Biology subjects

Perkins, N. D.

Publications and source records attributed to Perkins, N. D..

7 recordsLinked to original sources

bioRxiv2024

Domain Specific AI Segmentation of IMPDH2 Rod/Ring Structures in Mouse Embryonic Stem Cells

BackgroundInosine monophosphate dehydrogenase 2 (IMPDH2) is an enzyme that catalyses the rate limiting step of guanine nucleotides. In mouse embryonic stem cells (ESCs) IMPDH2 is held as large multi-protein complexes known as rod-ring (RR) structures that dissociate when ESCs differentiate. Manual analysis of RR structures from confocal microscopy images, although possible, is not feasible on a large scale due to the quantity of RR structures present in each field of view. To address this analysis bottleneck, we have created a fully automatic RR image classification pipeline to segment, characterise and measure feature distributions of these structures in ESCs. ResultsWe find that this model can automatically segment images with a Dice score of over 80% for both rods and rings for in-domain images compared to expert annotation, with a slight drop to 70% for datasets out of domain. Important feature measurements derived from these segmentations show high agreement with the measurements derived from expert annotation, achieving an R2 score of over 90% for counting the number of rings and rods over the dataset. ConclusionsWe have established for the first time a quantitative baseline for RR distribution in pluripotent ESCs and have made a pipeline available for training to be applied to other models in which RR remain an open topic of study.

bioinformatics↗

bioRxiv2022

Disruption of HIF1A translational control attenuates the HIF-dependent hypoxic response and solid tumour formation in vivo

Adaptation to reduced oxygen availability is mediated by the hypoxia-inducible factor (HIF) family of transcription factors. The activity and availability of HIF proteins is primarily driven by the stability of the HIF alpha subunits. However, it is becoming increasingly apparent that preferential translation of HIF1 mRNA is also necessary for full activation of the HIF1-dependent hypoxic response. Consequently, the mechanisms controlling HIF1 translation are of equivalent importance to the proline hydroxylase-dependent degradation pathways. Here we investigate the role of the 5UTR of the HIF1 mRNA in controlling preferential translation of endogenous HIF1 in hypoxic cells. CRISPR/Cas9-mediated genetic deletion of the 5 UTR of HIF1 results in reduced HIF1 levels following hypoxia, without alteration in mRNA or protein stability. HIF1 mRNA lacking the 5UTR was efficiently translated in adequately oxygenated cells but this was inhibited during hypoxia, consistent with the global block on protein synthesis. The HIF1 translational defect observed in cells missing the 5UTR led to reduced viability in hypoxic conditions in vitro and an impaired ability to form solid tumours in murine xenografts. Prevention of preferential HIF1 translation limits the duration and intensity of the HIF-dependent hypoxic response and disrupts the formation of solid tumours. Together these results demonstrate the importance of translation control over HIF1 and suggest that strategies to inhibit preferential HIF1 protein translation in hypoxic cancer cells will be an effective strategy to limit the growth of solid hypoxic tumours.

cancer biology↗

bioRxiv2021

Regulation of CHK1 inhibitor resistance by a c-Rel and USP1 dependent pathway

We previously discovered that deletion of c-Rel in the E-Myc mouse model of lymphoma results in earlier onset of disease, a finding that contrasted with the expected function of this NF-{kappa}B subunit in B-cell malignancies. Here we report that c-rel -/- E{micro}-Myc cells have an unexpected and major defect in the CHK1 pathway, with almost undetectable levels of CHK1 and CLSPN protein leading to therapeutic resistance to the highly specific CHK1 inhibitor (CHK1i) CCT244747. Similar downregulation of CHK1 levels was also seen in CCT244747 resistant U20S osteosarcoma cells. Further investigation revealed that downregulation of the deubiquitinase USP1 is responsible, at least in part, for these effects. Importantly, we demonstrate that c-rel -/- E{micro}-Myc lymphoma cells survive though upregulation of compensatory PI3K/AKT pathway activity. Moreover, targeting this pathway with Pictilisib (GDC-0941) effectively killed c-rel -/- E{micro}-Myc in vivo, while having no effect on wild type E-Myc cells. This data reveals an NF-{kappa}B regulated pathway controlling CHK1 activity in cancer cells and identifies a potential mechanism for both acquiring and overcoming CHK1i resistance in cancer patients.

cancer biology↗

bioRxiv2020

Temporal modulation of the NF-κB RelA network in response to different types of DNA damage

Different types of DNA damage can initiate phosphorylation-mediated signalling cascades that result in stimulus specific pro- or anti-apoptotic cellular responses. Amongst its many roles, the NF-{kappa}B transcription factor RelA is central to these DNA damage response pathways. However, we still lack understanding of the co-ordinated signalling mechanisms that permit different DNA damaging agents to induce distinct cellular outcomes through RelA. Here, we use label-free quantitative phosphoproteomics to examine the temporal effects of exposure of U2OS cells to either etoposide (ETO) or hydroxyurea (HU) by monitoring the phosphorylation status of RelA and its protein binding partners. Although few stimulus-specific differences were identified in the constituents of phosphorylated RelA interactome after exposure to these DNA damaging agents, we observed subtle, but significant, changes in their phosphorylation states, as a function of both type and duration of treatment. The DNA double strand break (DSB)-inducing ETO invoked more rapid, sustained responses than HU, with regulated targets primarily involved in transcription, cell division and canonical DSB repair. Kinase substrate prediction of ETO-regulated phosphosites suggest abrogation of CDK1 and ERK1 signalling, in addition to the known induction of ATM/ATR. In contrast, HU-induced replicative stress mediated temporally dynamic regulation, with phosphorylated RelA binding partners having roles in rRNA/mRNA processing and translational initiation, many of which contained a 14-3-3{varepsilon} binding motif, and were putative substrates of the dual specificity kinase CLK1. Our data thus point to differential regulation of key cellular processes and the involvement of distinct signalling pathways in modulating DNA damage-specific functions of RelA.

biochemistry↗

bioRxiv2019

Parallel siRNA screens to identify kinase and phosphatase modulators of NF-κB activity following DNA damage

DNA damage, such as that experienced by people undergoing chemotherapy, can directly activate NF-{kappa}B signalling which in turn can lead to resistance to genotoxic stress. NF-{kappa}B signalling is highly regulated by phosphorylation, but the enzymes required for these processes remain largely unknown. Identifying those enzymes responsible for regulating NF-{kappa}B activity may yield attractive targets for new clinical therapies, as well as provide the basis for better understanding of signalling network crosstalk. Here we present datasets from two independent RNAi screens using a stable NF-{kappa}B reporter U2OS cell line with the aim of identifying enzymes that alter NF-{kappa}B activity in response to DNA damage following etoposide and ionising radiation treatments. Although we observed high internal validity and specificity to NF-{kappa}B modulation within the screens, there was a striking dissimilarity between the results of the two different screens. These data therefore provide a cautionary lesson regarding the use of RNAi screening but also provide new candidates for kinase and phosphatase regulation of NF-{kappa}B activity in response to genotoxic stress.

cell biology↗

bioRxiv2019

A pseudo-meiotic centrosomal function of TEX12 in cancer

Cell division by meiosis involves an extraordinary chromosome choreography including pairing, synapsis and crossing over between homologous chromosomes1, 2. The many meiosis-specific genes involved in these processes also constitute a latent toolbox of chromosome remodelling and recombination factors that may be exploited through aberrant expression in cancer3, 4. Here, we report that TEX12, a structural protein involved in meiotic chromosome synapsis5-7, is aberrantly expressed in human cancers, with high TEX12 levels correlating with poor prognosis. We find that TEX12 knock-down causes proliferative failure in multiple cancer cell lines, and confirm its role in the early stages of oncogenesis through murine cancer models. Remarkably, somatically expressed TEX12 localises to centrosomes, leading to altered centrosome number and structure, features associated with cancer development. Further, we identify TEX12 in meiotic centrin-rich bodies, likely precursors of the mitotic centrosome, suggesting that this may represent an additional cellular function of TEX12 in meiosis that has been previously overlooked. Thus, we propose that an otherwise meiotic function of TEX12 in centrosome duplication is responsible for promoting oncogenesis and cellular proliferation in cancer, which may be targeted for novel cancer therapeutics and diagnostics.

cell biology↗

bioRxiv2018

Regulation of checkpoint kinase signalling and tumorigenesis by the NF-κB regulated gene, CLSPN

Inhibition of the tumour promoting activities of NF-{kappa}B by cell signalling pathways has been proposed as a natural mechanism to limit the development of cancer. However, there has been a lack of evidence for these effects in vivo. Here we report that RelAT505A mice, where a CHK1 targeted Thr505 phosphosite is mutated to alanine, display earlier onset of MYC driven lymphoma than wild type littermates. We describe a positive feedback loop in which the NF-{kappa}B subunits RelA and c-Rel, in a manner dependent upon RelA Thr505 phosphorylation, drive the expression of the ATR checkpoint kinase regulator Claspin in response to DNA replication stress in cancer cells. This in turn is required for maintenance of CHK1 activity. Loss of a single allele of the Clspn gene in mice is sufficient to drive earlier tumorigenesis and low levels of CLSPN mRNA expression are associated with worse survival in some forms of human cancer. We propose that loss of this pathway early in tumorigenesis promotes cancer development through increased genomic instability. However, in malignant cancer cells it can help promote their addiction to the checkpoint kinase signalling required for the maintenance of genomic integrity. Importantly, disruption of this pathway leads to resistance of cells to treatment with CHK1 inhibitors. Claspin expression could therefore act as a biomarker for responsiveness of patients to CHK1 inhibitors and provide a potential pathway for the development of tumour resistance.

cancer biology↗