Problems arising during DNA replication require the activation of the ATR–CHK1 pathway to ensure the stabilization and repair of the forks, and to prevent the entry into mitosis with unreplicated ...genomes. Whereas the pathway is essential at the cellular level, limiting its activity is particularly detrimental for some cancer cells. Here we review the links between replication stress (RS) and cancer, which provide a rationale for the use of ATR and Chk1 inhibitors in chemotherapy. First, we describe how the activation of oncogene-induced RS promotes genome rearrangements and chromosome instability, both of which could potentially fuel carcinogenesis. Next, we review the various pathways that contribute to the suppression of RS, and how mutations in these components lead to increased cancer incidence and/or accelerated ageing. Finally, we summarize the evidence showing that tumors with high levels of RS are dependent on a proficient RS-response, and therefore vulnerable to ATR or Chk1 inhibitors.
•The resolution of RS can happen during S, G2 or M phases.•Pathways involved in the resolution of RS are related to human disease.•Whereas low levels of RS can fuel carcinogenesis, high levels are toxic for cancer cells.•ATR and CHK1 inhibitors should be directed to cancers with high levels of RS.
...the percentage of biologics license applications (BLAs) granted approval by the Food and Drug Administration (FDA) has increased during the last decade, reaching 28% in 2021 4. Can we develop ...senomorphic drugs to modulate the senescence-associated secretory program (SASP)? ...while SA-β-galactosidase activity is a useful hallmark of senescence in research studies, a clinical biomarker for senescent cells is still needed. ...targeting MYC has been an important aim for the cancer research community for decades. ...one of the main scientific revolutions that is inevitably transforming cancer research is the advance of computational approaches.
Chemical inhibitors of the deubiquitinase USP7 are currently being developed as anticancer agents based on their capacity to stabilize P53. Regardless of this activity, USP7 inhibitors also generate ...DNA damage in a p53‐independent manner. However, the mechanism of this genotoxicity and its contribution to the anticancer effects of USP7 inhibitors are still under debate. Here we show that, surprisingly, even if USP7 inhibitors stop DNA replication, they also induce a widespread activation of CDK1 throughout the cell cycle, which leads to DNA damage and is toxic for mammalian cells. In addition, USP7 interacts with the phosphatase PP2A and supports its active localization in the cytoplasm. Accordingly, inhibition of USP7 or PP2A triggers very similar changes of the phosphoproteome, including a widespread increase in the phosphorylation of CDK1 targets. Importantly, the toxicity of USP7 inhibitors is alleviated by lowering CDK1 activity or by chemical activation of PP2A. Our work reveals that USP7 limits CDK1 activity at all cell cycle stages, providing a novel mechanism that explains the toxicity of USP7 inhibitors through untimely activation of CDK1.
SYNOPSIS
Inhibitors of the USP7 deubiquitinase are being developed as anticancer agents due to their capacity to stabilize p53. Here, the finding that USP7 suppresses CDK1 activity throughout the cell cycle, this leading to DNA damage in replicating cells, reveals a novel mechanism of USP7 inhibitor toxicity.
USP7 inhibition leads to a widespread increase in CDK1 activity throughout the cell cycle.
USP7 interacts with PP2A and is required for maintenance of PP2A cytoplasmic localization.
Inhibition of USP7 or PP2A triggers similar changes in the phosphoproteome.
The toxicity of USP7 inhibitors is mitigated by chemicals that inhibit CDK1 or activate PP2A.
The finding that USP7 inhibitors trigger widespread untimely CDK1 activation throughout the cell cycle represents another new toxicity mechanism of these potential anticancer drugs.
The telomere repeat-binding factor 1 (TERF1, referred to hereafter as TRF1) is a component of mammalian telomeres whose role in telomere biology and disease has remained elusive. Here, we report on ...cells and mice conditionally deleted for TRF1. TRF1-deleted mouse embryonic fibroblasts (MEFs) show rapid induction of senescence, which is concomitant with abundant telomeric gamma-H2AX foci and activation of the ATM/ATR downstream checkpoint kinases CHK1 and CHK2. DNA damage foci are rescued by both ATM and ATM/ATR inhibitors, further indicating that both signaling pathways are activated upon TRF1 deletion. Abrogation of the p53 and RB pathways bypasses senescence but leads to chromosomal instability including sister chromatid fusions, chromosome concatenation, and occurrence of multitelomeric signals (MTS). MTS are also elevated in ATR-deficient MEFs or upon treatment with aphidicolin, two conditions known to induce breakage at fragile sites, suggesting that TRF1-depleted telomeres are prone to breakage. To address the impact of these molecular defects in the organism, we deleted TRF1 in stratified epithelia of TRF1(Delta/Delta)K5-Cre mice. These mice die perinatally and show skin hyperpigmentation and epithelial dysplasia, which are associated with induction of telomere-instigated DNA damage, activation of the p53/p21 and p16 pathways, and cell cycle arrest in vivo. p53 deficiency rescues mouse survival but leads to development of squamous cell carcinomas, demonstrating that TRF1 suppresses tumorigenesis. Together, these results demonstrate that dysfunction of a telomere-binding protein is sufficient to produce severe telomeric damage in the absence of telomere shortening, resulting in premature tissue degeneration and development of neoplastic lesions.
Due to their capability to transport chemicals or proteins into target cells, cell‐penetrating peptides (CPPs) are being developed as therapy delivery tools. However, and despite their interesting ...properties, arginine‐rich CPPs often show toxicity for reasons that remain poorly understood. Using a (PR)n dipeptide repeat that has been linked to amyotrophic lateral sclerosis (ALS) as a model of an arginine‐rich CPP, we here show that the presence of (PR)n leads to a generalized displacement of RNA‐ and DNA‐binding proteins from chromatin and mRNA. Accordingly, any reaction involving nucleic acids, such as RNA transcription, translation, splicing and degradation, or DNA replication and repair, is impaired by the presence of the CPPs. Interestingly, the effects of (PR)n are fully mimicked by protamine, a small arginine‐rich protein that displaces histones from chromatin during spermatogenesis. We propose that widespread coating of nucleic acids and consequent displacement of RNA‐ and DNA‐binding factors from chromatin and mRNA accounts for the toxicity of arginine‐rich CPPs, including those that have been recently associated with the onset of ALS.
Synopsis
Arginine‐rich cell penetrating peptides (CPPs) can target therapeutic agents into cells, but often exhibit toxicity. Here, this is shown to originate from a generalized coating of cellular nucleic acids, affecting the physiological interactions with their binding factors.
Arginine‐rich CPPs coat cellular nucleic acids, leading to a generalized displacement of DNA‐ and RNA‐binding factors from chromatin and mRNA.
The impact of ALS‐associated (PR)20 peptides on translation is due to interactions with mRNA that prevent the assembly of 80S ribosomes.
The effects of arginine‐rich CPPs are recapitulated by protamine, the protein with the highest percentage of arginine within the animal proteome.
Non‐coding oligonucleotides and heparin alleviate the cellular effects of arginine‐rich peptides.
The toxic side effects of basic peptides used as pharmaceutic delivery tools, originates from their protamine‐like coating of nucleic acids.
Class I phosphoinositide 3-kinases are enzymes that generate 3-poly-phosphoinositides at the cell membrane following transmembrane receptor stimulation. Expression of the phosphoinositide 3-kinase β ...(PI3Kβ) isoform, but not its activity, is essential for early embryonic development. Nonetheless, the specific function of PI3Kβ in the cell remains elusive. Double-strand breaks (DSB) are among the most deleterious lesions for genomic integrity; their repair is required for development. We show that PI3Kβ is necessary for DSB sensing, as PI3Kβ regulates binding of the Nbs1 sensor protein to damaged DNA. Indeed, Nbs1 did not bind to DSB in PI3Kβ-deficient cells, which showed a general defect in subsequent ATM and ATR activation, resulting in genomic instability. Inhibition of PI3Kβ also retarded the DNA repair but the defect was less marked than that induced by PI3Kβ deletion, supporting a kinase-independent function for PI3Kβ in DNA repair. These results point at class I PI3Kβ as a critical sensor of genomic integrity.
Replicative stress during embryonic development influences ageing and predisposition to disease in adults. A protective mechanism against replicative stress is provided by the licensing of thousands ...of origins in G1 that are not necessarily activated in the subsequent S-phase. These 'dormant' origins provide a backup in the presence of stalled forks and may confer flexibility to the replication program in specific cell types during differentiation, a role that has remained unexplored. Here we show, using a mouse strain with hypomorphic expression of the origin licensing factor mini-chromosome maintenance (MCM)3 that limiting origin licensing in vivo affects the functionality of hematopoietic stem cells and the differentiation of rapidly-dividing erythrocyte precursors. Mcm3-deficient erythroblasts display aberrant DNA replication patterns and fail to complete maturation, causing lethal anemia. Our results indicate that hematopoietic progenitors are particularly sensitive to replication stress, and full origin licensing ensures their correct differentiation and functionality.
Post-translational modification of the DNA replication machinery by ubiquitin and SUMO plays key roles in the faithful duplication of the genetic information. Among other functions, ubiquitination ...and SUMOylation serve as signals for the extraction of factors from chromatin by the AAA ATPase VCP. In addition to the regulation of DNA replication initiation and elongation, we now know that ubiquitination mediates the disassembly of the replisome after DNA replication termination, a process that is essential to preserve genomic stability. Here, we review the recent evidence showing how active DNA replication restricts replisome ubiquitination to prevent the premature disassembly of the DNA replication machinery. Ubiquitination also mediates the removal of the replisome to allow DNA repair. Further, we discuss the interplay between ubiquitin-mediated replisome disassembly and the activation of CDK1 that is required to set up the transition from the S phase to mitosis. We propose the existence of a ubiquitin–CDK1 relay, where the disassembly of terminated replisomes increases CDK1 activity that, in turn, favors the ubiquitination and disassembly of more replisomes. This model has important implications for the mechanism of action of cancer therapies that induce the untimely activation of CDK1, thereby triggering premature replisome disassembly and DNA damage.
Trying to kill cancer cells by generating DNA damage is by no means a new idea. Radiotherapy and genotoxic drugs are routinely used in cancer therapy. More recent developments also explored the ...potential of targeting the DNA damage response (DDR) in order to increase the toxicity of radio- and chemo- therapy. Chk1 inhibitors have pioneered studies in this regard. Interestingly, early studies noted that Chk1 inhibitors were particularly toxic for p53-deficient cells. The model proposed for this observation was that this effect was due to the simultaneous abrogation of the G2 (Chk1) and G1 (p53) checkpoints. We here challenge this view, and propose a model where the toxicity of Chk1 inhibitors is rather due to the fact that these compounds generate high loads of replicative stress (RS) during S-phase, which are further boosted by the less restrictive S-phase entry found in p53-deficient cells. This new model implies that the particular toxicity of Chk1 inhibitors might not be restricted to p53-deficient cells, but could be extended to other mutations that promote a promiscuous S-phase entry. In addition, this rationale also implies that the same effect should also be observed for other molecules that target the RS-response (RSR), such as inhibitors of the Chk1-activating kinase ATR.
► The ATR-dependent RSR is distinct from the ATM-dependent DDR, and should be studied independently. ► RSR inhibition might be particularly toxic for a subset of tumors. ► ATR inhibitors promote p53-independent cell death.
Protein methylation is an important modification beyond epigenetics. However, systems analyses of protein methylation lag behind compared to other modifications. Recently, thermal stability analyses ...have been developed which provide a proxy of a protein functional status. Here, we show that molecular and functional events closely linked to protein methylation can be revealed by the analysis of thermal stability. Using mouse embryonic stem cells as a model, we show that Prmt5 regulates mRNA binding proteins that are enriched in intrinsically disordered regions and involved in liquid-liquid phase separation mechanisms, including the formation of stress granules. Moreover, we reveal a non-canonical function of Ezh2 in mitotic chromosomes and the perichromosomal layer, and identify Mki67 as a putative Ezh2 substrate. Our approach provides an opportunity to systematically explore protein methylation function and represents a rich resource for understanding its role in pluripotency.
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