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1
Hara, Takahiko, and Kosuke Tanegashima. "CXCL14 antagonizes the CXCL12-CXCR4 signaling axis." Biomolecular Concepts 5, no. 2 (2014): 167–73. http://dx.doi.org/10.1515/bmc-2014-0007.
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AbstractCXCL12 and CXCL14 are evolutionarily conserved members of the CXC-type chemokine family. CXCL12 binds specifically to the G-protein-coupled receptor CXCR4 to induce the migration of primordial germ cells, hematopoietic stem cells, and inflammation-associated immune cells. In addition, CXCL12-CXCR4 signaling is often enhanced in malignant tumor cells and facilitates increased proliferation as well as metastasis. Although macrophage migration inhibitory factor and extracellular ubiquitin interact with CXCR4 as agonistic factors, CXCL12 was believed to be the sole chemokine ligand for CXCR4. However, a very recent report revealed that CXCL14 binds to CXCR4 with high affinity and efficiently inhibits CXCL12-mediated chemotaxis of hematopoietic progenitor and leukemia-derived cells. CXCL14 does not directly cross-compete with CXCL12 for the CXCR4 binding but instead inactivates CXCR4 via receptor internalization. Because both CXCL12 and CXCL14 are expressed during embryogenesis and brain development in mice, these two chemokines could function in an interactive fashion. We propose that the CXCL14 gene has been conserved from fish to man due to its role in fine-tuning the strength of CXCL12-mediated signal transduction. In addition to its biological implications, the above finding will be important for designing anti-cancer compounds targeting the CXCL12-CXCR4 signaling axis. In fact, a stabilized dimeric peptide containing the C-terminal 51–77 amino acid residues of CXCL14 has been shown to have stronger CXCL12 antagonistic activity than full-length CXCL14.
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Urbantat, Ruth M., Peter Vajkoczy, and Susan Brandenburg. "Advances in Chemokine Signaling Pathways as Therapeutic Targets in Glioblastoma." Cancers 13, no. 12 (2021): 2983. http://dx.doi.org/10.3390/cancers13122983.
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With a median patient survival of 15 months, glioblastoma (GBM) is still one of the deadliest malign tumors. Despite immense efforts, therapeutic regimens fail to prolong GBM patient overall survival due to various resistance mechanisms. Chemokine signaling as part of the tumor microenvironment plays a key role in gliomagenesis, proliferation, neovascularization, metastasis and tumor progression. In this review, we aimed to investigate novel therapeutic approaches targeting various chemokine axes, including CXCR2/CXCL2/IL-8, CXCR3/CXCL4/CXCL9/CXCL10, CXCR4/CXCR7/CXCL12, CXCR6/CXCL16, CCR2/CCL2, CCR5/CCL5 and CX3CR1/CX3CL1 in preclinical and clinical studies of GBM. We reviewed targeted therapies as single therapies, in combination with the standard of care, with antiangiogenic treatment as well as immunotherapy. We found that there are many antagonist-, antibody-, cell- and vaccine-based therapeutic approaches in preclinical and clinical studies. Furthermore, targeted therapies exerted their highest efficacy in combination with other established therapeutic applications. The novel chemokine-targeting therapies have mainly been examined in preclinical models. However, clinical applications are auspicious. Thus, it is crucial to broadly investigate the recently developed preclinical approaches. Promising preclinical applications should then be investigated in clinical studies to create new therapeutic regimens and to overcome therapy resistance to GBM treatment.
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Chu, Tianci, Lisa B. E. Shields, Yi Ping Zhang, Shi-Qing Feng, Christopher B. Shields, and Jun Cai. "CXCL12/CXCR4/CXCR7 Chemokine Axis in the Central Nervous System: Therapeutic Targets for Remyelination in Demyelinating Diseases." Neuroscientist 23, no. 6 (2017): 627–48. http://dx.doi.org/10.1177/1073858416685690.
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The chemokine CXCL12 plays a vital role in regulating the development of the central nervous system (CNS) by binding to its receptors CXCR4 and CXCR7. Recent studies reported that the CXCL12/CXCR4/CXCR7 axis regulates both embryonic and adult oligodendrocyte precursor cells (OPCs) in their proliferation, migration, and differentiation. The changes in the expression and distribution of CXCL12 and its receptors are tightly associated with the pathological process of demyelination in multiple sclerosis (MS), suggesting that modulating the CXCL12/CXCR4/CXCR7 axis may benefit myelin repair by enhancing OPC recruitment and differentiation. This review aims to integrate the current findings of the CXCL12/CXCR4/CXCR7 signaling pathway in the CNS and to highlight its role in oligodendrocyte development and demyelinating diseases. Furthermore, this review provides potential therapeutic strategies for myelin repair by analyzing the relevance between the pathological changes and the regulatory roles of CXCL12/CXCR4/CXCR7 during MS.
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Zhou, Weiqiang, Shanchun Guo, Mingli Liu, Matthew E. Burow, and Guangdi Wang. "Targeting CXCL12/CXCR4 Axis in Tumor Immunotherapy." Current Medicinal Chemistry 26, no. 17 (2019): 3026–41. http://dx.doi.org/10.2174/0929867324666170830111531.
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Chemokines, which have chemotactic abilities, are comprised of a family of small cytokines with 8-10 kilodaltons. Chemokines work in immune cells by trafficking and regulating cell proliferation, migration, activation, differentiation, and homing. CXCR-4 is an alpha-chemokine receptor specific for stromal-derived-factor-1 (SDF-1, also known as CXCL12), which has been found to be expressed in more than 23 different types of cancers. Recently, the SDF-1/CXCR-4 signaling pathway has emerged as a potential therapeutic target for human tumor because of its critical role in tumor initiation and progression by activating multiple signaling pathways, such as ERK1/2, ras, p38 MAPK, PLC/ MAPK, and SAPK/ JNK, as well as regulating cancer stem cells. CXCL12/CXCR4 antagonists have been produced, which have shown encouraging results in anti-cancer activity. Here, we provide a brief overview of the CXCL12/CXCR4 axis as a molecular target for cancer treatment. We also review the potential utility of targeting CXCL12/CXCR4 axis in combination of immunotherapy and/or chemotherapy based on up-to-date literature and ongoing research progress.
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Khare, Tripti, Marc Bissonnette, and Sharad Khare. "CXCL12-CXCR4/CXCR7 Axis in Colorectal Cancer: Therapeutic Target in Preclinical and Clinical Studies." International Journal of Molecular Sciences 22, no. 14 (2021): 7371. http://dx.doi.org/10.3390/ijms22147371.
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Chemokines are chemotactic cytokines that promote cancer growth, metastasis, and regulate resistance to chemotherapy. Stromal cell-derived factor 1 (SDF1) also known as C-X-C motif chemokine 12 (CXCL12), a prognostic factor, is an extracellular homeostatic chemokine that is the natural ligand for chemokine receptors C-X-C chemokine receptor type 4 (CXCR4), also known as fusin or cluster of differentiation 184 (CD184) and chemokine receptor type 7 (CXCR7). CXCR4 is the most widely expressed rhodopsin-like G protein coupled chemokine receptor (GPCR). The CXCL12–CXCR4 axis is involved in tumor growth, invasion, angiogenesis, and metastasis in colorectal cancer (CRC). CXCR7, recently termed as atypical chemokine receptor 3 (ACKR3), is amongst the G protein coupled cell surface receptor family that is also commonly expressed in a large variety of cancer cells. CXCR7, like CXCR4, regulates immunity, angiogenesis, stem cell trafficking, cell growth and organ-specific metastases. CXCR4 and CXCR7 are expressed individually or together, depending on the tumor type. When expressed together, CXCR4 and CXCR7 can form homo- or hetero-dimers. Homo- and hetero-dimerization of CXCL12 and its receptors CXCR4 and CXCR7 alter their signaling activity. Only few drugs have been approved for clinical use targeting CXCL12-CXCR4/CXCR7 axis. Several CXCR4 inhibitors are in clinical trials for solid tumor treatment with limited success whereas CXCR7-specific inhibitors are still in preclinical studies for CRC. This review focuses on current knowledge of chemokine CXCL12 and its receptors CXCR4 and CXCR7, with emphasis on targeting the CXCL12–CXCR4/CXCR7 axis as a treatment strategy for CRC.
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Lin, Yuanqiang, Qingjie Ma, Lin Li та Hui Wang. "The CXCL12–CXCR4 axis promotes migration, invasiveness, and EMT in human papillary thyroid carcinoma B-CPAP cells via NF-κB signaling". Biochemistry and Cell Biology 96, № 5 (2018): 619–26. http://dx.doi.org/10.1139/bcb-2017-0074.
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Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy involving local and distant metastasis. It is known that CXC chemokine ligand 12 (CXCL12) interacts specifically with CXC chemokine receptor 4 (CXCR4) to guide the migration of PTC cells. However, the signaling pathway downstream of the CXCL12–CXCR4 axis in PTC is not fully understood. In the present study, high expression of CXCR4 was detected in 38 out of 82 specimens of PTC, and the expression level of CXCR4 significantly correlated with the stage of PTC. Additionally, the roles of the CXCL12–CXCR4 axis in the migration, invasion, and epithelial–mesenchymal transition (EMT) of B-CPAP cells were investigated in vitro. The motility and invasiveness were significantly enhanced in CXCR4-overexpressing B-CPAP cells with CXCL12 treatment. Moreover, the CXCL12–CXCR4 axis promoted the EMT process, as evidenced by a decreased level of E-cadherin and increased expressions of N-cadherin and vimentin. Furthermore, the CXCL12–CXCR4 axis activated the nuclear factor kappa-B (NF-κB) signaling pathway, whereas BAY11-7082, an IκB phosphorylation inhibitor, counteracted CXCL12–CXCR4-induced migration, invasion, and EMT processes in B-CPAP cells. In conclusion, the CXCL12–CXCR4 axis promotes the migration, invasion, and EMT processes in B-CPAP cells, at least partly, by activating the NF-κB signaling pathway.
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Smit, Martine J., Géraldine Schlecht-Louf, Maria Neves, et al. "The CXCL12/CXCR4/ACKR3 Axis in the Tumor Microenvironment: Signaling, Crosstalk, and Therapeutic Targeting." Annual Review of Pharmacology and Toxicology 61, no. 1 (2021): 541–63. http://dx.doi.org/10.1146/annurev-pharmtox-010919-023340.
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Elevated expression of the chemokine receptors CXCR4 and ACKR3 and of their cognate ligand CXCL12 is detected in a wide range of tumors and the tumor microenvironment (TME). Yet, the molecular mechanisms by which the CXCL12/CXCR4/ACKR3 axis contributes to the pathogenesis are complex and not fully understood. To dissect the role of this axis in cancer, we discuss its ability to impinge on canonical and less conventional signaling networks in different cancer cell types; its bidirectional crosstalk, notably with receptor tyrosine kinase (RTK) and other factors present in the TME; and the infiltration of immune cells that supporttumor progression. We discuss current and emerging avenues that target the CXCL12/CXCR4/ACKR3 axis. Coordinately targeting both RTKs and CXCR4/ACKR3 and/or CXCL12 is an attractive approach to consider in multitargeted cancer therapies. In addition, inhibiting infiltrating immune cells or reactivating the immune system along with modulating the CXCL12/CXCR4/ACKR3 axis in the TME has therapeutic promise.
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Pansy, Katrin, Julia Feichtinger, Barbara Ehall, et al. "Deregulated CXCR4-CXCL12 Signaling Impacts on the Pathogenesis of DLBCL." Blood 134, Supplement_1 (2019): 1519. http://dx.doi.org/10.1182/blood-2019-130029.
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Introduction: The interaction of the chemokine receptor CXCR4 and its ligand CXCL12 appears to be implicated in many important biological processes such as proliferation, survival, migration, and/or invasion. Furthermore, it is important for normal leukocyte trafficking. Deregulation of this axis is frequently observed in several hematologic malignancies. In diffuse large B cell lymphomas (DLBCL), the CXCR4-CXCL12 axis is still largely unexplored and published data are contradictive. Hence, we comprehensively studied the CXCR4-CXCL12-axis in our DLBCL cohort as well as the effects of CXCR4 antagonists on lymphoma cell lines in vitro. Methods: We determined the CXCR4 and CXCL12 expression levels in NGCB- and GCB-DLBCL consisting of primary and transformed follicular lymphomas (n=77 in total), the corresponding bone marrow samples (n=63) and non-neoplastic germinal center-B cells (GC-B, n=5) serving as non-malignant control. To investigate the effects of CXCR4 antagonists in vitro and their function in regulation of important pathways (JNK, ERK1/2 and NF-κB) known to be involved in lymphomagenesis, we treated lymphoma cell lines with three different CXCR4 antagonists, AMD070, AMD3100 and WK1 (a novel nicotinic acid derivative of AMD070 - synthesized by us), followed by functional assays and gene expression profile. Results: CXCR4 was 140-fold higher expressed in DLBCL compared to non-neoplastic GC-B cells. Interestingly, higher CXCR4 expression correlated to a clinically advanced stage, to bone marrow infiltration and worse cancer-specific survival in DLBCL. Further expression analysis by using the corresponding bone marrow biopsies demonstrated that CXCL12 expression correlated to the lymphoma infiltration rate and that CXCR4 expression was reduced in remission under therapy. Moreover, two CXCR4 antagonists - AMD070 and especially WK1 - exerted pro-apoptotic effects on CXCR4 positive lymphoma cells in vitro and induced the expression of certain pro-apoptotic genes in CXCR4 positive cell lines. Remarkably, these effects were more pronounced for the WK1. Finally, WK1 treatment resulted in reduced expression of JNK-, ERK1/2- and NFκB/BCR-target genes. Conclusion: Our data demonstrate that the CXCR4-CXCL12 axis is involved in the pathogenesis of DLBCL. Since CXCR4 antagonists exert pro-apoptotic effects and impact lymphoma relevant pathways, they represent interesting molecules to develop novel therapeutic agents. Disclosures No relevant conflicts of interest to declare.
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Tanegashima, Kosuke, Kenji Suzuki, Yuki Nakayama, et al. "CXCL14 is a natural inhibitor of the CXCL12-CXCR4 signaling axis." FEBS Letters 587, no. 12 (2013): 1731–35. http://dx.doi.org/10.1016/j.febslet.2013.04.046.
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Karpova, Darja, Julie K. Ritchey, Matthew S. Holt, et al. "Continuous blockade of CXCR4 results in dramatic mobilization and expansion of hematopoietic stem and progenitor cells." Blood 129, no. 21 (2017): 2939–49. http://dx.doi.org/10.1182/blood-2016-10-746909.
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Key Points Prolonged inhibition of CXCR4/CXCL12 signaling results in exceptional mobilization along with an expansion of the BM HSPC pool. Reversible inhibition of the CXCR4/CXCL12 axis may represent a novel strategy to restore damaged BM.
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Barbieri, Federica, Stefano Thellung, Roberto Würth, et al. "Emerging Targets in Pituitary Adenomas: Role of the CXCL12/CXCR4-R7 System." International Journal of Endocrinology 2014 (2014): 1–16. http://dx.doi.org/10.1155/2014/753524.
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Chemokines are chemotactic regulators of immune surveillance in physiological and pathological conditions such as inflammation, infection, and cancer. Several chemokines and cognate receptors are constitutively expressed in the central nervous system, not only in glial and endothelial cells but also in neurons, controlling neurogenesis, neurite outgrowth, and axonal guidance during development. In particular, the chemokine CXCL12 and its receptors, CXCR4 and CXCR7, form a functional network that controls plasticity in different brain areas, influencing neurotransmission, neuromodulation, and cell migration, and the dysregulation of this chemokinergic axis is involved in several neurodegenerative, neuroinflammatory, and malignant diseases. CXCR4 primarily mediates the transduction of proliferative signals, while CXCR7 seems to be mainly responsible for scavenging CXCL12. Importantly, the multiple intracellular signalling generated by CXCL12 interaction with its receptors influences hypothalamic modulation of neuroendocrine functions, although a direct modulation of pituitary functioningviaautocrine/paracrine mechanisms was also reported. Both CXCL12 and CXCR4 are constitutively overexpressed in pituitary adenomas and their signalling induces cell survival and proliferation, as well as hormonal hypersecretion. In this review we focus on the physiological and pathological functions of immune-related cyto- and chemokines, mainly focusing on the CXCL12/CXCR4-7 axis, and their role in pituitary tumorigenesis. Accordingly, we discuss the potential targeting of CXCR4 as novel pharmacological approach for pituitary adenomas.
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Liou, Angela, Cristina Delgado-Martin, David T. Teachey, and Michelle L. Hermiston. "The CXCR4/CXCL12 Axis Mediates Chemotaxis, Survival, and Chemoresistance in T-Cell Acute Lymphoblastic Leukemia." Blood 124, no. 21 (2014): 3629. http://dx.doi.org/10.1182/blood.v124.21.3629.3629.
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Abstract Background: While multi-agent chemotherapy has led to remarkable improvements in survival for children with T-cell acute lymphoblastic leukemia (T-ALL), outcomes remain dismal for the ~ 20% of patients that fail therapy. The goal of this study was to elucidate biological mechanisms contributing to chemoresistance in T-ALL. We hypothesized that chemoresistance owes not only to the aberrant signaling pathways intrinsic to the leukemia cells but also to contributions from an abnormal microenvironment in which they reside. The chemokine receptor ligand pair CXCR4/CXCL12 is important for normal leukocyte trafficking and deregulation is frequently observed in several hematologic malignancies. However, the role this axis plays in T-ALL is largely unknown. Here, we test the hypothesis that CXCR4/CXCL12 creates a sanctuary microenvironment that promotes T-ALL survival and provides protection from chemotherapy. Methods: A panel of T-ALL cell lines and primary patient samples expanded in NOD/SCID/c null (NSG) mice were cultured in the presence or absence of the CXCR4 antagonist, AMD3100, with or without chemotherapy. The human bone marrow stromal cell line, HS27a, which constitutively expresses the CXCR4 ligand CXCL12, was used to recapitulate the tumor microenvironment ex vivo. Transwell migration and modified pseudo-emperipolesis assays were used to examine CXCL12-mediated chemotaxis. Multi-parameter flow cytometry was used to evaluate the impact of activation of the CXCR4/CXCL12 axis on signaling networks, cell survival, and chemotherapy resistance. Results: We found CXCR4 membrane expression on all T-ALL cell lines and xenografts samples tested. Incubation with the CXCR4 ligand CXCL12 resulted in activation of survival signaling cascades (PI3K/AKT and MAPK), an effect blocked with AMD3100. Using a transwell system, we found dose dependent chemotaxis of T-ALL lymphoblasts to CXCL12 that was prevented by AMD3100. T-ALL cells also migrated into HS27a stromal cells in a CXCR4/CXCL12 dependent fashion. In addition, co-culturing T-ALL xenografts cells with HS27a imparted a survival advantage that was promptly eliminated by AMD3100 exposure. To test whether the CXCR4/CXCL12 microenvironment contributed to chemotherapy resistance, T-ALL xenografts cells were co-cultured ex vivo in the presence or absence of HS27a stromal cells and bortezomib or dexamethasone. Stromal cells conferred a marked chemoprotective effect that was specifically blocked by AMD3100. This highlights that stroma-mediated chemoresistance in xenograft samples is in part due to stromal-cell mediated activation of the CXCR4/CXCL12 axis. Conclusions: This study provides evidence for a T-ALL microenvironment that exploits the CXCR4/CXCL12 axis for leukemic cell recruitment, enhanced cell survival, and chemotherapy protection. Our findings also implicate the stroma as a major contributor to chemotherapy resistance in primary expanded patient samples partially due to the activation of the CXCR4/CXCL12 axis. These results further suggest that targeting the stroma through inhibition of this axis may be of therapeutic benefit in patients with chemotherapy resistant T-ALL. Disclosures No relevant conflicts of interest to declare.
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Liepelt, Anke, and Frank Tacke. "Stromal cell-derived factor-1 (SDF-1) as a target in liver diseases." American Journal of Physiology-Gastrointestinal and Liver Physiology 311, no. 2 (2016): G203—G209. http://dx.doi.org/10.1152/ajpgi.00193.2016.
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The chemokine stromal cell-derived factor-1 (SDF-1) or CXCL12 is constitutively expressed in healthy liver. However, its expression increases following acute or chronic liver injury. Liver sinusoidal endothelial cells (LSEC), hepatic stellate cells (HSC), and malignant hepatocytes are important sources of SDF-1/CXCL12 in liver diseases. CXCL12 is able to activate two chemokine receptors with different downstream signaling pathways, CXCR4 and CXCR7. CXCR7 expression is relevant on LSEC, while HSC, mesenchymal stem cells, and tumor cells mainly respond via CXCR4. Here, we summarize recent developments in the field of liver diseases involving this chemokine and its receptors. SDF-1-dependent signaling contributes to modulating acute liver injury and subsequent tissue regeneration. By activating HSC and recruiting mesenchymal cells from bone marrow, CXCL12 can promote liver fibrosis progression, while CXCL12-CXCR7 interactions endorse proregenerative responses in chronic injury. Moreover, the SDF-1 pathway is linked to development of hepatocellular carcinoma (HCC) by promoting tumor growth, angiogenesis, and HCC metastasis. High hepatic CXCR4 expression has been suggested as a biomarker indicating poor prognosis of HCC patients. Tumor-infiltrating myeloid-derived suppressor cells (MDSC) also express CXCR4 and migrate toward CXCL12. Thus CXCL12 inhibition might not only directly block HCC growth but also modulate the tumor microenvironment (angiogenesis, MDSC), thereby sensitizing HCC patients to conventional or emerging novel cancer therapies (e.g., sorafenib, regorafenib, nivolumab, pembrolizumab). We herein summarize the current knowledge on the complex interplay between CXCL12 and CXCR4/CXCR7 in liver diseases and discuss approaches on the therapeutic targeting of these axes in hepatitis, fibrosis, and liver cancer.
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Kim, Junchul, Sang-Woo Lee, and Kyungpyo Park. "CXCR4 Regulates Temporal Differentiation via PRC1 Complex in Organogenesis of Epithelial Glands." International Journal of Molecular Sciences 22, no. 2 (2021): 619. http://dx.doi.org/10.3390/ijms22020619.
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CXC-chemokine receptor type 4 (CXCR4), a 7-transmembrane receptor family member, displays multifaceted roles, participating in immune cell migration, angiogenesis, and even adipocyte metabolism. However, the activity of such a ubiquitously expressed receptor in epithelial gland organogenesis has not yet been fully explored. To investigate the relationship between CXCL12/CXCR4 signaling and embryonic glandular organogenesis, we used an ex vivo culture system with live imaging and RNA sequencing to elucidate the transcriptome and protein-level signatures of AMD3100, a potent abrogating reagent of the CXCR4-CXCL12 axis, imprinted on the developing organs. Immunostaining results showed that CXCR4 was highly expressed in embryonic submandibular gland, lung, and pancreas, especially at the periphery of end buds containing numerous embryonic stem/progenitor cells. Despite no significant increase in apoptosis, AMD3100-treated epithelial organs showed a retarded growth with significantly slower branching and expansion. Further analyses with submandibular glands revealed that such responses resulted from the AMD3100-induced precocious differentiation of embryonic epithelial cells, losing mitotic activity. RNA sequencing analysis revealed that inhibition of CXCR4 significantly down-regulated polycomb repressive complex (PRC) components, known as regulators of DNA methylation. Treatment with PRC inhibitor recapitulated the AMD3100-induced precocious differentiation. Our results indicate that the epigenetic modulation by the PRC-CXCR12/CXCR4 signaling axis is crucial for the spatiotemporal regulation of proliferation and differentiation of embryonic epithelial cells during embryonic glandular organogenesis.
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Kim, Junchul, Sang-Woo Lee, and Kyungpyo Park. "CXCR4 Regulates Temporal Differentiation via PRC1 Complex in Organogenesis of Epithelial Glands." International Journal of Molecular Sciences 22, no. 2 (2021): 619. http://dx.doi.org/10.3390/ijms22020619.
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CXC-chemokine receptor type 4 (CXCR4), a 7-transmembrane receptor family member, displays multifaceted roles, participating in immune cell migration, angiogenesis, and even adipocyte metabolism. However, the activity of such a ubiquitously expressed receptor in epithelial gland organogenesis has not yet been fully explored. To investigate the relationship between CXCL12/CXCR4 signaling and embryonic glandular organogenesis, we used an ex vivo culture system with live imaging and RNA sequencing to elucidate the transcriptome and protein-level signatures of AMD3100, a potent abrogating reagent of the CXCR4-CXCL12 axis, imprinted on the developing organs. Immunostaining results showed that CXCR4 was highly expressed in embryonic submandibular gland, lung, and pancreas, especially at the periphery of end buds containing numerous embryonic stem/progenitor cells. Despite no significant increase in apoptosis, AMD3100-treated epithelial organs showed a retarded growth with significantly slower branching and expansion. Further analyses with submandibular glands revealed that such responses resulted from the AMD3100-induced precocious differentiation of embryonic epithelial cells, losing mitotic activity. RNA sequencing analysis revealed that inhibition of CXCR4 significantly down-regulated polycomb repressive complex (PRC) components, known as regulators of DNA methylation. Treatment with PRC inhibitor recapitulated the AMD3100-induced precocious differentiation. Our results indicate that the epigenetic modulation by the PRC-CXCR12/CXCR4 signaling axis is crucial for the spatiotemporal regulation of proliferation and differentiation of embryonic epithelial cells during embryonic glandular organogenesis.
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Ghosh, Manik C., Gary D. Collins, Bolormaa Vandanmagsar, et al. "Activation of Wnt5A signaling is required for CXC chemokine ligand 12–mediated T-cell migration." Blood 114, no. 7 (2009): 1366–73. http://dx.doi.org/10.1182/blood-2008-08-175869.
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Abstract Chemokines mediate the signaling and migration of T cells, but little is known about the transcriptional events involved therein. Microarray analysis of CXC chemokine ligand (CXCL) 12−treated T cells revealed that Wnt ligands are significantly up-regulated during CXCL12 treatment. Real-time polymerase chain reaction and Western blot analysis confirmed that the expression of noncanonical Wnt pathway members (eg, Wnt5A) was specifically up-regulated during CXCL12 stimulation, whereas β-catenin and canonical Wnt family members were selectively down-regulated. Wnt5A augmented signaling through the CXCL12-CXCR4 axis via the activation of protein kinase C. Moreover, Wnt5A expression was required for CXCL12–mediated T-cell migration, and rWnt5A sensitized human T cells to CXCL12-induced migration. Furthermore, Wnt5A expression was also required for the sustained expression of CXCR4. These results were further supported in vivo using EL4 thymoma metastasis as a model of T-cell migration. Together, these data demonstrate that Wnt5A is a critical mediator of CXCL12-CXCR4 signaling and migration in human and murine T cells.
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Yang, Ping, Yae Hu, and Quansheng Zhou. "The CXCL12-CXCR4 Signaling Axis Plays a Key Role in Cancer Metastasis and is a Potential Target for Developing Novel Therapeutics against Metastatic Cancer." Current Medicinal Chemistry 27, no. 33 (2020): 5543–61. http://dx.doi.org/10.2174/0929867326666191113113110.
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Metastasis is the main cause of death in cancer patients; there is currently no effective treatment for cancer metastasis. This is primarily due to our insufficient understanding of the metastatic mechanisms in cancer. An increasing number of studies have shown that the C-X-C motif chemokine Ligand 12 (CXCL12) is overexpressed in various tissues and organs. It is a key niche factor that nurtures the pre-metastatic niches (tumorigenic soil) and recruits tumor cells (oncogenic “seeds”) to these niches, thereby fostering cancer cell aggression and metastatic capabilities. However, the C-X-C motif chemokine Receptor 4 (CXCR4) is aberrantly overexpressed in various cancer stem/progenitor cells and functions as a CXCL12 receptor. CXCL12 activates CXCR4 as well as multiple downstream multiple tumorigenic signaling pathways, promoting the expression of various oncogenes. Activation of the CXCL12-CXCR4 signaling axis promotes Epithelial-Mesenchymal Transition (EMT) and mobilization of cancer stem/progenitor cells to pre-metastatic niches. It also nurtures cancer cells with high motility, invasion, and dissemination phenotypes, thereby escalating multiple proximal or distal cancer metastasis; this results in poor patient prognosis. Based on this evidence, recent studies have explored either CXCL12- or CXCR4-targeted anti-cancer therapeutics and have achieved promising results in the preclinical trials. Further exploration of this new strategy and its potent therapeutics effect against metastatic cancer through the targeting of the CXCL12- CXCR4 signaling axis may lead to a novel therapy that can clean up the tumor microenvironment (“soil”) and kill the cancer cells, particularly the cancer stem/progenitor cells (“seeds”), in cancer patients. Ultimately, this approach has the potential to effectively treat metastatic cancer.
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Bachelerie, Françoise. "CXCL12/CXCR4-Axis Dysfunctions: Markers of the Rare Immunodeficiency Disorder WHIM Syndrome." Disease Markers 29, no. 3-4 (2010): 189–98. http://dx.doi.org/10.1155/2010/475104.
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The WHIM syndrome features susceptibility to human Papillomavirus infection-induced warts and carcinomas, hypogammaglobulinemia, recurrent bacterial infections, B and T-cell lymphopenia, and neutropenia associated with retention of senescent neutrophils in the bone marrow (i.e. myelokathexis). This rare disorder is mostly linked to inherited heterozygous autosomal dominant mutations in the gene encodingCXCR4, a G protein coupled receptor with a unique ligand, the chemokine CXCL12/SDF-1. Some individuals who have full clinical forms of the syndrome carry a wild typeCXCR4gene. In spite of this genetic heterogeneity, leukocytes from WHIM patients share in common dysfunctions of the CXCR4-mediated signaling pathway upon exposure to CXCL12. Dysfunctions are characterized by impaired desensitization and receptor internalization, which are associated with enhanced responses to the chemokine. Our increasing understanding of the mechanisms that account for the aberrant CXCL12/CXCR4-mediated responses is beginning to provide insight into the pathogenesis of the disorder. As a result we can expect to identify markers of the WHIM syndrome, as well as other disorders with WHIM-like features that are associated with dysfunctions of the CXCL12/CXCR4 axis.
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Zoughlami, Younes, Carlijn Voermans, Kim Brussen, et al. "Regulation of CXCR4 conformation by the small GTPase Rac1: implications for HIV infection." Blood 119, no. 9 (2012): 2024–32. http://dx.doi.org/10.1182/blood-2011-06-364828.
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Abstract The chemokine receptor CXCR4 is a critical regulator of cell migration and serves as a coreceptor for HIV-1. The chemokine stromal cell derived factor-1, also known as CXCL12, binds to CXCR4 and exerts its biologic functions partly through the small guanosine triphosphate hydrolase (GTPase) Rac1 (ras-related C3 botulinum toxin substrate 1). We show in different cell types, including CD34+ hematopoietic stem and progenitor cells, that inhibition of Rac1 causes a reversible conformational change in CXCR4, but not in the related receptors CXCR7 or CCR5. Biochemical experiments showed that Rac1 associates with CXCR4. The conformational change of CXCR4 on Rac1 inhibition blocked receptor internalization and impaired CXCL12-induced Gαi protein activation. Importantly, we found that the conformation adopted by CXCR4 after Rac1 inhibition prevents HIV-1 infection of both the U87-CD4-CXCR4 cell line and of primary peripheral blood mononuclear cells. In conclusion, our data show that Rac1 activity is required to maintain CXCR4 in the responsive conformation that allows receptor signaling and facilitates HIV-1 infection; this implies that Rac1 positively regulates CXCR4 function and identifies the Rac1-CXCR4 axis as a new target for preventing HIV-1 infection.
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Kumari, Anju, Aya Ludin, Karin Golan, et al. "PGE2 Promotes BM Hematopoietic Stem Cell Retention Via Stromal Lactate Production, cAMP and CXCL12/CXCR4 Regulation." Blood 124, no. 21 (2014): 771. http://dx.doi.org/10.1182/blood.v124.21.771.771.
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Abstract The CXCR4/CXCL12 axis is essential for retention and protection from DNA damage of quiescent hematopoietic stem cells (HSC) in their bone marrow (BM) niches. Murine CXCR4+ HSC tightly adhere to BM stromal cells which functionally express cell surface CXCL12. Stress induces secretion of CXCL12 by BM stromal cells and its release to the circulation, mediating hematopoietic stem and progenitor cell (HSPC) egress, recruitment and clinical mobilization. Previously, we reported that Prostaglandin E2 (PGE2), highly produced by COX-2+ BM αSMA+ monocyte/macrophages, upregulates surface CXCR4 expression on enriched human CD34+ HSPC and their CXCL12 induced motility via cAMP activation in vitro. PGE2 inhibits intracellular reactive oxygen species (ROS) generation in HSPC and also increases membrane bound CXCL12 expression by BM stromal cells leading to HSC adhesion to their niche supporting cells in vivo, overall contributing to BM stem cell retention. We also found that elevation in cAMP activation promotes CXCL12 secretion from BM stromal cells, and another report has recently shown that lactate signaling via its major receptor HCA-1 inhibits cAMP. Thus, we hypothesized that the major metabolite lactate, cAMP and PGE2 cross-regulate BM stem cell retention by modulating the CXCR4/CXCL12 axis. We found that both hematopoietic stem cells and BM stromal cells functionally express the lactate receptor HCA-1. Stimulation with PGE2 elevated lactate production by BM stromal cells and stimulation with a HCA-1 receptor agonist, or with lactate, both elevated membrane bound expression of CXCL12 on BM stromal cells. Moreover, since cAMP is elevated by PGE2 signaling whereas lactate signaling was shown to inhibit cAMP, we tested the role of cAMP in CXCL12 expression and secretion by BM stromal cells. We found that in vitro the cAMP enhancer forskolin increased CXCR4 expression by HSPC and in vivo forskolin administration reduced membrane bound CXCL12 levels and elevated CXCL12 secretion as expected. Conversely, in vivo forskolin co-administered with lactate, elevated membrane bound CXCL12 levels and reduced CXCL12 secretion, indicating that lactate limits cAMP elevation and promotes surface CXCL12 expression by BM stromal cells. In accordance,inhibition of cAMP under PGE2 stimulation both in vitro and in vivo, augmented membrane bound CXCL12 expression and inhibited CXCR4 upregulation, mimicking the effects of lactate. We found that PGE2 administration in vivo resulted in reduced CXCR4 expression on primitive BM HSPCs however in vitro PGE2 elevated CXCR4 expression on enriched HSPC. Our results suggest that PGE2 signaling in vivo induces secretion of the metabolite lactate by BM stromal cells, increasing membrane bound CXCL12 expression and reducing expression of CXCR4 on HSPC via cAMP inhibition. Importantly, repeated in vivo administration of PGE2, lactate or its receptor HCA-1 agonist (once daily for 2 days), all reduced CXCR4 expression and steady state egress of HSPC to the bloodcirculation. Thus, PGE2 via downstream lactate secretion acts as a BM stem cell retaining factor. In accordance, we found that in vivo inhibition of PGE2 production by repeated (once daily for five days) injections of COX-2 inhibitors, such as Meloxicam led to HSPC mobilization. This mobilization was abrogated by co-administration of lactate, suggesting that in vivo inhibition of meloxicam induced CXCL12 secretion and release by lactate prevents HSPC mobilization. We found that in vivo COX-2 inhibition reduced membrane expression of CXCL12 by BM stromal cells and elevated surface CXCR4 expression by BM HSPC in a ROS dependent manner. Moreover, neutralization of CXCR4 or CXCL12 by specific antibodies, or ROS by its scavenger NAC, all blocked meloxicam induced stem and progenitor cell mobilization. These results reveal that COX-2 inhibition increased BM CXCL12 secretion and its release to the blood, upregulated CXCR4 leading to HSPC mobilization in a ROS and CXCL12 dependent manner. In conclusion, our results reveal that PGE2 enhances both cAMP elevation and lactate secretion by BM stromal cells in the vicinity of hematopoietic stem cells. Lactate acts in an autocrine manner modulating surface CXCL12 expression by BM niche cells and reduced CXCR4 expression by hematopoietic stem cells via inhibition of cAMP, promoting retention and preservation of hematopoietic stem cells in their BM niches. Disclosures No relevant conflicts of interest to declare.
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Fukuda, Seiji, Hal E. Broxmeyer та Louis M. Pelus. "Flt3 ligand and the Flt3 receptor regulate hematopoietic cell migration by modulating the SDF-1α(CXCL12)/CXCR4 axis". Blood 105, № 8 (2005): 3117–26. http://dx.doi.org/10.1182/blood-2004-04-1440.
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AbstractFlt3 ligand (FL) enhances hematopoietic cell proliferation and facilitates hematopoietic stem cell mobilization in vivo, while the stromal-derived factor 1α (SDF-1α, CXC ligand 12 [CXCL12])/CXC receptor 4 (CXCR4) axis is critical for their homing and trafficking. We investigated if FL and its receptor, Flt3, functionally interact with CXCL12/CXCR4 to regulate hematopoietic cell migration. FL stimulated chemokinetic activity when used alone, but synergistically enhanced short-term migration of CD34+ cells, Ba/F3 cells expressing human Flt3 (Ba/F3-Flt3), and human RS4;11 acute leukemia cells, induced by CXCL12. Moreover, overexpression of constitutively activated internal tandem duplication (ITD)–Flt3 mutants in Ba/F3 cells dramatically enhanced migration toward CXCL12. In Ba/F3-Flt3 cells, synergistic cell migration to FL plus CXCL12 was associated with enhanced phosphorylation of mitogen-activated protein kinase p42/p44 (MAPKp42/p44), cyclic adenosine monophosphate response element binding protein (CREB), and Akt, and was partially inhibited by pretreatment of cells with selective inhibitors for MAPKp42/p44, protein kinase A (PKA), or phosphatidylinositol 3–kinase (PI3-kinase), implicating these pathways in migration to FL plus CXCL12. In contrast, prolonged exposure of CD34+ or Ba/F3-Flt3 cells to FL down-regulated CXCR4 expression, inhibited CXCL12-mediated phosphorylation of MAPKp42/p44, CREB, and Akt, and impaired migration toward CXCL12. These findings suggest that FL/Flt3 may facilitate hematopoietic cell migration/homing and mobilization by enhancing or inhibiting CXCL12/CXCR4 signaling pathways and that the FL/Flt3 axis participates in trafficking of normal and transformed hematopoietic cells.
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Yi, Dan, Bin Liu, Ting Wang, et al. "Endothelial Autocrine Signaling through CXCL12/CXCR4/FoxM1 Axis Contributes to Severe Pulmonary Arterial Hypertension." International Journal of Molecular Sciences 22, no. 6 (2021): 3182. http://dx.doi.org/10.3390/ijms22063182.
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Endothelial autocrine signaling is essential to maintain vascular homeostasis. There is limited information about the role of endothelial autocrine signaling in regulating severe pulmonary vascular remodeling during the onset of pulmonary arterial hypertension (PAH). In this study, we employed the first severe pulmonary hypertension (PH) mouse model, Egln1Tie2Cre (Tie2Cre-mediated disruption of Egln1) mice, to identify the novel autocrine signaling mediating the pulmonary vascular endothelial cell (PVEC) proliferation and the pathogenesis of PAH. PVECs isolated from Egln1Tie2Cre lung expressed upregulation of many growth factors or angiocrine factors such as CXCL12, and exhibited pro-proliferative phenotype coincident with the upregulation of proliferation-specific transcriptional factor FoxM1. Treatment of CXCL12 on PVECs increased FoxM1 expression, which was blocked by CXCL12 receptor CXCR4 antagonist AMD3100 in cultured human PVECs. The endothelial specific deletion of Cxcl12(Egln1/Cxcl12Tie2Cre) or AMD3100 treatment in Egln1Tie2Cre mice downregulated FoxM1 expression in vivo. We then generated and characterized a novel mouse model with endothelial specific FoxM1 deletion in Egln1Tie2Cre mice (Egln1/Foxm1Tie2Cre), and found that endothelial FoxM1 deletion reduced pulmonary vascular remodeling and right ventricular systolic pressure. Together, our study identified a novel mechanism of endothelial autocrine signaling in regulating PVEC proliferation and pulmonary vascular remodeling in PAH.
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Burger, Meike, Tanja Hartmann, Myriam Krome, et al. "Small peptide inhibitors of the CXCR4 chemokine receptor (CD184) antagonize the activation, migration, and antiapoptotic responses of CXCL12 in chronic lymphocytic leukemia B cells." Blood 106, no. 5 (2005): 1824–30. http://dx.doi.org/10.1182/blood-2004-12-4918.
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Abstract Growth and survival of chronic lymphocytic leukemia (CLL) B cells are favored by interactions between CLL and nontumoral accessory cells. CLL cells express CXCR4 chemokine receptors that direct leukemia cell chemotaxis. Marrow stromal cells or nurselike cells constitutively secrete CXCL12, the ligand for CXCR4, thereby attracting and rescuing CLL B cells from apoptosis in a contact-dependent fashion. Therefore, the CXCR4-CXCL12 axis represents a potential therapeutic target in CLL. We evaluated the most active CXCR4-specific antagonists (T140, TC14012, TN14003) for their capacity to inhibit CXCL12 responses in CLL cells. T140, or its analogs, inhibited actin polymerization, chemotaxis, and migration of CLL cells beneath stromal cells. CXCL12-induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) was abolished by CXCR4 antagonists. TC14012 and TN14003 antagonized the antiapoptotic effect of synthetic CXCL12 and stromal cell-mediated protection of CLL cells from spontaneous apoptosis. Furthermore, we found that stromal cells protected CLL cells from chemotherapy-induced apoptosis. Treatment with CXCR4 antagonists resensitized CLL cells cultured with stromal cells to fludarabine-induced apoptosis. These findings demonstrate that CXCR4 blocking agents effectively antagonize CXCL12-induced migratory and signaling responses and stromal protection of CLL cells from spontaneous or fludarabine-induced apoptosis. As such, small molecular CXCR4 antagonists may have activity in the treatment of patients with this disease. (Blood. 2005;106:1824-1830)
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Dumont, S. N., M. I. Aslam, J. C. McAuliffe, et al. "CXCR4-CXCL12 axis: Pivotal role as a metastatic mediator in small cell sarcoma." Journal of Clinical Oncology 27, no. 15_suppl (2009): 10569. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.10569.
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10569 Background: The chemokine CXCL12, and its receptor CXCR4, are expressed in over 23 different types of cancers, and have been associated with the metastatic phenotype and inferior clinical outcomes. Given the poor prognosis after failure of front-line therapy for patients with metastatic Ewing's sarcoma (EWS) and rhabdomyosarcoma (RMS), we examined the functional role of CXCR4 in these small cell sarcoma in vitro. Methods: Human EWS and RMS cell lines and tissue were used to study CXCR4 expression and activation. Immunoblotting techniques were used to evaluate CXCR4 activation and inhibition. Cell viability, cell cycle, apoptosis, chemosensitivity, migration, and invasion assays were utilized to assess the effects of the small peptide CXCR4 antagonist, CTCE-9908 (Chemokine Therapeutics) on cultured cells. Results: CXCR4 was highly expressed on 46 % of human RMS tumor samples. CXCR4 underwent phosphorylation after stimulation with CXCL12 in EWS and RMS cell lines with downstream activation of Akt, p42/44 MAPK, JAK2 and PLCγ1 in select cell lines. CTCE-9908 was able to specifically inhibit downstream signaling of Akt, p42/44 MAPK and JAK2. Decrease in cell proliferation (20–30%, p < .05), increase in cell apoptosis (20–40 %, p < .05) and cell cycle arrest was also observed with CXCR4 blockade. CTCE-9908 significantly inhibited migration and invasion (68–93 %, p < .05) in our cell lines. Conclusions: CXCR4-CXCL12 axis may be important in EWS and RMS metastasis. These results provide evidence that CTCE-9908 may be a novel therapy for these sarcomas. No significant financial relationships to disclose.
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Runyan, Cheyenne L., Stacia Z. McIntosh, Marlie M. Maestas, Kelsey E. Quinn, Ben P. Boren, and Ryan L. Ashley. "CXCR4 signaling at the ovine fetal–maternal interface regulates vascularization, CD34+ cell presence, and autophagy in the endometrium†." Biology of Reproduction 101, no. 1 (2019): 102–11. http://dx.doi.org/10.1093/biolre/ioz073.
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Abstract Placenta development is characterized by extensive angiogenesis and vascularization but if these processes are compromised placental dysfunction occurs, which is the underlying cause of pregnancy complications such as preeclampsia and intrauterine growth restriction. Dysregulation of placental angiogenesis has emerged as one of the main pathophysiological features in the development of placental insufficiency and its clinical consequences. The signaling axis initiated by chemokine ligand 12 (CXCL12) and its receptor CXCR4 stimulates angiogenesis in other tissues, and may be central to placental vascularization. We hypothesized that CXCL12-CXCR4 signaling governs the pro-angiogenic placental microenvironment by coordinating production of central angiogenic factors and receptors and regulates endometrial cell survival essential for placental function and subsequent fetal longevity. The CXCR4 antagonist, AMD3100, was used to elucidate the role of CXCL12-CXCR4 signaling regarding uteroplacental vascular remodeling at the fetal–maternal interface. On day 12 postbreeding, osmotic pumps were surgically installed and delivered either AMD3100 or PBS into the uterine lumen ipsilateral to the corpus luteum. On day 20, endometrial tissues were collected, snap-frozen in liquid nitrogen, and uterine horn cross sections preserved for immunofluorescent analysis. In endometrium from ewes receiving AMD3100 infusion, the abundance of select angiogenic factors was diminished, while presence of CD34+ cells increased compared to control ewes. Ewes receiving AMD3100 infusion also exhibited less activation of Akt/mTOR signaling, and elevated LC3B-II, a marker of cellular autophagy in endometrium. This study suggests that CXCL12-CXCR4 signaling governs placental homeostasis by serving as a critical upstream mediator of vascularization and cell viability, thereby ensuring appropriate placental development.
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Saha, Achinto, Songyeon Ahn, Jorge Blando, Fei Su, Mikhail G. Kolonin, and John DiGiovanni. "Proinflammatory CXCL12–CXCR4/CXCR7 Signaling Axis Drives Myc-Induced Prostate Cancer in Obese Mice." Cancer Research 77, no. 18 (2017): 5158–68. http://dx.doi.org/10.1158/0008-5472.can-17-0284.
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Chen, Guang, Si-Meng Chen, Xiang Wang, Xiao-Fei Ding, Jian Ding, and Ling-Hua Meng. "Inhibition of Chemokine (CXC Motif) Ligand 12/Chemokine (CXC Motif) Receptor 4 Axis (CXCL12/CXCR4)-mediated Cell Migration by Targeting Mammalian Target of Rapamycin (mTOR) Pathway in Human Gastric Carcinoma Cells." Journal of Biological Chemistry 287, no. 15 (2012): 12132–41. http://dx.doi.org/10.1074/jbc.m111.302299.
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CXCL12/CXCR4 plays an important role in metastasis of gastric carcinoma. Rapamycin has been reported to inhibit migration of gastric cancer cells. However, the role of mTOR pathway in CXCL12/CXCR4-mediated cell migration and the potential of drugs targeting PI3K/mTOR pathway remains unelucidated. We found that CXCL12 activated PI3K/Akt/mTOR pathway in MKN-45 cells. Stimulating CHO-K1 cells expressing pEGFP-C1-Grp1-PH fusion protein with CXCL12 resulted in generation of phosphatidylinositol (3,4,5)-triphosphate, which provided direct evidence of activating PI3K by CXCL12. Down-regulation of p110β by siRNA but not p110α blocked phosphorylation of Akt and S6K1 induced by CXCL12. Consistently, p110β-specific inhibitor blocked the CXCL12-activated PI3K/Akt/mTOR pathway. Moreover, CXCR4 immunoprecipitated by anti-p110β antibody increased after CXCL12 stimulation and Gi protein inhibitor pertussis toxin abrogated CXCL12-induced activation of PI3K. Further studies demonstrated that inhibitors targeting the PI3K/mTOR pathway significantly blocked the chemotactic responses of MKN-45 cells triggered by CXCL12, which might be attributed primarily to inhibition of mTORC1 and related to prevention of F-actin reorganization as well as down-regulation of active RhoA, Rac1, and Cdc42. Furthermore, rapamycin inhibited the secretion of CXCL12 and the expression of CXCR4, which might form a positive feedback loop to further abolish upstream signaling leading to cell migration. Finally, we found cells expressing high levels of cxcl12 were sensitive to rapamycin in its activity inhibiting migration as well as proliferation. In summary, we found that the mTOR pathway played an important role in CXCL12/CXCR4-mediated cell migration and proposed that drugs targeting the mTOR pathway may be used for the therapy of metastatic gastric cancer expressing high levels of cxcl12.
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Devi, Sapna, Yilin Wang, Weng Keong Chew, et al. "Neutrophil mobilization via plerixafor-mediated CXCR4 inhibition arises from lung demargination and blockade of neutrophil homing to the bone marrow." Journal of Experimental Medicine 210, no. 11 (2013): 2321–36. http://dx.doi.org/10.1084/jem.20130056.
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Blood neutrophil homeostasis is essential for successful host defense against invading pathogens. Circulating neutrophil counts are positively regulated by CXCR2 signaling and negatively regulated by the CXCR4–CXCL12 axis. In particular, G-CSF, a known CXCR2 signaler, and plerixafor, a CXCR4 antagonist, have both been shown to correct neutropenia in human patients. G-CSF directly induces neutrophil mobilization from the bone marrow (BM) into the blood, but the mechanisms underlying plerixafor-induced neutrophilia remain poorly defined. Using a combination of intravital multiphoton microscopy, genetically modified mice and novel in vivo homing assays, we demonstrate that G-CSF and plerixafor work through distinct mechanisms. In contrast to G-CSF, CXCR4 inhibition via plerixafor does not result in neutrophil mobilization from the BM. Instead, plerixafor augments the frequency of circulating neutrophils through their release from the marginated pool present in the lung, while simultaneously preventing neutrophil return to the BM. Our study demonstrates for the first time that drastic changes in blood neutrophils can originate from alternative reservoirs other than the BM, while implicating a role for CXCR4–CXCL12 interactions in regulating lung neutrophil margination. Collectively, our data provides valuable insights into the fundamental regulation of neutrophil homeostasis, which may lead to the development of improved treatment regimens for neutropenic patients.
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Beider, Katia, Evgenia Rosenberg, Hanna Bitner, et al. "S1P Modulator FTY720 Targets Multiple Myeloma Cell Proliferation and Stromal Interactions Via CXCR4/CXCL12 and mTOR Pathways." Blood 124, no. 21 (2014): 4707. http://dx.doi.org/10.1182/blood.v124.21.4707.4707.
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Abstract Introduction: Multiple myeloma (MM) is an incurable hematological malignancy characterized by proliferation of malignant plasma cells in the bone marrow (BM). Interactions between MM cells and BM milieu facilitate disease progression and therapy resistance. Chemokine receptor CXCR4 and its cognate ligand CXCL12 are implicated in these processes and are associated with poor prognosis. Sphingosine-1-phosphate (S1P) pathway is involved in cancer progression, including oncogenesis, cell survival and cell migration, therefore representing an attractive target for anti-cancer therapy. FTY720 (fingolimod) is a modulator of S1P signaling system that exhibit immunosuppressive and anti-cancer properties. The role of S1P system and FTY720 modulator in MM is less defined. The aim of this study was to explore the functional consequences of possible cross-talk between the CXCR4/CXCL12 and the S1P axes in MM cells and to evaluate the effect of S1P targeting with FTY720 as potential anti-MM therapeutic strategy. Results: The partners of the S1P pathway (S1P receptor 1 and sphingosine kinase 1 (SPHK1)) and CXCL12 chemokine were found to be co-expressed in MM cell lines and primary BM samples from MM patients. Increased mRNA levels of SPHK1 and CXCL12 were detected in MM BM samples (n=24) comparing to BM from healthy donors (n=7) (p<0.01). In vitro treatment of MM cell lines (n=6) with FTY720 modulator resulted in time- and dose-dependent cell death (IC50 2.8 – 5.3 µM). Further characterization of cell death mechanisms revealed that FTY720 treatment induced MM cell apoptosis with mitochondrial involvement, cytochrome C release and caspase 3 activation. Interestingly, suppressive potential of FTY720 negatively correlated with CXCR4 expression on MM cells. Enforced expression of CXCR4 reduced the sensitivity to FTY720, whereas silencing of endogenous CXCL12 increased the sensitivity of MM cells to FTY720-mediated cell death. These results suggested the CXCR4 axis to be directly regulated by S1P pathway. In support, we have found that FTY720 treatment significantly reduced CXCR4-dependent MM cell adhesion to fibronectin and abrogated MM migration toward CXCL12. Activation of signaling pathways, such as MAPK and Akt, in response to CXCL12 stimulation was also fully blocked by FTY720 pre-treatment. In addition to functional suppression, FTY720 directly and profoundly reduced CXCR4 cell-surface levels in a dose-dependent manner. Importantly, none of the suppressive effects of FTY720 (neither apoptosis, nor migration or adhesion inhibition) were dependent on protein phosphatase 2A (PP2A) activation, suggesting alternative mechanism of action. To further investigate down-stream molecular machinery involved in FTY720-mediated CXCR4 targeting in MM cells, the intra-cellular levels of different signaling mediators were evaluated. We identified the mTOR pathway to be regulated by CXCR4 and targeted by FTY720. FTY720 treatment suppressed mTOR signaling in MM cells, as demonstrated by de-phosphorylation of p70S6K and S6. Forced expression of CXCR4 and interaction with BM stromal cells antagonized with FTY720-mediated apoptosis and prevented FTY720-induced S6 de-phosphorylation. While, combination of FTY720 with mTOR inhibitor RAD001 resulted in significantly increased cell death, effectively abrogating CXCR4- and stroma-dependent resistance to FTY720 and suppressing mTOR signaling in MM cells. Finally, in a recently developed novel xenograft model of CXCR4-dependent systemic MM with BM involvement, in vivo FTY720 effectively reduced tumor burden in two third of the treated mice, decreasing both the levels of M protein in blood and the number of MM cells in BM. Conclusions: Taken together, our findings demonstrate cross talk between S1P and CXCR4/CXCL12 signaling pathways that may be of importance for MM cell survival and localization of the MM cells in CXCL12-expressing protective niches in the BM. Moreover, this is, to our knowledge, the first evidence that CXCR4 can be directly targeted with FTY720 modulator, thus restricting the tumor-promoting activities of S1P and CXCR4/CXCL12 axes. In addition, mTOR pathway was recognized as down-stream molecular partner being involved in FTY720-mediated anti-myeloma activities. Combining FTY720 with mTOR inhibitors may thus serve as promising novel therapeutic strategy in MM. Disclosures Peled: BioLineRx: Research Funding.
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Vitale, Candida, Valentina Griggio, Chiara Riganti та ін. "Targeting HIF-1α Regulatory Pathways as a Strategy to Hamper Tumor-Microenvironment Interactions in CLL". Cancers 13, № 12 (2021): 2883. http://dx.doi.org/10.3390/cancers13122883.
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The hypoxia-inducible factor 1 (HIF-1) and the CXCL12/CXCR4 axis regulate the interaction of chronic lymphocytic leukemia cells and the tumor microenvironment. However, the interconnections occurring between HIF-1 and the CXCL12/CXCR4 axis are not fully elucidated. Here, we demonstrate that the CXCL12/CXCR4 axis plays a pivotal role in the positive regulation of the α subunit of HIF-1 (HIF-1α) that occurs in CLL cells co-cultured with stromal cells (SC). Inhibitors acting at different levels on CXCR4 downstream signalling counteract the SC-induced HIF-1α upregulation in CLL cells, also hindering the SC-mediated pro-survival effect. HIF-1α inhibition also exerts off-tumor effects on the SC component, inducing the downregulation of target genes, including CXCL12. Consistently, our data show that pretreatment of leukemic cells and/or SC with idelalisib effectively abrogates the SC-mediated survival support. A combined on-tumor and off-tumor inhibition of HIF-1α was also observed in idelalisib-treated patients, who showed, along with a downregulation of HIF-1α target genes in leukemic cells, a significant decrease in CXCL12 serum concentration and changes in the bone marrow microenvironment. Our data demonstrate that the targeting of HIF-1α or its regulatory pathways acts at the tumor- and SC-level, and may be an appealing strategy to overcome the microenvironment-mediated protection of CLL cells.
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Qin, Liang, An-min Chen, Feng-jing Guo, Qing Yang, and Hui Liao. "CXCL12/CXCR4 axis promotes vasculogenic mimicry via PI3K/AKT signaling in osteosarcoma." Journal of Orthopaedic Translation 7 (October 2016): 128. http://dx.doi.org/10.1016/j.jot.2016.06.170.
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Liang, Zhongxing, Joann Brooks, Margaret Willard, et al. "CXCR4/CXCL12 axis promotes VEGF-mediated tumor angiogenesis through Akt signaling pathway." Biochemical and Biophysical Research Communications 359, no. 3 (2007): 716–22. http://dx.doi.org/10.1016/j.bbrc.2007.05.182.
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Wang, Xiao-Hui, Shu-Feng Zhang, Hai-Ying Wu, Jian Gao, Xu-Hui Wang, and Tian-Hui Gao. "SOX17 inhibits proliferation and invasion of neuroblastoma through CXCL12/CXCR4 signaling axis." Cellular Signalling 87 (November 2021): 110093. http://dx.doi.org/10.1016/j.cellsig.2021.110093.
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Lepsenyi, Mattias, Nader Algethami, Amr A. Al-Haidari та ін. "CXCL2-CXCR2 axis mediates αV integrin-dependent peritoneal metastasis of colon cancer cells". Clinical & Experimental Metastasis 38, № 4 (2021): 401–10. http://dx.doi.org/10.1007/s10585-021-10103-0.
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AbstractPeritoneal metastasis is an insidious aspect of colorectal cancer. The aim of the present study was to define mechanisms regulating colon cancer cell adhesion and spread to peritoneal wounds after abdominal surgery. Mice was laparotomized and injected intraperitoneally with CT-26 colon carcinoma cells and metastatic noduli in the peritoneal cavity was quantified after treatment with a CXCR2 antagonist or integrin-αV-antibody. CT-26 cells expressed cell surface chemokine receptors CXCR2, CXCR3, CXCR4 and CXCR5. Stimulation with the CXCR2 ligand, CXCL2, dose-dependently increased proliferation and migration of CT-26 cells in vitro. The CXCR2 antagonist, SB225002, dose-dependently decreased CXCL2-induced proliferation and migration of colon cancer cells in vitro. Intraperitoneal administration of CT-26 colon cancer cells resulted in wide-spread growth of metastatic nodules at the peritoneal surface of laparotomized animals. Laparotomy increased gene expression of CXCL2 at the incisional line. Pretreatment with CXCR2 antagonist reduced metastatic nodules by 70%. Moreover, stimulation with CXCL2 increased CT-26 cell adhesion to extracellular matrix (ECM) proteins in a CXCR2-dependent manner. CT-26 cells expressed the αV, β1 and β3 integrin subunits and immunoneutralization of αV abolished CXCL2-triggered adhesion of CT-26 to vitronectin, fibronectin and fibrinogen. Finally, inhibition of the αV integrin significantly attenuated the number of carcinomatosis nodules by 69% in laparotomized mice. These results were validated by use of the human colon cancer cell line HT-29 in vitro. Our data show that colon cancer cell adhesion and growth on peritoneal wound sites is mediated by a CXCL2-CXCR2 signaling axis and αV integrin-dependent adhesion to ECM proteins.
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Schajnovitz, Amir, Alexander Kalinkovich, Kfir Lapid та ін. "Human and Murine β-Defensin-Derived Peptides Induce Rapid Mobilization Of Murine Hematopoietic Stem and Progenitor Cells Via Activation Of CXCR4 Signaling and CXCL12 Release". Blood 122, № 21 (2013): 890. http://dx.doi.org/10.1182/blood.v122.21.890.890.
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Abstract Background Rapid mobilization of hematopoietic stem and progenitor cells (HSPCs) from the bone marrow (BM) to the peripheral blood by anti-CXCR4 agents such as AMD3100 is a complex process, which requires CXCL12 secretion, activation of proteolytic enzymes and supporting cellular compartments (Dar et. al, Leukemia 2011). Notably, components of innate immune system were also shown to be involved (Ratajczak et. al, Leukemia 2010). Human β-defensin-3 (hBD3) is an antimicrobial peptide possessing also anti-CXCR4 effects on human T cells in vitro (Feng et. al, JI 2006), suggesting its HSPC mobilizing potential. Here, we describe a novel approach for targeting CXCR4 in vivo by utilizing β-defensin-derived peptides, resulting in rapid HSPC mobilization. Results While AMD3100 blocked CXCL12-mediated signaling and migration of enriched BM mononuclear cells (MNCs) in vitro, we unexpectedly detected rapid phosphorylation of AKT, p38 and ERK1/2 in BM stromal cells (BMSCs). Interestingly, single administration of AMD3100 to mice resulted in enhancement of CXCR4 phosphorylation within minutes in both BM residing mesenchymal stem/progenitor cells (MSCs) and HSPCs, thus suggesting a CXCR4 agonistic activity. Aiming to test HSPC mobilizing potential of hBD3 and avoiding potential toxicity of systemic administration, we synthesized short linear peptides, comprising the C-terminal parts of hBD3 and the murine ortholog β-defensin-14 (mBD14), as well as a cyclic peptide of hBD3, comprising the same amino acids as the linear one, to serve as a control. All full-length β-defensins and tested peptides (both linear and cyclic) specifically bound CXCR4 (demonstrated by docking approach and anti-CXCR4 antibody competition assay) and efficiently blocked CXCL12-mediated activity of enriched BM MNCs in vitro including cell migration and CXCR4-dependent HIV infection. Intriguingly, full-length β-defensins and derived linear peptides (but not cyclic) revealed a strong stimulatory effect on BMSCs in vitro: triggering phosphorylation of AKT, p38 and ERK1/2 along with enhancing secretion of functional CXCL12. Administration of linear peptides to mice led to a fast activation of CXCR4 signaling in BMSCs and MSCs as well as in HSPCs accompanied by CXCL12 release to the circulation, increased activity of proteolytic enzymes and consequent rapid mobilization of progenitors as well as long-term repopulating stem cells. In addition, linear peptides augmented AMD3100-induced rapid mobilization. Importantly, the control cyclic peptide, which bound CXCR4 but failed to activate BMSCs in vitro, did not induce HSPC mobilization in vivo. Moreover, it inhibited both steady-state egress and AMD3100-induced mobilization of HSPCs. A series of in vivo inhibitory analyses confirmed dependence of hBD3- and mBD14-derived peptide-induced HSPC mobilization on the activation of CXCL12/CXCR4 axis and revealed involvement of uPA and JNK signaling as well as ROS generation. Conclusions Our study demonstrated for the first time the capability of β-defensin-derived peptides to activate in vivo CXCL12/CXCR4 signaling in both hematopoietic and non-hematopoietic BM cells, leading to rapid HSPC mobilization. We suggest that activation of CXCR4 signaling in non-hematopoietic BM cells is crucial for inducing HSPC mobilization. Accordingly, CXCR4-binding agents capable of triggering CXCR4 signaling in non-hematopoietic BM cells in vitro, would induce rapid HSPC mobilization. The results presented here help to better understand the mechanisms of rapid HSPC mobilization and have the potential of improving existing clinical protocols to increase the yield of HSPC harvest for transplantation. Disclosures: Scadden: Fate Therapeutics: Consultancy, Equity Ownership.
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Komorowski, Marcin, Joanna Tisonczyk, Agnieszka Kolakowska, Ryszard Drozdz, and Danuta Kozbor. "Modulation of the Tumor Microenvironment by CXCR4 Antagonist-Armed Viral Oncotherapy Enhances the Antitumor Efficacy of Dendritic Cell Vaccines against Neuroblastoma in Syngeneic Mice." Viruses 10, no. 9 (2018): 455. http://dx.doi.org/10.3390/v10090455.
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The induction of antitumor immune responses in tumor-bearing hosts depends on efficient uptake and processing of native or modified tumors/self-antigens by dendritic cells (DCs) to activate immune effector cells, as well as the extent of the immunosuppressive network in the tumor microenvironment (TME). Because the C-X-C motif chemokine receptor 4 (CXCR4) for the C-X-C motif chemokine 12 (CXCL12) is involved in signaling interactions between tumor cells and their TME, we used oncolytic virotherapy with a CXCR4 antagonist to investigate whether targeting of the CXCL12/CXCR4 signaling axis in murine neuroblastoma cells (NXS2)-bearing syngeneic mice affects the efficacy of bone marrow (BM)-derived DCs loaded with autologous tumor cells treated with doxorubicin for induction of immunogenic cell death. Here, we show that CXCR4 antagonist expression from an oncolytic vaccinia virus delivered intravenously to mice with neuroblastoma tumors augmented efficacy of the DC vaccines compared to treatments mediated by a soluble CXCR4 antagonist or oncolysis alone. This study is the first demonstration that modulating the tumor microenvironment by an armed oncolytic virus could have a significant impact on the efficacy of DC vaccines, leading to the generation of effective protection against neuroblastoma challenge.
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Kim, Bo-Reum, Seung-Hyun Jung, A.-Reum Han та ін. "CXCR4 Inhibition Enhances Efficacy of FLT3 Inhibitors in FLT3-Mutated AML Augmented by Suppressed TGF-β Signaling". Cancers 12, № 7 (2020): 1737. http://dx.doi.org/10.3390/cancers12071737.
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Given the proven importance of the CXCL12/CXCR4 axis in the stroma–acute myeloid leukemia (AML) interactions and the rapid emergence of resistance to FLT3 inhibitors, we investigated the efficacy and safety of a novel CXCR4 inhibitor, LY2510924, in combination with FLT3 inhibitors in preclinical models of AML with FLT3-ITD mutations (FLT3-ITD-AML). Quizartinib, a potent FLT3 inhibitor, induced apoptosis in FLT3-ITD-AML, while LY2510924 blocked surface CXCR4 without inducing apoptosis. LY2510924 significantly reversed stroma-mediated resistance against quizartinib mainly through the MAPK pathway. In mice with established FLT3-ITD-AML, LY2510924 induced durable mobilization and differentiation of leukemia cells, resulting in enhanced anti-leukemia effects when combined with quizartinib, whereas transient effects were seen on non-leukemic blood cells in immune-competent mice. Sequencing of the transcriptome of the leukemic cells surviving in vivo treatment with quizartinib and LY2510924 revealed that genes related to TGF-β signaling may confer resistance against the drug combination. In co-culture experiments of FLT3-ITD-AML and stromal cells, both silencing of TGF-β in stromal cells or TGF-β-receptor kinase inhibitor enhanced apoptosis by combined treatment. Disruption of the CXCL12/CXCR4 axis in FLT3-ITD-AML by LY2510924 and its negligible effects on normal immunocytes could safely enhance the potency of quizartinib, which may be further improved by blockade of TGF-β signaling.
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Simon, Anna, Dagmar Wider, Marie Follo, et al. "Targeting The Protective Microenvironment In Multiple Myeloma (MM): An Analysis Of The CXCL12/CXCR4-Axis and Its Inhibitors AMD3100 and Nox-A12 Combined With Antimyeloma Substances, Such As Pomalidomide and Carfilzomib." Blood 122, no. 21 (2013): 3851. http://dx.doi.org/10.1182/blood.v122.21.3851.3851.
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Abstract Introduction The interaction of malignant plasma cells (PC) with the bone marrow (BM) microenvironment is crucial for MM pathogenesis. The CXCL12/CXCR4-axis plays a key role in this cross-talk.CXCL12 induces homing of normal hematopoietic progenitor cells to the BM and also drives MM cells to their protective niche. The binding of CXCL12 to CXCR4 directly promotes PC survival and facilitates environment-mediated drug resistance, which ultimately renders antimyeloma substances ineffective. Therefore, targeting the CXCL12/CXCR4-axis in combination with the use of new antimyeloma therapeutics represents a promising approach in developing new treatment strategies. Methods Comparative analysis of three different CXCR4 antibody clones (12G5, 44717, 4G10) and their interaction with the CXCR4 inhibitor AMD3100 was performed by flow cytometry. The effect of CXCL12 on CXCR4 expression using the human MM cell lines (MMCLs) U266 and L363, with and without AMD3100, was assessed by flow- and image cytometry. CXCR4 downstream pathways were analysed by western blot. Pomalidomide and the specific proteasome inhibitor carfilzomib were tested in various concentrations, with and without AMD3100, using MMCLs (U266, L363) and BM samples from MM patients (n=4, BM infiltration >50%), with and without M210B4 stroma support. Cell viability was evaluated by trypan blue- and PI/annexin staining. Treatment effects on the expression of CD138, CD38 and CXCR4 were detected by flow- and image cytometry. The combination of carfilzomib with AMD3100 or NOX-A12 is currently being analysed regarding synergistic cytotoxicity and drug resistance. NOX-A12 is a PEGylated mirror-image oligonucleotide (Spiegelmer®) that binds to CXCL12 (stromal cell-derived factor-1, SDF-1) with high affinity, thereby inhibiting CXCL12 signalling on both its receptors, CXCR4 and CXCR7. The effect of NOX-A12 is of eminent interest when compared to AMD3100 and with use of various antimyeloma agents. Results AMD3100 treatment of U266 cells reduced CXCR4 expression with the commonly used antibody (ab)-clone 12G5 by 44-fold and with clone 44717 by 5-fold. The binding of the ab-clone 4G10 (FITC- and PE-coupled) was not influenced by AMD3100 at the concentrations tested (20-200 µl/ml), making 4G10 the most reliable for CXCR4 analysis. We also established a protocol for image cytometry that allows imaging of high cell throughputs, assessing viability and the expression of intra- and extracellular CXCR4 with no need for transfection. Human U266 cells showed high extra- and intracellular CXCR4-expression, whereas the lower expression of CXCR4 in L363 cells was confirmed. CXCL12 induced a notable decrease in extracellular CXCR4 and increased intracellular CXCR4 expression, confirming the reliability of our image cytometry protocol. AMD3100 alone inhibited cell proliferation (p<0.01) in U266 monoculture at different time points (t1=24h, t2=3d), although it was not cytotoxic. In terms of CXCR4 expression, CXCL12-induced CXCR4 internalization was inhibited by AMD3100, even after long incubation periods (t=3d). The use of carfilzomib on MMCLs confirmed prior cytotoxic concentrations (20-100nM), whereas L363 cells were even more sensitive than U266 cells. M210B4 co-culture induced CXCR4 expression and tumor cell protection, however was not able to completely induce resistance to carfilzomib: use of 100nM carfilzomib remained substantially cytotoxic, decreasing overall cell numbers, CD138 positive cells and CXCR4-expression in U266 cells. Co-cultivation of MM patient-derived BM cells with M210B4 stroma substantially reduced cell apoptosis as described (Zlei,Engelhardt, Ex Hematol 2007, Udi, Engelhardt, BJH 2013). When primary MM cells were grown without stroma support, AMD3100 did not enhance pomalidomide-induced cytotoxic effects; however, when primary MM cells were co-cultured with M210B4, pomalidomide (100nM) and AMD3100 (50µM) inhibited cell proliferation, and the combination was significantly more effective than AMD3100 alone (p=0.018). Conclusion Our findings stress the importance of CXCL12 and its receptor CXCR4 in MM progression and environment-mediated drug resistance. Analysis of the CXCL12/CXCR4-axis, of its inhibitors AMD3100 and NOX-A12 as well as their combined effects with antimyeloma substances may pave the way to novel therapeutic strategies. Disclosures: No relevant conflicts of interest to declare.
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Murphy, Philip T., Brendan p. Power, Patrick D. Thornton, and Judith H. Harmey. "Regulation of B-Cell Chronic Lymphocytic Leukaemia Cell Survival and Migration by the VEGF/SEMA3A Axis." Blood 112, no. 11 (2008): 2083. http://dx.doi.org/10.1182/blood.v112.11.2083.2083.
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Abstract B-cell Chronic Lymphocytic Leukaemia (B-CLL) is characterized by the accumulation of B-CLL lymphocytes in the blood, marrow and secondary lymphoid tissues. B-CLL cells have a long survival owing to alterations in the normal pathways of apoptosis. In the marrow and lymphoid tissues CLL cells are in close contact with stromal cells that constitute distinct microenvironments. The secretion of the CXCR4 ligand, CXCL12, by stromal cells attracts B-CLL cells and provides protection from spontaneous or induced apoptosis. Studies in other cell types have shown VEGF signalling is involved in regulating CXCR4 expression levels. The aim of this study was to examine VEGF receptor expression and the role of VEGF signalling in cell survival and CXCR4 expression. Expression levels of CXCR4 and VEGF receptors, VEGFR-1 and -2, in CLL samples were determined by flow cytometry. Expression of the VEGFR co-receptor, Neuropilin-1 (NRP1), was examined by Western blot. Cell migration towards CXCL12 was assessed using CoStar Transwell chambers (5mm pore size). Informed consent was received from all patients. VEGFR1 and VEGFR2 positive cells in 22 patient samples ranged from 0–38% and 1.5–83% respectively. NRP1 expression was detected in all samples analysed thus far (n=6). The NRP1 ligand, SEMA3A, a competitive inhibitor of VEGF binding to NRP1, decreased CXCR4 expression in patient CLL cells (n=8, p&lt;0.05). This decrease in CXCR4 levels correlated with reduced migration of SEMA3A treated CLL cells towards CXCL12 (88 +/− 12.7% of control levels, n=8, p&lt;0.05). Treatment of CLL cells with the VEGFR signalling inhibitor SU5416 decreased CLL cell survival which correlated with VEGFR1 expression levels (n=16, R=0.745, p&lt;0.01) but not with VEGFR2 expression levels. These results show that signalling through the VEGF co-receptor NRP1 plays an important role in regulating CXCR4 levels in CLL cells, as well as CLL cell migration along a CXCL12 gradient. Our results also suggest that VEGF signalling through VEGFR1 may be particularly important in regulating CLL cell survival. Thus, the VEGFR/NRP1 signalling pathway may represent an important therapeutic target in B-CLL.
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Chen, Ivy X., Vikash P. Chauhan, Jessica Posada, et al. "Blocking CXCR4 alleviates desmoplasia, increases T-lymphocyte infiltration, and improves immunotherapy in metastatic breast cancer." Proceedings of the National Academy of Sciences 116, no. 10 (2019): 4558–66. http://dx.doi.org/10.1073/pnas.1815515116.
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Metastatic breast cancers (mBCs) are largely resistant to immune checkpoint blockade, but the mechanisms remain unclear. Primary breast cancers are characterized by a dense fibrotic stroma, which is considered immunosuppressive in multiple malignancies, but the stromal composition of breast cancer metastases and its role in immunosuppression are largely unknown. Here we show that liver and lung metastases of human breast cancers tend to be highly fibrotic, and unlike primary breast tumors, they exclude cytotoxic T lymphocytes (CTLs). Unbiased analysis of the The Cancer Genome Atlas database of human breast tumors revealed a set of genes that are associated with stromal T-lymphocyte exclusion. Among these, we focused onCXCL12as a relevant target based on its known roles in immunosuppression in other cancer types. We found that the CXCL12 receptor CXCR4 is highly expressed in both human primary tumors and metastases. To gain insight into the role of the CXCL12/CXCR4 axis, we inhibited CXCR4 signaling pharmacologically and found that plerixafor decreases fibrosis, alleviates solid stress, decompresses blood vessels, increases CTL infiltration, and decreases immunosuppression in murine mBC models. By deletingCXCR4in αSMA+cells, we confirmed that these immunosuppressive effects are dependent on CXCR4 signaling in αSMA+cells, which include cancer-associated fibroblasts as well as other cells such as pericytes. Accordingly, CXCR4 inhibition more than doubles the response to immune checkpoint blockers in mice bearing mBCs. These findings demonstrate that CXCL12/CXCR4-mediated desmoplasia in mBC promotes immunosuppression and is a potential target for overcoming therapeutic resistance to immune checkpoint blockade in mBC patients.
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Jung, Young Yun, Jae-Young Um, Acharan S. Narula, et al. "Identification of Matrine as a Novel Regulator of the CXCR4 Signaling Axis in Tumor Cells." International Journal of Molecular Sciences 21, no. 13 (2020): 4731. http://dx.doi.org/10.3390/ijms21134731.
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Matrine, a quinolizidine alkaloid, is commonly employed for treating various viral and inflammatory disorders. Here, we have evaluated matrine for its activity on C-X-C chemokine receptor type 4 (CXCR4) and matrix metalloproteinases (MMP-9/2) expression, and its potential to affect tumor metastasis and invasion. The effects of matrine on CXCR4, MMP-9/2, and nuclear factor κB (NF-κB) activation in lung (A549), prostate (DU145), and pancreas (MIA PaCa-2) cells were investigated by diverse techniques. The expression level of CXCR4 and MMP-9/2 was analyzed by western blot analysis and reverse transcription polymerase chain reaction. NF-κB activation was also evaluated by western blot analysis, electrophoretic mobility shift assay as well as immunocytochemical experiments. Furthermore, we monitored cell invasion and metastasis activities by wound healing and Boyden chamber assays. We noted that matrine induced a down-regulation of CXCR4 and MMP-9/2 at both protein and mRNA levels. In addition, matrine negatively regulated human epidermal growth factor receptor 2 (HER2) and C-X-C Motif Chemokine Ligand 12 (CXCL12)-induced CXCR4 expression. Moreover, NF-κB suppression by matrine led to inhibition of metastatic potential of tumor cells. Our results suggest that matrine can block the cancer metastasis through the negative regulation of CXCR4 and MMP-9/2 and consequently it can be considered as a potential candidate for cancer therapy.
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Vitale, Candida, Valentina Griggio, Chiara Riganti, et al. "The Mevalonate Metabolic Pathway and the CXCL12/CXCR4 Axis Reciprocally Interact and Are Implicated in Fludarabine Resistance of Chronic Lymphocytic Leukemia Cells." Blood 124, no. 21 (2014): 833. http://dx.doi.org/10.1182/blood.v124.21.833.833.
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Abstract BACKGROUND: Treatment of fludarabine-resistant chronic lymphocytic leukemia (CLL) patients is an unmet clinical need. Fludarabine resistance in CLL depends on intrinsic molecular features of the tumor cells, and on bidirectional interactions occurring between CLL cells and stromal cells (SC) of the tumor microenvironment. One of the main players of SC-induced fludarabine resistance is the CXCL12/CXCR4 axis. CXCR4 is a G protein-coupled receptor constitutively expressed on CLL cells. The binding of CXCR4 with CXCL12 activates the Ras/ERK1-2/Akt and the RhoA-dependent signalling pathways. To be active transducers Ras and RhoA need to undergo a post-translational modification (i.e. isoprenylation) by means of small molecules produced by the mevalonate (Mev) pathway. We have recently demonstrated that the Mev pathway is more active in IGHV unmutated than in mutated CLL cells, and is amenable to pharmacological manipulation by statins (i.e. simvastatin [Sim]). It is currently unknown whether the Mev pathway and its pharmacological targeting are implicated in the modulation of the CXCL12/CXCR4 axis and in the SC-induced fludarabine resistance of CLL cells. AIM: The aim of this study was to investigate the reciprocal interactions between the Mev pathway and the CXCL12/CXCR4 axis, in order to identify potential targets to counteract the constitutive and SC-induced fludarabine resistance of CLL cells. METHODS: Immuno-magnetically purified patient-derived CLL cells were cultured alone or with murine SC (M2-10B4 cell line). In selected experiments, cell cultures were exposed to human recombinant CXCL12 (100 μg/ml), CXCR4 inhibitor AMD3100 (5 μg/ml), fludarabine (F-ara-A, 10 μM), Sim (1 μM), ERK1-2 kinase inhibitor PD98059 (10 μM), RhoA kinase inhibitor Y276 (10 μM), HIF-1α inhibitor YC-1 (10 μM). The activity of the Mev pathway was measured by the quantification of metabolites [i.e. cholesterol and farnesyl pyrophosphate (FPP)] produced by CLL cells after 24 h incubation with 1 μCi of [3H]acetate. Ras and RhoA activities were evaluated measuring their GTP-bound fraction, taken as an index of the G-protein activation, respectively by pull-down assay and by an ELISA based assay. ERK1-2 and HIF-1α phosphorylation were evaluated by Western Blot. RhoA kinase, Akt and HIF-1α activities were measured with specific immunoassay kit. The amount of CXCL12 in culture supernatants was assessed by ELISA assay. Cell viability was determined by Annexin-V/propidium Iodide immunostaining and flow cytometry analysis. RESULTS: Co-culture with SC upregulated the Mev pathway activity of CLL cells, as shown by the increased production of cholesterol and FPP. This SC-induced increase in the Mev pathway activity was followed by the activation of the downstream Ras/ERK1-2 and RhoA/RhoA kinase signalling, the upregulation of the pro-survival factor Akt, and an increase in the transcriptional activity of HIF-1α. These biological and molecular effects were identically observed when CLL cells were exposed to recombinant CXCL12, and were completely abrogated by the CXCR4 antagonist AMD3100, thus showing the key role of the CXCL12/CXCR4 axis in the SC-induced modulation of the Mev pathway and the downstream Ras/ERK1-2 and RhoA/RhoA kinase signalling. On the other hand, blocking the Mev pathway by Sim and targeting ERK1-2 kinases, RhoA kinase and HIF-1α by specific small-molecule inhibitors significantly reduced the constitutive activity and the SC-induced upregulation of the Ras/ERK1-2 and RhoA/RhoA kinase signal transduction. A regulatory role of the Mev pathway on the CXCL12/CXCR4 axis was observed not only in CLL cells but also in SC, as shown by the significant reduction in CXCL12 secretion by SC exposed to Sim. In the last set of experiments, we found that the inhibition of the Mev pathway by Sim potentiated the direct cytotoxic effect of fludarabine against CLL cells. Even more importantly, both Sim and the downstream HIF-1α inhibitor YC-1 were capable of counteracting the SC-mediated protection of CLL cells from fludarabine-induced cytotoxicity. CONCLUSIONS: Our data demonstrate that the Mev pathway has a regulatory role on the CXCL12/CXCR4 axis and on the SC-mediated protective effects toward spontaneous and fludarabine-induced CLL cell death. The upstream inhibition of the Mev pathway and the downstream targeting of HIF-1α are promising strategies to circumvent fludarabine resistance in CLL. Disclosures Boccadoro: Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees.
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Wright, Kathryn, Kumudika de Silva, Karren M. Plain, et al. "Mycobacterial infection-induced miR-206 inhibits protective neutrophil recruitment via the CXCL12/CXCR4 signalling axis." PLOS Pathogens 17, no. 4 (2021): e1009186. http://dx.doi.org/10.1371/journal.ppat.1009186.
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Pathogenic mycobacteria actively dysregulate protective host immune signalling pathways during infection to drive the formation of permissive granuloma microenvironments. Dynamic regulation of host microRNA (miRNA) expression is a conserved feature of mycobacterial infections across host-pathogen pairings. Here we examine the role of miR-206 in the zebrafish model ofMycobacterium marinuminfection, which allows investigation of the early stages of granuloma formation. We find miR-206 is upregulated following infection by pathogenicM.marinumand that antagomir-mediated knockdown of miR-206 is protective against infection. We observed striking upregulation ofcxcl12aandcxcr4bin infected miR-206 knockdown zebrafish embryos and live imaging revealed enhanced recruitment of neutrophils to sites of infection. We used CRISPR/Cas9-mediated knockdown ofcxcl12aandcxcr4bexpression and AMD3100 inhibition of Cxcr4 to show that the enhanced neutrophil response and reduced bacterial burden caused by miR-206 knockdown was dependent on the Cxcl12/Cxcr4 signalling axis. Together, our data illustrate a pathway through which pathogenic mycobacteria induce host miR-206 expression to suppress Cxcl12/Cxcr4 signalling and prevent protective neutrophil recruitment to granulomas.
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Hinton, Cimona V., Shalom Avraham, and Hava Karsenty Avraham. "Role of the CXCR4/CXCL12 signaling axis in breast cancer metastasis to the brain." Clinical & Experimental Metastasis 27, no. 2 (2008): 97–105. http://dx.doi.org/10.1007/s10585-008-9210-2.
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Quinn, K. E., A. K. Ashley, L. P. Reynolds, A. T. Grazul-Bilska, and R. L. Ashley. "Activation of the CXCL12/CXCR4 signaling axis may drive vascularization of the ovine placenta." Domestic Animal Endocrinology 47 (April 2014): 11–21. http://dx.doi.org/10.1016/j.domaniend.2013.12.004.
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Andrés-Benito, Pol, Mònica Povedano, Raúl Domínguez, et al. "Increased C-X-C Motif Chemokine Ligand 12 Levels in Cerebrospinal Fluid as a Candidate Biomarker in Sporadic Amyotrophic Lateral Sclerosis." International Journal of Molecular Sciences 21, no. 22 (2020): 8680. http://dx.doi.org/10.3390/ijms21228680.
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Sporadic amyotrophic lateral sclerosis (sALS) is a fatal progressive neurodegenerative disease affecting upper and lower motor neurons. Biomarkers are useful to facilitate the diagnosis and/or prognosis of patients and to reveal possible mechanistic clues about the disease. This study aimed to identify and validate selected putative biomarkers in the cerebrospinal fluid (CSF) of sALS patients at early disease stages compared with age-matched controls and with other neurodegenerative diseases including Alzheimer disease (AD), spinal muscular atrophy type III (SMA), frontotemporal dementia behavioral variant (FTD), and multiple sclerosis (MS). SWATH acquisition on liquid chromatography-tandem mass spectrometry (LC–MS/MS) for protein quantitation, and ELISA for validation, were used in CSF samples of sALS cases at early stages of the disease. Analysis of mRNA and protein expression was carried out in the anterior horn of the lumbar spinal cord in post-mortem tissue of sALS cases (terminal stage) and controls using RTq-PCR, and Western blotting, and immunohistochemistry, respectively. SWATH acquisition on liquid chromatography-tandem mass spectrometry (LC–MS/MS) revealed 51 differentially expressed proteins in the CSF in sALS. Receiver operating characteristic (ROC) curves showed CXCL12 to be the most valuable candidate biomarker. We validated the values of CXCL12 in CSF with ELISA in two different cohorts. Besides sALS, increased CXCL12 levels were found in MS but were not altered in AD, SMA, and FTD. Therefore, increased CXCL12 levels in the CSF can be useful in the diagnoses of MS and sALS in the context of the clinical settings. CXCL12 immunoreactivity was localized in motor neurons in control and sALS, and in a few glial cells in sALS at the terminal stage; CXCR4 was in a subset of oligodendroglial-like cells and axonal ballooning of motor neurons in sALS; and CXCR7 in motor neurons in control and sALS, and reactive astrocytes in the pyramidal tracts in terminal sALS. CXCL12/CXCR4/CXCR7 axis in the spinal cord probably plays a complex role in inflammation, oligodendroglial and astrocyte signaling, and neuronal and axonal preservation in sALS.
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Young, Kira, Barbara Conley, Diana Romero, et al. "BMP9 regulates endoglin-dependent chemokine responses in endothelial cells." Blood 120, no. 20 (2012): 4263–73. http://dx.doi.org/10.1182/blood-2012-07-440784.
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Abstract BMP9 signaling has been implicated in hereditary hemorrhagic telangiectasia (HHT) and vascular remodeling, acting via the HHT target genes, endoglin and ALK1. This study sought to identify endothelial BMP9-regulated proteins that could affect the HHT phenotype. Gene ontology analysis of cDNA microarray data obtained after BMP9 treatment of primary human endothelial cells indicated regulation of chemokine, adhesion, and inflammation pathways. These responses included the up-regulation of the chemokine CXCL12/SDF1 and down-regulation of its receptor CXCR4. Quantitative mass spectrometry identified additional secreted proteins, including the chemokine CXCL10/IP10. RNA knockdown of endoglin and ALK1 impaired SDF1/CXCR4 regulation by BMP9. Because of the association of SDF1 with ischemia, we analyzed its expression under hypoxia in response to BMP9 in vitro, and during the response to hindlimb ischemia, in endoglin-deficient mice. BMP9 and hypoxia were additive inducers of SDF1 expression. Moreover, the data suggest that endoglin deficiency impaired SDF1 expression in endothelial cells in vivo. Our data implicate BMP9 in regulation of the SDF1/CXCR4 chemokine axis in endothelial cells and point to a role for BMP9 signaling via endoglin in a switch from an SDF1-responsive autocrine phenotype to an SDF1 nonresponsive paracrine state that represses endothelial cell migration and may promote vessel maturation.
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Waldschmidt, Johannes M., Dagmar Wider, Stefan J. Müller, et al. "PIM1-Mediated CXCR4 Phosphorylation: A Potentially Class-Distinct Therapeutic Target of Next Generation Proteasome Inhibitors in Multiple Myeloma." Blood 128, no. 22 (2016): 2774. http://dx.doi.org/10.1182/blood.v128.22.2774.2774.
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Abstract Introduction: The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment is fundamental to MM pathogenesis. Cell adhesion-mediated drug resistance (CAM-DR) is regulated by adhesion receptors on MM cells such as CXCR4, CXCR7, CD49d and CD44. We and others have previously reported that CAM-DR towards drugs like bortezomib, pomalidomide or vorinostat may be dissolved by combining these novel agents with the CXCR4 inhibitor plerixafor. Different than expected, additional treatment with plerixafor in corresponding experiment however did not rescue the cytotoxic effects of the second generation proteasome inhibitor carfilzomib. We hypothesized that carfilzomib itself interferes with the CXCR4-CXCL12 axis in myeloma. Prior reports in AML and CLL indicate that PIM1-mediated CXCR4 phosphorylation at the position S339 is an essential step for CXCR4 recirculation to the cell surface and its function as CXCL12 receptor (Grundler et al. 2009, Decker et al. 2014). In this project, we therefore examined the effects of carfilzomib on the PIM1-CXCR4 axis as a not yet described, potentially class-distinct mechanism of action of this second generation proteasome inhibitor. Methods: U266, RPMI-8226, L363, MOLT-4, NCI-H929 and the stromal cell line M2-10B4 were utilized. Bortezomib (1, 10, 20, 50, 100nM), carfilzomib (20, 50, 100nM) and plerixafor (10, 50, 100μM) were used based on previous studies and are well comparable to clinically relevant doses. CXCL12 stimulation was performed with human recombinant CXCL12 (30nM). For combination studies, cells were preincubated with plerixafor (50µM). Viability was quantified by propidium iodide and annexinV-FITC using flow cytometry. For quantitative real-time PCR and Western blots, U266 monocultured cells were treated with a carfilzomib pulse (t=1h), were allowed to recover for 20 hours, starved for 4 hours and stimulated with CXCL12 for 15 minutes (n=4). PIM-1 mRNA transcript levels were assessed in U266 control vs. U266 treated with a carfilzomib pulse (100nM, t=1h) by qPCR. Data was analyzed according to the "delta-delta-CT method" based on the relative expression of PIM-1 vs. GAPDH. Results were normalized to the mean of the control samples. Results: FACS analyses determined a substantial decrease of CD138 and CXCR4 surface expression in a dose-dependent manner after 1h carfilzomib treatment of U266 cells. Further assessment of downstream signaling revealed that carfilzomib treatment significantly reduces CXCR4 phosphorylation at S339 without changing total levels of CXCR4 (Figure A) or total levels of ERK or pERK (not shown), excluding a general inhibition of phosphorylation or protein synthesis by carfilzomib. Following the hypothesis that CXCR4 is potentially phosphorylated by PIM1 kinase, we assessed the impact of carfilzomib on PIM-1 protein levels: PIM-1 kinase protein was significantly reduced in a dose-dependent manner along with the levels of pCXCR4 in response to increasing doses of carfilzomib (0-100nM, Figure B). To further investigate a possible direct interference at the mRNA level, we evaluated PIM-1 mRNA levels after 1h carfilzomib, confirming substantially reduced PIM-1 RNA transcripts (Figure C). Different from carfilzomib and in line with prior observations (Shay et al. 2005), bortezomib was shown to increase protein levels of PIM-1 (data not shown). Side-by-side comparative assays of bortezomib vs. carfilzomib in terms of reduced CXCR4 expression, decreased CXCR4 phosphorylation and PIM-1 levels on mRNA and protein level are currently ongoing and will be presented at the meeting. Conclusions: Similar to previous reports on ixazomib reducing PIM-1 on protein and mRNA levels by inhibiting the tumor-suppressive microRNA miR33b (Tian et al. 2012), this work provides a potentially distinct mechanism of action of the second generation proteasome inhibitor carfilzomib on the PIM1-CXCR4 axis and identifies PIM-1 as a valid target to overcome CAM-DR in multiple myeloma. Figure Carfilzomib overcomes stroma protection due to PIM-1 kinase inhibition. Figure. Carfilzomib overcomes stroma protection due to PIM-1 kinase inhibition. Disclosures Engelhardt: Janssen: Research Funding; Amgen: Research Funding; MSD: Research Funding; Celgene: Research Funding.
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Honczarenko, Marek, Yi Le, Aleksandra M. Glodek, et al. "CCR5-binding chemokines modulate CXCL12 (SDF-1)–induced responses of progenitor B cells in human bone marrow through heterologous desensitization of the CXCR4 chemokine receptor." Blood 100, no. 7 (2002): 2321–29. http://dx.doi.org/10.1182/blood-2002-01-0248.
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Although the SDF-1 (CXCL12)/CXCR4 axis is important for B-cell development, it is not yet clear to what extent CC chemokines might influence B lymphopoiesis. In the current study, we characterized CC chemokine receptor 5 (CCR5) expression and function of primary progenitor B-cell populations in human bone marrow. CCR5 was expressed on all bone marrow B cells at levels between 150 and 200 molecules per cell. Stimulation of bone marrow B cells with the CCR5-binding chemokine macrophage inflammatory protein 1β (MIP-1β; CCL4) did not cause chemotaxis, but CCL4 was able to trigger potent calcium mobilization responses and activation of the mitogen-activated protein kinase (MAPK) pathway in developing B cells. We also determined that CCR5-binding chemokines MIP-1α (CCL3), CCL4, and RANTES (CCL5), specifically by signaling through CCR5, could affect all progenitor B-cell populations through a novel mechanism involving heterologous desensitization of CXCR4. This cross-desensitization of CXCR4 was manifested by the inhibition of CXCL12-induced calcium mobilization, MAPK activation, and chemotaxis. These findings indicate that CCR5 can indeed mediate biologic responses of bone marrow B cells, even though these cell populations express low levels of CCR5 on their cell surface. Thus, by modulation of CXCR4 function, signaling through CCR5 may influence B lymphopoiesis by affecting the migration and maturation of B-cell progenitors in the bone marrow microenvironment.
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Karpova, Darja, Julie Ritchey, Matthew Holt, et al. "Expansion and Maintenance of Hematopoietic Stem and Progenitor Cells in Course of Long-Term Inhibition of CXCR4/CXCL12 Signaling." Blood 128, no. 22 (2016): 2648. http://dx.doi.org/10.1182/blood.v128.22.2648.2648.
Full textAbstract:
Abstract During the past two decades peripheral blood stem cells have become the favored graft source for HSCT with 80 % of allogeneic and almost 100 % of autologous HSCT performed with mobilized blood. The critical role of the interaction between the chemokine receptor CXCR4 and its chief ligand CXCL12 for retention and migration of hematopoietic stem and progenitor cells (HSPC) has been well established. Interference with CXCR4/CXCL12 signalling iscurrentlybeing exploited as a strategy to mobilize HSPC indirectly with the most clinically relevant mobilizing agent to date, G-CSF as well as directly with the bicyclam CXCR4 antagonist Plerixafor (AMD3100).In this study, qualitative and quantitative effects of long-term pharmacologic inhibition of CXCR4/CXCL12 axis within the HSPC compartment were investigated in healthy C57BL/6 mice using the non-peptidic small molecule CXCR4 antagonists Plerixafor and ALT1188 along with the Protein-EpitopeMimeticsInhibitor POL5551. Up to 12-14 fold higher mobilization efficiency was achieved by applying the antagonists via two weeks of continuous infusion (up to 8-10x104 CFU-C and LSK/ml) as compared to bolus treatment (4-6x103 CFU-C and LSK/ml) or 5-day course of G-CSF (3-6x103 CFU-C/ml).Despite dramatic increase in numbers of circulating HSPC, the BM HSPC pool dis not decrease; in fact it expanded up to 2-4-fold compared to steady state reservoir (sham-operated control mice) as measured by immunophenotypical (LSK SLAM) and functional (e.g. serial competitive transplantation) properties of the cells. Thus, in contrast to genetically CXCR4 ablatedHSPC, the reversible long-term blockade of the receptor did not diminish the long-term repopulating capacity of HSPC. Cell cycle analysis showed a 2-3-fold increase in cycling activity of BM HSPC: only 10-20% of LSK and 30-40 % of LSK SLAM cells were found to be quiescent (in G0 phase of the cell cycle) after two weeks of CXCR4 antagonist infusion versus 50-60 % of LSK and 70 % of LSK SLAM found in G0 under homeostatic conditions. This increased proliferation was very similar to the one induced transiently at day 3 G-CSF treatmentand would conceivably explain the sustained mobilization without concomitant depletion of the BM HSPC pool. Profiling of differentially treated BM HSC (LSK SLAM) via microarray analysis did not reveal substantial effects of CXCR4 inhibitor infusion on the expression signature. Ofnote, major cytological changes typically associated with G-CSF induced mobilization, e.g. depletion of bone lining osteoblast lineage cells and macrophages, were not detected in continuous infusion of POL5551 exposed BM suggesting limitedeffects within the BM niche compartment. Moreover analysis of the BM HSPC after different washout periods at the end of continuous infusion treatment revealed a rapid (within 1-3 days after discontinuation of infusion) reestablishment of steady state HSPC numbers in the BM.Our data suggest that prolonged pharmacologic blockade of the CXCR4/CXCL12 axis using multiple small molecule inhibitorsrepresents an approach thatreleasesHSPCwith efficiency superiorto any other knownmobilization strategybut also may serve as an effective method induce cell cycling and thus expand BM HSPCs. Figure Competitive transplantation of POL5551 treated andcontrol BM (n=5 recipients per group, mean±SEM) Figure. Competitive transplantation of POL5551 treated andcontrol BM (n=5 recipients per group, mean±SEM) Disclosures Levesque: GlycoMimetics: Equity Ownership.