Published OnlineFirst December 14, 2018; DOI: 10.1158/1541-7786.MCR-18-0575 Genome Maintenance Molecular Cancer Research The Antitumor Drugs Trabectedin and Lurbinectedin Induce Transcription-Dependent Replication Stress and Genome Instability Emanuela Tumini1, Emilia Herrera-Moyano1, Marta San Martín-Alonso1, Sonia Barroso1, Carlos M. Galmarini2, and Andres Aguilera1 Abstract R-loops are a major source of replication stress, DNA tedin and lurbinectedin increased the presence of Rad52 foci, a damage, and genome instability, which are major hallmarks marker of DNA damage, in an R-loop–dependent manner. In of cancer cells. Accordingly, growing evidence suggests that R- addition to providing new insights into the mechanisms of loops may also be related to cancer. Here we show that R-loops action of these drugs, our study reveals that R-loops could be play an important role in the cellular response to trabectedin targeted by anticancer agents. Given the increasing evidence (ET743), an anticancer drug from marine origin and its deriv- that R-loops occur all over the genome, the ability of lurbi- ative lurbinectedin (PM01183). Trabectedin and lurbinecte- nectedin and trabectedin to act on them may contribute to din induced RNA–DNA hybrid-dependent DNA damage in enhance their efficacy, opening the possibility that R-loops HeLa cells, causing replication impairment and genome insta- might be a feature shared by specific cancers. bility. We also show that high levels of R-loops increase cell sensitivity to trabectedin. In addition, trabectedin led to Implications: The data presented in this study provide the new transcription-dependent FANCD2 foci accumulation, which concept that R-loops are important cellular factors that con- was suppressed by RNase H1 overexpression. In yeast, trabec- tribute to trabectedin and lurbinectedin anticancer activity. Introduction (TAM). Indeed, both drugs inhibit the transcription of selected cytokines (e.g., CCL2, IL6, IL8, PTX3) by TAMs abrogating their Cancer therapy greatly relies on the use of small molecules that protumoral properties and modifying the tumor microenviron- directly perturb the DNA metabolism. Trabectedin (ET743) is one ment (9, 10). of such molecules. It has been initially derived from the sea squirt Tumor cells are commonly associated with genome instability. Ecteinascidia turbinata and is now produced synthetically, being One important natural source of instability is represented by R- currently used in therapy of patients with advanced soft tissue loops, a three-stranded nucleic acid structure consisting of an sarcoma (1) and platinum-sensitive ovarian cancer (2). Different RNA–DNA hybrid and the displaced single-stranded DNA of the derivatives of trabectedin have been synthesized, among them original DNA duplex (11). This structure could fulfill physiologic lurbinectedin (PM01183), which is presently under evaluation in roles as it occurs during immunoglobulin class switching recom- phase II and III clinical trials. bination, plasmid or mitochondrial DNA replication, and tran- One of the most important features of the mechanism of action scription regulation. However, they are an important source of of trabectedin and lurbinectedin is their inhibitory effect on DNA damage, genome instability, and replication stress, which transcription of protein-coding genes, which is mediated by relates R-loops with a number of neurodegenerative disorders and different means. First, they preferentially bind to specific DNA cancer (12, 13). Interestingly, the loss of function of the breast triplets that are often present on transcription recognition sites. In cancer susceptibility genes BRCA1 and BRCA2, which have been this way they could directly prevent loading of transcription shown to increase R-loops and R-loop-dependent DNA damage factors onto chromatin (3). Moreover, both drugs were shown (14, 15), is also associated with an increased sensitivity to tra- to induce degradation of the RNA polymerase II (RNAPII) bectedin (16). In a similar way, dysfunctions of the Fanconi through the ubiquitin–proteasome pathway (4–8). Of note, these anemia (FA) pathway that increased sensitivity to such agent effects are also observed in tumor-associated macrophages (5) also cause accumulation of R-loops (17, 18). In addition, there are some similarities between R-loops and trabectedin adducts worth being considered. The head-to-tail 1 Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER, binding of three trabectedin molecules to three adjacent opti- CSIC-Universidad Pablo de Olavide-Universidad de Sevilla, Seville, Spain. 2PharmaMar, Colmenar Viejo, Spain. mal DNA binding sites changes the DNA duplex conformation from the B-form to an intermediate between the A- and the B- Note: Supplementary data for this article are available at Molecular Cancer form, which strongly resembles the conformation of an RNA– Research Online (http://mcr.aacrjournals.org/). DNA hybrid (19). Moreover, R-loops that accumulate in the Corresponding Author: Andres Aguilera, Universidad de Sevilla; Av. Americo absence of the putative RNA–DNA helicases AQR or SETX Vespucio 24, Seville 41092, Spain. Phone: 34954468372; E-mail: [email protected] require the action of nucleotide excision repair (NER) to be doi: 10.1158/1541-7786.MCR-18-0575 processed into double-strand breaks (DSB; ref. 20) as well as Ó2018 American Association for Cancer Research. trabectedin adducts (21). www.aacrjournals.org 773 Downloaded from mcr.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst December 14, 2018; DOI: 10.1158/1541-7786.MCR-18-0575 Tumini et al. For these reasons, in this study we have further explored the Immunofluorescence and EdU labeling mechanism of action of trabectedin and lurbinectedin, in partic- Immunofluorescence was performed as previously described ular whether it could be partially mediated or affected by R-loops. (25). Briefly, cells were fixed with 3.7% formaldehyde in PBS for We show that the DNA damage and replicative stress caused by 15 minutes, permeabilized with 0.5% Triton X-100, and blocked trabectedin and lurbinectedin is indeed partially dependent on R- with 3% BSA in PBS for 1 hour. The coverslips were then incubated loops, as demonstrated by molecular and genetic assays using with primary antibodies diluted 1:500 in 3% BSA in PBS for both human cell lines and the yeast Saccharomyces cerevisiae as a 2 hours followed by 1 hour incubation with 1:1,000 diluted model eukaryotic system. Our findings suggest that R-loops could secondary antibodies and nuclei counterstaining with 1 mg/mL be targets of anticancer agents and given the evidence that R-loops DAPI in PBS. For FANCD2 immunolabeling, before fixation cells occur all over the genome (22), this opens the possibility that were pre-permeabilized with 0.25% Triton X-100 in PBS for 1 tumoral cells that accumulate R-loops may be more suitable for minute on ice. S9.6 immunofluorescence was performed as trabectedin and lurbinectedin treatment. previously described (20). For the EdU labeling, cells were pulse-labeled with 10 mmol/L EdU for 20 minutes before fixation. Materials and Methods EdU staining was performed with a Click-iT EdU Alexa Fluor 555 Imaging Kit (Invitrogen) according to manufacturer's instruc- Cell culture and treatments tions. Random images were acquired with a 63Â or 40Â objective HeLa cells were purchased from European Collection of Cell and foci or nuclear intensity were scored using the MetaMorph Cultures (ECACC), HeLa-TR (23), and HeLa HB-GFP (14) software. At least 100 cells were scored for each condition. were previously generated in our laboratory. All cell lines were maintained in DMEM medium, supplemented with 10% heat- DNA combing inactivated FCS, cultured at 37C in a humidified atmosphere DNA combing was performed as previously described (23). containing 5% CO , and routinely tested for mycoplasma using 2 Briefly, DNA fibers were extracted from cells in agarose plugs MycoAlert Mycoplasma Detection Kit (Lonza). HeLa HB-GFP immediately after CldU labeling and were stretched on silanized cells medium was supplemented with doxycycline at the final coverslips. DNA molecules were counterstained with an autoanti- concentration of 2 mg/mL to induce HB-GFP expression. Specific ssDNA antibody (DSHB, 1:500) and an anti-mouse IgG coupled genes were knocked down using ON-TARGET SMARTpool siRNA to Alexa Fluor 647 (A21241; Molecular Probes; 1:50). CldU and from Dharmacon. Transient transfection of siRNA was performed IdU were detected with BU1/75 (AbCys; 1:20) and an anti-rat IgG using DharmaFECT 1 (Dharmacon), according to the manufac- coupled to Alexa Fluor 488 (A21470; Molecular Probes; 1:50) or turer's instructions. The following plasmids were used for trans- B44 (Becton Dickinson; 1:20) anti-BrdU antibodies and an anti- fection: pcDNA3 (Invitrogen) (RNH1À) and pcDNA3-RNaseH1 mouse IgG coupled to Alexa Fluor 546 (A21123; Molecular (RNH1þ), containing the full-length RNase H1 cloned into Probes; 1:50), respectively. DNA fibers were analyzed on a Leica pcDNA3 (24). Lipofectamine 2000 (Invitrogen) was used for DM6000 microscope equipped with a DFC390 camera (Leica). plasmid transfection. Assays were performed 48 or 72 hours after Data acquisition was performed with LAS AF (Leica). Fork velocity transfection. Trabectedin (ET743) and lurbinectedin (PM01183) was calculated as previously described (26). Replication asym- were provided from Pharmamar. Cordycepin (C3394) and metry was calculated by dividing