Lartesertib

Harnessing the targeting potential of differential radiobiological effects of photon versus particle radiation for cancer treatment

Jinhua Zhang1,2,3 | Jing Si1,2,3 | Lu Gan1,2,3 | Rong Zhou4 | Menghuan Guo5 | Hong Zhang1,2,3

Abstract

Radiotherapy is one of the major modalities for malignancy treatment. High linear energy transfer (LET) charged‐particle beams, like proton and carbon ions, exhibit favourable depth‐dose distributions and radiobiological enhancement over conventional low‐LET photon irradiation, thereby marking a new era in high precision medicine. Tumour cells have developed multicomponent signal transduction net- works known as DNA damage responses (DDRs), which initiate cell‐cycle check- points and induce double‐strand break (DSB) repairs in the nucleus by nonhomologous end joining or homologous recombination pathways, to manage ionising radiation (IR)‐induced DNA lesions. DNA damage induction and DSB repair pathways are reportedly dependent on the quality of radiation delivered. In this review, we summarise various types of DNA lesion and DSB repair mechanisms, upon irradiation with low and high‐LET radiation, respectively. We also analyse factors influencing DNA repair efficiency. Inhibition of DNA damage repair pathways and dysfunctional cell‐cycle checkpoint sensitises tumour cells to IR. Radio‐ sensitising agents, including DNA–PK inhibitors, Rad51 inhibitors, PARP inhibitors, ATM/ATR inhibitors, chk1 inhibitors, wee1 kinase inhibitors, Hsp90 inhibitors, and PI3K/AKT/mTOR inhibitors have been found to enhance cell killing by IR through interference with DDRs, cell‐cycle arrest, or other cellular processes. The cotreatment of these inhibitors with IR may represent a promising therapeutic strategy for cancer.

KEYW OR DS
DNA damage, DNA repair, particle therapy, photon therapy, radiosensitizer

1 | IONISING RADIATION‐ BASED THERAPEUTICS

In general terms, the global incidence of the malignant tumour has in- creased year on year, and seriously threatens human health. Approxi- mately, 18.1 million new cancer cases and 9.6 million cancer deaths were reported globally in 2018 (Bray et al., 2018; Ferlay et al., 2019). Conventional photon radiation, such as X‐rays and γ‐rays are crucial treatment modalities for patients with cancer, and are often combined with systemic treatments, and applied to both neoadjuvant and defini- tive settings (Hartfiel et al., 2019). Charged particles, such as proton and carbon‐ion radiotherapy (RT) are an emerging option for cancer therapy, and have had an enormous breakthrough in this field. An estimated 150,000 patients have undergone proton beam treatment (Vitti & Parsons, 2019), and more than 20,000 patients have received carbon‐ion therapy worldwide, for a wide range of cancers (Durante & Debus, 2018). Charged‐particle RT shows favourable physical properties, with a maximum energy deposition at the end of the ion range, that is, the spread‐out‐Bragg peak. This characteristic allows for an ideal dose dis- tribution to target tumours, while minimising damage in adjacent healthy tissues (Hartfiel et al., 2019; Saager, Peschke, Brons, Debus, & Karger, 2018). Furthermore, in contrast to conventional photon‐based RT, carbon‐ion beams exhibit advantageous biological effectiveness in tumour therapy, such as increased relative biological effectiveness (RBE; Durante & Debus, 2018), oxygenation independence (Chew et al., 2019), reduced dependence on the cell‐cycle (Masunaga et al., 2008), and irreparable complex DNA damage (Sai et al., 2015).
One of the main parameters for evaluating the RBE of RT, is linear energy transfer (LET). This describes the energy transferred by ionising radiation (IR) along its path, and depends on the type of radiation (Vitti &Parsons, 2019). Remarkably, protons are usually low‐LET radiation, and their biological effects are approximately identical to photons (the RBE is 1.1 ; Saager et al., 2018), whereas the biological effects of carbon ions are more pronounced than protons (the RBE is ∼2.3; Si et al., 2019). Unlike low‐LET IR, which produces sparse ionising events randomly distributed throughout the cell to eventually induce less complex DNA damage (Lorat, Timm, Jakob, Taucher‐Scholz, & Rübe, 2016), high‐LET IR pre- dominantly deposits a high energy density at the end of the range (the Bragg Peak), causing more extensive damage, and often, fateful biolo- gical consequences (Liu et al., 2018; Zhao et al., 2017). High‐LET radia- tion contributes to clustered DNA damage, a DNA lesion complex that includes two or more individual lesions within a few helical DNA turns, which is largely responsible for the deleterious effects of high energy IR (Park, Peoples, Madugundu, Sanche, & Wagner, 2013; Q. Zhang et al., 2016). Previous studies have shown that most DNA lesions caused by heavy‐ion beams are single‐strand breaks (SSBs) and double‐strand breaks (DSBs) complexes. These lesions occur in close proximity to each other, leading to highly complex DNA damage. Moreover, DNA lesions induced by X‐rays are mostly single SSBs or DSBs, and are typically distant to each other (Q. Zhang et al., 2016). Even though both types of IR are considered low‐LET radiation, the complexity of DNA damage caused by protons and photons may differ (Fontana et al., 2015). Proton irradiation of hepatocellular carcinoma (HCC) cells has shown a con- tinuous activation of DNA damage repair proteins over time, reflecting the formation of more complex DSBs by protons, when compared with X‐rays (Choi et al., 2019). Collectively, these observations suggest that the higher the LET, the denser the ionisation events, resulting in a more complex induction of DNA damage.

2 | DNA DAMAGE AND REPAIR MECHANISMS

It is accepted that IR induces a variety of different types of DNA damage, including base damage, base loss (Vitti & Parsons, 2019), SSBs, DSBs, and DNA interstrand cross links (Parplys, Petermann, Petersen, Dikomey, & Borgmann, 2012). To safeguard genome sta- bility and integrity, cells have developed a sophisticated signalling network known as the DNA damage response (DDR), which initiates cell‐cycle checkpoints or induces cell death, to counteract variousDNA lesions (Li et al., 2017). If repair is successful, cells are released from cell‐cycle arrest to enter another cell cycle (Jiaranuchart, Kaida, Onozato, Harada, & Miura, 2018). Any mis‐repaired or unrepaired DSBs cause genome rearrangements, and lead to developmental defects, accelerated aging, cell death, mutagenic events, and cancer (Symington & Gautier, 2011; Xing et al., 2015).
The DDR signalling pathway commences with the activation of two specific kinases, ataxia telangiectasia mutated (ATM) and ATM‐and RAD3‐related (ATR), which sense DNA damage. Both cooperate with the MRN complex (MRE11–RAD50–NBS1) and the Rad17/9‐ 1‐1 complex (Rad17; Rad9–Rad1–Hus1), respectively (Burger, Ketley, & Gullerova, 2019; Stadler & Richly, 2017). Both kinases relay signals to downstream kinases such as Chk1/2, to trigger its activation, re- sulting in the inactivation of CDK/cyclin complexes, and consequently cell‐cycle arrest (Cassimere, Mauvais, & Denicourt, 2016; Parplys et al., 2012). Although ATM mainly activates Chk2, and Chk1 is acti- vated by ATR, there is considerable crosstalk between these two pathways, as they share many substrates (Patties et al., 2019; Qiu, Oleinick, & Zhang, 2018). Notably, ATM initiates cellular signalling cascade in response to DSBs, whereas ATR is activated by interrupted replication forks and extensive lesions in SSBs. This collective initiation/activation alleviates cellular genotoxic stress (Stadler & Richly, 2017; Yang et al., 2015).

2.1 | Cell‐cycle progression following RT

Cell‐cycle progression is a pivotal deterministic factor for IR‐induced DNA damage; both G1 and G2 checkpoints are activated to create time for extensive DNA repair, before cell‐cycle re‐entry (Jiaranuchart et al., 2018; Vitti & Parsons, 2019). The G1 checkpoint primarily stops damaged DNA from being replicated. Irradiation in- duces DSBs and the subsequent activation of ATM/ATR and Chk2 kinases, resulting in p53 accumulation. This induces the transcrip- tional activation of p21/WAF1/CIP1, which is a member of the CIP/ KIP family of cyclin dependant kinase (CDK) inhibitors. It binds to and suppresses activation of CDK2/4, thereby arresting tumour cells at the G1/S boundary. This process is mediated in a p53‐dependent manner (Cassimere et al., 2016; Manila, Kaida, Nakahama, & Miura, 2018). In parallel, this delayed transcriptional response is accom- panied by a transient and rapid p53‐independent response, which blocks S‐phase entry via promotion of the CDC25A phosphatase, which removes CDK2 inhibitory phosphorylation (Cassimere et al., 2016). Equally, the G2 checkpoint generally delays cells with DNA sequences that are improperly methylated or where chromosomes do not match up with the spindle mechanism (Girdhani, Sachs, & Hlatky, 2013). Activated ATM and ATR induce the phosphorylation and activation of Chk1/2 kinases, resulting in phosphorylation of CDC25C at Ser216, thereby inhibiting CDC2 kinase activity, and blocking activation of the CDK1/cyclin B complex. Finally, cells arrest at the G2/M boundary (Figure 1; Manila et al., 2018; Yan et al., 2015).
Cell‐cycle arrest is usually a sign that cells are repairing DNA damage, but is not necessarily a predictor of successful repair (Hartfiel et al., 2019). Different radiation approaches may provoke their own char- acteristic cell‐cycle modulation programmes. Generally, more pronounced delays in S‐ and G2‐phases are observed with increased LET (Fournier & Taucher‐Scholz, 2004). Increasing evidence indicates that cells irradiated with high‐LET particles, exhibit a more pronounced G2/M phase arrest and apoptosis, than those irradiated with con- ventional photon‐based RT (Fujisawa et al., 2015; Yang et al., 2015).
Studies show that carbon‐ion radiation induces a persistent G2/M block in PC3 cells at lower doses (2 Gy), in comparison with X‐rays (5 Gy), while in Caco‐2 cells, the G2/M arrest was transient after exposure to X‐rays (2 and 5 Gy), but permanent after irradiation with carbon ions (2 Gy; Suetens et al., 2016). G2 checkpoint arrest was increased in lung cancer cells by ∼2.3 times by carbon‐ion exposure, in relation to X‐ray exposure at the same physical dose (5 Gy), indicating carbon ions are more biologically effective than X‐rays, and induce more DNA lesions (Ma et al., 2015). Similarly, when analysing the cell‐cycle distribution of human lung cancer cells, it was observed that therapeutic protons (62 MeV), predominantly induced a G1 phase block (~2 fold increase), while cells after carbon‐ion irradiation (62 MeV) were blocked in the G2/M phase (~1.5–2 fold increase; Keta et al., 2017). Glioblastoma cells exposed to low energy proton irradiation (5.7 MeV) showed higher and more persistent induction of G2 arrest, than X‐ray irradiated controls (120 kV; ~1.5–2.5 fold; Moertel et al., 2004). Altogether, variations in cell‐cycle progression following IR appears to be dependent on the tumour type, photon or particle type and the radiation LET.

2.2 | γ‐H2AX/53BP1 foci as a marker of DSBs

Many DDR signalling or repair proteins assemble rapidly to DSBs sites, and are detected as DNA damage foci. To explore the repair kinetics of IR‐induced DSBs, the time course of phosphorylated his- tone variant H2AX (γ‐H2AX)/p53‐binding protein 1 (53BP1) DNA repair foci, which is phosphorylated or recruited near the end of DSBs, is usually monitored by immunofluorescence (Jiaranuchart et al., 2018). As a DNA damage sensor protein, γ‐H2AX strongly promotes the recruitment of DSB repair proteins, and is essential for effective repair by maintaining chromatin structural changes (Mueck, Rebholz, Harati, Rodemann, & Toulany, 2017; Zhao et al., 2017). The number of γ‐H2AX foci formed after irradiation is directly propor- tional to the number of DSBs formed, and their dephosphorylation is closely associated with DSB repair (Oonishi et al., 2012). Similarly, 53BP1 serves multiple roles in mammalian DNA damage repair, mediating repair pathway decisions of cells, rapidly participating in the repair of DSBs in heterochromatin in the G1 phase, and pro- tecting damaged DNA ends from excessive end resection (Sollazzo et al., 2017; Sunada, Nakanishi, & Miki, 2018). In brief, γ‐H2AX or 53BP1 foci represent DSB sites, and both serve as surrogate marker for DSBs.
Different H2AX phosphorylation levels occur in response to different particles. Previous studies have shown that both therapeutic protons and carbon ions increase γ‐H2AX expression, however, carbon ions enhance γ‐H2AX phosphorylation levels more than therapeutic protons (Keta et al., 2017). In addition, it is generally accepted that the higher the LET, the longer the foci persist in the cell nucleus. Unlike the foci induced by low‐LET‐radiation, which are subject to fast repair, high‐ LET radiation generates highly complex, large γH2AX/53BP1 focus clusters, with increased irregularity, and slower elimination (Sollazzo et al., 2017). Recent studies have demonstrated that large γ‐H2AX foci are characteristic of high‐LET IR, and are associated with the induction of clustered DSB lesions that are not easy to repair (Girdhani et al., 2013; Lopez et al., 2019). This phenomenon could be explained by the agglomeration of individual foci; a significant increase in γ‐H2AX foci was observed after exposure to proton radiation, relative to X‐ray radiation (Girdhani et al., 2013). Moreover, in human glioblastoma cell lines, the number of initial DSB lesions induced by carbon‐ion radiation (1.2–2.4 GeV) were smaller than photons (6 MV), while the foci of carbon‐ion radiation were much larger and contained more γ‐H2AX molecules than photons induced (Lopez et al., 2019). Similarly, in human pancreatic cancer cell lines, carbon‐ion beams (290 MeV) induced larger numbers, and increased sizes of γ‐H2AX foci, when compared with X‐rays (200 kV) at iso‐effective doses (Oonishi et al., 2012). Further- more, unlike irradiation with plateau protons or X‐ray photons, which induce small and circular shaped γ‐H2AX and 53BP1 foci, the foci in- duced by Bragg‐peak protons are larger, and more irregularly shaped.
Equally, DNA repair foci resolution was delayed by 2–6 hr after ex- posure to Bragg‐peak protons, which may have been due to the gen- eration of greater DNA damage in closer proximity (Oeck et al., 2018). Collectively, the size and frequency of radiation‐induced foci appears to vary with cell characteristics and radiation quality.

2.3 | DNA damage repair pathways

Base excision repair (BER), nucleotide excision repair (NER), mismatch repair (Iliakis, Murmann, & Soni, 2015), and translesion DNA synthesis are indispensable mechanisms for repairing damaged bases and SSBs (Bobkova et al., 2018). Among the different IR‐induced DNA lesions, DSBs are the most detrimental in the nucleus, with DNA repair capacity related to tumour radio‐resistance (Liu et al., 2018). It was reported that the majority of IR‐induced DSBs are effectively resolved, either through error free homologous recombination (HR) or error‐ prone nonhomologous end joining (NHEJ; Gerelchuluun et al., 2015).
Notably, the Ku‐independent alternative NHEJ pathway, as a backup to abrogated canonical NHEJ (c‐NHEJ) and HR, is also used by cells to remove DSBs (Toulany, 2019); however, it is even more error prone than c‐NHEJ (Lopez et al., 2019). c‐NHEJ is the primary pathway of choice for DSB repair in eukaryotic cells. The initial step involves Ku70/80 heterodimers sensing and binding to broken DNA ends, followed by the loading of DNA–PKcs onto the DNA–Ku complex (Jiaranuchart et al., 2018). Furthermore, the endonuclease, Artemis is recruited and forms a complex with the autophosphorylated DNA–PKcs to improve joining efficiencies through the endonucleolytic cleavage of 5′ and 3′ DNA overhangs (Gerodimos, Chang, Watanabe, & Lieber, 2017). Lastly, DNA ligase IV (Lig IV) in complex with XRCC4 stimulated by XLF, religate the processed DNA ends (Brouwer et al., 2016). It has been shown that the paralogue of XRCC4 and XLF (PAXX), an accessory c‐NHEJ factor, strongly interacts with the Ku complex, and functionally cooperates with XLF to stabilise c‐NHEJ machinery (Fig- ure 2a; Xing et al., 2015). Whereas an early step in alternative NHEJ is the generation of 3′ overhangs via a process of DNA end resection which requires MRN complex and CtIP (Sallmyr & Tomkinson, 2018).
Poly (ADP‐ribose) polymerase‐1 (PARP‐1), together with XRCC1‐Lig IIIα or Lig 1, eventually bind to DNA ends to initiate end‐joining via an alternative NHEJ mechanism, that does not require c‐NHEJ factors (Figure 2b; Newman et al., 2015; Vitti & Parsons, 2019). HR depends on the presence of a sister chromatid or homologous chromosome as a repair template and is therefore cell‐cycle‐ dependent (Sharma et al., 2020). During this process, the DSB end is recognised by the MRN complex, and is followed by MRN‐ and CtIP dependent DNA end resection (Grosse et al., 2014). Thereafter, single‐stranded DNA (ssDNA) tails are stabilised by the ssDNA‐ binding complex, RPA, which is subsequently exchanged by Rad51, forming a nucleoprotein filament, necessary for homology search, and strand invasion into the adjacent intact sister chromatid (Ger- elchuluun et al., 2015; Mueck et al., 2017). Ultimately, Rad54 facil- itates DNA synthesis related to branch migration, by dissociating Rad51 from the heteroduplex DNA (Figure 2c; Iliakis et al., 2015).

2.4 | DSBs repair pathway choice following IR

DNA damage induction and DSB repair pathways are reportedly different, upon photon and/or charged‐particle irradiation. The repair process via NHEJ or HR pathway may be controlled by the cell‐cycle phase, the complexity of DNA damage, repair protein proficiencies, and so on (Taleei, 2019). Cell‐cycle progression is a decisive factor in the choice of repair pathway. NHEJ operates throughout cell‐cycle phases, while HR exists only in late S and G2 phases (Dong et al., 2018; Jiaranuchart et al., 2018). Moreover, it is accepted that the occurrence and quality of IR‐induced DSBs depend on the radiation‐related ionisation density, which potentially exerts a significant impact on the subsequent choice of DSB repair pathway (Gerelchuluun et al., 2015). NHEJ and HR may compete with each other, especially during S/G2 phases, but studies have shown that less complex DSBs are preferentially repaired by the NHEJ process, while more complex DSBs, containing multiple damaged sites, cause a switch in repair from NHEJ to HR, or an alternative NHEJ pathway to ensure genomic stability (Grosse et al., 2014; Taleei, 2019).
NHEJ is the primary mechanism for DSB repair, especially in re- sponse to clinical photon beams. The inhibition of DNA–PKcs profoundly sensitises mouse embryonic fibroblast cells and nasopharyngeal carcinoma SUNE‐1 cells to X‐rays, by severely impairing DSB repair (Dong et al., 2017). This observation agrees with previous reports using lung adenocarcinoma and glioblastoma cells (Fontana et al., 2015). These studies suggest a relevant role of the c‐NHEJ pathway in photon‐ damaged DNA repair. However, conflicting reports also suggest that DNA repair pathway choice, specifically following proton beams ther- apy, may actually be different (Table 1). Given these opposing findings, more definitive evidence of the appropriate DNA repair pathway choice after proton radiation, must be investigated. With increased DNA damage, the repair efficiency of NHEJ stea- dily diminishes (Fontana et al., 2015), and pathways involved in DNA end resection are activated (Yajima et al., 2013). It is believed that complex DSBs induced by heavy‐ion irradiation are repaired by the HR pathway (Zhou et al., 2013). Studies have shown that complex DNA damage was not repaired by the NHEJ pathway when cells were ex- posed to iron ions at 2 Gy doses (Okayasu et al., 2006). When compared with photons, carbon‐ion irradiation caused more severe DNA damage where repair was less efficient, resulting in longer‐lasting cell‐ cycle delays, predominantly in the G2 phase, and concomitant augmented apoptosis in U87 and LN299 cells (two human glioblastoma cell lines). Moreover, the HR pathway appears to be more relevant for DSB repair after carbon‐ion beams, versus photons in PTEN proficient U87 cells (Lopez et al., 2019), in agreement with reports in Chinese hamster cells (Gerelchuluun et al., 2015), and hematopoietic mature and im- mature cells (Rall et al., 2015). Approximately, 85% of carbon‐ion‐ induced DSBs in particle tracks, are subject to resection in G2 cells. Moreover, ∼20–40% of G1 cells exhibit DNA end resection signalling (alternative to the NHEJ pathway; Yajima et al., 2013). A possible ex- planation for the particular importance of HR after high‐LET radiation, is that the high yields of small DNA DSBs fragments (<40 bp), prevent the Ku protein from effectively binding to the ends of one dsDNA fragment at the same time, thereby causing inefficient repair of densely clustered DSBs by NHEJ (Lopez et al., 2019). 3 | FACTORS AFFECTING DNA REPAIR EFFICIENCIES 3.1 | Complexity and location of DNA lesions Increasingly, evidence has revealed that genomes are not uniformly reparable, despite a global cellular competence towards DNA repair (Hewitt et al., 2012; X. Zhang et al., 2016). Research has suggested that the repair efficiency of DNA damage induced by heavy‐ion beams is lower than X‐rays, and most of the irreparable damage was clustered DNA damage, which pushed cells into senescence. In contrast, DNA damage induced by X‐rays was mostly repaired within 24 hr, and the remained repair‐resistant DNA damage was in preferential correlation with telomeric DNA, and the telomere‐ favoured persistent DNA damage contributes to X‐rays induced cellular senescence (X. Zhang et al., 2016). Therefore, both the complexity and location of DNA damage affects its repair efficiencies. DSB repair induced by IR operates in higher order chromatin environments (Timm, Lorat, Jakob, Taucher‐Scholz, & Rube, 2018). Chromatin reorganisation and decondensation play vital roles in regulating the cell's ability to repair DNA damage. Studies have shown that local chromatin organisation in and around damaged sites, undergo transient remodelling to promote access of repair factors, and the subsequent clearance of DNA lesions. This is fol- lowed by restoration of original chromatin structure when the repair is complete (Stadler & Richly, 2017). Low‐LET IR induces single DNA molecule damage throughout the nucleus, but nearly all eu- chromatic and heterochromatic DSBs are efficiently repaired within 24 hr, without visible chromatin decompaction. In contrast, high ionising densities from high‐LET IR causes clustered DNA damage, and triggers continuous rearrangements in chromatin architecture along particle trajectories, which may eventually disturb nucleus structural and functional organisation (Lorat et al., 2015; Timm et al., 2018). Moreover, other studies have shown that the dimension of high‐LET IR‐induced clustered DNA damage, largely relies on chromatin packing densities; multiple DSBs occur in highly compacted heterochromatin where lesions are processed with slower kinetics, whereas large heterochromatic DSB fractions re- mained unrepaired (Lorat et al., 2015). These data provide evidence that differences in the spacing and quantity of DSBs in clustered lesions, affect DNA repair efficiencies and may account for different radiobiological outcomes. 3.2 | The quiescent state Cellular quiescence is a reversible cellular state that allows cells to re‐enter and exit the proliferative cycle (J. Zhang et al., 2019). It is believed that quiescent tumour cells are more resistant to IR, due to their large hypoxic fraction and greater repair abilities, when compared with cycling tumour cells (Masunaga et al., 2008; Pei et al., 2017). DNA replication is one of the fundamental bio- logical processes that is tightly regulated to ensure the accurate and complete transmission of genetic information to daughter cells. The faithful replication of the genome is frequently chal- lenged through stresses originating exogenously or endogenously (Boyer, Walter, & Sorensen, 2016). Radiation therapy exerts its effect mainly through interference with DNA replication and in- duction of replication stress, which ends up generating DNA damage (Kwon et al., 2013). The quiescence status of cancer cells does not replicate their genome and is widely considered to be an essential protective mechanism that minimises endogenous stress caused by DNA replication (Mohrin et al., 2010). The mammalian ribonucleotide reductase subunit, p53R2, is required for both mitochondrial DNA replication and DNA repair in quiescent cells (Pontarin, Ferraro, Bee, Reichard, & Bianchi, 2012). Equally, previous studies have suggested that the ROS/RAC2/P38 MAPK feedback loop is associated with the radio‐resistance of quiescent cells, to 100 kV X‐rays (Pei et al., 2017). Furthermore, following γ‐ray irradiation, the radio‐sensitivity of pimonidazole‐unlabelled quiescent tumour cells (oxygenated cell fraction) was higher than the total cell fraction (quiescent plus proliferating cells; Masunaga et al., 2013). The difference in radio‐sensitivity between the total and quiescent‐cell populations after γ‐ray irradiation was markedly reduced with carbon‐ ion beams (290 MeV), especially with a greater LET value (Masunaga et al., 2008). Hence, carbon‐ion beams with a higher LET are promising treatment modalities for tumour and in- tratumour quiescent‐cell control. 4 | STRATEGIES TO ENHANCE RADIO‐ SENSITIVITY TO RT Although improvements have been made to potentiate radio‐ therapeutic cancer treatments, radio‐resistance remains a concern. It has been reported that suppression of DNA damage repair pathways and dysfunctional cell‐cycle checkpoints increases the radio‐ sensitivity of tumour cells (Lee & Okayasu, 2018). 4.1 | Sensitisation of tumours to RT through regulatory repair pathways Targeting DNA damage repair pathways may have potential for the radio‐sensitisation of tumour cells. As described, the two major pathways for DSB repair include HR and NHEJ, which are the main targets for radio‐sensitisation. It has been shown that inhibiting one or both pathways could induce radio‐sensitisation in irradiated tumour cells (Lee & Okayasu, 2018). 4.1.1 | Targeting the NHEJ pathway The kinase activity of DNA‐dependent protein kinase (DNA–PK) plays a crucial role in the NHEJ repair pathway and early‐stage cell‐cycle arrest, postirradiation (Dong et al., 2017; Dong et al., 2018). DNA–PK is a valid therapeutic target in tumour cells, where silencing of the kinase results in increased radio‐sensitivity and DSBs. Studies have shown that DNA–PKcs depletion markedly increased HCC sensitivity to proton beam irradiation, via accumulation of un- repairable DNA damage and apoptosis induction (Choi et al., 2019). Moreover, somewhat similar results were recorded in tumour cells cotreated with DNA–PK inhibitors, and photon or carbon‐ion beams (Table 2). Notably, DNA–PK inhibitor M3814 and DNA‐repair in- hibitor DT01 in combination with RT entered first‐in‐human trials of tumour patients (Harnor, Brennan, & Cano, 2017; Le Tourneau et al., 2016). In addition to DNA–PK inhibitors, nimotuzumab, a monoclonal antibody specifically targeting epidermal growth factor receptor (EGFR), suppressed the IR‐induced activation of DNA–PKcs by blocking the PI3K‐Akt pathway, thereby enhancing tumour cell radio‐sensitivity (Qu et al., 2013). DNA Ligase IV is responsible for sealing of DSBs during NHEJ. Inhibiting Ligase IV could cause the accumulation of unrepaired breaks in the genome, thereby serving as a strategy toward the treatment of cancer. Recent studies have suggested that SCR7, as a putative inhibitor of NHEJ, blocked Ligase IV‐mediated end‐joining by interfering with its DNA binding and significantly enhanced the sensitivity of cancer cells towards radiation (Srivastava et al., 2012). XRCC4 is a nuclear phosphoprotein that stimulates Ligase IV end‐joining activity, possibly via facilitating the recruitment of Ligase IV to DNA ends. Previous studies have shown that FBXW7 (a haplo‐insufficient tumour suppressor) facilitates NHEJ repair via K63‐linked polyubiquitylation of XRCC4 and FBXW7 depletion (MLN4924) sensitises cancer cells to radiation (Q. Zhang et al., 2016). Moreover, adenovirus expressing the truncated XRCC4 protein was demonstrated to sensitise ER‐negative MDA‐MB‐231 human breast cancer cells to IR, presumably through interference with the functional activity of Ligase IV, leading to the defection of NHEJ machinery (Jones et al., 2005). Meanwhile, there is evidence to suggest that adenoviral‐mediated mda‐7 (melanoma differentiation‐ associated gene‐7) downregulates the expression of NHEJ pathway components Ku70, XRCC4, and DNA Ligase IV and radio‐sensitises human lung carcinoma A549 cells (Nishikawa et al., 2004). Human polynucleotide kinase/phosphatase (PNKP) is a dual‐ function enzyme with 5′‐DNA kinase and 3′‐DNA phosphatase ac- tivities. It functions in various pathways including BER, DNA SSB repair, and NHEJ (Zolner et al., 2011), making it an attractive therapeutic target. Cells lacking PNKP show marked sensitivity to IR. The prostate cancer cell line, PC‐3 undergoes enhanced G2/M cell‐cycle arrest and significant cell death, via the formation of apoptotic bodies and nucleosomal DNA ladders after treatment with carbon‐ion radiation and the PNKP inhibitor, A12B4C3 (Srivastava, Sarma, & Chaturvedi, 2018). It was also reported that A12B4C3 or short‐ hairpin RNA (shRNA)‐mediated PNKP depletion, enhanced A549, and MDA‐MB‐231 cell radio‐sensitivity by a factor of two (Freschauf et al., 2009). In summary, targeting the NHEJ repair pathway may be a suitable approach to sensitise radioresistant tumour cells to IR. 4.1.2 | Targeting the HR pathway Similar to NHEJ, targeting the HR pathway could facilitate ther- apeutic gain for cancer therapy. Replication protein A1 (RPA1) is a heterotrimeric ssDNA‐binding protein that participates in DNA replication and is required for each of the three major DNA repair pathways: NER, BER, and DSBs repair. It is found that RPA loss either by shRNA‐mediated silencing or chemical inhibition (HAMNO) im- pairs the survival and self‐renewing potential of GSCs (glioblastoma cancer stem‐like cells) while increasing their sensitivity to IR by im- pairing the DNA repair capacity of GSCs (Pedersen, Anne, Elbaek, Vitting‐Serup, & Hamerlik, 2020). Depletion of RPA1 increases CNE‐ 2R (human radioresistant nasopharyngeal carcinoma cell line) andTE‐1R (radioresistant oesophageal cancer cells) cell radio‐sensitivity to X‐rays by impairing Rad51 recruitment to damage sites, con- tributing to G2/M phase arrest (Di, Sanyuan, Hong & Dahai, 2014; Z. Zhang et al., 2018). Rad51 is a central enzyme in DSB repair in the HR pathway, suggesting a therapeutic strategic role for Rad51 in cancer therapy. Increasing evidence suggests that the Rad51 inhibitor, RI‐1 suppresses cervical cancer cell growth by attenuating the cell‐cycle transition from G0/G1 to S phase, via cyclin D1 and p21 (Chen, Cai, Li, & Wu, 2017). Moreover, T0070907, a peroxisome proliferator activator receptor γ inhibitor, impairs the repair of IR‐induced DSBs by efficiently decreasing Rad51 protein levels, enhancing IR‐induced mitotic catastrophe, resulting in the poor survival of IR‐treated cervical cancer cells (An, Yu, & Park, 2016). Similarly, the radio‐ sensitising effects of Rad51 inhibition were demonstrated in glioblastoma stem‐like cells (Balbous et al., 2016), leukaemic T‐cells (Havelek et al., 2014), and osteosarcoma cells (Q. Liu et al., 2015). Furthermore, Rad51 and PARP inhibitor combinations sensitise tu- mour cells to proton radiation. Early evidence has suggested that PARP (olaparib) and RAD51 (B02) inhibitors result in limited cyto- toxic (alone or in combination) effects, but augmented and persistent DNA damage, prolonged cell‐cycle blockage, and delayed apoptosis when cells are exposed to protons (Wera, Lobbens, Stoyanov, Lucas, & Michiels, 2019). PARP‐1 is a nuclear enzyme that facilitates BER and DNA strand break repair by modifying key proteins. PARP inhibition causes synthetic lethality in cancer cells with dysfunctional BRCA1/2, which typically encode for key repair proteins in the HR pathway (Hirai et al., 2016; Luo et al., 2019). As a result of this discovery, PARP1 inhibitors (PARPi) are promising radio‐sensitisers in treating patients with BRCA1/2‐mutant tumours. Several third generation PRPAi's, including olaparib (AZD2281), AG14361, talazoparib (BMN673), niraparib (MK‐4827), and veliparib (ABT‐888), have been developed to sensitise tumour cells to IR (Table 3). Olaparib (AZD2281), the first PARPi to gain US Food and Drug Administration approval, radio‐ sensitised tumour cells irradiated with photons, protons and carbon ions, with levels of tumour radio‐sensitivity associated with muta- tions in HR repair genes (Cesaire et al., 2019). Hence, PARPi's could be applicable to a wide therapeutic range of LET radiation therapies, as a radio‐sensitiser through its DNA repair effects. Furthermore, fluzoparib, a novel PARPi, significantly increases the radio‐sensitivity of non‐small cell lung cancer (NSCLC) cells without BRCA1/2 muta- tions, through underlying mechanisms involved in enhanced apoptosis, G2/M phase arrest, increased IR‐induced DNA lesions, and delayed DDR activity, which breaks the definition that only patients with mutated BRCA1/2 could benefit from PARPi, thus extend the clinical application to more solid tumours and benefit more patients (Luo et al., 2019). Taken together, these data suggest that inhibitors disrupting DNA repair pathways could be promising options in in- creasing the therapeutic effect of RT. 4.1.3 | Targeting both NHEJ and HR pathways Suppression of both NHEJ and HR repair pathways induces greater radio‐sensitisation than impairment of either repair pathway alone. The heat shock protein 90 (Hsp90) is a chaperone that stabilises and regulates the function of tumour growth‐associated proteins. The inhibition of Hsp90 is believed to be a useful therapeutic strategy for cancer. TAS‐116, a novel Hsp90 inhibitor, selectively sensitises hu- man cancer cells to both X‐rays and carbon‐ion radiation, by de- creasing Rad51, Ku70, and DNA–PKcs expression, which are crucial players in the NHEJ and HR repair pathways (Lee et al., 2017). Moreover, in human lung adenocarcinoma cells exposed to carbonions, the Hsp90 inhibitor, PU‐H71 induced persistent γ‐H2AX foci, reduced RAD51 foci, and phosphorylated DNA–PKcs, with little or no sensitising effects on normal human fibroblast cells (Lee et al., 2016). Similarly, PU‐H71 increased radio‐sensitivity in a murine osteosarcoma cell line (LM8) to not only X‐rays, but also carbon‐ion irradiation, while only a minimal sensitising effect was observed in normal human fibroblasts (Li, Matsumoto, Furusawa, & Kamada, 2016), suggesting PUH‐71 is a potential cancer‐specific radio- sensitizer, via suppression of HR and NHEJ repair pathways. Fur- thermore, Hsp90 inhibitors also alter cell‐cycle distribution by disrupting cell‐cycle checkpoint proteins, thereby increasing tumour cell radio‐sensitivity. The combination of 17‐allylamino‐17‐ demethoxygeldanamycin (17‐AAG) and X‐rays, released G2/M phase arrest, which resulted in human oral squamous cell carcinoma LMF4 cells harbouring mis‐repaired DNA damage, moving on to G1 phase, thus enhancing tumour cell sensitivity to radiation (Musha et al., 2012). Unfortunately, 17‐AAG has limited uses due to poor phar- maceutical properties. BIIB021, an orally available Hsp90 inhibitor, enhances the radio‐sensitivity of oesophageal squamous cell carci- noma cell lines, by reducing the expression of radioresistant proteins (EGFR, Akt1, and Raf‐1), increasing apoptotic cells, and enhancing G2 arrest (Wang et al., 2014), in agreement with reports on head and neck squamous cell carcinoma cell lines (Yin et al., 2010). Moreover, studies have shown that the novel high affinity Hsp90 inhibitor, AT13387 disrupts cell‐cycle regulation, and results in G2/M arrest and S‐phase fraction reduction, thus potentiating radiation effects in squamous cell carcinoma and adenocarcinoma cells (Spiegelberg et al., 2015). Furthermore, the combination of TAS‐116 with X‐rays or carbon ions stimulates pronounced G2/M phase arrest, by cdc25c inhibition in HeLa cells (Lee et al., 2017). Akt1 facilitates the accumulation of DNA–PKcs at DNA damage sites, and promotes DNA–PKcs kinase activity for efficient NHEJ‐ mediated DSB repair (Toulany et al., 2012). Studies have shown that API‐59CJ‐OH, an AKT pathway inhibitor, enhances the radio‐ sensitivity of NSCLC cells, most likely through inhibition of DNA–PKcs‐dependent DSB repair (Toulany et al., 2008). Akt1 is also a regulatory component in the HR repair of DSBs, in a Rad51‐ dependent manner (Mueck et al., 2017). These results support the notion that Akt1 could act as an excellent target to selectively im- prove radio‐sensitivity in tumour cells. Therefore, inhibitors targeting both NHEJ and HR pathways are promising candidate radio‐ sensitisers, likely to be more effective than agents influencing one DSB repair pathway only. 4.2 | Sensitisation of tumours to RT through dysfunctional cell‐cycle checkpoints The more severe the DNA lesion induced by IR, the longer the cells are arrested in a specific cell‐cycle phase. Dysfunction of cell‐cycle checkpoint with chemicals, small interference RNA, or cell‐cycle kinase inhibitors, usually sensitises cancer cells to IR (C. Liu, Nie, Wang, & Mao, 2019). P27cip1 is essential for G1 checkpoint arrest, as it prevents cells from entering S‐phase after IR. The phosphorylation of p27cip1 at Ser140 by ATM maintains its stability after exposure to IR. The inhibition of this stabilisation, by replacing endogenous p27Kip1 with a Ser140 phospho‐mutant (S140A), increases the radio‐ sensitivity of tumour cells (Cassimere et al., 2016). Accumulating evidence shows that G1 checkpoints are dysre- gulated in most tumour cells, suggesting p53 inactivation or muta- tion. DNA damaged cells that lack normal p53 function may thus become solely dependent on the G2 checkpoint for survival (Lee et al., 2019; Manila et al., 2018). The abrogation of p53‐independent G2 checkpoint function is an attractive strategy for specific radio‐ sensitisation of cancer cells, especially for cells harbouring defective p53 functions. 4.2.1 | ATM/ATR inhibitors ATM/ATR kinases are essential for the activation of G2 checkpoints. As vital components of the DDR pathway, ATM/ATR activation ultimately results in cell‐cycle block at the G2/M phase, and the pro- motion of DNA repair (Tu et al., 2018). Thus, ATM/ATR inhibition is an attractive radio‐sensitising strategy for tumour therapy. Inhibition of ATM by KU‐55933 overcomes radio‐resistance in glioblastoma cancer stem cells, by reducing G2 checkpoint activation to effectively abrogate enhanced DSB repair proficiency, following X‐ray (sensitiser enhancement ratios = 2.6–3.5; Carruthers et al., 2015). Moreover, the ATR inhibitor, VX‐970 preferentially blocks ATR‐Ckh1‐CDC25A signalling, abrogating the G2 checkpoint, and delaying DSB resolution after irradiation in triple‐negative breast cancer (TNBC) cells, when compared with normal breast epithelial cells (Tu et al., 2018). These observations suggest VX‐970 is a tumour‐specific radio‐sensitiser for TNBC. Other ATR inhibitors including, AZD6738 (Vendetti et al., 2018), VE822 (Fokas et al., 2012), and M6620 (Tu et al., 2018) have also exhibited radio‐sensitising effects in tumour cells. In addition, VE‐821, a novel and specific ATR inhibitor, was reported to enhance sensitivity to low‐LET radiation in pancreatic cancer cells (Prevo et al., 2012), in agreement with reports in human promyelocytic leukaemic HL‐60 cells (p53‐negative; Salovska et al., 2014). Other studies have demonstrated that VE‐821, in combination with carbon ions, provides effective tumour controls by inducing the formation of multiple micronuclei, and abrogating G2/M cell‐cycle arrest (Fujisawa et al., 2015). Taken together, ATR inhibition is promising in improving radio‐sensitivity, no matter what level of LET radiation is adminis- tered (high or low). 4.2.2 | Chk1 inhibitors Chk1 is an evolutionarily conserved serine/threonine kinase that serves an important role at G2 checkpoints. Several Chk1 inhibitors have been developed and applied in combination with radiation for cancer therapeutics. UCN‐01, one of the first generation Chk1 inhibitors, was shown to radio‐sensitise p53‐deficient tumour cells by abrogating G2 blockage (Bridges et al., 2016). However, radio‐ sensitising effects and UCN‐01 cytotoxicity were lower than other Chk1 inhibitors, such as MK‐8776 (Suzuki, Yamamori, Bo, Sakai, & Inanami, 2017). Moreover, the potent Chk1 inhibitor SAR‐020106, sensitises human glioblastoma cells to X‐rays, via inhibition of the p53‐independent G2 checkpoint (Patties et al., 2019). CCT244747, an orally bioavailable Chk1 inhibitor also enhances the radio‐ sensitivity of bladder, head, and neck cancer cell lines, by abrogating IR‐induced G2 block, and prematurely entering mitosis (Patel et al., 2017). Furthermore, MK‐8776, a novel selective Chk1 kinase inhibitor, radio‐sensitises p53‐defective human tumour cells by me- chanisms that include abrogation of the G2 phase block, and suppression of DSB repair in a p53‐dependent manner (Bridges et al., 2016). However, Suzuki et al. (2017) suggested that MK‐8776 exerts a radio‐sensitising effect through the enhancement of aberrant mitosis and mitotic catastrophe rather than affect DNA repair activity. Notably, MK‐8776 abrogated the IR‐induced G2 checkpoints but to a smaller degree compared to the wee1 inhibitor, MK‐1775 (Bridges et al., 2016). 4.2.3 | Wee1 kinase inhibitors The Chk1 could activate Wee1 kinase to block G2/M transition and prevent cells with damaged DNA from entering mitosis (Qiu et al., 2018). Studies have shown that Wee1 kinase inhibition abrogates IR‐induced G2 arrest, and causes irradiated cancer cells harbouring unrepaired DNA lesions, to prematurely pass into mitosis, resulting in DSB accumulation in M‐phase cells, and mitotic catastrophe (Ma et al., 2015; Patel et al., 2019). These data indicate that Wee1 inhibition is an alternative strategy for the radio‐sensitisation of tu- mour cells. The potent Wee1 inhibitor, MK‐1775 (AZD1775), improved radio‐sensitivity of NSCLC cells to X‐rays and carbon ions, by abrogating Wee1 phospho‐Cdk1‐mediated G2 checkpoint acti- vation, postirradiation (Ma et al., 2015). Similar results using photons were also observed in human cervical cancer cell lines (Lee et al., 2019), and glioblastoma cell lines (Sarcar et al., 2011). Notably, MK1775 was reported to induce unscheduled replication initiation and DNA lesions in S phase, while p21 protected cancer cells against these effects. Studies have shown that loss of p21, sensitised tumour cells to combined treatments of MK1775 and X‐rays (Hauge, Macurek, & Syljuasen, 2019). As p21 expression can be epigenetically downregulated in human cancer, p21 levels may be another therapeutic consideration when implementing Wee1 inhibition in the cancer treatment. 4.2.4 | PI3K/AKT/mTOR inhibitors Targeting the PI3K/AKT/mTOR signalling pathway represents a promising therapeutic strategy for cancer therapy. Recent studies have proposed that dual PI3K/mTOR inhibition sensitises radio- resistant oral squamous cell carcinoma (OSCC) cells to IR, by sup- pressing the AKT/mTOR axis, and promoting G1 phase arrest by downregulating cyclin D1 and CDK4 activity (Yu et al., 2017). In HeLa cells, the insulin‐like growth factor I receptor (IGF‐IR) is putatively implicated in the radiation‐induced G2/M checkpoint via the PI3K/ AKT pathway, without affecting DSB repair activities, in part by controlling Chk1 localisation between the nucleus and cytoplasm. An IGF‐IR inhibitor (NVP‐AEW541) caused delayed release from radiation‐induced G2 arrest (Manila et al., 2018). The mTORC1‐ specific inhibitor, RAD001, increased the sensitivity of SCC4 (human tongue squamous cell carcinoma) cells to X‐rays, by suppressing mTOR signalling and inducing G2/M cell‐cycle arrest through the Chk1/CDC25C/CDC2‐cyclinB1 pathway (Sunada et al., 2018). However, mTORC1‐targeting therapies may have limited effectiveness in antitumour activities due to increased AKT signalling feed- back activation. The dual mTORC1/2 inhibitor, AZD2014, may be a better radiosensitizer than RAD001. Studies have shown that AZD2014 radio‐sensitises OSCC cells by inhibiting the AKT/mTOR axis, and inducing cell‐cycle arrest at G1 and G2/M phases, by disrupting cyclin D1‐CDK4 and cyclin B1‐CDC2 complex activation (Yu et al., 2016). Taken together, DDR modulation is an attractive ther- apeutic strategy for the treatment of radioresistant solid tumours. It is not surprising that radio‐sensitisers exhibit differential radio‐sensitising effects to low/high‐LET radiation since different DNA repair pathway is activated in response to the types of DNA damage induced by low/high‐LET radiation. For example, gemcitabine increased the radio‐sensitivity of human multiple myeloma cells to low‐LET γ‐rays, but not high‐LET particles (Lee & Okayasu, 2018). Equally, radio‐sensitisation was found in human colon ade- nocarcinoma cell line WiDr for the combination of antimetabolite 2‐deoxyglucose and synchronous photon irradiation. A similar setup for carbon‐ion irradiation showed no radio‐sensitisation, but complemented with each other for additive effects (Hasse et al., 2019). In addition, studies have shown that in human OSCC cells, 17‐AAG has synergistic effects with X‐rays on cell lethality, but not with carbon‐ion beams (Musha et al., 2012). In summary, there is an urgent need to develop strategies to improve the efficacy of high‐ LET radiation therapy. 5 | CONCLUSIONS AND OUTLOOK Currently, RT is widely used for clinical cancer therapy. When com- pared with photon‐induced individual DNA lesions, which are re- paired preferentially by the NHEJ pathway, charged‐particle radiation causes clustered DNA damage, that are preferentially re- paired by HR, or the alternative NHEJ pathway. Moreover, repair of DNA lesions induced by IR is correlated with the radio‐resistance of tumours, to some extent. Although various radio‐sensitising agents have been investigated, some of them cannot be applied to the all kind of radiation quality. Hence, it is important to explore more efficient approaches in improving the efficacy of both low‐ and high‐LET radiation. Two radiation types may interact with each other. Accumulating evidence indicates that exposure to mixed radiation beams causes higher levels of complex chromosomal aberrations, when compared with the simple addition of single mixed‐beam components (Chenget al., 2018; Staaf et al., 2013). It was reported that high‐LET IR (α particles) and low‐LET IR (X‐rays) interact to induce more DNA damage and less DNA repair than expected from an additive action (Cheng et al., 2018; Sollazzo et al., 2017). 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