Boveri and beyond: Chromothripsis and genomic instability from mitotic errors.

Mitotic cell division is tightly monitored by checkpoints that safeguard the genome from instability. Failures in accurate chromosome segregation during mitosis can cause numerical aneuploidy, which was hypothesized by Theodor Boveri over a century ago to promote tumorigenesis. Recent interrogation of pan-cancer genomes has identified unexpected classes of chromosomal abnormalities, including complex rearrangements arising through chromothripsis. This process is driven by mitotic errors that generate abnormal nuclear structures that provoke extensive yet localized shattering of mis-segregated chromosomes. Here, we discuss emerging mechanisms underlying chromothripsis from micronuclei and chromatin bridges, as well as highlight how this mutational cascade converges on the DNA damage response. A fundamental understanding of these catastrophic processes will provide insight into how initial errors in mitosis can precipitate rapid cancer genome evolution.

Mitotic clustering of pulverized chromosomes from micronuclei.

Complex genome rearrangements can be generated by the catastrophic pulverization of missegregated chromosomes trapped within micronuclei through a process known as chromothripsis1-5. As each chromosome contains a single centromere, it remains unclear how acentric fragments derived from shattered chromosomes are inherited between daughter cells during mitosis6. 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.

Epigenetic dysregulation from chromosomal transit in micronuclei.

Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers1-4, but whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei5,6 and subsequent rupture of the micronuclear envelope7 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.

Chromothripsis drives the evolution of gene amplification in cancer.

Focal chromosomal amplification contributes to the initiation of cancer by mediating overexpression of oncogenes1-3, 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.

SETD2 safeguards the genome against isochromosome formation.

Isochromosomes are mirror-imaged chromosomes with simultaneous duplication and deletion of genetic material which may contain two centromeres to create isodicentric chromosomes. Although isochromosomes commonly occur in cancer and developmental disorders and promote genome instability, mechanisms that prevent isochromosomes are not well understood. We show here that the tumor suppressor and methyltransferase SETD2 is essential to prevent these errors. Using cellular and cytogenetic approaches, we demonstrate that loss of SETD2 or its epigenetic mark, histone H3 lysine 36 trimethylation (H3K36me3), results in the formation of isochromosomes as well as isodicentric and acentric chromosomes. These defects arise during DNA replication and are likely due to faulty homologous recombination by RAD52. These data provide a mechanism for isochromosome generation and demonstrate that SETD2 and H3K36me3 are essential to prevent the formation of this common mutable chromatin structure known to initiate a cascade of genomic instability in cancer.

Structural variation cooperates with permissive chromatin to control enhancer hijacking-mediated oncogenic transcription.

Structural variants (SVs) involving enhancer hijacking can rewire chromatin topologies to cause oncogene activation in human cancers, including hematologic malignancies; however, because of the lack of tools to assess their effects on gene regulation and chromatin organization, the molecular determinants for the functional output of enhancer hijacking remain poorly understood. Here, we developed a multimodal approach to integrate genome sequencing, chromosome conformation, chromatin state, and transcriptomic alteration for quantitative analysis of transcriptional effects and structural reorganization imposed by SVs in leukemic genomes. We identified known and new pathogenic SVs, including recurrent t(5;14) translocations that cause the hijacking of BCL11B enhancers for the allele-specific activation of TLX3 in a subtype of pediatric leukemia. Epigenetic perturbation of SV-hijacked BCL11B enhancers impairs TLX3 transcription, which are required for the growth of t(5;14) leukemia cells. By CRISPR engineering of patient-derived t(5;14) in isogenic leukemia cells, we uncovered a new mechanism whereby the transcriptional output of SV-induced BCL11B enhancer hijacking is dependent on the loss of DNA hypermethylation at the TLX3 promoter. Our results highlight the importance of the cooperation between genetic alteration and permissive chromatin as a critical determinant of SV-mediated oncogene activation, with implications for understanding aberrant gene transcription after epigenetic therapies in patients with leukemia. Hence, leveraging the interdependency of genetic alteration on chromatin variation may provide new opportunities to reprogram gene regulation as targeted interventions in human disease.

Mechanistic origins of diverse genome rearrangements in cancer.

Cancer genomes frequently harbor structural chromosomal rearrangements that disrupt the linear DNA sequence order and copy number. To date, diverse classes of structural variants have been identified across multiple cancer types. These aberrations span a wide spectrum of complexity, ranging from simple translocations to intricate patterns of rearrangements involving multiple chromosomes. Although most somatic rearrangements are acquired gradually throughout tumorigenesis, recent interrogation of cancer genomes have uncovered novel categories of complex rearrangements that arises rapidly through a one-off catastrophic event, including chromothripsis and chromoplexy. Here we review the cellular and molecular mechanisms contributing to the formation of diverse structural rearrangement classes during cancer development. Genotoxic stress from a myriad of extrinsic and intrinsic sources can trigger DNA double-strand breaks that are subjected to DNA repair with potentially mutagenic outcomes. We also highlight how aberrant nuclear structures generated through mitotic cell division errors, such as rupture-prone micronuclei and chromosome bridges, can instigate massive DNA damage and the formation of complex rearrangements in cancer genomes.

Biphasic burrowing in Atlantic hagfish (Myxine limosa).

Myxine limosa is a burrowing species of hagfish that occurs in the western North Atlantic in areas with muddy substrate and at depths generally greater than 100 meters. Burrowing of M. limosa has been observed from submersibles, but little is known about the behavior of these animals within the substrate or the biomechanical mechanisms involved. Here, we investigated burrowing in M. limosa by observing individuals as they burrowed through transparent gelatin. A photoelastic setup using crossed polarizers allowed us to visualize stress development in the gelatin as the hagfish moved through it. We found that M. limosa created U-shaped burrows in gelatin using a stereotyped, two-phase burrowing behavior. In the first ('thrash') phase, hagfish drove their head and their anterior body into the substrate using vigorous sinusoidal swimming movements, with their head moving side-to-side. In the second ('wriggle') phase, swimming movements ceased, with propulsion coming exclusively from the anterior, submerged portion of body. The wriggle phase involved side-to-side head movements and movements of the submerged part of the body that resembled the internal concertina strategy used by caecilians and uropeltid snakes. The entire burrowing process took on average 7.6 min to complete and ended with the hagfish's head protruding from the substrate and the rest of its body generally concealed. Understanding the burrowing activities of hagfishes could lead to improved understanding of sediment turnover in marine benthic habitats, new insights into the reproductive behavior of hagfishes, or even inspiration for the design of burrowing robots.

Chromosomal instability drives metastasis through a cytosolic DNA response.

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.

Restoration of DNA repair mitigates genome instability and increases productivity of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are the primary host for manufacturing of therapeutic proteins. However, productivity loss is a major problem and is associated with genome instability, as chromosomal aberrations reduce transgene copy number and decrease protein expression. We analyzed whole-genome sequencing data from 11 CHO cell lines and found deleterious single-nucleotide variants in DNA repair genes. Comparison with primary Chinese hamster cells confirmed DNA repair to be compromised in CHO. Correction of key DNA repair genes by single-nucleotide variant reversal or expression of intact complementary DNAs successfully improved DNA repair and mitigated karyotypic instability. Moreover, overexpression of intact copies of LIG4 and XRCC6 in a CHO cell line expressing secreted alkaline phosphatase mitigated transgene copy loss and improved protein titer retention. These results show that correction of DNA repair genes yields improvements in genome stability in CHO, and provide new opportunities for cell line development for sustainable protein expression.

Cellular and genomic approaches for exploring structural chromosomal rearrangements.

Human chromosomes are arranged in a linear and conserved sequence order that undergoes further spatial folding within the three-dimensional space of the nucleus. Although structural variations in this organization are an important source of natural genetic diversity, cytogenetic aberrations can also underlie a number of human diseases and disorders. Approaches for studying chromosome structure began half a century ago with karyotyping of Giemsa-banded chromosomes and has now evolved to encompass high-resolution fluorescence microscopy, reporter-based assays, and next-generation DNA sequencing technologies. Here, we provide a general overview of experimental methods at different resolution and sensitivity scales and discuss how they can be complemented to provide synergistic insight into the study of human chromosome structural rearrangements. These approaches range from kilobase-level resolution DNA fluorescence in situ hybridization (FISH)-based imaging approaches of individual cells to genome-wide sequencing strategies that can capture nucleotide-level information from diverse sample types. Technological advances coupled to the combinatorial use of multiple methods have resulted in the discovery of new rearrangement classes along with mechanistic insights into the processes that drive structural alterations in the human genome.

Targeting of Nrf2 induces DNA damage signaling and protects colonic epithelial cells from ionizing radiation.

Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a key transcriptional regulator for antioxidant and anti-inflammation enzymes that binds to its endogenous inhibitor protein, Kelch-like ECH (erythroid cell-derived protein with CNC homology)-associated protein 1, in the cytoplasm under normal conditions. Various endogenous or environmental oxidative stresses, such as ionizing radiation (IR), can disrupt the Nrf2-Kelch-like ECH-associated protein 1 complex. This allows Nrf2 to translocate from the cytoplasm into the nucleus to induce transcription of heme oxygenase-1 and other cytoprotective enzymes through binding to antioxidant responsive elements. However, how Nrf2 protects cells from IR-induced damage remains unclear. Here, we report that Nrf2 activation by the synthetic triterpenoids, bardoxolone methyl (BARD) and 2-cyano-3,12-dioxooleana-1,9 (11)-dien-28-oic acid-ethyl amide, protects colonic epithelial cells against IR-induced damage, in part, by enhancing signaling of the DNA damage response. Pretreatment with BARD reduced the frequency of both G1 and S/G2 chromosome aberrations and enhanced the disappearance of repairosomes (C-terminal binding protein interacting protein, Rad51, and p53 binding protein-1 foci) after IR. BARD protected cells from IR toxicity in a Nrf2-dependent manner. The p53 binding protein-1 promoter contains three antioxidant responsive elements in which Nrf2 directly binds following BARD treatment. In addition, 2-cyano-3,12-dioxooleana-1,9 (11)-dien-28-oic acid-ethyl amide provided before exposure to a lethal dose of whole-body irradiation protected WT mice from DNA damage and acute gastrointestinal toxicity, which resulted in improved overall survival. These results demonstrate that Nrf2 activation by synthetic triterpenoids is a promising candidate target to protect the gastrointestinal tract against acute IR in vitro and in vivo.

Induction of chromosome-specific micronuclei and chromothripsis by centromere inactivation.

Chromothripsis describes the catastrophic fragmentation of individual chromosomes followed by its haphazard reassembly into a derivative chromosome harboring complex rearrangements. This process can be initiated by mitotic cell division errors when one or more chromosomes aberrantly mis-segregate into micronuclei and acquire extensive DNA damage. Approaches to induce the formation of micronuclei encapsulating random chromosomes have been used; however, the eventual reincorporation of the micronucleated chromosome into daughter cell nuclei poses a challenge in tracking the chromosome for multiple cell cycles. Here we outline an approach to genetically engineer stable human cell lines capable of efficient chromosome-specific micronuclei induction. This strategy, which targets the CENP-B-deficient Y chromosome centromere for inactivation, allows the stepwise process of chromothripsis to be experimentally recapitulated, including the mechanisms and timing of chromosome fragmentation. Lastly, we describe the integration of a selection marker onto the micronucleated Y chromosome that enables the diverse genomic rearrangement landscape arising from micronuclei formation to be interrogated.

Non-homologous end joining shapes the genomic rearrangement landscape of chromothripsis from mitotic errors.

Mitotic errors generate micronuclei entrapping mis-segregated chromosomes, which are susceptible to catastrophic fragmentation through chromothripsis. The reassembly of fragmented chromosomes by error-prone DNA double-strand break (DSB) repair generates diverse genomic rearrangements associated with human diseases. How specific repair pathways recognize and process these lesions remains poorly understood. Here we use CRISPR/Cas9 to systematically inactivate distinct DSB repair pathways and interrogate the rearrangement landscape of fragmented chromosomes. Deletion of canonical non-homologous end joining (NHEJ) components substantially reduces complex rearrangements and shifts the rearrangement landscape toward simple alterations without the characteristic patterns of chromothripsis. Following reincorporation into the nucleus, fragmented chromosomes localize within sub-nuclear micronuclei bodies (MN bodies) and undergo ligation by NHEJ within a single cell cycle. In the absence of NHEJ, chromosome fragments are rarely engaged by alternative end-joining or recombination-based mechanisms, resulting in delayed repair kinetics, persistent 53BP1-labeled MN bodies, and cell cycle arrest. Thus, we provide evidence supporting NHEJ as the exclusive DSB repair pathway generating complex rearrangements from mitotic errors.

Polycythemia as an Underlying Cause of Digital Gangrene: A Rare and Unusual Case Presentation.

Digital gangrene is frequently encountered in patients who have diabetes with peripheral vascular compromise, with or without superimposed infection. Preoperative laboratory values and radiographic images are important to determine a proper course of action. Equally important is a thorough history taking to confirm or rule out systemic entities and preexisting conditions that can aggravate or predispose one to the development of digital gangrene. A patient with diabetes presented with a rare and unusual case of digital gangrene, as he clinically had strong pedal pulses. Preoperative workup revealed a suspicion of polycythemia, which was subsequently confirmed. The patient underwent several days of phlebotomy until his hemoglobin and hematocrit levels were brought down to optimized levels before a digital amputation was performed. He went on to heal uneventfully, and he is currently being closely followed by oncology/hematology colleagues with periodic phlebotomy.

Epigenetic centromere identity is precisely maintained through DNA replication but is uniquely specified among human cells.

Centromere identity is defined and maintained epigenetically by the presence of the histone variant CENP-A. How centromeric CENP-A position is specified and precisely maintained through DNA replication is not fully understood. The recently released Telomere-to-Telomere (T2T) genome assembly containing the first complete human centromere sequences provides a new resource for examining CENP-A position. Mapping CENP-A position in clones of the same cell line to the T2T assembly identified highly similar CENP-A position after multiple cell divisions. In contrast, centromeric CENP-A epialleles were evident at several centromeres of different human cell lines, demonstrating the location of CENP-A enrichment and the site of kinetochore recruitment vary among human cells. Across the cell cycle, CENP-A molecules deposited in G1 phase are maintained in their precise position through DNA replication. Thus, despite CENP-A dilution during DNA replication, CENP-A is precisely reloaded onto the same sequences within the daughter centromeres, maintaining unique centromere identity among human cells.

Non-homologous end joining shapes the genomic rearrangement landscape of chromothripsis from mitotic errors.

Errors in mitosis can generate micronuclei that entrap mis-segregated chromosomes, which are susceptible to catastrophic fragmentation through a process termed chromothripsis. The reassembly of fragmented chromosomes by error-prone DNA double-strand break (DSB) repair generates a spectrum of simple and complex genomic rearrangements that are associated with human cancers and disorders. How specific DSB repair pathways recognize and process these lesions remains poorly understood. Here we used CRISPR/Cas9 to systematically inactivate distinct DSB processing or repair pathways and interrogated the rearrangement landscape of fragmented chromosomes from micronuclei. Deletion of canonical non-homologous end joining (NHEJ) components, including DNA-PKcs, LIG4, and XLF, substantially reduced the formation of complex rearrangements and shifted the rearrangement landscape toward simple alterations without the characteristic patterns of cancer-associated chromothripsis. Following reincorporation into the nucleus, fragmented chromosomes localize within micronuclei bodies (MN bodies) and undergo successful ligation by NHEJ within a single cell cycle. In the absence of NHEJ, chromosome fragments were rarely engaged by polymerase theta-mediated alternative end-joining or recombination-based mechanisms, resulting in delayed repair kinetics and persistent 53BP1-labeled MN bodies in the interphase nucleus. Prolonged DNA damage signaling from unrepaired fragments ultimately triggered cell cycle arrest. Thus, we provide evidence supporting NHEJ as the exclusive DSB repair pathway generating complex rearrangements following chromothripsis from mitotic errors.

Light-activated macromolecular phase separation modulates transcription by reconfiguring chromatin interactions.

Biomolecular condensates participate in the regulation of gene transcription, yet the relationship between nuclear condensation and transcriptional activation remains elusive. Here, we devised a biotinylated CRISPR-dCas9-based optogenetic method, light-activated macromolecular phase separation (LAMPS), to enable inducible formation, affinity purification, and multiomic dissection of nuclear condensates at the targeted genomic loci. LAMPS-induced condensation at enhancers and promoters activates endogenous gene transcription by chromatin reconfiguration, causing increased chromatin accessibility and de novo formation of long-range chromosomal loops. Proteomic profiling of light-induced condensates by dCas9-mediated affinity purification uncovers multivalent interaction-dependent remodeling of macromolecular composition, resulting in the selective enrichment of transcriptional coactivators and chromatin structure proteins. Our findings support a model whereby the formation of nuclear condensates at native genomic loci reconfigures chromatin architecture and multiprotein assemblies to modulate gene transcription. Hence, LAMPS facilitates mechanistic interrogation of the relationship between nuclear condensation, genome structure, and gene transcription in living cells.

De novo pyrimidine synthesis is a targetable vulnerability in IDH mutant glioma.

Mutations affecting isocitrate dehydrogenase (IDH) enzymes are prevalent in glioma, leukemia, and other cancers. Although mutant IDH inhibitors are effective against leukemia, they seem to be less active in aggressive glioma, underscoring the need for alternative treatment strategies. Through a chemical synthetic lethality screen, we discovered that IDH1-mutant glioma cells are hypersensitive to drugs targeting enzymes in the de novo pyrimidine nucleotide synthesis pathway, including dihydroorotate dehydrogenase (DHODH). We developed a genetically engineered mouse model of mutant IDH1-driven astrocytoma and used it and multiple patient-derived models to show that the brain-penetrant DHODH inhibitor BAY 2402234 displays monotherapy efficacy against IDH-mutant gliomas. Mechanistically, this reflects an obligate dependence of glioma cells on the de novo pyrimidine synthesis pathway and mutant IDH's ability to sensitize to DNA damage upon nucleotide pool imbalance. Our work outlines a tumor-selective, biomarker-guided therapeutic strategy that is poised for clinical translation.