Visualization of a DNA-PK/PARP1 complex

The DNA-dependent protein kinase (DNA-PK) and Poly(ADP-ribose) polymerase-1 (PARP1) are critical enzymes that reduce genomic damage caused by DNA lesions. They are both activated by DNA strand breaks generated by physiological and environmental factors, and they have been shown to interact. Here, we report in vivo evidence that DNA-PK and PARP1 are equally necessary for rapid repair. We purified a DNA-PK/PARP1 complex loaded on DNA and performed electron microscopy and single particle analysis on its tetrameric and dimer-of-tetramers forms. By comparison with the DNA-PK holoenzyme and fitting crystallographic structures, we see that the PARP1 density is in close contact with the Ku subunit. Crucially, PARP1 binding elicits substantial conformational changes in the DNA-PK synaptic dimer assembly. Taken together, our data support a functional, in-pathway role for DNA-PK and PARP1 in double-strand break (DSB) repair. We also propose a NHEJ model where protein–protein interactions alter substantially the architecture of DNA-PK dimers at DSBs, to trigger subsequent interactions or enzymatic reactions

Radiotherapy biobanking : current landscape, opportunities, challenges, and future aspirations

This work was supported by the National Cancer Research Institute (NCRI) Clinical and Translational Radiotherapy Research Working Group (CTRad), which was established in 2009 by six of the NCRI's funding partners. We gratefully acknowledge Carolyn Chan and Julie Stock (NCRI) for their assistance in collecting the responses to the questionnaire sent to CTRad membership. Open Access via the Wiley OA AgreementPeer reviewedPublisher PD

Ovarian Cancers Harbour Defects in Non-Homologous End Joining Resulting in Resistance to Rucaparib

Abstract Purpose: DNA damage defects are common in ovarian cancer and can be used to stratify treatment. Although most work has focused on homologous recombination (HR), DNA double-strand breaks are repaired primarily by nonhomologous end joining (NHEJ). Defects in NHEJ have been shown to contribute to genomic instability and have been associated with the development of chemoresistance. Experimental Design: NHEJ was assessed in a panel of ovarian cancer cell lines and 47 primary ascetic-derived ovarian cancer cultures, by measuring the ability of cell extracts to end-join linearized plasmid monomers into multimers. mRNA and protein expression of components of NHEJ was determined using RT-qPCR and Western blotting. Cytotoxicities of cisplatin and the PARP inhibitor rucaparib were assessed using sulforhodamine B (SRB) assays. HR function was assessed using γH2AX/RAD51 foci assay. Results: NHEJ was defective (D) in four of six cell lines and 20 of 47 primary cultures. NHEJ function was independent of HR competence (C). NHEJD cultures were resistant to rucaparib (P = 0.0022). When HR and NHEJ functions were taken into account, only NHEJC/HRD cultures were sensitive to rucaparib (compared with NHEJC/HRC P = 0.034, NHEJD/HRC P = 0.0002, and NHEJD/HRD P = 0.0045). The DNA-PK inhibitor, NU7441, induced resistance to rucaparib (P = 0.014) and HR function recovery in a BRCA1-defective cell line. Conclusions: This study has shown that NHEJ is defective in 40% of ovarian cancers, which is independent of HR function and associated with resistance to PARP inhibitors in ex vivo primary cultures. Clin Cancer Res; 23(8); 2050–60. ©2016 AACR.</jats:p

Determining the Potential of DNA Damage Response (DDR) Inhibitors in Cervical Cancer Therapy

Cisplatin-based chemo-radiotherapy (CRT) is the standard treatment for advanced cervical cancer (CC) but the response rate is poor (46–72%) and cisplatin is nephrotoxic. Therefore, better treatment of CC is urgently needed. We have directly compared, for the first time, the cytotoxicity of four DDR inhibitors (rucaparib/PARPi, VE-821/ATRi, PF-477736/CHK1i and MK-1775/WEE1i) as single agents, and in combination with cisplatin and radiotherapy (RT) in a panel of CC cells. All inhibitors alone caused concentration-dependent cytotoxicity. Low ATM and DNA-PKcs levels were associated with greater VE-821 cytotoxicity. Cisplatin induced ATR, CHK1 and WEE1 activity in all of the cell lines. Cisplatin only activated PARP in S-phase cells, but RT activated PARP in the entire population. Rucaparib was the most potent radiosensitiser and VE-821 was the most potent chemosensitiser. VE-821, PF-47736 and MK-1775 attenuated cisplatin-induced S-phase arrest but tended to increase G2 phase accumulation. In mice, cisplatin-induced acute kidney injury was associated with oxidative stress and PARP activation and was prevented by rucaparib. Therefore, while all inhibitors investigated may increase the efficacy of CRT, the greatest clinical potential of rucaparib may be in limiting kidney damage, which is dose-limiting

Common cancer-associated imbalances in the DNA damage response confer sensitivity to single agent ATR inhibition

ATR is an attractive target in cancer therapy because it signals replication stress and DNA lesions for repair and to S/G2 checkpoints. Cancer-specific defects in the DNA damage response (DDR) may render cancer cells vulnerable to ATR inhibition alone. We determined the cytotoxicity of the ATR inhibitor VE-821 in isogenically matched cells with DDR imbalance. Cell cycle arrest, DNA damage accumulation and repair were determined following VE-821 exposure. Defects in homologous recombination repair (HRR: ATM, BRCA2 and XRCC3) and base excision repair (BER: XRCC1) conferred sensitivity to VE-821. Surprisingly, the loss of different components of the trimeric non-homologous end-joining (NHEJ) protein DNA-PK had opposing effects. Loss of the DNA-binding component, Ku80, caused hypersensitivity to VE-821, but loss of its partner catalytic subunit, DNA-PKcs, did not. Unexpectedly, VE-821 was particularly cytotoxic to human and hamster cells expressing high levels of DNA-PKcs. High DNA-PKcs was associated with replicative stress and activation of the DDR. VE-821 suppressed HRR, determined by RAD51 focus formation, to a greater extent in cells with high DNA-PKcs. Defects in HRR and BER and high DNA-PKcs expression, that are common in cancer, confer sensitivity to ATR inhibitor monotherapy and may be developed as predictive biomarkers for personalised medicine

Targeting the DNA Double Strand Break Repair Machinery in Prostate Cancer

Regardless of the achievable remissions with first line hormone therapy in patients with prostate cancer (CaP), the disease escapes the hormone dependent stage to a more aggressive status where chemotherapy is the only effective treatment and no treatment is curative. This makes it very important to identify new targets that can improve the outcome of treatment. ATM and DNA-PK are the two kinases responsible for signalling and repairing double strand breaks (DSB). Thus, both kinases are pertinent targets in CaP treatment to enhance the activity of the numerous DNA DSB inducing agents used in CaP treatment such as ionizing radiation (IR). Colony formation assay was used to assess the sensitivity of hormone dependent, p53 wt (LNCaP) and hormone independent p53 mutant (PC3) CaP cell lines to the cytotoxic effect of IR and Doxorubicin in the presence or absence of Ku55933 and NU7441 which are small molecule inhibitors of ATM and DNA-PK, respectively. Flow cytometry based methods were used to assess the effect of the two inhibitors on cell cycle, apoptosis and H2AX foci formation. Neutral comet assay was used to assess the induction of DNA DSBs. Ku55933 or NU7441 alone increased the sensitivity of CaP cell lines to the DNA damaging agents, however combining both inhibitors together resulted in further enhancement of sensitivity. The cell cycle profile of both cell lines was altered with increased cell death, DNA DSBs and H2AX foci formation. This study justifies further evaluation of the ATM and DNA-PK inhibitors for clinical application in CaP patients. Additionally, the augmented effect resulting from combining both inhibitors may have a significant implication for the treatment of CaP patients who have a defect in one of the two DSB repair pathways

Phase I, Dose-Escalation, Two-Part Trial of the PARP Inhibitor Talazoparib in Patients with Advanced Germline BRCA1/2 Mutations and Selected Sporadic Cancers

Talazoparib inhibits PARP catalytic activity, trapping PARP1 on damaged DNA and causing cell death in BRCA1/2-mutated cells. We evaluated talazoparib therapy in this two-part, phase I, first-in-human trial. Antitumor activity, MTD, pharmacokinetics, and pharmacodynamics of once-daily talazoparib were determined in an open-label, multicenter, dose-escalation study (NCT01286987). The MTD was 1.0 mg/day, with an elimination half-life of 50 hours. Treatment-related adverse events included fatigue (26/71 patients; 37%) and anemia (25/71 patients; 35%). Grade 3 to 4 adverse events included anemia (17/71 patients; 24%) and thrombocytopenia (13/71 patients; 18%). Sustained PARP inhibition was observed at doses ≥0.60 mg/day. At 1.0 mg/day, confirmed responses were observed in 7 of 14 (50%) and 5 of 12 (42%) patients with BRCA mutation–associated breast and ovarian cancers, respectively, and in patients with pancreatic and small cell lung cancer. Talazoparib demonstrated single-agent antitumor activity and was well tolerated in patients at the recommended dose of 1.0 mg/day

PedGenie: meta genetic association testing in mixed family and case-control designs

<p>Abstract</p> <p>Background-</p> <p>PedGenie software, introduced in 2006, includes genetic association testing of cases and controls that may be independent or related (nuclear families or extended pedigrees) or mixtures thereof using Monte Carlo significance testing. Our aim is to demonstrate that PedGenie, a unique and flexible analysis tool freely available in Genie 2.4 software, is significantly enhanced by incorporating meta statistics for detecting genetic association with disease using data across multiple study groups.</p> <p>Methods-</p> <p>Meta statistics (chi-squared tests, odds ratios, and confidence intervals) were calculated using formal Cochran-Mantel-Haenszel techniques. Simulated data from unrelated individuals and individuals in families were used to illustrate meta tests and their empirically-derived p-values and confidence intervals are accurate, precise, and for independent designs match those provided by standard statistical software.</p> <p>Results-</p> <p>PedGenie yields accurate Monte Carlo p-values for meta analysis of data across multiple studies, based on validation testing using pedigree, nuclear family, and case-control data simulated under both the null and alternative hypotheses of a genotype-phenotype association.</p> <p>Conclusion-</p> <p>PedGenie allows valid combined analysis of data from mixtures of pedigree-based and case-control resources. Added meta capabilities provide new avenues for association analysis, including pedigree resources from large consortia and multi-center studies.</p