ABSTRACT
The cell surface glycoprotein P-cadherin is highly expressed in a number of malignancies, including those arising in the epithelium of the bladder, breast, esophagus, lung, and upper aerodigestive system. PCA062 is a P-cadherin specific antibody-drug conjugate that utilizes the clinically validated SMCC-DM1 linker payload to mediate potent cytotoxicity in cell lines expressing high levels of P-cadherin in vitro, while displaying no specific activity in P-cadherin-negative cell lines. High cell surface P-cadherin is necessary, but not sufficient, to mediate PCA062 cytotoxicity. In vivo, PCA062 demonstrated high serum stability and a potent ability to induce mitotic arrest. In addition, PCA062 was efficacious in clinically relevant models of P-cadherin-expressing cancers, including breast, esophageal, and head and neck. Preclinical non-human primate toxicology studies demonstrated a favorable safety profile that supports clinical development. Genome-wide CRISPR screens reveal that expression of the multidrug-resistant gene ABCC1 and the lysosomal transporter SLC46A3 differentially impact tumor cell sensitivity to PCA062. The preclinical data presented here suggest that PCA062 may have clinical value for treating patients with multiple cancer types including basal-like breast cancer.
Subject(s)
Antineoplastic Agents, Immunological/pharmacology , Biomarkers, Tumor , Cadherins/genetics , Immunoconjugates/pharmacology , Neoplasms/genetics , Amino Acid Sequence , Animals , Antibody-Dependent Cell Cytotoxicity/immunology , Antineoplastic Agents, Immunological/chemistry , Antineoplastic Agents, Immunological/pharmacokinetics , Binding Sites , Cadherins/chemistry , Cadherins/metabolism , Cell Line, Tumor , Disease Models, Animal , Drug Resistance, Neoplasm , Gene Expression , Humans , Immunoconjugates/chemistry , Immunoconjugates/pharmacokinetics , Immunohistochemistry , Macaca fascicularis , Mice , Models, Molecular , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , Protein Binding , Protein Transport , Rats , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
Numerous lines of evidence suggest that the polypeptide hormone prolactin (PRL) may contribute to breast and prostate tumorigenesis through its interactions with the prolactin receptor (PRLR). Here, we describe the biologic properties of LFA102, a humanized neutralizing monoclonal antibody directed against the extracellular domain of PRLR. This antibody was found to effectively antagonize PRL-induced signaling in breast cancer cells in vitro and in vivo and to block PRL-induced proliferation in numerous cell line models, including examples of autocrine/paracrine PRL activity. A single administration of LFA102 resulted in regression of PRL-dependent Nb2-11 tumor xenografts and significantly prolonged time to progression. Finally, LFA102 treatment significantly inhibited PRLR signaling as well as tumor growth in a carcinogen-induced, estrogen receptor-positive rat mammary cancer model as a monotherapy and enhanced the efficacy of the aromatase inhibitor letrozole when administered in combination. The biologic properties of LFA102, elucidated by the preclinical studies presented here, suggest that this antibody has the potential to be a first-in-class, effective therapeutic for the treatment of PRL-dependent cancers.
Subject(s)
Antibodies, Monoclonal, Humanized/administration & dosage , Antibodies, Monoclonal/administration & dosage , Breast Neoplasms/drug therapy , Neoplasms, Hormone-Dependent/drug therapy , Receptors, Prolactin/immunology , Animals , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Proliferation/drug effects , Female , Humans , MCF-7 Cells , Mice , Molecular Targeted Therapy , Neoplasms, Hormone-Dependent/metabolism , Neoplasms, Hormone-Dependent/pathology , Prolactin/metabolism , Rats , Receptors, Prolactin/antagonists & inhibitors , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: Chk1 kinase is a critical regulator of both S and G(2)-M phase cell cycle checkpoints in response to DNA damage. This study aimed to evaluate the biochemical, cellular, and antitumor effects of a novel Chk1 inhibitor, CHIR124. EXPERIMENTAL DESIGN: CHIR-124 was evaluated for its ability to abrogate cell cycle checkpoints, to potentiate cytotoxicity, and to inhibit Chk1-mediated signaling induced by topoisomerase I poisons in human tumor cell line and xenograft models. RESULTS: CHIR-124 is a quinolone-based small molecule that is structurally unrelated to other known inhibitors of Chk1. It potently and selectively inhibits Chk1 in vitro (IC(50) = 0.0003 micromol/L). CHIR-124 interacts synergistically with topoisomerase poisons (e.g., camptothecin or SN-38) in causing growth inhibition in several p53-mutant solid tumor cell lines as determined by isobologram or response surface analysis. CHIR-124 abrogates the SN-38-induced S and G(2)-M checkpoints and potentiates apoptosis in MDA-MD-435 breast cancer cells. The abrogation of the G(2)-M checkpoint and induction of apoptosis by CHIR-124 are enhanced by the loss of p53. We have also shown that CHIR-124 treatment can restore the level of cdc25A protein, which is normally targeted by Chk1 for degradation following DNA damage, indicating that Chk1 signaling is suppressed in the presence of CHIR-124. Finally, in an orthotopic breast cancer xenograft model, CHIR-124 potentiates the growth inhibitory effects of irinotecan by abrogating the G(2)-M checkpoint and increasing tumor apoptosis. CONCLUSIONS: CHIR-124 is a novel and potent Chk1 inhibitor with promising antitumor activities when used in combination with topoisomerase I poisons.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Drug Synergism , Protein Kinases/metabolism , Quinolines/administration & dosage , Quinuclidines/administration & dosage , Topoisomerase I Inhibitors , Animals , Antineoplastic Agents/pharmacology , Apoptosis , Cell Line, Tumor , Checkpoint Kinase 1 , Enzyme Inhibitors/pharmacology , Humans , Inhibitory Concentration 50 , Mice , Mice, SCID , Models, Chemical , Neoplasm Transplantation , Random AllocationABSTRACT
Tezacitabine is a nucleoside analogue characterized by a dual mechanism of action. Following intracellular phosphorylation, the tezacitabine diphosphate irreversibly inhibits ribonucleotide reductase, while the tezacitabine triphosphate can be incorporated into DNA during replication or repair, resulting in DNA chain termination. In the present study we have investigated the effect of the combination of tezacitabine and 5-fluorouracil (5-FU) or 5-fluoro-2'-deoxyuridine (FUdR) on HCT 116 human colon carcinoma cells and xenografts. We used response surface analysis (RSA) and clonogenic assay to evaluate combination effects of tezacitabine and 5-FU. Tezacitabine is antagonistic when combined with 5-FU in the RSA assay and does not effect the clonogenicity of HCT 116 cells when compared with cells treated with 5-FU alone. However, when combined sequentially with FUdR, tezacitabine leads to potentiation of cell killing in the clonogenic assay, additivity in the RSA assay, and increased apoptosis when compared to FUdR alone, suggesting that cytotoxicity of fluoropyrimidines such as FUdR that have more DNA-directed effects can be potentiated by tezacitabine. We also report that oral administration of the fluoropyrimidine capecitabine, an oral prodrug of 5-FU, in combination with tezacitabine shows statistically significant additivity in the HCT 116 xenograft model. This interaction may be explained by the finding that tezacitabine elevates activity of thymidine phosphorylase (TP), the enzyme required for activation of the capecitabine prodrug in tumors. Our results provide evidence that tezacitabine enhances the DNA-directed effects of fluoropyrimidines in human colon cancer cells and may modulate the antitumor activity of fluoropyrimidines.
Subject(s)
Colonic Neoplasms/pathology , DNA/drug effects , Deoxycytidine/analogs & derivatives , Floxuridine/pharmacology , Fluorouracil/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Colonic Neoplasms/enzymology , DNA Damage , Deoxycytidine/pharmacology , Humans , Thymidine Phosphorylase/metabolism , Thymidylate Synthase/metabolism , Transplantation, HeterologousABSTRACT
Chk1 is a key regulator of the S and G2/M checkpoints and is activated following DNA damage by agents such as the topoisomerase I inhibitor camptothecin (CPT). It has been proposed that Chk1 inhibitors used in combination with such a DNA damaging agent to treat tumors would potentiate cytotoxicity and increase the therapeutic index, particularly in tumors lacking functional p53. The aim of this study was to determine whether gene expression analysis could be used to inform lead optimization of a novel series of Chk1 inhibitors. The candidate small-molecule Chk1 inhibitors were used in combination with CPT to identify potential markers of functional Chk1 inhibition, as well as resulting cell cycle progression, using cDNA-based microarrays. Differential expression of several of these putative marker genes was further validated by RT-PCR for use as a medium-throughput assay. In the presence of DNA damage, Chk1 inhibitors altered CPT-dependent effects on the expression of cell cycle and DNA repair genes in a manner consistent with a Chk1-specific mechanism of action. Furthermore, differential expression of selected marker genes, cyclin E2, EGR1, and DDIT3, was dose dependent for Chk1 inhibition. RT-PCR results for these genes following treatment with a panel of Chk1 inhibitors showed a strong correlation between marker gene response and the ability of each compound to abrogate cell cycle arrest in situ following CPT-induced DNA damage. These results demonstrate the utility of global expression analysis to identify surrogate markers, providing an alternative method for rapid compound characterization to support advancement decisions in early drug discovery.
Subject(s)
Cell Cycle/drug effects , Gene Expression Profiling/methods , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , Transcription, Genetic/drug effects , Biomarkers/analysis , Camptothecin/pharmacology , Cell Cycle/genetics , Checkpoint Kinase 1 , DNA Damage/genetics , Dose-Response Relationship, Drug , Humans , Protein Kinase Inhibitors/chemistry , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
PURPOSE: The ectopically expressed and deregulated fibroblast growth factor receptor 3 (FGFR3) results from a t(4;14) chromosomal translocation that occurs in approximately 15% of multiple myeloma (MM) patients and confers a particularly poor prognosis. This study assesses the antimyeloma activity of CHIR-258, a small-molecule inhibitor of multiple receptor tyrosine kinases that is currently in phase I trials, in a newly developed FGFR3-driven preclinical MM animal model. EXPERIMENTAL DESIGN: We developed an orthotopic MM model in mice using a luciferase-expressing human KMS-11-luc line that expresses mutant FGFR3 (Y373C). The antimyeloma activity of CHIR-258 was evaluated at doses that inhibited FGFR3 signaling in vivo in this FGFR3-driven animal model. RESULTS: Noninvasive bioluminescence imaging detected MM lesions in nearly all mice injected with KMS-11-luc cells, which were mainly localized in the spine, skull, and pelvis, resulting in frequent development of paralysis. Daily oral administration of CHIR-258 at doses that inhibited FGFR3 signaling in KMS-11-luc tumors in vivo resulted in a significant inhibition of KMS-11-luc tumor growth, which translated into a significant improvement in animal survival. CONCLUSIONS: Our data provide a relevant preclinical basis for clinical trials of CHIR-258 in FGFR3-positive MM patients.
Subject(s)
Benzimidazoles/pharmacology , Multiple Myeloma/drug therapy , Quinolones/pharmacology , Receptor, Fibroblast Growth Factor, Type 3/antagonists & inhibitors , Receptor, Fibroblast Growth Factor, Type 3/biosynthesis , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Growth Processes/drug effects , Cell Line, Tumor , Enzyme Inhibitors/pharmacology , Humans , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/metabolism , Mice , Mice, SCID , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/metabolism , Multiple Myeloma/enzymology , Phosphorylation/drug effects , Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor, Fibroblast Growth Factor, Type 3/blood , Receptor, Fibroblast Growth Factor, Type 3/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Basic fibroblast growth factor (or FGF-2) has been shown to be a potent stimulator of retinal ganglion cell (RGC) axonal growth during development. Here we investigated if FGF-2 upregulation in adult RGCs promoted axon regrowth in vivo after acute optic nerve injury. Recombinant adeno-associated virus (AAV) was used to deliver the FGF-2 gene to adult RGCs providing a sustained source of this neurotrophic factor. FGF-2 gene transfer led to a 10-fold increase in the number of axons that extended past 0.5 mm from the lesion site compared to control nerves. Detection of AAV-mediated FGF-2 protein in injured RGC axons correlated with growth into the distal optic nerve. The response to FGF-2 upregulation was supported by our finding that FGF receptor-1 (FGFR-1) and heparan sulfate (HS), known to be essential for FGF-2 signaling, were expressed by adult rat RGCs. FGF-2 transgene expression led to only transient protection of injured RGCs. Thus the effect of this neurotrophic factor on axon extension could not be solely attributed to an increase in neuronal survival. Our data indicate that selective upregulation of FGF-2 in adult RGCs stimulates axon regrowth within the optic nerve, an environment that is highly inhibitory for regeneration. These results support the hypothesis that key factors involved in axon outgrowth during neural development may promote regeneration of adult injured neurons.
Subject(s)
Fibroblast Growth Factor 2/genetics , Growth Cones/metabolism , Nerve Regeneration/genetics , Optic Nerve Injuries/therapy , Retina/growth & development , Retinal Ganglion Cells/metabolism , Animals , Cell Differentiation/genetics , Cell Survival/genetics , Disease Models, Animal , Female , Fibroblast Growth Factor 2/physiology , Gene Expression Regulation, Developmental/genetics , Gene Transfer Techniques , Genetic Therapy , Genetic Vectors/genetics , Genetic Vectors/pharmacology , Genetic Vectors/therapeutic use , Growth Cones/ultrastructure , Heparitin Sulfate/metabolism , Nerve Regeneration/drug effects , Optic Nerve Injuries/metabolism , Optic Nerve Injuries/physiopathology , Rats , Rats, Sprague-Dawley , Receptor Protein-Tyrosine Kinases/drug effects , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, Fibroblast Growth Factor, Type 1 , Receptors, Fibroblast Growth Factor/drug effects , Receptors, Fibroblast Growth Factor/metabolism , Retina/cytology , Retina/metabolism , Retinal Ganglion Cells/cytology , Retinal Ganglion Cells/drug effects , Up-Regulation/geneticsABSTRACT
PURPOSE: To develop a small-animal model of choroidal neovascularization (CNV) by injecting adeno-associated virus (AAV)-VEGF into the subretinal space (SRS) of rats. METHODS: An adeno-associated viral vector encoding human VEGF(165) was injected into the subretinal space (SRS) of Sprague-Dawley or Long Evans rats. Expression of VEGF was identified by RT-PCR and immunohistochemistry. Physiological and pathologic changes in the retina and choroid were evaluated by electroretinography, fluorescein angiography, light microscopy, and three-dimensional reconstruction of serial sections. RESULTS: Green fluorescent protein (GFP) and VEGF were expressed for at least 20 months in the retina and retinal pigment epithelium (RPE). Histologic sections showed extensive subretinal neovascularization, degenerating photoreceptors, and proliferating RPE at 5 weeks to 20 months after injection of AAV-VEGF. At 2 to 12 months after injection, leaking blood vessels were detected by fluorescein angiography. Electroretinogram a- and b-wave amplitudes were significantly decreased during this time. Three-dimensional reconstruction of serial sections demonstrated that choroidal blood vessels penetrated Bruch's membrane, one of them splitting into three branches in the SRS. In the current model, CNV was produced in 95% of the animals tested (19/20). It persisted for more than 20 months, a necessary requirement for modeling the development of CNV in age-related macular degeneration (AMD). CONCLUSIONS: In this study, a highly reproducible animal model of long-lasting CNV was developed. This model is being used to test antiangiogenic molecules to reduce or inhibit CNV and could be extended to primates.
Subject(s)
Choroidal Neovascularization/etiology , Choroidal Neovascularization/metabolism , Dependovirus/genetics , Endothelial Growth Factors/biosynthesis , Intercellular Signaling Peptides and Proteins/biosynthesis , Lymphokines/biosynthesis , Animals , Choroidal Neovascularization/pathology , Disease Models, Animal , Electroretinography , Endothelial Growth Factors/genetics , Fluorescein Angiography , Gene Expression , Genetic Vectors , Green Fluorescent Proteins , Immunoenzyme Techniques , Indicators and Reagents/metabolism , Intercellular Signaling Peptides and Proteins/genetics , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Lymphokines/genetics , Rats , Rats, Long-Evans , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Recombinant adeno-associated viral (rAAV) vectors containing an improved tetracycline (tet) system of transcriptional regulation are an efficient strategy for the control of long-term therapeutic gene expression. In vivo studies with the original tet-off and tet-on vectors, while promising, have failed to demonstrate complete repression in the uninduced state. To address this issue, we incorporated the tTS(kid) fusion of the tet repressor and a KRAB-derived transcriptional silencer into the tet-on system in the context of rAAV vectors. The tTS(kid) repressor and rtTA activator were expressed constituitively from a regulator vector, and the repressor and an erythropoietin (Epo) transgene were expressed inducibly via a second vector. Following intramuscular co-injection of these vectors, we observed repeated induction of serum Epo protein following drug administration and undetectable levels after its withdrawal. Four cycles of regulation were achieved over a 32-week period. Thus, the tet-on system plus the tTS(kid) repressor delivered via nonpathogenic rAAV vectors is a powerful tool for controlling the in vivo expression of therapeutic transgenes. In a clinical setting, the repressor could provide a mechanism for abolishing transgene expression if it were no longer needed or if the safety of a patient became compromised.