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Histone H2AX plays a key role in DNA damage signalling in the surrounding regions of DNA double-strand breaks (DSBs)1,2. In response to DNA damage, H2AX becomes phosphorylated on serine residue 139 (known as γH2AX), resulting in the recruitment of the DNA repair effectors 53BP1 and BRCA13–6. Here, by studying resistance to poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2-deficient mammary tumours7,8, we identify a novel function for γH2AX in orchestrating drug-induced replication fork degradation. Mechanistically, γH2AX-dependent replication fork degradation is elicited by the inhibition of CtIP-mediated fork protection. As a result, H2AX loss restores replication fork stability and increases chemoresistance in BRCA1/2-deficient tumour cells without restoring homology-directed DNA repair, as highlighted by the lack of DNA damage-induced RAD51 foci. Furthermore, in the attempt to discover acquired genetic vulnerabilities, we find that ATM inhibition overcomes PARP inhibitor (PARPi) resistance in H2AX-deficient tumours by interfering with CtIP-mediated fork protection of stalled forks. In summary, our results demonstrate a novel role for H2AX in replication fork biology in BRCA-deficient tumours and establish a function of H2AX separable from its classical role in DNA damage signalling and DSB repair.

Supplementary Information

U87/Centrin2-EGFP/H2B-mCherry/puro cells were seeded on dishes with cover slip bottoms, treated with AZ32 (3 uM), and irradiated (5 Gy). Time-lapse videos were recorded intermittently (every 7 min) over 16 hrs. Treatment results in 11% aberrant mitoses (see Fig. 3F).

U87/shp53/Centrin2-EGFP/H2B-mCherry/shp53 cells were seeded on dishes with cover slip bottoms and irradiated (5 Gy). Time-lapse videos were recorded intermittently (every 7 min) over 16 hrs. Treatment results in 12% aberrant mitoses (see Fig. 3F).

U87/Centrin2-EGFP/H2B-mCherry/puro cells were seeded on dishes with cover slip bottoms and irradiated (5 Gy). Time-lapse videos were recorded intermittently (every 7 min) over 16 hrs. Treatment results in 2.7% aberrant mitoses (see Fig. 3F).

Inhibition of ataxia-telangiectasia mutated (ATM) during radiotherapy of glioblastoma multiforme (GBM) may improve tumor control by short-circuiting the response to radiation-induced DNA damage. A major impediment for clinical implementation is that current inhibitors have limited central nervous system (CNS) bioavailability; thus, the goal was to identify ATM inhibitors (ATMi) with improved CNS penetration. Drug screens and refinement of lead compounds identified AZ31 and AZ32. The compounds were then tested in vivo for efficacy and impact on tumor and healthy brain. Both AZ31 and AZ32 blocked the DNA damage response and radiosensitized GBM cells in vitro. AZ32, with enhanced blood–brain barrier (BBB) penetration, was highly efficient in vivo as radiosensitizer in syngeneic and human, orthotopic mouse glioma model compared with AZ31. Furthermore, human glioma cell lines expressing mutant p53 or having checkpoint-defective mutations were particularly sensitive to ATMi radiosensitization. The mechanism for this p53 effect involves a propensity to undergo mitotic catastrophe relative to cells with wild-type p53. In vivo, apoptosis was >6-fold higher in tumor relative to healthy brain after exposure to AZ32 and low-dose radiation. AZ32 is the first ATMi with oral bioavailability shown to radiosensitize glioma and improve survival in orthotopic mouse models. These findings support the development of a clinical-grade, BBB-penetrating ATMi for the treatment of GBM. Importantly, because many GBMs have defective p53 signaling, the use of an ATMi concurrent with standard radiotherapy is expected to be cancer-specific, increase the therapeutic ratio, and maintain full therapeutic effect at lower radiation doses. Mol Cancer Ther; 17(8); 1637–47. ©2018 AACR.

Cancers overcome replicative immortality by activating either telomerase or an alternative lengthening of telomeres (ALT) mechanism. ALT occurs in ~ 25% of high-risk neuroblastomas and relapse or progression in patients with ALT neuroblastoma during or after front-line therapy is frequent and almost uniformly fatal. Temozolomide + irinotecan is commonly used as salvage therapy for neuroblastoma. Patient-derived cell-lines and xenografts established from patients with relapsed ALT neuroblastoma demonstrated de novo resistance to temozolomide + irinotecan (as SN-38 in vitro, P

Background AZD0156 is an oral inhibitor of ATM, a serine threonine kinase that plays a key role in DNA damage response (DDR) associated with double-strand breaks. Topoisomerase-I inhibitor irinotecan is used clinically to treat colorectal cancer (CRC), often in combination with 5-fluorouracil (5FU). AZD0156 in combination with irinotecan and 5FU was evaluated in preclinical models of CRC to determine whether low doses of AZD0156 enhance the cytotoxicity of irinotecan in chemotherapy regimens used in the clinic. Methods Anti-proliferative effects of single-agent AZD0156, the active metabolite of irinotecan (SN38), and combination therapy were evaluated in 12 CRC cell lines. Additional assessment with clonogenic assay, cell cycle analysis, and immunoblotting were performed in 4 selected cell lines. Four colorectal cancer patient derived xenograft (PDX) models were treated with AZD0156, irinotecan, or 5FU alone and in combination for assessment of tumor growth inhibition (TGI). Immunofluorescence was performed on tumor tissues. The DDR mutation profile was compared across in vitro and in vivo models. Results Enhanced effects on cellular proliferation and regrowth were observed with the combination of AZD0156 and SN38 in select models. In cell cycle analysis of these models, increased G2/M arrest was observed with combination treatment over either single agent. Immunoblotting results suggest an increase in DDR associated with irinotecan therapy, with a reduced effect noted when combined with AZD0156, which is more pronounced in some models. Increased TGI was observed with the combination of AZD0156 and irinotecan as compared to single-agent therapy in some PDX models. The DDR mutation profile was variable across models. Conclusions AZD0156 and irinotecan provide a rational and active combination in preclinical colorectal cancer models. Variability across in vivo and in vitro results may be related to the variable DDR mutation profiles of the models evaluated. Further understanding of the implications of individual DDR mutation profiles may help better identify patients more likely to benefit from treatment with the combination of AZD0156 and irinotecan in the clinical setting.

The ATM inhibitor AZD1390 disrupts cellular responses to ionizing radiation (IR) and is a potent radiosensitizer being tested in clinical trials. Here, we evaluated, the effects of AZD1390 on radiation sensitivity and DNA damage repair pathways in glioblastoma (GBM) cells and patient derived xenografts (PDXs). AZD1390 (30 nM and higher) suppressed IR (5 Gy)-induced phosphorylation of ATM-Serine1981 and downstream phosphorylation sites on Kap1, Chk2 and H2AX in U251 cells and multiple PDXs. Consistent with enhanced DNA damage, AZD1390 increased IR induced G2/M arrest in U251 (80.6% with AZD1390/IR vs. 64.6% with IR, p= 0.01), GBM43 (61.9% vs. 25.7%, p= 0.01) and GBM39 (40.9% vs. 25.4%, p= 0.01). Moreover, in a clonogenic survival assay, AZD1390 sensitized U251 cells to 5 Gy IR (0.24% survival with AZD1390/IR vs. 2.3% with IR alone, p=0.01). In a reporter-based analysis of DNA repair capacity, ATM inhibition resulted in a 40 to 60% reduction in homologous recombination (HR) and modest but significant decrease in micro-homology mediated end joining (MMEJ) and gap fill-in synthesis in U251 cells but had no effect on non-homologous end joining, translesion synthesis, nucleotide or base excision repair pathways. Comparing effects of AZD1390 on repair in GBM14 (TP53-wt) and GBM43 (TP53-mutant), similar results were observed except that decreased MMEJ was seen only in GBM43 (0.03± 0.01% vs. 0.07± 0.01% in control, p=0.002). Intriguingly, RAD51 knockdown (to disrupt HR) sensitized U251 and GBM43 but not GBM14. The efficacy of AZD1390 ± IR was studied in vivo in 10 PDXs. IR was delivered to orthotopic tumors using opposed lateral 225 kVp beams. AZD1390 (20 mg/kg PO) was given just prior to each radiation dose (2 Gy x 5 fractions). AZD1390 monotherapy was mostly ineffective, IR alone was reasonably efficacious with an average 1.8 ± 0.1-fold-increase in survival relative to sham radiation (survival ratio) across all 10 models. IR/AZD1390 treatment resulted in significant survival extension relative to IR alone in 6 of 10 models. Analysis of the survival benefit of combination therapy compared to IR alone across the entire cohort of PDXs was statistically marginal (average survival ratio 2.3± 0.3 vs. 1.8 ± 0.1 with IR, p=0.08). However, when stratified by TP53 status, combination therapy was significantly more effective than IR (mean survival ratio 2.3 ± 0.1 vs. 1.6 ± 0.2 with IR alone, p=0.02) in TP53-mutant PDXs, where all 5 models benefited. In contrast, TP53-wt group had no benefit (mean survival ratio 2.2 ± 0.5 vs. 2.0 ± 0.2, p=0.61), GBM39 was only TP53-wt PDX that benefited from the combination. In conclusion, AZD1390 is an effective radio-sensitizer that causes disruption in HR and potentially other DNA repair pathways. Interestingly, in vivo radiosensitizing effects are mostly restricted to TP53-mutant GBM PDXs. Understandingmechanism of resistance in the context of different TP53 backgrounds remains an important future direction. Citation Format: Shiv K. Gupta, Jiajia Chen, Daniel J. Laverty, Surabhi Talele, Brett L. Carlson, Ann C. Mladek Tuma, Danielle Burgenske, Gaspar J. Kitange, Petra Hamerlik, Zachary D. Nagel, Stephen T. Durant, William F. Elmquist, Jann N. Sarkaria. AZD1390 radio-sensitizes p53-mutant GBM via disrupting homology directed DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2598.

BackgroundPersonalised medicine strategies may improve outcomes in pancreatic ductal adenocarcinoma (PDAC), but validation of predictive biomarkers is required. Having developed a clinical trial to assess the ATR inhibitor, AZD6738, in combination with gemcitabine (ATRi/gem), we investigated ATM loss as a predictive biomarker of response to ATRi/gem in PDAC.MethodsThrough kinase inhibition, siRNA depletion and CRISPR knockout of ATM, we assessed how ATM targeting affected the sensitivity of PDAC cells to ATRi/gem. Using flow cytometry, immunofluorescence and immunoblotting, we investigated how ATRi/gem synergise in ATM-proficient and ATM-deficient cells, before assessing the impact of ATM loss on ATRi/gem sensitivity in vivo.ResultsComplete loss of ATM function (through pharmacological inhibition or CRISPR knockout), but not siRNA depletion, sensitised to ATRi/gem. In ATM-deficient cells, ATRi/gem-induced replication catastrophe was augmented, while phospho-Chk2-T68 and phospho-KAP1-S824 persisted via DNA-PK activity. ATRi/gem caused growth delay in ATM-WT xenografts in NSG mice and induced regression in ATM-KO xenografts.ConclusionsATM loss augments replication catastrophe-mediated cell death induced by ATRi/gem and may predict clinical responsiveness to this combination. ATM status should be carefully assessed in tumours from patients with PDAC, since distinction between ATM-low and ATM-null could be critical in maximising the success of clinical trials using ATM expression as a predictive biomarker.

ATM inhibitors, such as 7, have demonstrated the antitumor potential of ATM inhibition when combined with DNA double-strand break-inducing agents in mouse xenograft models. However, the properties of 7 result in a relatively high predicted clinically efficacious dose. In an attempt to minimize attrition during clinical development, we sought to identify ATM inhibitors with a low predicted clinical dose (50 mg) and focused on strategies to increase both ATM potency and predicted human pharmacokinetic half-life (predominantly through the increase of volume of distribution). These efforts resulted in the discovery of 64 (AZD0156), an exceptionally potent and selective inhibitor of ATM based on an imidazo[4,5- c]quinolin-2-one core. 64 has good preclinical phamacokinetics, a low predicted clinical dose, and a high maximum absorbable dose. 64 has been shown to potentiate the efficacy of the approved drugs irinotecan and olaparib in disease relevant mouse models and is currently undergoing clinical evaluation with these agents.

While radiotherapy (RT) is fundamental for the treatment of brain tumors, irradiation of the brain frequently causes devastating effects on cognitive function and quality of life. DNA damage within neural stem cells (NSC) is a key factor in the pathogenesis of radiation-induced cognitive dysfunction. The ataxia telangiectasia mutated (ATM) kinase is a central protein in the DNA damage response and a critical determinant of tumor cell survival after radiation. ATM inhibition potently radiosensitizes preclinical models of GBM in vitro and in vivo. A novel, brain penetrant ATM inhibitor AZD1390, which is predicted to achieve brain tumor concentrations in the range of 1-5nM, is currently in early phase clinical evaluation in combination with RT. In marked contrast to observations in tumor models, genetic knockdown of ATM has radioprotective effects on NSC in vitro; the proposed mechanism is via suppression of p53 mediated apoptosis. The purpose of this study was to investigate the impact of AZD1390 on survival responses and mode of death in NSCs exposed to RT in vitro and in vivo. NSCs were derived from the telencephalon of E13 mouse embryos. Cells were treated with AZD1390 (0.1-10nM) 1 hour prior to ionizing radiation (IR; 0-5 Gy). Mode and timing of cell death was interrogated using IncuCyte live cell analysis to measure proliferation, cytotoxicity and apoptosis up to 72 hours post-IR. Cell viability and neurosphere formation assays were also used to measure radiation sensitivity in vitro. C57BL/6 mice received 20Gy hemibrain irradiation +/- 7-day treatment with AZD1390 (10mg/kg). Immunohistochemistry for Ki67 and Sox2 was used to assess effects on NSC in the subventricular zone (SVZ) 50 days post-irradiation. In vitro AZD1390 (1-10nM) inhibited ATM kinase function within 1 hour, evidenced by abrogation of KAP1 and p53 phosphorylation. NSCs primarily undergo apoptosis in response to IR. AZD1390 at 1 and 3nM significantly reduced apoptosis in irradiated NSCs (ratios of annexin V area under the curve 1.95 and 2 respectively); 10 nM had no effect on this parameter. Proliferation rates and cell viability after radiation were preserved at all drug concentrations. AZD1390 at 1nM did not modulate radiation effects on neurosphere formation whereas at 10nM a radiosensitizing effect was observed (ratio of SF[3Gy]=0.25). In vivo, IR decreased the number of Ki67 positive proliferating cells (92% reduction) and Sox2-positive cells (24% reduction) in the SVZ after 50 days; these effects were not exacerbated by addition of AZD1390. Acute effects (24 hours post-IR) are under investigation. We demonstrate in vitro that AZD1390 has radioprotective effects on NSCs at clinically achievable concentrations. In vivo, treatment with AZD1390 did not enhance the effects of radiation on NSCs in the SVZ. In the context of its profound radiosensitizing effects on GBM models, the absence of radiosensitization of NSCs both in vitro and in vivo strengthens the rationale for evaluating AZD1390 in combination with RT in GBM patients. Citation Format: Rodrigo Guttierez-Quintana, David J. Walker, Mark R. Jackson, Natividad Gomez-Roman, Sandeep Chahal, Stephen T. Durant, Anthony J. Chalmers. Radiotherapy in combination with the brain penetrant ATM inhibitor AZD1390 does not exacerbate radiation toxicity of neural stem cells in vitro or in vivo [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-017.

PARP inhibitors (PARPi) enhance radiation sensitivity in multiple cancer models, both in vitro and in vivo. Our observation that the radiosensitizing properties of PARPi are most pronounced in rapidly proliferating cells is reflected in early phase clinical trial data showing exacerbation of acute radiation toxicity in rapidly proliferating tissues such as oropharyngeal and esophageal mucosa. Lack of radiosensitization in late responding, slowly proliferating normal tissues indicates that PARPi may be more effectively combined with radiation therapy (RT) in patients with brain tumors. We are therefore evaluating the oral PARPi olaparib in combination with RT and/or temozolomide (TMZ) in the treatment of glioblastoma (GBM), the most prevalent and most aggressive primary brain tumor. Patients with GBM experience very poor outcomes in terms of median survival (c.1 year) and neurocognitive decline caused primarily by RT. Olaparib was initially evaluated in combination with daily low-dose TMZ in patients with recurrent GBM in the OPARATIC trial. Pharmacokinetic studies revealed that olaparib penetrates both core and margin regions of GBM, indicating that the BBB is significantly disrupted throughout these tumors. Olaparib could be safely combined with daily TMZ (75 mg/m2), but intermittent olaparib dosing (150 mg three days per week) was required to avoid dose-limiting hematological toxicity. Early phase testing of the olaparib-radiotherapy combination is now underway in three populations of patients with newly diagnosed GBM. Patients aged >65 with MGMT unmethylated GBM are being recruited to a randomized, placebo-controlled phase II study (PARADIGM) after a phase I dose escalation study showed that olaparib (200 mg twice daily) was extremely well tolerated when combined with brain irradiation (40 Gray in 15#). Good performance status patients aged Citation Format: Anthony J. Chalmers, Rodrigo Gutierrez-Quintana, David J. Walker, Karin Williams, Duncan Forster, Mark R. Jackson, Sarah Derby, Jon Stobo, Lorna Sweeting, Caroline Kelly, Stephen Durant, Kaye J. Williams. Enhancing the therapeutic ratio for glioblastoma by combining radiation therapy with PARP inhibitors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-006.

The structure-specific nuclease human flap endonuclease-1 (hFEN1) plays a key role in DNA replication and repair and may be of interest as an oncology target. We present the crystal structure of inhibitor-bound hFEN1, which shows a cyclic N-hydroxyurea bound in the active site coordinated to two magnesium ions. Three such compounds had similar IC50 values but differed subtly in mode of action. One had comparable affinity for protein and protein-substrate complex and prevented reaction by binding to active site catalytic metal ions, blocking the necessary unpairing of substrate DNA. Other compounds were more competitive with substrate. Cellular thermal shift data showed that both inhibitor types engaged with hFEN1 in cells, and activation of the DNA damage response was evident upon treatment with inhibitors. However, cellular EC50 values were significantly higher than in vitro inhibition constants, and the implications of this for exploitation of hFEN1 as a drug target are discussed.

The DNA damage response (DDR) safeguards genome stability and promotes cellular survival by counteracting deleterious consequences of DNA damage. Deficiencies in DDR pathways contribute to genome instability, a hallmark of cancer development, and targeting the DDR is a promising anti-cancer therapy strategy. We describe here in vitro and in vivo studies allowing mechanistic understanding of the benefit of combining our DDR kinase inhibitors (DDRi) targeting ATM (AZD0156), ATR (AZD6738) and DNA-PK (AZD7648) with the PARP inhibitor, olaparib, in homologous recombination-deficient backgrounds. Using the break-induced replication (BIR) assay, which measures homologous-recombination repair (HRR) of DNA double-strand breaks resembling broken replication forks, we observed that ATRi decreases this type of homologous recombination repair by up to 50%, while inhibiting DNA-PK or ATM showed no effect. This provides a mechanistic rationale for combining ATRi with compounds that cause replication-fork collapse, such as PARPi. Indeed, combination of olaparib with the ATRi in BRCA1 mutant UWB1.289 cells enhanced olaparib sensitivity a thousand-fold (GI50 from 590 nM to 0.6 nM). This correlates with the complete tumour growth inhibitions (TGI) observed for the ATRi + olaparib combination in the BRCA2 mutant HBCx-10 triple negative breast cancer PDX model and is consistent with a synergistic increase of genome instability measured by metaphase spread analysis (total aberrations: olaparib; 11, ATRi; 8, combination; 35). DNA-PKi, however, did not synergise with olaparib, showing little enhancement of olaparib single-agent treatment (GI50 from 0.59 μM to 0.33 μM) and no significant impact on the total amount of chromosomal aberrations (total aberrations: olaparib; 11, DNA-PKi ; 3, combination; 14), suggesting that DNA-PKi alone or in combination with olaparib have little impact on BRCA1 mutated tumours. In ATM KO FaDu xenografts, ATRi or DNA-PKi single-agent treatment resulted in TGI, with complete tumour regressions when combined. Focusing on the olaparib combination, metaphase spread analysis of ATRi-treated FaDu ATM KO cells showed an enhancement of DNA replication-dependent chromatid breaks (1.3 vs 0.2 breaks/spread in ATM WT), while DNA-PKi treatment predominantly caused chromosome breaks (1.7 vs 0.2 breaks/spread in ATM WT) that are not dependent on replication. When combined with olaparib, ATRi acted synergistically and in a manner similar to that observed in the BRCA1 mutant cells, while the DNA-PKi + olaparib combination was additive. These data suggest that enhanced olaparib sensitisation of ATM KO cells by ATRi or DNA-PKi combinations occurs through different mechanisms. Together, these findings provide mechanistic differentiation of our DDRi in specific genetic backgrounds. This work informs how to clinically position these agents to benefit the right patients not only as single-agent treatments, but also in combination. Citation Format: Josep V. Forment, Paul Wijnhoven, Antonio Ramos-Montoya, Jacqueline Fok, Valeria Follia, Mercedes Vazquez-Chantada, Rajesh Oderda, Zena Wilson, Alan Lau, Elisabetta Leo, Stephen Durant, Elaine Cadogan, Mark J. O'Connor. Mechanistic differentiation of targeted DDR agent combinations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 626.

[Image: see text] We report the discovery of a novel series of 3-cinnoline carboxamides as highly potent and selective ataxia telangiectasia mutated (ATM) kinase inhibitors. Optimization of this series focusing on potency and physicochemical properties (especially permeability) led to the identification of compound 21, a highly potent ATM inhibitor (ATM cell IC(50) 0.0028 μM) with excellent kinase selectivity and favorable physicochemical and pharmacokinetics properties. In vivo, 21 in combination with irinotecan showed tumor regression in the SW620 colorectal tumor xenograft model, superior inhibition to irinotecan alone. Compound 21 was selected for preclinical evaluation alongside AZD0156.

Poor survival rates of patients with tumors arising from or disseminating into the brain are attributed to an inability to excise all tumor tissue (if operable), a lack of blood-brain barrier (BBB) penetration of chemotherapies/targeted agents, and an intrinsic tumor radio-/chemo-resistance. Ataxia-telangiectasia mutated (ATM) protein orchestrates the cellular DNA damage response (DDR) to cytotoxic DNA double-strand breaks induced by ionizing radiation (IR). ATM genetic ablation or pharmacological inhibition results in tumor cell hypersensitivity to IR. We report the primary pharmacology of the clinical-grade, exquisitely potent (cell IC50, 0.78 nM), highly selective [>10,000-fold over kinases within the same phosphatidylinositol 3-kinase–related kinase (PIKK) family], orally bioavailable ATM inhibitor AZD1390 specifically optimized for BBB penetration confirmed in cynomolgus monkey brain positron emission tomography (PET) imaging of microdosed 11C-labeled AZD1390 (Kp,uu, 0.33). AZD1390 blocks ATM-dependent DDR pathway activity and combines with radiation to induce G2 cell cycle phase accumulation, micronuclei, and apoptosis. AZD1390 radiosensitizes glioma and lung cancer cell lines, with p53 mutant glioma cells generally being more radiosensitized than wild type. In in vivo syngeneic and patient-derived glioma as well as orthotopic lung-brain metastatic models, AZD1390 dosed in combination with daily fractions of IR (whole-brain or stereotactic radiotherapy) significantly induced tumor regressions and increased animal survival compared to IR treatment alone. We established a pharmacokinetic-pharmacodynamic-efficacy relationship by correlating free brain concentrations, tumor phospho-ATM/phospho-Rad50 inhibition, apoptotic biomarker (cleaved caspase-3) induction, tumor regression, and survival. On the basis of the data presented here, AZD1390 is now in early clinical development for use as a radiosensitizer in central nervous system malignancies.

Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase I inhibitor topotecan. Thus, we here establish that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib reflects delayed engagement of homologous recombination at DNA-replication-fork associated single-ended double-strand breaks (DSBs), allowing some to be subject to toxic NHEJ. Preventing DSB ligation by NHEJ, or enhancing homologous recombination by BRCA1-A complex disruption, suppresses this toxicity, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions. Notably, suppressor mutations in ATM-mutant backgrounds are different to those in BRCA1-mutant scenarios, suggesting new opportunities for patient stratification and additional therapeutic vulnerabilities for clinical exploitation., Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. Here, the authors provide evidence that these hypersensitivities reflect a crucial role for ATM at damaged replication forks being to prevent toxic DNA end-joining leading to chromosome fusions and cell death.

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) regulates the basal and stress-inducible expression of a battery of genes encoding key components of the glutathione-based and thioredoxin-based antioxidant systems, as well as aldo-keto reductase, glutathione S- transferase, and NAD(P)H:quinone oxidoreductase-1 drug-metabolizing isoenzymes along with multidrug-resistance-associated efflux pumps. It therefore plays a pivotal role in both intrinsic resistance and cellular adaptation to reactive oxygen species (ROS) and xenobiotics. Activation of Nrf2 can, however, serve as a double-edged sword because some of the genes it induces may contribute to chemical carcinogenesis by promoting futile redox cycling of polycyclic aromatic hydrocarbon metabolites or confer resistance to chemotherapeutic drugs by increasing the expression of efflux pumps, suggesting its cytoprotective effects will vary in a context-specific fashion. In addition to cytoprotection, Nrf2 also controls genes involved in intermediary metabolism, positively regulating those involved in NADPH generation, purine biosynthesis, and the β-oxidation of fatty acids, while suppressing those involved in lipogenesis and gluconeogenesis. Nrf2 is subject to regulation at multiple levels. Its ability to orchestrate adaptation to oxidants and electrophiles is due principally to stress-stimulated modification of thiols within one of its repressors, the Kelch-like ECH-associated protein 1 (Keap1), which is present in the cullin-3 RING ubiquitin ligase (CRL) complex CRL Keap1 . Thus modification of Cys residues in Keap1 blocks CRL Keap1 activity, allowing newly translated Nrf2 to accumulate rapidly and induce its target genes. The ability of Keap1 to repress Nrf2 can be attenuated by p62/sequestosome-1 in a mechanistic target of rapamycin complex 1 (mTORC1)-dependent manner, thereby allowing refeeding after fasting to increase Nrf2-target gene expression. In parallel with repression by Keap1, Nrf2 is also repressed by β-transducin repeat-containing protein (β-TrCP), present in the Skp1–cullin-1–F-box protein (SCF) ubiquitin ligase complex SCF β-TrCP . The ability of SCF β-TrCP to suppress Nrf2 activity is itself enhanced by prior phosphorylation of the transcription factor by glycogen synthase kinase-3 (GSK-3) through formation of a DSGIS-containing phosphodegron. However, formation of the phosphodegron in Nrf2 by GSK-3 is inhibited by stimuli that activate protein kinase B (PKB)/Akt. In particular, PKB/Akt activity can be increased by phosphoinositide 3-kinase and mTORC2, thereby providing an explanation of why antioxidant-responsive element-driven genes are induced by growth factors and nutrients. Thus Nrf2 activity is tightly controlled via CRL Keap1 and SCF β-TrCP by oxidative stress and energy-based signals, allowing it to mediate adaptive responses that restore redox homeostasis and modulate intermediary metabolism. Based on the fact that Nrf2 influences multiple biochemical pathways in both positive and negative ways, it is likely its dose–response curve, in terms of susceptibility to certain degenerative disease, is U-shaped. Specifically, too little Nrf2 activity will lead to loss of cytoprotection, diminished antioxidant capacity, and lowered β-oxidation of fatty acids, while conversely also exhibiting heightened sensitivity to ROS-based signaling that involves receptor tyrosine kinases and apoptosis signal-regulating kinase-1. By contrast, too much Nrf2 activity disturbs the homeostatic balance in favor of reduction, and so may have deleterious consequences including overproduction of reduced glutathione and NADPH, the blunting of ROS-based signal transduction, epithelial cell hyperplasia, and failure of certain cell types to differentiate correctly. We discuss the basis of a putative U-shaped Nrf2 dose–response curve in terms of potentially competing processes relevant to different stages of tumorigenesis.

As discussed in the previous chapter, the rationale for inhibition of ATM as a therapeutic strategy in cancer is both scientifically sound and well explored. The use of experimental models and, thereafter, the availability of tool compounds to inhibit the target, has allowed the role of ATM in cell signalling to be refined and has highlighted the potential utility of ATM inhibition for therapeutic intervention. The role of ATM as the central DNA damage response (DDR) protein, the high sensitivity of cells from A-T patients, who lack functional ATM, to IR and DNA damaging chemotherapy, and the consequences of knocking down ATM in otherwise proficient cells, have been well described and support ATM as a pharmaceutical target of interest. The somewhat atypical nature of ATM (a member of the PIKK family of kinases), combined with the size of the protein, have brought some unique challenges and opportunities to the discovery of inhibitors of ATM. The development of robust, high-throughput biochemical assays for ATM inhibition has proved challenging, thereby requiring the establishment of less conventional assays to facilitate drug discovery efforts. However, the availability of early compounds that were shown to share features of ATM loss (i.e. bringing about sensitisation of cells to IR induced cell damage and death), helped advance the process and over the past decade the research into ATM inhibition has advanced as the quality of available inhibitors has improved. In this chapter, we will explore the evolution of ATM inhibitors from crude but effective tools, through highly selective tool compounds and ultimately to the development of compounds with potential clinical utility as therapeutics for cancer patients.

Inhibition of ataxia-telangiectasia mutated (ATM) during radiotherapy of glioblastoma multiforme (GBM) may improve tumor control by short-circuiting the response to radiation-induced DNA damage. A major impediment for clinical implementation is that current inhibitors have limited central nervous system (CNS) bioavailability; thus, the goal was to identify ATM inhibitors (ATMi) with improved CNS penetration. Drug screens and refinement of lead compounds identified AZ31 and AZ32. The compounds were then tested in vivo for efficacy and impact on tumor and healthy brain. Both AZ31 and AZ32 blocked the DNA damage response and radiosensitized GBM cells in vitro. AZ32, with enhanced blood–brain barrier (BBB) penetration, was highly efficient in vivo as radiosensitizer in syngeneic and human, orthotopic mouse glioma model compared with AZ31. Furthermore, human glioma cell lines expressing mutant p53 or having checkpoint-defective mutations were particularly sensitive to ATMi radiosensitization. The mechanism for this p53 effect involves a propensity to undergo mitotic catastrophe relative to cells with wild-type p53. In vivo, apoptosis was >6-fold higher in tumor relative to healthy brain after exposure to AZ32 and low-dose radiation. AZ32 is the first ATMi with oral bioavailability shown to radiosensitize glioma and improve survival in orthotopic mouse models. These findings support the development of a clinical-grade, BBB-penetrating ATMi for the treatment of GBM. Importantly, because many GBMs have defective p53 signaling, the use of an ATMi concurrent with standard radiotherapy is expected to be cancer-specific, increase the therapeutic ratio, and maintain full therapeutic effect at lower radiation doses. Mol Cancer Ther; 17(8); 1637–47. ©2018 AACR.

AZD1390 is a novel, highly selective, brain-penetrant, potent inhibitor of the Ataxia Telangiectasia Mutated (ATM) kinase. ATM is activated in response to double-strand breaks (DSBs) and coordinates cellular responses to ionising radiation and other insults. Radiation is the mainstay of treatment for patients with brain tumors, and glioma cells are exquisitely sensitive to ATM inhibition. Therefore, AZD1390 is in clinical development in combination with radiation therapy for the treatment of patients with Glioblastoma Multiforme (GBM) and brain metastases from solid tumours (NCT03423628). [1] The aim of this work was to develop a translational PK/PD-efficacy model for AZD1390 that would enable the project team to assess the extent/duration of inhibition of target engagement required and help to predict the optimal dose and regimen of AZD1390 to treat patients with brain malignancies in combination with radiation. Time-course data of pharmacokinetics (PK), pharmacodynamics (PD) and tumor growth inhibition from mouse NCI-H2228 brain tumor orthotopic model studies were used to build a PK-PD/Efficacy or survival model. ATM activation is induced by IR treatment, becoming transiently phosphorylated within minutes of IR exposure and dissipates over a 24-hour period. This model quantitatively and dynamically integrates AZD1390 brain PK to the rate and extent of inhibition of phosphorylation of ATM, a proximal PD marker of ATM kinase activation caused by radiation induced DNA damage in the tumor and rate of induction of cell death (determined by bioluminescence signalling as a measure of tumor growth). In parallel, we also modelled cell cycle profiles in GBM cell lines to complement the in-vivo modelling work. The pATM time-course relative to IR radiation (2 Gy) was modelled using AZD1390 concentrations in the brain tumor and the corresponding pATM inhibition, which was then linked to efficacy by estimating tumor cell kill rate via exponential tumor growth model. The free brain concentration of AZD1390 that resulted in half-maximal inhibition (EC50) of pATM after radiation was estimated to be 0.8 nM (range 0.4 to 1.6 nM). Significant tumor regression in orthotopic tumor model was observed at doses >5 mg/kg. An average pATM inhibition over 24h in the mouse GBM survival model was in the range of 44% (5 mg/kg QD) to 88% (20 mg/kg BD). The % overall survival at 5 mg/kg QD and 20 mg/kg BD dose were 56% and 100%, respectively. Conclusion: The translation of mouse survival data to clinical schedules is unknown, thus efficacious dose was anchored to pATM inhibition required to deliver significant tumor regression. References: 1. Durant ST et al., The brain-penetrant clinical ATM inhibitor AZD1390 radiosensitizes and improves survival of preclinical brain tumor models. Sci. Adv. 2018;4. Citation Format: Venkatesh Pilla Reddy, Andy Sykes, Nicola Colclough, Stephen T. Durant, Lenka Oplustil O'Connor, Matthias Hoch, Nuria Buil Bruna, Serena De Vita, Melinda Merchant, Martin Pass. A preclinical PK/PD model based on a mouse glioblastoma survival model for AZD1390, a novel, brain-penetrant ATM kinase inhibitor, to predict the inhibition of DNA damage response induced by radiation and the human efficacious dose [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4868.

Background: AZD0156 is an oral inhibitor of ATM, a serine threonine kinase that plays a key role in DNA damage response associated with DNA double strand breaks. Topoisomerase-I inhibitors like irinotecan induce single-strand DNA breaks, which are converted to double-strand breaks during DNA replication. Thus the combination of AZD0156 and irinotecan is a rational combination for clinical use. Irinotecan is used clinically to treat a variety of malignancies, including colorectal cancer (CRC), usually in combination with 5-fluorouracil (5FU) as FOLFIRI. An ongoing phase 1 clinical trial is evaluating AZD0156 in combination with single-agent irinotecan and FOLFIRI in patients with refractory cancers (NCT02588105). The purpose of this study is to evaluate AZD0156 in combination with irinotecan and 5FU in preclinical models of CRC to help inform clinical use. Methods: Anti-proliferative effects of single-agent AZD0156 and combination therapy with SN38 (active metabolite of irinotecan) and 5FU were evaluated in CRC cell lines using the Cell-Titer Glo assay. Immunoblotting and cell cycle analysis were performed to determine the mechanism of enhanced combination effects. Four CRC patient derived xenograft (PDX) models were treated with AZD0156, irinotecan, and 5FU alone and in combination for assessment of tumor growth inhibition (TGI). Results: An enhanced antiproliferative effect was observed with the combination treatment over either single agent. A more significant synergistic effect was demonstrated with the combination of AZD0156 and SN38 as compared with the combination of AZD0156 and 5FU. Cell cycle data demonstrated enhanced cell cycle arrest with combination therapy as compared to single agents. Immunoblotting results suggest a decrease in phosphorylated gamma-H2AX in cell lines treated with combination therapies. Increased TGI was observed in CRC PDX models treated with the combination of AZD0156 and irinotecan as compared to single-agent therapy in 3 of 4 models. There was not a significant change in TGI with the addition of 5FU for triplet therapy in the majority of models. Conclusions: The combination of AZD0156 with irinotecan is synergistic in in vitro models and is associated with increased TGI in CRC PDX in vivo models. The addition of 5FU to AZD0156 and irinotecan did not result in increased TGI as compared to doublet therapy in CRC PDX models, though did not decrease the AZD0156/irinotecan combination effect. An ongoing clinical trial is evaluating this combination in patients with cancers refractory to standard treatments (NCT02588105). Citation Format: S. Lindsey Davis, Marina I. Schlaepfer, Stacey M. Bagby, Sarah J. Hartman, Betelehem W. Yacob, Tonia Tse, Dennis M. Simmons, Jennifer R. Diamond, Christopher H. Lieu, Alexis D. Leal, Elaine B. Cadogan, Gareth D. Hughes, Stephen T. Durant, Wells A. Messersmith, Todd M. Pitts. Ataxia telangiectasia mutated (ATM) kinase inhibitor AZD0156 in combination with 5-fluorouracil and irinotecan in preclinical models of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4720.

Flap endonuclease-1 (FEN1) is a highly conserved metallonuclease and is the main human flap endonuclease involved in the recognition and cleavage of single-stranded 5' overhangs from DNA flap structures. The involvement of FEN1 in multiple DNA metabolism pathways and the identification of FEN1 overexpression in a variety of cancers has led to interest in FEN1 as an oncology target. In this article, we describe the development of a 1536-well high-throughput screening assay based on the change in fluorescence polarization of a FEN1 DNA substrate labeled with Atto495 dye. The assay was subsequently used to screen 850 000 compounds from the AstraZeneca compound collection, with a Z' factor of 0.66 ± 0.06. Hits were followed up by IC50 determination in both a concentration-response assay and a technology artifact assay.

Activation of the p53 tumour suppressor protein in response to DNA damage leads to apoptosis or cell-cycle arrest. Enzymatic modifications are widely believed to affect and regulate p53 activity. We describe here a level of post-translational control that has an important functional consequence on the p53 response. We show that the protein arginine methyltransferase (PRMT) 5, as a co-factor in a DNA damage responsive co-activator complex that interacts with p53, is responsible for methylating p53. Arginine methylation is regulated during the p53 response and affects the target gene specificity of p53. Furthermore, PRMT5 depletion triggers p53-dependent apoptosis. Thus, methylation on arginine residues is an underlying mechanism of control during the p53 response.

The vast majority of cancers commandeer the activity of telomerase - the remarkable enzyme responsible for prolonging cellular lifespan by maintaining the length of telomeres at the ends of chromosomes. Telomerase is only normally active in embryonic and highly proliferative somatic cells. Thus, targeting telomerase is an attractive anti-cancer therapeutic rationale currently under investigation in various phases of clinical development. However, previous reports suggest that an average of 10-15% of all cancers lose the functional activity of telomerase and most of these turn to an Alternative Lengthening of Telomeres pathway (ALT). ALT-positive tumours will therefore not respond to anti-telomerase therapies and there is a real possibility that such drugs would be toxic to normal telomerase-utilising cells and ultimately select for resistant cells that activate an ALT mechanism. ALT exploits certain DNA damage response (DDR) components to counteract telomere shortening and rapid trimming. ALT has been reported in many cancer subtypes including sarcoma, gastric carcinoma, central nervous system malignancies, subtypes of kidney (Wilm's Tumour) and bladder carcinoma, mesothelioma, malignant melanoma and germ cell testicular cancers to name but a few. A recent heroic study that analysed ALT in over six thousand tumour samples supports this historical spread, although only reporting an approximate 4% prevalence. This review highlights the various methods of ALT detection, unravels several molecular ALT models thought to promote telomere maintenance and elongation, spotlights the DDR components known to facilitate these and explores why certain tissues are more likely to subvert DDR away from its usually protective functions, resulting in a predictive pattern of prevalence in specific cancer subsets.

Ionizing radiation induces delayed genomic instability in human cells, including chromosomal abnormalities and hyperrecombination. Here, we investigate delayed genome instability of cells exposed to UV radiation. We examined homologous recombination-mediated reactivation of a green fluorescent protein (GFP) gene in p53-proficient human cells. We observed an approximately 5-fold enhancement of delayed hyperrecombination (DHR) among cells surviving a low dose of UV-C (5 J/m2), revealed as mixed GFP+/- colonies. UV-B did not induce DHR at an equitoxic (75 J/m2) dose or a higher dose (150 J/m2). UV is known to induce delayed hypermutation associated with increased oxidative stress. We found that hypoxanthine phosphoribosyltransferase (HPRT) mutation frequencies were approximately 5-fold higher in strains derived from GFP+/- (DHR) colonies than in strains in which recombination was directly induced by UV (GFP+ colonies). To determine whether hypermutation was directly caused by hyperrecombination, we analyzed hprt mutation spectra. Large-scale alterations reflecting large deletions and insertions were observed in 25% of GFP+ strains, and most mutants had a single change in HPRT. In striking contrast, all mutations arising in the hypermutable GFP+/- strains were small (1- to 2-base) changes, including substitutions, deletions, and insertions (reminiscent of mutagenesis from oxidative damage), and the majority were compound, with an average of four hprt mutations per mutant. The absence of large hprt deletions in DHR strains indicates that DHR does not cause hypermutation. We propose that UV-induced DHR and hypermutation result from a common source, namely, increased oxidative stress. These two forms of delayed genome instability may collaborate in skin cancer initiation and progression.

The molecular mechanism by which foreign DNA integrates into the human genome is poorly understood yet critical to many disease processes, including retroviral infection and carcinogenesis, and to gene therapy. We hypothesized that the mechanism of genomic integration may be similar to transposition in lower organisms. We identified a protein, termed Metnase, that has a SET domain and a transposase/nuclease domain. Metnase methylates histone H3 lysines 4 and 36, which are associated with open chromatin. Metnase increases resistance to ionizing radiation and increases nonhomologous end-joining repair of DNA doublestrand breaks. Most significantly, Metnase promotes integration of exogenous DNA into the genomes of host cells. Therefore, Metnase is a nonhomologous end-joining repair protein that regulates genomic integration of exogenous DNA and establishes a relationship among histone modification, DNA repair, and integration. The data suggest a model wherein Metnase promotes integration of exogenous DNA by opening chromatin and facilitating joining of DNA ends. This study demonstrates that eukaryotic transposase domains can have important cell functions beyond transposition of genetic elements.

It is now clear that large DNA-binding proteins have evolved in mammals to orchestrate the relatively ancient process of DNA recombinational repair. These proteins are recruited to accurately repair DNA double strand breaks (DSBs)--the frequent, potentially lethal and mutagenic lesions in the genomes of all organisms. An essential mammalian regulator of DSB repair is BRCA1. Heterozygous BRCA1 mutations predispose individuals to breast, ovarian and other secondary cancers. BRCA1-defective cells exhibit reduced DSB repair, sensitivity to a wide range of DNA damaging agents, genomic instability and defects in the S-phase checkpoint, transcription and chromatin remodelling. DSBs can be repaired by RAD51/RPA-dependent homologous recombination (HR) or DNA-PK-dependent non-homologous end-joining (NHEJ). Both of these pathways can be imprecise and mutagenic. BRCA1 plays a central role in promoting accurate repair by both HR and NHEJ. Consistent with recent evidence, we have assembled a novel cell-cycle-dependent model in which DNA-PK inhibits RPA in S-phase of the cell cycle, while BRCA1 inhibits the exonuclease processivity of the MRE11/RAD50/NBS1 (MRN) complex and facilitates the removal of RPA in S and G2 phase. This model provides an explanation for how BRCA1 promotes accurate DSB repair during various phases of the cell cycle and also accounts for the dual effects that BRCA1 and MRN activity have upon DNA repair and S-phase arrest.

Mismatch repair (MMR) proteins repair mispaired DNA bases and have an important role in maintaining the integrity of the genome [1]. Loss of MMR has been correlated with resistance to a variety of DNA-damaging agents, including many anticancer drugs [2]. How loss of MMR leads to resistance is not understood, but is proposed to be due to loss of futile MMR activity and/or replication stalling [3,4]. We report that inactivation of MMR genes ( MLH1 , MLH2 , MSH2 , MSH3 , MSH6 , but not PMS1) in isogenic strains of Saccharomyces cerevisiae led to increased resistance to the anticancer drugs cisplatin, carboplatin and doxorubicin, but had no effect on sensitivity to ultraviolet C (UVC) radiation. Sensitivity to cisplatin and doxorubicin was increased in mlh1 mutant strains when the MLH1 gene was reintroduced, demonstrating a direct involvement of MMR proteins in sensitivity to these DNA-damaging agents. Inactivation of MLH1 , MLH2 or MSH2 had no significant effect, however, on drug sensitivities in the rad52 or rad1 mutant strains that are defective in mitotic recombination and removing unpaired DNA single strands. We propose a model whereby MMR proteins – in addition to their role in DNA-damage recognition – decrease adduct tolerance during DNA replication by modulating the levels of recombination-dependent bypass. This hypothesis is supported by the finding that, in human ovarian tumour cells, loss of hMLH1 correlated with acquisition of cisplatin resistance and increased cisplatin-induced sister chromatid exchange, both of which were reversed by restoration of hMLH1 expression.

After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years.

Poly(ADP-ribose) glycohydrolase (PARG) is the only enzyme known to catalyse hydrolysis of the O-glycosidic linkages of ADP-ribose polymers, thereby reversing the effects of poly(ADP-ribose) polymerases. PARG deficiency leads to cell death whilst PARG depletion causes sensitisation to certain DNA damaging agents, implicating PARG as a potential therapeutic target in several disease areas. Efforts to develop small molecule inhibitors of PARG activity have until recently been hampered by a lack of structural information on PARG. We have used a combination of bio-informatic and experimental approaches to engineer a crystallisable, catalytically active fragment of human PARG (hPARG). Here, we present high-resolution structures of the catalytic domain of hPARG in unliganded form and in complex with three inhibitors: ADP-ribose (ADPR), adenosine 5′-diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD) and 8-n-octyl-amino-ADP-HPD. Our structures confirm conservation of overall fold amongst mammalian PARG glycohydrolase domains, whilst revealing additional flexible regions in the catalytic site. These new structures rationalise a body of published mutational data and the reported structure-activity relationship for ADP-HPD based PARG inhibitors. In addition, we have developed and used biochemical, isothermal titration calorimetry and surface plasmon resonance assays to characterise the binding of inhibitors to our PARG protein, thus providing a starting point for the design of new inhibitors.

Metnase is a human protein with methylase (SET) and nuclease domains that is widely expressed, especially in proliferating tissues. Metnase promotes non-homologous end-joining (NHEJ), and knockdown causes mild hypersensitivity to ionizing radiation. Metnase also promotes plasmid and viral DNA integration, and topoisomerase IIα (TopoIIα)-dependent chromosome decatenation. NHEJ factors have been implicated in the replication stress response, and TopoIIα has been proposed to relax positive supercoils in front of replication forks. Here we show that Metnase promotes cell proliferation, but it does not alter cell cycle distributions, or replication fork progression. However, Metnase knockdown sensitizes cells to replication stress and confers a marked defect in restart of stalled replication forks. Metnase promotes resolution of phosphorylated histone H2AX, a marker of DNA double-strand breaks at collapsed forks, and it co-immunoprecipitates with PCNA and RAD9, a member of the PCNA-like RAD9–HUS1–RAD1 intra-S checkpoint complex. Metnase also promotes TopoIIα-mediated relaxation of positively supercoiled DNA. Metnase is not required for RAD51 focus formation after replication stress, but Metnase knockdown cells show increased RAD51 foci in the presence or absence of replication stress. These results establish Metnase as a key factor that promotes restart of stalled replication forks, and implicate Metnase in the repair of collapsed forks.

DNA double-strand breaks (DSBs) are repaired by nonhomologous end-joining (NHEJ) and homologous recombination (HR). The NHEJ/HR decision is under complex regulation and involves DNA-dependent protein kinase (DNA-PKcs). HR is elevated in DNA-PKcs null cells, but suppressed by DNA-PKcs kinase inhibitors, suggesting that kinase-inactive DNA-PKcs (DNA-PKcs-KR) would suppress HR. Here we use a direct repeat assay to monitor HR repair of DSBs induced by I-SceI nuclease. Surprisingly, DSB-induced HR in DNA-PKcs-KR cells was 2- to 3-fold above the elevated HR level of DNA-PKcs null cells, and approximately 4- to 7-fold above cells expressing wild-type DNA-PKcs. The hyperrecombination in DNA-PKcs-KR cells compared to DNA-PKcs null cells was also apparent as increased resistance to DNA crosslinks induced by mitomycin C. ATM phosphorylates many HR proteins, and ATM is expressed at a low level in cells lacking DNA-PKcs, but restored to wild-type level in cells expressing DNA-PKcs-KR. Several clusters of phosphorylation sites in DNA-PKcs, including the T2609 cluster, which is phosphorylated by DNA-PKcs and ATM, regulate access of repair factors to broken ends. Our results indicate that ATM-dependent phosphorylation of DNA-PKcs-KR contributes to the hyperrecombination phenotype. Interestingly, DNA-PKcs null cells showed more persistent ionizing radiation-induced RAD51 foci (but lower HR levels) compared to DNA-PKcs-KR cells, consistent with HR completion requiring RAD51 turnover. ATM may promote RAD51 turnover, suggesting a second (not mutually exclusive) mechanism by which restored ATM contributes to hyperrecombination in DNA-PKcs-KR cells. We propose a model in which DNA-PKcs and ATM coordinately regulate DSB repair by NHEJ and HR.

Non-homologous DNA end-joining (NHEJ) is a major pathway of double strand break (DSB) repair in human cells. Here we show that vanillin (3-methoxy-4-hydroxybenzaldehyde)--a naturally occurring food component and an acknowledged antimutagen, anticlastogen and anticarcinogen--is an inhibitor of NHEJ. Vanillin blocked DNA end-joining by human cell extracts by directly inhibiting the activity of DNA-PK, a crucial NHEJ component. Inhibition was selective and vanillin had no detectable effect on other steps of the NHEJ process, on an unrelated protein kinase or on DNA mismatch repair by cell extracts. Subtoxic concentrations of vanillin did not affect the ATM/ATR-dependent phosphorylation of Chk2 or the S-phase checkpoint response after ionising radiation. They significantly potentiated the cytotoxicity of cisplatin, but did not affect sensitivity to UVC. A limited screen of structurally related compounds identified two substituted vanillin derivatives that were 100- and 50-fold more potent than vanillin as DNA-PK inhibitors. These compounds also sensitised cells to cisplatin. The inhibition of NHEJ is consistent with the antimutagenic and other biological properties of vanillin, possibly altering the balance between DSB repair by NHEJ and homologous recombination.

MSH2 and MLH1 have a central role in correcting mismatches in DNA occurring during DNA replication and have been implicated in the engagement of apoptosis induced by a number of cytotoxic anticancer agents. The function of MLH1 is not clearly defined, although it is required for mismatch repair (MMR) and engagement of apoptosis after certain types of DNA damage. In order to identify other partners of MLH1 that may be involved in signalling MMR or apoptosis, we used human MLH1 in yeast two-hybrid screens of normal human breast and ovarian cDNA libraries. As well as known partners of MLH1 such as PMS1, MLH3 and MBD4, we identified the carboxy terminus of the human c-MYC proto-oncogene as an interacting sequence. We demonstrate, both in vitro by yeast two-hybrid and GST-fusion pull-down experiments, as well as in vivo by coimmunoprecipitation from human tumour cell extracts, that MLH1 interacts with the c-MYC protein. We further demonstrate that the heterodimeric partner of c-MYC, MAX, interacts with a different MMR protein, MSH2, both in vitro and in vivo. Using an inducible c-MYC-ER™ fusion gene, we show that elevated c-MYC expression leads to an increased HGPRT mutation rate of Rat1 cells and an increase in the number of frameshift mutants at the HGPRT locus. The effect on HGPRT mutation rate is small (2–3-fold), but is consistent with deregulated c-MYC expression partially inhibiting MMR activity.

p53 Activity is of critical importance in suppressing human cancer formation, highlighted by the fact that the majority of human tumors have defective or inactive p53. In normal cells, p53 is held at low levels in a latent form and, following DNA damage, is stabilized which usually results in apoptosis or cell cycle arrest. Most functions of p53 can be ascribed to its role as a sequence specific transcription factor, and an extensive repertoire of downstream responsive genes have been identified. p53 activity is influenced by post-translational modifications, including phosphorylation and lysine methylation. Most recently, arginine methylation mediated by PRMT5, has been identified as an additional and important p53 modification. DNA damage induced p53 arginine methylation impacts on the biochemical properties and functional outcome of the p53 response.