Transient genomic instability drives tumorigenesis through accelerated clonal evolution.

Abnormal numerical and structural chromosome content is frequently found in human cancer. To test the role of aneuploidy in tumor initiation and progression, we generated mice with random aneuploidies by transient induction of polo-like kinase 4 (Plk4), a master regulator of centrosome number. Short-term chromosome instability (CIN) from transient Plk4 induction resulted in formation of aggressive T-cell lymphomas in mice with heterozygous inactivation of one p53 allele and accelerated tumor development in the absence of p53. Transient CIN increased the frequency of lymphoma-initiating cells with a specific karyotype profile, including trisomy of chromosomes 4, 5, 14, and 15 occurring early in tumorigenesis. Tumor development in mice with chronic CIN induced by an independent mechanism (through inactivation of the spindle assembly checkpoint) gradually trended toward a similar karyotypic profile, as determined by single-cell whole-genome DNA sequencing. Overall, we show how transient CIN generates cells with random aneuploidies from which ones that acquire a karyotype with specific chromosome gains are sufficient to drive cancer formation, and that distinct CIN mechanisms can lead to similar karyotypic cancer-causing outcomes.

Genetic instability from a single S phase after whole-genome duplication.

Diploid and stable karyotypes are associated with health and fitness in animals. By contrast, whole-genome duplications-doublings of the entire complement of chromosomes-are linked to genetic instability and frequently found in human cancers1-3. It has been established that whole-genome duplications fuel chromosome instability through abnormal mitosis4-8; however, the immediate consequences of tetraploidy in the first interphase are not known. This is a key question because single whole-genome duplication events such as cytokinesis failure can promote tumorigenesis9. Here we find that human cells undergo high rates of DNA damage during DNA replication in the first S phase following induction of tetraploidy. Using DNA combing and single-cell sequencing, we show that DNA replication dynamics is perturbed, generating under- and over-replicated regions. Mechanistically, we find that these defects result from a shortage of proteins during the G1/S transition, which impairs the fidelity of DNA replication. This work shows that within a single interphase, unscheduled tetraploid cells can acquire highly abnormal karyotypes. These findings provide an explanation for the genetic instability landscape that favours tumorigenesis after tetraploidization.

cGAS-STING drives the IL-6-dependent survival of chromosomally instable cancers.

Chromosomal instability (CIN) drives cancer cell evolution, metastasis and therapy resistance, and is associated with poor prognosis1. CIN leads to micronuclei that release DNA into the cytoplasm after rupture, which triggers activation of inflammatory signalling mediated by cGAS and STING2,3. These two proteins are considered to be tumour suppressors as they promote apoptosis and immunosurveillance. However, cGAS and STING are rarely inactivated in cancer4, and, although they have been implicated in metastasis5, it is not known why loss-of-function mutations do not arise in primary tumours4. Here we show that inactivation of cGAS-STING signalling selectively impairs the survival of triple-negative breast cancer cells that display CIN. CIN triggers IL-6-STAT3-mediated signalling, which depends on the cGAS-STING pathway and the non-canonical NF-κB pathway. Blockade of IL-6 signalling by tocilizumab, a clinically used drug that targets the IL-6 receptor (IL-6R), selectively impairs the growth of cultured triple-negative breast cancer cells that exhibit CIN. Moreover, outgrowth of chromosomally instable tumours is significantly delayed compared with tumours that do not display CIN. Notably, this targetable vulnerability is conserved across cancer types that express high levels of IL-6 and/or IL-6R in vitro and in vivo. Together, our work demonstrates pro-tumorigenic traits of cGAS-STING signalling and explains why the cGAS-STING pathway is rarely inactivated in primary tumours. Repurposing tocilizumab could be a strategy to treat cancers with CIN that overexpress IL-6R.

Targeted assembly of ectopic kinetochores to induce chromosome-specific segmental aneuploidies.

Cancer cells display persistent underlying chromosomal instability, with individual tumour types intriguingly exhibiting characteristic subsets of whole, and subchromosomal aneuploidies. Few methods to induce specific aneuploidies will exist, hampering investigation of functional consequences of recurrent aneuploidies, as well as the acute consequences of specific chromosome mis-segregation. We therefore investigated the possibility of sabotaging the mitotic segregation of specific chromosomes using nuclease-dead CRISPR-Cas9 (dCas9) as a cargo carrier to specific genomic loci. We recruited the kinetochore-nucleating domain of centromere protein CENP-T to assemble ectopic kinetochores either near the centromere of chromosome 9, or the telomere of chromosome 1. Ectopic kinetochore assembly led to increased chromosome instability and partial aneuploidy of the target chromosomes, providing the potential to induce specific chromosome mis-segregation events in a range of cell types. We also provide an analysis of putative endogenous repeats that could support ectopic kinetochore formation. Overall, our findings provide new insights into ectopic kinetochore biology and represent an important step towards investigating the role of specific aneuploidy and chromosome mis-segregation events in diseases associated with aneuploidy.

A kinesin-based approach for inducing chromosome-specific mis-segregation in human cells.

Various cancer types exhibit characteristic and recurrent aneuploidy patterns. The origins of these cancer type-specific karyotypes are still unknown, partly because introducing or eliminating specific chromosomes in human cells still poses a challenge. Here, we describe a novel strategy to induce mis-segregation of specific chromosomes in different human cell types. We employed Tet repressor or nuclease-dead Cas9 to link a microtubule minus-end-directed kinesin (Kinesin14VIb) from Physcomitrella patens to integrated Tet operon repeats and chromosome-specific endogenous repeats, respectively. By live- and fixed-cell imaging, we observed poleward movement of the targeted loci during (pro)metaphase. Kinesin14VIb-mediated pulling forces on the targeted chromosome were counteracted by forces from kinetochore-attached microtubules. This tug-of-war resulted in chromosome-specific segregation errors during anaphase and revealed that spindle forces can heavily stretch chromosomal arms. By single-cell whole-genome sequencing, we established that kinesin-induced targeted mis-segregations predominantly result in chromosomal arm aneuploidies after a single cell division. Our kinesin-based strategy opens the possibility to investigate the immediate cellular responses to specific aneuploidies in different cell types; an important step toward understanding how tissue-specific aneuploidy patterns evolve.

Aneuploidy renders cancer cells vulnerable to mitotic checkpoint inhibition.

Selective targeting of aneuploid cells is an attractive strategy for cancer treatment1. However, it is unclear whether aneuploidy generates any clinically relevant vulnerabilities in cancer cells. Here we mapped the aneuploidy landscapes of about 1,000 human cancer cell lines, and analysed genetic and chemical perturbation screens2-9 to identify cellular vulnerabilities associated with aneuploidy. We found that aneuploid cancer cells show increased sensitivity to genetic perturbation of core components of the spindle assembly checkpoint (SAC), which ensures the proper segregation of chromosomes during mitosis10. Unexpectedly, we also found that aneuploid cancer cells were less sensitive than diploid cells to short-term exposure to multiple SAC inhibitors. Indeed, aneuploid cancer cells became increasingly sensitive to inhibition of SAC over time. Aneuploid cells exhibited aberrant spindle geometry and dynamics, and kept dividing when the SAC was inhibited, resulting in the accumulation of mitotic defects, and in unstable and less-fit karyotypes. Therefore, although aneuploid cancer cells could overcome inhibition of SAC more readily than diploid cells, their long-term proliferation was jeopardized. We identified a specific mitotic kinesin, KIF18A, whose activity was perturbed in aneuploid cancer cells. Aneuploid cancer cells were particularly vulnerable to depletion of KIF18A, and KIF18A overexpression restored their response to SAC inhibition. Our results identify a therapeutically relevant, synthetic lethal interaction between aneuploidy and the SAC.

Inhalable Textile Microplastic Fibers Impair Airway Epithelial Differentiation.

Rationale: Microplastics are a pressing global concern, and inhalation of microplastic fibers has been associated with interstitial and bronchial inflammation in flock workers. However, how microplastic fibers affect the lungs is unknown. Objectives: Our aim was to assess the effects of 12 × 31 µm nylon 6,6 (nylon) and 15 × 52 µm polyethylene terephthalate (polyester) textile microplastic fibers on lung epithelial growth and differentiation. Methods: We used human and murine alveolar and airway-type organoids as well as air-liquid interface cultures derived from primary lung epithelial progenitor cells and incubated these with either nylon or polyester fibers or nylon leachate. In addition, mice received one dose of nylon fibers or nylon leachate, and, 7 days later, organoid-forming capacity of isolated epithelial cells was investigated. Measurements and Main Results: We observed that nylon microfibers, more than polyester, inhibited developing airway organoids and not established ones. This effect was mediated by components leaching from nylon. Epithelial cells isolated from mice exposed to nylon fibers or leachate also formed fewer airway organoids, suggesting long-lasting effects of nylon components on epithelial cells. Part of these effects was recapitulated in human air-liquid interface cultures. Transcriptomic analysis revealed upregulation of Hoxa5 after exposure to nylon fibers. Inhibiting Hoxa5 during nylon exposure restored airway organoid formation, confirming Hoxa5's pivotal role in the effects of nylon. Conclusions: These results suggest that components leaching from nylon 6,6 may especially harm developing airways and/or airways undergoing repair, and we strongly encourage characterization in more detail of both the hazard of and the exposure to microplastic fibers.

Transcriptional differences between vesicular hand eczema and atopic dermatitis.

BACKGROUND: Transcriptome analyses of vesicular hand eczema (VHE) indicated a large overlap with atopic dermatitis (AD). However, differentially expressed genes (DEGs) that differentiate VHE from AD are unknown. OBJECTIVE: To identify distinctive transcriptional features of VHE in comparison to AD. METHODS: We re-analysed RNA sequencing data of 10 lesional palmar VHE epidermal biopsies and performed DEG analyses. We adjusted the obtained DEG results of 57 lesional whole AD skin biopsies of the upper extremities or trunk to our criteria. Up- and down-regulated DEGs in both skin diseases, VHE-only, AD-only, and opposite regulated DEGs were identified. Enrichment analyses and Chi-squared tests were conducted to test for differences in gene set enrichment between both skin diseases. RESULTS: Comparing 3028 DEGs in VHE (1645 up; 1383 down) with 5391 DEGs in AD (3842 up; 1549 down), revealed 1516 shared DEGs (1179 up; 337 down) and 1512 DEGs unique to VHE (466 up, 1046 down). Interferon signalling and necroptosis were significantly more prominent in VHE compared to AD. Downregulated genes identified only in VHE (like DNASE1L2, KRT2, KRT9 and KRT25) indicate an aberrant epidermal differentiation. CONCLUSION: Our study indicates a common pathophysiology between VHE and AD, but also reveals transcriptional differences between VHE and AD.

The H3.3K27M oncohistone affects replication stress outcome and provokes genomic instability in pediatric glioma.

While comprehensive molecular profiling of histone H3.3 mutant pediatric high-grade glioma has revealed extensive dysregulation of the chromatin landscape, the exact mechanisms driving tumor formation remain poorly understood. Since H3.3 mutant gliomas also exhibit high levels of copy number alterations, we set out to address if the H3.3K27M oncohistone leads to destabilization of the genome. Hereto, we established a cell culture model allowing inducible H3.3K27M expression and observed an increase in mitotic abnormalities. We also found enhanced interaction of DNA replication factors with H3.3K27M during mitosis, indicating replication defects. Further functional analyses revealed increased genomic instability upon replication stress, as represented by mitotic bulky and ultrafine DNA bridges. This co-occurred with suboptimal 53BP1 nuclear body formation after mitosis in vitro, and in human glioma. Finally, we observed a decrease in ultrafine DNA bridges following deletion of the K27M mutant H3F3A allele in primary high-grade glioma cells. Together, our data uncover a role for H3.3 in DNA replication under stress conditions that is altered by the K27M mutation, promoting genomic instability and potentially glioma development.

IL-1ß Induces a Proinflammatory Fibroblast Microenvironment that Impairs Lung Progenitors' Function.

Chronic obstructive pulmonary disease (COPD) is characterized by a persistent inflammatory state in the lungs and defective tissue repair. Although the inflammatory response in patients with COPD is well characterized and known to be exaggerated during exacerbations, its contribution to lung injury and abnormal repair is still unclear. In this study, we aimed to investigate how the inflammatory microenvironment affects the epithelial progenitors and their supporting mesenchymal niche cells involved in tissue repair of the distal lung. We focused on IL-1ß, a key inflammatory mediator that is increased during exacerbations of COPD, and used an organoid model of lung epithelial cells and fibroblasts to assess the effect of IL-1ß treatment on these cells' transcriptome and secreted factors. Whereas direct treatment of the lung organoids with IL-1ß promoted organoid growth, this switched toward inhibition when it was added as fibroblast pretreatment followed by organoid treatment. We then investigated the IL-1ß-driven mechanisms in the fibroblasts and found an inflammatory response related to (C-X-C motif) ligand (CXCL) chemokines; we confirmed that these chemokines were responsible for the impaired organoid growth and found that targeting their C-X-C chemokine receptors 1/2 (CXCR1/2) receptors or the IL-1ß intracellular signaling reduced the proinflammatory response and restored organoid growth. These data demonstrate that IL-1ß alters the fibroblasts' state by promoting a distinct inflammatory response, switching their supportive function on epithelial progenitors toward an inhibitory one in an organoid assay. These results imply that chronic inflammation functions as a shift toward inhibition of repair, thereby contributing to chronic inflammatory diseases like COPD.

Chromosomal Instability Characterizes Pediatric Medulloblastoma but Is Not Tolerated in the Developing Cerebellum.

Medulloblastoma is a pediatric brain malignancy that consists of four transcriptional subgroups. Structural and numerical aneuploidy are common in all subgroups, although they are particularly profound in Group 3 and Group 4 medulloblastoma and in a subtype of SHH medulloblastoma termed SHHα. This suggests that chromosomal instability (CIN), the process leading to aneuploidy, is an important player in medulloblastoma pathophysiology. However, it is not known if there is ongoing CIN in medulloblastoma or if CIN affects the developing cerebellum and promotes tumor formation. To investigate this, we performed karyotyping of single medulloblastoma cells and demonstrated the presence of distinct tumor cell clones harboring unique copy number alterations, which is suggestive of ongoing CIN. We also found enrichment for processes related to DNA replication, repair, and mitosis in both SHH medulloblastoma and in the highly proliferative compartment of the presumed tumor cell lineage-of-origin, the latter also being sensitive to genotoxic stress. However, when challenging these tumor cells-of-origin with genetic lesions inducing CIN using transgenic mouse modeling, we found no evidence for large chromosomal aberrations in the cerebellum or for medulloblastoma formation. We therefore conclude that without a background of specific genetic mutations, CIN is not tolerated in the developing cerebellum in vivo and, thus, by itself is not sufficient to initiate medulloblastoma.

Parental DNA Methylation States Are Associated with Heterosis in Epigenetic Hybrids.

Despite the importance and wide exploitation of heterosis in commercial crop breeding, the molecular mechanisms behind this phenomenon are not completely understood. Recent studies have implicated changes in DNA methylation and small RNAs in hybrid performance; however, it remains unclear whether epigenetic changes are a cause or a consequence of heterosis. Here, we analyze a large panel of over 500 Arabidopsis (Arabidopsis thaliana) epigenetic hybrid plants (epiHybrids), which we derived from near-isogenic but epigenetically divergent parents. This proof-of-principle experimental system allowed us to quantify the contribution of parental methylation differences to heterosis. We measured traits such as leaf area, growth rate, flowering time, main stem branching, rosette branching, and final plant height and observed several strong positive and negative heterotic phenotypes among the epiHybrids. Using an epigenetic quantitative trait locus mapping approach, we were able to identify specific differentially methylated regions in the parental genomes that are associated with hybrid performance. Sequencing of methylomes, transcriptomes, and genomes of selected parent-epiHybrid combinations further showed that these parental differentially methylated regions most likely mediate the remodeling of methylation and transcriptional states at specific loci in the hybrids. Taken together, our data suggest that locus-specific epigenetic divergence between the parental lines can directly or indirectly trigger heterosis in Arabidopsis hybrids independent of genetic changes. These results add to a growing body of evidence that points to epigenetic factors as one of the key determinants of hybrid performance.

METHimpute: imputation-guided construction of complete methylomes from WGBS data.

BACKGROUND: Whole-genome bisulfite sequencing (WGBS) has become the standard method for interrogating plant methylomes at base resolution. However, deep WGBS measurements remain cost prohibitive for large, complex genomes and for population-level studies. As a result, most published plant methylomes are sequenced far below saturation, with a large proportion of cytosines having either missing data or insufficient coverage. RESULTS: Here we present METHimpute, a Hidden Markov Model (HMM) based imputation algorithm for the analysis of WGBS data. Unlike existing methods, METHimpute enables the construction of complete methylomes by inferring the methylation status and level of all cytosines in the genome regardless of coverage. Application of METHimpute to maize, rice and Arabidopsis shows that the algorithm infers cytosine-resolution methylomes with high accuracy from data as low as 6X, compared to data with 60X, thus making it a cost-effective solution for large-scale studies. CONCLUSIONS: METHimpute provides methylation status calls and levels for all cytosines in the genome regardless of coverage, thus yielding complete methylomes even with low-coverage WGBS datasets. The method has been extensively tested in plants, but should also be applicable to other species. An implementation is available on Bioconductor.

Epigenetic basis of morphological variation and phenotypic plasticity in Arabidopsis thaliana.

Epigenetics is receiving growing attention in the plant science community. Epigenetic modifications are thought to play a particularly important role in fluctuating environments. It is hypothesized that epigenetics contributes to plant phenotypic plasticity because epigenetic modifications, in contrast to DNA sequence variation, are more likely to be reversible. The population of decrease in DNA methylation 1-2 (ddm1-2)-derived epigenetic recombinant inbred lines (epiRILs) in Arabidopsis thaliana is well suited for studying this hypothesis, as DNA methylation differences are maximized and DNA sequence variation is minimized. Here, we report on the extensive heritable epigenetic variation in plant growth and morphology in neutral and saline conditions detected among the epiRILs. Plant performance, in terms of branching and leaf area, was both reduced and enhanced by different quantitative trait loci (QTLs) in the ddm1-2 inherited epigenotypes. The variation in plasticity associated significantly with certain genomic regions in which the ddm1-2 inherited epigenotypes caused an increased sensitivity to environmental changes, probably due to impaired genetic regulation in the epiRILs. Many of the QTLs for morphology and plasticity overlapped, suggesting major pleiotropic effects. These findings indicate that epigenetics contributes substantially to variation in plant growth, morphology, and plasticity, especially under stress conditions.

Rate, spectrum, and evolutionary dynamics of spontaneous epimutations.

Stochastic changes in cytosine methylation are a source of heritable epigenetic and phenotypic diversity in plants. Using the model plant Arabidopsis thaliana, we derive robust estimates of the rate at which methylation is spontaneously gained (forward epimutation) or lost (backward epimutation) at individual cytosines and construct a comprehensive picture of the epimutation landscape in this species. We demonstrate that the dynamic interplay between forward and backward epimutations is modulated by genomic context and show that subtle contextual differences have profoundly shaped patterns of methylation diversity in A. thaliana natural populations over evolutionary timescales. Theoretical arguments indicate that the epimutation rates reported here are high enough to rapidly uncouple genetic from epigenetic variation, but low enough for new epialleles to sustain long-term selection responses. Our results provide new insights into methylome evolution and its population-level consequences.

Features of the Arabidopsis recombination landscape resulting from the combined loss of sequence variation and DNA methylation.

The rate of meiotic crossing over (CO) varies considerably along chromosomes, leading to marked distortions between physical and genetic distances. The causes underlying this variation are being unraveled, and DNA sequence and chromatin states have emerged as key factors. However, the extent to which the suppression of COs within the repeat-rich pericentromeric regions of plant and mammalian chromosomes results from their high level of DNA polymorphisms and from their heterochromatic state, notably their dense DNA methylation, remains unknown. Here, we test the combined effect of removing sequence polymorphisms and repeat-associated DNA methylation on the meiotic recombination landscape of an Arabidopsis mapping population. To do so, we use genome-wide DNA methylation data from a large panel of isogenic epigenetic recombinant inbred lines (epiRILs) to derive a recombination map based on 126 meiotically stable, differentially methylated regions covering 81.9% of the genome. We demonstrate that the suppression of COs within pericentromeric regions of chromosomes persists in this experimental setting. Moreover, suppression is reinforced within 3-Mb regions flanking pericentromeric boundaries, and this effect appears to be compensated by increased recombination activity in chromosome arms. A direct comparison with 17 classical Arabidopsis crosses shows that these recombination changes place the epiRILs at the boundary of the range of natural variation but are not severe enough to transgress that boundary significantly. This level of robustness is remarkable, considering that this population represents an extreme with key recombination barriers having been forced to a minimum.

Rebalancing the motor circuit restores movement in a Caenorhabditis elegans model for TDP-43 toxicity.

Amyotrophic lateral sclerosis can be caused by abnormal accumulation of TAR DNA-binding protein 43 (TDP-43) in the cytoplasm of neurons. Here, we use a C. elegans model for TDP-43-induced toxicity to identify the biological mechanisms that lead to disease-related phenotypes. By applying deep behavioral phenotyping and subsequent dissection of the neuromuscular circuit, we show that TDP-43 worms have profound defects in GABA neurons. Moreover, acetylcholine neurons appear functionally silenced. Enhancing functional output of repressed acetylcholine neurons at the level of, among others, G-protein-coupled receptors restores neurotransmission, but inefficiently rescues locomotion. Rebalancing the excitatory-to-inhibitory ratio in the neuromuscular system by simultaneous stimulation of the affected GABA- and acetylcholine neurons, however, not only synergizes the effects of boosting individual neurotransmitter systems, but instantaneously improves movement. Our results suggest that interventions accounting for the altered connectome may be more efficient in restoring motor function than those solely focusing on diseased neuron populations.

MDM2 amplification in rod-shaped chromosomes provides clues to early stages of circularized gene amplification in liposarcoma.

Well-differentiated liposarcoma (WDLS) displays amplification of genes on chromosome 12 (Chr12) in supernumerary ring or giant marker chromosomes. These structures have been suggested to develop through chromothripsis, followed by circularization and breakage-fusion-bridge (BFB) cycles. To test this hypothesis, we compared WDLSs with Chr12 amplification in rod-shaped chromosomes with WDLSs with rings. Both types of amplicons share the same spectrum of structural variants (SVs), show higher SV frequencies in Chr12 than in co-amplified segments, have SVs that fuse the telomeric ends of co-amplified chromosomes, and lack interspersed deletions. Combined with the finding of cells with transient rod-shaped structures in tumors with ring chromosomes, this suggests a stepwise process starting with the gain of Chr12 material that, after remodeling which does not fit with classical chromothripsis, forms a dicentric structure with other chromosomes. Depending on if and when telomeres from other chromosomes are captured, circularized or linear gain of 12q sequences will predominate.

IFNγ protects motor neurons from oxidative stress via enhanced global protein synthesis in FUS-associated amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis type 6 (ALS6) is a familial subtype of ALS linked to Fused in Sarcoma (FUS) gene mutation. FUS mutations lead to decreased global protein synthesis, but the mechanism that drives this has not been established. Here, we used ALS6 patient-derived induced pluripotent stem cells (hIPSCs) to study the effect of the ALS6 FUSR521H mutation on the translation machinery in motor neurons (MNs). We find, in agreement with findings of others, that protein synthesis is decreased in FUSR521H MNs. Furthermore, FUSR521H MNs are more sensitive to oxidative stress and display reduced expression of TGF-ß and mTORC gene pathways when stressed. Finally, we show that IFNγ treatment reduces apoptosis of FUSR521H MNs exposed to oxidative stress and partially restores the translation rates in FUSR521H MNs. Overall, these findings suggest that a functional IFNγ response is important for FUS-mediated protein synthesis, possibly by FUS nuclear translocation in ALS6.