Programmed cell death-1 (PD-1) is an immune receptor mainly expressed on activated CD4+ and CD8+ T cells or peripheral B cells [109]. Interaction of PD-1 and its ligand PD-L1 is critical to control the immune response, providing its binding constitutes an immune inhibiting checkpoint which leads to immune evasion. PD-L1 can be induced in cancer cells by a variety of stimuli such as T cell interferon gamma production or ionizing radiation (IR) [110] among others. The radiation-dependent PD-L1 activation seems to be related to DSBs and DNA repair response pathways and synergistically enhances antitumor immunity if applied together with immune checkpoint inhibitors [111]. This enhanced antitumor immunity seems to be related to increased mutational burden which increases neoantigen repertoire and tumor infiltrating lymphocytes (TILs) [112].

Interestingly, several studies also reported increased intratumoral CD8<sup>+</sup> T cell infiltration and interferon production after PARP inhibition [99,113]. Nevertheless, this increased presence of antitumor immunity and TILs can be counterbalanced by PARPi-induced expression of PD-L1 which subsequently activates the PD-1/PD-L1 immune checkpoint pathway [114].

Taken together, these observations provide further rationale for the combinatorial uses of DNA damaging agents, PARP inhibitors, and immune checkpoint blockade, in order to increase the benefits of the enhanced antitumor immunity avoiding the immunosuppressive effects of PD-L1 overexpression (Figure 7).

**Figure 7.** PARP inhibitors together with immune checkpoint inhibitors potentiate antitumor immune-mediated response. Activated T lymphocytes react against tumor antigens. The inhibition of PARP increases the number of lymphocytes infiltrating the tumor after the upregulation of chemokines, promoting an immune response mediated by CTLs. In spite PARP inhibitors modulate positively the upregulation of PDL-1 (favouring tumor scape from immune control) the anti-CTLA4 activates T cells to promote an antitumor response. Anti PD1/PDL-1 reverses CTL inhibition provoked by PARP inhibitor –induced PDL-1 expression. In this way, anti-PD1/PDL-1 can synergize with PARP inhibitors to ammeliorate antitumor immune response.

#### **3. PARylation in Autophagy**

One of the main characteristics of the metabolically hyperactive cancer cells is their high demand for nutrients and oxygen from their microenvironment. This makes them vulnerable to the deficiency of both oxygen (hypoxia) and nutrients (starvation). Both situations can induce metabolic stress leading to PARP-1 over activation; during these situations, cells must activate the autophagic pathway as a response to adapt and survive. In this context it has been shown that one of the first consequences of autophagy is the activation of poly-(ADP-ribosyl)ation. The combined effect of PARP-1 activity leading to PAR modification of AMPK (the main cell energy sensor) and the nutritional status (measured via mTORC1 activation) are fundamental during the first stages of autophagy [115].

The autophagyc pathway is present in all eukaryotic cells. It consists on a "self-eating" process necessary for the maintenance of the cell homeostasis. Through this lysosomal-dependent pathway different cellular organelles, portions of the cytosol and chaperone-associated cargoes are enfolded in double-membrane spheroids called autophagosomes, being then hydrolyzed by lysosomal enzymes [116]. Different cellular stresses can fire the autophagic response: hypoxia, pH variation, mitochondrial ROS production, DNA damage, intracellular pathogens, unfolded proteins or endoplasmic reticulum (ER) stress among others.

Expanding evidence indicates how autophagy is induced after DNA breakage. Ataxia Telangiectaxia Mutated (ATM) has been described as an important link between DNA Damage Response (DDR) and the induction of autophagy [117]. In response to DNA damage by mitochondrial ROS, external toxins or irradiation, ATM is auto phosphorylated within a Mre11, Rad50 and Nbs1 (MRN) multiprotein complex that binds DSBs. Once active ATM induces the activity of AMPk and its target tuberous sclerosis protein (TSC2), this leads to the inhibition of mTORC1, promoting the formation of autophagosomes dependent of Unc-51 like autophagy activating kinase (ULK1) [118]. This is how autophagy acts as a catabolic pathway of rapid energy recovery that the cell will use to grow and expand. However, this rapid acquisition of energy has two sides; on the one hand, it responds to a pro-survival role as long as the levels of autophagy remains biologically sustainable, this way, the degradation of excess vital components of the cell is avoided; but autophagy might also become a process of cell death due to the accumulation of autophagosomes degrading essential cellular structure;, this process is called autophagic cell death (ACD).

As we know, PARPs are important guardians of the genome integrity and they have been shown to link DNA damage response with the activation of autophagy [115,119]. Autophagy is also initiated in several settings of response to chemotherapy and mediated by PARP-1. Doxorubicin treatment leads to over-activation of PARP-1, followed by ATP and NAD+ depletion, triggering the non-toxic accumulation of autophagosomes [19]. In response to methylnitronitrosoguanidine (MNNG) double knockout Bax−/−Bak−/−MEFs activate PARP-1, reducing intracellular ATP levels and activating AMPk pathway and mTORC1 down-regulation. Suppression of AMPk pathway blocks MNNG-induced autophagy and enhances cell death [19]. The same results were found in a nasopharyngeal carcinoma model exploring over-activation of PARP-1, upon ionizing radiation there is a PARylation-dependent energy depletion and up-regulation of AMPk and ULK1 pathways [120]. All these studies have demonstrated that autophagy should be contemplated as a target in cancer during the induction of DNA damage and consequently, new strategies based on the synthetic lethality concept during PARPi must be explored.

In addition to DNA damage, nutrient deprivation can be considered as the most physiological stimulus to induce reversible autophagy. Recent studies of our group have implicated PARP-1 in autophagy induced by nutrient starvation [115,121]. The absence of PARP-1 or the use of PARP inhibitors (PJ34, DPQ and Olaparib) delay starvation-induced autophagy. Mitochondrial ROS accumulation derived from starvation promoted DNA damage and PARylation signaling from PARP-1 activation, which triggers ATP depletion, AMPk activation and mTORC1 inhibition. The lack of PARP-1 activity compensates ATP depletion compromising the cascade AMPk/mTORC1 inhibition/ autophagy. Starved PARP-1-deficient/inhibited cells showed increased apoptotic cell death [115]. In order to analyze the in vivo consequences of PARP-1 ablation on autophagy both PARP-1+/+ and PARP-1−/−pups were starved for short periods of time, concluding that PARP-1−/−neonates display a deficient liver autophagy response following acute starvation, showing that PARP-1 activity and PAR formation are key players in the decision of the cell to engage autophagy [115].

The absence of PARP-1 compromises the activation of AMPk, more precisely the isoform AMPkα, reducing phosphorylation on Thr172 [121,122]. A nuclear subpopulation of AMPkα was detected in the breast cancer cell line MCF7, where it forms a stable complex with PARP-1; moreover the activation of nuclear AMPkα requires two essential events: Firstly, starvation-induced ROS must be imported to the nucleus to generate DNA damage and PARP-1 activation. Secondly, PARP-1 modifies by PARylation the nuclear population of AMPkα. Energy depletion associated with starvation was increased during non-lethal over-activation of PARP-1 in response to ROS, resulting in a feedback loop that favored the interaction between PARP-1/AMPkα and promoted the activation of LKB1. The mechanism of modulation by PARylation of LKB1 is not clearly understood but using PARPi or during specific assays with siRNA PARP-1, LKB1 activation is compromised [121].

In non-starved cells, PARP-1 forms a complex with AMPK; after starvation PARP-1 is activated and PARylation disrupt the PARP-1/AMPK complex, this triggers the nuclear export of PARylated AMPkα to the cytoplasm. This significant PARylated AMPkα population, is recognized by the active form of LKB1 promoting the phosphorylation on regulator site Thr172 of AMPkα. Again, a positive feedback loop takes place between the cytosolic energy depletion and PARylated AMPkα to potentiate the activation of the cytosolic AMPkα population by LKB1. The presence of covalently PARylated LKB1 has not been demonstrated but the effect of PARPi using PJ34 or olaparib suggested the implication of PAR on LKB1. These data place the nucleus (a classical cellular component not associated with autophagy) and the interaction PARP-1 and AMPk as an initial sensor of the metabolic alterations derived from perturbations in the nutritional extracellular status, not necessarily related with the alterations in genomic integrity. PARylation could be considered as an alert indicator of changes in the energy balance in cancer cells (Figure 8).

**Figure 8.** Regulation of autophagy by PARylation. (1) PARP1 forms a complex with AMPKα in nucleus. (**A**) PARP activation conditions: During starvation-induced autophagy, ROS release leads DNA damage and PARP1 overactivation. Self-PARylated PARP1 interact with AMPKα1 subunit (2). The complex is disrupted and PAR-AMPKα is exported to cytosol (3). The presence of PAR-AMPK and the continuous absence of amino acids and ATP depletion favor total activation of AMPKα population by LKB1, inhibition of mTORC1, interaction PAR-phospho-AMPK/ULK1, and autophagosome formation (4). LKB1 activity is presumably modified in a PARylation-dependent manner. (**B**) PARP inhibited: Starvation-induced ROS production was abrogated after PARP inhibition. Following AMPKα1/PARP1 interaction (1) the AMPKα1 subunit is not PARylated and AMPK nuclear export is inhibited (2 and 3). In spite of nutrient and energy depletion, AMPKα is inactive; mTORC1 is partially activated and interacts with ULK1, favoring its inhibition (4). Finally, the autophagosomes production will be delayed.

#### **4. Cancer Initiating Cells**

Cancer Stem Cells (CSCs) are defined as the tumor cell subpopulation that present remarkably high plasticity, considering "plasticity" as the ability of pluripotent cells to induce trans-differentiation. The first evidence of cancer initiating stem-like cells was described in 1994 by Lapidot et al. and Caceres-Cortes et al. after injecting different leukemia cell subpopulations in mice [123]. Through the years, CSCs were determined in a wide multitude of cancers like breast [124], gliomas [125], prostate [126], melanoma [127], lung [128,129], colon [130,131], pancreas [132], head and neck squamous cell [133], liver [134] and renal carcinoma [135].

CSCs are a very aggressive subpopulation of cancer cells characterized by their self-renewal capacity, tumor initiation ability, resistance to chemo- and radiotherapy and multi-lineage differentiation (stemness) [125,136,137]. The presence of this subpopulation in tumors is considered to be a marker of bad prognosis and therefore they have been proposed as target in cancer therapy.

For several years, it has been known that reprogramming processes are led by specific genetic programs. Yamanaka et al. demonstrated that the ectopic expression of four genes (Oct4, Sox2, c-Myc and KLF4) in mouse fibroblast transforms them in embryogenic stem cells. These cells, denominated by the authors as induced-pluripotent stem cells (iPSCs), were able to generate cells from any germ layer [138]. This breakthrough made Shiya Yamanaka win the Noble Price of Medicine in 2012. Today, these genes are known as Yamanaka factors and several researches link the expression of these genes with cancer [138–140]. In fact, the risk of tumor development is the most important limitation in the application of iPS cell-based therapy. Tumor cells present processes of trans-differentiation and dedifferentiation or reprogramming acquiring features of CSCs [138].

In 2012, it was demonstrated that PARP-1 is involved in the reprograming process, promoting iPSCs. An intense PARylation was detected in iPSCs. Moreover, PARP-1 knockdown reduced the capacity of iPSCs generation after Yamanaka factors overexpression. It has been found that c-Myc directly regulates PARP-1 expression and PARylation. Over-expression of PARP-1 compensates the knockdown of c-Myc in reprogramming in MEFs [141]. In 2009, Gao et al. demonstrated that PARP-1 PARylates and controls Sox2 levels, regulating its activity. They found that PARylated Sox2 increases its stability and therefore induces the expression of its target gene FGF4. PARP inhibition has been observed reverting this scenario [142,143]. To reprogram cells to iPSCs by Yamanaka factors, an epigenetic remodeling is also necessary. PARP-1 regulates modification in histones that alter the chromatin pattern driving to pluripotent cell phenotype. In this line, PARP-1 is necessary to promote the access to chromatin of Oct4 [144].

Telomerase (TERT) is a reverse transcriptase necessary for telomeres elongation in embryonic stem cells. This retrotranscriptase is highly expressed in tumors and the expression is related with stemness capacity. KLF4 directly interacts with the promoter of TERT. PARP-1 interacts with KLF4 and mediates the expression of TERT in CSCs. In fact, PARP-1 suppression dramatically reduces the recruitment of KLF4 to the promoter of TERT, reducing its expression [145]. C-Myc has also been related with TERT expression, suggesting another possible relationship between PARP-1 and telomerase [146].

Due to the high resistance to radio and chemotherapy presented by CSCs, many studies focused their interest in this subpopulation as a target for future treatment. In glioblastoma (GBM), one of the most aggressive malignancies, the presence of CSCs was proposed as the main cause of tumor relapse [138]. Furthermore, reprogramming events driving the formation of endothelial cells from tumor cells have been described [147]. The importance of CSCs in this kind of tumors led Vescovi el al. to propose the differentiation of CSCs as a target, with the aim of reducing the malignancy of this brain cancer [148]. They found that glioma stem cells treated with bone morphogenetic proteins (BMPs), cytokines belonging to TGF-β superfamily, differentiated CSCs to non-stem glioma cells. BMPs interact with membrane receptors inducing differentiation through SMADs signaling pathway. It has been demonstrated that PARP-1 negatively regulates this pathway at different levels. Ectopic expression of PARP-1 suppresses the signaling mediated by BMP. On the contrary, knockdown of PARP-1 promotes BMP-mediated differentiation. Furthermore, PARG, an enzyme responsible for PAR degradation, plays a positive role in the pathway [149,150]. On the other hand, SMAD signaling activation can be a double-edged sword in GBM because the stimulation of this pathway with TGF-β induces the expression of leukemia inhibitory factor (LIF), a cytokine that induces maintenance of "stemness" capacity [151].

#### **5. Conclusions and Perspectives**

The PARP superfamily consist of a group of proteins characterized by the presence of what is called a "PARP signature" on their sequences. Their main characteristic activity is the process referred to as PARylation. Through NAD+ and ATP consumption, ADP-Ribose is generated and then transferred as poly or mono ADP-ribose to different target factors. These modifications alter their activity or stability having relevant implications on the cellular metabolism. Moreover, excessive PARylation has an impact itself: free PAR can function as a signaling molecule and its synthesis may blunt NAD+ and ATP levels producing cell death.

PAR synthesis is activated mostly during DNA damage, altered DNA configuration or when PARP present some posttranscriptional modifications. As we summarized in this review, PARP activation has key repercussions on the cell fate affecting processes like DNA repair, transcriptional regulation, DNA remodeling, hypoxic response, epithelial mesenchymal transition, angiogenesis, autophagy, inflammation and cancer stem cell programming. All these processes lead to changes in survival, proliferation, differentiation, or even malignant transformation. Considering the relevance of the previously enumerated processes, it is easy to understand the relevance of PARP during cancer development. Cells overexpressing PARP will be more likely to repair DNA damage induced by genotoxic agents, they will adapt better to hypoxia and will be prone to produce metastasis through angiogenesis and EMT. Four PARP inhibitors have been already approved by the FDA (olaparib, rucaparib, niraparib and talazoparib) and they are used today as a result of their ability to generate "synthetic lethality" on BRCA 1/2 mutated tumors (see [152] for a review). However, ongoing advanced clinical trials will most likely expand their prescription as is the case for the combination of PARP inhibitors with classical therapies (Table 1) and with anti-angiogenic treatment (Table 2).

Currently, one of the limitations facing this therapeutic option is considering mostly on the BRCA mutated cells as HR deficient. Knowing that more than 100 genes are involved on this DNA repair pathway, it looks likely that BRCA proficient cells could be still HR defective due to other mutations. Measuring the whole genomic instability within a tumor by surveying the loss of genetic heterozygosity, telomeric allelic imbalance or the extent of somatic mutations, could be a more precise approach to define PARP inhibition sensibility, making more patients candidate of this therapeutic approach.

Another important limitation that needs to be worked out is the emergence of resistances, especially during PARP inhibition in monotherapy (Figure 9).

**Figure 9.** Summary of PARP inhibition approaches during cancer treatments, different possible outcomes and ways designed to overcome the possible appearance of resistances.

*Cancers* **2020**, *12*, 739

**Table 2.** Di fferent clinical trials on cancer treatment combining PARP inhibition with anti-angiogenic strategies.

The generation of a selective force (like the caused by a drug treatment) in a highly mutagenic context can lead to the selection of resistant clones. They will then reproduce, forming a relapsed tumor resistant to the original treatment. To avoid this undesired consequence, the selective pressure can be reduced by spacing the treatments or by combining it with inhibitors for different targets, generating a constant pressure over the tumor while generating reduced selective forces.

Another way to avoid resistance is to study the genetic background and predict the more probable resistance mechanisms to arise, then combining the PARP inhibitor with secondary inhibitors for the factors related with the expected resistance. It is known that HR restoration, checkpoints activation, stalled DNA protection or drug efflux among others, can lead to PARP inhibition resistance. Combining PARP blockade with inhibitors for the possible ways of scape the treatment is being tested in clinical trials in order to make PARP inhibition more effective.

Based also in the studies summarized here, we propose that PARPi is an expansive field that may have therapeutic value beyond synthetic lethality. To this end, a precise comprehension of the implications of the different PARPs with PARylation in the complex tumor ecology is needed, including analysis of PARylation in both tumor and associated non-tumor cells, using single cell analysis and its consequences in tumor adaptation to hostile conditions. In this context, we can affirm that the use of PARP inhibitors against cancer treatment is not just a promising field but a reality and the challenge exists to widen their use by identifying new properties and deepening the role of PARP in tumor biology.

**Funding:** This work was supported by Ministerio de Economía y Competividad, Spanish Ministry of Economy and Competitiveness SAF2012-40011-C02-01, SAF2015-70520-R, RTICC RD12/0036/0026, RTI2018-098968-B-I00, CIBER Cáncer ISCIII CB16/12/00421 and Fundación Domingo Martínez to FJO. JMMC, is recipient of a FPI fellowship from Spanish Ministerio de Ciencia; MFC and EZM are recipients of FPU fellowships from the Spanish Ministerio de Ciencia.

**Conflicts of Interest:** The authors declare "no conflict of interests".

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### **ATM-Deficient Cancers Provide New Opportunities for Precision Oncology**

**Nicholas R. Jette 1, Mehul Kumar 1, Suraj Radhamani 1, Greydon Arthur 1, Siddhartha Goutam 1, Steven Yip 2, Michael Kolinsky 3, Gareth J. Williams 1, Pinaki Bose <sup>1</sup> and Susan P. Lees-Miller 1,\***

Received: 14 February 2020; Accepted: 12 March 2020; Published: 14 March 2020

**Abstract:** Poly-ADP ribose polymerase (PARP) inhibitors are currently used in the treatment of several cancers carrying mutations in the breast and ovarian cancer susceptibility genes *BRCA1* and *BRCA2*, with many more potential applications under study and in clinical trials. Here, we discuss the potential for extending PARP inhibitor therapies to tumours with deficiencies in the DNA damage-activated protein kinase, Ataxia-Telangiectasia Mutated (ATM). We highlight our recent findings that PARP inhibition alone is cytostatic but not cytotoxic in ATM-deficient cancer cells and that the combination of a PARP inhibitor with an ATR (ATM, Rad3-related) inhibitor is required to induce cell death.

**Keywords:** ATM; olaparib; ATR; PARP; PARP inhibitor; prostate cancer; pancreatic cancer; lung cancer

#### **1. PARP and PARP Inhibitors**

Genome instability, characterized by the accumulation of mutations and chromosomal alterations in the genome, is both a hallmark and a driver of cancer [1,2]. Yet the same genomic alterations that predispose a cell to cancer may also render cells susceptible to targeted therapies. Accordingly, a goal of precision oncology is to achieve better cancer control by targeting therapy to specific genetic defects or aberrations in the tumour, while causing less damage to normal tissue and consequently, fewer side-effects. One of the most dramatic examples of success in this area has been the use of poly-ADP-ribose polymerase (PARP) inhibitors in the treatment of patients with tumours that harbour inactivating mutations in the breast and ovarian cancer susceptibility genes, *BRCA1* and *BRCA2*.

PARP was identified in the 1960s as an enzyme that metabolizes nicotinamide adenine dinucleotide, NAD<sup>+</sup> [3]. Early studies indicated that inhibition of PARP blocked repair of DNA strand breaks and PARP inhibitors were soon considered as potential radiation sensitizers [4]. The first PARP inhibitor, a simple analogue of nicotinamide was generated in 1971 [5] and, given the reported roles of PARP in cell death and ischemia as well as DNA repair, there was increasing interest in the clinical applications of PARP inhibition [6,7]. PARP is now established as one of a family of poly-ADP polymerases of which PARPs 1, 2 and 3 have roles in the DNA damage response [8]. Of these, PARP 1 is the most well-studied and the one to which we will refer in this review. PARP1 binds avidly to ends of DNA that occur at single and double DNA strand breaks and then, in a process called PARylation, auto-modifies to create long polymers of poly-ADP-ribose (PAR). PAR chains interact with proteins involved in DNA

repair and other pathways, recruiting them to sites of DNA damage [9]. Importantly, PARylation also serves to disengage PARP from DNA ends [10]. Most PARP inhibitors in use today prevent PARylation, leading to trapping of PARP at DNA ends [11]. It was originally proposed that PARP inhibition blocked base excision repair and single strand break repair pathways thus increasing reliance on BRCA-dependent repair. However, this model has been challenged [12] and more recent studies have shown that olaparib reduces cell proliferation by inducing replication stress [13] and that olaparib sensitivity is due to engagement of homologous recombination repair (HRR) at replication forks [14].

In 2005, two seminal papers were published demonstrating that breast cancer cells with siRNA depletion of BRCA1 or BRCA2 were exquisitely sensitive to the PARP inhibitor NU1025 [15,16]. *BRCA1* and *BRCA2* are cancer predisposition genes that are inactivated in ~25% of inherited breast cancers, ~15% of all ovarian cancers and several other cancers, suggesting that PARP inhibitors might have potential in treating a wide-range of patients with BRCA-deficient tumours [17]. The PARP inhibitor AZD2881, also known as olaparib or LynparzaTM, showed promise in mouse models of breast cancer [18] and quickly moved into clinical trials, showing anti-tumour activity in BRCA-mutated cancers, even in phase I studies [19]. Olaparib, the first PARP inhibitor to gain regulatory approval, is now FDA-approved in advanced ovarian [20], breast [21], pancreatic [22] and prostate cancers [23], with the PARP inhibitors rucaparib [24], niraparib [25] and talazoparib [26] also FDA-approved in varying indications [17,27].

BRCA1 and 2 play critical roles in detection, signalling and repair of DNA double strand breaks (DSBs) via the HRR pathway. HRR is active in S phase at stalled replication forks and in G2 phase of the cell cycle after DSBs have been resected to contain long ssDNA overhangs on their 3' ends [28]. These long regions of ssDNA are bound by replication protein A (RPA) and BRCA2 plays a role in the replacement of RPA with RAD51, the protein that initiates strand invasion and the search for a homologous DNA sequence during HRR [28]. BRCA1 interacts with BRCA2 via the PALB2 protein, and is recruited to DNA damage-induced foci where it participates in activating DNA repair and cell signalling pathways [29]. Given the encouraging early results showing PARP inhibitor sensitivity in BRCA-deficient cells, screens were initiated to identify other proteins that when knocked down with siRNA might confer sensitivity to PARP inhibitors [30–32]. One of these was Ataxia Telangiectasia Mutated (ATM).

#### **2. ATM**

ATM is a member of the phosphatidylinositol-3 kinase-like (PIKK) family of serine/threonine protein kinases with critical roles in the cellular response to DNA damaging agents, such as ionizing radiation (IR), that produce DSBs [33]. Like other members of the PIKK family, ATM is a large protein of over 350 kDa that is composed of an extended N-terminal region containing multiple HEAT (Huntingtin, Elongation factor 3, A subunit of protein phosphatase 2A and mammalian Target of rapamycin) repeats and a C-terminal kinase domain that has amino acid similarity to phosphatidyl inositol-3 kinase (PI3K) and is flanked and stabilized by conserved FRAP–ATM–TRRAP (FAT) and FAT-C domains. Generation of DSBs and/or changes in chromatin structure lead to activation of ATM and its autophosphorylation on serine 1981 [34]. Activated ATM phosphorylates a multitude of downstream targets including p53, checkpoint kinase 2 (Chk2) and histone H2AX [35]. Indeed, phospho-proteomics studies have identified hundreds of PIKK-dependent, DNA damage-induced phosphorylation events in cells [36,37]. Consistent with its role in the repair of IR-induced DSBs, cell lines with loss or inactivation of ATM are radiation sensitive, have cell cycle checkpoint defects [38] and have defects in slow repair of complex DNA damage lesions and DSBs in the context of heterochromatin [39]. Recently, roles in preventing premature aging [40] and in reactive oxygen sensing [41] have also been reported.

Germline inactivation of both copies of the *ATM* gene causes Ataxia-Telangiectasia (A–T), a devastating childhood condition characterized by ataxia (wobbly gait), telangiectasia (blood vessel abnormalities) and progressive neurodegeneration, particularly in the cerebellum, that renders its victims wheelchair-bound. A–T patients also have immune defects and cancer predisposition and usually succumb to their condition in their early twenties [42]. Accordingly, cell lines derived from A–T

patients and ATM knock out mice are hypersensitive to IR and other chemotherapeutic agents [43,44], raising the possibility that cancers with loss of ATM may be more sensitive to DNA damaging agents than their ATM-proficient counterparts [45].

#### **3. Targeting ATM-Deficient Cancers**

Genome sequencing has revealed that *ATM* is mutated in a variety of human cancers, including mantle cell lymphoma (MCL), colorectal, lung and prostate cancers. Analysis of *ATM* mutation frequency in The Cancer Genome Atlas (TCGA) cohort using c-Bioportal [46,47] indicates that ATM is mutated in approximately 5% of all cancers, with some, such as MCL, with a much higher mutation frequency of ~40% (Figure 1A). Similarly, ATM is mutated in ~20% of colorectal and uterine cancers and approximately 10% of prostate and lung cancers (Figure 1A). The vast majority of these mutations are missense mutations and are scattered throughout the coding region (Figure 1B and [45]). An exception is R377C/H, which occurred in 74 of the 2263 (~3%) of the cancers queried (Figure 1B), and has been identified as a cancer mutation hotspot [48,49]. The R337C/H mutation was prominent in colorectal cancer, but not in prostate, lung or pancreatic cancer (Figure 1C–F). Although the functional consequences of this and most other *ATM* mutations is not known, given that in A–T many mutations in *ATM* induce protein truncation, protein destabilization and resulting loss of function [50], combined with the fact that siRNA-mediated loss of ATM in cancer cell lines results in PARP inhibitor sensitivity [30–32], it seems likely that many cancers with *ATM* mutation that lead to loss of function could be candidates for PARP inhibitor treatment.

Given that *ATM* is mutated or lost in over 40% of MCL [51], we and others examined the effects of ATM loss on PARP inhibitor sensitivity in human lymphoma cell lines that lack ATM protein expression. These cell lines were more sensitive to olaparib than their ATM-proficient counterparts in both cell line and animal models [52–54]. Moreover, the ATM kinase inhibitor KU55933 enhanced sensitivity to olaparib in ATM-proficient cells indicating that ATM kinase activity protects from PARP inhibitor sensitivity [53]. Similar results were observed in gastric cancer cell lines [55], and in colorectal cancer cell lines with shRNA depletion of ATM [56]. Deletion of ATM in mouse models of lung cancer and pancreatic cancer also induced sensitivity to PARP inhibitors and/or DNA damaging agents, as did inhibitors of the related protein kinase ATR (ATM and Rad3-related) [57,58].

We observed that in MCL, gastric and colorectal cancer cell lines with loss or down regulation of ATM, sensitivity to olaparib was enhanced when *TP53* was also mutated or deleted [52,53,55,56]. However, in mouse models, both *TP53*-proficient and deficient cells were sensitive to olaparib [58]. Therefore, the effect of p53 status on PARP inhibitor sensitivity requires further clarification. Although co-mutation of both *ATM* and *TP53* is rare [59], co-mutation has been shown to occur in 2–3% of non-small cell lung cancer where it increases tumour mutation burden and correlates with better response to immune checkpoint therapy [60], suggesting additional opportunities for targeted therapy for ATM-deficient tumours.

To address the mechanism of olaparib-induced cell sensitivity in human cells lacking ATM, we used CRISPR/Cas9 to delete ATM from the p53-proficient lung adenocarcinoma cell line, A549. In keeping with recent findings, olaparib alone reduced cell proliferation [13], but surprisingly, did not induce cell death [61]. Rather, olaparib was found to induce reversible G2 arrest in ATM-deficient A549 cells [61]. Since the related protein kinase ATR plays a critical role in the G2 checkpoint [62], and given ATM-deficient cells are sensitive to ATR inhibitors [58,63–65], we asked whether inhibition of ATR using VE-821 [66,67], would ablate G2 arrest and induce cell death in olaparib-treated ATM-deficient cells. This was indeed found to be the case. Combined treatment with olaparib plus ATR inhibition with VE821 induced cell death only in ATM-deficient A549 cells, suggesting that patients with ATM-deficient tumours could benefit from a combination of PARP and ATR inhibitors [61].

**Figure 1.** Frequency of Ataxia-Telangiectasia Mutated (*ATM)* mutations in human cancer. (**A**) *ATM* was queried against all entries in the curated non-redundant data set on c-Bioportal (references [46,47]) accessed January 2020. Duplicate studies were removed and copy number variations are not included. The frequency of *ATM* alteration in various cancers is shown. (**B**) ATM is a 3056 amino acid protein consisting of a N-terminal TAN (telomere length maintenance and DNA damage repair) domain (residues 7–165), and a C-terminal kinase domain (residues 2714–2961) flanked by FAT (2097–2488) and FATC (3205–3055) domains. The location of mutations in ATM from all samples in the curated non-redundant data site available on c-Bioportal (references [46,47]), accessed January 2020 (duplicate sets removed and copy number variation not include) is shown. Mutations were distributed across the entire the coding region however, one mutation R337C/H was detected in 74 out of 2263 samples, across all cancers. (**C**) The R337C/H mutation was frequent in bowel cancer (22 out of 331 samples) but less so in lung (panel **D**), prostate (panel **E**) and pancreatic cancers (Panel **F**).

*ATM* is frequently mutated in prostate cancer [68], as well as somatic and hereditary forms of pancreatic cancers [69,70], suggesting that patients with these cancers might also benefit from treatment with a PARP inhibitor. Nevertheless, PROFOUND, a phase III trial that examined the clinical efficacy of olaparib versus standard treatment (abiraterone acetate/enzalutamide) in patients with metastatic, castration-resistant prostate cancer (mCRPC) and HRR gene alterations, revealed less impressive rates of radiographic progression free survival benefit with olaparib in patients with *ATM* alterations, in contrast to patients with other HRR gene alterations (e.g., *BRCA2*), based upon exploratory, hypothesis-generating gene-by-gene subgroup analysis [23]. Thus, it appeared necessary to consider therapeutic approaches that may enhance the efficacy of PARP inhibition in ATM-deficient cancers.

We therefore examined whether the combination of olaparib plus an ATR inhibitor would be effective in cell line models of prostate and pancreatic cancer. We depleted ATM from the prostate cancer cell line PC-3 using CRISPR/Cas9 and found that although olaparib reduced cell proliferation, ATM-deficient cells did not undergo apoptosis unless olaparib was combined with an ATR inhibitor, either VE-821, as in [61] or AZD6738 [71], an ATR inhibitor in clinical trials [72]. Significantly, olaparib and AZD6738 had little effect on ATM-proficient cells either alone or in combination [71]. Similar results were seen with the pancreatic cancer cell line, Panc 10.05, in which ATM was depleted by shRNA [71]. Thus, the combination of PARP and ATR inhibitors could be beneficial in a number of ATM-deficient cancers, including lung, prostate and pancreatic [71]. While A549 has wild type p53 [73], PC-3 are *TP53* null [74] and Panc 10.05 contain a homozygous mutation at I225N [75,76], therefore these data suggest that the sensitivity of ATM-deficient cells to the combination of PARP inhibitor and ATR inhibitor is not dependent on p53 status.

Our results also highlight the importance of the method used to assess cell viability in determining sensitivity of a cell line to a particular agent. Although ATM-deficient cells were highly sensitive to olaparib in clonogenic survival assays and the number of viable cells was decreased compared to ATM-proficient cells measured using the trypan blue exclusion assay, analysis of sub-G1 DNA or annexin staining did not reveal evidence of cell death [61,71]. Rather, olaparib-treated, ATM-deficient cells underwent reversible G2 arrest, and did not undergo cell death until an ATR inhibitor was also added [61,71]. Moreover, similar results have been seen in a patient-derived xenograft model of *BRCA*-mutant high-grade serous ovarian cancer, suggesting that PARP inhibition also increases reliance on ATR-dependent G2 arrest in BRCA-deficient cells [77], thus the combination of PARP and ATR inhibition may have benefits in other HDR-deficient cancers.

#### **4. ATM Mutation Versus Loss of Function: Identifying Patients Who May Benefit from PARP Inhibitor Treatment**

To date, most work from our lab and others has centred on the effects of olaparib on cell lines or mice in which ATM has been deleted [52–54,57,58,61,71] or inhibited with KU55933 [53,55]. An important difference is that in cancer, ATM is mutated, but the effects of these mutations on ATM function are, for the most part, unknown. As shown in Figure 1, literally hundreds of mutations have been identified in ATM and these mutations are scattered throughout the coding region. Apart from R337H/C, a hotspot mutation prevalent in colorectal cancer, other individual mutations are seen less frequently and their effects on ATM function is not known. Recently, three papers describe cryo-electron microscopy structures of Tel1, the well-conserved ATM homolog in lower eukaryotes, showing that ATM forms an autoinhibited dimer [78–80], providing insight into the conformational changes necessary for ATM activation. Three of these structures provide atomic models of the C-terminal kinase domain, and one, from a thermophilic fungus, also provides an atomic model of the majority of the N-terminal heat repeat domain in open and closed conformations. These structures provide the molecular basis to begin to understand cancer-associated *ATM* mutations. Initial analyses predict many cancer-associated mutations in the kinase domain are likely to impact ATM activity or protein folding [78,79], and the equivalent residue to R337 appears to stabilize the packing of two helices in the N-terminal domain, with the R337C/H mutation possibly destabilizing this region of ATM [78]. The methods developed to purify wildtype ATM homologs pave the way for more comprehensive studies to test the effect of cancer-associated mutations on ATM activities and stability.

For the treatment of cancer patients based on *ATM* status, it will be critical to identify those patients who have mutations that impact ATM activity or stability and are therefore most likely to benefit from PARP/ATR inhibitor combination treatment. This may be challenging by DNA sequencing alone, given the number of mutations identified and that their effects on ATM function are, for the most part, not known. That being said, preliminary clinical trials have shown promising, if not controversial results. In an initial trial of 60 men with mCRPC, 5 were shown to have mutation of *ATM* and 4 responded to olaparib [81]. A subsequent larger trial in a similar patient population demonstrated 7 of 19 patients with ATM mutation met at least one response criteria [82]. In contrast, a multicentre retrospective review of patients with mCRPC treated with olaparib showed no patients with *ATM* mutations achieved a PSA response and had significantly worse progression-free survival (PFS) and overall survival compared to *BRCA*-mutated patients. The addition of olaparib to paclitaxel failed to improve overall survival over paclitaxel alone in Asian patients with recurrent gastric cancer, both in the overall patient population as well as those with low or absent ATM expression, as determined by immunohistochemistry (IHC) [83]. Our findings that olaparib alone is cytostatic in ATM-deficient cells and that PARP and ATR inhibitors need to be combined to kill ATM-deficient cells is in line with these findings and suggest that this combinatorial approach could improve long-term survival in patients.

Despite the large number of uncharacterized mutations in *ATM* in cancer, many mutations known to impact ATM function, such as those resulting in A–T, frequently induce protein destabilization [50]. Therefore, determining the presence of ATM protein in cancer patient biopsy samples through IHC or other approaches may prove useful. Interestingly, although data from TCGA reveals that the *ATM* gene is mutated in 12–14% of patient samples with lung adenocarcinoma, another study has reported that ~40% of lung adenocarcinoma patients have low ATM protein expression by IHC [84]. A possible explanation for the apparent difference between *ATM* gene alteration and ATM protein expression could be methylation of the *ATM* promoter leading to transcription silencing [85]. Indeed, our analysis of methylation data in TCGA datasets revealed significant negative correlations between *ATM* promoter methylation and *ATM* gene expression in prostate adenocarcinoma (*p* = 1.962 <sup>×</sup> 10<sup>−</sup>7, Spearman's rho = −0.23), lung adenocarcinoma (*p* = 0.001159, Spearman's rho = −0.15) and colon adenocarcinoma (*p* = 0.0372, Spearman's rho = −0.13) (Figure 2). These findings suggest that ATM promoter methylation may play a role in the regulation of *ATM* gene expression in these cancer types.

Another possibility for stratifying patients who will benefit from a combination of PARP and ATR inhibitors would be to use a surrogate marker of ATM functionality, either through the use of phosphospecific antibodies to ATM itself or its downstream targets or an RNA signature specific to loss of ATM functionality. Indeed, the search for identifying tumours which exhibit "BRCAness" or HRR deficiency is an area of active investigation [27,86]. Answers to these questions and more may become apparent over the next few years as PARP inhibitor therapy is tested in more patients with defects in DNA damage response genes.

**Figure 2.** *ATM* promoter methylation and *ATM* gene expression in adenocarcinomas. Scatter plots showing the correlation between methylation beta values of the *ATM* promoter probe cg01756564 and *ATM* gene expression in TCGA datasets of (**A**) prostate adenocarcinoma (PRAD, *n* = 496), (**B**) colon adenocarcinoma (COAD, *n* = 276) and (**C**) lung adenocarcinoma (LUAD, *n* = 454). Spearman's rho values are indicated in the top right. Grey region of linear fit indicates 95% confidence interval. Asterisks in the top left indicate significance. \* = *p* < 0.05, \*\* = *p* <0.01, \*\*\* = *p* < 0.001.

#### **5. Concluding Remarks**

In conclusion, basic research into DNA damage repair biochemistry led to the identification of PARP, the inhibition of which, almost 40 years later, is showing great promise in the treatment of *BRCA*-deficient ovarian, breast, prostate and pancreatic cancer patients [87–89]. Work from our lab and others discussed here has shown that PARP inhibitors may also have potential in treating patients with ATM-deficient tumours. Our recent studies have revealed that in ATM-deficient cancer cell lines, olaparib is cytostatic not cytotoxic and that combination of olaparib with an ATR inhibitor is needed

to induce cell death [61,71]. Moreover, similar results were seen in models of BRCA-mutant ovarian cancer, suggesting that inhibition of ATR potentiates the effects PARP inhibition in *BRCA*-deficient cells [77], thus the combination of PARP and ATR inhibition may have benefits in other HDR-deficient cancers. It will be exciting to see whether this finding will also apply to other PARP inhibitors and, most importantly, in the clinic for improving outcomes for cancer patients. Indeed, several clinical trials using a PARP inhibitor in combination with an ATR inhibitor are ongoing (Table 1).

**Table 1.** List of ongoing clinical trials combining a poly-ADP ribose polymerase (PARP) inhibitor with an ATR inhibitor. Information obtained from https://clinicaltrials.gov, accessed March 9 2020.

It is also possible that ongoing preclinical studies will reveal new synthetic lethal interactions within the DNA damage response. Indeed, inhibitors to other proteins in the DNA damage response are being developed [27]. Identification of alternative DNA damage response genes to target in cancer could be useful as therapies in their own right and also in cases where tumours become resistant to other therapies, such as is currently observed in *BRCA*-deficient tumours treated with PARP inhibitors [90,91]. Finally, ATM has widespread cellular roles outside the DNA damage response, including roles in cellular redox signalling [41] and regulation of autophagy [92,93], apoptosis and other cell death pathways [94–98], further increasing potential opportunities to target ATM-deficient cells with novel therapies.

**Author Contributions:** Conceptualization, N.R.J., M.K. (Michael Kolinsky), S.P.L.-M.; investigation, N.R.J., M.K. (Mehul Kumar), S.R., G.A., S.G., S.Y., M.K. (Michael Kolinsky), G.J.W., P.B., S.P.L.-M.; writing—original draft preparation, N.R.J., S.R., G.A., S.G., S.Y., M.K. (Michael Kolinsky), S.P.L.-M.; writing—review and editing, N.R.J., M.K. (Mehul Kumar), S.R., G.A., S.G., S.Y., M.K. (Mehul Kumar), G.J.W., P.B., S.P.L.-M.; funding acquisition, S.P.L.-M. All authors have read and agreed to the published version of the manuscript.

**Funding:** S.P.L.-M. thanks the Alberta Cancer Foundation, the Cancer Research Society and the Pancreatic Cancer Society of Canada for support. Work in the S.P.L.-M. and G.J.W. laboratories is also supported by NIH program project grant CA92584.

**Conflicts of Interest:** S.Y. has received honoraria and/or consulting fees from Janssen, Pfizer, Roche, BMS, Merck, AstraZeneca, Bayer, and Novartis. M.K. (Michael Kolinsky) has accepted honoraria and/or consulting fees from Janssen, Ipsen, Astellas, BMS, Merck, AstraZeneca, Bayer, and travel support from Novartis. N.R.J., M.K. (Mehul Kumar), S.R., G.A., S.G., G.J.W., P.B. and S.P.L.-M. declare no conflict of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## **The Controversial Roles of ADP-Ribosyl Hydrolases MACROD1, MACROD2 and TARG1 in Carcinogenesis**

#### **Karla L.H. Feijs 1,\*, Christopher D.O. Cooper <sup>2</sup> and Roko Žaja <sup>1</sup>**

Received: 15 January 2020; Accepted: 27 February 2020; Published: 5 March 2020

**Abstract:** Post-translational modifications (PTM) of proteins are crucial for fine-tuning a cell's response to both intracellular and extracellular cues. ADP-ribosylation is a PTM, which occurs in two flavours: modification of a target with multiple ADP-ribose moieties (poly(ADP-ribosyl)ation or PARylation) or with only one unit (MARylation), which are added by the different enzymes of the PARP family (also known as the ARTD family). PARylation has been relatively well-studied, particularly in the DNA damage response. This has resulted in the development of PARP inhibitors such as olaparib, which are increasingly employed in cancer chemotherapeutic approaches. Despite the fact that the majority of PARP enzymes catalyse MARylation, MARylation is not as well understood as PARylation. MARylation is a dynamic process: the enzymes reversing intracellular MARylation of acidic amino acids (MACROD1, MACROD2, and TARG1) were discovered in 2013. Since then, however, little information has been published about their physiological function. MACROD1, MACROD2, and TARG1 have a 'macrodomain' harbouring the catalytic site, but no other domains have been identified. Despite the lack of information regarding their cellular roles, there are a number of studies linking them to cancer. However, some of these publications oppose each other, some rely on poorly-characterised antibodies, or on aberrant localisation of overexpressed rather than native protein. In this review, we critically assess the available literature on a role for the hydrolases in cancer and find that, currently, there is limited evidence for a role for MACROD1, MACROD2, or TARG1 in tumorigenesis.

**Keywords:** ADP-ribosylation; cancer; macrodomain; ADP-ribosyl hydrolase; PARP; ARTD; MACROD1; MACROD2; TARG1

#### **1. ADP-Ribosylation Reactions**

Post-translational modifications of proteins can have a myriad of consequences: changing interactomes, stability, localisation, or activity are just a few examples. To date, more than 200 types of modification are known [1]. The addition of ADP-ribose moieties to a protein was first reported in the 1960s when chains of (poly)ADP-ribose (PAR) were identified on target proteins, termed poly(ADP-ribosyl)ation (PARylation) [2]. Since then, PARylation has been intensively studied, leading to the identification of crucial roles in the repair of DNA damage and to the development of specific inhibitors that are utilised in the clinic to treat diverse cancers [3]. In contrast, much less is known about its smaller sibling, mono(ADP-ribosyl)ation (MARylation), where only single ADP-ribose units (MAR) are conjugated into proteins. In 2008, it was first noted that PARP10/ARTD10 has MARylation rather than PARylation activity [4] and, lately, it has become clear that only the minority of PARP enzymes are capable of PARylation [5]. Moreover, not only protein, but also DNA has been identified as a target for ADP-ribosylation catalysed by PARP3 [6,7]. Following PARP1 and PARP2, PARP3 has also been described as a potential therapeutic target in certain cancers [8–11]. The most recent addition to the spectrum of ADP-ribosylated molecules is RNA, which can be modified in vitro by multiple PARPs [12].

One of the best-studied but still barely understood MARylating enzymes is PARP10. PARP10 was initially identified as an interactor of MYC [13] and, later, of ubiquitinated proliferating cell nuclear antigen (PCNA) [14,15]. Its overexpression leads to cell death in HeLa cells through an unknown mechanism [16]. In contrast, in non-transformed RPE-1 cells, overexpression leads to stimulation of cell growth [17]. It has been given a role in replication and DNA damage [15,17,18] as well as mitochondrial metabolism [19], but it is not clear what the relevant substrates of this enzyme are. Potential substrates were identified using protein microarrays [20], which was performed for the PARylating PARP2 [21]. However, partially due to a lack of antibodies at that time, these modifications were not verified to occur in cells. PARP10 was also reported to be a regulator of NF-kB signaling [22]. PARP14 functions as a co-activator of STAT6 and has been described as an anti-cancer and anti-inflammatory target, reviewed in more detail elsewhere [23,24]. PARP16 is located at the endoplasmic reticulum (ER) and plays a role in the unfolded protein response [25,26]. The most recent function identified for MARylation is its role in the anti-viral defence. PARP7, PARP10, and PARP12 gene expression is induced by interferon-α and ADP-ribosylation mediated by these PARPs may function to counteract viral proliferation [27,28]. In addition to enzymes of the PARP family, several sirtuins (SIRTs) appear to be able to ADP-ribosylate substrates in addition to their better-studied NAD+-dependent deacylase activity [29,30]. The first research showing that SIRTs possess MARylation activity described it as a very weak activity [31]. Other papers, focusing on single enzymes, showed that the mitochondrial SIRT4 MARylates glutamate dehydrogenase to downregulate insulin secretion [32]. The nuclear SIRT6 modifies PARP1 [33] and BAF10 [34] to regulate their activities and, lastly, the nucleolar SIRT7 was reported to MARylate histones p53 and ELK4 [35]. To date, very little information is available about the MARylating activity of the SIRTs. The functional consequences of MARylation in normal cell physiology, thus, appear to be very diverse, reviewed extensively elsewhere [36]. Herein, we focus on the enzymes reversing the intracellular MARylation reaction: the macrodomain-containing hydrolases.

#### **2. Macrodomains**

Intimately linked to ADP-ribosylation is a protein structural module known as the macrodomain, named for the unusually large histone macroH2A from which it was identified [37]. The macrodomain is a protein fold formed around a central β-sheet, flanked by α-helices that forms a pocket where ADP-ribose can bind [38,39]. These pockets are slightly different between the macrodomain-containing proteins with the consequence that the macrodomain-containing protein family can be subdivided. Some macrodomains bind to PAR and others bind to MAR [40–43].

Macrodomains not only serve as binding modules to mediate protein interactions depending on ADP-ribosylation, but a number of them have catalytic activity. Poly(ADP-ribosyl)glycohydrolase (PARG) is able to degrade PAR chains by cleaving the glycosidic bond between adjacent ADP-ribose moieties. However, it cannot remove the final ADP-ribose attached to the protein [44]. Three other proteins, MACROD1, MACROD2, and TARG1, are not active toward PAR but are capable of removing the ADP-ribose moiety connected to the protein partner, presumably by cleaving an ester bond between ADP-ribose and an acidic receptor amino acid [45–47]. They can, therefore, both reverse any MARylation as generated by PARP enzymes, and also remove the last ADP-ribose left behind by PARG. Of the 12 human macrodomain-containing proteins known today, 11 proteins were identified by sequence homology [39,43]. The macrodomain of PARG was only recognised after the structure had been solved [44], which indicates the possibility that more macrodomain-containing proteins remain to be identified. A recent bioinformatics approach has suggested that the largely uncharacterised protein C12orf4 harbours a macrodomain, or at least a macro-like domain, but this remains to be confirmed experimentally [48].

The macrodomain is an evolutionary well-conserved module [39]. For instance, recent work has shown that certain alpha-viruses encode a protein with a macrodomain-like fold [49], which displays catalytic activity towards MARylated proteins [50–52]. The existence of such proteins enhances the evidence connecting existing links between MARylation and immunity [53]. In this review, we will focus specifically on potential oncogenic functions of the mono(ADP-ribosyl)hydrolases MACROD1, MACROD2, and TARG1. It should also be noted, however, that other macrodomain-containing proteins such as PARG and CHD1L (ALC1) have been previously linked closely to cancer [54,55].

#### **3. Macrodomain-Containing Hydrolases: MACROD1, MACROD2, and TARG1**

MACROD1, MACROD2, and TARG1 are relatively small proteins (Table 1) without any notable other domains beside the macrodomain. The first catalytic activity of MACROD1, MACROD2, and TARG1 that was recognised was their deacetylation of *O-*acetyl-ADP-ribose (*O*AADPR) to generate acetyl and ADP-ribose [56,57]. *O*AADPR arises as a by-product of SIRT-mediated deacetylation in which the acetyl group is transferred onto ADP-ribose while releasing nicotinamide, and may have important biological functions [58]. Two years later, three labs reported the activity of MACROD1, MACROD2, and TARG1 in removing ADP-ribose from protein substrates, which was proposed to be limited to removal of ADP-ribose from acidic amino acids [43,45–47]. Serine and arginine MARylation were suggested to be removed by ARH3 and ARH1, respectively [59–62], which are structurally different proteins that do not contain a macrodomain. In 2017, MACROD1, MACROD2, and TARG1 were reported as being capable of removing ADP-ribose from both modified single-stranded DNA [63] and, in 2018, also being capable of removing ADP-ribose from MARylated single-stranded RNA [12]. Lastly, in 2019, the hydrolysis of α-NAD was added to the list of their in vitro activities [64].

To date, it is not known which of these activities is relevant for their functionality in cells, either in normal physiology nor in pathological conditions. This is predominantly due to the fact that it is not well known how prevalent their in vitro substrates are in cells or what their cellular functions may be. This is partially due to the fact that antibodies or modules recognising MARylation have been developed only recently [65–67]. Before these technical developments, it was not possible to confirm with antibodies that MARylation of proteins takes place in cells, and, hence, reversal mediated by these macrodomain-containing proteins could also not be assessed. Currently, it is still not straightforward to estimate endogenous MARylation levels in cells, as no inhibitors were available for MACROD1, MACROD2, or TARG1. During experimental procedures such as cell fixation or lysis, MARylation could possibly be removed by these enzymes and, therefore, go undetected. One study was performed to identify MACROD1 inhibitors using an AlphaScreen assay, where inhibitors in the micromolar range were identified. These can serve as lead compounds for further optimisation, but are likely not specific enough yet to be used for studies in cells due to their other activities at this concentration [68]. Another study identified an allosteric inhibitor of macrodomain 2 of PARP14, which blocks its binding to ADP-ribose. However, it was not tested whether the identified compound might inhibit one of the macrodomains with catalytic activity [69].

 upon (WB) is smaller [70].

The roles of MACROD1, MACROD2, and TARG1 in normal cell biological processes are not well understood. They appear to be expressed and localised in different tissues and intracellular locations, which clearly gives them unique roles to play despite their biochemical similarity. This also explains why loss of TARG1 alone can lead to a neurodegenerative phenotype [45]. MACROD1 resides in mitochondria [40,70,71], but, depending on the tag used for overexpression, will reside exclusively in the nucleus instead [70]. MACROD2 displays a more diffuse cytoplasmic/nuclear distribution [70,75] and overexpressed TARG1 appears to be present in nucleoplasm, nucleoli, and cytoplasmic stress granules [14,70]. TARG1 and MACROD1 are expressed throughout the different tissues with an enrichment of MACROD1 in skeletal muscle at both the protein and RNA level [70]. MACROD2 has, thus far, only been detected in human neuroblastoma cells and in mouse cortical neurons [70,76]. Several reports have correlated polymorphisms or deletions within the *MACROD2* gene with autism-spectrum disorders [77–80]. It is not clear, however, whether the *MACROD2* gene product itself or surrounding genes are responsible for this association even though elevated protein expression in neurons supports a potential brain-specific function of MACROD2 [76]. However, a long non-coding RNA has been identified within an intron of the *MACROD2* gene, which is more highly expressed in most tissues investigated than the *MACROD2* mRNA, and, thereby, potentially confuses correlations of mutations in the *MACROD2* gene and phenotypes [81]. In another genome-wide association study, *MACROD2* was identified as a factor influencing vascular-adhesion protein-1 (VAP-1) levels, which the authors confirmed by knockdown of MACROD2. This leads to lower VAP-1 expression in adipocytes, presumably through the transcriptional regulation of VAP-1 [82]. MACROD2 was also reported to leave the nucleus upon DNA damage, dependent on phosphorylation by ATM [75], whereas TARG1 localises from nucleoli to nuclear sites of damage [14], which both have an unclear functional relevance. More data may be found on MACROD1, which is also known as leukaemia-related protein 16 or LRP16. MACROD1 has been attributed to a number of functions in the nucleus, such as co-activation of the androgen receptor [83], counteracting PARP7-mediated MARylation in the nucleus [84,85], and activation of NF-kB signalling [86–88]. MACROD1 was also reported as an enhancer of oestrogen receptor signalling [89], and is upregulated after stimulation of cells with oestrogen [72,74,90]. No regulatory factors have been identified yet for MACROD2 and TARG1. A BioID interaction screen, which identifies proteins in close proximity to the protein of interest [91], identified many proteins involved in nuclear/cytoplasmic and mitochondrial nucleic acid metabolism as interactors of TARG1 and MACROD1, respectively [70]. Whether these proteins are MARylated and serve as a substrate of TARG1/MACROD1 remains to be determined. It is possible that, despite spatio-temporal restrictions, MACROD1 and TARG1 in their respective compartments are involved in similar signalling networks, converging on the regulation of cellular nucleic acids. The physiological functions of the three enzymes remains elusive. Most puzzling perhaps is that, at the moment, despite the mitochondrial localisation of MACROD1, the majority of reports describe nuclear functions. Future work will need to address this apparent discrepancy.

#### **4. Mono(ADP-ribosyl)ation in Cancer**

The post-translational modification poly(ADP-ribosyl)ation has been intimately linked to cancer before [92,93], as have other post-translational modifications such as phosphorylation [94]. In BRCA1/BRCA2-deficient patients, PARP1 inhibitors have been shown effective specifically against the tumour cells applied in the clinic. However, this is one of the rare examples of a synthetic lethal interaction [95]. Better understanding of the processes regulated by MARylation will provide opportunities for further drug development, as is exemplified by current research into the potential of PARP14 as a drug target [24]. Little is known about the potential role of the mono(ADP-ribosyl)hydrolases in cancer. *MACROD1*, *MACROD2*, and *OARD1* (TARG1) exhibit mutations only in 0.9%, 2.6%, and 1% of cancer patient samples, respectively, from over 1000 samples in the cBioPortal curated dataset [96]. The fact that *MACROD2* mutation rates are twice the rates seen for *MACROD1* and *OARD1* likely reflects that the *MACROD2* gene is larger (Figure 1), and is

located at a known fragile region [97]. A specific recurring deletion of exon 6 has been observed in esophageal squamous cell carcinoma and gastric cancer [98]. For *MACROD1*, a *RUNX-MACROD1* fusion was identified in leukaemia [99] and, for *MACROD2*, a *PDGFRA-MACROD2* fusion in a pleomorphic sarcoma [100]. Despite the low number of identified mutations in patient tumour samples, several reports have correlated *MACROD1* or *MACROD2* expression levels with the clinical outcome, as described in the next paragraphs.

**Figure 1.** Overview of the *MACROD1*, *MACROD2*, and *OARD1* gene structure.

The gene structure of *MACROD1*, *MACROD2*, and *OARD1* is shown schematically. More lncRNAs are present. However, only RPS10P2-201 is displayed since it has been shown to be relevant RPS10P2-201 [81]. CDS = coding sequence. UTR = untranslated region.

#### **5. MACROD1 in Cancer**

A number of studies have addressed a potential oncogenic function of MACROD1 and started deciphering the molecular mechanism underlying observed effects. MACROD1 expression was shown to be upregulated by oestrogen [73,74], which leads to several studies of the role of MACROD1 in tumours with a differential oestrogen status. MACROD1 overexpression in the oestrogen and progesterone receptor positive Ishikawa cells, derived from an endometrial cancer, had no effect on cell proliferation. It did, however, enhance the invasiveness of these cells as measured by transwell assays [101]. Mechanistically, the authors propose a mechanism wherein MACROD1, dependent on oestrogen, blocks recruitment of ERα to the E-cadherin promoter, which lowers E-cadherin expression and, through this, enhances invasiveness. shRNA-mediated knockdown of MACROD1 achieved the opposite effect by enhancing E-cadherin expression [101]. This implies that MACROD1 can be an important factor in metastasis. These findings appear to contradict an earlier report where overexpression of MACROD1 in MCF7 cells, which are oestrogen-responsive breast cancer cells, showed an effect on cell growth. It led to enhanced proliferation [74]. A later report studying MACROD1 in 293T cells showed that knockdown of MACROD1 sensitized cells to TNFα-induced

apoptosis [86]. From these studies, it is, thus, not clear what effect loss or gain of MACROD1 has on cell physiology. Recent work has demonstrated that fusing an N-terminal tag, such as GFP, to MACROD1 leads to a nuclear localisation, whereas C-terminally labelled and an endogenous protein appear to be exclusively localised in mitochondria [70]. Mass spectrometry datasets have also detected MACROD1 in the mitochondria [102]. This does not exclude the possibility that, under specific circumstances, MACROD1 may re-localise to the nucleus. In pathogenic conditions, such as the presence of a *RUNX-MACROD1* fusion protein that was identified in leukaemia [99], the protein likely also localises to the nucleus instead of the mitochondria, as the RUNX fusion will mask the mitochondrial targeting sequence, comparable to the localisation after labelling with an N-terminal GFP tag. Unfortunately, some of the studies investigating MACROD1 have either used N-terminally tagged fusion constructs [84,85,88], or have not stated clearly how the fusion proteins were generated [103]. Furthermore, the majority of applied antibodies show multiple bands in the western blot and, hence, are not suitable for immunohistochemistry (IHC) or immunofluorescence (IF) (Table 2). It will be worthwhile to repeat some of the studies of MACROD1 s molecular function in carcinogenesis to clarify whether unlabeled MACROD1 overexpression leads to enhanced cell growth, or whether this effect depends on the tumour background and also to study knockdown/knockout systems more thoroughly.

Despite these technical challenges, multiple studies have correlated tumour growth with MACROD1 expression in either xenograft models or in patient cohorts. Overexpression of MACROD1 in the hepatocellular carcinoma lines HepG2 and MHCC-97L leads to a decrease in cell growth and metastatic potential, as measured by transwell assays [103]. Furthermore, when delivered into nude mice, cells overexpressing MACROD1 lead to a decreased tumour volume and have a lower metastatic potential compared to cells without overexpressed MACROD1 [103]. With the information available, it cannot be distinguished whether this is a genuine effect or an artefact due to forced nuclear localisation of the overexpression construct [103]. In a similar set of experiments in the pancreatic carcinoma cell lines, Panc1, CFPAC1, Bxpc3, SW1990, AsPC1, and HPDE6-C7, opposite results were achieved [105]. Knockdown of MACROD1 leads to enhanced apoptosis and decreased cell growth. However, only one shRNA construct was used, so any effects seen could be potentially off-target. Cells overexpressing the same MACROD1 construct as in the study described before [103] grew faster and were more resistant to apoptosis. Xenograft experiments show the same trend. Cells lacking MACROD1 had a lower tumour-forming potential and higher survival rate. Cells overexpressing MACROD1 had a higher tumour volume and a lower survival rate [105]. The authors do not comment on the opposing effects in these two tumour types, but agree that larger-scale studies are required to verify these findings. A third study shows IHC of lung tumour samples with an antibody of an unknown source and specificity [104]. High MACROD1 expression, as measured by IHC, was reported to correlate with a negative outcome in colorectal carcinoma and in gastric carcinoma. However, the antibody used was generated by the authors and not validated by a western blot [106,107]. CRISPR-mediated *MACROD1* knockout rhabdomyosarcoma cells [70] and *MACROD1* knockout mice appear viable [71], which makes it unlikely that loss of MACROD1 has a drastic growth inhibitory or developmental effect.

In conclusion, most of the data available on an oncogenic function of MACROD1 rely on poorly characterised antibodies, unclear overexpression constructs or a single shRNA construct. The majority of these studies would need to be reproduced with a more thorough characterisation and description of the materials used to be able to draw deeper conclusions. Several studies agree that MACROD1 expression can be induced by oestrogen [73,74,89,90,101], but it remains unclear what the effect of this overexpression is on cells. The *RUNX-MACROD1* fusion identified in leukaemia may provide an important hint at a potential pathologic function. It is possible that the physiological mitochondrial localisation of MACROD1 can turn into a pathogenic nuclear one, where it aberrantly acts as a transcriptional activator. It will be interesting to see whether more instances can be identified where such fusions are present. Alternatively, other masking events may occur in cells, such as binding by interaction partners or PTMs, which, thereby, redirects the protein to the nucleus for a physiological function.

#### **6. MACROD2 in Cancer**

A number of analyses suggest MACROD2 may play a potential role in cancer. MACROD2 copy number is increased in three different tamoxifen-resistant MCF7 breast cancer cell lines, prompting the authors to analyse MACROD2 expression in patient samples. In oestrogen receptor-positive tissues of breast cancer patients with a recorded tamoxifen-resistance, however, three patients displayed a decreased copy number, whereas the other two patients showed an increase. Using IHC with a custom antibody, varying levels of MACROD2 were detected in primary and secondary tumour tissues of these patients, collectively showing that MACROD2 may be overexpressed in cancer tissues. MCF7 and T47D cells with exogenously overexpressed MACROD2 grow faster than control cells in media containing tamoxifen, which implies that MACROD2 confers tamoxifen-resistance to the cells and appear to be stimulated by tamoxifen, as demonstrated by faster growth in medium with tamoxifen than without [110]. Conversely, tamoxifen-resistant MCF7 clones with shRNA-mediated knockdown of MACROD2 become more sensitive even though it does not completely reverse resistance [110]. This may be mediated by the activation of oestrogen-regulated genes in response to tamoxifen in cells overexpressing MACROD2. Lastly, cells stably expressing shRNA to knockdown MACROD2

grow markedly slower in nude mice [110]. The authors argue that, as a cancer specific fragile site, the MACROD2 gene can be lost, but this fragility also allows for amplification in the specific case of ER-positive breast cancers treated with tamoxifen to incur resistance. A more recent study showed that, in approximately one-third of colorectal carcinomas investigated, heterozygous or homozygous losses were mapped within the *MACROD2* gene in which the majority are intragenic microdeletions mapping to a region in exons 4 to 5 [111]. To test whether MACROD2 deficiency can promote tumourigenesis, *MACROD2* knockout was introduced into an adenomatous polyposis coli protein (Apc) mouse model. Mutations in the APC tumour suppressor are a major driver of sporadic colorectal cancers, where it could be shown that even haploinsufficiency of *MACROD2* leads to more and larger adenoma formation [111]. Furthermore, human cells transplanted into nude mice displayed increased tumour growth when lacking *MACROD2*, but a reduced tumour growth when MACROD2 was overexpressed [111]. The underlying mechanism was suggested to be impaired PARP1 activity in the *MACROD2*-/- cells, which leads to increased sensitivity of DNA damage and, ultimately, causes enhanced chromosomal instability [111,112].

These findings appear paradoxical with the previous report, where loss of MACROD2 impairs cell growth from a loss of resistance to tamoxifen. *MacroD2* knockout mice do not show altered survival rates after sub-lethal irradiation compared to wildtype mice, which indicates that loss of MACROD2 alone is not sufficient to drive tumourigenesis triggered by DNA damage [109]. It is not clear whether loss or overexpression of MACROD2 contributes to tumourigenesis, or whether both can drive tumour growth dependent on conditions, such as the functionality of the DNA damage repair systems or the oestrogen receptor status. In an investigation of stage-III colon cancer, MACROD2 expression determined by immunohistochemistry was found to correlate with poor survival [113]. A human protein atlas antibody (HPA049076) was used for this work, which has been retracted by the company in the meantime and did not appear to be validated in any way, such as western blotting or siRNA-mediated knockdown. It is, thus, not clear whether this antibody recognises MACROD2 at all, or whether it recognises additional proteins, which may be upregulated in the samples analysed. Altogether, it appears that loss of MACROD2 as such is not sufficient to drive tumourigenesis, but may have an additive effect in models prone to tumour formation such as loss of APC in colorectal cells. More studies are urgently needed to clarify the exact role of MACROD2 in both the onset of cancer as well as in the response of existing tumours to therapies.

#### **7. TARG1 in Cancer**

The only phenotype associated with TARG1 expression is neurodegeneration, occurring due to a mutation that leads to a truncated protein with a disrupted macrodomain, and, thereby, loss of catalytic activity [45]. It is not clear whether this neurodegeneration is a result of a potentially toxic truncated or unfolded protein or from loss of hydrolase activity. Knockdown of TARG1 leads to a decrease in 293T cell proliferation and a slight increase in senescence in U2OS cells, which are derived from an osteosarcoma [45]. CRISPR-mediated knockdown of TARG1 does not influence HeLa or U2OS proliferation [14,70], which leaves it unclear in which setting TARG1 is required for cell growth. Overexpression does not lead to changes in cell proliferation [14,70]. We could not identify any data linking TARG1 to cancer, nor see elevated expression or mutation in databases such as COSMIC. Based on expression levels in databases and these experimental results, at this stage, it does appear unlikely that TARG1 is involved in cancer even though further experimentation is required.

#### **8. Conclusions**

Despite the presence of several publications reporting a correlation between MACROD1 or MACROD2 levels and cancer development or progression, it is not clear at the moment that they have a causative role. MACROD2 is potentially relevant in ER-positive, tamoxifen resistant breast cancers where it may confer resistance to treatment. However, larger cohorts of patient samples need to be analysed to further substantiate these initial findings. Loss of *MACROD2* in an APC null background potentially stimulates tumour formation. However, loss of *MacroD2* alone does not have any effect in a knockout mouse model. Detailed studies are required to determine how loss of MACROD2 might cooperate with loss of APC to drive cancer. MACROD1 levels are upregulated by oestrogen with unclear consequences for cells. Yet, it may down-regulate E-cadherin and, thereby, promote metastasis. If confirmed, this may be an attractive therapeutic target intending to keep the cancer dormant and prevent metastasis.

Conflicting data show that overexpression and knockdown of MACROD1 have no effect, no stimulus, and no inhibition of cell growth, which implies that, perhaps, inhibiting MACROD1 may not have detrimental effects for the whole organism but rather may be dependent on the tumour background and, thus, may represent a valid drug target. The effects observed may, however, be dependent on the constructs used as well as on the specific cell-types and have to be studied in more detail. Future work will have to dissect in which context loss or gain of MACROD1 may be driving aspects of cancer growth. The recent development and characterisation of more specific antibodies, the ongoing improvements of mass spectrometric measurements of MARylation, and also the attempts at making specific inhibitors for both transferases and hydrolases should allow a more detailed analysis of their (patho-)physiological function in the near future. The partially paradoxical findings described will undoubtedly be clarified with better validated tools to determine the extent to which MACROD1, MACROD2, and TARG1 are relevant for tumourigenesis in order to establish their potential as drug candidates.

**Author Contributions:** Writing, review, and editing: K.L.H.F., C.D.O.C., and R.Ž. All authors have read and agreed to the published version of the manuscript.

**Funding:** The START Program of the Medical Faculty, RWTH Aachen University to K.L.H.F. (10/18) and to R.Z (13/20) funded this research.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## **The Development of Rucaparib**/**Rubraca**®**: A Story of the Synergy Between Science and Serendipity**

#### **Nicola J Curtin**

Professor of Experimental Cancer Therapeutics, Translational and Clinical Research Institute, Newcastle University Centre for Cancer, Faculty of Medical Sciences, Medical School, Paul O'Gorman Building, Newcastle University, Newcastle upon Tyne NE2 4HH, UK<EMAIL_ADDRESS>or<EMAIL_ADDRESS>Tel.: +44-0-191-208-4415

Received: 15 January 2020; Accepted: 26 February 2020; Published: 29 February 2020

**Abstract:** The poly(ADP-ribose) polymerase (PARP) inhibitor, Rubraca®, was given its first accelerated approval for BRCA-mutated ovarian cancer by the FDA at the end of 2016, and further approval by the FDA, EMA and NICE followed. Scientists at Newcastle University initiated the early stages, and several collaborations with scientists in academia and the pharmaceutical industry enabled its final development to the approval stage. Although originally considered as a chemo- or radiosensitiser, its current application is as a single agent exploiting tumour-specific defects in DNA repair. As well as involving intellectual and physical effort, there have been a series of fortuitous occurrences and coincidences of timing that ensured its success. This review describes the history of the relationship between science and serendipity that brought us to the current position.

**Keywords:** PARP; drug development; synthetic lethality; clinical trials

#### **1. Introduction: Rationale for the Development of PARP Inhibitors**

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) have been the most significant addition to the armoury for the treatment of ovarian cancer since the introduction of platinum therapy in the 1970s and represent a paradigm shift in the way cancers may be treated. This is the story of the development of the first PARPi to enter anticancer clinical trials, Rubraca®, formerly known as rucaparib or AG014699, the nomenclature used here largely reflects the name of the drug at the time of use. The development of Rubraca was the result of a substantial intellectual and physical effort by many individuals working as a team but it is hard to avoid the conclusion that luck has played a significant role, and this story describes how serendipity has contributed to the final success. This review describes the development of rucaparib in the context of other advances in the understanding of DNA repair and highlights the lucky breaks along the way.

The development of PARPi that ultimately led to rucaparib began in Newcastle University in 1990 but the story begins before that. The first observation that suggested the existence of PARP was the profound NAD<sup>+</sup> depletion following exposure of cells to ethyleneimine in 1956 [1]. Coming so soon after the discovery of DNA, this effect was not attributed to the effect of DNA methylation, rather an impact on cellular metabolism. The product ADP-ribose polymers, or poly(ADP-ribose) (PAR), was identified in 1963 and at first thought to be some new kind of nucleic acid [2], with elegant experiments a few years later showing the time course of the disappearance of NAD<sup>+</sup> and the corresponding appearance of polymers and nicotinamide [3]. The first suggestion of the involvement of PARP in DNA repair was made by Edward Miller in 1975 [4]. To test this hypothesis, Purnell and Whish [5] developed the first inhibitors, which were based on the catalytic mechanism of PARP and the observation that the by-product, nicotinamide, exerts some feedback inhibition. These early benzamide inhibitors included 3-amino benzamide (3AB), which is still used as a PARPi today. The pivotal study, conducted by Barbara Durkacz in Sydney Shall's lab, was published in 1980, showing that 3AB prevented the repair of DNA and increased cytotoxicity following exposure to the DNA methylating agent, DMS [6].

#### **2. Early Studies in Newcastle**

Barbara Durkacz moved to Newcastle University in the mid-1980s to establish PARP-related studies, where she was joined by Mike Purnell for the synthesis of inhibitors. However, it was the appointment of Hilary Calvert as the new director of the Cancer Research Unit in Newcastle, and his collaboration with Bernard Golding, Professor of Organic Chemistry at Newcastle University, that really got inhibitor development going. They established a drug development programme in October 1990, with funding from the North of England Cancer Research Campaign. Other members of the team were Roger Griffin (chemist), Herbie Newell (pharmacologist) and me (biologist/biochemist). PARP was one of three initial targets for drug discovery, tapping into Barbara Durkacz's expertise. A succession of post-graduate chemistry students embarked on inhibitor synthesis guided by Roger Griffin and Bernard Golding. However, to establish any kind of structure–activity relationship (SAR) a robust, reproducible and quantitative activity assay was needed and herein lay the problem. The results using a published assay [7,8] were variable in the extreme, with replicates wildly different from each other, and no better than random. Months were spent trying to generate inhibition data until finally, in exasperation, I decided to try to get to the bottom of the problem. The assay involved incubating permeabilised cells with 32P-NAD<sup>+</sup> and broken DNA to activate the cellular PARP then precipitating the polymer with TCA, collecting it onto filters, washing surplus NAD<sup>+</sup> away and counting the acid-precipitated radioactivity. Investigating each step of the assay to identify the source of the irreproducibility by taking replicates at different stages in the assay revealed that it was nothing to do with the cell permeabilisation or collection of the product but was the reaction itself. A eureka moment led me to discover a very simple solution: switching to plastic tubes. Because of the charged nature of NAD<sup>+</sup> and the polymer we had been siliconising glass tubes for the reaction to negate the charge on the glass and prevent binding. It turned out that the siliconising agent itself was causing highly variable interference with the activity of the enzyme. By switching to plastic this interference was removed but another problem was identified, i.e., the poor thermal conductivity of the plastic, which was overcome by a pre-warming step. Thereafter, this assay provided the robust, reproducible and quantitative data needed for SAR generation. This is the first example of the synergy of science and serendipity (#1).

The next example of serendipity (#2) came on the chemistry side. Inhibitors were designed to incorporate the nicotinamide pharmacophore, which was thought to make major interactions within the catalytic site of PARP. Studies with other NAD-dependent enzymes suggested that binding was favoured when the carboxamide was anti to the aromatic ring. In 3AB, it can freely rotate to the cis-orientation, meaning that binding is relatively weak (Ki = 10 μM, IC50 = 30 μM). Parallel SAR studies led by Judy Sebolt-Leopold at Warner Lambert and an "analogue by catalogue" approach in Kunihiro Ueda's lab identified that compounds where the carboxamide group was held anti to the aromatic ring, by constraining it through incorporation into a ring structure, had increased potency [9,10]. Our approach was to hold the carboxamide in the anti- conformation through hydrogen bonding with an oxazole or imidazole group. However, during the synthesis of the target compound, 2-methylbenzoxazole-4-carboxamide, a molecular rearrangement occurred generating 8-Hydroxy-2-methylquinazolin-4[3H]-one, NU1025, where the carboxamide was constrained in the desired orientation through incorporation into a ring [11] (Figure 1). NU1025 (Ki = 48 nM IC50 = 400 nM) had comparable activity to compounds identified by the Sebolt-Leopold and Ueda groups and was substantially more potent than the compound we had been aiming to make, which had an IC50 of 10 μM. This lucky accident therefore gave us our first hit compound and allowed us to explore the cellular effects of PARP inhibition.

**Figure 1.** Accidental synthesis of first "hit" compound, NU1025. During the attempted synthesis of 2-methylbenzoxazole-4-carboxamide, which could exist in the desired anti- conformation or the syn- conformation (left), a molecular rearrangement occurred, resulting in NU1025, a much more potent compound.

My first PhD student, Karen Bowman, was tasked not only with determining the potency of the compounds against PARP activity (aided and abetted by Louise Pemberton who also synthesised some of the early inhibitors) but also with evaluating the ability of NU1025 and the benzimidazole, NU1064 (2-methylbenzimidazole-4-carboxamide, IC50 = 1.1 μM) to increase the cytotoxicity of a variety of anticancer drugs. She confirmed that PARP inhibition increases the cytotoxicity of DNA alkylating agents using temozolomide and its active derivative, MTIC. She identified that it was the DNA repair phase that was critical, rather than the DNA damage induction phase as NU1025 was as active when it was added after a 20 min pulse with MTIC as when the 2 compounds were given simultaneously. Karen also confirmed the radiosensitisation data that had been generated with the early benzamides. She showed that PARP inhibition was more effective at preventing recovery from potentially lethal damage than radiosensitising exponentially growing cells. Potentially lethal recovery is when cells are given a high dose of irradiation and their survival is estimated immediately or after a recovery period where they are allowed to repair the DNA damage. This is significant because the non-replicating fraction of a tumour is generally radioresistant. We were unable to see any sensitisation effect of NU1025 or NU1064 on antimetabolite-induced cytotoxicity [12]. Using NU1025, we were the first to demonstrate that PARP inhibition increased the DNA damage and cytotoxicity caused by topoisomerase I poisons, but not topoisomerase II poisons [13]. We continued synthesising new compounds and testing them for PARP activity with the next major step in potency being the synthesis of the benzimidazole NU1085 (2-(4-hydroxyphenyl)-benzimidazole-4-carboxamide Ki = 6 nM, IC50 = 80 nM) by Sheila Srinivasan [14]. Since our compounds were numbered sequentially, this means that our 25th compound NU1025 was 200x more potent than 3AB and our 85th compound was 1000x more potent.

The generation and characterization of PARP-knockout mice that both were viable and fertile by three groups independently in the late 1990s [15–17], initially caused us to think that PARP was non-essential, and so inhibiting it would not compromise the viability of cancer cells. However, I recalled the data of Satoh and Lindahl [18] demonstrating that nuclear extracts depleted of PARP were able to repair nicked DNA but nuclear extracts with PARP but incubated either in the absence of NAD<sup>+</sup> or presence of 3AB could not. These authors proposed that binding of PARP to the DNA break caused an obstruction unless it could auto poly (ADP-ribosylate) and dissociate to allow access of repair proteins to fill in and re-seal the gap. This was the first demonstration of PARP "trapping", although this phrase was not coined until at least 20 years later [19]. These observations meant inhibited PARP was likely to be more effective in inhibiting DNA repair than no PARP enzyme. The ability of cells derived from these mice to generate PAR led to the discovery of PARP2 [20] and subsequently other

PARPs by structural analogy [21], with the original most abundant PARP subsequently being called PARP1. It is important to note here that PARP1 is the most abundant PARP and most important in terms of DNA repair, with PARP2 having a similar role. Most inhibitors inhibit both PARP1 and 2, and the double knockout of PARP1 and PARP2 is lethal [22].

#### **3. Advanced Compound Development and Pre-clinical Studies Resulting from the Newcastle–Agouron Collaboration**

Our next piece of good luck (serendipity #3) was to meet Zdenek Hostomsky at the 9th NCI/EORTC annual meeting in Amsterdam in 1996. He was proposing to initiate a PARP programme at Agouron Pharmaceuticals (in La Jolla, California, USA), a company specialising in crystal structure-based drug design. He had obtained the plasmid containing the construct for the PARP1 catalytic domain from Gilbert deMurcia and the co-ordinates of its crystal structure from Georg Schultz [23]. All he needed was highly potent inhibitors. We struck up an agreement and gave him NU1085 to co-crystalise with the PARP1 catalytic domain. Initial promising results meant that their 6 month pilot funding to Newcastle was extended for a further 2 years to support two post-doctoral chemists and a technician, Alex White, Sarah Mellor and Richard Davies, and two post-doctoral biologists and a technician, Carol Delaney, Chris Calabrese and Lan Zhen Wang. At Agouron, analysis of the interactions made between the inhibitor and the PARP catalytic domain by Bob Almassy led the lead chemist on the project, Steve Webber, to propose tricyclic compounds with the carboxamide constrained within a 7 membered ring. Synthetic medicinal chemistry at Agouron was undertaken by Don Skalitzky and Stacie Canan, working with Steve Webber. Karen Maegley ran the biochemical evaluation, with cell-based target inhibition and cytotoxicity assays being done by Jianke Li. Ted Boritzki and Bob Kumpf led the conventional and computational pharmacology, respectively at Agouron in collaboration with Chris Calabrese and Huw Thomas in Newcastle. At the end of the 2 year period, several potent tricyclic indole and benzimidazole inhibitors had been made at Agouron and they extended the funding to Newcastle for a further 3 years to cover the in vitro and in vivo pre-clinical evaluation of these inhibitors, as well as the development of pharmacodynamic biomarkers until the clinical trial started in 2003.

Extensive investigation of the cellular activity of these tricyclic compounds in terms of their ability to enhance the cytotoxicity of the DNA methylating agent, temozolomide, and the topoisomerase I poison, topotecan was undertaken in Newcastle, principally in two colorectal cell lines, LoVo and SW620 by Lan Wang and Suzanne Kyle, with Jianke Li investigating the effects in A549 lung cancer cells. My group have always maintained the highest possible stringency regarding the cultivation of cells, confirming they are mycoplasma free by routine regular testing (2 monthly intervals) and handling each cell line separately (including reagents) to eliminate the possibility of cross-contamination. Although these studies preceded routine authentication, our stocks have been authenticated since and all human cell lines are either purchased new and placed in an authenticated bank at first or second passage or authenticated and then placed in the authenticated bank. In vivo efficacy and pharmacokinetic/pharmacodynamic (PK/PD) studies were also undertaken by Chris Calabrese working with Huw Thomas and Mike Batey [24]. The first potential clinical candidate was AG14361 (1-(4-((dimethylamino)methyl)phenyl)-8,9-dihydro-2,7,9a-triazabenzo[cd]azulen-6(7H)-one: Ki < 5 nM) [25–29]. AG14361 caused profound sensitisation of temozolomide and was able to overcome temozolomide resistance due to defects in DNA mismatch repair [30]. The enhancement of topotecan and ionising radiation cytotoxicity in vitro was less pronounced but again the prevention of recovery from potentially lethal irradiation was substantial. In vivo radiosensitisation studies were conducted in Manchester by Kaye Williams and Ian Stratford [29]. Investigations in NIH 3T3 cells from PARP1-knockout mice enabled my PhD student, Lisa Smith, co-supervised by a topoisomerase expert, Caroline Austin, to confirm that topotecan was more cytotoxic in PARP-null cells and sensitisation by AG14361 was indeed due to PARP1 inhibition [31].

Investigation of in vivo activity against SW620 and Lovo xenografts revealed some interesting data. AG14361 caused an approximately 3-fold increase in temozolomide-induced anticancer activity against Lovo xenografts, but the combination caused sustained complete tumour regressions of SW620 xenografts. This was curious because in cell cultures, Lovo cells were the most sensitised to temozolomide by AG14361 and SW620 cells were not sensitised at all. SW620 cells were particularly sensitive to temozolomide alone (due to functional mismatch repair and lack of methylguanine methyltransferase). This led to another example of the synergy of science of serendipity: the discovery of the vasoactivity of this class of compounds. I reasoned that the discrepancy between the in vivo and in vitro effects meant that the tumour regression had to be something to do with the tumour micro-environment. Nicotinamide was known to be a vasodilator [32], and so I hypothesised that AG14361, which, like all PARPi contains a nicotinamide pharmacophore, might improve the delivery of temozolomide to more of the tumour cells. In collaboration with Kaye Williams and Ian Stratford in Manchester, we showed that indeed AG14361 did increase the areas perfused in the tumour, a further example of the synergy of science and serendipity (#4). [29]. This effect may have also contributed to the enhancement of the anticancer activity of the topoisomerase I poison, irinotecan, by AG14361, which was greater in vivo than in vitro. Similar vasoactive effects were reported by others with olaparib, which was accompanied by increased radiosensitivity of tumour xenografts due to better oxygenation [33]. Vasoactivity may contribute to the in vivo radiosensitisation by AG14361 but we have not investigated whether this is the case. It is possible therefore that it is a class effect and that PARPi may increase the anticancer activity of other drugs by increasing delivery to the tumour. It should be noted that these experiments were done with subcutaneous xenografts and the effect on in situ tumours may be somewhat different, especially as PARPi in the clinic have not been reported to cause hypotension except rarely in the elderly population [34], as might be expected from a vasodilator. Nevertheless, there may be effects that are specific to the tumour vasculature, but this has never been investigated.

The exciting data with AG14361 led to a proposal for a clinical trial with this compound being designed at a meeting of the team early 2000 and drafted for submission to CRUK's New Agent Committee. The teams in Newcastle (CR Calabrese, AH Calvert, NJ Curtin, BW Durkacz, BT Golding, RJ Griffin and DR Newell), Manchester (J Monaghan, I Stratford, and K Williams) and Agouron (R Almassy, T Boritzki, S Canan-Koch, L DiMolfetto, G Furman, Z Hostomsky, A Johnston, R Kumpf, J Li, K Maegley, D. Skalitzky, SE Webber and K Zhang) all contributed to the proposal. Meanwhile, there had been continued evaluation of a number of potential inhibitors. Many of the studies that had originally been conducted with AG14361 were also replicated with several other compounds, in vitro by Suzanne Kyle and Lan-Zhen Wang and in vivo by Chris Calabrese and Huw Thomas. Computational studies by Bob Kumpf suggested a fluoro substituent at the 8 position would improve activity with the ultimate compound being 8-fluoro-5-(4-((methylamino)methyl)phenyl)-2,3,4,6-tetrahydro-1*H*-azepino[5,4,3-*cd*]indol-1-one, or AG014447. AG014447 and/or the phosphate salt—called AG014699—had been included in the pre-clinical in vitro and in vivo studies and was selected for clinical investigation [35]. AG014699 (rucaparib) was indeed more potent than AG14361 in in vivo studies and, whereas 10 mg/kg AG14361 caused complete tumour regressions in combination with temozolomide, 1 mg/kg AG014699 was equally effective [29,35]. It also had a similar vasoactive effect [36]. Pharmacokinetic (PK) studies with AG14699, conducted by Huw Thomas and Mike Batey, showed that although it was cleared quite quickly from the bloodstream, it accumulated in tumour xenografts and, at the efficacious dose, it suppressed PARP activity in the tumour by >50% for 24 h. The clinical trial proposal was re-written with AG014699 data, with input from the team above. The involvement of the team who had developed and studied the drug pre-clinically ensured that the first clinical trial was as rationally designed, with a full understanding of the pre-clinical data as it is possible to have and supported by translational pharmacodynamic, as well as PK measurements. The proposal was submitted to CRUK's New Agent

committee in 2001, with a successful outcome. AG014699, now called rucaparib, was the first PARPi to be given to a patient with cancer in May 2003.

#### **4. The First Clinical Trial and Supporting Science**

Based on the pre-clinical data, the target dose clinically (PARP inhibitory dose: PID) was one that inhibited PARP activity by >50% in a surrogate tissue (lymphocytes) for at least 24 h. To support this pharmacodynamically guided clinical trial, we started adapting the 32P NAD<sup>+</sup> incorporation assay we had used to measure PARP activity and inhibition in permeabilised cells as part of the early inhibitor development. In these endeavours, we were blessed with two more items of good fortune. Firstly, experiments conducted early in 2002 by Suzanne Kyle revealed that PARP inhibition was not lost as soon as the drug was removed Figure 2A), meaning that pre-treated cells could be harvested and processed without loss of inhibition. This was an instance of real serendipity (# 5), as it is quite rare to see such persistence. Secondly, at the same time, Suzanne showed that PARP activity was maintained in cells that had been cryopreserved at -80 ◦C for up to 14 weeks. More importantly, rucaparib's inhibition of PARP activity was incredibly stable: cells that had been pre-treated with rucaparib could be harvested and cryopreserved for up to 14 weeks without loss of inhibition, an even more remarkable example of serendipity (#6); Figure 2B.

**Figure 2.** Inhibition of cellular poly (ADP-ribose) polymerase (PARP) activity. (**A**) PARP inhibition by increasing concentrations of AG14699 (rucaparib) following pre-treatment of L1210 cells (30 min) with drug prior to harvesting (red symbols and line) compared with drug added to the permeabilised cells in the reaction mixture (blue). Data are the mean and SD of three replicates from a representative experiment. (**B**) Stability of PARP inhibition by rucaparib with storage. Cells were exposed to rucaparib for 30 min prior to harvesting and cryopreservation. PARP activity was measured by 32P NAD<sup>+</sup> incorporation. Previously unpublished data.

We then started harvesting lymphocytes from healthy volunteers (i.e., our colleagues in the lab). However, we soon discovered that PARP activity in human lymphocytes is orders of magnitude lower than in cultured cells and we would have needed approximately 50 ml blood pre- and post-treatment with rucaparib to be able to detect inhibition reliably. This is where yet another example of good fortune came in (serendipity #7). Alex Burkle had recently moved to Newcastle with his post-doc Ragen Pfeiffer and they had developed a much more sensitive immunoblot assay using the 10H antibody to the product, PAR [37]. They shared this technology with us and Ruth Plummer, who was my clinical fellow at that time, validated this pharmacodynamic (PD) assay to GCLP standard. It was initially intended that she would carry out the rucaparib Phase I trial. However, there was a substantial delay in the start of the trial, partially due to the take-over of Agouron by Warner Lambert (in January 1999) that was followed less than a year later by the take-over of Warner Lambert by Pfizer, who did not at that time have an oncology programme. Instead of conducting the rucaparib trial, Ruth used the PD assay to monitor the effects of temozolomide on PARP activity in patients with metastatic melanoma instead [38]. It was my next clinical fellow, Chris Jones, who conducted the trial under the guidance of Ruth Plummer and Hilary Calvert, the principal and chief investigators, respectively. Chris made further improvements to the assay and monitored PARP inhibition in the lymphocytes and tumour biopsies of patients treated with rucaparib [39]. Using this assay, another student of mine, Thomas Zaremba, set out to study PARP1 genomics, expression and activity in healthy volunteers and cancer patients with the aim of determining a) whether PARP genotype contributed to reduced PARP activity and the likelihood of developing cancer and b) whether PARP1 genotype, expression or activity contributed to unexpected toxicities in patients treated with temozolomide or radiotherapy. He did not manage to answer these questions but what he did find was that a) only 20% of the variation in PARP activity is due to the variation in PARP1 expression, b) that men have on average 40% more PARP activity than women and c) that it is androgen-driven, suggesting post-translational modification or co-activators/repressors that are androgen regulated [40]. The question this raises is: when used as a chemo- or radiosensitiser, should PARPi doses be lower in women?

The first trial of rucaparib was a dose escalation trial in cohorts of 3 patients with a variety of solid tumours receiving 1, 2, 4, 8 and 12 mg/m<sup>2</sup> in combination with half-dose temozolomide (100 mg/m2). There was a dose-dependent decrease in PARP activity, with the desired ≥50% inhibition of PARP activity for 24 h being consistently achieved above 8 mg/m2. In the absence of increased toxicity, 12 mg/m<sup>2</sup> was established as the safe dose to begin escalation of the temozolomide up to full dose (200 mg/m2) in melanoma patients consenting to a pre-treatment and post-treatment biopsy. There was a dose-dependent decrease in PARP activity in the tumours too, with approx. 90% inhibition 4–8 h after administration of 12 mg/m2. Escalating the dose to 18 mg/m2 led to increased haematological toxicity and 12 mg/m2 was established as the recommended Phase 2 dose to take forward in combination with temozolomide. Unfortunately, although this combination may have shown an improvement on historical data of response to temozolomide alone in patients with melanoma, there was also more haematological toxicity [41]. This may have doomed rucaparib to the same failure to progress beyond phase II studies, as with other DNA repair inhibitors such as the MGMT inhibitors O6 benzylguanine and Patrin [42], had it not been for the next and most significant example of serendipity on this project.

#### **5. Discovery of the Synthetic Lethality of PARPi**

The synergy between science and serendipity (#8) reached its peak with the discovery of synthetic lethality in BRCA mutant and other cancers defective in homologous recombination DNA repair (HRR). This came about through lucky coincidences with timing of various discoveries and meetings as described below. At the end of 2001, Herbie Newell was invited to speak at Sheffield University about the drug development programme in Newcastle University. Present at this meeting was Thomas Helleday, who had been recruited to Sheffield from Tomas Lindahl's lab. Tomas Lindahl (co-recipient of the Nobel prize for chemistry 2015 for his work on DNA repair) had a long-standing interest in PARP, its function and the effects of inhibitors. Tomas Lindahl had noted that in the presence of PARP inhibitors, there was an increase in recombination events. This led him to propose that the negative charge on the ADP-ribose polymer helped to repel negatively charged DNA from sites of DNA breakage to avoid unwanted recombination events [43]. Thomas Helleday had investigated further and found that cells lacking XRCC1 (the scaffold protein recruited to DNA breaks by ADP-ribose polymers [44], as well as those lacking PARP1, accumulated more γH2AX foci (a marker of DNA damage) and RAD51 foci (a marker of HRR). As a result of Herbie's talk in Sheffield Thomas requested some of our more potent and selective PARPi. We gave him some NU1025 and invited him to give a talk in Newcastle to present his work in January 2002. He convincingly showed that XRCC1 defects had similar effects in increasing HRR compared to PARP1 deletion or inhibition with NU1025. This suggested the polymers were not acting to inhibit recombination but instead a failure of BER led to an increase in HRR. He proposed that cell lacking BER/PARP function would be dependent on HRR for survival and as such cells lacking HRR function may be dependent on PARP activity for survival. He tested his hypothesis in cells defective in HRR (IRS1 lacking XRCC2 and irs1SF lacking XRCC3), which were killed by concentrations of NU1025 that did not kill wt or XRCC2/3 corrected cells.

By this time, I had widened my interest in DNA repair and as well as leading the pre-clinical biological studies on PARPi, I also led the DNA-PK and ATM inhibitor projects at Newcastle. As a result of this, I attended a meeting early in 2002, where Ashok Venkitaraman described the data supporting the key role of BRCA2 in HRR. BRCA 1 and BRCA 2 were discovered in the early 1990s as breast cancer susceptibility genes but at first it was not clear why. Their function in DNA repair began to emerge towards the end of that decade and it was confirmed that they played key roles in HRR around the turn of the millennium [45–48]. More reading around the topic convinced me that our PARP inhibitors might have therapeutic potential in BRCA mutant hereditary breast cancer, which I put to the drug development team in May 2002. The BRCA2 mutant Capan-1 pancreatic cells along with the BRCA wt BxPC-3 pancreatic cells, as control, were duly purchased and their sensitivity to AG14361 was determined by Suzanne Kyle. Frustratingly, these initial experiments, conducted in July 2002, did not show that the BRCA mutant cells were more sensitive. However, experiments that had been conducted 3 to 4 months previously in wt (AA8) and the HRD derivative (XRCC3 mutant: irs1SF) Chinese hamster ovary cells had indeed shown a very marked difference in the sensitivity of the wt and HRD cells (Figure 3). To this day, I am not sure why the data were so disappointing with the Capan-1 cells as we got more impressive results both in vitro and in vivo with this cell line using rucaparib (see later). Maybe it was the design of the experiment and the exposure period (24 h) was insufficient, as less than a complete cell cycle, or the moribund cells were lost during harvesting or maybe the BxPC-3 cells were also HRD.

**Figure 3.** AG14361 is more cytotoxic in XRCC3 mutant irs1SF cells compared to wild-type AA8 Chinese hamster ovary cells (**A**) but BRCA2 mutant Capan-1 cells are not more sensitive than BRCA wild-type BxPC-3 pancreatic cancer cells (**B**). Cells were exposed to increasing concentrations of AG14361 for 24 h prior to seeding for colony formation in fresh medium. The insert shows an extended concentration range for pancreatic cell lines. Previously unpublished data pooled from three independent experiments.

We communicated our findings to Thomas Helleday, with whom we had established a collaboration, in the autumn of 2002. By Spring 2003, he had obtained the VC8 BRCA2 mutant derivatives of Chinese hamster lung fibroblast V79 cells, which he later shared with us. Suzanne got her first result with these cells in June 2003 and they were so impressive we could hardly believe it. She replicated the result several times and confirmed that 98.5% of BRCA2 mutant cells were killed by 10 μM AG14361, a concentration that had no significant effect on the viability of either parental wt cells or the BRCA2-corrected cells. Conducting in vivo experiments with these cells proved more of a

challenge. The V-C8 cells would not grow subcutaneously and, conversely, the V79 cells generated very haemorrhagic tumours that rapidly made the mice unwell such that they had to be humanely killed. However, Huw Thomas eventually succeeded in growing the V-C8 cells and the BRCA2 corrected V-C8.B2 cells intramuscularly. These experiments showed that treatment of mice with AG14361 caused a complete regression of the BRCA2 mutant tumour but growth of the BRCA2 corrected tumour was unaffected. The data generated by Suzanne and Huw went into the paper that was published in Nature in 2005 [49]. These key experiments, which have led to the current use of PARPi clinically, were actually undertaken outside of the remit of the drug development programme. This was because at Herbie Newell's suggestion, the rest of the drug development management team at Newcastle had decided that PARP was no longer to be considered a drug development project to be discussed at subsequent meetings in June 2003 as the clinical trial (albeit in combination with temozolomide) was underway.

The data with the BRCA mutant and HRD models, where a tumour-specific defect in DNA repair is exploited by inhibiting a complementary DNA repair pathway were indeed ground-breaking. The findings completely changed the way we think about treating cancer, from trying to overcome some property of the tumour that gave it an advantage to exploiting a vulnerability. Indeed, the search for additional examples of synthetic lethality has become somewhat of a holy grail. However, I believe PARP inhibition in HRD is unlikely to be bettered as an example of synthetic lethality for 2 reasons: (1) there is a very high level of DNA SSB, that depend on PARP for their repair, due to the endogenous ROS produced by normal metabolism, and this is increased in cancer by virtue of the associated inflammation, and (2) HRR defects are relatively common in cancer, not only due to BRCA mutations. This is yet another example of the synergy of science and serendipity (#9)

Understanding the importance of the synthetic lethality of PARPi in HRD cancers, Thomas Helleday was keen to patent our findings before publication. Following meetings with Cancer Research Technology, we undertook work to protect rucaparib and these data were included in the patent (WO 2005/012305 A2) filed 16th April 2004.

#### **6. The Clinical Development of Rubraca**®**as A Single Agent and Predictive Biomarker Development in Ovarian Cancer**

During this time, the Phase I clinical trial of rucaparib with temozolomide was ongoing and there was clinical interaction with Pfizer who later conduct a single agent dose escalation study with rucaparib. This study was led by Hilary Calvert and Ruth Plummer, who recruited Yvette Drew, a clinical fellow, in 2006 to undertake the trial. In parallel, Yvette conducted pre-clinical and translational studies with me. The translational studies included taking blood samples from the patients before and at intervals after rucaparib adminiatration. These samples were used to determine the rucaparib PK and also PARP1 genotype, protein levels and inhibition by rucaparib in the patients' PBMCs (lymphocytes).She also undertook studies in a panel of BRCA wt and mutant human cancer cell lines to confirm the differential sensitivity we had seen in the matched Chinese hamster cells (AA8/irs1SF and V-C8/V-C8.B2). This panel included the UACC3199, which had epigenetically silenced BRCA1 due to promoter methylation and the Capan-1 cells that had been disappointing in the AG14361 experiments. Such studies were needed because there were reports that other BRCA mutant human cancer cells were insensitive to PARPi, including AG14361 [50,51]. Fortunately, rucaparib sensitivity was found to be substantially greater in the BRCA mutant cells (including the Capan-1 cells) than the wt and, importantly, the BRCA silenced cells were comparable with the mutant ones in this respect [52].

At the same time, I had established an interaction with a gynaecological surgeon, Richard Edmondson, who had a number of trainees keen to undertake lab-based projects for MD or PhD. This turned out to be yet another example of the synergy between science and serendipity (#10). I was aware that BRCA mutation/HRD was a determinant of sensitivity to cisplatin/carboplatin from discussions I had had with Paul Harkin at a meeting in 2003. The standard of care for ovarian cancer is surgery followed by carboplatin + paclitaxel chemotherapy with an initial response rate of approximately 60%. Knowing that only approximately 15%–25% of ovarian cancer is associated with BRCA mutations, that left 35%–45% of the responses unaccounted for. I reasoned that they must be HRD for some other reason and that we could test this hypothesis by measuring γH2AX and RAD51 foci. γH2AX foci are formed at collapsed replication forks and DNA DSBs and RAD51 coats the SS DNA to form the nucleoprotein filament needed to invade the complementary DNA on the sister chromatid during HRR resulting in RAD51 foci. Therefore, γH2AX foci indicate the lesions generated by PARP inhibition and RAD51 foci indicate when HRR occurs. We first met with Pfizer representatives (including Zdenek Hostomsky and Gerrit Los) in September 2006 to suggest that Yvette Drew, the clinical fellow undertaking the trial of single agent rucaparib, would not only work with cell lines to establish the spectrum of rucaparib sensitivity (described above) but also to develop an assay for HRR function, based on measuring γH2AX and RAD51 foci. We also proposed to evaluate the HRR status in primary cultures of ovarian cancer ascites collected during primary/debulking surgery with another clinical fellow Asima Mukhopadhyay, a trainee gynaecological oncology surgeon supervised by Richard Edmondson and me. Thanks to Zdenek Hostomsky, ever our PARP champion even though he had been diverted to other projects, I secured funding from Pfizer in 2008 to support these studies with an additional technician, Evan Mulligan, to help with planned in vivo efficacy studies..

These studies led to two key papers published in 2010/11 [52,53]. The data Yvette generated in the cell lines not only demonstrated the difference in rucaparib cytotoxicity between HRD and HRR functional cells but also that RAD51 foci were a useful discriminant/potential biomarker of HRR function. That is, the induction of RAD51 foci in the HRD cells was not significant whereas there was a significant increase in the BRCA wt cells. Looking at γH2AX foci really brought it home to us how much endogenous damage cells sustain without the addition of exogenous genotoxic agents. The level of the foci after only 24 h of inhibition of endogenous damage repair with rucaparib was the same as the standard radiotherapy fraction of 2 Gy. After some significant work to establish the optimum conditions to grow primary cultures from ascites, Asima successfully translated this HRR function assay to the ascites cultures and her initial studies showed that approximately 60% of the primary cultures were indeed HRD and that this corresponded to a greater rucaparib-induced inhibition of cell growth. This was the first demonstration that >50% of ovarian cancers are HRD and, at significantly less expense than similar findings by the TCGA, which was published the following year [54].

The in vivo experiments in mice bearing BRCA1 mutant (MDA-MB-436) or BRCA2 mutant (Capan-1) xenografts, conducted by Yvette Drew and Evan Mulligan, showed that rucaparib caused significant tumour growth delay and this was more pronounced when the treatment period was 5x weekly for 6 weeks compared with daily x10. This was not entirely unexpected from an understanding of the underlying mechanism whereby to kill the cells PARP must be inhibited as the cell progresses through S-phase. These findings were recapitulated in the clinical study that Yvette was conducting where responses were seen when the patients switched from daily treatment for 5 days every 3 weeks to continuous dosing [55]. This study, initiated in 2007, was the first single-agent trial of rucaparib, sponsored by CRUK and Pfizer. It began very cautiously, starting at low dose (escalating from 4 to 18 mg/m2 i.v) on an intermittent schedule (daily for 5 days every 3 weeks) based on the trial in combination with temozolomide. During the study, there was a switch to an oral formulation and daily dosing, starting at 92 mg (equivalent to 18 mg/m2 based on an oral bioavailability of approximately 30% and average body surface area of 1.7 m2) once a day for 7 days and escalating to 600 mg 2x/day continuously. Many more responses were seen once dosing was continuous. It is not altogether clear whether it was the increase in the dose or the intensity of the schedule that was critical. Unfortunately, this rather slow start allowed rucaparib to be overtaken by olaparib, which went into its first clinical trial as a single agent and the dose was escalated more quickly, as there was no history of combination toxicity data to suggest a cautious approach was necessary. As a result, the first publication showing that the pre-clinical data on the synthetic lethality of PARPi in HRD/BRCA mutant cancer was translatable to the clinic was with olaparib [56].

In a way, this result could have been avoided with more careful scrutiny of the pre-clinical data. Pre-clinically much lower (at least 10x) concentrations (in cell cultures) and doses (in tumour xenograft

studies in mice) were needed to sensitise cells and tumours to temozolomide than were needed for single agent activity in BRCA mutated/HRD cancer cells. Furthermore, increasing the dose of rucaparib to one that was active against BRCA mutant xenografts was so toxic that the mice had to be humanely killed a few days into the drug administration [29,35]. These differences are easily understood by reference to the mode of action of the PARPi in the different scenarios. Because DNA integrity is so important, the cell has more than adequate levels of repair enzymes to cope with endogenous and most environmental DNA damage. Couple this with the fact that single base lesions and SSB are the commonest form of endogenous lesions, and these are dependent on PARP for their repair, and it is easy to see that cells will have a surplus of PARP to cope with daily levels of DNA damage. In order for the damage to overwhelm this repair capacity and cause death in cells lacking the back-up pathway of HRR, there has to be a total suppression of PARP activity and relatively high levels of drug are needed to achieve this. In addition, PARP must be inhibited long enough for all tumour cells to enter S-phase with unrepaired DNA lesions and so long/continuous exposures are necessary. In the alternative scenario where, e.g., temozolomide, induces orders of magnitude greater numbers of base lesions and SSBs, it is not necessary to inhibit their repair completely to render the remaining DNA damage sufficient to kill cells and so much lower levels and shorter duration of PARP inhibition are needed. Also, when used as a single agent, the PARPi is exploiting a tumour-specific defect, and side effects are not expected so higher doses can be used. When the PARPi is synergistic with the cytotoxic in cancer cells the combination is likely to also be synergistically toxic to normal tissues so only lower doses are tolerated.

The observation that continuous therapy was much better than intermittent led us to investigate how durable PARP inhibition by rucaparib was. This study, initiated in 2011 in collaboration with and support from Zdenek Hostomsky and Gerrit Los, was partially based on our observations in the first clinical trial: as the dose of rucaparib was increased PARP activity was suppressed in PBMCs for 24 h after the 30 min infusion, even though the drug was no longer detectable in the plasma. Indeed, at the 12 mg/m2 dose, PARP activity was still way below pre-dose levels on the Monday after the last Friday dose in many patients [39]. Pre-clinically we found that rucaparib accumulated above the extracellular concentration and was retained after drug removal in cancer cells and that PARP was inhibited for at least 72 h after a 30 min exposure. In in vivo studies, a single oral dose of rucaparib inhibited PARP activity in xenografts for at least 7 days even though it was cleared from the blood within 48 h and was undetectable in the tumour within 72 h. Moreover, tumour growth inhibition following weekly dosing with rucaparib was equivalent to daily dosing [57]. This, I believe, is another example of serendipity (#11) ensuring the continuous suppression of PARP activity necessary for antitumour activity as a single agent. It is possible that this might be exploited clinically by weekly dosing schedules.

Pfizer initiated a Phase I study in combination with carboplatin alone or with paclitaxel or pemetrexed or a combination of cyclophosphamide and epirubicin NCT01009190 in November 2009 [58]. There was little pre-clinical rationale for such a study, as PARPi do not sensitise cells to either pemetrexed (an antifolate), cyclophosphamide (DNA cross-linking agent), epirubicin (DNA intercalating topoisomerase II poison) or paclitaxel (an antitubulin agent, not a DNA damaging agent) and the data with cisplatin or carboplatin are variable and cell line dependent, most probably additive in HRD cells only [59]. The triple combination arms were dropped during the study and the MTD in combination with carboplatin (AUC 5) was 240 mg one daily (carboplatin day 1, rucaparib days 1–14 of a 21 day cycle) due largely to haematological toxicities [58]. Thus, the MTD for rucaparib in combination with carboplatin (with which is does not synergise) is approximately four times higher than with temozolomide and 2.5x lower than as a single agent.

#### **7. Studies That Led to FDA Approval**

In June 2011, Pfizer licenced rucaparib to Clovis oncology and in November, study 10, (NCT01482715) a phase I–II trial of oral rucaparib as a single agent, was initiated [60,61]. By this time, Hilary Calvert had moved from Newcastle to UCL and he supported another clinician, Rebecca

Kristeleit, who led the study. The Phase I component was a dose escalation of the oral formulation, based on the intermittent vs. continuous dosing study described above, in patients with solid tumours to establish the recommended phase II dose (RP2D) based on tolerability and efficacy. The RP2D of 600 mg 2x daily was then used to treat women with BRCA1/2 mutated high-grade serous ovarian cancer as the Phase II part of the study. The objective response rate was 60% and the toxicities were generally mild with only 5 out of 98 patients having to discontinue treatment due to an adverse event.

Ongoing studies with other PARPi clearly demonstrated that BRCA mutation was not synonymous with a response in ovarian cancer [62], with cases with BRCA mutations in the non-responder group as well as cases without BRCA mutations in the responder group. Indeed, our earlier studies (described above) clearly indicated that BRCA mutation significantly underestimated the HRD and PARPi sensitive population. Several companies were developing predictive biomarkers of HRD and we shared some of our samples that we had previously characterised by the functional assay (by this time, our collection had grown substantially) with Tom Harding at Clovis and Foundation Medicine from 2011 to 2013. However, it seemed that the normal cellular component (that died off during culture leaving us with just the cancer cells to classify) diluted the cancer component too much for the samples to be of much use. Nevertheless, based on the observation that BRCA mutations result in substantial genome-wide loss of heterozygosity (LOH), Foundation Medicine [63] developed a genomic profiling test to identify HRD independent of BRCA mutation to be used as a companion diagnostic in the Ariel 2 trial NCT01891344 [64]. Similar tests have been developed by Myriad. Although these tests represent genetic "scarring" caused by an HRR defect at some time during the cancer's development, but not necessarily at the time of treatment, they are substantially less challenging than functional assays, which by their nature require viable fresh tumour tissue. Screening for mutations in known HRR genes has also been used.