RESUMO
Neuroblastoma is a childhood cancer of the sympathetic nervous system that accounts for approximately 10% of all paediatric oncology deaths. To identify genetic risk factors for neuroblastoma, we performed a genome-wide association study (GWAS) on 2,251 patients and 6,097 control subjects of European ancestry from four case series. Here we report a significant association within LIM domain only 1 (LMO1) at 11p15.4 (rs110419, combined P = 5.2 × 10(-16), odds ratio of risk allele = 1.34 (95% confidence interval 1.25-1.44)). The signal was enriched in the subset of patients with the most aggressive form of the disease. LMO1 encodes a cysteine-rich transcriptional regulator, and its paralogues (LMO2, LMO3 and LMO4) have each been previously implicated in cancer. In parallel, we analysed genome-wide DNA copy number alterations in 701 primary tumours. We found that the LMO1 locus was aberrant in 12.4% through a duplication event, and that this event was associated with more advanced disease (P < 0.0001) and survival (P = 0.041). The germline single nucleotide polymorphism (SNP) risk alleles and somatic copy number gains were associated with increased LMO1 expression in neuroblastoma cell lines and primary tumours, consistent with a gain-of-function role in tumorigenesis. Short hairpin RNA (shRNA)-mediated depletion of LMO1 inhibited growth of neuroblastoma cells with high LMO1 expression, whereas forced expression of LMO1 in neuroblastoma cells with low LMO1 expression enhanced proliferation. These data show that common polymorphisms at the LMO1 locus are strongly associated with susceptibility to developing neuroblastoma, but also may influence the likelihood of further somatic alterations at this locus, leading to malignant progression.
Assuntos
Proteínas de Ligação a DNA/genética , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Neuroblastoma/genética , Oncogenes/genética , Fatores de Transcrição/genética , Alelos , Linhagem Celular Tumoral , Proliferação de Células , Cromossomos Humanos Par 11/genética , Variações do Número de Cópias de DNA/genética , Progressão da Doença , Europa (Continente)/etnologia , Duplicação Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Genoma Humano/genética , Genômica , Genótipo , Humanos , Proteínas com Domínio LIM , Neuroblastoma/patologia , Razão de Chances , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Taxa de SobrevidaRESUMO
B-cell maturation antigen (BCMA, also termed TNFRSF17) is an attractive therapeutic target due to its restricted expression on normal and malignant plasma cells (PC). GSK2857916 (or J6M0-MMAF) is a BCMA-specific antibody conjugated to the microtubule-disrupting agent monomethyl auristatin F (MMAF) via a protease-resistant linker. To evaluate the clinical potential of this agent, tumour cells from seventy multiple myeloma (MM) patients were assessed for BCMA expression by immunohistochemistry and flow cytometry. All patients tested expressed BCMA, at varying levels, and both surface and intracellular expression were observed. BCMA expression is maintained through relapse, extramedullary spread and in residual disease post therapy. BCMA levels may also be prognostically useful as higher levels of BCMA were associated with poorer outcomes, even taking into account genetic risk. We observed rapid internalization of surface BCMA and newly expressed protein by 1 h, suggesting a mechanism for J6M0-MMAF activity even with low surface antigen. J6M0-MMAF mediated cytotoxicity of MM cells varied with dose and antigen levels, with clonogenic progenitors killed at lower doses than mature cells. In comparison, J6M0-MMAF killing of primary CD138(+) myeloma cells occurred with slower kinetics. Our observations support BCMA to be a promising therapeutic target in MM for novel therapies such as J6M0-MMAF.
Assuntos
Antígeno de Maturação de Linfócitos B/antagonistas & inibidores , Imunoconjugados/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antígeno de Maturação de Linfócitos B/genética , Antígeno de Maturação de Linfócitos B/metabolismo , Medula Óssea/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citometria de Fluxo , Seguimentos , Expressão Gênica , Humanos , Imunoconjugados/farmacologia , Imuno-Histoquímica , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/mortalidade , Plasmócitos/metabolismo , Plasmócitos/patologia , PrognósticoRESUMO
B-cell maturation antigen (BCMA), highly expressed on malignant plasma cells in human multiple myeloma (MM), has not been effectively targeted with therapeutic monoclonal antibodies. We here show that BCMA is universally expressed on the MM cell surface and determine specific anti-MM activity of J6M0-mcMMAF (GSK2857916), a novel humanized and afucosylated antagonistic anti-BCMA antibody-drug conjugate via a noncleavable linker. J6M0-mcMMAF specifically blocks cell growth via G2/M arrest and induces caspase 3-dependent apoptosis in MM cells, alone and in coculture with bone marrow stromal cells or various effector cells. It strongly inhibits colony formation by MM cells while sparing surrounding BCMA-negative normal cells. J6M0-mcMMAF significantly induces effector cell-mediated lysis against allogeneic or autologous patient MM cells, with increased potency and efficacy compared with the wild-type J6M0 without Fc enhancement. The antibody-dependent cell-mediated cytotoxicity and apoptotic activity of J6M0-mcMMAF is further enhanced by lenalidomide. Importantly, J6M0-mcMMAF rapidly eliminates myeloma cells in subcutaneous and disseminated mouse models, and mice remain tumor-free up to 3.5 months. Furthermore, J6M0-mcMMAF recruits macrophages and mediates antibody-dependent cellular phagocytosis of MM cells. Together, these results demonstrate that GSK2857916 has potent and selective anti-MM activities via multiple cytotoxic mechanisms, providing a promising next-generation immunotherapeutic in this cancer.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígeno de Maturação de Linfócitos B/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Imunotoxinas/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Linfócitos B/imunologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Fatores Imunológicos/imunologia , Fatores Imunológicos/uso terapêutico , Imunotoxinas/imunologia , Lenalidomida , Camundongos , Camundongos SCID , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Talidomida/análogos & derivados , Talidomida/imunologia , Talidomida/uso terapêuticoRESUMO
BACKGROUND: We previously reported the identification of ONC201/TIC10, a novel small molecule inducer of the human TRAIL gene that improves efficacy-limiting properties of recombinant TRAIL and is in clinical trials in advanced cancers based on its promising safety and antitumor efficacy in several preclinical models. METHODS: We performed a high throughput luciferase reporter screen using the NCI Diversity Set II to identify TRAIL-inducing compounds. RESULTS: Small molecule-mediated induction of TRAIL reporter activity was relatively modest and the majority of the hit compounds induced low levels of TRAIL upregulation. Among the candidate TRAIL-inducing compounds, TIC9 and ONC201/TIC10 induced sustained TRAIL upregulation and apoptosis in tumor cells in vitro and in vivo. However, ONC201/TIC10 potentiated tumor cell death while sparing normal cells, unlike TIC9, and lacked genotoxicity in normal fibroblasts. Investigating the effects of TRAIL-inducing compounds on cell signaling pathways revealed that TIC9 and ONC201/TIC10, which are the most potent inducers of cell death, exclusively activate Foxo3a through inactivation of Akt/ERK to upregulate TRAIL and its pro-apoptotic death receptor DR5. CONCLUSION: These studies reveal the selective activity of ONC201/TIC10 that led to its selection as a lead compound for this novel class of antitumor agents and suggest that ONC201/TIC10 is a unique inducer of the TRAIL pathway through its concomitant regulation of the TRAIL ligand and its death receptor DR5.
Assuntos
Antineoplásicos/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Genes Reporter , Compostos Heterocíclicos de 4 ou mais Anéis/química , Humanos , Imidazóis , Luciferases/metabolismo , Mutagênicos/toxicidade , Regiões Promotoras Genéticas/genética , Piridinas , Pirimidinas , Bibliotecas de Moléculas Pequenas/química , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Neuroblastoma is a childhood cancer that is often fatal despite intense multimodality therapy. In an effort to identify therapeutic targets for this disease, we performed a comprehensive loss-of-function screen of the protein kinome. Thirty kinases showed significant cellular cytotoxicity when depleted, with loss of the cell cycle checkpoint kinase 1 (CHK1/CHEK1) being the most potent. CHK1 mRNA expression was higher in MYC-Neuroblastoma-related (MYCN)-amplified (P < 0.0001) and high-risk (P = 0.03) tumors. Western blotting revealed that CHK1 was constitutively phosphorylated at the ataxia telangiectasia response kinase target site Ser345 and the autophosphorylation site Ser296 in neuroblastoma cell lines. This pattern was also seen in six of eight high-risk primary tumors but not in control nonneuroblastoma cell lines or in seven of eight low-risk primary tumors. Neuroblastoma cells were sensitive to the two CHK1 inhibitors SB21807 and TCS2312, with median IC(50) values of 564 nM and 548 nM, respectively. In contrast, the control lines had high micromolar IC(50) values, indicating a strong correlation between CHK1 phosphorylation and CHK1 inhibitor sensitivity (P = 0.0004). Furthermore, cell cycle analysis revealed that CHK1 inhibition in neuroblastoma cells caused apoptosis during S-phase, consistent with its role in replication fork progression. CHK1 inhibitor sensitivity correlated with total MYC(N) protein levels, and inducing MYCN in retinal pigmented epithelial cells resulted in CHK1 phosphorylation, which caused growth inhibition when inhibited. These data show the power of a functional RNAi screen to identify tractable therapeutical targets in neuroblastoma and support CHK1 inhibition strategies in this disease.
Assuntos
Neuroblastoma/tratamento farmacológico , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/farmacologia , Apoptose/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Humanos , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/patologia , Proteínas Nucleares/análise , Proteínas Oncogênicas/análise , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro , Fase S/efeitos dos fármacosRESUMO
Inhibition of centromere-associated protein-E (CENP-E) has demonstrated preclinical anti-tumor activity in a number of tumor types including neuroblastoma. A potent small molecule inhibitor of the kinesin motor activity of CENP-E has recently been developed (GSK923295). To identify an effective drug combination strategy for GSK923295 in neuroblastoma, we performed a screen of siRNAs targeting a prioritized set of genes that function in therapeutically tractable signaling pathways. We found that siRNAs targeted to extracellular signal-related kinase 1 (ERK1) significantly sensitized neuroblastoma cells to GSK923295-induced growth inhibition (p = 0.01). Inhibition of ERK1 activity using pharmacologic inhibitors of mitogen-activated ERK kinase (MEK1/2) showed significant synergistic growth inhibitory activity when combined with GSK923295 in neuroblastoma, lung, pancreatic and colon carcinoma cell lines. Synergistic growth inhibitory activity of combined MEK/ERK and CENP-E inhibition was a result of increased mitotic arrest and apoptosis. There was a significant correlation between ERK1/2 phosphorylation status in neuroblastoma cell lines and GSK923295 growth inhibitory activity (r = 0.823, p = 0.0006). Consistent with this result we found that lung cancer cell lines harboring RAS mutations, which leads to oncogenic activation of MEK/ERK signaling, were significantly more resistant than cell lines with wild-type RAS to GSK923295-induced growth inhibition (p = 0.047). Here we have identified (MEK/ERK) activity as a potential biomarker of relative GSK923295 sensitivity and have shown the synergistic effect of combinatorial MEK/ERK pathway and CENP-E inhibition across different cancer cell types including neuroblastoma.
Assuntos
Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Sarcosina/análogos & derivados , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Biomarcadores Tumorais , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Humanos , Irinotecano , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neuroblastoma/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Interferência de RNA , RNA Interferente Pequeno , Sarcosina/farmacologia , TemozolomidaRESUMO
In recent years, there has been considerable interest in mAb-based induction of costimulatory receptor signaling as an approach to combat cancer. However, promising nonclinical data have yet to translate to a meaningful clinical benefit. Inducible T-cell costimulator (ICOS) is a costimulatory receptor important for immune responses. Using a novel clinical-stage anti-ICOS immunoglobulin G4 mAb (feladilimab), which induces but does not deplete ICOS+ T cells and their rodent analogs, we provide an end-to-end evaluation of the antitumor potential of antibody-mediated ICOS costimulation alone and in combination with programmed cell death protein 1 (PD-1) blockade. We demonstrate, consistently, that ICOS is expressed in a range of cancers, and its induction can stimulate growth of antitumor reactive T cells. Furthermore, feladilimab, alone and with a PD-1 inhibitor, induced antitumor activity in mouse and humanized tumor models. In addition to nonclinical evaluation, we present three patient case studies from a first-time-in-human, phase I, open-label, dose-escalation and dose-expansion clinical trial (INDUCE-1; ClinicalTrials.gov: NCT02723955), evaluating feladilimab alone and in combination with pembrolizumab in patients with advanced solid tumors. Preliminary data showing clinical benefit in patients with cancer treated with feladilimab alone or in combination with pembrolizumab was reported previously; with example cases described here. Additional work is needed to further validate the translation to the clinic, which includes identifying select patient populations that will benefit from this therapeutic approach, and randomized data with survival endpoints to illustrate its potential, similar to that shown with CTLA-4 and PD-1 blocking antibodies. Significance: Stimulation of the T-cell activation marker ICOS with the anti-ICOS agonist mAb feladilimab, alone and in combination with PD-1 inhibition, induces antitumor activity across nonclinical models as well as select patients with advanced solid tumors.
Assuntos
Instituições de Assistência Ambulatorial , Anticorpos Monoclonais , Humanos , Animais , Camundongos , Anticorpos Monoclonais/farmacologia , Inibidores de Checkpoint Imunológico , Imunoglobulina G , Inibição PsicológicaRESUMO
Immune checkpoint inhibitors demonstrate clinical activity in many tumor types, however, only a fraction of patients benefit. Combining CD137 agonists with these inhibitors increases anti-tumor activity preclinically, but attempts to translate these observations to the clinic have been hampered by systemic toxicity. Here we describe a human CD137xPD-L1 bispecific antibody, MCLA-145, identified through functional screening of agonist- and immune checkpoint inhibitor arm combinations. MCLA-145 potently activates T cells at sub-nanomolar concentrations, even under suppressive conditions, and enhances T cell priming, differentiation and memory recall responses. In vivo, MCLA-145 anti-tumor activity is superior to immune checkpoint inhibitor comparators and linked to recruitment and intra-tumor expansion of CD8 + T cells. No graft-versus-host-disease is observed in contrast to other antibodies inhibiting the PD-1 and PD-L1 pathway. Non-human primates treated with 100 mg/kg/week of MCLA-145 show no adverse effects. The conditional activation of CD137 signaling by MCLA-145, triggered by neighboring cells expressing >5000 copies of PD-L1, may provide both safety and potency advantages.
Assuntos
Ligante 4-1BB/agonistas , Anticorpos Biespecíficos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Linfócitos T CD8-Positivos/efeitos dos fármacos , Inibidores de Checkpoint Imunológico/farmacologia , Ligante 4-1BB/imunologia , Animais , Anticorpos Biespecíficos/imunologia , Antígeno B7-H1/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos , Humanos , Inibidores de Checkpoint Imunológico/imunologia , Tolerância Imunológica/efeitos dos fármacos , Memória Imunológica/efeitos dos fármacos , Imunoterapia , Ativação Linfocitária/efeitos dos fármacosRESUMO
Immune cell functions are regulated by co-inhibitory and co-stimulatory receptors. The first two generations of cancer immunotherapy agents consist primarily of antagonist antibodies that block negative immune checkpoints, such as programmed cell death protein 1 (PD1) and cytotoxic T lymphocyte protein 4 (CTLA4). Looking ahead, there is substantial promise in targeting co-stimulatory receptors with agonist antibodies, and a growing number of these agents are making their way through various stages of development. This Review discusses the key considerations and potential pitfalls of immune agonist antibody design and development, their differentiating features from antagonist antibodies and the landscape of agonist antibodies in clinical development for cancer treatment.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Neoplasias/imunologia , Neoplasias/terapia , Animais , Antígeno CTLA-4/antagonistas & inibidores , Humanos , Imunoterapia/métodos , Terapia de Alvo Molecular/métodos , Receptor de Morte Celular Programada 1/antagonistas & inibidoresRESUMO
A better characterization of T-cell subsets in the microenvironment of classical Hodgkin lymphoma (cHL) would help to develop immunotherapies. Using multicolor flow cytometry, we identified in 6 of 43 cHL tissue samples a previously unrecognized subset of CD8 T cells coexpressing CXCR5 and inducible T-cell costimulator (ICOS) molecules (CD8CXCR5+ICOS+). These cells shared phenotypic features with follicular helper T (TFH) cells including low CCR7 expression together with high expression of B-cell lymphoma-6, programmed cell death 1, B and T lymphocyte attenuator, CD200, and OX40. They had deficient cytotoxicity, low interferon-γ secretion, and common functional properties with intratumoral CD4+ TFH cells, such as production of interleukin-4 (IL-4), IL-21, CXCL13, and capacity to sustain B cells. Gene profiling analysis showed a significant similarity between the signatures of CD8CXCR5+ICOS+ T cells and CD4+ TFH cells. Benign lymphadenitis tissues (n = 8) were devoid of CD8CXCR5+ICOS+ cells. Among the 35 B-cell lymphoma tissues analyzed, including follicular lymphomas (n = 13), diffuse large cell lymphomas (n = 12), marginal zone lymphomas (MZLs; n = 3), mantle cell lymphomas (n = 3), and chronic lymphocytic leukemias (n = 4), only 1 MZL sample contained CD8CXCR5+ICOS+ cells. Lymphoma tumors with CD8CXCR5+ICOS+ cells shared common histopathological features including residual germinal centers, and contained high amounts of activated CD8CXCR5-ICOS+ cells. These data demonstrate a CD8 T-cell differentiation pathway leading to the acquisition of some TFH similarities. They suggest a particular immunoediting process with global CD8 activation acting mainly, but not exclusively, in HL tumors.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Regulação Neoplásica da Expressão Gênica , Doença de Hodgkin/metabolismo , Proteína Coestimuladora de Linfócitos T Induzíveis/biossíntese , Proteínas de Neoplasias/biossíntese , Receptores CXCR5/biossíntese , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Diferenciação Celular , Citocinas/metabolismo , Feminino , Doença de Hodgkin/patologia , Humanos , MasculinoRESUMO
Apoptosis is a cell suicide program that is initiated after cells are exposed to cytotoxic stresses including UV, IR irradiation, chemotherapeutic drugs, hypoxia, serum deprivation and TRAIL. Caspases are the central components of this process. In mammals, caspases involved in apoptotic responses are classified into two groups according to their function and structure. The first group is termed initiator caspases (caspase-2, 8, 9, 10) that contain N-terminal adapter domains which allow for auto-cleavage and activation of downstream caspases. The second group is termed effector or executioner caspases (caspase-3, 6, 7) that lack N-terminal adapter domains and are cleaved and activated by initiator caspases. Lakhani et al. (Science 2006, 311:847-51) have reported that caspase-3 and -7 regulate mitochondrial events in the apoptotic pathway. In this journal club, we summarize the results of the article and include some open questions left in the study.
Assuntos
Apoptose , Caspase 3/fisiologia , Caspase 7/fisiologia , Mitocôndrias/metabolismo , Animais , Caspase 3/genética , Caspase 7/genética , Camundongos , Camundongos KnockoutRESUMO
Hypoxia induces Hif-1alpha and selects for loss of wild-type p53 function, both of which can promote tumor cell survival. We evaluated the ability of TRAIL to induce apoptosis of human tumor cell lines exposed to hypoxia. H460 lung cancer cells express low levels of Hif-1alpha, stabilize wild-type p53 during hypoxia, and undergo TRAIL-induced apoptosis. In U2OS osteosarcoma or PA1 ovarian teratocarcinoma cells, high levels of Hif-1alpha and low levels of stable p53 are detected during hypoxia, and cells undergo low levels of TRAIL-induced apoptosis as compared to H460 cells. H460 cells are sensitized to TRAIL-induced apoptosis, whereas U2OS are protected, and little apoptosis is observed in relatively TRAIL-resistant PA1 during hypoxia. Forced expression of Hif-1alpha is also surprisingly a potent inducer of apoptosis in wild-type p53 expressing H460 cells and further promotes TRAIL-induced apoptosis. TRAIL-sensitive wild-type p53-expressing HCT116 colon carcinoma cells modestly elevate Hif-1alpha levels and are equally or slightly more sensitive to TRAIL during hypoxia. In contrast, p53-null HCT116 have higher levels of Hif-1alpha during normoxia and are extremely sensitive to TRAIL, but are protected from TRAIL-induced apoptosis during hypoxia. We hypothesize that a hypoxic tumor microenvironment may alter sensitivity to TRAIL, which may be impacted by Hif-1alpha levels and p53 status. These findings suggest that particular attention to hypoxic regions of tumors and sensitizers to hypoxia-induced cell death may be required to optimize therapeutic combinations using TRAIL.
Assuntos
Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/farmacologia , Apoptose/efeitos dos fármacos , Hipóxia Celular/fisiologia , Neoplasias do Colo/tratamento farmacológico , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Células HCT116 , Humanos , Fator 1 Induzível por Hipóxia/genética , Fator 1 Induzível por Hipóxia/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
PURPOSE: To assess the immunologic effects of dabrafenib and trametinib in vitro and to test whether trametinib potentiates or antagonizes the activity of immunomodulatory antibodies in vivo. EXPERIMENTAL DESIGN: Immune effects of dabrafenib and trametinib were evaluated in human CD4(+) and CD8(+) T cells from healthy volunteers, a panel of human tumor cell lines, and in vivo using a CT26 mouse model. RESULTS: Dabrafenib enhanced pERK expression levels and did not suppress human CD4(+) or CD8(+) T-cell function. Trametinib reduced pERK levels, and resulted in partial/transient inhibition of T-cell proliferation/expression of a cytokine and immunomodulatory gene subset, which is context dependent. Trametinib effects were partially offset by adding dabrafenib. Dabrafenib and trametinib in BRAF V600E/K, and trametinib in BRAF wild-type tumor cells induced apoptosis markers, upregulated HLA molecule expression, and downregulated certain immunosuppressive factors such as PD-L1, IL1, IL8, NT5E, and VEGFA. PD-L1 expression in tumor cells was upregulated after acquiring resistance to BRAF inhibition in vitro. Combinations of trametinib with immunomodulators targeting PD-1, PD-L1, or CTLA-4 in a CT26 model were more efficacious than any single agent. The combination of trametinib with anti-PD-1 increased tumor-infiltrating CD8(+) T cells in CT26 tumors. Concurrent or phased sequential treatment, defined as trametinib lead-in followed by trametinib plus anti-PD-1 antibody, demonstrated superior efficacy compared with anti-PD-1 antibody followed by anti-PD-1 plus trametinib. CONCLUSION: These findings support the potential for synergy between targeted therapies dabrafenib and trametinib and immunomodulatory antibodies. Clinical exploration of such combination regimens is under way.
Assuntos
Antineoplásicos/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Imidazóis/farmacologia , Oximas/farmacologia , Piridonas/farmacologia , Pirimidinonas/farmacologia , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno CTLA-4/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Humanos , Fatores Imunológicos/farmacologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos BALB C , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an antitumor protein that is in clinical trials as a potential anticancer therapy but suffers from drug properties that may limit efficacy such as short serum half-life, stability, cost, and biodistribution, particularly with respect to the brain. To overcome such limitations, we identified TRAIL-inducing compound 10 (TIC10), a potent, orally active, and stable small molecule that transcriptionally induces TRAIL in a p53-independent manner and crosses the blood-brain barrier. TIC10 induces a sustained up-regulation of TRAIL in tumors and normal cells that may contribute to the demonstrable antitumor activity of TIC10. TIC10 inactivates kinases Akt and extracellular signal-regulated kinase (ERK), leading to the translocation of Foxo3a into the nucleus, where it binds to the TRAIL promoter to up-regulate gene transcription. TIC10 is an efficacious antitumor therapeutic agent that acts on tumor cells and their microenvironment to enhance the concentrations of the endogenous tumor suppressor TRAIL.
Assuntos
Antineoplásicos/farmacologia , Núcleo Celular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ativação Transcricional , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Efeito Espectador/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Proteína Forkhead Box O3 , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Glioblastoma/enzimologia , Glioblastoma/genética , Glioblastoma/patologia , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Humanos , Imidazóis , Camundongos , Modelos Biológicos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Piridinas , Pirimidinas , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Oncogenic Myc alters mitochondrial metabolism, making it dependent on exogenous glutamine (Gln) for cell survival. Accordingly, Gln deprivation selectively induces apoptosis in MYC-overexpressing cells via unknown mechanisms. Using MYCN-amplified neuroblastoma as a model, we identify PUMA, NOXA, and TRB3 as executors of Gln-starved cells. Gln depletion in MYC-transformed cells induces apoptosis through ATF4-dependent, but p53-independent, PUMA and NOXA induction. MYC-transformed cells depend on both glutamate-oxaloacetate transaminase and glutamate dehydrogenase to maintain Gln homeostasis and suppress apoptosis. Consequently, either ATF4 agonists or glutaminolysis inhibitors potently induce apoptosis in vitro and inhibit tumor growth in vivo. These results reveal mechanisms whereby Myc sensitizes cells to apoptosis, and validate ATF4 agonists and inhibitors of Gln metabolism as potential Myc-selective cancer therapeutics.
Assuntos
Fator 4 Ativador da Transcrição/fisiologia , Apoptose , Glutamina/metabolismo , Neuroblastoma/metabolismo , Proteínas Proto-Oncogênicas c-myc/fisiologia , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Ácido Amino-Oxiacético/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Caspases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neuroblastoma/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Proteína X Associada a bcl-2/metabolismoRESUMO
FDG (18F-deoxy-glucose) is the current gold standard for PET imaging. FLT (3'-deoxy-3'-(18F-fluorothymidine), a PET imaging marker of proliferation, has been proposed as an alternative to FDG for the assessment of therapeutic response. We examined the therapeutic predictive value of FLT-PET and FDG-PET using CALU-6, a human, p53-null, non-small cell lung cancer cell line with comparison of combined targeted therapy, TRAIL and sorafenib, versus combined conventional chemotherapy, docetaxel and cisplatin. CALU-6 tumor-bearing nu/nu mice (n=46) were evaluated in 3 therapeutic trials measuring FLT and FDG prediction of tumor response at 72 h following initiation of daily combination therapy with targeted agents, TRAIL (200 µg i.v.) and sorafenib (30 mg/kg i.p.) and compared to conventional chemotherapeutics cisplatin (3 mg/kg i.p.) and docetaxel (7.5 mg/kg i.p.). PET imaging response was compared to morphological and histological indicators of therapeutic response, including decreased vascularity (in vivo AngioSense imaging and anti-CD31 staining), slowed tumor growth (caliper measurements), decreased cellular proliferation (Ki-67 staining) and increased apoptosis (TUNEL staining). Decreases in tumor accumulation of FLT (FLTMAX -30%, p=0.03) at 72 h post treatment were observed in response to TRAIL and sorafenib combination therapy resulting in smaller, less vascular, more apoptotic tumors. No similar reduction in tumor accumulation of FLT (FLTMAX -2%, p=0.67) was observed 72 h following initiation of cisplatin and docetaxel combination therapy, despite histological and morphological evidence of drug response. In contrast, tumor imaging with FDG did demonstrate a decrease in accumulation in both treatment groups, -21% (p=0.30) in response to cisplatin/docetaxel and -8% (p=0.59) in response to TRAIL/sorafenib, but did not reach statistical significance. FLT, but not FDG, is predictive of therapeutic response to the targeted regimen TRAIL/sorafenib. However, FLT-PET may not predict therapeutic response to DNA damaging agents in p53-null tumors, likely due to loss of cell cycle control of thymidine kinase 1 (TK1). Thus, tumor imaging response by FLT may be limited in human tumors without functional p53.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico por imagem , Carcinoma Pulmonar de Células não Pequenas/genética , Radioisótopos de Flúor , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/genética , Tomografia por Emissão de Pósitrons , Proteína Supressora de Tumor p53/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzenossulfonatos/farmacologia , Benzenossulfonatos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Modelos Animais de Doenças , Radioisótopos de Flúor/metabolismo , Radioisótopos de Flúor/normas , Fluordesoxiglucose F18/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Niacinamida/análogos & derivados , Compostos de Fenilureia , Tomografia por Emissão de Pósitrons/normas , Piridinas/farmacologia , Piridinas/uso terapêutico , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Sorafenibe , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Lapatinib, a dual HER2/EGFR (human epidermal growth factor receptor 2/epidermal growth factor receptor) inhibitor, is a recently approved targeted therapy for metastatic breast cancer. Because lapatinib enhances the efficacy of the chemotherapeutic agent capecitabine in breast cancer patients, we tested whether lapatinib also enhances the activity of anticancer agents in colorectal cancer. We found that lapatinib improved the proapoptotic effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and two TRAIL receptor agonists, the antibodies mapatumumab and lexatumumab. Tumors from mice treated with a combination of lapatinib and TRAIL exhibited more immunostaining for cleaved caspase-8, a marker of the extrinsic cell death pathway, than did tumors from mice treated with lapatinib or TRAIL alone. Furthermore, combination therapy suppressed tumor growth more effectively than either agent alone. Lapatinib up-regulated the proapoptotic TRAIL death receptors DR4 and DR5, leading to more efficient induction of apoptosis in the presence of TRAIL receptor agonists. This activity of lapatinib was independent of EGFR and HER2. The off-target induction of DR5 by lapatinib resulted from activation of the c-Jun amino-terminal kinase (JNK)/c-Jun signaling axis. This activity of lapatinib on TRAIL death receptor expression and signaling may confer therapeutic benefit when increased doses of lapatinib are used in combination with TRAIL receptor-activating agents.
Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Apoptose , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Eletroforese em Gel de Poliacrilamida , Células HCT116 , Células HT29 , Humanos , Imuno-Histoquímica , Lapatinib , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tumor hypoxia is an inherent impediment to cancer treatment that is both clinically significant and problematic. In this study, we conducted a cell-based screen to identify small molecules that could reverse the apoptotic resistance of hypoxic cancer cells. Among the compounds, we identified were a structurally related group that sensitized hypoxic cancer cells to apoptosis by inhibiting the kinases GSK-3ß and cyclin-dependent kinase (CDK) 1. Combinatorial inhibition of these proteins in hypoxic cancer cells and tumors increased levels of c-Myc and decreased expression of c-IAP2 and the central hypoxia response regulator hypoxia-inducible factor (HIF) 1α. In mice, these compounds augmented the hypoxic tumor cell death induced by cytotoxic chemotherapy, blocking angiogenesis and tumor growth. Taken together, our findings suggest that combinatorial inhibition of GSK-3ß and CDK1 augment the apoptotic sensitivity of hypoxic tumors, and they offer preclinical validation of a novel and readily translatable strategy to improve cancer therapy.
Assuntos
Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteína Quinase CDC2/antagonistas & inibidores , Hipóxia Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tiazóis/farmacologia , Adenocarcinoma/irrigação sanguínea , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/enzimologia , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Apoptose/fisiologia , Proteína Quinase CDC2/fisiologia , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/transplante , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/enzimologia , Sinergismo Farmacológico , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/fisiologia , Glicogênio Sintase Quinase 3 beta , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Proteínas Inibidoras de Apoptose/fisiologia , Irinotecano , Camundongos , Camundongos Nus , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/fisiologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-myb/fisiologia , Pirimidinas/uso terapêutico , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Tiazóis/uso terapêutico , Proteína Supressora de Tumor p53/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In human neuroblastoma, amplification of the MYCN gene predicts poor prognosis and resistance to therapy. Because hypoxia contributes to aggressive tumor phenotypes, predominantly via two structurally related hypoxia inducible factors, HIF-1α and HIF-2α, we examined hypoxia responses in MYCN-amplified neuroblastoma cells. We demonstrate here that HIF-1α, but not HIF-2α, is preferentially expressed in both MYCN-amplified neuroblastoma cells and primary tumors in comparison to samples without MYCN amplification. Our results showed that interplay between N-Myc and HIF-1α plays critical roles in neuroblastoma. For example, high levels of N-Myc override HIF-1α inhibition of cell cycle progression, enabling continued proliferation under hypoxia. Furthermore, both HIF-1α and N-Myc are essential for the Warburg effect (aerobic glycolysis) in neuroblastomas by activating the transcription of multiple glycolytic genes. Of note, expressions of Phosphoglycerate Kinase 1 (PGK1), Hexokinase 2 (HK2), and Lactate Dehydrogenase A (LDHA) were each significantly higher in MYCN-amplified neuroblastomas than in tumors without MYCN amplification. Interestingly, MYCN-amplified neuroblastoma cells are "addicted" to LDHA enzymatic activity, as its depletion completely inhibits tumorigenesis in vivo. Thus, our results provide mechanistic insights explaining how MYCN-amplified neuroblastoma cells contend with hypoxic stress and paradoxically how hypoxia contributes to neuroblastoma aggressiveness through combinatorial effects of N-Myc and HIF-1α. These results also suggest that LDHA represents a novel, pharmacologically tractable target for neuroblastoma therapeutics.
Assuntos
Genes myc , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Animais , Ciclo Celular/genética , Hipóxia Celular/genética , Progressão da Doença , Feminino , Amplificação de Genes , Células HCT116 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Desidrogenase/biossíntese , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Lactato Desidrogenase 5 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neuroblastoma/patologia , Transplante HeterólogoRESUMO
We have identified TNFSF10 (TRAIL) as a p53-transcriptional target gene. There are two p53 DNA-binding sites in the human TNFSF10 promoter region, at 346 and 625 bp upstream of the transcription start site. A human p53-expressing adenovirus (Ad-p53) induced TRAIL mRNA and protein expression in HCT116 p53-/- human colon cancer cells. A human TRAIL-promoter reporter assay showed increased luciferase activity with the promoter vector that contains two p53 DNA-binding motifs,following Ad-p53 infection, compared to the control adenovirus infection. Using HCT116 cells, gene silencing of TNFSF10 by siRNA suppressed caspase 3 and 7 activity, even after treatment with the DNA-damaging chemotherapeutic agent adriamycin. TRAIL protein expression was elevated in adriamycin-treated breast cancer cells. In vivo, TRAIL expression was induced in mouse natural killer cells at 24 hours after systemic treatment with 5-Fluorouracil. p53-dependent TRAIL induction in natural killer cells after chemotherapy exposure provides a link between the tumor suppressor p53 and the host immune response during cancer therapy as well as a paracrine-mediated cell-extrinsic death response. Our findings provide new mechanistic insights into the signaling of p53-dependent cell death and tumor suppression, including the involvement of the host immune system and natural killer cells in vivo in the anti-tumor efficacy of chemotherapy.