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

Abstract

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 chromosomes1,2,3,4,5. Here we examine the mechanism underlying chromothripsis and kataegis by 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 the 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.

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Fig. 1: The effect of TREX1 on telomere-crisis-induced rearrangements.
Fig. 2: TREX1 promotes chromothripsis.
Fig. 3: APOBEC3B induces kataegis during telomere crisis.
Fig. 4: TREX1 and APOBEC3B determine genome instability during telomere crisis.

Data availability

All sequencing data pertaining to this study have been deposited with the European Nucleotide Archive database under primary accession no. PRJEB23723 and secondary accession no. ERP105494. All other data supporting the findings of this study are available within the article and its supplementary information files and from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

All code used in this study is available at the Wellcome Sanger Institute GitHub page (https://github.com/cancerit) or by request to the authors (A.C., P.J.C.).

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Acknowledgements

We thank S. Dewhurst for insightful comments on this manuscript and N. Saini for generating the logo data. Research reported in this publication was supported by grants from the National Cancer Institute (no. R35CA210036), the Starr Cancer Consortium (grant no. I9-A9-047) and from the Breast Cancer Research Foundation to T.d.L. T.d.L. is an American Cancer Society Rose Zarucki Trust Research Professor. D.A.G. is supported by the National Institutes of Health Intramural Research Program Project (no. Z1AES103266). J.M. is supported by grants from the National Cancer Institute (no. R00CA212290), an MSK Cancer Center Support Grant/Core Grant (no. P30 CA008748), the Starr Cancer Consortium (grant no. I12-0030), the V Foundation for Cancer Research and a Pew Biomedical Scholar Fellowship.

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Authors and Affiliations

Authors

Contributions

J.M., T.d.L. and P.J.C. conceived and designed the study. J.M., A.C., A.D., K.C. and E.T. performed the experiments. J.M., A.C., D.A.G., L.J.K., P.J.C. and T.d.L. analyzed the data. J.M. and T.d.L. wrote the manuscript with contributions from P.J.C., A.C. and D.A.G. All authors approved the final manuscript.

Corresponding authors

Correspondence to John Maciejowski, Peter J. Campbell or Titia de Lange.

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T.d.L. is a member of the scientific advisory board of Calico Life Sciences. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 TREX1 affects CN alterations but not cell viability after telomere crisis.

a, Summary of the number of post-crisis T2p1 and TREX1 KO clones analyzed from independent telomere crisis experiments, the frequency of simple and complex CN changes detected (1×) and the number of clones selected for 30x WGS. Note that many more TREX1 KO than T2p1 clones were analyzed by 30x WGS. This does not reflect greater survival and does not introduce a bias. Uninduced T2p1 clones were not analyzed by 30x WGS since they were shown to lack chromothripsis previously (ref. 4). b, Plot showing percentage of annexin V positive cells 72 hours after dox treatment of two batches of T2p1 and three batches of TREX1 KO lines. Bars represent mean and s.d. from n = 2 independent experiments. P values derived from Student’s t test. (ns: not significant). c. Plating efficiency of T2p1 and TREX1 KO cells after dox induction. Cells were seeded in 96 well plates at different cell number per well and scored for positive wells two weeks later. Bars represent mean and s.d. from n = 3 independent experiments. P values derived from Student’s t test. The numbers below the graph refer to the experiments shown in (a). d, Immunoblot of the lack of cGAS protein in T2p1 cells. HEK293, MCF10A, MCF10A CGAS KO16 are shown for comparison. The protein STING is present in all cell lines.

Source data

Extended Data Fig. 2 Circos plots of genome alterations in T2p1 post-crisis clones.

a, Four T2p1 post-crisis clones with complex events identified by 1x WGS were analyzed by 30x WGS and their associated genome plots are shown. Circos plots show somatic mutations including substitutions (outermost, dots represent 6 mutation types: C>A, blue; C>G, black; C>T, red; T>A, grey; T>C, green; T>G, pink), indels (the second outer circle, color bars represent five types of indels: complex, grey; insertion, green; deletion other, red; repeat-mediated deletion, light red; microhomology-mediated deletion, dark red) and rearrangements (innermost, lines representing different types of rearrangements: tandem duplications, green; deletions, orange; inversions, blue; translocations: grey). The number of detected base substitutions, indels, and rearrangements are shown to the right of each panel. b, Genomic information on two post-crisis TREX1 KO clones displayed as in (a).

Extended Data Fig. 3 Examples of chromothripsis-like and Local Jump events in TREX1 KO post-crisis clones.

Three TREX1 KO post-crisis subclones with complex (Z142) events, simple (T108) events or no rearrangements (T101) identified by 1x WGS were analyzed by 30x WGS. DNA CN profiles and rearrangement joins were obtained from Battenberg analysis of 30x target coverage genomic sequencing data. Annotation as in Fig. 1a. Variant allele frequency tracks are shown below the chromosome ideograms. Examples show a chromothripsis-like event in Z142, a local n jump in T108, and a local 3 jump in T101.

Extended Data Fig. 4 Clonal evolution in post-crisis clones M2dox120, A3B1590 and M2dox121.

a, Chromothripsis and rainfall plot of clone M2dox120 involving chromosomes 1 and 3. b, Chromothripsis and rainfall plot of clone A3B1590 involving chromosomes 1 and 11. c, Chromothripsis and rainfall plot of clone M2dox121 involving chromosomes 9 and 12. Evidence of parallel crises manifested as chromothripsis affecting two distinct regions on separate chromosomes in (a) and (b). Evidence for sequential events affecting the same derivative chromosome in (c). Top of each plot: the arcs represent the two ends of rearrangements. Arcs are grouped from top to bottom by the type of rearrangement orientation as follows: deletion (D;+ -); tandem duplication (TD; -+); tail-tail (TT;++); head-head (HH; —). The bottom of each plot shows filled circles which represent positions of point mutations colored by mutation type. The Y-axis shows the distance of each mutation to the next on the same chromosome, with the respective axis on the left-hand side of the graph.

Extended Data Fig. 5 Gene Editing of APOBEC3B.

a, Relative APOBEC3A mRNA levels (normalized to GAPDH) in U937 and RPE1 cells determined by qRT-PCR showing that APOBEC3A is not expressed in the telomere crisis cell system. Bars represent mean and s.d. from n = 3 independent experiments. b, Schematic of the APOBEC3B locus showing landmarks relevant to CRISPR editing. sgRNA sequences used for CRISPR editing are shown below. Protospacer adjacent motifs are marked in red. c, APOBEC3B amino acid sequence showing exon boundaries, catalytic domains, and predicted gene disruption from CRISPR editing. d, PCR screening identifies clones harboring at least one copy of a CRISPR-generated inversion in the APOBEC3B locus. Clones used for subsequent experiments are marked in red. e, PCR screening confirms biallelic disruption of the endogenous APOBEC3B locus. Clones used for subsequent experiments are marked in red. f, Immunoblot for APOBEC3B and γ-tubulin shows absence of APOBEC3B in 4 CRISPR-edited clones. Clones #15 and #26 were selected for further this study. Asterisk marks a cross-reacting polypeptide. Blot is representative of n = 3 independent experiments. g, Proliferation of the APOBEC3B CRISPR KO clones with and without doxycycline induction of TRF2-DN. Data from n = 1 experiment.

Source data

Extended Data Fig. 6 1x and 30x WGS information on APOBEC3B KO post-crisis clones.

a, Summary of the number of APOBEC3B KO clones isolated from independent telomere crisis experiments, the frequency of simple and complex CN changes detected (1×) and the number of clones selected for 30x WGS. Parallel information on the T2p1 (Extended Data Fig. 1a) is provided for comparison. b, and c, Information of complex events in APOBEC3B KO clones as in Fig. 2b and Fig. 2c. Parallel information on T2p1 and TREX1 KO post crisis clones is provided (from Fig. 2). d, Bar plot displaying the number of CN changes associated with the complex events indicated in APOBEC3B KO clones together with T2p1 and TREX1 KO information (from Fig. 2d). P values derived from ANOVA (ns: not significant).

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Maciejowski, J., Chatzipli, A., Dananberg, A. et al. APOBEC3-dependent kataegis and TREX1-driven chromothripsis during telomere crisis. Nat Genet 52, 884–890 (2020). https://doi.org/10.1038/s41588-020-0667-5

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