SMALL MOLECULE INHIBITORS OF DNA-PK
FOR TUMOR SENSITIZATION TO ANTICANCER THERAPY

In the cell there exist many types of DNA lesions. The double-strand breaks (DSBs) are probably the most deleterious form of DNA damage because they are more difficult to repair than other types of lesions. They can be caused by exogenous sources (ionizing radiation and various chemical agents) but also by endogenous events (cellular metabolic processes, mainly producing reactive oxygen species, such as cellular respiration, inflammation, hypoxia-reperfusion) (1-4). When the lesion is not correctly repaired, it may result in genomic instability and cell death, thus greatly affecting the organism's development and predisposing it to cancer and other disorders. It is necessary for mammalian cells to have a repair system that is able to identify and efficiently respond to DNA insults (1, 2). Cells have therefore developed a network of cellular mechanisms called the DNA damage response (DDR). DDR includes a set of damage tolerance processes, cell-cycle checkpoints and DNA repair pathways which allow for continuous monitoring of DNA integrity and function, thus preventing the appearance of deleterious mutations and rearrangements (5, 6).

Cancer cells have frequently compromised some components of DDR pathways. The use of essential pathway inhibitors could induce synthetic lethality; normal cells would not be affected. Another promising approach to the use of DNA-repair inhibitors in treatment is the overcoming of the resistance to DNA-damaging therapeutics. In both approaches, individual tumor testing of the efficiency of DSB repair pathways would undoubtedly be of great benefit.

In eukaryotes there are two main complementary mechanisms for DSB reparation, non-homologous end-joining (NHEJ) and homologous recombination (HR) (7). While HR utilizes homologous sequences from an undamaged chromatid to effective reparation, NHEJ has no apparent requirement for extensive sequence homology (8).

The presence of a DNA lesion is signalled by the major regulators of the DDR, the phosphatidylinositol-3 kinase-related kinases (PIKKs), a family of Ser/Thr-protein kinases that are structurally different from classical protein kinases (9, 10). PIKKs are also referred to as class IV of PI3Ks (phosphatidylinositol-3 kinases). PIKKs and PI3Ks are functionally distinct but have a related origin, and PIKKs contain a kinase domain with motifs typical for the PI3K family (10). The PIKK family includes six members that have varied biological functions. Of these, ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) play a prominent role in DNA damage repair and checkpoint response (9). DNA-PKcs and ATM answer mainly to DNA double-strand breaks, while ATR is triggered by single-strand breaks and DNA replication blocks (11).

ATAXIA-TELANGIECTASIA MUTATED AND
ATAXIA-TELANGIECTASIA AND RAD3-RELATED

ATM and ATR are large kinases that share many functional and biochemical similarities and have a strong preference for phosphorylation of serine or threonine residues whichare followed by glutamine (12). The name of ATM is derived from human autosomal recessive neurodegenerative disease, Ataxia telangiectasia, where the gene which encodes this protein kinase is mutated, resulting in neurodegeneration, immune dysfunction, radiosensitivity and cancer predisposition (13).

ATM has a critical role in cancer suppression and DNA double-strand break repair. Initial studies showed that ATM is in inactive form in unharmed cells, but DNA damage or treatment with chemical agents causes an increase in its cellular activity. The inactive dimer is activated by auto-phosphorylation on serine 1981 resulting in disassociation of the dimer into active monomers. It was reported that the histone acetyl-transferase, TIP60, is important for ATM activation (14).

Another protein kinase acting in concordance with ATM is ATR. It has been found that ATR is responsible for the phosphorylation of certain ATM substrates. In DSB response, ATM is essential for the immediate, rapid phase, whereas the main role of ATR occurs in the later stages of response. ATR also responds to stalled replication forks and hypoxia. Moreover, it was shown (15) to preferentially bind to UV-light-damaged DNA. Another important feature of ATR is its requirement of an accessory protein, ATR-interacting protein (ATRIP). ATRIP is one of the immediate substrates once ATR has been activated, and they depend on each other for stability (16).

DNA-DEPENDENT PROTEIN KINASE

The focus of this review is, however, on the next member of the phosphatidylinositol-3-kinase superfamily. This kinase is abundantly expressed in almost all mammalian cells. DNA-PK consists of a large catalytic subunit, DNA-PKcs, and a heterodimeric protein Ku. DNA-PKcs is one of the largest (465 kDa) kinases identified to date and it is a kinase that is absolutely dependent on DNA binding to be active (17). Ku protein forms a ring structure that completely encircles the DNA. Its name is derived from the surname of the autoimmune disorder patient in whom the complex was discovered as an autoantigen. The Ku heterodimer is composed of the Ku70 (73 kDa) and Ku80 (86 kDa) subunits. It has a high affinity for the ends of double-stranded DNA. Cells that lack Ku or DNA-PKcs are radiosensitive and defective in DSB repair (18, 19).

DNA-PK has been implicated in a variety of processes, from the activation of innate immunity to the regulation of gene expression, but its primary role in cellular metabolism is to initiate NHEJ (19). In addition, recent findings by Kong et al. suggest DNA-PK may regulate the homeostasis of cell proliferation as well as having an important role in regulating the gene response to feeding/insulin stimulation. DNA-PK regulates fatty acid synthesis by modulating the protein expression of fatty acid synthase (2). There is also proof that DNA-PK influences or interacts with the p53 and p21 activities which lead to cellular senescence and apoptosis (20). These studies reveal that DNA-PK has more important roles than originally thought (2).

DNA-PK has a central function in the DSB response, where it is the main regulator of the NHEJ which phosphorylates itself and other DNA damage response and repair protein (20, 21). The enzymatic activity of DNA-PK is required for NHEJ because eliminating the kinase activity of DNA-PK makes cells highly sensitive to agents that induce DSBs (17). Conversely, cells characterized by resistance to DNA damaging agents show increased levels of DNA-PKcs (20). In DNA DSBs, broken ends are in most cases incompatible and their biochemical configuration can be various, so a simple ligation step cannot be performed (22).

DNA DAMAGE RESPONSE SYSTEM

Eukaryotic cells have developed complex mechanisms that are activated after exposure to DNA-damaging agents and other cellular stresses. One of the initiating events in cellular response to DSBs in chromatin is rapid phosphorylation of histone H2AX on serine 139 to generate γH2AX in the vicinity of DSB. DNA-PKcs, ATM and ATR are able to catalyze this process. Further DSB processing is associated with a characteristic modification and relocalization of the DDR proteins to subnuclear structures called 'foci'. γH2AX was shown to attract a number of proteins that are associated with DNA damage response, including Nbs1, 53BP1, Mdc1, Tip60 and others. The formation of γH2AX foci is now generally accepted as a quantitative marker of DSBs and can be conveniently detected by immunofluorescence microscopy (23).

DSBs are repaired in eukaryotes by mechanisms based on HR and NHEJ. As DNA-PK is the main regulator of NHEJ, let us take a moment to consider this process. The mechanism of NHEJ is rather uncomplicated and predominates in the G0 and G1 phase (18). It is a process where DNA ends are directly joined together without a specific request for sequence homology and is error-prone, in contrast to HR (24). Proteins necessary in the process include Ku70 and Ku86 (also called Ku80), Artemis, XRCC4 (the X-ray cross complementing protein 4), XLF (the XRCC4-like factor) and DNA ligase IV (17).

In the first step the protein Ku70/Ku86 comes in contact with DNA ends at DSB sites. Then DNA-PKcs is recruited to the DSB through its interaction with Ku and DNA (17). This binding is required for the activation of serine/threonine kinase activity. Autophosphorylation of DNA-PKcs leads to a dissociation of the DNA – Ku – DNA-PKcs complex. Endonuclease Artemis may be involved at this stage. Artemis interacts with DNA-PKcs and this complex is responsible for resecting DNA termini (removal of 5´ and trimming of long 3´ overhangs) (25). Artemis is activated by ATM and allows DNA double-strand ends to be rejoined by non-homologous end-joining (24). At the final stage of the process, the ligatable nicks on each strand are ligated by the XRCC4-DNA-ligase IV complex (22). In both NHEJ and HR, the MRN complex plays a crucial role. It figures as a sensor of DSBs and thus the complex is involved in the initial processing. The complex contains the proteins Mre11, Rad50, and Nbs1 (24).

Mre11 plays a main role in DNA metabolism. The 3´– 5´ exonuclease and ssDNA endonuclease activities are enhanced when Mre11 is in the holocomplex. The Mre11 complex is important for the promotion of the DSB end resection. It underlies the initiation of checkpoint responses and is required for the initiation of HR (26).

Protein Ku is ideal for initiating diverse types of DSB end repairs because it can bind to a variety of double-stranded end structures, such as ends with 3´and 5´overhangs, hairpin ends and blunt ends (17). Although it does not possess enzymatic activity, it is indispensable in aligning and protecting DNA ends. Interestingly, this protein is also associated with telomeres. Ku protects its ends from being recognized as double strand breaks (27).

DNA-PK INHIBITORS

Chemotherapy and radiotherapy are often associated with severe side effects. To improve the response of these therapies, it would be favorable if the tumor cells could be sensitized to anticancer agents or radiation. Successful treatment is currently based on killing the rapidly dividing cells and is therefore more toxic to fast-growing cancer cells (28).

The ability of cancer cells to repair DNA lesions is an important element of their sensitivity to chemo- or radio-therapy (29). The capability of DNA-PK to recognize and initiate the repair of DNA damage can protect cancer cells from the cytotoxic effects of DNA-damaging cancer therapies (30). It has been observed that overactivation of DNA-PK, which can occur in cancers, results in increased DNA damage resistance. For example, the upregulation of DNA-PK activity in isolates of B-cell chronic lymphocytic leukemia disrupts DNA damage-induced apoptosis (31); or resistance to mitoxantrone in HL-60/MX2 cells is due to an increase in DNA-PK expression and could be reverted by DNA-PK inhibition (32). Elevated DNA-PKcs expression has also been reported in gastric, hepatocellular and ovarian cancer (33). On the other hand, human cell lines with a defective DNA-PK function (cells lacking Ku or the DNA-PKcs) are hypersensitive to agents that evoke DNA double-strand breaks. Importantly, inhibition of DNA-PK has been demonstrated to enhance the cytotoxicity of ionizing radiation (IR) and a number of DSB-inducing agents in vitro as discussed in detail in the following paragraphs. By blocking DNA DSB repair, selective DNA-PK inhibitors have the potential to serve as chemo- and radio- potentiators in cancer treatment (31, 34). Due to the high rate of proliferation and metabolic activity in cancer cells, DNA-PK´s active participation in the regulation of homeostasis of cell proliferation proposes additional mechanisms by which DNA-PK inhibition can target these cells (20).

The specific DNA-PK inhibitors currently known are limited by poor solubility and high metabolic lability in vivo, thus meaning a short serum half-life (20). Construction of new compounds based on existing drugs is the most important strategy to improve drug efficacy, pharmacokinetic parameters and to reduce toxicity (35). In the ideal case, chemo- or radio-sensitizer should have no intrinsic toxicity and preferentially sensitize tumor cells to chemical agents or radiation. Currently available inhibitors of DNA-PK have various degrees of selectivity and can affect also other enzymes of phosphatidylinositol-3 kinases superfamily (Fig. 1).

Fig. 1. Mode of action of DNA-PK inhibitors in the context of phosphatidylinositol-3 kinase-like kinases. DNA-dependent protein kinase (DNA-PK) catalytic subunit, ataxia-telangiectasia mutated (ATM), and ataxia-telangiectasia and Rad3-related (ATR) play a prominent role in DNA damage repair and cell cycle arrest. These kinases are structurally related to a broader family of phosphatidylinositol-3 kinases (PI3K), which canonical representatives are activated during growth factors (GF) signalling through receptor tyrosinekinases (RTK). Small molecule inhibitors of DNA-PK have various degrees of selectivity towards DNA-PK; their effect may combine inhibition of more representatives of PI3Ks or be exclusively attributed to inhibition of DNA-PKcs.

PI3K INHIBITORS WITH ACTIVITY AGAINST
PIKK, INCLUDING DNA-PK

Wortmannin and its derivatives PX-866 and PWT-458

One of the first identified inhibitors, wortmannin, was isolated in 1957. It was defined as a potent non-competitive irreversible inhibitor of PI3K in 1993, but it is also active against PIKK family members, including DNA-PK (35, 36). It is a furanosteroid metabolite of the fungi Penicillium funiculosum and has an IC50 in the nanomolar range (5 nM for PI3K and equipotent for DNA-PK) (20). The inhibitor causes a covalent modification of Lys802 and conformational reshuffle in the ATP binding site (35, 37). Pastwa et al. found that non-cytotoxic concentration of wortmannin enhanced the cytotoxic effect of etoposide three-fold, and approximately five-fold when combined with cisplatin in human glioblastoma cells MO59. Wortmannin increased DSB levels (in a concentration-dependent manner) and G2/M arrest as well as increasing the accumulation of cells in the S-phase. It also inhibited DSB repair (38). Wortmannin has also proven to be an effective radiosensitizer, although it has poor solubility in aqueous solutions, lacks specificity, and has exhibited toxicity in vivo. These properties limit the clinical application of this compound (20), and for this reason a series of semisynthetic analogues of wortmannin were prepared.

Ihle et al. (39) developed the compound PX-866, created by opening the furan ring at the C-20 position. PX-866 offered several improvements over wortmannin, such as increased stability and biological activity and reduced toxicity. PX-866 was shown to be active as a single drug against ovarian (OvCar-3), colon (HT29), and non-small cell lung cancer (A549) xenografts (39). Ihle et al. further discovered that PX-866 potentiates in vivo growth inhibition induced by ionizing radiation in OvCar-3 human tumor xenografts and also increased the antitumor activity of cisplatin in A549 non-small cell lung cancer (NSCLC) xenografts (35, 39). PX-866 had considerable efficacy in preclinical studies against head and neck squamous cell cancer (HNSCC) xenografts. An additive effect was found in vivo with docetaxel in the same mice model, as well as during Phase II trial in human patients (40).

PWT-458, pegylated-17-hydroxywortmannin with significant improvements over wormannin itself, was discovered by Yu et al. This drug was active against PTEN-negative U87MG glioma, NSCLC A549 and the VHL-negative renal cell carcinoma (RCC) A498 xenograft tumors grown in nude mice. It also enhanced the antitumor efficacy of paclitaxel in glioma and NSCLC A549 xenografts. In the NSCLC model, the combination of PWT-458 and paclitaxel attained a complete arrest of tumor growth, whereas either agent alone only reduced growth. Yu et al. also revealed the synergizing effect of PWT-458 and Intron-A in RCC A498, where an impressive tumor regression was achieved. Expansion of this tumor in mice is accompanied by extensive tumor blood vessel growth. A further observation made in this same study was that the combination therapy resulted in reduced tumor vasculature (41).

LY294002 and its prodrug SF1126

LY294002 is a morpholine derivate of the plant flavonoid quercetin. It is a non specific ATP-competitive inhibitor of PI3K and has also been shown to display comparable inhibitory activity against DNA-PK (20, 33). in vitro and in vivo studies found that this compound has a number of potential effects, such as cell growth inhibition, antiangiogenic activity, G1 phase cell cycle arrest and radiosensitivity induction. Interesting inhibitory effect of compounds LY294002 and wortmannin was tested by Sun et al. They studied mechanism of action of Rumex acetosa extract that evokes vasorelaxation in endothelial cells. When the inhibitors were used, vasorelaxation was attenuated. Thus this process is activated via PI3K/Akt pathway (42).

The inhibitor binds reversibly to the kinase domain of DNA-PK (10). Gupta et al. combined LY294002 with radiation in the human bladder cancer cell line T24. They found that LY294002 has a statistically significant synergistic effect leading to enhanced radiation cell killing (43). The chemosensitizing effect was also tested in combination with cisplatin in glioblastoma cells (44) and pancreatic cells, both in vitro and in vivo (45). Combination therapy showed a synergistic effect against human pancreatic xenografts; it markedly increased cleavage of caspase-3 and cytoplasmic histone-associated DNA fragments (45). Cisplatin with LY294002 caused a significant decrease in the pancreatic cell proliferation of U343 and U87 but apoptotic cell death was increased only in U343 cell lines (44). However, due to its undesirable properties, as in wortmannin, and the rapid metabolic clearance (1 h), its effects cannot be clinically assessed in humans. Nevertheless, LY294002 has proven to be a productive lead compound. Compounds with more favorable properties have been produced through biochemical modifications with LY294002 as a template (20).

To avoid the physicochemical disadvantages of LY294002, the prodrug SF1126 was created. Its purpose was to increase solubility and to target tumors through binding to specific integrins. It has demonstrated more a favorable antiangiogenic and antitumor efficiency in vivo without serious toxicity, thus making it a more suitable candidate for potential use in treatment. Indeed, it has now successfully finished phase I clinical trials (10). The efficacy of the drug was tested in patients with advanced solid tumors and B-cell malignancies. It showed promising preliminary antitumor effects both alone and in combination with rituximab in one patient with therapy resistant chronic lymphocytic leukemia. Furthermore, initial anti-lymphoma activity was observed in a patient with diffuse large B-cell lymphoma. The drug was well tolerated and the most common treatment emergent adverse events were gastrointestinal. Even though inhibition of PI3K is known to cause peripheral tissue insulin resistance by enhancing B-cell sensitivity to glucose and thus increasing insulin secretion as compensation, this effect is temporary and does not lead to any significant disturbance in glucose homeostasis. SF1126 was used in a patient with pancreas cancer and had no effect on normal skin epidermis compared to tumor tissue (46).

However, wortmannin and its derivatives PX-866 and PWT-458, as well as LY294002 and its prodrug SF1126, are nonselective PI3K inhibitors. It is therefore impossible to assign antitumor and sensitizing effects to the inhibition of DNA-PK only. The majority of the observed effects are probably related to the inhibition of survival and proliferation signals through the receptor-PI3K-Akt-(mTOR) pathway, another promising target of future anticancer therapy (47).

SELECTIVE DNA-PK INHIBITORS

NU7441 and NU7026

LY294002 was used as a starting point for the synthesis of the more potent and selective DNA-PK inhibitor NU7441 (2-N-morpholino-8-dibenzothiophenyl-chromen-4-one; KU-57788). This inhibitor showed excellent selectivity across the PIKKs and unrelated kinases. In cell lines it showed strong inhibition of DNA-PK and acted as a chemopotentiator in vitro and also in vivo (31). Doxorubicin and ionizing radiation (IR) induced DSBs persisted for a prolonged period of time in the presence of NU7441 (20). Zhao et al. reported preclinical assessment of this inhibitor using Chinese hamster ovary cells. The potentiation of IR and the etoposide effects were achieved as a result of DNA-PK inhibition. Furthermore, NU7441 increased the persistence of γH2AX foci after IR-induced or etoposide-induced DNA damage. It also prolonged G2/M arrest. Zhao et al. also examined the chemosensitization and radiosensitization in human colon cancer cells LoVo and SW620. They found that NU7441 at a low, non-cytotoxic concentration (1 µmol/l) significantly enhanced the cytotoxicity of IR, doxorubicin and etoposide in both cell lines. In SW620 cells, the compound caused a sizeable increase in doxorubicin-induced cytotoxicity of between two- to three-fold depending on the concentration of the cytostatic. NU7441 caused a marked potentiation of the IR effect in both cell lines. LD90 in SW620 decreased from 4 Gy to 1.1 Gy thanks to NU7441, and in LoVo from 3 Gy to 1 Gy. In the in vivo study, NU7441 enhanced the antitumor activity of etoposide in a human colon cancer SW620 xenograft model (48). Ciszewski et al. studied the sensitizing effect of NU7441 in combination with doxorubicin or ionizing radiation in the treatment of three human breast cancer cell lines (MCF-7, MDA-MB-231 and T47D). NU7441 significantly increased the sensitivity to doxorubicin three- to sixteen-fold, to IR four- to twelve-fold. NU7441 caused accumulation of cells in the G2/M phase of the cell cycle (49). In comparison with the ATM inhibitor KU-55933, NU7441 exhibited a greater degree of radiosensitization in breast carcinoma cells MCF7. Reduced clearance of γH2AX associated with increased sensitivity to IR was observed. There was no significant effect when these compounds were combined (50). Tichy et al. investigated the reaction to ionizing radiation in the human leukemic T-lymphocyte cell line MOLT-4 preincubated with NU7441. They found that combination therapy caused increased phosphorylation of H2AX as well as the induction of apoptosis. The difference in the percentage of apoptotic cells in the group treated with IR (48%) and IR combined with NU7441 (27%) was almost twofold (51). The same compound was tested in human cancer cells proficient (M059-Fus1) and deficient (M059 J) in DNA-PK. The inhibitor was combined with doxorubicin or IR and caused chemo- and radio-potentiation only in M059-Fus1. Doxorubicin- and IR-induced DNA DSBs was measured by gH2AX foci (52).

LY294002 was also used in the development of NU7026 (2-(morpholin-4-yl)-benzo[h]chromen-4-one). This compound is approximately six-fold more potent than LY294002 as a DNA-PK inhibitor, and at least seventy-fold more selective for DNA-PK than other PI3Ks (53). It also exhibits excellent selectivity, as its IC50 are 0.19 – 0.28 µM for DNA-PK, > 2.4 µM for phosphoinositide 3-kinase, and > 100 µM for ATM and ATR (33, 54). NU7026 increased the cytotoxicity of IR and topoisomerase II poisons (idarubicin, daunorubicin, doxorubicin, etoposide, amsacrine and mitoxantrone) using K562 leukemia cells (55) and HL-60 and HL-60/MX2 leukemia cells (32). Also, the radiosensitizing effect of NU7026 was observed in N87 gastric cancer cells coupled with increased levels of DNA damage and cell-cycle arrest in G2/M (56). Hisatomi et al. demonstrated the potentiating effect of NU7026 in combination with etoposide on M059K and ATL cells (57). Despite the promising in vitro studies, preclinical in vivo pharmacokinetic results showed that the drug is rapidly cleared from circulation (10).

IC86621 and IC87361

Other compounds based on the LY294002 structure have been tested, namely IC86621 (58) and IC87361 (59). IC86621, a selective DNA PK inhibitor, had no activity against the related protein kinases ATM and ATR even at concentrations of 100 µM. It also inhibited p110β subunits of phosphatidylinositol 3-kinase. IC86621 significantly enhanced the cytotoxicity of two DSB inducing agents - bleomycin and etoposide. Interestingly, it did not sensitize the cells to the effect of doxorubicin. The study also reported radiosensitizing properties of the compound independent of cell type (colorectal, ovarian, pancreatic, bronchoalveolar carcinoma, leukemia cells). All tested cancer cell lines exhibited an increase in radiosensitivity ranging from 1.5- to 4.2-fold (58). Shinohara et al. determined whether IC87361 is able to sensitize cancer cells to ionizing radiation in vitro. They treated the melanoma cell line B16F0 and lung cancer cells LCC with an inhibitor or IR 6 Gy alone and then both IC87361 and 6 Gy. The ionizing radiation had a moderate inhibition effect in both cell lines, but the inhibitor alone had no significant response. In contrast, combination treatment revealed significant reduction in the survival of LCC and B16F0 cells (59).

SU11752

Ismail et al. screened a three-substituted indolin-2-ones library and found SU11752, which behaved as a competitive inhibitor for the ATP-site in DNA-PK. In comparison with wortmannin, this compound was a more selective inhibitor of DNA-PK and did not affect ATM kinase or PI3-kinase activity. SU11752 showed an ability to impede DNA double-strand break repair in a human glioblastoma cell line following exposure to IR (five-fold) or in calicheamicin-induced cells. Nevertheless, SU11752 has a relatively weak binding to DNA-PK, therefore it is not likely to be suitable for clinical trials. On the other hand, Ismail et al. believe that the compound may serve as an excellent starting point for the development of new specific DNA-PK inhibitors which can be useful in combination with chemo- and radio-therapy (28, 37).

IC486241 (ICC)

Davidson et al. reported an ability of the novel specific DNA-PKcs inhibitor, IC486241 (ICC), to synergize the cytotoxicity of DNA damaging agents in diverse breast cancer cell lines - MCF7, BT-20 and MDA-MB-436. They observed that ICC significantly increased the cytotoxicity of doxorubicin and cisplatin and this synergy was found at nontoxic concentrations of ICC (1 – 2 µM). Treatment with doxorubicin caused phosphorylation of DNA-PKcs, with the response to DNA damage being reduced in the presence of ICC. Moreover, in contrast with doxorubicin alone, treatment of BT-20 cell line by doxorubicin in combination with ICC revealed a decrease in γH2AX. This is caused by the reversible blocking of the ATP binding pocket of DNA-PKcs by ICC, and thus autophosphorylation and phosphorylation of other DNA-PKcs substrates such as H2AX is prevented. Davidson et al. concluded that the increased cytotoxicity in the combination treatment was the result of decreased DNA-PKcs activity and increased damage of DNA resulting from inhibition of the NHEJ pathway (60). Whether the downregulation of gH2AX phosphorylation is solely an effect of DNA-PK inhibition or whether ICC has an additional inhibitory effect on ATM remains an open question, since no data on ICC selectivity have been published. In another study, Davidson et al. examined the efficacy of ICC in combination with DNA damaging chemotherapeutic agents in colon cancer cell lines HCT-116 and HT-29. When the inhibitor was added simultaneously with irinotecan, cisplatin and oxaliplatin, the IC50 of the platinum compounds were marginally reduced. However, when the ICC was combined with SN38 (the active metabolite of irinotecan), the cytotoxic effect was highly synergistic. The compound inhibited the autophosphorylation of DNA-PKcs and also slightly decreased γH2AX. Cell cycle analysis showed significant G2/M arrest after 24h exposure to SN38 and SN38 together with ICC (61).

COMPOUNDS WITH MIXED ACTIVITY,
INCLUDING DNA-PK INHIBITION

Caffeine

Block et al. showed efficient inhibition properties of caffeine towards DNA-PK activity in vitro. DNA-PK is inhibited by caffeine through a mixed non-competitive mechanism; it can bind to both DNA-PK and DNA-PK-ATP complexes but with unequal affinity. They found that while caffeine inhibits DNA-PK-dependent substrate phosphorylation, autophosphorylation of DNA-PK is only weakly inhibited in tissue culture cells. However, the compound is known to inhibit primarily ATM and ATR (62).

Vanillin

Vanillin is known as a naturally occurring food component and an acknowledged agent with anti-mutagenic effects. Durant and Karran reported that vanillin inhibits DNA repair by NHEJ and acts as a selective inhibitor of DNA-dependent protein kinase. Moreover, they found that vanillin does not significantly impair the ATM/ATR-dependent kinase activity required for Chk-2 phosphorylation (8). Vanillin possess anti-carcinogenic activity such as effective anti-mutagens (63), and has shown its anti-metastatic potential in vivo against 4T1 breast cancer cells (64), the colon cancer cell line HCT116 in vitro (65) and has exhibited more cytolytic and cytostatic effects on the colorectal cancer cell line HT-29 than on the normal human fibroblasts (63). Vanillin is able to sensitize the A2780 human ovarian carcinoma cell line and TK6 lymphoblasts and make them susceptible to the effects of cisplatin. A significant effect of vanillin on radiation sensitivity was not observed (8).

NK314

Hisatomi et al. presented NK314, a synthetic benzo[c]phenanthridine alkaloid, as an inhibitor with specificity for topoisomerase II alpha. However, they also report inhibition of DNA DSB repair mediated by inducing the degradation of DNA-PKcs. NK314 not only induces DNA damage by itself, but in some cell lines also prolongs repair of radiation induced DSBs and acts as a dual inhibitor of Top2a and DNA-PK. They investigated the effects of NK314 on DNA damage response with irradiation and it showed potent antitumor activity against adult T-cell leukemia-lymphoma. Inhibitory effect of cell growth by the compound was weak on normal peripheral blood mononuclear cells (PBMCs). NK314 induced dose-dependent G2/M arrest whereas PBMCs were kept in G0/G1 phase (57). NK314 is currently in clinical trials for the treatment of this type of leukemia (20).

CC-115

Mortensen et al. (66) described the preclinical characterization of CC-115. It is an orally active dual inhibitor of DNA-PK and mTOR (mammalian target of rapamycin) kinase (TORK). The compound showed antitumor activity and an ability to induce apoptosis in a panel of tumor cell lines. Furthermore, dose-dependent tumor growth inhibition was demonstrated in numerous solid tumor xenografts (66). Inhibition of DNA-PK caused NHEJ disruption, inhibition of substrates Ku80, XRCC4 and autophosphorylation of DNA-PK. Tsuji et al. also established HR pathway inhibition by indirectly inhibiting ATM (67). Raymon et al. tested antiproliferative activity in mice using U87MG xenografts. CC-115 displayed dose-dependent inhibition of tumor growth and the survival of mice with implanted glioblastoma was prolonged (68). The inhibitor is now in a phase 1 clinical trial for the treatment of advanced malignancies (NCT01353625).

CONCLUSION

The study of DNA-PK inhibition is a very promising target in anticancer research owing to the fact that inhibition of this kinase makes the cells more sensitive to ionizing radiation and chemotherapy, as summarised in Table 1. It is beyond any doubt that inhibition of DNA-PK will affect also non-malignant cells, but the evidence indicates that some types of tumour cells will be much more sensitive compared to normal tissues: This was observed in vitro, but more importantly in vivo during tests on animal cancer models (usually xenografts) or during clinical trials, where sensitisation to cisplatin, docetaxel, paclitaxel or etoposide was observed after treatment with DNA-PK inhibitors. Moreover, since DNA-PK strengthens chemotherapy and radiotherapy resistance, DNA-PK inhibition shows considerable promise in the fight against tumor resistance.

Table 1. Inhibitors used in combination therapy.

However, DNA-PK inhibitors are frequently limited by poor pharmacokinetics. There is great potential for studying and developing new compounds with improved properties and this is a key step for obtaining an effective anticancer drug.

Acknowledgements: This study was supported by grants of Charles University, GAUK project no. 932516, SVV-260397/2017 and Progres Q40.

Conflict of interests: None declared.

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