Zalypsis(PM00104)isapotentinducerof γ-H2AXfociand...

Zalypsis (PM00104) is a potent inducer of γ-H2AX foci andreveals the importance of the C ring of trabectedin fortranscription-coupled repair inhibition

Josée Guirouilh-Barbat, Smitha Antony,and Yves Pommier

Laboratory of Molecular Pharmacology, Center for CancerResearch, National Cancer Institute, NIH, Bethesda, Maryland

AbstractZalypsis (PM00104) is a novel tetrahydroisoquinoline al-kaloid related to trabectedin [ecteinascidin 743 (Et743)].Et743 and PM00104 have similar A and B rings but differin their C rings. The present study shows that Et743 andPM00104 differ in at least two ways: in their DNA bindingproperties and nucleotide excision repair (NER) dependen-cy for cellular targeting. DNase I footprinting shows thatthe two drugs bind DNA differentially. We also found that,in contrast to Et743, the antiproliferative activity ofPM00104 does not depend on transcription-coupledNER. Accordingly, PM00104 induces γ-H2AX foci withthe same efficiency in NER-deficient or NER-proficientcells. Moreover, the formation of γ-H2AX foci is replica-tion dependent for PM00104, whereas it is both transcrip-tion and replication dependent in the case of Et743. Thesefindings show the importance of the C ring structure oftetrahydroisoquinoline ecteinascidin derivatives for NERtargeting. Finally, PM00104 exerts antiproliferative activ-ity at nanomolar concentrations and induces γ-H2AX re-sponse in two Ewing's sarcoma cell lines, suggestingthat γ-H2AX could serve as a pharmacodynamic biomark-er for the clinical development of PM00104. [Mol CancerTher 2009;8(7):2007–14]

IntroductionPM00104 (Zalypsis) is a novel semisynthetic tetrahydroiso-quinoline alkaloid. This family of compounds includes jor-umycin, obtained from a pacific nudibranch, the family of

renieramycins, which are isolated from sponges, and the ec-teinascidins isolated from a Caribbean tunicate. These com-pounds are potent cytotoxic agents and display highantitumor and antimicrobial activities (1, 2). Consistentwith other members of this family, PM00104 exhibits strongin vitro and in vivo antitumor activity in a wide variety ofsolid and hematologic tumor cell lines and human trans-plantable breast, gastric, prostate, and renal xenografted tu-mors. PM00104 has now entered phase I clinical trials forthe treatment of solid tumors and lymphoma.

PM00104 is chemically related to ecteinascidin 743 (Et743;trabectedin; Fig. 1A). However, PM00104 was chemicallyengineered, whereas Et743 is a natural product initially pu-rified from extracts of the tunicate Ecteinascidia turbinata in1990 (3). Structurally, PM00104 and Et743 consist of similarA and B ring structures but differ in their C ring (Fig. 1A).This chemical difference is important because the C ring ofEt743 has been proposed to directly interact with the nucle-otide excision repair (NER) endonuclease, XPG (4, 5).

Et743 is registered as Yondelis and approved for the treat-ment of soft tissue sarcomas. Et743 is also being studied inphase III trial for ovarian cancer and in phase II trials forbreast, prostate, and pediatric sarcomas. Et743 alkylatesthe N2 position of guanines in the DNA minor groove(6, 7). Nuclear magnetic resonance–based studies indicatesthat the A and B subunits of Et743 are responsible for DNArecognition and bonding (8), whereas the C ring is projectedout of the minor groove, making limited contacts with theDNA (6, 7, 9). A unique characteristic of Et743 is its selec-tive poisoning of the transcription-coupled NER (TC-NER;refs. 5, 10, 11). This was revealed by the finding that a coloncarcinoma cell line (HCT116-ER-5) selected for resistance toEt743 was deficient for the NER factor, XPG (5), and thatTC-NER–deficient cells were resistant to Et743 (5, 10, 11).This mechanism of action sets Et743 apart from the “clas-sic” DNA alkylating agents, such as platinum derivatives,which tend to be inactive in TC-NER–overexpressing cells(12, 13).

The aim of the present study was to compare PM00104 andEt743 with regard to DNA binding and NER dependencyand to uncover pharmacodynamic biomarkers for the clinicaldevelopment of PM00104. To that effect, we report our inves-tigation of the sequence-specific DNA binding of PM00104 incomparison with Et743, and the contribution of NER for theantiproliferative activity of the drugs. Finally, based on theclinical data available showing activity of PM00104 in sarco-mas and the recent finding that Et743 induces γ-H2AX (14–16), we determined whether the induction of DNA damageby PM00104 could be measured using γ-H2AX as a pharma-codynamic marker (17) in human Ewing's sarcoma cell lines.

Received 6/25/08; revised 4/10/09; accepted 5/15/09; publishedOnlineFirst 7/7/09.

Grant support: Intramural Research Program, Center for Cancer Research,National Cancer Institute, NIH.

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 toindicate this fact.

Note: J. Guirouilh-Barbat and S. Antony contributed equally to this work.

Requests for reprints: Yves Pommier, National Cancer Institute, NIH, 37Convent Drive, Building 37, Room 5068, NIH, Bethesda, MD 20892-4255.Phone: 301-496-5944; Fax: 301-402-0752. E-mail: [email protected]

Copyright © 2009 American Association for Cancer Research.

doi:10.1158/1535-7163.MCT-09-0336

Mol Cancer Ther 2009;8(7). July 2009

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Published Online First on July 7, 2009 as 10.1158/1535-7163.MCT-09-0336

on May 19, 2018. © 2009 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst July 7, 2009; DOI: 10.1158/1535-7163.MCT-09-0336

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Materials and MethodsDrugs, Chemicals, and Enzymes

Et743 and PM00104 were provided by Pharmamar. Drugstock solutions were made in DMSO at 10 mmol/L, and fur-ther dilutions were made in DMSO immediately before use.T4 polynucleotide kinase and polyacrylamide-bis were pur-chased from Invitrogen. DNA Quick Spin columns werepurchased from Roche Diagnostics. [γ-32P]dATP was pur-chased from New England Nuclear. Aphidicolin was pur-chased from Sigma Chemical Co.Cell Culture

All cell lines were maintained in DMEM containing 10%FCS. Xeroderma pigmentosum group D (XPD) and their

stably complemented counterparts XPD-c were providedby Dr. Kenneth Kraemer (Basic Research Laboratory, Centerfor Cancer Research, National Cancer Institute, NIH,Bethesda, MD). XPF, XPG, HCT116, and wild-typeGM00637 were obtained from the Coriell Cell Repository(Camden, NJ). Human colon carcinoma HCT116 Et743-re-sistant derivative was generated as described (5). Ewing'ssarcoma cells TC32 and TC71 were obtained from Dr. JavedKhan (Advanced Technology Center, National Cancer Insti-tute, NIH, Gaithersburg, MD).DNase I Footprinting

The 161-bp DNA fragment from pBluescript SK(−) phage-mid DNA (Stratagene) was generated by PCR amplification

Figure 1. Comparative DNA bind-ing profiles of PM00104 and Et743.A, chemical structures of Et743 andPM00104. B, representative DNase Ifootprinting experiment. The DNAsubstrate [5′-end–labeled PvuII/Hin-dIII fragment of pBluescript SK(−)phagemid DNA (pSK)] was reactedin the absence (No drug) or presenceof 1 or 10 μmol/L of Et743 orPM00104 for 30 min at 37°C. Sam-ples were then subjected to either notreatment (0) or treatment with in-creasing concentrations of DNase Ifor 1 min at 25°C. Reactions werestopped with 15 mmol/L EDTA (finalconcentration). DNA fragments wereseparated in denaturing polyacryl-amide gels. Lane G + A, purine lad-der after formic acid treatment.Numbers on the right correspond tothe DNA sequence indicated in C.C, differential DNase I cutting in thepresence of 10 μmol/L PM00104(red line) and Et743 (blue line). Nega-tive and positive values correspond todrug-protected sites and enhancedcleavage, respectively. Verticalscales are in units of ln(fa)-ln(fc),where fa is the fractional cleavage atany bond in the presence of the drugand fc is the fractional cleavage of thesame bond in the control, given close-ly similar extents of overall digestion.The results are displayed on a loga-rithmic scale and the DNA sequenceis indicated at the bottom. Numberson the right of B correspond to thatsequence.

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as described (18). Reaction products were separated by elec-trophoresis in 1.5% agarose gels made in 1× Tris-borateEDTA buffer. The 161-bp fragment (pSK) was eluted fromthe gel slice using the Qiaex II kit (Qiagen, Inc.). The pSKDNA was 5′-end labeled by using T4 polynucleotide kinasewith [γ-32P]dATP for 1 h at 37°C. Reactions were stopped byincubating the samples for 10 min at 70°C. To retain the 5′-radiolabel in only one of the strands, samples were cleavedwith HindIII restriction endonuclease in supplied NE buffer2 (New England Biolabs) for 1 h at 37°C. Labeled mixtureswere subsequently centrifuged through mini Quick SpinDNA columns to remove the unincorporated radiolabel.

For the DNase I footprinting experiments, labeled pSKwas reacted in the absence (control) or presence of 1 or10 μmol/L of Et743 or PM00104 in 10 mmol/L Tris-Cl (pH7.5), 50 mmol/L KCl, 5 mmol/L MgCl2, 0.1 mmol/L EDTA,and 15 μg/mL bovine serum albumin (final concentrations).After incubation for 30 min at 37°C, samples were subjectedto either no treatment (−) or treatment with several concen-trations of DNase I (0.005, 0.01, 0.05, or 0.1 unit for controland 0.05, 0.1, 0.5, or 1 unit for drug treated) in a 10 μL reac-tion for 1 min at 25°C (6). Reactions were stopped with15 mmol/L EDTA (final concentration) and DNA sequenc-ing loading buffer (80% formamide, 10 mmol/L NaOH,1 mmol/L EDTA, 0.1% xylene cyanol, and 0.1% bromphenolblue). Samples were heated to 90°C and immediately loadedinto DNA sequencing gels (7% polyacrylamide; 19:1 acryl-amide:bis) containing 7 mol/L urea in Tris-borate EDTAbuffer. Electrophoreses were at 2,500 V (60 W) for 4 h. Imag-ing and quantitation were done with a PhosphorImager us-ing ImageQuant software (Molecular Dynamics).3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium

Bromide Assays

Cells were seeded at a density of 1,000 per well in 96-wellmicrotiter plates. Et743 or PM00104 was added the next day.After 72 h, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide (MTT) was added to each well (0.5 mg/mL;Sigma-Aldrich), and plates were maintained at 37°C for 4 h.The medium was then discarded and DMSO was addedto each well to lyse the cells. Absorbance was measured at450 nm using a multiwell spectrophotometer (Emax, Molec-ular Devices).Confocal Microscopy

Cells were grown 1 d before drug treatment in Nuncchamber slides (Nalgene). Following treatment, the mediumwas aspirated out and cells were washed in PBS. Cells wereimmediately fixed and permeabilized by a 20-min incuba-tion at room temperature with 2% paraformaldehyde anda 5-min incubation in ice-cold 70% ethanol. To block non-specific binding, cells were incubated with 8% bovine serumalbumin for 1 h at room temperature. Cells were stained for90 min with mouse anti-γ-H2AX antibody (1:1,000 dilution;Upstate Biotechnology). Fluorescent secondary antibody(Alexa Fluor 488, 1:800; Molecular Probes) was then addedfor 45 min. All incubations were in 1% bovine serum albu-min at room temperature and followed by three PBSwashes. Nuclei were stained with propidium iodide (0.05mg/mL) and RNase A (0.5 mg/mL) for 10 min at 37°C.

Slides were mounted using Vectashield mounting liquid(Vector Labs) and visualized using a Nikon Eclipse TE-300confocal laser scanning microscope system. Images werecaptured and stored as TIFF files. For each sample in eachexperiment, 50 to 200 cells were scored. The quantificationof staining intensities was made with Adobe Photoshop 7.0and normalized to the number of analyzed cells.

ResultsPM00104 Exhibits Differential DNase I Footprinting in

Comparison with Et743

Because PM00104 and Et743 are derived from a similarchemical scaffold (A-B rings), which is known to form cova-lent bonding with DNA (6, 19) but differ in their appendedC rings (see Fig. 1A for structures), we evaluated whetherthe differences in C ring affected DNase I footprinting (6).As illustrated in Fig. 1B, PM00104 produced a clear foot-print with suppression of cleavage at the drug binding sitesand enhancement of cleavage in the flanks of the drug-bound DNA (6, 20). This indicates that PM00104 is a se-quence-specific DNA binding agent. Comparison of theDNA cleavage patterns obtained in the presence ofPM00104 and Et743 showed DNA binding at several similarsites with differences in the relative intensities (Fig. 1B andC). At 1 μmol/L, the DNase I footprinting of PM00104seemed generally stronger for PM00104 than for Et743(Fig. 1B). At 10 μmol/L, both PM00104 and Et743 gave clearfootprints. Some binding sites, however, were stronger forPM00104 (Fig. 1C). These results show that PM00104 bindsDNA at specific sites and exhibits both similarities and dif-ferences compared with Et743.

Figure 2. The antiproliferative activity of PM00104 is not influenced bythe XPD-NER status. Cells were exposed to the indicated concentrationsof Et743 or PM00104 for 72 h and analyzed by MTTassay. Open symbols,NER-deficient XPD cells; closed symbols, XPD-complemented counter-part, XPD-c. Squares, Et743; circles, PM00104.

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The Antiprol i ferative Activity of PM00104 Is

Independent of NER

Because Et743 is selective for TC-NER–proficient cells(5, 10), we tested the antiproliferative activity of PM00104in NER-deficient XPD cells and their XPD-complementedcounterparts, XPD-c (Fig. 2). As previously described (5),XPD-complemented cells are ∼4-fold more sensitive toEt743 than XPD cells. Remarkably, NER functionality hadno effect on the activity of PM00104 (Fig. 2). The XPD-deficient and XPD-complemented cell lines were equallysensitive to PM00104 with IC50 in the subnanomolar range(0.2 nmol/L). These results suggested that, in contrast toEt743, PM00104 acts independently of NER.

Next, we tested the antiproliferative activity of PM00104in the Et743-resistant cell line (HCT116-ER-5) derived fromthe colon carcinoma cell line HCT116 (5). Resistance of theHCT116-ER-5 cells has been linked to inactivation of theNER nuclease XPG (5). When tested for the antiproliferativeactivity of PM00104, HCT116-ER-5 exhibited a slight resis-tance compared with the parental HCT116 (Fig. 3, left).However, because those HCT116-ER-5 cells were selectedin the presence of Et743, it is possible that other alterationsbesides XPG inactivation could contribute to their partialresistance to PM00104. To test this further, we analyzedtwo other NER-deficient cell lines, XPG and XPF, previous-ly reported to be resistant to Et743 (5). Those XPG and XPFcells showed no resistance to PM00104 when comparedwith wild-type fibroblasts GM00637 (Fig. 3, right). Thus,the combination of these results indicates that, by contrastto Et743, the antiproliferative activity of PM00104 is inde-pendent of NER.

Induction of Replication-Dependent γ-H2AX Foci by

PM00104

Phosphorylation of H2AX, one of the histone H2A var-iants, on Ser139 (referred to as γ-H2AX) is one of the mostsensitive and reliable markers for DNA damage response(17, 21). Because Et743 was recently found to induceγ-H2AX response (14–16), and we found the Et743-inducedγ-H2AX response more intense in NER-proficient cells(14, 15), we investigated whether PM00104 also inducedγ-H2AX foci. We compared the induction of γ-H2AX inXPD and XPD-complemented cells. Figure 4A shows thatPM00104 and Et743 induced γ-H2AX foci in both XPDand XPD-complemented cells. However, Et743 induced sig-nificantly more γ-H2AX foci in XPD-complemented than inXPD cells, whereas PM00104 had the same efficiency in bothcell lines. Figure 4B shows the quantification of several ex-periments and that the intensity of γ-H2AX staining afterEt743 was increased by 9.0 ± 0.1–fold and 4.1 ± 0.6–foldin XPD-c and XPD cells, respectively. In contrast, the effectof PM00104 on γ-H2AX induction was statistically similarin XPD-complemented and XPD cells (3.7 ± 0.7–fold and2.3 ± 0.7–fold, respectively). Those results show that the in-duction of γ-H2AX foci by PM00104 in NER-proficient orNER-deficient cells is comparable with the induction ob-served after Et743 treatment of NER-deficient cells.

Single-cell analyses for γ-H2AX response to PM00104consistently showed that some cells remained negative,whereas neighboring cells were positive for γ-H2AX (seeFig. 4A). We had previously observed a similar phenome-non for γ-H2AX induction by camptothecins and the inde-noisoquinolines (inhibitors of topoisomerase I) and related

Figure 3. Comparative antiproliferative activity of PM00104 in Et743-resistant cells. Left, HCT116-ER-5 cells selected for resistance to Et743 (5) arecross-resistant to PM00104; right, XPF and XPG fibroblasts (both NER deficient) are not cross-resistant to PM00104. Cells were exposed to the indicatedconcentrations of PM00104 for 72 h and analyzed by MTT assay.



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this selective response to the conversion of the topoisome-rase I cleavage complexes into replication-induced DNAdamage in S-phase cells (18, 22). To investigate whetherPM00104-induced γ-H2AX foci were also replication depen-dent, cells were treated with PM00104 in the presence or ab-sence of the DNA polymerase inhibitor aphidicolin (22). Therepresentative pictures shown in Fig. 4C show that aphidico-lin almost completely inhibited the appearance of γ-H2AXfoci. Quantitation of repeated independent experimentsshows marked inhibition of the γ-H2AX response (Fig. 4D)in cells treated simultaneously with PM00104 and aphidico-lin. From these results, we conclude that PM00104-inducedγ-H2AX foci are replication dependent and probably corre-spond to replication-associated DNA damage.Activity of PM00104 in Ewing's Sarcoma Cells

Because Et743 and PM00104 are both active in soft tissuesarcomas, we compared the potency of PM00104 and Et743

in Ewing's sarcoma cell lines. First, we did MTT assays intwo Ewing's sarcoma cell lines, TC32 and TC71. As shownon Fig. 5, both Et743 and PM00104 are highly potent inboth cell lines with IC50 at the subnanomolar range. TheIC50s were 0.04 and 0.15 nmol/L for Et743 and PM00104,respectively.

Because γ-H2AX is in development as a pharmacody-namic biomarker (17, 18), we looked for the induction ofγ-H2AX foci in the Ewing's sarcoma cells treated withPM00104 or Et743. Time course experiments with10 nmol/L drug concentrations showed that both drugs in-duced γ-H2AX foci within 2 hours of exposures (Fig. 6, left).With PM00104, the γ-H2AX foci appeared later, and likewith Et743, the γ-H2AX signal induced by PM00104 in-creased with drug exposure. Dose-response experiments af-ter 4-hour drug treatments showed that 1 nmol/L PM00104was sufficient to induce detectable γ-H2AX (representative

Figure 4. γ-H2AX foci induced byPM00104 are independent of NERand are replication associated.A and B, γ-H2AX foci in XPD andXPD-c cells treated with 10 nmol/LEt743 or PM00104 for 6 h. A, repre-sentative pictures. B, fold increaseof the intensity ofγ-H2AX stainingnormalized to nontreated cells (arbi-trary units). Columns, averagesof five independent experiments;bars, SE. C and D, inhibition ofPM00104-induced γ-H2AX by theDNA polymerase inhibitor aphidicolin(APD). XPD-c cells were preincu-bated with 1 μmol/L aphidicolin for15 min. PM00104 (10 nmol/L) wasthen added for an additional 6 h.C, representative pictures. D, foldincrease of the intensity of γ-H2AXstaining normalized to untreated cells(arbitrary units). Columns, averageof three independent experiments;bars, SE.



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pictures in Fig. 6, right). The γ-H2AX signal increased withdrug concentrations for both drugs. At equal concentration,the γ-H2AX staining was consistently more intense withEt743 than with PM00104. Those experiments show thatγ-H2AX foci are induced both by PM00104 and Et743 andthat Et743 is generally more potent to induce γ-H2AX focithan PM00104 in Ewing's sarcoma cells.

DiscussionPM00104 is in phase I clinical trials. Because it is structur-ally related to Et743, we compared both compounds andinvestigated whether histone γ-H2AX could be used as abiomarker for clinical pharmacodynamic assessment. Thepresent findings show that PM00104 differs from Et743 inat least two ways: in its DNA binding properties and NERdependency for cellular targeting. We also show thatPM00104 is effective in various cell lines, including Ewing'ssarcoma cells, at nanomolar concentrations and that his-tone γ-H2AX is induced by PM00104 at pharmacologicconcentrations.

Previous studies have implicated the A and B rings ofEt743 as being primarily responsible for its minor grooveDNA binding, whereas the C ring protrudes from theDNA minor groove (see Fig. 1A; refs. 4–6, 11). Accordingly,we find that alteration of the C ring, as in PM00104, doesnot prevent the ability of the drug to bind DNA (seeFig. 1B and C). Furthermore, PM00104 and Et743 exhibitoverall similarity and sequence-specific differences in theirDNA footprinting profiles (Fig. 1B). David-Cordonnieret al. (23) also showed that analogues of Et743 modifiedon the C ring retain DNA binding ability. Our DNase I foot-printing experiments (see Fig. 1) show sequence-specific

differences between PM00104 and Et743, which agrees withthe results of David-Cordonnier et al. (23) who also ob-served slight differences in the binding properties of threeother C ring–modified analogues of Et743. Thus, our pres-ent findings suggest that chemical differences in the C ringdo influence the interactions of PM00104 with DNA. It isplausible that such differences between PM00104 andEt743 may translate into differential effects on chromatinand on the interactions of PM00104 with other nuclear pro-teins and transcription factors.

Several studies have suggested the importance of the Cring of Et743 for cellular activity. For instance, replacingthe C ring of Et743 by a tetrahydro-β-carboline reduces anti-tumor activity (24). Saframycin A, which is structurally re-lated to ecteinascidins but lacks the C unit, has lowerantitumor activity than ecteinascidins (7, 24). However,modifications of the C ring can be done without total lossof biological activity. For instance, phthalascidin (Pt650) isas efficient as Et743 in a panel of 36 cancer cell lines (25).Other analogues of Et743 display very different biologicalactivities (23). Here, we show that PM00104 retains potentantiproliferative activity at nanomolar concentrations.

Our next question was whether the NER selectivity ofEt743 (5, 10) was shared by PM00104. This was of particularinterest because the C ring of Et743, which protrudes out-side of the DNA minor groove (7), has been proposed tomake the key contact with the NER complex (4, 5). We com-pared the activities of PM00104 and Et743 in an isogenicpair of XPD-deficient and XPD-complemented cells and intwo other cell lines deficient for NER, which we had previ-ously used to establish the TC-NER–dependent selectivityof Et743 (5). Our results show that the activity of NERhad no effect on the antiproliferative activity of PM00104(see Figs. 2 and 3). Those findings provide further evidencefor the importance of the C ring of Et743 for specific NERpoisoning (4).

The similar activity of PM00104 in NER-proficient andNER-deficient cells suggests that the repair of the DNA le-sions generated by PM00104 is not dependent on the NERpathway. In that respect, PM00104 differs from both Et743(which tends to be more effective in NER-proficient cells;see above) and from platinum derivatives. Indeed, platinumadducts are repaired by the NER, and a common cause ofresistance to platinum derivatives is due to increased NERactivity in tumor cells (12, 13). It is therefore plausible thatPM00104 could be used in platinum-resistant tumors or incombination with platinum derivatives.

Histone γ-H2AX, the phosphorylated form of H2AX, hasrecently emerged as potential clinical biomarker because itcan be detected with high sensitivity and specificity usingantibodies and immunofluorescence (14–17, 22). Our studyshows γ-H2AX activation at pharmacologic concentrationsof PM00104. We recently reported that the increased activityof Et743 in NER-proficient cells was correlated to anincreased formation of γ-H2AX in those cells (see Fig. 4;ref. 14). Here, we show that the induction of γ-H2AX fociby PM00104 is independent of NER (see Fig. 4). We alsoshow that γ-H2AX activation is detectable at nanomolar

Figure 5. Antiproliferative activity of PM00104 in Ewing's sarcomacells. Ewing's sarcoma TC32 and TC71 cells were incubated with the in-dicated concentrations of PM00104 and Et743 for 72 h and analyzed byMTT assay. Closed symbols, TC71 cells; open symbols, TC32 cells.Circles, PM00104; squares, Et743.



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pharmacologic concentrations of PM00104 in Ewing's sarco-ma cells (see Fig. 6). Thus, PM00104 behaves as an “Et743-like” molecule without the NER-dependent component.Lacking the NER-dependent component of Et743 may pro-vide some benefit for PM00104. Indeed, loss of heterozygos-ity and impaired expression of several NER factors havebeen reported in sporadic solid tumors (5, 26) and in sarco-ma (27). In such tumors, PM00104 may remain potentiallyas active as Et743. In addition, PM00104 might be less toxictoward the normal cells, which are NER proficient. Finally,from a translational standpoint, our experiments indicatethat γ-H2AX can be considered as a pharmacodynamic bio-marker for the upcoming clinical development of PM00104.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank the Laboratory of Molecular Pharmacology research team and inparticular Dr. Kurt Kohn for suggestions during the course of the presentstudies and the Pharmamar team and in particular Drs. Carmen Cuevas andJosé Fernandez Sousa-Faro for kindly providing PM00104 and Et743.

References

1. Lane JW, Estevez A, Mortara K, Callan O, Spencer JR, Williams RM.Antitumor activity of tetrahydroisoquinoline analogues 3-epi-jorumycinand 3-epi-renieramycin G. Bioorg Med Chem Lett 2006;16:3180–3.

2. Scott JD, Williams RM. Chemistry and biology of the tetrahydroisoqui-noline antitumor antibiotics. Chem Rev 2002;102:1669–730.

3. Rinehart KL, Holt TG, Fregeau NL, et al. Bioactive compounds fromaquatic and terrestrial sources. J Nat Prod 1990;53:771–92.

4. Herrero AB, Martin-Castellanos C, Marco E, Gago F, Moreno S. Cross-talk between nucleotide excision and homologous recombination DNArepair pathways in the mechanism of action of antitumor trabectedin.Cancer Res 2006;66:8155–62.

5. Takebayashi Y, Pourquier P, Zimonjic DB, et al. Antiproliferative activityof ecteinascidin 743 is dependent upon transcription-coupled nucleotide-excision repair. Nat Med 2001;7:961–6.6. Pommier Y, Kohlhagen G, Bailly C, Waring M, Mazumder A, Kohn KW.DNA sequence- and structure-selective alkylation of guanine N2 in theDNA minor groove by ecteinascidin 743, a potent antitumor compoundfrom the Caribbean tunicate Ecteinascidia turbinata. Biochemistry 1996;35:13303–9.

7. Zewail-Foote M, Hurley LH. Ecteinascidin 743: a minor groove alkylatorthat bends DNA toward the major groove. J Med Chem 1999;42:2493–7.

8. Moore BM II, Seaman FC, Hurley LH. NMR-based model of an ecteinas-cidin 743-DNA adduct. J Am Chem Soc 1997;119:5475–6.9. Sakai R, Rinehart KL, Guan Y, Wang AH. Additional antitumor ecteinas-cidins from a Caribbean tunicate: crystal structures and activities in vivo.Proc Natl Acad Sci U S A 1992;89:11456–60.10. Erba E, Bergamaschi D, Bassano L, et al. Ecteinascidin-743 (ET-743),a natural marine compound, with a unique mechanism of action. Eur JCancer 2001;37:97–105.

11. Zewail-Foote M, Li VS, Kohn H, Bearss D, Guzman M, Hurley LH. Theinefficiency of incisions of ecteinascidin 743-DNA adducts by the UvrABCnuclease and the unique structural feature of the DNA adducts can beused to explain the repair-dependent toxicities of this antitumor agent.Chem Biol 2001;8:1033–49.

12. Furuta T, Ueda T, Aune G, Sarasin A, Kraemer KH, Pommier Y.

Figure 6. Induction of γ-H2AX atpharmacologic concentrations ofPM00104 and Et743 in Ewing's sar-coma cells. Representative picturesof γ-H2AX staining in TC71 cells incu-bated with Et743 or PM00104 for theindicated times and concentrations.



2013




Transcription-coupled nucleotide excision repair as a determinant ofcisplatin sensitivity of human cells. Cancer Res 2002;62:4899–902.

13. Reed E. ERCC1 and clinical resistance to platinum-based therapy. ClinCancer Res 2005;11:6100–2.

14. Guirouilh-Barbat J, Pommier Y. Et-743 induced DNA double strandbreaks: a link between histone γ-H2AX, transcription coupled nucleotideexcision repair and Mre11 endonuclease [abstract number 4642]. 2006,p. 1090.

15. Guirouilh-Barbat J, Redon C, Pommier Y. Transcription-coupled DNAdouble-strand breaks are mediated via the nucleotide excision repair andthe Mre11-Rad50-Nbs1 complex. Mol Biol Cell 2008;19:3969–81.

16. Soares DG, Escargueil AE, Poindessous V, et al. Replication andhomologous recombination repair regulate DNA double-strand break for-mation by the antitumor alkylator ecteinascidin 743. Proc Natl Acad SciU S A 2007;104:13062–7.

17. Bonner WM, Redon CE, Dickey JS, et al. γH2AX and cancer. Nat RevCancer 2008;8:957–67.

18. Antony S, Agama KK, Miao ZH, et al. Novel indenoisoquinolines NSC725776 and NSC 724998 produce persistent topoisomerase I cleavagecomplexes and overcome multidrug resistance. Cancer Res 2007;67:10397–405.

19. Guan Y, Sakai R, Rinehart KL, Wang AH. Molecular and crystal struc-tures of ecteinascidins: potent antitumor compounds from the Caribbeantunicate Ecteinascidia turbinata. J Biomol Struct Dyn 1993;10:793–818.

20. Bailly C, Cuthbert AW, Gentle D, Knowles MR, Waring MJ. Sequence-selective binding of amiloride to DNA. Biochemistry 1993;32:2514–24.

21. Redon C, Pilch D, Rogakou E, Sedelnikova O, Newrock K, Bonner W.Histone H2A variants H2AX and H2AZ. Curr Opin Genet Dev 2002;12:162–9.

22. Furuta T, Takemura H, Liao ZY, et al. Phosphorylation of histone H2AXand activation of Mre11, Rad50, and Nbs1 in response to replication-de-pendent DNA double-strand breaks induced by mammalian DNA topoi-somerase I cleavage complexes. J Biol Chem 2003;278:20303–12.

23. David-Cordonnier MH, Gajate C, Olmea O, et al. DNA and non-DNAtargets in the mechanism of action of the antitumor drug trabectedin.Chem Biol 2005;12:1201–10.

24. Sakai R, Jares-Erijman EA, Manzanares I, Silva Elipe MV, Rinehart KL.Ecteinascidins: putative biosynthetic precursors and absolute stereochem-istry. J Am Chem Soc 1996;118:9017–23.

25. Martinez EJ, Corey EJ, Owa T. Antitumor activity- and gene expres-sion-based profiling of ecteinascidin Et 743 and phthalascidin Pt 650.Chem Biol 2001;8:1151–60.

26. Castro MA, Mombach JC, de Almeida RM, Moreira JC. Impaired ex-pression of NER gene network in sporadic solid tumors. Nucleic Acids Res2007;35:1859–67.

27. Le Morvan V, Longy M, Bonaiti-Pellie C, et al. Genetic polymorphismsof the XPG and XPD nucleotide excision repair genes in sarcoma patients.Int J Cancer 2006;119:1732–5.



2014




Published OnlineFirst July 7, 2009.Mol Cancer Ther Josée Guirouilh-Barbat, Smitha Antony and Yves Pommier transcription-coupled repair inhibitionreveals the importance of the C ring of trabectedin for

-H2AX foci andγZalypsis (PM00104) is a potent inducer of

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