Synovial sarcoma (SS) is a subtype of soft tissue sarcoma (STS) that accounts for 5–10% of all STS cases. SS typically arises in the para-articular regions, and approximately 30% of cases occur in adolescents and young adults [1]. Genetically, SYT-SSX1 or SYT-SSX2 fusion transcripts have been detected in most SS; biphasic SS has an SYT–SSX1 fusion transcript, and monophasic SS has SYT–SSX2 [2] [3]. Although treatment with doxorubicin, ifosfamide, eribulin, trabectedin, and pazopanib have been established as chemotherapeutic options for advanced SS [4], these drugs do not offer adequate therapeutic efficacy. Research on therapeutic approaches for SS have been slow due to the rarity of the disease, emphasizing the necessity for novel innovative treatments.

Poly (ADP-ribose) polymerase enzymes (PARPs) bind to sites of DNA damage and mark single-strand breaks (SSBs) by synthesizing poly (ADP-ribose) chains. Notably, these poly (ADP-ribose) chains recruit numerous scaffold proteins and DNA repair enzymes to repair breaks [5]. PARP1 was initially reported as a regulator of base excision repair. However, it is also involved in homologous recombination (HR), nonhomologous end-joining, and microhomology-mediated end-joining repairs [5]. Recently, selective PARP1/2 inhibitors have been reported to exert therapeutic effects in BRCA1/2-mutation positive breast cancer, ovarian cancer, and HR repair deficient (HRD) ovarian cancer. Additionally, PARP inhibitors exert therapeutic effects in neuroendocrine tumors, such as small cell lung cancer (SCLC) and Ewing's sarcoma (EWS), which do not harbor BRCA1/2 mutations [6].

Notably, a PARP inhibitor talazoparib exerts anticancer effect by strongly inhibiting PARP1 and PARP2. Recent findings indicate that talazoparib strongly inhibits PARP16, a mono-ADP-ribosyltransferase, in addition to PARP1 and 2, thus broadening its target spectrum [7]. Preclinical studies indicate a potential interaction between PARP and the androgen receptor, which primarily fuels prostate cancer cell proliferation. Inhibition of the androgen receptor is linked to increased PARP activity and reduced expression of HRR genes, therefore, this relationship provides a scientific rationale for simultaneously inhibiting both targets [8] [9] [10] [11]. Moreover, talazoparib combined with enzalutamide as the first-line treatment of homologous recombination repair (HRR)-deficient prostate cancer significantly prolonged progression-free survival (PFS) in a phase 3 clinical trial [12]. Furthermore, talazoparib showed high efficacy and significantly extended PFS in patients with advanced breast cancer harboring germline BRCA mutations [13]. Approximately 70% of breast cancer with germline BRCA1 mutations are identified in triple-negative breast cancer, which is characterized by poor patient outcomes due to its limited therapeutic options [14] [15], talazoparib has the promising potential for this disease. Thus, talazoparib is a drug that has shown its efficacy in clinical trials in multiple cancer types and has been reported in combination therapy with other drugs, which is expected to lead to further development of its clinical application [16] [17] [18].

Although the efficacy of talazoparib has been demonstrated in several cancers, its effects on SS have not yet been clarified. Therefore, this study aimed to investigate the efficacy of talazoparib and the potential additive effects of talazoparib and other DNA repair mechanism inhibitors in SS.

To investigate the antitumor mechanism of talazoparib in SS, we measured cell apoptosis using annexin V- 4',6-diamidino-2-phenylindole (DAPI) assay. In Aska-SS and HS-SY2 cells with high PARP16 expression, there was a dose-dependent increase in the number of annexin V-positive apoptotic cells and the total number of DAPI-positive dead cells after 72 h of talazoparib treatment. In contrast, treatment with even 1 µM of talazoparib resulted in only a slight increase in apoptosis and total dead cells in SW982 and Yamato-SS cells with low PARP16 expression (Fig. 2a, 2b, Figure S4a). Viable cells, annexin V and DAPI negative cells, were decreased in a dose-dependent manner by talazoparib treatment in Aska-SS and HS-SY2 cells, however, the difference was minimal in SW982 and Yamato-SS cells (Figure S4b).

Mechanistically, talazoparib increased cleaved PARP expression and γH2AX phosphorylation (indicative of double-stranded DNA breaks [DSBs]) in Aska-SS and HS-SY2 cells, on the other hand, talazoparib had little effect on cleaved PARP expression and γH2AX phosphorylation in SW982 and Yamato-SS cells (Fig. 2c). Overall, these results indicate that talazoparib exerts anticancer effects against SS cells with high PARP16 expression by inducing apoptosis and DNA damage. However, the effects of talazoparib may be limited in SS cells with low PARP16 expression.

In addition to PARP inhibition, targeting other DNA damage response (DDR) proteins, such as WEE1, ataxia telangiectasia and rad3-related protein (ATR), checkpoint kinase 1 (CHK1), has recently emerged as a promising therapeutic strategy in other types of cancers [19] [20] [21]. Therefore, we investigated the effects of other DNA damage inhibitors, including WEE1, CHK1, and ATR inhibitors, against SS cells. Notably, the WEE1 inhibitor adavosertib, CHK1 inhibitor prexasertib, and ATR inhibitor ceralasertib inhibited cell viability in the four SS cell lines (Fig. 3a), although there were differences in sensitivity among the cell lines. Considering that the efficacies of some ATR inhibitors in several types of cancers have been investigated in clinical trials [22] and that the ATR inhibitor VX970 exerted antitumor effects in SS via DNA damage [23], we focused on the ATR inhibitor ceralasertib. Celarasertib treatment inhibited ATR phosphorylation and induced the cleavage of PARP and γH2AX, suggesting that celarasertib induced DNA damage and cell apoptosis (Fig. 3b). Collectively, these results indicate that DNA repair response inhibitors suppress cell proliferation and may be effective against SS.

Since the additive effects of talazoparib combined with ATR inhibitors in SS remains unclear, therefore, we investigated the additive effects of talazoparib and ceralasertib on SS cells. As shown in Fig. 1d, the sensitivity of SS cells to talazoparib varied between the high and low PARP16-expressing groups, with HS-SY2 and Aska-SS demonstrating high sensitivity and SW982 and Yamato-SS showing low sensitivity. Therefore, HS-SY2 and Aska-SS, which exhibited high sensitivity to talazoparib, were treated with low concentrations of talazoparib, whereas SW982 and Yamato-SS, which exhibited low sensitivity to talazoparib, were treated with high concentrations of talazoparib in combination with ceralasertib. Notably, cell viability assay showed that combined treatment with talazoparib and ceralasertib exerted additive inhibitory effect on the viability of three of the four SS cell lines (Fig. 4a). The combined effect of PARP inhibitor olaparib and ceralasertib showed the similar antitumor effect to that of talazoparib and ceralasertib (Figure S5). Western blot analysis revealed that talazoparib or ceralasertib monotherapy increased the expression of cleaved PARP and γH2AX. Additionally, talazoparib and ceralasertib combined therapy further increased the expression of cleaved PARP and γH2AX in Aska-SS and HS-SY2 cells, indicating the additive antitumor effect of the combination (Fig. 4b). Furthermore, cell apoptosis was assessed to confirm the additive effects of talazoparib and ceralasertib, using annexin-based assay. Importantly, the number of apoptotic cells increased following combined treatment with talazoparib and ceralasertib (Fig. 4c). Furthermore, the total number of DAPI-positive dead cells were also increased in the combination treatment compared to treatment with talazoparib or ceralasertib alone and viable cells decreased in the combination treatment compared to treatment with talazoparib or ceralasertib alone (Figure S6a, b). Overall, combined therapy with talazoparib and ceralasertib exerts antitumor effects against SS cells through DNA damage and apoptosis pathways, suggesting a potential treatment strategy for SS.

Recently, there has been advances in the development of cancer immunotherapy and precision medicine, significantly improving the prognosis of some cancers. However, treatments for synovial sarcoma are limited, indicating the need for novel treatment strategies. In the present study, treatment with the PARP inhibitor talazoparib inhibited the growth of SS cells, particularly through the expression of PARP16. Talazoparib induced apoptosis and DNA damage in SS cells, and combined treatment with the ATR inhibitor ceralasertib exerted a superior antitumor effect. To the best of our knowledge, this is the first study to elucidate the potential of talazoparib and DDR pathway inhibitors, including ATR, CHK1, and WEE1, in SS cells.

Genomic instability and mutations have been identified as hallmarks of cancer [24]. Typically, human cells repair DNA instabilities or abnormalities via the DDR pathway. The most common form of DNA damage is damage to one strand of the DNA double helix, either by SSBs or chemical modification of DNA bases. Damaged lesions are repaired using base excision repair pathways, nucleotide excision repair pathways, mismatch repair pathways, and crucial enzymes. Although SSBs are relatively easily repaired, DSBs pose a considerable threat to DNA integrity and require complex repair processes such as non-homologous end joining and homologous recombination. Therefore, various DDR pathways are involved in DNA damage repair. PARPs play pivotal roles in SSB repair [25]. SSBs are recognized by PARP1 and PARP2, which promote the formation of poly (ADP-ribose) chains at the SSB site or adjacent protein regions and recruit base excision repair proteins. Olaparib, a PARP inhibitor, was initially approved for the treatment of ovarian cancer with BRCA mutations. Subsequently, several other PARP inhibitors have been developed, expanding their indications to include breast cancer, prostate cancer, pancreatic cancer, and other malignancies [26]. Talazoparib has also been clinically examined for breast and prostate cancers, with further expansion of its indications to other types of cancers. Selective PARP1 inhibitors have recently been developed. PARP1 have been reported to regulate the repair of DNA single-strand breaks generated directly, or during base excision repair [27] [28]. Therefore, a PARP1 selective inhibitor, such as saruparib, may reduce adverse events for patients, and clinical trials with PARP inhibitors are currently ongoing. In the future, selective PARP1 inhibitors may show clinical benefit with less toxicities.

In the present study, talazoparib inhibited the proliferation and viability SS cells with high PARP16 expression by inducing DNA damage and apoptosis. In a previous report, an association between PARP16 and mono(ADP-ribosyl)ation (MARylation) was reported. MARylation is a type of cellular protein modification that involves the addition of a single molecule, ADP-ribose, to a target protein. Enzymes such as PARP are involved in this process, which is involved in various physiological functions of cells such as DNA repair, transcriptional regulation, signal transduction, and stress response. The siRNA-based screen with human ovarian cancer cell lines in the previous study confirmed that PARP16 has a significant effect on MARylation of cytoplasmic and ribosomal proteins. Inhibition of PARP16 markedly reduced MARylation of ribosomal proteins and promoted protein synthesis. This inappropriate promotion of protein synthesis by inhibition of PARP16 suppressed the proliferation of ovarian cancer cells in cell culture and suppressed tumor growth in in vivo mouse model [29]. These findings indicate that suppression of PARP16 affects cell proliferation.

In this study, talazoparib combined with the ATR inhibitor ceralasertib exerted superior antitumor effect, suggesting that this combination therapy may be effective even for SS cells with low sensitivity to talazoparib. Our study confirmed the underlying mechanism of talazoparib monotherapy and talazoparib and ceralasertib combined therapy in SS. In previous studies, combination therapy with DNA-damaging drugs, such as topotecan, trabectedin, and temozolomide, and ATR, ATM, and PARP inhibitors, showed synergistic effects against sarcoma, melanoma, SCLC, non-small cell lung cancer, and bladder, ovarian, and pancreatic cancers [30]. Clinical trials have indicated the efficacy of topotecan and ATR inhibitors against SCLC [31] [32], and a combination of talazoparib and WEE1 inhibitors was effective against SCLC, myelodysplastic syndrome, and acute myelogenous leukemia [33] [7]. Several preclinical studies have demonstrated the efficacy of DDR inhibitors in SS, including selective DNA-PK inhibitors and ATR inhibitors [23] [34] [35]. Thus, the potential of combination therapies targeting DNA repair mechanisms has been reported, however, the additive effects of talazoparib combined with ATR inhibitors in SS remains unclear.

ATR is a kinase responsible for orchestrating cellular responses to SSBs, DSBs, and replication stress, and serves as a crucial factor in the DNA repair mechanism [36]. Clinical research targeting ATR has identified various promising cancer treatments, including ATR inhibitors such as ceralasertib, berzosertib, elimusertib, camonsertib, and tuvusertib [22]. Although clinical studies are yet to demonstrate the efficacy of ATR inhibitors for SS, a preclinical study reported the effect of the ATR inhibitor berzosertib against SS [23]. Combined treatment with berzosertib and PARP inhibitors or cisplatin exerted synergistic therapeutic effects on cell viability; however, the underlying mechanism of talazoparib against SS remains unclear. Clinical studies of combined PARP inhibitor and ATR inhibitor have been reported in SCLC, and ovarian cancer [37] [38] [39]. In those reports, the effects varied by cancer type, and the major adverse events identified were fatigue, nausea, anorexia, and hematologic toxicity. However, these adverse events were tolerable, and it was concluded that combination therapy with a PARP inhibitor and an ATR inhibitor was tolerable. The PARP inhibitor used in these clinical trials was olaparib, which is in advanced development, and clinical trials regarding combination therapy with talazoparib and an ATR inhibitor are considered an issue for further study.

Despite the promising findings, this study had several limitations. Although we demonstrated that talazoparib induced apoptosis and DNA damage in SS cells, further research is necessary to elucidate the detailed signaling pathways and other antitumor effects, such as cellular senescence. Additionally, in vivo experiments are necessary to facilitate the application of the　combined therapy in humans, as only in vitro experiments were performed in this study. Moreover, although we demonstrated the potential of PARP16 in predicting the efficacy of talazoparib, further investigation in large cohorts is necessary.

Conclusively, talazoparib inhibits the viability and proliferation of SS cells by inducing apoptosis and DNA damage. Overall, this study provides valuable information for the development of novel treatment strategies for SS, particularly focusing on the effectiveness of talazoparib and the additive effects of ATR inhibitors.

Gene expression of PARP1 and PARP16 in SS was evaluated using the Gene Expression Omnibus (GEO) repository (profiles: GDS2736/219034_at, GDS2736/208644_at, https://www.ncbi.nlm.nih.gov/geoprofiles/), a public database for gene expression. Gene expression of PARP16 in various cancer types was evaluated using the data form CCLE (https://sites.broadinstitute.org/ccle).