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Chromothripsis drives the evolution of gene amplification in cancer

A Publisher Correction to this article was published on 01 March 2021

This article has been updated

Abstract

Focal chromosomal amplification contributes to the initiation of cancer by mediating overexpression of oncogenes1,2,3, 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.

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Fig. 1: Chromothripsis drives ecDNA formation and amplification as DMs early during drug resistance development in a PARP- and NHEJ-dependent manner.
Fig. 2: DM structure is optimized by chromothripsis during adaptation to increased selection pressure.
Fig. 3: DMs preferentially integrate into broken DNA sites near chromosome ends and form ectopic HSRs.
Fig. 4: HSRs fragment in interphase bridges and form micronuclei and DMs through chromothripsis and NHEJ repair.

Data availability

Paired-end WGS data are available at the European Nucleotide Archive (ENA), accession number ERP107458. In-situ Hi-C sequencing data are available at the Gene Expression Omnibus (GEO), accession number GSE119825. RNA sequencing data are available at GEO, accession number GSE119979. TCGA database can be accessed at https://portal.gdc.cancer.gov/. Source data are provided with this paper.

Code availability

A pseudocode of the simulation performed in Extended Data Fig. 4i–l is provided in Supplementary Fig. 3. Code can be found at https://github.com/sfbrunner/chromothripsis-gene-amp-cancer.

Change history

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Acknowledgements

This work was funded by grants from the US National Institutes of Health (R35 GM122476 to D.W.C.), the Wellcome Trust (WT088340MA to P.J.C.), the US National Institutes of Health (K99 CA218871 to P.L.), the Swiss National Science Foundation (P2SKP3-171753 to S.F.B.), the Ludwig Institute for Cancer Research (D.W.C. and B.R.), an MSK Cancer Center Core Grant from the NIH (P30 CA 008748 to R.Y.), the National Institute of General Medical Sciences (P41GM103412, R24GM137200 to M.H.E.), and the High End Instrumentation Award (S10OD021784 to M.H.E.). D.W.C. and B.R. receive salary support from the Ludwig Institute for Cancer Research. We thank A. Shiau for providing access to the CQ1 spinning disk confocal system and N. Shimizu for providing the Colo320-DM-GFP cell line.

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Authors

Contributions

O.S. and D.W.C. conceived the project and wrote the manuscript. O.S. designed, performed, and analysed all experiments unless otherwise specified here. O.S. and P.L. performed HSR evolution experiments. O.S. and Y.N.-A. tested drug treatment concentrations, O.S. and D.H.K. performed live-cell imaging and performed DM integration experiments following Cas9 expression. R.Y. obtained human DNA samples and performed FISH of human biopsies. O.S. and J.S.Z.L. designed sgRNA for Cas9 experiments. S.F.B., P.J.C., and O.S. performed and analysed the DNA sequencing experiments. O.S., S.F.B., and Y.S. analysed the RNA sequencing data. O.S. and M.Y. performed the HiC experiments. O.S., R.F., and B.R. analysed the HiC sequencing data. G.A.C. and O.S. performed the CLEM experiments. O.S., G.A.C., and M.H.E. analysed the CLEM experiments. All authors provided input on the manuscript. D.W.C., P.J.C., and O.S. supervised all aspects of the work.

Corresponding authors

Correspondence to Peter J. Campbell or Don W. Cleveland.

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The authors declare no competing interests.

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Peer review information Nature thanks Jan Korbel, Roel Verhaak and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Genomics and transcriptomics before and after methotrexate resistance.

a, Representative DNA-FISH images showing parental HeLa karyotype of chromosome 5 found in the five primary clones used in the study. The parental der(3p5q) is shown in the top left image. b, Colony assay showing methotrexate sensitivity of the parental HeLa and five derivative clones. c, Representative DNA-FISH images (of the indicated independent experiments n displayed below each image) of surviving cells and colonies of naive HeLa cells treated with methotrexate at indicated concentrations for the indicated times. Increased DHFR signals over wild-type (>3 signals) were frequently observed, and DHFR aggregates indicative of HSR formation (with chromosome 5 interphase bridge detected in some cases – outlined with white dashed line in top right image) or DHFR+ DMs (dispersed signal) were found in surviving colonies of 100-200 cells at day 17. d, RNA expression and DNA copy number levels plotted on the linear maps of chromosome 5 of six methotrexate-resistant clones with no DHFR amplification. Representative DNA-FISH images are displayed (of at least 10 different chromosome spreads from each clone). e, Linear regression comparing DNA copy number and RNA expression levels of DHFR in methotrexate-resistant HeLa clones. f, Principal component analysis (PCA) of naive and resistant HeLa clones (with DMs or without DHFR amplification). Source data

Extended Data Fig. 2 Formation of DMs through chromothripsis in methotrexate-resistant cells.

a, Representative DNA-FISH of metaphase spread prepared from clone PD29424h (see Fig. 1c) showing DHFR amplified in DMs (marked with white circles). Alignment of the normal chromosome 5 (DHFR+) and shorter chromothriptic chromosome 5 (DHFR-) is presented. Representative DNA-FISH from a metaphase spreads hybridized with chromosome 5 paint probe (green) and BAC probe (RP11-958F12, red, ~30 Mb away from DHFR) found on both the normal chromosome 5 (single location) and shorter chromothriptic chromosome 5 (multiple dispersed locations) is also presented. b, i, k, m, n, Copy number, allelic ratio, and structural variation profiles of indicated samples. c, Representative FISH images from clone PD29425d showing a normal chromosome 5 (DHFR+, RP11-958F12 positive), a shorter chromothriptic chromosome 5 (DHFR-, RP11-958F12 positive), and a DM (DHFR+, RP11-958F12 negative). See panel b for BAC probe target. d, Representative DNA-FISH image of metaphase spreads prepared from clone PD29427k and hybridized with BAC RP11-314L7 (labels fragment #1 of the DM as shown in Fig. 1d) and DHFR locus probes. Insets show the co-localization of the non-contiguous genomic locations within the DMs. e, Representative FACS analysis using propidium iodide staining showing cell cycle distribution in asynchronized and mitotic clones prepared for Hi-C experiments. For gating strategy see Supplementary Fig. 2. f, In situ Hi-C map of chromosome 5 (65-110 Mb) of asynchronized and mitotic cells from methotrexate-resistant clone PD29427k. Interactions between distant chromosome locations (preserved also in the mitotic sample where topologically associated domains are erased) are circled in black (above the diagonal red line) and presented natively (below the line). g, Reconstruction of the circular map of the DM present in clone PD29427k. Numbers represent the four DM segments appearing in panel f (notice that segment 3 is rearranged). h, List of the genes found on the DM (with RNA expression relative to naive HeLa cells). Numbers represent the four DM segments appearing in panel f. j, Representative DNA-FISH from clone PD29425l showing co-localization of DHFR (green) and BAC 33C19 probes signals. See panel i for BAC probe target. l, Representative DNA-FISH image of metaphase spreads prepared from clone PD29425g (see Fig. 1e) hybridized with chromosome 5 paint (green) and DHFR locus (red) probes. Inset shows the DHFR positive DM. Notice only one chromosome 5 and one der(3p5q) are present. o, p, Representative DNA-FISH image of metaphase spreads prepared from clones PD45714a (o, see Fig. 1f) and PD45725b (p, see Fig. 1g) hybridized with chromosome 5 paint (green) and DHFR locus (red) probes showing DHFR amplification in DMs. q, List of DM+ HeLa clones showing size, copy number, and number of non-contiguous fragments. r, Colony assay of naive HeLa cells treated with DNA repair inhibitors (control – untreated). Images are representative of two independent experiments.

Extended Data Fig. 3 BRAFV600E amplification in patients with drug-resistant colorectal cancer.

a, c, Treatment and biopsies collection timeline of patients 1 and 2. b, d, Representative FISH images (of entire section stained) showing BRAF amplification in biopsies from post-treatment biopsies from patients 1 and 2. Probes used are listed in the methods section.

Extended Data Fig. 4 DMs are numerically and structurally unstable.

a, Strategy used to isolate DM or HSR positive clones from a heterogeneous population of methotrexate-resistant HeLa cells. b, Left: Average number of DHFR+ DMs in three clones derived from a methotrexate-resistant HeLa population was determined using DNA-FISH. Analyses with indicated p-values above plots were performed using Student t-test. *P < 0.05, **P < 0.01, ***P < 0.001 are p-values calculated using one-way ANOVA. Error bars represent mean ± s.e.m., number of spreads examined for each condition is written below each graph. Right: Representative DNA-FISH image of metaphase spreads from clone PD29429f (640 nM) containing 46 DHFR+ DMs as determined by DNA-FISH. Inset shows a representative DHFR positive DM. c, Average intensity of DHFR signal in DMs relative to the intensity of the endogenous DHFR signal on chromosome 5 from the same spread, as determined by DNA-FISH using a probe (RP11-90A9) specific for the DHFR locus. Analyses with indicated p-values above plots were performed using Student t-test. **P < 0.01 and ***P < 0.001 are p-values calculated using one-way ANOVA. Error bars represent mean ± s.e.m. Representative DNA-FISH image of chromosome spread from which insets are shown in Fig. 2b is presented. d, Copy number, allelic ratio, and structural variation profiles of indicated samples. e, Read counts in structural variation breakpoints in sample PD29429i showing potential different DM subspecies forming through chromothripsis. f, Breakpoint PCR using primers specific for three different rearrangements detected using WGS. For gel source data, see Supplementary Information Fig. 1. g, Percentage of DM-positive cells exposed to low or high methotrexate concentrations with large micronuclei, as determined by DNA-FISH. Results represent an average of 3 clones per methotrexate concentration (as seen in Extended Data Fig. 4a), error bars represent mean ± s.d. Representative DNA-FISH image of a cell with a large micronucleus is provided. 40 nM: n = 147, 130, 98; 640 nM: n = 74, 216, 133. h, Analysis of DM inheritance to two daughter cells after one cell division. Cells were seeded at low dilution and 24 h later DNA-FISH on interphase daughter cells from PD29429h (40 nM) or PD29429i (640 nM) was performed using a DHFR probe. DM numbers in each daughter cell were counted and plotted on an x-y plot (n = 54 daughters per condition). i, Schematic explaining the steps of a simulation testing the effect of harbouring DMs with more than one DHFR gene. jl, In-silico simulation showing that cells containing a 2-copy DHFR DM have a selection advantage (i), with increased DM content (j), and that 2-copy DHFR DMs are selected over DMs with a single copy of DHFR (k). Plotted is the median and first and third quartile (grey ribbon). For simulation pseudocode see Supplementary Information Fig. 3. Source data

Extended Data Fig. 5 DMs integrate into ectopic chromosomes following DNA damage.

ac, Percentage of DM integration as detected using DNA-FISH with probes against chromosome 5 (green) and DHFR (red) in clone PD29429i after ionizing irradiation (a), doxorubicin (b), or transfection with nucleases specific for a region near the DHFR locus (c). Representative images of DNA-FISH of DMs integrated into ectopic chromosomes are presented below each graph. d, Percentage of ectopic HSRs detected in DM-positive clones treated with increased methotrexate concentration (x2.5 fold higher) with or without (control, 4 clones) the addition of ABT-888 (PARPi, 7 clones) and NU7026 (NHEJi, 4 clones) for 3 weeks. Means ± s.e.m. are presented. control: n = 107, 96, 46, 164; PARPi: n = 67, 247, 94, 128, 17, 130; NHEJi: n = 51, 45, 28, 38. Representative FISH images of ectopic HSRs at the end of chromosome (78% of the cases) or in the middle of chromosomes (22% of the cases) are presented. e, Percentage of GFP+ HSRs detected in colo320DM-GFP cell line treated with DMSO (control) or with ABT-888 (PARPi) or olaparib (PARPi) for 2 weeks. Means ± s.e.m. of three independent experiments are shown. p-values *P < 0.05 and **P < 0.001 calculated using one-way ANOVA are presented. control: n = 26, 47, 33; ABT-888: n = 42, 44, 37; Olaparib: n = 29, 32, 34. Representative fluorescent images of GFP+ DMs (from control) and GFP+ HSRs (from PARPi treated cells) are presented. f, Percentage of chromosome spreads in which large DHFR+ DMs were detected using DNA-FISH when PD29429i was exposed to 1,500 nM methotrexate only (control) or with addition of 15μM ABT-888 (PARPi) or 10 μM NU7026 (DNA-PKi) for 3 weeks. Means ± s.e.m. of three independent experiments are presented. * p-value <0.001 calculated using one-way ANOVA and Tukey’s Multiple Comparison Test. Number of spreads scored per condition/experiment: 1,500 nM MTX (52, 53, 49), 1,500 nM MTX + ABT-888 (50, 76, 55), 1,500 nM MTX + NU7026 (57, 16, 42). Representative image (of three independent experiments as shown in the plot to the left) of a chromosome spread containing multiple normal sized and large DMs is presented. g, Percentage of chromosome spreads in which large myc+ DMs were detected using DNA-FISH in colo320DM-GFP cells with indicated types of treatment for 2 weeks (DMSO served as control, PARP inhibitors used were 15μM ABT-888 and 10 μM Olaparib, hydroxyurea used at 100 μM). Representative image of a chromosome spread containing multiple normal sized and large DMs (white arrows) is presented. Means ± s.e.m. of three independent experiments with *P < 0.05 calculated using one-way ANOVA are presented. control: n = 22, 22, 21; ABT-888: n = 26, 19, 21; Olaparib: n = 20, 22, 21; hydroxyurea: n = 20, 22, 20; hydroxyurea+ABT-888: n = 19, 22, 24; hydroxyurea+Olaparib: n = 20, 22, 22. h, Quantification of the interaction frequency between DM or HSR sequences and the rest of the genome in mitotic or asynchronous cells. Linear chromosome maps (chromosomes 1-X, excluding chromosome 5) are presented from beginning to end (p arm to q arm direction), and the normalized interaction frequency is presented. i, Interaction frequency as a function of distance from the centromere (R - pearson correlation). j, Fold-change (relative to cells with no amplification) of DHFR sequences interaction in DM+ and HSR+ with centre of chromosomes (50% of sequences in chromosome centre) and with chromosome ends (25% of sequences in each chromosome end). Means ± s.d. and P values calculated using two-sided paired t-test are presented. Source data

Extended Data Fig. 6 Increased selective pressure drives the transition of intra- to extrachromosome amplification through chromothripsis.

a, Experimental outline of a parental HSR+ clone treated with increasing methotrexate concentrations. b, Representative FISH image of 23 metaphase spreads prepared from the methotrexate-resistant HSR+ clone PD29426a hybridized with chromosome 5 paint (green) and DHFR locus (red) probes. Inset shows the DHFR repeat located on the 5q arm of the derivative 3p5q chromosome. c, DNA copy number, rearrangement, and allelic ratio profile of clone PD29426a showing two copy number jumps flanked by head-to-head and tail-to-tail inversions corresponding to two BFB cycles. d, Schematic depicting the order of events during the two break-fusion-bridge (BFB) cycles that led to the formation of the HSR in clone PD29426a. e, RNA expression presented on the linear chromosome 5 map showing increased gene expression from the HSR region. f, In situ Hi-C map of chromosome 5 (65-110 Mb) of mitotic cells from methotrexate-resistant clone PD29426a, showing the HSR in which there is higher interaction within the regions with copy number jumps. g, In situ Hi-C map of chromosome 5 (65-110 Mb) of asynchronized cells from HSR+ clone PD29426a. TADs are visible throughout this region and increased interactions are observed in the HSR region (increased red coloration, top left quadrant). h, Cell cycle analysis using propidium iodide staining showing cell cycle distribution in asynchronized cells and mitotic cells of clone PD29426a. For gating strategy see Supplementary Information Fig. 2. i, Representative FISH image (of two independent clones, total of 26 spreads imaged) of metaphase spreads prepared from a DM subclone (PD29426h) derived from the HSR+ clone PD29426a. Spreads were hybridized with chromosome 5 paint (green) and DHFR locus (red) probes. Inset shows the DHFR positive DMs. j, DNA copy number, rearrangement, and allelic ratio profile of clone PD29426h. The DM is composed of fragments with varying copy number states derived from the original HSR region, connected by multiple rearrangements.

Extended Data Fig. 7 Chromothripsis and kataegis in gene amplification.

a, Representative FISH image (of 12 spreads imaged) of metaphase spreads prepared from clone PD29427p and hybridized with chromosome 5 paint (green) and DHFR locus probes, revealing the existence of a DHFR+ HSR. b, Top: Copy number profile of a methotrexate-resistant clone (PD29427p) presenting an HSR profile resulting from 3 BFB cycles with overlaid complex rearrangements. Rearrangements are presented on top: TD – tandem duplication, D – deletion, HH – head-to-head inversion, TT – tail-to-tail inversion. Inter-mutation distance (i.m.d) and allelic ratio (bottom, blue/red dots) are also presented. Bottom: RNA expression profile of clone PD29427p, relative to control naive HeLa cells, presented on the linear map of chromosome 5 and matching the DNA copy number plotted above. c, Representative FISH image (of 6 spreads imaged) of metaphase spreads prepared from clone PD29427p and hybridized with chromosome 5 paint (green) and BAC probe 314L7 (see chromosomal location indicated in panel b) probes. Inset shows probe 314L7 signal is flanking the DHFR signal within the HSR. d, Rainfall plots showing the inter-mutation distance (i.m.d.) within chromosome 5 of each DM clone. e, Structural variations and inter-mutation distance (i.m.d.) of three kataegis positive DM clones.

Extended Data Fig. 8 Characterization of steps leading to HSR fragmentation and DM formation.

a, Representative FISH images (of 464 spreads imaged from 8 different time points as shown in Fig. 4a) showing chromosome 5 (green) and DHFR (red) probes in HSR+ clone PD29428e at basal methotrexate concentration (60nM, top), and following treatment with increased methotrexate (500 nM, middle and bottom) in which HSR fragments of variable sizes and DMs can be detected. b, Colony assay of clone PD29428e (HSR+) resistant to 60 nM and treated with 500 nM methotrexate. Numbers of cells seeded are indicated and colonies were fixed and stained at day 18. c, Number of cells counted (automatically) using the CQ1 microscope in 10 min interval during 48 h of filming of PD29428e cells treated with 60 nM or 500 nM or without (dFBS) methotrexate. Cell numbers were normalized to the initial number present at time point 0. P-value calculated using one-way ANOVA. d, Measurement of the HSR length in clone PD29428e after ten days of exposure to higher methotrexate concentration. The length of the HSR was normalized to the length of the normal chromosome 5 from the same spread. Mean ± s.e.m. of n = 15 per group and p-value calculated using two-tailed t-test are presented. Insets showing length of the HSR at day 10 after exposure to basal (left) or increased (right) methotrexate concentrations (scale is 180 Mb, equivalent to chromosome 5 size). e, Measurement of the HSR length in clone PD29428e during a two-week exposure to higher methotrexate concentration. The length of the HSR was normalized to the length of the normal chromosome 5 from the same spread. Means ± s.e.m. of 15 HSR lengths per each time point (except day 14 – 16 HSRs scored). p-values calculated using one-way ANOVA and * denotes significant (range of P < 0.05-0.001) change in HSR length between each point of 8-14 days and each point of 0-6 days. f, Percentage of spreads containing dicentric HSR+ chromosomes in clone PD29428e exposed to basal or increased methotrexate concentration for 10 days. Mean ± s.d. of three independent experiments (60nM: n = 42, 30, 33; 500nM: 23, 33, 29) and p-value calculated using two-tailed t-test are presented. g, Percentage of spreads containing dicentric HSR+ chromosomes in clone PD29428e after increasing methotrexate concentration for the indicated times. day 0: n = 98; day 2: n = 41; day 4: n = 39; day 6: n = 63; day 8: n = 53; day 10: n = 55; day 12: n = 66; day 14: n = 123. Inset shows representative image (from 538 spreads imaged) of a chromatid-type dicentric chromosome with one fused end, and chromosome type fusion with two distinct centromeres. Percentages indicate frequency range of each dicentric type throughout the two-week experiment. h, Abnormalities identified in PD29428e cells treated without or with increased methotrexate concentration (60 nM and 500 nM, respectively) for 11 days during 48 h filming. Mean ± s.d. of three independent experiments and p-values (*P < 0.01 and *P < 0.001) calculated using two-way ANOVA are presented. Division scored: 60nM: n = 115, 108, 95; 500nM: n = 51, 50, 42. Bridges (anaphase bridges), micronuclei (MN), bridges+MN, death, and multipolar divisions were scored. Insets from Supplementary Video 1 showing anaphase bridges, micronuclei, and also interphase bridges are presented. i, Analysis as performed in (h) looking at events occurring during the division of the daughter cells generated from the parental cells scored in h. Division scored: 60nM: n = 100, 90, 66; 500nM: n = 85, 76, 66. j, Top: Percentage of nuclei presenting DHFR+ nuclear bulges indicative of bridge rupture in PD29428e HSR+ clone treated with increased methotrexate (500nM) over the course of 12 days. Mean ± s.d. of three independent experiments (60nM: n = 144, 221, 204; 500nM: n = 163, 218, 214) and p-value calculated using two-tailed t-test are presented. Bottom: Representative image of DHFR+ (red) labelled nuclear bulge. k, Representative image of the data in Fig. 4d. l, Copy number profile of a methotrexate-resistant clone (PD45727b) presenting an HSR profile resulting from multiple BFB cycles (marked with red arrows) with overlaid complex rearrangements. A representative DNA-FISH image (of 20 spreads imaged) showing dicentric HSRs is presented. DMs were observed in low frequency under this condition. m, Copy number profile of clone PD45727b subjected to increased methotrexate concentration (sample PD45728b) presenting additional rearrangements and formation of multiple DMs (as seen in the representative DNA-FISH image from 10 spreads imaged, white circles). TD – tandem duplication, D – deletion, HH – head-to-head inversion, TT – tail-to-tail inversion. Source data

Extended Data Fig. 9 Chromothripsis drives resolution of KIF15 intrachromosomal amplification through telomere capture.

a, Copy number profile of chromosome 3 showing a rearranged 3p region with KIF15 amplification (copy number = 15) and a translocation of a telomeric fragment from a lost chromosome 1. b, Representative DNA-FISH images (of 33 spreads imaged) from STLC resistant clones with (PD45759a) or without (PD45760a) KIF15 amplification showing the translocation of the telomeric chromosome 1 fragment to the end of a chromosome 3 arm. c, Representative DNA-FISH images (of 20 spreads imaged) for chromosome 3 (green) and KIF15 (RP11-659N22, red) showing normal KIF15 in PD45760a (STLC resistant without KIF15 amplification, 3 KIF15 copies labelled with a, b, and c) and in PD45759a (STLC resistant with KIF15 amplification found on two rearranged chromosomes marked with * and originating from chromosome c as seen in PD45760a). d, Representative DNA-FISH image (from 25 spreads imaged) showing from PD45759a showing the rearranged chromosome containing KIF15 amplification and telomeric region of chromosome 1 at the tip of the chromosome. e, Proposed order of events leading to KIF15 amplification and resolution of the dicentric state through a chromothriptic event.

Extended Data Fig. 10 Structure of the interphase chromosome bridge.

a, Snapshots from live-cell imaging (Supplementary Video 2) of PD29428e cells exposed to increased methotrexate concentration (500nM) for 11 days, labelled with SiR-DNA label, and filmed for 48 h (of 944 cell divisions filmed, as shown in Extended Data Fig. 8h-i). Frames are at 10-min intervals. An initial DNA+ bridge forms from which a DNA fragments is maintained as a micronucleus following bridge rupture. b, Representative image of PD29428e cells exposed to increased methotrexate concentration (500nM) for 10 days and analysed using DNA-FISH probes for chromosome 5 centromere (green) and DHFR (red), showing micronuclei within interphase DNA bridges (representative of 34 bridges). c, Immunofluorescent image analysis of zeocin treated cancer cell lines showing formation of micronuclei within interphase DNA bridges (representative images of 85 bridges with micronuclei from the four cell lines tested. Number of cells scored: 293T control: n = 99; 293T zeocin: n = 116; DLD1 control: n = 88; DLD1 zeocin: n = 107; HeLa control: n = 126; HeLa zeocin: n = 412; HT29 zeocin: n = 25; T47d zeocin: n = 73). d, e, CLEM images (of a total of 5 images taken) of an anaphase bridge (d, red arrow marks aberrant nuclear envelope at midbody region) and an interphase bridge (e, red arrow marks constriction in DNA bridge at midbody site). NE – nuclear envelope. f, Representative immunofluorescence images (of 43 different bridges) showing AuroraB localization in interphase DNA bridges. g, CLEM of an abnormally shaped micronucleus found in PD29428e cells exposed to increased methotrexate concentration (500 nM) for 10 days in which micronuclei form following interphase bridge rupture (representative of 2 micronuclei imaged). Source data

Extended Data Fig. 11 Similarities between DHFR and oncogene amplifications.

a, b, Summary of the overall structural variant events found across all samples analysed in the methotrexate-resistant HeLa cohort (a, 18 clones) and in patients with glioblastoma cancer from the TCGA database (b, 41 patients). c, Amplifications (> = 10 copy numbers) of either of 11 indicated genes in patients found in the TCGA database. d, Distribution of rearrangements in chromosomes containing specific amplifications. Near equal distribution of rearrangements suggest events are random as expected in chromothripsis. TD – tandem duplication, D – deletion, HH – head-to-head inversion, TT – tail-to-tail inversion. e, Copy number plots showing the mean copy number gain presented on the linear chromosome map in methotrexate-resistant HeLa cells (chromosome 5) and TCGA samples presenting oncogene amplifications. The percentage of each type of rearrangement found in chromosomes with amplifications is presented. Red – tandem duplications, Blue – head-head inversions, Green – tail-tail inversions, Purple – deletions. f, Microhomology (red), non-templated (teal) sequences, or direct end-joining (yellow), found at breakpoints in chromosomes with specific oncogene amplifications from TCGA patients. The number of homologous or non-homologous bases at breakpoints are shown on the x-axis. The number of breakpoints showing each kind of junction is shown on the y-axis. g, Schematic illustrating a roadmap to gene amplification in cancer cells. Source data

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-3, including the raw gels, gating strategy and pseudocode.

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Reporting Summary

Supplementary Table 1

Patient information used for whole genome sequencing.

Video 1

Live imaging of HSR+ clone PD29428e exposed to increased methotrexate – formation of interphase bridge. Clone PD29428e was exposed to increased methotrexate concentration (500nM) for 11 days, labeled with SiR-DNA label, and filmed for 48 hours. Frames are at 10-minute intervals. See Extended Data Fig. 8h-i for details.

Video 2

Live imaging of HSR+ clone PD29428e exposed to increased methotrexate – DNA fragments from interphase bridge form micronucleus. Clone PD29428e was exposed to increased methotrexate concentration (500nM) for 11 days, labeled with SiR-DNA label, and filmed for 48 hours. Frames are at 10-minute intervals. See Extended Data Fig. 10a for details.

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Shoshani, O., Brunner, S.F., Yaeger, R. et al. Chromothripsis drives the evolution of gene amplification in cancer. Nature 591, 137–141 (2021). https://doi.org/10.1038/s41586-020-03064-z

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