Identification of a Novel SYK/C-MYC/MALAT1 Signaling Pathway and Its Potential Therapeutic Value in Ewing Sarcoma

Published OnlineFirst March 23, 2017; DOI: 10.1158/1078-0432.CCR-16-2185

Cancer Therapy: Preclinical Clinical Cancer Research Identiﬁcation of a Novel SYK/c-MYC/MALAT1 Signaling Pathway and Its Potential Therapeutic Value in Ewing Sarcoma Haibo Sun1,2, De-Chen Lin2, Qi Cao2, Brendan Pang3, David D. Gae1, Victor Kwan Min Lee3, Huey Jin Lim3, Ngan Doan4, Jonathan W. Said4, Sigal Gery2, Marilynn Chow5, Anand Mayakonda6, Charles Forscher2, Jeffrey W. Tyner5, and H. Phillip Koefﬂer2,6,7

Abstract

Purpose: Ewing sarcoma (EWS) is a devastating soft tissue Results: SYK was identified as a candidate actionable target sarcoma affecting predominantly young individuals. Tyrosine through both high-throughput screens. SYK was highly phosphor- kinases (TK) and associated pathways are continuously activated ylated in the majority of EWS cells, and SYK inhibition by a variety in many malignancies, including EWS; these enzymes provide of genetic and pharmacologic approaches markedly inhibited candidate therapeutic targets. EWS cells both in vitro and in vivo. Ectopic expression of SYK Experimental Design: Two high-throughput screens (a siRNA rescued the cytotoxicity triggered by SYK-depletion associated library and a small-molecule inhibitor library) were performed in with the reactivation of both AKT and c-MYC. A long noncoding EWS cells to establish candidate targets. Spleen tyrosine kinase RNA, MALAT1, was identified to be dependent on SYK-mediated (SYK) phosphorylation was assessed in EWS patients and cell signaling. Moreover, c-MYC, a SYK-promoted gene, bound to the lines. SYK was inhibited by a variety of genetic and pharmaco- promoter of MALAT1 and transcriptionally activated MALAT1, logical approaches, and SYK-regulated pathways were investigat- which further promoted the proliferation of EWS cells. ed by cDNA microarrays. The transcriptional regulation of Conclusions: This study identifies a novel signaling involving MALAT1 was examined by ChIP-qPCR, luciferase reporter, and SYK/c-MYC/MALAT1 as a promising therapeutic target for the qRT-PCR assays. treatment of EWS. Clin Cancer Res; 23(15); 4376–87. 2017 AACR.

Introduction effective therapies are needed. Many pediatric solid tumors have activation of tyrosine kinases (TK), which play important roles in Ewing sarcoma is an aggressive soft tissue malignancy of Ewing sarcoma biology (6, 7). For example, EWS–FLI1 fusion children and adolescents, which is characterized by the chromo- protein promotes the activities of TKs, including FAK, PDGFR, and somal translocation leading EWS to fuse to FLI1 (1–4). Although IGF1R (8–11). Notably, targeting IGF1R by either small-molecule about 70% of children with Ewing sarcoma can be cured by inhibitors or antibodies has enhanced patients' survival in several surgery and chemotherapy either with or without radiotherapy, clinical trials (12). only 30% of those with metastasis can be cured (5). Thus, new Spleen tyrosine kinase (SYK) is a nonreceptor TK that is highly expressed in hematopoietic cells and regulates cellular adaptive immune responses (13). SYK also promotes cancer cell survival in 1Immunogenetics and Transplantation Laboratory, Department of Surgery, leukemia and pediatric retinoblastoma (14). Small-molecule University of California San Francisco, San Francisco, California. 2Department inhibitors of SYK (PRT062607 and GS-9973) have shown anti- of Medicine, Cedars-Sinai Medical Center, UCLA School of Medicine, Los neoplastic properties in these tumor types (15–17). 3 Angeles, California. Department of Pathology, National University Hospital In this study, two unbiased high-throughput screens, a TK- Singapore, Singapore. 4Department of Pathology and Laboratory Medicine, UCLA School of Medicine, Los Angeles, California. 5Department of Cell, Devel- focused siRNA library (18) and a small-molecule inhibitor library opmental & Cancer Biology, Knight Cancer Institute, Oregon Health & Science (19), were performed to identify signaling pathways which were University, Portland, Oregon. 6Cancer Science Institute of Singapore, National valuable for therapeutic interventions. Through a series of func- University of Singapore, Singapore. 7National University Cancer Institute, tional investigations, we established a novel signaling pathway National University Hospital Singapore, Singapore. involving SYK/c-MYC/MALAT1 in the setting of Ewing sarcoma Note: Supplementary data for this article are available at Clinical Cancer biology, and further showed its potential for therapeutic inter- Research Online (http://clincancerres.aacrjournals.org/). vention for this pediatric malignancy. J.W. Tyner and H.P. Koefﬂer share last authorship of this article. Corresponding Authors: Haibo Sun, University of California San Francisco, 45 Materials and Methods Castro Street, San Francisco, CA 94114. E-mail: [email protected]; and De- Reagents, kits, and antibodies Chen Lin, [email protected] The following reagents and antibodies were used: GS-9973 doi: 10.1158/1078-0432.CCR-16-2185 (MedKoo Sciences); siRNA pools targeting MALAT1 (Dharma- 2017 American Association for Cancer Research. con); antibodies against SYK (#2712), p-SYK (#2710 for

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treatment and cell viability assays were performed as described Translational Relevance previously (22). Although several molecular targets have been identified in Ewing sarcoma, their clinical trials have yet to show success. Apoptosis and cell-cycle assays Through high-throughput screens using both siRNA and Cell apoptosis was measured using propidium iodide (PI) and fi small-molecule inhibitor libraries, we identi ed SYK as an Annexin V (BD Biosciences) double staining and was assessed on In vitro important progrowth kinase in EWS. therapeutic win- a LSRII flow cytometer (BD Biosciences). Cell-cycle analysis was fi dows of two SYK-speci c inhibitors (PRT062607 and GS- performed by PI staining (Sigma-Aldrich) for DNA content fol- 9973) in EWS were similar to those of chronic lymphocytic lowed by flow cytometric analysis. All flow cytometry data were leukemia (CLL) cells. We further noted that forced activation analyzed using FlowJo software (Tree Star). of SYK was able to rescue the anti-EWS effects of SYK inhibitors. Thus, our data indicate that targeting SYK by selective small-molecule inhibitors holds the potential for treating Clonogenic assay EWS. In addition, we found SYK hyperphosphorylation in Cells were seeded in 12-well plates and treated with the indi- EWS, providing a basis for evaluating phosphorylated SYK as a cated doses of drugs. Two weeks after the treatment, colonies were fi potential biomarker in EWS. xed with 70% ethanol, stained with 0.1% methylene blue (Sigma), and positive colonies were counted using open CFU software (http://opencfu.sourceforge.net/).

immublotting), p-AKT (#4060), MEK (#4694) and p-MEK Immunoblotting and IHC (#9122), EZH2 (#5246; Cell Signaling Technology); p-SYK Protein lysates were resolved by SDS-PAGE, transferred to fl (SAB4503839 for IHC), and b-actin (A5316; Sigma). Other polyvinylidene di uoride membrane (Merck Millipore), and reagents included: Imprint RNA Immunoprecipitation (RIP) Kit followed by immunoblotting procedures as described previously fl (Sigma); Pierce Magnetic ChIP Kit (ThermoScientific); BioT trans- (22). IHC was performed as described previously (23). Brie y, fi fection reagent (Bioland Scientific); anti-rabbit IgG and anti- snap-frozen sections were xed in 100% acetone at 4 C, and p- mouse conjugated HRP antibodies (BD Biosciences); Dual-Lucif- SYK antibody was applied using standard immunoperoxidase erase Reporter Assay System (Promega). Small-molecule inhibi- techniques in a Sequenza semiautomatic stainer (Thermo Scien- fi tors were either purchased or generously provided by the sources ti c). p-SYK expression was scored using H-score method as outlined in Supplementary Table S1. described previously (21).

High-throughput screens with both siRNA and small-molecule Chromatin immunoprecipitation and RNA-IP 6 inhibitor libraries A total of 2 10 cells for each chromatin immunoprecipita- fi siRNA screens were performed in SKNMC, A673, TC32, tion (ChIP) reaction were xed with formaldehyde and ChIP TC71, SKES1, EW8, CADO-ES1, and EWS-502 cell lines. Briefly, experiments were performed using MAGnify Chromatin Immu- 500 cells/well were seeded in 96-well plates and incubated with noprecipitation System (Invitrogen) according to the manufac- siRNA transfection mixtures for 96 hours. All siRNAs were from turer's instructions. The precipitated DNA samples were quanti- fi Dharmacon RNAi Technologies and the RaPID Assays (Sigma) ed by qRT-PCR, and the data were expressed as the percentage of and other siRNA experiments were performed as described input DNA. Primers used for the ChIP-q-PCR are listed in Sup- 6 previously (18). Small-molecule inhibitor screening was per- plementary Table S2. For RNA-IP, lysate prepared from 2 10 m formed in SKNMC, A673, TC32, TC71, SKES1, EW8, TTC-446, cells were immunoprecipitated using 5 g of either normal rabbit CADO-ES1 cell lines, and the cell viability was calculated on the IgG, or anti-EZH2 antibody according to the manufacturer's basis of an algorithm previously described (19, 20). Briefly, 200 instructions. Immunoprecipitation of EZH2-recruited RNA was cells/well were seeded in 384-well plates and incubated with measured by qRT-PCR using GAPDH as a control gene. indicated small-molecule inhibitors by seven serial of concen- trations (three times of dilutions between dosages, ranging CRISPR-Cas9 guide RNA design from 10 nmol/L to 10 mmol/L) for 72 hours. At the end point, The LentiCRISPRv2 virus vector was obtained from Addgene, MTS assay was used to measure the cell viability. IC50 values and the guide RNAs were designed on the basis of the Optimized were calculated from a third-order polynomial curve fitting to CRISPR Design service engine (http://crispr.mit.edu/). Vector the data points by an in-house analytic framework as we have subcloning was described previously (24). Guide RNA sequences described recently (19). and primers for vector subcloning are listed in Supplementary Table S2. Cell culture, drug treatment, and cell viability assays Ewing sarcoma cell lines (SKNMC, A673, TC32, TC71, SKES1, Lentiviral infections EW8, TTC-446, EWS502, and CADO-ES1) were kindly provided Lentiviral shSYKs and control vectors were generous gifts from by Dr. Kimberly Stegmaier (Harvard Medical School, Boston, MA) Dr. Kimberly Stegmaier (25). Lentiviral shc-MYC and control and Dr. Stephen L. Lessnick (University of Utah, Salt Lake City, vectors were obtained from Addgene (26). Cells were transduced UT), and were grown in DMEM (Corning) supplemented with with viral particles in the presence of 8 mg/mL polybrene for 16 10% FBS, penicillin, and streptomycin. The identity of all cell lines hours followed by replacement of the lentivirus-containing media was recently verified by short tandem repeat analysis (21). All cells with fresh media. Two days after infection, puromycin (2 mg/mL) have been tested with no mycoplasma contamination. Drug was added for 3 days to eliminate uninfected cells.

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Dual luciferase reporter assay Line Encyclopedia (CCLE, http://www.broadinstitute.org/ccle; MALAT1 promoter regions (1010 bp, 700 to þ310 bp) were ref. 28), respectively. Differences between two groups were ana- amplified using DNA from TC71 cells as templates, and the PCR lyzed using either paired or unpaired two-tailed Student t test. products were cloned into pGL3B luciferase vector (Promega). One-way ANOVA was used for comparisons among multiple This vector was transfected into cells, luciferase activity was groups (, P < 0.05; , P < 0.01; , P < 0.001). Overlaps of assessed 48 hours after transfection using the Dual-Luciferase gene lists were identified using program VENN (http://bioinfor Reporter Assay System according to the manufacturer's instruc- matics.psb.ugent.be/webtools/Venn/). Statistical significance of tions (Promega), and the ratio of Firefly/Renilla luciferase activ- overlapping was determined using c2 tests with one degree of ities (RLU) was calculated. Primers for the construction of the freedom and Yates correction as previously described (29). luciferase expressing vector (pGL3B-MALAT1) are listed in Sup- plementary Table S2. Results qRT-PCR High-throughput siRNA and small-molecule inhibitor library Total RNA was extracted using RNeasy Isolation Kit (Qiagen). screens identify SYK as a novel oncogenic kinase in Ewing qRT-PCR was performed with standard procedures as described sarcoma previously (21). Briefly, cDNA was generated using qScript cDNA To screen unbiasedly for potential novel oncogenic kinases Synthesis Kit (Quanta Biosciences) and qRT-PCR was performed in Ewing sarcoma, we tested eight Ewing sarcoma cell lines by on CFX96 qPCR System (Bio-Rad). Expression of each gene was transfecting a kinase-specific siRNA library (set of four siRNAs D C normalized to GAPDH and quantified using 2 ( t) method. targeting each gene) against 94 known kinases on a gene-per- Primers for qRT-PCR are listed in Supplementary Table S2. gene basis followed by the measurement of cell viability. As shown in Fig. 1A, the top 20 genes ranked by median inhibition cDNA microarray and data analysis rate values (MV) included a number of known oncogenic cDNA microarray and data analysis were performed as kinases in Ewing sarcoma, such as ROR1, KIT, FGFR, PTK described previously (21). Briefly, RNA was reverse transcribed families (PTK-2 and 7), Src families (FGR and SRMS), ERBB4, and hybridized on Affymetrix GeneChip HGU133 plus 2. The and EGFR (9, 30–37), which strongly supported the method- whole transcriptome expression data were obtained using the ology and effectiveness of our high-throughput screen. Impor- robust multichip average method (https://www.bioconductor. tantly, we identified novel targets including SYK and related org). Gene-set enrichment analysis (GSEA) was performed using proteins (SYK and ZAP70), BTK, ABL1, FLT4, and MATK. Of GSEA v2.07 tool (http://www.broad.mit.edu/gsea/) with msigdb. interest, MV of siSYK ranked the second and significantly v4.0 to identify significantly enriched gene sets. cDNA microarray suppressed the growth of three of the Ewing sarcoma cell lines data are available at the GEO repository website (GSE93677). examined (Fig. 1A and B). To complement the siRNA library screen, we performed an Animal models independent high-throughput approach testing a panel of 116 Animal experiments were approved by the Cedars-Sinai Insti- small-molecule inhibitors that are either FDA approved or in tutional Animal Care and Use Committee (IACUC). Seven-week- clinical trials. This compound library included inhibitors target- old female athymic nude mice (Crl:NU(NCr)-Foxn1nu) were ing well-established kinase pathways (e.g., RTK-MAPK, JAK, PI3K/ obtained from Charles River Laboratories (San Diego, CA) and AKT, PKC, IkK, AURK, and CDKs), as well as a variety of non- inoculated subcutaneously in both flanks with a suspension of kinase prosurvival factors including HSP70/90 proteins, BCL2 TC71 cells (2.0 106) in Matrigel. Five days after injection, when and BET families, WNT/b-catenin, and sonic hedgehog pathways, the tumor xenografts were noted to be growing, mice were and so on (Supplementary Table S1). The IC50 was determined for randomly divided into two groups and orally treated with either each compound across eight Ewing sarcoma cell lines. The specific vehicle [0.6% (w/v) aqueous Pluronic F-68, n ¼ 8] or GS-9973 (20 anti–Ewing sarcoma effects of the compounds were further deter- mg/kg, n ¼ 8), thrice weekly 4 weeks. Tumor volumes were mined by the comparison of the IC50 values in Ewing sarcoma cell measured with calipers, and were calculated using the following lines to those in 151 bone marrow aspirate samples tested using formula: volume (mm3) ¼ [width (mm)]2 length (mm)/2. the same platform that we previously published (Fig. 1C; Sup- Mice were sacrificed at the end of the fifth week from the date of plementary Table S3; ref. 19). For those compounds newly cell injection, and the tumors were dissected and weighted. included in this study, comparably low IC50s, based on their previous reports in other cancers, were also listed (Supplementary c-MYC and H3K27ac ChIP-seq analysis Table S4; refs. 16, 38–41). As shown in Fig. 1C, the targets of top c-MYC ChIP-seq uniform peaks on MALAT1 genomic region ranked chemicals included protein kinase C (PKC), AURK, PI3K, was analyzed using CistromeFinder system (http://cistrome.org/ B-RAF/VEGFR, EGFR/JAK2, and SYK. As SYK stood out as a novel finder) and ENCODE project (27) on 10 human cells. H3K27ac candidate in both screens, our further studies focused on this histone mark was analyzed using ENCODE project. The results kinase in Ewing sarcoma. Immunoblotting assays showed that were visualized further by UCSC Genome Browser (https:// although total SYK protein was expressed relatively weakly in genome.ucsc.edu/cgi-bin/hgGateway). Ewing sarcoma, the kinase was prominently phosphorylated in Ewing sarcoma cells compared with leukemic cells which have Statistical analysis been shown to have hyperactivation of SYK (ref. 42; Fig. 1E). In The mRNA expression level of MALAT1 from various types of addition, IHC staining of p-SYK on 35 primary Ewing sarcoma primary cancer tissues and cell lines were examined by analyzing samples revealed that 40% of samples showed strong phosphor- Expression Project for Oncology ExpO dataset (https://www.ncbi. ylation of this kinase (Fig. 1D; Supplementary Fig. S2; and nlm.nih.gov/geo/query/acc.cgi?acc=GSE2109) and Cancer Cell Supplementary Table S5).

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A B D Negative + Positive siRNA SKNMC A673 TC32 TC71 SKES1 EW8 TTC-446 CADO Median ROR1 SKES1 SYK ZAP70 BTK SURVIVIN KIT FGFR3 ABL1 DDR1 PTK2 EPHA6 PTK7 40X FGR ERBB4 TC32 SRMS FLT4 LMTK3 DDR2 MATK ++ Positive EGFR PTK2B H Score value STYK1 0–99 MUSK 100–199 TYRO3 TC71 20% FER 200–300 n BMX = 7 ALK 40% TNK2 n = 14 EPHB3 FRK 40% FYN n = 14 ITK LMTK2 ERBB2 PTK9L LCK EPHB6 RET PDGFRB FGFR2 C IC50 (nmol/L) of EWS cell lines ABL2 IGF1R Ranking BM Con SKNMC A673 TC32 TC71 SKES1 EW8 CADO EWS502 Drug name Target JAK3 TXK 1 CHIR-265 B-RAF/VEGFR TEK 2 PI-103 PI3K ERBB3 TNK1 3 VX-680 pan-Aurora EPHA1 4 EPHA7 AZD-1152 Aurora B MERTK 5 Go6976 PKC ROR2 FES 6 MLN-8054 Aurora A FGFR4 7 MET PKC-412 PKC TEC 8 Erlotinib EGFR/JAK2V617F KDR MST1R 9 Staurosporin wide range of targets` EPHA4 10 TYK2 CHIR-258 wide range of targets SRC 11 N/A Elesclomol Hsp90/apoptosis PTK6 EPHB4 12 N/A PRT062607 SYK JAK1 13 N/A BMS-345541 IKK PDGFRA KRAS 14 N/A GDC-0941 PI3K NTRK2 FGFR1 15 N/A XL-880 MET, VEGFR2,KDR ROS1 …… …… JAK2 …… …… EPHB2 LTK 54 GW-786034 VEGFR/c-KIT EPHA2 CSK 55 Sorafenib VEGFR, PDGFR, RAF LYN …… …… AATK …… …… FLT1 TP53RK 115 linifanib VEGFR/PDGFR/KDR/CSF1R HCK 116 NTRK1 Nilotinib BCR-ABL/KIT/LCK/EPHA/DDR EPHB1 TIE Most resistant Most sensitive EPHA3 YES1 CSF1R E EWS Leukemia EPHA8 Con SKNMC TC71 SKES1 TC32 A673 TTCEW8 KG1 U973 NB4 HL60 HL60R BLK NTRK3 AXL p-SYK FLT3 RYK PTK9 SYK EPHA5 NRAS INSR b-Actin Most pro-proliferate Most anti-proliferative

Figure 1. High-throughput siRNA and small-molecule inhibitor library screens identiﬁed SYK as a candidate therapeutic target in Ewing sarcoma. A, Heatmap of inhibition rate values of all siRNAs in eight Ewing sarcoma cell lines. "CADO," CADO-ES1. Arrows highlight SYK and ZAP70 tyrosine kinases. B, Bar graphs of cell viability upon transfection of different siRNAs (same as A) in SKES1, TC32, and TC71 cell lines. Values represent percentage of mean (normalized to the median value

on the plate) SD (n ¼ 3). C, Heatmap of IC50s of small-molecule inhibitors against eight Ewing sarcoma cell lines. The left column showed the pooled results from 151 leukemia bone marrow samples (19). BM Con, bone marrow control. The color scale is the same as A. A full list of the small-molecule inhibitors is shown in Supplementary Table S1. MV, median inhibition rate value. D, Representative IHC photos of p-SYK expression in Ewing sarcoma tissues, with pie charts showing H-scoring. E, p-SYK and SYK expression in Ewing sarcoma and acute myeloid leukemia cell lines examined by immunoblotting. b-Actin was used as a loading control.

SYK promotes the malignant phenotype of Ewing (Fig. 2A). Either of the two selective SYK inhibitors (PRT062607 sarcoma cells and GS-9973), dose-dependently inhibited the viability of Ewing To determine the biological signiﬁcance of SYK in Ewing sarcoma cells (mean IC50s approximately 3.8 and 4.6 mmol/L, sarcoma, it was inhibited through a variety of genetic and chem- respectively; Fig. 2B). In addition, through blocking the phos- ical approaches. Consistent with the earlier screening results, phorylation of SYK (Fig. 2D), these inhibitors markedly sup- knockdown of SYK by two independent shRNAs signiﬁcantly pressed AKT phosphorylation (Fig. 2F), clonogenic growth decreased the proliferation of Ewing sarcoma cell lines (Fig. 2C), and induced massive cell death (Fig. 2E) as well as

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SKES1 TC71 TC32 A G H 1.5 Scramble sh 1 sh 2 Scramble sh 1 sh 2 Scramble sh 1 sh 2 SKES1 EV EV SYK SYK *** SYK ecto 1.0 * endo p-SYK β-Actin 0.5 Absorbance 0.8 Con 1.5 Con 1.5 Con β-Actin SKES1 shSYK 1 shSYK 1 shSYK 1 0.0 0.6 shSYK 2 shSYK 2 shSYK 2 0 1 2 3 4 5 1.0 1.0 Day 0.4 TC71 EV SYK 1.0 0.5 0.5 EV *** 0.2 ecto 0.8 Absorbance

Absorbance SYK Absorbance p-SYK endo 0.6 ** 0.0 0.0 0.0 01234 5 012345 543210 0.4 Days Days Days β-Actin B Absorbance 0.2 TC71 125 125 0.0 0 1 2 3 4 5 100 I 100 ecto Day SYK 75 75 endo Con shSYK1 50 50 2.0 TTC-466 TC71 TTC-466 TC71 p-AKT SYK 25 CADO-ES SKES1 CADO-ES SKES1 shSYK1+ SYK TC32 SKNMC 25 TC32 SKNMC 1.5 A673 EW8 A673 EW8 AKT Cell viability (% of con) 0 Cell viability (% of con) 0 1.0 0 0.01 0.1 1 10 0 0.01 0.1 1 10 p-MEK μ μ PRT062607 ( mol/L) GS-9973 ( mol/L) 0.5 C MEK Absorbance 600 800 TC32 1,000 TC71 SKES1 0.0 β-Actin 0 1 2 3 4 5 800 600 400 Con SYK shSYK 1 SYK+shSYK 1 Day 600 400 400 J 100 200 100 EV EV 200 * * 200 * SYK SYK Colony numbers Colony numbers Colony numbers 75 *** 75 *** 0 0 0 ** *** DMSO GS GS PRT DMSO GS GS PRT DMSO GS GS PRT 50 50 ** 25 25

TC32 TC71 SKES1 (% of con) D Cell viability SKES1 TC71 DMSO GS PRT DMSO GS PRT DMSO GS PRT 0 0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 p-SYK p-SYK p-SYK PRT062607 (μmol/L) PRT062607 (μmol/L) SYK K SYK SYK 100 100 EV EV p-AKT p-AKT p-AKT * 75 SYK 75 SYK *** *** AKT AKT AKT *** β-Actin β-Actin β-Actin 50 *** 50 ** 25 25 (% of con) (% of (% of con) 80 80 SKES1 Cell viability TC71 E 40 TC32 TC71 SKES1 Cell viability 0 0 30 60 60 0 2 4 6 8 10 0 2 4 6 8 10 GS-9973 (μmol/L) GS-9973 (μmol/L) 20 40 40 M NC 10 20 20 1.5 KO-SYK (% of Con) (% of (% of Con) (% of Con)

Apoptotic cells Apoptotic KO-SYK+SYK Apoptotic cells 0 Apoptotic cells 0 0 1.0 Con GS PRT Con GS PRT Con GS PRT L DMSO+Serum Serum+GS-9973 NC KO1 KO2 KO3 Combo F 0.5

DMSO 7 15 30 60 7 15 30 60 Time (min) SYK Absorbance

p-AKT 0.0 β-Actin 543210 AKT Days

Figure 2. SYK activation promotes Ewing sarcoma malignancy. A, Ewing sarcoma cells stably expressing either SYK-speciﬁc shRNAs (sh 1 and 2) or scrambled shRNA control (Scramble), and cell proliferation was measured by MTT assay (bottom). Knockdown was evaluated by immunoblotting (top). Con, control cells. B, Cell growth of eight Ewing sarcoma cell lines treated by SYK inhibitors was measured by MTT assay. C, Clonogenic assay of three Ewing sarcoma cell lines treated by SYK inhibitors. Bar graphs displayed colony numbers. GS (GS-9973), 1 and 2 mmol/L; PRT (PRT062607), 1 mmol/L. D, Ewing sarcoma cells were treated with either GS-9973 (2 and 4 mmol/L) or PRT062607 (2 mmol/L) for 24 hours, and immunoblotting was conducted to evaluate the expression of indicated proteins and phospho(p)-proteins. b-Actin was used as a loading control. E, Ewing sarcoma cells were treated with 5 mmol/L of GS-9973 or PRT062607 for 24 hours, and Annexin V/PI assays were conducted to evaluate cell apoptosis. Bar graphs displayed the percentage of apoptotic cells (positive Annexin V þ PI). Con, Control. F, TC71 cells were serum-starved overnight, treated with either DMSO control or GS-9973 (2 mmol/L) for 2 hours followed by addition of medium containing 10% FBS for indicated durations, and the activation of AKT was measured by immunoblotting. G and H, SKES1 and TC71 cells were transiently transfected with either empty plasmid (EV) or plasmid encoding myristoylated SYK, and cell proliferation was measured by MTT assay (right). Immunoblotting was performed to determine p-SYK expression. Ecto, ectopically expressed SYK; Endo, endogenously expressed SYK. I, TC71 cells stably expressing indicated shRNAs were transiently transfected with myristoylated SYK or control vectors, and subjected to immunoblotting (left) and MTT assay (right). J and K, SKES1 and TC71 cells were transiently transfected with either myristoylated SYK or empty vector (EV) for 24 hours, followed by treatment with increasing dose of PRT062607 (0, 0.25, 0.5, 1, 2 mmol/L; J)or GS-9973 (0, 1.25, 2.5, 5, 10 mmol/L; K), and cell proliferation was measured by MTT assay. L, CRISPR/Cas9 gene editing efﬁciency of TC71 cells with different SYK guide RNAs (gRNA) was evaluated by immunoblotting. KO1, gRNA-1; KO2, gRNA-2; KO3, gRNA-3; Combo, mix of 3 gRNAs; M, TC71 cells with either SYK knockout (mix of 3 gRNAs) or nontargeting control gRNA (NC) were transiently transfected with either myristoylated SYK or empty vector, and cell proliferationwas measured by MTT assay.

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0.00001, c2 tests with one degree of freedom and Yates correction; Fig. 4A; Supplementary Table S6, random qRT-PCR validation results were shown in Fig. 4C and D). Interestingly, we noticed a long noncoding RNA, known as Metastasis Asso- ciated Lung Adenocarcinoma Transcript 1 (MALAT1), among the top-ranked downregulated genes (Fig. 4B). MALAT1 has been implicated in multiple physiologic processes, and it is highly expressed in many types of cancers (48). Analysis of a Pan-cancer expression microarray dataset (49) showed that MALAT1 expression was ranked third highest in Ewing sarcoma among all 18 different types of sarcomas (Fig. 4E; Supplemen- tary Table S7; ref. 49). In addition, CCLE dataset showed that MALAT1 expression was the eighth highest in Ewing sarcoma among 37 different types of cancer cell lines (Fig. 4F; Supple- mentary Table S8). These results together suggest that MALAT1 is highly expressed both in Ewing sarcoma tumor tissues and cell lines. To validate that MALAT1 expression is dependent on SYK-activated signaling, we inhibited SYK either by shRNA Figure 3. knockdown (Fig. 4G), CRISPR/Cas9 knockout (Fig. 4H), or SYK inhibitor (GS-9973) significantly suppressed Ewing sarcoma xenograft small-molecule inhibitors (2 mmol/L, 24 hours; Fig. 4I), and growth. A and B, Nude mice engrafted with TC71 cells were treated by oral confirmed the consistent and marked decrease of MALAT1 gavage with either vehicle or GS-9973 (20 mg/kg). Tumor weights at completion of the study (A) and the resected tumors (B)areshown.C, levels. In contrast, ectopic expression of active SYK enhanced Tumor tissues were dissected at completion of the study, protein was the expression levels of MALAT1 (Fig. 4J). extracted, immunoblotted, and probed with indicated antibodies. Data (A)representmean SD. Oncogenic functions of MALAT1 in Ewing sarcoma We next examined the role of MALAT1 in Ewing sarcoma through siRNA-mediated knockdown, and noticed that it potent- cell-cycle arrest (Supplementary Fig. S3). Importantly, ectopic ly inhibited cell proliferation (Fig. 5A and B). Furthermore, expression of the myristoylated (constitutively active) form of silencing MALAT1 robustly induced cell apoptosis (Fig. 5C) and SYK promoted cell proliferation (Fig. 2G and H), enhanced G1 cell-cycle arrest (Fig. 5D) in Ewing sarcoma cells, with con- phosphorylation of both AKT and MEK (Fig. 2I, left), and comitant downregulation of cyclin D1 level and upregulation of completely or partly rescued the cytotoxicity triggered by either p27kip1 and p21cip1 levels (Fig. 5E). shRNA knockdown or chemical inhibition of SYK (Fig. 2I–K). To confirm further the ontarget effect of these loss-of-function experi- MALAT1 is transcriptionally activated through SYK/c-MYC ments, CRISPR/Cas9–mediated gene editing was used to disrupt pathway endogenous SYK expression, which again significantly decreased We next sought to determine the mechanisms underlying the Ewing sarcoma cell proliferation (Fig. 2L and M), and was rescued upregulation of MALAT1 by signaling mediated by SYK. by the restoration of active SYK (Fig. 2M). MALAT1 was recently reported to be transcriptionally activated We next examined the anti–Ewing sarcoma property of the SYK by c-FOS in renal cell carcinoma (50). In addition to c-FOS, inhibitor (GS-9973) in a murine xenograft model. As shown bioinformatics analysis predicted that 20 additional transcrip- in Fig. 3A and B, GS-9973 treatment significantly suppressed tion factors, including c-MYC, had the potential to bind to the tumor growth. GS-9973 also potently decreased the levels of p- promoter region of MALAT1 (50). Interestingly, our microarray SYK, p-AKT, and p-MEK in the tumor cells (Fig. 3C), in accordance data showed inhibition of SYK significantly downregulated the with our observations obtained in vitro. expression of c-MYC (Supplementary Table S6). GSEA analysis further identified that c-MYC downstream targets (51) were Long noncoding RNA MALAT1 is upregulated by SYK-mediated significantly enriched in those genes altered by SYK inhibition signaling in Ewing sarcoma (Supplementary Fig. S1E). Given that SYK activation was To understand better the role of SYK in Ewing sarcoma, we observed to induce expression of c-MYC in hematopoietic cell performed whole transcriptome profiling of TC71 cells after lines (52), we hypothesized that a novel signaling axis involv- SYK inhibition through either shRNA knockdown or exposure ing SYK/c-MYC/MALAT1 might be operative in Ewing sarcoma to GS-9973 (2 mmol/L, 24 hours), and compared the results to cells. To address this hypothesis, we analyzed public c-MYC the control cells. SYK inhibition globally altered gene transcrip- ChIP-seq data using both CistromeFinder and ENCODE pro- tion; and GSEA analysis revealed that genes associated with jects. Of note, two major c-MYC–binding peaks spanning genomic-unstable Ewing sarcoma phenotype (43), cell cycle MALAT1 promoter were identified across different cell types (44, 45), and NFkB pathway (46) were significantly enriched in (Fig. 6A; Supplementary Table S9), supporting the positive SYK knockdown cells (Supplementary Fig. S1A–S1C), and regulation of c-MYC on MALAT1 transcription. In addition, genes associated with cell metastasis (47) were significantly strong H3K27ac signals (predictive of active enhancer and enriched in cells treated with the SYK inhibitor (Supplementary promoter) were detected to be surrounding MALAT1 promoter. Fig. S1D). Venn diagram analysis showed that 426 and 1,126 Both c-MYC–binding peaks almost perfectly fitted into genes were codownregulated and coupregulated, respectively, H3K27ac "dip" (Fig. 6A) strongly suggesting that c-MYC occu- by both shRNA- and GS-9973–mediated approaches (P < pies the MALAT1 promoter and recruits cofactors and histone

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A CD DMSO SYKi 1.2 Scramble shSYK SYKi shSYK 1.2 1.0 1.0 Downregulated genes Upregulated genes 0.8 P < 0.0001 P < 0.0001 0.8 0.6 0.6 0.4 0.4 0.2 0.2 Relative mRNA level Relative mRNA level 0.0 0.0

PTN PTN MEST RND3 RND3 MMP1 RASA1ZBED3SUSD5 MESTMMP1 RASA1ZBED3SUSD5 E MAK2P7 MAK2P7 B DMSO SYKi Scramble shSYK Gene Gene

TAOK1 MAGEA12 level (log2) DDX17 MMP1 CCDC171 MALAT1 SPEN RARRES2 MALA1 Expression EWS FN1 TCEA3 DLEU2 PTN 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 LRRN3 LOC40004 VEZF1 MEST EGR1 SLC38A4 F DHX9 SERPINF1 LONRF1 COL1A2 FN1 SUSD5 ELK4 RND3 13 FN1 PTN C3 LXN SMC4 ZNF738 12 OSBPL8 PTN PAPPA LOXL1-AS ANKRD36B CTSZ 11 THAP6 MALAT1 NKX3-1 NEFL KLHL5 MAGEA6 DSP CCDC155 10 ZFHX3 IQCG C1orf43 EFHC2 MALAT1 OVAAL 9 MALAT1 NEFL MAP1B ZNF430 FN1 C14orf159 8 LRRN3 CCL28 EWS ABCD3 COL3A1 STOX2 MAGEA3 MALA1 Expression level (log2) PRKDC COL3A1 1 2 3 4 5 6 7 8 9 10 11 1213141516 1718192021222324252627282930 31323334353637 TET2 NKX2-3 MYCBP2 TNFAIP6 HNRNPA2B1 COL3A1 TPR NEFL G H NOTCH2NL CRABP1 Scramble shSYK NC KO-SYK PAPPA CCL28 SFPQ TP63 1.2 TC71 1.2 TC32 1.2 TC71 1.2 TC32 TPR SYNPO2 MKI67 CCND2 1.0 1.0 1.0 1.0 PPM1L TNFAIP6 HIP1 CALD1 0.8 0.8 0.8 0.8 RBM5 MALAT1 PRRC2C MAP1B SNX5 HSPA2 0.6 0.6 0.6 0.6 COL3A1 S100A6 ZNF704 ZNF573 0.4 0.4 0.4 0.4 WASL DLX1 HELLS PFKFB2 0.2 0.2 0.2 0.2 NID2 MALAT1 Relative RNA level RNA Relative level RNA Relative CEP135 POPDC3 0.0 0.0 Relative RNA level 0.0 Relative RNA level PURB CYGB 0.0 COL3A1 COL6A1 SYK MALAT1 SYK MALAT1 SYK MALAT1 SYK MALAT1 LINC00622 APOE PABPC1L COL1A2 OLFM1 MAP1B ANKRD13C ALDH1A1 I DMSO GS-9973 PRT062607 J Empty vector SYK MIR3682 ZNF430 1.2 1.2 RUSC1-AS1 CHD7 TC71 TC32 80 150 MUM1 BCHE RASEF MALAT1 1.0 1.0 60 100 DYNC1H1 NA TC71 TC32 ASH1L SYNPO2 0.8 0.8 40 PRRC2C MALAT1 50 NASP ZNF614 0.6 20 PAPPA SHISA6 0.6 ARMCX4 DCX 0.4 0.4 2.0 10 MALAT1 JAG2 8 1.5 6 Least Most 0.2 0.2 1.0 4

Relative RNA level Relative RNA level Relative RNA level 0.5 Relative RNA level 2 expressed expressed 0.0 0.0 0.0 0 MALAT1 MALAT1 SYK MALAT1 SYK MALAT1

Figure 4. Long noncoding RNA MALAT1 is upregulated by SYK in Ewing sarcoma cells. A, Venn diagrams showing genes codownregulated and coupregulated by SYK inhibitor GS-9973 (SYK i) and SYK knockdown (shSYK) in TC71 cells. B, Heatmaps showing top 70 downregulated genes upon either GS-9973 treatment or SYK knockdown in TC71 cells. Results of two replicas from each group are shown. C and D, Eight genes that showed signiﬁcant downregulation by either GS-9973 treatment or SYK knockdown as measured by cDNA microarray, were randomly selected, and mRNA level of each gene was evaluated by qRT-PCR. E, Bar graph showing normalized MALAT1 RNA expression from patients with one of 18 different types of sarcoma that was previously reported RNA array dataset (49). Cancer types are shown in Supplementary Table S7. F, Bar graph showing RNA level of MALAT1 among over 1,000 cancer cell lines representing 37 cancer types. Data were obtained from CCLE database. Cancer types are shown in Supplementary Table S8. G–J, mRNA levels of SYK and MALAT1 were examined by qRT-PCR in TC71 and TC32 cells upon indicated treatment.

modifiers to activate MALAT1 transcription. To validate this confirmed by the luciferase reporter assay (Fig. 6D and E). observation experimentally, ChIP-qPCR and dual luciferase Importantly, ectopic expression of c-MYC significantly induced reporter assays were performed. A set of primer pairs was the promoter activity, whereas silencing of either c-MYC or SYK designed to tile across the promoter regions (700 to þ310 produced an opposite effect (Fig. 6D and E; Supplementary bp) of MALAT1 (Fig. 6B). ChIP-qPCR results revealed that c- Fig. S4). In addition, the suppressive effect of SYK inhibition MYC bound more avidly to the promoter of MALAT1 in Ewing was rescued by ectopic expression of c-MYC (Fig. 6E). Con- sarcoma cells compared with either IgG or FLI1 antibody sistent with this model, silencing of SYK downregulated the (recognizing EWS-FLI1) negative controls (Fig. 6C). In addi- expression of both c-MYC and MALAT1 (Fig. 6F), and c-MYC tion, the transcriptional activity of MALAT1 promoter was knockdown significantly inhibited MALAT expression (Fig. 6G

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A Scramble siMALAT1 SKES1 TC32 TC71 1.0 1.0 1.0

0.8 0.8 0.8

0.6 0.6 0.6 ** ** 0.4 0.4 *** 0.4 0.2 0.2 0.2 Relative mRNA level Relative mRNA level 0.0 0.0 Relative mRNA level 0.0

B 1.5 Scramble 1.5 Scramble 2.0 Scramble siMALAT1 siMALAT1 siMALAT1 1.5 1.0 1.0 SKES1 TC32 TC71 1.0 0.5 0.5

Absorbance 0.5 Absorbance Absorbance * * 0.0 * * 0.0 0.0 * 012345 012345 012345 Days Days Days

Figure 5. C Scramble siMALAT1 Silencing of MALAT1 inhibited Ewing sarcoma 40 SKES1 40 TC32 30 TC71 cell growth. A–E, Ewing sarcoma cells were transiently transfected with scrambled siRNA 30 *** 30 *** control (Scramble) or a set of 4 siRNAs against 20 *** MALAT1 (siMALAT1) for 24 hours, and the knockdown efﬁciency was evaluated by qRT- 20 20 PCR (A); cell proliferation was measured by MTT 10 (% of total) 10 10 (% of total) assay (B); cell apoptosis was evaluated by (% of total) Apoptotic cells Apoptotic cells Annexin V/PI assays (C); cell cycle was measured Apoptotic cells by PI staining (D); p27kip1, p21cip1, and cyclin D1 0 0 0 protein levels were measured by immunoblotting. b-Actin was used as a loading G0/G1 S G2 G2 control (E). Data (A–C) represent mean SD of D three independent experiments. SKES1 TC32 TC71 100 100 100

80 80 80

60 60 60 Cells Cells 40 40 Cells 40 (% of total) (% of total) (% of total) 20 20 20

0 0 0

E SKES1 TC32 TC71 Con si Con si Con si Cyclin D1

p27kip1

p21cip1

β-Actin

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B C IgG EWS-FLI1 c-MYC MALAT1 promoter -700 0 +310 6 8 MALAT1 TC32 -609 PR1 -501 7 5 TC71 -516 PR2 -416 6 -444 PR3 -339 4 5 -196 PR4 -66 3 NS 4 PR5 85 NS 10 3 NS PR6 226 2 Primer pairs 134 2 203 PR7 300 1 1 Relative fold change Relative fold change 0 0 PR1 PR2 PR3 PR4 PR5 PR6 PR7 PR1 PR2 PR3 PR4 PR5 PR6 PR7

DETC71 TC71 F NS 1.5 1.5 10 10 NC KO-SYK NC KO-SYK 8 8 TC71 TC32 6 6 1.0 1.0 4 4 2 2 0.010 0.010 NS 0.5 0.5 0.008 NS 0.008 0.006 0.006 Relative mRNA level Relative mRNA level Relative mRNA RLV (Luc/Ren) RLV (Luc/Ren) 0.004 0.004 0.0 0.0 0.002 0.002 SYK c-MYC MALAT1 SYK c-MYC MALAT1 0.000 0.000 pGL3B + + pGL3B + + pGL3B-MALAT1 + + + + pGL3B-MALAT1 + + + + + Renilla + + + + + + Renilla + + + + + + + KO-SYK + KO-SYK + + G H c-MYC c-MYC + Con + + TC71 TC32 1.2 Empty vectors + + + Empty vectors + + shc-MYC PRT062607 + sh MYC + 1.0 c-MYC 0.8 0.6 β-Actin 0.4 0.2

Relative mRNA level 0.0 TC71 TC32

Figure 6. MALAT1 is transcriptionally activated by SYK/c-MYC regulatory pathways. A, c-MYC ChIP-seq uniform peaks and H3K27ac histone marks on MALAT1 genomic region were analyzed by CistromeFinder and ENCODE projects, respectively, and were visualized by UCSC Genome Browser. B, Diagram displaying the positions of each primer pair on the promoter (700 to þ310 bp) of MALAT1 designed for ChIP-q-PCR experiments. C, Bar graphs showing the relative fold change of ChIP-qPCR results in TC71 and TC32 cells. D and E, Transient transcriptional activation of the luciferase gene was measured using dual-luciferase reporter assay. Renilla vector was cotransfected into TC71 cells along with different combinations of vectors. Bar graphs displaying RLV (relative luciferase value to Renilla value, Luc/Ren). F, qRT-PCR results showing the levels of MALAT1 and c-MYC upon SYK knockout by CRISPER/CAS9. G and H, TC71 and TC32 cells were stably infected with shc-MYC lentivirus, and expression of c-MYC and MALAT1 was measured by immunoblotting (G) and qRT-PCR (H), respectively. b-Actin was used as a loading control. Data (C–F, H) represent mean SD of three independent experiments.

and H). These results indicate that SYK-mediated signaling Discussion enhanced the expression of c-MYC, which further transcrip- Although several potential molecular targets have been iden- tionally activated MALAT1 through direct binding to its tiﬁed in Ewing sarcoma, their clinical trials aimed at these targets promoter. have yet to show success (53). Through high-throughput screens Taken together, our results suggest that a novel oncogenic using both siRNA and small-molecule inhibitor libraries, we signaling involving SYK/c-MYC/MALAT1 contribute to the malig- conﬁrmed a number of previously reported Ewing sarcoma nancy of Ewing sarcoma, which provide potential therapeutic actionable candidates such as ERBB4, EGFR, ROR1, KIT, and targets to treat this malignancy (Fig. 7).

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Figure 7. A schematic shows a novel SYK/c-MYC/MALAT1 signaling pathway in Ewing sarcoma cells.

FGFRs (9, 30–37). Most importantly, we identified SYK as an sarcoma cells (Fig. 5E). In agreement with our findings, depletion kip1 important progrowth kinase in Ewing sarcoma through unbiased of MALAT1 caused G1 arrest with the upregulation of p53, p27 , integrative approaches. and p21cip1 in human fibroblasts (58). Most recently, Malakar Chronic lymphocytic leukemia (CLL) is strongly addicted to and colleagues showed that MALAT1 transcriptionally activated SYK-mediated prosurvival pathway and is considered as one of cyclin D1 in hepatocellular carcinoma cells (59). These data the most sensitive malignancies for SYK inhibition. An in vitro suggest that MALAT1 controls G1 cell-cycle transition in multiple study of PRT062607 shows that 64% (27 of 42 samples) of CLL cell types by modulating the expression of important cell-cycle primary cells had IC50s higher than 3 mmol/L (16). Another SYK regulators. inhibitor, GS-9973, is currently under assessment in a phase II CLL Although the mechanism underlying cancer-specific overex- clinical trial (NCT01799889). In vitro viability study of 14 primary pression of MALAT1 is poorly understood, previous bioinformat- CLL samples showed that GS-9973 had a mean IC50 of 3.7 mmol/L ic analysis predicted that a number transcription factors, including (17, 54). Our results demonstrated a similar sensitivity to these c-FOS and c-MYC, could interact with the promoter region of two SYK inhibitors compared with CLL cells. We further noted MALAT1 (50). Importantly, our analysis of publicly available c- that forced activation of SYK rescued the anti–Ewing sarcoma MYC ChIP-seq data identified strong c-MYC occupancy on effects of the SYK inhibitors. Taken together, our data indicate that MALAT1 promoter region. ChIP-qPCR and luciferase reporter targeting SYK by selective small-molecule inhibitors holds the assays confirmed that c-MYC directly interacted with this region, potential for treating Ewing sarcoma. and enhanced the transcriptional activity of this lncRNA. Inter- Our immunoblotting result noted that albeit total SYK protein estingly, c-MYC is a SYK-regulated gene in hematopoietic cells levels were low in Ewing sarcoma cells, the kinase was promi- (52, 60), and our results also showed that silencing of SYK nently phosphorylated in these cells compared with leukemic downregulated c-MYC expression in Ewing sarcoma cells. More- lines which are known to have hyperactivation of SYK (ref. 42; Fig. over, luciferase assay demonstrated that forced expression of c- 1E). These results were further supported by IHC staining of p-SYK MYC promoted the transcription of MALAT1 regardless of SYK on 35 primary Ewing sarcoma tissues, which revealed that 40% of activity. Together, these results support the model that SYK samples had strong phosphorylation of SYK (Fig. 1D; Supple- elevates the transcription of MALAT1 through regulating c-MYC. mentary Table S5). How does SYK become hyperactivated in EZH2 is a histone methyl transferase subunit of the Polycomb Ewing sarcoma cells? One possible mechanism is through SRC repressor complex, which is either recurrently mutated or highly family kinase-dependent activation (13). Attas and colleagues expressed in many cancers (61). Recent studies have shown that showed that macrophages derived from hck / fgr / lyn / mice, EZH2 helps EWS-FLI1 to drive tumor growth and metastasis in which lose Src-family kinases in myeloid leukocytes, markedly Ewing sarcoma cells (62). MALAT1 interacts with EZH2, and the reduced SYK activation (55). Interestingly, recent studies reported oncogenic activities of MALAT1 were inhibited by EZH2 depletion that SRC kinases are also highly activated in Ewing sarcoma cells, in renal cancer (50). Therefore, we hypothesized that MALAT1 and and inhibition of SRC efficiently decreased Ewing sarcoma cell EZH2 might also cooperate in Ewing sarcoma cells. Importantly, growth (9, 36), which was confirmed by our high-throughput RNA-IP assay revealed that MALAT1 interacted directly with EZH2 approaches (Fig. 1A and C). in both TC71 and TC32 cells (Supplementary Fig. S5). Although Activated SYK results in phosphorylation of tyrosine residues of further investigations are needed to elucidate the biological sig- downstream targets including PKC, ERK, AKT, and NF-kBin nificance of this interaction, our findings highlight a strong hematopoietic cells (14). In agreement with these observations, oncogenic role of MALAT1 in Ewing sarcoma biology. our high-throughput screens found that Ewing sarcoma cells were In summary, through integrative molecular and cellular also dependent on PKC, ERK, and AKT for survival. Moreover, approaches, the current study elucidates a novel oncogenic signal- cDNA microarray analysis suggested that NF-kB pathway was ing axis involving SYK, c-MYC, and MALAT1 in the setting of Ewing suppressed by SYK inhibition in Ewing sarcoma cells. In addition, sarcoma biology (Fig. 7). Importantly, our results strongly suggest our immunoblotting assays showed that SYK inhibitors decreased that targeting SYK-mediated signaling may serve as a promising the phosphorylation of both AKT and ERK. Together, these results therapeutic strategy for the treatment of Ewing sarcoma patients. suggest that SYK activates multiple important prosurvival pathways in Ewing sarcoma, and future studies will determine the Disclosure of Potential Conflicts of Interest biological significance underlying these signals activated by SYK. No potential conflicts of interest were disclosed. Our work demonstrated that a LncRNA, MALAT1, is deregulated by SYK-mediated signaling. MALAT1 is a known oncoRNA Authors' Contributions overexpressed in many different cancer types (48, 56, 57). We Conception and design: H. Sun, H.J. Lim, S. Gery, H.P. Koeffler observed that MALAT1 knockdown induced G1 arrest in Ewing Development of methodology: H. Sun, J.W. Said

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Acquisition of data (provided animals, acquired and managed patients, Excellence initiative, and the Singapore Ministry of Health's National Med- provided facilities, etc.): H. Sun, D.-C. Lin, Q. Cao, B. Pang, V.K.M. Lee, ical Research Council under its Singapore Translational Research (STaR) J.W. Said, M. Chow, J.W. Tyner Investigator Award. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H. Sun, B. Pang, J.W. Said, A. Mayakonda, J.W. Tyner, H.P. Koeffler Grant Support Writing, review, and/or revision of the manuscript: H. Sun, D.-C. Lin, D.-C. Lin was supported by Donna and Jesse Garber Awards for Cancer D.D. Gae, D.-C. Lin, C. Forscher, J.W. Tyner Research and the National Center for Advancing Translational Sciences Administrative, technical, or material support (i.e., reporting or organizing UCLA CTSI Grant UL1TR000124. This research is also partially supported data, constructing databases): D.-C. Lin, S. Gery, M. Chow by the RNA Biology Center, Cancer Science Institute of Singapore, NUS Study supervision: D.-C. Lin, H.P. Koeffler (MOE2014-T3-1-006). Minor edits in figure clarity: D.D. Gae The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Acknowledgments advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate We thank Dr. Kimberly Stegmaier and Dr. Stephen L. Lessnick for their this fact. generous help with the reagents and cell lines. This research was supported by the Alan B. Slifka Foundation, the National Research Foundation Singa- Received August 31, 2016; revised October 5, 2016; accepted March 21, 2017; pore and the Singapore Ministry of Education under the Research Centres of published OnlineFirst March 23, 2017.

References 1. Tirode F, Surdez D, Ma X, Parker M, Le Deley MC, Bahrami A, et al. 17. Sharman J, Hawkins M, Kolibaba K, Boxer M, Klein L, Wu M, et al. An Genomic landscape of Ewing sarcoma defines an aggressive subtype open-label phase 2 trial of entospletinib (GS-9973), a selective spleen with co-association of STAG2 and TP53 mutations. Cancer Discov tyrosine kinase inhibitor, in chronic lymphocytic leukemia. Blood 2014;4:1342–53. 2015;125:2336–43. 2. Riggi N, Stamenkovic I. The biology of Ewing sarcoma. Cancer Lett 18. Bicocca VT, Chang BH, Masouleh BK, Muschen M, Loriaux MM, Druker 2007;254:1–10. BJ, et al. Crosstalk between ROR1 and the Pre-B cell receptor promotes 3. Brohl AS, Solomon DA, Chang W, Wang J, Song Y, Sindiri S, et al. The survival of t(1;19) acute lymphoblastic leukemia. Cancer Cell 2012; genomic landscape of the Ewing sarcoma family of tumors reveals recurrent 22:656–67. STAG2 mutation. PLoS Genet 2014;10:e1004475. 19. Tyner JW, Yang WF, Bankhead AIII, Fan G, Fletcher LB, Bryant J, et al. Kinase 4. Crompton BD, Stewart C, Taylor-Weiner A, Alexe G, Kurek KC, Calicchio pathway dependence in primary human leukemias determined by rapid ML, et al. The genomic landscape of pediatric Ewing sarcoma. Cancer inhibitor screening. Cancer Res 2013;73:285–96. Discov 2014;4:1326–41. 20. Jiang YY, Lin DC, Mayakonda A, Hazawa M, Ding LW, Chien WW, et al. 5. Castex MP, Rubie H, Stevens MC, Escribano CC, de Gauzy JS, Gomez- Targeting super-enhancer-associated oncogenes in oesophageal squamous Brouchet A, et al. Extraosseous localized Ewing tumors: improved outcome cell carcinoma. Gut 2016. [Epub ahead of print]. with anthracyclines–the French society of pediatric oncology and interna- 21. Sun H, Lin DC, Cao Q, Guo X, Marijon H, Zhao Z, et al. CRM1 inhibition tional society of pediatric oncology. J Clin Oncol 2007;25:1176–82. promotes cytotoxicity in Ewing sarcoma cells by repressing EWS-FLI1- 6. Macy ME, Sawczyn KK, Garrington TP, Graham DK, Gore L. Pediatric dependent IGF-1 signaling. Cancer Res 2016;76:2687–97. developmental therapies: interesting new drugs now in early-stage clinical 22. Sun H, Hattori N, Chien W, Sun Q, Sudo M, GL EL, et al. KPT-330 has trials. Curr Oncol Rep 2008;10:477–90. antitumour activity against non-small cell lung cancer. Br J Cancer 2014; 7. Rossler J, Geoerger B, Taylor M, Vassal G. Small molecule tyrosine kinase 111:281–91. inhibitors: potential role in pediatric malignant solid tumors. Curr Cancer 23. Lin DC, Xu L, Chen Y, Yan H, Hazawa M, Doan N, et al. Genomic and Drug Targets 2008;8:76–85. functional analysis of the E3 ligase PARK2 in glioma. Cancer Res 8. Tanaka M, Yamazaki Y, Kanno Y, Igarashi K, Aisaki K, Kanno J, et al. Ewing's 2015;75:1815–27. sarcoma precursors are highly enriched in embryonic osteochondrogenic 24. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, et al. progenitors. J Clin Invest 2014;124:3061–74. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 9. Crompton BD, Carlton AL, Thorner AR, Christie AL, Du J, Calicchio ML, 2014;343:84–7. et al. High-throughput tyrosine kinase activity profiling identifies FAK as a 25. Carnevale J, Ross L, Puissant A, Banerji V, Stone RM, DeAngelo DJ, et al. SYK candidate therapeutic target in Ewing sarcoma. Cancer Res 2013;73: regulates mTOR signaling in AML. Leukemia 2013;27:2118–28. 2873–83. 26. Lin CH, Jackson AL, Guo J, Linsley PS, Eisenman RN. Myc-regulated 10. Scotlandi K, Benini S, Sarti M, Serra M, Lollini PL, Maurici D, et al. Insulin- microRNAs attenuate embryonic stem cell differentiation. EMBO J like growth factor I receptor-mediated circuit in Ewing's sarcoma/periph- 2009;28:3157–70. eral neuroectodermal tumor: a possible therapeutic target. Cancer Res 27. Consortium EP. An integrated encyclopedia of DNA elements in the 1996;56:4570–4. human genome. Nature 2012;489:57–74. 11. Uren A, Merchant MS, Sun CJ, Vitolo MI, Sun Y, Tsokos M, et al. Beta- 28. Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S, platelet-derived growth factor receptor mediates motility and growth of et al. The Cancer Cell Line Encyclopedia enables predictive modelling of Ewing's sarcoma cells. Oncogene 2003;22:2334–42. anticancer drug sensitivity. Nature 2012;483:603–7. 12. Arnaldez FI, Helman LJ. New strategies in Ewing sarcoma: lost in trans- 29. Ihara M, Meyer-Ficca ML, Leu NA, Rao S, Li F, Gregory BD, et al. Paternal lation? Clin Cancer Res 2014;20:3050–6. poly (ADP-ribose) metabolism modulates retention of inheritable sperm 13. Mocsai A, Ruland J, Tybulewicz VL. The SYK tyrosine kinase: a crucial player histones and early embryonic gene expression. PLoS Genet 2014;10: in diverse biological functions. Nat Rev Immunol 2010;10:387–402. e1004317. 14. Geahlen RL. Getting Syk: spleen tyrosine kinase as a therapeutic target. 30. Ordonez JL, Amaral AT, Carcaboso AM, Herrero-Martin D, del Carmen Trends Pharmacol Sci 2014;35:414–22. Garcia-Macias M, Sevillano V, et al. The PARP inhibitor olaparib enhances 15. Zhang J, Benavente CA, McEvoy J, Flores-Otero J, Ding L, Chen X, et al. A the sensitivity of Ewing sarcoma to trabectedin. Oncotarget 2015;6: novel retinoblastoma therapy from genomic and epigenetic analyses. 18875–90. Nature 2012;481:329–34. 31. Mendoza-Naranjo A, El-Naggar A, Wai DH, Mistry P, Lazic N, Ayala FR, 16. Spurgeon SE, Coffey G, Fletcher LB, Burke R, Tyner JW, Druker BJ, et al. The et al. ERBB4 confers metastatic capacity in Ewing sarcoma. EMBO Mol Med selective SYK inhibitor P505-15 (PRT062607) inhibits B cell signaling and 2013;5:1019–34. function in vitro and in vivo and augments the activity of fludarabine in 32. Agelopoulos K, Richter GH, Schmidt E, Dirksen U, von Heyking K, Moser B, chronic lymphocytic leukemia. J Pharmacol Exp Ther 2013;344:378–87. et al. Deep sequencing in conjunction with expression and functional

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SYK Inhibition in Ewing Sarcoma

analyses reveals activation of FGFR1 in Ewing sarcoma. Clin Cancer Res 47. Chandran UR, Ma C, Dhir R, Bisceglia M, Lyons-Weiler M, Liang W, et al. 2015;21:4935–46. Gene expression profiles of prostate cancer reveal involvement of multiple 33. Druker BJ. Taking aim at Ewing's sarcoma: is KIT a target and will imatinib molecular pathways in the metastatic process. BMC Cancer 2007;7:64. work? J Natl Cancer Inst 2002;94:1660–1. 48. Yoshimoto R, Mayeda A, Yoshida M, Nakagawa S. MALAT1 long non- 34. Potratz J, Tillmanns A, Berning P, Korsching E, Schaefer C, Lechtape B, et al. coding RNA in cancer. Biochim Biophys Acta 2016;1859:192–9. Receptor tyrosine kinase gene expression profiles of Ewing sarcomas reveal 49. Baird K, Davis S, Antonescu CR, Harper UL, Walker RL, Chen Y, et al. Gene ROR1 as a potential therapeutic target in metastatic disease. Mol Oncol expression profiling of human sarcomas: insights into sarcoma biology. 2016;10:677–92. Cancer Res 2005;65:9226–35. 35. DuBois SG, Marina N, Glade-Bender J. Angiogenesis and vascular targeting 50. Hirata H, Hinoda Y, Shahryari V, Deng G, Nakajima K, Tabatabai ZL, et al. in Ewing sarcoma: a review of preclinical and clinical data. Cancer Long noncoding RNA MALAT1 promotes aggressive renal cell carcinoma 2010;116:749–57. through Ezh2 and interacts with miR-205. Cancer Res 2015;75:1322–31. 36. Guan H, Zhou Z, Gallick GE, Jia SF, Morales J, Sood AK, et al. Targeting Lyn 51. O'Donnell KA, Yu D, Zeller KI, Kim JW, Racke F, Thomas-Tikhonenko A, inhibits tumor growth and metastasis in Ewing's sarcoma. Mol Cancer Ther et al. Activation of transferrin receptor 1 by c-Myc enhances cellular 2008;7:1807–16. proliferation and tumorigenesis. Mol Cell Biol 2006;26:2373–86. 37. Greve B, Sheikh-Mounessi F, Kemper B, Ernst I, Gotte M, Eich HT. Survivin, 52. Minami Y, Nakagawa Y, Kawahara A, Miyazaki T, Sada K, Yamamura H, a target to modulate the radiosensitivity of Ewing's sarcoma. Strahlenther et al. Protein tyrosine kinase Syk is associated with and activated by the IL-2 Onkol 2012;188:1038–47. receptor: possible link with the c-myc induction pathway. Immunity 1995; 38. Bair JS, Palchaudhuri R, Hergenrother PJ. Chemistry and biology of 2:89–100. deoxynyboquinone, a potent inducer of cancer cell death. J Am Chem 53. Subbiah V, Anderson P. Targeted therapy of Ewing's sarcoma. Sarcoma Soc 2010;132:5469–78. 2011;2011:686985. 39. Buontempo F, Chiarini F, Bressanin D, Tabellini G, Melchionda F, Pession 54. Burke RT, Meadows S, Loriaux MM, Currie KS, Mitchell SA, Maciejewski P, A, et al. Activity of the selective IkappaB kinase inhibitor BMS-345541 et al. A potential therapeutic strategy for chronic lymphocytic leukemia by against T-cell acute lymphoblastic leukemia: involvement of FOXO3a. Cell combining Idelalisib and GS-9973, a novel spleen tyrosine kinase (Syk) Cycle 2012;11:2467–75. inhibitor. Oncotarget 2014;5:908–15. 40. Siegel MB, Liu SQ, Davare MA, Spurgeon SE, Loriaux MM, Druker BJ, et al. 55. Fitzer-Attas CJ, Lowry M, Crowley MT, Finn AJ, Meng F, DeFranco AL, et al. Small molecule inhibitor screen identifies synergistic activity of the bro- Fcgamma receptor-mediated phagocytosis in macrophages lacking the Src modomain inhibitor CPI203 and bortezomib in drug resistant myeloma. family tyrosine kinases Hck, Fgr, and Lyn. J Exp Med 2000;191:669–82. Oncotarget 2015;6:18921–32. 56. Wu Y, Huang C, Meng X, Li J. Long noncoding RNA MALAT1: insights into 41. Faria CC, Golbourn BJ, Dubuc AM, Remke M, Diaz RJ, Agnihotri S, et al. its biogenesis and implications in human disease. Curr Pharm Des Foretinib is effective therapy for metastatic sonic hedgehog medulloblas- 2015;21:5017–28. toma. Cancer Res 2015;75:134–46. 57. Gutschner T, Hammerle M, Diederichs S. MALAT1–a paradigm for long 42. Cheng S, Coffey G, Zhang XH, Shaknovich R, Song Z, Lu P, et al. SYK noncoding RNA function in cancer. J Mol Med 2013;91:791–801. inhibition and response prediction in diffuse large B-cell lymphoma. 58. Tripathi V, Shen Z, Chakraborty A, Giri S, Freier SM, Wu X, et al. Long Blood 2011;118:6342–52. noncoding RNA MALAT1 controls cell cycle progression by regulating the 43. Ferreira BI, Alonso J, Carrillo J, Acquadro F, Largo C, Suela J, et al. Array expression of oncogenic transcription factor B-MYB. PLoS Genet 2013;9: CGH and gene-expression profiling reveals distinct genomic instability e1003368. patterns associated with DNA repair and cell-cycle checkpoint pathways in 59. Malakar P, Shilo A, Mogilavsky A, Stein I, Pikarsky E, Nevo Y, et al. Long Ewing's sarcoma. Oncogene 2008;27:2084–90. noncoding RNA MALAT1 promotes hepatocellular carcinoma develop- 44. Georges SA, Biery MC, Kim SY, Schelter JM, Guo J, Chang AN, et al. ment by SRSF1 up-regulation and mTOR activation. Cancer Res 2016; Coordinated regulation of cell cycle transcripts by p53-Inducible micro- 77:1155–67. RNAs, miR-192 and miR-215. Cancer Res 2008;68:10105–12. 60. Wossning T, Herzog S, Kohler F, Meixlsperger S, Kulathu Y, Mittler G, et al. 45. Whitfield ML, Sherlock G, Saldanha AJ, Murray JI, Ball CA, Alexander Deregulated Syk inhibits differentiation and induces growth factor-inde- KE, et al. Identification of genes periodically expressed in the human pendent proliferation of pre-B cells. J Exp Med 2006;203:2829–40. cell cycle and their expression in tumors. Mol Biol Cell 2002;13: 61. Kim KH, Roberts CW. Targeting EZH2 in cancer. Nat Med 2016;22:128–34. 1977–2000. 62. Richter GH, Plehm S, Fasan A, Rossler S, Unland R, Bennani-Baiti IM, et al. 46. Hinata K, Gervin AM, Jennifer Zhang Y, Khavari PA. Divergent gene EZH2 is a mediator of EWS/FLI1 driven tumor growth and metastasis regulation and growth effects by NF-kappa B in epithelial and mesenchy- blocking endothelial and neuro-ectodermal differentiation. Proc Natl Acad mal cells of human skin. Oncogene 2003;22:1955–64. Sci U S A 2009;106:5324–9.

www.aacrjournals.org Clin Cancer Res; 23(15) August 1, 2017 4387

Identification of a Novel SYK/c-MYC/MALAT1 Signaling Pathway and Its Potential Therapeutic Value in Ewing Sarcoma

Haibo Sun, De-Chen Lin, Qi Cao, et al.

Clin Cancer Res 2017;23:4376-4387. Published OnlineFirst March 23, 2017.

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