DNA topoisomerase IIß inhibition blocks DNA end resection and synergizes with PARPi in BRCA1-deficient models

DNA topoisomerase IIß inhibition blocks DNA end resection and synergizes with PARPi in BRCA1-deficient models Rosa Camarillo a,b,1,2 , Rosario Prados-Carvajal a,b,1 , Andr´ es Cruz-García a,b,3 , Guillermo Rodríguez-Real a,b,4 , Andrea Herencia-Ropero c,d , Violeta Serra c , Sonia Jimeno a,b,* , Pablo Huertas a,b,* a Facultad de Biología, Universidad de Sevilla, Sevilla 41080, Spain b Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER, Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, Sevilla 41092, Spain c Experimental Therapeutics Group, Vall d ′ Hebron Institute of Oncology, Barcelona, Spain d Department of Biochemistry and Molecular Biology, Autonomous University of Barcelona, Barcelona, Spain ARTICLE INFO Keywords: Recombination Topoisomerase II DNA-end resection BRCA1 Cancer Personalized medicine ABSTRACT DNA end resection is a critical step that governs how a broken chromosome will be repaired. As such, it is heavily regulated by multiple cellular signals and processes. Alterations in the regulation of DNA end resection have consequences for cell survival upon exposure to cytotoxic agents, including those used during cancer chemotherapy. Here, we identified several small molecules that affect the process of DNA end resection. Among them, we focus on determining the mode of action of merbarone, a DNA topoisomerase II inhibitor. We uncover a role of the topoisomerase IIβ isoform in the full processing of DNA breaks. Moreover, we show that the effect of merbarone is affected by the formation of G4 quadruplexes and that BRCA1-deficient cancer cells are sensitive to merbarone. Strikingly, this sensitivity can be partially suppressed in cell lines expressing hypomorphic versions of BRCA1 lacking exon 11, a hypomorph that has been linked to PARPi-resistance. Using cellular models, we show that PARPiand merbarone-resistant BRCA1 exon 11 mutant cells, but not wildtype BRCA1 cells, are sensitive to the combination of both drugs. Finally, we show that combination of merbarone and the PARPi olaparib has a mild antitumor effect in a PARPi-resistant PDX model bearing a BRCA1 exon 11 mutation. 1. Introduction Genomes have an intrinsic level of instability as a natural consequence of the exposure to cellular and environmental threats [1]. To minimize the accumulation of mutations, cells have developed mechanisms that detect, signal and repair different kinds of DNA damage [2]. Among the different DNA lesions, Double Strand Breaks (DSBs) are the most cytotoxic and most difficult to repair. DSB repair is a complex process that can be achieved by several different mechanisms, collectively categorized into two branches depending on whether or not an homologous template has to be used: Homologous Recombination (HR; [3]) or Non-Homologous End Joining (NHEJ; [4]). The choice between repair pathways occurs at the level of DNA-end resection [5]. This mechanism consists of 5 ´to 3 ´degradation of one strand at each side of the break, producing ssDNA, that is immediately covered by the RPA protein complex [5,6]. Resected DNA ends inhibit NHEJ, but are needed Abbreviations: IR, Irradiation; MER, Merbarone; PDX, Patient Derived Xenografts; PYR, Pyridostatin; TOPOII α , Topoisomerase II α ; TOPOIIβ, Topoisomerase IIβ; NHEJ, Non-Homologous End Joining; HR, Homologous Recombination; MMEJ, Microhomology-Mediated End Joining. * Correspondence to: CABIMER, Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, Sevilla 41092, Spain. E-mail addresses: [email protected] (S. Jimeno), [email protected] (P. Huertas). 1 *These authors equally contributed to this manuscript. 2 Present address: MRC Laboratory of Medical Sciences, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN. 3 Present address: Institute of Clinical Genetics, University Hospital Carl Gustav Carus, National Center for Tumor Diseases, German Cancer Research Center, Dresden, Germany. 4 Present address: University of Cologne, Faculty of Medicine and University Hospital Cologne, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf; Center for Molecular Medicine Cologne; CECAD Center of Excellence on Cellular Stress Responses in Aging-Associated Diseases, 50931 Cologne, Germany. Contents lists available at ScienceDirect DNA Repair journal homepage: www.elsevier.com/locate/dnarepair https://doi.org/10.1016/j.dnarep.2025.103866 DNA Repair 152 (2025) 103866 Available online 27 June 2025 1568-7864/© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/bync-nd/4.0/ ). (although to different extents) for HR and Microhomology-Mediated End Joining (MMEJ) [5]. DNA transactions usually require the movement of molecular machines on DNA. Due to the double stranded nature of the DNA molecule, in many cases, such processes create topological changes, known as DNA supercoiling, in their wake [7]. Normally, DNA supercoiling is eliminated by a specific class of enzymes, known as topoisomerases, that during their normal cycle create transient breaks in the DNA and later religate them after supercoiling has been eliminated without the need to engage the DNA repair machinery [8]. Topoisomerases are divided into two categories: type I, enzymes that transiently create single strand breaks and type II, enzymes that transiently produce double strand breaks [9]. Two isoforms of topoisomerase II exist in human cells, termed TOPOII α and TOPOIIβ [10]. These two isoforms display similar structural features and catalytic properties, but their distinct functional activities are not yet understood [10]. As DNA topoisomerases maintain the topology of DNA, their participation in DNA repair cannot be underestimated. Indeed, topoisomerase II has been implicated in the cellular response to DNA damage. For instance, radiation response is altered in topoisomerase II-depleted cells [11]. Moreover, TOPOIIβ has been reported to play a role in promoting DSB repair following peroxide damage [12]. A previous study in human fibroblasts suggested a role for TOPOIIβ in UV-induced DNA excision repair [13]. It has also been reported that abasic DNA lesions can recruit both TOPOII α and TOPOIIβ to those specific DNA lesions [14]. Moreover, an increase in DNA damage accumulation was observed in TOPOIIβ knock-out cells after treatment with genotoxic agents [15]. It has also been demonstrated that TOPOIIβ is essential in Ku70-dependent and PARP1-dependent DSB repair pathways in primary cerebellar granule neurons [16]. Indeed, the decreasing activity of TOPOIIβ with age has been associated with its possible role in the decline of DNA repair activity in neurons during aging [17]. Moreover, the isoforms TOPOII α and TOPOIIβ have distinct expression patterns across different cancer types. TOPOII α is frequently upregulated in rapidly proliferating tumor cells, such as those found in breast, ovarian, and lung cancers [18,19]. Conversely, the expression of TOPOIIβ tends to be more heterogeneous and context-dependent, with certain tumors exhibiting elevated levels compared to normal tissues, while others display reduced or negligible expression (source TCGA). Importantly, the topoisomerase II poisons (small molecules that trap topoisomerases in the middle of the catalytic cycle, hence creating DSBs) or catalytic inhibitors (that do not create breaks) are commonly used to treat both hematological and solid tumors [20-22]. Unfortunately, the treatment with topoisomerase poisons could lead to the appearance of genomic translocations, which in turn can lead to secondary malignancies [22]. Proper detection and repair of DSBs ensures that most chemical and physical changes on DNA have little consequence as they are very efficiently removed by DNA repair mechanisms. Indeed, if DSBs are not properly repaired, this can lead to mutagenesis, genome instability and diseases such as cancer [23,24]. As a matter of fact, increased mutagenesis is a common hallmark of cancer. Given the importance of DNA repair in tumor progression, there are many anti-cancer therapies based on the reduced capability of some tumor cells to cope with an exogenous source of DNA damage due to defective repair. Indeed, all radiotherapies and many targeted and general chemotherapies are effective only because cancer cells are more sensitive to DNA damaging agents. A paradigmatic example is the targeted therapy using Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) for BRCA1or BRCA2-related tumors [25], that are currently approved for treatment of ovarian, prostate, breast and pancreatic cancer. Unfortunately, the development of resistance to PARPis is common and a big concern [26]. Therefore, it is very important to discover new drugs that might impair a specific route of DSB repair in order to improve treatment outcomes from both monotherapy or combination therapy for different tumor-types. To discover new drugs that may impact DNA repair by HR, we have used three drug-libraries (containing a total of 552 compounds) to carry out a screen for new drugs that cause changes in DNA resection efficiency (provoking either upor down-regulation of this process). We identified 13 drugs that decrease DNA resection efficiency and 26 drugs with the opposite effect. Among them, we have further studied the effect of the Topoisomerase II catalytic inhibitor, merbarone (MER), and we have demonstrated that TOPOIIβ is involved in proper DNA-end resection upon DNA damage. Furthermore, we have observed that MER is especially efficient in blocking resection in a BRCA1-defective background. Interestingly, BRCA1-mutant cells that have developed resistance to PARPi become re-sensitised to PARPi when co-treated with MER. Similar results, albeit to a more limited extent, are observed in vivo in BRCA1-defective patient-derived xenograft (PDX) mouse models resistant to PARPi. 2. Experimental procedures 2.1. Cell lines and growth conditions U2OS and MEF cell lines were grown in DMEM (Sigma-Aldrich). MDA-MB-436 complemented with RAD51B and expressing either wildtype BRCA1, a BRCA1 exon 11 mutant or lacZ as a negative control were provided by AstraZeneca and were grown in RPMI (SigmaAldrich). In all cases, media were supplemented with 10 % fetal bovine serum (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), 100 units/ ml penicillin and 100 μ g/ml etomycin (Sigma-Aldrich). Cells were routinely screened for mycoplasma infection. All cells were last authenticated in June 2024 by the GenePrint® 10 System (Promega, Madison, WI, USA), and data were analyzed using genemapper® id-x v1.2 software (Applied Biosystems, Waltham, MA, USA) at the genomic core facility of the Instituto de Investigaciones Biomedicas SolsMorreale. 2.2. Screening In all cases, 6000 U2OS cells were plated per well in 96-well plates (6005550, PerkinElmer). One day after seeding, control wells were transfected with CtIP siRNA. The medium was changed after 6 h. 72 h after seeding, cells were treated for 1 h either with the indicated drugs (1 μ M) from three Selleckchem libraries used in this study (L3000, L1400 and L2900, Selleckchem) or with the controls DMSO, H2O or MLN2449. Then, cells were irradiated with 10 Gy and kept at 37ºC for 1 h. Plates were then washed once with PBS followed by treatment with preextraction buffer (25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl2, 300 mM sucrose and 0.2 % Triton X-100) for 5 min on ice. Then, cells were fixed with 4 % paraformaldehyde (Santa Cruz) for 15 min on ice. After two washes with PBS, cells were blocked with blocking solution (5 % FBS in PBS) for 1 h at RT. Primary antibodies against RPA and ϒ-H2AX (Table S2) were incubated for 2 h at RT, washed with PBS and then incubated with secondary antibodies (Table S3) for 1 h at RT in the dark. Then, cells were washed with PBS, stained with Hoechst (861405, Sigma) and washed with PBS. Finally, plates were imaged using an ImageXpress Micro (Molecular Devices) at 40X magnification, and blue, green and Texas red filters were used to detect nuclei, ϒ-H2AX and RPA signals respectively. Images were analyzed, and the number of RPA foci per cell was quantified automatically with the Image MetaExpress Software (Molecular Devices). To facilitate the comparison between experiments, this ratio was normalized with each DMSO or H 2 O control. Conditions that skewed the balance towards increased HR repair resulted in a fold increase above 1.5. In contrast, a net decrease of this ratio (values below 0.85) represented an imbalance towards NHEJ. Data represents a minimum of two sets of experiments. 2.3. siRNAs, plasmids and transfections siRNA duplexes were obtained from Sigma-Aldrich or Dharmacon (Table S1) and were transfected using RNAiMax Lipofectamine Reagent R. Camarillo et al. DNA Repair 152 (2025) 103866 2 Mix (Life Technologies), according to the manufacturer’s instructions. Plasmid transfection of U2OS cells was carried out using FUGENE 6 Transfection Reagent (Promega) according to the manufacturer’s protocol. 2.4. HR and NHEJ analysis U2OS cells bearing a single copy integration of the reporters DR-GFP [27], SA-GFP (SSA)[28] or EJ5-GFP (NHEJ)[28] were used to analyze the different DSB repair pathways. In all cases, 50,000 cells were plated in 6-well plates in duplicate. One day after seeding, cells were transfected with the indicated siRNA and the medium was replaced with fresh media 24 h later. The next day, each duplicate culture was infected with lentiviral particles containing the I-SceI–BFP expression construct at MOI 10 using 8 µg/ml polybrene in 1.5 ml of DMEM. Then, cells were left to grow for an additional 24 h before changing the medium for fresh DMEM. One day later, cells were washed with PBS, trypsinized, neutralized with DMEM, centrifuged for 5 min at 700 g, fixed with 4 % (w/v) paraformaldehyde for 20 min and collected by centrifugation. Then, cell pellets were washed once with PBS before resuspension in 150 µl of PBS. Samples were analysed with a BD FACSAria with the BD FACSDiva Software v5.0.3. Four different parameters were considered: side scatter (SSC), forward scatter (FSC), blue fluorescence (407 nm violet laser BP, Filter 450/40), green fluorescence (488 nm blue laser BP Filter 530/30). Finally, the number of green cells from at least 10,000 events positives for blue fluorescence (infected with the I-SceI–BFP construct) was scored. The average of both duplicates was calculated for each sample of every experiment. To facilitate the comparison between experiments, this ratio was normalized with siRNA control. At least four independent experiments were carried out for each condition and the average and standard deviation is represented. 2.5. SDS-PAGE and western blot analysis Protein extracts were prepared in 2 ×Laemmli buffer (4 % SDS, 20 % glycerol, 125 mM Tris-HCl, pH 6.8) and passed 10 times through a 0.5 mm needle–mounted syringe to reduce viscosity. Proteins were resolved by SDS-PAGE and transferred to low fluorescence PVDF membranes (Immobilon-FL, Millipore). Membranes were blocked with Odyssey Blocking Buffer (LI-COR) and blotted with the appropriate primary antibody and infra-red dyed secondary antibodies (LI-COR) (Table S2, S3). Antibodies were prepared in blocking buffer supplemented with 0.1 % Tween-20. Membranes were air-dried in the dark and scanned in an Odyssey Infrared Imaging System (LI-COR), and images were analysed with ImageStudio software (LI-COR). 2.6. Immunofluorescence and microscopy For RPA foci visualization, U2OS cells depleted for different proteins, or MEF cells, were seeded on coverslips. For the experiment with merbarone (MER; 5 μ M) or dexrazoxane (1 μ M), drug was added to the plates 30 min before irradiation. At 1 h after irradiation (10 Gy), coverslips were washed once with PBS followed by treatment with pre-extraction buffer (25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl 2 , 300 mM sucrose and 0.2 % Triton X-100) for 5 min on ice. Cells were fixed with 4 % paraformaldehyde (w/v) in PBS for 20 min. Following two washes with PBS, cells were blocked for 1 h with 5 % FBS in PBS, co-stained with the appropriate primary antibodies (Table S2) in blocking solution overnight at 4ºC or for 2 h at room temperature, washed again with PBS and then co-immunostained with the appropriate secondary antibodies (Table S3) in blocking buffer. After washing with PBS and dried with ethanol 70 % and 100 % washes, coverslips were mounted into glass slides using Vectashield mounting medium with DAPI (Vector Laboratories). RPA foci immunofluorescences were analysed using a Leica Fluorescence microscope. 2.7. SMART (single-molecule analysis of resection tracks) SMART was performed as described [29]. Briefly, cells were grown in the presence of 10 μ M BrdU for 24 h. Pretreatment with inhibitors was performed as described in the previous section. Cultures were then irradiated (10 Gy) and harvested after 1 h. Cells were embedded in low-melting agarose (Bio-Rad), followed by DNA extraction. DNA fibers were stretched on silanized coverslips, and immunofluorescence was carried out to detect BrdU (Table S2). Samples were observed with a Nikon NI-E microscope, and images were taken and processed with the NIS ELEMENTS Nikon Software. For each experiment, at least 200 DNA fibers were analyzed, and the length of the fibers was measured with Adobe Photoshop CS4. For the topoisomerase II catalytic inhibition or mock treatment, the drugs were added 1 h prior to irradiation. 2.8. Cell cycle analysis Cells were trypsinized and fixed with cold 70 % ethanol overnight, incubated with 250 μ g/ml RNase A (Sigma) and 10 μ g/ml propidium iodide (Fluka) at 37ºC for 30 min and analysed with a FACSCalibur (BD). Cell cycle distribution data were further analysed using ModFit LT 3.0 software (Verity Software House Inc). 2.9. Immunoprecipitations U2OS cells expressing GFP or GFP-PIF1 were harvested in lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5 % NP-40, 1x protease inhibitors [Roche] and 1x phosphatase inhibitor cocktail 1 [Sigma]) and incubated for 30 mins on ice with Benzonase (70746–3, Sigma). Protein extract (1 mg) was mixed with 35 µL of washed magnetic anti-GFP beads (GFP-Trap_M, Chromotek) and incubated 2 h at 4ºC with gentle rocking. For the incubation NP-40 concentration is reduced to 0.2 %. Beads were then washed 3 times with wash buffer (10mMTrisHCl pH 7.5, 150mMNaCl, 0.5 mM EDTA, 0.01 % NP-40, 1x protease inhibitors [Roche] and 1x phosphatase inhibitor cocktail 1 [Sigma]), and the precipitate was eluted in SDS sample buffer by boiling the beads and loaded onto a gel. 2.10. Clonogenic cell survival assays Clonogenic assays were performed by seeding 3.000 MDA-MB-436 cells into 24-well plates in triplicate. Merbarone as added to the culture medium at various concentrations (2.5 µM, 5 µM, 10 µM, 15 µM, 20 µM, and 25 µM). After incubating the cells for 2 h, they were treated with either olaparib (AZD2281, AstraZeneca) at concentrations of 0.1 µM, 0.33 µM, or 1 µM, or with vehicle control (DMSO). Cells were then incubated at 37◦C for a period of 14 days to allow colony formation and for drugs to act. Once colonies had formed, cells were stained using a solution containing 0.5 % crystal violet (Merck) and 20 % ethanol (Merck). After staining, the solution was removed, and the plates were rinsed with water to wash off excess dye. The stained plates were scanned, and the dye was solubilized by adding 300 µL of 10 % acetic acid to each well. The plates were then shaken for 15 min to fully dissolve the dye. Next, 100 µL of the resulting solution was transferred to a 96-well plate with a clear bottom, and the optical density (OD) was measured at 590 nm. A blank was subtracted from the OD readings as a control to correct for any background signal. To normalize the data, the well coverage was standardized to the DMSO control for each plate and multiplied by 100 to calculate the percentage coverage relative to DMSO. The dose-response curves were plotted using GraphPad Prism, and non-linear regression (log(inhibitor) vs. normalized response - variable slope) was used to determine the IC50 values R. Camarillo et al. DNA Repair 152 (2025) 103866 3 2.11. Generation of PDX models and in vivo treatment experiments A fresh tumor sample of 30–60 mm 3 from a patient with a triple negative breast cancer harboring a mutation in exon 11 of BRCA1 was collected for implantation into the lower flank of 6-week-old female athymic HsdCpb:NMRI-Foxn1nu (Janvier) mice. This procedure was performed under a protocol approved by the Ethics Committee for Clinical Research (PR(AG)484/2017) and an associated written informed consent. Experiments were conducted following the European Union’s animal care directive (2010/63/EU) and were approved by the Ethical Committee of Animal Experimentation of the Vall d’Hebron Research Institute. After implantation, animals were continuously supplemented with 1.5 µM 17β-estradiol (Sigma-Aldrich) in their drinking water. Upon growth of the engrafted tumors, flash-frozen and formalin-fixed, paraffin embedded (FFPE) samples were taken for genotyping and histological analyses. To evaluate the sensitivity to the drugs, tumor-bearing mice were equally distributed into treatment groups with tumors ranging 100–300 mm. Olaparib was administered orally (p.o) six times per week in 10 % v/v DMSO, 10 % w/v Kleptose [HP-β-CD] at 100 mg/kg. Merbarone was administered p.o five times per week in 10 % v/v DMSO, 40 % v/v PEG400, 5 % v/v Tween 80 at 40 mg/kg. The combination was administered following the same schedules as in the monotherapy arms. Tumor growth was measured with caliper bi-weekly from first day of treatment. In all experiments, mouse weight was recorded twice weekly. Tumor volume was calculated as V =4 π /3⋅L⋅l2, “L” being the largest diameter and “l” the smallest. Mice were euthanized according to humane endpoints, e.g. when tumors reached 1500 mm or in case of severe weight loss, in accordance with institutional guidelines. The antitumor activity in therapy-resistant tumors was determined by comparing individual tumor volume at 21 days to its baseline: % tumor volume change =(V 21days -V initial )/V initial ⋅ 100. For therapy sensitive tumors, the best response was defined as the minimum value of % tumor volume change sustained for at least 10 days. To classify the overall response of each PDX, we modified the RECIST (mRECIST) criteria, to be based on the mean % tumor volume change: complete response (CR), best response<-95 %; partial response (PR), −95 %<best response<-30 %; stable disease (SD), −30 %<best response<+20 %, progressive disease (PD), best response>+20 %. 2.12. Statistical analysis Statistical significance was determined with a Student’s t-test or ANOVA, as indicated, using PRISM software (Graphpad Software Inc.). Statistically significant differences were labelled with one, two or three asterisks if P <0.05, P <0.01 or P <0.001, respectively. 3. Results 3.1. Screening for drugs that affect DNA end resection Given the impact of DNA repair pathways in cancer development and evolution, we carried out a screen to identify drugs that might have an impact in DNA repair pathway choice. Due to its importance in this process, we focused on the DNA end resection step. Therefore, we set up a system to measure RPA foci formation as a proxy for DNA end resection using 96-well plates and high-throughput microscopy in U2OS cells after treating cells with three different drug libraries that contained either FDA-approved drugs or natural compounds (see Material and Methods for details) (Fig. 1A). After 1 h of drug treatment, we exposed cells to 10 Gy of γ–irradiation and incubated them for an extra hour to allow resection to proceed. Then, we carried out immunofluorescence experiments to measure the accumulation of RPA at sites of DNA double strand breaks (DSBs). To perform the screen, we included several controls in each plate: first, the vehicles of the drugs used (either DMSO or water), which were used to normalize the results in each case; the neddylation inhibitor drug (MLN2449) that we have previously demonstrated stimulates DNA resection [30] and, finally, we also included an siRNA depleting the bona fide DNA resection factor, CtIP [5]. The experiment was repeated independently twice. We ranked the effect on RPA foci formation of the 552 drugs tested, and we observed that 61 increased DNA end resection (Fig. 1B, green ellipse) whereas 62 decreased DNA resection (Fig. 1B, red ellipse). The selected candidates (those with RPA foci formation efficiency over 1.5 or below 0.85 of the levels obtained with the mock treatment in both repeats) are listed in Table 1. A representation of the numeric results obtained for those candidates and of the indicated controls (green for MLN2449 and red for siRNA against CtIP) are shown in Fig. 1C. Homologous recombination and thus, DNA resection, occurs mainly during S and G2 phases of the cell cycle so, in order to avoid false positives due to the accumulation of cells in either G1 or S/G2 and confirm a real effect in the DNA resection process, we studied the cell cycle profile after the treatment with each of the selected candidate drugs and observed no significant changes (Suppl. Fig. S1A). Then, we repeated the RPA foci experiments upon treatment with the selected candidates in a selective, more controlled, condition. We used computer-scoring of RPA foci in immunofluorescence images. As can be seen in Fig. 1D, we were able to reproduce most, but not all, of the effects obtained in the screen and those results were statistically significant. The induction of DNA damage can be considered one of the most successful approaches in cancer treatment. That is why many drugs that cause DNA damage are used as chemotherapeutic agents. Therefore, deregulation of components of the DDR (i.e DNA repair) may contribute to increase the effectiveness of several genotoxic agents used as cancer treatments [31]. Not surprisingly, most of the candidates which induced a defect in DNA end resection came from the anti-cancer library. They perform their anti-neoplastic activity by inhibiting key proteins in different pathways, for example: Histone deacetylase (mocetinostat), c-Met (SU11274, BMS777607), proteasome (MLN2238), Chk1/2 (AZD7762), Akt (GSK690693), PI3K (Cal-101) or topoisomerase I (irinotecan HCl Trihydrate) or Topoisomerase II (idarubicin HCl, merbarone). The inhibition of topoisomerases is of great interest for cancer treatment because they can artificially maintain the DNA strand breaks induced by topoisomerases [20,22]. Therefore, topoisomerase inhibitors block the repair of the break and promote DNA damage which, ultimately, would lead to cell death. Indeed, etoposide (VP-16) and doxorubicin are well known anticancer agents [21]. Furthermore, recently it has been observed that one of the isoforms of type I topoisomerase (TOP3α ) is involved in HR [32]. Strikingly, two of the selected drugs that reduced DNA end resection were bona fide topoisomerase II inhibitors (idarubicin and merbarone), suggesting that topoisomerase activity might be required for fully proficient DNA end resection. Both drugs block topoisomerase enzyme activity using different mechanisms. Idarubicin HCl is an anthracycline and it is considered a TOPOII poison because it prevents TOPOII turnover after inducing the DSB, therefore generating DNA damage [33]. Furthermore, it has been approved as a therapeutic agent for certain types of leukemia [34]. In contrast, merbarone (MER) is a catalytic inhibitor of TOPOII and it blocks TOPOII activity before DNA cleavage, so it does not create DNA damage by itself [21]. Since the generation of DSBs at the same time of TOPOII catalytic inhibition could mask the real role of TOPOII in DNA resection after IR-induced formation of breaks, we decided to focus on merbarone as a potential DNA end resection inhibitor and to study the putative role of topoisomerase II in resection. 3.2. Topoisomerase IIβ is required for DNA end resection In agreement with our screen (Fig. 1C-D), MER addition mildly, but significatively, reduced the accumulation of RPA at DNA break sites, upon exposure of cells to 10 Gy IR (Fig. 2A). We confirmed that this effect was due to topoisomerase inhibition by using an alternative small R. Camarillo et al. DNA Repair 152 (2025) 103866 4 (caption on next page) R. Camarillo et al. DNA Repair 152 (2025) 103866 5 molecule with the same effect, dexrazoxane (Fig. 2A). As the cell cycle is a major regulator of DNA end resection, that is limited to S and G2 phases of the cell cycle, we repeated the experiment in cells stained with CENPF. CENPF accumulates during S and G2, rendering three classes of cells: negative, for G1 cells; low, for cells in S phase; and high for cells in G2. As shown in Fig. 2B, the reduction in RPA foci number was observed both in S and G2 cells, but more significantly in the latter. Next, and in order to check whether only resection initiation was impaired or if resection processivity was also compromised, we did SMART experiments (single molecule analysis of resection tracks), a high-resolution technique that measures the actual length of resected DNA in individual DNA fibers [29]. As can be seen in Fig. 2C, inhibition of topoisomerase II catalytic activity by MER, strongly constrained the processivity of resection in IR-induced breaks, as the length of resected DNA was reduced by half in the presence of this small molecule. Again, as is the case for the RPA foci phenotype, this effect was not due to a change in the cell cycle profile of the cells exposed to MER (Suppl. Fig. S1B). As we have already stated in the Introduction, there are two isoforms of topoisomerase II in human cells (TOPOII α and TOPOIIβ) [9,10]. To understand if either or both topoisomerase II isoforms are involved in DNA end resection, we analyzed the putative contribution of each of them to the resection defect phenotype. As seen in Fig. 2D, cells transfected with different siRNAs against TOPOIIβ, but not TOPOII α , showed reduced RPA foci. For an example of depletion efficiency of the protein using the siRNAs see Suppl. Fig. S2A. This effect was not associated to cell cycle changes (Fig. 2E and Suppl. Fig. S2B). We confirmed the involvement in DNA end resection of TOPOIIβ by repeating the RPA foci formation assay in mouse embryonic fibroblasts (MEFs) derived from a TOPOIIβ knockout mouse (Fig. 2F) and by measuring the length of resected DNA after irradiation using SMART experiments in the same cells (Fig. 2G). Therefore, we conclude that TOPOIIβ plays a role in DNA end resection. Despite the lack of a clear RPA defective phenotype observed after TOPOII α depletion with siRNAs, more experiments would be needed to be able to rule out a role for TOPOII α in the DNA end resection process. DNA end resection is an obligatory step in homology-driven DSB repair. Moreover, DNA resection is thought to block NHEJ. Given the defect observed in DNA end resection when TOPOIIβ is depleted, we decided to study the possible effect of TOPOIIβ depletion in DNA recombination, or in NHEJ outcome. As expected, depletion of TOPOIIβ caused a mild defect in Single Strand Annealing (SSA), that, while not statistically significant, shows a clear trend (Fig. 3A). SSA is a simple, Rad51-independent, type of homology-driven repair that relies almost exclusively on DNA end resection [3]. Also, and mimicking the depletion of resection factors such as CtIP, downregulation of TOPOIIβ slightly increased NHEJ, although not in a statistically significant manner (Fig. 3B). Surprisingly, when we measured Rad51-dependent gene conversion no effect was observed upon TOPOIIβ depletion (Fig. 3C). Hence, altogether, our data support a role for TOPOIIβ in the choice between DSB repair pathways, by affecting the extent of DNA end resection, although additional roles downstream of the recombination pathway are likely. Interestingly, re-analysis of a previous genome-wide screen for factors altering the balance between DSB repair pathway choice agreed with the idea that TOPOIIβ, but not TOPOII α , skewed the balance toward NHEJ [35]. 3.3. The effect of Merbarone in DNA end resection is related to PIF1 helicase Recently, several studies have emerged pointing to a role for Topoisomerases in the homeostasis and function of G4s in human cells (reviewed in 34). Given this connection, we wanted to check if there might be a relationship between G4-stabilization and the DNA resection phenotype observed when topoisomerase II is blocked or depleted. Thus, we decided to combine MER with the depletion of PIF1, as a means of stabilizing G4s. As shown in Fig. 4A, an epistatic effect in the RPA foci formation phenotype is observed when we combine MER addition with PIF1 depletion, in agreement with PIF1 and TOPOIIβ working in the same genetic pathway. Moreover, the overexpression of PIF1, which reduces G4 formation, suppressed the DNA end resection defect caused by MER addition (Fig. 4B). Beyond the genetic interaction, we discovered a physical interaction between PIF1 and TOPOIIβ. Using a PIF1GFP fusion protein, we were able to co-immunoprecipitate TOPOIIβ protein (Fig. 4C), suggesting both proteins act together. 3.4. Topoisomerase inhibition synergizes with the resection defect upon BRCA1 depletion With the results presented here, we envision a scenario in which TOPOIIβ is needed to complete DNA end resection over G4s structures, or other atypical DNA structures, within the same genetic pathway as PIF1 helicase. Interestingly, we have shown that PIF1 is also involved with BRCA1 for resection over G4s structure [36], but also it has been shown that TOPOIIβ interacts with and it is regulated by the BRCA1-BARD1 complex during transcription [37]. Thus, we decided to explore the relationship between topoisomerase II inhibition and BRCA1 depletion. Interestingly, the effect in DNA end resection (measured by RPA foci accumulation) after the combination of MER treatment and BRCA1 depletion by shRNA gave a strong synergistic effect (Fig. 5A). Similar results were obtained by inhibiting Topoisomerase II using dexrazoxane (Fig. 5A), reinforcing the idea that TOPOII becomes extremely important for DNA end resection when BRCA1 is absent. To see if this might be relevant in a clinical setup in BRCA1-deficient cancers, we analyzed the possible combined toxic effect of MER with the PARPi olaparib. This drug has been shown to very efficiently and selectively kill HR-defective tumors [38]. We used the breast cancer cell line MDA-MB-436, defective in BRCA1, complemented with different vectors: either a plasmid bearing a wildtype copy of BRCA1 (BRCA1 WT), a plasmid expressing a version of BRCA1 mutated at exon 11 (X11), or a control plasmid harboring lacZ (lacZ). Additionally, our cells always express an ectopic version of RAD51B, as this gene has been shown to be mutated in this genetic background. These cells are sensitive to olaparib if carrying lacZ, whereas they are resistant when a wildtype copy of the BRCA1 is stably transfected into them (Suppl. Fig. S3). Along the same lines, BRCA1-defective tumor cells expressing lacZ were also more sensitive to MER treatment (Fig. 5B). One of the major clinical challenges in the use of olaparib for treating BRCA1-deficient tumors is the appearance of resistance to the drug [26]. Indeed, numerous compounds have undergone thorough investigation when used in conjunction with PARPi to try to overcome the resistance observed in patients [39-41]. Such resistance can be acquired by different means, including the elimination of NHEJ proteins such as Fig. 1. Screening to look for drugs that affect RPA foci formation efficiency (A) Scheme of the workflow of the screening. (B) Representation of the results of the screening. The effect on RPA foci of each drug was normalized to control, treated with DMSO or water depending on the small molecule, and ranked. Red and green ellipses show those treatments that significatively reduced or increase RPA foci formation, respectively. (C) Numeric results of the positive candidates selected from the screening. The number of RPA foci was normalized to the control sample and represented in a logarithmic scale. The media and the standard deviation of the two repeats performed is represented. Cells treated with the neddylation inhibitor MLN2449 (green bar) or siRNA against CtIP (red bar) were used as positive controls. (D) Cells pre-treated for 1 h with the indicated drug or with DMSO or water as a control were irradiated (10 Gy), collected after 1 h and visualized by immunofluorescence to analyze the presence of RPA foci. The mean of the number of RPA foci per cell was normalized to control. The average and standard deviation of three independent experiments is shown. Statistical significance was determined with a Student’s t-test. One, two or three asterisk represents p <0.05, p <0.01 or p<0.005, respectively. R. Camarillo et al. DNA Repair 152 (2025) 103866 6 53BP1, or re-expression of hypomorphic versions of BRCA1 [26]. Among these is the expression of BRCA1 bearing a deletion in exon 11 [26]. Interestingly, stable expression of an exon 11 deleted version of BRCA1 not only completely abolished sensitivity to olaparib (Suppl. Fig. S3) but also alleviated sensitivity to MER (Fig. 5B), suggesting both drugs might cause cytotoxicity in a similar way in BRCA1-deficient backgrounds. Next, we decided to test the putative synergistic effect of both compounds in different BRCA1-backgrounds. As seen in Fig. 5C, in Table 1 Selected candidates description. DRUG FDA STATUS TARGET PATHWAY INFORMATION Arbutin Approved Tyrosinase Proteases Tyrosinase inhibitor Hordenine Not approved   It inhibits the uptake of norepinephrine in mammals. Melanogenesis inhibitor by suppressing cAMP production (S)−10Hydroxycamptothecin Approved Topoisomerase DNA Damage DNA topoisomerase I inhibitor with potent anti-tumor activity. Lisinopril Approved RAAS Endocrinology & Hormones Angiotensin-converting enzyme (ACE) inhibitor. Renin-Angiotensinaldosterone system (RAAS) inhibitor. Deforolimus (Ridaforolimus) Not approved mTOR PI3K/Akt/mTOR Selective mTOR inhibitor Obatoclax mesylate (GX15–070) Not approved Bcl2 Autophagy Bcl2 inhibitor Mocetinostat (MGCD0103) Not approved HDAC Potent HDAC inhibitor SU11274 Not approved c-Met Met inhibitor Dimesna Approved   Uroprotective agent used to decrease urotoxicity. Idarubicin HCl Approved TOPOII DNA topoisomerase II (topo II α ) inhibitor. CAL−101 (GS−1101) Approved PI3K PI3K/Akt/mTOR Selective PI3K class I inhibitor. BMS 777607 Not approved Axl Met-related inhibitor for c-Met, Axl, Ron and Tyro3 MLN2238 Approved Proteasome Proteases Inhibits the chymotrypsin-like proteolytic (β5) site of the 20S proteasome (-)-Epigallocatechin gallate Not approved DNA Methyltransferase, HER2, Telomerase,EGFR, Fatty Acid Synthase DNA Damage Inhibitor of telomerase and DNA methyltransferase. EGCG blocks the activation of EGF receptors and HER−2 receptors. ECGG inhibits fatty acid synthase and glutamate dehydrogenase activity. AZD7762 Not approved CHK Novel CHK1 and CHK2 inhibitor Irinotecan HCl Trihydrate (Campto) Approved TOPOI DNA Damage Topoisomerase I inhibitor. Chrysophanic acid (Chrysophanol) Not approved EGFR EGFR/mTOR pathway inhibitor. GW4064 Not approved FXR Agonist of farnesoid X receptor (FXR) VS−5584 (SB2343) Not approved PI3K PI3K/mTOR inhibitor for mTOR, PI3K α /β/δ/γ Anagrelide HCl Approved PDE Metabolism Treatment of essential thrombocytosis Sotrastaurin (AEB071) Not approved PKC TGF-beta/Smad Potent selective pan-PKC inhibitor and highly inhibits PKCθ CNX−2006 Not approved EGFR Mutant-selective EGFR inhibitor AZ628 Not approved RAF Wild-type c-RAF and B-RAF V600E inhibitor Sirtinol Not approved Sirtuin Specific SIRT1 and SIRT2 inhibtor BAY 11–7082 (BAY 11–7821) Not approved E2 conjugating Inhibits TNF α -induced IκB α and inactivates the E2-conjugating enzymes Ubc (ubiquitin conjugating) 13, UbcH7 and the E3 ligase LUBA Cabozantinib malate (XL184) Approved TAM Receptor,VEGFR Protein Tyrosine Kinase Potent VEGFR2 inhibitor that also inhibits c-Met, Ret, Kit, Flt−1/3/4, Tie2, and AXL IWP−2 Not approved Wnt/beta-catenin Inhibitor of Wnt processing and, selective blockage of Porcn-mediated Wnt palmitoylation. GF109203X Not approved PKC Potent PKC inhibitor PCI−32765 (Ibrutinib) Approved BTK Angiogenesis Inhibitor of the enzyme Bruton’s tyrosine kinase (Btk). CHIR−99021 (CT99021) HCl Not approved GSK−3GSK−3 α and GSK−3β inhibitor GSK690693 Not approved Akt Pan-Akt inhibitor targeting Akt1, Akt2 and Akt3 GSK2606414 Not approved PERK Selective PERK inhibitor Dacomitinib (PF299804, PF−00299804) Not approved EGFR Pan-ErbB inhibitor against ErbB1, ErbB2 and ErbB4 Dalcetrapib (JTT−705) Approved CETP Metabolism rhCETP inhibitor Torin 2 Not approved mTOR/ATM/ATR mTOR inhibitor. Inhibition of ATM/ATR/DNA-PK Romidepsin (FK228, Depsipeptide) Not approved HDAC Potent HDAC1 and HDAC2 inhibitor Merbarone Not approved TOPOII Selective topoisomerase II inhibitor R. Camarillo et al. DNA Repair 152 (2025) 103866 7 a BRCA1-proficient background, MER sensitivity was not increased when combined with different concentrations of olaparib, in agreement with the cells being resistant to both treatments. In contrast, in a BRCA1-deficient background, cells were more sensitive to MER (compare Figs. 5C and 5D), but we did not observe any further sensitization to olaparib (Fig. 5D), likely reflecting the fact that those cells are already sensitive to either treatment alone. Note that only low doses of olaparib were used here, as higher concentrations killed all cells. Strikingly, when we analyzed the combinatorial effect of both drugs in the olapariband MER-resistant exon11 BRCA1 background, a new picture emerged. Indeed, BRCA1 exon 11 deleted cells were sensitive to a combination of both drugs, in a dose-dependent manner (Fig. 5E). However, when no olaparib was added, exon 11 cells were as resistant to MER as cells expressing wildtype BRCA1 (Fig. 5B, compare also Figs. 5C and 5E, DMSO). We wondered if our cellular results translated to in vivo models. We used a Patient-Derived Xenograft (PDX) model from a triple negative breast cancer patient harboring a pathogenic variant in exon 11 of BRCA1, which has been described to confer partial resistance to olaparib by re-expressing an exon 11-deleted hypomorphic protein. In this case, as seen in Fig. 5F, the treatment with olaparib stabilizes the size of the tumor but is not able to eliminate it. MER only treatment had very little effect on tumor growth. Interestingly, we observed a mild but consistent reduction in tumor volume when both drugs were combined (Fig. 5F). Unfortunately, MER treatment was very toxic to mice, likely due to the high doses used, as previously reported in phase I clinical trials in patients [42]. Thus, we had to stop the treatment after three months due to humane reasons. Fig. 2. Merbarone effect on DNA resection (A) Cells pre-treated with the topoisomerase II inhibitors merbarone, dexrazoxane or DMSO as a control were irradiated (10 Gy), collected after 1 h and the presence of RPA foci visualized by immunofluorescence. The average and standard deviation of three independent experiments is shown in the left side. Representative images on the right side. (B) Same as A but represented individually in G1, S or G2 cells according to the levels of CENPF. (C) Media of the length of the resected DNA obtained by the SMART protocol on Merbarone-treated cells. The media and the standard deviation of three experiments is shown. (D) Cells transfected with four different siRNAs against TOPOII α or TOPOIIβ or a control sequence (siNT) were irradiated with 10 Gy, collected after 1 h and the presence of RPA foci visualized by immunofluorescence. Other details as in Fig. 2A. (E) Same as (D), but represented individually in G1, S and G2 according to the levels of CENPF (F) Same as A but in MEFs wildtype or knockout for TOP2β. (G) Media of the length of the resected DNA obtained by the SMART protocol on TOPOIIβ knockout cells. The media and the standard deviation of three experiments is shown. In all cases, statistical significance was determined with a Student’s t-test. One, two or three asterisk represents p <0.05, p <0.01 or p <0.005, respectively. R. Camarillo et al. DNA Repair 152 (2025) 103866 8 4. Discussion Our study highlights the intricate relationship between topoisomerase II β (TOPOIIβ) and DNA end resection, a crucial step in the homologous recombination (HR) pathway of DNA double-strand break (DSB) repair. Through a comprehensive drug screen, we identified several compounds that modulate DNA end resection efficiency. Among these, the topoisomerase II catalytic inhibitor, merbarone (MER), emerged as a particularly significant player. Our data underscore an important role specifically for the isoform TOPOIIβ, but not TOPOII α , in maintaining efficient DNA end resection and DSB repair. The observed differences may arise from their distinct functional roles. TOPOII α is indispensable for resolving topological DNA constraints occurring during DNA replication and transcription, whereas TOPOIIβ is likely to play a more specialized role in DNA repair and the maintenance of genome stability. Along these lines, other studies indicate that TOPOIIβ is necessary not only for the formation of DNA breaks but also for their proper repair, emphasizing the importance of this isoform in maintaining genomic stability and preventing chromosomal abnormalities [43]. These observations support our hypothesis that TOPOIIβ could be crucial in the resection of DNA ends and, therefore, in the repair of DSBs. This distinction is particularly relevant when considering the therapeutic Fig. 3. TOPOIIβ depletion affects DSB repair pathways. (A) Effect of TOPOIIβ depletion in the Single Strand Annealing (SSA) reporter SA-GFP. The induction of a DSB by I-SceI located between two repeats in direct orientation will render GFP positive cells only when intramolecular SSA takes place. CtIP depletion was used as a control (B) Same as (A), but using the NHEJ reporter EJ5-GFP. In this case, two I-SceI-induced DSBs could be repaired by conservative or mutagenic NHEJ granting the accumulation of functional GFP. (C) Same as (A), but using the gene conversion reporter DR-GFP. In this case, the I-SceI-induced DSB could be repaired by conservative gene conversion using a close by truncated GFP sequence. In all cases, statistical significance was determined with a Student’s t-test. One, two or three asterisk represents p <0.05, p <0.01 or p <0.005, respectively. R. Camarillo et al. DNA Repair 152 (2025) 103866 9