In a search for mediators of the p53 tumor suppressor pathway, which induces pleiotropic and often antagonistic cellular responses, we identified the long noncoding RNA (lncRNA) NEAT1. NEAT1 is an essential architectural component of paraspeckle nuclear bodies, whose pathophysiological relevance remains unclear. Activation of p53, pharmacologically or by oncogene-induced replication stress, stimulated the formation of paraspeckles in mouse and human cells. Silencing Neat1 expression in mice, which prevents paraspeckle formation, sensitized preneoplastic cells to DNA-damage-induced cell death and impaired skin tumorigenesis. We provide mechanistic evidence that NEAT1 promotes ATR signaling in response to replication stress and is thereby engaged in a negative feedback loop that attenuates oncogene-dependent activation of p53. NEAT1 targeting in established human cancer cell lines induced synthetic lethality with genotoxic chemotherapeutics, including PARP inhibitors, and nongenotoxic activation of p53. This study establishes a key genetic link between NEAT1 paraspeckles, p53 biology and tumorigenesis and identifies NEAT1 as a promising target to enhance sensitivity of cancer cells to both chemotherapy and p53 reactivation therapy.

DNA-dependent protein kinase (DNA-PK) is a critical player in the DNA damage response (DDR) and instrumental in the non-homologous end-joining pathway (NHEJ) used to detect and repair DNA double-strand breaks (DSBs). We demonstrate that the potent and highly selective DNA-PK inhibitor, AZD7648, is an efficient sensitizer of radiation- and doxorubicin-induced DNA damage, with combinations in xenograft and patient-derived xenograft (PDX) models inducing sustained regressions. Using ATM-deficient cells, we demonstrate that AZD7648, in combination with the PARP inhibitor olaparib, increases genomic instability, resulting in cell growth inhibition and apoptosis. AZD7648 enhanced olaparib efficacy across a range of doses and schedules in xenograft and PDX models, enabling sustained tumour regression and providing a clear rationale for its clinical investigation. Through its differentiated mechanism of action as an NHEJ inhibitor, AZD7648 complements the current armamentarium of DDR-targeted agents and has potential in combination with these agents to achieve deeper responses to current therapies.

DNA double-strand breaks (DSBs) are perhaps the most toxic of all DNA lesions, with defects in the DNA damage response to DSBs being associated with various human diseases. Although it is known that DSB repair pathways are tightly regulated by ubiquitylation, we do not yet have a comprehensive understanding of how deubiquitylating enzymes (DUBs) function in DSB responses. Here, by carrying out a multi-dimensional screening strategy for human DUBs, we identify several with hitherto unknown links to DSB repair, the G2/M DNA-damage checkpoint and genome-integrity maintenance. Phylogenetic analyses reveal functional clustering within certain DUB subgroups, suggesting evolutionally conserved functions and/or related modes-of action. Furthermore, we establish that the DUB UCHL5 regulates DSB resection and repair by homologous recombination through protecting its interactor, NFRKB, from degradation. Collectively our findings extend the list of DUBs promoting the maintenance of genome integrity, and highlight their potential as therapeutic targets for cancer.

Emerging data demonstrate homologous recombination (HR) defects in castration-resistant prostate cancers, rendering these tumours sensitive to PARP inhibition. Here we demonstrate a direct requirement for the androgen receptor (AR) to maintain HR gene expression and HR activity in prostate cancer. We show that PARP-mediated repair pathways are upregulated in prostate cancer following androgen-deprivation therapy (ADT). Furthermore, upregulation of PARP activity is essential for the survival of prostate cancer cells and we demonstrate a synthetic lethality between ADT and PARP inhibition in vivo. Our data suggest that ADT can functionally impair HR prior to the development of castration resistance and that, this potentially could be exploited therapeutically using PARP inhibitors in combination with androgen-deprivation therapy upfront in advanced or high-risk prostate cancer.

PAXX was identified recently as a novel nonhomologous end-joining DNA repair factor in human cells. To characterize its physiological roles, we generated Paxx-deficient mice. Like Xlf−/− mice, Paxx−/− mice are viable, grow normally, and are fertile but show mild radiosensitivity. Strikingly, while Paxx loss is epistatic with Ku80, Lig4, and Atm deficiency, Paxx/Xlf double-knockout mice display embryonic lethality associated with genomic instability, cell death in the central nervous system, and an almost complete block in lymphogenesis, phenotypes that closely resemble those of Xrcc4−/− and Lig4−/− mice. Thus, combined loss of Paxx and Xlf is synthetic-lethal in mammals.

The poly (ADP-ribose) polymerase (PARP) inhibitor olaparib is FDA approved for the treatment of BRCA-mutated breast, ovarian and pancreatic cancers. Olaparib inhibits PARP1/2 enzymatic activity and traps PARP1 on DNA at single-strand breaks, leading to replication-induced DNA damage that requires BRCA1/2-dependent homologous recombination repair. Moreover, DNA damage response pathways mediated by the ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia mutated and Rad3-related (ATR) kinases are hypothesised to be important survival pathways in response to PARP-inhibitor treatment. Here, we show that olaparib combines synergistically with the ATR-inhibitor AZD6738 (ceralasertib), in vitro, leading to selective cell death in ATM-deficient cells. We observe that 24 h olaparib treatment causes cells to accumulate in G2-M of the cell cycle, however, co-administration with AZD6738 releases the olaparib-treated cells from G2 arrest. Selectively in ATM-knockout cells, we show that combined olaparib/AZD6738 treatment induces more chromosomal aberrations and achieves this at lower concentrations and earlier treatment time-points than either monotherapy. Furthermore, single-agent olaparib efficacy in vitro requires PARP inhibition throughout multiple rounds of replication. Here, we demonstrate in several ATM-deficient cell lines that the olaparib and AZD6738 combination induces cell death within 1-2 cell divisions, suggesting that combined treatment could circumvent the need for prolonged drug exposure. Finally, we demonstrate in vivo combination activity of olaparib and AZD6738 in xenograft and PDX mouse models with complete ATM loss. Collectively, these data provide a mechanistic understanding of combined PARP and ATR inhibition in ATM-deficient models, and support the clinical development of AZD6738 in combination with olaparib.

SummaryRepair of DNA double-strand breaks is crucial for maintaining genome integrity and is governed by post-translational modifications such as protein ubiquitylation. Here, we establish that the deubiquitylating enzyme USP4 promotes DNA-end resection and DNA repair by homologous recombination. We also report that USP4 interacts with CtIP and the MRE11-RAD50-NBS1 (MRN) complex and is required for CtIP recruitment to DNA damage sites. Furthermore, we show that USP4 is ubiquitylated on multiple sites including those on cysteine residues and that deubiquitylation of these sites requires USP4 catalytic activity and is required for USP4 to interact with CtIP/MRN and to promote CtIP recruitment and DNA repair. Lastly, we establish that regulation of interactor binding by ubiquitylation occurs more generally among USP-family enzymes. Our findings thus identify USP4 as a novel DNA repair regulator and invoke a model in which ubiquitin adducts regulate USP enzyme interactions and functions.

AZD6738 (ceralasertib) is a potent and selective orally bioavailable inhibitor of ataxia telangiectasia and rad3-related (ATR) kinase. ATR is activated in response to stalled DNA replication forks to promote G2/M-cell cycle checkpoints and fork restart. Here, we found AZD6738 modulated CHK1 phosphorylation and induced ATM-dependent signaling (pRAD50) and the DNA damage marker γH2AX. AZD6738 inhibited breakinduced replication (BIR) and homologous recombination repair (HRR). In vitro sensitivity to AZD6738 was elevated in, but not exclusive to, cells with defects in the ATM-pathway or that harbor putative drivers of replication stress such as CCNE1amplification. This translated to in vivo anti-tumor activity, with tumor control requiring continuous dosing and free plasma exposures which correlated with induction of pCHK1, pRAD50, and γH2AX. AZD6738 showed combinatorial efficacy with agents associated with replication fork stalling and collapse such as carboplatin and irinotecan and the PARP inhibitor olaparib. These combinations required optimisation of dose and schedules in vivo and showed superior anti-tumor activity at lower doses compared to that required for monotherapy. Tumor regressions required at least 2 days of daily dosing of AZD6738 concurrent with carboplatin, while twicedaily dosing was required following irinotecan. In a BRCA2-mutant patient-derived triple-negative breast cancer (TNBC) xenograft model, complete tumor regression was achieved with 3-5 days of daily AZD6738 per week concurrent with olaparib. Increasing olaparib dosage or AZD6738 dosing to twice-daily allowed complete tumor regression even in a BRCA wild-type TNBC xenograft model. These preclinical data provide rationale for clinical evaluation of AZD6738 as a monotherapy or combinatorial agent.