Nicola Curtin - Academia.edu (original) (raw)

Papers by Nicola Curtin

Cancer Research, May 1, 2008

406 An important determinant of the susceptibility of cancer cells to DNA damaging anti-cancer th... more 406 An important determinant of the susceptibility of cancer cells to DNA damaging anti-cancer therapeutics is the ability of the cells to repair the DNA damage inflicted upon them. It has therefore been proposed that inhibition of DNA repair processes could lead to the potential therapeutic endpoints of radio- and chemosensitisation. A key kinase involved in the detection, signaling and repair of DNA double strand breaks (DSBs) is the DNA-dependent protein kinase (DNA-PK). DNA-PK is intimately involved in the repair of DNA DSBs by the process of non-homologous end joining (NHEJ). Inhibition of DNA-PK activity through the probe compounds LY294002 (non-selective, IC50 1.5uM) and NU7441 (selective; IC50=13nM) has previously been shown to lead to cellular radio- and chemo-sensitisation. Through the generation of a focused compound library based around NU7441 to improve this compound’s overall biopharmaceutical properties, we have identified the compound KU-0060648. This agent exhibits 20-1000 fold selectivity for DNA-PK over related PIKK enzymes and PI3K family members. KU-0060648 was found not to radio-potentiate DNA-PKcs deficient cells, further highlighting its selective nature in vitro. KU-0060648 was able to inhibit the serine-2056 auto-phosphorylation site of DNA-PKcs with an IC50 of 60nM in FaDu cells and abrogated the phosphorylation of H2AX in response to ionizing radiation (IR). Doses of as little as 100nM were found to radiosensitise FaDu cells (Enhancement Ratio at 10% survival of 2.3), using clonogenic assays as an endpoint. KU-0060648 was able to radiopotentiate 2Gy of IR after only 2 hours of exposure, highlighting its role in disrupting the NHEJ dependent fast component of DNA DSB repair. KU-0060648 was also found to preferentially kill BRCA2-defective and ATM-defective cells in combination with IR or etoposide, suggesting that DNA repair defective tumour cells may provide a therapeutic advantage for this class of agent. Pharmacokinetic profiling of KU-0060648 in mice revealed that the compound showed 100% bioavailability, with a t1/2 of 142 minutes after p.o. dosing. In a fractionated IR (5x2Gy) FaDu xenograft model, KU-0060648, dosed at either 10 or 25mg/kg qd 30 minutes before IR caused significant growth delay compared to the control 5x2Gy group. PK/PD relationships were established to define the exposures and level of DNAPK inhibition required for effective radiosensitisation. Our results to date support the further in vivo evaluation of this novel class of agent as a radiosensitiser prior to clinical evaluation.

Biochemical Society Transactions, Jan 6, 2023

Methods in molecular biology, 2011

This chapter describes some of the techniques in use in our laboratories for the investigation of... more This chapter describes some of the techniques in use in our laboratories for the investigation of PARP inhibitors in clinical medicine. More specifically, we are involved in investigating the utility of PARP inhibitors in the treatment of hematopoietic malignancies. We are also actively investigating the properties of the PARP systems in cell biology. We begin the chapter with a very brief history of the invention and use of PARP inhibitors. We then explain the underlying logic of the use of PARP inhibitors either in combination with chemo- or radiotherapy or as single agents used alone. We then provide in full detail the protocols that we use to study PARP inhibitors in cell biology to identify patients that should be susceptible to PARP inhibitor treatment and to manage and investigate these patients throughout their treatment.

Methods in molecular biology, 2017

Cancer drug discovery and develogment, 2018

ATR is a highly versatile player in the DNA damage response (DDR) that signals DNA damage via CHK... more ATR is a highly versatile player in the DNA damage response (DDR) that signals DNA damage via CHK1 phosphorylation to the S and G2/M cell cycle checkpoints and to promote DNA repair. It is activated by ssDNA, principally occurring due to replication stress that is caused by unrepaired endogenous DNA damage or induced by a variety of anticancer chemotherapy and ionizing radiation. Since an almost ubiquitous feature of cancer cells is loss of G1 control, e.g., through p53 mutation, it is thought that their greater dependence on S and G2/M checkpoint function may render them more susceptible to ATR inhibition. ATR promotes homologous recombination DNA repair and inter-strand cross-link repair. Impairment of ATR function by genetic means or with inhibitors increases the sensitivity of cells to a wide variety of DNA damaging chemotherapy and radiotherapy, with the greatest sensitization observed with gemcitabine and cisplatin. Early inhibitors developed in the 1990s were weak and non-specific but the encouraging data led to the development of more potent and specific inhibitors. We review here the pre-clinical chemo- and radiosensitisation data obtained with these inhibitors that has led to the entry into clinical trial, the potential to combine ATR inhibitors with other DNA repair modulators, and identification of single-agent ATR inhibitor cytotoxicity in cells with activated oncogenes and particular defects in the DDR that may result in greater replication stress or dependence on ATR for survival.

Pharmacology & Therapeutics, Mar 1, 2020

The DNA damage response (DDR) machinery is responsible for detecting DNA damage, pausing the cell... more The DNA damage response (DDR) machinery is responsible for detecting DNA damage, pausing the cell cycle and initiating DNA repair. Ataxia telangiectasia and Rad3-related (ATR) protein is a key kinase at the heart of the DDR, responsible for sensing replication stress (RS) and signalling it to S and G2/M checkpoints to facilitate repair. In cancer, loss of G1 checkpoint control and activation of oncogenes that drive replication, result in cancer cells more likely to enter S phase with increased RS. These cancer cells become more reliant on their S and G2/M checkpoints, making this an attractive anti-cancer target. Targeting ATR is the focus of many oncology drug pipelines with a number of potent, selective ATR inhibitors developed, four (M6620, M4344, AZD6738 and BAY1895344) are currently in clinical development. Here we summarise the pre-clinical data supporting the use of ATR inhibitors as monotherapy and in combination with chemotherapy, radiotherapy and novel targeted agents such as PARP inhibitors. We discuss the current clinical trial data and the challenges of taking ATR inhibitors into the clinic and of identifying biomarkers to aid patient selection.

British Journal of Cancer, Oct 1, 2011

Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to m... more Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to mutations in the BRCA1 or BRCA2 genes. Cisplatin chemotherapy or poly(ADP-ribose) polymerase (PARP) inhibitors were tested for these tumors in clinical trials. In a screen for novel drugs that selectively kill BRCA2-defective cells, we identified 6-thioguanine (6TG), which induces DNA double-strand breaks (DSB) that are repaired by HR. Furthermore, we show that 6TG is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. Spontaneous BRCA1-defective mammary tumors gain resistance to PARP inhibitors through increased P-glycoprotein expression. Here, we show that 6TG efficiently kills such BRCA1-defective PARP inhibitor–resistant tumors. We also show that 6TG could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene. Although HR is reactivated in PARP inhibitor–resistant BRCA2-defective cells, it is not fully restored for the repair of 6TG-induced lesions. This is likely to be due to several recombinogenic lesions being formed after 6TG. We show that BRCA2 is also required for survival from mismatch repair–independent lesions formed by 6TG, which do not include DSBs. This suggests that HR is involved in the repair of 6TG-induced DSBs as well as mismatch repair–independent 6TG-induced DNA lesion. Altogether, our data show that 6TG efficiently kills BRCA2-defective tumors and suggest that 6TG may be effective in the treatment of advanced tumors that have developed resistance to PARP inhibitors or platinum-based chemotherapy. Cancer Res; 70(15); 6268–76. ©2010 AACR.

Up to 50% of epithelial ovarian cancers (EOC) display defects in the homologous recombination (HR... more Up to 50% of epithelial ovarian cancers (EOC) display defects in the homologous recombination (HR) pathway. We sought to determine the ramifications of the homologous recombination–deficient (HRD) status on the clinicopathologic features, chemotherapy response, and survival outcomes of patients with EOCs. HR status was determined in primary cultures from ascitic fluid in 50 chemotherapy-naïve patients by a functional RAD51 immunofluorescence assay and correlated with in vitro sensitivity to the PARP inhibitor (PARPi), rucaparib. All patients went on to receive platinum-based chemotherapy; platinum sensitivity, tumor progression, and overall survival were compared prospectively in HR-competent versus HRD patients. Compared with HR-competent patients, the HRD group was predominantly serous with a higher median CA125 at presentation. HRD was associated with higher ex vivo PARPi sensitivity and clinical platinum sensitivity. Median follow-up duration was 14 months; patients in the HRD g...

European Journal of Cancer, 2020

ePoster, 2019

Introduction/Background In the phase III LACC cervical cancer trial, shorter disease-free surviva... more Introduction/Background In the phase III LACC cervical cancer trial, shorter disease-free survival and overall survival was observed in patients treated with minimally invasive radical hysterectomy in comparison to traditional open surgery. One hypothesis for this difference is an adverse effect of the prolonged exposure to the hypoxic, CO2-rich, acidotic environment created during minimally invasive surgery. Greater cervical tumour hypoxia-inducible factor 1α (HIF-1α) indicating tissue hypoxia has previously been shown to be a strong independent prognostic marker in early stage cervical cancer. This pre-clinical study aimed to evaluate the effect of hypercapnia, hypoxia and pH on cervical cancer cell growth. Methodology Three cervical cancer cell lines (Hela, SiHa, C33ca) were incubated in variable CO2(5–20%) and O2(5%–20%) conditions, in an InvivO2 incubator with HEPES medium for 3 doubling times, standardised for humidity and temperature. Growth rates were assessed by Sulforhodamine B colorimetric (SRB) assays with measurement of pH in spent media. Matched controls were incubated in routine physiological conditions. Results There was a 1.5-fold difference in doubling time of cell lines under physiological conditions with doubling times ranging from 22 to 34 hours. Incubation in a hypercapnic environment persistently resulted in growth inhibition across all three cell lines at all seeding densities, with mean growth inhibition of 45.0% (SD 16.0%) at 20% CO2. Inhibition of growth was greatest in C33a, the most rapidly dividing cell line with mean reduction in growth of 55.5%. pH remained within physiological range falling by a mean of 0.45 to a minimum of 7.12. Conclusion Hypercapnia results in growth inhibition in cervical cancer cell lines, independent of acidosis. It is unlikely that exposure to the CO2 rich intra-operative environment during minimally invasive surgery is responsible for the poor prognosis reported in the LACC trial. Disclosure Nothing to disclose.

Comprehensive Medicinal Chemistry III, 2017

The DNA damage response (DDR), comprising DNA repair and cell-cycle checkpoint pathways, is an at... more The DNA damage response (DDR), comprising DNA repair and cell-cycle checkpoint pathways, is an attractive target for cancer therapy. DDR inhibitors have been developed to (i) overcome DDR-mediated resistance to DNA-damaging anticancer therapy and (ii) exploit DDR dysfunction in cancer by targeting complementary pathways. The latter approach, exemplified by poly(ADP-ribose) polymerase (PARP) inhibitors in tumors with BRCA1 or BRCA2 mutations, is predicted to be more tumor-selective.

Cancer Research, 2015

Background The NAD+-requiring enzyme 3-phosphoglycerate dehydrogenase (PHGDH) diverts glycolytic ... more Background The NAD+-requiring enzyme 3-phosphoglycerate dehydrogenase (PHGDH) diverts glycolytic flux into serine production and folate metabolism by catalyzing the oxidization of 3-phosphoglycerate to phosphohydroxypyruvate. The PHGDH gene is located on chromosome 1(1p12), a region frequently amplified in melanoma and certain breast cancer forms. PHGDH knockdown in cells with amplified PHGDH or overexpressing PHGDH at the protein level resulted in cell growth inhibition. In addition ectopic overexpression of PHGDH in a non-tumorigenic cell line induced morphological changes characteristic of transformation. Therefore, inhibitors of PHGDH may be therapeutically valuable. No PHGDH inhibitors have been reported to date, our aim is to develop PHGDH inhibitors targeting the cofactor (and substrate) binding site. Methods Full length PHGDH and the catalytic subunit (sPHGDH) were expressed in, and isolated from E. coli. PHGDH was found to co-purify with its cofactor, making it necessary to...

Nature Reviews Clinical Oncology, 2019

Cancers

Cisplatin-based chemo-radiotherapy (CRT) is the standard treatment for advanced cervical cancer (... more 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 accumulatio...

Five PARP inhibitors (PARPi) are approved for cancer treatment, they exploit cancer-specific defe... more Five PARP inhibitors (PARPi) are approved for cancer treatment, they exploit cancer-specific defects in homologous recombination repair (HRR) to selectively kill tumour cells. Continuous PARP inhibition is required for single-agent anticancer activity. PARPi are also being investigated with ATR inhibitors clinically. We previously showed rucaparib caused prolonged PARP inhibition. Here we aimed to determine if this property was unique to rucaparib or common to other PARPis and the implications for scheduling with an ATR inhibitor (VE-821). Durability of PARP inhibition was determined at 0, 1, 24, 48 and 72 h after a 1 h pulse of 1μM of rucaparib, olaparib, niraparib, talazoparib or pamiparib in IGROV-1 (human ovarian cancer) cells. Inhibition of PARP was sustained to a variable degree with all inhibitors, but reduced with time. Rucaparib caused the most persistent inhibition of PARP activity, which was maintained at ≥75% for 72 h after drug withdrawal. In contrast, only 12% inhibiti...

Cells

Ataxia telangiectasia and Rad-3 related kinase (ATR) signals DNA lesions and replication stress (... more Ataxia telangiectasia and Rad-3 related kinase (ATR) signals DNA lesions and replication stress (RS) to the S and G2/M checkpoints and DNA repair pathways making it a promising target to exploit the dysregulated DNA damage response in cancer. ATR inhibitors (ATRi) are under clinical investigation as monotherapy and in combination with other anticancer agents. Molecular determinants of sensitivity to ATRi are common in ovarian cancer, suggesting the therapeutic potential of ATRi. We investigated the cytotoxicity of the ATRi, VE-821, in a panel of human ovarian cancer cell lines. High grade serous (HGS) cell lines were significantly more sensitive to VE-821 than non-HGS (p ≤ 0.0001) but previously identified determinants of sensitivity (TP53, ATM and BRCA1) were not predictive. Only low RAD51 (p = 0.041), TopBP1 (p = 0.026) and APOBEC3B (p = 0.015) protein expression were associated with increased VE-821 sensitivity. HGS cells had increased levels of RS (pRPASer4/8 and γH2AX nuclear i...

Assess the baseline expression of the genes in the Oncology Biomarker Panel across the panel of o... more Assess the baseline expression of the genes in the Oncology Biomarker Panel across the panel of ovarian and cervical cancer cell lines. This data will then be compared to the sensitivity of the cell lines to DDR inhibitors. The expression data will be correlated with the sensitivity of the cell lines to see whether determinants of sensitivity described in the literature translate. A hierarchy of genes will be determined. It is expected that the cells that are more sensitive to DDR inhibitors will have decreased expression of the genes known to be determinants of sensitivity.