Tumor and Stem Cell Biology Research

Published OnlineFirst September 17, 2012; DOI: 10.1158/0008-5472.CAN-12-0614

Cancer Tumor and Stem Cell Biology Research

OTX2 Represses Myogenic and Neuronal Differentiation in Medulloblastoma Cells

Ren-Yuan Bai1, Verena Staedtke1, Hart G. Lidov3, Charles G. Eberhart2, and Gregory J. Riggins1

Abstract The brain development transcription factor OTX2 is overexpressed and/or genomically amplified in most medulloblastomas, but the mechanistic basis for its contributions in this setting are not understood. In this study, we identified OTX2 as a transcriptional repressor and a gatekeeper of myogenic and neuronal differentiation in medulloblastoma cells. OTX2 binds to the MyoD1 core enhancer through its homeobox domain, and the remarkable repressor activity exhibited by the homeobox domain renders OTX2 transcriptionally repressive. RNA interference–mediated attenuation of OTX2 expression triggered myogenic and neuronal differentiation in vitro and prolonged the survival in an orthotopic medulloblastoma mouse model. Conversely, inducing myogenic conversion of medulloblastoma cells led to the loss of OTX2 expression. In medullomyoblastoma, a medulloblastoma subtype containing muscle elements, myogenic cells share cytogenetic signatures with the primitive tumor cells and OTX2 expression was lost in the differentiated myogenic cells. Thus, OTX2 functions via its homeobox domain as a suppressor of differentiation, and the loss of OTX2 expression is linked to the myogenesis in medullomyoblastoma. Together, our findings illustrate the origin of muscle cells in medullomyoblastomas and the oncogenic mechanism of OTX2 as a repressor of diverse differentiating potential. Cancer Res; 72(22); 1–14. 2012 AACR.

Introduction is featured with rhabdomyoblastic elements immunoreactive Medulloblastoma, the most common type of pediatric brain to desmin, myoglobin, and fast myosin (3). malignancy, is an aggressive primitive neuroectodermal tumor In some medulloblastomas, driver mutations have been fi arising from the cerebellum. It presents a significant cause of identi ed. For example, Sonic Hedgehog (SHH) pathway muta- cancer-related death in children and current treatment of tions are mainly associated with the nodular/desmoplastic and radio-chemotherapy could impair children's development and anaplastic medulloblastomas, which presumably arise from cause long-term adverse effects (1). In addition to the general the granule neuron precursor cells (GNPC; ref. 2). Activating classic medulloblastoma designation, current World Health mutations in the WNT pathway contribute to about 7% to 15% Organization (WHO) classification recognizes 4 variants: des- of medulloblastoma and mainly present as the classic medul- moplastic/nodular medulloblastoma, medulloblastoma with loblastoma (2). Recently, this group of medulloblastoma has extensive nodularity anaplastic medulloblastoma, and large been determined of originating outside the cerebellum from cell medulloblastoma (2). The most common classic medul- the cells of the dorsal brainstem (4). Among the majority of loblastoma usually arises in the vermis of the cerebellum, lacks medulloblastomas that are non-SHH and non-WNT types, the distinctive features, and is composed of densely packed small driver genetic alterations and tumor origin are largely unde- fi fi MYC undifferentiated cells. A secondary description termed medul- ned, whereas the overexpression and/or ampli cation of OTX2 lomyoblastoma exists for the medulloblastoma with variously and represent one of the main oncogenic features (2, 5). Orthodenticle Homeobox 2 OTX2 differentiated myogenic cells mixed with the tumor cells (2). It ( ), a member of a highly conserved family of the bicoid-like homeobox transcription factors, controls brain morphogenesis (6). During embryogenesis, OTX2 is required for specification and regionalization of Authors' Affiliations: Departments of 1Neurosurgery and 2Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland; and the developing brain and is expressed in restricted areas of the 3Department of Pathology, Children's Hospital Boston, Harvard Medical forebrain and midbrain and throughout the posterior cerebel- School, Boston, Massachusetts lum, particularly, within the external granular layer and the Note: Supplementary data for this article are available at Cancer Research emerging internal granular layer (6, 7). The abundant expres- Online (http://cancerres.aacrjournals.org/). sion of OTX2 in developing brain is silenced in adult rodent Corresponding Authors: Ren-Yuan Bai or Gregory J. Riggins, Ludwig brain except for the pineal gland and ventral tegmental area Collaborative Laboratory, Department of Neurosurgery, Johns Hopkins University, Koch Building Rm. 257, 1550 Orleans Street, Baltimore, MD (VTA) neurons (8, 9). OTX2 controls neuron subtype identity by 21231. Phone: 410-502-2905; Fax: 410-502-5559; E-mail: [email protected] antagonizing molecular and functional features of the dorsal– or [email protected] lateral VTA (10). In the ventral midbrain and dorsal thalamus, doi: 10.1158/0008-5472.CAN-12-0614 OTX2 controls the identity of neuronal progenitors and is 2012 American Association for Cancer Research. required to suppress the progenitors from generating various

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neuronal cell types during the brain development (11, 12). description. Different hypotheses include the possibilities that These findings indicate the pivotal repressor role of OTX2 in tumor myoblasts arise from recruitment and migration of controlling the fate and differentiation of various progenitors mesenchymal or multipotent endothelial cells or that medul- in the early developing brain. lomyoblastoma is a variant of malignant teratomas (3). More Although OTX2 expression in children and adults is still recently, it has been speculated that medullomyoblastoma found in the retina, OTX2 is mainly silenced in the central myoblasts are a result of the primitive neuroectodermal cells nervous system and the other organs in human (13). Genomic undergone myogenic differentiation (3, 25). copy gains and amplification have been identified in various In this study, we aimed to investigate the molecular mech- frequencies ranging from 1% to 21% by different methods and anism of OTX2 in medulloblastoma and shed light as to why studies (5, 14, 15). Several laboratories have reported the medullomyoblastoma contains muscle cells and how OTX2 overexpression of OTX2 in more than 60% of medulloblasto- contributes to this process. Starting with the genes that are mas (13, 16, 17). The sustained expression of OTX2 in the transcriptionally regulated by OTX2 in medulloblastoma, we tumorigenic stem cells and progenitors could possibly achieve built a molecular model of OTX2 that supports its function as a the similar function of maintaining the primitive status of gatekeeper of myogenic and neuronal differentiation in medul- those medulloblastoma-generating cells. This function could loblastoma. Turning off OTX2 expression can cause medullo- be highly relevant, as medulloblastoma is regarded as primitive blastoma cells to progress toward myogenic and neuronal neuroectodermal tumor (PNET), a type of embryogenic tumor, differentiation. Thus, inducing differentiation by interfering and is believed to originate from aberrant neural stem/pro- with OTX2-mediated transcriptional repression could offer genitor cells. therapeutic benefit, as shown by the survival extension in the The significance of OTX2 as a medulloblastoma oncogene is orthotopic medulloblastoma mouse model. underscored by the recent whole-exon sequencing and copy number analysis of medulloblastoma genomes (15). The geno- Materials and Methods mic amplification frequency of OTX2 gives statistical support Cell lines and cell culture to the notion that this alteration is selected as a driver Cos 1, HeLa, mouse neuroblastoma cell line Neuro2a, and mutation, and beside MYC, OTX2 is likely only 1 of the 2 mouse myoblast cell line C2C12 were acquired from American amplified oncogenes in medulloblastoma. Overexpression of Type Culture Collection. The following medulloblastoma cell OTX2 in the mouse hindbrain resulted in the accumulation of lines were used in the study were obtained from the Duke proliferative clusters of cells in the cerebellar white matter and University Brain Tumor Center (Durham, NC) or indicated in dorsal brainstem of postnatal mice (18). In addition, knock- the references: D283Med (D283; ref. 26); D341Med (D341), down by OTX2 siRNA or suppression of OTX2 promoter by 9-cis D425Med (D425), D487Med (D487), D556Med (D556), DAOY, retinoic acid (RA) slowed medulloblastoma tumor growth in MCD1, UW228-2 (UW228), and Mhh-Med-1 (Mhh1; ref. 27). All mice (5, 19), suggesting that OTX2 expression sustains tumor cells were maintained in Dulbeccos' Modified Eagles' Media growth. Recently, Bunt and colleagues reported that induced (DMEM) supplemented with 10% FBS and antibiotics. All cells overexpression of OTX2 in medulloblastoma cells leads to G1 were kept in frozen stocks upon reception and were not arrest in cell cycle and senescence-like phenotype (20), where- additionally authenticated. as, however, an inducible knockdown of OTX2 activated cell- cycle regulators and neuronal genes (21). Beside genomic Plasmids and constructs amplification as a means of OTX2 activation, the majority of OTX2-L or OTX2 was subcloned into pCDNA3.1 and trans- medulloblastomas overexpress OTX2 through an unknown fected into Cos1 cells by Lipofectamine 2000 (Invitrogen). mechanism. Despite its strong presence as a potential onco- OTX2 was fused with 3 copies of FLAG sequence on the N- gene, the basic underlying molecular mechanism of OTX2 in terminus and subcloned in pCMV-TAG-2B (Stratagene). This medulloblastoma tumorigenesis remains to be defined. construct was transfected in D425 cells by electroporation During the course of the present study, we determined that at 200 V/25 ms with a GenePulser (Bio-Rad) and selected by OTX2 is involved in differentiation and hypothesized that 1 mg/mL of geneticin. Single clones were selected by limiting OTX2 was additionally involved in the closely related medul- dilution. Homeobox triple mutant OTX2-3M and OTX2-HD-3M lomyoblastoma, in part, due to the large and unusual regions of (R89G, P133T, and P134A) were generated, which lost the DNA- differentiated muscle cells within this tumor. Medullomyo- binding affinity of the homeobox domain as described before blastoma makes up 3% to 5% of all medulloblastoma cases and (28). OTX2-HD contains the HD domain (aa 35–95). OTX2-DHD the features of this tumor may provide important clues to the was created by deleting the aa 35–95 of human OTX2 cDNA. pathogenesis of this embryonal tumors (22, 23). The distin- Human MyoD core enhancer sequence was cloned into guishing feature of medullomyoblastoma is the presence of pGL3-P vector containing SV40 minimal promoter and lucif- cells showing muscle differentiation among the primitive erase to create the pMyoD-CER construct (29). The promoter neuroectodermal tumor cells that characterize medulloblas- region of human synapsin I (408 to þ47), with or without the tomas (3). These myogenic elements in medullomyoblastoma Re1 sequence as previously described (30), was cloned in pGL3 can be found in various differentiation phases, ranging from luciferase construct (Promega). Plasmids were transfected into myoblastic single cells to well-developed striated muscle struc- HeLa or Neuro2a cells by Lipofectamine 2000 or into D425 cells ture (24). The origin of the muscle components in medullo- by electroporation as described above. pRL-CMV from Pro- myoblastoma has been an interesting question since its first mega encoding Renilla luciferase was cotransfected as the

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transfection control. After 36 hours, a luciferase assay was buffer, and sonicated for 1 minute at 30% power output by a conducted with the Dual-Luciferase Reporter Assay System VC505 sonicater (Sonic & Materials Inc.). Rabbit anti-OTX2 from Promega using the FB12 luminometer (Zylux). At least 3 antibody or a control rabbit antibody was used for immuno- independent experiments were carried out. Luciferase activity precipitation. PCR reactions were carried out with primers was normalized to Renilla luciferase activity. targeting the indicated regions. siRNA knockdowns Immunofluorescence staining The following siRNA duplexes were ordered from Integrated Paraffin-embedded sections of confirmed cases of medullo- DNA Technologies: OTX2_562: rGrGrArUrArUrGrCrUrGrGr- myoblastomas, MMB1–3, were obtained from the Department CrUrCrArArCrUrUrCrCrUrACT/rArGrUrArGrGrArArGrUrUr- of Pathology of the Children's Hospital Boston (Boston, MA), GrArGrCrCrArGrCrArUrArUrCrCrUrU, OTX2_758: rCrCrArCr- and MMB4 was obtained from the Department of Pathology of UrGrArUrUrGrCrUrUrGrGrArUrUrArUrArArGGA/rUrCrCrUr- Johns Hopkins Hospital (Baltimore, MD). Mouse brain tumor UrArUrArArUrCrCrArArGrCrArArUrCrArGrUrGrGrUrU, and sections as well as human normal brain sample were embed- REST_502: rGrCrArGrArArUrCrUrGrArArGrArArCrArGrUrUr- ded in paraffin. Slides were deparaffinized using a standard UrGrUGC/rGrCrArCrArArArCrUrGrUrUrCrUrUrCrArGrArUr- procedure and treated with antigen retrieval citrate solution UrCrUrGrCrUrU. Randomized siRNA control was purchased (BioGenex). The immunofluorescence staining using anti- from Ambion. In each experiment, 10 mg siRNA was transfected OTX2 or anti-desmin antibodies followed the procedure into 2 million cells by electroporation as described above. described before (19). Inducible OTX2 knockdown by short hairpin RNA (shRNA) was achieved with the V2THS_87164 lentivirus construct of Coculturing of C2C12 and D425-GFP Open Biosystems. OTX2 shRNAmir in pTRIPZ were transfected D425 cells were transfected with pCDNA3.1-GFP and along with SPAX2 and pMD.G in 293T cells by Lipofectamine a polyclonal population (D425-GFP) was selected with 2000 (Invitrogen). Virus was harvested after 48 hours and 0.2 mg/mL zeocin. Equal proportions of D425-GFP and infected D425 cells by incubating with 8 mg/mL polybrene C2C12 cells were mixed and grown on chamber slides with (Sigma). Control cells were infected with lentivirus carrying the medium containing 10% FBS for 2 days and later with differ- same construct but lacking the OTX2-shRNAmir sequence. entiation medium containing 2% horse serum for 4 to 6 days. Cells were selected by puromycin and transcription of shRNA- The cells were then fixed and stained as described above. mir were induced by adding 0.1 mg/mL doxycycline and assessed by the expression of red fluorescent protein (RFP). FISH Human BAC clone RP11-1085N6 from Invitrogen was used Isolation of RNA and reverse transcription-PCR for detecting OTX2 locus and was directly labeled with Spec- Total RNA was isolated by SV Total RNA Isolation Kit trumGreen-dUTP. Prelabeled SpectrumOrange c-MYC (Promega). cDNA was made by cDNA Synthesis System (Invi- (8q24.12-q24.13) probe was purchased from Abbott Molecular trogen). PCR was carried out using Platinum Taq polymerase Vysis. FISH on paraffin sections followed the procedure as (Invitrogen) for 27 cycles using gene-specific custom primer. described before (32). FISH signals were assessed by an Axio- plan2 Imaging microscope by Zeiss with the Isis FISH Imaging Antibodies and Western blotting System V5.4 by MetaSystem GmbH. The combination of Cells were lysed in lysis buffer as described before, and immunofluorescent staining and FISH was carried out accord- immunostaining was conducted according to standard pro- ing to Nolen and colleagues with modifications (33). Briefly, the cedure (31). The following antibodies were used in this study: paraffin sections were deparaffinized in xylene and rehydrated mouse anti-OTX2 against the human OTX2 aa1–289 by an ethanol series. Then sections were incubated overnight at (MAB1979) and anti-b-TubIII clone Tuj1 (R&D Systems); rab- 70C in citrate antigen retrieval buffer (BioGenex), rinsed in bit anti-OTX2 against the human full-length OTX2 (AB9566) PBS, and incubated in 0.5 mg/mL pepsin at 37C for 3 minutes. and anti-synapsin I (Millipore); mouse anti-MyoG clone F5D, Sections were stained by mouse anti-desmin antibody and mouse anti-Gal4, rabbit anti-MYH H-300 and anti-GAPDH DyLight649 (Cy5) anti-mouse secondary antibody from Vector (Santa Cruz Biotech); rabbit anti-REST (Millipore); rabbit and Lab with the procedure described above. Subsequently, the mouse anti-FLAG (Sigma); mouse anti-desmin antibody clone sections were fixed for 10 minutes in ice cold Clarke's fixative (3 D33 (Dako), rabbit anti-desmin antibody clone Y66 (Millipore); parts 100% ethanol, 1 part glacial acetic acid), then washed in and mouse anti-p21 (Cell Signaling Technology). Mouse anti- 95% ethanol, dehydrated in 100% ethanol, and dried in the dark. REST antibody was kindly provided by D.J. Anderson. Signals To proceed with FISH, sections were rehydrated in water and were visualized by the SuperSignal chemiluminescent system processed as described above. Fluorescent signals were ana- (Pierce). lyzed and photographed with a fluorescent microscope equipped with 40,6-diamidino-2-phenylindole (DAPI), fluores- Chromatin immunoprecipitation cein isothiocyanate (FITC), TRITC/Orange, and Cy5 filters. The OTX2 chromatin immunoprecipitation (ChIP) assay was conducted with the ChIP Assay Kit according to the BeadArray expression profiling manufacturer's instructions (Upstate). Briefly, D425 cells were Total RNA was isolated and subjected to reverse transcrip- first cross-linked with 1% formalin for 15 minutes, lysed by SDS tion and amplification using recommended procedures from

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Illumina. In the Johns Hopkins Illumina core laboratory led by Myogenic differentiation is governed by a group of basic- Dr. Christopher Cheadle, samples were loaded onto a Human- helix-loop-helix (b-HLH) DNA-binding proteins, in which Ref-8 Beadarray slide, which contains 8 identical arrays with MyoD is regarded as the master regulator capable of inducing probes for 22,000 transcripts as annotated by the NCI cDNA MyoG and initiating the myogenic pathway (34). Myosin annotation. Data were analyzed by the BeadStudio Gene proteins such as MYH3 and MYL4 observed in our data are Expression Module program of Illumina. found either in embryonic or mature muscle cells. MyoD engages the cell-cycle machinery to activate cell-cycle with- Measurement of cell growth drawal before myogenic differentiation. p21/CDKN1A, p57/ Cells were plated in 96-well, flat-bottomed plates and mixed CDKN1C, and GADD45g are downstream targets of MyoD with 10% of WST-1 solution (Alexis). After incubation for 1 to 2 pathway that are responsible for cell-cycle arrest. The induc- hours at 37 C, the plates were measured by a Victor3 plate tion of the apoptotic initiator caspase-9 indicates apoptosis in reader at an absorbance of 450 nm (PerkinElmer). Wells filled the cells with OTX2 knockdown. with WST-1 and media were used as blank controls. Neuronal markers such as the neuron-specific b-tubulin III (b-TubIII/TUBB4), neurofilament 3 (NEF3), NCAM1, and dou- Animal experiments blecortex (DCX) were also induced (Fig. 1B; Supplementary nu nu Female athymic nude mice (NCr- / ) of 4- to 5-week-old Table S1), reflecting the activation of neuronal differentiation were purchased from the Frederick National Laboratory for subsequent to OTX2 knockdown. Cancer Research of NCI, MD. For the implantation procedure, At the protein level, myogenic differentiation factors MyoG, mice were anesthetized via intraperitoneal injection of keta- myosin heavy chain (MYH), downstream target p21, as well as mine/xylazine. On a stereotactic frame, 1 million D425 cells neuronal markers b-TubIII and synapsin I, were induced after transfected with empty vector or with doxycycline-inducible 48 hours of OTX2 knockdown by OTX2_562 siRNA, as shown by shRNAmir construct targeting on OTX2 were injected in a Western blotting (Fig. 1C). The inductions of MyoD and MEF2C prepared burr hole 2 mm lateral to the sagittal suture and 1 mm were confirmed by reverse transcription (RT)-PCR (Fig. 1C). anterior to the coronal suture, at a depth of 3 mm below the Use of OTX2_758 siRNA targeting a different region of the dura at a rate of 1 mL/min. Eight days after the implantation, OTX2 mRNA confirmed these results (Supplementary Fig. S2A). fi mice were gavaged with 50 mg/kg doxycycline daily for the rst A similar induction of differentiation following OTX2 knock- 3 days and once every 5 days for the rest of the period. down was also observed in the other OTX2-positive medulloblastoma cell lines, D283, D458, and D341 (Fig. 1D; Supple- Results mentary Fig. S2B and S2C). Re1-silencing transcription factor OTX2 knockdown led to induction of myogenic genes in (REST/NRSF) is a major suppressor of neurogenesis, which is medulloblastoma cells turned off after neural stem/progenitor cells undergo neural D425, a medulloblastoma cell line with genomic amplifica- differentiation (35, 36). We did not observe any significant tion of OTX2 locus (5), expresses the short isoform of OTX2 and change in REST protein with both OTX2_562 and OTX2_758 is among a panel of OTX2-positive medulloblastoma cell lines siRNA. The knockdown of OTX2 led to a dramatic decrease of (Supplementary Fig. S1A and S1B). OTX2 knockdown by siRNA cell growth (Fig. 1E), consistent with a previous report (16). in D425 cells led to extensive induction of myogenic pathways. In Fig. 1A, we compared transcripts in D425 cells transfected Association of OTX2 with MyoD core enhancer with a control siRNA with those transfected by OTX2 siRNA The expression of MyoD is tightly controlled by 3 regulatory (OTX2_562) at 36, 48, and 72 hours, on the Illumina gene units. The 258-bp core enhancer region (CER) located 20 kb expression bead array HumanRef-8 containing probes for relative to the MyoD transcriptional start site regulates the 22,000 transcripts. The regulated genes were defined as 2-fold initial expression of MyoD in muscle precursors and deter- change with a minimal expression of 50 in the array counts. In mines the tissue-specific expression (29). The distal regulatory this single experiment, the significance as well as the false- region (DDR) at around 5 kb is required for the maintenance positive counts were determined on the basis of the consis- of MyoD expression in muscle cells (37). The sequence sur- tency in the time course of 24, 48, and 72 hours. It revealed 277 rounding the core promoter is termed as the proximal regu- transcripts with >2-fold induction and 137 transcripts sup- latory region (PRR). MyoD core enhancer contains 4 E-box pressed by more than 50%, with possible replicates corrected. motives, which could provide binding sites for the E-box The largest and clearly obvious functional cluster of genes that proteins during heterodimerization with b-HLH myogenic were activated was a group related to myogenic differentiation. factors (29). The regulation of tissue-specific expression by These genes included MyoD1 (MyoD), MyoG, MYH3, MYL4 p21, the 258-bp CER of MyoD can be conveyed by a construct GADD45g, MEF2C and p57, all of which were markedly induced combining CER with a heterologous promoter (29). In this after 36 hours of OTX2 knockdown. Apoptotic gene caspase-9 study, we used a CER/SV40 minimal promoter luciferase was also induced and a moderate sub-G1 fraction indicating construct (pMyoD-CER) in D425 cells, where it showed the apoptotic cells was observed by flow cytometry (Supple- significant activity that reveals myogenic potential (Fig. mentary Fig. S1C). Fifty of the 277 transcripts, which were 1F, control siRNA). OTX2 knockdown activated pMyoD-CER increased at least 2-fold, were closely related to skeletal, substantially further (Fig. 1F, OTX2 siRNA), indicating that cardiac, and smooth muscle function or growth (Supplemen- the MyoD CER is likely involved in the activation of MyoD by tary Table S1). OTX2 knockdown.

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C D425 cells with E G A OTX2 siRNA induced muscle

OTX2 siRNA -OTX2 Input control differentiation genes and cell–cycle arrest 100 α Con 48 72 96 h CER α-OTX2 12,000 80 -15k 0 h α-MyoG 10,000 36 h 60 α-MYH -10k 8,000 48 h 40 α-p21 72 h Growth % DRR 6,000 α-β-TubΙΙΙ 20 PRR

Array counts Array 4,000 α-Syn I 48 72 96 48 72 96 h 5′UTR 2,000 α-REST D425 D283 WB: α α OTX2 0 -GAPDH OTX2 OTX2 -OTX2 siRNA siRNA γ 9 2C 21 5 MyoD1 RT DES F p p57 MyoD MyoD MyoG MYH3 MYL4 DD4 ME A MEF2C RT G Caspase CER -15k -10k DRR PRR 5’UTR GAPDH RT B D F H pGL3-P OTX2 siRNA induced 200 D283 cells with neuronal differentiation markers pMyoD-CER 100 OTX2 siRNA 150 1,000 0 h con 48 96 h α-OTX2 50 800 36 h 100 48 h α-MYH 600 50 72 h Normalized iuciferase 0 α-β-TubΙΙΙ

400 Normalized luciferase α-p21 0 VP16 VP16 Array counts control OTX2 200 siRNA siRNA VP16-OTX2 VP16-OTX2- 3M α-REST * VP16-OTX2 0 pGL3-P pMyoD-CER α β-TubΙΙΙ NEF3 DCX MAP1A -GAPDH MyoD-CER Psv Luc MyoD-CER Psv Luc OT X2 VP 16

Figure 1. OTX2 knockdown activates myogenic pathway in medulloblastoma cells and the association of OTX2 with the core enhancer of MyoD locus. A, OTX2 knockdown activated myogenic pathway genes and caspase-9. D425 cells were transfected with OTX2 siRNA (OTX2_562) or with randomized control siRNA. Total RNA was isolated from the cells after 36, 48, or 72 hours of transfection. RNA of the control was made after 48 hours of the transfection with control siRNA. Gene expression was analyzed by Illumina Beadarray technology and selected sets of genes involved in muscle differentiation are displayed in the graph. GADD45g, growth arrest and DNA damage–inducible gamma; MEF2C, MADS box transcription enhancer factor 2; MYH3, myosin heavy polypeptide 3, skeletal muscle; MYL4, myosin light polypeptide 4; MyoD (MyoD1), myogenic determination/myogenic factor 3; MyoG, myogenin/ myogenic factor 4; p21, CDKN1A; p57, CDKN1C. B, neuronal differentiation markers are activated by OTX2 siRNA in D425 cells. As described in A, selected sets of genes involved in neuronal differentiation are displayed. b-TubIII, b-tubulin III; DCX, doublecortex (doublecortin) transcript variant 3; NEF3, neurofilament 3. C, D425 cells were transfected with OTX2_562 siRNA or a random sequence control siRNA (Con). Western blotting (white background) or RT-PCR (black background) was carried out at 48, 72, and 96 hours for the indicated targets or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) normalization control. , a nonspecific band. MYH, skeletal and cardiac myosin heavy chain isoforms. D, in a similar experiment as C, medulloblastoma cell line D283 was transfected with OTX2_562 siRNA or a random sequence control siRNA (con). E, OTX2 siRNA reduces growth in D425 cells and D283 cells. D425 and D283 cells were treated by control siRNA and OTX2 siRNAs (OTX2_562) and incubated for 48, 72, or 96 hours. Viable cells were quantified by WST-1 reagent and graphed as percentage of the control siRNA cells. F, knockdown of OTX2 by siRNA activates the reporter construct of MyoD core enhancer (CER). D425 cells were first transfected with OTX2_562 siRNA or control siRNA for 36 hours. Subsequently, a luciferase construct (pMyoD-CER) containing the 258-bp MyoD CER and a SV40 minimal promoter was transfected and luciferase activity was determined after 24 hours. G, ChIP of OTX2 with MyoD CER. ChIP was conducted with D425 cells using control rabbit antibody or OTX2 antibody. Bound DNA fragments were detected by PCR with primers encompassing the indicated regions of MyoD locus. H, OTX2 homeobox domain mediates the interaction with MyoD CER. OTX2 or OTX2 homeobox triple mutant OTX2-3M (R89G, P133T, and P134A) was cloned in fusion with VP16 transactivation domain and cotransfected with vector pGL3-P or pMyoD-CER in D425 cells. Luciferase activity was measured and normalized.

In ChIP with an anti-OTX2 antibody to determine the ability with the pMyoD-CER construct, as this can serve as an potential in vivo association of OTX2 with the MyoD CER, indirect readout of the association of OTX2 with the MyoD CER DRR, PRR, and/or various positions in the MyoD locus, OTX2 (38). VP16-OTX2 was capable of activating pMyoD-CER sig- showed afﬁnity only with CER (Fig. 1G). While OTX2 displayed niﬁcantly above VP16 vector alone, whereas the VP16-OTX2- no intrinsic transactivity when fused with Gal4-binding 3M construct containing triple DNA-binding mutations in the domain (Gal4-BD) in D425 cells (Fig. 1H), we fused OTX2 with homeobox domain (HD) lost this transactivity (Fig. 1H). This the VP16 transactivation domain to test its transactivation result indicates that attachment of OTX2 protein with MyoD

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CER is mediated by the DNA-binding property of the OTX2 HD. taining the HD remained transcriptionally inactive (data not It is well established that the homeoprotein Msx1 represses shown). In contrast, the deletion of the homeobox domain MyoD by directly binding to CER, presumably in cooperation rendered OTX2 potently transactive, suggesting that HD was with histone H1b (39). In a mammalian 2-hybrid assay, OTX2 crucial for the ability of OTX2 to suppress transcription. showed no binding with the MyoD or H1b protein (Supple- Figure 2B shows the result of coexpressing Gal4-MyoD and mentary Fig. S3A), suggesting that OTX2 does not affect MyoD various Gal4-OTX2 constructs in the 1-hybrid transactivation function via direct protein–protein interaction. assay. This experiment reveals that Gal4-OTX2, Gal4-OTX2-HD (Gal4-HD), and Gal4-OTX2-HD-3M (Gal4-HD-3M) were capa- OTX2 HD functions as a transcriptional repressor ble of suppressing the transactivity of Gal4-MyoD, whereas We used a 1-hybrid system that uses the binding properties Gal4-OTX2-DHD (Gal4-DHD) in combination further elevated of the Gal4 DNA-binding domain to recruit a Gal4-OTX2 fusion the transactivating potential. The Gal4 DNA binding domain protein onto a Gal4-binding luciferase reporter. The full-length (aa 1–147) is reported to homodimerize and be recruited by the OTX2 fusion protein, as well as the OTX2-3M mutant and OTX2 Gal4-binding sites located in the luciferase reporter (40). HD alone (OTX2-HD), appeared transcriptionally inert in the Because OTX2 and MyoD can be brought into physical prox- Gal4 1-hybrid system in D425 (Fig. 2A). Among a series of Gal4- imity with this system as a heterodimer, the repression of the OTX2 deletion mutants, we observed that all constructs con- transcriptional activity of MyoD by OTX2 can be observed. The

A OTX2 HD B OTX2 HD 200 +Gal4-MyoD 60 Gal4-BD Gal4-BD D ΔH Gal4-seq Luc Gal4-seq Luc r r - 150 o 2 2 t to X -3M X Gal4- c c T D D T 40 Gal4-BD ve ve O H H O 100 MyoD -MyoD -OTX2 20 50 -ΔHD Normalized luciferase Normalized

Normalized luciferase Normalized 0 0 -HD

HD aa1-147 HD Gal4- OTX2 OTX2 Vector OTX2- Δ Δ OTX2- 3M Vector Vector HD Gal4-HD -3M Gal4-HD Δ Gal4- Gal4-OTX2 - WB: α-Gal4 +Gal4-MyoD OTX2- C MyoD HD D E Gal4-BD 200 M 3 Gal4-seq Luc - D D D H ΔH 150 r -H - - HD Gal4-BD D D D to o D OTX2 c o o y o MyoD OTX2 3xF- e v My My M My OTX2-3M *** 100 -ΔHD α -MYH HD 50 HD-3M *** α OTX2-ΔHD Normalized luciferase Normalized -FLAG 0

HD α Δ -GAPDH Vector D283 cells Gal4-MyoD -HD-3M Gal4-MyoD Gal4-MyoD -HD Gal4-MyoD -OTX2-

WB: α-Gal4

Figure 2. OTX2 functions as a transcriptional repressor of MyoD. A, OTX2 HD mediates transcriptional repression. OTX2, OTX2-3M, OTX2-HD (HD), or OTX2 deleted of HD (OTX2-DHD) were cloned in fusion with Gal4 DNA–binding domain (BD, aa1–147) and cotransfected in D425 cells along with a luciferase reporter construct containing 4 Gal4-binding sites (Gal4-seq). B, Gal4-OTX2 represses the transactivity of Gal4-MyoD in D425 cells. Gal4-MyoD was transfected along with empty vector or with indicated Gal4-OTX2 constructs. Right, anti-Gal4 Western blotting (WB) showed the expression levels of transfected constructs. C, fusion protein of MyoD and OTX2-HD (HD) showed repressed transcription activity. OTX2-HD (HD), OTX2-HD-3M (HD-3M), or OTX2-DHD were cloned in fusion with Gal4-MyoD and tested for their transactivities in D425 cells. D, fusion of OTX2-HD decreases the myogenic potential of MyoD protein in D283 medulloblastoma cells. 3 FLAG-tagged MyoD, MyoD-OTX2-HD (MyoD-HD), MyoD-OTX2-HD-3M (MyoD-HD-3M), or MyoD-OTX2-DHD (MyoD-DHD) was transiently transfected in D283 cells for 2 days. Cell lysates were blotted for myogenic marker MYH. E, a summary of the OTX2 mutants used in the study. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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similar repression of MyoD transactivity was displayed when expressing endogenous wild-type REST, pSyn was signifi- Gal4-MyoD was fused with OTX2-HD or OTX2-HD-3M and, cantly repressed in comparison to pSyn-DRe1 (Fig. 3D, left conversely, fusion of Gal4-MyoD with OTX2-DHD was able to side). In contrast, pSyn and pSyn-DRe1 displayed the same further enhance the transactivity of Gal4-MyoD (Fig. 2C). These levels of activity in Neuro2a cells, where no functional REST experiments all support the hypothesis that OTX2 suppresses is expressed (44). Our results indicate that REST is not a MyoD transcription and that this suppression is mediated by its direct target of OTX2-mediated suppression, and the induc- homeobox domain. tion of neuronal markers by OTX2 siRNA knockdown It has been previously described that the transactivity of revealed the transcriptional suppression mediated by OTX2 Gal4-MyoD is a good indicator of the in vivo myogenic potential unrelated to REST activity, whereas REST can function as an of MyoD regarding its induction of myogenic pathway (41). We independent suppressor of neuronal differentiation in asso- transiently overexpressed 3FLAG-tagged MyoD constructs in ciation with HDAC (model in Fig. 3E). However, to suppress D283 cells rather than in D425 cells as the latter did not neuronal differentiation, both OTX2 and REST are needed as transfect well with full-length MyoD. In D283, MyoD-OTX2- revealed by the fact that knockdown of OTX2 and REST HD (MyoD-HD) and MyoD-OTX2-HD-3M (MyoD-HD-3M) individually was sufficient for the induction of neuronal fusion proteins showed only a marginal induction of the genes in D425 cells (Fig. 3A and B). myogenic marker MYH, whereas the wild-type MyoD and MyoD-OTX2-DHD (MyoD-DHD) displayed high levels of MYH OTX2 expression and myogenic conversion in D425 cells induction and reflected similar myogenic potential (Fig. 2D). are mutually exclusive Similar results were obtained with pMyo-CER in D425 cells We next examined the relationship of OTX2 expression (Supplementary Fig. S3B). Therefore, OTX2, in particular its and potential myogenic differentiation of medulloblastoma HD, can suppress the myogenic potential of MyoD of inducing cells. It has been shown that the neural stem cells can endogenous myogenic gene expression in medulloblastoma undergo myogenic conversion when cocultured with cells and fibroblasts. mouse C2C12 myoblasts (45). Whether medulloblastoma The transcription suppression mediated by OTX2 cells propagated in serum-containing media are able to be appeared to be independent of histone deacetylases (HDAC). induced into similar myogenic conversion has been Incubating D425 cells with trichostatin A (TSA), an HDAC unknown before. D425 cells usually grow semi-adherently inhibitor, did not activate the myogenic pathway as indi- in single-cell form mixed with floating clusters in suspen- cated by the absence of MYH expression, in contrast to the sion form. We first generated a stable polyclonal D425 induction of neuronal marker b-TubIII and synapsin I by population transfected with pCDNA3.1-GFP and cocul- TSA (Fig. 3A). Neuronal differentiation is known to be tured it with C2C12 cells in equal proportions on chamber controlled by REST in association with HDAC (42). REST slides. After being cultured in the low-serum differentiation mainly represses the terminal neuronal differentiation genes condition for 6 days, a small population of GFP-positive such as SCG10, sodium channel type II, synapsin I, b-TubIII, myotubes emerged among the myotubes derived from neurofilament 3 (150 kD), synaptophysin, and glutamate C2C12 myoblasts, along with undifferentiated D425-GFP receptor through its specificbindingtotheRe1 consensus cells remaining in round single-cell form (Fig. 4A). The sequence within the promoters of these genes (43). In GFP-positive multinucleated cells (green) were stained general, REST is expressed in stem cells and non-neuronal positive with MYH or desmin antibody (red) and displayed cells and is downregulated for induction and maintenance of similar morphology and alignment with surrounding myo- the neuronal phenotype (35). In our study, siRNA-mediated tubes, which indicated the myogenic conversion of D425- knockdown of REST induced neuronal markers directly GFP cells. These GFP-positive myotubes have lost the OTX2 repressed by REST but failed to have any impact on medul- expression, as revealed by the negative nuclear staining loblastoma cell growth. Among the genes induced by OTX2 with OTX2 antibody (Fig. 4B). To rule out the possible siRNA knockdown, some are subject to Re1-mediated REST fusion of D425 and C2C12 cells, we incubated D425-GFP repression, such as b-TubIII and synapsin I,whereasothers and C2C12-RFP cells and observed similar transformation such as MAP1A and DCX are not known targets of REST of D425 cells (Supplementary Fig. S4A). These data indicate (Supplementary Table S1). The promoter constructs with that D425 medulloblastoma cells undergoing myogenic (pSyn) or without (pSyn-DRe1) the REST-binding Re1 conversion lose OTX2 expression. sequence have been described before (30). In D425 control Next, we created stable D425-OTX2-shRNA lines by infecting cells, we observed the expected repression of pSyn and de- D425 with lentivirus carrying a doxycycline-inducible OTX2- repression of pSyn-DRe1 (Fig. 3C, left side). Knockdown of shRNA knockdown construct. After incubating with doxycy- OTX2 by siRNA resulted in a marked increase of both pSyn cline for 7 to 14 days, staining with muscle marker desmin and and pSyn-DRe1 (760-fold) activities, which lifted pSyn activ- neuronal marker b-tubulin III revealed diverse populations ity well above the level of the de-repressed pSyn-DRe1 (104- undergone myogenic or/and neuronal differentiation, with the fold) in D425 control cells (Fig. 3C, right side). Therefore, the appropriate multinucleated myogenic or network-forming transcriptionalrepressiononpSynbyOTX2isnotmediated neuronal features (Fig. 4C). Similar results of OTX2 knockdown by Re1 sequence and should be independent of REST. The in D283 cells are shown in Supplementary Fig. S4B and S4C. specificity of the REST-mediated repression via Re1 is con- The effective doxycycline-induced knockdown of OTX2 and the firmed by the assay in HeLa and Neuro2D cells. In HeLa cells resulted inductions of MyoD and desmin in D425 cells are

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A B C 1,000 - + TSA con 48 96 h α−βTubΙΙΙ α-REST 800 α 100 -syn I α−βTubΙΙΙ 600

50 α-MYH α-syn I Growth 400 %

α-OTX2 α-GAPDH 0 Fold of luciferase 200 48 72 96 h D425 cells D425 cells with 0 REST siRNA n 1 n 1 tor Re ΔRe Δ pSy - pSy - Vector n Vec n pSy pSy D425 control D425 OTX2 siRNA

D 200 Hela E Neuro2A Neuronal HDAC 150 A 2 terminal ro REST la u differentiation e e H N 100 α HD -REST OTX2 F

Fold of luciferase 50 T myo- MyoD diff. CER 0 n n MyoD locus Re1 pSy Δ pSy ΔRe1 Vector - Vector - n n pSy pSy

Figure 3. OTX2 represses neuronal markers independently of REST. A, TSA induces neuronal differentiation but not muscle differentiation. D425 cells were incubated with 300 nmol/L of HAC inhibitor TSA for 48 hours and subjected to Western blotting of neuronal and muscle differentiation markers. B, siRNA knockdown of REST induces neuronal differentiation marker b-tubulin III and synapsin I but shows no effect on cell growth. D425 cells were transfected with control siRNA or REST siRNA in a time course of 48 and 96 hours. Western blot analyses revealed the knockdown of REST and the induction of b-tubulin III and synapsin I. C, OTX2 knockdown greatly increased the activity of synapsin promoter with and without the REST-binding site, Re1 motif. Synapsin I promoter with (pSyn) or without (pSyn-DRe1) the 23-bp Re1 motif was cloned in pGL3 luciferase construct. D425 cells treated with control siRNA or OTX2_562 siRNA for 36 hours were transfected with pGL3 vector, pSyn, or pSyn-DRe1 for another 36 hours. D, a control experiment of pSyn and pSyn-DRe1 showed the Re1 speciﬁcity. Neuro2D cells do not express functional REST and HeLa cells do. pSyn-DRe1 activity was de-repressed compared with pSyn in HeLa cells, whereas both constructs showed similar activities in Neuro2D cells. Anti-REST Western blotting conﬁrmed the REST statues in these cells. E, schematic of a proposed model of OTX2 action in medulloblastoma. The homeobox domain of OTX2 acts directly on the core enhancer region (CER) of MyoD to suppress muscle differentiation. Both OTX2 and REST/HDAC are required for the repression of neuronal differentiation markers. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

confirmed in Fig. 5A. Thus, the knockdown of OTX2 led to shRNA tumors (Fig. 5C, upper pictures and the color framed myogenic and neuronal phenotypes in D425 and D283 medul- magnifications on the right side). loblastoma cells. Mutually exclusive expression of OTX2 and myogenic OTX2 knockdown led to myogenic induction and marker in medullomyoblastomas prolonged survival in orthotopic xenografts We obtained 4 medullomyoblastoma samples (#1–4) as The D425-OTX2-shRNA cells were implanted in the frontal paraffin-embedded slides. The limited slide number of MMB4 lobe of athymic nude mouse brain and the animals were fed only allowed anti-OTX2 3,30-diaminobenzidine (DAB) and with doxycycline 8 days following the implantation. Previous hematoxylin and eosin (H&E) staining, in which the myoblasts report using D425 cells that were stably transfected with are distinguished by rhabdoid cytoplasm stained in red by OTX2-shRNA showed moderate survival extension com- eosin (Supplementary Fig. S5). Immunostaining with OTX2 pared with the control (5). In Fig. 5B, in vivo induction of antibody revealed that all 4 medullomyoblastomas expressed OTX2 knockdown showed a marked survival benefitinmice, OTX2 protein (Fig. 6; Supplementary Fig. S5). On immunoflu- extending the mean survival from 26 days of the control orescent staining of MMB1–3, the tumor cells with nuclear animals to 79 days. Because of the reported potential anti- expression of OTX2 presented a distribution distinct to the tumor activity of doxycycline (46, 47), the control animals desmin-positive myogenic/myoblastic cells, some of which were equally treated with doxycycline. Staining of the par- displayed multinucleated structure and characteristic muscle affin-embedded brain slides with anti-OTX2 antibody striations. Desmin-positive cells in those medullomyoblasto- showed reduced OTX2 expression by OTX2 knockdown (Fig. mas showed various phases of myogenic differentiation, from 5C). Myogenic marker desmin was negative in the control single cells with single nucleus to the ones undergone nuclear D425 tumor but induced in cells within the D425-OTX2- fusion and myotube alignment. Overall, myogenic populations

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A GFP α-MYH-TX Merged Merged+DAPI

D425-GFP & α-MYH-TX

D425-GFP & α-Des-TX

D425-GFP & α-OTX2-TX

GFP α-OTX2-TX Merged Merged+DAPI C

D425-OTX2- shRNA

α-Des-Green+DAPI Bright α-β-TubIII-Green+DAPI Bright

Figure 4. Mutual exclusion of myogenic differentiation and OTX2 expression. A and B, loss of OTX2 expression by myogenic conversion of D425 cells. A, GFP-transfected D425 cells were cocultured with C2C12 mouse myoblasts in differentiation medium. Cells were stained with myogenic marker MYH or desmin (Des) antibody and Texas Red (TX) secondary antibody. B, expression of OTX2 was visualized by OTX2 antibody and TX secondary antibody. Thirty GFP-positive cells with myotube morphology were examined and none of them showed significant OTX2 staining. White arrowheads indicate the multiple nuclei in the myogenic D245 cells, which were stained by DAPI (merge þ DAPI). It is worth noting that the polyclonal D425-GFP cells expressed GFP in various levels, which remained OTX2-positive when maintained in the undifferentiated single-cell form. C, OTX2 knockdown activated myogenic and neuronal differentiation in D425 cells. D425 cells infected with lentivirus of doxycycline (Dox)-inducible OTX2-shRNA construct were incubated with 0.1 mg/mL Dox on surface coated with poly-D-lysine for 10 days. Desmin or b-tubulin III staining visualized the cells that had undergone myogenic or neuronal differentiation. The white arrowhead indicates a desmin-positive D425 cell with 3 nuclei. Brightfield pictures with phase contrast are shown next to the immunofluorescent staining. Control D425 cells infected with mock lentivirus did not show positive staining of desmin and b-tubulin III (data not shown). in MMB1 and 2 are more pronounced than in MMB3. Among blastoma samples (Fig. 7A). It is worth noting that these these myogenic cells, OTX2 expression was either nondetect- sections often contain cells with incomplete nuclei. To further able or significantly reduced (Fig. 6). assess the cytogenetic features of the myogenic populations, paraffin sections were first stained with desmin antibody in Myogenic cells share the same cytogenetic signature with combination with Cy5 secondary antibody and then hybridized medullomyoblastoma tumor cells with OTX2 probe labeled with SpectrumGreen and c-MYC The origin of the myogenic element among the primitive probe labeled with SpectrumOrange. The subsequent hybrid- neuroectodermal tumor cells in medullomyoblastoma has ization procedure inevitably impaired the desmin immunoflu- been under speculation since its first description in 1933 orescent signals, despite the treatment with the Clark's fixative. (3). To address this issue, we investigated cytogenetic features Nonetheless, clear immunofluorescent signals of desmin were including OTX2 and c-MYC genomic copy number in myogenic retained in MMB1 and 2, where the myogenic populations are and primitive neuroectodermal tumor cells, as the often most pronounced. Figure 7B and Supplementary Fig. S6A amplified MYC could serve as a marker of medulloblastoma showed the desmin-positive myoblasts with 2 copies of OTX2 tumor cells to distinguish them from the possible normal tissue (green) and 3 to 5 copies of c-MYC (white) in MMB1. In MMB2, 3 population in the primary tumor. The round-shaped neuroec- copies of OTX2 and 4 to 6 copies of c-MYC were found in todermal medulloblastoma cells can be recognized by small desmin-positive myoblasts (Fig. 7C; Supplementary Fig. S6B). sizes with scant cytoplasm in relation to the pronounced Thus, the desmin-positive myoblasts in these examined medul- nuclei. FISH with OTX2 and c-MYC probes revealed that the lomyoblastoma samples share the same key cytogenetic sig- primitive tumor cells in MMB1 and 3 carry 2 copies of OTX2, natures with the primitive neuroectodermal tumor cells and whereas MMB2 has 3 copies of OTX2 gene and c-MYC copy therefore should have arisen from the same origin. The loss of numbers were increased at low levels in all 3 medullomyo- OTX2 expression may occur in certain tumor cell populations

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A Con. OTX2-shRNA B Con +Dox n = 7 - + - + Dox 100 P = 0.0003 α-OTX2 OTX2-shRNA +Dox n = 7 α 100 -Des Con.+Dox Con w/o Dox n = 4 β OTX2-shRNA 50 OTX2-shRNA w/o Dox n = 4 a- TubIII +Dox 50 α-GAPDH Percent survival

MyoD1 RT of growth % 0 0 GAPDH RT 6 days with 0.1 μg/mL Dox 0 25 50 75 100 125 150 Days after tumor implantation C Con. +Dox OTX2-shRNA+Dox T T

α-Des

B B T T

α-OTX2

B B

Figure 5. OTX2 knockdown induced myogenic marker in D425 xenograft tumor. A, D425 cells were infected with lentivirus of doxycycline (Dox)- inducible OTX2-shRNA. Four days after incubation with Dox, induction of the indicated genes was analyzed by Western blotting and RT-PCR. Control cells were infected with the control lentivirus of vector lacking OTX2-shRNA. B, survival curve of the nude mice implanted intracranially with D425 cells infected with control lentiviral construct or lentiviral construct with Dox-inducible OTX2-shRNA. Solid lines indicate the groups implanted with control (Con) and OTX2-shRNA cells treated with Dox after 8 days of implantation. Dashed lines indicate the untreated (w/o DOX) mice implanted with control and OTX2-shRNA cells. The mean survival of Con þ Dox, OTX2-shRNA þ Dox, Con w/o Dox, and OTX2-shRNA w/o Dox are 26, 79, 21, and 26.5 days, respectively. C, parafﬁn-embedded brain samples of control D425 xenograft or OTX2 knockdown D425 xenograft were stained by desmin (Des) or OTX2 antibody. Objective of 100 was used, and the areas with colored frames were further magniﬁed and are shown on the right side of the picture. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

during tumor progression, which following the model of OTX2 Discussion knockdown in various medulloblastoma cell lines can lead to OTX2 is involved in deﬁning vertebrate brain structure extensive myogenic differentiation along with the induction of during embryogenesis (6). On molecular level, OTX2 controls neuronal markers of those tumor cells. neuron subtype identity as a repressing factor in coordination

MMB #1 MMB #2 MMB #3

Figure 6. Mutually exclusive expression of OTX2 and α-OTX2-TX myogenic marker desmin in α -Des-FITC medullomyoblastomas. Parafﬁn- embedded MMB1–3 were co- stained with rabbit OTX2 antibody and mouse desmin (Des) antibody and subsequently with anti-rabbit Texas Red (TX) and anti-mouse FITC secondary antibodies. DAPI α-OTX2-TX staining was merged in the bottom α-Des-FITC to indicate the nuclei, where OTX2 DAPI is normally located and markedly absent in the desmin-positive cells.

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A Metaphase MMB #1 MMB #2 MMB #3

OTX2-Green OTX2-Green/MYC-Red OTX2-Green/MYC-Red OTX2-Green/MYC-Red

B MMB #1 C MMB #2

OTX2-Green/MYC-White/Des-IF-Red/DAPI OTX2-Green/MYC-White Des-IF-Red/DAPI

Figure 7. Myogenic cells share the same cytogenetic signatures with the primitive medulloblastoma tumor cells in medullomyoblastomas. A, hybridization of a normal human leukocyte in metaphase with the OTX2 probe showed 2 copies of OTX2 locus (left picture). Paraffin-embedded samples of MMB1–3 were hybridized with OTX2 probe labeled in green and c-MYC probe labeled in orange and rendered in red color. DAPI staining revealed the nuclei. B and C, MMB1 and 2 samples were first stained with mouse desmin antibody and anti-mouse Cy5 (red) secondary antibody. Subsequently, the sections were hybridized with OTX2 and c-MYC probes, and the nuclei were visualized by DAPI (blue). OTX2 signals were rendered in green and c-MYC in white. Yellow arrows indicate the myogenic cells with desmin staining. Enlarged images of the 2 nuclei indicated by the yellow arrows in B are displayed on the right side of B. with other transcriptional regulators (9, 11, 12, 48). In this elongated myotubes in the differentiation media. While the study, we identified that the OTX2 HD domain mediates impact of 0.1 mg/mL doxycycline, a concentration used in this transcriptional repression of OTX2 in medulloblastoma cells study, is not obvious in myogenic conversion, the higher independently of its DNA-binding ability. It is possible that this concentrations above 1 to 2 mg/mL doxycycline clearly repression is mediated by a binding partner with OTX2 HD. HD impaired the myogenic conversion in C2C12 cells. Thus, the includes aa 35–95 of OTX2, distinct from the en1-like motif (aa Tet-on system could carry potential risk of reducing myogenic 151–158) identified as the docking site of Tle4 repressor (49) differentiation especially at higher doxycycline concentration. and could serve as the binding site of other transcriptional The origin of the muscle elements in medullomyoblastoma regulators, such as the TALE-homeodomain transcriptional has been debated for many years (3, 25). The very few genetic coactivator Meis2 (50). Given the HDAC-independent nature of features of some medullomyoblastomas described so far the OTX2 repression of myogenic pathway in medulloblastoma include the alterations in 17q and low-level c-MYC amplifica- cells, in contrast to the REST/HDAC-dependent suppression of tion (3). In this study, with all 4 medullomyoblastomas expres- neuronal markers mediated also by OTX2, an unknown repressing OTX2, MMB1–3 were investigated on a cytogenetic level sor independent of HDAC could be recruited by OTX2 HD and and MMB1 and 2 were further examined for myogenic marker act as a key element in controlling the multiple differentiation desmin. We showed that in both medullomyoblastomas, the pathways in medulloblastoma cells. Further understanding of same cytogenetic signatures of OTX2 and c-Myc copy number this molecular mechanism could be of therapeutic significance profile exist in desmin-positive myogenic cells and primitive for medulloblastoma. neuroectodermal cells. OTX2 knockdown in D425 and other Recently, it was reported that the Tet-on doxycycline-induc- medulloblastoma cells led to myogenic and neuronal differ- ible knockdown of OTX2 activated cell-cycle regulators and entiation, closely resembling the myogenic and neuronal ele- neuronal genes (21). As our microarray experiment was carried ments mixed in medullomyoblastomas (24, 25). Immunofluo- out with the transient transfection of OTX2 siRNA, and the rescent staining of MMB1–3 revealed the mutually exclusive knockdown experiments can be affected by different factors nature of OTX2 expression in undifferentiated cells and such as the various knockdown efficacies and culture condi- myogenic phenotypes, which was modeled in vitro in Fig. 4 tions, we assessed the potential impact of doxycycline in by the loss of OTX2 expression following myogenic conversion myogenic differentiation by using the mouse C2C12 myoblasts of D425 cells cocultured with C2C12 myoblasts. Thus, the in light of this new report (Supplementary Fig. S7). C2C12 cells differentiated myogenic cells appear to share common can maintain the fibroblast-like phenotype with the growth origin with the primitive neuroectodermal cells and the loss media and undergo myogenic differentiation into multinuclear of OTX2 expression in certain primitive populations within

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medullomyoblastoma could have triggered the myogenic dif- pase-9, were induced. Flow cytometry revealed a minor pop- ferentiation and the formation of striated muscle elements. ulation (10.53%) of dead cells upon OTX2 knockdown in D425 Microenvironmental factors, genomic, or epigenetic instability cells. Thus, it is likely that therapeutic benefits observed with of the tumor could all possibly contribute to the loss of OTX2 OTX2 knockdown in vitro and in the mouse xenograft model expression in some tumor cell populations. When cocultured were the results of a combination of antiproliferation and pro- with the myoblast or injected in the mouse muscle, neural stem apoptotic effects. Our previous study evaluated the possible cells can differentiate to myotubes (45). In our coculturing use of RA, a natural regulator of neural development and a assay of D425 cells with C2C12 myoblasts, D425 cells could repressor of OTX2 promoter, in treating OTX2-positive medul- differentiate to myotubes bearing multinuclear features and loblastoma xenograft tumor in mice (19). We determined that muscle markers, accompanied by the loss of OTX2 expression. although RA was capable of repressing the OTX2 expression, Although the potential factors triggering this kind of myogenic inducing neuronal differentiation, and suppressing the growth differentiation remain undetermined, these data indicated the of medulloblastoma cells, the opposing basic fibroblast growth general myogenic potential of some medulloblastoma cells and factor (bFGF) signaling in the brain could render RA ineffec- a reprogramming in gene expression that silenced OTX2. tive. In this line of investigation, an approach more targeted on During the normal brain development, OTX2 regulates the the HD-mediated transcriptional repression of OTX2, possibly fate and represses the differentiation of OTX2-expressing a small-molecule drug, could offer a better chance of thera- neural progenitors through a network of transcription factors peutic success. and external effectors (10–12). OTX2 is expressed in a wide- spread pattern in forebrain, midbrain, and hindbrain at least Disclosure of Potential Conflicts of Interest till P13 in the rat brain, and becomes silenced or largely No potential conflicts of interest were disclosed. reduced after P30 with the exception of the pineal gland and – VTA neuron (7 9). In the rat cerebellum, OTX2 expression was Authors' Contributions observed from E16 through P18 and remained weakly detect- Conception and design: R.-Y. Bai able until P30, with expression in EGL and germinal layer from Development of methodology: R.-Y. Bai, V. Staedtke Acquisition of data (provided animals, acquired and managed patients, P2 to P18 (8). This window of OTX2 expression corresponds provided facilities, etc.): R.-Y. Bai, V. Staedtke, H.G. Lidov, C.G. Eberhart with the period of brain development till the completion of Analysis and interpretation of data (e.g., statistical analysis, biostatistics, neuronal differentiation. During that time frame, OTX2 guides computational analysis): R.-Y. Bai, V. Staedtke, C.G. Eberhart Writing, review, and/or revision of the manuscript: R.-Y. Bai, C.G. Eberhart, neural progenitors/precursors through their differentiation G.J. Riggins into specific neuron subtypes, such as the glutamatergic Administrative, technical, or material support (i.e., reporting or orga- neurons in the thalamus and meso-diencephalic dopaminergic nizing data, constructing databases): R.-Y. Bai Study supervision: R.-Y. Bai, G.J. Riggins (mdDA) neurons, in a highly coordinated manner together Other: Identified, confirmed diagnosis and contributed case material - medul- with a complex network of transcriptional regulators (12, 48). loblastoma specimens of an unusual type, H.G. Lidov In the normal brain development, this may prevent the display of the myogenic potential existing in the neural stem cells/ Acknowledgments progenitors until the neuronal terminal differentiation, where- The authors thank Raluca Yonescu for excellent cytogenetic work, Jennifer Edwards for her contribution in SAGE analysis, Dr. Christopher Cheadle of the as the loss of the aberrant expression of OTX2 in medullo- Johns Hopkins Illumina core laboratory for Illumina microarray analysis, and the blastoma cells might give rise to the myogenic differentiation technical advice and help by Betty Tyler, I-Mei Siu, Gary Gallia, and Colette M. in medullomyoblastoma. apRhys. The finding that OTX2 represses transcription via its HD and blocks differentiation in the OTX2-positive medulloblastoma Grant Support provides an opportunity for therapies designed to interfere This project was supported by NIH grant R01 NS052507, the Virginia and D.K. Ludwig Fund for Cancer Research, and the Children's Cancer Foundation. G.J. with OTX2 and trigger differentiation and growth arrest in the Riggins is the recipient of the Irving J. Sherman M.D. Research Professorship. tumor. The inducible OTX2 knockdown in the intracranial The costs of publication of this article were defrayed in part by the payment of D425 xenograft tumor resulted in a marked improvement of page charges. This article must therefore be hereby marked advertisement in animal survival, indicating a promising therapeutic potential. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Following OTX2 knockdown, genes responsible for growth Received February 15, 2012; revised July 27, 2012; accepted September 6, 2012; arrest such as p21 and p57, as well as apoptotic gene cas- published OnlineFirst September 17, 2012.

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OF14 Cancer Res; 72(22) November 15, 2012 Cancer Research

OTX2 Represses Myogenic and Neuronal Differentiation in Medulloblastoma Cells

Ren-Yuan Bai, Verena Staedtke, Hart G. Lidov, et al.

Cancer Res Published OnlineFirst September 17, 2012.

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