RESUMO
The RAS-regulated RAF-MEK1/2-ERK1/2 signalling pathway is activated in cancer due to mutations in RAS proteins (especially KRAS), BRAF, CRAF, MEK1 and MEK2. Whilst inhibitors of KRASG12C (lung adenocarcinoma) and BRAF and MEK1/2 (melanoma and colorectal cancer) are clinically approved, acquired resistance remains a problem. Consequently, the search for new inhibitors (especially of RAS proteins), new inhibitor modalities and regulators of this pathway, which may be new drug targets, continues and increasingly involves cell-based screens with small molecules or genetic screens such as RNAi, CRISPR or protein interference. Here we describe cell lines that exhibit doxycycline-dependent expression KRASG12V or BRAFV600E and harbour a stably integrated EGR1:EmGFP reporter gene that can be detected by flow cytometry, high-content microscopy or immunoblotting. KRASG12V or BRAFV600E-driven EmGFP expression is inhibited by MEK1/2 or ERK1/2 inhibitors (MEKi and ERKi). BRAFi inhibit BRAFV600E-driven EmGFP expression but enhance the response to KRASG12V, recapitulating paradoxical activation of wild type RAF proteins. In addition to small molecules, expression of iDab6, encoding a RAS-specific antibody fragment inhibited KRASG12V- but not BRAFV600E-driven EmGFP expression. Finally, substitution of EmGFP for a bacterial nitroreductase gene allowed KRASG12V or BRAFV600E to drive cell death in the presence of a pro-drug, which may allow selection of pathway inhibitors that promote survival. These cell lines should prove useful for cell-based screens to identify new regulators of KRAS- or BRAF-dependent ERK1/2 signalling (drug target discovery) as well as screening or triaging 'hits' from drug discovery screens.
Assuntos
Proteínas Proto-Oncogênicas B-raf , Proteínas Proto-Oncogênicas p21(ras) , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sistema de Sinalização das MAP Quinases , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular Tumoral , Mutação , Proteínas ras/genética , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Mutations and gene amplifications that confer drug resistance emerge frequently during chemotherapy, but their mechanism and timing are poorly understood. Here, we investigate BRAFV600E amplification events that underlie resistance to the MEK inhibitor selumetinib (AZD6244/ARRY-142886) in COLO205 cells, a well-characterized model for reproducible emergence of drug resistance, and show that BRAF amplifications acquired de novo are the primary cause of resistance. Selumetinib causes long-term G1 arrest accompanied by reduced expression of DNA replication and repair genes, but cells stochastically re-enter the cell cycle during treatment despite continued repression of pERK1/2. Most DNA replication and repair genes are re-expressed as cells enter S and G2; however, mRNAs encoding a subset of factors important for error-free replication and chromosome segregation, including TIPIN, PLK2 and PLK3, remain at low abundance. This suggests that DNA replication following escape from G1 arrest in drug is more error prone and provides a potential explanation for the DNA damage observed under long-term RAF-MEK-ERK1/2 pathway inhibition. To test the hypothesis that escape from G1 arrest in drug promotes de novo BRAF amplification, we exploited the combination of palbociclib and selumetinib. Combined treatment with selumetinib and a dose of palbociclib sufficient to reinforce G1 arrest in selumetinib-sensitive cells, but not to impair proliferation of resistant cells, delays the emergence of resistant colonies, meaning that escape from G1 arrest is critical in the formation of resistant clones. Our findings demonstrate that acquisition of MEK inhibitor resistance often occurs through de novo gene amplification and can be suppressed by impeding cell cycle entry in drug.
RESUMO
The RAS-regulated RAF-MEK1/2-ERK1/2 signalling pathway is frequently de-regulated in human cancer. Melanoma in particular exhibits a high incidence of activating BRAFV600E/K and NRASQ61L/K mutations and such cells are addicted to the activity of these mutant oncoproteins. As a result three different BRAF inhibitors (BRAFi) have now been approved for BRAFV600E/K- mutant melanoma and have transformed the treatment of this disease. Despite this, clinical responses are typically transient as tumour cells develop resistance. These resistance mechanisms frequently involve reinstatement of ERK1/2 signalling and BRAFi are now deployed in combination with one of three approved MEK1/2 inhibitors (MEKi) to provide more durable, but still transient, clinical responses. Furthermore, inhibitors to ERK1/2 (ERK1/2i) have also been developed to counteract ERK1/2 signalling. However, recent studies have suggested that BRAFi/MEKi and ERK1/2i resistance can arise through activation of a parallel signalling pathway leading to activation of ERK5, an unusual protein kinase that contains both a kinase domain and a transcriptional transactivation domain. Here we review the evidence supporting ERK5 as a mediator of BRAFi/MEKi and ERK1/2i resistance. We also review the challenges in targeting ERK5 signalling with small molecules, including paradoxical activation of the transcriptional transactivation domain, and discuss new therapeutic modalities that could be employed to target ERK5.
RESUMO
The nonclassical extracellular signal-related kinase 5 (ERK5) mitogen-activated protein kinase pathway has been implicated in increased cellular proliferation, migration, survival, and angiogenesis; hence, ERK5 inhibition may be an attractive approach for cancer treatment. However, the development of selective ERK5 inhibitors has been challenging. Previously, we described the development of a pyrrole carboxamide high-throughput screening hit into a selective, submicromolar inhibitor of ERK5 kinase activity. Improvement in the ERK5 potency was necessary for the identification of a tool ERK5 inhibitor for target validation studies. Herein, we describe the optimization of this series to identify nanomolar pyrrole carboxamide inhibitors of ERK5 incorporating a basic center, which suffered from poor oral bioavailability. Parallel optimization of potency and in vitro pharmacokinetic parameters led to the identification of a nonbasic pyrazole analogue with an optimal balance of ERK5 inhibition and oral exposure.
Assuntos
Proteína Quinase 7 Ativada por Mitógeno , Pirróis , Proliferação de Células , Pirróis/farmacologiaRESUMO
Inhibitor of kappa B (IκB) kinase ß (IKKß) has long been viewed as the dominant IKK in the canonical nuclear factor-κB (NF-κB) signalling pathway, with IKKα being more important in non-canonical NF-κB activation. Here we have investigated the role of IKKα and IKKß in canonical NF-κB activation in colorectal cells using CRISPR-Cas9 knock-out cell lines, siRNA and selective IKKß inhibitors. IKKα and IKKß were redundant for IκBα phosphorylation and turnover since loss of IKKα or IKKß alone had little (SW620 cells) or no (HCT116 cells) effect. However, in HCT116 cells IKKα was the dominant IKK required for basal phosphorylation of p65 at S536, stimulated phosphorylation of p65 at S468, nuclear translocation of p65 and the NF-κB-dependent transcriptional response to both TNFα and IL-1α. In these cells, IKKß was far less efficient at compensating for the loss of IKKα than IKKα was able to compensate for the loss of IKKß. This was confirmed when siRNA was used to knock-down the non-targeted kinase in single KO cells. Critically, the selective IKKß inhibitor BIX02514 confirmed these observations in WT cells and similar results were seen in SW620 cells. Notably, whilst IKKα loss strongly inhibited TNFα-dependent p65 nuclear translocation, IKKα and IKKß contributed equally to c-Rel nuclear translocation indicating that different NF-κB subunits exhibit different dependencies on these IKKs. These results demonstrate a major role for IKKα in canonical NF-κB signalling in colorectal cells and may be relevant to efforts to design IKK inhibitors, which have focused largely on IKKß to date.
Assuntos
Neoplasias Colorretais/metabolismo , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/genética , Sistemas CRISPR-Cas , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Técnicas de Inativação de Genes , Células HCT116 , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/genética , Interleucina-1alfa/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/genética , Fosforilação/genética , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Transfecção , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Cells must adapt to changes in their environment to maintain cell, tissue and organismal integrity in the face of mechanical, chemical or microbiological stress. Nuclear factor-κB (NF-κB) is one of the most important transcription factors that controls inducible gene expression as cells attempt to restore homeostasis. It plays critical roles in the immune system, from acute inflammation to the development of secondary lymphoid organs, and also has roles in cell survival, proliferation and differentiation. Given its role in such critical processes, NF-κB signalling must be subject to strict spatiotemporal control to ensure measured and context-specific cellular responses. Indeed, deregulation of NF-κB signalling can result in debilitating and even lethal inflammation and also underpins some forms of cancer. In this review, we describe the homeostatic feedback mechanisms that limit and 're-set' inducible activation of NF-κB. We first describe the key components of the signalling pathways leading to activation of NF-κB, including the prominent role of protein phosphorylation and protein ubiquitylation, before briefly introducing the key features of feedback control mechanisms. We then describe the array of negative feedback loops targeting different components of the NF-κB signalling cascade including controls at the receptor level, post-receptor signalosome complexes, direct regulation of the critical 'inhibitor of κB kinases' (IKKs) and inhibitory feedforward regulation of NF-κB-dependent transcriptional responses. We also review post-transcriptional feedback controls affecting RNA stability and translation. Finally, we describe the deregulation of these feedback controls in human disease and consider how feedback may be a challenge to the efficacy of inhibitors.
Assuntos
Retroalimentação Fisiológica , Inflamação/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Animais , Sobrevivência Celular/genética , Regulação da Expressão Gênica , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Inflamação/genética , Fosforilação , Estabilidade de RNA/genéticaRESUMO
The RAS-regulated RAF-MEK1/2-ERK1/2 pathway promotes cell proliferation and survival and RAS and BRAF proteins are commonly mutated in cancer. This has fuelled the development of small molecule kinase inhibitors including ATP-competitive RAF inhibitors. Type I and type I½ ATP-competitive RAF inhibitors are effective in BRAFV600E/K-mutant cancer cells. However, in RAS-mutant cells these compounds instead promote RAS-dependent dimerisation and paradoxical activation of wild-type RAF proteins. RAF dimerisation is mediated by two key regions within each RAF protein; the RKTR motif of the αC-helix and the NtA-region of the dimer partner. Dimer formation requires the adoption of a closed, active kinase conformation which can be induced by RAS-dependent activation of RAF or by the binding of type I and I½ RAF inhibitors. Binding of type I or I½ RAF inhibitors to one dimer partner reduces the binding affinity of the other, thereby leaving a single dimer partner uninhibited and able to activate MEK. To overcome this paradox two classes of drug are currently under development; type II pan-RAF inhibitors that induce RAF dimer formation but bind both dimer partners thus allowing effective inhibition of both wild-type RAF dimer partners and monomeric active class I mutant RAF, and the recently developed "paradox breakers" which interrupt BRAF dimerisation through disruption of the αC-helix. Here we review the regulation of RAF proteins, including RAF dimers, and the progress towards effective targeting of the wild-type RAF proteins.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Quinases raf/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/fisiologia , Estrutura Secundária de Proteína/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/fisiologia , Quinases raf/química , Quinases raf/metabolismoRESUMO
Autophagy and cap-dependent mRNA translation are tightly regulated by the mechanistic target of rapamycin complex 1 (mTORC1) signalling complex in response to nutrient availability. However, the regulation of these processes, and mTORC1 itself, is different during mitosis, and this has remained an area of significant controversy; for example, studies have argued that autophagy is either repressed or highly active during mitosis. Recent studies have shown that autophagy initiation is repressed, and cap-dependent mRNA translation is maintained during mitosis despite mTORC1 activity being repressed. This is achieved in large part by a switch from mTORC1- to cyclin-dependent kinase 1 (CDK1)-mediated regulation. Here, we review the history and recent advances and seek to present a unifying model to inform the future study of autophagy and mTORC1 during mitosis.
Assuntos
Autofagia/fisiologia , Proteína Quinase CDC2/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Mitose/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Fosforilação/fisiologia , Biossíntese de Proteínas , Transdução de Sinais/fisiologiaRESUMO
ERK5 is a protein kinase that also contains a nuclear localisation signal and a transcriptional transactivation domain. Inhibition of ERK5 has therapeutic potential in cancer and inflammation and this has prompted the development of ERK5 kinase inhibitors (ERK5i). However, few ERK5i programmes have taken account of the ERK5 transactivation domain. We have recently shown that the binding of small molecule ERK5i to the ERK5 kinase domain stimulates nuclear localisation and paradoxical activation of its transactivation domain. Other kinase inhibitors paradoxically activate their intended kinase target, in some cases leading to severe physiological consequences highlighting the importance of mitigating these effects. Here, we review the assays used to monitor ERK5 activities (kinase and transcriptional) in cells, the challenges faced in development of small molecule inhibitors to the ERK5 pathway, and classify the molecular mechanisms of paradoxical activation of protein kinases by kinase inhibitors.
Assuntos
Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Animais , Núcleo Celular/metabolismo , Glutationa/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Inflamação , Fatores de Transcrição MEF2/metabolismo , Modelos Moleculares , Fosforilação , Conformação Proteica , Domínios Proteicos , Fator de Transcrição AP-1/metabolismo , Ativação TranscricionalRESUMO
The dual protein kinase-transcription factor, ERK5, is an emerging drug target in cancer and inflammation, and small-molecule ERK5 kinase inhibitors have been developed. However, selective ERK5 kinase inhibitors fail to recapitulate ERK5 genetic ablation phenotypes, suggesting kinase-independent functions for ERK5. Here we show that ERK5 kinase inhibitors cause paradoxical activation of ERK5 transcriptional activity mediated through its unique C-terminal transcriptional activation domain (TAD). Using the ERK5 kinase inhibitor, Compound 26 (ERK5-IN-1), as a paradigm, we have developed kinase-active, drug-resistant mutants of ERK5. With these mutants, we show that induction of ERK5 transcriptional activity requires direct binding of the inhibitor to the kinase domain. This in turn promotes conformational changes in the kinase domain that result in nuclear translocation of ERK5 and stimulation of gene transcription. This shows that both the ERK5 kinase and TAD must be considered when assessing the role of ERK5 and the effectiveness of anti-ERK5 therapeutics.
Assuntos
Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Inflamação/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/genética , Modelos Moleculares , Mutação , Conformação Proteica , Domínios Proteicos , Inibidores de Proteínas Quinases/farmacologia , Transcrição GênicaRESUMO
The RAS-regulated RAF-MEK1/2-ERK1/2 signaling pathway is frequently deregulated in cancer due to activating mutations of growth factor receptors, RAS or BRAF. Both RAF and MEK1/2 inhibitors are clinically approved and various ERK1/2 inhibitors (ERKi) are currently undergoing clinical trials. To date, ERKi display two distinct mechanisms of action (MoA): catalytic ERKi solely inhibit ERK1/2 catalytic activity, whereas dual mechanism ERKi additionally prevents the activating phosphorylation of ERK1/2 at its T-E-Y motif by MEK1/2. These differences may impart significant differences in biological activity because T-E-Y phosphorylation is the signal for nuclear entry of ERK1/2, allowing them to access many key transcription factor targets. Here, we characterized the MoA of five ERKi and examined their functional consequences in terms of ERK1/2 signaling, gene expression, and antiproliferative efficacy. We demonstrate that catalytic ERKi promote a striking nuclear accumulation of p-ERK1/2 in KRAS-mutant cell lines. In contrast, dual-mechanism ERKi exploits a distinct binding mode to block ERK1/2 phosphorylation by MEK1/2, exhibit superior potency, and prevent the nuclear accumulation of ERK1/2. Consequently, dual-mechanism ERKi exhibit more durable pathway inhibition and enhanced suppression of ERK1/2-dependent gene expression compared with catalytic ERKi, resulting in increased efficacy across BRAF- and RAS-mutant cell lines.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/análise , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Animais , Humanos , Masculino , Camundongos , Camundongos Nus , FosforilaçãoRESUMO
Since nuclear envelope breakdown occurs during mitosis in metazoan cells, it has been proposed that macroautophagy must be inhibited to maintain genome integrity. However, repression of macroautophagy during mitosis remains controversial and mechanistic detail limited to the suggestion that CDK1 phosphorylates VPS34. Here, we show that initiation of macroautophagy, measured by the translocation of the ULK complex to autophagic puncta, is repressed during mitosis, even when mTORC1 is inhibited. Indeed, mTORC1 is inactive during mitosis, reflecting its failure to localize to lysosomes due to CDK1-dependent RAPTOR phosphorylation. While mTORC1 normally represses autophagy via phosphorylation of ULK1, ATG13, ATG14, and TFEB, we show that the mitotic phosphorylation of these autophagy regulators, including at known repressive sites, is dependent on CDK1 but independent of mTOR. Thus, CDK1 substitutes for inhibited mTORC1 as the master regulator of macroautophagy during mitosis, uncoupling autophagy regulation from nutrient status to ensure repression of macroautophagy during mitosis.
Assuntos
Autofagia/fisiologia , Proteína Quinase CDC2/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Mitose/fisiologia , Células A549 , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Células HCT116 , Células HEK293 , Células HT29 , Células HeLa , Humanos , Lisossomos/metabolismo , Masculino , Fosforilação/fisiologia , Transdução de Sinais/fisiologiaRESUMO
BRAF and MEK1/2 inhibitors are effective in melanoma but resistance inevitably develops. Despite increasing the abundance of pro-apoptotic BIM and BMF, ERK1/2 pathway inhibition is predominantly cytostatic, reflecting residual pro-survival BCL2 family activity. Here, we show that uniquely low BCL-XL expression in melanoma biases the pro-survival pool towards MCL1. Consequently, BRAF or MEK1/2 inhibitors are synthetic lethal with the MCL1 inhibitor AZD5991, driving profound tumour cell death that requires BAK/BAX, BIM and BMF, and inhibiting tumour growth in vivo. Combination of ERK1/2 pathway inhibitors with BCL2/BCL-w/BCL-XL inhibitors is stronger in CRC, correlating with a low MCL1:BCL-XL ratio; indeed the MCL1:BCL-XL ratio is predictive of ERK1/2 pathway inhibitor synergy with MCL1 or BCL2/BCL-w/BCL-XL inhibitors. Finally, AZD5991 delays acquired BRAFi/MEKi resistance and enhances the efficacy of an ERK1/2 inhibitor in a model of acquired BRAFi + MEKi resistance. Thus combining ERK1/2 pathway inhibitors with MCL1 antagonists in melanoma could improve therapeutic index and patient outcomes.
Assuntos
Apoptose , Sistema de Sinalização das MAP Quinases , Melanoma/patologia , Terapia de Alvo Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Compostos Macrocíclicos/farmacologia , Camundongos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteína bcl-X/metabolismoRESUMO
Extracellular regulated kinase 5 (ERK5) signalling has been implicated in driving a number of cellular phenotypes including endothelial cell angiogenesis and tumour cell motility. Novel ERK5 inhibitors were identified using high throughput screening, with a series of pyrrole-2-carboxamides substituted at the 4-position with an aroyl group being found to exhibit IC50 values in the micromolar range, but having no selectivity against p38α MAP kinase. Truncation of the N-substituent marginally enhanced potency (â¼3-fold) against ERK5, but importantly attenuated inhibition of p38α. Systematic variation of the substituents on the aroyl group led to the selective inhibitor 4-(2-bromo-6-fluorobenzoyl)-N-(pyridin-3-yl)-1H-pyrrole-2-carboxamide (IC50 0.82⯵M for ERK5; IC50â¯>â¯120⯵M for p38α). The crystal structure (PDB 5O7I) of this compound in complex with ERK5 has been solved. This compound was orally bioavailable and inhibited bFGF-driven Matrigel plug angiogenesis and tumour xenograft growth. The selective ERK5 inhibitor described herein provides a lead for further development into a tool compound for more extensive studies seeking to examine the role of ERK5 signalling in cancer and other diseases.
Assuntos
Antineoplásicos/farmacologia , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Disponibilidade Biológica , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Camundongos , Camundongos Nus , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismoRESUMO
Acquired resistance to MEK1/2 inhibitors (MEKi) arises through amplification of BRAFV600E or KRASG13D to reinstate ERK1/2 signalling. Here we show that BRAFV600E amplification and MEKi resistance are reversible following drug withdrawal. Cells with BRAFV600E amplification are addicted to MEKi to maintain a precise level of ERK1/2 signalling that is optimal for cell proliferation and survival, and tumour growth in vivo. Robust ERK1/2 activation following MEKi withdrawal drives a p57KIP2-dependent G1 cell cycle arrest and senescence or expression of NOXA and cell death, selecting against those cells with amplified BRAFV600E. p57KIP2 expression is required for loss of BRAFV600E amplification and reversal of MEKi resistance. Thus, BRAFV600E amplification confers a selective disadvantage during drug withdrawal, validating intermittent dosing to forestall resistance. In contrast, resistance driven by KRASG13D amplification is not reversible; rather ERK1/2 hyperactivation drives ZEB1-dependent epithelial-to-mesenchymal transition and chemoresistance, arguing strongly against the use of drug holidays in cases of KRASG13D amplification.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Amplificação de Genes/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias/genética , Inibidores de Proteínas Quinases/uso terapêutico , Suspensão de Tratamento , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoRESUMO
MEK1/2 inhibitors are clinically approved for the treatment of BRAF-mutant melanoma, where they are used in combination with BRAF inhibitors, and are undergoing evaluation in other malignancies. Acquired resistance to MEK1/2 inhibitors, including selumetinib (AZD6244/ARRY-142866), can arise through amplification of BRAFV600E or KRASG13D to reinstate ERK1/2 signalling. We have found that BRAFV600E amplification and selumetinib resistance are fully reversible following drug withdrawal. This is because resistant cells with BRAFV600E amplification become addicted to selumetinib to maintain a precise level of ERK1/2 signalling (2%-3% of total ERK1/2 active), that is optimal for cell proliferation and survival. Selumetinib withdrawal drives ERK1/2 activation outside of this critical "sweet spot" (~20%-30% of ERK1/2 active) resulting in a p57KIP2-dependent G1 cell cycle arrest and senescence or expression of NOXA and cell death with features of autophagy; these terminal responses select against cells with amplified BRAFV600E. ERK1/2-dependent p57KIP2 expression is required for loss of BRAFV600E amplification and determines the rate of reversal of selumetinib resistance. Growth of selumetinib-resistant cells with BRAFV600E amplification as tumour xenografts also requires the presence of selumetinib to "clamp" ERK1/2 activity within the sweet spot. Thus, BRAFV600E amplification confers a selective disadvantage or "fitness deficit" during drug withdrawal, providing a rationale for intermittent dosing to forestall resistance. Remarkably, selumetinib resistance driven by KRASG13D amplification/upregulation is not reversible. In these cells ERK1/2 reactivation does not inhibit proliferation but drives a ZEB1-dependent epithelial-to-mesenchymal transition that increases cell motility and promotes resistance to traditional chemotherapy agents. Our results reveal that the emergence of drug-addicted, MEKi-resistant cells, and the opportunity this may afford for intermittent dosing schedules ("drug holidays"), may be determined by the nature of the amplified driving oncogene (BRAFV600E vs. KRASG13D), further exemplifying the difficulties of targeting KRAS mutant tumour cells.
RESUMO
Deregulated NF-κB signalling is implicated in the pathogenesis of numerous human inflammatory disorders and malignancies. Consequently, the NF-κB pathway has attracted attention as an attractive therapeutic target for drug discovery. As the primary, druggable mediator of canonical NF-κB signalling the IKKß protein kinase has been the historical focus of drug development pipelines. Thousands of compounds with activity against IKKß have been characterised, with many demonstrating promising efficacy in pre-clinical models of cancer and inflammatory disease. However, severe on-target toxicities and other safety concerns associated with systemic IKKß inhibition have thus far prevented the clinical approval of any IKKß inhibitors. This review will discuss the potential reasons for the lack of clinical success of IKKß inhibitors to date, the challenges associated with their therapeutic use, realistic opportunities for their future utilisation, and the alternative strategies to inhibit NF-κB signalling that may overcome some of the limitations associated with IKKß inhibition.
RESUMO
The RAS-regulated RAF-MEK1/2-ERK1/2 signalling pathway is de-regulated in a variety of cancers due to mutations in receptor tyrosine kinases (RTKs), negative regulators of RAS (such as NF1) and core pathway components themselves (RAS, BRAF, CRAF, MEK1 or MEK2). This has driven the development of a variety of pharmaceutical agents to inhibit RAF-MEK1/2-ERK1/2 signalling in cancer and both RAF and MEK inhibitors are now approved and used in the clinic. There is now much interest in targeting at the level of ERK1/2 for a variety of reasons. First, since the pathway is linear from RAF-to-MEK-to-ERK then ERK1/2 are validated as targets per se. Second, innate resistance to RAF or MEK inhibitors involves relief of negative feedback and pathway re-activation with all signalling going through ERK1/2, validating the use of ERK inhibitors with RAF or MEK inhibitors as an up-front combination. Third, long-term acquired resistance to RAF or MEK inhibitors involves a variety of mechanisms (KRAS or BRAF amplification, MEK mutation, etc.) which re-instate ERK activity, validating the use of ERK inhibitors to forestall acquired resistance to RAF or MEK inhibitors. The first potent highly selective ERK1/2 inhibitors have now been developed and are entering clinical trials. They have one of three discrete mechanisms of action - catalytic, "dual mechanism" or covalent - which could have profound consequences for how cells respond and adapt. In this review we describe the validation of ERK1/2 as anti-cancer drug targets, consider the mechanism of action of new ERK1/2 inhibitors and how this may impact on their efficacy, anticipate factors that will determine how tumour cells respond and adapt to ERK1/2 inhibitors and consider ERK1/2 inhibitor drug combinations.
Assuntos
Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Quinases/metabolismo , Animais , Humanos , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
BRAF is a cytoplasmic protein kinase, which activates the MEK-ERK signalling pathway. Deregulation of the pathway is associated with the presence of BRAF mutations in human cancer, the most common being V600E BRAF, although structural rearrangements, which remove N-terminal regulatory sequences, have also been reported. RAF-MEK-ERK signalling is normally thought to occur in the cytoplasm of the cell. However, in an investigation of BRAF localisation using fluorescence microscopy combined with subcellular fractionation of Green Fluorescent Protein (GFP)-tagged proteins expressed in NIH3T3 cells, surprisingly, we detected N-terminally truncated BRAF (ΔBRAF) in both nuclear and cytoplasmic compartments. In contrast, ΔCRAF and full-length, wild-type BRAF (WTBRAF) were detected at lower levels in the nucleus while full-length V600EBRAF was virtually excluded from this compartment. Similar results were obtained using ΔBRAF tagged with the hormone-binding domain of the oestrogen receptor (hbER) and with the KIAA1549-ΔBRAF translocation mutant found in human pilocytic astrocytomas. Here we show that GFP-ΔBRAF nuclear translocation does not involve a canonical Nuclear Localisation Signal (NLS), but is suppressed by N-terminal sequences. Nuclear GFP-ΔBRAF retains MEK/ERK activating potential and is associated with the accumulation of phosphorylated MEK and ERK in the nucleus. In contrast, full-length GFP-WTBRAF and GFP-V600EBRAF are associated with the accumulation of phosphorylated ERK but not phosphorylated MEK in the nucleus. These data have implications for cancers bearing single nucleotide variants or N-terminal deleted structural variants of BRAF.
RESUMO
Disruption of protein folding in the endoplasmic reticulum (ER) causes ER stress. Activation of the unfolded protein response (UPR) acts to restore protein homeostasis or, if ER stress is severe or persistent, drive apoptosis, which is thought to proceed through the cell intrinsic, mitochondrial pathway. Indeed, cells that lack the key executioner proteins BAX and BAK are protected from ER stress-induced apoptosis. Here we show that chronic ER stress causes the progressive inhibition of the extracellular signal-regulated kinase (ERK1/2) signalling pathway. This is causally related to ER stress since reactivation of ERK1/2 can protect cells from ER stress-induced apoptosis whilst ERK1/2 pathway inhibition sensitises cells to ER stress. Furthermore, cancer cell lines harbouring constitutively active BRAFV600E are addicted to ERK1/2 signalling for protection against ER stress-induced cell death. ERK1/2 signalling normally represses the pro-death proteins BIM, BMF and PUMA and it has been proposed that ER stress induces BIM-dependent cell death. We found no evidence that ER stress increased the expression of these proteins; furthermore, BIM was not required for ER stress-induced death. Rather, ER stress caused the PERK-dependent inhibition of cap-dependent mRNA translation and the progressive loss of pro-survival proteins including BCL2, BCLXL and MCL1. Despite these observations, neither ERK1/2 activation nor loss of BAX/BAK could confer long-term clonogenic survival to cells exposed to ER stress. Thus, ER stress induces cell death by at least two biochemically and genetically distinct pathways: a classical BAX/BAK-dependent apoptotic response that can be inhibited by ERK1/2 signalling and an alternative ERK1/2- and BAX/BAK-independent cell death pathway.