Biology subjects

Barr, A. R.

Publications and source records attributed to Barr, A. R..

13 recordsLinked to original sources

bioRxiv2025

Wee1 opposes APC/C(Cdh1) activity to promote S-phase entry

Wee1 phosphorylates and inhibits CDK activity to inhibit mitotic entry and establish a G2 DNA damage checkpoint. Consequently, Wee1 inhibitors are in clinical trials, developed to be synthetically lethal in TP53 mutant tumours that become reliant on a Wee1-mediated DNA damage checkpoint. However, Wee1 inhibitors have efficacy in TP53 wild-type tumours and many trials have been terminated due to high levels of toxic side-effects, suggesting that Wee1 has unknown functions. Here, we show that Wee1 promotes cell cycle re-entry from quiescence (G0) by opposing the activity of the E3 ubiquitin ligase, APC/CCdh1. Wee1 phosphorylates Cdh1 (FZR1) at key residues that mediate the interaction between Cdh1 and APC/C. Cells with loss-of-function of Wee1 during G0/G1 have delayed S-phase entry, an impaired G1/S transition, abnormal S-phase accumulation of the CDK inhibitor p21 and enter a p21-dependent G2 arrest. Reduced expression of APC/CCdh1 or p21 renders cells more sensitive to acute Wee1 inhibition and both pathways are downregulated in acquired Wee1 inhibitor resistance. Our study reveals a new cell cycle control mechanism that has implications for how Wee1 inhibitors should be used in the clinic.

cell biology↗

bioRxiv2025

Structural Identification of Major Molecular Determinants for Phosphotyrosine Recognition in Tyrosine Kinases Reveals Tumour Promoting and Suppressive Functions

Protein tyrosine kinases activate signaling pathways by catalyzing the phosphorylation of tyrosine residues in their substrates. Mounting evidence suggests that, in addition to recognizing phosphorylated tyrosine (pTyr) residues through specific phosphobinding modules, many protein kinases selectively recognize pTyr directly adjacent to the tyrosine residue they phosphorylate and catalyze the formation of twin pTyr-pTyr sites. Here, we demonstrate the importance of this phosphopriming-driven twin pTyr signaling in promoting cell cycle progression through the cell cycle-inhibitory protein p27Kip1. We identify, structurally resolve, and tune two distinct molecular determinants driving the selective recognition of pTyr directly N- and C-terminal to the target phospho-acceptor tyrosine site. We further show structural and biochemical conservation in this recognition, and identify cancer-associated alterations to these determinants that are unable to recognize phosphoprimed substrates. Finally, using an in vivo mouse model of leukemia we show that Bcr-Abl mutants unable to recognize phosphoprimed substrates paradoxically result in enhanced tumor development and progression. These data indicate that Bcr-Abl, like other proto-oncogenes such as Ras or Myc, engages both pro- and anti-oncogenic programs - but in the case of Bcr-Abl, this is accomplished through a mechanism involving traditional and phosphoprimed substrate recognition.

biochemistry↗

bioRxiv2025

Multimodal imaging reveals a lysosomal drug reservoir that drives heterogeneous distribution of PARP inhibitors

For all drugs, effective target engagement requires sufficient intracellular concentrations of drug to be reached, but whether tumour heterogeneity impacts drug distribution and efficacy is poorly studied. PARP inhibitors have transformed treatment of high-grade serous ovarian carcinoma (HGSOC), but resistance remains a clinical hurdle in this highly heterogeneous tumour type. We developed a patient-derived explant multi-modal imaging pipeline, which demonstrated that cell-intrinsic PARP inhibitor accumulation is highly variable, both between patients and within tumours. Spatial transcriptomics revealed enrichment of apoptotic and lysosomal signatures in high-drug regions. Rucaparib, an intrinsically fluorescent PARP inhibitor, accumulates heterogeneously at the single-cell level, with rucaparib-high cells demonstrating increased drug response relative to rucaparib low. Mechanistically, lysosomal sequestration creates a rucaparib reservoir that determines drug levels in the nucleus. Perturbation of lysosomal content altered intracellular levels of weak base PARP inhibitors rucaparib and niraparib, but not olaparib. Together these data suggest that lysosomes act as a reservoir for a subset of PARP inhibitor drugs to improve drug response.

cancer biology↗

bioRxiv2025

Inhibition of cytosolic DNA sensing and transposon activity safeguards pluripotency

Transposable elements (TEs) are mobile DNA sequences that make up a sizeable fraction of mammalian genomes yet are often tightly repressed by transcriptional and epigenetic mechanisms. During early development, epigenetic reprogramming selectively loosens TE repression, and TE transcription actively contributes to embryogenesis. This raises the question: how can embryos and embryonic stem cells (ESCs) tolerate TE expression without incurring widespread inflammation or DNA damage? Here, we reveal multiple mechanisms that prevent innate immune activation by TE-derived cytosolic DNA, including reduced cGAS/STING expression and signalling, dampening of Type I interferon responses by pluripotency factors, and post-transcriptional restriction of retrotransposition. These layers of protection are essential, as experimental perturbation triggers loss of ESC self-renewal and pluripotency. Our data explain how early development can be permissive to TE expression while safeguarding against harmful effects of TE activity.

developmental biology↗

bioRxiv2024

Pro-survival roles for p21(Cip1/Waf1) in Non-Small Cell Lung Cancer

Quiescence is a reversible state of proliferative arrest, distinct from senescence. While cancer is a disease of dysregulated proliferation, cancer cells can retain the ability to enter quiescence which confers advantages to tumour cells by protecting them from chemotherapy or by allowing metastasis to distant sites. Multiple mechanisms exist to induce and maintain quiescence that are not yet fully understood. Here, we show that high expression of the CDK inhibitor p21Cip1/Waf1 correlates with a poor prognosis in TP53 wild-type, but not TP53 mutant, non-small cell lung cancer (NSCLC) patients. Using quantitative single-cell imaging of genetically-engineered NSCLC reporter cell lines, we show that TP53 wild-type NSCLC cells can enter a p21-dependent spontaneous quiescent state, downstream of replication stress. Furthermore, p21 expression confers survival advantages to TP53 wild-type NSCLC cells, both under normal proliferation and in response to chemotherapy. We also show that p21 can promote tumour relapse by allowing cells to recover from both G1 and G2 arrest states after drug removal. Together, our data suggest that targeting p21 function in TP53 wild-type tumours could lead to better outcomes for chemotherapy treatment in NSCLC patients. Statement of SignificanceWe show that TP53WT Non-Small Cell Lung Cancer cells can enter a p21-dependent spontaneous quiescent state and that p21 maintains the viability of NSCLC cells, is chemoprotective and can promote tumour relapse.

cancer biology↗

bioRxiv2023

The oscillation of mitotic kinase governs cell cycle latches in mammalian cells

The mammalian cell cycle alternates between two phases: S-G2-M with high levels of A- and B-type cyclin-dependent kinases (CycA,B:CDK); and G1 with persistent degradation of CycA,B by Cdh1-activated APC/C (anaphase promoting complex/cyclosome). Because CDKs phosphorylate and inactivate Cdh1, these two phases are mutually exclusive. This toggle switch is flipped from G1 to S by cyclin-E (CycE:CDK), which is not degraded by Cdh1:APC/C; and from M to G1 by Cdc20:APC/C, which is not inactivated by CycA,B:CDK. After flipping the switch, cyclin E is degraded and Cdc20:APC/C 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 one mechanistic explanation for how whole genome doubling can arise, a common event in tumorigenesis that can drive tumour evolution.

systems biology↗

bioRxiv2022

Cell overgrowth during G1 arrest triggers an osmotic stress response and chronic p38 activation to promote cell cycle exit

Cell size and the cell cycle are intrinsically coupled and abnormal increases in cell size are associated with senescence. The mechanism by which overgrowth primes cells to exit the cell cycle remains unclear. We investigate this using CDK4/6 inhibitors that arrest cell cycle progression in G0/G1 and are used to treat ER+/HER2-metastatic breast cancer. We demonstrate that long-term CDK4/6 inhibition promotes cellular overgrowth during the G0/G1 arrest, causing widespread proteome remodeling and p38-p53-p21-dependent cell cycle exit. Cell cycle exit is triggered by two waves of p21 induction. First, overgrowth during a G0/G1 arrest induces an osmotic stress response, producing the first wave of p21 induction. Second, when CDK4/6 inhibitors are removed, a fraction of cells escape G0/G1 arrest and enter S-phase where overgrowth-driven replication stress results in a second wave of p21 induction that causes cell cycle withdrawal from G2, or the subsequent G1. This could explain why cellular hypertrophy is associated with senescence and why CDK4/6 inhibitors have long-lasting anti-proliferative effects in patients.

cell biology↗

bioRxiv2022

Patterns of interdivision time correlations reveal hidden cell cycle factors

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.

systems biology↗

bioRxiv2021

Genomic hallmarks of cellular dormancy in cancer and therapeutic implications

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. We developed methodology to robustly identify this state from transcriptomic signals and characterised 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/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, and propose a G0 arrest transcriptional signature that is linked with therapeutic resistance and can be used to further study and clinically track this state.

cancer biology↗

bioRxiv2021

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.

cell biology↗

bioRxiv2021

Conserved Cdk inhibitors show unique structural responses to tyrosine phosphorylation

Balanced proliferation-quiescence decisions are vital during normal development and in tissue homeostasis and their dysregulation underlies tumorigenesis. Entry into proliferative cycles is driven by Cyclin/Cyclin-dependent kinases (Cdks). Conserved Cdk inhibitors (CKIs), p21Cip1/Waf1, p27Kip1 and p57Kip2, bind to Cyclin/Cdks and inhibit Cdk activity. p27 tyrosine phosphorylation, in response to mitogenic signalling, promotes activation of CyclinD/Cdk4 and CyclinA/Cdk2. Tyrosine phosphorylation is conserved in p21 and p57, although the number of sites differs. We use molecular dynamics simulations to compare the structural changes in Cyclin/Cdk/CKI trimers induced by single and multiple tyrosine phosphorylation in CKIs and their impact on CyclinD/Cdk4 and CyclinA/Cdk2 activity. Despite shared structural features, CKI binding induces distinct structural responses in Cyclin/Cdks and the predicted effects of CKI tyrosine phosphorylation on Cdk activity are not conserved across CKIs. Our analyses suggest how CKIs may have evolved to be sensitive to different inputs to give context-dependent control of Cdk activity.

cell biology↗

bioRxiv2019

A high-content RNAi screen reveals multiple roles for long noncoding RNAs in cell division

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, linc00899, was selected for in-depth studies due to the robust mitotic delay observed upon its depletion. Transcriptome analysis of linc00899-depleted cells together with gain-of-function and rescue experiments across multiple cell types identified the neuronal microtubule-binding protein, TPPP/p25, as a target of linc00899. Linc00899 binds the genomic locus of TPPP/p25 and suppresses its transcription through a cis-acting mechanism. In cells depleted of linc00899, the consequent upregulation of TPPP/p25 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.

cell biology↗

bioRxiv2018

Cyclin A triggers Mitosis either via Greatwall or Cyclin B

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.

cell biology↗