Publications by authors named "Simon J Cook"

83 Publications

Inhibition of RAF dimers: it takes two to tango.

Biochem Soc Trans 2021 Feb;49(1):237-251

Signalling Programme, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, U.K.

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.
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http://dx.doi.org/10.1042/BST20200485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7924995PMC
February 2021

CDK1, the Other 'Master Regulator' of Autophagy.

Trends Cell Biol 2021 Feb 30;31(2):95-107. Epub 2020 Nov 30.

Signalling Laboratory, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT, UK. Electronic address:

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.
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http://dx.doi.org/10.1016/j.tcb.2020.11.001DOI Listing
February 2021

Small molecule ERK5 kinase inhibitors paradoxically activate ERK5 signalling: be careful what you wish for….

Biochem Soc Trans 2020 10;48(5):1859-1875

Signalling Laboratory, Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, U.K.

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.
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http://dx.doi.org/10.1042/BST20190338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609025PMC
October 2020

Paradoxical activation of the protein kinase-transcription factor ERK5 by ERK5 kinase inhibitors.

Nat Commun 2020 03 13;11(1):1383. Epub 2020 Mar 13.

Signalling Laboratory, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT, UK.

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.
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http://dx.doi.org/10.1038/s41467-020-15031-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7069993PMC
March 2020

Macroautophagy is repressed during mitosis - seeing is believing.

Autophagy 2020 04 20;16(4):775-776. Epub 2020 Feb 20.

Signalling Laboratory, The Babraham Institute, Cambridge, UK.

For the last two decades there has been wide ranging debate about the status of macroautophagy during mitosis. Because metazoan cells undergo an "open" mitosis in which the nuclear envelope breaks down, it has been proposed that macroautophagy must be inhibited to maintain genome integrity. While many studies have agreed that the number of autophagosomes is greatly reduced in cells undergoing mitosis, there has been no consensus on whether this reflects decreased autophagosome synthesis or increased autophagosome degradation. Reviewing the literature we were concerned that many studies relied too heavily on autophagy assays that were simply not appropriate for a relatively brief event such as mitosis. Using highly dynamic omegasome markers we have recently shown unequivocally that autophagosome synthesis is repressed at the onset of mitosis and is restored once cell division is complete. This is accomplished by CDK1, the master regulator of mitosis, taking over the function of MTORC1, to ensure autophagy is repressed during mitosis.
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http://dx.doi.org/10.1080/15548627.2020.1725405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7138195PMC
April 2020

The empirical basis for modelling glacial erosion rates.

Nat Commun 2020 02 6;11(1):759. Epub 2020 Feb 6.

School of Geography, Geology and the Environment, Keele University, Staffordshire, ST5 5BG, UK.

Glaciers are highly effective agents of erosion that have profoundly shaped Earth's surface, but there is uncertainty about how glacial erosion should be parameterised in landscape evolution models. Glacial erosion rate is usually modelled as a function of glacier sliding velocity, but the empirical basis for this relationship is weak. In turn, climate is assumed to control sliding velocity and hence erosion, but this too lacks empirical scrutiny. Here, we present statistically robust relationships between erosion rates, sliding velocities, and climate from a global compilation of 38 glaciers. We show that sliding is positively and significantly correlated with erosion, and derive a relationship for use in erosion models. Our dataset further demonstrates that the most rapid erosion is achieved at temperate glaciers with high mean annual precipitation, which serve to promote rapid sliding. Precipitation has received little attention in glacial erosion studies, but our data illustrate its importance.
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http://dx.doi.org/10.1038/s41467-020-14583-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005307PMC
February 2020

Dual-Mechanism ERK1/2 Inhibitors Exploit a Distinct Binding Mode to Block Phosphorylation and Nuclear Accumulation of ERK1/2.

Mol Cancer Ther 2020 02 20;19(2):525-539. Epub 2019 Nov 20.

Signalling Laboratory, The Babraham Institute, Babraham Research Campus, Cambridge, United Kingdom.

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.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-0505DOI Listing
February 2020

An mTORC1-to-CDK1 Switch Maintains Autophagy Suppression during Mitosis.

Mol Cell 2020 01 13;77(2):228-240.e7. Epub 2019 Nov 13.

Signalling Laboratory, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK. Electronic address:

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.
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http://dx.doi.org/10.1016/j.molcel.2019.10.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6964153PMC
January 2020

Targeting melanoma's MCL1 bias unleashes the apoptotic potential of BRAF and ERK1/2 pathway inhibitors.

Nat Commun 2019 11 14;10(1):5167. Epub 2019 Nov 14.

Signalling Programme, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT, UK.

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-X 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-X inhibitors is stronger in CRC, correlating with a low MCL1:BCL-X ratio; indeed the MCL1:BCL-X ratio is predictive of ERK1/2 pathway inhibitor synergy with MCL1 or BCL2/BCL-w/BCL-X 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.
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http://dx.doi.org/10.1038/s41467-019-12409-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6856071PMC
November 2019

Identification of a novel orally bioavailable ERK5 inhibitor with selectivity over p38α and BRD4.

Eur J Med Chem 2019 Sep 25;178:530-543. Epub 2019 May 25.

Newcastle Drug Discovery, Northern Institute for Cancer Research, School of Chemistry, Bedson Building, Newcastle University, Newcastle Upon Tyne, NE1 7RU, UK. Electronic address:

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 IC values in the micromolar range, but having no selectivity against p38α MAP kinase. Truncation of the N-substituent marginally enhanced potency (∼3-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 (IC 0.82 μM for ERK5; IC > 120 μM 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.
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http://dx.doi.org/10.1016/j.ejmech.2019.05.057DOI Listing
September 2019

MEK1/2 inhibitor withdrawal reverses acquired resistance driven by BRAF amplification whereas KRAS amplification promotes EMT-chemoresistance.

Nat Commun 2019 05 2;10(1):2030. Epub 2019 May 2.

Signalling Programme, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT, UK.

Acquired resistance to MEK1/2 inhibitors (MEKi) arises through amplification of BRAF or KRAS to reinstate ERK1/2 signalling. Here we show that BRAF amplification and MEKi resistance are reversible following drug withdrawal. Cells with BRAF 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 p57-dependent G1 cell cycle arrest and senescence or expression of NOXA and cell death, selecting against those cells with amplified BRAF. p57 expression is required for loss of BRAF amplification and reversal of MEKi resistance. Thus, BRAF amplification confers a selective disadvantage during drug withdrawal, validating intermittent dosing to forestall resistance. In contrast, resistance driven by KRAS 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 KRAS amplification.
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http://dx.doi.org/10.1038/s41467-019-09438-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497655PMC
May 2019

Over-expressed, N-terminally truncated BRAF is detected in the nucleus of cells with nuclear phosphorylated MEK and ERK.

Heliyon 2018 Dec 20;4(12):e01065. Epub 2018 Dec 20.

Leicester Cancer Research Centre, Clinical Sciences Building, University of Leicester, Leicester Royal Infirmary, Leicester LE2 7LX, UK.

BRAF is a cytoplasmic protein kinase, which activates the MEK-ERK signalling pathway. Deregulation of the pathway is associated with the presence of mutations in human cancer, the most common being , 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 (BRAF) were detected at lower levels in the nucleus while full-length BRAF 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-BRAF and GFP-BRAF 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 .
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http://dx.doi.org/10.1016/j.heliyon.2018.e01065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6304467PMC
December 2018

Targeting IKKβ in Cancer: Challenges and Opportunities for the Therapeutic Utilisation of IKKβ Inhibitors.

Cells 2018 Aug 23;7(9). Epub 2018 Aug 23.

Signalling Laboratory, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK.

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 IKKβ protein kinase has been the historical focus of drug development pipelines. Thousands of compounds with activity against IKKβ 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 IKKβ inhibition have thus far prevented the clinical approval of any IKKβ inhibitors. This review will discuss the potential reasons for the lack of clinical success of IKKβ 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 IKKβ inhibition.
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http://dx.doi.org/10.3390/cells7090115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162708PMC
August 2018

ERK1/2 inhibitors: New weapons to inhibit the RAS-regulated RAF-MEK1/2-ERK1/2 pathway.

Pharmacol Ther 2018 07 16;187:45-60. Epub 2018 Feb 16.

Signalling Programme, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, England, United Kingdom. Electronic address:

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.
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http://dx.doi.org/10.1016/j.pharmthera.2018.02.007DOI Listing
July 2018

De-RSKing ERK - regulation of ERK1/2-RSK dissociation by phosphorylation within a disordered motif.

FEBS J 2018 01;285(1):42-45

Signalling Programme, The Babraham Institute, Cambridge, UK.

The protein kinases ERK1/2 and RSK associate in unstimulated cells but must separate to target other substrates. In this issue, Gógl et al. show that phosphorylation of RSK by active ERK1/2 culminates in the formation of an intramolecular charge clamp between Lys729 and the phosphate group on Ser732. This promotes the dissociation of ERK1/2 from RSK allowing them to engage with other targets.
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http://dx.doi.org/10.1111/febs.14344DOI Listing
January 2018

Calcium phosphate particles stimulate interleukin-1β release from human vascular smooth muscle cells: A role for spleen tyrosine kinase and exosome release.

J Mol Cell Cardiol 2018 02 20;115:82-93. Epub 2017 Dec 20.

Signalling Programme, Babraham Institute, Babraham, Cambridge CB22 3AT, UK. Electronic address:

Aims: Calcium phosphate (CaP) particle deposits are found in several inflammatory diseases including atherosclerosis and osteoarthritis. CaP, and other forms of crystals and particles, can promote inflammasome formation in macrophages leading to caspase-1 activation and secretion of mature interleukin-1β (IL-1β). Given the close association of small CaP particles with vascular smooth muscle cells (VSMCs) in atherosclerotic fibrous caps, we aimed to determine if CaP particles affected pro-inflammatory signalling in human VSMCs.

Methods And Results: Using ELISA to measure IL-1β release from VSMCs, we demonstrated that CaP particles stimulated IL-1β release from proliferating and senescent human VSMCs, but with substantially greater IL-1β release from senescent cells; this required caspase-1 activity but not LPS-priming of cells. Potential inflammasome agonists including ATP, nigericin and monosodium urate crystals did not stimulate IL-1β release from VSMCs. Western blot analysis demonstrated that CaP particles induced rapid activation of spleen tyrosine kinase (SYK) (increased phospho-Y525/526). The SYK inhibitor R406 reduced IL-1β release and caspase-1 activation in CaP particle-treated VSMCs, indicating that SYK activation occurs upstream of and is required for caspase-1 activation. In addition, IL-1β and caspase-1 colocalised in intracellular endosome-like vesicles and we detected IL-1β in exosomes isolated from VSMC media. Furthermore, CaP particle treatment stimulated exosome secretion by VSMCs in a SYK-dependent manner, while the exosome-release inhibitor spiroepoxide reduced IL-1β release.

Conclusions: CaP particles stimulate SYK and caspase-1 activation in VSMCs, leading to the release of IL-1β, at least in part via exosomes. These novel findings in human VSMCs highlight the pro-inflammatory and pro-calcific potential of microcalcification.
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http://dx.doi.org/10.1016/j.yjmcc.2017.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823844PMC
February 2018

Use of multi-criteria decision analysis to identify potentially dangerous glacial lakes.

Sci Total Environ 2018 Apr 19;621:1453-1466. Epub 2017 Oct 19.

Université Paris-Dauphine, LAMSADE-CNRS, 75775 Paris Cedex 16, France; Université de Nice, ESPACE-CNRS, F-06204 Nice Cedex 03, France.

Glacial Lake Outburst Floods (GLOFs) represent a significant threat in deglaciating environments, necessitating the development of GLOF hazard and risk assessment procedures. Here, we outline a Multi-Criteria Decision Analysis (MCDA) approach that can be used to rapidly identify potentially dangerous lakes in regions without existing tailored GLOF risk assessments, where a range of glacial lake types exist, and where field data are sparse or non-existent. Our MCDA model (1) is desk-based and uses freely and widely available data inputs and software, and (2) allows the relative risk posed by a range of glacial lake types to be assessed simultaneously within any region. A review of the factors that influence GLOF risk, combined with the strict rules of criteria selection inherent to MCDA, has allowed us to identify 13 exhaustive, non-redundant, and consistent risk criteria. We use our MCDA model to assess the risk of 16 extant glacial lakes and 6 lakes that have already generated GLOFs, and found that our results agree well with previous studies. For the first time in GLOF risk assessment, we employed sensitivity analyses to test the strength of our model results and assumptions, and to identify lakes that are sensitive to the criteria and risk thresholds used. A key benefit of the MCDA method is that sensitivity analyses are readily undertaken. Overall, these sensitivity analyses lend support to our model, although we suggest that further work is required to determine the relative importance of assessment criteria, and the thresholds that determine the level of risk for each criterion. As a case study, the tested method was then applied to 25 potentially dangerous lakes in the Bolivian Andes, where GLOF risk is poorly understood; 3 lakes are found to pose 'medium' or 'high' risk, and require further detailed investigation.
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http://dx.doi.org/10.1016/j.scitotenv.2017.10.083DOI Listing
April 2018

ERK1/2 signalling protects against apoptosis following endoplasmic reticulum stress but cannot provide long-term protection against BAX/BAK-independent cell death.

PLoS One 2017 20;12(9):e0184907. Epub 2017 Sep 20.

Signalling Laboratory, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT, United Kingdom.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0184907PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5607168PMC
October 2017

Control of cell death and mitochondrial fission by ERK1/2 MAP kinase signalling.

FEBS J 2017 12 18;284(24):4177-4195. Epub 2017 Jun 18.

Signalling Programme, The Babraham Institute, Cambridge, UK.

The ERK1/2 signalling pathway is best known for its role in connecting activated growth factor receptors to changes in gene expression due to activated ERK1/2 entering the nucleus and phosphorylating transcription factors. However, active ERK1/2 also translocate to a variety of other organelles including the endoplasmic reticulum, endosomes, golgi and mitochondria to access specific substrates and influence cell physiology. In this article, we review two aspects of ERK1/2 signalling at the mitochondria that are involved in regulating cell fate decisions. First, we describe the prominent role of ERK1/2 in controlling the BCL2-regulated, cell-intrinsic apoptotic pathway. In most cases ERK1/2 signalling promotes cell survival by activating prosurvival BCL2 proteins (BCL2, BCL-x and MCL1) and repressing prodeath proteins (BAD, BIM, BMF and PUMA). This prosurvival signalling is co-opted by oncogenes to confer cancer cell-specific survival advantages and we describe how this information has been used to develop new drug combinations. However, ERK1/2 can also drive the expression of the prodeath protein NOXA to control 'autophagy or apoptosis' decisions during nutrient starvation. We also describe recent studies demonstrating a link between ERK1/2 signalling, DRP1 and the mitochondrial fission machinery and how this may influence metabolic reprogramming during tumorigenesis and stem cell reprogramming. With advances in subcellular proteomics it is likely that new roles for ERK1/2, and new substrates, remain to be discovered at the mitochondria and other organelles.
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http://dx.doi.org/10.1111/febs.14122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6193418PMC
December 2017

Visualization of Endogenous ERK1/2 in Cells with a Bioorthogonal Covalent Probe.

Bioconjug Chem 2017 06 12;28(6):1677-1683. Epub 2017 May 12.

Astex Pharmaceuticals , 436 Cambridge Science Park, Cambridge CB4 0QA, U.K.

The RAS-RAF-MEK-ERK pathway has been intensively studied in oncology, with RAS known to be mutated in ∼30% of all human cancers. The recent emergence of ERK1/2 inhibitors and their ongoing clinical investigation demands a better understanding of ERK1/2 behavior following small-molecule inhibition. Although fluorescent fusion proteins and fluorescent antibodies are well-established methods of visualizing proteins, we show that ERK1/2 can be visualized via a less-invasive approach based on a two-step process using inverse electron demand Diels-Alder cycloaddition. Our previously reported trans-cyclooctene-tagged covalent ERK1/2 inhibitor was used in a series of imaging experiments following a click reaction with a tetrazine-tagged fluorescent dye. Although limitations were encountered with this approach, endogenous ERK1/2 was successfully imaged in cells, and "on-target" staining was confirmed by over-expressing DUSP5, a nuclear ERK1/2 phosphatase that anchors ERK1/2 in the nucleus.
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http://dx.doi.org/10.1021/acs.bioconjchem.7b00152DOI Listing
June 2017

RNA-binding proteins ZFP36L1 and ZFP36L2 promote cell quiescence.

Science 2016 Apr;352(6284):453-9

Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Cambridge CB22 3AT, UK.

Progression through the stages of lymphocyte development requires coordination of the cell cycle. Such coordination ensures genomic integrity while cells somatically rearrange their antigen receptor genes [in a process called variable-diversity-joining (VDJ) recombination] and, upon successful rearrangement, expands the pools of progenitor lymphocytes. Here we show that in developing B lymphocytes, the RNA-binding proteins (RBPs) ZFP36L1 and ZFP36L2 are critical for maintaining quiescence before precursor B cell receptor (pre-BCR) expression and for reestablishing quiescence after pre-BCR-induced expansion. These RBPs suppress an evolutionarily conserved posttranscriptional regulon consisting of messenger RNAs whose protein products cooperatively promote transition into the S phase of the cell cycle. This mechanism promotes VDJ recombination and effective selection of cells expressing immunoglobulin-μ at the pre-BCR checkpoint.
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http://dx.doi.org/10.1126/science.aad5978DOI Listing
April 2016

Tumor cells with KRAS or BRAF mutations or ERK5/MAPK7 amplification are not addicted to ERK5 activity for cell proliferation.

Cell Cycle 2016 ;15(4):506-18

a Signalling Laboratory; The Babraham Institute ; Cambridge , UK.

ERK5, encoded by MAPK7, has been proposed to play a role in cell proliferation, thus attracting interest as a cancer therapeutic target. While oncogenic RAS or BRAF cause sustained activation of the MEK1/2-ERK1/2 pathway, ERK5 is directly activated by MEK5. It has been proposed that RAS and RAF proteins can also promote ERK5 activation. Here we investigated the interplay between RAS-RAF-MEK-ERK and ERK5 signaling and studied the role of ERK5 in tumor cell proliferation in 2 disease-relevant cell models. We demonstrate that although an inducible form of CRAF (CRAF:ER*) can activate ERK5 in fibroblasts, the response is delayed and reflects feed-forward signaling. Additionally, oncogenic KRAS and BRAF do not activate ERK5 in epithelial cells. Although KRAS and BRAF do not couple directly to MEK5-ERK5, ERK5 signaling might still be permissive for proliferation. However, neither the selective MEK5 inhibitor BIX02189 or ERK5 siRNA inhibited proliferation of colorectal cancer cells harbouring KRAS(G12C/G13D) or BRAF(V600E). Furthermore, there was no additive or synergistic effect observed when BIX02189 was combined with the MEK1/2 inhibitor Selumetinib (AZD6244), suggesting that ERK5 was neither required for proliferation nor a driver of innate resistance to MEK1/2 inhibitors. Finally, even cancer cells with MAPK7 amplification were resistant to BIX02189 and ERK5 siRNA, showing that ERK5 amplification does not confer addiction to ERK5 for cell proliferation. Thus ERK5 signaling is unlikely to play a role in tumor cell proliferation downstream of KRAS or BRAF or in tumor cells with ERK5 amplification. These results have important implications for the role of ERK5 as an anti-cancer drug target.
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http://dx.doi.org/10.1080/15384101.2015.1120915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5056618PMC
December 2016

Maternal DNA Methylation Regulates Early Trophoblast Development.

Dev Cell 2016 Jan;36(2):152-63

Epigenetics Programme, Babraham Institute, Cambridge CB22 3AT, UK; Centre for Trophoblast Research, University of Cambridge, Cambridge CB2 3EG, UK; The Wellcome Trust Sanger Institute, Cambridge CB10 1SA, UK.

Critical roles for DNA methylation in embryonic development are well established, but less is known about its roles during trophoblast development, the extraembryonic lineage that gives rise to the placenta. We dissected the role of DNA methylation in trophoblast development by performing mRNA and DNA methylation profiling of Dnmt3a/3b mutants. We find that oocyte-derived methylation plays a major role in regulating trophoblast development but that imprinting of the key placental regulator Ascl2 is only partially responsible for these effects. We have identified several methylation-regulated genes associated with trophoblast differentiation that are involved in cell adhesion and migration, potentially affecting trophoblast invasion. Specifically, trophoblast-specific DNA methylation is linked to the silencing of Scml2, a Polycomb Repressive Complex 1 protein that drives loss of cell adhesion in methylation-deficient trophoblast. Our results reveal that maternal DNA methylation controls multiple differentiation-related and physiological processes in trophoblast via both imprinting-dependent and -independent mechanisms.
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http://dx.doi.org/10.1016/j.devcel.2015.12.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729543PMC
January 2016

MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road.

Nat Rev Cancer 2015 Oct;15(10):577-92

Signalling Laboratory, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK.

The role of the ERK signalling pathway in cancer is thought to be most prominent in tumours in which mutations in the receptor tyrosine kinases RAS, BRAF, CRAF, MEK1 or MEK2 drive growth factor-independent ERK1 and ERK2 activation and thence inappropriate cell proliferation and survival. New drugs that inhibit RAF or MEK1 and MEK2 have recently been approved or are currently undergoing late-stage clinical evaluation. In this Review, we consider the ERK pathway, focusing particularly on the role of MEK1 and MEK2, the 'gatekeepers' of ERK1/2 activity. We discuss their validation as drug targets, the merits of targeting MEK1 and MEK2 versus BRAF and the mechanisms of action of different inhibitors of MEK1 and MEK2. We also consider how some of the systems-level properties (intrapathway regulatory loops and wider signalling network connections) of the ERK pathway present a challenge for the success of MEK1 and MEK2 inhibitors, discuss mechanisms of resistance to these inhibitors, and review their clinical progress.
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http://dx.doi.org/10.1038/nrc4000DOI Listing
October 2015

Identification of DYRK1B as a substrate of ERK1/2 and characterisation of the kinase activity of DYRK1B mutants from cancer and metabolic syndrome.

Cell Mol Life Sci 2016 Feb 7;73(4):883-900. Epub 2015 Sep 7.

Signalling Laboratory, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT, UK.

The dual-specificity tyrosine-phosphorylation-regulated kinase, DYRK1B, is expressed de novo during myogenesis, amplified or mutated in certain cancers and mutated in familial cases of metabolic syndrome. DYRK1B is activated by cis auto-phosphorylation on tyrosine-273 (Y273) within the activation loop during translation but few other DYRK1B phosphorylation sites have been characterised to date. Here, we demonstrate that DYRK1B also undergoes trans-autophosphorylation on serine-421 (S421) in vitro and in cells and that this site contributes to DYRK1B kinase activity. Whilst a DYRK1B(S421A) mutant was completely defective for p-S421 in cells, DYRK1B inhibitors caused only a partial loss of p-S421 suggesting the existence of an additional kinase that could also phosphorylate DYRK1B S421. Indeed, a catalytically inactive DYRK1B(D239A) mutant exhibited very low levels of p-S421 in cells but this was increased by KRAS(G12V). In addition, selective activation of the RAF-MEK1/2-ERK1/2 signalling pathway rapidly increased p-S421 in cells whereas activation of the stress kinases JNK or p38 could not. S421 resides within a Ser-Pro phosphoacceptor motif that is typical for ERK1/2 and recombinant ERK2 phosphorylated DYRK1B at S421 in vitro. Our results show that DYRK1B is a novel ERK2 substrate, uncovering new links between two kinases involved in cell fate decisions. Finally, we show that DYRK1B mutants that have recently been described in cancer and metabolic syndrome exhibit normal or reduced intrinsic kinase activity.
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http://dx.doi.org/10.1007/s00018-015-2032-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4735261PMC
February 2016

Stimulating translational research: several European life science institutions put their heads together.

Trends Mol Med 2015 Sep 5;21(9):525-7. Epub 2015 Aug 5.

EU-Life Translational Working Group; Centre for Genomic Regulation (CRG), Dr Aiguader 88, 08003 Barcelona, Spain; Universitat Pompeu Fabra (UPF), Barcelona, Spain.

Translational research leaves no-one indifferent and everyone expects a particular benefit. We as EU-LIFE (www.eu-life.eu), an alliance of 13 research institutes in European life sciences, would like to share our experience in an attempt to identify measures to promote translational research without undermining basic exploratory research and academic freedom.
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http://dx.doi.org/10.1016/j.molmed.2015.07.002DOI Listing
September 2015

DYRK1A-mediated Cyclin D1 Degradation in Neural Stem Cells Contributes to the Neurogenic Cortical Defects in Down Syndrome.

EBioMedicine 2015 17;2(2):120-34. Epub 2015 Jan 17.

Department of Developmental Biology, Instituto de Biología Molecular de Barcelona, CSIC, and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 08028 Barcelona, Spain.

Alterations in cerebral cortex connectivity lead to intellectual disability and in Down syndrome, this is associated with a deficit in cortical neurons that arises during prenatal development. However, the pathogenic mechanisms that cause this deficit have not yet been defined. Here we show that the human DYRK1A kinase on chromosome 21 tightly regulates the nuclear levels of Cyclin D1 in embryonic cortical stem (radial glia) cells, and that a modest increase in DYRK1A protein in transgenic embryos lengthens the G1 phase in these progenitors. These alterations promote asymmetric proliferative divisions at the expense of neurogenic divisions, producing a deficit in cortical projection neurons that persists in postnatal stages. Moreover, radial glial progenitors in the Ts65Dn mouse model of Down syndrome have less Cyclin D1, and Dyrk1a is the triplicated gene that causes both early cortical neurogenic defects and decreased nuclear Cyclin D1 levels in this model. These data provide insights into the mechanisms that couple cell cycle regulation and neuron production in cortical neural stem cells, emphasizing that the deleterious effect of DYRK1A triplication in the formation of the cerebral cortex begins at the onset of neurogenesis, which is relevant to the search for early therapeutic interventions in Down syndrome.
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http://dx.doi.org/10.1016/j.ebiom.2015.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4484814PMC
December 2016

Epigenetic memory of the first cell fate decision prevents complete ES cell reprogramming into trophoblast.

Nat Commun 2014 Nov 26;5:5538. Epub 2014 Nov 26.

1] Epigenetics Programme, The Babraham Institute, Cambridge CB22 3AT, UK [2] Centre for Trophoblast Research, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK [3] Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Tennis Court Road, Cambridge CB2 1QR, UK.

Embryonic (ES) and trophoblast (TS) stem cells reflect the first, irrevocable cell fate decision in development that is reinforced by distinct epigenetic lineage barriers. Nonetheless, ES cells can seemingly acquire TS-like characteristics upon manipulation of lineage-determining transcription factors or activation of the extracellular signal-regulated kinase 1/2 (Erk1/2) pathway. Here we have interrogated the progression of reprogramming in ES cell models with regulatable Oct4 and Cdx2 transgenes or conditional Erk1/2 activation. Although trans-differentiation into TS-like cells is initiated, lineage conversion remains incomplete in all models, underpinned by the failure to demethylate a small group of TS cell genes. Forced expression of these non-reprogrammed genes improves trans-differentiation efficiency, but still fails to confer a stable TS cell phenotype. Thus, even ES cells in ground-state pluripotency cannot fully overcome the boundaries that separate the first cell lineages but retain an epigenetic memory of their ES cell origin.
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http://dx.doi.org/10.1038/ncomms6538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263130PMC
November 2014

Intrinsic and acquired resistance to MEK1/2 inhibitors in cancer.

Biochem Soc Trans 2014 Aug;42(4):776-83

*Signalling Programme, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, U.K.

Recent clinical data with BRAF and MEK1/2 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase 1/2] inhibitors have demonstrated the remarkable potential of targeting the RAF-MEK1/2-ERK1/2 signalling cascade for the treatment of certain cancers. Despite these advances, however, only a subset of patients respond to these agents in the first instance, and, of those that do, acquired resistance invariably develops after several months. Studies in vitro have identified various mechanisms that can underpin intrinsic and acquired resistance to MEK1/2 inhibitors, and these frequently recapitulate those observed clinically. In the present article, we review these mechanisms and also discuss recent advances in our understanding of how MEK1/2 inhibitor activity is influenced by pathway feedback.
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http://dx.doi.org/10.1042/BST20140129DOI Listing
August 2014

The increase in BIK expression following ERK1/2 pathway inhibition is a consequence of G₁ cell-cycle arrest and not a direct effect on BIK protein stability.

Biochem J 2014 May;459(3):513-24

*Signalling Programme, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, U.K.

BIK (BCL2-interacting killer) is a pro-apoptotic BH3 (BCL2 homology domain 3)-only protein and a member of the BCL2 protein family. It was proposed recently that BIK abundance is controlled by ERK1/2 (extracellular-signal-regulated kinase 1/2)-catalysed phosphorylation, which targets the protein for proteasome-dependent destruction. In the present study, we examined ERK1/2-dependent regulation of BIK, drawing comparisons with BIM(EL) (BCL2-interacting mediator of cell death; extra long), a well-known target of ERK1/2. In many ERK1/2-dependent tumour cell lines, inhibition of BRAF(V600E) (v-raf murine sarcoma viral oncogene homologue B1, V600E mutation) or MEK1/2 (mitogen-activated protein kinase/ERK kinase 1/2) had very little effect on BIK expression, whereas BIM(EL) was strongly up-regulated. In some cell lines we observed a modest increase in BIK expression; however, this was not apparent until ~16 h or later, whereas BIM(EL) expression increased rapidly within a few hours. Although BIK was degraded by the proteasome, we found no evidence that this was regulated by ERK1/2 signalling. Rather, the delayed increase in BIK expression was prevented by actinomycin D, and was accompanied by increases in BIK mRNA. Finally, the delayed increase in BIK expression following ERK1/2 inhibition was phenocopied by a highly selective CDK4/6 (cyclin-dependent kinases 4 and 6) inhibitor, which caused a strong G₁ cell-cycle arrest without inhibiting ERK1/2 signalling. In contrast, BIM(EL) expression was induced by ERK1/2 inhibition, but not by CDK4/6 inhibition. We conclude that BIK expression is not subject to direct regulation by the ERK1/2 pathway; rather, we propose that BIK expression is cell-cycle-dependent and increases as a consequence of the G₁ cell-cycle arrest which results from inhibition of ERK1/2 signalling.
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http://dx.doi.org/10.1042/BJ20131346DOI Listing
May 2014