RESUMEN
Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1-5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia-reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.
Asunto(s)
Regeneración Nerviosa , Humanos , Neoplasias/tratamiento farmacológico , Regeneración Nerviosa/efectos de los fármacos , Isoformas de Proteínas/agonistas , Transducción de Señal/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase I/química , Fosfatidilinositol 3-Quinasa Clase I/efectos de los fármacos , Cardiotónicos/farmacología , Animales , Biocatálisis/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Neuritas/efectos de los fármacos , Daño por Reperfusión/prevención & control , Compresión Nerviosa , Proliferación Celular/efectos de los fármacosRESUMEN
A number of regulatory factors are recruited to chromatin by specialized RNAs. Whether RNA has a more general role in regulating the interaction of proteins with chromatin has not been determined. We used proteomics methods to measure the global impact of nascent RNA on chromatin in embryonic stem cells. Surprisingly, we found that nascent RNA primarily antagonized the interaction of chromatin modifiers and transcriptional regulators with chromatin. Transcriptional inhibition and RNA degradation induced recruitment of a set of transcriptional regulators, chromatin modifiers, nucleosome remodelers, and regulators of higher-order structure. RNA directly bound to factors, including BAF, NuRD, EHMT1, and INO80 and inhibited their interaction with nucleosomes. The transcriptional elongation factor P-TEFb directly bound pre-mRNA, and its recruitment to chromatin upon Pol II inhibition was regulated by the 7SK ribonucleoprotein complex. We postulate that by antagonizing the interaction of regulatory proteins with chromatin, nascent RNA links transcriptional output with chromatin composition.
Asunto(s)
Cromatina/metabolismo , ARN/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , Masculino , Ratones , Nucleosomas/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , Unión Proteica/fisiología , Proteómica/métodos , ARN Polimerasa II/metabolismo , Transcripción Genética/fisiología , Factores de Elongación Transcripcional/metabolismoRESUMEN
Despite the crucial role of RAF kinases in cell signaling and disease, we still lack a complete understanding of their regulation. Heterodimerization of RAF kinases as well as dephosphorylation of a conserved "S259" inhibitory site are important steps for RAF activation but the precise mechanisms and dynamics remain unclear. A ternary complex comprised of SHOC2, MRAS, and PP1 (SHOC2 complex) functions as a RAF S259 holophosphatase and gain-of-function mutations in SHOC2, MRAS, and PP1 that promote complex formation are found in Noonan syndrome. Here we show that SHOC2 complex-mediated S259 RAF dephosphorylation is critically required for growth factor-induced RAF heterodimerization as well as for MEK dissociation from BRAF. We also uncover SHOC2-independent mechanisms of RAF and ERK pathway activation that rely on N-region phosphorylation of CRAF. In DLD-1 cells stimulated with EGF, SHOC2 function is essential for a rapid transient phase of ERK activation, but is not required for a slow, sustained phase that is instead driven by palmitoylated H/N-RAS proteins and CRAF. Whereas redundant SHOC2-dependent and -independent mechanisms of RAF and ERK activation make SHOC2 dispensable for proliferation in 2D, KRAS mutant cells preferentially rely on SHOC2 for ERK signaling under anchorage-independent conditions. Our study highlights a context-dependent contribution of SHOC2 to ERK pathway dynamics that is preferentially engaged by KRAS oncogenic signaling and provides a biochemical framework for selective ERK pathway inhibition by targeting the SHOC2 holophosphatase.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Quinasas raf/química , Quinasas raf/metabolismo , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Línea Celular Tumoral , Edición Génica , Técnicas de Inactivación de Genes , Humanos , Fosforilación , Multimerización de Proteína , Proteínas ras/metabolismoRESUMEN
Alterations to the gene encoding the EZH2 (KMT6A) methyltransferase, including both gain-of-function and loss-of-function, have been linked to a variety of haematological malignancies and solid tumours, suggesting a complex, context-dependent role of this methyltransferase. The successful implementation of molecularly targeted therapies against EZH2 requires a greater understanding of the potential mechanisms by which EZH2 contributes to cancer. One aspect of this effort is the mapping of EZH2 partner proteins and cellular targets. To this end we performed affinity-purification mass spectrometry in the FAB-M2 HL-60 acute myeloid leukaemia (AML) cell line before and after all-trans retinoic acid-induced differentiation. These studies identified new EZH2 interaction partners and potential non-histone substrates for EZH2-mediated methylation. Our results suggest that EZH2 is involved in the regulation of translation through interactions with a number of RNA binding proteins and by methylating key components of protein synthesis such as eEF1A1. Given that deregulated mRNA translation is a frequent feature of cancer and that eEF1A1 is highly expressed in many human tumours, these findings present new possibilities for the therapeutic targeting of EZH2 in AML.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Leucemia Mieloide Aguda/metabolismo , Espectrometría de Masas , Mapeo de Interacción de Proteínas , Secuencia de Aminoácidos , Proteínas Portadoras/química , Proteínas Portadoras/aislamiento & purificación , Línea Celular Tumoral , Biología Computacional/métodos , Proteína Potenciadora del Homólogo Zeste 2/química , Proteína Potenciadora del Homólogo Zeste 2/aislamiento & purificación , Ontología de Genes , Células HL-60 , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Metilación , Modelos Moleculares , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas , Flujo de TrabajoRESUMEN
BACKGROUND: Therapies targeting estrogenic stimulation in estrogen receptor-positive (ER+) breast cancer (BC) reduce mortality, but resistance remains a major clinical problem. Molecular studies have shown few high-frequency mutations to be associated with endocrine resistance. In contrast, expression profiling of primary ER+ BC samples has identified several promising signatures/networks for targeting. METHODS: To identify common adaptive mechanisms associated with resistance to aromatase inhibitors (AIs), we assessed changes in global gene expression during adaptation to long-term estrogen deprivation (LTED) in a panel of ER+ BC cell lines cultured in 2D on plastic (MCF7, T47D, HCC1428, SUM44 and ZR75.1) or in 3D on collagen (MCF7) to model the stromal compartment. Furthermore, dimethyl labelling followed by LC-MS/MS was used to assess global changes in protein abundance. The role of target genes/proteins on proliferation, ER-mediated transcription and recruitment of ER to target gene promoters was analysed. RESULTS: The cholesterol biosynthesis pathway was the common upregulated pathway in the ER+ LTED but not the ER- LTED cell lines, suggesting a potential mechanism dependent on continued ER expression. Targeting the individual genes of the cholesterol biosynthesis pathway with siRNAs caused a 30-50 % drop in proliferation. Further analysis showed increased expression of 25-hydroxycholesterol (HC) in the MCF7 LTED cells. Exogenous 25-HC or 27-HC increased ER-mediated transcription and expression of the endogenous estrogen-regulated gene TFF1 in ER+ LTED cells but not in the ER- LTED cells. Additionally, recruitment of the ER and CREB-binding protein (CBP) to the TFF1 and GREB1 promoters was increased upon treatment with 25-HC and 27-HC. In-silico analysis of two independent studies of primary ER+ BC patients treated with neoadjuvant AIs showed that increased expression of MSMO1, EBP, LBR and SQLE enzymes, required for cholesterol synthesis and increased in our in-vitro models, was significantly associated with poor response to endocrine therapy. CONCLUSION: Taken together, these data provide support for the role of cholesterol biosynthesis enzymes and the cholesterol metabolites, 25-HC and 27-HC, in a novel mechanism of resistance to endocrine therapy in ER+ BC that has potential as a therapeutic target.
Asunto(s)
Antineoplásicos Hormonales/farmacología , Vías Biosintéticas , Neoplasias de la Mama/metabolismo , Colesterol/biosíntesis , Resistencia a Antineoplásicos , Estrógenos/metabolismo , Receptores de Estrógenos/metabolismo , Antineoplásicos Hormonales/uso terapéutico , Biomarcadores , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ésteres del Colesterol/metabolismo , Resistencia a Antineoplásicos/genética , Femenino , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Fenotipo , Pronóstico , Proteoma , Proteómica/métodos , Interferencia de ARN , Transcriptoma , Resultado del TratamientoRESUMEN
Most studies of cohesin function consider the Stromalin Antigen (STAG/SA) proteins as core complex members given their ubiquitous interaction with the cohesin ring. Here, we provide functional data to support the notion that the SA subunit is not a mere passenger in this structure, but instead plays a key role in the localization of cohesin to diverse biological processes and promotes loading of the complex at these sites. We show that in cells acutely depleted for RAD21, SA proteins remain bound to chromatin, cluster in 3D and interact with CTCF, as well as with a wide range of RNA binding proteins involved in multiple RNA processing mechanisms. Accordingly, SA proteins interact with RNA, and R-loops, even in the absence of cohesin. Our results place SA1 on chromatin upstream of the cohesin ring and reveal a role for SA1 in cohesin loading which is independent of NIPBL, the canonical cohesin loader. We propose that SA1 takes advantage of structural R-loop platforms to link cohesin loading and chromatin structure with diverse functions. Since SA proteins are pan-cancer targets, and R-loops play an increasingly prevalent role in cancer biology, our results have important implications for the mechanistic understanding of SA proteins in cancer and disease.
Asunto(s)
Estructuras R-Loop , ARN , ARN/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina , Factor de Unión a CCCTC/metabolismo , CohesinasRESUMEN
Antibody combinations targeting cell surface receptors are a new modality of cancer therapy. The trafficking and signalling mechanisms regulated by such therapeutics are not fully understood but could underlie differential tumour responses. We explored EGFR trafficking upon treatment with the antibody combination Sym004 which has shown promise clinically. Sym004 promoted EGFR endocytosis distinctly from EGF: it was asynchronous, not accompanied by canonical signalling events and involved EGFR clustering within detergent-insoluble plasma mebrane-associated tubules. Sym004 induced lysosomal degradation independently of EGFR ubiquitylation but dependent upon Hrs/Tsg101 that are required for the formation of intraluminal vesicles (ILVs) within late endosomes. We propose Sym004 cross-links EGFR physically triggering EGFR endocytosis and incorporation onto ILVs and so Sym004 sensitivity correlates with EGFR numbers available for binding, rather than specific signalling events. Consistently Sym004 efficacy and potentiation of cisplatin responses correlated with EGFR surface expression in head and neck cancer cells. These findings will have implications in understanding the mode of action of this new class of cancer therapeutics.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos , Antineoplásicos , Endocitosis/efectos de los fármacos , Transporte de Proteínas , Membrana Celular/metabolismo , Células Cultivadas , Proteínas de Unión al ADN , Complejos de Clasificación Endosomal Requeridos para el Transporte , Endosomas/metabolismo , Receptores ErbB/metabolismo , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/etiología , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Lisosomas/metabolismo , Fosfoproteínas , Receptores de Superficie Celular , Factores de TranscripciónRESUMEN
Combination of CDK4/6 inhibitors and endocrine therapy improves clinical outcome in advanced oestrogen receptor (ER)-positive breast cancer, however relapse is inevitable. Here, we show in model systems that other than loss of RB1 few gene-copy number (CN) alterations are associated with irreversible-resistance to endocrine therapy and subsequent secondary resistance to palbociclib. Resistance to palbociclib occurred as a result of tumour cell re-wiring leading to increased expression of EGFR, MAPK, CDK4, CDK2, CDK7, CCNE1 and CCNE2. Resistance altered the ER genome wide-binding pattern, leading to decreased expression of 'classical' oestrogen-regulated genes and was accompanied by reduced sensitivity to fulvestrant and tamoxifen. Persistent CDK4 blockade decreased phosphorylation of tuberous sclerosis complex 2 (TSC2) enhancing EGFR signalling, leading to the re-wiring of ER. Kinome-knockdown confirmed dependency on ERBB-signalling and G2/M-checkpoint proteins such as WEE1, together with the cell cycle master regulator, CDK7. Noteworthy, sensitivity to CDK7 inhibition was associated with loss of ER and RB1 CN. Overall, we show that resistance to CDK4/6 inhibitors is dependent on kinase re-wiring and the redeployment of signalling cascades previously associated with endocrine resistance and highlights new therapeutic networks that can be exploited upon relapse after CDK4/6 inhibition.
Asunto(s)
Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Receptores de Estrógenos/genética , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/terapia , Línea Celular Tumoral , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/metabolismo , Resistencia a Antineoplásicos/genética , Femenino , Fulvestrant/administración & dosificación , Humanos , Células MCF-7 , Ratones Endogámicos BALB C , Ratones Desnudos , Interferencia de ARN , Receptores de Estrógenos/metabolismo , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Tamoxifeno/administración & dosificación , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Mass spectrometry enables the unbiased characterization of protein complexes. The success of this approach and the amount of information that can be retrieved are highly dependent on the achieved purity of the protein complex to be analyzed. Here we describe a modified tandem affinity purification (moTAP) approach which can be used to isolate the tumor necrosis factor receptor 1 signaling complex for subsequent analysis by liquid chromatography-tandem mass spectrometry. Indeed, this approach can easily be adapted to the isolation of other membrane-bound and intracellular signaling complexes. This methodology allows for a highly sensitive analysis and characterization of complex components, including posttranslational modifications and for the identification of novel complex components.
Asunto(s)
Cromatografía de Afinidad/métodos , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Complejos Multiproteicos/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Reactivos de Enlaces Cruzados/química , Humanos , Complejos Multiproteicos/aislamiento & purificación , Receptores Tipo I de Factores de Necrosis Tumoral/aislamiento & purificaciónRESUMEN
Acquired drug resistance impacts the majority of patients being treated with tyrosine kinase inhibitors (TKIs) and remains a key challenge in modern anti-cancer therapy. The lack of clinically effective therapies to overcome resistance represents an unmet need. Understanding the signalling that drives drug resistance will facilitate the development of new salvage therapies to treat patients with secondary TKI resistance. In this study, we utilise mass spectrometry to characterise the global phosphoproteomic alterations that accompany the acquisition of resistance to two FDA-approved TKIs, pazopanib and dasatinib, in the A204 rhabdoid tumour cell line. Our analysis finds that only 6% and 9.7% of the quantified phosphoproteome is altered upon the acquisition of pazopanib and dasatinib resistance, respectively. Pazopanib resistant cells display elevated phosphorylation in cytoskeletal regulatory pathways while dasatinib resistant cells show an upregulation of the insulin receptor/IGF-1R signalling pathway. Drug response profiling rediscovers several previously reported vulnerabilities associated with pazopanib and dasatinib resistance and identifies a new dependency to the second generation HSP90 inhibitor NVP-AUY-922. This study provides a useful resource detailing the candidate signalling determinants of acquired TKI resistance; and reveals a therapeutic approach of inhibiting HSP90 function as a means of salvage therapy to overcome pazopanib and dasatinib resistance. SIGNIFICANCE: Pazopanib and dasatinib are tyrosine kinase inhibitors (TKIs) approved for the treatment of multiple cancer types. Patients who are treated with these drugs are prone to the development of drug resistance and consequently tumour relapse. Here we use quantitative phosphoproteomics to characterise the signalling pathways which are enriched in cells that have acquired resistance to these two drugs. Furthermore, targeted drug screens were used to identify salvage therapies capable of overcoming pazopanib and dasatinib resistance. This data advances our understanding of the mechanisms of TKI resistance and highlights candidate targets for cancer therapy.
Asunto(s)
Dasatinib/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de Neoplasias/metabolismo , Neoplasias , Fosfoproteínas/metabolismo , Pirimidinas/farmacología , Sulfonamidas/farmacología , Línea Celular Tumoral , Humanos , Indazoles , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Transducción de Señal/efectos de los fármacosRESUMEN
The linear-ubiquitin chain assembly complex (LUBAC) modulates signalling via various immune receptors. In tumour necrosis factor (TNF) signalling, linear (also known as M1) ubiquitin enables full gene activation and prevents cell death. However, the mechanisms underlying cell death prevention remain ill-defined. Here, we show that LUBAC activity enables TBK1 and IKKε recruitment to and activation at the TNF receptor 1 signalling complex (TNFR1-SC). While exerting only limited effects on TNF-induced gene activation, TBK1 and IKKε are essential to prevent TNF-induced cell death. Mechanistically, TBK1 and IKKε phosphorylate the kinase RIPK1 in the TNFR1-SC, thereby preventing RIPK1-dependent cell death. This activity is essential in vivo, as it prevents TNF-induced lethal shock. Strikingly, NEMO (also known as IKKγ), which mostly, but not exclusively, binds the TNFR1-SC via M1 ubiquitin, mediates the recruitment of the adaptors TANK and NAP1 (also known as AZI2). TANK is constitutively associated with both TBK1 and IKKε, while NAP1 is associated with TBK1. We discovered a previously unrecognized cell death checkpoint that is mediated by TBK1 and IKKε, and uncovered an essential survival function for NEMO, whereby it enables the recruitment and activation of these non-canonical IKKs to prevent TNF-induced cell death.
Asunto(s)
Quinasa I-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Células A549 , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas , Células HeLa , Humanos , Ratones Noqueados , Fosforilación/efectos de los fármacos , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal/efectos de los fármacos , Ubiquitinación/efectos de los fármacosRESUMEN
Resistance to endocrine therapy remains a major clinical problem in breast cancer. Genetic studies highlight the potential role of estrogen receptor-α (ESR1) mutations, which show increased prevalence in the metastatic, endocrine-resistant setting. No naturally occurring ESR1 mutations have been reported in in vitro models of BC either before or after the acquisition of endocrine resistance making functional consequences difficult to study. We report the first discovery of naturally occurring ESR1 Y537C and ESR1 Y537S mutations in MCF7 and SUM44 ESR1-positive cell lines after acquisition of resistance to long-term-estrogen-deprivation (LTED) and subsequent resistance to fulvestrant (ICIR). Mutations were enriched with time, impacted on ESR1 binding to the genome and altered the ESR1 interactome. The results highlight the importance and functional consequence of these mutations and provide an important resource for studying endocrine resistance.