RESUMO
Elesclomol is an investigational drug that exerts potent anticancer activity through the elevation of reactive oxygen species (ROS) levels and is currently under clinical evaluation as a novel anticancer therapeutic. Here we report the first description of selective mitochondrial ROS induction by elesclomol in cancer cells based on the unique physicochemical properties of the compound. Elesclomol preferentially chelates copper (Cu) outside of cells and enters as elesclomol-Cu(II). The elesclomol-Cu(II) complex then rapidly and selectively transports the copper to mitochondria. In this organelle Cu(II) is reduced to Cu(I), followed by subsequent ROS generation. Upon dissociation from the complex, elesclomol is effluxed from cells and repeats shuttling elesclomol-Cu complexes from the extracellular to the intracellular compartments, leading to continued copper accumulation within mitochondria. An optimal range of redox potentials exhibited by copper chelates of elesclomol and its analogs correlated with the elevation of mitochondrial Cu(I) levels and cytotoxic activity, suggesting that redox reduction of the copper triggers mitochondrial ROS induction. Importantly the mitochondrial selectivity exhibited by elesclomol is a distinct characteristic of the compound that is not shared by other chelators, including disulfiram. Together these findings highlight a unique mechanism of action with important implications for cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Cobre/metabolismo , Hidrazinas/farmacologia , Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Cobre/química , Humanos , Mitocôndrias/efeitos dos fármacos , Neoplasias/metabolismo , Oxirredução/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Systemic chemotherapy using two-drug platinum-based regimens for the treatment of advanced stage non-small cell lung cancer (NSCLC) has largely reached a plateau of effectiveness. Accordingly, efforts to improve survival and quality of life outcomes have more recently focused on the use of molecularly targeted agents, either alone or in combination with standard of care therapies such as taxanes. The molecular chaperone heat shock protein 90 (Hsp90) represents an attractive candidate for therapeutic intervention, as its inhibition results in the simultaneous blockade of multiple oncogenic signaling cascades. Ganetespib is a non-ansamycin inhibitor of Hsp90 currently under clinical evaluation in a number of human malignancies, including NSCLC. Here we show that ganetespib potentiates the cytotoxic activity of the taxanes paclitaxel and docetaxel in NSCLC models. The combination of ganetespib with paclitaxel, docetaxel or another microtubule-targeted agent vincristine resulted in synergistic antiproliferative effects in the H1975 cell line in vitro. These benefits translated to improved efficacy in H1975 xenografts in vivo, with significantly enhanced tumor growth inhibition observed in combination with paclitaxel and tumor regressions seen with docetaxel. Notably, concurrent exposure to ganetespib and docetaxel improved antitumor activity in 5 of 6 NSCLC xenograft models examined. Our data suggest that the improved therapeutic indices are likely to be mechanistically multifactorial, including loss of pro-survival signaling and direct cell cycle effects resulting from Hsp90 modulation by ganetespib. Taken together, these findings provide preclinical evidence for the use of this combination to treat patients with advanced NSCLC.
Assuntos
Antineoplásicos/administração & dosagem , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Paclitaxel/administração & dosagem , Taxoides/administração & dosagem , Triazóis/administração & dosagem , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Docetaxel , Combinação de Medicamentos , Feminino , Humanos , Camundongos , Camundongos SCID , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Elesclomol is a first-in-class investigational drug currently undergoing clinical evaluation as a novel cancer therapeutic. The potent antitumor activity of the compound results from the elevation of reactive oxygen species (ROS) and oxidative stress to levels incompatible with cellular survival. However, the molecular target(s) and mechanism by which elesclomol generates ROS and subsequent cell death were previously undefined. The cellular cytotoxicity of elesclomol in the yeast S. cerevisiae appears to occur by a mechanism similar, if not identical, to that in cancer cells. Accordingly, here we used a powerful and validated technology only available in yeast that provides critical insights into the mechanism of action, targets and processes that are disrupted by drug treatment. Using this approach we show that elesclomol does not work through a specific cellular protein target. Instead, it targets a biologically coherent set of processes occurring in the mitochondrion. Specifically, the results indicate that elesclomol, driven by its redox chemistry, interacts with the electron transport chain (ETC) to generate high levels of ROS within the organelle and consequently cell death. Additional experiments in melanoma cells involving drug treatments or cells lacking ETC function confirm that the drug works similarly in human cancer cells. This deeper understanding of elesclomol's mode of action has important implications for the therapeutic application of the drug, including providing a rationale for biomarker-based stratification of patients likely to respond in the clinical setting.
Assuntos
Antineoplásicos/farmacologia , Hidrazinas/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cobre/farmacologia , DNA Mitocondrial/genética , Ensaios de Seleção de Medicamentos Antitumorais , Transporte de Elétrons/efeitos dos fármacos , Humanos , Hidrazinas/química , Hidrazinas/uso terapêutico , Melanoma/tratamento farmacológico , Mutação/genética , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Targeted inhibition of the molecular chaperone Hsp90 results in the simultaneous blockade of multiple oncogenic signaling pathways and has, thus, emerged as an attractive strategy for the development of novel cancer therapeutics. Ganetespib (formerly known as STA-9090) is a unique resorcinolic triazolone inhibitor of Hsp90 that is currently in clinical trials for a number of human cancers. In the present study, we showed that ganetespib exhibits potent in vitro cytotoxicity in a range of solid and hematologic tumor cell lines, including those that express mutated kinases that confer resistance to small-molecule tyrosine kinase inhibitors. Ganetespib treatment rapidly induced the degradation of known Hsp90 client proteins, displayed superior potency to the ansamycin inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), and exhibited sustained activity even with short exposure times. In vivo, ganetespib showed potent antitumor efficacy in solid and hematologic xenograft models of oncogene addiction, as evidenced by significant growth inhibition and/or regressions. Notably, evaluation of the microregional activity of ganetespib in tumor xenografts showed that ganetespib was efficiently distributed throughout tumor tissue, including hypoxic regions >150 µm from the microvasculature, to inhibit proliferation and induce apoptosis. Importantly, ganetespib showed no evidence of cardiac or liver toxicity. Taken together, this preclinical activity profile indicates that ganetespib may have broad application for a variety of human malignancies, and with select mechanistic and safety advantages over other first- and second-generation Hsp90 inhibitors.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Triazóis/farmacologia , Animais , Antineoplásicos/efeitos adversos , Apoptose/efeitos dos fármacos , Benzoquinonas/efeitos adversos , Benzoquinonas/farmacologia , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Cristalografia por Raios X , Feminino , Células HL-60 , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Coração/efeitos dos fármacos , Coração/fisiologia , Humanos , Células K562 , Lactamas Macrocíclicas/efeitos adversos , Lactamas Macrocíclicas/farmacologia , Masculino , Camundongos , Camundongos Nus , Camundongos SCID , Neoplasias/metabolismo , Neoplasias/patologia , Coelhos , Ratos , Ratos Sprague-Dawley , Triazóis/efeitos adversos , Triazóis/química , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
There is accumulating evidence that dysregulated JAK signaling occurs in a wide variety of cancer types. In particular, mutations in JAK2 can result in the constitutive activation of STAT transcription factors and lead to oncogenic growth. JAK kinases are established Hsp90 client proteins and here we show that the novel small molecule Hsp90 inhibitor ganetespib (formerly STA-9090) exhibits potent in vitro and in vivo activity in a range of solid and hematological tumor cells that are dependent on JAK2 activity for growth and survival. Of note, ganetespib treatment results in sustained depletion of JAK2, including the constitutively active JAK2(V617F) mutant, with subsequent loss of STAT activity and reduced STAT-target gene expression. In contrast, treatment with the pan-JAK inhibitor P6 results in only transient effects on these processes. Further differentiating these modes of intervention, RNA and protein expression studies show that ganetespib additionally modulates cell cycle regulatory proteins, while P6 does not. The concomitant impact of ganetespib on both cell growth and cell division signaling translates to potent antitumor efficacy in mouse models of xenografts and disseminated JAK/STAT-driven leukemia. Overall, our findings support Hsp90 inhibition as a novel therapeutic approach for combating diseases dependent on JAK/STAT signaling, with the multimodal action of ganetespib demonstrating advantages over JAK-specific inhibitors.
Assuntos
Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Janus Quinase 2/metabolismo , Neoplasias Experimentais/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Triazóis/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Genes cdc , Camundongos , Neoplasias Experimentais/patologia , Células Tumorais CultivadasRESUMO
The aberrant overexpression of Wilms tumor 1 (WT1) in myeloid leukemia plays an important role in blast cell survival and resistance to chemotherapy. High expression of WT1 is also associated with relapse and shortened disease-free survival in patients. However, the mechanisms by which WT1 expression is regulated in leukemia remain unclear. Here, we report that heat shock protein 90 (Hsp90), which plays a critical role in the folding and maturation of several oncogenic proteins, associates with WT1 protein and stabilizes its expression. Pharmacologic inhibition of Hsp90 resulted in ubiquitination and subsequent proteasome-dependant degradation of WT1. RNAi-mediated silencing of WT1 reduced the survival of leukemia cells and increased the sensitivity of these cells to chemotherapy and Hsp90 inhibition. Furthermore, Hsp90 inhibitors 17-AAG [17-(allylamino)-17-demethoxygeldanamycin] and STA-9090 significantly reduced the growth of myeloid leukemia xenografts in vivo and effectively down-regulated the expression of WT1 and its downstream target proteins, c-Myc and Bcl-2. Collectively, our studies identify WT1 as a novel Hsp90 client and support the crucial role for the WT1-Hsp90 interaction in maintaining leukemia cell survival. These findings have significant implications for developing effective therapies for myeloid leukemias and offer a strategy to inhibit the oncogenic functions of WT1 by clinically available Hsp90 inhibitors.
Assuntos
Regulação Leucêmica da Expressão Gênica , Proteínas de Choque Térmico HSP90/metabolismo , Leucemia Mieloide/genética , Proteínas WT1/genética , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Benzoquinonas/farmacologia , Benzoquinonas/uso terapêutico , Linhagem Celular Tumoral , Etoposídeo/farmacologia , Feminino , Inativação Gênica , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/química , Humanos , Lactamas Macrocíclicas/farmacologia , Lactamas Macrocíclicas/uso terapêutico , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/metabolismo , Camundongos , Camundongos SCID , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Triazóis/uso terapêutico , Proteínas WT1/química , Proteínas WT1/metabolismoRESUMO
The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35-60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies.
Assuntos
Cromatografia Líquida/métodos , Proteoma , Espectrometria de Massas em Tandem/métodos , Reprodutibilidade dos TestesRESUMO
A major unmet need in LC-MS/MS-based proteomics analyses is a set of tools for quantitative assessment of system performance and evaluation of technical variability. Here we describe 46 system performance metrics for monitoring chromatographic performance, electrospray source stability, MS1 and MS2 signals, dynamic sampling of ions for MS/MS, and peptide identification. Applied to data sets from replicate LC-MS/MS analyses, these metrics displayed consistent, reasonable responses to controlled perturbations. The metrics typically displayed variations less than 10% and thus can reveal even subtle differences in performance of system components. Analyses of data from interlaboratory studies conducted under a common standard operating procedure identified outlier data and provided clues to specific causes. Moreover, interlaboratory variation reflected by the metrics indicates which system components vary the most between laboratories. Application of these metrics enables rational, quantitative quality assessment for proteomics and other LC-MS/MS analytical applications.
Assuntos
Cromatografia Líquida/métodos , Cromatografia Líquida/normas , Proteômica/métodos , Proteômica/normas , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massas em Tandem/normas , Animais , Galinhas , Proteínas do Ovo/análise , Laboratórios , Proteoma/análise , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/análise , SoftwareRESUMO
Optimal performance of LC-MS/MS platforms is critical to generating high quality proteomics data. Although individual laboratories have developed quality control samples, there is no widely available performance standard of biological complexity (and associated reference data sets) for benchmarking of platform performance for analysis of complex biological proteomes across different laboratories in the community. Individual preparations of the yeast Saccharomyces cerevisiae proteome have been used extensively by laboratories in the proteomics community to characterize LC-MS platform performance. The yeast proteome is uniquely attractive as a performance standard because it is the most extensively characterized complex biological proteome and the only one associated with several large scale studies estimating the abundance of all detectable proteins. In this study, we describe a standard operating protocol for large scale production of the yeast performance standard and offer aliquots to the community through the National Institute of Standards and Technology where the yeast proteome is under development as a certified reference material to meet the long term needs of the community. Using a series of metrics that characterize LC-MS performance, we provide a reference data set demonstrating typical performance of commonly used ion trap instrument platforms in expert laboratories; the results provide a basis for laboratories to benchmark their own performance, to improve upon current methods, and to evaluate new technologies. Additionally, we demonstrate how the yeast reference, spiked with human proteins, can be used to benchmark the power of proteomics platforms for detection of differentially expressed proteins at different levels of concentration in a complex matrix, thereby providing a metric to evaluate and minimize pre-analytical and analytical variation in comparative proteomics experiments.
Assuntos
Cromatografia Líquida/métodos , Cromatografia Líquida/normas , Técnicas de Laboratório Clínico/normas , Espectrometria de Massas/métodos , Espectrometria de Massas/normas , Proteínas de Saccharomyces cerevisiae/análise , Saccharomyces cerevisiae/metabolismo , Biomarcadores/metabolismo , Humanos , Proteômica/normasRESUMO
Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low mug/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma.
Assuntos
Proteínas Sanguíneas/análise , Espectrometria de Massas/métodos , Biomarcadores/sangue , Análise Química do Sangue/métodos , Humanos , Modelos Lineares , Espectrometria de Massas/normas , Proteoma/análise , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Avaliação da Tecnologia BiomédicaRESUMO
MLN944 (XR5944) is a novel bis-phenazine that has demonstrated exceptional efficacy against a number of murine and human tumor models. The drug was reported originally as a dual topoisomerase I/II poison, but a precise mechanism of action for this compound remains to be determined. Several lines of evidence, including the marginal ability of MLN944 to stabilize topoisomerase-dependent cleavage, and the sustained potency of MLN944 in mammalian cells with reduced levels of both topoisomerases, suggest that other activities of the drug exist. In this study, we show that MLN944 intercalates into DNA, but has no effect on the catalytic activity of either topoisomerase I or II. MLN944 displays no significant ability to stimulate DNA scission mediated by either topoisomerase I or II compared with camptothecin or etoposide, respectively. In addition, yeast genetic models also point toward a topoisomerase-independent mechanism of action. To examine cell cycle effects, synchronized human HCT116 cells were treated with MLN944, doxorubicin, camptothecin, or a combination of the latter two to mimic a dual topoisomerase poison. MLN944 treatment was found to induce a G(1) and G(2) arrest in cells that is unlike the typical G(2)-M arrest noted with known topoisomerase poisons. Finally, transcriptional profiling analysis of xenograft tumors treated with MLN944 revealed clusters of regulated genes distinct from those observed in irinotecan hydrochloride (CPT-11)-treated tumors. Taken together, these findings suggest that the primary mechanism of action of MLN944 likely involves DNA binding and intercalation, but does not appear to involve topoisomerase inhibition.
Assuntos
Camptotecina/análogos & derivados , Substâncias Intercalantes/farmacologia , Fenazinas/farmacologia , Animais , Antígenos de Neoplasias , Camptotecina/farmacologia , Catálise/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Análise por Conglomerados , DNA/química , DNA/metabolismo , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA , Relação Dose-Resposta a Droga , Fase G1/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Perfilação da Expressão Gênica , Células HCT116 , Humanos , Substâncias Intercalantes/química , Irinotecano , Masculino , Camundongos , Camundongos Nus , Mitose/efeitos dos fármacos , Mutação , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Fenazinas/química , Transplante Heterólogo , Ensaios Antitumorais Modelo de Xenoenxerto , Leveduras/efeitos dos fármacos , Leveduras/enzimologia , Leveduras/genéticaRESUMO
A genetic approach utilizing the yeast Saccharomyces cerevisiae was used to identify the target of antifungal compounds. This analysis led to the identification of small molecule inhibitors of RNA polymerase (Pol) III from Saccharomyces cerevisiae. Three lines of evidence show that UK-118005 inhibits cell growth by targeting RNA Pol III in yeast. First, a dominant mutation in the g domain of Rpo31p, the largest subunit of RNA Pol III, confers resistance to the compound. Second, UK-118005 rapidly inhibits tRNA synthesis in wild-type cells but not in UK-118005 resistant mutants. Third, in biochemical assays, UK-118005 inhibits tRNA gene transcription in vitro by the wild-type but not the mutant Pol III enzyme. By testing analogs of UK-118005 in a template-specific RNA Pol III transcription assay, an inhibitor with significantly higher potency, ML-60218, was identified. Further examination showed that both compounds are broad-spectrum inhibitors, displaying activity against RNA Pol III transcription systems derived from Candida albicans and human cells. The identification of these inhibitors demonstrates that RNA Pol III can be targeted by small synthetic molecules.
Assuntos
Antifúngicos/farmacologia , Inibidores Enzimáticos/farmacologia , RNA Polimerase III/antagonistas & inibidores , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Candida albicans/genética , Células Cultivadas , Relação Dose-Resposta a Droga , Farmacorresistência Fúngica/genética , Humanos , Dados de Sequência Molecular , Peso Molecular , Mutação/genética , Subunidades Proteicas/genética , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , RNA de Transferência/biossíntese , RNA de Transferência/genética , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/genética , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
Although the biochemical targets of most drugs are known, the biological consequences of their actions are typically less well understood. In this study, we have used two whole-genome technologies in Saccharomyces cerevisiae to determine the cellular impact of the proteasome inhibitor PS-341. By combining population genomics, the screening of a comprehensive panel of bar-coded mutant strains, and transcript profiling, we have identified the genes and pathways most affected by proteasome inhibition. Many of these function in regulated protein degradation or a subset of mitotic activities. In addition, we identified Rpn4p as the transcription factor most responsible for the cell's ability to compensate for proteasome inhibition. Used together, these complementary technologies provide a general and powerful means to elucidate the cellular ramifications of drug treatment.