Abstract

Simple Summary

   This study focuses on finding improved treatment strategies for
   advanced or metastatic synovial sarcoma, which is a rare and aggressive
   type of soft tissue sarcoma. The researchers tested a combination of
   two drugs, peposertib and doxorubicin, to see if they could work
   together to kill synovial sarcoma cells more efficiently. The
   experiments conducted in cultured cancer cells and mouse models of
   synovial sarcoma demonstrated that when these drugs were used together,
   they had a significantly stronger effect against cancer cells compared
   to using either drug alone. It is noteworthy that the combination could
   successfully overcome resistance to doxorubicin monotherapy in a
   patient-derived tumor model. This study also shed light on the
   underlying molecular mechanisms of this combination effect. Overall,
   the findings suggest that combining peposertib with doxorubicin could
   be a promising treatment option for synovial sarcoma patients in the
   future.

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: synovial sarcoma, DNA repair, NHEJ, DNA-PK, peposertib

1. Introduction

   Synovial sarcoma (SS) is a rare but highly malignant type of soft
   tissue sarcoma (STS), and accounts for 5–10% of all STS [[36]1,[37]2].
   In >95% of cases, SS is associated with a chromosomal translocation
   involving genes encoding the SWI-SNF complex component SS18 (formerly
   SYT) and a SSX transcriptional repressor. The translocation results in
   the formation of an in-frame fusion involving SS18 with SSX1, SSX2 and,
   less frequently, SSX4. This fusion event gives rise to SS18-SSX
   proteins, which play a pivotal role in driving the process of
   sarcomagenesis and hold essential diagnostic significance
   [[38]3,[39]4,[40]5,[41]6].

   The current recommended approach for treating localized synovial
   sarcoma continues to be surgical removal of the tumor with clear
   margins, supplemented by radiotherapy and/or chemotherapy, which are
   determined based on individual patient and tumor characteristics.
   [[42]1,[43]7]. More than 50% of SS patients develop metastatic disease,
   which is treated mostly with anthracycline-based chemotherapy regimens
   and palliative intent [[44]2,[45]8]. Doxorubicin in monotherapy, or in
   combination with ifosfamide, is considered as the SOC in first-line
   with average response rates of ~20% and ~30%, respectively, although
   this is not based on prospective randomized SS specific studies due to
   the rarity of the disease and the fact that STS have been clinically
   treated as one type of disease for a long time [[46]2,[47]5,[48]9].

   Anthracyclines such as doxorubicin exert their cytotoxic effects by
   inhibiting type II topoisomerase (Topo II) enzymes, which are essential
   for DNA replication and transcription [[49]10]. The mechanism of action
   of Topo II inhibitors involves the stabilization of the cleavage
   complex formed by Topo II and DNA, preventing the enzyme from
   re-ligating the break and releasing the DNA [[50]11]. The stabilized
   cleavage complex then becomes a cytotoxic intermediate, ultimately
   leading to DNA double strand breaks (DSB) [[51]12]. In mammalian cells,
   the nonhomologous-end-joining (NHEJ) repair pathway is critical for
   repairing Topo II-mediated DNA damage [[52]11,[53]13]. DNA-dependent
   protein kinase (DNA-PK) is an enzyme that belongs to the
   serine/threonine kinase family, and plays a crucial role in NHEJ
   mediated repair. It works in collaboration with five other factors,
   namely Ku70, Ku80, XRCC4, ligase IV, and Artemis [[54]13,[55]14]. The
   identification of DNA-PK’s crucial role in DNA damage repair has
   brought attention to the potential use of DNA-PK inhibitors to impede
   the repair process and increase the effectiveness of DNA-damaging
   agents [[56]14,[57]15]. Peposertib is an orally bioavailable inhibitor
   of DNA-PK, exhibiting strong potency and selectivity. In preclinical
   models, it has been shown to significantly enhance the antitumor
   effects of ionizing radiation and DNA double-strand break-inducing
   agents, including anthracyclines like doxorubicin [[58]14]. In the
   clinic, peposertib is currently being evaluated in combination with
   various DNA damaging agents, including external beam radiotherapy
   ([59]NCT04555577; [60]NCT04533750), radiopharmaceuticals
   ([61]NCT05868174; [62]NCT04750954; [63]NCT04071236), chemotherapies
   ([64]NCT03983824; [65]NCT04092270; [66]NCT05711615), and the ATR
   inhibitor tuvusertib ([67]NCT05687136).

   Consequently, we put forth the hypothesis that peposertib in
   conjunction with Topo II inhibitors will yield superior outcomes
   compared to individual agent treatments in synovial sarcoma models. In
   this study, we present compelling preclinical evidence of the
   synergistic antitumor effects achieved by combining peposertib with
   Topo II inhibitors, with a particular emphasis on doxorubicin, in human
   synovial sarcoma tumor models.

2. Materials and Methods

2.1. Reagents and Cell Culture

   Peposertib was synthesized at Merck Healthcare KGaA, Darmstadt,
   Germany. Doxorubicin and Etoposide were purchased from Sigma-Aldrich.
   For in vitro experiments, drugs were solubilized in DMSO to create
   stock solutions, which were then frozen and stored at −20 °C until
   needed. The concentration of DMSO in the media did not exceed 0.1%
   (v/v).

   SYO-1 cells were kindly provided by Okayama University, Okayama City,
   Japan, and the HS-SY-II cell line was purchased from RIKEN cell bank.
   Both cell lines were cultured in Dulbecco’s Modified Eagle Medium
   (DMEM) + 10% fetal calf serum at 37 °C with 10% CO[2]. Cell line
   identity was confirmed by short tandem repeats (STR) analyses,
   mycoplasma and bacterial contamination was excluded. Expression of the
   pathognomonic SS18-SSX fusion gene in both cell lines was confirmed by
   Western blotting.

2.2. Cell Viability Assay

   For viability and combination matrix assays, SYO-1 and HS-SY-II cells
   were plated at 3000 and 7500 cells per well, respectively, in 96-well
   plates. The next day, cells were treated with serial dilution of drugs
   using a Tecan D300e Digital Despenser, and DMSO concentration was
   normalized in all wells. At 168 h (7 days) following the drug
   treatment, the effect on cell growth or viability was assessed with
   Resazurin assay according to the manufacturer’s protocol and a
   fluorescent signal was recorded (560Ex/590Em) using a Tecan Infinite
   M200 plate reader.

   Dose response curve and IC[50] values were generated using Graphpad
   Prism (v9.0.0), while for the combination matrix, synergism was
   analyzed with Loewe’s additivity model using GeneData Screener Software
   (Version 19.0.5) and graphed using Combenefit software (v.2.021)
   [[68]16].

2.3. Immunoblotting

   Cells were harvested and lysed in RIPA buffer: 20 mM Tris-HCl (pH 7.5),
   150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate,
   2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1
   µg/mL leupeptin (Cell Signaling Technology, Danvers, MA, USA)
   supplemented with both protease and phosphatase inhibitors (Roche
   Diagnostics, Rotkreuz, Switzerland). To ensure lysis, cells were
   sonicated with Diagenode Bioruptor Plus for 10 min (30 s on/30 s off
   cycle) at 4 °C and subsequently centrifuged at 4 °C at 13,000 rpm.
   Protein concentration was determined by the BCA protein assay (Thermo
   Fisher Scientific, Waltham, MA, USA) and an equal amount of protein was
   mixed with 4x NuPAGE LDS Sample Buffer (Invitrogen, Waltham, MA, USA)
   and 10× NuPAGE Reducing Agent (Invitrogen). Samples were heated at 70
   °C for 10 min prior separation on NuPAGE 4–12% BisTris Mini Protein
   gels (Invitrogen) and NuPAGE MOPS SDS Running Buffer (Invitrogen).
   Proteins were subsequently transferred to polyvinylidene fluoride
   (PVDF) membrane via iBlot Dry Blotting System (Thermo Fisher
   Scientific, Waltham, MA, USA). Membranes were incubated with the
   appropriate antibodies and imaged with Bio-rad ChemiDoc Imaging System
   using Western Lighting Plus ECL (PerkinElmer, Waltham, MA, USA). The
   list of antibodies and their sources can be found in [69]Supplementary
   Table S2.

2.4. IncuCyte Live Cell Imaging

   SYO-1 and HS-SY-II cells were plated in 96-well plates and incubated
   overnight before drugs and IncuCyte Annexin V red reagent (Essen
   Bioscience, Ann Arbor, MI, USA) was added the next day to label
   apoptotic cells in real time. Cells were imaged using 10× objective in
   Incucyte S3 device at 2 h intervals for 7 days. Relative apoptosis
   events were determined by the number of Annexin V red counts per mm^2
   normalized to percent confluence.

2.5. RNA Extraction, NanoString nCounter Assay and the Analysis

   Total RNA was isolated using RNAqueous™-4PCR Total RNA Isolation Kit
   (Invitrogen) according to the manufacturer’s protocol and its
   concentration was measured with a Qubit 4.0 fluorometer. For the gene
   expression analysis, 100 ng of isolated total RNA were assessed using
   the nCounter PanCancer Pathway Panel (NanoString, Seattle, WA, USA)
   according to the manufacturer’s instruction.

   Data analysis and processing were conducted by integrating three
   independent quantification methods: DESeq2, Limma, and nSolver, with
   corresponding R packages (DESeq2 and limma). Background correction was
   performed by subtracting the value of “mean + 2× standard deviation”
   obtained from the negative controls from the raw counts. Further,
   adjusted raw counts were normalized to the geometric mean of six
   positive controls in each sample, followed by normalization using the
   geometric mean of 40 internal reference genes. Data exclusion criterion
   was set at a minimum of 30 counts of mRNA template. For multiple
   testing correction, the Bonferroni-Hochberg method was utilized,
   applying an adjusted p-value threshold of p = 0.05. Moreover, a
   log2-fold change (log2FC) threshold of 1 was selected as the criterion
   for identifying differentially regulated genes. A gene was classified
   as differentially expressed if it met the threshold criteria in at
   least two out of three quantification methods.

   Pathway enrichment analysis was performed using Fisher’s exact test
   with a significant threshold of p-value = 0.05, since nCounter
   PanCancer Pathway Panel comes with 770 genes.

2.6. Animal Studies

   In vivo efficacy data were generated in subcutaneous human
   cell-line-derived xenograft and patient-derived xenograft (PDX) models.
   For human cell-line-derived xenograft tumors, 5 million SYO-1 cells
   were injected subcutaneously (s.c) in 1:1 (v:v) DPBS/Matrigel Basement
   Membrane Matrix into the right flanks of female 8–10 weeks old H2d Rag2
   [C;129P2-H2^d-TgH(II2rg)^tm1Brn-TgH(Rag2)^tm1AltN4] mice (Taconic
   Biosciences). The study was randomized into groups (N = 10/group) of
   equal mean tumor volume (TV) prior to treatment. All studies were
   approved by the local animal welfare authority (Regierungspräsidium
   Darmstadt, Hesse, Germany; experimental license number DA4/Anz.1040).

   The efficacy studies in the synovial sarcoma PDX models CTG-1173 and
   CTG-2004 were performed at Champions Oncology according to the
   guidelines of the Institutional Animal Care and Use Committee (IACUC)
   of Champions Oncology. For these PDX studies, stock mice were
   bilaterally implanted with fragments from each of the 2 Champions
   TumorGraft^® models CTG-1173, CTG-2004. After tumors reached 1000–1500
   mm^3, they were harvested, and tumor fragments were implanted s.c. in
   the left flank of female study mice. Tumor growth was monitored twice a
   week using digital calipers, and TV was calculated using the formula
   (0.52 × [length × width^2]). When TV reached approximately 150–300
   mm^3, animals were matched by tumor size and assigned into vehicle
   control or treatment groups (n = 8/group), and dosing was initiated on
   d0 up to d52 (CTG-1173) or up to d34 (CTG-2004) or until mean TV in one
   group reached 1500 mm^3. Tumor size and body weight were measured twice
   a week. Histopathological and molecular analyses were performed at
   Champions Oncology, and data was reviewed at Merck Healthcare KGaA.

   For all in vivo studies, peposertib was formulated in vehicle (0.5%
   Methocel, 0.25% Tween20, 300 mmol/L sodium citrate buffer, pH 2.5 and
   administered orally. Doxorubicin or pegylated liposomal doxorubicin
   formulated for intravenous administration in 5% (50 mg/mL) glucose
   solution was injected into the tail vein once weekly at the indicated
   dose.

3. Results

3.1. Peposertib Enhances the Cytotoxicity of Topo II Inhibitors in Synovial
Sarcoma Cell Lines

   We selected two SS18:SSX fusion positive synovial sarcoma cell lines,
   SYO-1 and HS-SY-II, to evaluate if DNA-PK inhibition by peposertib
   could enhance the cytotoxic effects of Topo II inhibitors, such as
   doxorubicin or etoposide. Single agent treatment with either
   doxorubicin or etoposide revealed comparable cell-killing activity in
   both cell lines while peposertib had notable cytotoxicity (IC50 = 18µM
   in SYO-1 and 21µM in HS-SY-II) only at concentrations far above those
   previously reported to be achievable in a clinical setting [[70]17]
   ([71]Supplementary Figure S1A–C). Multi-dose combinatorial response
   matrices over various concentration ranges demonstrated that concurrent
   treatment of peposertib with either doxorubicin or etoposide
   substantially decreased the viability of both cell lines as assessed by
   an Alamar Blue viability assay. By systematically mapping the
   combination matrix data onto the Loewe model, the presence of
   synergistic effects (BLISS score >2.0) between peposertib and the two
   Topo II inhibitors was confirmed ([72]Figure 1A,B and [73]Supplementary
   Figure S1D,E). This synergy was particularly evident when considering
   the sublethal concentration ranges pertinent to individual monotherapy
   treatments. For a subsequent confirmatory study, we selected a
   sublethal peposertib concentration of 1µM co-administered with
   increasing concentrations of doxorubicin. This resulted in a pronounced
   reduction of the doxorubicin IC[50] from 19 nM to 0.9 nM (22-fold) in
   SYO-1 and from 21 nM to 0.4 nM (52-fold) in HS-SY-II cells ([74]Figure
   1C,D). A comparable effect was observed when 1 µM of peposertib was
   combined with etoposide ([75]Supplementary Figure S1F,G).

Figure 1.

   [76]Figure 1
   [77]Open in a new tab

   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.

   To corroborate the findings obtained from the Alamar Blue viability
   assay, we utilized Incucyte^® live cell imaging to monitor alterations
   in cell growth and death (apoptotic cells detected by Annexin V Red
   staining) throughout a 168 h duration. We focused on the combination
   treatment of 1μM peposertib with two concentrations of doxorubicin, 1
   nM and 5 nM. In the scenario of the low-dose combination (1 nM
   doxorubicin with 1µM peposertib), a qualitative assessment of
   bright-field images overlayed with Annexin V Red staining unveiled a
   notable decrease in cell density at 72 or 96 h subsequent to
   co-administration of peposertib and doxorubicin, as compared to the
   individual treatments ([78]Figure 2A, [79]Supplementary Figure S2A,B).
   This observed decrease in cell density was accompanied by a minor
   elevation of Annexin V staining in SYO-1 cells and moderate increase in
   HS-SY-II cells ([80]Figure 2B,C). In contrast, the co-administration of
   5 nM doxorubicin and peposertib resulted in a pronounced augmentation
   of Annexin V-positive cells in both cell lines ([81]Figure 2A and
   [82]Supplementary Figure S2A). Quantitative analyses of the Incucyte^®
   bright-field images confirmed both the cytostatic and cytotoxic natures
   of the low-dose and high-dose treatment regimens ([83]Figure 2B,C and
   [84]Supplementary Figure S2B).

Figure 2.

   [85]Figure 2
   [86]Open in a new tab

   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^®.

3.2. Peposertib Synergizes with Doxorubicin to Inactivate DNA Repair Pathways
and Activate p53 Tumor Suppressor Genes Simultaneously

   In order to study the underlying molecular mechanism driving the
   observed synergistic effects, we opted to employ the NanoString
   nCounter^® PanCancer pathways panel, which allowed us to scrutinize
   transcriptomic alterations in 770 genes across 13 cancer-associated
   canonical intracellular signaling pathways. We collected total RNA from
   SYO-1 cells that had been treated with either single agent or
   combination treatment for 24, 72, and 168 h ([87]Figure 3A). We used an
   adjusted p-value of 0.05 and log2FC of 1 as a threshold to define
   whether a gene was differentially regulated in response to treatment.
   Panel assessment of single-agent treatment with either doxorubicin or
   peposertib revealed that only very few genes exhibit differential
   regulation throughout the 168 h of treatment ([88]Supplementary Figure
   S3A–C). By contrast, co-treatment with doxorubicin and peposertib
   consistently impacted a greater set of genes across the
   cancer-associated signaling pathways, particularly after 72 h and 168 h
   treatment ([89]Figure 3B and [90]Supplementary Figure S3A). To be
   precise, we observed that a total of 66 genes exhibited significant
   upregulation, whereas 30 genes displayed significant downregulation
   upon combined treatment at the 168 h timepoint. A subsequent pathway
   enrichment analysis revealed that the majority of genes that were
   differentially regulated in the combined treatment are associated with
   p53-signaling and DNA damage repair ([91]Figure 3C). Heatmap
   visualization of p53-associated genes further revealed the
   time-dependent effects and synergistic impact of the combined treatment
   on the p53 pathway ([92]Figure 3D).

Figure 3.

   [93]Figure 3
   [94]Open in a new tab

   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.

3.3. Concurrent Administration of Peposertib and Doxorubicin Triggers p53
Mediated Apoptosis in Synovial Sarcoma Cell Lines

   Taking into account the data from our gene expression analysis and the
   mechanisms of action of both drugs, we hypothesized that disruptions in
   DNA damage repair pathways and p53-mediated signaling constitute a
   central theme underlying the mode of action of the synergistic effects.
   To further investigate this, we collected whole cell lysates from SYO-1
   and HS-SY-II cells treated with single-agents or combinations for 24 h
   and immunoblotted selected markers of DNA damage and repair signaling.
   Using γ-H2A.x as a generic proxy for DNA damage, it was observed that
   the combination of low-dose doxorubicin (1 nM) and peposertib did not
   induce levels of DNA damage different from the control treatments
   ([95]Figure 4A,B). When a higher concentration of 5 nM doxorubicin was
   combined with peposertib, the evident occurrence of apoptosis described
   earlier in the manuscript is accompanied by a noticeable increase in
   γ-H2A.x, an indicator for accumulation of unrepaired DNA damage
   ([96]Figure 4A,B). Elevated levels of phosphorylated p53 (Ser15) were
   detected in both cell lines, concomitant with an increase in protein
   expression of the cyclin-dependent kinase inhibitor p21, a
   transcriptional target of p53 ([97]Figure 4A,B). This finding is
   consistent with the gene expression analysis data ([98]Figure 3), which
   demonstrated strong upregulation of p53-dependent genes including
   CDKN1A, the gene encoding p21. Additionally, our Western blot analysis
   showed that Checkpoint kinase 2 (Chk2), a crucial mediator of various
   cellular responses to genotoxic stress, was phosphorylated (Thr68) upon
   treatment with the combination therapy.

Figure 4.

   [99]Figure 4
   [100]Open in a new tab

   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 [101]Figure S5

3.4. Peposertib Enhances the Anti-Tumor Activity of Doxorubicin In Vivo

   Next, we went on to investigate if peposertib is able to enhance the
   anti-tumor efficacy of doxorubicin in vivo in human xenograft models.
   In the clinic, doxorubicin can be administered in its pure, unmodified
   state, or as a pegylated liposomal formulation. The pegylated liposomal
   doxorubicin (PLD) formulation leverages advanced encapsulation
   techniques to optimize drug delivery and appears to have a favorable
   toxicity profile with better cardiac safety and less myelosuppression
   in the clinic [[102]18]. We first compared the anti-tumor activity and
   tolerability of conventional doxorubicin with PLD in monotherapy and in
   combination with peposertib in mice harboring established subcutaneous
   SYO-1 xenografts. As monotherapies, doxorubicin or PLD were
   intravenously administered once weekly at equivalent doses of 2 mg/kg.
   For the dual therapy approach, peposertib was given orally two times a
   day (BID) with an 8 h interval between the two administrations,
   spanning across 4 days. This regimen commenced 24 h following the
   intravenous delivery of doxorubicin or PLD. A scheme of a weekly
   treatment cycle is provided in [103]Supplementary Figure S4A. In this
   initial experiment, mice received four cycles of treatment. While
   doxorubicin monotherapy resulted in a moderate but statistically
   significant (p = 0.003) delay of tumor growth, treatment with PLD had a
   statistically significant superior anti-tumor efficacy (p < 0.001), and
   resulted in tumor regressions during the initial phase of treatment
   ([104]Figure 5A). The addition of peposertib to conventional
   doxorubicin treatment significantly enhanced the anti-tumor activity
   and resulted in tumor regression during the course of the 4 weeks of
   treatment, but tumors started to regrow approximately 2 weeks after
   treatment stopped ([105]Figure 5A). In contrast, when peposertib was
   combined with PLD treatment, durable anti-tumor responses were
   observed, and most tumors were still in regression even 60 days after
   the treatment was stopped ([106]Figure 5A). Importantly, all
   administered treatments exhibited excellent tolerance, as indicated by
   clinical symptoms and body weight alterations in the treated animals
   ([107]Figure 5B). This outcome not only reaffirmed the synergistic
   anti-tumor effects of doxorubicin and peposertib observed in vitro, but
   also highlighted the potential for an improved therapeutic window with
   the combination of peposertib and PLD, as evidenced in this tumor
   model. In a subsequent study, we investigated the dose-dependency of
   the anti-tumor effect of peposertib in combination with PLD. Mice
   harboring established subcutaneous SYO-1 xenografts were treated with
   once-weekly PLD, followed by 4 days treatment with either 50 mg/kg
   peposertib once daily (QD), 100 mg/kg QD, or 100 mg/kg BID. Peposertib
   monotherapy at the highest dose of 100 mg/kg BID administered for 4
   days/week and PLD monotherapy were included as control treatments in
   this experiment. The animals enrolled in this study underwent
   randomization across the various treatment groups with a higher mean
   tumor volume than in the previous study to facilitate a more effective
   comparison of tumor regression across the different dose groups.
   Consistent with the results from our in vitro investigations, we did
   not detect substantial anti-tumor effects from peposertib monotherapy,
   and the anti-tumor activity of PLD was in line with our previous study
   ([108]Figure 5C). The combination of PLD and 50 mg/kg QD demonstrated
   only marginal superiority over PLD monotherapy, but the addition of 100
   mg/kg QD peposertib significantly enhanced the anti-tumor activity
   compared to PLD monotherapy (p < 0.0001, [109]Figure 5C and
   [110]Supplementary Figure S4B). The strongest anti-tumor activity was
   observed with co-treatment of PLD and 100 mg/kg BID peposertib,
   resulting in long-lasting tumor regression for up to 100 days post
   treatment start ([111]Figure 5C) It is noteworthy that two tumors in
   this group relapsed approximately 40 days after cessation of treatment
   and began to undergo rapid regrowth. Upon a re-challenge with the same
   treatment regimen, both tumors promptly regressed again, indicating
   that tumor cells did not acquire resistance to the therapy during the
   initial treatment period ([112]Supplementary Figure S4C).

Figure 5.

   [113]Figure 5
   [114]Open in a new tab

   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.

   To enhance the clinical relevance of our findings, we opted for two
   patient-derived synovial sarcoma xenograft models and conducted
   efficacy studies. The model CTG-1173 was derived from a lung metastasis
   of a 24-year-old female patient diagnosed with stage IV synovial
   sarcoma. Prior to acquisition of tissue for the establishment of the
   PDX model, the patient underwent two rounds of chemotherapy, involving
   a regimen containing doxorubicin. However, the patient exhibited no
   response to this treatment. The second model, CTG-2004, originated from
   a lung metastasis of a 30-year-old female patient diagnosed with stage
   IV synovial sarcoma. Molecular analysis carried out at Champions
   Oncology unveiled the existence of a SS18-SSX1 fusion in the CTG-1173
   tumors, and the presence of SS18-SSX1 and SS18-SSX4 fusions in the
   CTG-2004 tumors, thus solidifying the categorization of the tumor type
   as synovial sarcoma. It is important to highlight that both tumors
   exhibited TP53 wildtype status, and lacked significant copy number
   alterations in any of the tested oncogenes. For a more comprehensive
   characterization, additional clinical, histological, and molecular data
   of the models can be found in [115]Supplementary Table S1. For the
   efficacy study, female mice harboring subcutaneously implanted tumors
   were randomized into four groups and treated with 6 cycles of the
   treatment scheme that was applied in the previous studies
   ([116]Supplementary Figure S4A). In CTG-2004, peposertib monotherapy
   had no effect on tumor growth compared to vehicle treatment, but PLD
   monotherapy significantly delayed tumor growth during the treatment
   period (p < 0.0001). Upon cessation of treatment, tumors started to
   regrow rapidly. In contrast, the combination treatment resulted in
   statistically superior (p < 0.0001) anti-tumor efficacy and tumor
   shrinkage during the treatment period ([117]Figure 6A). As in the
   previous studies, all administered treatments exhibited excellent
   tolerance, as indicated by clinical symptoms and body weight
   ([118]Figure 6B). In CTG-1173 xenografts, neither peposertib nor PLD in
   monotherapy significantly inhibited tumor growth ([119]Figure 6C). The
   resistance to PLD exhibited by these tumors aligns with the lack of
   responsiveness observed in the patient, who did not show an objective
   response to a doxorubicin containing chemotherapy regimen. This
   chemotherapy treatment was administered to the patient during clinical
   care before the extraction of tissue for the establishment of the
   model. Importantly, when peposertib was added to PLD, tumor growth was
   significantly blocked (p < 0.0001) throughout the treatment duration,
   in contrast to all control treatments ([120]Figure 6C). This finding
   underscores that resistance to doxorubicin-containing therapy can
   eventually be overcome by simultaneous inhibition of DNA-PK. Also, in
   this study, all treatments exhibited excellent tolerance ([121]Figure
   6D).

Figure 6.

   [122]Figure 6
   [123]Open in a new tab

   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.

4. Discussion

   In the present study, we present compelling evidence that the cytotoxic
   activity of Topo II inhibitors can be significantly augmented in
   synovial sarcoma cell lines by the potent and selective DNA-PK
   inhibitor peposertib. This holds significant importance, considering
   that advanced or metastatic synovial sarcoma presents a challenging
   clinical scenario characterized by limited treatment options and
   suboptimal outcomes [[124]2].

   The results observed in vitro using two widely recognized SS18:SSX
   fusion gene positive synovial sarcoma cell lines indicate not only an
   additive, but a synergistic anti-tumor activity of the Topo II
   inhibitors doxorubicin and etoposide when combined with peposertib
   ([125]Figure 1 and [126]Supplementary Figure S1). Considering possible
   clinical application, the tumor cell-specific synergistic activity
   holds significance within the framework of establishing a therapeutic
   window for potential clinical implementation of this combined treatment
   strategy. At a mechanistic level, we have shown that the primary
   cellular outcome for synovial sarcoma cells treated with the
   combination involves the initiation of apoptosis, likely through a
   p53-mediated pathway. ([127]Figure 2, [128]Figure 3 and [129]Figure 4).
   The pivotal tumor suppressor p53 assumes a crucial function in the
   cellular reaction to DNA damage by triggering cell cycle arrest and/or
   apoptosis. [[130]19,[131]20,[132]21]. Our NanoString gene expression
   analysis and Western blotting experiments reveal p53 hyperactivation
   when doxorubicin and peposertib are administered concurrently, as
   evidenced by Ser15 phosphorylation of p53 and the increase in mRNA and
   protein expression of the cyclin-dependent kinase inhibitor p21, a
   transcriptional target of p53 ([133]Figure 3 and [134]Figure 4).
   Similar induction of the p53 pathway after the treatment with
   peposertib and other DNA damaging agent such as irradiation has been
   reported before [[135]22]. Several other studies showed that DNA DSBs
   induce phosphorylation of p53 at Ser15 by Ataxia-telangiectasia mutated
   (ATM) [[136]23,[137]24]. ATM additionally triggers Chk2 activation,
   leading to the phosphorylation of p53 at Ser20 which, in turn, disrupts
   MDM2 binding and leads to stabilization of p53 [[138]25]. NHEJ is the
   primary cellular pathway to repair DNA DSBs induced by Topo II
   inhibitors. It is noteworthy that it has been previously demonstrated
   that DNA-PK negatively regulates ATM activity, and thus ATM may become
   hyperactivated as a compensatory mechanism in the presence of a
   pharmacological inhibitor of DNA-PK [[139]26]. We hypothesize that ATM
   hyperactivation (indicated by elevated pChk2 levels) and the subsequent
   intensified p53 signaling could mechanistically account for the
   significant induction of apoptosis in the two synovial sarcoma cell
   lines following the simultaneous administration of doxorubicin and
   peposertib ([140]Figure 2). This hypothesis is further supported by the
   results from our gene expression analyses in SYO-1 cells ([141]Figure
   3), which indicated significant transcriptional changes related to
   p53-dependent pathways, DNA damage repair pathways, and cell cycle and
   proliferation pathways exclusively in the combination treatment groups.
   In contrast to numerous other types of tumors, synovial sarcomas seem
   to exhibit a low occurrence of TP53 gene mutations ( < 10%)
   [[142]21,[143]27,[144]28]. This observation suggests that a strategy of
   inducing p53-mediated apoptosis through the proposed combination holds
   merit as a valid approach in treating synovial sarcoma. Although we
   could not find studies specifically addressing the p53-dependent
   antitumor activity of a combination therapy involving a Topo II
   inhibitor and a DNA-PK inhibitor in solid tumor models, our hypothesis
   is supported by research conducted on acute myeloid leukemia (AML)
   cells. In their study, Haines et al. demonstrated that peposertib
   sensitized AML cells with a functional p53 pathway, but not
   p53-deficient cells, to Topo II inhibitors [[145]22]. Although the
   study utilized small panels of p53 functional and dysfunctional cell
   lines instead of isogenic p53-wt/p53-null pairs, distinct responses
   were observed based on the p53 status. Additional studies with larger
   cell line panels, including functional characterization of p53-mediated
   apoptosis induction, are needed to further establish this hypothesis.
   Crucially, the in vitro results were substantiated by significant
   anti-tumor effects from the combined treatment approach when applied to
   mice carrying subcutaneously implanted tumors. ([146]Figure 5 and
   [147]Figure 6). We were able to establish a well-tolerated treatment
   regimen for the combined application using liposomal, but also
   unmodified, doxorubicin ([148]Figure 5A–D). The combination of PLD and
   peposertib exhibited notably enhanced anti-tumor efficacy compared to
   unmodified doxorubicin at equivalent doses. This could lead to an
   improved therapeutic window, a crucial aspect to contemplate if these
   findings are to be translated into a clinical context. The efficacy
   studies conducted in two PDX models of synovial sarcoma, which
   accurately represent human metastatic disease, provide further
   substantiation for the potential of this combination therapy in human
   synovial sarcoma. Particularly noteworthy is the PDX model CTG-1173,
   which originated from a young patient with metastatic disease who
   showed no response to a clinical regimen containing doxorubicin prior
   to tissue extraction for model establishment. Our study confirmed this
   doxorubicin resistance, as evidenced by the lack of significant
   efficacy of PLD monotherapy at the tested dose. However, the
   combination with peposertib successfully surmounted the treatment
   resistance ([149]Figure 6C).

   While advancements in genomic profiling have provided insights into
   potential therapeutic targets in synovial sarcoma, translating these
   discoveries into effective clinical interventions has proven
   challenging. The inadequacy of current treatment approaches is
   highlighted by the limited improvements in overall survival rates with
   existing therapies over the last decades [[150]1,[151]29]. Innovative
   approaches that exploit the unique molecular characteristics of
   synovial sarcoma, such as targeting SS18-SSX fusion gene-related
   pathways, epigenetic modifications, and immunomodulation, offer a
   compelling avenue for therapeutic innovation, but it remains elusive if
   these will demonstrate clinical benefit and result in regulatory
   approvals for this rare indication [[152]30,[153]31]. Thus, doxorubicin
   remains an established primary systemic treatment in a palliative
   context, yielding response rates that span from 16% to 27% along with a
   median survival of around 18 months from the commencement of first-line
   systemic therapy [[154]2]. We and others have previously demonstrated
   that selective pharmacological inhibition of DNA-PK is able to
   synergistically enhance the anti-tumor activity of anthracyclines,
   including doxorubicin, in a large variety of cancer cell lines in vitro
   [[155]14] and xenograft models in vivo. The latter includes breast
   cancer models [[156]32,[157]33], ovarian cancer models
   [[158]34,[159]35] and leiomyosarcoma models [[160]36], indications
   where anthracyclines constitute a crucial component of palliative
   treatment. Moreover, a clinical phase 1/2 study is currently underway
   to assess the safety and efficacy of peposertib in conjunction with PLD
   for patients with ovarian cancer ([161]NCT04092270). Additionally, a
   recent study has been published with the intention of investigating the
   combination of PLD with peposertib in patients with leiomyosarcoma
   ([162]NCT05711615). Our data expands the range of possible tumor
   indications, implying a scenario where the combination therapy could
   potentially provide added benefit for patients with synovial sarcoma.

5. Conclusions

   This study presents compelling evidence that combining Topo II
   inhibitors with the DNA-PK inhibitor, peposertib, can significantly
   enhance their effectiveness in well characterized preclinical models of
   synovial sarcoma. This has important implications for the treatment of
   advanced or metastatic synovial sarcoma, which currently lacks
   effective treatment options.

Acknowledgments