Atypical Chemokine Receptor Research Articles

Discovery and Development of First-in-Class ACKR3/CXCR7 Superagonists for Platelet Degranulation Modulation.

The atypical chemokine receptor 3 (ACKR3), formerly known as CXC-chemokine receptor 7 (CXCR7), has been postulated to regulate platelet function and thrombus formation. Herein, we report the discovery and development of first-in-class ACKR3 agonists, which demonstrated superagonistic properties with Emax values of up to 160% compared to the endogenous reference ligand CXCL12 in a β-arrestin recruitment assay. Initial in silico screening using an ACKR3 homology model identified two hits, C10 (EC50 19.1 μM) and C11 (EC50 = 11.4 μM). Based on these hits, extensive structure-activity relationship studies were conducted by synthesis and testing of derivatives. It resulted in the identification of the novel thiadiazolopyrimidinone-based compounds 26 (LN5972, EC50 = 3.4 μM) and 27 (LN6023, EC50 = 3.5 μM). These compounds are selective for ACKR3 versus CXCR4 and show metabolic stability. In a platelet degranulation assay, these agonists effectively reduced P-selectin expression by up to 97%, suggesting potential candidates for the treatment of platelet-mediated thrombosis.

Structural snapshot of a β-arrestin-biased receptor

Atypical chemokine receptor subtype 3 (ACKR3), a chemokine receptor, couples selectively to β-arrestins (βarrs) but not to G proteins despite having seven transmembrane (7TM) helix architecture. Yen et al. present cryogenic-electron microscopy (cryo-EM) structures of agonist-bound ACKR3, elucidating a distinct chemokine-binding mechanism, and offering a structural template to probe the transducer-coupling bias at this receptor.

Endothelial ACKR3 drives atherosclerosis by promoting immune cell adhesion to vascular endothelium

Background and Aims : Genome wide association studies revealed a strong association between the CXCL12 gene locus and cardiovascular diseases, highlighting its receptors CXCR4 and atypical chemokine receptor-3 (ACKR3) as targets for CVD research. Although vascular CXCR4 is well studied in atherosclerosis, the exact role of ACKR3 remains elusive. Therefore, the aim of this project is to decipher the role of vascular ACKR3 in atherosclerosis.

Structures of atypical chemokine receptor 3 reveal the basis for its promiscuity and signaling bias.

Both CXC chemokine receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) are activated by the chemokine CXCL12 yet evoke distinct cellular responses. CXCR4 is a canonical G protein–coupled receptor (GPCR), whereas ACKR3 is intrinsically biased for arrestin. The molecular basis for this difference is not understood. Here, we describe cryo-EM structures of ACKR3 in complex with CXCL12, a more potent CXCL12 variant, and a small-molecule agonist. The bound chemokines adopt an unexpected pose relative to those established for CXCR4 and observed in other receptor-chemokine complexes. Along with functional studies, these structures provide insight into the ligand-binding promiscuity of ACKR3, why it fails to couple to G proteins, and its bias toward β-arrestin. The results lay the groundwork for understanding the physiological interplay of ACKR3 with other GPCRs.

Effects of Small Molecule Ligands on ACKR3 Receptors.

Chemokines such as stromal derived factor 1 and their G protein coupled receptors are well-known regulators of the development and functions of numerous tissues. C-X-C motif chemokine ligand 12 (CXCL12) has two receptors: C-X-C chemokine motif receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3). ACKR3 has been described as an atypical “biased” receptor because it does not appear to signal through G proteins and, instead, signals solely through the β-arrestin pathway. In support of this conclusion, we have shown that ACKR3 is unable to signal through any of the known mammalian Gα isoforms and have generated a comprehensive map of the Gα activation by CXCL12/CXCR4. We also synthesized a series of small molecule ligands which acted as selective agonists for ACKR3 as assessed by their ability to recruit β-arrestin to the receptor. Using select point mutations, we studied the molecular characteristics that determine the ability of small molecules to activate ACKR3 receptors, revealing a key role for the deeper binding pocket composed of residues in the transmembrane domains of ACKR3. The development of more selective ACKR3 ligands should allow us to better appreciate the unique roles of ACKR3 in the CXCL12/CXCR4/ACKR3-signaling axis and better understand the structural determinants for ACKR3 activation.SIGNIFICANCE STATEMENTWe are interested in the signaling produced by the G protein coupled receptor atypical chemokine receptor 3 (ACKR3), which signals atypically. In this study, novel selective ligands for ACKR3 were discovered and the site of interactions between these small molecules and ACKR3 was defined. This work will help to better understand the unique signaling roles of ACKR3.

Conformational selection guides β-arrestin recruitment at a biased G protein-coupled receptor.

G protein-coupled receptors (GPCRs) recruit β-arrestins to coordinate diverse cellular processes, but the structural dynamics driving this process are poorly understood. Atypical chemokine receptors (ACKRs) are intrinsically biased GPCRs that engage β-arrestins but not G proteins, making them a model system for investigating the structural basis of β-arrestin recruitment. Here, we performed nuclear magnetic resonance (NMR) experiments on 13CH3-ε-methionine-labeled ACKR3, revealing that β-arrestin recruitment is associated with conformational exchange at key regions of the extracellular ligand-binding pocket and intracellular β-arrestin-coupling region. NMR studies of ACKR3 mutants defective in β-arrestin recruitment identified an allosteric hub in the receptor core that coordinates transitions among heterogeneously populated and selected conformational states. Our data suggest that conformational selection guides β-arrestin recruitment by tuning receptor dynamics at intracellular and extracellular regions.

Emerging Roles of the Atypical Chemokine Receptor 3 (ACKR3) in Cardiovascular Diseases.

Chemokines, and their receptors play a crucial role in the pathophysiology of cardiovascular diseases (CVD). Chemokines classically mediate their effects by binding to G-protein-coupled receptors. The discovery that chemokines can also bind to atypical chemokine receptors (ACKRs) and initiate alternative signaling pathways has changed the paradigm regarding chemokine-related functions. Among these ACKRs, several studies have highlighted the exclusive role of ACKR3, previously known as C-X-C chemokine receptor type 7 (CXCR7), in CVD. Indeed, ACKR3 exert atheroprotective, cardioprotective and anti-thrombotic effects through a wide range of cells including endothelial cells, platelets, inflammatory cells, fibroblasts, vascular smooth muscle cells and cardiomyocytes. ACKR3 functions as a scavenger receptor notably for the pleiotropic chemokine CXCL12, but also as a activator of different pathways such as β-arrestin-mediated signaling or modulator of CXCR4 signaling through the formation of ACKR3-CXCR4 heterodimers. Hence, a better understanding of the precise roles of ACKR3 may pave the way towards the development of novel and improved therapeutic strategies for CVD. Here, we summarize the structural determinant characteristic of ACKR3, the molecules targeting this receptor and signaling pathways modulated by ACKR3. Finally, we present and discuss recent findings regarding the role of ACKR3 in CVD.

P1600: ANTIGENS OF THE DUFFY BLOOD GROUP SYSTEM AND RISK OF COVID-19 INFECTION

Background: The Duffy blood group antigens are transmembrane glycoproteins, represented by two specificities (Fya and Fyb), also known as atypical chemokine receptor 1 (ACKR1). These structures are located not only on the red blood cell membrane, but also on the vascular endothelium, alveoli, epithelium of the renal tubules, and cerebellar neurons. The biological role of the Duffy system is to bind inflammatory chemokines and inhibit excessive leukocyte activation. The antigens also serve as receptors for viruses and malaria plasmodia to enter the cell. Therefore, the study of the participation of the Duffy blood group system in the development of COVID-19 infection is of particular interest. Aims: The aim of study was to compare the distribution of Duffy blood group antigens in individuals with and without COVID-19. Methods: The distribution of antigens of the Duffy blood group system was studied in 329 donors of blood components using equipment and reagents from BioRad (USA), OrthoClinicalDiagnostics (USA). The study was held from February 2020 to February 2021 (before the start of vaccination). 71 people had COVID-19, 258 didn’t. Age ranged from 20 to 63 years, median 38 years. Reliability of the diagnosis of COVID-19 is confirmed by a positive PCR test result and/or the presence of class G antibodies to the SARS-Cov2 virus. Statistical processing was performed using Fisher’s exact test. Results: The Fya antigen was detected in 75.2% of those who did not become ill and in 60.6% of those who had COVID-19 (p<0.05). The frequency of Fyb in donors with and without a new coronavirus infection was comparable, 78.9% and 77.5% respectively. The disease was less common in the individuals with the Fya phenotype. The COVID-19 incidence in them was 18.1%, while without the Fya antigen it reached 30.4% (p=0.02). Carriage of the Fyb antigen had no effect on the incidence of the disease, 21.9% in individuals with the presence of Fyb and 20.5% without it. Image:Summary/Conclusion: The Fya antigen showed a protective effect against COVID-19, its absence increased the risk of the disease by 1.5 times. The obtained results indicate the impact of blood group systems on the risk of COVID-19 infection, but the issue requires further study.

Abstract 6165: DARC/ACKR1 expression is associated with immune landscape changes among triple negative breast tumors

Abstract Recent work defining the immune landscape across breast cancer (BC) subtypes have revealed that TNBC tumors have increased immunogenicity, and these increases in immune cell infiltration are associated with better prognosis and outcomes. Our recent work has proposed the Duffy Antigen Receptor for Chemokines (DARC), as a potential driver of immune regulation in breast tumors. DARC/ACKR1 is an atypical chemokine receptor that promiscuously binds both pro-inflammatory CC and pro-angiogenic CXC class chemokines and plays a role in chemokine gradient establishment both in circulation, and at sites of inflammation via chemokine transcytosis. In the BC context, we and others have reported DARC/ACKR1 protein expression on breast tumor epithelial cells by IHC and have also shown a range of gene expression in breast tumors from bulk RNAseq data in the TCGA cohort. Specifically, alongside CIBERSORT deconvolution of tumor associated immune cell populations, we have shown a strong positive correlation between DARC/ACKR1 gene expression and total tumor-associated leukocyte (TAL) abundance across all BC subtypes. To investigate the role of DARC/ACKR1 specifically in the TNBC tumor microenvironment (TME), we have analyzed (1) in silico bulk RNAseq data from two independent TNBC cohorts, (2) imaging mass cytometry data from TNBC patients and (3) murine model of DARC/ACKR1 BC progression in a basal-like BC transgenic mouse model. Our in silico data shows strong positive correlation of DARC/ACKR1 expression and TAL abundance, when DARC/ACKR1 expression is both dichotomized and analyzed as a continuous variable. Immune cell populations increasing with DARC/ACKR1 expression include B cell, T cell, monocyte and macrophage populations, which was consistent with our previous findings across all BC subtypes. IMC analysis of TNBC FFPE sections to determine protein expression of various immune and structural markers on the single-cell level between DARC/ACKR1 high or low expressing tumors show cells from DARC/ACKR1 high expressing tumors clustered distinctly from DARC/ACKR1 low expressing tumors. Image analysis also revealed increased infiltration among our DARC/ACKR1 high tumors. Finally, our murine model of DARC/ACKR1 in TNBC, shows that tumor development followed similar disease progression as human BC, following early through late-stage disease. DARC/ACKR1 deficient mice were found to have larger tumor volume and more palpable tumors, where DARC/ACKR1 expressing mice show co-localization of DARC and immune cell expression. This is the first work to describe the role of DARC/ACKR1 in the TNBC TME in bulk tumor transcriptomic data, spatial single-cell protein level data, and murine BC tumor model. Further investigation of factors that drive immune response in TNBC tumors, like DARC/ACKR1, may lead the way to novel therapeutic options or biomarkers for better outcomes for women with TNBC. Citation Format: Rachel Martini, Kiel Telesford, Brittany Lord, Hiranmayi Ravichandran, Olivier Elemento, Nancy Manley, Michele Monteil, Lisa Newman, Upender Manne, Clayton Yates, Melissa B. Davis. DARC/ACKR1 expression is associated with immune landscape changes among triple negative breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6165.

Abstract LB032: CXCR7 drives prostate cancer growth through AURKA activation

Abstract Prostate cancer (PCa) is the most commonly diagnosed cancer among males in the United States. The mainstay treatment for metastatic PCa patients is androgen-deprivation therapies, including high-affinity androgen receptor antagonists, such as enzalutamide. Unfortunately, most patients develop resistance and relapse with castration-resistant PCa (CRPC). We have previously reported up-regulation of CXCR7, an atypical chemokine receptor, in CRPC. CXCR7 interacts with beta-arrestin (ARRB2), a cytoplasmic scaffold protein, and translocates to the cytoplasm to induce signal transduction and PCa progression. However, much remains to be known regarding CXCR7-driven downstream signaling and its therapeutic targeting in advanced PCa. To unveil CXCR7-downstream signaling, we utilized RNA-seq to profile gene expression. Gene ontology analyses revealed remarkable enrichment of CXCR7-regulated genes in G2/M cell cycle progression. Comprehensive phospho-proteomics analysis of CXCR7-knockdown PCa cells showed markedly reduced phosphorylation of AURKA substrates. AURKA, a serine/threonine-protein kinase, is a key regulator of mitosis and G2/M progression and is frequently upregulated in CRPC and the late-stage neuroendocrine PCa (NEPC). Activated AURKA experiences autophosphorylation and phosphorylates downstream substrates, e.g., TACC3, that mediate proper cell division. Immunoblotting confirmed that CXCR7 depletion reduces phosphorylation of AURKA and TACC3. Co-immunoprecipitation revealed protein-protein interaction between CXCR7 and AURKA, which is mediated by ARRB2. Accordingly, ARRB2-knockdown similarly inhibits AURKA phosphorylation and arrests cell proliferation. Immunofluorescence coupled with confocal imaging demonstrated that CXCR7 is packed into clathrin-coated vesicles and locates in the plasma membrane, cytoplasm, and perinuclear Golgi complex, which surrounds the centrosomal AURKA and ARRB2. We found that CXCR7 translocates to the Golgi complex along microtubule-dependent trafficking. Finally, we found that pharmacological inhibitors of AURKA abolish CXCR7-driven PCa cell proliferation in vitro and tumor growth in vivo. Conclusions: Our results describe for the first time that the CXCR7-ARRB2 complex interacts with AURKA and promotes its activation. Our data suggest that AURKA-targeting is a promising therapeutic strategy for CRPC and NEPC with CXCR7 up-regulation. Citation Format: Galina Gritsina, Zhuoyuan Lin, Ka Wing Fong, Xiaodong Lu, Changsheng Zhao, Jindan Yu. CXCR7 drives prostate cancer growth through AURKA activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB032.

Abstract 5435: CCRL2 affects the sensitivity of MDS and secondary AML to azacitidine

Abstract INTRODUCTION: We recently found that the atypical chemokine receptor, CCRL2 promotes the growth of MDS and secondary AML (sAML) while CCRL2 knockdown inhibits their growth. The aim of the current study is to investigate if CCRL2 regulates pathways associated with the development of resistance to hypomethylating agents (HMA), commonly used drugs in MDS and occasionally sAML. MATERIALS AND METHODS: We used lentivirus-mediated transduction of MDS92, MDS-L and TF-1 MDS/sAML cells to suppress CCRL2 expression. Two different shRNA constructs were used. We performed RNA sequencing and gene-set enrichment analysis of CCRL2 knocked down (KD) and wild-type (WT) TF-1 cells. We measured DNA methyl-transferases’ expression by western blot, CD11b and CD71 expression was measured in MDS and sAML cells to assess cell differentiation. Apoptosis was measured by Annexin V/PI staining, and clonogenicity by methylcellulose assays. CD34+ cells were sorted from bone marrow aspirates of MDS patients before the initiation of treatment with HMA by using magnetic beads and measurement of CCRL2 was performed by flow cytometry. Response to HMA in MDS patients was assessed by 6 months of treatment based on the International Working Group response criteria. RESULTS: CCRL2 KD cells demonstrated suppression of pathways associated with PRC2 complex activity, histone modification, and DNA methylation. CCRL2 KD also lead to a more prominent degradation of DNMT1, DNMT3A and DNTM3B under azacitidine treatment. CCRL2 increased apoptosis in response to 0.5 and 1 μM azacitidine (P&amp;lt;0.010) and MDS-L cells (P&amp;lt;0.010 with both sh1 and sh2). Similarly, CCRL2 knockdown increased morphologic differentiation with both 0.5 and 1 μΜ azacitidine (P&amp;lt;0.010) as well as increased the clonogenic inhibition caused by azacitidine (P&amp;lt;0.05). In order to analyze the effect CCRL2 clinically, we analyzed CCRL2 expression in CD34+ cells from patients undergoing HMA treatment. Non-responders to HMA (progressive disease) express higher levels of CCRL2 compared to CD34+ cells from responders (complete remission, partial remission or stable disease) (P=0.020). DISCUSSION: Our analysis suggests that CCRL2 regulates the expression of genes associated with induction of PRC2-mediated histone modification and DNA methylation in sAML cells. CCRL2 suppression also increased the sensitivity of MDS and sAML cells to azacitidine. In addition, increased CCRL2 expression in MDS cells is associated with worse response to HMA. These data suggest that targeting CCRL2 has therapeutic potential in MDS and sAML. Citation Format: Theodoros Karantanos, Patric Teodorescu, Brandy Perkins, Marios Arvanitis, Ilias Christodolou, Christopher Esteb, W. Brian Dalton, Tania Jain, Amy E. DeZern, Lukasz P. Gondek, Mark J. Levis, Gabriel Ghiaur, Richard J. Jones. CCRL2 affects the sensitivity of MDS and secondary AML to azacitidine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5435.

Endothelial ACKR3 drives atherosclerosis by promoting immune cell adhesion to vascular endothelium

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft SFB1123 European Research Council AdG_692511 Background Genome wide association studies revealed a strong association between the CXCL12 gene locus and cardiovascular diseases, highlighting its receptors CXCR4 and atypical chemokine receptor-3 (ACKR3) as targets for CVD research. Although vascular CXCR4 is well studied in atherosclerosis, the exact role of ACKR3 remains elusive. Therefore, the aim of this project is to decipher the role of vascular ACKR3 in atherosclerosis. Methods Vascular endothelium (Bmx-cre) specific Ackr3 deficient mice on Apoe-/- background were fed with a cholesterol-rich diet. Lesions in the aortic roots and arches were analyzed via H&amp;E staining. The phenotype was further characterized employing ex-vivo perfusion and intravital microscopy for cell adhesion and Evans-blue injection for endothelial permeability. Signaling studies are performed in human coronary artery endothelial cells (HCAECs). Results Endothelial Ackr3 deficiency resulted in significantly smaller atherosclerotic lesions, accompanied by a more stable plaque phenotypes (more lesional collagen and smooth muscle cell content, smaller necrotic cores). Endothelial-Ackr3-/- mice revealed a reduction in plaque macrophage content and ICAM positive endothelial cells in atherosclerotic lesions. Moreover, immune cell adhesion on carotid arteries lacking endothelial ACKR3 was decreased whilst endothelial permeability was not affected. ACKR3 silencing in TNF-stimulated HCAECs decreased adhesion molecule expression and downregulated MAPK signaling and NF-kB p65 phosphorylation. Endothelial cells in atherosclerotic lesions also revealed decreased phospho-NF-kB p65 expression in Ackr3 deficient mice. Conclusion Collectively, our findings indicate that arterial endothelial ACKR3 fuels atherosclerosis by mediating endothelium-immune cell adhesion, most likely through inflammatory MAPK and NF-kB pathways resulting in increased adhesion molecule expression.

Selective Elimination of Senescent Fibroblasts by Targeting the Cell Surface Protein ACKR3.

The accumulation of senescent cells in aging tissues is associated with age-related diseases and functional decline. Thus, senolysis, a therapy aimed at rejuvenation by removing senescent cells from the body, is being developed. However, this therapy requires the identification of membrane surface antigens that are specifically expressed on senescent cells for their selective elimination. We showed that atypical chemokine receptor 3 (ACKR3), a receptor of the CXC motif chemokine 12 (CXCL12) implicated in cancer, inflammation, and cardiovascular disorders, is selectively expressed on the surface of senescent human fibroblasts but not on proliferating cells. Importantly, the differential presence of ACKR3 enabled the isolation of senescent cells by flow cytometry using anti-ACKR3 antibodies. Furthermore, antibody-dependent cellular cytotoxicity assays revealed that cell surface ACKR3 preferentially sensitizes senescent but not dividing fibroblasts to cell injury by natural killer cells. Conclusively, the selective expression of ACKR3 on the surface of senescent cells allows the preferential elimination of senescent cells. These results might contribute to the future development of novel senolysis approaches.

The Extended N-Terminal Domain Confers Atypical Chemokine Receptor Properties to CXCR3-B.

The chemokine receptor CXCR3 plays a critical role in immune cell recruitment and activation. CXCR3 exists as two main isoforms, CXCR3-A and CXCR3-B, resulting from alternative splicing. Although the two isoforms differ only by the presence of an N-terminal extension in CXCR3-B, they have been attributed divergent functional effects on cell migration and proliferation. CXCR3-B is the more enigmatic isoform and the mechanisms underlying its function and signaling remain elusive. We therefore undertook an in-depth cellular and molecular comparative study of CXCR3-A and CXCR3-B, investigating their activation at different levels of the signaling cascades, including G protein coupling, β-arrestin recruitment and modulation of secondary messengers as well as their downstream gene response elements. We also compared the subcellular localization of the two isoforms and their trafficking under resting and stimulated conditions along with their ability to internalize CXCR3-related chemokines. Here, we show that the N-terminal extension of CXCR3-B drastically affects receptor features, modifying its cellular localization and preventing G protein coupling, while preserving β-arrestin recruitment and chemokine uptake capacities. Moreover, we demonstrate that gradual truncation of the N terminus leads to progressive recovery of surface expression and G protein coupling. Our study clarifies the molecular basis underlying the divergent effects of CXCR3 isoforms, and emphasizes the β-arrestin-bias and the atypical nature of CXCR3-B.

The Role of Atypical Chemokine Receptor D6 (ACKR2) in Physiological and Pathological Conditions; Friend, Foe, or Both?

Chemokines exert crucial roles in inducing immune responses through ligation to their canonical receptors. Besides these receptors, there are other atypical chemokine receptors (ACKR1–4) that can bind to a wide range of chemokines and carry out various functions in the body. ACKR2, due to its ability to bind various CC chemokines, has attracted much attention during the past few years. ACKR2 has been shown to be expressed in different cells, including trophoblasts, myeloid cells, and especially lymphoid endothelial cells. In terms of molecular functions, ACKR2 scavenges various inflammatory chemokines and affects inflammatory microenvironments. In the period of pregnancy and fetal development, ACKR2 plays a pivotal role in maintaining the fetus from inflammatory reactions and inhibiting subsequent abortion. In adults, ACKR2 is thought to be a resolving agent in the body because it scavenges chemokines. This leads to the alleviation of inflammation in different situations, including cardiovascular diseases, autoimmune diseases, neurological disorders, and infections. In cancer, ACKR2 exerts conflicting roles, either tumor-promoting or tumor-suppressing. On the one hand, ACKR2 inhibits the recruitment of tumor-promoting cells and suppresses tumor-promoting inflammation to blockade inflammatory responses that are favorable for tumor growth. In contrast, scavenging chemokines in the tumor microenvironment might lead to disruption in NK cell recruitment to the tumor microenvironment. Other than its involvement in diseases, analyzing the expression of ACKR2 in body fluids and tissues can be used as a biomarker for diseases. In conclusion, this review study has tried to shed more light on the various effects of ACKR2 on different inflammatory conditions.

Stimulation of the atypical chemokine receptor 3 (ACKR3) by a small-molecule agonist attenuates fibrosis in a preclinical liver but not lung injury model

Atypical chemokine receptor 3 (ACKR3, formerly CXC chemokine receptor 7) is a G protein-coupled receptor that recruits β-arrestins, but is devoid of functional G protein signaling after receptor stimulation. In preclinical models of liver and lung fibrosis, ACKR3 was previously shown to be upregulated after acute injury in liver sinusoidal and pulmonary capillary endothelial cells, respectively. This upregulation was linked with a pro-regenerative and anti-fibrotic role for ACKR3. A recently described ACKR3-targeting small molecule agonist protected mice from isoproterenol-induced cardiac fibrosis. Here, we aimed to evaluate its protective role in preclinical models of liver and lung fibrosis. After confirming its in vitro pharmacological activity (i.e., ACKR3-mediated β-arrestin recruitment and receptor binding), in vivo administration of this ACKR3 agonist led to increased mouse CXCL12 plasma levels, indicating in vivo interaction of the agonist with ACKR3. Whereas twice daily in vivo administration of the ACKR3 agonist lacked inhibitory effect on bleomycin-induced lung fibrosis, it had a modest, but significant anti-fibrotic effect in the carbon tetrachloride (CCl4)-induced liver fibrosis model. In the latter model, ACKR3 stimulation affected the expression of several fibrosis-related genes and led to reduced collagen content as determined by picro-sirius red staining and hydroxyproline quantification. These data confirm that ACKR3 agonism, at least to some extent, attenuates fibrosis, although this effect is rather modest and heterogeneous across various tissue types. Stimulating ACKR3 alone without intervening in other signaling pathways involved in the multicellular crosstalk leading to fibrosis will, therefore, most likely not be sufficient to deliver a satisfactory clinical outcome.

Structural Basis of Nanobody Induced ACKR3 Inhibition

When bound to their small, secreted chemotactic cytokine ligands (chemokines), the G protein‐coupled receptor (GPCR) subfamily known as chemokine receptors (CKRs) can promote cell proliferation, survival, and migration towards increasing chemokine concentration gradients. Their strong association with these phenotypic hallmarks of cancer and their upregulation in cancer cells has made these receptors a topic of extensive research in the cancer field. Recent findings show upregulation of the atypical chemokine receptor 3 (ACKR3) in a wide variety of cancers including various breast, lung, and brain cancers, suggesting the involvement of ACKR3 in cancer pathogenesis. Acting as a scavenger receptor for a narrow range of chemokine ligands (CXCL12/CXCL11), binding of ACKR3 to a cognate ligand induces non‐canonical β‐arrestin dependent signaling, which can lead to receptor‐ligand internalization, and cause a unique signaling cascade to directly promote cell proliferation, cell growth, and angiogenesis. Moreover, silencing of ACKR3 in murine models has been shown to significantly decrease the growth and invasiveness of breast, lung, and endothelial cancer. The development of therapeutics capable of selectively inhibiting ACKR3 has now become an area of active interest. Despite this drive, the characteristic promiscuity of the CKR network and a lack of understanding in ACKR3’s mechanism of inhibition has left all ACKR3 antagonists to date with varying degrees of partial agonism, low specificity, or low stability. Here we identify and characterize a new antagonist of ACKR3, turning to recombinant variable domain monomers from the unique heavy‐chain only camelid antibodies or “nanobodies”. These 13 kDa nanobodies have highly conserved structures with the exception of three structural loops which encode the nanobody complimentary‐determining regions (CDRs) involved in antigen recognition and protein binding. This intricate binding epitope allows nanobodies to uniquely target complex GPCR epitopes, offering increased receptor specificity and selectivity versus small molecules and traditional antibody therapeutics. Using NMR spectroscopy, we have resolved the solution structure of the nanobody VUN701, an ACKR3 antagonist capable of completely ablating ACKR3 signaling at nanomolar concentrations. The structure of this inhibitor contains an uncharacteristically well‐ordered extended beta‐hairpin in the CDR3 region. In combination with cell‐based assays and molecular modeling, this analysis has allowed us to develop an atomic view of the VUN701‐ACKR3 interface. These findings shed light on the mechanism behind ACKR3 inhibition and drive forward the development of highly specific lead compounds for cancer therapy.

Seven structures of atypical chemokine receptor 3 reveal the molecular bases for its promiscuity and signaling bias

Atypical chemokine receptor 3 (ACKR3) is a seven transmembrane (TM) spanning heptahelical receptor that is involved in different stages of cancer progression. Unlike canonical G protein‐coupled receptors (GPCRs), ACKR3 does not couple to heterotrimeric G proteins when activated by its endogenous ligand, CXCL12. It is therefore a naturally occurring fully arrestin biased receptor. ACKR3 however desensitizes normally and contributes to cancer progression through arrestin‐mediated signaling pathways and/or by active sequestration of chemokine ligands, which blocks CXCL12‐mediated downregulation of CXCR4. Here, we report seven cryo‐electron microscopic structures of ACKR3 bound to three different agonistic ligands, including CXCL12, a potent CXCL12 variant (CXCL12LRHQ), and a small molecule agonist (CCX662). Although the chemokines bind to ACKR3 in a truly unique way, all three ligands still extend deep in the orthosteric pocket. In all the structures, the TM spans of ACKR3 assume a configuration like those of active GPCRs, although the cytoplasmic pocket is more compact in ACKR3. In addition, compared to other chemokine receptors, the structure of the intracellular loop 2 (ICL2) of ACKR3 is quite different and lacks a kink at the cytoplasmic end of TM4. This is predicted to prevent ACKR3 from binding heterotrimeric G proteins in a canonical way, thereby blocking nucleotide exchange. In summary, our structures provide insight into the promiscuous recognition of ACKR3 relative to other chemokine receptors, and into its distinct functions relative to other canonical chemokine receptors. The structure models may also guide future drug discovery efforts targeting ACKR3 for cancer therapy.

Regulation of Endothelial Cell function by Integration of Gαi and β‐arrestin signaling at Atypical Chemokine Receptor 3

Atypical Chemokine receptor 3 (ACKR3), also known as C‐X‐C chemokine receptor type 7 (CXCR7), is a G protein‐coupled receptor (GPCR) implicated in several physiological processes including leukocyte trafficking, cancer, and angiogenesis. Commonly co‐expressed with C‐X‐C chemokine receptor type 4 (CXCR4), ACKR3 is classified as an β‐arrestin‐biased “scavenger” receptor due to its lack of G‐protein signaling and its ability to efficiently internalize and degrade its ligands, CXCL11 and CXCL12. However, much is still unknown regarding ACKR3 signaling mechanisms and its implications on cellular physiology. Here we describe ongoing studies to characterize the mechanisms that underlie ACKR3 signaling and endothelial cell function. We demonstrated two ACKR3 specific agonists, WW36 and WW38, promote proliferation and migration of human umbilical vein endothelial cells (HUVECs). Surprisingly, we found that ACKR3‐promoted proliferation and migration was sensitive to pertussis toxin (PTX), suggesting the involvement of Gαi/o proteins at a receptor which does not canonically signal via G proteins. We also observed similar sensitivity to PTX on ERK activation in HUVECs. We hypothesized that these results may in part be explained by the recently described coordination of Gαi and β‐arrestin (βarr) to promote Gαi:βarr complex‐specific downstream signaling. In support of this hypothesis, we demonstrated the formation of a Gαi:βarr complex upon ACKR3 activation in a dose‐dependent manner. To characterize the necessary components in Gαi and βarr, we first developed active proximal polar core mutant and proximal finger loop βarr mutant and tested these at the prototypical class B Vasopressin 2 Receptor. Both mutants lead to considerable decrease in complex formation with the finger loop mutation nearly abolishing our split signal. Within the Gαi subfamily, we observed considerable differences in the ability to form complex, with Gαi2 and Gαi3 promoting less complex formation than Gαi1 and Gαo. We developed several Gαi mutants, hypothesizing that residues that differ between Gαi isoforms may impact Gαi:βarr formation. Using NanoBRET we demonstrated that these mutants impair complex formation, suggesting that they may be involved either via direct contacts with βarr or impacting Gαi conformation such that complex formation is no longer as favorable. These studies reveal a novel role for Gαi:βarr complexes in mediating ACKR3 function that contribute to endothelial cell biology. Additionally, these findings support an emerging paradigm in which G proteins and βarr coordinate their signaling downstream of GPCRs.

Determining the Requirements for Gαi:β‐arrestin Complex Formation at G Protein‐Coupled Receptors

G protein‐coupled receptors (GPCRs) comprise the largest class of transmembrane receptors and are targeted by nearly a third of prescription drugs. Canonical GPCR signaling involves pathways mediated by G proteins and β‐arrestins (βarrs). While these have historically been thought of as separate, our lab has demonstrated that the inhibitory G protein Gα subunit (Gαi) and βarr form complexes following receptor activation. We have also shown that Gαi:βarr complex formation occurs even if the receptor signals through other classes of G proteins or lacks G protein activity altogether, as in the case of endogenously βarr‐biased Atypical Chemokine Receptor 3 (ACKR3). To expand on this work, we used a split nano‐luciferase assay to examine Gαi:βarr complex formation at βarr‐biased mutants of two ‘balanced’ receptors, which exhibit both G protein and βarr signaling— the type 1 Angiotensin II Receptor (AT1R) and D2 dopamine Receptor (D2R). Consistent with our results with ACKR3, we found that mutation of these other receptors to abrogate G protein‐mediated signaling did not eliminate Gαi:βarr association. Observing this at multiple additional receptors further supported the idea that G protein activity is not required for Gαi:βarr complex formation. To add to our characterization of the determinants of Gαi:βarr association, we then sought to explore how different mechanisms of βarr activation may contribute to complex formation at ACKR3. To accomplish this, we utilized previously described polar core, proximal finger loop, and lipid mutant βarr constructs. The polar core and proximal finger loop mutants achieve constitutive activation by different mechanisms, while the lipid mutant’s ability to be recruited to and bind at the plasma membrane is deficient; these result in decreased Gαi:βarr association at Vasopressin Receptor 2 (V2R). We compared these mutants’ ability to associate with Gαi at ACKR3. Our constitutively active βarr constructs showed differential abilities to form Gαi:βarr complexes compared to wild‐type βarr, suggesting that certain methods of βarr activation may be more relevant in Gαi:βarr complex formation than others. Additional insights into the determinants of Gαi:βarr association in the context of receptor, G protein, and βarr activity will enhance our understanding of the Gαi:βarr complex and its importance in GPCR signaling, potentially informing future development of therapeutic agents targeting GPCRs.