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Targeting TRIM37-driven centrosome dysfunction in 17q23-amplified breast cancer

Nature volume 585, pages 447–452 (2020)Cite this article

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Abstract

Genomic instability is a hallmark of cancer, and has a central role in the initiation and development of breast cancer1,2. The success of poly-ADP ribose polymerase inhibitors in the treatment of breast cancers that are deficient in homologous recombination exemplifies the utility of synthetically lethal genetic interactions in the treatment of breast cancers that are driven by genomic instability3. Given that defects in homologous recombination are present in only a subset of breast cancers, there is a need to identify additional driver mechanisms for genomic instability and targeted strategies to exploit these defects in the treatment of cancer. Here we show that centrosome depletion induces synthetic lethality in cancer cells that contain the 17q23 amplicon, a recurrent copy number aberration that defines about 9% of all primary breast cancer tumours and is associated with high levels of genomic instability4,5,6. Specifically, inhibition of polo-like kinase 4 (PLK4) using small molecules leads to centrosome depletion, which triggers mitotic catastrophe in cells that exhibit amplicon-directed overexpression of TRIM37. To explain this effect, we identify TRIM37 as a negative regulator of centrosomal pericentriolar material. In 17q23-amplified cells that lack centrosomes, increased levels of TRIM37 block the formation of foci that comprise pericentriolar material—these foci are structures with a microtubule-nucleating capacity that are required for successful cell division in the absence of centrosomes. Finally, we find that the overexpression of TRIM37 causes genomic instability by delaying centrosome maturation and separation at mitotic entry, and thereby increases the frequency of mitotic errors. Collectively, these findings highlight TRIM37-dependent genomic instability as a putative driver event in 17q23-amplified breast cancer and provide a rationale for the use of centrosome-targeting therapeutic agents in treating these cancers.

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Fig. 1: PLK4 inhibition is synthetically lethal with TRIM37 amplification.
Fig. 2: PLK4 inhibition triggers mitotic catastrophe in TRIM37-amplified cancer cells.
Fig. 3: PCM sequestration by TRIM37 drives mitotic catastrophe in acentrosomal cells.
Fig. 4: TRIM37 overexpression delays centrosome separation in late G2 phase, and promotes mitotic errors.

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Data availability

Data that support the findings of this study are available from the corresponding authors upon reasonable request. Source data are provided with the paper.

References

  1. Burrell, R. A., McGranahan, N., Bartek, J. & Swanton, C. The causes and consequences of genetic heterogeneity in cancer evolution. Nature 501, 338–345 (2013).

    Article  ADS  CAS  Google Scholar 

  2. Kalimutho, M. et al. Patterns of genomic instability in breast cancer. Trends Pharmacol. Sci. 40, 198–211 (2019).

    Article  CAS  Google Scholar 

  3. Lord, C. J. & Ashworth, A. PARP inhibitors: synthetic lethality in the clinic. Science 355, 1152–1158 (2017).

    Article  ADS  CAS  Google Scholar 

  4. Curtis, C. et al. The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature 486, 346–352 (2012).

    Article  CAS  Google Scholar 

  5. Dawson, S.-J., Rueda, O. M., Aparicio, S. & Caldas, C. A new genome-driven integrated classification of breast cancer and its implications. EMBO J. 32, 617–628 (2013).

    Article  CAS  Google Scholar 

  6. Ali, H. R. et al. Genome-driven integrated classification of breast cancer validated in over 7,500 samples. Genome Biol. 15, 431 (2014).

    Article  Google Scholar 

  7. Wong, Y. L. et al. Reversible centriole depletion with an inhibitor of Polo-like kinase 4. Science 348, 1155–1160 (2015).

    Article  ADS  CAS  Google Scholar 

  8. Moyer, T. C., Clutario, K. M., Lambrus, B. G., Daggubati, V. & Holland, A. J. Binding of STIL to Plk4 activates kinase activity to promote centriole assembly. J. Cell Biol. 209, 863–878 (2015).

    Article  CAS  Google Scholar 

  9. Lambrus, B. G. et al. p53 protects against genome instability following centriole duplication failure. J. Cell Biol. 210, 63–77 (2015).

    Article  CAS  Google Scholar 

  10. Cuella-Martin, R. et al. 53BP1 integrates DNA Repair and p53-dependent cell fate decisions via distinct mechanisms. Mol. Cell 64, 51–64 (2016).

    Article  CAS  Google Scholar 

  11. Meitinger, F. et al. 53BP1 and USP28 mediate p53 activation and G1 arrest after centrosome loss or extended mitotic duration. J. Cell Biol. 214, 155–166 (2016).

    Article  CAS  Google Scholar 

  12. Fong, C. S. et al. 53BP1 and USP28 mediate p53-dependent cell cycle arrest in response to centrosome loss and prolonged mitosis. eLife 5, e16270 (2016).

    Article  Google Scholar 

  13. Lambrus, B. G. et al. A USP28–53BP1–p53–p21 signaling axis arrests growth after centrosome loss or prolonged mitosis. J. Cell Biol. 214, 143–153 (2016).

    Article  CAS  Google Scholar 

  14. Ciriello, G. et al. Comprehensive molecular portraits of invasive lobular breast cancer. Cell 163, 506–519 (2015).

    Article  CAS  Google Scholar 

  15. Pereira, B. et al. The somatic mutation profiles of 2,433 breast cancers refines their genomic and transcriptomic landscapes. Nat. Commun. 7, 11479 (2016).

    Article  ADS  CAS  Google Scholar 

  16. Monni, O. et al. Comprehensive copy number and gene expression profiling of the 17q23 amplicon in human breast cancer. Proc. Natl Acad. Sci. USA 98, 5711–5716 (2001).

    Article  ADS  CAS  Google Scholar 

  17. Pärssinen, J., Kuukasjärvi, T., Karhu, R. & Kallioniemi, A. High-level amplification at 17q23 leads to coordinated overexpression of multiple adjacent genes in breast cancer. Br. J. Cancer 96, 1258–1264 (2007).

    Article  Google Scholar 

  18. Sinclair, C. S., Rowley, M., Naderi, A. & Couch, F. J. The 17q23 amplicon and breast cancer. Breast Cancer Res. Treat. 78, 313–322 (2003).

    Article  CAS  Google Scholar 

  19. Balestra, F. R., Strnad, P., Flückiger, I. & Gönczy, P. Discovering regulators of centriole biogenesis through siRNA-based functional genomics in human cells. Dev. Cell 25, 555–571 (2013).

    Article  CAS  Google Scholar 

  20. Oegema, K., Davis, R. L., Lara-Gonzalez, P., Desai, A. & Shiau, A. K. CFI-400945 is not a selective cellular PLK4 inhibitor. Proc. Natl Acad. Sci. USA 115, E10808 (2018).

    Article  CAS  Google Scholar 

  21. Mason, J. M. et al. Functional characterization of CFI-400945, a Polo-like kinase 4 inhibitor, as a potential anticancer agent. Cancer Cell 26, 163–176 (2014).

    Article  CAS  Google Scholar 

  22. Kawakami, M. et al. Polo-like kinase 4 inhibition produces polyploidy and apoptotic death of lung cancers. Proc. Natl Acad. Sci. USA 115, 1913–1918 (2018).

    Article  CAS  Google Scholar 

  23. Bhatnagar, S. et al. TRIM37 is a new histone H2A ubiquitin ligase and breast cancer oncoprotein. Nature 516, 116–120 (2014).

    Article  ADS  CAS  Google Scholar 

  24. Branon, T. C. et al. Efficient proximity labeling in living cells and organisms with TurboID. Nat. Biotechnol. 36, 880–887 (2018).

    Article  CAS  Google Scholar 

  25. Roux, K. J., Kim, D. I., Raida, M. & Burke, B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J. Cell Biol. 196, 801–810 (2012).

    Article  CAS  Google Scholar 

  26. Oughtred, R. et al. The BioGRID interaction database: 2019 update. Nucleic Acids Res. 47, D529–D541 (2019).

    Article  CAS  Google Scholar 

  27. Wang, W., Xia, Z.-J., Farré, J.-C. & Subramani, S. TRIM37, a novel E3 ligase for PEX5-mediated peroxisomal matrix protein import. J. Cell Biol. 216, 2843–2858 (2017).

    Article  CAS  Google Scholar 

  28. Densham, R. M. et al. Human BRCA1–BARD1 ubiquitin ligase activity counteracts chromatin barriers to DNA resection. Nat. Struct. Mol. Biol. 23, 647–655 (2016).

    Article  CAS  Google Scholar 

  29. Ben-David, U. et al. Genetic and transcriptional evolution alters cancer cell line drug response. Nature 560, 325–330 (2018).

    Article  ADS  CAS  Google Scholar 

  30. Silkworth, W. T., Nardi, I. K., Paul, R., Mogilner, A. & Cimini, D. Timing of centrosome separation is important for accurate chromosome segregation. Mol. Biol. Cell 23, 401–411 (2012).

    Article  CAS  Google Scholar 

  31. Kaseda, K., McAinsh, A. D. & Cross, R. A. Dual pathway spindle assembly increases both the speed and the fidelity of mitosis. Biol. Open 1, 12–18 (2012).

    Article  Google Scholar 

  32. Zhang, Y. et al. USP44 regulates centrosome positioning to prevent aneuploidy and suppress tumorigenesis. J. Clin. Invest. 122, 4362–4374 (2012).

    Article  CAS  Google Scholar 

  33. Meitinger, F. et al. TRIM37 controls cancer-specific vulnerability to PLK4 inhibition. Nature https://doi.org/10.1038/s41586-020-2710-1 (2020).

  34. Di, Z. et al. Ultra high content image analysis and phenotype profiling of 3D cultured micro-tissues. PLoS ONE 9, e109688 (2014).

    Article  ADS  Google Scholar 

  35. Booij, T. H. et al. Development of a 3D tissue culture-based high-content screening platform that uses phenotypic profiling to discriminate selective inhibitors of receptor tyrosine kinases. J. Biomol. Screen. 21, 912–922 (2016).

    Article  CAS  Google Scholar 

  36. Sandercock, A. M. et al. Identification of anti-tumour biologics using primary tumour models, 3-D phenotypic screening and image-based multi-parametric profiling. Mol. Cancer 14, 147 (2015).

    Article  Google Scholar 

  37. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    Article  CAS  Google Scholar 

  38. Wang, L., Wang, S. & Li, W. RSeQC: quality control of RNA-seq experiments. Bioinformatics 28, 2184–2185 (2012).

    Article  CAS  Google Scholar 

  39. Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

    Article  CAS  Google Scholar 

  40. Firat-Karalar, E. N. & Stearns, T. in Methods in Cell Biology vol. 129 (eds Basto, R. & Oegema, K.) 153–170 (Academic, 2015).

  41. Mi, H. et al. PANTHER version 11: expanded annotation data from Gene Ontology and Reactome pathways, and data analysis tool enhancements. Nucleic Acids Res. 45, D183–D189 (2017).

    Article  CAS  Google Scholar 

  42. Mi, H., Muruganujan, A., Casagrande, J. T. & Thomas, P. D. Large-scale gene function analysis with the PANTHER classification system. Nat. Protocols 8, 1551–1566 (2013).

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by a Cancer Research UK Career Development Fellowship C52690/A19270 (to J.R.C.), and by National Institutes of Health grants R01GM114119 and R01GM133897, an American Cancer Society Scholar grant RSG-16-156-01-CCG, and an American Cancer Society Mission Boost Grant MBG-19-173-01-MBG (to A.J.H.). Z.Y.Y. is supported by the National Science Scholarship from A*STAR, Singapore. J.R.C. holds a Lister Institute Research Prize Fellowship. The Wellcome Centre for Human Genetics is supported by Wellcome grant 090532/Z/09/Z. The C.J.L. and A.N.J.T laboratories are funded by NC3Rs (NC/P001262/1), Breast Cancer Now funding to the Breast Cancer Now Toby Robins Research Centre (CTR-Q4-Y2), private donations to the ICR Development Office, and NHS funding to the NIHR Biomedical Research Centres at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London, and the Royal Marsden Hospital. We also thank R. Peat for breast cancer cell lines.

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Author notes

  1. These authors contributed equally: Zhong Y. Yeow, Bramwell G. Lambrus

Authors and Affiliations

  1. Medical Research Council (MRC) Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

    Zhong Y. Yeow, Mary-Anne Durin & J. Ross Chapman

  2. Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK

    Zhong Y. Yeow, Mary-Anne Durin, Daniela Moralli, Catherine M. Green & J. Ross Chapman

  3. Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Bramwell G. Lambrus, Kevin H. Zhan, Lauren T. Evans, Phillip M. Scott, Thao Phan, Elizabeth Park, Lorena A. Ruiz & Andrew J. Holland

  4. The Breast Cancer Now Toby Robins Breast Cancer Research Centre, The Institute of Cancer Research, London, UK

    Rebecca Marlow, Eleanor G. Knight, Daniela Novo, Syed Haider, Andrew N. J. Tutt & Christopher J. Lord

  5. The Breast Cancer Now Unit, King’s College London, London, UK

    Rebecca Marlow, Luned M. Badder & Andrew N. J. Tutt

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Contributions

Z.Y.Y. and B.G.L. designed, performed and analysed the majority of the experiments, and prepared the figures. M.-A.D., D.M. and C.M.G. assisted with associated cytogenetic experiments. K.H.Z. performed and analysed centrosomal intensity quantification experiments. L.T.E. generated the cell lines and analysed the movies for the data in Fig. 4b, c. P.M.S. created and performed the imaging of the DLD-1 cell line that expresses CEP192–mNeonGreen. T.P. analysed centrosome separation in 17q23-amplified and non-17q23-amplified cell lines. E.P. created the PLK4AS TP53−/− RPE-1 cells that express EB3–mNeonGreen, TUBG1–TagRFP and H2B–iRFP. L.A.R. analysed PCM foci in mitotic MDA-MB-436 and DLD-1 cells. R.M., E.G.K., L.M.B., D.N. and S.H. conducted and analysed 3D tumour cell line and PDO experiments, under the supervision of A.N.J.T. and C.J.L. J.R.C. and A.J.H. conceived and co-supervised the study, designed experiments, and analysed the data. Z.Y.Y., B.G.L., J.R.C. and A.J.H. co-wrote the manuscript.

Corresponding authors

Correspondence to J. Ross Chapman or Andrew J. Holland.

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Competing interests

C.J.L. received research funding from AstraZeneca, Merck KGaA and Artios; received consultancy, SAB membership or honoraria payments from Syncona, Sun Pharma, Gerson Lehrman Group, Merck KGaA, Vertex, AstraZeneca, Tango, 3rd Rock, Ono Pharma and Artios; and has stock in Tango and Ovibio. None of these is directly relevant to the work published and/or discussed in this Article. C.J.L. is also a named inventor on patents describing the use of DNA repair inhibitors and stands to gain from the development as part of the ICR Rewards to Inventors scheme. A.N.J.T received research funding from AstraZeneca and Merck KGaA that is not directly relevant to work published or discussed in this Article. A.N.J.T. also receives payments from the ICR Rewards to Inventors scheme associated with patents describing the use of DNA repair inhibitors and stands to gain from their development.

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Peer review information Nature thanks Renata Basto, Sarah McClelland 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 TRIM37 overexpression in HCT116 and RPE-1 cells recapitulates synthetic lethality with centrosome loss.

Related to Fig. 1. a, Top, centrosome number distribution in interphase MCF-7 cells at various times after addition of centrinone (PLK4i) (125 nM). Mean ± s.e.m. Bottom, representative images of centrosome staining (centrioles labelled by centrin, and PCM labelled by CEP192). n = 3 biological replicates, each comprising >100 cells. b, Representative data of a 14-day clonogenic survival assay of MCF-7 and RPE-1 cells with the indicated genotypes treated with DMSO (control) or centrinone (PLK4i) (125 nM). n = 3 biological replicates. c, Immunoblot showing TRIM37 protein levels in two WT MCF-7 clones stably expressing control vector or a TRIM37-targeting sgRNA. β-Actin, loading control. Representative data; n = 3 biological replicates. For gel source data, see Supplementary Fig. 1. d, Representative data of a 10-day clonogenic survival of indicated MCF-7 cell lines treated with DMSO (control) or centrinone (PLK4i) (125 nM). e, Quantification of n = 3 biological replicates in d. P values, unpaired two-tailed t-test. Mean ± s.e.m. f, Immunoblot of lysates prepared from WT and TP53−/− HCT116 cells expressing a control (eGFP) or TRIM37 transgene. MCF-7 cells were used as a reference for TRIM37 protein overexpression in a 17q23-amplified cell line. β-Actin, loading control. Representative data; n = 3 biological replicates. For gel source data, see Supplementary Fig. 1. g, Representative data of a 14-day clonogenic survival assay of HCT116 cells treated with DMSO (control) or centrinone (PLK4i) (125 nM). h, Quantification of n = 3 biological replicates in g. P values, unpaired two-tailed t-test. Mean ± s.e.m. i, Immunoblot showing doxycycline-induced GST or TRIM37 expression in PLK4AS TP53−/− RPE-1 cells. β-Actin, loading control. Representative data; n = 3 biological replicates. For gel source data, see Supplementary Fig. 1. j, Representative data of a 14-day colony survival assay of PLK4AS TP53−/− RPE-1 cells expressing doxycycline-inducible GST (control) or TRIM37 transgenes, treated with DMSO (control) or 3MB-PP1 (3MB). AS, analogue sensitive. k, Quantification of n = 3 biological replicates in j. P values, unpaired two-tailed t-test. Mean ± s.e.m. l, Representative images of PLK4AS TP53−/− RPE-1 cells in j. Scale bars, 100 μm.

Source data

Extended Data Fig. 2 Inhibitor selectivity for PLK4—and not other kinases—is required for the synthetic lethal killing of cells overexpressing TRIM37.

a, Representative data of a 10-day clonogenic survival of indicated MCF-7 cell lines treated with DMSO (control), centrinone, CFI-400945 or ZM447439. Data acquired in parallel to experiment in Fig. 1c, d. b, Quantification of a, n = 3 biological replicates. P values, unpaired two-tailed t-test. Mean ± s.e.m. c, Left, representative flow cytometric analysis of DNA content in MCF-7 cells treated with DMSO (control), centrinone, CFI-400945 or ZM447439 for 3 d. Right, quantification of the percentage of cells with >4N DNA content (polyploidy). n = 3 biological replicates. Mean ± s.e.m.

Source data

Extended Data Fig. 3 Additional characterization of TRIM37 expression and synthetic lethality in breast cancer cell lines and patient-derived organoids (PDOs).

Related to Fig. 1. a, Immunoblot showing TRIM37 protein levels in the indicated 17q23-amplified cell lines (MDA-MB-361, BT474 and MCF-7) and non-17q23-amplified cell lines (BT549, MDA-MB-231 and MDA-MB-436) expressing control or TRIM37-targeting shRNA. β-Actin, loading control. Representative data; n = 3 biological replicates. For gel source data, see Supplementary Fig. 1. b, Clonogenic survival of 17q23-amplified and non-17q23-amplified cell lines treated with DMSO (control) or centrinone (PLK4i) (125 nM). Representative data; n = 3 biological replicates. c, Images of DMSO- or PLK4i-treated MDA-MB-361 and BT474 cells expressing control vector or TRIM37-targeting shRNA. Scale bars, 200 μm. Representative data; n = 3 biological replicates. d, Left, Representative flow cytometric DNA content analysis in DMSO- or PLK4i-treated MDA-MB-361 and BT474 cells. Percentages of sub-G1 events are indicated. Right, percentage of sub-G1 cells across n = 3 biological replicates. P values, unpaired two-tailed t-test. Mean ± s.e.m. e, TRIM37 gene expression in PDOs. Gene expression is reported as a z-score derived from RNA-seq data sets across n = 22 independent biological samples. f, Viability of patient-derived breast tumour organoids following a 14-d exposure to the indicated concentrations of centrinone. Data from n = 2 biological replicates are shown. Mean ± s.e.m. g, Immunoblot showing TRIM37 protein levels in PDOs. β-Actin, loading control. Data from n = 1 biological replicate. For gel source data, see Supplementary Fig. 1. h, Viability of 3D cultures of the indicated cell lines following a 14-d exposure to the indicated concentrations of centrinone. Left, n = 2 biological replicates, Mean ± s.e.m. Right, n = 4 technical replicates, Mean ± s.e.m.

Source data

Extended Data Fig. 4 TRIM37 localizes to centrosomes, where it interacts with, and regulates the abundance of PCM proteins.

Related to Fig. 3. a, Immunoblot showing TRIM37 and biotinylated proximity interactors. Ponceau-stained blot indicates loading. Data are from a single experiment performed in duplicate. For gel source data, see Supplementary Fig. 1. b, Gene ontology analysis of mass spectrometry data. c, Thresholded mass spectrometry results displaying the top 30 proximity interactors by spectral count. Interactors were filtered to isolate those with >2 × more peptides in the mTurbo-TRIM37 sample compared to control. d, Left, immunofluorescence of TRIM37 in TRIM37+/+ and TRIM37−/− RPE-1 cells. Scale bars, 5 μm. Right, quantification of TRIM37 intensity at the centrosome in RPE-1 cells. n = 3 biological replicates, each comprising >40 cells. P values, unpaired two-tailed t-test. Mean ± s.e.m. e, Immunofluorescence of biotin-labelled proteins in mTurbo cell lines. Representative data; n = 3 biological replicates. Scale bars, 5 μm. f, Co-immunoprecipitation showing the interaction of TRIM37 with CEP192. Representative data; n = 3 biological replicates. For gel source data, see Supplementary Fig. 1. g, Immunoblot showing the levels of TRIM37 and PCM components in non-17q23-amplified versus 17q23-amplified cell lines. β-Actin, loading control. Representative data; n = 3 biological replicates. For gel source data, see Supplementary Fig. 1.

Source data

Extended Data Fig. 5 TRIM37 suppresses microtubule nucleation by the centrosome and supresses the formation of non-centrosomal PCM foci.

Related to Fig. 3. a, Microtubule regrowth following nocodazole washout in control vector- or TRIM37-shRNA-expressing MCF-7 mitotic cells. Representative images from b. n = 3 biological replicates. Scale bars, 5 μm. b, Quantification of microtubule regrowth following nocodazole washout in control vector- or TRIM37-shRNA-expressing MCF-7 mitotic cells. n = 3 biological replicates, each with >25 cells. P values, unpaired two-tailed t-test. Mean ± s.e.m. c, Quantification of centrosomal EB1 intensity following nocodazole washout in control vector- or TRIM37-shRNA-expressing MCF-7 mitotic cells. n = 3 biological replicates, each with >25 cells. P values, unpaired two-tailed t-test. Mean ± s.e.m. d, Representative images of mitotic PCM foci in acentrosomal RPE-1 cells described in Fig. 3d. n = 3 biological replicates. Scale bars, 5 μm. e, Left, representative images of mitotic PCM foci in acentrosomal MDA-MB-436 and DLD-1 cells. Scale bars, 5 μm. Right, quantification of mitotic PCM foci in centrinone-treated MDA-MB-436 and DLD-1 cells that lacked centrosomes. n ≥ 3 biological replicates, each comprising ≥ 84 cells for DLD-1 cells and ≥ 6 cells for MDA-MB-436 cells. Mean ± s.e.m. f, Quantification of CEP192 foci area in TRIM37+/+ versus TRIM37−/− RPE-1 cells in d. n = 3 biological replicates, each comprising >20 cells. P values, unpaired two-tailed t-test. Mean ± s.e.m. g, Representative images for spindle length analysis in indicated MCF-7 cells described in Fig. 3g. n = 3 biological replicates. Scale bars, 5 μm.

Source data

Extended Data Fig. 6 Non-centrosomal PCM foci nucleate microtubules and contribute to spindle assembly in DLD-1 cells.

Related to Fig. 3. a, Representative time-lapse images of mitosis in DMSO-treated control DLD-1 cells. n = 3 biological replicates. Scale bar, 5 μm. b, Representative time-lapse images of PCM foci formation during mitosis in acentrosomal DLD-1 cells. Scale bar, 5 μm. n = 3 biological replicates. Arrows indicate PCM foci. c, Representative time-lapse images of microtubule nucleation from centrosomes in the mitotic spindle of DMSO-treated control DLD-1 cells. n = 3 biological replicates. Scale bar, 5 μm. d, Representative time-lapse images of microtubule nucleation from PCM foci incorporated into the mitotic spindle of acentrosomal DLD-1 cells. n = 3 biological replicates. Scale bar, 5 μm. e, Representative time-lapse images of microtubule nucleation from a PCM focus before its incorporation into the mitotic spindle of the acentrosomal cell shown in d. n = 3 biological replicates. Scale bar, 1 μm.

Extended Data Fig. 7 Non-centrosomal PCM foci nucleate microtubules and contribute to spindle assembly in RPE-1 cells.

Related to Fig. 3. a, Representative time-lapse images of mitosis in DMSO-treated control PLK4AS TP53−/− RPE-1 cells. n = 3 biological replicates. Scale bar, 5 μm. b, Representative time-lapse images of PCM foci formation during mitosis in acentrosomal PLK4AS TP53−/− RPE-1 cells. n = 3 biological replicates. Scale bar, 5 μm. Arrows indicate PCM foci. c, Representative time-lapse images of microtubule nucleation from centrosomes in the mitotic spindle of DMSO-treated control PLK4AS TP53−/− RPE-1 cells. n = 3 biological replicates. Scale bars, 5 μm. d, Representative time-lapse images of microtubule nucleation from PCM foci incorporated into the mitotic spindle of acentrosomal PLK4AS TP53−/− RPE-1 cells. n = 3 biological replicates. Scale bar, 5 μm. e, Representative time-lapse images of microtubule nucleation from a PCM focus before its incorporation into the mitotic spindle in acentrosomal PLK4AS TP53−/− RPE-1. n = 3 biological replicates. Scale bar, 1 μm.

Extended Data Fig. 8 Depletion of CEP192 in RPE-1 cells recapitulates the synthetic lethal mitotic phenotypes observed in high-TRIM37 expressing cells.

Related to Fig. 3. a, Immunoblot showing the CEP192 levels in indicated control and CEP192-depleted PLK4AS TP53−/− RPE-1 cells. α-Tubulin, loading control. For gel source data, see Supplementary Fig. 1. b, Quantification of mitotic centrosomal CEP192 signal in the same cells as described in a. n = 3 biological replicates, each comprising >30 cells. P values, unpaired two-tailed t-test. Mean ± s.e.m. c, Representative images of centrosomal CEP192 in the same cells as described in a. Scale bars, 5 μm. d, Quantification of the percentage of 3MB-PP1 (3MB)-treated PLK4AS TP53−/− RPE-1 cells with acentrosomal mitotic CEP192 PCM foci. n = 3 biological replicates, each comprising >30 cells. P values, unpaired two-tailed t-test. Mean ± s.e.m. e, Representative time-lapse images of mitotic progression in DMSO- or 3MB-PP1 (3MB)-treated control and CEP192-depleted PLK4AS TP53−/− RPE-1 cells. Cells are labelled with H2B-iRFP and tagRFP-tubulin. n = 3 biological replicates.

Source data

Extended Data Fig. 9 Cell cycle regulation of TRIM37 expression.

Related to Fig. 4. a, Schematic of the experimental protocol used for cell cycle synchronization. Samples were subjected to dual flow cytometry staining of phospho-histone Ser10 (pH3) to mark mitotic cells and propidium iodide (PI) to determine synchronization efficiency. M, mitotic phase. b, Flow cytometric DNA content analysis of samples collected according to a. Left, RPE-1. Right, MCF-7. Async, asynchronous. c, Mitotic index of cell cycle samples as determined by the percentage of pH3-positive cells with 4N DNA content. n = 3 biological replicates. Mean ± s.e.m. d, Immunoblot showing endogenous TRIM37, cyclin A and pH3 in samples analysed in b. β-Actin, loading control. For gel source data, see Supplementary Fig. 1. e, RT-qPCR analysis indicating relative TRIM37 mRNA expression in RPE-1 cells analysed in b. Data were normalized to TRIM37 mRNA expression in asynchronous cells. n = 3 biological replicates. Mean ± s.e.m.

Source data

Extended Data Fig. 10 TRIM37 overexpression delays centrosome maturation in G2/M phase.

Related to Fig. 4. a, Quantification of centrosomal α-tubulin intensity from time-lapse movies of dividing MCF-7 cells expressing either control vector or TRIM37-targeting shRNA. Quantification of >20 cells. Mean ± s.e.m. b, Quantification of the distance between the two centrosomes at NEBD in MCF-7 cells. n = 3 biological replicates, each comprising >12 cells. P values, unpaired two-tailed t-test. Mean ± s.e.m. c, Quantification of centrosomal α-tubulin intensity from time-lapse movies of dividing RPE-1 tet-on TRIM37 cells. Quantification of >20 cells. Mean ± s.e.m. d, Representative time-lapse images of centrosome maturation in MCF-7 cells. n = 3 biological replicates. Scale bars, 5 μm. e, Representative time-lapse images of centrosome maturation in RPE-1 tet-on TRIM37 cells. n = 3 biological replicates. Scale bars, 5 μm.

Source data

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-3. Supplementary Figure 1 contains the uncropped gel blots. Supplementary Figure 2 shows validation of RPE-1 knockout cell lines. Immunoblot showing the levels of USP28, 53BP1 and p53 in RPE-1 cells with indicated genotypes. β-Actin, loading control. Data is representative of n = 3, biological replicates. Supplementary Figure 3 contains the flow cytometry gating strategies for ploidy, sub-G1 and cell cycle analyses. Data is representative of n = 3, biological replicates.

Reporting Summary

Supplementary Data 1

Primary mass-spectrometry experimental data.

Video 1

DMSO-treated MCF-7; TP53-/- control cell undergoing normal division, as shown in Figure 2d. H2B-iRFP in red, and tagRFP-tubulin in green. Images are taken in 3 µm sections; one stack was imaged every 5 minutes; displayed at 8 frames per second. Data is representative of n = 3, biological replicates.

Video 2

PLK4i-treated MCF-7; TP53-/- control cell undergoing mitotic slippage, as shown in Figure 2d. H2B-iRFP in red, and tagRFP-tubulin in green. Images are taken in 3 µm sections; one stack was imaged every 5 minutes; displayed at 8 frames per second. Data is representative of n = 3, biological replicates.

Video 3

DMSO-treated MCF-7; TP53-/- shTRIM37 cell undergoing normal division, as shown in Figure 2d. H2B-iRFP in red, and TagRFP-tubulin in green. Images are taken in 3 µm sections; one stack was imaged every 5 minutes; displayed at 8 frames per second. Data is representative of n = 3, biological replicates.

Video 4

PLK4i-treated MCF-7; TP53-/- shTRIM37 cell undergoing normal division, as shown in Figure 2d. H2B-iRFP in red, and TagRFP-tubulin in green. Images are taken in 3 µm sections; one stack was imaged every 5 minutes; displayed at 8 frames per second. Data is representative of n = 3, biological replicates.

Video 5

DMSO-treated DLD-1; CEP192-mNeonGreen control cell undergoing normal division, as shown in Extended Data Fig. 6a. CEP192-mNeonGreen in green, EB1-TagRFP in red, and H2B-iRFP in magenta. Images are taken in 1 µm sections; one stack was imaged every 5 minutes; displayed at 5 frames per second. Data is representative of n = 3, biological replicates.

Video 6

PLK4i-treated DLD-1 CEP192-mNeonGreen cell undergoing division in the absence of centrioles, as shown in Extended Data Fig. 6b. CEP192-mNeonGreen in green, EB1-TagRFP in red, and H2B-iRFP in magenta. Images are taken in 1 µm sections; one stack was imaged every 5 minutes; displayed at 5 frames per second. Data is representative of n = 3, biological replicates.

Video 7

Microtubule nucleation from centrosomes in a DMSO-treated DLD-1; CEP192-mNeonGreen control cell, as shown in Extended Data Fig. 6c. CEP192-mNeonGreen in green, and EB1-TagRFP in black. Images were taken every 2 seconds; displayed at 5 frames per second. Data is representative of n = 3, biological replicates.

Video 8

Microtubule nucleation from acentrosomal PCM foci in a PLK4i-treated DLD-1; CEP192-mNeonGreen cell, as shown in Extended Data Fig. 6d. CEP192-mNeonGreen in green, and EB1-TagRFP in black. Images were taken every 2 seconds; displayed at 5 frames per second. Data is representative of n = 3, biological replicates.

Video 9

Microtubule nucleation from an acentrosomal PCM focus that has no yet incorporated into the mitotic spindle in a PLK4i-treated DLD-1; CEP192-mNeonGreen cell, as shown in Extended Data Fig. 6e. CEP192-mNeonGreen in green, and EB1-TagRFP in black. Images were taken every 2 seconds; displayed at 5 frames per second. Data is representative of n = 3, biological replicates.

Video 10

DMSO-treated RPE-1; PLK4AS; TP53-/- control cell undergoing normal division, as shown in Extended Data Fig. 7a. EB3-mNeonGreen in green, TUBG1-TagRFP in red, and H2B-iRFP in magenta. Images were taken in 1 µm sections; one stack was imaged every 5 minutes; displayed at 5 frames per second. Data is representative of n = 3, biological replicates.

Video 11

3MB-PP1-treated RPE-1; PLK4AS; TP53-/- cell undergoing division in the absence of centrioles, as shown in Extended Data Fig. 7b. EB3-mNeonGreen in green, TUBG1-TagRFP in red, and H2B-iRFP in magenta. Images were taken in 1 µm sections; one stack was imaged every 5 minutes; displayed at 5 frames per second. Data is representative of n = 3, biological replicates.

Video 12

Microtubule nucleation from centrosomes in a DMSO-treated RPE-1; PLK4AS; TP53-/- control cell, as shown in Extended Data Fig. 7c. TUBG1-TagRFP in green, and EB3-mNeonGreen in black. Images were taken every 2 seconds; displayed at 5 frames per second. Data is representative of n = 3, biological replicates.

Video 13

Microtubule nucleation from acentrosomal PCM foci in a 3MB-PP1-treated RPE-1; PLK4AS; TP53-/- cell, as shown in Extended Data Fig. 7d. TUBG1-TagRFP in green, and EB3-mNeonGreen in black. Images were taken every 2 seconds; displayed at 5 frames per second. Data is representative of n = 3, biological replicates.

Video 14

Microtubule nucleation from an acentrosomal PCM focus that had not yet incorporated into the mitotic spindle in a 3MB-PP1-treated RPE-1; PLK4AS; TP53-/- cell, as shown in Extended Data Fig. 7e. TUBG1-TagRFP in green, and EB3-mNeonGreen in black. Images are taken every 2 seconds; displayed at 5 frames per second. Data is representative of n = 3, biological replicates.

Video 15

DMSO-treated RPE-1; PLK4AS; TP53-/- control cell undergoing normal division, as quantified in Figure 3j and shown in Extended Data Fig. 8e. H2B-iRFP in red, and EGFP-tubulin in green. Images are taken in 3 µm sections; one stack was imaged every 5 minutes; displayed at 8 frames per second. Data is representative of n = 3, biological replicates.

Video 16

3MB-PP1-treated RPE-1; PLK4AS; TP53-/- control cell undergoing normal division, as quantified in Figure 3j and shown in Extended Data Fig. 8e. H2B-iRFP in red, and EGFP-tubulin in green. Images are taken in 3 µm sections; one stack was imaged every 5 minutes; displayed at 8 frames per second. Data is representative of n = 3, biological replicates.

Video 17

DMSO-treated RPE-1; PLK4AS; TP53-/- shCEP192 cell undergoing normal division, as quantified in Figure 3j and shown in Extended Data Fig. 8e. H2B-iRFP in red, and EGFP-tubulin in green. Images are taken in 3 µm sections; one stack was imaged every 5 minutes; displayed at 8 frames per second. Data is representative of n = 3, biological replicates.

Video 18

3MB-PP1-treated RPE-1; PLK4AS; TP53-/- shCEP192 cell undergoing mitotic slippage, as quantified in Figure 3j and shown in Extended Data Fig. 8e. H2B-iRFP in red, and EGFP-tubulin in green. Images are taken in 3 µm sections; one stack was imaged every 5 minutes; displayed at 8 frames per second. Data is representative of n = 3, biological replicates.

Video 19

MCF-7 control cell undergoing normal division, as shown in Extended Data Fig. 10d. H2B-iRFP in red, and EGFP-tubulin in green. Images are taken in 3 µm sections; one stack was imaged every 5 minutes; displayed at 8 frames per second. Data is representative of n = 3, biological replicates.

Video 20

MCF-7 shTRIM37 cell undergoing normal division, as shown in Extended Data Fig. 10d. H2B-iRFP in red, and EGFP-tubulin in green. Images are taken in 3 µm sections; one stack was imaged every 5 minutes; displayed at 8 frames per second. Data is representative of n = 3, biological replicates.

Video 21

RPE-1; TP53-/-; tet-on TRIM37 dox(-) cell undergoing normal division, as shown in Extended Data Fig. 10e. H2B-iRFP in red, and EGFP-tubulin in green. Images are taken in 3 µm sections; one stack was imaged every 5 minutes; displayed at 8 frames per second. Data is representative of n = 3, biological replicates.

Video 22

RPE-1; TP53-/-; tet-on TRIM37 dox(+) cell undergoing delayed centrosome maturation and mitotic slippage, as shown in Extended Data Fig. 10e. H2B-iRFP in red, and EGFP-tubulin in green. Images are taken in 3 µm sections; one stack was imaged every 5 minutes; displayed at 8 frames per second. Data is representative of n = 3, biological replicates.

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Yeow, Z.Y., Lambrus, B.G., Marlow, R. et al. Targeting TRIM37-driven centrosome dysfunction in 17q23-amplified breast cancer. Nature 585, 447–452 (2020). https://doi.org/10.1038/s41586-020-2690-1

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