Genome-Wide Screens Reveal that Resveratrol Induces Replicative Stress in Human Cells.

Resveratrol is a natural product associated with wide-ranging effects in animal and cellular models, including lifespan extension. To identify the genetic target of resveratrol in human cells, we conducted genome-wide CRISPR-Cas9 screens to pinpoint genes that confer sensitivity or resistance to resveratrol. An extensive network of DNA damage response and replicative stress genes exhibited genetic interactions with resveratrol and its analog pterostilbene. These genetic profiles showed similarity to the response to hydroxyurea, an inhibitor of ribonucleotide reductase that causes replicative stress. Resveratrol, pterostilbene, and hydroxyurea caused similar depletion of nucleotide pools, inhibition of replication fork progression, and induction of replicative stress. The ability of resveratrol to inhibit cell proliferation and S phase transit was independent of the histone deacetylase sirtuin 1, which has been implicated in lifespan extension by resveratrol. These results establish that a primary impact of resveratrol on human cell proliferation is the induction of low-level replicative stress.

The interplay of homology-directed repair pathways in the repair of zebularine-induced DNA-protein crosslinks in Arabidopsis.

DNA-protein crosslinks (DPCs) are highly toxic DNA lesions represented by proteins covalently bound to the DNA. Persisting DPCs interfere with fundamental genetic processes such as DNA replication and transcription. Cytidine analog zebularine (ZEB) has been shown to crosslink DNA METHYLTRANSFERASE1 (MET1). Recently, we uncovered a critical role of the SMC5/6-mediated SUMOylation in the repair of DPCs. In an ongoing genetic screen, we identified two additional candidates, HYPERSENSITIVE TO ZEBULARINE 2 and 3, that were mapped to REGULATOR OF TELOMERE ELONGATION 1 (RTEL1) and polymerase TEBICHI (TEB), respectively. By monitoring the growth of hze2 and hze3 plants in response to zebularine, we show the importance of homologous recombination (HR) factor RTEL1 and microhomology-mediated end-joining (MMEJ) polymerase TEB in the repair of MET1-DPCs. Moreover, genetic interaction and sensitivity assays showed the interdependency of SMC5/6 complex, HR, and MMEJ in the homology-directed repair of MET1-DPCs in Arabidopsis. Altogether, we provide evidence that MET1-DPC repair in plants is more complex than originally expected.

Combined inhibition of RAD51 and CHK1 causes synergistic toxicity in cisplatin resistant cancer cells by triggering replication fork collapse.

The therapeutic efficacy of the anticancer drug cisplatin is limited by acquired drug resistance. Cisplatin forms DNA crosslinks, that, if not removed, lead to replication stress. Due to this, the DNA damage response (DDR) gets activated regulating cell cycle arrest, DNA repair, cell death or survival. This makes DDR components promising targets for the development of new therapeutic approaches aiming to overcome acquired drug resistance. To this end, cisplatin-resistant bladder cancer cells were analyzed regarding their sensitivity to combination treatments with selected pharmacological DDR inhibitors. Synergistic cytolethal effects were achieved after combined treatment with low to moderate doses of the non-genotoxic RAD51-inhibitor (RAD51i) B02 and CHK1-inhibitor (CHK1i) PF477736. This effect was also found in cisplatin resistant tumor cells of other origin as well as with other RAD51i and CHK1i. Combined treatments promoted decelerated replication, S-phase blockage, accumulation of DNA strand breaks, DDR activation and stimulation of apoptotic cell death as compared to mono-treatment, which is independent of the expression of RAD51, CHK1, and PrimPol. Based on these data, we suggest combined inhibition of RAD51 and CHK1 to overcome acquired cisplatin resistance of malignant cells. We propose that the molecular mechanism of this synergistic toxicity relies on a simultaneous inactivation of two key DNA damage tolerance pathways regulating replication fork restart, thereby circumventing the activation of alternative compensatory mechanisms and, in consequence, eventually effectively triggering apoptotic cell death by replication fork collapse.

Nuclear pCHK1 as a potential biomarker of increased sensitivity to ATR inhibition.

Excessive genomic instability coupled with abnormalities in DNA repair pathways induces high levels of 'replication stress' when cancer cells propagate. Rather than hampering cancer cell proliferation, novel treatment strategies are turning their attention towards targeting cell cycle checkpoint kinases (such as ATR, CHK1, WEE1, and others) along the DNA damage response and replicative stress response pathways, thereby allowing unrepaired DNA damage to be carried forward towards mitotic catastrophe and apoptosis. The selective ATR kinase inhibitor elimusertib (BAY 1895344) has demonstrated preclinical and clinical monotherapy activity; however, reliable predictive biomarkers of treatment benefit are still lacking. In this study, using gene expression profiling of 24 cell lines from different cancer types and in a panel of ovarian cancer cell lines, we found that nuclear-specific enrichment of checkpoint kinase 1 (CHK1) correlated with increased sensitivity to elimusertib. Using an advanced multispectral imaging system in subsequent cell line-derived xenograft specimens, we showed a trend between nuclear phosphorylated CHK1 (pCHK1) staining and increased sensitivity to the ATR inhibitor elimusertib, indicating the potential value of pCHK1 expression as a predictive biomarker of ATR inhibitor sensitivity. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

RAD51: Beyond the break.

As the primary catalyst of homologous recombination (HR) in vertebrates, RAD51 has been extensively studied in the context of repair of double-stranded DNA breaks (DSBs). With recent advances in the understanding of RAD51 function extending beyond DSBs, the importance of RAD51 throughout DNA metabolism has become increasingly clear. Here we review the suggested roles of RAD51 beyond HR, specifically focusing on their interplay with DNA replication and the maintenance of genomic stability, in which RAD51 function emerges as a double-edged sword.

Hydrostatic pressure prevents chondrocyte differentiation through heterochromatin remodeling.

Articular cartilage protects and lubricates joints for smooth motion and transmission of loads. Owing to its high water content, chondrocytes within the cartilage are exposed to high levels of hydrostatic pressure, which has been shown to promote chondrocyte identity through unknown mechanisms. Here, we investigate the effects of hydrostatic pressure on chondrocyte state and behavior, and discover that application of hydrostatic pressure promotes chondrocyte quiescence and prevents maturation towards the hypertrophic state. Mechanistically, hydrostatic pressure reduces the amount of trimethylated H3K9 (K3K9me3)-marked constitutive heterochromatin and concomitantly increases H3K27me3-marked facultative heterochromatin. Reduced levels of H3K9me3 attenuates expression of pre-hypertrophic genes, replication and transcription, thereby reducing replicative stress. Conversely, promoting replicative stress by inhibition of topoisomerase II decreases Sox9 expression, suggesting that it enhances chondrocyte maturation. Our results reveal how hydrostatic pressure triggers chromatin remodeling to impact cell fate and function.This article has an associated First Person interview with the first author of the paper.

Firing of Replication Origins Is Disturbed by a CDK4/6 Inhibitor in a pRb-Independent Manner.

Over the last decade, CDK4/6 inhibitors (palbociclib, ribociclib and abemaciclib) have emerged as promising anticancer drugs. Numerous studies have demonstrated that CDK4/6 inhibitors efficiently block the pRb-E2F pathway and induce cell cycle arrest in pRb-proficient cells. Based on these studies, the inhibitors have been approved by the FDA for treatment of advanced hormonal receptor (HR) positive breast cancers in combination with hormonal therapy. However, some evidence has recently shown unexpected effects of the inhibitors, underlining a need to characterize the effects of CDK4/6 inhibitors beyond pRb. Our study demonstrates how palbociclib impairs origin firing in the DNA replication process in pRb-deficient cell lines. Strikingly, despite the absence of pRb, cells treated with palbociclib synthesize less DNA while showing no cell cycle arrest. Furthermore, this CDK4/6 inhibitor treatment disturbs the temporal program of DNA replication and reduces the density of replication forks. Cells treated with palbociclib show a defect in the loading of the Pre-initiation complex (Pre-IC) proteins on chromatin, indicating a reduced initiation of DNA replication. Our findings highlight hidden effects of palbociclib on the dynamics of DNA replication and of its cytotoxic consequences on cell viability in the absence of pRb. This study provides a potential therapeutic application of palbociclib in combination with other drugs to target genomic instability in pRB-deficient cancers.

Replicative Stress Coincides with Impaired Nuclear DNA Damage Response in COX4-1 Deficiency.

Cytochrome c oxidase (COX), a multimeric protein complex, is the final electron acceptor in the mitochondrial electron transfer chain. Primary COX deficiency, caused by mutations in either mitochondrial DNA or nuclear-encoded genes, is a heterogenous group of mitochondrial diseases with a wide range of presentations, ranging from fatal infantile to subtler. We previously reported a patient with primary COX deficiency due to a pathogenic variant in COX4I1 (encoding the common isoform of COX subunit 4, COX4-1), who presented with bone marrow failure, genomic instability, and short stature, mimicking Fanconi anemia (FA). In the present study, we demonstrated that accumulative DNA damage coincided primarily with proliferative cells in the patient's fibroblasts and in COX4i1 knockdown cells. Expression analysis implicated a reduction in DNA damage response pathways, which was verified by demonstrating impaired recovery from genotoxic insult and decreased DNA repair. The premature senescence of the COX4-1-deficient cells prevented us from undertaking additional studies; nevertheless, taken together, our results indicate replicative stress and impaired nuclear DNA damage response in COX4-1 deficiency. Interestingly, our in vitro findings recapitulated the patient's presentation and present status.

Recurrent chromosomal imbalances provide selective advantage to human embryonic stem cells under enhanced replicative stress conditions.

Human embryonic stem cells (hESCs) and embryonal tumors share a number of common features, including a compromised G1/S checkpoint. Consequently, these rapidly dividing hESCs and cancer cells undergo elevated levels of replicative stress, inducing genomic instability that drives chromosomal imbalances. In this context, it is of interest that long-term in vitro cultured hESCs exhibit a remarkable high incidence of segmental DNA copy number gains, some of which are also highly recurrent in certain malignancies such as 17q gain (17q+). The selective advantage of DNA copy number changes in these cells has been attributed to several underlying processes including enhanced proliferation. We hypothesized that these recurrent chromosomal imbalances become rapidly embedded in the cultured hESCs through a replicative stress driven Darwinian selection process. To this end, we compared the effect of hydroxyurea-induced replicative stress vs normal growth conditions in an equally mixed cell population of isogenic euploid and 17q + hESCs. We could show that 17q + hESCs rapidly overtook normal hESCs. Our data suggest that recurrent chromosomal segmental gains provide a proliferative advantage to hESCs under increased replicative stress, a process that may also explain the highly recurrent nature of certain imbalances in cancer.

Hes1 deficiency causes hematopoietic stem cell exhaustion.

The transcriptional repressor Hairy Enhancer of Split 1 (HES1) plays an essential role in the development of many organs by promoting the maintenance of stem/progenitor cells, controlling the reversibility of cellular quiescence, and regulating both cell fate decisions. Deletion of Hes1 in mice results in severe defects in multiple organs and is lethal in late embryogenesis. Here we have investigated the role of HES1 in hematopoiesis using a hematopoietic lineage-specific Hes1 knockout mouse model. We found that while Hes1 is dispensable for steady-state hematopoiesis, Hes1-deficient hematopoietic stem cells (HSCs) undergo exhaustion under replicative stress. Loss of Hes1 upregulates the expression of genes involved in PPARγ signaling and fatty acid metabolism pathways, and augments fatty acid oxidation (FAO) in Hes1 f/f Vav1Cre HSCs and progenitors. Functionally, PPARγ targeting or FAO inhibition ameliorates the repopulating defects of Hes1 f/f Vav1Cre HSCs through improving quiescence in HSCs. Lastly, transcriptome analysis reveals that disruption of Hes1 in hematopoietic lineage alters expression of genes critical to HSC function, PPARγ signaling, and fatty acid metabolism. Together, our findings identify a novel role of HES1 in regulating stress hematopoiesis and provide mechanistic insight into the function of HES1 in HSC maintenance.

MYC-Induced Replicative Stress: A Double-Edged Sword for Cancer Development and Treatment.

MYC is a transcription factor that controls the expression of a large fraction of cellular genes linked to cell cycle progression, metabolism and differentiation. MYC deregulation in tumors leads to its pervasive genome-wide binding of both promoters and distal regulatory regions, associated with selective transcriptional control of a large fraction of cellular genes. This pairs with alterations of cell cycle control which drive anticipated S-phase entry and reshape the DNA-replication landscape. Under these circumstances, the fine tuning of DNA replication and transcription becomes critical and may pose an intrinsic liability in MYC-overexpressing cancer cells. Here, we will review the current understanding of how MYC controls DNA and RNA synthesis, discuss evidence of replicative and transcriptional stress induced by MYC and summarize preclinical data supporting the therapeutic potential of triggering replicative stress in MYC-driven tumors.

Translesion Synthesis or Repair by Specialized DNA Polymerases Limits Excessive Genomic Instability upon Replication Stress.

DNA can experience "replication stress", an important source of genome instability, induced by various external or endogenous impediments that slow down or stall DNA synthesis. While genome instability is largely documented to favor both tumor formation and heterogeneity, as well as drug resistance, conversely, excessive instability appears to suppress tumorigenesis and is associated with improved prognosis. These findings support the view that karyotypic diversity, necessary to adapt to selective pressures, may be limited in tumors so as to reduce the risk of excessive instability. This review aims to highlight the contribution of specialized DNA polymerases in limiting extreme genetic instability by allowing DNA replication to occur even in the presence of DNA damage, to either avoid broken forks or favor their repair after collapse. These mechanisms and their key regulators Rad18 and Polθ not only offer diversity and evolutionary advantage by increasing mutagenic events, but also provide cancer cells with a way to escape anti-cancer therapies that target replication forks.

The LAMMER kinase is involved in morphogenesis and response to cell wall- and DNA-damaging stresses in Candida albicans.

Dual specificity LAMMER kinase has been reported to be conserved across species ranging from yeasts to animals and has multiple functions. Candida albicans undergoes dimorphic switching between yeast cells and hyphal growth forms as its key virulence factors. Deletion of KNS1, which encodes for LAMMER kinase in C. albicans, led to pseudohyphal growth on YPD media and defects in filamentous growth both on spider and YPD solid media containing 10% serum. These cells exhibited expanded central wrinkled regions and specifically reduced peripheral filaments. Among the several stresses tested, the kns1Δ strains showed sensitivity to cell-wall and DNA-replicative stress. Under fluorescent microscopy, an increase in chitin decomposition was observed near the bud necks and septa in kns1Δ cells. When the expression levels of genes for cell wall integrity (CWI) and the DNA repair mechanism were tested, the kns1 double-deletion cells showed abnormal patterns compared to wild-type cells; The transcript levels of genes for glycosylphosphatidylinositol (GPI)-anchored proteins were increased upon calcofluor white (CFW) treatment. Under DNA replicative stress, the expression of MluI-cell cycle box binding factor (MBF)-targeted genes, which are expressed during the G1/S transition in the cell cycle, was not increased in the kns1 double-deletion cells. This strain showed increased adhesion to the surface of an agar plate and zebrafish embryo. These results demonstrate that Kns1 is involved in dimorphic transition, cell wall integrity, response to DNA replicative stress, and adherence to the host cell surface in C. albicans.

Multiple roles of glyoxalase 1-mediated suppression of methylglyoxal glycation in cancer biology-Involvement in tumour suppression, tumour growth, multidrug resistance and target for chemotherapy.

Glyoxalase 1 (Glo1) is part of the glyoxalase system in the cytoplasm of all human cells. It catalyses the glutathione-dependent removal of the endogenous reactive dicarbonyl metabolite, methylglyoxal (MG). MG is formed mainly as a side product of anaerobic glycolysis. It modifies protein and DNA to form mainly hydroimidazolone MG-H1 and imidazopurinone MGdG adducts, respectively. Abnormal accumulation of MG, dicarbonyl stress, increases adduct levels which may induce apoptosis and replication catastrophe. In the non-malignant state, Glo1 is a tumour suppressor protein and small molecule inducers of Glo1 expression may find use in cancer prevention. Increased Glo1 expression is permissive for growth of tumours with high glycolytic activity and is thereby a biomarker of tumour growth. High Glo1 expression is a cause of multi-drug resistance. It is produced by over-activation of the Nrf2 pathway and GLO1 amplification. Glo1 inhibitors are antitumour agents, inducing apoptosis and necrosis, and anoikis. Tumour stem cells and tumours with high flux of MG formation and Glo1 expression are sensitive to Glo1 inhibitor therapy. It is likely that MG-induced cell death contributes to the mechanism of action of current antitumour agents. Common refractory tumours have high prevalence of Glo1 overexpression for which Glo1 inhibitors may improve therapy.

Uncoupling fork speed and origin activity to identify the primary cause of replicative stress phenotypes.

In growing cells, DNA replication precedes mitotic cell division to transmit genetic information to the next generation. The slowing or stalling of DNA replication forks at natural or exogenous obstacles causes "replicative stress" that promotes genomic instability and affects cellular fitness. Replicative stress phenotypes can be characterized at the single-molecule level with DNA combing or stretched DNA fibers, but interpreting the results obtained with these approaches is complicated by the fact that the speed of replication forks is connected to the frequency of origin activation. Primary alterations in fork speed trigger secondary responses in origins, and, conversely, primary alterations in the number of active origins induce compensatory changes in fork speed. Here, by employing interventions that temporally restrict either fork speed or origin firing while still allowing interrogation of the other variable, we report a set of experimental conditions to separate cause and effect in any manipulation that affects DNA replication dynamics. Using HeLa cells and chemical inhibition of origin activity (through a CDC7 kinase inhibitor) and of DNA synthesis (via the DNA polymerase inhibitor aphidicolin), we found that primary effects of replicative stress on velocity of replisomes (fork rate) can be readily distinguished from primary effects on origin firing. Identifying the primary cause of replicative stress in each case as demonstrated here may facilitate the design of methods to counteract replication stress in primary cells or to enhance it in cancer cells to increase their susceptibility to therapies that target DNA repair.

DNA replication stress: from molecular mechanisms to human disease.

The genome of proliferating cells must be precisely duplicated in each cell division cycle. Chromosomal replication entails risks such as the possibility of introducing breaks and/or mutations in the genome. Hence, DNA replication requires the coordinated action of multiple proteins and regulatory factors, whose deregulation causes severe developmental diseases and predisposes to cancer. In recent years, the concept of "replicative stress" (RS) has attracted much attention as it impinges directly on genomic stability and offers a promising new avenue to design anticancer therapies. In this review, we summarize recent progress in three areas: (1) endogenous and exogenous factors that contribute to RS, (2) molecular mechanisms that mediate the cellular responses to RS, and (3) the large list of diseases that are directly or indirectly linked to RS.

Loss of Wilms tumor 1 protein is a marker for apoptosis in response to replicative stress in leukemic cells.

A remaining expression of the transcription factor Wilms tumor 1 (WT1) after cytotoxic chemotherapy indicates remaining leukemic clones in patients. We determined the regulation and relevance of WT1 in leukemic cells exposed to replicative stress and DNA damage. To induce these conditions, we used the clinically relevant chemotherapeutics hydroxyurea and doxorubicin. We additionally treated cells with the pro-apoptotic kinase inhibitor staurosporine. Our data show that these agents promote apoptosis to a variable extent in a panel of 12 leukemic cell lines and that caspases cleave WT1 during apoptosis. A chemical inhibition of caspases as well as an overexpression of mitochondrial, anti-apoptotic BCL2 family proteins significantly reduces the processing of WT1 and cell death in hydroxyurea-sensitive acute promyelocytic leukemia cells. Although the reduction of WT1 correlates with the pharmacological efficiency of chemotherapeutics in various leukemic cells, the elimination of WT1 by different strategies of RNA interference (RNAi) does not lead to changes in the cell cycle of chronic myeloid leukemia K562 cells. RNAi against WT1 does also not increase the extent of apoptosis and the accumulation of γH2AX in K562 cells exposed to hydroxyurea. Likewise, a targeted genetic depletion of WT1 in primary oviduct cells does not increase the levels of γH2AX. Our findings position WT1 as a downstream target of the apoptotic process that occurs in response to cytotoxic forms of replicative stress and DNA damage.

The Protective Role of Dormant Origins in Response to Replicative Stress.

Genome stability requires tight regulation of DNA replication to ensure that the entire genome of the cell is duplicated once and only once per cell cycle. In mammalian cells, origin activation is controlled in space and time by a cell-specific and robust program called replication timing. About 100,000 potential replication origins form on the chromatin in the gap 1 (G1) phase but only 20⁻30% of them are active during the DNA replication of a given cell in the synthesis (S) phase. When the progress of replication forks is slowed by exogenous or endogenous impediments, the cell must activate some of the inactive or "dormant" origins to complete replication on time. Thus, the many origins that may be activated are probably key to protect the genome against replication stress. This review aims to discuss the role of these dormant origins as safeguards of the human genome during replicative stress.

DNA replication and cancer: From dysfunctional replication origin activities to therapeutic opportunities.

A dividing cell has to duplicate its DNA precisely once during the cell cycle to preserve genome integrity avoiding the accumulation of genetic aberrations that promote diseases such as cancer. A large number of endogenous impacts can challenge DNA replication and cells harbor a battery of pathways to promote genome integrity during DNA replication. This includes suppressing new replication origin firing, stabilization of replicating forks, and the safe restart of forks to prevent any loss of genetic information. Here, we describe mechanisms by which oncogenes can interfere with DNA replication thereby causing DNA replication stress and genome instability. Further, we describe cellular and systemic responses to these insults with a focus on DNA replication restart pathways. Finally, we discuss the therapeutic potential of exploiting intrinsic replicative stress in cancer cells for targeted therapy.

Balancing self-renewal against genome preservation in stem cells: How do they manage to have the cake and eat it too?

Stem cells are endowed with the awesome power of self-renewal and multi-lineage differentiation that allows them to be major contributors to tissue homeostasis. Owing to their longevity and self-renewal capacity, they are also faced with a higher risk of genomic damage compared to differentiated cells. Damage on the genome, if not prevented or repaired properly, will threaten the survival of stem cells and culminate in organ failure, premature aging, or cancer formation. It is therefore of paramount importance that stem cells remain genomically stable throughout life. Given their unique biological and functional requirement, stem cells are thought to manage genotoxic stress somewhat differently from non-stem cells. The focus of this article is to review the current knowledge on how stem cells escape the barrage of oxidative and replicative DNA damage to stay in self-renewal. A clear statement on this subject should help us better understand tissue regeneration, aging, and cancer.