Common fragile sites (CFS) are specific genomic regions prone to chromosomal instability under conditions of DNA replication stress. CFSs manifest as breaks, gaps, and constrictions on metaphase chromosomes under mild replication stress. These replication-sensitive CFS regions are preferentially unstable during cancer development, as reflected by their association with copy number variants (CNVs) frequently arise in most tumor types. Over the years, it became clear that a combination of different characteristics underlies the enhanced sensitivity of CFSs to replication stress. As of today, there is a strong evidence that the core fragility regions along CFSs overlap with actively transcribed large genes with delayed replication timing upon replication stress. Recently, the mechanistic basis for CFS instability was further extended to regions which span topologically associated domain (TAD) boundaries, generating a fragility signature composed of replication, transcription and genome organization. The presence of difficult-to-replicate AT-rich repeats was one of the early features suggested to characterize a subgroup of CFSs. These long stretches of AT-dinucleotide have the potential to fold into stable secondary structures which may impede replication fork progression, leaving the region under-replicated. Here, we focus on the molecular mechanisms underlying repeat instability at CFSs and on the proteins involved in the resolution of secondary structure impediments arising along repetitive sequence elements which are essential for the maintenance of genome stability.

DNA replication is a complex process 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 mutated HRAS activation leads to increased topoisomerase 1 (TOP1) expression, causing aberrant replication fork acceleration and DNA damage by decreasing RNA-DNA hybrids or R-loops. In these cells, restoration of TOP1 expression or mild replication inhibition rescues the perturbed replication and reduces DNA damage. Furthermore, TOP1 or RNaseH1 overexpression induces accelerated replication and DNA damage, highlighting the importance of TOP1 equilibrium in regulating R-loop homeostasis to ensure faithful DNA replication and genome integrity. Altogether, our results dissect a mechanism of oncogene-induced DNA damage by aberrant replication fork acceleration.

BACKGROUND: Antisense oligonucleotide- based drugs for splicing modulation were recently approved for various genetic diseases with unmet need. Here we aimed to generate skipping over exon 23 of the CFTR transcript, to eliminate the W1282X nonsense mutation and avoid RNA degradation induced by the nonsense mediated mRNA decay mechanism, allowing production of partially active CFTR proteins lacking exon 23. METHODS: ∼80 ASOs were screened in 16HBEge W1282X cells. ASO candidates showing significant exon skipping were assessed for their W1282X allele selectivity and the increase of CFTR protein maturation and function. The effect of a highly potent ASO candidates was further analyzed in well differentiated primary human nasal epithelial cells, derived from a W1282X homozygous patient. RESULTS: ASO screening led to identification of several ASOs that significantly decrease the level of CFTR transcripts including exon 23. These ASOs resulted in significant levels of mature CFTR protein and together with modulators restore the channel function following free uptake into these cells. Importantly, a highly potent lead ASOs, efficiently delivered by free uptake, was able to increase the level of transcripts lacking exon 23 and restore the CFTR function in cells from a W1282X homozygote patient. CONCLUSION: The highly efficient exon 23 skipping induced by free uptake of the lead ASO and the resulting levels of mature CFTR protein exhibiting channel function in the presence of modulators, demonstrate the ASO therapeutic potential benefit for CF patients carrying the W1282X mutation with the objective to advance the lead candidate SPL23-2 to proof-of-concept clinical study.

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.

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.

Gene therapy offers great promise for cystic fibrosis which has never been quite fulfilled due to the challenges of delivering sufficient amounts of the CFTR gene and expression persistence for a sufficient period of time in the lungs to have any effect. Initial trials explored both viral and non-viral vectors but failed to achieve a significant breakthrough. However, in recent years, new opportunities have emerged that exploit our increased knowledge and understanding of the biology of CF and the airway epithelium. New technologies include new viral and non-viral vector approaches to delivery, but also alternative nucleic acid technologies including oligonucleotides and siRNA approaches for gene silencing and gene splicing, described in this review, as presented at the 2019 annual European CF Society Basic Science meeting (Dubrovnik, Croatia). We also briefly discuss other emerging technologies including mRNA and CRISPR gene editing that are advancing rapidly. The future prospects for genetic therapies for CF are now diverse and more promising probably than any time since the discovery of the CF gene.

Common fragile sites (CFSs) are genomic regions prone to breakage under replication stress conditions recurrently rearranged in cancer. Many CFSs are enriched with AT-dinucleotide rich sequences (AT-DRSs) which have the potential to form stable secondary structures upon unwinding the double helix during DNA replication. These stable structures can potentially perturb DNA replication progression, leading to genomic instability. Using site-specific targeting system, we show that targeted integration of a 3.4 kb AT-DRS derived from the human CFS FRA16C into a chromosomally stable region within the human genome is able to drive fragile site formation under conditions of replication stress. Analysis of >1300 X chromosomes integrated with the 3.4 kb AT-DRS revealed recurrent gaps and breaks at the integration site. DNA sequences derived from the integrated AT-DRS showed in vitro a significantly increased tendency to fold into branched secondary structures, supporting the predicted mechanism of instability. Our findings clearly indicate that intrinsic DNA features, such as complexed repeated sequence motifs, predispose the human genome to chromosomal instability.

Common fragile sites (CFSs) are specific genomic regions in normal chromosomes that exhibit genomic instability under DNA replication stress. As replication stress is an early feature of cancer development, CFSs are involved in the signature of genomic instability found in malignant tumors. The landscape of CFSs is tissue-specific and differs under different replication stress inducers. Nevertheless, the features underlying CFS sensitivity to replication stress are shared. Here, we review the events generating replication stress and discuss the unique characteristics of CFS regions and the cellular responses aimed to stabilizing these regions.

DNA replication stress is one of the early drivers enabling the ongoing acquisition of genetic changes arising during tumorigenesis. As such, it is a feature of most pre-malignant and malignant cells. In this review article, we focus on the early events initiating DNA replication stress and the preferential sensitivity of common fragile sites (CFSs) to this stress. CFSs are specific genomic regions within the normal chromosomal structure, which appear as gaps and breaks in the metaphase chromosomes of cells grown under mild replication stress conditions. The main characteristics predisposing CFSs to instability include late replication timing, delayed replication completion, failure to activate additional origins, origin paucity along large genomic regions, collision between replication and transcription complexes along large genes, and the presence of AT-dinucleotide rich sequences. The contribution of these features to instability at CFSs during early cancer development is discussed.

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.

Premature termination codons (PTC) originate from nucleotide substitution introducing an in-frame PTC. They induce truncated, usually non-functional, proteins, degradation of the PTC containing transcripts by the nonsense-mediated decay (NMD) pathway and abnormal exon skipping. Readthrough compounds facilitate near cognate amino-acyl-tRNA incorporation, leading potentially to restoration of a functional full-length protein. Splicing mutations can lead to aberrantly spliced transcripts by creating a cryptic splice site or destroying a normal site. Most mutations result in disruption of the open reading frame and activation of NMD. Antisense oligonucleotides are single stranded short synthetic RNA-like molecules chemically modified to improve their stability and ability to recognize their target RNAs and modify the splice site. This review focuses on recent developments in therapies aiming to improve the health of CF patients carrying nonsense or splicing mutations.

Common fragile sites (CFSs) are chromosomal regions characterized as hotspots for breakage and chromosomal rearrangements following DNA replication stress. They are preferentially unstable in pre-cancerous lesions and during cancer development. Recently CFSs were found to be tissue- and even oncogene-induced specific, thus indicating an unforeseen complexity. Here we review recent developments in CFS research that shed new light on the molecular basis of their instability and their importance in cancer development.

Human pluripotent stem cells (hPSCs) frequently acquire chromosomal aberrations, including aneuploidy, during culture. Recently, we identified a replication stress-based mechanism leading to ongoing chromosomal instability in aneuploid hPSCs that may also operate during the initiation of instability in diploid cells.

Human pluripotent stem cells (hPSCs) frequently acquire chromosomal aberrations such as aneuploidy in culture. These aberrations progressively increase over time and may compromise the properties and clinical utility of the cells. The underlying mechanisms that drive initial genomic instability and its continued progression are largely unknown. Here, we show that aneuploid hPSCs undergo DNA replication stress, resulting in defective chromosome condensation and segregation. Aneuploid hPSCs show altered levels of actin cytoskeletal genes controlled by the transcription factor SRF, and overexpression of SRF rescues impaired chromosome condensation and segregation defects in aneuploid hPSCs. Furthermore, SRF downregulation in diploid hPSCs induces replication stress and perturbed condensation similar to that seen in aneuploid cells. Together, these results suggest that decreased SRF expression induces replicative stress and chromosomal condensation defects that underlie the ongoing chromosomal instability seen in aneuploid hPSCs. A similar mechanism may also operate during initiation of instability in diploid cells.

Cystic fibrosis (CF; OMIM #219700) is a common autosomal recessive disease. The spectrum and frequency of CFTR mutations vary significantly in different populations and ethnic groups. A genetic epidemiological study was conducted in the indigenous ethnic group of people known as the Karachais. They live in the Republic of Karachay-Cherkessia, which lies in the northwest of Russia's North Caucasus region. Karachai's are Turkic-speaking and consist of 194 thousand people (approximately 40% of the population of the Republic). Molecular genetic analysis was performed in 10 unrelated Karachai families with CF patients from three districts in the Republic. A high frequency of W1282X mutation was found (18 of 20 mutant alleles): eight patients were homozygous for the W1282X mutation, and two were compound heterozygous (the second alleles were R1066C and R709X). Analysis for 13 common CF mutations in the sample of 142 healthy Karachais identified two 1677delTA and two W1282X mutation carriers. Thus, the most common CFTR mutation, F508del, was not detected among the CF patients or in healthy Karachais. The most frequent mutation among Karachai patients is W1282X (90%). Its frequency in healthy Karachais is approximately 0.007. Haplotype analysis using the CFTR intragene DNA markers IVS1CA, IVS6aGATT, IVS8CA and IVS17bCA showed that the origins of the W1282X mutation in Karachay-Cherkessia and the Eastern European part of Russia are different.

The presence of dormant, microscopic cancerous lesions poses a major obstacle for the treatment of metastatic and recurrent cancers. While it is well-established that microRNAs play a major role in tumorigenesis, their involvement in tumor dormancy has yet to be fully elucidated. We established and comprehensively characterized pairs of dormant and fast-growing human osteosarcoma models. Using these pairs of mouse tumor models, we identified three novel regulators of osteosarcoma dormancy: miR-34a, miR-93, and miR-200c. This report shows that loss of these microRNAs occurs during the switch from dormant avascular into fast-growing angiogenic phenotype. We validated their downregulation in patients' tumor samples compared to normal bone, making them attractive candidates for osteosarcoma therapy. Successful delivery of miRNAs is a challenge; hence, we synthesized an aminated polyglycerol dendritic nanocarrier, dPG-NH2, and designed dPG-NH2-microRNA polyplexes to target cancer. Reconstitution of these microRNAs using dPG-NH2 polyplexes into Saos-2 and MG-63 cells, which generate fast-growing osteosarcomas, reduced the levels of their target genes, MET proto-oncogene, hypoxia-inducible factor 1α, and moesin, critical to cancer angiogenesis and cancer cells' migration. We further demonstrate that these microRNAs attenuate the angiogenic capabilities of fast-growing osteosarcomas in vitro and in vivo. Treatment with each of these microRNAs using dPG-NH2 significantly prolonged the dormancy period of fast-growing osteosarcomas in vivo. Taken together, these findings suggest that nanocarrier-mediated delivery of microRNAs involved in osteosarcoma tumor-host interactions can induce a dormant-like state.

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.

Oncogene expression can lead to replication stress and genome instability. Recently, we identified oncogene-induced fragile sites (FSs) and revealed that the landscape of recurrent fragility in the same cell type is dynamic. This implies an additional level of complexity in the molecular basis of recurrent fragility in cancer.