*2.4.2 RAD51*

RAD51 is another key protein in the HR pathway that is also associated with prognosis and treatment response in various cancers. A growing number of studies demonstrate that RAD51 protein is overexpressed in many cancers including breast, prostate, bladder, pancreas, and lung, and this overexpression can up-regulate HR activity and result in resistance to DNA-damaging drugs [88–91]. Increased expression of RAD51 has also been reported in sarcoma patients [92, 93]. Du et al. conducted a study to explore the relationship between RAD51 expression and resistance to radio- or chemotherapy in osteosarcoma [93]. They suppressed the expression of RAD51 using shRNA and found increased sensitivity to chemotherapy and radiation in osteosarcoma cell lines through induction of cell cycle arrest and apoptosis [93]. Hannay et al., investigated the association between RAD51 expression and resistance to chemotherapy in STS patients [92]. They evaluated the RAD51 expression in 62 human primary recurrent and metastatic STS samples [92]. Only 3 tumor samples showed no RAD51 expression, while most of them had overexpressed RAD51 expression levels [92]. They showed that siRNA-mediated RAD51 targeting resulted in STS sensitivity to doxorubicin [92]. Overall, these studies highlighted the significance of RAD51 in chemoresistance and suggested that RAD51 could be considered as a prognostic factor or even a therapeutic target for treatment of sarcoma patients.

### **2.5 Non-homologous end joining (NHEJ) pathway**

The NHEJ pathway is another important pathway responsible for repairing DSBs [94]. Unlike HR, NHEJ directly re-ligates two broken DNA strands without requiring a homologous sequence as an undamaged template, which makes this pathway prone to making errors [94]. NHEJ is initiated by binding of KU70/80 proteins, followed by recruitment of other key factors such as DNA-dependent protein kinases (DNA-PKcs), XRCC4, XLF, LIG4, and PAXX (a newly identified NHEJ component) [95] to complete the repair process [96]. Loss of the key factors involved in this pathway is positively correlated with increased genomic instability and sensitivity to DNA damaging chemotherapy drugs [94]. However, the over-activation of NHEJ has also been reported to be associated with increased genomic instability and tumorigenesis due to error-prone and inappropriate repair [94]. Thereby, both loss and over-activation of NHEJ factors have found to be associated with increased cancer incidence [94]. Moreover, a large number of studies have shown that differential expression of key NHEJ factors has significant impacts on the treatment response and overall prognosis in different types of cancers [94, 97–101].

Several studies have shown the significance of NHEJ components in sarcomas. For example, in a study on Ewing sarcoma patients, Kyriazoglou et al., has reported that NHEJ and HR genes are significantly up-regulated in comparison with healthy blood donors [102]. They analyzed the expression levels of 15 genes in 32 cases of Ewing sarcoma using Real-time PCR. XRCC5, XRCC6, Polm, LIG4 from the NHEJ pathway and RAD51, RAD52, RAD54, BRCA2, and FRANCD from the HR pathways have found to be significantly up-regulated in Ewing sarcoma patients [102]. In another study, Ma et al. investigated the role of PAXX protein in chemoresistance in osteosarcoma [96]. They found a significant positive correlation between enhanced PAXX-KU70 interaction and NHEJ efficiency and resistance to doxorubicin and cisplatin [96]. They also showed that PAXX deficiency re-sensitizes osteosarcoma cells to the chemotherapy drugs, which provides evidence that PAXX protein could be considered as a target for treatment of chemoresistant osteosarcoma patients [96]. Additionally study conducted by Hu et al., demonstrated the significance of KU80 expression in radiosensitivity of osteosarcoma cells [103]. They have shown that shRNA-mediated suppression of KU80 protein sensitized U2OS osteosarcoma cells to radiation through shortening of telomere length [103]. Taken together, key NHEJ factors have important roles in cancer progression, drug resistance, and patient's prognosis [94], which makes them interesting for further research regarding their prognostic and therapeutic potential in sarcomas. However, targeting NHEJ remains challenging as little is known about the inhibitors. Several PI3K inhibitors such as wortmannin and LY94002 (**Figure 1**) are being used for therapeutic intervention of DNA-PKcs in the NHEJ pathway [104].

### **2.6 ATR/CHK1 DNA damage sensors in DNA repair pathways**

## *2.6.1 ATR*

Ataxia-telangiectasia and Rad3-related protein (ATR) is one of the most upstream DDR kinases and belongs to the phosphatidylinositol 3-kinase-related kinase (PIKK) protein family [105]. ATR is a serine/threonine-protein kinase activated in response to a broad spectrum of DNA damage, including DSBs and various DNA lesions that interfere with replication [106]. In response to DNA damage, several proteins are phosphorylated at Ser/Thr-Glu motifs and additional sites in response to DNA damage by ATR [107]. ATR phosphorylates its major downstream effector checkpoint kinase 1 (CHK1) and prevents the entry of cells with damaged

**153**

patients.

*2.6.2 CHK1*

*DNA Repair Defects in Sarcomas*

*DOI: http://dx.doi.org/10.5772/intechopen.94881*

preclinical and clinical studies [112].

or incompletely replicated DNA into mitosis from the G2 phase of the cell cycle [108]. This regulation is particularly apparent in cells with a defective G1 checkpoint, a common cancer cell feature because of p53 mutations [109, 110]. ATR also suppresses replication stress (RS) by inhibition of extra origin firing, particularly in cells with activated oncogenes [111]. Therefore, ATR could be an ideal therapeutic target in cancer. Currently, ATR inhibitors have been developed and are used either as single agents or in combination with radiotherapy or chemotherapy in both

Several preclinical studies demonstrate that ATR could be a therapeutic target in sarcomas [113–117]. Laroche-clary et al., designed a study to investigate the antitumor effects of ATR inhibition in STS [113]. They treated STS cell lines with ATR inhibitor VE-822 either as a single agent or in combination with gemcitabine as a chemotherapeutic drug [113]. The data demonstrated significant synergist effects between these two drugs [113]. They found considerable cell growth suppression, apoptosis induction, and increased γH2AX expression after combined treatment of STS cells with VE-822 and gemcitabine in a higher efficacy than either agent alone [113]. Furthermore, they performed *in vivo* experiments on a patient-derived xenografts (PDXs) of undifferentiated pleomorphic sarcoma and found significant tumor growth suppression and increased PFS after treatment with combination of VE-822 and gemcitabine [113]. Taken together, this study highlighted the importance of ATR in STS and showed that ATR inhibition in combination with chemotherapy is efficacious in pre-clinical models. In another study, a series of parallel high-throughput siRNA screens were performed by Jones et al., in synovial sarcoma tumor cells and the results were compared with more than 130 non-synovial sarcoma tumor cells to get better insights into genetic dependencies and potential therapeutic targets in synovial sarcoma [114]. The analysis revealed a significant reliance of synovial sarcoma tumor cells on ATR protein kinase activity [114]. Furthermore, they showed that ATR inhibition will result in significant anti-tumor effects in synovial sarcoma *in vitro* and *in vivo* [114]. They also performed combination treatments with cisplatin and PARP inhibitors and found higher tumorsuppressive effects than either agent alone [114]. In summary, this study presented ATR protein alteration as a key factor in synovial sarcoma progression and proposed a novel therapeutic potential for synovial sarcoma patients [114]. The role of ATR protein was also demonstrated in Ewing sarcoma [115]. Nieto-Soler et al., designed a study to investigate the importance of ATR pathway in Ewing sarcoma [115]. They showed that Ewing sarcoma tumors that have high levels of RS are significantly dependent on ATR pathway [115]. Furthermore, they treated Ewing sarcoma cell lines and mice models with two independent ATR inhibitors and found considerable anti-tumor effects both *in vitro* and *in vivo* [115]. Collectively, this study highlighted the dependency of Ewing sarcoma to ATR pathway and identified ATR inhibition as a promising therapeutic strategy in Ewing sarcoma with high levels of RS [115]. Future preclinical studies and subsequent clinical trials will provide with additional reliable data on the effectiveness of ATR inhibition in sarcoma to translate this therapeutic approach into clinic as a possible treatment approach for sarcoma

As mentioned above, checkpoint kinase 1 (CHK1) is the major downstream effector of ATR [108]. CHK1, a serine/threonine-specific protein kinase, plays an essential role in preventing cell cycle progression when damaged DNA is being repaired [118, 119]. DNA damage is sensed by ATR, activated ATR phosphorylates and activates CHK1. CHK1 has several targets which all act to regulate cell cycle

#### *DNA Repair Defects in Sarcomas DOI: http://dx.doi.org/10.5772/intechopen.94881*

*DNA - Damages and Repair Mechanisms*

**2.5 Non-homologous end joining (NHEJ) pathway**

and overall prognosis in different types of cancers [94, 97–101].

The NHEJ pathway is another important pathway responsible for repairing DSBs [94]. Unlike HR, NHEJ directly re-ligates two broken DNA strands without requiring a homologous sequence as an undamaged template, which makes this pathway prone to making errors [94]. NHEJ is initiated by binding of KU70/80 proteins, followed by recruitment of other key factors such as DNA-dependent protein kinases (DNA-PKcs), XRCC4, XLF, LIG4, and PAXX (a newly identified NHEJ component) [95] to complete the repair process [96]. Loss of the key factors involved in this pathway is positively correlated with increased genomic instability and sensitivity to DNA damaging chemotherapy drugs [94]. However, the over-activation of NHEJ has also been reported to be associated with increased genomic instability and tumorigenesis due to error-prone and inappropriate repair [94]. Thereby, both loss and over-activation of NHEJ factors have found to be associated with increased cancer incidence [94]. Moreover, a large number of studies have shown that differential expression of key NHEJ factors has significant impacts on the treatment response

Several studies have shown the significance of NHEJ components in sarcomas. For example, in a study on Ewing sarcoma patients, Kyriazoglou et al., has reported that NHEJ and HR genes are significantly up-regulated in comparison with healthy blood donors [102]. They analyzed the expression levels of 15 genes in 32 cases of Ewing sarcoma using Real-time PCR. XRCC5, XRCC6, Polm, LIG4 from the NHEJ pathway and RAD51, RAD52, RAD54, BRCA2, and FRANCD from the HR pathways have found to be significantly up-regulated in Ewing sarcoma patients [102]. In another study, Ma et al. investigated the role of PAXX protein in chemoresistance in osteosarcoma [96]. They found a significant positive correlation between enhanced PAXX-KU70 interaction and NHEJ efficiency and resistance to doxorubicin and cisplatin [96]. They also showed that PAXX deficiency re-sensitizes osteosarcoma cells to the chemotherapy drugs, which provides evidence that PAXX protein could be considered as a target for treatment of chemoresistant osteosarcoma patients [96]. Additionally study conducted by Hu et al., demonstrated the significance of KU80 expression in radiosensitivity of osteosarcoma cells [103]. They have shown that shRNA-mediated suppression of KU80 protein sensitized U2OS osteosarcoma cells to radiation through shortening of telomere length [103]. Taken together, key NHEJ factors have important roles in cancer progression, drug resistance, and patient's prognosis [94], which makes them interesting for further research regarding their prognostic and therapeutic potential in sarcomas. However, targeting NHEJ remains challenging as little is known about the inhibitors. Several PI3K inhibitors such as wortmannin and LY94002 (**Figure 1**) are being

used for therapeutic intervention of DNA-PKcs in the NHEJ pathway [104].

Ataxia-telangiectasia and Rad3-related protein (ATR) is one of the most upstream DDR kinases and belongs to the phosphatidylinositol 3-kinase-related kinase (PIKK) protein family [105]. ATR is a serine/threonine-protein kinase activated in response to a broad spectrum of DNA damage, including DSBs and various DNA lesions that interfere with replication [106]. In response to DNA damage, several proteins are phosphorylated at Ser/Thr-Glu motifs and additional sites in response to DNA damage by ATR [107]. ATR phosphorylates its major downstream effector checkpoint kinase 1 (CHK1) and prevents the entry of cells with damaged

**2.6 ATR/CHK1 DNA damage sensors in DNA repair pathways**

**152**

*2.6.1 ATR*

or incompletely replicated DNA into mitosis from the G2 phase of the cell cycle [108]. This regulation is particularly apparent in cells with a defective G1 checkpoint, a common cancer cell feature because of p53 mutations [109, 110]. ATR also suppresses replication stress (RS) by inhibition of extra origin firing, particularly in cells with activated oncogenes [111]. Therefore, ATR could be an ideal therapeutic target in cancer. Currently, ATR inhibitors have been developed and are used either as single agents or in combination with radiotherapy or chemotherapy in both preclinical and clinical studies [112].

Several preclinical studies demonstrate that ATR could be a therapeutic target in sarcomas [113–117]. Laroche-clary et al., designed a study to investigate the antitumor effects of ATR inhibition in STS [113]. They treated STS cell lines with ATR inhibitor VE-822 either as a single agent or in combination with gemcitabine as a chemotherapeutic drug [113]. The data demonstrated significant synergist effects between these two drugs [113]. They found considerable cell growth suppression, apoptosis induction, and increased γH2AX expression after combined treatment of STS cells with VE-822 and gemcitabine in a higher efficacy than either agent alone [113]. Furthermore, they performed *in vivo* experiments on a patient-derived xenografts (PDXs) of undifferentiated pleomorphic sarcoma and found significant tumor growth suppression and increased PFS after treatment with combination of VE-822 and gemcitabine [113]. Taken together, this study highlighted the importance of ATR in STS and showed that ATR inhibition in combination with chemotherapy is efficacious in pre-clinical models. In another study, a series of parallel high-throughput siRNA screens were performed by Jones et al., in synovial sarcoma tumor cells and the results were compared with more than 130 non-synovial sarcoma tumor cells to get better insights into genetic dependencies and potential therapeutic targets in synovial sarcoma [114]. The analysis revealed a significant reliance of synovial sarcoma tumor cells on ATR protein kinase activity [114]. Furthermore, they showed that ATR inhibition will result in significant anti-tumor effects in synovial sarcoma *in vitro* and *in vivo* [114]. They also performed combination treatments with cisplatin and PARP inhibitors and found higher tumorsuppressive effects than either agent alone [114]. In summary, this study presented ATR protein alteration as a key factor in synovial sarcoma progression and proposed a novel therapeutic potential for synovial sarcoma patients [114]. The role of ATR protein was also demonstrated in Ewing sarcoma [115]. Nieto-Soler et al., designed a study to investigate the importance of ATR pathway in Ewing sarcoma [115]. They showed that Ewing sarcoma tumors that have high levels of RS are significantly dependent on ATR pathway [115]. Furthermore, they treated Ewing sarcoma cell lines and mice models with two independent ATR inhibitors and found considerable anti-tumor effects both *in vitro* and *in vivo* [115]. Collectively, this study highlighted the dependency of Ewing sarcoma to ATR pathway and identified ATR inhibition as a promising therapeutic strategy in Ewing sarcoma with high levels of RS [115]. Future preclinical studies and subsequent clinical trials will provide with additional reliable data on the effectiveness of ATR inhibition in sarcoma to translate this therapeutic approach into clinic as a possible treatment approach for sarcoma patients.

## *2.6.2 CHK1*

As mentioned above, checkpoint kinase 1 (CHK1) is the major downstream effector of ATR [108]. CHK1, a serine/threonine-specific protein kinase, plays an essential role in preventing cell cycle progression when damaged DNA is being repaired [118, 119]. DNA damage is sensed by ATR, activated ATR phosphorylates and activates CHK1. CHK1 has several targets which all act to regulate cell cycle

arrest [118]. Phosphorylation of the CDC25 dual specificity phosphatase family mediated by CHK1 causes phosphatase degradation, resulting in increased phosphorylation and inhibition of multiple cyclin dependent kinase (CDK) proteins, positive regulators of the cell cycle [119]. In addition to CDC25 phosphatases, WEE1 kinase is phosphorylated and activated by CHK1, subsequently leading to the inhibitory phosphorylation of CDK1 [120]. It is therefore logical that inhibitors of CHK1 in cancer treatment could facilitate cell cycle progression with damaged DNA and induce apoptosis [118].

Several preclinical studies and a few clinical studies demonstrate that CHK1 could be a therapeutic target in sarcoma treatment [115, 116, 121–124]. Larocheclary et al., conducted a study to investigate the role of CHK1 protein kinase in p53 mutant and wild-type STS [122]. They performed a systematic screening of a panel of 10 STS cell lines after combination treatment of CHK1 inhibitor (GDC-0575) with gemcitabine [122]. They showed that GDC-0575 induced apoptosis by abrogating DNA damage-induced S and G2–M checkpoints [122]. Moreover, they observed a synergistic or additive effect of GDC-0575 in combination with gemcitabine *in vitro* and *in vivo* in TP53-proficient but not in TP53-deficient sarcoma models [122]. Before conducting the mentioned study, they had analyzed the expression profile of a series of 339 complex genomics sarcomas and 108 translocation-related sarcomas, they showed that CHK1 expression is significantly associated with poor prognosis in sarcoma patients [125]. Moreover, they evaluated the efficacy of CHK1 inhibition in STS patients in a phase 1 clinical study with 3 STS patients (two with p53 mutation and one without p53 mutation) [122]. Two STS patients who had p53 mutation demonstrated promising response to the combination of gemcitabine and GDC-0575, while the other patient displayed no clinical benefit [122]. In conclusion, they provided pre-clinical and clinical evidence of the significance of CHK1 activity in STS and revealed that combination of CHK1 inhibitors with chemotherapy could be a promising treatment strategy for p53-mutant STS patients [122, 125]. There are also numbers of studies which have highlighted the important role of CHK1 activity in osteosarcoma progression and drug resistance and showed that CHK1 inhibitors either as a single agent or in combination with other drugs could be considered as a promising therapeutic target for treatment of osteosarcoma patients [126–129]. Regarding the role of CHK1 in Ewing sarcoma progression, some studies demonstrated that CHK1 protein is over-activated in Ewing sarcoma and showed that Ewing sarcoma cells are sensitive to CHK1 inhibitors either as a single agent or in combination with other drugs *in vivo* and *in vitro* [116, 121, 130, 131]. Further clinical investigations are needed to confirm whether treatment of sarcoma with CHK1 inhibition is efficacious therapeutic approach to improve sarcoma patient outcomes at a higher level of evidence.
