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The cellular response to DNA double-strand breaks (DSB) is principally the result of two competing repair pathways. The Tumour Protein P53 Binding Protein 1 (TP53BP1) gene product plays a central role in DSB repair pathway choice, promoting non-homologous end-joining (NHEJ) and counteracting homologous recombination (HR). As with all DNA damage response (DDR) proteins, cell-cycle regulation of 53BP1 is critical to its function. The inappropriate activity of 53BP1 in mitosis, during which most of the DDR is inactivated to preserve genome stability, has been shown to have adverse effects on cell division that are characteristic of cancers. We explored the consequences of replication stress, associated with oncogene activity, and the impact of aberrantly active 53BP1 in mitosis. This work reveals interference with normal mitotic mechanisms for detecting and resolving stalled replication intermediates; most evidently manifest as reduced mitotic DNA synthesis (MiDAS), and increased accumulation of acentric micronuclei and G1 nuclear bodies. We propose that the functional sequestration of TOPBP1 may be responsible for these chromosomal mis-segregation defects. The mechanism by which 53BP1 (together with RIF1 and MAD2L2) promote NHEJ remains elusive. Genome instability in HR-defective cancers, principally those harbouring a mutation in BRCA1, can be rescued by the concomitant loss of either 53BP1, RIF1 or MAD2L2. This phenomenon has relevance for the development of PARP (Poly(ADP-ribose) polymerase) inhibitor resistance, now being used in BRCA1-deficient breast, ovarian and prostate cancers based on the principle of ‘synthetic lethality’. As part of a search for other resistance mechanisms, we identified the shieldin complex (SHLD1/C20orf196 and SHLD2/FAM35A) that when lost suppressed PARP inhibitor sensitivity in BRCA1-deficient cancer cells. We demonstrate that SHLD1/2 represent the terminal end-effectors of the 53BP1-RIF1-MAD2L2 axis, supporting NHEJ activity. We identified three OB-fold domains in SHLD2/FAM35A, that bind single-stranded DNA and are necessary for NHEJ. Loss of SHLD1 or SHLD2 (SHLD1/2) caused a hyper-resection phenotype, and which, in the absence of BRCA1 was associated with rescuing RAD51 loading. Downregulation in SHLD1/2 expression was also associated with intrinsic and acquired resistance across a panel of BRCA1-deficient patient-derived xenograft models. Promisingly, this mechanism may impart an acquired vulnerability to cisplatin or radiotherapy. Thus, we have characterised a critical component of the 53BP1 axis which is deterministic in repair pathway choice and carries implications for the treatment of cancers., Wellcome Trust Clinical Fellowship 206721/Z/17/Z. Royal College of Surgeons US-UK Fulbright Scholarship 2015.

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.

International audience; 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.

Objectives Evidence of the accuracy of predictive tests in confirming the presence and grade of upper urinary tract urothelial carcinomas (UUTUC) is limited. We present the largest series evaluating the diagnostic value of pre- and intra-operative parameters in the detection of UUTUC. Materials and methods We retrospectively analysed records of patients who underwent diagnostic ureteroscopy between 2005 and 2014 for suspected UUTUC. Pre-operative workup included voided urine cytology and CT imaging. Intra-operative assessments involved ureteroscopy to directly visualise suspicious lesions, and where possible selective cytology and biopsy. Primary outcomes were the visualisation of UUTUC and histopathological confirmation of tumour. Results Hundred out of 160 (63 %) patients presenting with suspected upper tract malignancy had UUTUC. Voided and selective urine cytology and CT individually predicted UUTUC with a sensitivity/specificity of 63/67, 76/73, and 95/26 %, respectively. Forty out of 48 (83 %) patients who had abnormal CT and abnormal voided urine cytology had UUTUC, while 100 % of those with normal CT and normal voided cytology (investigated for ongoing symptoms) were normal. Comparing endoscopic biopsy to nephroureterectomy specimen grade, 19 (46 %), 18 (44 %), and 4 (10 %) were identical, upgraded, and downgraded, respectively. Conclusion Pre-operative investigations can predict UUTUCs. When these investigations were normal, the risk of UUTUC is negligible. In selective patients with abnormal investigations, ureteroscopy should be performed to confirm and predict the grade of UUTUC, in order to guide future management. Selective cytology is unlikely to significantly contribute to the diagnostic workup of UUTUC. Electronic supplementary material The online version of this article (doi:10.1007/s00345-016-1820-8) contains supplementary material, which is available to authorized users.