CXCR4 Agonist Research Articles

Biased agonists of the chemokine receptor CXCR3 differentially drive formation of Gαi and β‐arrestin complexes

G‐protein‐coupled receptors (GPCRs) are the most common cell surface receptor class and influence nearly every biological process within a cell. Approximately 30% of all FDA‐approved medications target GPCRs. Following receptor activation, G proteins and β‐arrestins are currently appreciated to be the two primary proximal intracellular signaling pathways that regulate GPCR signaling. It has been thought that G protein and β‐arrestin signaling pathways are largely discernable, evidenced in part by ‘biased agonists’ that can preferentially activate one of these pathways relative to the other. However, increased crosstalk between G protein and β‐arrestin pathways is beginning to be appreciated. We recently described the ability of Gα proteins to directly interact with β‐arrestins to form signaling scaffolds, with Gαi/o found to directly interact with β‐arrestin following GPCR activation regardless of canonical GPCR G protein coupling. We previously demonstrated in other work that all GPCRs tested, including the vasopressin type 2 receptor, β2‐adrenergic receptor, neurotensin receptor type 1, and the dopamine D1 and D2 receptors, formed a Gαi:β‐arrestin complex. Now that we appreciate this novel GPCR signaling paradigm, it is unclear if biased agonists that preferentially stimulate either G protein‐ or β‐arrestin‐dependent signaling differentially regulate Gαi:β‐arrestin interactions, if at all. To address this question, we utilized the chemokine receptor CXCR3 that binds three endogenous chemokines with high affinity. We demonstrate that these endogenous biased CXCR3 chemokines, CXCL9, CXCL10, and CXCL11, along with its synthetic biased agonists, VUF10661 and VUF11418, differentially form Gαi:β‐arrestin complexes. The ability of CXCR3 agonists to form Gαi:β‐arrestin complexes does not correlate highly with more well‐established GPCR signaling pathways including G protein recruitment, G protein signaling (e.g., regulation of cAMP), or β‐arrestin recruitment, suggesting that Gαi:β‐arrestin complex formation is distinct from conventional G protein or β‐arrestin signaling events. Additionally, differential Gαi:β‐arrestin complex formation was found to correlate with similar degrees of Gαi:β‐arrestin:CXCR3 “megaplex” formation, suggesting that formation of a long‐lived complex may depend upon close coupling of the two effectors at the receptor initially. This work provides further support for a separable Gαi:β‐arrestin signaling pathway and enhances our understanding of GPCR signaling and biased agonism.Support or Funding InformationNIH

A CXCR4 receptor agonist strongly stimulates axonal regeneration after damage.

ObjectiveTo test whether the signaling axis CXCL12α‐CXCR4 is activated upon crush/cut of the sciatic nerve and to test the activity of NUCC‐390, a new CXCR4 agonist, in promoting nerve recovery from damage.MethodsThe sciatic nerve was either crushed or cut. Expression and localization of CXCL12α and CXCR4 were evaluated by imaging with specific antibodies. Their functional involvement in nerve regeneration was determined by antibody‐neutralization of CXCL12α, and by the CXCR4 specific antagonist AMD3100, using as quantitative read‐out the compound muscle action potential (CMAP). NUCC‐390 activity on nerve regeneration was determined by imaging and CMAP recordings.ResultsCXCR4 is expressed at the injury site within the axonal compartment, whilst its ligand CXCL12α is expressed in Schwann cells. The CXCL12α‐CXCR4 axis is involved in the recovery of neurotransmission of the injured nerve. More importantly, the small molecule NUCC‐390 is a strong promoter of the functional and anatomical recovery of the nerve, by acting very similarly to CXCL12α. This pharmacological action is due to the capability of NUCC‐390 to foster elongation of motor neuron axons both in vitro and in vivo.InterpretationImaging and electrophysiological data provide novel and compelling evidence that the CXCL12α‐CXCR4 axis is involved in sciatic nerve repair after crush/cut. This makes NUCC‐390 a strong candidate molecule to stimulate nerve repair by promoting axonal elongation. We propose this molecule to be tested in other models of neuronal damage, to lay the basis for clinical trials on the efficacy of NUCC‐390 in peripheral nerve repair in humans.

Targeting CXCR4, VLA4, and CXCR2 for Hematopoietic Stem Cell Mobilization

Background: Hematopoietic stem cell (HSC) transplant is an essential treatment for a variety of blood disorders and malignancies. A key step in this procedure is the mobilization of donor stem cells. The most commonly used regimen for donor mobilization is a 5-day course of G-CSF. The length of this regimen coupled with the associated side effects emphasizes a need for superior alternatives. In recent years, there has been a growing understanding of mechanisms governing stem cell retention within the bone marrow niche. This has led to the development of new mobilization drugs that specifically target these processes. Two examples of previously described drugs that target mechanisms of stem cell retention are Plerixafor (a CXCR4 inhibitor already in clinical use), and truncated Gro-Beta (tGroβ; a CXCR2 agonist). Another potential target for inducing mobilization is disruption of the interaction between the VLA-4 integrin and its ligand VCAM-1. In this study, we evaluate the efficacy of novel VLA-4 inhibitors (VLA4i) alone and in combination with Plerixafor and/or tGroβ for the purposes of hematopoietic stem cell mobilization. Methods: We synthesized over 15 novel VLA-4 inhibitor molecules and tested their potency using soluble VCAM-1 binding assays. The 5 inhibitors determined to be most potent were then tested in vivo in DBA mice for their ability to mobilize HSCs alone and in combination with tGroβ and/or Plerixafor (n=5). HSC mobilization was measured in wild-type and splenectomized mice via flow cytometry to quantify the proportion of LSK (Lineage- Sca+ cKit+) cells as well as via Colony Forming Unit (CFU) assays. For competitive transplant, mobilized CD45.1+ BALB/c mouse blood (10 uL) was injected into lethally irradiated CD45.2+ BALB/c recipients alongside 2.5x105 CD45.2+ BALB/c bone marrow cells (n=10 / cohort). HSC engraftment was monitored monthly via flow cytometry for ratio of 45.1+ vs. 45.2+ cells in peripheral blood. Results: Firetagrast and BIO5192 are previously characterized VLA4i that have been administered to humans for indications unrelated to HSC mobilization. Our best VLA4i to date, LGB-2019, exhibited similar potency as BIO5192 in preventing the binding of sVCAM-1 to VLA-4 (IC50: 1.7nM) and was >200-fold more potent than firategrast. LGB-2019 showed increased aqueous solubility and mobilized 1.5-fold more murine LSK cells for a longer time period (peak HSC mobilization maintained for 4 hours) than BIO5192 when administered alone. Simultaneous injection of C57BL/6 mice with LGB-2019 (VLA4i), Plerixafor (CXCR4i) and tGro-β (CXCR2a) resulted in a synergistic increase in circulating CFUs (Fig. 1A; 9.8 x 103 CFUs/mL) and LSKs (Fig. 1B; 12.8 LSKs/uL) at 4 hours post-injection. In contrast, 5 days of G-CSF treatment mobilized approximately 3-fold and 8-fold less CFUs and LSKs, respectively (Fig. 1A-B). We saw no significant difference in mobilization for splenectomized vs. wildtype mice (23.4 x 103 CFUs/mL vs. 23.0 x 103 CFUs/mL) when mobilizing DBA/2 mice via VLA4i+CXCR4i+CXCR2a. Three months after competitive transplantation, blood obtained from BALB/c mice mobilized with the triple combination engrafted significantly better than blood obtained from mice treated with G-CSF or the dual combinations (Fig. 1C). Summary: New insights about the stem cell niche have allowed for the development of targeted drugs for the purposes of mobilization. Here, we show that a novel VLA-4 receptor inhibitor in combination with two other known mobilizers induces mobilization of hematopoietic stem and progenitor cells (CFU/LSK) at levels superior to the standard of care G-CSF and in a dramatically shortened time frame. Mouse transplant data also show superior engraftment in lethally irradiated recipients when using the triple cocktail regimen compared to the G-CSF mobilized graft. Secondary transplants are ongoing and will provide a more complete picture of primitive HSC mobilization and serial engraftment properties of the cells. Disclosures Rettig: WashU: Patents & Royalties: Patent Application 16/401,950. Karpova:WashU: Patents & Royalties: Patent Application 16/401,950. Ruminski:WahU: Patents & Royalties: Patent Application 16/401,950. Morrow:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. DiPersio:Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Equity Ownership; Incyte: Consultancy, Research Funding; Bioline Rx: Research Funding, Speakers Bureau; Macrogenics: Research Funding, Speakers Bureau; Karyopharm Therapeutics: Consultancy; Celgene: Consultancy; Amphivena Therapeutics: Consultancy, Research Funding; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; NeoImmune Tech: Research Funding.

Rapid and Robust Mobilization of CD34+ HSCs without G-CSF Following Administration of Mgta-145 Alone or in Combination with Plerixafor

Rapid and Robust Mobilization of CD34+ HSCs without G-CSF Following Administration of Mgta-145 Alone or in Combination with Plerixafor

Bone marrow-derived mesenchymal stem cells repair severe acute pancreatitis by secreting miR-181a-5p to target PTEN/Akt/TGF-β1 signaling

BackgroundSevere acute pancreatitis (SAP) is associated with high morbidity and mortality. Bone marrow mesenchymal stem cells (BMSCs) have shown obvious protective effect on SAP. However, little is known about the underlying mechanism. The objective of this study is to unravel the role and regulatory mechanism of miR-181a-5p in BMSCs-mediated pancreatic repair. MethodsBMSCs were isolated from Sprague-Dawley rats and characterized by flow cytometry and Oil Red O staining. Sodium taurocholate- and caerulein-induced models were used as SAP models in vivo and in vitro, respectively. Pancreatic injury were evaluated by H&E and histopathological analysis, as well as by measuring levels of amylase, lipase and cytokines. qRT-PCR and western blotting were performed to detect the level of miR-181a-5p and the protein levels of PTEN/Akt, respectively. ELISA was conducted to detect the levels of TNF-α, IL-1β, IL-6, angiopoietin, IL-4, IL-10 and TGF-β1. The apoptotic rate of AR42J cells was quantitated by concurrent staining with Annexin-V-FITC and PI. ResultsBMSCs significantly attenuated pancreatic injury in SAP rats by reducing inflammatory infiltration and necrosis, and this effect was abolished by CXCR4 agonist AMD3100. ADM3100 exhibited more severe pancreatic injury and decreased miR-181a-5p levels in the pancreas and serum compared to SAP group. Overexpression of miR-181a-5p in BMSCs (BMSCs-miR-181a-5p) markedly potentiated the protective effect of BMSCs by reducing histological damage and levels of amylase and lipase. Moreover, BMSCs-miR-181a-5p dramatically reduced levels of angiopoietin, TNF-α, IL-1β and IL-6, but induced the levels of IL-4 and IL-10. In caerulein-treated AR42J cells, co-culturing of BMSCs-miR-181a-5p alleviated caerulein-induced increase of amylase and lipase, and apoptosis via PTEN/Akt/TGF-β1 signaling. ConclusionBMSCs alleviate SAP and reduce inflammatory responses and apoptosis by secreting miR-181a-5p to target PTEN/Akt/TGF-β1 signaling. Hence, BMSCs-miR-181a-5p could serve as potential therapeutic target for SAP.

An Agonist of the CXCR4 Receptor Strongly Promotes Regeneration of Degenerated Motor Axon Terminals.

The activation of the G-protein coupled receptor CXCR4 by its ligand CXCL12α is involved in a large variety of physiological and pathological processes, including the growth of B cells precursors and of motor axons, autoimmune diseases, stem cell migration, inflammation, and several neurodegenerative conditions. Recently, we demonstrated that CXCL12α potently stimulates the functional recovery of damaged neuromuscular junctions via interaction with CXCR4. This result prompted us to test the neuroregeneration activity of small molecules acting as CXCR4 agonists, endowed with better pharmacokinetics with respect to the natural ligand. We focused on NUCC-390, recently shown to activate CXCR4 in a cellular system. We designed a novel and convenient chemical synthesis of NUCC-390, which is reported here. NUCC-390 was tested for its capability to induce the regeneration of motor axon terminals completely degenerated by the presynaptic neurotoxin α-Latrotoxin. NUCC-390 was found to strongly promote the functional recovery of the neuromuscular junction, as assayed by electrophysiology and imaging. This action is CXCR4 dependent, as it is completely prevented by AMD3100, a well-characterized CXCR4 antagonist. These data make NUCC-390 a strong candidate to be tested in human therapy to promote nerve recovery of function after different forms of neurodegeneration.

Targeting VLA4 integrin and CXCR2 mobilizes serially repopulating hematopoietic stem cells.

Mobilized peripheral blood has become the primary source of hematopoietic stem and progenitor cells (HSPCs) for stem cell transplantation, with a five-day course of granulocyte colony stimulating factor (G-CSF) as the most common regimen used for HSPC mobilization. The CXCR4 inhibitor, plerixafor, is a more rapid mobilizer, yet not potent enough when used as a single agent, thus emphasizing the need for faster acting agents with more predictable mobilization responses and fewer side effects. We sought to improve hematopoietic stem cell transplantation by developing a new mobilization strategy in mice through combined targeting of the chemokine receptor CXCR2 and the very late antigen 4 (VLA4) integrin. Rapid and synergistic mobilization of HSPCs along with an enhanced recruitment of true HSCs was achieved when a CXCR2 agonist was co-administered in conjunction with a VLA4 inhibitor. Mechanistic studies revealed involvement of CXCR2 expressed on BM stroma in addition to stimulation of the receptor on granulocytes in the regulation of HSPC localization and egress. Given the rapid kinetics and potency of HSPC mobilization provided by the VLA4 inhibitor and CXCR2 agonist combination in mice compared to currently approved HSPC mobilization methods, it represents an exciting potential strategy for clinical development in the future.

Mgta-145 in Combination with Plerixafor Mobilizes Large Numbers of Hematopoietic Stem Cells That Lead to Rapid Engraftment Following Autologous Transplantation in Nonhuman Primates

The majority of bone marrow transplants (BMT) utilize granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood (mPB) as the source of hematopoietic stem cells (HSCs). However, CD34+ harvest with G-CSF is variable and frequently unpredictable, often requires multiple apheresis sessions and is associated with significant side effects. Identification of a mobilizing regimen that consistently produces high numbers of engraftable CD34+ cells without the need for G-CSF would be ideal. We previously reported that MGTA-145, a CXCR2 agonist, when combined with the CXCR4 inhibitor, plerixafor, rapidly mobilizes CD34+ cells (Blood 2017 130:1920). In the current study, we evaluated the ability of MGTA-145 plus plerixafor to mobilize and effectively engraft HSCs in a pre-clinical nonhuman primate (NHP) stem cell transplantation animal model. Rhesus macaques were mobilized with a single dose of MGTA-145, plerixafor or MGTA-145 plus plerixafor versus a multi-dose regimen of G-CSF. Detailed immune profiling was performed at 0 through 24 hours post treatment. Within 4 hours of dosing, MGTA-145 plus plerixafor resulted in a 16-fold increase in the number of CD34+CD90+CD45RA- HSCs in the periphery (p=0.0003, n=11), which were previously shown to predict successful transplant as evidenced by rapid neutrophil and platelet recovery in an autologous NHP transplant model (Sci Trans Med 2017 9:1145). Next, NHPs mobilized with MGTA-145 plus plerixafor were leukapheresed and CD34+ cells enriched by positive selection. These cells were genetically modified and transplanted back into the same NHP conditioned with 1080 cGy (4 doses over 48 hours). A representative apheresis yield was 2.3 × 106 CD34+ and 0.9 × 106 HSCs per kg, and the final dose of gene-marked CD34+ and HSCs for transplant following 48 hours in culture was 1.7 × 106 and 0.8 × 106 per kg, respectively. GFP marking in vivo was consistent with the marking on the primitive HSCs prior to transplant. Neutrophil and platelet engraftment occurred at day 10 and 15 post-transplant, respectively (Figure 1). The study is ongoing, and additional follow-up data on all available animals will be presented. Co-administration of MGTA-145 plus plerixafor leads to both rapid and efficacious mobilization of CD34+ cells in the pre-clinical NHP large animal model. We found that MGTA-145 plus plerixafor mobilized grafts contain large numbers of CD34+CD90+CD45RA- HSCs, which are capable of long-term multilineage reconstitution, and preliminary results from autologous transplantation studies in NHPs demonstrate that MGTA-145 plus plerixafor mobilized HSCs lead to rapid neutrophil and platelet recovery. This study demonstrates, for the first time, that CD34+ cells mobilized with a novel regimen of MGTA-145 plus plerixafor can be collected via leukapheresis, subsequently gene modified and transplanted into NHPs, resulting in rapid engraftment.

Co-Administration of Mgta-145 and Plerixafor Rapidly Mobilizes High Numbers of Hematopoietic Stem Cells and Graft-Versus-Host Disease Inhibiting Monocytic Cells in Non-Human Primates

The majority of bone marrow transplants (BMTs) utilize granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood (mPB) as the source of hematopoietic stem cells (HSCs). Mobilization with G-CSF requires a multi-day dosing regimen and fails to mobilize a large proportion of CD34+CD90+CD45RA- HSCs, the cell type responsible for hematopoietic engraftment. Moreover, up to 80% of allogenic recipients will experience significant side effects such as acute graft-versus-host disease (GvHD), even though G-CSF mPB contains a small and variable number of immunosuppressive monocytes (Vendramin et al., BBMT 2014). Identification of a mobilizing regimen that consistently and rapidly produces high numbers of HSCs and immunosuppressive monocytes without the need for G-CSF would be ideal. We previously reported that MGTA-145, a CXCR2 agonist, rapidly mobilizes HSCs when combined with the CXCR4 inhibitor, plerixafor (Blood 2017 130:1920). In the present study, detailed profiling was performed at 0 through 24 hours after mobilization of non-human primates (NHPs) with a single dose of MGTA-145, plerixafor, or MGTA-145 plus plerixafor versus a multi-dose, five-day regimen of G-CSF. MGTA-145 plus plerixafor led to a 19-fold increase in number of CD34+CD90+CD45RA- HSCs within four hours of dosing (p As the risk of GvHD remains a significant clinical problem in the allogenic setting, MGTA-145 plus plerixafor may rapidly mobilize an advantageous graft relative to the standard of care, G-CSF, since MGTA-145 plus plerixafor results in significantly higher numbers of both engraftable HSCs and highly immunosuppressive monocytes.

MGTA-145 in Combination with Plerixafor Rapidly Mobilizes High Numbers of Hematopoietic Stem Cells and Graft-Versus-Host Disease Inhibiting Myeloid-Derived Suppressor Cells in Non-Human Primates

Background. The majority of bone marrow transplants (BMTs) are performed with granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood (mPB) as the source of hematopoietic stem cells (HSCs) for patients. Up to 80% of mPB allogeneic recipients, however, will experience graft-versus-host disease (GvHD). Despite higher levels of CD3+ T cells in mPB grafts compared to BM, the level of acute GvHD observed following transplant of HLA-matched mPB is comparable to HLA-matched BM. One explanation is that G-CSF mobilized grafts contain myeloid-derived suppressor cells (MDSCs) possessing potent immunosuppressive properties capable of inhibiting T cell proliferation in vitro. The percentage of MDSCs is variable in grafts mobilized with G-CSF and clinical data suggest that patients transplanted with mPB grafts that contain higher numbers of MDSCs may have better outcomes including lower rates of acute GvHD (Vendramin et al., BBMT 2014). Identification of a mobilizing regimen that consistently produces high numbers of HSCs and MDSCs may be preferred.We recently reported that MGTA-145 (GroβT), a CXCR2 agonist, when combined with the CXCR4 inhibitor, plerixafor, robustly mobilizes HSCs (Blood 2017 130:1920). In this study, non-human primates (NHPs) were mobilized with a single dose of MGTA-145, plerixafor, or MGTA-145/plerixafor versus a multi-dose regimen of G-CSF, and mPB was harvested to allow detailed immune profiling at 0 through 24 hours. We observed a significant and rapid increase in number of HSCs and CD34dim monocytes with potent in vitro and in vivo immunosuppressive properties.Results. MGTA-145/plerixafor consistently produced a 16-fold increase in number of CD34+CD90+CD45RA- HSCs within four hours of dosing (p=0.0003, n=11). Profiling of graft subsets from these primates also showed a 10-fold increase over baseline in the number of CD34dim monocytes at 4 hours post treatment (p<0.0001, n=11, Figure 1A) that corresponded to 2-3-fold higher frequency and number compared to G-CSF or plerixafor alone (p<0.01, n=2-5) and correlated with degree of HSC mobilization (p<0.0001). To determine if this monocytic cell population had immunosuppressive properties, CD34dim cells were sorted from peripheral blood of NHPs treated with MGTA-145/plerixafor and co-cultured with anti-CD2, anti-CD3 and anti-CD28-stimulated autologous T cells. MGTA-145/plerixafor CD34dim monocytes suppressed T cell proliferation, as measured by CFSE staining after four days.To assess whether these immunosuppressive monocytes may prevent GvHD, we developed a xenograft GvHD model in NSG mice. MGTA-145/plerixafor mPB (6 x 106 PBMCs) containing a high percentage of CD34dim monocytes were injected into sublethally irradiated NSG mice. This was compared to unmobilized primate PBMCs (6 x 106 PBMCs) containing relatively low numbers of CD34dim cells. At day 20, all mice (8/8) transplanted with unmobilized PBMCs had died of acute GvHD compared to none of the mice transplanted with MGTA-145/plerixafor mPB. Mice transplanted with unmobilized PBMCs also demonstrated 3-fold higher numbers of T-cells and increased T-cell activation compared to mice transplanted with MGTA-145/plerixafor mobilized PBMCs (p<0.01, n=6-8). At day 60 post-transplant, 7/8 mice remained alive (Figure 1B, p<0.0001). To assess whether this immunosuppressive effect is due to CD34dim monocytes, we sorted these cells and transplanted PBMCs depleted of CD34dim monocytes into NSG mice. In addition, experiments comparing the number and function of primate HSCs mobilized by MGTA-145/plerixafor or G-CSF alone using the NSG engraftment model and using autologous NHP transplant coupled with ex vivo HSC gene therapy are ongoing.Conclusions. Co-administration of MGTA-145/plerixafor in NHPs results in both rapid and efficacious mobilization of highly enriched HSCs and a CD34dim monocyte population with potent immunosuppressive activity compared to cells mobilized with plerixafor alone or with the current standard of care, G-CSF. The increased number of these immunosuppressive monocytes compared to G-CSF has the potential to reduce GvHD in the allogenic transplant setting. Thus, MGTA-145/plerixafor may offer an advantageous graft in the allogeneic setting where the risk of GvHD remains a significant clinical problem. IND-enabling studies of MGTA-145 are in progress to assess this regimen for mPB collection and transplant. [Display omitted] DisclosuresGoncalves:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Falahee:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Li:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Morrow:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Overexpression of CXCR4 synergizes with LL-37 in the metastasis of breast cancer cells

Chemokine receptor CXCR4 was involved in the progression of breast cancer to a metastatic phenotype, leading to the major cause of death in patients. A more in-depth understanding of signaling mechanism underlying CXCR4 is critical to develop effective therapies toward metastasis. Recently, the role of antimicrobial peptide LL-37 in contributing to the metastasis of breast cancer cells was observed. Clinical analysis of data herein demonstrated for the first time that overexpression of LL-37 and CXCR4 co-existed in human primary breast tumors with lymph node metastases. Further study disclosed that forced expression of CXCR4 led to the enhancement of pro-migratory signaling and migration rate induced by LL-37 in breast cancer cells. Moreover, LL-37 affected tumor microenvironment including induction of migration of mesenchymal stem cells and CXCR4-dependent capillary-like tubule formation. Functional analysis showed that LL-37 induced the internalization of CXCR4 through approaching Glu268, the residue of CXCR4, independent of the binding pocket (Asp171, Asp262, and Glu288) for CXCR4 inhibitor AMD3100, signifying that LL-37 is a distinct agonist of CXCR4. These results suggest the reciprocal roles of LL-37 and CXCR4 in promoting breast cancer cell migration and provide new insight into the design of CXCR4 inhibitor for intervention of metastatic breast cancer.

Lactobacillus rhamnosus GG protects the intestinal epithelium from radiation injury through release of lipoteichoic acid, macrophage activation and the migration of mesenchymal stem cells

ObjectiveLactobacillus rhamnosus GG (LGG), a probiotic, given by gavage is radioprotective of the mouse intestine. LGG-induced radioprotection is toll-like receptor 2 (TLR2) and cyclooxygenase-2 (COX-2)-dependent and is associated with the...

The Effect of Gliptins to Improve Functional Outcome after Stroke in Mice Is Mediated by the CXCR4/SDF-1α Pathway

Dipeptidyl peptidase-4 (DPP-4) inhibitors (gliptins) decrease hyperglycemia by inhibiting glucagon-like peptide-1 (GLP-1) cleavage. Gliptins can also improve stroke outcome in rodents independently of GLP-1R. However, the underlying mechanisms are unknown. Stromal cell-derived factor-1α (SDF-1α) is a DPP-4 substrate and CXCR4 agonist that promotes beneficial effects in injured brains. However, SDF-1α involvement in gliptin-mediated neuroprotection is unproven. We aimed to determine whether gliptins improve stroke outcome via SDF-1α/CXCR4 and identify additional targets behind the efficacy. Adult, male C57bl6/j mice (n=101) underwent transient focal cerebral ischemia. Linagliptin was administered for 3 days or 3 weeks. The CXCR4-antagonist AMD3100 was administered starting a day before ischemia. Stroke outcome was assessed by measuring upper-limb function and brain tissue damage. Levels of active GLP-1, gastric inhibitory polypeptide (GIP) and SDF-1α were quantified by ELISA in serum and brain. Mass spectrometry was used to identify additional gliptin-mediated effectors in brain tissue samples. Sustained, post-ischemic treatment with linagliptin reduced motor impairment (p003C0.01) and tissue damage (p003C0.05). Linagliptin increased serum levels of GLP-1 (p003C0.0001), GIP (p003C0.05) and SDF-1α (p003C0.05). However, only SDF-1α levels were increased in the brain (p003C0.001). Linagliptin also decreased the presence of neurogranin-derived peptides and peptides from an isoform of myelin basic protein (MBG, p003C0.05). The inhibition of SDF-1α/CXCR4 pathway diminished the positive effects of linagliptin on stroke outcome (not different from vehicle). We propose a gliptin-mediated neuroprotective mechanism via SDF-1α, which could affect Ca2+ homeostasis, altering neurogranin and MBG processing and potentially decreasing calpain activity. These results provide new insights into restorative gliptin-mediated effects against stroke. Disclosure F. Chiazza: Other Relationship; Self; Boehringer Ingelheim GmbH. H. Tammen: Consultant; Self; Boehringer Ingelheim GmbH. H. Pintana: Other Relationship; Self; Boehringer Ingelheim GmbH. G. Lietzau: Other Relationship; Self; Boehringer Ingelheim GmbH. M. Collino: None. T. Nystrom: Research Support; Self; AstraZeneca. Advisory Panel; Self; Eli Lilly and Company, Novo Nordisk A/S. Speaker's Bureau; Self; Boehringer Ingelheim GmbH. T. Klein: Employee; Self; Boehringer Ingelheim GmbH. V. Darsalia: Other Relationship; Self; Boehringer Ingelheim GmbH. C. Patrone: Other Relationship; Self; Boehringer Ingelheim GmbH.

The SDF1-CXCR4 Signals Regulate Adipose Tissue Expansion by Modulating Angiogenesis in Diet-Induced Obesity in Mice

Adipose tissue (AT) expands with angiogenesis in obesity. Mature vessels in the epididymal white adipose tissue (eWAT) are tightly wrapped with pericytes (PCs), which attenuates endothelial (ECs) proliferation. We found that obesity-associated increase of PDGF-B promoted detachment of PCs from nature vessels; thereby vessels were prone to neoangiogenesis. In this study, we aimed to clarify the role of stromal cell derived factor 1 (SDF1), another proangiogenic factor in the AT development in obesity. In the whole-mount immunofluorescence study of organ cultured eWAT, treatment with PDGF-B or SDF1 for 24 h significantly promoted detachment of PCs from vessels. The effect of SDF1 on PCs was reproduced by treatment with ATI2341, a potent CXCR4 agonist, but not by VUX11207, a CXCR7 agonist. Interestingly, PCs were well associated with vessels when tissue was treated with both PDGF-B and SDF1. In addition, the association was declined by preincubation with AMD3100, a CXCR4 antagonist or when AT was derived from Pdgfrb knockout mice. To further explore the in vivo effect of SDF1 in AT during obesity, we administrated Anagliptin (Ana), a DPP-4 inhibitor to C57BL/6J mice fed chow or HFD for 12 weeks, since SDF1 is a known substrate for DPP-4. The protein amount of SDF1 was significantly elevated in the eWAT of Ana-treated mice. Importantly, HFD-induced adipose tissue expansion was significantly attenuated with reduced vascular density and PCs detachment in the eWAT by Ana. To clarify the mechanism by which PCs attach even under HFD-fed condition by coexistence of SDF1 and PDGF-B, we isolated ECs, PCs, and macrophages (Mφ) from eWAT of mice. Expression of PDGFB was increased in Mφ and decreased in ECs by HFD, whereas these changes were attenuated and the gradient of PDGFB concentration between PCs and ECs was blunted in Ana-treated eWAT. These findings suggest that both PDGF-B and SDF1 coordinately regulate PCs detachments and adipose tissue expansion with obesity in mice. Disclosure T. Wada: Research Support; Self; Sanwa Kagaku Kenkyusho Co., Ltd.. E. Watanabe: None. Y. Kotera: None. Y. Onogi: None. H. Tsuneki: None. T. Sasaoka: None.

The effect of DPP-4 inhibition to improve functional outcome after stroke is mediated by the SDF-1\u03b1/CXCR4 pathway

BackgroundDipeptidyl peptidase-4 (DPP-4) inhibitors (gliptins) are approved drugs for the treatment of hyperglycemia in patients with type 2 diabetes. These effects are mainly mediated by inhibiting endogenous glucagon-like peptide-1 (GLP-1) cleavage. Interestingly, gliptins can also improve stroke outcome in rodents independently from GLP1. However, the underlying mechanisms are unknown. Stromal cell-derived factor-1α (SDF-1α) is a DPP-4 substrate and CXCR4 agonist promoting beneficial effects in injured brains. However, SDF-1α involvement in gliptin-mediated neuroprotection after ischemic injury is unproven. We aimed to determine whether the gliptin linagliptin improves stroke outcome via the SDF-1α/CXCR4 pathway, and identify additional effectors behind the efficacy.MethodsMice were subjected to stroke by transient middle cerebral artery occlusion (MCAO). linagliptin was administered for 3 days or 3 weeks from stroke onset. The CXCR4-antagonist AMD3100 was administered 1 day before MCAO until 3 days thereafter. Stroke outcome was assessed by measuring upper-limb function, infarct volume and neuronal survival. The plasma and brain levels of active GLP-1, GIP and SDF-1α were quantified by ELISA. To identify additional gliptin-mediated molecular effectors, brain samples were analyzed by mass spectrometry.ResultsLinagliptin specifically increased active SDF-1α but not glucose-dependent insulinotropic peptide (GIP) or GLP-1 brain levels. Blocking of SDF-1α/CXCR4 pathway abolished the positive effects of linagliptin on upper-limb function and histological outcome after stroke. Moreover, linagliptin treatment after stroke decreased the presence of peptides derived from neurogranin and from an isoform of the myelin basic protein.ConclusionsWe showed that linagliptin improves functional stroke outcome in a SDF-1α/CXCR4-dependent manner. Considering that Calpain activity and intracellular Ca2+ regulate neurogranin and myelin basic protein detection, our data suggest a gliptin-mediated neuroprotective mechanism via the SDF-1α/CXCR4 pathway that could involve the regulation of Ca2+ homeostasis and the reduction of Calpain activity. These results provide new insights into restorative gliptin-mediated effects against stroke.

CXCR4 inhibitor, MSX‐122 suppresses AOM‐induced colon cancer in Apc+/Min mouse

IntroductionIn prior studies, we showed that EGFR is required for tumor promotion by Western diet in the azoxymethane (AOM) model of sporadic colon cancer and the AOM/DSS model of inflammation‐associated colon cancer. We also demonstrated that ADAM17, the metalloprotease responsible for shedding membrane‐bound EGFR ligands, controls tumor development in both the AOM and the Apc+/mutant multiple intestinal neoplasia (Min) mouse model. Furthermore, we established that Sdf1a‐CXCR4 signals activated EGFR by an ADAM17‐dependent mechanism in colon cancer cells; and that Western diet up‐regulated Sdf1a‐CXCR4 and ADAM17 in colonic mucosa. We hypothesized that CXCR4 blockade might inhibit colon cancer development. The aim of this study was to test the ability of a novel CXCR4 antagonist, MSX‐122 (substituted benzylamine) to inhibit AOM‐promoted colon cancer in Apc+/Min mice. Min mice without AOM treatment mostly develop small intestinal tumors, whereas AOM‐treated Apc+/Min mice develop colon cancers (Suzui et al 2002).MethodsIn studies in cell culture, we examined the ability of MSX‐122 to block Sdf1a‐induced EGFR transactivation. In vivo, Apc+/Min mice were given AOM (10 mg i.p./kg body weight) and two weeks later mice were started on a Western diet (20% fat). Mice received MSX‐122 i.p. (2.5 mg/kg body weight, n=12) or vehicle (DMSO, n=13) three times weekly and were killed after 10 wks.ResultsMSX‐122 blocked EGFR transactivation by Sdf1a in HT29 colon cancer cells as assessed by reductions in pErbB2, pAKT and pERK. In vivo, MSX‐122 was well tolerated with mice showing comparable weight gain to vehicle‐treated mice. As expected, AOM induced colon tumors in Apc+/Min mice. MSX‐122 significantly reduced colon tumor incidence (92.3% vs. 50.0%, p=0.025) and cancer incidence (69.2% vs. 16.7%, p=0.015). MSX‐122 also reduced tumor burden as assessed by total tumor multiplicity (1.8 vs. 1.0 tumor per TBM; p=0.02), tumor volume (169.5 mm3 vs. 61.2 mm3, p&lt;0.023) and tumor proliferation as assessed by Ki67 staining (37.2±3.6% vs. 25.3±5.8%, p&lt;0.005). Cancer multiplicity also appeared to be decreased (1.6 vs. 1.0 tumor/TBM, p=0.17).ConclusionsMSX‐122 blocked EGFR transactivation by CXCR4 agonist Sdf1a in colon cancer cells. MSX‐122 also inhibited colon tumor development and progression in the AOM‐promoted Apc+/Min mouse model. We speculate that MSX‐122, which was well tolerated in a phase 1b clinical trial (Painter, W. 2015), could serve as a chemopreventive agent in individuals at increased risk for colon cancer.Support or Funding InformationNIH R01CA164124This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rapid Mobilization Reveals a Highly Engraftable Hematopoietic Stem Cell

Hematopoietic stem cell transplantation is a potential curative therapy for malignant and nonmalignant diseases. Improving the efficiency of stem cell collection and the quality of the cells acquired can broaden the donor pool and improve patient outcomes. We developed a rapid stem cell mobilization regimen utilizing a unique CXCR2 agonist, GROβ, and the CXCR4 antagonist AMD3100. A single injection of both agents resulted in stem cell mobilization peaking within 15min that was equivalent in magnitude to a standard multi-day regimen of granulocyte colony-stimulating factor (G-CSF). Mechanistic studies determined that rapid mobilization results from synergistic signaling on neutrophils, resulting in enhanced MMP-9 release, and unexpectedly revealed genetic polymorphisms in MMP-9 that alter activity. This mobilization regimen results in preferential trafficking of stem cells that demonstrate a higher engraftment efficiency than those mobilized by G-CSF. Our studies suggest a potential new strategy for the rapid collection of an improved hematopoietic graft.

Effects of cognate, non-cognate and synthetic CXCR4 and ACKR3 ligands on human lung endothelial cell barrier function

Recent evidence suggests that chemokine CXCL12, the cognate agonist of chemokine receptors CXCR4 and ACKR3, reduces thrombin-mediated impairment of endothelial barrier function. A detailed characterization of the effects of CXCL12 on thrombin-mediated human lung endothelial hyperpermeability is lacking and structure-function correlations are not available. Furthermore, effects of other CXCR4/ACKR3 ligands on lung endothelial barrier function are unknown. Thus, we tested the effects of a panel of CXCR4/ACKR3 ligands (CXCL12, CXCL11, ubiquitin, AMD3100, TC14012) and compared the CXCR4/ACKR3 activities of CXCL12 variants (CXCL12α/β, CXCL12(3–68), CXCL121, CXCL122, CXCL12-S-S4V, CXCL12-R47E, CXCL12-K27A/R41A/R47A) with their effects on human lung endothelial barrier function in permeability assays. CXCL12α enhanced human primary pulmonary artery endothelial cell (hPPAEC) barrier function, whereas CXCL11, ubiquitin, AMD3100 and TC14012 were ineffective. Pre-treatment of hPPAEC with CXCL12α and ubiquitin reduced thrombin-mediated hyperpermeability. CXCL12α-treatment of hPPAEC after thrombin exposure reduced barrier function impairment by 70% (EC50 0.05–0.5nM), which could be antagonized with AMD3100; ubiquitin (0.03–3μM) was ineffective. In a human lung microvascular endothelial cell line (HULEC5a), CXCL12α and ubiquitin post-treatment attenuated thrombin-induced hyperpermeability to a similar degree. CXCL12(3–68) was inefficient to activate CXCR4 in Presto-Tango β-arrestin2 recruitment assays; CXCL12-S-S4V, CXCL12-R47E and CXCL12-K27A/R41A/R47A showed significantly reduced potencies to activate CXCR4. While the potencies of all proteins in ACKR3 Presto-Tango assays were comparable, the efficacy of CXCL12(3–68) to activate ACKR3 was significantly reduced. The potencies to attenuate thrombin-mediated hPPAEC barrier function impairment were: CXCL12α/β, CXCL121, CXCL12-K27A/R41A/R47A > CXCL12-S-S4V, CXCL12-R47E > CXCL122 > CXCL12(3–68). Our findings indicate that CXCR4 activation attenuates thrombin-induced lung endothelial barrier function impairment and suggest that protective effects of CXCL12 are dictated by its CXCR4 agonist activity and interactions of distinct protein moieties with heparan sulfate on the endothelial surface. These data may facilitate development of compounds with improved pharmacological properties to attenuate thrombin-induced vascular leakage in the pulmonary circulation.

Ligand activation induces different conformational changes in CXCR3 receptor isoforms as evidenced by plasmon waveguide resonance (PWR)

The chemokine receptor CXCR3 plays important roles in angiogenesis, inflammation and cancer. Activation studies and biological functions of CXCR3 are complex due to the presence of spliced isoforms. CXCR3-A is known as a pro-tumor receptor whereas CXCR3-B exhibits anti-tumor properties. Here, we focused on the conformational change of CXCR3-A and CXCR3-B after agonist or antagonist binding using Plasmon Waveguide Resonance (PWR). Agonist stimulation induced an anisotropic response with very distinct conformational changes for the two isoforms. The CXCR3 agonist bound CXCR3-A with higher affinity than CXCR3-B. Using various concentrations of SCH546738, a CXCR3 specific inhibitor, we demonstrated that low SCH546738 concentrations (≤1 nM) efficiently inhibited CXCR3-A but not CXCR3-B’s conformational change and activation. This was confirmed by both, biophysical and biological methods. Taken together, our study demonstrates differences in the behavior of CXCR3-A and CXCR3-B upon ligand activation and antagonist inhibition which may be of relevance for further studies aimed at specifically inhibiting the CXCR3A isoform.

G Protein-Coupled Receptor Kinase 3 and Protein Kinase C Phosphorylate the Distal C-Terminal Tail of the Chemokine Receptor CXCR4 and Mediate Recruitment of β-Arrestin.

Phosphorylation of G protein-coupled receptors (GPCRs) is a key event for cell signaling and regulation of receptor function. Previously, using tandem mass spectrometry, we identified two phosphorylation sites at the distal C-terminal tail of the chemokine receptor CXCR4, but were unable to determine which specific residues were phosphorylated. Here, we demonstrate that serines (Ser) 346 and/or 347 (Ser-346/7) of CXCR4 are phosphorylated upon stimulation with the agonist CXCL12 as well as a CXCR4 pepducin, ATI-2341. ATI-2341, a Gαiβγ heterotrimer-biased CXCR4 agonist, induced more robust phosphorylation of Ser-346/7 compared with CXCL12. Knockdown of G protein-coupled receptor kinase (GRK) 2, GRK3, or GRK6 reduced CXCL12-induced phosphorylation of Ser-346/7 with GRK3 knockdown having the strongest effect, while inhibition of the conventional protein kinase C (PKC) isoforms, particularly PKCα, reduced phosphorylation of Ser-346/7 induced by either CXCL12 or ATI-2341. The loss of GRK3- or PKC-mediated phosphorylation of Ser-346/7 impaired the recruitment of β-arrestin to CXCR4. We also found that a pseudo-substrate peptide inhibitor for PKCζ effectively inhibited CXCR4 phosphorylation and signaling, most likely by functioning as a nonspecific CXCR4 antagonist. Together, these studies demonstrate the role Ser-346/7 plays in arrestin recruitment and initiation of receptor desensitization and provide insight into the dysregulation of CXCR4 observed in patients with various forms of WHIM syndrome.