Lurbinectedin Specifically Triggers the Degradation of Phosphorylated RNA Polymerase II and the Formation of DNA Breaks in Cancer Cells
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Published OnlineFirst September 14, 2016; DOI: 10.1158/1535-7163.MCT-16-0172 Small Molecule Therapeutics Molecular Cancer Therapeutics Lurbinectedin Specifically Triggers the Degradation of Phosphorylated RNA Polymerase II and the Formation of DNA Breaks in Cancer Cells Gema Santamaría Nunez~ 1, Carlos Mario Genes Robles2, Christophe Giraudon2, Juan Fernando Martínez-Leal1, Emmanuel Compe2,Fred eric Coin2, Pablo Aviles1, Carlos María Galmarini1, and Jean-Marc Egly2 Abstract We have defined the mechanism of action of lurbinectedin, a ubiquitin/proteasome machinery; and (iii) the generation of marine-derived drug exhibiting a potent antitumor activity DNA breaks and subsequent apoptosis. The finding that inhi- across several cancer cell lines and tumor xenografts. This drug, bition of Pol II phosphorylation prevents its degradation and currently undergoing clinical evaluation in ovarian, breast, and the formation of DNA breaks after drug treatment underscores smallcelllungcancerpatients,inhibits the transcription pro- the connection between transcription elongation and DNA cess through (i) its binding to CG-rich sequences, mainly repair. Our results not only help to better understand the high located around promoters of protein-coding genes; (ii) the specificity of this drug in cancer therapy but also improve our irreversible stalling of elongating RNA polymerase II (Pol II) understanding of an important transcription regulation mech- on the DNA template and its specific degradation by the anism. Mol Cancer Ther; 15(10); 1–14. Ó2016 AACR. Introduction derivatives, anthracyclines, etc.; ref. 10). Currently, several laboratories are developing inhibitors of cyclin-dependent Cancer cells aberrantly deregulate specific gene expression kinases (CDK) that have a critical role in regulating transcrip- programs with critical functions in cell differentiation, prolifer- tion initiation, pause release, and elongation (e.g., CDK7, ation, and survival (1). Differently from noncancer cells, those CDK8, or CDK9), the three main steps involved in RNA altered gene programs in cancer cells have a striking dependence synthesis (11, 12). Other approaches are inhibition of DNA on continuous active transcription. For example, small cell lung repair mechanisms (e.g., irinotecan, topotecan, olaparib; cancer (SCLC) cells are addicted to lineage-specific and proto- ref. 13) or chromatin remodeling (HDAC inhibitors or oncogenic transcription factors that support their growth (2–7). demethylating agents; refs. 14, 15). Although these compounds Similarly, triple-negative breast cancer (TNBC) is highly depen- have already entered clinical trials, the mechanisms by which dent on uninterrupted transcription of a specific key set of genes they disturb transcription as well as those driving to cancer cell (8, 9). Pharmacologic modulation of transcription of protein- death are far from being understood. coding genes may thus provide an approach to identify and treat Here, we describe the inhibition of transcription by lurbi- tumor types that are dependent on deregulated transcription for nectedin (PM01183; Fig. 1A), an anticancer agent that is being maintenance of their oncogenic state. evaluated in late-stage (phases II and III) clinical trials. Lurbi- Targeting DNA in tumor cells happened to be the most nectedin is structurally related to trabectedin, containing the explored therapeutic strategy to block DNA processing enzymes same pentacyclic skeleton of the fused tetrahydroisoquinoline such as those involved in transcription (e.g., cisplatin and rings, but differing by the presence of a tetrahydro-B-carboline replacing the additional tetrahydroisoquinoline of trabectedin. The pentacyclic skeleton is mostly responsible for DNA minor 1Cell Biology and Pharmacogenomics Department, Pharmamar SA, groove recognition and binding. Lurbinectedin reacts with the 2 Colmenar Viejo, Madrid, Spain. Department of Functional Genomics exocyclic amino group of guanines in the minor groove of DNA and Cancer, IGBMC, CNRS/INSERM/University of Strasbourg, C. U. Strasbourg, France. forming a covalent bond. The resulting adduct is additionally stabilized through the establishment of van der Waals interac- Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). tions and one or more hydrogen bonds with neighboring nucleotides in the opposite strand of the DNA double helix G. Santamaría Nunez~ and C.M. Genes Robles are first coauthors. (16). The additional tetrahydro b-carboline moiety protrudes Corresponding Author: C.M. Galmarini, Pharmamar SA, Avda de los Reyes 1, from the DNA minor groove and could be interacting directly Colmenar Viejo 28770, Madrid, Spain. Phone: 34 918466158; Fax: 34 918466001; with specific factors involved in DNA repair and transcription E-mail: [email protected] pathways. Indeed, it is possible that this part of the molecule doi: 10.1158/1535-7163.MCT-16-0172 interacts directly with TC-NER factors and could interfere with Ó2016 American Association for Cancer Research. the repair mechanism. In this sense, lurbinectedin is able to www.aacrjournals.org OF1 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst September 14, 2016; DOI: 10.1158/1535-7163.MCT-16-0172 Santamaría Nunez~ et al. attenuate the repair of specific nucleotide excision repair (NER) in the article have been authenticated in the laboratory in the last 6 substrates (17, 18). In addition to its activity in tumor cells, it months. was recently shown that lurbinectedin affects the inflammatory microenvironment, with a selective apoptotic-inducing effect Cell proliferation on mononuclear phagocytes and a specific inhibition of pro- Cell proliferation was studied by [3-(4,5-dimethythiazol-2-yl)- duction of inflammatory cytokines (19, 20). In this work, we 2,5-diphenyl] tetrazolium bromide (MTT) assays that were per- show that, following its specific target on CG-rich sequences formed following the manufacturer's instructions (MTT Cell located at promoters of protein-coding genes, lurbinectedin Proliferation Kit I; Roche Diagnostics). Briefly, cells were seeded induces the specific degradation of elongating (phosphorylat- in 96-well trays. Serial dilutions of lurbinectedin, PM030779, or ed) RNA polymerase II (Pol II) by the ubiquitin-proteasome PM120306 were added to the medium. Exposure to the drugs was fi machinery. This process occurs speci cally on activated genes maintained during 72 hours. Determination of IC50 values was and is associated with the formation of DNA breaks that drive performed by iterative nonlinear curve fitting using the Prism 5.0 tumor cells to apoptosis. Inhibition of Pol II phosphorylation statistical software (GraphPad). The data presented are the aver- prevents its degradation and the formation of DNA breaks. age of three independent experiments performed in triplicate. These investigations not only show how lurbinectedin causes a cascade of events on the transcription process that can explain DNA electrophoretic mobility shift assay its antiproliferative activity on tumor cells, but also improve The binding assay was performed with a 250 pb PCR product our understanding of the fate of Pol II when it encounters a from the human adiponectin gene. After incubation with lesion on the DNA. appropriate concentrations of the compounds at 25Cduring 1 hour, the DNA was subjected to electrophoresis in a 2% (w/v) Materials and Methods agarose/TAE gel, stained with ethidium bromide (Sigma) and photographed. Reagents Lurbinectedin was produced by PharmaMar through a semi- DNase I footprinting assays synthetic method. Z-Leu-Leu-Leu-al (MG-132), 5,6-dichloro- Radiolabeled AS/CGG and OS/CCG were bound to magnetic fl benzimidazole-1-a-D-ribofuranoside (DRB), and avopiridol beads (Dynabeads) and incubated for 30 minutes at room tem- were purchased from Sigma. The following antibodies were perature with the indicated drug concentrations (21). After exten- used for Western blotting: POLR2A (RPB1, Pol II) (clones N-20 sive washings, DNase I digestion was performed for 45 seconds at and H-224), POLR1A (RNA Pol I), POLRMT (B-1), CCNH room temperature. Purified nucleic acids were resolved on an 8% (Cyclin H) (B-1), CDK9 (C-20), ERCC2 (p80-TFIIH) denaturing Urea-polyacrylamide gel. (H-150), TP53 (FL-393) from Santa Cruz Biotechnology; TBP, GTF2H1 (TFIIH), POLR2D (RPB4, RNA Pol II), POLR2B (RPB2, In vitro transcription assays RNA Pol II), CRCP (RNA Pol III) from Abcam; CDK7 from Cell Run-off transcription assays were performed using recombi- – Signaling Technology; and anti phospho-Ser2Pol II (clone H5) nant TFIIB, TFIIE, TFIIF, TBP, TFIIH, and RNA pol II, as previously – and anti phospho-Ser5 Pol II (clone H14) from Covance. The described (22). following antibodies were used for chromatin immunoprecip- itation (ChIP) and immunoprecipitation (IP) experiments: RNA synthesis quantification in tumor cells Antibodies against phospho-Ser2 Pol II (clone 3E8) and phos- A549 (3.5 Â 104 cells/well), A673 (2.6 Â 105), HCT116 (1.8 pho-Ser2 Pol II (clone 3E10) were from Active Motif. Poly- Â 105), HeLa (1.5 Â 105), and MDA-MB-231 (2 Â 105)were clonal antibodies against POLR2A (H-224), CDK7 (C-19), seeded in 24-well plates and incubated with lurbinectedin or CDK9 (H-169), UBB (Ubiquitin clone FL-76 or clone A-5), vehicle (DMSO) for 30, 45, 60, and 90 minutes and pulsed with Biotin(33), and ERCC4 (XPF, clone H-300) were from Santa 5 mCi [3H] uridine (Perkin Elmer) for