Thèse Concevoir des Petites Protéines qui Inhibent Topbp1 dans le Cancer H/F

Doctorat.Gouv.Fr

DNA double-strand breaks (DSBs) are deleterious lesions that challenge genome integrity, because they cause insertion, deletion or chromosome rearrangements when mitosis or replication occur. Unrepaired DSBs predispose cells to malignant transformation and enhance tumorigenesis. To mitigate this threat, somatic mammalian cells rely on the activity of multiple DNA repair machineries that are tightly regulated throughout the cell cycle. Topoisomerase II-binding protein (TopBP1), a prototype of scaffold' protein, orchestrates DSB DNA repair throughout the cell cycle. It consists of nine well-folded BRCA1 C-terminal (BRCT) domains and several intrinsically disordered regions (IDRs) that are post-translationally modified. TopBP1 domains BRCT0/1/2, BRCT4/5 and BRCT7/8 bind to phosphorylated peptides. In S-phase, BRCT4/5 contributes to the localization of TopBP1 at stalled replication forks through its interaction with phosphorylated MDC1 SDT repeats (Yamane et al., Mol Cell Biol 2002 ; Wang et al., J Cell Biol 2011; Leung et al., Structure 2013). In mitosis, BRCT0/1/2 is essential for the recruitment of TopBP1 to DNA damage induced by irradiation (Leimbacher et al., Mol Cell 2019). Therefore, it interacts with the phosphorylated serines of MDC1 located just upstream from the first SDT repeat. The TopBP1 IDRs exhibit highly conserved motifs that are targets for kinases. The disordered region in between BRCT6 and BRCT7/8 is called the ATR Activating Domain (AAD), as it activates ATR at stalled replication forks to trigger phosphorylation of Chk1 and cell cycle arrest (Kumagai et al., Cell 2006). In mitosis, the disordered region located in between BRCT4/5 and BRCT6, further named L56, is necessary for TopBP1 foci formation after irradiation (De Marco Zompit et al., Nat Commun 2022). This region binds to several partners in mitosis, including CIP2A, an endogeneous inhibitory factor of the phosphatase PP2A. TopBP1 and CIP2A accumulation at mitotic DSBs are interdependent. Cells lacking CIP2A display increased radio-sensitivity and chromosomal instability. In our team, we are designing artificial binders of TopBP1 that modulate its activity in order to obtain novel molecules with anti-tumour properties, in collaboration with the team of J. Basbous at IGH (Montpellier) (Frattani et al., Mol Cell 2021).

In our team, we are designing artificial binders of TopBP1 that modulate its activity in order to obtain novel molecules with anti-tumour properties, in collaboration with the team of J. Basbous at IGH (Montpellier) (Frattani et al., Mol Cell 2021). First, we chose to target TopBP1 at stalled replication forks. Indeed, TopBP1 is a major ATR activator in S-phase. In a wide range of cancers, tumour cells depend on ATR to survive to high levels of DNA damage. ATR inhibitors have been developed for clinical use, either as a monotherapy or in combination with chemotherapy, to enhance cytotoxicity of drugs against tumours with high replicative stress (Lecona & Fernandez-Capetillo, Nat Rev Cancer 2018). However, their use in clinics is limited due to significant toxicity and acquired resistance. Targeting TopBP1 recruitment to stalled replication forks and/or activation of ATR could be novel alternatives to decrease the activity of ATR in tumour cells. A PhD student from the lab has already successfully designed single-chain antibody (or VHHs) that interact with different motifs of the disordered AAD region of TopBP1, in collaboration with the company Hybrigenics, and is currently characterizing the 3D structures of the complexes between AAD and these VHHs. In parallel, we propose to produce binders against the BRCT4/5 domain of TopBP1, in order to reduce the formation of TopBP1 foci at sites of replication stress. As BRCT4/5 is a folded domain, we will take advantage on the -Rep facility of I2BC to obtain several -Rep molecules against this TopBP1 domain. A-Rep are in general highly stable and specific binders (Urvoas et al., Trends Biotechnol 2012), which could perturb TopBP1 function in interphase.
Second, we propose to target TopBP1 at DSBs in mitosis. Indeed, deletion of the disordered region L56 completely disrupts TopBP1 recruitment to sites of chromosome breaks, which is critical for proper segregation of acentric chromosome fragments (De Marco Zompit et al., Nat Commun 2022). As cancer cells must frequently deal with remnants of replication problems from the preceding S-phase, at least a subset of these replication intermediates will be converted into DSBs in mitosis, and will rely on TopBP1 for their stabilization and/or repair. We aim at designing novel binders of L56, in order to reduce the formation of TopBP1 foci at mitotic DSBs. Therefore, two strategies will be used : first, the selection of VHHs against L56, in collaboration with the company Hybrigenics, as successfully performed in the case of TopBP1 AAD ; second, the in silico design of binders against L56 using the AI-based programs RFdiffusion (for backbone calculations), ProteinMPNN (for sequence assignment) and AlphaFold2 (for filtering) (Fox et al., Structure 2025). For this last task, we have initiated a collaboration with J.W. Rogers that has adapted the RFdiffusion approach to the design of binders against disordered regions (Vasquez Torres et al., Nature 2024).

The PhD student will focus on the engineering of mitotic binders of TopBP1. He/she will express and analyse by Nuclear Magnetic Resonance the disordered region L56 of TopBP1. He/she will characterize the interaction between L56 and the different single-chain antibodies selected by the company Hybrigenics. He/she will collaborate with J.W. Rogers to calculate artificial binders of L56 using cutting-edge AI approaches. He/she will analyse the interactions between L56 and these binders, including their regulations by mitotic phosphorylation events. The PhD student will take advantage of the AFMB protein production facility in Marseille to obtain a large number of binders, and will screen them at I2BC using different biophysical techniques including NMR, fluorescence anisotropy, BioLayer Interferometry and Isothermal Titration Calorimetry. He/she will attempt to crystallize the complexes between L56 and the best binders, and will propose mutations to increase the affinity between L56 and these binders. Chimeric bivalent binders will also be designed that target simultaneously two regions of L56, and the interactions between L56 and these bivalent binders will be analysed. The PhD student will also produce TopBP1 and its mitotic partner CIP2A in insect cells, will reconstitute the phospho-dependent complex between these two proteins in vitro, will characterize its 3D structure by cryoEM, and will check the capacity of the different binders to disrupt this interaction. Finally, the selected best binders will be expressed using lentiviruses in 293T cells endogeneously expressing Halo-TopBP1 by our collaborators, the team of J. Basbous at IGH (Montpellier), in order to observe their impact on TopBP1 foci formation and genomic stability.