ORIGINAL RESEARCH published: 03 September 2021 doi: 10.3389/fcell.2021.715126 Lysine Methyltransferase Inhibitors Impair H4K20me2 and 53BP1 Foci in Response to DNA Damage in Sarcomas, a Synthetic Lethality Edited by: César López-Camarillo, Strategy Universidad Autónoma de la Ciudad de México, Mexico Ignacio Campillo-Marcos 1,2,3† , Eva Monte-Serrano 1,2† , Elena Navarro-Carrasco 1,2† , Reviewed by: Raúl García-González 1,2† and Pedro A. Lazo 1,2* † Jorg Kobarg, 1 Molecular Mechanisms of Cancer Program, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior State University of Campinas, Brazil de Investigaciones Científicas (CSIC)-Universidad de Salamanca, Salamanca, Spain, 2 Instituto de Investigación Biomédica Olivier Binda, de Salamanca (IBSAL), Hospital Universitario de Salamanca, Salamanca, Spain, 3 Cancer Epigenetics Group, Josep Université Claude Bernard Lyon 1, Carreras Leukemia Research Institute (IJC), Barcelona, Spain France *Correspondence: Pedro A. Lazo Background: Chromatin is dynamically remodeled to adapt to all DNA-related pedro.lazo@csic.es processes, including DNA damage responses (DDR). This adaptation requires DNA † ORCID: and histone epigenetic modifications, which are mediated by several types of enzymes; Ignacio Campillo-Marcos orcid.org/0000-0002-7657-7127 among them are lysine methyltransferases (KMTs). Eva Monte-Serrano orcid.org/0000-0002-0865-2244 Methods: KMT inhibitors, chaetocin and tazemetostat (TZM), were used to study Elena Navarro-Carrasco their role in the DDR induced by ionizing radiation or doxorubicin in two human orcid.org/0000-0002-1533-8210 sarcoma cells lines. The effect of these KMT inhibitors was tested by the analysis of Raúl García-González orcid.org/0000-0003-4044-2543 chromatin epigenetic modifications, H4K16ac and H4K20me2. DDR was monitored by Pedro A. Lazo the formation of γH2AX, MDC1, NBS1 and 53BP1 foci, and the induction of apoptosis. orcid.org/0000-0001-8997-3025 Results: Chaetocin and tazemetostat treatments caused a significant increase of Specialty section: H4K16 acetylation, associated with chromatin relaxation, and increased DNA damage, This article was submitted to Molecular and Cellular Oncology, detected by the labeling of free DNA-ends. These inhibitors significantly reduced H4K20 a section of the journal dimethylation levels in response to DNA damage and impaired the recruitment of 53BP1, Frontiers in Cell and Developmental Biology but not of MDC1 and NBS1, at DNA damaged sites. This modification of epigenetic Received: 26 May 2021 marks prevents DNA repair by the NHEJ pathway and leads to cell death. Accepted: 16 August 2021 Conclusion: KMT inhibitors could function as sensitizers to DNA damage-based Published: 03 September 2021 therapies and be used in novel synthetic lethality strategies for sarcoma treatment. Citation: Campillo-Marcos I, Keywords: chaetocin, tazemetostat, ionizing radiation, doxorubicin, DNA repair, histone methylation, 53BP1 foci, Monte-Serrano E, H2AX foci Navarro-Carrasco E, García-González R and Lazo PA (2021) Lysine Methyltransferase Inhibitors Impair H4K20me2 INTRODUCTION and 53BP1 Foci in Response to DNA Damage in Sarcomas, a Synthetic Chromatin structure is dynamically remodeled by DNA and histone epigenetic modifications to Lethality Strategy. control and coordinate all DNA-based processes such as transcription, replication, recombination Front. Cell Dev. Biol. 9:715126. or DNA repair (Becker and Workman, 2013; Hauer and Gasser, 2017). These epigenetic doi: 10.3389/fcell.2021.715126 modifications are mediated by several chromatin modifiers and depend on the cellular context Frontiers in Cell and Developmental Biology | www.frontiersin.org 1 September 2021 | Volume 9 | Article 715126 Campillo-Marcos et al. KMT Inhibitors Impair DNA Damage Response (Ehrenhofer-Murray, 2004; Kouzarides, 2007; Kulis and Esteller, of the corresponding DDR pathway (Polo, 2015; Campillo- 2010; Dawson and Kouzarides, 2012). Some of these proteins that Marcos et al., 2021). These sequential processes involve changes modify, or bind to, histone modifications are often dysregulated in covalent modifications of histones (Polo, 2015), which are in cancer and have been used as drug targets in novel necessary for the recruitment of specific DNA repair factors. therapeutic strategies in oncology (Dawson and Kouzarides, In this context, histone acetylation plays an important role in 2012; Bates, 2020). response to DNA damage, since acetylated histones H3 and H4 Histone methylation is one of the most prevalent chromatin are recognized by chromatin remodelers and protein kinases modifications and plays a key role in the regulation, activation implicated in specific DDR pathways (Deem et al., 2012; Garcia- and silencing of genes in euchromatin, and is also associated Gonzalez et al., 2020). Among them, the acetylation of histone with heterochromatin condensation (Dawson and Kouzarides, H4 in K16 (H4K16ac) induced by DNA damage is also associated 2012). This dynamic covalent modification of histones occurs with chromatin relaxation (Murr et al., 2006; Garcia-Gonzalez in N-terminal lysine or arginine residues (Shilatifard, 2006; et al., 2020). However, other chromatin readers depend on DesJarlais and Tummino, 2016), and several inhibitors have specific histone methylations to be recruited to DNA damage been designed against lysine methyltransferases (KMTs) and sites, such as 53BP1 (Pei et al., 2011; Wakeman et al., 2012; Zhao demethylases (KDMs), which can be used to improve current et al., 2020), a protein involved in non-homologous end joining cancer treatments (Wang and Patel, 2013; Toh et al., 2017). (NHEJ) (Lottersberger et al., 2013; Panier and Boulton, 2014; Among them is chaetocin, which inhibits KMTs such as Shibata, 2017), a key DNA repair pathway in resting cells such as SUV39H1 (Greiner et al., 2005; Chaib et al., 2012; Lai et al., 2015) neurons or cancer stem cells. In this context, the dimethylation or G9a (Iwasa et al., 2010; Cherblanc et al., 2013). In addition of histone H4 in lysine 20 (H4K20me2), mediated by SET8 to its role as KMT inhibitor (Greiner et al., 2005), chaetocin is (Dulev et al., 2014) and NSD2/MMSET (Pei et al., 2012), is also a competitive inhibitor of the thioredoxin reductase, whose necessary for the recruitment of 53BP1 at locations with DNA function is to compensate the deleterious effect of reactive oxygen damage (Botuyan et al., 2006; Pei et al., 2011; Dulev et al., species (ROS) (Tibodeau et al., 2009). Other exogenous agents 2014). H4K20me2 stabilizes the interaction between chromatin such as ionizing radiation also facilitate ROS production (Jackson and 53BP1 in foci (Li et al., 2020), and facilitate DNA repair by the and Bartek, 2009; Ciccia and Elledge, 2010), causing single- and NHEJ pathway (Bunting et al., 2010; Panier and Boulton, 2014). double-strand breaks (SSBs and DSBs, respectively) (Ciccia and Due to the role of DDR in the maintenance of genome Elledge, 2010), and their accumulation can lead to cell death integrity and cellular homeostasis, defects in DNA repair (Ciccia and Elledge, 2010). Both effects contribute to chromatin pathways directly lead to the accumulation of SSBs and DSBs, relaxation and the generation of oxidative stress, which facilitate and the subsequent cell death. In fact, patients with mutations DNA damage (Isham et al., 2007; Tibodeau et al., 2009; Dixit et al., in several DDR pathways respond much better to treatment, 2014; He et al., 2017). In this context, chaetocin also induces cell becoming super responders (Wheeler et al., 2021). In this work, death in different types of tumors, including multiple myeloma we have studied the molecular base by which KMT inhibitors, (Isham et al., 2007), leukemia (Chaib et al., 2012), melanoma chaetocin and tazemetostat, impair DDR (32, 52) by mimicking a (Han et al., 2017), gliomas (Dixit et al., 2014; Ozyerli-Goknar DNA repair defect, which would allow their use as DNA damage et al., 2019), and gastric (Liao et al., 2019), ovarian (Li et al., sensitizers (53, 54) and become candidates for novel synthetic 2019) and non-small cell lung cancers (Liu et al., 2015). Another lethality strategies in sarcomas cells treated with either ionizing recent KMT inhibitor that targets EZH2 is tazemetostat, which is radiation (IR) (Lee et al., 2013) or doxorubicin (Maurel et al., clinically used in sarcomas (Italiano, 2020; Rothbart and Baylin, 2009; D’Ambrosio et al., 2020). 2020). Furthermore, PARP1 regulates NHEJ (Non-homologous end joining) (Couto et al., 2011; Caron et al., 2019), and its inhibitors, such as olaparib, also cause DNA damage (McMahon MATERIALS AND METHODS et al., 2016) and are used in sarcomas in combination with DNA damaging agents, such as ionizing radiation (IR) (Lee et al., 2013). Reagents and Inhibitors In turn, olaparib also sensitizes glioblastoma cells to treatment Doxorubicin (Ref. 16416646, Thermo-Fisher Scientific), olaparib with temozolomide (Lesueur et al., 2019; Higuchi et al., 2020; (AZD2281) (Ref. O-9201, LC Laboratories, Woburn, MA, Navarro-Carrasco and Lazo, 2021) and ovarian cancer cells with United States), chaetocin (Ref. C9492, Sigma-Aldrich Merck), BRCA mutations (Fong et al., 2010). tazemetostat (Ref. S7128, Selleckchem) and JMJD2 inhibitor SSBs and DSBs are specifically repaired by different DDR (5-carboxy-8HQ; Ref. 420201, Calbiochem, Merck-Millipore). mechanisms, which are strictly coordinated in order to All other reagents were from Sigma-Aldrich-Merck (Darmstadt, sequentially detect, identify, signal and repair specific DNA Germany) (Supplementary Table 1). lesions based on their type (d’Adda di Fagagna, 2008; Jackson and Bartek, 2009). Initially, all these DDR pathways require a Cell Lines and Culture local distortion of chromatin caused by the DNA lesion (Ball Two human sarcoma cell lines, U2OS (ATCC, HTB-96) from and Yokomori, 2011; Bakkenist and Kastan, 2015), which is an osteosarcoma and SK-LMS-1 (ATCC, HTB-88) from a necessary to trigger the sequential steps in the response, ranging leiomyosarcoma, were validated by and obtained from the from chromatin remodeling and DNA protection of damaged ATCC and grown as recommended by the supplier in DMEM sites, to the recognition of the type of damage and the activation supplemented with antibiotics, 10% FBS and 5 mM glutamine. Frontiers in Cell and Developmental Biology | www.frontiersin.org 2 September 2021 | Volume 9 | Article 715126 Campillo-Marcos et al. KMT Inhibitors Impair DNA Damage Response Both cell lines are mycoplasma free. Experiments with inhibitors NE, United States) (Sanz-Garcia et al., 2012; Monsalve et al., were performed in serum-deprived cells for 48 h to eliminate 2016; Campillo-Marcos and Lazo, 2019). All western blots were mitogenic signals. performed in triplicate and corresponds to the accompanying immunofluorescence figure. DNA Damage DNA damage was induced by treatment with different doses (0.5, Immunofluorescence and Confocal 1 or 3 Gy) of ionizing radiation using a Gammacell 1,000 Elite Microscopy irradiator (Theratronics, Ottawa, Canada) with a 137 Cs source. Cells were plated on 60 mm dishes, which included coverslips, Alternatively DNA damage was also induced by treatment with to be used for immunofluorescence experiments (Salzano doxorubicin (topoisomerase II inhibitor) or olaparib (PARP1 et al., 2014, 2015; Monsalve et al., 2016; Moura et al., 2018; inhibitor) (Sanz-Garcia et al., 2012; Campillo-Marcos and Lazo, Campillo-Marcos and Lazo, 2019). Cells on coverslips were 2019). These measurements were performed in serum deprived fixed with 3% paraformaldehyde for 30 min and treated with cells (0.5%) for 48 h to remove mitogenic signaling and promote 200 mM glycine solution for 15 min at room temperature DNA repair by the NHEJ pathway. Chaetocin (100 nM) or to eliminate the paraformaldehyde. Cells were permeabilized tazemetostat (80 nM) were added at 24 h after serum withdrawal with 0.2% Triton X-100 solution in PBS for 30 min and and cells were exposed to each KMT inhibitor for 24 h. When blocked with 1% BSA in PBS for 30 min at room temperature olaparib (10 µM) was combined with chaetocin, this PARP or overnight at 4◦ C. For the simultaneous detection of two inhibitor was added 3 h later than this KMT inhibitor (27 h after proteins, coverslips were sequentially incubated with the two deprivation of serum). In case of doxorubicin (3 µM), it was primary antibodies, followed by three washes for 10 min in added 22 h later than chaetocin o tazemetostat (46 h after serum PBS after each one. The incubation with their corresponding withdrawal), so that cells were exposed to this chemotherapeutic secondary antibodies (Table 1), was performed for 1 h at room drug for 2 h. Finally, DNA damage caused by IR was induced after temperature in darkness, and finally washed three times for 24 h of treatment with chaetocin or tazemetostat and 48 h after 10 min in PBS. Nuclei were stained with DAPI (40 , 60 -diamidino- deprivation of serum. 2-phenylindole) (Sigma), diluted 1:1,000 in PBS, for 15 min at room temperature and washed three times for 10 min in Cell Lysates and Histone Extraction PBS. Coverslips were mounted with Mowiol (Calbiochem-Merck, Cells were lysed with the RIPA lysis buffer (150 mM NaCl, Darmstadt, Germany). Images were acquired with a LEICA SP5 1.5 mM MgCl2 , 10 mM NaF, 4 mM EDTA, 50 mM Hepes, DMI-6000B confocal microscope (Leica), with the following 1% Triton X-100, 0.1% SDS, and 10% glycerol) supplemented lasers: Argon (488 nm), DPSS (561 nm) and UV Diode (405 nm). by phosphatases inhibitors (1 mM NaF and 1 mM sodium These images were captured with a 63.0× lens zoomed in 1.5– orthovanadate) and proteases inhibitors (1 mM PMSF, 10 µg/mL 3 × with a 1,024 × 1,024 frame and 600 Hz scanning speed aprotinin, and 10 µg/mL leupeptin). Acidic extracts of histones and pinhole (95.6 µm), lasers intensity and photomultipliers gain were prepared as previously reported (Shechter et al., 2007). All and offset were maintained constant for all samples examined. protein extracts were quantified using the Bradford protein assay Image analysis was performed with the ImageJ software1 . These (Bio-Rad; Hercules, CA, United States). Lysates were boiled at imaging experiments were independently performed three times, 100◦ C in Laemmli buffer for 5 min for gel loading. and one of these experiments was shown in its corresponding main or Supplementary Figure. The number of cells analyzed Antibodies in each independent experiment was obtained from different The antibodies used are listed in Table 1 and were diluted in fields within the same experiment and is was indicated in all box TBS-0.1% Tween20 or PBS-1% BSA for immunoblots and/or plots of each image. immunofluorescence assays, respectively. TUNEL Assays TUNEL assay (TdT-mediated dUTP Nick-End Labeling) (Roche) SDS-Page Electrophoresis and Western was used to label free DNA-ends in damaged DNA in cells. Blot Analysis The detection is based on the binding of fluorescein-12-dUTP Proteins were fractionated by SDS-Page vertical electrophoresis to the 30 -OH of the DNA strand and was detected by a and transferred to Immobilon-FL membranes (Millipore) that fluorescence microscope. Briefly, cells were cultured on glass were blocked with TBS-T buffer [25 mM Tris–HCl (pH 8.0), coverslips and fixed with 3% PFA in PBS for 15 min at room 50 mM NaCl and 2.5 mM KCl, 0.1% Tween-20] and 5% non- temperature. PFA was removed and 200 mM glycine was added fat dry milk, or 5% BSA (bovine serum albumin), for 1 h for 15 min. Cells were permeabilized with 0.2% triton X-100 at room temperature. Next, membranes were incubated with for 15 min and blocked with 1% BSA in PBS for 30 min the primary antibody overnight at 4 ◦ C, followed by three at room temperature or overnight at 4◦ C. Coverslips were washes of 10 min in TBS-T buffer. Afterward, membranes were incubated with 50 µl of TUNEL reaction mixture (prepared incubated with their corresponding secondary antibodies for 1 h according to the manufacturer instructions) for 1 h at 37◦ C in in darkness, followed by three washes with TBS-T buffer (10 min). darkness, followed by three washes for 10 min in PBS. Nuclei Finally, membrane signals were detected using the LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences; Lincoln, 1 https://imagej.nih.gov/ij/ Frontiers in Cell and Developmental Biology | www.frontiersin.org 3 September 2021 | Volume 9 | Article 715126 Campillo-Marcos et al. KMT Inhibitors Impair DNA Damage Response TABLE 1 | List of antibodies and applications. Primary antibodies Dilution Clone and/or reference Supplier (WB/IF) code 53BP1 Rabbit polyclonal -; 1/200 H300, sc-22760 Santa Cruz Biotechnology 53BP1 Rabbit polyclonal 1/500; 1/200 NB100-304 Novus Biologicals γH2AX Mouse monoclonal -; 1/200 Clone JBW301; 05-636 Millipore MDC1 Rabbit polyclonal 1/500; 1/200 ab11169 Abcam NBS1 Mouse monoclonal -; 1/200 611871 BD Biosciences NBS1 (Nibrin) Rabbit polyclonal 1/1000; - N 3162 Sigma-Aldrich PARP1 Mouse monoclonal 1/1000; - sc-8007 Santa Cruz Biotechnology β-actin Mouse monoclonal 1/2000; - AC15/A5441 Sigma-Aldrich Histone Rabbit monoclonal 1/500; 1/400 ab109463 Abcam H4-K16ac Histone Rabbit polyclonal 1/500; 1/100 9759 Cell Signaling H4-K20me2 H3 Rabbit polyclonal 1/1000; - 9175 Cell Signaling Cleaved caspase 3 Rabbit monoclonal 1/1000; - 5A1E/9664 Cell Signaling Secondary antibodies Dilution Reference code Supplier (WB/IF) Anti-mouse IgG (WB) Goat Anti-Mouse IgG, DyLight 680 (red) 1/10000; - 35518 Thermo Scientific Anti-rabbit IgG (WB) Goat Anti-Rabbit IgG, DyLight 800 (green) 1/10000; - 35571 Thermo Scientific Goat anti-Mouse IgG Goat anti-Mouse IgG linked to Cy3 (red) -; 1/1000 115-165-146 Jackson ImmunoResearch (IF) Goat anti-rabbit IgG (IF) Goat anti-rabbit IgG linked to Cy2 (green) -; 1/1000 111-225-144 Jackson ImmunoResearch were stained with DAPI (Sigma), diluted 1:1,000 in PBS, for 10 Salzano et al., 2015). Chaetocin significantly increased min at room temperature, and washed three times for 10 min the number of γH2AX foci with respect to control cells in PBS. Coverslips were mounted with Mowiol (Calbiochem- (Figures 1A–C). Furthermore, the combination of this inhibitor Merck, Darmstadt, Germany) on microscope slides. Samples with lower doses of IR (0.5 Gy), another source of oxidative stress, were visualized using a Leica TCS SP5 DMI-6000B confocal showed a synergic effect on the assembly of these foci (Figure 1C, microscope (Leica) and analyzed with the ImageJ software. As left). However, this effect was not observed with higher doses of western blots and immunofluerescences, TUNEL assays were IR (1 or 3 Gy) in U2OS osteosarcoma cells (Figures 1A–C) and independently performed in triplicate and one representative SK-LMS-1 leiomyosarcoma cells (Supplementary Figure 2). experiment for each cell line was selected for the main figure. The After the initial phosphorylation of histone H2AX, additional number of cells in each independent experiment is indicated in repair proteins are sequentially recruited to DSBs. One of the corresponding figure. these proteins is 53BP1 (Pei et al., 2011; Wakeman et al., 2012), which determines DNA repair by the NHEJ pathway Statistical Analysis (Mirman and de Lange, 2020; Zhao et al., 2020). We studied The IBM SPSS 25 statistics package was used for analysis. whether chaetocin interfered with the assembly of 53BP1 foci Statistical significance was calculated using Mann–Whitney U in serum-deprived cells. The formation of these 53BP1 foci was tests, which analyze differences between only two pairs of impaired in U2OS osteosarcoma cells treated with chaetocin samples, or Dunn’s multiple comparison tests whether more than independently of the dose of IR used (Figures 1B,C). No two samples were assessed at the same time, and not all of them differences in 53BP1 protein levels were detected by western blot were adjusted to a normal distribution (Bremer and Doerge, after chaetocin and/or IR treatments (Figure 1D), which rules 2009). For the study we followed the ASBC recommendations out a reduction of endogenous 53BP1 protein as the cause of foci (Pollard et al., 2019). loss. Similar results were obtained in SK-LMS-1 leiomyosarcoma cells (Supplementary Figure 2). Therefore, we concluded that chaetocin impairs the recruitment of 53BP1, but not of γH2AX, RESULTS to DSBs induced by IR. Chaetocin Induces DNA Damage by The Effect of Chaetocin on 53BP1 Foci Itself and Impairs the Recruitment of Formation Is Independent of the Agent 53BP1 at Damage Sites Used to Induce DNA Damage Initially, we tested whether chaetocin, a KMT inhibitor, was able Next, we ruled out that the effect of chaetocin on the assembly to induce DNA damage, which was detected by the formation of 53BP1 foci was dependent on the type of agent used to cause of γH2AX foci, a surrogate marker of DDR early steps (van DNA damage. For this aim, we tested the effect of chaetocin on Attikum and Gasser, 2005; Bekker-Jensen and Mailand, 2010; 53BP1 foci induced by doxorubicin, which targets topoisomerase Frontiers in Cell and Developmental Biology | www.frontiersin.org 4 September 2021 | Volume 9 | Article 715126 Campillo-Marcos et al. KMT Inhibitors Impair DNA Damage Response FIGURE 1 | Chaetocin impairs the assembly of 53BP1 foci in response to ionizing radiation (IR) in U2OS cells deprived of serum. (A) γH2AX and 53BP1 foci formation in response to different doses of IR. (B) Effect of chaetocin on γH2AX and 53BP1 foci formation after inducing DNA damage with different doses of IR. (C) Quantification of γH2AX (left) and 53BP1 (right) foci in response to chaetocin and/or IR. (D) The immunoblot shows that chaetocin and/or IR have no effect on 53BP1 protein levels. ns, not significant. *p < 0.05, **p < 0.01, and ***p < 0.001. These images only show the detail in one cell selected for presentation. The field images are shown in Supplementary Figure 1. Ctrl, control without IR. Frontiers in Cell and Developmental Biology | www.frontiersin.org 5 September 2021 | Volume 9 | Article 715126 Campillo-Marcos et al. KMT Inhibitors Impair DNA Damage Response II, or olaparib, a specific PARP1 inhibitor that is used in cancer by these drugs was determined. The KMT inhibitor chaetocin treatment by itself or in combination with other drugs as a interferes with the formation of 53BP1 foci, but did not affect form of synthetic lethality (Dungey et al., 2009; Tewari et al., γH2AX foci formation, as a result of treating cells with either 2015; Ramakrishnan Geethakumari et al., 2017; Robson et al., doxorubicin (Figure 2A) or olaparib (Figure 2B). These results 2017; Chabanon et al., 2019). Furthermore, olaparib is also lethal indicated that chaetocin alters the DNA damage response by in sarcomas in combination with other agents causing DNA impairing 53BP1 foci formation independently of the agent damage (Lee et al., 2013). Therefore, the effect of chaetocin and causing DSBs. Moreover, this effect occurs after the formation of doxorubicin or olaparib on γH2AX and 53BP1 foci induced γH2AX foci and previous to 53BP1 foci assembly. FIGURE 2 | Effect of chaetocin on the formation of γH2AX and 53BP1 foci after inducing DSBs with doxorubicin (Doxo) (A) and olaparib (Olap) (B) in U2OS cells deprived of serum mitogenic signals. The effect of chaetocin on these foci in response to IR in SK-LMS-1 cells is shown in Supplementary Figure 2. Quantifications of these nuclear foci in response to chaetocin and/or doxorubicin or olaparib are shown to the right. ns, not significant, ***p < 0.001. These images only show the detail in one cell selected for presentation. The field images are shown in Supplementary Figure 3. Ctrl, control without doxorubicin or olaparib, as appropriate. Frontiers in Cell and Developmental Biology | www.frontiersin.org 6 September 2021 | Volume 9 | Article 715126 Campillo-Marcos et al. KMT Inhibitors Impair DNA Damage Response FIGURE 3 | Chaetocin does not interfere with MDC1 foci formation and NBS1 accumulation after inducing DSBs by IR in U2OS cells deprived of serum. (A) Effect of chaetocin on MDC1 foci in response to IR (3 Gy). Quantification of MDC1 foci after treating cells with chaetocin, IR and their combination (center). The immunoblot reflects that no variations in MDC1 protein levels are induced by chaetocin and/or IR (right). ns, not significant. ***p < 0.001. (B) Effect of chaetocin on NBS1 accumulation in response to IR (3 Gy) in U2OS cells. Quantification of NBS1 nuclear fluorescence after treating cells with chaetocin, IR and their combination (center). The immunoblot reflects that no variations in NBS1 protein levels were induced by chaetocin and/or IR (right). ns, not significant, *p < 0.05, ***p < 0.001. Ctrl, control without IR. These images only show the detail in one cell selected for presentation. The field images are shown in Supplementary Figure 5. Ctrl, control without chaetocin. Chaet, chaetocin (100 nM). The lack of 53BP1 foci in response to the combination of IR independently of the post-irradiation time, but this protein was and chaetocin might be a consequence of a delay in the assembly diffusely accumulated in nuclei. However, the number and size of these foci caused by chaetocin, which might require longer of γH2AX residual foci (720 min post-IR) increased significantly times. Because of that, we performed a time curve after chaetocin (Supplementary Figure 4), and repair was stalled and did not treatment and/or IR exposure and analyzed both γH2AX and progress. These results demonstrated that DDR is not working 53BP1 foci formation at different time points. We observed efficiently in presence of chaetocin, implying that the sequential that 53BP1 foci did not assemble in response to chaetocin response, which depends on 53BP1, is blocked. Consequently, Frontiers in Cell and Developmental Biology | www.frontiersin.org 7 September 2021 | Volume 9 | Article 715126 Campillo-Marcos et al. KMT Inhibitors Impair DNA Damage Response FIGURE 4 | Effect of chaetocin on H4K20me2 induced by IR or doxorubicin. (A) Effect of chaetocin on H4K20me2 induced by IR in SK-LMS-1 cells. (B) Effect of chaetocin on H4K20me2 induced by doxorubicin in U2OS cells. Quantification of nuclear fluorescence associated with H4K20 dimethylation in response to chaetocin, IR, doxorubicin or their combination (center). Analysis of H4K20me2 levels by western blot (right). Ctrl, control without IR or doxorubicin, as appropriate. ns, not significant, ***p < 0.001. These images only show the detail in one cell selected for presentation. The field images are shown in Supplementary Figure 6. Ctrl, control without chaetocin. Chaet, chaetocin (100 nM). earlier proteins participating in this process, such as γH2AX, are IR. Among them is the assembly of MDC1 and NBS1 foci, accumulated at DNA damaged sites, whose repair is stalled. two proteins that are phosphorylated in response to IR and mediate the accumulation of 53BP1 at DSBs (Eliezer et al., 2009; MDC1 and NBS1, Intermediate DDR Lavin et al., 2015; Monsalve et al., 2016). To determine whether chaetocin impairs the formation of MDC1 and NBS1 foci, cells Proteins Between γH2AX and 53BP1 Foci were treated with chaetocin, DNA damage was induced by IR and Formation, Are Not Affected by the formation of MDC1 and NBS1 foci was studied. Chaetocin Chaetocin Treatment did not interfere with either MDC1 (Figure 3A) or NBS1 foci The defective formation of 53BP1 foci might be a consequence formation (Figure 3B) in response to IR, which are two sequential of a disruption of previous steps in the sequential response to steps between γH2AX and 53BP1 foci in DDR, and their protein Frontiers in Cell and Developmental Biology | www.frontiersin.org 8 September 2021 | Volume 9 | Article 715126 Campillo-Marcos et al. KMT Inhibitors Impair DNA Damage Response FIGURE 5 | Effect of chaetocin on the acetylation of histone H4 in lysine 16 (H4K16ac) in U2OS cells treated with IR. (A) Detection of H4K16ac by immunofluorescence. (B) Quantification of the increase in H4K16ac induced by IR. ns, not significant, ***p < 0.001. (C) Immunoblot to show the increase of H4K16ac levels by IR. These images only show the detail in one cell selected for presentation. The field images are shown in Supplementary Figure 8. Ctrl, control without chaetocin. Chaet, chaetocin (100 nM). levels were not affected. This suggests that this DDR failure is due DNA damage causes an early local relaxation of chromatin to the deficient recruitment of 53BP1 at locations with damaged that is associated with the acetylation of histone H4 in DNA as a consequence of chaetocin treatment. K16 (H4K16ac) (Li and Wang, 2017; Garcia-Gonzalez et al., 2020). Therefore, we also determined H4K16 acetylation levels, H4K20me2 Induced by DNA Damage Are which would have to decrease in order to enable 53BP1 to Impaired by Chaetocin Treatment bind to chromatin through H4K20me2 (Hsiao and Mizzen, One of the chromatin modifications that mediates the 2013; Lottersberger et al., 2013). As expected, these H4K16 recruitment of 53BP1 at DSBs is the dimethylation of the acetylation levels increased after inducing DNA damage by IR histone H4 at lysine 20 (H4K20me2), which is directly and, in a smaller degree, by chaetocin, since this modification recognized by the TUDOR domains of 53BP1 (Pei et al., is a very early step in DDR, but such high levels remain 2011; Wilson et al., 2016). Therefore, we tested the ability high in response to the combination of IR and chaetocin of chaetocin to modify H4K20me2 levels in response to IR (Figure 5). Based on these results and taking into account that or doxorubicin in U2OS and SK-LMS-1 sarcoma cells. The chaetocin impairs 53BP1 foci formation, we suppose that IR treatment of both cell lines with the combination of chaetocin increases H4K16ac levels in a transient manner, which facilitate and IR resulted in a significant reduction of H4K20me2 levels the assembly of 53BP1 foci just when such levels start to (Figure 4A, Supplementary Figure 7A). Furthermore, this decrease, whereas chaetocin, by itself or in combination with reduction in H4K20 dimethylation levels was also observed in IR, not only reduces H4K20me2 levels, but also interferes response to doxorubicin and chaetocin treatments (Figure 4B, with H4K16 deacetylation. Altogether, these data reinforce the Supplementary Figure 7B). This effect on H4K20me2 would idea that KMT inhibitors like chaetocin involve changes in explain why this KMT inhibitor only affects the accumulation of the histone epigenetic code that interfere with DNA repair by 53BP1, but not of γH2AX, NBS1 or MDC1, at damaged sites. the NHEJ pathway. Frontiers in Cell and Developmental Biology | www.frontiersin.org 9 September 2021 | Volume 9 | Article 715126 Campillo-Marcos et al. KMT Inhibitors Impair DNA Damage Response FIGURE 6 | Effect of tazemetostat (TZM) on H4K20me2 levels in response to DNA damage induced by doxorubicin. (A) Effect of tazemetostat on the levels of H4K20me2 in U2OS cells deprived of serum. The tazemetostat effect on γH2AX and 53BP1 is shown in Figure 7. (B) Effect of tazemetostat on the levels of H4K20me2 in SK-LMS-1 cells. The tazemetostat effect on γH2AX and 53BP1 is shown in Supplementary Figure 9. ***p < 0.001. Ctrl, control without doxorubicin. Tazemetostat Impairs H4K20me2 the cells to DNA damage (Karakashev et al., 2020). Therefore, we tested whether tazemetostat could also impair the H4K20me2 as DNA Damage Response Induced by part of the DDR in sarcoma cells treated with doxorubicin. The Doxorubicin treatment with tazemetostat significantly reduced the levels of Tazemetostat is a KMT inhibitor targeting EZH2 (Italiano et al., H4K20me2 in response to doxorubicin in U2OS (Figure 6A) and 2018) and G9a (Soumyanarayanan and Dymock, 2016; Dockerill SK-LMS-1 (Figure 6B) cells as well as inhibited the formation of et al., 2020) that has been approved for the treatment of sarcomas 53BP1 foci induced by doxorubicin in U2OS cells (Figure 7) and (Italiano et al., 2018; Gounder et al., 2020) and sensitizes ovarian SK-LMS-1 cells (Supplementary Figure 9). Frontiers in Cell and Developmental Biology | www.frontiersin.org 10 September 2021 | Volume 9 | Article 715126 Campillo-Marcos et al. KMT Inhibitors Impair DNA Damage Response FIGURE 7 | Effect of tazemetostat on γH2AX and 53BP1 foci in response to doxorubicin treatment in U2OS osteosarcoma cells deprived of serum. Effect of tazemetostat on the formation of γH2AX (red) and 53BP1 (green) foci. The detail images selected indicated by boxes are shown to the right. Graphs at the bottom show the quantification of γH2AX and 53BP1 foci. ns, not significant. ***p < 0.001. Ctrl, control without doxorubicin. JMJD2 Inhibitor Does Not Alter the the consequences of treatment with a lysine demethylase (KDM) inhibitor, JMJD2i, on 53BP1 foci formation. Our results showed Formation of 53BP1 Foci Induced by that these foci were correctly formed after the combination of DNA Damage JMJD2i and different doses of IR (Figure 8). This result supports 53BP1 is an essential mediator protein in NHEJ pathway and its our hypothesis that the effect of chaetocin and tazemetostat recruitment at damage sites depends on covalent modifications on 53BP1 foci formation is modulated by variations of histone in specific histones residues and repair proteins. Since chaetocin methylation patterns, since high methylation levels associated or tazemetostat, KMT inhibitors, impair the accumulation of with the treatment with JMJD2i facilitates the recruitment of 53BP1 at damage sites in response to DNA damage, we studied 53BP1 in response to DNA damage. Frontiers in Cell and Developmental Biology | www.frontiersin.org 11 September 2021 | Volume 9 | Article 715126 Campillo-Marcos et al. KMT Inhibitors Impair DNA Damage Response FIGURE 8 | The assembly of 53BP1 foci is not affected by JMJD2i treatment, which inhibits lysine demethylation, after inducing DSBs with IR in U2OS cells. (A) γH2AX and 53BP1 foci formation in response to JMJD2 inhibitor and/or IR. (B) Quantification of γH2AX and 53BP1 foci after JMJD2i treatment and inducing DNA damage by IR. ns, not significant, ***p < 0.001. The field images are shown in Supplementary Figure 10. Ctrl, control without IR. Chaetocin and Tazemetostat Facilitate were combined with doxorubicin in both U2OS (Figure 9A) and DNA Damage Induced by Doxorubicin SK-LMS-1 cells (Figure 9B). A consequence of KMT inhibition would be a facilitation of chromatin relaxation, which makes tumor cells more susceptible Chaetocin and Tazemetostat Promote to undergo DNA damage. Therefore, we tested whether these two Cell Death in Response to DNA Damage KMT inhibitors, chaetocin and tazemetostat, could really cause Induction DNA damage by themselves or in cooperation with doxorubicin. Next, we analyzed the effect of these inhibitors, chaetocin and The accumulation of DNA damage was detected by labeling tazemetostat, on the induction of apoptosis by detection of nuclear free-DNA ends in a TUNEL assay. These two KMT the cleavage of caspase 3 and PARP1. PARP1 is cleaved by inhibitors by themselves caused a minor, but significant increase activated caspase 3 and can be detected as a smaller protein in DNA damage, which reached their maximum effect when they with a specific antibody for this cleaved fragment. Because of Frontiers in Cell and Developmental Biology | www.frontiersin.org 12 September 2021 | Volume 9 | Article 715126 Campillo-Marcos et al. KMT Inhibitors Impair DNA Damage Response FIGURE 9 | Effect of chaetocin and tazemetostat on the induction of DNA damage detected in TUNEL assays in U2OS (A) and SK-LMS-1 (B) sarcoma cells deprived of serum. DNA damage was detected by labeling free DNA ends. *p < 0.05, **p < 0.01, and ***p < 0.001. Ctrl, control without doxorubicin. that, the cleavage of caspase-3 and PARP1 was determined in was induced by these drugs in cooperation with doxorubicin. both sarcoma cell lines. Cleaved caspase 3 was detected after This effect of chaetocin on PARP1 processing induced by ionizing chaetocin or tazemetostat treatments either by themselves or in radiation was further confirmed in U2OS and SK-LMS-1 cell lines combination with doxorubicin at different time points in both (Supplementary Figure 11). cell lines (Figures 10A,B). Chaetocin by itself was able to induce caspase activation at shorter times than tazemetostat, whose effect required a longer time and the cooperation of doxorubicin. The DISCUSSION processing of PARP1 as a result of caspase activation was similarly determined (Figures 10C,D). Chaetocin was also more effective Genomic instability contributes to cancer development. at a shorter time than tazemetostat and PARP1 processing that However, the generation of excessive DNA damage by treatment Frontiers in Cell and Developmental Biology | www.frontiersin.org 13 September 2021 | Volume 9 | Article 715126 Campillo-Marcos et al. KMT Inhibitors Impair DNA Damage Response FIGURE 10 | Chaetocin and tazemetostat cause an activation of caspases detected by the processed caspase 3 and PARP1 in doxorubicin treated cells. Effect of chaetocin and tazemetostat on processed caspase 3 after treatment with doxorubicin in U2OS (A) and SK-LMS-1 (B) cells deprived of serum. Effect of chaetocin and tazemetostat on PARP1 cleavage after treatment with doxorubicin in U2OS (C) and SK-LMS-1 (D) cells deprived of serum. The cleavage of PARP1 in response to chaetocin is shown in Supplementary Figure 11. combinations can lead to a loss of tumor cell viability. In this The other is the sequential accumulation and phosphorylation of context, approaches promoting DNA damage accumulation histone γH2AX, MDC1, and NBS1 at DNA damaged locations in tumor cells can be exploited in novel cancer therapeutic (Lou et al., 2006; Eliezer et al., 2009; Liu et al., 2012; Lavin strategies of synthetic lethality. Among potential targets are et al., 2015; Monsalve et al., 2016). In this context, we have those proteins involved in different DDR pathways either by demonstrated that the inhibition of KMTs interfered with combining them in tumors with mutations in a DDR gene, DDR and the NHEJ pathway, which require the spatial and such as BRCA1 (Fong et al., 2010) or WRN (Chan et al., 2019; temporal coordination of different DNA repair factors and Kategaya et al., 2019; Lieb et al., 2019), or by pharmacological a local dynamic remodeling of chromatin. Furthermore, the targeting of another DDR pathway, such as olaparib, which manipulation of epigenetic modifications mediated by the KMT targets PARP1 (Tewari et al., 2015; Leichman et al., 2016; inhibitors chaetocin and tazemetostat did not alter earlier steps Srinivasan et al., 2017). An alternative is to simultaneously target in DDR, such as γH2AX, MDC1 and NBS1 foci, suggesting that two different DDR pathways or associated processes, such as KMT inhibitors work at later steps in DDR. chromatin remodeling, with drugs to promote tumor cell death. The dimethylation of histone H4 in lysine 20 (H4K20me2) Based on that, KMTs are good candidates, since they regulate stabilizes the interaction between chromatin and 53BP1 in foci chromatin compaction. If these KMT inhibitors were combined (Li et al., 2020). Our study shows that chaetocin and tazemetostat with DNA-based treatments, such as IR or doxorubicin, which strongly reduce H4K20me2 levels and consequently interfere are commonly used in sarcomas, the impact on DNA damage with DDR due to the impairment of 53BP1 recruitment to could be significantly higher and thus lethal for the tumor cell. damage locations. Chaetocin is more efficient than tazemetostat The recruitment of 53BP1 to chromatin in response to DSBs in impairing H4K20me2, which is necessary for DDR by the depends on two major components. One is the balance of NHEJ pathway. This effectiveness of chaetocin is likely to be epigenetic modifications such as H4K16ac (Murr et al., 2006; a consequence of its less specific inhibition of KMTs, while Garcia-Gonzalez et al., 2020), H2AK15ub (Fradet-Turcotte et al., tazemetostat has a more specific target, EZH2 (Brach et al., 2017; 2013) and H4K20me2 (Jacquet et al., 2016; Wilson et al., 2016). Italiano et al., 2018; Rothbart and Baylin, 2020). Furthermore, Frontiers in Cell and Developmental Biology | www.frontiersin.org 14 September 2021 | Volume 9 | Article 715126 Campillo-Marcos et al. KMT Inhibitors Impair DNA Damage Response Han et al., 2017; He et al., 2017; Li et al., 2019; Ozyerli-Goknar et al., 2019; Wen et al., 2019). This effect of chaetocin on ROS production and its lower KMT specificity make it more toxic than tazemetostat. Due to the DDR impairment caused by KMT inhibitors, chaetocin or tazemetostat, DNA damage is accumulated and leads to tumor death. The treatment with these two KMT inhibitors caused an increase in DNA damage, higher when cells were treated with IR or doxorubicin, which was detected by labelling of free DNA-ends in TUNEL assays, and an increase in processed PARP1 and caspase-3, which indicates that apoptosis has been activated in tumor cells. All these results are consistent with the role of these KMT inhibitors in facilitating cell death (Duriez and Shah, 1997; Soldani and Scovassi, 2002). Therefore, the combination of a KMT inhibitor with current treatments based on DNA damage in sarcomas, such as ionizing radiation or doxorubicin, could be useful to develop novel strategies that sensitize tumor cells (Figure 11) and, at the same time, might contribute to reduce the dose of these toxic treatments. The consequence would be a reduction of treatment side effects that would lead to an improvement in patient quality of life and life expectancy. CONCLUSION The inhibition of KMTs by chaetocin or tazemetostat causes a significant reduction of H4K20me2 levels, which impairs the recruitment and formation of 53BP1 foci induced by DNA FIGURE 11 | Diagram showing the induction of DNA damage by radiation or damage. As a consequence of this defective DDR, there is doxorubicin, and the effect of KMT inhibitors on DDR. Both inhibitors promote an accumulation of DNA damage that promotes tumor cell the generation of DNA damage, impair its repair and thus contribute to death. Based on these effects, we propose that new therapeutic facilitate tumor cell death. The SUV39H1, EZH2, SETD8, and NSD2, which mediate H4 lysine methylation, would be the most probable candidate approaches against tumor cells could be based on the use of these enzymes inhibited by chaetocin and/or tazemetostat and explain why KMT inhibitors in synthetic lethality strategies to treat cancer. H4K20me2 levels decrease in presence of both KMT inhibitors. DATA AVAILABILITY STATEMENT the acetylation of H4K16 has to be removed in order to The original contributions presented in the study are included facilitate the dimethylation of H4K20 after the generation of in the article/Supplementary Material, further inquiries can be DNA damage (Hsiao and Mizzen, 2013; Tang et al., 2013). directed to the corresponding author/s. However, H4K16ac levels were still high in response to IR and chaetocin, making more difficult that H4K20 can be methylated and impairing 53BP1 recruitment to DNA damage locations. AUTHOR CONTRIBUTIONS Apart from the modulation of these epigenetic modifications, IC-M performed the experiments, analyzed the data, and the disruption of H2AK15 ubiquitination and its writer, the wrote the manuscript. RG-G, EN-C, and EM-S performed the RNF168 ubiquitin ligase, could also indirectly contribute to the experiments. PL designed and coordinated this work, analyzed impairment of 53BP1 foci formation. However, further studies the data, and wrote the manuscript. All authors contributed to should be performed in order to confirm this hypothesis. the article and approved the submitted version. Along with their effect on H4K20me2, both chaetocin and tazemetostat cause DNA damage by themselves, but this effect is particularly higher in the case of chaetocin. This effect is a likely FUNDING consequence of the generation of oxidative stress by chaetocin, which is a competitive substrate, and inhibitor, of the thioredoxin IC-M was supported by FPI-MINECO-FSE contract (BES-2014- reductase (Tibodeau et al., 2009) leading to an increase of reactive 067721). EM-S was funded by Consejería de Educación-Junta de oxygen species (ROS), which are directly related to DNA damage Castilla y León (CSI004-18). EN-C was funded by FPI-MINECO- and promote cell death (Bruner et al., 2000; Hoeijmakers, 2001; FSE contract (BES-2017-080543). RG-G was supported by van Gent et al., 2001; Isham et al., 2007; Chaib et al., 2012; Consejería de Educación-Junta de Castilla y León. This work Frontiers in Cell and Developmental Biology | www.frontiersin.org 15 September 2021 | Volume 9 | Article 715126 Campillo-Marcos et al. KMT Inhibitors Impair DNA Damage Response was supported by grants from Agencia Estatal de Investigación- SUPPLEMENTARY MATERIAL Ministerio de Ciencia, Innovación y Universidades-FEDER (SAF2016-75744-R, PID2019-105610RB-I00), and Consejería de The Supplementary Material for this article can be found Educación de la Junta de Castilla y León (CSI264P20, CLC-2017- online at: https://www.frontiersin.org/articles/10.3389/fcell.2021. 01) to PL. 715126/full#supplementary-material REFERENCES d’Adda di Fagagna, F. (2008). Living on a break: cellular senescence as a DNA- damage response. Nat. Rev. Cancer 8, 512–522. Bakkenist, C. J., and Kastan, M. B. (2015). Chromatin perturbations during the D’Ambrosio, L., Touati, N., Blay, J. Y., Grignani, G., Flippot, R., Czarnecka, DNA damage response in higher eukaryotes. DNA Repair 36, 8–12. doi: 10. A. M., et al. (2020). Doxorubicin plus dacarbazine, doxorubicin plus ifosfamide, 1016/j.dnarep.2015.09.002 or doxorubicin alone as a first-line treatment for advanced leiomyosarcoma: Ball, A. R. Jr., and Yokomori, K. (2011). Damage site chromatin: open or closed? a propensity score matching analysis from the European Organization for Curr. Opin. Cell Biol. 23, 277–283. doi: 10.1016/j.ceb.2011.03.012 Research and Treatment of Cancer Soft Tissue and Bone Sarcoma Group. Bates, S. E. (2020). Epigenetic therapies for cancer. N. Engl. J. Med. 383, 650–663. Cancer 126, 2637–2647. doi: 10.1002/cncr.32795 doi: 10.1056/NEJMra1805035 Dawson, M. A., and Kouzarides, T. (2012). Cancer epigenetics: from mechanism to Becker, P. B., and Workman, J. L. (2013). Nucleosome remodeling and epigenetics. therapy. Cell 150, 12–27. doi: 10.1016/j.cell.2012.06.013 Cold Spring Harb. Perspect. Biol. 5:a017905. doi: 10.1101/cshperspect.a017905 Deem, A. K., Li, X., and Tyler, J. K. (2012). Epigenetic regulation of genomic Bekker-Jensen, S., and Mailand, N. (2010). Assembly and function of DNA double- integrity. Chromosoma 121, 131–151. doi: 10.1007/s00412-011-0358-1 strand break repair foci in mammalian cells. DNA Repair 9, 1219–1228. doi: DesJarlais, R., and Tummino, P. J. (2016). Role of histone-modifying enzymes 10.1016/j.dnarep.2010.09.010 and their complexes in regulation of chromatin biology. Biochemistry 55, Botuyan, M. V., Lee, J., Ward, I. M., Kim, J. E., Thompson, J. R., Chen, J., et al. 1584–1599. doi: 10.1021/acs.biochem.5b01210 (2006). Structural basis for the methylation state-specific recognition of histone Dixit, D., Ghildiyal, R., Anto, N. P., and Sen, E. (2014). Chaetocin-induced ROS- H4-K20 by 53BP1 and Crb2 in DNA repair. Cell 127, 1361–1373. doi: 10.1016/ mediated apoptosis involves ATM-YAP1 axis and JNK-dependent inhibition of j.cell.2006.10.043 glucose metabolism. Cell Death Dis. 5:e1212. doi: 10.1038/cddis.2014.179 Brach, D., Johnston-Blackwell, D., Drew, A., Lingaraj, T., Motwani, V., Warholic, Dockerill, M., Gregson, C., and Dh, O. D. (2020). Targeting PRC2 for the treatment N. M., et al. (2017). EZH2 inhibition by tazemetostat results in altered of cancer: an updated patent review (2016 - 2020). Expert Opin. Ther. Pat. 31, dependency on B-cell activation signaling in DLBCL. Mol. Cancer Ther. 16, 119–135. doi: 10.1080/13543776.2021.1841167 2586–2597. doi: 10.1158/1535-7163.MCT-16-0840 Dulev, S., Tkach, J., Lin, S., and Batada, N. N. (2014). SET8 methyltransferase Bremer, M., and Doerge, R. M. (2009). Statistics at the Bench: A Step-By Step activity during the DNA double-strand break response is required for Handbook for Biologists. New York: Cold Spring Harbor Laboratory Press. recruitment of 53BP1. EMBO Rep. 15, 1163–1174. doi: 10.15252/embr. Bruner, S. D., Norman, D. P., and Verdine, G. L. (2000). Structural basis for 201439434 recognition and repair of the endogenous mutagen 8-oxoguanine in DNA. Dungey, F. A., Caldecott, K. W., and Chalmers, A. J. (2009). Enhanced Nature 403, 859–866. doi: 10.1038/35002510 radiosensitization of human glioma cells by combining inhibition of poly(ADP- Bunting, S. F., Callen, E., Wong, N., Chen, H. T., Polato, F., Gunn, A., et al. ribose) polymerase with inhibition of heat shock protein 90. Mol. Cancer Ther. (2010). 53BP1 inhibits homologous recombination in Brca1-deficient cells by 8, 2243–2254. doi: 10.1158/1535-7163.MCT-09-0201 blocking resection of DNA breaks. Cell 141, 243–254. doi: 10.1016/j.cell.2010.0 Duriez, P. J., and Shah, G. M. (1997). Cleavage of poly(ADP-ribose) polymerase: a 3.012 sensitive parameter to study cell death. Biochem. Cell Biol. 75, 337–349. Campillo-Marcos, I., García-González, R., Navarro-Carrasco, E., and Lazo, P. A. Ehrenhofer-Murray, A. E. (2004). Chromatin dynamics at DNA replication, (2021). The human VRK1 chromatin kinase in cancer biology. Cancer Lett. 503, transcription and repair. Eur. J. Biochem. 271, 2335–2349. doi: 10.1111/j.1432- 117–128. doi: 10.1016/j.canlet.2020.12.032 1033.2004.04162.x Campillo-Marcos, I., and Lazo, P. A. (2019). Olaparib and ionizing radiation trigger Eliezer, Y., Argaman, L., Rhie, A., Doherty, A. J., and Goldberg, M. (2009). The a cooperative DNA-damage repair response that is impaired by depletion of the direct interaction between 53BP1 and MDC1 is required for the recruitment VRK1 chromatin kinase. J. Exp. Clin. Cancer Res. 38:203. doi: 10.1186/s13046- of 53BP1 to sites of damage. J. Biol. Chem. 284, 426–435. doi: 10.1074/jbc. 019-1204-1 M807375200 Caron, M. C., Sharma, A. K., O’Sullivan, J., Myler, L. R., Ferreira, M. T., Rodrigue, Fong, P. C., Yap, T. A., Boss, D. S., Carden, C. P., Mergui-Roelvink, M., Gourley, A., et al. (2019). Poly(ADP-ribose) polymerase-1 antagonizes DNA resection C., et al. (2010). Poly(ADP)-ribose polymerase inhibition: frequent durable at double-strand breaks. Nat. Commun. 10:2954. doi: 10.1038/s41467-019-10 responses in BRCA carrier ovarian cancer correlating with platinum-free 741-9 interval. J. Clin. Oncol. 28, 2512–2519. doi: 10.1200/JCO.2009.26.9589 Chabanon, R. M., Muirhead, G., Krastev, D. B., Adam, J., Morel, D., Garrido, Fradet-Turcotte, A., Canny, M. D., Escribano-Díaz, C., Orthwein, A., Leung, C. C., M., et al. (2019). PARP inhibition enhances tumor cell-intrinsic immunity in Huang, H., et al. (2013). 53BP1 is a reader of the DNA-damage-induced H2A ERCC1-deficient non-small cell lung cancer. J. Clin. Invest. 129, 1211–1228. Lys 15 ubiquitin mark. Nature 499, 50–54. doi: 10.1038/nature12318 doi: 10.1172/JCI123319 Garcia-Gonzalez, R., Morejon-Garcia, P., Campillo-Marcos, I., Salzano, M., and Chaib, H., Nebbioso, A., Prebet, T., Castellano, R., Garbit, S., Restouin, A., Lazo, P. A. (2020). VRK1 phosphorylates Tip60/KAT5 and is required for et al. (2012). Anti-leukemia activity of chaetocin via death receptor-dependent H4K16 acetylation in response to DNA damage. Cancers 12:2986. doi: 10.3390/ apoptosis and dual modulation of the histone methyl-transferase SUV39H1. cancers12102986 Leukemia 26, 662–674. doi: 10.1038/leu.2011.271 Gounder, M., Schoffski, P., Jones, R. L., Agulnik, M., Cote, G. M., Villalobos, Chan, E. M., Shibue, T., McFarland, J. M., Gaeta, B., Ghandi, M., Dumont, N., V. M., et al. (2020). Tazemetostat in advanced epithelioid sarcoma with loss et al. (2019). WRN helicase is a synthetic lethal target in microsatellite unstable of INI1/SMARCB1: an international, open-label, phase 2 basket study. Lancet cancers. Nature 568, 551–556. doi: 10.1038/s41586-019-1102-x Oncol. 21, 1423–1432. doi: 10.1016/S1470-2045(20)30451-4 Cherblanc, F. L., Chapman, K. L., Brown, R., and Fuchter, M. J. (2013). Chaetocin Greiner, D., Bonaldi, T., Eskeland, R., Roemer, E., and Imhof, A. (2005). is a nonspecific inhibitor of histone lysine methyltransferases. Nat. Chem. Biol. Identification of a specific inhibitor of the histone methyltransferase SU(VAR)3- 9, 136–137. doi: 10.1038/nchembio.1187 9. Nat. Chem. Biol. 1, 143–145. doi: 10.1038/nchembio721 Ciccia, A., and Elledge, S. J. (2010). The DNA damage response: making it safe to Han, X., Han, Y., Zheng, Y., Sun, Q., Ma, T., Zhang, J., et al. (2017). Chaetocin play with knives. Mol. Cell 40, 179–204. doi: 10.1016/j.molcel.2010.09.019 induces apoptosis in human melanoma cells through the generation of reactive Couto, C. A., Wang, H. Y., Green, J. C., Kiely, R., Siddaway, R., Borer, C., et al. oxygen species and the intrinsic mitochondrial pathway, and exerts its anti- (2011). PARP regulates nonhomologous end joining through retention of Ku at tumor activity in vivo. PLoS One 12:e0175950. doi: 10.1371/journal.pone. double-strand breaks. J. Cell Biol. 194, 367–375. doi: 10.1083/jcb.201012132 0175950 Frontiers in Cell and Developmental Biology | www.frontiersin.org 16 September 2021 | Volume 9 | Article 715126 Campillo-Marcos et al. KMT Inhibitors Impair DNA Damage Response Hauer, M. H., and Gasser, S. M. (2017). Chromatin and nucleosome dynamics in Li, L., and Wang, Y. (2017). Cross-talk between the H3K36me3 and H4K16ac DNA damage and repair. Genes Dev. 31, 2204–2221. doi: 10.1101/gad.307702. histone epigenetic marks in DNA double-strand break repair. J. Biol. Chem. 292, 117 11951–11959. doi: 10.1074/jbc.M117.788224 He, J., Chen, X., Li, B., Zhou, W., Xiao, J., He, K., et al. (2017). Chaetocin Li, Z., Bao, J., Qi, Y., and Zhang, J. Z. H. (2020). Computational approaches to induces cell cycle arrest and apoptosis by regulating the ROS-mediated ASK- studying methylated H4K20 recognition by DNA repair factor 53BP1. Phys. 1/JNK signaling pathways. Oncol. Rep. 38, 2489–2497. doi: 10.3892/or.2017. Chem. Chem. Phys. 22, 6136–6144. doi: 10.1039/c9cp05635a 5921 Li, Z., Huang, L., Wei, L., Hou, Z., Ye, W., and Huang, S. (2019). Chaetocin Higuchi, F., Nagashima, H., Ning, J., Koerner, M. V. A., Wakimoto, H., and Cahill, induces caspase-dependent apoptosis in ovarian cancer cells via the generation D. P. (2020). Restoration of temozolomide sensitivity by PARP inhibitors in of reactive oxygen species. Oncol. Lett. 18, 1915–1921. doi: 10.3892/ol.2019.1 mismatch repair deficient glioblastoma is independent of base excision repair. 0507 Clin. Cancer Res 26, 1690–1699. doi: 10.1158/1078-0432.CCR-19-2000 Liao, X., Fan, Y., Hou, J., Chen, X., Xu, X., Yang, Y., et al. (2019). Identification of Hoeijmakers, J. H. (2001). Genome maintenance mechanisms for preventing Chaetocin as a Potent non-ROS-mediated anticancer drug candidate for gastric cancer. Nature 411, 366–374. doi: 10.1038/35077232 Cancer. J. Cancer 10, 3678–3690. doi: 10.7150/jca.32803 Hsiao, K. Y., and Mizzen, C. A. (2013). Histone H4 deacetylation facilitates 53BP1 Lieb, S., Blaha-Ostermann, S., Kamper, E., Rippka, J., Schwarz, C., Ehrenhofer- DNA damage signaling and double-strand break repair. J. Mol. Cell Biol. 5, Wolfer, K., et al. (2019). Werner syndrome helicase is a selective vulnerability 157–165. doi: 10.1093/jmcb/mjs066 of microsatellite instability-high tumor cells. eLife 8:e43333. doi: 10.7554/eLife. Isham, C. R., Tibodeau, J. D., Jin, W., Xu, R., Timm, M. M., and Bible, K. C. 43333 (2007). Chaetocin: a promising new antimyeloma agent with in vitro and in vivo Liu, J., Luo, S., Zhao, H., Liao, J., Li, J., Yang, C., et al. (2012). Structural activity mediated via imposition of oxidative stress. Blood 109, 2579–2588. mechanism of the phosphorylation-dependent dimerization of the MDC1 doi: 10.1182/blood-2006-07-027326 forkhead-associated domain. Nucleic Acids Res. 40, 3898–3912. doi: 10.1093/ Italiano, A. (2020). Targeting epigenetics in sarcomas through EZH2 inhibition. nar/gkr1296 J. Hematol. Oncol. 13:33. doi: 10.1186/s13045-020-00868-4 Liu, X., Guo, S., Liu, X., and Su, L. (2015). Chaetocin induces endoplasmic Italiano, A., Soria, J. C., Toulmonde, M., Michot, J. M., Lucchesi, C., Varga, A., reticulum stress response and leads to death receptor 5-dependent apoptosis in et al. (2018). Tazemetostat, an EZH2 inhibitor, in relapsed or refractory B-cell human non-small cell lung cancer cells. Apoptosis 20, 1499–1507. doi: 10.1007/ non-Hodgkin lymphoma and advanced solid tumours: a first-in-human, open- s10495-015-1167-4 label, phase 1 study. Lancet Oncol. 19, 649–659. doi: 10.1016/S1470-2045(18)30 Lottersberger, F., Bothmer, A., Robbiani, D. F., Nussenzweig, M. C., and de 145-1 Lange, T. (2013). Role of 53BP1 oligomerization in regulating double-strand Iwasa, E., Hamashima, Y., Fujishiro, S., Higuchi, E., Ito, A., Yoshida, M., et al. break repair. Proc. Natl. Acad. Sci. U.S.A. 110, 2146–2151. doi: 10.1073/pnas. (2010). Total synthesis of (+)-chaetocin and its analogues: their histone 1222617110 methyltransferase G9a inhibitory activity. J. Am. Chem. Soc. 132, 4078–4079. Lou, Z., Minter-Dykhouse, K., Franco, S., Gostissa, M., Rivera, M. A., Celeste, doi: 10.1021/ja101280p A., et al. (2006). MDC1 maintains genomic stability by participating in the Jackson, S. P., and Bartek, J. (2009). The DNA-damage response in human biology amplification of ATM-dependent DNA damage signals. Mol. Cell. 21, 187–200. and disease. Nature 461, 1071–1078. doi: 10.1038/nature08467 doi: 10.1016/j.molcel.2005.11.025 Jacquet, K., Fradet-Turcotte, A., Avvakumov, N., Lambert, J. P., Roques, C., Maurel, J., Lopez-Pousa, A., de Las Penas, R., Fra, J., Martin, J., Cruz, J., et al. Pandita, R. K., et al. (2016). The TIP60 complex regulates bivalent chromatin (2009). Efficacy of sequential high-dose doxorubicin and ifosfamide compared recognition by 53BP1 through direct H4K20me binding and H2AK15 with standard-dose doxorubicin in patients with advanced soft tissue sarcoma: acetylation. Mol. Cell. 62, 409–421. doi: 10.1016/j.molcel.2016.03.031 an open-label randomized phase II study of the Spanish group for research on Karakashev, S., Fukumoto, T., Zhao, B., Lin, J., Wu, S., Fatkhutdinov, N., et al. sarcomas. J. Clin. Oncol. 27, 1893–1898. (2020). EZH2 inhibition sensitizes CARM1-high, homologous recombination McMahon, M., Frangova, T. G., Henderson, C. J., and Wolf, C. R. (2016). Olaparib, proficient ovarian cancers to PARP inhibition. Cancer Cell 37, 157.e6–167.e6. monotherapy or with ionizing radiation, exacerbates DNA damage in normal doi: 10.1016/j.ccell.2019.12.015 tissues: insights from a new p21 reporter mouse. Mol. Cancer Res. 14, 1195– Kategaya, L., Perumal, S. K., Hager, J. H., and Belmont, L. D. (2019). Werner 1203. doi: 10.1158/1541-7786.MCR-16-0108 syndrome helicase is required for the survival of cancer cells with microsatellite Mirman, Z., and de Lange, T. (2020). 53BP1: a DSB escort. Genes Dev. 34, 7–23. instability. iScience 13, 488–497. doi: 10.1016/j.isci.2019.02.006 doi: 10.1101/gad.333237.119 Kouzarides, T. (2007). Chromatin modifications and their function. Cell 128, Monsalve, D. M., Campillo-Marcos, I., Salzano, M., Sanz-Garcia, M., Cantarero, 693–705. doi: 10.1016/j.cell.2007.02.005 L., and Lazo, P. A. (2016). VRK1 phosphorylates and protects NBS1 from Kulis, M., and Esteller, M. (2010). DNA methylation and cancer. Adv. Genet. 70, ubiquitination and proteasomal degradation in response to DNA damage. 27–56. doi: 10.1016/B978-0-12-380866-0.60002-2 Biochim. Biophys. Acta Mol. Cell Res. 1863, 760–769. doi: 10.1016/j.bbamcr. Lai, Y. S., Chen, J. Y., Tsai, H. J., Chen, T. Y., and Hung, W. C. (2015). The 2016.02.005 SUV39H1 inhibitor chaetocin induces differentiation and shows synergistic Moura, D. S., Campillo-Marcos, I., Vazquez-Cedeira, M., and Lazo, P. A. (2018). cytotoxicity with other epigenetic drugs in acute myeloid leukemia cells. Blood VRK1 and AURKB form a complex that cross inhibit their kinase activity and Cancer J. 5:e313. doi: 10.1038/bcj.2015.37 the phosphorylation of histone H3 in the progression of mitosis. Cell Mol. Life Lavin, M. F., Kozlov, S., Gatei, M., and Kijas, A. W. (2015). ATM-dependent Sci. 76, 2591–2611. doi: 10.1007/s00018-018-2746-7 phosphorylation of all three members of the mrn complex: from sensor to Murr, R., Loizou, J. I., Yang, Y. G., Cuenin, C., Li, H., Wang, Z. Q., et al. (2006). adaptor. Biomolecules 5, 2877–2902. doi: 10.3390/biom5042877 Histone acetylation by Trrap-Tip60 modulates loading of repair proteins and Lee, H. J., Yoon, C., Schmidt, B., Park, D. J., Zhang, A. Y., Erkizan, H. V., et al. repair of DNA double-strand breaks. Nat. Cell. Biol. 8, 91–99. doi: 10.1038/ (2013). Combining PARP-1 inhibition and radiation in Ewing sarcoma results ncb1343 in lethal DNA damage. Mol. Cancer Ther. 12, 2591–2600. doi: 10.1158/1535- Navarro-Carrasco, E., and Lazo, P. A. (2021). VRK1 depletion facilitates the 7163.MCT-13-0338 synthetic lethality of temozolomide and olaparib in glioblastoma cells. Front. Leichman, L., Groshen, S., O’Neil, B. H., Messersmith, W., Berlin, J., Chan, E., Cell Dev. Biol. 9:683038. doi: 10.3389/fcell.2021.683038 et al. (2016). Phase II study of olaparib (AZD-2281) after standard systemic Ozyerli-Goknar, E., Sur-Erdem, I., Seker, F., Cingoz, A., Kayabolen, A., Kahya- therapies for disseminated colorectal cancer. Oncologist 21, 172–177. doi: 10. Yesil, Z., et al. (2019). The fungal metabolite chaetocin is a sensitizer for 1634/theoncologist.2015-0319 pro-apoptotic therapies in glioblastoma. Cell Death Dis. 10:894. doi: 10.1038/ Lesueur, P., Lequesne, J., Grellard, J. M., Dugue, A., Coquan, E., Brachet, P. E., s41419-019-2107-y et al. (2019). Phase I/IIa study of concomitant radiotherapy with olaparib Panier, S., and Boulton, S. J. (2014). Double-strand break repair: 53BP1 comes into and temozolomide in unresectable or partially resectable glioblastoma: OLA- focus. Nat. Rev. Mol. Cell Biol. 15, 7–18. doi: 10.1038/nrm3719 TMZ-RTE-01 trial protocol. BMC Cancer 19:198. doi: 10.1186/s12885-019-5 Pei, D., Zhang, Y., and Zheng, J. (2012). Regulation of p53: a collaboration between 413-y Mdm2 and Mdmx. Oncotarget 3, 228–235. doi: 10.18632/oncotarget.443 Frontiers in Cell and Developmental Biology | www.frontiersin.org 17 September 2021 | Volume 9 | Article 715126 Campillo-Marcos et al. KMT Inhibitors Impair DNA Damage Response Pei, H., Zhang, L., Luo, K., Qin, Y., Chesi, M., Fei, F., et al. (2011). MMSET regulates Tewari, K. S., Eskander, R. N., and Monk, B. J. (2015). Development of olaparib histone H4K20 methylation and 53BP1 accumulation at DNA damage sites. for BRCA-deficient recurrent epithelial ovarian cancer. Clin. Cancer Res. 21, Nature 470, 124–128. doi: 10.1038/nature09658 3829–3835. doi: 10.1158/1078-0432.CCR-15-0088 Pollard, D. A., Pollard, T. D., and Pollard, K. S. (2019). Empowering statistical Tibodeau, J. D., Benson, L. M., Isham, C. R., Owen, W. G., and Bible, K. C. methods for cellular and molecular biologists. Mol. Biol. Cell 30, 1359–1368. (2009). The anticancer agent chaetocin is a competitive substrate and inhibitor doi: 10.1091/mbc.E15-02-0076 of thioredoxin reductase. Antioxid. Redox. Signal. 11, 1097–1106. doi: 10.1089/ Polo, S. E. (2015). Reshaping chromatin after DNA damage: the choreography of ARS.2008.2318 histone proteins. J. Mol. Biol. 427, 626–636. doi: 10.1016/j.jmb.2014.05.025 Toh, T. B., Lim, J. J., and Chow, E. K. (2017). Epigenetics in cancer stem cells. Mol. Ramakrishnan Geethakumari, P., Schiewer, M. J., Knudsen, K. E., and Kelly, W. K. Cancer 16:29. doi: 10.1186/s12943-017-0596-9 (2017). PARP inhibitors in prostate cancer. Curr. Treat. Options. Oncol. 18:37. van Attikum, H., and Gasser, S. M. (2005). The histone code at DNA breaks: a guide doi: 10.1007/s11864-017-0480-2 to repair?. Nat. Rev. Mol. Cell Biol. 6, 757–765. Robson, M., Im, S. A., Senkus, E., Xu, B., Domchek, S. M., Masuda, N., et al. van Gent, D. C., Hoeijmakers, J. H., and Kanaar, R. (2001). Chromosomal stability (2017). Olaparib for metastatic breast cancer in patients with a germline BRCA and the DNA double-stranded break connection. Nat. Rev. Genet. 2, 196–206. mutation. N. Engl. J. Med. 377, 523–533. doi: 10.1056/NEJMoa1706450 doi: 10.1038/35056049 Rothbart, S. B., and Baylin, S. B. (2020). Epigenetic therapy for epithelioid sarcoma. Wakeman, T. P., Wang, Q., Feng, J., and Wang, X. F. (2012). Bat3 facilitates H3K79 Cell 181:211. doi: 10.1016/j.cell.2020.03.042 dimethylation by DOT1L and promotes DNA damage-induced 53BP1 foci at Salzano, M., Sanz-Garcia, M., Monsalve, D. M., Moura, D. S., and Lazo, P. A. G1/G2 cell-cycle phases. EMBO J. 31, 2169–2181. doi: 10.1038/emboj.2012.50 (2015). VRK1 chromatin kinase phosphorylates H2AX and is required for foci Wang, Z., and Patel, D. J. (2013). Small molecule epigenetic inhibitors targeted formation induced by DNA damage. Epigenetics 10, 373–383. doi: 10.1080/ to histone lysine methyltransferases and demethylases. Q. Rev. Biophys. 46, 15592294.2015.1028708 349–373. doi: 10.1017/S0033583513000085 Salzano, M., Vazquez-Cedeira, M., Sanz-Garcia, M., Valbuena, A., Blanco, S., Wen, C., Wang, H., Wu, X., He, L., Zhou, Q., Wang, F., et al. (2019). ROS-mediated Fernandez, I. F., et al. (2014). Vaccinia-related kinase 1 (VRK1) confers inactivation of the PI3K/AKT pathway is involved in the antigastric cancer resistance to DNA-damaging agents in human breast cancer by affecting DNA effects of thioredoxin reductase-1 inhibitor chaetocin. Cell Death Dis. 10:809. damage response. Oncotarget 5, 1770–1778. doi: 10.18632/oncotarget.1678 doi: 10.1038/s41419-019-2035-x Sanz-Garcia, M., Monsalve, D. M., Sevilla, A., and Lazo, P. A. (2012). Vaccinia- Wheeler, D. A., Takebe, N., Hinoue, T., Hoadley, K. A., Cardenas, M. F., Hamilton, related Kinase 1 (VRK1) is an upstream nucleosomal kinase required for the A. M., et al. (2021). Molecular features of cancers exhibiting exceptional assembly of 53BP1 foci in response to ionizing radiation-induced DNA damage. responses to treatment. Cancer Cell 39, 1–16. doi: 10.1016/j.ccell.2020.10.015 J. Biol. Chem. 287, 23757–23768. doi: 10.1074/jbc.M112.353102 Wilson, M. D., Benlekbir, S., Fradet-Turcotte, A., Sherker, A., Julien, J. P., McEwan, Shechter, D., Dormann, H. L., Allis, C. D., and Hake, S. B. (2007). Extraction, A., et al. (2016). The structural basis of modified nucleosome recognition by purification and analysis of histones. Nat. Protoc. 2, 1445–1457. doi: 10.1038/ 53BP1. Nature 536, 100–103. doi: 10.1038/nature18951 nprot.2007.202 Zhao, B., Rothenberg, E., Ramsden, D. A., and Lieber, M. R. (2020). The molecular Shibata, A. (2017). Regulation of repair pathway choice at two-ended DNA double- basis and disease relevance of non-homologous DNA end joining. Nat. Rev. strand breaks. Mutat. Res. 803-805, 51–55. doi: 10.1016/j.mrfmmm.2017.07. Mol. Cell Biol. 21, 765–781. doi: 10.1038/s41580-020-00297-8 011 Shilatifard, A. (2006). Chromatin modifications by methylation and ubiquitination: Conflict of Interest: The authors declare that the research was conducted in the implications in the regulation of gene expression. Annu. Rev. Biochem. 75, absence of any commercial or financial relationships that could be construed as a 243–269. doi: 10.1146/annurev.biochem.75.103004.142422 potential conflict of interest. Soldani, C., and Scovassi, A. I. (2002). Poly(ADP-ribose) polymerase-1 cleavage during apoptosis: an update. Apoptosis 7, 321–328. doi: 10.1023/a: Publisher’s Note: All claims expressed in this article are solely those of the authors 1016119328968 and do not necessarily represent those of their affiliated organizations, or those of Soumyanarayanan, U., and Dymock, B. W. (2016). Recently discovered EZH2 and the publisher, the editors and the reviewers. Any product that may be evaluated in EHMT2 (G9a) inhibitors. Future Med. Chem. 8, 1635–1654. doi: 10.4155/fmc- this article, or claim that may be made by its manufacturer, is not guaranteed or 2016-0096 endorsed by the publisher. Srinivasan, G., Sidhu, G. S., Williamson, E. A., Jaiswal, A. S., Najmunnisa, N., Wilcoxen, K., et al. (2017). Synthetic lethality in malignant pleural Copyright © 2021 Campillo-Marcos, Monte-Serrano, Navarro-Carrasco, García- mesothelioma with PARP1 inhibition. Cancer Chemother. Pharmacol. 80, 861– González and Lazo. This is an open-access article distributed under the terms 867. doi: 10.1007/s00280-017-3401-y of the Creative Commons Attribution License (CC BY). The use, distribution or Tang, J., Cho, N. W., Cui, G., Manion, E. M., Shanbhag, N. M., Botuyan, M. V., reproduction in other forums is permitted, provided the original author(s) and the et al. (2013). Acetylation limits 53BP1 association with damaged chromatin copyright owner(s) are credited and that the original publication in this journal to promote homologous recombination. Nat. Struct. Mol. Biol. 20, 317–325. is cited, in accordance with accepted academic practice. No use, distribution or doi: 10.1038/nsmb.2499 reproduction is permitted which does not comply with these terms. Frontiers in Cell and Developmental Biology | www.frontiersin.org 18 September 2021 | Volume 9 | Article 715126