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The CIP2A–TOPBP1 axis safeguards chromosome stability and is a synthetic lethal target for BRCA-mutated cancer

Nature Cancer volume 2, pages 1357–1371 (2021)Cite this article

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Abstract

BRCA1/2-mutated cancer cells adapt to the genome instability caused by their deficiency in homologous recombination (HR). Identification of these adaptive mechanisms may provide therapeutic strategies to target tumors caused by the loss of these genes. In the present study, we report genome-scale CRISPR–Cas9 synthetic lethality screens in isogenic pairs of BRCA1- and BRCA2-deficient cells and identify CIP2A as an essential gene in BRCA1- and BRCA2-mutated cells. CIP2A is cytoplasmic in interphase but, in mitosis, accumulates at DNA lesions as part of a complex with TOPBP1, a multifunctional genome stability factor. Unlike PARP inhibition, CIP2A deficiency does not cause accumulation of replication-associated DNA lesions that require HR for their repair. In BRCA-deficient cells, the CIP2A–TOPBP1 complex prevents lethal mis-segregation of acentric chromosomes that arises from impaired DNA synthesis. Finally, physical disruption of the CIP2A–TOPBP1 complex is highly deleterious in BRCA-deficient tumors, indicating that CIP2A represents an attractive synthetic lethal therapeutic target for BRCA1- and BRCA2-mutated cancers.

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Fig. 1: CIP2A loss is synthetically lethal with BRCA1 or BRCA2 deficiency.
Fig. 2: CIP2A-null cells do not have increased loads of HR-engaging DNA lesions.
Fig. 3: CIP2A prevents acentric chromosome segregation.
Fig. 4: CIP2A colocalizes with TOPBP1 on mitotic structures.
Fig. 5: The CIP2A–TOPBP1 interaction is essential in BRCA2−/− cells.
Fig. 6: Disruption of the CIP2A–TOPBP1 complex causes genome instability and lethality in BRCA-deficient cells.
Fig. 7: CIP2A prevents S phase-associated DNA lesions from causing acentric chromosome mis-segregation.
Fig. 8: Therapeutic proof of concept.

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Data availability

Source data for Figs. 1–8 and Extended Data Figs. 1–10 have been provided. Illumina sequencing data for the screens were deposited at the National Center for Biotechnology Information (accession no. PRJNA753451). Further information on research design is available in the Nature Research Reporting Summary linked to this article. All other data supporting the findings of the present study are available from the corresponding author on reasonable request.

Code availability

The CCA code is also available at Zenodo (https://doi.org/10.5281/zenodo.5149154).

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Acknowledgements

We dedicate this work to the memory of our friend and colleague Silvia Emma Rossi. We thank members of the Durocher lab for helpful discussions and A. Jeanrenaud for technical help. We also thank J. Moffat for his generous sharing of the TKO sgRNA libraries and R. Greenberg for the U2OS 2-6-5 cell line. We thank K. Chan at the NBCC (LTRI) for sequencing. We also thank the Repare in vivo pharmacology team (A. Roulston, A. Bonneau-Fortin, M.-È. Leclaire and S. Fournier) for their help with tumor xenograft assays. S.A. was supported by a Banting post-doctoral fellowship and S.E.R. was supported by a fellowship from Fondazione AIRC per la Ricerca sul Cancro. Y.X. holds, and A.A.Q. and D.S. held, post-doctoral fellowships from the Canadian Institutes for Health Research (CIHR). Work in M.S.’s lab was supported by grants from the Swiss National Foundation (grant no. 310030_189141) and the Groeber Foundation. D.D. is a Canada Research Chair (Tier I) and work in his lab was supported by grants from the CIHR (grant no. FDN143343) and Canadian Cancer Society (grant no. 705644), with additional support from the Krembil Foundation and Repare Therapeutics.

Author information

Author notes

  1. Sylvie M. Noordermeer

    Present address: Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands

  2. Nicole Hustedt

    Present address: Lonza AG, Visp, Switzerland

  3. These authors contributed equally: Salomé Adam, Silvia Emma Rossi.

  4. Deceased: Silvia Emma Rossi.

Authors and Affiliations

  1. Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada

    Salomé Adam, Silvia Emma Rossi, Nathalie Moatti, Yibo Xue, Timothy F. Ng, Alejandro Álvarez-Quilón, Dheva Setiaputra, Sylvie M. Noordermeer, Nicole Hustedt, Rachel K. Szilard, Natasha Chaudhary, Meagan Munro, Henrique Melo & Daniel Durocher

  2. Department of Gynecology, University Hospital and University of Zurich, Schlieren, Switzerland

    Mara De Marco Zompit & Manuel Stucki

  3. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    Timothy F. Ng & Daniel Durocher

  4. Repare Therapeutics, St-Laurent, Quebec, Canada

    Alejandro Álvarez-Quilón, Jessica Desjardins, Vivek Bhaskaran, Giovanni Martino, Toshiro K. Ohsumi, Artur Veloso, Shou Yun Yin, Robert Papp, Jordan T. F. Young & Michael Zinda

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Contributions

S.A. and S.E.R. contributed equally to the present study and carried out the bulk of the experimental work, writing and revising drafts, as well as preparing figures. N.M., M.M.Z., Y.X., T.F.N., A.A.Q., D.S. and S.M.N. provided additional data and reagents. J.D. undertook the BRCA1 screen shown in the present study. V.B., G.M., S.Y.Y. and R.P. undertook the animal and pharmacokinetic studies. S.M.N. undertook the original BRCA1 synthetic lethal screen. T.K.O. provided CCA. N.H. provided the first validation of the BRCA–CIP2A synthetic lethality. R.K.S., N.C. and M.M. provided technical assistance that included key reagent generation and, for R.K.S., manuscript editing. A.V. and H.M. provided bioinformatics expertise. J.T.F.Y. supervised J.D. M.Z. supervised all authors based at Repare Therapeutics. M.S. supervised M.M.Z. D.D. conceived the project, supervised the project, wrote and revised the manuscript, prepared figures and acquired funding for the present study.

Corresponding author

Correspondence to Daniel Durocher.

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Competing interests

D.D. is a shareholder and advisor of Repare Therapeutics. A.A.Q., J.D., V.B., G.M., S.Y.Y., R.P., J.T.F.Y., A.V. and M.Z. are employees of Repare Therapeutics. T.K.O. was an employee of Repare Therapeutics. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Cancer thanks Dipanjan Chowdhury, Joanna Loizou 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

Extended Data Fig. 1 Data supporting CIP2A as a protein recognizing mitotic DNA lesions.

a, Immunoblotting of whole-cell extracts of RPE1-hTERT p53-/- Cas9 cells, parental (WT) or BRCA1-/-, expressing the indicated sgRNAs and either a virus expressing an sgRNA-resistant FLAG-CIP2A (CIP2A*) or an empty virus (EV). Lysates were probed for FLAG, CIP2A and tubulin (loading control). Representative of two immunoblots. b, Immunoblot examining CIP2A levels in isogenic and BRCA-deficient cells in the DLD1 and RPE1-hTERT p53-/- Cas9 (RPE1-hTERT) backgrounds. Tubulin was used as a loading control. Immunoblotting was performed once. c, Clonogenic survival assay of RPE1-hTERT p53-/- Cas9 cells of the indicated genotype following treatment with camptothecin (CPT). Data presented as the mean ± S.D. n = 4 independent clonogenic assays apart from the 15 nM dose where n = 3; analyzed with extra sum-of-squares F test and compared to WT. ∗∗∗p = 1.27 × 10−7 (#1), 1.68 × 10−6 (#6). d, e, Analysis of sister chromatid exchanges (SCEs) in cells of the indicated genotype. Quantitation is shown in (d) as cell-based data; bars represent the mean ± S.D. n = 31, 30, 31, 30, 30 aggregated from 3 independent metaphase spreads; analyzed with Kruskall-Wallis non-parametric test, followed by Dunn’s multiple comparisons, compared to WT. n.s.= 0.4398, 0.6574, ∗∗∗p = 1.97 × 10−13, 1.18 × 10−13. Representative micrographs of metaphase spreads are shown in (e). Arrowheads indicate an SCE event. f, Immunoblotting for CIP2A in DLD1 WT and CIP2A knockout clones. Tubulin was used as a loading control. Representative of 3 independent immunoblots. g, Analysis of RAD51 foci in EdU+ cells in RPE1-hTERT (WT) and indicated CIP2A-/- clones. Left, aggregate cell-based values. Right, mean of each independent experiment. Bars represent the mean ± S.D. n = 60 aggregated from 3 independent immunostainings; analyzed with Kruskall-Wallis non-parametric test, followed by Dunn’s multiple comparisons, compared to WT; n.s.= 0.5542, 1.0000, 1.0000. All scale bars = 10 μm. Statistical analyses and full blots are found in Source Data ED Fig. 1.

Source data

Extended Data Fig. 2 CIP2A promotes genome integrity of BRCA-deficient cells.

a, b, Colocalization analysis of CIP2A and γH2AX in DLD1 cells pre- and post-IR (2 Gy) using Squassh (size parameter). Quantitation is shown in (a). Threshold for colocalization is 0.5 (dashed line). Data points represent cells analysed and bars represent the mean ± 95% C.I. n = 15 aggregated from 3 independent immunostainings. Representative micrographs are shown in (b). UT, untreated. c, Representative micrographs of the experiment shown in Fig. 3f. d, Quantitation of radial chromosomes (left) and chromatid breaks (right) in metaphase spreads from DLD1 cells upon transduction of virus expressing the indicated sgRNAs along with Cas9. The bars represent the mean ± S.D. n = 30 aggregated from 3 independent experiments with 10 metaphases scored per experiment; analyzed with Kruskall-Wallis non-parametric test, followed by Dunn’s multiple comparisons, compared to sgAAVS1; n.s.=1.0000, ∗p = 0.0107 (radials); ∗∗∗p = 0.0001, 0.0044 (breaks). e, Representative micrographs of the experiment presented in Fig. 3g. Arrowheads indicate chromosome aberrations. f, Representative micrographs of the experiment shown in Fig. 3h. MNi are indicated with white triangles. Scale bar = 10 μm. Statistical analyses are found in Source Data ED Fig. 2.

Source data

Extended Data Fig. 3 CIP2A acts in mitosis with TOPBP1.

a, Correlation network based on Pearson’s correlation of gene-level NormZ derived from the genotoxic dataset shown in ref. 28. b, Colocalization analysis by Squassh of the experiment shown in Fig. 4b using size as parameter. Each point represents a mitotic cell analysed, bars represent the mean ± 95% C.I. n = 15 aggregated from 3 independent immunostainings. c, Representative micrographs of the experiment quantitated in Fig. 4c. Nocodazole-treated U2OS cells previously transfected with the indicated siRNAs were fixed 1 h post-X-irradiation (2 Gy) and processed for immunofluorescence with the indicated antibodies. Scale bar = 10 μm. d, Competitive growth assays in DLD1 Cas9 cells (WT) or an isogenic BRCA2-/- counterpart transduced with virus expressing the indicated sgRNAs. Data presented as mean ± S.D. n = 3 independent growth assays; analyzed with two-way ANOVA with repeated measures and Šídák’s multiple comparisons testing. ∗∗p = 0.0067 (d18), n.s.=0.46359. Statistical analyses are found in Source Data ED Fig. 3.

Source data

Extended Data Fig. 4 CIP2A and TOPBP1 localize to under-replicated DNA in mitotic cells.

a, Additional micrographs of CIP2A/TOPBP1 structures observed in mitotic cells after treatment with low-dose aphidicolin. Relates to Fig. 4e. Shown are curved (upper panels) and straight (lower panels) filaments. Representative of n = 3 independent immunostainings. b, Micrographs of U2OS parental (WT) and MDC1-/- anaphase cells that were either treated with aphidicolin (400 nM) for 16 h or left untreated. Quantitation of this experiment is shown in Fig. 4f. c, d, Colocalization analysis of TOPBP1 and CIP2A foci in U2OS parental (WT) or MDC1-/- cells using Squassh, size parameter. Representative micrographs (of two independent immunostainings) are shown in (c) quantitation shown in (d). Threshold for colocalization is 0.5 (dashed line). Bar represents the mean ± 95% C.I. n = 24 (WT) or 23 (MDC1-/-). e, Colocalization analysis of RPA and CIP2A in early prophase DLD1 BRCA2-/- cells by Squassh, size parameter. Threshold for colocalization is 0.5 (dashed line). Bar represents the mean ± 95% C.I. n = 15 aggregated from 3 independent immunostainings. Refers to Fig. 4h. f, Quantitation of spontaneous RPA and CIP2A foci in early prophase DLD1 BRCA2-/- cells. Data points are cell-based data, bar represents the mean ± S.D. n = 32 cells aggregated from 3 independent immunostainings. Analyzed with a Mann-Whitney test; U value (normal approximation)=-6.49202, ∗∗∗p = 1.30 × 10−14. g, Representative micrographs of the experiment shown in Fig. 4i with additional MDA-MB-436 cells showing elongation of CIP2A-TOPBP1 filaments during mitosis. h, Immunoblot of lysates from cells used in the experiment shown in Fig. 4j. Lysates were probed for GFP, CIP2A and tubulin (loading control). Representative of n = 2 independent immunoblots. Full blots are provided in Blot Source Data ED Fig. 4. All scale bars = 10 µm. Statistical analyses are found in Source Data ED Fig. 4.

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Extended Data Fig. 5 CIP2A interacts with TOPBP1.

a, Co-immunoprecipitation of CIP2A with TOPBP1. Whole-cell extracts from U2OS parental (WT) or MDC1-/- (KO) cells were subjected to immunoprecipitation with normal mouse IgG or a CIP2A antibody and were then immunoblotted with the indicated antibodies. Representative of two independent experiments. b, Micrographs of the LacR/LacO assay assessing the interaction between endogenous CIP2A and TOPBP1 variants fused to FLAG-LacR shown in Fig. 5f. Representative of 3 independent immunostainings. Scale bar = 10 µm. c, LacR/LacO assay assessing the interaction between endogenous CIP2A and TOPBP1 variants fused to FLAG-LacR. Data presented as the mean values ± S.D. n = 3 independent immunostainings; analyzed with one-way ANOVA, followed by multiple comparisons Dunnet test, all compared to FLAG-LacR; ∗∗∗p = 1 × 10−15. d, Alanine scanning of TOPBP1 (830-851) residues by yeast two-hybrid assay with CIP2A (1-560). Five residues that abolish the TOPBP1-CIP2A interaction when mutated to alanine were identified. AD, activation domain; BD, Gal4 DNA binding domain. Expression of proteins was verified by immunblotting but not shown. Representative of n = 2 independent sets of transformations. e, CIP2A and TOPBP1 interact directly. Upper: 1 μg of GST or GST-CIP2A (1-560) were separated by SDS-PAGE and stained with Coomassie. Lower blot: GST pull-down experiment with either GST or GST-CIP2A (1-560) incubated with either MBP, MBP-TOPBP1756–999 or MBP-TOPBP1756–999-3A. Bound proteins were processed for immunoblotting with an anti-MBP antibody. The CIP2A/TOPBP1 interaction has been observed > 5 times; the loss of interaction by the 3 A mutation is representative of two independent pulldown assays. Full blots are provided in Blot Source Data ED Fig. 5. Statistical analyses are found in Source Data ED Fig. 5.

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Extended Data Fig. 6 Data supporting that the CIP2A-TOPBP1 interaction promotes HR-deficient cell viability.

a, Immunoblots of whole-cell extracts from DLD1 parental (WT) or BRCA2-/- cells transduced with the indicated HA-tagged TOPBP1-expressing lentivirus or an empty virus that expresses an HA epitope (EV(HA)). Lysates were probed with antibodies to HA or tubulin (loading control). FL, full-length. Representative of n = 3 independent transductions. b, Micrographs of DLD1 cells transduced with the indicated virus that were arrested in mitosis with a 16 h treatment with nocodazole, exposed to a 2 Gy IR dose and processed for immunofluorescence with the indicated antibodies 1 h later. Relates to the experiment quantitated in Fig. 5h. Representative of three immunostainings. c, Representative images of crystal violet stains of the clonogenic survival experiment presented in Fig. 5i. Representative of three clonogenic assays. d, Representative micrographs of the experiment presented in Fig. 5j showing DAPI-stained cells to monitor micronucleation (labeled with arrowheads). Representative of three immunostainings. Scale bars =10 μm. Full blots are provided in Source Data ED Fig. 6.

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Extended Data Fig. 7 Disruption of the TOPBP1-CIP2A interaction is lethal in BRCA-deficient cells.

a, DLD1 BRCA2-/- cells transduced with either an empty virus containing only the destabilization domain (DD; EV, left) or a virus encoding B6L (right) were treated with AS1 (1 μM) for the indicated periods or left untreated (UT). Shown are micrographs of mitotic cells stained with antibodies to CIP2A, γH2AX or FLAG (labeling the DD). Quantitation is shown in Fig. 6b. Representative of three independent immunostainings. b, Anti-FLAG immunoblots of whole-cell extracts derived from DLD1 parental (WT) or BRCA2-/- cells untreated or treated with either Shield-1 (S1) or Aqua-Shield-1 (AS1) for 72 h. KAP1 is used as a loading control. Representative of three independent immunostainings. c, Representative images of the clonogenic survival experiment presented in Fig. 6f. Representative of three independent clonogenic experiments. d, Anti-FLAG immunoblots of whole-cell extracts from RPE1-hTERT p53-/- Cas9 parental (WT) or BRCA1-/- cells treated with AS1 for 72 h. Histone H2A is used as a loading control. Representative of two independent immunoblots. e, Clonogenic survival of RPE1-hTERT p53-/- Cas9 parental (WT) or BRCA1-/- cells following expression of B6L for the indicated periods of time prior to replating without AS1. Data presented as mean ± S.D. n = 3 clonogenic assays; analyzed with two-way ANOVA with repeated measures and Šídák’s multiple comparisons. n.s.=0.99907, ∗∗p = 0.0076 (d4) for WT and BRCA1-/- respectively. f, Additional representative micrographs from the experiment shown in Fig. 7c. Left panel shows a normal anaphase cell; right panel shows a cell with a lagging centromere-positive chromosome. All scale bars = 10 µm. Statistical analyses and full blots are found in Source Data ED Fig. 7.

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Extended Data Fig. 8 The CIP2A-TOPBP1 interaction does not impact ATR activation but promotes viability of MDA-MB-436 cells.

a, Immunoblots assessing CHK1 S345 phosphorylation in DLD1 cells transduced with either an empty virus (EV) that expresses the DD domain alone or a virus expressing DD-tagged B6L following induction with 1 μM AS1. Cells were treated with hydroxyurea (HU) at the indicated doses for 24 h before harvesting. Representative of two independent transductions. b, Immunoblots assessing CHK1 S345 and RPA2 pS33 phosphorylation in whole-cell lysates of RPE1-hTERT p53-/- Cas9 parental (WT) or independent CIP2A-/- clones. Cells were treated with camptothecin (CPT; 1 μM) for the indicated times prior to harvesting. Representative of two independent immunoblots. c, Anti-FLAG immunoblots of whole-cell extracts from MDA-MB-436 cells treated with Aqua-Shield-1 (AS1) for 72 h. KAP1 is used as loading control. Representative of three independent immunoblots. d, Proliferation curves for MDA-MB-436 cells upon B6L stabilization by AS1 treatment (1 μM). Cells were transduced with an empty virus (EV) as control. Representative of three proliferation assays, analyzed with a Mann-Whitney test; U value (normal approximation)= -5.612486, -5.163978. e, Quantitation of independent proliferation experiments described in (d). Data presented as the mean values at endpoint ± S.D. n = 3 proliferation assays; analyzed with a t-test, assuming data is normally distributed, ∗∗p = 0.0038. f, Quantitation of micronuclei (MNi)-positive cells in MDA-MB-436 cells transduced with an either empty virus (EV) or B6L-expressing virus following addition of AS1. Data presented as the mean ± S.D. n = 3 independent MNi counts; analyzed with two-way ANOVA with repeated measures and Šídák’s multiple comparisons testing. ∗∗∗p = 0.00015. Statistical analyses and full blots are found in Source Data ED Fig. 8.

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Extended Data Fig. 9 Probing the role of PP2A inhibition by CIP2A in the maintenance of genome integrity in BRCA-deficient cells.

a, Immunoblots showing levels of B56α (left), B56γ (middle) and of B6L and the destabilization domain (DD) from the empty virus (EV) (right) after 72 h of siRNA treatment and 48 h of AS1 induction. Representative of two independent immunostainings. b, Quantitation of micronuclei (MNi) in DLD1 BRCA2-/- cells stably transduced with a B6L-encoding lentivirus and transfected with control (siCTRL) or B56α and B56γ-targeting siRNAs. B6L expression was induced by AS1 addition for 48 h. Data presented as the mean ± S.D. n = 3 independent MNi counts; analyzed with a two-tailed t-test, assuming data is normally distributed, **p = 0.0031, **p = 0.0072. c, Representative micrographs of the experiment in (b). Arrowheads indicate micronuclei. d, LacR/LacO assay assessing the interaction between endogenous CIP2A, HA-tagged B56α or B56γ and FLAG-LacR-TOPBP1. Data presented as the intensity of the signal at the LacR-TOPBP1 focus over background (dashed line). Bars represent the mean ± S.D. aggregated from two independent localization assays measuring 15 cells per experiment. e, Representative micrographs of the experiment in (d). Statistical analyses and full blots are found in Source Data ED Fig. 9.

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Extended Data Fig. 10 Data supporting proof-of-concept disruption of CIP2A-TOPBP1 interaction.

a, Determination of the EC50 growth inhibitory concentration of AS1 in DLD1 BRCA2-/- cells transduced with lentivirus expressing either the DD domain alone (EV) or B6L. Data is presented as the mean ± SEM. n = 3 technical replicates; analyzed with extra sum-of-squares F test. ***p = 1 × 10−15. b, Pharmacokinetic analysis of AS1 free plasma concentration over a 24 h period in mice. Data is presented as the mean of values from n = 3 animals. c, Analysis of cell viability by CellTiter GLo of the indicated DLD1 cell lines following celastrol (left) or TD-52 (right) treatments at the indicated concentrations. Data presented as the mean values ± S.D. n = 3 independent drug sensitivity assays; analyzed with extra sum-of-squares F test. Statistical analyses are found in Source Data ED Fig. 10.

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Adam, S., Rossi, S.E., Moatti, N. et al. The CIP2A–TOPBP1 axis safeguards chromosome stability and is a synthetic lethal target for BRCA-mutated cancer. Nat Cancer 2, 1357–1371 (2021). https://doi.org/10.1038/s43018-021-00266-w

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