Chromothripsis and ecDNA initiated by N4BP2 nuclease fragmentation of cytoplasm-exposed chromosomes

Abstract

Genome instability, including chromothripsis, is a hallmark of cancer. Cancer cells frequently contain micronuclei-small, nucleus-like structures formed by chromosome missegregation-that are susceptible to rupture, exposing chromatin to cytoplasmic nucleases. Through an unbiased, imaging-based small interfering RNA screen that targeted all 204 known and putative human nucleases, we identified a previously uncharacterized cytoplasmic endonuclease, NEDD4-binding protein 2 (N4BP2), that enters ruptured micronuclei and initiates DNA damage, leading to chromosome fragmentation. N4BP2 promoted genome rearrangements (including chromothripsis), formation of extrachromosomal DNA (ecDNA) in drug-induced gene amplification, tumorigenesis, and tumor cell proliferation in an induced model of human high-grade glioma. Analysis of more than 10,000 human cancer genomes revealed elevated N4BP2 expression to be predictive of chromothripsis and copy number amplifications, including ecDNA.

Figure 1.. Imaging-based screen for human nucleases that fragment micronucleated chromosomes.

(A, B) Schematic of siRNA-mediated screen for human nucleases inducing DNA damage and chromosome fragmentation in micronuclei (MN). Dld1-Y^MN cells allow for inducible micronucleation of Y chromosome (see also Fig. S1A). The MN of MN-induced (for 72 hours) Dld1-Y^MN cells transfected with siRNAs targeting 204 nucleases (see Table S1) were scored for the reduction of DNA damage (using a DNA double strand breaks marker γH2AX) compared to non-silencing (NS) siRNA (negative control, high γH2AX) and γH2AX inhibitor wortmannin (positive control, low γH2AX) (γH2AX immunofluorescence, primary assay shown in [A]). Next, mitotic spreads from MN-induced Dld1-Y^MN cells, transfected with the siRNAs targeting hits identified in a primary assay, were analyzed for the presence of fragmented Y chromosomes using DNA FISH probes against Y chromosome (Y chromosome DNA FISH, secondary assay, shown in [B]). (C) DNA damage in Y chromosome-containing micronuclei with abnormal nuclear envelope in MN-induced Dld1-Y^MN cells. Y chromosome paint probe (green), γH2AX (red), Lamin B 1 (magenta). Scale bar, 5 μm. (D) Distribution of γH2AX signal in micronuclei normalized to non-silencing control (NS, dashed line) for each siRNA of siRNA library (blue rhombuses). Reduction of γH2AX upon wortmannin treatment was used as a baseline. Pink box highlights candidate genes with the strongest γH2AX reduction (zoomed in on top), N4BP2 nuclease shown as red rhombus; TREX1 and APE1 are shown as black rhombuses. Mean±SEM, n=3 independent experiments. (E, F) Representative images (E) and quantification (F) of DNA damage accumulation (detected with γH2AX, red) in micronuclei with ruptured nuclear envelope (indicated by GFP-NLS efflux from the micronucleus). Scale bar, 2 μm. Mean±SEM, n=3 independent experiments. (G) Immunoblot of N4BP2 using lysates from Dld1-Y^MN cells of indicated genotypes. Short and long exposures are shown. Tubulin was used as a loading control. (H) Quantification of metaphase spreads from MN-induced Dld1-Y^MN cells of indicated genotypes containing fragmented Y chromosome. n=3 independent clones per each genotype. (I) Representative images of metaphase spreads in Dld1-Y^MN, N4BP2 knockout (KO) clones, TREX1 KO clones in Dld1-Y^MN, after induction of MN for 72 hours. Y chromosome paint probe (green), Y centromere probe (red). Yellow arrows point at the intact Y chromosome, red arrows: Y centromere fragments, white arrows: Y chromosome fragments. Scale bars, 20 μm, 5 μm. (C, E, I) DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue).

Figure 2.. Fragmentation of chromosomes exposed to N4BP2.

(A) Representative immunofluorescence (IF) images of MN-induced Dld1-Y^MN cells showing localization of endogenous N4BP2 nuclease (green) forming droplet-like foci in MN with extensive DNA damage (γH2AX, red). Scale bars, 10 μm and 2 μm, as indicated. (B) Percentage of γH2AX-positive ruptured MN in GFP-NLS-expressing Dld1-Y^MN cells upon indicated siRNA treatments. Mean±SEM, n=3 independent experiments. (C) Percentage of γH2AX-positive ruptured MN in GFP-NLS-expressing Dld1-Y^MN cells of the indicated genotypes. See (S4A) for a scheme of experiment. Mean±SEM, n=3 independent experiments. (D) Percentage of γH2AX-positive intact MN in GFP-NLS-expressing Dld1-Y^MN cells of the indicated genotypes. See (S4A) for a scheme of experiment.Mean±SEM, n=3 independent experiments. (E) Representative IF images of GFP-NLS-expressing U2OS cells, showing localization of endogenous N4BP2 nuclease within ruptured micronuclei (identified by the absence of GFP-NLS, orange arrows) with extensive DNA damage (γH2AX, red). MN were induced by nocodazole. White arrows indicate intact, GFP-NLS-positive micronuclei. Scale bars, 10 μm and 2 μm, as indicated. (F) Representative IF images of Dld1-Y^MN cells with induced MN, transfected as indicated, showing localization of N4BP2-GFP (green) inside γH2AX (red)–positive MN. Scale bars, 5 μm and 1 μm, as indicated. (G) Live-cell imaging of U2OS cells expressing N4BP2-GFP and RFP-NLS, MN were induced with nocodazole. N4BP2-GFP enters ruptured MN (identified by the loss of RFP-NLS, occurs at 15 min time point) and gradually accumulates in MN. Scale bar, 10 μm and 2 μm, as indicated. (H) (Top) Schematic of the approach used to target N4BP2-GFP into intact nuclei and micronuclei in N4BP2 KO Dld1-Y^MN cells. (Bottom) Representative images of DNA damage accumulation (γH2AX, red) upon targeting N4BP2-GFP-NLS into intact nuclei vs cytoplasmic N4BP2 (N4BP2-GFP). Scale bar, 5 μm. (I) (Top) Schematic of the approach used to target N4BP2-GFP into intact nuclei and micronuclei in U2OS cells. (Bottom) Representative images of mitotic spreads upon N4BP2-GFP-NLS expression for 0h, 24h and 48h. Chromosome fragments are indicated by green arrowheads. Numbers indicate each individual fragment in the zoomed-in area. Scale bar, 5 μm. Quantifications are shown in (J). (J) Quantifications of fragmented mitotic spreads (percentage of all spreads) in U2OS cells upon N4BP2-GFP-NLS expression for 0h, 24h and 48h. n=3 independent experiments per each condition, minimum 300 cells per each experiment. See schematic of experiment and images of representative mitotic spreads in (I). (K) Quantifications of percentage of cells with MN in WT U2OS cells (white column) vs 24h and 48h of N4BP2-GFP overexpression (red columns). Pie charts below each corresponding condition show percentage of ruptured (blue, numbers) and intact (white) MN. N=3 independent experiments, 219 cells. See (S6G) for GFP control. (L) Percentage of Y chromosome-positive nuclei in long-term cultured (40 passages) wild type (WT) and N4BP2 knockout (KO) Dld1-Y^MN cells. Schematic of the experiment is shown in (S7A,B). n=3 independent clones per each genotype, ≥200 cells per clone. See also (S7).

Figure 3.. N4BP2 promotes ecDNA generation.

(A) Generation of HeLa cells that carry DHFR-encoding double minute/ecDNA providing resistance to high methotrexate (high MTX) concentrations. MTX-resistant HeLa clone with amplified DHFR gene within homogenously staining region (HSR, orange) was treated with high MTX concentration (640 nM) for 10 days, which provided selection pressure and promoted double minutes/ecDNA formation (shown as orange circles). Cells were transfected with indicated siRNAs at day 1 and day 5. N4BP2 is depicted as yellow circle, ruptured nuclear envelope as green dashed line. (B) Representative image of N4BP2-GFP localizing on a chromatin bridge (yellow arrows) in HeLa clone with amplified DHFR gene, zoomed-in view shown below. Scale bars, 10 μm and 2 μm, as indicated. (C) Representative image of HeLa cells from (A) showing co-localization of N4BP2-GFP with γH2AX (red) in the chromatin bridge. Scale bars, 10 μm and 2 μm, as indicated. (D) Immunoblot of N4BP2 using lysates of HeLa cells from (A), transfected with indicated siRNAs. Short and long exposures are shown. Tubulin was used as a loading control. (E) Percentage of HeLa cells from (A) with ecDNA, after induction of ecDNA formation with high (640 nM) concentration of MTX for 14 days, treated with indicated siRNAs. n=605 cells from 3 independent experiments. (F) Representative DNA-FISH images demonstrating ecDNA-positive mitotic spreads (marked with yellow arrows) upon treatment with NS siRNA vs ecDNA-negative mitotic spreads in N4BP2 siRNA condition. Scale bars 5 μm and 20 μm.

Figure 4.. N4BP2 initiates genome rearrangements and chromothripsis.

(A) Schematic of a strategy used to assess genome rearrangements induced by N4BP2. Low levels of N4BP2-GFP or GFP were transiently expressed in Dld1-Y^MN, N4BP2 KO cells, micronuclei were induced by adding DOX/IAA for 72h. After single cell sorting (based on green GFP signal) and clonal expansion, clonal isolates were analyzed by a set of methods (shown below). FISH, Fluorescence In Situ Hybridization; SKY, Spectral Karyotyping; WGS, Whole Genome Sequencing; OGM, Optical Genome Mapping. (B) Y chromosome status and chromosomal rearrangements observed in clonal isolates produced in (A). (C-E) Representative FISH images (C) of a normal Y chromosome without visible defects on metaphase spreads of Dld1-Y^MN N4BP2 KO clonal isolate following transient GFP expression (left) and an example of derivative Y chromosomes (also shown with SKY analysis (D) and long read WGS (E) from Dld1-Y^MN, N4BP2 KO clonal isolate obtained after transient expression of N4BP2-GFP as shown in (A) (right panels). Y chromosome paint probes are green and Y centromere probes are red. DAPI is shown in blue. (F, G) WGS-enabled reconstruction of a pre-existing fusion [in a parental clone, (F)] between a nearly full length (196.8 Mb) chromosome 3 (Chr3) and an extra copy of the first 41.7 Mb of the p-arm of chromosome 1 (Chr1), producing a large, 238.5 Mb fusion chromosome. (G) N4BP2-GFP-derived clone, in which the first 41.7 Mb of the p arm of Chr1 had undergone focal chromothripsis with insertions at 3 different locations of pieces of Chr3 mapped adjacent to the initial Chr1/Chr3 fusion site. (H-K) Assessment of the role of N4BP2 in genome/chromosomal rearrangements in cancer using pan-cancer genomics. (H) Independent effect size (x-axis) of chromothripsis and copy number segmentation (y-axis) on N4BP2 expression in a multivariate regression model. (I) Chromothripsis is enriched in tumor samples with N4BP2 copy number gains, while compared with the association of TP53 mutation status (J) (WT=no mutation, MUT=LOH, homozygous deletion or driver point mutation/indel) and chromothripsis status across TCGA (two-sided Fisher’s exact test). (K) Example of structural variant profile from samples with high expression of N4BP2, exhibiting characteristic signs of ecDNA. Arcs indicate rearrangements between two regions of the genome, TLOC=translocation (green), INV=inversion (brown), DUP=tandem duplication (purple), DEL=deletion (red).

Figure 5.. N4BP2-induced DNA damage and ecDNA formation in induced gliomas.

(A) Schematic of induced human high-grade glioma generation. Induced pluripotent stem cells (iPSCs) with a combination of driver mutations commonly altered in human glioblastomas were genetically modified (WT, N4BP2 KO, TREX1 KO) and differentiated into neural/glial progenitor cells, to be intracranially injected into mouse brain to form primary tumors. (B) Schematic of the approach used to induce MN in iNPCs and primary tumor spheres [generated as shown in (A)], followed by immunofluorescence analysis of fixed cells (C, D; S12D, E) and DNA-FISH analysis of metaphase spreads (E; S12F). (C) Percentage of γH2AX-positive MN in the primary tumor spheres in the absence of N4BP2 or TREX1. n=3 independent experiments per each genotype, 970 cells. 3 independent experiments per each condition. (D) Quantification of the fraction of γH2AX-positive ruptured micronuclei (aberrant Lamin B1 staining) in primary tumor spheres in the presence and absence of N4BP2 or TREX1. 2 independent experiments per each genotype, n=300 micronuclei (control), n=350 micronuclei (N4BP2 KO) and n=320 micronuclei (TREX KO). 3 independent experiments per each condition. (E) Quantification of metaphase spreads containing ecDNA in primary tumor spheres in the presence and absence of N4BP2 or TREX1. 3 independent experiments per each genotype, n=77 spreads (control), n=406 spreads (N4BP2 KO) and n=197 spreads (TREX KO). 3 independent experiments per each condition. (F) Representative images of metaphase spreads in iHGG model showing ecDNA (yellow arrowheads), genotypes as indicated. Scale bar, 10 μm. (B, C) DNA was stained with DAPI (blue). (G) Quantification of tumor area on mouse brain sections in the presence and absence of N4BP2 or TREX1 (n = 5 mice for control, n = 5 mice for N4BP2 KO, and n = 4 mice for TREX1 KO) (H) Number of dividing tumor cells (Ki67-positive, normalized to wild type) in the presence or absence of N4BP2 or TREX1. Sample sizes: n = 3 mice (control), n = 4 mice (N4BP2 KO), and n = 6 mice (TREX1 KO). (I) Representative composite images of brain sections showing iHGG tumors, with human nuclei (HuNuc) stained in purple and tumor boundaries indicated by dashed lines. Genotypes as indicated.