RESUMEN
BACKGROUND: Minibeam radiation therapy is an experimental radiation therapy utilizing an array of parallel submillimeter planar X-ray beams. In preclinical studies, minibeam radiation therapy has been shown to eradicate tumors and cause significantly less damage to normal tissue compared to equivalent radiation doses delivered by conventional broadbeam radiation therapy, where radiation dose is uniformly distributed. METHODS: Expanding on prior studies that suggested minibeam radiation therapy increased perfusion in tumors, we compared a single fraction of minibeam radiation therapy (peak dose:valley dose of 28 Gy:2.1 Gy and 100 Gy:7.5 Gy) and broadbeam radiation therapy (7 Gy) in their ability to enhance tumor delivery of PEGylated liposomal doxorubicin and alter the tumor microenvironment in a murine tumor model. Plasma and tumor pharmacokinetic studies of PEGylated liposomal doxorubicin and tumor microenvironment profiling were performed in a genetically engineered mouse model of claudin-low triple-negative breast cancer (T11). RESULTS: Minibeam radiation therapy (28 Gy) and broadbeam radiation therapy (7 Gy) increased PEGylated liposomal doxorubicin tumor delivery by 7.1-fold and 2.7-fold, respectively, compared to PEGylated liposomal doxorubicin alone, without altering the plasma disposition. The enhanced tumor delivery of PEGylated liposomal doxorubicin by minibeam radiation therapy is consistent after repeated dosing, is associated with changes in tumor macrophages but not collagen or angiogenesis, and nontoxic to local tissues. Our study indicated that the minibeam radiation therapy's ability to enhance the drug delivery decreases from 28 to 100 Gy peak dose. DISCUSSION: Our studies suggest that low-dose minibeam radiation therapy is a safe and effective method to significantly enhance the tumor delivery of nanoparticle agents.
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DYRK1A (dual-specificity tyrosine phosphorylation-regulated kinase 1a) is highly expressed in glioma, an aggressive brain tumor, and has been proposed as a therapeutic target for cancer. In the current study, we have used an optimized and validated time-resolved fluorescence energy transfer (TR-FRET)-based DYRK1A assay for high-throughput screening (HTS) in 384-well format. A small-scale screen of the FDA-approved Prestwick drug collection identified the ß-carboline, harmine, and four related analogs as DYRK1A inhibitors. Hits were confirmed by dose response and in an orthogonal DYRK1A assay. Harmine's potential therapeutic use has been hampered by its off-target activity for monoamine oxidase A (MAO-A) which impacts multiple nervous system targets. Selectivity profiling of harmine and a broader collection of analogs allowed us to map some divergent SAR (structure-activity relationships) for the DYRK1A and MAO-A activities. The panel of harmine analogs had varying activities in vitro in glioblastoma (GBM) cell lines when tested for anti-proliferative effects using a high content imaging assay. In particular, of the identified analogs, harmol was found to have the best selectivity for DYRK1A over MAO-A and, when tested in a glioma tumor xenograft model, harmol demonstrated a better therapeutic window compared to harmine.
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Antineoplásicos/farmacología , Inhibidores de la Monoaminooxidasa , Neoplasias , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Carbolinas , Harmina/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Monoaminooxidasa , Inhibidores de la Monoaminooxidasa/farmacología , Quinasas DyrKRESUMEN
Transforming growth factor beta-activated kinase 1 (TAK1) has been implicated for its role in inflammatory signaling and as an important mediator of cellular apoptosis and necroptosis in various cell types. Our recent discovery of a first-in-class, potent and selective TAK1 inhibitor, takinib, represents a novel pharmacological tool to evaluate TAK1's role in cancer. In this study we evaluated the potential therapeutic capacity of TAK1 inhibition on tumor growth and on tumor microenvironment remodeling. In a screen of 16 cancer cell lines, takinib in combination with tumor necrosis factor (TNF) was found to induce cell death (>20%) in 6 out of 16 cell lines. Furthermore, knocking out of TAK1 in MDA-MB-231 cells dramatically increased their sensitization to TNF-mediated apoptosis. In vivo xenographs of MDA-MB-231 TAK1KO tumors displayed delayed tumor growth and increased overall survival compared to TAK1WT controls. Histological and proteomic analysis of TAK1KO tumors showed altered angiogenic signaling and inflammatory signaling via immune cells. Overall, these findings suggest that the targeting of TAK1 in immune mediated cancers may be a novel therapeutic axis.
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Irinotecan treats a range of solid tumors, but its effectiveness is severely limited by gastrointestinal (GI) tract toxicity caused by gut bacterial ß-glucuronidase (GUS) enzymes. Targeted bacterial GUS inhibitors have been shown to partially alleviate irinotecan-induced GI tract damage and resultant diarrhea in mice. Here, we unravel the mechanistic basis for GI protection by gut microbial GUS inhibitors using in vivo models. We use in vitro, in fimo, and in vivo models to determine whether GUS inhibition alters the anticancer efficacy of irinotecan. We demonstrate that a single dose of irinotecan increases GI bacterial GUS activity in 1 d and reduces intestinal epithelial cell proliferation in 5 d, both blocked by a single dose of a GUS inhibitor. In a tumor xenograft model, GUS inhibition prevents intestinal toxicity and maintains the antitumor efficacy of irinotecan. Remarkably, GUS inhibitor also effectively blocks the striking irinotecan-induced bloom of Enterobacteriaceae in immune-deficient mice. In a genetically engineered mouse model of cancer, GUS inhibition alleviates gut damage, improves survival, and does not alter gut microbial composition; however, by allowing dose intensification, it dramatically improves irinotecan's effectiveness, reducing tumors to a fraction of that achieved by irinotecan alone, while simultaneously promoting epithelial regeneration. These results indicate that targeted gut microbial enzyme inhibitors can improve cancer chemotherapeutic outcomes by protecting the gut epithelium from microbial dysbiosis and proliferative crypt damage.
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Microbioma Gastrointestinal/efectos de los fármacos , Glucuronidasa/antagonistas & inhibidores , Glucuronidasa/efectos de los fármacos , Animales , Antineoplásicos Fitogénicos/farmacología , Bacterias/efectos de los fármacos , Modelos Animales de Enfermedad , Disbiosis/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Femenino , Glucuronidasa/metabolismo , Humanos , Irinotecán/farmacología , Ratones , Ratones Desnudos , Neoplasias/tratamiento farmacológicoRESUMEN
Immune challenge of invading macrophages at sites of infection is associated with release of TNF, which triggers a local cytokine storm as part of the normal inflammatory response. Whereas this response maybe beneficial in fighting off infections, similar responses triggered in autoimmune diseases contribute significantly to the underlying damaging pathology associated with these diseases. Here we show that Takinib, a highly discriminatory inhibitor of transforming growth factor Beta- activated kinase 1 (TAK1), selectively and potently reduces TNF production in pro-inflammatory THP-1 macrophages. A complete survey of 110 cytokines, showed robust loss of proinflammatory cytokine responsiveness to lipopolysaccharide (LPS) and interferon gamma (IFNγ) challenge in response to Takinib. The mechanisms of action of Takinib was recapitulated in TAK1 KO macrophages. TAK1 KO cells showed significant loss of TNF production as well as release of IL-6 in response to LPS challenge. Furthermore, Takinib blocked the ability of exogenously added LPS to promote phosphorylation of, c-Jun, p38 protein kinases as well as downstream transcription factors regulated by nuclear factor κ-light-chain-enhancer of activated B cells (NFκB). In a mouse LPS challenge model, Takinib significantly reduced TNF serum levels. Our findings demonstrate that Takinib has utility in the treatment inflammatory disease by locally suppressing TNF production from invading macrophages.
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Quinasas Quinasa Quinasa PAM/genética , Macrófagos/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Femenino , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Targeted therapeutics that are initially effective in cancer patients nearly invariably engender resistance at some stage, an inherent challenge in the use of any molecular-targeted drug in cancer settings. In this study, we evaluated resistance mechanisms arising in metastatic melanoma to MAPK pathway kinase inhibitors as a strategy to identify candidate strategies to limit risks of resistance. To investigate longitudinal responses, we developed an intravital serial imaging approach that can directly visualize drug response in an inducible RAF-driven, autochthonous murine model of melanoma incorporating a fluorescent reporter allele (tdTomatoLSL). Using this system, we visualized formation and progression of tumors in situ, starting from the single-cell level longitudinally over time. Reliable reporting of the status of primary murine tumors treated with the selective MEK1/2 inhibitor (MEKi) trametinib illustrated a time-course of initial drug response and persistence, followed by the development of drug resistance. We found that tumor cells adjacent to bundled collagen had a preferential persistence in response to MEKi. Unbiased transcriptional and kinome reprogramming analyses from selected treatment time points suggested increased c-Kit and PI3K/AKT pathway activation in resistant tumors, along with enhanced expression of epithelial genes and epithelial-mesenchymal transition downregulation signatures with development of MEKi resistance. Similar trends were observed following simultaneous treatment with BRAF and MEK inhibitors aligned to standard-of-care combination therapy, suggesting these reprogramming events were not specific to MEKi alone. Overall, our results illuminate the integration of tumor-stroma dynamics with tissue plasticity in melanoma progression and provide new insights into the basis for drug response, persistence, and resistance.Significance: A longitudinal study tracks the course of MEKi treatment in an autochthonous imageable murine model of melanoma from initial response to therapeutic resistance, offering new insights into the basis for drug response, persistence, and resistance. Cancer Res; 78(2); 542-57. ©2017 AACR.
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Biomarcadores de Tumor/genética , Resistencia a Antineoplásicos , Microscopía Intravital/métodos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Melanoma/patología , Inhibidores de Proteínas Quinasas/farmacología , Piridonas/farmacología , Pirimidinonas/farmacología , Animales , Apoptosis/efectos de los fármacos , Estudios de Casos y Controles , Proliferación Celular/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Estudios Longitudinales , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Ratones , Mutación , Pronóstico , Transducción de Señal , Células Tumorales CultivadasRESUMEN
The cellular and organismal phenotypic response to a small-molecule kinase inhibitor is defined collectively by the inhibitor's targets and their functions. The selectivity of small-molecule kinase inhibitors is commonly determined in vitro, using purified kinases and substrates. Recently, competitive chemical proteomics has emerged as a complementary, unbiased, cell-based methodology to define the target landscape of kinase inhibitors. Here, we evaluated and optimized a competitive multiplexed inhibitor bead mass spectrometry (MIB/MS) platform using cell lysates, live cells, and treated mice. Several clinically active kinase inhibitors were profiled, including trametinib, BMS-777607, dasatinib, abemaciclib, and palbociclib. MIB/MS competition analyses of the cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors abemaciclib and palbociclib revealed overlapping and unique kinase targets. Competitive MIB/MS analysis of abemaciclib revealed 83 target kinases, and dose-response MIB/MS profiling revealed glycogen synthase kinase 3 alpha and beta (GSK3α and ß) and Ca2+/calmodulin-dependent protein kinase II delta and gamma (CAMKIIδ and γ) as the most potently inhibited. Cell-based and in vitro kinase assays show that in contrast to palbociclib, abemaciclib directly inhibits GSK3α/ß and CAMKIIγ/δ kinase activity at low nanomolar concentrations. GSK3ß phosphorylates ß-catenin to suppress WNT signaling, while abemaciclib (but not palbociclib or ribociclib) potently activates ß-catenin-dependent WNT signaling. These data illustrate the power of competitive chemical proteomics to define kinase target specificities for kinase inhibitors, thus informing clinical efficacy, dose-limiting toxicities, and drug-repurposing efforts.Implications: This study uses a rapid and quantitative proteomics approach to define inhibitor-target data for commonly administered therapeutics and provides a cell-based alternative to in vitro kinome profiling. Mol Cancer Res; 16(2); 333-44. ©2017 AACR.
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Aminopiridinas/administración & dosificación , Bencimidazoles/administración & dosificación , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Proteómica/métodos , Vía de Señalización Wnt/efectos de los fármacos , Aminopiridinas/farmacología , Animales , Bencimidazoles/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Espectrometría de Masas , RatonesRESUMEN
Activation of the Hedgehog (Hh) pathway effector GLI1 is linked to tumorigenesis and invasiveness in a number of cancers, with targeting of GLI1 by small molecule antagonists shown to be effective. We profiled a collection of GLI antagonists possessing distinct mechanisms of action for efficacy in phenotypic models of inflammatory and non-inflammatory breast cancer (IBC and non-IBC) that we showed expressed varying levels of Hh pathway mediators. Compounds GANT61, HPI-1, and JK184 decreased cell proliferation, inhibited GLI1 mRNA expression and decreased the number of colonies formed in TN-IBC (SUM149) and TNBC (MDA-MB-231 and SUM159) cell lines. In addition, GANT61 and JK184 significantly down-regulated GLI1 targets that regulate cell cycle (cyclin D and E) and apoptosis (Bcl2). GANT61 reduced SUM149 spheroid growth and emboli formation, and in orthotopic SUM149 tumor models significantly decreased tumor growth. We successfully utilized phenotypic profiling to identify a subset of GLI1 antagonists that were prioritized for testing in in vivo models. Our results indicated that GLI1 activation in TN-IBC as in TNBC, plays a vital role in promoting cell proliferation, motility, tumor growth, and formation of tumor emboli.
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Compuestos Heterocíclicos con 2 Anillos/farmacología , Neoplasias Inflamatorias de la Mama/tratamiento farmacológico , Piridinas/farmacología , Pirimidinas/farmacología , Tiazoles/farmacología , Proteína con Dedos de Zinc GLI1/biosíntesis , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Neoplasias Inflamatorias de la Mama/metabolismo , Neoplasias Inflamatorias de la Mama/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Terapia Molecular Dirigida , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Extracellular expression of heat shock protein 90 (eHsp90) by tumor cells is correlated with malignancy. Development of small molecule probes that can detect eHsp90 in vivo may therefore have utility in the early detection of malignancy. We synthesized a cell impermeable far-red fluorophore-tagged Hsp90 inhibitor to target eHsp90 in vivo. High resolution confocal and lattice light sheet microscopy show that probe-bound eHsp90 accumulates in punctate structures on the plasma membrane of breast tumor cells and is actively internalized. The extent of internalization correlates with tumor cell aggressiveness, and this process can be induced in benign cells by overexpressing p110HER2. Whole body cryoslicing, imaging, and histology of flank and spontaneous tumor-bearing mice strongly suggests that eHsp90 expression and internalization is a phenomenon unique to tumor cells in vivo and may provide an "Achilles heel" for the early diagnosis of metastatic disease and targeted drug delivery.
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Neoplasias de la Mama/patología , Colorantes Fluorescentes/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Endocitosis , Espacio Extracelular/metabolismo , Genes erbB-2 , Xenoinjertos , Humanos , RatonesRESUMEN
To maintain tolerance, autoreactive B cells must regulate signal transduction from the BCR and TLRs. We recently identified that dendritic cells and macrophages regulate autoreactive cells during TLR4 activation by releasing IL-6 and soluble CD40 ligand (sCD40L). These cytokines selectively repress Ab secretion from autoreactive, but not antigenically naive, B cells. How IL-6 and sCD40L repress autoantibody production is unknown. In this work, we show that IL-6 and sCD40L are required for low-affinity/avidity autoreactive B cells to maintain tolerance through a mechanism involving receptor cross-talk between the BCR, TLR4, and the IL-6R or CD40. We show that acute signaling through IL-6R or CD40 integrates with chronic BCR-mediated ERK activation to restrict p-ERK from the nucleus and represses TLR4-induced Blimp-1 and XBP-1 expression. Tolerance is disrupted in 2-12H/MRL/lpr mice where IL-6 and sCD40L fail to spatially restrict p-ERK and fail to repress TLR4-induced Ig secretion. In the case of CD40, acute signaling in B cells from 2-12H/MRL/lpr mice is intact, but the chronic activation of p-ERK emanating from the BCR is attenuated. Re-establishing chronically active ERK through retroviral expression of constitutively active MEK1 restores tolerance upon sCD40L, but not IL-6, stimulation, indicating that regulation by IL-6 requires another signaling effector. These data define the molecular basis for the regulation of low-affinity autoreactive B cells during TLR4 stimulation; they explain how autoreactive but not naive B cells are repressed by IL-6 and sCD40L; and they identify B cell defects in lupus-prone mice that lead to TLR4-induced autoantibody production.
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Autoanticuerpos/biosíntesis , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Nefritis Lúpica/metabolismo , Receptor Cross-Talk/inmunología , Receptor Toll-Like 4/fisiología , Animales , Subgrupos de Linfocitos B/patología , Antígenos CD40/metabolismo , Ligando de CD40/metabolismo , Núcleo Celular/enzimología , Núcleo Celular/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Femenino , Tolerancia Inmunológica/genética , Nefritis Lúpica/enzimología , Nefritis Lúpica/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Ratones Transgénicos , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Receptores de Antígenos de Linfocitos B/fisiología , Receptores de Interleucina-6/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
The ability to induce Ab responses to pathogens while maintaining the quiescence of autoreactive cells is an important aspect of immune tolerance. During activation of TLR4, dendritic cells (DCs) and macrophages (MFs) repress autoantibody production through their secretion of IL-6 and soluble CD40L (sCD40L). These soluble mediators selectively repress B cells chronically exposed to Ag, but not naive cells, suggesting a means to maintain tolerance during TLR4 stimulation, yet allow immunity. In this study, we identify TNF-α as a third repressive factor, which together with IL-6 and CD40L account for nearly all the repression conferred by DCs and MFs. Similar to IL-6 and sCD40L, TNF-α did not alter B cell proliferation or survival. Instead, it reduced the number of Ab-secreting cells. To address whether the soluble mediators secreted by DCs and MFs functioned in vivo, we generated mice lacking IL-6, CD40L, and TNF-α. Compared to wild-type mice, these mice showed prolonged anti-nuclear Ab responses following TLR4 stimulation. Furthermore, adoptive transfer of autoreactive B cells into chimeric IL-6(-/-) × CD40L(-/-) × TNF-α(-/-) mice showed that preplasma cells secreted autoantibodies independent of germinal center formation or extrafollicular foci. These data indicate that in the absence of genetic predisposition to autoimmunity, loss of endogenous IL-6, CD40L, and TNF-α promotes autoantibody secretion during TLR4 stimulation.
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Autoanticuerpos/biosíntesis , Células Dendríticas/inmunología , Tolerancia Inmunológica , Macrófagos/inmunología , Células Plasmáticas/inmunología , Células Madre/inmunología , Traslado Adoptivo , Animales , Antígenos Nucleares/genética , Antígenos Nucleares/inmunología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Ligando de CD40/deficiencia , Células Cultivadas , Células Dendríticas/metabolismo , Tolerancia Inmunológica/genética , Interleucina-6/deficiencia , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Ratones Noqueados , Ratones Transgénicos , Células Plasmáticas/metabolismo , Células Plasmáticas/trasplante , Quimera por Radiación/inmunología , Células Madre/metabolismo , Receptor Toll-Like 4/fisiología , Factor de Necrosis Tumoral alfa/deficienciaRESUMEN
CASE: A 3-year-old boy underwent emergency external ventricular drainage and excision of a fourth ventricle anaplastic ependymoma. A week later, the child was given a ventriculo-peritoneal shunt. Fourteen days after shunting, the child developed a subphrenic abscess and acute cholecystitis that required surgery. RESULTS: A Staphylococcus epidermidis was isolated both from the ventricular catheter and CSF and from the subphrenic abscess and the gallbladder. To our knowledge, this is the first report of cholecystitis evolving as a descending shunt infection. The current literature related with this unique complication is briefly reviewed.