RESUMEN
Non-Hodgkin lymphomas (NHLs) make up the majority of lymphoma diagnoses and represent a very diverse set of malignancies. We sought to identify kinases uniquely up-regulated in different NHL subtypes. Using multiplexed inhibitor bead-mass spectrometry (MIB/MS), we found Tyro3 was uniquely up-regulated and important for cell survival in primary effusion lymphoma (PEL), which is a viral lymphoma infected with Kaposi's sarcoma-associated herpesvirus (KSHV). Tyro3 was also highly expressed in PEL cell lines as well as in primary PEL exudates. Based on this discovery, we developed an inhibitor against Tyro3 named UNC3810A, which hindered cell growth in PEL, but not in other NHL subtypes where Tyro3 was not highly expressed. UNC3810A also significantly inhibited tumor progression in a PEL xenograft mouse model that was not seen in a non-PEL NHL model. Taken together, our data suggest Tyro3 is a therapeutic target for PEL.
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Linfoma no Hodgkin/enzimología , Linfoma de Efusión Primaria/enzimología , Terapia Molecular Dirigida , Proteoma/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Apoptosis/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Linfocitos B/virología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Schwannomas are common, highly morbid and medically untreatable tumors that can arise in patients with germ line as well as somatic mutations in neurofibromatosis type 2 (NF2). These mutations most commonly result in the loss of function of the NF2-encoded protein, Merlin. Little is known about how Merlin functions endogenously as a tumor suppressor and how its loss leads to oncogenic transformation in Schwann cells (SCs). Here, we identify nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-inducing kinase (NIK) as a potential drug target driving NF-κB signaling and Merlin-deficient schwannoma genesis. Using a genomic approach to profile aberrant tumor signaling pathways, we describe multiple upregulated NF-κB signaling elements in human and murine schwannomas, leading us to identify a caspase-cleaved, proteasome-resistant NIK kinase domain fragment that amplifies pathogenic NF-κB signaling. Lentiviral-mediated transduction of this NIK fragment into normal SCs promotes proliferation, survival, and adhesion while inducing schwannoma formation in a novel in vivo orthotopic transplant model. Furthermore, we describe an NF-κB-potentiated hepatocyte growth factor (HGF) to MET proto-oncogene receptor tyrosine kinase (c-Met) autocrine feed-forward loop promoting SC proliferation. These innovative studies identify a novel signaling axis underlying schwannoma formation, revealing new and potentially druggable schwannoma vulnerabilities with future therapeutic potential.
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Neurilemoma/genética , Neurofibromatosis 2/genética , Neurofibromina 2/genética , Proteínas Serina-Treonina Quinasas/genética , Animales , Comunicación Autocrina/genética , Carcinogénesis/genética , Caspasa 1/genética , Proliferación Celular/genética , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Factor de Crecimiento de Hepatocito/genética , Humanos , Ratones , Terapia Molecular Dirigida , FN-kappa B/genética , Neurilemoma/complicaciones , Neurilemoma/tratamiento farmacológico , Neurilemoma/patología , Neurofibromatosis 2/complicaciones , Neurofibromatosis 2/tratamiento farmacológico , Neurofibromatosis 2/patología , Complejo de la Endopetidasa Proteasomal/genética , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-met/genética , Células de Schwann , Transducción de Señal/genética , Quinasa de Factor Nuclear kappa BRESUMEN
RATIONALE: Cystic fibrosis (CF) lung disease is caused by the loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) combined with hyperactivation of the epithelial sodium channel (ENaC). In the lung, ENaC is responsible for movement of sodium. Hyperactivation of ENaC, which creates an osmotic gradient that pulls fluid out of the airway, contributes to reduced airway hydration, causing mucus dehydration, decreased mucociliary clearance, and recurrent acute bacterial infections. ENaC represents a therapeutic target to treat all patients with CF independent of their underlying CFTR mutation. OBJECTIVES: To investigate the in vitro and in vivo efficacy of SPX-101, a peptide mimetic of the natural regulation of ENaC activity by short palate, lung, and nasal epithelial clone 1, known as SPLUNC1. METHODS: ENaC internalization by SPX-101 in primary human bronchial epithelial cells from healthy and CF donors was assessed by surface biotinylation and subsequent Western blot analysis. SPX-101's in vivo therapeutic effect was assessed by survival of ß-ENaC-transgenic mice, mucus transport in these mice, and mucus transport in a sheep model of CF. MEASUREMENTS AND MAIN RESULTS: SPX-101 binds selectively to ENaC and promotes internalization of the α-, ß-, and γ-subunits. Removing ENaC from the membrane with SPX-101 causes a significant decrease in amiloride-sensitive current. The peptide increases survival of ß-ENaC-transgenic mice to greater than 90% with once-daily dosing by inhalation. SPX-101 increased mucus transport in the ß-ENaC mouse model as well as the sheep model of CF. CONCLUSIONS: These data demonstrate that SPX-101 promotes durable reduction of ENaC membrane concentration, leading to significant improvements in mucus transport.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/uso terapéutico , Fibrosis Quística/tratamiento farmacológico , Bloqueadores del Canal de Sodio Epitelial/uso terapéutico , Canales Epiteliales de Sodio/uso terapéutico , Depuración Mucociliar/efectos de los fármacos , Mucosa Respiratoria/efectos de los fármacos , HumanosRESUMEN
Neural crest induction involves the combinatorial inputs of the FGF, BMP and Wnt signaling pathways. Recently, a two-step model has emerged where BMP attenuation and Wnt activation induces the neural crest during gastrulation, whereas activation of both pathways maintains the population during neurulation. FGF is proposed to act indirectly during the inductive phase by activating Wnt ligand expression in the mesoderm. Here, we use the chick model to investigate the role of FGF signaling in the amniote neural crest for the first time and uncover a novel requirement for FGF/MAPK signaling. Contrary to current models, we demonstrate that FGF is required within the prospective neural crest epiblast during gastrulation and is unlikely to operate through mesodermal tissues. Additionally, we show that FGF/MAPK activity in the prospective neural plate prevents the ectopic expression of lateral ectoderm markers, independently of its role in neural specification. We then investigate the temporal participation of BMP/Smad signaling and suggest a later involvement in neural plate border development, likely due to widespread FGF/MAPK activity in the gastrula epiblast. Our results identify an early requirement for FGF/MAPK signaling in amniote neural crest induction and suggest an intriguing role for FGF-mediated Smad inhibition in ectodermal development.
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Factores de Crecimiento de Fibroblastos/metabolismo , Gástrula/embriología , Estratos Germinativos/embriología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Cresta Neural/embriología , Transducción de Señal/fisiología , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Embrión de Pollo , Ectodermo/embriología , Electroporación , Técnica del Anticuerpo Fluorescente , Estratos Germinativos/metabolismo , Procesamiento de Imagen Asistido por Computador , Hibridación in Situ , Mesodermo/metabolismo , Microscopía , Modelos Biológicos , Pirroles , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Proteínas Smad/metabolismoRESUMEN
Regulatory transcription factors of the Pax family play fundamental roles in the function of multipotent cells during vertebrate development, post-natal regeneration, and cancer. Pax7 and its homologue Pax3 are important players in neural crest and muscle development. Both genes are coexpressed in various tissues and are thought to provide similar, but not identical, functions. The mechanisms that allow specific regulation of Pax7 remain largely unknown. Here, we report for the first time that Pax7 is regulated by SUMOylation. We identify the interaction of Pax7 with Ubc9, the SUMO conjugating enzyme, and reveal that SUMOylation machinery is enriched in neural crest precursors and plays a critical role in NC development. We demonstrate that Pax7 becomes SUMOylated and identify an essential role for lysine 85 (K85) in Pax7-SUMOylation. Despite high conservation surrounding K85 amongst Pax genes, we were unable to identify SUMOylation of other Pax proteins tested, including Pax3. Using a non-SUMOylatable Pax7 variant (K85 X R), we demonstrate that SUMOylation is essential for the function of Pax7 in neural crest development, C2C12 myogenic differentiation, and transcriptional transactivation. Our study provides new mechanistic insight into the molecular regulation of Pax7's function by SUMOylation in neural crest and muscle development.
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Cresta Neural/metabolismo , Factor de Transcripción PAX7/metabolismo , Secuencia de Aminoácidos , Animales , Diferenciación Celular , Línea Celular , Embrión de Pollo , Pollos , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Desarrollo de Músculos , Mutagénesis Sitio-Dirigida , Mioblastos/citología , Mioblastos/metabolismo , Factor de Transcripción PAX3 , Factor de Transcripción PAX7/química , Factor de Transcripción PAX7/genética , Factores de Transcripción Paired Box/metabolismo , Unión Proteica , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Alineación de Secuencia , Sumoilación , Transfección , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
Grade IV glioma, formerly known as glioblastoma multiforme (GBM) is the most aggressive and lethal type of brain tumor, and its treatment remains challenging in part due to extensive interpatient heterogeneity in disease driving mechanisms and lack of prognostic and predictive biomarkers. Using mechanistic inference of node-edge relationship (MINER), we have analyzed multiomics profiles from 516 patients and constructed an atlas of causal and mechanistic drivers of interpatient heterogeneity in GBM (gbmMINER). The atlas has delineated how 30 driver mutations act in a combinatorial scheme to causally influence a network of regulators (306 transcription factors and 73 miRNAs) of 179 transcriptional "programs", influencing disease progression in patients across 23 disease states. Through extensive testing on independent patient cohorts, we share evidence that a machine learning model trained on activity profiles of programs within gbmMINER significantly augments risk stratification, identifying patients who are super-responders to standard of care and those that would benefit from 2 nd line treatments. In addition to providing mechanistic hypotheses regarding disease prognosis, the activity of programs containing targets of 2 nd line treatments accurately predicted efficacy of 28 drugs in killing glioma stem-like cells from 43 patients. Our findings demonstrate that interpatient heterogeneity manifests from differential activities of transcriptional programs, providing actionable strategies for mechanistically characterizing GBM from a systems perspective and developing better prognostic and predictive biomarkers for personalized medicine.
RESUMEN
The neural crest is a migratory population of embryonic cells with a tremendous potential to differentiate and contribute to nearly every organ system in the adult body. Over the past two decades, an incredible amount of research has given us a reasonable understanding of how these cells are generated. Neural crest induction involves the combinatorial input of multiple signaling pathways and transcription factors, and is thought to occur in two phases from gastrulation to neurulation. In the first phase, FGF and Wnt signaling induce NC progenitors at the border of the neural plate, activating the expression of members of the Msx, Pax, and Zic families, among others. In the second phase, BMP, Wnt, and Notch signaling maintain these progenitors and bring about the expression of definitive NC markers including Snail2, FoxD3, and Sox9/10. In recent years, additional signaling molecules and modulators of these pathways have been uncovered, creating an increasingly complex regulatory network. In this work, we provide a comprehensive review of the major signaling pathways that participate in neural crest induction, with a focus on recent developments and current perspectives. We provide a simplified model of early neural crest development and stress similarities and differences between four major model organisms: Xenopus, chick, zebrafish, and mouse.
Asunto(s)
Inducción Embrionaria , Regulación del Desarrollo de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Cresta Neural/embriología , Placa Neural/embriología , Vía de Señalización Wnt , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Embrión de Pollo , Transición Epitelial-Mesenquimal , Factores de Crecimiento de Fibroblastos/metabolismo , Ratones , Morfogénesis , Receptores Notch/metabolismo , Proteínas Wnt/metabolismo , Xenopus laevis/embriología , Pez Cebra/embriologíaRESUMEN
Melanoma is characterized by oncogenic mutations in pathways regulating cell growth, proliferation, and metabolism. Greater than 80% of primary melanoma cases harbor aberrant activation of the mitogen-activated protein kinase kinase/extracellular-signal-regulated kinase (MEK/ERK) pathway, with oncogenic mutations in BRAF, most notably BRAF V600E, being the most common. Significant progress has been made in BRAF-mutant melanoma using BRAF and MEK inhibitors; however, non-V600 BRAF mutations remain a challenge with limited treatment options. We report the case of an individual diagnosed with stage III BRAF D594G-mutant melanoma who experienced an extraordinary response to the ERK1/2 inhibitor ulixertinib as fourth-line therapy. Ulixertinib was obtained via an intermediate expanded access protocol with unique flexibility to permit both single-agent and combination treatments, dose adjustments, breaks in treatment to undergo surgery, and long-term preventive treatment following surgical resection offering this patient the potential for curative treatment.
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Melanoma , Neoplasias Cutáneas , Aminopiridinas , Línea Celular Tumoral , Humanos , Sistema de Señalización de MAP Quinasas , Melanoma/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf , Pirroles , Neoplasias Cutáneas/tratamiento farmacológico , Melanoma Cutáneo MalignoRESUMEN
Mutations in MAPT (microtubule-associated protein tau) cause frontotemporal dementia (FTD). MAPT mutations are associated with abnormal tau phosphorylation levels and accumulation of misfolded tau protein that can propagate between neurons ultimately leading to cell death (tauopathy). Recently, a p.A152T tau variant was identified as a risk factor for FTD, Alzheimer's disease, and synucleinopathies. Here we used induced pluripotent stem cells (iPSC) from a patient carrying this p.A152T variant to create a robust, functional cellular assay system for probing pathophysiological tau accumulation and phosphorylation. Using stably transduced iPSC-derived neural progenitor cells engineered to enable inducible expression of the pro-neural transcription factor Neurogenin 2 (Ngn2), we generated disease-relevant, cortical-like glutamatergic neurons in a scalable, high-throughput screening compatible format. Utilizing automated confocal microscopy, and an advanced image-processing pipeline optimized for analysis of morphologically complex human neuronal cultures, we report quantitative, subcellular localization-specific effects of multiple kinase inhibitors on tau, including ones under clinical investigation not previously reported to affect tau phosphorylation. These results demonstrate the potential for using patient iPSC-derived ex vivo models of tauopathy as genetically accurate, disease-relevant systems to probe tau biochemistry and support the discovery of novel therapeutics for tauopathies.
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Glutamatos/metabolismo , Procesamiento de Imagen Asistido por Computador , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Neuronas/patología , Proteómica , Tauopatías/patología , Proteínas tau/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biomarcadores/metabolismo , Línea Celular , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteínas Quinasas/metabolismo , Piridinas/química , Piridinas/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Inhibition of the HER2/ERBB2 receptor is a keystone to treating HER2-positive malignancies, particularly breast cancer, but a significant fraction of HER2-positive (HER2+) breast cancers recur or fail to respond. Anti-HER2 monoclonal antibodies, like trastuzumab or pertuzumab, and ATP active site inhibitors like lapatinib, commonly lack durability because of adaptive changes in the tumor leading to resistance. HER2+ cell line responses to inhibition with lapatinib were analyzed by RNAseq and ChIPseq to characterize transcriptional and epigenetic changes. Motif analysis of lapatinib-responsive genomic regions implicated the pioneer transcription factor FOXA1 as a mediator of adaptive responses. Lapatinib in combination with FOXA1 depletion led to dysregulation of enhancers, impaired adaptive upregulation of HER3, and decreased proliferation. HER2-directed therapy using clinically relevant drugs (trastuzumab with or without lapatinib or pertuzumab) in a 7-day clinical trial designed to examine early pharmacodynamic response to antibody-based anti-HER2 therapy showed reduced FOXA1 expression was coincident with decreased HER2 and HER3 levels, decreased proliferation gene signatures, and increased immune gene signatures. This highlights the importance of the immune response to anti-HER2 antibodies and suggests that inhibiting FOXA1-mediated adaptive responses in combination with HER2 targeting is a potential therapeutic strategy.
RESUMEN
BACKGROUND: In healthy lungs, epithelial sodium channel (ENaC) is regulated by short, palate, lung, and nasal clone 1 (SPLUNC1). In cystic fibrosis (CF), ENaC is hyperactivated in part due to a loss of SPLUNC1 function. We have developed SPX-101 to replace the lost function of SPLUNC1 in the CF lung. METHODS: Expression of SPLUNC1 was determined in sputum from healthy and CF donors. Stability of SPLUNC1, S18 (the ENaC regulatory domain of SPLUNC1), and SPX-101 was determined in sputum from CF donors and towards neutrophil elastase. Activity of SPX-101 after exposure to CF sputum was determined in airway epithelial cells from CF donors and in the ßENaC transgenic mouse model. RESULTS: SPLUNC1 protein expression is significantly reduced in CF as compared to healthy sputum. SPLUNC1 is rapidly degraded in CF sputum as well as by a number of individual proteases known to be found in the sputum. SPX-101, but not S18, is stable in CF sputum. Finally, SPX-101 retains its ability to internalize ENaC, regulate airway surface liquid height, and increase survival of ßENaC mice after exposure to CF sputum. CONCLUSIONS: Our results demonstrate that SPX-101, but not SPLUNC1 or S18, is stable in CF sputum. These results support the therapeutic development of SPX-101 for the treatment of cystic fibrosis.
Asunto(s)
Fibrosis Quística/metabolismo , Bloqueadores del Canal de Sodio Epitelial/farmacología , Canales Epiteliales de Sodio/metabolismo , Glicoproteínas/metabolismo , Fosfoproteínas/metabolismo , Mucosa Respiratoria , Animales , Células Cultivadas , Descubrimiento de Drogas , Humanos , Transporte Iónico/efectos de los fármacos , Transporte Iónico/fisiología , Elastasa de Leucocito/metabolismo , Ratones , Ratones Transgénicos , Depuración Mucociliar/efectos de los fármacos , Péptidos/metabolismo , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/enzimología , Mucosa Respiratoria/metabolismo , Esputo/metabolismoRESUMEN
Screening of an inhibitor library targeting kinases and epigenetic regulators identified several molecules having antiproliferative synergy with extraterminal domain (BET) bromodomain (BD) inhibitors (JQ1, OTX015) in triple-negative breast cancer (TNBC). GSK2801, an inhibitor of BAZ2A/B BDs, of the imitation switch chromatin remodeling complexes, and BRD9, of the SWI/SNF complex, demonstrated synergy independent of BRD4 control of P-TEFb-mediated pause-release of RNA polymerase II. GSK2801 or RNAi knockdown of BAZ2A/B with JQ1 selectively displaced BRD2 at promoters/enhancers of ETS-regulated genes. Additional displacement of BRD2 from rDNA in the nucleolus coincided with decreased 45S rRNA, revealing a function of BRD2 in regulating RNA polymerase I transcription. In 2D cultures, enhanced displacement of BRD2 from chromatin by combination drug treatment induced senescence. In spheroid cultures, combination treatment induced cleaved caspase-3 and cleaved PARP characteristic of apoptosis in tumor cells. Thus, GSK2801 blocks BRD2-driven transcription in combination with BET inhibitor and induces apoptosis of TNBC. IMPLICATIONS: Synergistic inhibition of BDs encoded in BAZ2A/B, BRD9, and BET proteins induces apoptosis of TNBC by a combinatorial suppression of ribosomal DNA transcription and ETS-regulated genes.
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Proteínas Cromosómicas no Histona/genética , Proteínas del Tejido Nervioso/genética , Receptores de Superficie Celular/genética , Factores de Transcripción/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Azepinas/farmacología , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Sinergismo Farmacológico , Femenino , Humanos , Indolizinas/farmacología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Polimerasa II/genética , ARN Ribosómico/genética , Receptores de Superficie Celular/antagonistas & inhibidores , Sulfonas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Triazoles/farmacología , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Background: Meningiomas are the most common primary brain tumor in adults, and somatic loss of the neurofibromatosis 2 (NF2) tumor suppressor gene is a frequent genetic event. There is no effective treatment for tumors that recur or continue to grow despite surgery and/or radiation. Therefore, targeted therapies that either delay tumor progression or cause tumor shrinkage are much needed. Our earlier work established mammalian target of rapamycin complex mTORC1/mTORC2 activation in NF2-deficient meningiomas. Methods: High-throughput kinome analyses were performed in NF2-null human arachnoidal and meningioma cell lines to identify functional kinome changes upon NF2 loss. Immunoblotting confirmed the activation of kinases and demonstrated effectiveness of drugs to block the activation. Drugs, singly and in combination, were screened in cells for their growth inhibitory activity. Antitumor drug efficacy was tested in an orthotopic meningioma model. Results: Erythropoietin-producing hepatocellular receptor tyrosine kinases (EPH RTKs), c-KIT, and Src family kinase (SFK) members, which are biological targets of dasatinib, were among the top candidates activated in NF2-null cells. Dasatinib significantly inhibited phospho-EPH receptor A2 (pEPHA2), pEPHB1, c-KIT, and Src/SFK in NF2-null cells, showing no cross-talk with mTORC1/2 signaling. Posttreatment kinome analyses showed minimal adaptive changes. While dasatinib treatment showed some activity, dual mTORC1/2 inhibitor and its combination with dasatinib elicited stronger growth inhibition in meningiomas. Conclusion: Co-targeting mTORC1/2 and EPH RTK/SFK pathways could be a novel effective treatment strategy for NF2-deficient meningiomas.
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Antineoplásicos/farmacología , Biomarcadores de Tumor/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Meníngeas/patología , Meningioma/patología , Neurofibromina 2/deficiencia , Receptores de la Familia Eph/antagonistas & inhibidores , Animales , Apoptosis , Proliferación Celular , Humanos , Neoplasias Meníngeas/tratamiento farmacológico , Neoplasias Meníngeas/metabolismo , Meningioma/tratamiento farmacológico , Meningioma/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Receptores de la Familia Eph/genética , Receptores de la Familia Eph/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Multiplexed small molecule inhibitors covalently bound to Sepharose beads (MIBs) were used to capture functional kinases in luminal, HER2-enriched and triple negative (basal-like and claudin-low) breast cancer cell lines and tumors. Kinase MIB-binding profiles at baseline without perturbation proteomically distinguished the four breast cancer subtypes. Understudied kinases, whose disease associations and pharmacology are generally unexplored, were highly represented in MIB-binding taxonomies and are integrated into signaling subnetworks with kinases that have been previously well characterized in breast cancer. Computationally it was possible to define subtypes using profiles of less than 50 of the more than 300 kinases bound to MIBs that included understudied as well as metabolic and lipid kinases. Furthermore, analysis of MIB-binding profiles established potential functional annotations for these understudied kinases. Thus, comprehensive MIBs-based capture of kinases provides a unique proteomics-based method for integration of poorly characterized kinases of the understudied kinome into functional subnetworks in breast cancer cells and tumors that is not possible using genomic strategies. The MIB-binding profiles readily defined subtype-selective differential adaptive kinome reprogramming in response to targeted kinase inhibition, demonstrating how MIB profiles can be used in determining dynamic kinome changes that result in subtype selective phenotypic state changes.
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BACKGROUND: Most novel cancer therapeutics target kinases that are essential to tumor survival. Some of these kinase inhibitors are associated with cardiotoxicity, whereas others appear to be cardiosafe. The basis for this distinction is unclear, as are the molecular effects of kinase inhibitors in the heart. METHODS AND RESULTS: We administered clinically relevant doses of sorafenib, sunitinib (cardiotoxic multitargeted kinase inhibitors), or erlotinib (a cardiosafe epidermal growth factor receptor inhibitor) to mice daily for 2 weeks. We then compared the effects of these 3 kinase inhibitors on the cardiac transcriptome using RNAseq and the cardiac kinome using multiplexed inhibitor beads coupled with mass spectrometry. We found unexpectedly broad molecular effects of all 3 kinase inhibitors, suggesting that target kinase selectivity does not define either the molecular response or the potential for cardiotoxicity. Using in vivo drug administration and primary cardiomyocyte culture, we also show that the cardiosafety of erlotinib treatment may result from upregulation of the cardioprotective signal transducer and activator of transcription 3 pathway, as co-treatment with erlotinib and a signal transducer and activator of transcription inhibitor decreases cardiac contractile function and cardiomyocyte fatty acid oxidation. CONCLUSIONS: Collectively our findings indicate that preclinical kinome and transcriptome profiling may predict the cardiotoxicity of novel kinase inhibitors, and suggest caution for the proposed therapeutic strategy of combined signal transducer and activator of transcription/epidermal growth factor receptor inhibition for cancer treatment.
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Antineoplásicos/toxicidad , Receptores ErbB/antagonistas & inhibidores , Clorhidrato de Erlotinib/toxicidad , Perfilación de la Expresión Génica , Cardiopatías/inducido químicamente , Corazón/efectos de los fármacos , Indoles/toxicidad , Miocardio/enzimología , Niacinamida/análogos & derivados , Compuestos de Fenilurea/toxicidad , Inhibidores de Proteínas Quinasas/toxicidad , Proteómica , Pirroles/toxicidad , Factor de Transcripción STAT3/antagonistas & inhibidores , Animales , Cardiotoxicidad , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ecocardiografía , Receptores ErbB/metabolismo , Ácidos Grasos/metabolismo , Femenino , Corazón/diagnóstico por imagen , Cardiopatías/diagnóstico por imagen , Cardiopatías/enzimología , Cardiopatías/genética , Ratones , Terapia Molecular Dirigida , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/enzimología , Niacinamida/toxicidad , Oxidación-Reducción , Mapas de Interacción de Proteínas , Ratas Sprague-Dawley , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Sorafenib , Sunitinib , Factores de TiempoRESUMEN
BACKGROUND: Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Prognosis remains poor despite multimodal therapy. Developing alternative treatments is essential. Drugs targeting kinases within the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) effectors of receptor tyrosine kinase (RTK) signaling represent promising candidates. METHODS: We previously developed a non-germline genetically engineered mouse model of GBM in which PI3K and MAPK are activated via Pten deletion and KrasG12D in immortalized astrocytes. Using this model, we examined the influence of drug potency on target inhibition, alternate pathway activation, efficacy, and synergism of single agent and combination therapy with inhibitors of these 2 pathways. Efficacy was then examined in GBM patient-derived xenografts (PDX) in vitro and in vivo. RESULTS: PI3K and mitogen-activated protein kinase kinase (MEK) inhibitor potency was directly associated with target inhibition, alternate RTK effector activation, and efficacy in mutant murine astrocytes in vitro. The kinomes of GBM PDX and tumor samples were heterogeneous, with a subset of the latter harboring MAPK hyperactivation. Dual PI3K/MEK inhibitor treatment overcame alternate effector activation, was synergistic in vitro, and was more effective than single agent therapy in subcutaneous murine allografts. However, efficacy in orthotopic allografts was minimal. This was likely due to dose-limiting toxicity and incomplete target inhibition. CONCLUSION: Drug potency influences PI3K/MEK inhibitor-induced target inhibition, adaptive kinome reprogramming, efficacy, and synergy. Our findings suggest that combination therapies with highly potent, brain-penetrant kinase inhibitors will be required to improve patient outcomes.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Resistencia a Antineoplásicos , Glioblastoma/tratamiento farmacológico , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Animales , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Ratones , Fosforilación , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Targeting the dysregulated BRAF-MEK-ERK pathway in cancer has increasingly emerged in clinical trial design. Despite clinical responses in specific cancers using inhibitors targeting BRAF and MEK, resistance develops often involving nongenomic adaptive bypass mechanisms. Inhibition of MEK1/2 by trametinib in patients with triple-negative breast cancer (TNBC) induced dramatic transcriptional responses, including upregulation of receptor tyrosine kinases (RTK) comparing tumor samples before and after one week of treatment. In preclinical models, MEK inhibition induced genome-wide enhancer formation involving the seeding of BRD4, MED1, H3K27 acetylation, and p300 that drives transcriptional adaptation. Inhibition of the P-TEFb-associated proteins BRD4 and CBP/p300 arrested enhancer seeding and RTK upregulation. BRD4 bromodomain inhibitors overcame trametinib resistance, producing sustained growth inhibition in cells, xenografts, and syngeneic mouse TNBC models. Pharmacologic targeting of P-TEFb members in conjunction with MEK inhibition by trametinib is an effective strategy to durably inhibit epigenomic remodeling required for adaptive resistance.Significance: Widespread transcriptional adaptation to pharmacologic MEK inhibition was observed in TNBC patient tumors. In preclinical models, MEK inhibition induces dramatic genome-wide modulation of chromatin, in the form of de novo enhancer formation and enhancer remodeling. Pharmacologic targeting of P-TEFb complex members at enhancers is an effective strategy to durably inhibit such adaptation. Cancer Discov; 7(3); 302-21. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 235.
Asunto(s)
Antineoplásicos/uso terapéutico , Elementos de Facilitación Genéticos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Factor B de Elongación Transcripcional Positiva/antagonistas & inhibidores , Piridonas/uso terapéutico , Pirimidinonas/uso terapéutico , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Azepinas/farmacología , Azepinas/uso terapéutico , Proteínas de Ciclo Celular , Línea Celular Tumoral , Metilación de ADN , Receptor con Dominio Discoidina 1/genética , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Epigénesis Genética , Femenino , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Humanos , Ratones Endogámicos BALB C , Ratones SCID , Terapia Molecular Dirigida , Proteínas Nucleares/antagonistas & inhibidores , Factor B de Elongación Transcripcional Positiva/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , Piridonas/farmacología , Pirimidinonas/farmacología , Interferencia de ARN , Factores de Transcripción/antagonistas & inhibidores , Triazoles/farmacología , Triazoles/uso terapéutico , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The characterization of kinases as oncogenic drivers has led to more than 30 FDA-approved targeted kinase inhibitors for cancer treatment. Unfortunately, these therapeutics fail to have clinical durability because of adaptive responses from the kinome and transcriptome that bypass inhibition of the targeted pathway. In our recent work, we describe a method to prevent these adaptive responses at an epigenetic level, generating a durable response to kinase inhibition.
RESUMEN
Therapeutics that target ERBB2, such as lapatinib, often provide initial clinical benefit, but resistance frequently develops. Adaptive responses leading to lapatinib resistance involve reprogramming of the kinome through reactivation of ERBB2/ERBB3 signaling and transcriptional upregulation and activation of multiple tyrosine kinases. The heterogeneity of induced kinases prevents their targeting by a single kinase inhibitor, underscoring the challenge of predicting effective kinase inhibitor combination therapies. We hypothesized that, to make the tumor response to single kinase inhibitors durable, the adaptive kinome response itself must be inhibited. Genetic and chemical inhibition of BET bromodomain chromatin readers suppresses transcription of many lapatinib-induced kinases involved in resistance, including ERBB3, IGF1R, DDR1, MET, and FGFRs, preventing downstream SRC/FAK signaling and AKT reactivation. Combining inhibitors of kinases and chromatin readers prevents kinome adaptation by blocking transcription, generating a durable response to lapatinib, and overcoming the dilemma of heterogeneity in the adaptive response.
Asunto(s)
Neoplasias de la Mama/metabolismo , Resistencia a Antineoplásicos/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Receptor ErbB-2/metabolismo , Transducción de Señal/fisiología , Antineoplásicos/farmacología , Western Blotting , Línea Celular Tumoral , Femenino , Humanos , Lapatinib , Espectrometría de Masas , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Proteómica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The central role of the BRAF-MEK-ERK pathway in controlling cell fate has made this pathway a primary target for deregulated activation in cancer. BRaf is activated by Ras proteins allowing Ras oncogenes to constitutively activate the pathway. Activating BRaf mutations are also frequent in several cancers, being the most common oncogenic mutation in thyroid carcinoma and melanoma. There are currently two inhibitors, vemurafenib and dabrafenib, approved for treatment of malignant melanoma having activating BRaf mutations. Concurrent administration of BRAF and MAP-ERK kinase (MEK) inhibitor (trametinib) is significantly more active in patients with BRAF-mutant melanoma than either single agent alone, but progression to resistance ultimately occurs by different mechanisms that increase the activation of extracellular signal-regulated kinase (ERK). Such adaptive changes in tumor cell signaling networks allow bypass of targeted oncoprotein inhibition. This is true with targeted inhibitors for BRaf and MEK as well as specific inhibitors for AKT, mTOR, and many receptor tyrosine kinases such as EGF receptor (EGFR) and HER2. It is this adaptive response to targeted kinase inhibitors that contributes to the failure of single-agent kinase inhibitors to have durable responses. This failure is seen in virtually all cancers treated with single-agent kinase inhibitors, most of which are not as dependent on a single signaling pathway such as BRaf-MEK-ERK in melanoma. Thus, understanding the breadth of adaptive reprogramming responses to specific targeted kinase inhibition will be critical to develop appropriate combination therapies for durable clinical responses.