Accurate replication of DNA requires stringent regulation to ensure genome integrity. In human cells, thousands of origins of replication are coordinately activated during S phase, and the velocity of replication forks is adjusted to fully replicate DNA in pace with the cell cycle. Replication stress induces fork stalling and fuels genome instability. The mechanistic basis of replication stress remains poorly understood despite its emerging role in promoting cancer. Here we show that inhibition of poly(ADP-ribose) polymerase (PARP) increases the speed of fork elongation and does not cause fork stalling, which is in contrast to the accepted model in which inhibitors of PARP induce fork stalling and collapse. Aberrant acceleration of fork progression by 40% above the normal velocity leads to DNA damage. Depletion of the treslin or MTBP proteins, which are involved in origin firing, also increases fork speed above the tolerated threshold, and induces the DNA damage response pathway. Mechanistically, we show that poly(ADP-ribosyl)ation (PARylation) and the PCNA interactor p21 (p21) are crucial modulators of fork progression. PARylation and p21 act as suppressors of fork speed in a coordinated regulatory network that is orchestrated by the PARP1 and p53 proteins. Moreover, at the fork level, PARylation acts as a sensor of replication stress. During PARP inhibition, DNA lesions that induce fork arrest and are normally resolved or repaired remain unrecognized by the replication machinery. Conceptually, our results show that accelerated replication fork progression represents a general mechanism that triggers replication stress and the DNA damage response. Our findings contribute to a better understanding of the mechanism of fork speed control, with implications for genomic (in)stability and rational cancer treatment.

Both Myc and Ras oncogenes impact cellular metabolism, deregulate redox homeostasis and trigger DNA replication stress (RS) that compromises genomic integrity. However, how are such oncogene‐induced effects evoked and temporally related, to what extent are these kinetic parameters shared by Myc and Ras, and how are these cellular changes linked with oncogene‐induced cellular senescence in different cell context(s) remain poorly understood. Here, we addressed the above‐mentioned open questions by multifaceted comparative analyses of human cellular models with inducible expression of c‐Myc and H‐RasV12 (Ras), two commonly deregulated oncoproteins operating in a functionally connected signaling network. Our study of DNA replication parameters using the DNA fiber approach and time‐course assessment of perturbations in glycolytic flux, oxygen consumption and production of reactive oxygen species (ROS) revealed the following results. First, overabundance of nuclear Myc triggered RS promptly, already after one day of Myc induction, causing slow replication fork progression and fork asymmetry, even before any metabolic changes occurred. In contrast, Ras overexpression initially induced a burst of cell proliferation and increased the speed of replication fork progression. However, after several days of induction Ras caused bioenergetic metabolic changes that correlated with slower DNA replication fork progression and the ensuing cell cycle arrest, gradually leading to senescence. Second, the observed oncogene‐induced RS and metabolic alterations were cell‐type/context dependent, as shown by comparative analyses of normal human BJ fibroblasts versus U2‐OS sarcoma cells. Third, the energy metabolic reprogramming triggered by Ras was more robust compared to impact of Myc. Fourth, the detected oncogene‐induced oxidative stress was due to ROS (superoxide) of non‐mitochondrial origin and mitochondrial OXPHOS was reduced (Crabtree effect). Overall, our study provides novel insights into oncogene‐evoked metabolic reprogramming, replication and oxidative stress, with implications for mechanisms of tumorigenesis and potential targeting of oncogene addiction.

the whole E13,5 brain and in the olfactory bulbs (OB) of E18,5 brain (Fig. 1b, Extended Data Fig. 1d, e). Also, neural stem cells (NSCs) isolated from Ambra1 cKO mice show increased levels of several cell-cycle regulatory proteins (Fig. 1c, Extended Data Fig. 1f, g), together with higher clonogenic potential and replication rate (Fig 1d, Extended Data Fig. 1h). Strikingly, levels of cyclin D1 and D2 proteins and phosphorylated pRb (S807/811) are highly increased both ex and in vivo (Fig. 1c, e, Extended Data Fig. 1g, i-m), suggesting an AMBRA1dependent Cyclin D modulation. Indeed, consistent with our previous results 7 , we find in neural ex vivo and in vitro cell lines that AMBRA1 directly binds and regulates the stability of N-Myc, via the phosphatase PP2A, thereby controlling Cyclin D1 and D2 transcription (Extended Data Fig. 1n-r). Moreover, we noticed that both cyclin D1 and D2 are highly resilient to proteasomal degradation in Ambra1-deficiency conditions (Fig. 1f, Extended Data Fig. 2a, b). In line with the fact that both Myc and D-type cyclins positively regulate G1/S transition 10,11 , Ambra1 cKO NSCs show a shorter G1 phase with faster entry into, and longer residence in S phase (Extended Data Fig. 2c). By reducing cyclin D/CDK kinase activity we could restore proliferation to wt levels (Extended Data Fig. 2d), highlighting the importance of accelerated G1/S transition in the AMBRA1depleted driven phenotype. Additionally, we found that due to Ambra1 deficiency, deregulated cell cycle progression is followed by increased cell death, a phenotype rescued upon cyclin D/CDK activity inhibition (Extended Data Fig. 2e, f). Of note, Ambra1 deficiency in neurodevelopment promotes staminal niche

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.

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.

Medulloblastomas are the most common, and often fatal, paediatric brain tumours that feature high genomic instability, frequent infection by human cytomegalovirus (HCMV) and resistance to radiation and chemotherapy. The causes of the pronounced chromosomal instability and its potential links with HCMV infection and/or resistance to genotoxic therapies remain largely unknown. To address these issues, here we have combined immunohistochemical analysis of a series of 25 paediatric medulloblastomas, complemented by medulloblastoma cell culture models including experimental HCMV infection. Using eight established immunohistochemical markers to assess the status of the DDR machinery, we found pronounced endogenous DNA damage signalling (cH2AX marker) and robust constitutive activation of both the ATM-Chk2 and ATR-Chk1 DNA damage checkpoint kinase cascades, yet unexpectedly modest p53 tumour suppressor activation, across our medulloblastoma cohort. Most tumours showed high proliferation (Ki67 marker), variable oxidative DNA damage (8-oxoguanine lesions) and formation of 53BP1 nuclear 'bodies', the latter indicating (along with ATR-Chk1 signalling) endogenous replication stress. The bulk of the clinical specimens also showed expression of HCMV immediate early and late proteins, in comparative analyses using three immunohistochemical protocols. Cell culture experiments validated the chronic endogenous replication stress in medulloblastoma cell lines and showed sharply differential, intriguing responses of normal cells and medulloblastoma cells to HCMV infection, including differential subcellular mislocalization and enhancement of replication stress-related 53BP1 body formation, the latter in cell-non-autonomous manner. Overall, our results strongly indicate that Abbreviations 53BP1, p53-binding protein 1; ATCC, American Type Culture Collection; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia mutated and Rad3 related; Chk1, checkpoint kinase 1; Chk2, checkpoint kinase 2; COX-2, cyclooxygenase-2; DDR, DNA damage response; FBS, fetal bovine serum; FISH, fluorescence in situ hybridization; GFAP, glial fibrillary acidic protein; HCMV, human cytomegalovirus; IDH, isocitrate dehydrogenase; IE72, immediate early 72; Ini-1, integrase interactor 1; MAP-2, microtubule-associated protein 2; MDC1, mediator of DNA damage checkpoint protein 1; MOI, multiplicity of infection; NGS, next-generation sequencing; Olig2, oligodendrocyte transcription factor 2; PBS, phosphate-buffered saline; PGE2, prostaglandin E2; SHH, sonic hedgehog; WNT, wingless; γH2AX, phosphorylated histone H2AX. in human medulloblastomas, the DDR checkpoint barrier is widely activated, at least in part due to replication stress. Furthermore, we propose that unorthodox p53 defects other than mutations may allow high proliferation despite the ongoing checkpoint signalling and that the highly prevalent HCMV may impact the medulloblastoma host cell replication stress and DNA repair. Collectively, the scenario we report here likely fuels genomic instability ...

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Mammalian cells have mechanisms to counteract the effects of metabolic and exogenous stresses, many of that would be mutagenic if ignored. Damage arising during DNA replication is a major source of mutagenesis. The extent of damage dictates whether cells undergo transient cell cycle arrest and damage repair, senescence or apoptosis. Existing dogma defines these alternative fates as distinct choices. Here we show that immortalised breast epithelial cells are able to survive prolonged S phase arrest and subsequently re-enter cycle after many days of being in an arrested, senescence-like state. Prolonged cell cycle inhibition in fibroblasts induced DNA damage response and cell death. However, in immortalised breast epithelia, efficient S phase arrest minimised chromosome damage and protected sufficient chromatin-bound replication licensing complexes to allow cell cycle re-entry. We propose that our observation could have implications for the design of drug therapies for breast cancer.