Claspin: From replication stress and DNA damage responses to cancer therapy

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Claspin: From replication stress and DNA damage responses to cancer therapy Diana Azenhaa,b,c, Maria Celeste Lopesa,b, Teresa C. Martinsb,c,* a

Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal Center for Neuroscience and Cell Biology and Institute for Biomedical Imaging and Life Sciences (CNC.IBILI), University of Coimbra, Coimbra, Portugal c Portuguese Institute for Oncology at Coimbra, Coimbra, Portugal. *Corresponding author: e-mail address: [email protected] b

Contents 1. Introduction 2. Claspin, DNA replication, and replication stress 2.1 Claspin and DNA replication 2.2 Claspin and the replication stress response 3. Claspin and the DNA damage response 3.1 Claspin and checkpoint activation in response to DNA damage 3.2 Claspin and DNA repair 4. Claspin as a target for chemo- and radio-sensitization 4.1 Inhibitors targeting RSR 4.2 Inhibitors targeting DNA repair pathways 4.3 A rationale for targeting Claspin 5. Conclusions and perspectives Conflict of Interest Funding References

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Abstract Cancer is still one of the major causes of death worldwide. Radiation therapy and chemotherapy remain the main treatment modalities in cancer. These therapies exert their effect mainly through interference with DNA replication and induction of DNA damage. It is believed that one way of improving the efficacy of cancer treatment will be to inhibit the replication stress and DNA damage responses and promote mitotic catastrophe of cancer cells. So far, the majority of the efforts have focused central players of checkpoint responses, such as ATR and CHK1, and DNA damage repair, such as PARPs. Being a key player in the replication stress response, checkpoint activation, and the DNA damage response, Claspin constitutes an attractive therapeutic target in cancer, namely

Advances in Protein Chemistry and Structural Biology ISSN 1876-1623 https://doi.org/10.1016/bs.apcsb.2018.10.007

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for radio- and chemo-sensitization. In this review, we will go through Claspin functions in the replication stress and DNA damage responses and will discuss how Claspin can be targeted in cancer treatment, as well as the effects of Claspin inhibition.

1. Introduction Cancer is the second cause of mortality worldwide and was responsible for 8.8 million deaths in 2015. WHO predictions point to a 70% increase in the number of new cases for the next 20 years (http://www.who.int/ mediacentre/factsheets/fs297/en). Radiotherapy (RT) and chemotherapy (CT), along with surgery, are the main treatment modalities for cancer. These therapies exert their effect mainly through infliction of DNA damage. In addition, conventional CT also aims to inhibit DNA replication and induce replication stress, which also ends up generating DNA damage. As such, it is believed that improving the efficacy of conventional cancer treatment will majorly depend on a finer understanding of the biological response of tumor cells to genotoxic stress, as well as on the identification of molecular changes of tumor cells that affect these responses and may be used either as biomarkers of these responses, or as therapeutic targets (e.g., Kahn, Tofilon, & Camphausen, 2012). Therapeutic approaches targeting the replication stress response (RSR) and the DNA damage response (DDR) are believed to preferentially sensitize cancer cells to therapy because these cells present a higher proliferative stress and genomic instability than normal cells (e.g., Boyer, Walter, & Sørensen, 2016; Carrassa & Damia, 2017). The majority of the efforts developed so far have focused on players of cell cycle checkpoints (e.g., ATR, CHK1) and the DNA damage response (e.g., PARP) (e.g., Raleigh & Haas-Kogan, 2013; Syljua˚sen, Hasvold, Hauge, & Helland, 2015). Survival of cells exposed to RT or to the genotoxic agents used in CT is dependent on several biological processes, such as cell cycle checkpoint activation and DNA damage repair. Cell cycle checkpoints are barriers located at critical points of the cell cycle that monitor and ensure correct timing and sequence of all molecular events that occur during proliferation. These checkpoints are activated in response to stressful stimuli, such as DNA damage and replication stress, and result in cell cycle arrest, which provides the cell time to try to repair DNA lesions and resume the cell cycle, or the activation of cell death mechanisms, when damage is irreparable or too

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extensive ( Jones & Petermann, 2012). One key protein involved in the DDR and replication stress is Claspin. Claspin was first identified in Xenopus egg extracts as an essential mediator of ATR-Chk1 checkpoint activation following replication blocks and/or UV exposure (Kumagai & Dunphy, 2000). Human Claspin is a nuclear protein that is encoded by a gene (CLSPN) located on chromosome 1p34.3 (Kumagai & Dunphy, 2000). Claspin monitors DNA replication and, in the presence of replication problems, triggers cell cycle checkpoints. Claspin seems to bridge checkpoint responses and DNA damage repair through several pathways. Being an important component of the RSR and the DDR, it is conceivable that Claspin may constitute a putative therapeutic target in cancer, namely for radio- and chemo-sensitization.

2. Claspin, DNA replication, and replication stress 2.1 Claspin and DNA replication Faithfull transmission of genetic information depends on the successful replication of DNA and chromosome duplication. Thus, one of the primary sources of genomic instability is replication stress, which may originate from endogenous (e.g., oncogene-induced stress, secondary DNA structures, repetitive DNA sequences, collisions with the transcriptional machinery, and limiting nucleotides), or exogenous sources (e.g., DNA lesions induced by exogenous genotoxic agents) (e.g., Boyer et al., 2016) and is related to stalling of replication forks (Forment & O’Connor, 2018). DNA replication involves several key events that take place in specific moments of the cell cycle. Replication initiates in late mitosis and the G1 phase of the cell cycle through the assembly of the pre-replication complexes (pre-RCs) at thousands of replication origins. These complexes are constituted by the ORC (origin recognition complex), Cdc6, CDT1, and the Mcm 2–7 helicase. Then, at the G1-S phase transition, the Mcm helicase is activated and the pre-initiation complexes are formed through recruitment of several partners, namely TopB1, Treslin, Cdc45, GINS, RECQL4, Mcm10, and the Claspin-Tim-Tipin complex. This is followed by the activation of the CMG (Cdc45-Mcm-GINS) helicase complex and the initiation of DNA synthesis at replication origins (origin firing) in the S phase. The activated CMG helicase complex unwinds the DNA strands, giving rise to structures called replication forks, an event that promotes recruitment of several other proteins, such as RPA, which binds to single-stranded DNA (ssDNA) and stabilizes the fork. At this time, the

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tension imposed by DNA unwinding is relieved by the action of topoisomerases, which first cut and then re-anneal the DNA strand(s). After origin firing, there is also the recruitment of the ATR-ATRIP complex, Claspin, the 9-1-1 complex and CHK1, whose function is the limitation of firing of the origins. It is during the S phase that DNA polymerases (pol) ε e δ replicate the genome. In the G2 phase of the cell cycle, replication is finished and the Mcm helicase and DNA polymerases are unloaded from the DNA (Boyer et al., 2016; Hsieh & Peng, 2017). Claspin has a clear role in DNA replication (Fig. 1) and interacts with numerous proteins involved in the process, such as Tim, Tipin, Mcm proteins, DNApol α, δ, ε, Cdc7 kinase, and Cdc45 helicase (Broderick, Rainey, Santocanale, & Nasheuer, 2013; Errico & Costanzo, 2012; Lee, Gold, Shevchenko, Shevchenko, & Dunphy, 2005; Sar et al., 2004; Sercin & Kemp, 2011; Uno & Masai, 2011). Claspin is one of the integral components of the replisome, which is loaded onto replication origins, and directly monitors DNA synthesis during the S phase (Lee et al., 2005; Sar et al., 2004; Smits, Cabrera, Freire, & Gillespie, 2018; Uno & Masai, 2011). Association of Claspin with chromatin is dependent on the pre-initiation complex and Cdc45, an observation that suggests that binding of Claspin to chromatin is closely linked to the initiation of DNA replication and to opening of the DNA strands (Smits et al., 2018). In addition, Claspin seems to have a role in the initiation of DNA replication during unperturbed S phases through the recruitment of Cdc7, this way facilitating phosphorylation of Mcm proteins (Yang et al., 2016). These phosphorylation events are crucial for the initiation of replication, as they trigger the assembly of the initiation complex and origin firing. Therefore, recruitment of Cdc7 to pre-formed pre-RCs, by Claspin, may constitute a major determinant for replication timing regulation (Yang et al., 2016). It is possible that Claspin may provide an additional layer of regulation in DNA replication through regulation of the access of Cdc7 to its key substrates (Masai, Yang, & Matsumoto, 2017). Claspin seems to be required for normal fork progression, as its silencing induces a significant decrease in replication rates (Petermann, Helleday, & Caldecott, 2008). It is possible that Claspin facilitates fork progression by preventing prolonged stalling of replication forks (Errico & Costanzo, 2012). Claspin also has a role in the maintenance of replication fork stability (Scorah & McGowan, 2009). The yeast homolog, Mrc1, is also required for normal fork progression, traveling with the replication fork (Hodgson, Calzada, & Labib, 2007; Lou et al., 2008; Osborn & Elledge, 2003; Szyjka, Viggiani, & Aparicio, 2005; Tourrie`re, Versini, Cordo´n-Preciado,

Fig. 1 Role of Claspin in DNA replication. Claspin interacts with several other proteins during DNA replication. In G1/S transition, Claspin is complexed with Tim and Tipin and recruited to the DNA, being one of the components of the pre-initiation complexes. It is an integral component of the replisome, which is loaded onto replication origins in the S phase. Claspin has a role in the initiation of DNA replication and opening of the DNA strands, and promotes origin firing, through recruitment of Cdc7, which phosphorylates MCM proteins. During the S phase of the cell cycle, Claspin, together with ATR and CHK1, also has a role in the regulation of origin firing and in maintenance of replication fork stability. In addition, Claspin, through interaction with DNA pol ε, is responsible for coupling polymerization and unwinding of the DNA leading strand at replication forks, during the elongation process.

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Alabert, & Pasero, 2005). Mrc1, through the interaction with Cdc45, Mcm proteins and DNA pol ε, appears to couple strand polymerization with unwinding of the double strand during replication in unperturbed and stressed cells (Lou et al., 2008). Claspin is believed to have a similar role through its interaction with DNA pol ε and Cdc45 (Bando et al., 2009; Katou et al., 2003; Komata, Bando, Araki, & Shirahige, 2009; Lou et al., 2008; Nedelcheva et al., 2005). A model have emerged in which And1, a component of the replication progression complex, together with Tipin, couples helicase and DNA pol α activities on the lagging strand DNA template, while Claspin, through interaction with DNA pol ε, is responsible for coupling polymerization and the unwinding on the leading strand at replication forks (Errico & Costanzo, 2012). When replication forks stall, And1 interacts with Claspin, promoting its association with CHK1 and recruitment of Claspin-CHK1 complexes to the sites of damage, this way facilitating CHK1 phosphorylation by ATR and checkpoint activation (Hao et al., 2015).

2.2 Claspin and the replication stress response Cells suffer replicative stress when there is persistent stalling of replication forks. Sensing of stalled forks involves coating of ssDNA stretches by RPA, as well as the action of several proteins that are part of the replication complex, such as TopB1, Nbs1, Rad17, and the 9-1-1 complex (Tourrie`re & Pasero, 2007). The signal is then channeled to different response pathways by mediator proteins, such as Claspin (Kumagai & Dunphy, 2000; Tourrie`re & Pasero, 2007). It has been shown that Claspin is immediately recruited to chromatin upon replication fork slowdown (Koundrioukoff et al., 2013). Prolonged replication stress may trigger several cellular responses that will ensure that DNA replication is completed before mitosis. One of these responses is the activation of dormant origins of replication (Forment & O’Connor, 2018). Replication of eukaryotic genomes requires licensing of thousands of origins of replication during the G1 phase of the cell cycle. However, not all licensed origins will fire during the subsequent S phase (some will stay dormant) and not all active origins will fire at the same time (early origins will fire earlier than late origins of replication). When there is persistent stalling of an active replication fork, this can be compensated by firing of a dormant neighboring origin to ensure that the region containing the paused replisome is replicated (Techer, Koundrioukoff, Nicolas, & Debatisse, 2017). Nevertheless, to avoid

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DNA re-replication, origin firing must be tightly regulated. CHK1 and Claspin are involved in the inhibition of origin firing, but through different pathways. CHK1 acts primarily through phosphorylation of Cdc25A, which promotes its degradation in an ubiquitin-dependent fashion, and by preventing the interaction between Treslin and TopB1 (Hsieh & Peng, 2017; Scorah & McGowan, 2009). In turn, Claspin functions through a not yet elucidated pathway that does not involve CDK2/Cdc25 (Scorah & McGowan, 2009). It is established that Claspin participates in the regulation of origin firing through its interaction with Cdc7 (Fig. 2a), and the activation of the ATR-CHK1 checkpoint, which is involved in the inhibition of origin firing, among other outcomes (GonzalezBesteiro & Gottifredi, 2015). Cdc7 is a key serine/threonine kinase

Fig. 2 Functions of Claspin in replication stress response. The replicative stress responses (RSR) is triggered when replication forks stall. Claspin has several roles in RSR: (a) it participates in the regulation of origin firing, both through activation of CHK1, and, independently of CHK1, through interaction with Cdc7; (b) it has an adaptor role in the activation of the intra-S phase and G2/M checkpoints mediated by the ATRCHK1 axis; (c) together with Tim and Tipin, it is a component of the replication fork protection complex, which maintains and monitors replication fork stability, activates the ATR-CHK1-mediated checkpoints, and triggers the process of translesion synthesis; (d) it regulates the expression of common fragile sites, which constitute a source of genomic instability; and (e) it contributes for fork recovery through maintenance of sister chromatid cohesion. When these protective mechanisms do not operate effectively, replication stress may lead to extensive DNA damage and cell death.

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implicated in the initiation of DNA replication, namely through the phosphorylation of Mcm proteins, which results in the activation of the replisome (Deegan & Diffley, 2016). Claspin seems to promote Cdc7 and Mcm2 interaction through recruitment of Cdc7 to the pre-RC (Yang et al., 2016), where Mcm2 is phosphorylated and activated, and to facilitate the initiation of replication (Azenha, Lopes, & Martins, 2017). Simultaneously, cells may also trigger the RSR (Fig. 2b). This pathway is activated whenever there are high levels of RPA-coated ssDNA stretches. RPA-ssDNA mediates recruitment of ATR to the region and indirectly activate ATR through its obligatory partner, ATRIP (Hsieh & Peng, 2017). RPA-coated ssDNA stretches also facilitate loading of the 9-1-1 complex to primer-template junctions (Marechal & Zou, 2013), where it forms a complex with RHINO, TopB1, and Claspin, which contributes to the full activation of ATR (Hsieh & Peng, 2017). Activated ATR then phosphorylates different targets, including CHK1, a pivotal kinase for the triggering of the DDR and replication checkpoint activation (Zhao & Piwnica-Worms, 2001), which will be described later. CHK1 recruitment to stalled replication forks is promoted by several protein complexes, namely Tim-Tipin, Tipin-TPA32, Tipin-Claspin, and Claspin-Rad9 complexes (Hsieh & Peng, 2017). Claspin is an essential mediator of CHK1 activation, bringing ATR and CHK1 together (Chini & Chen, 2003; Kumagai & Dunphy, 2000). Specifically in the case of replication stress, activation of the ATRCHK1 pathway will help to ensure that there is a steady supply of dNTPs for DNA synthesis. Activation of the ATR-CHK1 axis leads to regulation of the activity of an enzyme that is required for the generation of dNTPs, the ribonucleotide reductase (RNR). Activated ATR will allow the degradation of cyclin F, which is involved in the ubiquitin-mediated degradation of RMM2, one of the components of RNR, thus maintaining RNR levels and increasing the production of dNTPs (D’Angiolella et al., 2012). The ATR-CHK1 pathway also contributes to RMM2 accumulation through the stabilization of E2F1, a transcription factor that promotes expression of RMM2 (Buisson, Boisvert, Benes, & Zou, 2015). Human Claspin is a DNA binding protein that associates with branched DNA structures that can arise at stalled replication forks, sites of DNA damage, and unwound DNA during replication, in a highly specific and strong manner (Kumagai & Dunphy, 2000; Sar et al., 2004; Uno & Masai, 2011). Claspin was found to be a component of “the replication fork protection complex,” along with Tim and Tipin (Fig. 2c). This complex participates

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in several replication-related functions such as maintenance and monitoring of replication fork stability, through the activation of both the ATR-CHK1 pathway and the process of translesion DNA synthesis (TLS), which is mediated by mono-ubiquitinated PCNA and allows the continuous replication of DNA through different types of DNA lesions. PCNA is an important component of replication forks. Upon DNA damage or replication stress, it is either mono- or poly-ubiquitinated. Mono-ubiquitination of PCNA relies on Rad18 and Rad6 and is thought to facilitate its interaction with translesion DNA polymerases, which allow for the continuous advance of replication forks on damaged DNA and provide an avoidant mechanism for single-strand (SSBs) and double-strand breaks (DSBs) (Lopes, Foiani, & Sogo, 2006). Claspin, together with Tim, promotes ubiquitination of PCNA, facilitating the recruitment of Rad18 to PCNA (Scorah & McGowan, 2009; Yang, Shiotani, Classon, & Zou, 2008; Yang & Zou, 2009). PCNA ubiquitination seems to be triggered by the uncoordinated action of DNA polymerases and the Mcm helicase (Chang, Lupardus, & Cimprich, 2006). Given the role of Claspin in the coordination of the action of these enzymes (Errico & Costanzo, 2012), it is possible that one of the functions of Claspin in monitoring of DNA replication and in RSR is sensing the uncoupling of the activities of these enzymes and the transduction of this information onto PCNA, promoting its ubiquitination. The ATR-CHK1 pathway also plays an important role in fork protection. In order to replication to resume after replicative stress, stalled forks have to maintain their structure and replication competency (Dungrawala et al., 2015). Stalled forks can be remodeled by proteins that have a role in their restart and repair (Neelsen & Lopes, 2015). However, these remodeling activities must be kept under tight control, as excessive remodeling can result in fork collapse and breakage and, consequently, in DSBs (Deh
e & Gaillard, 2017). ATR-CHK1 prevents excessive fork remodeling, as well as excessive generation of ssDNA, and cleavage of the stalled forks by endonucleases (Forment & O’Connor, 2018). In the budding yeast, the Claspin homolog, Mrc1, was shown to maintain fork integrity by promoting the binding of Cdc45 with replisomes (Katou et al., 2003). Additionally, Mrc1 is able to physically interact with DNA pol ε and with components of the Mcm helicase complex, a feature that suggests that it may maintain replication fork stability through coupling of helicase and polymerase activities (Katou et al., 2003; Lou et al., 2008). Claspin may be involved in a similar process (Fig. 2c) since it was shown that the Claspin-Tim-Tipin complex is able to regulate movement of the Mcm helicase complex

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(Gaillard, Garcia-Muse, & Aguilera, 2015). Uncoupling of the activities of the Mcm helicase and the DNA polymerases leads to the appearance of long stretches of ssDNA in between these enzymes, which is a trigger of the RSR. In these situations, restraining of the movement of the Mcm helicase also helps to keep the amounts of ssDNA under control. In addition, RPA coats the ssDNA ends protecting them from nucleolytic processing and ensuring the stability of stalled replication forks (Gaillard et al., 2015). When these protective mechanisms are not able to maintain ssDNA at low levels, there may be the massive collapse of replication forks throughout the genome, leading to extensive DNA breaks and cell death (replication catastrophe) (Toledo, Neelsen, & Lukas, 2017). Through its role in the activation of the ATR-CHK1 pathway, Claspin also has a protective role on genome replication through chromosomal fragile site monitoring (Fig. 2d). Chromosomal fragile sites are highly unstable and recombinogenic regions of the genome that are prone to break under stress and difficult to replicate, thereby contributing to genomic instability and cancer development (Dillon, Burrow, & Wang, 2010). The ATRCHK1 pathway monitors and guarantees the proper replication of these regions. This pathway has a crucial role in maintenance of chromosomal fragile site stability, by steadying stalled replication forks and avoiding fork collapse (Casper, Nghiem, Arlt, & Glover, 2002; Durkin, Arlt, Howlett, & Glover, 2006; Franchitto, 2013; Franchitto & Pichierri, 2014; Koundrioukoff et al., 2013). It has been shown that Claspin silencing resulted in genomic abnormalities in the form of specific fragile site expression (Focarelli et al., 2009). Finally, Claspin additionally contributes for fork recovery through maintenance of sister chromatid cohesion (Fig. 2e), as holding sister chromatids together, which occurs immediately after fork passage, contributes to fork repair (Tourrie`re & Pasero, 2007). Replication stress is a common event in cancer cells. Overexpression or activation of oncogenes associated with cell proliferation results in replicative stress, due to conflicts between replication and transcription, increased topological tensions, and also shortage of dNTPs (Macheret & Halazonetis, 2015), and increase the probability of persistent stalling of replication forks with consequent DNA damage (Debatisse, Le Tallec, Letessier, Dutrillaux, & Brison, 2012). As such, oncogene-induced replication stress constitutes an important source of genomic instability. Nevertheless, as excessive DNA damage may put cell survival at risk, cancer cells need to balance genomic instability with cell survival and proliferation. Interestingly, while mutations in components of the DDR are common, inactivation

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of RSR genes is rare, which suggests that tumor cells require a functional RSR to survive (Lecona & Fernandez-Capetillo, 2014). Therefore, it is conceivable that the proteins involved in the RSR, including Claspin, may constitute good therapeutic targets in cancer.

3. Claspin and the DNA damage response 3.1 Claspin and checkpoint activation in response to DNA damage The main target for the biologic effects of ionizing radiation (IR) and genotoxic drugs is the DNA. The most common forms of the induced DNA damage are single-base damage and SSBs. However, these lesions are usually readily repaired by the cell and are, thus, of little biologic importance when cell viability is concerned. The most lethal DNA lesions are DSBs, which are highly correlated with apoptosis induction (Raleigh & Haas-Kogan, 2013). Interestingly, replication stress also generates DSBs, particularly one-ended breaks (Hsieh & Peng, 2017). In the presence of DNA lesions, the cells activate the DDR, an evolutionary conserved signal transduction network that has evolved to prevent genomic instability and promote survival ( Jones & Petermann, 2012; Raleigh & Haas-Kogan, 2013). In the first steps of the DDR, cell cycle checkpoints are activated and promote cell cycle arrest, giving the cell time to try to repair the DNA. If DNA is successfully repaired, the cell resumes the cell cycle (checkpoint recovery); if damage is irreparable, apoptosis, or senescence is triggered. Alternatively, cells may adapt (checkpoint adaptation). Depending on the cell cycle phase, distinct checkpoints are activated (Fig. 3). In G1/S, checkpoint responses involve the activation of the ATM-CHK2-p53 pathway, providing the cell time to repair any DNA damage before replication. As for the S phase, the intra-S phase checkpoint is mainly mediated by the ATR-Claspin-CHK1 pathway and also involves activation of DNA-PK and Wee1. These proteins are able to delay origin firing and, this way, give the cell time to deal with unrepaired DNA damage, or with DNA damage that have resulted from replication stress, and prevent under-replication of DNA. Finally, the G2/M checkpoint is primarily dependent on the activation of CHK1, Claspin, MYT1, and Wee1, which work to delay mitotic entry, and, thus, give the cell one last chance to repair DNA damage before cell division. Failure to repair DNA damage, or to complete DNA replication, and mitotic entry may later result in mitotic catastrophe and cell death (O’Connor, 2015).

Fig. 3 See figure legend on next page.

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In general, DDR responses are composed by a common set of finely regulated steps, which include sensing of DNA damage, transduction of the signal to and recruitment of repair effectors to the lesion site, and the final repair of DNA damage. The ATM/CHK2 and the ATR/Claspin/CHK1 axis are the key elements of DDR. DSBs usually activate the ATM/CHK2 pathway, after sensing of the damage by the MRE11/RAD50/Nbs1 (MRN) complex. ATM then activates CHK2 to promote cell cycle arrest at G1 through phosphorylation and stabilization of p53, which induces expression of cell cycle inhibitors, such as p21. In turn, SSBs and all kinds of damage that threaten DNA replication (e.g., base adducts, interstrand crosslinks, DSBs), lead to activation of ATR. As described for replication stress, ssDNA stretches that arise from these lesions are tagged by RPA, which activates ATR-ATRIP. These structures can also be generated in DSBs through resection of the break ends, and upon activation of nucleotide excision repair (NER) (Li, Pearlman, & Hsieh, 2016). The ATR-ATRIP complex is responsible for the phosphorylation and activation of CHK1. As already mentioned, Claspin has a crucial scaffold role in CHK1 activation, bringing together ATR and CHK1, this way allowing for successful CHK1 phosphorylation by ATR and an adequate checkpoint response (Chini & Chen, 2003; Clarke & Clarke, 2005; Kumagai & Dunphy, 2000; Zhao & Piwnica-Worms, 2001). Activated CHK1 then triggers the intra-S and G2/M checkpoints. In the S phase, CHK1 phosphorylates Cdc25A, promoting its degradation by the proteasome, which, in turn, leads to a decrease in the activity of CDK2. In G2/M, CHK1 phosphorylates Cdc25B, also promoting its degradation, and this way blocks activation of CDK/Cyclin Fig. 3 Claspin and the DNA damage response. In the presence of DNA damage, one of the main roles of Claspin is to promote CHK1-mediated activation of intra-S phase and G2/M checkpoints. Both these checkpoints are triggered by the presence of RPA-coated ssDNA stretches, which activate the ATR-ATRIP complex that then activates CHK1. In the intra-S phase checkpoint, further activation of DNA-PK and Wee1 will allow delay of origin firing and prevent under-replication of DNA. With regard to G2/M checkpoint, activation of CHK1, Claspin, MYT1, and Wee1 is responsible for the induction of cell cycle arrest, this way providing the cell time to repair damaged DNA. Upon DNA damage, Claspin also seems to bridge checkpoint responses with several DNA repair pathways, including HR, NHEJ, NER, MMR, and the FANC/BRCA pathway. In addition, Claspin seems to act as a switch that determines cell fate after DNA damage: its activation ensures checkpoint activation, cell cycle arrest, and DNA repair, whereas its degradation promotes checkpoint recovery (when DNA is successfully repaired), or apoptosis or senescence (when the cell fails to repair the DNA). Degradation of Claspin can also promote checkpoint adaptation.

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B and arrests the cell in G2 (Dai & Grant, 2010; Sørensen et al., 2003). CHK1 can also activate Wee1, whose function is the inhibition of CDK1, thus arresting the cells at the G2/M transition and preventing the cells with DNA damage from entering mitosis (Lee, Kumagai, & Dunphy, 2001).

3.2 Claspin and DNA repair In addition to its pivotal role in triggering cell cycle checkpoints, Claspin also seems to play a role in the activation of DNA damage repair (Fig. 3). It has been shown that Claspin can be activated by DNA damage, particularly DSBs, even in the absence of replication stress. It seems that Claspin is able to bind to chromatin in a replication-independent fashion when damage opens access to the DNA (Yoo, Jeong, & Dunphy, 2006). Available data suggest that Claspin may bridge checkpoint activation and different DNA repair pathways, namely homologous recombination (HR), nonhomologous end joining (NHEJ), mismatch repair (MMR), NER, and the FANC/BRCA pathway. Claspin seems to mediate physical interactions between components of the DNA damage checkpoint and repair mechanisms promoting crosstalk between these pathways (Azenha et al., 2017). Different DDR pathways are triggered in response to distinct DNA lesions (Table 1). Nevertheless, these different pathways may compensate one another when the dedicated pathway is inactive, and may either work independently or co-ordinately to repair different types of damage (Hosoya & Miyagawa, 2014; O’Connor, 2015). The response to DNA lesions will also depend on the cell cycle status of the cell. For instance, in the presence of DSBs cells may trigger either HR or NEHJ. As, in HR, the presence of an undamaged sister chromatid is required, which will Table 1 Types of DNA damage and pathways involved in their repair. Type of DNA damage Repair pathway triggered

Single-strand breaks (SSBs)

Base excision repair (BER)

Double-strand breaks (DSBs)

Homologous recombination (HR) Non-homologous end joining (NHEJ)

Bulky adducts

Nucleotide excision repair (NER) Translesion synthesis (TLS)

Nucleotide mutations, substitutions, deletions, insertions

Mismatch repair (MMR)

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serve as the template for repair, this pathway will only be elicited in cells that have replicated the DNA. In turn, G1 cells will rely on NHEJ to repair DSBs. As already mentioned, the most lethal DNA lesions are DSBs, which may be repaired by HR or NHEJ. HR is the most reliable DSB repair pathway, as it assures accurate repair of the lesion. In contrast, NHEJ, although still effective, is less accurate and may lead to genomic instability (O’Connor, 2015). In HR, DSBs are initially sensed by the MRN complex, which promotes tethering and stabilization of the broken ends and activates ATM (Carrassa & Damia, 2017; Lavin, Kozlov, Gatei, & Kijas, 2015). Activated ATM then phosphorylates H2AX, promoting binding of MDC1, which serves to amplify the signal through recruitment of additional MRN and ATM molecules (Durocher, 2009). MRE11, with the help of CtIP and Exo1, then resects the DNA, resulting in a long 30 ssDNA strand that will acquire the ability to invade the homologous sequence of the other strand to form a D-loop intermediate (strand invasion). D-loop formation requires binding of Rad51 to ssDNA and the formation of a presynaptic filament, an event that requires previous binding of the ssDNA by RPA and recruitment of several proteins (Rad52, BRCA1/2, and Rad51 paralogs) that have a role in the replacement of RPA by Rad51, and the stabilization of the filament (Williamson, Williamson, & Lees-Miller, 2009). The short 30 -DNA ends are extended by Exo1 or DNA2, an event that requires the interaction with BLM, MRN, and RPA, and the second DNA end is captured by annealing to the extended D-loop, leading to the formation of Holliday junctions. These crossed-strand structures are later resolved by MMS4 and MUS81 by non-crossing over or crossing over. In the first case, the Holliday junctions disengage and DNA strand pairing is followed by gap filling. In the second situation, the crossing over is followed by gap filling (Hsieh & Peng, 2017; Williamson et al., 2009). BRCA1 is a key protein in HR, promoting recruitment of BRCA2 to the DSB, which then facilitates loading of Rad51 onto RPA-coated ssDNA overhangs. BRCA1 also associates with CtIP, an interaction that is required for the formation of ssDNA at DSB ends, and the control of the initiation and speed of DNA end resection (Hsieh & Peng, 2017; Williamson et al., 2009). In addition, BRCA1 has an important regulating role in HR, promoting the ubiquitination of CtIP (Williamson et al., 2009). Of note, And1, which promotes Claspin function in checkpoint activation, is required for HR, having a role in the regulation of CtIP activity in DNA end resection (Li, Li, Wu, Han, & Zhu, 2017). In turn, end resection

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and resulting RPA-coated ssDNA stretches will trigger checkpoint activation through the ATR-Claspin-CHK1 axis. Both Claspin and BRCA1 were shown to mediate CHK1 activation through phosphorylation in response to IR-induced DSBs (Lin, Li, Stewart, & Elledge, 2004; Yoo et al., 2006). Claspin directly interacts with BRCA1 and acts upstream BRCA1 (Lin et al., 2004; Yoo et al., 2006), and seems to have a role in recruitment and phosphorylation of BRCA1 (Lin et al., 2004). Claspin may help to recruit BRCA1 to damaged sites on chromatin, where it helps in the activation of both the intra-S phase and the G2-phase checkpoints after exposure to IR through regulation of CHK1 phosphorylation and activation (Lin et al., 2004; Yoo et al., 2006). Activated CHK1 then mediates checkpoint enforcement and DNA repair by HR through regulation of the interaction between BRCA2 and Rad51 (Bahassi et al., 2008; Liu et al., 2000; Sørensen et al., 2005; Yarden, Pardo-Reoyo, Sgagias, Cowan, & Brody, 2002). This role of BRCA1 in checkpoint activation seems to rely on its E3 ligase activity, and its ubiquitination and nuclear uptake of Claspin, as well as maintenance of chromatin-bound Claspin levels (Sato et al., 2012; Yoo et al., 2006). Indeed, different types of ubiquitination can regulate protein function by degradation, by changes in their interaction partners, or by alterations in intracellular localization and changes in activities (Liu, Li, & Lu, 2016). The role of Claspin in NHEJ is not as straightforward. However, it is well established that there is a functional and perhaps physical interaction between Claspin and a key protein of NHEJ, the DNA-PKcs (Lin, Shih, Shang, Matsunaga, & Chen, 2014). NHEJ is composed of five major steps: (1) recognition of damaged DNA ends by Ku proteins; (2) recruitment of DNA-PKcs and Artemis by Ku70/80 complex; (3) end processing; (4) fill in of the gaps or end bridging; and (5) ligation. DNA-PKcs forms a complex with Artemis and, through phosphorylation, activates its endonuclease activity, so that Artemis is able to deal with the 50 and 30 overhangs and hairpins. After DNA end processing, the resulting ends are filled in through new DNA synthesis by DNA pol μ and λ, which were found to associate with the Ku/DNA/XRCC4/DNA ligase IV complex. The final ligation step is carried out by the PNK/XRCC4/DNA ligase IV/XLF complex (Williamson et al., 2009). DNA-PKcs seems to be important in tethering the DNA ends together during NHEJ and protecting them from inappropriate end processing. Afterward, DNA-PKcs undergoes autophosphorylation, which leads to its dissociation from DNA ends, facilitating the access of downstream effectors to the ends of the DSB. Therefore, its main functions in

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NHEJ seem to be the protection of DNA ends and regulation of the access of other proteins to DSBs, this way regulating progression of this repair pathway (Williamson et al., 2009). In addition, DNA-PKcs is also involved in RSR, being phosphorylated by ATR (Yajima, Lee, & Chen, 2006). It has been shown that DNA-PKcs can interact directly with CHK1 and that this association promotes DNA-PKcs function in NHEJ (Goudelock, Jiang, Pereira, Russell, & Sanchez, 2003). In addition, DN-PKcs stimulates Claspin expression and stabilizes CHK1-Claspin complexes during checkpoint responses (Lin et al., 2014). Therefore, it is possible that in the presence of DSBs, particularly breaks that induce replication fork collapse in the G1-phase of the cell cycle, activation of ATR leads to phosphorylation and activation of DNA-PKcs, which then stimulates Claspin expression, this way contributing to the phosphorylation and activation of CHK1. Another important function of DNA-PKcs seems to be the stabilization of ClaspinCHK1 complexes, which may favor CHK1 activation, which, in turn, will promote the function of DNA-PKcs in NHEJ. Claspin may also bridge cell cycle checkpoint activation with NER (global genome NER), a pathway mainly involved in the repair of bulky DNA lesions, caused by modified nucleotides that distort the structure of the double helix of DNA, such as pyrimidine dimers (Hosoya & Miyagawa, 2014; O’Connor, 2015). NER encompasses several steps. Detection of DNA lesions seems to be performed by XPC, which forms a trimeric complex with HR23B and CEN2, XPA, and the DDB1/ DDB2 complex. DDB2 is part of a functional CUL4A-based ubiquitin ligase through its interaction with DBB1. The DDB1/DDB2 complex is responsible for the ubiquitination of several substrates including DDB2 itself, XPC and histones H2A, H3, and H4. Its activity facilitates NER, as the ubiquitination of XPC enhances its affinity for DNA, and that of histones facilitates access of repair proteins to damaged DNA. Ubiquitination of DDB2 quickly targets this protein to proteasomal degradation, and, consequently promotes displacement of the DDB1/DDB2 complex from the lesion, a feature that also increases the accessibility of other proteins to the site of damage (Vrouwe & Mullenders, 2009). Afterward, the DNA helicase TFIIH, RPA and two endonucleases, XPF-ERCC1 and XPG, are recruited to the site of damage, by XPC/HR23B, where they are responsible for the excision of the DNA stretch harboring the lesion, allowing for re-synthesis of chromosomal DNA for the re-establishment of the original state of chromatin (Vrouwe & Mullenders, 2009). TFIIH mediates initial unwinding of DNA, which is followed by binding of

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RPA, XPA, and XPG, allowing for full opening around the lesion. XPG and XPF-ERCC1 are involved in dual incision 30 and 50 of the lesion, respectively. In addition, XPG is required to stabilize the open DNA bubble containing the lesion, promoting binding, and 50 incision by XPF-ERCC1. Dual incision and removal of the lesion are followed by gap filling and ligation (repair synthesis). Repair synthesis requires RPA, RF-C, PCNA, DNA pol ε (in S phase cells) and δ (in replicating and quiescent cells), and Ligase I. Finally, the 50 end of the newly synthesized DNA is ligated to the original sequence by Ligase I (in the case of nick sealing) or ERCC1-LigIIIα (for ligation of NER-induced breaks) (Vrouwe & Mullenders, 2009). Interestingly, NER proteins can bridge this pathway to cell cycle checkpoint activation and the DDR. As already mentioned, RPA has a key role in the activation of ATR during NER through binding to the ssDNA stretches formed during excision of the DNA lesion. These RPA containing ssDNA stretches resemble structures formed when replication forks stall and therefore trigger ATR signaling via a common mechanism (Vrouwe & Mullenders, 2009). One of the outcomes of ATR activation is cell cycle arrest through stabilization of p53, which promotes expression of cell cycle inhibitors, such as p21. Of note, p53 can also induce expression of both DDB2 and XPC. Thus, through activation of p53, ATR may enhance NER ability after DNA damage (Vrouwe & Mullenders, 2009). Claspin has been shown to interact with key proteins of NER (Fig. 3), namely XPC, DDB1, and DDB2 (Prætorius-Ibba et al., 2007). Claspin is required for the CUL4A-mediated DDB2 turnover, as well as for recruitment of DDB2 to the damage site (Prætorius-Ibba et al., 2007). Thus, Claspin seems to have a role in facilitating the access of NER machinery to damaged DNA. It seems that upon UV exposure, Claspin associates with DDB2 and promotes its recruitment to the DNA lesion, this way promoting XPC binding and the ubiquitination of NER proteins, including DDB2 itself. Later degradation of DDB2 then facilitates access of downstream NER effectors to the site of damage. DNA interstrand crosslinks are highly toxic lesions because of their ability to block DNA synthesis (Guervilly, Mac
e-Aim
e, & Rosselli, 2008). Although the mechanisms of removal of interstrand crosslinks are still not fully understood, they seem to involve proteins of different DNA repair pathways, such as NER, TLS, HR, and MMR. Triggering of either of these pathways is preceded by activation of the FANC-BRCA pathway (Guervilly et al., 2008; Lopez-Martinez, Liang, & Cohn, 2016). In response to DNA damage, the ATR/CHK1 pathway is triggered, leading to the

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phosphorylation of several components of the FA core complex. The FA core complex is composed of eight FANC proteins (FANCA, B, C, E, F, G, L, and M) and mono-ubiquitinates the FANCD2/FANCI complex, allowing its recruitment to the sites of damage. Although the function of this complex is not fully elucidated, it is believed that it is responsible for the orchestration of the recruitment of downstream effectors to the lesion (e.g., BRCA2, BRCA1, BRIP1, PALB2, Rad51C, SLX4, XPF, and Rad51) (Guervilly et al., 2008; Lopez-Martinez et al., 2016). Interestingly, FA proteins also seem to have a role in replication fork protection and recovery after stalling induced by different genomic stressors. For instance, FANCD2 is able to recruit the nuclease FAN1, BLM, FANCJ, and BRCA2 to promote fork recovery in a core complex-independent fashion (LopezMartinez et al., 2016). The FANCD2/FANCI complex may additionally have a role in the resolution of lesions arising from common fragile sites, ensuring a correct segregation of chromosomes during mitosis (LopezMartinez et al., 2016). It has been shown that Rad9, Rad17, and ATR are required for optimal activation of FANCD2, and that the CHK1/ Claspin complex is necessary for FANCD2 mono-ubiquitination and its assembly in subnuclear foci in response to DNA damage. These functions of Claspin and CHK1 are independent of ATR. Interestingly, these two proteins are not required for the DNA damage-independent roles of FANCD2 (Guervilly et al., 2008). It has been proposed that the CHK1/ Claspin complex may participate in the phosphorylation of other FANC proteins, namely components of the FA core complex, such as FANCE, and, this way, either promote the ubiquitin ligase activity of the FA core complex over FANCD2, or stabilize the interaction between the FA core complex and FANCD2 (Guervilly et al., 2008). Of note, Claspin inhibition was shown to lead to fragile site expression (Focarelli et al., 2009). Thus, together with the FANCD2/FANCI complex, Claspin may have a role in the protection of cells against recombinatory events that may originate from fragile sites. The MMR pathway deals with several replication errors, including mismatch base-pairing, nucleotide insertions, and nucleotide deletions, as well as with a subset of DNA lesions caused by SN1 DNA alkylators, 6-thioguanine, fluoropyrimidines, cisplatin, UV light, and carcinogens that form DNA adducts (Li et al., 2016). In brief, MMR can be divided in four steps: (1) recognition of the mismatch by MSHs (mainly MutSα (MSH2MSH6 complexes) in humans); (2) recruitment of MLHs (particularly MutLα (MLH1-PMS2 complexes) in humans) by ATP-bound MSHs,

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which bridge mismatch recognition to DNA strand scission; (3) excision of the DNA strand with the mismatch, which involves the 30 -endonuclease activity of PMS2 and 50 -excision by Exo1; and (4) gap filling by the replicative polymerase using the remaining DNA strand as template (MartinLo´pez & Fishel, 2013; Reyes, Schmidt, Kolodner, & Hombauer, 2015). Strand discrimination is thought to be based on the presence of nicks or gaps in the daughter DNA strand (e.g., 5´or 30 ends of Okazaki fragments or ribonucleotides misincorporated by DNA polymerases during replication). Alternatively, it was proposed that PCNA or CMG could also serve as a signal for strand discrimination, as they are both loaded onto to DNA with a specific orientation and polarity (Reyes et al., 2015). To prevent mutations due to DNA replication errors that escape proofreading by DNA pol ε and δ, MMR must repair the newly synthesized DNA strand before the next round of DNA replication. Thus, MMR must be coupled to DNA replication, which was proposed to involve the interaction between MutLα and PCNA (Reyes et al., 2015). In addition, it has been shown that epigenetic modifications, such as the histone H3K36me3 modification, can promote recruitment of MutSα to chromatin, which could facilitate interaction with PCNA at the replication fork (Li et al., 2013). MMR is also coupled to checkpoint activation and apoptosis induction, particularly upon exposure of cells to SN1 DNA methylators, which lead to generation of O6-methylguanine (MeG), causing MeG-C and MeG-T mispairs (Liu et al., 2010). G2-checkpoint activation through the ATR-CHK1 axis, in these circumstances, appears to require the action of MutSα and MutLα upstream of ATR. It has been demonstrated that both these complexes interact with several proteins of the ATR-CHK1 pathway, including Claspin, and that recruitment of the MMR complexes precedes that of proteins involved in the ATR-CHK1 axis (Liu et al., 2010). This is conceivable with a scenario in which MutSα binds to DNA, forming a clamp that slides along the DNA double helix tracking for mismatches, and, when a mismatch is found, promotes recruitment of MutLα. Interaction between the two complexes may somehow promote recruitment of proteins of the ATR-CHK1 axis and activation of this pathway, to which Claspin belongs. Interaction of Claspin with the MMR complexes is thought to either be indirect (probably as part of CHK1-Claspin complexes) or to depend on post-translational modifications (Liu et al., 2010). Activation of this checkpoint seems to be important for the induction of cell cycle arrest for MMR to proceed (Adamson et al., 2005).

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Taking all these data into account, Claspin seems to have a role in bridging checkpoint responses with different pathways of DNA repair, as required. In general, Claspin seems to function in the initial steps of these pathways, namely in the recruitment of the repair machinery and in the processing of DNA at the site of damage before repair. The ability of Claspin to differentially activate one or another repair pathway, based on the type of injury the DNA has suffered, may rely on site-specific phosphorylations, which is thought to be a way of increasing the functional diversity of proteins (Yoo et al., 2006). It has been shown that specific phosphorylation of Claspin in distinct residues is crucial in the discrimination between different DNA damage structures (Yoo et al., 2006). Finally, Claspin may also have a pivotal role on cell fate decisions. It is not only important for checkpoint activation and cell cycle arrest, allowing for DNA repair to proceed, but also for subsequent decisions of life and death. If the cell is able to repair the DNA damage, checkpoint responses must be terminated in order to the cell to resume the cell cycle and divide. Checkpoint recovery requires Claspin degradation mediated by βTrCP-SCF ubiquitin ligase complex and Plk1 (Mailand, Bekker-Jensen, Bartek, & Lukas, 2006; Mamely et al., 2006; Peschiaroli et al., 2006). In turn, when cell cycle arrest is too prolonged or the DNA damage irreparable, cell senescence, or apoptosis, respectively, must be induced in order to prevent genomic instability. These processes also require Claspin degradation, which is dependent on the action of caspases 3 and 7 (particularly caspase 7) and the proteasome (Clarke, Bennett, & Clarke, 2005; Semple, Smits, Fernaud, Mamely, & Freire, 2007). Nevertheless, some cells may still proceed to mitosis even in the presence of DNA damage, a phenomenon known as checkpoint adaptation, which may constitute a source of genomic instability and promote carcinogenesis. This process also requires Plk1-mediated degradation of Claspin, as a means to inactivate Chk1 (Syljua˚sen, Jensen, Bartek, & Lukas, 2006; Yoo, Kumagai, Shevchenko, Shevchenko, & Dunphy, 2004). Of note, senescence may also promote carcinogenesis and senescent cells may constitute a source of tumor recurrences (Magdalou, Lopez, Pasero, & Lambert, 2014).

4. Claspin as a target for chemo- and radio-sensitization Cancer cell features that are not shared by healthy cells constitute ideal targets for therapy because they can be exploited to specifically kill cancer

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cells. During malignant transformation, oncogene activation (e.g., amplification of cyclin D1 or E) promotes cell proliferation and cell survival. In addition, cancer cells tend to loose proliferation breaks, namely cell cycle checkpoints (e.g., deficiencies in pRB), and to be exposed to high levels of reactive oxygen species, which cause DNA damage. Therefore, replication stress is a hallmark of cancer. Conventional CT and RT aim to enhance replicative stress, through the induction of DNA damage, and consequent cell death. Loss of functional checkpoints sensitize tumor cells to these therapies. It is believed that further increasing replicative stress to levels that the cell is not able to cope with may constitute an adequate therapeutic approach. Another important aspect of cancer cells is the loss of one or more DDR pathways, leading to a greater dependency on the other pathways (Dobbelstein & Sørensen, 2015; O’Connor, 2015). This feature may also create therapeutic opportunities, namely through the use of DDR inhibitors. The goal, now, is to identify RSR and DDR targets that may increase the therapeutic index over standard CT and RT and devise ways to effectively select cancers that will be susceptible to RSR- and DDRtargeted drugs.

4.1 Inhibitors targeting RSR With regard to RSR, the main focus of drug development has been the ATR-CHK1 and the Wee1-CDK1/2 pathways. CHK1, ATR, and Wee1 inhibitors abrogate the G2 checkpoint and promote DNA damage in the S phase, a feature that seems to contribute to the cytotoxicity of these inhibitors (Syljua˚sen et al., 2015). Inhibitors of ATR, CHK1, and Wee1 are currently being investigated in clinical trials (reviewed in Carrassa & Damia, 2017). Cancer cells commonly present a deregulated G1-checkpoint, due to mutations of the p53/pRb pathway, and, thus, rely more on the G2/checkpoint for survival after RSR and DNA damage. These increased reliance on this checkpoint turn cancer cells more sensitive to inhibition of the ATR/ CHK1 axis. This inhibition appears to increase the cytotoxic activity of the major classes of DNA-damaging agents used in cancer treatment (Carrassa & Damia, 2017). Two ATR inhibitors (AZD6738, VX-970) are currently under trial. VX-970 (Vortex Pharmaceuticals) was shown to markedly enhance cell death induced by DNA damage in some cancer types, while normal cells were spared (Charrier et al., 2011). The effect was even more pronounced in cells deficient in p53 or ATM (Reaper et al., 2011). VX-970 further sensitized non-small cell lung cancer cells to the effects of cisplatin,

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gemcitabine, etoposide, and oxiplatin (Hall et al., 2014). As for AZD6738 (Astra Zeneca), it was shown to be efficient as a single agent in p53- and ATM-deficient tumors, and to sensitize tumor cells to a variety of cytotoxic drugs, PARP inhibitors, and IR in vitro (Kim et al., 2017; Mohni et al., 2015). ATR inhibition was shown to be synthetically lethal with p53, XRCC1, ERCC1, POLD1, ARID1A, CHK1, and ATM. Cells with defective BER and HR pathways, or with high expression of DNA-PKcs, were also shown to be more sensitive to ATR inhibitors (Carrassa & Damia, 2017). Several CHK1 inhibitors are also currently under clinical trials: e.g., GDC-0575, LY2606368 (prexasertib), LY880070, and SRA737. These drugs showed anti-cancer synergy with drugs that generate replication stress and replication-dependent DNA damage, such as antimetabolites (Daud et al., 2015; Infante et al., 2017; Walton et al., 2016). Synthetic lethality has been observed with c-Myc and cyclin D1 overexpression, as well as deficiencies in HR pathway. In addition, CHK1 inhibitors may be useful in combination treatments using drugs that target pro-survival and proliferative pathways, such as ibrutinib (BTK inhibitor) and p38/MEK inhibitors, and the HR pathway (e.g., PARP inhibitors). Combination of CHK1 inhibitors with inhibitors of other players of RSR (e.g., ATR, Wee1) also seems to have a synthetic lethal effect (Carrassa & Damia, 2017). For instance, combination of CHK1 and ATR inhibitors caused replication fork arrest, accumulation of ssDNA, replication collapse, and synergistic cell death (Sanjiv et al., 2016). Finally, the most potent and selective Wee1 inhibitor available so far is AZD1775 (Astra Zeneca). It has been shown to sensitize tumor cells to the cytotoxic effects of several DNA-damaging drugs, including antimetabolites, topoisomerase inhibitors, and DNA crosslinking agents (Do et al., 2015; Do, Doroshow, & Kummar, 2013), and to be more effective in p53-deficient cells (Hirai et al., 2009; Leijen et al., 2016; Rajeshkumar et al., 2011) and cells with defects in DNA repair pathways (Do et al., 2013).

4.2 Inhibitors targeting DNA repair pathways Cancer cells are characterized by increased genomic instability due to deficiencies in DNA repair pathways and DNA recombination genes. It has been shown that therapeutic efficacy is influenced by the ability of cancer cells to repair treatment-induced DNA damage. Therefore, it is believed that drugs that target components of DNA repair pathways may sensitize cancer cells to conventional therapy. DNA repair deficiencies of cancer cells

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have been exploited therapeutically using synthetic lethal approaches, which preferentially target cancer cells, while sparing healthy cells, thus reducing side effects. For instance, PARP inhibitors (PARPi) are being used to exploit BRCA deficiencies in cancer cells. PARPs are proteins involved in DNA damage recognition and binding to SSBs, and triggering of different DNA repair pathways, including BER, HR, NER, NHEJ, and MMR, through recruitment of repair proteins (Gavande et al., 2016). PARP1, in particular, is involved in the repair of SSBs. BRCAs, in turn, are important for the repair of DSBs, as already described. PARP inhibition is synthetically lethal to BRCA-deficient cancer cells due to the accumulation of SBBs, which end up being converted in DSBs during DNA replication. In BRCA-deficient cells, these lesions cannot be repaired efficiently and eventually lead to cell death. The first PARPi to be approved for clinical use was Olaparib (AZD-2281, Astra Zeneca), which is used in ovarian cancer treatment in women carrying mutations in BRCAs and that have had at least three lines of prior CT. Olaparib is currently under study in clinical trials as monotherapy or part of combination therapies for treatment of different cancers, namely breast, pancreatic, prostate, and colorectal cancer. Other compounds under investigation are Talazoparib (Pfizer) and Rucaparib (Clovis Oncology). Talazoparib seems to be the most potent of these three compounds and to selectively target cells with mutations in BRCA1/2 or PTEN (Cardnell et al., 2013; Murai et al., 2014). NER inhibitors are also being investigated. Many agents of conventional chemotherapy, such as platinum salts, impart their clinical efficacy via induction of bulky DNA damage, which is repaired by NER. Therefore, NER has an important impact in the efficacy of these therapies and may constitute a good target for therapy. Presently, the main focus has been on DNA verification proteins, such as RPA and XPA, but also the ERCC1-XPF complex (Gavande et al., 2016). As RT remains the mainstay in treatment of several cancers and its effects are mainly mediated through generation of DSBs, another focus of research is HR and NHEJ DNA repair pathways. With regard to NHEJ, efforts are being made to develop inhibitors that target different steps of the pathway: (1) DNA-PK (e.g., Wortmannin, LY294002, NU7026, and NU7441), and Ku70/80 inhibitors, which target termini recognition and end bridging; (2) artemis, a key player in DNA end processing; and (3) ligases I, III, and IV, which are involved in DNA ligation. Although NHEJ is the major pathway for repair of IR-induced DSBs, as it may operate throughout the cell cycle,

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the success of inhibitors targeting this pathway has still been limited (Gavande et al., 2016). As for HR, the focus has been on Rad51 due to its key role in the repair and tolerance to CT- and IR-induced DSBs, the high expression of this protein in many tumors, and the reliance of cancer cells on higher Rad51 activity to deal with replication stress. Rad51 inhibitors are still in early phases of development. The approaches under test use two different strategies: (1) exploitation of Rad51 overexpression in cancer by further increasing Rad51 expression to toxic levels and promoting recombination events (e.g., RS-1) and (2) inhibition of Rad51 DNA strand exchange activity through disruption of its ability to bind ssDNA (e.g., BO2, halenaquinone, DIDS), or blockade of protein-protein interaction sites (e.g., IBR2, IBR20). In general, all these compounds were shown to be effective in cancer cell lines, and RS-1 also in a xenograft animal model (Alagpulinsa, Ayyadevara, & Shmookler Reis, 2014; Budke et al., 2013, 2012; Ishida et al., 2009; Mason et al., 2015, 2014; Takaku et al., 2011; Zhu et al., 2015, 2013). Finally, BER also constitutes an attractive target in cancer therapy because this pathway seems to be deregulated in a variety of cancers (Wallace, 2014). The number of potential targets is small because there are few proteins involved in this pathway and because of the convergence of the pathway after the generation of the apurinic/apyrimidinic site (AP-site). The first step of BER is the recognition of the modified base by a DNA glycosylase. Thereafter, the glycosidic bond is hydrolyzed, resulting in an abasic site that is recognized by APE1. The phosphodiester bond 50 of the abasic site is nicked generating 30 OH and 50 deoxyribose-phosphate termini. From this moment on, the pathway can diverge and repair can be performed by one of two mechanisms: long-patch or short-patch repair (Gavande et al., 2016). Efforts to target BER have mainly focused APE1 and DNA pol β, a polymerase that can act in both BER pathways. With regard to APE1, a large number of inhibitors are being studied (reviewed in Al-safi, Odde, Shabaik, & Neamati, 2012), which may affect the redox activity of APE1 (but not its DNA repair activity; e.g., E3330 and analogs) or be specific for its DNA repair activity (e.g., CRT0044876, NSC332395, gossypol) (Kelley, Georgiadis, & Fishel, 2012; Kitada et al., 2003; Madhusudan et al., 2005; Rai et al., 2012; Ren et al., 2015). A number of putative DNA pol β inhibitors have also been identified (e.g., NSC666715, NSC124854), which sensitize cancer cells to the effects of temozolomide ( Jaiswal et al., 2011, 2009, 2015). However, studies are still in a very early stage.

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4.3 A rationale for targeting Claspin CT- and RT-induced cell death rely on the attempt of cells to divide in the presence of DNA damage, which leads to mitotic catastrophe or, sometimes, replicative senescence. Cancer cells commonly become resistant to apoptosis induction and, therefore, treatment-induced apoptosis is not believed to be a major response of these cells to CT and RT. Further inhibition of cell cycle checkpoints will also impair cell cycle arrest, this way preventing effective repair of DNA damage induced by genotoxic therapy. Being a key component of the ATR-CHK1 axis, Claspin may constitute an adequate therapeutic target in this context. Not only because of its functions in CHK1 activation but also due to Claspin’s CHK1-independent functions, both in RSR and DDR, including the activation of relevant DNA repair pathways. Suppressing Claspin function may sensitize cancer cells to death in several ways. Claspin inactivation will prevent checkpoint activation (Fig. 4a) through the ATR-CHK1 axis in response to replication stress and DNA damage. As a consequence, cells will not be able to arrest the cell cycle in G2/M and repair the damage. This is even more relevant in p53-deficient cells, which are unable to activate the G1/S checkpoint. Given that p53 also contributes to the intra-S phase and G2/M checkpoints, by maintenance of cell cycle arrest through the action of p21 and repression of cyclin B, cells that loose p53 activity will majorly rely on the ATR-CHK1 pathway to induce intra-S phase and G2/M arrest upon DNA damage, and will be more reliant on the corresponding checkpoints (both mediated by ATR-ClaspinCHK1) for cell survival and maintenance of genome integrity. Therefore, abrogation of this checkpoint may selectively sensitize p53-deficient tumor cells to genotoxic agents, while sparing surrounding healthy cells (Syljua˚sen et al., 2015). The use of Claspin inhibitors will also abrogate its role in monitoring DNA replication, and will thus prevent smooth fork progression, and may promote uncoupling of polymerization and unwinding of the leading strand at replication forks, which may further increase the replication stress levels of tumor cells. As already described, Claspin has a relevant role in the regulation of replication timing (Fig. 4b), namely through recruitment of Cdc7 to pre-RCs and promotion of Mcm protein phosphorylation (Yang et al., 2016). Therefore, inhibition of Claspin, similarly to that of Cdc7, may halt DNA replication, extending the duration of the S phase, which eventually leads to cell death, particularly in p53-deficient cells (Montagnoli, Moll, & Colotta, 2010, 2004). In addition, this interference

Fig. 4 A model for the effects of Claspin inhibitors. Inhibition of Claspin may sensitize cancer cells to cell death in different ways: (a) inability to activate the intra-S phase and G2/M checkpoints as CHK1 phosphorylation by ATR is prevented; (b) arrest in DNA replication due to impaired recruitment of Cdc7 and ineffective phosphorylation and activation of Mcm proteins; (c) impairment of TLS, both through inhibition of Ccd7 recruitment, which will prevent recruitment of Rad18 (and indirectly that of DNA pol μ) to stalled replication forks, and blockade of PCNA mono-ubiquitination; (d) promotion of fragile site expression, which may lead to accumulation of DNA damage and consequent mitotic catastrophe; and (e) compromised repair of DNA damage, this way promoting genomic instability and shifting cell fate toward cell death.

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with Cdc7 recruitment may also have a negative effect on TLS (Fig. 4c), as Cdc7 was shown to be required to recruitment of Rad18 to stalled replication forks, and, thus, indirectly, to recruitment of translesion DNA pol μ (Yamada et al., 2013). Of note, inhibition of Claspin may also compromise TLS through an impairment of PCNA ubiquitination (Fig. 4c). These actions of Claspin will impede continuity of DNA replication and fork progression through DNA lesions. Claspin inactivation can also promote common fragile site instability (Fig. 4d). When these protective mechanisms, mediated by Claspin, are not active, massive collapse of replication forks may occur, leading to accumulation of DNA damage and mitotic catastrophe. Finally, Claspin seems to lie at a hub that links checkpoint activation and triggering of different DNA repair pathways (Fig. 4e). As such, Claspin inhibition may also compromise repair of DNA damage, contributing to excessive genomic instability in cancer cells, unbalancing the fragile equilibrium between genomic instability and cell proliferation and survival presented by cancer cells, and shifting this balance in favor of cell death. Besides using Claspin inhibitors in the context of synthetic lethality, exploring cancer cells’ deficiencies (e.g., p53 or other G1-checkpoint mediators), one can also envisage the use of Claspin inhibitors in combined targeted therapies. This way, even cells with a functional p53 axis/G1 checkpoint could be targeted, through “pharmaceutically-induced synthetic lethality”. Combination of Claspin inhibitors with inhibitors of p53, ATM, or CHK2 would simultaneously abrogate G1, intra-S, and G2/S checkpoints, thus miming cell intrinsic deficiencies. Another interesting approach is to simultaneously shut off checkpoint activation and relevant pathways of DNA repair. This kind of combinatory approaches is already being tested for other targets. For instance, it has been observed that: (1) blockade of an ERK-mediated compensatory signaling pathway after CHK1 inhibition enhanced cell killing (Dent et al., 2011); (2) combined PARP and CHK1 or ATR inhibition promoted cell death either isolated or in combination with RT or CT (Peasland et al., 2011; Vance et al., 2011); (3) Wee1 inhibitors potentiated the effects of CHK1 inhibitors (Davies et al., 2011); and (4) simultaneous inactivation of ATR and CHK1 increased DNA damage accumulation and cell death (Sanjiv et al., 2016). With regard to combined therapies targeting checkpoint activation and DNA repair pathways, it is interesting to note that inhibition of Claspin may also impact triggering of some of these pathways. Further understanding of the role of Claspin in activation/modulation of DNA damage repair pathways would be very important in this context.

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Notably, Claspin inhibition, in contrast to other agents that directly or indirectly interfere with p53 activity, may also sensitize cells to undergo apoptosis, as it has been shown that Claspin degradation is required for apoptosis induction (Clarke et al., 2005; Semple et al., 2007). Nevertheless, Claspin degradation may also allow cells with unrepaired DNA to resume the cell cycle and complete mitosis (checkpoint adaptation) (Syljua˚sen et al., 2006; Yoo et al., 2004), a phenomenon that may relate to resistance to genotoxic therapy. This is a potential concern in using Claspin inhibitors, in as much as checkpoint adaptation may also increase genomic instability and, thus, promote tumor development. The use of Claspin inhibitors may also be interesting because it was demonstrated that cancer stem cells are particularly sensitive to G2-checkpoint inhibitors (Bartucci et al., 2012; Bertrand et al., 2014; Signore et al., 2014; Venkatesha et al., 2012; Wang et al., 2012; Wu et al., 2012). Indeed, cancer stem cells are known to be more resistant to treatment with genotoxic agents due to their ability activate DNA damage repair mechanisms (MaugeriSacca, Bartucci, & De Maria, 2014). Radiation-resistance in these cells seems to be associated with fast activation of key DDR players, such as ATM, CHK2, CHK1, and Rad17. Interestingly, inhibition of CHK1 abrogated resistance of cancer stem cells to IR and CT (Bao et al., 2006; Bartucci et al., 2012). It would be interesting to study the effect of Claspin inhibitors in cancer stem cells in the context of chemo- and radio-sensitization. Importantly, some tumors present mutations in G2-checkpoint proteins that result in decreased protein expression (Bertoni et al., 1999). This may also be true for Claspin, as oncovirus-induced carcinogenesis requires Claspin elimination (Koganti et al., 2014; Spardy et al., 2009; Studach et al., 2010). In addition, Claspin mutations and genetic variants were found in cancer patients that may impact its expression and/or function (Azenha, Lopes, & Martins, 2012; Erkko, Pylkas, Karppinen, & Winqvist, 2008; Madeira et al., 2012; Madeira, Gonc¸alves, Ferreira, Lacerda, & Martins, 2008; Wang et al., 2008; Zhang et al., 2009). Reduced expression/function of Claspin may further sensitize tumor cells to the action of specific inhibitors, which may allow for the usage of lower doses of these chemicals with fewer side effects (Syljua˚sen et al., 2015). Alternatively, there are G2 checkpoint proteins that may be overexpressed in human cancers (Cole et al., 2011; Iorns et al., 2009; Lin et al., 2004; Magnussen et al., 2012; Mir et al., 2010; Parikh et al., 2014). This is thought to reflect a compensatory mechanism that the cell uses to cope with high replication stress (Lecona & Fernandez-Capetillo, 2014; Sørensen & Syljua˚sen, 2012). Notably, these cells present increased sensitivity to selective inhibitors (Cole et al., 2011;

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Hoglund et al., 2011) and may thus still constitute good targets for inhibitory therapy (Syljua˚sen et al., 2015). Indeed, CHK1 overexpression is thought to reflect a dependency of cancer cells on RSR, which makes CHK1 a good target for therapy (Forment & O’Connor, 2018). Again, Claspin may also constitute a good candidate target, as its overexpression is associated with resistance to IR in lung cancer brain metastasis and it has been demonstrated that depletion of Claspin was associated with enhanced radiosensitivity in lung cancer cells and with increased survival of xenograft models treated with IR (Choi et al., 2014).

5. Conclusions and perspectives Conventional CT and RT increase cancer cell replication stress by inducing DNA damage and thereby promote cancer cell death. However, the prolonged replicative stress caused by conventional therapies has the ability to promote genomic instability and may end up promoting cell adaptation to treatment. Recently, research has focused on the development of small molecule inhibitors of specific proteins that target components of the signaling cascades that are triggered by replication stress and DNA damage. Inactivation of players of RSR and DDR with pharmacological agents may further increase the replicative stress that is characteristic of cancer cells and promote cancer cell death. Some compounds have already reached advances stages of preclinical and clinical trials. Nevertheless, there are still several other targets and approaches that can be exploited. We propose that Claspin may constitute an adequate therapeutic target in cancer treatment. Due to its key role both in RSR and in the activation of different pathways of DNA damage repair, Claspin inactivation may block checkpoint activation and DNA repair, forcing the cell to proceed through the cell cycle and enter mitosis with extensive DNA damage, which will eventually lead to mitotic catastrophe, apoptosis, or senescence induction. Of note, degradation of Claspin also sensitizes cells to apoptosis. Drugs targeting Claspin could be used in monotherapy regimens in settings of synthetic lethality, as demonstrated for CHK1 (e.g., tumors with p53 deficiencies), in combination with conventional cancer therapy (further promoting replication stress and DNA damage), and/or in multidrug regimens, in combination with other targeted drugs, directed either to other components of RSR or DDR, or to other validated hallmarks of cancer, as shown for PARPi (Liu et al., 2014). In order for the genetic deficiencies of cancer cells to be effectively exploited through targeted inhibitory therapies, adequate patient selection

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is needed. Analysis of patients’ genetic background beyond TP53 status may constitute a good starting point to infer the efficacy of RSR and DDR inhibitors in the context of synthetic lethality approaches, since several gene-drug interactions have already been described in this context (Table 2). In addition, as already explained, oncogene activation is associated

Table 2 Genetic alterations that may impact response to RSR inhibitors. Genetic changes Response to RSR inhibitors

APOBEC3A/B overexpression

Sensitivity to ATR (possibly CHK1) inhibitors

ARID1A loss

Sensitivity to ATR and CHK1 inhibitors

ATM loss or inactivation

Sensitivity to ATR and CHK1 inhibitors

ATRX loss

Sensitivity to ATR inhibitors

CCNE1 overexpression

Sensitivity to ATR inhibitors

CDC25A loss

Sensitivity to ATR and CHK1 inhibitors

CDK2 loss

Sensitivity to Wee1 inhibitors

CHK1 overexpression

Sensitivity to ATR and CHK1 inhibitors

CUL1 loss

Sensitivity to Wee1 inhibitors

DAXX loss

Sensitivity to ATR inhibitors

E2F1 overexpression or activation

Sensitivity to ATR, CHK1 and Wee1 inhibitors

EWS/FLI1 translocation

Sensitivity to ATR inhibitors

EWS/ERG translocation

Sensitivity to ATR inhibitors

Histone H3 variants (K36me3)

Sensitivity to ATR, CHK1 and Wee1 inhibitors

KDM4A overexpression

Sensitivity to ATR, CHK1 and Wee1 inhibitors

KRAS activating mutations

Sensitivity to ATR inhibitors

MYC overexpression

Sensitivity to CHK1 inhibitors

RAD51 loss

Sensitivity to ATR and CHK1 inhibitors

SETD2 loss or inactivation

Sensitivity to ATR, CHK1 and Wee1 inhibitors

SKP2 loss TP53 loss or inactivation

Sensitivity to Wee1 inhibitors Sensitivity to ATR (possibly Wee1) inhibitors

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with increased replicative stress and, possibly, with augmented sensitivity to RSR inhibitors. As such, tumor characterization with regard to oncogene expression (e.g., CCNE1 and MYC overexpression, RAS mutations, among others) may also be useful in settings of inhibitory therapies. However, it is important to be aware of the limitations of genetic analysis performed in archival tumor samples collected at initial diagnosis, as these may not reflect the biology of the tumor at the time of implementation of inhibitory therapy. In fact, although oncogene-driven tumors usually experience replication stress during their evolution, many of them end up adapting to replication stress, a feature that may render inhibitory therapies targeting the RSR no longer effective. Therefore, other readouts of tumor biology (e.g., liquid biopsies) with regard to RSR and DDR that can be obtained at the time of treatment initiation must be identified. These readouts should also be adequate for the identification of mutations and other strategies associated with emerging resistances to the inhibitors throughout treatment. For instance, it has been observed that tumors with BRCA1/2 mutations treated with PARPi may end up developing resistance to these drugs through secondary mutations in BRCAs that restore the open reading frame and, thus, allow production of functional BRCA proteins with the ability to repair DNA damage through HR (Edwards et al., 2008; Sakai et al., 2008; Swisher et al., 2008); and through loss of 53BP1 expression or up-regulation of efflux pumps (Bouwman, Aly, Escandell, et al., 2010; Bunting et al., 2010; Jaspers et al., 2013; Rottenberg et al., 2008). Finally, one should be aware that, as DDR and RSR proteins are crucial for the maintenance of genome integrity, interfering with their functions may bring about an increase in the genomic instability of cells, including normal cells, an event that may have serious consequences. Therefore, one cannot loose sight of possible long-term consequences of these therapeutic approaches. Extensive knowledge on the mechanisms of action of the targeted players, as well as that of the corresponding inhibitors, both in preclinical and clinical settings, is required to minimize or obviate such unwanted adverse effects.

CONFLICT OF INTEREST The authors declare that there are no conflicts of interest.

FUNDING This work was partially supported by the Portuguese Foundation for Science and Technology (grant number SFRH/BD/73093/2010).

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