DNA-Dependent Protein Kinase Inhibitor Peposertib Potentiates the Cytotoxicity of Topoisomerase II Inhibitors in Synovial Sarcoma Models

Abstract

Synovial sarcoma is a rare and highly aggressive subtype of soft tissue sarcoma. The clinical challenge posed by advanced or metastatic synovial sarcoma, marked by limited treatment options and suboptimal outcomes, necessitates innovative approaches. The topoisomerase II (Topo II) inhibitor doxorubicin has remained the cornerstone systemic treatment for decades, and there is pressing need for improved therapeutic strategies for these patients. This study highlights the potential to enhance the cytotoxic effects of doxorubicin within well-characterized synovial sarcoma cell lines using the potent and selective DNA-PK inhibitor, peposertib. In vitro investigations unveil a p53-mediated synergistic anti-tumor effect when combining doxorubicin with peposertib. The in vitro findings were substantiated by pronounced anti-tumor effects in mice bearing subcutaneously implanted tumors. A well-tolerated regimen for the combined application was established using both pegylated liposomal doxorubicin (PLD) and unmodified doxorubicin. Notably, the combination of PLD and peposertib displayed enhanced anti-tumor efficacy compared to unmodified doxorubicin at equivalent doses, suggesting an improved therapeutic window-a critical consideration for clinical translation. Efficacy studies in two patient-derived xenograft models of synovial sarcoma, accurately reflecting human metastatic disease, further validate the potential of this combined therapy. These findings align with previous evidence showcasing the synergy between DNA-PK inhibition and Topo II inhibitors in diverse tumor models, including breast and ovarian cancers. Our study extends the potential utility of combined therapy to synovial sarcoma.

Keywords: DNA repair; DNA-PK; NHEJ; peposertib; synovial sarcoma.

Figure 1

Peposertib synergistically enhances the cytotoxicity of doxorubicin in synovial sarcoma cells. Overlay of Loewe synergy scores (A) SYO-1 and (B) HS-SY-II cells with combinations of doxorubicin and peposertib. Cell viability data were used to impute the synergy scores using Combenefit software. Potentiation of doxorubicin cytotoxicity by 1 µM peposertib on (C) SYO-1 and (D) HS-SY-II cells as measured using an Alamar Blue viability assay 168 h post treatment.

Figure 2

Combined treatment of peposertib and doxorubicin induces apoptotic cell death in synovial sarcoma cell lines. (A) Incucyte^® bright-field images of SYO-1 at 96 h post treatment at 10× magnification overlay with AnnexinV-Red staining. Images represent 3 independent replicates. Quantification of Annexin V-Red positive cells in (B) SYO-1 and (C) HS-SY-II over the course of treatment as monitored using the Incucyte^®.

Figure 3

Gene expression analysis revealed induction of p53- and DNA damage repair signaling upon combined treatment with doxorubicin and peposertib. (A) Scheme of the experimental setup for gene expression analysis in SYO-1 cells using the NanoString nCounter^® PanCancer panel. (B) Volcano plots highlighting differentially regulated genes (red dots) identified by limma upon combination treatment for 24, 72 and 168 h. The dotted horizontal and vertical lines indicate the adjusted p-value of 0.05 and log2FC of 1, respectively, whereas the names indicate differentially regulated genes identified by at least two methods. (C) Pathway enrichment analysis at 24, 72 and 168 h. Significantly enriched pathways were identified using Fisher’s exact test. Dashed line indicates the enrichment significance of the p-value (0.05). DE differentially expressed genes; non-DE not differentially expressed genes. (D) Heatmap showing expression levels of selected p53 related genes included in the Nanostring nCounter panel. Each column represents one biological replicate.

Figure 4

Peposertib in combination with doxorubicin activates p53 signaling and suppresses DNA repair. SYO-1 (A) and HS-SY-II (B) cells were exposed to doxorubicin, 1 μM peposertib or their combination for a period of 24 h. Protein lysates were analyzed by Western blotting using antibodies against phospho-Chk2 (T68), phospho-p53 (S15), p21, phospho-H2Ax (S139), and actin (loading control). Induction of p53 signaling pathway and accumulation of DNA-damage is observed in the combination groups with higher doxorubicin dose for both cell lines. The uncropped blots are shown in Figure S5

Figure 5

Peposertib enhances the anti-tumor activity of doxorubicin in subcutaneous SYO-1 xenograft tumors. (A,C): tumor growth of SYO-1 xenografts treated with vehicle, peposertib, doxorubicin, PLD or combinations of doxorubicin or PLD with peposertib (n = 10 for all groups, mean ± SEM). (B,D): relative body weight changes of SYO-1 tumor-bearing mice throughout the studies. Statistical analysis was performed using two-way ANOVA. In (A), comparison of 2 mg/kg doxorubicin and 2 mg/kg PLD treatment at day 31 reached statistical significance, p < 0.0001. Comparison of the 2 combination arms also revealed statistically significant differences at day 71, p < 0.0001. For (B), anti-tumor activity of both PLD + peposertib 100 mg/kg QD and PLD + 100 mg/kg BID was statistically significant compared to PLD monotherapy, p < 0.001.

Figure 6

Peposertib enhances the anti-tumor activity of doxorubicin in synovial sarcoma PDX models. Tumor growth of (A) CTG-2004 and (C) CTG-1173 PDX treated with vehicle, peposertib, PLD or a combination (n = 8 for all groups, mean ± SEM). (B,D): corresponding mouse bodyweight change over time. The p values were calculated by two-way ANOVA, **** p < 0.0001.