Assessment of DNA Double Strand Break Repair Activity Using High-throughput and Quantitative Luminescence-based Reporter Assays.

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is mediated through several mechanisms: homologous recombination (HR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and single strand annealing (SSA). Cellular assays are essential to measure the proficiency and modulation of these pathways in response to various stimuli. Here, we present a suite of extrachromosomal reporter assays that each measure the reconstitution of a nanoluciferase reporter gene by one of the four major DSBR pathways in cells. Upon transient transfection into cells of interest, repair of pathway-specific reporter substrates can be measured in under 24 h by the detection of Nanoluciferase (NanoLuc) luminescence. These robust assays are quantitative, sensitive, titratable, and amenable to a high-throughput screening format. These properties provide broad applications in DNA repair research and drug discovery, complementing the currently available toolkit of cellular DSBR assays.

Quantitative, titratable and high-throughput reporter assays to measure DNA double strand break repair activity in cells.

Repair of DNA damage is essential for the maintenance of genome stability and cell viability. DNA double strand breaks (DSBs) constitute a toxic class of DNA lesion and multiple cellular pathways exist to mediate their repair. Robust and titratable assays of cellular DSB repair (DSBR) are important to functionally interrogate the integrity and efficiency of these mechanisms in disease models as well as in response to genetic or pharmacological perturbations. Several variants of DSBR reporters are available, however these are often limited by throughput or restricted to specific cellular models. Here, we describe the generation and validation of a suite of extrachromosomal reporter assays that can efficiently measure the major DSBR pathways of homologous recombination (HR), classical nonhomologous end joining (cNHEJ), microhomology-mediated end joining (MMEJ) and single strand annealing (SSA). We demonstrate that these assays can be adapted to a high-throughput screening format and that they are sensitive to pharmacological modulation, thus providing mechanistic and quantitative insights into compound potency, selectivity, and on-target specificity. We propose that these reporter assays can serve as tools to dissect the interplay of DSBR pathway networks in cells and will have broad implications for studies of DSBR mechanisms in basic research and drug discovery.

Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models.

PURPOSE: DNA polymerase theta (Polθ, encoded by the POLQ gene) is a DNA repair enzyme critical for microhomology mediated end joining (MMEJ). Polθ has limited expression in normal tissues but is frequently overexpressed in cancer cells and, therefore, represents an ideal target for tumor-specific radiosensitization. In this study we evaluate whether targeting Polθ with novel small-molecule inhibitors is a feasible strategy to improve the efficacy of radiotherapy. EXPERIMENTAL DESIGN: We characterized the response to Polθ inhibition in combination with ionizing radiation in different cancer cell models in vitro and in vivo. RESULTS: Here, we show that ART558 and ART899, two novel and specific allosteric inhibitors of the Polθ DNA polymerase domain, potently radiosensitize tumor cells, particularly when combined with fractionated radiation. Importantly, noncancerous cells were not radiosensitized by Polθ inhibition. Mechanistically, we show that the radiosensitization caused by Polθ inhibition is most effective in replicating cells and is due to impaired DNA damage repair. We also show that radiosensitization is still effective under hypoxia, suggesting that these inhibitors may help overcome hypoxia-induced radioresistance. In addition, we describe for the first time ART899 and characterize it as a potent and specific Polθ inhibitor with improved metabolic stability. In vivo, the combination of Polθ inhibition using ART899 with fractionated radiation is well tolerated and results in a significant reduction in tumor growth compared with radiation alone. CONCLUSIONS: These results pave the way for future clinical trials of Polθ inhibitors in combination with radiotherapy.

Discovery, Characterization, and Structure-Based Optimization of Small-Molecule In Vitro and In Vivo Probes for Human DNA Polymerase Theta.

Human DNA polymerase theta (Polθ), which is essential for microhomology-mediated DNA double strand break repair, has been proposed as an attractive target for the treatment of BRCA deficient and other DNA repair pathway defective cancers. As previously reported, we recently identified the first selective small molecule Polθ in vitro probe, 22 (ART558), which recapitulates the phenotype of Polθ loss, and in vivo probe, 43 (ART812), which is efficacious in a model of PARP inhibitor resistant TNBC in vivo. Here we describe the discovery, biochemical and biophysical characterization of these probes including small molecule ligand co-crystal structures with Polθ. The crystallographic data provides a basis for understanding the unique mechanism of inhibition of these compounds which is dependent on stabilization of a "closed" enzyme conformation. Additionally, the structural biology platform provided a basis for rational optimization based primarily on reduced ligand conformational flexibility.

Drug-gene Interaction Screens Coupled to Tumor Data Analyses Identify the Most Clinically Relevant Cancer Vulnerabilities Driving Sensitivity to PARP Inhibition.

PARP inhibitors (PARPi) are currently indicated for the treatment of ovarian, breast, pancreatic, and prostate cancers harboring mutations in the tumor suppressor genes BRCA1 or BRCA2. In the case of ovarian and prostate cancers, their classification as homologous recombination repair (HRR) deficient (HRD) or mutated also makes PARPi an available treatment option beyond BRCA1 or BRCA2 mutational status. However, identification of the most relevant genetic alterations driving the HRD phenotype has proven difficult and recent data have shown that other genetic alterations not affecting HRR are also capable of driving PARPi responses. To gain insight into the genetics driving PARPi sensitivity, we performed CRISPR-Cas9 loss-of-function screens in six PARPi-insensitive cell lines and combined the output with published PARPi datasets from eight additional cell lines. Ensuing exploration of the data identified 110 genes whose inactivation is strongly linked to sensitivity to PARPi. Parallel cell line generation of isogenic gene knockouts in ovarian and prostate cancer cell lines identified that inactivation of core HRR factors is required for driving in vitro PARPi responses comparable with the ones observed for BRCA1 or BRCA2 mutations. Moreover, pan-cancer genetic, transcriptomic, and epigenetic data analyses of these 110 genes highlight the ones most frequently inactivated in tumors, making this study a valuable resource for prospective identification of potential PARPi-responsive patient populations. Importantly, our investigations uncover XRCC3 gene silencing as a potential new prognostic biomarker of PARPi sensitivity in prostate cancer. Significance: This study identifies tumor genetic backgrounds where to expand the use of PARPis beyond mutations in BRCA1 or BRCA2. This is achieved by combining the output of unbiased genome-wide loss-of-function CRISPR-Cas9 genetic screens with bioinformatics analysis of biallelic losses of the identified genes in public tumor datasets, unveiling loss of the DNA repair gene XRCC3 as a potential biomarker of PARPi sensitivity in prostate cancer.

VEGF pathway inhibition potentiates PARP inhibitor efficacy in ovarian cancer independent of BRCA status.

Poly ADP-ribose polymerase inhibitors (PARPi) have transformed ovarian cancer (OC) treatment, primarily for tumours deficient in homologous recombination repair. Combining VEGF-signalling inhibitors with PARPi has enhanced clinical benefit in OC. To study drivers of efficacy when combining PARP inhibition and VEGF-signalling, a cohort of patient-derived ovarian cancer xenografts (OC-PDXs), representative of the molecular characteristics and drug sensitivity of patient tumours, were treated with the PARPi olaparib and the VEGFR inhibitor cediranib at clinically relevant doses. The combination showed broad anti-tumour activity, reducing growth of all OC-PDXs, regardless of the homologous recombination repair (HRR) mutational status, with greater additive combination benefit in tumours poorly sensitive to platinum and olaparib. In orthotopic models, the combined treatment reduced tumour dissemination in the peritoneal cavity and prolonged survival. Enhanced combination benefit was independent of tumour cell expression of receptor tyrosine kinases targeted by cediranib, and not associated with change in expression of genes associated with DNA repair machinery. However, the combination of cediranib with olaparib was effective in reducing tumour vasculature in all the OC-PDXs. Collectively our data suggest that olaparib and cediranib act through complementary mechanisms affecting tumour cells and tumour microenvironment, respectively. This detailed analysis of the combined effect of VEGF-signalling and PARP inhibitors in OC-PDXs suggest that despite broad activity, there is no dominant common mechanistic inter-dependency driving therapeutic benefit.

DROSHA is recruited to DNA damage sites by the MRN complex to promote non-homologous end joining.

The DNA damage response (DDR) is the signaling cascade that recognizes DNA double-strand breaks (DSBs) and promotes their resolution via the DNA repair pathways of non-homologous end joining (NHEJ) or homologous recombination (HR). We and others have shown that DDR activation requires DROSHA; however, whether DROSHA exerts its functions by associating with damage sites, what controls its recruitment, and how DROSHA influences DNA repair remains poorly understood. Here, we show that DROSHA associates with DSBs independently of transcription. Neither H2AX, nor ATM or DNA-PK kinase activities are required for recruitment of DROSHA to break sites. Rather, DROSHA interacts with RAD50, and inhibition of the MRN complex by mirin treatment abolishes this interaction. MRN complex inactivation by RAD50 knockdown or mirin treatment prevents DROSHA recruitment to DSBs and, as a consequence, also prevents 53BP1 (also known as TP53BP1) recruitment. During DNA repair, DROSHA inactivation reduces NHEJ and boosts HR frequency. Indeed, DROSHA knockdown also increases the association of downstream HR factors such as RAD51 to DNA ends. Overall, our results demonstrate that DROSHA is recruited at DSBs by the MRN complex and directs DNA repair towards NHEJ.

MRE11-RAD50-NBS1 Complex Is Sufficient to Promote Transcription by RNA Polymerase II at Double-Strand Breaks by Melting DNA Ends.

The MRE11-RAD50-NBS1 (MRN) complex supports the synthesis of damage-induced long non-coding RNA (dilncRNA) by RNA polymerase II (RNAPII) from DNA double-strand breaks (DSBs) by an unknown mechanism. Here, we show that recombinant human MRN and native RNAPII are sufficient to reconstitute a minimal functional transcriptional apparatus at DSBs. MRN recruits and stabilizes RNAPII at DSBs. Unexpectedly, transcription is promoted independently from MRN nuclease activities. Rather, transcription depends on the ability of MRN to melt DNA ends, as shown by the use of MRN mutants and specific allosteric inhibitors. Single-molecule FRET assays with wild-type and mutant MRN show a tight correlation between the ability to melt DNA ends and to promote transcription. The addition of RPA enhances MRN-mediated transcription, and unpaired DNA ends allow MRN-independent transcription by RNAPII. These results support a model in which MRN generates single-strand DNA ends that favor the initiation of transcription by RNAPII.

Synthesis and Evaluation of Scalable D-A-D π-Extended Oligomers as p-Type Organic Materials for Bulk-Heterojunction Solar Cells.

The synthesis and characterization of four novel donor-acceptor-donor π-extended oligomers, incorporating naphtha(1-b)thiophene-4-carboxylate or benzo(b)thieno(3,2-g) benzothiophene-4-carboxylate 2-octyldodecyl esters as end-capping moieties, and two different conjugated core fragments, is reported. The end-capping moieties are obtained via a cascade sequence of sustainable organic reactions, and then coupled to benzo(c)(1,2,5)thiadiazole and its difluoro derivative as the electron-poor π-conjugated cores. The optoelectronic properties of the oligomers are reported. The novel compounds revealed good film forming properties, and when tested in bulk-heterojunction organic photovoltaic cell devices in combination with PC61BM, revealed good fill factors, but low efficiencies, due to their poor absorption profiles.

Functional transcription promoters at DNA double-strand breaks mediate RNA-driven phase separation of damage-response factors.

Damage-induced long non-coding RNAs (dilncRNA) synthesized at DNA double-strand breaks (DSBs) by RNA polymerase II are necessary for DNA-damage-response (DDR) focus formation. We demonstrate that induction of DSBs results in the assembly of functional promoters that include a complete RNA polymerase II preinitiation complex, MED1 and CDK9. Absence or inactivation of these factors causes a reduction in DDR foci both in vivo and in an in vitro system that reconstitutes DDR events on nucleosomes. We also show that dilncRNAs drive molecular crowding of DDR proteins, such as 53BP1, into foci that exhibit liquid-liquid phase-separation condensate properties. We propose that the assembly of DSB-induced transcriptional promoters drives RNA synthesis, which stimulates phase separation of DDR factors in the shape of foci.

Correction to: DNA Damage In situ Ligation followed by Proximity Ligation Assay (DI-PLA).

Work in Fabrizio d'Adda di Fagagna's laboratory is supported by the Associazione Italiana per la Ricerca sul Cancro, AIRC (application 12971), Cariplo Foundation (grant 2010.0818 and 2014-0812), Fondazione Telethon (GGP12059 and GGP17111), Association for International Cancer Research (AICR-Worldwide Cancer Research Rif. N. 14-1331).

DNA Damage In Situ Ligation Followed by Proximity Ligation Assay (DI-PLA).

Cells have evolved DNA repair mechanisms to maintain their genetic information unaltered and a DNA damage response pathway that coordinates DNA repair with several cellular events. Despite a clear role for DNA damage in the form of DNA double-strand breaks (DSBs) in several cellular processes, the most commonly used methods to detect DNA lesions are indirect, and rely on antibody-based recognition of DNA damage-associated factors, leaving several important questions unanswered. Differently, here we describe DNA damage In situ ligation followed by Proximity Ligation Assay (DI-PLA), that allows sensitive detection of physical DSBs in fixed cells, through direct labeling of the DSBs with biotinylated oligonucleotides, and subsequent signal amplification by PLA between biotin and a partner protein in the proximity of the DNA break.

Recent Advancements in DNA Damage-Transcription Crosstalk and High-Resolution Mapping of DNA Breaks.

Until recently, DNA damage arising from physiological DNA metabolism was considered a detrimental by-product for cells. However, an increasing amount of evidence has shown that DNA damage could have a positive role in transcription activation. In particular, DNA damage has been detected in transcriptional elements following different stimuli. These physiological DNA breaks are thought to be instrumental for the correct expression of genomic loci through different mechanisms. In this regard, although a plethora of methods are available to precisely map transcribed regions and transcription start sites, commonly used techniques for mapping DNA breaks lack sufficient resolution and sensitivity to draw a robust correlation between DNA damage generation and transcription. Recently, however, several methods have been developed to map DNA damage at single-nucleotide resolution, thus providing a new set of tools to correlate DNA damage and transcription. Here, we review how DNA damage can positively regulate transcription initiation, the current techniques for mapping DNA breaks at high resolution, and how these techniques can benefit future studies of DNA damage and transcription.

A damaged genome's transcriptional landscape through multilayered expression profiling around in situ-mapped DNA double-strand breaks.

Of the many types of DNA damage, DNA double-strand breaks (DSBs) are probably the most deleterious. Mounting evidence points to an intricate relationship between DSBs and transcription. A cell system in which the impact on transcription can be investigated at precisely mapped genomic DSBs is essential to study this relationship. Here in a human cell line, we map genome-wide and at high resolution the DSBs induced by a restriction enzyme, and we characterize their impact on gene expression by four independent approaches by monitoring steady-state RNA levels, rates of RNA synthesis, transcription initiation and RNA polymerase II elongation. We consistently observe transcriptional repression in proximity to DSBs. Downregulation of transcription depends on ATM kinase activity and on the distance from the DSB. Our study couples for the first time, to the best of our knowledge, high-resolution mapping of DSBs with multilayered transcriptomics to dissect the events shaping gene expression after DSB induction at multiple endogenous sites.

A novel single-cell method provides direct evidence of persistent DNA damage in senescent cells and aged mammalian tissues.

The DNA damage response (DDR) arrests cell cycle progression until DNA lesions, like DNA double-strand breaks (DSBs), are repaired. The presence of DSBs in cells is usually detected by indirect techniques that rely on the accumulation of proteins at DSBs, as part of the DDR. Such detection may be biased, as some factors and their modifications may not reflect physical DNA damage. The dependency on DDR markers of DSB detection tools has left questions unanswered. In particular, it is known that senescent cells display persistent DDR foci, that we and others have proposed to be persistent DSBs, resistant to endogenous DNA repair activities. Others have proposed that these peculiar DDR foci might not be sites of damaged DNA per se but instead stable chromatin modifications, termed DNA-SCARS. Here, we developed a method, named 'DNA damage in situ ligation followed by proximity ligation assay' (DI-PLA) for the detection and imaging of DSBs in cells. DI-PLA is based on the capture of free DNA ends in fixed cells in situ, by ligation to biotinylated double-stranded DNA oligonucleotides, which are next recognized by antibiotin anti-bodies. Detection is enhanced by PLA with a partner DDR marker at the DSB. We validated DI-PLA by demonstrating its ability to detect DSBs induced by various genotoxic insults in cultured cells and tissues. Most importantly, by DI-PLA, we demonstrated that both senescent cells in culture and tissues from aged mammals retain true unrepaired DSBs associated with DDR markers.

Mec1/ATR regulates the generation of single-stranded DNA that attenuates Tel1/ATM signaling at DNA ends.

Tel1/ATM and Mec1/ATR checkpoint kinases are activated by DNA double-strand breaks (DSBs). Mec1/ATR recruitment to DSBs requires the formation of RPA-coated single-stranded DNA (ssDNA), which arises from 5'-3' nucleolytic degradation (resection) of DNA ends. Here, we show that Saccharomyces cerevisiae Mec1 regulates resection of the DSB ends. The lack of Mec1 accelerates resection and reduces the loading to DSBs of the checkpoint protein Rad9, which is known to inhibit ssDNA generation. Extensive resection is instead inhibited by the Mec1-ad mutant variant that increases the recruitment near the DSB of Rad9, which in turn blocks DSB resection by both Rad53-dependent and Rad53-independent mechanisms. The mec1-ad resection defect leads to prolonged persistence at DSBs of the MRX complex that causes unscheduled Tel1 activation, which in turn impairs checkpoint switch off. Thus, Mec1 regulates the generation of ssDNA at DSBs, and this control is important to coordinate Mec1 and Tel1 signaling activities at these breaks.

Interplays between ATM/Tel1 and ATR/Mec1 in sensing and signaling DNA double-strand breaks.

DNA double-strand breaks (DSBs) are highly hazardous for genome integrity because they have the potential to cause mutations, chromosomal rearrangements and genomic instability. The cellular response to DSBs is orchestrated by signal transduction pathways, known as DNA damage checkpoints, which are conserved from yeasts to humans. These pathways can sense DNA damage and transduce this information to specific cellular targets, which in turn regulate cell cycle transitions and DNA repair. The mammalian protein kinases ATM and ATR, as well as their budding yeast corresponding orthologs Tel1 and Mec1, act as master regulators of the checkpoint response to DSBs. Here, we review the early steps of DSB processing and the role of DNA-end structures in activating ATM/Tel1 and ATR/Mec1 in an orderly and reciprocal manner.