Haplotype-resolved karyotype construction from Hi-C data using refLinker.

Chromosomal aberrations are prevalent in cancer genomes, yet it remains challenging to resolve the long-range structure of rearranged chromosomes. A key problem is to determine the chromosomal origin of rearranged genomic segments, which requires chromosome-length haplotype information. Here we describe refLinker, a new computational method for whole-chromosome haplotype inference using external reference panels and Hi-C. We show that refLinker ensures consistent long-range phasing accuracy in both diploid human genomes and aneuploid cancers, including regions with loss-of-heterozygosity and high-level focal amplification. We further demonstrate the feasibility of complex genome reconstruction using haplotype-specific Hi-C contacts, revealing new karyotype features in two widely studied cancer cell lines. Together, these findings provide a new framework for the complete resolution of long-range chromosome structure in complex genomes and highlight the unique advantages of Hi-C data for reconstructing cancer genomes with chromosome-scale continuity.

2D and 3D multiplexed subcellular profiling of nuclear instability in human cancer.

Nuclear atypia, including altered nuclear size, contour, and chromatin organization, is ubiquitous in cancer cells. Atypical primary nuclei and micronuclei can rupture during interphase; however, the frequency, causes, and consequences of nuclear rupture are unknown in most cancers. We demonstrate that nuclear envelope rupture is surprisingly common in many human cancers, particularly glioblastoma. Using highly-multiplexed 2D and super-resolution 3D-imaging of glioblastoma tissues and patient-derived xenografts and cells, we link primary nuclear rupture with reduced lamin A/C and micronuclear rupture with reduced lamin B1. Moreover, ruptured glioblastoma cells activate cGAS-STING-signaling involved in innate immunity. We observe that local patterning of cell states influences tumor spatial organization and is linked to both lamin expression and rupture frequency, with neural-progenitor-cell-like states exhibiting the lowest lamin A/C levels and greatest susceptibility to primary nuclear rupture. Our study reveals that nuclear instability is a core feature of cancer, and links nuclear integrity, cell state, and immune signaling.

Heritable transcriptional defects from aberrations of nuclear architecture.

Transcriptional heterogeneity due to plasticity of the epigenetic state of chromatin contributes to tumour evolution, metastasis and drug resistance1-3. However, the mechanisms that cause this epigenetic variation are incompletely understood. Here we identify micronuclei and chromosome bridges, aberrations in the nucleus common in cancer4,5, as sources of heritable transcriptional suppression. Using a combination of approaches, including long-term live-cell imaging and same-cell single-cell RNA sequencing (Look-Seq2), we identified reductions in gene expression in chromosomes from micronuclei. With heterogeneous penetrance, these changes in gene expression can be heritable even after the chromosome from the micronucleus has been re-incorporated into a normal daughter cell nucleus. Concomitantly, micronuclear chromosomes acquire aberrant epigenetic chromatin marks. These defects may persist as variably reduced chromatin accessibility and reduced gene expression after clonal expansion from single cells. Persistent transcriptional repression is strongly associated with, and may be explained by, markedly long-lived DNA damage. Epigenetic alterations in transcription may therefore be inherently coupled to chromosomal instability and aberrations in nuclear architecture.

ERα-associated translocations underlie oncogene amplifications in breast cancer.

Focal copy-number amplification is an oncogenic event. Although recent studies have revealed the complex structure1-3 and the evolutionary trajectories4 of oncogene amplicons, their origin remains poorly understood. Here we show that focal amplifications in breast cancer frequently derive from a mechanism-which we term translocation-bridge amplification-involving inter-chromosomal translocations that lead to dicentric chromosome bridge formation and breakage. In 780 breast cancer genomes, we observe that focal amplifications are frequently connected to each other by inter-chromosomal translocations at their boundaries. Subsequent analysis indicates the following model: the oncogene neighbourhood is translocated in G1 creating a dicentric chromosome, the dicentric chromosome is replicated, and as dicentric sister chromosomes segregate during mitosis, a chromosome bridge is formed and then broken, with fragments often being circularized in extrachromosomal DNAs. This model explains the amplifications of key oncogenes, including ERBB2 and CCND1. Recurrent amplification boundaries and rearrangement hotspots correlate with oestrogen receptor binding in breast cancer cells. Experimentally, oestrogen treatment induces DNA double-strand breaks in the oestrogen receptor target regions that are repaired by translocations, suggesting a role of oestrogen in generating the initial translocations. A pan-cancer analysis reveals tissue-specific biases in mechanisms initiating focal amplifications, with the breakage-fusion-bridge cycle prevalent in some and the translocation-bridge amplification in others, probably owing to the different timing of DNA break repair. Our results identify a common mode of oncogene amplification and propose oestrogen as its mechanistic origin in breast cancer.

A tubule-sheet continuum model for the mechanism of nuclear envelope assembly.

Nuclear envelope (NE) assembly defects cause chromosome fragmentation, cancer, and aging. However, major questions about the mechanism of NE assembly and its relationship to nuclear pathology are unresolved. In particular, how cells efficiently assemble the NE starting from vastly different, cell type-specific endoplasmic reticulum (ER) morphologies is unclear. Here, we identify a NE assembly mechanism, "membrane infiltration," that defines one end of a continuum with another NE assembly mechanism, "lateral sheet expansion," in human cells. Membrane infiltration involves the recruitment of ER tubules or small sheets to the chromatin surface by mitotic actin filaments. Lateral sheet expansion involves actin-independent envelopment of peripheral chromatin by large ER sheets that then extend over chromatin within the spindle. We propose a "tubule-sheet continuum" model that explains the efficient NE assembly from any starting ER morphology, the cell type-specific patterns of nuclear pore complex (NPC) assembly, and the obligatory NPC assembly defect of micronuclei.

Breakage of cytoplasmic chromosomes by pathological DNA base excision repair.

Chromothripsis is a catastrophic mutational process that promotes tumorigenesis and causes congenital disease1-4. Chromothripsis originates from aberrations of nuclei called micronuclei or chromosome bridges5-8. These structures are associated with fragile nuclear envelopes that spontaneously rupture9,10, leading to DNA damage when chromatin is exposed to the interphase cytoplasm. Here we identify a mechanism explaining a major fraction of this DNA damage. Micronuclei accumulate large amounts of RNA-DNA hybrids, which are edited by adenine deaminases acting on RNA (ADAR enzymes) to generate deoxyinosine. Deoxyinosine is then converted into abasic sites by a DNA base excision repair (BER) glycosylase, N-methyl-purine DNA glycosylase11,12 (MPG). These abasic sites are cleaved by the BER endonuclease, apurinic/apyrimidinic endonuclease12 (APE1), creating single-stranded DNA nicks that can be converted to DNA double strand breaks by DNA replication or when closely spaced nicks occur on opposite strands13,14. This model predicts that MPG should be able to remove the deoxyinosine base from the DNA strand of RNA-DNA hybrids, which we demonstrate using purified proteins and oligonucleotide substrates. These findings identify a mechanism for fragmentation of micronuclear chromosomes, an important step in generating chromothripsis. Rather than breaking any normal chromosome, we propose that the eukaryotic cytoplasm only damages chromosomes with pre-existing defects such as the DNA base abnormality described here.

Whole chromosome loss and genomic instability in mouse embryos after CRISPR-Cas9 genome editing.

Karyotype alterations have emerged as on-target complications from CRISPR-Cas9 genome editing. However, the events that lead to these karyotypic changes in embryos after Cas9-treatment remain unknown. Here, using imaging and single-cell genome sequencing of 8-cell stage embryos, we track both spontaneous and Cas9-induced karyotype aberrations through the first three divisions of embryonic development. We observe the generation of abnormal structures of the nucleus that arise as a consequence of errors in mitosis, including micronuclei and chromosome bridges, and determine their contribution to common karyotype aberrations including whole chromosome loss that has been recently reported after editing in embryos. Together, these data demonstrate that Cas9-mediated germline genome editing can lead to unwanted on-target side effects, including major chromosome structural alterations that can be propagated over several divisions of embryonic development.

Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing.

Genome editing has therapeutic potential for treating genetic diseases and cancer. However, the currently most practicable approaches rely on the generation of DNA double-strand breaks (DSBs), which can give rise to a poorly characterized spectrum of chromosome structural abnormalities. Here, using model cells and single-cell whole-genome sequencing, as well as by editing at a clinically relevant locus in clinically relevant cells, we show that CRISPR-Cas9 editing generates structural defects of the nucleus, micronuclei and chromosome bridges, which initiate a mutational process called chromothripsis. Chromothripsis is extensive chromosome rearrangement restricted to one or a few chromosomes that can cause human congenital disease and cancer. These results demonstrate that chromothripsis is a previously unappreciated on-target consequence of CRISPR-Cas9-generated DSBs. As genome editing is implemented in the clinic, the potential for extensive chromosomal rearrangements should be considered and monitored.

The Ubiquitin Ligase TRAIP: Double-Edged Sword at the Replisome.

In preparation for cell division, the genome must be copied with high fidelity. However, replisomes often encounter obstacles, including bulky DNA lesions caused by reactive metabolites and chemotherapeutics, as well as stable nucleoprotein complexes. Here, we discuss recent advances in our understanding of TRAIP, a replisome-associated E3 ubiquitin ligase that is mutated in microcephalic primordial dwarfism. In interphase, TRAIP helps replisomes overcome DNA interstrand crosslinks and DNA-protein crosslinks, whereas in mitosis it triggers disassembly of all replisomes that remain on chromatin. We describe a model to explain how TRAIP performs these disparate functions and how they help maintain genome integrity.

Acquired resistance to combined BET and CDK4/6 inhibition in triple-negative breast cancer.

BET inhibitors are promising therapeutic agents for the treatment of triple-negative breast cancer (TNBC), but the rapid emergence of resistance necessitates investigation of combination therapies and their effects on tumor evolution. Here, we show that palbociclib, a CDK4/6 inhibitor, and paclitaxel, a microtubule inhibitor, synergize with the BET inhibitor JQ1 in TNBC lines. High-complexity DNA barcoding and mathematical modeling indicate a high rate of de novo acquired resistance to these drugs relative to pre-existing resistance. We demonstrate that the combination of JQ1 and palbociclib induces cell division errors, which can increase the chance of developing aneuploidy. Characterizing acquired resistance to combination treatment at a single cell level shows heterogeneous mechanisms including activation of G1-S and senescence pathways. Our results establish a rationale for further investigation of combined BET and CDK4/6 inhibition in TNBC and suggest novel mechanisms of action for these drugs and new vulnerabilities in cells after emergence of resistance.

Mechanisms generating cancer genome complexity from a single cell division error.

The chromosome breakage-fusion-bridge (BFB) cycle is a mutational process that produces gene amplification and genome instability. Signatures of BFB cycles can be observed in cancer genomes alongside chromothripsis, another catastrophic mutational phenomenon. We explain this association by elucidating a mutational cascade that is triggered by a single cell division error-chromosome bridge formation-that rapidly increases genomic complexity. We show that actomyosin forces are required for initial bridge breakage. Chromothripsis accumulates, beginning with aberrant interphase replication of bridge DNA. A subsequent burst of DNA replication in the next mitosis generates extensive DNA damage. During this second cell division, broken bridge chromosomes frequently missegregate and form micronuclei, promoting additional chromothripsis. We propose that iterations of this mutational cascade generate the continuing evolution and subclonal heterogeneity characteristic of many human cancers.

Factors promoting nuclear envelope assembly independent of the canonical ESCRT pathway.

The nuclear envelope (NE) undergoes dynamic remodeling to maintain NE integrity, a process involving the inner nuclear membrane protein LEM2 recruiting CHMP7/Cmp7 and then ESCRT-III. However, prior work has hinted at CHMP7/ESCRT-independent mechanisms. To identify such mechanisms, we studied NE assembly in Schizosaccharomyces japonicus, a fission yeast that undergoes partial mitotic NE breakdown and reassembly. S. japonicus cells lacking Cmp7 have compromised NE sealing after mitosis but are viable. A genetic screen identified mutations that promote NE integrity in cmp7Δ cells. Unexpectedly, loss of Lem2 or its interacting partner Nur1 suppressed cmp7Δ defects. In the absence of Cmp7, Lem2 formed aggregates that appear to interfere with ESCRT-independent NE sealing. A gain-of-function mutation implicated a membrane and ESCRT-III regulator, Alx1, in this alternate pathway. Additional results suggest a potentially general role for unsaturated fatty acids in NE integrity. These findings establish the existence of mechanisms for NE sealing independent of the canonical ESCRT pathway.

The coordination of nuclear envelope assembly and chromosome segregation in metazoans.

The nuclear envelope (NE) is composed of two lipid bilayer membranes that enclose the eukaryotic genome. In interphase, the NE is perforated by thousands of nuclear pore complexes (NPCs), which allow transport in and out of the nucleus. During mitosis in metazoans, the NE is broken down and then reassembled in a manner that enables proper chromosome segregation and the formation of a single nucleus in each daughter cell. Defects in coordinating NE reformation and chromosome segregation can cause aberrant nuclear architecture. This includes the formation of micronuclei, which can trigger a catastrophic mutational process commonly observed in cancers called chromothripsis. Here, we discuss the current understanding of the coordination of NE reformation with chromosome segregation during mitotic exit in metazoans. We review differing models in the field and highlight recent work suggesting that normal NE reformation and chromosome segregation are physically linked through the timing of mitotic spindle disassembly.

Comprehensive analysis of chromothripsis in 2,658 human cancers using whole-genome sequencing.

Chromothripsis is a mutational phenomenon characterized by massive, clustered genomic rearrangements that occurs in cancer and other diseases. Recent studies in selected cancer types have suggested that chromothripsis may be more common than initially inferred from low-resolution copy-number data. Here, as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), we analyze patterns of chromothripsis across 2,658 tumors from 38 cancer types using whole-genome sequencing data. We find that chromothripsis events are pervasive across cancers, with a frequency of more than 50% in several cancer types. Whereas canonical chromothripsis profiles display oscillations between two copy-number states, a considerable fraction of events involve multiple chromosomes and additional structural alterations. In addition to non-homologous end joining, we detect signatures of replication-associated processes and templated insertions. Chromothripsis contributes to oncogene amplification and to inactivation of genes such as mismatch-repair-related genes. These findings show that chromothripsis is a major process that drives genome evolution in human cancer.

Mitotic CDK Promotes Replisome Disassembly, Fork Breakage, and Complex DNA Rearrangements.

DNA replication errors generate complex chromosomal rearrangements and thereby contribute to tumorigenesis and other human diseases. One mechanism that triggers these errors is mitotic entry before the completion of DNA replication. To address how mitosis might affect DNA replication, we used Xenopus egg extracts. When mitotic CDK (Cyclin B1-CDK1) is used to drive interphase egg extracts into a mitotic state, the replicative CMG (CDC45/MCM2-7/GINS) helicase undergoes ubiquitylation on its MCM7 subunit, dependent on the E3 ubiquitin ligase TRAIP. Whether replisomes have stalled or undergone termination, CMG ubiquitylation is followed by its extraction from chromatin by the CDC48/p97 ATPase. TRAIP-dependent CMG unloading during mitosis is also seen in C. elegans early embryos. At stalled forks, CMG removal results in fork breakage and end joining events involving deletions and templated insertions. Our results identify a mitotic pathway of global replisome disassembly that can trigger replication fork collapse and DNA rearrangements.

Human nuclear RNAi-defective 2 (NRDE2) is an essential RNA splicing factor.

The accurate inheritance of genetic material is a basic necessity in all domains of life and an unexpectedly large number of RNA processing factors are required for mitotic progression and genome stability. NRDE2 (nuclear RNAi defective-2) is an evolutionarily conserved protein originally discovered for its role in nuclear RNA interference (RNAi) and heritable gene silencing in Caenorhabditis elegans (C. elegans). The function of the human NRDE2 gene remains poorly understood. Here we show that human NRDE2 is an essential protein required for suppressing intron retention in a subset of pre-mRNAs containing short, GC-rich introns with relatively weak 5' and 3' splice sites. NRDE2 preferentially interacts with components of the U5 small nuclear ribonucleoprotein (snRNP), the exon junction complex, and the RNA exosome. Interestingly, NRDE2-depleted cells exhibit greatly increased levels of genomic instability and DNA damage, as well as defects in centrosome maturation and mitotic progression. We identify the essential centriolar satellite protein, CEP131, as a direct NRDE2-regulated target. NRDE2 specifically binds to and promotes the efficient splicing of CEP131 pre-mRNA, and depleting NRDE2 dramatically reduces CEP131 protein expression, contributing to impaired recruitment of critical centrosomal proteins (e.g., γ-tubulin and Aurora Kinase A) to the spindle poles during mitosis. Our work establishes a conserved role for human NRDE2 in RNA splicing, characterizes the severe genomic instability phenotypes observed upon loss of NRDE2, and highlights the direct regulation of CEP131 splicing as one of multiple mechanisms through which such phenotypes might be explained.

Cells Lacking the RB1 Tumor Suppressor Gene Are Hyperdependent on Aurora B Kinase for Survival.

Small cell lung cancer (SCLC) accounts for 15% of lung cancers and is almost always linked to inactivating RB1 and TP53 mutations. SCLC frequently responds, albeit briefly, to chemotherapy. The canonical function of the RB1 gene product RB1 is to repress the E2F transcription factor family. RB1 also plays both E2F-dependent and E2F-independent mitotic roles. We performed a synthetic lethal CRISPR/Cas9 screen in an RB1 -/- SCLC cell line that conditionally expresses RB1 to identify dependencies that are caused by RB1 loss and discovered that RB1 -/- SCLC cell lines are hyperdependent on multiple proteins linked to chromosomal segregation, including Aurora B kinase. Moreover, we show that an Aurora B kinase inhibitor is efficacious in multiple preclinical SCLC models at concentrations that are well tolerated in mice. These results suggest that RB1 loss is a predictive biomarker for sensitivity to Aurora B kinase inhibitors in SCLC and perhaps other RB1 -/- cancers. SIGNIFICANCE: SCLC is rarely associated with actionable protooncogene mutations. We did a CRISPR/Cas9-based screen that showed that RB1 -/- SCLC are hyperdependent on AURKB, likely because both genes control mitotic fidelity, and confirmed that Aurora B kinase inhibitors are efficacious against RB1 -/- SCLC tumors in mice at nontoxic doses.See related commentary by Dick and Li, p. 169.This article is highlighted in the In This Issue feature, p. 151.