RESUMO
Mating disruption of a flighted spongy moth, Lymantria dispar japonica (Motchulsky)(Lepidoptera: Lymantridae), with a synthetic version of its sex pheromone, (+)-disparlure ([7R,8S] -cis-7,8-epoxy-2- methyloctadecane), was tested in the forests in Japan. Pheromone trap catches and the percentage mating of tethered females were measured in the pheromone-treated and untreated control forests. The attraction of male moths to pheromone traps placed at a height of 1.5 m was significantly disrupted when the pheromone dispensers were placed at 1.5 m height, but many moths were captured in control plots. Mating of tethered females placed at 1.5 m was inhibited entirely, while 44% of females were mated in an untreated control forest. We report the first trial of mating disruption against a flighted spongy moth, and these results suggest that mating disruption with the synthetic sex pheromone appears promising for reducing damage caused by L. dispar japonica.
RESUMO
Here, we report the application of a long-read sequencer, PromethION, for analyzing human cancer genomes. We first conducted whole-genome sequencing on lung cancer cell lines. We found that it is possible to genotype known cancerous mutations, such as point mutations. We also found that long-read sequencing is particularly useful for precisely identifying and characterizing structural aberrations, such as large deletions, gene fusions, and other chromosomal rearrangements. In addition, we identified several medium-sized structural aberrations consisting of complex combinations of local duplications, inversions, and microdeletions. These complex mutations occurred even in key cancer-related genes, such as STK11, NF1, SMARCA4, and PTEN The biological relevance of those mutations was further revealed by epigenome, transcriptome, and protein analyses of the affected signaling pathways. Such structural aberrations were also found in clinical lung adenocarcinoma specimens. Those structural aberrations were unlikely to be reliably detected by conventional short-read sequencing. Therefore, long-read sequencing may contribute to understanding the molecular etiology of patients for whom causative cancerous mutations remain unknown and therapeutic strategies are elusive.
Assuntos
Adenocarcinoma/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Genes Neoplásicos , Sequenciamento Completo do Genoma/métodos , Linhagem Celular Tumoral , Aberrações Cromossômicas , Variações do Número de Cópias de DNA , Feminino , Perfilação da Expressão Gênica , Rearranjo Gênico , Técnicas de Genotipagem , Humanos , Masculino , Mutação , Transcrição GênicaRESUMO
Radiotherapy (RT) combined with immune checkpoint inhibitors has recently produced outstanding results and is expected to be adaptable for various cancers. However, the precise molecular mechanism by which immune reactions are induced by fractionated RT is still controversial. We aimed to investigate the mechanism of the immune response regarding multifractionated, long-term radiation, which is most often combined with immunotherapy. Two human esophageal cancer cell lines, KYSE-450 and OE-21, were irradiated by fractionated irradiation (FIR) daily at a dose of 3 Gy in 5 d/wk for 2 weeks. Western blot analysis and RNA sequencing identified type I interferon (IFN) and the stimulator of IFN genes (STING) pathway as candidates that regulate immune response by FIR. We inhibited STING, IFNAR1, STAT1, and IFN regulatory factor 1 (IRF1) and investigated the effects on the immune response in cancer cells and the invasion of surrounding immune cells. We herein revealed type I IFN-dependent immune reactions and the positive feedback of STING, IRF1, and phosphorylated STAT1 induced by FIR. Knocking out STING, IFNAR1, STAT1, and IRF1 resulted in a poorer immunological response than that in WT cells. The STING-KO KYSE-450 cell line showed significantly less invasion of PBMCs than the WT cell line under FIR. In the analysis of STING-KO cells and migrated PBMCs, we confirmed the occurrence of STING-dependent immune activation under FIR. In conclusion, we identified that the STING-IFNAR1-STAT1-IRF1 axis regulates immune reactions in cancer cells triggered by FIR and that the STING pathway also contributes to immune cell invasion of cancer cells.
Assuntos
Neoplasias Esofágicas , Imunidade , Fator Regulador 1 de Interferon , Fator de Transcrição STAT1 , Linhagem Celular/efeitos da radiação , Neoplasias Esofágicas/genética , Humanos , Imunidade/efeitos da radiação , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/metabolismo , Fator Regulador 1 de Interferon/efeitos da radiação , Interferon Tipo I , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/efeitos da radiação , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Receptor de Interferon alfa e beta/efeitos da radiação , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT1/efeitos da radiaçãoRESUMO
Cell line-derived xenograft (CDX) models created by implanting cancer cell lines into immunodeficient mice have contributed largely to the development of cancer drug therapies. However, cell lines often lose their original biological characteristics through many passages and cancer tissues in CDX models have many cancer cells and few cancer stromal cells, therefore CDX models are currently considered not suitable for predicting the results of clinical studies. Conversely, patient-derived xenograft (PDX) models are gaining importance, as human cancer biological characteristics and microenvironments are recreated by implanting tumor tissue into immunodeficient mice. These highly expected, evidently beneficial PDX models have been used in some basic research and are becoming more generalized. However, quality control and quality assurance criteria have not been established for them, and challenges and problems in the utilization of valuable PDX models in drug development have yet to be clarified. In this report, we conducted a questionnaire survey among researchers in Japanese academic institutions and pharmaceutical companies to understand the current status of PDX models in Japan. Based on the questionnaire results, we summarized the situations surrounding respondent's utilization and quality control in the development of anticancer drugs and proposed several measures to facilitate the utilization of PDX models in the development of anticancer drugs.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Modelos Animais de Doenças , Desenvolvimento de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Japão , Camundongos , Especificidade da EspécieRESUMO
Proper assembly of kinetochores (KTs) during mitosis is required for bipolar attachment of spindle microtubules (MTs) and the accumulation of spindle assembly checkpoint (SAC) components. Here we show that testis-expressed protein 14 (Tex14), which has been implicated in midbody function, is recruited to KTs by Plk1 in a Cdk1-dependent manner during early mitosis. Exclusion of Tex14 from kinetochores results in an inability to efficiently localize outer KT components, impaired KT-MT attachment, chromosome congression defects, and whole-chromosome instability. In addition, we demonstrate that phosphorylation of Tex14 by Plk1 during metaphase promotes APC(Cdc20)-mediated Tex14 degradation. Inhibition of this phosphorylation event causes retention of Tex14 at KTs and results in delayed metaphase-to-anaphase transition and chromosome segregation defects. Our findings identify Tex14 as an important mediator of KT structure and function and the fidelity of chromosome separation.
Assuntos
Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Microtúbulos/metabolismo , Fatores de Transcrição/fisiologia , Sítios de Ligação , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Segregação de Cromossomos , Células HEK293 , Células HeLa , Humanos , Cinetocoros/ultraestrutura , Microtúbulos/ultraestrutura , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Quinase 1 Polo-LikeRESUMO
In order to increase the success rate for developing new Cdc7 inhibitors for cancer therapy, we explored a new chemotype which can comply with the previously-constructed pharmacophore model. Substitution of a pyridine ring of a serendipitously-identified Cdc7 inhibitor 2b with a 3-methylpyrazole resulted in a 4-fold increase in potency and acceptable kinase selectivity, leading to the identification of thieno[3,2-d]pyrimidin-4(3H)-one as an alternative scaffold. Structure-activity relationship (SAR) study revealed that incorporation of a substituted aminomethyl group into the 2-position improved kinase selectivity. Indeed, a pyrrolidinylmethyl derivative 10c was a potent Cdc7 inhibitor (IC50=0.70nM) with high selectivity (Cdk2/Cdc7≥14,000, ROCK1/Cdc7=200). It should be noted that 10c exhibited significant time-dependent Cdc7 inhibition with slow dissociation kinetics, cellular pharmacodynamic (PD) effects, and COLO205 growth inhibition. Additionally, molecular basis of high kinase selectivity of 10c is discussed by using the protein structures of Cdc7 and Cdk2.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/química , Pirimidinonas/química , Tiofenos/síntese química , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/metabolismo , Humanos , Concentração Inibidora 50 , Cinética , Simulação de Acoplamento Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Pirimidinonas/síntese química , Pirimidinonas/farmacocinética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Relação Estrutura-Atividade , Tiofenos/química , Tiofenos/farmacocinética , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
Cell division cycle 7 (Cdc7) is a serine/threonine kinase that plays important roles in the regulation of DNA replication process. A genetic study indicates that Cdc7 inhibition can induce selective tumor-cell death in a p53-dependent manner, suggesting that Cdc7 is an attractive target for the treatment of cancers. In order to identify a new class of potent Cdc7 inhibitors, we generated a putative pharmacophore model based on in silico docking analysis of a known inhibitor with Cdc7 homology model. The pharmacophore model provided a minimum structural motif of Cdc7 inhibitor, by which preliminary medicinal chemistry efforts identified a dihydrothieno[3,2-d]-pyrimidin-4(1H)-one scaffold having a heteroaromatic hinge-binding moiety. The structure-activity relationship (SAR) studies resulted in the discovery of new, potent, and selective Cdc7 inhibitors 14a, c, e. Furthermore, the high selectivity of 14c, e for Cdc7 over Rho-associated protein kinase 1 (ROCK1) is discussed by utilizing a docking study with Cdc7 and ROCK2 crystal structures.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinonas/farmacologia , Humanos , Modelos Moleculares , Pirimidinonas/síntese química , Pirimidinonas/química , Relação Estrutura-AtividadeRESUMO
Centromere-associated protein-E (CENP-E) is a mitotic kinesin which plays roles in cell division, and is regarded as a promising therapeutic target for the next generation of anti-mitotic agents. We designed novel fused bicyclic CENP-E inhibitors starting from previous reported dihydrobenzofuran derivative (S)-(+)-1. Our design concept was to adjust the electron density distribution on the benzene ring of the dihydrobenzofuran moiety to increase the positive charge for targeting the negatively charged L5 loop of CENP-E, using predictions from electrostatic potential map (EPM) analysis. For the efficient synthesis of our 2,3-dihydro-1-benzothiophene 1,1-dioxide derivatives, a new synthetic method was developed. As a result, we discovered 6-cyano-7-trifluoromethyl-2,3-dihydro-1-benzothiophene 1,1-dioxide derivative (+)-5d (Compound A) as a potent CENP-E inhibitor with promising potential for in vivo activity. In this Letter, we discuss the design and synthetic strategy used in the discovery of (+)-5d and structure-activity relationships for its analogs possessing various fused bicyclic L5 binding moieties.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Óxidos S-Cíclicos/síntese química , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Imidazóis/síntese química , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Óxidos S-Cíclicos/química , Óxidos S-Cíclicos/farmacologia , Células HeLa , Humanos , Imidazóis/química , Imidazóis/farmacologia , Concentração Inibidora 50 , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Autophagy mediates the degradation of intracellular macromolecules and organelles within lysosomes. There are three types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. Heat shock protein 70.1 (Hsp70.1) exhibits dual functions as a chaperone protein and a lysosomal membrane stabilizer. Since chaperone-mediated autophagy participates in the recycling of â¼30% cytosolic proteins, its disorder causes cell susceptibility to stress conditions. Cargo proteins destined for degradation such as amyloid precursor protein and tau protein are trafficked by Hsp70.1 from the cytosol into lysosomes. Hsp70.1 is composed of an N-terminal nucleotide-binding domain (NBD) and a C-terminal domain that binds to cargo proteins, termed the substrate-binding domain (SBD). The NBD and SBD are connected by the interdomain linker LL1, which modulates the allosteric structure of Hsp70.1 in response to ADP/ATP binding. After the passage of the Hsp70.1-cargo complex through the lysosomal limiting membrane, high-affinity binding of the positive-charged SBD with negative-charged bis(monoacylglycero)phosphate (BMP) at the internal vesicular membranes activates acid sphingomyelinase to generate ceramide for stabilizing lysosomal membranes. As the integrity of the lysosomal limiting membrane is critical to ensure cargo protein degradation within the acidic lumen, the disintegration of the lysosomal limiting membrane is lethal to cells. After the intake of high-fat diets, however, ß-oxidation of fatty acids in the mitochondria generates reactive oxygen species, which enhance the oxidation of membrane linoleic acids to produce 4-hydroxy-2-nonenal (4-HNE). In addition, 4-HNE is produced during the heating of linoleic acid-rich vegetable oils and incorporated into the body via deep-fried foods. This endogenous and exogenous 4-HNE synergically causes an increase in its serum and organ levels to induce carbonylation of Hsp70.1 at Arg469, which facilitates its conformational change and access of activated µ-calpain to LL1. Therefore, the cleavage of Hsp70.1 occurs prior to its influx into the lysosomal lumen, which leads to lysosomal membrane permeabilization/rupture. The resultant leakage of cathepsins is responsible for lysosomal cell death, which would be one of the causative factors of lifestyle-related diseases.
RESUMO
Centromere-associated protein-E (CENP-E), a mitotic kinesin that plays an important role in mitotic progression, is an attractive target for cancer therapeutic drugs. For the purpose of developing novel CENP-E inhibitors as cancer therapeutics, we investigated a fused bicyclic compound identified by high throughput screening, 4-oxo-4,5-dihydrothieno[3,4-c]pyridine-6-carboxamide 1a. Based on this scaffold, we designed inhibitors for efficient binding at the L5 site in CENP-E utilizing homology modeling as well as electrostatic potential map (EPM) analysis to enhance CENP-E inhibitory activity. This resulted in a new lead, 5-bromoimidazo[1,2-a]pyridine 7, which showed potent CENP-E enzyme inhibition (IC50: 50nM) and cellular activity with accumulation of phosphorylated histone H3 in HeLa cells. Our homology model and EPM analysis proved to be useful tools for the rational design of CENP-E inhibitors.
Assuntos
Amidas/síntese química , Compostos Bicíclicos com Pontes/química , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Imidazóis/síntese química , Piridinas/síntese química , Amidas/química , Amidas/metabolismo , Sítios de Ligação , Proteínas Cromossômicas não Histona/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Imidazóis/química , Imidazóis/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Piridinas/química , Piridinas/metabolismo , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
Normal epithelial cells exert their competitive advantage over RasV12-transformed cells and eliminate them into the apical lumen via cell competition. However, the internal or external factors that compromise cell competition and provoke carcinogenesis remain elusive. In this study, we examine the effect of sequential accumulation of gene mutations, mimicking multi-sequential carcinogenesis on RasV12-induced cell competition in intestinal epithelial tissues. Consequently, we find that the directionality of RasV12-cell extrusion in Wnt-activated epithelia is reversed, and transformed cells are delaminated into the basal lamina via non-cell autonomous MMP21 upregulation. Subsequently, diffusively infiltrating, transformed cells develop into highly invasive carcinomas. The elevated production of MMP21 is elicited partly through NF-κB signaling, blockage of which restores apical elimination of RasV12 cells. We further demonstrate that the NF-κB-MMP21 axis is significantly bolstered in early colorectal carcinoma in humans. Collectively, this study shows that cells with high mutational burdens exploit cell competition for their benefit by behaving as unfit cells, endowing them with an invasion advantage.
Assuntos
Competição entre as Células , NF-kappa B , Animais , Cães , Humanos , Células Madin Darby de Rim Canino , Transdução de Sinais , Carcinogênese , Metaloproteinases da Matriz SecretadasRESUMO
Serine/threonine kinase, cell division cycle 7 (CDC7) is critical for initiating DNA replication. TAK-931 is a specific CDC7 inhibitor, which is a next-generation replication stress (RS) inducer. This study preclinically investigates TAK-931 antitumor efficacy and immunity regulation. TAK-931 induce RS, generating senescence-like aneuploid cells, which highly expressed inflammatory cytokines and chemokines (senescence-associated secretory phenotype, SASP). In vivo multilayer-omics analyses in gene expression panel, immune panel, immunohistochemistry, RNA sequencing, and single-cell RNA sequencing reveal that the RS-mediated aneuploid cells generated by TAK-931 intensively activate inflammatory-related and senescence-associated pathways, resulting in accumulation of tumor-infiltrating immune cells and potent antitumor immunity and efficacy. Finally, the combination of TAK-931 and immune checkpoint inhibitors profoundly enhance antiproliferative activities. These findings suggest that TAK-931 has therapeutic antitumor properties and improved clinical benefits in combination with conventional immunotherapy.
Assuntos
Proteínas de Ciclo Celular , Neoplasias , Humanos , Proteínas de Ciclo Celular/metabolismo , Inibidores de Checkpoint Imunológico , Proteínas Serina-Treonina Quinases/metabolismo , Aneuploidia , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
BACKGROUND & AIMS: Inherited mutations in the BRCA2 tumor suppressor have been associated with an increased risk of pancreatic cancer. To establish the contribution of Brca2 to pancreatic cancer we developed a mouse model of pancreas-specific Brca2 inactivation. Because BRCA2-inactivating mutations cause defects in repair of DNA double-strand breaks that result in chromosomal instability, we evaluated whether Brca2 inactivation induced instability in pancreatic tissue from these mice and whether associated pancreatic tumors were hypersensitive to DNA damaging agents. METHODS: We developed mouse models that combined pancreas-specific Kras activation and Trp53 deletion with Brca2 inactivation. Development of pancreatic cancer was assessed; tumors and nonmalignant tissues were analyzed for chromosomal instability and apoptosis. Cancer cell lines were evaluated for sensitivity to DNA damaging agents. RESULTS: In the presence of disrupted Trp53, Brca2 inactivation promoted the development of premalignant lesions and pancreatic tumors that reflected the histology of human disease. Cancer cell lines derived from these tumors were hypersensitive to specific DNA damaging agents. In contrast, in the presence of KrasG12D, Brca2 inactivation promoted chromosomal instability and apoptosis and unexpectedly inhibited growth of premalignant lesions and tumors. CONCLUSIONS: Trp53 signaling must be modified before inactivation of the Brca2 wild-type allele, irrespective of Kras status, for Brca2-deficient cells to form tumors.
Assuntos
Proteína BRCA2/genética , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Supressora de Tumor p53/genética , Animais , Apoptose/genética , Proteína BRCA2/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/fisiopatologia , Instabilidade Cromossômica/genética , Quebras de DNA de Cadeia Dupla , Modelos Animais de Doenças , Deleção de Genes , Regulação Neoplásica da Expressão Gênica/fisiologia , Camundongos , Camundongos Knockout , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/fisiopatologia , Mutação Puntual , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Lesões Pré-Cancerosas/fisiopatologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Cell division cycle 7 (CDC7), a serine/threonine kinase, plays important roles in DNA replication. We developed a highly specific CDC7 inhibitor, TAK-931, as a clinical cancer therapeutic agent. This study aimed to identify the potential combination partners of TAK-931 for guiding its clinical development strategies. Unbiased high-throughput chemical screening revealed that the highest synergistic antiproliferative effects observed were the combinations of DNA-damaging agents with TAK-931. Functional phosphoproteomic analysis demonstrated that TAK-931 suppressed homologous recombination repair activity, delayed recovery from double-strand breaks, and led to accumulation of DNA damages in the combination. Whole-genome small interfering RNA library screening identified sensitivity-modulating molecules, which propose the experimentally predicted target cancer types for the combination, including pancreatic, esophageal, ovarian, and breast cancers. The efficacy of combination therapy in these cancer types was preclinically confirmed in the corresponding primary-derived xenograft models. Thus, our findings would be helpful to guide the future clinical strategies for TAK-931.
Assuntos
Neoplasias , Reparo de DNA por Recombinação , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , DNA , Dano ao DNA , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteínas Serina-Treonina QuinasesRESUMO
Tumor heterogeneity underlies resistance to tyrosine kinase inhibitors (TKI) in lung cancers harboring EGFR mutations. Previous evidence suggested that subsets of preexisting resistant cells are selected by EGFR-TKI treatment, or alternatively, that diverse acquired resistance mechanisms emerge from drug-tolerant persister (DTP) cells. Many studies have used bulk tumor specimens or subcloned resistant cell lines to identify resistance mechanism. However, intratumoral heterogeneity can result in divergent responses to therapies, requiring additional approaches to reveal the complete spectrum of resistance mechanisms. Using EGFR-TKI-resistant cell models and clinical specimens, we performed single-cell RNA-seq and single-cell ATAC-seq analyses to define the transcriptional and epigenetic landscape of parental cells, DTPs, and tumor cells in a fully resistant state. In addition to AURKA, VIM, and AXL, which are all known to induce EGFR-TKI resistance, CD74 was identified as a novel gene that plays a critical role in the drug-tolerant state. In vitro and in vivo experiments demonstrated that CD74 upregulation confers resistance to the EGFR-TKI osimertinib and blocks apoptosis, enabling tumor regrowth. Overall, this study provides new insight into the mechanisms underlying resistance to EGFR-TKIs. SIGNIFICANCE: Single-cell analyses identify diverse mechanisms of resistance as well as the state of tolerant cells that give rise to resistance to EGFR tyrosine kinase inhibitors.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Inibidores de Proteínas Quinases/farmacologia , Análise de Célula Única , Animais , Antígenos de Diferenciação de Linfócitos B/genética , Apoptose/genética , Linhagem Celular Tumoral , Sequenciamento de Cromatina por Imunoprecipitação , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal , Receptores ErbB/antagonistas & inibidores , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/metabolismo , Camundongos , Análise de Célula Única/métodos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Single-cell level analysis is powerful tool to assess the heterogeneity of cellular components in tumor microenvironments (TME). In this study, we investigated immune-profiles using the single-cell analyses of endoscopically- or surgically-resected tumors, and peripheral blood mononuclear cells from gastric cancer patients. Furthermore, we technically characterized two distinct platforms of the single-cell analysis; RNA-seq-based analysis (scRNA-seq), and mass cytometry-based analysis (CyTOF), both of which are broadly embraced technologies. Our study revealed that the scRNA-seq analysis could cover a broader range of immune cells of TME in the biopsy-resected small samples of tumors, detecting even small subgroups of B cells or Treg cells in the tumors, although CyTOF could distinguish the specific populations in more depth. These findings demonstrate that scRNA-seq analysis is a highly-feasible platform for elucidating the complexity of TME in small biopsy tumors, which would provide a novel strategies to overcome a therapeutic difficulties against cancer heterogeneity in TME.
Assuntos
Análise de Célula Única , Neoplasias Gástricas/patologia , Microambiente Tumoral , Adulto , Biópsia , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , RNA-Seq , Neoplasias Gástricas/genéticaRESUMO
Amplification and/or overexpression of human epidermal growth factor receptor 2 (HER2) are observed in 15-20% of breast cancers (HER2+ breast cancers), and anti-HER2 therapies have significantly improved prognosis of patients with HER2+ breast cancer. One resistance mechanism to anti-HER2 therapies is constitutive activation of the phosphoinositide 3-kinase (PI3K) pathway. Combination therapy with small-molecule inhibitors of AKT and HER2 was conducted in HER2+ breast cancer cell lines with or without PIK3CA mutations, which lead to constitutive activation of the PI3K pathway. PIK3CA mutations played important roles in resistance to single-agent anti-HER2 therapy in breast cancer cell lines. Combination therapy of a HER2 inhibitor and an AKT inhibitor, as well as other PI3K pathway inhibitors, could overcome the therapeutic limitations associated with single-agent anti-HER2 treatment in PIK3CA-mutant HER2+ breast cancer cell lines. Furthermore, expression of phosphorylated 4E-binding protein 1 (p4EBP1) following the treatment correlated with the antiproliferative activities of the combination, suggesting that p4EBP1 may have potential as a prognostic and/or efficacy-linking biomarkers for these combination therapies in patients with HER2+ breast cancer. These findings highlight potential clinical strategies using combination therapy to overcome the limitations associated with single-agent anti-HER2 therapies in patients with HER2+ breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/genética , Fulvestranto/farmacologia , Fulvestranto/uso terapêutico , Lapatinib/farmacologia , Lapatinib/uso terapêutico , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Expressão Gênica , Humanos , Mutação , Fosforilação , Proteínas de Ligação a RNA/metabolismoRESUMO
In our pursuit of developing a novel, potent, and selective cell division cycle 7 (Cdc7) inhibitor, we optimized the previously reported thieno[3,2-d]pyrimidinone analogue I showing time-dependent Cdc7 kinase inhibition and slow dissociation kinetics. These medicinal chemistry efforts led to the identification of compound 3d, which exhibited potent cellular activity, excellent kinase selectivity, and antitumor efficacy in a COLO205 xenograft mouse model. However, the issue of formaldehyde adduct formation emerged during a detailed study of 3d, which was deemed an obstacle to further development. A structure-based approach to circumvent the adduct formation culminated in the discovery of compound 11b (TAK-931) possessing a quinuclidine moiety as a preclinical candidate. In this paper, the design, synthesis, and biological evaluation of this series of compounds will be presented.
Assuntos
Antineoplásicos/uso terapêutico , Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazolonas/uso terapêutico , Pirimidinas/uso terapêutico , Pirimidinonas/uso terapêutico , Quinuclidinas/uso terapêutico , Tiofenos/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Desenho de Fármacos , Descoberta de Drogas , Formaldeído/química , Humanos , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Pirazolonas/farmacologia , Pirimidinas/farmacologia , Pirimidinonas/síntese química , Pirimidinonas/metabolismo , Quinuclidinas/síntese química , Quinuclidinas/metabolismo , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Despite the worldwide approval of three generations of EGFR tyrosine kinase inhibitors (TKI) for advanced non-small cell lung cancers with EGFR mutations, no TKI with a broad spectrum of activity against all clinically relevant mutations is currently available. In this study, we sought to evaluate a covalent mutation-specific EGFR TKI, TAS6417 (also named CLN-081), with the broadest level of activity against EGFR mutations with a prevalence of ≥1%. Lung cancer and genetically engineered cell lines, as well as murine xenograft models were used to evaluate the efficacy of TAS6417 and other approved/in-development EGFR TKIs (erlotinib, afatinib, osimertinib, and poziotinib). We demonstrate that TAS6417 is a robust inhibitor against the most common EGFR mutations (exon 19 deletions and L858R) and the most potent against cells harboring EGFR-T790M (first/second-generation TKI resistance mutation). In addition, TAS6417 has activity in cells driven by less common EGFR-G719X, L861Q, and S768I mutations. For recalcitrant EGFR exon 20 insertion mutations, selectivity indexes (wild-type EGFR/mutant EGFR ratio of inhibition) favored TAS6417 in comparison with poziotinib and osimertinib, indicating a wider therapeutic window. Taken together, we demonstrate that TAS6417 is a potent EGFR TKI with a broad spectrum of activity and a wider therapeutic window than most approved/in-development generations of EGFR inhibitors. IMPLICATIONS: TAS6417/CLN-081 is a potent EGFR TKI with a wide therapeutic window and may be effective in lung cancer patients with clinically relevant EGFR mutations.
Assuntos
Antineoplásicos/farmacologia , Derivados de Benzeno/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Acrilamidas/farmacologia , Afatinib/farmacologia , Compostos de Anilina/farmacologia , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/farmacologia , Éxons/genética , Humanos , Indolizinas , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Mutação , Quinazolinas/farmacologiaRESUMO
Replication stress (RS) is a cancer hallmark; chemotherapeutic drugs targeting RS are widely used as treatments for various cancers. To develop next-generation RS-inducing anticancer drugs, cell division cycle 7 (CDC7) has recently attracted attention as a target. We have developed an oral CDC7-selective inhibitor, TAK-931, as a candidate clinical anticancer drug. TAK-931 induced S phase delay and RS. TAK-931-induced RS caused mitotic aberrations through centrosome dysregulation and chromosome missegregation, resulting in irreversible antiproliferative effects in cancer cells. TAK-931 exhibited significant antiproliferative activity in preclinical animal models. Furthermore, in indication-seeking studies using large-scale cell panel data, TAK-931 exhibited higher antiproliferative activities in RAS-mutant versus RAS-wild-type cells; this finding was confirmed in pancreatic patient-derived xenografts. Comparison analysis of cell panel data also demonstrated a unique efficacy spectrum for TAK-931 compared with currently used chemotherapeutic drugs. Our findings help to elucidate the molecular mechanisms for TAK-931 and identify potential target indications.