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Common fragile sites (CFSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Several features were suggested to underlie CFS instability, however, these features are prevalent across the genome. Therefore, the molecular mechanisms underlying CFS instability remain unclear. Here, we explore the transcriptional profile and DNA replication timing (RT) under mild replication stress in the context of the 3D genome organization. The results reveal a fragility signature, comprised of a TAD boundary overlapping a highly transcribed large gene with APH-induced RT-delay. This signature enables precise mapping of core fragility regions in known CFSs and identification of novel fragile sites. CFS stability may be compromised by incomplete DNA replication and repair in TAD boundaries core fragility regions leading to genomic instability. The identified fragility signature will allow for a more comprehensive mapping of CFSs and pave the way for investigating mechanisms promoting genomic instability in cancer. Common fragile sites are regions susceptible to replication stress and are prone to chromosomal instability. Here, the authors, by analyzing the contribution of 3D chromatin organization, identify and characterize a fragility signature and precisely map these fragility regions.

Genomic instability plays a key role in driving cancer development. It is already found in precancerous lesions and allows the acquisition of additional cancerous features. A major source of genomic instability in early stages of tumorigenesis is DNA replication stress. Normally, origin licensing and activation, as well as replication fork progression, are tightly regulated to allow faithful duplication of the genome. Aberrant origin usage and/or perturbed replication fork progression leads to DNA damage and genomic instability. Oncogene activation is an endogenous source of replication stress, disrupting replication regulation and inducing DNA damage. Oncogene-induced replication stress and its role in cancer development have been studied comprehensively, however its molecular basis is still unclear. Here, we review the current understanding of replication regulation, its potential disruption and how oncogenes perturb the replication and induce DNA damage leading to genomic instability in cancer.

Abstract DNA replication is a complex process that is tightly regulated to ensure faithful genome duplication, and its perturbation leads to DNA damage and genomic instability. Replication stress is commonly associated with slow and stalled replication forks. Recently, accelerated replication has emerged as a non-canonical form of replication stress. However, the molecular basis underlying fork acceleration is largely unknown. Here we show that increased topoisomerase 1 (TOP1) expression induces aberrant replication fork acceleration and DNA damage by decreasing RNA-DNA hybrids (R-loops). Degradation of R-loops by overexpression of RNaseH1 also accelerates replication and generates DNA damage. Furthermore, upregulation of TOP1 by activation of the mutated HRAS oncogene leads to fork acceleration and DNA damage in pre-senescent cells. In these cells, restoration of TOP1 expression level or mild replication inhibition rescues the perturbed replication and reduces DNA damage. These findings highlight the importance of TOP1 equilibrium in the regulation of R-loop homeostasis to ensure faithful DNA replication and genome integrity. Competing Interest Statement The authors have declared no competing interest.

Common fragile sties (CFSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Several features characterizing CFSs have been associated with their instability, however, these features are prevalent across the genome and do not account for all known CFSs. Therefore, the molecular mechanism underlying CFS instability remains unclear. Here, we explored the transcriptional profile and temporal order of DNA replication (replication timing, RT) of cells under replication stress conditions. We show that the RT of only a small portion of the genome is affected by replication stress, and that CFSs are enriched for delayed RT. We identified a signature for chromosomal fragility, comprised of replication stress-induced delay in RT of early/mid S-phase replicating regions within actively transcribed large genes. This fragility signature enabled precise mapping of the core fragility region. Furthermore, the signature enabled the identification of novel fragile sites that were not detected cytogenetically, highlighting the improved sensitivity of our approach for identifying fragile sites. Altogether, this study reveals a link between altered DNA replication and transcription of large genes underlying the mechanism of CFS expression. Thus, investigating the RT and transcriptional changes in cancer may contribute to the understanding of mechanisms promoting genomic instability in cancer. Footnotes * BK was updated as a corresponding author.

Chronic obstructive pulmonary disease (COPD) exacerbations are associated with increased risk of major adverse cardiovascular events (MACE) and mortality. Here, we investigate whether the safety and efficacy of aclidinium bromide differ due to exacerbation history in patients with COPD and increased cardiovascular risk. ASCENT-COPD was a Phase 4, multicenter, double-blind, randomized, placebo-controlled, parallel-group study of patients with moderate-to-very severe COPD and increased cardiovascular risk. Patients were randomized 1:1 to receive aclidinium or placebo twice daily for up to 3 years. Outcomes included time to first MACE and all-cause mortality over 3 years, exacerbation rate during the first year on-treatment, and change in baseline pre-dose forced expiratory volume in 1 second (FEV ) over 3 years. This pre-specified subgroup analysis compared outcomes in patients receiving aclidinium vs placebo. The comparison of patients with vs without an exacerbation history was added following a protocol amendment to increase enrollment in the primary study. Of 3589 patients, 2156 (60.1%) had ≥1 moderate or severe exacerbations in the prior year, compared with 1433 (39.9%) without prior exacerbations. Although patients with an exacerbation history had numerically higher rates of MACE and mortality regardless of treatment, aclidinium did not increase risk of MACE (≥1: hazard ratio [HR] 0.79, 95% confidence interval [CI]: 0.54-1.16; none: HR 1.27, 95% CI: 0.65-2.47; interaction =0.233) or all-cause mortality (≥1: HR 1.08, 95% CI: 0.81-1.43; none: HR 0.66, 95% CI: 0.36-1.22; interaction =0.154), regardless of exacerbation history. Aclidinium reduced the exacerbation rate vs placebo irrespective of exacerbation history (≥1: rate ratio [RR] 0.80, 95% CI: 0.68-0.94; none: RR 0.69, 95% CI: 0.54-0.89; interaction =0.340) and improved FEV (interaction =0.633). In patients with moderate-to-very severe COPD and increased cardiovascular risk, aclidinium did not increase risk of MACE or mortality and reduced exacerbation rate vs placebo, regardless of exacerbation history. ClinicalTrials.gov Identifier: NCT01966107.