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The cytoplasmic DNA sensor cGAS detects DNA in ruptured micronuclei and activates an innate immune response. Autoimmunity under surveillance Innate immune activation has been implicated in autoimmunity and cancer. Here, Andrew Jackson and colleagues provide evidence for an underlying mechanism whereby ruptured micronuclei, which result from endogenous or exogenous chromosomal damage, activate a cell-autonomous inflammatory response via the cytoplasmic DNA sensor cGAS. They conclude that cGAS recognition of micronuclei may be acting as a kind of immune surveillance system in cells. Elsewhere in this issue, Roger Greenberg and colleagues report a link between mitosis and DNA-damage-induced inflammatory signalling involving cGAS in cancer cells. DNA is strictly compartmentalized within the nucleus to prevent autoimmunity 1 ; despite this, cyclic GMP–AMP synthase (cGAS), a cytosolic sensor of double-stranded DNA, is activated in autoinflammatory disorders and by DNA damage 2 , 3 , 4 , 5 , 6 . Precisely how cellular DNA gains access to the cytoplasm remains to be determined. Here, we report that cGAS localizes to micronuclei arising from genome instability in a mouse model of monogenic autoinflammation, after exogenous DNA damage and spontaneously in human cancer cells. Such micronuclei occur after mis-segregation of DNA during cell division and consist of chromatin surrounded by its own nuclear membrane. Breakdown of the micronuclear envelope, a process associated with chromothripsis 7 , leads to rapid accumulation of cGAS, providing a mechanism by which self-DNA becomes exposed to the cytosol. cGAS is activated by chromatin, and consistent with a mitotic origin, micronuclei formation and the proinflammatory response following DNA damage are cell-cycle dependent. By combining live-cell laser microdissection with single cell transcriptomics, we establish that interferon-stimulated gene expression is induced in micronucleated cells. We therefore conclude that micronuclei represent an important source of immunostimulatory DNA. As micronuclei formed from lagging chromosomes also activate this pathway, recognition of micronuclei by cGAS may act as a cell-intrinsic immune surveillance mechanism that detects a range of neoplasia-inducing processes.

The authors report a link between mitosis, the formation of micronuclei and DNA-damage-induced cGAS-dependent inflammation. Cell cycle effects of combined radiation and genotoxic cancer therapy Ionizing radiation and genotoxic cancer therapy induce innate immunity mechanisms and lead to an increased production of inflammatory cytokines. The delayed nature of this response, which occurs a few days after treatment, is not well understood. Roger Greenberg and colleagues report a link between mitosis, the formation of micronuclei and DNA-damage-induced inflammatory signalling involving the pattern recognition receptor cGAS in cancer cells. The authors advise that temporal modulation of the cell cycle is important to consider in therapeutic approaches involving genotoxic agents and immune checkpoint blockers. Elsewhere in this issue, Andrew Jackson and colleagues provide evidence for an underlying mechanism whereby ruptured micronuclei activate a cell-autonomous inflammatory response via cGAS. Inflammatory gene expression following genotoxic cancer therapy is well documented, yet the events underlying its induction remain poorly understood. Inflammatory cytokines modify the tumour microenvironment by recruiting immune cells and are critical for both local and systemic (abscopal) tumour responses to radiotherapy 1 . A poorly understood feature of these responses is the delayed onset (days), in contrast to the acute DNA-damage responses that occur in minutes to hours. Such dichotomous kinetics implicate additional rate-limiting steps that are essential for DNA-damage-induced inflammation. Here we show that cell cycle progression through mitosis following double-stranded DNA breaks leads to the formation of micronuclei, which precede activation of inflammatory signalling and are a repository for the pattern-recognition receptor cyclic GMP–AMP synthase (cGAS). Inhibiting progression through mitosis or loss of pattern recognition by stimulator of interferon genes (STING)–cGAS impaired interferon signalling. Moreover, STING loss prevented the regression of abscopal tumours in the context of ionizing radiation and immune checkpoint blockade in vivo . These findings implicate temporal modulation of the cell cycle as an important consideration in the context of therapeutic strategies that combine genotoxic agents with immune checkpoint blockade.

Chromosomal instability is a hallmark of cancer that results from ongoing errors in chromosome segregation during mitosis. Although chromosomal instability is a major driver of tumour evolution, its role in metastasis has not been established. Here we show that chromosomal instability promotes metastasis by sustaining a tumour cell-autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol. This leads to the activation of the cGAS–STING (cyclic GMP-AMP synthase–stimulator of interferon genes) cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signalling. Genetic suppression of chromosomal instability markedly delays metastasis even in highly aneuploid tumour models, whereas continuous chromosome segregation errors promote cellular invasion and metastasis in a STING-dependent manner. By subverting lethal epithelial responses to cytosolic DNA, chromosomally unstable tumour cells co-opt chronic activation of innate immune pathways to spread to distant organs. In chromosomally unstable tumour cells, rupture of micronuclei exposes genomic DNA and activates the cGAS–STING cytosolic DNA-sensing pathway, thereby promoting metastasis. Chromosomal instability promotes metastasis The cGAS–STING cytosolic DNA-sensing pathway detects the presence of double-stranded DNA in the cytosol of cells, which triggers an inflammatory response. This pathway can be activated by foreign or cellular DNA. Lewis Cantley and colleagues show that the pathway is activated in human cancer cells with chromosomal instability. Improper segregation of chromosomes during cell division leads to the formation of unstable micronuclei, which burst and release their DNA into the cytosol. The resulting inflammatory response involves activation of NF-κB signalling and promotes metastasis in a STING-dependent manner. These findings link chromosomal instability to metastasis and may offer new avenues to preventing the spread of cancer to distant organs.

Focal chromosomal amplification contributes to the initiation of cancer by mediating overexpression of oncogenes 1 – 3 , and to the development of cancer therapy resistance by increasing the expression of genes whose action diminishes the efficacy of anti-cancer drugs. Here we used whole-genome sequencing of clonal cell isolates that developed chemotherapeutic resistance to show that chromothripsis is a major driver of circular extrachromosomal DNA (ecDNA) amplification (also known as double minutes) through mechanisms that depend on poly(ADP-ribose) polymerases (PARP) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Longitudinal analyses revealed that a further increase in drug tolerance is achieved by structural evolution of ecDNAs through additional rounds of chromothripsis. In situ Hi-C sequencing showed that ecDNAs preferentially tether near chromosome ends, where they re-integrate when DNA damage is present. Intrachromosomal amplifications that formed initially under low-level drug selection underwent continuing breakage–fusion–bridge cycles, generating amplicons more than 100 megabases in length that became trapped within interphase bridges and then shattered, thereby producing micronuclei whose encapsulated ecDNAs are substrates for chromothripsis. We identified similar genome rearrangement profiles linked to localized gene amplification in human cancers with acquired drug resistance or oncogene amplifications. We propose that chromothripsis is a primary mechanism that accelerates genomic DNA rearrangement and amplification into ecDNA and enables rapid acquisition of tolerance to altered growth conditions. Chromothripsis—a process during which chromosomes are ‘shattered’—drives the evolution of gene amplification and subsequent drug resistance in cancer cells.

Defects in the architecture or integrity of the nuclear envelope are associated with a variety of human diseases 1 . Micronuclei, one common nuclear aberration, are an origin for chromothripsis 2 , a catastrophic mutational process that is commonly observed in cancer 3 – 5 . Chromothripsis occurs after micronuclei spontaneously lose nuclear envelope integrity, which generates chromosome fragmentation 6 . Disruption of the nuclear envelope exposes DNA to the cytoplasm and initiates innate immune proinflammatory signalling 7 . Despite its importance, the basis of the fragility of the micronucleus nuclear envelope  is not known. Here we show that micronuclei undergo defective nuclear envelope assembly. Only ‘core’ nuclear envelope proteins 8 , 9 assemble efficiently on lagging chromosomes, whereas ‘non-core’ nuclear envelope proteins 8 , 9 , including nuclear pore complexes (NPCs), do not. Consequently, micronuclei fail to properly import key proteins that are necessary for the integrity of the nuclear envelope and genome. We show that spindle microtubules block assembly of NPCs and other non-core nuclear envelope proteins on lagging chromosomes, causing an irreversible defect in nuclear envelope assembly. Accordingly, experimental manipulations that position missegregated chromosomes away from the spindle correct defective nuclear envelope assembly, prevent spontaneous nuclear envelope disruption, and suppress DNA damage in micronuclei. Thus, during mitotic exit in metazoan cells, chromosome segregation and nuclear envelope assembly are only loosely coordinated by the timing of mitotic spindle disassembly. The absence of precise checkpoint controls may explain why errors during mitotic exit are frequent and often trigger catastrophic genome rearrangements 4 , 5 . The mitotic spindle prevents normal nuclear envelope assembly on missegregated chromosomes, leading to spontaneous envelope disruption of micronuclei and subsequent genome instability.

Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers 1 – 4 , but whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei 5 , 6 and subsequent rupture of the micronuclear envelope 7 profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice, as well as in cancer and non-transformed cells. Some of the changes in histone PTMs occur because of the rupture of the micronuclear envelope, whereas others are inherited from mitotic abnormalities before the micronucleus is formed. Using orthogonal approaches, we demonstrate that micronuclei exhibit extensive differences in chromatin accessibility, with a strong positional bias between promoters and distal or intergenic regions, in line with observed redistributions of histone PTMs. Inducing CIN causes widespread epigenetic dysregulation, and chromosomes that transit in micronuclei experience heritable abnormalities in their accessibility long after they have been reincorporated into the primary nucleus. Thus, as well as altering genomic copy number, CIN promotes epigenetic reprogramming and heterogeneity in cancer. Missegregated chromosomes that are sequestrated in micronuclei are subject to changes in histone modifications leading to abnormalities in chromatin accessibility that remain long after the chromosomes have been reincorporated into the primary nucleus.

Chromosome segregation errors during cell divisions generate aneuploidies and micronuclei, which can undergo extensive chromosomal rearrangements such as chromothripsis 1 – 5 . Selective pressures then shape distinct aneuploidy and rearrangement patterns—for example, in cancer 6 , 7 —but it is unknown whether initial biases in segregation errors and micronucleation exist for particular chromosomes. Using single-cell DNA sequencing 8 after an error-prone mitosis in untransformed, diploid cell lines and organoids, we show that chromosomes have different segregation error frequencies that result in non-random aneuploidy landscapes. Isolation and sequencing of single micronuclei from these cells showed that mis-segregating chromosomes frequently also preferentially become entrapped in micronuclei. A similar bias was found in naturally occurring micronuclei of two cancer cell lines. We find that segregation error frequencies of individual chromosomes correlate with their location in the interphase nucleus, and show that this is highest for peripheral chromosomes behind spindle poles. Randomization of chromosome positions, Cas9-mediated live tracking and forced repositioning of individual chromosomes showed that a greater distance from the nuclear centre directly increases the propensity to mis-segregate. Accordingly, chromothripsis in cancer genomes 9 and aneuploidies in early development 10 occur more frequently for larger chromosomes, which are preferentially located near the nuclear periphery. Our findings reveal a direct link between nuclear chromosome positions, segregation error frequencies and micronucleus content, with implications for our understanding of tumour genome evolution and the origins of specific aneuploidies during development. Using single-cell DNA sequencing after an error-prone mitosis in untransformed, diploid cell lines and organoids, chromosomes are shown to have different segregation error frequencies that result in non-random aneuploidy landscapes.

Genomic instability arising from defective responses to DNA damage 1 or mitotic chromosomal imbalances 2 can lead to the sequestration of DNA in aberrant extranuclear structures called micronuclei (MN). Although MN are a hallmark of ageing and diseases associated with genomic instability, the catalogue of genetic players that regulate the generation of MN remains to be determined. Here we analyse 997 mouse mutant lines, revealing 145 genes whose loss significantly increases ( n  = 71) or decreases ( n  = 74) MN formation, including many genes whose orthologues are linked to human disease. We found that mice null for Dscc1 , which showed the most significant increase in MN, also displayed a range of phenotypes characteristic of patients with cohesinopathy disorders. After validating the DSCC1 -associated MN instability phenotype in human cells, we used genome-wide CRISPR–Cas9 screening to define synthetic lethal and synthetic rescue interactors. We found that the loss of SIRT1 can rescue phenotypes associated with DSCC1 loss in a manner paralleling restoration of protein acetylation of SMC3. Our study reveals factors involved in maintaining genomic stability and shows how this information can be used to identify mechanisms that are relevant to human disease biology 1 . Genetic screening identifies a rich catalogue of regulators of micronucleus formation.

Complex genome rearrangements can be generated by the catastrophic pulverization of missegregated chromosomes trapped within micronuclei through a process known as chromothripsis 1 – 5 . As each chromosome contains a single centromere, it remains unclear how acentric fragments derived from shattered chromosomes are inherited between daughter cells during mitosis 6 . Here we tracked micronucleated chromosomes with live-cell imaging and show that acentric fragments cluster in close spatial proximity throughout mitosis for asymmetric inheritance by a single daughter cell. Mechanistically, the CIP2A–TOPBP1 complex prematurely associates with DNA lesions within ruptured micronuclei during interphase, which poises pulverized chromosomes for clustering upon mitotic entry. Inactivation of CIP2A–TOPBP1 caused acentric fragments to disperse throughout the mitotic cytoplasm, stochastically partition into the nucleus of both daughter cells and aberrantly misaccumulate as cytoplasmic DNA. Mitotic clustering facilitates the reassembly of acentric fragments into rearranged chromosomes lacking the extensive DNA copy-number losses that are characteristic of canonical chromothripsis. Comprehensive analysis of pan-cancer genomes revealed clusters of DNA copy-number-neutral rearrangements—termed balanced chromothripsis—across diverse tumour types resulting in the acquisition of known cancer driver events. Thus, distinct patterns of chromothripsis can be explained by the spatial clustering of pulverized chromosomes from micronuclei. The CIP2A–TOPBP1 complex tethers fragmented chromosomes from micronuclei for asymmetric mitotic inheritance, explaining distinct patterns of chromosome rearrangements in cancers and genomic disorders.