ABSTRACT
In beta-thalassemia, the mechanism driving ineffective erythropoiesis (IE) is insufficiently understood. We analyzed mice affected by beta-thalassemia and observed, unexpectedly, a relatively small increase in apoptosis of their erythroid cells compared with healthy mice. Therefore, we sought to determine whether IE could also be characterized by limited erythroid cell differentiation. In thalassemic mice, we observed that a greater than normal percentage of erythroid cells was in S-phase, exhibiting an erythroblast-like morphology. Thalassemic cells were associated with expression of cell cycle-promoting genes such as EpoR, Jak2, Cyclin-A, Cdk2, and Ki-67 and the antiapoptotic protein Bcl-X(L). The cells also differentiated less than normal erythroid ones in vitro. To investigate whether Jak2 could be responsible for the limited cell differentiation, we administered a Jak2 inhibitor, TG101209, to healthy and thalassemic mice. Exposure to TG101209 dramatically decreased the spleen size but also affected anemia. Although our data do not exclude a role for apoptosis in IE, we propose that expansion of the erythroid pool followed by limited cell differentiation exacerbates IE in thalassemia. In addition, these results suggest that use of Jak2 inhibitors has the potential to profoundly change the management of this disorder.
Subject(s)
Cell Differentiation , Erythroid Cells/pathology , Erythropoiesis , Janus Kinase 2/genetics , beta-Thalassemia/blood , Animals , Apoptosis , Cyclin-Dependent Kinases/genetics , Janus Kinase 2/antagonists & inhibitors , Mice , Spleen/pathologyABSTRACT
We report that TG101348, a selective small-molecule inhibitor of JAK2 with an in vitro IC50 of approximately 3 nM, shows therapeutic efficacy in a murine model of myeloproliferative disease induced by the JAK2V617F mutation. In treated animals, there was a statistically significant reduction in hematocrit and leukocyte count, a dose-dependent reduction/elimination of extramedullary hematopoiesis, and, at least in some instances, evidence for attenuation of myelofibrosis. There were no apparent toxicities and no effect on T cell number. In vivo responses were correlated with surrogate endpoints, including reduction/elimination of JAK2V617F disease burden assessed by quantitative genomic PCR, suppression of endogenous erythroid colony formation, and in vivo inhibition of JAK-STAT signal transduction as assessed by flow cytometric measurement of phosphorylated Stat5.
Subject(s)
Amino Acid Substitution , Disease Models, Animal , Janus Kinase 2/antagonists & inhibitors , Janus Kinase 2/genetics , Polycythemia Vera/drug therapy , Polycythemia Vera/enzymology , Protein Kinase Inhibitors/therapeutic use , Pyrrolidines/therapeutic use , Sulfonamides/therapeutic use , Animals , Bone Marrow Transplantation , Cell Line, Tumor , Colony-Forming Units Assay , Endpoint Determination , Flow Cytometry , Hematopoietic System/cytology , Hematopoietic System/drug effects , Humans , Janus Kinase 2/metabolism , Mice , Mice, Inbred C57BL , Phenylalanine/genetics , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/pharmacology , Pyrrolidines/pharmacokinetics , Signal Transduction/drug effects , Sulfonamides/pharmacokinetics , Survival Rate , Treatment Outcome , Valine/geneticsABSTRACT
Polycythemia Vera (PV) is a myeloproliferative disorder (MPD) that is commonly characterized by mutant JAK2 (JAK2V617F) signaling, erythrocyte overproduction, and a propensity for thrombosis, progression to myelofibrosis, or acute leukemia. In this study, JAK2V617F expression by human hematopoietic progenitors promoted erythroid colony formation and erythroid engraftment in a bioluminescent xenogeneic immunocompromised mouse transplantation model. A selective JAK2 inhibitor, TG101348 (300 nM), significantly inhibited JAK2V617F+ progenitor-derived colony formation as well as engraftment (120 mg/kg) in xenogeneic transplantation studies. TG101348 treatment decreased GATA-1 expression, which is associated with erythroid-skewing of JAK2V617F+ progenitor differentiation, and inhibited STAT5 as well as GATA S310 phosphorylation. Thus, TG101348 may be an effective inhibitor of JAK2V617F+ MPDs in clinical trials.
Subject(s)
Cell Differentiation/drug effects , Erythroid Precursor Cells/enzymology , Erythroid Precursor Cells/pathology , Janus Kinase 2/antagonists & inhibitors , Polycythemia Vera/enzymology , Polycythemia Vera/pathology , Protein Kinase Inhibitors/pharmacology , Adult , Aged , Amino Acid Substitution , Animals , Base Sequence , Erythroid Precursor Cells/drug effects , Female , Humans , Janus Kinase 2/genetics , Male , Mice , Middle Aged , Molecular Sequence Data , Phenylalanine/genetics , Protein Kinase Inhibitors/chemistry , Signal Transduction/drug effects , Stem Cell Transplantation , Valine/geneticsABSTRACT
Age-related macular degeneration (AMD) is one of the leading causes of loss of vision in the industrialized world. Attenuating the VEGF signal in the eye to treat AMD has been validated clinically. A large body of evidence suggests that inhibitors targeting the VEGFr pathway may be effective for the treatment of AMD. Recent studies using Src/YES knockout mice suggest that along with VEGF, Src and YES play a crucial role in vascular leak and might be useful in treating edema associated with AMD. Therefore, we have developed several potent benzotriazine inhibitors designed to target VEGFr2, Src, and YES. One of the most potent compounds is 4-chloro-3-{5-methyl-3-[4-(2-pyrrolidin-1-yl-ethoxy)phenylamino]benzo[1,2,4]triazin-7-yl}phenol ( 5), a dual inhibitor of both VEGFr2 and the Src family (Src and YES) kinases. Several ester analogues of 5 were prepared as prodrugs to improve the concentration of 5 at the back of the eye after topical administration. The thermal stability of these esters was studied, and it was found that benzoyl and substituted benzoyl esters of 5 showed good thermal stability. The hydrolysis rates of these prodrugs were studied to analyze their ability to undergo conversion to 5 in vivo so that appropriate concentrations of 5 are available in the back-of-the-eye tissues. From these studies, we identified 4-chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]amino}-1,2,4-benzotriazin-7-yl)phenyl benzoate ( 12), a topically administered prodrug delivered as an eye drop that is readily converted to the active compound 5 in the eye. This topically delivered compound exhibited excellent ocular pharmacokinetics and poor systemic circulation and showed good efficacy in the laser induced choroidal neovascularization model. On the basis of its superior profile, compound 12 was advanced. It is currently in a clinical trial as a first in class, VEGFr2 targeting, topically applied compound for the treatment of AMD.
Subject(s)
Macular Degeneration/drug therapy , Ophthalmic Solutions/therapeutic use , Phenols/therapeutic use , Prodrugs/therapeutic use , Triazines/therapeutic use , Administration, Topical , Animals , Choroidal Neovascularization/drug therapy , Clinical Trials as Topic , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Design , Eye/drug effects , Eye/radiation effects , Lasers , Mice , Mice, Knockout , Models, Molecular , Molecular Structure , Ophthalmic Solutions/chemistry , Ophthalmic Solutions/pharmacokinetics , Phenols/chemistry , Phenols/pharmacokinetics , Prodrugs/chemistry , Prodrugs/pharmacokinetics , Structure-Activity Relationship , Triazines/chemistry , Triazines/pharmacokinetics , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , src-Family Kinases/antagonists & inhibitorsABSTRACT
We describe the identification of [7-(2,6-dichlorophenyl)-5-methylbenzo [1,2,4]triazin-3-yl]-[4-(2-pyrrolidin-1-ylethoxy)phenyl]amine (3), a potent, orally active Src inhibitor with desirable PK properties, demonstrated activity in human tumor cell lines and in animal models of tumor growth.
Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Pyrrolidines/chemical synthesis , Triazines/chemical synthesis , src-Family Kinases/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Cytochrome P-450 Enzyme Inhibitors , Dogs , Enzyme Inhibitors/pharmacokinetics , Half-Life , Humans , Isoenzymes/antagonists & inhibitors , Lung Neoplasms/drug therapy , Mice , Mice, Nude , Models, Molecular , Molecular Conformation , Neoplasm Transplantation , Protein Binding , Pyrrolidines/pharmacology , Rats , Structure-Activity Relationship , Triazines/pharmacologyABSTRACT
Antagonists of alphavbeta3 and alphavbeta5 disrupt angiogenesis in response to bFGF and VEGF, respectively. Here, we show that these alphav integrins differentially contribute to sustained Ras-extracellular signal-related kinase (Ras-ERK) signaling in blood vessels, a requirement for endothelial cell survival and angiogenesis. Inhibition of FAK or alphavbeta5 disrupted VEGF-mediated Ras and c-Raf activity on the chick chorioallantoic membrane, whereas blockade of FAK or integrin alphavbeta3 had no effect on bFGF-mediated Ras activity, but did suppress c-Raf activation. Furthermore, retroviral delivery of active Ras or c-Raf promoted ERK activity and angiogenesis, which anti-alphavbeta5 blocked upstream of Ras, whereas anti-alphavbeta3 blocked downstream of Ras, but upstream of c-Raf. The activation of c-Raf by bFGF/alphavbeta3 not only depended on FAK, but also required p21-activated kinase-dependent phosphorylation of serine 338 on c-Raf, whereas VEGF-mediated c-Raf phosphorylation/activation depended on Src, but not Pak. Thus, integrins alphavbeta3 and alphavbeta5 differentially regulate the Ras-ERK pathway, accounting for distinct vascular responses during two pathways of angiogenesis.
Subject(s)
Integrin alphaVbeta3/metabolism , Integrins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Neovascularization, Physiologic , Receptors, Vitronectin/metabolism , ras Proteins/metabolism , Animals , Chick Embryo , Enzyme Activation , Fibroblast Growth Factor 2/metabolism , Focal Adhesion Protein-Tyrosine Kinases , Integrin alphaVbeta3/antagonists & inhibitors , Integrins/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Receptors, Vitronectin/antagonists & inhibitors , Signal Transduction/physiology , Vascular Endothelial Growth Factor A/metabolism , p21-Activated Kinases , src-Family Kinases/metabolismSubject(s)
Endothelial Growth Factors/antagonists & inhibitors , Lymphokines/antagonists & inhibitors , Neoplasms/drug therapy , Receptors, Vascular Endothelial Growth Factor/therapeutic use , Recombinant Fusion Proteins/therapeutic use , Animals , Apoptosis/drug effects , Breast Neoplasms/drug therapy , Colorectal Neoplasms/drug therapy , Female , Humans , Intercellular Signaling Peptides and Proteins , Mice , Neoplasms/blood supply , Neoplasms, Experimental/blood supply , Neoplasms, Experimental/drug therapy , Neovascularization, Pathologic/prevention & control , Transplantation, Heterologous , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Raf kinases have been linked to endothelial cell survival. Here, we show that basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) differentially activate Raf, resulting in protection from distinct pathways of apoptosis in human endothelial cells and chick embryo vasculature. bFGF activated Raf-1 via p21-activated protein kinase-1 (PAK-1) phosphorylation of serines 338 and 339, resulting in Raf-1 mitochondrial translocation and endothelial cell protection from the intrinsic pathway of apoptosis, independent of the mitogen-activated protein kinase kinase-1 (MEK1). In contrast, VEGF activated Raf-1 via Src kinase, leading to phosphorylation of tyrosines 340 and 341 and MEK1-dependent protection from extrinsic-mediated apoptosis. These findings implicate Raf-1 as a pivotal regulator of endothelial cell survival during angiogenesis.
Subject(s)
Apoptosis , Endothelium, Vascular/cytology , Neovascularization, Physiologic , Proto-Oncogene Proteins c-raf/metabolism , Animals , Cell Survival , Cells, Cultured , Chick Embryo , Endothelial Growth Factors/pharmacology , Endothelium, Vascular/drug effects , Enzyme Activation , Enzyme Inhibitors/pharmacology , Fibroblast Growth Factor 2/pharmacology , Flavonoids/pharmacology , Humans , Intercellular Signaling Peptides and Proteins/pharmacology , Lymphokines/pharmacology , MAP Kinase Kinase 1 , Mitochondria/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinases/metabolism , Neovascularization, Pathologic , Neovascularization, Physiologic/drug effects , Phosphorylation , Point Mutation , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Protein Transport , Proto-Oncogene Proteins B-raf , Proto-Oncogene Proteins c-raf/chemistry , Proto-Oncogene Proteins c-raf/genetics , Signal Transduction , Umbilical Veins , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , p21-Activated Kinases , src-Family Kinases/antagonists & inhibitors , src-Family Kinases/metabolismABSTRACT
Efforts to influence the biology of blood vessels by gene delivery have been hampered by a lack of targeting vectors specific for endothelial cells in diseased tissues. Here we show that a cationic nanoparticle (NP) coupled to an integrin alphavbeta3-targeting ligand can deliver genes selectively to angiogenic blood vessels in tumor-bearing mice. The therapeutic efficacy of this approach was tested by generating NPs conjugated to a mutant Raf gene, ATPmu-Raf, which blocks endothelial signaling and angiogenesis in response to multiple growth factors. Systemic injection of the NP into mice resulted in apoptosis of the tumor-associated endothelium, ultimately leading to tumor cell apoptosis and sustained regression of established primary and metastatic tumors.
Subject(s)
Genetic Therapy/methods , Nanotechnology , Neoplasms, Experimental/therapy , Neovascularization, Pathologic/therapy , Proto-Oncogene Proteins c-raf/genetics , Receptors, Vitronectin/metabolism , Adenosine Triphosphate/metabolism , Animals , Apoptosis , Cations , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Gene Targeting , Gene Transfer Techniques , Genetic Vectors , Humans , In Situ Nick-End Labeling , Ligands , Lipids , Melanoma, Experimental/blood supply , Melanoma, Experimental/pathology , Melanoma, Experimental/therapy , Mice , Mice, Inbred BALB C , Mutation , Neoplasm Metastasis , Neoplasm Transplantation , Neoplasms, Experimental/blood supply , Neoplasms, Experimental/pathology , Neovascularization, Pathologic/pathology , Proto-Oncogene Proteins c-raf/metabolism , Random Allocation , Tumor Cells, CulturedABSTRACT
Vascular endothelial growth factor (VEGF) promotes vascular permeability (VP) and neovascularization, and is required for development. We find that VEGF-stimulated Src activity in chick embryo blood vessels induces the coupling of focal adhesion kinase (FAK) to integrin alpha(v)beta5, a critical event in VEGF-mediated signaling and biological responsiveness. In contrast, FAK is constitutively associated with beta1 and beta3 integrins in the presence or absence of growth factors. In cultured endothelial cells, VEGF, but not basic fibroblast growth factor, promotes the Src-mediated phosphorylation of FAK on tyrosine 861, which contributes to the formation of a FAK/alpha(v)beta5 signaling complex. Moreover, formation of this FAK/alpha(v)beta5 complex is significantly reduced in pp60c-src-deficient mice. Supporting these results, mice deficient in either pp60c-src or integrin beta5, but not integrin beta3, have a reduced VP response to VEGF. This FAK/alpha(v)beta5 complex was also detected in epidermal growth factor-stimulated epithelial cells, suggesting a function for this complex outside the endothelium. Our findings indicate that Src can coordinate specific growth factor and extracellular matrix inputs by recruiting integrin alpha(v)beta5 into a FAK-containing signaling complex during growth factor-mediated biological responses.
Subject(s)
Endothelial Growth Factors/pharmacology , Integrins/metabolism , Lymphokines/pharmacology , Protein-Tyrosine Kinases/metabolism , Receptors, Vitronectin , Signal Transduction/physiology , src-Family Kinases/metabolism , Amino Acid Sequence , Animals , Cells, Cultured , Chick Embryo , Chorion/cytology , Chorion/enzymology , Endothelium, Vascular/cytology , Endothelium, Vascular/embryology , Endothelium, Vascular/enzymology , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Humans , Integrins/genetics , Kidney/cytology , Mice , Mice, Knockout , Molecular Sequence Data , Neovascularization, Physiologic/physiology , Phosphorylation , Protein Structure, Tertiary , Protein-Tyrosine Kinases/chemistry , Rabbits , Signal Transduction/drug effects , Tyrosine/metabolism , Umbilical Veins/cytology , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , src-Family Kinases/geneticsABSTRACT
As cancer cells undergo metastasis--invasion and migration of a new tissue--they penetrate and attach to the target tissue's basal matrix. This allows the cancer cell to pull itself forward into the tissue. The attachment is mediated by cell-surface receptors known as integrins, which bind to components of the extracellular matrix. Integrins are crucial for cell invasion and migration, not only for physically tethering cells to the matrix, but also for sending and receiving molecular signals that regulate these processes.