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Intrinsically disordered proteins can phase separate from the soluble intracellular space, and tend to aggregate under pathological conditions. The physiological functions and molecular triggers of liquid demixing by phase separation are not well understood. Here we show in vitro and in vivo that the nucleic acid-mimicking biopolymer poly(ADP-ribose) (PAR) nucleates intracellular liquid demixing. PAR levels are markedly induced at sites of DNA damage, and we provide evidence that PAR-seeded liquid demixing results in rapid, yet transient and fully reversible assembly of various intrinsically disordered proteins at DNA break sites. Demixing, which relies on electrostatic interactions between positively charged RGG repeats and negatively charged PAR, is amplified by aggregation-prone prion-like domains, and orchestrates the earliest cellular responses to DNA breakage. We propose that PAR-seeded liquid demixing is a general mechanism to dynamically reorganize the soluble nuclear space with implications for pathological protein aggregation caused by derailed phase separation. Intrinsically disordered proteins can phase separate from the soluble intracellular space. Here the authors show that the nucleic acid-mimicking biopolymer poly(ADP-ribose) (PAR) nucleates intracellular liquid demixing and orchestrates the earliest cellular responses to DNA breakage.

Although protein ADP-ribosylation is involved in diverse biological processes, it has remained a challenge to identify ADP-ribose acceptor sites. Here, we present an experimental workflow for sensitive and unbiased analysis of endogenous ADP-ribosylation sites, capable of detecting more than 900 modification sites in mammalian cells and mouse liver. In cells, we demonstrate that Lys residues, besides Glu, Asp and Arg residues, are the dominant in vivo targets of ADP-ribosylation during oxidative stress. In normal liver tissue, we find Arg residues to be the predominant modification site. The cellular distribution and biological processes that involve ADP-ribosylated proteins are different in cultured cells and liver tissue, in the latter of which the majority of sites were found to be in cytosolic and mitochondrial protein networks primarily associated with metabolism. Collectively, we describe a robust methodology for the assessment of the role of ADP-ribosylation and ADP-ribosyltransferases in physiological and pathological states. ADP-ribosylation is a reversible post-translational protein modification involved in many cellular processes. Here the authors describe a sensitive approach for the analysis of ADP-ribosylation sites under physiologic conditions and identify lysine residues as in vivo targets of ADP-ribosylation.

Induction of DNA double-strand breaks (DSBs) in ribosomal DNA (rDNA) repeats is associated with ATM-dependent repression of ribosomal RNA synthesis and large-scale reorganization of nucleolar architecture, but the signaling events that regulate these responses are largely elusive. Here we show that the nucleolar response to rDNA breaks is dependent on both ATM and ATR activity. We further demonstrate that ATM- and NBS1-dependent recruitment of TOPBP1 in the nucleoli is required for inhibition of ribosomal RNA synthesis and nucleolar segregation in response to rDNA breaks. Mechanistically, TOPBP1 recruitment is mediated by phosphorylation-dependent interactions between three of its BRCT domains and conserved phosphorylated Ser/Thr residues at the C-terminus of the nucleolar phosphoprotein Treacle. Our data thus reveal an important cooperation between TOPBP1 and Treacle in the signaling cascade that triggers transcriptional inhibition and nucleolar segregation in response to rDNA breaks. DNA double-strand breaks in ribosomal DNA repeats is associated with repression of ribosomal RNA synthesis. Here the authors reveal a cooperation between TOPBP1 and Treacle in the signaling cascade that triggers transcriptional inhibition and nucleolar segregation in response to rDNA breaks.

BackgroundT-cell engagers are dependent on crosslinking of tumor cells with T cells and the activation of endogenous T-cells for their mechanism-of-action (MoA). As a result, such dose-response relationships are difficult to predict before a phase 1 trial begins. To overcome this prediction limitation from the bench to the clinic, and to take into consideration the heterogeneity of multiple MoA factors in the human tumor microenvironment, innovative quantitative modeling approaches are required. Here, we describe a quantitative systems pharmacology (QSP) model developed to support the first-in-human trial design for CDR404, a highly potent and highly specific antibody-based T-cell engager that binds bivalently to a MAGE-A4 peptide displayed on HLA-A*02:01 on cancer cells and monovalently to CD3 on T-cells.MethodsThe QSP model for CDR404 focused on describing the drug PK and dynamics of binding to MAGE-A4/HLA-A*02:01 and CD3 in the tumor compartment. Receptor turnover (synthesis and degradation) was also included in the model. Measured binding KDs as well as MAGE-A4 peptide/HLA-A*02:01 copy numbers on cancer cells were used to inform the model development.ResultsBy fitting the binding-related parameters, the model was able to describe the in vitro cytotoxicity dose responses for four PBMC donors at different effector-to-target ratios for two MAGE-A4+/HLA-A02:01+ human cancer cell lines (NCI-H1703: squamous lung cancer and A375: melanoma). Differences in target cell lysis from donors correlated with varying percentages of CD8 T cells among the donors. Trimer formation corresponding to in vitro IFNγ release was also predicted by the model. Following calibration to in vitro data, the model for cancer patients was developed. The impact of variability in MAGE-A4 expression levels in the tumor on trimer formation was explored through sensitivity analysis. Dose selection for the first in human study was based on doses that predicted to have similar trimer formation as the cytotoxicity assays. The model predicted Phase 1 trial starting dose for CDR404, and doses potentially associated with anti-tumor responses in patients will be presented at the meeting.ConclusionsQSP modeling is a powerful approach which guides the clinical trial design through integrating biophysics data, in vitro functional data, preclinical PK and MoA-related target biology. The developed QSP model will become an integrated part of the clinical development program and can be updated with emerging clinical data during the Phase 1 trial to facilitate discovery of a safe and therapeutic dose range for CDR404.Ethics ApprovalAnimal studies were performed in compliance with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. The animal care and use program is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), which assures compliance with accepted standards for the care and use of laboratory animals.

BackgroundSquamous non-small cell lung cancer (SQ-NSCLC) is the 2nd most common type of lung cancer. Given the paucity of actionable oncogene drivers, and lack of efficacy from multiple therapies in the Lung-MAP trial, there is a high unmet need in SQ-NSCLC to develop effective 2nd-line immunotherapies for patients with disease progression after immune checkpoint inhibitors (ICI).The melanoma antigen gene A4 (MAGE-A4) is exclusively expressed in cancer and absent in somatic tissues. MAGE-A4-derived peptides presented on HLA molecules at the cell surface recently emerged as a novel therapeutic opportunity. Thus, the two key objectives of this study were to: 1). Evaluate MAGE-A4 expression in human SQ-NSCLC; 2). Demonstrate the anti-cancer activity of CDR404, an antibody-based bispecific and bivalent T-cell engager targeted against MAGE-A4230–239 peptide in vitro and in vivo xenograft models of SQ-NSCLC.MethodsMAGE-A4 mRNA prevalence and expression in SQ-NSCLC was analyzed using the TCGA database (https://www.cancer.gov/tcga). Protein expression of MAGE-A4 was confirmed using immunohistochemistry (IHC) in fifty FFPE human SQ-NSCLC samples (clone E7O1U).CDR404 target cell killing in the presence of human PBMCs was assessed using the human SQ-NSCLC cell line NCI-H1703. HLA-A*02:01+MAGE-A4neg cancer cells were used as controls. To exclude reactivity of CDR404 in healthy tissues, HLA-A*02:01+ primary cells presenting peptides with high MAGE-A4 similarity were co-cultured with human PBMCs. In vivo activity of CDR404 in SQ-NSCLC was evaluated with an NCI-H1703 xenograft model in NSG mice.ResultsSQ-NSCLC had the highest MAGE-A4 mRNA expression levels among solid cancers in the TCGA database. IHC showed positive MAGE-A4 staining in 28/50 (56%) of SQ-NSCLC samples. In vitro, CDR404 showed efficient target cell lysis across all effector-to-target ratios tested. Similarly, simultaneous target engagement and resulting synapse formation induced T cell activation and secretion of cytolytic molecules in an effector-to-target ratio-dependent fashion. No reactivity was observed using co-cultured HLA-A*02:01+MAGE-A4neg cancer cells. Lack of T cell activation/cytolytic molecule release in the presence of HLA-A*02:01+ primary cells confirmed the specificity profile of CDR404. In vivo, treatment with four different doses of CDR404 induced complete tumor regression in the SQ-NSCLC NCI-H1703 xenograft model.ConclusionsThe high MAGE-A4 expression levels and the highly specific anti-cancer cell activity of CDR404 make it a highly attractive immunotherapy for development post-progression on ICI for patients with HLA-A*02:01+ SQ-NSCLC. A multi-tumor phase 1 trial of CDR404, including SQ-NSCLC, is expected to begin in 2024 with prospective patient selection for both HLA-A*02:01 and tumor MAGE-A4.Ethics ApprovalAnimal studies were performed in compliance with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. The animal care and use program is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), which assures compliance with accepted standards for the care and use of laboratory animals.

The chromatin structure is important for recognition and repair of DNA damage. Many DNA damage response proteins accumulate in large chromatin domains flanking sites of DNA double-strand breaks. The assembly of these structures—usually termed DNA damage foci—is primarily regulated by MDC1, a large nuclear mediator/adaptor protein that is composed of several distinct structural and functional domains. Here, we are summarizing the latest discoveries about the mechanisms by which MDC1 mediates DNA damage foci formation, and we are reviewing the considerable efforts taken to understand the functional implication of these structures.

Chromosome breakage elicits transient silencing of ribosomal RNA synthesis, but the mechanisms involved remained elusive. Here we discover an in trans signalling mechanism that triggers pan-nuclear silencing of rRNA transcription in response to DNA damage. This is associated with transient recruitment of the Nijmegen breakage syndrome protein 1 (NBS1), a central regulator of DNA damage responses, into the nucleoli. We further identify TCOF1 (also known as Treacle), a nucleolar factor implicated in ribosome biogenesis and mutated in Treacher Collins syndrome, as an interaction partner of NBS1, and demonstrate that NBS1 translocation and accumulation in the nucleoli is Treacle dependent. Finally, we provide evidence that Treacle-mediated NBS1 recruitment into the nucleoli regulates rRNA silencing in trans in the presence of distant chromosome breaks. DNA damage induces silencing of ribosomal RNA (rRNA) transcription. Stucki and colleagues reveal that rRNA silencing is an ATM-dependent pan-nuclear response to irradiation, in which the nucleolar protein Treacle targets DNA-damage protein NBS1 to nucleoli.

The MRE11–RAD50–NBS1 (MRN) complex accumulates at sites of DNA double‐strand breaks in large chromatin domains flanking the lesion site. The mechanism of MRN accumulation involves direct binding of the Nijmegen breakage syndrome 1 (NBS1) subunit to phosphorylated mediator of the DNA damage checkpoint 1 (MDC1), a large nuclear adaptor protein that interacts directly with phosphorylated H2AX. NBS1 contains an FHA domain and two BRCT domains at its amino terminus. Here, we show that both of these domains participate in the interaction with phosphorylated MDC1. Point mutations in key amino acid residues of either the FHA or the BRCT domains compromise the interaction with MDC1 and lead to defects in MRN accumulation at sites of DNA damage. Surprisingly, only mutation in the FHA domain, but not in the BRCT domains, yields a G2/M checkpoint defect, indicating that MDC1‐dependent chromatin accumulation of the MRN complex at sites of DNA breaks is not required for G2/M checkpoint activation. Here, Stucki and colleagues analyse how the DNA repair complex MRE11‐RAD50‐NBS1 is retained at sites of DNA damage. They report that both the FHA domain and the tandem BRCT domain of NBS1 are required for the interaction with phosphorylated MDC1, which results in the accumulation of MRN at DSBs. Surprisingly, only mutation in the FHA domain, but not mutation in the tandem BRCT domain, yields a G2/M checkpoint defect, indicating that accumulation of the MRN complex at sites of DSBs is not required for G27/M checkpoint activation.