Synthetic lethality from the combination of a histone methyltransferase, SUV39H2 inhibitor and a poly (ADP-ribose) polymerase inhibitor for uterine leiomyosarcoma

Synthetic lethality from the combination of a histone methyltransferase, SUV39H2 inhibitor and a poly (ADP-ribose) polymerase inhibitor for uterine leiomyosarcoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synthetic lethality from the combination of a histone methyltransferase, SUV39H2 inhibitor and a poly (ADP-ribose) polymerase inhibitor for uterine leiomyosarcoma Yusuke Toyohara, Kenbun Sone, Kohei Kumegawa, Yoko Yamamoto, Ryuta Hachijo, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3928088/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Uterine leiomyosarcoma (uLMS) has a poor prognosis owing to its high recurrence rate and resistance to chemotherapy. Therefore, novel therapeutic targets for uLMS need to be discovered. SUV39H2 is a histone methyltransferase that promotes the repair of double-stranded DNA breaks by recruiting phosphorylated H2AX (γH2AX). In this study, we investigated the potential therapeutic targets of SUV39H2 in uLMS and the mechanism of synthetic lethality between PARP inhibitors and SUV39H2 inhibitors, OTS186935. Methods: First, we analyzed the mRNA and protein expression of SUV39H2 in clinical tissues of uLMS, normal myometrium, and leiomyomas using real-time polymerase chain reaction and immunohistochemistry, respectively. Next, we conducted drug sensitivity assays for OTS186935 alone and in combination with olaparib, a poly (ADP-ribose) polymerase inhibitor, using uLMS cell lines, SK-LMS-1 and SK-UT-1. We conducted an annexin assay to investigate the mechanisms of cellular death. We performed Western blotting, immunofluorescence, and chromatin immunoprecipitation sequencing (ChIP-seq) to investigate γH2AX following OTS186935 treatment in addition to in vivo experiments using nude mice with subcutaneously implanted uLMS. Results: SUV39H2 expression was significantly increased in uLMS compared to that in normal myometrium and leiomyomas. OTS186935 decreased cell viability in both cell lines, and its combination with olaparib resulted in synthetic lethality in SK-UT-1 cells (combination index = 0.87). Annexin assay revealed that the combination therapy induced apoptosis. After treatment with OTS186935, γH2AX accumulation decreased. ChIP-seq also showed downregulated γH2AX following OTS186935 treatment. Notably, the combination with OTS186935 and PARP inhibitor was significantly more effective in vivo . Conclusion: OTS186935 inhibits double-stranded DNA break repair as evidenced by γH2AX downregulation through ChIP-seq and other assays. OTS186935 combined with olaparib induces synthetic lethality in patients with uLMS. SUV39H2 uterine leiomyosarcoma γH2AX PARP inhibitor synthetic lethality Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Uterine leiomyosarcoma (uLMS) is a soft tissue sarcoma that arises from the mesenchymal tissue of the uterus [ 1 ]. uLMS is a major subtype of uterine sarcoma and accounts for 63% of cases of uterine sarcomas; this is followed by endometrial stromal sarcoma (21%), adenosarcoma (6%), and other types [ 2 ]. However, uLMS is rare, with an incidence of 0.36–1.7 per 100,000 women [ 1 , 3 , 4 ]. Primary total hysterectomy and bilateral salpingo-oophorectomy are recommended to treat uLMS [ 1 ]. The 5-year survival rates for stages I, II, III, and IV uLMS are 76%, 60%, 45%, and 29%, respectively [ 5 ]. Although advanced uLMS is generally treated with doxorubicin-based chemotherapy [ 6 ], uLMS has a high recurrence rate that ranges from 53–71%, resulting in poor prognosis [ 7 ]. Molecular targeted therapies such as pazopanib, eribulin, and trabectedin have been approved for uLMS [ 8 – 10 ]. However, response rates remain low and have not led to significant improvements in prognosis [ 8 – 10 ]. Recently, poly (ADP-ribose) polymerase (PARP) inhibitors for uLMS have been introduced, and phase II clinical trials have begun [ 11 , 12 ] because 25% of patients with uLMS had homologous recombination repair defects (HRD) [ 13 ]. In patients with HRD, PARP inhibitors can be used because HRD cells rely on alternative repair mechanisms such as non-homologous end joining (NHEJ) and single-strand break repair, which play a role in PARP [ 14 ]. However, the acquisition of resistance to PARP inhibitors has become a concern. Soft tissue sarcomas are broadly classified into simple and complex karyotypes; uLMS is a complex karyotype [ 15 ]. Simple karyotypes have specific chromosomal translocations and point mutations that can be molecular targets for therapy. In contrast, multiple genetic abnormalities, such as TP53 and RB , cause genomic instability and make it difficult to identify molecular therapeutic targets [ 15 , 16 ]. Moreover, uLMS is classified as a cancer with low genomic copy number abnormalities and genomic mutation rates compared with other cancers [ 17 ]. Therefore, novel therapeutic strategies for uLMS are comprised of different aspects in addition to specific genomic targets. Epigenetic studies focus on cancer research to elucidate carcinogenesis, cancer metastasis, and novel therapeutic strategies. Epigenetics reversibly regulates gene expression without altering the DNA sequence. Chromatin remodeling regulates gene expression and is involved in the transcriptional activity and repression of each gene. Epigenetics consists of three main components: DNA methylation, histone modification, and non-coding RNA-associated gene silencing. Lysine, arginine, serine, and threonine modify histone tails, which are responsible for transcriptional activity and repression [ 18 ]. The aberrant regulations of histone modification are thought to contribute to cancer development [ 18 , 19 ]. In 2020, tazemetostat, an histone methyltransferases, EZH2 inhibitor, was approved by the Food and Drug Administration for treatment-resistant epithelioid sarcoma and follicular lymphoma with EZH2 mutations. Therefore, the clinical application of histone methyltransferase inhibitors has attracted attention as a novel therapeutic strategy for cancers. Our group previously investigated histone methyltransferases and showed its efficacy on gynecological cancers [ 20 – 22 ]. Moreover, the efficacy of histone methyltransferases combined with PARP inhibitors has been reported [ 23 , 24 ]. Using EZH2 and PARP inhibitors, Karakashev et al. reported that CARM1 induces a synthetic lethal mechanism in high-grade serous ovarian cancers that highly express CARM1 [ 24 ]. Histone methyltransferase combined with PARP inhibitors enhanced the efficacy of PARP inhibitors and may provide insights to overcome PARP inhibitor resistance. Suppressor license of variegation 3–9 homolog 2 (SUV39H2), which is a histone methyltransferase, is responsible for regulating gene expression by catalyzing the trimethylation of histone H3 lysine 9 (H3K9me3). SUV39H2 is upregulated in normal cells of the cerebellum and testes and is involved in cell cycle regulation and cell differentiation [ 25 ]. When DNA damage occurs, phosphorylated H2AX (γH2AX) is induced, activating double-strand DNA break repair mechanisms. SUV39H2 is reportedly involved in the recruitment γH2AX, and that suppression of SUV39H2 reduces γH2AX activity [ 26 ]. In addition, SUV39H1/2 is required to homologous recombination repair (HRR) and when SUV39H1/2 defect, cells appear to be HRD [ 27 ]. Because of its high expression in multiple cancers [ 28 – 30 ] and its involvement in DNA break repair mechanisms, SUV39H2 has been used as a novel therapeutic target. Specific inhibitors of SUV39H2 have been developed (OTS186935) [ 31 ] and have shown efficacy in in vitro and in vivo experiments on breast cancer. This study aimed to evaluate the efficacy of an SUV39H2 inhibitor alone and in combination with a PARP inhibitor against uLMS. A novel epigenetics-focused target for uLMS was used to identify a novel therapeutic approach. Methods Clinical tissue samples and ethical statement Clinical tissue specimens for gene expression analysis and immunohistochemical staining (IHC) were obtained during gynecological surgeries performed at the University of Tokyo Hospital. For gene expression analysis, specimens were collected in 5–10-mm squares, flash-frozen in liquid nitrogen, and stored in a − 80°C freezer until use for mRNA extraction. Specimens used for IHC were 4-µm thick slices of formalin-fixed paraffin-embedded (FFPE) specimens. Cell lines SK-LMS-1 (ATCC, Manassas, VA, USA; Cat. # HTB-88) and SK-UT-1 (Cat. # HTB-114) were used as uLMS cell lines and were cultivated in Eagle’s minimum essential medium (E-MEM, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, MA, USA) and 1% penicillin/streptomycin (FUJIFILM Wako Pure Chemical Corporation) in an incubator at 37°C with 5% of CO 2 . The cell lines were subjected to FASMAC Short Tandem Repeat analysis for cell authentication. The MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland) was used to confirm that the cells were mycoplasma-free. Total RNA extraction and mRNA expression analysis of clinical tissue specimens and cell lines Clinical specimens and RLT buffer with 1% 2-mercaptoethanol (FUJIFILM Wako Pure Chemical Corporation) were mixed in MagNA Lyser Green Beads (Roche Diagnostics, Indianapolis, IN, USA) using a MagNA Lyser (Roche, Basel, Switzerland) (6,500 rpm for 70 s) followed by centrifugation (14,000 rpm for 3 min). The supernatant was collected, and total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Total RNA was extracted from the cell lines using the RNeasy Mini Kit (Qiagen). Complementary DNA was synthesized by reverse transcription using ReverTra Ace (Toyobo, Osaka, Japan). Real-time quantitative polymerase chain reaction (RT-qPCR) was performed on a QuantStudio 1 Real-Time PCR System (Thermo Fisher Scientific) using KOD SYBR qPCR Mix (Toyobo). mRNA expression levels of tissue samples and cell lines were confirmed with normalized by 2 −ΔΔCT method. ACTB was evaluated as the internal control. Table S1 shows the primer sequences. IHC After deparaffinization and antigen activation by heat (95°C, 20 min), anti-SUV39H2 antibody (Abcam, Cambridge, UK; Cat. # ab190870; 1:200) was added to the slides and incubated overnight at 4°C. Secondary antibody reactions and nuclear staining were performed using hematoxylin. Each specimen was evaluated using the IHC score, which is the sum of the proportion score (PS) and intensity score (IS). PS was calculated as the proportion of positive cells as follows: 0 = 0%; 1 = 67%. IS was graded as 0 (negative), 1 (weak), 2 (intermediate), and 3 (strong). Two raters independently evaluated the IHC scores, and the mean was calculated for each IHC score. Drug sensitivity assay Tazemetostat/EPZ-6438 (MedChemExpress, NJ, USA), an EZH2 inhibitor, OTS186935 (MedChemExpress), and olaparib (MedChemExpress), a PARP inhibitor, were used in this study. Each inhibitor was dissolved in 0.1% of dimethyl sulfoxide DMSO (Sigma-Aldrich). For the drug sensitivity assay, SK-LMS-1 and SK-UT-1 cells were pre-cultured in 96- or 48-well plates on the day before adding the inhibitor. On the next day (day 0), the medium was replaced with new medium diluted to the target concentration of the inhibitors. After 72 h (day 3) or 96 h (day 4) of incubation, cell viability was evaluated using cell counting kit-8 (DOJINDO, Kumamoto, Japan), while absorbance was measured with a microplate reader (BioTek, VT, USA). Three independent experiments were performed. The wells incubated with 0.1% DMSO were used as controls, and the ratio of absorbance at the desired concentration was calculated, with the absorbance of the control as 100%. The combination index (CI) was employed [ 32 ]. The CI, which was the half-maximal inhibitory concentration (IC50) of two drugs combined versus the IC50 of a single drug, was calculated as follows: CI = (D1)/(Dx)1 + (D2)/(Dx) 2 (D1) and (D2) = IC50 concentrations of drug 1 and drug 2, respectively, when combined (Dx) 1, (Dx) 2 = concentration of the respective IC50 of drug 1 and drug 2 as single agents The obtained CIs were classified as synergism (CI 1). Colony formation assay Colony formation assay was performed to verify the effects of inhibitors on long-term cultures. Six-well plates were pre-cultured with SK-LMS-1 and SK-UT-1 cells on the day before day 0. On day 0, the medium was replaced with new medium diluted to the target concentration of the inhibitors. The medium was replaced every 3 days, and the cells were fixed and stained on days 10–12. The number of colonies, defined as > 50 cells, was counted under a microscope. Three wells were analyzed for each concentration. Western blotting For protein extraction, the cell pellet was suspended with sodium dodecyl sulfate (SDS) lysis buffer (0.1M Tris-HCl pH 7.5, 10% Glycerol [FUJIFILM Wako], 2% SDS[FUJIFILM Wako Pure Chemical Corporation]), and the supernatant was collected by centrifugation (15,000 rpm, 10 min, 4°C) after heating at 95℃ for 5 min. Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad, Hercules, CA, USA) were used for SDS-PAGE, and the separated samples were transferred via a semi-dry transfer method using Trans-Blot Turbo Mini PVDF Transfer Packs (Bio-Rad). Amersham ECL Select (Cytiva, MA, USA) or SuperSignalTM West Femto (Thermo Fisher Scientific) were used as chemiluminescent reagents, and detection was performed using ImageQuant LAS 4000 (GE Healthcare Life Sciences, NJ, USA). Antibodies listed in Table S2 were used for primary antibody reactions. All reactions were performed overnight at 4°C. Secondary antibody reactions were performed using anti-mouse IgG HRP-linked antibody (Cell Signaling Technology, MA, USA; Cat. #7076, 1:3000) and anti-rabbit IgG horseradish peroxidase-linked antibodies (Cell Signaling Technology; Cat. #7074,1:3000). Cell cycle analysis For cell cycle analysis, cells were fixed using 70% ethanol after incubation with the target concentrations of the inhibitors. Following nucleic acid staining with propidium iodide (Sigma-Aldrich), the fluorescence intensity of the cells was determined using a BD FACSCalibur HG Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Cell cycle analysis was performed using the FlowJo software version 16 (FlowJo LLC, OR, USA) with default settings for automatic analysis. Three independent experiments were performed. Annexin assay After incubation with the target concentrations of the inhibitors, the centrifuged cell pellets were assayed using the FITC Annexin V Apoptosis Detection Kit II (BD Biosciences) according to the manufacturer’s instructions. Detection was performed using a BD FACSCalibur HG Flow Cytometer (BD Biosciences), and analysis to classify cells into early and late apoptosis was conducted using FlowJo software version 16 (FlowJo LLC). The percentages of early and late apoptotic cell populations were summed to calculate the percentage of apoptotic cells. All experiments were performed in triplicate under all conditions. Cell immunofluorescence staining Doxorubicin (Cayman Chemical Company) was used to induce DNA damage. uLMS cell lines were incubated on MICROCOVER GLASS (Matsunami Glass Ind., Ltd., Osaka, Japan) on 35-mm plates with the target concentrations of inhibitors until the targeted time, and fixation was performed with 4% paraformaldehyde phosphate buffer solution (FUJIFILM Wako Pure Chemical Corporation). After permeabilization with 0.05% Tween20 (Sigma-Aldrich) for 5 min twice and 0.5% TritonX-100 (Sigma-Aldrich) for 10 min, the cells were incubated in 3% bovine serum albumin (BSA, FUJIFILM Wako Pure Chemical Corporation) for 60 min at 25℃. Primary antibodies were diluted in 3% BSA and incubated overnight at 4°C in a wet box. Anti-phospho-histone H2A.X (Ser139) antibody (Cat. # JBW301, Lot# 3761799, 1:1000) was used. After cell permeabilization, cells were reacted with secondary antibody and 4',6-diamidino-2-phenylindole for nuclear staining for 60 min at 25°C. Goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody (Alexa Fluor 568, Thermo Fisher Scientific; Cat. # A11011, 1:1000) and donkey anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody (Alexa Fluor 488, Thermo Fisher Scientific; Cat. # A-21202, 1:1,000) were used. After washing the cells on the glass, the cover glass was encapsulated and observed under a confocal fluorescence microscope (LSM880; Carl Zeiss Co., Ltd., Oberkochen, Germany). Imaging was performed using ImageJ 1.53. Then, relative intensity of γH2AX of each cell was estimated by the default setting of ImageJ. RNA sequencing (RNA-seq) Total RNA was extracted using the abovementioned method and used for library preparation through the SMARTer Stranded Total RNA Sample Prep Kit (TaKaRa Bio, Tokyo, Japan). The library was prepared using an RNA Sample Prep Kit (TaKaRa Bio). Libraries were sequenced using a NextSeq 550 (Illumina, San Diego, CA, USA) with paired-end reads. Chromatin immunoprecipitation (ChIP) sequencing After culture, 5 × 10 6 uLMS cells were collected using 0.25% trypsin-EDTA (FUJIFILM Wako Pure Chemical Corporation). Cell pellets were cross-linked with 1% formaldehyde solution (FUJIFILM Wako Pure Chemical Corporation) for 10 min at 37°C and discontinued with 0.125 M glycine (FUJIFILM Wako Pure Chemical Corporation) for 5 min at 25°C. After lysing the cells in lysis buffer (Table S3 ), DNA fragmentation was performed by sonicating with a Covaris S220 (Covaris, Woburn, MA, USA) for 200 cycles for 10 min. The fragmented DNA underwent ChIP and incubated overnight at 4℃ with Dynabeads Protein G (Thermo Fisher Scientific) with 2.5 µg of anti-gamma H2A.X (phospho S139) antibody (Abcam, Cat. #ab2893, Lot:GR3446449-2). Next, we washed the beads with low salt immune complex wash buffer, high salt immune complex wash buffer, and LiCl immune complex wash buffer (Table S3 ) at 4°C for 5 min followed by washing with TE buffer (Table S3 ) for 5 min at 25°C. The fragments were extracted using an elution buffer (Table S3 ). Libraries were prepared using ThruPLEX DNA-Seq Kit (TaKaRa Bio). Sequencing was performed by paired-end reads using NextSeq 550 using the same method as that used for RNA-seq. Sequencing Data Analysis For RNA-seq analysis, sequence reads were trimmed to extract adaptor sequences using Skewer (v0.2.2) [ 33 ] and mapped to the hg38 genome using spliced transcript alignment to a reference (v.2.7.8a) [ 34 ]. Mapped reads were counted using feature Counts (v.2.0.10) [ 35 ]. Integrative Genomic Viewer (v2.11.1) [ 36 ] was used to visualize the mapped reads. In the chromatin immunoprecipitation sequencing (ChIP-seq) analysis, raw reads were aligned to hg38 using bowtie2 (v.2.4.2) [ 37 ]. Multi-aligned reads and PCR copies were eliminated using Picard (v.2.25.3; http://broadinstitute.github.io/picard/ ). Bedtools (v2.30.0) [ 38 ] was used to filter reads that overlapped with the blacklist of the Encyclopedia of DNA Elements (ENCODE). MACS2 (v 2.2.7.1) [ 39 ] was used to call peaks with the parameter ‘--keep-dup auto -q 0.1’. Ngsplot was used to visualize peak signals within a range of ± 1,000 bp around the γH2AX peaks in the doxorubicin-treated cells [ 40 ]. In vivo experiments Animal experiments were performed in accordance with the ARRIVE guidelines 2.0 and approved by the Animal Experiment Committee of the Graduate School of Medicine, University of Tokyo (Approval No.-H20-243). We used 5-week-old immunodeficient BALB/cAJcl-nu/nu mice purchased from CLEA Japan, Inc. Mice were maintained on a 12-h day/night cycle and kept in a clean environment with adequate food and water. Cages were maintained with wooden shavings with fewer than five mice per cage. SK-UT-1 was seeded (diluted with 50 uL of Matrigel [Corning, NY, USA] and 100 uL of E-MEM [FUJIFILM Wako Pure Chemical Corporation]) at 5x10 6 cells each in the dorsal subcutaneous tissue of nude mice under inhalation anesthesia sedation. Tumor volume was calculated using the following formula: tumor volume = (tumor short diameter) 2 × (tumor long diameter)/2. Treatment was initiated when the tumor diameter reached 5 mm. Drugs were administered intraperitoneally as follows: DMSO for the control group (DMSO group), OTS186935 10 mg/kg for the OTS186935 group, olaparib 50 mg/kg for the olaparib group, and OTS186935 10 mg/kg + olaparib 50 mg/kg for the combination group. Six mice were used in each group. Drugs were dissolved in 10% DMSO and PEG300 in 200 µL of saline. Tumor measurements were performed once every 2–3 days and finally evaluated on day 14 after drug administration. Statistical analysis For statistical analyses, each experiment was independently performed at least three times, and data were calculated as the mean ± standard deviation (SD). Student’s t-test was used to analyze data. Differences were considered statistically significant at P < 0.05. A one-sided test was used for the mRNA expression analysis of clinical samples, and a two-sided test was used for other samples. All statistical analyses were performed using Microsoft Excel and R (version 4.3.1). Results Expression analysis in clinical tissue samples and drug sensitivity assays mRNA expression levels of 11 histone methyltransferases were measured by RT-qPCR in clinical tissue samples from normal uterine myometrium (n = 4), uterine leiomyomas (n = 4), and uLMS (n = 6) (Table S4 ). Significant upregulation of EZH2 , SUV39H2 , and WHSC1 expression was observed in uLMS compared with normal uterine myometrium and uterine leiomyomas (Figs. 1 A, 1 B). Among the three histone methyltransferases, selective inhibitors of SUV39H2 and EZH2 were available. OTS186935 (MedChemExpress) and EPZ-6438 (tazemetostat, MedChemExpress) were analyzed for their efficacy against the uLMS cell lines SK-LMS-1 and SK-UT-1. EPZ-6438 failed to show efficacy against SK-LMS-1 and SK-UT-1, with an IC50 of 45.1 µM and 60.1 µM, respectively (Fig. S1 A). Meanwhile, OTS186935 showed drug sensitivity at relatively low concentrations, with an IC50 of 1.16 µM and 1.04 µM in SK-LMS-1 and SK-UT-1, respectively (Fig. 1 C). The expression levels of SUV39H2 in clinical tissue specimens were evaluated using IHC. As shown in Table S5 , 21 cases of uLMS, 5 cases of normal uterine myometrium, and 5 cases of uterine leiomyomas were evaluated. As shown in Fig. 1 D, the intensity score was classified as negative, weak, intermediate, and strong. uLMS showed a significant increase in the IHC score compared to normal uterine myometrium and uterine leiomyomas (Fig. 1 E and Fig. S1 B). Combination therapy with OTS186935 and olaparib To determine the mechanism of action of OTS186935, we performed Western blotting to detect the reaction of H3K9me3, which is catalyzed by SUV39H2 [ 31 ]. The results showed that OTS186935 decreased H3K9me3 expression (Fig. 2 A), indicating that OTS186935 inhibited SUV39H2. Next, we examined the combined effects of OTS186935 and olaparib on uLMS cell lines. As revealed by the cell viability assay, the CI was 0.99 for SK-LMS-1 and 0.87 for SK-UT-1 (Fig. 2 B and Fig. S2 ). The CI of SK-LMS-1 showed an additive effect, whereas that of SK-UT-1 showed a synergistic effect. To assess the drug sensitivity of the combination therapy in long-term cultures, a colony formation assay was performed (Fig. 2 C). SK-UT-1 showed a synergistic effect with the drug combination compared to SK-LMS-1, similar to the results of the cell viability assay. Elucidation of the synergistic effect in SK-UT-1 We investigated the mechanisms underlying the synergistic effect in SK-UT-1 compared with that in SK-LMS-1 and the cell cycle analysis results. After treatment with OTS186935 alone, the G2/M phase was significantly increased in both SK-LMS-1 and SK-UT-1 (G2/M phase in DMSO vs. OT186935, 17.2% vs. 27.4% in SK-LMS-1, p = 0.023; 33.4% vs. 40.9% in SK-UT-1, p = 0.008). However, when olaparib was added, there was no significant change in the cell cycle in either cell line (Fig. 3 A). Next, we evaluated the induction of apoptosis using an annexin assay; there was no significant increase in the percentage of apoptotic cells in SK-LMS-1 after combination therapy (Fig. S3 ). However, a significant increase in the percentage of apoptotic cells was observed when OTS186935 was combined with olaparib (Fig. 3 B). To confirm the induction of apoptosis by the combination therapy, Western blotting was performed to detect the accumulation of cleaved PARP. Similar to the results of the annexin assay, apoptosis was observed in SK-UT-1 cells after combination, which was not observed in SK-LMS-1 cells (Fig. 3 C). We hypothesized that the synergistic effects of OTS186935 and olaparib on SK-UT-1 cells may be related to factors involved in DNA damage/repair mechanisms. We investigated the DepMap Portal ( https://depmap.org/portal/ , accessed on 3/2/2022), which is a public database of cell lines, and found a deletion in 53BP1, which is a factor involved in double-stranded DNA break repair, in SK-UT-1 cells. Deletion of 53BP1 in SK-UT-1 cells caused a frameshift mutation, whereas that in SK-LMS-1 cells did not result in abnormalities. We actually conducted RNA-seq of SK-UT-1 cells, revealing p.T1060PfsTer36 of TP53BP1 (Fig. S4 ). Mechanism of OTS186935 on the DNA break repair pathway Next, we investigated the effects of OTS186935 on DNA repair. We used doxorubicin to induce DNA breaks in uLMS cells and investigated the reaction after treatment with OTS186935. We added 0.1% DMSO for negative control, 1 µM of OTS186935, 0.5 µM of doxorubicin, and OTS186935 1 µM + doxorubicin 0.5 µM to each of the uLMS cell lines. Doxorubicin treatment resulted in γH2AX accumulation, whereas the combination with OTS186935 suppressed γH2AX expression (Fig. 4 A). To confirm the suppression of γH2AX accumulation in the nucleus, cell immunofluorescence staining results were compared between 0.5 µM of doxorubicin and the addition of 1 µM of OTS186935. The results showed that OTS186935 suppressed γH2AX accumulation in the nucleus (Fig. 4 B). Moreover, ChIP-seq was performed on SK-UT-1 cells to further investigate the effects of OTS186935 on γH2AX. We performed ChIP-seq for the four conditions: 0.1% DMSO for negative control, 1 µM of OTS186935, 0.5 µM of Doxorubicin, and the combination of OTS186935 1 µM + doxorubicin 0.5 µM. Samples were then immunoprecipitated with anti-γH2AX antibody. The results showed that the addition of OTS186935 decreased the γH2AX signal, indicating that a decrease in the accumulation of γH2AX on the genome occurred and suggesting that OTS186935 inhibited double-strand DNA break repair (Figs. 4 C and 4 D). Effects of the combination of OTS186935 and Olaparib in in vivo experiments As shown in Figs. 5 A and B, the OTS186935 and combination groups showed a significant decrease in tumor volume compared to the DMSO group (p = 0.012). The decrease in tumor volume in the combination group was greater than that in the OTS186935 group (p = 0.59). Discussion In this study, SUV39H2 expression is increased in uLMS, suggesting that it may be involved in carcinogenesis, and that SUV39H2 inhibitors may be a novel therapeutic strategy for uLMS. We also demonstrated that the effects of the combination of OTS186935 and olaparib in uLMS cell lines and demonstrated its effectiveness in a 53BP1-deficient uLMS cell line. OTS186935 alone induced cell cycle arrest, whereas the addition of olaparib induced apoptosis and cell cycle arrest in 53BP1-deficient uLMS cells. OTS186935 also decreased the accumulation of γH2AX, suggesting that OTS186935 inhibited double-strand DNA repair. The prognosis of uLMS is poor owing to resistance to existing chemotherapies, hence the requirement of novel therapeutic strategies. We focused on a histone methyltransferase (SUV39H2) because it is related to carcinogenesis in various types of cancers [ 28 – 30 ]. In a previous report that investigated the relationship between EZH2 and melanoma and used 53 types of cell lines, sensitive cell lines for a histone methyltransferase inhibitor were defined as < 15 µM of IC50 [ 41 ]. The results of the drug sensitivity assay indicated that OTS186935 showed relatively high sensitivity to uLMS cell lines (Fig. 1 C). Additionally, in vivo experiments revealed the efficacy of OTS186935 in uLMS (Figs. 5 A and 5 B). Overall, these results suggest that OTS186935 is a novel therapeutic target for uLMS. Recently, PARP inhibitors have become increasingly important for the treatment of gynecological tumors with HRD as 25% of the patients with uLMS have HRD [ 13 ]. Case reports have used PARP inhibitors for uLMS, and phase II clinical trials of PARP inhibitors for uLMS have begun [ 11 , 12 , 42 ]. Although PARP inhibitors have become widely used, the acquisition of resistance to PARP inhibitors has become a critical issue in cancer treatment. HRR, wherein BRCA1/2 is mainly involved, and non-homologous end-joining (NHEJ), wherein 53BP1 is involved, act exclusively of each other. Therefore, in HRD, 53BP1-based NHEJ can be dominant, whereas when the mutation of NHEJ-related factors, such as 53BP1 , RIF1 , and REV7 are present, NHEJ dominance can be lost; this is one of the mechanisms by which HRR is activated even in BRCA mutations and how resistance to PARP inhibitors is acquired [ 14 ]. Strategies to resolve this resistance to PARP inhibitors are required, and combination therapies are being investigated. A phase II trial of temozolomide and olaparib combination therapy for uLMS has been conducted [ 43 ]. In other types of cancer, our group previously reported that the combination of LLY-507, an SMYD2 inhibitor, and olaparib showed additive effects in high-grade serous ovarian carcinoma, demonstrating the usefulness of the combination of a histone methyltransferase inhibitor and a PARP inhibitor [ 24 ]. Additionally, Karakashev et al. reported that in high-grade serous ovarian carcinoma with high CARM1 expression, EZH2 represses the MAD2L2 promoter, which promotes NEHJ, by histone methylation (H3K27me3). EZH2 inhibition induces NHEJ dominance, resulting in a synthetic lethal mechanism involving a PARP inhibitor [ 23 ]. Our results demonstrated the synthetic lethality of OTS186935 combined with olaparib in a 53BP1-deficient uLMS cell line. Although in vivo experiments did not show a significant difference between the OTS186935 and combination groups, the combination therapy tended to be superior to OTS186935 alone. The short duration of administration was assumed to be one reason for the lack of statistically significant differences. SUV39H2 is associated with DNA damage-induced γH2AX, and it was reported that suppression of SUV39H2 reduces the accumulation of γH2AX [ 26 ]. In addition, SUV39H1/2 is required to homologous recombination repair (HRR) and when SUV39H1/2 defect, cells appear to be HRD [ 27 ]. The hypothesis is that OTS186935 inhibits the double-strand DNA repair mechanism, which is represented by inhabitation of accumulation of γH2AX, while olaparib acts as an inhibitor of the single-strand repair mechanism. Furthermore, OTS186935 may exert inhibitory effects on HRR in cell lines wherein HRR is activated by the 53BP1 mutation because SUV39H2 is a key factor in HRR, thus inducing a synergistic effect (Fig. 6 ). This is the first study that focused on the therapeutic effect of an SUV39H2 inhibitor on uLMS as well as the first report that described the effect of an SUV39H2 inhibitor combined with a PARP inhibitor. This study had some limitations. Although we focused on 53BP1 for the differences between the two cell lines, other genes may have been involved in the synergistic effect; further validation is required to confirm the above mechanisms. Additionally, we observed a decrease in H3K9me3 levels after treatment with OTS186935; however, we did not observe a relationship between H3K9me3 and synthetic lethality. Conclusion OTS186935 showed efficacy on uLMS cell lines in vitro and in vivo . Combination therapy with olaparib resulted in synthetic lethality in 53BP1-deficient cell lines. We considered inhabitation of double-stranded DNA break repair by OTS186935, which suppressed γH2AX accumulation and induced synthetic lethality when combined with olaparib. Abbreviations DSB double-strand break ChIP-seq chromatin immunoprecipitation sequencing HRR homologous recombination repair HRD homologous recombination repair defects IHC immunohistochemical staining IS intensity score NHEJ non-homologous end joining PARP poly (ADP-ribose) polymerase γH2AX phosphorylated H2AX RT-qPCR real-time quantitative polymerase chain reaction PS proportion score RNA-seq RNA sequencing SD Standard division SSB single-strand break uLMS uterine leiomyosarcoma Declarations Ethics approval and consent to participate All patients were diagnosed by pathologists (certified as specialists by the Japanese Society of Pathology) at the University of Tokyo Hospital. This study was approved by The Human Genome, Gene Analysis Research Ethics Committee of the University of Tokyo and written informed consents were obtained from each patient (G0683-(26)). Consent for publication Not applicable Competing interests The authors declare that they have no competing interests Funding This study is partially supported by BRIDGE Author Contribution Yusuke Toyohara: Data curation, investigation, formal analyses, methodology, visualization, writing (original draft), and writing (review and editing). Kenbun Sone: conceptualization, data curation, funding acquisition, methodology, project administration, writing (original draft) and writing (review and editing). Kohei Kumegawa: Data curation, formal analysis, investigation, and methodology. Yoko Yamamoto: investigation, methodology, and writing (review and editing). Ryuta Hachijo: investigation, methodology, and writing (review and editing). Saki Tanimoto: Data curation, investigation, methodology, and writing (review and editing). Futaba Inoue: data curation, investigation, methodology, and writing (review and editing). Asako Kukita: Data curation, investigation, methodology, and writing (review and editing). Asympti Taguchi: data curation, investigation, methodology, and writing (review and editing); Masako Ikemura: investigation and writing (review and editing). Yuichiro Miyamoto: Supervision and writing (review and editing). Michihiro Tanikawa: Methodology, supervision, and writing (review and editing). Takayuki Iriyama: Supervision and writing (review and editing). Mayuyo Mori-Uchino: Supervision and writing (review and editing). Ryuji Hamamoto: Supervision and writing (review and editing). Tetsuo Ushiku: Supervision and writing (review and editing). Katsuhiko Oda: Methodology, supervision, and writing (review and editing). Yasushi Hirota: supervision and writing (review and editing). Reo Maruyama: conceptualization, methodology, supervision, and writing (review and editing). Yutaka Osuga: Supervision and writing (review and editing). Acknowledgements The authors are grateful to Dr. Liying Yang (Project for Cancer Epigenomics, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan) for helping our sequencing analysis. The authors would like to thank Editage for the English language review. Availability of data and materials All data generated or analyzed in this study are included in the published article [and its supplementary information files] References Desar IME, Ottevanger PB, Benson C, van der Graaf WTA. Systemic treatment in adult uterine sarcomas. Crit Rev Oncol Hematol. 2018;122:10–20. https://doi.org/10.1016/j.critrevonc.2017.12.009 . Tropé CG, Abeler VM, Kristensen GB. Diagnosis and treatment of sarcoma of the uterus. A review. Acta Oncol. 2012;51:694–705. https://doi.org/10.3109/0284186X.2012.689111 . Van den Bosch T, Coosemans A, Morina M, Timmerman D, Amant F. Screening for uterine tumours. 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EZH2 Cooperates with DNA Methylation to Downregulate Key Tumor Suppressors and IFN Gene Signatures in Melanoma. J Invest Dermatol. 2020;140:2442–2454e5. Seligson ND, Kautto EA, Passen EN, Stets C, Toland AE, Millis SZ, et al. BRCA1/2 functional loss defines a targetable subset in leiomyosarcoma. Oncologist. 2019;24:973–9. https://doi.org/10.1634/theoncologist.2018-0448 . Ingham M, Allred JB, Chen L, Das B, Kochupurakkal B, Gano K, et al. Phase II study of olaparib and temozolomide for advanced uterine leiomyosarcoma (NCI protocol 10250). J Clin Oncol. 2023;41:4154–63. https://doi.org/10.1200/JCO.23.00402 . Alagoz M, Katsuki Y, Ogiwara H, Ogi T, Shibata A, Kakarougkas A, et al. SETDB1, HP1 and SUV39 promote repositioning of 53BP1 to extend resection during homologous recombination in G2 cells. Nucleic Acids Res. 2015;43:7931–44. https://doi.org/10.1093/nar/gkv722 . Additional Declarations No competing interests reported. Supplementary Files FigureS1.tif Fig. S1A. Drug sensitivity assays for EPZ-6438. SK-LMS-1 and SK-UT-1 cell lines were treated by Tazemetostat (1–100 µM) for 96 h. FigureS1B.tif Fig. S1B. Images of immunohistochemical staining results for SUV39H2 in clinical samples. Yellow bar indicates 50 µm. FigureS2.tif Fig. S2. Cell viability assay for OTS186935 + olaparib. This figure shows the same results with Fig. 2B. FigureS3.tif Fig. S3. Annexin assay of SK-LMS-1 treated with 0.1% DMSO, 0.5 µM OTS186935, 1 µM olaparib, and 0.5 µM OTS186935 + 1 µM olaparib for 48 h. Proportion of apoptotic cells were calculated as the sum of Q2 and Q3. * Significant difference was analyzed by Student’s t- test, two-sided. Asterisks indicate p-values < 0.05. Error bars indicate means ± SD. SD, standard deviation. FigureS4.tif Fig. S4. RNA-seq of SK-UT-1 showed a p.T1060PfsTer36 of TP53BP1 (red rectangle). 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15:47:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3928088/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3928088/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50814290,"identity":"0b64afc1-f22a-4e2c-871d-3cd8f7c02262","added_by":"auto","created_at":"2024-02-07 19:37:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":253192,"visible":true,"origin":"","legend":"\u003cp\u003eExpression analysis in clinical tissue samples and drug sensitivity assays\u003c/p\u003e\n\u003cp\u003e(A) RT-qPCR of 11 types of histone methyltransferases in uLMS, uterine normal myometrium, and uterine leiomyomas. mRNA expression levels were normalized by 2\u003csup\u003e−ΔΔCT\u003c/sup\u003e method, and relative expressions are shown. Red represents high expression in the heatmap. (B) Three genes that were overexpressed in uLMS are compared with those in uterine normal myometrium and uterine leiomyomas (p values were in SUV39H2, in EZH2, and in WHSC1). Significant difference was analyzed by Student’s \u003cem\u003et-\u003c/em\u003etest, one-sided. Asterisks indicate p-values \u0026lt; 0.05. (C) Drug sensitivity assays for OTS186935. SK-LMS-1, and SK-UT-1 after treatment with OTS186935 (0.1–10 µM) for 96 h. Error bars indicate means ± SD. (D) IHC of SUV39H2 in clinical samples. Representative images of the intensity score, which was defined as score 0 = negative; 1 = weak; 2 = intermediate; and 3 = strong. The yellow bar indicates 50 µm. (E) IHC score of SUV39H2 in uLMS, uterine normal myometrium, and uterine leiomyomas. Significant difference was analyzed by Student’s \u003cem\u003et-\u003c/em\u003etest, two-sided. Asterisks indicate p-values \u0026lt; 0.05. RT-qPCR, real-time quantitative polymerase chain reaction; IHC, immunohistochemical staining; SD, standard division.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3928088/v1/9159f3009a0b0c7e931e24ad.png"},{"id":50814289,"identity":"6ec7bb25-b35e-4754-adba-5add1ecf2400","added_by":"auto","created_at":"2024-02-07 19:37:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":204021,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of OTS186935 combined with olaparib\u003c/p\u003e\n\u003cp\u003e(A) Inhibition of SUV39H2 decreased H3K9me3 expression in both SK-LMS-1 and SK-UT-1 cell lines after treatment with 0.5–1 µM of OTS186935 or 0.1% of DMSO for 48 h. (B) Cell viability assay for OTS186935 combined with olaparib. Cell viability expressed as a heatmap. Combination index of SK-UT-1 and SK-LMS-1 were 0.99 and 0.87, respectively. (C) Colony formation assay for the combination of OTS186935 and olaparib. Cell lines were treated with 0.1–0.5 µM of OTS186935, 1 µM of olaparib, or 0.1% of DMSO for 12 days. Significant difference was analyzed by Student’s \u003cem\u003et-\u003c/em\u003etest, two-sided. Asterisks indicate p-values \u0026lt; 0.05. Error bars indicate means ± SD. CI, combination index; SD, standard division.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3928088/v1/eb4c8e36bab4cf12c5f67592.png"},{"id":50814292,"identity":"fe688074-0e41-4fcd-9143-af0095282fe8","added_by":"auto","created_at":"2024-02-07 19:37:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":145928,"visible":true,"origin":"","legend":"\u003cp\u003eElucidation of the mechanism of the synergistic effect in SK-UT-1\u003c/p\u003e\n\u003cp\u003e(A) Cell cycle assay of SK-LMS-1 and SK-UT-1 after treatment with 0.1% DMSO, 0.5 µM OTS186935, 1 µM olaparib, and 0.5 µM OTS186935 + 1 µM olaparib for 48 h. Error bars indicate means ±SD. (B) Annexin assay of SK-UT-1 treated with 0.1% DMSO, 0.5 µM OTS186935, 1 µM olaparib, and 0.5 µM OTS186935 + 1 µM olaparib for 48 h. Proportion of apoptotic cells were calculated as the sum of Q2 and Q3. Significant difference was analyzed by Student’s \u003cem\u003et-\u003c/em\u003etest, two-sided. Asterisks indicate p-values \u0026lt; 0.05. Error bars indicate means ± SD. (C) Western blotting of SK-LMS-1 and SK-UT-1 treated with 0.1% DMSO, 0.5 µM OTS186935, 1 µM olaparib, and 0.5 µM OTS186935 + 1 µM olaparib for 48 h. SD, standard deviation.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3928088/v1/451817d1ddba3c4b247ed77c.png"},{"id":50814295,"identity":"4181d4b8-d7e3-4b62-ac41-6d8c6da17205","added_by":"auto","created_at":"2024-02-07 19:37:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":201046,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism of OTS186935 on the DNA break repair pathway\u003c/p\u003e\n\u003cp\u003e(A) Western blotting of SK-LMS-1 and SK-UT-1 treated with 0.1% DMSO as negative control, 1 µM OTS186935, 0.5 µM doxorubicin for DNA damage induction, and 1 µM OTS186935 + 0.5 µM doxorubicin for 1 h. (B) Cell immunofluorescence staining of SK-LMS-1 and SK-UT-1 treated with 0.5 µM doxorubicin for DNA damage induction 1 µM OTS186935 + 0.5 µM doxorubicin for 1 h. Relative intensity of γH2AX in each cell was estimated with ImageJ. Significant difference was analyzed by Student’s \u003cem\u003et-\u003c/em\u003etest, two-sided. Asterisks indicate p-values \u0026lt; 0.05. Error bars indicate means ± SD. Yellow bars indicate 10 µm in images. (C) ChIP-seq for SK-UT-1 treated with 0.1% DMSO as negative control, 1 µM OTS186935, 0.5 µM doxorubicin for DNA damage induction, and 1 µM OTS186935 + 0.5 µM doxorubicin for 1 h. (C) Average γH2AX-ChIP-seqsignal in SK-UT-1 cells treated with 0.1% DMSO as negative control, 1 µM OTS186935, 0.5 µM doxorubicin for DNA damage induction, and 1 µM OTS186935 + 0.5 µM doxorubicin for 1 h. (Peak center +/- 1 kb) (D) Heatmaps of γH2AX-ChIP-seq in SK-UT-1 cells treated with 0.1% DMSO as negative control, 1 µM OTS186935, 0.5 µM doxorubicin for DNA damage induction, and 1 µM OTS186935 + 0.5 µM doxorubicin for 1 h. Heatmaps are sorted by γH2AX-ChIP-seq signal in descending order (Peak center +/- 1 kb). SD, standard deviation; ChIP-seq, chromatin immunoprecipitation sequencing; γH2AX, phosphorylated H2AX.\u003c/p\u003e","description":"","filename":"FIgure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3928088/v1/99371543e6eb6254e6bfccdc.png"},{"id":50814299,"identity":"4493fbfb-0392-49fc-aee3-5899774c90eb","added_by":"auto","created_at":"2024-02-07 19:37:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":217776,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of OTS186935 + olaparib \u003cem\u003ein vivo\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e(A) Mice bearing SK-UT-1 cells (n = 6 per group) were intraperitoneally treated once daily with DMSO as the control group (the DMSO group), OTS186935 10 mg/kg body weight as the OTS186935 group, olaparib 50 mg/kg as the olaparib group, and OTS186935 10 mg/kg + olaparib 50 mg/kg as the combination group for 14 days. Significant difference was analyzed by Student’s \u003cem\u003et-\u003c/em\u003etest, two-sided. Asterisks indicate p-values \u0026lt; 0.05. Error bars indicate means ± SD. (B) Images of tumors treated as above. SD, standard deviation.\u003c/p\u003e","description":"","filename":"FIgure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3928088/v1/8605588893f6c7a691034e13.png"},{"id":50814297,"identity":"edb505d4-7c0c-4b71-9f4a-55b11c29473c","added_by":"auto","created_at":"2024-02-07 19:37:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":57962,"visible":true,"origin":"","legend":"\u003cp\u003eHypothesis of mechanisms on the synthetic lethality of OTS186935 and olaparib\u003c/p\u003e\n\u003cp\u003eOTS186935 inhibits the double-strand DNA repair mechanism as represented by the inhibition of γH2AX accumulation, while olaparib acts as an inhibitor of the single-strand repair mechanism. OTS186935 may produce a greater inhibitory effect on double-stranded DNA repair in cell lines wherein HR is activated by the 53BP1 mutation, thus inducing a synergistic effect. SSB, single-strand break; DSB, double-strand break; HRR, homologous recombination repair; NHEJ, non-homologous end joining.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3928088/v1/959eab8b98b413f1a82886c5.png"},{"id":59824963,"identity":"8caffd9b-7c1f-437e-a04d-3d8045e892cd","added_by":"auto","created_at":"2024-07-08 05:30:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1878930,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3928088/v1/87c0840f-606e-4d1f-97bd-c0e5fa73ee77.pdf"},{"id":50814294,"identity":"1cf0db33-6a15-4251-ab8b-ba9c8182b371","added_by":"auto","created_at":"2024-02-07 19:37:07","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":61174,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S1A. Drug sensitivity assays for EPZ-6438. SK-LMS-1 and SK-UT-1 cell lines were treated by Tazemetostat (1–100 µM) for 96 h.\u003c/p\u003e","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-3928088/v1/e765737665e42ad0d8bc9c49.tif"},{"id":50814296,"identity":"1da10563-94dc-4f73-983e-709b1aac1c7b","added_by":"auto","created_at":"2024-02-07 19:37:07","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1537006,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S1B. Images of immunohistochemical staining results for SUV39H2 in clinical samples. Yellow bar indicates 50 µm.\u003c/p\u003e","description":"","filename":"FigureS1B.tif","url":"https://assets-eu.researchsquare.com/files/rs-3928088/v1/81cb625bce9e656f24756f68.tif"},{"id":50814291,"identity":"1887e824-a1b0-4bbb-8ba1-41d9a0aae5c8","added_by":"auto","created_at":"2024-02-07 19:37:06","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":64854,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S2. Cell viability assay for OTS186935 + olaparib. This figure shows the same results with Fig. 2B.\u003c/p\u003e","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-3928088/v1/d0a6d855b3d3a8fe9f9d687b.tif"},{"id":50814301,"identity":"766446fc-25b6-452a-ab69-8b35ad267fa9","added_by":"auto","created_at":"2024-02-07 19:37:07","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":146286,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S3. Annexin assay of SK-LMS-1 treated with 0.1% DMSO, 0.5 µM OTS186935, 1 µM olaparib, and 0.5 µM OTS186935 + 1 µM olaparib for 48 h. Proportion of apoptotic cells were calculated as the sum of Q2 and Q3. * Significant difference was analyzed by Student’s \u003cem\u003et-\u003c/em\u003etest, two-sided. Asterisks indicate p-values \u0026lt; 0.05. Error bars indicate means ± SD. \u0026nbsp;SD, standard deviation.\u003c/p\u003e","description":"","filename":"FigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-3928088/v1/85855d72710d717b88f9e3e5.tif"},{"id":50814300,"identity":"64f9be97-fee7-4799-89f5-dffcdad079a0","added_by":"auto","created_at":"2024-02-07 19:37:07","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":95878,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S4. RNA-seq of SK-UT-1 showed a p.T1060PfsTer36 of \u003cem\u003eTP53BP1\u003c/em\u003e (red rectangle).\u003c/p\u003e","description":"","filename":"FigureS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-3928088/v1/d28df523a1c783c5916b4716.tif"},{"id":50814298,"identity":"4543c9ba-51ef-4fc8-a3be-e5f0b3e5b70c","added_by":"auto","created_at":"2024-02-07 19:37:07","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":20975,"visible":true,"origin":"","legend":"","description":"","filename":"Table.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3928088/v1/46d880040ee6cb75ee8d90bf.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthetic lethality from the combination of a histone methyltransferase, SUV39H2 inhibitor and a poly (ADP-ribose) polymerase inhibitor for uterine leiomyosarcoma","fulltext":[{"header":"Background","content":"\u003cp\u003eUterine leiomyosarcoma (uLMS) is a soft tissue sarcoma that arises from the mesenchymal tissue of the uterus [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. uLMS is a major subtype of uterine sarcoma and accounts for 63% of cases of uterine sarcomas; this is followed by endometrial stromal sarcoma (21%), adenosarcoma (6%), and other types [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, uLMS is rare, with an incidence of 0.36\u0026ndash;1.7 per 100,000 women [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Primary total hysterectomy and bilateral salpingo-oophorectomy are recommended to treat uLMS [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The 5-year survival rates for stages I, II, III, and IV uLMS are 76%, 60%, 45%, and 29%, respectively [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Although advanced uLMS is generally treated with doxorubicin-based chemotherapy [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], uLMS has a high recurrence rate that ranges from 53\u0026ndash;71%, resulting in poor prognosis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Molecular targeted therapies such as pazopanib, eribulin, and trabectedin have been approved for uLMS [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, response rates remain low and have not led to significant improvements in prognosis [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Recently, poly (ADP-ribose) polymerase (PARP) inhibitors for uLMS have been introduced, and phase II clinical trials have begun [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] because 25% of patients with uLMS had homologous recombination repair defects (HRD) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In patients with HRD, PARP inhibitors can be used because HRD cells rely on alternative repair mechanisms such as non-homologous end joining (NHEJ) and single-strand break repair, which play a role in PARP [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, the acquisition of resistance to PARP inhibitors has become a concern.\u003c/p\u003e \u003cp\u003eSoft tissue sarcomas are broadly classified into simple and complex karyotypes; uLMS is a complex karyotype [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Simple karyotypes have specific chromosomal translocations and point mutations that can be molecular targets for therapy. In contrast, multiple genetic abnormalities, such as \u003cem\u003eTP53\u003c/em\u003e and \u003cem\u003eRB\u003c/em\u003e, cause genomic instability and make it difficult to identify molecular therapeutic targets [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Moreover, uLMS is classified as a cancer with low genomic copy number abnormalities and genomic mutation rates compared with other cancers [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, novel therapeutic strategies for uLMS are comprised of different aspects in addition to specific genomic targets.\u003c/p\u003e \u003cp\u003eEpigenetic studies focus on cancer research to elucidate carcinogenesis, cancer metastasis, and novel therapeutic strategies. Epigenetics reversibly regulates gene expression without altering the DNA sequence. Chromatin remodeling regulates gene expression and is involved in the transcriptional activity and repression of each gene. Epigenetics consists of three main components: DNA methylation, histone modification, and non-coding RNA-associated gene silencing. Lysine, arginine, serine, and threonine modify histone tails, which are responsible for transcriptional activity and repression [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The aberrant regulations of histone modification are thought to contribute to cancer development [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn 2020, tazemetostat, an histone methyltransferases, EZH2 inhibitor, was approved by the Food and Drug Administration for treatment-resistant epithelioid sarcoma and follicular lymphoma with EZH2 mutations. Therefore, the clinical application of histone methyltransferase inhibitors has attracted attention as a novel therapeutic strategy for cancers. Our group previously investigated histone methyltransferases and showed its efficacy on gynecological cancers [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Moreover, the efficacy of histone methyltransferases combined with PARP inhibitors has been reported [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Using EZH2 and PARP inhibitors, Karakashev et al. reported that CARM1 induces a synthetic lethal mechanism in high-grade serous ovarian cancers that highly express CARM1 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Histone methyltransferase combined with PARP inhibitors enhanced the efficacy of PARP inhibitors and may provide insights to overcome PARP inhibitor resistance.\u003c/p\u003e \u003cp\u003eSuppressor license of variegation 3\u0026ndash;9 homolog 2 (SUV39H2), which is a histone methyltransferase, is responsible for regulating gene expression by catalyzing the trimethylation of histone H3 lysine 9 (H3K9me3). SUV39H2 is upregulated in normal cells of the cerebellum and testes and is involved in cell cycle regulation and cell differentiation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. When DNA damage occurs, phosphorylated H2AX (γH2AX) is induced, activating double-strand DNA break repair mechanisms. SUV39H2 is reportedly involved in the recruitment γH2AX, and that suppression of SUV39H2 reduces γH2AX activity [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In addition, SUV39H1/2 is required to homologous recombination repair (HRR) and when SUV39H1/2 defect, cells appear to be HRD [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Because of its high expression in multiple cancers [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and its involvement in DNA break repair mechanisms, SUV39H2 has been used as a novel therapeutic target. Specific inhibitors of SUV39H2 have been developed (OTS186935) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and have shown efficacy in \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments on breast cancer.\u003c/p\u003e \u003cp\u003eThis study aimed to evaluate the efficacy of an SUV39H2 inhibitor alone and in combination with a PARP inhibitor against uLMS. A novel epigenetics-focused target for uLMS was used to identify a novel therapeutic approach.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eClinical tissue samples and ethical statement\u003c/h2\u003e \u003cp\u003eClinical tissue specimens for gene expression analysis and immunohistochemical staining (IHC) were obtained during gynecological surgeries performed at the University of Tokyo Hospital. For gene expression analysis, specimens were collected in 5\u0026ndash;10-mm squares, flash-frozen in liquid nitrogen, and stored in a \u0026minus;\u0026thinsp;80\u0026deg;C freezer until use for mRNA extraction. Specimens used for IHC were 4-\u0026micro;m thick slices of formalin-fixed paraffin-embedded (FFPE) specimens.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell lines\u003c/h2\u003e \u003cp\u003eSK-LMS-1 (ATCC, Manassas, VA, USA; Cat. # HTB-88) and SK-UT-1 (Cat. # HTB-114) were used as uLMS cell lines and were cultivated in Eagle\u0026rsquo;s minimum essential medium (E-MEM, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, MA, USA) and 1% penicillin/streptomycin (FUJIFILM Wako Pure Chemical Corporation) in an incubator at 37\u0026deg;C with 5% of CO\u003csub\u003e2\u003c/sub\u003e. The cell lines were subjected to FASMAC Short Tandem Repeat analysis for cell authentication. The MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland) was used to confirm that the cells were mycoplasma-free.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTotal RNA extraction and mRNA expression analysis of clinical tissue specimens and cell lines\u003c/h2\u003e \u003cp\u003eClinical specimens and RLT buffer with 1% 2-mercaptoethanol (FUJIFILM Wako Pure Chemical Corporation) were mixed in MagNA Lyser Green Beads (Roche Diagnostics, Indianapolis, IN, USA) using a MagNA Lyser (Roche, Basel, Switzerland) (6,500 rpm for 70 s) followed by centrifugation (14,000 rpm for 3 min). The supernatant was collected, and total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer\u0026rsquo;s instructions. Total RNA was extracted from the cell lines using the RNeasy Mini Kit (Qiagen). Complementary DNA was synthesized by reverse transcription using ReverTra Ace (Toyobo, Osaka, Japan).\u003c/p\u003e \u003cp\u003eReal-time quantitative polymerase chain reaction (RT-qPCR) was performed on a QuantStudio 1 Real-Time PCR System (Thermo Fisher Scientific) using KOD SYBR qPCR Mix (Toyobo). mRNA expression levels of tissue samples and cell lines were confirmed with normalized by 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method. \u003cem\u003eACTB\u003c/em\u003e was evaluated as the internal control. Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e shows the primer sequences.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eIHC\u003c/h2\u003e \u003cp\u003eAfter deparaffinization and antigen activation by heat (95\u0026deg;C, 20 min), anti-SUV39H2 antibody (Abcam, Cambridge, UK; Cat. # ab190870; 1:200) was added to the slides and incubated overnight at 4\u0026deg;C. Secondary antibody reactions and nuclear staining were performed using hematoxylin. Each specimen was evaluated using the IHC score, which is the sum of the proportion score (PS) and intensity score (IS). PS was calculated as the proportion of positive cells as follows: 0\u0026thinsp;=\u0026thinsp;0%; 1\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;1%; 2\u0026thinsp;=\u0026thinsp;1\u0026ndash;10%; 3\u0026thinsp;=\u0026thinsp;10\u0026ndash;33%; 4\u0026thinsp;=\u0026thinsp;33\u0026ndash;67%; and 5\u0026thinsp;=\u0026thinsp;\u0026gt;\u0026thinsp;67%. IS was graded as 0 (negative), 1 (weak), 2 (intermediate), and 3 (strong). Two raters independently evaluated the IHC scores, and the mean was calculated for each IHC score.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDrug sensitivity assay\u003c/h2\u003e \u003cp\u003eTazemetostat/EPZ-6438 (MedChemExpress, NJ, USA), an EZH2 inhibitor, OTS186935 (MedChemExpress), and olaparib (MedChemExpress), a PARP inhibitor, were used in this study. Each inhibitor was dissolved in 0.1% of dimethyl sulfoxide DMSO (Sigma-Aldrich). For the drug sensitivity assay, SK-LMS-1 and SK-UT-1 cells were pre-cultured in 96- or 48-well plates on the day before adding the inhibitor. On the next day (day 0), the medium was replaced with new medium diluted to the target concentration of the inhibitors. After 72 h (day 3) or 96 h (day 4) of incubation, cell viability was evaluated using cell counting kit-8 (DOJINDO, Kumamoto, Japan), while absorbance was measured with a microplate reader (BioTek, VT, USA). Three independent experiments were performed. The wells incubated with 0.1% DMSO were used as controls, and the ratio of absorbance at the desired concentration was calculated, with the absorbance of the control as 100%.\u003c/p\u003e \u003cp\u003eThe combination index (CI) was employed [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The CI, which was the half-maximal inhibitory concentration (IC50) of two drugs combined versus the IC50 of a single drug, was calculated as follows:\u003c/p\u003e \u003cp\u003eCI = (D1)/(Dx)1 + (D2)/(Dx) 2\u003c/p\u003e \u003cp\u003e(D1) and (D2)\u0026thinsp;=\u0026thinsp;IC50 concentrations of drug 1 and drug 2, respectively, when combined\u003c/p\u003e \u003cp\u003e(Dx) 1, (Dx) 2\u0026thinsp;=\u0026thinsp;concentration of the respective IC50 of drug 1 and drug 2 as single agents\u003c/p\u003e \u003cp\u003eThe obtained CIs were classified as synergism (CI\u0026thinsp;\u0026lt;\u0026thinsp;1), additive (CI\u0026thinsp;=\u0026thinsp;1), and antagonism (CI\u0026thinsp;\u0026gt;\u0026thinsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eColony formation assay\u003c/h2\u003e \u003cp\u003eColony formation assay was performed to verify the effects of inhibitors on long-term cultures. Six-well plates were pre-cultured with SK-LMS-1 and SK-UT-1 cells on the day before day 0. On day 0, the medium was replaced with new medium diluted to the target concentration of the inhibitors. The medium was replaced every 3 days, and the cells were fixed and stained on days 10\u0026ndash;12. The number of colonies, defined as \u0026gt;\u0026thinsp;50 cells, was counted under a microscope. Three wells were analyzed for each concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eFor protein extraction, the cell pellet was suspended with sodium dodecyl sulfate (SDS) lysis buffer (0.1M Tris-HCl pH 7.5, 10% Glycerol [FUJIFILM Wako], 2% SDS[FUJIFILM Wako Pure Chemical Corporation]), and the supernatant was collected by centrifugation (15,000 rpm, 10 min, 4\u0026deg;C) after heating at 95℃ for 5 min. Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad, Hercules, CA, USA) were used for SDS-PAGE, and the separated samples were transferred via a semi-dry transfer method using Trans-Blot Turbo Mini PVDF Transfer Packs (Bio-Rad). Amersham ECL Select (Cytiva, MA, USA) or SuperSignalTM West Femto (Thermo Fisher Scientific) were used as chemiluminescent reagents, and detection was performed using ImageQuant LAS 4000 (GE Healthcare Life Sciences, NJ, USA).\u003c/p\u003e \u003cp\u003eAntibodies listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e were used for primary antibody reactions. All reactions were performed overnight at 4\u0026deg;C. Secondary antibody reactions were performed using anti-mouse IgG HRP-linked antibody (Cell Signaling Technology, MA, USA; Cat. #7076, 1:3000) and anti-rabbit IgG horseradish peroxidase-linked antibodies (Cell Signaling Technology; Cat. #7074,1:3000).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCell cycle analysis\u003c/h2\u003e \u003cp\u003eFor cell cycle analysis, cells were fixed using 70% ethanol after incubation with the target concentrations of the inhibitors. Following nucleic acid staining with propidium iodide (Sigma-Aldrich), the fluorescence intensity of the cells was determined using a BD FACSCalibur HG Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Cell cycle analysis was performed using the FlowJo software version 16 (FlowJo LLC, OR, USA) with default settings for automatic analysis. Three independent experiments were performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnnexin assay\u003c/h2\u003e \u003cp\u003eAfter incubation with the target concentrations of the inhibitors, the centrifuged cell pellets were assayed using the FITC Annexin V Apoptosis Detection Kit II (BD Biosciences) according to the manufacturer\u0026rsquo;s instructions. Detection was performed using a BD FACSCalibur HG Flow Cytometer (BD Biosciences), and analysis to classify cells into early and late apoptosis was conducted using FlowJo software version 16 (FlowJo LLC). The percentages of early and late apoptotic cell populations were summed to calculate the percentage of apoptotic cells. All experiments were performed in triplicate under all conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell immunofluorescence staining\u003c/h2\u003e \u003cp\u003eDoxorubicin (Cayman Chemical Company) was used to induce DNA damage. uLMS cell lines were incubated on MICROCOVER GLASS (Matsunami Glass Ind., Ltd., Osaka, Japan) on 35-mm plates with the target concentrations of inhibitors until the targeted time, and fixation was performed with 4% paraformaldehyde phosphate buffer solution (FUJIFILM Wako Pure Chemical Corporation). After permeabilization with 0.05% Tween20 (Sigma-Aldrich) for 5 min twice and 0.5% TritonX-100 (Sigma-Aldrich) for 10 min, the cells were incubated in 3% bovine serum albumin (BSA, FUJIFILM Wako Pure Chemical Corporation) for 60 min at 25℃. Primary antibodies were diluted in 3% BSA and incubated overnight at 4\u0026deg;C in a wet box. Anti-phospho-histone H2A.X (Ser139) antibody (Cat. # JBW301, Lot# 3761799, 1:1000) was used. After cell permeabilization, cells were reacted with secondary antibody and 4',6-diamidino-2-phenylindole for nuclear staining for 60 min at 25\u0026deg;C. Goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) cross-adsorbed secondary antibody (Alexa Fluor 568, Thermo Fisher Scientific; Cat. # A11011, 1:1000) and donkey anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) highly cross-adsorbed secondary antibody (Alexa Fluor 488, Thermo Fisher Scientific; Cat. # A-21202, 1:1,000) were used. After washing the cells on the glass, the cover glass was encapsulated and observed under a confocal fluorescence microscope (LSM880; Carl Zeiss Co., Ltd., Oberkochen, Germany). Imaging was performed using ImageJ 1.53. Then, relative intensity of γH2AX of each cell was estimated by the default setting of ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing (RNA-seq)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using the abovementioned method and used for library preparation through the SMARTer Stranded Total RNA Sample Prep Kit (TaKaRa Bio, Tokyo, Japan). The library was prepared using an RNA Sample Prep Kit (TaKaRa Bio). Libraries were sequenced using a NextSeq 550 (Illumina, San Diego, CA, USA) with paired-end reads.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eChromatin immunoprecipitation (ChIP) sequencing\u003c/h2\u003e \u003cp\u003eAfter culture, 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e uLMS cells were collected using 0.25% trypsin-EDTA (FUJIFILM Wako Pure Chemical Corporation). Cell pellets were cross-linked with 1% formaldehyde solution (FUJIFILM Wako Pure Chemical Corporation) for 10 min at 37\u0026deg;C and discontinued with 0.125 M glycine (FUJIFILM Wako Pure Chemical Corporation) for 5 min at 25\u0026deg;C. After lysing the cells in lysis buffer (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), DNA fragmentation was performed by sonicating with a Covaris S220 (Covaris, Woburn, MA, USA) for 200 cycles for 10 min. The fragmented DNA underwent ChIP and incubated overnight at 4℃ with Dynabeads Protein G (Thermo Fisher Scientific) with 2.5 \u0026micro;g of anti-gamma H2A.X (phospho S139) antibody (Abcam, Cat. #ab2893, Lot:GR3446449-2). Next, we washed the beads with low salt immune complex wash buffer, high salt immune complex wash buffer, and LiCl immune complex wash buffer (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e) at 4\u0026deg;C for 5 min followed by washing with TE buffer (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e) for 5 min at 25\u0026deg;C. The fragments were extracted using an elution buffer (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Libraries were prepared using ThruPLEX DNA-Seq Kit (TaKaRa Bio). Sequencing was performed by paired-end reads using NextSeq 550 using the same method as that used for RNA-seq.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSequencing Data Analysis\u003c/h2\u003e \u003cp\u003eFor RNA-seq analysis, sequence reads were trimmed to extract adaptor sequences using Skewer (v0.2.2) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and mapped to the hg38 genome using spliced transcript alignment to a reference (v.2.7.8a) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Mapped reads were counted using feature Counts (v.2.0.10) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Integrative Genomic Viewer (v2.11.1) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] was used to visualize the mapped reads. In the chromatin immunoprecipitation sequencing (ChIP-seq) analysis, raw reads were aligned to hg38 using bowtie2 (v.2.4.2) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Multi-aligned reads and PCR copies were eliminated using Picard (v.2.25.3; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://broadinstitute.github.io/picard/\u003c/span\u003e\u003cspan address=\"http://broadinstitute.github.io/picard/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Bedtools (v2.30.0) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] was used to filter reads that overlapped with the blacklist of the Encyclopedia of DNA Elements (ENCODE). MACS2 (v 2.2.7.1) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] was used to call peaks with the parameter \u0026lsquo;--keep-dup auto -q 0.1\u0026rsquo;. Ngsplot was used to visualize peak signals within a range of \u0026plusmn;\u0026thinsp;1,000 bp around the γH2AX peaks in the doxorubicin-treated cells [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eexperiments\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAnimal experiments were performed in accordance with the ARRIVE guidelines 2.0 and approved by the Animal Experiment Committee of the Graduate School of Medicine, University of Tokyo (Approval No.-H20-243). We used 5-week-old immunodeficient BALB/cAJcl-nu/nu mice purchased from CLEA Japan, Inc. Mice were maintained on a 12-h day/night cycle and kept in a clean environment with adequate food and water. Cages were maintained with wooden shavings with fewer than five mice per cage. SK-UT-1 was seeded (diluted with 50 uL of Matrigel [Corning, NY, USA] and 100 uL of E-MEM [FUJIFILM Wako Pure Chemical Corporation]) at 5x10\u003csup\u003e6\u003c/sup\u003e cells each in the dorsal subcutaneous tissue of nude mice under inhalation anesthesia sedation. Tumor volume was calculated using the following formula: tumor volume = (tumor short diameter)\u003csup\u003e2\u003c/sup\u003e \u0026times; (tumor long diameter)/2. Treatment was initiated when the tumor diameter reached 5 mm. Drugs were administered intraperitoneally as follows: DMSO for the control group (DMSO group), OTS186935 10 mg/kg for the OTS186935 group, olaparib 50 mg/kg for the olaparib group, and OTS186935 10 mg/kg\u0026thinsp;+\u0026thinsp;olaparib 50 mg/kg for the combination group. Six mice were used in each group. Drugs were dissolved in 10% DMSO and PEG300 in 200 \u0026micro;L of saline. Tumor measurements were performed once every 2\u0026ndash;3 days and finally evaluated on day 14 after drug administration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eFor statistical analyses, each experiment was independently performed at least three times, and data were calculated as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Student\u0026rsquo;s t-test was used to analyze data. Differences were considered statistically significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. A one-sided test was used for the mRNA expression analysis of clinical samples, and a two-sided test was used for other samples. All statistical analyses were performed using Microsoft Excel and R (version 4.3.1).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eExpression analysis in clinical tissue samples and drug sensitivity assays\u003c/h2\u003e \u003cp\u003emRNA expression levels of 11 histone methyltransferases were measured by RT-qPCR in clinical tissue samples from normal uterine myometrium (n\u0026thinsp;=\u0026thinsp;4), uterine leiomyomas (n\u0026thinsp;=\u0026thinsp;4), and uLMS (n\u0026thinsp;=\u0026thinsp;6) (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Significant upregulation of \u003cem\u003eEZH2\u003c/em\u003e, \u003cem\u003eSUV39H2\u003c/em\u003e, and \u003cem\u003eWHSC1\u003c/em\u003e expression was observed in uLMS compared with normal uterine myometrium and uterine leiomyomas (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong the three histone methyltransferases, selective inhibitors of SUV39H2 and EZH2 were available. OTS186935 (MedChemExpress) and EPZ-6438 (tazemetostat, MedChemExpress) were analyzed for their efficacy against the uLMS cell lines SK-LMS-1 and SK-UT-1. EPZ-6438 failed to show efficacy against SK-LMS-1 and SK-UT-1, with an IC50 of 45.1 \u0026micro;M and 60.1 \u0026micro;M, respectively (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Meanwhile, OTS186935 showed drug sensitivity at relatively low concentrations, with an IC50 of 1.16 \u0026micro;M and 1.04 \u0026micro;M in SK-LMS-1 and SK-UT-1, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe expression levels of SUV39H2 in clinical tissue specimens were evaluated using IHC. As shown in Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e, 21 cases of uLMS, 5 cases of normal uterine myometrium, and 5 cases of uterine leiomyomas were evaluated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, the intensity score was classified as negative, weak, intermediate, and strong. uLMS showed a significant increase in the IHC score compared to normal uterine myometrium and uterine leiomyomas (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCombination therapy with OTS186935 and olaparib\u003c/h2\u003e \u003cp\u003eTo determine the mechanism of action of OTS186935, we performed Western blotting to detect the reaction of H3K9me3, which is catalyzed by SUV39H2 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The results showed that OTS186935 decreased H3K9me3 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), indicating that OTS186935 inhibited SUV39H2. Next, we examined the combined effects of OTS186935 and olaparib on uLMS cell lines. As revealed by the cell viability assay, the CI was 0.99 for SK-LMS-1 and 0.87 for SK-UT-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The CI of SK-LMS-1 showed an additive effect, whereas that of SK-UT-1 showed a synergistic effect. To assess the drug sensitivity of the combination therapy in long-term cultures, a colony formation assay was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). SK-UT-1 showed a synergistic effect with the drug combination compared to SK-LMS-1, similar to the results of the cell viability assay.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eElucidation of the synergistic effect in SK-UT-1\u003c/h2\u003e \u003cp\u003eWe investigated the mechanisms underlying the synergistic effect in SK-UT-1 compared with that in SK-LMS-1 and the cell cycle analysis results. After treatment with OTS186935 alone, the G2/M phase was significantly increased in both SK-LMS-1 and SK-UT-1 (G2/M phase in DMSO vs. OT186935, 17.2% vs. 27.4% in SK-LMS-1, p\u0026thinsp;=\u0026thinsp;0.023; 33.4% vs. 40.9% in SK-UT-1, p\u0026thinsp;=\u0026thinsp;0.008). However, when olaparib was added, there was no significant change in the cell cycle in either cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we evaluated the induction of apoptosis using an annexin assay; there was no significant increase in the percentage of apoptotic cells in SK-LMS-1 after combination therapy (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). However, a significant increase in the percentage of apoptotic cells was observed when OTS186935 was combined with olaparib (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm the induction of apoptosis by the combination therapy, Western blotting was performed to detect the accumulation of cleaved PARP. Similar to the results of the annexin assay, apoptosis was observed in SK-UT-1 cells after combination, which was not observed in SK-LMS-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eWe hypothesized that the synergistic effects of OTS186935 and olaparib on SK-UT-1 cells may be related to factors involved in DNA damage/repair mechanisms. We investigated the DepMap Portal (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://depmap.org/portal/\u003c/span\u003e\u003cspan address=\"https://depmap.org/portal/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed on 3/2/2022), which is a public database of cell lines, and found a deletion in 53BP1, which is a factor involved in double-stranded DNA break repair, in SK-UT-1 cells. Deletion of 53BP1 in SK-UT-1 cells caused a frameshift mutation, whereas that in SK-LMS-1 cells did not result in abnormalities. We actually conducted RNA-seq of SK-UT-1 cells, revealing p.T1060PfsTer36 of \u003cem\u003eTP53BP1\u003c/em\u003e (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMechanism of OTS186935 on the DNA break repair pathway\u003c/h2\u003e \u003cp\u003eNext, we investigated the effects of OTS186935 on DNA repair. We used doxorubicin to induce DNA breaks in uLMS cells and investigated the reaction after treatment with OTS186935. We added 0.1% DMSO for negative control, 1 \u0026micro;M of OTS186935, 0.5 \u0026micro;M of doxorubicin, and OTS186935 1 \u0026micro;M\u0026thinsp;+\u0026thinsp;doxorubicin 0.5 \u0026micro;M to each of the uLMS cell lines. Doxorubicin treatment resulted in γH2AX accumulation, whereas the combination with OTS186935 suppressed γH2AX expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm the suppression of γH2AX accumulation in the nucleus, cell immunofluorescence staining results were compared between 0.5 \u0026micro;M of doxorubicin and the addition of 1 \u0026micro;M of OTS186935. The results showed that OTS186935 suppressed γH2AX accumulation in the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eMoreover, ChIP-seq was performed on SK-UT-1 cells to further investigate the effects of OTS186935 on γH2AX. We performed ChIP-seq for the four conditions: 0.1% DMSO for negative control, 1 \u0026micro;M of OTS186935, 0.5 \u0026micro;M of Doxorubicin, and the combination of OTS186935 1 \u0026micro;M\u0026thinsp;+\u0026thinsp;doxorubicin 0.5 \u0026micro;M. Samples were then immunoprecipitated with anti-γH2AX antibody. The results showed that the addition of OTS186935 decreased the γH2AX signal, indicating that a decrease in the accumulation of γH2AX on the genome occurred and suggesting that OTS186935 inhibited double-strand DNA break repair (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of the combination of OTS186935 and Olaparib in\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003eexperiments\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B, the OTS186935 and combination groups showed a significant decrease in tumor volume compared to the DMSO group (p\u0026thinsp;=\u0026thinsp;0.012). The decrease in tumor volume in the combination group was greater than that in the OTS186935 group (p\u0026thinsp;=\u0026thinsp;0.59).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, SUV39H2 expression is increased in uLMS, suggesting that it may be involved in carcinogenesis, and that SUV39H2 inhibitors may be a novel therapeutic strategy for uLMS. We also demonstrated that the effects of the combination of OTS186935 and olaparib in uLMS cell lines and demonstrated its effectiveness in a 53BP1-deficient uLMS cell line. OTS186935 alone induced cell cycle arrest, whereas the addition of olaparib induced apoptosis and cell cycle arrest in 53BP1-deficient uLMS cells. OTS186935 also decreased the accumulation of γH2AX, suggesting that OTS186935 inhibited double-strand DNA repair.\u003c/p\u003e \u003cp\u003eThe prognosis of uLMS is poor owing to resistance to existing chemotherapies, hence the requirement of novel therapeutic strategies. We focused on a histone methyltransferase (SUV39H2) because it is related to carcinogenesis in various types of cancers [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In a previous report that investigated the relationship between EZH2 and melanoma and used 53 types of cell lines, sensitive cell lines for a histone methyltransferase inhibitor were defined as \u0026lt;\u0026thinsp;15 \u0026micro;M of IC50 [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The results of the drug sensitivity assay indicated that OTS186935 showed relatively high sensitivity to uLMS cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Additionally, \u003cem\u003ein vivo\u003c/em\u003e experiments revealed the efficacy of OTS186935 in uLMS (Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Overall, these results suggest that OTS186935 is a novel therapeutic target for uLMS.\u003c/p\u003e \u003cp\u003eRecently, PARP inhibitors have become increasingly important for the treatment of gynecological tumors with HRD as 25% of the patients with uLMS have HRD [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Case reports have used PARP inhibitors for uLMS, and phase II clinical trials of PARP inhibitors for uLMS have begun [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Although PARP inhibitors have become widely used, the acquisition of resistance to PARP inhibitors has become a critical issue in cancer treatment. HRR, wherein BRCA1/2 is mainly involved, and non-homologous end-joining (NHEJ), wherein 53BP1 is involved, act exclusively of each other. Therefore, in HRD, 53BP1-based NHEJ can be dominant, whereas when the mutation of NHEJ-related factors, such as \u003cem\u003e53BP1\u003c/em\u003e, \u003cem\u003eRIF1\u003c/em\u003e, and \u003cem\u003eREV7\u003c/em\u003e are present, NHEJ dominance can be lost; this is one of the mechanisms by which HRR is activated even in BRCA mutations and how resistance to PARP inhibitors is acquired [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eStrategies to resolve this resistance to PARP inhibitors are required, and combination therapies are being investigated. A phase II trial of temozolomide and olaparib combination therapy for uLMS has been conducted [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In other types of cancer, our group previously reported that the combination of LLY-507, an SMYD2 inhibitor, and olaparib showed additive effects in high-grade serous ovarian carcinoma, demonstrating the usefulness of the combination of a histone methyltransferase inhibitor and a PARP inhibitor [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Additionally, Karakashev et al. reported that in high-grade serous ovarian carcinoma with high \u003cem\u003eCARM1\u003c/em\u003e expression, \u003cem\u003eEZH2\u003c/em\u003e represses the \u003cem\u003eMAD2L2\u003c/em\u003e promoter, which promotes NEHJ, by histone methylation (H3K27me3). EZH2 inhibition induces NHEJ dominance, resulting in a synthetic lethal mechanism involving a PARP inhibitor [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Our results demonstrated the synthetic lethality of OTS186935 combined with olaparib in a 53BP1-deficient uLMS cell line. Although \u003cem\u003ein vivo\u003c/em\u003e experiments did not show a significant difference between the OTS186935 and combination groups, the combination therapy tended to be superior to OTS186935 alone. The short duration of administration was assumed to be one reason for the lack of statistically significant differences. SUV39H2 is associated with DNA damage-induced γH2AX, and it was reported that suppression of SUV39H2 reduces the accumulation of γH2AX [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In addition, SUV39H1/2 is required to homologous recombination repair (HRR) and when SUV39H1/2 defect, cells appear to be HRD [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The hypothesis is that OTS186935 inhibits the double-strand DNA repair mechanism, which is represented by inhabitation of accumulation of γH2AX, while olaparib acts as an inhibitor of the single-strand repair mechanism. Furthermore, OTS186935 may exert inhibitory effects on HRR in cell lines wherein HRR is activated by the \u003cem\u003e53BP1\u003c/em\u003e mutation because SUV39H2 is a key factor in HRR, thus inducing a synergistic effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This is the first study that focused on the therapeutic effect of an SUV39H2 inhibitor on uLMS as well as the first report that described the effect of an SUV39H2 inhibitor combined with a PARP inhibitor.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis study had some limitations. Although we focused on \u003cem\u003e53BP1\u003c/em\u003e for the differences between the two cell lines, other genes may have been involved in the synergistic effect; further validation is required to confirm the above mechanisms. Additionally, we observed a decrease in H3K9me3 levels after treatment with OTS186935; however, we did not observe a relationship between H3K9me3 and synthetic lethality.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOTS186935 showed efficacy on uLMS cell lines \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Combination therapy with olaparib resulted in synthetic lethality in 53BP1-deficient cell lines. We considered inhabitation of double-stranded DNA break repair by OTS186935, which suppressed γH2AX accumulation and induced synthetic lethality when combined with olaparib.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDSB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edouble-strand break\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eChIP-seq\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003echromatin immunoprecipitation sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHRR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehomologous recombination repair\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHRD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehomologous recombination repair defects\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eimmunohistochemical staining\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eintensity score\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNHEJ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enon-homologous end joining\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePARP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epoly (ADP-ribose) polymerase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eγH2AX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphorylated H2AX\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRT-qPCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereal-time quantitative polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eproportion score\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRNA-seq\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRNA sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandard division\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSSB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esingle-strand break\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003euLMS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003euterine leiomyosarcoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eAll patients were diagnosed by pathologists (certified as specialists by the Japanese Society of Pathology) at the University of Tokyo Hospital. This study was approved by The Human Genome, Gene Analysis Research Ethics Committee of the University of Tokyo and written informed consents were obtained from each patient (G0683-(26)).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study is partially supported by BRIDGE\u0026thinsp;\u0026lt;\u0026thinsp;programs for Bridging the gap between R\u0026amp;d and the IDeal society (society 5.0) and Generating Economic and social value\u0026gt;\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYusuke Toyohara: Data curation, investigation, formal analyses, methodology, visualization, writing (original draft), and writing (review and editing). Kenbun Sone: conceptualization, data curation, funding acquisition, methodology, project administration, writing (original draft) and writing (review and editing). Kohei Kumegawa: Data curation, formal analysis, investigation, and methodology. Yoko Yamamoto: investigation, methodology, and writing (review and editing). Ryuta Hachijo: investigation, methodology, and writing (review and editing). Saki Tanimoto: Data curation, investigation, methodology, and writing (review and editing). Futaba Inoue: data curation, investigation, methodology, and writing (review and editing). Asako Kukita: Data curation, investigation, methodology, and writing (review and editing). Asympti Taguchi: data curation, investigation, methodology, and writing (review and editing); Masako Ikemura: investigation and writing (review and editing). Yuichiro Miyamoto: Supervision and writing (review and editing). Michihiro Tanikawa: Methodology, supervision, and writing (review and editing). Takayuki Iriyama: Supervision and writing (review and editing). Mayuyo Mori-Uchino: Supervision and writing (review and editing). Ryuji Hamamoto: Supervision and writing (review and editing). Tetsuo Ushiku: Supervision and writing (review and editing). Katsuhiko Oda: Methodology, supervision, and writing (review and editing). Yasushi Hirota: supervision and writing (review and editing). Reo Maruyama: conceptualization, methodology, supervision, and writing (review and editing). Yutaka Osuga: Supervision and writing (review and editing).\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors are grateful to Dr. Liying Yang (Project for Cancer Epigenomics, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan) for helping our sequencing analysis. The authors would like to thank Editage for the English language review.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eAll data generated or analyzed in this study are included in the published article [and its supplementary information files]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDesar IME, Ottevanger PB, Benson C, van der Graaf WTA. Systemic treatment in adult uterine sarcomas. Crit Rev Oncol Hematol. 2018;122:10\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.critrevonc.2017.12.009\u003c/span\u003e\u003cspan address=\"10.1016/j.critrevonc.2017.12.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrop\u0026eacute; CG, Abeler VM, Kristensen GB. Diagnosis and treatment of sarcoma of the uterus. A review. 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Nucleic Acids Res. 2015;43:7931\u0026ndash;44. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/gkv722\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkv722\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"SUV39H2, uterine leiomyosarcoma, γH2AX, PARP inhibitor, synthetic lethality","lastPublishedDoi":"10.21203/rs.3.rs-3928088/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3928088/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUterine leiomyosarcoma (uLMS) has a poor prognosis owing to its high recurrence rate and resistance to chemotherapy. Therefore, novel therapeutic targets for uLMS need to be discovered.\u003c/p\u003e\n\u003cp\u003eSUV39H2 is a histone methyltransferase that promotes the repair of double-stranded DNA breaks by recruiting phosphorylated H2AX (γH2AX). In this study, we investigated the potential therapeutic targets of SUV39H2 in uLMS and the mechanism of synthetic lethality between PARP inhibitors and SUV39H2 inhibitors, OTS186935.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst, we analyzed the mRNA and protein expression of SUV39H2 in clinical tissues of uLMS, normal myometrium, and leiomyomas using real-time polymerase chain reaction and immunohistochemistry, respectively. Next, we conducted drug sensitivity assays for OTS186935 alone and in combination with olaparib, a poly (ADP-ribose) polymerase inhibitor, using uLMS cell lines, SK-LMS-1 and SK-UT-1. We conducted an annexin assay to investigate the mechanisms of cellular death. We performed Western blotting, immunofluorescence, and chromatin immunoprecipitation sequencing (ChIP-seq) to investigate γH2AX following OTS186935 treatment in addition to \u003cem\u003ein vivo\u003c/em\u003e experiments using nude mice with subcutaneously implanted uLMS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSUV39H2\u003c/em\u003e expression was significantly increased in uLMS compared to that in normal myometrium and leiomyomas. OTS186935 decreased cell viability in both cell lines, and its combination with olaparib resulted in synthetic lethality in SK-UT-1 cells (combination index = 0.87). Annexin assay revealed that the combination therapy induced apoptosis. After treatment with OTS186935, γH2AX accumulation decreased. ChIP-seq also showed downregulated γH2AX following OTS186935 treatment. Notably, the combination with OTS186935 and PARP inhibitor was significantly more effective \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOTS186935 inhibits double-stranded DNA break repair as evidenced by γH2AX downregulation through ChIP-seq and other assays. OTS186935 combined with olaparib induces synthetic lethality in patients with uLMS.\u003c/p\u003e","manuscriptTitle":"Synthetic lethality from the combination of a histone methyltransferase, SUV39H2 inhibitor and a poly (ADP-ribose) polymerase inhibitor for uterine leiomyosarcoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-07 19:37:01","doi":"10.21203/rs.3.rs-3928088/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"eb460a15-1759-47a4-9e04-81b6f811a343","owner":[],"postedDate":"February 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-03T14:38:34+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-07 19:37:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3928088","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3928088","identity":"rs-3928088","version":["v1"]},"buildId":"ApUGefWb6u5IBVtyqm6d5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}