Breakage-fusion-bridge Research Articles

Beyond the Chromosome: Recent Developments in Decoding the Significance of Extrachromosomal Circular DNA (eccDNA) in Human Malignancies.

Extrachromosomal circular DNA (eccDNA) is a form of a circular double-stranded DNA that exists independently of conventional chromosomes. eccDNA exhibits a broad and random distribution across eukaryotic cells and has been associated with tumor-related properties due to its ability to harbor the complete gene information of oncogenes. The complex and multifaceted mechanisms underlying eccDNA formation include pathways such as DNA damage repair, breakage-fusion-bridge (BFB) mechanisms, chromothripsis, and cell apoptosis. Of note, eccDNA plays a pivotal role in tumor development, genetic heterogeneity, and therapeutic resistance. The high copy number and transcriptional activity of oncogenes carried by eccDNA contribute to the accelerated growth of tumors. Notably, the amplification of oncogenes on eccDNA is implicated in the malignant progression of cancer cells. The improvement of high-throughput sequencing techniques has greatly enhanced our knowledge of eccDNA by allowing for a detailed examination of its genetic structures and functions. However, we still lack a comprehensive and efficient annotation for eccDNA, while challenges persist in the study and understanding of the functional role of eccDNA, emphasizing the need for the development of robust methodologies. The potential clinical applications of eccDNA, such as its role as a measurable biomarker or therapeutic target in diseases, particularly within the spectrum of human malignancies, is a promising field for future research. In conclusion, eccDNA represents a quite dynamic and multifunctional genetic entity with far-reaching implications in cancer pathogenesis and beyond. Further research is essential to unravel the molecular pathways of eccDNA formation, elucidate its functional roles, and explore its clinical applications. Addressing these aspects is crucial for advancing our understanding of genomic instability and developing novel strategies for tailored therapeutics, especially in cancer.

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.

Abstract 4334: Insight into the structure of breakage fusion bridge events and chromothripsis in cancer cell lines using long-read sequencing

Abstract Cervical cancer is caused by human papillomavirus (HPV) infection and is the most common cause of cancer death in low-resource countries. We characterized 18 cervical and 4 head and neck cancer cell lines using long-read DNA and RNA sequencing and identified the HPV types, HPV integration sites, and cancer driver mutations. This cell line panel represents the major molecular subtypes of cervical cancer. Structural variation analysis revealed recurrent chromosomal alteration and telomeric deletions associated with DNA inversions resulting from breakage-fusion-bridge (BFB) cycles. BFB is a common mechanism of chromosomal alteration in cancer that has not been studied using long-read sequencing. Analysis of the inversion sites revealed staggered ends consistent with exonuclease deletion of the DNA after breakage. Some BFB events are complex, involving interchromosomal or intrachromosomal insertions or rearrangements. In total, there were three different types of BFB events: Type I, II, and III. Type I BFB events have staggered ends but no additional insertion at the fusion site. Type II BFB events have an insertion of DNA from another chromosome at the junction, and Type III has an insertion of sequences from the same chromosome as the BFB. BFB cycles are thought to resolve with the addition of a telomere. However, a search for telomeric sequences did not identify any on chromosomes impacted by BFB. In many cancers, BFB events co-occur with chromothripsis; however, in our cervical cancers, only 1 out of 13 had chromosomal shattering near the BFB event, indicating that the mechanism of chromosome rearrangement may be different in cervical cancers. Five cell lines have a Chr11q BFB event, with YAP1/BIRC2/BIRC3 gene amplification. Analysis of 911 publicly available cervical cancer exomes showed that YAP1 amplification is associated with a 10-year earlier age of diagnosis of cervical cancer (x2, p<0.0001) and is three times more common in African Americans (x2=12, p=0.003). Therefore, cervical cancer patients with YAP1/BIRC2/BIRC3 amplification, especially those of African American ancestry, might benefit from anti-YAP1/BIRC2/BIRC3 therapies. Our panel provides models for all the major HPV types and subtypes of cervical cancer that will aid in developing new therapeutic approaches. In summary, we uncovered novel insights into the mechanisms and consequences of BFB cycles in cervical cancer using long-read sequencing. We are expanding long-read sequencing to other cancer cell lines to gain knowledge of events associated with BFB. Citation Format: Isabel Rodriguez, Nicole Rossi, Ayse Gokce Kesus, Yi Xie, Tanveer Ahmad, Asher Bryant, Hong Lou, Jesica Godinez Paredes, Rose Milano, Mikhail Kolmogorov, Sonam Tulsyan, Jazmyn Bess, Vera Mukhina, Daria Gaykalova, Laksh Malik, Kimberley J. Billingsley, Cornelis Blauwendraat, Lisa Mirabello, Michael Dean. Insight into the structure of breakage fusion bridge events and chromothripsis in cancer cell lines using long-read sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4334.

Abstract 5870: Taxane-based chemotherapy leads to acquired resistance through ecDNA-based amplification of multi-drug resistance genes

Abstract Focal amplification on extrachromosomal DNA (ecDNA) drives high gene copy number and intercellular genomic heterogeneity, which can fuel tumor adaptation and drug resistance. Targeted therapies have largely failed in patients with oncogene amplification, and patients whose tumors harbor ecDNA-based gene amplification have particularly worse survival compared to patients whose cancers harbor other genetic drivers. Due to the lack of approved therapies for patients with oncogene amplified cancers, many patients receive chemotherapy; however, resistance often develops through mechanisms such as ATP-binding cassette (ABC) transporter overexpression, rendering cross-resistance to other forms of chemotherapy. We examined whether ecDNA amplification in tumor cell lines drives ABC transporter overexpression to mediate resistance to paclitaxel, a commonly prescribed taxane chemotherapy for cancer patients. We selected non-amplified (DLD1 and SW48) and amplified cell lines, including COLO320DM and COLO320HSR (MYC amplified on ecDNA or chromosomal loci, respectively); SNU16 and H716, (FGFR2 and MYC amplified ecDNA); and H2170 (ERBB2 and MYC amplified on ecDNA). All cell lines developed paclitaxel resistance, with the majority (5/7) gaining focal amplification of ABCB1, which encodes P-glycoprotein (P-gp), a common multi-drug resistance transporter. Four models–COLO320DM, COLO320HSR, SNU16, and H2170—developed ABCB1 amplification on ecDNA; one non-amplified cell line (DLD1) acquired focal ABCB1 amplification via a breakage-fusion-bridge (BFB) mechanism, a distinct form of amplification from ecDNA. ecDNA-based ABCB1 amplifications exhibited hallmarks of ecDNA, including intercellular copy number heterogeneity and paclitaxel dose-dependent rapid evolution of ABCB1 copy number. We next assessed whether drug resistance was reversible in ABCB1 amplified cell lines following removal of paclitaxel. Reversal of resistance was observed in COLO320DM and COLO320HSR via loss of ABCB1 ecDNA within 4-6 weeks. However, in SNU16 and H2170 resistant cells, ABCB1 amplification was retained up to 12 weeks of drug removal, with apparent integration into MYC ecDNA, demonstrating that ecDNA-enabled plasticity can result in both short- and long-term paclitaxel resistance. Although targeted therapies have revolutionized treatment approaches for patients with actionable oncodriver alterations, most cancer patients do not have actionable drivers and most patients still receive chemotherapy at some point in their course of treatment. We demonstrated that ecDNA-based resistance to paclitaxel is common in cancer cell lines and may also drive resistance to chemotherapy in patients. Thus, ecDNA-directed therapies (ecDTx) may be an important treatment option for chemo-resistant patients, who currently do not have many effective therapeutic options. Citation Format: Kristen Holmes, Dean Perusse, Cory DuPai, Homa Hemmati, Julie Weiss, Claire Kuelbs, John Bibay, Joan Chen, Debbie Liao, Christian Hassig, Shailaja Kasibhatla. Taxane-based chemotherapy leads to acquired resistance through ecDNA-based amplification of multi-drug resistance genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5870.

Abstract 4400: Transcriptional dysregulation of chromosomes localized into the micronucleus

Abstract Chromosomal Instability (CIN) is a characteristic of the most aggressive and drug-resistant cancers. Chromosomally unstable cells form micronuclei (MN), a structure with an unstable nuclear membrane that spontaneously surrounds missegregating chromosomes upon mitotic exit. Several studies have demonstrated that CIN drives drug resistance and metastasis through the karyotypic diversity caused by copy number alterations. Beyond facilitating karyotypic diversity, CIN has recently been demonstrated to cause durable epigenetic alterations including reduction of the activating histone H3K27ac mark in chromosomes localized to micronuclei. This reduction in histone H3K27 acetylation persists after the relocalization of the missegregated chromosome to the primary nucleus (PN). Given the loss of transcription-associated epigenetic modifications, we hypothesized that MN would exhibit transcriptional impairment. Indeed, by using nascent RNA assays and immunofluorescence, we found that chromosomes localized into the MN are less transcriptionally active and exhibit lower levels of active RNA polymerase II (RNAP2). Transcriptional inhibitors targeting RNAP2 largely abrogate MN transcription. Conversely, class-specific histone deacetylase inhibitors (HDACi) cause increased hyperacetylation of the MN relative to the PN, which persists beyond MN reincorporation into the PN. We also hypothesized that transcription in MN depends on the manner of MN biogenesis. Preliminary data shows that MN formed from breakage-fusion-bridge (BFB) events may cause less transcriptionally active MN than ones generated from treatment with the MPS1 inhibitor, reversine. To better understand the mechanisms that regulate transcription in the MN, our ongoing work is focused on an unbiased investigation of the micronuclear transcriptome. Citation Format: Danguole Norkunaite, Duaa Al-Rawi, Samuel Bakhoum. Transcriptional dysregulation of chromosomes localized into the micronucleus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4400.

Insights into the mechanisms and structure of breakage-fusion-bridge cycles in cervical cancer using long-read sequencing

Cervical cancer is caused by human papillomavirus (HPV) infection, has few approved targeted therapeutics, and is the most common cause of cancer death in low-resource countries. We characterized 19 cervical and four head and neck cancer cell lines using long-read DNA and RNA sequencing and identified the HPV types, HPV integration sites, chromosomal alterations, and cancer driver mutations. Structural variation analysis revealed telomeric deletions associated with DNA inversions resulting from breakage-fusion-bridge (BFB) cycles. BFB is a common mechanism of chromosomal alterations in cancer, and our study applies long-read sequencing to this important chromosomal rearrangement type. Analysis of the inversion sites revealed staggered ends consistent with exonuclease digestion of the DNA after breakage. Some BFB events are complex, involving inter- or intra-chromosomal insertions or rearrangements. None of the BFB breakpoints had telomere sequences added to resolve the dicentric chromosomes, and only one BFB breakpoint showed chromothripsis. Five cell lines have a chromosomal region 11q BFB event, with YAP1-BIRC3-BIRC2 amplification. Indeed, YAP1 amplification is associated with a 10-year-earlier age of diagnosis of cervical cancer and is three times more common in African American women. This suggests that individuals with cervical cancer and YAP1-BIRC3-BIRC2 amplification, especially those of African ancestry, might benefit from targeted therapy. In summary, we uncovered valuable insights into the mechanisms and consequences of BFB cycles in cervical cancer using long-read sequencing.

Deciphering complex breakage-fusion-bridge genome rearrangements with Ambigram

Breakage-fusion-bridge (BFB) is a complex rearrangement that leads to tumor malignancy. Existing models for detecting BFBs rely on the ideal BFB hypothesis, ruling out the possibility of BFBs entangled with other structural variations, that is, complex BFBs. We propose an algorithm Ambigram to identify complex BFB and reconstruct the rearranged structure of the local genome during the cancer subclone evolution process. Ambigram handles data from short, linked, long, and single-cell sequences, and optical mapping technologies. Ambigram successfully deciphers the gold- or silver-standard complex BFBs against the state-of-the-art in multiple cancers. Ambigram dissects the intratumor heterogeneity of complex BFB events with single-cell reads from melanoma and gastric cancer. Furthermore, applying Ambigram to liver and cervical cancer data suggests that the BFB mechanism may mediate oncovirus integrations. BFB also exists in noncancer genomics. Investigating the complete human genome reference with Ambigram suggests that the BFB mechanism may be involved in two genome reorganizations of Homo Sapiens during evolution. Moreover, Ambigram discovers the signals of recurrent foldback inversions and complex BFBs in whole genome data from the 1000 genome project, and congenital heart diseases, respectively.

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.

Unintended Genomic Outcomes in Current and Next Generation GM Techniques: A Systematic Review.

Classical genetic engineering and new genome editing techniques, especially the CRISPR/Cas technology, increase the possibilities for modifying the genetic material in organisms. These technologies have the potential to provide novel agricultural traits, including modified microorganisms and environmental applications. However, legitimate safety concerns arise from the unintended genetic modifications (GM) that have been reported as side-effects of such techniques. Here, we systematically review the scientific literature for studies that have investigated unintended genomic alterations in plants modified by the following GM techniques: Agrobacterium tumefaciens-mediated gene transfer, biolistic bombardment, and CRISPR-Cas9 delivered via Agrobacterium-mediated gene transfer (DNA-based), biolistic bombardment (DNA-based) and as ribonucleoprotein complexes (RNPs). The results of our literature review show that the impact of such techniques in host genomes varies from small nucleotide polymorphisms to large genomic variation, such as segmental duplication, chromosome truncation, trisomy, chromothripsis, breakage fusion bridge, including large rearrangements of DNA vector-backbone sequences. We have also reviewed the type of analytical method applied to investigate the genomic alterations and found that only five articles used whole genome sequencing in their analysis methods. In addition, larger structural variations detected in some studies would not be possible without long-read sequencing strategies, which shows a potential underestimation of such effects in the literature. As new technologies are constantly evolving, a more thorough examination of prospective analytical methods should be conducted in the future. This will provide regulators working in the field of genetically modified and gene-edited organisms with valuable information on the ability to detect and identify genomic interventions.

A Central Role of Telomere Dysfunction in the Formation of a Unique Translocation within the Sub-Telomere Region Resulting in Duplication and Partial Trisomy

Telomeres play a major role in maintaining genome stability and integrity. Putative involvement of telomere dysfunction in the formation of various types of chromosomal aberrations is an area of active research. Here, we report a case of a six-month-old boy with a chromosomal gain encompassing the 11q22.3q25 region identified by SNP array analysis. The size of the duplication is 26.7 Mb and contains 170 genes (OMIM). The duplication results in partial trisomy of the region in question with clinical consequences, including bilateral renal dysplasia, delayed development, and a heart defect. Moreover, the karyotype determined by R-banding and chromosome painting as well as by hybridization with specific sub-telomere probes revealed the presence of an unbalanced t(9;11)(p24;q22.3) translocation with a unique breakpoint involving the sub-telomere region of the short arm of chromosome 9. The karyotypes of the parents were normal. Telomere integrity in circulating lymphocytes from the child and from his parents was assessed using an automated high-throughput method based on fluorescence in situ hybridization (FISH) with telomere- and centromere-specific PNA probes followed by M-FISH multicolor karyotyping. Very short telomeres, as well as an increased frequency of telomere loss and formation of telomere doublets, were detected in the child’s cells. Interestingly, similar telomere profiles were found in the circulating lymphocytes of the father. Moreover, an assessment of clonal telomere aberrations identified chromosomes 9 and 11 with particularly high frequencies of such aberrations. These findings strongly suggest that telomere dysfunction plays a central role in the formation of this specific unbalanced chromosome rearrangement via chromosome end-to-end fusion and breakage–fusion–bridge cycles.

Signatures of copy number alterations in human cancer

Gains and losses of DNA are prevalent in cancer and emerge as a consequence of inter-related processes of replication stress, mitotic errors, spindle multipolarity and breakage–fusion–bridge cycles, among others, which may lead to chromosomal instability and aneuploidy1,2. These copy number alterations contribute to cancer initiation, progression and therapeutic resistance3–5. Here we present a conceptual framework to examine the patterns of copy number alterations in human cancer that is widely applicable to diverse data types, including whole-genome sequencing, whole-exome sequencing, reduced representation bisulfite sequencing, single-cell DNA sequencing and SNP6 microarray data. Deploying this framework to 9,873 cancers representing 33 human cancer types from The Cancer Genome Atlas6 revealed a set of 21 copy number signatures that explain the copy number patterns of 97% of samples. Seventeen copy number signatures were attributed to biological phenomena of whole-genome doubling, aneuploidy, loss of heterozygosity, homologous recombination deficiency, chromothripsis and haploidization. The aetiologies of four copy number signatures remain unexplained. Some cancer types harbour amplicon signatures associated with extrachromosomal DNA, disease-specific survival and proto-oncogene gains such as MDM2. In contrast to base-scale mutational signatures, no copy number signature was associated with many known exogenous cancer risk factors. Our results synthesize the global landscape of copy number alterations in human cancer by revealing a diversity of mutational processes that give rise to these alterations.

Breakage in breakage–fusion–bridge cycle: an 80-year-old mystery

2021 marked the 80th anniversary of Barbara McClintock's pioneering article on the breakage-fusion-bridge (BFB) cycle. Of the three steps of the BFB cycle, breakage remains the least understood despite its major contribution to mutagenesis. We discuss recent findings shedding light on how chromatin bridges break in yeast and animal cells.

Chromosome 1q12 Instability Drives Both 1q21 Amplification and Arm-Length Deletions in Multiple Myeloma

IntroductionChromosome instability is a driver of copy number aberrations (CNAs) in cancer, and is a factor leading to tumor heterogeneity and resistance to therapy. In multiple myeloma (MM) CNAs involving 1q21 and p53 are high-risk secondary aberrations identified in increasing frequency in relapsed and refractory patients. The 1q21 region contains a large number of possible candidate genes which show amplification (amp) and/or deregulated expression important in myeloma pathogenesis; these genes include MCL1, MUC1, IL6R, BCL9, CKS1B, ANP32E, PSMD4 and ILF2, among others. Given that 1q21 copy number gains are one of the most frequent CNAs in all cancers, the mechanisms for the accumulation of CNAs of 1q21 in MM have important implications in cancer genomics. The most common mechanisms for CNAs of 1q21 in MM are direct and inverted duplications of 1q21, focal amplification of 1q12-23 by breakage-fusion-bridge (BFB) cycles, and unbalanced jumping translocations of 1q12 (JT1q12s) pericentromeric heterochromatin. JT1q12s translocate the entire 1q to the pericentromeric or telomeric regions of non-homologous receptor chromosomes (RCs). Unfortunately, in addition to an increase of 1q21 CNAs, collateral damage involving concomitant losses of arm-length segments of the RCs occurs in the unbalanced pericentromeric translocations. This type of loss may be an underappreciated mechanism for the accumulation of haploinsufficiency and loss of tumor suppressor genes in MM, which have been reported in other malignancies. In our cytogenetic database the most frequent losses of RCs associated with JT1q12 gains are losses of 16q, 19q, 1p, 6q, and 17p, respectively.MethodsTo investigate the cytogenetic impact and progression of these arm-level deletions in RCs we performed a comprehensive metaphase analysis of 50 patients showing segmental aneuploidies with 4 or more copies of 1q21. G-banding, metaphase FISH, and spectral karyotyping were used to identify the key transient and clonal structural aberrations of 1q12 resulting in concomitant segmental aneuploidies in RCs.ResultsThe losses of 16q (8 patients), 19q (7 patients), and 17p (3 patients), were associated with whole-arm JT1q12s to the pericentromeric regions of these RCs. Losses of 6q (6 patients) were associated with a spectrum of JT1q12s aberrations which included the entire 6q from 6q10 through 6q27. Strikingly, isochromosome 1q (i1q) formation, which results in the deletion of 1p and gain of 1q (14 patients) showed the highest range CNAs for 1q21 (4-10). The higher copy number of 1q21 in the i1q patients resulted from BFB cycles of the 1q12-23 amplicon involving sister chromatid fusions (SCFs) at 1q12 breakpoints in these patients. Segmental amplifications of regions harboring MYC and MET were identified hitchhiking on JT1q12s, resulting in cryptic insertions in non-homologous RCs. Unexpectedly, BFB cycle amp of MYC involving SCFs at 1q12 breakpoints identified a novel mechanism for the amp of MYC in MM.ConclusionIn this study the unbalanced pericentromeric translocation of 1q12 involving RCs 16q, 19q, 6q, and 17p provides evidence for recurring arm-length deletions in the cytogenetic progression of MM. Importantly, loss of 17p is currently recognized as a high risk-stratification marker by interphase FISH (iFISH), and has previously been shown to result from JT1q12s in a subset of patients. We suggest that the accumulation of other concomitant copy number gains of 1q21 and losses of RCs such as 16q, 19q, and 6q may provide new risk stratification markers in myeloma. DisclosuresDavies:Seattle Genetics: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Bristol-Myers: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Morgan:Takeda: Consultancy, Honoraria; Bristol Myers: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

A new assay capturing chromosome fusions shows a protection trade-off at telomeres and NHEJ vulnerability to low-density ionizing radiation.

Chromosome fusions threaten genome integrity and promote cancer by engaging catastrophic mutational processes, namely chromosome breakage–fusion–bridge cycles and chromothripsis. Chromosome fusions are frequent in cells incurring telomere dysfunctions or those exposed to DNA breakage. Their occurrence and therefore their contribution to genome instability in unchallenged cells is unknown. To address this issue, we constructed a genetic assay able to capture and quantify rare chromosome fusions in budding yeast. This chromosome fusion capture (CFC) assay relies on the controlled inactivation of one centromere to rescue unstable dicentric chromosome fusions. It is sensitive enough to quantify the basal rate of end-to-end chromosome fusions occurring in wild-type cells. These fusions depend on canonical nonhomologous end joining (NHEJ). Our results show that chromosome end protection results from a trade-off at telomeres between positive effectors (Rif2, Sir4, telomerase) and a negative effector partially antagonizing them (Rif1). The CFC assay also captures NHEJ-dependent chromosome fusions induced by ionizing radiation. It provides evidence for chromosomal rearrangements stemming from a single photon–matter interaction.

Copy number alterations identify a smoking-associated expression signature predictive of poor outcome in head and neck squamous cell carcinoma

Cigarette smoking is a risk factor for the development of head and neck squamous cell carcinoma (HNSCC), partially due to tobacco-induced large-scale chromosomal copy-number alterations (CNAs). Identifying CNAs caused by smoking is essential in determining how gene expression from such regions impact tumor progression and patient outcome. We utilized The Cancer Genome Atlas (TCGA) whole genome sequencing data for HNSCC to directly identify amplified or deleted genes correlating with smoking pack-year based on linear modeling. Internal cross-validation identified 35 CNAs that significantly correlated with patient smoking, independent of human papillomavirus (HPV) status. The most abundant CNAs were chromosome 11q13.3-q14.4 amplification and 9p23.1/9p24.1 deletion. Evaluation of patient amplicons reveals four different patterns of 11q13 gene amplification in HNSCC resulting from breakage-fusion-bridge (BFB) events. . Predictive modeling identified 16 genes from these regions that denote poorer overall and disease-free survival with increased pack-year use, constituting a smoking-associated expression signature (SAES). Patients with altered expression of signature genes have increased risk of death and enhanced cervical lymph node involvement. The identified SAES can be utilized as a novel predictor of increased disease aggressiveness and poor outcome in smoking-associated HNSCC.

Structural variant evolution after telomere crisis

Telomere crisis contributes to cancer genome evolution, yet only a subset of cancers display breakage-fusion-bridge (BFB) cycles and chromothripsis, hallmarks of experimental telomere crisis identified in previous studies. We examine the spectrum of structural variants (SVs) instigated by natural telomere crisis. Eight spontaneous post-crisis clones did not show prominent patterns of BFB cycles or chromothripsis. Their crisis-induced genome rearrangements varied from infrequent simple SVs to more frequent and complex SVs. In contrast, BFB cycles and chromothripsis occurred in MRC5 fibroblast clones that escaped telomere crisis after CRISPR-controlled telomerase activation. This system revealed convergent evolutionary lineages altering one allele of chromosome 12p, where a short telomere likely predisposed to fusion. Remarkably, the 12p chromothripsis and BFB events were stabilized by independent fusions to chromosome 21. The data establish that telomere crisis can generate a wide spectrum of SVs implying that a lack of BFB patterns and chromothripsis in cancer genomes does not indicate absence of past telomere crisis.

Chromothripsis drives the evolution of gene amplification in cancer

Focal chromosomal amplification is an important route to generating cancer through mediating over-expression of oncogenes1–3 or to developing cancer therapy resistance by increasing expression of a gene whose action diminishes efficacy of an anti-cancer drug. Here we used whole-genome sequencing of clonal isolates developing chemotherapeutic resistance to identify chromothripsis as a major driver of extrachromosomal DNA (ecDNA) amplification into circular double minutes (DMs) through PARP- and DNA-PKcs-dependent mechanisms. Longitudinal analyses revealed that DMs undergo continuing structural evolution to promote increased drug tolerance through additional chromothriptic events. In-situ Hi-C sequencing is used to demonstrate that DMs preferentially tether near chromosome ends where they re-integrate when DNA damage is present. Intrachromosomal amplifications formed initially under low-level drug selection undergo continuing breakage-fusion-bridge cycles, generating >100 megabase-long amplicons that we show become trapped within interphase bridges and then shattered, producing micronuclei that mediate DM formation. Similar genome rearrangement profiles linked to localized gene amplification are identified in human cancers with acquired drug resistance or with oncogene amplifications. We propose that chromothripsis is a primary mechanism accelerating genomic DNA amplification and which enables rapid acquisition of tolerance to altered growth conditions.

Abstract PO-017: Single-cell copy number heterogeneity tracing enabling cancer gene discovery

Abstract Aneuploidy, the phenomenon that genomes acquire or lose chromosomal fragments, has been causally implicated in a wide variety of human cancer. Defining which copy number alterations (CNAs) are pathogenic is an important goal of cancer research. However, data based on bulk samples cannot fully depict tumor heterogeneity and evolution, which occurs in single cells, and thus have limited power to discover CNAs useful for cancer diagnostics and therapeutics. Recent advances in single-cell DNA sequencing have enabled acquisition of single-cell copy number (SCCN) profiles in tens of thousands of cells, which potentiate reconstruction of copy number evolution lineage and discovery of novel cancer genes. However, current analytical approaches that infer clonal lineages are neither accurate, nor scalable to analyzing molecular features, particularly CNA profiles obtained from thousands of cells. Statistical routines have not been established to leverage lineage tracing results towards identifying cancer-related genes. Here, we present a Minimal Event Distance Aneuploidy Lineage Tree (MEDALT) algorithm that infers the evolution history of a cell population based on single-cell copy number (SCCN) profiles. In a MEDALT, each node represents a cell, each edge represents a kinship between two cells, arrows point towards younger cells, and the root represents a normal diploid cell. We also present a statistical routine named lineage speciation analysis, which facilitates discovery of fitness-associated alterations (FAAs) and genes from SCCN lineage trees. To evaluate MEDALT, we simulated copy number evolution under various CNA mechanisms such as genome doubling, breakage-fusion-bridge (BFB), etc. and spiked in FAAs. We found that MEDALT substantially improved accuracy of FAA identification over GISTIC and conventional phylogenetics methods such as maximum likelihood (ML), maximal parsimony (MP) and neighbor joining (NJ) trees. We applied our methods on the single-cell DNA-sequencing data acquired from 20 triple-negative breast cancer patients (TNBCs), 4 of which had longitudinal pre-, mid- and post- treatment samples. Most of the TNBC samples appeared to have developed through branched evolution via multiple parallel lineages with distinct mutation rates and DNA damage repair (DDR) loss based on the constructed MEDALT. Using our approaches, we discovered novel genes that are predictive of patient survival in TCGA breast cancer data and are functionally more essential than other control gene sets, based on the CRISPR-cas9 knockout screen data obtained from 27 breast cancer cell lines in the DepMap database. Significant benefits in lineage tracing and cancer gene discovery were also achieved, when applying our approaches on the SCCN profiles derived using InferCNV from the single-cell RNA sequencing data of a cohort of 20 multiple myeloma, head and neck cancer, oral squamous cell carcinoma and ovarian cancer patients. The source code of our study is available at https://github.com/KChen-lab/MEDALT. Citation Format: Fang Wang, Qihan Wang, Vakul Mohanty, Shaoheng Liang, Jinzhuang Dou, Jincheng Han, Darlan Minussi, Ruli Gao, Li Ding, Nicholas Navin, Ken Chen. Single-cell copy number heterogeneity tracing enabling cancer gene discovery [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-017.

Essentiality of CTNNB1 in Malignant Transformation of Human Embryonic Stem Cells under Long-Term Suboptimal Conditions

Human embryonic stem cells (hESCs) gradually accumulate abnormal karyotypes during long-term suboptimal culture, which hinder their application in regenerative medicine. Previous studies demonstrated that the activation of CTNNB1 might be implicated in this process. Hence, the hESC line with stably silenced CTNNB1 was established to further explore the role of CTNNB1 in the malignant transformation of hESCs. It was shown to play a vital role in the maintenance of the physiological properties of stem cells, such as proliferation, migration, differentiation, and telomere regulation. Furthermore, the malignant transformation of hESCs was induced by continuous exposure to 0.001 μg/ml mitomycin C (MMC). The results showed that CTNNB1 and its target genes, including proto-oncogenes CCND1 and C-MYC, were aberrantly upregulated in hESCs after MMC treatment. Moreover, the high expression of CTNNB1 accelerated cell transition from G0/G1 phase to the S phase and stimulated the growth of cells containing breakage-fusion-bridge (BFB) cycles. Conversely, CTNNB1 silencing inhibited these effects and triggered a survival crisis. The current data indicated that CTNNB1 is intimately associated with the physiological properties of stem cells; however, the aberrant expression of CTNNB1 is involved in the malignant transformation of hESCs, which might advance the process by facilitating telomere-related unstable cell proliferation. Thus, the aberrant CTNNB1 level might serve as a potential biomarker for detecting the malignant transformation of hESCs.

APOBEC3-dependent kataegis and TREX1-driven chromothripsis during telomere crisis.

Chromothripsis and kataegis are frequently observed in cancer and may arise from telomere crisis, a period of genome instability during tumorigenesis when depletion of the telomere reserve generates unstable dicentric chromosomes1–5. Here we examine the mechanism underlying chromothripsis and kataegis using an in vitro telomere crisis model. We show that the cytoplasmic exonuclease TREX1, which promotes the resolution of dicentric chromosomes4, plays a prominent role in chromothriptic fragmentation. In absence of TREX1, the genome alterations induced by telomere crisis primarily involve Breakage-Fusion-Bridge cycles and simple genome rearrangements rather than chromothripsis. Furthermore, we show that the kataegis observed at chromothriptic breakpoints is the consequence of cytosine deamination by APOBEC3B. These data reveal that chromothripsis and kataegis arise from a combination of nucleolytic processing by TREX1 and cytosine editing by APOBEC3B.