RESUMO
Immune checkpoint inhibitors have shown significant therapeutic responses against tumors containing increased mutation-associated neoantigen load. We have examined the evolving landscape of tumor neoantigens during the emergence of acquired resistance in patients with non-small cell lung cancer after initial response to immune checkpoint blockade with anti-PD-1 or anti-PD-1/anti-CTLA-4 antibodies. Analyses of matched pretreatment and resistant tumors identified genomic changes resulting in loss of 7 to 18 putative mutation-associated neoantigens in resistant clones. Peptides generated from the eliminated neoantigens elicited clonal T-cell expansion in autologous T-cell cultures, suggesting that they generated functional immune responses. Neoantigen loss occurred through elimination of tumor subclones or through deletion of chromosomal regions containing truncal alterations, and was associated with changes in T-cell receptor clonality. These analyses provide insight into the dynamics of mutational landscapes during immune checkpoint blockade and have implications for the development of immune therapies that target tumor neoantigens.Significance: Acquired resistance to immune checkpoint therapy is being recognized more commonly. This work demonstrates for the first time that acquired resistance to immune checkpoint blockade can arise in association with the evolving landscape of mutations, some of which encode tumor neoantigens recognizable by T cells. These observations imply that widening the breadth of neoantigen reactivity may mitigate the development of acquired resistance. Cancer Discov; 7(3); 264-76. ©2017 AACR.See related commentary by Yang, p. 250This article is highlighted in the In This Issue feature, p. 235.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/terapia , Resistencia a Medicamentos Antineoplásicos/imunologia , Neoplasias Pulmonares/terapia , Receptores de Antígenos de Linfócitos T/genética , Adulto , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antígenos de Neoplasias/imunologia , Antineoplásicos Imunológicos/farmacologia , Antígeno CTLA-4/genética , Antígeno CTLA-4/imunologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/imunologia , Estudos de Coortes , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Imunoterapia , Ipilimumab/farmacologia , Ipilimumab/uso terapêutico , Janus Quinase 1/genética , Janus Quinase 2/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Nivolumabe , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Targeting surface receptors overexpressed on cancer cells is one way to specifically treat cancer versus normal cells. Vintafolide (EC145), which consists of folate linked to a cytotoxic small molecule, desacetylvinblastine hydrazide (DAVLBH), takes advantage of the overexpression of folate receptor (FR) on cancer cells. Once bound to FR, vintafolide enters the cell by endocytosis, and the reducing environment of the endosome cleaves the linker, releasing DAVLBH to destabilize microtubules. Vintafolide has shown efficacy and improved tolerability compared with DAVLBH in FR-positive preclinical models. As the first FR-targeting drug to reach the clinic, vintafolide has achieved favorable responses in phase II clinical trials in FR-positive ovarian and lung cancer. However, some FR-positive patients in these clinical trials do not respond to vintafolide. We sought to identify potential biomarkers of resistance to aid in the future development of this and other FR-targeting drugs. Here, we confirm that high P-glycoprotein (P-gp) expression was the strongest predictor of resistance to DAVLBH in a panel of 359 cancer cell lines. Furthermore, targeted delivery of DAVLBH via the FR, as in vintafolide, fails to overcome P-gp-mediated efflux of DAVLBH in both in vitro and in vivo preclinical models. Therefore, we suggest that patients whose tumors express high levels of P-gp be excluded from future clinical trials for vintafolide as well as other FR-targeted therapeutics bearing a P-gp substrate. Mol Cancer Ther; 15(8); 1998-2008. ©2016 AACR.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Ácido Fólico/análogos & derivados , Expressão Gênica , Alcaloides de Vinca/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Análise por Conglomerados , Biologia Computacional/métodos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Receptores de Folato com Âncoras de GPI/antagonistas & inibidores , Ácido Fólico/farmacologia , Perfilação da Expressão Gênica , Humanos , Camundongos , Platina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Combination drug therapy is a widely used paradigm for managing numerous human malignancies. In cancer treatment, additive and/or synergistic drug combinations can convert weakly efficacious monotherapies into regimens that produce robust antitumor activity. This can be explained in part through pathway interdependencies that are critical for cancer cell proliferation and survival. However, identification of the various interdependencies is difficult due to the complex molecular circuitry that underlies tumor development and progression. Here, we present a high-throughput platform that allows for an unbiased identification of synergistic and efficacious drug combinations. In a screen of 22,737 experiments of 583 doublet combinations in 39 diverse cancer cell lines using a 4 by 4 dosing regimen, both well-known and novel synergistic and efficacious combinations were identified. Here, we present an example of one such novel combination, a Wee1 inhibitor (AZD1775) and an mTOR inhibitor (ridaforolimus), and demonstrate that the combination potently and synergistically inhibits cancer cell growth in vitro and in vivo This approach has identified novel combinations that would be difficult to reliably predict based purely on our current understanding of cancer cell biology. Mol Cancer Ther; 15(6); 1155-62. ©2016 AACR.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios de Triagem em Larga Escala/métodos , Neoplasias Experimentais/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Camundongos , Pirazóis/administração & dosagem , Pirazóis/farmacologia , Pirimidinas/administração & dosagem , Pirimidinas/farmacologia , Pirimidinonas , Distribuição Aleatória , Sirolimo/administração & dosagem , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Massively parallel sequencing approaches are beginning to be used clinically to characterize individual patient tumors and to select therapies based on the identified mutations. A major question in these analyses is the extent to which these methods identify clinically actionable alterations and whether the examination of the tumor tissue alone is sufficient or whether matched normal DNA should also be analyzed to accurately identify tumor-specific (somatic) alterations. To address these issues, we comprehensively evaluated 815 tumor-normal paired samples from patients of 15 tumor types. We identified genomic alterations using next-generation sequencing of whole exomes or 111 targeted genes that were validated with sensitivities >95% and >99%, respectively, and specificities >99.99%. These analyses revealed an average of 140 and 4.3 somatic mutations per exome and targeted analysis, respectively. More than 75% of cases had somatic alterations in genes associated with known therapies or current clinical trials. Analyses of matched normal DNA identified germline alterations in cancer-predisposing genes in 3% of patients with apparently sporadic cancers. In contrast, a tumor-only sequencing approach could not definitively identify germline changes in cancer-predisposing genes and led to additional false-positive findings comprising 31% and 65% of alterations identified in targeted and exome analyses, respectively, including in potentially actionable genes. These data suggest that matched tumor-normal sequencing analyses are essential for precise identification and interpretation of somatic and germline alterations and have important implications for the diagnostic and therapeutic management of cancer patients.
Assuntos
Análise Mutacional de DNA , Genômica , Mutação , Neoplasias/genética , Medicina de Precisão , Biologia Computacional , Exoma , Reações Falso-Positivas , Biblioteca Gênica , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Polimorfismo de Nucleotídeo Único , Estudos RetrospectivosRESUMO
PURPOSE: Mammalian target of rapamycin (mTOR) inhibition activates compensatory insulin-like growth factor receptor (IGFR) signaling. We evaluated the ridaforolimus (mTOR inhibitor) and dalotuzumab (anti-IGF1R antibody) combination. EXPERIMENTAL DESIGN: In vitro and in vivo models, and a phase I study in which patients with advanced cancer received ridaforolimus (10-40 mg/day every day × 5/week) and dalotuzumab (10 mg/kg/week or 7.5 mg/kg/every other week) were explored. RESULTS: Preclinical studies demonstrated enhanced pathway inhibition with ridaforolimus and dalotuzumab. With 87 patients treated in the phase I study, main dose-limiting toxicities (DLT) of the combination were primarily mTOR-related stomatitis and asthenia at doses of ridaforolimus lower than expected, suggesting blockade of compensatory pathways in normal tissues. Six confirmed partial responses were reported (3 patients with breast cancer); 10 of 23 patients with breast cancer and 6 of 11 patients with ER(+)/high-proliferative breast cancer showed antitumor activity. CONCLUSIONS: Our study provides proof-of-concept that inhibiting the IGF1R compensatory response to mTOR inhibition is feasible with promising clinical activity in heavily pretreated advanced cancer, particularly in ER(+)/high-proliferative breast cancer (ClinicalTrials.gov identifier: NCT00730379).
Assuntos
Anticorpos Monoclonais/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Sirolimo/análogos & derivados , Adulto , Idoso , Animais , Anticorpos Monoclonais Humanizados , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Humanos , Pessoa de Meia-Idade , Receptor IGF Tipo 1 , Receptores de Somatomedina/antagonistas & inibidores , Receptores de Somatomedina/imunologia , Receptores de Somatomedina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/administração & dosagem , Serina-Treonina Quinases TOR/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Dinaciclib is a potent CDK1, 2, 5 and 9 inhibitor being developed for the treatment of cancer. Additional understanding of antitumor mechanisms and identification of predictive biomarkers are important for its clinical development. Here we demonstrate that while dinaciclib can effectively block cell cycle progression, in vitro and in vivo studies, coupled with mouse and human pharmacokinetics, support a model whereby induction of apoptosis is a main mechanism of dinaciclib's antitumor effect and relevant to the clinical duration of exposure. This was further underscored by kinetics of dinaciclib-induced downregulation of the antiapoptotic BCL2 family member MCL1 and correlation of sensitivity with the MCL1-to-BCL-xL mRNA ratio or MCL1 amplification in solid tumor models in vitro and in vivo. This MCL1-dependent apoptotic mechanism was additionally supported by synergy with the BCL2, BCL-xL and BCL-w inhibitor navitoclax (ABT-263). These results provide the rationale for investigating MCL1 and BCL-xL as predictive biomarkers for dinaciclib antitumor response and testing combinations with BCL2 family member inhibitors.
Assuntos
Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neoplasias/metabolismo , Compostos de Piridínio/farmacologia , Proteína bcl-X/metabolismo , Compostos de Anilina/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Óxidos N-Cíclicos , Modelos Animais de Doenças , Diterpenos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Compostos de Epóxi/farmacologia , Feminino , Dosagem de Genes , Humanos , Indolizinas , Masculino , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Neoplasias/genética , Fenantrenos/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/genéticaRESUMO
UNLABELLED: Next-generation sequencing was used to identify Notch mutations in a large collection of diverse solid tumors. NOTCH1 and NOTCH2 rearrangements leading to constitutive receptor activation were confined to triple-negative breast cancers (TNBC; 6 of 66 tumors). TNBC cell lines with NOTCH1 rearrangements associated with high levels of activated NOTCH1 (N1-ICD) were sensitive to the gamma-secretase inhibitor (GSI) MRK-003, both alone and in combination with paclitaxel, in vitro and in vivo, whereas cell lines with NOTCH2 rearrangements were resistant to GSI. Immunohistochemical staining of N1-ICD in TNBC xenografts correlated with responsiveness, and expression levels of the direct Notch target gene HES4 correlated with outcome in patients with TNBC. Activating NOTCH1 point mutations were also identified in other solid tumors, including adenoid cystic carcinoma (ACC). Notably, ACC primary tumor xenografts with activating NOTCH1 mutations and high N1-ICD levels were sensitive to GSI, whereas N1-ICD-low tumors without NOTCH1 mutations were resistant. SIGNIFICANCE: NOTCH1 mutations, immunohistochemical staining for activated NOTCH1, and HES4 expression are biomarkers that can be used to identify solid tumors that are likely to respond to GSI-based therapies.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Antineoplásicos/farmacologia , Carcinoma Adenoide Cístico/genética , Inibidores de Proteases/farmacologia , Neoplasias de Mama Triplo Negativas/genética , Animais , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Apoptose/genética , Biomarcadores , Carcinoma Adenoide Cístico/tratamento farmacológico , Carcinoma Adenoide Cístico/metabolismo , Linhagem Celular Tumoral , Senescência Celular/efeitos dos fármacos , Óxidos S-Cíclicos/farmacologia , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Exoma , Feminino , Regulação Neoplásica da Expressão Gênica , Rearranjo Gênico , Genes myc , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos Moleculares , Mutação , Prognóstico , Inibidores de Proteases/administração & dosagem , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores Notch/antagonistas & inibidores , Receptores Notch/química , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiadiazóis/farmacologia , Resultado do Tratamento , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Activation of the phosphoinositide 3-kinase pathway is commonly observed in human prostate cancer. Loss of function of phosphatase and tensin homolog (PTEN) is associated with the activation of AKT and mammalian target of rapamycin (mTOR) in many cancer cell lines as well as in other model systems. However, activation of mTOR is also dependent of kinases other than AKT. Here, we show that activation of mTOR is not dependent on AKT in a prostate-specific PTEN-deficient mouse model of prostate cancer. Pathway bifurcation of AKT and mTOR was noted in both mouse and human prostate tumors. We demonstrated for the first time that cotargeting mTOR and AKT with ridaforolimus/MK-8669 and M1K-2206, respectively, delivers additive antitumor effects in vivo when compared to single agents. Our preclinical data suggest that the combination of AKT and mTOR inhibitors might be more effective in treating prostate cancer patients than current treatment regimens or either treatment alone.
RESUMO
Although we have made great progress in understanding the complex genetic alterations that underlie human cancer, it has proven difficult to identify which molecularly targeted therapeutics will benefit which patients. Drug-specific modulation of oncogenic signaling pathways in specific patient subpopulations can predict responsiveness to targeted therapy. Here, we report a pathway-based phosphoprofiling approach to identify and quantify clinically relevant, drug-specific biomarkers for phosphatidylinositol 3-kinase (PI3K) pathway inhibitors that target AKT, phosphoinositide-dependent kinase 1 (PDK1), and PI3K-mammalian target of rapamycin (mTOR). We quantified 375 nonredundant PI3K pathway-relevant phosphopeptides, all containing AKT, PDK1, or mitogen-activated protein kinase substrate recognition motifs. Of these phosphopeptides, 71 were drug-regulated, 11 of them by all three inhibitors. Drug-modulated phosphoproteins were enriched for involvement in cytoskeletal reorganization (filamin, stathmin, dynamin, PAK4, and PTPN14), vesicle transport (LARP1, VPS13D, and SLC20A1), and protein translation (S6RP and PRAS40). We then generated phosphospecific antibodies against selected, drug-regulated phosphorylation sites that would be suitable as biomarker tools for PI3K pathway inhibitors. As proof of concept, we show clinical translation feasibility for an antibody against phospho-PRAS40(Thr246). Evaluation of binding of this antibody in human cancer cell lines, a PTEN (phosphatase and tensin homolog deleted from chromosome 10)-deficient mouse prostate tumor model, and triple-negative breast tumor tissues showed that phospho-PRAS40(Thr246) positively correlates with PI3K pathway activation and predicts AKT inhibitor sensitivity. In contrast to phosphorylation of AKT(Thr308), the phospho-PRAS40(Thr246) epitope is highly stable in tissue samples and thus is ideal for immunohistochemistry. In summary, our study illustrates a rational approach for discovery of drug-specific biomarkers toward development of patient-tailored treatments.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Medicina de Precisão , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal , Animais , Basófilos/efeitos dos fármacos , Basófilos/enzimologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Biologia Computacional , Proteínas do Citoesqueleto/metabolismo , Ativação Enzimática/efeitos dos fármacos , Epitopos/imunologia , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Camundongos , Neoplasias/enzimologia , Neoplasias/patologia , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfoproteínas/metabolismo , Fosfosserina/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Hyperactivation of the Ras signaling pathway is a driver of many cancers, and RAS pathway activation can predict response to targeted therapies. Therefore, optimal methods for measuring Ras pathway activation are critical. The main focus of our work was to develop a gene expression signature that is predictive of RAS pathway dependence. METHODS: We used the coherent expression of RAS pathway-related genes across multiple datasets to derive a RAS pathway gene expression signature and generate RAS pathway activation scores in pre-clinical cancer models and human tumors. We then related this signature to KRAS mutation status and drug response data in pre-clinical and clinical datasets. RESULTS: The RAS signature score is predictive of KRAS mutation status in lung tumors and cell lines with high (> 90%) sensitivity but relatively low (50%) specificity due to samples that have apparent RAS pathway activation in the absence of a KRAS mutation. In lung and breast cancer cell line panels, the RAS pathway signature score correlates with pMEK and pERK expression, and predicts resistance to AKT inhibition and sensitivity to MEK inhibition within both KRAS mutant and KRAS wild-type groups. The RAS pathway signature is upregulated in breast cancer cell lines that have acquired resistance to AKT inhibition, and is downregulated by inhibition of MEK. In lung cancer cell lines knockdown of KRAS using siRNA demonstrates that the RAS pathway signature is a better measure of dependence on RAS compared to KRAS mutation status. In human tumors, the RAS pathway signature is elevated in ER negative breast tumors and lung adenocarcinomas, and predicts resistance to cetuximab in metastatic colorectal cancer. CONCLUSIONS: These data demonstrate that the RAS pathway signature is superior to KRAS mutation status for the prediction of dependence on RAS signaling, can predict response to PI3K and RAS pathway inhibitors, and is likely to have the most clinical utility in lung and breast tumors.
Assuntos
Perfilação da Expressão Gênica , Neoplasias/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/metabolismo , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Cetuximab , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Bases de Dados Genéticas , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas ras/antagonistas & inibidores , Proteínas ras/genéticaRESUMO
NOTCH signaling is deregulated in the majority of T-cell acute lymphoblastic leukemias (T-ALL) as a result of activating mutations in NOTCH1. Gamma secretase inhibitors (GSI) block proteolytic activation of NOTCH receptors and may provide a targeted therapy for T-ALL. We have investigated the mechanisms of GSI sensitivity across a panel of T-ALL cell lines, yielding an approach for patient stratification based on pathway activity and also providing a rational combination strategy for enhanced response to GSI. Whereas the NOTCH1 mutation status does not serve as a predictor of GSI sensitivity, a gene expression signature of NOTCH pathway activity does correlate with response, and may be useful in the selection of patients more likely to respond to GSI. Furthermore, inhibition of the NOTCH pathway activity signature correlates with the induction of the cyclin-dependent kinase inhibitors CDKN2D (p19(INK4d)) and CDKN1B (p27(Kip1)), leading to derepression of RB and subsequent exit from the cell cycle. Consistent with this evidence of cell cycle exit, short-term exposure of GSI resulted in sustained molecular and phenotypic effects after withdrawal of the compound. Combination treatment with GSI and a small molecule inhibitor of CDK4 produced synergistic growth inhibition, providing evidence that GSI engagement of the CDK4/RB pathway is an important mechanism of GSI action and supports further investigation of this combination for improved efficacy in treating T-ALL.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Óxidos S-Cíclicos/farmacologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Inibidores de Proteases/farmacologia , Receptor Notch1/antagonistas & inibidores , Proteína do Retinoblastoma/metabolismo , Tiadiazóis/farmacologia , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Inibidor de Quinase Dependente de Ciclina p19/biossíntese , Inibidor de Quinase Dependente de Ciclina p27 , Fase G1/efeitos dos fármacos , Fase G1/genética , Perfilação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptor Notch1/genética , Receptor Notch1/metabolismo , Fase S/efeitos dos fármacos , Fase S/genética , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica , TransfecçãoRESUMO
Nr2e3 is a photoreceptor-specific nuclear receptor believed to play a role in photoreceptor development, differentiation, and survival. Much research has focused on the interaction of Nr2e3 with other transcription factors in determining the milieu of target gene expression in photoreceptors of the neonatal and adult retina. To investigate the downstream targets of Nr2e3 and thereby shed light on the functional pathways relevant to photoreceptor development and maintenance, expression profiling was performed on retinas from two different mouse knockout lines, one containing a targeted disruption of the Nr2e3 gene (Nr2e3 -/-), the other containing a spontaneous null allele of the Nr2e3 locus (rd7). Using whole genome microarrays, mRNA expression profiles of retinas from the two mutant strains were compared to those of wildtype C57BL/6 mice over a time course that ranged from postnatal day (p) 2 to 6months of age (p180). Additionally, expression profiling was performed on retinal explants treated with a putative NR2E3 agonist. The molecular profiling of Nr2e3 -/- and rd7/rd7 retinas identified 281 putative Nr2e3-dependent genes that were differentially expressed between wildtype and mutant retinas during at least one time point. Consistent with previous reports that Nr2e3 is necessary for the repression of cone-specific genes, increased expression of cone-specific genes was observed in the mutant samples, thereby providing proof-of-concept for the microarray screen. Further annotation of these data sets revealed ten predominant functional classes involved in the Nr2e3-mediated development and/or maintenance of photoreceptors. Interestingly, differences in the expression of Nr2e3-dependent genes exhibited two distinct temporal patterns. One group of genes showed a sustained difference in expression as compared to wildtype over the entire time course of the study, whereas a second group showed only transient differences which were largest around p10. Comparison of gene expression changes in Nr2e3 -/- and rd7/rd7 retinas with those uncovered by treating retinal explants with a putative NR2E3 agonist revealed four genes that were down-regulated in mutant retinas that lack Nr2e3 function but were up-regulated in agonist-treated explants. These results strongly suggest that the four genes may be direct targets of Nr2e3. Our identification of two sets of Nr2e3-regulated genes provides further evidence of a dual role for Nr2e3 in specification of photoreceptor fate during development as well as photoreceptor maintenance in the adult.
Assuntos
Proteínas do Olho/metabolismo , Células Fotorreceptoras de Vertebrados/citologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Proteínas do Olho/genética , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Receptores Nucleares Órfãos , Receptores Citoplasmáticos e Nucleares/genética , Retina/citologiaRESUMO
Detecting and understanding the potential for off-target pharmacological effects is critical in the optimization of lead compounds in drug discovery programs. Compound-mediated activation of the pregnane X receptor (PXR; NR1I2), a key regulator for drug metabolism genes, is often monitored to avoid potential drug-drug interactions. Two structural analogs, MRL-1 and MRL-2, were determined to be equivalent PXR activators in trans-activation assays. To differentiate these two PXR activators, their transcriptional effects were examined in PXR-sufficient (LS180) and PXR-deficient (Caco-2) adenocarcinoma cell lines. Both compounds regulated drug-management genes (e.g. CYP3A4, CYP2B6, UGT1A1 and ABCB1) in LS180 cells, but not in PXR-deficient Caco-2 cells. The potency of MRL-1 and MRL-2 on PXR activation was again equivalent as revealed by a set of 113 genes that were regulated by four prototypical PXR agonists (rifampicin, ritonavir, troglitazone and dexamethasone) in the LS180 cells. The specificity of the PXR signature genes was supported by the enrichment of putative PXR binding sites uncovered by sequence-based promoter analyses. Interestingly, an additional off-target activity of MRL-2 was suggested where sterol response element binding protein binding sites were found enriched in a subset of PXR signature genes. These genes, involved in cholesterol and fatty acid synthesis, were significantly regulated by ritonavir, chlorpromazine and MRL-2, which were linked to the manifestation of phospholipidosis. The present study demonstrates the utility of our approach in the differentiation and selection of lead compounds for drug development.
Assuntos
Técnicas de Química Combinatória/métodos , Perfilação da Expressão Gênica/métodos , Regiões Promotoras Genéticas , Receptores de Esteroides/metabolismo , Benzopiranos/farmacologia , Células CACO-2 , Células Cultivadas , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Regulação da Expressão Gênica , Humanos , Inativação Metabólica/genética , Intestinos/efeitos dos fármacos , Modelos Biológicos , Receptor de Pregnano X , Rifampina/farmacologia , Especificidade por Substrato/genética , Ativação Transcricional/efeitos dos fármacosRESUMO
BACKGROUND: Targeted transport of messenger RNA and local protein synthesis near the synapse are important for synaptic plasticity. In order to gain an overview of the composition of the dendritic mRNA pool, we dissected out stratum radiatum (dendritic lamina) from rat hippocampal CA1 region and compared its mRNA content with that of stratum pyramidale (cell body layer) using a set of cDNA microarrays. RNAs that have over-representation in the dendritic fraction were annotated and sorted into function groups. RESULTS: We have identified 154 dendritic mRNA candidates, which can be arranged into the categories of receptors and channels, signaling molecules, cytoskeleton and adhesion molecules, and factors that are involved in membrane trafficking, in protein synthesis, in posttranslational protein modification, and in protein degradation. Previously known dendritic mRNAs such as MAP2, calmodulin, and G protein gamma subunit were identified from our screening, as were mRNAs that encode proteins known to be important for synaptic plasticity and memory, such as spinophilin, Pumilio, eEF1A, and MHC class I molecules. Furthermore, mRNAs coding for ribosomal proteins were also found in dendrites. CONCLUSION: Our results suggest that neurons transport a variety of mRNAs to dendrites, not only those directly involved in modulating synaptic plasticity, but also others that play more common roles in cellular metabolism.
Assuntos
Dendritos/metabolismo , Hipocampo/citologia , Células Piramidais/citologia , RNA Mensageiro/metabolismo , Animais , Hibridização In Situ/métodos , Técnicas In Vitro , Masculino , Análise em Microsséries/métodos , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/fisiologia , Transporte Proteico/genética , Células Piramidais/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Transdução de Sinais/genética , Sinapses/metabolismoRESUMO
MOTIVATION: Tissue-specific transcription factor binding sites give insight into tissue-specific transcription regulation. RESULTS: We describe a word-counting-based tool for de novo tissue-specific transcription factor binding site discovery using expression information in addition to sequence information. We incorporate tissue-specific gene expression through gene classification to positive expression and repressed expression. We present a direct statistical approach to find overrepresented transcription factor binding sites in a foreground promoter sequence set against a background promoter sequence set. Our approach naturally extends to synergistic transcription factor binding site search. We find putative transcription factor binding sites that are overrepresented in the proximal promoters of liver-specific genes relative to proximal promoters of liver-independent genes. Our results indicate that binding sites for hepatocyte nuclear factors (especially HNF-1 and HNF-4) and CCAAT/enhancer-binding protein (C/EBPbeta) are the most overrepresented in proximal promoters of liver-specific genes. Our results suggest that HNF-4 has strong synergistic relationships with HNF-1, HNF-4 and HNF-3beta and with C/EBPbeta. AVAILABILITY: Programs are available for use over the Web at http://rulai.cshl.edu/tools/dwe.
Assuntos
Algoritmos , Sítios de Ligação , Perfilação da Expressão Gênica/métodos , Alinhamento de Sequência/métodos , Análise de Sequência de Proteína/métodos , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Animais , Sequência Consenso , Sequência Conservada , Humanos , Fígado/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/fisiologia , SoftwareRESUMO
MOTIVATION: Many bioinformatic approaches exist for finding novel genes within genomic sequence data. Traditionally, homology search-based methods are often the first approach employed in determining whether a novel gene exists that is similar to a known gene. Unfortunately, distantly related genes or motifs often are difficult to find using single query-based homology search algorithms against large sequence datasets such as the human genome. Therefore, the motivation behind this work was to develop an approach to enhance the sensitivity of traditional single query-based homology algorithms against genomic data without losing search selectivity. RESULTS: We demonstrate that by searching against a genome fragmented into all possible reading frames, the sensitivity of homology-based searches is enhanced without degrading its selectivity. Using the ETS-domain, bromodomain and acetyl-CoA acetyltransferase gene as queries, we were able to demonstrate that direct protein-protein searches using BLAST2P or FASTA3 against a human genome segmented among all possible reading frames and translated was substantially more sensitive than traditional protein-DNA searches against a raw genomic sequence using an application such as TBLAST2N. Receiver operating characteristic analysis was employed to demonstrate that the algorithms remained selective, while comparisons of the algorithms showed that the protein-protein searches were more sensitive in identifying hits. Therefore, through the overprediction of reading frames by this method and the increased sensitivity of protein-protein based homology search algorithms, a genome can be deeply mined, potentially finding hits overlooked by protein-DNA searches against raw genomic data.
Assuntos
Algoritmos , Mapeamento Cromossômico/métodos , Fases de Leitura Aberta , Proteínas/análise , Proteínas/química , Alinhamento de Sequência/métodos , Análise de Sequência de Proteína/métodos , Sequência de Aminoácidos , Dados de Sequência Molecular , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Homologia de Sequência de AminoácidosRESUMO
A large family of neural protocadherin (Pcdh) proteins is encoded by three closely linked mammalian gene clusters (alpha, beta, and gamma). Pcdh alpha and gamma clusters have a striking genomic organization. Specifically, each "variable" exon is spliced to a common set of downstream "constant" exons within each cluster. Recent studies demonstrated that the cell-specific expression of each Pcdh gene is determined bya combination of variable-exon promoter activation and cis-splicing of the corresponding variable exon to the first constant exon. To determine whether there are other similarly organized gene clusters in mammalian genomes, we performed a genome-wide search and identified a large number of mammalian genes containing multiple variable first exons. Here we describe several clusters that contain about a dozen variable exons arrayed in tandem, including UDP glucuronosyltransferase (UGT1), plectin, neuronal nitric oxide synthase (NOS1), and glucocorticoid receptor (GR) genes. In all these cases, multiple variable first exons are each spliced to a common set of downstream constant exons to generate diverse functional mRNAs. As an example, we analyzed the tissue-specific expression profile of the mouse UGT1 repertoire and found that multiple isoforms are expressed in a tissue-specific manner. Therefore, this variable and constant genomic organization provides a genetic mechanism for directing distinct cell- and tissue-specific patterns of gene expression.
Assuntos
Éxons/genética , Regulação da Expressão Gênica/genética , Animais , Clonagem Molecular , Evolução Molecular , Perfilação da Expressão Gênica/métodos , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Proteínas de Filamentos Intermediários/genética , Camundongos , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/genética , Família Multigênica/genética , N-Acetilglucosaminiltransferases/genética , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo I , Especificidade de Órgãos/genética , Filogenia , Plectina , Ratos , Receptores de Glucocorticoides/genéticaRESUMO
The human multidrug resistance gene MDR3 encodes a P-glycoprotein that belongs to the ATP-binding cassette transporter family (ABCB4). MDR3 is a critical trans-locator for phospholipids across canalicular membranes of hepatocytes, evidenced by the fact that human MDR3 deficiencies result in progressive familial intrahepatic cholestasis type III. It has been reported previously that MDR3 expression is modulated by hormones, cellular stress, and xenobiotics. Here we show that the MDR3 gene is trans-activated by the farnesoid X receptor (FXR) via a direct binding of FXR/retinoid X receptor alpha heterodimers to a highly conserved inverted repeat element (a FXR response element) at the distal promoter (-1970 to -1958). In FXR trans-activation assays, both the endogenous FXR agonist chenodeoxycholate and the synthetic agonist GW4064 activated the MDR3 promoter. Deletion or mutation of this inverted repeat element abolished FXR-mediated MDR3 promoter activation. Consistent with these data, MDR3 mRNA was significantly induced by both chenodeoxycholate and GW4064 in primary human hepatocytes in time- and dose-dependent fashions. In conclusion, we demonstrate that MDR3 expression is directly up-regulated by FXR. These results, together with the previous report that the bile salt export pump is a direct FXR target, suggest that FXR coordinately controls secretion of bile salts and phospholipids. Results of this study further support the notion that FXR is a master regulator of lipid metabolism.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/biossíntese , Proteínas de Transferência de Fosfolipídeos , Receptores Citoplasmáticos e Nucleares/fisiologia , Ativação Transcricional , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Bases , Proteínas de Transporte , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Hepatócitos , Humanos , Ligantes , Proteínas de Membrana , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides , Fatores de Transcrição/metabolismoRESUMO
Human kininogen belongs to the plasma kallikreinkinin system. High molecular weight kininogen is the precursor for two-chain kinin-free kininogen and bradykinin. It has been shown that the two-chain kinin-free kininogen has the properties of anti-adhesion, anti-platelet aggregation, and anti-thrombosis, whereas bradykinin is a potent vasodilator and mediator of inflammation. In this study we show that the human kininogen gene is strongly up-regulated by agonists of the farnesoid X receptor (FXR), a nuclear receptor for bile acids. In primary human hepatocytes, both the endogenous FXR agonist chenodeoxycholate and synthetic FXR agonist GW4064 increased kininogen mRNA with a maximum induction of 8-10-fold. A more robust induction of kininogen expression was observed in HepG2 cells, where kininogen mRNA was increased by chenodeoxycholate or GW4064 up to 130-140-fold as shown by real time PCR. Northern blot analysis confirmed the up-regulation of kininogen expression by FXR agonists. To determine whether kininogen is a direct target of FXR, we examined the sequence of the kininogen promoter and identified a highly conserved FXR response element (inverted repeat, IR-1) in the proximity of the kininogen promoter (-66/-54). FXR/RXRalpha heterodimers specifically bind to this IR-1. A construct of a minimal promoter with the luciferase reporter containing this IR-1 was transactivated by FXR. Deletion or mutation of this IR-1 abolished FXR-mediated promoter activation, indicating that this IR-1 element is responsible for the promoter transactivation by FXR. We conclude that kininogen is a novel and direct target of FXR, and bile acids may play a role in the vasodilation and anti-coagulation processes.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Cininogênios/genética , Fatores de Transcrição/fisiologia , Ativação Transcricional , Sítios de Ligação , Northern Blotting , Carcinoma Hepatocelular/metabolismo , Ácido Quenodesoxicólico/farmacologia , DNA/metabolismo , Proteínas de Ligação a DNA/agonistas , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Isoxazóis/farmacologia , Neoplasias Hepáticas/metabolismo , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , RNA Mensageiro/análise , Receptores Citoplasmáticos e Nucleares , Receptores do Ácido Retinoico/fisiologia , Sequências Repetitivas de Ácido Nucleico , Receptores X de Retinoides , Fatores de Transcrição/agonistas , Transfecção , Células Tumorais CultivadasRESUMO
Angiopoietins are members of the vascular endothelial growth factor family. One family member, angiopoietin-like protein 3 (Angptl3), was recently shown to be predominantly expressed in the liver and to play an important role in regulating lipid metabolism. In this study, we show that the Angptl3 gene is a direct target of the liver X receptor (LXR). Mice fed a high cholesterol diet exhibited a significant increase in Angptl3 expression in the liver. Oral administration to mice of T0901317, a synthetic LXR-selective agonist, increases levels of plasma lipids and Angptl3 mRNA in the liver. Treatment of HepG2 cells with LXR selective agonists led to a dose-dependent increase of Angptl3 mRNA. Analysis of the DNA sequence just 5' of the Angptl3 transcriptional start site revealed the presence of several potential transcription factor binding sites, including that for LXR. When transfected into HepG2 cells, the promoter activity of Angptl3 was significantly induced by LXR- or retinoid X receptor-selective agonists. Mutation of the predicted LXR binding site (DR4 element) completely abolished the LXR agonist-mediated activation of the promoter. Together, these studies show that Angptl3 is transcriptionally regulated by LXR, and reveals a novel mechanism by which LXR may regulate lipid metabolism.