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1.
Clin Cancer Res ; 27(15): 4353-4366, 2021 08 01.
Article in English | MEDLINE | ID: mdl-34011558

ABSTRACT

PURPOSE: Combining radiotherapy (RT) with DNA damage response inhibitors may lead to increased tumor cell death through radiosensitization. DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break repair via the nonhomologous end joining (NHEJ) pathway. We hypothesized that in addition to a radiosensitizing effect from the combination of RT with AZD7648, a potent and specific inhibitor of DNA-PK, combination therapy may also lead to modulation of an anticancer immune response. EXPERIMENTAL DESIGN: AZD7648 and RT efficacy, as monotherapy and in combination, was investigated in fully immunocompetent mice in MC38, CT26, and B16-F10 models. Immunologic consequences were analyzed by gene expression and flow-cytometric analysis. RESULTS: AZD7648, when delivered in combination with RT, induced complete tumor regressions in a significant proportion of mice. The antitumor efficacy was dependent on the presence of CD8+ T cells but independent of NK cells. Analysis of the tumor microenvironment revealed a reduction in T-cell PD-1 expression, increased NK-cell granzyme B expression, and elevated type I IFN signaling in mice treated with the combination when compared with RT treatment alone. Blocking of the type I IFN receptor in vivo also demonstrated a critical role for type I IFN in tumor growth control following combined therapy. Finally, this combination was able to generate tumor antigen-specific immunologic memory capable of suppressing tumor growth following rechallenge. CONCLUSIONS: Blocking the NHEJ DNA repair pathway with AZD7648 in combination with RT leads to durable immune-mediated tumor control.


Subject(s)
Cell Line, Tumor/radiation effects , DNA-Activated Protein Kinase/antagonists & inhibitors , Interferon Type I/drug effects , Neoplasms/radiotherapy , Protein Kinase Inhibitors/pharmacology , Purines/pharmacology , Pyrans/pharmacology , Radiation-Sensitizing Agents/pharmacology , Triazoles/pharmacology , Animals , Mice
2.
Int J Oncol ; 47(2): 446-54, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26095475

ABSTRACT

The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signalling network is frequently de-regulated in breast cancer and has been shown to mediate resistance to anti-HER2 agents. Whilst constitutive activation of this pathway is emerging as a marker of sensitivity to various PI3K pathway inhibitors, activity of these agents in the clinic may be limited by the presence of feedback loops, leading to reactivation of receptor tyrosine kinases, such as HER2/HER3. To determine whether inhibition of HER2 could increase the efficacy of AZD5363, a novel AKT inhibitor, a panel of breast cancer cells was dosed with AZD5363 in combination with AZD8931, an inhibitor of EGFR/HER2/HER3 signalling. We show that the combined treatment resulted in synergistic growth inhibition and enhanced cell death, specifically in the HER2-amplified cell lines. Investigation of the mechanism by western blot analysis revealed that the addition of AZD8931 prevented the induction of HER2/HER3 phosphorylation induced by AZD5363 and resulted in concomitant inhibition of both the PI3K/AKT/mTOR and ERK signalling pathways and induction of apoptosis. Using the HCC1954 xenograft model, which is resistant to trastuzumab, we show that the combination of AZD5363 and AZD8931 is more efficacious than either agent alone, resulting in profound tumour regressions. We conclude that the activity of AZD5363 in HER2-amplified breast cancer cells is enhanced by the addition of AZD8931 and that dual targeting of AKT and EGFR/HER2/HER3 signalling is an attractive treatment option to be explored in the clinic.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Breast Neoplasms/drug therapy , Gene Amplification , Pyrimidines/administration & dosage , Pyrroles/administration & dosage , Quinazolines/administration & dosage , Receptor, ErbB-2/genetics , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Breast Neoplasms/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Resistance, Neoplasm/drug effects , Drug Synergism , Female , Humans , MCF-7 Cells , Mice , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Pyrimidines/pharmacology , Pyrroles/pharmacology , Quinazolines/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Receptor, ErbB-2/metabolism , Xenograft Model Antitumor Assays
3.
Int J Oncol ; 39(1): 271-8, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21537841

ABSTRACT

Vandetanib is a multi-targeted receptor tyrosine kinase inhibitor that is in clinical development for the treatment of solid tumours. This preclinical study examined the inhibition of two key signalling pathways (VEGFR-2, EGFR) at drug concentrations similar to those achieved in the clinic, and their contribution to direct and indirect antitumour effects of vandetanib. For in vitro studies, receptor phosphorylation was assessed by Western blotting and ELISA, cell proliferation was assessed using a cell viability endpoint, and effects on cell cycle determined using flow cytometry. For in vivo studies, Western blotting, ELISA and immunohistochemistry (IHC) were used to assess receptor phosphorylation. Cell culture experiments demonstrated that anti-proliferative effects of vandetanib resulted from inhibition of either EGFR or VEGFR-2 signalling in endothelial cells, but were associated with inhibition of EGFR signalling in tumour cells. Vandetanib inhibited both EGFR and VEGFR-2 signalling in normal lung tissue and in tumour xenografts. In a lung cancer model expressing an activating EGFR mutation, the activity of vandetanib was similar to that of a highly selective EGFR inhibitor (gefitinib), and markedly greater than that of a highly selective VEGFR inhibitor (vatalanib). These data suggest that at the plasma exposures achieved in the clinic, vandetanib will significantly inhibit both VEGFR-2 and EGFR signalling, and that both inhibition of angiogenesis and direct inhibition of tumour cell growth can contribute to treatment response.


Subject(s)
Antineoplastic Agents/pharmacology , ErbB Receptors/antagonists & inhibitors , Piperidines/pharmacology , Quinazolines/pharmacology , Signal Transduction/drug effects , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Endothelial Cells/drug effects , ErbB Receptors/metabolism , Female , Humans , Mice , Mice, SCID , Neoplasms/physiopathology , Phenotype , Vascular Endothelial Growth Factor Receptor-2/metabolism , Xenograft Model Antitumor Assays
4.
Mol Cancer Ther ; 10(5): 861-73, 2011 May.
Article in English | MEDLINE | ID: mdl-21441409

ABSTRACT

Cediranib is a potent inhibitor of the VEGF receptor (VEGFR)-2 and VEGFR-3 tyrosine kinases. This study assessed the activity of cediranib against the VEGFR-1 tyrosine kinase and the platelet-derived growth factor receptor (PDGFR)-associated kinases c-Kit, PDGFR-α, and PDGFR-ß. Cediranib inhibited VEGF-A-stimulated VEGFR-1 activation in AG1-G1-Flt1 cells (IC(50) = 1.2 nmol/L). VEGF-A induced greatest phosphorylation of VEGFR-1 at tyrosine residues Y1048 and Y1053; this was reversed by cediranib. Potency against VEGFR-1 was comparable with that previously observed versus VEGFR-2 and VEGFR-3. Cediranib also showed significant activity against wild-type c-Kit in cellular phosphorylation assays (IC(50) = 1-3 nmol/L) and in a stem cell factor-induced proliferation assay (IC(50) = 13 nmol/L). Furthermore, phosphorylation of wild-type c-Kit in NCI-H526 tumor xenografts was reduced markedly following oral administration of cediranib (≥1.5 mg/kg/d) to tumor-bearing nude mice. The activity of cediranib against PDGFR-ß and PDGFR-α was studied in tumor cell lines, vascular smooth muscle cells (VSMC), and a fibroblast line using PDGF-AA and PDGF-BB ligands. Both receptor phosphorylation (IC(50) = 12-32 nmol/L) and PDGF-BB-stimulated cellular proliferation (IC(50) = 32 nmol/L in human VSMCs; 64 nmol/L in osteosarcoma cells) were inhibited. In vivo, ligand-induced PDGFR-ß phosphorylation in murine lung tissue was inhibited by 55% following treatment with cediranib at 6 mg/kg but not at 3 mg/kg or less. In contrast, in C6 rat glial tumor xenografts in mice, ligand-induced phosphorylation of both PDGFR-α and PDGFR-ß was reduced by 46% to 61% with 0.75 mg/kg cediranib. Additional selectivity was showed versus Flt-3, CSF-1R, EGFR, FGFR1, and FGFR4. Collectively, these data indicate that cediranib is a potent pan-VEGFR kinase inhibitor with similar activity against c-Kit but is significantly less potent than PDGFR-α and PDGFR-ß.


Subject(s)
Protein Kinase Inhibitors/pharmacology , Quinazolines/pharmacology , Receptors, Platelet-Derived Growth Factor/antagonists & inhibitors , Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors , Animals , COS Cells , Cell Line, Tumor , Cell Proliferation/drug effects , Chlorocebus aethiops , HEK293 Cells , Humans , Ligands , Lung/drug effects , Mice , Mice, Nude , NIH 3T3 Cells , Phosphorylation/drug effects , Platelet-Derived Growth Factor/metabolism , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins c-kit/antagonists & inhibitors , Quinazolines/chemistry , Rats , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Signal Transduction/drug effects , Stem Cell Factor/metabolism , Xenograft Model Antitumor Assays , fms-Like Tyrosine Kinase 3/antagonists & inhibitors
5.
Clin Cancer Res ; 16(14): 3548-61, 2010 Jul 15.
Article in English | MEDLINE | ID: mdl-20606037

ABSTRACT

PURPOSE: Vascular endothelial growth factor (VEGF) signaling is key to tumor angiogenesis and is an important target in the development of anticancer drugs. However, VEGF receptor (VEGFR) expression in human cancers, particularly the relative expression of VEGFR-2 and VEGFR-3 in tumor vasculature versus tumor cells, is poorly defined. EXPERIMENTAL DESIGN: VEGFR-2- and VEGFR-3-specific antibodies were identified and used in the immunohistochemical analysis of human primary cancers and normal tissue. The relative vascular localization of both receptors in colorectal and breast cancers was determined by coimmunofluorescence with vascular markers. RESULTS: VEGFR-2 and VEGFR-3 were expressed on vascular endothelium but not on malignant cells in 13 common human solid tumor types (n > 400, bladder, breast, colorectal, head and neck, liver, lung, skin, ovarian, pancreatic, prostate, renal, stomach, and thyroid). The signal intensity of both receptors was significantly greater in vessels associated with malignant colorectal, lung, and breast than adjacent nontumor tissue. In colorectal cancers, VEGFR-2 was expressed on both intratumoral blood and lymphatic vessels, whereas VEGFR-3 was found predominantly on lymphatic vessels. In breast cancers, both receptors were localized to and upregulated on blood vessels. CONCLUSIONS: VEGFR-2 and VEGFR-3 are primarily localized to, and significantly upregulated on, tumor vasculature (blood and/or lymphatic) supporting the majority of solid cancers. The primary clinical mechanism of action of VEGF signaling inhibitors is likely to be through the targeting of tumor vessels rather than tumor cells. The upregulation of VEGFR-3 on tumor blood vessels indicates a potential additional antiangiogenic effect for dual VEGFR-2/VEGFR-3-targeted therapy.


Subject(s)
Endothelium, Vascular/metabolism , Neoplasms, Experimental/metabolism , Neoplasms/metabolism , Vascular Endothelial Growth Factor Receptor-2/biosynthesis , Vascular Endothelial Growth Factor Receptor-3/biosynthesis , Animals , Blotting, Western , Cell Line, Tumor , Endothelium, Vascular/pathology , Humans , Immunohistochemistry , Mice , Mice, Nude , Mice, SCID , NIH 3T3 Cells , Neoplasm Transplantation , Neoplasms/pathology , Neoplasms, Experimental/pathology
6.
Mol Cancer Ther ; 6(8): 2198-208, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17699717

ABSTRACT

The vascular endothelial growth factor-A (VEGF-A) signaling pathway, a key stimulant of solid tumor vascularization, is primarily dependent on the activation of the endothelial cell surface receptor VEGF receptor-2 (VEGFR-2). AZD2171 is an oral, highly potent small-molecule inhibitor of VEGFR tyrosine kinase activity that inhibits angiogenesis and the growth of human tumor xenografts in vivo. Here, we show pharmacodynamic changes in VEGFR-2 phosphorylation induced by AZD2171. In mouse lung tissue, a single dose of AZD2171 at 6 mg/kg inhibited VEGF-A-stimulated VEGFR-2 phosphorylation by 87% at 2 h with significant inhibition (>or=60%) maintained to 24 h. To examine inhibition of VEGFR-2 phosphorylation in tumor vasculature by immunohistochemistry, a comprehensive assessment of antibodies to various phosphorylation sites on the receptor was undertaken. Antibodies to the phosphotyrosine epitopes pY1175/1173 and pY1214/1212 were found suitable for this application. Calu-6 human lung tumor xenografts, from mice receiving AZD2171 or vehicle treatment (p.o., once daily), were examined by immunohistochemistry. A significant reduction in tumor vessel staining of phosphorylated VEGFR-2 (pVEGFR-2) was evident within 28 h of AZD2171 treatment (6 mg/kg). This effect preceded a significant reduction in tumor microvessel density, which was detectable following 52 h of AZD2171 treatment. These data show that AZD2171 is a potent inhibitor of VEGFR-2 activation in vivo and suggest that AZD2171 delivers therapeutic benefit in Calu-6 tumors by targeting vessels dependent on VEGFR-2 signaling for survival. In addition, this work highlights the utility of measuring either pY1175/1173 or pY1214/1212 on VEGFR-2 as a pharmacodynamic marker of VEGFR-2 activation.


Subject(s)
Antineoplastic Agents/pharmacology , Lung Neoplasms/blood supply , Neovascularization, Pathologic/drug therapy , Quinazolines/pharmacology , Signal Transduction/drug effects , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Xenograft Model Antitumor Assays , Animals , Antibodies, Neoplasm , Antibodies, Phospho-Specific , Female , Humans , Lung Neoplasms/pathology , Mice , Mice, Nude , Phosphorylation/drug effects , Phosphotyrosine/metabolism , Reproducibility of Results , Vascular Endothelial Growth Factor Receptor-2/metabolism
7.
Cancer Res ; 65(10): 4389-400, 2005 May 15.
Article in English | MEDLINE | ID: mdl-15899831

ABSTRACT

Inhibition of vascular endothelial growth factor-A (VEGF) signaling is a promising therapeutic approach that aims to stabilize the progression of solid malignancies by abrogating tumor-induced angiogenesis. This may be accomplished by inhibiting the kinase activity of VEGF receptor-2 (KDR), which has a key role in mediating VEGF-induced responses. The novel indole-ether quinazoline AZD2171 is a highly potent (IC50 < 1 nmol/L) ATP-competitive inhibitor of recombinant KDR tyrosine kinase in vitro. Concordant with this activity, in human umbilical vein endothelial cells, AZD2171 inhibited VEGF-stimulated proliferation and KDR phosphorylation with IC50 values of 0.4 and 0.5 nmol/L, respectively. In a fibroblast/endothelial cell coculture model of vessel sprouting, AZD2171 also reduced vessel area, length, and branching at subnanomolar concentrations. Once-daily oral administration of AZD2171 ablated experimental (VEGF-induced) angiogenesis in vivo and inhibited endochondral ossification in bone or corpora luteal development in ovary; physiologic processes that are highly dependent upon neovascularization. The growth of established human tumor xenografts (colon, lung, prostate, breast, and ovary) in athymic mice was inhibited dose-dependently by AZD2171, with chronic administration of 1.5 mg per kg per day producing statistically significant inhibition in all models. A histologic analysis of Calu-6 lung tumors treated with AZD2171 revealed a reduction in microvessel density within 52 hours that became progressively greater with the duration of treatment. These changes are indicative of vascular regression within tumors. Collectively, the data obtained with AZD2171 are consistent with potent inhibition of VEGF signaling, angiogenesis, neovascular survival, and tumor growth. AZD2171 is being developed clinically as a once-daily oral therapy for the treatment of cancer.


Subject(s)
Neoplasms/drug therapy , Neoplasms/enzymology , Protein Kinase Inhibitors/pharmacology , Quinazolines/pharmacology , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Administration, Oral , Animals , Biological Availability , Bone Development/drug effects , Cell Proliferation/drug effects , Corpus Luteum/drug effects , Corpus Luteum/growth & development , Endothelial Cells/drug effects , Endothelial Cells/enzymology , Endothelial Cells/metabolism , Extracellular Matrix Proteins , Female , Humans , Mice , Myosin Heavy Chains , Neoplasms/blood supply , Neoplasms/pathology , Nonmuscle Myosin Type IIB , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacokinetics , Proteins/antagonists & inhibitors , Quinazolines/pharmacokinetics , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/metabolism , Vascular Endothelial Growth Factor Receptor-3/antagonists & inhibitors , Xenograft Model Antitumor Assays
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