Publications by authors named "David Oxley"

72 Publications

Gβγ is a direct regulator of endogenous p101/p110γ and p84/p110γ PI3Kγ complexes in mouse neutrophils.

Sci Signal 2020 11 3;13(656). Epub 2020 Nov 3.

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

The PI3Kγ isoform is activated by Gi-coupled GPCRs in myeloid cells, but the extent to which the two endogenous complexes of PI3Kγ, p101/p110γ and p84/p110γ, receive direct regulation through Gβγ or indirect regulation through RAS and the sufficiency of those inputs is controversial or unclear. We generated mice with point mutations that prevent Gβγ binding to p110γ (RK552DD) or to p101 (VVKR777AAAA) and investigated the effects of these mutations in primary neutrophils and in mouse models of neutrophilic inflammation. Loss of Gβγ binding to p110γ substantially reduced the activation of both p101/p110γ and p84/p110γ in neutrophils by various GPCR agonists. Loss of Gβγ binding to p101 caused more variable effects, depending on both the agonist and cellular response, with the biggest reductions seen in PIP production by primary neutrophils in response to LTB4 and MIP-2 and in the migration of neutrophils during thioglycolate-induced peritonitis or MIP2-induced ear pouch inflammation. We also observed that p101 neutrophils showed enhanced p84-dependent ROS responses to MLP and C5a, suggesting that competition may exist between p101/p110γ and p84/p110γ for Gβγ subunits downstream of GPCR activation. GPCRs did not activate p110γ in neutrophils from mice lacking both the p101 and p84 regulatory subunits, indicating that RAS binding to p110γ is insufficient to support GPCR activation in this cell type. These findings define a direct role for Gβγ subunits in activating both of the endogenous PI3Kγ complexes and indicate that the regulatory PI3Kγ subunit biases activation toward different GPCRs.
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http://dx.doi.org/10.1126/scisignal.aaz4003DOI Listing
November 2020

Commissioning and Quality Assurance of a novel solution for respiratory-gated PBS proton therapy based on optical tracking of surface markers.

Z Med Phys 2020 Aug 20. Epub 2020 Aug 20.

Center for Proton Therapy, Paul Scherrer Institute, 5232 Villigen, Switzerland.

We present the commissioning and quality assurance of our clinical protocol for respiratory gating in pencil beam scanning proton therapy for cancer patients with moving targets. In a novel approach, optical tracking has been integrated in the therapy workflow and used to monitor respiratory motion from multiple surrogates, applied on the patients' chest. The gating system was tested under a variety of experimental conditions, specific to proton therapy, to evaluate reaction time and reproducibility of dose delivery control. The system proved to be precise in the application of beam gating and allowed the mitigation of dose distortions even for large (1.4cm) motion amplitudes, provided that adequate treatment windows were selected. The total delivered dose was not affected by the use of gating, with measured integral error within 0.15cGy. Analysing high-resolution images of proton transmission, we observed negligible discrepancies in the geometric location of the dose as a function of the treatment window, with gamma pass rate greater than 95% (2%/2mm) compared to stationary conditions. Similarly, pass rate for the latter metric at the 3%/3mm level was observed above 97% for clinical treatment fields, limiting residual movement to 3mm at end-exhale. These results were confirmed in realistic clinical conditions using an anthropomorphic breathing phantom, reporting a similarly high 3%/3mm pass rate, above 98% and 94%, for regular and irregular breathing, respectively. Finally, early results from periodic QA tests of the optical tracker have shown a reliable system, with small variance observed in static and dynamic measurements.
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http://dx.doi.org/10.1016/j.zemedi.2020.07.001DOI Listing
August 2020

Transition to naïve human pluripotency mirrors pan-cancer DNA hypermethylation.

Nat Commun 2020 07 22;11(1):3671. Epub 2020 Jul 22.

Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, EC1M 6BQ, London, UK.

Epigenetic reprogramming is a cancer hallmark, but how it unfolds during early neoplastic events and its role in carcinogenesis and cancer progression is not fully understood. Here we show that resetting from primed to naïve human pluripotency results in acquisition of a DNA methylation landscape mirroring the cancer DNA methylome, with gradual hypermethylation of bivalent developmental genes. We identify a dichotomy between bivalent genes that do and do not become hypermethylated, which is also mirrored in cancer. We find that loss of H3K4me3 at bivalent regions is associated with gain of methylation. Additionally, we observe that promoter CpG island hypermethylation is not restricted solely to emerging naïve cells, suggesting that it is a feature of a heterogeneous intermediate population during resetting. These results indicate that transition to naïve pluripotency and oncogenic transformation share common epigenetic trajectories, which implicates reprogramming and the pluripotency network as a central hub in cancer formation.
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http://dx.doi.org/10.1038/s41467-020-17269-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376100PMC
July 2020

Cell-Surface Proteomics Identifies Differences in Signaling and Adhesion Protein Expression between Naive and Primed Human Pluripotent Stem Cells.

Stem Cell Reports 2020 05 16;14(5):972-988. Epub 2020 Apr 16.

Epigenetics Programme, The Babraham Institute, Cambridge, UK; Wellcome-Medical Research Council Cambridge Stem Cell Institute, Cambridge, UK. Electronic address:

Naive and primed human pluripotent stem cells (hPSC) provide valuable models to study cellular and molecular developmental processes. The lack of detailed information about cell-surface protein expression in these two pluripotent cell types prevents an understanding of how the cells communicate and interact with their microenvironments. Here, we used plasma membrane profiling to directly measure cell-surface protein expression in naive and primed hPSC. This unbiased approach quantified over 1,700 plasma membrane proteins, including those involved in cell adhesion, signaling, and cell interactions. Notably, multiple cytokine receptors upstream of JAK-STAT signaling were more abundant in naive hPSC. In addition, functional experiments showed that FOLR1 and SUSD2 proteins are highly expressed at the cell surface in naive hPSC but are not required to establish human naive pluripotency. This study provides a comprehensive stem cell proteomic resource that uncovers differences in signaling pathway activity and has identified new markers to define human pluripotent states.
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http://dx.doi.org/10.1016/j.stemcr.2020.03.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7220956PMC
May 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

Pitfalls in the beam modelling process of Monte Carlo calculations for proton pencil beam scanning.

Br J Radiol 2020 Mar 6;93(1107):20190919. Epub 2020 Feb 6.

Centre for Proton Therapy, Paul Scherrer Institute, Villigen, Switzerland.

Objective: Monte Carlo (MC) simulations substantially improve the accuracy of predicted doses. This study aims to determine and quantify the uncertainties of setting up such a MC system.

Methods: Doses simulated with two Geant4-based MC calculation codes, but tuned to the beam data, have been compared. Different methods of MC modelling of a pre-absorber have been employed, either modifying the beam source parameters (descriptive) or adding the pre-absorber as a physical component (physical).

Results: After the independent beam modelling of both systems in water (resulting in excellent range agreement) range differences of up to 3.6/4.8 mm (1.5% of total range) in bone/brain-like tissues were found, which resulted from the use of different mean water ionisation potentials during the energy tuning process. When repeating using a common definition of water, ranges in bone/brain agreed within 0.1 mm and gamma-analysis (global 1%,1mm) showed excellent agreement (>93%) for all patient fields. However, due to a lack of modelling of proton fluence loss in the descriptive pre-absorber, differences of 7% in absolute dose between the pre-absorber definitions were found.

Conclusion: This study quantifies the influence of using different water ionisation potentials during the MC beam modelling process. Furthermore, when using a descriptive pre-absorber model, additional Faraday cup or ionisation chamber measurements with pre-absorber are necessary.

Advances In Knowledge: This is the first study quantifying the uncertainties caused by the MC beam modelling process for proton pencil beam scanning, and a more detailed beam modelling process for MC simulations is proposed to minimise the influence of critical parameters.
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http://dx.doi.org/10.1259/bjr.20190919DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066947PMC
March 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

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

Correction to: Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data.

Genome Biol 2019 02 22;20(1):43. Epub 2019 Feb 22.

Epigenetics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK.

Following publication of the original article [1], it was reported that the incorrect "Additional file 3" was published. The correct additional file is given below.
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http://dx.doi.org/10.1186/s13059-019-1656-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6385436PMC
February 2019

Evaluation of the ray-casting analytical algorithm for pencil beam scanning proton therapy.

Phys Med Biol 2019 03 18;64(6):065021. Epub 2019 Mar 18.

Centre for Proton Therapy, Paul Scherrer Institute, Villigen, Switzerland. Department of Physics, ETH Zurich, Zurich, Switzerland.

For pencil beam scanned (PBS) proton therapy, analytical dose calculation engines are still typically used for the optimisation process, and often for the final evaluation of the plan. Recently however, the suitability of analytical calculations for planning PBS treatments has been questioned. Conceptually, the two main approaches for these analytical dose calculations are the ray-casting (RC) and the pencil-beam (PB) method. In this study, we compare dose distributions and dosimetric indices, calculated on both the clinical dose calculation grid and as a function of dose grid resolution, to Monte Carlo (MC) calculations. The analysis is done using a comprehensive set of clinical plans which represent a wide choice of treatment sites. When analysing dose difference histograms for relative treatment plans, pencil beam calculations with double grid resolution perform best, with on average 97.7%/91.9% (RC), 97.9%/92.7% (RC, double grid resolution), 97.6%/91.0% (PB) and 98.6%/94.0% (PB, double grid resolution) of voxels agreeing within  ±5%/±  3% between the analytical and the MC calculations. Even though these point-to-point dose comparison shows differences between analytical and MC calculations, for all algorithms, clinically relevant dosimetric indices agree within  ±4% for the PTV and within  ±5% for critical organs. While the clinical agreement depends on the treatment site, there is no substantial difference of indices between the different algorithms. The pencil-beam approach however comes at a higher computational cost than the ray-casting calculation. In conclusion, we would recommend using the ray-casting algorithm for fast dose optimization and subsequently combine it with one MC calculation to scale the absolute dose and assure the quality of the treatment plan.
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http://dx.doi.org/10.1088/1361-6560/aafe58DOI Listing
March 2019

Contour scanning, multi-leaf collimation and the combination thereof for proton pencil beam scanning.

Phys Med Biol 2018 12 20;64(1):015002. Epub 2018 Dec 20.

Centre for Proton Therapy, Paul Scherrer Institute, Villigen, Switzerland. Physics Department, ETH Zürich, Zürich, Switzerland.

In proton therapy, the lateral fall-off is often used to spare critical organs. It is therefore crucial to improve the penumbra for proton pencil beam scanning. However, previous work has shown that collimation may not be necessary for depths of  >15 cm in water. As such, in this work we investigate the effectiveness of a thin multi leaf collimator (just thick enough to completely stop protons with ranges of  <15 cm in water) for energy layer specific collimation in patient geometries, when applied in combination with both grid and contour scanned PBS proton therapy. For this, an analytical model of collimated beam shapes, based solely on data available in the treatment planning system, has been included in the optimization, with the resulting optimised plans then being recalculated using Monte Carlo in order to most accurately simulate the full physics effects of the collimator. For grid based scanning, energy specific collimation has been found to reduce the V30 outside the PTV by 19.8% for an example patient when compared to the same pencil beam placement without collimation. V30 could be even reduced by a further 5.6% when combining collimation and contour scanning. In addition, mixed plans, consisting of contour scanning for deep fields (max range  >15 cm WER) and collimated contour scanning for superficial fields (<15 cm), have been created for four patients, by which V30 could be reduced by 0.8% to 8.0% and the mean dose to the brain stem by 1.5% to 3.3%. Target dose homogeneity however is not substantially different when compared to the best un-collimated scenario. In conclusion, we demonstrate the potential advantages of a thin, multi leaf collimator in combination with contour scanning for energy layer specific collimation in PBS proton therapy.
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http://dx.doi.org/10.1088/1361-6560/aaf2e8DOI Listing
December 2018

Log file based Monte Carlo calculations for proton pencil beam scanning therapy.

Phys Med Biol 2019 01 29;64(3):035014. Epub 2019 Jan 29.

Centre for Proton Therapy, Paul Scherrer Institute, Villigen, Switzerland. Department of Physics, ETH Zurich, Zurich, Switzerland.

Patient specific quality assurance is crucial to guarantee safety in proton pencil beam scanning. In current clinical practice, this requires extensive, time consuming measurements. Additionally, these measurements do not consider the influence of density heterogeneities in the patient and are insensitive to delivery errors. In this work, we investigate the use of log file based Monte Carlo calculations for dose reconstructions in the patient CT, which takes the combined influence of calculational and delivery errors into account. For one example field, 87%/90% of the voxels agree within  ±3% when taking either calculational or delivery uncertainties into account (analytical versus Monte Carlo calculation/Monte Carlo from planned versus Monte Carlo from log file). 78% agree when considering both uncertainties simultaneously (nominal field versus Monte Carlo from log files). We then show the application of the log file based Monte Carlo calculations as a patient specific quality assurance tool for a set of five patients (16 fields) treated for different indications. For all fields, absolute dose scaling factors based on the log file Monte Carlo agree within  ±3% to the measurement based absolute dose scaling. Relative comparison shows that more than 90% of the voxels agree within  ±  5% between the analytical calculated plan and the Monte Carlo based on log files. The log file based Monte Carlo approach is an end-to-end test incorporating all requirements of patient specific quality assurance. It has the potential to reduce the workload and therefore to increase the patient throughput, while simultaneously enabling more accurate dose verification directly in the patient geometry.
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http://dx.doi.org/10.1088/1361-6560/aaf82dDOI Listing
January 2019

Comment on 'Collimated proton pencil-beam scanning for superficial targets: impact of the order of range shifter and aperture'.

Phys Med Biol 2018 10 17;63(20):208001. Epub 2018 Oct 17.

Centre for Proton Therapy, Paul Scherrer Institute, Villigen, Switzerland. Department of Physics, ETH Zürich, Zurich, Switzerland.

We read with interest the study by Bäumer et al (2018 Phys. Med. Biol. 63 085020), in particular that their conclusions are in contrast to those of our earlier paper (Winterhalter et al 2018 Phys. Med. Biol. 63 025022), namely that positioning the collimating aperture downstream of the range shifter leads to a superior penumbra. In contrast, we found sharper penumbras for the PSI scanning Gantry when the aperture is positioned upstream of the range shifter. We have run additional Monte Carlo simulations with components derived from the paper of Bäumer et al (2018 Phys. Med. Biol. 63 085020), but without modifying the beam description. As such, we obtain a relative penumbra reduction of 13% if the aperture is positioned downstream of the ranges shifter, which lies well within the measured/calculated penumbra reductions of Bäumer et al (2018 Phys. Med. Biol. 63 085020) of 17%/11%. The conclusions of Bäumer et al (2018 Phys. Med. Biol. 63 085020) and our previous work are therefore complementary, given the differences in the thicknesses of the beam modifying devices used in the two works. In addition, our analysis implies that initial beam characteristics are less important in determining the best order of components.
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http://dx.doi.org/10.1088/1361-6560/aae0e1DOI Listing
October 2018

Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data.

Genome Biol 2018 03 15;19(1):33. Epub 2018 Mar 15.

Epigenetics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK.

Background: Whole-genome bisulfite sequencing (WGBS) is becoming an increasingly accessible technique, used widely for both fundamental and disease-oriented research. Library preparation methods benefit from a variety of available kits, polymerases and bisulfite conversion protocols. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing.

Results: We perform a comparative analysis of several commonly used pre- and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se is the main trigger of pronounced sequencing biases, and PCR amplification builds on these underlying artefacts. The majority of standard library preparation methods yield a significantly biased sequence output and overestimate global methylation. Importantly, both absolute and relative methylation levels at specific genomic regions vary substantially between methods, with clear implications for DNA methylation studies.

Conclusions: We show that amplification-free library preparation is the least biased approach for WGBS. In protocols with amplification, the choice of bisulfite conversion protocol or polymerase can significantly minimize artefacts. To aid with the quality assessment of existing WGBS datasets, we have integrated a bias diagnostic tool in the Bismark package and offer several approaches for consideration during the preparation and analysis of WGBS datasets.
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http://dx.doi.org/10.1186/s13059-018-1408-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856372PMC
March 2018

Contractile responses to endothelin-1 are regulated by PKC phosphorylation of cardiac myosin binding protein-C in rat ventricular myocytes.

J Mol Cell Cardiol 2018 04 19;117:1-18. Epub 2018 Feb 19.

Babraham Institute, Babraham Research Campus, Babraham, Cambridge CB223AT, UK; Laboratory of Experimental Cardiology, Department of Cardiovascular Sciences, KU Leuven, Belgium. Electronic address:

The shortening of sarcomeres that co-ordinates the pump function of the heart is stimulated by electrically-mediated increases in [Ca]. This process of excitation-contraction coupling (ECC) is subject to modulation by neurohormonal mediators that tune the output of the heart to meet the needs of the organism. Endothelin-1 (ET-1) is a potent modulator of cardiac function with effects on contraction amplitude, chronotropy and automaticity. The actions of ET-1 are evident during normal adaptive physiological responses and increased under pathophysiological conditions, such as following myocardial infarction and during heart failure, where ET-1 levels are elevated. In myocytes, ET-1 acts through ET- or ET-G protein-coupled receptors (GPCRs). Although well studied in atrial myocytes, the influence and mechanisms of action of ET-1 upon ECC in ventricular myocytes are not fully resolved. We show in rat ventricular myocytes that ET-1 elicits a biphasic effect on fractional shortening (initial transient negative and sustained positive inotropy) and increases the peak amplitude of systolic Ca transients in adult rat ventricular myocytes. The negative inotropic phase was ET receptor-dependent, whereas the positive inotropic response and increase in peak amplitude of systolic Ca transients required ET receptor engagement. Both effects of ET-1 required phospholipase C (PLC)-activity, although distinct signalling pathways downstream of PLC elicited the effects of each ET receptor. The negative inotropic response involved inositol 1,4,5-trisphosphate (InsP) signalling and protein kinase C epsilon (PKCε). The positive inotropic action and the enhancement in Ca transient amplitude induced by ET-1 were independent of InsP signalling, but suppressed by PKCε. Serine 302 in cardiac myosin binding protein-C was identified as a PKCε substrate that when phosphorylated contributed to the suppression of contraction and Ca transients by PKCε following ET-1 stimulation. Thus, our data provide a new role and mechanism of action for InsP and PKCε in mediating the negative inotropic response and in restraining the positive inotropy and enhancement in Ca transients following ET-1 stimulation.
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http://dx.doi.org/10.1016/j.yjmcc.2018.02.012DOI Listing
April 2018

Experimental validation of a deforming grid 4D dose calculation for PBS proton therapy.

Phys Med Biol 2018 03 1;63(5):055005. Epub 2018 Mar 1.

Paul Scherrer Institute (PSI), Center for Proton Therapy, 5232 Villigen PSI, Switzerland. Department of Physics, ETH Zurich, 8092 Zurich, Switzerland.

The aim of this study was to verify the temporal accuracy of the estimated dose distribution by a 4D dose calculation (4DDC) in comparison to measurements. A single-field plan (0.6 Gy), optimised for a liver patient case (CTV volume: 403cc), was delivered to a homogeneous PMMA phantom and measured by a high resolution scintillating-CCD system at two water equivalent depths. Various motion scenarios (no motion and motions with amplitude of 10 mm and two periods: 3.7 s and 4.4 s) were simulated using a 4D Quasar phantom and logged by an optical tracking system in real-time. Three motion mitigation approaches (single delivery, 6[Formula: see text] layered and volumetric rescanning) were applied, resulting in 10 individual measurements. 4D dose distributions were retrospectively calculated in water by taking into account the delivery log files (retrospective) containing information on the actually delivered spot positions, fluences, and time stamps. Moreover, in order to evaluate the sensitivity of the 4DDC inputs, the corresponding prospective 4DDCs were performed as a comparison, using the estimated time stamps of the spot delivery and repeated periodical motion patterns. 2D gamma analyses and dose-difference-histograms were used to quantify the agreement between measurements and calculations for all pixels with [Formula: see text]5% of the maximum calculated dose. The results show that a mean gamma score of 99.2% with standard deviation 1.0% can be achieved for 3%/3 mm criteria and all scenarios can reach a score of more than 95%. The average area with more than 5% dose difference was 6.2%. Deviations due to input uncertainties were obvious for single scan deliveries but could be smeared out once rescanning was applied. Thus, the deforming grid 4DDC has been demonstrated to be able to predict the complex patterns of 4D dose distributions for PBS proton therapy with high dosimetric and geometric accuracy, and it can be used as a valid clinical tool for 4D treatment planning, motion mitigation selection, and eventually 4D optimisation applications if the correct temporal information is available.
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http://dx.doi.org/10.1088/1361-6560/aaad1eDOI Listing
March 2018

The Ageing Brain: Effects on DNA Repair and DNA Methylation in Mice.

Genes (Basel) 2017 Feb 17;8(2). Epub 2017 Feb 17.

Centre for Ageing and Vitality, Human Nutrition Research Centre, Institute of Cellular Medicine, Newcastle University, Campus for Ageing and Vitality, Newcastle upon Tyne NE4 5PL, UK.

Base excision repair (BER) may become less effective with ageing resulting in accumulation of DNA lesions, genome instability and altered gene expression that contribute to age-related degenerative diseases. The brain is particularly vulnerable to the accumulation of DNA lesions; hence, proper functioning of DNA repair mechanisms is important for neuronal survival. Although the mechanism of age-related decline in DNA repair capacity is unknown, growing evidence suggests that epigenetic events (e.g., DNA methylation) contribute to the ageing process and may be functionally important through the regulation of the expression of DNA repair genes. We hypothesize that epigenetic mechanisms are involved in mediating the age-related decline in BER in the brain. Brains from male mice were isolated at 3-32 months of age. Pyrosequencing analyses revealed significantly increased methylation with ageing, which correlated inversely with expression. The reduced expression correlated with enhanced expression of methyl-CpG binding protein 2 and ten-eleven translocation enzyme 2. A significant inverse correlation between methylation at CpG-site2 and expression was also observed. BER activity was significantly reduced and associated with increased 8-oxo-7,8-dihydro-2'-deoxyguanosine levels. These data indicate that and expression can be epigenetically regulated, which may mediate the effects of ageing on DNA repair in the brain.
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http://dx.doi.org/10.3390/genes8020075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333064PMC
February 2017

Retinol and ascorbate drive erasure of epigenetic memory and enhance reprogramming to naïve pluripotency by complementary mechanisms.

Proc Natl Acad Sci U S A 2016 10 11;113(43):12202-12207. Epub 2016 Oct 11.

Epigenetics Programme, Babraham Institute, Cambridge CB22 3AT, United Kingdom; Wellcome Trust Sanger Institute, Hinxton CB10 1SA, United Kingdom

Epigenetic memory, in particular DNA methylation, is established during development in differentiating cells and must be erased to create naïve (induced) pluripotent stem cells. The ten-eleven translocation (TET) enzymes can catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidized derivatives, thereby actively removing this memory. Nevertheless, the mechanism by which the TET enzymes are regulated, and the extent to which they can be manipulated, are poorly understood. Here we report that retinoic acid (RA) or retinol (vitamin A) and ascorbate (vitamin C) act as modulators of TET levels and activity. RA or retinol enhances 5hmC production in naïve embryonic stem cells by activation of TET2 and TET3 transcription, whereas ascorbate potentiates TET activity and 5hmC production through enhanced Fe recycling, and not as a cofactor as reported previously. We find that both ascorbate and RA or retinol promote the derivation of induced pluripotent stem cells synergistically and enhance the erasure of epigenetic memory. This mechanistic insight has significance for the development of cell treatments for regenenerative medicine, and enhances our understanding of how intrinsic and extrinsic signals shape the epigenome.
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http://dx.doi.org/10.1073/pnas.1608679113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5086989PMC
October 2016

Exosomes bind to autotaxin and act as a physiological delivery mechanism to stimulate LPA receptor signalling in cells.

J Cell Sci 2016 10 24;129(20):3948-3957. Epub 2016 Aug 24.

Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK

Autotaxin (ATX; also known as ENPP2), the lysophospholipase responsible for generating the lipid receptor agonist lysophosphatidic acid (LPA), is a secreted enzyme. Here we show that, once secreted, ATX can bind to the surface of cell-secreted exosomes. Exosome-bound ATX is catalytically active and carries generated LPA. Once bound to a cell, through specific integrin interactions, ATX releases the LPA to activate cell surface G-protein-coupled receptors of LPA; inhibition of signalling by the receptor antagonist Ki1642 suggests that these receptors are LPAR1 and LPAR3. The binding stimulates downstream signalling, including phosphorylation of AKT and mitogen-activated protein kinases, the release of intracellular stored Ca and cell migration. We propose that exosomal binding of LPA-loaded ATX provides a means of efficiently delivering the lipid agonist to cell surface receptors to promote signalling. We further propose that this is a means by which ATX-LPA signalling operates physiologically.
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http://dx.doi.org/10.1242/jcs.184424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087657PMC
October 2016

Norbin Stimulates the Catalytic Activity and Plasma Membrane Localization of the Guanine-Nucleotide Exchange Factor P-Rex1.

J Biol Chem 2016 Mar 20;291(12):6359-75. Epub 2016 Jan 20.

From the Signalling Programme and

P-Rex1 is a guanine-nucleotide exchange factor (GEF) that activates the small G protein (GTPase) Rac1 to control Rac1-dependent cytoskeletal dynamics, and thus cell morphology. Three mechanisms of P-Rex1 regulation are currently known: (i) binding of the phosphoinositide second messenger PIP3, (ii) binding of the Gβγ subunits of heterotrimeric G proteins, and (iii) phosphorylation of various serine residues. Using recombinant P-Rex1 protein to search for new binding partners, we isolated the G-protein-coupled receptor (GPCR)-adaptor protein Norbin (Neurochondrin, NCDN) from mouse brain fractions. Coimmunoprecipitation confirmed the interaction between overexpressed P-Rex1 and Norbin in COS-7 cells, as well as between endogenous P-Rex1 and Norbin in HEK-293 cells. Binding assays with purified recombinant proteins showed that their interaction is direct, and mutational analysis revealed that the pleckstrin homology domain of P-Rex1 is required. Rac-GEF activity assays with purified recombinant proteins showed that direct interaction with Norbin increases the basal, PIP3- and Gβγ-stimulated Rac-GEF activity of P-Rex1. Pak-CRIB pulldown assays demonstrated that Norbin promotes the P-Rex1-mediated activation of endogenous Rac1 upon stimulation of HEK-293 cells with lysophosphatidic acid. Finally, immunofluorescence microscopy and subcellular fractionation showed that coexpression of P-Rex1 and Norbin induces a robust translocation of both proteins from the cytosol to the plasma membrane, as well as promoting cell spreading, lamellipodia formation, and membrane ruffling, cell morphologies generated by active Rac1. In summary, we have identified a novel mechanism of P-Rex1 regulation through the GPCR-adaptor protein Norbin, a direct P-Rex1 interacting protein that promotes the Rac-GEF activity and membrane localization of P-Rex1.
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http://dx.doi.org/10.1074/jbc.M115.686592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4813545PMC
March 2016

Elf5-centered transcription factor hub controls trophoblast stem cell self-renewal and differentiation through stoichiometry-sensitive shifts in target gene networks.

Genes Dev 2015 Dec 19;29(23):2435-48. Epub 2015 Nov 19.

Epigenetics Programme, The Babraham Institute, Cambridge CB22 3AT, United Kingdom; Centre for Trophoblast Research, University of Cambridge, Cambridge CB2 3EG, United Kingdom;

Elf5 is a transcription factor with pivotal roles in the trophoblast compartment, where it reinforces a trophoblast stem cell (TSC)-specific transcriptional circuit. However, Elf5 is also present in differentiating trophoblast cells that have ceased to express other TSC genes such as Cdx2 and Eomes. In the present study, we aimed to elucidate the context-dependent role of Elf5 at the interface between TSC self-renewal and the onset of differentiation. We demonstrate that precise levels of Elf5 are critical for normal expansion of the TSC compartment and embryonic survival, as Elf5 overexpression triggers precocious trophoblast differentiation. Through integration of protein interactome, transcriptome, and genome-wide chromatin immunoprecipitation data, we reveal that this abundance-dependent function is mediated through a shift in preferred Elf5-binding partners; in TSCs, Elf5 interaction with Eomes recruits Tfap2c to triply occupied sites at TSC-specific genes, driving their expression. In contrast, the Elf5 and Tfap2c interaction becomes predominant as their protein levels increase. This triggers binding to double- and single-occupancy sites that harbor the cognate Tfap2c motif, causing activation of the associated differentiation-promoting genes. These data place Elf5 at the center of a stoichiometry-sensitive transcriptional network, where it acts as a molecular switch governing the balance between TSC proliferation and differentiation.
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http://dx.doi.org/10.1101/gad.268821.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4691948PMC
December 2015

Fgf and Esrrb integrate epigenetic and transcriptional networks that regulate self-renewal of trophoblast stem cells.

Nat Commun 2015 Jul 24;6:7776. Epub 2015 Jul 24.

1] Epigenetics Programme, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK [2] Centre for Trophoblast Research, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK.

Esrrb (oestrogen-related receptor beta) is a transcription factor implicated in embryonic stem (ES) cell self-renewal, yet its knockout causes intrauterine lethality due to defects in trophoblast development. Here we show that in trophoblast stem (TS) cells, Esrrb is a downstream target of fibroblast growth factor (Fgf) signalling and is critical to drive TS cell self-renewal. In contrast to its occupancy of pluripotency-associated loci in ES cells, Esrrb sustains the stemness of TS cells by direct binding and regulation of TS cell-specific transcription factors including Elf5 and Eomes. To elucidate the mechanisms whereby Esrrb controls the expression of its targets, we characterized its TS cell-specific interactome using mass spectrometry. Unlike in ES cells, Esrrb interacts in TS cells with the histone demethylase Lsd1 and with the RNA Polymerase II-associated Integrator complex. Our findings provide new insights into both the general and context-dependent wiring of transcription factor networks in stem cells by master transcription factors.
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http://dx.doi.org/10.1038/ncomms8776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4525203PMC
July 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

Resetting transcription factor control circuitry toward ground-state pluripotency in human.

Cell 2014 Sep;158(6):1254-1269

Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK; Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK. Electronic address:

Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here, we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling, are phenotypically stable, and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors, TFCP2L1 or KLF4, has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground-state pluripotency in human cells.
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http://dx.doi.org/10.1016/j.cell.2014.08.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4162745PMC
September 2014

The nuclear exosome is active and important during budding yeast meiosis.

PLoS One 2014 11;9(9):e107648. Epub 2014 Sep 11.

Epigenetics Programme, The Babraham Institute, Cambridge, United Kingdom.

Nuclear RNA degradation pathways are highly conserved across eukaryotes and play important roles in RNA quality control. Key substrates for exosomal degradation include aberrant functional RNAs and cryptic unstable transcripts (CUTs). It has recently been reported that the nuclear exosome is inactivated during meiosis in budding yeast through degradation of the subunit Rrp6, leading to the stabilisation of a subset of meiotic unannotated transcripts (MUTs) of unknown function. We have analysed the activity of the nuclear exosome during meiosis by deletion of TRF4, which encodes a key component of the exosome targeting complex TRAMP. We find that TRAMP mutants produce high levels of CUTs during meiosis that are undetectable in wild-type cells, showing that the nuclear exosome remains functional for CUT degradation, and we further report that the meiotic exosome complex contains Rrp6. Indeed Rrp6 over-expression is insufficient to suppress MUT transcripts, showing that the reduced amount of Rrp6 in meiotic cells does not directly cause MUT accumulation. Lack of TRAMP activity stabilises ∼ 1600 CUTs in meiotic cells, which occupy 40% of the binding capacity of the nuclear cap binding complex (CBC). CBC mutants display defects in the formation of meiotic double strand breaks (DSBs), and we see similar defects in TRAMP mutants, suggesting that a key function of the nuclear exosome is to prevent saturation of the CBC complex by CUTs. Together, our results show that the nuclear exosome remains active in meiosis and has an important role in facilitating meiotic recombination.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0107648PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161446PMC
October 2015

Dictyostelium uses ether-linked inositol phospholipids for intracellular signalling.

EMBO J 2014 Oct 1;33(19):2188-200. Epub 2014 Sep 1.

Signalling Programme Babraham Research Campus, Cambridge, UK

Inositol phospholipids are critical regulators of membrane biology throughout eukaryotes. The general principle by which they perform these roles is conserved across species and involves binding of differentially phosphorylated inositol head groups to specific protein domains. This interaction serves to both recruit and regulate the activity of several different classes of protein which act on membrane surfaces. In mammalian cells, these phosphorylated inositol head groups are predominantly borne by a C38:4 diacylglycerol backbone. We show here that the inositol phospholipids of Dictyostelium are different, being highly enriched in an unusual C34:1e lipid backbone, 1-hexadecyl-2-(11Z-octadecenoyl)-sn-glycero-3-phospho-(1'-myo-inositol), in which the sn-1 position contains an ether-linked C16:0 chain; they are thus plasmanylinositols. These plasmanylinositols respond acutely to stimulation of cells with chemoattractants, and their levels are regulated by PIPKs, PI3Ks and PTEN. In mammals and now in Dictyostelium, the hydrocarbon chains of inositol phospholipids are a highly selected subset of those available to other phospholipids, suggesting that different molecular selectors are at play in these organisms but serve a common, evolutionarily conserved purpose.
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http://dx.doi.org/10.15252/embj.201488677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4282506PMC
October 2014

The immune system GTPase GIMAP6 interacts with the Atg8 homologue GABARAPL2 and is recruited to autophagosomes.

PLoS One 2013 17;8(10):e77782. Epub 2013 Oct 17.

The Babraham Institute, Cambridge, Cambridgeshire, United Kingdom.

The GIMAPs (GTPases of the immunity-associated proteins) are a family of small GTPases expressed prominently in the immune systems of mammals and other vertebrates. In mammals, studies of mutant or genetically-modified rodents have indicated important roles for the GIMAP GTPases in the development and survival of lymphocytes. No clear picture has yet emerged, however, of the molecular mechanisms by which they perform their function(s). Using biotin tag-affinity purification we identified a major, and highly specific, interaction between the human cytosolic family member GIMAP6 and GABARAPL2, one of the mammalian homologues of the yeast autophagy protein Atg8. Chemical cross-linking studies performed on Jurkat T cells, which express both GIMAP6 and GABARAPL2 endogenously, indicated that the two proteins in these cells readily associate with one another in the cytosol under normal conditions. The GIMAP6-GABARAPL2 interaction was disrupted by deletion of the last 10 amino acids of GIMAP6. The N-terminal region of GIMAP6, however, which includes a putative Atg8-family interacting motif, was not required. Over-expression of GIMAP6 resulted in increased levels of endogenous GABARAPL2 in cells. After culture of cells in starvation medium, GIMAP6 was found to localise in punctate structures with both GABARAPL2 and the autophagosomal marker MAP1LC3B, indicating that GIMAP6 re-locates to autophagosomes on starvation. Consistent with this finding, we have demonstrated that starvation of Jurkat T cells results in the degradation of GIMAP6. Whilst these findings raise the possibility that the GIMAPs play roles in the regulation of autophagy, we have been unable to demonstrate an effect of GIMAP6 over-expression on autophagic flux.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0077782PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3804274PMC
September 2014

A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation.

Genome Biol 2013 ;14(10):R119

Background: DNA methylation (5mC) plays important roles in epigenetic regulation of genome function. Recently, TET hydroxylases have been found to oxidise 5mC to hydroxymethylcytosine (5hmC), formylcytosine (5fC) and carboxylcytosine (5caC) in DNA. These derivatives have a role in demethylation of DNA but in addition may have epigenetic signaling functions in their own right. A recent study identified proteins which showed preferential binding to 5-methylcytosine (5mC) and its oxidised forms, where readers for 5mC and 5hmC showed little overlap, and proteins bound to further oxidation forms were enriched for repair proteins and transcription regulators. We extend this study by using promoter sequences as baits and compare protein binding patterns to unmodified or modified cytosine using DNA from mouse embryonic stem cell extracts.

Results: We compared protein enrichments from two DNA probes with different CpG composition and show that, whereas some of the enriched proteins show specificity to cytosine modifications, others are selective for both modification and target sequences. Only a few proteins were identified with a preference for 5hmC (such as RPL26, PRP8 and the DNA mismatch repair protein MHS6), but proteins with a strong preference for 5fC were more numerous, including transcriptional regulators (FOXK1, FOXK2, FOXP1, FOXP4 and FOXI3), DNA repair factors (TDG and MPG) and chromatin regulators (EHMT1, L3MBTL2 and all components of the NuRD complex).

Conclusions: Our screen has identified novel proteins that bind to 5fC in genomic sequences with different CpG composition and suggests they regulate transcription and chromatin, hence opening up functional investigations of 5fC readers.
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http://dx.doi.org/10.1186/gb-2013-14-10-r119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4014808PMC
September 2014

A novel DYRK1B inhibitor AZ191 demonstrates that DYRK1B acts independently of GSK3β to phosphorylate cyclin D1 at Thr(286), not Thr(288).

Biochem J 2014 Jan;457(1):43-56

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

DYRK1B (dual-specificity tyrosine phosphorylation-regulated kinase 1B) is amplified in certain cancers and may be an oncogene; however, our knowledge of DYRK1B has been limited by the lack of selective inhibitors. In the present study we describe AZ191, a potent small molecule inhibitor that selectively inhibits DYRK1B in vitro and in cells. CCND1 (cyclin D1), a key regulator of the mammalian G1-S-phase transition, is phosphorylated on Thr(286) by GSK3β (glycogen synthase kinase 3β) to promote its degradation. DYRK1B has also been proposed to promote CCND1 turnover, but was reported to phosphorylate Thr(288) rather than Thr(286). Using in vitro kinase assays, phospho-specific immunoblot analysis and MS in conjunction with AZ191 we now show that DYRK1B phosphorylates CCND1 at Thr(286), not Thr(288), in vitro and in cells. In HEK (human embryonic kidney)-293 and PANC-1 cells (which exhibit DYRK1B amplification) DYRK1B drives Thr(286) phosphorylation and proteasome-dependent turnover of CCND1 and this is abolished by AZ191 or DYRK1B RNAi, but not by GSK3β inhibitors or GSK3β RNAi. DYRK1B expression causes a G1-phase cell-cycle arrest, but overexpression of CCND1 (wild-type or T286A) fails to overcome this; indeed, DYRK1B also promotes the expression of p21CIP1 (21 kDa CDK-interacting protein 1) and p27KIP1 (CDK-inhibitory protein 1). The results of the present study demonstrate for the first time that DYRK1B is a novel Thr(286)-CCND1 kinase that acts independently of GSK3β to promote CCND1 degradation. Furthermore, we anticipate that AZ191 may prove useful in defining further substrates and biological functions of DYRK1B.
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http://dx.doi.org/10.1042/BJ20130461DOI Listing
January 2014