Imperial College London

DrAlexisBarr

Faculty of MedicineInstitute of Clinical Sciences

Advanced Research Fellow
 
 
 
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Contact

 

+44 (0)20 7594 7772a.barr Website

 
 
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Location

 

Office 6.12BLMS BuildingHammersmith Campus

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Summary

 

Publications

Publication Type
Year
to

39 results found

Dragoi C-M, Kaur E, Barr AR, Tyson JJ, Novák Bet al., 2024, The oscillation of mitotic kinase governs cell cycle latches in mammalian cells., J Cell Sci, Vol: 137

The mammalian cell cycle alternates between two phases - S-G2-M with high levels of A- and B-type cyclins (CycA and CycB, respectively) bound to cyclin-dependent kinases (CDKs), and G1 with persistent degradation of CycA and CycB by an activated anaphase promoting complex/cyclosome (APC/C) bound to Cdh1 (also known as FZR1 in mammals; denoted APC/C:Cdh1). Because CDKs phosphorylate and inactivate Cdh1, these two phases are mutually exclusive. This 'toggle switch' is flipped from G1 to S by cyclin-E bound to a CDK (CycE:CDK), which is not degraded by APC/C:Cdh1, and from M to G1 by Cdc20-bound APC/C (APC/C:Cdc20), which is not inactivated by CycA:CDK or CycB:CDK. After flipping the switch, cyclin E is degraded and APC/C:Cdc20 is inactivated. Combining mathematical modelling with single-cell timelapse imaging, we show that dysregulation of CycB:CDK disrupts strict alternation of the G1-S and M-G1 switches. Inhibition of CycB:CDK results in Cdc20-independent Cdh1 'endocycles', and sustained activity of CycB:CDK drives Cdh1-independent Cdc20 endocycles. Our model provides a mechanistic explanation for how whole-genome doubling can arise, a common event in tumorigenesis that can drive tumour evolution.

Journal article

Crozier L, Foy R, Adib R, Kar A, Holt JA, Pareri AU, Valverde JM, Rivera R, Weston WA, Wilson R, Regnault C, Whitfield P, Badonyi M, Bennett LG, Vernon EG, Gamble A, Marsh JA, Staples CJ, Saurin AT, Barr AR, Ly Tet al., 2023, CDK4/6 inhibitor-mediated cell overgrowth triggers osmotic and replication stress to promote senescence, Molecular Cell, Vol: 83, Pages: 4062-4077.e5, ISSN: 1097-2765

Abnormal increases in cell size are associated with senescence and cell cycle exit. The mechanisms by which overgrowth primes cells to withdraw from the cell cycle remain unknown. We address this question using CDK4/6 inhibitors, which arrest cells in G0/G1 and are licensed to treat advanced HR+/HER2- breast cancer. We demonstrate that CDK4/6-inhibited cells overgrow during G0/G1, causing p38/p53/p21-dependent cell cycle withdrawal. Cell cycle withdrawal is triggered by biphasic p21 induction. The first p21 wave is caused by osmotic stress, leading to p38- and size-dependent accumulation of p21. CDK4/6 inhibitor washout results in some cells entering S-phase. Overgrown cells experience replication stress, resulting in a second p21 wave that promotes cell cycle withdrawal from G2 or the subsequent G1. We propose that the levels of p21 integrate signals from overgrowth-triggered stresses to determine cell fate. This model explains how hypertrophy can drive senescence and why CDK4/6 inhibitors have long-lasting effects in patients.

Journal article

Wiggins BG, Wang Y-F, Burke A, Grunberg N, Vlachaki Walker JM, Dore M, Chahrour C, Pennycook BR, Sanchez-Garrido J, Vernia S, Barr AR, Frankel G, Birdsey GM, Randi AM, Schiering Cet al., 2023, Endothelial sensing of AHR ligands regulates intestinal homeostasis, Nature, Vol: 621, Pages: 821-829, ISSN: 0028-0836

Endothelial cells (ECs) line the blood and lymphatic vasculature, and act as an essential physical barrier, control nutrient transport, facilitate tissue immunosurveillance, and coordinate angiogenesis/ lymphangiogenesis1,2. In the intestine, dietary and microbial cues are particularly important in the regulation of organ homeostasis. However, whether enteric ECs actively sense and integrate such signals is currently unknown. Here, we show that the aryl hydrocarbon receptor (AHR) acts as a critical node for EC-sensing of dietary metabolites in adult mice and human primary ECs. We first established a comprehensive single-cell endothelial atlas of the mouse small intestine, uncovering the cellular complexity and functional heterogeneity of blood and lymphatic ECs. Analyses of AHR mediated responses at single-cell resolution identified tissue-protective transcriptional signatures and regulatory networks promoting cellular quiescence and vascular normalcy at steady state. Endothelial AHR-deficiency in adult mice resulted in dysregulated inflammatory responses, and the initiation of proliferative pathways. Furthermore, endothelial sensing of dietary AHR ligands was required for optimal protection against enteric infection. In human ECs, AHR signalling promoted quiescence and restrained activation by inflammatory mediators. Together, our data provide a comprehensive dissection of the impact of environmental sensing across the spectrum of enteric endothelia, demonstrating that endothelial AHR signalling integrates dietary cues to maintain tissue homeostasis by promoting EC quiescence and vascular normalcy.

Journal article

Weston WA, Barr AR, 2023, A cell cycle centric view of tumour dormancy, BRITISH JOURNAL OF CANCER, ISSN: 0007-0920

Journal article

Barr AR, 2023, A lack of commitment to proliferation, NATURE, Vol: 619, Pages: 257-258, ISSN: 0028-0836

Journal article

Wiecek AJ, Cutty SJ, Kornai D, Parreno-Centeno M, Gourmet LE, Tagliazucchi GM, Jacobson DH, Zhang P, Xiong L, Bond GL, Barr AR, Secrier Met al., 2023, Genomic hallmarks and therapeutic implications of G0 cell cycle arrest in cancer, Genome Biology, Vol: 24, ISSN: 1474-7596

BACKGROUND: Therapy resistance in cancer is often driven by a subpopulation of cells that are temporarily arrested in a non-proliferative G0 state, which is difficult to capture and whose mutational drivers remain largely unknown. RESULTS: We develop methodology to robustly identify this state from transcriptomic signals and characterise its prevalence and genomic constraints in solid primary tumours. We show that G0 arrest preferentially emerges in the context of more stable, less mutated genomes which maintain TP53 integrity and lack the hallmarks of DNA damage repair deficiency, while presenting increased APOBEC mutagenesis. We employ machine learning to uncover novel genomic dependencies of this process and validate the role of the centrosomal gene CEP89 as a modulator of proliferation and G0 arrest capacity. Lastly, we demonstrate that G0 arrest underlies unfavourable responses to various therapies exploiting cell cycle, kinase signalling and epigenetic mechanisms in single-cell data. CONCLUSIONS: We propose a G0 arrest transcriptional signature that is linked with therapeutic resistance and can be used to further study and clinically track this state.

Journal article

Hughes F, Barr A, Thomas P, 2023, Patterns of interdivision time correlations reveal hidden cell cycle factors, eLife, Vol: 11, ISSN: 2050-084X

The time taken for cells to complete a round of cell division is a stochastic process controlled, in part, by intracellular factors. These factors can be inherited across cellular generations which gives rise to, often non-intuitive, correlation patterns in cell cycle timing between cells of different family relationships on lineage trees. Here, we formulate a framework of hidden inherited factors affecting the cell cycle that unifies known cell cycle control models and reveals three distinct interdivision time correlation patterns: aperiodic, alternator and oscillator. We use Bayesian inference with single-cell datasets of cell division in bacteria, mammalian and cancer cells, to identify the inheritance motifs that underlie these datasets. From our inference, we find that interdivision time correlation patterns do not identify a single cell cycle model but generally admit a broad posterior distribution of possible mechanisms. Despite this unidentifiability, we observe that the inferred patterns reveal interpretable inheritance dynamics and hidden rhythmicity of cell cycle factors. This reveals that cell cycle factors are commonly driven by circadian rhythms, but their period may differ in cancer. Our quantitative analysis thus reveals that correlation patterns are an emergent phenomenon that impact cell proliferation and these patterns may be altered in disease.

Journal article

Yao G, Dhawan J, Barr AR, 2022, Editorial: Cellular dormancy-State determination and plasticity, FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY, Vol: 10, ISSN: 2296-634X

Journal article

Swadling JB, Warnecke T, Morris KL, Barr ARet al., 2022, Conserved Cdk inhibitors show unique structural responses to tyrosine phosphorylation, BIOPHYSICAL JOURNAL, Vol: 121, Pages: 2312-2329, ISSN: 0006-3495

Journal article

Barr AR, McClelland SE, 2022, Cells on lockdown: long-term consequences of CDK4/6 inhibition, EMBO JOURNAL, Vol: 41, ISSN: 0261-4189

Journal article

Thomsen I, Kunowska N, de Souza R, Moody A-M, Crawford G, Wang Y-F, Khadayate S, Whilding C, Strid J, Karimi MM, Barr AR, Dillon N, Sabbattini Pet al., 2021, RUNX1 regulates a transcription program that affects the dynamics of cell cycle entry of naive resting B cells, Journal of Immunology, Vol: 207, Pages: 2976-2991, ISSN: 0022-1767

RUNX1 is a transcription factor that plays key roles in hematopoietic development and in hematopoiesis and lymphopoiesis. In this article, we report that RUNX1 regulates a gene expression program in naive mouse B cells that affects the dynamics of cell cycle entry in response to stimulation of the BCR. Conditional knockout of Runx1 in mouse resting B cells resulted in accelerated entry into S-phase after BCR engagement. Our results indicate that Runx1 regulates the cyclin D2 (Ccnd2) gene, the immediate early genes Fosl2, Atf3, and Egr2, and the Notch pathway gene Rbpj in mouse B cells, reducing the rate at which transcription of these genes increases after BCR stimulation. RUNX1 interacts with the chromatin remodeler SNF-2-related CREB-binding protein activator protein (SRCAP), recruiting it to promoter and enhancer regions of the Ccnd2 gene. BCR-mediated activation triggers switching between binding of RUNX1 and its paralog RUNX3 and between SRCAP and the switch/SNF remodeling complex member BRG1. Binding of BRG1 is increased at the Ccnd2 and Rbpj promoters in the Runx1 knockout cells after BCR stimulation. We also find that RUNX1 exerts positive or negative effects on a number of genes that affect the activation response of mouse resting B cells. These include Cd22 and Bank1, which act as negative regulators of the BCR, and the IFN receptor subunit gene Ifnar1 The hyperresponsiveness of the Runx1 knockout B cells to BCR stimulation and its role in regulating genes that are associated with immune regulation suggest that RUNX1 could be involved in regulating B cell tolerance.

Journal article

Pennycook BR, Barr AR, 2021, Palbociclib-mediated cell cycle arrest can occur in the absence of the CDK inhibitors p21 and p27, Open Biology, Vol: 11, ISSN: 2046-2441

The use of CDK4/6 inhibitors in the treatment of a wide range of cancers is an area of ongoing investigation. Despite their increasing clinical use, there is limited understanding of the determinants of sensitivity and resistance to these drugs. Recent data have cast doubt on how CDK4/6 inhibitors arrest proliferation, provoking renewed interest in the role(s) of CDK4/6 in driving cell proliferation. As the use of CDK4/6 inhibitors in cancer therapies becomes more prominent, an understanding of their effect on the cell cycle becomes more urgent. Here, we investigate the mechanism of action of CDK4/6 inhibitors in promoting cell cycle arrest. Two main models explain how CDK4/6 inhibitors cause G1 cell cycle arrest, which differ in their dependence on the CDK inhibitor proteins p21 and p27. We have used live and fixed single-cell quantitative imaging, with inducible degradation systems, to address the roles of p21 and p27 in the mechanism of action of CDK4/6 inhibitors. We find that CDK4/6 inhibitors can initiate and maintain a cell cycle arrest without p21 or p27. This work clarifies our current understanding of the mechanism of action of CDK4/6 inhibitors and has implications for cancer treatment and patient stratification.

Journal article

Pennycook B, Barr A, 2021, Palbociclib-mediated cell cycle arrest can occur in the absence of the CDK inhibitors p21 and p27

The use of CDK4/6 inhibitors in the treatment of a wide range of cancers is an area of ongoing investigation. Despite their increasing clinical use, there is limited understanding of the determinants of sensitivity and resistance to these drugs. Recent data has cast doubt on how CDK4/6 inhibitors arrest proliferation, provoking renewed interest in the role(s) of CDK4/6 in driving cell proliferation. As the use of CDK4/6 inhibitors in cancer therapies becomes more prominent, an understanding of their effect on the cell cycle becomes more urgent. Here, we investigate the mechanism of action of CDK4/6 inhibitors in promoting cell cycle arrest. Two main models explain how CDK4/6 inhibitors cause G1 cell cycle arrest, which differ in their dependence on the CDK inhibitor proteins p21 and p27. We have used live and fixed single-cell quantitative imaging, with inducible degradation systems, to address the roles of p21 and p27 in the mechanism of action of CDK4/6 inhibitors. We find that CDK4/6 inhibitors can initiate and maintain a cell cycle arrest without p21 or p27. This work clarifies our current understanding of the mechanism of action of CDK4/6 inhibitors and has implications for cancer treatment and patient stratification.

Journal article

Pennycook BR, Vesela E, Peripolli S, Singh T, Barr AR, Bertoli C, de Bruin RAMet al., 2020, E2F-dependent transcription determines replication capacity and S phase length, NATURE COMMUNICATIONS, Vol: 11, ISSN: 2041-1723

Journal article

Pennycook BR, Barr AR, 2020, Restriction point regulation at the crossroads between quiescence and cell proliferation, FEBS Letters, Vol: 594, Pages: 2046-2060, ISSN: 0014-5793

The coordination of cell proliferation with reversible cell cycle exit into quiescence is crucial for the development of multicellular organisms and for tissue homeostasis in the adult. The decision between quiescence and proliferation occurs at the restriction point, which is widely thought to be located in the G1 phase of the cell cycle, when cells integrate accumulated extracellular and intracellular signals to drive this binary cellular decision. On the molecular level, decision-making is exerted through the activation of cyclin-dependent kinases (CDKs). CDKs phosphorylate the retinoblastoma (Rb) transcriptional repressor to regulate the expression of cell cycle genes. Recently, the classical view of restriction point regulation has been challenged. Here, we review the latest findings on the activation of CDKs, Rb phosphorylation and the nature and position of the restriction point within the cell cycle.

Journal article

Barr AR, 2020, Editorial, FEBS LETTERS, Vol: 594, Pages: 2029-2030, ISSN: 0014-5793

Journal article

Hegarat N, Crncec A, Rodriguez MFSP, Iturra FE, Gu Y, Busby O, Lang PF, Barr AR, Bakal C, Kanemaki MT, Lamond A, Novak B, Ly T, Hochegger Het al., 2020, Cyclin A triggers Mitosis either via the Greatwall kinase pathway or Cyclin B, The EMBO Journal, Vol: 39, Pages: 1-23, ISSN: 0261-4189

Two mitotic cyclin types, cyclin A and B, exist in higher eukaryotes, but their specialised functions in mitosis are incompletely understood. Using degron tags for rapid inducible protein removal, we analyse how acute depletion of these proteins affects mitosis. Loss of cyclin A in G2‐phase prevents mitotic entry. Cells lacking cyclin B can enter mitosis and phosphorylate most mitotic proteins, because of parallel PP2A:B55 phosphatase inactivation by Greatwall kinase. The final barrier to mitotic establishment corresponds to nuclear envelope breakdown, which requires a decisive shift in the balance of cyclin‐dependent kinase Cdk1 and PP2A:B55 activity. Beyond this point, cyclin B/Cdk1 is essential for phosphorylation of a distinct subset of mitotic Cdk1 substrates that are essential to complete cell division. Our results identify how cyclin A, cyclin B and Greatwall kinase coordinate mitotic progression by increasing levels of Cdk1‐dependent substrate phosphorylation.

Journal article

Stojic L, Lun ATL, Mascalchi P, Ernst C, Redmond AM, Mangei J, Barr AR, Bousgouni V, Bakal C, Marioni JC, Odom DT, Gergely Fet al., 2020, A high-content RNAi screen reveals multiple roles for long noncoding RNAs in cell division, Nature Communications, Vol: 11, Pages: 1-21, ISSN: 2041-1723

Genome stability relies on proper coordination of mitosis and cytokinesis, where dynamic microtubules capture and faithfully segregate chromosomes into daughter cells. With a high-content RNAi imaging screen targeting more than 2,000 human lncRNAs, we identify numerous lncRNAs involved in key steps of cell division such as chromosome segregation, mitotic duration and cytokinesis. Here, we provide evidence that the chromatin-associated lncRNA, linc00899, leads to robust mitotic delay upon its depletion in multiple cell types. We perform transcriptome analysis of linc00899-depleted cells and identify the neuronal microtubule-binding protein, TPPP/p25, as a target of linc00899. We further show that linc00899 binds TPPP/p25 and suppresses its transcription. In cells depleted of linc00899, upregulation of TPPP/p25 alters microtubule dynamics and delays mitosis. Overall, our comprehensive screen uncovers several lncRNAs involved in genome stability and reveals a lncRNA that controls microtubule behaviour with functional implications beyond cell division.

Journal article

Barr AR, Mansfeld J, 2019, FEBS Letters Special Issue: Cell Cycle Control, FEBS LETTERS, Vol: 593, Pages: 2803-2804, ISSN: 0014-5793

Journal article

Stojic L, Lun ATL, Mascalchi P, Ernst C, Redmond AM, Mangei J, Barr AR, Bousgouni V, Bakal C, Marioni JC, Odom DT, Gergely Fet al., 2019, A high-content RNAi screen reveals multiple roles for long noncoding RNAs in cell division

<jats:title>ABSTRACT</jats:title><jats:p>Genome stability relies on proper coordination of mitosis and cytokinesis, where dynamic microtubules capture and faithfully segregate chromosomes into daughter cells. The role of long noncoding RNAs (lncRNAs) in controlling these processes however remains largely unexplored. To identify lncRNAs with mitotic functions, we performed a high-content RNAi imaging screen targeting more than 2,000 human lncRNAs. By investigating major hallmarks of cell division such as chromosome segregation, mitotic duration and cytokinesis, we discovered numerous lncRNAs with functions in each of these processes. The chromatin-associated lncRNA,<jats:italic>linc00899,</jats:italic>was selected for in-depth studies due to the robust mitotic delay observed upon its depletion. Transcriptome analysis of<jats:italic>linc00899</jats:italic>-depleted cells together with gain-of-function and rescue experiments across multiple cell types identified the neuronal microtubule-binding protein,<jats:italic>TPPP/p25,</jats:italic>as a target of<jats:italic>linc00899</jats:italic>.<jats:italic>Linc00899</jats:italic>binds the genomic locus of<jats:italic>TPPP/p25</jats:italic>and suppresses its transcription through a<jats:italic>cis</jats:italic>-acting mechanism. In cells depleted of<jats:italic>linc00899,</jats:italic>the consequent upregulation of<jats:italic>TPPP/p25</jats:italic>alters microtubule dynamics and is necessary and sufficient to delay mitosis. Overall, our comprehensive screen identified several lncRNAs with roles in genome stability and revealed a new lncRNA that controls microtubule behaviour with functional implications beyond cell division.</jats:p>

Working paper

Hégarat N, Crncec A, Peredoa Rodri-guez MFS, Iturra FE, Gu Y, Lang PF, Barr AR, Bakal C, Kanemaki MT, Lamond AI, Novak B, Ly T, Hochegger Het al., 2018, Cyclin A triggers Mitosis either via Greatwall or Cyclin B

<jats:title>Abstract</jats:title><jats:p>Two mitotic Cyclins, A and B, exist in higher eukaryotes, but their specialised functions in mitosis are poorly understood. Using degron tags we analyse how acute depletion of these proteins affects mitosis. Loss of Cyclin A in G2-phase prevents the initial activation of Cdk1. Cells lacking Cyclin B can enter mitosis and phosphorylate most mitotic proteins, because of parallel PP2A:B55 phos-phatase inactivation by Greatwall kinase. The final barrier to mitotic establishment corresponds to nuclear envelope breakdown that requires a decisive shift in the balance of Cdk1 and PP2A:B55 activity. Beyond this point Cyclin B/Cdk1 is essential to phosphorylate a distinct subset mitotic Cdk1 substrates that are essential to complete cell division. Our results identify how Cyclin A, B and Greatwall coordinate mitotic progression by increasing levels of Cdk1-dependent substrate phos-phorylation.</jats:p>

Working paper

Stojic L, Lun ATL, Mangei J, Mascalchi P, Quarantotti V, Barr AR, Bakal C, Marioni JC, Gergely F, Odom DTet al., 2018, Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis, Nucleic Acids Research, Vol: 46, Pages: 5950-5966, ISSN: 0305-1048

Loss-of-function (LOF) methods such as RNA interference (RNAi), antisense oligonucleotides or CRISPR-based genome editing provide unparalleled power for studying the biological function of genes of interest. However, a major concern is non-specific targeting, which involves depletion of transcripts other than those intended. Little work has been performed to characterize the off-target effects of these common LOF methods at the whole-transcriptome level. Here, we experimentally compared the non-specific activity of RNAi, antisense oligonucleotides and CRISPR interference (CRISPRi). All three methods yielded non-negligible off-target effects in gene expression, with CRISPRi also exhibiting strong clonal effects. As an illustrative example, we evaluated the performance of each method for determining the role of an uncharacterized long noncoding RNA (lncRNA). Several LOF methods successfully depleted the candidate lncRNA but yielded different sets of differentially expressed genes as well as a different cellular phenotype upon depletion. Similar discrepancies between methods were observed with a protein-coding gene (Ch-TOG/CKAP5) and another lncRNA (MALAT1). We suggest that the differences between methods arise due to method-specific off-target effects and provide guidelines for mitigating such effects in functional studies. Our recommendations provide a framework with which off-target effects can be managed to improve functional characterization of genes of interest.

Journal article

Heldt FS, Barr AR, Cooper S, Bakal C, Novak Bet al., 2018, A comprehensive model for the proliferation-quiescence decision in response to endogenous DNA damage in human cells, Proceedings of the National Academy of Sciences of the United States of America, Vol: 115, Pages: 2532-2537, ISSN: 0027-8424

Human cells that suffer mild DNA damage can enter a reversible state of growth arrest known as quiescence. This decision to temporarily exit the cell cycle is essential to prevent the propagation of mutations, and most cancer cells harbor defects in the underlying control system. Here we present a mechanistic mathematical model to study the proliferation–quiescence decision in nontransformed human cells. We show that two bistable switches, the restriction point (RP) and the G1/S transition, mediate this decision by integrating DNA damage and mitogen signals. In particular, our data suggest that the cyclin-dependent kinase inhibitor p21 (Cip1/Waf1), which is expressed in response to DNA damage, promotes quiescence by blocking positive feedback loops that facilitate G1 progression downstream of serum stimulation. Intriguingly, cells exploit bistability in the RP to convert graded p21 and mitogen signals into an all-or-nothing cell-cycle response. The same mechanism creates a window of opportunity where G1 cells that have passed the RP can revert to quiescence if exposed to DNA damage. We present experimental evidence that cells gradually lose this ability to revert to quiescence as they progress through G1 and that the onset of rapid p21 degradation at the G1/S transition prevents this response altogether, insulating S phase from mild, endogenous DNA damage. Thus, two bistable switches conspire in the early cell cycle to provide both sensitivity and robustness to external stimuli.

Journal article

Stojic L, Lun A, Mangei J, Mascalchi P, Quarantotti V, Barr AR, Bakal C, Marioni JC, Gergely F, Odom DTet al., 2017, Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis

<jats:title>ABSTRACT</jats:title><jats:p>Loss-of-function (LOF) methods, such as RNA interference (RNAi), antisense oligonucleotides or CRISPR-based genome editing, provide unparalleled power for studying the biological function of genes of interest. When coupled with transcriptomic analyses, LOF methods allow researchers to dissect networks of transcriptional regulation. However, a major concern is nonspecific targeting, which involves depletion of transcripts other than those intended. The off-target effects of each of these common LOF methods have yet to be compared at the whole-transcriptome level. Here, we systematically and experimentally compared non-specific activity of RNAi, antisense oligonucleotides and CRISPR interference (CRISPRi). All three methods yielded non-negligible offtarget effects in gene expression, with CRISPRi exhibiting clonal variation in the transcriptional profile. As an illustrative example, we evaluated the performance of each method for deciphering the role of a long noncoding RNA (lncRNA) with unknown function. Although all LOF methods reduced expression of the candidate lncRNA, each method yielded different sets of differentially expressed genes upon knockdown as well as a different cellular phenotype. Therefore, to definitively confirm the functional role of a transcriptional regulator, we recommend the simultaneous use of at least two different LOF methods and the inclusion of multiple, specifically designed negative controls.</jats:p>

Working paper

Cooper S, Barr AR, Glen R, Bakal Cet al., 2017, NucliTrack: an integrated nuclei tracking application, Bioinformatics, Vol: 33, Pages: 3320-3322, ISSN: 1367-4803

Live imaging studies give unparalleled insight into dynamic single cell behaviours and fate decisions. However, the challenge of reliably tracking single cells over long periods of time limits both the throughput and ease with which such studies can be performed. Here, we present NucliTrack, a cross platform solution for automatically segmenting, tracking and extracting features from fluorescently labelled nuclei. NucliTrack performs similarly to other state-of-the-art cell tracking algorithms, but NucliTrack’s interactive, graphical interface makes it significantly more user friendly.

Journal article

Asghar US, Barr AR, Cutts R, Beaney M, Babina I, Sampath D, Giltnane J, Lacap JA, Crocker L, Young A, Pearson A, Herrera-Abreu MT, Bakal C, Turner NCet al., 2017, Single-cell dynamics determines response to CDK4/6 inhibition in triple-negative breast cancer, Clinical Cancer Research, Vol: 23, Pages: 5561-5572, ISSN: 1078-0432

Purpose: Triple-negative breast cancer (TNBC) is a heterogeneous subgroup of breast cancer that is associated with a poor prognosis. We evaluated the activity of CDK4/6 inhibitors across the TNBC subtypes and investigated mechanisms of sensitivity.Experimental Design: A panel of cell lines representative of TNBC was tested for in vitro and in vivo sensitivity to CDK4/6 inhibition. A fluorescent CDK2 activity reporter was used for single-cell analysis in conjunction with time-lapse imaging.Results: The luminal androgen receptor (LAR) subtype of TNBC was highly sensitive to CDK4/6 inhibition both in vitro (P < 0.001 LAR vs. basal-like) and in vivo in MDA-MB-453 LAR cell line xenografts. Single-cell analysis of CDK2 activity demonstrated differences in cell-cycle dynamics between LAR and basal-like cells. Palbociclib-sensitive LAR cells exit mitosis with low levels of CDK2 activity, into a quiescent state that requires CDK4/6 activity for cell-cycle reentry. Palbociclib-resistant basal-like cells exit mitosis directly into a proliferative state, with high levels of CDK2 activity, bypassing the restriction point and the requirement for CDK4/6 activity. High CDK2 activity after mitosis is driven by temporal deregulation of cyclin E1 expression. CDK4/6 inhibitors were synergistic with PI3 kinase inhibitors in PIK3CA-mutant TNBC cell lines, extending CDK4/6 inhibitor sensitivity to additional TNBC subtypes.Conclusions: Cell-cycle dynamics determine the response to CDK4/6 inhibition in TNBC. CDK4/6 inhibitors, alone and in combination, are a novel therapeutic strategy for specific subgroups of TNBC. Clin Cancer Res; 23(18); 5561–72.

Journal article

Barr AR, Cooper S, Heldt FS, Butera F, Stoy H, Mansfeld J, Novak B, Bakal Cet al., 2017, DNA damage during S-phase mediates the proliferation-quiescence decision in the subsequent G1 via p21 expression, Nature Communications, Vol: 8, Pages: 1-17, ISSN: 2041-1723

Following DNA damage caused by exogenous sources, such as ionizing radiation, the tumour suppressor p53 mediates cell cycle arrest via expression of the CDK inhibitor, p21. However, the role of p21 in maintaining genomic stability in the absence of exogenous DNA-damaging agents is unclear. Here, using live single-cell measurements of p21 protein in proliferating cultures, we show that naturally occurring DNA damage incurred over S-phase causes p53-dependent accumulation of p21 during mother G2- and daughter G1-phases. High p21 levels mediate G1 arrest via CDK inhibition, yet lower levels have no impact on G1 progression, and the ubiquitin ligases CRL4Cdt2 and SCFSkp2 couple to degrade p21 prior to the G1/S transition. Mathematical modelling reveals that a bistable switch, created by CRL4Cdt2, promotes irreversible S-phase entry by keeping p21 levels low, preventing premature S-phase exit upon DNA damage. Thus, we characterize how p21 regulates the proliferation-quiescence decision to maintain genomic stability.

Journal article

Chavali PL, Chandrasekaran G, Barr AR, Tatrai P, Taylor C, Papachristou EK, Woods CG, Chavali S, Gergely Fet al., 2016, A CEP215-HSET complex links centrosomes with spindle poles and drives centrosome clustering in cancer, NATURE COMMUNICATIONS, Vol: 7, ISSN: 2041-1723

Journal article

Barr AR, Heldt FS, Zhang T, Bakal C, Novak Bet al., 2016, A Dynamical Framework for the All-or-None G1/S Transition, CELL SYSTEMS, Vol: 2, Pages: 27-37, ISSN: 2405-4712

Journal article

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