RESUMEN
Members of the B-cell lymphoma 2 (BCL-2) gene family are attractive targets for cancer therapy as they play a key role in promoting cell survival, a long-since established hallmark of cancer. Clinical utility for selective inhibition of specific anti-apoptotic Bcl-2 family proteins has recently been realized with the Food and Drug Administration (FDA) approval of venetoclax (formerly ABT-199/GDC-0199) in relapsed chronic lymphocytic leukemia (CLL) with 17p deletion. Despite the impressive monotherapy activity in CLL, such responses have rarely been observed in other B-cell malignancies, and preclinical data suggest that combination therapies will be needed in other indications. Additional selective antagonists of Bcl-2 family members, including Bcl-XL and Mcl-1, are in various stages of preclinical and clinical development and hold the promise of extending clinical utility beyond CLL and overcoming resistance to venetoclax. In addition to direct targeting of Bcl-2 family proteins with BH3 mimetics, combination therapies that aim at down-regulating expression of anti-apoptotic BCL-2 family members or restoring expression of pro-apoptotic BH3 family proteins may provide a means to deepen responses to venetoclax and extend the utility to additional indications. Here, we review recent progress in direct and selective targeting of Bcl-2 family proteins for cancer therapy and the search for rationale combinations.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Sistemas de Liberación de Medicamentos/métodos , Neoplasias Hematológicas/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonamidas/uso terapéutico , Animales , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patología , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína bcl-X/antagonistas & inhibidores , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
Targeting chromatin binding proteins and modifying enzymes can concomitantly affect tumor cell proliferation and survival, as well as enhance antitumor immunity and augment cancer immunotherapies. By screening a small-molecule library of epigenetics-based therapeutics, BET (bromo- and extra-terminal domain) inhibitors (BETi) were identified as agents that sensitize tumor cells to the antitumor activity of CD8+ T cells. BETi modulated tumor cells to be sensitized to the cytotoxic effects of the proinflammatory cytokine TNF. By preventing the recruitment of BRD4 to p65-bound cis-regulatory elements, BETi suppressed the induction of inflammatory gene expression, including the key NF-κB target genes BIRC2 (cIAP1) and BIRC3 (cIAP2). Disruption of prosurvival NF-κB signaling by BETi led to unrestrained TNF-mediated activation of the extrinsic apoptotic cascade and tumor cell death. Administration of BETi in combination with T-cell bispecific antibodies (TCB) or immune-checkpoint blockade increased bystander killing of tumor cells and enhanced tumor growth inhibition in vivo in a TNF-dependent manner. This novel epigenetic mechanism of immunomodulation may guide future use of BETi as adjuvants for immune-oncology agents.
Asunto(s)
Antineoplásicos/administración & dosificación , Neoplasias Colorrectales/tratamiento farmacológico , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Nucleares/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , Animales , Apoptosis/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , FN-kappa B/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Checkpoint inhibitor therapy has been a breakthrough in cancer research, but only some patients with cancer derive substantial benefit. Although mechanisms underlying sensitivity and resistance to checkpoint inhibitors are being elucidated, the importance of organ-specific regulation of immunity is currently underappreciated. Here, we call for a greater understanding of tissue-specific immunoregulation, namely, "tissue-specific immunostats," to make advances in treatments for cancer. A better understanding of how individual organs at baseline regulate the immune system could enable an improved precision medicine approach to cancer immunotherapy. Cancer Discov; 8(4); 395-402. ©2018 AACR.
Asunto(s)
Sistema Inmunológico , Neoplasias/inmunología , Neoplasias/terapia , Animales , Humanos , Inmunoterapia , Ratones , Especificidad de Órganos , Medicina de PrecisiónRESUMEN
Advances in the fields of cancer initiating cells and high-throughput in vivo shRNA screens have highlighted a need to observe the growth of tumor cells in cancer models at the clonal level. While in vivo cancer cell growth heterogeneity in xenografts has been described, it has yet to be measured. Here, we tested an approach to quantify the clonal growth heterogeneity of cancer cells in subcutaneous xenograft mouse models. Using a high-throughput sequencing method, we followed the fate in vitro and in vivo of ten thousand HCT-116 cells individually tagged with a unique barcode delivered by lentiviral transduction. While growth in vitro was less homogeneous than anticipated, we still find that 95% of the final cells derived from 80% of the original cells. In xenografts, however, 95% of the retrieved barcoded cells originated from only 6% of the initially injected cells, an effect we term "clonal dominance". We observed this clonal dominance in two additional xenograft models (MDA-MB-468 and A2780(cis)) and in two different host strains (NSG and Nude). By precisely and reproducibly quantifying clonal cancer cell growth in vivo, we find that a small subset of clones accounts for the vast majority of the descendant cells, even with HCT-116, a cell line reported to lack a tumor-initiating compartment. The stochastic in vivo selection process we describe has important implications for the fields of in vivo shRNA screening and tumor initiating cells.
Asunto(s)
Transformación Celular Neoplásica , Lentivirus/genética , Animales , Proliferación Celular , Células Clonales/patología , Femenino , Células HCT116 , Humanos , Ratones , ARN Interferente Pequeño/genética , Transducción GenéticaRESUMEN
Sphingosine kinases (SPHKs) are enzymes that phosphorylate the lipid sphingosine, leading to the formation of sphingosine-1-phosphate (S1P). In addition to the well established role of extracellular S1P as a mitogen and potent chemoattractant, SPHK activity has been postulated to be an important intracellular regulator of apoptosis. According to the proposed rheostat theory, SPHK activity shifts the intracellular balance from the pro-apoptotic sphingolipids ceramide and sphingosine to the mitogenic S1P, thereby determining the susceptibility of a cell to apoptotic stress. Despite numerous publications with supporting evidence, a clear experimental confirmation of the impact of this mechanism on tumor cell viability in vitro and in vivo has been hampered by the lack of suitable tool reagents. Utilizing a structure based design approach, we developed potent and specific SPHK1/2 inhibitors. These compounds completely inhibited intracellular S1P production in human cells and attenuated vascular permeability in mice, but did not lead to reduced tumor cell growth in vitro or in vivo. In addition, siRNA experiments targeting either SPHK1 or SPHK2 in a large panel of cell lines failed to demonstrate any statistically significant effects on cell viability. These results show that the SPHK rheostat does not play a major role in tumor cell viability, and that SPHKs might not be attractive targets for pharmacological intervention in the area of oncology.
Asunto(s)
Neoplasias/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Permeabilidad Capilar/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Concentración 50 Inhibidora , Ratones , Neoplasias/genética , Neoplasias/patología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Interferencia de ARN , Carga Tumoral/efectos de los fármacos , Ensayo de Tumor de Célula Madre , Factor A de Crecimiento Endotelial Vascular/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Endoglin (ENG), a co-receptor for several TGFß-family cytokines, is expressed in dividing endothelial cells alongside ALK1, the ACVRL1 gene product. ENG and ACVRL1 are both required for angiogenesis and mutations in either gene are associated with Hereditary Hemorrhagic Telangectasia, a rare genetic vascular disorder. ENG and ALK1 function in the same genetic pathway but the relative contribution of TGFß and BMP9 to SMAD1/5/8 activation and the requirement of ENG as a co-mediator of SMAD phosphorylation in endothelial cells remain debated. Here, we show that BMP9 and TGFß1 induce distinct SMAD phosphorylation responses in primary human endothelial cells and that, unlike BMP9, TGFß only induces SMAD1/5/8 phosphorylation in a subset of immortalized mouse endothelial cell lines, but not in primary human endothelial cells. We also demonstrate, using siRNA depletion of ENG and novel anti-ENG antibodies, that ENG is required for BMP9/pSMAD1 signaling in all human and mouse endothelial cells tested. Finally, anti-ENG antibodies that interfere with BMP9/pSMAD1 signaling, but not with TGFß1/pSMAD3 signaling, also decrease in vitro HUVEC endothelial tube formation and inhibit BMP9 binding to recombinant ENG in vitro. Our data demonstrate that BMP9 signaling inhibition is a key and previously unreported mechanism of action of TRC105, an anti-angiogenic anti-Endoglin antibody currently evaluated in clinical trials.
Asunto(s)
Anticuerpos Antiidiotipos/farmacología , Antígenos CD/metabolismo , Endotelio Vascular/metabolismo , Factores de Diferenciación de Crecimiento/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Antígenos CD/genética , Apoptosis , Western Blotting , Movimiento Celular , Proliferación Celular , Células Cultivadas , Endoglina , Endotelio Vascular/citología , Citometría de Flujo , Factor 2 de Diferenciación de Crecimiento , Factores de Diferenciación de Crecimiento/genética , Humanos , Ratones , Fosforilación , Unión Proteica , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Smad/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Despite the prevalence of KRAS mutations in human cancers, there remain no targeted therapies for treatment. The serine-threonine kinase STK33 has been proposed to be required for the survival of mutant KRAS-dependent cell lines, suggesting that small molecule kinase inhibitors of STK33 may be useful to treat KRAS-dependent tumors. In this study, we investigated the role of STK33 in mutant KRAS human cancer cells using RNA interference, dominant mutant overexpression, and small molecule inhibitors. As expected, KRAS downregulation decreased the survival of KRAS-dependent cells. In contrast, STK33 downregulation or dominant mutant overexpression had no effect on KRAS signaling or survival of these cells. Similarly, a synthetic lethal siRNA screen conducted in a broad panel of KRAS wild-type or mutant cells identified KRAS but not STK33 as essential for survival. We also obtained similar negative results using small molecule inhibitors of the STK33 kinase identified by high-throughput screening. Taken together, our findings refute earlier proposals that STK33 inhibition may be a useful therapeutic approach to target human KRAS mutant tumors.
Asunto(s)
Neoplasias/enzimología , Neoplasias/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas ras/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas p21(ras) , Interferencia de ARNRESUMEN
Midkine (MDK) is a heparin-binding growth factor involved in growth, survival, migration, and differentiation of various target cells and dysregulation of MDK signaling is implicated in a variety of inflammatory diseases and cancers. Although MDK has been reported to act on endothelial cells and to have proangiogenic effects, the exact role of MDK in angiogenesis is poorly defined. Here, we report that MDK is actually a modulator of angiogenesis and that it can abrogate the vascular endothelial growth factor A (VEGF-A)-induced proliferation of human microvascular endothelial cells in vitro through the downregulation of proangiogenic cytokines and through the upregulation of the antiangiogenic factor, tissue inhibitor of metalloproteinase 2. Phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) and of downstream signaling molecules, such as phosphatidylinositol-3-kinase and mitogen-activated protein kinases, is also impaired. Moreover, MDK downregulates VEGF-A-induced neovascularization and vascular permeability in vivo. We propose a model in which MDK is a new modulator of the VEGF-A-VEGFR-2 axis.
Asunto(s)
Endotelio Vascular/metabolismo , Factores de Crecimiento Nervioso/fisiología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Western Blotting , Permeabilidad Capilar , Pollos , Membrana Corioalantoides/patología , Neovascularización de la Córnea/metabolismo , Endotelio Vascular/citología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Midkina , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Neoplasias/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Ratas , Transducción de Señal , Piel , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Factor de Crecimiento Transformador alfa/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Paracaspase (MALT1), a member of an evolutionarily conserved superfamily of caspase-like proteins, has been shown to bind and colocalize with the protein Bcl10 in vitro and, because of this association, has been suggested to be involved in the CARMA1-Bcl10 pathway of antigen-induced nuclear factor kappaB (NF-kappaB) activation. We demonstrate that primary T and B lymphocytes from paracaspase-deficient mice are defective in antigen-receptor-induced NF-kappaB activation, cytokine production, and proliferation. Paracaspase acts downstream of Bcl10 to induce NF-kappaB activation and is required for the normal development of B cells, indicating that paracaspase provides the missing link between Bcl10 and activation of the IkappaB kinase complex.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Linfocitos B/inmunología , Activación de Linfocitos , Linfoma de Células B de la Zona Marginal/metabolismo , FN-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Linfocitos T/inmunología , Animales , Formación de Anticuerpos , Antígenos CD/análisis , Proteína 10 de la LLC-Linfoma de Células B , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/fisiología , Linfocitos B/metabolismo , Linfocitos B/fisiología , Caspasas , Diferenciación Celular , División Celular , Supervivencia Celular , Células Cultivadas , Citocinas/metabolismo , Eliminación de Gen , Marcación de Gen , Guanilato-Quinasas , Quinasa I-kappa B , Linfoma de Células B de la Zona Marginal/química , Ratones , Ratones Endogámicos C57BL , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Proteínas de Neoplasias/química , Nucleósido-Fosfato Quinasa/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/fisiología , Linfocitos T/metabolismo , Linfocitos T/fisiología , TransfecciónRESUMEN
Engagement of the T-cell receptor (TCR) initiates a signaling cascade that ultimately results in activation of the transcription factor NF-kappaB, which regulates many T-cell functions including proliferation, differentiation and cytokine production. Herein we demonstrate that Rip2, a caspase recruitment domain (CARD)-containing serine/threonine kinase, plays an important role in this cascade and is required for optimal TCR signaling and NF-kappaB activation. Following TCR engagement, Rip2 associated with Bcl10, a CARD-containing signaling component of the TCR-induced NF-kappaB pathway, and induced its phosphorylation. Rip2-deficient mice were defective in TCR-induced NF-kappaB activation, interleukin-2 production, and proliferation in vitro and exhibited defective T-cell-dependent responses in vivo. The defect in Rip2-/- T-cells correlated with a lack of TCR-induced Bcl10 phosphorylation. Furthermore, deficiency in Bcl10-dependent NF-kappaB activation could be rescued in Rip2-/- embryonic fibroblasts by exogenous wild-type Rip2 but not a kinase-dead mutant. Together these data define an important role for Rip2 in TCR-induced NF-kappaB activation and T-cell function and highlight the significance of post-translational modification of Bcl10 by Rip2 in T-cell signaling.