RESUMO
Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.
Assuntos
Piperidinas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Animais , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Feminino , Humanos , Camundongos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Piperidinas/síntese química , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Pirazóis/síntese química , Pirimidinas/síntese química , Especificidade por Substrato , Transcrição Gênica/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Peptidase 7 Específica de Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Understanding the molecular pathways by which oncogenes drive cancerous cell growth, and how dependence on such pathways varies between tumors could be highly valuable for the design of anti-cancer treatment strategies. In this work we study how dependence upon the canonical PI3K and MAPK cascades varies across HER2+ cancers, and define biomarkers predictive of pathway dependencies. A panel of 18 HER2+ (ERBB2-amplified) cell lines representing a variety of indications was used to characterize the functional and molecular diversity within this oncogene-defined cancer. PI3K and MAPK-pathway dependencies were quantified by measuring in vitro cell growth responses to combinations of AKT (MK2206) and MEK (GSK1120212; trametinib) inhibitors, in the presence and absence of the ERBB3 ligand heregulin (NRG1). A combination of three protein measurements comprising the receptors EGFR, ERBB3 (HER3), and the cyclin-dependent kinase inhibitor p27 (CDKN1B) was found to accurately predict dependence on PI3K/AKT vs. MAPK/ERK signaling axes. Notably, this multivariate classifier outperformed the more intuitive and clinically employed metrics, such as expression of phospho-AKT and phospho-ERK, and PI3K pathway mutations (PIK3CA, PTEN, and PIK3R1). In both cell lines and primary patient samples, we observed consistent expression patterns of these biomarkers varies by cancer indication, such that ERBB3 and CDKN1B expression are relatively high in breast tumors while EGFR expression is relatively high in other indications. The predictability of the three protein biomarkers for differentiating PI3K/AKT vs. MAPK dependence in HER2+ cancers was confirmed using external datasets (Project Achilles and GDSC), again out-performing clinically used genetic markers. Measurement of this minimal set of three protein biomarkers could thus inform treatment, and predict mechanisms of drug resistance in HER2+ cancers. More generally, our results show a single oncogenic transformation can have differing effects on cell signaling and growth, contingent upon the molecular and cellular context.
Assuntos
Sistema de Sinalização das MAP Quinases , Neoplasias/genética , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptor ErbB-2/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Biologia Computacional , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Genes erbB-2 , Humanos , Sistema de Sinalização das MAP Quinases/genética , Mutação , Neoplasias/patologia , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismoRESUMO
We have recently identified glucose-regulated protein-78 (GRP78) as a relevant molecular target expressed in metastatic tumors by fingerprinting the circulating repertoire of antibodies from cancer patients. Here we design and evaluate a ligand-receptor system based on the tumor cell membrane expression of GRP78. We show that GRP78 binding peptide motifs target tumor cells specifically in vivo and in human cancer specimens ex vivo. Moreover, synthetic chimeric peptides composed of GRP78 binding motifs fused to a programmed cell death-inducing sequence can suppress tumor growth in xenograft and isogenic mouse models of prostate and breast cancer. Together, these preclinical data validate GRP78 on the tumor cell surface as a functional molecular target that may prove useful for translation into clinical applications.
Assuntos
Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Apoptose , Membrana Celular/metabolismo , Chaperona BiP do Retículo Endoplasmático , Humanos , Ligantes , Masculino , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Transplante de Neoplasias , Peptídeos/metabolismo , Neoplasias da Próstata/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão , Células Tumorais CultivadasRESUMO
We show that a membrane-associated protease, aminopeptidase A (APA), is upregulated and enzymatically active in blood vessels of human tumors. To gain mechanistic insight, we evaluated angiogenesis in APA null mice. We found that, although these mice develop normally, they fail to mount the expected angiogenic response to hypoxia or growth factors. We then isolated peptide inhibitors of APA from a peptide library and show that they specifically bind to and inhibit APA, suppress migration and proliferation of endothelial cells, inhibit angiogenesis, and home to tumor blood vessels. Finally, we successfully treated tumor-bearing mice with APA binding peptides or anti-APA blocking monoclonal antibodies. These data show that APA is a regulator of blood vessel formation, and can serve as a functional vascular target.
Assuntos
Células Endoteliais/fisiologia , Glutamil Aminopeptidase/metabolismo , Motivos de Aminoácidos , Animais , Vasos Sanguíneos , Divisão Celular , Hipóxia Celular/fisiologia , Movimento Celular , Embrião de Galinha , Inibidores Enzimáticos , Substâncias de Crescimento/metabolismo , Humanos , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Neoplasias/metabolismo , Neovascularização Patológica , Biblioteca de Peptídeos , Peptídeos/metabolismo , Ligação Proteica , Transplante Heterólogo/patologiaRESUMO
CD137 (4-1BB) is a co-stimulatory receptor on immune cells and Nectin-4 is a cell adhesion molecule that is overexpressed in multiple tumor types. Using a series of poly(ethylene glycol) (PEG)-based linkers, synthetic bicyclic peptides targeting CD137 were conjugated to Bicycles targeting Nectin-4. The resulting bispecific molecules were potent CD137 agonists that require the presence of both Nectin-4-expressing tumor cells and CD137-expressing immune cells for activity. A multipronged approach was taken to optimize these Bicycle tumor-targeted immune cell agonists by exploring the impact of chemical configuration, binding affinity, and pharmacokinetics on CD137 agonism and antitumor activity. This effort resulted in the discovery of BT7480, which elicited robust CD137 agonism and maximum antitumor activity in syngeneic mouse models. A tumor-targeted approach to CD137 agonism using low-molecular-weight, short-acting molecules with high tumor penetration is a yet unexplored path in the clinic, where emerging data suggest that persistent target engagement, characteristic of biologics, may lead to suboptimal immune response.
Assuntos
Neoplasias , Animais , Moléculas de Adesão Celular , Camundongos , Nectinas , Neoplasias/tratamento farmacológico , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
Multiple tumor types overexpress Nectin-4 and the antibody-drug conjugate (ADC), enfortumab vedotin (EV) shows striking efficacy in clinical trials for metastatic urothelial cancer, which expresses high levels of Nectin-4, validating Nectin-4 as a clinical target for toxin delivery in this indication. Despite excellent data in urothelial cancer, little efficacy data are reported for EV in other Nectin-4 expressing tumors and EV therapy can produce significant toxicities in many patients, frequently leading to discontinuation of treatment. Thus, additional approaches to this target with the potential to extend utility and reduce toxicity are warranted. We describe the preclinical development of BT8009, a "Bicycle Toxin Conjugate" (BTC) consisting of a Nectin-4-binding bicyclic peptide, a cleavable linker system and the cell penetrant toxin mono-methylauristatin E (MMAE). BT8009 shows significant antitumor activity in preclinical tumor models, across a variety of cancer indications and is well tolerated in preclinical safety studies. In several models, it shows superior or equivalent antitumor activity to an EV analog. As a small hydrophilic peptide-based drug BT8009 rapidly diffuses from the systemic circulation, through tissues to penetrate the tumor and target tumor cells. It is renally eliminated from the circulation, with a half-life of 1-2 hours in rat and non-human primate. These physical and PK characteristics differentiate BT8009 from ADCs and may provide benefit in terms of tumor penetration and reduced systemic exposure. BT8009 is currently in a Phase 1/2 multicenter clinical trial across the US, Canada, and Europe, enrolling patients with advanced solid tumors associated with Nectin-4 expression.
Assuntos
Carcinoma de Células de Transição , Imunoconjugados , Imunotoxinas , Ratos , Animais , Nectinas , Ciclismo , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Moléculas de Adesão Celular/metabolismo , Carcinoma de Células de Transição/tratamento farmacológicoRESUMO
Immunoconjugates targeting cell-surface antigens have demonstrated clinical activity to enable regulatory approval in several solid and hematologic malignancies. We hypothesize that a rigorous and comprehensive surfaceome profiling approach to identify osteosarcoma-specific cell-surface antigens can similarly enable development of effective therapeutics in this disease. Herein, we describe an integrated proteomic and transcriptomic surfaceome profiling approach to identify cell-surface proteins that are highly expressed in osteosarcoma but minimally expressed on normal tissues. Using this approach, we identified targets that are highly expressed in osteosarcoma. Three targets, MT1-MMP, CD276, and MRC2, were validated as overexpressed in osteosarcoma. Furthermore, we tested BT1769, an MT1-MMP-targeted Bicycle toxin conjugate, in osteosarcoma patient-derived xenograft models. The results showed that BT1769 had encouraging antitumor activity, high affinity for its target, and a favorable pharmacokinetic profile. This confirms the hypothesis that our approach identifies novel targets with significant therapeutic potential in osteosarcoma.
Assuntos
Neoplasias Ósseas , Osteossarcoma , Antígenos de Superfície , Antígenos B7 , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Humanos , Metaloproteinase 14 da Matriz , Osteossarcoma/metabolismo , Proteômica/métodosRESUMO
The molecular diversity of receptors in human blood vessels remains largely unexplored. We developed a selection method in which peptides that home to specific vascular beds are identified after administration of a peptide library. Here we report the first in vivo screening of a peptide library in a patient. We surveyed 47,160 motifs that localized to different organs. This large-scale screening indicates that the tissue distribution of circulating peptides is nonrandom. High-throughput analysis of the motifs revealed similarities to ligands for differentially expressed cell-surface proteins, and a candidate ligand-receptor pair was validated. These data represent a step toward the construction of a molecular map of human vasculature and may have broad implications for the development of targeted therapies.
Assuntos
Vasos Sanguíneos/fisiologia , Biblioteca de Peptídeos , Variação Genética , Humanos , Oligopeptídeos/química , Especificidade de Órgãos , Reprodutibilidade dos Testes , SoftwareRESUMO
BACKGROUND: CD137 (4-1BB) is an immune costimulatory receptor with high therapeutic potential in cancer. We are creating tumor target-dependent CD137 agonists using a novel chemical approach based on fully synthetic constrained bicyclic peptide (Bicycle®) technology. Nectin-4 is overexpressed in multiple human cancers that may benefit from CD137 agonism. To this end, we have developed BT7480, a novel, first-in-class, Nectin-4/CD137 Bicycle tumor-targeted immune cell agonist™ (Bicycle TICA™). METHODS: Nectin-4 and CD137 co-expression analyses in primary human cancer samples was performed. Chemical conjugation of two CD137 Bicycles to a Nectin-4 Bicycle led to BT7480, which was then evaluated using a suite of in vitro and in vivo assays to characterize its pharmacology and mechanism of action. RESULTS: Transcriptional profiling revealed that Nectin-4 and CD137 were co-expressed in a variety of human cancers with high unmet need and spatial proteomic imaging found CD137-expressing immune cells were deeply penetrant within the tumor near Nectin-4-expressing cancer cells. BT7480 binds potently, specifically, and simultaneously to Nectin-4 and CD137. In co-cultures of human peripheral blood mononuclear cells and tumor cells, this co-ligation causes robust Nectin-4-dependent CD137 agonism that is more potent than an anti-CD137 antibody agonist. Treatment of immunocompetent mice bearing Nectin-4-expressing tumors with BT7480 elicited a profound reprogramming of the tumor immune microenvironment including an early and rapid myeloid cell activation that precedes T cell infiltration and upregulation of cytotoxicity-related genes. BT7480 induces complete tumor regressions and resistance to tumor re-challenge. Importantly, antitumor activity is not dependent on continuous high drug levels in the plasma since a once weekly dosing cycle provides maximum antitumor activity despite minimal drug remaining in the plasma after day 2. BT7480 appears well tolerated in both rats and non-human primates at doses far greater than those expected to be clinically relevant, including absence of the hepatic toxicity observed with non-targeted CD137 agonists. CONCLUSION: BT7480 is a highly potent Nectin-4-dependent CD137 agonist that produces complete regressions and antitumor immunity with only intermittent drug exposure in syngeneic mouse tumor models and is well tolerated in preclinical safety species. This work supports the clinical investigation of BT7480 for the treatment of cancer in humans.
Assuntos
Imunoterapia/métodos , Neoplasias/tratamento farmacológico , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Animais , Humanos , Camundongos , Neoplasias/imunologia , Ratos , Microambiente TumoralRESUMO
BACKGROUND: In contrast to immune checkpoint inhibitors, the use of antibodies as agonists of immune costimulatory receptors as cancer therapeutics has largely failed. We sought to address this problem using a new class of modular synthetic drugs, termed tumor-targeted immune cell agonists (TICAs), based on constrained bicyclic peptides (Bicycles). METHODS: Phage libraries displaying Bicycles were panned for binders against tumor necrosis factor (TNF) superfamily receptors CD137 and OX40, and tumor antigens EphA2, Nectin-4 and programmed death ligand 1. The CD137 and OX40 Bicycles were chemically conjugated to tumor antigen Bicycles with different linkers and stoichiometric ratios of binders to obtain a library of low molecular weight TICAs (MW <8 kDa). The TICAs were evaluated in a suite of in vitro and in vivo assays to characterize their pharmacology and mechanism of action. RESULTS: Linking Bicycles against costimulatory receptors (e.g., CD137) to Bicycles against tumor antigens (e.g., EphA2) created potent agonists that activated the receptors selectively in the presence of tumor cells expressing these antigens. An EphA2/CD137 TICA (BCY12491) efficiently costimulated human peripheral blood mononuclear cells in vitro in the presence of EphA2 expressing tumor cell lines as measured by the increased secretion of interferon γ and interleukin-2. Treatment of C57/Bl6 mice transgenic for the human CD137 extracellular domain (huCD137) bearing EphA2-expressing MC38 tumors with BCY12491 resulted in the infiltration of CD8+ T cells, elimination of tumors and generation of immunological memory. BCY12491 was cleared quickly from the circulation (plasma t1/2 in mice of 1-2 hr), yet intermittent dosing proved effective. CONCLUSION: Tumor target-dependent CD137 agonism using a novel chemical approach (TICAs) afforded elimination of tumors with only intermittent dosing suggesting potential for a wide therapeutic index in humans. This work unlocks a new path to effective cancer immunotherapy via agonism of TNF superfamily receptors.
Assuntos
Neoplasias/tratamento farmacológico , Peptídeos Cíclicos/administração & dosagem , Receptor EphA2/agonistas , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/agonistas , Células A549 , Animais , Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Feminino , Células HT29 , Humanos , Células Jurkat , Camundongos , Camundongos Transgênicos , Neoplasias/genética , Neoplasias/imunologia , Células PC-3 , Biblioteca de Peptídeos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Receptores OX40/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The EphA2 receptor is found at high levels in tumors and low levels in normal tissue and high EphA2 expression in biopsies is a predictor of poor outcome in patients. Drug discovery groups have therefore sought to develop EphA2-based therapies using small molecule, peptide, and nanoparticle-based approaches (1-3). However, until now only EphA2-targeting antibody-drug conjugates (ADC) have entered clinical development. For example, MEDI-547 is an EphA2-targeting ADC that displayed encouraging antitumor activity in preclinical models and progressed to phase I clinical testing in man. Here we describe the development of BT5528, a bicyclic peptide ("Bicycle") conjugated to the auristatin derivative maleimidocaproyl-monomethyl auristatin E to generate the Bicycle toxin conjugate BT5528. The report compares and contrasts the Pharmacokinetics (PK) characteristics of antibody and Bicycle-based targeting systems and discusses how the PK and payload characteristics of different delivery systems impact the efficacy-toxicity trade off which is key to the development of successful cancer therapies. We show that BT5528 gives rise to rapid update into tumors and fast renal elimination followed by persistent toxin levels in tumors without prolonged exposure of parent drug in the vasculature. This fast in, fast out kinetics gave rise to more favorable toxicology findings in rats and monkeys than were observed with MEDI-547 in preclinical and clinical studies.Graphical Abstract: http://mct.aacrjournals.org/content/molcanther/19/7/1385/F1.large.jpg.
Assuntos
Antineoplásicos/farmacologia , Sistemas de Liberação de Medicamentos , Fibrossarcoma/tratamento farmacológico , Oligopeptídeos/química , Peptídeos Cíclicos/farmacologia , Receptor EphA2/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacocinética , Apoptose , Proliferação de Células , Feminino , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Humanos , Imunotoxinas/farmacocinética , Imunotoxinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Oligopeptídeos/administração & dosagem , Peptídeos Cíclicos/farmacocinética , Receptor EphA2/genética , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
For two decades, the antigen recognized by the Pathologische Anatomie Leiden-Endothelium (PAL-E) monoclonal antibody, a standard vascular endothelial cell marker, has remained elusive. Here, we used a combinatorial phage display-based approach ("epitope mapping") to select peptides binding to the original PAL-E antibody. We found that a subset of the selected panel of peptides had motifs with strong homology to an exposed site within the b1 domain of human neuropilin-1 (NRP-1). We confirmed peptide binding by ELISA and by surface plasmon resonance. We also showed that the PAL-E antigen colocalizes with NRP-1 staining in endothelial cells. Crystal structure of the b1 domain in NRP-1 suggests that the PAL-E binding site overlaps with a vascular endothelial growth factor (VEGF) binding site. Taken together, these results indicate that NRP-1 is an endothelial cell antigen recognized by the true PAL-E antibody. The consistent biochemical, morphologic, and functional features between the PAL-E antigen and NRP-1 support our interpretation. Given that NRP-1 is a VEGF receptor, these results explain the attributes of the PAL-E antibody as a marker of vascular permeability and angiogenesis.
Assuntos
Anticorpos Monoclonais/imunologia , Neuropilina-1/imunologia , Peptídeos/imunologia , Fator A de Crescimento do Endotélio Vascular/imunologia , Sequência de Aminoácidos , Sítios de Ligação de Anticorpos , Células Cultivadas , Técnicas de Química Combinatória , Células Endoteliais/citologia , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Humanos , Modelos Moleculares , Neuropilina-1/metabolismo , Biblioteca de Peptídeos , Peptídeos/metabolismo , Ressonância de Plasmônio de Superfície , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
We have previously identified ligands from combinatorial peptide libraries that target tumor vasculature after in vivo selection. These ligands bind to differentially expressed receptors in angiogenic vasculature such as alpha(v)beta3/alpha(v)beta5 integrins, aminopeptidase N, and aminopeptidase A. We hypothesized that we can use these ligands to target angiogenic vasculature in retinopathies. Pathological retinal angiogenesis in conditions such as diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration is a major cause of blindness for which current treatments are inadequate. Here we tested whether known tumor vasculature targeting peptide ligands displayed on bacteriophage particles would home to the proliferating blood vessels of the retina in a standard mouse model of retinopathy of prematurity. We found that activated retinal blood vessels share many of the endothelial and periendothelial cell receptors expressed in tumor vasculature. Furthermore, these vascular receptors--alpha(v) integrins and aminopeptidases--are accessible through the circulation and mediate phage homing and internalization to endothelial and periendothelial cells. Treatment of mice with a peptide containing a alpha(v)beta3/alpha(v)beta5-integrin targeting domain fused to a proapoptotic domain significantly reduced oxygen-induced retinal angiogenesis by selectively inducing activated endothelial cell apoptosis. Targeted proapoptotic peptides may prove useful in the management of angiogenic retinal diseases.
Assuntos
Hipóxia/complicações , Peptídeos/uso terapêutico , Doenças Retinianas/etiologia , Doenças Retinianas/terapia , Neovascularização Retiniana , Animais , Animais Recém-Nascidos , Bacteriófagos/genética , Bacteriófagos/metabolismo , Antígenos CD13/metabolismo , Modelos Animais de Doenças , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Feminino , Glutamil Aminopeptidase/metabolismo , Humanos , Integrina alfaVbeta3/metabolismo , Integrinas/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Transplante de Neoplasias , Peptídeos/genética , Peptídeos/metabolismo , Receptores de Vitronectina/metabolismo , Retina/anatomia & histologia , Retina/metabolismo , Retina/patologia , Doenças Retinianas/patologia , Transplante HeterólogoRESUMO
A collection of 60 cell lines derived from human tumors (NCI-60) has been widely explored as a tool for anticancer drug discovery. Here, we profiled the cell surface of the NCI-60 by high-throughput screening of a phage-displayed random peptide library and classified the cell lines according to the binding selectivity of 26,031 recovered tripeptide motifs. By analyzing selected cell-homing peptide motifs and their NCI-60 recognition patterns, we established that some of these motifs (a) are similar to domains of human proteins known as ligands for tumor cell receptors and (b) segregate among the NCI-60 in a pattern correlating with expression profiles of the corresponding receptors. We biochemically validated some of the motifs as mimic peptides of native ligands for the epidermal growth factor receptor. Our results indicate that ligand-directed profiling of tumor cell lines can select functional peptides from combinatorial libraries based on the expression of tumor cell surface molecules, which in turn could be exploited as "druggable" receptors in specific types of cancer.
Assuntos
Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Peptídeos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Análise por Conglomerados , Técnicas de Química Combinatória , Receptores ErbB/metabolismo , Humanos , Ligantes , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Biblioteca de Peptídeos , Reprodutibilidade dos TestesRESUMO
PI3K is frequently mutated in cancer and plays an important role in cell growth and survival. Heregulin (HRG)-mediated autocrine or paracrine signaling through the receptor tyrosine kinase ErbB3 potently activates the PI3K/AKT pathway and has been shown to mediate resistance to a wide variety of anticancer agents. Although PI3K functions downstream of HRG-ErbB3, it is unknown whether activating mutations in PI3K render HRG ineffective. If so, patients with PI3K mutations would not be expected to benefit from ErbB3-directed therapies. Here, we find that a subset of cell lines harboring activating PI3K mutations can be further growth-stimulated by HRG, and this effect is blocked by incubation with seribantumab (MM-121), a monoclonal anti-ErbB3 antibody. Although expression of mutant PI3K in wild-type PI3K cells frequently results in loss of HRG-stimulated growth, some cell lines continue to respond to HRG. In cell lines where HRG-stimulated growth is lost, this loss is invariably accompanied by a reduction in ErbB3 levels, a corresponding increase in basal phosphorylation levels of FOXO-family transcription factors, and a reduction in HRG-induced downstream signaling. Importantly, HRG-stimulated growth is partially rescued by re-expressing ErbB3. This response is blocked by seribantumab, indicating that ErbB3 levels rather than downstream signaling proteins limit HRG-stimulated growth in PI3K mutant cells. Overall, these results suggest that activating mutations in PI3K do not preclude potential benefit from ErbB3-directed therapy, but that it may be important to measure ErbB3 levels in patients with PI3K mutant cancers to determine if they would benefit.
Assuntos
Mutação , Neoplasias/genética , Neoplasias/metabolismo , Neuregulina-1/metabolismo , Fosfatidilinositol 3-Quinases/genética , Receptor ErbB-3/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Neoplasias/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Receptor ErbB-3/antagonistas & inibidores , Receptor ErbB-3/genética , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Antineoplásicos/uso terapêutico , Carcinoma/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Piperazinas/uso terapêutico , Fator de Crescimento Derivado de Plaquetas/fisiologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Neoplasias do Colo do Útero/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Benzamidas , Carcinoma/irrigação sanguínea , Sistemas de Liberação de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Mesilato de Imatinib , Camundongos , Proteínas de Neoplasias/fisiologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/enzimologia , Comunicação Parácrina/efeitos dos fármacos , Pericitos/efeitos dos fármacos , Pericitos/enzimologia , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Receptores do Fator de Crescimento Derivado de Plaquetas/fisiologia , Neoplasias do Colo do Útero/irrigação sanguíneaRESUMO
Crosstalk and compensatory circuits within cancer signaling networks limit the activity of most targeted therapies. For example, altered signaling in the networks activated by the ErbB family of receptors, particularly in ERBB2-amplified cancers, contributes to drug resistance. We developed a multiscale systems model of signaling networks in ERBB2-amplified breast cancer to quantitatively investigate relationships between biomarkers (markers of network activity) and combination drug efficacy. This model linked ErbB receptor family signaling to breast tumor growth through two kinase cascades: the PI3K/AKT survival pathway and the Ras/MEK/ERK growth and proliferation pathway. The model predicted molecular mechanisms of resistance to individual therapeutics. In particular, ERBB2-amplified breast cancer cells stimulated with the ErbB3 ligand heregulin were resistant to growth arrest induced by inhibitors of AKT and MEK or coapplication of two inhibitors of the receptor ErbB2 [Herceptin (trastuzumab) and Tykerb (lapatinib)]. We used model simulations to predict the response of ErbB2-positive breast cancer xenografts to combination therapies and verified these predictions in mice. Treatment with trastuzumab, lapatinib, and the ErbB3 inhibitor MM-111 was more effective in inhibiting tumor growth than the combination of AKT and MEK inhibitors and even induced tumor regression, indicating that targeting both ErbB3 and ErbB2 may be an improved therapeutic approach for ErbB2-positive breast cancer patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Modelos Biológicos , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Transdução de Sinais/fisiologia , Animais , Anticorpos Biespecíficos , Anticorpos Monoclonais Humanizados , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/fisiopatologia , Simulação por Computador , Retroalimentação Fisiológica/fisiologia , Feminino , Lapatinib , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Neuregulina-1 , Proteína Oncogênica v-akt/antagonistas & inibidores , Quinazolinas , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-3/antagonistas & inibidores , TrastuzumabRESUMO
PURPOSE: Sorafenib is a multi-targeted tyrosine kinase inhibitor with therapeutic efficacy in several malignancies. Sorafenib may exert its anti-neoplastic effect in part by altering vascular permeability and reducing intra-tumoral interstitial hypertension. As correlative science with a phase II study in patients with advanced soft-tissue sarcomas (STS), we evaluated the impact of this agent on intra-tumor interstitial fluid pressure (IFP), serum circulating biomarkers, and vascular density. PATIENTS AND METHODS: Patients with advanced STS with measurable disease and at least one superficial lesion amenable to biopsy received sorafenib 400 mg twice daily. Intratumoral IFP and plasma and circulating cell biomarkers were measured before and after 1-2 months of sorafenib administration. Results were analyzed in the context of the primary clinical endpoint of time-to-progression (TTP). RESULTS: In 15 patients accrued, the median TTP was 45 days (range 14-228). Intra-tumoral IFP measurements obtained in 6 patients at baseline showed a direct correlation with tumor size. Two patients with stable disease at two months had post-sorafenib IFP evaluations and demonstrated a decline in IFP and vascular density. Sorafenib significantly increased plasma VEGF, PlGF, and SDF1α and decreased sVEGFR-2 levels. Increased plasma SDF1α and decreased sVEGFR-2 levels on day 28 correlated with disease progression. CONCLUSIONS: Pretreatment intra-tumoral IFP correlated with tumor size and decreased in two evaluable patients with SD on sorafenib. Sorafenib also induced changes in circulating biomarkers consistent with expected VEGF pathway blockade, despite the lack of more striking clinical activity in this small series. TRIAL REGISTRATION: ClinicalTrials.gov NCT00330421.
Assuntos
Benzenossulfonatos/farmacologia , Biomarcadores Tumorais/sangue , Líquido Extracelular/efeitos dos fármacos , Piridinas/farmacologia , Sarcoma/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas Angiogênicas/análise , Proteínas Angiogênicas/sangue , Antineoplásicos , Benzenossulfonatos/administração & dosagem , Benzenossulfonatos/uso terapêutico , Permeabilidade Capilar/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Niacinamida/análogos & derivados , Compostos de Fenilureia , Pressão , Inibidores de Proteínas Quinases , Piridinas/administração & dosagem , Piridinas/uso terapêutico , Terapia de Salvação , Sorafenibe , Fatores de Crescimento do Endotélio Vascular/efeitos dos fármacosRESUMO
Patients with bilateral vestibular schwannomas associated with neurofibromatosis type 2 (NF2) experience significant morbidity such as complete hearing loss. We have recently shown that treatment with bevacizumab provided tumor stabilization and hearing recovery in a subset of NF2 patients with progressive disease. In the current study, we used two animal models to identify the mechanism of action of anti-vascular endothelial growth factor (VEGF) therapy in schwannomas. The human HEI193 and murine Nf2(-/-) cell lines were implanted between the pia and arachnoid meninges as well as in the sciatic nerve to mimic central and peripheral schwannomas. Mice were treated with bevacizumab (10 mg/kg/wk i.v.) or vandetanib (50 mg/kg/d orally) to block the VEGF pathway. Using intravital and confocal microscopy, together with whole-body imaging, we measured tumor growth delay, survival rate, as well as blood vessel structure and function at regular intervals. In both models, tumor vessel diameter, length/surface area density, and permeability were significantly reduced after treatment. After 2 weeks of treatment, necrosis in HEI193 tumors and apoptosis in Nf2(-/-) tumors were significantly increased, and the tumor growth rate decreased by an average of 50%. The survival of mice bearing intracranial schwannomas was extended by at least 50%. This study shows that anti-VEGF therapy normalizes the vasculature of schwannoma xenografts in nude mice and successfully controls the tumor growth, probably by reestablishing a natural balance between VEGF and semaphorin 3 signaling.
Assuntos
Inibidores da Angiogênese/farmacologia , Anticorpos Monoclonais/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neurilemoma/tratamento farmacológico , Piperidinas/farmacologia , Quinazolinas/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados , Bevacizumab , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/genética , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Nus , Neovascularização Patológica/tratamento farmacológico , Neurilemoma/irrigação sanguínea , Neurilemoma/genética , Neurofibromatose 2/tratamento farmacológico , Neurofibromatose 2/genética , Neurofibromina 2/deficiência , Neurofibromina 2/genéticaRESUMO
Lymphatic metastasis is a critical determinant of cancer prognosis. Recently, several lymphangiogenic molecules such as vascular endothelial growth factor (VEGF)-C and VEGF-D were identified. However, the mechanistic understanding of lymphatic metastasis is still in infancy. Nitric oxide (NO) plays a crucial role in regulating blood vessel growth and function as well as lymphatic vessel function. NO synthase (NOS) expression correlates with lymphatic metastasis. However, causal relationship between NOS and lymphatic metastasis has not been documented. To this end, we first show that both VEGF receptor-2 and VEGF receptor-3 stimulation activate eNOS in lymphatic endothelial cells and that NO donors induce proliferation and/or survival of cultured lymphatic endothelial cells in a dose-dependent manner. We find that an NOS inhibitor, L-NMMA, blocked regeneration of lymphatic vessels. Using intravital microscopy that allows us to visualize the steps of lymphatic metastasis, we show that genetic deletion of eNOS as well as NOS blockade attenuates peritumor lymphatic hyperplasia of VEGF-C-overexpressing T241 fibrosarcomas and decreases the delivery of metastatic tumor cells to the draining lymph nodes. Genetic deletion of eNOS in the host also leads to a decrease in T241 tumor cell dissemination to the lymph nodes and macroscopic lymph node metastasis of B16F10 melanoma. These findings indicate that eNOS mediates VEGF-C-induced lymphangiogenesis and, consequently, plays a critical role in lymphatic metastasis. Our findings explain the correlation between NOS and lymphatic metastasis seen in a number of human tumors and open the door for potential therapies exploiting NO signaling to treat diseases of the lymphatic system.