ABSTRACT
Cell cycle checkpoint intervention is an effective therapeutic strategy for cancer when applied to patients predisposed to respond and the treatment is well-tolerated. A critical cell cycle process that could be targeted is the mitotic checkpoint (spindle assembly checkpoint) which governs the metaphase-to-anaphase transition and insures proper chromosomal segregation. The mitotic checkpoint kinase Mps1 was selected to explore whether enhancement in genomic instability is a viable therapeutic strategy. The basal-a subset of triple-negative breast cancer was chosen as a model system because it has a higher incidence of chromosomal instability and Mps1 expression is up-regulated. Depletion of Mps1 reduces tumor cell viability relative to normal cells. Highly selective, extremely potent Mps1 kinase inhibitors were created to investigate the roles of Mps1 catalytic activity in tumor cells and normal physiology (PF-7006, PF-3837; Ki<0.5 nM; cellular IC50 2-6 nM). Treatment of tumor cells in vitro with PF-7006 modulates expected Mps1-dependent biology as demonstrated by molecular and phenotypic measures (reduced pHH3-Ser10 levels, shorter duration of mitosis, micro-nucleation, and apoptosis). Tumor-bearing mice treated with PF-7006 exhibit tumor growth inhibition concomitant with pharmacodynamic modulation of a downstream biomarker (pHH3-Ser10). Unfortunately, efficacy only occurs at drug exposures that cause dose-limiting body weight loss, gastrointestinal toxicities, and neutropenia. Mps1 inhibitor toxicities may be mitigated by inducing G1 cell cycle arrest in Rb1-competent cells with the cyclin-dependent kinase-4/6 inhibitor palbociclib. Using an isogenic cellular model system, PF-7006 is shown to be selectively cytotoxic to Rb1-deficient cells relative to Rb1-competent cells (also a measure of kinase selectivity). Human bone marrow cells pretreated with palbociclib have decreased PF-7006-dependent apoptosis relative to cells without palbociclib pretreatment. Collectively, this study raises a concern that single agent therapies inhibiting Mps1 will not be well-tolerated clinically but may be when combined with a selective CDK4/6 drug.
Subject(s)
Cell Cycle Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Animals , Apoptosis/drug effects , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Breast Neoplasms/diagnosis , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Cyclin-Dependent Kinase 6/metabolism , Female , G1 Phase Cell Cycle Checkpoints/drug effects , Histones/metabolism , Humans , Intestine, Small/pathology , Mice , Mice, SCID , Mitosis/drug effects , Phosphorylation , Piperazines/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein Kinase Inhibitors/toxicity , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , Pyridines/pharmacology , RNA Interference , RNA, Small Interfering/metabolism , Rats , Transplantation, HeterologousABSTRACT
We report the preclinical evaluation of PF-06463922, a potent and brain-penetrant ALK/ROS1 inhibitor. Compared with other clinically available ALK inhibitors, PF-06463922 displayed superior potency against all known clinically acquired ALK mutations, including the highly resistant G1202R mutant. Furthermore, PF-06463922 treatment led to regression of EML4-ALK-driven brain metastases, leading to prolonged mouse survival, in a superior manner. Finally, PF-06463922 demonstrated high selectivity and safety margins in a variety of preclinical studies. These results suggest that PF-06463922 will be highly effective for the treatment of patients with ALK-driven lung cancers, including those who relapsed on clinically available ALK inhibitors because of secondary ALK kinase domain mutations and/or brain metastases.
Subject(s)
Antineoplastic Agents/administration & dosage , Brain Neoplasms/drug therapy , Brain Neoplasms/secondary , Drug Resistance, Neoplasm/drug effects , Lactams, Macrocyclic/administration & dosage , Neoplasms/drug therapy , Receptor Protein-Tyrosine Kinases/genetics , Aminopyridines , Anaplastic Lymphoma Kinase , Animals , Antineoplastic Agents/pharmacology , Brain Neoplasms/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Lactams , Lactams, Macrocyclic/pharmacology , Mice , Mutation , NIH 3T3 Cells , Neoplasms/genetics , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/pharmacology , Pyrazoles , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Xenograft Model Antitumor AssaysABSTRACT
Oncogenic c-ros oncogene1 (ROS1) fusion kinases have been identified in a variety of human cancers and are attractive targets for cancer therapy. The MET/ALK/ROS1 inhibitor crizotinib (Xalkori, PF-02341066) has demonstrated promising clinical activity in ROS1 fusion-positive non-small cell lung cancer. However, emerging clinical evidence has shown that patients can develop resistance by acquiring secondary point mutations in ROS1 kinase. In this study we characterized the ROS1 activity of PF-06463922, a novel, orally available, CNS-penetrant, ATP-competitive small-molecule inhibitor of ALK/ROS1. In vitro, PF-06463922 exhibited subnanomolar cellular potency against oncogenic ROS1 fusions and inhibited the crizotinib-refractory ROS1(G2032R) mutation and the ROS1(G2026M) gatekeeper mutation. Compared with crizotinib and the second-generation ALK/ROS1 inhibitors ceritinib and alectinib, PF-06463922 showed significantly improved inhibitory activity against ROS1 kinase. A crystal structure of the PF-06463922-ROS1 kinase complex revealed favorable interactions contributing to the high-affinity binding. In vivo, PF-06463922 showed marked antitumor activity in tumor models expressing FIG-ROS1, CD74-ROS1, and the CD74-ROS1(G2032R) mutation. Furthermore, PF-06463922 demonstrated antitumor activity in a genetically engineered mouse model of FIG-ROS1 glioblastoma. Taken together, our results indicate that PF-06463922 has potential for treating ROS1 fusion-positive cancers, including those requiring agents with CNS-penetrating properties, as well as for overcoming crizotinib resistance driven by ROS1 mutation.
Subject(s)
Drug Resistance, Neoplasm/genetics , Lactams, Macrocyclic/pharmacology , Mutation/genetics , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , Pyrazoles/pharmacology , Pyridines/pharmacology , Aminopyridines , Animals , Carcinogenesis/drug effects , Carcinogenesis/pathology , Cell Proliferation/drug effects , Crizotinib , Crystallography, X-Ray , Disease Models, Animal , Drug Resistance, Neoplasm/drug effects , Glioma/pathology , Humans , Lactams , Lactams, Macrocyclic/chemistry , Mice , Models, Molecular , Signal Transduction/drug effectsABSTRACT
Noninvasive imaging has been widely applied for monitoring antiangiogenesis therapy in cancer drug discovery. In this report, we used different imaging modalities including high-frequency ultrasound (HFUS), dynamic contrast enhanced-MR (DCE-MR), and fluorescence molecular tomography (FMT) imaging systems to monitor the changes in the tumor vascular properties after treatment with γ-secretase inhibitor PF-03084014. Sunitinib was tested in parallel for comparison. In the MDA-MB-231Luc model, we demonstrated that antiangiogenesis was one of the contributing mechanisms for the therapeutic effect of PF-03084014. By immunohistochemistry and FITC-lectin perfusion assays, we showed that the vascular defects upon treatment with PF-03084014 were associated with Notch pathway modulation, evidenced by a decrease in the HES1 protein and by the changes in VEGFR2 and HIF1α levels, which indicates down-stream effects. Using a 3D power Doppler scanning method, ultrasound imaging showed that the% vascularity in the MDA-MB-231Luc tumor decreased significantly at 4 and 7 days after the treatment with PF-03084014. A decrease in the tumor vessel function was also observed through contrast-enhanced ultrasound imaging with microbubble injection. These findings were consistent with the PF-03084014-induced functional vessel changes measured by suppressing the K(trans) values using DCE-MRI. In contrast, the FMT imaging with the AngioSence 680EX failed to detect any treatment-associated tumor vascular changes. Sunitinib demonstrated an outcome similar to PF-03084014 in the tested imaging modalities. In summary, ultrasound and DCE-MR imaging successfully provided longitudinal measurement of the phenotypic and functional changes in tumor vasculature after treatment with PF-03084014 and sunitinib.
Subject(s)
Magnetic Resonance Imaging , Neovascularization, Pathologic/diagnostic imaging , Neovascularization, Pathologic/drug therapy , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Angiogenesis Inhibitors/administration & dosage , Cell Line, Tumor , Contrast Media/chemistry , Humans , Indoles/administration & dosage , Molecular Imaging , Neovascularization, Pathologic/pathology , Pyrroles/administration & dosage , Radiography , Sunitinib , Tetrahydronaphthalenes/administration & dosage , Ultrasonography , Valine/administration & dosage , Valine/analogs & derivatives , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Metastatic bladder cancer is a serious condition with a 5-year survival rate of approximately 14 %, a rate that has remained unchanged for almost three decades. Thus, there is a profound need to identify the driving mutations for these aggressive tumors to better determine appropriate treatments. Mutational analyses of clinical samples suggest that mutations in either the phosphoinositide-3 kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) or RAS/MEK/ERK pathways drive bladder cancer progression, although it remains to be tested whether the inhibition of either (or both) of these pathways can arrest PI3K/mTOR- or Ras-driven proliferation. METHODS: Herein, we used several bladder cancer cell lines to determine drug sensitivity according to genetic background and also studied mouse models of engrafted UM-UC-3 cells and patient-derived xenografts (PDXs) to test PI3K/mTOR and MEK inhibition in vivo. RESULTS: Inhibition of these pathways utilizing PF-04691502, a PI3K and mTOR inhibitor, and PD-0325901, a MEK inhibitor, slowed the tumor growth of PDX models of bladder cancer. The growth inhibitory effect of combination therapy was similar to that of the clinical maximum dose of cisplatin; mechanistically, this appeared to predominantly occur via drug-induced cytostatic growth inhibition as well as diminished vascular endothelial growth factor secretion in the tumor models. Kinase arrays of tumors harvested after treatment demonstrated activated p53 and Axl as well as STAT1 and STAT3. CONCLUSION: Taken together, these results indicate that clinically relevant doses of PF-04691502 and PD-0325901 can suppress bladder tumor growth in PDX models, thus offering additional potential treatment options by a precision medicine approach.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , MAP Kinase Kinase Kinases/antagonists & inhibitors , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , Urinary Bladder Neoplasms/drug therapy , Xenograft Model Antitumor Assays/methods , Aged, 80 and over , Animals , Benzamides/administration & dosage , Benzamides/pharmacology , Diphenylamine/administration & dosage , Diphenylamine/analogs & derivatives , Diphenylamine/pharmacology , Disease Models, Animal , Female , Humans , MAP Kinase Kinase Kinases/genetics , MAP Kinase Kinase Kinases/metabolism , MAP Kinase Signaling System/drug effects , Mice , Mice, Inbred NOD , Mice, SCID , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinase Inhibitors/administration & dosage , Pyridones/administration & dosage , Pyridones/pharmacology , Pyrimidines/administration & dosage , Pyrimidines/pharmacology , Random Allocation , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Urinary Bladder Neoplasms/enzymology , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathologyABSTRACT
We performed immunohistochemistry for macrophage colony-stimulating factor 1 receptor (also known as c-fms proto-oncogene product) on tissue microarrays of human nontumor lung, pulmonary squamous cell carcinomas (SCC) and adenocarcinomas (ADC), and breast and ovarian carcinomas using a commercially available anti-cFMS antibody. The specificity of the antibody was validated by Western blot and mass spectrometry analysis. Staining of cFMS was restricted to stromal fibroblasts in pulmonary SCC and ADC specimens and was not identified in tumor epithelium or epithelium and stromal cells of nontumor lung. Evaluation of pulmonary SCC (n=63) and ADC (n=71) specimens revealed stromal fibroblast cFMS staining in 60% (38 of 63) and 35% (25 of 71) of the tumor samples, respectively. A similar pattern of stromal fibroblast cFMS staining was observed in breast (n=21) and ovarian (n=50) carcinomas. It was reported that glucocorticoids induced cFMS expression in breast carcinomas and choriocarcinomas. To investigate whether stromal cFMS expression in lung cancers was associated with glucocorticoid signaling, glucocorticoid receptor protein distribution was evaluated in lung tissue microarrays by immunohistochemistry. Stromal fibroblast glucocorticoid receptor staining was only observed in 18% (2 of 11) of pulmonary SCC and 6% (1 of 17) of ADC specimens, suggesting that cFMS expression may not be directly mediated by glucocorticoids in stromal fibroblasts of lung cancers. The tumor stromal cell expression of cFMS in certain tumor types (lung, ovarian, and breast) suggests the potential for more diverse tumor therapeutic options and presents an attractive target for drug development.
Subject(s)
Adenocarcinoma/metabolism , Breast Neoplasms/metabolism , Carcinoma, Squamous Cell/metabolism , Fibroblasts/metabolism , Lung Neoplasms/metabolism , Lung/metabolism , Ovarian Neoplasms/metabolism , Receptor, Macrophage Colony-Stimulating Factor/metabolism , Adenocarcinoma/pathology , Aged , Breast Neoplasms/pathology , Carcinoma, Squamous Cell/pathology , Female , Humans , Immunohistochemistry , Lung/pathology , Lung Neoplasms/pathology , Male , Molecular Targeted Therapy , Ovarian Neoplasms/pathology , Proto-Oncogene Mas , Tissue Array AnalysisABSTRACT
The identification and quantitative evaluation of lung tumors in mouse models is challenging and an unmet need in preclinical arena. In this study, we developed a noninvasive contrast-enhanced microCT (µCT) method to longitudinally evaluate and quantitate lung tumors in mice. Commercially available µCT contrast agents were compared to determine the optimal agent for visualization of thoracic blood vessels and lung tumors in naïve mice and in non-small-cell lung cancer models. Compared with the saline control, iopamidol and iodinated lipid agents provided only marginal increases in contrast resolution. The inorganic nanoparticulate agent provided the best contrast and visualization of thoracic vascular structures; the density contrast was highest at 15 min after injection and was stable for more than 4 h. Differential contrast of the tumors, vascular structures, and thoracic air space by the nanoparticulate agent enabled identification of tumor margins and accurate quantification. µCT data correlated closely with traditional histologic measurements (Pearson correlation coefficient, 0.995). Treatment of ELM4-ALK mice with crizotinib yielded 65% reduction in tumor size and thus demonstrated the utility of quantitative µCT in longitudinal preclinical trials. Overall and among the 3 agents we tested, the inorganic nanoparticulate product was the best commercially available contrast agent for visualization of thoracic blood vessels and lung tumors. Contrast-enhanced µCT imaging is an excellent noninvasive method for longitudinal evaluation during preclinical lung tumor studies.
Subject(s)
Contrast Media , Disease Models, Animal , Lung Neoplasms/diagnostic imaging , X-Ray Microtomography/methods , Animals , Lung Neoplasms/pathology , Mice , Mice, Inbred C57BLABSTRACT
PIK3CA (phosphoinositide-3-kinase, catalytic, alpha polypeptide) mutations can help predict the antitumor activity of phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway inhibitors in both preclinical and clinical settings. In light of the recent discovery of tumor-initiating cancer stem cells (CSCs) in various tumor types, we developed an in vitro CSC model from xenograft tumors established in mice from a colorectal cancer patient tumor in which the CD133+/EpCAM+ population represented tumor-initiating cells. CD133+/EpCAM+ CSCs were enriched under stem cell culture conditions and formed 3-dimensional tumor spheroids. Tumor spheroid cells exhibited CSC properties, including the capability for differentiation and self-renewal, higher tumorigenic potential and chemo-resistance. Genetic analysis using an OncoCarta™ panel revealed a PIK3CA (H1047R) mutation in these cells. Using a dual PI3K/mTOR inhibitor, PF-04691502, we then showed that blockage of the PI3K/mTOR pathway inhibited the in vitro proliferation of CSCs and in vivo xenograft tumor growth with manageable toxicity. Tumor growth inhibition in mice was accompanied by a significant reduction of phosphorylated Akt (pAKT) (S473), a well-established surrogate biomarker of PI3K/mTOR signaling pathway inhibition. Collectively, our data suggest that PF-04691502 exhibits potent anticancer activity in colorectal cancer by targeting both PIK3CA (H1047R) mutant CSCs and their derivatives. These results may assist in the clinical development of PF-04691502 for the treatment of a subpopulation of colorectal cancer patients with poor outcomes.
Subject(s)
Antineoplastic Agents/pharmacology , Class I Phosphatidylinositol 3-Kinases/genetics , Colorectal Neoplasms/drug therapy , Mutation , Neoplastic Stem Cells/drug effects , Pyridones/pharmacology , Pyrimidines/pharmacology , Administration, Oral , Adult , Animals , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cell Proliferation/drug effects , Cells, Cultured , Class I Phosphatidylinositol 3-Kinases/metabolism , Colorectal Neoplasms/enzymology , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Humans , Male , Mice, SCID , Neoplastic Stem Cells/enzymology , Neoplastic Stem Cells/pathology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolism , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: We aimed to assess the biologic activity of PF-03084014 in breast xenograft models. The biomarkers for mechanism and patient stratification were also explored. EXPERIMENTAL DESIGN: The in vitro and in vivo properties of PF-03084014 were investigated. The mRNA expressions of 40 key Notch pathway genes at baseline or after treatment were analyzed to link with the antitumor efficacy of PF-03084014 in a panel of breast cancer xenograft models. RESULTS: In vitro, PF-03084014 exhibited activity against tumor cell migration, endothelial cell tube formation, and mammosphere formation. In vivo, we observed apoptosis, antiproliferation, reduced tumor cell self-renewal ability, impaired tumor vasculature, and decreased metastasis activity after the treatment of PF-03084014. PF-03084014 treatment displayed significant antitumor activity in 10 of the 18 breast xenograft models. However, the antitumor efficacy in most models did not correlate with the in vitro antiproliferation results in the corresponding cell lines, suggesting the critical involvement of tumor microenvironment during Notch activation. In the tested breast xenograft models, the baseline expressions of the Notch receptors, ligands, and the cleaved Notch1 failed to predict the antitumor response to PF-03084014, whereas several Notch pathway target genes, including HEY2, HES4, and HES3, strongly corresponded with the response with a P value less than 0.01. Many of the best molecular predictors of response were also significantly modulated following PF-03084014 treatment. CONCLUSIONS: PF-03084014 showed antitumor and antimetastatic properties via pleiotropic mechanisms. The Notch pathway downstream genes may be used to predict the antitumor activity of PF-03084014 and enrich for responders among breast cancer patients.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Biomarkers, Tumor/metabolism , Breast Neoplasms/metabolism , Tetrahydronaphthalenes/pharmacology , Valine/analogs & derivatives , Amyloid Precursor Protein Secretases/metabolism , Antineoplastic Agents/administration & dosage , Biomarkers, Tumor/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Cell Line, Tumor , Cluster Analysis , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Neoplasm Metastasis/drug therapy , Receptors, Notch/metabolism , Signal Transduction/drug effects , Tetrahydronaphthalenes/administration & dosage , Valine/administration & dosage , Valine/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
The expression of endogenous retrotransposable elements, including long interspersed nuclear element 1 (LINE-1 or L1) and human endogenous retrovirus, accompanies neoplastic transformation and infection with viruses such as HIV. The ability to engender immunity safely against such self-antigens would facilitate the development of novel vaccines and immunotherapies. In this article, we address the safety and immunogenicity of vaccination with these elements. We used immunohistochemical analysis and literature precedent to identify potential off-target tissues in humans and establish their translatability in preclinical species to guide safety assessments. Immunization of mice with murine L1 open reading frame 2 induced strong CD8 T cell responses without detectable tissue damage. Similarly, immunization of rhesus macaques with human LINE-1 open reading frame 2 (96% identity with macaque), as well as simian endogenous retrovirus-K Gag and Env, induced polyfunctional T cell responses to all Ags, and Ab responses to simian endogenous retrovirus-K Env. There were no adverse safety or pathological findings related to vaccination. These studies provide the first evidence, to our knowledge, that immune responses can be induced safely against this class of self-antigens and pave the way for investigation of them as HIV- or tumor-associated targets.
Subject(s)
AIDS Vaccines/administration & dosage , AIDS Vaccines/immunology , Cancer Vaccines/administration & dosage , Cancer Vaccines/immunology , DNA Transposable Elements/immunology , Endogenous Retroviruses/immunology , AIDS Vaccines/genetics , Adult , Amino Acid Sequence , Animals , Cancer Vaccines/genetics , DNA Transposable Elements/genetics , Disease Models, Animal , Endogenous Retroviruses/genetics , Endogenous Retroviruses/metabolism , Female , Humans , Macaca mulatta , Male , Mice , Mice, Inbred BALB C , Molecular Sequence Data , env Gene Products, Human Immunodeficiency Virus/genetics , env Gene Products, Human Immunodeficiency Virus/immunology , gag Gene Products, Human Immunodeficiency Virus/genetics , gag Gene Products, Human Immunodeficiency Virus/immunologyABSTRACT
The primary target organ for uptake of systemically administered phosphorothioate oligonucleotides is the kidney cortex and the proximal tubular epithelium in particular. To determine the effect of oligonucleotide uptake on renal function, a detailed renal physiology study was performed in cynomolgus monkeys treated with 10-40 mg/kg/week ISIS 113715 for 4 weeks. The concentrations of oligonucleotide in the kidney cortex ranged from 1400 to 2600 µg/g. These concentrations were associated with histologic changes in proximal tubular epithelial cells that ranged from the appearance of cytoplasmic basophilic granules to atrophic and degenerative changes at higher concentrations. However, there were no renal functional abnormalities as determined by the typical measurements of blood urea nitrogen, serum creatinine, creatinine clearance, or urine specific gravity. Nor were there changes in glomerular filtration rate, or renal blood flow. Specific urinary markers of tubular epithelial cell damage, such as N-acetyl-glucosaminidase, and α-glutathione-s-transferase were not affected. Tubular function was further evaluated by monitoring the urinary excretion of amino acids, ß(2)-microglobulin, or glucose. Renal function was challenged by administering a glucose load and by examining concentrating ability after a 4-h water deprivation. Neither challenge produced any evidence of change in renal function. The only change observed was a low incidence of increased urine protein/creatinine ratio in monkeys treated with ≥40 mg/kg/week which was rapidly reversible. Collectively, these data indicate that ISIS 113715-uptake by the proximal tubular epithelium has little or no effect on renal function at concentrations of 2600 µg/g.
Subject(s)
Epithelial Cells/drug effects , Kidney Cortex/metabolism , Kidney Tubules, Proximal/pathology , Oligoribonucleotides/pharmacokinetics , Animals , Blood Urea Nitrogen , Creatinine/blood , Creatinine/urine , Cytoplasm/metabolism , Dose-Response Relationship, Drug , Epithelial Cells/metabolism , Epithelial Cells/pathology , Glucose/metabolism , Kidney Function Tests , Kidney Tubules, Proximal/cytology , Kidney Tubules, Proximal/drug effects , Macaca fascicularis , Male , Oligoribonucleotides/administration & dosage , Oligoribonucleotides/toxicity , Proteins/metabolism , Tissue DistributionABSTRACT
The role of PI3K and MAPK pathways in tumor initiation and progression is well established; hence, several inhibitors of these pathways are currently in different stages of clinical trials. Recent studies identified a PI3K/mTOR (PF-04691502) and a MEK inhibitor (PD-0325901) with strong potency and efficacy in different cell lines and tumor models. PD-0325901, however, showed adverse effects when administered at or above MTD (maximum tolerated dose) in the clinic. Here, we show in preclinical models that PD-0325901 at doses well below MTD (sub-MTD 1.5 mg/kg SID) is still a potent compound as single agent or in combination with PF-04691502. We first observed that PD-0325901 at 1.5 mg/kg SID and in combination with PF-04691502 (7.5 mg/kg; SID) significantly inhibited growth of H460 (carry Kras and PIK3CA mutations) orthotopic lung tumors. Additionally, we tested efficacy of PD-0325901 in Kras(G12D-LSL) conditional GEMMs (genetically engineered mouse models) which are a valuable tool in translational research to study tumor progression. Intranasal delivery of adenoviruses expressing Cre recombinase (Adeno-Cre) resulted in expression of mutant Kras leading to development of tumor lesions in lungs including adenomatous hyperplasia, large adenoma, and adenocarcinoma. Similar to H460 tumors, PD-0325901 as single agent or in combination with PF-04691502 significantly inhibited growth of tumor lesions in lungs in Kras(G12D-LSL) mice when treatment started at adenocarcinoma stage (at 14 weeks post-Adeno-Cre inhalation). In addition, immunohistochemistry showed inhibition of pS6 (phosphorylated ribosomal S6) in the treated animals particularly in the combination group providing a proof of mechanism for tumor growth inhibition. Finally, m-CT imaging in live Kras(G12D-LSL) mice showed reduction of tumor burdens in PD-0325901-treated animals at sub-MTD dose. In conclusion, our data suggest that PD-0325901 at doses below MTD is still a potent compound capable of tumor growth inhibition where Kras and/or PI3K are drivers of tumor growth and progression.
Subject(s)
Adenocarcinoma/drug therapy , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Lung Neoplasms/drug therapy , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins p21(ras)/genetics , TOR Serine-Threonine Kinases/antagonists & inhibitors , Adenocarcinoma/enzymology , Adenocarcinoma of Lung , Animals , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Benzamides/administration & dosage , Cell Line, Tumor , Diphenylamine/administration & dosage , Diphenylamine/analogs & derivatives , Dose-Response Relationship, Drug , Heterozygote , Humans , Lung Neoplasms/enzymology , Maximum Tolerated Dose , Mice , Mice, Mutant Strains , Neoplasm Transplantation , Pyridones/administration & dosage , Pyrimidines/administration & dosageABSTRACT
Osteopontin (OPN), also known as SPP1 (secreted phosphoprotein), is an integrin binding glyco-phosphoprotein produced by a variety of tissues. In cancer patients expression of OPN has been associated with poor prognosis in several tumor types including breast, lung, and colorectal cancers. Despite wide expression in tumor cells and stroma, there is limited evidence supporting role of OPN in tumor progression and metastasis. Using phage display technology we identified a high affinity anti-OPN monoclonal antibody (hereafter AOM1). The binding site for AOM1 was identified as SVVYGLRSKS sequence which is immediately adjacent to the RGD motif and also spans the thrombin cleavage site of the human OPN. AOM1 efficiently inhibited OPNa binding to recombinant integrin αvß3 with an IC50 of 65 nM. Due to its unique binding site, AOM1 is capable of inhibiting OPN cleavage by thrombin which has been shown to produce an OPN fragment that is biologically more active than the full length OPN. Screening of human cell lines identified tumor cells with increased expression of OPN receptors (αvß3 and CD44v6) such as mesothelioma, hepatocellular carcinoma, breast, and non-small cell lung adenocarcinoma (NSCLC). CD44v6 and αvß3 were also found to be highly enriched in the monocyte, but not lymphocyte, subset of human peripheral blood mononuclear cells (hPBMCs). In vitro, OPNa induced migration of both tumor and hPBMCs in a transwell migration assay. AOM1 significantly blocked cell migration further validating its specificity for the ligand. OPN was found to be enriched in mouse plasma in a number of pre-clinical tumor model of non-small cell lung cancers. To assess the role of OPN in tumor growth and metastasis and to evaluate a potential therapeutic indication for AOM1, we employed a Kras(G12D-LSL)p53(fl/fl) subcutaneously implanted in vivo model of NSCLC which possesses a high capacity to metastasize into the lung. Our data indicated that treatment of tumor bearing mice with AOM1 as a single agent or in combination with Carboplatin significantly inhibited growth of large metastatic tumors in the lung further supporting a role for OPN in tumor metastasis and progression.
Subject(s)
Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/secondary , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Osteopontin/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Chemotactic Factors/chemistry , Chemotactic Factors/immunology , Chemotactic Factors/metabolism , Disease Models, Animal , Humans , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/metabolism , Integrins/metabolism , Mice , Mice, SCID , Molecular Sequence Data , Osteopontin/chemistry , Osteopontin/immunology , Protein Binding , Protein Stability , Thrombin/metabolismABSTRACT
Recent studies in several tumor models indicated that treatment with angiogenic inhibitors may trigger induction of metastasis to other organs. Here we investigated modes of resistance and invasion in several tumor cell lines including 4T1 (breast), H460 (lung) and Colo205 (colorectal) using sunitinib at doses comparable to clinically utilized regimen. In comparison with vehicle-treated tumors, sunitinib increased metastasis to lung in 4T1 tumors and to peritoneal lymph node in Colo205 tumors. However, the same treatment did not induce invasiveness in H460 tumors, further suggesting that accelerating metastasis during treatment with angiogenic inhibitors is tumor cell-type dependent. Interestingly, Crizotinib (a dual inhibitor of c-Met and ALK pathways) as single agent or in combination with sunitinib reduced metastasis in all models tested suggesting a role for c-Met/HGF pathway in intrinsic- or sunitinib-induced-metastasis. Moreover, ELISA data showed that while c-Met is highly enriched in tumor cells, HGF is secreted mainly by the stroma (mouse HGF) suggesting a paracrine fashion for c-Met pathway activation in the tumors. In conclusion, our findings indicate that sunitinib-induced metastasis is tumor cell-type dependent and further supports a rationale for combination of anti-angiogenics and c-Met inhibition in the clinic.
Subject(s)
Angiogenesis Inhibitors/adverse effects , Breast Neoplasms/pathology , Colorectal Neoplasms/pathology , Hepatocyte Growth Factor/metabolism , Indoles/adverse effects , Lung Neoplasms/secondary , Proto-Oncogene Proteins c-met/metabolism , Pyrroles/adverse effects , Angiogenesis Inhibitors/therapeutic use , Animals , Breast Neoplasms/blood supply , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Cell Line, Tumor , Colorectal Neoplasms/blood supply , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/metabolism , Enzyme-Linked Immunosorbent Assay , Humans , Indoles/therapeutic use , Lung Neoplasms/metabolism , Lymphatic Metastasis , Mice , Mice, Nude , Neoplasm Metastasis , Neovascularization, Pathologic/drug therapy , Pyrroles/therapeutic use , Signal Transduction/drug effects , SunitinibABSTRACT
PURPOSE: [(18)F]FLT (3'-Fluoro-3' deoxythymidine)-PET imaging was proposed as a tool for measuring in vivo tumor cell proliferation. The aim of this article was to validate the use of [(18)F]FLT-PET imaging for measuring xenograft proliferation and subsequent monitoring of targeted therapy. EXPERIMENTAL DESIGN: In exponentially growing xenografts, factors that could impact the outcome of [(18)F]FLT-PET imaging, such as nucleoside transporters, thymidine kinase 1, the relative contribution of DNA salvage pathway, and the ratio of FLT to thymidine, were evaluated. The [(18)F]FLT tracer avidity was compared with other proliferation markers. RESULTS: In a panel of proliferating xenografts, [(18)F]FLT or [(3)H]thymidine tracer avidity failed to reflect the tumor growth rate across different tumor types, despite the high expressions of Ki67 and TK1. When FLT was injected at the same dose level as used in the preclinical [(18)F]FLT-PET imaging, the plasma exposure ratio of FLT to thymidine was approximately 1:200. Thymidine levels in different tumor types seemed to be variable and exhibited an inverse relationship with the FLT tracer avidity. In contrast, high-dose administration of bromdeoxyuridine (BrdUrd; 50 mg/kg) yielded a plasma exposure of more than 4-fold higher than thymidine and leads to a strong correlation between the BrdUrd uptake and the tumor proliferation rate. In FLT tracer-avid models, [(18)F]FLT-PET imaging as a surrogate biomarker predicted the therapeutic response of CDK4/6 inhibitor PD-0332991. CONCLUSIONS: Tumor thymidine level is one of the factors that impact the correlation between [(18)F]FLT uptake and tumor cell proliferation. With careful validation, [(18)F]FLT-PET imaging can be used to monitor antiproliferative therapies in tracer-avid malignancies.
Subject(s)
Dideoxynucleosides , Neoplasms/diagnostic imaging , Positron-Emission Tomography , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacology , Bromodeoxyuridine/metabolism , Cell Cycle Checkpoints/drug effects , Cell Proliferation/drug effects , Cyclin-Dependent Kinases/antagonists & inhibitors , Dideoxynucleosides/pharmacokinetics , Disease Models, Animal , Humans , Mice , Mice, Nude , Mice, SCID , Neoplasms/drug therapy , Neoplasms/metabolism , Piperazines/administration & dosage , Piperazines/pharmacology , Pyridines/administration & dosage , Pyridines/pharmacology , Thymidine/metabolism , Xenograft Model Antitumor AssaysABSTRACT
OBJECTIVES: To evaluate the safety and persistence of injectable polymethylmethacrylate (PMMA) microspheres as a long-lasting urethral bulking agent in pigs. PMMA microspheres of 2 different diameters (40 and 125 µm) were tested to investigate the potential for migration and dislocation after injection. A similar product containing 40-µm PMMA microspheres has been used as an injectable wrinkle filler for >25 years and received Food and Drug Administration approval in 2006 (ArteFill). METHODS: A total of 22 female pigs received 4 submucosal implantations of PMMA microspheres, using either a cystoscope or a newly developed urethral injection device (UroScope). At death and necropsy at 8 days and 1, 3, and 6 months, the urethral injection site, liver, lung, spleen, and pelvic and iliac lymph nodes were processed for histologic examination and microsphere count using organ dissolution and microscopy. RESULTS: All injected submucosal blebs were still present at 6 months and showed no signs of inflammation. Tissue dissolution of the local lymph nodes and major organs demonstrated the transport of some of the 40-µm microspheres to the local lymph nodes and lung but not to the liver or spleen. In contrast, no 125-µm microspheres were detected in any distant organ. CONCLUSIONS: The submucosal implantation of 125-µm PMMA microspheres into the urethra provided a safe and persistent bulking effect in pigs. The positive results of the present study encourage additional investigation of 125-µm PMMA microspheres as a long-lasting bulking agent for the treatment of female stress urinary incontinence. Furthermore, a newly developed urethral injection device (UroScope) proved beneficial and cost-effective to facilitate the transurethral injections of 125-µm PMMA microspheres.
Subject(s)
Microspheres , Polymethyl Methacrylate/therapeutic use , Urinary Incontinence, Stress/therapy , Animals , Biocompatible Materials , Female , Foreign-Body Reaction/pathology , Injections , Materials Testing , Particle Size , Polymethyl Methacrylate/administration & dosage , Swine , Swine, MiniatureABSTRACT
Flow cytometry has many applications in clinical medicine allowing rapid and highly specific characterization of cells in solution (e.g., peripheral blood) or from dissociated tissues. The data generated from these analyses may be used to diagnose and monitor progression of disease as well as aid in prognostication of selected pathologic processes. In recent years, flow cytometric techniques have established a foothold in preclinical drug development providing an ability to identify and characterize both cell morphology and function, as well as to more clearly assign observed alterations in one or more cell attributes as intended or unexpected effects of new biopharmaceutical entities. The inclusion of flow cytometric evaluation assays (some described in this chapter) during preclinical drug development has increased and enhanced the detail of data generated to support the safety and efficacy of new biopharmaceuticals. Flow cytometry analyses used in preclinical drug development that are described in this chapter include immunophenotyping, peripheral blood cross-reactivity, binding activity and stability and cell receptor dynamics.
Subject(s)
Drug Design , Drug Evaluation, Preclinical/methods , Flow Cytometry/methods , Immunophenotyping/methods , Animals , Antibodies/immunology , Antibody Specificity , Antigens/immunology , Biomarkers/metabolism , Cell Line , Flow Cytometry/standards , Humans , Reproducibility of ResultsABSTRACT
OBJECTIVE: To identify and characterize a fully human antibody directed against B lymphocyte stimulator (BLyS), a tumor necrosis factor-related cytokine that plays a critical role in the regulation of B cell maturation and development. Elevated levels of BLyS have been implicated in the pathogenesis of autoimmune diseases. METHODS: A human phage display library was screened for antibodies against human BLyS. A human monoclonal antibody, LymphoStat-B, specific for human BLyS was obtained from the library screening and subsequent affinity optimization mutagenesis. The antibody was tested for inhibition of human BLyS in vitro and in an in vivo murine model. Additionally, the consequences of BLyS inhibition were tested in vivo by administration of LymphoStat-B to cynomolgus monkeys. RESULTS: LymphoStat-B bound with high affinity to human BLyS and inhibited the binding of BLyS to its 3 receptors, TACI, BCMA, and BLyS receptor 3/BAFF-R. LymphoStat-B potently inhibited BLyS-induced proliferation of B cells in vitro, and administration of LymphoStat-B to mice prevented human BLyS-induced increases in splenic B cell numbers and IgA titers. In cynomolgus monkeys, administration of LymphoStat-B resulted in decreased B cell representation in both spleen and mesenteric lymph nodes. CONCLUSION: A fully human monoclonal antibody has been isolated that binds to BLyS with high affinity and neutralizes human BLyS bioactivity in vitro and in vivo. Administration of this antibody to cynomolgus monkeys resulted in B cell depletion in spleen and lymph node. This antibody may prove therapeutically useful in the treatment of autoimmune diseases in humans.