Biology subjects

Saurin, A. T.

Publications and source records attributed to Saurin, A. T..

16 recordsLinked to original sources

bioRxiv2025

A perfusion-based model to explain how paclitaxel achieves tumour-selective killing

Paclitaxel (taxol) is a commonly used chemotherapeutic that stabilizes microtubules to inhibit chromosome segregation during mitosis. Although it is used effectively to treat a wide variety of solid tumour types, it also impairs healthy cell proliferation leading to severe dose-limiting toxicities. Newer anti-mitotic drugs have been developed but these have so far failed to offer the same clinical benefit as paclitaxel, which begs the question of why this drug targets tumour cells so effectively? Here we develop a mathematical model of paclitaxel penetration and retention within 3D tumour environments following periodic drug-on/drug-off regimes typically used in the clinic. Our model suggests that during the drug-free periods, dense poorly-perfused tissue can retain paclitaxel for much longer than well-perfused tissue. This is due to paclitaxels ability to bind strongly to microtubules, which causes slower drug-release from densely packed tissue. Assuming that tumour cells are generally dense and less perfused than proliferative healthy tissues, this simple model suggests that tumour-selective killing could be achieved later in each chemotherapy cycle when the drug has otherwise cleared the healthy cell compartments. We use our model to optimize dosing regimens to allow paclitaxel to selectively kill tumour spheroids of different size, whilst sparing well-perfused healthy cells. Together, our model suggests paclitaxel could target key distinguishing features of many solid tumours: their size, 3D geometry and perfusion status. It is important to validate these predictions in cell models because, if correct, they could be harnessed to optimize paclitaxel use, to predict and enhance tumour responsiveness, and to develop newer drugs that are preferentially retained for longer within solid tumours.

cancer biology↗

bioRxiv2025

The mitotic stopwatch limits the proliferation of whole genome doubled cells

AO_SCPLOWBSTRACTC_SCPLOWWhole genome doubling (WGD) is a frequent event in tumourigenesis that promotes chromosomal instability and tumour evolution. WGD is sensed indirectly due to the presence of extra centrosomes, which activate the PIDDosome to induce a p53-dependent G1-arrest. Here we uncouple WGD from centrosome amplification and show that p53 still arrests tetraploid cells, but via the mitotic stopwatch; a p53/53BP1/USP28-dependent pathway that causes G1-arrest following an extended mitotic delay. Mitotic timing is unaffected by WGD, but the threshold mitotic delay needed to invoke a G1-arrest is reduced. This sensitivity to the stopwatch mechanism is not associated with altered levels of mitotic stopwatch components, but instead, is associated with enhanced p21 concentrations prior to mitosis. Similar effects are observed in diploid cells treated with CDK4/6 inhibitors to double their size, implicating G1 delays and cell size as key determinants of stopwatch sensitivity. This ability of the mitotic stopwatch to arrest proliferation after increases to genome or cell size has important implications for the progression and treatment of cancer. It also demonstrates that the stopwatch pathway can sense more than just mitotic delays.

cell biology↗

bioRxiv2025

Cell enlargement causes mitotic errors and aneuploidy in cells that evade senescence after CDK4/6 inhibition

AO_SCPLOWBSTRACTC_SCPLOWCDK4/6 inhibitors (CDK4/6i) arrest the cell cycle in G1 leading to cellular overgrowth and p53-dependent senescence. They are used to treat metastatic HR+/HER2-breast cancer, but resistance is common, and this has been associated with TP53 loss and senescence evasion. We show here that enlarged CDK4/6i-treated cells that evade senescence mis-segregate chromosomes due to defective chromosomal alignment and a weakened mitotic checkpoint, leading to aneuploidy and DNA damage. The chromosome alignment errors are associated with impaired Sgo1 localisation to centromeres and defective sister chromatin cohesion during mitosis. Importantly, all these mitotic defects can be rescued by constraining cell size during the CDK4/6i-treatment, and specifically restoring cohesion rescues the chromosome segregation errors. Together, this demonstrates mechanistically how cell enlargement drives genetic and karyotypic change in cells that re-enter the cell cycle following CDK4/6 inhibition. This could help fuel the rapid emergence of chemotherapy-resistant clones, especially in p53-null cells that evade senescence to drive drug-resistance in patients.

cancer biology↗

bioRxiv2025

PHD1-dependent hydroxylation of RepoMan (CDCA2) on P604 modulates the control of mitotic progression

Prolyl-hydroxylases (PHDs) are oxygen sensing enzymes that mediate the hydroxylation of proline residues. In mammals, three PHD isoforms (PHD1-3) are responsible for proline hydroxylation of Hypoxia Inducible Factor (HIF) alpha, a key regulator of the hypoxia response. In the accompanying paper (Jiang et. al., 2025) we report development of a mass spectrometry-based method to reliably identify proline hydroxylation (OH-Pro) sites on proteins and use this to identify a PHD-dependent OH-Pro modification at Pro604 on the protein RepoMan (CDCA2), a regulatory subunit for protein phosphatase PP1{gamma}, with important roles in mitotic progression and cell viability. Here, we investigate the functional significance of hydroxylation of RepoMan at P604. During M phase, the PP1-RepoMan complex dephosphorylates Thr3 of Histone H3 (H3T3) on chromosomes arms to ensure the correct localisation of the chromosomal passenger complex (CPC) at centromeres. We show that siRNA depletion of PHD1, but not PHD2, increases H3T3 phosphorylation in prometaphase-arrested cells. In cells depleted of endogenous RepoMan, exogenous expression of wild type RepoMan, but not a RepoMan P604A mutant, restored normal H3T3 phosphorylation localisation in prometaphase arrested cells. RepoMan P604 is located proximal to the Short Linear Motifs (SLiMs) that function as binding sites for the serine/threonine Protein Phosphatase 2A (PP2A). The interaction of RepoMan and PP2A-B56{gamma} is reduced in cells expressing RepoMan P604A. Moreover, analyses in both fixed and live cells released from a prometaphase arrest, show that expression of the RepoMan P604A mutant delays completion of mitosis, results in defects in chromosome alignment and segregation and increases levels of cell death. These data support a role for PHD1-mediated prolyl hydroxylation in controlling progression through mitosis, acting, at least in part, via hydroxylation of RepoMan at P604 regulating the interaction of RepoMan with PP2A during chromosome alignment and thereby controlling the levels of Histone H3 phosphorylation at Thr3.

cell biology↗

bioRxiv2024

A chemical-genetic system to rapidly inhibit the PP2A-B56 phosphatase

AO_SCPLOWBSTRACTC_SCPLOWSerine-threonine phosphatases have been challenging to study because of the lack of specific inhibitors. Their catalytic domains are druggable, but these are shared or very similar between individual phosphatase complexes, precluding their specific inhibition. Instead, phosphatase complexes achieve specificity by interacting with short-linear motifs (SLiMs) in substrates or their binding partners. We develop here a chemical-genetic system to rapidly inhibit these interactions within the PP2A-B56 family. Drug-inducible recruitment of ectopic SLiMs ("directSLiMs") is used to rapidly block the SLiM-binding pocket on the B56 regulatory subunit, thereby displacing endogenous interactors and inhibiting PP2A-B56 activity within seconds. We use this system to characterise PP2A-B56 substrates during mitosis and to identify a novel role for PP2A-B56 in maintaining kinetochore-microtubule attachments at metaphase. The directSLiMs approach can be used to inhibit any other phosphatase, enzyme or protein that uses a critical SLiM-binding interface, providing a powerful strategy to inhibit and characterise proteins once considered ."undruggable".

cell biology↗

bioRxiv2024

Elraglusib induces cytotoxicity via microtubule destabilisation independently of GSK3 inhibition

AO_SCPLOWBSTRACTC_SCPLOWElraglusib (9-ING-41) is an ATP-competitive inhibitor of glycogen synthase kinase-3 (GSK3) with pre-clinical studies demonstrating broad activity against many tumour types. Promising early-phase clinical trial data led to FDA orphan drug status, and a randomized phase 2 study in combination with cytotoxic chemotherapy in pancreatic cancer has recently completed its recruitment. Similarly, single-agent responses in adult T-cell leukaemia/lymphoma and melanoma, and combination treatment data in several other tumour types have been encouraging. The elraglusib mechanism of action is unknown, but it is unlikely to act through GSK3 inhibition because cytotoxicity is observed below the IC50 for GSK3 and other small molecule GSK3 inhibitors do not produce cytotoxic effects, at least in lymphoma cells. We show here that elraglusib perturbs chromosomal alignment to cause a mitotic arrest in multiple tumour lines. This arrest is caused by direct microtubule depolymerisation, which prevents the attachment of kinetochores to microtubules. At clinically relevant doses, these mitotically arrested cells eventually undergo mitotic slippage, leading to gross chromosome missegregation, DNA damage and apoptosis. These effects explain the cytotoxicity of elraglusib because temporarily pausing cell cycle progression with the CDK4/6 inhibitor palbociclib abolishes any drug-induced genotoxicity and apoptosis. In summary, elraglusib acts as a potent direct microtubule destabilizer both in vitro and across multiple cancer types, resulting in mitotic arrest, DNA damage and apoptosis. These effects likely account for its broad pan-cancer activity, which does not rely upon GSK3 inhibition as they are not replicated by other GSK3 inhibitors.

cancer biology↗

bioRxiv2023

The search for CDK4/6 inhibitor biomarkers has been hampered by inappropriate proliferation assays

CDK4/6 inhibitors arrest the cell cycle in G1 and are used in combination with hormone therapy to treat advanced HR+/HER- breast cancer. To allow more effective use of these drugs in breast cancer, and to facilitate their use in other tumour types, biomarkers that can predict response are urgently needed. We demonstrate here that previous large-scale screens designed to identify the most sensitive tumour types and genotypes have misrepresented the responsive cell lines because of a reliance on ATP-based proliferation assays. When cells arrest in G1 following CDK4/6 inhibition, they continue to grow in size, producing more mitochondria and ATP. This cellular overgrowth masks an efficient arrest using metabolic ATP-based assays, but not if DNA-based assays are used instead. By comparing tumour cells using different assay types, we demonstrate that the lymphoma lines previously identified as the most responsive cell types, simply appear to respond the best because they fail to overgrow during the G1 arrest. Similarly, the CDK4/6 inhibitor abemaciclib appears to inhibit proliferation better than palbociclib, but this is because it also inhibits cell overgrowth through off-target effects. DepMap analysis of previous screening data using only the reliable assay types, demonstrates that palbociclib-sensitivity is associated with sensitivity to Cyclin D1, CDK4 and CDK6 knockout/knockdown, and resistance is associated with sensitivity to Cyclin E1, CDK2 and SKP2 knockout/knockdown. Furthermore, potential biomarkers of palbociclib-sensitivity are increased expression of Cyclin D1 (CCND1) and RB1, and reduced expression of Cyclin E1 (CCNE1) and CDKN2A. None of these associations are present when analysing DepMap using similar data from metabolic assays. This reinforces the importance of new screens to assess CDK4/6 inhibitors, and potentially other anti-cancer drugs, against a wide range of cell types using an appropriate proliferation assay. This would help to better inform clinical trials and to identify much needed biomarkers of response.

cancer 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

Oncogenic signals prime cancer cells for toxic cell growth during a G1 cell cycle arrest

A long-term goal in cancer research has been to inhibit the cell cycle in tumour cells without causing toxicity in proliferative healthy tissues. The best evidence that this is achievable is provided by CDK4/6 inhibitors, which arrest the cell cycle in G1, are well-tolerated in patients, and are effective in treating ER+/HER2-breast cancer. CDK4/6 inhibitors are effective because they arrest tumour cells more efficiently than some healthy cell types and, in addition, they affect the tumour microenvironment to enhance anti-tumour immunity. We demonstrate here another reason to explain their efficacy. Tumour cells are specifically vulnerable to CDK4/6 inhibition because during the G1 arrest, oncogenic signals drive toxic cell overgrowth. This overgrowth causes permanent cell cycle withdrawal by either preventing exit from G1 or by inducing replication stress and genotoxic damage during the subsequent S-phase and mitosis. Inhibiting or reverting oncogenic signals that converge onto mTOR can rescue this excessive growth, DNA damage and cell cycle exit in cancer cells. Conversely, inducing oncogenic signals in non-transformed cells can drive these toxic phenotypes and sensitize cells to CDK4/6 inhibition. Together, this demonstrates how oncogenic signals that have evolved to stimulate constitutive tumour growth and proliferation can be driven to cause toxic cell growth and irreversible cell cycle exit when proliferation is halted in G1.

cancer biology↗

bioRxiv2022

A bifunctional kinase-phosphatase module integrates mitotic checkpoint and error-correction signalling to ensure mitotic fidelity

Two major mechanisms have evolved to safeguard genome stability during mitosis: the mitotic checkpoint delays mitosis until all chromosomes have attached to microtubules, and the kinetochore-microtubule error-correction pathway keeps this attachment process free from errors. We demonstrate here that the optimal strength and dynamics of both processes is set by a kinase-phosphatase pair (PLK1-PP2A) that engage in negative feedback on the BUB complex. Uncoupling this homeostatic feedback to skew the balance towards PLK1 produces a strong checkpoint, weak microtubule attachments, and mitotic delays. Conversely, skewing the balance towards PP2A causes a weak checkpoint, strong microtubule attachments, and chromosome segregation errors. The number of MELT motifs on the KNL1 signalling scaffold sets the optimal levels of each enzyme, because engineering KNL1 to recruit too many BUB complexes increases KNL1-PLK1/PP2A levels, and enhances checkpoint/microtubule attachment strength. In contrast, recruiting too few BUB complexes lowers KNL1-PLK1/PP2A, and decreases checkpoint/microtubule attachment strength. Both of these situations are associated with chromosome segregation errors. Together, these data demonstrate how a single bifunctional kinase-phosphatase module integrates two major mitotic processes to help preserve genome stability.

cell biology↗

bioRxiv2021

CDK4/6 inhibitors induce replication stress to cause long-term cell cycle withdrawal

AO_SCPLOWBSTRACTC_SCPLOWCDK4/6 inhibitors arrest the cell cycle in G1-phase. They are approved to treat breast cancer and are also undergoing clinical trials against a range of other tumour types. To facilitate these efforts, it is important to understand why a cytostatic arrest in G1 causes long-lasting effects on tumour growth. Here we demonstrate that a prolonged G1-arrest following CDK4/6 inhibition downregulates replisome components and impairs origin licencing. This causes a failure in DNA replication after release from that arrest, resulting in a p53-dependent withdrawal from the cell cycle. If p53 is absent, then cells bypass the G2-checkpoint and undergo a catastrophic mitosis resulting in excessive DNA damage. These data therefore link CDK4/6 inhibition to genotoxic stress; a phenotype that is shared by most other broad-spectrum anti-cancer drugs. This provides a rationale to predict responsive tumour types and effective combination therapies, as demonstrated by the fact that CDK4/6 inhibition induces sensitivity to chemotherapeutics that also cause replication stress.

cancer biology↗

bioRxiv2019

A spatio-temporal mathematical model for the dynamics of paclitaxel in cell monolayers.

The authors have withdrawn their manuscript due to the discovery of fundamental issues with the implementation of the ODE/PDE model, whose output was shown in Figure 5 of the original version of this paper, rendering the results and conclusion unreliable. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

cancer biology↗

bioRxiv2019

Kinetochore phosphatases suppress autonomous kinase activity to control the spindle assembly checkpoint

AO_SCPLOWBSTRACTC_SCPLOWLocal phosphatase regulation is critical for determining when phosphorylation signals are activated or deactivated. A typical example is the spindle assembly checkpoint (SAC) during mitosis, which regulates kinetochore PP1 and PP2A-B56 activities to switch-off signalling events at the correct time. In this case, kinetochore phosphatase activation dephosphorylates MELT motifs on KNL1 to remove SAC proteins, including the BUB complex. We show here that, surprisingly, neither PP1 or PP2A are required to dephosphorylate the MELT motifs. Instead, they remove polo-like kinase 1 (PLK1) from the BUB complex, which can otherwise maintain MELT phosphorylation in an autocatalytic manner. This is their principle role in the SAC, because both phosphatases become redundant if PLK1 is inhibited or BUB-PLK1 interaction is prevented. Therefore, phosphatase regulation is critical for the SAC, but primarily to restrain and extinguish autonomous kinase activity. We propose that these circuits have evolved to generate a semi-autonomous SAC signal that can be synchronously silenced following kinetochore-microtubule tension.

cell biology↗

bioRxiv2019

Cyclin B1 scaffolds MAD1 at the corona to activate the spindle assembly checkpoint

The Cyclin B:CDK1 kinase complex is the master regulator of mitosis that phosphorylates hundreds of proteins to coordinate mitotic progression. We show here that, in addition to these kinase functions, Cyclin B also scaffolds a localised signalling pathway to help preserve genome stability. Cyclin B1 localises to an expanded region of the outer kinetochore, known as the corona, where it scaffolds the spindle assembly checkpoint (SAC) machinery by binding directly to MAD1. In vitro reconstitutions map the key binding interface to a few acidic residues in the N-terminus of MAD1, and point mutations in this region remove corona MAD1 and weaken the SAC. Therefore, Cyclin B1 is the long-sought-after scaffold that links MAD1 to the corona and this specific pool of MAD1 is needed to generate a robust SAC response. Robustness, in this context, arises because Cyclin B1-MAD1 localisation becomes MPS1-independent after the corona has been established. We demonstrate that this allows corona-MAD1 to persist at kinetochores when MPS1 activity falls, ensuring that it can still be phosphorylated on a key C-terminal catalytic site by MPS1. Therefore, this study explains how corona MAD1 generates a robust SAC signal and why stripping of this pool by dynein is essential for SAC silencing. It also reveals that the key mitotic kinase, Cyclin B1-Cdk1, scaffolds the pathway that inhibits its own degradation.

cell biology↗

bioRxiv2019

PP1 and PP2A use opposite phospho-dependencies to control distinct processes at the kinetochore

PP1 and PP2A-B56 are major serine/threonine phosphatase families that achieve specificity by colocalising with substrates. At the kinetochore, however, both phosphatases localise to an almost identical molecular space and yet they still manage to regulate unique pathways and processes. By switching or modulating the positions of PP1/PP2A-B56 at kinetochores, we show that their unique downstream effects are not due to either the identity of the phosphatase or its precise location. Instead, these phosphatases signal differently because their kinetochore recruitment can be either inhibited (PP1) or enhanced (PP2A) by phosphorylation inputs. Mathematical modelling explains how these inverse phospho-dependencies elicit unique forms of cross-regulation and feedback, which allows otherwise indistinguishable phosphatases to produce distinct network behaviours and control different mitotic processes. Therefore, the kinetochore uses PP1 and PP2A-B56 because their binding motifs respond to kinase inputs in opposite ways. Genome-wide motif analysis suggests that many other pathways also select for these same key features, implying that these similar catalytic enzymes may have diverged during evolution to allow opposite modes of phospho-regulation.

cell biology↗

bioRxiv2018

A molecular explanation for PP2A-B56 isoform specificity during mitosis

PP2A-B56 is a serine/threonine phosphatase complex that regulates several major mitotic processes, including sister chromatid cohesion, kinetochore-microtubule attachment and the spindle assembly checkpoint. We show here that these key functions are shared between B56 isoforms which localise differentially to either the centromere or kinetochore. The centromeric B56 isoforms rely on a specific interaction with Sgo2, whereas the kinetochore isoforms bind preferentially to BubR1 and other proteins containing an LxxIxE motif. The molecular basis for this differential localisation can be mapped to a small C-terminal region in B56 that regulates these key interactions and defines whether PP2A-B56 complexes bind to either the centromere or kinetochore. Together, this study describes how different PP2A-B56 complexes utilise isoform-specific interactions to control distinct processes during mitosis.

cell biology↗