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The Poly ADP Ribose Polymerase (PARP) enzymes and DNA repair

PARP, a family of nuclear enzymes, consists of 17 enzymes including PARP-1 and PARP-2 which play a critical role in DNA Base Excision Repair (BER) pathway. Following exposure to DNA damage, PARP-1 and PARP-2 are activated and catalyse the cleavage of Nicotinamide Adenine Dinucleotide (NAD+) to form Poly ADP-Ribose (PAR) polymer. These PAR polymers are added to DNA, histones and DNA repair proteins including PARP, and recruit the repair machinery to repair DNA damage (Weil and Chen, 2011; Yuan et al., 2011).

PARP inhibitors and synthetic lethality

Synthetic lethality is a cellular phenomenon in which the function of two different genes are simultaneously lost causing cell death, whereas cell death does not occur due to loss of either gene function alone. Dysfunctional HRR pathway and inhibition of PARP lead to synthetic lethality in cells (Weil and Chen, 2011; Yuan et al., 2011; Curtin, 2013; Stordal et al., 2013). Different types of PARP inhibitors such as olaparib, veliparib, niraparib and rucaparib inhibit PARP enzyme activity and hinder DNA repair via the BER pathway, resulting in multiple double-strand breaks normally repaired by the HRR pathway. The tumour cells with BRCA1/2 mutation or BRCAness status cannot efficiently repair these double-strand breaks, leading to cell death (Figure 1-13) (Turner and Ashworth, 2011; Lupo and Trusolino, 2014; Michels et

al., 2014). Another mode of action for PARP inhibitors is to trap PARP proteins at sites of

DNA damage, which is highly toxic to cells due to blockade of DNA replication and induction of a replication stress response. Research indicates that these trapped PARP-DNA complexes are more toxic than blocking PARP enzyme activity (Turner and Ashworth, 2011; Murai et al., 2012; Lupo and Trusolino, 2014; Livraghi and Garber, 2015). PARP inhibitors are cytotoxic and proficiently result in synthetic lethality in tumour cells with BRCA1/2 deficiencies or BRCAness more than normal cells (Underhill et al., 2010; Weil and Chen, 2011).

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Figure 1-13: The role of PARP inhibitors in synthetic lethality. Molecular pathways underlying PARP/BRCA synthetic lethality. Red dotted lines indicate processes impaired by PARP blockade in HR-defective cells. In the presence of PARP inhibitors, SSB repair is precluded and either PARP is trapped onto DNA (A) or unrepaired SSBs are converted to DSBs by collision with the replication machinery (B). In both cases, resultant replication fork damage requires operational HR for efficient restart (C). HR-deficient BRCA mutant cells redirect to alternative, error-prone DNA repair pathways (D), undergoing genomic instability and cell death (Lupo and Trusolino, 2014).

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Predictive biomarkers for response to PARP inhibitors

Up to now, several studies have investigated and highlighted the mutation and epigenetic modification of genes implicated in sensitivity to PARP inhibitors. Although germline mutations of BRCA1/BRCA2 (Breast Cancer 1/2 tumour suppressor genes) were considered as robust predictive biomarkers of PARP inhibitor sensitivity, results of clinical trial have shown that the clinical efficacy of PARP inhibitors is not restricted to these genes (Yuan et al., 2011; Brown et al., 2016). A BRCAness phenotype, a biomarker of sensitivity to PARP inhibitors, is a defective HRR status due to epigenetic hypermethylation of the BRCA1 promoter, somatic mutation of BRCA1/2 or dysfunctional mutations in other HRR pathway genes (Konstantinopoulos et al., 2010; Michels et al., 2014; Bowtell et al., 2015). Defects in PTEN (Turner and Ashworth, 2011; Stordal et al., 2013), deficiency or low expression of RAD51,

ATM, ATR, EMSY genes (Weil and Chen, 2011; Ihnen et al., 2013) as well as mutation and

reduced expression of YH2AX (Brown et al., 2016) are proposed as markers of BRCAness status. Furthermore, amplification of AURKA and EMCY genes (Sourisseau et al., 2010; Ihnen

et al., 2013), overexpression of Aurora kinase A and post-translational protein modification

can also be considered as conferring a BRCAness phenotype (Michels et al., 2014). Low expression of genes involved in HRR and response to platinum-based chemotherapy was also reported to be associated with sensitivity to rucaparib (Ihnen et al., 2013).

In regard to PTEN gene, controversial results have been reported amongst which some demonstrated no statistically significant association between PTEN mutation and sensitivity to PARP inhibitors (Ihnen et al., 2013), whereas others showed PTEN mutations sensitize tumour cells to PARP inhibitors through downregulation of RAD51 and impaired HRR (Mendes- Pereira et al., 2009; Turner and Ashworth, 2011; Weil and Chen, 2011; O'Sullivan et al., 2014). The EMCY gene plays a role in HRR as a BRCA2-binding partner and its amplification may result in inactivation and silencing of the BRCA2 pathway in sporadic ovarian cancer (Ihnen

et al., 2013; Michels et al., 2014). Furthermore, amplification or overexpression of the AURKA

gene is implicated to be associated with sensitivity to rucaparib due to inhibition of RAD51 recruitment to DNA double-strand breaks (Ihnen et al., 2013). Another protein reported to be involved in response to PARP inhibitors is FANCF protein which is an adaptor protein stabilising the interaction between FANCC/FANCE and FANCA/FANCG subcomplexes and plays a critical role in the correct assembly of Fanconi anemia, FA, core complex. The FA core complex is necessary for FANCD2 monoubiquitination localized with BRCA1, RAD51 and other DNA repair proteins (Taniguchi et al., 2003). It was also suggested that aberrant

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expression of ETS transcription factors shown in different cancers may repress BRCA1/2. Moreover, interaction of PARP1 binding protein PARPBP, known as PARI, with RAD51 at replication forks may result in inhibition of HRR (Michels et al., 2014). Due to the greater toxicity of trapping PARP-DNA complexes compared to inhibition of PARP enzymatic activity and clinical importance of PARP trapping, PARP expression levels or baseline activity of PARP may be considered as a biomarker for PARP inhibition (Brown et al., 2016).

Resistance to PARP inhibitors

Development of resistance to PARP inhibitors occurs through different mechanisms. Secondary mutations reversing the BRCA deficiency from a mutated reading frame to a normal sequence reading frame leads to resistance to PARP inhibitors. Reversion of mutation is a phenomenon occurring following selective pressure of drug treatment. Aberrant expression and activity of PARP, upregulation of efflux transporters such as p-glycoprotein, and loss of 53BP1 are other mechanisms implicated in resistance to PARP inhibitors (Weil and Chen, 2011; Lupo and Trusolino, 2014; Frey and Pothuri, 2015). Acquired resistance to PARP inhibitors resulting from a secondary mutation has been confirmed to occur in patients (Weil and Chen, 2011).

PARP inhibitors in ovarian cancer

Deficiencies in HRR occur in up to 50% of epithelial ovarian cancers (Mukhopadhyay et al., 2012). BRCA1/2 mutations are present in the germline of 70-85% of patients with inherited ovarian cancer regardless of histological subtypes, and account for 23% of HGSC. The rate of

BRCA1/2 mutation in sporadic ovarian cancer is low and low expression levels of BRCA1/2 is

likely to be an important characteristic of non-inherited ovarian cancer (Weil and Chen, 2011). Some studies have identified impaired HRR pathway status in ovarian cancer cell lines, indicating the possible sensitivity of ovarian cancer patients bearing deficiencies in the HRR pathway other than only BRCA1/2 mutation to PARP inhibitors (Weil and Chen, 2011; Yuan

et al., 2011; Rigakos and Razis, 2012; Ihnen et al., 2013; Stordal et al., 2013; Michels et al.,

2014). Amplification of the EMSY gene is present in about 20% of cases with HGSC and the

FANCF methylation was reported in 21% of ovarian cancer (Rigakos and Razis, 2012).

PARP inhibitors are now undergoing clinical trials as targeted therapy for different types of cancer including ovarian cancer. Rucaparib is currently evaluated as a monotherapy in phase II clinical trials for patients with BRCA-associated ovarian cancer and in combination with chemotherapy for advanced solid tumours. The use of olaparib and veliparib as single agents

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and in combination with chemotherapy is undergoing phases I and II clinical trials for patients with different types of cancer, including ovarian cancer (Anwar et al., 2015; Frey and Pothuri, 2015). ARIEL2, a phase II trial of rucaparib in platinum sensitive, relapsed HGSC, and ARIEL3, a phase II clinical trials of rucaparib maintenance therapy following a platinum treatment in relapsed HGSC and endometrioid ovarian cancer, are ongoing to define a molecular signature of HR dysfunction in ovarian cancer patients (Frey and Pothuri, 2015). Niraparib, CEP-9722 and E7016 are other new PARP inhibitors currently undergoing clinical trials as single agents and in combination with chemotherapy in advanced solid tumours (Anwar et al., 2015).

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1.10 Hypothesis and Aims