ABSTRACT
Most drugs used to treat viral disease target a virus-coded product. They inhibit a single virus or virus family, and the pathogen can readily evolve resistance. Host-targeted antivirals can overcome these limitations. The broad-spectrum activity achieved by host targeting can be especially useful in combating emerging viruses and for treatment of diseases caused by multiple viral pathogens, such as opportunistic agents in immunosuppressed patients. We have developed a family of compounds that modulate sirtuin 2, an NAD+-dependent deacylase, and now report the properties of a member of that family, FLS-359. Biochemical and x-ray structural studies show that the drug binds to sirtuin 2 and allosterically inhibits its deacetylase activity. FLS-359 inhibits the growth of RNA and DNA viruses, including members of the coronavirus, orthomyxovirus, flavivirus, hepadnavirus, and herpesvirus families. FLS-359 acts at multiple levels to antagonize cytomegalovirus replication in fibroblasts, causing modest reductions in viral RNAs and DNA, together with a much greater reduction in infectious progeny, and it exhibits antiviral activity in humanized mouse models of infection. Our results highlight the potential of sirtuin 2 inhibitors as broad-spectrum antivirals and set the stage for further understanding of how host epigenetic mechanisms impact the growth and spread of viral pathogens.
Subject(s)
Coronavirus Infections , Coronavirus , Animals , Mice , Antiviral Agents/pharmacology , Sirtuin 2/genetics , RNA, ViralABSTRACT
Reprogramming tumor infiltrating myeloid cells to elicit pro-inflammatory responses is an exciting therapeutic maneouver to improve anti-tumor responses. We recently demonstrated that a distinct microtubule-targeting drug, plinabulin-a clinical-stage novel agent-modulates dendritic cell maturation and enhances anti-tumor immunity. Here, we investigated the effects of plinabulin on macrophage polarization in vitro and in vivo. Plinabulin monotherapy induced significant tumor growth inhibition in mice bearing subcutaneous MC38 colon cancer. Importantly, the regressing tumors were characterized by an increase in M1-like/M2-like tumor-associated macrophages (TAM) ratio. The efficacy of plinabulin remained unaltered in T cell-deficient Rag2-/- mice, suggesting an important role of macrophages in driving the drug's anti-tumor effect. Exposure of murine and healthy human macrophages to plinabulin induced polarization toward the M1 phenotype, including increased expression of co-stimulatory molecules CD80, CD86 and pro-inflammatory cytokines IL-1ß, IL-6, and IL-12. M2-associated immunosuppressive cytokines IL-10 and IL-4 were reduced. This pro-inflammatory M1-like skewing of TAMs in response to plinabulin was dependent on the JNK pathway. Functionally, plinabulin-polarized human M1 macrophages directly killed HuT 78 tumor cells in vitro. Importantly, plinabulin induced a functional M1-like polarization of tumor infiltrating macrophages in murine tumors as well as in tumor samples from ovarian cancer patients, by preferentially triggering M1 proliferation. Our study uncovers a novel immunomodulatory effect of plinabulin in directly triggering M1 polarization and proliferation as well as promoting TAM anti-tumoral effector functions.
ABSTRACT
PURPOSE: Chemotherapy-induced neutropenia (CIN) increases the risk of infections and mortality in cancer patients. G-CSF therapies are approved for the treatment of CIN, but non-G-CSF therapies are needed to increase efficacy and minimize side effects. Plinabulin is an inhibitor of tubulin polymerization that ameliorates CIN caused in patients by the microtubule stabilizer docetaxel. The present study evaluates the potential of plinabulin to reduce neutropenia induced by chemotherapies of different classes in a manner not dependent on increasing G-CSF. METHODS: The anti-CIN benefits of plinabulin were tested in rodents co-treated with docetaxel, cyclophosphamide or doxorubicin. Effects on G-CSF levels were evaluated in tissues by immunoassay. Flow cytometry was utilized to test treatment effects on femur bone marrow cell counts from immunocompetent mice-bearing orthotopic 4T1 breast cancer tumors. RESULTS: Plinabulin alleviated neutropenia induced by microtubule stabilizing, DNA cross-linking and DNA intercalating chemotherapies, yet did not affect bone marrow or blood G-CSF levels. The number of lineage-/Sca1+/c-Kit+ (LSK) hematopoietic stem/progenitor cells (HSPC) in murine bone marrow collected 2 days after treatment was not affected by docetaxel monotherapy despite increased plasma G-CSF in this group. LSK cell number was, however, increased when plinabulin was combined with docetaxel, without affecting G-CSF. CONCLUSIONS: Results support the clinical testing of plinabulin as a non-G-CSF-based treatment for CIN associated with chemotherapies of different mechanisms. Results also support HSPC as a focal point for future mechanism-of-action work aimed at understanding the ability of plinabulin to reduce this serious side effect of cytotoxic therapy in cancer patients.
Subject(s)
Antineoplastic Agents/adverse effects , Diketopiperazines/pharmacology , Granulocyte Colony-Stimulating Factor/pharmacology , Neutropenia/chemically induced , Neutropenia/drug therapy , Animals , Antineoplastic Agents/pharmacology , Bone Marrow/drug effects , Breast Neoplasms/drug therapy , Cell Line, Tumor , Cyclophosphamide/pharmacology , Docetaxel/pharmacology , Doxorubicin/pharmacology , Female , Male , Mice , Rats , Rats, Sprague-DawleyABSTRACT
We have previously identified the interaction between mammalian V-ATPase a2-subunit isoform and cytohesin-2 (CTH2) and studied molecular details of binding between these proteins. In particular, we found that six peptides derived from the N-terminal cytosolic domain of a2 subunit (a2N1-402) are involved in interaction with CTH2 (Merkulova, Bakulina, Thaker, Grüber, & Marshansky, 2010). However, the actual 3D binding interface was not determined in that study due to the lack of high-resolution structural information about a-subunits of V-ATPase. Here, using a combination of homology modeling and NMR analysis, we generated the structural model of complete a2N1-402 and uncovered the CTH2-binding interface. First, using the crystal-structure of the bacterial M. rubber Icyt-subunit of A-ATPase as a template (Srinivasan, Vyas, Baker, & Quiocho, 2011), we built a homology model of mammalian a2N1-352 fragment. Next, we combined it with the determined NMR structures of peptides a2N368-395 and a2N386-402 of the C-terminal section of a2N1-402. The complete molecular model of a2N1-402 revealed that six CTH2 interacting peptides are clustered in the distal and proximal lobe sub-domains of a2N1-402. Our data indicate that the proximal lobe sub-domain is the major interacting site with the Sec7 domain of first CTH2 protein, while the distal lobe sub-domain of a2N1-402 interacts with the PH-domain of second CTH2. Indeed, using Sec7/Arf-GEF activity assay we experimentally confirmed our model. The interface formed by peptides a2N1-17 and a2N35-49 is involved in specific interaction with Sec7 domain and regulation of GEF activity. These data are critical for understanding of the cross-talk between V-ATPase and CTH2 as well as for the rational drug design to regulate their function.
Subject(s)
Drug Design , GTPase-Activating Proteins/chemistry , GTPase-Activating Proteins/metabolism , Monomeric GTP-Binding Proteins/metabolism , Vacuolar Proton-Translocating ATPases/metabolism , Amino Acid Sequence , Animals , Bacteria , Binding Sites , Mice , Models, Molecular , Protein Binding , Protein Conformation , Protein Subunits/chemistry , Protein Subunits/metabolismABSTRACT
Parathyroid hormone (PTH) affects the skeleton by acting on osteocytes (Ots) in bone through yet unclear mechanisms. We report that matrix metalloproteinase 14 (MMP14) expression/activity are increased in bones from mice with genetic constitutive activation (ca) of the PTH receptor 1 (PTH1R) in Ots (caPTH1ROt) and in bones from mice exposed to elevated PTH levels but not in mice lacking [conditional knockout (cKO)] the PTH1R in Ots (cKOPTH1ROt). Furthermore, PTH upregulates MMP14 in human bone cultures and in Ot-enriched bones from floxed control mice but not from cKOPTH1ROt mice. MMP14 activity increases soluble receptor activator of NF-κΒ ligand production, which in turn, stimulates osteoclast differentiation and resorption. Pharmacologic inhibition of MMP14 activity reduced the high bone remodeling exhibited by caPTH1ROt mice or induced by chronic PTH elevation and decreased bone resorption but allowed full stimulation of bone formation induced by PTH injections, thereby potentiating bone gain. Thus, MMP14 is a new member of the intricate gene network activated in Ots by PTH1R signaling that can be targeted to adjust the skeletal responses to PTH in favor of bone preservation.-Delgado-Calle, J., Hancock, B., Likine, E. F., Sato, A. Y., McAndrews, K., Sanudo, C., Bruzzaniti, A., Riancho, J. A., Tonra, J. R., Bellido, T. MMP14 is a novel target of PTH signaling in osteocytes that controls resorption by regulating soluble RANKL production.
Subject(s)
Bone Resorption/metabolism , Matrix Metalloproteinase 14/metabolism , Osteocytes/metabolism , Parathyroid Hormone/metabolism , RANK Ligand/biosynthesis , Signal Transduction/physiology , Animals , Bone Resorption/genetics , Cells, Cultured , Gene Regulatory Networks/physiology , Matrix Metalloproteinase 14/genetics , Mice , Mice, Knockout , Osteoclasts/cytology , Osteoclasts/metabolism , Osteocytes/cytology , Osteogenesis/physiology , Parathyroid Hormone/genetics , RANK Ligand/genetics , Receptor, Parathyroid Hormone, Type 1/genetics , Receptor, Parathyroid Hormone, Type 1/metabolismABSTRACT
Rho-associated kinase 2 (ROCK2) determines the balance between human T helper 17 (TH17) cells and regulatory T (Treg) cells. We investigated its role in the generation of T follicular helper (TFH) cells, which help to generate antibody-producing B cells under normal and autoimmune conditions. Inhibiting ROCK2 in normal human T cells or peripheral blood mononuclear cells from patients with active systemic lupus erythematosus (SLE) decreased the number and function of TFH cells induced by activation ex vivo. Moreover, inhibition of ROCK2 activity decreased the abundance of the transcriptional regulator Bcl6 (B cell lymphoma 6) and increased that of Blimp1 by reducing the binding of signal transducer and activator of transcription 3 (STAT3) and increasing that of STAT5 to the promoters of the genes Bcl6 and PRDM1, respectively. In the MRL/lpr murine model of SLE, oral administration of the selective ROCK2 inhibitor KD025 resulted in a twofold reduction in the numbers of TFH cells and antibody-producing plasma cells in the spleen, as well as a decrease in the size of splenic germinal centers, which are the sites of interaction between TFH cells and B cells. KD025-treated mice showed a substantial improvement in both histological and clinical scores compared to those of untreated mice and had reduced amounts of Bcl6 and phosphorylated STAT3, as well as increased STAT5 phosphorylation. Together, these data suggest that ROCK2 signaling plays a critical role in controlling the development of TFH cells induced by autoimmune conditions through reciprocal regulation of STAT3 and STAT5 activation.
Subject(s)
Lupus Erythematosus, Systemic/immunology , STAT3 Transcription Factor/immunology , STAT5 Transcription Factor/immunology , Signal Transduction/immunology , T-Lymphocytes, Helper-Inducer/immunology , rho-Associated Kinases/immunology , Adolescent , Adult , Aged , Animals , Disease Models, Animal , Female , Heterocyclic Compounds, 4 or More Rings , Humans , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/pathology , Male , Mice , Mice, Inbred MRL lpr , Middle Aged , Plasma Cells/immunology , Plasma Cells/pathology , Positive Regulatory Domain I-Binding Factor 1/genetics , Positive Regulatory Domain I-Binding Factor 1/immunology , Proto-Oncogene Proteins c-bcl-6/genetics , Proto-Oncogene Proteins c-bcl-6/immunology , STAT3 Transcription Factor/genetics , STAT5 Transcription Factor/genetics , Signal Transduction/genetics , T-Lymphocytes, Helper-Inducer/pathology , rho-Associated Kinases/geneticsABSTRACT
Rho-associated kinase 2 (ROCK2) regulates the secretion of proinflammatory cytokines and the development of autoimmunity in mice. Data from a phase 1 clinical trial demonstrate that oral administration of KD025, a selective ROCK2 inhibitor, to healthy human subjects down-regulates the ability of T cells to secrete IL-21 and IL-17 by 90% and 60%, respectively, but not IFN-γ in response to T-cell receptor stimulation in vitro. Pharmacological inhibition with KD025 or siRNA-mediated inhibition of ROCK2, but not ROCK1, significantly diminished STAT3 phosphorylation and binding to IL-17 and IL-21 promoters and reduced IFN regulatory factor 4 and nuclear hormone RAR-related orphan receptor γt protein levels in T cells derived from healthy subjects or rheumatoid arthritis patients. Simultaneously, treatment with KD025 also promotes the suppressive function of regulatory T cells through up-regulation of STAT5 phosphorylation and positive regulation of forkhead box p3 expression. The administration of KD025 in vivo down-regulates the progression of collagen-induced arthritis in mice via targeting of the Th17-mediated pathway. Thus, ROCK2 signaling appears to be instrumental in regulating the balance between proinflammatory and regulatory T-cell subsets. Targeting of ROCK2 in man may therefore restore disrupted immune homeostasis and have a role in the treatment of autoimmunity.
Subject(s)
CD4-Positive T-Lymphocytes/drug effects , Interleukin-17/metabolism , Interleukins/metabolism , Protein Kinase Inhibitors/pharmacology , STAT3 Transcription Factor/physiology , rho-Associated Kinases/antagonists & inhibitors , Administration, Oral , CD4-Positive T-Lymphocytes/metabolism , Cells, Cultured , Humans , Interleukin-17/genetics , Interleukins/genetics , Phosphorylation , Promoter Regions, Genetic , Protein Kinase Inhibitors/administration & dosage , STAT3 Transcription Factor/metabolism , Transcription, GeneticABSTRACT
BACKGROUND: Although the addition of epidermal growth factor receptor (EGFR) antibodies to various platinum-based chemotherapy regimens for non-small cell lung cancer (NSCLC) is being actively pursued in the clinic, rationale for the prioritization of specific regimens is lacking. MATERIALS AND METHODS: We evaluated the antitumor effects of necitumumab, a recombinant human IgG1 antibody targeting EGFR, in combination with cisplatin plus gemcitabine, pemetrexed, or paclitaxel in a panel of 9 subcutaneous tumor models of NSCLC established in nu/nu athymic mice. RESULTS: Necitumumab in combination with cisplatin/gemcitabine was particularly effective, although interestingly, the mechanisms underlying these benefits were model dependent. For example, increased tumor cell apoptosis contributed towards combination efficacy in the A549 model, in association with increased expression of hsa-miR-29b and reduced expression of antiapoptotic genes including DNA methyltransferase DNMT3B, commonly up-regulated in patients with NSCLC. Such inverse effects of combination therapy on DNMT3B and hsa-miR-29b expression were found in multiple models. Importantly, in the A549 model, hsa-miR-29b down-regulation of DMNT3b reduced promoter methylation of tumor suppressor genes such as Cell adhesion molecule 1 (CADM1), Ras associated (RalGDS/AF-6) domain family member 1 (RASSF1), and Fragile histidine triad gene (FHIT), increasing their expression. CONCLUSION: These results offer a preclinical rationale for combining an EGFR antibody with cisplatin/gemcitabine for patients with NSCLC, and provide potential molecular biomarkers for tailoring therapy.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , ErbB Receptors/antagonists & inhibitors , Lung Neoplasms/drug therapy , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/immunology , Base Sequence , Carcinoma, Non-Small-Cell Lung/genetics , Cell Line, Tumor , Cisplatin/administration & dosage , Cluster Analysis , DNA Methylation/drug effects , Deoxycytidine/administration & dosage , Deoxycytidine/analogs & derivatives , Drug Evaluation, Preclinical , ErbB Receptors/immunology , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lung Neoplasms/genetics , Mice , Mice, Nude , MicroRNAs/genetics , Promoter Regions, Genetic , RNA, Messenger/genetics , Signal Transduction , Tumor Suppressor Proteins/genetics , Xenograft Model Antitumor Assays , GemcitabineABSTRACT
BACKGROUND: Clinically relevant targets for developmental drug efficacy in animal models of cancer are critical yet understudied parameters. MATERIALS AND METHODS: Cetuximab, a chimeric antibody to epidermal growth factor receptor (EGFR), was administered to athymic mice bearing subcutaneous tumors established with 13 human colorectal cancer cell lines of varying biomarker status, defined by DNA sequencing and RT-PCR. RESULTS: If tumor growth inhibition is taken as a target, as is commonly done, then in contrast to the clinical situation where KRAS mutation strongly predicts for a lack of clinically meaningful benefit in colorectal cancer patients, cetuximab alone and in combination with irinotecan-based chemotherapy were efficacious in a similar proportion of KRAS wild-type and mutant models. It was only when tumor regression was utilized to define relevant efficacy that cetuximab monotherapy was efficacious in KRAS wild-type, but not mutant models. Adding cytotoxic therapy to cetuximab treatment increased tumor regression frequency in both genotypes to the point that once again the response was similar for KRAS wild-type and mutant models. CONCLUSION: Our data support shifting the threshold for claiming clinically relevant targeted therapy efficacy in subcutaneous xenograft models towards tumor regression, rather than tumor growth inhibition, focusing on the evaluation of tumor cells that are addicted to the pathways being targeted.
Subject(s)
Antibodies, Monoclonal/pharmacology , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Genes, ras , Mutation , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal, Humanized , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Biomarkers, Tumor/metabolism , Camptothecin/administration & dosage , Camptothecin/analogs & derivatives , Cetuximab , Colorectal Neoplasms/metabolism , Gene Dosage , Genes, erbB-1 , Humans , Irinotecan , Mice , Mice, Nude , Organoplatinum Compounds/administration & dosage , Oxaliplatin , Proto-Oncogene Proteins B-raf/genetics , Xenograft Model Antitumor AssaysABSTRACT
The benefits of inhibiting vascular endothelial growth factor (VEGF) signaling in cancer patients are predominantly attributed to effects on tumor endothelial cells. Targeting non-endothelial stromal cells to further impact tumor cell growth and survival is being pursued through the inhibition of additional growth factor pathways important for the survival and/or proliferation of these cells. However, recent data suggest that VEGF receptor (VEGFR)-specific inhibitors may target lymphatic vessels and pericytes in addition to blood vessels. Here, in fact, we demonstrate that DC101 (40 mg/kg, thrice a week), an antibody specific to murine VEGFR2, significantly reduces all three of these stromal components in subcutaneous (SKRC-29) and orthotopic (786-O-LP) models of renal cell carcinoma (RCC) established in nu/nu athymic mice. Sunitinib (40 mg/kg, once daily), a receptor tyrosine kinase inhibitor of VEGFR2 and other growth factor receptors, also caused significant loss of tumor blood vessels in RCC models but had weaker effects than DC101 on pericytes and lymphatic vessels. In combination, sunitinib did not significantly add to the effects of DC101 on tumor blood vessels, lymphatic vessels, or pericytes. Nevertheless, sunitinib increased the effect of DC101 on tumor burden in the SKRC-29 model, perhaps related to its broader specificity. Our data have important implications for combination therapy design, supporting the conclusion that targeting VEGFR2 alone in RCC has the potential to have pleiotropic effects on tumor stroma.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Carcinoma, Renal Cell/drug therapy , Indoles/pharmacology , Kidney Neoplasms/drug therapy , Pyrroles/pharmacology , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Animals , Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents/therapeutic use , Carcinoma, Renal Cell/blood supply , Carcinoma, Renal Cell/pathology , Disease Models, Animal , Drug Interactions , Female , Humans , Hypoxia-Inducible Factor 1/biosynthesis , Indoles/therapeutic use , Kidney Neoplasms/blood supply , Kidney Neoplasms/pathology , Lymphatic Vessels/drug effects , Lymphatic Vessels/pathology , Mice , Mice, Nude , Mutation , Neoplasm Transplantation , Neovascularization, Pathologic , Pericytes/drug effects , Pericytes/pathology , Pyrroles/therapeutic use , Stromal Cells/drug effects , Stromal Cells/pathology , Sunitinib , Tumor Burden , Tumor Cells, Cultured , Vascular Endothelial Growth Factor Receptor-2/immunology , Von Hippel-Lindau Tumor Suppressor Protein/geneticsABSTRACT
Microtubules are a well-validated target for anticancer therapy. Molecules that bind tubulin affect dynamic instability of microtubules causing mitotic arrest of proliferating cells, leading to cell death and tumor growth inhibition. Natural antitubulin agents such as taxanes and Vinca alkaloids have been successful in the treatment of cancer; however, several limitations have encouraged the development of synthetic small molecule inhibitors of tubulin function. We have previously reported the discovery of two novel chemical series of tubulin polymerization inhibitors, triazoles (Ouyang et al. Synthesis and structure-activity relationships of 1,2,4-triazoles as a novel class of potent tubulin polymerization inhibitors. Bioorg Med Chem Lett. 2005; 15:5154-5159) and oxadiazole derivatives (Ouyang et al. Oxadiazole derivatives as a novel class of antimitotic agents: synthesis, inhibition of tubulin polymerization, and activity in tumor cell lines. Bioorg Med Chem Lett. 2006; 16:1191-1196). Here, we report on the anticancer effects of a lead oxadiazole derivative in vitro and in vivo. In vitro, IMC-038525 caused mitotic arrest at nanomolar concentrations in epidermoid carcinoma and breast tumor cells, including multidrug-resistant cells. In vivo, IMC-038525 had a desirable pharmacokinetic profile with sustained plasma levels after oral dosing. IMC-038525 reduced subcutaneous xenograft tumor growth with significantly greater efficacy than the taxane paclitaxel. At efficacious doses, IMC-038525 did not cause substantial myelosuppression or peripheral neurotoxicity, as evaluated by neutrophil counts and changes in myelination of the sciatic nerve, respectively. These data indicate that IMC-038525 is a promising candidate for further development as a chemotherapeutic agent.
ABSTRACT
FMS-related tyrosine kinase receptor 3 (FLT3) is a class III receptor tyrosine kinase that holds considerable promise as a therapeutic target in hematologic malignancies. Current efforts directed toward the development of small-molecule tyrosine kinase inhibitors of FLT3 may be limited by off-target toxicities and the development of drug resistance. Target-specific antibodies could overcome these hurdles and provide additional mechanisms to enhance the antitumor efficacy of FLT3 inhibitors. IMC-EB10 is a novel antibody directed against FLT3. The binding of IMC-EB10 to FLT3 results in antiproliferative effects in vitro and in mouse models engrafted with human leukemia cells that harbor wild-type or constitutively activated FLT3. Future clinical trials will test these notions formally and will identify the most appropriate opportunities for this member of a new generation of antileukemic therapies.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents/therapeutic use , Leukemia/drug therapy , Protein Kinase Inhibitors/therapeutic use , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , Animals , Drug Delivery Systems , Humans , Immunoglobulin G/therapeutic use , Mice , Mutation , fms-Like Tyrosine Kinase 3/genetics , fms-Like Tyrosine Kinase 3/immunologyABSTRACT
In testing novel anticancer therapies, researchers strive to utilize models that reflect the human disease as much as feasible. In this regard, orthotopic models are frequently developed because cancer cells in these models form tumors in, and metastasize from, a tissue environment similar to the tissue of origin of the cancer cells. Here we adapted an orthotopic colorectal cancer model, in which HT-29 colorectal cancer cells form tumors in the rectal lining and metastasize to the para-aortic lymph nodes with high frequency. Firefly luciferase-expressing HT-29 cells were used in this model to realize the benefits of bioluminescence imaging (BLI). A combination of irinotecan, 5-fluorouracil (5-FU), and leucovorin (LV) (IFL) was used as a standard chemotherapeutic regimen positive control. BLI allowed for the demonstration of the effects of IFL on tumor growth in the rectal lining, with tumor weight measurements at the end of the study reflecting total tumor burden. BLI also allowed relatively easy demonstration of reduced tissue metastasis with IFL treatment, compared to more time-consuming histological techniques. It is concluded that the orthotopic colorectal cancer model approach described represents a valuable tool for validating treatment strategies in this indication.
Subject(s)
Antimetabolites, Antineoplastic/therapeutic use , Antineoplastic Agents, Phytogenic/therapeutic use , Camptothecin/analogs & derivatives , Colorectal Neoplasms , Fluorouracil/therapeutic use , Leucovorin/therapeutic use , Luminescent Measurements/methods , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Camptothecin/therapeutic use , Cell Culture Techniques , Cell Line, Tumor , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Female , Humans , Irinotecan , Mice , Mice, Inbred BALB C , Mice, Nude , Random Allocation , Treatment Outcome , Vitamin B Complex/therapeutic useABSTRACT
Cancer patients receiving epidermal growth factor receptor (EGFR) antibody therapy often experience an acneiform rash of uncertain etiology in skin regions rich in pilosebaceous units. Currently, this condition is treated symptomatically with very limited, often anecdotal success. Here, we show that a monoclonal antibody targeting murine EGFR, ME1, caused a neutrophil-rich hair follicle inflammation in mice, similar to that reported in patients. This effect was preceded by the appearance of lipid-filled hair follicle distensions adjacent to enlarged sebaceous glands. The cytokine tumor necrosis factor-alpha (TNFalpha), localized immunohistochemically to this affected region of the pilosebaceous unit, was specifically up-regulated by ME1 in skin but not in other tissues examined. Moreover, skin inflammation was reduced by cotreatment with the TNFalpha signaling inhibitor, etanercept, indicating the involvement of TNFalpha in this inflammatory process. Interleukin-1, a cytokine that frequently acts in concert with TNFalpha, is also involved in this process given the efficacy of the interleukin-1 antagonist Kineret. Our results provide a mechanistic framework to develop evidence-based trials for EGFR antibody-induced skin rash in patients with cancer.
Subject(s)
Antibodies, Monoclonal/adverse effects , Dermatitis/prevention & control , ErbB Receptors/immunology , Interleukin-1/antagonists & inhibitors , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antibodies, Monoclonal/therapeutic use , Dermatitis/etiology , Dermatitis/pathology , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , ErbB Receptors/antagonists & inhibitors , Etanercept , Exanthema/chemically induced , Exanthema/prevention & control , Female , Humans , Immunoglobulin G/pharmacology , Interleukin 1 Receptor Antagonist Protein/pharmacology , Interleukin-1/genetics , Interleukin-1/metabolism , Mice , Mice, SCID , Receptors, Tumor Necrosis Factor , Reverse Transcriptase Polymerase Chain Reaction , Skin/drug effects , Skin/metabolism , Skin/pathology , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolismABSTRACT
BACKGROUND: Rational strategies utilizing anticancer efficacy and biological principles are needed for the prioritization of specific combination targeted therapy approaches for clinical development, from among the many with experimental support. MATERIALS AND METHODS: Antibodies targeting epidermal growth factor receptor (EGFR) (cetuximab), insulin-like growth factor-1 receptor (IGF-IR) (IMC-A12) or vascular endothelial growth factor receptor 2 (VEGFR2) (DC101), were dosed alone or in combination, in 11 human tumor xenograft models established in mice. Efficacy readouts included the tumor burden and incidence of metastasis, as well as tumor active hypoxia inducible factor-1 (HIF-1), human VEGF and blood vessel density. RESULTS: Cetuximab and DC101 contributed potent and non-overlapping benefits to the combination approach. Moreover, DC101 prevented escape from IMC-A12 + cetuximab in a colorectal cancer model and cetuximab prevented escape from DC101 therapy in a pancreatic cancer model. CONCLUSION: Targeting VEGFR2 + EGFR was prioritized over other treatment strategies utilizing EGFR, IGF-IR and VEGFR2 antibodies. The criteria that proved to be valuable were a non-overlapping spectrum of anticancer activity and the prevention of resistance to another therapy in the combination.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Colorectal Neoplasms/drug therapy , Disease Models, Animal , ErbB Receptors/antagonists & inhibitors , Pancreatic Neoplasms/drug therapy , Receptor, IGF Type 1/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Animals , Antibodies, Monoclonal, Humanized , Antineoplastic Agents , Cell Line, Tumor , Cetuximab , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Drug Therapy, Combination , ErbB Receptors/immunology , ErbB Receptors/metabolism , Female , Humans , Mice , Mice, Nude , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Receptor, IGF Type 1/immunology , Receptor, IGF Type 1/metabolism , Vascular Endothelial Growth Factor Receptor-2/immunology , Vascular Endothelial Growth Factor Receptor-2/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Platelet-derived growth factor receptor beta (PDGFRbeta) is upregulated in most of solid tumors. It is expressed by pericytes/smooth muscle cells, fibroblast, macrophage, and certain tumor cells. Several PDGF receptor-related antagonists are being developed as potential antitumor agents and have demonstrated promising antitumor activity in both preclinical and clinical settings. Here, we produced a fully human neutralizing antibody, IMC-2C5, directed against PDGFRbeta from an antibody phage display library. IMC-2C5 binds to both human and mouse PDGFRbeta and blocks PDGF-B from binding to the receptor. IMC-2C5 also blocks ligand-stimulated activation of PDGFRbeta and downstream signaling molecules in tumor cells. In animal studies, IMC-2C5 significantly delayed the growth of OVCAR-8 and NCI-H460 human tumor xenografts in nude mice but failed to show antitumor activities in OVCAR-5 and Caki-1 xenografts. Our results indicate that the antitumor efficacy of IMC-2C5 is primarily due to its effects on tumor stroma, rather than on tumor cells directly. Combination of IMC-2C5 and DC101, an anti-mouse vascular endothelial growth factor receptor 2 antibody, resulted in significantly enhanced antitumor activity in BxPC-3, NCI-H460, and HCT-116 xenografts, compared with DC101 alone, and the trend of additive effects to DC101 treatment in several other tumor models. ELISA analysis of NCI-H460 tumor homogenates showed that IMC-2C5 attenuated protein level of vascular endothelial growth factor and basic fibroblast growth factor elevated by DC101 treatment. Finally, IMC-2C5 showed a trend of additive effects when combined with DC101/chemotherapy in MIA-PaCa-2 and NCI-H460 models. Taken together, these results lend great support to the use of PDGFRbeta antagonists in combination with other antiangiogenic agents in the treatment of a broad range of human cancers.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Neoplasms/drug therapy , Receptor, Platelet-Derived Growth Factor beta/immunology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibody Affinity/immunology , Cell Line, Tumor , Drug Synergism , Drug Therapy, Combination , Female , Flow Cytometry , HCT116 Cells , Humans , Mice , Mice, Nude , NIH 3T3 Cells , Neoplasms/pathology , Peptide Library , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Targeted therapy for cancer is shifting towards an approach of inhibiting multiple pathways, justified in part by the ability of cancer cells to overcome the inhibition of a single pathway. However the literature is replete with preclinical data supporting the anticancer potential of numerous combinations of targeted agents, making it difficult to select the combination strategies to invest in through clinical development. One characteristic of a combination strategy that can be utilized for prioritization is synergy. Synergy indicates that the effect of the combination is greater than that predicted from the monotherapy potencies. Here we describe a detailed method for establishing synergy between two treatments in vivo. We utilized this method to establish that antibodies targeting the epidermal growth factor receptor and vascular endothelial growth factor receptor-2 are synergistic with regard to antitumor effects, in a BxPC-3 subcutaneous xenograft model for pancreatic cancer.
Subject(s)
Antibodies/pharmacology , ErbB Receptors/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Xenograft Model Antitumor Assays/methods , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cell Line, Tumor , Drug Synergism , Humans , Mice , Neoplasms/drug therapy , Treatment Outcome , Tumor Burden/drug effectsABSTRACT
Vascular endothelial growth factor receptor (VEGFR) has recently been discovered on ovarian cancer cells, but its functional significance is unknown and is the focus of this study. By protein analysis, A2780-par and HeyA8 ovarian cancer cell lines expressed VEGFR-1 and HeyA8 A2774, and SKOV3ip1 expressed VEGFR-2. By in situ hybridization (ISH), 85% of human ovarian cancer specimens showed moderate to high VEGFR-2 expression, whereas only 15% showed moderate to high VEGFR-1 expression. By immunofluorescence, little or no VEGFR-2 was detected in normal ovarian surface epithelial cells, whereas expression was detected in 75% of invasive ovarian cancer specimens. To differentiate between the effects of tumor versus host expression of VEGFR, nude mice were injected with SKOV3ip1 cells and treated with either human VEGFR-2 specific antibody (1121B), murine VEGFR-2 specific antibody (DC101) or the combination. Treatment with 1121B reduced SKOV3ip1 cell migration by 68% (p < 0.01) and invasion by 72% (p < 0.01), but exposure to VEGFR-1 antibody had no effect. Treatment with 1121B effectively blocked VEGF-induced phosphorylation of p130Cas. In vivo treatment with either DC101 or 1121B significantly reduced tumor growth alone and in combination in the SKOV3ip1 and A2774 models. Decreased tumor burden after treatment with DC101 or 1121B correlated with increased tumor cell apoptosis, decreased proliferative index, and decreased microvessel density. These effects were significantly greater in the combination group (p < 0.001). We show functionally active VEGFR-2 is present on most ovarian cancer cells. The observed anti-tumor activity of VEGF-targeted therapies may be mediated by both anti-angiogenic and direct anti-tumor effects.
Subject(s)
Ovarian Neoplasms/pathology , Vascular Endothelial Growth Factor Receptor-2/physiology , Animals , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Apoptosis , Cell Proliferation , Crk-Associated Substrate Protein/physiology , Female , Humans , Mice , Ovarian Neoplasms/blood supply , Ovarian Neoplasms/therapy , Signal Transduction , Vascular Endothelial Growth Factor Receptor-2/analysis , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Xenograft Model Antitumor AssaysABSTRACT
A series of arylphthalazine derivatives were synthesized and evaluated as antagonists of VEGF receptor II (VEGFR-2). IM-094482 57, which was prepared in two steps from commercially available starting materials, was found to be a potent inhibitor of VEGFR-2 in enzymatic, cellular and mitogenic assays (comparable activity to ZD-6474). Additionally, 57 inhibited the related receptor, VEGF receptor I (VEGFR-1), and showed excellent exposure when dosed orally to female CD-1 mice.
Subject(s)
Phthalazines/pharmacokinetics , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Administration, Oral , Animals , Biological Availability , Female , Isoquinolines/chemical synthesis , Isoquinolines/pharmacokinetics , Mice , Mice, Inbred Strains , Phthalazines/administration & dosage , Phthalazines/chemical synthesis , Piperidines , Quinazolines , Vascular Endothelial Growth Factor Receptor-1/antagonists & inhibitorsABSTRACT
PURPOSE: To establish whether cetuximab, a chimeric IgG1 antibody targeting epidermal growth factor receptor, has the potential to restore responsiveness to oxaliplatin in preclinical cancer models, as has been shown with irinotecan in irinotecan refractory metastatic colorectal cancer patients. EXPERIMENTAL DESIGN: The effects of cetuximab and oxaliplatin, alone or in combination, were tested in vitro and in vivo using human colorectal cancer cell lines selected for oxaliplatin resistance, as well as parental control cell lines. Evaluations were made of subcutaneous xenograft tumor growth in nu/nu athymic mice, as well as activation of mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2) and AKT, expression of DNA repair genes, density of apurinic/apyrimidinic DNA damage, and accumulation of platinum-DNA adducts in vitro. RESULTS: Oxaliplatin + cetuximab efficacy in murine subcutaneous xenograft models was greater than that of monotherapies and independent of the responsiveness to oxaliplatin monotherapy. In vitro, cetuximab reduced expression of excision repair cross-complementation group 1 and XPF, which are key components of the nucleotide excision repair pathway involved in the excision of platinum-DNA adducts. In addition, cetuximab reduced expression of XRCC1, a component of the base excision repair pathway responsible for the repair of apurinic/apyrimidinic sites. Effects of cetuximab on DNA repair protein levels were downstream to effects on mitogen-activated protein kinase and AKT pathway activation. In line with effects on DNA repair protein expression, cetuximab increased the accumulation of platinum and apurinic/apyrimidinic sites on DNA during oxaliplatin treatment. CONCLUSIONS: Cetuximab has the potential to salvage the benefits of oxaliplatin in oxaliplatin-resistant colorectal cancer patients by reducing DNA repair capacity.