RESUMEN
BACKGROUND: Age-related macular degeneration (AMD), the most common cause of blindness in the developed world, usually affects individuals older than 60 years of age. The majority of visual loss in this disease is attributable to the development of choroidal neovascularization (CNV). Mononuclear phagocytes, including monocytes and their tissue descendants, macrophages, have long been implicated in the pathogenesis of neovascular AMD (nvAMD). Current therapies for nvAMD are based on targeting vascular endothelial growth factor (VEGF). This study is aimed at assessing if perturbation of chemokine signaling and mononuclear cell recruitment may serve as novel complementary therapeutic targets for nvAMD. METHODS: A promiscuous chemokine antagonist (BKT130), aflibercept treatment, or combined BKT130+aflibercept treatment was tested in an in vivo laser-induced model of choroidal neovascularization (LI-CNV) and in an ex vivo choroidal sprouting assay (CSA). Quantification of CD11b+ cell in the CNV area was performed, and mRNA levels of genes implicated in CNV growth were measured in the retina and RPE-choroid. RESULTS: BKT130 reduced the CNV area and recruitment of CD11b+ cells by 30-35%. No effect of BKT130 on macrophages' proangiogenic phenotype was demonstrated ex vivo, but a lower VEGFA and CCR2 expression was found in the RPE-choroid and a lower expression of TNFα and NOS1 was found in both RPE-choroid and retinal tissues in the LI-CNV model under treatment with BKT130. CONCLUSIONS: Targeting monocyte recruitment via perturbation of chemokine signaling can reduce the size of experimental CNV and should be evaluated as a potential novel therapeutic modality for nvAMD.
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Quimiocinas/antagonistas & inhibidores , Neovascularización Coroidal/tratamiento farmacológico , Monocitos/efectos de los fármacos , Anciano , Anciano de 80 o más Años , Animales , Antígeno CD11b/metabolismo , Movimiento Celular/efectos de los fármacos , Quimiocinas/metabolismo , Neovascularización Coroidal/etiología , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Femenino , Humanos , Rayos Láser , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Monocitos/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Ratas , Ratas Long-Evans , Receptores CCR2/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/uso terapéutico , Proteínas Recombinantes de Fusión/uso terapéutico , Retina/metabolismo , Retina/patología , Factor de Necrosis Tumoral alfa/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
CXCR4 expression in neuroblastoma tumors correlates with disease severity. In this study, we describe mechanisms by which CXCR4 signaling controls neuroblastoma tumor growth and response to therapy. We found that overexpression of CXCR4 or stimulation with CXCL12 supports neuroblastoma tumorigenesis. Moreover, CXCR4 inhibition with the high-affinity CXCR4 antagonist BL-8040 prevented tumor growth and reduced survival of tumor cells. These effects were mediated by the upregulation of miR-15a/16-1, which resulted in downregulation of their target genes BCL-2 and cyclin D1, as well as inhibition of ERK. Overexpression of miR-15a/16-1 in cells increased cell death, whereas antagomirs to miR-15a/16-1 abolished the proapoptotic effects of BL-8040. CXCR4 overexpression also increased miR-15a/16-1, shifting their oncogenic dependency from the BCL-2 to the ERK signaling pathway. Overall, our results demonstrate the therapeutic potential of CXCR4 inhibition in neuroblastoma treatment and provide a rationale to test combination therapies employing CXCR4 and BCL-2 inhibitors to increase the efficacy of these agents.Significance: These results provide a mechanistic rationale for combination therapy of CXCR4 and BCL-2 inhibitors to treat a common and commonly aggressive pediatric cancer.Cancer Res; 78(6); 1471-83. ©2017 AACR.
Asunto(s)
Neoplasias Encefálicas/patología , MicroARNs/metabolismo , Neuroblastoma/patología , Receptores CXCR4/metabolismo , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Ratones , MicroARNs/genética , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Péptidos/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chemokines and their receptors play critical roles in the progression of autoimmunity and inflammation. Typically, multiple chemokines are involved in the development of these pathologies. Indeed, targeting single chemokines or chemokine receptors has failed to achieve significant clinical benefits in treating autoimmunity and inflammation. Moreover, the binding of host atypical chemokine receptors to multiple chemokines as well as the binding of chemokine-binding proteins secreted by various pathogens can serve as a strategy for controlling inflammation. In this work, promiscuous chemokine-binding peptides that could bind and inhibit multiple inflammatory chemokines, such as CCL2, CCL5, and CXCL9/10/11, were selected from phage display libraries. These peptides were cloned into human mutated immunoglobulin Fc-protein fusions (peptibodies). The peptibodies BKT120Fc and BKT130Fc inhibited the ability of inflammatory chemokines to induce the adhesion and migration of immune cells. Furthermore, BKT120Fc and BKT130Fc also showed a significant inhibition of disease progression in a variety of animal models for autoimmunity and inflammation. Developing a novel class of antagonists that can control the courses of diseases by selectively blocking multiple chemokines could be a novel way of generating effective therapeutics.
RESUMEN
Purpose: The potential of the high-affinity CXCR4 antagonist BL-8040 as a monotherapy-mobilizing agent and its derived graft composition and quality were evaluated in a phase I clinical study in healthy volunteers (NCT02073019).Experimental Design: The first part of the study was a randomized, double-blind, placebo-controlled dose escalation phase. The second part of the study was an open-label phase, in which 8 subjects received a single injection of BL-8040 (1 mg/kg) and approximately 4 hours later underwent a standard leukapheresis procedure. The engraftment potential of the purified mobilized CD34+ cells was further evaluated by transplanting the cells into NSG immunodeficient mice.Results: BL-8040 was found safe and well tolerated at all doses tested (0.5-1 mg/kg). The main treatment-related adverse events were mild to moderate. Transient injection site and systemic reactions were mitigated by methylprednisolone, paracetamol, and promethazine pretreatment. In the first part of the study, BL-8040 triggered rapid and substantial mobilization of WBCs and CD34+ cells in all tested doses. Four hours postdose, the count rose to a mean of 8, 37, 31, and 35 cells/µL (placebo, 0.5, 0.75, and 1 mg/kg, respectively). FACS analysis revealed substantial mobilization of immature dendritic, T, B, and NK cells. In the second part, the mean CD34+ cells/kg collected were 11.6 × 106 cells/kg. The graft composition was rich in immune cells.Conclusions: The current data demonstrate that BL-8040 is a safe and effective monotherapy strategy for the collection of large amounts of CD34+ cells and immune cells in a one-day procedure for allogeneic HSPC transplantation. Clin Cancer Res; 23(22); 6790-801. ©2017 AACR.
Asunto(s)
Antígenos CD34/metabolismo , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Péptidos/administración & dosificación , Receptores CXCR4/antagonistas & inhibidores , Animales , Biomarcadores , Femenino , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Voluntarios Sanos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Humanos , Inmunofenotipificación , Leucaféresis , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Modelos Animales , Trasplante HomólogoRESUMEN
Functional role of CXCR4 in chronic myelogenous leukemia (CML) progression was evaluated. Elevated CXCR4 significantly increased the in vitro survival and proliferation in response to CXCL12. CXCR4 stimulation resulted in activation of extracellular signal-regulated kinase (Erk)-1/2, Akt, S6K, STAT3, and STAT5 prosurvival signaling pathways. In accordance, we found that in vitro treatment with CXCR4 antagonist BKT140 directly inhibited the cell growth and induced cell death of CML cells. Combination of BKT140 with suboptimal concentrations of imatinib significantly increased the anti-CML effect. BKT140 induced apoptotic cell death, decreasing the levels of HSP70 and HSP90 chaperones and antiapoptotic proteins BCL-2 and BCL-XL, subsequently promoting the release of mitochondrial factors cytochrome c and SMAC/Diablo. Bone marrow (BM) stromal cells (BMSC) markedly increased the proliferation of CML cells and protected them from imatinib-induced apoptosis. Furthermore, BMSCs elevated proto-oncogene BCL6 expression in the CML cells in response to imatinib treatment, suggesting the possible role of BCL6 in stroma-mediated TKI resistance. BKT140 reversed the protective effect of the stroma, effectively promoted apoptosis, and decreased BCL6 levels in CML cells cocultured with BMSCs. BKT140 administration in vivo effectively reduced the growth of subcutaneous K562-produced xenografts. Moreover, the combination of BKT140 with low-dose imatinib markedly inhibited tumor growth, achieving 95% suppression. Taken together, our data indicate the importance of CXCR4/CXCL12 axis in CML growth and CML-BM stroma interaction. CXCR4 inhibition with BKT140 antagonist efficiently cooperated with imatinib in vitro and in vivo. These results provide the rational basis for CXCR4-targeted therapy in combination with TKI to override drug resistance and suppress residual disease.
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Benzamidas/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Oligopéptidos/farmacología , Piperazinas/farmacología , Pirimidinas/farmacología , Receptores CXCR4/antagonistas & inhibidores , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/farmacología , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Expresión Génica , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proto-Oncogenes Mas , Receptores CXCR4/genética , Transducción de Señal/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Ensayo de Tumor de Célula Madre , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: CXCR4 plays an important role in the retention of stem cells within the bone marrow. BKT140 (4F-benzoyl-TN14003) is a 14-residue bio stable synthetic peptide, which binds CXCR4 with a greater affinity compared with plerixafor (4 vs. 84 nmol/L). Studies in mice demonstrated the efficient and superior mobilization and transplantation of stem cells collected with GCSF-BKT140, compared with those obtained when using stem cells obtained with each one of these mobilizing agent alone. These results have served as a platform for the present clinical phase I study. EXPERIMENTAL DESIGN: Eighteen patients with multiple myeloma who were preparing for their first autologous stem cell transplantation were included. Patients received a standard multiple myeloma mobilization regimen, consisting of 3 to 4 g/m(2) cyclophosphamide (day 0), followed by granulocyte colony-stimulating factor (G-CSF) at 5 µg/kg/d starting on day 5 and administered between 8 and 10 pm until the end of stem cell collection. A single injection of BKT140 (0.006, 0.03, 0.1, 0.3, and 0.9 mg/kg) was administered subcutaneously on day 10 in the early morning, followed by G-CSF 12 hours later. RESULTS: BKT140 was well tolerated at all concentrations, and none of the patients developed grade 3 and 4 toxicity. A single administration of BKT140 at the highest dose, 0.9 mg/kg, resulted in a robust mobilization and collection of CD34(+) cells (20.6 ± 6.9 × 10(6)/kg), which were obtained through a single apheresis. All transplanted patients received â¼5.3 × 10(6) CD34(+) cells/kg, which rapidly engrafted (n = 17). The median time to neutrophil and platelet recovery was 12 and 14 days, respectively, at the highest dose (0.9 mg/kg). CONCLUSIONS: When combined with G-CSF, BKT140 is a safe and efficient stem cell mobilizer that enabled the collection of a high number of CD34(+) cells in 1 and 2 aphaeresis procedures, resulting in successful engraftment.
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Antineoplásicos/farmacología , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Mieloma Múltiple/metabolismo , Mieloma Múltiple/terapia , Oligopéptidos/farmacología , Receptores CXCR4/antagonistas & inhibidores , Adulto , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Recuento de Células Sanguíneas , Línea Celular Tumoral , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Femenino , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Factor Estimulante de Colonias de Granulocitos/efectos adversos , Factor Estimulante de Colonias de Granulocitos/farmacología , Trasplante de Células Madre Hematopoyéticas , Humanos , Masculino , Ratones , Persona de Mediana Edad , Oligopéptidos/administración & dosificación , Oligopéptidos/efectos adversos , Unión Proteica , Receptores CXCR4/metabolismo , Resultado del TratamientoRESUMEN
Platelets are the terminal differentiation product of megakaryocytes (MKs). Cytokines, such as thrombopoietin (TPO), are known to influence different steps in MK development; however, the complex differentiation and platelet localization processes are not fully understood. MKs express the receptor CXCR4 and have been shown to migrate in response to CXCL12 and to increase their platelet production. In this study, we studied the role of CXCR4 in platelet production with the high affinity CXCR4 antagonist, BKT140. Single and sequential administration of BKT140 significantly increased the number of MKs and haematopoietic progenitors (HPCs) within the bone marrow (BM). Increased megakaryopoiesis was associated with increased platelet production. Single and sequential administration of BKT140 also increased the number of HPCs in the blood. In a model of 5-fluorouracil-induced thrombocytopenia, BKT140 significantly reduced the severity and duration of thrombocytopenia and cytopenia when administered before and after chemotherapy. Our results demonstrated that the CXCR4 antagonist, BKT140, mediated unique beneficial effects by stimulating megakaryopoiesis and platelet production. These results provide evidence for the possible therapeutic use of BKT140 for modulating platelet numbers in thrombocytopenic conditions.
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Plaquetas/citología , Plaquetas/efectos de los fármacos , Oligopéptidos/administración & dosificación , Receptores CXCR4/antagonistas & inhibidores , Trombopoyesis/efectos de los fármacos , Trombopoyesis/fisiología , Animales , Antineoplásicos/efectos adversos , Plaquetas/metabolismo , Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Femenino , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Recuento de Leucocitos , Células Progenitoras de Megacariocitos/citología , Células Progenitoras de Megacariocitos/efectos de los fármacos , Ratones , Recuento de Plaquetas , Trombocitopenia/inducido químicamente , Trombocitopenia/tratamiento farmacológico , Trombopoyetina/farmacologíaRESUMEN
PURPOSE: Chemokine axis CXCR4/CXCL12 is critically involved in the survival and trafficking of normal and malignant B lymphocytes. Here, we investigated the effect of high-affinity CXCR4 antagonist BKT140 on lymphoma cell growth and rituximab-induced cytotoxicity in vitro and in vivo. EXPERIMENTAL DESIGN: In vitro efficacy of BKT140 alone or in combination with rituximab was determined in non-Hodgkin lymphoma (NHL) cell lines and primary samples from bone marrow aspirates of patients with NHL. In vivo efficacy was evaluated in xenograft models of localized and disseminated NHL with bone marrow involvement. RESULTS: Antagonizing CXCR4 with BKT140 resulted in significant inhibition of CD20+ lymphoma cell growth and in the induction of cell death, respectively. Combination of BKT140 with rituximab significantly enhanced the apoptosis against the lymphoma cells in a dose-dependent manner. Moreover, rituximab induced CXCR4 expression in lymphoma cell lines and primary lymphoma cells, suggesting the possible interaction between CD20 and CXCR4 pathways in NHL. Primary bone marrow stromal cells (BMSC) further increased CXCR4 expression and protected NHL cells from rituximab-induced apoptosis, whereas BKT140 abrogated this protective effect. Furthermore, BKT140 showed efficient antilymphoma activity in vivo in the xenograft model of disseminated NHL with bone marrow involvement. BKT140 treatment inhibited the local tumor progression and significantly reduced the number of NHL cells in the bone marrow. Combined treatment of BKT140 with rituximab further decreased the number of viable lymphoma cells in the bone marrow, achieving 93% reduction. CONCLUSIONS: These findings suggest the possible role of CXCR4 in NHL progression and response to rituximab and provide the scientific basis for the development of novel CXCR4-targeted therapies for refractory NHL.
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Anticuerpos Monoclonales de Origen Murino/farmacología , Antígenos CD20/metabolismo , Antineoplásicos/farmacología , Linfoma no Hodgkin/metabolismo , Oligopéptidos/farmacología , Receptores CXCR4/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Médula Ósea/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Microambiente Celular/efectos de los fármacos , Humanos , Linfoma no Hodgkin/tratamiento farmacológico , Linfoma no Hodgkin/patología , Ratones , Rituximab , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVES: CXCR4/CXCL12 interactions promote non-small cell lung cancer (NSCLC) growth and dissemination. Furthermore, this axis might promote NSCLC resistance to chemotherapy and/or radiotherapy. Therefore, the CXCR4/CXCL12 axis constitutes an attractive therapeutic target for the treatment of NSCLC. We aimed to characterize the therapeutic efficacy of the novel CXCR4 antagonist BKT140 against human NSCLC. METHODS: We determined the CXCR4 expression in 5 NSCLC cell lines (H358, A549, H460, H1299, and L4). We then tested the colony-forming capacity and proliferation of these cells in the presence of CXCL12 and BKT140. Next, we measured the in vivo growth of A549 and H460 xenografts with or without BKT140 treatment. Finally, we examined, in vitro, the potential antiproliferative effect of BKT140 combined with cisplatin or paclitaxel and after irradiation of NSCLC cells. RESULTS: All tested cell lines expressed CXCR4 and showed increased colony formation in response to CXCL12 stimulation. BKT140 reduced the colony-forming capacity of NSCLC cells. Proliferation assays demonstrated both cytotoxic and cytostatic properties for this peptide. H460 cells were the most sensitive to BKT140 and A549 cells the least. Subcutaneous administration of BKT140 significantly delayed the development of H460 xenografts and showed a similar trend for A549 xenografts. Finally, the antiproliferative effects of BKT140 appears to be additive to those of chemotherapeutic drugs and radiotherapy. CONCLUSIONS: Targeting the CXCL12/CXCR4 axis with BKT140 attenuated NSCLC cells tumor growth and augmented the effects of chemotherapy and radiotherapy. Future research will benefit from delineating the downstream mechanism of BKT140 action and defining BKT140 susceptibility markers.
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Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Oligopéptidos/farmacología , Receptores CXCR4/antagonistas & inhibidores , Animales , Antineoplásicos/administración & dosificación , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quimiocina CXCL2/metabolismo , Quimioradioterapia , Cisplatino/administración & dosificación , Relación Dosis-Respuesta a Droga , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/radioterapia , Ratones , Ratones Desnudos , Oligopéptidos/administración & dosificación , Paclitaxel/administración & dosificación , Receptores CXCR4/metabolismo , Factores de Tiempo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: The chemokine receptor CXCR4 and its ligand CXCL12 are involved in the progression and dissemination of a diverse number of solid and hematological malignancies. Binding CXCL12 to CXCR4 activates a variety of intracellular signal transduction pathways that regulate cell chemotaxis, adhesion, survival, proliferation, and apoptosis. MATERIALS AND METHODS: Here, we demonstrate that the CXCR4 antagonist, 4F-benzoyl-TN14003 (BKT140), but not AMD3100, exhibits a CXCR4-dependent preferential cytotoxicity toward malignant cells of hematopoietic origin. BKT140 significantly and preferentially stimulated multiple myeloma apoptotic cell death. BKT140 treatment induced morphological changes, phosphatidylserine externalization, decreased mitochondrial membrane potential, caspase-3 activation, sub-G1 arrest, and DNA double-stranded breaks. RESULTS: In vivo, subcutaneous injections of BKT140 significantly reduced, in a dose-dependent manner, the growth of human acute myeloid leukemia and multiple myeloma xenografts. Tumors from animals treated with BKT140 were smaller in size and weights, had larger necrotic areas and high apoptotic scores. CONCLUSIONS: Taken together, these results suggest a potential therapeutic use for BKT140 in multiple myeloma and leukemia patients.