Pharmacological inhibition of EZH2 as a promising differentiation therapy in embryonal RMS
- Roberta Ciarapica†1Email author,
- Elena Carcarino†2,
- Laura Adesso†1,
- Maria De Salvo†1,
- Giorgia Bracaglia†1,
- Pier Paolo Leoncini1,
- Alessandra Dall’Agnese2,
- Federica Verginelli1,
- Giuseppe M Milano1,
- Renata Boldrini3,
- Alessandro Inserra4,
- Stefano Stifani5,
- Isabella Screpanti6,
- Victor E Marquez7,
- Sergio Valente8,
- Antonello Mai8,
- Pier Lorenzo Puri2, 9,
- Franco Locatelli1, 10,
- Daniela Palacios2 and
- Rossella Rota1Email author
© Ciarapica et al.; licensee BioMed Central Ltd. 2014
Received: 4 November 2013
Accepted: 12 February 2014
Published: 27 February 2014
Embryonal Rhabdomyosarcoma (RMS) is a pediatric soft-tissue sarcoma derived from myogenic precursors that is characterized by a good prognosis in patients with localized disease. Conversely, metastatic tumors often relapse, leading to a dismal outcome. The histone methyltransferase EZH2 epigenetically suppresses skeletal muscle differentiation by repressing the transcription of myogenic genes. Moreover, de-regulated EZH2 expression has been extensively implied in human cancers. We have previously shown that EZH2 is aberrantly over-expressed in RMS primary tumors and cell lines. Moreover, it has been recently reported that EZH2 silencing in RD cells, a recurrence-derived embryonal RMS cell line, favors myofiber-like structures formation in a pro-differentiation context. Here we evaluate whether similar effects can be obtained also in the presence of growth factor-supplemented medium (GM), that mimics a pro-proliferative microenvironment, and by pharmacological targeting of EZH2 in RD cells and in RD tumor xenografts.
Embryonal RMS RD cells were cultured in GM and silenced for EZH2 or treated with either the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) that induces EZH2 degradation, or with a new class of catalytic EZH2 inhibitors, MC1948 and MC1945, which block the catalytic activity of EZH2. RD cell proliferation and myogenic differentiation were evaluated both in vitro and in vivo.
Here we show that EZH2 protein was abnormally expressed in 19 out of 19 (100%) embryonal RMS primary tumors and cell lines compared to their normal counterparts. Genetic down-regulation of EZH2 by silencing in GM condition reduced RD cell proliferation up-regulating p21Cip1. It also resulted in myogenic-like differentiation testified by the up-regulation of myogenic markers Myogenin, MCK and MHC. These effects were reverted by enforced over-expression of a murine Ezh2, highlighting an EZH2-specific effect. Pharmacological inhibition of EZH2 using either DZNep or MC inhibitors phenocopied the genetic knockdown of EZH2 preventing cell proliferation and restoring myogenic differentiation both in vitro and in vivo.
These results provide evidence that EZH2 function can be counteracted by pharmacological inhibition in embryonal RMS blocking proliferation even in a pro-proliferative context. They also suggest that this approach could be exploited as a differentiation therapy in adjuvant therapeutic intervention for embryonal RMS.
KeywordsEZH2 Histone methyltransferase rhabdomyosarcoma Polycomb proteins Differentiation DZnep EZH2 catalytic inhibitors
Pediatric rhabdomyosarcoma (RMS) is a locally invasive soft-tissue sarcoma with a predisposition to metastasize that accounts for ~ 30% of all soft-tissue sarcomas (STS) and for 7-8% of all solid tumors in childhood . Embryonal RMS is the major histopathologic subtype, accounting for 60% of all RMS cases and, when nonmetastatic, shows a 5-year overall survival of 70% . Childhood cancer statistics show that the outcome for young patients with RMS has tremendously improved from 53% in 1975–1978 to 68% in 1979–1982 , but unfortunately current treatments for embryonal RMS in the metastatic form often do not respond to therapy. Indeed, metastatic or relapsed forms, even if they can undergo complete remission with secondary therapy, are often characterized by poor long-term prognosis and dismal outcome [4–6]. Moreover, children who relapse need to be closely monitored for a long time as anti-cancer therapy side effects may persist or develop months or years after treatment. Therefore, novel more specific and less toxic treatment approaches, such as molecular targeted therapies, are under study. Since RMS cells share characteristics of skeletal muscle precursors, the most reliable theory about the origin of RMS suggests that perturbations of the normal mesenchymal development of the skeletal muscle lineage might have a causative role . Consistently, results from some groups and ours recently suggest that a differentiation therapy seems to represent an alternative way to reduce the aggressiveness of cancer cells, not by exerting cytotoxicity but by restoring the differentiation fate of tumor cells [8–12]. Indeed, under specific treatments, RMS cells progress toward less proliferating myoblast-like cells that are capable to develop myotube-like structure. The methyltransferase Polycomb Group (PcG) protein Enhancer of zeste homolog 2 (EZH2), the catalytic factor of the Polycomb Repressor Complex 2 (PRC2), represses gene transcription by silencing target genes through methylation of histone H3 on lysine 27 (H3K27me3) and it has been shown to prevent cell differentiation and promote cell proliferation in several tissues . Increasing evidence demonstrates that EZH2 is not only aberrantly expressed in several types of human cancers, but often behaves as a molecular biomarker of poor prognosis [14–21]. EZH2 was clearly shown to act as a negative regulator of skeletal muscle differentiation favoring the proliferation of myogenic precursors [22–24]. This function results from an EZH2-dependent direct repression of genes related to myogenic differentiation . We previously reported that EZH2 is markedly expressed in the RMS context, both in cell lines and primary tumors compared to their normal counterparts . The first evidence of the role of EZH2 as a main player in the inability of RMS cells to undergo differentiation has been recently reported in vitro for the embryonal RMS cell line RD, established from a tumor recurrence, through EZH2 genetic silencing upon serum withdrawal .
Here, after having shown that EZH2 was de-regulated in a cohort of primary embryonal RMS, we evaluated whether it was possible to boost the differentiation capability of embryonal RMS RD cells after EZH2 inhibition even in serum-enriched culture conditions. As an additional promising approach, we investigated whether pharmacological inhibition of EZH2 in RD cells by either reducing its expression or catalytically inhibiting its activity might be detrimental for cancer cell proliferation both in vitro and in vivo. Our data demonstrate that EZH2 down-regulation restores the myogenic differentiation of RD cells with no need to reduce serum (cultured in growth medium), and that pharmacological inhibition of EZH2 is a feasible way to restrain the tumor-promoting potential in embryonal RMS.
Additional file 1: Supplementary Methods.
RD embryonal RMS cell line was obtained from American Type Culture Collection (Rockville, MD). A204 and RH18 embryonal RMS cell lines were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). Normal Human Skeletal Muscle cells (SkMC; myoblasts) were obtained from PromoCell (Heidelberg Germany).
Cells were lysed and assayed as previously reported . Briefly, cells were lysed in cytoplasm lysis buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.2 mM EDTA, 1 mM DTT), containing protease inhibitors, 0.5 mM phenylmethylsulfonylfluoride (PMSF) and 0.6% Nonidet P-40 (Sigma Chemical Co., St Louis, MO, USA). Lysates were centrifuged at 10.000 rpm 10 min at 4°C and the supernatants (cytoplasmic fractions) were split into aliquots and rapidly frozen. The nuclear pellet was washed in buffer A without Nonidet P-40 and finally resuspended in nuclear lysis buffer B (20 mM HEPES pH 7.9, 0.4 M NaCl, 2 mM EDTA, 1 mM DTT), containing protease inhibitors and 1 mM PMSF (Sigma Chemical Co., St Louis, MO, USA). Samples were incubated on ice 30 min and centrifuged at 13.000 rpm 10 min at 4°C; the supernatants (nuclear fractions) were split into aliquots and rapidly frozen or used for western blot analysis.
Western blotting was performed on whole-cell lysates and histone extracts as previously described [27, 28]. Briefly, cells were lysed in RIPA buffer (50 mM Tris–HCl pH7.4, 150 mM NaCl, 1 mM EDTA, 1% D.O.C. (Na), 0,1% SDS, 1% Triton X-100) containing protease inhibitors (Sigma Chemical Co., St Louis, MO, USA). Lysates were sonicated, incubated on ice 30 min and centrifugated at 10,000 g 20 min at 4°C. Supernatants were used as total lysates. Protein concentrations were estimated with the BCA protein assay (Pierce, Rockford, IL). EZH2 was detected using the EZH2 antibody (612666; Transduction LaboratoriesTM, BD, Franklin Lakes, NJ). Antibodies against Myogenin (F5D) and Myosin Heavy Chain (Meromyosin, MF20) were obtained from the Developmental Studies Hybridoma Bank at the University of Iowa (DSHB, Iowa City, IA). Antibodies against p21Cip1 (sc-397), β-actin (sc-1616) and all secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Antibodies against Troponin I (4002) were obtained from Cell Signaling (Beverly, MA). The antibody against the Topoisomerase IIβ was obtained from Sigma Aldrich (Sigma Chemical Co., St Louis, MO, USA). Antibody against against Histone 3 (H3), H3K27me3 (Lys27) and H3K4me3 (Lys4) were obtained from Millipore (EMD Millipore Corporation, Billerica, MA, USA). Antibody against α-tubulin (ab4074) was from Abcam (Cambridge, UK). All the antibodies were used in accordance with the manufacturer’s instructions.
Cells were harvested and washed twice with ice-cold Phosphate Buffered saline (PBS) 1X supplemented with 5 mM Sodium Butyrate and resuspended in Triton Extraction Buffer (TEB: PBS, 0.5% Triton X 100 (v/v)) containing 2 mM PMSF and 0.02% (w/v) NaN3 (107 cells/ml) and lysated on ice for 10 min. Lysates were centrifuged at 2000 rpm for 10 min at 4°C and the pellets were washed in half volume of TEB and centrifuged.Histones were extracted O/N at 4°C from pellets resuspended in 0.2 N HCl (4×107 cells/ml). Samples were then centrifuged and supernatants were used for western blot analysis.
Transient RNA interference
Cells were sequentially transfected by 2 subsequent rounds (24 h), to secure efficient cell silencing, with ON-TARGETplus SMART pool siRNA targeting different regions of the EZH2 transcript (L-004218-00) or non-targeting siRNA (control; D-001206-13), previously validated in other publications [14, 29, 30] (both from Dharmacon, Thermo Fisher Scientific, Lafayette, CO).
Real time qRT-PCR
Total RNA was extracted using TRizol (Invitrogen, Carlsbad, CA) and analyzed by real-time RT-qPCR for relative quantification of gene expression  using Taqman gene assays (Applied Biosystems, Life Technologies, Carlsbad, CA) for GAPDH (Hs99999905_m1), EZH2 (Hs01016789_m1), Myogenin (Hs01072232_m1), MCK (Hs00176490_m1) and p21 (Hs00355782_m1). For the relative quantification of Murine Ezh2 and MHC mRNA the SYBR-green method was used (Applied Biosystems, Life Technologies, Carlsbad, CA) with primers previously reported  or available on request. The values were normalized to the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. An Applied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosystems, Life Technologies, Carlsbad, CA) was used for measurements.
Murine Ezh2 over-expression
Flag-tagged murine Ezh2, cloned into the pMSCV retroviral vector (Addgene, Cambridge, MA) or control empty vector, both co-expressing the Green Fluorescent Protein (GFP) as reporter gene, were kindly obtained from G. Caretti. Phoenix ampho cells were obtained from ATCC and cultured in DMEM supplemented with 10% FBS (growth medium, GM).Transient transfection of Phoenix ampho cells were performed using lipofectamine reagent (Invitrogen, Carlsbad, MA) and viral particles were collected after 48 h. Supernatant containing viral particles were used to infect RD cells O/N in the presence of 8 ug/ml of polybrene.
Immunofluorescence for MHC detection
Immunofluorescence to visualize MHC was performed as previously described using the MF-20 antibody (Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, IA) . Briefly, cells were washed 3 times in PBS, fixed 10 min in 4% PFA and permealized 5 min with 0.2% Triton X-100 in PBS. After 30 min in PBS containing 3% bovine serum albumin, slides were incubated 1 h at room temperature with the MF-20 antibody against myosin heavy chain (MHC; Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, IA). After 2 washing in PBS, cells were treated with a rhodamine-conjugated secondary antibody (Millipore, Temecula, CA). After being counterstained with DAPI, chamber slides were mounted in GelMount (Biomeda, Foster City, CA, USA). Images were acquired with an Eclipse E600 fluorescence microscope, through LUCIA software version 4.81 (Nikon, Sesto Fiorentino, Firenze, Italy).
Cell cycle and apoptosis assays
Cells were transfected 24 h after seeding (Day 0) with siRNAs and after 24 h transfected again. Then, they were harvested and counted at the reported time points. For pharmacological treatments RD cells were treated with the S-adenosyl-L-homocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) and MC1945 for 24 h, 48 h, 72 h and 96 h. For cell cycle assay, cells were harvested by trypsinization at the indicated time points, washed in ice-cold PBS, fixed in 50% PBS and 50% acetone/methanol (1:4 v/v) for at least 1 h and, after removing alcoholic fixative, stained in the dark with a solution containing 50 μg/ml Propidium Iodide (PI) and 100 μg/ml RNase (Sigma) for 30 min at room temperature. For quantification of apoptosis, cells were harvested, washed twice with ice-cold PBS and stained in calcium-binding buffer with APC-conjugated Annexin V and 7-Aminoactinomycin D (7-AAD) using Annexin V apoptosis detection kit (BD Pharmingen, San Diego, CA), according to manufacturer’s recommendations. Samples were analyzed within 1 h. The stained cells were analyzed for both cell cycle and apoptosis by fluorescence-activated cell sorting using a FACSCantoII equipped with a FACSDiva 6.1 CellQuest software (Becton Dickinson Instrument, San Josè, CA).
Chromatin immunoprecipitation (ChIP)
ChIP assay was performed as previously described (70) with minor modifications. Briefly, chromatin was cross-linked in 1% formaldehyde for 15 min at room temperature and quenched by addition of glycine at 125 mM final concentration for 5 min at room temperature before being placed on ice. Cells were washed twice with ice-cold PBS containing 1 mM PMSF and 1X protease inhibitors, resuspended in ice-cold cell lysis buffer (10 mM Tris–HCl pH 8, 10 mM NaCl, 0.2% NP-40, 1 mM PMSF and 1X protease inhibitors) and incubated on ice for 20 minutes. After centrifugation at 4000 rpm for 5 min, nuclei were resuspended in ice-cold nuclear lysis buffer (50 mM TrisHCl pH 8.1; 10 mM EDTA; 1% SDS, 1 mM PMSF and 1X protease inhibitors) and left on ice for 10 min. Chromatin was then sonicated to an average fragment size of 200–300 bp using a Bioruptor and diluted ten times with IP dilution buffer (16.7 mM Tris–HCl pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 0.01%SDS, 1.1% Triton X-100, 1 mM PMSF and 1X protease inhibitors). Diluted chromatin was pre-cleared using protein G-agarose magnetic beads (Invitrogen) for 1 hour at 4°C and incubated with the corresponding antibodies O/N at 4°C. The following antibodies were used: anti-acetylated histone H3, anti-trimethyl Lysine 27 histone H3 and anti-trimethyl Lysine 4 histone H3 (EMD Millipore Corporation, Billerica, MA, USA) and anti-Ezh2 (Diagenode s.a. Liège, Belgium). Immunoprecipitated chromatin was recovered by incubation with protein G-agarose magnetic beads (Invitrogen, Carlsbad, CA) for 2 hours at 4°C. Beads were washed twice with low salt washing buffer (20 mM Tris–HCl pH8, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 150 mM NaCl), twice with high salt washing buffer (20 mM Tris–HCl pH8, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 500 mM NaCl) and twice with TE before incubating them with elution buffer (10 mM Tris–HCl pH8 1 mM EDTA, 1% SDS) for 30 minutes at 65°C. Cross-linking was then reverted O/N at 65°C and samples were treated with proteinase K for 2 hours at 42°C. The DNA was finally purified by phenol: chloroform extraction in the presence of 0.4 M LiCl and ethanol precipitated. Purified DNA was resuspended in 50 μl of water. Real-time PCR was performed on input samples and equivalent amounts of immunoprecipitated material with the SYBR Green Master Mix (Applied Biosystems, Life Technologies, Carlsbad, CA). Primer sequences are available on request.
Xenograft experiments and immunohistochemistry
Athymic 6-week-old female BALB/c nude mice (nu + \nu+) were purchased from Charles River. Procedures involving animals and their care were conformed to institutional guidelines that comply with national and international laws and policies (EEC Council Directive 86\609, OJ L 358, 12 December 1987). RD cell suspensions in PBS (10×106 cells in 100 μl) were injected subcutaneously into the posterior flanks of nude mice. When the tumors became palpable, i.e., about approximately 70–80 mm3, mice were intraperitoneally injected with MC1945 (2.5 mg/Kg) or control vehicle (DMSO) twice daily, 3 days per week for 3 weeks when mice were sacrificed. No visible signs of toxicity such as weight loss or behavioral change were seen with the compound dose and treatment timing used, as already reported [32, 33]. Tumor volume was measured by caliper with the following formula: tumor volume (mm3) = L × S2 × π/6 wherein L is the longest and S the shorter diameter and π/6 is a constant to calculate the volume of an ellipsoid, as described . Representative tumor growth data were obtained from 3 mice per treatment/group. In a parallel experiment, 3 mice per treatment/group were sacrificed 12 days after the first treatment, i.e. the exponential tumor growth phase, and xenografts removed after tumor volume measurement. Portions of the excised tumors embedded in paraffin were used for immunohistochemical analysis. Sections of 10 μm cut from xenograft blocks were stained with hematoxylin/eosin. Five μm serial sections were subjected to immunohistochemistry for the expression of EZH2 and Ki67 with methods and antibodies reported below for primary human RMS samples. The MF-20 antibody (DSHB, USA) was used to detect the expression of MHC. Counterstaining was carried out with Gill’s hematoxyline (Bio-Optica, MI, Italy). Sections were dehydrated and mounted in non-aqueous mounting medium. Images were acquired under an Eclipse E600 microscope (Nikon) through the LUCIA software, version 4.81 (Nikon) with a Nikon Digital Camera DXM1200F.
Immunohistochemistry on RMS primary tissues
Clinical and histopathological features of pediatric patients with embryonal rhabdomyosarcoma (RMS) (n=19)
Embryonal RMS n (%)
Orbit-genitourinary tract-head and neck$
< 5 cm
≥ 5 cm
Expression of markers
EZH2 (positive cells/microscopic field)
40 (range 29-44)
Ki67 (positive cells/microscopic field)
20 (range 17-29)
The Student’s t-test was done to assess the difference between various treatments. Statistical significance was set at a two-tailed P value less than 0.05. All analyses were performed with SPSS 11.5.1 for Windows Package (© SPSS, Inc., 1989–2002 and © LEADTOOLS 1991–2000, LEAD Technologies, Inc., Chicago, IL).
EZH2 protein is expressed in embryonal RMS primary tumors
Down-regulation of EZH2 reduces embryonal RMS cell proliferation
To define whether EZH2 was required to sustain embryonal RMS proliferation, as it occurs for other kind of human cancers [36, 37], cell proliferation of the established embryonal RMS cell line RD, derived from a tumor recurrence , and cultured in growth medium, i.e. supplemented with 10% serum, was evaluated upon EZH2 genetic silencing. After two consecutive rounds of RNA interference with siRNAs against EZH2, the level of EZH2 protein expression in RD cells decreased more than 80% starting from 24 h after the first siRNA transfection (Figure 2d). In this condition, EZH2 knockdown in RD cells resulted in 36 ± 6% and 48 ± 8% inhibition of cell proliferation at day 3 and 4, respectively, compared to cells treated with a non-targeting control siRNA (Figure 2c). We confirmed the anti-proliferative effect of EZH2 siRNA with MTT assay (Additional file 2: Figure S1). To ascertain that the growth inhibition was the result of a reduced activity of EZH2, we analyzed the methylation status of Lys 27 on histone H3. Moreover, the Lys 4, a residue not methylated by EZH2, was also evaluated for methylation. We observed a global decrease of trimethylated Lys 27 (H3K27me3), but not of trimethylated Lys 4 (H3K4me3) at day 3 post-EZH2 siRNA transfection (Figure 2e), suggesting that EZH2-dependent histone methylation was specifically impaired upon EZH2 siRNA. These results indicate that over-expressed EZH2 sustains proliferation in embryonal RMS cells.
Down-regulation of EZH2 is sufficient to restore embryonal RMS cell myogenic differentiation in growth medium
Pharmacological inhibition of EZH2 prevents embryonal RMS cell proliferation
Altogether, these findings clearly suggest that pharmacological inhibition of EZH2 affects the proliferative potential of embryonal RMS cells and phenocopies the cell-specific effect of siRNA-mediated EZH2 depletion.
Pharmacological inhibition of EZH2 restores myogenic differentiation of embryonal RMS cells even in the presence of growth medium
Pharmacological inhibition of EZH2 induces myogenic differentiation in embryonal RMS tumor xenografts
In the last decade, to trace the way for developing innovative anti-cancer therapies, several groups focused their pre-clinical research on the modulation of epigenetic regulators often aberrantly expressed in cancer. Due to the fact that epigenetic processes are key players in cell tissue specification during the embryonal life, this approach seems to be particularly captivating for those cancers, such as pediatric embryonal RMS, in which the pathogenic mechanisms involve the deregulation of genes controlling the lineage commitment . Among these, the histone methyltransferase EZH2 is a fundamental negative regulator of myogenic precursor differentiation by repressing the expression of myogenic genes through the H3K27me3 mark deposition on the promoters of myogenic genes [22, 28]. We recently reported that EZH2 transcripts were aberrantly expressed in both embryonal RMS primary tumors and in the RD cell line [25, 35]. In this study, we report that, as for transcripts, EZH2 protein is aberrantly over-expressed in 19 out of 19 embryonal RMS primary tumors compared to normal muscle tissues, thus indicating that the high level of expression of EZH2 is a common molecular lesion of embryonal RMS neoplasia.
Moreover, a recent report indicates that the RD cell line, derived from an embryonal RMS local recurrence and thus representative of an aggressive tumor , may reactivate muscle-specific genes and develop a partial recovery of myocyte phenotype following EZH2 knockdown when depleted of serum . We show here that it is possible to revert the tumor phenotype of the RD cell line by silencing EZH2 even under proliferative stimuli such as in a serum-enriched molecular context. The final result is the acquisition of a myogenic phenotype, by the de-repression of myogenic genes Myogenin and MCK, which can be rescued by the over-expression of a murine Ezh2 not targeted by the used siRNA oligos. More importantly, as a proof-of-concept we report that in these pro-proliferative conditions, pharmacological inhibition of EZH2 by two different approaches, i.e. by decreasing its availability or hampering its activity, is capable to prevent the proliferation and allow the recovery of myogenic differentiation of these cells in vitro and in vivo. In line with the inability of RD cells to undergo terminal differentiation in conditions that induce myotube formation in normal, non-tumorigenic, myoblasts (REF), low-serum differentiation medium did not potentiate the effect of EZH2 depletion/inactivation on the myogenic-like characteristics vs growth medium. Consistently, EZH2 expression is not modulated by serum deprivation in RD cells (data not shown). Small molecule inhibitors of histone methyltransferases are emerging  and a number of novel EZH2 inhibitors are under preclinical evaluation in other types of cancer [43–45].
Here we treated RD RMS cells with the prototype inhibitor of PRC2, deazaneplanocin A (DZNep), which acts through an indirect mechanism by reducing the level of EZH2 protein [17, 46]. Recently, DZNep has been reported to be effective in several preclinical studies favoring apoptosis and/or differentiation of tumor cells [39, 47–49]. We found that DZNep arrested RD proliferation in a dose-dependent manner with a concomitant down-regulation of EZH2 protein levels and a decrease in global levels of H3K27me3, while the levels of the other repressive mark H3K9me3 remained unchanged, suggesting an EZH2-specific effect at the doses utilized. Strikingly, in the same growth condition DZNep induced the appearance of MHC-positive multinucleated myotube-like structure in RD cells, accompanied by the activation of myogenic genes such as Myogenin and MCK, and with no signs of apoptosis. The observation that in RMS DZNep induces myogenic differentiation instead of apoptosis, the general effect that DZNep has in other human cancer, suggests that its inhibition toward EZH2 is quite specific being pro-differentiative. However, since DZNep may affect other methyltransferases, we enrolled in our study also two molecules belonging to a new class of catalytic inhibitors, validated against a panel of histone methyltransferases [32, 40], MC1948, which has been already validate as EZH2 inhibitor in myoblasts  and a new, more effective, derivative, MC1945. Both MC inhibitors phenocopied the effects of DZNep and EZH2 genetic depletion in vitro, indicating a common mechanism of action. More importantly we observed that MC1945 was able to restrain tumor growth of RD xenografts in nude mice inducing tumor cells differentiation in vivo. Pharmacological inhibition of EZH2 by using a new EZH2 inhibitor has been recently shown to induce anti-tumoral effects in malignant rhabdoid tumor (MRT) cells deleted for SMARCB1. Importantly, this result highlights the dependency of SMARCB1-mutant/deleted MRT tumorigenicity on EZH2. However, the Authors showed no effects of the inhibitor on SMARCB1-wild-type RD cells that were cultured in medium replenished with the drug on day 4 . Differently, we treated RD cells with new doses of inhibitors every day since this approach was defined as effective during preliminary experiments. As a consequence, in our experimental protocol tumor cells were in contact with fresh drug each 24 h. These diverse approaches could be responsible for the difference in the response to pharmacological inhibitors.
In summary, here we present a preclinical study in which the experimental evidence indicates that the pharmacological targeting of EZH2 might represent a way to reduce the aggressiveness of RMS, promoting a more differentiated phenotype and thus enlarging the scenery of the future clinical intervention to treat this type of tumors.
Collectively our data provide evidence that EZH2 abnormal over-expression is responsible for both sustaining proliferation and inhibiting myogenic differentiation of embryonal RMS. More importantly, our results indicate that pharmacological targeting of EZH2 might represent a potential feasible approach to be used as adjuvant treatment for making conventional therapy more effective on less aggressive and more differentiated RMS.
RC is a Junior Scientist and LA, GB, MDS, PPL and FV are PhD and doctoral fellows working on the transcriptional regulation of pediatric cancers in the Laboratory of Angiogenesis directed by RR. DP is a Junior Scientist and EC is a doctoral fellow working on the role of developmental pathway in rhabdomyosarcoma. AD is a doctoral fellow working on the developmental mechanisms in muscle cells in the Laboratory directed by PLP. GMM, RB and AI are Oncologist, Pathologist and Surgeon of the oncology group, respectively. SS is a PhD and Full Professor in Neuroscience and a Chercheur National; Fonds de la Recherche en Sante du Quebec. IS is a MD and Full Professor committed to the preclinical and clinical research against pediatric cancers. VEM and AM are MD and Full Professors in Biochemistry with long lasting experience in biochemical drugs production and testing. SV is a PhD working in the Laboratory directed by AM. PLP is a MD and Full Professor with long lasting experience in the study of skeletal muscle and soft tissue sarcomas. FL is an MD and Full Professor of Pediatrics and the Head of the Oncohematology Dept with a long standing experience in preclinical research and clinical management of pediatric tumor patients. RR is a PhD and the Head of the Laboratory of Angiogenesis with experience in mechanisms that regulate gene expression and cell growth in pediatric cancers.
Skeletal Muscle Cells
Growth factor-supplemented Medium
Enhancer of Zeste of Homologue 2
- PcG protein:
Polycomb Group protein
Polycomb Repressor Complex 2
small interfering RNA
Myosin Heavy Chain
Muscle Creatine Kinase
S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A
- MC1948 and MC1945:
catalytic EZH2 inhibitors
trimethylated lysine 27 on Histone 3
trimethylated lysine 4 on Histone 3
trimethylated lysine 9 on Histone 3.
We thank E. Giorda for FACS analysis. Myogenin (Wright WE), and MF20 (Fishman DA) antibodies were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. We wish to thank G. Caretti for the Ezh2 murine plasmid and control. SS is a Chercheur National of the Fonds de la Recherche en Santé du Quebec.
Associazione Italiana per la Ricerca sul Cancro (AIRC, 10338) and Italian Ministry of Health Ricerca Corrente (RR); Association for International Cancer Research (AICR-UK, 12–0168) (DP); AIRC 5 per mille (FL); NIH Intramural Research Program, National Cancer Institute, CCR (VEM); PRIN 2009PX2T2E, FIRB RBFR10ZJQT, and FP7 Project BLUEPRINT/282510 (AM).
- Loeb DM, Thornton K, Shokek O: Pediatric soft tissue sarcomas. Surg Clin North Am. 2008, 88 (3): 615-627. 10.1016/j.suc.2008.03.008.View ArticlePubMedPubMed CentralGoogle Scholar
- Sultan I, Ferrari A: Selecting multimodal therapy for rhabdomyosarcoma. Expert Rev Anticancer Ther. 2010, 10 (8): 1285-1301. 10.1586/era.10.96.View ArticlePubMedGoogle Scholar
- Smith MA, Seibel NL, Altekruse SF, Ries LA, Melbert DL, O’Leary M, Smith FO, Reaman GH: Outcomes for children and adolescents with cancer: challenges for the twenty-first century. J Clin Oncol. 2010, 28 (15): 2625-2634. 10.1200/JCO.2009.27.0421.View ArticlePubMedPubMed CentralGoogle Scholar
- Pappo AS, Anderson JR, Crist WM, Wharam MD, Breitfeld PP, Hawkins D, Raney RB, Womer RB, Parham DM, Qualman SJ, Grier HE: Survival after relapse in children and adolescents with rhabdomyosarcoma: A report from the Intergroup Rhabdomyosarcoma Study Group. J Clin Oncol. 1999, 17 (11): 3487-3493.PubMedGoogle Scholar
- Mazzoleni S, Bisogno G, Garaventa A, Cecchetto G, Ferrari A, Sotti G, Donfrancesco A, Madon E, Casula L, Carli M: Outcomes and prognostic factors after recurrence in children and adolescents with nonmetastatic rhabdomyosarcoma. Cancer. 2005, 104 (1): 183-190. 10.1002/cncr.21138.View ArticlePubMedGoogle Scholar
- Oberlin O, Rey A, Lyden E, Bisogno G, Stevens MC, Meyer WH, Carli M, Anderson JR: Prognostic factors in metastatic rhabdomyosarcomas: results of a pooled analysis from United States and European cooperative groups. J Clin Oncol. 2008, 26 (14): 2384-2389. 10.1200/JCO.2007.14.7207.View ArticlePubMedPubMed CentralGoogle Scholar
- Tapscott SJ, Thayer MJ, Weintraub H: Deficiency in rhabdomyosarcomas of a factor required for MyoD activity and myogenesis. Science. 1993, 259 (5100): 1450-1453. 10.1126/science.8383879.View ArticlePubMedGoogle Scholar
- Wang H, Garzon R, Sun H, Ladner KJ, Singh R, Dahlman J, Cheng A, Hall BM, Qualman SJ, Chandler DS, Croce CM, Guttridge DC: NF-kappaB-YY1-miR-29 regulatory circuitry in skeletal myogenesis and rhabdomyosarcoma. Cancer Cell. 2008, 14 (5): 369-381. 10.1016/j.ccr.2008.10.006.View ArticlePubMedGoogle Scholar
- Taulli R, Bersani F, Foglizzo V, Linari A, Vigna E, Ladanyi M, Tuschl T, Ponzetto C: The muscle-specific microRNA miR-206 blocks human rhabdomyosarcoma growth in xenotransplanted mice by promoting myogenic differentiation. J Clin Invest. 2009, 119 (8): 2366-2378.PubMedPubMed CentralGoogle Scholar
- Raimondi L, Ciarapica R, De Salvo M, Verginelli F, Gueguen M, Martini C, De Sio L, Cortese G, Locatelli M, Dang TP, Carlesso N, Miele L, Stifani S, Limon I, Locatelli F, Rota R: Inhibition of Notch3 signalling induces rhabdomyosarcoma cell differentiation promoting p38 phosphorylation and p21(Cip1) expression and hampers tumour cell growth in vitro and in vivo. Cell Death Differ. 2012, 19 (5): 871-881. 10.1038/cdd.2011.171.View ArticlePubMedGoogle Scholar
- Puri PL, Wu Z, Zhang P, Wood LD, Bhakta KS, Han J, Feramisco JR, Karin M, Wang JY: Induction of terminal differentiation by constitutive activation of p38 MAP kinase in human rhabdomyosarcoma cells. Genes Dev. 2000, 14 (5): 574-584.PubMedPubMed CentralGoogle Scholar
- MacQuarrie KL, Yao Z, Fong AP, Diede SJ, Rudzinski ER, Hawkins DS, Tapscott SJ: Comparison of genome-wide binding of MyoD in normal human myogenic cells and rhabdomyosarcomas identifies regional and local suppression of promyogenic transcription factors. Mol Cell Biol. 2013, 33 (4): 773-784. 10.1128/MCB.00916-12.View ArticlePubMedPubMed CentralGoogle Scholar
- Sparmann A, van Lohuizen M: Polycomb silencers control cell fate, development and cancer. Nat Rev Cancer. 2006, 6 (11): 846-856. 10.1038/nrc1991.View ArticlePubMedGoogle Scholar
- Bracken AP, Pasini D, Capra M, Prosperini E, Colli E, Helin K: EZH2 is downstream of the pRB-E2F pathway, essential for proliferation and amplified in cancer. EMBO J. 2003, 22 (20): 5323-5335. 10.1093/emboj/cdg542.View ArticlePubMedPubMed CentralGoogle Scholar
- Raaphorst FM, Meijer CJ, Fieret E, Blokzijl T, Mommers E, Buerger H, Packeisen J, Sewalt RA, Otte AP, van Diest PJ: Poorly differentiated breast carcinoma is associated with increased expression of the human polycomb group EZH2 gene. Neoplasia. 2003, 5 (6): 481-488.View ArticlePubMedPubMed CentralGoogle Scholar
- Varambally S, Dhanasekaran SM, Zhou M, Barrette TR, Kumar-Sinha C, Sanda MG, Ghosh D, Pienta KJ, Sewalt RG, Otte AP, Rubin MA, Chinnaiyan AM: The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature. 2002, 419 (6907): 624-629. 10.1038/nature01075.View ArticlePubMedGoogle Scholar
- Tan J, Yang X, Zhuang L, Jiang X, Chen W, Lee PL, Karuturi RK, Tan PB, Liu ET, Yu Q: Pharmacologic disruption of Polycomb-repressive complex 2-mediated gene repression selectively induces apoptosis in cancer cells. Genes Dev. 2007, 21 (9): 1050-1063. 10.1101/gad.1524107.View ArticlePubMedPubMed CentralGoogle Scholar
- Suva ML, Riggi N, Janiszewska M, Radovanovic I, Provero P, Stehle JC, Baumer K, Le Bitoux MA, Marino D, Cironi L, Marquez VE, Clement V, Stamenkovic I: EZH2 is essential for glioblastoma cancer stem cell maintenance. Cancer Res. 2009, 69 (24): 9211-9218. 10.1158/0008-5472.CAN-09-1622.View ArticlePubMedGoogle Scholar
- Kodach LL, Jacobs RJ, Heijmans J, van Noesel CJ, Langers AM, Verspaget HW, Hommes DW, Offerhaus GJ, van den Brink GR, Hardwick JC: The role of EZH2 and DNA methylation in the silencing of the tumour suppressor RUNX3 in colorectal cancer. Carcinogenesis. 2010, 31 (9): 1567-1575. 10.1093/carcin/bgq147.View ArticlePubMedPubMed CentralGoogle Scholar
- Kalushkova A, Fryknas M, Lemaire M, Fristedt C, Agarwal P, Eriksson M, Deleu S, Atadja P, Osterborg A, Nilsson K, Vanderkerken K, Oberg F, Jernberg-Wiklund H: Polycomb target genes are silenced in multiple myeloma. PLoS One. 2010, 5 (7): e11483-10.1371/journal.pone.0011483.View ArticlePubMedPubMed CentralGoogle Scholar
- Aad G, Abbott B, Abdallah J, Abdelalim AA, Abdesselam A, Abdinov O, Abi B, Abolins M, Abramowicz H, Abreu H, Acerbi E, Acharya BS, Ackers M, Adams DL, Addy TN, Adelman J, Aderholz M, Adomeit S, Adorisio C, Adragna P, Adye T, Aefsky S, Aguilar-Saavedra JA, Aharrouche M, Ahlen SP, Ahles F, Ahmad A, Ahmed H, Ahsan M, Aielli G, et al: Search for new particles in two-jet final states in 7 TeV proton-proton collisions with the ATLAS detector at the LHC. Physical review letters. 2010, 105 (16): 161801-View ArticlePubMedGoogle Scholar
- Caretti G, Di Padova M, Micales B, Lyons GE, Sartorelli V: The Polycomb Ezh2 methyltransferase regulates muscle gene expression and skeletal muscle differentiation. Genes Dev. 2004, 18 (21): 2627-2638. 10.1101/gad.1241904.View ArticlePubMedPubMed CentralGoogle Scholar
- Juan AH, Kumar RM, Marx JG, Young RA, Sartorelli V: Mir-214-dependent regulation of the polycomb protein Ezh2 in skeletal muscle and embryonic stem cells. Mol Cell. 2009, 36 (1): 61-74. 10.1016/j.molcel.2009.08.008.View ArticlePubMedPubMed CentralGoogle Scholar
- Wong CF, Tellam RL: MicroRNA-26a targets the histone methyltransferase Enhancer of Zeste homolog 2 during myogenesis. J Biol Chem. 2008, 283 (15): 9836-9843. 10.1074/jbc.M709614200.View ArticlePubMedGoogle Scholar
- Ciarapica R, Russo G, Verginelli F, Raimondi L, Donfrancesco A, Rota R, Giordano A: Deregulated expression of miR-26a and Ezh2 in rhabdomyosarcoma. Cell Cycle. 2009, 8 (1): 172-175. 10.4161/cc.8.1.7292.View ArticlePubMedGoogle Scholar
- Marchesi I, Fiorentino FP, Rizzolio F, Giordano A, Bagella L: The ablation of EZH2 uncovers its crucial role in rhabdomyosarcoma formation. Cell Cycle. 2012, 11 (20): 3828-3836. 10.4161/cc.22025.View ArticlePubMedPubMed CentralGoogle Scholar
- Ciarapica R, Annibali D, Raimondi L, Savino M, Nasi S, Rota R: Targeting Id protein interactions by an engineered HLH domain induces human neuroblastoma cell differentiation. Oncogene. 2009, 28 (17): 1881-1891. 10.1038/onc.2009.56.View ArticlePubMedGoogle Scholar
- Palacios D, Mozzetta C, Consalvi S, Caretti G, Saccone V, Proserpio V, Marquez VE, Valente S, Mai A, Forcales SV, Sartorelli V, Puri PL: TNF/p38alpha/polycomb signaling to Pax7 locus in satellite cells links inflammation to the epigenetic control of muscle regeneration. Cell Stem Cell. 2010, 7 (4): 455-469. 10.1016/j.stem.2010.08.013.View ArticlePubMedPubMed CentralGoogle Scholar
- Fan T, Jiang S, Chung N, Alikhan A, Ni C, Lee CC, Hornyak TJ: EZH2-dependent suppression of a cellular senescence phenotype in melanoma cells by inhibition of p21/CDKN1A expression. Mol Cancer Res. 2011, 9 (4): 418-429. 10.1158/1541-7786.MCR-10-0511.View ArticlePubMedPubMed CentralGoogle Scholar
- Chng KR, Chang CW, Tan SK, Yang C, Hong SZ, Sng NY, Cheung E: A transcriptional repressor co-regulatory network governing androgen response in prostate cancers. EMBO J. 2012, 31 (12): 2810-2823. 10.1038/emboj.2012.112.View ArticlePubMedPubMed CentralGoogle Scholar
- Wu Z, Lee ST, Qiao Y, Li Z, Lee PL, Lee YJ, Jiang X, Tan J, Aau M, Lim CZ, Yu Q: Polycomb protein EZH2 regulates cancer cell fate decision in response to DNA damage. Cell Death Differ. 2011, 18 (11): 1771-1779. 10.1038/cdd.2011.48.View ArticlePubMedPubMed CentralGoogle Scholar
- Valente S, Lepore I, Dell'Aversana C, Tardugno M, Castellano S, Sbardella G, Tomassi S, Di Maro S, Novellino E, Di Santo R, Costi R, Altucci L, Mai A: Identification of PR-SET7 and EZH2 selective inhibitors inducing cell death in human leukemia U937 cells. Biochimie. 2012, 94 (11): 2308-2313. 10.1016/j.biochi.2012.06.003.View ArticlePubMedGoogle Scholar
- Bray M, Driscoll J, Huggins JW: Treatment of lethal Ebola virus infection in mice with a single dose of an S-adenosyl-L-homocysteine hydrolase inhibitor. Antiviral Res. 2000, 45 (2): 135-147. 10.1016/S0166-3542(00)00066-8.View ArticlePubMedGoogle Scholar
- Vekony H, Raaphorst FM, Otte AP, van Lohuizen M, Leemans CR, van der Waal I, Bloemena E: High expression of Polycomb group protein EZH2 predicts poor survival in salivary gland adenoid cystic carcinoma. J Clin Pathol. 2008, 61 (6): 744-749.View ArticlePubMedGoogle Scholar
- Walters ZS, Villarejo-Balcells B, Olmos D, Buist TW, Missiaglia E, Allen R, Al-Lazikani B, Garrett MD, Blagg J, Shipley J: JARID2 is a direct target of the PAX3-FOXO1 fusion protein and inhibits myogenic differentiation of rhabdomyosarcoma cells. Oncogene. 2013, doi: 10.1038/onc.2013.46. [Epub ahead of print]Google Scholar
- Cao W, Ribeiro Rde O, Liu D, Saintigny P, Xia R, Xue Y, Lin R, Mao L, Ren H: EZH2 promotes malignant behaviors via cell cycle dysregulation and its mRNA level associates with prognosis of patient with non-small cell lung cancer. PLoS One. 2012, 7 (12): e52984-10.1371/journal.pone.0052984.View ArticlePubMedPubMed CentralGoogle Scholar
- Alimova I, Birks DK, Harris PS, Knipstein JA, Venkataraman S, Marquez VE, Foreman NK, Vibhakar R: Inhibition of EZH2 suppresses self-renewal and induces radiation sensitivity in atypical rhabdoid teratoid tumor cells. Neuro Oncol. 2013, 15 (2): 149-160. 10.1093/neuonc/nos285.View ArticlePubMedGoogle Scholar
- McAllister RM, Melnyk J, Finkelstein JZ, Adams EC, Gardner MB: Cultivation in vitro of cells derived from a human rhabdomyosarcoma. Cancer. 1969, 24 (3): 520-526. 10.1002/1097-0142(196909)24:3<520::AID-CNCR2820240313>3.0.CO;2-M.View ArticlePubMedGoogle Scholar
- Wang C, Liu Z, Woo CW, Li Z, Wang L, Wei JS, Marquez VE, Bates SE, Jin Q, Khan J, Ge K, Thiele CJ: EZH2 Mediates epigenetic silencing of neuroblastoma suppressor genes CASZ1, CLU, RUNX3, and NGFR. Cancer research. 2012, 72 (1): 315-324. 10.1158/0008-5472.CAN-11-0961.View ArticlePubMedGoogle Scholar
- Mai A, Valente S, Cheng D, Perrone A, Ragno R, Simeoni S, Sbardella G, Brosch G, Nebbioso A, Conte M, Altucci L, Bedford MT: Synthesis and biological validation of novel synthetic histone/protein methyltransferase inhibitors. ChemMedChem. 2007, 2 (7): 987-991. 10.1002/cmdc.200700023.View ArticlePubMedGoogle Scholar
- Lawlor ER, Thiele CJ: Epigenetic changes in pediatric solid tumors: promising new targets. Clin Cancer Res. 2012, 18 (10): 2768-2779. 10.1158/1078-0432.CCR-11-1921.View ArticlePubMedPubMed CentralGoogle Scholar
- Kubicek S, O'Sullivan RJ, August EM, Hickey ER, Zhang Q, Teodoro ML, Rea S, Mechtler K, Kowalski JA, Homon CA, Kelly TA, Jenuwein T: Reversal of H3K9me2 by a small-molecule inhibitor for the G9a histone methyltransferase. Molecular cell. 2007, 25 (3): 473-481. 10.1016/j.molcel.2007.01.017.View ArticlePubMedGoogle Scholar
- Qi W, Chan H, Teng L, Li L, Chuai S, Zhang R, Zeng J, Li M, Fan H, Lin Y, Gu J, Ardayfio O, Zhang JH, Yan X, Fang J, Mi Y, Zhang M, Zhou T, Feng G, Chen Z, Li G, Yang T, Zhao K, Liu X, Yu Z, Lu CX, Atadja P, Li E: Selective inhibition of Ezh2 by a small molecule inhibitor blocks tumor cells proliferation. Proc Natl Acad Sci U S A. 2012, 109 (52): 21360-21365. 10.1073/pnas.1210371110.View ArticlePubMedPubMed CentralGoogle Scholar
- McCabe MT, Ott HM, Ganji G, Korenchuk S, Thompson C, Van Aller GS, Liu Y, Graves AP, Della Pietra A, Diaz E, LaFrance LV, Mellinger M, Duquenne C, Tian X, Kruger RG, McHugh CF, Brandt M, Miller WH, Dhanak D, Verma SK, Tummino PJ, Creasy CL: EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature. 2012, 492 (7427): 108-112. 10.1038/nature11606.View ArticlePubMedGoogle Scholar
- Knutson SK, Wigle TJ, Warholic NM, Sneeringer CJ, Allain CJ, Klaus CR, Sacks JD, Raimondi A, Majer CR, Song J, Scott MP, Jin L, Smith JJ, Olhava EJ, Chesworth R, Moyer MP, Richon VM, Copeland RA, Keilhack H, Pollock RM, Kuntz KW: A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells. Nature chemical biology. 2012, 8 (11): 890-896.PubMedGoogle Scholar
- Simon JA, Lange CA: Roles of the EZH2 histone methyltransferase in cancer epigenetics. Mutat Res. 2008, 647 (1–2): 21-29.View ArticlePubMedGoogle Scholar
- Fiskus W, Wang Y, Sreekumar A, Buckley KM, Shi H, Jillella A, Ustun C, Rao R, Fernandez P, Chen J, Balusu R, Koul S, Atadja P, Marquez VE, Bhalla KN: Combined epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A and the histone deacetylase inhibitor panobinostat against human AML cells. Blood. 2009, 114 (13): 2733-2743. 10.1182/blood-2009-03-213496.View ArticlePubMedPubMed CentralGoogle Scholar
- Gannon OM, Merida de Long L, Endo-Munoz L, Hazar-Rethinam M, Saunders NA: Dysregulation of the repressive H3K27 trimethylation mark in head and neck squamous cell carcinoma contributes to dysregulated squamous differentiation. Clin Cancer Res. 2013, 19 (2): 428-441. 10.1158/1078-0432.CCR-12-2505.View ArticlePubMedGoogle Scholar
- Benoit YD, Witherspoon MS, Laursen KB, Guezguez A, Beausejour M, Beaulieu JF, Lipkin SM, Gudas LJ: Pharmacological inhibition of polycomb repressive complex-2 activity induces apoptosis in human colon cancer stem cells. Exp Cell Res. 2013, 319 (10): 1463-1470. 10.1016/j.yexcr.2013.04.006.View ArticlePubMedGoogle Scholar
- Knutson SK, Warholic NM, Wigle TJ, Klaus CR, Allain CJ, Raimondi A, Porter Scott M, Chesworth R, Moyer MP, Copeland RA, Richon VM, Pollock RM, Kuntz KW, Keilhack H: Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2. Proc Natl Acad Sci U S A. 2013, 110 (19): 7922-7927. 10.1073/pnas.1303800110.View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/139/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.