The PARP inhibitor olaparib enhances the sensitivity of Ewing sarcoma to trabectedin

Oncotarget18875 www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget/ Oncotarget, Vol. 6, No. 22 The PARP inhibitor olaparib enhances the sensitivity of Ewing sarcoma to trabectedin José Luis Ordóñez1,4,*, Ana Teresa Amaral1,4,*, Angel M. Carcaboso2, David Herrero- Martín3, María del Carmen García-Macías4, Vicky Sevillano4, Diego Alonso4, Guillem Pascual-Pasto2, Laura San-Segundo4, Monica Vila-Ubach2, Telmo Rodrigues4, Susana Fraile4, Cristina Teodosio4, Agustín Mayo-Iscar5, Miguel Aracil6, Carlos María Galmarini6, Oscar M. Tirado3, Jaume Mora2 and Enrique de Álava1,4 1 Laboratory of Molecular Pathology- Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocio/CSIC/ Universidad de Sevilla, Seville, Spain 2 Developmental Tumor Biology Laboratory. Preclinical Therapeutics and Drug Delivery Research Program, Hospital Sant Joan de Deu Barcelona, Spain 3 Sarcoma Research Group, Laboratori d’Oncología Molecular, Institut d’Investigació Biomèdica de Bellvitge (IDIBELL), L’Hospitalet de Llobregat, Barcelona, Spain 4 Centro de Investigación del Cáncer, Instituto de Biología Molecular y Celular del Cáncer/ Consejo Superior de Investigaciones Científicas-Universidad de Salamanca, Salamanca, Spain 5 Statistics and Operations Research Department-University of Valladolid, Spain 6 Cell Biology and Pharmacogenomics Department, Pharmamar, Madrid, Spain *These authors have contributed equally to this work Correspondence to: José Luis Ordóñez García, email: [email protected] Correspondence to: Enrique de Álava, email: [email protected] Keywords: ewing sarcoma, PARP inhibitor, trabectedin, DNA damage, PDX models Received: March 13, 2015 Accepted: May 13, 2015 Published: May 27, 2015 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. AbstrAct Recent preclinical evidence has suggested that Ewing Sarcoma (ES) bearing EWSR1-ETS fusions could be particularly sensitive to PARP inhibitors (PARPinh) in combination with DNA damage repair (DDR) agents. Trabectedin is an antitumoral agent that modulates EWSR1-FLI1 transcriptional functions, causing DNA damage. Interestingly, PARP1 is also a transcriptional regulator of EWSR1-FLI1, and PARPinh disrupts the DDR machinery. Thus, given the impact and apparent specificity of both agents with regard to the DNA damage/DDR system and EWSR1-FLI1 activity in ES, we decided to explore the activity of combining PARPinh and Trabectedin in in vitro and in vivo experiments. The combination of Olaparib and Trabectedin was found to be highly synergistic, inhibiting cell proliferation, inducing apoptosis, and the accumulation of G2/M. The drug combination also enhanced γH2AX intranuclear accumulation as a result of DNA damage induction, DNA fragmentation and global DDR deregulation, while EWSR1-FLI1 target expression remained unaffected. The effect of the drug combination was corroborated in a mouse xenograft model of ES and, more importantly, in two ES patient-derived xenograft (PDX) models in which the tumors showed complete regression. In conclusion, the combination of the two agents leads to a biologically significant deregulation of the DDR machinery that elicits relevant antitumor activity in preclinical models and might represent a promising therapeutic tool that should be further explored for translation to the clinical setting. This article has been corrected. Correction in: Oncotarget. 2017; 8:43592-43592. Oncotarget18876 www.impactjournals.com/oncotarget INtrODUctION ES is an aggressive developmental mesenchymal tumor [1]. As a result of multimodal therapy, including strategies from traditional chemotherapeutic agents, radiotherapy, and surgery, the current cure rate of ES patients with localized disease is 70% [2, 3]. However, the survival rate for patients with relapsed, multifocal/ metastatic disease is still less than 20%, mainly due to development of drug resistance. Recently, a retrospective study by our group revealed the prognostic value of chromosome 1 gain (1qG) in ES [4]. Within the upregulated candidate genes identified in 1qG ES, a particularly interesting gene was found: poly (ADP-ribose) polymerase-1 (PARP1) [4]. Other studies have recently described that PARP1 plays an important role in tumors bearing ETS fusions, such as prostate cancer and ES [5, 6]. PARP proteins play a relevant role in the regulation of the cell cycle, apoptosis, genomic stability, chromatin remodeling, transcription, tumor growth and even the induction of chromosomal translocations. PARP1 has been shown to act as a transcriptional regulator of EWSR1-FLI1, which has triggered interest in testing PARPinh in ES [6, 7]. However, the best known activity of PARP proteins is related to the mechanisms of DDR, especially regarding the Base Excision repair (BER) pathway [8]. In brief, the BER is the main pathway responsible for repairing single-strand breaks (SSB) of DNA. In the absence of an effective BER mechanism, SSB accumulation results in Double-Strand Breaks (DSB), the most lethal form of DNA damage. Globally, when the DDR machinery fails this produces both an increase in the frequency of acquired mutations and genomic instability and both these determine the appearance, evolution and progression of tumors. In fact, an active DDR machinery is essential for the physiology of the cell, ensuring its survival, and is an important mechanism of resistance to cytotoxic agents. Accordingly, the inhibition of the DDR in tumor cells provides an excellent therapeutic opportunity [9]. Inhibitors of the DDR machinery have been used successfully against tumor cells in monotherapy or combined with other agents in order to sensitize tumor cells to the cytotoxic activity of other drugs. Within this large group of drugs, to date PARPinh represents one of the most promising subgroup of agents [8, 9]. In this sense, it has recently been reported that ES cells are defective in DNA breakage repair systems, being very sensitive to PARPinh in combination with DNA damaging-drugs [10]. Trabectedin is a marine-derived compound with proven antitumor activity against several types of neoplasms, including sarcomas, ovarian, and breast cancer, among others, by inducing DNA breaks. Clinical trials have shown that Trabectedin is particularly active in sarcomas bearing translocations, namely myxoid Liposarcoma (ML), ES and Synovial Sarcoma [11, 12]. In ES, Grohar et al. showed that, similarly to the previous observation in ML, Trabectedin interferes directly with the chimeric protein EWSR1-FLI1 [12]. In fact, treatment with Trabectedin specifically deregulates the transcriptional activity of the chimeric protein [12-14]. Given the impact and apparent specificity of PARPinh and Trabectedin with regard to the DNA damage/DDR system and EWSR1-FLI1 activity in ES, we decided to explore the effects of the combination through a series of in vitro and in vivo studies. We report that the combination of Trabectedin and Olaparib is highly synergistic in ES cell lines, inducing major DNA damage in vitro and in vivo and causing a clinically significant degree of tumor regression in PDX) models of ES. rEsULts Es cells are especially sensitive to olaparib, which induces G2/M accumulation independently of the 1q status Initially, we checked the status of PARP1 in a panel of ES cell lines (Supplementary Table S1) using a home-made fluorescence in situ hybridization (FISH) probe specific for PARP1. We observed that ES cell lines previously described as 1qG in fact showed an amplification of PARP1 by FISH (Figure 1A). cDNA analysis showed that all ES cell lines express PARP1 with different mRNA levels (Figure 1B). Also, using a specific antibody for PARP1, by Western-blot we observed that all cell lines studied expressed PARP1 at similar levels, independently of the 1qG status, (Figure 1C-1D). We studied the sensitivity of ES cell lines to a group of PARPinh, including Olaparib, Veliparib and Iniparib. Olaparib was more active in inhibiting proliferation than the other two drugs assayed, with lower IC50 levels at 72 hours of exposure (high nM-low μM range, with a median of 1.995 ± 0.46μM). Veliparib was the second most effective agent, with IC50 levels of proliferation in the μM range, with a median of 14.14±2.75μM (approximately 7 fold higher than Olaparib). Finally, Iniparib was the least effective agent, showing IC50 levels also in the μM range but with a median of 74.95 ± 5.02μM (approximately 38-fold higher than Olaparib) (Figure 2A). Interestingly, we observed that after 72 hours of exposure to Olaparib IC50 levels were higher than those obtained after 6 days of treatment (Supplementary Figure S1A). Given that PARP1 is located on chromosome 1q, and in view of our previous results describing some ES tumors and cell lines with 1qG, we searched for a correlation between the status of 1q [Gained or Normal (N)] and Olaparib sensitivity (Supplementary Figure S1B). We observed a trend towards a higher sensitivity of 1qG cell lines to Olaparib, but it was not statistically significant, probably due to the low number of cell lines analyzed (Mann Whitney U test, p Oncotarget18877 www.impactjournals.com/oncotarget > 0,05). The correlation between the status of p53 (wildtype or mutated) and the sensitivity to Olaparib was not statistically significant either (Mann Whitney U test, p > 0.05) (Supplementary Figure S1C). We also studied the effects of Olaparib on the cell cycle profile using two ES cell lines, A673 (1qN) and A4573 (1qG) (Figure 2B). Both cell lines showed G2/M accumulation after treatment, even at low doses of Olaparib (Figure 2B). the combination of olaparib and trabectedin is highly synergistic in Es cell lines Having observed that Olaparib was much more cytotoxic than the other PARPinh, we studied the effects of the combination of Olaparib with Trabectedin. ES cell lines (n = 10) were exposed to different combinations of both agents at a constant ratio of 1:20.000 (Trabectedin:Olaparib) for 72 hours and Combination Indices (CIs) were determined according to [15, 16]. Interestingly, synergistic effects were observed in all but two cell lines (Table 1). We then studied the effects of this drug combination on apoptosis induction via caspase -3 and -7 activation after 48 hours of drug exposure as well as cell cycle effects after 24 hours of treatment in two cell lines, RM82 and TC71. We observed that the drug combination (250pM of Trabectedin and 5μM of Olaparib) increased the apoptotic rate in the TC71 cell line (Supplementary Figure S2A). In agreement with this, we observed cleaved PARP accumulation after treatment with the combination, especially at 24 hours, in the TC71 cell line (Supplementary Figure S2B). Furthermore a G2/M accumulation and the elimination of the G0/G1 phase were also observed after treatment with the drug combination (Supplementary Figure S2C). In the RM82 cell line, treatment with the drug combination only induced mild apoptosis (Supplementary Figure S2A). By contrast, no effects on cell migration after 72 hours of drug exposure were observed (Supplementary Figure S3). Figure 1: PArP1: gene status, mrNA and protein expression. A. FISH of PARP1. ES cell lines previously described as 1qG showed extra-copies of PARP1 (CADO and TC71 cell lines) whereas A673, described as 1qN, showed only two copies of PARP1 by FISH. Green and red dots represent PARP1 gene copies and 1q centromeres, respectively. Arrows also indicate the green dots (PARP1 gene copies). b. q-RT-PCR shows that PARP1 expression is heterogeneous among the ES cell lines studied. SJRH also express PARP1 at similar levels to the ES cell lines. A4573 (1qG) and A673 (1qN) are the cell lines with the highest expression of PARP1. The MCF7 breast cancer cell line was used as a positive control. c. Western blotting showed that ES cell lines express PARP1 under normal conditions. Here, breast cancer cell lines (MCF7 and MD-MB 23) were used as positive controls. D. Quantification of PARP1 protein levels with respect to β-actin expression using Image J software. Oncotarget18878 www.impactjournals.com/oncotarget the combination of olaparib and trabectedin enhances DNA damage while EWsr1-FLI1 activity is not deregulated DNA damage induction was evaluated by testing for the presence of intranuclear foci of γH2AX and 53BP1 after short (8 hours) and long (24 hours) drug exposures. Initially, cells were treated with Trabectedin (250pM) and/or Olaparib (5μM) for 8 hours. Trabectedin induced a stronger DSB accumulation than that induced by Olaparib alone (Student´s t-test, p < 0.05). Nevertheless, the combination of both agents resulted in a clear enhancement of DSB formation (Figure 3A and Supplementary Figure S4). After 24 hours of drug exposure, the drug combinations resulted in cumulative effects of DNA damage through the induction of DSB, even at the lowest drug concentrations used (125pM of Trabectedin and 2.5 μM) (Figure 3B). Interestingly, the combination did not affect EWSR1-FLI1 transcriptional activity (Supplementary Figure S5A). Moreover, DNA fragmentation assays revealed that both drugs were able to generate enough damage to induce DNA fragmentation (Figure 3C and Supplementary Figure S5B). Olaparib and/or trabectedin induce deregulation of the DDr machinery After demonstrating that the combination of Olaparib and Trabectedin induced DNA damage by DSB accumulation, we next analyzed the regulation of the DDR machinery in TC71 cells. To this end, we table 1: Proliferation assays show that the combination of Olaparib and trabectedin is synergistic in all Es cell lines with the exception of A4573 and WE68, where the cIs are almost additive (values close to 1). Cell lines CI Effects RDES 0.73 Synergism WE68 0.92 Nearly additive STAET-2.1 0.49 Synergism RM82 0.69 Synergism TC71 0.4 Synergism SK-N-MC 0.008 Synergism CADO 0.4 Synergism A673 0.77 Synergism A4573 0.93 Nearly additive STAET-1 0.64 Synergism SJRH 0.11 Synergism Oncotarget18879 www.impactjournals.com/oncotarget used low-density arrays (DDR PCR Array; PAHS-042Z, Qiagen). After 16.5 hours of treatment with Olaparib (5µM) and/or Trabectedin (250pM), RNA was extracted and the changes in the mRNA levels regarding DNA damage induction and DDR mechanisms were analyzed. Treatment with Trabectedin caused an overexpression of homologuous recombination (HR)-related genes such as BRCA1, BRCA2, RAD18, RAD52 and BRIP1 (BRCA1/2- associated molecule) as well as POLD3 (DNA polymerase delta subunit 3); NEIL3 (nei endonuclease VIII-like 3) involved in the BER pathway; RFC1 (replication factor C), which is involved in GC-NER DSB repair; OGG1; XRCC1, involved in DNA- and protein-binding and strand-break correction, and UNG, a DNA glycosylase involved in the BER pathway (Supplementary Tables S2, S3 and Supplementary Figure S6). Treatment with Olaparib induced the expression of the HR-related genes EXO1, RAD54L, RAD51B and RAD18, together with BRIP1, RFC1, PARP3, XRCC, POLD3 and PARP3 (Supplementary Tables S2, S3 and Supplementary Figure S7). The global deregulation of the DNA damage and repair machinery was clearly observed with the combined treatment, where the HR genes RAD54L, EXO1, PARP2, BRCA1, BRCA2, RAD18, RAD50, RAD51 and RPA1 were overexpressed (Supplementary Table S2, S3 and S4). Changes in expression in this array were highly Figure 2: PArPinh activity: proliferation and cell cycle analysis. A. IC50 of proliferation after 72 hours of drug exposure. ES cell lines and the SJRH cell line were more sensitive to Olaparib than to the other agents b.. Cell cycle analysis on A673 (1qN) and A4573 (1qG) after treatment with two different concentrations of Olaparib. Oncotarget18880 www.impactjournals.com/oncotarget correlated with DSB repair by the NER pathway (p value: 1.02E-23), the NHEJ pathway (p-value: 1,81E-23), the HR pathway (p-value: 4,41E-18) and the related BRCA1 and BRCA2 pathways (p-values 4,52E-18 and 2,24E-17) (Supplementary Table S4). Ingenuity Pathway Analysis (IPA) discriminated the most significant upregulated and downregulated molecules involved in the major DNA damage pathways. The combination treatment induced a stronger deregulation of the molecules involved in these pathways (Figure 4). the drug combination reduces tumor growth dramatically in an in vivo model In view of the results obtained in the in vitro study, we decided to test the antitumor effects of the Trabectedin and Olaparib combination in a NOD/SCID mouse xenograft model of ES. Xenografts were generated by subcutaneous injection of TC71 cells. Animals were randomized in 5 groups and treated for three weeks: 1) Control group: drug vehicles; 2) Trabectedin group: Trabectedin alone (0.15mg/Kg IV once a week five times per week); 3) Olaparib group: Olaparib (100mg/ Kg BID IP five times per week); 4) Combination1 group: Olaparib alone (100mg/Kg BID IP five times per week) for 1 week, and Olaparib plus Trabectedin (0.15mg/ Kg IV once a week) for the second and third week; 5) Combination 2 group: Olaparib (100mg/Kg BID IP) plus Trabectedin (0.15mg/Kg IV once a week). After three weeks of treatment, the mice were sacrificed and tumors were extracted, weighed, and evaluated by histopathology. The evolution of tumor volume after randomization (see Material and Methods section) and tumor weight Figure 3: DNA damage induction studies. (A and B) Images represent data obtained in the study of induction of DSB evaluated by the presence of intranuclear γH2AX and 53BP1 foci after treatment with Trabectedin and/or Olaparib in the TC71 cell line. A. Short-term DNA damage induction (8 hours of drug exposure). The combination of Trabectedin and Olaparib led to an increased presence of pH2AX and 53BP1 foci in the nucleus. b. Long-term DNA damage induction (24 hours of drug exposure). c. Image represents data obtained in the fragmentation assays after 24 – 48 hours of drug exposure. Although DNA fragmentation was also observed in the control (DMSO), Trabectedin and/or Olaparib induced higher DNA fragmentation. Here, DSB-induced DNA fragmentation can be seen by the presence of a smear just below the intact DNA (represented by *), whereas the smaller fragments (represented by **) are produced exclusively by apoptosis induction. In all cases, combination 1 refers to 125pM Trabectedin and 2.5µM Olaparib; combination 2 refers to 250pM Trabectedin and 5µM. Olaparib. Oncotarget18881 www.impactjournals.com/oncotarget at the end of the study (18 days after the treatments had started), shown in Figure 5A and 5B respectively, were evaluated in all groups. Although Trabectedin and Olaparib alone were able to reduce tumor volume as compared with the control groups, these differences were not statistically significant (Mann-Whitney U-test, p > 0.05). However, the combination of both drugs dramatically reduced tumor growth (Mann-Whitney U-test p < 0.05). In fact, the tumors were smaller at the end of the study than they were at the moment of randomization, before treatment. Histopathological analysis of representative tumors from each group of mice revealed significant morphological changes. The drug-induced morphological changes were characterized by the presence of ES cells with conspicuous nucleoli, granular chromatin, an enlarged cytoplasm and vacuoles (Figure 5C). Necrosis was observed in the animals of all groups, although it was significantly more extensive in the animals treated with the combination of drugs (t-test, p < 0.05) (Figure 5C and Supplementary Figure S8A). The necrotic/ viable tumor ratio was much higher in the treated groups (Supplementary Figure S8A, B). No signs of toxicity were observed with the combination treatment. the drug combination increases DNA damage induction and DDr activation in the in vivo model A representative number of tumor areas from tumors of all groups in the in vivo study were evaluated by Immunoflorescence (IF)/ Immunohistochemstry (IHC) in order to assess the effects of the treatments. To quantify the findings, we used the Ariol Image analysis system (Olympus). The following parameters were studied: apoptosis (fragmentation-late apoptosis and PARP cleavage) (TUNEL assay and cleaved PARP); proliferation (Ki67), DNA damage induction (γH2AX) and DDR activation (BRCA2). The proliferation index was higher in the controls as compared with all treated groups, and this was statistically significant (p < 0.05) (Figure 6, central panel A and Supplementary Figure S8B). These results are in agreement with the data from the TUNEL assays, where late apoptosis due to DNA fragmentation was mostly observed in representative tumor areas from animals treated with Trabectedin alone or in tumors from animals treated with the combination of drugs (Figure 6, upper Figure 4: Expression changes in DNA damage repair genes in Es cells from the tc71 cell line. Schematic representation of gene interactions in cells treated with the combination of Olaparib (5µM) and Trabectedin (250pM), obtained after performing IPA. Overexpressed genes are highlighted in red, and the down-regulated genes are highlighted in green. The central genes involved in HR machinery are highlighted in color, such as BRAC1, EXO1, RAD51 and BRCA2, among others. Oncotarget18882 www.impactjournals.com/oncotarget panel A). PARP cleavage was observed in tumors from the Olaparib group (Figure 6, lower panel A). Interestingly, DNA damage induction as assessed by pH2AX staining was much higher in animals from both combination groups (mean Combination 1: 60.975%, mean Combination 2 group: 68.09%) as compared with controls (mean 0.85%); Trabectedin (mean 11.13%) and Olaparib (mean 1.04%) (p < 0.05) (Figure 6B and Supplementary Figure S8C, S9). This observation correlated well with the overexpression of the HR effector molecule, BRCA2. Treatment with the combination of Olaparib and Trabectedin resulted in a much more intense and diffuse staining of BRCA2, especially in combination 2 (Combination1: mean 42.03%; Combination 2: mean 76.79% vs. mean control group 11.9%) (Supplementary Figure S8D). Additionally, BRCA2 expression was diffuse and intense in the case of tumors from the Trabectedin group (mean 63.84%) (Figure 5C and Supplementary Figure S8D, S10). the trabectedin-olaparib combination induces complete tumor regression in PDX models Because studies in PDX models are likely to be more predictive of patient response to treatment than conventional cell lines [17], we performed survival studies in two ES PDX models (HSJD-ES-004 and HSJDES-006) established from patient biopsies at the Sant Joan de Déu Hospital (HSJD, Barcelona, Spain). The clinical characteristics of the patients and the molecular details of both tumor models are shown in Table 2. The tumor response at the end of treatment is shown in Table 3. The most remarkable finding was that the Olaparib-Trabectedin combination achieved complete response (CR) in 100% of the tumors of both PDX models. In contrast, Olaparib treatment alone led to progressive disease (PD) in 100% of the tumors. Trabectedin alone achieved CR in 88% of the HSJD-ES-004 tumors and CR in 78% of the HSJD-ES-006 tumors. CRs in monotherapy were transient in most animals and did not persist until the end of the survival study (Figure 7A). In the HSJD-ES-004 model, the median survival of the controls was 14 days, as compared to 35 table 2: Details of the Es PDX models. Model code source of biopsy1 Age at biopsy Primary tumor Age at diagnosis Metastasis at diagnosis Demography Fusion gene stAG2 mutation p53 mutation Patient status HSJDES-0042 Relapse. Metastasis in the mediastinum 18 y T12 vertebral body 10 y No Male, white EWSR1- FLI1 negative negative NED3 HSJDES-006 Relapse. Metastasis in the lung 14 y Fibula 12 y No Male, white EWSR1- FLI1 negative negative AWD4 1 Tumor tissue was collected with informed consent under an Institutional Review Board-approved protocol. 2 Further details on this PDX model are published in [21], coded SJDES022-R. 3 No evidence of disease. 4 Alive with disease. table 3: Es PDX models: response to treatment (day 21). PDX Model treatment tumors (n) response1 cr Pr sD PD HSJD-ES-004 Control 10 0 0 0 10 Olaparib 9 0 0 0 9 Trabectedin 8 7 0 0 1 Combination 8 8 0 0 0 HSJD-ES-006 Control 10 0 0 0 10 Olaparib 10 0 0 0 10 Trabectedin 9 1 1 0 7 Combination 9 9 0 0 0 1 CR: Complete response (CR) was defined, as previously described in [34], as the disappearance of measurable tumor mass (<0.10 cm3) at the end of treatment (day 21); PR: Partial response (PR) was defined as a tumor volume regression ≥50% at the end of treatment (day 21) but with a measurable tumor size (≥0.10 cm3); SD: Stable disease (SD) was defined as <50% regression from the initial volume and a ≤25% increase in initial volume at the end of treatment (day 21). PD: Progressive disease (PD) was defined as <50% regression from the initial volume and a >25% increase in the initial volume at the end of treatment (day 21). Oncotarget18883 www.impactjournals.com/oncotarget days for the Olaparib group (p = 0.192), and 75 days for the Trabectedin group (p = 0.0 01). In the HSJD-ES-006 model, the median survival of the controls was 11 days, as compared to 17 days for the Olaparib group (p < 0.05), and 35 days for the Trabectedin group (p = 0.001). The combination therapy extended survival significantly, up to a value greater than the evaluation period (80 days), when compared with the control mice (p < 0.05 for both models; Figure 7B). DIscUssION Trabectedin induces DNA damage and cellular death in vitro and interrupts EWSR1-FLI1-dependent transcription [12]. Additionally, ES cells are sensitive to PARPinh, which disrupt the DDR system of tumor cells [7, 10]. Furthermore, PARP1, the main PARPinh target, also acts as a transcriptional regulator of EWSR1-FLI1 [6]. Accordingly, we formulated the hypothesis that the combination of Trabectedin with a PARPinh could interfere with the DDR system and might be able to inhibit EWSR1-FLI1, and could therefore be of interest as a therapeutic strategy. The results obtained in the present study support the clinical development of a combination of PARPinh with Trabectedin in ES. The relationship between PARP1 and ES has been documented for many years [18]. However, knowledge of the more specific and relevant role of this protein in ES is much more recent [6, 7]. Brenner et al. described that PARP1 interacts physically with EWSR1-FLI1 fusion, acting in a positive feed-back loop and hence regulating its own expression [6]. In a retrospective study, our group Figure 5: In vivo model using the tc71 cell line. A. Graphic representing the evolution of tumor growth in the different treatment groups. Tumor growth was clearly halted by the combination of Olaparib and Trabectedin (Combination2) and when Trabectedin began to be administered one week later (Combination 1). b. Graphic representation of mean tumor weights at the end of the study (18 days after treatment began to be administered) in each of the treatment groups. Tumor weight was clearly reduced, especially in the Combination 1 and 2 groups. c. Representative pictures showing (in columns from left to right) tumors extracted and 4X and 60X H&E representative areas of the tumors from each treatment group respectively. In the second column some necrotic areas are indicated among areas of living cells (*). In the third column, the tumors in the control group show characteristic small round cells with a round nucleus, regular chromatin, a clear nucleolus, scant and rare cytoplasm and a high number of mitotic figures (white arrows). 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