Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) are currently used to treat BRCA1/2 mutant cancers. Although PARPi sensitivity has been attributed to homologous recombination (HR) defects, other roles of HR factors have also been linked to response to PARPi, including replication fork protection. In this study, we investigated PARPi sensitivity in ovarian cancer patient-derived xenograft (PDX) models in relation to HR proficiency and replication fork protection. Analysis of BRCA1/2 status showed that in our cohort of 31 ovarian cancer PDX models 22.6% harbored a BRCA1/2 alteration (7/31), and 48.3% (15/31) were genomically unstable as measured by copy number alteration analysis. In vivo, PARPi olaparib response was measured in 15 selected PDX models. Functional assessment of HR using ex vivo irradiation-induced RAD51 foci formation identified all olaparib-sensitive PDX models, including four models without BRCA1/2 alterations. In contrast, replication fork protection or replication speed in ex vivo tumor tissue did not correlate with olaparib response. Targeted panel sequencing in olaparib-sensitive models lacking BRCA1/2 alterations revealed a MUS81 variant as a possible mechanism underlying PARPi sensitivity. Combined, we show that ex vivo RAD51 analysis effectively predicts in vivo olaparib response and revealed a subset of PARPi-sensitive, HR-deficient ovarian cancer PDX models, lacking a BRCA1/2 alteration.

• Publication type
• Journal Article MeSH

PARP inhibitors (PARPi), known for their ability to induce replication gaps and accelerate replication forks, have become potent agents in anticancer therapy. However, the molecular mechanism underlying PARPi-induced fork acceleration has remained elusive. Here, we show that the first PARPi-induced effect on DNA replication is an increased replication fork rate, followed by a secondary reduction in origin activity. Through the systematic knockdown of human DNA polymerases, we identify POLA1 as mediator of PARPi-induced fork acceleration. This acceleration depends on both DNA polymerase α and primase activities. Additionally, the depletion of POLA1 increases the accumulation of replication gaps induced by PARP inhibition, sensitizing cells to PARPi. BRCA1-depleted cells are especially susceptible to the formation of replication gaps under POLA1 inhibition. Accordingly, BRCA1 deficiency sensitizes cells to POLA1 inhibition. Thus, our findings establish the POLA complex as important player in PARPi-induced fork acceleration and provide evidence that lagging strand synthesis represents a targetable vulnerability in BRCA1-deficient cells.

• MeSH
• DNA-Directed DNA Polymerase metabolism   genetics   MeSH
• DNA Polymerase I MeSH
• DNA Primase * metabolism   genetics   MeSH
• DNA, Single-Stranded * metabolism   genetics   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Poly(ADP-ribose) Polymerase Inhibitors * pharmacology   MeSH
• BRCA1 Protein * metabolism   genetics   MeSH
• DNA Replication * drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• BRCA1 protein, human MeSH Browser
• DNA polymerase alpha-primase MeSH Browser
• DNA-Directed DNA Polymerase MeSH
• DNA Polymerase I MeSH
• DNA Primase * MeSH
• DNA, Single-Stranded * MeSH
• Poly(ADP-ribose) Polymerase Inhibitors * MeSH
• BRCA1 Protein * MeSH

Accurate and complete replication of genetic information is a fundamental process of every cell division. The replication licensing is the first essential step that lays the foundation for error-free genome duplication. During licensing, minichromosome maintenance protein complexes, the molecular motors of DNA replication, are loaded to genomic sites called replication origins. The correct quantity and functioning of licensed origins are necessary to prevent genome instability associated with severe diseases, including cancer. Here, we delve into recent discoveries that shed light on the novel functions of licensed origins, the pathways necessary for their proper maintenance, and their implications for cancer therapies.

• MeSH
• Humans MeSH
• Minichromosome Maintenance Proteins genetics   metabolism   MeSH
• Neoplasms * genetics   MeSH
• DNA Replication * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Minichromosome Maintenance Proteins MeSH

DNA synthesis of the leading and lagging strands works independently and cells tolerate single-stranded DNA generated during strand uncoupling if it is protected by RPA molecules. Natural alkaloid emetine is used as a specific inhibitor of lagging strand synthesis, uncoupling leading and lagging strand replication. Here, by analysis of lagging strand synthesis inhibitors, we show that despite emetine completely inhibiting DNA replication: it does not induce the generation of single-stranded DNA and chromatin-bound RPA32 (CB-RPA32). In line with this, emetine does not activate the replication checkpoint nor DNA damage response. Emetine is also an inhibitor of proteosynthesis and ongoing proteosynthesis is essential for the accurate replication of DNA. Mechanistically, we demonstrate that the acute block of proteosynthesis by emetine temporally precedes its effects on DNA replication. Thus, our results are consistent with the hypothesis that emetine affects DNA replication by proteosynthesis inhibition. Emetine and mild POLA1 inhibition prevent S-phase poly(ADP-ribosyl)ation. Collectively, our study reveals that emetine is not a specific lagging strand synthesis inhibitor with implications for its use in molecular biology.

• MeSH
• Chromatin MeSH
• DNA genetics   MeSH
• Emetine * pharmacology   MeSH
• DNA, Single-Stranded * MeSH
• DNA Replication MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• DNA MeSH
• Emetine * MeSH
• DNA, Single-Stranded * MeSH
• Okazaki fragments MeSH Browser

Viral infections enhance cancer risk and threaten host genome integrity. Although human cytomegalovirus (HCMV) proteins have been detected in a wide spectrum of human malignancies and HCMV infections have been implicated in tumorigenesis, the underlying mechanisms remain poorly understood. Here, we employed a range of experimental approaches, including single-molecule DNA fiber analysis, and showed that infection by any of the four commonly used HCMV strains: AD169, Towne, TB40E or VR1814 induced replication stress (RS), as documented by host-cell replication fork asymmetry and formation of 53BP1 foci. The HCMV-evoked RS triggered an ensuing host DNA damage response (DDR) and chromosomal instability in both permissive and non-permissive human cells, the latter being particularly relevant in the context of tumorigenesis, as such cells can survive and proliferate after HCMV infection. The viral major immediate early enhancer and promoter (MIEP) that controls expression of the viral genes IE72 (IE-1) and IE86 (IE-2), contains transcription-factor binding sites shared by promoters of cellular stress-response genes. We found that DNA damaging insults, including those relevant for cancer therapy, enhanced IE72/86 expression. Thus, MIEP has been evolutionary shaped to exploit host DDR. Ectopically expressed IE72 and IE86 also induced RS and increased genomic instability. Of clinical relevance, we show that undergoing standard-of-care genotoxic radio-chemotherapy in patients with HCMV-positive glioblastomas correlated with elevated HCMV protein markers after tumor recurrence. Collectively, these results are consistent with our proposed concept of HCMV hijacking transcription-factor binding sites shared with host stress-response genes. We present a model to explain the potential oncomodulatory effects of HCMV infections through enhanced replication stress, subverted DNA damage response and induced genomic instability.

• MeSH
• Cytomegalovirus * genetics   metabolism   MeSH
• Carcinogenesis genetics   MeSH
• Humans MeSH
• Genomic Instability MeSH
• DNA Damage * MeSH
• Promoter Regions, Genetic MeSH
• Virus Replication MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Poly(ADP-ribose) polymerase 1 (PARP1) is implicated in the detection and processing of unligated Okazaki fragments and other DNA replication intermediates, highlighting such structures as potential sources of genome breakage induced by PARP inhibition. Here, we show that PARP1 activity is greatly elevated in chicken and human S phase cells in which FEN1 nuclease is genetically deleted and is highest behind DNA replication forks. PARP inhibitor reduces the integrity of nascent DNA strands in both wild-type chicken and human cells during DNA replication, and does so in FEN1-/- cells to an even greater extent that can be detected as postreplicative single-strand nicks or gaps. Collectively, these data show that PARP inhibitors impede the maturation of nascent DNA strands during DNA replication, and implicate unligated Okazaki fragments and other nascent strand discontinuities in the cytotoxicity of these compounds.

• MeSH
• DNA genetics   MeSH
• DNA Repair MeSH
• Poly(ADP-ribose) Polymerase Inhibitors * pharmacology   MeSH
• Poly (ADP-Ribose) Polymerase-1 genetics   MeSH
• DNA Damage MeSH
• DNA Replication * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Poly(ADP-ribose) Polymerase Inhibitors * MeSH
• Poly (ADP-Ribose) Polymerase-1 MeSH

Ribosome biogenesis is an essential, energy demanding process whose deregulation has been implicated in cancer, aging, and neurodegeneration. Ribosome biogenesis is therefore under surveillance of pathways including the p53 tumor suppressor. Here, we first performed a high-content siRNA-based screen of 175 human ribosome biogenesis factors, searching for impact on p53. Knock-down of 4 and 35 of these proteins in U2OS cells reduced and increased p53 abundance, respectively, including p53 accumulation after depletion of BYSL, DDX56, and WDR75, the effects of which were validated in several models. Using complementary approaches including subcellular fractionation, we demonstrate that endogenous human WDR75 is a nucleolar protein and immunofluorescence analysis of ectopic GFP-tagged WDR75 shows relocation to nucleolar caps under chemically induced nucleolar stress, along with several canonical nucleolar proteins. Mechanistically, we show that WDR75 is required for pre-rRNA transcription, through supporting the maintenance of physiological levels of RPA194, a key subunit of the RNA polymerase I. Furthermore, WDR75 depletion activated the RPL5/RPL11-dependent p53 stabilization checkpoint, ultimately leading to impaired proliferation and cellular senescence. These findings reveal a crucial positive role of WDR75 in ribosome biogenesis and provide a resource of human ribosomal factors the malfunction of which affects p53.

• MeSH
• Cell Nucleolus genetics   metabolism   MeSH
• DEAD-box RNA Helicases metabolism   MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Humans MeSH
• Cell Adhesion Molecules metabolism   MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• RNA Precursors metabolism   MeSH
• Ribosomal Proteins * genetics   metabolism   MeSH
• Ribosomes genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• BYSL protein, human MeSH Browser
• DDX56 protein, human MeSH Browser
• DEAD-box RNA Helicases MeSH
• Nuclear Proteins MeSH
• Cell Adhesion Molecules MeSH
• Tumor Suppressor Protein p53 MeSH
• RNA Precursors MeSH
• Ribosomal Proteins * MeSH
• TP53 protein, human MeSH Browser

Autophagy is an evolutionarily conserved process that captures aberrant intracellular proteins and/or damaged organelles for delivery to lysosomes, with implications for cellular and organismal homeostasis, aging and diverse pathologies, including cancer. During cancer development, autophagy may play both tumour-supporting and tumour-suppressing roles. Any relationships of autophagy to the established oncogene-induced replication stress (RS) and the ensuing DNA damage response (DDR)-mediated anti-cancer barrier in early tumorigenesis remain to be elucidated. Here, assessing potential links between autophagy, RS and DDR, we found that autophagy is enhanced in both early and advanced stages of human urinary bladder and prostate tumorigenesis. Furthermore, a high-content, single-cell-level microscopy analysis of human cellular models exposed to diverse genotoxic insults showed that autophagy is enhanced in cells that experienced robust DNA damage, independently of the cell-cycle position. Oncogene- and drug-induced RS triggered first DDR and later autophagy. Unexpectedly, genetic inactivation of autophagy resulted in RS, despite cellular retention of functional mitochondria and normal ROS levels. Moreover, recovery from experimentally induced RS required autophagy to support DNA synthesis. Consistently, RS due to the absence of autophagy could be partly alleviated by exogenous supply of deoxynucleosides. Our results highlight the importance of autophagy for DNA synthesis, suggesting that autophagy may support cancer progression, at least in part, by facilitating tumour cell survival and fitness under replication stress, a feature shared by most malignancies. These findings have implications for better understanding of the role of autophagy in tumorigenesis, as well as for attempts to manipulate autophagy as an anti-tumour therapeutic strategy.

• MeSH
• Autophagy * MeSH
• Autophagosomes drug effects   metabolism   MeSH
• Models, Biological MeSH
• Stress, Physiological * drug effects   MeSH
• Camptothecin pharmacology   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Oncogenes * MeSH
• DNA Replication * drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Camptothecin MeSH

Research on repurposing the old alcohol-aversion drug disulfiram (DSF) for cancer treatment has identified inhibition of NPL4, an adaptor of the p97/VCP segregase essential for turnover of proteins involved in multiple pathways, as an unsuspected cancer cell vulnerability. While we reported that NPL4 is targeted by the anticancer metabolite of DSF, the bis-diethyldithiocarbamate-copper complex (CuET), the exact, apparently multifaceted mechanism(s) through which the CuET-induced aggregation of NPL4 kills cancer cells remains to be fully elucidated. Given the pronounced sensitivity to CuET in tumor cell lines lacking the genome integrity caretaker proteins BRCA1 and BRCA2, here we investigated the impact of NPL4 targeting by CuET on DNA replication dynamics and DNA damage response pathways in human cancer cell models. Our results show that CuET treatment interferes with DNA replication, slows down replication fork progression and causes accumulation of single-stranded DNA (ssDNA). Such a replication stress (RS) scenario is associated with DNA damage, preferentially in the S phase, and activates the homologous recombination (HR) DNA repair pathway. At the same time, we find that cellular responses to the CuET-triggered RS are seriously impaired due to concomitant malfunction of the ATRIP-ATR-CHK1 signaling pathway that reflects an unorthodox checkpoint silencing mode through ATR (Ataxia telangiectasia and Rad3 related) kinase sequestration within the CuET-evoked NPL4 protein aggregates.

• Keywords
• ATR pathway, BRCA1, BRCA2, DNA damage, NPL4, disulfiram, replication stress, targeted cancer therapy,
• MeSH
• Adaptor Proteins, Signal Transducing metabolism   MeSH
• Ataxia Telangiectasia Mutated Proteins antagonists & inhibitors   metabolism   MeSH
• Checkpoint Kinase 1 metabolism   MeSH
• Disulfiram pharmacology   MeSH
• DNA-Binding Proteins metabolism   MeSH
• Nuclear Proteins antagonists & inhibitors   metabolism   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Neoplasms metabolism   pathology   MeSH
• Alcohol Deterrents pharmacology   MeSH
• Protein Aggregation, Pathological chemically induced   MeSH
• DNA Damage drug effects   MeSH
• Valosin Containing Protein metabolism   MeSH
• Protein Aggregates drug effects   MeSH
• DNA Replication drug effects   MeSH
• Signal Transduction drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adaptor Proteins, Signal Transducing MeSH
• Ataxia Telangiectasia Mutated Proteins MeSH
• ATR protein, human MeSH Browser
• ATRIP protein, human MeSH Browser
• Checkpoint Kinase 1 MeSH
• CHEK1 protein, human MeSH Browser
• Disulfiram MeSH
• DNA-Binding Proteins MeSH
• Nuclear Proteins MeSH
• NPLOC4 protein, human MeSH Browser
• Alcohol Deterrents MeSH
• Valosin Containing Protein MeSH
• Protein Aggregates MeSH
• VCP protein, human MeSH Browser

The Wee1 inhibitor MK1775 (AZD1775) is currently being tested in clinical trials for cancer treatment. Here, we show that the p53 target and CDK inhibitor p21 protects against MK1775-induced DNA damage during S-phase. Cancer and normal cells deficient for p21 (HCT116 p21-/-, RPE p21-/-, and U2OS transfected with p21 siRNA) showed higher induction of the DNA damage marker γH2AX in S-phase in response to MK1775 compared to the respective parental cells. Furthermore, upon MK1775 treatment the levels of phospho-DNA PKcs S2056 and phospho-RPA S4/S8 were higher in the p21 deficient cells, consistent with increased DNA breakage. Cell cycle analysis revealed that these effects were due to an S-phase function of p21, but MK1775-induced S-phase CDK activity was not altered as measured by CDK-dependent phosphorylations. In the p21 deficient cancer cells MK1775-induced cell death was also increased. Moreover, p21 deficiency sensitized to combined treatment of MK1775 and the CHK1-inhibitor AZD6772, and to the combination of MK1775 with ionizing radiation. These results show that p21 protects cancer cells against Wee1 inhibition and suggest that S-phase functions of p21 contribute to mediate such protection. As p21 can be epigenetically downregulated in human cancer, we propose that p21 levels may be considered during future applications of Wee1 inhibitors.

• Keywords
• CDK activity, DNA damage, Wee1 kinase, cancer treatment, checkpoint kinase inhibition, p21 (Cip1/Waf1),
• MeSH
• Checkpoint Kinase 1 antagonists & inhibitors   MeSH
• Cyclin-Dependent Kinases antagonists & inhibitors   metabolism   MeSH
• Phosphorylation drug effects   MeSH
• HCT116 Cells MeSH
• Cyclin-Dependent Kinase Inhibitor p21 genetics   metabolism   MeSH
• S Phase Cell Cycle Checkpoints drug effects   MeSH
• Humans MeSH
• RNA, Small Interfering genetics   MeSH
• Neoplasms drug therapy   metabolism   MeSH
• DNA Damage drug effects   genetics   MeSH
• Cell Cycle Proteins antagonists & inhibitors   MeSH
• Antineoplastic Agents pharmacology   therapeutic use   MeSH
• Pyrazoles pharmacology   therapeutic use   MeSH
• Pyrimidinones pharmacology   therapeutic use   MeSH
• Transfection MeSH
• Protein-Tyrosine Kinases antagonists & inhibitors   MeSH
• Cell Survival drug effects   genetics   radiation effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• adavosertib MeSH Browser
• CDKN1A protein, human MeSH Browser
• Checkpoint Kinase 1 MeSH
• CHEK1 protein, human MeSH Browser
• Cyclin-Dependent Kinases MeSH
• Cyclin-Dependent Kinase Inhibitor p21 MeSH
• RNA, Small Interfering MeSH
• Cell Cycle Proteins MeSH
• Antineoplastic Agents MeSH
• Pyrazoles MeSH
• Pyrimidinones MeSH
• Protein-Tyrosine Kinases MeSH
• WEE1 protein, human MeSH Browser