*2.1.1 APE1/Ref-1*

One of the most important components of BER pathway is APE1/Ref-1 (apurinic/apyrimidinic endonuclease 1/redox factor-1). APE1/Ref-1 is a multifunctional protein involved in response to oxidative stress, cell cycle regulation, transcriptional activation, protein stability, apoptosis, and cell survival [16, 20]. The different functions of this protein can be categorized into two main activities: apurinic/apyrimidinic endonuclease activity and reduction–oxidation (redox) activity. The endonuclease activity allows APE1/Ref-1 protein to function as a DDR component in BER pathway by recognizing and cleavage of the abasic site [21]. The redox activity of APE1/Ref-1 gives it a critical transcriptional regulatory role in which enhances the activity of numerous transcription factors, including STAT3, NF-kB, HIF-1, and AP-1 [21, 22]. APE1/Ref-1 has a crucial role in maintaining cancer cells in a survival state through its DNA repair properties [23, 24]. Also, its redox function increases the activity of signaling pathways that are involved in promoting growth, migration, and survival in tumor cells as well as inflammation and angiogenesis in the tumor microenvironment [23, 25]. The overexpression of APE1/Ref-1 has been reported in many tumor types, and that change is associated with drug resistance, metastasis, cancer aggressiveness, and overall poor prognosis [16].

Several studies have shown that APE1/Ref-1 protein is overexpressed in sarcoma patients and is correlated with metastasis and lower survival rates [26–30]. The correlation between angiogenesis, as an important factor in tumor growth and metastasis, and APE1/Ref-1 in osteosarcoma was elucidated by the series of studies conducted by Wang et al. [26, 31–33]. They showed that transforming growth factor beta (TGFβ) is directly regulated by APE1/Ref-1 and its expression level was significantly reduced in APE1/Ref-1 deficient osteosarcoma cells [31]. TGFβ increases the chances of cancer metastasis through multiple mechanisms including immunosuppression, invasion, and angiogenesis [34]. They demonstrated that knocking down APE1/Ref-1 using specific siRNA in osteosarcoma led to down-regulation of TGFβ expression and suppression of angiogenesis *in vitro* based on human umbilical vein endothelial cells (HUVECs) in transwell and matrigel tube formation assays [31]. In addition, siRNA-mediated silencing of APE1/Ref-1 significantly suppressed tumor growth in xenograft mice models [31]. These experimental data indicated that APE1/Ref-1 promotes angiogenesis in osteosarcoma through a TGFβ-dependent pathway [31]. Additionally, they showed that the expression levels of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) are also regulated by APE1/Ref-1 [32]. However, the suppression of angiogenesis in APE1/ Ref-1 knockdown cells is not dependent on their transcriptional activity [32, 33]. Wang et al., also used siRNA against APE1/Ref-1 protein to investigate its inhibition in osteosarcoma [26]. They demonstrated that the siRNA-mediated inhibition of APE1/Ref-1 sensitized the osteosarcoma cells to DNA damaging agents: methyl methanesulfonate, H2O2, ionizing radiation, and chemotherapeutic agents [26]. Another study conducted by Xiao et al., investigated the association of APE1/Ref-1 polymorphisms with osteosarcoma [35]. They performed a 2-stage case–control study in a total of 378 osteosarcoma patients and 616 normal controls and concluded that the patients who have certain APE1/Ref-1 polymorphisms have lower APE1/Ref-1 expression and higher survival rates [35]. Over the past few years, small molecule inhibitors targeting APE1/Ref-1 have been developed and showed remarkable anti-tumor effects with limited toxicity in a variety of cancers, in both *in vitro* and *in vivo* models [36–40]. However, the efficacy of these inhibitors still needs to be investigated in sarcomas.

#### *2.1.2 PARP*

Poly (ADP-ribose) polymerase (PARP) is another important DDR protein involved in cell proliferation, differentiation, and transformation [41]. PARP has the ability to covalently add poly ADP-ribose (PAR) chains to target proteins and alter their functions [42]. This enzymatic activity gives PARP the capability of being involved in diverse set of cellular processes including DNA damage repair [41, 42]. PARP inhibitors have drawn a lot of attention in cancer research community based on their remarkable anti-tumor effects in HR-deficient cancers [43]. Studies on PARP function in DNA repair system have led to development of numbers of FDAapproved inhibitors for treatment of various solid tumors [44–46].

PARP protein is found to be playing a key role in sarcoma as well, highlighted by several preclinical and clinical studies conducted in recent years. A study designed by Park et al., underscored the association between PARP activity and poor prognosis in osteosarcoma patients and showed the efficacy of PARP inhibition in combination with chemotherapy in this disease [47]. They evaluated the expression level of DNA damage molecules in 35 osteosarcoma patients and found that the expression levels of PARP1, γH2AX, and The Breast Cancer Susceptibility genes (BRCA1 and BRCA2) are accompanied with shorter overall survival in these patients [47]. *In vitro* experiments on osteosarcoma cell lines demonstrated that the PARP inhibitor olaparib as a single agent could inhibit cell proliferation in a dose- and timedependent manner [47]. Moreover, the combination of olaparib with doxorubicin

**147**

*DNA Repair Defects in Sarcomas*

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

showed significant synergistic effects in osteosarcoma cells [47]. The *in vivo* experiments also validated the growth-suppressive effects of individual and co-treatment of olaparib and doxorubicin in orthotopic osteosarcoma mice models [47]. In osteosarcoma cells treated with the combination treatment of olaparib plus doxorubicin, flow cytometry analysis showed increased apoptosis as evident by increased expression levels of cleaved caspase 3, cleaved PARP1, BAX, and decreased levels of BCL2 [47]. The sensitivity of HR-deficient osteosarcoma cells to PARP inhibition is demonstrated in a study performed by Engert et al. [48]. They treated a panel of osteosarcoma cell lines with PARP inhibitor talazoparib alone and in combination with chemotherapeutic drugs (temozolomide (TMZ), SN-38, doxorubicin, cisplatin, methotrexate (MTX), etoposide/carboplatin) [48]. They found a direct correlation between HR repair deficiency and increased sensitivity of osteosarcoma cells to PARP inhibition [48]. All osteosarcoma cell lines harboring BRCA1/2 mutation for both alleles (so-called "BRCAness") have shown a significant reduction in cell growth following treatment with talazoparib (MG63, ZK-58, Saos-2, and MNNG-HOS) [48]. However, U2OS (osteosarcoma cells) that are heterozygous for BRCA2 mutation and carry one intact allele were resistant to PARP inhibition [48]. Furthermore, TMZ showed the highest anti-proliferative synergistic effect with the PARP inhibitor among other chemotherapeutic drugs, and this effect induced through apoptosis pathway as indicated by caspase activation, increased expression level of BAX and BAK, DNA fragmentation, and loss of mitochondrial membrane potential [48]. These findings suggested a promising potential for development of novel therapeutic strategies using PARP inhibitors in combination with conventional treatments in osteosarcoma patients with features of BRCAness (more discussed in Section 3.4.1). Likewise, a large number of preclinical studies on other types of sarcomas including Ewing sarcoma, chondrosarcoma, rhabdomyosarcoma, and other STS emphasized the effectiveness of PARP inhibition in combination with chemotherapy as a promising therapeutic strategy for treatment of sarcoma patients [49–53]. In a study conducted by Laroche et al., the anti-tumor effect of PARP inhibitor rucaparib in combination with a chemotherapy drug trabectedin was explored in a panel of STS cell lines and a mouse model of liposarcoma [54]. The data obtained from this study demonstrated that the combination of rucaparib and trabectedin synergistically inhibited cell growth and induced G2M cell cycle arrest, γH2AX intranuclear accumulation, and apoptosis *in vitro* [54]. They also carried out *in vivo* experiments and showed that this combination significantly suppressed tumor growth, increased the progression-free survival, and elevated the

percentage of tumor necrosis in the xenograft mice model [54].

Although preclinical studies have presented PARP inhibition as an effective treatment option in sarcoma, clinical trials have failed to demonstrate a promising clinical outcome in patients so far [55, 56]. Schafer et al. conducted a phase I/II clinical trial of PARP inhibitor talazoparib in combination with low-dose temozolomide in patients with refractory/recurrent solid tumors including sarcoma [55]. From April 2014 to January 2018, 40 patients (including 15 Ewing sarcoma, 4 osteosarcomas, 2 synovial sarcomas, and one rhabdomyosarcoma) were enrolled in this study and treated with talazoparib and temozolomide [55]. The data showed that this combination therapy was well tolerated; reversible neutropenia and thrombocytopenia were the primary dose-limiting toxicities (DLTs) [55]. However, no significant anti-tumor activity was observed in sarcoma patients [55]. Similarly, a phase II clinical trial of PARP inhibitor (olaparib) in refractory Ewing sarcoma patients has also failed to demonstrate a promising clinical outcome [56]. One possible explanation for this direct contrast between preclinical and clinical studies is that PARP inhibition could induce anti-tumor effects in *de novo* Ewing sarcoma but not in pretreated, chemoresistant patients [55, 56]. However, the limited number of

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

*DNA - Damages and Repair Mechanisms*

be investigated in sarcomas.

*2.1.2 PARP*

The correlation between angiogenesis, as an important factor in tumor growth and metastasis, and APE1/Ref-1 in osteosarcoma was elucidated by the series of studies conducted by Wang et al. [26, 31–33]. They showed that transforming growth factor beta (TGFβ) is directly regulated by APE1/Ref-1 and its expression level was significantly reduced in APE1/Ref-1 deficient osteosarcoma cells [31]. TGFβ increases the chances of cancer metastasis through multiple mechanisms including immunosuppression, invasion, and angiogenesis [34]. They demonstrated that knocking down APE1/Ref-1 using specific siRNA in osteosarcoma led to down-regulation of TGFβ expression and suppression of angiogenesis *in vitro* based on human umbilical vein endothelial cells (HUVECs) in transwell and matrigel tube formation assays [31]. In addition, siRNA-mediated silencing of APE1/Ref-1 significantly suppressed tumor growth in xenograft mice models [31]. These experimental data indicated that APE1/Ref-1 promotes angiogenesis in osteosarcoma through a TGFβ-dependent pathway [31]. Additionally, they showed that the expression levels of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) are also regulated by APE1/Ref-1 [32]. However, the suppression of angiogenesis in APE1/ Ref-1 knockdown cells is not dependent on their transcriptional activity [32, 33]. Wang et al., also used siRNA against APE1/Ref-1 protein to investigate its inhibition in osteosarcoma [26]. They demonstrated that the siRNA-mediated inhibition of APE1/Ref-1 sensitized the osteosarcoma cells to DNA damaging agents: methyl methanesulfonate, H2O2, ionizing radiation, and chemotherapeutic agents [26]. Another study conducted by Xiao et al., investigated the association of APE1/Ref-1 polymorphisms with osteosarcoma [35]. They performed a 2-stage case–control study in a total of 378 osteosarcoma patients and 616 normal controls and concluded that the patients who have certain APE1/Ref-1 polymorphisms have lower APE1/Ref-1 expression and higher survival rates [35]. Over the past few years, small molecule inhibitors targeting APE1/Ref-1 have been developed and showed remarkable anti-tumor effects with limited toxicity in a variety of cancers, in both *in vitro* and *in vivo* models [36–40]. However, the efficacy of these inhibitors still needs to

Poly (ADP-ribose) polymerase (PARP) is another important DDR protein involved in cell proliferation, differentiation, and transformation [41]. PARP has the ability to covalently add poly ADP-ribose (PAR) chains to target proteins and alter their functions [42]. This enzymatic activity gives PARP the capability of being involved in diverse set of cellular processes including DNA damage repair [41, 42]. PARP inhibitors have drawn a lot of attention in cancer research community based on their remarkable anti-tumor effects in HR-deficient cancers [43]. Studies on PARP function in DNA repair system have led to development of numbers of FDA-

PARP protein is found to be playing a key role in sarcoma as well, highlighted by several preclinical and clinical studies conducted in recent years. A study designed by Park et al., underscored the association between PARP activity and poor prognosis in osteosarcoma patients and showed the efficacy of PARP inhibition in combination with chemotherapy in this disease [47]. They evaluated the expression level of DNA damage molecules in 35 osteosarcoma patients and found that the expression levels of PARP1, γH2AX, and The Breast Cancer Susceptibility genes (BRCA1 and BRCA2) are accompanied with shorter overall survival in these patients [47]. *In vitro* experiments on osteosarcoma cell lines demonstrated that the PARP inhibitor olaparib as a single agent could inhibit cell proliferation in a dose- and timedependent manner [47]. Moreover, the combination of olaparib with doxorubicin

approved inhibitors for treatment of various solid tumors [44–46].

**146**

showed significant synergistic effects in osteosarcoma cells [47]. The *in vivo* experiments also validated the growth-suppressive effects of individual and co-treatment of olaparib and doxorubicin in orthotopic osteosarcoma mice models [47]. In osteosarcoma cells treated with the combination treatment of olaparib plus doxorubicin, flow cytometry analysis showed increased apoptosis as evident by increased expression levels of cleaved caspase 3, cleaved PARP1, BAX, and decreased levels of BCL2 [47]. The sensitivity of HR-deficient osteosarcoma cells to PARP inhibition is demonstrated in a study performed by Engert et al. [48]. They treated a panel of osteosarcoma cell lines with PARP inhibitor talazoparib alone and in combination with chemotherapeutic drugs (temozolomide (TMZ), SN-38, doxorubicin, cisplatin, methotrexate (MTX), etoposide/carboplatin) [48]. They found a direct correlation between HR repair deficiency and increased sensitivity of osteosarcoma cells to PARP inhibition [48]. All osteosarcoma cell lines harboring BRCA1/2 mutation for both alleles (so-called "BRCAness") have shown a significant reduction in cell growth following treatment with talazoparib (MG63, ZK-58, Saos-2, and MNNG-HOS) [48]. However, U2OS (osteosarcoma cells) that are heterozygous for BRCA2 mutation and carry one intact allele were resistant to PARP inhibition [48]. Furthermore, TMZ showed the highest anti-proliferative synergistic effect with the PARP inhibitor among other chemotherapeutic drugs, and this effect induced through apoptosis pathway as indicated by caspase activation, increased expression level of BAX and BAK, DNA fragmentation, and loss of mitochondrial membrane potential [48]. These findings suggested a promising potential for development of novel therapeutic strategies using PARP inhibitors in combination with conventional treatments in osteosarcoma patients with features of BRCAness (more discussed in Section 3.4.1). Likewise, a large number of preclinical studies on other types of sarcomas including Ewing sarcoma, chondrosarcoma, rhabdomyosarcoma, and other STS emphasized the effectiveness of PARP inhibition in combination with chemotherapy as a promising therapeutic strategy for treatment of sarcoma patients [49–53]. In a study conducted by Laroche et al., the anti-tumor effect of PARP inhibitor rucaparib in combination with a chemotherapy drug trabectedin was explored in a panel of STS cell lines and a mouse model of liposarcoma [54]. The data obtained from this study demonstrated that the combination of rucaparib and trabectedin synergistically inhibited cell growth and induced G2M cell cycle arrest, γH2AX intranuclear accumulation, and apoptosis *in vitro* [54]. They also carried out *in vivo* experiments and showed that this combination significantly suppressed tumor growth, increased the progression-free survival, and elevated the percentage of tumor necrosis in the xenograft mice model [54].

Although preclinical studies have presented PARP inhibition as an effective treatment option in sarcoma, clinical trials have failed to demonstrate a promising clinical outcome in patients so far [55, 56]. Schafer et al. conducted a phase I/II clinical trial of PARP inhibitor talazoparib in combination with low-dose temozolomide in patients with refractory/recurrent solid tumors including sarcoma [55]. From April 2014 to January 2018, 40 patients (including 15 Ewing sarcoma, 4 osteosarcomas, 2 synovial sarcomas, and one rhabdomyosarcoma) were enrolled in this study and treated with talazoparib and temozolomide [55]. The data showed that this combination therapy was well tolerated; reversible neutropenia and thrombocytopenia were the primary dose-limiting toxicities (DLTs) [55]. However, no significant anti-tumor activity was observed in sarcoma patients [55]. Similarly, a phase II clinical trial of PARP inhibitor (olaparib) in refractory Ewing sarcoma patients has also failed to demonstrate a promising clinical outcome [56]. One possible explanation for this direct contrast between preclinical and clinical studies is that PARP inhibition could induce anti-tumor effects in *de novo* Ewing sarcoma but not in pretreated, chemoresistant patients [55, 56]. However, the limited number of completed clinical trials of PARP inhibitors in sarcoma compared to other tumors hinders us from making a definite conclusion (**Table 2**). Future preclinical and clinical studies will shed more lights on the effectiveness of PARP inhibition as a possible treatment approach for sarcoma patients.
