ABSTRACT
The reported proof of principle study demonstrated the feasibility of local delivery of a c-Met inhibitor (VXc-140) in a subcutaneous xenograft tumor model. VXc-140 was formulated in a wafer delivery system for direct implantation into the tumor. Systemic and local tumor exposure of VXc-140 was analyzed. High tumor exposures coupled with fast release of compound were associated with significant tumor regression and reduction in tumor levels of phosphorylated c-Met. High VXc-140 tumor-to-plasma ratios (â¼42 at the tumor periphery) were achieved. The tumor response achieved (7/11 partial response) with VXc-140 with the local delivery in the wafer (4 mg over 15 days) was comparable to the regression observed (11/15 partial response) for VXc-140 in the oral delivery (â¼8 mg total administered once a day for 2 weeks). Notably, the plasma levels in animals implanted with VXc-140 wafers ranged from 2 to 4 µM, which, although higher than trough levels achieved with oral administration, were well below oral Cmax levels (â¼42 µM) suggesting that toxicities associated with Cmax exposure may be reduced or eliminated by local delivery. The high tumor to plasma exposure of VXc-140 and the efficacy observed with local wafer delivery warrants further exploration into the utility of local delivery.
Subject(s)
Antineoplastic Agents/pharmacology , Neoplasms/drug therapy , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Administration, Oral , Animals , Chemistry, Pharmaceutical/methods , Drug Delivery Systems/methods , Female , Heterografts/drug effects , Humans , Mice , Pilot ProjectsABSTRACT
Aberrant activation of signaling through the RAS-RAF-MEK-ERK (MAPK) pathway is implicated in numerous cancers, making it an attractive therapeutic target. Although BRAF and MEK-targeted combination therapy has demonstrated significant benefit beyond single-agent options, the majority of patients develop resistance and disease progression after approximately 12 months. Reactivation of ERK signaling is a common driver of resistance in this setting. Here we report the discovery of BVD-523 (ulixertinib), a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and ERK1/2 selectivity. In vitro BVD-523 treatment resulted in reduced proliferation and enhanced caspase activity in sensitive cells. Interestingly, BVD-523 inhibited phosphorylation of target substrates despite increased phosphorylation of ERK1/2. In in vivo xenograft studies, BVD-523 showed dose-dependent growth inhibition and tumor regression. BVD-523 yielded synergistic antiproliferative effects in a BRAFV600E-mutant melanoma cell line xenograft model when used in combination with BRAF inhibition. Antitumor activity was also demonstrated in in vitro and in vivo models of acquired resistance to single-agent and combination BRAF/MEK-targeted therapy. On the basis of these promising results, these studies demonstrate BVD-523 holds promise as a treatment for ERK-dependent cancers, including those whose tumors have acquired resistance to other treatments targeting upstream nodes of the MAPK pathway. Assessment of BVD-523 in clinical trials is underway (NCT01781429, NCT02296242, and NCT02608229). Mol Cancer Ther; 16(11); 2351-63. ©2017 AACR.
Subject(s)
Aminopyridines/administration & dosage , Melanoma/drug therapy , Protein Kinase Inhibitors/administration & dosage , Proto-Oncogene Proteins B-raf/genetics , Pyrroles/administration & dosage , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Resistance, Neoplasm/genetics , Humans , MAP Kinase Signaling System/drug effects , Melanoma/genetics , Melanoma/pathology , Mice , Mutation , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Xenograft Model Antitumor AssaysABSTRACT
This work demonstrates the application of a 3D culture system-Cells-in-Gels-in-Paper (CiGiP)-in evaluating the metabolic response of lung cancer cells to ionizing radiation. The 3D tissue-like construct-prepared by stacking multiple sheets of paper containing cell-embedded hydrogels-generates a gradient of oxygen and nutrients that decreases monotonically in the stack. Separating the layers of the stack after exposure enabled analysis of the cellular response to radiation as a function of oxygen and nutrient availability; this availability is dictated by the distance between the cells and the source of oxygenated medium. As the distance between the cells and source of oxygenated media increased, cells show increased levels of hypoxia-inducible factor 1-alpha, decreased proliferation, and reduced sensitivity to ionizing radiation. Each of these cellular responses are characteristic of cancer cells observed in solid tumors. With this setup we were able to differentiate three isogenic variants of A549 cells based on their metabolic radiosensitivity; these three variants have known differences in their metastatic behavior in vivo. This system can, therefore, capture some aspects of radiosensitivity of populations of cancer cells related to mass-transport phenomenon, carry out systematic studies of radiation response in vitro that decouple effects from migration and proliferation of cells, and regulate the exposure of oxygen to subpopulations of cells in a tissue-like construct either before or after irradiation.
Subject(s)
Cell Culture Techniques/methods , Lung Neoplasms/radiotherapy , A549 Cells , Cell Proliferation/radiation effects , Cell Survival/radiation effects , Humans , Hydrogels , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Lung Neoplasms/metabolism , Oxygen/metabolism , Paper , Radiation Tolerance , Tumor Hypoxia/radiation effectsABSTRACT
This work describes a 3D, paper-based assay that can isolate sub-populations of cells based on their invasiveness (i.e., distance migrated in a hydrogel) in a gradient of concentration of oxygen (O2). Layers of paper impregnated with a cell-compatible hydrogel are stacked and placed in a plastic holder to form the invasion assay. In most assays, the stack comprises a single layer of paper containing mammalian cells suspended in a hydrogel, sandwiched between multiple layers of paper containing only hydrogel. Cells in the stack consume and produce small molecules; these molecules diffuse throughout the stack to generate gradients in the stack, and between the stack and the bulk culture medium. Placing the cell-containing layer in different positions of the stack, or modifying the permeability of the holder to oxygen or proteins, alters the profile of the gradients within the stack. Physically separating the layers after culture isolates sub-populations of cells that migrated different distances, and enables their subsequent analysis or culture. Using this system, three independent cell lines derived from A549 cancer cells are shown to produce distinguishable migration behavior in a gradient of oxygen. This result is the first experimental demonstration that oxygen acts as a chemoattractant for cancer cells.
Subject(s)
Chemotaxis , Neoplasms/pathology , Oxygen/chemistry , Paper , Animals , Biological Assay , Cell Culture Techniques , Cell Line, Tumor , Cell Survival , Chemotactic Factors/chemistry , HEK293 Cells , Humans , Hydrogel, Polyethylene Glycol Dimethacrylate/chemistry , Male , Mice , Mice, Nude , Models, Theoretical , Neoplasm Invasiveness , Neoplasm Metastasis , Permeability , PhenotypeABSTRACT
Platinum-based DNA-damaging chemotherapy is standard-of-care for most patients with lung cancer but outcomes remain poor. This has been attributed, in part, to the highly effective repair network known as the DNA-damage response (DDR). ATR kinase is a critical regulator of this pathway, and its inhibition has been shown to sensitize some cancer, but not normal, cells in vitro to DNA damaging agents. However, there are limited in vivo proof-of-concept data for ATR inhibition. To address this we profiled VX-970, the first clinical ATR inhibitor, in a series of in vitro and in vivo lung cancer models and compared it with an inhibitor of the downstream kinase Chk1. VX-970 markedly sensitized a large proportion of a lung cancer cell line and primary tumor panel in vitro to multiple DNA damaging drugs with clear differences to Chk1 inhibition observed. In vivo VX-970 blocked ATR activity in tumors and dramatically enhanced the efficacy of cisplatin across a panel of patient derived primary lung xenografts. The combination led to complete tumor growth inhibition in three cisplatin-insensitive models and durable tumor regression in a cisplatin-sensitive model. These data provide a strong rationale for the clinical evaluation of VX-970 in lung cancer patients.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cisplatin/pharmacology , DNA Damage , Isoxazoles/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrazines/pharmacology , Animals , Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , Cell Line, Tumor , Cisplatin/administration & dosage , DNA/drug effects , DNA/genetics , Drug Synergism , Female , Humans , Mice , Mice, SCID , Phosphorylation , Protein Kinase Inhibitors/administration & dosage , Random Allocation , Signal Transduction , Xenograft Model Antitumor AssaysABSTRACT
The poor success rate of discovering new, effective chemotherapeutics for oncology may reflect the failure of targeting treatments to the more aggressive, tumorigenic cells of the malignancy. Data have now emerged from several laboratories, examining both liquid and solid primary tumor tissues, that implicate cancer stem cells (CSCs) as the 'master-driver' cellular population for tumorigenicity. Moreover, these putative CSCs appear relatively resistant to existing chemotherapeutic and radiation therapy. Several different cellular pathways have been identified as likely mechanisms causal for the underlying insensitivity of the CSCs to conventional therapy. Progress has been made in the isolation and expansion of these CSCs for constructing conventional high-throughput phenotypic screening campaigns. However, challenges remain in designing optimal proof-of-concept trials for the clinical development of compounds targeting the elimination of CSCs.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Discovery/methods , Drug Screening Assays, Antitumor/methods , Neoplasms/drug therapy , Neoplastic Stem Cells/drug effects , Animals , Humans , Neoplastic Stem Cells/cytology , Neoplastic Stem Cells/metabolismSubject(s)
Antineoplastic Agents/therapeutic use , Drug Discovery , Neoplasms/drug therapy , Animals , Antineoplastic Agents/pharmacology , Biomedical Research , Cell Line, Tumor , Drug Screening Assays, Antitumor , Humans , Neoplasms/metabolism , Neoplasms/pathology , Neoplastic Stem Cells/physiology , Oxidation-ReductionABSTRACT
Novel therapies for the treatment of solid tumors have generally failed to improve patient overall survival. These therapeutic approaches are typically focused on targeting signaling pathways implicated in cell growth and/or survival in order to shrink the malignant mass and achieve an objective clinical response; however, too often these responses are followed by eventual regrowth of the tumor. This clinical conundrum could be explained by the existence of a tumorigenic cell population that is relatively resistant to these therapies and retains pluripotent status in order to repopulate the original tumor and/or contribute to distant metastasis following treatment. Compelling data from liquid tumors, and more recently from studies focused on solid tumors, now support the existence of such tumorigenic cells (i.e., cancer stem cells) as a distinct subpopulation within the total tumor cell mass. These cancer stem cells (CSCs), as compared to the non-CSC population, have the ability to reconstitute the primary tumor phenotype when transplanted into recipient animals. In addition, data are beginning to emerge demonstrating that many standard-of-care chemotherapeutics are less effective in promoting cell death or cytostasis in these putative cancer stem cells as compared to effects in the non-stem cell cancerous cells. Therefore, targeting these locomotive drivers of tumors, the cancer stem cell population, should be considered a high priority in the continued pursuit of more effective cancer therapies.
Subject(s)
Antigens, Neoplasm/drug effects , Antineoplastic Agents/pharmacology , Cell Transformation, Neoplastic/drug effects , Drug Resistance, Neoplasm/drug effects , Neoplastic Stem Cells/pathology , Neovascularization, Pathologic/therapy , Signal Transduction/drug effects , Animals , Cell Transformation, Neoplastic/pathology , Drug Delivery Systems , Drug Design , Drug Resistance, Neoplasm/physiology , Humans , Neoplasm Proteins/antagonists & inhibitors , Neoplasms/pathology , Neoplastic Stem Cells/drug effects , Signal Transduction/physiologyABSTRACT
Mitogen-activated protein kinases (MAPKs) comprise a family of well-conserved serine/threonine kinases that control a vast array of physiological functions in a number of organisms ranging from yeast to mammals. Recently gene-targeting experiments have shed light on in vivo functions of MAPKs. In particular, embryos deficient in extracellular signal-regulated kinase (ERK) 2 lack mesoderm differentiation and placental angiogenesis. Knockout mice for c-Jun amino-terminal kinases have revealed roles for these kinases in neural apoptosis and activation/differentiation of T cells. Deletion of p38alpha MAPK results in angiogenic defects in the placenta and peripheral vessels. ERK5-deficient embryos are embryonic lethal due to defects in angiogenesis and cardiovascular development. Although these results have provided new insights for MAPK research, development and analysis of conditional knockout mice are required in order to investigate roles of MAPKs, especially, in other biological processes such as disease pathogenesis.
Subject(s)
Gene Targeting , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Animals , Embryo, Mammalian/abnormalities , Embryo, Mammalian/anatomy & histology , Embryo, Mammalian/enzymology , Embryo, Mammalian/physiology , MAP Kinase Signaling System/physiology , Mice , Mice, Knockout , PhenotypeABSTRACT
The extracellular signal-regulated kinase (ERK) is a component of the mitogen-activated protein kinase cascade. Exon 2 of erk2 was deleted by homologous recombination and resulted in embryonic lethality at embryonic day 6.5. erk2 mutant embryos did not form mesoderm and showed increased apoptosis but comparable levels of BrdUrd incorporation, indicating a defect in differentiation. erk2 null embryonic stem (ES) cells exhibited reduced total ERK activity upon serum stimulation, augmented ERK1 phosphorylation, and decreased downstream p90Rsk phosphorylation and activity; yet ES cell proliferation was unaffected. Mutant ES cells were capable of forming mesoderm; however, treatment of mutant ES cells with the mitogen-activated protein kinase kinase inhibitor PD184352 decreased total ERK activity and expression of the mesodermal marker brachyury, suggesting that ERK1 can compensate for ERK2 in vitro. Normal embryos at embryonic day 6.5 expressed activated ERK1/2 in the extraembryonic ectoderm, whereas erk2 mutant embryos had no detectable activated ERK1/2 in this region, suggesting that activated ERK1 was not expressed, and therefore cannot compensate for loss of ERK2 in vivo. These data indicate that ERK2 plays an essential role in mesoderm differentiation during embryonic development.
Subject(s)
Cell Differentiation/physiology , Mesoderm/cytology , Mitogen-Activated Protein Kinase 1/metabolism , Animals , Benzamides/pharmacology , Cell Differentiation/genetics , Cell Division , Cell Line , Enzyme Inhibitors/pharmacology , Female , Heterozygote , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/deficiency , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Stem CellsABSTRACT
Erk5 is a mitogen-activated protein kinase, the biological role of which is largely undefined. Therefore, we deleted the erk5 gene in mice to assess its function in vivo. Inactivation of the erk5 gene resulted in defective blood-vessel and cardiac development leading to embryonic lethality around embryonic days 9.5-10.5. Cardiac development was retarded largely, and the heart failed to undergo normal looping. Endothelial cells that line the developing myocardium of erk5-/- embryos displayed a disorganized, rounded morphology. Vasculogenesis occurred, but extraembryonic and embryonic blood vessels were disorganized and failed to mature. Furthermore, the investment of embryonic blood vessels with smooth muscle cells was attenuated. Together, these data define an essential role for Erk5 in cardiovascular development. Moreover, the inability of Erk5-deficient mice to form a complex vasculature suggests that Erk5 may play an important role in controlling angiogenesis.