Biology subjects

Stucki, M.

Publications and source records attributed to Stucki, M..

9 recordsLinked to original sources

bioRxiv2026

Chromatin adaptors and TOPBP1 condensates cooperate to organize ATM signaling

DNA damage response proteins frequently accumulate in biomolecular condensates, yet how these structures cooperate with classical adaptor-mediated recruitment mechanisms to organize DNA damage signaling remains poorly understood. Here, we identify Treacle and the ATM adaptor NBS1 as prominent components of TOPBP1 condensates and show that these structures activate ATM signaling in the absence of DNA damage. At rDNA breaks, Treacle recruits NBS1 and TOPBP1 through genetically separable interaction modules. Acute protein degradation further revealed that Treacle is required for condensate assembly, whereas TOPBP1 remains continuously required for condensate maintenance. Finally, we show that efficient ATM accumulation at IR-induced DNA double-strand breaks similarly depends on both NBS1 and TOPBP1, indicating that this two-component mechanism is not restricted to nucleolar DNA damage. Together, our findings support a two-component model in which adaptor proteins provide molecular specificity, whereas TOPBP1 condensates create the spatial organization required for robust ATM signaling.

cell biology↗

bioRxiv2025

CIP2A Mediates the Recruitment of the SLX4-MUS81-XPF Tri-Nuclease Complex in Mitosis and Protects Against Replication Stress

DNA replication stress frequently elicits problems in mitosis because incompletely replicated chromosomes or replication intermediates physically link sister chromatids together and prevent their proper segregation during anaphase. We and others recently discovered a mitotic role for the CIP2A oncoprotein, which is critically implicated in chromosome stability maintenance and chromosome fragment clustering during mitosis. In addition, in homologous recombination deficient (HRD) cells, CIP2A is essential and may thus constitute a new drug target in HRD cancers. Yet, the precise mechanisms by which CIP2A suppresses chromosomal instability during mitosis and thus allows for the survival of HRD cancer cells remain largely elusive. Here we characterize CIP2As role in DNA replication stress responses. We show that upon replication stress, wild-type cells show an elevated accumulation of CIP2A foci during mitosis, indicating its involvement in the mitotic response to replication stress. Defective DNA replication leads to the accumulation of under-replicated DNA, which can be carried into mitosis. We demonstrate that in the absence of CIP2A, cells fail to recruit the SLX4-MUS81-XPF (SMX) tri-nuclease complex to sites of under-replicated DNA in mitosis, resulting in a high incidence of lagging chromosomes during anaphase and subsequent micronuclei formation. In a subset of cell lines, we also observed CIP2A-dependent mitotic DNA synthesis (MiDAS) upon replication stress. However, our data suggest that MiDAS and SMX recruitment are not functionally linked. This novel role of CIP2A in managing under-replicated DNA may provide insights into the molecular mechanisms underlying therapeutic vulnerabilities in cancer cells.

cell biology↗

bioRxiv2025

ARID1A stabilises non-homologous end joining factors at DNA breaks induced by the G4 ligand pyridostatin

ARID1A is a subunit of the BAF chromatin remodelling complex that is frequently mutated in cancer. It is challenging to predict how ARID1A loss impacts cancer therapy response because it participates in many different cellular pathways. G quadruplex (G4) binding ligands, such as pyridostatin, have shown anticancer effects, but the pathways and genetic determinants involved in the response to G4 ligands are still not fully understood. Here, we show that ARID1A deficient cells are selectively sensitive to pyridostatin when compared with isogenic controls. Sensitivity to pyridostatin was apparent in ovarian and colorectal cancer cell line models, and in vivo studies suggest that G4 ligands hold promise for treating ARID1A deficient cancers. While we find that ARID1A impacts on pyridostatin-induced transcriptional responses, we find that pyridostatin-mediated toxicity in ARID1A-deficient cells is driven by defective DNA repair of topoisomerase-induced breaks. We show that ARID1A-deficient cells are unable to efficiently accumulate non-homologous end joining proteins on chromatin following pyridostatin exposure. These data uncover a role for ARID1A in the cellular response to G4 ligands, and link remodelling to G4 ligand-induced transcriptional and DNA damage responses.

molecular biology↗

bioRxiv2025

Treacle and MDC1 coordinate rDNA break repair by homologous recombination

The repair of DNA double-strand breaks in repetitive sequences is challenging because the abundance of potential templates for homology-directed repair (HDR) increases the risk of ectopic recombination and chromosome rearrangements. Relocalization of repair sites in repetitive sequences to a safe location prior to RAD51 loading has been observed in various organisms and is thought to limit aberrant recombination events. Here, we characterize this process in ribosomal DNA (rDNA) repeats, which reside within the nucleoli, specialized nuclear compartments dedicated to ribosome biogenesis. Induction of DSBs in rDNA triggers large-scale mobilization of damaged repeats from the nucleolar interior to the nucleolar periphery, where repair occurs. We show that this process is coordinated by the adaptor proteins Treacle and MDC1, which act sequentially to control nucleolar reorganization and repair factor recruitment. Treacle promotes rDNA mobilization through its role in nucleolar DNA damage signalling, whereas MDC1 acts downstream of nucleolar segregation to facilitate the recruitment of HDR factors at nucleolar caps. Following establishment of a {gamma}H2AX chromatin domain, MDC1-dependent RNF8-mediated chromatin ubiquitylation promotes BRCA1 recruitment via both the RNF168-dependent pathway and the RAP80-ABRAXAS complex. This in turn enables the sequential accumulation of PALB2 and RAD51 at nucleolar caps. Together, our findings demonstrate that RAD51 loading at rDNA breaks is tightly coupled to nucleolar reorganization and is mediated by dual RNF8-dependent signalling pathways, thereby ensuring that HDR is spatially restricted to the nucleolar periphery and reducing the risk of ectopic recombination between repetitive sequences.

cell biology↗

bioRxiv2024

MDC1 mediates Pellino recruitment to sites of DNA double-strand breaks

Ubiquitylation is critically implicated in the recognition and repair of DNA double-strand breaks. The adaptor protein MDC1 mediates the recruitment of the key DNA damage responsive E3 ubiquitin ligase RNF8 to the break sites. It does so by directly interacting with RNF8 in a phosphorylation-dependent manner that involves the RNF8 FHA domain, thus initiating targeted chromatin ubiquitylation at the break sites. Here, we report that MDC1 also directly binds to two additional E3 ubiquitin ligases, Pellino1 and 2, which were recently implicated in the DNA damage response. Through a combination of biochemical, biophysical and X-ray crystallographic approaches, we reveal the molecular details of the MDC1-Pellino complexes. Furthermore, we show that in mammalian cells, MDC1 mediates Pellino recruitment to sites of DNA double-strand breaks by a direct phosphorylation-dependent interaction between the two proteins. Taken together, our findings provide new molecular insights into the ubiquitylation pathways that govern genome stability maintenance.

cell biology↗

bioRxiv2023

Innate immune receptor C5aR1 regulates cancer cell fate and can be targeted to improve radiotherapy in tumours with immunosuppressive microenvironments

An immunosuppressive microenvironment causes poor tumour T-cell infiltration and is associated with reduced patient overall survival in colorectal cancer. How to improve treatment responses in these tumours is still a challenge. Using an integrated screening approach to identify cancer-specific vulnerabilities, we identify complement receptor C5aR1 as a druggable target which when inhibited improves radiotherapy even in tumours displaying immunosuppressive features and poor CD8+ T-cell infiltration. While C5aR1 is well-known for its role in the immune compartment, we find that C5aR1 is also robustly expressed on malignant epithelial cells, highlighting potential tumour-cell specific functions. C5aR1 targeting results in increased NF-{kappa}B-dependent apoptosis specifically in tumours and not normal tissues; indicating that in malignant cells, C5aR1 primarily regulates cell fate. Collectively, these data reveal that increased complement gene expression is part of the stress response mounted by irradiated tumours and that targeting C5aR1 can improve radiotherapy even in tumours displaying immunosuppressive features.

cancer biology↗

bioRxiv2021

CIP2A is a prime synthetic-lethal target for BRCA-mutated cancers

BRCA1/2-mutated cancer cells must adapt to the genome instability caused by their deficiency in homologous recombination. Identifying and targeting these adaptive mechanisms may provide new therapeutic strategies. Here we present the results of genome-scale CRISPR/Cas9-based synthetic lethality screens in isogenic pairs of BRCA1- and BRCA2-deficient cells that identified the gene encoding CIP2A as essential in a wide range of BRCA1- and BRCA2-mutated cells. Unlike PARP inhibition, CIP2A-deficiency does not cause accumulation of replication-associated DNA lesions that require homologous recombination for their repair. CIP2A is cytoplasmic in interphase but, in mitosis, accumulates at DNA lesions as part of a complex with TOPBP1, a multifunctional genome stability factor. In BRCA-deficient cells, the CIP2A-TOPBP1 complex prevents lethal mis-segregation of acentric chromosomes that arises from impaired DNA synthesis. Finally, physical disruption of the CIP2A-TOPBP1 complex is highly deleterious in BRCA-deficient cells and tumors, indicating that targeting this mitotic chromosome stability process represents an attractive synthetic-lethal therapeutic strategy for BRCA1- and BRCA2-mutated cancers.

cancer biology↗

bioRxiv2021

The CIP2A-TOPBP1 complex safeguards chromosomal stability during mitosis

The accurate repair of DNA double-strand breaks (DSBs), highly toxic DNA lesions, is crucial for genome integrity and is tightly regulated during the cell cycle. In mitosis, cells inactivate DSB repair in favor of a tethering mechanism that stabilizes broken chromosomes until they are repaired in the subsequent cell cycle phases. How this is achieved mechanistically is not yet understood, but the adaptor protein TOPBP1 is critically implicated in this process. Here, we identify CIP2A as a TOPBP1-interacting protein that regulates TOPBP1 localization specifically in mitosis. Cells lacking CIP2A display increased micronuclei formation, DSB repair defects and chromosomal instability. CIP2A is actively exported from the cell nucleus in interphase but, upon nuclear envelope breakdown at the onset of mitosis, gains access to chromatin where it forms a complex with MDC1 and TOPBP1 to promote TOPBP1 recruitment to sites of mitotic DSBs. Collectively, our data uncover CIP2A-TOPBP1 as a mitosis-specific genome maintenance complex.

cell biology↗

bioRxiv2020

Targeting C5aR1 Increases the Therapeutic Window of Radiotherapy

Engaging innate immune pathways is emerging as a productive way of achieving durable anti-tumor responses. However, systemic administration of these therapies can result in toxicity, deemed to be particularly problematic when combined with current standard-of-care cytotoxic treatments such as radiotherapy. Increasing the therapeutic window of radiotherapy may be achieved by using targeted therapies, however, few pre-clinical studies investigate both tumor and normal tissue responses in detail. Here we show that targeting innate immune receptor C5aR1 improves tumor radiation response while reducing radiation-induced normal tissue toxicity, thereby increasing the therapeutic window. Genetically or pharmacologically targeting C5aR1 increases both IL-10 expression in the small intestine and IL-10 secretion by tumor cells. Increased IL-10 attenuates RelA phosphorylation and increases apoptosis in tumor cells, leading to improved radiation responses in murine models. Of note, these radiosensitizing effects are tumor-specific since, in the gastrointestinal tract, targeting C5aR1 instead results in decreased crypt cell apoptosis reduced signs of histological damage and improved survival following total abdominal irradiation in mice. Furthermore, the potent and orally active C5aR1 inhibitor, PMX205, improves tumor radiation responses even in a context of reduced/absent CD8+ T cell infiltration. These data indicate that PMX205 can modulate cancer-cell intrinsic functions to potentiate anti-tumor radiation responses even in tumors displaying features of T-cell deficiency or exclusion. Finally, using a preclinical murine model allowing the simultaneous assessment of tumor and normal tissue radiation responses, we show that PMX205 treatment reduces histological and functional markers of small-bowel toxicity while affording a positive tumor response. Our data, therefore, suggest that targeting C5aR1 could be a promising approach for increasing the therapeutic window of radiotherapy.

cancer biology↗