POLQ seals post-replicative ssDNA gaps to maintain genome stability in BRCA-deficient cancer cells.

POLQ is a key effector of DSB repair by microhomology-mediated end-joining (MMEJ) and is overexpressed in many cancers. POLQ inhibitors confer synthetic lethality in HR and Shieldin-deficient cancer cells, which has been proposed to reflect a critical dependence on the DSB repair pathway by MMEJ. Whether POLQ also operates independent of MMEJ remains unexplored. Here, we show that POLQ-deficient cells accumulate post-replicative ssDNA gaps upon BRCA1/2 loss or PARP inhibitor treatment. Biochemically, cooperation between POLQ helicase and polymerase activities promotes RPA displacement and ssDNA-gap fill-in, respectively. POLQ is also capable of microhomology-mediated gap skipping (MMGS), which generates deletions during gap repair that resemble the genomic scars prevalent in POLQ overexpressing cancers. Our findings implicate POLQ in mutagenic post-replicative gap sealing, which could drive genome evolution in cancer and whose loss places a critical dependency on HR for gap protection and repair and cellular viability.

Loss of nuclear DNA ligase III reverts PARP inhibitor resistance in BRCA1/53BP1 double-deficient cells by exposing ssDNA gaps.

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, preclinical and clinical research with PARPi has revealed multiple resistance mechanisms, highlighting the need for identification of novel functional biomarkers and combination treatment strategies. Functional genetic screens performed in cells and organoids that acquired resistance to PARPi by loss of 53BP1 identified loss of LIG3 as an enhancer of PARPi toxicity in BRCA1-deficient cells. Enhancement of PARPi toxicity by LIG3 depletion is dependent on BRCA1 deficiency but independent of the loss of 53BP1 pathway. Mechanistically, we show that LIG3 loss promotes formation of MRE11-mediated post-replicative ssDNA gaps in BRCA1-deficient and BRCA1/53BP1 double-deficient cells exposed to PARPi, leading to an accumulation of chromosomal abnormalities. LIG3 depletion also enhances efficacy of PARPi against BRCA1-deficient mammary tumors in mice, suggesting LIG3 as a potential therapeutic target.

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.

Targeting ATR in Cancer Medicine.

As a key component of the DNA Damage Response, the Ataxia telangiectasia and Rad3-related (ATR) protein is a promising druggable target that is currently widely evaluated in phase I-II-III clinical trials as monotherapy and in combinations with other rational antitumor agents, including immunotherapy, DNA repair inhibitors, chemo- and radiotherapy. Ongoing clinical studies for this drug class must address the optimization of the therapeutic window to limit overlapping toxicities and refine the target population that will most likely benefit from ATR inhibition. With advances in the development of personalized treatment strategies for patients with advanced solid tumors, many ongoing ATR inhibitor trials have been recruiting patients based on their germline and somatic molecular alterations, rather than relying solely on specific tumor subtypes. Although a spectrum of molecular alterations have already been identified as potential predictive biomarkers of response that may sensitize to ATR inhibition, these biomarkers must be analytically validated and feasible to measure robustly to allow for successful integration into the clinic. While several ATR inhibitors in development are poised to address a clinically unmet need, no ATR inhibitor has yet received FDA-approval. This chapter details the underlying rationale for targeting ATR and summarizes the current preclinical and clinical landscape of ATR inhibitors currently in evaluation, as their regulatory approval potentially lies close in sight.

Translational reprogramming following UVB irradiation is mediated by DNA-PKcs and allows selective recruitment to the polysomes of mRNAs encoding DNA repair enzymes.

UVB-induced lesions in mammalian cellular DNA can, through the process of mutagenesis, lead to carcinogenesis. However, eukaryotic cells have evolved complex mechanisms of genomic surveillance and DNA damage repair to counteract the effects of UVB radiation. We show that following UVB DNA damage, there is an overall inhibition of protein synthesis and translational reprogramming. This reprogramming allows selective synthesis of DDR proteins, such as ERCC1, ERCC5, DDB1, XPA, XPD, and OGG1 and relies on upstream ORFs in the 5' untranslated region of these mRNAs. Experiments with DNA-PKcs-deficient cell lines and a specific DNA-PKcs inhibitor demonstrate that both the general repression of mRNA translation and the preferential translation of specific mRNAs depend on DNA-PKcs activity, and therefore our data establish a link between a key DNA damage signaling component and protein synthesis.

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.

Ataxia-Telangiectasia Mutated Loss-of-Function Displays Variant and Tissue-Specific Differences across Tumor Types.

PURPOSE: Mutations in the ATM gene are common in multiple cancers, but clinical studies of therapies targeting ATM-aberrant cancers have yielded mixed results. Refinement of ATM loss of function (LOF) as a predictive biomarker of response is urgently needed. EXPERIMENTAL DESIGN: We present the first disclosure and preclinical development of a novel, selective ATR inhibitor, ART0380, and test its antitumor activity in multiple preclinical cancer models. To refine ATM LOF as a predictive biomarker, we performed a comprehensive pan-cancer analysis of ATM variants in patient tumors and then assessed the ATM variant-to-protein relationship. Finally, we assessed a novel ATM LOF biomarker approach in retrospective clinical data sets of patients treated with platinum-based chemotherapy or ATR inhibition. RESULTS: ART0380 had potent, selective antitumor activity in a range of preclinical cancer models with differing degrees of ATM LOF. Pan-cancer analysis identified 10,609 ATM variants in 8,587 patient tumors. Cancer lineage-specific differences were seen in the prevalence of deleterious (Tier 1) versus unknown/benign (Tier 2) variants, selective pressure for loss of heterozygosity, and concordance between a deleterious variant and ATM loss of protein (LOP). A novel ATM LOF biomarker approach that accounts for variant classification, relationship to ATM LOP, and tissue-specific penetrance significantly enriched for patients who benefited from platinum-based chemotherapy or ATR inhibition. CONCLUSIONS: These data help to better define ATM LOF across tumor types in order to optimize patient selection and improve molecularly targeted therapeutic approaches for patients with ATM LOF cancers.

ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks.

It is generally thought that the DNA-damage checkpoint kinases, ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), work independently of one another. Here, we show that ATM and the nuclease activity of meiotic recombination 11 (Mre11) are required for the processing of DNA double-strand breaks (DSBs) to generate the replication protein A (RPA)-coated ssDNA that is needed for ATR recruitment and the subsequent phosphorylation and activation of Chk1. Moreover, we show that efficient ATM-dependent ATR activation in response to DSBs is restricted to the S and G2 cell cycle phases and requires CDK kinase activity. Thus, in response to DSBs, ATR activation is regulated by ATM in a cell-cycle dependent manner.

Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways.

Gene editing through repair of CRISPR-Cas9-induced chromosomal breaks offers a means to correct a wide range of genetic defects. Directing repair to produce desirable outcomes by modulating DNA repair pathways holds considerable promise to increase the efficiency of genome engineering. Here, we show that inhibition of non-homologous end joining (NHEJ) or polymerase theta-mediated end joining (TMEJ) can be exploited to alter the mutational outcomes of CRISPR-Cas9. We show robust inhibition of TMEJ activity at CRISPR-Cas9-induced double-strand breaks (DSBs) using ART558, a potent polymerase theta (PolÏ´) inhibitor. Using targeted sequencing, we show that ART558 suppresses the formation of microhomology-driven deletions in favor of NHEJ-specific outcomes. Conversely, NHEJ deficiency triggers the formation of large kb-sized deletions, which we show are the products of mutagenic TMEJ. Finally, we show that combined chemical inhibition of TMEJ and NHEJ increases the efficiency of homology-driven repair (HDR)-mediated precise gene editing. Our work reports a robust strategy to improve the fidelity and safety of genome engineering.

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.

Suppression of HIV-1 infection by a small molecule inhibitor of the ATM kinase.

Chemotherapy that is used to treat human immunodeficiency virus type-1 (HIV-1) infection focuses primarily on targeting virally encoded proteins. However, the combination of a short retroviral life cycle and high mutation rate leads to the selection of drug-resistant HIV-1 variants. One way to address this problem is to inhibit non-essential host cell proteins that are required for viral replication. Here we show that the activity of HIV-1 integrase stimulates an ataxia-telangiectasia-mutated (ATM)-dependent DNA damage response, and that a deficiency of this ATM kinase sensitizes cells to retrovirus-induced cell death. Consistent with these observations, we demonstrate that a novel and specific small molecule inhibitor of ATM kinase activity, KU-55933, is capable of suppressing the replication of both wild-type and drug-resistant HIV-1.

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.

Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy.

BRCA1 and BRCA2 are important for DNA double-strand break repair by homologous recombination, and mutations in these genes predispose to breast and other cancers. Poly(ADP-ribose) polymerase (PARP) is an enzyme involved in base excision repair, a key pathway in the repair of DNA single-strand breaks. We show here that BRCA1 or BRCA2 dysfunction unexpectedly and profoundly sensitizes cells to the inhibition of PARP enzymatic activity, resulting in chromosomal instability, cell cycle arrest and subsequent apoptosis. This seems to be because the inhibition of PARP leads to the persistence of DNA lesions normally repaired by homologous recombination. These results illustrate how different pathways cooperate to repair damage, and suggest that the targeted inhibition of particular DNA repair pathways may allow the design of specific and less toxic therapies for cancer.

Identification and optimisation of novel and selective small molecular weight kinase inhibitors of mTOR.

A pharmacophore mapping approach, derived from previous experience of PIKK family enzymes, was used to identify a hit series of selective inhibitors of the mammalian target of rapamycin (mTOR). Subsequent refinement of the SAR around this hit series based on a tri-substituted triazine scaffold has led to the discovery of potent and selective inhibitors of mTOR.

Selective inhibition of BRCA2-deficient mammary tumor cell growth by AZD2281 and cisplatin.

PURPOSE: To assess efficacy of the novel, selective poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor AZD2281 against newly established BRCA2-deficient mouse mammary tumor cell lines and to determine potential synergy between AZD2281 and cisplatin. EXPERIMENTAL DESIGN: We established and thoroughly characterized a panel of clonal cell lines from independent BRCA2-deficient mouse mammary tumors and BRCA2-proficient control tumors. Subsequently, we assessed sensitivity of these lines to conventional cytotoxic drugs and the novel PARP inhibitor AZD2281. Finally, in vitro combination studies were done to investigate interaction between AZD2281 and cisplatin. RESULTS: Genetic, transcriptional, and functional analyses confirmed the successful isolation of BRCA2-deficient and BRCA2-proficient mouse mammary tumor cell lines. Treatment of these cell lines with 11 different anticancer drugs or with gamma-irradiation showed that AZD2281, a novel and specific PARP inhibitor, caused the strongest differential growth inhibition of BRCA2-deficient versus BRCA2-proficient mammary tumor cells. Finally, drug combination studies showed synergistic cytotoxicity of AZD2281 and cisplatin against BRCA2-deficient cells but not against BRCA2-proficient control cells. CONCLUSION: We have successfully established the first set of BRCA2-deficient mammary tumor cell lines, which form an important addition to the existing preclinical models for BRCA-mutated breast cancer. The exquisite sensitivity of these cells to the PARP inhibitor AZD2281, alone or in combination with cisplatin, provides strong support for AZD2281 as a novel targeted therapeutic against BRCA-deficient cancers.

8-Biarylchromen-4-one inhibitors of the DNA-dependent protein kinase (DNA-PK).

The synthesis and biological evaluation of libraries of 8-biarylchromen-4-ones enabled the elucidation of structure-activity relationships for inhibition of the DNA-dependent protein kinase (DNA-PK), with 8-(3-(thiophen-2-yl)phenyl)chromen-4-one and 8-(3-(thiophen-3-yl)phenyl)chromen-4-one being especially potent inhibitors.

Novel alkoxybenzamide inhibitors of poly(ADP-ribose) polymerase.

We have previously described poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors based on a substituted benzyl-phthalazinone scaffold. As an alternative chemical template, a novel series of alkoxybenzamides were developed with restricted conformation through intramolecular hydrogen bond formation; the compounds exhibit low nM enzyme and cellular activity as PARP-1 inhibitors.

Preclinical evaluation of a potent novel DNA-dependent protein kinase inhibitor NU7441.

DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by ionizing radiation and topoisomerase II poisons, such as etoposide and doxorubicin. A major pathway for the repair of DSB is nonhomologous end joining, which requires DNA-dependent protein kinase (DNA-PK) activity. We investigated the therapeutic use of a potent, specific DNA-PK inhibitor (NU7441) in models of human cancer. We measured chemosensitization by NU7441 of topoisomerase II poisons and radiosensitization in cells deficient and proficient in DNA-PK(CS) (V3 and V3-YAC) and p53 wild type (LoVo) and p53 mutant (SW620) human colon cancer cell lines by clonogenic survival assay. Effects of NU7441 on DSB repair and cell cycle arrest were measured by gammaH2AX foci and flow cytometry. Tissue distribution of NU7441 and potentiation of etoposide activity were determined in mice bearing SW620 tumors. NU7441 increased the cytotoxicity of ionizing radiation and etoposide in SW620, LoVo, and V3-YAC cells but not in V3 cells, confirming that potentiation was due to DNA-PK inhibition. NU7441 substantially retarded the repair of ionizing radiation-induced and etoposide-induced DSB. NU7441 appreciably increased G(2)-M accumulation induced by ionizing radiation, etoposide, and doxorubicin in both SW620 and LoVo cells. In mice bearing SW620 xenografts, NU7441 concentrations in the tumor necessary for chemopotentiation in vitro were maintained for at least 4 hours at nontoxic doses. NU7441 increased etoposide-induced tumor growth delay 2-fold without exacerbating etoposide toxicity to unacceptable levels. In conclusion, NU7441 shows sufficient proof of principle through in vitro and in vivo chemosensitization and radiosensitization to justify further development of DNA-PK inhibitors for clinical use.

Pyranone, thiopyranone, and pyridone inhibitors of phosphatidylinositol 3-kinase related kinases. Structure-activity relationships for DNA-dependent protein kinase inhibition, and identification of the first potent and selective inhibitor of the ataxia telangiectasia mutated kinase.

Structure-activity relationships have been investigated for inhibition of DNA-dependent protein kinase (DNA-PK) and ATM kinase by a series of pyran-2-ones, pyran-4-ones, thiopyran-4-ones, and pyridin-4-ones. A wide range of IC50 values were observed for pyranones and thiopyranones substituted at the 6-position, with the 3- and 5-positions proving intolerant to substitution. Related pyran-2-ones, pyran-4-ones, and thiopyran-4-ones showed similar IC50 values against DNA-PK, whereas the pyridin-4-one system proved, in general, ineffective at inhibiting DNA-PK. Extended libraries exploring the 6-position of 2-morpholino-pyran-4-ones and 2-morpholino-thiopyrano-4-ones identified the first highly potent and selective ATM inhibitor 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (151C; ATM; IC50=13 nM) and revealed constrained SARs for ATM inhibition compared with DNA-PK. One of the most potent DNA-PK inhibitors identified, 2-(4-methoxyphenyl)-6-(morpholin-4-yl)pyran-4-one (16; DNA-PK; IC50=220 nM) effectively sensitized HeLa cells to the topoisomerase II inhibitor etoposide in vitro.