ORIGINAL RESEARCH published: 31 May 2022 doi: 10.3389/fendo.2022.876602 Novel Targeting of DNA Methyltransferase Activity Inhibits Ewing Sarcoma Cell Proliferation and Enhances Tumor Cell Sensitivity to DNA Damaging Drugs by Activating the DNA Damage Response Edited by: Camilla Cristalli 1*, Maria Cristina Manara 1, Sergio Valente 2, Evelin Pellegrini 1, Haim Werner, Alberto Bavelloni 1, Alessandra De Feo 1, William Blalock 3, Elisabetta Di Bello 2, Tel Aviv University, Israel David Piñeyro 4, Angelika Merkel 4, Manel Esteller 4,5,6,7, Oscar M. Tirado 8, Reviewed by: Antonello Mai 2,9 and Katia Scotlandi 1* Silvio Naviglio, 1 Laboratory of Experimental Oncology, IRCCS Istituto Ortopedico Rizzoli, Bologna, Italy, 2 Department of Drug Chemistry and University of Campania Luigi Vanvitelli, Italy Technologies, Faculty of Pharmacy and Medicine, Sapienza University of Rome, Rome, Italy, 3 Istituto di Genetica Molecolare-Luigi Guenther Richter, Luca Cavalli Sforza, UOS Bologna, Consiglio Nazionale delle Ricerche (IGM-CNR), Bologna, Italy, 4 Josep Carreras Leukaemia Charité Universitätsmedizin Berlin, Research Institute (IJC), Barcelona, Spain, 5 Centro de Investigación Biomedica en Red Cancer (CIBERONC), Madrid, Spain, 6 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain, 7 Physiological Sciences Department, School of Germany Medicine and Health Sciences, University of Barcelona (UB), Barcelona, Spain, 8 Sarcoma Research Group, Oncobell Program, *Correspondence: Bellvitge Biomedical Research Institute (IDIBELL), Centro de Investigación Biomedica en Red Cancer (CIBERONC), Barcelona, Camilla Cristalli Spain, 9 Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy camilla.cristalli@ior.it Katia Scotlandi katia.scotlandi@ior.it DNA methylation is an important component of the epigenetic machinery that regulates the malignancy of Ewing sarcoma (EWS), the second most common primary bone tumor Specialty section: This article was submitted to in children and adolescents. Coordination of DNA methylation and DNA replication is Cancer Endocrinology, critical for maintaining epigenetic programming and the DNMT1 enzyme has been a section of the journal Frontiers in Endocrinology demonstrated to have an important role in both maintaining the epigenome and Received: 15 February 2022 controlling cell cycle. Here, we showed that the novel nonnucleoside DNMT inhibitor Accepted: 19 April 2022 (DNMTi) MC3343 induces a specific depletion of DNMT1 and affects EWS tumor Published: 31 May 2022 proliferation through a mechanism that is independent on DNA methylation. Depletion Citation: of DNMT1 causes perturbation of the cell cycle, with an accumulation of cells in the G1 Cristalli C, Manara MC, Valente S, Pellegrini E, Bavelloni A, De Feo A, phase, and DNA damage, as revealed by the induction of gH2AX foci. These effects Blalock W, Di Bello E, Piñeyro D, elicited activation of p53-dependent signaling and apoptosis in p53wt cells, while in p53 Merkel A, Esteller M, Tirado OM, Mai A and Scotlandi K (2022) mutated cells, persistent micronuclei and increased DNA instability was observed. Novel Targeting of DNA Treatment with MC3343 potentiates the efficacy of DNA damaging agents such as Methyltransferase Activity Inhibits doxorubicin and PARP-inhibitors (PARPi). This effect correlates with increased DNA Ewing Sarcoma Cell Proliferation and Enhances Tumor Cell Sensitivity to damage and synergistic tumor cytotoxicity, supporting the use of the DNMTi MC3343 DNA Damaging Drugs by Activating as an adjuvant agent in treating EWS. the DNA Damage Response. Front. Endocrinol. 13:876602. Keywords: ewing sarcoma, epigenetic therapies, DNA methylation, DNMT inhibitors, DNA damage, Drug doi: 10.3389/fendo.2022.876602 synergism, doxorubicin, PARP inhibitors Frontiers in Endocrinology | www.frontiersin.org 1 May 2022 | Volume 13 | Article 876602 Cristalli et al. DNMTi MC3343 effects in EWS INTRODUCTION undifferentiated state, and inhibit the invasiveness of cells, thus supporting the therapeutic value of this approach (38). MC3343 is Ewing sarcoma (EWS), a malignancy of mesenchymal origin with reported to preferentially bind to DNA in CG-rich regions, and to propensity for metastasis, is the second most common primary destabilize the DNMT1–AdoM-et DNA complex and two close tumor of bone and soft tissue in children and young people. analogues of MC3343 were shown to degrade DNMTs via Although important insights into EWS pathogenesis have proteasome degradation (35, 37). Here, we demonstrated that emerged in recent years, EWS patient treatment is still confined MC3343 specifically inhibits DNMT1 expression and activity in to dense multidrug chemotherapy, and the prognosis of high-risk EWS cells, but it does not affect the methylation status of EWS cells. patients remains dismal, with a less than 40% of survival rate at 5 MC3343 slowed cell proliferation, induced DNA damage and cell years for patients with metastatic disease at diagnosis and for those death, and acted synergistically with other DNA damaging drugs, who relapse after first-line treatment (1–3). Thus, there is a strong such as doxorubicin and PARPi, thereby indicating its potential use demand from patients, families and physicians for additional as an interesting novel adjuvant therapeutic agent against EWS. therapies, which may derive only from further knowledge of the genetic and biological features of the tumor. From a genetic point of view, EWS is characterized by balanced chromosomal translocations in which a member of the FET gene family is fused RESULTS with an ETS transcription factor, with the most common fusion The Non-Nucleoside DNMTi MC3343 being EWS–FLI1 (85% of cases) (4). The tumor is a developmental Reduces DNMT1 Protein Levels and disease, genetically homogeneous (5–7) but epigenetically heterogeneous (8), with an increased level of epigenetic Enzyme Activity but Does Not Affect the complexity in individuals with metastatic disease (9). The EWS DNA Methylation Status of EWS Cells epigenome is increasingly being investigated to identify promising The efficacy of MC3343 was tested in 11 human patient-derived therapeutic targets. EWS-FLI1, the genetic hallmark of EWS and the EWS cell lines, including 8 conventional, and 3 novel cell lines from driving oncogene (10, 11), is a tumor-specific transcriptional factor, patient-derived xenografts (PDX). The IC50 values, calculated after responsible for massive epigenetic reprogramming, by inducing de 72h of cell exposure to the compound, were similar in all the cell novo Ewing-specific enhancers at GGAA microsatellites and by lines, ranging from 2 to 5 µM (Table 1). The agent induced a dose- altering the state of gene regulatory elements (12–14). The unique dependent reduction of the expression of DNMT1, but the effect on epigenetic signature of EWS could potentially be reverted by the DNMT3a was much milder (Figure 1A), indicating a more specific identification of novel agents targeting epigenetic mechanisms. mechanisms of action when compared to the conventional inhibitor Several promising agents for the treatment of this disease have of DNA methylation, 5AzadC, that was used as a control. been reported at the preclinical level, including inhibitors of histone Consistently, MC3343 was similar to or even more potent than deacetylases (HDACi) (15–17), lysine-specific histone demethylase 5AzadC in inhibiting DNMT enzyme activity (Figure 1B). 1A (LSD1i) (14, 18), DNA methyltransferases (DNMTi) (9, 19–21) However, when we used a genome-wide methyl-CpG array to and combinations of these agents (22, 23). DNA methylation is compare the DNA methylation status of three EWS cell lines essential for crucial biological processes, such as maintaining (TC-71, A-673 and PDX-EW#4-C) after 72h treatment with genome stability, embryonic development and cell differentiation either MC3343 or 5AzadC, a profound discrepancy between the (24, 25). Disruption of DNA methylation patterns represents a two drugs was observed. In fact, while cell exposure to 5AzadC common feature of many forms of cancer (26) and was proven to resulted in extensive reduction of CpG methylation in all three cell contribute to EWS pathogenesis (9, 27–29). In humans, DNA lines, cells treated with MC3343 revealed a methylation pattern methylation is catalyzed by members of the DNMT family of indistinct from that observed in untreated cells (Figure 1C). This enzymes that transfer a methyl group from S-adenosyl- indicates a different mechanism of action between the two methionine (AdoMet) to DNA cytosine C5 (30, 31). DNMTs are mainly classified in de novo methyltransferases, DNMT3s, and TABLE 1 | Cytotoxic effects of the DNMTi MC3343 in EWS cell lines. maintenance methyltransferase, DNMT1, required respectively to Cell line MC3343 IC50 mM establish and maintain genomic methylation. In addition, DNMT1 contains motifs that allow the enzyme to localize at replication foci TC-71 2.48 ± 0.66 during S phase (30), and the absence of DNMT1 was proved to LAP-35 5.89 ± 0.06 6647 3.99 ± 0.82 result in replication fork stalling and activation of the DNA damage SK-N-MC 2.96 ± 0.17 response (DDR) (32, 33), leading to cell cycle arrest, mitotic SK-ES-1 3.96 ± 0.56 catastrophe and through a mechanism independent of DNA IOR/CAR 3.83 ± 0.41 methylation. In this study, we evaluate the efficacy of MC3343, a A673 2.20 ± 0.23 RD-ES-1 5.39 ± 0.09 new quinoline-based DNMTi with high activity against cancer cells, PDX-EW#2-C 4.58 ± 0.84 including osteosarcoma (34–37). Pharmacologic manipulation of PDX-EW#4-C 2.28 ± 0.18 DNMTs, either through the use of conventional FDA-approved PDX-EW#5-C 3.05 ± 0.64 nucleoside inhibitor of DNA methylation, 5-aza-2’-deoxycytidine IC50 values (drug concentration inducing 50% of growth inhibition) were calculated after (5AzadC; decitabine), or through non-nucleoside inhibitors was 72h exposition to the drug in 11 EWS cell lines. The mean ± SE of three independent proven to be sufficient to stop tumor growth, reverse the experiments are reported. Frontiers in Endocrinology | www.frontiersin.org 2 May 2022 | Volume 13 | Article 876602 Cristalli et al. DNMTi MC3343 effects in EWS compounds that deserves to be further analyzed, because although The DNMT1 MC3343 Inhibitor 5AzadC is approved by the US FDA for the treatment of Synergistically Increases the Cytotoxicity myelodysplasia and other malignancies, it has demonstrated of Doxorubicin and PARPi in EWS Cells unsatisfactory results in the treatment of EWS patients (39). We then evaluated the impact of the DNMTi MC3343 on cytotoxicity induced by DNA damaging agents, such as DNMTi MC3343 Treatment Results in doxorubicin, a widely-used genotoxic drug that intercalates Reduced Proliferation of EWS Cells and between DNA base pairs a nd inhibits the enzyme Induction of a Genotoxic Stress Response topoisomerase II (45), and talazoparib, a PARPi that functions The depletion of DNMT1 leads to the removal of the enzyme by impairing DNA repair mechanisms (46, 47). The from replication forks, which was demonstrated to result in pretreatment of cells with the MC3343 increased the DNA strand breaks and the activation of genotoxic stress cytotoxicity of both doxorubicin and talazoparib (Figure 6A). pathway signaling, leading to further DNA damage, gH2AX Combination indexes ranged from moderate to strong synergism foci formation and cell death (40, 41). Thus, we analyzed the (Figure 6B) and the formation of micronuclei that are prone to impact of MC3343 treatment on EWS cells on these biological rupture (gH2AX positive) was significantly higher in combined processes. Using double labeling with propidium iodide to treatments (Figures 6C, D). mark S-phase cells and bromodeoxyuridine (BrdU) to label cells actively synthesizing DNA, we discovered that MC3343 treatment triggers a significant accumulation of cells in the G1 DISCUSSION (Figure 2). As expected, the percentage of cells in G1 phase was higher in the faster proliferating TC-71 and A-673 cells EWS is a developmental tumor that derives from the (doubling time: 18-20h) compared to LAP-35 and PDX- transformation of an undifferentiated mesenchymal stem cell EW#4-C (doubling time: 40-50h) (p < 0.05 Student’s t test). that is blocked during the differentiation process by the Besides effects on the cell cycle, MC3343 induced the occurrence of a reciprocal chromosomal translocation and formation of histone variant gH2AX foci, a characteristic forming of an EWS-ETS chimera. The quiet nature of the EWS marker of cells undergoing replication stress and DNA genome, the paucity of mutations, and the evidence that EWS- strand breaks (42) with much faster kinetics as compared to FLI1 can induce a complete reprogramming of gene expression 5AzadC. In fact, treatment of EWS cells with MC3343 led to an (12, 13) guided the attention of researchers to epigenetic induction of gH2AX foci starting after 3h with a peak at 6h; alterations as the main mechanisms capable of regulating while in cells treated with 5AzadC, the appearance of gH2AX progression of this disease. Among the different epigenetic foci formation occurred only after 24h and not in all cell lines mechanisms, DNA methylation was demonstrated to have an (Figure 3 and Supplementary Figure S1). We also evaluated important role leading to patterns of enhancer reprogramming the impact of MC3343 on genomic instability in four EWS cell that were shared by all EWS samples (9). The importance of lines. Control and treated cells were cultured for 6 to 24 hours, DNA methylation alterations in EWS tumorigenesis and and the impact on genome stability monitored by evaluating progression together with exciting outcomes in hematological micronuclei, a well-recognized marker of genomic instability malignancies (21, 48, 49), encouraged us to study the therapeutic (43) (Figures 4A, B). This effect was better represented in p53 potential of DNMT inhibitors in EWS. Unlike genetic mutated TC-71 and A-673 cells than in p53wt LA-P35 and alterations, DNA methylation is reversible, which makes it PDX-EW#4-C cells (p< 0.0001 Student’s t test). In addition, extremely interesting for therapeutic approaches. However, the percentage of cells with micronuclei following exposure to although several epi-drugs received FDA approval (39), MC3343 was maintained in p53 mutated cells even after epigenetic therapy is still in its early stages. 5-azacytidine (5- removing the compound for at least two cell doublings AZA) and 5AzadC, first synthesized by Piskala and Sorm (1964), (rescue for 48h-96h), indicating that in p53 mutated cells are known to be incorporated into DNA and to inhibit DNA (TC-71 and A-673) micronuclei are inherited and persist methylation, which has led to the use of Decitabine as a first line into the daughter cells. In contrast, when the p53 response is therapeutic agent for cancers in which epigenetic silencing of functional (LAP-35 and PDX-EW#4-C), exposure to MC3343 critical regulatory genes has occurred (50). Nevertheless, despite led to activation of p53-dependent signaling (Figure 5A) and their high efficacy, such drugs have demonstrated poor cells that were more prone to apoptosis, as demonstrated by bioavailability, chemical instability, and toxic side-effects in annexin labeling (Figure 5B). Representative western blottings phase I trials, supporting the need for development of other, indicated that inactivation of DNMT1 by MC3343 induced more effective and less toxic compounds. The quinoline phosphorylation of p53 on Ser15, a residue that is targeted by derivative MC3343 specifically targets DNMT1, and we have ATM and whose phosphorylation has been correlated with recently demonstrated that this novel non-nucleoside DNMTi accumulation of p53 and activation of its downstream targets affects osteosarcoma cell proliferation, induces osteoblastic in response to DNA damage (44). Moreover, upregulation of differentiation through specific re-expression of genes p21 and cleavage of pro-Caspase 3 and PARP1 were regulating this physiologic process and increased stable consistently observed in p53 wt MC3343-treated PDX- doxorubicin binding to DNA with fewer toxic effects on EW#4-C but not in p53 mut TC-71 cells. healthy cells than either 5-AZA or 5azadC (34). Here, we Frontiers in Endocrinology | www.frontiersin.org 3 May 2022 | Volume 13 | Article 876602 Cristalli et al. DNMTi MC3343 effects in EWS A B C FIGURE 1 | MC3343 efficiently reduces DNMT protein level and enzymatic activity in EWS cells but DNA methylation status is left unaltered. (A) Western blot analysis of DNMT1 and DNMT3a, in untreated (CTRL), MC3343-treated (3–10 mM), and 5azadC-treated (3–10 mM) TC-71, A-673, LAP-35 and PDX-EW#4-C cells. Equal protein loading was monitored by anti-GAPDH blotting. One representative immunoblot of three independent experiments is shown. (B) DNMT activity measured in EWS cells after MC3343 and 5azadC treatments expressed as OD (optical density)/hours/mg (*p < 0.05; **p < 0.01, ***p < 0.001, one-way ANOVA test). The mean ± SE of three independent experiments are reported. (C) Heatmap illustrating the difference of methylation between EWS cells treated with MC3343 and 5azadC. Frontiers in Endocrinology | www.frontiersin.org 4 May 2022 | Volume 13 | Article 876602 Cristalli et al. DNMTi MC3343 effects in EWS FIGURE 2 | MC3343 induces a G1 block in EWS cells. Cell-cycle analysis of four representative EWS cell lines after 24 h of exposure to MC3343 as determined by BrdU uptake. x-axis indicates DNA content (PI) and y-axis indicates BrdU incorporation (log10). In each panel, the different stages of the cell cycle are highlighted. Percentages of cells in each cell-cycle stage are shown in histograms on the right. Data represent the mean ± SE of at least three experiments. demonstrated that although MC3343 reduced DNMT1 protein MC3343. DNA damage led to the accumulation of cells with levels and enzymatic activity in all EWS cell lines tested, the persistent DNA instability in p53 mutated cells, as shown by compound had little effects on the DNA methylation status of increased micronuclei, while in wt p53 cells, which are most treated cells. This effect was remarkably different with respect to representative of the condition in EWS (7), MC3343 led to the 5AzadC as but similar to what was previously reported in studies activation of p53 and induction of apoptosis. In addition, in where DNMT1 knock-down was obtained by either antisense pharmacologic targeting of DNMT1 by MC3343 was found to oligonucleotides or siRNA approaches (51–53). The genetic potentiate the efficacy of cytotoxic agents, such as the inactivation of DNMT1 was found to activate a number of conventional chemotherapeutic doxorubicin and the PARPi genes involved in the cellular stress response and cell cycle talazoparib. Doxorubicin is a leading drug in the treatment of arrest by a DNA methylation-independent mechanism (54). In EWS but fails as a single agent; while PARPi were found to be EWS cells, disruption of DNMT1 induced by MC3343 resulted highly effective in preclinical conditions (55), but they have failed in the loss of cell proliferation, followed by progressive DNA to show impressive clinical benefits for patients with EWS (56), instability and cell death. Upon inactivation of DNMT1, cells suggesting the need to develop new strategies to maximize the arrest in the G1 phase, leading to blockage of DNA replication. effectiveness of these agents. We previously demonstrated that DNMT1 is normally recruited to the replication fork, and MC3343, as well as siRNA against DNMTs, was capable of previous data suggested that it is the absence of the DNMT1 increasing stable doxorubicin bonds with DNA, triggering protein from the fork, not the loss of its DNA methylation doxorubicin-induced DNA damage (34). Others have shown activity, that causes stalled forks and activation of the DNA that pretreatment with DNMTi also leads to enhanced tight damage response (52). Accordingly, we observed DNA double binding of PARP1 to chromatin, enhancing DNA damage and strand break (DSB) formation (gH2AX foci) after treatment with PARPi efficacy (57). In this paper we confirmed that the Frontiers in Endocrinology | www.frontiersin.org 5 May 2022 | Volume 13 | Article 876602 Cristalli et al. DNMTi MC3343 effects in EWS A B FIGURE 3 | MC3343 and 5azadC kinetics of DNA damage induction. (A) Merged immunofluorescence of TC-71 and A-673 staining for gH2AX (green) and DAPI (blue) after 3, 6 and 24h of treatment with DMSO, MC3343 or 5azadC. Digital images were acquired under identical conditions at the same time, using image analysis software (NIS Elements, Nikon). Representative images are shown. Scale bars, 20 µm. (B) DNA DSBs measured by levels of mean fluorescence intensity (MFI) of gH2AX positive nuclei in at least 20 different fields for each condition. (*p < 0.05; ***p < 0.001, one-way ANOVA test). combination of the DNMTi MC3343 and DNA damaging agents represents a valuable extra benefit for pediatric patients potentiates effects against EWS. with EWS. Taken together, we demonstrate in the present study that DNMT1 is essential for the proliferation and survival of EWS cells. Complete inactivation of DNMT1 by MC3343 results in a MATERIALS AND METHODS G1 phase arrest and triggers the activation of DDR. Very likely, p53 mutated cells that escape cell cycle arrest enter mitosis with Chemicals their damaged DNA largely unrepaired, which causes severe MC3343 was prepared as previously reported (36, 37). Stock abnormalities, culminating in cell death. In p53 wt cells, p53 solutions of 5-Aza-2′-deoxycytidine (5azadC) (A3656, Sigma- activation following replication disruption led to apoptosis. In Aldrich, St. Louis, MA, U.S.), Doxorubicin (D1515, Sigma- keeping with previous evidences (51–53), this response appears Aldrich) and Talazoparib (BMN673) (S7048, Selleckchem, to be independent of the effects of DNMT1 in DNA methylation. Houston, TX, U.S.) were prepared and stored according to the The fact that different methods of DNMT1 knockdown targeting manufacturer’s instructions. different regions of DNMT1 as well as pharmacological inhibition trigger the same response in a broad panel of cells DNMT Activity Assay in a Cell Context from both human and mouse supports the hypothesis that DNMT activity was quantified with an EpiQuick DNA depletion of DNMT1 is mainly responsible for the general Methyltransferase Activity/Inhibition Assay Kit (P-3001-1, cellular response reported here. The demonstration that Epigentek Inc., Farmingdale, NY, U.S.). Cells were treated with MC3343 treatment increases the efficacy of doxorubicin and MC3343 or 5azadC (3-10 µM) for 48 h, and 5µg of nuclear extracts, PARPi in killing EWS cells offers the possibility of reducing the isolated with EpiQuick Nuclear Extraction Kit (OP-0002-1, toxicity of these agents while improving therapy, which Epigentek Inc), were added to each reaction well, according to the Frontiers in Endocrinology | www.frontiersin.org 6 May 2022 | Volume 13 | Article 876602 Cristalli et al. DNMTi MC3343 effects in EWS A B FIGURE 4 | MC3343 treatment induces micronuclei formation. (A) Representative images of gH2AX (green) and DNA (Hoechst 33258, blue) staining of EWS cells following treatment with MC3343. The white arrows indicate micronuclei that contains DSBs (as indicated by DNA and gH2AX double-staining). Scale bars, 10 µm. (B) Percentage of micronuclei in EWS cells treated with MC3343 at the indicated times and after 48-96h of rescue with drug-free medium replacement. Data are presented as the mean percentage of micro-nucleated cells quantified from at least 20 random fields (mean ± SE; *p < 0.05, ***p < 0.001, one-way ANOVA test). manufacturer’s protocol. Absorbance was determined using a penicillin (20 U/mL) and streptomycin (100 µg/mL) microplate spectrophotometer at 450 nm (GloMax luminometer, (ECB3001D, Euroclone) in a 37°C humidified at 5% CO2. Promega, Madison, WI, U.S.). The data are expressed as the mean ± SE of three independent experiments. Cell Treatments To perform cell viability experiments, 2-5x105 cells were plated Cells Lines in 6-well dishes and treated after 24h with the indicated drugs for A panel of 11 EWS human cell lines were analyzed. A-673 (CRL- 72h, roughly equivalent to at least two doubling times of each cell 1598), SK-ES-1 (HTB-86), SK-N-MC (HTB-10), and RD-ES line, before Trypan blue (T8154, Sigma-Aldrich) vital cell (HTB-166) were provided by the American Type Culture counting or DNA methylation studies. In parallel, cells were Collection (ATCC, Manassas, VA, U.S.); TC-71 and 6647 were treated with medium containing dimethyl sulfoxide (DMSO) kindly provided by T.J. Triche (Children’s Hospital, Los Angeles, (D2438, Sigma-Aldrich) as a control. The highest final CA, U.S.). LAP-35 and IOR/CAR, were obtained in the concentration of DMSO in the medium was <0.005%, which Experimental Oncology Lab of the Rizzoli Institute as had no effect on cell growth. previously described (58, 59). The cell lines PDX-EW#2-C, In sequential drugs combination experiments, cells were PDX-EW#4-C and PDX-EW#5-C were obtained from patient- plated and pre-treated with MC3343 (3-5 µM) for 24h then derived xenografts (PDXs) as previously described (60). All cell the medium was replaced with the drugs at indicated lines were tested for mycoplasma contamination every 3 months concentrations (doxorubicin 3-30 ng/ml, talazoparib 1-10 nM) (LT07-318, MycoAlert Mycoplasma Detection Kit, Lonza, Basel, or fresh IMDM medium without drugs. Switzerland). Cell lines were authenticated (STR profiling) by analysis of the following loci: AMEL, D3S1358, TH01, D21S11, Flow Cytometry D18S51, D10S1248, D1S1656, D2S1338, D16S539, D22S1045, For cell cycle analysis after 24h of treatment, 5-bromo-2’- VWA, D8S1179, FGA, D2S441, D12S391, D19S433 and SE33 deoxyuridine (BrdU) (B5002, Sigma-Aldrich) labeling was used to (last control December 2017 and July 2018; POWERPLEX ESX assess cell proliferation. Cells were processed for indirect 17 Fast System, Promega, Madison, WI, U.S.). All cell lines were immunofluorescence using anti‐BrdU mAb (1:8, 347580, BD- immediately amplified to constitute liquid nitrogen stocks and Biosciences, Franklin Lakes, NJ, U.S.) primary antibody and anti‐ were never passaged for more than 1 month upon thawing. Cells mouse FITC secondary antibody (1:100, 31569, Thermo Fisher were maintained in Iscove’s Modified Dulbecco’s Medium Scientific, Waltham, MA, U.S.) and 20 mg/mL propidium iodide (ECB2072L, IMDM, Euroclone, Milan, Italy) supplemented (P4170, Sigma-Aldrich) for the analysis of DNA content prior to with 10% heat-inactivated FBS (ECS0180L, Euroclone), flow cytometry analysis (Sysmex, Partec GmbH Münster, Frontiers in Endocrinology | www.frontiersin.org 7 May 2022 | Volume 13 | Article 876602 Cristalli et al. DNMTi MC3343 effects in EWS A B FIGURE 5 | Activation of p53 signaling and induction of apoptosis in TP53 wild type cell lines. (A) TP53 mutated TC-71 cell line and TP53 wild-type PDX-EW#4-C cells were treated with 3µM MC3343 for 24h. Whole cell lysates were analyzed using western blot. Three independent experiments were performed and one representative immunoblot of p-ATM, p21, phospho-p53, p53, PARP and caspase-3 cleaved is shown (B) Representative flow cytometry scatter plots showing apoptosis of p53 mutated (TC-71 and A-673) and p53 wild-type (LAP-35 and PDX-EW#4-C) EWS cells stained with Annexin-V/7-AAD following 48h treatment with MC3343 (3-5 µM) or DMSO. Numbers at the corners represent the percentage of cells found in each quadrant (viable-lower left, early apoptotic-lower right, late apoptotic-upper right, and dead cells-upper left are indicated). Germany). Detection and quantification of apoptosis was obtained bisulfite conversion, 600 ng of input gDNA was required. by cell staining with Annexin V and 7-amino-actinomycin D (7- Bisulfite-modified gDNA was prepared using the EZ DNA AAD) (A1310, Thermo Fisher Scientific) using the PE Annexin V Methylation kit (Zymo Research) according to the detection kit I (Life Technologies Corporation, Monza, Italy) manufacturer’s instructions. Conversion reagent was added, according to the manufacturer’s instructions. Samples were followed by subsequent incubation in a thermocycler to analyzed on an Attune NxT Acoustic Focusing Cytometer (Life denature the samples. CT-converted DNA was washed and de- Technologies Corporation), and data were analyzed using Attune sulfonated with de-sulfonation buffer, after which the DNA was Cytometric 2.6 software (Life Technologies Corporation). Cells were washed again and eluted with 12 mL elution buffer. The whole- classified as early apoptotic, late apoptotic or dead if they were genome amplification process required 250 ng of input bisulfite- Annexin V+/7-AAD−, Annexin V+/7-AAD+ or Annexin V−/7- converted DNA (MA1) and created a sufficient quantity of DNA AAD+, respectively. (1,000X amplification) for use on a single BeadChip in the Infinium methylation assay (Illumina RPM and MSM). After DNA Extraction and Methylation Profiling amplification, the product was fragmented using a proprietary DNA was extracted from cell pellets by PureLink genomic DNA reagent (FMS), precipitated with 2-propanol (plus precipitating kit (K182001, Thermo Fisher Scientific). The DNA methylation reagent; PM1), and re-suspended in formamide-containing profiles of the samples were obtained using the Infinium hybridization buffer (RA1). The DNA samples were denatured Methylation EPIC Array (850 K). DNA samples were assessed for 20 min at 95°C and placed in a humidified container for a for their quality using a NanoDrop ® ND-2000 UV-Vis minimum of 16 h at 48°C, allowing CpG loci to hybridize with the Spectrophotometer (Thermo Fisher Scientific). The samples 50-mer capture probes. Following hybridization, the BeadChip/ were separated in agarose gels. Those with intact genomic DNA, Te-Flow chamber assembly was placed on a temperature- showing no smear in the gel, were selected for subsequent controlled Tecan flow-through chamber rack, and subsequent experiments. Intact genomic DNA was diluted to 50 ng/mL washing, extension, and staining were performed by adding based on Quant-iT PicoGreen (Invitrogen) quantitation. reagents to the Te-Flow chamber. For the allele-specific single- Concentrations were adjusted according to these results. For base extension assay, primers were extended by polymerase and Frontiers in Endocrinology | www.frontiersin.org 8 May 2022 | Volume 13 | Article 876602 Cristalli et al. DNMTi MC3343 effects in EWS A B C D FIGURE 6 | Synergism of combined treatments with DNA damaging drugs. (A) TC-71, A-673, LAP-35 and PDX-EW#4-C cells were treated for 24h with 3-5 µM MC3343 and, after wash-out, treated for 48h with 3-10 ng/mL of doxorubicin or 3-10 nM of talazoparib. Cell viability was measured by trypan blue vital cell counts and is expressed as the percentage of live cells with respect to the untreated control. Data are shown as mean ± SE of three independent experiments (*p < 0.05; **p < 0.01, ***p < 0.001, one-way ANOVA test). (B) Heatmap of drug interactions of sequential treatments with MC3343 and doxorubicin or talazoparib. Cell viability was assessed by trypan blue cell counts. Synergistic, additive, or antagonistic drug interactions were calculated by combination index (CI). (C) Merged immunofluorescence of EWS cells staining for gH2AX (green) and DNA (blue) after treatment with MC3343 and doxorubicin/talazoparib as indicated. Representative images are shown. Scale bars, 10 µm. (D) Percentage of micro-nucleated cells after sequential treatment with MC3343 and doxorubicin or talazoparib. Data are presented as mean percentage of micro-nucleated cells quantified from at least 20 random fields (mean ± SE; ***p < 0.001, one-way ANOVA test). labeled nucleotide mix (TEM), and then stained by repeated DNA Methylation Analysis application of STM (staining reagent) and ATM (anti-staining Methylation array data was processed with R statistical language reagent). After staining, the slides were washed with low-salt wash using methods from the Bioconductor. Data quality was assessed buffer (PB1), immediately coated with XC4, and imaged using the using the standard pipeline from the minfi package: Low quality iScan System (Illumina). The iScan System has a two-color (532 probes (detection pvalue > 0.01) and low-quality samples (> 0.05% nm/658 nm) confocal fluorescent scanner with 0.54 mm pixel low quality probes) were removed from the analysis and known resolution. The scanner excited the fluorophores generated during SNPs, sex chromosomes and cross-hybridizing probes were signal amplification/staining of the allele-specific (one color) excluded. The data was normalized using ‘ssNoob’ normalization extension products on the BeadChips. Image intensities were method and, after normalization, m-values and beta-values were extracted using Illumina’s GenomeStudio Software. calculated. Differential methylation analysis was performed with the Frontiers in Endocrinology | www.frontiersin.org 9 May 2022 | Volume 13 | Article 876602 Cristalli et al. DNMTi MC3343 effects in EWS limma package (~ 0 + treatment + cell line; single sites, p<0.01) and ANOVA. The data were considered statistically significant at p DMRcate (regions, fdr < 0.01). <0.05. Drug interactions were analyzed by the combination index (CI) calculated with the fractional product method based on that Immunofluorescence Analysis described by Chou et al. (61). Subclassification of CI values was Immunofluorescence was performed on adherent cells grown on used (0.1– 0.3 strong synergism, 0.3–0.7 synergism, 0.7–0.85 coverslips, fixed in cold methanol for 7 minutes at -20°C and moderate synergism, 0.85–0.9 slight synergism, 0.9–1.1 permeabilized with 0.15% Triton X-100 (T8787, Sigma-Aldrich) nearly additive). in PBS, blocked with 4% Bovine serum albumin (BSA) (A4503, Sigma-Aldrich)/PBS for 1 hour at room temperature and incubated with anti-phospho-histone H2A.X (Ser139) antibody DATA AVAILABILITY STATEMENT (1:100, 9718, Cell Signaling Technology, Danvers, MA, U.S). Nuclei were counterstained by bisbenzimide Hoechst 33258 (0.5 The data presented in the study have been deposited in NCBI's µg/ml, B2883, Sigma-Aldrich, St. Louis, MA, U.S.). Cell Gene Expression Omnibus accession number GSE202143 (https:// fluorescence was then evaluated using a Nikon Eclipse 90i www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE202143). microscope (Nikon Instruments, Florence, Italy). For quantification purposes, gH2AX spots and micronuclei were quantified from at least 20 random fields (~500 cells), ETHICS STATEMENT expressed as gH2AX mean fluorescence intensity (MFI) and percentage of cells with micronuclei respectively. Images The ethics committee of the Rizzoli Institute approved the study acquisition and processing were conducted using the NIS- (Prot. 0006167 2020/04/29; Prot. 0011371 2019/09/25) and the Elements A.R. 3,10 software. establishment of PDX models (Prot. 0009323 2016/04/22 and Prot. 0009164 2017/09/22). Western Blotting EWS cells were lysed with RIPA buffer (89900, Thermo Fisher Scientific) containing protease inhibitors (A32955, Thermo AUTHOR CONTRIBUTIONS Fisher Scientific) and phosphatase inhibitors (A32957, Thermo Fisher Scientific). Equivalent amounts of total cell lysates were Conception and design: CC and KS. Development of separated by 8-12% SDS-PAGE under denaturing conditions and methodology: CC, MM, SV, DP, ME, AMe, OT, AMa, KS. transferred onto nitrocellulose membrane (0.45 mm, Bio-Rad, Acquisition and analysis of data: CC, MM, EP, AB, AD, WB, Hercules, CA, U.S.). Membranes were incubated overnight with ED, DP, AMe, ME, OT. Writing, review, and/or revision of the the following primary antibodies: anti-DNMT1 (1:2000, A300- manuscript: KS, CC, and MM wrote the manuscript, and all 041A-M lot# 1, Bethyl Laboratories, Montgomery, TX, U.S), authors reviewed and edited the manuscript. Study supervision: anti-DNMT3a (1:1000, sc-20703 lot # L0412, Santa Cruz CC and KS. All authors contributed to the article and approved Biotechnology, Dallas, TX, U.S.), anti-phospho-ATM (S1981) the submitted version. (1:2000, ab36810 lot# gr3185919-27, Abcam, Cambridge, UK), anti-p21 (1:1000, sc-6246 lot # E0511, Santa Cruz Biotechnology), anti-phospho-p53 (Ser15) (1:2000, 9284 lot#9, FUNDING Cell Signaling Technology), anti-p53 (1:3000, MCA1701 lot #410, Bio-Rad), anti-PARP1 (1:5000, 9542 lot#15, Cell This work was supported by the Italian Ministry of Health (RF- Signaling Technology), anti-cleaved Caspase-3 (Asp175) 2016-02361373). (1:500, 9661 lot # 38, Cell Signaling Technology) and anti- GAPDH (1:10000, 2118 lot # 14, Cell Signaling Technology). After washes with 1x Tris-buffered saline/Tween20, the membranes were incubated with secondary anti-rabbit or anti- ACKNOWLEDGMENTS mouse antibodies conjugated to horseradish peroxidase (GE The research leading to these results has received funding from Healthcare, Piscataway, NJ, U.S.) and revealed by SuperSignal AIRC under IG 2019 - ID. 22805 project – P.I., Katia Scotlandi, West Pico PLUS Chemiluminescent Substrate (34580, Thermo and Ministry of University and Research under FISR2019_00374 Fisher Scientific). MeDyCa project – P.I. Antonello Mai. The authors would also thank Cristina Ghinelli for graphic support. Statistical Analyses All statistical analyses were performed using Prism version 7.0 (GraphPad Software, La Jolla, CA, US). IC50 (concentration required to inhibit cell proliferation by 50%) values were SUPPLEMENTARY MATERIAL calculated from the linear transformation of dose-response curves. Comparison between two groups were evaluated with The Supplementary Material for this article can be found online at: two-tailed Student’s t-tests. Experimental data including more https://www.frontiersin.org/articles/10.3389/fendo.2022.876602/ than 2 groups were analyzed using one-way or two-way full#supplementary-material Frontiers in Endocrinology | www.frontiersin.org 10 May 2022 | Volume 13 | Article 876602 Cristalli et al. DNMTi MC3343 effects in EWS REFERENCES 18. Welch D, Kahen E, Fridley B, Brohl AS, Cubitt CL, Reed DR. Small Molecule Inhibition of Lysine-Specific Demethylase 1 (LSD1) and Histone Deacetylase 1. Ingley KM, Maleddu A, Grange FL, Gerrand C, Bleyer A, Yasmin E, et al. (HDAC) Alone and in Combination in Ewing Sarcoma Cell Lines. PloS One Current Approaches to Management of Bone Sarcoma in Adolescent and (2019) 14:e0222228. doi: 10.1371/JOURNAL.PONE.0222228 Young Adult Patients. Pediatr Blood Cancer (2022) 69:e29442. doi: 10.1002/ 19. Nestheide S, Bridge JA, Barnes M, Frayer R, Sumegi J. Pharmacologic PBC.29442 Inhibition of Epigenetic Modification Reveals Targets of Aberrant Promoter 2. Whelan J, Le Deley MC, Dirksen U, Le TG, Brennan B, Gaspar N, et al. High- Methylation in Ewing Sarcoma. Pediatr Blood Cancer (2013) 60:1437–46. Dose Chemotherapy and Blood Autologous Stem-Cell Rescue Compared doi: 10.1002/pbc.24526 With Standard Chemotherapy in Localized High-Risk Ewing Sarcoma: 20. Park H-R, Jung W-W, Kim H-S, Park Y-K. Microarray-Based DNA Results of Euro-E.W.I.N.G.99 and Ewing-2008. J Clin Oncol (2018) Methylation Study of Ewing’s Sarcoma of the Bone. Oncol Lett (2014) 36:3110–9. doi: 10.1200/JCO.2018.78.2516 8:1613–7. doi: 10.3892/ol.2014.2322 3. Dirksen U, Brennan B, Le Deley MC, Cozic N, Van Den Berg H, Bhadri V, 21. Pappalardi MB, Keenan K, Cockerill M, Kellner WA, Stowell A, Sherk C, et al. et al. High-Dose Chemotherapy Compared With Standard Chemotherapy Discovery of a First-in-Class Reversible DNMT1-selective Inhibitor With and Lung Radiation in Ewing Sarcoma With Pulmonary Metastases: Results Improved Tolerability and Efficacy in Acute Myeloid Leukemia. Nat Cancer of the European Ewing Tumour Working Initiative of National Groups, 99 (2021) 2:1002–17. doi: 10.1038/S43018-021-00249-X Trial and Ewing 2008. J Clin Oncol (2019) 37:3192–202. doi: 10.1200/ 22. Hurtubise A, Bernstein ML, Momparler RL. Preclinical Evaluation of the JCO.19.00915 Antineoplastic Action of 5-Aza-2’-Deoxycytidine and Different Histone 4. Grünewald TGP, Cidre-Aranaz F, Surdez D, Tomazou EM, De Á lava E, Kovar Deacetylase Inhibitors on Human Ewing’s Sarcoma Cells. Cancer Cell Int H, et al. Ewing Sarcoma. Nat Rev Dis Prim (2018) 4:5. doi: 10.1038/s41572- (2008) 8:16. doi: 10.1186/1475-2867-8-16 018-0003-x 23. Garcı́a-Domı́nguez DJ, Hontecillas-Prieto L, Rodrı́guez-Nú ñez P, Pascual- 5. Solomon DA, Kim T, Diaz-Martinez LA, Fair J, Elkahloun AG, Harris BT, Pasto G, Vila-Ubach M, Garcı́a-Mejı́as R, et al. The Combination of et al. Mutational Inactivation of STAG2 Causes Aneuploidy in Human Epigenetic Drugs SAHA and HCI-2509 Synergistically Inhibits EWS-FLI1 Cancer. Sci (80- ) (2011) 333:1039–43. doi: 10.1126/science.1203619 and Tumor Growth in Ewing Sarcoma. Oncotarget (2018) 9:31397–410. 6. Tirode F, Surdez D, Ma X, Parker M, Le Deley MC, Bahrami A, et al. Genomic doi: 10.18632/oncotarget.25829 Landscape of Ewing Sarcoma Defines an Aggressive Subtype With Co- 24. Hu C, Liu X, Zeng Y, Liu J, Wu F. DNA Methyltransferase Inhibitors Association of STAG2 and TP53 Mutations. Cancer Discovery (2014) Combination Therapy for the Treatment of Solid Tumor: Mechanism and 4:1342–53. doi: 10.1158/2159-8290.CD-14-0622 Clinical Application. Clin Epigenet (2021) 13:166. doi: 10.1186/S13148-021- 7. Crompton BD, Stewart C, Taylor-Weiner A, Alexe G, Kurek KC, Calicchio 01154-X ML, et al. The Genomic Landscape of Pediatric Ewing Sarcoma. Cancer 25. Chen Z, Zhang Y. Role of Mammalian Dna Methyltransferases in Discovery (2014) 4:1326–41. doi: 10.1158/2159-8290.CD-13-1037 Development. Annual Rev Biochem (2020) 89:135–58. doi: 10.1146/ 8. de Alava E. Ewing Sarcoma, an Update on Molecular Pathology With annurev-biochem-103019 Therapeutic Implications. Surg Pathol Clin (2017) 10:575–85. doi: 10.1016/ 26. Berdasco M, Esteller M. Clinical Epigenetics: Seizing Opportunities for J.PATH.2017.04.001 Translation. Nat Rev Genet (2019) 20:109–27. doi: 10.1038/S41576-018- 9. Sheffield NC, Pierron G, Klughammer J, Datlinger P, Schönegger A, Schuster 0074-2 M, et al. DNA Methylation Heterogeneity Defines a Disease Spectrum in 27. Patel N, Black J, Chen X, Marcondes AM, Grady WM, Lawlor ER, et al. DNA Ewing Sarcoma. Nat Med (2017) 23:386–95. doi: 10.1038/nm.4273 Methylation and Gene Expression Profiling of Ewing Sarcoma Primary 10. Riggi N, Suvà ML, Stamenkovic I. Ewing’s Sarcoma. N Engl J Med (2021) Tumors Reveal Genes That are Potential Targets of Epigenetic Inactivation. 384:154–64. doi: 10.1056/NEJMRA2028910 Sarcoma (2012) 2012:498472. doi: 10.1155/2012/498472 11. Kovar H. Blocking the Road, Stopping the Engine or Killing the Driver? 28. Avigad S, Shukla S, Naumov I, Cohen IJ, Ash S, Meller I, et al. Aberrant Advances in Targeting EWS/FLI-1 Fusion in Ewing Sarcoma as Novel Methylation and Reduced Expression of RASSF1A in Ewing Sarcoma. Pediatr Therapy. Expert Opin Ther Targets (2014) 18:1315–28. doi: 10.1517/ Blood Cancer (2009) 53:1023–8. doi: 10.1002/pbc.22115 14728222.2014.947963 29. Gharanei S, Brini AT, Vaiyapuri S, Alholle A, Dallol A, Arrigoni E, et al. 12. Riggi N, Knoechel B, Gillespie SM, Rheinbay E, Boulay G, Suva ML, et al. Ews- RASSF2 Methylation is a Strong Prognostic Marker in Younger Age Patients FLI1 Utilizes Divergent Chromatin Remodeling Mechanisms to Directly With Ewing Sarcoma. Epigenetics (2013) 8:893–8. doi: 10.4161/epi.25617 Activate or Repress Enhancer Elements in Ewing Sarcoma. Cancer Cell 30. Jurkowska RZ, Jurkowski TP, Jeltsch A. Structure and Function of (2014) 26:668–81. doi: 10.1016/j.ccell.2014.10.004 Mammalian Dna Methyltransferases. ChemBioChem (2011) 12:206–22. 13. Tomazou EM, Sheffield NC, Schmidl C, Schuster M, Schonegger A, Datlinger doi: 10.1002/cbic.201000195 P, et al. Epigenome Mapping Reveals Distinct Modes of Gene Regulation and 31. Ravichandran M, Jurkowska RR, Jurkowski TP. Target Specificity of Widespread Enhancer Reprogramming by the Oncogenic Fusion Protein Mammalian DNA Methylation and Demethylation Machinery. Org Biomol EWS-FLI1. Cell Rep (2015) 10:1082–95. doi: 10.1016/j.celrep.2015.01.042 Chem (2018) 16:1419–35. doi: 10.1039/C7OB02574B. pubs.rsc.org. 14. Theisen ER, Selich-Anderson J, Miller KR, Tanner JM, Taslim C, Pishas KI, 32. Jin B, Robertson KD. DNA Methyltransferases, DNA Damage Repair, and et al. Chromatin Profiling Reveals Relocalization of Lysine-Specific Cancer. Adv Exp Med Biol (2013) 754:3–29. doi: 10.1007/978-1-4419-9967- Demethylase 1 by an Oncogenic Fusion Protein. Epigenetics (2021) 16:405– 2_1 24. doi: 10.1080/15592294.2020.1805678 33. Zhou D, Robertson KD. Role of DNA Methylation in Genome Stability. 15. Schmidt O, Nehls N, Prexler C, von Heyking K, Groll T, Pardon K, et al. Class (2016) 24:409–24. doi: 10.1016/B978-0-12-803309-8.00024-0 I Histone Deacetylases (HDAC) Critically Contribute to Ewing Sarcoma 34. Manara MC, Valente S, Cristalli C, Nicoletti G, Landuzzi L, Zwergel C, et al. A Pathogenesis. J Exp Clin Cancer Res (2021) 40:322. doi: 10.1186/S13046- Quinoline-Based Dna Methyltransferase Inhibitor as a Possible Adjuvant in 021-02125-Z Osteosarcoma Therapy. Mol Cancer Ther (2018) 17:1881–92. doi: 10.1158/ 16. Ban J, Aryee DN, Fourtouna A, van der Ent W, Kauer M, Niedan S, et al. 1535-7163.MCT-17-0818 Suppression of Deacetylase SIRT1 Mediates Tumor-Suppressive NOTCH 35. Zwergel C, Fioravanti R, Stazi G, Sarno F, Battistelli C, Romanelli A, et al. Response and Offers a Novel Treatment Option in Metastatic Ewing Novel Quinoline Compounds Active in Cancer Cells Through Coupled Dna Sarcoma. Cancer Res (2014) 74:6578–88. doi: 10.1158/0008-5472.CAN-14- Methyltransferase Inhibition and Degradation. Cancers (Basel) (2020) 12:447. 1736 doi: 10.3390/CANCERS12020447 17. Souza BK, da Costa Lopez PL, Menegotto PR, Vieira IA, Kersting N, 36. Valente S, Liu Y, Schnekenburger M, Zwergel C, Cosconati S, Gros C, et al. Abujamra AL, et al. Targeting Histone Deacetylase Activity to Arrest Cell Selective non-Nucleoside Inhibitors of Human DNA Methyltransferases Growth and Promote Neural Differentiation in Ewing Sarcoma. Mol Active in Cancer Including in Cancer Stem Cells. J Med Chem (2014) Neurobiol (2018) 55:7242–58. doi: 10.1007/s12035-018-0874-6 57:701–13. doi: 10.1021/jm4012627 Frontiers in Endocrinology | www.frontiersin.org 11 May 2022 | Volume 13 | Article 876602 Cristalli et al. DNMTi MC3343 effects in EWS 37. Gros C, Fleury L, Nahoum V, Faux C, Valente S, Labella D, et al. New Insights 53. Chen T, Hevi S, Gay F, Tsujimoto N, He T, Zhang B, et al. Complete on the Mechanism of Quinoline-Based DNA Methyltransferase Inhibitors. Inactivation of DNMT1 Leads to Mitotic Catastrophe in Human Cancer Cells. J Biol Chem (2015) 290:6293–302. doi: 10.1074/jbc.M114.594671 Nat Genet (2007) 39:391–6. doi: 10.1038/ng1982 38. Chik F, Szyf M. Effects of Specific DNMT Gene Depletion on Cancer Cell 54. Milutinovic S, Zhuang Q, Niveleau A, Szyf M. Knockdown of DNA Transformation and Breast Cancer Cell Invasion; Toward Selective DNMT Methyltransferase 1 Triggers an intra-S-phase Arrest of DNA Replication Inhibitors. Carcinogenesis (2011) 32:224–32. doi: 10.1093/CARCIN/BGQ221 and Induction of Stress Response Genes. J Biol Chem (2003) 278:14985–95. 39. de Nigris F, Ruosi C, Napoli C. Clinical Efficiency of Epigenetic Drugs doi: 10.1074/jbc.M213219200 Therapy in Bone Malignancies. Bone (2021) 143:115605. doi: 10.1016/ 55. Norris RE, Adamson PC, Nguyen VT, Fox E. Preclinical Evaluation of the J.BONE.2020.115605 PARP Inhibitor, Olaparib, in Combination With Cytotoxic Chemotherapy in 40. Knox JD, Araujo FD, Bigey P, Slack AD, Price GB, Zannis-Hadjopoulos M, Pediatric Solid Tumors. Pediatric Blood Cancer (2014) 61:145–50. et al. Inhibition of DNA Methyltransferase Inhibits DNA Replication. J Biol doi: 10.1002/pbc.24697 Chem (2000) 275:17986–90. doi: 10.1074/JBC.C900894199 56. Choy E, Butrynski JE, Harmon DC, Morgan JA, George S, Wagner AJ, et al. 41. Milutinovic S, Knox JD, Szyf M. Dna Methyltransferase Inhibition Induces the Phase II Study of Olaparib in Patients With Refractory Ewing Sarcoma Transcription of the Tumor Suppressor p21 WAF1/CIP1/Sdi1. J Biol Chem Following Failure of Standard Chemotherapy. BMC Cancer (2014) 14:1–6. (2000) 275:6353–9. doi: 10.1074/jbc.275.9.6353 doi: 10.1186/1471-2407-14-813/FIGURES/2 42. Furuta T, Takemura H, Liao ZY, Aune GJ, Redon C, Sedelnikova OA, et al. 57. Abbotts R, Topper MJ, Biondi C, Fontaine D, Goswami R, Stojanovic L, et al. Phosphorylation of Histone H2AX and Activation of Mre11, Rad50, and Nbs1 DNA Methyltransferase Inhibitors Induce a BRCAness Phenotype That in Response to Replication-Dependent DNA Double-Strand Breaks Induced Sensitizes NSCLC to PARP Inhibitor and Ionizing Radiation. Proc Natl by Mammalian DNA Topoisomerase I Cleavage Complexes. J Biol Chem Acad Sci USA (2019) 116:22609–18. doi: 10.1073/pnas.1903765116 (2003) 278:20303–12. doi: 10.1074/JBC.M300198200 58. Bagnara GP., Serra M, Giovannini M, Badiali M, Stella M, Montaldi A, et al 43. Guo X, Dai X, Wu X, Cao N, Wang X. Small But Strong: Mutational and Establishment and Characterization of a Primitive Neuroectodermal Tumor Functional Landscapes of Micronuclei in Cancer Genomes. Int J Cancer of Bone Continuous Cell Line (LAP-35). Inter J Cell Cloning (1990) 8:409–24. (2021) 148:812–24. doi: 10.1002/ijc.33300 doi: 10.1002/stem.5530080644 44. Canman CE, Lim DS, Cimprich KA, Taya Y, Tamai K, Sakaguchi K, et al. 59. Guerzoni C, Fiori V, Terracciano M, Manara MC, Moricoli D, Pasello M, et al. Activation of the ATM Kinase by Ionizing Radiation and Phosphorylation of CD99 Triggering in Ewing Sarcoma Delivers a Lethal Signal Through p53 P53. Science (1998) 281:1677–9. doi: 10.1126/SCIENCE.281.5383.1677 Pathway Reactivation and Cooperates With Doxorubicin. Clin Cancer Res 45. You F, Gao C. Topoisomerase Inhibitors and Targeted Delivery in Cancer (2015) 21:146–56. doi: 10.1158/1078-0432.CCR-14-0492 Therapy. Curr Top Med Chem (2019) 19:713–29. doi: 10.2174/ 60. Nanni P, Landuzzi L, Manara MC, Righi A, Nicoletti G, Cristalli C, et al. Bone 1568026619666190401112948 Sarcoma Patient-Derived Xenografts are Faithful and Stable Preclinical 46. Murai J, Huang SYN, Das BB, Renaud A, Zhang Y, Doroshow JH, et al. Models for Molecular and Therapeutic Investigations. Sci Rep (2019) Trapping of PARP1 and PARP2 by Clinical Parp Inhibitors. Cancer Res (2012) 9:12174. doi: 10.1038/s41598-019-48634-y 72:5588–99. doi: 10.1158/0008-5472.CAN-12-2753 61. Chou TC. The Median-Effect Principle and the Combination Index for 47. Min A, Im SA. Parp Inhibitors as Therapeutics: Beyond Modulation of Quantitation of Synergism and Antagonism(1991) (Accessed January 17, Parylation. Cancers (Basel) (2020) 12:394. doi: 10.3390/CANCERS 2022). 12020394 48. Cancer Genome Atlas Research Network, Ley TJ, Miller C, Ding L, Raphael BJ, Conflict of Interest: The authors declare that the research was conducted in the Mungall AJ, et al. Genome Atlas Research Network Genomic and Epigenomic absence of any commercial or financial relationships that could be construed as a Landscapes of Adult De Novo Acute Myeloid Leukemia. N Engl J Med (2013) potential conflict of interest. 368:2059–74. doi: 10.1056/NEJMoa1301689 49. Izzo F, Lee SC, Poran A, Chaligne R, Gaiti F, Gross B, et al. DNA Methylation Publisher’s Note: All claims expressed in this article are solely those of the authors Disruption Reshapes the Hematopoietic Differentiation Landscape. Nat Genet and do not necessarily represent those of their affiliated organizations, or those of (2020) 52:378–87. doi: 10.1038/S41588-020-0595-4 the publisher, the editors and the reviewers. Any product that may be evaluated in 50. Christman JK. 5-Azacytidine and 5-aza-2′-deoxycytidine as Inhibitors of this article, or claim that may be made by its manufacturer, is not guaranteed or DNA Methylation: Mechanistic Studies and Their Implications for Cancer endorsed by the publisher. Therapy. Oncogene (2002) 21:5483–95. doi: 10.1038/sj.onc.1205699 51. Milutinovic S, Brown SE, Zhuang Q, Szyf M. DNA Methyltransferase 1 Knock Copyright © 2022 Cristalli, Manara, Valente, Pellegrini, Bavelloni, De Feo, Blalock, Di Down Induces Gene Expression by a Mechanism Independent of DNA Bello, Piñeyro, Merkel, Esteller, Tirado, Mai and Scotlandi. This is an open-access Methylation and Histone Deacetylation. J Biol Chem (2004) 279:27915–27. article distributed under the terms of the Creative Commons Attribution License doi: 10.1074/jbc.M312823200 (CC BY). The use, distribution or reproduction in other forums is permitted, provided 52. Unterberger A, Andrews SD, Weaver ICG, Szyf M. Dna Methyltransferase 1 the original author(s) and the copyright owner(s) are credited and that the original Knockdown Activates a Replication Stress Checkpoint. Mol Cell Biol (2006) publication in this journal is cited, in accordance with accepted academic practice. No 26:7575–86. doi: 10.1128/MCB.01887-05 use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Endocrinology | www.frontiersin.org 12 May 2022 | Volume 13 | Article 876602