The antitumor drugs trabectedin and lurbinectedin induce transcription-dependent replication stress and genome instability

1 The antitumor drugs trabectedin and lurbinectedin induce 1 transcription-dependent replication stress and genome 2 instability 3 4 Emanuela Tumini 1, Emilia Herrera-Moyano 1, Marta San Martín-Alonso 1, 5 Sonia Barroso 1, Carlos M. Galmarini 2 and Andrés Aguilera 1 * 6 7 1 Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER, 8 CSIC-Universidad Pablo de Olavide-Universidad de Sevilla, Av. Américo 9 Vespucio 24, 41092 SEVILLE, Spain; 2 PharmaMar, Av. de los Reyes 1, 10 28770 Colmenar Viejo, Spain 11 12 *Corresponding author: Andrés Aguilera, Centro Andaluz de Biología 13 Molecular y Medicina Regenerativa-CABIMER; Av. Américo Vespucio 24, 14 41092 SEVILLE, Spain. Phone: +34 954468372. E-mail: [email protected] 15 16 Running title: ET743 and PM01183 and RNA-dependent DNA damage 17 18 Keywords 19 Trabectedin, lurbinectedin, R-loops, genome instability, cancer 20 21 Conflict of interests statement 22 Dr C.M. Galmarini is an employee and shareholder of PharmaMar. The 23 remaining authors declare no conflict of interest. 24 25 2 ABSTRACT 26 27 R-loops are a major source of replication stress, DNA damage and genome 28 instability, which are major hallmarks of cancer cells. Accordingly, growing 29 evidence suggests that R-loops may also be related to cancer. Here we 30 show that R-loops play an important role in the cellular response to 31 trabectedin (ET743), an anticancer drug from marine origin and its derivative 32 lurbinectedin (PM01183). Trabectedin and lurbinectedin induced RNA-DNA 33 hybrid-dependent DNA damage in HeLa cells, causing replication 34 impairment and genome instability. We also show that high levels of R-loops 35 increase cell sensitivity to trabectedin. In addition, trabectedin led to 36 transcription-dependent FANCD2 foci accumulation, which was suppressed 37 by RNase H1 overexpression. In yeast, trabectedin and lurbinectedin 38 increased the presence of Rad52 foci, a marker of DNA damage, in an R- 39 loop-dependent manner. In addition to providing new insights into the 40 mechanisms of action of these drugs, our study reveals that R-loops could 41 be targeted by anticancer agents. Given the increasing evidence that R- 42 loops occur all over the genome, the ability of lurbinectedin and trabectedin 43 to act on them may contribute to enhance their efficacy, opening the 44 possibility that R-loops might be a feature shared by specific cancers. 45 Implications: The data presented in this study provide the new concept that 46 R-loops are important cellular factors that contribute to trabectedin and 47 lurbinectedin anticancer activity. 48 49 50 3 51 Introduction 52 53 Cancer therapy greatly relies on the use of small molecules that directly 54 perturb the DNA metabolism. Trabectedin (ET743) is one of such molecules. 55 It has been initially derived from the sea squirt Ecteinascidia turbinata and is 56 now produced synthetically, being currently employed in therapy of patients 57 with advanced soft tissue sarcoma (1) and platinum-sensitive ovarian cancer 58 (2). Different derivatives of trabectedin have been synthesized, among them 59 lurbinectedin (PM01183), which is presently under evaluation in phase II and 60 III clinical trials. 61 One of the most important features of the mechanism of action of 62 trabectedin and lurbinectedin is their inhibitory effect on transcription of 63 protein-coding genes that is mediated by different means. First, they 64 preferentially bind to specific DNA triplets that are often present on 65 transcription recognition sites. In this way they could directly prevent loading 66 of transcription factors onto chromatin (3). Moreover, both drugs were shown 67 to induce degradation of the RNA polymerase II (RNAPII) through the 68 ubiquitin-proteasome pathway (4-8). Of note, these effects are also 69 observed in tumor-associated macrophages (TAMs). Indeed, both drugs 70 inhibit the transcription of selected cytokines (e.g. CCL2, IL6, IL8, PTX3) by 71 TAMs abrogating their protumoral properties and modifying the tumor 72 microenvironment (9, 10). 73 Tumor cells are commonly associated with genome instability. One 74 important natural source of instability is represented by R-loops, a three- 75 4 stranded nucleic acid structure consisting of an RNA-DNA hybrid and the 76 displaced single-stranded DNA of the original DNA duplex (11). This 77 structure could fulfill physiological roles as it occurs during immunoglobulin 78 class switching recombination, plasmid or mitochondrial DNA replication and 79 transcription regulation. On the other hand, they are an important source of 80 DNA damage, genome instability and replication stress, which relates R- 81 loops with a number of neurodegenerative disorders and cancer (12, 13). 82 Interestingly, the loss of function of the breast cancer susceptibility genes 83 BRCA1 and BRCA2, which have been shown to increase R-loops and R- 84 loop-dependent DNA damage (14, 15), is also associated with an increased 85 sensitivity to trabectedin (16). In a similar way, dysfunctions of the Fanconi 86 anemia pathway that increased sensitivity to such agent (5) also cause 87 accumulation of R-loops (17, 18). 88 In addition, there are some similarities between R-loops and trabectedin 89 adducts worth being considered. The head-to-tail binding of three 90 trabectedin molecules to three adjacent optimal DNA binding sites changes 91 the DNA duplex conformation from the B-form to an intermediate between 92 the A- and the B-form, which strongly resembles the conformation of an 93 RNA-DNA hybrid (19). Moreover, R-loops that accumulate in the absence of 94 the putative RNA-DNA helicases AQR or SETX require the action of 95 Nucleotide Excision Repair (NER) to be processed into Double Strand 96 Breaks (DSBs) (20) as well as trabectedin adducts (21). 97 For these reasons, in this study we have further explored the mechanism 98 of action of trabectedin and lurbinectedin, in particular whether it could be 99 partially mediated or affected by R-loops. We show that the DNA damage 100 5 and replicative stress caused by trabectedin and lurbinectedin is indeed 101 partially dependent on R-loops, as demonstrated by molecular and genetic 102 assays using both human cell lines and the yeast Saccharomyces cerevisiae 103 as a model eukaryotic system. Our findings suggest that R-loops could be 104 targets of anticancer agents and given the evidence that R-loops occur all 105 over the genome (22), this opens the possibility that tumoral cells that 106 accumulate R-loops may be more suitable for trabectedin and lurbinectedin 107 treatment. 108 109 110 Materials and Methods 111 112 Cell culture and treatments 113 HeLa cells were purchased from ECACC (European Collection of Cell 114 Cultures), HeLa-TR (24) and HeLa HB-GFP (14) were previously generated 115 in our laboratory. All cell lines were maintained in DMEM medium, 116 supplemented with 10% heat-inactivated fetal calf serum (FCS), cultured at 117 37ºC in a humidified atmosphere containing 5% CO2 and routinely tested for 118 Mycoplasma using MycoAlert Mycoplasma Detection Kit (Lonza). HeLa HB- 119 GFP cells medium was supplemented with doxycycline at the final 120 concentration of 2 g/ml to induce HB-GFP expression. Specific genes were 121 knocked down using ON-TARGET SMARTpool siRNA from Dharmacon. 122 Transient transfection of siRNA was performed using DharmaFECT 1 123 (Dharmacon) according to the manufacturer’s instructions. The following 124 plasmids were used for transfection: pcDNA3 (Invitrogen) (RNH1-) and 125 6 pcDNA3-RNaseH1 (RNH1+), containing the full length RNase H1 cloned into 126 pcDNA3 (23). Lipofectamine 2000 (Invitrogen, Carlsbad, CA) was used for 127 plasmid transfection. Assays were performed 48h or 72h after transfection. 128 Trabectedin (ET743) and lurbinectedin (PM01183) were provided from 129 Pharmamar. Cordycepin (C3394) and Neocarzinostatin (N9162) were 130 purchased from Sigma. 131 132 Single cell electrophoresis 133 Single cell electrophoresis or comet assays were performed as described 134 using a commercial kit (Trevigen, Gaithersburg, MD, USA) following the 135 manufacturer´s instructions. Comet tail moments were analyzed using Open 136 Comet software. At least 100 cells were scored in each experiment to 137 calculate the median of the tail moment, as reported (24). 138 139 Cell proliferation 140 HeLa cells were siRNA transfected to knock down the specified genes 141 and plated in 96-well plate. Following 72 hours, cells were treated with 142 increasing doses of trabectedin (ET743) for 24 hours and cell proliferation 143 was measured by the WST-1 reagent (Roche) following the manufacture´s 144 instructions. Absorbance was measured using microplate (ELISA) reader 145 VARIOSKAN FLASH (Thermo) at 450 nm with a reference wavelength of 146 690 nm. Absorbance values were normalized to the value of the untreated 147 and represented as arbitrary units (A.U.). 148 149 Antibodies 150 7 Antibodies anti-RNASEH1 (15606-1-AP) and anti-FANCD2 (sc-20022) 151 anti-53BP1 (NB100-304) were purchased from Proteintech, Santa Cruz and 152 Novus Biologicals, respectively. The S9.6 antibody was purified from the 153 hybridoma cell line HB-8730. 154 155 Immunofluorescence and EdU labelling 156 Immunofluorescence was performed as previously described (25). 157 Briefly, cells were fixed with 3.7% formaldehyde in PBS for 15 minutes, 158 permeabilized with 0.5% Triton X-100 and blocked with 3% bovine serum 159 albumin (BSA) in PBS for 1 hour. The coverslips were then incubated with 160 primary antibodies diluted 1:500 in 3% BSA in PBS for 2 hours followed by 1 161 hour incubation with 1:1000 diluted secondary antibodies and nuclei 162 counterstaining with 1 g/ml DAPI in PBS. For FANCD2 immunolabelling, 163 before fixation cells were pre-permeabilized with 0.25% Triton X-100 in PBS 164 for 1 minute on ice. S9.6 immunofluorescence was performed as previously 165 described (20). For the EdU labeling, cells were pulse-labeled with 10 M 166 EdU for 20 minutes before fixation. EdU staining was performed with a Click- 167 iT EdU Alexa Fluor 555 Imaging kit (Invitrogen) according to manufacturer’s 168 instructions. Random images were acquired with a 63X or 40X objective and 169 foci or nuclear intensity were scored using the MetaMorph software. At least 170 100 cells were scored for each condition. 171 172 DNA combing 173 DNA combing was performed as previously described (24). Briefly, DNA 174 fibers were extracted from cells in agarose plugs immediately after CldU 175 8 labeling and were stretched on silanized coverslips. DNA molecules were 176 counterstained with an autoanti-ssDNA antibody (DSHB, 1:500) and an anti- 177 mouse IgG coupled to Alexa 647 (A21241, Molecular Probes, 1:50). CldU 178 and IdU were detected with BU1/75 (AbCys, 1:20) and an anti-rat IgG 179 coupled to Alexa 488 (A21470, Molecular Probes, 1:50) or B44 (Becton 180 Dickinson, 1:20) anti-BrdU antibodies and an anti-mouse IgG coupled to 181 Alexa 546 (A21123, Molecular Probes, 1:50), respectively. DNA fibers were 182 analyzed on a Leica DM6000 microscope equipped with a DFC390 camera 183 (Leica). Data acquisition was performed with LAS AF (Leica). Fork velocity 184 was calculated as previously described (26). Replication asymmetry was 185 calculated by dividing (longest green tract –shortest green tract) by the 186 longest tract in divergent CIdU tracks. 187 188 Yeast ‘halo’ assay 189 Yeast cells of the wild-type W303 or the rad52  strains were plated in 190 presence of 0.003% SDS in order to increase yeast cell permeability. 191 Afterwards, paper filter disks were placed on the agar plates and 3 µL of 192 increasing amounts of trabectedin or lurbinectedin (or DMSO as carrier 193 control) were spotted onto them. After 3 to 6 days cells growth was 194 monitored, plates were scanned and the diameter of the growth inhibition 195 halo was measured. 196 197 Rad52 foci analysis in yeast cells 198 Wild-type W303 yeast cells were transformed in plates containing 199 doxycycline (at the final concentration of 10 g/ml in order to repress the 200 9 expression of ectopic RNase H1) with pWJ1344 plasmid coding the RAD52- 201 YFP fusion protein (27) and with pCM189-RNH1 with the RNH1 gene under 202 the tet promoter (28) or the empty vector pCM189 (29). Cells were grown 203 overnight with medium SC-L-U (Complete medium SC: 0.17% yeast nitrogen 204 base and 0.5% ammonium sulfate, 2% glucose, supplemented with amino 205 acids; –L and -U indicate the absence of leucine and uracil respectively) 206 containing 0.003% SDS and without doxycyclin. Mid-log cultures 207 overexpressing RNase H1 or not, were treated with increasing doses of 208 trabectedin or lurbinectedin during 2 hours at 30ºC with 200 rpm shaking. 209 Following fixation with 2.5% formaldehyde in 0.1M KHPO at pH 6.4 for 10 210 minutes, the cells were washed twice with KHPO 0.1M pH 6.6 and once with 211 KHPO 0.1M pH 7.4. Cells were permeabilized with ethanol 80% for 10 212 minutes and after centrifugation were resuspended in water containing DAPI 213 at the final concentration of 1 g/ml to counterstain the nuclei. Rad52–YFP 214 foci were scored in more than 200 nuclei from S-G2 mid-log cells, using a 215 Leica DC 350F microscope. 216 217 Statistical analysis 218 Statistical significance was determined by t-tests or the Mann-Whitney 219 test as specified. All tests performed were 2-tailed unless otherwise 220 specified and three levels of statistical significance were considered: *p ≤ 221 0.05, **p ≤ 0.01 and ***p ≤ 0.001. 222 223 224 Results 225 16 concentrations of the tested drug were placed on top of the plate. After 376 growth, halos of growth inhibition around the disks were formed with a 377 diameter that correlates with the levels of toxicity of the drug. As can be 378 seen in Fig. 5a, increasing concentrations of trabectedin lead to increased 379 yeast sensitivity, as detected by the size of the halo of growth inhibition 380 around the trabectedin spot. Importantly, this halo was largely increased in 381 rad52  yeast strains inactivated for homologous recombination DSB repair 382 in comparison with the wild-type strain, even at concentrations at which 383 trabectedin had no effect on wild-type cells (Fig. 5a). This result suggests 384 that recombinogenic DNA breaks accumulated after trabectedin treatment, 385 consistent with previous reports (21). To confirm this, we analyzed 386 recombinogenic DSBs by Rad52 foci, used as a way to detect DSB repair 387 centers (27). Consistently, trabectedin caused a significant increase of 388 Rad52 foci. Cells treated with 250 M trabectedin, showed a 3-fold increase 389 in the percentage of cells with Rad52 foci compared to the untreated control 390 (Fig. 5b). To assay whether Rad52 foci were dependent on RNA-DNA 391 hybrids, RNAse H1 was overexpressed in yeast cells by transformation with 392 an RNase H1-overexpressing plasmid. Notably, the increase in Rad52 foci 393 was suppressed by RNase H1 overexpression (Fig. 5b and Supplementary 394 Fig. S6a). These results indicate that trabectedin-induced breaks are linked 395 to the accumulation of RNA-DNA hybrids also in yeast cells. 396 Next, we assayed the effect of lurbinectedin on cell growth and genome 397 instability using the assays described above for trabectedin. As can be seen 398 in Fig. 5c, lurbinectedin affected yeast growth as determined by the size of 399 the growth inhibition halo formed around the increasing concentrations of the 400 17 chemical spotted on Whatman filter papers. Again, this effect was 401 significantly enhanced in rad52∆ strains, consistent with the idea that cell 402 growth was impaired due to the accumulation of unrepaired DSBs. 403 Accordingly, lurbinectedin treatment caused a 2-3-fold increase of cells with 404 Rad52 foci compared to untreated cells and this phenotype was fully 405 suppressed by RNase H1 overexpression (Fig. 5d and Supplementary Fig. 406 S6a). The decreased amount of Rad52 foci after overexpression of RNase 407 H1 in trabectedin- or lurbinectedin-treated cells, was not due to the reduction 408 of the protein levels, as confirmed by western blot (Supplementary Fig. S6b). 409 Consequently, lurbinectedin, as well as trabectedin, caused R-loop 410 dependent genome instability both in yeast and in human cells. This finding 411 suggests that their action is most likely common to many different cell types, 412 since they target molecular pathways that are well conserved across 413 evolution. 414 415 Discussion 416 417 Increasing evidence points out the role of RNA-DNA hybrids in the boost 418 of genome instability and replication stress that are major hallmarks of 419 cancer cells. This opens the possibility that such hybrids could occur at high 420 levels in some tumor cells and consequently could be used as therapeutic 421 targets. For this reason, it is of growing importance to address the 422 relationship between R-loops and the action of anticancer agents. 423 Trabectedin and lurbinectedin are two powerful drugs used in cancer 424 treatment whose ability to block cell proliferation is linked to its potential to 425 18 cause DNA breaks and inhibit transcription. In this study, we show that 426 trabectedin and lurbinectedin activities are favored by transcription and, at 427 least partially, they might be enhanced by R-loops. Trabectedin and 428 lurbinectedin treatment enhanced transcription-dependent genome instability 429 and replication stress (Figs. 1 and 2). Exposure to trabectedin or 430 lurbinectedin did not cause a global increase in R-loops as assayed by S9.6 431 immunofluorescence (Supplementary Fig. S3). However, these drugs 432 caused genome instability and replicative stress that were dependent on 433 transcription and RNA-DNA hybrids both in yeast and human cells. These 434 findings suggest that trabectedin and lurbinectedin toxicity is higher at sites 435 undergoing active transcription and R-loop accumulation. 436 It was previously demonstrated that trabectedin and lurbinectedin directly 437 bind DNA, forming adducts (6) and distort the double helix. Here we showed 438 that these DNA adducts compromise replication fork progression; indeed, we 439 found that trabectedin caused a profound impairment of DNA synthesis (Fig. 440 2a). To some extent, we can attribute this effect to R-loop formation, 441 because RNase H1 overexpression partially suppressed this DNA synthesis 442 defect. RNase H1 specifically cleaves the RNA strand of RNA-DNA hybrids 443 therefore, overexpression of this endoribonuclease counteracts the 444 accumulation of RNA-DNA hybrids. However, the rescue of the EdU 445 incorporation defect by RNase H1 overexpression was mild, which suggests 446 that only a subset of the trabectedin-induced replicative problems were 447 mediated by RNA-DNA hybrids. Analysis of DNA replication at the single 448 molecule level by DNA combing revealed that transcription inhibition with 449 cordycepin reduces the increase in fork asymmetry provoked by trabectedin 450 19 exposure (Fig. 2b), consistent with the conclusion that R-loops contribute to 451 fork stalling in trabectedin treated cells. In any case, we cannot exclude the 452 possibility that the inhibition of transcription by trabectedin could contribute 453 to RF asymmetry. 454 R-loop-dependent replication problems caused by trabectedin are 455 possibly counteracted by the Fanconi Anemia (FA) pathway. The FA 456 pathway has a key role in balancing replication stress, in particular when 457 inter-strand crosslinking agents such as Mitomycin C (MMC) cause 458 replication fork blockage (37) and has been shown to limit R-loop 459 accumulation and R-loop-dependent genome instability (17, 18). 460 Interestingly, FA is critical for the integrity of common fragile sites (CFSs), 461 since FANCD2, a central player of the FA pathway, is required for a safe 462 replication through R-loop-accumulating CFSs (38). Here, we found an 463 increased accumulation of FANCD2 foci after trabectedin treatment that, 464 importantly, was fully rescued by RNase H1 overexpression (Fig. 1c). This 465 phenotype was different to that caused by other genotoxic agents. Thus, 466 contrary to trabectedin, overexpression of RNase H1 in cells treated with the 467 radiomimetic agent neocarzinostatin (NCZ) exacerbated FANCD2 foci 468 accumulation (Supplementary Fig. S2). This may be explained if FANCD2 469 foci induced by NCZ are not RNA-DNA hybrid-dependent, and RNase H1 470 overexpression causes an additional type of replication stress. This finding 471 adds further support to the conclusion that trabectedin has a prominent and 472 specific action on R-loops where it preferentially causes DNA damage and 473 breaks able to block replication fork progression. Consistently, using a cell 474 line system where R-loops were stabilized by the ectopic expression of the 475 20 RNA-DNA hybrid binding domain of the RNase H1 enzyme fused to GFP, 476 and additionally increasing the basal levels of R-loop through the siRNA- 477 mediated depletion of THOC1 or BRCA2, we determined that cells with 478 higher levels of R-loops were more sensitive to trabectedin treatment (Fig. 479 3). 480 Importantly, we were able to recapitulate the phenotype of R-loop- 481 dependent genome instability caused by trabectedin and lurbinectedin in 482 yeast cells. Rad52 foci induced after treatment of either trabectedin or 483 lurbinectedin were rescued by RNase H1 overexpression (Fig. 5). This result 484 strongly suggests that the preferred action of these anti-tumoral agents on 485 DNA is independent of the eukaryotic system or cell type, arguing strongly in 486 favor of a preferential action on the R-loop-containing chromatin. In HeLa 487 cells exposed to these agents, the RNA-DNA hybrid-dependent DNA 488 damage phenotype was not so clear as it was in yeast cells. However, we 489 were able to show by comet assay and 53BP1 immunofluorescence that the 490 DNA damage induced by trabectedin or lurbinectedin was partially rescued 491 by RNase H1 overexpression (Fig. 4 and Supplementary Fig. S1). Besides, 492 we found that H2AX foci accumulation after trabectedin treatment was 493 further increased in cells overexpressing RNase H1 (Supplementary Fig. 494 S5a). However, even if phosphorylated H2AX on Ser139 is used as a DSB 495 marker, it also denotes replication stress, since it is extended through a large 496 chromatin domain as an early response to replication fork stalling in an ATR- 497 dependent process (39). RNase H1 overexpression could cause replication 498 stress and instability, likely because it hampers replication by altering 499 Okazaki fragment dynamics. Accordingly, DNA synthesis was impaired in 500 21 cells overexpressing RNase H1 (Fig. 2a). The double treatment with RNase 501 H1 overexpression and trabectedin might have an additive effect by their 502 impact on replication, enhancing the probability of ssDNA gaps or breaks 503 that would lead to increased cell death (Supplementary Fig. S5b). 504 The novel finding of an interplay between R-loops and trabectedin in 505 genome instability induction, is of particular interest because it could be 506 exploited for cancer cells in which R-loops might be a common feature. The 507 observation that trabectedin toxicity is augmented by the dysfunction in 508 pathway that have been shown to protect both from cancer and from R-loops 509 accumulation, such as the Fanconi anemia pathway and the BRCA1 and 510 BRCA2 tumor suppressor genes (14, 15, 17, 18), supports the idea that R- 511 loops might be a hallmark of specific types of cancer (40). It has been shown 512 that cancer patients with BRCA mutations exhibit improved clinical response 513 to trabectedin (16). It would be interesting in the future to check if R-loops 514 are increased in tumoral cells derived from such patients to strengthen our 515 hypothesis that a subset of tumors characterized by an higher accumulation 516 of R-loops are more suitable to be treated with trabectedin. 517 518 Disclosure of Potential Conflicts of interests 519 Dr C.M. Galmarini is an employee and shareholder of PharmaMar. 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