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Common fragile sites (CFSs) are difficult-to-replicate regions of eukaryotic genomes that are sensitive to replication stress and that require resolution by the MUS81–EME1 endonuclease to re-initiate POLD3-dependent DNA synthesis in early mitosis; this study defines the specific pathway of events causing the CFS fragility phenotype. Inbuilt chromosome fragility Common fragile sites (CFSs) are regions of eukaryotic genomes that are sensitive to DNA replication stressors. They are stably maintained and replicated in the human genome most of the time, but have been associated with some cancers and genetic disease. CFS 'expression' was shown recently to be a programmed event promoted by the MUS81 endonuclease, rather than disruption of the chromosome structure caused by mechanical forces in mitosis. Ian Hickson and colleagues have now defined the specific chain of events causing CFSs. They describe a pathway of unscheduled DNA synthesis regulated by the MUS81 endonuclease, the POLD3 subunit of DNA polymerase delta and the prophase pathway in early mitosis. Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis 1 . Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs) 2 , 3 , 4 , 5 . CFSs are difficult-to-replicate loci that manifest as gaps or breaks on metaphase chromosomes (termed CFS ‘expression’), particularly when cells have been exposed to replicative stress 6 . The MUS81–EME1 structure-specific endonuclease promotes the appearance of chromosome gaps or breaks at CFSs following replicative stress 7 , 8 , 9 . Here we show that entry of cells into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN + ) and replicative stress, we suggest that targeting this pathway could represent a new therapeutic approach.

The ability to accurately measure mutations is critical for basic research and identifying potential drug and chemical carcinogens. Current methods for in vivo quantification of mutagenesis are limited because they rely on transgenic rodent systems that are low-throughput, expensive, prolonged, and do not fully represent other species such as humans. Next-generation sequencing (NGS) is a conceptually attractive alternative for detecting mutations in the DNA of any organism; however, the limit of resolution for standard NGS is poor. Technical error rates (∼1 × 10−3) of NGS obscure the true abundance of somatic mutations, which can exist at per-nucleotide frequencies ≤1 × 10−7. Using duplex sequencing, an extremely accurate error-corrected NGS (ecNGS) technology, we were able to detect mutations induced by three carcinogens in five tissues of two strains of mice within 31 d following exposure. We observed a strong correlation between mutation induction measured by duplex sequencing and the gold-standard transgenic rodent mutation assay. We identified exposurespecific mutation spectra of each compound through trinucleotide patterns of base substitution. We observed variation in mutation susceptibility by genomic region, as well as by DNA strand. We also identified a primordial marker of carcinogenesis in a cancerpredisposed strain of mice, as evidenced by clonal expansions of cells carrying an activated oncogene, less than a month after carcinogen exposure. These findings demonstrate that ecNGS is a powerful method for sensitively detecting and characterizing mutagenesis and the early clonal evolutionary hallmarks of carcinogenesis. Duplex sequencing can be broadly applied to basic mutational research, regulatory safety testing, and emerging clinical applications.

Error-corrected next-generation sequencing (ecNGS) is an emerging technology with the potential to revolutionize the field of genetic toxicology. Here, we present recommendations from an expert working group convened to discuss potential applications, advantages and challenges associated with implementing ecNGS in nonclinical safety studies.Error-corrected next-generation sequencing (ecNGS) is an emerging technology with the potential to revolutionize the field of genetic toxicology. Here, we present recommendations from an expert working group convened to discuss potential applications, advantages and challenges associated with implementing ecNGS in nonclinical safety studies.

Chronic immunosuppressive therapies are crucial in organ transplantation but can increase the risk of opportunistic infections and cancer over time. We investigated immune status changes in 10 kidney transplant patients and 11 age-matched healthy adults using broad in vitro stimulation of subject-derived peripheral blood mononuclear cells followed by mass cytometry by time of flight over 6 mo. Overall, the immune cells of transplant patients exhibited increased CD8+ T cell activation and differentiation compared with healthy donors, with elevated CD8+ CD57+, MIP-1β, and interferon γ production (P < 0.05, P < 0.05, and P < 0.01, respectively). CD107a and granzyme B expression were increased in CD8+ T cells and CD56bright natural killer cells (P < 0.05 and P < 0.01, respectively), while T regulatory cells had decreased interleukin-10 production (P < 0.05). These changes indicated a proinflammatory environment influenced by induction therapy and ongoing maintenance drugs. Additionally, transplant recipients displayed signs of immune modulation, including decreased tumor necrosis factor α, interferon γ, and MIP-1β expression in γδT cells (P < 0.05 and P < 0.01), and reduced interleukin-17 and granulocyte-macrophage colony-stimulating factor expression in CD8+ T memory cell subsets (P < 0.05). The diverse functional changes underscore the importance of comprehensive immune status profiling for optimizing individual treatment strategies and developing better immunosuppressants that specifically target activated cell populations.

Chromosomal instability (CIN) is a hallmark of cancer, caused by persistent errors in chromosome segregation during mitosis. Aggressive cancers like high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) have a high frequency of CIN and TP53 mutations. Here, we show that inhibitors of the KIF18A motor protein activate the mitotic checkpoint and selectively kill chromosomally unstable cancer cells. Sensitivity to KIF18A inhibition is enriched in TP53 -mutant HGSOC and TNBC cell lines with CIN features, including in a subset of CCNE1 -amplified, CDK4–CDK6-inhibitor-resistant and BRCA1 -altered cell line models. Our KIF18A inhibitors have minimal detrimental effects on human bone marrow cells in culture, distinct from other anti-mitotic agents. In mice, inhibition of KIF18A leads to robust anti-cancer effects with tumor regression observed in human HGSOC and TNBC models at well-tolerated doses. Collectively, our results provide a rational therapeutic strategy for selective targeting of CIN cancers via KIF18A inhibition.

Chromosomal fragile sites (CFSs) are prone to breakage and thus can cause genomic instability. Hickson and colleagues demonstrate that the MUS81 endonuclease localizes to CFSs and mediates their processing, revealing that CFS cleavage is an active process. Fragile sites are chromosomal loci with a propensity to form gaps or breaks during early mitosis, and their instability is implicated as being causative in certain neurological disorders and cancers 1 . Recent work has demonstrated that the so-called common fragile sites (CFSs) often impair the faithful disjunction of sister chromatids in mitosis 2 . However, the mechanisms by which CFSs express their fragility, and the cellular factors required to suppress CFS instability, remain largely undefined. Here, we report that the DNA structure-specific nuclease MUS81–EME1 localizes to CFS loci in early mitotic cells, and promotes the cytological appearance of characteristic gaps or breaks observed at CFSs in metaphase chromosomes. These data indicate that CFS breakage is an active, MUS81–EME1-dependent process, and not a result of inadvertent chromatid rupturing during chromosome condensation. Moreover, CFS cleavage by MUS81–EME1 promotes faithful sister chromatid disjunction. Our findings challenge the prevailing view that CFS breakage is a nonspecific process that is detrimental to cells, and indicate that CFS cleavage actually promotes genome stability.

Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis (1). Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs) (2-5). CFSs are difficult-to-replicate loci that manifest as gaps or breaks on metaphase chromosomes (termed CFS 'expression'), particularly when cells have been exposed to replicative stress (6). The MUS81-EME1 structure-specific endonuclease promotes the appearance of chromosome gaps or breaks at CFSs following replicative stress (7-9). Here we show that entry of cells into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability ([CIN.sup.+]) and replicative stress, we suggest that targeting this pathway could represent a new therapeutic approach.

ABSTRACT The ability to accurately measure mutations is critical for basic research and identification of potential drug and chemical carcinogens. Current methods for in vivo quantification of mutagenesis are limited because they rely on transgenic rodent systems that are low-throughput, expensive, prolonged, and don’t fully represent other species such as humans. Next generation sequencing (NGS) is a conceptually attractive alternative for mutation detection in the DNA of any organism, however, the limit of resolution for standard NGS is poor. Technical error rates (~1×10−3) of NGS obscure the true abundance of somatic mutations, which can exist at pernucleotide frequencies ≤1×10−7. Using Duplex Sequencing, an extremely accurate error-corrected NGS (ecNGS) technology, we were able to detect mutations induced by 3 carcinogens in 5 tissues of 2 strains of mice within 31 days following exposure. We observed a strong correlation between mutation induction measured by Duplex Sequencing and the gold-standard transgenic rodent mutation assay. We identified exposure-specific mutation spectra of each compound through trinucleotide patterns of base substitution. We observed variation in mutation susceptibility by genomic region, as well as by DNA strand. We also identified the primordial signs of carcinogenesis in a cancer-predisposed strain of mice, as evidenced by clonal expansions of cells carrying an activated oncogene, less than a month after carcinogen exposure. These findings demonstrate that ecNGS is a powerful method for sensitively detecting and characterizing mutagenesis and the early clonal evolutionary hallmarks of carcinogenesis. Duplex Sequencing can be broadly applied to chemical safety testing, basic mutational research, and related clinical uses. SIGNIFICANCE STATEMENT Error-corrected next generation sequencing (ecNGS) can be used to rapidly detect and quantify the in vivo mutagenic impact of environmental exposures or endogenous processes in any tissue, from any species, at any genomic location. The greater speed, higher scalability, richer data outputs, as well as cross-species and cross-locus applicability of ecNGS compared to existing methods make it a powerful new tool for mutational research, regulatory safety testing, and emerging clinical applications. Competing Interest Statement CCV, LNW, TL and JJS, are employees and equity holders at TwinStrand Biosciences Incorporated. RRY is an employee of MilliporeSigma. At the time the study was conducted, RK was an employee of MilliporeSigma but is now an employee of EMD Serono. MilliporeSigma and EMD Serono are independent business units of Merck KGaA, Darmstadt Germany. SM is an employee of Amgen. MRF was an employee of Amgen at the time of the study and is currently an employee of Expansion Therapeutics. Footnotes * Text was reduced in length to suit journal requirements but no change to scientific content/conclusion. Small polish to grammar, syntax, organization to ready it for publication. * ABBREVIATIONS B[α]P Benzo[α]pyrene DS Duplex Sequencing ecNGS Error-Corrected Next Generation Sequencing ENU N-Ethyl-N-Nitrosourea MF Mutant Frequency SNV Single Nucleotide Variant TCR Transcription-Coupled Repair TGR Transgenic Rodent TPM Transcripts per million VC Vehicle Control VAF Variant Allele Frequency