Lurbinectedin Specifically Triggers the Degradation of Phosphorylated RNA Polymerase II and the Formation of DNA Breaks in Cancer Cells

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. 2016 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 2016 American Association for Cancer Research. the repair mechanism. In this sense, lurbinectedin is able to

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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 immunoprecipitation (ChIP) and immunoprecipitation (IP) experiments: RNA synthesis quantification in tumor cells Antibodies against phospho-Ser2 Pol II (clone 3E8) and phos- A549 (3.5 104 cells/well), A673 (2.6 105), HCT116 (1.8 pho-Ser2 Pol II (clone 3E10) were from Active Motif. Poly- 105), HeLa (1.5 105), and MDA-MB-231 (2 105)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 30 minutes in medium Cruz Biotechnology. Antibody against g-H2AX (ab2893) was supplemented with 10% FCS. Then, cells were rinsed twice in obtained from Abcam. POLR2A (1PB-7C2), PSMC5 (Sug-1, PBS and fixed with chilled 10% trichloroacetic acid for 10 clone 3SCO), and GTF2F2 (TFIIF b) antibodies were from minutes, and the monolayers were washed with ethanol and IGBMC. air-dried at room temperature. The precipitated macromole- cules were dissolved in 0.5 N NaOH and 0.1% SDS and diluted Cell lines in Ultima-Flo M (Perkin Elmer). The radioactivity was mea- The following cell lines were obtained from the ATCC in sured using a b-counter Hidex 300SL. 2007: A549 (lung adenocarcinoma; CCL-185), A673 (human muscle Ewing's sarcoma; CRL-1598), HCT-116 (human colo- Western blotting analysis rectal carcinoma; CCL-247), HT-29 (human colorectal carcino- Cell protein extracts were prepared following standard proce- ma; HTB-38), MCF7 (breast adenocarcinoma; HTB-22), MDA- dures in RIPA buffer in the presence of protease inhibitors MB-231 (breast adenocarcinoma; HTB-26), and PSN-1 (pan- (Complete, Roche Diagnostics) and phosphatase inhibitors creatic adenocarcinoma; CRM-CRL-3211). HeLa and HeLa (PhosStop, Roche Diagnostics). After quantification with the siXPF are cervical carcinoma cells and were obtained from Micro-BCA Protein Assay Kit (Thermo Scientific), 15 to 25 mgof Tebu-Bio. Dr. M. D'Incalci (Istituto Mario Negri) generously protein were separated by SDS-PAGE and transferred to PVDF provided IGROV1 and IGROV-ET ovarian cancer cell lines. All membranes (Immobilon-P; Millipore). After using appropriated cell lines were cultured in the medium recommended by the primary and secondary antibodies, blots were developed by a supplier supplemented with 10% FBS, 2 mmol/L L-glutamine, and peroxidase reaction using the ECL detection system (Amersham- penicillin–streptomycin mix (Sigma). None of the cell lines used G.E. Healthcare).

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Figure 1. Antiproliferative activity of lurbinectedin in several cancer cells. A, structure of lurbinectedin. B, antiproliferative activity of lurbinectedin in human lung (A549), Ewing sarcoma (A673), colon (HCT-116, HT-29), breast (MCF7, MDA-MB-231), cervix (HeLa), and pancreas (PSN-1) cancer cell lines. Cell viability was analyzed 72 hours after lurbinectedin treatment by MTT assay. C, in vivo antitumor activity of lurbinectedin. Treatment with the drug results in tumor growth inhibition in A549 xenograft models compared with vehicle-treated animals. Lurbinectedin treatment started at a tumor volume size of c. 150 mm3 and was intravenously administered in three cycles of three consecutive weekly doses at 0.18 mg/kg/day. Each point represents median values of n ¼ 10. D, survival curves of animals bearing A549 tumors after treatment with lurbinectedin. Drug treatment was associated with a statistically signiﬁcant increase in survival compared with placebo-treated mouse model. E, IGROV- and IGROV-ET–resistant cell viability was analyzed 72 hours after lurbinectedin treatment by MTT (&, IGROV; ~, IGROV-ET). F, binding to naked DNA. Increasing amounts of lurbinectedin and PM030779 were incubated with a DNA fragment of 250 bp, and the electrophoresis was run in 2% agarose-TAE. G, antiproliferative activity of lurbinectedin and its analogue PM030779 in human A549 lung cancer cells. Cell viability was analyzed 72 hours after treatment by MTT assay (*, Lur; &, PM030779).

Pol II half-life measurement have been treated with lurbinectedin. Whole-cell extracts were Pulse-chase analyses were carried out in cells that were pre- prepared using RIPA buffer (0.01 mol/L Tris–HCl, pH 8.0, 0.14 treated during 2 hours in DMEM cys/met medium and then mol/L NaCl, 1% Triton X-100, 0.1% Na Deoxycholate, 0.1% metabolically labeled with 100 mCi/mL 35S-methionine for 1 SDS), and Pol II was immunoprecipitated using a speciﬁc anti- hour in DMEM cys/met medium. After washing steps, cells body and resolved by SDS–PAGE. 35S-Pol II bands were

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quantified by phosphorimager analysis using ImageJ software CAGGAA-3' and rev 5'-TGAGATCGTCCAACTCAGCTGTCA-3'. (open source). Biotin-ChIP assays have been performed as previously described (23). Immunostaining A549 cells were treated with the appropriate concentration of NER assays lurbinectedin for 4 hours in the absence or presence of the OS/CCG DNA template (containing a single lurbinectedin transcription inhibitor 20 mmol/L DRB (preincubation of 1 hour), adduct; ref. 21) has been bound to magnetic beads (Dynabeads) washed, fixed (4% paraformaldehyde), permeabilized (0.5% and incubated with the indicated drug concentrations. Dual Triton X-100), and incubated with the primary anti-Pol II mono- incision assays were next carried out after addition of XPG, clonal antibody for 1 hour at 37C. Thereafter, the cells were XPF/ERCC1, XPC/hHR23B, RPA, XPA, and TFIIH. The reactions washed and incubated with the AlexaFluor 594 secondary goat were conducted as previously described (24). anti-mouse IgG (Invitrogen) for 30 minutes at 37C. Finally, the slides were incubated with Hoechst 33342 (Sigma) and mounted with Mowiol mounting medium. Pictures were taken with a Leica Comet assays DM IRM fluorescence microscope equipped with a 100x oil After lurbinectedin or PM030779 treatment, single-cell gel immersion objective and a DFC 340 FX digital camera (Leica). electrophoresis assay has been used (CometAssay; Trevigen) following the manufacturer's instructions. Experiments and pic- Immunoprecipitation tures analyses have been performed as previously described (25). A549 whole-cell extracts were made using RIPA buffer supplemented with PIC (Roche) and phosphatase inhibitors (Active Antitumor activity in xenograft murine models Motif). Antibodies were incubated with magnetic beads and lately All animal protocols were reviewed and approved according to protein whole-cell extract was added. Sequential washes were regional Institutional Animal Care and Use Committees. Mice done, and the resulting sample was loaded on SDS-PAGE for used in the following experiments were always female 4 to 6 weeks Western blotting. When Tandem Ubiquitin Binding Entity of age, 16 to 25 gr, athymic nude-Foxn1 nu/nu obtained from (TUBES 2) technology (Lifesensors) was used, equilibrated slurry Envigo (Italy). Mice were housed in individually ventilated cages was initially incubated with protein whole cell extract at 4C. After on a 12-hour light–dark cycle at 21 to 23oC and 40% to 60% extensive washing steps, the resulting sample was loaded on SDS- humidity. Mice were allowed free access to an irradiated diet and PAGE for Western blotting. sterilized water. Design, randomization, and monitoring of experiments (including body weights and tumor measurements) Subcellular protein fractionation were performed using NewLab Software v2.25.06.00 (NewLab A549 cells were incubated with the different compounds at Oncology). All mice were s.c. xenografted with A549 cancer cells 6 different time points, and subcellular protein fractionation was into their right flank with c. 3 10 cells in 0.2 mL of a mixture performed using the Subcellular Protein Fractionation Kit for (50:50; v:v) of Matrigel basement membrane matrix (Becton Cultured Cells (Thermo Scientific Waltham) following the man- Dickinson) and serum-free medium. When tumors reached 3 ufacturer's instructions. approximately 150 mm , mice were randomly assigned to treatment or control groups. Lurbinectedin was intravenously admin- Reverse transcription and quantitative PCR istered in three consecutive weekly doses (0.18 mg/kg/day), Total RNA was isolated using the RNeasy Mini Kit (QIAGEN) whereas the control animals received an equal volume of vehicle and reverse transcribed with SuperScript II reverse transcriptase with the same schedule. Caliper measurements of the tumor (Invitrogen). The quantitative PCR was done using the Lightcycler diameters were made twice weekly, and tumor volumes were 2 480 SYBR Green and the Lightcycler 480 (Roche Diagnostics). calculated according to the following formula: (a x b) /2, where a The primers used in the real-time PCR experiments were as and b were the longest and shortest diameters, respectively. follows: for RARb2: fw 5'-CCAGCAAGCCTCACATGTTTCCAA- Animals were humanely killed when their tumors reached 3,000 3 3' and rv 5'-TACACGCTCTGCACCTTTAGCACT-3'; for Glyceral- mm or if significant toxicity (e.g., severe body weight reduction) dehyde 3-phosphate dehydrogenase (GAPDH): fw 5'-TCGA- was observed. Differences in tumor volumes between treated and CAGTCAGCCGCATCTTCTTT-3' and rv 5'-ACCAAATCCGTT- control groups were evaluated using the Mann–Whitney U test. GACTCCGACCTT-3'. RARb2 mRNA levels represent the ratio Statistical analyses were performed by LabCat v8.0 SP1 (Innova- between values obtained from treated and untreated cells nor- tive Programming Associates, Inc.). malized against the housekeeping GAPDH mRNA. Chem-Seq assay Chromatin immunoprecipitation A549 cells treated with 300 nmol/L of PM120306 during 4 Cells were cross-linked at room temperature for 10 minutes hours were cross-linked with 1% formaldehyde at room temper- with 1% formaldehyde. Chromatin was isolated and sonicated ature for 10 minutes. After sonication, the soluble chromatin was (23). Samples were immunoprecipitated (IP) with antibodies at incubated with 80 mL of Streptavidin beads (Life technologies) 4C overnight, and protein G-Sepharose beads (Upstate) were overnight. After wash the captured DNA was eluted, de-cross- added, incubated 3 hours at 4C, and sequentially washed. DNA linked and purified by QiAquick Spin columns (Qiagen). The fragments were purified using the QIAquick PCR purification Kit DNA was used to construct a library and sequenced on an (QIAGEN) and analyzed by real-time PCR using sets of primers Illumina HiSeq 2500 system. The sequencing reads were aligned targeting RARb2 gene promoter: fw 5'-TGGTGATGTCAGAC- to the hg19 assembly by using Bowtie 1.0. The HT-seq data were TAGTTGGGTC-3' and rev 5'-GCTCACTTCCTACTACTTCTGT- visualized by preparing custom tracks for the UCSC (University of CAC-3'; and RARb2 exon 4: fw 5'-TCCAGCTGTCAGGAATGA- California Santa Cruz) genome browser (https://genome.ucsc.edu).

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Annotations were performed by using both MACS (http://liulab. bound (Fig. 2A, lanes 4–6). We also observed that the drug dfci.harvard.edu/MACS/; ref. 26) and Ensembl database Release interacted with the opposite strand (OS) through hydrogen bonds 75. CG motifs search was performed by using Hypergeometic and van der Waals forces (Fig. 2A, lanes 11–12). Optimization of Motif Enrichment (HOMER; http://homer.salk. To identify genomic binding sites of lurbinectedin in A549 edu; ref. 27). The pattern search of the motif CGG in the summit cells, we next performed a chemical affinity capture (Chem-Seq) (100 bp) of all the identified peaks was done using a home-made which measures the incorporation of the biotin-linked lurbinec- Java program. tedin analogue (PM120306; Supplementary Fig. S1B) within DNA followed by DNA sequencing. This analogue that binds Results guanine through its hydroxyl group (as lurbinectedin) was shown Lurbinectedin targets DNA to arrest cancer cell growth to exhibit antiproliferative properties similar to lurbinectedin We analyzed the effect of lurbinectedin on several human (Supplementary Fig. S1C and S1D, see below). Sonicated chro- cancer cell lines including lung (A549), Ewing sarcoma (A673), matin from cells treated by PM120306 was incubated with colon (HCT-116, HT-29), breast (MCF7, MDA-MB-231), cervix magnetic streptavidin beads to isolate the biotinylated DNA (HeLa), and pancreas (PSN-1), over a 72-hour period (Fig. 1B; fragments followed by massively parallel DNA sequencing. These Supplementary Table S1). Lurbinectedin showed a potent anti- sequences were used to reveal DNA regions enriched (peaks) in fi proliferative activity with IC50 values in the low nanomolar range PM120306-bound sites genome wide. We identi ed approxi- on all the cancer cell lines tested so far. We then performed mately 1,000 peaks in the sequenced DNA. Interestingly, we xenograft studies to check whether the antiproliferative effect of detected a high density of peaks in the vicinity (þ/- 10 Kb) of lurbinectedin translated into in vivo antitumor activity. A549 cells promoter-transcription start sites (TSS; Fig. 2B). We also observed were xenografted into the right flank of athymic nu/nu mice. Once that CpG-rich regions around TSS (Fig. 2C) overlapped with the the tumors reached c.150 mm3, mice were randomized into peaks previously identified (Fig. 2B). Knowing that the optimal groups (n ¼ 10) and either vehicle or lurbinectedin (0.18 mg/ binding site of lurbinectedin is the CGG triplet, we did a pattern kg/day) was intravenously administered in three consecutive search of this motif in all the identified peaks: 50% of them weekly doses. In those conditions, lurbinectedin presented anti- included at least one CGG triplet (data not shown). tumor activity with a statistically significant inhibition of tumor Altogether, our data show that lurbinectedin specifically targets growth (Fig. 1C). In control mice, mean tumor volumes at 0, 7, CG-rich regions that are largely located in areas surrounding and 14 days were 165 mm3, 582 mm3, and 1,575 mm3, respec- promoter regions. tively, whereas in lurbinectedin-treated mice, mean tumor volumes at the same time intervals were 170 mm3, 168 mm3, Lurbinectedin inhibits RNA synthesis and induces Pol II and 278 mm3, respectively (P ¼ 0.7, P ¼ 0.004, and P ¼ 0.002, degradation respectively). Lurbinectedin also induced an improvement of The inhibition of RNA synthesis by lurbinectedin was investi- mice survival (mean survival for control mice: 18 days vs. mean gated in A549 lung cancer cells (Fig. 3A) as well as in Ewing survival in lurbinectedin-treated mice: 47 days; P ¼ 0.001; Fig. sarcoma (A673), colon (HCT-116), cervix (HeLa), and breast 1D). No significant toxicity or body weight loss was observed in (MDA-MB-231) cancer cell lines (Supplementary Fig. S2A). All the treated animals (data not shown). cell lines were treated with lurbinectedin (30 nmol/L) over time 3 Interestingly, IGROV-ET ovarian cancer cells, overexpressing before being pulsed with [ H] uridine. Total RNA synthesis was P-glycoprotein and previously shown to be resistant to doxo- inhibited by around 40% after 30 minutes and almost 80% after rubicin, etoposide, and trabectedin (28), were less sensitive to 2-hour treatment in all the cell lines analyzed. We also investi- lurbinectedin (Fig. 1E). These data indicated that lurbinectedin gated whether lurbinectedin would affect RNA synthesis when needed to accumulate in the cell to exert its antiproliferative added to a well-defined in vitro transcription assay containing the activity. Band shift assays next demonstrated that the drug adenovirus major late promoter (AdMLP) as a template, in bound to DNA (Fig. 1F). Indeed, we observed a delay in addition to TFIIB, TBP, TFIIE, TFIIF, TFIIH basal transcription electrophoretic migration of a 250 bp DNA fragment treated factors, and RNA pol II (Pol II; ref. 21). The exposure of the DNA with lurbinectedin, while when treated with PM030779, a template to increasing amounts of lurbinectedin before adding structural analogue lacking the hydroxyl group at position the transcription machinery led to a progressive inhibition of RNA 21 that is involved in the covalent binding to guanines (ref. 29; synthesis (Fig. 3B). Supplementary Fig. S1A), DNA migrated as the control-untreat- Having observed that the drug induced RNA synthesis inhibi- ed fragment. We then analyzed the effect of either lurbinectedin tion, we next examined the fate of components involved in the or PM030779 on A549 lung cancer cells over a 72-hour period transcription process overtime after lurbinectedin treatment. In (Fig. 1G). Lurbinectedin showed a potent antiproliferative A549 whole-cell extracts, we observed a rapid disappearance of activity with IC50 values in the low nanomolar range, whereas both the hypo- (IIa) and hyper- (IIo) phosphorylated forms of PM030779 was inactive. Rpb1, the largest subunit of Pol II, in a time-dependent manner (Fig. 3C); Western blot also showed that the carboxy-terminal Lurbinectedin specifically targets DNA CG-rich regions domain (CTD contains 52 Serine and threonine rich hepta- We next searched for the target site of the drug using a DNase I repeats) of Rpb1 was phosphorylated on both Serine 5 and Serine footprinting assay. We designed a DNA template that contained a 2 (Fig. 3D), indicating that the drug treatment did not prevent the unique, high-affinity adduct-forming site (CGG triplet) in just one Pol II phosphorylation. Pol II disappearance was also observed in strand. We observed that lurbinectedin was, in fact, bound to both all the other human tumor cells so far tested (Supplementary Fig. strands (Fig. 2A). Lurbinectedin-bound DNA strand (AS) was S2B). We also noticed the disappearance of Rpb2, the second protected from DNase I digestion from nucleotides T-4 to Tþ6, largest subunit of Pol II, after 15 hours, whereas the amounts of being G0 the guanine to which lurbinectedin was covalently other subunits such as Rpb4 remained unchanged along the time

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Figure 2. Lurbinectedin targets GC-rich regions. A, DNase I footprinting on DNA template containing a unique drug-binding site. The AS/CGG (AS, left part) and OS/CCG (OS, right part) were incubated with increasing Lurbinectedin (Lur) concentrations and treated with DNase I. The positions of the protected nucleotides are indicated (G0 is the guanine that is covalently bound to the drug). B, chem-Seq analysis of A549 cells treated with biotinylated-lurbinectedin. Peaks distribution histogram in the vicinity of the transcription start site (TSS) þ/- 10 Kb. C, density of CG repeats around the nearest TSS of all peaks.

course (Fig. 3D). Remarkably, other RNA polymerases, such as Pol ically labeled with [35S]-methionine for 1 hour and then treated I (Rpa194 subunit), Pol III (Rpc9 subunit), or mitochondrial RNA with the drug. Cells were collected at different times, and Pol II Pol, remained present in the cell extracts several hours after drug was immunoprecipitated before being resolved by SDS–PAGE treatment (Fig. 3D). Western blot analyses showed that the drug and autoradiographed (Fig. 3F). Newly synthesized [35S]-Pol II did not affect other transcription factors, such as TFIIH (as was detected up to 6 hours in untreated cells, whereas in drug- visualized by the presence of CDK7 kinase and p62 subunits), treated cells, Pol II labeling was almost absent after the ﬁrst hour. TFIIF (b subunit), P-TEFb (CDK9 subunit) as well as TBP (a member of TFIID), all of them being involved in various steps Phosphorylation of Pol II is essential for its degradation of the RNA synthesis process (Fig. 3E). To further investigate the potential connection between the We next studied the turnover of Pol II following lurbinectedin phosphorylation and the degradation of Pol II after lurbinectedin treatment by conventional pulse chase. A549 cells were metabol- treatment, we pretreated A549 cells with 20 mmol/L of DRB. This

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Figure 3. Pol II degradation in lurbinectedin-treated A549 cells. A, kinetics of RNA synthesis. A549 cells were treated with lurbinectedin (Lur) or vehicle (DMSO) in normal growth medium for 0, 30, 45, 60, 90, and 120 minutes and pulsed with 5 mCi/mL [3H]uridine for 30 minutes. Data are mean SEM values (triplicates, n ¼ 1). B, in vitro transcription assay using AdMLP as a template in addition to all the basal transcription factor and Pol II. The DNA template was incubated with amounts of drug as indicated. In lane 6, D stands for DMSO. 309 nt indicates the size of the RNA transcript. C–E, Western blot analyses of extracts from A549 cell and collected at different time after lurbinectedin (Lur, 30 nmol/L) treatment. Different antibodies have been used to reveal the hyperphosphorylated (IIo) and hypophosphorylated (IIa) forms of the Rpb1 subunit of Pol II, Rpb1 phosphorylated on Serine 5 (S5-P) and Serine 2 (S2-P), Rpb2 and Rpb4 subunits of Pol II, Rpa194 subunit of RNA polymerase I (Pol I), Rpc9 subunit of RNA polymerase III (Pol III), mitochondrial RNA Polymerase (Mt Pol), as well as the CDK7 and p62 subunits of TFIIH, TFIIFb, and CDK9 subunit of P-TEFb. b-Tubulin (Tub) has been used as a control. F, turnover of Pol II as detected by conventional pulse chase. A549 cells were metabolically labeled with [35S]-methionine for 1 hour and then treated with either normal medium (NT) or supplemented with the drug (Lur, 30 nmol/L). Cells were collected at different times, and Pol II was immunoprecipitated with anti-POLR2A clone H-224 before being resolved by SDS–PAGE and autoradiographed. Graph depicts Pol II protein levels (au, arbitrary unit). Data are the mean SEM of two independent quantifications. agent inhibits the CDK7 kinase subunit of TFIIH that phosphor- have been observed when A549 cells were pretreated with flavo- ylates serine 5 of CTD of Pol II (Fig. 4A, lanes 1 and 3), thus piridol (Flv; Fig. 4A, lanes 6 and 7), a flavonoid that inhibits preventing Pol II elongation and, consequently, RNA synthesis. several cyclin-dependent kinases, including CDK9, that phos- Pretreatment of cells with DRB prevented the Rpb1 degradation phorylate serine 2 of CTD. In parallel, confocal microscopy induced by lurbinectedin (Fig. 4A, lanes 2 and 4). Similar effects revealed that the amount of Pol II (in green) strongly decreased

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ubiquitinated complex, as evidenced by the smear observable from 0.5 hour of treatment (Fig. 5A, lanes 2–4). Converse experiments showed that the immunoprecipitated fraction by TUBES (tandem ubiquitin binding entity) contained phosphorylated Pol II that was no more present after 2 hours of treatment (Fig. 5B, lanes 1–4). To further investigate whether Pol II degradation induced by lurbinectedin was dependent on the ubiquitin-proteasome system, A549 cells were pretreated with PYR-41, a chemical inhibitor of UBA1, an ubiquitin-activating enzyme (30) before adding the drug. In those conditions, the phosphorylated Pol II was accumulated (Fig. 5C, lanes 2 and 4), suggesting that mono-ubiquitination, one of the ﬁrst steps of the ubiquitin/proteasome degradation process, was needed for lurbinectedin activity. Pretreat- ment of cells with the proteasome inhibitor MG-132 similarly prevented drug-mediated degradation of Pol II (Fig. 5D, lanes 2–3 and 5–6).

The ubiquitin proteasome degradation process occurs at activated genes The above data prompted us to investigate if the degradation of phosphorylated (elongating) Pol II that started few hours after lurbinectedin treatment was occurring in transcriptionally active genes. Western blot analysis revealed that Pol II was mainly located in the chromatin-bound fraction and not in the soluble extract several hours after drug treatment (Fig. 6A, top and bottom panels). This suggested that the degradation pro- cessmighthavebeeninitiatedatthegenelevel.Theeffectofthe drug on gene expression in A549 cells was thus tested by monitoring the transcription of RARb2, a retinoic acid receptor responsive gene that contains CGG triplets that are highly favorable for lurbinectedin bonding and whose expression was induced by trans-retinoic acid (t-RA). While in untreated cells, RARb2 expression was peaking around 3 hours after t-RA treatment (Fig. 6B), in drug-treated cells, RARb2 expression was hardly initiated and completely abolished few hours after treatment. Figure 4. We next monitored the recruitment of the transcription Inhibition of Pol II phosphorylation prevents its degradation by lurbinectedin. A, machinery on RARb2 by using ChIP coupled to real-time PCR. A549 cells were pretreated for 1 hour with either DRB (25 mmol/L) or flavopiridol In untreated cells, ChIP assays showed that phosphorylated Pol II (Flv, 5 mmol/L) before the addition of lurbinectedin (Lur, 30 nmol/L) for 4 hours as well as TFIIH (as indicated by the presence of its CDK7 subunit) as indicated at the top of each figure. Cell extracts were then analyzed by was abundantly recruited at the RARb2 promoter 3 hours after t- Western blot using antibodies directed toward hyperphosphorylated (IIo) and RA treatment (Fig. 6C1); the time course paralleled the peak of hypophosphorylated (IIa) Rpb1 subunit of Pol II as well as Rpb1 subunit phosphorylated in Ser5 (S5-P) and in Ser2 (S2-P); NT, nontreated cells. B, in situ RNA synthesis (Fig. 6B). On the contrary, in drug-treated cells, we immunofluorescence microscopy of A549 tumor cells treated with lurbinectedin detected low amounts of Pol II and TFIIH (Fig. 6D1). Interest- (10 nmol/L) for 4 hours in the absence or presence of DRB (25 mmol/L; ingly, P-TEFb (detected by the presence of CDK9), which was preincubation of 1 hour). Control, control cells; DRB, DRB-treated cells; Lur, visible at the promoter and exon 4 of RARb2 in untreated cells, lurbinectedin-treated cells; DRBþLur, DRB pretreated cells followed by accumulated much more at exon 4 in lurbinectedin-treated cells lurbinectedin treatment. Pol II was detected with anti-POLR2A clone 1PB-7C2 (Fig. 6C2, D2 and C4, D4). In this case, CDK9 as well as antibody (green). Nuclei were counterstained with Hoechst-33342 (blue). Scale bar, 10 mm. phosphorylated Pol II (S2-P), which were present at 2 hours, disappeared at 3 hours; in contrast, they were still detected in untreated cells (Fig. 6C4), which was indicative of active RNA 4 hours after lurbinectedin treatment (Fig. 4B, panels control and synthesis at that time point (Fig. 6B). The differences in the ratio Lur), in clear contrast to what occurred upon DRB pretreatment between total Pol II and S2-P observed at exon 4 in untreated (Fig. 4B, panels DRB and DRBþLur). Altogether, lurbinectedin versus treated cells could be explained by the regulation of Pol II activity required Pol II phosphorylation. activity by some phospho/dephosphorylation processes (31). In We then investigated the mechanism of lurbinectedin- lurbinectedin-treated cells, ChIP-Ubi/reChIP-Pol II showed that induced Pol II degradation. A549 cells were treated with lurbi- an ubiquitination process was already engaged on the RARb2 as nectedin, and, using a Pol II antibody, we performed an opposed to untreated cells (Fig. 6C3 and 6D3); we also noticed immunoprecipitation from whole-cell extracts over time. The the presence of SUG1 (a subunit of the proteasome) at the immune-precipitated Pol II fraction appeared to be part of an promoter.

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Figure 5. Degradation of Pol II by the ubiquitin/ proteasome machinery. A, Western blot of immune-precipitate Pol II from whole-cell extracts from A549 cells treated with 30 nmol/L of lurbinectedin (Lur) during 0.5, 1, and 2 hours. The immunoprecipitated fraction has been analyzed by using antibodies against mono and polyubiquitin (top) and Pol II (bottom). B, isolated ubiquitinated proteins from the above A549 whole- cell extracts treated with 30 nmol/L of Lur at different times (top) using TUBES technology allowed to detect phosphorylated (S2-P) from Rpb1. C, A549 cells were pretreated with PYR-41 (PYR), an UBA1 inhibitor, for 6 hours before addition of Lur 30 nmol/L for 4 hours. Hypophosphorylated (Pol IIa) and hyperphosphorylated forms (Pol IIo) of Pol II were detected by Western blot. b-Tubulin (Tub) has been used as a control. D, A549 cells were pretreated with MG-132 (50 mmol/L) proteasome inhibitor for 1 hour as indicated (þ); DMSO (NT) or increasing amounts of Lur (10 and 30 nmol/L) were then added for 4 hours. Pol IIo and Pol IIa were detected by Western blot. b-Tubulin (Tub) was used as loading control.

The above data strongly suggest that Pol II degradation by the tional assays then showed that the absence of DNA breaks was not ubiquitin-proteasome machinery is initiated in actively transcrib- due to DRB itself because increasing concentrations of DRB did ing genes upon treatment with lurbinectedin, and only the phos- not inhibit in vitro NER (Supplementary Fig. S3). We should notice phorylated elongating Pol II is degraded in the process. that Comet assays also demonstrated that the level of DNA breaks induced by the drug was significantly lower in XPF-deficient cells DNA breaks are dependent on Pol II phosphorylation compared with XPF-proficient cells (Fig. 7E and F). Interestingly We also examined whether the NER pathway could eliminate XPF as well as g-H2AX was significantly present after drug treat- the lurbinectedin covalently bound to DNA triplet. A labeled DNA ment at exon 4 of RARb2 in lurbinectedin-treated cells compared template (containing a single lurbinectedin adduct) was incubat- with the untreated ones (Fig. 6C5 and 6D5). In addition, a ed with XPC/HR23B, TFIIH, XPA, RPA, XPG, and as indicated XPF/ bioChIP assay that measured the incorporation of biotinylated ERCC1; we did not observe the removal of the damaged oligo- dUTP within broken DNA (32) also showed the presence of DNA nucleotide, but rather unexpectedly, we detected several DNA breaks at exon 4 (Fig. 6C5 and 6D5). breaks on the opposite DNA strand (Fig. 7A) originated only Altogether, the above data strongly underline the connection when the XPF endonuclease was present (21). However, it was between Pol II elongation (following its phosphorylation), Pol II worthwhile to notice that phosphorylated histone H2AX degradation, and the generation of DNA breaks in cells treated (g-H2AX), a hallmark of DNA breaks, was detected in lurbinecte- with lurbinectedin. din-treated cell extracts that coincided with the disappearance of phosphorylated Pol II (S2-P; Fig. 7B, lanes 1–7). Furthermore, pretreatment of cells with MG-132 resulted in the accumulation of Discussion S2-P, a decrease in the presence of g-H2AX, and accumulation of Current research efforts in cancer therapy are aimed to XPF (Fig. 7B, lanes 8–9). disturb transcription, one of the fundamental processes DNA breaks were next evaluated in A549 cells using the Comet involved in the maintenance of the oncogenic state. Here, we assay, which is based on the neutral or alkaline lysis of labile DNA have studied the mechanism of action of lurbinectedin, an at sites of damage. DNA from cells that have accumulated DNA anticancer agent under clinical investigation with very prom- breaks appeared as fluorescent comets with tails of DNA frag- ising results, and defined the fate of Pol II upon genotoxic mentation or unwinding, whereas normal, undamaged DNA did attack. We first demonstrated the antiproliferative activity of not migrate far from the origin. In lurbinectedin-treated cells, we lurbinectedin in several cancer cell lines with IC50 values in low observed a clear increase in DNA strand breaks at 4 hours when nanomolar range (Fig. 1B); this potent activity was confirmed compared with untreated cells that were almost abolished in cells in human tumors xenografted in nude mice where lurbinecte- pretreated with either DRB or Flv (Figures 7C and D). It should be din induced a significant inhibition of tumor growth and noticed that Comet assays allow the detection of both single- improvement of mice survival (Fig. 1C and D). Molecular stranded (SSD) and double-stranded (DSB) DNA breaks. Addi- biology approaches next showed that lurbinectedin binds

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Figure 6. Recruitment of the transcription and ubiquitin-proteasome machinery on RARb2 activated gene in lurbinectedin-treated A549 cells. A, A549 cells treated with lurbinectedin (Lur; 30 nmol/L) at 0.5, 2, and 4 hours were fractionated in chromatin and the soluble nuclear fraction and analyzed to both Pol IIo and Pol IIa. B, relative t-RA induced RARb2 mRNA expression in lurbinectedin (Lur, 30 nmol/L) -treated and -untreated (Ctrl) A549 cells. RARb2 mRNA levels represent the ratio between values obtained from treated and untreated cells normalized against the housekeeping GAPDH mRNA. C and D, schematic representation of the RARb2 with the indicated amplicons designed at the promoter (Pr) including the RARb responsive elements (RARE), the TATA box and the TSS (þ1) and exon 4 (E; top). ChIP monitoring the t-RA–dependent occupancy of Pol II, Rpb1 subunit phosphorylated in Ser5 (S5-P), and in Ser2 (S2-P), CDK7, CDK9, Sug1, and ubiquitinated Pol II at the promoter (C1–C3 and D1–D3). ChIP/re-ChIP (Ubi/Pol II) identiﬁed the presence of ubiquitination in Pol II using an antiubiquitin antibody and then an anti–Pol II antibody; ChIP monitoring the t-RA–dependent occupancy of Pol II, Rpb1 subunit phosphorylated in Ser2 (S2-P), CDK9, XPF, and g-H2AX at Exon 4 of RARb2 locus from either vehicle (Ctrl) (C4 and C5) or lurbinectedin (Lur)-treated A549 cells (D4 and D5). DNA breaks (DB) were detected using antibodies directed toward Biotin-dUTP incorporated into the broken DNA. Error bars, the SD of three independent experiments.

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Figure 7. DNA breaks formation in lurbinectedin-treated A549 cells. A, NER assay in the absence (-) or presence (þ) of XPF endonuclease with the immobilized OS/CCG DNA template and increasing amounts (1, 10, 100 mmol/L) of lurbinectedin. The positions of the nucleotides are indicated in reference to the nucleotide that binds the drug. B, Western blotting of Ser2 phosphorylated Rbp1 (S2-P), g-H2AX, and XPF in A549 cells untreated (NT) or treated during 0.5, 1, 2, 3, 4, and 6 hours with lurbinectedin (30 nmol/L). C, representative images of damaged DNA in the neutral Comet assay in A549 cells untreated (Ctrl) or treated with lurbinectedin (Lur, 30 nmol/L) for 4 hours, in the presence or absence of DRB (20 mmol/L) or flavopiridol (Flv, 5 mmol/L) as transcription inhibitors. D, tail moment quantification of A549 cells treated with lurbinectedin (Lur) at different times (0.5, 1, 2, 4 hours) and in different conditions (DRB or Flv) using the TriTek CometScore Freeware software. The Comet assay was performed in neutral and alkaline conditions to distinguish between DSBs and SSBs/double strand breaks (DSBsþSSBs), respectively. For each condition, a minimum of 100 cells were analyzed, and the experiment was repeated in duplicate. E, representative images of neutral Comet assay (DSBs) performed with HeLa and HeLa-XPF (XPF-deficient) cells that were untreated (Ctrl) or treated with lurbinectedin (Lur, 30 nmol/L) for 4 hours. F, tail moment quantification of the DSBs in HeLa control and XPF-deficient cells treated with lurbinectedin (Lur, 30 nmol/L) for 4 hours. For the tail moment quantification, the TriTek CometScore Freeware software was used, a minimum of 100 cells were analyzed, and the experiment was repeated in duplicate. specifically to CG-rich sequences located in the vicinity of the formation of DNA breaks (Figs. 3C–E, 6D1 and D5, 7B–F). promoters of protein-coding genes (Fig. 2B). Further investiga- Based on these results, we propose lurbinectedin drive cancer tions demonstrated that lurbinectedin induced in tumor cells, a cells to apoptosis by the mechanism of action described in specific and irreversible degradation of Pol II that paralleled the Supplementary Fig. S4.

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Some of the CG-rich sequences targeted by lurbinectedin not by itself explain the Pol II degradation that also occurred (Fig. 2C) can be highly related to the regulation of gene following treatment of cells with a-amanitin, which binds with expression by constituting CpG islands that can be methylated high affinity to the Rpb1 subunit of Pol II. It thus seems that in to silence the corresponding gene. The GC-dependent (specific) both cases (binding of either lurbinectedin to DNA or a-amanitin binding preference of lurbinectedin, to areas surrounding the to Rpb1), degradation occurs because elongating Pol II is irre- protein-coding gene promoters, does not exclude the possibil- versibly stalled on the DNA template. As a consequence, the ity that the drug binds also to other CG-rich sites of the genome ubiquitin-proteasome degradation process will be engaged (41, targeted by transcription factors, such as SP1 (21, 33), or 42). In the case of lurbinectedin, this is characterized by the involved in rDNA transcription (34). At any rate, in tumor ubiquitination of Pol II (Fig. 5A and B) and the recruitment of cells, Pol II is very active and could be arrested by the drug Sug1 (Fig. 6D3) in the vicinity of the adduct site. already bound to the DNA. Indeed, Pol II degradation occurred Taking together, our results show how lurbinectedin, by once the CTD of its Rpb1 subunit was phosphorylated (Fig. 3C– inhibiting transcription, causes a cascade of events that can E). Certainly, Pol II phosphorylation by TFIIH (CDK7) and explain its potent effects on tumor cells. The relevance of Pol II P-TEFb (CDK9) allows elongation and therefore RNA synthesis degradation and formation of DNA breaks was finally con- (35). Neither Pol II transcription factor partners nor other RNA firmed in vivo (Supplementary Fig. S6). In these xenografted polymerases were degraded (Fig. 3B–C), underlining a second models, the significant inhibition of tumor growth observed level of specificityofthedrug.Atthesametime,Cometand after lurbinectedin treatment was correlated to both Pol II Biochip experiments evidenced the formation of DNA breaks in degradation and induction of DNA damage. Therefore, lurbi- lurbinectedin-treated cells (Figs. 6D5 and 7C–F) and the pres- nectedin may help in the treatment of tumors with transcrip- ence of g-H2AX (a DNA break marker) on the activated RARb2 tion addiction. In this regard, it was recently reported that, in gene (Figs. 6D5 and 7B). Surprisingly, inhibition of Pol II combination with doxorubicin, lurbinectedin has compelling phosphorylation with either DRB (an inhibitor of the TFIIH activity as a second-line treatment in patients with SCLC (43), a transcription initiation factor) or flavopiridol (an inhibitor of tumor type that could be most sensitive to transcription-target- the P-TEFb elongation factor) prevented its degradation (Fig. ing drugs (3–5, 7, 44, 45). Lurbinectedin has also showed 4A) and the formation of DNA breaks (Fig. 7C). Thus, it seems impressive activity in platinum-resistant ovarian cancer (46), that, when lurbinectedin is bound to the DNA, the elongating a subtype of ovarian cancer that has been related to gene (phosphorylated) Pol II is stalled in front of the lurbinectedin- expression alterations affecting different oncogenic pathways DNA adduct, and although DNA breaks appeared on the related to drug resistance and tumor microenvironment (47– genome (Fig. 6D5), it fails to promote the elimination of the 49). Targeting transcription addiction with lurbinectedin might lesion. Indeed, due to the specific interaction of lurbinectedin be similarly beneficial in the treatment of other tumor types to both DNA strands (16), DNA breaks could have resulted that are known to be dependent on transcription dysregulation from an incomplete DNA repair activity. It is likely that DNA (50, 51). repair factors that have been either carried by the elongating Pol In summary, here we have described the mode of action of II (36) or recruited following the conventional transcription lurbinectedin for cancer targeting. This drug inhibits the tran- coupled repair (TCR) mechanism (i.e., recruitment of NER scription process by (1) its binding to CG-rich sequences, mainly factors by the stalled Pol II in front of a lesion; refs. 37, 38) located around promoters of protein-coding genes; (2) the irre- fail to remove the lesion (Fig. 7A). Indeed, the endonuclease versible stalling of elongating RNA Pol II on the DNA template XPF, involved in the repair of DNA inter and intra-crosslinks and its specific degradation by the ubiquitin/proteasome machin- (39), was found colocalizing with g-H2AX at the RARb2 (Fig. ery; and (3) the generation of DNA breaks and subsequent 6D5). Moreover, XPF-deficient cells presented much lower apoptosis. These results also improved our understanding of an amounts of DNA breaks compared with their proficient coun- important transcription regulation mechanism. terparts (Fig. 7E and F). However, this does not exclude the possible involvement of other factors such as Topo IIa that was Disclosure of Potential Conflicts of Interest shown to be essential for AR nuclear receptor transactivation C.M. Galmarini and P. Aviles are Senior Managers and have ownership (40). Finally, and in agreement with our proposed model, the (including patents) in PharmaMar. G. Santamaría Nuñez and J.F. Martínez-Leal structural analogue of lurbinectedin that failed to bind DNA are PharmaMar employees. J.-M. Egly reports receiving commercial research (Fig. 1F) did not produce any degradation of Pol II as well as grant from PharmaMar. No potential conflicts of interest were disclosed by the DNA damage (Supplementary Fig. S5A), demonstrating that other authors. the DNA binding of the drug to the CG-rich regions is responsible for the disturbance of the transcription process. Similarly, Authors' Contributions ~ in P-glycoprotein–overexpressing IGROV-ET ovarian cancer Conception and design: G. Santamaría Nunez, C. Giraudon, P. Aviles, cells (28), the lower activity of lurbinectedin was correlated C.M. Galmarini, J.-M. Egly Development of methodology: G. Santamaría Nunez,~ C.M. Genes Robles, to the absence of RNA synthesis inhibition and Pol II degra- E. Compe, J.-M. Egly dation and to lower amounts of DNA breaks induced by the Acquisition of data (provided animals, acquired and managed patients, drug (Supplementary Fig. S5B). provided facilities, etc.): G. Santamaría Nunez,~ C.M. Genes Robles Our results can also be used to explain the regulation of Analysis and interpretation of data (e.g., statistical analysis, biostatistics, ~ transcription mechanisms after genotoxic insults. Indeed, we can computational analysis): G. Santamaría Nunez, C.M. Genes Robles, infer from them that, in the absence of DNA damage removal and C. Giraudon, J.F. Martínez-Leal, E. Compe, F. Coin, P. Aviles, C.M. Galmarini, J.-M. Egly subsequent restart of RNA synthesis, it is likely that there is a Writing, review, and/or revision of the manuscript: G. Santamaría Nunez,~ mechanism that restricts the amount of time that any stalled Pol II C.M. Genes Robles, C. Giraudon, J.F. Martínez-Leal, E. Compe, P. Aviles, can reside on an activated gene. However, a failure of TCR could C.M. Galmarini, J.-M. Egly

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Administrative, technical, or material support (i.e., reporting or organizing Grant Support data, constructing databases): C. Giraudon, J.F. Martínez-Leal J.M. Egly has been awarded with the following grants: ERC Advanced Study supervision: C. Giraudon, J.F. Martínez-Leal, P. Aviles, C.M. Galmarini, grant, l'Agence Nationale de la Recherche (N#ANR1 08MIEN-022-03), J.M. Egly l'Association de la Recherche contre le Cancer (SL22013060782), the Institut National du Cancer (INCA-2008-041), and Ligue Nationale contre Acknowledgments le Cancer. We thank María Jose Guillen-Navarro, María JoseMunoz,~ Jose Manuel The costs of publication of this article were defrayed in part by the Molina-Guijarro, Cathy Braun, and the IGBMC facilities for their expertise and payment of page charges. This article must therefore be hereby marked advertisement technical support. We also are grateful to Carmen Cuevas Marchante, Jose María in accordance with 18 U.S.C. Section 1734 solely to indicate Fernandez Sousa Faro, and Nicolas Le May for fruitful discussions. Sequencing this fact. and bioinformatics analysis (with the expertise of Tao Ye and Baptiste Bidon) were performed by the IGBMC Microarray and Sequencing platform, a member Received March 28, 2016; revised May 23, 2016; accepted June 15, 2016; of the "France Genomique" consortium (ANR-10-INBS-0009). published OnlineFirst September 14, 2016.

References 1. Hoadley KA, Yau C, Wolf DM, Cherniack AD, Tamborero D, Ng S, et al. nucleotide excision repair and show activity toward platinum-resistant Multiplatform analysis of 12 cancer types reveals molecular classification cells. Mol Cancer Ther 2011;10:1481–9. within and across tissues of origin. Cell 2014;158:929–44. 19. Allavena P, Belgiovine C, Liguori M, Bello E, Frapolli R, Galmarini CM, et al. 2. Dooley AL, Winslow MM, Chiang DY, Banerji S, Stransky N, Dayton TL, Lurbinectedin reduces tumor-associated macrophages and the production et al. Nuclear factor I/B is an oncogene in small cell lung cancer. Genes Dev of inflammatory cytokines, chemokines, and angiogenic factors in 2011;25:1470–5. preclinical models. AACR Annual Meeting 2016. New Orleans, 2016, 3. Jiang T, Collins BJ, Jin N, Watkins DN, Brock MV, Matsui W, et al. Achaete- pp. A1284 scute complex homologue 1 regulates tumor-initiating capacity in human 20. Vidal A, Munoz C, Guillen MJ, Moreto J, Puertas S, Martinez-Iniesta M, et al. small cell lung cancer. Cancer Res 2009;69:845–54. Lurbinectedin (PM01183), a new DNA minor groove binder, inhibits 4. Osborne JK, Larsen JE, Shields MD, Gonzales JX, Shames DS, Sato M, et al. growth of orthotopic primary graft of cisplatin-resistant epithelial ovarian NeuroD1 regulates survival and migration of neuroendocrine lung carci- cancer. Clin Cancer Res 2012;18:5399–411. nomas via signaling molecules TrkB and NCAM. Proc Natl Acad Sci U S A 21. Feuerhahn S, Giraudon C, Martinez-Diez M, Bueren-Calabuig JA, Galmar- 2013;110:6524–9. ini CM, Gago F, et al. XPF-dependent DNA breaks and RNA polymerase II 5. Pedersen N, Mortensen S, Sorensen SB, Pedersen MW, Rieneck K, Bovin LF, arrest induced by antitumor DNA interstrand crosslinking-mimetic alka- et al. Transcriptional gene expression profiling of small cell lung cancer loids. Chem Biol 2011;18:988–99. cells. Cancer Res 2003;63:1943–53. 22. Coin F, Oksenych V, Mocquet V, Groh S, Blattner C, Egly JM. Nucleotide 6. Rudin CM, Durinck S, Stawiski EW, Poirier JT, Modrusan Z, Shames DS, excision repair driven by the dissociation of CAK from TFIIH. Mol Cell et al. Comprehensive genomic analysis identifies SOX2 as a frequently 2008;31:9–20. amplified gene in small-cell lung cancer. Nat Genet 2012;44:1111–6. 23. Singh A, Compe E, Le May N, Egly JM. TFIIH subunit alterations causing 7. Voortman J, Lee JH, Killian JK, Suuriniemi M, Wang Y, Lucchi M, et al. Array xeroderma pigmentosum and trichothiodystrophy specifically disturb comparative genomic hybridization-based characterization of genetic several steps during transcription. Am J Hum Genet 2015;96:194–207. alterations in pulmonary neuroendocrine tumors. Proc Natl Acad Sci 24. Dubaele S, Proietti De Santis L, Bienstock RJ, Keriel A, Stefanini M, Van U S A 2010;107:13040–5. Houten B, et al. Basal transcription defect discriminates between xero- 8. Wang Y, Zhang T, Kwiatkowski N, Abraham BJ, Lee TI, Xie S, et al. CDK7- derma pigmentosum and trichothiodystrophy in XPD patients. Mol Cell dependent transcriptional addiction in triple-negative breast cancer. Cell 2003;11:1635–46. 2015;163:174–86. 25. Ostling O, Johanson KJ. Microelectrophoretic study of radiation-induced 9. Franco HL, Kraus WL. No Driver behind the Wheel? Targeting transcription DNA damages in individual mammalian cells. Biochem Biophys Res in cancer. Cell 2015;163:28–30. Commun 1984;123:291–8. 10. Damsma GE, Alt A, Brueckner F, Carell T, Cramer P. Mechanism of 26. Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, transcriptional stalling at cisplatin-damaged DNA. Nat Struct Mol Biol et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol 2008; 2007;14:1127–33. 9:R137. 11. Kwiatkowski N, Zhang T, Rahl PB, Abraham BJ, Reddy J, Ficarro SB, et al. 27. Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P, et al. Simple Targeting transcription regulation in cancer with a covalent CDK7 inhib- combinations of lineage-determining transcription factors prime cis-reg- itor. Nature 2014;511:616–20. ulatory elements required for macrophage and B cell identities. Mol Cell 12. Coin F, Egly JM. Revisiting the function of CDK7 in transcription by virtue 2010;38:576–89. of a recently described TFIIH kinase inhibitor. Mol Cell 2015;59:513–4. 28. Erba E, Bergamaschi D, Bassano L, Ronzoni S, Di Liberti G, Muradore I, 13. Pommier Y. Drugging topoisomerases: Lessons and challenges. ACS Chem et al. Isolation and characterization of an IGROV-1 human ovarian cancer Biol 2013;8:82–95. cell line made resistant to Ecteinascidin-743 (ET-743). Br J Cancer 14. Falkenberg KJ, Johnstone RW. Histone deacetylases and their inhibitors in 2000;82:1732–9. cancer, neurological diseases and immune disorders. Nat Rev Drug Discov 29. Marco E, David-Cordonnier MH, Bailly C, Cuevas C, Gago F. Further 2014;13:673–91. insight into the DNA recognition mechanism of trabectedin from the 15. Navada SC, Steinmann J, Lubbert M, Silverman LR. Clinical development differential affinity of its demethylated analogue ecteinascidin ET729 for of demethylating agents in hematology. J Clin Invest 2014;124:40–6. the triplet DNA binding site CGA. J Med Chem 2006;49:6925–9. 16. Bueren-Calabuig JA, Giraudon C, Galmarini CM, Egly JM, Gago F. Tem- 30. Yang Y, Kitagaki J, Dai RM, Tsai YC, Lorick KL, Ludwig RL, et al. Inhibitors of perature-induced melting of double-stranded DNA in the absence and ubiquitin-activating enzyme (E1), a new class of potential cancer thera- presence of covalently bonded antitumour drugs: Insight from molecular peutics. Cancer Res 2007;67:9472–81. dynamics simulations. Nucleic Acids Res 2011;39:8248–57. 31. Sanso M, Fisher RP. Pause, play, repeat: CDKs push RNAP II's buttons. 17. Romano M, Frapolli R, Zangarini M, Bello E, Porcu L, Galmarini CM, et al. Transcription 2013;4:146–52. Comparison of in vitro and in vivo biological effects of trabectedin, 32. Ju BG, Lunyak VV, Perissi V, Garcia-Bassets I, Rose DW, Glass CK, et al. A lurbinectedin (PM01183) and Zalypsis(R) (PM00104). Int J Cancer topoisomerase IIbeta-mediated dsDNA break required for regulated tran- 2013;133:2024–33. scription. Science 2006;312:1798–802. 18. Soares DG, Machado MS, Rocca CJ, Poindessous V, Ouaret D, Sarasin A, 33. Dynan WS, Tjian R. The promoter-specific transcription factor Sp1 binds to et al. Trabectedin and its C subunit modified analogue PM01183 attenuate upstream sequences in the SV40 early promoter. Cell 1983;35:79–87.

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Santamaría Nunez~ et al.

34. Peltonen K, Colis L, Liu H, Trivedi R, Moubarek MS, Moore HM, et al. A active treatment as second-line therapy in small cell lung cancer (SCLC). targeting modality for destruction of RNA polymerase I that possesses J Clin Oncol 33, 2015 (suppl; abstr 7509). anticancer activity. Cancer Cell 2014;25:77–90. 44. Christensen CL, Kwiatkowski N, Abraham BJ, Carretero J, Al-Shahrour F, 35. Compe E, Egly JM. TFIIH: When transcription met DNA repair. Nat Rev Mol Zhang T, et al. Targeting transcriptional addictions in small cell lung cancer Cell Biol 2012;13:343–54. with a covalent CDK7 inhibitor. Cancer Cell 2014;26:909–22. 36. Le May N, Fradin D, Iltis I, Bougneres P, Egly JM. XPG and XPF endonu- 45. Osada H, Tatematsu Y, Yatabe Y, Horio Y, Takahashi T. ASH1 gene is a cleases trigger chromatin looping and DNA demethylation for accurate speciﬁc therapeutic target for lung cancers with neuroendocrine features. expression of activated genes. Mol Cell 2012;47:622–32. Cancer Res 2005;65:10680–5. 37. Hanawalt PC, Spivak G. Transcription-coupled DNA repair: Two decades of 46. Poveda A, Berton-Rigaud D, Ray-Coquard I, Alexandre J, Provansal M, Soto progress and surprises. Nat Rev Mol Cell Biol 2008;9:958–70. A, et al. Lurbinectedin (PM01183), an active compound in platinum- 38. Laine JP, Egly JM. When transcription and repair meet: A complex system. resistant/refractory ovarian cancer (PRROC) patients: results of a two Trends Genet 2006;22:430–6. -stage, controlled phase II study. J Clin Oncol 2014;32:abstr 5505. 39. McNeil EM, Melton DW. DNA repair endonuclease ERCC1-XPF as a novel 47. Flemming A. Anticancer drugs: Finding the perfect combination. Nat Rev therapeutic target to overcome chemoresistance in cancer therapy. Nucleic Drug Discov 2015;14:13. Acids Res 2012;40:9990–10004. 48. Koti M, Siu A, Clement I, Bidarimath M, Turashvili G, Edwards A, et al. A 40. Haffner MC, Aryee MJ, Toubaji A, Esopi DM, Albadine R, Gurel B, et al. distinct pre-existing inﬂammatory tumour microenvironment is associated Androgen-induced TOP2B-mediated double-strand breaks and prostate with chemotherapy resistance in high-grade serous epithelial ovarian cancer gene rearrangements. Nat Genet 2010;42:668–75. cancer. Br J Cancer 2015;112:1215–22. 41. Beaudenon SL, Huacani MR, Wang G, McDonnell DP, Huibregtse JM. Rsp5 49. Mozzetti S, Martinelli E, Raspaglio G, Prislei S, De Donato M, Filippetti F, ubiquitin-protein ligase mediates DNA damage-induced degradation of et al. Gli family transcription factors are drivers of patupilone resistance in the large subunit of RNA polymerase II in Saccharomyces cerevisiae. Mol ovarian cancer. Biochem Pharmacol 2012;84:1409–18. Cell Biol 1999;19:6972–9. 50. Sandberg AA. Updates on the cytogenetics and molecular genetics of 42. Wilson MD, Harreman M, Svejstrup JQ. Ubiquitylation and degradation of bone and soft tissue tumors: Lipoma. Cancer Genet Cytogenet 2004;150: elongating RNA polymerase II: The last resort. Biochim Biophys Acta 93–115. 2013;1829:151–7. 51. Tomlins SA, Bjartell A, Chinnaiyan AM, Jenster G, Nam RK, Rubin MA, et al. 43. Forster M, Calvo E, Olmedo Garcia ME, Lopez Criado MP, Moreno V, Soto- ETS gene fusions in prostate cancer: from discovery to daily clinical practice. Matos A, et al. Lurbinectedin (PM01183) with doxorubicin (DOX), an Eur Urol 2009;56:275–86.

OF14 Mol Cancer Ther; 15(10) October 2016 Molecular Cancer Therapeutics

Lurbinectedin Specifically Triggers the Degradation of Phosphorylated RNA Polymerase II and the Formation of DNA Breaks in Cancer Cells

Gema Santamaría Nuñez, Carlos Mario Genes Robles, Christophe Giraudon, et al.

Mol Cancer Ther Published OnlineFirst September 14, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-16-0172

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