Abstract

BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. To understand the resistance mechanisms, we conducted whole-genome CRISPR–Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. We identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show that C20orf196 and FAM35A form a complex, ‘Shieldin’ (SHLD1/2), with FAM35A interacting with single-stranded DNA through its C-terminal oligonucleotide/oligosaccharide-binding fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining by restricting DSB resection and to counteract homologous recombination by antagonizing BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitizes BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance.

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Acknowledgements

The authors thank all S.P.J. laboratory members for support and advice, and Cambridge colleagues N. Lawrence for OMX super-resolution microscopy support and R. Butler for help with computational image analyses and programming. The authors also thank S. Selivanova and S. Hough for help with plasmid amplification, sample preparation and tissue culture maintenance, K. Dry for extensive editorial assistance, F. Muñoz-Martinez for assistance with CRISPR–Cas9 knockout generation, L. Radu for assistance with protein purification, C. Lord (Institute of Cancer Research, London) for SUM149PT cells, D. Durocher (University of Toronto, Canada) for U2OS LacSceIII cells, F. Alt (Harvard University, USA) for CH12F3 cells and 53bp1 knockout CH12F3 cell clones, T. Honjo (Kyoto University, Japan) for permission to use the CH12F3 cell line, and J. Serrat in the Jacobs lab for technical assistance. The SPJ lab is largely funded by a Cancer Research UK (CRUK) Program Grant, C6/A18796, and a Wellcome Trust (WT) Investigator Award, 206388/Z/17/Z. Core infrastructure funding was provided by CRUK grant C6946/A24843 and WT grant WT203144. S.P.J. receives a salary from the University of Cambridge. H.D. is funded by WT Clinical Fellowship 206721/Z/17/Z. TWC was supported by a Cambridge International Scholarship. D.P. is funded by Cancer Research UK studentship C6/A21454. The P.B. lab is supported by the Emmy Noether Program (BE 5342/1-1) from the German Research Foundation and a Marie Curie Career Integration Grant from the European Commission (630763). The L.P. lab is funded by the WT (investigator award 104641/Z/14/Z) and the Medical Research Council (project grant MR/N000161/1). The C.C. lab was supported with funding from CRUK. The J.J. lab was supported by the European Research Council grant ERC-StG 311565, The Dutch Cancer Society (KWF) grant KWF 10999, and the Netherlands Organization for Scientific Research (NWO) as part of the National Roadmap Large-scale Research Facilities of the Netherlands, Proteins@Work (project no. 184.032.201 to the Proteomics Facility of the Netherlands Cancer Institute). The L.D. lab is funded by the Institut Pasteur, the Institut National du Cancer (no. PLBIO16-181) and the European Research Council (starting grant agreement no. 310917). W.W. is part of the Pasteur–Paris University (PPU) International PhD program and this project received funding from the CNBG company, China. Q.W. is funded by the Wellcome Trust (200814/Z/16/Z ). The V.S. lab work was funded by the Instituto de Salud Carlos III (ISCIII), an initiative of the Spanish Ministry of Economy and Innovation partially supported by European Regional Development FEDER Funds (PI17-01080 to VS), the European Research Area-NET, Transcan-2 (AC15/00063), a non-commercial research agreement with AstraZeneca UK, and structural funds from the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR, 2017 SGR 540) and the Orozco Family. V.S. received a salary and travel support to C.C.’s lab from ISCIII (CP14/00228, MV15/00041) and the FERO Foundation.

Author information

Author notes

  1. These authors contributed equally: Ting-Wei Will Chiang, Chloe Lescale, Inge de Krijger.

Affiliations

  1. The Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK

    • Harveer Dev
    • , Ting-Wei Will Chiang
    • , Domenic Pilger
    • , Julia Coates
    • , Matylda Sczaniecka-Clift
    • , Mareike Herzog
    • , Jonathan Lam
    • , Mukerrem Demir
    • , Gabriel Balmus
    • , Rimma Belotserkovskaya
    • , Yaron Galanty
    •  & Stephen P. Jackson
  2. Academic Urology Group, Department of Surgery, Cambridge University Hospitals NHS Foundation Trust, Addenbrooke’s Hospital, Cambridge, UK

    • Harveer Dev
  3. Genome Integrity, Immunity and Cancer Unit, Department of Immunology, Department of Genomes and Genetics, Institut Pasteur, Paris, France

    • Chloe Lescale
    • , Wenming Wei
    •  & Ludovic Deriano
  4. Division of Oncogenomics, The Netherlands Cancer Institute, Plesmanlaan , Amsterdam, the Netherlands

    • Inge de Krijger
    •  & Jacqueline J. L. Jacobs
  5. Department of Oncology and Cancer Research UK Cambridge Institute, Li Ka Shing Centre, University of Cambridge, Cambridge, UK

    • Alistair G. Martin
    • , Abigail Shea
    • , Alejandra Bruna
    •  & Carlos Caldas
  6. Institute of Molecular Biology (IMB), Mainz, Germany

    • Matthias Ostermaier
    •  & Petra Beli
  7. Department of Biochemistry, University of Cambridge, Cambridge, UK

    • Qian Wu
    •  & Luca Pellegrini
  8. Wellcome Trust Sanger Institute, Hinxton, UK

    • Fengtang Yang
    • , Beiyuan Fu
    •  & Gabriel Balmus
  9. AstraZeneca, Waltham, MA, USA

    • Zhongwu Lai
  10. Vall d’Hebron Institute of Oncology, Barcelona, Spain

    • Violeta Serra
  11. AstraZeneca, Cambridge, UK

    • Mark J. O’Connor

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Contributions

T.W.C. and S.P.J. conceived the project and T.W.C. initiated the project by performing the CRISPR–Cas9 screens, with M.H. doing the bioinformatic analyses. L.D. supervised, and C.L. and W.W. performed CSR and Igh locus instability experiments. J.J. supervised, and I.K. performed recombinant MAD2L2 co-IPs, MAD2L2 IRIF/IB, pS4/8-RPA2 IB and telomere fusion experiments. M.O. and P.B. performed mass spectrometry. J.C. performed and analysed clonogenic survival experiments and random plasmid integration assay. M.D. and M.S. generated human knockout cell lines and M.S. performed in vitro pulldown experiments. J.L. carried out oligonucleotide interaction studies. D.P. performed end-resection assays. T.W.C. and R.B. generated RPE1 p53ko, RPE1 p53ko/BRCAko and p53ko/BRCA1ko/53BP1ko cell lines. Y.G. and S.P.J. supervised the above. G.B. performed IR survivals in mouse cells, mouse sgRNA cloning, and FISH with F.Y. and B.F. L.P. performed structural analysis and FAM35A modelling. Q.W. performed purification of bacterially expressed recombinant FAM35A proteins and EMSA. A.M., A.S., A.B. and C.C. performed patient-derived xenograft experiments on PARPi-induced resistance. V.S., M.O’C. and Z.L. established, performed, analysed and characterized PDXs in the experiments on intrinsic PARPi resistance. H.D. assisted with many of the above, and devised and performed all other experiments. H.D., Y.G. and S.P.J. wrote the manuscript with input from all others. L.D., J.J., Y.G. and S.P.J. supervised the project.

Competing interests

S.P.J. receives some research funding from AstraZeneca and is a named inventor on patents describing the use of PARP inhibitors in cancer therapy. V.S.’s laboratory receives research funding support from AstraZeneca. M.O. and Z.L. are employees and shareholders of AstraZeneca.

Corresponding authors

Correspondence to Ludovic Deriano or Jacqueline J. L. Jacobs or Yaron Galanty or Stephen P. Jackson.

Integrated supplementary information

  1. Supplementary Figure 1 Whole Genome CRISPR screen data and validation studies.

    a, Schematic of the one vector lentiviral GeCKOv2 system. b, Histograms of sgRNA representation of GeCKOv2 (GKv2) library A (left panel) and B (right panel). Inset: cumulative distribution of sequencing reads. The number of sequencing reads for the 10th and 90th sgRNA percentiles are indicated by the dashed vertical blue lines and text labels. The representation of sgRNAs is indicated by the fold-difference between the 10th and 90th percentile. c, Representative surviving clones after treatment with PARP inhibitors, representative of 2 independent experiments. Top panel: naïve, un-transduced SUM149PT cells; bottom panel: GeCKOv2 library-transduced cells. d, Distributions of sgRNA frequencies arising in different conditions; Box and whisker plot with centre line at median, box limits at 25th/75th centiles and whiskers ±1.5xIQR; n=3 technical replicates. e, sgRNA enrichments after treatments with the indicated drugs; each dot represents one sgRNA, with coloured dots representing the indicated target genes. f, Verification of BRCA1 mutant SUM149PT, BRCA1ko RPE1 and siRNAs and shRNAs used in this paper, by immunoblot or RT-qPCR (bars represent means; one experiment performed in triplicate). g, Clonogenic survival assay using the indicated siRNAs in BRCA1-proficient cells (WT); lower panel shows AUC. Bars represent mean ± SEM, one-way Anova; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant (p≥0.05); n=3 independent experiments, with individual data points plotted over bars; statistical source data including the precise p values can be found in Supplementary Table 5. h, Genotypes of human knockout clones used in this work confirmed by Topo-cloning and Sanger sequencing. i, Cell cycle profiles of cells transfected with the indicated siRNAs used in this work (bars represent means derived from two independent experiments). All immunoblots are representative of two independent experiments; unprocessed scans of immunoblots are shown in Supplementary Fig 8.

  2. Supplementary Figure 2 C20orf196/FAM35A interactions and localisation to DNA damage sites.

    a, Co-localisation quantification of FAM35A/derivatives GFP-fusions with mCherry-LacR-C20orf196. Horizontal bars represent means, one-way Anova; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant (p≥0.05); n=3 independent experiments; statistical source data including the precise p values can be found in Supplementary Table 5. b, C-terminus of FAM35A interacts with C20orf196 in cells (without normalisation). c, Immunoprecipitation-mass spectrometric analysis of protein interactors of GFP-C20orf196 or GFP-FAM35A (MAD2L2 is detected in both; far left and middle left panels). GFP-C20orf196 co-immunoprecipitates with Flag-MAD2L2 in HEK293 cells (middle right IB panel). Endogenous MAD2L2 co-immunoprecipitates with GFP-FAM35A and GFP-C20orf196 (far right IB panel) in HEK293 cells. d, Live-cell imaging of GFP-FAM35A or GFP-C20orf196 transiently expressed in U2OS cells stably expressing RFP-53BP1. Recruitment of GFP-FAM35A and GFP-C20orf196 to laser tracks was visible 30 min after laser micro-irradiation; representative image from 3 independent experiments. e, GFP-FAM35A and GFP-FAM35A N-terminus co-localise with 53BP1 in IRIF by super-resolution microscopy; histogram of n=11 cells per condition. f, Depletion of FAM35A or C20orf196 does not affect 53BP1 IRIF (U2OS cells). g, as in f but for MAD2L2 IRIF. Bars represent means derived from 2 independent experiments, with individual data points plotted over bars. h, Depletion of FAM35A or C20orf196 does not affect MAD2L2 protein levels. All immunoblots are representative of two independent experiments; unprocessed scans of immunoblots are shown in Supplementary Fig 8.

  3. Supplementary Figure 3 DNA damage response and IRIF factor dependencies of FAM35A and C20orf196.

    a, Minimal variation of doxycycline induced GFP-FAM35A (U2OS) and GFP-C20orf196 (RPE1) in cells treated with the indicated siRNAs. Immunoblots shown are representative of two independent experiments with unprocessed scans of immunoblots in Supplementary Fig 8. b, Representative images of GFP-FAM35A (left panel) and GFP-C20orf196 (right panel) IRIF in γH2AX positive cells quantified in Fig 2e. Scale bar 10µm. c, Depletion of PTIP does not affect GFP-C20orf196 or GFP-FAM35A IRIF. Bars represent mean ± SEM, one-way Anova; ns=not significant (p≥0.05); n=3 independent experiments, with individual data points plotted over bars; statistical source data can be found in Supplementary Table 5. Scale bar 10µm. d, Camptothecin induced GFP-FAM35A foci. Scale bar 10µm. e, Representative images of GFP-FAM35A derivatives with/without pre-extraction ±IR; d-e representative of 2 independent experiments. Scale bar 10µm. f, Representative images of GFP-FAM35A N-terminus IRIF dependencies quantified in Fig 2f. Scale bar 10µm.

  4. Supplementary Figure 4 FAM35A and C20orf196 directly affect class switch recombination.

    a, Clonogenic survival assay following IR treatment using wild-type, Fam35ako or C20orf196ko mouse ES cells (right panel shows AUC). Bars represent means ± SEM, one-way Anova; n=3 independent experiments, with individual data points plotted over bars. b, Genotypes of CH12-Cas9 cell knockout clones used in CSR assays confirmed by Topo-cloning and Sanger sequencing. c, Flow cytometry profiles showing the percentage of IgA+ cells for indicated CH12-Cas9 cell clones (genotypes) after 3 days stimulation with anti-CD40, IL-4 and TGF-β. Cell clone numbers are indicated; representative of 3 independent experiments. d, CSR assay in C20orf196ko cells complemented with C20orf196. Bars represent means ± SEM, one-way Anova; n=3 independent experiments, with individual data points plotted over bars. e, CH12-Cas9 clones were plated at 50,000 cells/ml and counted after 3 days stimulation with anti-CD40, IL4, and TGF-β. Bars represent means ± SEM, one-way Anova; n=3 independent experiments, with individual data points plotted over bars. For a, d and e, *p<0.05, ***p<0.001, ****p<0.0001, ns=not significant (p≥0.05); statistical source data including the precise p values can be found in Supplementary Table 5. f, Igh, α germ-line transcripts (αGLT) and Aid mRNA were quantified by semi-quantitative RT–PCR using 2.5-fold serial dilutions of cDNA made from CH12-Cas9 cells and indicated CH12-Cas9 knockout cell clones after 2 days stimulation with anti-CD40, IL4, and TGF-β. Hprt was used as a control for transcript expression. Immunoblots are representative of two independent experiments with unprocessed scans of immunoblots in Supplementary Fig 8.

  5. Supplementary Figure 5 Effects of FAM35A and C20orf196 on telomere fusions, DNA binding and DNA-end resection.

    a, Telomere fusion assay as shown in Fig 4b but complemented with shRNA resistant human C20orf196. Bars represent means derived from 2 independent experiments with ≥1300 chromosomes counted per condition, and individual data points plotted over bars; source data can be found in Supplementary Table 5. b, qRT-PCR of mouse (left) and human (right) transcripts in MEFs. Bars represent means from one experiment performed in triplicate. c, FRAP of GFP-RPA1 in stably expressing U2OS cells, depleted of FAM35A or C20orf196. Points represent mean ± 95% confidence intervals; residence time calculated as previously described50; n=28 independent experiments (siCTRL), n=22 (siFAM35A) and n=30 (siC20orf196). d, Structure of yeast RPA1 (yRPA1) with ssDNA. e, Coomassie stained SDS-PAGE gel showing the bacterial purified FAM35A variants used in EMSAs. Immunoblots are representative of two independent experiments with unprocessed scans of immunoblots in Supplementary Fig 8. f, GFP-FAM35A W489/W640A is able to interact with mCherry-LacR-C20orf196 in cells; representative of two independent experiments, scale bar 10µm. g, Overexpression of FAM35A or derivatives does not sensitise wild-type cells to olaparib, adjacent panel shows AUC. Bars represent means from one experiment performed in triplicate.

  6. Supplementary Figure 6 FAM35A and C20orf196 functions relating to homologous recombination.

    a, Representative images for quantifications of GFP-FAM35A presented in Fig 6a; scale bar 10µm. b, Representative images for quantifications presented in Fig 6b; scale bar 10µm. c, Representative images of FAM35A and C20orf196 effects on DNA-end resection in wild-type and BRCA1ko cells as measured by RPA nuclear intensity (after pre-extraction) following camptothecin treatment in the indicated genotypes, quantified in Supplementary Fig 6d; scale bar 10µm. d, Quantification of nuclear RPA intensity; n=5 independent experiments, except WT siAbraxas and BRCA1ko siCtIP (n=2) and BRCA1ko/FAM35Ako (n=4), with individual data points plotted over bars. e, Quantification of BRCA2 accrual at laser micro-irradiated RPE1 cells with the indicated genotypes for the representative images presented in Fig 6d. n=3 independent experiments, with individual data points plotted over bars. f, Gating strategy employed for TLR assay. g, AUC for clonogenic survival assay presented in Fig 6g. N=4 independent experiments, except BRCA1ko/FAM35Ako +FAM35A and +N-terminus where n=3, and +C-terminus where n=2; with individual data points plotted over bars. h, FAM35A and 53BP1 effects on olaparib resistance in BRCA1ko cells are not additive as measured by clonogenic survival assay (left panel), AUC (right panel). N=4 independent experiments, except BRCA1ko/53BP1ko/FAM35Ako where n=2; with individual data points plotted over bars. In d, e, g and h, bars represent mean ± SEM, one-way Anova; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant (p≥0.05); statistical source data including the precise p values can be found in Supplementary Table 5.

  7. Supplementary Figure 7 Tumour growth curves in mice and cell sensitivities of SHLD mutant cells to DNA damaging agents.

    a, Tumour growth curves of PDX mice cohorts treated with vehicle or olaparib in Fig 7a; points are means, with lines representing s.d. for each of cohorts 1-4. b, AUC for clonogenic survival assay presented in Fig 7c. N=3 independent experiments except BRCA1ko and C20orf196ko where n=4, and BRCA1ko/FAM35Ako where n=2. c, AUC for clonogenic survival assay presented in Fig 7d. N=3 independent experiments except WT where n=6 and BRCA1ko/FAM35Ako where n=2. b-c Bars represent mean ± SEM, one-way Anova; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant (p≥0.05). Individual data points plotted over bars; statistical source data including the precise p values can be found in Supplementary Table 5. d, GFP-FAM35A foci are not affected by depletion of FANCD2; representative images (left panel) and quantification (right panel). Bars represent means from 2 independent experiments, with individual data points plotted over bars. Scale bar 10µm. e, Graphical summary of SHLD2FAM35A domains and their function.

  8. Supplementary Figure 8

    Uncropped blots

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https://doi.org/10.1038/s41556-018-0140-1