HS-binding proteins have, however, been defined in only a few cases. We describe here a synthetic methodology that can rapidly provide a library of well-defined HS oligosaccharides. It is based on the use of modular disaccharides to assemble several selectively protected tetrasaccharides that were subjected to selective chemical modifications such as regioselective O- and N-sulfation and selective de-sulfation. A number of the resulting compounds were subjected to enzymatic modifications by 3-O-sulfotransferases-1 (3-OST1) to provide 3-O-sulfated derivatives. The various approaches for diversification allowed one tetrasaccharide to be converted into 12 differently sulfated derivatives. By employing tetrasaccharides with different backbone compositions, a library of 47 HS-oligosaccharides was prepared and the resulting compounds were used to construct a HS microarray. The ligand requirements of a number of HS-binding proteins including fibroblast growth factor 2 (FGF-2), and the chemokines CCL2, CCL5, CCL7, CCL13, CXCL8, and CXCL10 were examined using the array. Although all proteins recognized multiple compounds, they exhibited clear differences in structure-binding characteristics. The HS microarray data guided the selection of compounds that could interfere in biological processes such as cell proliferation. Although the library does not cover the entire chemical space of HS-tetrasaccharides, the binding data support a notion that changes in cell surface HS composition can modulate protein function.

Differential structural remodelling of heparan sulfate by chemokines: the role of chemokine oligomerization.

Chemokines control the migration of cells in normal physiological processes and in the context of disease such as inflammation, autoimmunity and cancer. Two major interactions are involved: (i) binding of chemokines to chemokine receptors, which activates the cellular machinery required for movement; and (ii) binding of chemokines to glycosaminoglycans (GAGs), which facilitates the organization of chemokines into haptotactic gradients that direct cell movement. Chemokines can bind and activate their receptors as monomers; however, the ability to oligomerize is critical for the function of many chemokines in vivo Chemokine oligomerization is thought to enhance their affinity for GAGs, and here we show that it significantly affects the ability of chemokines to accumulate on and be retained by heparan sulfate (HS). We also demonstrate that several chemokines differentially rigidify and cross-link HS, thereby affecting HS rigidity and mobility, and that HS cross-linking is significantly enhanced by chemokine oligomerization. These findings suggest that chemokine-GAG interactions may play more diverse biological roles than the traditional paradigms of physical immobilization and establishment of chemokine gradients; we hypothesize that they may promote receptor-independent events such as physical re-organization of the endothelial glycocalyx and extracellular matrix, as well as signalling through proteoglycans to facilitate leukocyte adhesion and transmigration.

The Anti-inflammatory Protein TSG-6 Regulates Chemokine Function by Inhibiting Chemokine/Glycosaminoglycan Interactions.

TNF-stimulated gene-6 (TSG-6) is a multifunctional protein secreted in response to pro-inflammatory stimuli by a wide range of cells, including neutrophils, monocytes, and endothelial cells. It has been shown to mediate anti-inflammatory and protective effects when administered in disease models, in part, by reducing neutrophil infiltration. Human TSG-6 inhibits neutrophil migration by binding CXCL8 through its Link module (Link_TSG6) and interfering with the presentation of CXCL8 on cell-surface glycosaminoglycans (GAGs), an interaction that is vital for the function of many chemokines. TSG-6 was also found to interact with chemokines CXCL11 and CCL5, suggesting the possibility that it may function as a broad specificity chemokine-binding protein, functionally similar to those encoded by viruses. This study was therefore undertaken to explore the ability of TSG-6 to regulate the function of other chemokines. Herein, we demonstrate that Link_TSG6 binds chemokines from both the CXC and CC families, including CXCL4, CXCL12, CCL2, CCL5, CCL7, CCL19, CCL21, and CCL27. We also show that the Link_TSG6-binding sites on chemokines overlap with chemokine GAG-binding sites, and that the affinities of Link_TSG6 for these chemokines (KD values 1-85 nm) broadly correlate with chemokine-GAG affinities. Link_TSG6 also inhibits chemokine presentation on endothelial cells not only through a direct interaction with chemokines but also by binding and therefore masking the availability of GAGs. Along with previous work, these findings suggest that TSG-6 functions as a pluripotent regulator of chemokines by modulating chemokine/GAG interactions, which may be a major mechanism by which TSG-6 produces its anti-inflammatory effects in vivo.

Examining Roles of Glycans in Chemokine-Mediated Dendritic-Endothelial Cell Interactions.

Interactions between glycosaminoglycans (GAGs) and chemokines play a critical role in multiple physiological and pathological processes, including tumor metastasis and immune-cell trafficking. During our studies examining the genetic importance of the GAG subtype known as heparan sulfate (HS) on lymphatic endothelial cells (LECs), we established a repertoire of methods to assess how HS affects chemokine-mediated cell-cell interactions. In this chapter, we describe methods for monitoring migration and adhesion interactions of dendritic cells (DCs), the most potent antigen-presenting cells, with LECs. We will also report a methodology to assess chemokine-receptor interactions while incorporating approaches to target HS in the system. This includes in situ methods to visualize and quantify direct interactions between chemokines and chemokine receptors on DC surfaces, and how targeting HS produced by LECs or even DCs affects these interactions. These methods enable the mechanistic and functional characterization of GAG-chemokine interactions in cell-based studies that model physiologic interactions ex vivo. They may also be used to obtain novel insights into GAG-mediated biological processes.

The dependence of chemokine-glycosaminoglycan interactions on chemokine oligomerization.

Both chemokine oligomerization and binding to glycosaminoglycans (GAGs) are required for their function in cell recruitment. Interactions with GAGs facilitate the formation of chemokine gradients, which provide directional cues for migrating cells. In contrast, chemokine oligomerization is thought to contribute to the affinity of GAG interactions by providing a more extensive binding surface than single subunits alone. However, the importance of chemokine oligomerization to GAG binding has not been extensively quantified. Additionally, the ability of chemokines to form different oligomers has been suggested to impart specificity to GAG interactions, but most studies have been limited to heparin. In this study, several differentially oligomerizing chemokines (CCL2, CCL3, CCL5, CCL7, CXCL4, CXCL8, CXCL11 and CXCL12) and select oligomerization-deficient mutants were systematically characterized by surface plasmon resonance to determine their relative affinities for heparin, heparan sulfate (HS) and chondroitin sulfate-A (CS-A). Wild-type chemokines demonstrated a hierarchy of binding affinities for heparin and HS that was markedly dependent on oligomerization. These results were corroborated by their relative propensity to accumulate on cells and the critical role of oligomerization in cell presentation. CS-A was found to exhibit greater chemokine selectivity than heparin or HS, as it only bound a subset of chemokines; moreover, binding to CS-A was ablated with oligomerization-deficient mutants. Overall, this study definitively demonstrates the importance of oligomerization for chemokine-GAG interactions, and demonstrates diversity in the affinity and specificity of different chemokines for GAGs. These data support the idea that GAG interactions provide a mechanism for fine-tuning chemokine function.

Dual targeting of the chemokine receptors CXCR4 and ACKR3 with novel engineered chemokines.

The chemokine CXCL12 and its G protein-coupled receptors CXCR4 and ACKR3 are implicated in cancer and inflammatory and autoimmune disorders and are targets of numerous antagonist discovery efforts. Here, we describe a series of novel, high affinity CXCL12-based modulators of CXCR4 and ACKR3 generated by selection of N-terminal CXCL12 phage libraries on live cells expressing the receptors. Twelve of 13 characterized CXCL12 variants are full CXCR4 antagonists, and four have Kd values <5 nm. The new variants also showed high affinity for ACKR3. The variant with the highest affinity for CXCR4, LGGG-CXCL12, showed efficacy in a murine model for multiple sclerosis, demonstrating translational potential. Molecular modeling was used to elucidate the structural basis of binding and antagonism of selected variants and to guide future designs. Together, this work represents an important step toward the development of therapeutics targeting CXCR4 and ACKR3.

Chemokine and chemokine receptor structure and interactions: implications for therapeutic strategies.

The control of cell migration by chemokines involves interactions with two types of receptors: seven transmembrane chemokine-type G protein-coupled receptors and cell surface or extracellular matrix-associated glycosaminoglycans. Coordinated interaction of chemokines with both types of receptors is required for directional migration of cells in numerous physiological and pathological processes. Accumulated structural information, culminating most recently in the structure of a chemokine receptor in complex with a chemokine, has led to a view where chemokine oligomers bind to glycosaminoglycans through epitopes formed when chemokine subunits come together, while chemokine monomers bind to receptors in a pseudo two-step mechanism of receptor activation. Exploitation of this structural knowledge has and will continue to provide important information for therapeutic strategies, as described in this review.

Multiple glycosaminoglycan-binding epitopes of monocyte chemoattractant protein-3/CCL7 enable it to function as a non-oligomerizing chemokine.

The interaction of chemokines with glycosaminoglycans (GAGs) facilitates the formation of localized chemokine gradients that provide directional signals for migrating cells. In this study, we set out to understand the structural basis and impact of the differing oligomerization propensities of the chemokines monocyte chemoattractant protein (MCP)-1/CCL2 and MCP-3/CCL7 on their ability to bind GAGs. These chemokines provide a unique comparison set because CCL2 oligomerizes and oligomerization is required for its full in vivo activity, whereas CCL7 functions as a monomer. To identify the GAG-binding determinants of CCL7, an unbiased hydroxyl radical footprinting approach was employed, followed by a focused mutagenesis study. Compared with the size of the previously defined GAG-binding epitope of CCL2, CCL7 has a larger binding site, consisting of multiple epitopes distributed along its surface. Furthermore, surface plasmon resonance (SPR) studies indicate that CCL7 is able to bind GAGs with an affinity similar to CCL2 but higher than the non-oligomerizing variant, CCL2(P8A), suggesting that, in contrast to CCL2, the large cluster of GAG-binding residues in CCL7 renders oligomerization unnecessary for high affinity binding. However, the affinity of CCL7 is more sensitive than CCL2 to the density of heparan sulfate on the SPR surfaces; this is likely due to the inability of CCL7 to oligomerize because CCL2(P8A) also binds significantly less tightly to low than high density heparan sulfate surfaces compared with CCL2. Together, the data suggest that CCL7 and CCL2 are non-redundant chemokines and that GAG chain density may provide a mechanism for regulating the accumulation of chemokines on cell surfaces.

Lymphatic specific disruption in the fine structure of heparan sulfate inhibits dendritic cell traffic and functional T cell responses in the lymph node.

Dendritic cells (DCs) are potent APCs essential for initiating adaptive immunity. Following pathogen exposure, trafficking of DCs to lymph nodes (LNs) through afferent lymphatic vessels constitutes a crucial step in the execution of their functions. The mechanisms regulating this process are poorly understood, although the involvement of certain chemokines in this process has recently been reported. In this study, we demonstrate that genetically altering the fine structure (N-sulfation) of heparan sulfate (HS) specifically in mouse lymphatic endothelium significantly reduces DC trafficking to regional LNs in vivo. Moreover, this alteration had the unique functional consequence of reducing CD8(+) T cell proliferative responses in draining LNs in an ovalbumin immunization model. Mechanistic studies suggested that lymphatic endothelial HS regulates multiple steps during DC trafficking, including optimal presentation of chemokines on the surface of DCs, thus acting as a co-receptor that may function "in trans" to mediate chemokine receptor binding. This study not only identifies novel glycan-mediated mechanisms that regulate lymphatic DC trafficking, but it also validates the fine structure of lymphatic vascular-specific HS as a novel molecular target for strategies aiming to modulate DC behavior and/or alter pathologic T cell responses in lymph nodes.

A general method for site specific fluorescent labeling of recombinant chemokines.

Chemokines control cell migration in many contexts including development, homeostasis, immune surveillance and inflammation. They are also involved in a wide range of pathological conditions ranging from inflammatory diseases and cancer, to HIV. Chemokines function by interacting with two types of receptors: G protein-coupled receptors on the responding cells, which transduce signaling pathways associated with cell migration and activation, and glycosaminoglycans on cell surfaces and the extracellular matrix which organize and present some chemokines on immobilized surface gradients. To probe these interactions, imaging methods and fluorescence-based assays are becoming increasingly desired. Herein, a method for site-specific fluorescence labeling of recombinant chemokines is described. It capitalizes on previously reported 11-12 amino acid tags and phosphopantetheinyl transferase enzymes to install a fluorophore of choice onto a specific serine within the tag through a coenzyme A-fluorophore conjugate. The generality of the method is suggested by our success in labeling several chemokines (CXCL12, CCL2, CCL21 and mutants thereof) and visualizing them bound to chemokine receptors and glycosaminoglycans. CXCL12 and CCL2 showed the expected co-localization on the surface of cells with their respective receptors CXCR4 and CCR2 at 4 °C, and co-internalization with their receptors at 37 °C. By contrast, CCL21 showed the presence of large discrete puncta that were dependent on the presence of both CCR7 and glycosaminoglycans as co-receptors. These data demonstrate the utility of this labeling approach for the detection of chemokine interactions with GAGs and receptors, which can vary in a chemokine-specific manner as shown here. For some applications, the small size of the fluorescent adduct may prove advantageous compared to other methods (e.g. antibody labeling, GFP fusion) by minimally perturbing native interactions. Other advantages of the method are the ease of bacterial expression, the versatility of labeling with any maleimide-fluorophore conjugate of interest, and the covalent nature of the fluorescent adduct.

The chemokine receptor CCR1 is constitutively active, which leads to G protein-independent, ß-arrestin-mediated internalization.

Activation of G protein-coupled receptors by their associated ligands has been extensively studied, and increasing structural information about the molecular mechanisms underlying ligand-dependent receptor activation is beginning to emerge with the recent expansion in GPCR crystal structures. However, some GPCRs are also able to adopt active conformations in the absence of agonist binding that result in the initiation of signal transduction and receptor down-modulation. In this report, we show that the CC-type chemokine receptor 1 (CCR1) exhibits significant constitutive activity leading to a variety of cellular responses. CCR1 expression is sufficient to induce inhibition of cAMP formation, increased F-actin content, and basal migration of human and murine leukocytes. The constitutive activity leads to basal phosphorylation of the receptor, recruitment of ß-arrestin-2, and subsequent receptor internalization. CCR1 concurrently engages Gαi and ß-arrestin-2 in a multiprotein complex, which may be accommodated by homo-oligomerization or receptor clustering. The data suggest the presence of two functional states for CCR1; whereas receptor coupled to Gαi functions as a canonical GPCR, albeit with high constitutive activity, the CCR1·ß-arrestin-2 complex is required for G protein-independent constitutive receptor internalization. The pertussis toxin-insensitive uptake of chemokine by the receptor suggests that the CCR1·ß-arrestin-2 complex may be related to a potential scavenging function of the receptor, which may be important for maintenance of chemokine gradients and receptor responsiveness in complex fields of chemokines during inflammation.

Emerging concepts and approaches for chemokine-receptor drug discovery.

IMPORTANCE OF THE FIELD: Chemokine receptors are most noted for their role in cell migration. However, inappropriate utilization or regulation of these receptors is implicated in many inflammatory diseases, cancer and HIV, making them important drug targets. AREAS COVERED IN THIS REVIEW: Allostery, oligomerization and ligand bias are presented as they pertain to chemokine receptors and their associated pathologies.Specific examples of each are described from the recent literature and their implications are discussed in terms of drug discovery efforts targeting chemokine receptors. WHAT THE READER WILL GAIN: Insight into the expanding view of the multitude of pharmacological variables that need to be considered or that may be exploited in chemokine receptor drug discovery. TAKE HOME MESSAGE: Since 2007, two drugs targeting chemokine receptors have been approved by the FDA, Maraviroc for preventing HIV infection and Mozobil™ for hematopoietic stem cell mobilization. While these successes permit optimism for chemokine receptors as drug targets, only recently has the complexity of this system begun to be appreciated. The concepts of allosteric inhibitors, biased ligands and functional selectivity raise the possibility that drugs with precisely-defined properties can be developed. Other complexities such as receptor oligomerization and tissue-specific functional states of receptors also offer opportunities for increased target and response specificity, although it will be more challenging to translate these ideas into approved therapeutics compared to traditional approaches.

Elucidating the CXCL12/CXCR4 signaling network in chronic lymphocytic leukemia through phosphoproteomics analysis.

BACKGROUND: Chronic Lymphocytic Leukemia (CLL) pathogenesis has been linked to the prolonged survival and/or apoptotic resistance of leukemic B cells in vivo, and is thought to be due to enhanced survival signaling responses to environmental factors that protect CLL cells from spontaneous and chemotherapy-induced death. Although normally associated with cell migration, the chemokine, CXCL12, is one of the factors known to support the survival of CLL cells. Thus, the signaling pathways activated by CXCL12 and its receptor, CXCR4, were investigated as components of these pathways and may represent targets that if inhibited, could render resistant CLL cells more susceptible to chemotherapy. METHODOLOGY/PRINCIPAL FINDINGS: To determine the downstream signaling targets that contribute to the survival effects of CXCL12 in CLL, we took a phosphoproteomics approach to identify and compare phosphopeptides in unstimulated and CXCL12-stimulated primary CLL cells. While some of the survival pathways activated by CXCL12 in CLL are known, including Akt and ERK1/2, this approach enabled the identification of additional signaling targets and novel phosphoproteins that could have implications in CLL disease and therapy. In addition to the phosphoproteomics results, we provide evidence from western blot validation that the tumor suppressor, programmed cell death factor 4 (PDCD4), is a previously unidentified phosphorylation target of CXCL12 signaling in all CLL cells probed. Additionally, heat shock protein 27 (HSP27), which mediates anti-apoptotic signaling and has previously been linked to chemotherapeutic resistance, was detected in a subset (approximately 25%) of CLL patients cells examined. CONCLUSIONS/SIGNIFICANCE: Since PDCD4 and HSP27 have previously been associated with cancer and regulation of cell growth and apoptosis, these proteins may have novel implications in CLL cell survival and represent potential therapeutic targets. PDCD4 also represents a previously unknown signaling target of chemokine receptors; therefore, these observations increase our understanding of alternative pathways to migration that may be activated or inhibited by chemokines in the context of cancer cell survival.

Phosphoproteomic analysis of chemokine signaling networks.

Chemokines induce a number of intracellular signaling pathways by activating second messengers (e.g. calcium) and phosphorylation cascades in order to mediate a myriad of functions including cell migration, survival and proliferation. Although there is some degree of overlap in chemokine receptor-mediated pathway activation, different chemokines will often elicit distinct signaling events. Factors such as cell type, receptor expression levels, G protein availability, and disease state will also influence the signaling response from chemokine-induced receptor activation. Improvements in mass spectrometry, enrichment strategies, and database search programs for identifying phosphopeptides have made phosphoproteomics an accessible biological tool for studying chemokine-induced phosphorylation cascades. Although signaling pathways involved in chemokine-mediated migration have been fairly well characterized, less is known regarding other signaling cascades elicited by chemokines (e.g. to induce proliferation) or the potential for distinct pathway activation in a disease state such as cancer. CXCL12(SDF-1)/CXCR4 signaling has been shown to play an important role in the survival of chronic lymphocytic leukemia (CLL) cells, and thus provides a good system for exploring chemokine signaling, particularly in the interest of survival pathway activation. In this chapter, we describe the use of immobilized metal affinity chromatography (IMAC) phosphopeptide enrichment followed by reversed-phase liquid chromatography and tandem mass spectrometry (LC-MS/MS) analysis for exploring CXCL12-mediated signaling in human CLL patient cells.

Chemokines and cancer: migration, intracellular signalling and intercellular communication in the microenvironment.

Inappropriate chemokine/receptor expression or regulation is linked to many diseases, especially those characterized by an excessive cellular infiltrate, such as rheumatoid arthritis and other inflammatory disorders. There is now overwhelming evidence that chemokines are also involved in the progression of cancer, where they function in several capacities. First, specific chemokine-receptor pairs are involved in tumour metastasis. This is not surprising, in view of their role as chemoattractants in cell migration. Secondly, chemokines help to shape the tumour microenvironment, often in favour of tumour growth and metastasis, by recruitment of leucocytes and activation of pro-inflammatory mediators. Emerging evidence suggests that chemokine receptor signalling also contributes to survival and proliferation, which may be particularly important for metastasized cells to adapt to foreign environments. However, there is considerable diversity and complexity in the chemokine network, both at the chemokine/receptor level and in the downstream signalling pathways they couple into, which may be key to a better understanding of how and why particular chemokines contribute to cancer growth and metastasis. Further investigation into these areas may identify targets that, if inhibited, could render cancer cells more susceptible to chemotherapy.