Jihane Basbous

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.

Replication stress triggers extensive remodelling of nuclear architecture to safeguard genome stability, but how this reorganization is coordinated remains unclear. Here we identify TopBP1 condensates as central regulators of nuclear actin remodelling during this response. Sustained replication stress promotes TopBP1 condensate formation in S phase at both the nuclear interior and the nuclear periphery. We show that these condensates drive nuclear F-actin assembly through ATR-mTOR-N-WASP-dependent nucleation, while separately stabilizing actin filaments through an mTOR-dependent, ATR-independent mechanism involving cofilin inhibition. Nuclear F-actin, in turn, is required for proper TopBP1 condensate positioning, revealing a reciprocal relationship between actin network organization and condensate localization. At the nuclear periphery, TopBP1 condensates further require Lamin B1 and stabilize Emerin, coupling nuclear envelope integrity to the spatial organization of DNA repair. Notably, this TopBP1-dependent nuclear actin programme is enhanced in oxaliplatin-resistant colorectal cancer cells, where it promotes survival under chemotherapy. Our findings establish TopBP1 condensates as key architectural hubs that integrate replication stress signaling with nuclear actin dynamics and envelope organization, and identify TopBP1 condensation as a potential therapeutic target in chemo-resistant cancer.

In cancer cells, ATR signaling is crucial to tolerate the intrinsically high damage levels that normally block replication fork progression. Assembly of TopBP1, a multifunctional scaffolding protein, into condensates is required to amplify ATR kinase activity to the levels needed to coordinate the DNA damage response and manage DNA replication stress. Many ATR inhibitors are tested for cancer treatment in clinical trials, but their overall effectiveness is often compromised by the emergence of resistance and toxicities. In this proof-of-concept study, we propose to disrupt the ATR pathway by targeting TopBP1 condensation. First, we screened a molecule-based library using a previously developed optogenetic approach and identified several TopBP1 condensation inhibitors. Among them, AZD2858 disrupted TopBP1 assembly induced by the clinically relevant topoisomerase I inhibitor SN-38, thereby inhibiting the ATR/Chk1 signaling pathway. We found that AZD2858 exerted its effects by disrupting TopBP1 self-interaction and binding to ATR in mammalian cells, and by increasing its chromatin recruitment in cell-free Xenopus laevis egg extracts. Moreover, AZD2858 prevented S-phase checkpoint induction by SN-38, leading to increased DNA damage and apoptosis in a colorectal cancer cell line. Lastly, AZD2858 showed a synergistic effect in combination with the FOLFIRI chemotherapy regimen in a spheroid model of colorectal cancer.

Human topoisomerase IIβ binding protein 1 (TopBP1) is a scaffold protein involved in DNA replication initiation, DNA repair, transcription regulation, and checkpoint activation. TopBP1 forms nuclear condensates that act as a molecular switch to amplify ATR activity and promote the activation of the checkpoint effector kinase Chk1. In cancer cells, ATR activity is crucial to tolerate the intrinsically high level of DNA lesions and obstacles that block replication fork progression. Thus, ATR inhibitors are currently tested in clinical trials, often in combination with chemotherapy drugs. However, resistance and toxicity are still major issues. The weak interactions that hold TopBP1 condensates together are highly sensitive to changes in the cellular milieu, suggesting that small molecules may alter the formation of TopBP1 condensates. Here, we developed a high-throughput screening system to identify TopBP1 condensation modulators. This system allowed us to identify FDA-approved drugs, including thimerosal and quinacrine, that inhibit TopBP1 condensation and block the activation of ATR/Chk1 signaling. Mechanistically, quinacrine impaired TopBP1’s ability to associate with chromatin, thereby interfering with its capacity to form condensates. Furthermore, quinacrine enhanced the therapeutic efficacy of 5-fluorouracil and irinotecan, components of the clinically used FOLFIRI regimen in a mouse model of peritoneal carcinomatosis from colorectal cancer.

In cancer cells, ATR signaling is crucial to tolerate the intrinsically high damage levels that normally block replication fork progression. Assembly of TopBP1, a multifunctional scaffolding protein, into condensates is required to amplify ATR kinase activity to the levels needed to coordinate the DNA damage response and manage DNA replication stress. Many ATR inhibitors are tested for cancer treatment in clinical trials, but their overall effectiveness is oven compromised by the emergence of resistance and toxicities. In this proof-of-concept study, we propose to disrupt the ATR pathway by targeting TopBP1 condensation. First, we screened a molecule-based library using a previously developed optogenetic approach and identified several TopBP1 condensation inhibitors. Amongst them, AZD2858 disrupted TopBP1 assembly induced by the clinically relevant topoisomerase I inhibitor SN-38, thereby inhibiting the ATR/Chk1 signaling pathway. We found that AZD2858 exerted its effects by disrupting TopBP1 self-interaction and binding to ATR in mammalian cells, and by increasing its chromatin recruitment n cell-free Xenopus laevis egg extracts. Moreover, AZD2858 prevented S-phase checkpoint induction by SN-38, leading to increased DNA damage and apoptosis in a colorectal cancer cell line. Lastly, AZD2858 showed synergistic effect in combination with the FOLFIRI chemotherapy regimen in a spheroid model of colorectal cancer.

Fanconi Anemia (FA) is a rare inherited recessive disorder characterized by bone marrow failure, congenital abnormalities, and a predisposition to myeloid leukemia and some solid cancers. FA is caused by mutations in one of the 22 so-called FANC genes, with two thirds of FA patients being FANCA mutants. FANC proteins cooperate within the FANC/BRCA pathway, a DNA repair system essential for resolving DNA interstrand crosslinks (ICLs) in S-phase. The canonical understanding of FA pathogenesis involves genome instability and impaired cellular proliferation due to DNA damage accumulation and stress response overactivation. However, recent data shows FANCA has a function in ribosome biogenesis and translation, which prompts a reassessment of FANCA’s role(s) in cellular physiology and FA pathogenesis. We reasoned that unveiling the multiple interactions of the FANCA protein would shed light on these potentially new FANCA functions. By combining immunoprecipitation and BioID approaches, in three different cell lines and with different in silico analysis methods, we constructed lists of FANCA interactome proteins of different levels of selectivity and assembled them into a novel multilayered FANCA protein-protein interaction (PPI) landscape. Strikingly, we find that FANCA associates with proteins involved in processes distinct from DNA repair, such as translation, ribosome biogenesis, mRNA splicing, nucleocytoplasmic transport, chromatin remodeling, and the TCA cycle. We then analyzed the protein content of polysome profiling fractions and found that FANCA loss may affect ribosomal protein stoichiometry within ribosomal subunits. Overall, our data expands FANCA’s PPI network far beyond the previously reported interactions. Importantly, we confirm the biological significance of low-selectivity interactions and propose to re-evaluate their importance in proteomic studies. The study of previously unsuspected FANCA functions, such as translation, could provide new therapeutic targets in FA and therefore improve patient care.

Biomolecular condensates are large assemblies of proteins and nucleic acids that form distinct compartments inside the cell without being surrounded by a membrane. They form through multivalent interactions, are not stereospecifically defined, and can scale with component addition. By concentrating specific biomolecules at specific times and cellular locations, condensates play key roles in many processes, such as transcription, RNP assembly, cell cycle, DNA repair, and stress responses. Condensate biology greatly benefited from systematic analyses of their composition. However, condensates often have heterogenous sizes and are built on interaction networks that include stable and labile components. They also have highly variable compositions and dynamics. Their purification thus represents a significant challenge, and it necessitates extensive testing and adaptation of techniques originally designed for other applications. This article aims to synthesize the existing empirical knowledge on the extraction and purification of cellular condensates and analyze the challenges inherent to this field.

During mitosis, the microtubule depolymerase KIF2C, the tumor suppressor BRCA2, and the kinase PLK1 contribute to the control of kinetochore-microtubule attachments. Both KIF2C and BRCA2 are phosphorylated by PLK1, and BRCA2 phosphorylated at T207 (BRCA2-pT207) serves as a docking site for PLK1. Reducing this interaction results in unstable microtubule-kinetochore attachments. Here we identified that KIF2C also directly interacts with BRCA2-pT207. Indeed, the N-terminal domain of KIF2C adopts a Tudor/PWWP/MBT fold that unexpectedly binds to phosphorylated motifs. Using an optogenetic platform, we found that KIF2C forms membrane-less organelles that assemble through interactions mediated by this phospho-binding domain. KIF2C condensation does not depend on BRCA2-pT207 but requires active Aurora B and PLK1 kinases. Moreover, it concentrates PLK1 and BRCA2-pT207 in an Aurora B-dependent manner. Finally, KIF2C depolymerase activity promotes the formation of KIF2C condensates, but strikingly, KIF2C condensates exclude tubulin: they are located on microtubules, especially at their extremities. Altogether, our results suggest that, during the attachment of kinetochores to microtubules, the assembly of KIF2C condensates amplifies PLK1 and KIF2C catalytic activities and spatially concentrates BRCA2-pT207 at the extremities of microtubules. We propose that this novel and highly regulated mechanism contributes to the control of microtubule-kinetochore attachments, chromosome alignment, and stability.

In cancer cells, ATR signaling is crucial to tolerate the intrinsically high damage levels that normally block replication fork progression. Assembly of TopBP1, a multifunctional scaffolding protein, into condensates is required to amplify ATR kinase activity to the levels needed to coordinate the DNA damage response and manage DNA replication stress. Many ATR inhibitors are tested for cancer treatment in clinical trials, but their overall effectiveness is oven compromised by the emergence of resistance and toxicities. In this proof-of-concept study, we propose to disrupt the ATR pathway by targeting TopBP1 condensation. First, we screened a molecule-based library using a previously developed optogenetic approach and identified several TopBP1 condensation inhibitors. Amongst them, AZD2858 disrupted TopBP1 assembly induced by the clinically relevant topoisomerase I inhibitor SN-38, thereby inhibiting the ATR/Chk1 signaling pathway. We found that AZD2858 exerted its effects by disrupting TopBP1 self-interaction and binding to ATR in mammalian cells, and by increasing its chromatin recruitment n cell-free Xenopus laevis egg extracts. Moreover, AZD2858 prevented S-phase checkpoint induction by SN-38, leading to increased DNA damage and apoptosis in a colorectal cancer cell line. Lastly, AZD2858 showed synergistic effect in combination with the FOLFIRI chemotherapy regimen in a spheroid model of colorectal cancer. Graphical