RESUMEN
KRAS-mutant colorectal cancers are resistant to therapeutics, presenting a significant problem for â¼40% of cases. Rapalogs, which inhibit mTORC1 and thus protein synthesis, are significantly less potent in KRAS-mutant colorectal cancer. Using Kras-mutant mouse models and mouse- and patient-derived organoids, we demonstrate that KRAS with G12D mutation fundamentally rewires translation to increase both bulk and mRNA-specific translation initiation. This occurs via the MNK/eIF4E pathway culminating in sustained expression of c-MYC. By genetic and small-molecule targeting of this pathway, we acutely sensitize KRASG12D models to rapamycin via suppression of c-MYC. We show that 45% of colorectal cancers have high signaling through mTORC1 and the MNKs, with this signature correlating with a 3.5-year shorter cancer-specific survival in a subset of patients. This work provides a c-MYC-dependent cotargeting strategy with remarkable potency in multiple Kras-mutant mouse models and metastatic human organoids and identifies a patient population that may benefit from its clinical application. SIGNIFICANCE: KRAS mutation and elevated c-MYC are widespread in many tumors but remain predominantly untargetable. We find that mutant KRAS modulates translation, culminating in increased expression of c-MYC. We describe an effective strategy targeting mTORC1 and MNK in KRAS-mutant mouse and human models, pathways that are also commonly co-upregulated in colorectal cancer.This article is highlighted in the In This Issue feature, p. 995.
Asunto(s)
Neoplasias Colorrectales/genética , Factor 4E Eucariótico de Iniciación/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/efectos de los fármacos , Inhibidores mTOR/farmacología , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Animales , Neoplasias Colorrectales/metabolismo , Modelos Animales de Enfermedad , Factor 4E Eucariótico de Iniciación/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Fascin is an actin binding and bundling protein that is not expressed in normal epithelial tissues but overexpressed in a variety of invasive epithelial tumors. It has a critical role in cancer cell metastasis by promoting cell migration and invasion. Here we report the crystal structures of fascin in complex with a series of novel and potent inhibitors. Structure-based elaboration of these compounds enabled the development of a series with nanomolar affinities for fascin, good physicochemical properties and the ability to inhibit fascin-mediated bundling of filamentous actin. These compounds provide promising starting points for fascin-targeted anti-metastatic therapies.
Asunto(s)
Antineoplásicos/síntesis química , Proteínas Portadoras/antagonistas & inhibidores , Diseño de Fármacos , Proteínas de Microfilamentos/antagonistas & inhibidores , Pirazoles/química , Piridinas/química , Quinolonas/química , Antineoplásicos/metabolismo , Sitios de Unión , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Humanos , Concentración 50 Inhibidora , Proteínas de Microfilamentos/metabolismo , Simulación del Acoplamiento Molecular , Estructura Terciaria de Proteína , Pirazoles/metabolismo , Piridinas/metabolismo , Quinolonas/metabolismo , Relación Estructura-ActividadRESUMEN
Glioblastoma (GBM) is an aggressive and incurable primary brain tumor that causes severe neurologic, cognitive, and psychologic symptoms. Symptoms are caused and exacerbated by the infiltrative properties of GBM cells, which enable them to pervade the healthy brain and disrupt normal function. Recent research has indicated that although radiotherapy (RT) remains the most effective component of multimodality therapy for patients with GBM, it can provoke a more infiltrative phenotype in GBM cells that survive treatment. Here, we demonstrate an essential role of the actin-myosin regulatory kinase myotonic dystrophy kinase-related CDC42-binding kinase (MRCK) in mediating the proinvasive effects of radiation. MRCK-mediated invasion occurred via downstream signaling to effector molecules MYPT1 and MLC2. MRCK was activated by clinically relevant doses per fraction of radiation, and this activation was concomitant with an increase in GBM cell motility and invasion. Furthermore, ablation of MRCK activity either by RNAi or by inhibition with the novel small-molecule inhibitor BDP-9066 prevented radiation-driven increases in motility both in vitro and in a clinically relevant orthotopic xenograft model of GBM. Crucially, treatment with BDP-9066 in combination with RT significantly increased survival in this model and markedly reduced infiltration of the contralateral cerebral hemisphere.Significance: An effective new strategy for the treatment of glioblastoma uses a novel, anti-invasive chemotherapeutic to prevent infiltration of the normal brain by glioblastoma cells.Cancer Res; 78(22); 6509-22. ©2018 AACR.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Proteína Quinasa de Distrofia Miotónica/antagonistas & inhibidores , Actinas/química , Animales , Antineoplásicos/farmacología , Neoplasias Encefálicas/radioterapia , Miosinas Cardíacas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Femenino , Glioblastoma/radioterapia , Humanos , Ratones , Ratones Desnudos , Microscopía Fluorescente , Cadenas Ligeras de Miosina/metabolismo , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Miosinas/química , Invasividad Neoplásica , Fenotipo , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
The myotonic dystrophy-related Cdc42-binding kinases MRCKα and MRCKß contribute to the regulation of actin-myosin cytoskeleton organization and dynamics, acting in concert with the Rho-associated coiled-coil kinases ROCK1 and ROCK2. The absence of highly potent and selective MRCK inhibitors has resulted in relatively little knowledge of the potential roles of these kinases in cancer. Here, we report the discovery of the azaindole compounds BDP8900 and BDP9066 as potent and selective MRCK inhibitors that reduce substrate phosphorylation, leading to morphologic changes in cancer cells along with inhibition of their motility and invasive character. In over 750 human cancer cell lines tested, BDP8900 and BDP9066 displayed consistent antiproliferative effects with greatest activity in hematologic cancer cells. Mass spectrometry identified MRCKα S1003 as an autophosphorylation site, enabling development of a phosphorylation-sensitive antibody tool to report on MRCKα status in tumor specimens. In a two-stage chemical carcinogenesis model of murine squamous cell carcinoma, topical treatments reduced MRCKα S1003 autophosphorylation and skin papilloma outgrowth. In parallel work, we validated a phospho-selective antibody with the capability to monitor drug pharmacodynamics. Taken together, our findings establish an important oncogenic role for MRCK in cancer, and they offer an initial preclinical proof of concept for MRCK inhibition as a valid therapeutic strategy.Significance: The development of selective small-molecule inhibitors of the Cdc42-binding MRCK kinases reveals their essential roles in cancer cell viability, migration, and invasive character. Cancer Res; 78(8); 2096-114. ©2018 AACR.
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Antineoplásicos/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Descubrimiento de Drogas , Proteína Quinasa de Distrofia Miotónica/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/uso terapéutico , Pirimidinas/uso terapéutico , Pirroles/uso terapéutico , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Carcinoma de Células Escamosas/enzimología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Ratones , Ratones Desnudos , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Neoplasias Cutáneas/enzimología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
While numerous studies have aimed to develop strategies to inhibit the development and progression of atherosclerosis, recent attention has focussed on the regression of pre-existing atherosclerotic plaques. As important regulator of total body cholesterol homeostasis, the liver X receptor (LXR) could possibly be an important target to induce regression. Here, we describe the effect of LXR activation by the synthetic agonist T0901317 on lesion regression in different mouse models with early fatty streak lesions or advanced collagen-rich lesions. Although T0901317 caused a dramatic increase in plasma (V)LDL levels in low-density lipoprotein (LDL) receptor knockout mice, no further increase in lesion size was observed, which points to beneficial LXR activity in the vascular wall. In normolipidemic C57BL/6 mice with cholate diet-induced atherosclerotic lesions, T0901317 treatment improved plasma lipoprotein levels and induced lesion regression (-43%, p<0.05). Apolipoprotein E (APOE) reconstitution in APOE knockout mice by means of bone marrow transplantation dramatically improved plasma lipoprotein profiles and resulted in a marked regression of initial (-45%, p<0.001) and advanced lesions (-23%, p<0.01). Atherosclerosis regression was associated with a decrease in the absolute macrophage content (-84%, p<0.001). T0901317 supplementation further decreased the size of early (-71%, p<0.001 vs baseline; -48%, p<0.01 vs chow diet alone) and more advanced atherosclerotic lesions (-36%, p<0.001 and -17%, p=0.06 respectively). In conclusion, our study highlights the potential of LXR agonist T0901317 to stimulate removal of macrophages from atherosclerotic lesions ultimately leading to a highly significant plaque regression of both early and advanced atherosclerotic lesions.
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Hidrocarburos Fluorados/uso terapéutico , Macrófagos/efectos de los fármacos , Receptores Nucleares Huérfanos/agonistas , Placa Aterosclerótica/tratamiento farmacológico , Sulfonamidas/uso terapéutico , Animales , Apolipoproteínas E/genética , Trasplante de Médula Ósea , Recuento de Células , VLDL-Colesterol/sangre , Dieta , Modelos Animales de Enfermedad , Femenino , Hidrocarburos Fluorados/administración & dosificación , Hidrocarburos Fluorados/farmacología , Receptores X del Hígado , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Placa Aterosclerótica/sangre , Placa Aterosclerótica/patología , Receptores de LDL/genética , Índice de Severidad de la Enfermedad , Sulfonamidas/administración & dosificación , Sulfonamidas/farmacología , Triglicéridos/sangreRESUMEN
The purpose of the study was to examine the potential of inhibition of cathepsin S as a treatment for autoimmune diseases. A highly selective cathepsin S inhibitor, CSI-75, was shown to upregulate levels of the cathepsin S substrate, invariant chain Lip10, in vitro as well as in vivo in C57Bl/6 mice after oral administration. Functional activity of the compound was shown by a reduction in the OVA-specific response of OVA-sensitized splenocytes from C57Bl/6 mice as well as from OVA-TCR transgenic mice (DO11.10). Since these studies revealed a selective suppression of the Th1 and Th17 cytokines causing a shift to Th2, CSI-75 was tested in the murine HC-gp39-immunization model. Indeed, CSI-75 specifically reduced the circulating HC-gp39-specific IgG2a in these mice indicating selective inhibition of the Th1 type of response in vivo. The importance of especially the Th1 and Th17 cell subsets in the pathology of autoimmune diseases, renders CatS inhibition a highly interesting potential therapeutic treatment of autoimmune diseases. Therefore, CSI-75 was tested in a murine model of multiple sclerosis (i.e. experimental autoimmune encephalomyelitis (EAE)) in a semi-therapeutic setting (ie. oral treatment after initial sensitization to antigen). Finally, in a murine model with features resembling rheumatoid arthritis (the collagen-induced arthritis (CIA) model), CSI-75 was tested in a therapeutic manner (after disease development). CSI-75 caused a significant reduction in disease score in both disease models, indicating a promising role for CatS inhibitors in the area of therapeutic treatments for autoimmune diseases.
Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Catepsinas/antagonistas & inhibidores , Piperidinas/uso terapéutico , Inhibidores de Proteasas/uso terapéutico , Piridinas/uso terapéutico , Administración Oral , Animales , Células Presentadoras de Antígenos/efectos de los fármacos , Artritis Experimental/tratamiento farmacológico , Enfermedades Autoinmunes/inmunología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Piperidinas/administración & dosificación , Inhibidores de Proteasas/administración & dosificación , Piridinas/administración & dosificación , Células TH1/fisiologíaRESUMEN
OBJECTIVE: Liver X receptors (LXRs) have previously been implicated in the regulation of inflammation and have, in general, been ascribed an antiinflammatory role. This study was therefore undertaken to explore the biologic mechanisms of LXRs in vivo and in vitro in an experimental inflammatory arthritis model. METHODS: Male DBA/1 mice were immunized with type II collagen and treated from an early or established stage of arthritis with 2 different concentrations of the LXR agonists T1317 and GW3965 or vehicle control. The mice were monitored for articular inflammation and cartilage degradation by scoring for clinical signs of arthritis, histologic examination of the joints, and analysis of serum cytokine and antibody levels. In vitro, primary human monocytes and T cells were cultured in the presence of GW3965 or T1317, and the concentrations of proinflammatory cytokines were measured by multiplex assay. RESULTS: Contrary to expectations, LXR agonism with the use of 2 discrete, specific molecular entities led to substantial exacerbation of articular inflammation and cartilage destruction in this murine collagen-induced arthritis model. This was associated ex vivo with elevated cytokine expression, with enhanced Th1 and Th17 cellular responses, and with elevated collagen-specific autoantibody production. In vitro, LXR agonists, in concert with lipopolysaccharide, promoted cytokine and chemokine release from human monocytes, and similar effects were observed in a T cell-macrophage coculture model that closely recapitulates the pathways that drive synovial cytokine release. CONCLUSION: Since LXRs are present in rheumatoid arthritis (RA) synovium, these results suggest that LXR-mediated pathways could exacerbate the chronic inflammatory response typical of RA.
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Artritis Experimental/metabolismo , Cartílago Articular/metabolismo , Proteínas de Unión al ADN/agonistas , Inflamación/metabolismo , Receptores Citoplasmáticos y Nucleares/agonistas , Animales , Artritis Experimental/patología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Cartílago Articular/patología , Células Cultivadas , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Humanos , Hidrocarburos Fluorados/farmacología , Inflamación/patología , Interleucina-17/metabolismo , Interleucina-1alfa/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos/farmacología , Receptores X del Hígado , Masculino , Ratones , Ratones Endogámicos DBA , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Monocitos/patología , Receptores Nucleares Huérfanos , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal/fisiología , Sulfonamidas/farmacologíaRESUMEN
Several studies have shown that in the arterial wall hepatocyte growth factor/scatter factor (HGF/SF) is expressed by smooth muscle cells (SMCs) but acts on endothelial cells, not SMCs. Other studies, however, have indicated that SMCs can respond to HGF/SF. We have reinvestigated expression and activity of HGF/SF and its receptor MET in arterial SMC and endothelial cell cultures and in whole arteries after superficial or deep injury or atherogenesis. High-density cultures of SMCs produced HGF/SF but did not express MET, whereas SMCs, at the leading edge of injured cultures, expressed both ligand and receptor and showed a dramatic motility and growth response to HGF/SF. In line with these results, HGF/SF and MET expression was undetectable in the media of uninjured carotid arteries but was induced after deep arterial injury in areas of SMC migration in the neointima. Strong MET expression was also observed in the SMCs of the atherosclerotic lesions of homozygous apoE(-/-) mice, whereas HGF/SF was expressed by macrophage-derived foam cells. These results demonstrate that MET is induced in migrating and proliferating SMCs and that HGF/SF and MET are key mediators of the SMC response in atherogenesis.
Asunto(s)
Arterias/lesiones , Aterosclerosis/fisiopatología , Factor de Crecimiento de Hepatocito/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Animales , Western Blotting , Movimiento Celular/fisiología , Células Cultivadas , Endotelio Vascular/metabolismo , Humanos , Inmunohistoquímica , Ratones , Músculo Liso Vascular/metabolismo , Técnicas de Cultivo de ÓrganosRESUMEN
The time-course of endothelial regrowth and functional recovery following polytetrafluoroethylene filament-induced endothelial denudation in vivo was studied in the left common carotid artery of the mouse. This technique does not result in any intimal hyperplasia, enabling the investigation of endothelial function without any confounding effect of intimal thickening. Endothelial coverage was assessed histologically, and functional recovery was assessed as restoration of receptor-mediated, endothelium-dependent relaxation to acetylcholine in vitro. Re-endothelialization of the carotid artery was complete within 8 days of denudation. However, relaxations to acetylcholine, which are mediated by endothelium-derived nitric oxide, were only partially restored 10 days after the procedure. At this time point, arterial responses to either phenylephrine, the receptor-independent endothelium-dependent dilator cyclopiazonic acid, or the nitric oxide donor diethylamine NONOate, were not significantly different to controls. At 25 days after denudation, acetylcholine-evoked responses remained significantly depressed compared to controls but at 90 days full recovery was observed. These data indicate that following mechanical denudation of the mouse carotid artery, although endothelial re-growth is complete within 8 days, recovery of endothelial cell function - assessed as the ability of the regenerated endothelium to mediate acetylcholine-stimulated relaxation - remains impaired for a prolonged period.