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Small Molecule Therapeutics

Lurbinectedin Specifically Triggers theDegradation of Phosphorylated RNA PolymeraseII and the Formation of DNA Breaks inCancer CellsGema Santamaría Nu~nez1, Carlos Mario Genes Robles2, Christophe Giraudon2,Juan Fernando Martínez-Leal1, Emmanuel Compe2, Fr�ed�eric Coin2, Pablo Aviles1,Carlos María Galmarini1, and Jean-Marc Egly2

Abstract

We have defined the mechanism of action of lurbinectedin, amarine-derived drug exhibiting a potent antitumor activityacross several cancer cell lines and tumor xenografts. This drug,currently undergoing clinical evaluation in ovarian, breast, andsmall cell lung cancer patients, inhibits the transcription pro-cess through (i) its binding to CG-rich sequences, mainlylocated around promoters of protein-coding genes; (ii) theirreversible stalling of elongating RNA polymerase II (Pol II)on the DNA template and its specific degradation by the

ubiquitin/proteasome machinery; and (iii) the generation ofDNA breaks and subsequent apoptosis. The finding that inhi-bition of Pol II phosphorylation prevents its degradation andthe formation of DNA breaks after drug treatment underscoresthe connection between transcription elongation and DNArepair. Our results not only help to better understand the highspecificity of this drug in cancer therapy but also improve ourunderstanding of an important transcription regulation mech-anism. Mol Cancer Ther; 15(10); 1–14. �2016 AACR.

IntroductionCancer cells aberrantly deregulate specific gene expression

programs with critical functions in cell differentiation, prolifer-ation, and survival (1). Differently from noncancer cells, thosealtered gene programs in cancer cells have a striking dependenceon continuous active transcription. For example, small cell lungcancer (SCLC) cells are addicted to lineage-specific and proto-oncogenic transcription factors that support their growth (2–7).Similarly, triple-negative breast cancer (TNBC) is highly depen-dent on uninterrupted transcription of a specific key set of genes(8, 9). Pharmacologic modulation of transcription of protein-coding genes may thus provide an approach to identify and treattumor types that are dependent on deregulated transcription formaintenance of their oncogenic state.

Targeting DNA in tumor cells happened to be the mostexplored therapeutic strategy to block DNA processing enzymessuch as those involved in transcription (e.g., cisplatin and

derivatives, anthracyclines, etc.; ref. 10). Currently, severallaboratories are developing inhibitors of cyclin-dependentkinases (CDK) that have a critical role in regulating transcrip-tion initiation, pause release, and elongation (e.g., CDK7,CDK8, or CDK9), the three main steps involved in RNAsynthesis (11, 12). Other approaches are inhibition of DNArepair mechanisms (e.g., irinotecan, topotecan, olaparib;ref. 13) or chromatin remodeling (HDAC inhibitors ordemethylating agents; refs. 14, 15). Although these compoundshave already entered clinical trials, the mechanisms by whichthey disturb transcription as well as those driving to cancer celldeath are far from being understood.

Here, we describe the inhibition of transcription by lurbi-nectedin (PM01183; Fig. 1A), an anticancer agent that is beingevaluated in late-stage (phases II and III) clinical trials. Lurbi-nectedin is structurally related to trabectedin, containing thesame pentacyclic skeleton of the fused tetrahydroisoquinolinerings, but differing by the presence of a tetrahydro-B-carbolinereplacing the additional tetrahydroisoquinoline of trabectedin.The pentacyclic skeleton is mostly responsible for DNA minorgroove recognition and binding. Lurbinectedin reacts with theexocyclic amino group of guanines in the minor groove of DNAforming a covalent bond. The resulting adduct is additionallystabilized through the establishment of van der Waals interac-tions and one or more hydrogen bonds with neighboringnucleotides in the opposite strand of the DNA double helix(16). The additional tetrahydro b-carboline moiety protrudesfrom the DNA minor groove and could be interacting directlywith specific factors involved in DNA repair and transcriptionpathways. Indeed, it is possible that this part of the moleculeinteracts directly with TC-NER factors and could interfere withthe repair mechanism. In this sense, lurbinectedin is able to

1Cell Biology and Pharmacogenomics Department, Pharmamar SA,Colmenar Viejo, Madrid, Spain. 2Department of Functional Genomicsand Cancer, IGBMC, CNRS/INSERM/University of Strasbourg, C. U.Strasbourg, France.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

G. Santamaría Nu~nez and C.M. Genes Robles are first coauthors.

Corresponding Author: C.M. Galmarini, Pharmamar SA, Avda de los Reyes 1,Colmenar Viejo 28770, Madrid, Spain. Phone: 34 918466158; Fax: 34 918466001;E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-16-0172

�2016 American Association for Cancer Research.

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attenuate the repair of specific nucleotide excision repair (NER)substrates (17, 18). In addition to its activity in tumor cells, itwas recently shown that lurbinectedin affects the inflammatorymicroenvironment, with a selective apoptotic-inducing effecton mononuclear phagocytes and a specific inhibition of pro-duction of inflammatory cytokines (19, 20). In this work, weshow that, following its specific target on CG-rich sequenceslocated at promoters of protein-coding genes, lurbinectedininduces the specific degradation of elongating (phosphorylat-ed) RNA polymerase II (Pol II) by the ubiquitin-proteasomemachinery. This process occurs specifically on activated genesand is associated with the formation of DNA breaks that drivetumor cells to apoptosis. Inhibition of Pol II phosphorylationprevents its degradation and the formation of DNA breaks.These investigations not only show how lurbinectedin causes acascade of events on the transcription process that can explainits antiproliferative activity on tumor cells, but also improveour understanding of the fate of Pol II when it encounters alesion on the DNA.

Materials and MethodsReagents

Lurbinectedin was produced by PharmaMar through a semi-synthetic method. Z-Leu-Leu-Leu-al (MG-132), 5,6-dichloro-benzimidazole-1-a-D-ribofuranoside (DRB), and flavopiridolwere purchased from Sigma. The following antibodies wereused for Western blotting: POLR2A (RPB1, Pol II) (clones N-20and H-224), POLR1A (RNA Pol I), POLRMT (B-1), CCNH(Cyclin H) (B-1), CDK9 (C-20), ERCC2 (p80-TFIIH)(H-150), TP53 (FL-393) from Santa Cruz Biotechnology; TBP,GTF2H1 (TFIIH), POLR2D (RPB4, RNA Pol II), POLR2B (RPB2,RNA Pol II), CRCP (RNA Pol III) from Abcam; CDK7 from CellSignaling Technology; and anti–phospho-Ser2Pol II (clone H5)and anti–phospho-Ser5 Pol II (clone H14) from Covance. Thefollowing antibodies were used for chromatin immunoprecip-itation (ChIP) and immunoprecipitation (IP) experiments:Antibodies against phospho-Ser2 Pol II (clone 3E8) and phos-pho-Ser2 Pol II (clone 3E10) were from Active Motif. Poly-clonal antibodies against POLR2A (H-224), CDK7 (C-19),CDK9 (H-169), UBB (Ubiquitin clone FL-76 or clone A-5),Biotin(33), and ERCC4 (XPF, clone H-300) were from SantaCruz Biotechnology. Antibody against g-H2AX (ab2893) wasobtained from Abcam. POLR2A (1PB-7C2), PSMC5 (Sug-1,clone 3SCO), and GTF2F2 (TFIIF b) antibodies were fromIGBMC.

Cell linesThe following cell lines were obtained from the ATCC in

2007: A549 (lung adenocarcinoma; CCL-185), A673 (humanmuscle Ewing's sarcoma; CRL-1598), HCT-116 (human colo-rectal carcinoma; CCL-247), HT-29 (human colorectal carcino-ma; HTB-38), MCF7 (breast adenocarcinoma; HTB-22), MDA-MB-231 (breast adenocarcinoma; HTB-26), and PSN-1 (pan-creatic adenocarcinoma; CRM-CRL-3211). HeLa and HeLasiXPF are cervical carcinoma cells and were obtained fromTebu-Bio. Dr. M. D'Incalci (Istituto Mario Negri) generouslyprovided IGROV1 and IGROV-ET ovarian cancer cell lines. Allcell lines were cultured in the medium recommended by thesupplier supplementedwith 10%FBS, 2mmol/L L-glutamine, andpenicillin–streptomycin mix (Sigma). None of the cell lines used

in the article have been authenticated in the laboratory in the last 6months.

Cell proliferationCell proliferation was studied by [3-(4,5-dimethythiazol-2-yl)-

2,5-diphenyl] tetrazolium bromide (MTT) assays that were per-formed following the manufacturer's instructions (MTT CellProliferation Kit I; Roche Diagnostics). Briefly, cells were seededin 96-well trays. Serial dilutions of lurbinectedin, PM030779, orPM120306were added to themedium. Exposure to the drugs wasmaintained during 72 hours. Determination of IC50 values wasperformed by iterative nonlinear curve fitting using the Prism 5.0statistical software (GraphPad). The data presented are the aver-age of three independent experiments performed in triplicate.

DNA electrophoretic mobility shift assayThe binding assay was performed with a 250 pb PCR product

from the human adiponectin gene. After incubation withappropriate concentrations of the compounds at 25�C during1 hour, the DNA was subjected to electrophoresis in a 2% (w/v)agarose/TAE gel, stained with ethidium bromide (Sigma) andphotographed.

DNase I footprinting assaysRadiolabeled AS/CGG and OS/CCG were bound to magnetic

beads (Dynabeads) and incubated for 30 minutes at room tem-perature with the indicated drug concentrations (21). After exten-sive washings, DNase I digestion was performed for 45 seconds atroom temperature. Purified nucleic acids were resolved on an 8%denaturing Urea-polyacrylamide gel.

In vitro transcription assaysRun-off transcription assays were performed using recombi-

nant TFIIB, TFIIE, TFIIF, TBP, TFIIH, and RNA pol II, as previouslydescribed (22).

RNA synthesis quantification in tumor cellsA549 (3.5 � 104 cells/well), A673 (2.6 � 105), HCT116 (1.8

� 105), HeLa (1.5 � 105), and MDA-MB-231 (2 � 105) wereseeded in 24-well plates and incubated with lurbinectedin orvehicle (DMSO) for 30, 45, 60, and 90 minutes and pulsed with5 mCi [3H] uridine (Perkin Elmer) for 30 minutes in mediumsupplemented with 10% FCS. Then, cells were rinsed twice inPBS and fixed with chilled 10% trichloroacetic acid for 10minutes, and the monolayers were washed with ethanol andair-dried at room temperature. The precipitated macromole-cules were dissolved in 0.5 N NaOH and 0.1% SDS and dilutedin Ultima-Flo M (Perkin Elmer). The radioactivity was mea-sured using a b-counter Hidex 300SL.

Western blotting analysisCell protein extracts were prepared following standard proce-

dures in RIPA buffer in the presence of protease inhibitors(Complete, Roche Diagnostics) and phosphatase inhibitors(PhosStop, Roche Diagnostics). After quantification with theMicro-BCA Protein Assay Kit (Thermo Scientific), 15 to 25 mg ofprotein were separated by SDS-PAGE and transferred to PVDFmembranes (Immobilon-P; Millipore). After using appropriatedprimary and secondary antibodies, blots were developed by aperoxidase reaction using the ECL detection system (Amersham-G.E. Healthcare).

Santamaría Nu~nez et al.

Mol Cancer Ther; 15(10) October 2016 Molecular Cancer TherapeuticsOF2




Pol II half-life measurementPulse-chase analyses were carried out in cells that were pre-

treated during 2 hours in DMEM cys�/met� medium and thenmetabolically labeled with 100 mCi/mL 35S-methionine for 1hour in DMEM cys�/met� medium. After washing steps, cells

have been treated with lurbinectedin. Whole-cell extracts wereprepared using RIPA buffer (0.01 mol/L Tris–HCl, pH 8.0, 0.14mol/L NaCl, 1% Triton X-100, 0.1% Na Deoxycholate, 0.1%SDS), and Pol II was immunoprecipitated using a specific anti-body and resolved by SDS–PAGE. 35S-Pol II bands were

Figure 1.

Antiproliferative activity of lurbinectedin in several cancer cells.A, structure of lurbinectedin.B, antiproliferative activity of lurbinectedin in human lung (A549), Ewingsarcoma (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 afterlurbinectedin treatment by MTT assay. C, in vivo antitumor activity of lurbinectedin. Treatment with the drug results in tumor growth inhibition in A549xenograft models comparedwith vehicle-treated animals. Lurbinectedin treatment started at a tumor volume size of c. 150mm3 andwas intravenously administeredin 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 A549tumors after treatment with lurbinectedin. Drug treatment was associated with a statistically significant increase in survival compared with placebo-treated mousemodel. E, IGROV- and IGROV-ET–resistant cell viability was analyzed 72 hours after lurbinectedin treatment by MTT (&, IGROV; ~, IGROV-ET). F, binding tonakedDNA. Increasing amounts of lurbinectedin and PM030779were incubatedwith a DNA fragment of 250 bp, and the electrophoresiswas 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 treatmentby MTT assay (*, Lur; &, PM030779).

Lurbinectedin Induces Degradation of RNA Pol II

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quantified by phosphorimager analysis using ImageJ software(open source).

ImmunostainingA549 cells were treated with the appropriate concentration of

lurbinectedin for 4 hours in the absence or presence of thetranscription inhibitor 20mmol/LDRB (preincubation of 1 hour),washed, fixed (4% paraformaldehyde), permeabilized (0.5%Triton X-100), and incubated with the primary anti-Pol II mono-clonal antibody for 1 hour at 37�C. Thereafter, the cells werewashed and incubated with the AlexaFluor 594 secondary goatanti-mouse IgG (Invitrogen) for 30 minutes at 37�C. Finally, theslides were incubated with Hoechst 33342 (Sigma) andmountedwithMowiol mountingmedium. Pictures were taken with a LeicaDM IRM fluorescence microscope equipped with a 100x oilimmersion objective and a DFC 340 FX digital camera (Leica).

ImmunoprecipitationA549 whole-cell extracts were made using RIPA buffer supple-

mented with PIC (Roche) and phosphatase inhibitors (ActiveMotif). Antibodieswere incubatedwithmagnetic beads and latelyprotein whole-cell extract was added. Sequential washes weredone, and the resulting sample was loaded on SDS-PAGE forWestern blotting. When Tandem Ubiquitin Binding Entity(TUBES 2) technology (Lifesensors) was used, equilibrated slurrywas initially incubatedwith proteinwhole cell extract at 4�C. Afterextensive washing steps, the resulting sample was loaded on SDS-PAGE for Western blotting.

Subcellular protein fractionationA549 cells were incubated with the different compounds at

different time points, and subcellular protein fractionation wasperformed using the Subcellular Protein Fractionation Kit forCultured Cells (Thermo Scientific Waltham) following the man-ufacturer's instructions.

Reverse transcription and quantitative PCRTotal RNA was isolated using the RNeasy Mini Kit (QIAGEN)

and reverse transcribed with SuperScript II reverse transcriptase(Invitrogen). The quantitative PCRwas done using the Lightcycler480 SYBR Green and the Lightcycler 480 (Roche Diagnostics).The primers used in the real-time PCR experiments were asfollows: for RARb2: fw 5'-CCAGCAAGCCTCACATGTTTCCAA-3' and rv 5'-TACACGCTCTGCACCTTTAGCACT-3'; for Glyceral-dehyde 3-phosphate dehydrogenase (GAPDH): fw 5'-TCGA-CAGTCAGCCGCATCTTCTTT-3' and rv 5'-ACCAAATCCGTT-GACTCCGACCTT-3'. RARb2 mRNA levels represent the ratiobetween values obtained from treated and untreated cells nor-malized against the housekeeping GAPDH mRNA.

Chromatin immunoprecipitationCells were cross-linked at room temperature for 10 minutes

with 1% formaldehyde. Chromatin was isolated and sonicated(23). Samples were immunoprecipitated (IP) with antibodies at4�C overnight, and protein G-Sepharose beads (Upstate) wereadded, incubated 3 hours at 4�C, and sequentially washed. DNAfragments were purified using the QIAquick PCR purification Kit(QIAGEN) and analyzed by real-time PCR using sets of primerstargeting RARb2 gene promoter: fw 5'-TGGTGATGTCAGAC-TAGTTGGGTC-3' and rev 5'-GCTCACTTCCTACTACTTCTGT-CAC-3'; and RARb2 exon 4: fw 5'-TCCAGCTGTCAGGAATGA-

CAGGAA-3' and rev 5'-TGAGATCGTCCAACTCAGCTGTCA-3'.Biotin-ChIP assays have been performed as previously described(23).

NER assaysOS/CCG DNA template (containing a single lurbinectedin

adduct; ref. 21) has been bound to magnetic beads (Dynabeads)and incubated with the indicated drug concentrations. Dualincision assays were next carried out after addition of XPG,XPF/ERCC1, XPC/hHR23B, RPA, XPA, and TFIIH. The reactionswere conducted as previously described (24).

Comet assaysAfter lurbinectedin or PM030779 treatment, single-cell gel

electrophoresis assay has been used (CometAssay; Trevigen)following the manufacturer's instructions. Experiments and pic-tures analyses have been performed as previously described (25).

Antitumor activity in xenograft murine modelsAll animal protocols were reviewed and approved according to

regional Institutional Animal Care and Use Committees. Miceused in the following experimentswere always female 4 to6weeksof age, 16 to 25 gr, athymic nude-Foxn1 nu/nu obtained fromEnvigo (Italy). Mice were housed in individually ventilated cageson a 12-hour light–dark cycle at 21 to 23oC and 40% to 60%humidity. Mice were allowed free access to an irradiated diet andsterilized water. Design, randomization, and monitoring ofexperiments (including body weights and tumor measurements)were performed using NewLab Software v2.25.06.00 (NewLabOncology). All mice were s.c. xenografted with A549 cancer cellsinto their right flank with c. 3 � 106 cells in 0.2 mL of a mixture(50:50; v:v) of Matrigel basement membrane matrix (BectonDickinson) and serum-free medium. When tumors reachedapproximately 150 mm3, mice were randomly assigned to treat-ment or control groups. Lurbinectedin was intravenously admin-istered in three consecutive weekly doses (0.18 mg/kg/day),whereas the control animals received an equal volume of vehiclewith the same schedule. Caliper measurements of the tumordiameters were made twice weekly, and tumor volumes werecalculated according to the following formula: (a x b)2/2, where aand b were the longest and shortest diameters, respectively.Animals were humanely killed when their tumors reached 3,000mm3 or if significant toxicity (e.g., severe body weight reduction)was observed. Differences in tumor volumes between treated andcontrol groups were evaluated using the Mann–Whitney U test.Statistical analyses were performed by LabCat v8.0 SP1 (Innova-tive Programming Associates, Inc.).

Chem-Seq assayA549 cells treated with 300 nmol/L of PM120306 during 4

hours were cross-linked with 1% formaldehyde at room temper-ature for 10 minutes. After sonication, the soluble chromatin wasincubated with 80 mL of Streptavidin beads (Life technologies)overnight. After wash the captured DNA was eluted, de-cross-linked and purified by QiAquick Spin columns (Qiagen). TheDNA was used to construct a library and sequenced on anIllumina HiSeq 2500 system. The sequencing reads were alignedto the hg19 assembly by using Bowtie 1.0. The HT-seq data werevisualized by preparing custom tracks for the UCSC (University ofCalifornia Santa Cruz) genome browser (https://genome.ucsc.edu).






Annotations were performed by using both MACS (http://liulab.dfci.harvard.edu/MACS/; ref. 26) and Ensembl database Release75. CG motifs search was performed by using HypergeometicOptimization of Motif Enrichment (HOMER; http://homer.salk.edu; ref. 27). The pattern search of the motif CGG in the summit(100 bp) of all the identified peaks was done using a home-madeJava program.

ResultsLurbinectedin targets DNA to arrest cancer cell growth

We analyzed the effect of lurbinectedin on several humancancer cell lines including lung (A549), Ewing sarcoma (A673),colon (HCT-116, HT-29), breast (MCF7, MDA-MB-231), cervix(HeLa), and pancreas (PSN-1), over a 72-hour period (Fig. 1B;Supplementary Table S1). Lurbinectedin showed a potent anti-proliferative activity with IC50 values in the low nanomolar rangeon all the cancer cell lines tested so far. We then performedxenograft studies to check whether the antiproliferative effect oflurbinectedin translated into in vivo antitumor activity. A549 cellswere xenografted into the rightflank of athymic nu/numice.Oncethe tumors reached c.150 mm3, mice were randomized intogroups (n ¼ 10) and either vehicle or lurbinectedin (0.18 mg/kg/day) was intravenously administered in three consecutiveweekly doses. In those conditions, lurbinectedin presented anti-tumor activity with a statistically significant inhibition of tumorgrowth (Fig. 1C). In control mice, mean tumor volumes at 0, 7,and 14 days were 165 mm3, 582 mm3, and 1,575 mm3, respec-tively, whereas in lurbinectedin-treated mice, mean tumorvolumes at the same time intervals were 170 mm3, 168 mm3,and 278 mm3, respectively (P ¼ 0.7, P ¼ 0.004, and P ¼ 0.002,respectively). Lurbinectedin also induced an improvement ofmice survival (mean survival for control mice: 18 days vs. meansurvival in lurbinectedin-treated mice: 47 days; P ¼ 0.001; Fig.1D). No significant toxicity or body weight loss was observed inthe treated animals (data not shown).

Interestingly, IGROV-ET ovarian cancer cells, overexpressingP-glycoprotein and previously shown to be resistant to doxo-rubicin, etoposide, and trabectedin (28), were less sensitive tolurbinectedin (Fig. 1E). These data indicated that lurbinectedinneeded to accumulate in the cell to exert its antiproliferativeactivity. Band shift assays next demonstrated that the drugbound to DNA (Fig. 1F). Indeed, we observed a delay inelectrophoretic migration of a 250 bp DNA fragment treatedwith lurbinectedin, while when treated with PM030779, astructural analogue lacking the hydroxyl group at position21 that is involved in the covalent binding to guanines (ref. 29;Supplementary Fig. S1A), DNA migrated as the control-untreat-ed fragment. We then analyzed the effect of either lurbinectedinor PM030779 on A549 lung cancer cells over a 72-hour period(Fig. 1G). Lurbinectedin showed a potent antiproliferativeactivity with IC50 values in the low nanomolar range, whereasPM030779 was inactive.

Lurbinectedin specifically targets DNA CG-rich regionsWe next searched for the target site of the drug using a DNase I

footprinting assay. We designed a DNA template that contained aunique, high-affinity adduct-forming site (CGG triplet) in just onestrand.Weobserved that lurbinectedinwas, in fact, bound to bothstrands (Fig. 2A). Lurbinectedin-bound DNA strand (AS) wasprotected from DNase I digestion from nucleotides T-4 to Tþ6,being G0 the guanine to which lurbinectedin was covalently

bound (Fig. 2A, lanes 4–6). We also observed that the druginteractedwith the opposite strand (OS) throughhydrogenbondsand van der Waals forces (Fig. 2A, lanes 11–12).

To identify genomic binding sites of lurbinectedin in A549cells, we next performed a chemical affinity capture (Chem-Seq)which measures the incorporation of the biotin-linked lurbinec-tedin analogue (PM120306; Supplementary Fig. S1B) withinDNA followed by DNA sequencing. This analogue that bindsguanine through its hydroxyl group (as lurbinectedin) was shownto exhibit antiproliferative properties similar to lurbinectedin(Supplementary Fig. S1C and S1D, see below). Sonicated chro-matin from cells treated by PM120306 was incubated withmagnetic streptavidin beads to isolate the biotinylated DNAfragments followed bymassively parallel DNA sequencing. Thesesequences were used to reveal DNA regions enriched (peaks) inPM120306-bound sites genome wide. We identified approxi-mately 1,000 peaks in the sequenced DNA. Interestingly, wedetected a high density of peaks in the vicinity (þ/- 10 Kb) ofpromoter-transcription start sites (TSS; Fig. 2B).We also observedthat CpG-rich regions around TSS (Fig. 2C) overlapped with thepeaks previously identified (Fig. 2B). Knowing that the optimalbinding site of lurbinectedin is the CGG triplet, we did a patternsearch of this motif in all the identified peaks: 50% of themincluded at least one CGG triplet (data not shown).

Altogether, our data show that lurbinectedin specifically targetsCG-rich regions that are largely located in areas surroundingpromoter regions.

Lurbinectedin inhibits RNA synthesis and induces Pol IIdegradation

The inhibition of RNA synthesis by lurbinectedin was investi-gated in A549 lung cancer cells (Fig. 3A) as well as in Ewingsarcoma (A673), colon (HCT-116), cervix (HeLa), and breast(MDA-MB-231) cancer cell lines (Supplementary Fig. S2A). Allcell lines were treated with lurbinectedin (30 nmol/L) over timebefore being pulsed with [3H] uridine. Total RNA synthesis wasinhibited by around 40% after 30 minutes and almost 80% after2-hour treatment in all the cell lines analyzed. We also investi-gated whether lurbinectedin would affect RNA synthesis whenadded to a well-defined in vitro transcription assay containing theadenovirus major late promoter (AdMLP) as a template, inaddition to TFIIB, TBP, TFIIE, TFIIF, TFIIH basal transcriptionfactors, and RNA pol II (Pol II; ref. 21). The exposure of the DNAtemplate to increasing amounts of lurbinectedin before addingthe transcriptionmachinery led to a progressive inhibition of RNAsynthesis (Fig. 3B).

Having observed that the drug induced RNA synthesis inhibi-tion, we next examined the fate of components involved in thetranscription process overtime after lurbinectedin treatment. InA549 whole-cell extracts, we observed a rapid disappearance ofboth the hypo- (IIa) and hyper- (IIo) phosphorylated forms ofRpb1, the largest subunit of Pol II, in a time-dependent manner(Fig. 3C); Western blot also showed that the carboxy-terminaldomain (CTD contains 52 Serine and threonine rich hepta-repeats) of Rpb1was phosphorylated on both Serine 5 and Serine2 (Fig. 3D), indicating that the drug treatment did not prevent thePol II phosphorylation. Pol II disappearance was also observed inall the other human tumor cells so far tested (Supplementary Fig.S2B). We also noticed the disappearance of Rpb2, the secondlargest subunit of Pol II, after 15 hours, whereas the amounts ofother subunits such as Rpb4 remained unchanged along the time






course (Fig. 3D). Remarkably, other RNApolymerases, such as PolI (Rpa194 subunit), Pol III (Rpc9 subunit), ormitochondrial RNAPol, remained present in the cell extracts several hours after drugtreatment (Fig. 3D). Western blot analyses showed that the drugdid not affect other transcription factors, such as TFIIH (asvisualized by the presence of CDK7 kinase and p62 subunits),TFIIF (b subunit), P-TEFb (CDK9 subunit) as well as TBP (amember of TFIID), all of them being involved in various stepsof the RNA synthesis process (Fig. 3E).

We next studied the turnover of Pol II following lurbinectedintreatment by conventional pulse chase. A549 cells were metabol-

ically labeled with [35S]-methionine for 1 hour and then treatedwith the drug. Cells were collected at different times, and Pol IIwas immunoprecipitated before being resolved by SDS–PAGEand autoradiographed (Fig. 3F). Newly synthesized [35S]-Pol IIwas detected up to 6 hours in untreated cells, whereas in drug-treated cells, Pol II labeling was almost absent after the first hour.

Phosphorylation of Pol II is essential for its degradationTo further investigate the potential connection between the

phosphorylation and the degradation of Pol II after lurbinectedintreatment, we pretreated A549 cells with 20 mmol/L of DRB. This

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 areindicated (G0 is the guanine that is covalently bound to the drug). B, chem-Seq analysis of A549 cells treated with biotinylated-lurbinectedin. Peaks distributionhistogram in the vicinity of the transcription start site (TSS) þ/- 10 Kb. C, density of CG repeats around the nearest TSS of all peaks.






agent inhibits the CDK7 kinase subunit of TFIIH that phosphor-ylates serine 5 of CTD of Pol II (Fig. 4A, lanes 1 and 3), thuspreventing Pol II elongation and, consequently, RNA synthesis.Pretreatment of cells with DRB prevented the Rpb1 degradationinduced by lurbinectedin (Fig. 4A, lanes 2 and 4). Similar effects

have been observed when A549 cells were pretreated with flavo-piridol (Flv; Fig. 4A, lanes 6 and 7), a flavonoid that inhibitsseveral cyclin-dependent kinases, including CDK9, that phos-phorylate serine 2 of CTD. In parallel, confocal microscopyrevealed that the amount of Pol II (in green) strongly decreased

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 normalgrowthmedium for 0, 30, 45, 60, 90, and 120minutes and pulsedwith 5 mCi/mL [3H]uridine for 30minutes. Data aremean� SEM values (triplicates, n¼ 1).B, in vitrotranscription assay using AdMLP as a template in addition to all the basal transcription factor and Pol II. The DNA template was incubated with amounts ofdrug 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 fromA549 cell and collected atdifferent 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 RNApolymerase 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 weremetabolically labeled with [35S]-methionine for 1 hour and then treated with either normal medium (NT) or supplemented with the drug (Lur, 30 nmol/L).Cellswere collected at different times, andPol II was immunoprecipitatedwith anti-POLR2AcloneH-224beforebeing resolvedby SDS–PAGE andautoradiographed.Graph depicts Pol II protein levels (au, arbitrary unit). Data are the mean � SEM of two independent quantifications.






4 hours after lurbinectedin treatment (Fig. 4B, panels control andLur), in clear contrast to what occurred upon DRB pretreatment(Fig. 4B, panels DRB and DRBþLur). Altogether, lurbinectedinactivity required Pol II phosphorylation.

We then investigated the mechanism of lurbinectedin-induced Pol II degradation. A549 cells were treated with lurbi-nectedin, and, using a Pol II antibody, we performed animmunoprecipitation from whole-cell extracts over time. Theimmune-precipitated Pol II fraction appeared to be part of an

ubiquitinated complex, as evidenced by the smear observablefrom 0.5 hour of treatment (Fig. 5A, lanes 2–4). Converseexperiments showed that the immunoprecipitated fraction byTUBES (tandem ubiquitin binding entity) contained phosphor-ylated 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 bylurbinectedin was dependent on the ubiquitin-proteasome sys-tem, A549 cells were pretreated with PYR-41, a chemical inhibitorof UBA1, an ubiquitin-activating enzyme (30) before adding thedrug. In those conditions, the phosphorylated Pol II was accu-mulated (Fig. 5C, lanes 2 and 4), suggesting that mono-ubiqui-tination, one of the first steps of the ubiquitin/proteasome deg-radation process, was needed for lurbinectedin activity. Pretreat-ment of cells with the proteasome inhibitor MG-132 similarlyprevented drug-mediated degradation of Pol II (Fig. 5D, lanes 2–3and 5–6).

The ubiquitin proteasome degradation process occurs atactivated genes

The above data prompted us to investigate if the degradationof phosphorylated (elongating) Pol II that started few hoursafter lurbinectedin treatment was occurring in transcriptionallyactive genes. Western blot analysis revealed that Pol II wasmainly located in the chromatin-bound fraction and not in thesoluble extract several hours after drug treatment (Fig. 6A, topand bottom panels). This suggested that the degradation pro-cess might have been initiated at the gene level. The effect of thedrug on gene expression in A549 cells was thus tested bymonitoring the transcription of RARb2, a retinoic acid receptorresponsive gene that contains CGG triplets that are highlyfavorable for lurbinectedin bonding and whose expression wasinduced by trans-retinoic acid (t-RA). While in untreated cells,RARb2 expression was peaking around 3 hours after t-RAtreatment (Fig. 6B), in drug-treated cells, RARb2 expressionwas hardly initiated and completely abolished few hours aftertreatment.

We next monitored the recruitment of the transcriptionmachinery on RARb2 by using ChIP coupled to real-time PCR.In untreated cells, ChIP assays showed that phosphorylated Pol IIaswell as TFIIH (as indicated by the presence of its CDK7 subunit)was abundantly recruited at the RARb2 promoter 3 hours after t-RA treatment (Fig. 6C1); the time course paralleled the peak ofRNA synthesis (Fig. 6B). On the contrary, in drug-treated cells, wedetected low amounts of Pol II and TFIIH (Fig. 6D1). Interest-ingly, P-TEFb (detected by the presence of CDK9), which wasvisible at the promoter and exon 4 of RARb2 in untreated cells,accumulated much more at exon 4 in lurbinectedin-treated cells(Fig. 6C2, D2 and C4, D4). In this case, CDK9 as well asphosphorylated Pol II (S2-P), which were present at 2 hours,disappeared at 3 hours; in contrast, they were still detected inuntreated cells (Fig. 6C4), which was indicative of active RNAsynthesis at that time point (Fig. 6B). The differences in the ratiobetween total Pol II and S2-P observed at exon 4 in untreatedversus treated cells could be explained by the regulation of Pol IIactivity by some phospho/dephosphorylation processes (31). Inlurbinectedin-treated cells, ChIP-Ubi/reChIP-Pol II showed thatan ubiquitination process was already engaged on the RARb2 asopposed to untreated cells (Fig. 6C3 and 6D3); we also noticedthe presence of SUG1 (a subunit of the proteasome) at thepromoter.

Figure 4.

Inhibition of Pol II phosphorylation prevents its degradation by lurbinectedin. A,A549 cellswere pretreated for 1 hour with either DRB (25 mmol/L) or flavopiridol(Flv, 5 mmol/L) before the addition of lurbinectedin (Lur, 30 nmol/L) for 4 hoursas indicated at the top of each figure. Cell extracts were then analyzed byWestern blot using antibodies directed toward hyperphosphorylated (IIo) andhypophosphorylated (IIa) Rpb1 subunit of Pol II as well as Rpb1 subunitphosphorylated in Ser5 (S5-P) and in Ser2 (S2-P); NT, nontreated cells. B, in situimmunofluorescencemicroscopy of A549 tumor cells treatedwith lurbinectedin(10 nmol/L) for 4 hours in the absence or presence of DRB (25 mmol/L;preincubation of 1 hour). Control, control cells; DRB, DRB-treated cells; Lur,lurbinectedin-treated cells; DRBþLur, DRB pretreated cells followed bylurbinectedin treatment. Pol II was detected with anti-POLR2A clone 1PB-7C2antibody (green). Nuclei were counterstained with Hoechst-33342 (blue). Scalebar, 10 mm.






The above data strongly suggest that Pol II degradation by theubiquitin-proteasomemachinery is initiated in actively transcrib-ing genes upon treatment with lurbinectedin, and only the phos-phorylated elongating Pol II is degraded in the process.

DNA breaks are dependent on Pol II phosphorylationWe also examined whether the NER pathway could eliminate

the lurbinectedin covalently bound toDNA triplet. A labeledDNAtemplate (containing a single lurbinectedin adduct) was incubat-edwithXPC/HR23B, TFIIH,XPA,RPA,XPG, andas indicatedXPF/ERCC1; we did not observe the removal of the damaged oligo-nucleotide, but rather unexpectedly, we detected several DNAbreaks on the opposite DNA strand (Fig. 7A) originated onlywhen the XPF endonuclease was present (21). However, it wasworthwhile to notice that phosphorylated histone H2AX(g-H2AX), a hallmark of DNA breaks, was detected in lurbinecte-din-treated cell extracts that coincided with the disappearance ofphosphorylated Pol II (S2-P; Fig. 7B, lanes 1–7). Furthermore,pretreatment of cellswithMG-132 resulted in the accumulationofS2-P, a decrease in the presence of g-H2AX, and accumulation ofXPF (Fig. 7B, lanes 8–9).

DNA breaks were next evaluated in A549 cells using the Cometassay, which is based on the neutral or alkaline lysis of labile DNAat sites of damage. DNA from cells that have accumulated DNAbreaks appeared as fluorescent comets with tails of DNA frag-mentation or unwinding, whereas normal, undamaged DNA didnot migrate far from the origin. In lurbinectedin-treated cells, weobserved a clear increase in DNA strand breaks at 4 hours whencompared with untreated cells that were almost abolished in cellspretreated with either DRB or Flv (Figures 7C andD). It should benoticed that Comet assays allow the detection of both single-stranded (SSD) and double-stranded (DSB) DNA breaks. Addi-

tional assays then showed that the absence ofDNAbreakswas notdue to DRB itself because increasing concentrations of DRB didnot inhibit in vitroNER (Supplementary Fig. S3).We shouldnoticethat Comet assays also demonstrated that the level of DNA breaksinduced by the drug was significantly lower in XPF-deficient cellscompared with XPF-proficient cells (Fig. 7E and F). InterestinglyXPF as well as g-H2AX was significantly present after drug treat-ment at exon 4 of RARb2 in lurbinectedin-treated cells comparedwith the untreated ones (Fig. 6C5 and 6D5). In addition, abioChIP assay that measured the incorporation of biotinylateddUTP within broken DNA (32) also showed the presence of DNAbreaks at exon 4 (Fig. 6C5 and 6D5).

Altogether, the above data strongly underline the connectionbetween Pol II elongation (following its phosphorylation), Pol IIdegradation, and the generation of DNA breaks in cells treatedwith lurbinectedin.

DiscussionCurrent research efforts in cancer therapy are aimed to

disturb transcription, one of the fundamental processesinvolved in the maintenance of the oncogenic state. Here, wehave studied the mechanism of action of lurbinectedin, ananticancer agent under clinical investigation with very prom-ising results, and defined the fate of Pol II upon genotoxicattack. We first demonstrated the antiproliferative activity oflurbinectedin in several cancer cell lines with IC50 values in lownanomolar range (Fig. 1B); this potent activity was confirmedin human tumors xenografted in nude mice where lurbinecte-din induced a significant inhibition of tumor growth andimprovement of mice survival (Fig. 1C and D). Molecularbiology approaches next showed that lurbinectedin binds

Figure 5.

Degradation of Pol II by the ubiquitin/proteasome machinery. A, Westernblot of immune-precipitate Pol II fromwhole-cell extracts from A549 cellstreated with 30 nmol/L of lurbinectedin(Lur) during 0.5, 1, and 2 hours. Theimmunoprecipitated fraction has beenanalyzed by using antibodies againstmono and polyubiquitin (top) and Pol II(bottom). B, isolated ubiquitinatedproteins from the above A549 whole-cell extracts treated with 30 nmol/L ofLur at different times (top) using TUBEStechnology allowed to detectphosphorylated (S2-P) from Rpb1.C, A549 cells were pretreated withPYR-41 (PYR), an UBA1 inhibitor, for 6hours before addition of Lur 30 nmol/Lfor 4 hours. Hypophosphorylated (PolIIa) and hyperphosphorylated forms(Pol IIo) of Pol II were detected byWestern blot. b-Tubulin (Tub) has beenused as a control. D, A549 cells werepretreated with MG-132 (50 mmol/L)proteasome inhibitor for 1 hour asindicated (þ); DMSO (NT) or increasingamounts of Lur (10 and 30 nmol/L)were then added for 4 hours. Pol IIo andPol IIa were detected by Western blot.b-Tubulin (Tub) was used as loadingcontrol.






Figure 6.

Recruitment of the transcription and ubiquitin-proteasome machinery on RARb2 activated gene in lurbinectedin-treated A549 cells. A, A549 cells treatedwith 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 representthe ratio between values obtained from treated and untreated cells normalized against the housekeeping GAPDH mRNA. C and D, schematic representation ofthe RARb2 with the indicated amplicons designed at the promoter (Pr) including the RARb responsive elements (RARE), the TATA box and the TSS (þ1) andexon 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 antiubiquitinantibody 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) weredetected using antibodies directed toward Biotin-dUTP incorporated into the broken DNA. Error bars, the SD of three independent experiments.






specifically to CG-rich sequences located in the vicinity of thepromoters of protein-coding genes (Fig. 2B). Further investiga-tions demonstrated that lurbinectedin induced in tumor cells, aspecific and irreversible degradation of Pol II that paralleled the

formation of DNA breaks (Figs. 3C–E, 6D1 and D5, 7B–F).Based on these results, we propose lurbinectedin drive cancercells to apoptosis by the mechanism of action described inSupplementary Fig. S4.

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/CCGDNAtemplate and increasing amounts (1, 10, 100mmol/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 ofA549 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 Freewaresoftware. 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 Cometassay (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 momentquantification, the TriTek CometScore Freeware software was used, a minimum of 100 cells were analyzed, and the experiment was repeated in duplicate.






Some of the CG-rich sequences targeted by lurbinectedin(Fig. 2C) can be highly related to the regulation of geneexpression by constituting CpG islands that can be methylatedto silence the corresponding gene. The GC-dependent (specific)binding preference of lurbinectedin, to areas surrounding theprotein-coding gene promoters, does not exclude the possibil-ity that the drug binds also to other CG-rich sites of the genometargeted by transcription factors, such as SP1 (21, 33), orinvolved in rDNA transcription (34). At any rate, in tumorcells, Pol II is very active and could be arrested by the drugalready bound to the DNA. Indeed, Pol II degradation occurredonce the CTD of its Rpb1 subunit was phosphorylated (Fig. 3C–E). Certainly, Pol II phosphorylation by TFIIH (CDK7) andP-TEFb (CDK9) allows elongation and therefore RNA synthesis(35). Neither Pol II transcription factor partners nor other RNApolymerases were degraded (Fig. 3B–C), underlining a secondlevel of specificity of the drug. At the same time, Comet andBiochip experiments evidenced the formation of DNA breaks inlurbinectedin-treated cells (Figs. 6D5 and 7C–F) and the pres-ence of g-H2AX (a DNA break marker) on the activated RARb2gene (Figs. 6D5 and 7B). Surprisingly, inhibition of Pol IIphosphorylation with either DRB (an inhibitor of the TFIIHtranscription initiation factor) or flavopiridol (an inhibitor ofthe P-TEFb elongation factor) prevented its degradation (Fig.4A) and the formation of DNA breaks (Fig. 7C). Thus, it seemsthat, when lurbinectedin is bound to the DNA, the elongating(phosphorylated) Pol II is stalled in front of the lurbinectedin-DNA adduct, and although DNA breaks appeared on thegenome (Fig. 6D5), it fails to promote the elimination of thelesion. Indeed, due to the specific interaction of lurbinectedinto both DNA strands (16), DNA breaks could have resultedfrom an incomplete DNA repair activity. It is likely that DNArepair factors that have been either carried by the elongating PolII (36) or recruited following the conventional transcriptioncoupled repair (TCR) mechanism (i.e., recruitment of NERfactors by the stalled Pol II in front of a lesion; refs. 37, 38)fail to remove the lesion (Fig. 7A). Indeed, the endonucleaseXPF, involved in the repair of DNA inter and intra-crosslinks(39), was found colocalizing with g-H2AX at the RARb2 (Fig.6D5). Moreover, XPF-deficient cells presented much loweramounts of DNA breaks compared with their proficient coun-terparts (Fig. 7E and F). However, this does not exclude thepossible involvement of other factors such as Topo IIa that wasshown to be essential for AR nuclear receptor transactivation(40). Finally, and in agreement with our proposed model, thestructural analogue of lurbinectedin that failed to bind DNA(Fig. 1F) did not produce any degradation of Pol II as well asDNA damage (Supplementary Fig. S5A), demonstrating thatthe DNA binding of the drug to the CG-rich regions is respon-sible for the disturbance of the transcription process. Similarly,in P-glycoprotein–overexpressing IGROV-ET ovarian cancercells (28), the lower activity of lurbinectedin was correlatedto the absence of RNA synthesis inhibition and Pol II degra-dation and to lower amounts of DNA breaks induced by thedrug (Supplementary Fig. S5B).

Our results can also be used to explain the regulation oftranscription mechanisms after genotoxic insults. Indeed, we caninfer from them that, in the absence of DNA damage removal andsubsequent restart of RNA synthesis, it is likely that there is amechanism that restricts the amount of time that any stalled Pol IIcan reside on an activated gene. However, a failure of TCR could

not by itself explain the Pol II degradation that also occurredfollowing treatment of cells with a-amanitin, which binds withhigh affinity to the Rpb1 subunit of Pol II. It thus seems that inboth cases (binding of either lurbinectedin toDNAora-amanitinto Rpb1), degradation occurs because elongating Pol II is irre-versibly stalled on the DNA template. As a consequence, theubiquitin-proteasome degradation process will be engaged (41,42). In the case of lurbinectedin, this is characterized by theubiquitination of Pol II (Fig. 5A and B) and the recruitment ofSug1 (Fig. 6D3) in the vicinity of the adduct site.

Taking together, our results show how lurbinectedin, byinhibiting transcription, causes a cascade of events that canexplain its potent effects on tumor cells. The relevance of Pol IIdegradation and formation of DNA breaks was finally con-firmed in vivo (Supplementary Fig. S6). In these xenograftedmodels, the significant inhibition of tumor growth observedafter lurbinectedin treatment was correlated to both Pol IIdegradation and induction of DNA damage. Therefore, lurbi-nectedin may help in the treatment of tumors with transcrip-tion addiction. In this regard, it was recently reported that, incombination with doxorubicin, lurbinectedin has compellingactivity as a second-line treatment in patients with SCLC (43), atumor type that could be most sensitive to transcription-target-ing drugs (3–5, 7, 44, 45). Lurbinectedin has also showedimpressive activity in platinum-resistant ovarian cancer (46),a subtype of ovarian cancer that has been related to geneexpression alterations affecting different oncogenic pathwaysrelated to drug resistance and tumor microenvironment (47–49). Targeting transcription addiction with lurbinectedin mightbe similarly beneficial in the treatment of other tumor typesthat are known to be dependent on transcription dysregulation(50, 51).

In summary, here we have described the mode of action oflurbinectedin for cancer targeting. This drug inhibits the tran-scription process by (1) its binding to CG-rich sequences, mainlylocated around promoters of protein-coding genes; (2) the irre-versible stalling of elongating RNA Pol II on the DNA templateand its specific degradation by the ubiquitin/proteasomemachin-ery; and (3) the generation of DNA breaks and subsequentapoptosis. These results also improved our understanding of animportant transcription regulation mechanism.

Disclosure of Potential Conflicts of InterestC.M. Galmarini and P. Aviles are Senior Managers and have ownership

(including patents) in PharmaMar. G. Santamaría Nuñez and J.F. Martínez-Lealare PharmaMar employees. J.-M. Egly reports receiving commercial researchgrant from PharmaMar. No potential conflicts of interest were disclosed by theother authors.

Authors' ContributionsConception and design: G. Santamaría Nu~nez, C. Giraudon, P. Aviles,C.M. Galmarini, J.-M. EglyDevelopment of methodology: G. Santamaría Nu~nez, C.M. Genes Robles,E. Compe, J.-M. EglyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. Santamaría Nu~nez, C.M. Genes RoblesAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. Santamaría Nu~nez, C.M. Genes Robles,C. Giraudon, J.F. Martínez-Leal, E. Compe, F. Coin, P. Aviles, C.M. Galmarini,J.-M. EglyWriting, review, and/or revision of the manuscript: G. Santamaría Nu~nez,C.M. Genes Robles, C. Giraudon, J.F. Martínez-Leal, E. Compe, P. Aviles,C.M. Galmarini, J.-M. Egly






Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. Giraudon, J.F. Martínez-LealStudy supervision: C. Giraudon, J.F. Martínez-Leal, P. Aviles, C.M. Galmarini,J.M. Egly

AcknowledgmentsWe thank María Jos�e Guillen-Navarro, María Jos�e Mu~noz, Jose Manuel

Molina-Guijarro, Cathy Braun, and the IGBMC facilities for their expertise andtechnical support. We also are grateful to CarmenCuevasMarchante, Jos�eMaríaFern�andez Sousa Faro, and Nicolas Le May for fruitful discussions. Sequencingand bioinformatics analysis (with the expertise of Tao Ye and Baptiste Bidon)were performed by the IGBMCMicroarray and Sequencing platform, amemberof the "France G�enomique" consortium (ANR-10-INBS-0009).

Grant SupportJ.M. Egly has been awarded with the following grants: ERC Advanced

grant, l'Agence Nationale de la Recherche (N#ANR1 08MIEN-022-03),l'Association de la Recherche contre le Cancer (SL22013060782), theInstitut National du Cancer (INCA-2008-041), and Ligue Nationale contrele Cancer.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 28, 2016; revised May 23, 2016; accepted June 15, 2016;published OnlineFirst September 14, 2016.

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Published OnlineFirst September 14, 2016.Mol Cancer Ther Gema Santamaría Nuñez, Carlos Mario Genes Robles, Christophe Giraudon, et al. DNA Breaks in Cancer CellsPhosphorylated RNA Polymerase II and the Formation of Lurbinectedin Specifically Triggers the Degradation of

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