High-fidelity DNA replication is critical for the faithful transmission of genetic information to daughter cells. Following genotoxic stress, specialized DNA damage tolerance pathways are activated ...to ensure replication fork progression. These pathways include translesion DNA synthesis, template switching and repriming. In this Review, we describe how DNA damage tolerance pathways impact genome stability, their connection with tumorigenesis and their effects on cancer therapy response. We discuss recent findings that single-strand DNA gap accumulation impacts chemoresponse and explore a growing body of evidence that suggests that different DNA damage tolerance factors, including translesion synthesis polymerases, template switching proteins and enzymes affecting single-stranded DNA gaps, represent useful cancer targets. We further outline how the consequences of DNA damage tolerance mechanisms could inform the discovery of new biomarkers to refine cancer therapies.
Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disease caused by a truncated lamin A protein (progerin) that drives cellular and organismal decline. HGPS patient-derived fibroblasts ...accumulate genomic instability, but its underlying mechanisms and contribution to disease remain poorly understood. Here, we show that progerin-induced replication stress (RS) drives genomic instability by eliciting replication fork (RF) stalling and nuclease-mediated degradation. Rampant RS is accompanied by upregulation of the cGAS/STING cytosolic DNA sensing pathway and activation of a robust STAT1-regulated interferon (IFN)-like response. Reducing RS and the IFN-like response, especially with calcitriol, improves the fitness of progeria cells and increases the efficiency of cellular reprogramming. Importantly, other compounds that improve HGPS phenotypes reduce RS and the IFN-like response. Our study reveals mechanisms underlying progerin toxicity, including RS-induced genomic instability and activation of IFN-like responses, and their relevance for cellular decline in HGPS.
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•Replication stress in progerin-expressing cells is caused by fork stalling and degradation•Progerin activates cGAS/STING pathway and a robust STAT1-regulated IFN-like response•Replication stress and IFN-like response contribute to HGPS cellular aging phenotypes•Calcitriol reduces replication stress and IFN-like response, rejuvenating HGPS cells
Kreienkamp et al. reveal mechanisms underlying cellular decline in the premature aging disease Hutchinson-Gilford progeria syndrome. Progerin, the mutant protein that causes this disease, elicits replication stress and a cell-intrinsic innate immune response. The study identifies strategies, such as calcitriol, that rescue these phenotypes and rejuvenate progeria cells.
Accurate DNA replication is constantly threatened by DNA lesions arising from endogenous and exogenous sources. Specialized DNA replication stress response pathways ensure replication fork ...progression in the presence of DNA lesions with minimal delay in fork elongation. These pathways broadly include translesion DNA synthesis, template switching, and replication fork repriming. Here, we discuss recent advances toward our understanding of the mechanisms that regulate the fine-tuned balance between these different replication stress response pathways. We also discuss the molecular pathways required to fill single-stranded DNA gaps that accumulate throughout the genome after repriming and the biological consequences of using repriming instead of other DNA damage tolerance pathways on genome integrity and cell fitness.
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Replication stress response pathways allow DNA replication to tolerate obstacles with minimal delay in fork elongation. In this review, Quinet et al. discuss the molecular contexts in which cells choose repriming over template switching and translesion synthesis to tolerate these obstacles and the consequences of this choice on genome integrity.
PRIMPOL repriming allows DNA replication to skip DNA lesions, leading to ssDNA gaps. These gaps must be filled to preserve genome stability. Using a DNA fiber approach to directly monitor gap ...filling, we studied the post-replicative mechanisms that fill the ssDNA gaps generated in cisplatin-treated cells upon increased PRIMPOL expression or when replication fork reversal is defective because of SMARCAL1 inactivation or PARP inhibition. We found that a mechanism dependent on the E3 ubiquitin ligase RAD18, PCNA monoubiquitination, and the REV1 and POLζ translesion synthesis polymerases promotes gap filling in G2. The E2-conjugating enzyme UBC13, the RAD51 recombinase, and REV1-POLζ are instead responsible for gap filling in S, suggesting that temporally distinct pathways of gap filling operate throughout the cell cycle. Furthermore, we found that BRCA1 and BRCA2 promote gap filling by limiting MRE11 activity and that simultaneously targeting fork reversal and gap filling enhances chemosensitivity in BRCA-deficient cells.
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•Two temporally distinct pathways fill ssDNA gaps in human cells•RAD18, PCNA monoubiquitination, and REV1-POLζ promote gap filling in G2•UBC13, RAD51, and REV1-POLζ promote gap filling in S•BRCA1 and BRCA2 promote gap filling by limiting MRE11 activity in S and G2
PRIMPOL generates ssDNA gaps, which must be filled to maintain genome stability. Tirman et al. show that two distinct pathways fill ssDNA gaps throughout the cell cycle in human cells and that BRCA proteins promote gap filling by limiting MRE11 activity. Disruption of these pathways enhances genome instability and chemosensitivity.
DNA replication forks are constantly challenged by DNA lesions induced by endogenous and exogenous sources. DNA damage tolerance mechanisms ensure that DNA replication continues with minimal effects ...on replication fork elongation either by using specialized DNA polymerases, which have the ability to replicate through the damaged template, or by skipping the damaged DNA, leaving it to be repaired after replication. These mechanisms are evolutionarily conserved in bacteria, yeast, and higher eukaryotes, and are paramount to ensure timely and faithful duplication of the genome. The
Prim
ase and DNA-directed
Pol
ymerase (PRIMPOL) is a recently discovered enzyme that possesses both primase and polymerase activities. PRIMPOL is emerging as a key player in DNA damage tolerance, particularly in vertebrate and human cells. Here, we review our current understanding of the function of PRIMPOL in DNA damage tolerance by focusing on the structural aspects that define its dual enzymatic activity, as well as on the mechanisms that control its chromatin recruitment and expression levels. We also focus on the latest findings on the mitochondrial and nuclear functions of PRIMPOL and on the impact of loss of these functions on genome stability and cell survival. Defining the function of PRIMPOL in DNA damage tolerance is becoming increasingly important in the context of human disease. In particular, we discuss recent evidence pointing at the PRIMPOL pathway as a novel molecular target to improve cancer cell response to DNA-damaging chemotherapy and as a predictive parameter to stratify patients in personalized cancer therapy.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Acute treatment with replication-stalling chemotherapeutics causes reversal of replication forks. BRCA proteins protect reversed forks from nucleolytic degradation, and their loss leads to ...chemosensitivity. Here, we show that fork degradation is no longer detectable in BRCA1-deficient cancer cells exposed to multiple cisplatin doses, mimicking a clinical treatment regimen. This effect depends on increased expression and chromatin loading of PRIMPOL and is regulated by ATR activity. Electron microscopy and single-molecule DNA fiber analyses reveal that PRIMPOL rescues fork degradation by reinitiating DNA synthesis past DNA lesions. PRIMPOL repriming leads to accumulation of ssDNA gaps while suppressing fork reversal. We propose that cells adapt to repeated cisplatin doses by activating PRIMPOL repriming under conditions that would otherwise promote pathological reversed fork degradation. This effect is generalizable to other conditions of impaired fork reversal (e.g., SMARCAL1 loss or PARP inhibition) and suggests a new strategy to modulate cisplatin chemosensitivity by targeting the PRIMPOL pathway.
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•Multiple cisplatin doses suppress reversed fork degradation in BRCA-deficient cells•The PRIMPOL adaptive response suppresses fork reversal and leads to ssDNA gaps•The ATR kinase regulates the PRIMPOL-mediated adaptive response•Impaired fork reversal shifts the balance toward PRIMPOL-mediated repriming
Reversed replication forks are degraded in BRCA-deficient cells. Quinet et al. show that treatment with multiple cisplatin doses promotes PRIMPOL repriming while suppressing fork reversal and preventing degradation. These studies identify a new function of PRIMPOL in the replication stress response and explain how cells adapt to multiple drug doses.
DNA replication is essential for cell survival, and cancer cells employ diverse DNA damage tolerance mechanisms to cope with replication stress, including Translesion DNA Synthesis (TLS), Template ...Switching (TS) and fork reversal, and re-priming. TLS utilizes lower fidelity DNA polymerases to directly bypass replication obstacles, TS involves use of the complementary sister chromatid to replicate past roadblocks, and re-priming engages an enzyme that re-initiates DNA synthesis downstream of the DNA lesion. Fork reversal is one form of TS, in which the complementary daughter strands reanneal, forming a reversed fork substrate and repositioning the DNA obstacle ahead of the replication machinery. While fork reversal is typically classified as a high-fidelity mechanism of DDT, these replication intermediates must be protected from nucleolytic processing. In cells lacking the breast cancer susceptibility proteins 1 or 2 (BRCA1 or BRCA2), these reversed replication forks are extensively degraded by nucleases. Mutations in the BRCA1 and BRCA2 genes confer an increased lifetime risk of breast and ovarian cancers, as the BRCA proteins play essential roles in preserving genome stability. Despite extensive nucleolytic degradation at reversed replication forks in the absence of BRCA1 or BRCA2, BRCA-deficient cells can activate specialized fork recovery pathways to cope with replication stress and promote resumption of DNA synthesis and cell survival. BRCA2-deficient cancer cells rely on a MUS81/POLD3-dependent fork recovery mechanism, but this same pathway does not mediate fork recovery in BRCA1-deficient cancer cells. The central aims of my thesis project are to determine the mechanism of replication fork recovery in BRCA1-deficient cancer cells and to investigate whether this pathway can be targeted to modulate cell survival and chemosensitivity. I determined that the RAD18, UBC13, and PALB2 proteins promote fork recovery in BRCA1-deficient cancer cells. RAD18 and UBC13 catalyze PCNA ubiquitination, leading to recruitment of downstream TLS polymerases or TS factors, respectively. I also demonstrate that PCNA ubiquitination is directly involved in replication fork recovery in BRCA1-deficient cancer cells. PALB2 is a canonical DNA repair protein that is also recruited to damaged chromatin in the absence of BRCA1, and my work suggests that this protein participates in fork recovery following replication stress. I show that RAD18 and PCNA ubiquitination loss lead to decreased viability of BRCA1-deficient cancer cells, and inhibition of UBC13 increases sensitivity of BRCA1-mutated ovarian cancer cells to replication stress. I also link this RAD18/UBC13/PALB2-dependent pathway of fork recovery to clinical outcomes in BRCA1-deficient ovarian cancers, as higher pathway expression is associated with worsened Progression-Free Survival. Taken together, these data highlight the potential clinical relevance of targeting fork recovery in BRCA1-deficient tumors as a therapeutic strategy and suggest possible predictive utility of measuring levels of these fork recovery factors in cancer.
Abstract BRCA1/2 proteins function in genome stability by promoting repair of double-stranded DNA breaks through homologous recombination and by protecting stalled replication forks from nucleolytic ...degradation. In BRCA1/2-deficient cancer cells, extensively degraded replication forks can be rescued through distinct fork recovery mechanisms that also promote cell survival. Here, we identified a novel pathway mediated by the E3 ubiquitin ligase RAD18, the E2-conjugating enzyme UBC13, the recombination factor PALB2, the E3 ubiquitin ligase RNF168 and PCNA ubiquitination that promotes fork recovery in BRCA1- but not BRCA2-deficient cells. We show that this pathway does not promote fork recovery by preventing replication fork reversal and degradation in BRCA1-deficient cells. We propose a mechanism whereby the RAD18–UBC13–PALB2–RNF168 axis facilitates resumption of DNA synthesis by promoting re-annealing of the complementary single-stranded template strands of the extensively degraded forks, thereby allowing re-establishment of a functional replication fork. We also provide preliminary evidence for the potential clinical relevance of this novel fork recovery pathway in BRCA1-mutated cancers, as RAD18 is over-expressed in BRCA1-deficient cancers, and RAD18 loss compromises cell viability in BRCA1-deficient cancer cells.
Poly ADP ribose polymerase inhibitors (PARPi) are most effective in BRCA1/2 mutated ovarian tumors. Better treatments are needed for homologous recombination HR-proficient cancer, including CCNE1 ...amplified subtypes. We have shown that histone deacetylase inhibitors (HDACi) sensitize HR-proficient ovarian cancer to PARPi. In this study, we provide complementary preclinical data for an investigator-initiated phase 1/2 clinical trial of the combination of olaparib and entinostat in recurrent, HR-proficient ovarian cancer.
We assessed the in vitro effects of the combination of olaparib and entinostat in SKOV-3, OVCAR-3 and primary cells derived from CCNE1 amplified high grade serous ovarian cancer (HGSOC) patients. We then tested the combination in a SKOV-3 xenograft model and in a patient-derived xenograft (PDX) model.
Entinostat potentiates the effect of olaparib in reducing cell viability and clonogenicity of HR-proficient ovarian cancer cells. The combination reduces peritoneal metastases in a SKOV-3 xenograft model and prolongs survival in a CCNE1 amplified HR-proficient PDX model. Entinostat also enhances olaparib-induced DNA damage. Further, entinostat decreases BRCA1, a key HR repair protein, associated with decreased Ki-67, a proliferation marker, and increased cleaved PARP, a marker of apoptosis. Finally, entinostat perturbs replication fork progression, which increases genome instability.
Entinostat inhibits HR repair by reducing BRCA1 expression and stalling replication fork progression, leading to irreparable DNA damage and ultimate cell death. This work provides preclinical support for the clinical trial of the combination of olaparib and entinostat in HR-proficient ovarian cancer and suggests potential benefit even for CCNE1 amplified subtypes.
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Over 80% of women with high-grade serous ovarian cancer (HGSOC) develop tumor resistance to chemotherapy and die of their disease. There are currently no FDA-approved agents to improve sensitivity to ...first-line platinum- and taxane-based chemotherapy or to PARP inhibitors. Here, we tested the hypothesis that expression of growth arrest-specific 6 (GAS6), the ligand of receptor tyrosine kinase AXL, is associated with chemotherapy response and that sequestration of GAS6 with AVB-S6-500 (AVB-500) could improve tumor response to chemotherapy and PARP inhibitors. We found that GAS6 levels in patient tumor and serum samples collected before chemotherapy correlated with ovarian cancer chemoresponse and patient survival. Compared with chemotherapy alone, AVB-500 plus carboplatin and/or paclitaxel led to decreased ovarian cancer-cell survival
and tumor burden
. Cells treated with AVB-500 plus carboplatin had more DNA damage, slower DNA replication fork progression, and fewer RAD51 foci than cells treated with carboplatin alone, indicating AVB-500 impaired homologous recombination (HR). Finally, treatment with the PARP inhibitor olaparib plus AVB-500 led to decreased ovarian cancer-cell survival
and less tumor burden
. Importantly, this effect was seen in HR-proficient and HR-deficient ovarian cancer cells. Collectively, our findings suggest that GAS6 levels could be used to predict response to carboplatin and AVB-500 could be used to treat platinum-resistant, HR-proficient HGSOC. IMPLICATIONS: GAS6/AXL is a novel target to sensitize ovarian cancers to carboplatin and olaparib. Additionally, GAS6 levels can be associated with response to carboplatin treatment.