Systematic identification of anticancer drug targets reveals a nucleus-to-mitochondria ROS-sensing pathway.

Multiple anticancer drugs have been proposed to cause cell death, in part, by increasing the steady-state levels of cellular reactive oxygen species (ROS). However, for most of these drugs, exactly how the resultant ROS function and are sensed is poorly understood. It remains unclear which proteins the ROS modify and their roles in drug sensitivity/resistance. To answer these questions, we examined 11 anticancer drugs with an integrated proteogenomic approach identifying not only many unique targets but also shared ones-including ribosomal components, suggesting common mechanisms by which drugs regulate translation. We focus on CHK1 that we find is a nuclear H2O2 sensor that launches a cellular program to dampen ROS. CHK1 phosphorylates the mitochondrial DNA-binding protein SSBP1 to prevent its mitochondrial localization, which in turn decreases nuclear H2O2. Our results reveal a druggable nucleus-to-mitochondria ROS-sensing pathway-required to resolve nuclear H2O2 accumulation and mediate resistance to platinum-based agents in ovarian cancers.

The TRESLIN-MTBP complex couples completion of DNA replication with S/G2 transition.

It has been proposed that ATR kinase senses the completion of DNA replication to initiate the S/G2 transition. In contrast to this model, we show here that the TRESLIN-MTBP complex prevents a premature entry into G2 from early S-phase independently of ATR/CHK1 kinases. TRESLIN-MTBP acts transiently at pre-replication complexes (preRCs) to initiate origin firing and is released after the subsequent recruitment of CDC45. This dynamic behavior of TRESLIN-MTBP implements a monitoring system that checks the activation of replication forks and senses the rate of origin firing to prevent the entry into G2. This system detects the decline in the number of origins of replication that naturally occurs in very late S, which is the signature that cells use to determine the completion of DNA replication and permit the S/G2 transition. Our work introduces TRESLIN-MTBP as a key player in cell-cycle control independent of canonical checkpoints.

Checkpoint-mediated DNA polymerase ε exonuclease activity curbing counteracts resection-driven fork collapse.

DNA polymerase ε (Polε) carries out high-fidelity leading strand synthesis owing to its exonuclease activity. Polε polymerase and exonuclease activities are balanced, because of partitioning of nascent DNA strands between catalytic sites, so that net resection occurs when synthesis is impaired. In vivo, DNA synthesis stalling activates replication checkpoint kinases, which act to preserve the functional integrity of replication forks. We show that stalled Polε drives nascent strand resection causing fork functional collapse, averted via checkpoint-dependent phosphorylation. Polε catalytic subunit Pol2 is phosphorylated on serine 430, influencing partitioning between polymerase and exonuclease active sites. A phosphormimetic S430D change reduces exonucleolysis in vitro and counteracts fork collapse. Conversely, non-phosphorylatable pol2-S430A expression causes resection-driven stressed fork defects. Our findings reveal that checkpoint kinases switch Polε to an exonuclease-safe mode preventing nascent strand resection and stabilizing stalled replication forks. Elective partitioning suppression has implications for the diverse Polε roles in genome integrity maintenance.

ATR Protects the Genome against R Loops through a MUS81-Triggered Feedback Loop.

R loops arising during transcription induce genomic instability, but how cells respond to the R loop-associated genomic stress is still poorly understood. Here, we show that cells harboring high levels of R loops rely on the ATR kinase for survival. In response to aberrant R loop accumulation, the ataxia telangiectasia and Rad3-related (ATR)-Chk1 pathway is activated by R loop-induced reversed replication forks. In contrast to the activation of ATR by replication inhibitors, R loop-induced ATR activation requires the MUS81 endonuclease. ATR protects the genome from R loops by suppressing transcription-replication collisions, promoting replication fork recovery, and enforcing a G2/M cell-cycle arrest. Furthermore, ATR prevents excessive cleavage of reversed forks by MUS81, revealing a MUS81-triggered and ATR-mediated feedback loop that fine-tunes MUS81 activity at replication forks. These results suggest that ATR is a key sensor and suppressor of R loop-induced genomic instability, uncovering a signaling circuitry that safeguards the genome against R loops.

CHK1 Inhibitor Blocks Phosphorylation of FAM122A and Promotes Replication Stress.

While effective anti-cancer drugs targeting the CHK1 kinase are advancing in the clinic, drug resistance is rapidly emerging. Here, we demonstrate that CRISPR-mediated knockout of the little-known gene FAM122A/PABIR1 confers cellular resistance to CHK1 inhibitors (CHK1is) and cross-resistance to ATR inhibitors. Knockout of FAM122A results in activation of PP2A-B55α, a phosphatase that dephosphorylates the WEE1 protein and rescues WEE1 from ubiquitin-mediated degradation. The resulting increase in WEE1 protein expression reduces replication stress, activates the G2/M checkpoint, and confers cellular resistance to CHK1is. Interestingly, in tumor cells with oncogene-driven replication stress, CHK1 can directly phosphorylate FAM122A, leading to activation of the PP2A-B55α phosphatase and increased WEE1 expression. A combination of a CHK1i plus a WEE1 inhibitor can overcome CHK1i resistance of these tumor cells, thereby enhancing anti-cancer activity. The FAM122A expression level in a tumor cell can serve as a useful biomarker for predicting CHK1i sensitivity or resistance.

Replication Stress Induces ATR/CHK1-Dependent Nonrandom Segregation of Damaged Chromosomes.

Nonrandom DNA segregation (NDS) is a mitotic event in which sister chromatids carrying the oldest DNA strands are inherited exclusively by one of the two daughter cells. Although this phenomenon has been observed across various organisms, the mechanism and physiological relevance of this event remain poorly defined. Here, we demonstrate that DNA replication stress can trigger NDS in human cells. This biased inheritance of old template DNA is associated with the asymmetric DNA damage response (DDR), which derives at least in part from telomeric DNA. Mechanistically, we reveal that the ATR/CHK1 signaling pathway plays an essential role in mediating NDS. We show that this biased segregation process leads to cell-cycle arrest and cell death in damaged daughter cells inheriting newly replicated DNA. These data therefore identify a key role for NDS in the maintenance of genomic integrity within cancer cell populations undergoing replication stress due to oncogene activation.

The F-Box Domain-Dependent Activity of EMI1 Regulates PARPi Sensitivity in Triple-Negative Breast Cancers.

The BRCA1-BRCA2-RAD51 axis is essential for homologous recombination repair (HRR) and is frequently disrupted in breast cancers. PARP inhibitors (PARPis) are used clinically to treat BRCA-mutated breast tumors. Using a genetic screen, we identified EMI1 as a modulator of PARPi sensitivity in triple-negative breast cancer (TNBC) cells. This function requires the F-box domain of EMI1, through which EMI1 assembles a canonical SCF ubiquitin ligase complex that constitutively targets RAD51 for degradation. In response to genotoxic stress, CHK1-mediated phosphorylation of RAD51 counteracts EMI1-dependent degradation by enhancing RAD51's affinity for BRCA2, leading to RAD51 accumulation. Inhibition of RAD51 degradation restores HRR in BRCA1-depleted cells. Human breast cancer samples display an inverse correlation between EMI1 and RAD51 protein levels. A subset of BRCA1-deficient TNBC cells develop resistance to PARPi by downregulating EMI1 and restoring RAD51-dependent HRR. Notably, reconstitution of EMI1 expression reestablishes PARPi sensitivity both in cellular systems and in an orthotopic mouse model.

CHK1 controls zygote pronuclear envelope breakdown by regulating F-actin through interacting with MICAL3.

CHK1 mutations could cause human zygote arrest at the pronuclei stage, a phenomenon that is not well understood at the molecular level. In this study, we conducted experiments where pre-pronuclei from zygotes with CHK1 mutation were transferred into the cytoplasm of normal enucleated fertilized eggs. This approach rescued the zygote arrest caused by the mutation, resulting in the production of a high-quality blastocyst. This suggests that CHK1 dysfunction primarily disrupts crucial biological processes occurring in the cytoplasm. Further investigation reveals that CHK1 mutants have an impact on the F-actin meshwork, leading to disturbances in pronuclear envelope breakdown. Through co-immunoprecipitation and mass spectrometry analysis of around 6000 mouse zygotes, we identified an interaction between CHK1 and MICAL3, a key regulator of F-actin disassembly. The gain-of-function mutants of CHK1 enhance their interaction with MICAL3 and increase MICAL3 enzymatic activity, resulting in excessive depolymerization of F-actin. These findings shed light on the regulatory mechanism behind pronuclear envelope breakdown during the transition from meiosis to the first mitosis in mammals.

SLFN11 Blocks Stressed Replication Forks Independently of ATR.

SLFN11 sensitizes cancer cells to a broad range of DNA-targeted therapies. Here we show that, in response to replication stress induced by camptothecin, SLFN11 tightly binds chromatin at stressed replication foci via RPA1 together with the replication helicase subunit MCM3. Unlike ATR, SLFN11 neither interferes with the loading of CDC45 and PCNA nor inhibits the initiation of DNA replication but selectively blocks fork progression while inducing chromatin opening across replication initiation sites. The ATPase domain of SLFN11 is required for chromatin opening, replication block, and cell death but not for the tight binding of SLFN11 to chromatin. Replication stress by the CHK1 inhibitor Prexasertib also recruits SLFN11 to nascent replicating DNA together with CDC45 and PCNA. We conclude that SLFN11 is recruited to stressed replication forks carrying extended RPA filaments where it blocks replication by changing chromatin structure across replication sites.

Chemo-Phosphoproteomic Profiling with ATR Inhibitors Berzosertib and Gartisertib Uncovers New Biomarkers and DNA Damage Response Regulators.

The ATR kinase protects cells against DNA damage and replication stress and represents a promising anti-cancer drug target. The ATR inhibitors (ATRi) berzosertib and gartisertib are both in clinical trials for the treatment of advanced solid tumors as monotherapy or in combination with genotoxic agents. We carried out quantitative phospho-proteomic screening for ATR biomarkers that are highly sensitive to berzosertib and gartisertib, using an optimized mass spectrometry pipeline. Screening identified a range of novel ATR-dependent phosphorylation events, which were grouped into three broad classes: (i) targets whose phosphorylation is highly sensitive to ATRi and which could be the next generation of ATR biomarkers; (ii) proteins with known genome maintenance roles not previously known to be regulated by ATR; (iii) novel targets whose cellular roles are unclear. Class iii targets represent candidate DNA damage response proteins and, with this in mind, proteins in this class were subjected to secondary screening for recruitment to DNA damage sites. We show that one of the proteins recruited, SCAF1, interacts with RNAPII in a phospho-dependent manner and recruitment requires PARP activity and interaction with RNAPII. We also show that SCAF1 deficiency partly rescues RAD51 loading in cells lacking the BRCA1 tumor suppressor. Taken together these data reveal potential new ATR biomarkers and new genome maintenance factors.

The Hammer and the Dance of Cell Cycle Control.

Cell cycle checkpoints secure ordered progression from one cell cycle phase to the next. They are important to signal cell stress and DNA lesions and to stop cell cycle progression when severe problems occur. Recent work suggests, however, that the cell cycle control machinery responds in more subtle and sophisticated ways when cells are faced with naturally occurring challenges, such as replication impediments associated with endogenous replication stress. Instead of following a stop and go approach, cells use fine-tuned deceleration and brake release mechanisms under the control of ataxia telangiectasia and Rad3-related protein kinase (ATR) and checkpoint kinase 1 (CHK1) to more flexibly adapt their cell cycle program to changing conditions. We highlight emerging examples of such intrinsic cell cycle checkpoint regulation and discuss their physiological and clinical relevance.

14-3-3ε augments OGT stability by binding with S20-phosphorylated OGT.

The relationship between O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and mitosis is intertwined. Besides the numerous mitotic OGT substrates that have been identified, OGT itself is also a target of the mitotic machinery. Previously, our investigations have shown that Checkpoint kinase 1 (Chk1) phosphorylates OGT at Ser-20 to increase OGT levels during cytokinesis, suggesting that OGT levels oscillate as mitosis progresses. Herein we studied its underlying mechanism. We set out from an R17C mutation of OGT, which is a uterine carcinoma mutation in The Cancer Genome Atlas. We found that R17C abolishes the S20 phosphorylation of OGT, as it lies in the Chk1 phosphorylating consensus motif. Consistent with our previous report that pSer-20 is essential for OGT level increases during cytokinesis, we further demonstrate that the R17C mutation renders OGT less stable, decreases vimentin phosphorylation levels and results in cytokinesis defects. Based on bioinformatic predictions, pSer-20 renders OGT more likely to interact with 14-3-3 proteins, the phospho-binding signal adaptor/scaffold protein family. By screening the seven isoforms of 14-3-3 family, we show that 14-3-3ε specifically associates with Ser-20-phosphorylated OGT. Moreover, we studied the R17C and S20A mutations in xenograft models and demonstrated that they both inhibit uterine carcinoma compared to wild-type OGT, probably due to less cellular reproduction. Our work is a sequel of our previous report on pS20 of OGT and is in line with the notion that OGT is intricately regulated by the mitotic network.

The luciferase-based in vivo protein-protein interaction assay revealed that CHK1 promotes PP2A and PME-1 interaction.

Protein phosphatase 2A (PP2A) is an essential serine/threonine protein phosphatase, and its dysfunction is involved in the onset of cancer and neurodegenerative disorders. PP2A functions as a trimeric holoenzyme whose composition is regulated by the methyl-esterification (methylation) of the PP2A catalytic subunit (PP2Ac). Protein phosphatase methylesterase-1 (PME-1) is the sole PP2Ac methylesterase, and the higher PME-1 expression is observed in various cancer and neurodegenerative diseases. Apart from serving as a methylesterase, PME-1 acts as a PP2A inhibitory protein, binding directly to PP2Ac and suppressing its activity. The intricate function of PME-1 hinders drug development by targeting the PME-1/PP2Ac axis. This study applied the NanoBiT system, a bioluminescence-based protein interaction assay, to elucidate the molecular mechanism that modulates unknown PME-1/PP2Ac protein-protein interaction (PPI). Compound screening identified that the CHK1 inhibitors inhibited PME-1/PP2Ac association without affecting PP2Ac methylation levels. CHK1 directly phosphorylates PP2Ac to promote PME-1 association. Phospho-mass spectrometry identified multiple phospho-sites on PP2Ac, including the Thr219, that affect PME-1 interaction. An anti-phospho-Thr219 PP2Ac antibody was generated and showed that CHK1 regulates the phosphorylation levels of this site in cells. On the contrary, in vitro phosphatase assay showed that CHK1 is the substrate of PP2A, and PME-1 hindered PP2A-mediated dephosphorylation of CHK1. Our data provides novel insights into the molecular mechanisms governing the PME-1/PP2Ac PPI and the triad relationship between PP2A, PME-1, and CHK1.

CHK1-CDC25A-CDK1 regulate cell cycle progression and protect genome integrity in early mouse embryos.

After fertilization, remodeling of the oocyte and sperm genomes is essential to convert these highly differentiated and transcriptionally quiescent cells into early cleavage-stage blastomeres that are transcriptionally active and totipotent. This developmental transition is accompanied by cell cycle adaptation, such as lengthening or shortening of the gap phases G1 and G2. However, regulation of these cell cycle changes is poorly understood, especially in mammals. Checkpoint kinase 1 (CHK1) is a protein kinase that regulates cell cycle progression in somatic cells. Here, we show that CHK1 regulates cell cycle progression in early mouse embryos by restraining CDK1 kinase activity due to CDC25A phosphatase degradation. CHK1 kinase also ensures the long G2 phase needed for genome activation and reprogramming gene expression in two-cell stage mouse embryos. Finally, Chk1 depletion leads to DNA damage and chromosome segregation errors that result in aneuploidy and infertility.

Replication Catastrophe: When a Checkpoint Fails because of Exhaustion.

Proliferating cells rely on the so-called DNA replication checkpoint to ensure orderly completion of genome duplication, and its malfunction may lead to catastrophic genome disruption, including unscheduled firing of replication origins, stalling and collapse of replication forks, massive DNA breakage, and, ultimately, cell death. Despite many years of intensive research into the molecular underpinnings of the eukaryotic replication checkpoint, the mechanisms underlying the dismal consequences of its failure remain enigmatic. A recent development offers a unifying model in which the replication checkpoint guards against global exhaustion of rate-limiting replication regulators. Here we discuss how such a mechanism can prevent catastrophic genome disruption and suggest how to harness this knowledge to advance therapeutic strategies to eliminate cancer cells that inherently proliferate under increased DNA replication stress.

ASPM promotes ATR-CHK1 activation and stabilizes stalled replication forks in response to replication stress.

ASPM is a protein encoded by primary microcephaly 5 (MCPH5) and is responsible for ensuring spindle position during mitosis and the symmetrical division of neural stem cells. We recently reported that ASPM promotes homologous recombination (HR) repair of DNA double strand breaks. However, its potential role in DNA replication and replication stress response remains elusive. Interestingly, we found that ASPM is dispensable for DNA replication under unperturbed conditions. However, ASPM is enriched at stalled replication forks in a RAD17-dependent manner in response to replication stress and promotes RAD9 and TopBP1 loading onto chromatin, facilitating ATR-CHK1 activation. ASPM depletion results in failed fork restart and nuclease MRE11-mediated nascent DNA degradation at the stalled replication fork. The overall consequence is chromosome instability and the sensitization of cancer cells to replication stressors. These data support a role for ASPM in loading RAD17-RAD9/TopBP1 onto chromatin to activate the ATR-CHK1 checkpoint and ultimately ensure genome stability.

In silico and structure-based evaluation of deleterious mutations identified in human Chk1, Chk2, and Wee1 protein kinase.

Checkpoint kinases Chk1, Chk2, Wee1 are playing a key role in DNA damage response and genomic integrity. Cancer-associated mutations identified in human Chk1, Chk2, and Wee1 were retrieved to understand the function associated with the mutation and also alterations in the folding pattern. Therefore, an attempt has been made to identify deleterious effect of variants using in silico and structure-based approach. Variants of uncertain significance for Chk1, Chk2, and Wee1 were retrieved from different databases and four prediction servers were employed to predict pathogenicity of mutations. Further, Interpro, I-Mutant 3.0, Consurf, TM-align, and have (y)our protein explained were used for comprehensive study of the deleterious effects of variants. The sequences of Chk1, Chk2, and Wee1 were analyzed using Clustal Omega, and the three-dimensional structures of the proteins were aligned using TM-align. The molecular dynamics simulations were performed to explore the differences in folding pattern between Chk1, Chk2, Wee1 wild-type, and mutant protein and also to evaluate the structural integrity. Thirty-six variants in Chk1, 250 Variants in Chk2, and 29 in Wee1 were categorized as pathogenic using in silico prediction tools. Furthermore, 25 mutations in Chk1, 189 in Chk2, and 14 in Wee1 were highly conserved, possessing deleterious effect and also influencing the protein structure and function. These identified mutations may provide underlying genetic intricacies to serve as potential targets for therapeutic inventions and clinical management.

Proapoptotic activity of JNK-sensitive BH3-only proteins underpins ovarian cancer response to replication checkpoint inhibitors.

Recent studies indicate that replication checkpoint modulators (RCMs) such as inhibitors of CHK1, ATR, and WEE1 have promising monotherapy activity in solid tumors, including platinum-resistant high grade serous ovarian cancer (HGSOC). However, clinical response rates are generally below 30%. While RCM-induced DNA damage has been extensively examined in preclinical and clinical studies, the link between replication checkpoint interruption and tumor shrinkage remains incompletely understood. Here we utilized HGSOC cell lines and patient-derived xenografts (PDXs) to study events leading from RCM treatment to ovarian cancer cell death. These studies show that RCMs increase CDC25A levels and CDK2 signaling in vitro, leading to dysregulated cell cycle progression and increased replication stress in HGSOC cell lines independent of homologous recombination status. These events lead to sequential activation of JNK and multiple BH3-only proteins, including BCL2L11/BIM, BBC3/PUMA and the BMF, all of which are required to fully initiate RCM-induced apoptosis. Activation of the same signaling pathway occurs in HGSOC PDXs that are resistant to poly(ADP-ribose) polymerase inhibitors but respond to RCMs ex vivo with a decrease in cell number in 3-dimensional culture and in vivo with xenograft shrinkage or a significantly diminished growth rate. These findings identify key cell death-initiating events that link replication checkpoint inhibition to antitumor response in ovarian cancer.

Reciprocal regulation of p21 and Chk1 controls the cyclin D1-RB pathway to mediate senescence onset after G2 arrest.

Senescence is an irreversible withdrawal from cell proliferation that can be initiated after DNA damage-induced cell cycle arrest in G2 phase to prevent genomic instability. Senescence onset in G2 requires p53 (also known as TP53) and retinoblastoma protein (RB, also known as RB1) family tumour suppressors, but how they are regulated to convert a temporary cell cycle arrest into a permanent one remains unknown. Here, we show that a previously unrecognised balance between the cyclin-dependent kinase (CDK) inhibitor p21 and the checkpoint kinase Chk1 controls cyclin D-CDK activity during G2 arrest. In non-transformed cells, p21 activates RB in G2 by inhibiting cyclin D1 complexed with CDK2 or CDK4. The resulting G2 exit, which precedes the appearance of senescence markers, is associated with a mitotic bypass, Chk1 downregulation and reduction in the number of DNA damage foci. In p53/RB-proficient cancer cells, a compromised G2 exit correlates with sustained Chk1 activity, delayed p21 induction, untimely cyclin E1 re-expression and genome reduplication. Conversely, Chk1 depletion promotes senescence by inducing p21 binding to cyclin D1- and cyclin E1-CDK complexes and downregulating CDK6, whereas knockdown of the checkpoint kinase Chk2 enables RB phosphorylation and delays G2 exit. In conclusion, p21 and Chk2 oppose Chk1 to maintain RB activity, thus promoting the onset of senescence induced by DNA damage in G2.

Functions, Regulation, and Therapeutic Implications of the ATR Checkpoint Pathway.

The ATR (ATM and rad3-related) pathway is crucial for proliferation, responding to DNA replication stress and DNA damage. This critical signaling pathway is carefully orchestrated through a multistep process requiring initial priming of ATR prior to damage, recruitment of ATR to DNA damage lesions, activation of ATR signaling, and, finally, modulation of ATR activity through a variety of post-translational modifications. Following activation, ATR functions in several vital cellular processes, including suppression of replication origin firing, promotion of deoxynucleotide synthesis and replication fork restart, prevention of double-stranded DNA break formation, and avoidance of replication catastrophe and mitotic catastrophe. In many cancers, tumor cells have increased dependence on ATR signaling for survival, making ATR a promising target for cancer therapy. Tumor cells compromised in DNA repair pathways or DNA damage checkpoints, cells reliant on homologous recombination, and cells with increased replication stress are particularly sensitive to ATR inhibition. Understanding ATR signaling and modulation is essential to unraveling which tumors have increased dependence on ATR signaling as well as how the ATR pathway can best be exploited for targeted cancer therapy.