Aneuploidy is a hallmark of cancer and underlies genetic disorders characterized by severe developmental defects, yet the molecular mechanisms explaining its effects on cellular physiology remain elusive. Here we show, using a series of human cells with defined aneuploid karyotypes, that gain of a single chromosome increases genomic instability. Next-generation sequencing and SNP-array analysis reveal accumulation of chromosomal rearrangements in aneuploids, with break point junction patterns suggestive of replication defects. Trisomic and tetrasomic cells also show increased DNA damage and sensitivity to replication stress. Strikingly, we find that aneuploidy-induced genomic instability can be explained by the reduced expression of the replicative helicase MCM2-7. Accordingly, restoring near-wild-type levels of chromatin-bound MCM helicase partly rescues the genomic instability phenotypes. Thus, gain of chromosomes triggers replication stress, thereby promoting genomic instability and possibly contributing to tumorigenesis.

Perturbed DNA replication in early stages of cancer development induces chromosomal instability preferentially at fragile sites. However, the molecular basis for this instability is unknown. Here, we show that even under normal growth conditions, replication fork progression along the fragile site, FRA16C, is slow and forks frequently stall at AT-rich sequences, leading to activation of additional origins to enable replication completion. Under mild replication stress, the frequency of stalling at AT-rich sequences is further increased. Strikingly, unlike in the entire genome, in the FRA16C region additional origins are not activated, suggesting that all potential origins are already activated under normal conditions. Thus, the basis for FRA16C fragility is replication fork stalling at AT-rich sequences and inability to activate additional origins under replication stress. Our results provide a mechanism explaining the replication stress sensitivity of fragile sites and thus, the basis for genomic instability during early stages of cancer development.

No abstract

Common fragile sites are specific genomic loci that form constrictions and gaps on metaphase chromosomes under conditions that slow, but do not arrest, DNA replication. These sites have been shown to have a role in various chromosomal rearrangements in tumors. Different DNA damage response proteins were shown to regulate fragile site stability, including ataxia-telangiectasia and Rad3-related (ATR) and its effector Chk1. Here, we investigated the role of ataxia-telangiectasia mutated (ATM), the main transducer of DNA double-strand break (DSB) signal, in this regulation. We demonstrate that replication stress conditions, which induce fragile site expression, lead to DNA fragmentation and recruitment of phosphorylated ATM to nuclear foci at DSBs. We further show that ATM plays a role in maintaining fragile site stability, which is revealed only in the absence of ATR. However, the activation of ATM under these replication stress conditions is ATR independent. Following conditions that induce fragile site expression both ATR and ATM phosphorylate Chk1, suggesting that both proteins regulate fragile site expression probably via their effect on Chk1 activation. Our findings provide new insights into the interplay between ATR and ATM pathways in response to partial replication inhibition and in the regulation of fragile site stability.

Background: Antisense oligonucleotide (ASO)-based drugs for splicing modulation were recently approved for various genetic diseases with unmet need. Here we aimed to develop an ASO-based splicing modulation therapy for Cystic Fibrosis (CF) patients carrying the 3849 + 10 kb C-to-T splicing mutation in the CFTR gene. Methods:We have screened, in FRT cells expressing the 3849 + 10 kb C-to-T splicing mutation, ~30 2 -O-Methyl-modified phosphorothioate ASOs, targeted to prevent the recognition and inclusion of a cryptic exon generated due to the mutation. The effect of highly potent ASO candidates on the splicing pattern, protein maturation and CFTR function was further analyzed in well differentiated primary human nasal and bronchial epithelial cells, derived from patients carrying at least one 3849 + 10 kb C-to-T allele.Results: A highly potent lead ASO, efficiently delivered by free uptake, was able to significantly increase the level of correctly spliced mRNA and completely restore the CFTR function to wild type levels in cells from a homozygote patient. This ASO led to CFTR function with an average of 43% of wild type levels in cells from various heterozygote patients. Optimized efficiency of the lead ASO was further obtained with 2 -Methoxy Ethyl modification (2 MOE). Conclusion:The highly efficient splicing modulation and functional correction, achieved by free uptake of the selected lead ASO in various patients, demonstrate the ASO therapeutic potential benefit for CF patients carrying splicing mutations and is aimed to serve as the basis for our current clinical development.